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AMERICAN

MALACOLOGICAL

BULLETIN

SMITHS?^

^tWO5?003

-^^aries

Journal of the American Malacological Society

hUp://www. malacological. org

i

VOLUME 23 December 27, 2007 number 1/2

The Publications of the American Malacological Union/Society.

EUGENE V. CO AN and ALAN R. RABAT 1

Taxonomic occurrences of gastropod spermatozeugmata and non-stylommatophoran

spermatophores updated. ROBERT ROBERTSON 11

A developmental perspective on evolutionary innovation in the radula of the predatory
neogastropod family Muricidae.

GREGORY S. HERBERT, DIDIER MERLE, and CARLOS S. GALLARDO 17

Phylogenetic relationships of the columbellid taxa Cotoiiopsis and Cosrnioconcha

(Neogastropoda: Buccinoidea: Columbellidae). HELENA FORTUNATO 33

Family Pseudolividae (Caenogastropoda, Muricoidea): A polyphyletic taxon.

LUIZ RICARDO L. SIMONE 43

Gastropod mating systems: An introduction to the symposium. JANET L. LEONARD 79

Reproductive behavior of the dioecious tidal snail Cerithidea rliizophornrwn
(Gastropoda: Potamididae).

MAYA TAKEUCHI, HARUMI OHTAKI, and KIYONORI TOMIYAMA 81

Causes of variation in sex ratio and modes of sex determination in the Mollusca — an overview.

YOICHIYUSA

continued on back cover

Cover photo: Radula of Chicopiiwntus laqueatus (Sowerby, 1841 ) from Herbert et al.

AMERICAN MALACOLOGICAL BULLETIN

Kenneth M. Brown, Editor-iii-Chief
Department of Biological Sciences
Louisiana State Lhiiversity
Baton Rouge, Louisiana 70803

USA

BOARD OF EDITORS

Cynthia D. Trowbridge, Managing Editor

Department of Zoology

Oregon State University

Corvallis, Oregon 97331

USA

lattice Voltzow
Department of Biology
University of Scranton
Scranton, Pennsylvania 18510-4625
USA

Robert H. Cowie

Center for Conservation Research and Training

University of Hawaii

3050 Maile Way, Gilmore 408

Honolulu, Hawaii 96822-2231

USA

Carole S. Hickman

University of California Berkeley

Department of Integrative Biology

3060 VLSB #3140

Berkeley, California 94720

USA

Timothy A. Pearce

Carnegie Museum of Natural History
4400 Forbes Avenue
Pittsburgh, Pennsylvania 15213-4007
USA

Paula M. Mikkelsen
Paleontological Research Institution
1259 Trumansburg Road
Ithaca, New York 14850-1313
USA

Alan I. Kohn
Department of Zoology
Box 351800

University of Washington
Seattle, Washington 98195
USA

Dianna Padilla

Department of Ecology and Evolution
Stony Brook University
Stony Brook, New York 11749-5245
USA

Roland C. Anderson
The Seattle Aquarium
1483 Alaskan Way
Seattle, Washington 98101
USA

The American Malacological Bulletin is the scientific journal of the American Malacological Society, an international society of professional,
student, and amateur malacologists. Complete information about the Society and its publications can be found on the Society's website:
http://www.malacological.org

AMERICAN MALACOLOGICAL SOCIETY MEMBERSHIP

MEMBERSHIP INFORMATION: Individuals are invited to com-
plete the membership application available at the end of this issue.

SUBSCRIPTION INFORMATION: Institutional subscriptions are
available at a cost of $65 plus postage for addresses outside the
USA.

Further information on dues, postage fees (for members outside
the U.S.), and payment options can be found on the Membership
Application at the end of this issue.

ALL MEMBERSHIP APPLICATIONS, SUBSCRIPTION ORDERS,
AND PAYMENTS should be sent to the Society Treasurer:

Dawn E. Dittman

Tunison Laboratory of Aquatic Science
3075 Gracie Rd.

Cortland, New York 13045-9357
USA

CHANGE OF ADDRESS INFORMATION should be sent to the
Society Secretary:

Paul Callomon
Department of Malacology

The Academy of Natural Sciences of Philadelphia
1900 Benjamin Franklin Parkway
Philadelphia, Pennsylvania 19103-1195
USA

INFORMATION FOR CONTRIBUTIONS is available on-line and
appears at the end of this issue.

MANUSCRIPT SUBMISSION, CLAIMS, AND PERMISSIONS TO

REPRINT JOURNAL MATERIAL should be sent to the Editor-in-Chief:

Kenneth M. Brown, Editor-in-Chief

Department of Biological Sciences

Louisiana State University

Baton Rouge, Louisiana 70803

USA

Voice: 225-578-1740 • Fax: 225-578-2597
E-mail: kmbrown@lsu.edu

AMERICAN MALACOLOGICAL BULLETIN 23(1/2)
AMER. MALAC. BULL.

ISSN 0740-2783

Copyright © 2007 by the American Malacological Society

AMERICAN MALACOLOGICAL BULLETIN CONTENTS VOLUME 23 I NUMBER l/2

The Publications of the American Malacological Union/Society.

EUGENE V. GOAN and ALAN R. RABAT 1

Taxonomic occurrences of gastropod spermatozeugmata and non-stylommatophoran

spermatophores updated. ROBERT ROBERTSON 11

A developmental perspective on evolutionary innovation in the radula of the predatory
neogastropod family Muricidae.

GREGORY S. HERBERT, DIDIER MERLE, and CARLOS S. GALLARDO 17

Phylogenetic relationships of the columbellid taxa Cotouopsis and Cosmiocoucha

(Neogastropoda: Buccinoidea: Columbellidae). HELENA EORTUNATO 33

Family Pseudolividae (Caenogastropoda, Muricoidea): A polyphyletic taxon.

LUIZ RICARDO L. SIMONE 43

Gastropod mating systems: An introduction to the symposium. JANET L. LEONARD 79

Reproductive behavior of the dioecious tidal snail Cerithidea rhizophoranun
(Gastropoda: Potamididae).

MAYA TAKEUCHI, HARUMI OHTAKI, and KI YONORI TOMIYAMA 81

Causes of variation in sex ratio and modes of sex determination in the Mollusca — an overview.

YOICHIYUSA 89

Poecilogony and larval ecology in the gastropod genus Alderia. PATRICK J. KRUG 99

Food intake, growth, and reproduction as affected by day length and food availability in the
pond snail Lymnaea stagnalis.

ANDRIES TER MAAT, COR ZONNEVELD, J. ARJAN G.M. DE VISSER, RENE F. JANSEN,

KORA MONTAGNE-WAJER, and JORIS M. KOENE 113

Phally polymorphism and reproductive biology in Arioliwax (Ariolimax) buttotii (Pilsbry and
Vanatta, 1896) (Stylommatophora: Arionidae).

JANET L. LEONARD, JANE A. WESTFALL, and JOHN S. PEARSE 121

A review of mating behavior in slugs of the genus Deroceras (Pulmonata: Agriolimacidae).

HEIKEREISE 137

Reproductive biology and mating conflict in the simultaneously hermaphroditic land snail

Arianta arhustorum. BRUNO BAUR 157

A literature database on the mating behavior of stylommatophoran land snails and slugs.

ANGUS DAVISON and PETER MORDAN 173

The function of dart shooting in helicid snails. RONALD CHASE 183

Meeting Announcement 190

Melbourne Romaine Carriker: 25 February 1915 - 25 February 2007 An Appreciation.

CLEMENT L. COUNTS, III, ROBERT S. PREZANT, and J. EVAN WARD 191

Financial Report 195

Index to Vol. 23 197

Membership Form 201

Information for Contributors 203

1

\

i

1

I

j

Amer. Malac. Bull. 23; 1-10

The Publications of the American Malacological Union/Society

Eugene V. Coan^ and Alan R. Kabat^

‘ Santa Barbara Museum of Natural History, 2559 Puesta del Sol Road, Santa Barbara, California 93105, U.S.A.,
gene.coan@sierraclub.org

“ Museum of Comparative Zoology, Hai"vard University, Cambridge, Massachusetts 02138, U.S.A., alankabat@aol.com

Abstract: This paper documents the publications of the American Malacological Society, and its predecessor, the American Malacological
Union, from 1931 through 2007. Information on the dates of publication is included, based primarily on library receipt records. Several
publications were erroneously dated as to year, which has resulted in new taxa described in those publications, as well as abstracts and
articles, being attributed to the wrong year.

Key words: AMS, AMU, publication dates, bibliography

The American Malacological Union (AMU), now the
American Malacological Society (AMS), founcied in 1931,
issued reports containing abstracts and papers presented at
its annual meetings from its earliest years. At first these
reports were published in The Nautilus. Shortly thereafter,
reports in The Nautilus were supplemented by a printed
report mailed to all members after the annual meeting that
included a membership list. By 1941, The Nautilus no longer
included meeting reports. The titles of the AMU’s reports
changed several times over the years (Bieler and Kabat 1991 -
present). During World War II, when no meetings were
held, reports were issued to ensure communication and
continuity.

In 1971, the AMU’s publication became known as the
Bulletin. In 1983, the Bulletin became a peer-reviewed sci-
entific journal, the American Malacological Bulletin, with an
increasing number of original articles. By 1985, the American
Malacological Bulletin no longer included reports on and
abstracts of papers presented at the annual meeting, which
were published only in the program and abstracts volume
issued to meeting participants, with the organizer of each
meeting generally responsible for its publication. By this
time, the membership list was no longer included in the
annual report, and was instead published as part of, or in
connection with, the organization’s newsletter.

The American Malacological Union Newsletter began in
1968 and was issued irregularly. Its name changed to the
AMU News in 1984, to American Malacological Newsletter in
1996, thence to American Malacological Society Newsletter in
1999, and finally to the Newsletter of the American Malaco-
logical Society in 2003. The newsletter is now circulated
chiefly electronically and is available on the AMS website:
http://www.malacological.org/. In 2003, the membership list
moved from the newsletter to become a separate, electroni-
cally distributed publication.

The purpose of this paper is to provide the publication
dates of AMU/AMS publications. In many cases, incorrect
dates were printed on them, and some issues were misnum-
bered. As a result, abstracts, papers, and even new taxa that
appear in this series are sometimes misdated in subsequent
literature.

Although the AMU/AMS and its publications have been
documented in three histories (Murray 1999, Teskey 1964,
1982), three indices (Anonymous 1966, Counts 1988, Teskey
1975), and one listing (Anonymous 1981), none of these
provided fully accurate or complete dates for the AMU/AMS
publications. The Annual Report for 1967 (Report No. 34:
84) provided the dates of publication for the reports from
1961 through 1966; we are unaware of any other similar
listings.

The reports appearing in The Nautilus from 1931 to
1941 are easily dated using the compilation of Coan and
Harasewych (1993).

The individual reports and bulletins contain various
kinds of dates. The most common is the date the included
membership list was prepared, herein indicated by “Mem-
bership List.” These cannot be taken as publication dates,
although some other sources have used them, because the
report was often printed and mailed weeks or months after
the membership list was prepared. These dates, however, are
included here for comparison and to indicate that the pub-
lication could not have been issued any earlier.

Some reports are simply dated, without further indica-
tion as to what this date means (herein “Stated Date”). Start-
ing with volume 18 of the American Malacological Bulletin,
the printed date, for the first time, gives both the month and
day, and is intended and assumed to be the publication date,
based on receipt of mailed copies within one or two weeks.
In earlier years, however, these dates were demonstrably off
by a year. In some cases, the printed dates are labeled as

2

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

being specifically a publication date or mailing date, essen-
tially the same thing. In the mid-1990s, several issues did not
have the year on the cover or on the table of contents, but
the year was included on the spine; such data, of course, are
lost when the journal issues are bound, as is the case for most
libraries.

It is instructive to compare any printed dates with the
dates the publication was received in libraries, most of which
stamp the date of receipt on each copy upon arrival. How-
ever, this is also not entirely conclusive, because there may
be delays between receipt of the mail and date stamping.
Moreover, some institutions with malacologists on their
staffs did not separately subscribe to the AMU/AMS publi-
cations, and those institutions instead obtained their copies
through donations from these persons, often months or
years after publication. It appears that for some years the
reports were sent by airmail to Europe and by bulk mail
within the U.S., accounting for The Natural History Mu-
seum (London) having several of the earliest receipt dates.

We have examined the sets of AMU/ AMS publications
in the Academy of Natural Sciences of Philadelphia (ANSP),
Library of Congress (LOG), National Museum of Natural
History (USNM), Harvard University Museum of Compara-
tive Zoology (MCZ), California Academy of Sciences (CAS),
University of Maryland (UMd), University of Washington
Fisheries and Oceanography Library (UW), and The Natural
History Museum, London (BMNH). Three colleagues pro-
vided us with information on the sets at the Field Museum
of Natural History (FMNH), the American Museum of
Natural History (AMNH), and the Carnegie Museum of
Natural History (CMNH). When these dates are consistently
later than the published dates — sometimes much later — it is
clear that the stated publication dates must be incorrect. If
no date is given for a particular institution for a given year,
this means that the institution ( 1 ) lacks the volume in ques-
tion; (2) has the volume, but there is no date stamp; or (3)
has the volume, but the date stamp is several weeks, months,
years, or even decades later. In several cases, one of us (EVC)
noted the date of receipt of his personal copies.

We have also included the three “Special Publications "
which were issued in the 1980s as supplements to the Ameri-
can Malacological Bulletin. However, as these were not in-
cluded in the regular subscription, many libraries did not
order them.

When we are certain or fairly certain about publication
dates, these are indicated with a single date. In other cases,
we present the evidence thus far available, which in many
cases indicates that the Bulletins were issued the year follow-
ing the meeting, or months, or even a year after the internal
date.

For reference, the place and dates of the meetings
through 1985 are given, along with the number of the meet-

ing. After the publications stopped covering the meetings,
this information is not provided here, but is readily available
online (Coan and Rabat 1996-present). Also indicated are
the meetings of the Pacific Division of the AMU from its
inception in 1948 until its separation as the Western Society
of Malacologists in 1968.

The International Code of Zoological Nometiclature
(ICZN 1999) emphasizes that editors and publishers should
keep track of dates of publication in journals that may have
new taxa or other nomenclatural acts:

“Recommendation 2 1C. Specification of date. An
editor or publisher should state the date of publi-
cation of a work, and of each component part of a
serial publication, and of any work issued in parts.

In a volume made up of parts brought out sepa-
rately, the day of publication of each part, and the
exact pages, plates, maps, etc. that constitute it,
should be specified” (p. 23).

This paper is designed to bring the AMU/AMS publications
into retroactive compliance with the Code.

In the recent catalog of family and higher names in the
Gastropoda, and elsewhere, the subfamily names Pseudo-
melatominae Morrison and the (unavailable) Lophiotomi-
nae and Crassispirinae Morrison are dated from 1 December
1965, the date printed on AMU Annual Reports for 1965
(Bouchet and Rocroi 2005: 57, 101, 144, 256), but this report
was not actually published until 28 February 1966, as evi-
denced in the list below. Four papers by Morrison in AMU
publications are misdated in the bibliography of his works
by Rosewater (1984), including this paper on the Turridae.

From the list below, it is clear that publication dates
have been incorrectly stated, even in recent years. It is ob-
vious that in spite of a “publication date” of 30 December
1976, on the cover of the report for 1976, a January 1977
publication/mailing date is more likely because no one re-
ceived this issue until early February 1977. Fortunately, no
new taxa appear to be included. Similarly, American Mala-
cological Bidletin 3(1) is dated December 1984, but no insti-
tutions received it until mid-Februai-y 1985, and a late Janu-
ary or early February 1985 publication date is more likely.
No new taxa were included in this issue. The issue AMB 8( 1 )
was dated August 1990, but it appears not be have been
received anywhere before early October, so a publication
date of mid- to late-September seems more probable. Again,
no new taxa were included in this issue. However, AMB
17(1/2), indicated as having been published in December
2002, appears to have actually been issued in February 2003.
This issue contains new taxa of marine and terrestrial gas-
tropods, as well as a species of fossil rostroconch, and all of
these should evidently be dated 2003 rather than 2002.

AMERICAN MALACOLOGICAL SOCIETY PUBLICATIONS

3

ANNUAL REPORTS AND BULLETIN

Notes

* Publication dates of The Nautilus from Coan and Harasewych (1993).
n/a = not applicable.

* Issue numbers printed on some ot the Bulletins, but incorrectly numbered and not corresponding either to the number
of the meeting or to the sequential number of separately issued bulletins.

The Nautilus; American Malacologkal Union [Report]

Meeting

Publication

Earliest Date of
Publication

Later Receipt Dates

Comments

1 (Philadelphia,

The Nautilus 45{\): 1-5

13 July 1931

n/a

Publication date: Coan

Pennsylvania, 30 April-2
May 1931)
n/a

“Members of the American

1 April 1932

n/a

and Harasewych
(1993)

Date on cover

2 (Washington, D.C.,

Malacological Union,” 5
pages

The Nautilus 46(1): 1-3

23 July 1932

n/a

Publication date: Coan

26-28 May 1932)

“Mrs. Imogene C.

1932

n/a

and Harasewych
(1993)

Undated, presumably

3 (Cambridge,

Robertson’s Rambling
Notes”, 1 1 pages
The Nautilus 47(1): 37-44

16 June 1933

n/a

issued in 1932
Publication date: Coan

Massachusetts, 25-27
May 1933)

4 (Stanford, California,

The Nautilus 48(2): 72

15 October 1934

n/a

and Harasewych
(1993)

Publication date: Coan

25-28 June 1934)

Report, 12 pages

ANSP = 16 October 1934

n/a

and Harasewych
(1993)

Stated date = 1 August

5 (Buffalo, New York,

The Nautilus 49(2): 62-63

8 November 1935

n/a

1934

Publication date: Coan

27-29 lune 1935)

Report, 10 pages, 1 plate

not known; possibly late

n/a

and Harasewych
(1993)

[no data available]

6 (St. Petersburg, Florida,

The Nautilus 51(1): 33-36

1935 or 1936
14 July 1936

n/a

Publication date: Coan

21-24 April 1936)

The American

MCZ = 21 October 1936

n/a

and Idarasewych
(1993)

Membership List = 1

7 (Ann Arbor, Michigan,

Malacological Union
[Report], 14 pages
The Nautilus 51(2): 68-71

22 October 1937

n/a

September 1936
Publication date: Coan

3-5 August 1937)

8 (Havana, Cuba, 1-6

The American

Malacological Union
[Report], 16 pages
The Nautilus 52(2): 66-72

MCZ = 26 February 1938
28 October 1938

n/a

and Harasewych
(1993)

Membership List = 1
January 1938

Publication date: Coan

August 1938)

The American
Malacological Union
[Report], 20 pages,
frontispiece

MCZ = 28 April 1939

and Harasewych
(1993)

Membership List = 1
January 1939

4

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Earliest Date of

Meeting

Publication

Publication

Later Receipt Dates

Comments

9 (Toronto, Ontario,

The Nautilus 53(1); 36;

21 July 1939; 20 October

n/a

Publication date; Coan

Canada, 20-23 June

The Nautilus 53(2);

1939

and Harasevvych

1939)

68-72

(1993)

The American

MCZ = 2 January 1940

Membership List =

Malacological Union
[Report], 16 pages

November 1939

10 (Philadelphia,

The Nautilus 54(1); 35-37

23 July 1940

n/a

Publication date; Coan

Pennsylvania, 17-21 June

and Harasevvych

1940)

(1993)

The American

MCZ = 17 January 1941

Membership List =

Malacological Union
[Report], 18 pages

December 1940

1 1 (Rockland and

The Nautilus 55(2); 70-72

24 October 1941

n/a

Publication date; Coan

Thomaston, Maine,

and Harasewych

26-29 August 1941 )

(1993)

The American

Probably 1942

Membership List =

Malacological Union
[Report], 44 pages

January 1942

1942-1943 - No meetings

News Bulletin and Annual Report

Earliest Date of

Meeting

Publication

Publication

Later Receipt Dates

Comments

1944 - No meeting

News Bulletin and Annual

Probably 1944

Membership List =

Report, 24 pages,
frontispiece

January 1944

1945 - No meeting

News Bulletin and Annual

MCZ = 26 November

Membership List =

Report, 21 pages,
frontispiece

1945

October 1945

12 (Washington, D.C.,

News Bulletin and Annual

MCZ = 22 April 1947;

Membership List =

14-16 August 1946)

Report, 22 pages,
frontispiece

ANSP = 22 April 1947

April 1947

13 (Pacific Grove,

News Bulletin and Annual

CMNH = 20 February

Membership List =

California, 18-21 June
1947)

Report, 32 pages

1948

December 1947

14 (Pittsburgh,

News Bulletin and Annual

MCZ = 9 April 1949

ANSP = 18 April

Membership List =

Pennsylvania, 25-27
August 1948); 1, Pacific
Division (Los Angeles,
California, 10-11 April
1948)

Report, 36 pages

1949

March 1949

15 (Coral Gables, Florida,

News Bulletin and Annual

MCZ = 1 February 1950

ANSP = 6 February

Membership List =

16-18 June 1949); 2,
Pacific Division (Long
Beach, California, 14-16

Report, 36 pages

1950

November 1949

June 1949)

16 (Chicago, Illinois, June

News Bulletin and Annual

MCZ = 26 January 1951

ANSP = 1 February

Membership List =

14-16, 1950); 3, Pacific
Division (Santa Barbara,
California, 7-9 April
1950)

Report, 39 pages

1951

December 1950

AMERICAN MALACOLOGICAL SOCIETY PUBLICATIONS

5

Earliest Date of

Meeting

Publication

Publication

Later Receipt Dates

Comments

17 (Buffalo, New York,

News Bulletin and Annual

28 lanuary 1952 [“date

ANSP = 10 March

Membership List =

22-24 August 1951); 4,

Report, 41 pages

based on annotation by

1952; MCZ = 17

December 1951

Pacific Division

Morrison on reprint in

March 1952

(Oakland, California,

MNHN” (Bouchet and

22-24 lune 1951)

Rocroi 2005: 333)]

Annual Report

Earliest Date of

Meeting

Publication

Publication

Later Receipt Dates

Comments

18 (Cambridge,

Annual Report, 44 + [i]

MCZ = 4 February 1953;

Memlaership List = 31

Massachusetts, 20-22

pages

ANSP = 4 February

December 1952

August 1952); 5, Pacific
Division (Los Angeles,
California, 20-22 lime

1953

[inside front cover]

1952)

19 (Lawrence, Kansas,

Annual Report, 51 pages

MCZ = 15 March 1954;

Stated Date /

25-27 lune 1953); 6,

ANSP =15 March 1954

Membership List = 31

Pacific Division (Pacific
Grove, California, 12-13
lune 1953)

December 1953

20 (Durham, New

Annual Report, 44 pages

MCZ = 21 January 1955

ANSP = 26 January

Membership List = 31

Hampshire, 16-18
August 1954); 7, Pacific
Division (Los Angeles,
California, 18-19 June
1954)

1955

December 1954

21 (New York, New York,

Annual Reports 22*, 58

ANSP = 16 April 1956

MCZ = 1 7 April

Stated Date and

26-29 lune 1955); 8,

pages

1956

Membership List = 31

Pacific Division
(Stanford University,
California, 15-16 July
1955)

December 195

22 (San Diego, California,

Annual Reports 23*, 52

n/a

LOC = 23 February

Stated Date and

11-14 July 1956); 9,

pages

1958 [late]

Membership List, 31

Pacific Division (same)

December 1956

23 (Yale University, New

Annual Reports 24*, 56

UMd = 28 March 1958

Stated Date = 1 January

Haven, Connecticut,
16-19 July 1957); 10,
Pacific Division (Santa
Barbara, California, 30
May-1 June 1957)

pages

1958

24 (Ann Arbor, Michigan,

Annual Reports 25*, 73

UMd = 11 February 1959

Stated Date = 1 January

2-6 September 1958); 11,
Pacific Division
(Berkeley, California,
27-29 lune 1958)

pages

1959

25 (Philadelphia,

Annual Reports 26*, 79

UMd = 26 February 1960

Stated Date = 1 January

Pennsylvania, 30 June-3

pages

1960; meeting

July 1959); 12, Pacific

misnumbered as “26"

Division (Redlands,
California, 9-12 July
1959)

on cover and title page

6

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Meeting

Publication

Earliest Date of
Publication

Later Receipt Dates

26 (Montreal, Quebec,
Canada, 9-12 August
1960); 13, Pacific
Division (Pacific Grove,
California, 22-25 lune
1960)

Annual Reports 27*, 76
pages

EVC = February 1961

BMNH = 4
October 1961;
LOC = 2
November 1961

27 (Washington, D.C.,

Annual Reports 28*, 81

12 December 1961

BMNH = 8 lanuary

19-23 lune 1961); 14,
Pacific Division (Goleta,
California, 28 Iune-1
I Lily 1961)

pages

(Report for 1967, 34:
84)

1962; LOC = 10
lanuary 1962

28 (St. Petersburg, Florida,

Annual Reports 29*, 68

21 December 1962

LOC = 23 lanuary

31 |uly-3 August 1962);
15, Pacific Division
(Pacific Grove,
California, 27-30 lune
1962)

pages

(Report for 1967, 34:
84)

1963; BMNH =
28 lanuary 1963

29 (Buffalo, New York,

Annual Reports 30*, 83

1 lanuary 1964 (Report

LOC = 23 lanuary

18-21 lune 1963); 16,
Pacific Division (Goleta,
California, 26-29 lune
1963)

pages

for 1967, 34: 84)

1964; BMNH =
14 February
1964; UMd = 14
February 1964

30 (New Orleans,

Annual Reports 31*, 102

14 December 1964

LOC = 31

Louisiana, 21-24 luly
1964); 17, Pacific
Division (Pacific Grove,
California, 18-21 lune
1964)

pages

(Report for 1967, 34:
84)

December 1964

31 (New York, New York,

Annual Reports 32*, 122

28 February 1966 (Report

LOC = 3 March

20-23 lune 1965); 18,
Pacific Division (San
Diego, California, 24-27
lune 1965)

pages

for 1967, 34: 84)

1966; UMd = 7
March 1966

32 (Chapel Hill, North

Annual Reports 33*, 120

28 February 1967 (Report

LOC = 8 March

Carolina, 22-27 August
1966); 19, Pacific
Division (Seattle,
Washington, 19-22 lune
1966)

pages

for 1967, 34: 84)

1967; UMd = 16
March 1967

33 (Ottawa, Ontario,

Annual Reports 34*, 119

Mailing Date: 20 March

BMNH = 2 May

Canada, 31 Iuly-6
August 1967); 20, Pacific
Division (Pacific Grove,
California, 28-30 lune
1967)

pages

1968

1968

34 (Corpus Christi, Texas,

Annual Reports 35*, 97

Mailing Date = 27

UMd = 20 lanuary

15-19 luly 1968); 21,
Pacific Division (Pacific
Grove, California, 19-22
lune 1968)

pages

December 1968

1969

35 (Marinette, Wisconsin,

Annual Reports 36*, 96

Mailing Date = 19

USNM = 13

21-23 luly 1969)

pages

December 1969

January 1970

Comments

Stated Date = 1 lanuary
1961; meeting
misnumbered as “27”
on cover and title
page

Stated Date = 1
December 1961;
meeting misnumbered
as “28” on cover and
title page

Stated Date = 1
December 1962;
meeting misnumbered
as “29” on cover and
title page

Stated Date = 1
December 1963

Stated Date = 1
December 1964

Stated Date = 1
December 1965

Stated Date = 1
December 1966

Stated Date = 1
December 1967

Stated Date = 1
December 1968

Stated Date = 1
December 1969

AMERICAN MALACOLOGICAL SOCIETY PUBLICATIONS

7

Meeting

Publication

Earliest Date of
Publication

Later Receipt Dates

Comments

36 (Key West, Florida,

Annual Reports 37*, 112

Mailing Date = 18

UMd = 8 March

Stated Date = 1

16-20 luly 1970)

pages

February 1971

1971; FMNH =
23 March 1971

December 1970

Bulletin

Meeting

Publication

Earliest Date of
Publication

Later Receipt Dates

Comments

37 (Cocoa Beach, Florida,
15-19 July 1971)

Bulletiit, 70 pages

ANSP = 2 March 1972

FMNH = 8 March
1972

Stated Date = February
1972

38 (Galveston, Texas, 9-14
July 1972)

Bulletin, 64 pages

Publication Date = 23
March 1973

USNM = 30 March
1973

39 (Newark and Greenville,
Delaware, 24-28 June
1973)

Bulletin, 69 pages

Publication Date = 22
May 1974

ANSP = 28 May
1974; UMd = 28
May 1974

40 (Springfield,
Massachusetts, 3-7
August 1974)

Bulletin, 94 pages

Index, 1934-1974, [ii] -t 57
PP

Publication Date = May
1975

Probably issued with the
preceding in 1975

ANSP = 6 June
1975; UMd = 10
June 1975

undated

41 (San Diego, California,

Bulletin, 94 pages -i- inside

Publication date = 30

UMd = 20 February

22-26 lime 1975)

back cover

January 1976

1976

42 (Columbus, Ohio, 2-6
August 1976)

Bulletin, 89 pages

Publication date = 30
December 1976

UW = 9 February
1977; FMNH =
14 February 1977

43 (Naples, Florida, 10-15
July 1977)

Bulletin, 1 18 pages

BMNH = 24 April 1978

USNM = 28 April
1978; UMd = 28
April 1978

Membership List = 15
October 1977

44 (Wilmington, North
Carolina, 16-21 July 1978)

Bulletin, 86 pages

BMNH = 28 March 1979

USNM = 29 March
1979

Membership List = 15
October 1978

45 (Corpus Christi, Texas,
5-11 August 1979)

Bulletin, 86 pages

EVC = March 1980

USNM = 20 March
1980; BMNH =
21 March 1980

Membership List = 1
November 1979

46 (Louisville, Kentucky,

Bulletin, 94 pages

USNM = 19 March 1981;

LOC = 21 March

Membership List = 15

19-25 July 1980)

BMNH = 19 March
1981

1981

October 1980

47 (Ft. Lauderdale, Florida,

Bulletin, 76 pages

BMNH = 15 February

LOC = 23 February

Membership List = 20

19-25 July 1981)

1982

1982; ANSP = 23
February 1982;
UMd = 23
February 1982

October 1981

48 (New Orleans,

American Malacological

LOC = 28 June 1983

USNM = 17 July

Stated Date = July 1983

Louisiana, 19-23 July
1982)

Bulletin 1: 136 pages

1983

[cover]; May 1983
[1st page and Counts
(1988)]

49 (Seattle, Washington,

American Malacological

BMNH = 30 March 1984

ANSP = 3 April

Stated Date = February

7-13 August 1983)

Bulletin 2: 127 pages

1984; USNM = 3
April 1984

1984

50 (Norfolk, Virginia,

American Malacological

BMNH = 18 February

FMNH = 28

Stated Date = December

22-27 July 1984)

Bulletin 3(1): 1-133

1985

February 1985;
USNM = 28
February 1985

1984

8

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

American Malacological Bulletin

Meeting

Publication

Earliest Date of
Publication

Latest Date of
Publication

Comments

n/a

American Malacological
Bulletin 3(2): [ii]
+135-272

BMNH = 17 July 1985

FMNH = 18 luly
1985; LOG = 18
July 1985; UMd =
18 July 1985

Stated Date =

June 1985

n/a

American Malacological
Bulletin, Special Edition
1: 116 pages

(not known; presumably
on or after July 1985)

LISNM = 7 August
1986 [late];
BMNH = 24
August 1987 [late]

Stated Date =

July 1985

51 (Kingston, Rhode
Island, 28 July-2 August
1985)

American Malacological
Bulletin 4(1): 1-147

CAS = 5 March 1986

UW = 14 March
1986

Stated Date =
1986

February

Note: the 1985 meeting was the last one to be reported upon in detail in the American Malacological Bulletin. For additional
information about subsequent AMU/AMS meetings, see Coan and Rabat (1996-present).

American Malacological Bulletin

PuJilication

Earliest Date of Publication

Latest Date of Publication

Comments

American Malacological Bulletin,
Special Edition 2: 239 pages

(not Icnown; presumably on or
after June 1986)

FMNH = 1 May 1987 [late];
BMNH = 24 August 1987
[late]

Stated Date = June 1986

American Malacological Bidletin

FMNH = 12 September 1986

AMNH = 15 September 1986

Stated Date = August 1986

4(2): 149-247

American Malacological Bidletin,
Special Edition 3: 74 pages

(not known; presumably on or
after October 1986)

USNM = 24 April 1987 [late]

Stated Date = October 1986

American Malacological Bulletin
5(1): 1-152

BMNH = 11 February 1987;
AMNH = 11 February 1987;
FMNH = 11 February 1987

ANSP = 12 February 1987

Stated Date = January 1987

American Malacological Bulletin

BMNH = 14 July 1987

UW = 15 July 1987

Stated Date = June 1987

5(2): 153-306

American Malacological Bulletin

BMNFI = 25 February 1988

FMNH = 1 March 1988

Stated Date = January 1988

6(1): 1-164

American Malacological Bulletin
6(2): 165-305

October 1988

USNM = 10 October 1988

Stated Date = October 1988 [as
“July 1988” in Counts
(1988); corrected to October
in AMB 7(1): 89

American Malacological Bulletin
7(1): 1-91

BMNH = 24 May 1989; ANSP
= 24 May 1989

USNM = 26 May 1989; UMd =
26 May 1 989

Stated Date = April 1989

American Malacological Bulletin

UW = 23 March 1990

FMNH = 26 March 1990

Stated Date = February 1990

7(2): 93-175

American Malacological Bulletin

UW = 10 October 1990

BMNH = 12 October 1990

Stated Date = August 1990

8(1): 1-96

American Malacological Bulletin
8(2): 97-182

BMNH = 31 May 1991; ANSP
= 31 May 1991

UW = 3 June 1991; UMd = 3
June 1991

Stated Date = April 1991

American Malacological Bulletin
9(1): 1-[104]

BMNH = 30 December 1991

UW = 10 January 1992; ANSP =
11 January 1992

Issue 9(2): 219 gives
“December 1991”

American Malacological Bulletin
9(2): 105-219

EVC = August 1992

BMNH = 3 September 1992;
ANSP = 3 September 1992

Page 219 gives “August 1992”

American Malacological Bulletin
10(1): 1-[112]

USNM = 18 February 1993

ANSP = 19 February 1993

Issue 10(2): 295 gives
“February 1993”

AMERICAN MALACOLOGICAL SOCIETY PUBEICATIONS

9

Publication

Earliest Date of Publication

Latest Date of Publication

Comments

American Malacological Bulletin
10(2); iv + 113-295

ANSP = 8 December 1993

USNM = 9 December 1993

Preface gives “July 1993”; page
295 gives “November 1993”

American Malacological Bulletin

USNM = 30 December 1994;

Page iii gives “December 1994,”

11(1): iii -1- l-[86] pages

ANSP = 30 December 1994

as does issue 11(2): 211

American Malacological Bulletin

ANSP = 8 September 1995

USNM = 11 September 1995

Page 211 gives “August 1995”

11(2): ii + 87-[212]

American Malacological Bulletin
12(1-2); ii -t 1-[156]

AMNH = 24 January 1997

ANSP = 28 January 1997

I^age 155 gives “September
1996”; issue 13(1/2); 154
gives “October 1996”; spine
gives “1996”

American Malacological Bulletin
13(1/2): ii -t 1-[156]

AMNH = 10 January 1997

ANSP = 13 January 1997

Page 154 gives “December
1996”; spine gives “1996”

American Malacological Bulletin

ANSP = 5 January 1998

FMNH = 7 January 1998; LOC

Issue 14(2): 234 gives

14(1); i + 1-86

= 8 January 1998

“December 1997”

American Malacological Bulletin

LOC 25 January 1999

AMNH = 28 January 1999

Stated Date “December 1998”

14(2): ii -I- 87-234 pages

American Malacological Bidletin
15(1); iii -t 1-111

ANSP = 15 November 1999

AMNH = 16 November 1999

Stated Date “1999”; issue 15(2):
208 gives “October 1999”

American Malacological Bidletin
15(2); i -1- [113]-208

BMNH = 22 January 2001

AMNH = 29 [anuary 2001

Page 208 gives “December
2000”

American Malacological Bulletin
16(1/2): iii -1- 1-266 pages

EVC = 7 November 2001

Page 266 gives “September
2001”

American Malacological Bulletin

BMNH = 26 February 2003

ANSP = 3 March 2003; AMNH

Stated Date = “December

17(1/2): iii -h 1-165

= 4 March 2003

2002”; to be mailed January
2003 [e-mail, 3 Januaiy 2003
from A. Valdes to R. Bieler]

American Malacological Bulletin
18(1/2): i + 174 pages

7 May 2004

Publication date given on front
cover

American Malacological Bulletin
19( 1/2); i -1- 150 pages

14 October 2004

Publication date given on front
cover

American Malacological Bulletin
20( 1/2): ii -t- 165 pages

27 April 2005

Publication date given on front
cover

American Malacological Bulletin
21( 1/2); i -H 131 pages

9 February 2006

Publication date given on front
cover

American Malacological Bidletin
22( 1/2): V -1- 176 pages

26 March 2007

Publication date given on front
cover

OTHER PUBLICATIONS

Anonymous. 1940. H. A. Pilshry: Scientific coiitribiitions made from
1882 to 1939. American Malacological Union. 63 pages. (This
includes a short biography of Pilsbry, and may have been
edited by his colleague, H. B. Baker].

How to Collect Shells

The predecessor to this stand-alone work was included in the re-
port of the Eleventh Annual Meeting of the American Mala-
cological Union as full-length papers by F. C. Baker, H. van
der Schalie, W. (. Clench, B. R. Bales, T. Burch, (. S. Schwen-
gel, and T. L. McGinty [with one additional page of discus-
sion], 1942. Methods of collecting and preserving Mollusca.
Symposium papers. American Malacological Union, Eleventh
Annual Meeting, [Report]: 5-37.

Abbott, R. T., G. M. Moore, 1. S. Schwengel, and M. C. Teskey, eds.
1955 [March 31]. How to Collect Shells {A Symposium), 1st ed.
American Malacological Union, Buffalo, New York, [iv] -I- 75
+ [5] pp.

LaRocc]ue, A., ed. 1961. How to Collect Shells {A Symposium), 2nd
ed. American Malacological Union, Marinette, Wisconsin,
iv -I- 92 -H [5] pp.

Abbott, R. T., M. K. Jacobson and M. C. Teskey, eds. 1966. How to
Collect Shells [A Symposium), 3rd ed. American Malacological
Union, Marinette, Wisconsin, iv + 101 -I- [5] pp.

Jacobson, M. K., ed. 1974. How to Study and Collect Shells. {A
Symposium), 4th ed. American Malacological Union, Wrights-
ville Beach, North Carolina, [iv] -I- 107 pp.

Sherborn, C. D. and E. R. Sykes, 1906. [Reprint of] Museum Bolt-
enianum by P. F. Roding, 1798, [iii] -I- viii -I- 199 pp. [subse-

10

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

quently reprinted by the American Malacological Union,
1986],

Sturm, C. F., T. A. Pearce, and A. Valdes, eds. 2006 [25 Inly], The
Molliisks: A Guide to Their Study, Collection, and Preservation.
Boca Raton, Florida: Universal Publishers, xiii + 445 pp.

ACKNOWLEDGMENTS

We thank R. Bieler (LMNH), P. Mikkelsen (AMNH),
and T. A. Pearce (CMNH) for providing us with informa-
tion about the dates of receipt of AMU/AMS publications in
their institutional libraries. We also thank C. L. Sturm, lr.>
for information on the editions of How to Collect Shells and
R. E. Petit, J. Voltzow, and an anonymous reviewer for their
helpful reviews of an earlier draft of this paper.

introduced by loseph P. E. Morrison (December 17, 1906 -
December 2, 1983). The Nautilus 98(1): 1-9.

Teskey, M. C. 1964. Flistory of the American Malacological Union.
The Shelletter of Shells and Their Neighbors [California] 23: 7.

Teskey, M. C. 1975. Index, 1934 through 1974. American Malaco-
logical Union, 57 pp. [This index probably mailed with the
Bulletin for 1975, but was not published as an issue or as a
numbered supplement of that serial).

Teskey, M. C. 1982. Half-century of AMU. Bulletin of the American
Malacological Union for 1981: [iii]-[v].

Accepted: 5 February 2007

LITERATURE CITED

Anonymous. 1966. Abstract index (1949-1965) [and] author index,
abstracts, 1949-1965. American Malacological Union, Annual
Reports for 1965: 105-122.

Anonymous. 1981. Miscellanea. Annual Reports and Bulletins of
the American Malacological Lhiion. Malacological Review 14:
67-112.

Anonymous. 1989. [Correction]. American Malacological Bulletin
7(1): 89.

Bieler, R. and A. R. Rabat. 1991. Malacological lournals and News-
letters, 1773-1990. The Nautilus 105(2): 39-61, periodically
updated version available at: http://fml.fieldmuseum.org/
collections/search.cgi?dest=mjl

Bouchet, P. and l.-P. Rocroi. 2005. Classification and nomenclator
of gastropod families. Malacologia 47(1-2): 1-397.

Coan, E. V. and M. G. Harasewych. 1993. Publication dates of The
Nautilus. The Nautilus 106(4): 174-180.

Coan, E. V. and A. R. Rabat. 1996. Annotated catalog of malaco-
logical meetings, including symposia and workshops in mala-
cology. American Malacological Bulletin 13(1-2): 129-148, pe-
riodically updated version available at: http://erato.acnatsci
.org/ams/pdfs/symposia.pdf

Counts, C. L., 111. 1988. Index to the American Malacological Bul-
letin: 1983 to 1988. Volumes 1 through 6, Special Edition
numbers 1-3. American Malacological Bulletin 6(2): 219-305
[see also anonymous correction in 7(1): 89, 1989].

ICZN [International Commission on Zoological Nomenclature].
1999. International Code of Zoological Nomenclature. London,
England (International Trust for Zoological Nomenclature),
xxix -r 306 pp.

Murray, H. D. 1999. History {evolution) of the American Malaco-
logical Union (Society). Privately published, San Antonio,
Texas. 15 pp. [distributed at the 1999 meeting of the American
Malacological Society, Pittsburgh, Pennsylvania].

Rosewater, 1. 1984. A bibliography and list of taxa of Mollusca

Amer. Malac. Bull 23: 11-16

Taxonomic occurrences of gastropod spermatozeugmata and
non-stylommatophoran spermatophores updated

Robert Robertson’*^

Department of Malacology, The Academy of Natural Sciences of Philadelphia, 1900 Benjamin Franklin Parkway,

Philadelphia, Pennsylvania 19103-1 195, U.S.A., hhandrrconch@aol.com

Abstract: Spermatozeugmata, not to be confused with spermatophores, that also transfer sperm, are compound structures (parasperms
with attached eusperms) known only in certain “mesogastropods”: Loxonematoidea? (Abyssochrysidae), Littorinoidea (Littorinidae),
Triphoroidea (Triphoridae and Cerithiopsidae), Tonnoidea (Ranellidae), Janthinoidea (Epitoniidae and Janthinidae), and doubtlully
Cypraeoidea (Cypraeidae). This pattern of taxonomic occurrence does not match that of any other character known, their morphology is
diverse, and it is concluded that the spermatozeugmata in these taxa are not all homologous and that, like spermatophores, they have
evolved repeatedly. Littorinid spermatozeugmata have frequently been studied after fixation and shrinkage of the parasperms (“nurse cells”),
during which the eusperms drop off. Spermatozeugmata are not a synapomorphy linking the Triphoroidea and Janthinoidea. Records of
spermatophores (except in the “pulmonate” suborder Stylommatophora) since Robertson (1989) are updated.

Key words: sperm transfer, inferred homoplasy, euspermatozoa, paraspermatozoa, nurse cells

SPERMATOZEUGMATA

Spermatozeugmata (singular: spermatozeugma) are not
to be confused with spermatophores (Robertson 1989) al-
though both transfer sperm. Spermatozeugmata are of in-
tracellular origin and consist of single paraspermatozoa
(Hodgson 1997, Buckland-Nicks et al. 2000) with numerous
euspermatozoa (fertile sperm) attached externally by their
acrosomes. Parasperm are also called apyrene sperm and
nurse cells. They are known only in “prosobranchs”, but do
not all become spermatozeugmata. Spermatophores are se-
creted extracellularly and contain the eusperms. Spermato-
phores occur spioradically in many major groups within all
gastropods, including many stylommatophorans. Sperma-
tozeugmata are even more sporadic and are known only in
certain “mesogastropods”:

Incertae sedis (Loxonematoidea?): Abyssochrysidae:
Abyssochrysos (Healy 1989).

Littorinoidea: Littorinidae: Littoraria (Reinke 1911, as
“Littorina”)y Littorina, Littoraria (Reinke 1912, latter as “Lif-
torina”), Littorina (Ankel 1930: 599, 600; Linke 1933), Lit-
toraria (Woodard 1942a, 1942b, Lenderking 1954, all as ‘'Lit-
torina”, latter: nurse cell as “spermatophore”), Melarhaphe
(Battaglia 1952, as “Littorina”), Littoraria (Marcus and Mar-
cus 1963, as “Littorina”), Bembicium (Bedford 1965), Cen-
chritis (as “Tectarius”), Tectarins (as “Echininus”), Littoraria
(as “Littorina”), Nodilittorina (in part as “Littorina” (all
Borkowski 1971), Littorina (Buckland-Nicks 1973, Buck-

Current address: 510 Homestead Ave., Haddonfield, New lersey
08033, U.S.A.

land-Nicks and Chia 1977), Nodilittorina (Jordan and
Ramorino 1975, as “Littorina”), Littoraria (Reid 1986, Healy
and Jamieson 1993, Buckland-Nicks et al. 2000).

Triphoroidea [including “Cerithiopsoidea”]: Triphori-
dae: Triplwra (Houston 1985), Viriola (Healy 1987, 1990).
Cerithiopsidae: Cerithiopsis (Fretter and Graham 1962,
Houston 1985), Seda (Houston 1985, Healy 1990).

Cypraeoidea?: Cypraeidae?: Erronea? (Healy 1986a, as
“Cypraea”).

Tonnoidea: Ranellidae: Fusitriton (Buckland-Nicks et al
1982).

Janthinoidea [“Epitonioidea”]: Epitoniidae: Epitoniiiin
(Ankel 1926, 1938: 6-9, 1958, all as “Scala”, Fretter 1953, as
“Clathrus”), Opalia (Bulnheim 1962a, 1962b), Epitoniuni
(Nishiwaki 1964, Tochimoto 1967, Bulnheim 1968, Nishi-
waki and Tochimoto 1 969), Opalia, Epitoniuni (Melone et al.
1978, 1980, latter as “Scala”), Epitoniuni (Robertson 1983a,
1983b, Collin 2000, as “Nituiiscala”). fanthinidae: Jantliina
(Ankel 1926, 1930, Laursen 1953, Graham 1954, Wilson and
Wilson 1956, as “lantliina”). Curiously, two epitoniid spe-
cies have dimorphic spermatozeugmata (Nishiwaki and To-
chimoto 1969).

The all-“mesogastropod” taxonomic pattern of occur-
rence of spermatozeugmata is non-congruent with any other
character known. Robertson ( 1985, 1989: table 1 ) and Collin
(1997, 2000) reported the congruent occurrences of up to
five non-homologoLis characters, suggesting that the taxa are
related. I'he non-congruence of spermatozeugmata and their
varied morphologies suggest that they are not homologous
between the superfamilies listed above. The .same descriptive
name in different taxa does not make a character homolo-

11

12

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

gous. The compound origin and nature of spermatozeug-
mata could well have originated by homoplasy. The Tri-
phoridae appear to have both spermatozeugmata and
spermatophores, perhaps in different genera, but this needs
confirmation.

The distinctive nurse cells of littorinids are believed to
be parasperms. They are characteristic in being spherical to
oblong, with or without projecting rods. In the Littoraria
subgenus Pahistoriua the nurse cells are elongate or fusi-
form, having a pseudotrich. Reid has not seen and reported
attached eusperms since 1986, perhaps because since then he
has consistently studied littorinid parasperms after fixation
and shrinkage. As Reinke (1912), Woodard (1942b), Marcus
and Marcus (1963), Jordan and Ramorino ( 1975), and Healy
and Jamieson (1993) have observed, the acrosomes are at-
tached weakly and the eusperms drop off easily. Perhaps all
littorinids have these evanescent “spermatozeugmata”. A
cypraeid [Erronea] appears to have dimorphic parasperms:
vermitorm ones as well as spherical, littorinid-like “nurse
cells.” Eusperms are semi-attached only to the latter, and
these “could be considered a form of ‘spermatozeugmata’”
(Healy 1986a).

Healy (1987) stated that the spermatozeugmata of Vi-
riola (Triphoridae) are “very mobile”, but in 1990 he stated
that they and those of Seila (Cerithiopsidae) are “capable of
only slow movement.” Original observations on the behav-
ior of living janthinoidean spermatozeugmata are included
in Wilson and Wilson (1956), Nishiwaki and Tochimoto
(1969), Melone et al. (1980), and Robertson (1983a). Uni-
formly, there seems to be pseudocopulation. They are not
“vigorously mobile,” swimming with “considerable speed”
on their “relatively long journeys.”

Eretter (1953), Healy (1986b, 1987, 1990, 1994), and
Niitzel (1998) believed that because of their spermatozeug-
mata the Triphoridae and Cerithiopsidae are related to the
Janthinoidea. They are similar in being large and containing
numerous axonemes, but otherwise they are morphologi-
cally different in the three groups. Niitzel (1998: 2) went so
far as to suggest that spermatozeugmata are the “most con-
vincing synapomorphy.” Overlooking Niitzel’s monograph,
Collin (2000) reviewed most of the characters traditionally
used to support the “Ptenoglossa” (including the Eulimidae
but excluding the Architectonicidae, neither of which has
known spermatozeugmata). There appear to be no other
possible synapomorphies. Other literature on the Triphoroi-
dea and Janthinoidea bears this out: Pruvot-Eol (1925,
1952), Johansson (1947, as “Scala”, 1953), Risbec (1953),
Graham (1954), Marcus and Marcus (1963), Houston
(1985), Houbrick ( 1987), and Collin (2004). “Ptenoglossan”
radulae are diverse morphologically. If there is only one
supposed “synapomorphy” linking two superfamilies, the
validity of it may be questioned. Thus, I agree with Ponder

(1998) that it is improbable that Triphoroidea and Janthi-
noidea are closely related.

SPERMATOPHORES

Records in non-stylommatophorans since Robertson
(1989):

Neritoidea: Neritiliidae: Pisulina (Kano and Kase 2002),
Neritilia (Kano et al 2001, Kano and Kase 2003). Neritidae:
Clithon, Neritina [as “Neriptewn” and “Vittina”] (Starmiihl-
ner 1970), Neritina, Septaria (Starmiihlner 1974), Clithon,
Neritina, Septaria (Starmiihlner 1976), Clithon, Neritina
(Starmiihlner 1983, 1984), Nerita, Neritilia, Neritina, Pu-
perita (Starmiihlner 1988), Septaria (Haynes and Wawra
1989), Nerita, Neritina (Houston 1990), Theliostyla (Zehra
and Perveen 1991), Nerita, Piiperita, Clithon, Neritina, Sep-
taria, Neritilia (Starmiihlner 1993), Nerita (Sasaki 1998),
Septaria (Haynes 2001), Septaria, Neritina (Haynes 2005).
Phenacolepadidae: Cinnalepeta? (Sasaki 1998).

Campaniloidea: Plesiotrochidae: Plesiotrochiis (Hou-
brick 1990). [Placement: Healy 1993].

Cerithioidea: Cerithiidae: Bittium? Bittiolnin (Houbrick
1993), Cerithinni (Houbrick 1992). Dialidae?: Diala? (Pon-
der 1991). Melanopsidae (as “Thiaridae; Melanopsinae”):
Fauniis (Houbrick 1991b). Paludomidae (including Thiari-
dae, s. /. ): Lavigeria (Michel 1995), Tanganyicia (West 1997,
Strong and Glaubrecht 2002), Tiphobia, Paramelania, Lim-
notrochus, Chytra, Tanganyicia, Stanleya, Mysorelloides, Rey-
nwndia, Lavigeria, Spekia, Stornisia (Glaubrecht and Strong
2004). Pleuroceridae: Elimia (Jones and Branson 1964, as
“Mudalia”), Semisulcospira (Nakano and Nishiwaki 1989).
Potamididae: Terebralia (Houbrick 1991a). Scaliolidae:
Finella (Ponder 1994). Thiaridae, s. /.: Vinnndu (Michel
2004). Thiaridae, s. s.: Thiara (Glaubrecht and Strong 2004). ®

Turritellidae: Tiirritella (Kennedy 1995).

Pterotracheoidea: Atlantidae: Atlanta (Jamieson and
Newman 1989). Carinariidae: Pterosoma (Lalli and Gilmer
1989). The “spermatophores” in Atlantidae reported by
Tesch (reference in Robertson 1989) are actually the egg
cases of a pleustonic insect, Halobates (Seapy 1996).

Vermetoidea: Vermetidae: Dendroponia, Serpidorbis,
Vernietns (Calvo and Templado 2005).

Pyramidelloidea: Pyramidellidae: Pyramidella? (Ponder
1987), Boonea (Wise 2001), Fargoa (Robertson 1996), lolaea
(Hori and Kuroda 2001 ), Odostoniella (Schander et al. 1999),
Parthenina (Hori and Kuroda 2002).

Cavolinioidea: Cavoliniidae: Diacria (Lalli and Gilmer
1989). Limacinidae: Limacina (Lalli and Gilmer 1989).

|1

Clionoidea?: Pneumodermatidae?: Criicibranchaea? ’
(Lalli and Gilmer 1989).

GASTROPOD SPERMATOZEUGMATA AND SPERMATOPHORES

13

Hedylopsoidea: Parhedylidae: PoutoJjedylc, Unela
(Poizat 1989).

Aeolidioidea: Aeolidiidae: Aeolidiella (Haase and Karls-
son 2000).

Spermatophore presence can he difficult to ascertain,
and a “spermatophore hursa” in a pallial oviduct may not
always receive one (despite my assumption to the contrary in
1989: 362, A7). In Robertson (1989) I reported spermato-
phores in the cerithioidean Litiopidae, hut Houhrick ob-
served only bursae in them. Glaubrecht and Strong (2004)
inferred the spermatophore-forming organ in male paludo-
mid cerithioideans.

ACKNOWLEDGEMENTS

This study would not have been possible without access
to the magnificent Ewell Sale Stewart Library of The Acad-
emy of Natural Sciences of Philadelphia.

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Accepted: 14 May 2007

Amer. Maine. Bull. 23: 17-32

A developmental perspective on evolutionary innovation in the radula of the
predatory neogastropod family Muricidae"^

Gregory S. Herbert\ Didier Merle^, and Carlos S. Gallardo^

' Department of Geology, University of South Florida, Tampa, Florida, U.S.A., gherbert@cas.usf.edu

^ Unite de Paleontologie, Departement Histoire de la Terre, Museum national d’Histoire naturelle, UMR 5143 CNRS, 8, rue Buffon,
75005 Paris, France, dmerle@mnhn.fr

^ Institute de Zoologia, Universidad Austral de Chile, Casilla 567, Valdivia, Chile, cgallard@uach.cl

Abstract: The neogastropod family Muricidae includes a diverse set ot radular bauplane, including a beaked, three-dimensional form, a
flattened-pentacusped form, and a third “dagger” type in which the central rachidian cusp is massive and elongate. Examination of the
radular ontogenies of representatives of five muricid subfamilies reveals that several species undergo changes in radular form during
ontogeny on a scale comparable to the evolutionary differences between higher taxa. The species Concholepas concholepas (Bruguiere, 1789)
(Rapaninae) and Trophoii geversianiis (Pallas, 1774) (Trophoninae) begin ontogeny with a tliree-dimensional rachidian characteristic of the
Ocenebrinae or Muricopsinae but end with the dagger rachidian typical of their respective subfamilies. Young individuals of Vitularia
salebrosa (King and Broderip, 1832) (Muricopsinae?) also have a three-dimensional rachidian but shift to a double-dagger morphology by
adulthood. Chicoreus (Phyllonotiis) pomiim (Gmelin, 1791) (Muricinae) has a typical flattened muricine rachidian as an adult but possesses
a “buccinoid”-like rachidian just after hatching. Urosalpinx cinerea (Say, 1822) (Ocenebrinae), was unique among the species examined in
exhibiting no ontogenetic changes in radular form. The occurrence of two radular bauplane within the same individual snail during
ontogeny suggests great potential for rapid, convergent evolution of adult features through simple changes in developmental timing. A
three-dimensional rachidian, for example, could be retained into adulthood through paedomorphosis in any lineage possessing the
three-dimensional-to-dagger ontogeny. Systematic assignments of muricids based solely on radular features should be reexamined.

Key words: muricid, rachidian, bauplan, ontogeny, heterochrony

The radulae of a number of gastropod species undergo
small to large-scale changes in the number, type, and struc-
tural complexity of teeth between the pre-metamorphic lar-
val stage, when the radula first forms, and maturation (ref-
erences in Fujioka 1984a, Page and Willan 1988, Nybakken
1990, Waren 1990). A controversial but long-standing idea is
that such changes in development play a dominant role in
the evolutionary origins of new characters, or innovations,
and, hence, in the origin ot higher taxa (reviewed in Gould
1977). Rather than requiring widespread alteration of struc-
tural genes controlling morphology, innovation may derive
simply through small-scale changes in regulatory genes con-
trolling the rate and/or timing of development, i.e., het-
erochrony. Size changes associated with heterochronic evo-
lution may also provide a catalyst for extensive innovation
throughout the organism. Changes in skeletal structure, for
example, often evolve to compensate for the detrimental
by-products of developmental miniaturization (see refer-
ences in Hanken 1985, Hanken and Wake 1993).

With few exceptions (e.g., Guralnick and Lindberg

1999), however, molluscan biologists have yet to investigate
radular evolution from the perspective of development. In
the present study, we document radular ontogenies in sev-
eral taxa belonging to the predatory gastropod family Mu-
ricidae and examine the possibility that the evolution of
developmental timing has played a central role in the re-
peated origins of subfamily-level radular bauplane within
the Muricidae. Phylogenetic studies are necessary to test the
hypothesis that heterochronic mechanisms have been in-
volved in the evolution of any particular structure, but on-
togenetic analyses presented herein are a necessary first step.

BACKGROUND

Radular bauplane in the Muricidae

Two nomenclatural schemes have been utilized in the
past to describe the basic structural types (referred to
throughout this paper as “bauplane”) of the muricid rachid-
ian teeth and to delineate the muricid subfamilies. The first

* From the symposium “Relationships of the Neogastropoda” presented at the meeting of the American Malacological Society, held 31 iuly-
4 August 2004 at Sanibel Island, Florida.

17

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

system, established by Arakawa (1962, 1964, 1965) and Wu
(1965, 1968, 1973), groups muricids according to the num-
ber of cusps on the rachidian tooth. These workers subdi-
vided muricid radulae into two classes — a complex “pen-
tacusped” rachidian having a central cusp, two lateral cusps,
and two marginal cusps, and a simplified “tricusped” rachid-
ian having only the central and two lateral cusps (Fig. 1).
Fujioka (1985a) later added a third class for taxa having a
“monocusped” rachidian (i.e., only a central cusp) and rec-
ognized a number of intermediate classes as well. As noted
by Kool (1987), however, this system suffered from the
rather arbitrary manner in which it was applied and is no
longer used. For example, some authors counted only major
cusps but others counted both cusps and denticles. The vari-
able size, position, and number of cusps and denticles in
different muricids make this system impossible to apply un-
ambiguously (Kool 1987).

An alternative system, built upon in this paper, was
developed by Yokes (1971), Radwin and D’Attilio (1971,
1976), and D’Attilio (1980, 1991) and is based largely upon
variation in the morphology of the most prominent struc-
ture on the rachidian — the central cusp — rather than the
number of cusps on the tooth. In the “flattened” type, the
rachidian is broad, with all the cusps lying in the same plane
and the central cusp being slightly longer than either of the

laterals (Fig. 2M). This type occurs in almost all species
presently assigned to the subfamilies Muricinae, Typhinae,
Tripterotyphinae, and Haustrinae, as well as in many species
currently assigned to the Trophoninae and the Miirexsid-
Muricopsis genus group of the Muricopsinae (Radwin and
D’Attilio 1971, 1976, Yokes 1971) (Figs. 2A-L). Because the
Muricinae predate all other muricid subfamilies by at least
20 million years (see Yokes 1971, 1990, 1992, 1994, Garvie
1991, 1992, Marko and Yermeij 1999, Merle 1999, Yermeij
and Carlson 2000), and because the anatomical condition of
the Muricinae is likely primitive within the Muricidae (Ha-
rasewych 1984), the flattened rachidian type of muricines
and other muricids is presumably the plesiomorphic condi-
tion for the family.

In a second type, which Yokes (1971) referred to as
“three-dimensional” or “3-D,” the rachidian base is narrow
(Fig. 3J) and rectangular, with a short, beak-like central cusp
that projects up to 90 degrees away from the rachidian base
and up to 45 degrees away from either lateral cusp (Fig. 31).
Yokes (1971) and Radwin and D’Attilio (1971) have used
terms such as “triangular harrow,” “cowl-like,” and “fang-
like” to describe this type as well. A 3-D rachidian type
characterizes Yokes’ (1971) Murexiella genus group of the
Muricopsinae (= Favartia/Pygmaepterys subclade of Merle
and Houart 2003), some species of Murkopsis (see Radwin

Figure 1. Pentacusped, tricusped, and monocusped rachidian bauplane of Arakawa (1962, 1964, 1965), Wu (1965, 1968, 1973), and Fujioka
( 1985a). A. The pentacusped rachidian characterized by the rapanine Neothais harpa (Conrad, 1837), locality: Maui, Hawaiian Islands, scale
bar = 50 pm. B. The tricusped rachidian characterized by the ergalataxine Crania crassiilnata (Hedley, 1915), locality: Gulf of Carpentaria,
northern Australia, scale bar = 50 pm. C. The monocusped rachidian characterized by the rapanine Dmpella data Blainville, 1832, Kauai,
Hawaiian Islands, scale bar = 20 pm. D-F, penta-, tri-, and monocusped rachidia, modified from Fujioka (1985a, fig. 8).

DEVELOPMENT AND EVOLUTION OE THE MURICID RADULA

19

Figure 2. The “flattened” rachidian bauplan. A-B. The muricine Aspella indentata Carpenter, 1857, locality; Mira Mar, north of Manzanillo,
Mexico, scale bar = 50 pm. C-D. The muricine Miirex brevispina var. macgilUvrayi Dohrn, 1862, locality: Papau New Guinea, scale bar =
100 pm. E-F. The “trophonine” Xymetwpsis bucdneiis (Lamarck, 1816), locality; Tierra del Fuego, Argentina, scale bar = 50 pm. G-H. The
haustrine Haiistrmn haustoriiim (Gmelin, 1791), locality; New Zealand, scale bar = 100 pm. 1-1. The muricopsine Miircxsul octagoinis Quoy
and Gaimard, 1833, locality: New Zealand, scale bar = 50 pm. K-L. The typhine Typhisahi graiidis (A. Adams, 1855), locality: Golfo tie
Tehuantepec, Mexico, scale bar = 50 pm. M. The muricine rachidian bauplan, modified from Vokes (1971, fig. 2a).

and D’Attilio 1976), and the Ocenebra-Ocinebrina genus
group of the Ocenebrinae {sensu Vermeij and Vokes 1997).
Other muricid taxa that possess a beak-like central cusp
include the putative-muricopsines Vitularia Swainson, 1840;
Acanthotrophon Hertlein and Strong, 1951; and Bizetiella
Radwin and D’Attilio, 1972; and the muricine Chicopinnatiis
laqueatus (Sowerby, 1841) (Figs. 3A-H).

In addition to these two general categories of rachidia,

we recognize a third — a “dagger” rachidian — for muricids
having a flattened rachidian but modified with a more mas-
sive and consicierably more elongate central cusp and much
weaker lateral and marginal cusps (Fig. 41). Fujioka’s
(1985a) “monocusped” rachidian (Figs. 1C, IF) is an ex-
treme form of this third type. Muricids possessing a dagger-
type rachidian include most rapanine and ergalataxine spe-
cies as well as the “I7nn's-like” ocenebrines (c.g., Nticella

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Florida, scale bar = 50 |am. C-D. The muricopsine Caribiella alveata (Kiener, 1842), locality. Discovery Bay, Jamaica, scale bar = 20 pm.
E-F. The muricopsine Acanthotrophon sorenseni Hertlein and Strong, 1951, locality: Gulf of California, Mexico, scale bar = 20 pm. G-H.
The muricine Chicopmnatiis laqueatus (Sowerby, 1841), locality: Orote Point, Guam, Oceania, scale bar = 20 pm. I-J. The three-dimensional
rachidian bauplan, modified from Yokes (1971, fig. 2b).

Roding, 1798; Acanthimi Fischer de Waldheim, 1807; etc.,
see Vermeij and Yokes 1997), Trophon geversianus (Pallas,
1774) (type species of the type genus of the Trophoninae),
and several prohlematic South American genera {e.g.. Chorus
Gray, 1847; Xanthochoriis Fischer, 1884) (Figs. 4A-H).

Researchers have long relied upon these bauplane to
assign species to subfamilies and reconstruct muricid phy-
logeny based on the assumption that the radula is a conser-
vative character complex relative to features of the shell and
operculum (Yokes 1964, 1971, Radwin and D’Attilio 1971,
1976, fiouart 1992; see additional references in Kool 1987).
Yokes (1971), for example, regarded the subfamily Ocene-
brinae as phylogenetically nested within the Muricopsinae
on the basis of both groups sharing a 3-D rachidian, despite
the fact that other morphological features, such as those of
the early shell whorls and operculum, support different re-
lationships (Yokes 1971, Kool 1993a, Merle 1999, Yermeij
and Carlson 2000). Using the same logic, Radwin and
D’Attilio (1976) assigned the genus Vitiilaria to the Muri-
copsinae, although shell and opercular characters are diffi-

cult to reconcile with this classification (see Yokes 1967,
1977, 1986). Most recently, Bouchet and Houart (1996) re-
assigned the muricine Chicoreus gubbi (Reeve, 1849) to the
Ocenebrinae as a new genus Chicocenebra Bouchet and
Houart, 1996 based solely on its having a 3-D rachidian,
even though its classification as a member of the Muricinae
had not been questioned previously when only the shell
morphology of this species was known.

Previous studies of radular development and evolution
in muricids

If bauplan-level transformations in the muricid radula
can occur as a result of small-scale changes in developmental
timing, then radular features may be less conservative and,
thus, less phylogenetically informative, than is currently
thought. At present, the best answer to this question conies
from a series of seminal papers by Fujioka (1982, 1984a,
1984b, 1985a, 1985b) on the radulae of the subfamilies
Rapaninae and Ergalataxinae. Fujioka found that the lateral
cusps, marginal cusps, and intermediate and marginal den-

DEVELOPMENT AND EVOLUTION OF THE MURICID RADULA

21

Figure 4. The dagger rachidian bauplan. A-B. The ergalataxine Cronia (Cronia) avellana (Reevem, 1846), locality: western Australia, scale
bar = 100 pm. C-D. The rapanine Agnewia tritoniformis (Blainville, 1833), locality: Manly, New South Wales, Australia, scale bar = 50 pm.
E-F. The ocenebrine Nucella ostrina Gould, 1852, locality: Monterey, California, scale bar = 20 pm. G-H. The ergalataxine? Xaiithochoriis
cassidiformis Blainville, 1824, locality: Metri Bay, Chile, scale bar = 50 pm. I. The ergalataxine rachidian bauplan, modified from Fujioka
(1985a, fig. 8).

tides in many species of these two subfamilies become pro-
gressively atrophied during ontogeny, while the central cusp
becomes longer and its base becomes wider.

Rapanines, however, begin ontogeny with a pen-
tacusped rachidian and typically end at the pentacupsed or
tricusped stage, whereas many of the ergalataxines studied
begin ontogeny at the tricusped stage and end with a tri-
cusped or monocusped rachidian. Under the assumption
that new characters, such as atrophication, are only added to
the end of ontogeny, Fujioka reasoned that the less atrophied
pentacusped rachidia of rapanines is the relatively primitive
condition and that the ergalataxine condition evolved hy
peramorphic heterochrony (extended atrophication) of this
ancestral ontogeny. Phylogenies published recently for the
Rapaninae generally support this evolutionary scenario with
ergalataxines depicted as a nested clade within the Rapaninae
{i.e., Kool 1993a, Vermeij and Carlson 2000, but see Tan
2003).

More recently, DiSalvo ( 1988) and DiSalvo and Carriker
(1994) documented ontogenetic changes in the rachidian
tooth morphology of the rapanine muricid Coticholepas con-
cholepas (Bruguiere, 1789). This species was found to change
from a 3-D rachidian in early post-metamorphic animals to
a dagger-type rachidian in small sub-adults. Although these
workers did not comment on the evolutionary significance
of their observations or document the exact size at which
this transition occurs, it suggests to us that the 3-D and
dagger rachidia in adults could potentially evolve rapidly
from one to the other through heterochronic processes.

Focus of the present study

A 3-D rachidian was reported in none of the many
juvenile rapanine species studied hy Fujioka, which makes its
more recently reported occurrence in the rapanine Coticho-
lepas suspect. Thus, the initial goal of the present study was
to test the results of DiSalvo and Carriker by collecting new

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

early post-larval specimens for Concholepas concholepas,
verifying their taxonomic identity, and re-examining their
early stage radulae using SEM.

The second focus of this paper was to examine the on-
togenies of muricids outside of the Rapaninae and Ergal-
ataxinae. With the exception of Houart’s (1992) illustration
of the radular ontogeny of the muricine Chicoreus (Triplex)
torrefactiis (Sowerby, 1841), there have been no attempts to
document ontogenetic series in non-rapanine or non-
ergalataxine muricids. Our present study of new and previ-
ously collected material allows us to examine for the first
time the ontogenies of species representing the Trophoni-
nae, Ocenebrinae, a second species of the Muricinae, and a
putative member of the Muricopsinae.

The third focus of this study was to investigate whether
large-scale morphological shifts occur during the earliest on-
togenetic stages of development, i.e., between larval meta-
morphosis and juveniles around 10 mm shell length. Eujioka
examined no radulae from early post-metamorphic stage
individuals to small juveniles 3 mm in shell length. Eor more
than half the species Eujioka studied, he examined no juve-
niles smaller than 10 mm in length, and many of his smallest
“juveniles” were greater than 25 mm in shell length. DiSal-
vo’s work, in contrast, suggested that changes during earliest
ontogeny [i.e., less than 10 mm shell length) may be sub-
stantial (DiSalvo 1988, DiSalvo and Carriker 1994) and pos-
sibly be related to changes in mode of predation and feeding
behavior that can occur within this size range (see discussion
section). This study documents the endpoints of the entire
ontogenetic sequences beginning with rachidia in earliest
post-metamorphic individuals, material permitting.

MATERIALS AND METHODS
Radula preparation

Radulae were recovered from late juvenile and adult
alcohol-preserved and dried specimens by dissolving dis-
sected proboscis tissues in a concentrated solution of potas-
sium hydroxide (KOH) for 1-3 days. Radular ribbons, visible
with the naked eye, were collected with forceps, rinsed in a
series of hot distilled-water washes, fixed to aluminum tabs
with double-sided conductive tape, air dried, and gold
coated (40 nm thickness) for scanning electron microscopy.

Radulae of early juvenile snails were recovered from
dried and alcohol preserved specimens by gently crushing
the larval shells in a shallow petri dish filled with a concen-
trated solution of KOH. After two hours, the dish was heated
to 90°C to reverse any precipitation of KOH crystals that
might have formed on the radular ribbon. Because they were
too small to be collected with forceps, radulae were removed
from the KOH solution using a dropper partially filled with

warm distilled water. This was done to dilute the KOH so-
lution collected with the radula and prevent later precipita-
tion of KOH crystals on the radular ribbon during drying.
The dilute KOH solution with the radula was then trans-
ported by dropper through two rinses of hot distilled water
in separate dishes for additional dilution. After two rinses,
cleansed radulae were transferred in distilled water by drop-
per to an aluminum tab coated with double-sided conduc-
tive tape and air dried. Aluminum tabs with radulae were
gold coated (40 nm thickness). All radulae were examined
with an ISI DS-130 scanning electron microscope at the
University of California, Davis’s Eacility for Advanced
Instrumentation.

Radula Terminology

Throughout the remainder of this paper, we use the
standard terminology illustrated by Radwin and D’Attilio
(1976) and Kool (1987, 1993a) to refer to parts of the ra-
chidian radular tooth.

RESULTS

Subfamily RAPANINAE

Concholepas concholepas (Bruguiere, 1789)

(Figs. 5A-F)

Material examined

Nineteen juvenile to sub-adult specimens of Concho-
lepas concholepas ranging in shell length from 11 to 30 mm I
were collected in March 2001 from Mehuin, 70 km north of
Valdivia, Chile, and placed in 50% ethanol for later dissec- '
tion. An additional nine early post-metamorphic individuals
ranging from 1.7 to 3.9 mm were captured between Septem- i
ber 1999 and lanuary 2000 and between January 2000 and
August 2000 from artificial collection plates installed at Las
Cruces (coast of Santiago Province), Chile, and stored in
50% ethanol. ^

Ontogeny

In early post-metamorphic juveniles with shell lengths
ranging from 1.7 to 3.9 mm, rachidian widths are 20 pm and
have a 3-D rachidian morphology (Figs. 5A-D). Most have a ;
single marginal denticle, although the largest individual in ;
this class had two in one marginal area and one in the other ■
(Fig. 5A). The rachidian base end-point is marked by a |’
prominent marginal cusp that runs parallel to the rachidian
base. Behind this marginal cusp is a shorter marginal cusp
that is poorly developed and bud-like. The two cusps com-
bined loosely resemble the double marginal cusps of the 1
Ocenebrinae. :■

In young animals with shell lengths between 11 and 13 j

DEVELOPMENT AND EVOLUTION OF THE MURICID RADULA

23

Figure 5. Radular ontogeny of the rapanine muricid Concholepas coiiciwlepas. A-D. Front and lateral views of rachidia of early post-
metamorphic individuals ranging from 1.7 to 3.9 mm in shell length, locality: Las Cruces, Chile, scale bars = 10 pm. E-F. Front and lateral
views of rachidia of a small juvenile with shell length of 15 mm, locality: Mehuin, Chile, scale bar = 50 pm.

mm, the rachidian tooth increases to approx. 100 pm in
width and assumes the dagger-type morphology (Figs. 5E-
F). The lateral cusps are turned outwardly at their distal
ends, marginal cusp number increases to two or three, and
outer lateral denticles begin to appear. The rachidian base

begins to develop a small, rounded lateral extension at both
ends, which may be homologous with the hud-like second
marginal cusp observed in the early post-metamorphic ju-
veniles. The radular ontogeny of Concholepas is essentially
stabilized at the dagger-type rachidian by a shell length of 1 1

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

mm with little modification afterwards. Large adults may
reach shell lengths upwards of 125 mm. Radulae of larger
adult animals are figured elsewhere (Kool 1987, 1993b, Di-
Salvo 1988) for comparison.

Remarks

DiSalvo (1988) and DiSalvo and Carriker (1994) illus-
trated the rachidian tooth morphology of the pediveliger
stage (1.6- 1.9 mm shell length) of the rapanine Concholepas
coticholepas from neustonic pediveliger larvae reared from
egg capsules captured at sea and hatched in the lab. The
present study confirms their data showing this early stage to
have a 3-D nrchidian. Possession of a 3-D-type rachidian
during any stage of ontogeny in a rapanine is unusual be-
cause previous studies of radular ontogeny of rapanines and
a nested subclade within the Rapaninae, the Ergalataxinae,
have reported only the flat rachidian type at any stage of
development (Fujioka 1984a, 1984b, 1985a). However, given
the size range of juvenile rapanines examined by Fujioka, it
is possible that this stage of ontogeny was overlooked.

The transition from 3-D to dagger-type rachidian oc-
curs after metamorphosis (DiSalvo, pers. comm.) and be-
tween shell lengths of 4 and 11 mm (this study).

Subfamily TROPHONINAE

Trophon geversianus (Pallas, 1774)

(Figs. 6A-D)

Material examined

Research material for Trophon geversianus (Pallas, 1774)
was obtained from dried and alcohol-preserved specimens in
the personal collection of E. H. Yokes (Tulane University).
This material included ten dried pre-hatched juveniles, all
less than 2 mm in shell length, removed from a single egg
capsule collected at a beach at Rio Grande, Tierra del Euego,
Argentina. Although not yet hatched, the animals appear to
have undergone metamorphosis, as indicated by the initia-
tion of teleoconch (adult) sculpture. Also sampled were
twenty alcohol-preserved adult specimens (all > 30mm)
from Bahia El Pescador, south of Puerto Piramides, in the
northeastern part of Golfo Nuevo, Argentina. This collection
was also from the collection of E. H. Yokes.

Ontogeny

In post-metamorphic, pre-hatched juveniles of this spe-
cies, the rachidian tooth is approx. 10 pm in width and has
a 3-D morphology (Figs. 6A-B). The intermediate denticle is
long and has an attachment site on the rachidian base inde-
pendent of the lateral cusp but closer to the lateral than the
central cusp. The basal region is rectangular and deep, with
a strong marginal cusp and a second bud-like cusp closer to
the radular ribbon.

Rachidian teeth in adults sampled are approx. 200 pm in
width and exhibit a dagger- type morphology (Figs. 6C-D).
The intermediate denticle is shorter than in early ontogeny
(only one-fifth the height of the lateral cusp) and fused with
the lateral cusp instead of having a separate attachment site
on the rachidian base. The outer edges of the lateral cusps
possess small serrations or outer lateral denticles. The basal
end-point is rectangular but shallow and marked by a single
marginal cusp. The second bud-like cusp of early ontogeny
is obsolete or nearly so.

Remarks

Several authors have published line drawings and scan-
ning electron micrographs of the radulae of adult Trophon
geversianus, including Radwin and D’Attilio (1976), Kool
( 1993a), and Pastorino (2002). The present study is the first
to document the morphology of the rachidia in early post-
metamorphic individuals.

Subfamily OCENEBRINAE

Urosalpinx cinerea (Say, 1822)

(Eigs. 7A-D)

Material examined

Specimens of the ocenebrine muricid Urosalpinx cinerea
were obtained in May 2001 from intertidal barnacle, mussel,
and bryozoan-encrusted rocks from a jetty in San Erancisco
Bay in Burlingame, California, USA. This species is native to
the western Atlantic, but was introduced to the eastern Pa-
cific nearly a century ago (Radwin and D’Attilio 1976).
Twenty large adults (20-30 mm), including both males and
females, were collected at the Burlingame locality and pre-
served immediately in 75% ethanol for later dissection. An-
other 10 individuals were placed in a single aquarium with
recirculating seawater and provided with barnacles for food.
Within days, adult snails deposited egg capsules on tank
walls. After approx, six weeks, several hundred hatchlings
(1. 5-2.0 mm shell length) emerged from the capsules as
crawl-away juveniles and began drilling barnacles provided
and cannibalizing one another by drilling. Approximately 50
hatchlings were harvested immediately and preserved in
75% ethanol.

Ontogeny

Animals ~2.0 mm in shell length possess rachidia 10 pm
in width with a 3-D morphology (Pigs. 7A-B). Adult rachidia
are larger (approx. 100 pm) but nearly identical in shape
(Pigs. 7C-D).

Remarks

Radwin and D’Attilio (1976) and Kool (1993b) illus-
trated the adult radula of Urosalphix cinerea, but there have

DEVELOPMENT AND EVOLUTION OE THE MURICID RADULA

25

Figure 6. Radular ontogeny of the trophonine muricid Twphon geversiauiis. A-B. Front and lateral views of rachidia ot early post-
metamorphic (pre-hatched) individuals with shell lengths of 1.5 to 2.0 mm, locality: Tierra del Fuego, Argentina, scale bars = 10 pm. C-D.
Front and lateral views of rachidia of adult specimen with shell length of 30 mm, locality: Golfo Nuevo, Argentina, scale bar = 100 pm.

been no studies of radular morphology during early ontog-
eny. Carriker (1969) figured the mature radula of a subspe-
cies, Urosalpinx cinerea var. etterae Baker, 1955, including
eight scanning electron micrographs of radulae from various
angles and two light micrographs of a rasping radula in the
process of drilling a shell. The radula of this subspecies ap-
pears to differ from that of the nominate form in having one
or two extra marginal denticles.

Subfamily MURICOPSINAE?

Vitularia salebrosa (King and Broderip, 1832)

(Eigs. 8A-I)

Material examined

One dried juvenile specimen of Vitularia salebrosa (9.9
mm shell length) and ten dried adult specimens (30-50 mm
shell length) from various tropical eastern Pacific localities
were obtained from the personal research collections of E. H.

Vokes and G. 1. Vermeij. No other specimens of this species
were available at the time the study was conducted.

Ontogeny

At the small juvenile stage (one 9.9 mm specimen), the
rachidian is just over 5 pm in width and resembles the 3-D
type in having a short, beak-like central cusp (Eigs. 8A-C).
However, the rachidian base is Hat ratber than rectangular, a
condition typically associated with flattened rachidia. Adja-
cent to the central cusp are three pairs of short, conical
“cusps.” The intermediate denticle is unusual for the 3-D
type in projecting further from the rachidian base than the
adjacent “lateral” cusp. The attachment site for the denticle
is separate from the lateral cusp as in some muricopsines.
The outermost cusp sits far from the margin endpoint,
which curves into a pseudo-cusp. The rachidian ot a larger
specimen (31 mm shell length) differs in having a slightly
longer outermost cusp.

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Figure 7. Radular ontogeny of the ocenebrine miiricid Urosalpinx cinerea. A-B. Front and lateral views of rachidia of early post-
metamorphic individuals with shell lengths of 1.5 to 2.0 mm, locality: San Francisco Bay, California, scale bar = 5 pm. C-D. Front and lateral
views of rachidia of adult specimen with shell length of 25 mm, locality: San Francisco Bay, California, scale bar = 50 pm.

The largest individuals of Vitularia salehrosa available to
us (40 to 50 mm specimens) failed to produce a radula in
eight of the nine specimens we examined (89%). A single
radula from a shell 50 mm in length shows rachidian teeth to
be approx. 70 pm in width with long, tusk-like outermost
(marginal?) cusps situated far from the base endpoint (Figs.
8G-I). The overall morphology of the central cusp is roughly
of the 3-D rachidian type, but the outermost cusps are mas-
sive and elongated as in the typical dagger-type rachidian.

Remarks

D’Attilio’s ( 1991 ) investigation of the radula of Vitularia
salehrosa showed that this species has at least two different
radular morphotypes, including a “normal” rachidian with
seven cusps and a second rachidian with only three cusps,
which he described as “extremely aberrant resembling noth-
ing else known to me.” The latter morphotype has one sub-
obsolete central cusp and two massive, incurved, tusk-like
lateral cusps. D’Attilio did not provide information on the

sizes of the “normal” and “aberrant” rachidia, but the pres-
ent data suggest they could represent end-members of a
single ontogenetic sequence.

A second “aberrant” feature of the radula of Vitularia
salehrosa is its occasional absence. D’Attilio (1991) reported
that his own efforts to recover a radula from this species
were successful only twice out of ten total attempts, which is
similar to the success rate of one out of nine attempts re-
ported in this study. This species is parasitic on oysters and
attacks by pushing the proboscis between the valve margins,
aided initially by drilling (G. S. Herbert and G. P. Dietl, pers.
obs.). Attacking oysters at the edge could result in amputa-
tion of the proboscis and radular loss when the oyster closes
its valves. Another possibility is that the radula is used only
to initiate an edge-drilled hole, and afterwards, the animal
reabsorbs used teeth and stops forming new ones as it begins
a parasitic existence. A major group of muricid parasites, the
coralliophilines, lacks a radula, but these are obligate para-
sites, whereas V. salehrosa is not.

DEVELOPMENl' AND EVOLUTION OF THE MURICID RADULA

27

Figure 8. Radular ontogeny of the muricopsine? muricid Vitularin saicbwsa. A-C. Front and lateral views of racliida of small juvenile with
shell length of 9.9 mm, Fig. C shows worn portion of radula presumably used in feeding, locality: Venado Beach, Panama, scale bar = 5
pm. D-F. Front and lateral views of rachidia of medium-sized juvenile 31 mm in shell length, locality: Venado Beach, Panama, scale bar
= 20 pm. G-I. Front and lateral views of rachidia of mature specimen with shell length of 50 mm, locality: Panama, scale bar = 50 pm.

We place this species tentatively in the Muricopsinae
after Radwin and D’Attilio (1976), although this assignment
was and remains controversial due to shell and opercular
similarities of this genus to some members of the Ocenebri-
nae (Vokes 1986). In a cladistic analysis of the Muricidae
based on morphological characters of the shell, ovocapsules,

and radula (D. Merle and G. S. Herbert, unpubl. obs.),
Vitularia is unec]ui vocally placed outside the Muricopsinae
and may prove to be a sister group ol the Ocenehrinae.
Further phylogenetic investigations are necessary to clarify
the systematic position of this problematic genus within the
Muricidae.

28

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Subfomily MURICINAE

Chicoreus (Phyllonotus) ponium (Gmelin, 1791)

(Figs. 9A-D)

Material examined

Ten adult specimens of the muricine muricid Chicoreus
(Phyllonotus) poniurn were collected from shallow subtidal
seagrass beds in December 2002 in St. loseph’s Bay, Florida,
USA and transferred to aquaria, where they were monitored
and fed regularly. Females deposited communal masses of
egg capsules, and offspring hatched within several weeks.
Several hundred individuals (1.0-1. 5 mm shell length)
hatched with a brief pediveliger stage of approx. 24 hours
before absorbing the velum and using only the foot for lo-
comotion. After a week, many juveniles began cannibalizing
one another by drilling. These fully metamorphosed juve-
niles were collected then and preserved in 75% ethanol. Five
adults (60-70 mm shell length), including males and females.

were also preserved in ethanol after being relaxed in a 7.5%
isotonic solution of magnesium chloride.

Ontogeny

Early post-metamorphic juveniles (1.0-1. 5 mm shell
length) have a rachidian that is 15 pm wide and has only
three cusps of similar lengths and lying in the same plane,
with intermediate denticles usually absent (Figs. 9A-B). Each
rachidian of an adult snail is roughly 200 pm in width, and
has a wider base, more massive cusps, a new intermediate
denticle, and a more elongate central cusp (Figs. 9C-D).

Remarks

Radwin and Wells (1968) and Radwin and D’Attilio
(1976) figured line drawings of the mature radula of
Chicoreus (Phyllonotus) ponnim. There are no other pub-
lished illustrations of the early post-metamorphic or juvenile
stage radular morphologies of this species.

Figure 9. Radular ontogeny of the muricine Chicoreus (Phyllonotus) ponnim. A-B. Front views of rachidia showing irregular presence of
intermediate denticle in an early post-metamorphic individual with shell length of 1.5 mm, locality: St. loseph’s Bay, Florida, scale bar =
10 pm. C-D. Front and lateral views of rachidia of mature individual with shell length 63 mm, locality: St. Joseph’s Bay, Florida, scale
bar = 100 pm.

DEVELOPMENT AND EVOLUTION OF THE MURICID RADULA

29

The absence of intermediate denticles and overall ap-
pearance of the rachidian in young individuals of Phyllono-
tus pomum gives this radular element a generalized neogas-
tropod or “buccinoid” appearance. Similar rachidia occur in
buccinids but also in olivids, melongenids, and turbinellid
neogastropods, for example. Most interesting is the fact that
this rachidian type has not been documented previously
within the Muricidae. It is the first-known link in rachidian
form between muricids and non-muricids.

DISCUSSION

Fujioka (1982, 1984a, 1984b, 1985a, 1985b) was the first
to report that the muricid radula has the capacity to undergo

radical transformation between subfamily-level bauplane
during ontogeny. He found, specifically, that the ergalatax-
ine monocusped rachidian likely evolved through peramor-
phic heterochrony, i.c., extension of an ancestral rapanid
ontogeny characterized by progressive reduction of all but
the central cusp. With the exception of a brief treatment ot
the radular ontogeny of the muricine Chicoreus (Triplex)
torrefactiis by Houart (1992), however, we have, until now,
known nothing of the ontogenies of the radulae of other
muricids.

The present study demonstrates that major transforma-
tions between radular bauplane are nearly pervasive within
the Muricidae, with transformations occurring in four ol the
five subfamilies studied (Fig. 10). The ontogeny ot Phyllo-
notiis pomum also links the seemingly disparate rachidian

Rachidian type:

Three-Dimensional

Dagger

Concholepas concholepas

Stages

Trophon geversianus

Young

Adult

Urosalpinx cinerea

Vitularia salebrosa

Rachidian type:

Buccinoid

Flattened

Chicoreus (Phyllonotus) pomum

Figure 10. Generalized patterns of radular ontogeny in five species of muricid gastropod. A-B. Most of the species studied herein begin
ontogeny with the three-dimensional rachidian tooth as found in the Ocenebrinae and Muricopsinae but end with a dagger rachidian typical
of the Rapaninae and Ergalataxinae as well as some Trophoninae and the Thais-Wke Ocenebrinae (;.e., Nucella, Acanthina, etc.). C-D. One
species, Phyllonotus pomum, begins ontogeny with a generalized neogastropod (“buccinoid”) rachidian and shifts to a flattened, pentacusped
rachidian with intermediate denticles typical of the Muricidae.

30

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

morphologies of muricid and non-muricid neogastropods,
and suggests that the primitive, pentacusped rachidian of the
Muricidae evolved through extension of development.
Equally interesting is the widespread occurrence of the 3-D
rachidian that, until now, has been considered a defining
trait of only the Muricopsinae and the Ocenebrinae. Data
presented or reviewed in this paper demonstrate that the
3-D rachidian also occurs in at least some species of the
Muricinae [Chicopinnatus and Chicoreus (Triplex)], Rapani-
nae (Concholepas), and Trophoninae (Trophon).

Feeding observations for one of the species studied, the
rapanine Concholepas concholepas, suggest that the 3-D ra-
chidian could be a functional specialization for scraping
against hard substrata, whereas the dagger-type rachidian
that occurs later in ontogeny could be specialized for non-
drilling modes of prey subjugation. Individuals of C. con-
cholepas drill shelled prey or rasp rock surfaces in search of
algae exclusively during their early post-metamorphosis
stage, when the animal has the 3-D rachidian, but shift to
attacking prey by stabbing or lacerating the soft parts with
the radula through natural orifices by the time they reach 10
to 15 mm in shell length, when it has the dagger-type ra-
chidian (Castilla et al. 1979, Paine and Suchanek 1983,
DiSalvo 1982, 1988, Dye 1991, DiSalvo and Carriker 1994).

Muricids that possess a 3-D rachidian as adults, i.e.,
most species of the Ocenebrinae and Muricopsinae, also use
drilling as their primary or exclusive mode of attack (G. S.
Herbert, pers. obs.), again tying this rachidian bauplan to a
specific drilling function. Ocenebrine and muricopsine mu-
ricids also tend to be exceedingly small relative to other taxa
in the Muricidae and, thus, most similar ecologically to ju-
venile rather than large adult individuals of Concholepas con-
cholepas. It may well be that young or small muricids lack the
power to incapacitate prey using faster techniques, such as
toxins or the brute force of chipping and prying (Herbert
2004), thus requiring slower methods for feeding such as
drilling (Diet! and Herbert 2005) and, hence, a specialized
radular type. Studies using computer modeling of the vari-
ous radular morphologies within the Muricidae will be nec-
essary to understand the exact functional basis of these radu-
lar types.

It is striking that essentially the same ontogenetic trend
toward a more flattened rachidian base and elongation of
just one or two cusps occurs in species that differ at all stages
in important details of cusp number, position, and shape.
Such differences suggest that basic structural similarities
(i.e., 3-D vs. flattened vs. dagger forms) among adult ra-
chidia may be the result of independent innovation. Once
the first 3-D-to-dagger ontogenetic trajectoiy evolved, any
descendent lineages possessing this generalized ontogeny
would have had the opportunity to retain the 3-D rachidian

into adulthood independently through evolutionary trunca-
tion or a slowing of development. For these reasons, it is
important to revisit past systematic assignments based on
the bauplane that are the focus of the present study (see
background section).

The repeated evolution of new traits, or innovations,
has been a central theme of the muricid radiation (Vermeij
1998, 2001, Marko and Vermeij 1999, Vermeij and Carlson
2000), perhaps more so than in any other neogastropod
clade. Although this phenomenon has been examined in the
past from the standpoint of extrinsic factors, such as envi-
ronmental conditions (e.g., temperature, productivity) and
community dynamics (e.g., presence of incumbents, compe-
tition intensity), the ontogenetic data presented herein point
to a complementary process, namely the evolution of devel-
opmental timing. Major morphological transformations
during ontogeny increase the amount of phenotypic varia-
tion in the population upon which natural selection can act
and, thus, the intrinsic capacity of a species to create new
characters or transform existing ones. They can also reduce
genetic constraints on repetitive innovation by allowing
morphologies or structures already present at one stage of
development to shift to later (or earlier) stages through
small-scale changes in the rate or timing of development.

ACKNOWLEDGMENTS

The authors would like to thank Emily Vokes, Geerat
Vermeij, and Gregory Dietl for providing some of the ma-
terial used in this study; Louis DiSalvo for providing useful
information about the biology of Concholepas concholepas;
Maroniae Oleson for assisting us with lab and field work;
and Jerry Harasewych for kindly inviting us to participate in
the Relationships of the Neogastropoda symposium at the
2004 American Malacological Society meeting at Sanibel. We
also thank Geerat Vermeij and two anonymous reviewers for
providing helpful comments on earlier drafts of this paper.

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Accepted: 26 September 2006

Amer. Make. Bull 23: 33-42

Phylogenetic relationships of the columbellid taxa Cotonopsis and Cosmioconcha
(Neogastropoda: Buccinoidea: Columbellidae)'^

Helena Fortunate^

Smithsonian Tropical Research Institute, Center for Tropical Paleoecology and Anthropology, P.O. Box 0843-00153,

Balboa, Panama RP, fortunae@si.edu

Abstract: Phylogenetic reconstructions are still lacking for many molluscan groups, making evolutionary inferences much weaker. The
genera Cotonopsis Olsson, 1942 and Cosmioconcha Dali, 1913 are part of the so called Strombina group, and as such have been used as
models to study patterns of speciation and extinction brought about by the rise of the Central American gateway. Earlier work, based on
a few species of each genus, pointed towards a very close relationship of these genera, which prompted a complete cladistic analysis,
including all species of both genera to evaluate the level of relationship. Cladistic analyses based on shell morphology support the
monophyly of the group composed by Cotonopsis -I- Cosmioconcha. Cotonopsis as currently defined is paraphyletic and includes Cosmio-
concha. Cotonopsis (Tuirina) keeps its constituency and may retain its subgeneric status. Cotonopsis sensu stricto should he redefined to
include part of Cosmioconcha. Cosmioconcha should be subdivided into two groups. One of these groups should be included in Cotonopsis
sensu stricto. The second group should be given subgeneric status. Cotonopsis has a much earlier time of origination and most probably
derives from Cosmioconcha. Obtained results give support to some of the evolutionaiy patterns documented earlier tor the Neogene
molluscan faunas of tropical America and contribute to a better understanding of the Plio-Pleistocene divergence and turnover events
related to the rise of the Panamanian land bridge.

Key words: gastropods, phylogeny, columbellids, morphology

The family Columbellidae is one of the most diverse and
abundant shallow-water gastropod groups. The family has
undergone rapid radiation, with over 400 species having
evolved since the Danian Paleocene (Keen 1971, Abbott
1974, Radwin 1977, Tracey et al. 1993). The Strombina group
sensu Jung, 1989, is one of the best known columbellid taxa,
as it has been used as a model system to study evolutionary
trends in species composition, diversity, and ecological pat-
terns related to the Neogene rise of the Panama land bridge
(VermeiJ 1978, Jackson et al. 1993, 1996, Fortunato 1998,

1999). Despite these studies, only recently have the phylo-
genetic relationships of these taxa been investigated (de-
Maintenon 1994, 1999, 2005, Fortunato and Jung 1995,

Fortunato 1998). Cotonopsis Olsson, 1942, and Cosmio-
concha (Dali, 1913) are among the genus-level taxa that be-
long to this group. They are abundant and include mostly
recent species with a predominantly tropical American
distribution.

In his latest revision, Jung (1989) included Cotonopsis
but excluded Cosmioconcha from the Strombina group.

Work on the anatomy as well as preliminary cladistic analy-
ses based on a subset of taxa (Fortunato and Jung 1995)
confirmed Radwin’s ( 1977) hypothesis of a possible relation-

ship between Cosmioconcha and Strombina Morch, 1852
based on radular and shell morphology. These results
pointed to a veiy close relationship between Cotonopsis and
Cosmioconcha. The objective of this paper it is to investigate
the phylogenetic relationships of these genera, including all
fossil and living species, based on shell morphology in order
to better understand their history and evolution.

MATERIALS AND METHODS

This analysis includes all known fossil and Recent spe-
cies of the genera Cotonopsis and Cosmioconcha (Table 1).
Cotonopsis is a very young genus (Jung 1989), with the first
known species dating from the early Pliocene of Ecuador.
Most of the diversity within Cotonopsis developed during the
Plio-Pleistocene turnover, around the time of formation of
the Panama land barrier. Cotonopsis includes 18 species
grouped in two subgenera. Only one species is known ex-
clusively as a fossil. Of the 17 living species, 13 inhabit the
eastern Pacific basin (Jung 1989), two were reported from
the Caribbean region (Houbrick 1983, Petuch 1988, Fortunato
2002b), one was described from the west coast of Afi'ica (Emer-
son 1993), and a fourth species was found in the Andaman Sea
(Kosuge et al. 1998, Kronenberg and DeJeker 1998, 1999).

From the symposium “Relationships of the Neogastropoda” presented at the meeting of the American Malacological Society, held 31
July-4 August 2004 at Sanibel Island, Florida.

' Current address: Institut ftir Geowissenchaften Universitat Kiel, Ludwig-Meyn-Strasse 10, D-24118 Kiel, Germany.

33

34

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Table 1. Taxa included in the phylogenetic analyses. Stratigraphic and geographic ranges are given only for ingroup taxa. LM, late Miocene;
EP, early Pliocene; LP, late Pliocene. See Table 2 for more information on species of the genus Cosmioconcha. Extinct species indicated by

an n.

Species

Status

Stratigraphic

range

Geographic range

Nassarius hiteostoma (Broderip & Sowerby,

Outgroup

1829)

Nassarius antillannn Orbigny, 1842
Canthanis ringens (Reeve, 1846)

Outgroup

Outgroup

Latirus amceutncus (Reeve, 1847)
Cotonopsis {Cotonopsis) panacostaricensis

Outgroup
Type of species of

LP — Recent

Eastern Pacific (Costa

(Olsson, 1942)

Cotonopsis

Rica — Colombia)

Cotonopsis (Cotonopsis) edentida (Dali, 1908)

Olsson, 1942

Recent

Eastern Pacific (G. of

Cotonopsis (Cotonopsis) argentea (Houbrick,

Recent

California — Panama)
Caribbean (Dominican Republic)

1983)

Cotonopsis (Cotonopsis) crassiparva (lung.

Recent

Eastern Pacific (Galapagos Is.)

1989)

Cotonopsis (Cotonopsis) deroyae (Emerson 8t

Recent

Eastern Pacific (Galapagos Is.)

D’Attilio, 1969)

Cotonopsis (Cotonopsis) aff. deroyae (Emerson

Recent

Eastern Pacific (Peru)

& D’Attilio, 1969)

Cotonopsis (Cotonopsis) esnieraldensis (Olsson,

EP

Eastern Pacific (Ecuador)

1964)*

Cotonopsis (Cotonopsis) jaliscana (lung, 1989)

Recent

Eastern Pacific (Mexico)

Cotonopsis (Cotonopsis) inendozana (Shasky,

Recent

Eastern Pacific (Mexico — El Salvador)

1970)

Cotonopsis (Cotonopsis) skoghindae (lung,

Recent

Eastern Pacific (Gulf of California)

1989)

Cotonopsis (Cotonopsis) siiteri (lung, 1989)

Recent

Eastern Pacific (Gulf of

Cotonopsis (Cotonopsis) aff. suteri (lung,

Recent

California — Mexico)

Eastern Pacific (Mexico — Costa Rica)

1989)

Cotonopsis (Cotonopsis) phiiketensis (Kosuge,

Recent

Andaman Sea (Phuket Is.)

Roussy & Muangman, 1998)

Cotonopsis (Cotonopsis) lindae (Petuch, 1988)

Recent

Caribbean (Barbados)

Cotonopsis (Cotonopsis) njonfilsi Emerson,

Recent

Western Africa (Senegal)

1993

Cotonopsis (Turrina) hirundo (Gaskoin, 1852)

Pleistocene —

Eastern Pacific (Gulf of

Cotonopsis (Turrina) radwini (lung, 1989)

Recent

Recent

California — Ecuador)

Eastern Pacific (Mexico — Panama)

Cotonopsis (Turrina) tiirrita (G. B. Sowerby I,

Recent

Eastern Pacific (El Salvador —

1832)

Cosmioconcha modesta (Powys, 1835)

Type species of

Recent

Colombia)

Eastern Pacific (El Salvador —

Cosmioconcha pahneri (Dali, 1913)

Cosmioconcha
Dali, 1913

LM — Recent

Ecuador)

Eastern Pacific (Gulf of

Cosmioconcha parvula (Dali, 1913)

Recent

California — Panama)
Eastern Pacific (Gulf of

Cosmioconcha rehderi (Hertlein & Strong,

Recent

Galifornia — Panama )

Eastern Pacific (Mexico — Ecuador)

1951)

Cosmioconcha pergracilis (Dali, 1913)

Recent

Eastern Pacific (Mexico)

Cosmioconcha nitens (C. B. Adams, 1850)

Recent

Caribbean (Cuba, Puerto Rico)

Cosmioconcha calliglypta (Dali & Simpson,

Recent

Caribbean (Florida, Texas, Puerto

1901)

Rico)

PHYLOGENY OF COTONOPSIS AND COSMIOCONCHA

35

The earliest known Cosmioconclia species dates from the
middle Miocene. Cosinioconcha was first described as a
subgemis of Amphissa H. & A. Adams, 1853 (Dali, 1913).
Radwin (1978) elevated it to generic rank. Cosmiocouchn
includes seven described species, two inhabiting the
Caribbean Sea and five the eastern Pacific region (Table 2,
Figs. 1-2). Recent patterns of diversity and abundance of
this taxon indicate a radiation similar to other paciphile
genera.

Outgroup taxa were selected from Nassariidae, Buccini-
dae, and Fasciolariidae. Four common taxa from three buc-
cinoidean families were selected as outgroups: Nassariidae —
Nassarius luteostoma (Broderip & Sowerby, 1829) and N.
autillarum d’Orbigny, 1842; Buccinidae — CatJtlmrus ringens
(Reeve, 1846); Fasciolariidae — Latirus concentricus (Reeve,
1847). These taxa were selected based on availability and not
on the presumption of close phylogenetic relationship.

Forty- two qualitative characters were identified (Ap-
pendix 1). Shell sculpture is one of the most characteristic
elements of this group, and provides numerous diagnostic
characters. Fourteen characters code for type and sculptural
details of the teleoconch and body whorl. Presence of shoul-
der, constriction, inflation, and angulation of the whorls, as
well as presence and strength of humps were also coded.
Apertural elements (thickness, denticles, apertural and colu-
mellar calluses and plicae, parietal ridge) used in traditional
taxonomy of this group of gastropods are included here as
well. Other characters are general shell shape, type of spire,
type and depth of suture, and the relation between the total
height and the height of the body whorl. All taxa were coded
from direct observation.

MacClade 3.0 (Maddison and Maddison 1992) was used
to create the data matrix of 25 taxa and 42 morphological
characters (Appendix 2). The
heuristic search in PAUP 4.0b 10
(Swofford 2001 ) was used for the
analyses, using a random addi-
tion sequence with ten repli-
cate searches performed. All
characters were unordered and
weighted equally. Glade support
was assessed through a bootstrap
procedure (100 bootstrap repli-
cates with 10 random addition
sequences). Tree support was
determined using Bremer decay
analysis (Bremer 1994) in which
progressively longer trees are
saved and their consensus calcu-
lated in order to see how many
more steps are required to col-
lapse branches.

RESULTS

Cladistic analyses of the data matrix in Appendix 2
yielded six most-parsimonious trees (F=218 steps, Cl=0.303,
Rl=0.513, and RG=0.155). Only the strict consensus tree
(Fig. 3) is presented here (the 50% majority rule consensus
tree shows exactly the same topology).

The ingroup is monophyletic in all trees, consisting of a
single clade grouping all Cotonopsis and Cosmioconclia spe-
cies. This clade is defined by fusiform shells with high spire
and mostly un-sculptured earlier teleoconch whorls, body
whorl mostly un-sculptured, apertures with moderately
thickened outer lips, and a well developed, recurved anterior
canal.

The genus Cotonopsis, as traditionally constructed, is
paraphyletic and includes the polyphyletic Cosmioconclia.
The subgenus Cotonopsis [Turrina) emerges as a monophy-
letic crown group. Cosmioconclia species are grouped in two
separate clades within Cotonopsis. One clade, which contains
the type species of Cosmioconclia, emerges in an unresolved
trichotomy with a small clade containing the type species of
Cotonopsis and a large clade that includes Cotonopsis, the
remaining Cosmioconclia, and Cotonopsis (Tiirriiui).

All species assigned to Cotonopsis sensu stricto emerge
as a grade that also includes a small clade of three species of
Cosmioconclia, including its type species. These species have
mostly stout shells with axially sculptured late spire whorls
and well defined cords at the base of the body whorl. I'hey
have broad apertures with denticles and thin outer lip edges.

This grade (all Cotonopsis sensu stricto -t- three Cosniio-
conclia taxa) has several smaller groupings. Its base is weakly
resolved with several Cotonopsis species branching succes-

Table 2. Synopsis of species belonging to the genus Cosmioconclia Dali, 1913. *, type species.

Genus

Species

Author & Year

Synonyms

Cosmioconclia

modesta *

Powys, 1835

Buccinum modestiim Powys,

1835; Stromhina lacvistriata
Li, 1930

palmeri

Dali, 1913

parvula

Dali, 1913

relideri

Hertlein & Strong, 1951

pergmcilis

Dali, 1913

nitens

C. B. Adams, 1850

Fusiis nitens C. B. Adams, 1850;

Coliimbella (Astyris) perpicta
Dali 8c Simpson, 1901;
Mitrclla perpicta (Dali 8c
Simpson) Woodring, 1928

calliglypta

Dali & Simpson, 1901

Anacliis calliglypta Dali 8c

Simpson, 1901

36

AMERICAN MALACOLOGICAL BULLETIN

23 • 1/2 • 2007

10mm

5mm

L

Figure 1. Species of Cosmwconcha Dali, 1913. A-C Cosnnocoucha modesta (NMB 17442). D-F Cosmiocoucha pahnen (NMB 17793) G-1
Cosmiocoiicha parvida (NMB H18181 ). 1-L Cosmwconcha rehden (NMB H18182). M-O Cosmwconcha mtens (NMB 18567). Views are front,
rear, and from right side. All specimens belong to the Gibson-Smith Recent collection housed at the Naturhistorisches Museum Basel,

Switzerland.

PHYLOGENY OF COTONOPSIS AND COSMIOCONCHA

37

Figure 2. Species of Cosmiocondia Dali, 1913, protoconchs. A-R Cosniioconcha modesta. C-D Cosmioconcha palweri. E-F Cosmioconcha
parvula. G-H Cosmioconcha rehderi. I-l Cosmioconcha nitens. Same specimens as in Fig. 1.

sively. Among these are C. inouftlsi and C. lindae, an African
and a Caribbean species respectively. The next branch has
two small subclades, one with three Cosmioconcha and a
second one joining two Cotonopsis taxa. Next to diverge is
the Cotonopsis living in the Andaman Sea, followed by an-
other small group formed by two eastern Pacific Cotonopsis.
The last grouping of this grade joins a Caribbean and two
eastern Pacific Cotonopsis taxa.

The two other groups are sister clades and are located as
crown groups. One of these clades groups, but does not
resolve, the three Cotonopsis (Tiirrina) taxa. The second
group is composed by four Cosniioconcha species, among
which appear the two Caribbean species. Species of the
crown clades have slender shells, almost no axial ornamen-
tation on the spire whorls, and narrower apertures with a

thicker edge. The species of Cosmioconcha have more convex
spires, numerous denticles on the ap>erture, and a collar like
band below the spire suture. Cotonopsis {Tnrrina) taxa are
characterized by taller, straight-sided shells and a well de-
veloped thickening behind the outer lip. Results of the
Bremer decay analysis are plotted onto the strict consensus
tree (Fig. 3).

The first round resulted in 284 trees with 219 steps or
less. These trees support the monophyly of the entire in-
group as well as the monophyly of both crown clades, i.e.
Cotonopsis (Tnrrina) and the four Cosmioconcha species.
Two small groups that appear among the basal branches
(one joining three eastern Pacific Cosmioconcha, and another
with two Cotonopsis sensu stricto taxa) are equally supported
here. The second round of the decay yielded 6,278 trees, 220

38

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Latirus concentricus
Cantharus ringens
Nassarius luteostoma
Nassarius antillarum
Cotonopsis (Cotonopsis) jaliscana
Cotonopsis (Cotonopsis) att.deroyae
Cotonopsis (Cotonopsis) suteri
Cotonopsis (Cotonopsis) monfiist
Cotonopsis (Cotonopsis) lindae
Cotonopsis (Cotonopsis) edentula
Cotonopsis (Cotonopsis) crassiparva
Cotonopsis (Cotonopsis) panacostaricensis
Cosmiconcha palmeri
Cosmiconcha modesta
Cosmiconcha rehderi
Cotonopsis (Cotonopsis) phuketensis
Cotonopsis (Cotonopsis) esmeraidensis
Cotonopsis (Cotonopsis) aft. suteri
Cotonopsis (Cotonopsis) argentea
Cotonopsis (Cotonopsis) mendozana
Cotonopsis (Cotonopsis) skoglundae
Cotonopsis (Cotonopsis) deroyae
Cotonopsis (Turrina) turrita
Cotonopsis (Turrina) hirundo
Cotonopsis (Turrina) radwini
Cosmiconcha pergraciiis
Cosmiconcha parvula
Cosmiconcha nitens
Cosmiconcha calliglypta

Figure 3. Strict consensus tree of the six most parsimonious cladograms. Numbers below branch nodes are Bremer support values, i.e., the
number of extra steps necessary to collapse that node. Nodes without values collapse with one extra step. Numbers above branch nodes are
bootstrap support values for that node.

steps or less. The ingroup is still monophyletic but only the
group with the two Caribbean Cosmioconcha species is sup-
ported. The third round yielded 117,655 trees, 221 steps or
less. It supports the monophyly of the ingroup but there is
no resolution. The fourth round of decay analyses overflowed
the memory with over 500,000 trees. The monophyly of the
ingroup is still supported here. The bootstrap support for some
of the clades is plotted in the strict consensus tree (Fig. 3).

DISCUSSION

The main objective of this work was to re-evaluate all
species that have traditionally been assigned to the genera
Cotonopsis and Cosmioconcha, in order to assess their rela-
tionships and the true constituency of these genera. Earlier
analyses based on a selected subset of species from each of
these genera (Fortunato and lung 1995) suggested a close
relationship of these taxa, confirming Radwin’s (1977) hy-
pothesis of a relationship between Cosmioconcha and the

Strombina group of which Cotonopsis is part (Jung 1989).
Our objective was to test this relationship, including in the
analysis all known species currently included in both genera.

The results of this study indicate that Cotonopsis -l- Cos-
mioconcha form a monophyletic group. Cotonopsis as it was
initially defined by Jung ( 1989) is paraphyletic and contains
Cosmioconcha. Of the two subgenera of Cotonopsis, only
Cotonopsis (Turrina) is monophyletic and retains its entire
constituency. Cotonopsis sensu stricto, as currently con-
structed, is paraphyletic. Its status as a monophyletic taxon
could be restored only by synonymizing both Cosmioconcha
and Turrina. Alternatively, inclusion of more closely related
outgroups might alter the rooting of the tree. Rooting at
Position A (Fig. 3) would be required to retain monophyletic
Cotonopsis and Cosmioconcha as sister taxa although the ma-
jority of species currently assigned to Cosmioconcha would
still emerge Cotonopsis.

Species assigned to Cosmioconcha are divided into two
groups. The first group is composed of three eastern Pacific

PHYLOGENY OF COTONOPSIS AND COSMIOCONCHA

39

species, and is located near the base of the tree, among
Cotonopsis sensu stricto taxa. This group includes the type
species, Cosmioconcha modesta. The second group is one of
the crown subclades and unites two Caribbean and two east-
ern Pacific species. This group is sister of Cotonopsis {Tiirrina).

Within the grade Cotonopsis sensu stricto, C. monftlsi, a
deep water species from West Africa, and C. lindne, a shallow
water species from Barbados, form adjacent branches but are
flanked by eastern Pacific species from California, Mexico,
and Peru that have no known fossil record. It is tempting to
speculate about the possible existence of geminate pairs [i.e.,
closely related taxa separated by a barrier (Jordan 1908)]
among the extinct fossil ancestors of these taxa. These rela-
tionships also suggest an earlier radiation of American spe-
cies, probably from the eastern Pacific towards the Atlantic
before the closure of the Panamanian Strait. Unfortunately,
none of these species have a known fossil record which could
help calibrate the time of such radiation. However, both the
geographic distribution of these species, and the fact that
several of the following taxa (within the context of this tree
topology) have fossils dating back to the middle Miocene
[i.e., Cosmioconcha palmeri (Dali, 1913)) suggest that such a
radiation may have taken place during the middle Miocene.
It is also reasonable to assume the possible existence of fossil
lineages yet to be found. Molecular studies could provide an
alternative tool to elucidate these relationships.

Three eastern Pacific species of Cosmioconcha^ including
the type species, emerge as a clade. The stem species, C.
palmeri, has the oldest fossil record of all the species in this
study, being known from the middle Miocene deposits of
Darien [Radwin, 1977; Panama Paleontological Project
(PPP) data]. Based on these results, it is reasonable to as-
sume that Cotonopsis sensu stricto is much older than pos-
tulated by Jung (1989) in his revision of the Strombina
group. Jung indicated an early Pliocene age for Cotonopsis,
based on the occurrence of Cotonopsis esmeraldensis (Olsson,
1964) in the early Pliocene of Ecuador. Results of the present
analysis indicate that Cosmioconcha is part of Cotonopsis
sensu stricto, thus moving the time of origination of this
genus most probably to middle Miocene.

Another closely related small clade unites the Recent
Cotonopsis (Cotonopsis) crassiparva and a late Pliocene Coto-
nopsis (Cotonopsis) panacostaricensis (Olsson, 1942), the type
species of Cotonopsis.

Cotonopsis (Cotonopsis) phnketensis (Kosuge et ah,
1998), a shallow water species from the Andaman Sea, is the
second species in this genus with a distribution outside of
tropical America. There are infreciuent reports of plankto-
trophic larvae crossing the central Pacific barrier (Scheltema,
1978). Most Cotonopsis have planktotrophic larvae (excep-
tions are C. jaliscana, C. esmeraldensis, and C. argentea; au-
thorities in Table 1 ) able to spend a considerable amount of
time in the plankton (Fortunate 2002a). Again, the lack of

fossil data precludes the dating of this dispersal event. Nev-
ertheless, the presence of an early Pliocene species within a
sister clade indicates that it may date back to the early
Pliocene, at the very least. Here again, molecular data would
be useful to help resolve these events.

The next clade comprises Cotonopsis esmeraldensis (early
Pliocene of Ecuador) and a recent eastern Pacific species, C.
aff snteri. This is probably a case of speciation with a switch
in developmental mode, as C. esmeraldensis is a non-
planktotroph whereas its sister species has planktonic lar-
vae. Cotonopsis esmeraldensis is the only extinct taxon in the
analyzed data set. The basal species of this clade, C. pnkhe-
tensis, is also a planktotroph. A trans-isthmian event in the
history of the group is documented in the next branch of this
phylogenetic tree. Cotonopsis argentea, a non planktotroph
taxon found in deep water of the Dominican Republic coast
is the sister taxon of two eastern Pacific species (C. mendo-
zana and C. skoghmdae).

The crown of the tree is composed by two subclades
with a relatively strong Bremer and bootstrap support. The
stem taxon is Cotonopsis deroyae. One of the groups includes
the three Cotonopsis (Tnrrina) species, confirming the com-
position and monophyly of this subgenus. The second
group, composed by four Cosmioconcha species, documents
another trans-isthmian event: C. nitens and C. calliglypta are
shallow water taxa inhabiting the Caribbean Sea that di-
verged from an eastern Pacific taxon.

Based on the obtained results and the phylogenetic re-
construction presented here, Cotonopsis sensu stricto, as
presently understood, represents a grade that includes sev-
eral Cosmioconcha taxa (i.e., C. palmeri, C. modesta, and C.
rehderi), among them the type species of Cosntioconcha. All
have stout shells with high spires, axially sculptured early
teleoconch whorls, body whorls with strong cords on the
base, and wide apertures.

The four “Cosmioconcha" species that constitute one of
the crown groups of the tree are not closely related to the
type species of Cosmioconcha. These species are characterized
by smaller fusiform shells, absence of sculpture on the early
teleoconch, absent or weak cords on the body whorl, and
narrow apertures. The character “presence of a collar-like
band below the suture”, traditionally used to unify Cosmio-
concha taxa is not reliable and should not be given more
value than any other morphological character.

Cotonopsis is a taxon that reflects the pulse of origina-
tion that occurred in the eastern Pacific at the Pleio- Pliocene
boundary. Most of the recognized taxa originated during the
last two million years, probably along the shallow waters of
the eastern Pacific coast. Unfortunately, the stratigraphic rec-
ord of the eastern Pacific region is not very well preserved
(Coates etal. 1992, Jackson et al. 1993, 1996) and there is no
fossil record for most of the known species. Cosmioconcha
also originated in this region and has a fossil record that

40

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

dates back to the middle Miocene. Based on the phylogenetic
reconstruction presented here it is reasonable to assume that
Cotonopsis derives from a Cosmioconcha-like ancestor. The
group then radiates and speciates with the documented in-
crease in species diversity towards the recent, a pattern well
documented for the entire Strombina group (lung 1989, Jack-
son et al. 1993, 1996).

The Strombina group has been used as a model system
to document patterns of diversification during the Neogene
rise of the Panamanian isthmus. Phylogenetic inferences
have started to give historical support to earlier studies. The
taxa studied here are part of this group and the results con-
firm the validity of the evolutionaiy patterns documented
earlier (Jackson et al. 1993, 1996, Portunato and Jung 1995,
Portunato 1998, 1999). It is also reasonable to assume the
existence of fossil lineages and even Recent taxa yet to be
found that could contribute to a better understanding of the
natural history of the molluscan fauna of the region and its
relationships.

AKNOWLEDGMENTS

This work was presented at the molluscan phylogeny
symposium organized by M. G. Harasewych during the AMS
meetings, 2004. 1 thank the institutions that loaned materials
for this study. A. Velarde, J. Jara, M. Alvarez, P. Rodriguez,
and the Urraca’s crew helped with field collections and labo-
ratory work. STRTs digital and SEM laboratory personnel
helped with the illustrations. This work was supported by
the Scholarly Studies and the Walcott programs of the
Smithsonian Institution.

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41

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pelagic veliger lar\'ae and the evolution of marine prosobranchs
gastropods. In: B. Battaglia 1 A. Beardmore, eds., Marine Organ-
isms. Plenum Publishing Corporation. New York, New York.

Swofford, D. L. 2001. PAUP: Phylogenetic Analysis Using Parsimony,
Version 4.0bl0. Illinois Natural History Survey, Champaign,
Illinois.

Tracey, S., ). A. Todd, and D. H. Erwin. 1993. Gastropoda. In:
M. J. Benton, ed.. The Fossil Record 2, Chapman and Hall, London.
Vermeij, G. I. 1978. Biogeography and Adaptation. Harvard Univer-
sity Press, Gambridge, Massachusetts.

Accepted: 27 March 2007

Appendix 1. Character and character state list.

1- Shell shape: (0) fusiform (elongate, spire high); (1) strombiform, spire low; (2) buccinoid (stout, spire tapering); (3) columbelloid
(stout, spire high)

2- Shape of spire whorls: (0) straight sided; (1) straight going to convex; (2) straight going to concave

3- Depth of suture: (0) shallow; (1) impressed; (2) incised

4- Shoulder on spire whorls: (0) absent; (1) present, inconspicuous; (2) present, strong

5- Number of whorls in protoconch: (0) <2; (1) 2-3; (2) >3

6- Axial sculpture on early teleoconch whorls: (0) absent; (1) present, inconspicuous and subordinate; (2) present, well developed

7- Spiral sculpture on early teleoconch whorls: (0) absent; (1) present, inconspicuous and subordinate; (2) present, well developed

8- Axial sculpture on late spire whorls: (0) absent; (1) present, inconspicuous and subordinate; (2) present, well developed

9- Spiral sculpture on late spire whorls: (0) absent; (1) present, inconspicuous and subordinate; (2) present, well developed

10- Spiral sculpture on body whorl: (0) absent; (1) present, inconspicuous and subordinate; (2) present, well developed

11- Axial sculpture on body whorl: (0) absent; (1) present, inconspicuous and subordinate; (2) present, well developed

12- Shoulder on body whorl: (0) absent; (1) present

13- Cords on base of body whorl: (0) absent; (1) present, weak; (2) present, well developed

14- Concavity on central part of body whorl: (0) absent; (1) present

15- Constriction on lower part of body whorl: (0) inconspicuously constricted; (1) strongly constricted

16- Inflation of body whorl: (0) not inflated; (1) inflated

17- Type of sculpture on early vs. late spire whorls: (0) same; (1) different

18- Shape of aperture: (0) broad; (1) narrow; (2) slit-like

19- Thickness of outer lip: (0) not thickened; (1) slightly thickened; (2) conspicuous thickness

20- Teeth on inner surface of outer lip: (0) absent; (1) present, small and inconspicuous; (2) present, strongly developed

21- Number of teeth on inner surface of outer lip: (0) none; (1) few (1-5); (2) numerous (>5)

22- Posterior canal: (0) absent; (1) present, inconspicuous; (2) present, well developed

23- Apertural callus: (0) absent; (1) present, as a slight thickness; (2) present, continuous, well developed

24- Columellar denticles: (0) absent; (1) present;

25- Parietal callus: (0) absent; (1) present, slightly thickened; (2) present, well developed

26- Parietal denticles: (0) absent; (1) present

27- Parietal ridge: (0) absent; (1) present, small and inconspicuous; (2) present, well developed

28- Sinus on outer lip: (0) absent; (1) present

29- Flaring of outer lip: (0) absent; (1) present

30- Length of anterior canal: (0) short; (1) intermediate; (2) long

31- Width of anterior canal: 90) wide; (1) narrow

32- Extension of adapical part of outer lip (aperture edge at suture): (0) outer lip not extended; (1) outer lip somewhat extended after
suture

33- Shape of anterior canal: (0) slightly curved; (1) strongly curved; (2) straight

34- Notch of anterior canal (at the end): (0) shallow; (1) deep depression

35- Thickening behind outer lip: (0) absent; (1) present, slight thickness; (2) present, well developed

36- Dorsal hump: (0) absent; (1) present, slight thickness; (2) present, well developed

37- Edge of outer lip: (0) sharp; (1) rounded

38- Hump on left side of outer lip: (0) absent; (1) present, slight thickness; (2) present, well developed

39- Repeated thickenings behind outer lip: (0) absent; (1) present

40- Plicae on columella: (0) absent; (1) present

41- Relation aperture height/total height: (0) aperture <‘/2 total shell height; (1) aperture much smaller than */: total shell height; (2)
aperture bigger than Vi but smaller than Va total shell height

42- Gollar-like band below spire suture: (0) absent; (1) present

42

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Appendix 2. Character matrix used for analyses.
Species

Characters

Nassariiis luteostoma

2111221222

2220120012

2220202010

0022101001

00

Nassarius aiitilinnim

2111222222

2120120002

2220202010

0022101001

00

Canthams ringens

2102021222

2220120001

2211112010

0012200000

00

Lati nis co iicen trie us

0202021222

2120110001

2010100012

2020000002

10

Cotoiwpsis (Cotoiwpsis) argentea

0100020200

1010100111

1011101002

0011100000

00

Cotonopsis (Cotoiwpsis) crassiparva

0120120211

0020110022

1120202011

0011100000

00

Cotoiwpsis (Cotoiwpsis) deroye

0100120011

0010100121

1120102002

1011100000

00

Cotoiwpsis (Cotoiwpsis) edentula

0120120000

0020111010

0110101012

0111100000

00

Cotoiwpsis (Cotoiwpsis) jaliscaiia

0121020000

0110111121

2120102010

0000100000

00

Cotonopsis (Cotonopsis) mendozana

0100120000

0010111021

1020201010

0011100000

00

Cotoiwpsis (Cotonopsis) panacostaricensis

0120120211

1020110011

2120102012

0011100000

00

Cotonopsis (Cotoiwpsis) skoginiidae

0100?22021

0010001012

2020201002

1011100000

00

Cotoiwpsis (Cotonopsis) suteri

0111210000

0120111021

1120121002

0010100000

00

Cotonopsis (Cotoiwpsis) esmeraldensis

0100020111

0120111122

1020101002

0111101000

10

Cotoiwpsis (Cotonopsis) aff. deroye

olomoloo

1100111011

2110102000

0000101000

10

Cotoiwpsis (Cotonopsis) atf. suteri

0110120111

0120101011

1220001001

0010100000

00

Cotonopsis (Cotonopsis) phuketensis

0120110010

0020101111

2020100001

0001100000

00

Cotoiwpsis (Cotonopsis) lindae

0100110100

0020111012

2211102001

0101100000

00

Cotonopsis (Cotonopsis) inonfilsi

0121212011

0120100022

2111101000

1121101000

00

Cotonopsis (Turrina) turrita

0000100010

0010001120

0210102002

0001200100

00

Cotonopsis (Turrina) hiriindo

0000100010

0010101020

0210001012

1011200000

00

Cotonopsis (Turrina) radwini

0000100010

0010111020

1210102010

0011200000

00

Cosmioconcha palineri

0220120010

0120111032

2221200011

0011100000

01

Cosmioconcha nwdesta

3220101011

0010001022

2220201010

0001200000

01

Cosmioconcha rehderi

3200220210

2020011031

1220201011

0121200000

11

Cosmioconcha nitens

0210100010

0010001021

2110100000

0021100000

11

Cosmioconcha calliglypta

0200110110

0010011021

2110100000

0021101000

11

Cosmioconcha parvula

0200100011

0010101022

2110100002

0001101000

01

Cosmioconcha pergracilis

0200200010

0010101021

1110100002

0001101000

01

Ainer. Maine. Bull. 23: 43-78

Family Pseudolividae (Caenogastropoda, Muricoidea): A polyphyletic taxon’^

Luiz Ricardo L. Simone

Museu de Zoologia da Universidade de Sao Paulo, Cx. Postal 42494, 04299-970 Sao Paulo, SP, Brazil, lrsimone@usp.br

Abstract; A detailed morphological study was performed on the following taxa normally considered to belong to the family Pseudolividae:
(1) Zemira australis (Sowerby, 1833) from Australia; (2) Fulmcntuin ancilla (Hanley, 1859) from South Africa; and (3) Melnpiinii lineatum
(Lamarck, 1822) from South Africa. Two additional species of pseudolivids, Bentlwbia atafona Simone, 2003 and B. complexirhyiia Simone,
2003, from Brazil and New Zealand respectively, are considered. Two other muricoideans are included in this study: ( 1 ) Nassodonta dorri
(Watteblet, 1886) [Nassariidae] from Vietnam (morphological study also included) and (2) Sirntiis senegaleusis (Gmelin, 1791) (Muricidae)
from Brazil (published elsewhere). Both species are outgroups, but operationally included as part of the ingroup in order to test the
monophyly of the Pseudolividae. In particular, N. dorri has a shell very similar to a pseudolivid. A complete taxonomical and morphological
treatment of each species is included, as a scenario of a formal phylogenetic analysis. Additional outgroups considered include a pool of
Tonnoidea (the root) and Conoidea. The cladogram is: (Tonnoidea (Conoidea {{Beutlwhia atafona-B. complexirhyna) {Nassodonta dorri
(Zemira australis (Fulmentiim ancilla {Siratiis senegalensis-Melapium lineatum))))))). Analyses of each important character and of the
cladogram were performed. Some of the conclusions include that the family Pseudolividae, as presently understood, is polyphyletic, as it

would include a nassariid {N. dorri) and a muricid (S. senegaleusis).
Key words: Neogastropoda, polyphyly, morphology, phylogeny

The taxon Pseudolividae Fisher, 1884, had been previ-
ously used by several researchers {e.g., Cossmann 1901, Goli-
kov and Starobogatov 1975, Squires 1989), but it was better
defined as a family by Kantor (1991), based on anatomical
features of basal neogastropods. The family reunites genera
previously considered as belonging to several other families,
including, e.g., Cancellariidae {Bentlwbia Dali, 1889), Buc-
cinidae {Buccinorbis Conrad, 1865), and Olividae (Melapiwn
Adams and Adams, 1853; PseudoUva Swainson, 1840; Sylvn-
nocochlis Melvill, 1903; Zemira Adams and Adams, 1853).
This taxonomy was followed by some researchers {e.g., Ver-
meij and DeVries 1997, Bouchet and Vermeij 1998, Pacaud
and Schnetler 1999, Nielsen and Frassinetti 2003). More-
over, Vermeij (1997, 1998) revised the family Pseudo-
lividae, including fossil species, establishing its origin in the
late Cretaceous. A more complete history of the concept of
the family can also be found in that paper. However,
some authors still considered the family as a subtaxon of
Olividae {e.g., Hayes 1994, Smith 1998) (Pseudolivinae).
Although our knowledge of pseudolivid species is relatively
rich, particularly with regard to anatomy (e.g.. Ponder
and Darragh 1975, Kantor 1991, Simone 2003), the defini-
tion of the family remains unclear, and no phylogenetic
analysis has yet been performed, other than that of Kantor
(1991).

The main difficulty in studying pseudolivids is finding
preserved animals. Pseudolivids are normally rare and found
in deep waters, which precludes obtaining a large set of
samples for an extensive anatomical study. Although the
pseudolivids are more abundant as fossils, with about a hun-
dred species (Vermeij 1998), they are relatively poor in di-
versity in the Recent fauna, with about 10-15 living species.
As about a third of the species are available for study, be-
longing to the different branches of the family, a study of
them appears to be worthwhile, at least in terms of testing
the monophyly of the group and identifying anatomical
characters that would better define it.

This paper is part of a larger project on the phylogenetic
definition of the Caenogastropoda based on detailed
morphology, this time focusing the Pseudolividae. One of
the genera, Bentlwbia Dali, 1889, was published else-
where (Simone 2003), and the species of remaining genera
are included herein.

Nassodonta dorri (Watteblet, 1886), from Vietnam, one
of the few freshwater neogastropods known, belonging to
the family Nassariidae, has a shell similar in morphology
to those of pseudolivids (Kantor and Kilburn 2001). This
taxon is also included in this study to test the monophyly
of the Pseudolividae, as the shell characters certainly can
converge.

* From the symposium “Relationships of the Neogastropoda” presented at the meeting of the American Malacological Society, held
31 luly-4 August 2004 at Sanibel Island, Florida.

43

44

AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

MATERIALS AND METHODS

Most specimens used in this study belong to institu-
tional collections. The specimens were dissected by standard
techniques, under a microscope, with the specimens im-
mersed in water. Some organs such as the oviduct and
foregut were processed by standard histological tech-
niques to obtain serial sections 5 pm in thickness and stained
using Mallory trichrome. Hard structures, such as shells,
radulae, and jaws were also examined using the SEM in the
“Laboratorio de Microscopia Eletronica do Museu de Zoo-
logia da Universidade de Sao Paulo”. The descriptive part of
this paper provides a complete description of the first
species; the remaining species are described under a com-
parative aspect, with most of similar features omitted. This
measure is adopted for decreasing the length of this paper
and for optimizing the data. The same approach is adopted
in the figures. A detailed list of examined species follows
each species description.

The section on comparative morphology is organized as
a phylogenetic analysis. The account on each character be-
gins with abbreviated descriptive sentence followed by
plesiomorphic and derived conditions(s); also included are
Cl and RI (consistency and retention indices, respectively)
and values for the character under the most parsimonious
hypothesis. Following the apomorphic state(s), a list of ter-
minal taxa with the apomorphic condition is presented.
Hundreds of characters were selected, based on the exam-
ined samples. Those that emerged as autapomorphic, highly
variable, or overlapping, were selected but not included in
the cladistic analysis. The remaining characters were orga-
nized into states, coded, polarized comparing with out-
groups, and a cladistic analysis was performed.

Other previously studied Caenogastropoda were
selected as outgroups. They are mainly the following; Ceri-
thioidea (Simone 2001), Stromboidea (Simone 2005),
Cypraeoidea (Simone 2004a),

Calyptraeoidea (Simone 2002),
and architaenioglossans (Si-
mone 2004b). In the discus-
sion, some specific outgroup
taxa are mentioned, based
upon observed or published
data. However, in the matrix of
characters (Table 1 ) only 4 taxa
are shown, the ground plan of
the Conoidea and Tonnoidea.

The ground plan of these su-
perfamilies are chosen in the
sense of being representative;
however, the final result is the
same if the ground plan were

substituted by any one of the 64 (terminal) species present in
those papers. Two additional species are included, Siratiis
senegalensis (Gmelin, 1790) [Muricidae], based on unpub-
lished observations from another, ongoing study, and the
nassariid Nassodonta dorri, mentioned above. Each charac-
ter, state, and polarization is justified in the discussion sec-
tion which includes a concise explanation when warranted.

The discussion of each character is also based on the
analysis of the resulting tree (Figs. 1-2) although the matrix
of characters (Table 1) and the subsequent tree (Figs. 1-2)
are shown only in the following section.

The synapomorphies of the ingroup (superfamily auta-
pomorphies) are preserved in the present paper, because
they are the main concern as referred to in the introduction.
The ingroup autapomorphies are the basis for a better es-
tablishment of a still imprecise taxon. They confirm the
internal position of some possible “outgroups” such as
tonnoideans and conoideans. They will be useful in the on-
going phylogenetic study of the entire order Caenogas-
tropoda as the ground plan of the superfamily. Additionally,
they are in agreement with some phylogenetic approaches
used in studies of other groups [e.g., Yeates 1992, Pinna 1996).

Some multistate characters are here analyzed under an
additive (ordered) approach. In each case, the additive
concept is justified in the discussion and is always based
on the ontogeny, or because each state represents a clear
modification of the preceding one. Additionally, each addi-
tive multistate character was also analyzed as non-additive,
and any fortuitous change in the result and/or indices are
also reported. The cladistic analysis was performed with the
aid of the computer program “Tree Gardner 2.2” (Ramos
1997), which basically works as an interface of the Hennig86
(Farris 1988). The used algorithm was “ie”. The computer
program PAUP was also used, mainly to determine the ro-
bustness of the nodes. Both programs presented the same
result.

Table 1. Matrix of characters of ingroup and two outgroups (bottom).

Taxon/character

1

2

3

12345

67890

12345

67890

12345

67890

1234

Bentiwbia atafona

10101

00121

10001

00011

21010

20101

1111

Benthobia complexirhyna

10101

00121

10001

00011

21010

20101

1111

Zemira australis

11010

01120

01111

10011

01121

11101

0111

Melapium Uneatum

00100

13010

01111

mil

11121

21010

0011

Fiihnentiim ancilla

11110

00010

01101

11011

21111

11110

1111

Nassodonta dorri

10110

01011

00111

10111

?1110

2??01

0?11

Siratus senegalensis

00010

12000

01111

11111

mil

OHIO

0211

Conoidea

00100

00000

00001

00001

00010

00001

0011

Tonnoidea

00000

00000

00000

00000

00000

00000

0000

PSEUDOLIVIDAE: A POLYPHYLETIC TAXON

45

Figure 1. Single most parsimonious tree based on the data matrix in Table 1, with three
outgroups operationally analyzed as part of the ingroup (Conoidea, Siratiis, and Nassodonta).
Length: 62; Cl = 66; R1 = 69. Each symbol indicates a synapomorphy supporting each node
(only the homoplastic autapomorphies are shown) as follows: full sc]uare = non-homoplastic
synapomorphy; circle = convergence; empty square = reversion.

Figure 2. Single most parsimonious tree (same of Fig. 1, excluding both more basal out-
groups) (Length; 62; Cl = 66; R1 = 69), with the nodes numbered. The gray branches
represent the non-pseudolivid taxa, left branch a member of the Nassariidae, right branch a
member of the Muricidae. The black branches represent the taxa mostly considered to be
Pseudolividae, showing the polyphyletic nature of the taxon.

SYSTEMATICS

Genus Zemim Adams and Adams,
1853

(Type species Eburna niistmlis, by
monotypy)

Zemim australis (Sowerby, 1833)
(Figs. 3A-F, 4A-B, 5A-7H)

Synonymy; see Ponder and Dar-
ragh 1975: 101.

Complement:

Zemira australis: Ponder and Dar-
ragh 1975: 89-97, 101-104
(text figs. 1, 2, pi. 7 fig. 1-2.
pi. 8 figs. 12-24); Smith 1998:
835-836 (fig. 15.165-C).

Description

Shell (Figs. 3A-C, 3F). Fusiform,
pale brown, opaque. Protoconch,
spherical, smooth, opaque, of about
one whorl; boundary between proto-
conch and teleoconch unclear. Spire
pointed, about half of length of body
whorl. Suture well-marked by a subsu-
tural, concave, wide groove, from pro-
toconch up to outer lip; surface of
groove smooth, external edge elevated,
forming a low carina. Remaining re-
gions sculptured by uniform, spiral,
narrow furrows, about nine in pen-
ultimate whorl, about 10 in body
whorl; one of these furrows, located
between middle and anterior thirds of
body whorl, deeper and wider (Figs.
3B-C). No umbilicus except a narrow
furrow in inferior third around inner
lip (Fig. 3A). Peristome oval, white,
glossy (Figs. 3A, 3F). Canal short
and narrow, left edge truncate, right
edge wanting, as continuation of outer
lip. Outer lip simple, cutting edge,
rounded; very short tooth between
middle and inferior thirds correspon-
dent to deeper spiral furrow of body
whorl (Figs. 3C, 3F); wide notch in su-
perior region, at some distance from
suture correspondent to sub-sutural
Carina. Inner lip simple, callus narrow,
slightly more transparent than inner
region of peristome.

46

AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

Figure 3. Shells and opercula. A-F, Zemira australis, AMS 333288. A-C, shell (specimen #1 ),
female, apertural, dorsal and lateral views, length = 18.6 mm; D-E, operculum, outer and
inner views, arrow indicates separation of two scar regions, scale bar = 2 mm; F, detail of
aperture closed by operculum (specimen #4), showing labral tooth and opercular sculpture.
G-I, Nassodonta dorri, MZSP 53533; G-H, shell, apertural and dorsal views, length = 13.6
mm; I-|, operculum, outer and inner views, length = 5 mm.

Head-foot (Figs. 5A, 5C, 5F).
Head weakly protruded, bilobed, with
single region pigmented by dark
brown, the rest pale cream. Tentacles
located close to each other and close to
median line; tentacles’ base very wide,
flat, flap-like, outer edge rounded; dis-
tal half of tentacles marked by abrupt
narrowing of the base, narrow, taper-
ing gradually; tentacles’ tip rounded.
Foot broad, of about half whorl when
retracted. Sole oval, edges thick and
rounded. Anterior furrow of pedal
glands deep, straight, thick superior
and inferior edges, not reaching lat-
eral-anterior end. Lateral region of the
sole of the foot dearly extending be-
yond remaining dorsal regions of foot,
division marked by a shallow longitu-
dinal furrow lying somewhat in middle
region between sole edge and dorsal
region of foot (Fig. 5A). Opercular pad
elliptical, almost as wide as the dorsal
surface of the foot; possessing clear,
median, oblique difference in levels
(Fig. 5C); posterior half of this division
forming small area with different, iri-
descent color. Columellar muscle thick,
of about one half whorl. Male with large
penis in posterior-right region behind
the right tentacle described below.

Operculum (Figs. 3D-F). Elliptical,
horny, pale to reddish brown. Nucleus
sub-terminal, located closer to interior-
inner edge. Outer surface with normal
concentric growth lines, and series of
radial lines produced by minute, aligned
scales located on the growth lines, from
nucleus to edges (Fig. 3F). Low carina
running at some distance from inferior
and inner edges, from nucleus up to
middle level of inner edge. Inner sur-
face glossy. Scar elliptical, occupying
about 2/3 of inner area, somewhat dis-
located closer to inner edge. Scar having
two different levels of about the same
area, one superior and another inferior;
both separated by a wide chevron,
marking a low step (Fig. 3E, arrow); a
small notch in region where the chev-
ron touches outer scar edge.

FSEUDOLIVIDAE: A POLYPHYLEI'IC TAXON

47

Mantle organs (Figs. 5B, 5D).
Mantle edge simple, thick. Siphon
small, not extending beyond mantle
edge. Osphradinm about 1/3 the width
of the pallial cavity and V4 of its length.
Osphradinm filaments tall, central re-
gion scalloped by 5 folds in the left and
6 folds in the right filaments (Fig. 5B:
os). Osphradinm filaments widely at-
tached along mantle roof. Osphradinm
anterior end curved to the left, with
left filaments clearly smaller than right
filaments; remaining osphradial re-
gions with somewhat symmetrical fila-
ments (left filaments slightly smaller).
Very narrow area between osphradinm
and gill. Ctenidial vein narrow, dislo-
cated weakly beyond left gill edge, to-
wards right edge of osphradinm. Gill
slightly longer than the osphradinm
and of about the same width; its ante-
rior end broadly pointed, located
closer to the mantle edge, far from an-
terior end of osphradinm; posterior
gill end located slightly posterior to
that of osphradinm. Afferent gill vessel
very narrow, lying at a short distance
from the right edge of the gill. Between
the gill and the right edge of pallial
cavity there is an area ec]iiivalent in
width to that of the gill. The hypo-
branchial gland is thin, greenish beige,
covering most of the area between the
gill and the rectum, including the left
and ventral surfaces of the rectum; the
anterior region of the hypobranchial
gland tapers gradually. Rectum nar-
row, running along the right edge of
the pallial cavity (Figs. 5B, 5D-E).
Anus simple, sessile, locateci between
middle and anterior thirds of pallial
cavity. Pallial gonodncts located be-
tween rectum and pallial fioor, de-
scribed below.

Visceral mass (Fig. 5D). Anterior
whorl mostly occupied by stomach,
kidney, and pericardium (Figs. 5E,
6E). Digestive gland greenish brown,
located along inferior region of each
visceral whorl, covering middle diges-
tive tubes and also two whorls poste-

Figure 4. Scanning electron micrographs of radulae. A-B, Zcmira iwstralis, scale bars = 30
pm. C-D, Fnimeiitum ancilla, scale bars = 50 pm. E-F, Melapiiiiu liiicalnm, scale bars = 50
pm. G-I, Nassodonta dorri, scale bars = 30 pm.

48

AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

*1

Figure 5. Zeniira australis anatomy. A, head-foot, male, frontal view. B, pallial cavity roof, transverse section at middle level of osphradium.
C, foot, detail of opercular pad, dorsal view, operculum removed. D, pallial cavity, ventral- inner view, and visceral mass, male. E, region
of kidney, ventral view, ventral wall of kidney and pericardium removed, anterior membrane partially deflected to right. F, head and
haemocoel, ventral view, foot removed. Scale bars = 1 mm. Abbreviations listed in section with figure captions.

PSEUDOLIVIDAE: A POLYPHYLETIC TAXON

49

rior to stomach. Gonad pale beige, lying along superior and
columellar surfaces of visceral whorls posterior to stomach.

Circulatory and excretory systems (Fig. 5E). Pericar-
dium located just posterior to gill, along the left anterior
region of the visceral mass (Fig. 5D). Auricle small, trian-
gular, attached to anterior surface of pericardium, with the
ctenidial vein entering from the left and the connection to
kidney at its right end. Auricle connected to anterior surface
of ventricle. Ventricle very large, filling most of pericardium
volume. Aortas located along posterior region of the ven-
tricle; anterior aorta about 4 times larger than posterior
aorta, and located ventral to it. Kidney occupies about 1/3 of
pallial cavity volume, located along middle and right regions
of the anterior end of the visceral mass. Nephridial gland
triangular in section, broader anteriorly, gradually narrow-
ing posteriorly; lying along the dorsal region of the reno-
pericardial wall. Renal lobe occupying most of the kidney’s
interior volume, presenting two flaps of similar thickness,
fused along right region; ventral flap shorter (about half of
dorsal flap), intestine running through it; dorsal flap occu-
pying most of renal dorsal surface. Afferent renal vessel
large, running from the haemocoel, covering right side of
nephropore, with some branches inserted in inner surface of
dorsal flap of renal lobe.

Digestive system (Figs. 5F-8A). Proboscis relatively
short (about 1/3 of haemocoel length) (Figs. 5F, 7A: pb).
Mouth transverse along proboscis tip. Buccal cavity with
pair of broad and tall lateral folds, each one dividing within
a short distance, one branch running to the odontophore
tube, the other to the esophagus (Fig. 5F). Ventral surface
between buccal folds with a clear, low, flat, chitinous plat-
form (Fig. 7F: ol). Odontophore oval, about half the length
of the proboscis (Figs. 7A, 7E). Odontophore tube connect-
ing it with buccal cavity. Odontophore muscles (Figs. 6A-D,
7E-F): ml, several small muscle fibers connecting buccal
mass to adjacent inner surface of proboscis; mj, pair of peri-
buccal muscles and protractor of odontophore, origin thin
within dorsal wall of oral cavity, running along odontophore
tube becoming thicker, inserting into outer surface of carti-
lages, externally to m6 and medially to m4, in two branches,
one anterior and another posterior, posterior branch about
twice the size of and longer than the anterior branch; m2,
pair of retractor muscles of odontophore, originating in ven-
tral surface of haemocoel, in region just posterior to pro-
boscis (when retracted), running dorsally, with median fi-
bers running through nerve ring, inserting into posterior
surface of odontophore, part into m5 and part into m4
regions close to median line; m2a, auxiliary of m2, being
single and running between both m2, attached to ventral
surface of anterior aorta; its fibers apparently originated ven-
tral to nerve ring, not passing through it (Figs. 6A-D); m3.

pair of thin dorsal protractor muscles of odontophore, origi-
nating in anterior-dorsal end of odontophore tube, at its
juncture with the esophagus, running posteriorly, covering
dorsal surface of odontophore tube, inserting into odonto-
phore middle-dorsal surface (Figs. 7E-F); m4, strong pair of
dorsal tensor radular muscles, originating in odontophore
cartilages along a line surrounding their ventral surface, run-
ning towards dorsal surrounding lateral surface of cartilages,
inserting laterally along radular sac into the region near the
buccal cavity; m5, pair of secondaiy dorsal tensor muscles of
radula, originating in posterior and medial regions of the
cartilages, running dorsal and medial as continuation from
ni4, inserting into radular sac near the buccal cavity along-
side and medial to m4 insertion; m6, thin horizontal muscle,
uniting both odontophore cartilages, with about 3/4 of car-
tilage length, inserting along a line into the ventral and ex-
ternal surfaces of the cartilages, at a short distance from their
inner-ventral edge, starting at the anterior end of the carti-
lages and ending just before their posterior c]uarter (Figs.
6B-C); ml la, pair of ventral tensor muscles of radula, thin,
somewhat broad, originating partly in the posterior-ventral
end of cartilages and partly in the m2 insertion, running
anteriorly covering m6, inserting into ventral edge of radula
and subradular cartilage, and some inner portion preceding
this (Fig. 6D); ml4, pair of ventral protractor muscles of
odontophore, originating along ventral surface of oral fube
and tube of odontophore, running posteriorly at short dis-
tance from median line, covering central surface of odonto-
phore, inserting into posterior-ventral surface of odonto-
phore, close to m2 insertion (Fig. 7E). Other non-muscular
odontophore structures: sc, subradular cartilage, expanding
in exposed region of radula into buccal cavity, covering
neighboring surface of radula (Figs. 6A, 6D, 7F); oc, odon-
tophore cartilages, somewhat elliptical, flat, with medial-
ventral edge slightly straighter than outer edge, posterior
region clearly narrow (Figs. 6B, 6C); br, subradular mem-
brane, covering inner surface of subradular cartilage and
radula, m4, ni5, and ml la inserfions. Radula (Figs. 4A-B):
rachidian toofh with short transverse base, spanning about
1/3 of radular ribbon, 3 long, tall (about 2/3 of base length),
sharp pointed cusps somewhat ecyiidistant from each other,
central cusp symmetrical, outer cusps weakly turned out-
wardly; between rachidian and lateral teeth a distance
equivalent to 1/3 of rachidian width; lateral tooth hook-like,
base broad (equivalent to 2/3 of rachidian base width),
gradually narrowing up to sharply pointed tip, height about
1.5 that of rachidian; straight to weakly curved inwardly.
Salivary glands clustering along anterior region of valve of
Leiblein and ventral ganglia of nerve ring, attaching to lateral
surface of the anterior esophagus just anterior to the valve of
Leiblein (Figs. 7A-B); their ducts very narrow, totally at-

50

AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

Figure 6. Zernim australis anatomy. A, odontophore, dorsal view, anterior odontophore tube removed. B, same, outer layer of muscles and
radular apparatus removed and only partially shown (rs). C, odontophore, ventral view, only its right side shown, posterior muscles
deflected. D, same, outer view, outer layer of muscles and membrane partially removed. E, midgut, ventral view as in situ, some adjacent
structures also shown. Scale bars = 1 mm. Abbreviations listed in section with figure captions.

tached to anterior esophagus wall and to lateral wall of oral
tube; opening very small (Fig. 7F: sa), into anterior region of
lateral folds of buccal cavity, somewhat ventrally, just within
the anterior end of a narrow furrow surrounding the ventral
edge of the odontophore tube folds. Anterior esophagus
with somewhat thick walls, length ec]uivalent to that of
odontophore, inner surface with lateral, longitudinal, low,
and flat folds that become narrower posteriorly. Between
these folds are secondary, low, narrow folds (Fig. 7F). Valve

of Leiblein with about 1/4 of odontophore volume, anterior
region with a transverse, white band into which long cilia
insert, middle and posterior regions pale beige, correspond-
ing to a tall inner gland occupying most of inner surface;
oblique furrow (by pass) present, separating all valve regions
(Figs. 7A-B); inner surface smooth, not glandular, bordered
by pair of low and veiy narrow folds that diverge in its
anterior region, and continuous with middle esophagus
folds posteriorly. Middle esophagus about half as long as

PSEUDOLIVIDAE: A POLYPHYLETIC TAXON

51

Figure 7. Zemira australis anatomy. A, foregut, extended, ventral view. B, region of the valve of Leiblein, its oblique furrow (vf) seen by
transparency. C, gland of Leiblein partially uncoiled, some adjacent structures also shown. D, transition between middle and posterior
esophagus and duct of gland of Leiblein, opened longitudinally to expose inner tokis. E, buccal mass, left view. F, buccal mass, anterior
region opened longitudinally and deflected to expose inner surfaces. Scale bars = 1 mm. Abbreviations listed in section with figure captions.

52

AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

anterior esophagus (Fig. 7A: em), walls thin; inner surface
with longitudinal, low, narrow folds, a pair of close folds
larger (Eig. 7D), running towards duct of gland of Leiblein.
Gland of Leiblein triangular in situ (Figs. 5F, 7A), long and
somewhat flat if uncoiled (Fig. 7C), becoming about as long
as posterior esophagus; anterior aorta crossing between
middle and posterior thirds of this gland. Duct of gland of
Leiblein long and narrow (about as long as middle esopha-
gus, and about half of its diameter) (Figs. 7A, 7D); having
two origins, one sub-terminal in anterior end of the gland,
the other in a portion more posterior (Fig. 7C); these two
ducts unite within a short distance, remaining duct having
pair of tall, longitudinal, narrow folds (continuation from
larger folds of middle esophagus); these folds separate a
narrow, white, multi-lobed secondary gland from a smooth,
narrow area; this secondary gland occupies about 2/3 of duct
volume, ending abruptly before the duct’s insertion into the
esophagus (Fig. 7D). Posterior esophagus (Figs. 7A, 7D; ep)
about twice as long as the anterior esophagus, inner surface
with narrow longitudinal folds, some low, others taller (cov-
ering lower folds) diverging and coalescing randomly; these
folds disappearing abruptly before stomach. Stomach
spherical, blind sac, about half the width of adjacent visceral
whorl. Esophagus enters stomach along its left anterior re-
gion (Figs. 6E, 8A: st), intestine originates to the right of the
esophageal insertion. Gastric inner surface (Fig. 8A) mostly
smooth, except for a pair ot low, narrow folds that run along
its left surface, from the esophageal insertion, disappearing
gradually into the posterior gastric surface. Duct to digestive
gland single, wide, located between esophageal insertion and
intestinal origin (Fig. 6E). Intestine with tall, dorsal,
smooth, long and triangular platform adjacent to the stom-
ach (Fig. 8A); left edge of this platform alongside a band of
longitudinal, narrow folds; right edge of this platform serv-
ing as insertion of several transversal folds, each about twice
as wide as the longitudinal folds, becoming gradually ob-
lic]ue, surrounding ventral surface of intestine, ending at left
edge of band with longitudinal folds. Inner surface of intes-
tine, beyond this platform, with only longitudinal folds, very
close to each other, filling inner surface totally. Intestine
runs almost straight anteriorly, crossing through anterior
region of digestive gland, kidney lobe, and right edge of
pallial cavity (Figs. 5E, 6E). Rectum and anus described
above (pallial cavity).

Genital system. Male (Figs. 5A, 8B). Visceral vas def-
erens begins a half whorl before anterior end of testis.
Within a short distance it becomes a very broad, intensely
coiled seminal vesicle, occupying about half of adjacent vis-
ceral whorl (Fig. 5D). Seminal vesicle located in the ventral
surface of last whorl of the visceral mass, posterior to kidney;
becomes narrow at some distance posterior from pallial cav-
ity, running about 1/6 whorl. Prostate gland relatively nar-

row, running along right edge of pallial cavity ventral to
rectum, visceral vas deferens inserting posteriorly (Fig. 5D:
pt); walls thick-glandular; no apertures to pallial cavity; in-
ner lumen surrounded by muscle fibers. Prostate spans
about Vs pallial cavity length, gradually becoming narrow,
crossing to pallial floor. This region in the pallial cavity floor
with thick, muscular walls, slightly convolute up to penis
base (Fig. 8B). Penis slightly larger than half of pallial cavity
volume, stubby, dorso-ventrally flat (Figs. 5A, 8B); base
broad, with a large, broad right fold covering base of right
tentacle; then twisting, remaining tall, flat and thick, nar-
rowing gradually up to bluntly pointed tip (Fig. 8B). Pallial
vas deferens within the integument, becoming penis duct.
Penis duct running approximately along penis center, very
narrow, weakly coiled. Penis aperture apical, very small.

Female (Figs. 8C-F). Visceral oviduct very narrow, run-
ning along middle region of columellar surface of the last
whorl of the visceral mass, about W whorl preceding pallial
cavity, gradually becoming thicker, inserting into pallial ovi-
duct without clear separation. Posterior region of pallial ovi-
duct protruding into kidney, having a narrow zigzag. Albu-
men (whitish) and capsule (beige) glands adjacent, albumen
gland spanning posterior 1/5 of pallial oviduct. Seminal re-
ceptacle very small, located between albumen and capsule
glands (Fig. 8C); flat to rounded; duct very narrow, attached
along the dorsal surface of the pallial oviduct, opening into
the vaginal furrow between the albumen and capsule glands.
Capsule gland with flat lumen, vaginal furrow running along
its left edge, with surface smooth (Fig. 8D). Female pore
wide, protruded, with thick edges (Figs. 8E-F: fp). Bursa
copulatrix small, short, located along left side of the distal,
detached portion of the pallial oviduct (Fig. 8F: be); with
thick muscular walls; its aperture turned anteriorly, occupy-
ing about 2/3 of total female pore; inner surface with low,
wide, longitudinal folds. Capsule gland aperture narrow,
situated to the right of the bursa aperture; its walls thick
muscular, protruding inside the chamber of the terminal
atrium of the capsule gland. Terminal atrium, with thin
walls, located between capsule gland anterior and female
pore. Female pore with several wide, longitudinal folds. No
cement gland in foot sole.

Central nervous system (Figs. 8G-H). Relatively well-
concentrated. No distinction between pleural and cerebral
ganglia. Cerebral ganglia broadly connected to each other.
Pedal ganglia slightly smaller than cerebro-pleural ganglia;
pedal commissure broad, but narrower than cerebral com-
missure. Cerebro-pedal and pleuro-pedal connectives short,
but distinguishable. Pair of buccal ganglia small, located
close to posterior edge of the cerebral ganglia.

Measurements of shells (in mm). AMS C333288;
?1 = 18.6 by 12.0; 62 = 19.3 by 12.2; 63 = 16.6 by 9.8;
54 = 14.1 by 8.7.

PSEUDOLIVIDAE: A POLYPHYLETIC TAXON

53

Figure 8. Zemira australis anatomy. A, stomach, ventral view, its ventral wall and adjacent region of intestine (in) and esophagus (es) opened
longitudinally, inner surface exposed. B, penis and adjacent region of its base, ventral view, penis partially deflected, its duct (pd) seen by
transparency. C, pallial oviduct, dorsal view, detail of its posterior region. D, pallial oviduct, transversal section though its middle region.
E, pallial oviduct, entire ventral view, including its portion in kidney chamber. F, same, detail of its anterior end, ventral wall removed,
region of pore opened longitudinally and deflected to show its parts. G, central nervous system, ventral, slightly right oblique view, esophageal
passage (es) shown by arrow. H, same, dorsal, slightly left oblique view. Scale bars = 1 mm. Abbreviations listed in section with figure captions.

Distribution. East coast of Australia.

Habitat. Sandy, from 3 to 146 m depth (Beechey 2005).
Material examined. AUSTRALIA. New South Wales;
Sydney (Shelf Benthic Survey col.), 1.6 km east of Malabar

outlet, 33°58.250'S 1 5 1° 1 7.000'E, 66 m depth, AMS
C092091, 3? (Sta. 027653, SBS3, 26/jii/1973), 2.3 km east of
Malabar outlet, 33°59.450'S 151°16.800'E, 66 m depth, AMS
C333288, 3d, 6? (Sta. 002609E, SBS5, 25/x/1973), 2.6 km

54

AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

east of Cape Banks, 33°59.500'S I5I°I6.740'E, 66 m depth,
AMS C406792, Id (Sta. 038772, SBS25, 26/1/1973).

Genus Fidmentiim Eischer, 1884

(Type species Biiccinwn sepimentum Rang, 1832,
by monotypy)

Fulmentum ancilla (Hanley, 1859)

(Figs. 9A-E, 4C-D, 10A-13F)

PseiidoUva ancdla: Kantor 1991: 31-34 (figs. 12, 13);
Hayes 1994: 77-78.

Sylvanocoddis ancdla: Lorenz 1989: 16-18.

Fiilmentwn ancdla: Vermei) 1998: 60.

Description

Shell (Figs. 9A-B, 9E). Large (about 60 mm), heavy,
biconical. Color brown to beige. Periostracum velvet-like,
rust colored, partially eroded. Spire conical, aperture span-
ning 70% of shell length. Protoconch of IVi whorls, white,
smooth, to wealdy reticulated in some areas, border with
teleoconch unclear. Teleoconch of 5-6 whorls, each whorl
with a weakly concave shoulder. Axial sculpture limited to
growth lines. A wide spiral furrow runs along the anterior
third of the shell, forming a low, broad labral tooth. Umbi-
licus absent. Aperture elliptical, posterior end pointed.
Siphonal canal wide, open, very short. Inner lip smooth,
callus narrow and low except for a small, low node at some
distance from posterior end. Outer lip with simple edge,
thickened at the shoulder and in region of the labral spine.
Thickening of the outer lip and the callus of the inner lip
located just posterior to the spiral furrow on the previous
whorl.

Head-foot (Figs. 9E, lOB-C, llA). Head not protruded.
Color mostly pale beige, with dark brown, coalescent (form-
ing wide transversal bands) spots in head and dorsal surface
of foot (Fig. 9E). Tentacles relatively short, located close to
each other; basal half clearly thicker than distal half. Eyes
located along outer edge of tentacles at middle level, proxi-
mal to their constriction; terminal portion of tentacle very
short (Figs. lOB-C). Rhynchostome located ventral to and
between the tentacles. Foot spans Vi whorl when retracted;
sole simple; anterior edge with deep furrow that contains the
pedal glands. Dorsal and ventral edges of this furrow rela-
tively thick and rounded; both ends of anterior edge
rounded. Columellar muscle spans Vi whorl, thick; posterior
end with a wide, rounded component, and a small (about
10% of origin area) projection located along its left end.
Haemocoel about 1/3 head-foot width along its anterior half,
posterior half becomes very narrow and turned to the left
(Fig. IOC).

Operculum (Figs. 9C-D). Elliptical, horny, brown, oc-
cupying entire shell aperture. Nucleus terminal, inferior.
Outer surface with concentric growth lines and narrow un-

dulations. Inner surface glossy. Scar occupying about 80% of
inner surface, approx, central, slightly displaced closer to
inner and superior edges. Scar divided into approx, two
similar sized areas by oblique line. Inferior region broadly
pointed, with wide free projection; triangular, wide furrow
running along middle region of this projection, starting in
the apex, finishing in the scar.

Mantle organs (Figs. lOA, lOE). Mantle edge simple,
thick, transversally banded. Siphon relatively long (about
half of pallial cavity length), edges simple (Figs. 9E, lOA);
mantle edge surrounding its base. Pallial cavity with about
Wi whorls. Osphradium about % pallial cavity length and
about '/4 of its width, with curved, symmetrical filaments;
anterior and posterior ends rounded; each filament relatively
tall, projecting laterally; dorsal edge connected to mantle
along Vi its length the rest supported by a rod; ventral edge
thin, with small notch located approximately in central re-
gion; right filaments covering ctenidial vein and part of gill
filament bases (Fig. lOE: os). Gill about 3/4 of pallial cavity
length and 1/4 of its width; anterior end pointed, posterior
to that of osphradium; ctenidial vein extending a short dis-
tance anterior to gill end; posterior gill end rounded, touch-
ing pericardium. Gill filaments triangular and tall, curved to
right; rod relatively broad; filament tip rounded, preceded by
narrow region. Ctenidial vein narrow, lying along left edge of
gill. Afferent gill vessel very narrow, lying at some distance
from apparent right gill edge. Hypobranchial gland some-
what tall, whitish, covering entire area between gill and rec-
tum (about V2 pallial cavity width), anterior end at level of
anus. Rectum relatively narrow, running along right edge of
pallial cavity. Anus detached, situated between middle and
anterior thirds of pallial cavity, with small papilla on its right
side. Genital ducts lying between rectum and right edge of
cavity, described below.

Visceral mass (Fig. lOA). Spans about three whorls.
Reno-pericardial structures occupy anterior half whorl.
Stomach located just posterior to reno-pericardial area. Di-
gestive gland greenish cream, occupying all whorls, sur-
rounding stomach. Gonad same color as digestive gland,
occupying superior and columellar surface of each visceral
whorl, terminating a short distance posterior to kidney.

Circulatory and excretory systems (Fig. lOD). Pericar-
dium about '/4 of kidney volume. Heart similar to that of
Zeniira. Kidney somewhat trapezoid, spanning half
whorl. Nephridial gland flat, thicker anteriorly, lying along
entire dorsal half of reno-pericardial membrane. Kidney lobe
similar to that of Zemira. Afferent renal vessel large, with
branches running between folds of both kidney lobe flaps,
covering right side of nephropore, connected to membrane
between kidney and pallial cavities, producing small urinary
cavity.

Digestive system (Figs. 1 1A-12E). Rhynchostome forms
a small opening located ventral to and between both cephalic

PSEUDOLIVIDAE: A POLYPHYLETIC TAXON

55

Figure 9. Shells, opercula, and living specimens. A-E, Fiihuentuiu aiidihr, A-B, shell, NMNH E5279, female, apertural and dorsal views,
length = 64.5 mm. C-D, Operculum, outer and inner views. E, Live, crawling specimen from Jeffreys Bay, South Africa, photo courtesy of
Brian Hayes. F-K, Melapium Uneatum. F-G, shell, NMNH V9979, male, apertural and dorsal views, 2 egg capsules attached in anterior region
of inner lip (total length = 26,2 mm). H, detail of egg capsules attached to shell, NMNH 59733, portions ot both capsules removed, showing
young specimens inside. E), dorsal and apertural views ot a young specimen extracted from an egg capsule shown in the preceding figure,
scale bar = 2 mm. K, live, crawling specimen from off Algoa Bay, South Africa, photo courtesy of Brian Hayes.

56

AMERICAN MALACOLOGICAL BULLETIN

23 • 1-2 • 2007

Figure 10. Fulmentum nncilla anatomy. A, pallial cavity and visceral mass, male, ventral-inner view. B, head-foot, male, frontal view. C, head
and haemocoel, ventral view, foot and columellar muscle removed. D, kidney and pericardium, ventral view, ventral wall removed, renal
membrane with pallial cavity (km) deflected anteriorly (right in fig.). E, Transverse section through pallial cavity roof, at mid-length of the
osphradium. Scale bars = 2 mm. Abbreviations listed in section with figure captions.

PSEUDOLIVIDAE: A POLYPHYLETIC TAXON

57

ad sa

me

Figure 11. Fuhnentum ancilla anatomy. A, foregut partially uncoiled, ventral view, adjacent region of head also shown. B, buccal mass, left
view, esophagus opened longitudinally. C, odontophore, dorsal view, some muscles deflected. D, same, dorsal view, superficial layers of
muscles and membrane reflected, aorta shown as in situ. Scale bars = 2 mm. Abbreviations listed in section with figure captions.

tentacles (Fig. IOC). Proboscis short (less than 1/3 of hae-
mocoel length) and broad (about haemocoel width) (Figs.
IOC, llA); walls thick, muscular. Proboscis retractor
muscles forming a main paair, one in each side, left retractor
muscle slightly more dorsal than right, originating at middle
level of haemocoel latero-ventral region, running towards

middle level of p^roboscis, inserting along its distal half (Fig.
IOC: rm); several accessory proboscis retractor muscles
along dorsal surface, thinner than main retractor muscles,
originating in a virtual line connecting both main retractor
muscles, surrounding dorsal haemocoelic wall. Mouth a
transversal slit on proboscis tip. Oral tube with thick walls.

58

AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

about half the length of the odontophore. Oral cavity wide,
with a pair of low, broad, dorsal folds, each occupying about
1/3 of the dorsal surface and 1/3 of area between them; each
dorsal fold with rounded anterior end, at short distance
from mouth (Fig. 1 IB); remaining fold characters similar to
those of Zemira. Ventral chitinous platform present (Fig.
IIB, ad). Odontophore about same length as proboscis
(when extended), protruding beyond proboscis into haemo-
coel when retracted (Figs. IOC, llA). Odontophore organi-
zation and musculature similar to that of Zemira, with fol-
lowing distinctions. Odontophore muscles (Figs. 11B-12A):
m2 a single pair inserted into posterior region of m4, by side
of radular sac; m2a absent; m5 pair wider and thicker; m6
with anterior end at some distance from that of odonto-
phore cartilages (Fig. 10); ml la pair broader (Fig. IID).
Odontophore cartilages elliptical, with posterior region simi-
lar to anterior region (Fig. 12A). Radula (Figs. 4C-D): ra-
chidian tooth with broad base (about 60% of radular rib-
bon), close to adjacent teeth, with 3 tall (about 1/2 base
width), triangular, sharply pointed cusps, somewhat equi-
distant and separated from each other; distance between
rachidian and lateral teeth area equivalent to half of rachid-
ian base width; lateral tooth with base 60% of rachidian
width, 2 tall (height equivalent to base width), triangular,
sharply pointed cusps, one on each side of the tooth, inner
cusp slightly smaller than outer cusp. Anterior esophagus
shorter than odontophore, wall thick muscular, with some
muscles connecting its latero-ventral region to the adjacent
region of the haemocoel floor, sometimes passing through
the salivary glands; inner surface with a pair of lateral folds,
as continuations from buccal cavity folds, gradually narrow-
ing (Figs. IIA-B: ae). Salivary glands paired, each an ellip-
tical, separated mass located along each side of valve of
Leiblein and close to the nerve ring (Figs. 1 lA-B, 12B: sg).
Salivary ducts very narrow, originating in the anterior end of
the gland, penetrating the lateral walls of the anterior
esophagus within a very short distance; running within this
wall up to salivary aperture. Salivary aperture very small,
located in lateral edge of buccal cavity dorsal folds, at mid-
level, just within the posterior end of a narrow and shallow
furrow running anteriorly, surrounding antero-lateral edge
of dorsal folds (Fig. IIB; sa). Accessory salivary gland
single, elliptical, internally hollow, situated within the hae-
mocoel near the middle region of odontophore’s ventral
surface. Anterior region of gland gradually narrowing with-
out clear division from its duct. Duct long and very narrow,
equal to the length of odontophore, lying along ventral sur-
face of the odontophore, odontophore tube and oral tube
(Figs. IIA-B: ae); opening of duct very small, in median
region of the ventral surface of the oral tube, just posterior
to the mouth (Fig. IIB: ad). Valve of Leiblein with about
half size of odontophore, inner organization similar to that

of Zemira, with narrow folds of the oblique furrow disap-
pearing gradually at anterior and posterior ends (Figs. IIA-
B, 12B: vl). Middle esophagus, narrow, roughly equal in
length to the anterior esophagus, walls thin, inner surface
smooth (Figs. IIA-B: em). Gland of Leiblein brown, long,
triangular, anterior region wide, narrowing gradually to-
wards posterior, posterior tip narrow and rounded; anterior
aorta crossing through this gland between middle and ante-
rior thirds (Figs. IOC, 1 lA: gl). The portion of the duct of the
Gland of Leiblein that is free from the gland is relatively long
(about half the length of the middle esophagus) (Figs. IIA,
12C: Id); inner surface with a longitudinal, white gland in its
dorsal side, and a smooth, thin region on the ventral side;
lacking transverse septa within (Fig. 12C). Posterior esopha-
gus narrow, about three times as long as anterior esophagus,
wall thin, inner surface smooth or with narrow longitudinal
folds, close to each other (Figs. 11 A; ep; 12D-E: es). Stomach
trapezoidal, weakly dorso-ventrally flattened, occupies about
half of visceral whorl volume, is situated about 1/4 whorl
posterior to kidney (Figs. 12D-E); esophagus joins the stom-
ach at left-anterior end, the intestinal joins the stomach to
the right of the esophagus, and is slightly wider. Duct to
digestive gland single, located a short distance posterior to
esophageal insertion and intestinal origin; duct with very
wide and flat base, with branches running from opposite
sides after short distance. Gastric walls thick muscular. Gas-
tric inner surface with transversal, low, broad, somewhat
irregular folds (Fig. 12E). Intestine almost straight, weakly
sigmoid, running anteriorly (Figs. lOD, 12D-E); inner sur-
face full of low, narrow, closely spaced longitudinal folds; A
larger pair of adjacent folds run along the left side of the
stomach, gradually disappearing into rectum. Intestine
passes initially through digestive gland, then through the
ventral flap of the kidney lobe. Rectum and anus as de-
scribed above (pallial organs).

Genital system. Male (Figs. lOA-B, 13A-B). Testis as
described above (visceral mass). Seminal vesicle very large,
occupying the columellar surface of almost the entire last
whorl of the visceral mass, forming a relatively narrow, ex-
tremely convoluted tube (Fig. lOA: sv). Seminal vesicle
abruptly terminates near the pallial cavity, giving rise to a
very narrow vas deferens that crosses the afferent renal vessel
dorsally, and becomes exposed in the pallial cavity, lying
along the posterior and right pallial cavity edges, gradually
becoming thicker (Fig. lOA). No prostate gland differen-
tiable. Vas deferens descends to pallial floor at mid length of
the pallial cavity. The portion of the vas deferens lying on
pallial floor is open (furrow); with tall, thick edges (Fig.
lOB), becoming convoluted near the base of the penis. Penis
of about 1/4 of pallial cavity volume (Fig. lOB); twisted
inwards in basal region, middle region slightly broader, con-
stricting gradually up to narrow, rounded tip. Penis duct

PSEUDOLIVIDAE: A POLYPHYLETIC TAXON

59

Figure 12. Fuhnentiim ancilla anatomy. A, odontophore, dorsal view, most of the superficial membrane and muscles removed, radular sac
deflected to right, both cartilages (oc) deflected from each other, left ni5 turned downwards. B, region of the valve of Leiblein (vl), ventral
view. C, region of duct of the gland of Leiblein (Id), ventral view, most tubes opened longitudinally. 1), midgut as in situ, ventral view,
position of kidney indicated. E, same, most tubes opened longitudinally to expose inner surface. Scale bars = 1 mm. Abbreviations listed
in section with figure captions.

open (a furrow), lying on inner penis edge, relatively deep,
runs up to penis tip (Fig. 13A). Terminal papilla in penis tip,
about 1/6 of penis length, located inside a chamber formed
by terminal portion of penis (Fig. 13B).

Female (Figs. 13C-D). Visceral structures similar to
those of males. Visceral oviduct very narrow, running along
the middle of the columellar surface of the visceral mass.
Visceral oviduct inserting in left side of pallial oviduct, an-

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

Figure 13. FiilnienUuii ancilla anatomy. A, penis and adjacent region of its base, dorsal view. B, detail of penis tip, showing, partially by
transparency, terminal papilla. C, pallial oviduct, ventral view, detail of its terminal region mostly opened longitudinally, walls reflected. D,
entire pallial oviduct, ventral view as in situ, some adjacent structures also shown, a transverse section at indicated level also shown. E,
central nervous system, ventral view, esophageal passage indicated (es). F, same, dorsal view. Scale bars A, D = 2 mm; others = 1 mm.
Abbreviations listed in section with figure captions.

PSEUDOLIVIDAE: A POLYPHYLETIC TAXON

61

terior to albumen gland, opening to vaginal duct. Albumen
gland whitish, about 'A length of pallial oviduct, forming a
blind-sac with thick walls and flat lumen that is continuous
with the lumen of the capsule gland. Seminal receptacle
triangular, located between albumen and capsule glands,
close to right-dorsal side, no apparent duct, opening directly
to vaginal duct between both glands. Capsule gland beige,
inner lumen broad and flat (as wide as gland). Vaginal duct
lying all along capsule gland left edge, separated from it by a
low fold of dorsal lamina ot capsule gland. Capsule gland
laminae terminating close to genital pore, without forming a
vestibule. Female pore protruded, rounded, located to right
of anus. Female pore walls thick muscular, inner lumen flat,
curved from left to right, then to left again, expanding
gradually, leading to pore (Fig. 13C). Female pore edges
thick, preceded by longitudinal, broad folds. No cement
gland in sole of foot.

Central nervous system (Figs. 13E-F). Located at base
of proboscis, a short distance ventral to the rhynchostome.
Very concentrated, practically no individual ganglia distin-
guishable. Cerebro-pleural ganglia widely connected to each
other along median line. Both also widely connected to pedal
ganglia, no connective distinguishable. Pedal ganglia of
about the same size as the cerebro-pleural ganglia. Commis-
sure between both pedal ganglia relatively narrow and very
short (both pedal ganglia maintained in contact). Pair of
buccal ganglia small, close to each other, located oblic^uely
(left ganglion slightly more anterior) in dorsal region of
cerebro-pleural ganglia; connective with cerebral ganglia
narrow and short, left connective a little longer and joining
another secondary nerve, running anteriorly. Supra- and
subesophageal ganglia about half the size of the main gan-
glia, located near and ventral to right cerebral ganglion; sub-
esophageal ganglion connected to cerebral ganglion by a
narrow and short connective; supra-esophageal ganglion
connected to subesophageal ganglion by a broad and very
short connective, and also to left cerebral ganglion by a
narrow and short connective.

Measurements of shells (in mm). NMSA E5279:
64.5 by 35.0.

Distribution. South Africa.

Habitat. Rocks and coarse sand, from 32 to 81 m depth.

Material examined. SOUTH AFRICA. Western Cape;
93 km SE of Mossel Bay, 68.4 m depth, NMSA E2770, 1 $
(no shell) (Exch. C. Marais col., 24/vi/1988), SW of Mossel
Bay, Agulhas Bank, 81 m depth, NMSA E5279, 1 9 (Exch. C.
Marais col., xi/1988); Struis Bay, 34°47.2'S 20°08.6'E, 32 m
depth, NMSA S3578, Id (no shell) (dredged Sardinopsis,
08/vi/1991).

Discussion. Fulmentu?n ancilln has been mostly referred
in the genus Pseiidoliva or Sylvanocochlis Melvill, 1903, for
which it is type species. Recognition of Fidmentiim is based
on the arguments of Vermeij (1998: 60), who considered

both genera {Sylvanococldis and Fidmentum) as synonyms.
Kantor’s (1991) anatomical description was used as the
ground plan for the anatomical study of this species. Results
of the present study generally agree with Kantor’s data; the
few cfifferent points include the presence of transversal dis-
tinct folds in the hyp)obranchial gland found in his speci-
mens (his fig. 12D), but lacking in those examined here.

Genus Melapiiim Adams and Adams, 1853

(Type species Pyrida lineata, by subsequent designation
of Cossmann, 1901)

Melapium lineatum (Lamarck, 1822)

(Figs. 9F-K, 4E-F, 14A-17F)

Melapium lineatum: Liltved 1985: 9; Kantor 1991: 39-41
(figs. lA-B, 2B, 17, 18); Hayes 1994: 77-78; Vermeij
1998: 75.

Description

Shell (Figs. 9F-K). Of medium size (about 30 mm),
rounded; wider than long. Walls thick. Color cream, with
narrow axial bands, dark beige, with irregularly intercalated
longer and shorter bands, mainly concentrated in a band
along the middle of the body whorl; canal white, with purple
pigmentation within the anterior edge. Protoconch flat,
dome-shaped, of 1 ‘A whorls; transition to teleoconch indis-
tinct (Figs. 9G-J). Spire flat, low, weakly elevated, with about
3 whorls. Suture relatively deep. Body whorl very large, sur-
rounding almost completely the penultimate whorl. Surface
glossy, lacking sculpture except for weakly visible growth
lines. Anterior region with carina surrounding left edge of
canal, projected forwards. Aperture rounded (Fig. 9F), lo-
cated close to suture, peristome white, gradually becoming
orange in interior regions. Outer lip simple, semi-circular;
edge thick, rounded. Inner lip bearing thick callus, cover-
ing roughly half of the ventral surface. Canal short, broad,
relatively deep, projected forwards. Young specimens (about
2 whorls) antero-posteriorly longer, outline elliptical (Figs.
91-1); outer surface opaque, sculptured by a net ot thin and
very narrow reticulation of spiral and axial lines (Fig. 91).

Head-foot (Figs. 9K, 14A-B, 14 D, 15B). Head weakly
protruded, socket-like; basal region of head as short flap.
Tentacles located in both ends of this flap; each tentacle long
and narrow, with a broader region just above base; tip
pointed. Color mostly beige-cream, with bluish band flanked
by a narrow bands of red and yellow surround the dorsal
surface of foot at its margins (Fig. 9K); tentacles with
pale base and orange middle and distal regions. Eyes located
on both ends of head-flap, below the base of each tentacle
(Figs. 14A, 14D). Rhynchostome in the form of a transversal
slit is located between the tentacles (Fig. 14D). Foot very
wide and broad; thick in center, gradually becoming thinner
toward the periphery, uniformly in all directions. Furrow of

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

Figure 14. Melapiurn lineatiim anatomy. A, head-foot, male, frontal view. B, same, posterior view. C, pallial cavity and visceral mass, male, [;

ventral-inner view. D, head and haemocoel, ventral view, foot and columellar muscle removed. E, Transverse section through pallial cavity |i

roof, at mid-length of the osphradium. Scale bars = 2 mm. Abbreviations listed in section with figure captions. [j

pedal glands very thin, restricted to anterior half of foot edge
(Lig. 14A: pg). Columellar muscle of about 1/3 whorl, having
broad main flap in middle and right regions; secondary flap
taller and longer, at its left end, projected deeper, weakly
coiled (Fig. 12A: cm). Male penis relatively small, originating
far removed posteriorly from right tentacle (described below).
Haemocoel oval, broad, weakly curved to left (Fig. 14D).

Operculum. Absent.

Mantle organs (Figs. 14C, 14E). Mantle border simple.

wide, thick, unpigmented. Pallial cavity broad and short
(just over Vi whorl). Siphon long (equal in length to pallial
cavity) and slender; edges simple; inner surface with low
transversal folds. Siphon base with pair of reinforcements
extending as low folds beyond siphon base, parallel to
mantle border, longer on right, about 1/3 of mantle border
length, gradually diminishing. Osphradium slightly longer
than '/2 pallial cavity length and about 1/5 of its width;
anterior end rounded, posterior end pointed. Osphradium

PSEUDOLIVIDAE: A POLYPHYLETIC TAXON

63

filaments tall, symmetrical, mostly free from attachment to
mantle roof (connected mainly to osphradial ganglion),
forming a longitudinal concavity along this ganglion. Each
filament extends ventrally about twice the diameter of the
osphradium ganglion, its edge reinforced by a rod that is
weaker along inner regions closer to the ganglion. Gill ellip-
tical and broad, slightly shorter than pallial cavity and about
half as wide, slightly curved to left. Anterior and posterior
ends broadly pointed, anterior end slightly forward of os-
phradium margin, and separated from it by a low, broad fold
of the siphonal base. Posterior end of gill extends beyond
posterior end of osphradium, reaching the pericardium. Gill
filaments triangular, relatively low, apex at or slightly to the
right of center, rounded; rod broad, lying along left edge,
extending slightly beyond the membranous portion of the
filament. Ctenidial vein and afferent gill vessel narrow (af-
ferent slightly narrower), running along respective gill edges.
Hypobranchial gland moderately thick, cream colored, cov-
ering most of the area between the gill and rectum (-1/3 of
pallial roof), becoming narrow in the region anterior to the
anus, surrounding right edge of gill. Rectum relatively broad,
running along the right edge of the pallial cavity for about
2/3 of its length. Anus detached, located between middle and
anterior thirds of pallial cavity; with small papilla along its
left edge. Genital ducts lying between rectum and pallial
floor, described below.

Visceral mass (Fig. 14C). Of about 2Vi whorls, rapidly
enlarging and almost involute. Right portion of visceral
structures encroaching into the right posterior portion of the
pallial cavity. Kidney triangular, spanning ~Vi whorl along
its right border, partially located inside the middle and right
portion of the posterior pallial cavity. Pericardium located
slightly to the left of the middle of the posterior portion of
the pallial cavity. Stomach located about 1/3 whorl posterior
to kidney, occupying about half of adjacent visceral whorl
volume. Digestive gland orange, extending from apex to kid-
ney, surrounding middle digestive tubes. Gonad within right
and columellar surfaces of visceral mass, of the same color as
the digestive gland all along its length. Ovary surrounded
entirely by digestive gland, becoming internal to it.

Circulatory and excretory systems (Figs. 14C, 15A).
Heart volume about 1/3 kidney volume; characters similar to
those described for Zemira, auricle small, entirely attached
along antero-dorsal region of pericardium; ventricle very
large, intensely muscular. Anterior aorta 5-6 times thicker
than posterior aorta. About half of the kidney encroaches
into the pallial cavity roof Nephridial gland relatively thick
(thicker anteriorly), covering entire middle and dorsal re-
gion of reno-pericardial membrane. Renal lobe similar to
Zernira, but with more developed transverse, irregular folds;
ventral and dorsal flaps of renal lobe with tall folds interca-
lating with each other successively along their right region.

Rectum passes through ventral flap of renal lobe. Dorsal flap
of renal lobe hard and yellowish; ventral flap whitish
and softer. Renal efferent vessel very large (almost as large
as anterior aorta); running from posterior end of haemocoel,
penetrating intro renal chamber with left side broadly
attached to middle region of pericardium, before giving rise
to multiple branches that become progressively smaller and
insert between renal lobe folds. Nephropore a transversal
slit, with muscular edges, located in middle of reno-pallial
membrane; internally free from folds or vessels inserting
close to it.

Digestive system (Figs. 15B-16E). Proboscis relatively
short (about 1/3 of haemocoel length) and narrow (about
half of its width) (Figs. 14D, 15B). Several narrow proboscis
retractor muscles surround it almost completely, but more
concentrated in dorsal-right region, originating in anterior
and middle surfaces of haemocoel (Fig. 15B). Proboscis wall
very thick in rhynchodeal region, relatively thin in buccal
mass region (Fig. 15D). Mouth transverse in proboscis tip.
Oral tube relatively long (about same length as odonto-
phore) and narrow; inner surface with a broad pair of dorso-
lateral folds, each fold with about 1/3 of oral tube surface,
anterior end of each fold rounded, narrowing gradually pos-
teriorly. A shallow area between both folds is equivalent to
that of each fold in width (Figs. 15D-E). A longitudinal
platform runs along the ventral surface medially, flat, thick,
with weakly elevated lateral edges; remaining surface of oral
tube smooth (Fig. 15E; ol). Odontophore tube forms exten-
sion of the oral tube (Fig. 15E: oo), ventral platform runs
along it up to odontophore; walls thin muscular; length
equivalent to that of odontophore.

Odontophore oval, about 1/3 of proboscis length.
Odontophore organization and muscles somewhat similar to
those described for Zernira., distinctions are as follows:
Odontophore muscles (Figs. 15E-16D): m2 pair narrow and
thin, also inserting in ni4 posterior region; m2a absent; me
pair very wide, inserting in ventral surface of cartilages sur-
rounding at some distance the ni6 insertion (Figs. 16A-C);
m3l, a thin layer of longitudinal muscles covering the dorsal
surface of the odontophore tube from the antero-dorsal edge
of the odontophore to the intersection between the odon-
tophore tube and esophagus; m3t, a thin layer of transverse
muscle fibers covering posterior and ventral surfaces of
odontophore (Fig. 16B), posterior to m3a; m4 pair thinner,
originated from posterior edge of cartilages, with fibers
splayed to form fan, surrounding cartilages inner surface;
m5, weakly differentiable from m4, appearing as median
continuation of m4; m6, thin and relatively narrow, anterior
region at level of anterior end of cartilages, posterior end
about at middle level of cartilages, inserting into ventro-
medial edge of cartilages, except in posterior region where
they become gradually wider, inserting in outer cartilages

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

Figure 15. Melapiuin lineatiim anatomy. A, anterior region of visceral mass, ventral view, pericardial and renal ventral wall removed, efferent
renal vessel (rv) opened longitudinally, rectum (rt) sectioned transversally. B, head and foregut, ventral view, most structures partially
uncoiled. C, valve of Leiblein opened longitudinally, its inner structures (gland and valve) removed, only its oblique furrow (vf) remaining.
D, proboscis and buccal mass, left view, proboscis opened longitudinally. E, buccal mass, left view, esophagus and odontophore tube (oo)
sectioned longitudinally. Scale bars = 1 mm. Abbreviations listed in section with figure captions.

PSEUDOLIVIDAE: A POLYPHYEETIC TAXON

65

Figure 16. Melapium Uneatum anatomy. A, odontophore, dorsal view, odontophore tube connecting it to the oral tube opened longitu-
dinally. B, same, ventral view, superficial layer of membrane and muscles removed, circular muscle (me) sectioned and reflected. C, same,
ventral tensor muscle (ml la) sectioned and reflected. D, same, ventral view, radular sac totally removed and reflected downward and to
the left, both cartilages and most muscles reflected. E, midgut as in situ, ventral view, topology of adjacent structures also shown. Scale
bars = 1 mm. Abbreviations listed in section with figure captions.

surface (Figs. 16C-D); m8, pair of short muscles on dorsal
edge of cartilages (mostly probably part of m4), running
attached to dorsal-anterior edge of cartilages, from their
anterior region to their middle level (Fig. 16D); ml la pair
very wide and thin, originating on m4-m5 posterior region
(Fig. 16B). Odontophore cartilages (oc) thin and antero-

posteriorly long; anterior end broadly pointed; jtosterior end
rounded, uniform in width along their length (Figs. 16C-D).
Subradular cartilage (Fig. 16A; sc) relatively narrow, cover-
ing only part of odontophore portion exposed in buccal
cavity. Radular sac relatively short, slightly longer than
odontophore. Radula teeth (Figs. 4E-F): rachidian tooth

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

wide, spanning 70% of radular width, base wide, boomer-
ang-shaped, with lateral ends rounded, broader than central
area, somewhat arched, with 3 pointed, closely spaced, tri-
angular cusps ec]Lial in size located in central area, with
length equivalent to transverse width of base. Cusps aligned
and joined in a single, projected, flat base. Space between
rachidian and lateral tooth narrow. Lateral tooth hook-like,
base about ’A of rachidian width, gradually narrowing and
curving inwards distally, base disposed somewhat obliquely,
tip sharply pointed. Anterior esophagus originating anterior
to odontophore tube, separated from it by a tall septum;
inner surface with pair of lateral folds (continuation from
folds of oral tube), smooth (Figs. 15B, 15E: ea); three times
the length of the odontophore. Salivary glands relatively
large, as two separated masses, clustered around valve of
Leiblein and nerve ring (Figs. 14D, 15B). Salivary ducts very
narrow, originating in middle of each gland’s median sur-
face, running anteriorly close to anterior esophagus, pen-
etrating the anterior esophageal walls at some distance from
oral tube, at the level of the odontophore; then both ducts
run within the esophageal walls and inside the dorsal folds of
the oral tube; salivary aperture small, in middle region of
dorsal folds anterior end (Fig. 15E: sa). Valve of Leiblein
with about 1/4 of odontophore volume, inner organization
similar to that of Zemira, but differs in the oblique furrow
being wider and shorter, undergoing the entire torsional
rotation within the valve walls, ending just beyond the
middle portion of the valve and at the same level as it started
in region preceding valve (Figs. 15B-C). Middle esophagus
(Fig. 15B: em) broad, about twice the length of the anterior
esophagus, internally 3/4 filled by a whitish gland that lacks
an inner septum. This gland lies along a region of esophagus,
its anterior and posterior ends forming short blind-sacs that
are located at some distance from the anterior and posterior
ends of the middle esophagus.

Beyond this gland is a hollow furrow that is separated
from the glandular region by a pair of tall and narrow folds.
These folds connect with the bases of the blind sacs at both
ends of the gland. The remaining middle esophagus (ante-
rior and posterior to gland) a simple tube with thin walls
[Kantor (1991: 39) referred to this gland as accessory gland].
Gland of Leiblein long and irregularly coiled, somewhat flat
(Fig. 15B: gl); width roughly uniform along its length, except
for a broader anterior region close to its juncture with the
esophagus and very slender, hollow, distal region, with a
small, rounded tip. Duct of the gland of Leiblein runs all
along its inner surface (Fig. 15B: Id), with a short proximal
portion of the duct free of the gland, its inner surface simple,
with 7-8 narrow, longitudinal folds. Posterior esophagus as
long as middle esophagus, anterior half narrow, inner sur-
face smooth, expands abruptly after passing through the
diaphragm dividing the haemocoel from the visceral cavity
(Fig. 15B: ep), developing 10-12 tall, glandular longitudinal

folds, somewhat separated from each other. Stomach occu-
pies about half of the whorl volume, forming inflated curve
(Fig. 16E); two ducts to lead to the digestive gland, the duct
closer to the esophageal insertion is narrow, bifurcating at a
short distance from its origin, turned postero-ventrally; the
other duct is closer to the intestine, broad, longer antero-
posteriorly, turned antero-ventrally. The inner gastric sur-
face is mostly smooth, except some low folds continuing
from the posterior esophagus that gradually diminish
posterior to esophageal duct to digestive gland. Intestine
(Fig. 16E: in) slightly broader than the esophagus, initially
running parallel to it. There is no clear demarcation between
the intestine and the stomach. In a short distance, the in-
testine broadens to become as wide as the stomach and
curves abruptly toward the right, lying on the posterior renal
wall (Fig. 16E). Rectum and anus as described above (pal-
lial organs).

Genital system. Development (Figs. 9F, 9H, 17C).
There is evidence to suggest direct development, as most
specimens, including males, carry a pair of large egg capsules
attached to the anterior region of inner lip (Fig. 9F). Both
capsules are equivalent in size and each contains a single
specimen with 3 or more whorls (Fig. 9H). Each capsule is
long, somewhat cylindrical, with both ends rounded. The
capsule wall is flexible, heavy, thin but strong, pale beige in
color, and opaque, not transparent. Capsules are attached by
a short and wide peduncle, located longitudinally along the
side (Fig. 17C). Both capsules remain side by side, situated
transversally across the anterior half of the shell’s inner lip.

Young specimens removed from capsules show com-
plete development, no operculum and appear to have the
capacity for crawling (Figs. 9IT-1). Nothing but viscous yolk
was found inside capsules, except occasionally a granulose
soft tissue surrounding young specimens in early develop-
ment (Fig. 17C).

Male (Figs. 14A, 17B). Testes located along the colu-
mellar surface of the visceral whorls. Seminal vesicle situated
~Vi whorl posterior to pallial cavity, consists of a highly
convoluted narrow tube that is Vi the width of the visceral
mass width and runs along the middle, columellar region
(Fig. 14C: sv). A narrower region of the seminal vesicle ad-
jacent to the pericardium and afferent renal vessel opens into
the middle region of the posterior-ventral end of pallial cav-
ity. Pallial vas deferens gradually becomes open and glandu-
lar forming the prostate gland. Prostate gland narrow,
opened as a thick walled furrow, running along ventral sur-
face of rectum for about Vi pallial cavity length (Fig. 14C:
pt). Pallial vas deferens gradually becoming narrow, crosses
to right corner of the pallial floor, as superficial, relatively
narrow, protruded furrow, surrounding columellar muscle
by a distance equivalent to 1/4 of pallial cavity length;
abruptly turning left on pallial floor and entering the base of
the penis. Penis long, somewhat flattened; proximal half of

PSEUDOLIVIDAE: A POLYPHYEETIC TAXON

67

Figure 17. Melapium lineatum anatomy. A, pallial oviduct and some adjacent structures, ventral view. B, penis and adjacent region ot its
base, dorsal view, penis reflected upward. C, anterior region of shell, anterior view, partially showing the siphonal canal and a pair of egg
capsules, with rectangular hole cut. D, visceral mass, female, transverse section through middle region of the penultimate whorl. E, central
nervous system, ventral, right oblique view. F, same, dorsal, left obliciue view, visceral ganglion (vg) included. Scale bars = 1 mm.
Abbreviations listed in section with figure captions.

uniform width, with furrow running along its posterior
edge; middle region with an irregular surface, furrow cross-
ing to opposite penis edge; distal half about % as wide as
proximal half, with furrow along its anterior edge; penis tip
blunt, rounded, furrow leading to a small sub-terminal
papilla (Eig. 17B).

Female (Eig. 17A). Ovary occupies entire surface of vis-

ceral whorls, covering entirely digestive gland, is thinner
along the columellar surface (Eig. 17D). Visceral oviduct
very narrow, running along middle ventral surface of visceral
mass, running along the left edge of the kidney before en-
tering the left edge of the pallial twiduct, between its poste-
rior and middle thirds. Pallial oviduct with about same
length of pallial cavity, protruding into the anterior region of

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

the kidney and separated from mantle border by a distance
ec]uivalent to 'A of pallial cavity length. The albumen gland,
a small whitish appendix, comprises about 1/6 the size of the
pallial oviduct and is located in the left half of the posterior
pallial oviduct, ventral to the kidney, joining the capsule
gland through a wide anterior opening. The seminal recep-
tacle is equal in size to the albumen gland, and located
alongside it, consisting of small, rounded lobes, several very
narrow, iridescent ducts that run from posterior to anterior
along left its edge and connect to the posterior end of the
capsule gland. The capsule gland occupies most of the pal-
lial oviduct, is yellowish and somewhat flattened dorso-
ventrally, consisting of a pair of thick glandular lamellae.
The female pore is sub-terminal, situated to the left of the
anterior-most portion of the capsule gland at the end of a
tall, wide, terminal papilla. Terminal papilla is situated
slightly posterior and to the right of the anus, and is pre-
ceded by a small, hollow, thick-walled chamber continuous
with the capsule gland lumen. There are no signs of a cement
gland in sole of the foot.

Central nervous system (Eigs. 17E-E). The nerve ring is
highly concentrated, and it is difficult to distinguish the
ganglia and connectives. Its total volume occupies roughly
1/10 of the haemocoel. The pedal ganglia are equivalent in
size to the cerebral-pleural ganglia. The passage of the
esophagus is narrow. A pair of buccal ganglia are located
close to the cerebral ganglia. Supra and sub-esophageal gan-
glia are also located close to the nerve ring. The visceral
ganglion is located posteriorly at a distance equivalent to the
nerve ring length.

Measurements (in mm). NMNH S6362: 24.7 by 22.7;
NMNH 59733: 26.5 by 23.6; NMNH V1899: 20.9 by 22.3;
NMNH V9979: 26.2 by 24.0.

Distribution. South Africa.

Habitat. Eine sand.

Material examined. SOLITH AERICA. Eastern Cape
(dredged R.V. Meiring Naude); Transkei, off Mbotyi,
31°29'02”S 29°45'04"E, 48-50 m depth 3 2, NMNH V9978,
(sta. F5, viii/1981 ); off East London, 33°06.2'S 27°52.4'E, 70
m depth. Id, NMNH V9979 (sta. XX34, 16/vii/1984). West-
ern Cape; Agulhas Bank, E of Martha Point, 34°29.5'S
20°33.3'E, 28 m depth. Id, NMNH S6362 (NMDP CC13,
7/iv/1991), 34°29.5'S 20°32.9'E, 24-28 m depth. Id, NMNH
59733 (MMDP CC14, 7/iv/1991 ); South Cape, south of Cape
Infanta, 35°38'S 20°50'E, 90 m depth, Id, NMNH V1899
(MMDP sta. A16590D, 30/ix/1994).

Discussion. The genus Melapium was removed from
Pseudolividae by Kantor (1991) and Vermeij (1998), based
on anatomical and conchological peculiarities. One of the
more conspicuous differences is the lack of spiral groove at
the outer lip and the prominent entrance of the siphonal
canal. Kantor ( 1991 ) erected a new family, the Melapiidae, to

include this genus. Vermeij (1998), on the other hand, ar-
gued that Melapium can be placed in the Strepturidae. As
several authors still include this genus in the Pseudolividae
(OBIS 2004), this assignment is tentatively maintained here.

This study was built upon the anatomical description of
Kantor ( 1991), but expanded to include developmental data
and the different strategy of carrying the egg capsules.

Fimrily Nassariidae

Genus Nassodontn H. Adams, 1867

(Type species Nassa insignis H. Adams, 1867, by mono-

typy)

Nassodonta dorri (Watteblet, 1886)

(Figs. 3G-J, 4G-I, 18A-19D)

Canidia dorri Watteblet, 1886: 56-57 (pi. 4, fig.2).

Nassodonta dorri: Kantor and Kilburn 2001: 99-104
(figs. 1-21).

Description

Shell (Figs. 3G-H). Fusiform, up to 15 mm; color green-
ish beige, with some sparse, weak, pale brown chevrons on
the body whorl. Walls very thick. Protoconch eroded. Spire
of about 4 convex whorls; suture deep; last two spire whorls
with strong axial nodular threads, gradually disappearing
towards body whorl. Body whorl mostly smooth except for
growth lines; ventral region with about 5 wide, uniformly
spaced, spiral furrows along its anterior half, the inferior
furrow widest, encircling the siphonal canal. These furrows
continue onto the dorsal surface of body whorl. The poste-
rior furrow is wider, producing a small projection on the
outer lip. Aperture elliptical, peristome whitish. Inner lip
smooth, thick, lacking callus. Outer lip thick, blunt, with 2
or 3 low teeth along its central area. Canal wide, short,
simple. Other details in Kantor and Kilburn (2001: 101).

The following description is based on re-hydrated semi-
mummified specimens.

Head-foot (Figs. 18A-B). Head-foot consists of IVi
conical whorls. Head not obvious and inlaid, marked only by
presence of tentacles. Tentacles about Vi as long as the foot,
the proximal % clearly broader than distal 'A. Eyes are well
developed, located at outer sides tentacle prior to narrower
region. The broad optical nerve is easily seen by transpar-
ency, running along the center of each tentacle. Tentacle
bases adjacent. Each tentacle located close to each other,
running parallel like a socket. Rhynchostome very narrow,
located just ventral to region between tentacle bases. Foot
wide, ample, simple, occupying about ’A of shell volume
when retracted. Retraction is umbrella-like, producing a
ventral concavity. The anterior furrow of pedal glands is
surrounded by thick edges that are restricted to the anterior
border of the foot, weakly expanding laterally. Ventral ii
mantle insertion far posterior to pedal edges. Columellar

PSEUDOLIVIDAE: A POLYPHYLETIC TAXON

69

Figure 18. Nassodonta dorri anatomy. A, head-foot, male, frontal view. B, head and haemocoel, ventral view, foot and columellar muscle
removed. C, pallial cavity, male, ventral-inner view. D, penis and adjacent region of its base, ventral view, penis partially reflected, portions
of penis duct (pd) seen by transparency. E, buccal mass, left view. Scale bars = 1 mm. Abbreviations listed in section with figure captions.

muscle simple, of about % whorl. Male with large penis
inserted posterior to right tentacle, at short distance from
median line. Haemocoel elliptical, relatively wide, oblic]ue.

Operculum (Eigs. 31-1). Corneous, pale brown, ellipti-
cal, occupying entire aperture. Nucleus sub-terminal, ante-
rior. Outer sculpture concentric, with weak spiral growth
lines. Inner surface glossy; scar elliptical, deep, closer to in-
ner edge, occupying about half of opercular area. Other de-
tails in Kantor and Kilburn (2001:101, fig. 14).

Mantle organs (Fig. 18C). Mantle border simple, some-
what thick, whitish. Siphon thick, short, conical, with simple
edges, about Vi of pallial cavity length. Pallial cavity spans
about 1 whorl. Osphradium long, elliptical, with an area
ec][uivalent to V3 of gill area, left filaments about halt the size
of the right filaments; anterior end rounded, located at base
of siphon; posterior end pointed. Gill spans about Vi of
pallial roof area, adjacent to the osphradium. Its anterior end
is pointed, but broadens at first rapidly then gradually until

70

AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

the posterior Vi of the pallial cavity then narrowing gradu-
ally. Gill filaments are low, triangular, and have a central
apex. The roof of the pallial cavity separates the gill from the
rectum, which is narrow and runs along the right edge of the
pallial cavity. The anus is simple, at the distal end of a short,
detached portion of the rectum, at the level of the anterior
end of the gill.

Visceral mass. Not seen.

Circulatory and excretory systems. Not seen.

Digestive system (Eigs. 18B, 18E-19D). The proboscis
is narrow, thin walled, about % of the head-foot length
(Eig. 18B: pb) with a thick muscular, sphincter-like base
(Fig. 19A). Proboscis and buccal mass relatively short, with
about Vi remaining in the retracted proboscis. Odontophore
about Vi of proboscis length, about 'A protruding beyond
the proboscis in retracted position. The oral tube with is
about Vi the odontophore length and width, its inner surface
smooth and simple. The odontophore and esophagus detach
from each other just posterior to oral tube, but are bound to
each other by a series of narrow lateral muscles (mt). Odon-
tophore muscles (Figs. 18E-19D): ml, small muscles con-
necting lateral edge of odontophore tube with adjacent re-
gion of esophagus; m2, strong pair of buccal mass retractor
muscles, originating from ventral surface of haemocoel, run-
ning anteriorly, mostly attached to ventral surface of the
proboscis, inserting into the posterior end of the cartilages;
m2a, pair of strong accessory buccal mass retractor muscles
and accessory dorsal tensors of the radula (Figs. I9C-D),
originating in same region as m2, running medially along-
side the m2 pair, attached to the ventral surface of the pro-
boscis, becoming broader after penetration into odonto-
phore, inserting along lateral surfaces of radular sac; m3,
superficial and thin pair of muscles, originating in dorsal
region of m2a pair, in a region just anterior to their pen-
etration into the odontophore, running along superficial
membrane covering odontophore, splaying along anterior
region of this membrane; m4, pair of strong dorsal tensor
muscles of radula, originating in medial, dorsal, and poste-
rior surfaces of cartilages, running anteriorly, inserting into
the radular sac with m2a; m4d, narrow pair of accessory
dorsal muscles of radula, having same parameters as m4 but
running more dorsal, originating more dorsal and anterior
to ni4 origin, and inserting more medially, covering m4/m2a
insertion; m5, pair of ventro-medial dorsal tensor muscles of
the radula, originating on the outer-ventral surface of the
posterior end of the cartilages (opposed to m4 and ni4a
origins), running anteriorly, covering the ventral surface of
m4/m2a, inserting along the radular sac with m4/m2a; m6,
thin, horizontal muscle, connecting both cartilages along
their median-ventral edges, from a region just posterior to
cartilages join, spanning roughly half the length of the odon-

tophore, narrow anteriorly, gradually broadening posteri-
orly. mj, a pair of narrow odontophore protractor muscles,
originating along the odontophore anterior tube (that con-
nects odontophore to oral tube), gradually becoming thicker ;
posteriorly, inserting in odontophore cartilages along the
middle region of their dorsal edge; me, circular muscle op-
posing m6, inserting along outer-dorsal edge of both carti-
lages (opposite to m6), surrounding dorsally all inner
muscles (except ml la); ml la, pair of narrow ventral tensor |
muscles of the radula, originating on the median-dorsal re-
gion of the cartilages, just anterior to m4 origin, running ;
anteriorly, covering me, inserting into the ventral end of the
subradular membrane (Fig. 19C). Subradular cartilage (sc) is
a convex membrane covering the anterior region of the ;
odontophore, protrudes into the buccal cavity (Fig. 19G).
Odontophore cartilages (oc) are paired, long, narrow, flat,
slightly broader anteriorly (Figs. 19B-C); their posterior ends <
rounded, narrow; fused along ventral edge along the anterior
*/4 of their length. Radula about IV2 times odontophore
length. Radular teeth (Figs. 4G-I): Rachidian arched, flat,
spans Vi of radular ribbon width; with -10 sharp, pointed
cusps along the cutting edge, somewhat similar to each
other, slightly shorter towards tooth margins, separated
from each other by space equivalent to their size. Secondary,
irregularly disposed, small cusps are sometimes present, ]
mainly along the median region; lateral edge straight, 'A of
rachidian width; posterior edge concave, well separated from
neighboring tooth. Lateral teeth with a rectangular base
about % as wide as the rachidian tooth, separated from
rachidian tooth by a gap about 1/8 of the radular ribbon
width. Lateral teeth are situated oblicjuely as if continuations
of the rachidian tooth, each with -5 broad cusps along its
cutting edge. Cusps similar in size except for outermost
cusp, which is about 3 times the length and slightly broader
than remaining cusps. Outermost cusp curved inward. In-
nermost cusp with up to five very small secondary cusps
along its inner edge. Other details of radula as reported in
Kantor and Kilburn (2001: 101-102, figs. 15-20). Salivary
glands are whitish, amorphous, and restricted to the ante- |
rior region of haemocoel, surrounding the proboscis base 1
and nerve ring. Neither their ducts nor the accessory salivary ^
glands were found (Fig. 18B: sg). The anterior esophagus !,
(Fig. 18B: ea) is narrow, originating from the oral tube (ot) |
after a distance equivalent to half the length of the odonto- |j
phore. 3-4 pairs of narrow, well-separated slender pairs of
jugal muscles connect the lateral surface of anterior esopha-
gus to the lateral surface of odontophore tube (Fig. 18E:
ml). Inner surface of anterior esophagus smooth. Anterior
esophagus length equivalent to that of the proboscis. Valve
of Leiblein about V4 odontophore volume in size, located just
posterior to nerve ring, at the ventral base of the proboscis

PSEUDOLIVIDAE: A POLYPHYLETIC TAXON

71

D

Figure 19. Nassodonta dorri
anatomy. A, foregut, ventral view,
proboscis (pb) opened longi-
tudinally, head region also shown.
B, odontophore cartilages, dorsal
view, insertion of some muscles
also shown. C, odontophore, ven-
tral view, superficial layer of
membrane and muscles partially
removed, structures of posterior
region partially reflected. D, same,
dorsal view, superficial layer of
membrane and muscles removed,
ventral portion of radula par-
tially reflected forwards. Scale
bars = 1 mm. Abbreviations listed
in section with figure captions.

(Fig. 18B). the gland of Leiblein was not found. The poste-
rior esophagus is narrow, running along the left side of the
haemocoel. Midgut not examined. Rectum and anus are
described above (pallial cavity).

Genital system. Male (Figs. 18A, I8D). Visceral struc-
tures not examined. Prostate gland narrow, closed (tubular),
running ventral to and to the right of the rectum for 3/4 of
its length (Fig. 18C: pt) before narrowing and abruptly

72

AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

crossing to the pallial floor. The pallial vas deferens is broad-
est just after descending to the pallial floor, narrows and
forms a zigzag on the surface of the pallial floor leading to
the penis base (Fig. 18D: vd). The penis is about half as long
as the head-foot (Fig. 18A), its base twisted and oval in
section. The penis widens somewhat abruptly at mid-length
forming a blunt bulge along its right edge. The median and
distal thirds of the penis are flat (Fig. 18D), gradually taper-
ing to a pointed tip. The penis is duct narrow, running
medially to the penis tip, where it opens.

Female. No well-preserved female examined. Visceral
structures not seen. The pallial oviduct is whitish, closed
(tubular), about twice the width or the rectum, and appar-
ently lacks an anterior bursa copulatrix. The sole of the foot
of females with a thick walled, whitish, glandular cement
gland situated medially in the anterior half of the foot, as
deep as half the foot width.

Central nervous system. Only pedal ganglia well-
preserved, located in ventral region of proboscis base close to
each other and to median line (Fig. 18B: pu). Each ganglion
occupies a volume about 1/8 of the odotophore. Statocysts
with large statolith, each located in the ventral haemocoel
surface; partly immersed in the salivary glands and partly in
the local pedal musculature.

Measurements of shells (in mm). MZSP 53533: dl:
13.6 by 8.6; 62: 12.0 by 7.7; S3: 12.0 by 8.0; ?4: 14.2 by 8.3.

Distribution. Vietnam.

Habitat. Brackish, practically freshwater muddy
environment.

Material examined. VIETNAM; Kolan, MZSP 53533, 2
shells, 3 d , 2 $ re-hydrated soft parts, Gemert private collec-
tion, 3 shells (beach of river).

Discussion. The shell of Nassodonta is easily confused
with that of pseudolivids, being virtually identical to that of
the genus Macron H. and A. Adams, 1853. It differs concho-
logically mainly in having a shorter spire. As discussed by
Vermeij (1998: 70-71), Macron was referred to the Nassari-
idae. Based on its anatomy, Nassodonta undoubtedly belongs
to the Nassariidae-Bucdnidae, mainly on the basis of odon-
tophore features.

CHARACTERS

SheU

1. Shell spiral furrow at last whorl: 0 = absent; 1 =
present {Zeinira, Fulnientiiin, Benthobia, Nas-
sodonta) (Cl = 50; RI = 50).

The spiral furrow dividing the shell body whorl
traces the position of the labral tooth on the outer
lip, although this tooth is not well-developed in all
species. It is normally present as a shallow, oblique
furrow between the middle and anterior thirds of
the body whorl. This furrow is one of the more

conspicuous pseudolivid shell features, and has
been called as “pseudolivid groove” (Vermeij 1998).

However, some taxa considered to be pseudo-
livids, such as Melapiinn, lack this furrow, while a
furrow and labral tooth is present in other genera
that unquestionably belong to other families. Some
examples include: Acanthina Waldheim, 1807 (Mu-
ricidae); Lencozonia Gray, 1847 (Fasdolariidae);
Ancilla Lamarck, 1799 (Olividae); and Bivetiella
Wenz, 1938 (Cancellariidae). These indicate the
high degree of convergence in this character.

2. Tooth on outer lip: 0 = absent; 1 = present (Zemira,
Fulinentinn) (Cl = 50; RI = 0).

Although the furrow normally is associated with
a tooth at the outer lip, also called labral tooth, this
character only refers to a well-developed one, and
not to a small projection. Apparently, the tooth at
the outer lip is associated with predation on bi-
valves, serving to separate the valves. In the present
study, it is equally parsimonious to consider state 1
as supporting node 4 reverting in node 6 or as a
convergence between Zemira and Fnlmentum, the
first hypothesis is shown in the Fig. 1.

3. Determinate growth: 0 = present; 1 = practically
absent (Melapinm, Fulmentnm, Nassodonta, Bentho-
bia) (Cl = 33; RI = 0).

Determinate growth is the development of a dif-
ferentiated peristome when the animal becomes
mature. This feature is well explored by Vermeij
and Signor (1992) and is here applied. As determi-
nate growth is present in most of higher Caenogas-
tropods (Simone 2000), it is considered plesiomor-
phic for neogastropods, and its absence, i.e., the
non-determinate growth is here considered a de-
rived reversal. As some species considered in state 1
have a weak thickness of the outer lip, the word
“practically” is introduced.

Head-foot

4. Cephalic tentacles: 0 = separated {Melapinm, Ben-
thobia); 1 = joined to each other {Nassodonta, Ful-
mentnm, Zemira, Siratus) (Cl = 50; RI = 66).

The cephalic tentacles placed together, close to
the median line. This kind of modification is dif-
ferent from the normal feature in higher caenogas-
tropods, which possess the state 0.

5. Foot posterior furrow: 0 = absent; 1 = present {Ben-
thobia) (Cl = 100; RI = 100).

The conspicuous posterior furrow on the sole of
the foot is restrict to the genus Benthobia and may
be a character of the genus. It is discussed in Simone
(2003).

FSEUDOLIVIDAE: A POLYPHYLETIC TAXON

73

6. Columellar muscle: 0 = simple; 1 = double (with a
siphonal branch) [Siratus, Melaphiui) (Cl = 100;
R1 = 100).

The posterior region of the columellar muscle is
normally a simple and broad flap. However, in the
species listed under sttrte 1, this region is bifid, hav-
ing a wider left branch and another slender and
taller right branch (Figs. 14A-B: cm). This feature
has been commonly found in muricids according to
my experience, and is related to an anterior furrow
internally on each whorl, maintained by the sipho-
nal canal, into which the right branch fits.

7. Operculum nucleus: 0 = terminal {Fiiliuentuni,
Benthobia)-., 1 = sub-terminal {Nassodouta, Zemira);
2 = almost central (Siratus); 3 = absent (Melapiiim)
(Cl = 75; RI = 0; not additive).

An operculum with a terminal nucleus is a
modified condition in gastropods, but among the
neogastropods, this is the plesiomorphic condition.
The remaining states, including the loss of the oper-
culum, are considered further modifications. While
the loss of the operculum is certainly a different
phenomenon from the position of the nucleus,
these are joined because of the non-additive condi-
tion. This is mathematically equivalent to consider-
ing the loss as a separate character. With respect to
the state allocation in the cladogram, it is equally
parsimonious to consider state 1 as supporting node
3, then reversing in Fulmentiim, or a convergence be-
tween Nassodonta and Zemira. The first hypiothesis is
shown in the Fig. 1.

Pallial organs

8. Siphon: 0 = long; 1 = short, almost inconspicuous
(Benthobia, Zemira) (Cl = 50; RI = 50).

The siphon is a modification of the mantle bor-
der and is distinct from the development of a si-
phon in the shell. There are taxa that possess a
siphon in the shell, yet lack a developed siphon in
the mantle, as, e.g., Stromboidea (Simone 2005) and
Cerithioidea (Simone 2001), while other taxa pos-
sess a well-developed siphon at mantle border, but
lack any special modification in the shell, as, e.g.,
Calyptraeoidea (Simone 2002). The long and ex-
ploratory pallial siphon is the rule in Hypsogas-
tropoda, and is considered to be plesiomorphic in
neogastropods. State 1 is considered a reduction.

9. Osphradium length relative to gill length: 0 =
shorter than half (Siratus); 1 = longer than half
(Melapium, Fidmentum, Nassodonta); 2 - almost
same length (Zemira, Benthobia) (Cl = 50; RI = 33;
additive).

Although the states are optimized as additive,
based on ontogeny, identical results and indices
are produced when the character is considered not
additive.

10. Osphradium: 0 = symmetrical; 1 = asymmetrical
(Benthobia, Nassodonta) (Cl = 50; R 1= 50).

Symmetry refers to the left and right filaments
being symmetrical about the axis of the osphradial
gangilion, with the filaments of both sides similar
sized. It is equally parsimonious to consider state 1
as convergent between node 2 and Nassodonta, or
supporting node 1 and reverting in node 4; the first
hypothesis is shown in the Fig. 1.

11. Osphradium with monopectinate anterior por-
tion: 0 = absent; 1 = present (Benthobia) (Cl = 100;
RI = 100).

Osphradium characters (9-11) are normally con-
nected to reduction in body size. The smaller the
animal, the larger, proportionally, is the osphra-
dium. The same can be concluded with regard to
the asymmetry of the osphradium filaments.
Smaller animals tend to have the left filaments
smaller than the right ones. The loss of the left
filaments (character 11) can be considered as the
extreme of this tendency to miniaturization. In the
case of Benthobia, the monopectinate condition is
only present in the anterior portion of the osphra-
dium (Simone 2003: figs. 7B, 9B, llA, 12A).

Digestive system

12. Ventral chitinous platform within the oral tube:
0 = absent; 1 = present (Melapium, Zemira, Siratus,
Fidmentum) (Cl = 100; RI = 100).

The chitinous platform is a relatively thick lon-
gitudinal band located along the ventral surface of
the oral tube. It starts close to the point where the
odontophore enters the oral tube, and ends close to
the mouth. This platform is particularly well devel-
oped in muricids that 1 have examined, but it is also
present in the above listed other species (Figs. 7F,
15E: ol). The function of this structure is unknown,
but normally the salivary glands open in the
middle level of its lateral edges, which suggests a
relationship between the glands and the chitinous
platform. This reinforcement of the inner surface of
the oral tube may possibly be linked to contact with
the radular teeth, seiwing to avoid self-injury. There
does not appear to be a direct relationship with the
jaws, which are located on the dorsal surface of the
oral tube, and are normally absent in neogastropods.

1 3. Length of odontophore horizontal muscle ( m6): 0 =
about hall the length of the cartilages; 1 = almost

74

AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

the same length as the cartilages [Nassodonta,
Fulmentwn, Melapium, Zemira, Siratus) (Cl = 100;
R1 = 100).

The horizontal muscle (m6) connects both odon-
tophore cartilages to each other along their ventral
edge. In neogastropods, however, this muscle is
normally thin and tends to become longer, almost
as long as the cartilages. This feature is explored in
this character (Eigs. 6B, I2A, 16C-D, 19B).

14. law muscle (mj): 0 = thin, as a flap; 1 = as a separate
band {Stratus, Zemira, Melapium, Nassodonta)
(Cl = 50; RI = 66).

The jaw muscles (mj) are modified in neogas-
tropods because of the greater development of the
odontophore tube, which makes the connection be-
tween it and the oral tube. This modification may
be responsible for the further alteration of state 1.
Although the neogastropods normally lack jaws, as
is the case in the species examined, this name is here
maintained in order to indicate the homology of
this structure with those of the remaining caeno-
gastropods.

15. Odontophore ventral tensor muscle of radula
(ml la-m4v): 0 = absent; 1 = present (all taxa in this
study) (Cl = 100; RI = 100).

16. Dorsal tensor muscles of radula m4 and ni5: 0 =
separated from each other; 1 = continuous with
each other {Siratus, Fulmentum, Melapium, Zemira,
Nassodonta) (Cl = 100; RI = 100).

17. Connection of m4 with inner surface of cartilages:
0 = absent; 1 = present {Siratus, Melapium, Fulmen-
tum) (Cl = 100; RI = 100).

18. Odontophore cartilage outline: 0 = elliptical; 1 =
elongated {Siratus, Melapium, Nassodonta) (Cl =
50; RI = 50).

19. The odontophore of the muricoideans (broad
sense) is different from those of the remaining cae-
nogastropods in two main features. The first is the
tendency to elongation, which results in odonto-
phores that may be as long as the proboscis. They
extend into the haemocoel when the proboscis is
retracted. Another difference is the development of
ventral tensor muscles of the radula. This muscle
pair is present in archaeogastropods, but is practi-
cally lost in caenogastropods. The muricoideans re-
vert to this condition, re-acquiring the ventral ten-
sor muscles from a modification ot the dorsal ones.
The modifications resulting from these tendencies
are explored in above characters (15-18). Odonto-
phore tube connecting odontophore to the oral
tube: 0 = absent or very short; 1 = long (all taxa in
this study) (Cl = 100; RI = 100).

The odontophore tube is a separate structure
from the well-known “oral tube” (Simone 2003, fig.
7G: oo) (Eigs. 15D, 18E: oo). This muscular tube
connects the odontophore to the oral tube. Elonga-
tion of this tube is another common character of
the muricoideans, in which the buccal mass struc-
tures become V-shaped, with the mouth at the ver-
tex of this “V”.

20. Esophageal origin: 0 = posterior to odontophore;
1 = anterior-dorsal to odontophore (buccal mass
V-shaped) (all taxa in this study) (Cl = 100;
RI = 100).

In most caenogastropods, the buccal mass and
esophagus are linear, i.e., the origin of the esopha-
gus is in the posterior region of the odontophore.
As noted above, the buccal mass and esophagus
may be V-shaped in the muricoideans, with the
anterior esophagus and the elongate odontophore
running parallel to each other.

21. Accessory salivary gland: 0 = absent {Zemira); 1 =
paired {Siratus, MelapiunF); 2 = unpaired {Fulmen-
tum, Benthobia) (? = Nassodonta) (Cl = 66; RI = 50;
not additive).

As the accessory salivary gland has been consid-
ered as a synapomorphy of the Neogastropoda
(Ponder 1974, Haszprunar 1988), its absence is con-
sidered plesiomorphic. This condition in Zemira is
most likely a reversion. Kantor (1991) had consid-
ered, however, two apomorphic states of the acces-
sory salivary gland, absent and the single (unpaired)
condition.

The presence (state 2) in Fulmentum can be con-
sidered an autapomorphy for this taxon (conver-
gent with node 2) or supporting node 5. The second
hypothesis is shown in the Fig. 1, but in this hy-
pothesis the pair of accessory salivary gland origi-
nated from a single gland.

22. Valve of Leiblein: 0 = absent; 1 = present (all taxa in
this study) (Cl = 100; RI = 100).

23. Valve of Leiblein oblique furrow: 0 = absent; 1 =
present {Zemira, Melapium, Fubnentum, Nas-
sodonta, Siratus) (Cl = 100; RI = 100).

The valve of Leiblein is considered to be another
synapomorphy of the Neogastropoda (Haszprunar
1988), and is certainly present in most muricoide-
ans. As nothing similar can currently be attributed
to the Conoidea, the presence of this valve is here
considered to be an apomorphic state (character
22), supporting a branch uniting muricoideans and
cancellarioideans. Its internal organization, on the
other hand, has not been studied in detail. Com-
parisons at this level are very difficult, as informa-

PSEUDOLIVIDAE: A POLYPHYLETIC TAXON

75

tion is inadequate. Certainly, the valve of Leiblein is
a complex structure, the function of which is un-
certain. In some species, the valve of Leiblein has a
transverse furrow that can be considered as homolo-
gous to the bypass shown by Ponder (1974; fig. 3), a
way for the food pass directly to the middle esophagus
without passing through the valve. This condition is
considered apomorphic (Pigs. 7B, 12B, 15C), being
one of the synapomorphies of the node 3.

24. Gland of Leiblein: 0 = absent; 1 = present (all); 2 ==
elongated {Zemira, Melapium) (Cl = 66; RI = 0;
additive).

The gland of Leiblein is another synapomorphy
of the Neogastropoda (Ponder 1974; Haszprunar
1988). It is further modified in several taxa, includ-
ing its disappearance and its modification into a
venom gland in the conoideans. The modification
explored here is the elongated form. In this pattern,
the gland is stored inside the haemocoel intensely
coiled. It is shown in Pigs. 7C and 15B artificially
uncoiled. The additive condition is based on ontog-
eny, as very young specimens possess a shorter
gland, however, nothing changes in the result or
indices if the character is considered not additive.

25. Gland of Leiblein duct: 0 = short; 1 = long (about
half the length of the middle esophagus) {Siratus,
Zemira, Melapium, Fulmentum) (G1 = 100; RI = 100).

26. Gland of Leiblein duct: 0 = with transversal septa
(Siratus); 1 = glandular (Zemira, Fulmentum);
2 - simple (Melapium, Benthobia, Nassodonta)
(GI = 40; RI = 25; additive).

The gland of Leiblein characters (24-26) are
based on the hypothesis that this gland is a modi-
fication of the middle esophageal gland, present in
the higher mesogastropods (Naticoidea, Cyprae-
oidea, Tonnoidea). In these taxa, the esophageal
gland consists of a series of transverse septa. Some-
thing similar is also found in some muricoideans,
but reduced and located in the duct of gland of
Leiblein, further supporting the link between these
two structures. The presence of such septa in the
gland duct is considered plesiomorphic. The glan-
dular condition of the duct is considered as an in-
termediate step for a simple-duct condition. This is
the reason for considering the states in an additive
optimization; however, if considered not additive,
the resulted cladogram is the same, but the indices
change to Cl = 50 and RI = 0.

27. Stomach form: 0 = a simple curve; 1 = with a di-
lated chamber posterior to esophageal insertion
(Fulmentum, Melapium, Zemira, Siratus) (Cl = 100;
RI = 100).

28. Number of stomach ducts to the digestive glands:

0 = 2 (Melapium); 1 = 1 (Benthobia, Fulmetitum,
Zemira, Siratus) (?= Nassodonta) (Cl = 50; RI = 0).

The stomach of normally carnivorous neogastro-
pods, is, in most taxa, a simple curve. However,
some taxa have developed a more complex stomach
that is considered to be apomorphic herein. The
number of the ducts leading to the digestive gland
tends to be simplified, form a pair, as normal in
lower caenogastropods, to a single duct.

29. Anal papilla: 0 = absent; 1 = present (Siratus, Me-
lapium, Fulmentum) (Cl = 100; RI = 100).

The anal papilla is not a conspicuous structure,
but is present along the dorsal margin of the anus in
above mentioned species (figs. lOA, 14C: al), as
well as in the remaining muricids examined. It most
likely represents a synapomorphy.

Genital system

30. Penis duct: 0 = open (a furrow) (Siratus, Melapium,
Fulmentum); 1 = closed (a tube) (Zemira, Nas-
sodonta, Benthobia) (Cl = 50; RI = 66).

31. Penis retractile terminal broad papilla: 0 = absent;

1 = present (Benthobia, Fulmentum) (Cl = 50;
RI = 50).

The male genital system is not normally well
preserved, as most of the visceral structures are lost
or not extracted without damage. This is the case of
the present sample. However, comparisons were
made among those species for which material was
available. The opened condition (character 30) is
considered plesiomorphic, based on the condition
found in most basal caenogastropods, but the
closed (tubular) condition commonly occurs
throughout the caenogastropods as convergences.

32. Bursa copulatrix: 0 = a blind sac; 1 = terminal, as
continuation of oviduct (Benthobia, Fulmentum,
Zemira); 2 - absent (Siratus) (not additive)
(Cl = 66; RI = 0).

The polarization of this character is also based
on the condition normally found in other caeno-
gastropods, mainly higher mesogastropods. Al-
though several interesting differences in the female
genital system were found, all appeared to be auta-
pomorphic, except for the condition of the bursa
copulatrix.

Central nervous system

33. Nerve ring ganglia: 0 = ganglia separated; 1 = gan-
glia almost fused (all) (Cl = 100; RI = 100).

34. Buccal ganglia: 0 = close to buccal mass; 1 = close to
neiwe ring (all) (Cl = 100; RI = 100).

76

AMERICAN MALACOLOGICAL BULLETIN 23 • 1-2 • 2007

Both central nervous system characters are po-
larized based on the remaining caenogastropods, in
such the ganglia are clearly separated from each
other (character 33), and the paired buccal ganglia
are located far from the nerve ring and closer to the
buccal mass (34). Both conditions are further modi-
fied in the above mentioned taxa.

TAXONOMY

The cladogram based on the set of characters shown in
the Table 1 is depicted in figures 1 and 2. Character polarity
is based mainly on Tonnoideans, i.e., Tonna galea (Linne,
1758) and T. maculosa (Dillwyn, 1817) (Simone 1995), as
well as other species still under study. Based on this scenario,
the Conoidea share seven synapomorphies with the ingroup,
the more important being determinate shell growth (char-
acter 3), the presence of the odontophore ventral tensor
muscle of radula (15), the gland of Leiblein (character 24),
closure of penis duct (character 30), and the adaptations of
the nerve ring (characters 33, 34).

The ingroup also is supported by eight synapomorphies
(node 1 ), the more important being the spiral furrow in the
last shell whorl (character 1), the elongation of the osphra-
dium (9), the odontophore tube (19), the valve of Leiblein
(22), the further modification of the gland of Leiblein duct
(26), and the reduction of the stomach ducts (28).

Node 2 represents the genus Beuthobia, based on data
from Simone (2003). Although five species had been studied,
only two are included here because of the completeness of
data. This node is supported by 9 synapomorphies, two are
non-homoplastic; the posterior furrow of the foot (character
5) and the monopectinate condition of the anterior region of
the osphradium (11). The features are convergent with other
branches of the cladogram. Among the more notable are: the
shortness of the siphon (8), the osphradium equal in length
to the gill (9), a single accessory salivaiy gland (21) and the
terminal papilla of the penis.

Node 3, the remaining ingroup, is supported by six
synapomorphies, the more conspicuous are: the close situ-
ation of the cephalic tentacles (character 4), the sub-terminal
condition of the opercular nucleus (7), the length of the
horizontal muscle of odontophore (13), and the oblique fur-
row of the valve of Leiblein (23).

The next dichotomy separates the nassariid Nassodouta
dorri from the remaining ingroup taxa (node 4). The tax-
onomy of the genus Nassodouta has been analyzed by Kantor
and Kilburn (2001), who provided a history and additional
comments.

However, there is a remarkable similarity in shell char-
acters with the pseudolivid taxa. As pointed by Kantor and

Kilburn (2001: fig. 13), Nassodouta also possesses a deep
basal spiral furrow in the anterior region of body whorl,
mostly associated with a labral tooth. On the other hand, the
anatomical characters such as the radula and the degree of
fusion between both odontophore cartilages clearly show the
nassariid nature of the Nassodouta.

Node 4 is supported by five synapomorphies, the more
interesting being: the tooth at outer shell lip (character 2)
that reverts in the node 6, the ventral chitinous platform in
the oral tube (12), further elongation of the duct of the gland
of Leiblein (25) and the dilatation of the stomach (27).

Node 5 unites the remaining ingroup species except
Zemira australis, and is supported by four synapomorphies,
most noteworthy being the single accessory salivary gland
(character 21) and the anal papilla (29).

Node 6 is supported by five synapomorphies, and unites
Melapium, that mostly is considered a pseudolivid, with the
muricid Stratus seuegaleusis. Of the synapomorphies for this
node, the more important are the double condition of the
columellar muscle (character 6) and the paired state of the
accessory salivary glands.

Based on this scenario, the formal family Pseudolividae,
in the present sense, is not monophyletic. Pour of the in-
cluded genera {Beuthobia, Zemira, Fubueutum, and Mela-
pium) are mixed with a nassariid (Nassodouta) and a muri-
cid (Siratus). Although the studied set of species represents
only a subset of the genera included in the Pseudolividae, it
appears to be is sufficient to demonstrate the polyphyletic
nature of the taxon. Since the type species of Pseudoliva
Swainson, 1840, the type genus of the family Pseudolividae,
P. crassa (Gmelin, 1791), was not studied, no definite con-
clusions about the taxonomy of Pseudolividae can yet be
reached. However, at least the present concept of the family
level taxon has been shown as lacking phylogenetic support.

Among the species studied, Fulmentum aucilla is closest
to Pseudoliva. Some authors consider this species to belong
to Pseudoliva (e.g., Kantor 1991, Hayes 1994). If P. crassa is
dose to F. aucilla, the cladogram indicates that it, and with
Melapium, could be considered as belonging to Muricidae
or, at least, a sister taxon of that family. According to this
tree topology, the genera Zemira and Beuthobia could be
placed in other families. Kantor (1991: 34) provided some
anatomical information on Pseudoliva zebriua A. Adams,
1853, which shares similarities with F. aucilla, mainly with
respect to foregut characters. This can further indicate a
close relationship between Fulmentum and Pseudoliva.

The phylogenetic analysis by Kantor (1991: fig. 19)
shows four synapomorphies supporting the monophyly of
the Pseudolividae (except Melapium). Three of the synapo-
morphies (three teeth per radular row, a short free portion
of the duct of salivary gland, and the anal gland) occur
commonly in the Muricoidea. A fourth synapomorphy (ac-

PSEUDOLIVIDAE; A POLYPHYLETIC TAXON

77

cessory esophageal gland) is a distinctive character, but the
variability of form and position of this gland, and its pres-
ence in several other muricoideans, suggest the possibility of
convergence. While these four anatomical characters are the
basis for recognizing the family Pseudolividae, none of them
emerged as important synapomorphies in the present study.
Similar arguments can be made with regard to the seven
synapomorphies proposed for Melapiwn in that paper.

The outcome of the present analysis shows Pseudo-
lividae, as currently understood, to be polyphyletic, contra-
dicting previously published results, and indicating that the
present concept of this taxon must be reevaluated. Most
probably some of the genera now assigned to Pseudolividae
will be found to belong to other families, while the name
Pseudolividae is apparently only applicable only to the gen-
era Pseudoliva and Fiilmentwn.

The definitions and limits of the families of the muri-
coideans only can be refined after a much wider analysis,
including samples of much more representatives.

CONCLUSIONS

( 1 ) The family Pseudolividae, in the present concept, is
polyphyletic and must be not used as a formal
taxon.

(2) Detailed morphology is valuable for comparative
studies, as all examined species differ greatly in
most structures.

(3) The genera Zemira, Fuhnentum, and Melapiinn
share synapomorphies with the genus Siratns (Mu-
ricidae). These taxa also share further synapomor-
phies with the Nassodonta (Nassariidae), being
separated by them from Benthobia.

(4) No special taxonomical re-arrangement is proposed
because of weakness of definition of the Muricoidea
families.

ACKNOWLEDGMENTS

I thank R. N. Kilburn, Natal Museum, South Africa
(NMSA), for the loan of specimens of Fuhnentum and Me-
lapium; Winston Ponder and Ian Loch, Australian Museum,
for the loan of Zemira, and Leo van Cermet, Netherlands,
for the gift and loan of Nassodonta. This study had a gov-
ernmental support of the Fapesp (Fundac^ao de Amparo a
Pesquisa do Estado de Sao Paulo), process number 04/
10793-9.

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Golikov, A. N. and Y. I. Starobogatov. 1975. Systematics of proso-
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Haszprunar, G. 1988. On the origin and evolution of major gas-
tropod groups, with special reference to the Streptoneura.
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Hayes, B. 1994. Two rare South African shells belonging to the
Olividae family. World Shells 11: 77-78.

Kantor, Y. I. 1991. On the morphology and relationships of some
oliviform gastropods. Ruthenica 1: 17-52.

Kantor Y. 1. and R. N. Kilburn. 2001. Rediscovery of Canidia dorri
Watteblet, 1886, with discussion of its systematic position
(Gastropoda: Neogastropoda: Nassariidae: Nassodonta). The
Nautilus 115: 99-104.

Liltved, W. R. 1985. Melapiuni lineatum (Lamarck, 1822). Strand-
loper 214: 9.

Lorenz, Ir., F. 1989. Conoscete Sylvanocochlis ancilla (Hanley,
1859)? Conchiglia 21: 16-18.

Nielsen, S. N. and D. Frassinetti 2003. New and little known species
of Pseudolividae (Gastropoda) from the Tertiary of Chile. The
Nautilus 117: 91-96.

OBIS Indo-Pacific Molluscan Database. 2004. Available at http://
data.acnatsci.org/obis 17 August 2005.

Pacaud, ). M. and K. I. Schnetler. 1999. Revision of the gastropod
family Pseudolividae from the Paleocene of West Greenland
and Denmark. Bulletin of the Geologieal Society of Denmark 46:
53-67.

Pinna, M. C. C. 1996. A phylogenetic analysis of the Asian catfish
families Sisoridae, Akysidae, and Amblycipitidae, with a hy-
pothesis on the relationships of the Neotropical Aspredinidae
(Teleostei, Ostariophysi). Field Zoology 84: 1-83.

Ponder, W. F. 1974. lire origin and evolution of the Neogas-
tropoda. Malacologia 12: 295-338.

Ponder, W. F. and T. A. Darragh. 1975. The genus Zemira H. and
A. Adams (Mollusca: Neogastropoda). Journal of the Malaco-
logical Society of Australia 3: 89-105.

Ramos, T. C. 1997. Tree Gardner, version 2.2. Distributed by the
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Simone, L. R. L. 1995. Anatomical study on Tonna galea (Linne,
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Simone, L. R. L. 2000. Filogenia das familias de Caenogastropoda
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Simone, L. R. L. 2001. Phylogenetic analyses of Cerithioidea (Mol-
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389.

Accepted: 28 March 2007

FIGURE CAPTIONS

In the figures, the following abbreviations are used:
aa, anterior aorta; ac, auricle connection with kidney cham-
ber; ad, accessory salivary gland duct; ae, accessory salivary
gland; af, afferent gill vessel; ag, albumen gland; al, anal
papilla; an, anus; ao, posterior aorta; at, vaginal atrium; au,
auricle; ba, bursa copulatrix aperture; be, bursa copulatrix;
bg, buccal ganglion; bm, buccal mass; br, subradular mem-
brane; ce, cerebral-pleural ganglia; eg, capsule gland; cm,
columellar muscle; cp, capsule; cv, ctenidial vein; dc, dorsal
chamber of buccal mass; dd, duct to digestive gland; df.

dorsal fold of buccal mass; dg, digestive gland; di, diaphragm
membrane separating haemocoel from visceral cavity; ea,
anterior esophagus; ef, esophageal folds; em, middle esopha-
gus; ep, posterior esophagus; es, esophagus; ey, eye; fp, fe-
male pore; fs, foot sole; ft, foot; gi, gill or gill filament; gl,
gland of Leiblein; gm, gill muscle; gp, pedal ganglion; he,
head; hg, hypobranchial gland; in, intestine; ir, insertion of
m4 in tissue on radula (to); is, insertion of m5 in subradular
membrane; kc, membrane between kidney and pericardium;
kd, dorsal chamber of kidney; ki, kidney; kl, kidney dorsal
lobe; km, membrane between kidney and pallial cavity; kv,
ventral lobe of kidney; Id, duct of gland of Leiblein; Ig,
secondary gland of duct of gland of Leiblein; ml to ml 4,
extrinsic and intrinsic odontophore muscles; mb, mantle
border; me, circular muscles of odontophore; mf, muscle
fibers; mj, jaws, buccal, and oral tube muscles; mo, mouth;
ne, nephropore; ng, nephridial gland; nr, nerve ring; nv,
nerve; oa, opercular pad; oc, odontophore cartilage; od,
odontophore; of, odontophore cartilage fusion; oi, opercular
insertion; ol, oral tube ventral chitinous platform; oo, odon-
tophore tube connecting to oral tube; op, operculum; os,
osphradium; ot, oral tube; oy, ovary; pa, penis aperture; pb,
proboscis; pc, pericardium; pd, penis duct; pe, penis; pf,
penis furrow; pg, pedal glands anterior furrow; pp, penis
papilla; pt, prostate; pu, pedal ganglion; py, pallial cavity; ra,
radula; rd, seminal receptacle duct; rh, rhynchostome; rm,
retractor muscle of proboscis; rn, radular nucleus; rs, radu-
lar sac; rt, rectum; rv, renal efferent vessel; sa, salivary gland
aperture at oral tube; sc, subradular cartilage; sd, salivaiy
duct; se, septum between esophagus and odontophore in
buccal mass; sg, salivary gland; sh, shell siphon canal; si,
siphon or siphon insertion; sp, supra-esophageal ganglion;
sr, seminal receptacle; st, stomach; su, subesophageal gan-
glion; sv, seminal vesicle; sy, statocyst; te cephalic tentacle;
tg, integument; to, tissue on middle region of radula pre-
ceding buccal cavity; ts, testis; va, vaginal duct; vd, vas def-
erens; ve, ventricle; vf, oblique furrow of valve of Leiblein;
vg, visceral ganglion; vn, visceral nerve; vl, valve of Leiblein;
VO, visceral oviduct.

Arner. Maine. Bull 23: 79-80

Gastropod mating systems: An introduction to the symposium’^

Janet L. Leonard

Joseph M. Long Marine Laboratory, University of California-Santa Cruz, Santa Cruz, California 95060, U.S.A., jlleonar@ucsc.edu
Key words: reproduction, sexual selection, sexual behavior

Sex is what organisms are all about, and gastropods are
no exception. The sexual behavior and reproductive biology
of gastropods has fascinated naturalists from earliest times.
The aerial mating behavior of Umax niaximus Linnaeus,
1758, the chains of copulating Aplysia Linnaeus, 1767, the
love dart of Helix (Linnaeus, 1758), the egg cases of whelks
and naticids, and the delicate gelatinous egg masses of nudi-
branchs have been objects of wonder and speculation for
centuries and the more we learn about such phenomena, the
more marvelous they seem. Our childish pleasure at the
delicacy and symmetry of a moon snail’s egg collar is only
enhanced by the understanding of its importance in allowing
eggs to develop on muddy substrata; our astonishment at the
length of the entwined penes that suspend a pair of mating
L. maximus is only intensified by consideration ot the con-
flicting pressures of natural and sexual selection that must
have produced the phenomenon. The papers in this volume
provide a wealth of new pleasures both by describing new
and fascinating observations in gastropod sexual biology and
by providing deeper insights into some of the more familiar
systems.

The term mating system is shorthand for the species-
typical reproductive behavior of a species: that is, who mates,
when they mate, who is successful and why. The mating
system is a product of both natural and sexual selection and
is, in a sense, the grand finale to the life history of a species.
Understanding the mating system of a species requires
knowledge of many aspects of its ecology, physiology, and
behavior. There is perhaps no species, including our own,
for which the mating system is completely understood.
However, in recent decades tremendous progress has been
made in understanding mating systems from the standpoint
of behavioral and evolutionary ecology. Modern mating sys-
tems theory views the mating system as the outcome of
selection acting on selfish individuals who may have con-
flicting interests, even as they come together to produce and
perhaps, rear, their offspring. Much of this work has dealt
with humdrum and boring taxa such as birds, mammals.

and insects but application of what Eric Charnov ( 1982) has
termed, “selection thinking” to invertebrates, including gas-
tropods, came early, with the publication of Mike Ghiselin’s
(1974) book, “The Economy of Nature and the Evolution of
Sex” and George Williams’s (1975) “Sex and Evolution”.
However, it has taken time for malacologists to embrace
sexual selection and mating systems theory for a variety of
reasons; many gastropods are hermaphrodites and the ap-
plication of sexual selection theory to hermaphrodites is not
entirely straightforward (see review in Leonard 2006); Dar-
win’s idea that gastropods lacked the sensory and mental
capacity to choose mates has been very influential and has
had much intuitive appeal. However, it has been shown that
the hermaphroditic basommatophoran, BuUiuis truncatus
(Audouin, 1826) can discriminate among mates based on
their infection status and that this differs with the genotype
of the chooser (Webster and Gower, 2006).

Over the last three decades, gradually and one by one, a
variety of laboratories have begun to explore the sexual bi-
ology of gastropods as models for testing predictions of mat-
ing system theory and to use mating systems theory to un-
derstand the biology of the gastropods they are interested in.
The immediate stimulus for the current symposium came
from the realization that a certain critical mass has been
reached and that it was time to bring together a selection of
these workers from around the world to compare notes and
provide an overview into the diversity of gastropod biology
and gastropod research. The joint meeting at Asilomar
seemed to be the ideal opportunity and the resulting sym-
posium, “Gastropod Mating Systems” on the morning of
lime 27, 2005 consisted of nine talks, covering a wide variety
of topics from the genetics of sex ratio (Yusa, this volume),
to reproductive physiology (Ter Maat et al. and Mayeri) and
paternal care (Grosberg). Three of the talks dealt with pro-
sobranchs; two with opisthobranchs and four with pulmo-
nates; one with the basommatophoran, Lymnaca stagnalis
(Linnaeus, 1758), and three with stylommatophorans. Two
of the talks are unfortunately not represented in this volume:

* From the symposium “Gastropod Mating Systems” presented at the joint meeting ot the American Malacological Society and Western
Society of Malacologists, held 26-30 June 2005 at Asilomar, Pacific Grove, California.

79

80

AMERICAN MALACOLOGICAL BULLETIN

23 • 1/2 • 2007

“Mating systems and family conflicts in a marine
snail” by Rick Grosberg, Center for Population Bi-
ology, University of California-Davis

and

“Mating and egg-laying behavior in Aplysia -
Pheromones and neural mechanisms” by Earl May-
eri, Department of Physiology, University of Cali-
fornia-San Francisco

However, we have added two important papers from authors
who were not able to attend the symposium: a review of the
mating system and reproductive biology of Arianta arbiisto-
riim (Linnaeus, 1758) by Bruno Baur of the University of
Basel and a review of work on dart-shooting in helicids by
Ronald Chase of McGill LIniversity. Several of the papers (by
Yusa, Reise, Davison, Baur, and Chase) represent important
reviews and syntheses of previously published work while
others (e.g., Takeuchi etal, Krug, and Leonard et al.) present
new data. The paper by Ter Maat et al. provides an impor-
tant comparison of field and laboratory data on reproductive
allocation. While this volume may not convey a sense of the
beauty and magical atmosphere of the Asilomar Conference
Grounds, the papers presented here will provide a sense of
the many stimulating directions and developments in the
new field of gastropod mating systems research.

ACKNOWLEDGEMENTS

I would like to thank Peter Roopnarine of the Western
Society of Malacologists and Dianna Padilla of the American
Malacological Society for support of the symposium and all
the participants who made it so stimulating and enjoyable.
Thanks are also due to the California State Parks system
which maintains the Asilomar facility and makes it available
for such meetings.

LITERATURE CITED

Charnov, E. L. 1982. The Theory of Sex Allocation. Princeton Uni-
versity Press, Princeton, New lersey.

Ghiselin, M. T. 1974. The Economy of Nature and the Evolution of
Sex. University of California Press, Berkeley, California.
Leonard, J. L. 2006. Sexual selection: Lessons from hermaphrodite
mating systems. Integrative and Comparative Biology 46: 349-
367.

Webster, |. P. and C. M. Gower. 2006. Mate choice, frequency
dependence, and the maintenance of resistance to parasitism
in a simultaneous hermaphrodite. Integrative and Comparative
Biology 46: 407-418.

Williams, G. C. 1975. Sex and Evolution. Princeton University
Press, Princeton, New lersey.

Amer. Maine. Bull. 23: 81-87

Reproductive behavior of the dioecious tidal snail Cerithidea rhizophorarum
(Gastropoda: Potamididae)’^

Maya Takeuchi, Harumi Ohtaki, and Kiyonori Tomiyama

Department of Earth and Environmental Sciences, Faculty of Science, Kagoshima University, Korimoto, Kagoshima, 890-0065, lapan,
cococoro2001@yahoo.co.jp

Abstract: The dioecious snail Cerithidea rhizophorarum (Adams, 1855) is distributed along the coasts of the western Pacific up to the
Tohoku district, northern Honshu, Japan. It inhabits reed grasslands and mangrove forests with Kaudelia candel and Hibiscus hauiaho trees
on a mud flat located at the mouth of Atagogawa River in Kiire. Studies of mating and tree climbing behaviors of the species were conducted
at this site from April 2000 to May 2003. Mating behavior was observed in Inly and August 2002. The time of commencement, termination,
and duration were recorded for each copulation. The peak of matings during daytime was seen at 1 to 2, and 5 hours before the lowest tide
and during nighttime, between 1 hour before and after lowest tide. However, mating rarely occurred on cloudy days. Climbing behavior
was observed in an area of 100 square meters where only K. candel trees existed. The number ot snails on trees was counted, and daily
activity of the snails on trees was monitored in summer and winter, hourly throughout the day. The snails were mainly found on mud from
spring to summer but frequently climbed up the tree at particular times during summer. Most individuals were on trees and motionless
during winter.

Key words: reproduction, dioecious snail, climbing behavior, gastropod

Molluscs have two types of mating systems: dioecy and
hermaphroditism (simultaneous hermaphrodite, protandric
hermaphrodite, and protogynous hermaphrodite). These di-
vergent patterns of mating systems are found even in the
same taxonomic classes. Bisexual reproduction is common
in molluscs, and the pattern of fertilization in most classes is
internal, but external fertilization is found in some classes.
Hermaphroditic species can be divided into ones that can
self-fertilize and ones that cannot. Hence molluscs have
many divergences in the reproduction pattern and may be
the phylum with the most variable reproductive patterns in
the animal kingdom. How could such various reproductive
strategies evolve?

There are many reports of mating behavior in hermaph-
rodites such as Pulmonata and Opisthobranchia, but it is
rarely reported in dioecious Prosobranchia. A clearer under-
standing of the mating behavior of this group would be one
of the keys to solve the evolution of various reproductive
strategies in molluscs.

The dioecious prosobranch snail Cerithidea rhizophora-
rum (Adams, 1855) commonly inhabits tidal flats in eastern
Asia. In the tidal flat of the Atagogawa River, Kiire-Cho,
Kagoshima, mating by shell mounting was observed (Ohtaki
et al. 2001). Ohtaki et al. (2001) also reported mating be-
havior, but it was incomplete. In this study, several aspects of

mating behavior of C. rhizophorarum were examined in the
field including duration of copulation both in daytime and
nighttime.

MATERIALS AND METHODS

Study site

The tidal flat is at the mouth of Atagogawa River flow-
ing through Kiire-Cho, Kagoshima-city. This river is located
by the Nisseki oil camp, and it joins Yahata River in this
point. A small mangrove forest consisting of Kaudelia candel
and Hibiscus hamabo, at the northern limit of mangrove
distribution in the West Pacific, covers this tidal flat. Some
species of gastropod, such as Cerithideopsilla djadjariensis (K.
Martin, 1899), Cerithideopsilla cingulata (Gmelin, 1791), Ba-
tillaria multiformis (Lischke, 1869), Batillaria cumiugi
(Crosse, 1862), Clypeomorus coraliuui (Kiener, 1834), Reti-
cimassa festiva (Powy, 1833), Clithou oualauieusis (Lesson,
1831), and Clithou faba (Sowerby, 1836) inhabit the tidal
flat. We established three study sites (A, B, C) 60 m from the
shore of Atagogawa River.

Size distribution

We collected 100 Cerithidea rhizophorarum with a net
(1 mm mesh) at random at the three stations (A, B, C) and

* From the symposium “Gastropod Mating Systems” presented at the joint meeting of the American Malacological Society and Western
Society of Malacologists, held 26-30 June 2005 at Asilomar, Pacific Grove, California.

81

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

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Figure 1. Seasonal change at each
station in the width-frequency distri-
bution in Cerithidea rhizophomrum.

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REPRODUCTIVE BEHAVIOR OF CERITHIDEA

83

2001

Apr.

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84

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

2002

Apr.

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REPRODUCTIVE BEHAVIOR OF CERITHIDEA

85

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Figure 1. (continued)

Station A

Station B

StationC

measured their shell width in situ every month from April
2000 through May 2003.

1. Copulation frequency

Snail mating can be categorized as face-to-face mating
or shell-mounting mating (Asami 1998). Cerithidea rliizo-
phorarum mates by shell mounting (Ohtaki et al. 2001).
Ohtaki et al. (2001) reported that copulatory behavior was
observed from the middle of June to the middle of August
and peak of initial time of copulation was before lowest tide.
In this study, we counted the number of individuals and
copulating couples along the route every 15 minutes on 4
July and 21 July 2001, 12 and 25 June, 12 and 13 July, and
10 August 2002 in daytime, and 12 and 13 July 2002 at night.
While sampling the transect eveiy 15 minutes, we placed
little flags marked with an identification number near each
pair of C. rhizophomruni in the act of coupling. When the
pair of C. rhizophorarurn separated, it was assumed that the
copulation was terminated. The length between the begin-
ning and the end of the copulation was checked and noted.
We used a headlamp for night searches. On the same day, we
picked 100 copulation pairs at random and marked upper
snails with a permanent marker. We brought copulating
pairs back to laboratory and measured them (both weight
and shell height). Their shells were broken, and we deter-
mined their sex by examining the reproductive gland.

2. Climbing behavior

Cerithidea rhizophorarurn is known as an intertidal snail
species, but it does climb trees. This behavior was observed
in a small mangrove forest of the Atagogawa River (Waka-
matu and Tomiyama 2000, Ohtaki et al. 2002). In this study,
we observed snails on the trees from May 2000 to May 2003,
2 hours after the lowest tide of spring tides every month.

RESULTS

Size distribution

During this period no large change in the number of
Cerithidea rhizophorarurn was noticed (Fig. 1). Newly re-

cruited juveniles (3-6 mm in shell width) of C. rhizophora-
ruin were found in higher tidal zones than the other pot-
amidid and batillariid species. The distribution of juveniles
of C. rhizophorarurn was limited to lower tidal zones.

Copulation frequency

The largest number of copulations was observed be-
tween one and two hours before the lowest tide in the af-
ternoon and two hours before and after the lowest tide at
night (Fig. 2). The mean shell width of upper individuals was
10.96 ± 2.36 mm (mean ± SD, N - 100, range = 8.6-13.0
mm) and 11.72 ± 29 mm (mean ± SD, N = 100, range =
8.8-13.9 mm) for lower individuals (Fig. 3). There was no
significant correlation between the shell width of upper and
lower individuals {R^ = 0.082), but upper individuals were
significantly smaller than lower ones (Student’s f-test,
P < 0.05). From the reproductive organs of the 100 pairs,
65% were male (upper) - female (lower) pairs, 31% were
male - male pairs, 1% female - female pair, and 3% female
(upper) - male (lower) pairs were observed. The mean width
of individuals with male reproductive organs was 11.49 ±
2.69 mm (mean ± SD; N - 69, range = 8.8-13.9 mm). There
was no relation in shell width between the females and the
males (f-test; P > 0.05).

Climbing behavior

There were clear seasonal changes in total number of
individuals of Cerithidea rhizophorarum on the trunk of the
mangrove tree Kandelia candel (Fig. 4). In 2000, 1071 snails
were observed on trees in May, 1355 in June, 601 in July, 46
snails in August, and in September the number of snails
increased to 1041. The following year, from September to
December, the number of snails climbing up the trunk of
mangrove trees was at its peak, with the number decreasing
from February through March. This tendency was seen every
year at the same period.

DISCUSSION

Wakamatsu and Tomiyama (2000) reported that re-
cruitment of Cerithidea rhizophorarum at this site was so

86

AMERICAN MALACOLOGICAL BULLETIN 23 • 111- 2007

Jul.4th 2001

12 T
10
8
6
4
2
0

Jul21st 2001

9:45

17:30

Figure 2. Daily changes in percent-
age of copulating individuals in the
population. Arrows indicate the
lowest tides.

Jun. 12th 2002

10:00 12:00 14:00 16:00

Jun.25th 2002

12 T

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upper shell width (mm]

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= 0.0048

0 A i 1 1 1 1

0 0.5 I 1.5 2 2-6

upper wet weight 1^

shell width |mm|

Figure 3. A, Relationship between
shell width of upper individuals and
lower individuals. R~ = 0.0033, P >
0.05, N - 100. B, Frequency of shell
width of upper individuals and
lower individuals. Solid bar, upper
individuals; open bar, lower indi-
viduals; N = 100. C, Relationship
between shell width of upper indi-
viduals and lower individuals. =
0.0048, P > 0.05, N = 100. D, Fre-
quency of shell width of male and
female. Solid bar, male; open bar,
female; N = 100.

Figure 4. Seasonal changes in total
number of individuals of Cerithidea
rhizophorarum on the trunk of the
mangrove tree Kaiidelia cmidel (May
2000 - May 2003).

REPRODUCTIVE BEHAVIOR OE CERITHIDEA

87

small that it could not be detected. But in this study, newly
recruited juveniles (3-6 mm) appeared from November 2001
to March 2002, and they grew to 10 mm in length from
March to Inly 2002. However, new recruitment was not
observed every year. Wakamatsu and Tomiyama (2000) and
Ohtaki et al. (2001) suggested that C. rhizophomnu}i had
deceased in numbers at this site. We conclude that the de-
crease in population is due to imposex caused by TBT. Erom
the results of proportions of copulation pairs, it appears
males mount females.

More male-to-male (31%) than female-to-female
couples were observed (1%), supporting the hypothesis of
Wakamatsu and Tomiyama (2000) and Ohtaki et al (2001).

The peak frequency of copulation was generally before
the lowest tide in daytime. Copulation was most frequently
observed during the 3-6 hours around the time of the lowest
tide and mostly during 1-2 hours around the time of the
lowest tide at night. As we did not observe copulation when
it had begun to rain, rain may block the copulation of Ceri-
thidea rhizophorarum.

The population of Cerithidea rhizophorarum on trees
increased at the highest tide and decreased at the lowest tide.
This might be the daily rhythm to avoid the water. Snails
were observed on trees from the beginning of spring to lune
and descended from trees in luly to August. These rhythms
synchronize with the copulation period. The population on
the trees increased in September, and decreased in winter.
Climbing trees at the beginning of winter could be in prepa-
ration for hibernation.

ACKNOWLEDGEMENTS

We thank Janet Leonard for her help and the invitation
to present this study at the symposium, and all participants
at the symposium.

LITERATURE CITED

Asami, T., R. H. Cowie, and K. Ohbayashi. 1998. Evolution of
mirror images by sexually asymmetric mating behavior in her-
maphroditic snails. The American Naturalist 152: 225-236.

Ohtaki, H., E. Maki, and K. Tomiyama. 2001. Seasonal changes in
distribution and reproduction behavior of Cerithidea rhizo-
phorarum. Venus 60: 199-21.

Ohtaki, H., E. Maki, and K. Tomiyama. 2002. Climbing behavior of
Cerithidea rhizophorarum. Venus 61: 215-223.

Wakamatsu, A. and K. Tomiyama. 2007. Seasonal changes in size
distribution of Potamididae (Gastropoda) on Mangrove tidal
flat. Venus 59: 225-243.

Accepted; 9 May 2007

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Amer. Malac. Bull. 23: 89-98

Causes of variation in sex ratio and modes of sex determination in the
Mollusca — an overview"^

Yoichi Yusa

Faculty of Science, Nara Women’s University, Nara 630-8506, lapan, yusa@cc.nara-wu.ac.jp

Abstract: The mechanisms for variation in the primary and apparent sex ratios, from both theoretical and empirical perspectives, are
reviewed. A series of experiments on the sex ratios and mode of sex determination in the apple snail Poniacca canaliculnta (Lamarck, 1822)
show that broods have highly variable sex ratios even though the sex ratios of populations are 1:1. I suggest that the mechanism responsible
for this pattern is oligogenic sex determination, i.e., sex determination by a small number of genes. Two other molluscan groups, the
protandric oysters of the genus Cmssostrea Sacco, 1897, and mussels of the genus Mytilus Linnaeus, 1758 also show variable sex ratios. In
both cases, the number of genes responsible for the variation appears to be small.

Key words: sex-determining gene, genetic mechanism, genetic sex determination, molluscs, Pouiacea caualiculata

Fisher ( 1930) was the first to show that the sex ratio of
a population should be 1:1 if producing a son or a daughter
requires an equal cost. Because each offspring inherits half of
its genetic material from its mother and half from the father,
the members of the sex in short supply will have a higher
expectancy of genetic contribution to the next generation
than members of the sex in excess. Therefore, the genetic
tendencies that produce members of the sex in short supply
will be selected by natural selection, until an equal sex ratio
is realized in the population.

Since Fisher’s theory, studies on sex ratio have expanded
along three major lines. The first line is the extension of this
theory to include cases for which its premises do not hold,
such as local mate competition or local resource competition
(Hamilton 1967, Trivers and Willard 1973, Clarke 1978).
The second is to treat sexuality in general such as sex allo-
cation in simultaneous hermaphrodites (Charnov 1982).
The third treats the conflict between individuals or between
genes for reproduction, such as the worker-queen conflict in
the Hymenoptera (Trivers and Hare 1976) or conflict be-
tween nuclear and cytoplasmic genes (Werren and Beuke-
boom 1998). Overall, these theoretical studies have fueled
many empirical studies, and together they have advanced
our understanding of sex ratio or, more generally, of evolu-
tionary patterns (Hardy 2002).

On the other hand, fewer studies have been done on
mechanisms of sex-ratio variation. This is probably because
of the belief that studying evolutionarily stable sex ratios
does not require exact knowledge of the genetic background
producing them. However, evolutionarily stable sex ratios

are not independent of the underlying mechanisms although
they are not fully constrained by the mechanism as evi-
denced by the presence of large sex- ratio variations under
chromosomal sex determination (West and Sheldon 2002).

The sex-determining mechanism is one of the factors
affecting sex ratios. However, other genetic or non-genetic
factors such as sex-ratio genes, cytoplasmic sex factors, or
environmental factors may also affect the sex ratio. On the
other hand, the importance of sex determination lies not
only in its relevance to sex ratios but also to the problem of
sex itself. After all, what sex is and how sex is determined are
two of the fundamental questions in biology. Recent studies
have succeeded in identifying sex-determining genes {Sry in
mammals, Sinclair et al. 1990; DMY in a fish, Matsuda et al.
2002). However, most information comes from a limited
number ot model organisms, and the wide variety of sex-
determining mechanisms in many organisms have not been
studied. One of the few exceptions is the insightful work on
sex-determining mechanisms and its evolution by Bull
(1983).

Experimental studies on sex ratio are generally easy to
conduct. After all, to study the sex ratio of a population one
has only to count the numbers of males and females at an
appropriate stage of the life cycle. Considering this, it is
surprising how few studies have been done to elucidate the
genetic background that produces sex-ratio bias in organ-
isms other than vertebrates and insects. The Mollusca — the
most diverse animal taxon in terms ot both the number of
species and the modes of life, except for the Arthropoda — is
no exception. To date, studies on the genetic mechanism

* From the symposium “Gastropod Mating Systems” presented at the joint meeting of the American Malacological Society and Western
Society of Malacologists, held 26-30 lune 2005 at Asilomar, Pacific Grove, California.

89

90

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

producing various sex ratios in the Mollusca are limited to
only a few groups, such as the oysters of the genus Cras-
sostrea Sacco, 1897 (Haley 1977, Guo et al. 1998), the mus-
sels of the genus Mytiliis Linnaeus, 1758 (Saavedra et al.
1997, Kenchington et al. 2002), and the apple snail Pomacea
caiialiculata (Lamarck, 1822) (Yusa and Suzuki 2003, Yusa
2004b, 2006, 2007).

The purpose of this paper is to review studies on the
mechanisms that produce sex ratios and the modes of sex
determination in molluscs. I do not treat the adaptive sig-
nificance of sex ratios in detail as there are many good pa-
pers such as Hamilton (1967), Charnov (1982), and Hardy
(2002).

DEFINITION OF SEX RATIO

I define the sex ratio as the proportion of males / (males
-t- females). 1 consider the sex ratio in populations consisting
mainly of male and female individuals. Thus, in this review
I include sex-changing molluscs, as each individual is either
male or female at a time. I do not consider simultaneous
hermaphrodites, except for treating them as a factor biasing
the sex ratio. Charnov (1982) provides the theoretical frame-
work of sex allocation in simultaneous hermaphrodites.
Studies on sexual issues in simultaneously hermaphroditic
molluscs are reviewed in Leonard (1991), Baur (1998), and
other contributions from this symposium.

Sex ratios can be measured at different stages of the life
cycle of an organism. Sex ratio at fertilization or when sex is
determined is called the primary sex ratio. Sex ratio at birth
is called the secondary sex ratio. Sex ratios at later stages can
also be considered, such as sex ratio at sexual maturity or
that of individuals available for mating (operational sex ra-
tio; Emlen and Oring 1977). In this review, I mainly treat the
primary sex ratio. Other sex ratios are referred to as “appar-
ent sex ratios.”

Sex ratios can be considered at the population level
(population sex ratio) or within each brood (brood or off-
spring sex ratio). These two are not always the same. For
instance, in infinite populations, parents producing any
brood sex ratios are equally adaptive although population
sex ratio will become 0.5 by Fisherian selection (Williams
1979).

MECHANISMS THAT MAY AFFECT APPARENT
SEX RATIOS

Data on sex ratios are often taken from a field popula-
tion as the proportion of males in adult individuals, without
any information on sex differences in mortality, size, or
other features. Thus, almost inevitably, many factors poten-

tially affect the apparent sex ratio. To determine the primary
sex ratio, well-controlled experiments, where confounding
factors are eliminated, are ideal. However, it is often impos-
sible to conduct such experiments, or the field data them-
selves may be the goal of a study (Takeuchi et al. 2007). Even
so, many factors may potentially bias the apparent sex ratio
(Table 1).

Misidentification of sex

Many molluscs show sexual dimorphism in (i) the shape
of the shell or the soft parts (some unionoids, Dillon 2000;
the ampullariids Marisa corniiarietis [Linnaeus, 1758],
Demian and Ibrahim 1972; and Pomacea canaliculata, Caz-
zaniga 1990); (ii) body color (the ampullariid Marisa cor-
niiarietis, Demian and Ibrahim 1972); or (iii) body size (am-
pullariids and vivipariids, Dillon 2000; assimineid snails
Assiminea japonica Martens, 1877 and Angiistassiminea cas-
tanea [Westerlund, 1883], Kurata and Kikuchi 2000). In
such cases, the sex may be identified by external morphology
without sacrificing the animals. However, the external mor-
phology is often unreliable in identifying sex, and inspection
of the gonads or other reproductive organs is preferable
whenever possible. Even if gonads are examined, misidenti-
fication may occur, especially when only a small amount can
be excised for inspection to keep the animals alive (Bauer
1987).

Various sampling biases

Males and females may differ in habitat use, behavior
(such as mobility and activity patterns), conspicuousness (in
terms of color or brightness), or body size. For instance, in

Table 1. Mechanisms that may affect apparent and primary sex
ratios.

1. Mechanisms for apparent sex ratios
Misidentification of sex

Sampling biases due to differential habitat use, behavior, etc.
Differential mortality (embryonic, juvenile, or later)
Differential age at maturity
2 Mechanisms for primay sex ratios
Sex-ratio genes and cytoplasmic factors
Sexual system
Parthenogenesis
Sex change

Simultaneous hermaphroditism
Mode of sex determination
Environment
Sex-determining genes
Heterogamety
Oligogenes
Polygenes

MOLLUSCAN SEX RA'l lO AND SEX DETERMINATION

91

the vivipariid Vivipariis ater (Cristofori and Ian, 1832), fe-
males tend to hibernate earlier than males; thus, the appar-
ent sex ratio of the population in autumn is male-biased
(Keller and Ribi 1993). Different habitat use has been sug-
gested as a cause of female-biased sex ratios in another vivi-
pariid, Sinotaia qiiadmta historica (Gould, 1859) (Hirai et al.
2004). In many snails females grow larger than males (e.g.,
Pomacea cnnaliculata, Cazzaniga 1990; Assiminea japonica,
Kurata and Kikuchi 2000) although in some species males
are larger {Angustassiminea castanea, Kurata and Kikuchi
2000). Sexual dimorphism in size affects the apparent sex
ratio if researchers sample larger individuals more often than
smaller ones.

Differential mortality

The sex ratio at birth may be different from the primary
sex ratio if the hatching rate differs between the sexes. This
effect is especially important when the hatching rate is low.
However, the effect of differential hatching rate among egg
masses can be assessed by studying the correlation between
hatching rate and the secondary sex ratio. If there is no
significant correlation, then the differential hatching rates
are not responsible for the sex-ratio variation among egg
masses (Yusa and Suzuki 2003).

Differential mortality in juvenile or adult stages may
also skew the sex ratio at later stages (e.g., Sinotaia quadrata
historica, Hirai et al. 2004; Basycou carica [Gmelin, 1791],
Avise et al. 2004). In addition, if age at maturity differs
between the sexes, the sex that matures earlier will normally
outnumber the sex that matures later due to the higher
mortality of the latter before reaching sexual maturity.

MECHANISMS RESPONSIBLE FOR PRIMARY
SEX RATIOS

There are three general categories of mechanisms that
may affect primary sex ratios (Table 1 ); sex-ratio genes and
cytoplasmic factors, the sexual system (gonochoric, her-
maphroditic, or parthenogenetic), and the mode of sex de-
termination.

Sex-ratio genes and cytoplasmic factors

Sex-ratio genes are nuclear genes that are expressed in
the parents (the father, the mother, or both) and control the
sex ratio of the offspring. For example, X-chromosome drive
genes in Drosophila skew the proportion of X-carrying
sperm during meiosis or fertilization (Hamilton 1967,
Stouthamer et al. 2002).

Cytoplasmic sex factors or distorters are genetic ele-
ments present in the cytoplasm, such as the bacterial genus
Wolbachia (Stouthamer et al. 2002). They often distort the

host’s sex ratio towards female, because the parasite is usu-
ally inherited only through the female lineage and hence
female-biased sex ratios are advantageous to them. Cytoplas-
mic sex factors are not known in Mollusca, and a prelimi-
nary trial to detect them was unsuccessful in Pomacea catia-
liculata (Yusa 2006). This does not necessarily mean,
however, that all molluscs are free from these factors. A
possible candidate, for example, is the paternally inherited
(M) mitochondria of Mytihis spp. (Saavedra et al. 1997,
Sutherland et al. 1998, Zouros 2000, Kenchington et al. 2002,
Cao et al. 2004) and unionoid bivalves (Dillon 2000).

Sexual system

Parthenogenesis

Molluscs with highly female-biased sex ratios often turn
out to reproduce parthenogenetically. For instance, in the
freshwater snail Potamopyrgus antipodarwn (Gray, 1843),
the sex ratio varies from 0-50% among populations (Wal-
lace 1992). In most populations the sex ratio is female-biased
(Lively 1992). The populations with extremely low sex ratios
consist of triploid females that reproduce parthenogeneti-
cally, and populations with ratios that are approximately 0.5
consist of diploid sexuals (Lively 1992, Wallace 1992). In
some populations, parthenogenetic and sexual individuals
coexist; these populations have intermediate sex ratios
(Lively 1992, Jokela et al. 1997).

Freshwater clams of the genus Corbicula Megerle von
Miihlfeld, 1811 are predominantly hermaphrodites, repro-
ducing by self-fertilization. However, maternal genes are ex-
truded from the oocyte during the first meiotic division, so
that fertilized eggs have only the paternal nuclear genome
(androgenesis; Komaru et al. 1997, Ishibashi et al. 2003).
Because the offspring have the same genome as the parent
through non-reductional sperm, this represents a special
case of parthenogenesis.

Sex change

Sex change is either protandrous (first mature as male
and then change sex to female) or protogynous (female to
male). The occurrence of sex change has two major ettects
on sex ratio. First, the brood sex ratio, when followed as a
time series, is biased towards the first-maturing sex when
they are young, and then skews towards the later-maturing
sex as a direct consec^uence of sex change, as shown in Cras-
sostrea gigas (Thunberg, 1793) (Guo et al. 1998). Secondly,
sex change affects the sex ratio of the population as well. The
sex ratio should be distorted towards the first developing sex
in sex changers (Charnov and Bull 1989). In fact, several
studies have reported male-biased sex ratios in protandrous
molluscs (the oysters Crassostrea spp., Haley 1977, Guo et al.
1998; the pearl oyster Pinclada mazatlanica lameson, 1901,
Arnaud-Haond el al. 2003; the slipper shell of the genus

92

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Crepidiila Lamarck, 1799, Hoagland 1978, Collin 1995,
Richard et al. 2006).

Sex ratio may vary seasonally or spatially in protandrous
molluscs. For instance, in Crepidida convexa Say, 1822, the
sex ratio is male-biased from fall to spring, when new re-
cruits become sexually mature as males, then female-biased
in summer when many of them change sex to female
(Hoagland 1978). Hoagland also reported positive correla-
tions between adult density and sex ratio in many local
populations of Crepidula foniicata (Linnaeus, 1758) ancf C.
convexa. This is not due to adaptive sex-ratio adjustment in
response to variable density, but rather to variation in the
number of recruits among sites; sites with many recruits
have higher densities and, after the recruits mature as males,
have higher proportions of males.

Simultaneous hermaphroditism

In some organisms, simultaneous hermaphrodites co-
exist with males (androdioecy), females (gynodioecy), or
both (trioecy). In the Mollusca, coexistence of gonochoric
individuals and simultaneous hermaphrodites has been re-
ported (unionoid bivalves, Dillon 2000; the freshwater pearl
mussel Margaritifera margaritifera [Linnaeus, 1758], Bauer
1987; Mytihis spp., Saavedra et al. 1997). When the propor-
tion of hermaphrodites is small and the population consists
mainly of gonochoric individuals, the sex ratios are nearly
0.5 in most species studied so far (for unionoid bivalves, see
table 2.2 in Dillon 2000; in Mytilus, Saavedra et al. 1997). In
the freshwater mussel, Margaritifera margaritifera, the pro-
portion of males in the population consisting of males, fe-
males, and hermaphrodites is nearly 0.5 (Bauer 1987). Be-
cause females can change sex to hermaphrodites and vice
versa, the equal proportion of males and non-males suggests
that a simple genetic mechanism such as heterogamety is
involved (Bauer 1987).

MODE OF SEX DETERMINATION

Environmental sex determination

In the case of gonochoric organisms, an individual’s sex
is determined environmentally, genetically, or both. Envi-
ronmental sex determination can result from factors such as
temperature, food availability, daylength, or the presence of
adult individuals (Bull 1983). Well-known molluscan ex-
amples are slipper shells of the genus Crepidida, in which
small individuals that settle on larger individuals mature as
males, whereas solitary ones mature as females (Hoagland
1978). These small males may change sex, but there are also
true males which do not have the ability to change sex (Coe
1936; although Hoagland raises some doubt about this). As

an adaptive response, sex ratios in organisms with environ-
mental sex determination are expected to be biased towards
the sex developing in the worse conditions (e.g., poor nu-
trients; Charnov and Bull 1989).

Genetic sex determination

Sex-determining genes can be distinguished by the
number of genes into two-factor, oligogenic and polygenic
systems. Sex determination by heterogamety, such as XY
(male heterogamety) or ZW (female heterogamety), is a
well-known system involving two genetic factors. The ma-
jority of gonochoric molluscs with known sex-determining
systems are XY (gastropods, Nakamura 1986; some bivalves,
Allen et al. 1986, Guo and Allen 1994). Examples of XO sex
determination, with XX females and XO males, are wide-
spread in the Neritidae (Nakamura 1986).

In species of the freshwater snails Viviparus Montfort,
1819, both XY and ZW sex determinations are known to
occur, although ZW is the majority (Barsiene et al. 2000).
This has an important implication that one sex-determining
system [e.g., XY) can evolve from another (ZW) relatively
easily.

Under heterogamety, the expected brood and popula-
tion sex ratios are both 0.5, with little variation expected
under the binomial distribution unless other mechanisms
affect the sex ratios. In accordance with this, the sex ratio is
0.5 in most molluscan populations where data are available
(e.g., in the unionoid bivalves and in freshwater “proso-
branchs”, Dillon 2000), although female-biased sex ratios are
reported in some “prosobranchs” such as Pomatiopsis cin-
cinnatiensis (Lea, 1840) and Goniobasis (also referred to as
Elimia H. & A. Adams, 1854) semicarinata (Say, 1829) (Dil-
lon 2000). Unfortunately, due to the lack of sufficient infor-
mation, the reason for the female-bias is unknown.

Oligogenic sex determination depends upon a small
number of genes. For instance, in the platyfish and wood
lemming, sex is determined by three genetic factors, X, Y,
and W (Bull 1983). Under oligogenic sex determination,
offspring sex ratios vary among different pairs of parents.
For instance, in the platyfish, mating between an XX female
and a YY male produces all sons (XY males), whereas mating
between a WX female and an XY male produces only one-
quarter sons (Bull 1983).

Polygenic sex determination is a system in which many
genes are involved in determining an individual’s sex. Each
gene has only a minor effect on sex determination, and
hence, sex-ratio variation. Models of polygenic sex determi-
nation are proposed by Buhner and Bull (1982) and Bull
(1983). There are no molluscan examples of polygenic sex
determination.

MOLLUSCAN SEX RATIO AND SEX DETERMINATION

93

SEX-RATIO VARIATION AND SEX DETERMINATION
IN POMACEA CANALICULATA

The apple snail Pomacea canalicidata (Ampullariidae) is
a South American freshwater snail introduced into many
Asian countries, including Taiwan, the Philippines, Thai-
land, Vietnam, lapan, and China (Naylor 1996, Yusa and
Wada 1999). It is a serious rice pest (Naylor 1996) as well as
a keystone species controlling the function of wetland eco-
systems (Carlsson et al. 2004). Because no effective control
methods have been developed, sex-ratio variation in P. cana-
liculata has been studied to explore novel genetic control
methods (Yusa 2004a, 2004b).

Potnacea canaliadata is gonochoric. Although there is a
report that P. canalicidata can change sex (Keawjam and
Upatham 1990), later authors have failed to confirm this.
Sex ratios in populations in rice fields are often female-
biased (Banpavichit et al. 1994, Tanaka et al. 1999), but these
may be due to differential survival or growth rates between
the sexes.

To study the sex ratio at birth, we removed up to 80
hatchlings from each egg mass, and reared them for 50 days
or more, until individuals began developing their reproduc-
tive organs (Yusa and Suzuki 2003). We dissected all snails
to identify the sexes based on the presence or absence of the
testis (for males) or the albumen gland (for females). We
used egg masses collected for three months duly, August,
and September) in organic rice fields where no chemicals
had been sprayed, and those laid by laboratory-reared pairs.

Sex ratios varied greatly among egg masses from nearly
all males to nearly all females (Fig. 1). In many egg masses,
the sex ratio was significantly different from 0.5. For in-
stance, in July the sex ratio was significantly different (P <
0.05 by binomial test) from 0.5 in 15 out of 27 egg masses.
This proportion is much higher than expected by chance
(only 1 or 2 out of 27 egg masses are expected to be statis-
tically significant at P - 0.05). Likewise, the sex ratio was
significantly biased in 13 of 25 egg masses in August, and in
7 of 27 in September.

Irrespective of the large variation among egg masses, the
average sex ratio was close to 0.5 in all three months (mean
± SD = 0.47 ± 0.20 in July; 0.53 ± 0.25 in August; and 0.47
± 0.18 in September). Thus, brood sex ratios of Pomacea
canalicidata were highly variable, yet population sex ratios
were unbiased. We repeated similar experiments using egg
masses of laboratory-reared mating pairs originating from
two different populations. The results were nearly identical:
large variations in brood sex ratios and unbiased population
sex ratios were detected in all eight experiments conducted
(Yusa and Suzuki 2003).

Survival was high (94-98%) during the rearing period

Sex ratio (proportion of males)

Figure 1. Brood sex ratio (proportion of males) of field-collected
egg masses in the apple snail Pomacea canalicidata (after Yusa and
Suzuki 2003). Black areas indicate egg masses whose sex ratios are
significantly different from 0.5 (P < 0.05 by binomial test); gray
areas indicate those that are not significant.

and did not expilain the variation in brood sex ratio. Hatch-
ing rate was low, but there was no correlation laetween
hatching rate and sex ratio in any experimental series. A
possible factor responsible for variation in sex ratio was egg
weight. The relationship between the average weight of an
egg and the sex ratio was negative in all eight experimental
series, and the relationship was significant in three experi-
mental series. Heavier eggs tended to produce female snails
whereas lighter ones produced males. Although the meaning
of this relationship is unknown, the effect was too small to
explain all of the variation.

Environmental effects on sex ratio in
Pomacea canalicidata

To study the effects of environment on brood sex ratio,
I used a split-brood design, where each brood was split into
two (or sometimes three) groups of nearly equal numbers of
hatchlings, and each group was reared under two different
conditions for each environmental factor (Yusa 2(K)4b). 1
tested factors such as the presence of adult males or females,

94

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

food availability, temperature, age of the parents, size of the
aquarium (as an index of crowding), and indoor or outdoor
conditions (with different daylengths).

Among the environmental factors studied, none had a
significant effect on brood sex ratio. On the other hand,
brood sex ratios were significantly different among different
egg masses or parents in all experiments. These results sug-
gest that environmental sex determination does not occur
and that the variation in sex ratio is under genetic influence
in this species.

Genetics of sex-ratio variation in Pomacea canalkulata

To investigate the genetics of sex-ratio variation, I stud-
ied parent-offspring regressions and sib correlations of sex
ratio (Yusa 2006). for parent-offspring regressions, the sex
ratio of each brood was regressed to the sex ratio of its
father’s siblings or that of its mother’s siblings. Correlations
in sex ratios between sisters and between brothers were also
investigated.

There were significant positive relationships in brood
sex ratio in the female lineage: the regression between off-
spring sex ratio and the sex ratio of the mother’s siblings
(slope = 0.28) and the correlation between the offspring sex
ratios of two sisters (r = 0.41) were both significant (P <
0.05). On the other hand, father-offspring regression (slope
= 0.10) and the correlation between two brothers (r = -0.13)
were not significant (Yusa 2006).

Thus, the results suggest inheritance of sex-ratio varia-
tion through the female lineage. However, the regression or
correlation coefficients were too low to postulate cytoplas-
mic sex factors or maternal genes, which may show coeffi-
cients of nearly 1.0 for mother-offspring regression and cor-
relation between sisters (Table 2; Yusa 2006). The results
were not congruent with typical sex-ratio genes or sex-
determining polygenes with additive effects, which will show
coefficients of nearly 0.5 for both male and female lineages
(Table 2). A further study suggests that the variation in sex
ratio is due to sex determination by a small number of genes
(oligogenic sex determination; Yusa, 2007).

Oligogenic sex determination means the coexistence of

multiple sex-determining genes in a population. It may
occur during the evolutionary transition from one sex-
determining system to another, or it may result from a mix-
ture of two or more populations with different sex-
determining systems. In Pomacea canaliculata, XY sex
determination is reported in some Japanese populations
(von Brand et al. 1990) but not in Brazil (Mercado Laczko
and Lopretto 1998). This discrepancy suggests that different
sex-determining systems may coexist in P. canalkulata, as
they do in the frog Rana rugosa Schlegel (Nishioka et al.
1994).

GENETICS OE SEX DETERMINATION IN
OTHER MOLLUSCS

Sex determination in Crassostrea

Oysters of the genus Crassostrea change sex. In general,
they are protandrous hermaphrodites, but the details of
the sexual system differ among species and experimental
conditions.

In Crassostrea virginica (Gmelin, 1791), the majority of
individuals change sex from male to female, but a small
proportion of individuals may change from female to male
(Haley 1977). Pure males and pure females also exist. Haley
proposed an additive 3-loci model to explain the difference
in brood sex ratio and patterns of sex between families, with
m being the allele for maleness and / for femaleness. Under
this hypothesis the sex of an individual is determined by
ratio of m:f alleles, such that those with 3-6 nis are true
males, those with 2 m’s are protandrous hermaphrodites, 1
}n are females (possibly protogynous hermaphrodites), and
no m are true females. To my knowledge, this is the only
study suggesting oligogenic sex determination in the Mol-
lusca. However, both the experiments and the hypothesis
have been criticized by later authors (Guo et al. 1998; Yusa
and Suzuki 2003).

In Crassostrea gigas, individuals are either protandrous
hermaphrodites or true males. In a study of parental effects
on the sex ratio, Guo et al. ( 1998) found that the sex ratio of

Table 2. Expected regression or correlation coefficients of brood sex ratios under various genetic systems and those actually obtained in
Pomacea canaliculata (after Yusa 2006). The genes are supposed to have additive effects.

Genetic system

Mother-offspring

regression

Father-offspring

regression

Correlation
between sisters

Correlation
between brothers

Cytoplasmic sex factors
Sex-ratio genes (biparental)

Polygenic sex determination

Data obtained in P. canaliculata (mean ± SE)

1

0.5

0.5

0.28 ±0.13

0

0.5

0.5

0.10 ± 0.16

1

0.25

0.25

0.41 ± 0.18

0

0.25

0.25

-0.13 ± 0.19

MOLLUSCAN SEX RATIO AND SEX DETERMINATION

95

individuals at one year of age was dependent on the father
and independent of the mother. They proposed that the sex
is determined by a single locus, with M and F genes, and
individuals of MF genotype become males and those with FF
genotype are protandrous hermaphrodites.

Sex-ratio variation and doubly uniparental inheritance
of mitochondria in Mytilus spp.

In the mussels Mytilus ediilis Linnaeus, 1758, Mytilus
trossulus (Gould, 1850), and Mytilus galloproviuciulis La-
marck, 1819, brood sex ratios vary from all males to all
females (Saavedra et al. 1997, Kenchington et al. 2002). Ir-
respective of this variation, the average of brood sex ratios
(population sex ratio) is nearly 0.5 (Saavedra et al. 1997,
Zouros 2000, Kenchington et al. 2002). Such highly variable
brood sex ratios under equal population sex ratios are simi-
lar to the sex-ratio patterns in Pomacea canaliculata. The
major difference between the mussels and the apple snail is
that in the mussels sex ratio appears to be dependent only on
the mother and independent of the father (Saavedra et al.
1997, Zouros 2000, Kenchington et al. 2002; but see below),
whereas in the apple snail both parents contribute equally to
the sex-ratio variation (Yusa 2007).

In mussels, sex-ratio variation involves a phenomenon
called “doubly uniparental inheritance” (DUl) of mitochon-
dria (Saavedra et al. 1997, Zouros 2000, Kenchington et al.
2002). There are two types of mitochondria, M and E. The M
types are transmitted only from the father to the sons
through sperm, and the E types from the mother to both
sons and daughters through eggs. DUI has been demon-
strated in five families of bivalves: Mytilidae, Unionidae,
Hyriidae, Margaritiferidae, and Veneridae (Kenchington et
al. 2002, Cogswell et al. 2006, Walker et al. 2006).

Saavedra et al. ( 1997) postulate a masculizing factor, W,
in M-type mitochondria. This is supported by the observa-
tions that (i) some fathers who fail to pass the M genome to
their offspring have few sons and many daughters (Saavedra
et al. 1997), and that (ii) M mitochondria are passed into
female embryos as well as male embryos. However, in the
female embryos they appear to be outnumbered by F mito-
chondria whereas in the male embryos M mitochondria
preferentially enter into the supposed germ line (Sutherland
et al. 1998, Cao et al. 2004, Cogswell et al. 2006). These
observations suggest that embryos with many M types in the
germ line will develop the testis and become males. Because
some fathers fail to pass the M genome to their offspring, at
least some part of genetic sex-ratio variation depends on the
father. However, this effect is small and the major source of
sex-ratio variation depends on the mother (Saavedra et al.
1997, Kenchington et al. 2002).

The fact that most genetic variation in the sex ratio

depends on the mother suggests that some factor from the
mother controls the behavior of M mitochondria. Saavedra et
al. (1997) postulate that there is a feminizing factor Z, and
the amount of the factor in the eggs depends on the mother.
Kenchington et al. (2002) further postulate that the factor is
controlled by a pair of alleles (Z and z) at a single locus. This
allows only three genotypes of females: ZZ, Zz, and zz. The
ZZ mothers produce almost all sons, Zz mothers produce
both sons and daughters at a fixed ratio, and zz mothers
produce all daughters (Kenchington et al. 2002). However,
the simple 2-factor model of a feminizing factor Z does not
fully explain the variable brood sex ratios observed in Myti-
lus. Continuous but highly variable sex ratios probably re-
quire more factors, at least some of which may be genetic, as
suggested by the pedigree experiments (Kenchington et al.
2002).

In SLimmaiy, there appears to be a masculizing factor W
in M mitochondria in Mytilus spp. Some genetic variation
exists in the fathers’ ability to pass the M genome to the
offspring. Also there may be another sex-determining factor
Z in the eggs, which may control the action of W. The
genetics of Z factor is unknown, but probably more than two
genes or alleles are necessary to explain the large variation in
the sex ratio. The genetics of sex-ratio variation in Mytilus
therefore seems to be oligogenic.

CONCLUSION AND FUTURE DIRECTION

Sex ratio is a basic property of a population and hence
has direct relevance to the fitness of the individual control-
ling the sex ratio of the brood. The sex-determining system
may not be fixed, but rather may be fairly labile and subject
to evolutionary changes. For example, XY and ZW sex de-
termination coexist in Viviparus; there appears to be oligo-
genic sex determination in Potnacea caualiculata, and possi-
bly in Mytilus spp.

Studies on variation in sex ratio, such as the ones on
Pomacea canaliculata, are very easy to conduct — just rear the
hatchlings and determine their sexes. Also, studies on sex
chromosomes are not difficult, yet very few studies have
been done in molluscs, especially outside gastropods. Con-
sidering the wide variety of molluscan taxa and their life
histories, 1 suspect that studies may lead to unanticipated
findings.

At the same time, novel techniques are useful. For in-
stance, Avise et al. (2004) developed molecular sex identifi-
cation techniques using microsatellite loci in the whelk Busy-
con carica. Also, artificial formation of triploid individuals by
cytochalasin B treatment was successful in elucidating de-
tailed mechanisms of XY sex determination, such as Y being

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

dominant to X in the dwarf surfclam Mulinia lateralis (Say,
1822) (Guo and Allen 1994), or sex determination by X:au-
tosome ratio in soft-shelled clam Mya arenaria (Allen et al.
1986). Such techniques will be powerful tools to study the
genetics of sex determination and sex-ratio variation in
molluscs.

ACKNOWLEDGMENTS

I thank Jan Leonard for organizing and inviting me to
the symposium on gastropod mating systems, and speakers
and attendants there for fruitful discussion. Thanks are also
due to Ryo Ishibashi, Jan Leonard, Mayu Obata, Janice Volt-
zow, Shigeyuki Yamato, and two anonymous reviewers for
comments on various versions of the manuscript; Narisato
Hirai for useful information; and Rob Dillon and Keiji Wada
for encouragement and discussion.

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Accepted: 9 May 2007

Amer. Maine. Bull. 23: 99- 1 1 1

Poecilogony and larval ecology in the gastropod genus Alderia"^

Patrick J. Krug

Department of Biological Sciences, California State University, Los Angeles, California 90032-8201, U.S.A., pkrug@calstatela.edu

Abstract: The gastropod genus Alderia (Allman, 1845) (Opisthobranchia; Sacoglossa) contains a rare case of poecilogony, or variable larval
development within a species. This paper reviews the alternative larval morphs and dispersal strategies expressed by Alderia spp., and
presents new data on larval ecology, environmentally induced changes in development, and rates of metamorphosis for larvae differing in
age and life history. Recent morphological and molecular analyses revealed a ciTptic poecilogonous species in the previously monotypic
genus Alderia. The newly described Alderia willowi Krug et ai, 2007 occurs south of Bodega Harbor, California, and was the subject of all
prior studies by Krug and co-workers. Unlike its strictly planktotrophic congener Alderia modesta (Loven, 1844), A. willowi produces either
small feeding larvae that have a 30-day pre-competent period or large larvae that need not feed to metamorphose. Individuals can vary the
developmental mode of their offspring, with a variable proportion switching from lecithotrophy (prevalent in summer) to planktotrophy
in winter or spring; a similar shift is induced in some adults upon transfer to the laboratory. In a second dispersal polymorphism, a variable
percentage of lecithotrophic larvae undergo spontaneous metamorphosis within 2 days of hatching, while their siblings disperse until
induced to settle by carbohydrate cues from the host algae Vaiicheria spp. The percentage of spontaneous metamorphosis is uncorrelated
with fecundity and is generally between 15-30%, a possible product of stabilizing selection on this bet-hedging dispersal strategy. Despite
their different ages, competent larval morphs produced by alternative developmental pathways are similar in size, swimming behavior, and
responses to dissolved settlement cues. However, competent planktotrophic larvae and older lecithotrophic larvae initiated metamorphosis
faster after settlement than newly hatched lecithotrophic larvae, suggesting a link between planktonic period and habitat choice. Although
rare, poecilogonous species like A. willowi offer special insights into the evolutionary causes and ecological consequences of alternative life
histories.

Key words: bet-hedging, cryptic species, dispersal polymorphism, larval settlement, sacoglossan

A major djfference between marjne and terrestrjal life
historjes is the dichotomy between feeding and non-feeding
modes of larval development among marine invertebrates
(Strathmann 1990, 1993). Most species produce either a
large number of small larvae that must feed in order to attain
competence (planktotrophy) or fewer, larger larvae that can
metamorphose without feeding (lecithotrophy) (Wray and
Raff 1991, Levin and Bridges 1995). Ecologically similar and
related species often differ in developmental mode for rea-
sons that remain unclear, and there is no theory specifying
which selective regimes should favor lecithotrophy over
planktotrophy (Todd et al. 1998, McEdward 2000). Phylo-
genetic studies have demonstrated that transitions between
developmental modes have occurred numerous times in di-
verse groups such as molluscs (Duda and Palumbi 1999,
Meyer 2003, Collin 2004), echinoderms (Hart et al. 1997,
Hart 2000, Jeffrey et al. 2003), and urochordates (Hadfield et
al. 1995). Planktotrophy is presumed to be ancestral to leci-
thotrophy because complex feeding structures, once lost, are
rarely regained via adaptive evolution (Strathmann 1978,

* From the symposium “Gastropod Mating Systems” presented at
the joint meeting of the American Malacological Society and West-
ern Society of Malacologists, held 26-30 June 2005 at Asilomar,
Pacific Grove, California.

1985, 1993, Wray 1995). Both the fossil record and phylo-
genetic hypotheses generally support this one-way trend to-
wards non-planktotrophic development (Hansen 1982), al-
though species with nurse eggs may be an exception (Collin
2004). The forces that drive transitions to non-feeding larvae
are poorly understood, however, and the dearth of interme-
diate stages or organisms expressing multiple forms of de-
velopment within a species have impaired attempts to un-
derstand this aspect of life-histoiy evolution.

Although causal explanations remain elusive, the eco-
logical and evolutionary consequences of different develop-
mental strategies are profound (Perron 1986, Pechenik
1999). The paleontological record for molluscs indicates that
species with planktotrophic larvae have greater geological
longevity and broader geographical ranges than those exhib-
iting lecithotrophy, which speciate at a higher rate (Hansen
1978, 1980, 1982, Jablonksi 1986). Species with lecithotro-
phic or direct development are more prone to extinction,
but often show greater local adaptation (Hansen 1978, San-
ford et al. 2003). The prolonged pelagic period of most feed-
ing larvae makes them effective vectors for gene flow, and
planktotrophic taxa generally exhibit panmictic populations
(Palumbi 1995, Caley et al. 1996). The continuous influx of
alleles from distant populations can prevent planktotrophic
species from adapting to local conditions (Vermeij 1982).

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Intraspecific phylogeographic breaks occur in planktotro-
phic species but are usually small in magnitude compared to
the highly structured populations often observed within spe-
cies that have non-feeding larvae with reduced dispersal po-
tential (Kyle and Boulding 2000, Wares et al 2001, Collin
2003).

An improved understanding of how selection drives de-
velopmental evolution may come from studies of rare spe-
cies that display intraspecific polymorphism in larval devel-
opment. Termed “poecilogony”, this phenomenon appears
perplexingly restricted to spionid polychaetes and opistho-
branch molluscs (Giard 1905, Hoagland and Robertson
1988, Bouchet 1989, Ghia et al 1996). Molecular studies
discredited other purported examples of variable develop-
ment as cryptic species. True cases of poecilogony can in-
form as to the ecological significance of alternative dispersal
strategies (Levin 1984a, Levin and Huggett 1990, Levin and
Bridges 1994), and may indicate when natural selection fa-
vors one larval mode over another. Such species may also
provide insight into the mechanisms underlying develop-
mental transitions, such as allocation of maternal resources
and gene expression (Villinski et al 2002, Marsh and Fiel-
man 2005).

Some opisthobranchs have been classified as poecilogo-
nous because offspring can metamorphose either before or
after hatching from benthic egg masses. These may be more
constructively termed “dispersal polymorphisms”; they do
not constitute variable development as there is no difference
in egg size or larval trophic mode. Such dimorphisms can
result if the egg mass matrix retains or releases larvae ac-
cording to season (e.g., Elysia timida Risso, 1818; Marin and
Ros 1989), adult nutritional state [Tenellia adspersa Nord-
mann, 1845; Chester 1996), or experimental conditions
{Berghia veiriiciconiis Costa, 1864; Carroll and Kempf 1990).
In the cephalaspidean Haniinaea caUidegenita Gibson and
Ghia, 1989, some larvae attain competence prior to hatching
and undergo intracapsular metamorphosis triggered by an
inducer in the egg jelly, while siblings become competent
after hatching and settle in response to benthic biofilms
(Gibson and Ghia 1989). Dispersal polymorphisms can also
result from differences in settlement cue requirements
among pelagic larvae (Mackay and Doyle 1978, Raimondi
and Keough 1990, Toonen and Pawlik 1994), but this has
received little attention as a potentially important adaptation
in marine life histories.

Truly variable development is expressed in species
where developmental mode differs between offspring due to
variation in egg size or pre-hatching consumption of nurse
eggs or extra-zygotic yolk. In all recognized cases, this in-
volves alternative developmental pathways that produce ei-
ther feeding larvae that ingest planktonic food after release
or larvae that can metamorphose without feeding after re-

lease from parental brood structures or benthic egg masses.
One definition of “lecithotrophy” applies to larvae that are
incapable of ingesting particulate food (Ghia 1974); other
definitions include larvae that do not need to feed to com-
plete metamorphosis, whether or not they can (Thompson
1967). The larger larval morph of most poecilogonous spe-
cies can feed, either by ingesting nurse eggs prior to hatching
or facultatively capturing phytoplankton if food is available
(Gibson and Gibson 2004, Botello and Krug 2006, Pernet
and McArthur 2006). In this review, I use Thompson’s
(1967) definition of “lecithotrophic.”

It is noteworthy that no example of variable develop-
ment involves a free-spawning organism; among poly-
chaetes, eggs are retained in structures associated with adult
tubes, and in sacoglossan opisthobranchs, within benthic egg
masses. Although there are reported examples of poecil-
ogony among sacoglossans that include pelagic lecithotro-
phy and direct benthic development (usually through en-
capsulated metamorphosis), these likely constitute examples
of dispersal polymorphisms as described above, or cryptic
species (e.g.., ''Elysia cauze” later recognized as three distinct
species; Glark and Goetzfried 1978, Glark 1984, 1994).

Research into poecilogony has been historically con-
founded by the prevalence of cryptic species in the taxa that
contain well-documented examples of variable development,
namely polychaetes and opisthobranchs (Grassle and Grassle
1976, Glark 1984, Hoagland and Robertson 1988, Morrow et
al 1992, Glark 1994, Ghia et al 1996, Schulze et al 2000,
Kruse et al 2003). Confirmation of variable development
requires molecular data or breeding experiments to demon-
strate that individuals differing in larval trophic mode are
conspecifics. Among polychaetes, such data exist for Boccar-
dia proboscidea (Gibson et al 1999, Gibson and Gibson
2004), populations of Streblospio benedicti from the east
coast of North America (Levin 1984b, Levin et al 1991,
Schulze et al 2000), and Pygospio elegans (Morgan et al
1999). Among opisthobranchs in the order Sacoglossa, three
poecilogonous species have been confirmed by molecular
data or laboratory crosses. In breeding studies, planktotro-
phic and direct-developing specimens of Elysia chlorotica
Gould, 1870 from different populations produce viable hy-
brid offspring (West et al 1984). The Caribbean species
Costasiella ocellifem Simroth, 1895 was reported as a cryptic
species complex (Miles and Clark 2002), expressing plank-
totrophy or benthic development in different populations,
but recent data indicate these populations comprise a single
species and that individuals differing in development co-
occur and share mitochondrial haplotypes (Ellingson and
Krug, unpubl. obs.).

The third example of poecilogony among sacoglossans
was reported from southern California among sea slugs in
the genus Alderia (Allman, 1845), a member of the cerata-

I

POECILOGONY AND CRYPTIC SPECIES IN ALDERIA

101

bearing family Limapontiidae (Krug 1998). Alderia spp. are
specialized herbivores that feed solely upon the algae
Vaiicheria spp. (Ochrophyta: Vaucheriales), found on mud-
flats in temperate estuaries throughout the northern hemi-
sphere (Trowbridge 2002). The type species A. modesta
(Loven, 1844) was described from northern Europe and later
reported from both coasts of North America (Engel et al.
1940, Hand and Steinberg 1955, Hartog 1959, Bleakney and
Bailey 1967, Clark 1975, Vader 1981, Bleakney 1988, Trow-
bridge 1993, 2002, Martynov et al. 2006). Planktotrophy was
the sole developmental mode reported from the western
Atlantic (Clark 1975), eastern Atlantic (Engel et al 1940,
Seelemann 1967), western Pacific (Chernyshev and Chaban
2005), and eastern Pacific as far south as Monterey, Califor-
nia (Hand and Steinberg 1955, Trowbridge 1993, Gibson
and Chia 1994). Populations south of Monterey were re-
cently described as a new species A. willowi Krug et al, 2007,
based on morphology, molecular data, and the expression of
both lecithotrophy and planktotrophy (Ellingson and Krug
2006, Krug et al. 2007).

This paper will focus on Alderia spp., reviewing prior
studies that used A. willowi as a model to study swimming
and settlement behavior of alternative larval morphs and
recent work on cryptic speciation in the genus. New data will
be presented to address the following research questions: ( 1 )
How do reproductive and larval characteristics for the poe-
cilogonous A. willowi compare with those of its planktotro-
phic congener A. modestal (2) Does environment affect the
type of larvae produced by A. willowP. (3) Does the percent-
age of spontaneous metamorphosis co-vaiy with fecundity
or does it remain similar across populations of A. willowi?
(4) Do newly competent planktotrophic larvae show an ac-
celerated rate of metamorphosis similar to that of older leci-
thotrophic larvae?

MATERIALS AND METHODS
Study sites and taxa

Specimens of Alderia spp. and algae were collected at
low tide on mudflats from study sites along the west coast of
the U.S.A. (Fig. 1). The algal host in southern California is
recognized as Vaucheria loiigicaidis (Abbott and Hollenberg
1976); however, this nominal taxon likely comprises a cryp-
tic species complex, as there has been no detailed taxonomic
assessment of Vaucheria spp. from the northeastern Pacific.
I refer to algae from southern California as \7. loiigicaidis but,
when referring to the whole coast, use Vaucheria spp. to
reflect taxonomic uncertainty in this genus.

Clutch and larval characteristics of Alderia spp.

For Alderia modesta, adults were collected in Bodega
Harbor, California, in September and October 2004-2005.

Egg masses collected within 8 hr of deposition were isolated
in dishes of 0.22-pm filtered seawater (FSW) until hatching
commenced. The diameters of eggs and capsules were mea-
sured from high-resolution digital photographs of egg
strings removed from the egg mass, calibrating pixel number
per pm with photographs of a hemocytometer grid at the
same magnification. Data for planktotrophic and lecithotro-
phic development in Alderia willowi are from Krug (1998).

Changes in developmental mode: field surveys and
laboratory experiments

Surveys of developmental mode were carried out in San
Diego ( 1996-1999, 2003-2004), the Upper Newport Bay Eco-
logical Reseiwe (2003-2004), and a man-made wetland ad-
jacent to the Los Angeles Harbor (2003-2004). Specimens
were isolated in petri dishes of seawater for clutch deposition
overnight, and clutches were scored for developmental mode
1-2 days later based on egg size (Krug 1998). The proportion
of the population producing lecithotrophic clutches was
compared for March vs. August of 1996-1999 for San Diego,
the only site for which such data were available each year,
using a Mann-Whitney U-test (as implemented by StatView
statistical software package).

Adults collected from Los Angeles were typed for de-
velopmental mode of their first clutch (February 2004).
Those producing lecithotrophic eggs {n -20) were placed on
a patch of Vaucheria longicaulis from which all slugs had
been removed; algae and slugs were maintained in an incu-
bator at 22 °C on a 14:10 light:dark cycle. Egg masses de-
posited overnight were collected and typed for developmen-
tal mode every 1-2 days for 3 weeks. The proportion of
lecithotrophic clutches (y) was regressed against time (x)
using a Model 1 regression in StatView.

Spontaneous metamorphosis in clutches of
field-collected adults

Adult slugs were collected in San Diego (August 1999),
San Francisco (September 2004), and from three sites within
Tomales Bay: south Tomales, September 2003; Cow Land-
ing, September 2004, and Walker Creek, September 2004.
Slugs were transported to the lab, isolated, and their first
clutch harvested. Egg masses were kept in FSW until hatch-
ing (5-6 days) and scored for (1) egg number and (2) the
percentage of spontaneous metamorphosis occurring in the
first 2 days post-hatching (Krug 2001). The null hypothesis
of no variation in the percentage of spontaneous metamor-
phosis between sites was evaluated using a non-parametric
Kruskal-Wallis test. A Spearman Rank Correlation test was
used to compare fecundity and percentage of spontaneous
metamorphosis for the five sites.

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Figure 1. Map of collection sites for Alderia spp. from the northeastern Pacific. Inset shows
close-up of the area between San Francisco Bay and Bodega Harbor, where both species
seasonally co-occur and have their southern (A. modesta) or northern (A. wdlowi) range
limits.

Rate of metamorphosis in larvae
of different ages and
developmental modes

Planktotrophic larvae of Alderia
willowi were cultured as in Krug and
Zimmer (2000, 2004) and used in as-
says after 32 days. Larvae {n = 15 per
dish, 6 replicates per age class) were
exposed to live Vaucheria longicaulis
and scored for settlement and meta-
morphosis starting 2 hr after algae
were added and at 12 hr intervals
thereafter. Settlement is a reversible
attachment to the bottom, whereas
metamorphosis is an irreversible
transformation into a juvenile slug.
Cumulative percentages of metamor-
phosis were not normally distributed
after transformation and were, thus,
compared after 24 and 48 hr within
each age class using a Wilcoxon Signed
Rank test; no difference would indi-
cate that most metamorphosis oc-
curred in the first 24 hr whereas a dif-
ference would indicate a delayed
metamorphic response. These assays
were run concurrently with the experi-
ments in Botello and Krug (2006), a
study from which data for lecithotro-
phic larvae of A. willowi aged 1, 2 and
4 days post-hatching were taken; the
data for planktotrophic larvae were
not previously reported.

RESULTS AND DISCUSSION

Poecilogony and cryptic species in
the genus Alderia

The first report of lecithotrophy
in '"Alderia modesta” was also a south-
ern range extension of ~500 km for
this previously monotypic genus
(Krug 1998). The global phylogeogra-
phy of Alderia was recently studied by
sampling estuaries throughout the
northeastern Pacific (Fig. 1), one site
in the western Pacific, and one in the
eastern Atlantic. Slugs were typed for
developmental mode and morphology
of the dorsum and radula, and por-
tions of two mitochondrial genes (cy-

POECILOGONY AND CRYPTIC SPECIES IN ALDERIA

103

tochrome oxidase I (COI) and I6S ribosomal subunit) were
sequenced and analyzed (Ellingson and Krug 2006). Most
specimens south of Bodega Elarbor, Calitornia, U.S.A. com-
prise a poecilogonous cryptic species, Alderia willowi (Krug
et al. 2007). It is distinguished from A. niodesta by a humped
dorsum covered with fused patches ol dark pigment (Figs.
2A-B), the seasonal production of lecithotrophic larvae, and
the morphology of its radula and egg masses. The congener
A. modesta occurs seasonally in San Francisco Bay and from
Tomales Bay north throughout the Pacific and north Atlan-
tic. Alderia modesta has a smooth, speckled dorsum (Figs.
2C-D) and expresses only planktotrophic development.

Molecular data discriminated clearly between the sister
species, which form reciprocally monophyletic clades 18-

24% divergent in COI and 2.9% divergent in 16S (Tamura-
Nei distances) (Ellingson and Krug 2006). All prior publi-
cations by Krug and co-workers on "Alderia aiodesia" in fact
concerned its cryptic sister species A. willowi. Though nomi-
nally conspedfic, A. modesta from the north Atlantic are
substantially divergent (10-12% in COI) from Pacific A.
modesta. Populations in the two oceans have likely been
allopatric since the early Pleistocene, supporting the hypoth-
esis of Bleakney (1988) that trans-Arctic gene flow in A.
modesta ceased when glaciation covered the Arctic sea. How-
ever, given the lack of morphological and developmental
differences between ocean basins, A. modesta may still be one
biological species; breeding studies and further sampling of
Atlantic sites are needed to test this hypothesis. Speciation in

Figure 2. Photographs of individuals of Alderia spp. from the northeastern Pacific. A, Dorsal view of Alderia willowi, recently described from
California. A distinctive morphological feature of this species is that the dorsum is raised into a hump, with a yellow stripe extending down
the midline; the dark pigmentation is also fused into continuous patches. B, Side view of the same specimen, which had just been
hypodermically inseminated; the white bulge on the foot (Sp) is a subcutaneous sperm deposit. C, Dorsal view of Alderia niodesta, found
on eastern and western coasts of the Atlantic and Pacific. Its dorsum is smooth, often shield-shaped, and covered in speckles of dark
pigment. This individual had recently fed on Vaucheria longicaulis and the green cytoplasm is visible within digestive diverticula ramifying
throughout the body, including the cerata. The penis, extending from the right side of the head, is tipped with a stylet (St) for piercing the
body wall of mates during hypodermic insemination. D, Interspecific mating attempt, with a specimen of A. niodesta probing a smaller
specimen of A. willowi with its penis (P). Green digestive diverticula can be seen eneivating the penis of A. niodesta. The humped dorsum
(H) of A. willowi is evident. All photos to same scale; scale bar = 800 pm.

104

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Table 1. Clutch characteristics of Alderia willowi, a poecilogonous species occurring south of Bodega Harbor, California, and its planko-
trophic congener Alderia modesta, which occurs from San Francisco Bay northwards. For A. willowi, adults were collected from the Northern
Wildlife Preserve in Mission Bay, San Diego, from lanuary to April in 1996 and 1997; plankotrophic and lecithotrophic clutches were reared
at 25°C (Krug 1998). Data for A. modesta were obtained in September 2005 for specimens from Bodega Harbor. Data are means ± SD.

Alderia willowi

Alderia modesta

Lecithotrophic

Planktotrophic

Plankotrophic

Encapsulated period (days)

Eggs per clutch
Egg diameter (pm)

Egg capsule diameter (pm)

Maximum larval shell dimension at hatching (pm)

5.4±0.6(h = 30)
32 ± 12(;; = 30)
105±5(n = 328)
247 ±31 {11 = 328}
186±9(« = 282)

3.0 ±0.5 (« = 30)
311 ± 134(n = 30)
68±4(» = 517)
121 ± 12 (« = 517)
116 ± 8 (h = 160)

4.7 ± 1.2 (n= 18)
463 ± 117 {n = 15)
78±4(« = 75)
127 ± 10(h = 75)
124 ±8 (n = 45)

100

80

60

40

20

fd San Diego
I Newport
INI Los Angeles

Mar

Apr May Jun

1999

Jul Aug

given in Table 1. This table is adapted
from Krug (1998) to reflect the new
species status of A. willowi and to in-
clude comparative data for the true A.
modesta. The larger planktotrophic
clutches of A. modesta took longer to
develop and hatch than the smaller
planktotrophic clutches of A. wdlowi.
Mean adult size was greater for indi-
viduals of A. modesta (range: 5.5 - 23.1
mg, n = 6 populations) than for A.
willowi (range: 0.9 - 5.8 mg, n = 6
populations). In both species about
80% of the variance in fecundity is due
to adult size (Ellingson and Krug
2006).

2003

2004

Figure 3. Seasonal variation in the proportion of adult Alderia willowi producing lecithotro-
phic clutches in three southern Californian estuaries. Expression of lecithotrophy generally
only drops below 80% from October to April (shaded).

the Pacific pre-dated colonization of the Atlantic, indicating
a Pacific origin for the genus Alderia (Ellingson and Krug
2006).

Reproductive and larval characteristics of Alderia spp.

Developmental data for planktotrophic larvae of Alderia
modesta, and for both clutch types in Alderia willowi, are

Environmentally cued change in
development in Alderia willowi

Populations of Alderia willowi
were surt^eyed in southern California
starting in 1996, and showed a consis-
tent seasonal trend. In 1999 (San
Diego) and 2003-2004 (3 field sites),
clutches were 80-100% lecithotrophic
from May-September, but typically
less than 80% lecithotrophic from Oc-
tober-April (Fig. 3). The San Diego
population had the most extreme

shifts; from 1996-1999, the population

was 100% lecithotrophic in August
but only 55 ± 25% SD lecithotrophic in March of those years
(Mann-Whitney U-test, Z = -2.3, P < 0.05). The switch to
planktotrophy was roughly coincident with seasonal drops
in sea-surface temperature and salinity. Populations from
Newport Bay and Los Angeles Harbor exhibited a similar
trend, but a higher proportion of adults remained leci-
thotrophic from fall to spring (Fig. 3).

POECILOGONY AND CRYPTIC SPECIES IN ALDERIA

105

Some slugs expressing lecithotrophy in the field
switched to planktotrophy when held in the laboratory for
1-3 weeks (Eig. 4). Adults were maintained on patches of
Vancheria Jongicaulis in the laboratory and laid typical num-
bers of clutches; the change in their egg size was thus not due
to starvation, which can also trigger a switch to planktotro-
phy (Krug 1998). The proportion of planktotrophic clutches
increased over time (Eig. 4, and results of a regression of the
effect of time, x, on % planktotrophy, y: y = -2.80x -t- 93.69;
Fj 9 = 28.18, P = 0.0005, r = 0.73). The change in larval type
was interpreted as an adult response to the lab environment;
reverse transitions to lecithotrophy have not been success-
fully induced, suggesting the laboratory mimics winter con-
ditions. Siblings reared from the egg under laboratory con-
ditions exhibited either developmental mode and rarely
changed between modes (N. Smolensky and P. Krug, un-
publ. obs.). Slugs transitioning between development modes
in the laboratory sometimes produced clutches of interme-
diate-sized eggs, first reported in Krug ( 1998); such clutches
are produced by field-collected slugs during transitional
months, when the populations are switching either from or
towards lecithotrophy (unpubl. data). These data suggest
seasonal changes in development reflect changes within in-
dividuals, not just between generations within a population.

Together, the evidence suggests that individuals respond
to fluctuating conditions by varying their egg size and,
hence, mode of development of their larvae. Seasonal shifts
in development have not been reported among other poe-
cilogonous species, in which larval type varies by location
(West et al. 1984, Levin and Huggett 1990, Levin and Creed
1991, Morgan et al. 1999, Miles and Clark 2002). There is no
other example of a poecilogonous species in which indi-
vidual adults facultatively change the larval type of their
offspring, making Alderia willowi a unique model system for
investigating the ecological forces and
biochemical mechanisms underlying
alternative developmental modes.

The life-history strategies of
planktotrophy and lecithotrophy
substantially alter the hatching
characteristics and dispersal potential
of larvae. Lecithotrophy doubles the
encapsulated period of larvae in Alde-
ria willowi and reduces fecundity by an
order of magnitude (Table 1). How-
ever, lecithotrophic larvae are compe-
tent to metamorphose even before
hatching (Krug 2001, Botello and
Krug 2006), reducing their total devel-
opment time from about 35 days
(planktotrophy) to 5 days and pre-
sumably decreasing larval mortality.

450

300 -

150 -

San Diego
1999

100 ^

60

40

20 -

20

Days since collection

Figure 4. Change in developmental mode expressed by adult Al-
deria willowi following collection trom the field. Adults initially
produced lecithotrophic larvae when first collected and were main-
tained on patches of Vaitcheria longiaudis in the laboratory for
three subsequent weeks. Data are the percentage of lecithotrophic
clutches produced on a given day out ot the total number of egg
masses deposited by adult specimens (n = 25).

Lecithotrophic veligers are rare in plankton samples above
patches of Vaucheria spp. (D. Willette and P. Krug, unpubl.
obs.), suggesting most metamorphose at or before release.
Alternation of developmental modes is thus one dispersal
polymorphism, producing long-lived versus short-term lar-
vae in A. willowi.

Spontaneous metamorphosis and bet-hedging dispersal
strategies in Alderia willowi

A second dispersal polymorphism occurs within leci-
thotrof)hic clutches oi Alderia willowi: 0-90% of larvae spon-
taneously metamorphose prior to emergence or within 2
days of hatching, while their siblings delay settlement until

O eggs/clutcli

^ % spontaneous
metamorphosis

r 45

30

f

T3

O

3

of

3

<T>

O

O

■3

3"

San Francisco
2004

south Tomales
Bay 2003

Cow Landing
2004

Walker Creek
2004

Figure 5. Spontaneous metamorphosis among lecithotrophic laiwac of Alderia willowi from
different years and field sites. Adults were collected from San Diego (August 1999), southern
Tomales Bay (September 2003), and from San Francisco Bay, Cow Landing, and Walker
Creek (August 2004). Data are percentages of spontaneous metamorphosis (mean ± SE)
occurring over the first 48 hr post-hatching, in the absence of any inductive substratum.

106

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

contacting the host alga (Krug 2001 ). In many species, larvae
are more likely to metamorphose spontaneously with age, a
“desperate laiwa” effect (Gibson 1995, Toonen and Pawlik
2001). The case of A. willowi stands in contrast: veligers that
do not spontaneously metamorphose die over the next 14
days if denied access to the host, contradicting the hypoth-
esis that non-feeding larvae should lose settlement specificity
with age.

Although this trait was highly variable among individu-
als, the San Diego population hovered around a mean of
roughly 25% spontaneous metamorphosis over four years
(Krug 2001). Data from four additional populations, sur-
veyed from August to September of 1999, 2003 or 2004,
showed a similar trend (Fig. 5). The proportion of sponta-
neous metamorphosis varied significantly among popula-
tions (results of a Kruskal-Wallis test: H = 11.02, P < 0.05)
but was uncorrelated with fecundity (Spearman Rank Cor-
relation: P > 0.30). Local conditions were highly variable, as
reflected in the mean fecundity of different populations, but
good conditions did not produce higher levels of spontane-
ous metamorphosis (Fig. 5). Stabilizing selection could
maintain a mean 15-30% spontaneous metamorphosis at
most sites and times, even under optimal conditions. The
highest mean value (San Diego, 1999) was due to two slugs
that produced clutches with >80% spontaneous metamor-
phosis, reflecting the variable nature of this trait.

Spontaneous metamorphosis among newly hatched lar-
vae acts as a bet-hedging dispersal strategy, retaining some
offspring from each clutch in the parental habitat while al-
lowing the remainder to potentially locate a new algal patch.
Bet-hedging is a strategy that raises the geometric mean fit-
ness of a genotype by lowering variation in reproductive
success year to year (Seger and Brockmann 1987, Phillipi
and Seger 1989, Hopper 1999). A middle-of-the-road ap-
proach, bet-hedging genotypes trade the benefit of produc-
ing a high recruitment cohort under good conditions against
the risk of no reproductive success in bad seasons. This
makes it likely that some offspring will survive, regardless of
environmental fluctuations. Over many generations, such a
genotype has a higher relative growth rate than one that fails
to reproduce under any given set of conditions.

In most populations of Alderia willowi, about a quarter
of lecithotrophic larvae metamorphose with an abbreviated
planktonic period or none at all; these larvae are likely to
survive if local conditions remain favorable in the natal habi-
tat patch. Their siblings disperse until cued to metamor-
phose by contact with a new patch of algae, and may survive
if conditions in the parental habitat deteriorate. Such a strat-
egy maximizes the chance that some offspring will survive,
whether local patches of Vaucheria spp. persist or die back.
The proportion of spontaneous metamorphosis was also
phenotypically plastic, decreasing in response to adult star-

vation (Krug 2001). Strategies that vary the spatial or tem-
poral distribution of offspring are known for other taxa in-
cluding plants (Payne and Maun 1981, Telenius and
Torstensson 1989, Imbert 1999), mammals (Gaines and Mc-
Clenaghan 1980), and insects (Harrison 1980, Bradford and
Roff 1993, Zera and Denno 1997, Langellotto and Denno
2001), and may be a common evolutionary response to fluc-
tuating environments (Giesel 1976, McPeek and Holt 1992,
Kawecki and Stearns 1993, Chia et al. 1996).

Effects of developmental pathway on larval swimming
and settlement behavior

From a larval biologist’s perspective, poecilogony is a
chance to explore how conspecific larval morphs produced
by divergent developmental pathways compare behaviorally,
especially in their response to physical and chemical stimuli
during habitat choice. Studies of other poecilogonous spe-
cies have focused on the ecological effects of different strat-
egies on adult population dynamics (Levin and Huggett
1990, Morgan et al. 1999), but not on larval behavior. Vari-
able development in Alderia willowi provides the opportu-
nity to compare competent larvae differing greatly in age and
in trophic mode.

Krug and Zimmer (2000, 2004) compared physical
properties (size, passive sinking rate) as well as swimming
behavior for precompetent planktotrophic larvae and both
types of competent larvae (Table 2). Quantitative motion
analysis revealed the swimming paths of planktotrophic lar-
vae grow straighter and are increasingly directed downwards
as larvae mature; competent stages are larger, and sink and
swim faster than early stages. When planktotrophic larvae
attain competence, they are the same size as 1-day old leci-
thotrophic larvae, and sink and swim at a similar speed
(Table 2). Both competent morphs also share the same
shadow response, suggesting behaviors are conserved across
developmental programs (Krug and Zimmer 2004). The pri-
mary difference in the two morphs is that lecithotrophic
larvae swim downward in straighter paths, resulting in a
greater net rate of displacement towards the bottom. Mod-
eling efforts indicate differences in swimming behavior
could produce a 5-fold greater rate of contact with the bot-
tom for lecithotrophic larvae under natural flow conditions.

If mature planktotrophic larvae tend to encounter dis-
solved cues while suspended in the water column, stronger
behavioral responses might adaptively increase their odds of
recruitment. In contrast, because most lecithotrophic larvae
hatch in or near suitable juvenile habitat, selective pressure
to respond to dissolved cues could be relaxed for this morph.
To test these hypotheses, both types of competent larvae
were exposed to algal extract or field-collected conditioned
seawater (CSW) from within algal patches. Swimming be-
haviors were quantified through video motion analysis for

POECILOGONY AND CRYPTIC SPECIES IN ALDERIA

107

Table 2. Physical characteristics and swimming behavior of planktotrophic and lecithotrophic larvae of varying ages in Alderia willowi (Krug
and Zimmer 2004). Data are mean (± SE) larval size, sinking rate, and swimming behvaiors for larvae differing in age and developmental
mode. NGD is a ratio of the linear distance from the first to the last point of a given path to the actual distance traveled; a value of zero
indicates a circle, whereas a ratio of 1.0 represents a straight line. Vertical speed is a measure of larval movement in the Y-dimension only.
Speed is a non-directional scalar, whereas net vertical displacement rate is a vector with negative values indicating downward movement.
Behaviors were quantified in the dark using an IR-sensitive video camera.

Planktotrophic Lecithotrophic

8-d old 32-d old l-d old 4-d old

Shell size (pm)

126 ± 1

194 ± 1

194 ± 1

194 ± 1

Gravitational fall velocity (mm/s)

-0.90 ± 0.09

-1.59 ±0.12

-1.52 ± 0.10

-0.99 ± 0.10

Swimming speed (mm/s)

0.85 ± 0.05

1.03 ± 0.09

1.21 ±0.04

1.32 ±0.07

NGD ratio

0.83 ± 0.03

0.84 ± 0.02

0.92 ± 0.01

0.90 ± 0.03

Vertical speed (mm/s)

0.42 ± 0.06

0.65 ± 0.07

0.92 ± 0.06

0.99 ±0.12

Net vertical displacement rate (mm/s)

-0.10 ±0.08

-0.40 ±0.12

-0.78 ± 0.09

-0.73 ± 0.21

larvae suspended in the water or moving along the bottom of
an experimental chamber. Larvae of both types respond
similarly to dissolved cues, turning more frequently and
staying close to where the cue is initially perceived (Krug and
Zimmer 2000). Larvae suspended off the bottom signifi-
cantly decrease their speed, swimming in slow helices instead
of the straight lines seen in seawater controls. Along the
bottom, larvae swim in tight circles or hop, making frequent
contact with the substrate (Krug and Zimmer 2000). Such
behaviors should increase the chance of contacting a point
source of soluble cues (Tamburri et al. 1996). Little differ-
ence is evident between the two morphs, indicating that
selection has maintained a suite of behaviors in relatively
non-dispersing larvae that should enhance opportunities for
host colonization.

Upon contact with the host alga or after exposure to
dissolved cues, larvae attach to the substrate with the pro-
podium (settlement) and begin metamorphosis. Competent
larvae of both morphs are equally host specific, settling in
response to Vaucheria longicaulis but not other macroalgae
(Krug and Zimmer 2004). When tested with lecithotrophic
larvae, host specificity in Alderia willowi is higher than that
of any other sacoglossan studied to date, with >90% meta-
morphosis on V. longicaulis but no significant response (0-
10%) to 16 alternative algae or mudflat sediments (Krug
2001). Specificity for V. longicaulis does not diminish with
age (Krug 2001). Such specificity is not found among anas-
pidean opisthobranchs, which can be stenophagous as adults
but settle less specifically as larvae; >30% of Aplysia califor-
nica larvae metamorphosed on 10 different macroalgae and
>25% of Aplysia oculifera settled on 4 of 12 tested macroal-
gae (Pawlik 1989, Plant et al. 1995). Sacoglossans restricted
to a single genus or species of host algae often settle specifi-
cally on the obligate adult food, reflecting a high degree of

coevolution (West et al. 1984, Krug 2001, P. Krug, unpubl.
obs.).

Dispersal period and rate of metamorphosis

Larvae of Alderia willowi vary in their rate of metamor-
phosis according to age. Larvae of all ages settle within 1-2 hr
of exposure to Vaucheria longicaidis, but young lecithotro-
phic larvae (1-2 days post-hatching) delay metamorphosis
relative to older larvae (Botello and Krug 2006). About 50%
of 1 - or 2-day old lecithotrophic larvae complete metamor-
phosis after 24 hr, but 90-100% response requires 48 hr (Fig.
6). In contrast, 4-day old lecithotrophic larvae metamor-
phose at an accelerated rate, with >90% of metamorphosis
occurring in the first 24 hr of exposure to the host (Fig. 6).
Young larvae rarely metamorphose in the first 12 hr after
settlement, but a substantial fraction of older lecithotrophic
larvae metamorphosed in <12 hr (Botello and Krug 2006).
Previously unpublished data show that competent plankto-
trophic larvae behaved like older lecithotrophic larvae, with
most metamorphosis occurring in the first 24 hr (Fig. 6).
Thus, in both morphs, longer-lived larvae metamorphose
faster after settling on the host.

The increased rate of metamorphosis among older leci-
thotrophic larvae may be a response to diminishing yolk
reserves; 1-2 day old larvae can energetically afford to delay
metamorphosis for 12-24 hr while evaluating their sur-
roundings, whereas some older larvae die after 5 days (Krug
2001). The results for competent planktotrophic larvae can
be similarly interpreted as an adaptive response, as larvae
that have survived a perilous month at sea are under strong
pressure to locate a patch of Vaucheria without further risk
of planktonic mortality. Because competent planktotrophic
larvae were fed prior to the assay, they were not energetically
constrained as were the 4-d old lecithotrophic larvae; it

108

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

□ l-d 0

pn 2-d r lecithotrophic

■ 4-d J

d 32-d planktotrophic

24 48

Time since exposure to host alga (h)

Figure 6. Rate of metamorphosis for different larval morphs ot
Alderia willowi. Cumulative percentages of metamorphosis follow-
ing exposure to the adult host alga are plotted for lecithotrophic
larvae of varying ages (data from Botello and Krug 2006) vs. com-
petent, 32-day old planktotrophic larvae. For each age class, percent
metamorphosis after 24 vs. 48 hr was compared using a Wilcoxon
Signed-Rank test to determine if less metamorphosis occurred dur-
ing the first 24 hr period; *, P < 0.05.

therefore appears that metamorphic rate varies according to
the life history of a larva, as well as to individual energy
levels. Larvae differing widely in age thus have similar pat-
terns of host specificity and settlement behaviors, but differ
in rate of metamorphosis depending on an individual’s dis-
persal history.

CONCLUSIONS AND FUTURE DIRECTIONS

The developmental plasticity previously attributed to
Alderia modesta in fact occurs within its sister species, Alde-
ria willowi. Specimens of A. willowi can vary the develop-
mental mode of their larvae, alternating seasonally between
lecithotrophy and planktotrophy, which is unprecedented
among poecilogonous polychaetes and gastropods. Ongoing
research aims to unravel the environmental cues that trigger
changes in development, which in turn may shed light on
the evolutionary forces that favor lecithotrophy in the south-
ern species. Within lecithotrophic clutches of A. willowi, a
second dispersal polymorphism exists: some larvae sponta-
neously metamorphose at hatching, while their siblings dis-
perse until stimulated to metamorphose by dissolved or sur-
face-bound carbohydrates from the host alga Vaiicheria
longicaidis. The proportion of spontaneous metamorphosis
is highly variable between individuals but the population
mean rarely exceeds 25%, even when conditions are optimal.
Stabilizing selection might maintain this level to produce a

bet-hedging effect, with a quarter of all offspring recruiting
into the parental population and the rest dispersing until an
algal patch is encountered. The alternative developmental
pathways in Alderia willowi converge to make a similar com-
petent larva; although differing in age by >30 days, both
morphs swim and sink at similar rates, and alter their swim-
ming behavior in response to habitat cues in ways predicted
to increase the likelihood of successful recruitment. The rate
of metamorphosis, however, changes according to the de-
velopmental history and energy level of a given larva.

As biology moves into the proteomic era, poecilogonous
species offer the chance to investigate proximal mechanisms
such as changes in gene regulation and maternal effects that
underlie alternative developmental pathways. Transitions
between developmental modes have occurred frequently in
many invertebrate taxa, yet stable expression of multiple
developmental morphs is vanishingly rare, a paradox waiting
to be resolved. The study of poecilogonous species like Al-
deria willowi should provide a more complete understanding
of how the interplay between adult and larval ecology shapes
adaptive evolution of marine life histories.

ACKNOWLEDGEMENTS

I thank a cadre of talented students including D. Wil-
lette, R. Ellingson, V. Rodriguez, E. Hidalgo, J. Asif, N. Smo-
lensky, and G. Botello for much assistance and enthusiasm.
I thank ]. Leonard for the invitation to participate in the
2005 AMS/WSM symposium, and many colleagues for dis-
cussions over the years, including L. Angeloni, R. Burton, R.
Collin, D. Eernisse, N. Holland, D. Jacobs, L. Levin, A.
Manzi, J. Pawlik, B. Pernet, R. Toonen, C. Trowbridge, A.
Valdez, D. Zacherl, D. Zimmer and C. Zimmer. Two anony-
mous reviewers and J. Voltzow provided comments that
greatly improved this manuscript. Access to field sites was
provided by I. Kay through the Natural Reserve Office of the
University of California (San Diego), B. Shelton (Newport
Bay), and M. Schaadt (Los Angeles Harbor). This paper was
supported by awards from the National Science foundation
(OCE 02-42272 and HRD 03-17772).

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Accepted: 9 May 2007

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Amer. Maine. Bull. 23; 113-120

Food intake, growth, and reproduction as affected by day length and food
availability in the pond snail Lymnaea stagnalis^

Andries Ter Maat*’ *, Cor Zonneveld', J. Arjan G.M. de Visser^, Rene F. Jansen’,

Kora Montagne-Wajer’, and Joris M. Koene’

' Faculty of Earth and Life Sciences, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
^ Laboratory of Genetics, Wageningen Lhiiversity, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands

Abstract: With the aim of integrating the physiology and evolutionary ecology of Lymnaea stagnalis (Linnaeus, 1758), we studied the effects
of day length and food availability on the energy budget. Snails were assigned to two different photoperiods and three levels of food
availability. The snails were kept individually, and food consumption, growth, and egg production were measured for about 2 months.
Snails could nearly compensate for a one-day starvation period by increasing the rate of food-intake. I lowever, food-intake rates did not
increase further after a starvation period of 2 days. Growth was well described by the Von Bertalanffy growth eejuation. The ultimate size
of snails kept under medium-day conditions (MD; light:dark = 12:12 h) was not affected by food availability. By contrast, the ultimate size
of snails kept under long-day conditions (LD; light;dark = 16:8 h) depended on food availability; those fed the lowest quantities grow the
least. Dry-weight densities (dry weight/wet weight) of MD snails were considerably above those of LD snails. In MD snails, food availabilify
did not appreciably affect dry-weight density. By contrast, in LD snails, dry-weight density decreased with decreasing food availability. The
reproductive output of LD snails declined with declining foocf availability, but was 2 to 4 times that of MD snails. The difference in
reproductive output was largely accounted for by the difference in stored energy, i.e. diy-weight cfensity. To gauge the extent to which the
conclusions from our laboratory work applied to free-living snails, a field study was conducted. The wild-caught snails’ dry-weight density
was also lowest in long-day conditions when most eggs were laid. However, the dry-weight densities during medium and short days were
lower than the dry-weight densities of laboratory animals under LD conditions. Thus, in the field, snails stored less energy than in the
laboratory.

Key words: allocation, food availability, growth, reproduction

Ecological studies on the energetic costs of growth and
reproduction have far-reaching implications for understand-
ing the functioning of animals in relation to their environ-
ment {e.g., Dillon 2000). Physiological studies focus on the
underlying regulatory processes of growth and reproduction.
The latter approach has resulted in a vast knowledge of the
basic mechanisms of regulation of growth and reproduction
(reviewed in Chase 2002). However, the integration of eco-
logical and physiological knowledge is often hampered be-
cause the choice of experimental animal is determined by
several considerations that rarely coincide. As a result, few if
any experimental animals exist that are thoroughly studied
from both perspectives. The aim of the present paper is to fill
this gap in knowledge of the physiological ecology of the
pond snail Lymnaea stagnalis (Linnaeus, 1758).

The great pond snail Lymnaea stagnalis is a pulmonate

Current address: Max-Planck-Institut fiir Ornithologie, Seewie-
sen, 82319 Seewiesen, Germany, termaat@orn.mpg.de

gastropod belonging to the suborder Basommatophora and
the family of the Lymnaeidae. This simultaneous hermaph-
rodite occurs commonly in European lakes, ponds, and
ditches and can be easily collected in the field {e.g.,
Puurtinen et al. 2004) where it has an annual life cycle. Eggs
are laid in masses containing between 50 and 1 50 eggs, de-
pending on the individual’s body size (Koene et al 2007).
Offspring can be produced via self-fertilization and cross-
fertilization; when allosperm has been received, there is a
preference for outcrossing (Cain 1956, Knott et al. 2003).
Large populations can also be cultured in the laboratory
under semi-natural conditions (Van Der Steen et al. 1969).
These laboratory conditions allow for extensive control over
external factors (e.g., food, temperature, light, etc.) as well as
experimental manipulations (e.g., De Visser et al. 1994) and
neurophysiological experiments (e.^., De Boer et al. 1997).
As a result, the species has become a widely used, physi-
ological model system for research focusing on neuronal and
endocrinological control mechanisms (e.g., El Filali et al.
2006; reviewed in Chase 2002).

* From the symposium “Gastropod Mating Systems” presented at the joint meeting of the American Malacological Society and Western
Society of Malacologists, held 26-30 )une 2005 at Asilomar, Pacific Grove, California.

113

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

The organs and cell groups producing hormones that
regulate growth (Geraerts 1976) and reproduction (Geraerts
and Algera 1976, Geraerts and Bohlken 1976, De Boer et al.
1997) are known, as well as the amino acid sequences of
these hormones (Ebberink et al. 1985, Vreugdenhil et nl.
1985, Smit et al. 1988, De Lange et al. 1997, Jimenez et al.
2004). For example, growth is regulated by a growth hor-
mone produced by the light green cells (LGC). Egg laying is
triggered by the discharge of the neurosecretory caudo-
dorsal cells (GDC; Ter Maat et al. 1989). These neurons are
electrically coupled and during a discharge, a massive
amount ot the egg-laying hormone is released. This hor-
mone, called CDCH, has been fully characterized (Geraerts
et al. 1985, Jimenez et al. 2004) and gives rise to ovula-
tion, which results within two hours in an egg mass being
oviposited.

Despite the detailed knowledge about the neuro-
endocrinological regulation of growth and reproduction in
this species, surprisingly little is known about the function-
ing of these processes in relation to the animal’s environ-
ment. Environmental variables, like day length and food
availability, are known to affect the allocation of energy to
growth and reproduction [e.g., Scheerboom 1978, Bohlken
and loosse 1982). Lfowever, no detailed studies have been
performed to quantify these environmental factors. Such
studies should be quite relevant for an understanding of the
hormonal regulation of growth and reproduction. To bring
these two fields of research closer together, in the present
study we focused on the interaction between photoperiod
and food availability in the allocation ot energy to growth
and reproduction.

MATERIALS AND METHODS
Experimental design

Two laboratory experiments were performed, one under
medium-day conditions (MD: light:dark = 12:12 h; duration
80 days), the other under long-day conditions (LD: light:
dark = 16:8 h; duration 57 days). The adult snails, taken
from a mass culture bred under standard conditions, i.e.,
MD (Van der Steen et al. 1969), were kept individually in
perforated polyethylene beakers with a lid (460 ml). At the
start of the experiment, the snails were adult. The shell
lengths of the MD animals were 24.40 ± 1.55 mm, the LD
animals 22.71 ± 0.91 mm. Group size was 20 in the MD
experiment and 15 in the LD experiment. The perforated
beakers were placed in a tank with continuous water ex-
change using Amsterdam tap water through Cu-free piping.
The water temperature was kept at 17.5 ± 0.5 °C. Beakers
were changed every 3 or 4 weeks. Coprophagy was not
prevented.

In both experiments, three levels of food availability
were studied by varying the frequency at which food was
supplied. The snails in group 1 received lettuce in excess of
their requirements every day, those in group 2 at two sub-
sequent days followed by one day of starvation, and those in
group 3 every third day followed by two days of starvation.

Measurements of food consumption, growth, and egg
production were made. A broad-leaved variety of lettuce was
used as food. From the flat parts of the leaves, where only
small vascular bundles are present, circular discs were
punched with a surface area of 19.6 cm^. Snails were either
provided with 2 discs or starved. After 24 h the remaining
lettuce was removed from the jars, spread out on a Perspex
plate, and covered with a glass plate. The plates were sub-
sequently recorded on a video tape, and recordings were
digitized to determine the surface area. The difference be-
tween the area provided and remaining was used as a mea-
sure for consumption.

Every two weeks the shell heights were measured with a
caliper to the nearest 0. 1 mm. At the end of the experiment,
snails were frozen in liquid nitrogen. After thawing, the shell
was separated from the body, and the wet weight of the body
was determined. The shell and body were freeze-dried, after
which they were weighed to the nearest 0.1 mg. Dry weight
density (i.e., the ratio of dry weight to wet weight, expressed
as a percentage) was used as a measure of consumption.

Egg masses were collected every day. The egg masses
were stored in 70% ethanol until the number of eggs per egg
mass was counted.

Data analysis

Von Bertalanffy growth curves were fitted to the data on
shell heights for individual snails. The Von Bertalanffy
growth curve is given by the equation:

h(t) = - (h,, - hjexp{-gt} (1)

where h(t) denotes the current shell height; h^, the ultimate
shell height, which may eventually be reached if the snail is
kept under constant conditions for a long period; h^^, the
shell height at the start of the experiment; and g, the Von
Bertalanffy growth coefficient. The use of the Von Bertalan-
ffy curve has previously been shown to be appropriate for
this species (Zonneveld and Kooijman 1989).

For regression analyses we assumed a normally distrib-
uted scatter with homogeneous variance around the model
curves. Given this assumption, maximum likelihood esti-
mates are given by the least squares method. To obtain the
least squares estimates of the parameters, we used the Gauss-
Newton method (Richter and Sondgerath 1990). Standard
deviations of the parameters were estimated according to the
large sample theory of maximum likelihood estimators (Cox

ENERGY BUDGET OE THE POND SNAIL

1 15

and Hinkley 1974). Comparisons between groups were made
using Tukey’s post-hoc test.

Field study

For nearly two years, from 29 August 2002 to 14 April
2004 we collected a total of 564 Lymnaea stagnalis specimens
from two ditches in the Eempolder near Amsterdam, the
Netherlands. The Eempolder is a protected landscape en-
closed by dikes consisting of pastures separated by ditches.
Samples were taken in all months of the year, and on each
sampling date we tried to collect a representative sample of
both adults and juveniles. Immediately after collection, the
animals were weighed and shell length was measured. The
snails were subsequently dissected and the shell, albumen
gland, and prostate gland were removed and weighed (these
data will be published elsewhere, Koene et ai, unpuhl. data).
The soft body parts were freeze dried and weighed. All ani-
mals were checked for parasites. All year round, almost 50%
of the snails collected in the field are infected with one or
more species of parasites, among which were Trichobilharzia
ocellata, Echinostoma revoliitum, Opisthioglyphe nmae, Hy-
poderaeum conoidiim, Diplostomiim spathaceurn, and Pseii-
doechinoparyphium echinatum (Loy and Haas 2001, De Jong-
Brink and Koene 2005, Koene et al„ unpuhl. data). In the
current paper we present data on the dry weight density of
individuals not containing parasites (N = 283).

RESULTS

Growth and food availability

Von Bertalanffy growth curves were fitted to the mea-
sured shell heights (Table 1 ). The growth rate parameter g in
equation 1 is a measure for the curvature of the growth
curve. If the time constant g~' is larger than the duration of
the experiment, the curvature will be barely observable;
hence, it can be very difficult to estimate this parameter. This

Table 1. Means (and standard deviations) of parameter estimates
of Von Bertalanffy growth curves. Abbreviations: fy, ultimate shell
height; hj,, initial shell height; g, the Von Bertalanffy growth coef-

ficient; n, sample

size.

Day Food

length regimen

hg (SD) cm

h|3 (SD) cm

g (SD) d '

II

MD 1

3.45 (0.22)

2.25 (0.20)

0.0394 (0.018)

20

2

3.53 (0.28)

2.25 (0.17)

0.0284 (0.0077)

20

3

3.43 (0.53)

2.25 (0.18)

0.0145 (0.0050)

13

LD 1

3.33 (0.29)

1.86 (0.12)

0.0493 (0.014)

15

2

3.35 (0.27)

1.99 (0.084)

0.0321 (0.010)

15

3

2.62 (0.18)

2.03 (0.12)

0.0307 (0.011)

15

situation applied to only 7 MD snails of group 3, which were
the slowest growing snails. Standard deviations are based on
the estimates of the parameters for the individual snails.

The growth coefficient g was significantly affected by
day length and food availability (two way ANOVA; day
length: F = 15.6, df = 1, 92, P < 0.001; food availability: F -
25.4, df = 2, 92, P < 0.001), but there seemed to be no
interaction between these two factors (F = 2.0, df = 1, 92, F
= 0.14). The growth coefficient was larger in ED snails than
in MD snails, indicating that the LD animals grew faster. The
growth coefficient decreased with decreasing food availabil-
ity. In LD conditions, the animals had slower growth. Also,
limited food supply led to slower growth rates.

Food availability bad no effect on the ultimate length in
MD snails (one way ANOVA, F = 0.28, df - 2, 50, P > 0.5).
In LD snails, food availability also had no effect on the
ultimate lengths of groups 1 and 2, but snails in group 3
remained much smaller (P < 0.001). The ultimate shell
heights of groups 1, 2, and 3 of MD snails differed slightly
from those of group 1 and 2 of LD snails (one way ANOVA,
F = 5.6, df = 1, 68, 0.01 < P < 0.05). In conclusion, when
food was abundant, day length had a small effect on the
ultimate size attained, even though this length was reached
later by LD animals. However, when food was limited, LD
animals grew more slowly and reached a smaller ultimate
size.

Dry weights consist of structural body mass and stored
energy. The dry weight density, i.e., dry weight per wet
weight (without shell), can be used to compare the amounts
of stored energy in different groups. Diy-weight densities of
MD and LD snails at the three levels of food availability are
shown (Fig. lA). A two-way ANOVA showed that MD and
LD snails reacted similarly to food availability (interaction
between day length and food availability: F = 2.3, df - 2, 99,
P > 0.05). The MD snails had higher dry-weight densities
than LD snails (F = 293.0, df = \, 99, P < 0.0001 ). There was
a significant overall effect of food availability (F = 4.05, df —
2, 99, P < 0.05); the lowest dry-weight densities occurred in
the animals receiving the least amount of food and vice versa
(Tukey at F = 0.05).

In summary, food availability determines only growth
rate and not ultimate size in medium-day animals; however,
under long-day length conditions, food availability was a
major determinant of final size as well as growth rate. Stored
energy was higher with higher food availability in both
groups.

Food consumption and fecundity

At each level of food availability, the fecundity of LD
animals was higher than that of MD animals. Also, when
food was present on two out of three days, fewer eggs were
laid on the day when no food was present. However, when

116

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Figure 1. Dry weight densities under LD (Long Day, 16:8 h light:
dark) and MD (Medium Day, 12:12 h light:dark) conditions in the
laboratory and in the field. A. Laboratory snails. Data are shown for
animals that received food on all days of the schedule. LD animals
had lower dry weight densities than did MD animals (P < 0.001;
Student’s r-test). B. Dry weight densities of wild-caught snails. Dur-
ing the year, a minimum was reached during summer.

food was provided on one day out of three, egg production
was ec]ual (MD) or lower (LD) than on the days the animals
went without food. Table 2 provides the data on consump-
tion and oviposition on each of the three days of the food
cycle. The pattern of egg production was established during
the first three-day cycle and persisted throughout the experi-
ment. In the MD experiment, 19, 17, and 10 snails of group
1, 2, and 3, respectively, produced at least 1 egg mass. In the
LD experiment, all of the snails produced egg masses. The
average interval between oviposition bouts depended on
food availability. In LD animals, the mean intervals were
2.13 (SD = 0.26), 2.41 (SD = 0.36), and 4.18 (SD = 0.89)
days for groups 1, 2, and 3, respectively. In MD animals the
intervals were 9.85 (SD = 9.66), 16.77 (SD = 6.49), and 36.89
(SD = 26.35). Animals that did not lay any eggs were ex-
cluded from the analyses; this occurred only in the MD
group as follows: group I, ii - 1 snail; group 2: n = 2; group
3: n = 10. The differences between food regimes were sig-
nificant in both LD (Kruskal-Wallis, H = 31.6, df = 2, P <
0.0001 ) and MD conditions (H = 13.7, df = 2, P < 0.001 ). LD
animals at all three levels of food availability laid more eggs
and egg masses than their MD counterparts. These results
are in keeping with earlier studies on the effects of daylength
(Bohlken and Joosse 1982).

Egg masses shown in Table 2 for a certain day were
collected at the end of that day of the food cycle. Snails that
were fed every day {i.e., group 1) showed no preference tor
any day of the food cycle to oviposit (MD: Chi-st]uare, x“ =
1.46, P > 0.01; LD = 0.536, P > 0.01). Snails of group 2
laid the fewest egg masses on the day they were starved.
However, MD snails laid the most egg masses on the second
day of the food cycle (x^ = 45.2, P < 0.01 ), whereas LD snails

Table 2. Lettuce consumption, egg mass, and egg production on
each day of the feeding protocol. Data for MD (Medium Day) and
LD (Long Day) animals. Means and standard deviations are given.
There were three feeding schedules: 1, food every day; 2, food on
days 1 and 2; 3, food on day 1 only. The data from the days the
animals went without food are in italics.

Lettuce consumption (cm“ per day per animal)

Day

length

Food

regimen

Day 1

Day 2

Day 3

MD

1

14.94 (3.27)

14.50 (3.45)

13.79 (3.39)

2

23.71 (3.44)

17.86 (3.10)

0.00 (0.00)

3

21.12 (4.09)

0.00 (0.00)

0.00 (0.00)

LD

1

16.35 (3.20)

16.14 (4.06)

16.09 (2.58)

2

22.99 (5.18)

18.07 (5.02)

0.00 (0.00)

3

14.47 (2.76)

0.00 (0.00)

0.00 (0.00)

Eggs per day per animal

Day

Food

length

regimen

Day 1

Day 2

Day 3

MD

1

13.62 (7.79)

15.39 (11.42)

16.31 (12.53)

2

10.51 (9.35)

17.23 (12.31)

4.33 (5.99)

3

1.53 (2.65)

2.59 (3.86)

1.42 (3.17)

LD

1

41.29 (27.14)

37.77 (17.61)

38.86 (18.50)

2

36.79 (14.75)

27.05 (11.8)

7.49 (6.85)

3

8.82 (6.53)

15.16(8.07)

15.50 (8.94)

Egg masses per day per animal

Day

Food

length

regimen

Day 1

Day 2

Day 3

MD

1

0.15 (0.08)

0.17 (0.10)

0.18 (0.12)

2

0.11 (0.09)

0.18 (0.11)

0.04 (0.05)

3

0.02 (0.03)

0.03 (0.04)

0.01 (0.03)

LD

1

0.50 (0.22)

0.47 (0.14)

0.46 (0.21)

2

0.59 (0.17)

0.49 (0.18)

0.19(0.13)

3

0.18 (0.15)

0.27 (0.13)

0.30 (0.15)

laid the most egg masses on the first day (x^ = 57.1, P <
0.01 ). No preference could be demonstrated for MD snails of
group 3 (x" = 1.75, P < 0.01), probably due to the small
number of egg masses that were laid. In LD group 3 snails,
most egg masses were laid on the days they were starved
(X“ = 9.66, P < 0.01).

Because the data in Table 2 are not independent, the
results should be interpreted with caution. Nevertheless, we
think that the differences between the groups can be attrib-
uted to the food availability per se. In both groups that were
fed every day there was not even a slight indication of a
periodicity, whereas in all other groups the periodicity was
pronounced, with the exception of MD group 3, where very
few egg masses were laid. The patterns of egg laying during
the three-day cycle did not change during the experiment.

ENERGY BUDGET OF THE POND SNAIL

117

I'he relationship between egg production and consump-
tion is shown (Fig 2). In groups 1 and 2, the slope of the fit
was 2.247 and 2.211 eggs X cm" lettuce. In group 3, many
animals did not lay eggs and there was no correlation be-
tween consumption and egg production. Combining all
three groups yielded a slope of 1.419. In ED animals, all of
which laid eggs, the slope of overall fit was 2.242 eggs per
cm" lettuce. We conclude that an egg yield of about 2.2 eggs
per cm^ lettuce is a reasonable estim,ate.

Dry weight density in the field

Adults were present throughout the year and two gen-
erations partially overlap during the spring. Dry- weight den-
sities were determined for 283 unparasitized specimens of
Lymnaea stagnalis collected over the course of the year (Fig.
1). Dry- weight density varied during the year and was lower
in summer, the season when most eggs are produced. The
overall level of dry-weight density was lower in the field than
in the laboratory. This was true for both long and short-day
length conditions.

DISCUSSION

Food consumption

After starvation, individuals of Lymnaea stagnalis had
higher consumption rates than snails that were fed continu-
ously. Thus snails of group 2 appeared able to compensate

Figure 2. Relationship between food consumption and egg pro-
duction in snails reared under LD (Long Day, circles) and MD
(Medium Day, squares) conditions with the three feeding cycles.
White fill, regimen 3; intermediate fill, regimen 2; dark fill, regimen
1. The lines are fitted to all the data of the MD and LD groups,
respectively.

for the day they were starved. This explains the relatively
slight differences in growth and reproduction.

Growth

Bohlken and loosse ( 1982) also studied the effects of day
length on growth and reproduction in Lymnaea stagnalis.
They reared a few hundred snails in one large tank under the
same LD and MD conditions we used. We fitted growth
curves to the data of Bohlken and loosse ( 1982), yielding the
following parameter estimates: for MD snails, h^ = 3.54 cm,
g = 0.014 d^’; for LD snails, h^ = 2.97 cm, g = 0.017 d^\ The
estimate for the growth rate parameter of MD snails corre-
sponds to the one we determined for group 3 snails. The
ultimate shell height for MD snails is ec]ual to the one we
found in the present experiment for the three groups; for LD
snails it is between that of groups 2 and 3. This comparison
suggests that in the experiment of Bohlken and Joosse
( 1982), food consumption was as limited as in our group 3.
Data on the growth of snails that were kept individually and
fed a limited amount of lettuce are provided by Geraerts
(1976) and Bohlken et al. (1984). Growth curves were fitted
to their data on control snails. In both cases, values of the
growth rate parameter are in agreement with those found in
the present study for the best-fed snails (0.04 < growth rate
< 0.06). Also, they were much higher than those reported by
Bohlken and Joosse (1982). Apparently, snails kept in isola-
tion have much better feeding conditions than do snails kept
in large groups.

We found no differences between the ultimate shell
heights of groups 1, 2, and 3 of MD snails. Thus the main-
tenance costs in these groups should eventually be identical.
Because the rate of food intake was not identical, the less fed
groups must allocate less energy to reproduction. Indeed,
MD snails of group 2 produced fewer eggs than those of
group 1, while the size differences between the snails were
small throughout the experiment.

According to Kooijman (1993), the Von Bertalanffy
growth coefficient decreases with increasing maximum stor-
age capacity, because the animal has to build up the energy
stores. The larger these stores, the longer it takes to build
them up. LD snails had lower dry-weight densities than MD
snails, so in all likelihood LD snails had less stored energy. In
accordance with this prediction, LD snails had the higher
Von Bertalanffy growth rates.

Input from the tentacles on the LGC, which produce a
growth hormone related to insulin, may provide one way in
which environmental factors could influence growth (Rou-
bos and Van der Wal-Divendal 1982, Smit et al. 1988).

Timing of oviposition in relation to the food cycle

In both MD and LD snails, the timing of oviposition
seemed to depend on the availability of food. However, MD

118

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

and LD snails reacted differently to the presence or absence
of food. Both LD and MD snails suppressed egg laying when
food was absent. Under LD conditions, egg-laying of group

2 was maximal on the first day after starvation. Under MD
conditions, egg-laying in group 2 was maximal on the sec-
ond day after starvation. A comparison with group 3 is not
feasible because so few snails laid eggs. However, the fact
that in LD snails in group 3 egg-laying was maximal on the
second day of starvation is remarkable. Because snails of
group 3 were starved for two days, the reduced egg laying on
the day that the snails were fed might reflect a time-budget
problem: the snails had no time to oviposit since they were
busy feeding. However, this suggestion is not supported by
the following observation. Snails of group 2 laid most
egg masses on one of the days they were fed, whereas LD
snails of group 3 laid most egg masses on one of the days
they were starved. Yet the food-intake rates of groups 2 and

3 were equal. Hence we conclude that there was no time-
budget conflict between egg-laying and feeding during the
experiment.

A number of factors are known to affect the excitability
of the egg-laying, hormone-producing CDC’s, and hence egg
laying. A discharge of this cell cluster is an all-or-nothing
phenomenon; between discharges, the CDC remain electri-
cally silent. One trigger for egg laying is a transfer from dirty
to clean water (Clean Water Stimulus: Ter Maat et al. 1983).
The present experiments suggest that food availability also
affects the excitability of the CDC’s, although indirectly,
which is in agreement with electrophysiological findings
(Ter Maat et al. 1982). Reduced food availability does not
affect the size of the egg mass but does cause an increase of
the interval between the deposition of successive egg masses.
Before oviposition, energy allocated to reproduction is
stored in various glands, including the albumen gland. The
fact that both day length and food availability affect the
reproductive rate but not the size of an egg mass suggests
that oviposition is likely to occur if the gland’s contents pass
a certain threshold. This suggests that the filling of the al-
bumen gland (and/or other glands) influences the excitabil-
ity of the CDC’s, possibly via the activation of stretch recep-
tors. This idea is supported by the finding that in Lymnaea
stagnalis albumen glands are heavier when the animals go
longer without egg-laying (Koene and Ter Maat 2004). In
contrast, in the garden snail Helix aspersa (Muller, 1774) the
number of ripe oocytes in the ovotestis provides a permissive
signal for the occurrence of egg-laying (Antkowiak and
Chase 2003). We think that in I. stagnalis, storage of pack-
aging material for the eggs is the critical factor in egg laying.
Given these findings, it would be interesting to study
whether egg-laying and the excitability of the CDCs depends
on sensory signals from the albumen gland or from other
accessoiy organs containing packaging material.

Comparison of MD and LD snails

Average somatic and reproductive production rate was a
function of the average rate of food intake. Although such
relationships are difficult to interpret, there was a clear dif-
ference between MD and LD snails. With regard to somatic
production, however, there is no difference. There was a very
clear difference in the reproductive output of MD and LD
snails. At the same average food-intake rate, LD snails pro-
duced about 20 eggs per day more than MD snails. Quali-
tatively, this agrees with the observation that the daily egg
production rate in LD snails was about 2 to 3 times that of
MD snails.

If somatic production was more or less the same in LD
and MD snails, how were LD snails able to maintain a much
higher reproductive rate? LD snails had lower dry weights
and therefore probably fewer energy reserves. The difference
in dry weight acquired during the experiment for MD and
LD snails, as calculated from the dry weight density and the
volume increase, was about 0.12 g. The dry weight of eggs,
including the capsule in which the eggs are embedded, is
about 0.15 mg per egg (Zonneveld and Kooijman 1989).
Hence the difference in acquired dry weight is equivalent to
about 800 eggs. The experiment lasted for approximately 60
days, during which LD snails produced on the average 20
eggs per day more than did MD snails (1200 eggs more
during the experiment). The difference in dry weight ex-
plains about 70% of this difference in egg production. Thus,
we conclude that the difference in the rate of egg production
was largely due to a decrease in energy reserves of LD snails
compared to MD snails.

In the field, Lymnaea stagnalis is essentially an annual
species, breeding in the summer season. Light conditions
similar to our MD treatment are experienced in autumn and
spring, during the juvenile period. It is advantageous to have
large energy stores during this period, because food avail-
ability will be low and unpredictable. Light conditions simi-
lar to our LD treatment are experienced in the field in late
spring and in the summer. Lood availability will be predict-
ably high in this period so there should be no need to have
large energy stores. To maximize the reproductive output,
the energy stores should be depleted. Our experiments were
performed at a constant day length, while in the field, day
length gradually increases to LD conditions with the onset of
the summer. Hemminga et al. (1985) showed that snails
indeed draw on their energy reserves after a change from a
short to a long day length.

In conclusion, the current study shows that the high rate
of reproduction under long day conditions can be main-
tained by keeping stores at a low level. In contrast, medium
day animals invest more in storage, and lay eggs only when
energy storage is above a certain level. The data on size and
dry weight density in the field show a similar relationship

ENERGY BUDGET OE THE POND SNAIL

119

with day length as the laboratory data, in that long days are
associated with high fecundity and low energy storage. How-
ever, a major difference between field and laboratory data is
that dry weight densities are higher in the laboratory, pre-
sumably due to differences in feeding conditions.

ACKNOWLEDGEMENTS

Garool Popelier, Tim Smelik, Rene Thijssen, and Anton
Pieneman assisted in the practical work. Wim Van der Steen,
Bas Kooijman, and Ger Ernsting provided comments on
draft versions of the manuscript. C. Zonneveld was sup-
ported by the Netherlands Foundation for Biological Re-
search, and J.M. Koene was supported by the Research
Council for Earth and Life Sciences (ALW); both agencies
are financed by the Netherlands Organization for Scientific
Research (NWO).

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Accepted: 9 March 2007

Amer. Maine. Bull. 23: 121-135

Phally polymorphism and reproductive biology in Ariolimax (Ariolimax) buttoni
(Pilsbry and Vanatta, 1896) (Stylommatophora: Arionidae)"^

Janet L. Leonard', Jane A. WestfalP, and John S. Pearse'

' Joseph M. Long Marine Laboratory, University of California-Santa Cruz, Santa Cruz, California, 95060, U.S.A., jlleonar@ucsc.edu
^ Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506-5802, U.S.A.

Abstract: Phally polymorphism, whereby some individuals in a hermaphroditic species have a complete functional penis (euphally),
whereas others lack a penis (aphally) or have a reduced, non-functional penis (hemiphally), has evolved many times in pulmonate
gastropods. Since the discovery of apophallation (penis amputation) in Arioliiiiax Mdrch, 1860, aphallates in this genus have been attributed
to apophallation. In laboratory studies in Ariolimax (Ariolimax) Imttom (Pilsbry and Vanatta, 1896), we found aphally in juveniles as well
as in individuals reared in isolation to adulthood, demonstrating that the aphaliate condition is not always due to apophallation. Some
aphallate individuals reared in isolation from hatching produced eggs and viable hatchlings, providing the first demonstration of uniparental
reproduction in this species. Egg-to-egg generation time in laboratory-reared individuals ranged from 10 months to more than 24 months.
Anatomical data also elucidate the reproductive cycle of this species. Four reproductive states have been identified by the appearance of the
reproductive system. Spring and early summer populations consist of individuals in the immature and intermediate reproductive states. The
hypertrophied state was found from autumn until early spring. Egg-laying occurred in the laboratory in the fall and winter. Copulation
consists of unilateral or simultaneously reciprocal intromissions and occurred in the laboratory between February and September. Very long
copulations (more than 7 h) are more frequent than in other species of Ariolimax. Phally polymorphism, uniparental reproduction, and
the variation in generation time should play important roles in determining the variance of mating success and the potential for sexual
selection in this hermaphroditic species.

Key words: gastropod, genitalia, aphally, life history, self-fertilization

Banana slugs, giant stylommatophoran slugs of the ge-
nus Ariolimax Morch, 1860, are common and conspicuous
members of temperate rain forests and other mesic habitats
of the northwestern coast of North America. Although ba-
nana slugs are well-known and popular with the general
public, remarkably little is known about their biology (but
see Harper 1988, Leonard et al. 2002, Cody 2006, Pearson et
al. 2006). In Ariolimax, as in stylommatophorans in general,
taxonomy has been based on genital characters (Pilsbry
1948). Eberhard (1985) suggested that where genital char-
acters have evolved rapidly enough to distinguish species
(and even subspecies), sexual selection has played an impor-
tant role in the evolution of the group. The genus Ariolimax
offers a particularly good opportunity to test this hypothesis
since it consists of a small number of taxa (Rotb and Sade-
ghian 2003, Pearse et al. 2007), most of which are found in
coastal Central California in c]uite similar habitats, but
which have very divergent genitalia and sexual behavior
(Leonard et al. 2002, 2005). Accurate information about the
reproductive biology of Ariolimax spp. is necessary to per-
form such a test.

One of the most mysterious aspects of Ariolimax biology
is the existence of aphallate individuals and the extent to
which these can be explained by apophallation. Apophalla-
tion is a behavior first observed in Ariolimax (Meadarioii)
californicus (Cooper, 1872) (Heath 1916, Leonard et al.
2002) and subsequently in Ariolimax (Meadarioii) dolicho-
phalhts Mead, 1943 (Mead 1942, 1943, Harper 1988, Leo-
nard et al. 2002) whereby the penis is sometimes chewed oft
at the end of copulation in these simultaneous hermaphro-
dites. Since Heath (1916) most authors have been content to
explain aphallate individuals of Ariolimax as the product of
apophallation. However, Heath himself expressed doubt,
saying, “years ago I visited Hog Island in Tomales Bay, and
found over 400 specimens ... every one of the specimens was
totally lacking a penis or any sign of one.” (quoted in Mead,
1943, p. 685). Mead (1943) later collected in the same area,
finding no slugs at Hog Island and an entirely phallate popu-
lation nearby. Pauli (1951) examined the gross anatomy of
Ariolimax buttoni (Pilsbry and Vanatta, 1896) (= A. colum-
bianus (Gould in Binny, 1951 ), see below) at Mills College in
Oakland, California and found that of the 27 adult speci-

From the symposium “Gastropod Mating Systems” presented at the joint meeting of the American Malacological Society and Western
Society of Malacologists, held 26-30 June 2005 at Asilomar, Pacific Grove, California.

121

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

mens she dissected, none had a penis. More recently, Roth
(2004) reviewed anatomical descriptions of ArioUmax in de-
tail, and suggested that the descriptions are inconsistent with
what would be expected if a penis were amputated during
copulation. He argued that apophallation should sever both
the penis and vas deferens, leaving unconnected remnants of
both and an intact penis retractor muscle, whereas Pilsbry
and Vanatta’s ( 1896) drawing of Aphallariou Imttoni showed
an intact vas deferens connected to the genital atrium by a
short bulb with no penis and no penis retractor muscle. Roth
(2004) concluded that the descriptions were more consistent
with a phally polymorphism. Such phally polymorphisms
are well-known and widespread in pulmonates, including
stylommatophorans (Tompa 1984, Pokryszko 1987, Baur
and Chen 1993, Doums and Jarne 1996, Viard et al. 1997,
Doums et nl. 1998, Gomez 2001), and there are even in-
stances of a phally polymorphism having lead to an errone-
ous splitting ot a single species into two genera (e.g., Lace
1992).

Here, we present previously unpublished observations
on aphallate and euphallate individuals in two populations
of ArioUmax (ArioUmax) buttoni and data showing aphally in
individuals which cannot have been involved in apophalla-
tion because they were reared in isolation to adulthood.
These observations demonstrate that this species has a phally
polymorphism, with both aphallate and euphallate individu-
als in some populations. We also present laboratory obser-
vations on the sexual behavior and life cycle of A. buttoui
and document uniparental reproduction in this species.

MATERIALS AND METHODS

Taxonomy

The animals used in this study are ArioUmax (ArioU-
max) buttoni. This species was first described by Pilsbry and
Vanatta (1896) from a large series of aphallic ariolimacines
from Oakland, California as a new genus. Subsequently, this
taxon was synonymized to ArioUmax (ArioUmax) coUimbi-
anus (Gould in Binny, 1951 ) (Waste 1940, Mead 1943, Pils-
bry 1948) in the expectation that the aphally could be at-
tributed to apophallation during copulation. This species
was considered to be the only ariolimacine to include macu-
late individuals and was stated to have a range extending
from Tuolomne County, Monterey County, the eastern
shore of San Francisco Bay, and the city of San Francisco in
California, north to southeastern Alaska (Pilsbry 1948, Roth
and Sadeghian 2003). Recent molecular evidence suggests
that A. columbiauus, as defined by Mead (1943) and Pilsbry
(1948), is not monophyletic but rather that populations of
ArioUmax north of Mendocino County, California (the true
A. coUimbiamis, since the species was described from speci-
mens collected near the Columbia River, see discussion in

Pilsbry 1948) are evolutionarily distinct from the more
southern populations (Leonard et al. 2005, Pearse et al.
2005). The name ArioUmax buttoni (Pilsbry & Vanatta,
1896) has been revived to designate the southern clades for-
merly included in A. columbiamis (Pearse et al. 2007). Ario-
Umax buttoui and A. columbiamis do not even represent
sister clades (Pearse et al. 2007). Like A. columbiamis, A.
buttoui may be either maculate or immaculate. ArioUmax
(Meadariou) brachyphalliis Mead, 1943 is sympatric with A.
buttoui in San Francisco but all individuals used in this study
from San Francisco have been identified by molecular mark-
ers as belonging to A. buttoui. There are no reports of sym-
patric ariolimacines in Alameda, Sacramento, Mendocino,
or Marin counties.

Animals

Mills College animals

Specimens were collected from March 1951 to February
1952 in two locations; the Mills College campus (37° 46’
41”N, 122° 10’ 49”W) and the nearby Leona Heights Park
(37° 47’ 31”N, 122° 10’ 41”W) in Oakland, Alameda County,
California. Approximately 24 animals from each location
were housed in the laboratory in terraria filled with damp
earth and fed with lettuce. Other animals were dissected
shortly after collection. j

UCSC animals ■'

Specimens were collected from five locations in Central j
California: (i) a wooded area of Mount Parnassus on the [
campus of the University of California, San Francisco, San !j
Francisco County (37°45’ 38”N; 122° 27’ 28”W); (ii) a levee I
of Staten Island in the Cosumnes River, Sacramento County |
(38° 15’ 56”N, 121° 26’ 31”W); (hi) near Comptche, Men-
docino County (39° 15’ 54” N, 122° 35’ 24” W); and two |
locations in Marin County, namely (iv) near Muir Woods in fi
central Marin County (37° 53’ 07”N, 122° 32’ 26”W) and (v) |j
the west side of Tomales Bay (38° 10’ 25”N, 122° 55’ 26”W) j
in western Marin County. Animals were maintained in the
laboratory as groups or individuals in plastic boxes as de-
scribed elsewhere (Leonard et al. 2002). I

Anatomical studies

Mills College animals

A total of 67 individuals of ArioUmax buttoui, ranging
from 16 to 55 grams in weight, were dissected. Three slugs |
were dissected at the beginning and middle of each month
from March to May 1951. Five slugs were dissected every
two weeks from October 1951 to February 1952. Two of the |
slugs were taken from the animals kept in terraria whereas '
the other three were collected from the field. The day before
dissection, the slugs were weighed and the next day they j
were drowned, dried to remove excess mucus, pinned to a |

PHALLY POLYMORPHISM IN ARIOLIMAX BUTTONI

123

dissecting tray and dissected in water. A longitudinal inci-
sion was made on the right side from the caudal pore to the
mouth, the dorsal skin retracted, and the genitalia freed by
severing connective tissue and the protractor muscles of fhe
tentacles. After observation of the intact condition, the skin
was cut around the atrium and the reproductive system was
laid out separately for measurement. The data recorded
were: a) the color and degree of development of the repro-
ductive system; b) the position of the vas deferens and the
presence or absence of a penis and/or terminal bulb on the
vas deferens; c) the approx, width of the vagina and oviduct;
d) the approx, length and wicith of the spermatheca; e) the
approx, length and width of the ovotestis; and f) the approx,
length of the albumen gland.

UCSC Animals

Anatomical studies were conducted with Ariolimax but-
toni derived from two populations (Staten Island and To-
males Bay) and reared as described by Leonard et al. (2002).
Three of the individuals were collected from the field and
then maintained in the laboratory until their deaths, at
which time they were frozen. The other individuals were
hatched in the laboratory and frozen when slightly over 30
months of age. They were thawed just before dissection,
pinned to a dissecting tray, and dissected under water. An
incision was made through the body wall with scissors along
the left side, just above the foot, and the dorsal body wall
peeled back to expose the digestive system and genital or-
gans, and the albumen gland, ovotestes, and male and female
parts of the genital organs teased apart. The condition of the
spermatotheca and the presence or absence of the penis was
noted. If absent, the presence or absence of a penis stub was
noted as well as the course of the vas deferens. The male and
female portions of the genital organs were sketched for most
specimens; in a few cases they were measured and photo-
graphed through the dissecting microscope, using an
Olympus Camedia C-3040 digital camera. Upon comple-
tion, the dissected animals were preserved and archived in
10% formalin.

Rearing studies

Two sets of rearing studies have been conducted with
Ariolimax buttoni. The first series was conducted at Mills
College (Mills College animals) from 1951 to 1952 (Westfall
1960). Eggs and juveniles were maintained in terraria filled
with damp earth along with the parents. They were fed let-
tuce leaves. In total 17 juveniles ranging in age from 1 day to
19 weeks were taken for anatomical study.

The second rearing study was conducted at the Long
Marine Laboratory of the University of California-Santa
Cruz (UCSC animals) from 2003 to 2006. Slugs were col-
lected from Staten Island and Tomales Bay and held in

group boxes. Eggs laid in tall and early winter 2003-2004
were maintained as described previously (Leonard et al.
2002) and hatchlings weighed when found and transferred
to individual plastic containers. All containers were cleaned
and lettuce added weekly. After the juveniles reached 5 g in
weight, dry cat food was also given. Slugs were transferred to
larger containers as necessary as they grew. They were
weighed weekly until 6 mo and biweekly thereafter.

All hatchlings were reared in isolation until lime 2004
when, as part of a mating study (Leonard, et al. unpubl.
obs.), 40 individuals from the Staten Island group and 40
from the Tomales Bay group were randomly assigned to a
mating treatment. Eight individuals, each from Staten Island
and Tomales Bay parents, were assigned to the single animal
(continued isolation) treatment. Equal numbers of individu-
als from Tomales Bay and Staten Island parents were also
assigned to each of four other rearing conditions: a) paired
with an individual from the same source population; b)
paired with an individual from the other source population;
c) in a group box with three others from the same source
population; or d) in a group box with one individual from
the same source population and two from the other source
population. All of the individuals derived from the Staten
Island population were spotted whereas all of the Tomales
Bay descendants were immaculate. Redwood bark mulch
was added to each box to encourage egg-laying. Slugs were
weighed biweekly and cleaned and fed weekly (as in Leonard
et al. 2002), so that all boxes were checked for eggs at least
weekly. Clutches found were treated as described above.
Hatchlings were weighed when found and preserved in 95%
ethanol for future genetic analysis.

Behavioral observations

All observations on sexual behavior reported here for
Ariolimax biittotii (see Table 3) resulted from casual obser-
vations of UCSC animals held in group boxes. Where ani-
mals were observed to be copulating when the box was
opened for cleaning, notes were made and/or the behavior
was videotaped as time allowed. Where possible, observa-
tions were continued for 30 min after the end of the copu-
lation. The copulation of Mendocino individuals on April
10, 2001 was noted but not followed for any length of time.

RESULTS

Phally status

Mills College animals

Only 7 of the 67 Mills College slugs (4 of the 55 slugs
collected on the Mills College campus, and 3 of the 12 slugs
collected at Leona Park), were found to have a penis and, of

124

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

those, one only had a penis fragment (Table 1 ). In 59 of the
60 individuals lacking a penis, the vas deferens ended
abruptly in the atrial mesentery (fig. 1). In 20 of these 59
individuals, there was a small bulb at the end of the vas
deferens as described by Pilsbry and Vanatta (1896). The
apical termination of the vas deferens in the individual with
a fragment of a penis was on the penis fragment. This speci-
men was in the immature reproductive state (see below).
None of the 17 Mills College juveniles hatched in the labo-
ratory had a penis.

UCSC atiimals

A total of 16 individuals were dissected (Table 2). All of
the individuals derived from Tomales Bay populations and
the individuals collected from UCSf and Central Marin had
a fully developed penis with an apical retractor muscle and
a long vas deferens that terminated in the penis (Eig. 2B).
None of the 10 individuals derived from the Staten Island
population had a penis (fig. 2A), including two individuals
that spent their entire lives in isolation. Of these 10 indi-
viduals, 5 had a small stub where the penis would be in a
euphallate individual (fig. 2 A) whereas the remaining 5
lacked any trace of a penis (fig. 1). All 10 individuals lacked
a penial retractor muscle and 9/10 had a blind termination
of the vas deferens. In one individual the vas deferens ended
in a bulb as was seen in many of the Mills College animals
(Table 1). The vas deferens connected to the base of the
penial stub in one individual.

Reproductive development

Mills College aiiii?iah

Measurements and appearance of the individual organs
of the reproductive tract for the 67 dissected adult Mills
College slugs are given in Westfall ( 1952). On the basis of the
color and appearance of the organs and the length of the
albumen gland, four reproductive states were identified: 1)

immature, 2) intermediate (or “sperm producing”), 3) hy- i
pertrophied (or “egg-laying”), and 4) “old” (Appendix). The
reproductive state in the dissected slugs varied with season ' j
(fig. 3). In the spring most (10/12) individuals were in the
intermediate state (fig. 3) with an albumen gland of small to !

intermediate size. In contrast, in the fall and winter there was 7
a clear division into two groups of individuals: those in the ^ •
immature state with a very small albumen gland and those in !i
the hypertrophied state with a very large albumen gland. |!
Slugs collected in October and kept in captivity were also i’
found to be more often in the hypertrophied state as the date
of dissection moved from November to february. -j.

There was no relationship between season or reproduc- i '
tive state and the presence of a penis, four of the individuals : |
with a penis were in the immature reproductive state, one in
the intermediate state, and two others in the hypertrophied |
state (Appendix; fig. 3). In addition, there was no relation- j
ship between body weight and the presence of a penis; the
second smallest and third largest animals dissected had a
penis (fig. 4). Each reproductive state included individuals
with a broad range of weights although the smallest animals
were in the immature state and the largest in the hypertro-
phied state (fig. 4).

The reproductive tract was not distinguishable in juve-
niles for two weeks after hatching. After two weeks, the
organs of the reproductive system were in the immature .
state with a hair-like hermaphroditic duct, leading from a ■'
small, white, smooth, lobed ovotestis and a small albumen
gland. After a short distance this duct divided into a free
oviduct and a vas deferens. These ducts were straight and
hair-like in younger specimens but began to show more
coiling in older specimens. In older juveniles the vagina and
vas deferens terminated jointly in the atrium (as in fig. 1)
but there was no indication of a penis even at 19 weeks of age
when other reproductive organs were well developed. Juve-
nile slugs of 15-19 weeks of age weighed between 3 and

Table 1. Summary of morphological data on adult Arioliinax biittoni from Mills College in Oakland, California

Phally status

Number
of slugs

Weight
(mean ± SD)

Number in
immature state*

Number in
intermediate state*

Number in

hypertrophied

state*

Number with state unclear

Penis present

6

32.12 1 129 g

3

0

2

1 (state questionable)

Penis fragment

1

37.5 g

1

0

0

No penis but bulb on

20

32.4 1 9.08 g

2

9

8

1 (state questionable)

end of vas deferens
No penis and no bulb

39

29.3 ± 5.62 g

18

13

8

Unknown

Weight (mean ± SD)
Total

1

67

24.5 g

28.42 ± 6.61 g
24

30.49 ± 6.27 g
22

33.31 ± 9.52 g
18

( insufficient information )
32.53 ± 9.91 g
3

* See Appendix for definition of reproductive states.

PHALLY POLYMORPHISM IN ARIOLIMAX BUTTONI

125

Albumen gland

Ovotestis

Hermaphroditic duct with spermatozoa
Ovisperm duct with eggs

Vas deferens

Spermatheca
Free oviduct

Vagina

Genital orifice

Figure 1. Drawing of a dissected aphallate individual ot Ariolimax
biittoni which had been observed to lay eggs before dissection;
hypertrophied reproductive state (see Appendix) with sperm
present in the hermaphroditic duct and the oviduct distended with
eggs. The hermaphroditic duct proceeds from the ovotestis (=her-
maphroditic gland = gonad) carrying both eggs and sperm. After
the albumen gland, the term ovisperm duct is used for a convoluted
conjoined vas deferens and oviduct. After the vas deferens branches
off (to the penis in euphallate specimens, see Figure 2B) to end near
the atrium here, the ovisperm duct continues as the free oviduct to
the point of attachment of the spermatheca (=bursa copulatrix =
gametolytic gland) with the vagina. The free oviduct is usually a
wide, convoluted duct while the vagina is a short, straight tube
from the spermatheca to the atrium, and is divided into two por-
tions by a small, thick, annular muscle (intrinsic muscle of the
vagina).

6.3 g (Westfall 1960). The juvenile slugs all showed the
immature reproductive state (Appendix), except that the
very first juvenile examined, dissected at four weeks of age
(representative weight, 0.7 g, Westfall 1960), was found to
have a relatively large, loosely lobed, ovotestis. The reason
for the different appearance of this one individual is not
known.

UCSC animals

The spermatheca was reddish in most individuals from
Staten Island. All Tomales Bay animals had a small sper-
matheca and in 3 of the 4, it was pale in color. While all of
the isolated, dissected animals from Staten Island (3/8), laid
one or more clutches of eggs, none of the isolated Tomales
Bay animals laid eggs (see below).

Egg-laying

Results on egg-laying and hatching in the Mills College
animals have been published elsewhere (Westfall 1960). In
UCSC animals, egg-laying by animals hatched in the fall-
winter of 2003-2004 from Staten Island and Tomales Bay
parents, began in October 2004 and continued until late
February 2005. Egg-laying then paused for this group of
slugs until mid-August 2005 when it resumed and continued
until late February 2006. However, only in one box of four
individuals did egg production occur in both seasons (2004-
2005 and 2005-2006).

Three individuals from Staten Island parents, isolated
since hatching, laid eggs (only two were dissected and listed
in Table 2). One individual, from a clutch of 13 November
2003, produced three clutches for a total ot 89 eggs: 32 eggs
on 13 December 2004 (Clutch lA), 13 eggs on 20 December-
2004 (Clutch IB), and 45 eggs on 9 February 2005 (Ckrtch
1C). This individual was found dead on 4 August 2005. All
three clutches developed normally and produced a total of
45 viable hatchlings. Clutch lA produced 21 hatchlings be-
tween 31 lanuary and 7 Febrrrary 2005. Clutch IB produced
6 hatchlings from three eggs between 3 and 9 February 2005:
one egg produced a single hatchling, one produced twins,
and one produced triplets. The production of more than one
hatchling fror-n an egg has also been seen in other species of
Ariolimax (Leonard et ai, unpubl. data; B. Miller, pers.
coirrm.). Clutch IC produced 18 hatchlings 24-28 March
2005.

The second individual (from a clutch found 28 Novem-
ber 2003) laid a total of 120 eggs, 101 which hatched: a) 28
eggs found 12 October 2005, 16 of which hatched; b) 17 eggs
found 18 November 2005, 18 of which hatched, including
one set of twins; c) 56 eggs found 8 December 2005, 50 ot
which hatched; and d) 19 eggs found 17 February 2006, 17
of which hatched. The third individual, from a clutch of 13
November 2003 [a clutch mate of the first individual
(above)], was found with a clutch of 80 eggs on 8 December
2005, which produced 66 hatchlings, and a clutch of 56 eggs
on 19 lanuary 2006 which pr oduced 55 hatchlings. The latter-
two individuals sui-vived until frozen on 22 June 2006, and
were found to be aphallate when dissected (Table 2).

A box of four individuals, two from Staten Island par-
ents and two fror-n Tomales Bay parents produced a clutch of
17 eggs on 14 October 2004. The oldest individual in the box

126

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Table 2. Summary of dissections of Ariolimax biittoni held at UCSC at Santa Cruz, California

Population

Isolate/Group-held/Collected?

Penis

Vas deferens

Spermatheca

Egg laying

Central Marin

Collected as adult

Very large bulbous

Connects to penis

Never laid eggs in
lab

UCSF

Collected as adult

Very thick

Connects to penis

Sac-like

Never laid eggs in
lab

Staten Island

Collected as adult

No trace of penis

Blind termination

Reddish, flaccid

Never laid eggs in
lab

Laid fertile eggs

Staten Island

Reared in isolation throughout
life

Small penial stub

Blind termination

Reddish

Staten Island

Reared in isolation throughout
life

Small penial stub

Blind termination

Reddish, sac-like

Laid fertile eggs

Staten Island

Laboratory reared, paired at 6
months of age

Splayed penial stub

Blind termination

Dark red

3 clutches of fertile
eggs from pair

Staten Island

Laboratory reared, paired at 6
months of age

No trace of penis

Ends in genital pore

3 clutches of fertile
eggs from pair

Staten Island

Laboratory reared, paired at 6
months of age

No trace of penis

Connects to base of
penial stub near
atrium

Red, flaccid

Fertile eggs laid by
pair

Staten Island

Laboratoiy reared, grouped at
6 months of age

No trace of penis;
1 cm of vagina
everted

Blind termination

Large

Several clutches of
fertile eggs laid
by group

Staten Island

Laboratory reared, grouped at
6 months of age

Small penial bulb

Blind termination

Dark reddish

Several clutches of
fertile eggs laid
by group

Staten Island

Laboratory reared, grouped at
6 months of age

No trace of penis

Ends in bulb

Several clutches of
fertile eggs laid
by group

Staten Island

Laboratory reared, grouped at
6 months of age

No trace of penis;
0.5 cm of genital
pore everted

Blind termination

Large, flaccid, red

Several clutches of
fertile eggs laid
by group

Tomales Bay

Reared in isolation throughout
life

Penis present

Connects to penis

Flabby, small

Never laid eggs

Tomales Bay

Reared in isolation throughout
life

Penis present

Connects to penis

Reddish, moderately
small

Never laid eggs

Tomales Bay

Reared in isolation throughout
life

Penis present

Connects to penis

Small, pale

Never laid eggs

Tomales Bay

Laboratory reared, paired at 6
months ot age

Penis present

Connects to penis

Small, creamy, firm

Never laid eggs

came from a clutch of 13 November 2004, so the parent was
no more than 1 1 months old at the time of egg-laying. This
clutch produced 3 hatchlings. On 1 1 November 2004, a
clutch of 29 eggs was found in a box containing a pair of
individuals, both from Tomales Bay parents, one from a
clutch found 30 December 2003 and the other from a clutch
found 2 January 2004. This means that the individual laying
the eggs could not have been older than 10.5 months from
the date the egg was laid. Eourteen of the 29 eggs in this
clutch hatched. A second clutch of 12 eggs found in this box
on 13 December 2004 produced 6 hatchlings. On 24 No-
vember 2004 the Staten Island (spotted) individual of a pair
of one Staten Island (spotted) and one Tomales Bay (im-

maculate) slug, was found laying eggs. A total of 54 eggs were
found in the box at that time, although it is not clear that
they were all laid by the spotted mother. Lorty-one
hatchlings were produced from this clutch. The spotted slug
was hatched from a clutch found 16 January 2004 and so was
approx. 10 months and eight days in age (from the egg).
Nine more eggs were found in this box on 24 November
2004 and this clutch produced 2 hatchlings.

Sexual Behavior

Little is known about sexual behavior in Ariolimax hiit-
toni. To date, we have never observed a complete sexual
interaction from courtship to the termination of copulation

PHALLY POLYMORPHISM IN ARIOLIMAX BUTTONI

127

Figure 2. Photographs of dissections of
aphallate (A) and euphallate (B) individu-
als of AriaUmax buttoiii. Individual A was
laboratory reared from Staten Island par-
ents. Individual B was laboratory reared
from Tomales Bay parents. AVD, ascend-
ing vas deferens; DVD, descending vas def-
erens; P, penis; PR, penis stub; V, vagina;
location of atrium.

B

E

100
90
80
70
60
50
40
30
20
10
0

3-Feb-51 25-Mar-51 14-May-51 3-lul-51 22-Aug-51 ll-Oct-51 30-Nov-51 19-lan-52 9-Mar-52

■ hyper with penis
□ hypertrophied

♦ interm with penis
0 intermediate

• immature with penis
0 immature

□ ■

□

CD

□ E

□

o ^ ♦

0

ooo

x>

o

>

»

0 8 0 8 0 t °

Figure 3. Relationship between reproduc-
tive state (Appendix), season, and albumen
gland length in 66 individuals Ariolimax
buttoni.

in this species. However, UCSC animals maintained in the
laboratory in groups have occasionally been found in copula.
Ariolimax buttoni, like A. cohimbianiis, and unlike other spe-
cies of Ariolimax, has both spotted (maculate) and unspotted
(immaculate) individuals, and three of the copulating pairs
observed in this study involved both a maculate and an
immaculate individual (Fig. 5; Table 3). Copulations be-
tween maculate and immaculate individuals are also seen in
A. cohimbianus (Cody 2006).

In the laboratory, cop>ulations have been observed from
February through early September. Copulation was observed
on 11 or 12 (see below) occasions involving animals from 5
different populations (Table 3). In one case, a pair of slugs
from Central Marin was found copulating unilaterally at
11:07 pm on 5 September 2002 (by JSP), observed until
12:30 am 6 September 2002, and then left alone overnight.
What was apparently the same pair of slugs, in the same
position, was found copulating when the box was next
checked at 2:30 pm on 6 September 2002 and the pair finally
separated at 10 pm that evening. We cannot say with con-
fidence whether this represents one or two copulations. If we
count it as two copulations, of the 12 copulations that have
been observed, 7 involved simultaneously reciprocal copu-
lation between the members of a pair whereas 5 were uni-

lateral. If the interactions of 5-6 September 2002 are counted
as one copulation, then 7/1 1 copulations observed have been
simultaneously reciprocal. In three cases, the termination of
the copulation was observed and in all of these cases, the
copulation was unilateral when first observed. Of these, one
terminated 62 min after the copulation was noticed, one
terminated 98 min after first observed and one (5-6 Septem-
ber 2002) terminated 7.5 h after observation resumed on the
second day; 22 h and 53 min after the pair was first seen
copulating. Reciprocal copulations may become unilateral
when one penis is withdrawn considerably earlier than the
other (Leonard et al. 2002). The available data (Table 3)
show that copulations in Ariolimax buttoni are often very
long. In 3/12 observations copulation lasted more than 7 h,
and in one case (5-6 September 2002), the interaction may
have lasted almost 23 h. In the unilateral copulation on 5
February 2001, between two immaculate individuals col-
lected from the UCSF campus, when the pair were first seen,
one slug had intromission as a male and the second slug
(acting as female) had its penis completely everted and rest-
ing against the body of its partner. The end ot the penis was
greatly enlarged in the form of a broad bulb. As the second
slug withdrew its penis, the bulb gradually deflated and the
penis involuted from the tip. In the observations of 5-6

128

AMERICAN MALACOLOGICAL BULLETIN 23 - 112 - 2007

100

90

•g 80

E

^ 70
? 60

■ hyper with penis
□ hypertrophied

♦ interm with penis
o intermediate

• immature with penis
o immature

50

-Ri-

V 30

E

I 20

<

10

6>8 0°

® O 0

10

20

30 40

Body Weight (gm)

50

60

Figure 4. Relationship between reproduc-
tive state (Appendix), body weight, and al-
bumen gland length in 66 individuals of
Ariolimax buttoni.

Table 3. Copulations
Population

of Ariolimax buttoni from
Date

1 different populations in
Time first observed

California.

Observations ended

Type of intromission

UCSF

2/5/2001

16:05

17:43

Unilateral

Mendocino

4/10/2001

17:45

17:46

Simultaneously reciprocal

Mendocino

5/3/2001

17:30

20:29

Simultaneously reciprocal

Mendocino

5/17/2001

21:05

22:23

Simultaneously reciprocal

Central Marin

711712002

13:15

15:17

Simultaneously reciprocal

Central Marin

9/5/2002

23:07

24:30

Unilateral, spotted male

Central Marin

9/6/2002

14:30

22:00

Unilateral, spotted male

UCSF

4/8/2003

18:31

26:01

Simultaneously reciprocal

UCSF

5/5/2003

11:52

23:35

Simultaneously reciprocal

Tomales Bay

5/6/2003

16:04

18:28

Unilateral

Tomales Bay

6/19/2003

15:45

16:47

Unilateral

Tomales Bay

8/8/2003

13:44

18:30

Simultaneously reciprocal

September 2002 one slug was seen to contact the penis with
its mouth on several occasions but there was no evidence of
chewing on the penis.

DISCUSSION

Phally Polymorphism

Since Heath’s (1916) description of apophallation in
Ariolimax califomicus, the tendency has been to explain ab-
sence of a penis in this genus in this way (Mead 1942, 1943).
However, Heath himself expressed doubt of this interpreta-
tion (cited in Mead 1943, see above). The current study was
stimulated by the observation that all 27 individuals of An'o-
Iwiax collected on the Mills College campus for an anatomi-
cal study lacked a penis (Pauli 1951). This is a higher inci-
dence of aphally than would be expected from the 5%
apophally rate reported by Heath (1916) for A. califoniiais.
Only 7 of 67 slugs collected from Mills College in the current

study had a penis. In all cases the aphallate slugs were lacking
not only a penis but all penial musculature, and the vas
deferens was connected to tissue at or near the atrium (Lig.
1), terminating in a bulb in many cases, which is not what
one would expect from apophallation (see discussion by
Roth 2004). Further evidence that aphally in this population
is not derived from apophallation during copulation comes
from the observation that none of a series of sexually im-
mature laboratory-reared slugs from Mills College, which
were up to 4 months old, showed any signs of development
of a penis. This led to the hypothesis that aphally occurred
naturally in some individuals of A. buttoni, and that an in-
nate phally polymorphism rather than apophallation was
largely responsible for the presence or absence of a penis in
this and perhaps other species of Ariolimax.

This hypothesis was confirmed by the observation that
in individuals derived from the Staten Island population,
aphally was found even in two individuals that were reared
in isolation from the egg to the age of 30 months and had

PHALLY POLYMORPHISM IN ARIOLIMAX BUTTONI

129

both laid eggs. Aphally in these individuals could not be due
to either apophallation or sexual immaturity. Moreover, the
anatomy of these aphallic isolates was consistent both with
that of other Staten Island individuals that were group-
housed as adults and with that of the Mills College animals.
The aphallates are characterized by either the complete ab-
sence of a penis (Fig. 1 ) or reduction of the penis to a small
stub (Fig. 2A); in both cases, the penial retractor muscle is
absent and the vas deferens ends blindly in a mesentery or in
the neighborhood of the atrium. The description of dissec-
tion of a freshly apophallate A. dolichophallus by Mead
(1942) suggests, however, that the distinctions between
aphally as described in Fig. 1 and 2, and a healed apophallate
individual, are subtle.

The anatomy of Ariolimax biittoiii, as described by Pils-
bry and Vanatta (1896), and the aphallate individuals from
Mills College described here (Fig. 1, also Pauli 1951) and the
Staten Island animals (Fig. 2A), is very consistent with the
descriptions and illustrations of aphallic individuals in other
stylommatophorans (Watson 1934, Riedel 1955, Tompa
1984, Pokryszko 1987, see discussion in Roth 2004). Phally
polymorphism, in which a hermaphroditic population or
species consists of a mixture of individuals with normal pe-
nes (euphallic individuals) and individuals with either mark-
edly reduced penes (hemiphallic individuals) or no penis at
all (aphallic individuals) is widespread in the Pulmonata and
has evolved many times (see discussion in Duncan 1975,
Tompa 1984, Pokryszko 1990, Lace 1992, Schrag and Read
1992, Viard et al. 1997, Doums et nl. 1998, Backeljau et al.
2001 ). The results presented in this study offer a clear dem-
onstration of phally polymorphism in A. Inittoni.

Aphally seems to be common in at least some popula-
tions of Ariolimax buttoni. Pilsbry and Vanatta (1896, dis-
cussion in Pilsbry 1948) examined a large collection of in-
dividuals lacking a penis from Oakland, and Heath (Mead
1942, cited above) dissected 400 aphallate specimens from
Hog Island in Tomales Bay. Westfall (1952) also reported
that of 85 specimens collected at Mills College in the spring
of 1952 (subsequent to the work described here), only 9 had
a penis, of which 3 were in the hypertrophied state and 6 in
the immature reproductive state, in the terminology used
here. It is characteristic of phally polymorphisms that the
percentage of a given morph varies widely from one area to
another and from season to season (Lace 1992). Baur and
Chen ( 1993) found that the frequency of aphallic individuals
in populations of Chrondrina avenacea (Bruguiere) varied
from 0.9% to 89.2% in the vicinity of Basel, Switzerland. The
tendency for the frequency of phally morphs to vary with
environmental conditions {e.g., Watson 1934, Schrag and
Read 1992, Baur et al. 1993) may explain why Mead (1942,
1943) found only euphallate individuals in the vicinity of
Hog Island whereas Heath had found only aphallate indi-
viduals at Hog Island earlier. In this study, all four individu-

als derived from populations near Tomales Bay were euphal-
late (Table 2).

The results presented here, then, demonstrate that
phally polymorphism is present in Ariolimax buttoni, show-
ing that only 7/67 Mills College and 4/16 UCSC, including
0/10 Staten Island, slugs had a penis, and leave open the
question of whether apophallation occurs in this species.
Aphallate individuals have also been found in a population
oi Ariolimax (Meadarion) brachyphallus from Hillsborough,
San Mateo City, CA (Pearse, impubl. data). Wright (1938)
stated that 415 individuals of Ariolimax californicus he ex-
amined did not have a penis, or other “male parts” whereas
248 individuals were found to have normal, “or regenerat-
ing” penes. Wright also stated that the aphallic individuals
were the result of apophallation but provided no evidence to
support this, nor did he provide details of the anatomy or
even information as to the source of these specimens.
Wright’s observation suggests that an anatomical study of
that species is needed to determine whether aphally as well as
apophallation occurs. It is possible that phally polymor-
phism will be found to be more widespread in Ariolimax.

The adaptive significance of phally polymorphism is not
entirely clear. Local Mate Competition theory (Charnov

1982) predicts that allocation to male function in hermaph-
rodites should be reduced where few sexual partners are
available, suggesting that aphally should be more common
where self-fertilization is common or population densities
are low. Baur et al. ( 1993) hypothesized that aphally evolves
in populations that typically self-fertilize. In both cases,
aphally is predicted to be associated with a capacity for uni-
parental reproduction and the role of environmental factors
in influencing the ratio of aphallic to euphallic offspring is
hypothesized to be an adaptation to colonizing new habitats
(Schrag and Read 1992, but see Baur et al. 1993)

Uniparental Reproduction

Uniparental reproduction, either by self-fertilization, as
has been widely assumed, and well-documented in some
cases, or by apomixis, as suggested by some authors (Mc-
Cracken and Selander 1980, Foltz et al. 1982a, Hoffmann

1983) is widespread, although not universal in stylommato-
phorans (Tompa 1984, Heller 2001). Within a genus, some
species may be obligate outcrossers whereas others readily
self-fertilize (Foltz et al. 1982b, Reise 2002). Mead (1942)
reared Ariolimax in isolation for as long as two years without
the production of any eggs but considered the question of
uniparental reproduction in Ariolimax still open. Here we
report that A. buttoni can produce viable offspring with high
rates of hatching success without cross-fertilization. Unipa-
rental reproduction has also been observed in A. dolicho-
phallus but with low hatching success (Miller and Sinervo
2007). Westfall (1952) reported that in Mills College ani-
mals, the development of the ovotestis did not differ be-

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

tween euphallic and aphallic individuals; that is the qualita-
tive degree of sperm vs. egg production did not appear to
depend on phally status. The production of sperm obseiwed
in aphallate individuals in histological studies (Westfall,
1952), suggests that self-fei'tilization could occur in A. Init-
toni. Similar results have been reported in the genus Zoni-
toides Lehman, 1862 (Watson 1934). Resolution of the ques-
tion of whether these offspring are the result of selfmg or
apomixis will rec]uire genetic analysis.

Sexual Behavior

Of the observations reported in Table 3, about half of
the pairs had reciprocal intromissions and the other halt had
unilateral intromissions, in which one slug was acting as a
female and the other as a male. Copulation lasted more than
7 h after being first observed in 3 of 12 observations, and in
one case (5-6 September 2002) the interaction may have
lasted almost 23 h after detection (see above; Table 3). Thus,
copulation in Ariolimax biittoni often lasts more than the
two hours typical of simultaneously reciprocal intromissions
of A. dolicliophaUus and much longer than the brief, unilat-
eral intromissions of A. califorincus (Leonard et al. 2002). In
long reciprocal copulations such as those of A. dolicJiophallus
(Leonard et al. 2002) it is not unusual for one individual to
withdraw the penis long before the other (Leonard, unpub-
lished observation). Therefore, it seems likely that copula-
tions between euphallate A. buttoni (Figs. 5A-C) are nor-
mally long and simultaneously reciprocal. The unilateral
intromissions observed here may have been the end of si-
multaneously reciprocal copulations or they may have in-
volved copulation with a partner that lacked a penis (below).

Life History

The life histories of Ariolimax spp. are poorly known.
The observations reported here provide the most detailed
picture available on sexual development and the reproduc-
tive cycle for any species ot' Ariolimax. We found that, in the
laboratory, Ariolimax buttoni may live more than 30 months.
This is consistent with previous reports that individuals of A.
californicus and A. dolicliophaUus, collected as adults, sur-
vived more than 18 months in the laboratory (Leonard et al.
2002). The anatomical and histological data obtained in the
Mills College study indicate that, in Oakland populations,
individuals may live more than a year in the field, since two
classes of individual were found in fall and winter dissec-
tions; spring and summer specimens showed a gradual in-
crease in albumen gland length, suggesting the maturation of
a single cohort (Fig. 3). This fits well with field data (Pearson
et al. 2006) for identifiable individuals in a population of A.
buttoni in Orinda, Contra Costa Cty, CA that showed a life
span of approx. 2 years.

The data also provide a clear picture of the phenology of
Ariolimax buttoni. Both UCSC (Leonard et al. unpublished)

and Mills College animals held in the laboratory (Westfall
1952, 1960) laid eggs in the fall and winter as suggested for
Ariolimax by Mead (1942). Mills College animals with red-
dish-brown ovotestes, a spermatheca filled with reddish
fluid, and a yellow albumen gland larger than the ovotestis
(the hypertrophied reproductive state), believed to represent
the egg-laying stage, were first seen in a dissection of 22
October 1951 and then seen regularly until February 1952
(Fig. 3). Egg-laying and juvenile growth from individuals in
the Mills College portion of the study have been reported
elsewhere (Westfall 1960). Westfall (1960) reported a mini-
mum time to hatching of 23 days and a maximum of 2
months for Mills College slugs, which brackets the 47-54
days found for UCSC animals (Leonard et al. unpublished).
In A. dolichophallus, hatching time ranged from 51-55 days
and in A. californicus from 46-81 days (Leonard et al. 2002).
These hatching times all reflect laboratory conditions and
will probably depend strongly on temperature, making it
likely that hatching times in the field will be somewhat
longer. In the spring and summer, animals were found to be
in the immature and intermediate reproductive states,
whereas in fall and winter all three states were found (Fig. 3).
This is consistent with data from Pauli (1951) who noticed
that the reproductive system was more often in the imma-
ture state from February to April, whereas hypertrophy of
the reproductive tract was predominant in A. buttoni col-
lected from September to the middle of February. In field
observations, juveniles of A. buttoni weighing less than 1
gram appeared in lanuaiy (Pearson et al. 2006).

Since laboratory data demonstrate that Ariolimax but-
toni can live more than one year, with some individuals
laying eggs at less than one year of age while some clutch
mates reared under the same conditions do not lay eggs until
over two years of age (see below), it seems likely that not all
individuals will reproduce in their first year, perhaps ac-
counting for the occurrence of individuals in the immature
and intermediate reproductive states throughout the year in
this study. Protiindrous development of the ovotestis is typi-
cal of stylommatophorans (Tompa 1984) and has been re-
ported for A. californicus (Gottfried and Dorfman 1970),
making it seem probable that development in A. buttoni may
involve a progression from the immature reproductive state
through the intermediate male state to the hypertrophied
female state, and then the old reproductive state, although
the intermediate state may be heterogeneous. The interme-
diate reproductive state is the state characterized by massive
sperm production (Westfall 1952; Appendix) and one would
expect it to be associated with copulation. Our observations
suggest that in A. buttoni may become sexually mature and
begin copulation as early as the summer of their first year
and lay eggs that same fall. The first specimen found to have
an albumen gland greater than 20 mm in length in the Mills
College study was dissected in April 1951 (Fig. 3). The first

PHALLY POLYMORPHISM IN ARIOLIMAX BUTTONI

131

Figure 5. A. A copulating pair of
euphallate individuals of ArioUmax
biittoni. Both individuals were col-
lected from the UCSF campus and
held in a group hox; found copulat-
ing when the box was opened for
cleaning. B. The same pair of indi-
viduals later in same copulation,
with at least one penis inflated. C.
One individual of same copulation
with short, rigid withdrawn penis
still everted after withdrawal from
partner.

individual found to have a reddish and enlarged sper-
matheca (= bursa copulatrix = gametolytic gland) was dis-
sected 17 October 1951. The spermatheca, often termed the
gametolytic gland (Tompa 1984, Gomez 2001), serves to
digest both alio- and autosperm in stylommatophorans, as
well as stray oocytes and excess secretions. The reddish color
of the spermatheca may, therefore, indicate copulation and/
or egg-laying. Individuals from the Staten Island population
that were reared in isolation but had laid eggs had a reddish
spermatheca (Table 2) but one of the individuals from To-

males Bay, also reared in isolation but having never laid eggs,
also had a reddish spermatheca. Laboratory observations
from UCSC animals (Table 3) show copulations occurring
between February and September. This is consistent with
field observations from A. doUchophallus in which observa-
tions of copulating pairs ranged from February to mid-
October (Leonard et al. 2002).

Laboratory data from the UCSC animals indicate that
the rate of maturation, at least in terms of egg-laying, varies
widely. The age at first egg-laying has varied from slightly

132

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

over 10 months to more than 24 months among individuals
that have laid at least one dutch and many animals in the
study had not yet laid eggs at 30 months of age (Leonard et
al. unpublished), as Mead (1942) also reported. Gottfried
and Dorfman ( 1970), reported that individuals from a popu-
lation oi ArioUmax californicus from Portola Valley, San Ma-
teo Go., California, reared in the laboratory, showed protan-
drous gonadal maturation with individuals having immature
gonads at body weights of less than 10 g and individuals of
20-30 g and approx. 12 months of age having “maturing
male phase” gonads. The first “intersex” gonads appeared at
a body weight of 40-45 g and an approx, age of 24 months
and fully female gonads were associated with a body weight
of 55-60 g in weight and 36 months of age. These data are
similar to those from individuals in the current study (Staten
Island isolates. Table 2) that first laid eggs at two years of age,
although there was in general no clear relationship between
weight and reproductive state in the Mills College animals
(Table 1; Figure 4). In a study of identifiable individuals in
the field, Pearson et al. (2006) found a correlation between
age and weight only up to 20 g for A. hiittoni. Gottfried and
Dorfman’s (1970) data differ substantially from the results
obtained for A. caUfoniicus by Leonard et al (2002) using the
same rearing conditions as used for the UCSC animals in the
current study, where lab-hatched individuals were found to
begin copulating at 8 months of age, laying eggs as early as
50 weeks of age, and achieving a much higher growth rate
than reported by Gottfried and Dorfman ( 1970). Lab-reared
A. califortiicus were observed to copulate as early as eight
months of age (from the egg) and to lay eggs at 12 months
(Leonard et al. 2002) and A. (ArioUmax) stramineus Hemp-
hill, 1891 and A. (Meadarion) hrachyphallus Mead, 1943
have also been observed to lay eggs at less than 12 mo of age
in the laboratory (Leonard et al. unpublished). In contrast.
Mead ( 1942, p. 116) reported that the genital system was still
“very minute and completely non-functional” in A. bracliy-
phalliis at the end of one year and that sexual maturation
(ability to copulate as a male and to receive sperm but not
lay eggs) was reached in A. doliclwphallus at approx. 18 mo.
Miller and Sinervo (2007) reported large variance in growth
of A. dolichophallus in the laboratory, as seen by Leonard et
al. (2002) for A. caUfoniicus. This may be associated with
variance in sexual development as seen here in A. buttoni.

Reproduction in stylommatophorans appears to be
strongly influenced by environmental factors (Potts 1975,
Tompa 1984, South 1992, Gomot de Vautleury 2001 ). More-
over, growth rates may vary greatly among clutch mates
reared under the same conditions (Shibata and Rollo 1988,
Leonard et al. 2002, Miller and Sinervo 2007), and, as seen in
the current study, the age at first egg-laying may vary by
more than 12 months among clutch mates. Consequently,

variation from year to year and site to site would not be
unexpected in ArioUmax buttoni. However, in general the life
cycle seems to involve egg-laying in fall and winter with
hatching in late winter to early spring, and some individuals
copulating as early as the late summer or fall of their first
year and laying eggs in the late fall or winter. Overwintering
individuals may copulate in the spring or summer of the
second year and lay eggs that fall or winter. Whether regres-
sion of gonads occurs in A. buttoni (see discussion in Mead
1942, Westfall 1952) is not clear. Also, we do not know
whether an individual will lay eggs in successive years. Some
stylommatophorans have an iteroparous life cycle, with
cycles of gonadal development and reproduction in succes-
sive years whereas other species have a pattern whereby in-
dividuals have a more or less prolonged semelparous life
cycle (perhaps over several years) and gonadal development
is not reversible once sexually mature (see discussion in
Heller 2001). The available data do not allow us to distin-
guish between these models for A. buttoni, or other species
of ArioUmax.

Sexual Selection and the Sexual Biology of
ArioUmax buttoni

In order to understand the potential for sexual selection,
it is important to understand the age at first reproduction,
the duration of the reproductive life span, the type of mating
behavior, and the potential for uniparental reproduction.
The potential strength of sexual selection is measured by the
variance in reproductive success among individuals (see re-
view in Leonard 2006), and one factor that would tend to
increase the potential for sexual selection in a species is a
skewed sex ratio. While in simultaneous hermaphrodites the
operational sex ratio, or sex ratio at the time of mating, is
considered to be 1:1, if there were a delay between mating
and egg-laying there may be a skew in breeding sex ratio
(Arnold and Duvall 1994). In ArioUmax, A. californicus have
been observed to copulate as young as 8 months of age and
to begin egg laying at 11.5 months of age (Leonard et al.
2002). This delay suggests that not all individuals that copu-
late will lay eggs, creating a potential skew between the num-
ber of individuals in the population that sire young and
those that are mothers of young, which would tend to in-
crease the variance in reproductive success among individu-
als and the potential for sexual selection. The range in age at
first egg-laying seen in A. buttoni in the current study (from
10 to more than 24 months) further suggests that variance in
female reproductive success will be substantial. Phally poly-
morphism may also contribute to skewed breeding or even
operational sex ratios in that aphallates will be unable to
copulate as males with other individuals although they may
be able to copulate as females (but see Reise 2007). The
anatomy of aphallic individuals in A. buttoni, with the vas

PHALLY POLYMORPHISM IN ARIOLIMAX BUTTONI

133

deferens ending blindly or in a bulb seems to be inconsistent
with transfer of sperm to conspecifics. However, observa-
tions of copulation in the female role by apophallate A.
doUchophalliis (Leonard et al. 2002) suggest that aphallate
individuals may be able to copulate as females, receiving
sperm from euphallate partners. In the stylommatophoran.
Vertigo pusilla O.F. Muller, 1774, Pokryzsko (1990) found
that aphallates copulated as females with euphallate indi-
viduals. In the basommatophoran Biiliinis truucatus, in
which self-fertilization is the predominant mode of repro-
duction, phally status (euphallic or aphallic) was not corre-
lated with offspring heterozygosity, indicating that aphallic
individuals are as likely to have their eggs fertilized by a
conspecific as were euphallic individuals (Doums et al. 1996,
Viard et al 1997). In a mixed population of aphallates and
euphallates with outcrossing, the euphailates should have
greater potential reproductive success in the male role, in-
creasing variance in reproductive success and the potential
for sexual selection. However, if reproduction in A. buttoni
is predominantly uniparental, the opportunity for sexual se-
lection would be limited.

Sexual behavior also offers indications of the strength ot
sexual selection. The very long (>7 h) copulations observed
in this study (Table 3) suggest that sexual behavior in Ario-
limax buttoni may involve higher expense and/or risk than in
species with shorter copulations. Sexual selection in her-
maphrodites has been hypothesized to involve resolution
through reciprocity between members of a pair of a sexual
conflict over sexual role (Axelrod and Hamilton 1981, Leo-
nard 1990, recent reviews by Michiels 1998, Leonard 2005,
2006). In A. dolichophalhis the mating system is based on
simultaneously reciprocal copulation between the members
of a pair (Leonard et al 2002). In A. califoniiciis intromis-
sions are unilateral but occur in bouts and the members of
a pair are hypothesized to alternate roles during these bouts,
creating reciprocity (Leonard et al. 2002). In the data pre-
sented here, about half of the observed copulations were
simultaneously reciprocal (Fig. 5A) and since in other spe-
cies of Ariolimax simultaneously reciprocal copulations of-
ten involve a period of unilateral intromission after one
individual withdraws (Leonard et al. 2002, Leonard and
Pearse, uupubl. obs.), we hypothesize that simultaneously
reciprocal copulations will be found to be the rule in A.
buttoni where both individuals are euphallate.

If reciprocity were important to the mating system, as
predicted by the Hermaphrodite’s Dilemma model (Leonard
1990), then individuals able to mate in only one sexual role,
as is assumed to be the case for aphallates (but see Westfall
1952), should be less desirable as mating partners than eu-
phallates. If on the other hand, the reciprocity seen between
euphallates is “unconditional” and an artifact of both her-
maphrodites being eager to act as males as predicted by

Michiels (1998, see discussion in Leonard 2005), then aphal-
lates should be as desirable as euphallates as sexual partners.
Ariolimax buttom, offers an exciting opportunity to distin-
guish between these models. Additionally, the capacity for
uniparental reproduction should allow hermaphrodites to
avoid Game of Chicken conditions in the Hermaphrodite’s
Dilemma (Leonard 1990). Therefore, in a species with the
option of uniparental reproduction, the Hermaphrodite’s
Dilemma predicts that reciprocity will be conditional on the
partner’s behavior (see Leonard 2005). The evidence tor
phally polymorphism and uniparental reproduction in A.
buttoni creates an interesting opportunity to test the predic-
tions of theory. Genetic studies will be rec]uired to distin-
guish between hypotheses and to measure the variance in
reproductive success and hence, potential for sexual selec-
tion in Ariolimax.

ACKNOWLEDGEMENTS

We wish to thank Dr. Barry Roth for his help and en-
couragement with this study and our efforts with Ariolimax
generally, Erin Serra and Ed Whisler for alerting us to the
Staten Island population of A. buttoni and help in collecting
there, Earl Mayeri for help in collecting at UCSF, Vicki
Pearse for help in collecting in Marin County, Renate
Eberle for collecting in Mendocino County, and Mallory
Hoover and Gina Scott for help in preparation of Figure 1 .
The paper greatly benefited horn the comments of anony-
mous reviewers.

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Accepted: 9 May 2007

APPENDIX

Definitions of Reproductive States in Ariolimax buttoni

(Histological data from Westfall 1952)

1. Immature State: Reproductive system located anteriorly,
close to atrium; ovotestis (=hermaphroditic gland = gonad),
white, often transparent; small, thin hermaphroditic duct;

spermatheca (=bursa copulatrix = gametolytic gland) flat-
tened, transparent; albumen gland < 20 mm in length, usu-
ally smaller than the ovotestes, made up of small, white
lobes, dose to and alongside ovotestis. Histologically, sper-
matozoa absent; abundant spermatogonia and spermato-
cytes, only occasional groups of spermatids; germinal epi-
thelium well-formed, lumen without much connective
tissue; fair number of eggs in various stages of development
and degeneration. The reproductive system in the immature
state resembles that of juvenile slugs.

2. Intermediate State. Ovotestis smooth, creamy, solid;
greatly expanded hermaphroditic duct; spermatheca, color-
less; albumen gland white or sometimes yellow, appearance
as immature state. Histologically, cross section with sperm
predominant in ovotestes, characteristically arranged in or-
derly fashion around inner periphery of each acinus; sperm
massed in hermaphroditic duct; a few eggs in various stages
of development; large oocytes protrude from inner walls of
the acini; oocytes easily distinguished by large amount of
cytosome in proportion to size of distinct, round, clear
nucleus; nucleolus of oocyte usually stains darkly; degener-
ating oocytes fairly common, appear yolky, usually irregular
in outline.

3. Hypertrophied State. Ovotestis dark, reddish brown with
granular appearance; hermaphroditic duct, convoluted and
yellow, pushing ovotestis and albumen glands posteriorly,
filled with masses of spermatozoa; spermatheca bulges with
red fluid; albumen gland very large (> 20 mm), larger than
ovotestis, extending posteriorly, filling up much of the body
cavity. The one slug taken for dissection while egg-laying was
in the hypertrophied state with oviduct distended with eggs
and masses of sperm in hermaphroditic duct. Histologically,
ovotestis cross section somewhat ragged, acini noticeably
shrunken and detached, separated by wide spaces within
ovotestis membranes; all stages of spermatogenesis present,
although spermatozoa fewer than in intermediate stage and
no longer arrayed in strikingly regular fashion of the inter-
mediate state; eggs large, relatively rare; most ova degener-
ating, apparently left over from spawning. Portion of
hermaphroditic duct visible in ovotestis cross sections
shrunken, sperm masses visible only in smears under the
microscope.

4. “Old” State. “Six dehydrated and wrinkled specimens
from a moist terrarium which were dissected had atrophied
reproductive organs with straight ducts, but in all cases the
hermaphroditic gland was a light reddish-brown color. In
three of these specimens the spermatheca seemed white and
cloudy. Smears on microscope slides showed that the sper-
mathecae were filled with spermatozoa” (Westfall 1952, p.
46).

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Amer. Maine. Bull. 23: 137-156

A review of mating behavior in slugs of the genus Deroceras
(Pulmonata: Agriolimacidae)’^

Heike Reise

State Museum of Natural History Gorlitz, PF 300 154, 02806 Gorlitz, Germany, Heike.Reise@smng.smwk.sachsen.de

Abstract: The genus Deroceras Rafinesque, 1820 (the largest genus of terrestrial slugs) shows a high diversity ol penis morphologies and
mating behaviors. The function of most ot the appending external and internal penial structures, some ol them truly bizarre, is largely
unknown. This paper reviews mating behavior ancf reproduction, based on data on 16 species from the literature and from unpublished
observations. I analyze patterns common to all Deroceras species and differences among species. The general mating pattern consists of a
long courtship with mutual stroking with a sarcobelum, a sudden penis eversion, and external sperm exchange (copulation). I distinguish
also precourtship and withdrawal phases. Sperm exchange is usually very quick but, in a few species, occupies a considerable proportion
of the total mating duration. Mutual sperm exchange is the rule. Species differences involve the durations of certain mating phases, presence
and nature of initial trail following, nature and intensity of stroking (including the degree of contact with the sarcobelum), aggressiveness
of courtship behavior, and the timing of the penial gland eversion. I hypothesize that the radiation of mating behaviors and associated
structures has been driven by an arms race resulting from conflicting interests of mating partners over sperm donation and use. This could
also have increased the rate of speciation in Deroceras. There are indications of the presence of sperm competition and conflicting interests
between mating partners: individuals mate repeatedly, can store and digest sperm, and simultaneously use sperm from different mating
partners for fertilization. Some details of mating behavior also indicate conflict. The timing of the penial gland eversion after sperm
exchange suggests a manipulation akin to the role of love darts in helicid snails. Finally, some recommendations for studying mating
behavior in Deroceras are given.

Key words: courtship, genital morphology, partner manipulation, sexual conflict, simultaneous hermaphrodite

Deroceras Rafinesque, 1820 is the largest genus of ter-
restrial slugs (over 100 known species), and comprises the
major part of the slug family Agriolimacidae (Wiktor 2000).
It is Holarctic with most species restricted to the Palaearctic,
although a few synanthropic species have been introduced to
most other continents. The widespread pest species have
been comparatively well studied. Fdowever, there are many
species with apparently small geographic ranges, and for a
number of species not much more than the morphology and
type locality is known. There is no well-supported phylogeny
of Deroceras available except for the separation of six species
into the subgenus Liolytopelte Simroth, 1901.

Deroceras slugs are externally rather uniform (each spe-
cies has some externally identical congeners), and most of
the internal anatomy also varies very little among species.
Almost the only species-specific characters are provided by
the penial morphology: there is a wide variety of appending
and internal structures (side pockets, glands, folds, pilasters,
etc.), but their functions are largely unknown.

The diversity of penial morphologies is accompanied by
a diversity of mating behaviors, and even sibling species can
differ considerably (Gerhardt 1935, Reise 1995, 2001, Wiktor

* From the symposium “Gastropod Mating Systems” presented at
the joint meeting of the American Malacological Society and
Western Society of Malacologists, held 26-30 Rine 2005 at Asilo-
mar, Pacific Grove, California.

2000). I review here the mating behavior ot Deroceras and
indicate which patterns are consistent in all species and
which vary among or within species. The elaborate mating
behavior and the diversity of penial structures, including
rather extravagant and bizarre structures, caused Reise
(2001 ) to hypothesize that the diversity of mating behaviors
and associated genital structures is driven by an evolutionary
arms race between male and female functions in these si-
multaneous hermaphrodites.

The background to this hypothesis is that at least some
species of Deroceras are able to selt-fertilize and/or to mate
repeatedly (Rymzhanov and Schileyko 1991, Rymzhanov
1994, Reise 1996, 1997, 2001, Lebovitz 1998, Wiktor 2000
and references he cites on p. 375). Moreover, they may si-
multaneously use sperm from different mating partners for
fertilization of a single clutch (H. Reise, B. Zimdars, M.
Scheibe, J. Sauer, and C. Matthieu, unpubl. obs. on Dero-
ceras panormitanum (Lessona and Pollonera, 1882)). A re-
ceiver might thus not use the donor’s sperm and instead use
sperm from another (earlier or later) donor or its own sperm
to fertilize its eggs (unused ejaculates may be digested in the
bursa copulatrix). It is even possible that individuals try not
to donate sperm in some matings if it would be better to
invest the ejaculate in a higher-quality partner or if there are
indications that this partner would only digest the ejaculate
(Leonard 1991, Michiels 1998). A sexual conilict could arise
if partners attempit to avoid one of the sexual roles ( male or

137

138

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

female) or if sperm donors can manipulate their partners to
use this batch of donated sperm to fertilize more eggs.
Counter-adaptations would lead to an evolutionary arms
race; this might drive rapid diversification and the develop-
ment of bizarre genital structures and mating behaviors.
Evolutionary arms races driven by sexual conflict have been
convincingly shown in gonochorists (Rice 2000), and Leon-
ard (1990, 2006), Michiels (1998), and Michiels and Koene
(2006) have proposed that sexual conflict may also be strong
in simultaneous hermaphrodites. Convincing evidence for
an arms race between sperm-donating and sperm-receiving
functions in simultaneous hermaphrodites comes from a
comparative analysis of love-dart shooting and receiving or-
gans in helicid snails (Koene and Schulenburg 2005). There
are also indications of intraspecific coevolution of male and
female reproductive traits in the terrestrial snail Arianta ar-
hnstorum (Linnaeus, 1758) (Beese et ah 2006).

My review is based on data on
mating behavior from 16 species
(Table 1). These data include my
own published and unpublished ob-
servations as well as descriptions by
others. The published descriptions
vary considerably in quality. Some
are based on single or very few
chance observations in the field and
provide little information. Some oth-
ers do not specify sample sizes. My
own unpublished observations (and
those of M. Benke and I. Schulze)
cited in this paper are based on labo-
ratory observations of the mating be-
havior of wild-collected or labora-
tory-bred individuals. Animals were
kept isolated for a few days prior to
being put together and then kept un-
der at least periodic observation for a
few hours until they did or did not
start to mate; thus my observations
often include early precourtship be-
havior. In all species at least some
matings were video-recorded.

Comparisons are hampered by
inconsistent or unclear definitions of
mating phases (see section on general
mating pattern) and by uncertain
species identities; these have also led
to misunderstandings between au-
thors. A particularly good example is
Garrick’s (1938) description of the
mating behavior of Deroceras agreste
(Linnaeus, 1758) (probably what we

now know as D. reticulatum (Muller, 1774), see below),
based on one field observation in Scotland. The author
seems to have misunderstood the sarcobelum as the penis,
and he reported that it was inserted into the partner’s genital
aperture. Consequently, he interpreted the entire courtship
as copulation and saw discrepancies with the copulation
time given for this species by Taylor (1902-1907). He may
possibly have been confused by Heath’s (1916) description
of the mating behavior of Ariolimax californicus (Gould in A.
Binney, 1851), an arionid slug with penis intromission. The
genus Deroceras was called Agriolimax Morch, 1865 at this
time, and a misspelling in the reference list implies that he
confused the similar generic names and thought to see what
he expected.

The commonest example of uncertain species identity is
that in older papers Deroceras reticulatum was usually not
distinguished from D. agreste, so that it is unclear which

Table 1. Sources of data on mating in

Deroceras. (*: species identity uncertain).

Subgenus Deroceras s.s.

D. agreste (Linnaeus, 1758)

Gerhardt 1933, 1934’*', H. Reise, unpubl. obs.
( 1 mating)

D. fatrense Macha, 1981

Reise, unpubl. obs. (S12 matings)

D. gorgonhim Wiktor et al, 1994

Reise et al. 2007

D. laeve (Muller, 1774)

Karlin and Bacon 196U, Rymzhanov 1994*,
Barker 1999*

D. lombricoides (Morelet, 1845)'^

Simroth 1891, Castillejo et al. 1989

D. nitidum (Morelet, 1845)

Castillejo et al. 1989

D. panormitanuin (Lessona and

Gerhardt 1939, Quick 1960, Webb 1961, 1965,

Pollonera, 1882)

Barker 1999, Reise and Hutchinson 2001b,
Benke et al. 2005, Benke 2006, H. Reise,
M. Scheibe, J. Sauer and C. Matthieu,
unpubl. obs. (^60 complete matings)

D. planarioides (Simroth, 1910)

Gerhardt 1939*

D. praecox Wiktor, 1966

Reise 1995, unpubl. obs. (^29 matings)

D. rethiinnonensis de Winter

Wiktor 1994

and Butot, 1986

D. reticulatum (Muller, 1774)

Simroth 1885, Gerhardt 1933*, 1934, Wiktor
1960, Karlin and Bacon 1961, Webb 1961,
1965, Nicholas 1984, Barker 1999, H. Reise,
unpubl. obs. (^2 matings)

D. rodnae Grossu and Lupu, 1965

Reise 1995, 1997, unpubl. obs.
(^33 matings)

D. sturanyi (Simroth, 1894)

Gerhardt 1936* — as “D. laeve", Kosihska 1980,
Rymzhanov 1994, H. Reise and C. Natusch,
unpubl. obs. (6 matings)

D. turciciuri (Simroth, 1894)

Gerhardt 1935* — as “Deroceras aff. turcicum",
H. Reise, unpubl. obs. (^7 matings)

Subgenus Liolytopelte

D. bureschi (Wagner, 1934)

Wiktor 1983, 2000

D. caucasicum (Simroth, 1901)

Rymzhanov and Schileyko 1991

MA^riNG BEHAVIOR IN DEROCERAS

139

species were observed (Wiktor 2000). Gerhardt’s (1933,
1934, 1936, 1939) valuable descriptions of the mating be-
havior of several Deroceras species were hampered by uncer-
tain species determinations (Gerhardt 1934, 1939, Wiktor
1960). In 1933, he published descriptions for ^Agrioliniax
agrestis" and "Agriolimax lacvis\ but later corrected their
identities to “Deroceras reticiilatuni” and “Deroceras agreste"\
respectively (Gerhardt 1934), which is how I will refer to his
1933 descriptions. Then in 1936 Gerhardt described the mat-
ing behavior of a species that he thought to be the real
Deroceras laeve (Muller, 1774). However, he later expressed
some uncertainty about the species identity (Gerhardt 1939).
It seems probable that he was indeed wrong again: all details
that Gerhardt (1936) provided about his D. laeve — time of
occurrence, body color, sarcobelum shape and its use during
courtship, bulbous shape of everted penial mass, and un-
usually long copulation — fit very well with the externally
hard-to-distinguish D. sturanyi (Simroth, 1894), a species of
which malacologists were hardly aware at that time and with
which D. laeve has often been confused (Quick 1960). Later
descriptions of matings of D. laeve with which one might
compare are sparse: Karlin and Bacon (1961) repeated Ger-
hardt’s (1936) statement that its courtship is similar to that
of D. reticulatiim but stress that the partners have less inti-
mate contact. However, they do not mention copulation,
which they probably would have done had they observed it.
Barker (1999) just repeated information provided by Ger-
hardt (1936) and Karlin and Bacon (1961), so one has to
wonder to what extent Barker observed the mating of D.
laeve. Rymzhanov’s ( 1994) description of the mating behav-
ior of D. laeve from Kazakhstan differs in almost every aspect
from the earlier papers. The origin that Rymzhanov (1994)
proposed for the aphallic individuals in his population (apo-
phallation) differs from what we know of their origin in
Europe, so Kazakh D. laeve may well be a distinct species.
Thus, although four publications claim to describe the mat-
ing behavior of D. laeve, there are no reliable data. In this
paper I will consider the species studied by Gerhardt ( 1936)
as D. sturanyi and refer to the one described by Rymzhanov
(1994) as “Kazakh D. laeve”. The papers of Karlin and Bacon
(1961) and Barker (1999) will be assumed to pertain to D.
laeve, because D. sturanyi is unknown in North America and
New Zealand, but this should be viewed as provisional.

I do not include other genera of the Agriolimacidae
because at least some are considerably different in their geni-
tal anatomy and thus possibly also in their mating behavior.
Besides, almost nothing is known about them. The only
published description of mating behavior in another agrio-
limacid genus concerns a species of Eurcopenis Castillejo and
Wiktor, 1983 (Rodriguez et al. 1989), morphologically the
most similar genus to Deroceras, of which it had formerly
been classified as a subgenus.

Casual observations suggest that mating often occurs
during early morning, but no one has carried out systematic
observations throughout 24 hours, so I do not attempt to
review this aspect of mating behaviour. Mating slugs are
often observed in the open, but again there are no systematic
observations of what proportion of matings in the wild are
hidden, for instance under leaves.

GENITAL MORPHOLOGY

There are discrepancies in how to describe the relative
position of parts of molluscan genitalia. Sometimes, the
parts further away from the genital pore are called “proxi-
mal” ie.g., Castillejo et al. 1989, Rymzhanov 1994, Reise

1997, 2001, Hausdorf 1998, Barker 1999, Reise and Hutchin-
son 2001a) and sometimes “distal” [e.g.. Quick 1960, Webb
1965, Nicholas 1984, Backeljau and De Bruyn 1990, Reise
1995). Some authors use “anterior” and “posterior”, “basal”
and “terminal”, or other terms relating to the approximate
positions of the organs in the animal at rest and their ori-
entations {e.g.. Mead 1943, Webb 1961, Sirgel 1973, Rilhle

1998, Tompa 1984, Wiktor 2000). However, as not all sec-
tions of the genital tract are orientated anterior-posterior in
animals at rest, I will use the terms proximal and distal and
apply the first definition, which is that used in medicine:
parts of the genitalia nearer to the genital pore are further
away from the body centre (when not everted) and thus
distal. Parts further away from the genital pore, and thus
nearer to the body centre, are proximal (Fig. 1).

In the genital tract of Deroceras (Fig. 1 ), the penis has a
more or less sac-like shape, but can consist of one or more
chambers and may have side pockets and diverse append-
ages. In at least one species, D. laeve, the penis is often

Figure 1. Schematic genital tract of Deroceras panonuitaniun [alter
fig. 5.1 in South (1992)].

140

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

reduced (hemiphallic) or entirely lacking (aphallic) (Simroth
1884, Wiktor 1973, 2000, Tompa 1984).

Most species have a more-or-less finger-like penial
gland appending somewhere near the proximal end of the
penis. This is also called the trifid or penial appendage {e.g..
Quick 1960, Runham and Hunter 1970, Runham 1978) or
flagellum {e.g., Simroth 1885, Gerhardt 1933, 1935, Wiktor
1960, Webb 1961 ); it may or may not be homologous to the
flagellum of helicid snails (Sirgel 1973, Nicholas 1984, Haus-
dorf 1998). The name “penial gland” seems justified because
there are indications for secretory activity (Sirgel 1973, Ni-
cholas 1984, Benke et al. 2005, Benke 2006). Interspecifically,
the penial gland varies widely in size, can be branched or
unbranched, and lobed or smooth (Eig. 2). In some species,
particularly Dewceras gorgonium Wiktor et al.., 1994 (Fig.
2F), it is extremely large and tree-like. The number of
branches can vary intraspecifically (Wiktor 2000, Benke
2006).

rhe lumen of the penis also contains diverse structures.
The most important is the sarcobelum (or stimulator;
“Reizkbrper” of earlier German authors), located in the dis-
tal, swollen part of the penis. It is a conical or tongue-like
structure, solid but with a central blood sinus, and consists
of muscle, glandular, and connective tissue cells in a collagen
matrix (Nicholas 1984). The sarcobelum plays an important
role during courtship when it is pushed outside the genital
orifice by the eversion of the distal part of the penis (the

sarcobelum itself does not evaginate). Its surface has longi-
tudinal ridges with a strongly ciliated epithelium, and there
are gland cells in the sarcobelum and the surrounding inner
penial wall (Sirgel 1973, Els 1978, Nicholas 1984). This and
its use during courtship suggest that this organ transfers
secretions onto the mating partner. Shape and size of the
sarcobelum vary considerably among species (Fig. 3). Spe-
cies of the subgenus Liolytopelte have a calcareous plate at
the base of the sarcobelum (Fig. 3D).

The walls of other penial protuberances have also been
reported to contain glands (Wiktor 2000). However, the
function of these and most other penial structures is largely
unknown.

The sac-like bursa copulatrix (also called “sper-
matheca”) opens, via the bursa trunk, into the genital atrium
(Nicholas 1984) or base of the penis (Hausdorf 1998, Wiktor
2000). It is a lytic organ and digests excess sperm as well as
some other secretions (Nicholas 1984, Tompa 1984), prob-
ably including other components of ejaculates.

GENERAL MATING PATTERN

The mating pattern of Deroceras consists of four main
phases (Fig. 4).

(i) Precourtship phase: the partners encounter and in-
vestigate each other.

Figure 2. Penis diversity in Dewceras. A, D.
reticulatiim. B, D. muioicum Wiktor et al,
1994. C, D. ikaria Reischiitz, 1983. D, D.
christae Riihle, 1998. E, D. adolphi Wiktor,
1998. F. D. gorgonium. G, D. helicoidale. H,
D. glandulosum (Simroth, 1904). I, D. gi-
iistianiun Wiktor, 1998. 1, D. oertzeni (Sim-
roth, 1889). K, L, D. praecox. L, pocket at
proximal end of penis — different perspec-
tive of same specimen as K. Sources of
drawings: A, H, Wiktor 2000; B, Wiktor et
al. 1994; C, I, Wiktor 2001; D, G, Rahle
1998; E, I, Wiktor 1998; F, Reise et al. 2007;
K, L, Reise and Hutchinson 2001a. Repro-
duced by permission of the Museum and
Institute of Zoology of the Polish Academy
of Sciences (A, D, H), the Museum of Zo-
ology Dresden (B, G, E, I), the Natural His-
tory Museum of Crete (C, 1), Springer (F),
The Association of Polish Malacologists
(K. L).

MATING BEHAVIOR IN DEROCERAS

141

Figure 3. Variability of sarcobelum shapes
in Dewccms. Conical sarcobelum: A, D. gi-
ustianmu Wiktor, 1998. B, D. relhimiuvieii-
sis. C, D. laevc. With calcareous plate at
base of sarcobelum (cp): D, D. (Lio-
lytopeltc) caiicaskiim. Flat sarcobelum: E,
D. rodnac from SE Poland. F, D. subagreste
(Simroth, 1892). G, D. histumdatiim Wik-
tor, 2000 (sarcobelum consisting of two
lobes; here protruded through the genital
opening). Sources of drawings: A, Wiktor,
1998; B, Wiktor 2001; C, D, F, G, Wiktor
2000. Reproduced by permission of the
Museum of Zoology Dresden (A), the
Natural History Museum of Crete (B), the
Museum and Institute of Zoology of the
Polish Academy of Sciences (C, D, F, G).

Precourtship Courtship Copulation Withdrawal

Figure 4. General mating pattern of Deroceras [after Reise (1995)].

(ii) Courtship phase: both partners have their sarcobe-
lum protruded from the genital opening and as-
sume a position with their genital pores facing each
other, forming a circle or yin-yang configuration.

(iii) Copulation phase: the slugs evert their penes, en-
twine them, and mutually transfer the ejaculates
from penis to penis (there is no intromission).

(iv) Withdrawal phase: the penes are retracted together
with the attached sperm masses.

I consider the beginning of precourtship as the moment
when two slugs start to show clear signs of interest: investi-
gating each other with tentacles or mouth, circling, or trail
following. I refrain from the synonymous term “recognition
phase” {e.g., Reise 1995) because this implies too restricted a
function. 1 define courtship as starting when both partners
have everted their sarcobela. The separation of precourtship
and courtship seems reasonable in the majority of species in
which the behaviors during these two phases clearly differ.
However, there are species with less clear-cut separations: for
example, in Deroceras gorgonium the partners may not evert
their sarcobela at roughly the same time and they may re-
tract them repeatedly (Reise et al. 2007). For this reason, and
to enable comparisons with other authors, I also use the

term “precopulatory phase”, meaning precourtship + court-
ship. Barker (1999) occasionally used this term without
clearly defining it.

I define the beginning of copulation as when penis ever-
sion starts (excluding the earlier partial eversion of the distal
penis that protrudes the sarcobelum). Copulation ends and
withdrawal starts when the genitalia lose contact with the
partners. Withdrawal ends when the genitalia are fully re-
tracted into the body (but not necessarily into the original
position within the body, which can take much longer:
Webb 1961, Nicholas 1984). Behavior related to mating may
continue after withdrawal: during mating, particularly dur-
ing copulation, the partners secrete abundant mucus, which
covers the mating substratum (Simroth 1885, Gerhardt
1933, Wiktor 1960, 2000, Karlin and Bacon 1961, Kosihska
1980, Reise 1995), and this is often eaten by one or both
partners after withdrawal (Kosihska 1980, Wiktor 2000).
Also, slugs have been observed to lick otf penial gland se-
cretion received during copulation (see copulation section).

There are some discrepancies in the distinction of mat-
ing phases and their nomenclature by different authors. This
complicates interspecific comparisons. For example, Cas-
tillejo et al. (1989) called the entire mating (except precourt-
ship) “copulation”, whereas Rymzhanov and Schileyko
(1991) called it (including precourtship) “courtship”, as ap-
parently did Barker (1999). At one point in his paper
Rymzhanov ( 1994) applied a Russian term for “mating play”
to the entire mating, as do Rymzhanov and Schileyko ( 1991 )
but at another point he restricted this term to only the
precopulation phase.

Often, particularly in older publications, mating is di-
vided into only two phases: the behavior before copulation
and the copulation. The first part has been called the nuptial
dance (Wiktor, 1960, Pilsbry 1948), Vorspiel (/.c., foreplay:
Simroth 1885, 1891, Gerhardt 1933, 1935, 1939, 1940) or

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

courtship (Webb 1961, Tompa 1984). It is often unclear
whether the descriptions of courtship include only courtship
behavior in my strict sense or also precourtship. Often little
attention has been paid to this precourtship phase, either
because the behavior was not recognized as early mating, or
because observations started only at courtship (usually the
case with field observations). However, in some cases pre-
courtship was clearly considered as a first part of courtship,
as by Karlin and Bacon (1961) who used the term “posi-
tional movements”, or it was distinguished as a separate
“recognition” phase (Kosihska 1980, Reise 1995, 1997, Wik-
tor 2000). This is then followed by the “stimulation phase”,
“mating dance” or “excitatory movements” (courtship in my
strict sense; Karlin and Bacon 1961, Wiktor 2000) or “court-
ship” and “pre-copulation” (Kosihska 1980). Rymzhanov
(1994) distinguished three different precopulatory phases:
recognition (for the first, short mutual investigations), fol-
lowing (for apparent trail-following behavior, but including
some time when sarcobela are already everted), and circling.

The copulation, also called coition (Karlin and Bacon
1961, Webb 1961) or Begattung (Gerhardt 1933, 1936), has
also not been clearly defined. While almost all authors seem
to agree that copulation starts when the major parts of the
penes begin eversion and entwine, I know of no publication
clearly defining the ending of the copulation and withdrawal
phases. In only a few papers has withdrawal been distin-
guished as a phase (Reise 1995, 1997, Wiktor 2000); it was
called “postcopulation” in Deroceras stiiranyi and Kazakh D.
laeve (Kosihska 1980, Rymzhanov 1994).

Matings are sometimes broken off, primarily during the
precourtship or early courtship phases (Kosihska 1980,
Rymzhanov 1994, Reise 1995, Wiktor 2000, M. Benke, pers.
comm.); this suggests that the function of the long courtship
is not mate choice but there could be an influence of court-
ship on sperm exchange or on its subsequent use. A retreat
from courtship into precourtship behavior is also possible;
that is, one or both slugs retract their sarcobelum but evert
it again later. This seems to happen to different degrees
in different species (see comments above on Deroceras
gorgonium).

Sometimes more than two slugs are involved in pre-
courtship and/or courtship (Simroth 1885, Gerhardt 1933,
1935, Wiktor 1960, Karlin and Bacon 1961, Kosihska 1980,
Rymzhanov and Schileyko 1991, Reise 1995, unpubl. obs.).
Either all slugs (usually three, but Rymzhanov and Schileyko
[1991] saw up to seven) start mating behavior more or less
simultaneously, or one individual is attracted by a mating
couple. Rymzhanov and Schileyko (1991) stated that the
precourtship phase is omitted in such cases, and that par-
ticipating individuals had usually mated already, but Reise et
al. (2007) observed trail following with alternating partici-
pation. The participation of additional individuals in a

courtship can lead to apparent confusion and seems to delay
the mating process (Rymzhanov and Schileyko 1991, H.
Reise, unpubl. obs.). Never were more than two slugs ob-
served to be involved in a copulation (Karlin and Bacon
1961, H. Reise, unpubl. obs.); either courtship was broken
off by all slugs (and two slugs might start again later) or one
partner would leave earlier (H. Reise, unpubl. obs.) or would
not participate in copulation (Simroth 1885).

Individuals will usually mate again after a few days (H.
Reise, unpubl. obs.). However, Rymzhanov and Schileyko
(1991) and Rymzhanov (1994) observed that Deroceras
stiiranyi would mate only twice, and that in D. caiicasicwn
(Simroth, 1901) the third and fourth courtships would not
lead to copulation. This does not agree with my own obser-
vations on various other species of Deroceras; this discrep-
ancy might have to do with species differences or with meth-
odology. I found that animals will remate in the laboratory
more than twice if isolated for several days between matings,
but they stop showing interest in mating at a later stage of
adulthood, irrespective of whether they have mated already
or not. Rymzhanov and Schileyko (1991) used field-
collected specimens so they did not know the slugs’ ages and
possibly not their full mating histories.

TIMING

Even allowing for uncertain or differing definitions of
each phase in different publications, species clearly differ
considerably in the absolute and relative durations of mating
phases (Table 2). These timing differences can act as efficient
precopulatory isolation mechanisms (Reise 1995, Wiktor
2000). For example, individuals from allopatric populations
of Deroceras rodnae Grossu and Lupu, 1965 and Deroceras
praecox Wiktor, 1966 court with each other in the labora-
tory, but there is no overlap of the species-specific durations
of courtship. The slug with the shorter courtship phase (D.
praecox) proceeds to the copulation phase [i.e., everts the
penis) when its partner is still at early courtship. Because
there is no receptive partner (i.e., not another everted penis
to entwine with), the D. praecox individual retracts its penis
together with its own ejaculate, and mating is broken off
(Reise 1995).

In most species in which mating has been observed, the
courtship phase (or precopulatory phase) lasts much longer
than the copulation and takes up most of the mating. The
shortest known courtships, in Deroceras reticulatum and D.
praecox, take about 15-20 min (but courtship can take longer
in both species) and the longest courtship takes more than 7
h (D. gorgonium; the entire precopulatory phase may take 9
h: Reise et al. 2007).

The copulation is usually very brief compared to the

MATING BEHAVIOR IN DEROCERAS

143

Table 2. Duration of different mating phases in Deroceras.

Species

Precourtship

Courtship

Copulation

Withdrawal

Reference

D. agreste

>48 min

30 s

H. Reise, unpubl. obs.

>20->50 min

>60 s

Gerhardt 1933, 1934

D. gorgonium

95-276 min

145-434 min

18-25 s

Reise et al. 2007

D. laeve

up to 60 min

Barker 1999

Kazakh D. laeve

60-70 min

30-50 s

long (apophallation)

Rymzhanov 1994

(including

precourtship?)

D. lombricoides

up to >60 s

long

Simroth 1891

D. pauormitamim

>10-15 min

Webb 1961

>20-30 min

>10-15 min

Barker 1999

up to >45 min

<3-12.5 min

usually 3-5 min

Gerhardt 1939

ca. 50-80 min

ca. 3-12 min

H. Reise, unpubl. obs.

0-28 min (mean =

44-107 min

0.8-9. 5 min (mean =

0.7-5. 1 min (mean =

Benke 2006

12.5 min)

(mean = 66.7

2.7 min; without

2.6 min)

min)

penial gland
eversion )

D. planarioides

ca. 60 min

<10 s until start of

Gerhardt 1939

penis retraction

D. praecox

20-60 min

30-60 s

Reise 1995

D. rethimnonensis

ca. 30 min

Wiktor 1994

D. reticulatum

30- >90 min

<15 s

Simroth 1885

>45 min

28-49 s

Gerhardt 1933, 1934

up to >70 min

ca. 30 s

Wiktor 1960

30-almost 120

<60 s

Karlin and Bacon

min

1961

15-36 min

“seconds, or at most a

Webb 1961

few minutes”

65 min

ca. 30 s

Nicholas 1984

30-75 min

ca. 30 s

Barker 1999

D. rodnae

95-200 min

30-60 s

Reise 1995

D. sturanyi

usually >10 min

usually 30-40

“a few hours”

“some time”

Kosihska 1980

min (up to
70 min)

15-120 min

52-169 min

60-144 min

1-70 min

H. Reise and C.

Natusch, unpubl.
obs.

>19-71 min

Gerhardt 1936

26-71 min

140-168 min

Rymzhanov 1994

D. turcicum

longer than D.

very fast

Gerhardt 1935

reticulatum

up to >240 min

ca. 20 s

H. Reise, unpubl. obs.

D. caucasicum

90-210 min’^

3-4 min

Rymzhanov and

Schileyko 1991

* refers to precourtship and courtship

precopulatory phase, and penis eversion starts rather sud-
denly, often almost explosively. At one extreme, copulation
lasts only about 20 s in Deroceras turcicum (Simroth, 1894)
(Gerhardt 1935, H. Reise, unpubl. obs.) and perhaps only 10
s in Deroceras planarioides (Simroth, 1910) (exact end of
copulation unclear; Gerhardt 1939). However, copulation
can last considerably longer and, at least in one species, can

even take longer than the courtship (D. stiiraiiyi has a 26-71
min courtship and a 60-168 min copulation: Kosihska 1980,
Rymzhanov 1994, H. Reise and C. Natusch, pers. obs.).

The durations depend also on temperature (Wiktor
1960, 2000, Karlin and Bacon 1961, Kosihska 1980, Rymzha-
nov and Schileyko 1991) and possibly humidity and light
regime (Rymzhanov and Schileyko 1991 ). There is also con-

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AMERICAN MALACOLOGICAL BULLETIN 1^' Ml- 2007

siderable intraspecific variability (Table 2). Karlin and Bacon
(1961) observed that even in couples of Deroceras reticiila-
ttiin mating at the same time, the duration of precourtship
varied by a factor of three.

PRECOURTSHIP PHASE

The initial mating behavior, lasting until eversion of
both sarcobela, is the phase with the least published infor-
mation about it, because casual observations are usually
made when mating has already started. Probably in all spe-
cies the partners initially investigate each other with their
tentacles and mouth and eat the partner’s body mucus
(Kosihska 1980, Rymzhanov 1994, Reise 1995, Wiktor
2000).

Many species show some degree of trail following,
which can constitute a major part of the precourtship phase.
This behavior has been described as simple directional fol-
lowing of one slug along the mucus trail of another to catch
up with a potential mating partner (Wareing 1986, Wiktor
2000) and has sometimes also been called a chase (Gerhardt
1933, Webb 1961). However, my own observations indicate
that trail following during the recognition phase is a com-
plex behavioral pattern involving the active participation of
both partners. Usually the partner following keeps very close
to the leader. In Deroceras panortnitanum and other species
with pronounced trail following, the tail is flattened laterally
and becomes a flag-like structure (Fig. 5); it is slightly lifted
up above the ground, and waves side-to-side, either in front
of the follower’s head or between its tentacles, thus contrib-
uting to the occasional contacts between tail and tentacles. If
the follower falls behind, the leader seemingly tends to wait
tor it, tail waving. It would be interesting to conduct experi-
ments in the dark to test whether tail waving acts as a visual
stimulus, but it might also emit a chemical attractant.

Trail following has been observed in Deroceras panor-

Figure 5. Tail enlargement during trail following in Deroceras
panormitaniim.

mitaniun (H. Reise, unpubl. obs., Benke 2006, but see be-
low), D. gorgonium (although little pronounced, Reise et al
2007) and D. sturanyi (Kosihska 1980, Rymzhanov 1994). It
was described as a regular component of mating in D. re-
ticulatum by Gerhardt (1933), Quick (1960), and Wareing
( 1986), but Webb ( 1961 ) and Barker ( 1999) indicated that it
occured only occasionally. I observed that it did not occur in
D. rodnae and D. praecox (Reise 1995, 1997); Barker (1999)
stated that it did not occur in D. laeve; but Rymzhanov
(1994) did describe it in Kazakh D. laeve. The fact that it was
not mentioned by Gerhardt ( 1936, 1939) for D. planarioides
and D. sturanyi nor by Nicholas (1984) for D. reticulatwn is
more difficult to interpret.

The idea that trail following serves for catching up with
a potential mate implies that the follower is the more active,
mating-initiating partner. However, the tail waving indicates
that the leader’s role can be much more interactive than has
been assumed. Moreover, in Deroceras panonnitamifii al-
most always, and in D. gorgonium usually, it is the leader that
extrudes its sarcobelum first (Benke 2006, Reise et al. 2007,
H. Reise, unpubl. obs.). Moreover, later in courtship, at least
in D. gorgonium, this trail-leading partner is also the first to
exhibit each successive behavioral pattern. In Kazakh D.
laeve, the leader is the first to touch the partner during
courtship and to retract its penis after copulation (Rymzha-
nov 1994). Thus, if there is a partner with an initiating role,
it is probably the leading slug. However, this might differ
interspecifically, because the follower everts its sarcobelum
first in D. caucasicurn (Rymzhanov and Schileyko 1991 ) and
D. sturanyi (Rymzhanov 1994).

By the end of precourtship, the partners form an open
circle with their heads towards the partner’s tail and genital
pores pointing towards the inside of the circle, and they
often begin circling. In trail-following couples, this position
is reached by the leading slug finally crawling in a bow back
towards the follower, usually towards the latter’s tail. This is
often when the second partner or both partners evert the
sarcobelum and start courtship.

The behavior and duration of the precourtship phase
vary considerably, not only between species (Table 2) but
also within species, probably owing to variation in the mo-
tivation to mate (Benke 2006, Reise et al. 2007, H. Reise,
unpubl. obs.). Slugs in which isolation is likely to have gen-
erated a high motivation to mate tend to abridge the pre-
courtship phase and may even move on to courtship soon
after first contact (see below). This supports the suppositions
that precourtship serves to assess a partner’s readiness to
mate (Wiktor 2000) and that some behavioral patterns
might also aim at motivating a partner that does not show
initial interest. A role in species recognition is also possible
(Wiktor 2000).

Intraspecific variability might be the reason for some

MATING BEHAVIOR IN DEROCERAS

145

discrepancies in descriptions of precourtship by ditferent
authors. For instance, trail following usually occurs in Dew-
ceras stiiranyi but can be skipped by some couples (Rymzha-
nov 1994). Probably more often, however, these differences
may be caused by incomplete observations or differing opin-
ions about whether a behavior should be considered part of
mating. In D. patiormitanum, for example, Webb (1961,
1965) and Barker (1999), based on observations of unspeci-
fied numbers of couples from France and New Zealand,
reported that mating starts with circling (which occurs after
trail following) and Gerhardt (1939, based on about 30
couples from Wales) even expressly stressed that there was
no trail following. However, in the more than 150 matings of
D. panormitanum from the UK, Belgium, the Netherlands,
and Germany that I and my co-workers have watched, this
behavioral pattern was almost always present, although oc-
casionally very brief.

COURTSHIP PHASE

Courtship begins when both partners protrude their
sarcobela; this is usually a rapid process. Eversion happens
simultaneously (Wiktor 2000) or one soon after the other
(Wiktor 2000, H. Reise, unpubl. obs.). In D. panormitaniun
this is a rather fixed process: as described above, the slug
leading the trail following turns back towards the follower,
and they then form a circle. Most often, the first slug everts
at the moment of turning back, and the second slug everts
soon after formation of the circle. However, sometimes the
second sarcobelum has everted shortly before the circle is
formed, or sometimes the first sarcobelum everts shortly
after circle formation (Benke 2006, H. Reise, unpubl. obs.).
In contrast, sarcobelum eversion in D. gorgonium is quite
variable: often one slug everts long before the other and may
retract repeatedly. However, eversion can also be almost si-
multaneous in this species, and the temporary retractions do
not always occur. This causes a highly variable duration of
the precourtship (95-276 min) and courtship phases (145-
434 min) in this species. However, the duration of the entire
precopulatory phase varies much less than each component
phase (about 7-9 h); so a long precourtship is followed by a
short courtship and vice versa (Reise et al. 2007).

Kosihska ( 1980) reported that the sarcobela are “gener-
ally, although not always” everted during courtship of Dero-
cerns stiiranyi. It is unclear whether she meant that one or
both partners might not evert the sarcobelum at all during
courtship (which would be unique amongst Deroceras), or
whether it had already been everted earlier.

Stroking the partner with the sarcobelum, or at least
apparent efforts to use it to touch the partner, are the most
prominent aspects of courtship. As soon as the sarcobela are

everted, the slugs direct them towards their partners, and by
then they have formed a configuration with their genital
pores facing towards one another; often this is a circular
configuration. Sooner or later, the partners position them-
selves more and more into a yin-yang position: each head is
at the partner’s side and the genital openings thus lie close to
each other (Fig. 4B-C). A tight yin-yang is the position for
copulation. In all species, slugs get progressively closer dur-
ing courtship and stroking intensity progressively increases
but stops just before copulation.

There are large interspecific differences in the duration
of the courtship phase (see section on timing), the intensity
and speed of circling, the position of the partners towards
each other, the way of stroking, and to what extent there is
an aggressive component. I now discuss these in turn.

All species show some circling during most of the court-
ship (Barker [1999] stated that there is no circling in Dero-
ceras laeve, but see the introduction section about my
doubts). Circling is almost always clockwise and more or less
around a central point which hardly moves. If each partner
follows the other’s tail, they form a circle; if each crawls
towards the other’s right side, they assume the yin-yang
position; in both cases I term the movement “circling”. Oc-
casionally, one partner may leave the position and circle one
or two turns around its own axis or around a slower partner,
but it will always return into the original circle or yin-yang
configuration (Wiktor 1960, Rymzhanov and Schileyko
1991, H. Reise, unpubl. obs.).

Most species start with a circle and then slowly change
towards yin-yang, as in Deroceras reticiilatum (Gerhardt
1933, Webb 1961, H. Reise, unpubl. obs.), D. stiiranyi
(Kosihska 1980), and D. caiicasiciiin (Rymzhanov and Schi-
leyko 1991). Others are in the yin-yang position from a very
early stage (D. rodiiae, D. praecox, Deroceras fatrense Macha,
1981: Reise [1995, 1997, unpubl. obs.]). Deroceras gorgoniiini
seems unusual in that almost the entire courtship consists of
two alternating behavioral patterns: individuals wave their
sarcobela whilst remaining stationary (see below) and then
circle half a revolution (so that each slug ends up in its
partner’s former position).

The speed of circling generally decreases later in court-
ship. Kosihska ( 1980) distinguished two phases of courtship
in Deroceras stiiranyi: (i) a “quick circular dance” in a larger
circle, with the mouth and sarcobelum touching the part-
ner’s tail, lasting usually for 20-30 min, but sometimes more
than 1 h and (ii) a “slow circular dance” in a smaller circle,
lasting about 10 min and occasionally interrupted by 40-65
s bouts in the yin-yang position when their sarccabela and
mouths touch the area around the other’s genital opening.
During the “quick dance”, the partners needed usually 20-60
sec to complete one circle, and during the “slow dance”,
60-90 s. The decrease of circling speed in D. stiiranyi was also

146

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

noted by Gerhardt (1939) and Rymzhanov (1994), although
Rymzhanov’s timings are contradictory: first 80-95 s per
revolution, and later 68-75 s. Specimens of Kazakh D. laeve
take 1.5-2. 5 min during early courtship and 4.5-5 min later
(Rymzhanov 1994). Also in D. caucasicum, circling is faster
at the beginning (2 min per circle) than later (10-12 min)
(Rymzhanov and Schileyko 1991).

All sarcobela, particularly the larger ones, are very ma-
neuverable organs and seem adapted for touching the part-
ner and for transferring a secretion onto its body. It would
make adaptive sense also if they had a chemosensory func-
tion in assessing the physiological state of the partner, but
there is no evidence for this. As there is an enormous diver-
sity ot sarcobelum shapes and sizes (Fig. 3), it is not sur-
prising that also their use varies considerably. Species with
large, flat, tongue-like sarcobela appear particularly efficient
at transferring secretions. Such sarcobela are usually laid flat
onto the partner and stroked along its body. The large area
of such sarcobela and their extremely flexible movements
ensure that much body surface is covered. Deroceras fatrense
and D. rodnae stroke mainly the partner’s back and side,
most often around the mantle, using the underside of the
sarcobelum (although some individuals of D. rodnae also
often use the narrow edge). Deroceras praecox strokes mainly
the partner’s lower flank and the sole using the upper side of
the sarcobelum (Reise 1995, 1997, unpubl. obs.). There are
many other species that have a flat sarcobelum, but their
mating behavior has not been observed. So, it remains to be
examined whether large flat sarcobela consistently stroke
more intensely and closely than others (but see descrip-
tions of D. caiicasicum and Deroceras lomhricoides (Morelet,
1845) below).

The majority of species have conical sarcobela, varying
from rather short, stout organs to long, pointed, fmger-like
ones. The medium-long sarcobela of Deroceras reticulatiun,
D. agreste, Deroceras nitidnm (Morelet, 1845), and D. stu-
ranyi are used to stroke the partners during almost the entire
courtship, but the limited length and maneuverability per-
mit touching only the facing flank of the partner, and the
conical shape does not allow very broad contact. The rather
aggressive D. panormitaniim touches considerably less fre-
quently and less intensely during the first part of courtship
than other species with similar sarcobela (see below).

Short bump-like sarcobela such as in some Deroceras
laeve (Wiktor 2000) surely cannot stroke as well as longer
ones and must reach the partner only when it is very close.
This is one reason why I suspect that published descriptions
of D. laeve mating behavior (Gerhardt 1936, Karlin and Ba-
con 1961) have dealt with different species (see also the
introduction section). In Kazakh D. laeve, Rymzhanov
(1994) seems to have seen more touching with the mouth
than with the sarcobelum. Detailed descriptions of the

courtship of species with such rudimentary sarcobela would
be highly valuable.

At the other extreme, Deroceras gorgonium has a very
long, slim sarcobelum with a sharply pointed tip. There is
almost no body contact during early courtship (which can
last for several hours), and the sarcobelum, stretched out
perpendicular to the body, merely waves in front of the
partner’s face. Only later do the partners get closer, but even
then for much of the time the sarcobela touch each other or
the partner’s body just with their tips. We have occasionally
observed transfer of secretion droplets via the tip of the
sarcobelum (Reise et al. 2007). Maybe the sarcobela are so
long in this species to bridge the long distance between part-
ners and to apply the secretion, and animals might keep so
far apart to avoid receiving the secretion. Secretion droplets
have also been observed on the sarcobela of courting D.
caucasicnm (S. Leonov, pers. comm.).

There are a few species for which the stroking behavior
is unusual in some way, and reexamination would be desir-
able. Rymzhanov and Schileyko (1991) describe the mating
of Deroceras [Liolytopelte] caiicasicum from some introduced
Kazakh populations (Wiktor 2000) where the sarcobelum is
used for intense stroking but, judging from their published
figures, the partners seem to take up the yin-yang position
only shortly before copulation. Their figures show a large flat
sarcobelum covering rather large parts of the partner’s body.
However, there are contradictoiy opinions about whether
the sarcobelum is like this (Rymzhanov and Schileyko 1991,
Likharev and Wiktor 1980) or conical (Wiktor 2000, S. Le-
onov, personal communication: photographs of mating
couples from the Crimea). This might reflect geographical
differences between populations or indicate different taxa.

The sarcobelum of Deroceras lombricoides is also flat, but
unusually thin, wide and very short (Wiktor 2000). At ever-
sion, it resembles the arionid ligula (Wiktor 2000), and one
wonders how this fold-like structure can stroke a partner.
However, a huge flat lobe is pressed onto the partner’s back
during courtship (fig. XI in Simroth 1891, Castillejo et al.
1989), so it seems that the bulky, distal part of the penis is
everted through the genital pore together with the horse-
shoe-like fold mounted upon it.

The courtship of Deroceras (Liolytopelte) biireschi (Wag-
ner, 1934) also appears to be unusual, although the scanty
description is based on only a single field observation (Wik-
tor 1983, personal communication). The “inconspicuous”
sarcobelum (Wiktor 2000) is so small that it is not even
mentioned in an earlier anatomical description (Wiktor
1983) and hardly recognizable on the genital drawings (Wik-
tor 1983, 2000). During courtship, it is protruded together
with an everted fmger-like penial appendix assumed to be
homologous to the penial gland (Wiktor 2000), and the
partners are described as stroking each other with this struc-

MATING BEHAVIOR IN DEROCERAS

147

ture rather than with the sarcobelum (Wiktor 2000, fig. 36).
This would be the only known case in Deroceras where a
penial appending structure is already everted during court-
ship and used for stroking. The calcareous plate of Lio-
lytopelte seems not to be used during courtship except that
partners of D. caucasicurn lick the mucus off it, perhaps to
prepare it for sperm exchange (Rymzhanov and Schileyko
1991; see copulation section). However, this behavior was
not mentioned for D. bureschi, the only other species of
Liolytopelte whose mating behavior has been observeci (Wik-
tor 1983, 2000).

The sarcobelum of Deroceras turdcum is very polymor-
phic, varying from conical to a flat tongue, and from short
to rather long (Reise and Hutchinson 2001a). The observa-
tions of mating (Gerhardt 1935, H. Reise, unpubl. obs.)
indicate much similarity to D. reticidatuni. However, it
would be interesting to test whether stroking intensity and
efficiency vary intraspecifically with the shape and size of this
organ.

Slugs also differ in how close they get during courtship
and whether there is an aggressive component in the behav-
ior. While some species (particularly the ones with a large,
flat sarcobelum ) are very close from the beginning and show
no, or hardly any, aggression (e.g., Deroceras rodnae and D.
praecox), other species keep very distant {e.g., D. gorgonium,
see above), and some regularly exchange biting attacks dur-
ing the early courtship phase. Occasional biting has been
observed in D. reticulatum (Webb 1961; Karlin and Bacon
[1961] mentioned “pugnacious” strikes with the head, prob-
ably for slime feeding), D. agreste (Gerhardt 1933), and D.
gorgonium (Reise et al. 2007).

In the particularly aggressive species Deroceras panormi-
taniim, the aggression prevents them from getting dose until
the later stages of courtship. In this species partners initially
exchange vigorous bites whenever they get closer or one tries
to touch the other with its sarcobelum. Strikes onto the
flank, tail, sarcobelum or head are often recognizable as bites
rather than mere “licking”, and the partner usually reacts
with a short backward movement, frequently followed by an
attack in response. The movements of the stretched sarco-
bela look like fencing matches in which the partners try to
stroke but not to be stroked. However, bites and strokes
often do not hit the partner, and many bouts of such ag-
gression look like ritualized duels. As they do during trail
following in this species, the tails seem to play an important
role in the early phase of courtship: slightly lifted up from
the ground and still enlarged, they are often waved just in
front of the partner’s face, possibly distracting the partner’s
biting attacks from more sensitive genital and head regions.
There is strong tail lashing during phases of mutual attacks
and particularly when being bitten. However, after a while,
the partners slowly become less aggressive, their separation

decreases, and the sarcobela finally stroke as intensely as in
other species such as D. reticulatum (H. Reise, unpubl. obs.).
Deroceras planarioides also exhibits much aggression and tail
lashing (Gerhardt 1939).

Just before the start of copulation, the sarcobela are
slightly contracted and point more or less upwards (Simroth
1885, Gerhardt 1933, Wiktor 1960). The genital openings
and the bases of the sarcobela are pressed against those of
the partner (Gerhardt 1933, 1936, 1939, Nicholas 1984,
Rymzhanov 1994, Reise et al. 2007). Mouth and tentacles
may “fumble” around the genital pore {e.g., Gerhardt 1939,
Webb 1961, H. Reise, unpubl. obs.). The anterior parts of
the bodies swell, lie slightly over onto their left, and the
mantles are pulled backwards. Although circling stops, the
partners may entwine the anterior parts of their bodies in an
even tighter position just before penis eversion. Usually, par-
ticularly when one partner shows some apparent reluctance,
the slugs take up this position repeatedly, but the “reluctant”
partner will always loosen the contact again before mutual
penis eversion finally begins (Nicholas 1984, Reise et al.
2007). This is probably what Castillejo et al. ( 1989) observed
in Deroceras nitidum and interpreted as two different, alter-
nating kinds of stimulation: sarcobela opposite one another
(the figures imply that tentacle and sarcobelum touch areas
near the genital pore) alternating with stroking the partner’s
flank.

Kosihska (1980) reports a short transitional stage in
Deroceras sturanyi: the sarcobelum and the “remaining parts
of the copulatory organs” are everted and retracted before
copulation starts. There must be some intraspecific variabil-
ity, because we observed this only in one out of six matings
(H. Reise and C. Natusch, unpubl. obs.). Short, partial penis
eversion preceding copulation was also observed in D. re-
ticidatwii (Nicholas 1984).

During courtship, the ejaculate is assembled within the
penis. In Deroceras reticulatum, sperm starts flowing from its
storage site (the ductus hermaphroditicus) 10 minutes after
the start of courtship, and it first appears in the penis after
20 minutes. Ten minutes later, all sperm has arrived in the
penis, and within a further 10 minutes the prostate secretion
has completed the sperm package (Nicholas 1984). In D.
panormitamim, sperm was not found in the penis 10 min-
utes after the start of courtship, but there was an ejaculate in
a specimen killed after 30 minutes (Benke 2006). Nothing is
known about other species. However, it would be particu-
larly interesting to compare these data with those from spe-
cies with very short or very long courtships.

COPULATION

Copulation is the phase of sperm exchange and lasts
from the start of penis eversion to the moment when the

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AMERICAN MALACOLOGICAL BULLETIN U- Ml' 2007

genitalia lose contact with the partner. The two everted pe-
nes swell to several times their normal size (Nicholas 1984).
They appear as a bulbous, bluish transparent mass lying
between the partners, and it is hard to distinguish compo-
nent parts.

The speed of penis eversion and the overall duration of
copulation vary considerably. Most common is a sudden,
sometimes explosive, eversion and a very short copulation
compared to the ciuration of courtship. Some species, such
as Deroceras gorgouiwn and D. turdcum, reach maximum
eversion within one second (Reise et al. 2007, H. Reise, un-
publ. obs.). The high speed requires video-recording to dis-
criminate the sequence of events (although video may some-
times not suffice because of the difficulty in distinguishing
the parts of the penis; rapid killing of couples at different
stages of copulation is thus also helpful). For this reason,
most published descriptions give only an overall duration of
copulation, and it is often not clear whether this includes a
part of or the entire withdrawal.

As they evert, the penes entwine, in some species more
tightly than in others, and they press against each other in an
apparently species-specific way. The entwinement is
achieved by a sickle-like curve of the everted penial bags. The
shape of the penis must matter considerably for a successful
“embrace” and sperm exchange. The proximal ends of some
penes may even be spiral (c.g.. Figs. 2K-L), which might
facilitate close entwinement. The extreme is Deroceras heli-
coidale Riihle, 1998 with its spectacularly prolonged, helically
coiled, penial bag (fig. 2G, Wiktor 2000), but nothing is
known of its copulation. In this and many other species, the
curve of the penis is evident even when retracted, but in
other species the curve is generated only on eversion as a
result of the insertion site of the penial retractor muscle
(Simroth 1885, Gerhardt 1939, Wiktor 1960). Rymzhanov
( 1994) stresses that Kazakh D. laeve differs from other Dero-
ceras in that the penes are pressed less intensely against each
other; he does not mention any entwinement.

There is no spermatophore, and the ejaculate is trans-
fered as an amorphous soft mass. Simroth (1885) described
the ejaculate found in the penis of Deroceras reticiilatwn
before sperm exchange as fine strings rolled up into round-
ish bodies and surrounded by a mucous layer. At least in this
species, the sperm are indeed packed in several discrete
bundles and wrapped by several layers of secretions pro-
duced by the prostate (Nicholas 1984; her thesis also details
secretion activities of other parts of the genital tract at suc-
cessive mating stages). Simroth’s assumption ( 1885) that the
sperm mass of Deroceras is a precursor of spermatophores is
incompatible with current knowledge of the phylogeny
(Hausdorf 1998). Rather, it is probably an adaptation to a
copulatory system with external sperm exchange (Nicholas
1984).

At copulation, the ejaculate is everted together with the
donor's penis and transferred onto the surface of the receiv-
er’s everted penis; both partners donate and receive simul-
taneously. The retraction of the penis then takes the ejaculate
with it (Webb 1961, 1965, Nicholas 1984, Reise 1995, un-
publ. obs., Wiktor 2000, Benke 2006). In the few species for
which details of sperm exchange are known, it happens at
the peak of penis eversion and thus very early in copulation;
this is irrespective of how long the entire copulation lasts
(Reise 1995, 1997 [Deroceras praecox, D. rodnae, D. fatrense],
Benke 2006 [D. patiormitanum], Reise et al. 2007 [D. gorgo-
niuin] ). Even in D. sturanyi, a species with an extremely long
copulation phase, sperm exchange happens early in copula-
tion, although the transfer is slightly slower in this species
(Gerhardt 1936). It remains to be investigated what happens
during the rest of the copulation in such species with long
copulations. Spasms on the body surface of D. sturanyi
(Kosihska 1980) indicate activities of internal organs. In
other taxa, copulations continuing after sperm exchange
have been supposed to represent efforts of donors to prevent
sperm digestion and thus help the sperm reach the sperm
storage site (Michiels 1998).

The ejaculate seems to be transferred from a particular
area of the donor’s penial wall onto a particular area of the
recipient’s penial wall. In Deroceras gorgoniiiniy the ejaculate
is “slapped” onto the partner’s penis (onto an area on the
wall just distal to the base of the sarcobelum), and the do-
nating part of the penis slackens immediately after this. In D.
reticidatum, the ejaculate is collected at the base of the penial
gland in the donor’s penis and transferred onto the folds in
the proximal part of the receiver’s penis (Webb 1961, Ni-
cholas 1984).

The majority of Deroceras species have one or more
glandular penial side pockets (Sirgel 1973, Wiktor 2000). At
least in D. praecox, D. patiormitanum, and D. gorgonium
these are everted during copulation (Webb 1961, 1965,
Benke 2006, Reise et al. 2007, H. Reise, unpubl. obs.). Their
functions are largely unknown, but, at least in some species,
one pocket seems to serve as an ejaculate-holding bag prior
to transfer, probably to ensure a more exact positioning of
the ejaculate onto the partner’s penis. The function of the
additional bags occuring in a number of species is unknown.
Most is known about D. panormitanum. The penis of this
species has two side pockets: the longer one (“terminal lobe”
of Webb 1961, 1965, “longer penial diverticle” of Sirgel
1973) is everted at the beginning of copulation and slackens
immediately after maximum eversion; the shorter one (“me-
dial lobe” of Webb 1961, 1965, “shorter penial diverticle” of
Sirgel 1973) is everted slightly later (Benke 2006), or con-
siderably later (Webb 1961), pressed onto the partner’s
shorter pocket and remains inflated for a longer time, during
which pumping movements of the penial mass can be ob-

MATING BEHAVIOR IN DEROCERAS

149

served (Benke 2006). The longer pocket is filled with the
sperm mass during courtship (Sirgel 1973, Benke 2006) and
it also adds its own, probably holocrine, secretions (Sirgel
1973). At eversion, the curved longer pocket is laid around
the base of the receiver’s sarcobelum, to which the ejaculate
is then attached (Benke 2006). Little or no sperm can be
found in the shorter side pocket (Sirgel 1973, Benke 2006).
This has a different internal structure from the longer pocket
and produces two different types of secretions (Sirgel 1973).
Sirgel (1973) assumes that these secretions are added to the
ejaculate during the eversion process, but while it is still in
the lumen of the penis. In that case, it is be puzzling why this
shorter pocket is everted later than the other one and re-
mains inflated for so long. However, this secretion must be
the reason why Webb (1961) thought that the ejaculate is
transferred with the second, shorter pocket. The latter was
also reported by Barker (1999) but the perfect agreement
between these publications suggests that Barker based his
account on Webb’s.

During courtship of Deroceras caucasicwn, the sperm
mass is put into, and wrapped by, an albumen membrane
extended like a hammock within the donor’s penis between
two nodular appendices. This package is then laid onto the
receiver’s calcareous plate during copulation (Rymzhanov
and Schileyko 1991). This is apparently the function of the
plate, whose presence defines the subgenus Liolytopeltc.

In Deroceras paiwrmitanmn, D. caitcaskiim, D. reticida-
twn, D. sturanyi, and Kazakh D. Ineve, the ejaculate is trans-
ported through the vas deferens into the penis before copu-
lation starts (Simroth 1885, Wiktor 1960, Webb 1961,
Nicholas 1984, Rymzhanov and Schileyko 1991, Rymzhanov
1994, Benke 2006). However, Gerhardt (1933) and Reise
(1995) reported seeing the ejaculate being expelled through
the vas deferens during copulation of D. agreste and D. rod-
nae respectively. Gerhardt (1939) reported this also for D.
pauormitamim, which is in conflict with other, more thor-
ough, studies of this species based on rapid killing at differ-
ent mating stages (Webb 1961, 1965, Benke 2006). This calls
for a critical reexamination in D. agreste and D. rodnae using
the rapid-killing technique.

The ejaculate is visible either during the entire process
of sperm exchange or at least when the slugs separate. In at
least some species, the ejaculate forms a longish package,
sometimes stretched so much that one end still sticks out of
the genital orifice when the penis is fully retracted. The color
of ejaculates varies between white and yellow (Gerhardt
1933, 1936, 1939, Wiktor 1960, H. Reise, unpubl. obs.); it is
white in Deroceras praecox but yellow in its sibling species D.
rodnae (Reise 1995).

Species that have an appending penial gland (at the
proximal end of the penis. Fig. 1 ) evert it during copulation
(Simroth 1885, Gerhardt 1933, 1939, Webb 1961, 1965, Ni-

cholas 1984, Castillejo et al. 1989, Reise 1995, Wiktor 2000,
Reise et al. 2007. Runham (1978) wrongly wrote that it is
everted during courtship in Deroceras reticidatam, and Wik-
tor (1983, 2000) suggested this for D. bnresclii — see section
on courtship). In species in which the gland is sufficiently
large, it usually spreads over the partner’s body (Fig. 6;
Webb 1961, 1965, Nicholas 1984, Castillejo et al. 1989, Reise
and Hutchinson 2001b, Benke et al. 2005, Benke 2006, H.
Reise, unpubl. obs.). This eversion is striking in species with
a large gland {e.g., D. panonnitanuw), which can span most
of the body length. The gland is always everted for only a
short time and retraction starts immediately after full ever-
sion (Webb 1961, Reise 1995, unpubl. obs., Benke 2006).
However, the timing varies between species. In some species
with a very sudden and quick copulation, the partners evert
their glands more or less simultaneously along with the full
penis eversion and sperm exchange (Reise 1995, unpubl.
obs.). However, in D. gorgoiuwn, where copulation is also
very quick, video recording showed that the highly branched
glands are everted immediately after the ejaculate has been
transferred onto the receiver’s penis (Reise et al. 2007). Simi-
larly, Wiktor (1960) and Webb (1961) observed that in D.
retkidatuni the gland is everted at the end of copulation
when other parts of the penis are collapsing. In D. panor-
nutanum, the comparatively slow copulation makes it even
clearer that gland eversion is after sperm exchange, when the
other parts of the penis have begun to retract (Fig. 5; Webb
1961, Reise and Hutchinson 2001b, Benke et al. 2005, Benke
2006). Moreover, the second partner begins to evert its gland
only when the first partner has finished, or almost finished,
retracting its penis and gland and often has already started to
crawl away. Particularly in the latter case, some or all fingers
of the gland may miss their target and spread on the ground
(H. Reise, unpubl. obs.). Benke (2006) observed intervals of
10-72 s (mean = 32 s) between the eversions of each part-
ner’s gland, but she did not find a clear difference in the

Figure 6. Eversion of the penial gland in Deroceras panormitaimm.
A, Maximum eversion of the left slug’s gland. B, Eversion of the
right slug’s gland; the partner has already retracted and is crawling
away. Drawn from two video frames.

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success of first and second gland eversions: out of 35 mat-
ings, Benke observed full or partial misses for the first ever-
sion in four cases and for the second in three cases.

The only report that the penial gland of one partner
does not evert during a copulation is from two matings (out
of 27) of Deroceras paiiormitainim (Benke 2006, personal
communication). The penial gland of one of these slugs was
still filled 120 min after copulation, indicating that the se-
cretion had been produced but not transfered. The penial
gland of the other slug, killed after 30 min, was empty; it
might not have produced a secretion or eversion might have
been overlooked. It can be difficult to spot the eversion if the
gland spreads underneath the partner’s body, as appears to
be common in D. gorgonium (Reise et al. 2007). Nothing is
known about how species with a very small penial gland,
such as D. agreste, use it.

The function of the penial gland is not known. Webb
(1961) assumed that it functions for “semen-securing and
retaining” during retraction, but there is no evidence for this
(Nicholas 1984, Benke 2006, H. Reise, unpubl. obs.). How-
ever, the observation that it is everted only after sperm ex-
change in Deroceras panorinitamim has prompted the hy-
pothesis that it transfers a secretion to manipulate the
partner physiologically so as to increase the chance of pa-
ternity (Reise and Hutchinson 2001b; cf. the proven function
ot the dart in Cornu aspersum [Muller, 1774]; Koene and
Chase 1998, Rogers and Chase 2001, 2002, Landolfa et al
2001, see also the discussion section). The secretion would
thus act as an allohormone (Koene and Ter Maat 2001).
Histological studies and direct observations have shown that
the penial gland is filled up during early courtship and that
this secretion is transferred onto the partner’s skin (Nicholas
1984, Benke et al. 2005, Benke 2006). Observations that mat-
ing partners in D. panormitamim try to lick off this secretion
from their own body just after copulation (Benke 2006)
might be efforts to escape manipulation by the partner. At
least some of the secretion accumulating in the penial gland
before copulation is transferred from the prostate (Nicholas
1984), but there are also indications of aprocrine secretion in
the penial gland itself (Sirgel 1973, Nicholas 1984, Benke
2006).

Although the usual mode in Deroceras is simultaneous
mutual sperm exchange, unilateral sperm transfer occurs
occasionally. In D. rodnae, Reise ( 1995) observed a few such
cases associated with unusual behavior of the sperm donor.
The donor slug tried to keep contact with the recipient by
following, with genitalia still partly everted, but the recipient
fully retracted its genitalia and showed no further interest. I.
Schulze (pers. comm.) did not find an ejaculate in 6 out of
105 individuals of D. panormitamim killed immediately after
copulation. Benke (2006) killed couples of the same species
10-90 min after copulation and found that in 9 out of 39

individuals there was no ejaculate in the penial sac (where
the received sperm mass should have been). At least 4 of the
partners of these 9 slugs had one sperm package in their
penial sac as well as another one in the long side pocket; that
is, one ejaculate that they had just received and one prepared
for donation. Gerhardt (1933) reported ejaculates of D. re-
ticulatum which missed the receiver and ended up on the
ground (I. Schulze [pers. comm.] observed another in-
stance). Such lost ejaculates, or partial ejaculates, are also
implied by Nicholas’ (1984) statement that after copulation
D. reticnlatiim eat remaining sperm along with accumulated
mucus. Cases of unilateral sperm exchange where an ejacu-
late was produced but not donated successfully (either kept
by the sperm owner or lost during transfer) might represent
accidents. Alternatitively, they might be caused by one part-
ner deliberately not performing one of the two sexual roles.
Cheating on the reciprocal sperm exchange by avoiding do-
nating sperm is an unlikely explanation, because this partner
has paid much of the cost of the male role by having pro-
duced an ejaculate. However, deliberate sperm rejection by a
receiver would be more plausible. Sperm rejection (or eat-
ing) might be advantageous over sperm digestion in the
bursa copulatrix if the ejaculate contains manipulating sub-
stances causing costs to the receiver.

Kosihska (1980) observed several couples of Deroceras
stiiranyi in which only one partner fully everted the penis,
and she concluded that only one partner received sperm.
However, I doubt that any sperm transfer took place because
this must surely require not only a donating but also a re-
ceiving penis. This is also relevant to the question of whether
aphallic indviduals of D. laeve can receive sperm from an
euphallic individual (see sections on genital morphology and
withdrawal). The only other recorded cases of unilateral pe-
nis eversion are from mixed couples of two different species,
D. praecox and D. rodnae (Reise 1995; see section on timing).

Webb (1961) noted that copulating Deroceras reticida-
tiim “invariably” take up their own ejaculate or a mixture of
both partners’ ejaculates, but he did not indicate what evi-
dence prompted this conclusion.

The duration of the copulation phase varies remarkably
between species (Table 2). Gerhardt (1939) classified Dero-
ceras into two groups: (1) species with short copulations
during which partners entwine further: D. reticulatum, D.
agreste, D. turcicum, and D. pJanarioides; (2) species with
long copulations during which partners do not entwine fur-
ther: D. panormitamim, D. lombricoides, and D. stiiranyi
(which he called D. laeve, see introduction; mistakenly, on p.
199, he listed D. reticulatum and D. agreste instead). Dero-
ceras fatrense, D. gorgonium, D. praecox, D. rodnae, and Ka-
zakh D. laeve should now be added to the fast group. Ger-
hardt’s ( 1939) hypothesis of a consistent association between
speed of copulation and further entwining during copula-

MATING BEHAVIOR IN DEROCERAS

151

tion must now be rejected: D. prciecox, a species with a very
fast copulation, does not entwine further. Instead, the part-
ners are even pushed apart by the everting penial mass (H.
Reise, unpubl. obs.). I observed the same phenomenon in
video-recorded copulations of D. tiircicum (H. Reise, un-
publ. obs.), which is in disagreement with Gerhardt ( 1935).
However, he might have worked with a different species (he
called his slugs “D. aff. tiircicum”).

The species with “fast” copulations form a rather ho-
mogeneous group with a copulation time of less than one
minute. In contrast, the “long” copulations of the remaining
species vary from three minutes to several hours and their
intraspecific variability is much higher: about 3-12 min in D.
panormitanum and 60-148 min in D. sturnnyi (Table 2).
Examination of additional species may well establish more of
a continuum between the groups with short and long copu-
lations, but even then the variation in duration would re-
main to be explained.

WITHDRAWAL

Irrespective of whether the copulation is short or long,
penis retraction is usually a rather fast and straightforward
process once started (Gerhardt 1935, 1936, 1939, Reise 1995,
unpubl. obs.) although it is much slower than the eversion.
There are a few exceptions: (i) delayed or only partial re-
traction by one of the partners in rare cases of unilateral
sperm exchange (see preceding section); (ii) late eversion of
the penial gland in Deroceras paiiormitamim when the main
part of the penis is already retracting (see preceding section);
(iii) one partner remaining inactive at the mating site with
fully everted penis in D. sturanyi (sometimes) and Kazakli D.
Ineve (always), with or without apophallation (see below).
Because eversion of the penial gland probably plays an im-
portant role and overlaps other components of copulation, I
include gland eversion as part of the copulation phase and
define the end of the copulation phase (and the start of the
withdrawal phase) as when the genitalia no longer have any
contact with the mating partner. As a consequence, penis
withdrawal and sperm uptake may start before the with-
drawal phase.

The most common mode seems to be that both partners
withdraw more or less simultaneously, performing intense
pumping and rocking movements with the anterior body. As
soon as the genitals untangle, the slugs separate and may
crawl away from each other before finishing withdrawal.
Usually the partners show no further interest in each other,
with the exceptions (i) and (iii) mentioned in the preceding
paragraph. The sarcobelum is the last part to disappear in-
side. Some ejaculate may stick to the last genital parts to
remain everted, and then the slug may turn its head to its

genital orifice and apparently push the sperm mass in (Reise
1995). However, Benke (2006) observed two cases in Dero-
ceras panoriiiitaniini where one partner tried to eat ejaculate
sticking to its own, not yet fully retracted, genitalia. So the
question of whether the slugs are assisting uptake of the
ejaculate or eating it {cf. Karlsson and Haase 2002) needs
reinvestigation. Eating could also be a last resort if the up-
take is going to fail, similar to when the ejaculate has been
lost onto the ground.

Some unusual behaviors during the withdrawal phases
have been reported in Deroceras sturanyi and Kazakh D.
laeve. Probably in many matings of D. sturanyi, only one
partner retracts its penis immediately after copulation and
crawls away. The other partner remains motionless at the
mating site for some time with its penis still everted and
slowly retracts it later. Kosihska (1980) apparently observed
this in all matings but unfortunately does not tell how many
she observed or how much later the second partner re-
tracted. We found a very high variability in the mating be-
havior of D. sturanyi collected in Germany, only about 200
km away from Kosihska’s population. In only one out of six
full matings did one partner retract its penis considerably
later (70 min) than the other. In four cases, both partners
retracted more or less simultaneously, with not more than
one minute difference. In one pair, one partner left only the
sarcobelum everted, which was retracted after 25 min (H.
Reise and C. Natusch unpubl. obs.). Rymzhanov (1994) and
Gerhardt (1936) did not observe delayed retraction in any of
the matings of 18 D. sturanyi from Kazakhstan or three
matings of D. sturanyi from Germany.

Rymzhanov (1994) has reported retraction by only one
partner as a regular pattern in matings of 12 Kazach Dero-
ceras laeve. Moreover, the penis of the slug remaining at the
mating site was not retracted later but bitten off by its owner
and finally eaten by the other slug, which returned to the
mating site and assisted amputation by pulling. Apophalla-
tion had previously been reported only from Arioliniax
(Morch, 1860), in which the penis may also be eaten by the
partner (Leonard et al. 2002). These slugs are from a differ-
ent family, Arionidae. They copulate by mutual or unilateral
penis intromission and one penis (or both) is occasionally
bitten off, usually after a period of struggle and pulling by
the owner (implying that the penis gets trapped in the part-
ner’s genital tract (Mead 1943, Harper 1988, Heath in Pils-
bry 1948; 710-711). Rymzhanov’s (1994) observation of Ka-
zach D. laeve is even more remarkable because the penis is
not trapped in the partner’s genital tract, and because apo-
phallation is the rule rather than the exception. It seems hard
to imagine a reason why an individual should voluntarily
initiate amputation of its own penis; if it were the metabolic
cost of keeping the organ when it will not be used again, it
is puzzling why the slug does not eat it itself. One possible

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explanation might be that the amputee has been manipu-
lated by the partner by transfer of a secretion which inhibits
retraction ability. The partner might be interested not only
in gaining an additional food source but also in restraining
it from remating and thus preventing sperm competition
and/or shifting sex allocation towards the female function
( the external mode of sperm exchange in Deroceras makes it
improbable that individuals without a penis can donate or
even receive sperm, in contrast with ArioUmax where aphal-
late individuals can still receive sperm because there is in-
tromission [Leonard et al. 2002]). However, this explanation
would only be plausible if the amputee has been able to take
up the received ejaculate without penis retraction. Rymzha-
nov (1994) reported that the amputee was always the fol-
lower during precourtship trail following, so its fate was
fixed at the start of mating. His paper further suggests that
this apophallation is the origin of aphallic and hemiphallic
individuals in D. laeve (see section on genital morphology).
However, in German and North American populations of D.
laeve, individuals that have grown up in isolation are often
aphallic (Barth 2001, V. Barth and H. Reise, unpubl. obs.,
Reise and Hutchinson 2002, Jordaens et al. 2006). The phe-
nomenon and the species identity are worthy of further in-
vestigation.

After full withdrawal, there is usually some eating of
mucus by one or both partners. They may return to the
mating site and lick the mucus-covered ground (Kosihska
1980, Nicholas 1984, Rymzhanov 1994, H. Reise, unpubl.
obs.) or lick their own body surface (Benke 2006, H. Reise,
unpubl. obs.), but there is much intraspecific variation. The
function of this behavior is unknown. The mucus might
simply serve as a nutritious substance. But a slug licking its
own body might be tiying to consume, and thus inactivate,
an allohormone transferred by the partner by its sarcobelum
during courtship or by its penial gland during copulation
(see those sections).

Full withdrawal of all genital parts into their original
position takes much longer than the withdrawal phase. The
retraction of the penial gland of Deroceras reticulatam takes
75 minutes according to Nicholas (1984) and “several
hours” according to Webb (1961), but it might well take
more time in species with more highly branched or longer
penial glands.

There is some controversy about the fate of the sperm
after penis retraction. Because the sperm is transfered during
copulation onto the penis, immediately after retraction it
must lie somewhere in the main penial bag (Nicholas 1984,
Benke 2006, Reise et al. 2007. It must then move via the
atrium towards the bursa copulatrix and oviduct. However,
there is an array of opinions, some better supported than
others, about how long the ejaculate remains in the penis,
whether the sperm has to enter the bursa copulatrix or not

before proceeding to a sperm storage site, and whether it
migrates along the female or male groove of the spermovi-
duct (Webb 1961 Sirgel 1973, Nicholas 1984, Tompa 1984,
Rymzhanov and Schileyko 1991, Wiktor 2000).

DISCUSSION

Despite the mixed quality of observations and the small
proportion of species examined, considerable variation in
the mating behavior of Deroceras is already apparent. How-
ever, the extent to which this variation is intraspecific or
interspecific is not always clear, and there is a need to in-
vestigate the intraspecific variation between and within
populations. The genus contains many nominate species of
similar morphology; mating behavior can sometimes pro-
vide a suite of extra characters to help resolve such taxo-
nomic problems. Because mating behavior is a potential iso-
lating mechanism, it will also be interesting to study mating
behavior across the contact zones of closely related species
with parapatric distributions.

Mating in Deroceras involves very complex copulatory
organs and behavioral patterns. After a comparatively long
time of caressing with the sarcobela, copulation usually be-
gins very suddenly and sperm transfer occurs often, or
maybe always, at the very beginning of copulation. This
demands perfect coordination and penis alignment between
the partners. It seems plausible that slight discrepancies in
the preceding courtship phase {e.g., due to different shape
and movement of the sarcobelum) or during sperm transfer
{e.g., due to shape differences of the penes and thus less
perfect entwining) can impair sperm exchange. I have often
observed difficulties between conspecific mating partners
from different populations, which might be caused by such
slight differences (Reise 2001, unpubl. obs.). In other taxa,
intraspecific variation of sexual behavior among populations
is common, and morphological, behavioral, and other traits
that determine mate recognition {e.g., pheromones) may
evolve quickly and play a significant role in allopatric spe-
ciation (Arnquist and Danielsson 1999, Verell 1999). If, as
hypothesized in the introduction, sexual conflict between
mating partners drives rapid evolution of penial morphology
and mating behavior in Deroceras, interpopulation incom-
patibilities, rapid speciation, and many species might be the
consequence.

There is so far little evidence addressing the importance
of sexual conflict in Deroceras. The unusual behavior of
sperm donors in rare cases of unilateral sperm transfer in D.
rodnae (see section on copulation) might be interpreted as a
preference for the female (sperm receiving) role and for
occasional cheating in a mating system based on reciprocity
(according to the hermaphrodite’s dilemma model: Leonard

MATING BEHAVIOR IN DEROCERAS

153

1990, 1991, 2005). Both the preference and the cheating
suggest partner conflict over sexual roles. However, the cases
of unilateral sperm transfer when both ejaculates had been
produced might represent sperm rejection, i.e. preference of
the male role. The preference for one of the two sexual roles
has been repeatedly predicted for mating systems of simul-
taneous hermaphrodites, but it is controversial which factors
should decide the prefered role (reviewed by Anthes et al.
[2006]).

Michiels ( 1998) has suggested that a preference for the
female role could result in elaborate behavior to stimulate
the partner into donating sperm and to assess its readiness to
do so; until reciprocity is assured, no sperm should be do-
nated. Although we know almost nothing of the mechanisms
of stimulation or assessment, this might explain the veiy
long courtships in Deroceras and some species of Umax Lin-
naeus, 1758 (Gerhardt 1933), which surely entail higher
costs than short courtships (Baur 1998). Assurance of reci-
procity and/or manipulation of the mating partner into ac-
cepting sperm have been proposed as an explanation of
elaborate courtship in a nudibranch (Karlsson and Haase
2002). A further possibility is that the length and vigor of
courtship have been sexually selected as honest signals of the
partner’s condition and thus of its genetic quality. Although
complete mate rejection seems to be rare once courtship has
started, “weak” courtship behavior might make it more
likely that sperm exchange is unilateral, or might reduce how
much sperm is donated or the partner’s use of donated
sperm.

The length of courtship contrasts with the extremely fast
penis eversion and sperm transfer in some species of Dero-
ceras. Tight physical contact ot penes during copulation
might ensure reciprocal sperm exchange {cf. the entwined
penes in Limax\ Michiels 1998). However, a possible conse-
quence of the rapidity of copulation is that once an indi-
vidual has everted its sperm mass with its penis, it may have
no chance to withhold its ejaculate, should it realize that the
partner is not going to donate. This conflicts with Davison et
fl/.’s (2005) assumption that in systems with simultaneous
reciprocal mating, cheating is possible only after intromis-
sion (meaning sperm transfer). The ability to cheat before
sperm exchange might be a peculiarity of groups with ex-
ternal penis-to-penis sperm transfer. It remains unclear what
has favored the evolution of the rapid penis eversion and
sperm transfer; the advantage of being slightly quicker to
evert than the partner seems unlikely to be in unilaterally
snatching the partner’s ejaculate, because the penis has to be
fully everted before the ejaculate becomes available. It seems
more compatible with a preference for the sperm-donating
role.

Another possible cause of partner conflict, widely as-
sumed to exist in gonochorists as well as hermaphrodites.

concerns control over fertilization. Sperm donors should
endeavor to ensure that their sperm fertilizes the partner’s
eggs. If the manipulations to achieve this, or the loss of
control itself, involve a cost to the sperm receiver, counter-
adaptations are expected. In Deroceras the most promising
evidence of a manipulation comes from the appending pe-
nial gland in species in which it is everted only alter sperm
exchange. This cannot serve sperm exchange but might ma-
nipulate the partner into using the donor’s sperm, as do
allohormones transferred by the love dart of Cornu aspersum
(see section on copulation). However, other functions are
also possible, such as increasing the number of eggs avaiktble
for fertilization as might be the case in Lynmaea stagiialis
(Linnaeus, 1758) (Koene et al. 2006) or delaying subsec]uent
mating by an antiaphrodisiac effect {cf. Andersson et al.
2004), or marking the partner so as to prevent repeated
mating with the same mate (cf. Ivy et al. 2005).

The unusual external mode of sperm exchange could
also indicate sexual conflict over control of egg fertilization:
Emberton ( 1994) hypothesized that the extremely prolonged
intertwining of penes of some polygyrid and Liinax species
represent male efforts to place the ejaculate as far away from
the partner’s gametolytic bursa copulatrix as possible. The
evolution from penis intromission into the bursa trunk to-
wards external transfer from penis to penis has evolved at
least four times independently within the pulmonates (Em-
berton 1994). However, Solem ( 1974) thought that elaborate
genital structures, including very prolonged penes, evolved
to enhance species recognition — that is, as an isolation
mechanism. I tend to agree with Emberton ( 1994) and think
that circumvention ot female control may have driven the
abandonment ot direct sperm transfer into the bursa copu-
latrix. However, I do not agree that a longer penis would be
even better in circumventing the bursa copulatrix than a
moderately long one. Allosperm is believed to travel up the
spermoviduct, and thus to pass the entrance of the bursa
trunk, irrespective of penis length. Another possible expla-
nation for the very prolonged penes of polygyrids and Umax
is that they have been sexually selected as condition-
dependent cues used to assess the desirability of the mating
partner. In contrast, most of the various extravagant penial
structures in some Deroceras species (Fig. 2) seem unlikely to
be reliably condition-dependent (their size is still rather
trivial compared with that of the body), but they remain
most explicable by some form of arms race.

There are many other aspects of mating behavior and
morphology that one might examine to discover indications
of partner conflicts in Deroceras: the role of the (probably
secretion-transferring) sarcobelum, the function of addi-
tional penial side pockets (besides ejaculate-holding), the
processes occuring after sperm exchange before penis with-
drawal, the occasionally long-delayed penis withdrawal in D.

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j

stumnyi, and apophallation in Kazakh D. laeve. Other sug-
gestions for future research are the study of mating in species
that represent morphological or behavioral extremes, and
the relating of behavior to morphology in species poly-
morphic for a penial structure (such as the highly variable
sarcobelum in D. turciciim: Reise and Hutchinson 2001a).
Several other fruitful lines of research depend on the devel-
opment of a molecular-based phylogeny.

Finally, I will make the following recommendations for
studies of mating behavior in Deroceras. (i) Observations
should include all mating phases, which normally requires
introducing individuals to each other in the laboratory, (ii)
Records should be kept of matings that cease before copu-
lation, in particular noting at what stage this happens and
the size and mating histories of the individuals involved, (hi)
Individuals should be followed so as to reveal correlations in
which partner takes which role at different stages, (iv) Note
whether both partners donate and receive an ejaculate, and,
if not, whether both partners have manufactured sperm
packages, (v) Direct observations should be complemented
by video-recording and rapid killing of couples at successive
mating stages, (vi) The studies should include several pairs
and, preferably, more than one population.

ACKNOWLEDGEMENTS

Many thanks to Claudia Natusch, Marleen Scheibe, Jo-
sephine Sauer, and Christiane Matthieu, who helped with
the observations of mating behavior of Deroceras pariormi-
ta)ium and to Bettina Zimdars for assistance with some of
the figures and the reference list. I am very indebted to John
M. C. Hutchinson for many valuable discussions and com-
ments on several versions of the manuscript, for help with
the figures, and for correcting the English. Many thanks also
to Andrzej Wiktor, Joris Koene, Tim Pearce, Janice Voltzow
and an annonymous referee for vaJuable comments on the
manuscript. I am grateful to Mandy Benke, Ines Schulze and
Sergey Leonov for permission to incorporate their observa-
tions prior to publication and to Andrzej Wiktor and Wolf-
gang Riihle for permission to use their drawings for Figures
2 and 3. Janet L. Leonard patiently answered my questions
about banana slugs’ mating systems; she also invited me to
talk at the AMS conference, thus initiating this paper.

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Accepted: 31 October 2006

Ainer. Maine. Bull. 23: 157-172

Reproductive biology and mating conflict in the simultaneously hermaphroditic
land snail Arianta arhustorum^

Bruno Baur

Department of Environmental Sciences, Section of Conservation Biology, University of Basel, St. lohanns-Vorstadt 10,

CH-4056 Basel, Switzerland, Bruno. Baur@unibas.ch

Abstract: This review summarizes the present knowledge on the reproductive biology, mating system, sperm competition, sex allocation,
and mating conflict in the simultaneously hermaphroditic land snail Arianta arbustoriini (Linnaeus, 1758) (Helicidae). Field studies and
controlled laboratory experiments indicate that mating is random with respect to shell size. However, subtle effects of inbreeding (reduced
hatching success of eggs and viability of juveniles) and outbreeding were found. Individuals of A. arhiistorum mate repeatedly with different
partners and store viable sperm for more than one year. Spermatophore transfer is highly reciprocal, but the number of sperm they contain
(800,000-4,000,000) is not necessarily equal. Snails need 3-4 weeks to replenish their autosperm reseiwes after a successful copulation. Sperm
are monomorphic. However, there is considerable among-population — and to a minor extent — within-population variation in total sperm
length. Sperm utilization patterns in double-mated individuals of A. arbmtonim revealed striking differences among individuals. There is
a huge variation in the structure of the spermatheca, which consists of 2-9 blind tubules. Different lines of evidence suggest that the snails
might be able to store and expel sperm stored in single tubules and thus promote a selective fertilization of eggs (cryptic female choice).
Maternal investment in eggs is considerable. Snails mated 1, 2, or 3 times showed that irrespective of the number of matings the individuals
devoted >95% of the resources into the female function.

Key words: Gastropoda, mating system, reproductive behavior, sex allocation, sperm competition

Traditional models of sexual selection explain the evo-
lution of secondary sexual traits (mainly in males) and pref-
erences for reproductive partners disjrlaying such traits
(mainly in females; Andersson 1994). Although these models
differ in how sexual selection operates, they all propose that
partner choice increases both average male and average fe-
male fitness in a population (Pizzari and Snook 2003). Re-
cent theoretical and empirical work, however, has stressed
that sexual conflict may be a potential broker of sexual se-
lection. When the fitness interests of females and males di-
verge, a reproductive strategy that increases the fitness of one
sex may decrease the fitness of the other sex. In this way
unequal reproductive investments per gamete (egg versus
sperm) lead to sexual conflicts (Trivers 1972). In recent
years, experimental evidence for sexual conflicts has been
reported in a variety of gonochoristic animals (Chapman
et al. 2003, Arnqvist and Rowe 2005). Mechanisms of sexual
conflicts are of particular interest in simultaneous hermaph-
rodites (Michiels 1998).

In hermaphrodites, selection for female traits cannot be
independent from selection for male traits of the same in-
dividual. Sexual selection for traits related to mate attraction
is assumed to be weaker in hermaphrodites (Greeff and
Michiels 1999a), but because many simultaneous hermaph-

rodites mate repeatedly and store sperm for long periods,
they are affected by forces similar to those leading to com-
plicated mating strategies and sperm competition in animals
with separate sexes (Charnov 1996, Michiels 1998, Angeloni
et al 2003). However, unlike gonochoristic species, simul-
taneous hermaphrodites have an additional reproductive
strategy; they can adjust the ratio of resources invested into
reproduction in the female role versus the male role, de-
pending on current selection pressures and environmental
conditions (Charnov 1982). Sex allocation complicates
sexually selected strategies because any increased investment
in one sexual role results in a decreased investment in the
other.

Pulmonate gastropods are excellent study organisms for
determining the mechanisms and effects of sperm competi-
tion and sex-specific reproductive allocation on both the
fitness of snails and the conflict between male and female
function within an individual (Baur and Baur 2000). Al-
though the theoretical framework for sexual conflicts in si-
multaneous hermaphrodites already exists, such conflicts
have rarely been examined experimentally in terrestrial gas-
tropods (for an exception see Chase 2007).

The aim of this review is to summarize the present
knowledge on the reproductive biology, mating system,

* From the symposium “Gastropod Mating Systems” presented at the joint meeting of the American Malacological Society and Western
Society of Malacologists, held 26-30 fune 2005 at Asilomar, Pacific Grove, California.

157

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

sperm competition, sex allocation, and mating conflict in the
pulmonate land snail Arinnta arbiistorwn (Linnaeus, 1758).
The strength of this model system comes from the possibility
of conducting field experiments and of studying the behav-
ior of individual snails under controlled conditions; the abil-
ity to produce laboratory crosses with relative ease; and the
availability of well-established techniques {e.g., to estimate
the number of sperm delivered and the number of eggs
produced [Locher and Baur 1997], and to measure sperm
length, sperm velocity, motility and longevity [Minoretti
and Baur 2006]; two novel non-invasive techniques for iso-
lating DNA and a set of microsatellite markers which can be
used for paternity analysis [Locher and Baur 2001, Arm-
bruster et al. 2005]). By compiling the existing information,
this review should stimulate future studies on mating sys-
tems and reproductive behavior in simultaneously hermaph-
roditic gastropods.

NATURAL HISTORY

Members of the species Arianta arbiistorwn are simul-
taneously hermaphroditic, helicid land snails common in
moist habitats of northwestern and central Europe, living at
elevations of up to 2700 m above sea level in the Alps (Ker-
ney and Cameron 1979). Adult shell size diminishes with
increasing elevation (population means of shell breadth
ranging from 13 to 23 mm; Burla and Stahel 1983, Baur
1984a, Baur and Raboud 1988). The species shows a high
phenotypic variability in shell and soft body color that is
related to habitat and elevation (Burla and Stahel 1983, Git-
tenberger 1991, Gosteli 2005). Variation in shell color is
continuous between large brown morphs, occurring mainly
in forests in the lowlands, and small yellow morphs in alpine
grasslands at high elevation. Population density is highly
variable in A. arbustorum, ranging from 0.01 to 1 1 adults per
m^ (Baur 1986a, 1988a). On mountain slopes, A. arbiistorwn
can be continuously distributed over large areas and thereby
reach huge population sizes (Baur 1986a). In contrast, partly
or completely isolated populations consisting of only a few
individuals can persist in small patches of marginal habitat
(Baur 1993a, Ak(,:akaya and Baur 1996). Information on pat-
terns of daily movement and dispersal in A. arbustorum can
be found in Baur (1986a), A. Baur and B. Baur (1990, 1992,
1993), Baur et al (1997), and Kleewein (1999).

Arianta arbustorum has determinate growth. Sexual ma-
turity is attained after the completion of shell growth. How-
ever, matings involving individuals that had not yet finished
their shell growth have been observed in a few cases (Baur
1984b). Age at maturity increases from 2 years in snails
living in the lowlands to 5 years in individuals in alpine
populations, but median adult longevity (3-3.5 years; maxi-
mum 14 years) and adult survival rates (0.5-0. 7 per year) are

approximately the same at all elevations (Baur and Raboud
1988). Individuals of A. arbustorum mate repeatedly during
the reproductive season (Baur 1988b). In the field, snails
deposit one to three egg batches, each consisting of 20-60
eggs, per reproductive season (Baur and Raboud 1988, Baur
1990a, B. Baur and A. Baur 1993). During tbe winter snails
hibernate in leaf litter or soil (Terhivuo 1978).

Arianta arbustorum exhibits large interpopulational ge-
netic variation (Arter 1990, Haase et al 2003) and shows
considerable variation in behavior and rate of parasitic in-
festation (Baur 1986b, 1994a, Baur and Gosteli 1986, Baur
and Baur 2005). In some populations, individuals of A. ar-
biistorurn are infested by the mite Riccardoella limacum
which sucks blood and lives in the snails’ lungs. Prevalence
of mite infection ranged from 45.8 to 77.8% in four natural
populations of A. arbustorum while in seven other popula-
tions no infected snails were found (Baur and Baur 2005).
Information on density-dependent growth and potential
predators on A. arbustorum is summarized in Reichardt et al
(1985). Aspects of food choice in A. arbustorum have been
examined by Speiser and Rowell-Rahier (1991, 1993) and
Speiser et al (1992).

MODE OF REPRODUCTION

Arianta arbustorum was long believed to reproduce ex-
clusively by cross-fertilization (Lang 1904). Chen (1994)
reared 44 individuals in isolation from the subadult stage
and recorded their reproductive performance for 1, 2, or 3
years under laboratory conditions after they had attained
sexual maturity. The snails produced eggs without being
mated, however, in a significantly lower number than did
mated control snails. In their first reproductive season, only
1 egg hatched out of 284 eggs (hatching success 0.4%) pro-
duced by 33 snails. In tbe second reproductive season, 32 of
the 671 eggs laid by 29 snails hatched (hatching success
4.8%). In the third reproductive season, 23 of the 191 eggs
laid by 6 snails hatched (hatching success 12.0%). The per-
centages of snails that laid fertile eggs were 3.0% (1 of 33
snails), 31.0% (9 of 29 snails), and 33.3% (2 of 6 snails),
respectively, for the 3 years after maturation. The number of
hatchlings produced by unmated snails was 1-2% of that
produced by mated snails of the same age. This indicates that
A. arbustorum is able to self-fertilize, but with a great reduc-
tion in fitness. Cross-fertilization is the dominant mode of
reproduction in this species. Self-fertilization might occur
only if no mating partners were available over long periods.

MATING PATTERNS

Size-assortative mating is a common pattern in natural
populations of many invertebrate and vertebrate species.

REPRODUCTIVE BIOLOGY OF ARIANTA ARBUSTORUM

159

Hermaphroditic land snails would greatly enhance their re-
productive success by choosing large mates since female fe-
cundity (number of clutches, clutch size, and egg size) is
positively correlated with shell size (Wolda 1963, Baur
1988a, Baur and Raboud 1988). Ridley ( 1983) suggested that
size-assortative mating patterns should occur in simulta-
neously hermaphroditic land snails with reciprocal fertiliza-
tion and size-related female fecundity. He argued that all
individuals invest substantially (all their eggs) in mating, so
there will be selection for careful mate choice. In another
approach, Parker (1983) proposed a model for indiscrimi-
nate mate choice (random mating). This should occur when
there is little variance in mate c]uality in both sexes, and/or
when search costs for mates are high {e.g., low encounter
rates due to low population densities or low mobility). Baur
(1992a) examined mating patterns in natural populations of
Helix poniatia Linnaeus, 1758 and Ariauta arbiistoniin. In a
large population of H. pomntia (700-1000 individuals) in
Sweden, snails showed a slight (but non-significant) ten-
dency towards size-assortative mating, whereas mating in a
SLibalpine population of A. arbustorwn in Switzerland was
random with respect to size. Baur (1992a) also conducted
controlled choice experiments to test whether individuals
of A. arbustorwn discriminate between mating partners of
different sizes and whether a large shell size might be of
advantage in groups of courting snails to increase mating
success. In mate-choice tests with snails of different shell
size, pairs formed randomly with respect to size. Courtship
was neither hindered nor prolonged in pairs with large size
differences. In the second experiment, a large A. arbustorum
was placed close to two courting conspecifics (both smaller).
The larger snail interfered with the courting snails, but in
general did not displace one of them. Courtship progressed
to copulation only if one of the three snails ceased to court;
this was independent of the size of the individual. Thus, a
large shell did not increase mating success. Time-constraints
of locomotory activity and high costs of searching for a mate
can explain the prevalence of random mating patterns in
simultaneously hermaphroditic land snails (Baur 1992a).

Mating has also been reported to be random with re-
spect to shell size, shell color, and banding pattern in Cepaea
nemoralis (Linnaeus, 1758) (Schilder 1950, Schnetter 1950,
Lamotte 1951, Wolda 1963). Mating was also random be-
tween resident and introduced individuals of Helix pomatia
( Woyciechowski and Lomnicki 1977). Mate-choice tests
with Arianta arbustorum from geographically isolated popu-
lations in Sweden and Switzerland revealed that snails from
three populations preferred to mate with snails from their
population of origin although no interpopulational differ-
ences in latency or duration of courtship were found ( Baur
and Baur 1992a). Mating preferences could partly be ex-
plained by differences in mating propensity in two of the

three populations, but not in matings between a Swedish
and a Swiss population. Cross-breeding demonstrated a high
degree of reproductive compatibility between these two dis-
tant populations. In contrast, pairs involving individuals
from two distant Swiss populations had a reduced fertility.
The experimental results indicate effects of outbreeding de-
pression between distant populations of A. arbustorum.
However, the extent of outbreeding depression seems not to
be related to the geographical distance between populations.

Mating between closely related individuals can incur
substantial fitness costs (i.e., inbreeding depression). For si-
multaneous hermaphrodites such as Arianta arbustorum,
inbreeding depression is regarded as the most important
selective force acting against self-fertilization, and maintain-
ing outcrossing (Ghiselin 1969, Jarne and Charlesworth
1993). B. Baur and A. Baur (1997) performed mate-choice
tests to examine whether individuals of A. arbustorum dis-
criminate between full-sibs and non-sibs from the same
population and whether incestuous matings reduce the
snails’ subsequent reproductive success. Full-siblings (Fj)
were raised under laboratory conditions. Snails mated ran-
domly with respect to the degree of relatedness, indicating a
lack of inbreeding avoidance by selective mating. Snails that
mated with full-sibs did not differ in number of eggs, hatch-
ing success of eggs, or number of offspring produced from
those mated with unrelated conspecifics. In another breed-
ing experiment with A. arbustorum from a different popu-
lation, Chen ( 1993) compared the reproductive performance
of snails that mated with full-sibs to that of snails that mated
with unrelated partners. Again, there was no difference
in the number ot eggs produced. However, eggs of inbred
snails showed a lower hatching success (30.4%) than those of
OLitbred snails (48.5%). Thus, inbred snails had fewer
hatchlings. Furthermore, inbred offspring reared in the gar-
den had a higher mortality rate than outbred offspring
reared in the same environment, but no difference was
found when offspring from both groups were kept in the
laboratory. This result supports the hypothesis that cross-
fertilization in simultaneous hermaphrodites is maintained
by inbreeding depression. It also shows that the extent of
negative inbreeding effects vary among populations and en-
vironments in which the snails are kept.

MULTIPLE PATERNITY IN THE WILD

The genetic background of shell polymorphism, includ-
ing ground color and banding pattern, is well studied in
Arianta arbustormn (Oldham 1934, Cook and King 1966).
One locus controls the ground color of the shell, which may
be yellow or brown (brown being dominant). Thus, shell
color can be used as a genetic marker to analyze paternity in

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Figure 1. Copulating pair of Arianta arbustonun in an alpine grass-
land in Switzerland (photo: B. Baur).

broods of double-mated individuals of known genotype be-
cause shell color is already distinctly expiressed in hatchlings.
Twenty-two adult A. arbustonim with yellow shells were col-
lected in a pasture on Mt. Raimeux (Jura mountains, Swit-
zerland) at an elevation of 1290 m (B. Baur 1994b). This
population is dimorphic with respect to shell color. Two
estimates revealed very similar results: 27.7% (n = 94) of the
adult A. arbustonim had yellow shells on 20 May 1993 and
27.4% [n = 124) on 24 July 1993. Assuming Hardy-
Weinberg equilibrium, the frequency of the allele “brown” is
estimated to p = 0.475 and that of the allele “yellow” to q =
0.525. Of the 22 adult A. arbustonim, 19 produced 34
clutches with fertilized eggs in the laboratory. Nine of the 34
clutches (26.5%) deviated significantly from Mendelian ra-
tios of single paternity, jrroviding evidence for multiple pa-
ternity (B. Baur 1994b). This figure may underestimate the
actual frequency of multiple paternity because repeated mat-
ings with snails of the same genotype will produce results
that are indistinguishable from the broods of single matings.
Several snails deposited 2 or 3 clutches. Considering the total
number of offspring produced by single snails during 65
days, the progeny of 12 of 19 individuals (63.2%) deviated
significantly from Mendelian ratios of a single copulation.

SPERM COMPETITION

Spierm competition, the competition between sperma-
tozoa from different males to fertilize the eggs of a single
female during one reproductive cycle, is a form of male-male
competition that occurs between insemination and fertiliza-

tion (Parker 1970). It is a part of intrasexual selection. Sperm
competition has been solely treated as a male-male conflict
over many years, with females being an inert arena in which
the conflict occurs. More recently, female processes that af-
fect male reproductive success and that occur after copula-
tion have received increasing attention: cryptic female choice
and selective sperm use (Eberhard 1996, see below).

Courtship and dart shooting

During courtship, many helicid snails attempt to pierce
the body walls of their mating partners with mucus-coated
calcareous darts (Koene and Schulenburg 2005). The mucus
covering the dart induces conformational changes in the
female reproductive tract of the recipient, closing off the
entrance to the gametolytic bursa copulatrix. In Helix as-
persa O. F. Muller, 1774, a pulmonate land snail with obli-
gate dart shooting, individuals that were hit by darts stored
significantly more sperm than did snails that were missed
(Landolfa et al. 2001, Rogers and Chase 2001, 2002, Landolfa
2002, see also Chase 2007).

In Arianta arbustonim, dart shooting is not an obliga-
toiy element of courtship behavior. Baminger et al. (2000)
examined dart shooting in relation to different aspects of
sperm competition (allosperm storage and autosperm deliv-
ery) in three natural populations of A. arbustonim in the
Eastern Alps, Austria. Twenty-six (30.2%) of the 86 copu-
lating snails used their darts. There was no reciprocity in dart
shooting: individuals shot their darts independently of the
behavior of their mating partners. Further, the occurrence of
dart shooting was related neither to the number of sperm
delivered nor to the number of sperm received from the
partner. Finally, the occurrence of dart shooting was not
influenced by the amount of allosperm stored from pre-
vious matings. In laboratory matings of A. arbustonim from
Mt. Raimeux, Swiss Jura mountains, 50% of the copulating
individuals pushed or tried to push a dart into their partners
(Bojat and Haase 2002). Dart recipients did not store more
sperm than snails not hit by the dart. This indicates that
the importance of dart shooting for sperm storage varies
among species of snails and even among populations of the
same species.

Spermatophore formation and sperm transfer

In Arianta arbustonim, the spermatophore has a distinc-
tive form consisting of head filament, sperm container, and
a 2-3 cm long tail. The spermatophore is formed in the
epiphallus (head filament and sperm container) and in the
flagellum (tail) during copulation (Hofmann 1923).
Baminger and Haase (2001) examined formation and filling
of spermatophores by collecting spermatophores in mating

REPRODUCTIVE BIOLOGY OF ARIANTA ARBUSTORUM

161

pairs of A. arbustonim at certain intervals after the beginning
of copulation. Two minutes after penis intromission there
was no trace of a spermatophore. After 5 min, a spermato-
phore without tail was visible in two of four individuals.
After 10 min, each snail contained a complete, albeit small,
spermatophore consisting of head filament, container, and
fully-developed tail. The part of the spermatophore that
contained the sperm was increasing in size until shortly
before the spermatophore was transferred (approx. 90 min
after penis intromission). Spermatophore formation was
initiated more or less synchronously in mating partners
shortly after the beginning of copulation. However, the
growth and final size of the spermatophore were not ad-
justed between the mating partners. These observations are
supported by the finding that the numbers of sperm trans-
ferred by two mating snails are not correlated (Baur et al.
1998). This also indicates that sperm trading (sensu Leonard
1991) does not occur in A. arbustonim (see also number of
sperm delivered).

The form of the spermatophore in Arianta arbustonim
is very similar to that in Helix pomatia (Lind 1973), except
that the head of the spermatophore is proportionally shorter
and not filamentous in the latter species (Hofmann 1923).
The tail, with its spiral cross section, especially suggests that
the function is identical in both species (Baminger and
Haase 2001 ). After spermatophore transfer, only sperm that
migrate through the tail, which reaches deep into the vagina,
can bypass the digesting bursa copulatrix and reach the sper-
moviduct through which they travel to the spermatheca,
where they are stored. The head filament probably guaran-
tees that the spermatophore is not pushed too far up into the
diverticulum, preventing the tail from getting too close to
the entrance of the bursal duct. The dissolution of the sper-
matophore in the female tract of the receiver takes more
than one week in A. arbustonim (Haase and Baur 1995).

Number of sperm delivered

Sperm number, in some cases, is an important deter-
minant for achieving successful fertilization in sperm com-
petition (Birkhead and Moller 1998). Theoretical models
and empirical evidence from various studies suggest that,
fundamentally, numerical superiority is an adaptive strategy
for sperm competition (Parker 1990a, 1990b, Birkhead and
Moller 1998). However, males may incur a substantial cost
in the production of ejaculates and spermatophores (Dews-
bury 1982, Nakatsuru and Kramer 1982). Parker’s model
(1990a, 1990b) predicts that males faced with an increased
risk of sperm competition should maximize the prospects of
fertilization by inseminating more sperm per ejaculate. For
example, owing to sperm storage from previous copulations,
mating with a nonvirgin partner may result in a higher risk
of sperm competition than mating with a virgin partner.

Experimental evidence for adjustment of ejaculate size with
respect to mating history has been provided for insects, sala-
manders, rats, and humans (Birkhead and Moller 1998). In
other species, however, males do not adjust the size of their
ejaculates to the mating history (e.g., in zebra finches, Birk-
head and Moller 1998).

An adjustment of the number of sperm released is also
a prerequisite for sperm trading. In simultaneous hermaph-
rodites, a sexual conflict may arise when there is a difference
in potential fitness gain between the role of the sperm donor
and that of the sperm receiver (Charnov 1979, Leonard
1991). Hermaphroditic individuals in a population would
benefit from mating primarily in the more fitness-enhancing
sexual role, leading to a conflict of interest between two
prospective mating partners (Charnov 1979). Gamete trad-
ing might have evolved to resolve the sexual conflict in si-
multaneous hermaphrodites (Leonard 1991). The gamete-
trading model is based on the premise that the preferred role
for a simultaneous hermaphrodite will be the one that con-
trols fertilization (Leonard 1991). In particular, this model
predicts that where the female function controls fertilization,
the mating system will be based on sperm trading.

Baur et al ( 1998) examined whether individuals of Ari-
anta arbustonim adjust sperm release according to the po-
tential risk of sperm competition incurred with a virgin or
nonvirgin mating partner and whether sperm trading occurs
in mating pairs. In controlled mating trials, focal snails were
allowed to copulate with virgin or nonvirgin partners to
simulate a different risk of sperm competition in a given
mating. The number of sperm transferred ranged from
802,620 to 3,968,800 (mean = 2,185,100; u = 91), but was
related neither to the mating history of the partner nor to the
duration of the copulation. This indicates that individuals of
A. arbustonim are not able to adjust sperm expenditure to
the mating history of the partner. Furthermore, the numher
of sperm transferred was correlated neither with the size of
the donor nor with the size of the recipient. There was,
however, a high degree of reciprocity in spermatophore
transfer: in 45 of the 46 mating pairs investigated both part-
ners delivered a spermatophore that contained spermatozoa.
In contrast, the numbers of sperm transferred by the two
mating partners were not correlated. This indicates that
sperm trading does not occur in A. arbustonim.

Locher and Baur (2000a) examined the effect of in-
creased risk of sperm competition on male and female re-
productive traits in individuals of Arianta arbustonim. In a
laboratory experiment snails were exposed to mucous trails
of conspecifics, simulating a high risk of sperm competition.
Courtship behavior, spermatophore size, and number of
sperm delivered were not influenced by a higher risk of
sperm competition. However, snails constantly exposed to
mucous trails of conspecifics deposited more egg batches

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than snails denied any cues from conspecific mucous trails.
This indicates that A. arbustoriim does not respond to ex-
perimentally increased cues from conspecifics, which were
assumed to mimic a high risk of sperm competition by de-
livering more sperm.

Number of matings and intermating interval

Multiple mating is common in helicid snails. Individu-
als of Helix poniatia, Cepaea nemoralis, and Arianta arbiis-
toriun have been observed to mate repeatedly with different
partners in the course of a reproductive season, resulting in
multiple-sired broods (Wolda 1963, Murray 1964, B. Baur
1988b, 1994b). Individuals of Helix powatia copulated 2-6
times per year in a Danish population (Lind 1988) and 2-4
times in a German population (Tischler 1973). Individuals
of H. aspersa copulated on average 3 times (maximum 7
times) in a British population (Learnley 1993, 1996). Actual
records on the number of matings in natural A. arbustorum
populations are not available. Video-recording of A. arbus-
torujH kept in groups of six snails under laboratory condi-
tions revealed that individuals copulated between 0 and 3
times in a period of 58 days (N. Minoretti, pers. comm.).
Lurthermore, paternity analysis in egg batches of A. arbus-
torum sampled in a natural population indicated that at least
63% of the snails used sperm from two or more mates to
fertilize their eggs (B. Baur 1994b).

The few data available on mating frequency in gastro-
pods suggest that terrestrial gastropods copulate less fre-
quently than freshwater and marine gastropods (Baur 1998).
In intertidal and terrestrial gastropods the reproductive ac-
tivity is limited by favorable environmental conditions. The
long-lasting courtship and mating behavior of terrestrial gas-
tropods may exceed the period favorable for locomotory
activity (the high risk of desiccation may incur a significant
cost of mating). Lreshwater and marine habitats, however,
may provide temporally more constant conditions favorable
for mating activities than terrestrial habitats. Other explana-
tions for the relatively small number of copulations in ter-
restrial pulmonates include the costs of producing mucus
during mating, the production of spermatophores and darts
(in some species), and the large number of sperm delivered
during a copulation, which may result in sperm depletion.

Locher and Baur (1999) showed that individuals of An-
anta arbustorum needed at least 8 days to replenish their
sperm reserves after a successful copulation. Lurthermore,
the number of sperm delivered in the second copulation
increased with an increasing intermating interval from 6 to
29 days. This finding suggests that the number of sperm
delivered increases with even longer intermating intervals.
Hiinggi et al. (2002) examined the size of the spermatophore
and the number of sperm delivered in two groups of A.

arbustoru?n that remated either after 3-4 weeks or after 7-8
weeks. The results indicated that A. arbustorum entirely re-
plenishes its autosperm reserves within 3-4 weeks after a
successful copulation.

Sperm size and quality

Much interest has also been focused on theory concern-
ing the significance of the size and quality of sperm (e.g.,
Parker 1982, Parker and Begon 1993, Pizzari and Birkhead
2002). The size of sperm may influence their power and
swimming speed as well as longevity because of changes in
the energetic demands of longer or shorter flagella. Lor ex-
ample, in echinoids that use broadcast spawning, there is
evidence that sperm velocity and longevity covary between
and within species (Levitan 1993). Levitan (2000) has shown
that in a sea urchin, sperm velocity and longevity are traded
off against each other. In taxa with sperm storage organs,
sperm length may determine the ability to reach the storage
organs first and to move to the ovum from the storage
organs once ovulation takes place. However, assuming a
fixed resource budget, smaller sperm may allow males to
produce more gametes, which may be adaptive if sperm
compete numerically (Parker 1982). Confounding variables,
such as the morphology and biochemistry of the female repro-
ductive tract, might also affect sperm form and function.

Besides the size and number of sperm transferred, the
quality of ejaculates (proportion of live, morphologically
normal spermatozoa and motility of spermatozoa) might be
important in determining the outcome of sperm competi-
tion. Indeed, recent studies in gonochoristic animals reveal
substantial intraspecific variation in sperm motility and lon-
gevity. This variation may function in postcopulatory sexual
selection. A synthesis of the available literature indicates that
these sperm-quality traits affect fertilization success and that
they are important in both sperm competition and cryptic
female choice (Snook 2005).

Molluscan sperm are characterized by a remarkable ar-
ray of morphological features (Thompson 1973, Anderson
and Personne 1976, Hodgson et al. 1996). The interspecific
variation in sperm morphology is frequently used as a taxo-
nomic character. Data on sperm length are available for
several gastropod species. Spermatozoa of terrestrial pulmo-
nates are among the longest of the molluscs [e.g., 850 pm in
Helix pomatia; Thompson 1973). However, intraspecific
variation in sperm length and quality has not been analyzed
in any hermaphroditic gastropod species.

Minoretti and Baur (2006) developed techniques to
measure sperm length, sperm velocity, percentage motility,
and longevity of sperm in Arianta arbustorum. They exam-
ined variation in sperm length in individuals from four
natural populations and variation in velocity, motility, and

REPRODUCTIVE BIOLOGY OF ARIANTA ARBUSTORUM

163

longevity of sperm in two populations. Sperm ot A. arbiis-
toriim are monomorphic. Like other pulmonates, A. arhus-
torum produces extremely long sperm. Independent of shell
size, sperm length differed among populations ( mean values
of populations: 878, 898, 913, and 939 pm) and — to a minor
extent — even among individuals within populations. Mean
sperm length of a snail was not correlated with the number
of sperm delivered in a spermatophore. Individual snails
showed consistent sperm length in successive matings. The
mean sperm velocity was not influenced by shell size, nor did
it differ between populations. However, mean sperm velocity
differed among individual snails (range 52-112 pm/s). Per-
centage motility and longevity of sperm differed between
snails from different populations but were not affected by
shell size. No correlations were found between length, ve-
locity, percentage motility, and longevity of sperm. Thus,
individual snails differed in sperm quality. This interindi-
vidual variation may partly explain differences in fertiliza-
tion success.

Sperm precedence

Sperm precedence is the differential sperm usage from
consecutive matings (mating order effect). It is typically
measured as the proportion of eggs fertilized by the second
of two mates (the P2 value). Patterns of sperm utilization
were investigated in double-mated individuals of Arianta
arbiistorum (B. Baur 1994b). In particular, the effects of
delay between copulations (range 9-380 days) and size of the
sperm donor on sperm precedence (P2) were examined.
Using shell color as a genetic marker, paternity was analyzed
in 132 broods produced by 35 snails that had mated with
two partners of different genotypes. Sperm precedence (P2)
was influenced by the time between the two matings
when the mating delay exceeded 70 days (one reproduc-
tive season). In the first brood of snails that mated twice
within 70 days, P2 averaged 0.34, indicating precedence of
sperm from the first mate. In contrast, P2 averaged 0.76 in
broods of snails that remated in the following season,
indicating a decreased viability of sperm from the first mate.
The size of sperm-donating individuals had no effect on the
fertilization success of their sperm in the first brood pro-
duced after the second copulation. Analysis of long-term
sperm utilization in 23 snails that laid 3-9 batches over 2
years revealed striking differences among individuals. Five
snails (21.7%) exhibited precedence of sperm from the first
mate throughout, 8 snails (34.8%) showed precedence of
sperm from the second mate throughout, whereas 10 snails
(43.5%) exhibited sperm mixing in successive batches. This
indicates that different mechanisms might be involved in
creating the observed inter- individual variation in sperm
precedence.

FEMALE ROLE IN SPERM COMPETITION:
CRYPTIC FEMALE CHOICE

Until recently, most research concentrated on male as-
pects of sperm competition in gonochoristic animals. In the
past few years, there has been increasing interest in the pos-
sibility that females influence the outcome of sperm com-
petition by cryptic female choice and selective sperm use
(Eberhard 1996). Females might be able to discriminate be-
tween and differentially utilize the sperm of different males,
a process referred to as ‘sperm choice’ (Birkhead 1998).
There are broad and narrow definitions of “sperm choice”;
some authors make it synonymous with “cryptic female
choice” (see Eberhard 2000, Kempenaers et nl. 2000, Pitnick
and Brown 2000). Cryptic female choice has been defined as
nonrandom paternity biases resulting from female morphol-
ogy, physiology or behavior that occur after coupling (Pit-
nick and Brown 2000). This definition ascribes to sperm
choice any biases in paternity owing to the way females
handle sperm, regardless ot the specific mechanism or evo-
lutionary causes, and regardless of proximate control. The
only relevant consideration for this definition is whether a
female-mediated process generates sexual selection on
males. A general problem with cryptic female choice is that
it is difficult to rule out the direct influence from males (e.g.,
Edvardsson and Arnqvist 2000).

In simultaneously hermaphroditic pulmonates the role
of the female duct is to receive sperm from a copulating
partner, to store the sperm, to provide a site for fertilization,
to form the egg capsule, and to digest sperm and remnants
of the spermatophore so as to absorb nutritional fluids re-
ceived with the ejaculate.

Variation in the morphology of the female
reproductive tract

Rapid evolution of reproductive traits has been attrib-
uted to sexual selection arising from interactions between
the sexes {e.g., Eberhard 1996). Inter- and intraspecific stud-
ies in gonochoristic animals revealed a covariation between
sperm characteristics and the size of the female reproductive
tract, indicating an evolutionary divergence, which is con-
sistent with the theory of post-copulatory sexual selection. In
Arianta arbustonun, like other terrestrial pulmonates, the
enormous variation in structure and morphology ot the
spermatheca, fertilization chamber, and sperm digesting or-
gan could have evolved in response to different levels of
sperm competition and/or cryptic female choice (Baur
1998). Baminger and Haase (2000) examined the variability
of the distal genitalia involved in spermatophore production,
reception, and manipulation in 1 1 3 adult individuals of A.
arbiistorum from six natural populations. In particular,
Baminger and Haase (2000) asked whether the variation in

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genitalia is related to the intensity of sexual selection, mea-
sured as local population density. The size of the genitalia
was unexpectedly inversely related to population density,
probably because of an increased inhibitory effect of snail
mucus. Patterns of variation of female and male charac-
ters did not differ. However, the influence of sexual selection
on genitalia size and variance could not be unambiguously
determined.

Beese et al (2006a) c]uantified the variation in male and
female reproductive traits among six natural populations of
Arianta arbiistormn and examined the covariation in inter-
acting traits. There was a significant among-population
variation in spermatophore volume, number of sperm trans-
ferred, and sperm length, as well as in volume of the sperm
storage organ (spermatheca) and number of tubules, but not
in spermathecal length. Lurthermore, there was no relation-
ship between sperm size and spermathecal length. There
was, however, a positive association between the number of
sperm transferred and spermathecal volume. This result sug-
gests that the same post-copulatory mechanisms that operate
in gonochorists drive the correlated evolution of reproduc-
tive characters in hermaphrodites.

The wall of the spermatophore received is dissolved in
the bursa tract diverticulum (Beese et al 2006b). The di-
gested material is taken up by epithelial cells.

Organ for sperm storage

Individuals of Arianta arbustorum are able to store vi-
able sperm from different mating partners for more than 1
year (Baur 1988b). The morphology of the sperm storage
organ (spermatheca) may influence the outcome ot sperm
competition in A. arbustorum, as shown in insects (Simmons
and Siva-Jothy 1998). Arianta arbustorum shows a consid-
erable variation in the structure of the spermatheca (Haase
and Baur 1995, Beese and Baur 2006). It consists of two to
nine blind tubules uniting to a common duct, which opens
into the fertilization chamber (Haase and Baur 1995,
Baminger and Haase 1999, Baminger et al 2000). The mus-
culature surrounding the spermathecal tubules is arranged
in a complex three dimensional network (Bojat et al 2001a,
2001b, 2001c). If there were a selective activation of the
muscles of each tubule (which has not yet been examined),
this would allow the animal to expel sperm stored in single
tubules and thus promotes a selective fertilization of eggs.
The ciliation of the common duct is probably responsible for
the distribution of incoming sperm among the tubules.

Bojat and Haase (2002) assessed the amount of allo-
sperm stored in the spermatheca in relation to the structure
of the spermatheca (number of spermathecal tubules) in 18
individuals of Arianta arbustorum that had copulated once.
Snails differed in patterns of sperm storage: two individuals
used 100% of their spermathecal tubules, two used 80%,

three 75%, two 66.7%, one 50%, two 40%, three 33.3%, two
25%, and one used 20%. The main tubule always contained
sperm (51-100% of the total amount of sperm stored, i.e.,
more than all lateral tubules combined). The amount of
sperm stored was not correlated with the volume of the
received spermatophore. However, the amount of sperm
stored was positively correlated with the number of sper-
mathecal tubules. This suggests that the female role of the
receiver controls the number of sperm stored.

Baminger and Haase (1999) examined whether the
variation in number of spermathecal tubules and the
amount ot allosperm stored are influenced by the risk of
sperm competition, as indicated by the local density of adult
Arianta arbustorum in six natural populations in the Eastern
Alps, Austria. The number of spermathecal tubules ranged
from two to nine. However, the six populations did not
differ in either the mean number of spermathecal tubules or
the cumulative length of the tubules. Individuals from dif-
ferent populations did not differ in the amount of sperm
stored, and the amount of sperm stored was not correlated
with population density. This indicates that the risk of sperm
competition does not affect the number of spermathecal
tubules. However, it is still not known whether individuals in
high-density populations store allosperm from more differ-
ent mating partners than those in low-density populations.

A histochemical analysis of the spermatheca of Arianta
arbustorum revealed polysaccharides in the periphery of con-
nective tissue and muscle cells (Bojat et al 2003). Polysac-
charides were differentially distributed in the epithelial cells
of the fertilization chamber and spermatheca, indicating re-
gions that differed in physiological activity. In general, the
concentration of polysaccharides including glycogen in-
creased towards the blind end of the spermathecal tubules.
Lipids were more or less equally distributed in the epithe-
lium along the tubules. Polysaccharides and lipids have nu-
tritive function. The highly active epithelium could provide
nutrients for the stored and active spermatozoa (Rogers and
Chase 2002). However, an exchange of substances between
epithelium and spermatozoa has not been observed.

BENEFITS OF MULTIPLE MATING FOR FEMALES

In many animals, males are selected to mate as many
times as possible to maximize their reproductive success
(Bateman 1948, Trivers 1972). For females, in contrast, the
advantage of multiple mating is not so obvious. The rela-
tively small number of ova produced by a female could be
fertilized by sperm from one or very few male ejaculates,
especially when the female can store sperm or has a short
reproductive period. Moreover, possible costs of mating
should select against unnecessary matings. While females of
some species mate only once in their lives, multiple mating

REPRODUCTIVE BIOLOGY OF ARIANTA ARBUSTORUM

165

by females is generally very common. Several hypotheses
have been suggested to explain the adaptive significance of
multiple mating by females (Birkhead and Moller 1998).
Among them, the hypothesis of sperm replenishment is the
most straightforward explanation for multiple mating by
females. The underlying mechanism could vary: the sperm
received from one male may not be enough to fertilize all the
eggs produced by a female, the viability of sperm stored may
decrease with time, or a mate may have transferred sperm of
low quality. Another hypothesis predicts genetic advantages
(multiple mating with different partners may lead to mul-
tiple paternity and thus increase the genetic variability
among the offspring of a brood). Furthermore, females may
enjoy nutritional benefits from repeated matings by receiv-
ing nutrients with the spermatophore. These hypotheses are
not mutually exclusive.

Chen and Baur (1993) examined reproductive traits
over two years in individuals of Arianta arbiistonim that
copulated several times per year (snails kept in pairs), in
individuals that copulated twice (once at the beginning of
each year) or once (at the beginning of the first year), and in
individuals prevented from copulation (snails kept isolated).
Copulations were not always reciprocally successful: 3 of 57
snails (5.3%) failed to produce fertile eggs although their
mates reproduced successfully. Similarly, 2 of 15 pairs
(13.3%) failed to reproduce successfully. Snails allowed to
mate repeatedly within each season tended to lay more eggs
than snails that mated once per year. However, the number
of hatchlings did not differ significantly between the two
treatment groups because eggs laid by snails allowed to mate
repeatedly had a lower hatching success. Snails that remated
in the second year laid more eggs with a higher hatching
success, and thus produced more hatchlings, than snails that
mated only once at the beginning of the first year. Snails that
were prevented from mating produced a few hatchlings (by
self-fertilization) in the second year; their reproductive suc-
cess was less than 1% of that of mated snails. These results
suggest that multiple mating is also adaptive for the female
function of A. arhnstonuu by increasing female fecundity
and fertility and serving as a hedge against unsuccessful
copulations.

Egg production in several species of stylommatophoran
gastropods is stimulated by mating behavior and/or sub-
stances derived from male ejaculate (Takeda 1983, Bride et
al. 1991 ). In Helix aspersa, mating increases the synthesis and
release of a dorsal body hormone essential for vitellogenesis,
ovulation, and egg-laying (Saleuddin et al 1991). Whether
this activation of the dorsal body is direct, under either
neural or hormonal control, or indirect under gonadal in-
fluence, is not known (Saleuddin et al. 1991 ). B. Baur and A.
Baur (1992b) examined experimentally whether extended
courtship display or repeated copulation in the course of a

reproductive season stimulates egg production in Arianta
arbiistorinn. Clutch size decreased in successive egg batches
of individuals that copulated once at the beginning of the
reproductive season (a seasonal decrease in clutch size was
also observed in A. arbnstoruin kept in field cages; Baur
1990a). Repeated copulation, however, was found to in-
crease clutch size, while courtship display did not affect egg
production. Repeated copulation neither accelerated the on-
set of egg laying nor increased the hatching success of eggs.
These results suggest that reciprocal intromission and/or re-
ceipt of a spermatophore, but not the long-lasting courtship
behavior, stimulates egg production in A. arbustorum (B.
Baur and A. Baur 1992b).

MATERNAL INVESTMENT AND EGG PROVISIONING

Parental investment may often be critical to the survival
and growth of young, but the larger the investment per
offspring, the lower the number of offspring that can be
produced. Several models have been developed to predict the
optimal size of offspring under different environmental con-
ditions. Although the models make different predictions,
these are based on the assumption that egg size is a reliable
measure of the amount and quality of resources invested in
each offspring (i.e., larger eggs are supposed to contain more
organic material). A. Baur (1994) examined the within- and
between-clutch variation in egg size and nutrient content of
Arianta arbnstornni. The volume of single eggs ranged from
5.5 to 26.3 mm’ (grand mean 13.4 mm’). The overall range
of the nitrogen concentration of the eggs was 3. 1-5.0%
(grand mean 4.1%), and that of the carbon concentration
28.6-34.9% (32.2%). The nitrogen concentration indicates
that eggs of A. arbustorum have a protein concentration of
25.5%. The within-clutch variation in egg size expressed by
the coefficient ot variation averaged 11.1% for egg volume
and 8.3% for dry weight. Corresponding values for the con-
centrations of N and C were 3.7 and 1.6%. Thus, egg size was
in general more variable than the nutrient concentration ot
the eggs. Considering mean clutch values, the nutrient con-
tents (in mg) scaled isometrically with egg size.

Successive studies showed that not only do egg and
clutch size vary seasonally but also the protein and carbon
concentrations of the eggs do so (A. Baur and B. Baur 1997).
Furthermore, seasonal changes in egg size and egg provi-
sioning occur among populations (Baur and Baur 1998).

Apart from increasing egg size, maternal nutrition can
be enhanced by providing hatchlings with food. Alexander
(1974) suggested that if parents were unable to increase their
investment in young through increasing egg size, an alter-
native strategy is to increase clutch size and allow some
siblings to consume others (the icebox effect). The optimum

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clutch size can be found by calculating the clutch size leading
to maximum brood productivity, taking into account the
effects of sibling cannibalism and possible trade-offs.

Maternal provision of trophic eggs of different types to
hatchlings is widespread among marine gastropods. Numer-
ous species of prosobranch snails normally produce trophic
eggs, which serve as the first food for their progeny (B. Baur
1992b, 1994c). The consumption of trophic eggs can be
facultative (may not occur in all egg capsules of a species) or
obligate. A similar form of food provisioning has evolved in
terrestrial gastropods. Hatchlings of various species of her-
bivorous land snails cannibalize sibling eggs (Baur 1992b).
Emerging juveniles of Arianta arbustorum first eat their own
egg shells and then the eggs of unhatched siblings, including
those with tully developed embryos (Baur and Baur 1986).
Egg cannibalism occurs exclusively during the hatchling
stage (due to an age-specific occurrence of digestive en-
zymes), juvenile and adult snails being herbivorous (Baur
1987a). Cannibalistic hatchlings eat only conspecific eggs
(Baur 1988c, 1988d) and do not discriminate between sib
and non-sib eggs (;.c., eggs from neighboring batches; Baur
1987b). Furthermore, newly hatched snails discriminate nei-
ther between fertilized and unfertilized conspecific eggs nor
between eggs with well-developed embryos and eggs with
less advanced embryos (Baur 1993b). Significant benefits
accrue to cannibalistic hatchlings of A. arbustorum. Labora-
tory experiments demonstrated that newly hatched snails fed
a cannibalistic diet during their first 10 days of life increased
in wet weight 2.6 times as much as siblings fed on lettuce
(Baur 1990b). In this experiment, egg consumption within
10 days ranged from 0.7 to 4.0 eggs per individual and in-
crease in weight of cannibalistic hatchlings was positively
correlated with the number of eggs consumed. Diet did not
affect hatchling survival during the first 10 days, but it did
influence future survival: 66.6% of the individuals initially
fed on eggs attained adulthood compared to 38.0% of those
fed on lettuce. Cannibalistic hatchlings tended to complete
shell growth more rapidly and thus became sexually mature
earlier than non-cannibalistic ones, but the two groups did
not differ in adult shell size. Thus, a cannibalistic diet during
the hatchling stage will give accelerated growth and higher
survival.

The extent of within-batch egg cannibalism depends
primarily on the hatching asynchrony of the eggs and on the
hatchlings’ propensity for cannibalism (Baur and Baur 1986,
B. Baur 1994c). Under natural conditions, the hatching
asynchrony and, as a consequence, the extent of egg canni-
balism will depend also upon the type of oviposition
(batches or scattered eggs), on the spatial heterogeneity of
egg-laying sites, and on climatic conditions. The great varia-
tion between populations in propensity for cannibalism sug-

gests different costs and benefits of egg cannibalism in dif-
ferent situations (B. Baur 1994c).

SEX ALLOCATION

Reproductive resource allocation is a fundamental as-
pect of life history with profound ecological and evolution-
ary consequences. Allocation decisions in hermaphroditic
plants and animals are particularly interesting because indi-
viduals can potentially maximize reproductive success
through a wide variety of different strategies. Thus, a key
observation for testing sex allocation theory in simultaneous
hermaphrodites is the proportion of resources devoted to
male vs. female function (Charnov 1982). The specific allo-
cation strategy followed by a hermaphrodite may affect the
extent of sexual selection and mating behavior (Michiels
1998). Most models of sex allocation are based on the con-
cept of male and female gain curves (the relationship be-
tween relative investment in either male or female gamete
production and resulting reproductive success; Charnov
et al. 1976, Charnov 1982). These models have received sub-
stantial empirical support {e.g., in coral reef fishes [Fisher
1981, Fischer and Petersen 1987] and in a polychaete worm
[Sella 1990]. All these hermaphrodites have external fertil-
ization. Many hermaphroditic invertebrates, however, have
some form of copulation, sperm storage, and internal fertil-
ization (Michiels 1998).

More recently, models of sex allocation for outbreeding
hermaphrodites with internal fertilization and sperm storage
have been developed (Charnov 1996, Greeff and Michiels
1999b). These models consider how various aspects of
sperm competition, such as mating frequency, sperm diges-
tion, and different mechanisms of sperm displacement affect
sex allocation in simultaneous hermaphrodites. The models
predict that a reduced mating rate leads to a reduction in
resources allocated to the male function (Charnov 1996,
Greeff and Michiels 1999b), while sperm digestion leads to
an increase in allocation to the male function (Greeff and
Michiels 1999b).

Tocher and Baur (2000b) examined the effect of mating
frequency on male and female reproductive output (number
of sperm delivered and eggs deposited) and on the resources
allocated to the male and female function (dry mass, nitro-
gen, and carbon contents of spermatophores and eggs) in
individuals of Arianta arbustorum. Virgin snails were al-
lowed to mate once, twice, or three times within a period of
51 days. Snails from the three treatment groups did not
differ in shell volume. The total number of sperm delivered
increased from 3,098,000 in snails that copulated once to
5,001,000 in snails that copulated twice, and to 6,849,000 in
individuals with three copulations. Considering the number

REPRODUCTIVE BIOLOGY OF ARIANTA ARBUSTORUM

167

of sperm delivered per copulation, however, there was no
difference between snails with different numbers of copula-
tions. Female reproductive output was not influenced by the
number of copulations. Snails that copulated once, twice, or
three times produced the same number of egg batches (3.9,
3.8, and 4.3, respectively) and the same number of eggs
(113.7, 116.2, and 116.3) within one reproductive season.
Considering snails from all treatment groups, there was a
positive correlation between the total number of sperm de-
livered and the number of eggs produced. This indicates that
individuals that delivered many sperm generally produced a
large number of eggs.

In this experiment, the dry mass of spermatophores
transferred averaged 0.91 mg (Locher and Baur 2000b).
Spermatophores had a nitrogen concentration of 12.05%,
indicating that a spermatophore on average contained 0.11
mg nitrogen. The dry mass of single eggs averaged 2.14 mg
with a nitrogen concentration of 4.02%. Thus, one egg on
average contained 0.086 mg nitrogen. The relative allocation
to the male function (expressed as percentage of the total dry
mass or amount of nitrogen devoted to the male function)
increased with increasing number of copulations (dry mass:
0.58% in snails that copulated once, 0.91% in snails that
copulated twice, and 1.66% in individuals that copulated
three times; nitrogen: 1.72%, 2.68%, and 4.72%, respec-
tively). FFowever, independently of the measure chosen, re-
productive allocation was highly female-biased. In none of
the measures used did the average proportion of resources
devoted to the male function reach 5% of the total allocation
in snails that copulated three times. Considering individual
snails, the maximum nitrogen allocation to the male func-
tion was 13.35% in snails that copulated three times. Thus,
only a minor part of the resources available for reproduction
were devoted to the male function. Furthermore, snail size
did not affect the relative reproductive allocation to male or
female function. The finding that an increased mating fre-
quency leads to an increased allocation to the male function
was predicted by Charnov (1996) and Greeff and Michiels
(1999b), even though their models considered much higher
mating frequencies (5-50 or even an infinite number of
copulations). Taking into account the short period of activ-
ity of snails living in subalpine populations, the assumed
range of 1-3 copulations per reproductive season might be
reasonable for the animals studied.

The female skew in sex allocation found in Arianta ar-
bustorum was much larger than predicted by the models of
Charnov ( 1996) and Greeff and Michiels ( 1999b). A possible
explanation for this pronounced female skew could be in-
complete estimates of reproductive allocation to either func-
tion. In simultaneously hermaphroditic land snails like A.
arbustorum, each mating in the male role carries costs, which
include the cost of courtship behavior (e.g., the optional dart

shooting), spermatophore and sperm production, and other
possible costs associated with mating. During the long-
lasting courtship, gastropods produce huge amounts of mu-
cus, an energetically expensive behavior (Davies et nl. 1990).
To my knowledge no numerical estimate of the costs of
courtship behavior is available for any terrestrial gastropod.
Furthermore, it is not clear whether the costs of courtship
can be entirely assigned to the male function. For several
reasons repeated mating might also be advantageous for the
female function (see above).

A fundamental assumption of sex allocation theory in
simultaneous hermaphrodites is a trade-off between male
and female function, i.e., the animal has a fixed amount of
resources to allocate between the genders (Charnov 1982).
Locher and Baur (2000a, 2000b) did not find any trade-off
between the two functions. In contrast, a positive relation-
ship between the resources allocated to the male and female
functions was recorded. This result could be explained by a
condition-dependent allocation and/or a “good genes” sce-
nario. With regard to quality, any genes that affect the qual-
ity of spermatozoa and ova in the same direction would lead
to a positive association between the two (Schlichting and
Delesalle 1997). In hermaphroditic pulmonates such as Ari-
auta arbustoriim, spermatozoa and ova are produced simul-
taneously in the same organ, the so-called ovotestis. It would
be most interesting to disentangle possible associations be-
tween the quality of spermatozoa and ova in these snails.

Trade-offs in resource allocation may not occur or may
be less pronounced under favorable conditions. Under
stressful conditions, such as limited food supply, high tem-
perature, or drought, the energy intake might be carefully
shared among different functions, including reproduction.
Locher and Baur (2002) examined the effect of nutritional
stress on mating behavior and male and female reproductive
output (dry mass and nitrogen contents of spermatophores,
sperm delivered, and eggs deposited) in individuals of Ari-
anta arbustonnu kept under three different food regimes:
ample (100%), restricted (50%), and extremely restricted
(25%) food supply. Independent of the extent of nutritional
stress, 10-12% of the resources taken up were invested in
reproductive output (both gender functions together) and
88-90% in maintenance (including feces and excretion).
Courtship and copulation behavior was affected by nutri-
tional stress. Except for one pair, snails with an extremely
restricted food supply did not mate. Individuals with re-
stricted food supply tended to court longer and copulated
for a shorter period than individuals with ample food sup-
ply. Nutritional stress did not affect the number of sperm
delivered. However, snails with a restricted food supply pro-
duced fewer eggs. Thus, snails kept under nutritional stress
invested relatively more resources in the male function than
in the female function. Nevertheless, the absolute reproduc-

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live output remained highly female biased (>95% in all ex-
perimental groups).

In hermaphroditic snails a reduced supply of protein
and calcium might affect growth and alter the allocation to
either sexual function. Wacker and Baur (2004) tested this
hypothesis by maintaining subadult Arianta arbiistorum on
artificial diets composed of single compounds (particular
amino acids, carbohydrates, fatty acids, minerals, vitamins)
on an agar-based diet. Snails fed a high protein diet grew
faster and reached adulthood earlier than individuals fed a
low protein diet. Different calcium contents did not affect
shell growth, but increased mortality when the calcium con-
tent of the food was low. Furthermore, diet-related differ-
ences in mating propensity were found.

CONCLUDING REMARKS

Much recent research effort has been directed at ex-
plaining sex-specific differences in reproductive strategies
and sexual selection in gonochoristic animals. Simultaneous
hermaphroditism imposes evolutionary constraints on re-
producing individuals that are different from those in gono-
choristic species. Yet, simultaneous hermaphrodites, in par-
ticular puhnonate gastropods, have received little attention
with respect to mating strategies and sexual selection. This
review summarizes the present knowledge of several aspects
of sexual selection in Arianta arbiistoruni. Several other gas-
tropod species may be well-suited for further studies on
mating strategies and sexual selection. There are many topics
that remain largely unexplored and there is much to be
learned in this most interesting animal group.

ACKNOWLEDGMENTS

This review summarizes the research of the “Arianta
group” in our institute over the past 15 years. Anette Baur
contributed a significant part of the research presented. I am
grateful to her and all the Master- and Ph.D. -students
and collaborators involved in the different projects. I also
thank Anette Baur, Kathleen Beese, Michael T. Ghiselin,
Nichole Minoretti, and an anonymous reviewer for con-
structive comments on the manuscript. The research has
received financial support from the Swiss National Science
Foundation.

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Accepted; 9 March 2007

Ainer. Maine. Bull. 23: 173-181

A literature database on the mating behavior of stylommatophoran land snails
and slugs'^

Angus Davison^ and Peter Mordan^

* Institute of Genetics, School of Biology, University of Nottingham, Queens Medical Centre, Nottingham, NG7 2UH, UK,
angus.davison@nottingham.ac.Lik

^ Department of Zoology, The Natural History Museum, London, SW7 5BD, UK, pbni@nhm.ac.uk

Abstract: Stylommatophoran land snails and slugs generally mate by shell-mounting or face-to-face. Although phylogenetic evidence
suggests that the mating position has remained more or less constant throughout the evolution of most lineages, other aspects of mating
behavior and associated reproductive characters are highly variable. Along with other gastropods, therefore, stylommatophoran land snails
and slugs could be particularly useful in trying to understand sex and sex allocation theory in hermaphrodites. It is often difficult, however,
to compare mating behavior in different species because the literature is difficult to access or reports have not been tormally published. Here
we review studies on the mating behavior of snails and slugs, with the additional aim of creating a central access point and database for
use as a resource by those interested in stylommatophoran mating behavior. As we maintain the database, updated versions will be made
available at http://www.molluscs.org.

Key words: love darts, mollusc, sexual conflict, reciprocal mating, simultaneous hermaphrodite

The mating behavior of a wide variety of stylommato-
phoran land snails has been observed, but the descriptions
are often within texts that are not easily accessible, or cannot
be searched electronically. Many malacologists have also
made their own informal observations of mating behavior,
but do not publish them for lack of time, or because they are
not perceived to be of sufficient worth on their own. Because
there have been no recent reviews of the mating behavior of
snails and slugs, we set out to collect as many observations
together as possible, both formal and informal. Such an
approach has already proved useful in trying to understand
how so-called “love” darts evolved (Davison et al. 2005).
Differences in mating behavior have also been used to un-
derstand the distribution of chiral variation (or asymmetry)
among different taxonomic groups (Asami et al. 1998) and
the evolution of external sperm exchange (Emberton 1994).
The aim of this brief review, therefore, is to create a starting
point for a compilation of data on the mating behavior of
stylommatophoran snails and slugs.

Although there are some exceptions {e.g., the elongated
penes and external fertilization of Umax maximiis Linnaeus,
1758), mating in the majority of land snails and slugs can be
classified as either face-to-face or shell-mounting (Figure 1;
Asami et al. 1998). The vast majority of species are also
simultaneous hermaphrodites. In theory, therefore, four dif-
ferent modes of mating are possible because sex is also

either simultaneous reciprocal (both individuals are male
and female at the same time) or unilateral (each individual
has a defined role as male or female during a single round of
mating):

Face-to-face, simultaneous reciprocal;

Face-to-face, unilateral;

Shell-mounting, simultaneous reciprocal;

Shell-mounting, unilateral.

When two individuals mate unilaterally, they often
switch roles after one round of mating — male becomes
female and female becomes male. The frec]uency with which
this occurs is difficult to assess because it requires extended
observations, and also the frequency of mate switching de-
pends upon the condition (or desire) of each snail (Koene
and Ter Maat 2005). The problem is further complicated
because often the most efficient means to make laboratory
observations of mating behavior is to isolate individuals for
some time before bringing them together. For a variety of
possible reasons {e.g., availability of seminal fluid), isolated
individuals are more likely to switch mates after one round
of mating (Koene and Ter Maat 2005).

Another concern is whether sperm or spermatophore
transfer is always reciprocal if mating is reciprocal; from an
evolutionary point of view, the reciprocal exchange of sperm
is just as important. In some species of Siiccinea Draparnaud,

* From the symposium “Gastropod Mating Systems” presented at the joint meeting of the American Malacological Society and Western
Society of Malacologists, held 26-30 fime 2005 at Asilomar, Pacific Grove, California.

173

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Figure 1. A, Items marnwratus Ferussac, 1801 (Helicidae) mating
lace-to-face and simultaneous reciprocally. Horizontal field width
approximately 6 cm. Photo by A. Davison, in the laboratory. B,
Ligmis fasciatus Pilsbry, 1912 (lower snail, acting as male) and
Orthaliciis floridensis Pilsbry, 1899 (upper snail, acting as female)
(Orthalicidae) mating by shell-mounting. Specimens in hardwood
hammock in the Redlands of southern Dade County, Florida. Im-
age reproduced with the kind permission of the photographer, Phil
Poland, ppolandl@tampabay.rr.com. Vertical field width approx.
7 cm.

1801 that mate reciprocally by shell-mounting, sperm trans-
fer is sometimes unilateral (lordaens et al. 2005), whereas
other species such as Arianta arbustorum Linnaeus, 1758
have a high reciprocity (Baur 1998). In species that develop
as males before becoming simultaneous hermaphrodites,
such as Lissachatina (Achatina) fiilica Bowdich, 1822, sperm
transfer may frequently be unilateral (Tomiyama 1993).

We surveyed the formal literature on the mating behav-
ior of land snails and slugs and then classified each species as
to whether it mates face-to-face, by shell-mounting, simul-
taneous reciprocally, or unilaterally (Table 1). We also in-
cluded informal observations, when available (mating posi-
tion and, to a lesser extent, reciprocity can be scored from
photographs), and tried to identify video recordings of mat-
ing behavior (Table 2). One striking, immediately apparent
result is that face-to-face mating is exclusively associated
with simultaneous reciprocal mating. Snails and slugs in
three monophyletic groups mate face-to-face and simulta-
neous reciprocally: the Helicoidea, Limacoidea, and Philo-
mycidae (Davison et al 2005).

One other reason to study mating behavior is to under-
stand the evolution of “love” darts. Despite the attention
that greets each advance, little is known about the use of
darts outside of Cantareus aspersus {Helix aspersa Muller,
1774) (Koene and Chase 1998a, 1998b, Landolfa et al. 2001,
Rogers and Chase 2001, Rogers and Chase 2002, Koene and

Schulenberg 2005, Chase 2006), except that there is consid-
erable variation in the timing of the use of the dart, its
morphology, and the number used in different species (Ash-
ford 1883, Tompa 1980, Baminger et al. 2000, Koene and
Schulenberg 2005). At the extreme of the spectrum, some
species of Eiihadra Pilsbry, 1890 repeatedly stab a dart
(-3000 times) during “foreplay” prior to mating (]. Koene
and S. Chiba, personal communication). Opinions also vary
over what constitutes a dart. Although some might contend
that an “amatorial” organ is a dart, we argue that while darts
and amatorial organs may (or may not) have similar func-
tions, they are clearly distinguishable because only the
former is a “hard, calcified or chitinous organ that is used to
pierce a partner during mating” (Davison et al. 2005).

There have been very few formal observations of dart
use. The only photographs that we are aware of showing
darts “in use” are of C. aspersus (Koene and Chase 1998a,
1998b, Landolfa et al. 2001, Rogers and Chase 2001, Rogers
and Chase 2002, Koene and Schulenberg 2005), Cepaea
nemoralis (Davison et al. 2005), and Trichotoxon heynemanni
(Schilthuizen 2005). It would therefore be useful if creating
a database also stimulated malacologists to record dart use
and to publicize their efforts. We have recently used infor-
mation in the database to show that dart-bearing species are
confined to the same three monophyletic groups mentioned
above (the Helicoidea, Limacoidea, and Philomycidae) and
that they all mate face-to-face and simultaneous reciprocally.
However, there is no evidence that the relationship is causal
(Davison et al. 2005).

Although there are still some large clades of snails for
which there have been few observations of mating behavior,
an interesting dichotomy has emerged between invariant
mating position and other highly variable reproductive char-
acters. Some species have head warts (Binder 1977, Takeda
1982, Ealkner 1993), penial stimulators (Reise 2004), or
amatorial organs (Panha 1987), whereas individuals in other
species bite off the penis of their partner (Leonard et al.
2002) or entwine their penes before exchanging sperm ex-
ternally (Quick 1960). Although it has been known that
some families tend to have the same shell shape (Cain 1977),
the strong and (almost) invariant correlation between mat-
ing position and shell shape has mostly been overlooked
(Asami et al. 1998, Davison et al. 2005).

Our attention is drawn to the exceptions. In the helicoid
group, species of Amphidromus Albers, 1850 have high-
spired shells but still mate reciprocally, even between chi-
rally-reversed individuals (M. Schilthuizen, personal com-
munication). As they also lack darts, it is tempting to
speculate that the shell-shape change and lack of darts are in
some way associated, but there is no firm evidence. Clausiild
snails are interesting because some species mate unilaterally
whereas others mate reciprocally; there is even within-
species variation (Nordsieck 2005a, 2005b). Finally, Oreohe-

MATING BEHAVIOR OF SNAILS AND SLUGS

175

Table 1. An ovei-view of the literature on tlie mating behavior of stylommatophoran land snails and slugs. Mating behaviour: FF,
face-to-face; I, idiosyncratic; SM, shell-mounting; SR, simultaneous reciprocal; U, unilateral (includes sequential unilateral mating); ?, not
known. Shell shape: H, high-spired; L, low-spired; S, slug or semi-slug. Darts: N, dart and art-sac absent; Y, dart or dart-sac present.

Shell

Family Genus Mating shape Darts References

Helicoidea

Bradybaenidae

Bradybaeua Beck, 1837

FF

SR

L

Y

Asami et nl. 1998

Euhadra Pilsbry, 1890

FF

SR

L

Y

Takeda and Tsuruoka 1979, Azuma
1995, Asami ct al. 1998, S.

Chiba, A. Davison, ). Koene,
pers. obs.

Camaenidae

Mandarina Pilsbiy, 1895

FF

SR

L

N

S. Chiba & A. Davison, pers. obs.

Caracolus Montfort, 1810

FF

SR

L

N

Howell-Rivero 1950, Webb 1970b,
1974

Polydontes Montfort, 1810

7

?

L

N

Webb 1970b, 1974

Pleurodonte Fischer von Waldheim, 1807

FF

SR

L

N

Sanchez Munoz 2005a

Satsuma A. Adams, 1868

FF

SR

L

N

Abbott 1989, Azuma 1995

Amphidronms Albers, 1850

FF

SR

H

N

Schilthuizen and Davison 2005, M.
Schilthuizen, pers. comm.

Zachrysia Pilsbry, 1894

FF

SR

L

N

Howell-Rivero 1946, Sanchez
Munoz 2005b

Helicidae

Cepaea Held, 1837

FF

SR

L

Y

Beaumont 1988

Cantareiis Risso, 1826

FF

SR

L

Y

Giusti and Lepri 1980-1981,
Adamo and Chase 1988, Giusti
and Andreini 1988

Theba Risso, 1826

FF

SR

L

Y

Giusti and Andreini 1988

Arianta Leach in Turton, 1831

FF

SR

L

Y

Hofmann 1923, Locher and Baur
2000

Helix Linnaeus, 1758

FF

SR

L

Y

Meisenheimer 1907, leppesen 1976,
Lind 1976, Giusti and Lepri
1980-1981, Chung 1987

Tacheocampylaea Pfeiffer, 1877

FF

SR

L

Y

Giusti and Lepri 1980-1981

Eobania P. Hesse, 1913

FF

SR

L

Y

Giusti and Lepri 1980-1981

Iherus Montfort, 1810

FF

SR

L

Y

Rabaneda-Bueno et al 2004, this
paper

Helminthoglyptidae Cepolis Montfort, 1810

FF

SR

L

Y

Webb 1942, 1952a

Helminthoglypta Ancey, 1887

FF

SR

L

Y

Webb 1942, 1952a, van der Laan
1971, van der Laan 1980

Hiimboldtiana Ihering, 1892

FF

SR

L

Y

Webb 1980b

Monadenia Pilsbry, 1895
Sotwrella Pilsbry, 1900

FF

FF

SR

SR

L

L

Y

Webb 1952b
Webb 1980a, 1990

Polyinita Beck, 1837

FF

SR

L

Y

Moreno 1950, Tur et al. 2002

Hygromiidae

Cochlkella Ferussac, 1820

FF

SR

H

Y

Schileyko and Menkhorst 1997,
Asami et al. 1998

Monacha Fitzinger, 1833

FF

SR

L

Y

Storey 2005

Halolinmohelix Germain, 1913

FF

SR

L

Y

Block 1968a

Polygyridae^

Allogona Pilsbry, 1939

FF

SR

L

N

Webb 1948a, Emberton 1994,
Atkinson 2005

Ashmimella Pilsbry & Cockerell, 1899

FF

SR

L

N

Webb 1954a, Emberton 1994

Cryptomastix Pilsbry, 1939

FF

SR

L

N

Webb 1970c, Emberton 1994

Mesodon Rafmesque in Ferussac, 1821

FF'

SR

L

N

Webb 1954b, Emberton 1991

Neohelix Ihering, 1892

FF

SR

L

N

Webb 1952d, Emberton 1994

Polygyra Say, 1818

FF'

SR

L

N

Archer 1933, Emberton 1994,
Webb 1994a, 1994b

176

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Table 1. Continued
Family

Genus

Mating

Shell

shape

Darts

References

Stenotrema Rafmesque, 1819

FF‘

SR

L

N

Webb 1947, 1948b, Emberton 1994

Tnlobopsis Pilsbry, 1939

FF

SR

L

N

Webb 1965, Emberton 1994

Triodopsis Rafmesque, 1819

FF

SR

L

N

Webb 1948a, 1959, Emberton 1994

Vespencola Pilsbry, 1939

FF

SR

L

N

Webb 1970a, Emberton 1994

Limacoidea

Agriolimacidae

Dewceras Rafmesque, 1820

FF

SR

S

N

Reise 1995, Reise 2004

Arionidae

Arion Ferussac, 1819

FF

SR

S

N

Adams 1910, Quick 1946, Davis
1977

Geomalaciis Allman, 1842

FF

?

S

N

Platts and Speight 1988

ArioUmax Morch, 1859

FF

SR/U

S

N

Leonard et al. 2002

Ariophantidae

Arioplumta Desmoulins, 1829

FF

SR

L

Y

Dasen 1933

Hemiplecta Albers, 1850

FF

SR

L

N

S. Panha, pers. comm.

Macrochlamys {Syama) Godwin-Austen, 1908

FF

SR

L

N

S. Panha, pers. comm.

Micropannarion Simroth, 1893

FF

?

S

N

Liew Thor Seng, pers. comm.

Gasti'odontidae

Oxychilus Fitzinger, 1833

FF

SR

L

N

Rodriguez and Gomez 1999

Limacidae

Umax Linnaeus, 1758

I'

SR

S

N

Chase 1952, Quick 1960, Langlois
1965, Baur 1998

Limaciis Lehmann, 1864

FF

SR

S

N

Barker 1999

Milacidae

Tandonia (Milax) Lesson & Pollonera, 1882

FF

SR

s

N

Quick 1960

Trochomorphidae

Bertia Ancey, 1887

FF

SR?

L

N

Menno Schilthuizen, pers. comm.

Urocyclidae

Trichotoxon Simroth, 1889

FF

SR

s

Y

Bernard Verdcourt, pers. comm.

Gymnarion Pilsbry, 1919

FF

SR

L

N

Binder 1977

Sheldonia Ancey, 1887

FF

SR

L

N

Herbert and Kilburn 2004

ElisoUmax Cockerell, 1893

FF

SR

S

N

Herbert and Kilburn 2004

Vitrinidae

Semilimax Agassiz, 1845

FF

SR

s

Y

Kunkel 1933

Vitrinobracliiiim Kunkel, 1929

FF

SR

s

N

Kunkel 1933

Zonitidae

Mesomphix Rafmesque, 1819

FF

SR

L

N

Webb 1952c

Ventridens Binney & Bland, 1869

FF

SR

L

Y

Pilsbry 1946, Webb 1948c

Other

Acavidae

Helicoplumta Westerlund, 1886

SM

U

L

N

George Williams, pers. comm.

Achatinidae

Achatina Lamarck, 1799

SM

SR

H

N

Tomiyama 1994, 1996

Archachatiua Albers, 1850

SM

SR

H

N

Plummer 1975

Cerastidae

Zebrinops Thiele, 1931

FF

SR

H

N

Block 1968b

Ceriidae

Gerion Roding, 1798

?

U

H

N

Woodruff 1978

Chondrinidae

Solatopupa Pilsbry, 1917

?

u

H

N

Boato and Rasotto 1987

Clausiliidae

Albinaria Vest, 1867

SM

SR

H

N

Schilthuizen and Lombaerts 1995,
Menno, Schilthuizen, pers. comm.

Enpbacdiisa O. Boettger, 1877

SM

U

H

N

Asami et al 1998

Liichupliaediisa Pilsbry, 1901

SM

u

H

N

Asami et al. 1998

Stereophaedusa O. Boettger, 1877

SM

u

H

N

Asami et al 1998

(Alopilinae)

Agathylla H. & A. Adams, 1855

SM

SR

H

N

Nordsieck 1969, 2005a, 2005b

Cochlodina Ferussac, 1821

SM

U

H

N

Nordsieck 2005a, 2005b

Ddima Hartmann, 1842

SM

SR

H

N

Nordsieck 1969, 2005a, 2005b

Herilla H. & A. Adams, 1855

SM

SR

H

N

Nordsieck 2005a, 2005b

Medora H. & A. Adams, 1855

SM

SR

H

N

Nordsieck 2005a, 2005b

(Baleinae)

Balea Gray, 1824

SM

U

H

N

Nordsieck 2005a, 2005b

(Clausiliinae)

Lacbiiaria Hartmann, 1842

SM

u

H

N

Nordsieck 2005a, 2005b

Mncrogastra Hartmann, 1841

SM

u

H

N

Nordsieck 2005a, 2005b

Claiisilia Draparnaud, 1805

SM

u

H

N

Nordsieck 2005a, 2005b

Ruthenica Lindholm, 1924

SM

u

LI

N

Nordsieck 2005a, 2005b

Neostyriaca A. Wagner, 1920

SM

u

H

N

Nordsieck 2005a, 2005b

MA'riNG BEHAVIOR OF SNAILS AND SLUGS

177

Table 1. Continued
Family

Genus

Mating

Shell

shape

Darts

References

Discidae

Aiiguispira Morse, 1864

SM‘

SR

L

N

Webb 1968b

Enidae

Mastiis Beck, 1837

SM

SR

H

N

Paramakelis and Mylonas 2002,
Paramakelis, pers. comm.

Haplotrematidae

Haplotrema Ancey, 1881

SM

U

L

N

Webb 1943

Oreohelicidae

Oreohelix Pilsbry, 1904

SM

u

L

N

Webb 1951

Orthalicidae

Liguus Montfort, 1810

SM

u

H

N

Davidson 1965, Poland 2005

Partulidae

Partula Ferussac, 1819

SM

u

H

N

Lipton and Murray 1979

Philomycidae

Philomyciis Rafinesque, 1820

FF

SR

S

Y

Webb 1968a

Rhytididae

ParypJumta Albers, 1850

SM

u

L

N

Stringer et al. 2003

Spiraxidae

Eiiglandina Fischer tk Crosse, 1870

SM

LI

H

N

Cook 1985

Strophocheilidae

Stwpliocheiliis Spix, 1827

SM

U

H

N

Wiswell and Browning 1967

Succineidae

Catiuella Pease, 1870

SM'

SR

H

N

Webb 1977a

Oxyloma Westerlund, 1885

SM

U/SR

H

N

Webb 1977a, 1977b, 1977c

Sucdma Draparnaud, 1801

SM

U/SR

H

N

Rieper 1912, Hecker 1965, Webb
1977a, Jackiewicz 1980,
Villalobos et al. 1995, lordaens
et al. 2005

Valloniidae

Valloinn Risso, 1826

FF

SR?

L

N

Barker 1999

Vertiginidae

Vertigo Miiller, 1774

SM

U?

H

N

Barker 1999

‘ External sperm exchange; sperm is deposited on the male’s everted penis without intromission (see Emberton 1994)
” See Emberton ( 1994) for details of mating in other Polygyrid species (mostly papers by Webb).

Table 2. Species for which there are videos showing mating behavior.

Mating

Taxon

behavior

Film-maker

Ariolimax dolichophalltis Mead, 1943

Face-to-face

Brooke Miller, UC Santa Cruz (miller@biology.ucsc.edu)
http://bio.research.ucsc.edu/grad/weaver/Pages/proiect.html

Cantareus aspersus Mtiller, 1774

Face-to-face

Joris Koene, Vrije Universiteit (joris.koene@falw.vu.nl)
Ronald Chase, McGill University (ronald.chase@mcgill.ca)

Deroceras sp.

Face-to-face

Heike Reise, Staatliches Museum fur Naturkunde Gorlitz
( Heike.Reise@smng.smwk.sachsen.de

http://www.malacsoc.org.Uk/Malacological%20Bulletin/BLlLL43/king2.htm#dive

Eultadra sandai Kobelt, 1879

Face-to-face

Nishi Hirotaka (movie archives of animal behaviour)
http://zoo2.zool.kyoto-u.ac.jp/ethol/

Satsuma amanoi Kuroda, 1960

Face-to-face

Nishi Hirotaka (movie archives of animal behaviour)
http://zoo2.zool.kyoto-u.ac.jp/ethol/

Mastiis pupa Linnaeus, 1758

Shell-mounting

Aris Paramakelis, University of Crete (parmakel@nhmc.uoc.gr)

lix Pilsbty, 1904 is another intriguing genus because it mates
by shell-mounting (Webb 1951). Although its phylogeny is
unclear (Wade et al. 2006), we predict that it will either fall
within the helicoid group or be basal to it.

We expect that observations of mating behavior will
grow over the coming years and so will continue to update
the database of mating behavior (updated versions at http;//

www.molluscs.org). We welcome any data that we have in-
advertently excluded, including single photographs.

ACKNOWLEDGEMENTS

We would like to thank Glenn Webb for making so
many observations over so many years. This paper would

178

AMERICAN MALACOLOGICAL BULLETIN 25 '111- 2007

not have been possible without his contribution. Thanks also
to comments received from two anonymous referees.

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MATING BEHAVIOR OF SNAILS AND SLUGS

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Accepted: 9 March 2007

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Amer. Maine. Bull 23: 183-189

The function of dart shooting in helicid snails’^

Ronald Chase

Department of Biology, McGill University, 1205 Ave. Docteur Penfield, Montreal, Quebec, H3A IBl Canada, ronald.chase@mcgill.ca

Abstract: Some stylommatophoran species, including several helicid snails common to Europe and North America, drive sharp, calcareous
darts into their sexual partners prior to copulation. Why any animal would treat a prospective mate in this manner has been the subject
of considerable speculation. One widely held belief is that the dart stimulates the partner. Here, 1 review evidence showing that this
hypothesis, along with several others, is almost certainly incorrect. On the other hand, there is strong empirical support for the idea that
the dart increases the reproductive fitness of the successful shooter by promoting the survival and utilization of its sperm. How the dart
works to produce this effect is an open question; current evidence indicates that it injects a chemical agent into the recipient and that this
substance contracts the female tract in such a manner as to facilitate the passage of allosperm to the spermatheca. Although successful dart
shooting clearly benefits the shooter, there is little evidence to suggest either a cost or a benefit to the recipient.

Key words: sexual selection, sperm competition, courtship, sexual conflict, Cantareus aspersus

According to recent phylogenetic studies (Koene and
Schulenburg 2005, Davison et al. 2006), a dart is used in only
4-9 families within the Stylommatophora, which comprises
approx. 60 families of snails and slugs in total. It is note-
worthy that all the dart-bearing families mate in a simulta-
neous, reciprocal manner (Davison et al. 2006). Most of the
research on molluscan darts has been done on helicid snails,
in particular Cantareus aspersus (Muller, 1774; formerly He-
lix aspersa). Therefore, the present review relates specifically
to C. aspersus unless otherwise noted.

The dart, like other reproductive structures, differs
greatly in size and shape among species (Koene and Schu-
lenburg 2005). Individuals of a few species even possess mul-
tiple darts, all of which are released in a single courtship
episode. In Cantareus aspersus, the dart is sharply pointed; it
has a fluted shaft and a corona by which it attaches to the
dart sac while in storage (Fig. 1). The dart from the first
shooter is released about 30 min before the initiation of
courtship (Fig. 2). About 25 min later, the second animal
releases its dart. Copulation (mutual intromission) ensues
after a further 10 min (Chung 1987, Adamo and Chase
1988). Although the act of releasing the dart is often de-
scribed as “shooting,” in fact the dart does not travel
through the air. It is forcefully externalized, but its corona
remains lightly attached to the dart sac until it is pulled away
after the tip becomes embedded in the partner’s skin. Ap-
proximately one-half of the darts strike the body wall of the
partner and remain lodged there for hours, whereas the rest
of the darts either miss the intended target altogether or
strike only weakly, then fall out. In the former cases, the dart

is retracted. Significantly, copulation occurs regardless of the
fate of either partner’s dart.

From appearances, it would seem that the dart is harm-
ful, but this has not been proven. Although I have observed
hundreds of matings, I have never seen any reaction to the
dart apart from a momentary reflexive withdrawal of the body.
Never has an animal sulfered a noticeable long-term effect,
let alone death. However, I have seen one dart penetrate cleanly
through the head of its target (Fig. 1 in Chase and Blanchard
2006) and another dart lodge in the cerebral ganglion.

The possibility of interactions between the two dart
shooting events ot a courtship was examined in a recent
study (Chase and Vaga 2006). We found that neither the
timing, accuracy, nor location of the second shot was influ-
enced by the success or failure of the first shot. This result,
and others, indicates that dart shooting is not a source of
conflict during the mating process: the protracted courtships
cannot be interpreted as attempts to shoot without being
shot. Rather, each snail appears to be interested only in
getting off the best possible shot, evidently one that pen-
etrates deeply near the genital pore, for reasons to be ex-
plained below. Although we found no evidence for direct
costs of dart receipt, the possibility of indirect, post-
copulatory costs remains a possibility; this too will be dis-
cussed below.

FOUR FALSIFIED HYPOTHESES

The striking behavior ot dart shooting has occasioned
numerous commentaries through the ages, with no paucity

* From the symposium “Gastropod Mating Systems” presented at the joint meeting of the American Malacological Society and Western
Society of Malacologists, held 26-30 June 2005 at Asilomar, Pacific Grove, California.

183

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Figure 1. Photograph of a dart from Cantareiis aspersiis. The co-
rona remains attached to the tubercle of the dart sac until the dart
strikes its target. As the dart is expelled, mucus collects in the fluted
cavities created by the vanes.

of speculation as to its function. Here, I briefly review several
hypotheses which, although once plausible, may now be re-
jected (see also Landolfa 2002).

1. Sexual stimulation

Swammerdamm described the dart in the mid seven-
teenth century, but he was apparently unaware of its role in
reproduction (for references, see Kothbauer 1988). The first
written account of dart shooting is by Maupertuis (1753).
Maupertuis’ interpretation of the dart’s function was pre-
cisely that of most modern authors, namely that it stimulates
the partner to proceed with mating. Kothbauer (1988) has
drawn our attention to the fact that Maupertuis’ view of dart
shooting in snails, as well as that of many subsequent au-
thors, corresponds to the main idea behind Eros, the Greek
god who was able to cause other gods to fall in love by
shooting them with arrows. Indeed, although I have no evi-
dence, I suspect that the ancient Greeks created Eros after
observing Cantarens aspersiis (or perhaps Helix pomatia Lin-
naeus, 1758) shooting darts in their gardens. Maupertuis
asserted that the snail’s use of the dart is necessary and
justifiable due to the snails’ lethargic disposition, but he
argued that for humans to use similar violent means to
arouse passions would be immoral.

An immediate objection to the idea that the dart’s func-
tion is to stimulate the partner is that by the time the dart is
shot, i.e., late in the courtship ritual, the partner is already
highly aroused; indeed the partner is nearly ready to shoot its
own dart. Hence, at this point, there is no need to further
stimulate the partner.

Several empirical studies have examined whether the
receipt of a dart does, in fact, quicken the activity of the
targeted partner. Adamo and Chase (1988) found that the
interval between the two dart shots was slightly reduced
when the first shot hit the partner compared to when it

Figure 2. An individual of Cantareiis aspersiis photographed at the
moment of dart release. Because this dart did not penetrate the
partner, it was retracted by the shooter and digested. The peculiar
squeezed appearance of the shooter’s tentacles is the consequence
of elevated hydrostatic pressure. Note also the everted genital ap-
paratus of the intended target snail. The photograph was digitally
edited to eliminate background and to enhance contrast; the dart
was colored white. Shell length of the upper snail is ca. 27 mm.
Original photograph by Shelley Adamo; photograph reproduced
with permission of Oxford University Press.

missed the partner; these data suggested a stimulatory effect
of the dart. However, other studies (e.g., Lind 1976, Helix
pomatia) reported either an absence of stimulation or an
actual diminution in the level of arousal after a snail was hit
by a dart. Additionally, in a recent study with a large sample
size and strictly defined measures, we found no significant
effect of successful dart shooting on the interval between
dart shots (Chase and Vaga 2006). Nor did we find that the
outcome of either dart shot affected the duration of court-
ship (measured from the first dart shot to intromission).

2. Species recognition

This hypothesis (Diver 1940) is built on the fact that
snails lack an auditory sense and have essentially no vision,
leaving only touch and chemosensation as instruments by
which to distinguish conspecifics from heterospecifics. The
hypothesis is effectively disproven by the fact that snails
show no reluctance to mate even when untouched by their
partner’s dart. An alternative, and likely, means of identify-
ing conspecifics is through the extensive body contacts that
occur during courtship.

THE FUNCTION OF DART SHOOTING

185

3. A gift of calcium

Charnov (1979) proposed that the dart is a gift of cal-
cium that can be used to promote the development of off-
spring. It is true, of course, that young snails require an
ample supply of calcium to grow their shells, and that en-
vironmental sources of the mineral may be limited. On logi-
cal grounds, however, it would seem to be a poor strategy to
give away as much calcium by shooting a dart as one is likely
to get by receiving a dart. In any case, the amount of calcium
that can be effectively transmitted to the offspring from a
donated dart is too small to make an appreciable difference
(Koene and Chase 1998a).

4. A signal of intention

In an attempt to solve the enigma of the dart, Leonard
(1992) advanced the idea that snails shoot darts to signal
their readiness to deliver sperm to their partner. This hy-
pothesis grew out of earlier work in which she claimed that
the male role is the less preferred role in the helicid mating
system because the fate of donated sperm is uncertain. Thus,
she argued, snails would rather mate as females. The evolu-
tion of the dart provided an honest signal of a snail’s inten-
tion to donate sperm, thus inducing the partner to recipro-
cate and allowing both snails to benefit. Leonard’s bold
hypothesis, however, has been contested on both theoretical
and empirical grounds. First, the assumed preference for the
female role is untenable because, over time, the fitness of
male actors and female actors is exactly equal (Greeff and
Michiels 1998). Second, several of Leonard’s specific predic-
tions have been falsified (Adamo and Chase 1996). Critically,
snails that do not receive darts nevertheless intromit and
they deliver full spermatophores to their non-shooting or
poorly shooting partners (Rogers and Chase 2001 , Chase and
Vaga 2006).

ONE SUPPORTED HYPOTHESIS

The only hypothesis to receive consistent empirical sup-
port states that successful dart shooting enhances male fit-
ness by allowing more of the shooter’s sperm to become
stored in the recipient’s spermathecal sacs, hereafter referred
to as the sperm-loading hypothesis. As first proposed by
Chung (1987) and later elaborated upon by Adamo and
Chase (1996), the sperm-loading hypothesis treats dart
shooting as a male manipulative device while ignoring fe-
male interests, but I discuss the female point of view below.
In addition, neither Chung (1987) nor Adamo and Chase
(1996) explicitly referred to the concept of sperm competi-
tion, i.e., competition between males to fertilize eggs, al-
though it is in this context that the hypothesis is correctly
placed today. Helicid snails are ideal participants for sperm

competitions because they mate promiscuously, they store
sperm for long periods of time, and they fertilize internally
(Chase 2002).

In helicid snails, sperm are packaged inside a spermato-
phore for transfer during copulation. After the spermato-
phore is delivered to the partner, the sperm leave the sper-
matophore and migrate to the storage site, a structure
known as the fertilization pouch-spermathecal complex
(FPSC). Along the way, digestive enzymes typically digest
about 99.98% of the received allosperm (Lind 1973, Rogers
and Chase 2001).

Strong evidence in favor of the sperm-loading hypoth-
esis came from the study of Rogers and Chase (2001 ). Virgin
snails were mated one time only with partners that either hit
them with their darts or missed them with their darts. Seven
days after the mating, the former virgin was dissected and
the FPSC was removed. Allosperm in the FPSC were labeled
using a fluorescent DNA stain, then counted. Snails that
were hit by a dart stored 1 1 6% more sperm than snails that
were missed (Rogers and Chase 2001 ). Because helicid snails
can produce multiple egg clutches from the sperm of a single
donor (Chen and Baur 1993), the results of this experiment
imply a fitness advantage to the successful dart shooter be-
cause its sperm should remain available for a larger number
of clutches than would be the case if its dart had missed.

To see whether the increased sperm storage that we
observed after a single mating would provide an advantage
when the successful shooter competed with a second sperm
donor, we conducted competitive mating trials in which one
donor hit the recipient with his dart and the other donor
missed. The order of hits and misses was balanced. After the
matings, we waited for eggs to be laid and then genotyped
the twice- mated mother, each of the two potential fathers,
and a randomly chosen sample of the offspring. Note that if
the sperm used for fertilization were selected by a raffle-like
process, then the donor that has managed to store the most
sperm will be the most successful father. Thus, we predicted
that the successful dart shooter would father more offspring
than the unsuccessful shooter. The experiment was con-
ducted twice, with slightly different conditions, but with very
similar results (Landolfa et al. 2001 , Rogers and Chase 2002).
Successful dart shooting significantly improved paternity in
this competitive mating situation (Fig. 3). In Cantareus as-
persus, sperm from the first donor is used preferentially over
that from the second donor, regardless of the success or
failure of dart shooting (Evanno et al. 2005, Chase and Blan-
chard 2006). As a consequence of this phenomenon, known
as first donor precedence, the influence of the dart is most
pronounced with respect to the second donor. The paternity
of the second donor increased from 17%, when its dart
missed and the first donor’s dart hit, to 39% when its dart hit

186

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

90

First donor hit First donor missed

Second donor missed Second donor hit

Figure 3. Successful dart shooting increases reproductive success in
competitive matings. Snails mated with two spierm donors before
producing offspring. One donor hit with its dart, the other missed.
Paternities were determined by allozyme genotyping; means ± SE
are shown. The effects of the dart are most evident with respect to
the second donor ( 17% paternity vs. 39% paternity), owing to first
sperm precedence. Data are from Rogers and Chase (2002).

and the first donor’s dart missed (Fig. 3; Rogers and Chase
2002).

UNRESOLVED QUESTIONS

While considerable progress has been made in imder-
stanciing the function of the dart, several questions remain
concerning its mechanism of action and its evolution.

1. How does the dart influence sperm utilization?

We suppose that receipt of a dart triggers events in a
signaling pathway that ultimately produces effects in the
organs that receive sperm. A complete account of the dart’s
mechanism of action will require descriptions of each step in
the signaling pathway. Here, I focus on the initial signal,
which is conveyed by the dart. Although the dart itself is a
hard structure that will elicit responses in mechanosensory
neurons when it penetrates the body wall, the possibility that
the dart carries a chemical signal must also be considered
because the dart is covered with mucus when it is expelled by
the shooter. The presence of mucous glands specifically as-
sociated with the dart sac is a consistent feature of the dart-

bearing stylommatophoran species (Koene and Schulenburg
2005). The mucus produced by these glands could be used
simply to lubricate the jrassage of the dart out of the animal.
However, several pieces of evidence suggest that it contains
one or more chemical components that are essential effec-
tors of the dart’s function. First, the fluted structure of the
dart itself (Fig. 1) can be seen as an adaptation to increase
the amount of mucus that can be loaded onto the dart. In
Cantareiis aspersiis, the dart carries about 2 mg of mucus
(Chung 1986). Second, quantitative analysis has revealed
that the effect of dart shooting on sperm storage and pater-
nity are both significantly dependent on the shell volume of
the recipient (Rogers and Chase 2001, 2002). The smaller the
recipient snail, the larger the dart’s effect. One interpretation
of this relationship is that the potency of the dart is dimin-
ished in larger animals due to chemical dilution. Third,
when mucus from the dart gland is applied to the female
reproductive tract in vitro, contractions occur that cause a
reconfiguration of the tract at the critical junction between
the organ that receives the spermatophore (the bursa tract
diverticulum) and the sperm digestive gland (the bursa
copulatrix) (Koene and Chase 1998b). These mucus-induced
contractions could allow more sperm to escape enzymatic
digestion as they travel from the safety of the spermatophore
to the safety of the spermatheca.

Based on the observations summarized above, it is rea-
sonable to suppose that the dart functions by injecting mu-
cus into the recipient and that molecules present in the
mucus cause temporary structural changes in the female
tract. According to this idea, the dart serves only to convey
and inject the chemical agent. To test the hypothesis, Katrina
Blanchard and I conducted an experiment to determine
whether the dart works by a mechanical means or a chemical
means (Chase and Blanchard 2006). As in the experiments
described above (Landolfa et al. 2001, Rogers and Chase
2002), we arranged for a snail to receive sperm from two
donors before producing offspring. In this experiment, how-
ever, none of the snails shot darts. Instead, we poked the
eventual mother with a hypodermic needle as soon as we
detected the partner’s intention to dart-shoot. In one of the
two matings, we injected saline through the needle; in the
other mating, we injected an extract of the dart gland mucus.
Thus, in both cases, the mother received mechanical stimu-
lation, but only in the latter case did she receive chemical
stimulation. On average, snails delivering sperm in associa-
tion with injections of mucus fathered 2.3 times the number
of babies as did competing snails that delivered sperm to the
same mother in association with injections of saline (Chase
and Blanchard 2006). This result provides strong evidence
that the dart works largely or entirely by injecting mucus,
not simply by rupturing the skin. We cannot exclude the

'I'HE FUNCTION OF DART SHOOTING

187

possibility, however, that skin rupture alone may have a
small effect on paternity.

2. What is the identity of the bioactive molecule(s) in
the dart’s mucus?

The next step will be to identify the bioactive mol-
ecule(s) in the dart’s mucus. Because gastropod molluscs
often use peptides as neurotransmitters and hormones
(Chase 2002), and because peptides have been found in the
secretions of the mucous gland (Bcirnchen 1967, Chung
1986), the effective agent is likely to belong to this class of
molecule. To identify the molecule requires an efficient bio-
assay. At the present time, a modified procedure to count
stored allosperm in once-mated individuals offers the best
opportunity. By adopting an approach based on the succes-
sive fractionation of mucous extracts, and by using sperm
counts as the bioassay, it should be possible to identify the
molecule(s) of interest.

3. Does dart shooting either benefit or harm
the recipient?

While successful dart shooting almost certainly benefits
the shooter, it is not known whether it either benefits or
harms the recipient. Benefits to the recipient would occur in
either of two scenarios: ( 1 ) if successful dart shooting were
associated with genes that provide superior viability (the
“good genes” model) or (2) if the ability to shoot success-
fully were heritable, in which case the offspring of the re-
cipient would have a fitness advantage (the “sexy sons”
model). Although there is as yet no evidence bearing on
these possibilities, suitable tests could be conducted. To test
the “good genes” model, it will be necessary to compare the
longevity, or viability, of successful and unsuccessful shoot-
ers. To test the “sexy sons” model, it needs to be shown that
variability in dart structure, mucous content, or dart-
shooting behavior is heritable. Until one of these tests pro-
vides positive evidence of a benefit to the recipient (and
none will be easy to perform), it is not unreasonable to
assume a benefit to the shooter alone, in which case dart
shooting could be characterized as “male manipulation”
(Adamo and Chase 1996).

Rather than benefiting the recipient, the dart could be
costly if it reduced the recipient’s control over the process of
fertilization, if it damaged tissue, or if it increased the
chances of infection via the wound. However, none of these
possible long-term effects has been documented, and short-
term negative effects appear negligible (Chase and Vaga
2006). Thus, current evidence indicates that while successful
dart shooting benefits the shooter, its consequences for the
recipient are neutral.

Bearing in mind that the recipient is the female partner
in this drama, and assuming for the moment that successful

dart shooting is associated with high quality genes, it has
been proposed (Landolfa 2002) that females perceive the
successful dart shot as an indicator of good genes and that
they therefore “choose” to store and use sperm from the
successful shooter. This would amount to mate selection,
but with the choice being made after copulation, i.e., in a
“cryptic” manner (Eberhard 1996). It is conceivable that the
female function could sort the sperm from various donors to
different spermathecal sacs, and later, prior to fertilization,
she could selectively release the sperm belonging to the high-
est-quality donor (see Bojat et al. 2001). Attractive though
this idea may be, there is no evidence to support it. Further-
more, as noted above, we recently found that injections of
mucus through a needle can replicate the benefits to male
fitness that ordinarily follow from successful dart shooting.
Thus, if females are choosing, they could only be doing so on
the basis of the mucus. The use of any other trait, including
any present in the shooting behavior, the dart structure, or
the sperm, is excluded by the design ot the aforementioned
experiment.

4. How did the dart evolve?

The steps in the evolution of the dart apparatus are
difficult to imagine and probably impossible to confirm.
There are many types of “accessory” or “auxiliary” structures
associated with the stylommatophoran penis (Tompa 1984).
These structures comprise two major groups: ( 1 ) the sarco-
belum, a fleshy club-like appendage, and (2) the gypsobe-
lum, a hard, sharp instrument, of which the dart is just one
example. Although glands are certainly associated with ac-
cessory structures in many species, it would be usetul to
learn the full extent ot this association. It glands were in-
variably associated with accessory structures, then this would
support my contention that the primary adaptation is the
evolution of a bioactive agent capable of influencing pater-
nity. In ancestral cases, the substance might have been se-
creted, unaided, out the genital pore. Subsequently, different
lineages may have independently evolved accessory struc-
tures to improve the efficiency of delivery of the secretion
product. Alternatively, if there were taxa that possess either
a sarcobelum or a gypsobelum but no gland, then perhaps
the accessory structure itself is able to enhance paternity. As
mentioned earlier, our experimental evidence in Cantareiis
nspersus is insufficient to rule out this possibility (Chase and
Blanchard 2006). In this latter scenario, the glandular prod-
uct would be a secondary development that increased the
power of the manipulation.

If, in fact, the recipient of a dart sutlers a cost to its
female function, then an evolutionary arms race (Parker
1979) may evolve in which adaptations are selected that on
the one hand maximize the dart’s efficacy and on the other
hand minimize the extent of the harm caused by it. Koene

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

and Schulenburg (2005) recently reported results that are
consistent with this picture. From a phylogenetic analysis,
they found an association between a multi-component mea-
sure of the dart’s shape and a multi-component measure of
the “complexity” of the bursa tract diverticulum. Species
that have small darts have short diverticula, whereas species
with large darts or highly curved darts have long diverticula.
This result can be interpreted in light of the fact that longer
diverticula make it more difficult for sperm to escape safely
(Lind 1973). Thus, it would appear that, as species evolved,
the female function selected longer diverticula to defend
itself from the harmful effects of more powerful darts. How-
ever, until a specific cost of dart receipt is documented,
c]uestions relating to sexual conflict and its attendant an-
tagonistic coevolution with respect to the dart will remain
controversial.

CONCLUSION

Substantial progress has been made in recent years on
the question of why snails shoot darts. In Cantareus aspersus,
the dart is used to increase the survival and storage of the
shooter’s sperm in the recipient’s spermathecal sacs. As a
consequence, successful shooters have greater reproductive
success. The phenomenon can be characterized as one of
post-copulatory sexual selection in the context of intense
sperm competition. To describe it in such terms would have
been impossible prior to the bloom of sexual selection
theory in the mid-twentieth century, thus explaining, I be-
lieve, why it took so long for the riddle of the dart to be
solved. Not until empirical work on birds and insects had
made known the details of post-copulatory sexual selection
in those animals, and theoreticians had elaborated a general
context able to accommodate other taxa, could one have
imagined the dart’s hidden function. Studies on species
other than C. aspersus are needed to generalize the findings
that are summarized in this paper and to provide insights
into the dart’s evolution.

ACKNOWLEDGMENTS

The author’s research is funded by the Natural Sciences
and Engineering Research Council of Canada. I thank Ka-
trina Blanchard for pertinent discussions.

LITERATURE CITED

Adamo, S. A. and R. Chase. 1988. Courtship and copulation in the
terrestrial snail Helix aspersa. Canadian Journal of Zoology 66:
1446-1453.

Adamo, S. A. and R. Chase. 1996. Dart shooting in helicid snails:
An “honest” signal or an instrument of manipulation? Journal
of Theoretical Biology 180: 77-80.

Bojat, N. C., M. Dtirrenberger, and M. Haase. 2001. The sper-
matheca in the land snail, Arianta arbustoruni (Pulmonata:
Stylommatophora): Muscle system and potential role in sexual
selection. Invertebrate Biology 120: 217-226.

Bdrnchen, M. 1967. Untersuchungen zur Sekretion der fmgerfor-
migen Drtisen von Helix pornatia. Zeitschrift fur Zellforschung
78: 402-426.

Charnov, E. L. 1979. Simultaneous hermaphroditism and sexual
selection. Proceedings of the National Academy of Sciences USA
76: 2480-2484.

Chase, R. 2002. Behavior and its Neural Control in Gastropod Mol-
luscs. Oxford University Press, New York.

Chase, R. and K. C. Blanchard. 2006. The snail’s love-dart delivers
mucus to increase paternity. Proceedings of the Royal Society
(B) 273: 1471-1475.

Chase, R. and K. Vaga. 2006. Independence, not conflict, charac-
terizes dart shooting and sperm exchange in a hermaphroditic
snail. Behavioral Ecology and Sociobiology 59: 32-739.

Chen, X. and B. Baur. 1993. The effect of multiple mating on
female reproductive success in the simultaneously hermaph-
roditic land snail Arianta arbustoruni. Canadian Journal of
Zoology 71: 2431-2436.

Chung, D. J. D. 1986. Stimulation of genital eversion in the land
snail Helix aspersa by extracts of the glands of the dart appa-
ratus. Journal of Experimental Zoology 238: 129-139.

Chung, D. 1. D. 1987. Courtship and dart shooting behavior of the
land snail Helix aspersa. The Veliger 30: 24-39.

Davison, A., C. M. Wade, P. B. Mordan, and S. Chiba. 2006. Sex
and darts in slugs and snails (Mollusca: Gastropoda: Stylom-
matophora). Journal of Zoology 267: 329-338.

Diver, C. 1940. The problem of closely related species living in the
same area. In: l.S. Huxley, ed.. The New Systematics. Claren-
don Press, Oxford, UK. Pp. 303-328.

Eberhard, W. G. 1996. Female Control: Sexual Selection by Cryptic
Female Choice. Princeton University Press, Princeton, New
Jersey.

Evanno, G., L. Madec, J.-F. Arnaud. 2005. Multiple paternity and
postcopulatory sexual selection in a hermaphrodite: What in-
fluences sperm precedence in the garden snail Helix aspersal
Molecular Ecology 14: 805-812.

Greeff, J. M. and N. K. Michiels. 1998. Sperm digestion and recip-
rocal sperm transfer can drive hermaphrodite sex allocation to
equality. American Naturalist 153: 421-430.

Koene, J. M. and R. Chase. 1998a. The love dart of Helix aspersa is
not a gift of calcium. Journal of Molluscan Studies 64: 75-80.

Koene, I. M. and R. Chase. 1998b. Changes in the reproductive
system of the snail Helix aspersa caused by mucus from the
love dart. Journal of Experimental Biology 201: 2313-2319.

Koene, J. M. and H. Schulenburg. 2005. Shooting darts: Co-
evolution and counter-adaptation in hermaphroditic snails.
BMC Evolutionary Biology 5: 25.

Kothbauer, V. H. 1988. Uber Liebespfeile, Schnecken und Welt-

THE FUNCTION OF DART SHOOTING

189

bilder. Annalen des Nanirliistorisches Museums in Wien 90B:
163-169.

Landolfa, M. A. 2002. On the adaptive function of the love dart of
Helix aspersa. The Veliger 45: 231-249.

Landolfa, M. A., D. M. Green, and R. Chase. 2001. Dart shooting
influences paternal reproductive success in the snail Helix as-
persa (Pulmonata, Stylommatophora). Behavioral Ecology 12;
773-777.

Leonard, J. L. 1992. The “love-dart” in helicid snails: A gift of
calcium or a firm commitment? Journal of Theoretical Biology
159: 513-521.

Lind, H. 1973. The functional significance of the spermatophore
and the fate of spermatozoa in the genital tract of Helix po-
matia (Gastropoda: Stylommatophora). Journal of Zoology
169: 39-64.

Lind, H. 1976. Causal and functional organization of the mating
behaviour sequence in Helix pomatia (Pulmonata: Gas-
tropoda). Behaviour 59: 162-202.

Maupertuis, P. L. M. de. 1753. Vanis Physique. In: G. Olms, ed..
Oeuvres, Vol 2. 1965. Verlagsbuchhandlung, Hildesheim, Ger-
many.

Parker, G. A. 1979. Sexual selection and sexual conflict. In: M. S
Blum and N. A. Blum, eds.. Sexual Selection and Reproductive
Competition in Insects. Academic Press, New York. Pp. 123-
166.

Rogers, D. W. and R. Chase. 2001. Dart receipt promotes sperm
storage in the garden snail Helix aspersa. Behavioral Ecology
and Sociobiology 50: 122-127.

Rogers, D. W. and R. Chase. 2002. Determinants of paternity in the
garden snail Helix aspersa. Behavioral Ecology and Sociobiology
52: 289-295.

Tompa, A. S. 1984. Land snails (Stylommatophora). Jti: A. S.
Tompa, N. H. Verdonk, and 1. A. M. Van den Biggelaar, eds..
The Mollusca, Vol. 7. Academic Press, Orlando, Florida. Pp.
45-140.

Accepted: 1 July 2006

MEETING ANNOUNCEMENT
74th Annual Meeting of the American Malacological Society

Carbondale, Illinois

The American Malacological Society will hold its 74*'^ annual meeting in Carbondale, Illinois from June
29-July 3, 2008. The venue will be the Southern Illinois University Student Center, which houses an auditorium,
several ballrooms and meeting rooms, and a number of restaurants and coffee shops. The conference will begin
with an icebreaker on Sunday evening. Special events will include an outdoor reception at Blue Sky Vineyard
(www.blueskyvineyard.com) on Monday night, a poster session and the AMS Auction of molluscan miscellany on
Tuesday night, and a barbecue banquet (with vegetarian options) at the 17* Street Bar & Grill Warehouse,
Southern Illinois’ most unique banquet facility (www2.muiphysboro.com/community/restaurant/17thstreet.html)
on Wednesday night. There are persistent rumors of a dance party on Tuesday or Wednesday night as well. The
special sessions and symposia will include:

• a land snail conservation symposium and workshop in honor of the late Leslie Hubricht, organized by Kathryn
Perez (University of North Carolina-Chapel Hill/Duke University), Jay Cordeiro (NatureServe), Jochen Gerber
(Field Museum of Natural History), and Kevin Roe (Iowa State University)

• a symposium on molluscan taxonomy in the 21st century, organized by Benoit Dayrat (UC Merced)

• a special session on cephalopod biology organized by Dr. Frank Anderson, Dr. Christine Huffard (Monterey
Bay Aquarium Research Institute), and Dr. Elizabeth Shea (Delaware Museum of Natural History)

On Thursday, two field trips will introduce meeting participants to two wonderful mollusk habitats in
southern Illinois. Participants will be able to take a tour of the Larue Pine Hills/Otter Pond Research Natural Area,
a fantastic area of limestone bluffs and outcrops (and home of Euchemotrema hubrichti, the conference mascot)
or a trip to local aquatic habitats to search for freshwater bivalves and gastropods.

Visitors to Carbondale usually travel through either St. Louis or Chicago, though Memphis is also an option.
There is a convenient shuttle service from St. Louis Lambert International Airport to Carbondale. From Chicago
or Memphis, you can take a train — The City of New Orleans, which stops in Carbondale on its Chicago-to-New
Orleans route. American Connections regional commuter flights arrive at the Williamson County airport (618-
993-3353) several times daily. The airport is located 16 miles east of the SIU campus.

We look forward to seeing you in Carbondale, Illinois in 2008!

Frank E. (Andy) Anderson, President (2008)

American Malacological Society
feander@siu.edu

190

Amer. Malac. Bull. 23: 191-194

Melbourne Romaine Carriker: 25 February 1915 - 25 February 2007
An Appreciation

Clement L. Counts, IIl‘, Robert S. Prezant^, and J. Evan Ward^

' Department of Biological Sciences, Richard A. Henson School of Science and Technology, Salisbury University,

Salisbury, Maryland 21801-6861, U.S.A., clcounts@salisbury.edu

^ College of Science and Mathematics, Montclair State University, Montclair, New lersey 07043, U.S.A., prezantr@mail.montclair.edu
^ Department of Marine Sciences, University of Connecticut, 1080 Shennecossett Road, Groton, Connecticut 06340, U.S.A.,
evan.ward@uconn.edu

On 25 February 2007, our mentor, colleague, and great
friend, Melbourne Romaine Carriker died at Lewes, Dela-
ware. It was his ninety-second birthday. He was surrounded
by his children and grandchildren.

Mel’s life was as eventful and full as his scientific career.
He was born 25 February 1915 to Melbourne Armstrong
Carriker, Jr. and Myrtle Carmella Carriker on the family
coffee plantation, Vista Nieve, near Santa Marta, Colombia.
Mel detailed his boyhood experiences on the plantation in
his memoir Vista Nieve (Carriker 2000). In 1925, at the age
of ten, Mel participated in his first biological expedition
accompanying his father, a world-class ornithologist and en-
tomologist, to the eastern slope of the Andes.

The plantation was sold in 1927. After the sale, the
family moved to Tom’s River, New Jersey, and Mel’s father
became a curator of birds at the Academy of Natural Sci-
ences of Philadelphia (ANSP). Mel attended the public
schools and graduated from high school in 1934. In 1934
and early 1935, Mel and his father returned to the Andes in
Bolivia on another ornithological expedition (Carriker, Jr.
2006). During the steamship trip, Mel demonstrated his re-
markable abilities on the dance floor, exhibiting such skill
that other dancers stopped to watch him and his partner.
These displays were attributed to lessons provided by Mel’s
mother in Tom’s River (Castillo and Holyoak 2004). This
journey to the Andes was epic with train travel to the Alto
Plano, a steamer across Lake Titicaca, and brushes with Bo-
livian troops fighting a war with Argentina (Carriker 2005,
Carriker, Jr. 2006). It was during this expedition that Mel
contracted malaria.

Mel entered Rutgers University in 1935 and he majored
in agricultural research and minored in zoology, graduating
with honors and a B.S. in Zoology in 1938. Mel noted, by
playing a few minutes in a varsity match, he lettered in water
polo. It was Mel’s aim to become an ornithologist but in
1938, his undergraduate advisor, Thurlow C. Nelson, offered
him a position to study population movements of oyster

larvae in Barnegat Bay, New Jersey. In fall 1938, he entered
the University of Wisconsin and there earned a Master of
Philosophy and, then, a Doctor of Philosophy degree in June
1943. During summers from 1938 through 1941, Mel re-
turned to Great Bay, New Jersey, and in the summer of 1942
he was placed in charge of the Oyster Investigation Labora-
tory at Bivalve, New Jersey. These experiences launched his
research on Mollusca. Mel joined the graduate student group
of Lowell E. Noland to study Lymnaea stagnalis (Linne,
1758), the snail vector for swimmer’s itch in humans. His
doctoral dissertation focused on radular and digestive
anatomy, physiology, and function of L. stagnalis.

During 1939 at Wisconsin, Mel met Meriel Roosevelt
McAllister, known as Scottie. Following graduation from
Wisconsin, Mel entered the Naval Officers Training program
at Harvard College in June 1943 and emerged an Ensign in
the United States Naval Reseiwe. On 17 October 1943, he
and Scottie were married in Richmond, Virginia, at a cer-
emony officiated by Scottie’s uncle. Mel and Scottie would
have four sons: Eric, Bruce, Neal, and Robert. Mel was or-
dered for further training at Fort Schuyler, New York, fol-
lowed by training in Miami, Florida. Mel was then ordered
to the Aleutian Islands to serve aboard a small patrol craft
with a crew of 60 men and 5 officers. Since the Japanese had
been absent for several months, there was little to do but
make patrols, during which his duties were standing watch
and burning obsolete codes. Eventually, he was promoted to
Lieutenant (junior grade) and was made executive officer
(second-in-command). Between patrols, Mel collected mu-
ricid gastropods and their blood sera from the Aleutian wa-
ters for shipment. Mel laughed that the seamen thought this
behavior was odd but forgave him because he was, after all,
an officer, so odd behavior was expected. Mel placed these
Alaskan collections in the alcohol-preserved collections at
ANSP in the mid-1980s. Eventually his ship was sent to Pearl
Harbor for escort duty, including escorting barges filled with
pineapples. At the war’s end, Mel was ordered to report for

191

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AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

duty aboard a destroyer, patrolling off the Philippines, and
became a civilian again on 25 December 1945.

Mel and his family moved in with his mother at Belmar,
New Jersey, though he spent some time at Madison, Wis-
consin, publishing his dissertation. Although Mel had five
offers for positions, he was persuaded by Thurlow Nelson to
return to Rutgers and became a Lecturer of Zoology in 1946.
Mel came to regret taking the position since many of the
faculty remembered him as an undergraduate and still
thought of him as such. Then as an Assistant Professor at
Rutgers, he developed a graduate course in estuarine ecology
and participated in field courses where students and Mel’s
colleagues from geology and botany studied one of three
transects across the state. During 1947-1951, Mel, Thurlow
Nelson, and Harold Haskin conducted studies on Merce-
nnria mercenaria (Linne, 1758) with a view to commercial-
ization. Nelson and Haskin worked in Delaware Bay while
Mel worked on Little Egg Harbor, New Jersey. By 1954, it
became evident that Rutgers had room for only two marine
biologists and Mel opted to accept a position as Assistant
Professor at the University of North Carolina at Chapel Hill.
During 1954 and 1955, Mel conducted research on oysters
and clams on Gardner’s Island, New York under the spon-
sorship of the U.S. Fish and Wildlife Service in cooperation
with Victor Loosanoff.

While at UNC, Mel spent 1956 to 1960 doing research
at North Carolina Institute of Fisheries Research. He also
cooperated at the National Marine Fisheries Service Labo-
ratory at Morehead City and the Duke University Marine
Laboratory at Beaufort, North Carolina. During these sum-
mers, Mel focused his research on gastropods that drilled
oysters. The chair of the department, Charles Jenner, headed
both the limnology and marine ecology divisions of the de-
partment and undervalued Mel’s contributions to the point
that Mel was dismissed in 1961.

Mel then accepted a position at the U.S. Fish and Wild-
life Service. Mel and his family moved to Easton, Maryland
in the fall of 1961 and he took up his position at the Bureau
of Commercial Fisheries Laboratory at Oxford, Maryland.
As Chief of the Shellfish Mortality Program, Mel was in
charge of research on MSX, the parasitic disease of Cras-
sostrea virginica Gmelin, 1791 that was gaining a substantial
hold on oyster populations in Chesapeake Bay. However,
funding was problematic and frustrating. Just as Mel was
beginning at Oxford, he was offered the position as director
of the newly established Systematics-Ecology Program at the
Marine Biological Laboratory at Woods Hole, Massachu-
setts.

The Systematics-Ecology Program operated successfully
between 1962 and 1972 to study the flora and fauna of the
western North Atlantic. Mel developed the keys to Woods
Hole Region with Ralph 1. Smith (Smith 1964). Mel believed

that the accurate identification of species was central to good
ecological practice. In furtherance of this belief, Mel devel-
oped and supervised the publication of the series Keys to the
Flora and Fauna of the Northeast Atlantic Coast for the
National Marine Fisheries Service. During this time, Mel
also served on the Northeastern Regional Council, as-
sembled by the American Institute of Biological Sciences to
study bioscience research to be conducted on a manned
Earth-orbiting space station (Olive and Beem 1967). Mel
famously made a motion picture of the drilling behavior
of Urosalpinx cinerea (Say, 1822) that has since been placed
on the Internet (http://www.iwf.de/iwf/do/mkat/details
.aspx?Signatur=C-l- 13067), along with amplified recordings
of the rasping of the radula of muricids as they drilled
through oyster shell. By 1972, federal funding was becoming
scarce and Mel accepted a full professorship at the College of
Marine Studies of the University of Delaware.

Mel (Fig. 1) was responsible for helping to lay out the
new Harry L. Cannon Laboratory. He was instrumental in
developing the shellfisheries program, and a new species of
ameba found in the tanks was named in his honor (Ovalopo-
diin carrikeri Sawyer, 1980). Mel taught graduate courses in
malacology and supervised the research of doctoral students
(see Carriker on AMS web site: Table 1) and master’s stu-
dents (AMS web site: Table 2). Mel also recruited experts in
marine ecology who presented summer graduate courses.
Mel served on the doctoral and master’s committees of over
150 individuals since 1951 (AMS web site: Table 3).

Mel’s research, over six decades, concentrated on the
biology of Crassostrea virginica and its predator Urosalpinx
cinerea. Mel believed that the biology and ecology of preda-
tor and prey were entwined and that one could not be un-
derstood without knowledge of the other. How does U. ci-
nerea penetrate the shell of C. virginical How do newly
hatched U. cinerea find C. virginical What are the structures
and physiology of U. cinerea that allow it to bore a hole
through the shell of C. virginica and other bivalves? Mel
employed everything from simple field observations to x-ray
microanalysis. His observations included sound recordings
of the rasping of U. cinerea and cinematography (which can
still be viewed on a web site). He was also among the first to
apply scanning electron microscopy to the microstructure of
the radula of U. cinerea and the shells of C. virginica and
Mytilus edulis (Linnaeus, 1758). Mel identified the accessory
boring organ (ABO) of the drills U. cinerea and Eupleura
caudate (Say, 1822) and, through anatomical, histological,
and histochemical methods, elucidated their structure and
function in penetration of bivalve shells. Mel was able to link
the shape of a bore hole with the snails that produced it even
in paleontological specimens (Carriker and Yochelson
1968). Mel’s studies also made use of histochemistry, and he
examined the elemental analysis of major and minor trace

MELBOURNE R. CARRIKER-AN APPRECIATION

193

Figure 1. Mel Carriker in his laboratory early in his career at the
University of Delaware, College of Marine Studies (now Graduate
College of Marine and Earth Studies). University of Delaware stock
photograph.

elements in oyster shell using a proton probe, developed by
Charles P. Swann. Mel also studied chemoreception by U.
cinerea and E. caudata with his student Betsy Brown and
post-doctoral fellows Leslie G. Williams and Dan Rittschof.
Mel continued to exercise his interest in estuarine pollution
and its effects on the benthos, the invasion of coastal waters
by exotic species and the impact of those invasions on com-
mercially-valuable molluscan species. Mel summarized
much of the results of his long study of oysters in Kennedy
et al. (1996) and Mercenaria rnercenarin in Kraeuther and
Castagna (2001).

Mel served in many scientific organizations. Mel was
particularly active in the National Shellfisheries Association
(NSA), and in 1998 the Association founded a student re-
search grant in his name. In the NSA, he served as Treasurer,
Secretary, Vice-President, President, and Editor of the Pro-

ceedings of the National Shellfisheries Association. Mel was
named an Honored Life Member of the Association in 1991.
Mel was instrumental in the transformation of the Proceed-
ings of the National Shellfisheries Association into the Journal
of Shellfish Research. In 2005, Mel published a histoiy of the
association: The Taming of the Oyster. Mel was also active in
the American Malacological Society in which he served as
Vice-President, President, Member of Council, and was
named an Honorary Life Member. Mel was instrumental in
the transformation of the Bulletin of the American Malaco-
logical Union into the American Malacological Bnlletin and
served as a founding Associate Editor. At the most recent
meeting of the AMS, a Carriker Student Research Grant
program was also founded. He was a member of the Institute
of Malacology, which publishes Malacologia, and he long
served on the editorial board of the Quarterly Review of
Biology. Other professional societies included the American
Society of Zoologists, the New England Estuarine Research
Federation (in which he was an Honorary Life Member),
and the Atlantic Estuarine Research Federation.

Mel retired from the University of Delaware in 1985 at
the age of 70 and was named Professor Emeritus. A sympo-
sium was held in his honor on the Lewes campus at which
many of his friends and colleagues presented papers (Prezant
and Counts 1985). Mel was so esteemed among his students
that in 2001, to honor his 85*'" birthday, his students sur-
prised Mel with “Carrikerfest”, a celebration of his life to date
[http://darc.cms.udel.edu/carrikerfest200 1 /cfestindex.html] .
To further honor his contributions, his students and the
university presented him with the Carriker Contemplative
Garden just next to the shellfisheries laboratory. Mel, who
walked to work on a daily basis, continued working at Lewes
until two days before he suffered his stroke. LDuring his
emeritus years, Mel continued to submit annual activities
reports to the Dean’s office, although he was no longer re-
c]uired to do so. Dr. Nancy Targett, Dean of the College,
noted that Mel had more productive years in retirement
than some faculty members aspire to during their active
career. During his retirement, Mel published 31 papers, 4
book chapters, and 4 books. Mel served as president of the
Deliiware Partners in the Americas in which he worked for
closer scientific cooperation between the University and
Panama. He also actively served the Association of Marine
Laboratories of the Caribbean. By 2000, Mel had published
over 160 professional papers (AMS web site: Table 4) and
reports, 45 abstracts, and more than 255 presentations at
scientific meetings, a significant portion during his “retire-
ment” years. Mel continued to participate in professional
meetings throughout his professional life (Fig. 2). As was
true throughout his entire career, Mel deeply respected his
students and colleagues. This has been recognized by the
National Shellfisheries Association and the American Mala-

194

AMERICAN MALACOLOGICAL BULLETIN 23 • 1/2 • 2007

Figure 2. Mel Carriker at the March 2006 MidAtlantic Malacolo-
gists meeting at the Delaware Museum of Natural History. Photo
by Robert Robertson.

cological Society and, joining those professional organiza-
tions in memorializing Mel’s dedication to his students, the
University of Delaware, College of Marine and Earth Studies
has now established the Melbourne R. Carriker Student Eel-
lowship Endowment.

Mel will be remembered for his many professional and
scientific accomplishments but those of us who were hon-
ored to be his friends and students will always treasure the
warmth of his friendship, encouragement, high standard of
professional conduct, and devotion to the advancement of
science. All of us who knew Mel Carriker are better for it.

ware, Peggy Conlon, and staff for assistance in assembling
Mel’s records and bibliography.

Note: A full listing of Mel’s graduate students (including
thesis and dissertation topics), a listing of those graduate
students who had Mel sit on their research committees, and
his complete bibliography can be found at the web site of the
American Malacological Society: http://www.malacological
•org/

LITERATURE CITED

Carriker, M. A., Jr. 2006. Experiences of an Ornithologist Along the
Highways and Byways of Bolivia: Collecting Birds in an Isolated,
Magnificent Land in the Nineteen Thirties. M. R. Carriker and
R. C. Dalgleish, eds. AuthorHouse, Bloomington, Indiana.
Carriker, M. R. 2000. Vista Nieve: The Remarkable True Adventures
of an Early Twentieth Century Naturalist and His Eamily in
Colombia, South America. Blue Mantle Press, Rio Hondo,
Texas.

Carriker, M. R. 2005. Taming of the oyster: A history of evolving
shellfisheries and the National Shellfisheries Association. S. E.
Shumway, ed. Sheridan Press, Hanover, Pennsylvania.
Carriker, M. R. and E. L. Yochelson. 1968. Recent gastropod bore-
holes and Ordovician cylindrical borings. United States Geo-
logical Survey Professional Paper 593B: B1-B23.

Castillo, W. and S. S. Holyoak. 2004. Interview with Melbourne R.
Carriker. Rutgers Oral History Archives, History Department,
Rutgers, The State University, New Brunswick, New Jersey
[available at http://oralhistory.rutgers.edu/Interviews/
carriker_melbourne.html] .

Kennedy, V. S., R. I. E. Newell, and A. F. Eble, eds. 1996. The
Eastern Oyster Crassostrea virginica. Maryland Sea Grant Col-
lege, College Park, Maryland.

Kraeuther, J. and M. Castagna, eds. 2001. Biology of the Hard Clam.
Elsevier, New York.

Olive, J. R. and D. R. Beem, eds. 1967. Bioscience Research During
Earth-Orbiting Missions: Manned Earth-Orbiting Space Station.
American Institute of Biological Sciences, Washington, D.C.
NASr-132.

Prezant, R. S. and C. L. Counts, III, eds. 1985. Perspectives in
Malacology. American Malacological Bidletin Special Edition
No. 1. iii + 116 pp.

Smith, R. I. 1964. Keys to Marine Invertebrates of the Woods Hole
Region. Systematics-Ecology Program, Marine Biological
Laboratory, Woods Hole, Massachusetts.

ACKNOWLEDGEMENTS

We thank Dr. Robert Robertson (ANSP, retired) for
photographs of Mel and Dr. Nancy Targett, Dean, Graduate
College of Marine and Earth Sciences, University of Dela-

AMERICAN MALACOLOGICAL SOCIETY, INC
FINANCIAL REPORT
General Accounts
2005 Income and Expenses

TOTAL ASSETS (January 1, 2005)

$170,765.82

INCOME

$34,426.78

Membership Dues

14,025.00

Membership Dues (2002)

20.00

Membership Dues (2003)

489.00

Membership Dues (2004)

1,622.00

Membership Dues (2005)

8,806.00

Membership Dues (2006)

2,031.00

Membership Dues (2007)

1,009.00

Membership Dues (2008)

48.00

Interest and Dividends from Endowment

3,632.21

Capital Gains Distribution

10.98

Life Membership Fund

275.74

Symposium & Student Fund

3,345.49

Publications Income

9,481.36

AMB Subscriptions

2,129.00

AMB Page Charges

4,055.00

AMB Back Issues

608.00

AMB Reprint Charges

1,641.00

AMB Postage & Misc. Income

1,048.36

Donations

1,516.00

Symposium Endowment Fund

255.00

Student Endowment Fund

1,161.00

Shell Museum Huiricane Fund

100.00

Income from Annual Meeting

5,772.21

EXPENSES

$36,856.79

Treasurer and Secretai7 Office Expenses

172.33

Affiliate Memberships

225.00

Banking & Credit Card Fees

681.77

Incorporation & Registration Fees

95.00

Insurance/Bond Fees

552.00

Website Expenses

886.76

Annual Meeting Deposit & Symposium 1

Expenses

7,400.00

Publication Expenses

12,225.83

AMB (20 (1/2))

10,901.68

Reprints

372.82

Managing Editor Travel

859.78

Postage

63.55

Overpayment refunds

28.00

Student Research Grants

3,000.00

Travel Expenses for Officers

3,618.10

Student Paper Awards

500.00

Hurricane Relief

7,500.00

NET LOSS in 2005

$2,430.01

TOTAL ASSETS (December 31, 2005)

$170,097.82

**lncliides capital gains and losses in endowment portfolios which fluctuate with
the market.

AMERICAN MALACOLOGICAL SOCIETY, INC
FINANCIAL REPORT
General Accounts
2006 Income and Expenses

TOTAL ASSETS (January 1 , 2006) 1 70,097.82

INCOME $58,370.04

Membership Dues

13,783.00

Membership Dues (2004)

80.00

Membership Dues (2005)

737.00

Membership Dues (2006)

8,608.00

Membership Dues (2007)

2,575.00

Membership Dues (2008)

1,687.00

Membership Dues (2009)

48.00

Membership Dues (2010)

48.00

Interest and Dividends from Endowment

4,087.54

Life Membership Fund

310.49

Symposium & Student Fund

3,777.05

Publications Income

9,682.89

AMB Subscriptions

5,176.27

AMB Page Charges

1,896.00

AMB Back Issues

96.00

AMB Reprint Charges

533.00

AMB and Book Royalties

1,511.62

AMB Postage & Misc. Income

470.00

Donations

3,769.00

Symposium Endowment Fund

20.00

Student Endowment Fund

300.00

Student Fund from Auction

3,449.00

Income from Annual Meeting

27,047.61

Field trip income

395.00

Registration and other income

26,652.61

EXPENSES

$40,499.50

Treasurer and Secretary Office Expenses

339.51

Affiliate Memberships

325.00

Banking & Credit Card Fees

840.19

Incorporation & Registration Fees

45.00

Insurance/Bond Fees

1,037.00

Website Expenses

110.35

Annual Meeting & Symposium Expenses

23,450.64

Publication Expenses

12,369.12

AMB (21(1/2))

8,817.63

Reprints

658.97

Sturm Book

1,365.08

Managing Editor Travel

1,474.93

Misc. postage etc

52.51

Student Research Grants

2,000.00

Travel Expenses for Officers

3,363.66

Student Paper Awards

500.00

Net Gain in 2006

$13,989.57

TOTAL ASSETS (December 3 1 , 2006)

$193,587.74

**Includes capital gains and losses in endowment portfolios which fluctuate with

the market.

INDEX TO VOLUME 23

Baur, B. 23: 157
Chase, R. 23: 183
Coan, E. V. 23: 1
Counts, C. L., Ill 23: 191
Davison, A. 23: 173
Fortunato, H. 23: 33
Gallardo, C. S. 23: 17
Herbert, G. S. 23: 17
De Visser, J. A. G. M. 23: 113
Jansen, R. F. 23: 113

Abyssochrysidae 23: 1 1
Abyssochrysos 23: 11
Acnnthina 23: 20, 72
Acanthotrpphon 23: 19
Acavidae 23: 176
Achntina 23: 176
Achatinidae 23: 176
adolphi, Deroceras 23: 140
adspersa, Tenellia 23: 100
Aeolidiella 23: 13
Aeolidiidae 23: 13
Aeolidioidea 23: 13
Agathylla 23: 176
agreste, Deroceras 23: 138
agrestis, Agriolimax 23: 139
Agriolimacidae 23: 137, 176
Agriolimax 23: 138
Albinaria 23: 176
Alderia 23: 99
Allogona 23: 175
Alopiinae 23: 177
alveata, Caribiella 23: 20
atnanoi, Satsuma 23: 177
Arnphidromus 23: 174
Amphissa 23: 35
Ampullariidae 23: 93
Ancilla 23: 72
ancilla, Fidmentum 23: 43
ancilla, Pseudoliva 23: 54

AUTHOR INDEX

Rabat, A. R. 23: 1
Koene, J. M. 23: 113
Krug, P. J. 23: 99
Leonard, J. L. 23: 79, 121
Merle, D. 23: 17
Montagne-Wajer, K. 23: 113
Mordan, P. 23: 173
Ohtaki, H. 23: 81
Pearse, J. S. 23: 121
Prezant, R. S. 23: 191

Reise, H. 23: 137
Robertson, R. 23: 1 1
Simone, L. R. L. 23: 43
Takeuchi, M. 23: 81
Ter Maat, A. 23: 1 1 3
Tomiyama, K. 23: 81
Ward, J. E. 23: 191
Westfall, J. A. 23: 121
Yusa, Y. 23: 89
Zonneveld, C. 23: 113

PRIMARY MOLLUSCAN TAXA INDEX

[first occurrence in each paper recorded, new taxa in bold]

ancilla, Sylvanocochlis 23: 54
Anguispira 23: 177
antillarum, Nassariiis 23: 34
antipodariim, Potamopyrgiis 23: 91
Aplysia 23: 79

arbiistoriim, Arianta 23: 80, 138, 157,
174

Archachatina 23: 176
Architectonicidae 23: 12
arenaria. My a 23: 96
argentea, Cotonopsis 23: 34
Arianta 23: 175
Ariolimax 23: 121, 176
Arion 23: 176

Arionidae 23: 121, 151, 176

Ariopha?ita 23: 176

Ariopbantidae 23: 176

Ashnmnella 23: 175

aspersa. Helix 23: 118, 160, 174, 183

aspersum. Cornu 23: 150

aspersus, Cantareus 23: 174, 183

atafoua, Benthobia 23: 43

ater, Viviparus 23: 91

Atlanta 23: 12

Atlantidae 23: 12

australis, Eburna 23: 45

australis, Zemira 23: 43

avellana, Crouia 23: 21

avenacea, Chroudritia 23: 129

Balea 23: 176
Baleinae 23: 176
Basommatophora 23: 113
Bemhiciuni 23: 1 1
Benthobia 23: 43
Bertta 23: 176

bistimulatuin, Deroceras 23: 141

Bittiolum 23: 12

Bittium 23: 12

Bivetiella 23: 72

Bizetiella 23: 19

Boonea 23: 12

bracbyphallus, Ariolimax 23: 122
Bradybaeua 23: 175
Bradybaenidae 23: 175
brevispina, Murex 23: 19
buccitieus, Xymenopsis 23: 19
Buccinidae 23: 35, 43
Buccinoidea 23: 33
Buccinorbis 23: 43
Buccinuni 23: 54
bureschi, Deroceras 23: 138
buttoni, Aphallarion 23: 122
buttoni, Ariolimax 23: 121
Caenogastropoda 23: 43
californica, Aplysia 23: 107
califonucus, Ariolimax 23: 121, 138
callidegeuita, Plamiuaea 23: 100
calliglypta, Anachis 23: 35

197

calliglypta, Cosmioconcha 23; 34
Calyptraeoidea 23: 43
Camaenidae 23: 175
Campaniloidea 23: 12
canaliculata, Pomacea 23; 89
Cancellariidae 23: 43
Cantareus 23: 175
Caracohis 23: 175
caricOy Busycon 23: 91
Carinariidae 23: 12
cassidifonnis, Xanthochorus 23: 21
castauea, Angustassiminea IX. 90
Catinella 23: 177
caiicasicum, Deroceras 23: 138
caudate, Eiipleura 23; 192
cauze, Elysia 23: 100
Cavoliniidae 23: 12
Cavolinioidea 23: 12
Cencliritis 23: 1 1
Cepaea 23; 175
Cepolis 23: 175
Cerastidae 23: 176
Ceriidae 23: 176
Cerion 23: 176
Cerithiidae 23: 12
Cerithioidea 23: 12, 43
Cerithiopsidae 23: 1 1
Cerithiopsis 23: 1 1
Cerithiopsoidea 23: 11
Cerithiwn 23; 12
Chicocenebra 23: 20
Chicopinnatus 23: 30
Chicoreiis 23: 30
chlorotica, Elysia 23; 100
Chondrinidae 23: 176
Chorus 23; 20
christae, Deroceras 23; 140
Chytra 23: 12

cincinnatiensis, Pomatiopsis 23: 92
cinerea, Urosalpinx 23: 17, 192
cingulata, Cerithideopsilla 23; 81
Cinnalepeta 23: 12
Clathrus 23: 1 1
Clausilia 23: 176
Clausiliidae 23: 176
Clausiliinae 23: 177
Clionoidea 23: 12
Clithou 23: 12
Cochlicella 23: 175
Cochlodina 23: 176

Columbellidae 23: 33
colwnbianus, Ariolimax 23: 121
complexirhyna, Benthobia 23: 43
concentricus, Latirus 23: 34
concholepas, Concholepas 23: 17
Conoidea 23: 43
convexa, Crepidula 23: 92
coraliuru, Clypeomorus 23; 81
Corbicida 23: 91
coruuarietis, Marisa 23: 90
Cosmioconcha 23: 33
Cotonopsis 23: 33
crassa, Pseudoliva 23: 76
crassiparva, Cotonopsis 23: 34
Crassispirinae 23: 2
Crassostrea 23: 89
crassidnata, Cronia 23: 18
Crepidula 23: 92
Crucibranchaea 23: 12
Cryptomastix 23; 175
cumingi, Batillaria 23: 81
Cypraea 23: 1 1
Cypraeidae 23: 1 1
Cypraeoidea 23: 11, 43
Delinia 23: 176
Dendropoma 23: 12
Deroceras 23: 137, 176
deroyae, Cotonopsis 23: 34
Diacria 23: 12
Dtala 23: 12
Dialidae 23: 12
Discidae 23: 177

djadjariensis, Cerithideopsilla 23: 81

dolichophallus, Ariolimax 23: 121, 177

dorri, Canidia 23: 68

dorri, Nassodonta 23; 43

Echininus 23: 1 1

edentula, Cotonopsis 23: 34

edulis, Mytilus 23: 192, 195

e/rtfrt, Drupella 23; 18

Elimia 23: 12, 92

Elisolimax 23: 176

Enidae 23: 177

Eobania 23: 175

Epitoniidae 23: 11

Epitonioidea 23: 1 1

Epitonium 23: 11

Ergalataxinae 23: 20

Erronea 23: 11

esmeraldensis, Cotonopsis 23: 34

Euglandina 23: 177
Euhadra 23: 174
Eulimidae 23: 12
Euphaedusa 23: 176
faba, Clithon 23: 81
Fargoa 23: 12
fasciatus, Liguus 23: 174
Fasciolariidae 23: 35, 72
fatrense, Deroceras 23: 138
Faunus 23: 12
Favartia 23: 18
festiva, Reticunassa 23; 81
Finella 23: 12

floridensis, Orthalicus 23; 174
fornicata, Crepidula 23: 92
fulica, Lissachatina 23: 174
Fulmentum 23: 54
Furcopenis 23: 139
Fusitriton 23: 1 1
galea, Tonna 23; 76
galloprovincialis, Mytilus 23: 95
Gastrodontidae 23: 176
Gastropoda 23: 81
Geomalacus 23: 176
geversianus, Trophon 23: 17
gigas, Crassostrea 23: 91
giustianum, Deroceras 23: 140
glandulosum, Deroceras 23: 140
gorgonium, Deroceras 23: 138, 140
grandis, Typhisala 23: 19
gubbi, Chicoreus 23: 20
Gymnarion 23: 176
Halolimnohelix 23: 175
Haplotrefua 23: 177
Haplotrematidae 23: 177
harpa, Neothais 23: 18
haustorium, Haustrum 23: 19
Haustrinae 23: 18
Hedylopsoidea 23: 13
Helicidae 23; 157, 174
helicoidale, Deroceras 23: 140
Helicoidea 23: 174
Helicophanta 23: 176
He/ix 23: 79, 175
Helminthoglypta 23: 175
Elelminthoglyptidae 23: 175
Hemiplecta 23: 176
23: 176

heynemanni, Trichotoxon 23: 174
hirundo, Gotonopsis 23: 34

198

Hiunboldtiana 23: 175
Hygromiidae 23: 175
Hypsogastropoda 23: 73
Hyriidae 23: 95
lanthina 23: 1 1
Iherns 23: 175
ikaria, Deroceras 23: 140
indentata, Aspella 23: 19
insignis, Nassa 23: 68
lolaea 23: 12

jaliscana, Cotonopsis 23: 34
Janthina 23: 11
Janthinidae 23: 1 1
Janthinoidea 23: 1 1
japotiica, Assimiiiea 23: 90
Laciniaria 23: 176
laeve, Deroceras 23: 138
laevigata, Strombina 23: 35
laevis, Agriolimax 23: 139
laqiieatus, Chicopinnatus 23: 19
lateralis, Midinia 23: 96
Lavigeria 23: 12
Leucozonia 23: 72
Liguiis 23: 177
Limaddae 23: 176
Limacina 23: 12
Limacinidae 23: 12
Limacoidea 23: 174
Limacus 23: 176
Limapontiidae 23: 101
Umax 23: 153, 176
Limnotrochus 23: 12
lindae, Cotonopsis 23: 34
lineata, Pyrula 23: 61
lineatwn, Melapium 23: 43
Liolytopelte 23: 137
Litiopidae 23: 13
Littoraria 23: 1 1
Littorina 23: 1 1
Littorinidae 23: 1 1
Littorinoidea 23: 1 1
lombricoides, Deroceras 23: 138
Lophiotominae 23: 2
Loxonematoidea 23: 1 1
Luchuphaediisa 23: 176
hiteostoina, Nassarins 23: 34
Lymnaeidae 23: 113
Macrochlamys 23: 176
Macrogastra 23: 176
Macron 23: 72

maculosa, Tonna 23: 76
Mandarina 23: 175
margaritifera, Margaritifera 23: 92
Margartiferidae 23: 95
marmoratus, Iberus 23: 174
Mastus 23: 177
maximus, Umax 23: 79, 173
mazatlantica, Pinctada 23: 91
Medora 23: 176
Melanopsidae 23: 12
Melanopsinae 23: 12
Melapiidae 23: 68
Melapium 23: 43
Melarliaplie 23: 1 1
mendozana, Cotonopsis 23: 34
mercenaria, Mercenaria 23: 192
Mesodon 23: 175
Mesomphix 23: 176
Microparmarion 23: 176
Milacidae 23: 176
minoicmn, Deroceras 23: 140
modesta, Alderia 23: 99
modesta, Cosmioconcha 23: 34
modestinn, Buccimim 23: 35
Monacha 23: 175
Monadenia 23: 175
monfilsi, Cotonopsis 23: 34
Mndalia 23: 12
multiformis, Batillaria 23: 81
Murexiella 23: 18
Miirexsul 23: 18
Muriddae 23: 17, 43
Muridnae 23: 17
Muricoidea 23: 43
Muricopsinae 23: 17
Muricopsis 23: 18
Mysorelloides 23: 12
Mytildae 23: 95
Mytilns 23: 89
Nassariidae 23: 35, 43
Nassodonta 23: 68
Naticoidea 23: 75
nemoralis, Cepaea 23: 159, 174
Neogastropoda 23: 33, 74
Neoheltx 23: 175
Neostyriaca 23: 176
Neripteron 23: 12
Nerita 23: 12
Neritidae 23: 12
Neritilia 23: 12

Neritiliidae 23: 12

Neritina 23: 12

Neritoidea 23: 12

tiitens, Cosmioconcha 23: 34

nitens, Fiisiis 23: 35

Nitidiscala 23: 11

iiitidiim, Deroceras 23: 138

Nodilittorina 23: 1 1

Af»a’/d 23: 19

ocellifera, Costasiella 23: 100

Ocenebra 23: 19

Ocenebrinae 23: 17

Ocinebrina 23: 19

octagonus, Mitrcxsul 23: 19

oculifera, Aplysia 23: 107

Odostomella 23: 12

oertzeni, Deroceras 23: 140

Olividae 23: 43

Opaha 23: 1 1

Opisthobranchia 23: 99

Oreoheliddae 23: 177

Oreohelix 23: 174

Orthaliddae 23: 174

ostrina, Niicella 23: 21

oualaniensis, Clithon 23: 81

Oxychilus 23: 176

Oxyloma 23: 177

palmeri, Cosmioconcha 23: 34

Paludomidae 23: 12

Palnstorina 23: 12

panacostaricensis, Cotonopsis 23: 34

panormitaniim, Deroceras 23: 137

Paramelania 23: 12

Parhedylidae 23: 13

Parthettina 23: 12

Partula 23: 177

Partulidae 23: 177

parvula, Cosmioconcha 23: 34

Paryphanta 23: 177

pergracilis, Cosmioconcha 23: 34

perpicta, Cohimhella 23: 35

perpicta, Mitrella 23: 35

perriigata, Urosalpinx 23: 20

Phenacolepadidae 23: 12

Philomyddae 23: 174

Philomycns 23: 177

pluiketensis, Cotonopsis 23: 34

Pisiilina 23: 12

planarioides, Deroceras 23: 138
Plesiotrochidae 23: 12

199

Plesiotrochus 23: 12
Pleuroceridae 23: 12
Pleuwdonte 23: 175
Pneumodermatidae 23: 12
Polydontes 23: 175
Polygyra 23: 175
Polygyridae 23: 175
Polyniita 23: 175
pomatia, Helix 23: 159, 184
potman, Chicoreus 23: 17
ponmrn, Phyllonotiis 23: 17
Pontohedyle 23: 13
Potamididae 23: 12, 81
praecox, Deroceras 23: 138, 140
Pseudoliva 25: 43
Pseudolividae 23: 43
Pseudolivinae 23: 43
Pseudomelatominae 23: 2
Ptenoglossa 23: 12
Pterosoma 23: 12
Pterotracheoidea 23: 12
Pulmonata 23: 137
papa, Mastus 23: 177
Puperita 23: 12
pusilla, Vertigo 23: 133
Pygmaepterys 23: 18
Pyramidella 23: 12
Pyramidellidae 23: 12
Pyramidelloidea 23: 12
quadrata, Sinotaia 23: 91
radwini, Cotonopsis 23: 34
Ranellidae 23: 1 1
Rapaninae 23: 17
rehderi, Cosmioconcha 23: 34
rethimnotiensis, Deroceras 23: 138
reticidatum, Deroceras 23: 138
Reymondia 23: 12
rhizophorarum, Cerithidea 23: 81
Rhytididae 23: 177
ringens, Cantharns 23: 34
rodnae, Deroceras 23: 138
Ruthenica 23: 176
Sacoglossa 23: 99
salebrosa, Vitidaria 23: 17
sandai, Eiiliadra 23: 1 77
Satsuma 23: 175
Scala 23: 1 1
Scaliolidae 23: 12
Seda 23: 1 1

semicarinata, Goniobasis 23: 92
Semilimax 23: 176
Semisukospira 23: 12
senegaletisis, Siratus 23: 43
sepimentam, Biiccinum 23: 54
Sept aria 23: 12
Serpulorbis 23: 12
Sheldonia 23: 176
Siratus 23: 72

skoglundae, Cotonopsis 23: 34
SolatopHpa 23: 176
Sonorella 23: 175
sorenseni, Acanthotrophon 23: 20
Spekia 23: 12
Spiraxidae 23: 177
stagnalis, Lynniaea 23: 79, 113, 153,
191

Stanleya 23: 12
Stenotrema 23: 176
Stereophaedusa 23: 176
Stonnsia 23: 12
straniineus, Ariolimax 23: 132
Strepturidae 23:

Strotnbina 23: 33

Stromboidea 23: 44

Strophocheilidae 23: 177

Strophocheihis 23: 177

stiiranyi, Deroceras 23: 138

Stylommatophora 23: 11, 121, 183

subagreste, Deroceras 23: 141

Succinea 23: 173

Succineidae 23: 177

suteri, Cotonopsis 23: 34

Sylvanocochlis 23: 43

Tacheocampylaea 23: 175

Tandonia 23: 176

Tanganyicia 23: 12

Tectarius 23: 1 1

Terebralia 23: 12

Thais 23: 19

Theba 23: 175

Theliostyla 23: 12

Thiara 23: 12

Thiaridae 23: 12

timida, Elysia 23: 100

Tiphobia 23: 12

Tonnoidea 23: 11, 43

torrefactus, Chicoreus 23: 22

Trichotoxoti 23: 176

Trilobopsis 23: 176
Triodopsis 23: 176
Triphora 23: 11
Triphoridae 23: 11
Triphoroidea 23: 11
Triplex 23: 30
Tripterotyphinae 23: 18
tritoniformis, Agnewia 23: 21
Trochomorphidae 23: 176
Trophon 23: 30
Trophoninae 23: 17
trossulus, Mytilus 23: 95
truncatiis, Biilinus 23: 79, 133
turcicum, Deroceras 23: 138
Turrina 23: 33
turrita, Cotonopsis 23: 34
Turritella 23: 12
Turritellidae 23: 12
Typhinae 23: 18
Unela 23: 13
Unionidae 23: 95
Urocyclidae 23: 176
Vallonia 23: 177
Valloniidae 23: 177
Veneridae 23: 95
Ventridens 23: 176
Vermetidae 23: 12
Vermetoidea 23: 12
Vermetus 23: 12
verrucicornis, Berghia 23: 100
Vertiginidae 23: 177
Vertigo 23: 177
Vespericola 23: 176
Viruindu 23: 12

virginica, Crassostrea 23: 94, 192

Viriola 23: 11

Vitrinidae 23: 176

Vitrinobrachiwn 23: 176

Vittina 23: 12

Vitularia 23: 19

Viviparus 23: 92

willowi, Alderia 23: 99

Xanthochorus 23: 20

Zachrysia 23: 175

zebrina, Pseudoliva 23: 76

Zebrinops 23: 176

Zemira 23: 43

Zonitidae 23: 176

Zonitoides 23: 130

200

THE AMERICAN MALACOLOGICAL SOCIETY

Dr. Dawn E. Dittman, Treasurer

Tunison Laboratory of Aquatic Science
3075 Oracle Rd., Cortland, NY 13045-9357, U.S.A.

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THE AMERICAN MALACOLOGICAL SOCIETY

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Donovan, D. A., J. P- Danko, and T. H. Carefoot. 1999.
Functional significance of shell sculpture in gastropod
molluscs: Test of a predator-deterrent hypothesis in
Ceratostoma foliatum (Gmelin). Journal of Experimental
Marine Biology and Ecology 236: 235-251.

Seed, R. 1980. Shell growth and form in the Bivalvia. In:
D. C. Rhoads and R. A. Lutz, eds.. Skeletal Growth of
Aquatic Organisms. Plenum Press, New York. Pp. 23-67.
Yonge, C. M. and T. E. Thompson. 1976. Living Marine Mol-
luscs. William Collins Son and Co., Ltd., London.

Dali, W. H. 1889. Reports on the results of dredging, under
the supervision of Alexander Agassiz, in the Gulf of
Mexico (1877-78) and in the Caribbean Sea (1879-80),
by the United States Coast Survey Steamer “Blake,” Lieu-
tenant-Commander C. D. Sigsbee, U. S. N., and Com-
mander J. R. Bartlett, U. S. N., commanding. Report on
the Mollusca, Pt. 2: Gastropoda and Scaphopoda. Bulle-
tin of the Museum of Comparative Zoology 18: 1-492, pis.
10-40.

Orbigny, A, d’. 1835-46. Voyage dans I’AmOnque
Meridionale (le Bresil, la Republique Orientale de
rUruguay, la Republique Argentine, la Patagonie, la
Republique du Chili, la Republique de Bolivia, la
Republique du Peroii), execute pendant les annees 1826,
1827, 1828, 1829, 1830, 1831, 1832 et 1833. Vol. 5, Part 3
[Mollusques) . Bertrand, Paris. Dates of publication: pp.
1-48, [1835], pp. 49-184 [1836], pp. 185-376 [1837], pp.
377-408 [1840], pp. 409-488 [1841], pp. 489-758 -H pis.
1-85 [1846].

Hurd, J. C. 1974. Systematics and Zoogeography of the Union-
acean Mollusks of the Coosa River Drainage of Alabama,
Georgia and Tennessee. Ph.D. Dissertation, Auburn Uni-
versity, Alabama.

U.S. Environmental Protection Agency. 1990. Forest riparian
habitat survey. Available at: http://www.epa.gov/waterat-
las/geo/iil6_usmap.html 25 January 2003.

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204

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1

I

Poecilogony and larval ecology in the gastropod genus Alderia. PATRICK J. KRUG 99

Food intake, growth, and reproduction as affected by day length and food availability in the
pond snail Lymnaea stagnalis.

ANDRIES TER MAAT, COR ZONNEVELD, J. ARJAN G.M. DE VISSER, RENE E. JANSEN,

KORA MONTAGNE-WAJER, and JORIS M. KOENE 113

Phally polymorphism and reproductive biology in Ariolimax (Ariolimax) buttoni (Pilsbry and
Vanatta, 1896) (Stylommatophora: Arionidae).

JANET L. LEONARD, JANE A. WESTEALL, and JOHN S. PEARSE 121

A review of mating behavior in slugs of the genus Derocems (Pulmonata: Agriolimacidae).

HEIKEREISE 137

Reproductive biology and mating conflict in the simultaneously hermaphroditic land snail

Arianta arbustorum . BRUNO BAUR 157

A literature database on the mating behavior of stylommatophoran land snails and slugs.

ANGUS DAVISON and PETER MORDAN 173

The function of dart shooting in helicid snails. RONALD CHASE 183

Meeting Announcement 190

Melbourne Romaine Carriker: 25 February 1915 - 25 February 2007 An Appreciation.

CLEMENT L. COUNTS, III, ROBERT S. PREZANT, and J. EVAN WARD 191

Financial Report 195

Index to Vol. 23 197

Membership Form 201

Information for Contributors 203

SMITHSONIAN INSTITUTION LIBRARIES

6^,

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mocL

AMERICAN

MALACOLOGICAL

BULLETIN

Journal of the American Malacological Society

http:// WWW. malacological.org

Introduction to the symposium “Cephalopods: A behavioral perspective.”

JENNIFER A. MATHER

Chambered nautilus (Nautilus pompilius pouipiJius) responds to underwater vibrations.

CHRISTIAN P. SOUCIER and JENNIFER A. BASIL

Octopus sucker-arm coordination in grasping and manipulation. FRANK W. GRASSO

Short-term pain for long-term gain: A hypothetical role tor the mantle in coleoid

cephalopod circulation. ALISON J. KING and SHELLEY A. ADAMO

A new approach to octopuses’ body pattern analysis: A framework for taxonomy and

behavioral studies. TATIANA. S. LEITE and JENNIFER A. MATHER

Observations of deep-sea octopodid behavior from undersea vehicles. JANET R. VOIGHT

To boldly go where no mollusc has gone betore: Personality, play, thinking, and

consciousness in cephalopods. JENNIFER A. MATHER

Freshwater snails (Mollusca: Gastropoda) from the Commonwealth of Dominica with a
discussion of their roles in the transmission ot parasites.

WILL K. REEVES, ROBERT T. DILLON, JR., and GREGORY A. DASCH

continued on hack cover

. 1

. 3

13

25

31

43

51

59

Cover photo: Graneledone loubin, 1918 from Baby Bare, Cascadia Basin, North Pacific Ocean trom Voight

AMERICAN MALACOLOGICAL BULLETIN

Kenneth M. Brown, Editor-in-Chief
Department of Biological Sciences
Louisiana State University
Baton Rouge, Louisiana 70803
USA

Janice Voltzow
Department of Biology
University of Scranton
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USA

Robert H, Cowie

Center for Conservation Research and Training

University of Hawaii

3050 Maile Way, Gilmore 408

Honolulu, Hawaii 96822-2231

USA

Carole S. Hickman

University of California Berkeley

Department of Integrative Biology

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Timothy A. Pearce

Carnegie Museum of Natural History
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BOARD OF EDITORS

Cynthia D. Trowbridge, Managing Editor

Department of Zoology

Oregon State University

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Paula M. Mikkelsen
Paleontological Research Institution
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Alan J. Kohn
Department of Zoology
Box 351800

University of Washington
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Dianna Padilla

Department of Ecology and Evolution
Stony Brook University
Stony Brook, New York 11749-5245
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Roland C. Anderson
The Seattle Aquarium
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Seattle, Washington 98101
USA

The American Malacological Bulletin is the scientific journal of the American Malacological Society, an international society of professional,
student, and amateur malacologists. Complete information about the Society and its publications can be found on the Society’s website:
http://www.malacologkal.org

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AMERICAN MALACOLOGICAL BULLETIN 24(1/2)
AMER. MALAC. BULL.

ISSN 0740-2783

Copyright © 2008 by the American Malacological Society

AMERICAN MALACOLOGICAL BULLETIN

CONTENTS^ Volume 24 | number 1/2

A APR ^ ’ -J —

\ MBRAR'SSx^

Introduction to the symposium “Cephalopods: A behavioral perspective.”

JENNIFER A. MATHER 1

Chambered nautilus {Nautilus pompilius ponipilius) responds to underwater vibrations.

CHRISTIAN P. SOUCIER and JENNIFER A. BASIL 3

Octopus sucker-arm coordination in grasping and manipulation. FRANK W. GRASSO 13

Short-term pain for long-term gain: A hypothetical role for the mantle in coleoid

cephalopod circulation. ALISON J. KING and SHELLEY A. ADAMO 25

A new approach to octopuses’ body pattern analysis: A framework for taxonomy and

behavioral studies. TATIANA. S. ELITE and JENNIFER A. MATHER 31

Observations of deep-sea octopodid behavior from undersea vehicles. JANET R. VOIGHT 43

To boldly go where no mollusc has gone before: Personality, play, thinking, and

consciousness in cephalopods. JENNIFER A. MATHER 51

Freshwater snails (Mollusca: Gastropoda) from the Commonwealth of Dominica with a
discussion of their roles in the transmission of parasites.

WILL K. REEVES, ROBERT T. DILLON, JR., and GREGORY A. DASCH 59

Patterns of activity cycles in juvenile California two-spot octopuses (Octopus bimaculoides).

DAVID L. SINN 65

Discovery of the South African polyplacophoran Stenosemus siwplicissiiuus (Thiele, 1906)

(Mollusca, Polyplacophora, Ischnochitonidae) in the Southern Ocean.

ENRICO SCHWABE 71

Imposex level and penis malformation in Hexaplex trunculus from the Tunisian coast.

YOUSSEF LAHBIB, SAMI ABIDLI, and NAJOUA TRIGUI EL MENIF 79

Threatened Bliss Rapids snail’s susceptibility to desiccation: Potential impact from hydroelectric

facilities. DAVID C. RICHARDS and TRISTAN D. ARRINGTON 91

Field observations of the nocturnal mantle-flap lure of Latiipsilis teres. ANDREW LEE RYPEL 97

Meta-analysis of the relationship between salinity and molluscs in tidal river estuaries ot
southwest Florida, U.S.A. PAUL A. MONTAGNA, ERNEST D. ESTEVEZ,

TERRY A. PALMER, and MICHAEL S. FLANNERY 101

Research Note: Giant African snail, Achatiua fulica, as a snail predator.

WALLACE M. MEYER HI, KENNETH A. HAYES, and AMANDA L. MEYER 117

Research Note: Life history and host fish identification for Fusconaia burkei and Pleurobetna
strodeanum (Bivalvia: Unionidae). MEGAN P. WHITE, HOLLY N. BLALOCK-HEROD,
and PAUL M. STEWART 121

Index to Vol. 24 127

Membership Form 130

Information for Contributors 132

Meeting Announcement 134

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Amer. Malac. Bull. 24: 1

Introduction to the symposium “Cephalopods: A behavioral perspective”

Jennifer A. Mather

Department of Psychology, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta TIK 3M4, Canada, mather@uleth.ca

Behavior is not an area we usually associate with mol-
luscs, and one tends to think instead of vertebrates, espe-
cially mammals. Yet when we do think of molluscan behav-
ior, it is the cephalopods that come to mind. With their large
centralized brain, reputed high intelligence, efficient physi-
ology, and complex motor output, cephalopods have an ex-
cellent basis for complex behavior. Despite this capacity,
cephalopod behavior is little known and not well explored,
and the authors in this symposium, especially the paper
collection, attempt to shine light into various corners with a
wide variety of cephalopod subjects.

One of the simplest aspects of behavior is sensory re-
ception, and one of the ‘simplest’ systems and most mollus-
can-general is that found in Nautilus Linnaeus, 1758. Soucier
and Basil discuss a pioneering laboratory investigation of
tactile sensitivity in the nautiloids; clearly these deep-sea ani-
mals should rely on non-visual information much of the
time, but only their chemical sensing has been well investi-
gated. Now that their mechanical reception has been estab-
lished, further research will no doubt look more at its use in
natural situations and the limits of and receptors for its sensitivity.

The programming of motor output, the root ot behav-
ior, is similarly simple on the surface. Grasso has tackled the
motor output of suckers of Octopus Cuvier, 1797 and
their combinations to produce actions on the environment.
While movement ought to be simple, the use and coordina-
tion of hundreds of suckers turns out to be, as befits the
complexity of neural support of the suckers, both complex
and variable. How much of this programming is central and
how much peripheral as well as how the ‘reflex’ arm control
system can perform such complex maneuvers remains to be
investigated; again the foundation has been laid for further
investigation.

Behavior is linked to the underlying physiology of the
animal, and the thoughtful paper by King and Adamo makes
sense of the paradoxes in the combination of Sepia Linnaeus,
1758 cuttlefish linkage of mantle contraction and blood cir-
culation. The motor action of mantle contraction has a ma-
jor effect on the circulation of blood through this area, and
the authors evaluate why the particular patterns of blood

* From the symposium “Cephalopods: A behavioral perspective”
presented at the joint meeting of the American Malacological So-
ciety and Western Society of Malacologists, held 29 luly to 3 August
2006 in Seattle, Washington.

flow during this major event occur. More of such behavior-
physiological linkage is needed, and the King and Adamo
paper is a welcome start.

One unique, coleoid cephalopod motor system is re-
sponsible for the chromatophore system that produces skin
patterns and colors, but its complexity means that it is often
characterized only informally. Leite and Mather use a com-
puterized data analysis approach to build tbe repertoire of
one Octopus species. Such characterization offers insight into
the neural production of patterns and pattern complexity on
the skin; in addition, this approach may assist us in taxo-
nomic investigation of the species complex of Octopus vul-
garis Cuvier, 1797.

Behavior gives us insights into physiology and ecology
of animals, and behavior of deep-sea octopods in underwa-
ter videos is the subject of the paper by Voigt. Because hu-
mans are veiy limited in their activities in the deep sea,
cephalopod research has focused on the easily available near-
shore and near-surface species of Octopus, Sepia, and Loligo
Lamarck, 1798. Thus, Voight’s insight into how these deep-
sea and little-known animals behave is particularly welcome.

The most complex areas of behavior are the emergent
aspects such as play, personality, and cognition, studied
mainly in Octopus so far. Mather covers the research in these
areas and suggests that we have much to learn about the
intelligence, cognitive capacity, and even possible conscious-
ness in cephalopods. She challenges us to look at behavior of
molluscs, particularly in but not limited to cephalopods, for
greater underlying subtly and complexity than we have as-
sumed so far.

In addition to these published papers, other symposium
participants presented work on a range of interesting aspects
of cephalopod behavior. Huffard discussed octopus mating
strategies for Abdopus Norman and Finn, 2001; Cosgrove
discussed the brooding behavior of Enteroctopus Rochebrune
and Mabille, 1889. Again with Enteroctopus dofleiui (Wiilker,
1910) as a model, Lyons and Scheel discussed the ecological
impact and movement of octopuses in their natural envi-
ronment. Finally, Williams looked at the chemical defenses
of hatchling Hapalochlaena Robson, 1929, and Bush dis-
cussed why deep-sea squid might ink into the dark.

1 would like to thank Roland Anderson of the Seattle
Aquarium, for requesting the symposium and assisting in its
assembly, and tbe helpful reviewers and patient authors who
worked through all the revisions.

1

/f> *

Amer. Maine. Bull. 24: 3-11

Chambered nautilus {Nautilus pompilius pompilius) responds to
underwater vibrations'^

Christian P. Soucier' and Jennifer A. Basil

Evolution, Ecology, and Behavior Program, City University of New York-Graduate Center, Department of Biology, CUNY Brooklyn
College, 2900 Bedford Avenue, Brooklyn, New York 11210, U.S.A., CSoucier@brooklyn.cuny.edu and IBasil@brooklyn.cuny.edu

Abstract: The deep-water cephalopod Nautilus pompilius pompilius Linnaeus, 1738 may benefit from detecting potential signals such as
mechanical and acoustical stimuli in its dark habitat where visual information is often limited. Here we examined whether specimens of
chambered nautilus are capable of responding to waterborne vibration — a sensory mechanism that has yet to be investigated. We measured
the ventilation rate of animals responding to a vibrating bead that produced a range of displacements and velocities. We found that
nautiluses do indeed respond to underwater acoustical stimuli, decreasing their ventilation in the presence of a vibratory stimulus.
Vibrations resulting from large-bead displacements and high source-velocities caused the animals to decrease their ventilation the most.
Stimuli <20 cm from the animals caused a further reduction in their ventilation rates than those at greater distances. 'I'hese nocturnal
animals, living in dark conditions where visual information is often limited, may benefit from including vibrations in the suite of stimuli
to which they can respond.

Key words: cephalopods, acoustics, behavior, ventilation, source-displacement

Organisms must cope with a variety of stimuli in the
marine environment, and the ability to process this infor-
mation may contribute to both survival and reproduction.

' Because the marine environment is dominated by mechani-
i cal and acoustical energies, such as water currents or vibra-
tions that may eventually be converted to sound waves, it is
a reasonable assumption that many organisms, including
Nautilus pompilius pompilius Linnaeus, 1758, may benefit
from the ability to detect and respond to these varying types
I of stimuli.

In the last three decades, researchers have identified the
variety of sensory systems that contribute to the survival and
functional ecology of the chambered nautilus {e.g., Rudel-
mann and Tu 1997). Nautilus pompilius pompilius has served
as a model in studies of olfaction, vision, and equilibrium
reception. Nautiluses, although predominantly chemotactic,
are capable of using many sensory systems to complete basic
survival tasks {vision: Muntz 1991, 1994a, 1994b, eiiiiilibrium
j reception: Budelmann 1977, Neumeister and Budelmann
j 1997, olfaction: Basil et al. 2000, 2002, 2005). Here we dem-
j onstrate that Nautilus pompilius is also capable of detecting
and responding to underwater vibrational stimuli.

!| Nautilus pompilius is considered to be one of the oldest

' Present Address: 333 East 102nd Street, Suite 726, New York, New
York 10029, U.S.A.

members of the class Cephalopoda (phylum Mollusca).
Presently, the genus represents less than 1% of the entire
cephalopod assemblage (Wood and O’Dor 2000). Nautiluses
are the only extant hard-shelled cephalopod, and are there-
fore commonly used as a modern analog of the ellesmero-
ceratids, an ancestral lineage that dates back ca. 500 Ma
(Ward 1987, Wray ct al. 1995, Ward and Saunders 1997).
Nautiluses are bottom dwellers but are not completely re-
stricted to the sediment (nektobenthic). They make daily
vertical migrations at dawn and dusk along coral reef slopes
throughout the Indo-Pacific, including the Philippines, Pa-
lau, Fiji, Papua New Guinea, Australia, Samoa, and Tonga
(Ward 1987, O’Dor et al. 1993). Nautiluses have limited
visual abilities and detect light wavelengths only shorter than
650 nm, with the most efficient absorption occurring at 467
nm (Muntz 1986). They also inhabit a primarily aphotic
environment and are commonly found at depths of 150-300
m. Because the internal environment of their shell is resis-
tant to pressure change, nautiluses dwell in depths up to 803
m before shell implosion occurs (Saunders and Landman
1987, Iordan et al. 1988).

Nautiluses are slow moving and non-visual, and in gen-
eral their life history strategies differ greatly from their
highly visual relatives, octopuses, squids, and cuttlefish (sub-
class Coleoidea), which typically live at shallower depths
although not exclusively. Aside from life-history strategies.

From the symposium “Cephalopods: A behavioral perspective” presented at the joint meeting of the American Malacological Society and
Western Society of Malacologists, held 29 luly to 3 August 2006 in Seattle, Washington.

3

4

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

nautiloids and coleoids differ in external morphology as
well. Coleoids typically possess 8-10 appendages (arms and/
or tentacles), all of which are lined with mechanoreceptors
and chemoreceptors with the latter occurring particularly
within the suckers (Hanlon and Messenger 1996, Cheng and
Caldwell 2000, Messenger 2001). Nautiloids have 90-94 ten-
tacles that are typically covered with mechanoreceptor and
chemosensory cells (Hamada et al. 1978, Fukada 1987, Ruth
et al. 2002). Nautiloids also have a gas-filled external shell
that is sectioned into chambers. Coleoids possess highly de-
veloped eyes with lenses that form distinct images. The eyes
of Nautilus lack a lens but are capable of forming images and
capturing light in dark environments, including biolumines-
cence (Muntz 1994a, 1994b). Given the vast ecological and
morphological differences between coleoids and nautiloids,
it is a reasonable prediction that each group would use sen-
sory systems, such as vibration detection, differently.

Sources of sound in the ocean include seismic activity,
storm events, man-made contributions, and biological ac-
tivity. For an animal to identify sound as a stimulus, it must
extract a signal from the ambient sound environment or,
more informally, from background noise (Rogers and Cox
1988). Sound emission can originate from many different
sources, but all sound production begins in a similar fashion:
a longitudinal, propagating mechanical wave is generated by
a change in volume, physical oscillation, or movement. Dis-
turbances from a change in volume that originate from a
single pole, such as a pulsating sphere or the inflation of a
teleost swim bladder, are referred to as monopole sources.
Dipole sources result from a disturbance in the medium in
which the volume of the source remains constant but the
signal has two points of origin. Typical examples of dipole
sources are spheres that vibrate between two points or the
sinusoidal movements of a fish moving through the water
column (Kalmijn 1988, Coombs 1994).

The acoustic fields created by these sources can be di-
vided into two components: near-field (or local-flow field)
and far-field. Stimuli associated with local-flow fields are
dominated by particle velocity, displacement, and accelera-
tion, whereas stimuli associated with the far-field can be
more accurately measured in scalar c]uantities such as pres-
sure and density that reflect only the magnitude of the signal.
Non-pelagic animals that live in ocean bottoms, coral reefs,
intertidal areas, etc., operate primarily in the local-flow field
simply because sound waves do not have adec]uate space to
radiate from the source. Pelagic animals frec]uently operate
within both fields and have sensory systems adapted for
detection within each field that are dependent on their spa-
tial location at any given time (Bleckmann 1994). An ex-
ample of the latter would be fishes that possess both lateral-
line systems and otoliths, which serve as overlapping sensory
systems. The lateral line detects low-frequency stimuli within

only a few body lengths of the source, whereas the otolith
organs and other components of the inner ear respond to
acoustic reception from the outer reaches of the local-flow
field well into the far-field (Kalmijn 1988, Braun et al. 2002).
A similar model could be applied to nautiluses. A plausible
mechanism might be that the immediate source (i.e., a group
of snapping shrimp) could be detected through mechanore-
ceptors located on certain tentacles (Ruth et al. 2002) while
the progression of the wave through the remainder of the
near-field into the far-field could be detected by equilibrium
receptor organs such as statocysts (Budelmann 1988, Rogers
and Cox 1988, Neumeister and Budelmann 1997).

Williamson (1988) tested vibration sensitivity in the
northern octopus Eledone cirwsa (Lamarck, 1798) and de-
termined that the hair-cell sensitivity within the statocyst of
the octopus was three or four orders of magnitude less sen-
sitive than what average fishes can detect. The statocyst of E.
cirrosa is therefore not considered to be an auditory organ
compared to the auditory or far-field detection systems of
fishes, although its threshold sensitivities were similar to
those of other aquatic invertebrates. More importantly, these
results demonstrated that this organ is sensitive to biologi-
cally relevant vibrations. Additional studies have suggested
that less sensitive vibration thresholds may enhance coleoid
survival by lessening the effect of intense acoustic emissions
that odontocete predators use to disorient their prey
(Moynihan 1985) and that vibration sensitivity need not be
confined to the statocyst, indicating that certain mechano-
receptors may be sensitive to vibration as well (Williamson
1988).

It is this line of logic that suggests that Nautilus may
detect underwater vibration. The statocysts of nautiluses are
more primitive than those of coleoids. Perhaps the extreme
external morphological differentiation between nautiluses
and coleoids has prevented the evolution of such a complex
organ due to space or phylogenetic constraints. Additionally,
and perhaps more acoustically relevant, there is the gas-filled
external shell of the chambered nautilus. Although this shell
and its chambers are thought primarily to compensate for
buoyancy, principles of underwater acoustics dictate that the
shell may also double as a resonating chamber, thereby po-
tentially nullifying the need for the development of a more
complex receptor organ.

MATERIALS AND METHODS

Animals

Eleven wild-caught, adult individuals of Nautilus pom-
pilius, originally collected in the Philippines and purchased
through Sea-Dwelling Creatures™, California, were housed
in a re-circulating system at the Aquatic Research and En-

A/A [/r/L!7S VIBRATION DETECTION

5

vironmental Assessment Center (AREAC) at Brooklyn Col-
lege of the City University of New York. The animals were
divided into two groups and kept separately in a closed
system that consisted of two 530-L polyethylene tanks filled
with artificial sea water (Instant Ocean™). Both tanks were
connected in tandem to a 94.8-L biofilter that contained
aeration and filtration media. The animals were kept at con-
stant temperature of 17 °C and at sa-
linities between 32 and 34 psu. Tilapia
fish heads (Oreocliroums niloticus edu-
ardianus) were used as a primary food
source, and rations were administered
every third day. Daily checks of water
quality (temperature, salinity, dis-
solved oxygen, pH, calcium, alkalinity,
ammonia, nitrite, nitrate, and phos-
phate) were conducted to monitor the
system and maintain the health of the
animals. Trace elements in the form of
a calcium/alkalinity liquid buffer sys-
tem (B-Ionic™) were added on a
weekly basis.

Small and large source-displacement
experiments

Experimental apparatus

In two source-displacement experi-
ments (Small Source-Displacement
Experiment [SSDE] and Large Source-
Displacement Experiment [LSDE]), the
experimental arena was a rectangular
Plexiglas™ tank (51 cm long X 25.4 cm
wide X 31.7 cm tall), containing -30
cm standing water (Fig. 1). To control
for ambient background noise, an in-
sulated and isolated basement room
was selected to run the trials. Within
the room, the tank was placed on a
vibration-absorption table constructed
from a granite slab ( 1 5 1 cm x 56 cm x
3 cm). The slab was placed on 12
tennis balls that were separately set in
plastic rings and spaced evenly across
a metal desk (73.5 cm x 77 cm x
115 cm).

Two digital cameras (Sony Digital
Handycam, model DCR-VXIOOO)
mounted on tripods recorded each
trial and provided both top and side
views. One camera was positioned 1 .5 m
in front of the long-axis of the tank

and the other was placed 1 m above the tank. Visual contact
between animals and observers and inadvertent cuing was
prevented by placing a removable blind along three sides of
the tank and maintaining a minimal distance of 3 m from the
uncovered portion of the tank. One fluorescent light bulb was
used overhead to illuminate the apparatus, and experimenters
did not move in front of the apparatus during the trials.

Robyn Crook, 2005

Figure 1. Experimental setup for source-displacement experiments. A, top-view camera; B,
oscilloscope; C, laptop computer; D, side-view camera; E, vibration absorption table; F,
experimental tank with animal; G, wall mount with mini-shaker and shaft/bead.

6

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

Vibrating stimulus

A dipole source was created by mounting a spherical
acrylic bead (18.95 mm in SSDE and 9.44 mm in LSDE) to
an aluminum shaft ( 17 cm in length and 2 mm in diameter)
that was bent at a 90° angle and attached to a mini-shaker
(Bruel and Kjaer vibration exciter, model 4810). The mini-
shaker was fixed to a wall-mounted frame and positioned
inside of the tank, such that the bead was located in the
middle. Pulse trains were delivered using a laptop computer,
and signal outputs were monitored with an oscilloscope
(Tenma, model 72-320). Displacement values were based on
existing literature (Williamson 1988, Klages et al. 2002) and
divided into two overlapping ranges that were presented in
separate experiments. This format was chosen to minimize
habituation to the stimulus and to prevent stress resulting
from extended trial times necessaiy to present the entire
range of displacements. The smaller values were tested in the
SSDE and ranged from 0.01 to 0.13 mm, whereas the larger
values were tested in the LSDE and ranged from 0.08 to 1.12
mm. For the Large Source-Displacement Experiment, a ste-
reo receiver (Kenwood, model VR-615) was used to amplify
the signal, thereby increasing the source displacement.

Stimulus signals were created using SigGenRP v.4.4
stimulus design software from Tucker-Davis Technologies.
Stimulus presentations were compiled and edited using
CoolEdit Pro v.2.1 from Syntrillium Software Corpoi'ation
recently renamed Adobe Audition v.1.5. Each of the stimu-
lus pulse trains was 5 s long and included ten 2-ms clicks of
the same amplitude, separated by nine 0.553-s intervals of
silence. Clicks are defined as short, intense bursts of energy
that encompass a wide range of frequencies. Stimulus pulses
and their respective source-displacements were measured
and calibrated prior to the experiment using a Metrolight
laser micrometer (model Alpha X03). All pulse trains were
presented only once in each of the trial sequences. Their
presentation orders were determined using a random num-
ber generator.

Experim ental p raced 1 1 res

Trials were conducted on separate days between the
hours of 1100 and 1800. The experimental tank was filled
with conditioned seawater from the home tank to ensure
that each animal was constantly exposed to uniform and
familiar olfactory cues. Seven animals were used in the SSDE
and five animals were used in the LSDE, three of which were
the same (repeated-measures within-subject design; Myers
and Well 2003). Animals were transported from the home
tank in covered buckets, gently transferred to the test arena,
and allowed to habituate for 10 min prior to the start of
experimental trials. Following habituation, video recording
commenced and individuals were subjected to a 5-min con-
trol period during which time no vibrational pulses were

administered. The control period was followed by a 5-min
“stimulus package” that began with 20 s of baseline silence
and continued with the presentation of 1 1 randomly ordered
pulse trains that were separated by 20 s of silence.

Treatment order (control first, stimulus second) was I
not altered between trials because it was unclear how long
the effect of the stimulus on the behavior of the animals, if
any, would last. If the stimuli were to be presented before the ,
control in these initial experiments, any continuing effect on '
the behavior of the animals would reduce the legitimacy of
the control data. After trial completion, video recording was
stopped and animals were returned to their home tank. The
test aquarium was rinsed thoroughly between trials with :
fresh water to remove any residual individual olfactory cues.

Frequency-sensitivity experiment

Experimental apparatus

The experimental arena was similar to that of the SSDE
and LSDE with the exception that a smaller, rectangular
Plexiglas™ tank (41 cm X 21 cm x 26.8 cm) containing
-25 cm standing water was used. Additionally, four foam
pads that measured 14.5 cm in height were used to absorb
background vibration, and only one camera, placed 1.5 m in
front of the long axis of the tank, was used.

Vibrating stimulus

Stimulus frequencies were generated in an identical
fashion to that described previously in the SSDE section.
Stimulus presentations were compiled and edited using
CoolEdit Pro v.2.1 from Syntrillium Software Corporation
(Adobe Audition v.1.5). The 5-min stimulus package con-
sisted of 11 randomly ordered frequencies (10, 50, 75, 100,
150, 200, 300, 400, 500, 750, and 1000 Hz) that were chosen
based on existing literature and by determining which fre-
quencies might be most prevalent in the animal’s natural
habitat (Williamson 1988, Klages et al. 2002). A 0.37 mm
bead displacement was used for all frequencies so corre-
sponding source-velocities could later be determined. This
value was chosen based on results from the LSDE that re-
vealed that this displacement value caused a large decrease in
nautilus ventilation rate and was large enough to eliminate
concerns of background interference. Each frequency emis-
sion was 5 s long and was separated by 20 s of silence. A
selected frequency was included only once per trial sequence
and the presentation orders of the frequencies were deter-
mined with a random number generator.

Experimental procedures

See Experimental procedures from the previous experi-
ment for habituation procedures. Eight animals were used in
the frequency-sensitivity experiment (FSE), and trials con-
sisted of a 5-min control period (silence) and a 5-min stimu-

NAUTILUS VIBRATION DETECTION

7

lus-set presentation consisting of 1 1 randomly ordered fre-
quencies. The presentation of the treatment category
(control or stimulus) was alternated between trials, and a
5-min buffer period (silence) was inserted between treat-
ments to control for order effects.

Data collection and behavioral analysis

Data were collected from the video recordings by two
independent “blind” observers using a Sony DHR-1000 digi-
tal video-cassette recorder. A suite of five typical Nautilus
behaviors ( Basil et al. 2005 ) was identified prior to the ex-
periment but no a priori assumptions were made about
whether those behaviors would be evident or about their
magnitude and polarity. Trials were subdivided into 5-s bins
and individual behavioral measurements were recorded in
real time for each bin. Typical behaviors such as rocking,
touching the bottom of the tank (not just resting on the
^ bottom), tentacle extension (expressed as a percentage of
j body length), and the “cat’s whiskers” foraging posture
; were not detected in any of the trials. Ventilation rate was a
consistent and robust measure of response and has been
used as an experimental measure for other cephalopods
(King and Adamo 2006) and, hence, will be the focus of all
our analyses.

Ventilation rate was defined as the number of com-
pleted respirations per 5-s interval and is abbreviated as
ventilation rate/5s or VR. This behavior was recorded by
! observing the area of the mantle cavity bilaterally located
1 posterior to the eye or by minor vertical oscillations ot the
l! entire animal produced by water expulsion through the hy-
j| ponome (Fig. 2). A completed respiration was defined as
j either { 1 ) the period between one closure of the mantle to
the next or (2) the deviation in movement of the animal
; from a standing position to a position either slightly above
or below, and then the return to the initial standing position,

! which has proven to be another reliable indicator of venti-
lation in these animals (Basil et al. 2005).

I

Statistical analysis

A repeated-measures within-subject design was used for
I all three experiments (Myers and Well 2003). Paired samples
I Student’s f-tests were used to compare ventilation rates of
animals between treatments to determine if exposure to a
'll vibratory stimulus had any effect on behavior. Both control
p and stimulus periods were 5-min long and data were col-
lected in 5-s intervals or time bins. Data for each time bin
were combined and averaged for each treatment and tor
i| each animal.

j Additional analyses were then performed on data that
|! were divided into categories based on the spatial and tem-
!| poral response of the animals. Two “distance” categories

Dorsal

A

n

t

e

r

1

o

r

Sheath

Digital

tentacle

Preocular

tentacle

Postocular

tentacle

Ventral

pore

(KbUiker’s canal)

Hyponome

P

0
s
t
e
r

1
o
r

Figure 2. Lateral view of Nautilus pompilius pouipilius, depicting
various external components with emphasis on the location (near
the rhinophore and the epidermal pore that connects Kolliker’s
canal with the left statocyst) of the mantle cavity that was used to
count ventilation rates.

were created: responses of animals <20 cm and >20 cm from
the source. Spearman’s Rank correlation tests were used to
examine the correlation between distance from the source
and ventilatory behavior. In instances where the same ani-
mal was used in more than one experiment, a single mean
ventilation rate was used to prevent pseudoreplication. This
was not possible for analyses that examined potential effects
of distance from the source on ventilation rate, as animals
that participated in more than one experiment often occu-
pied both distance categories, therefore requiring that the
trial averages be separated for analysis.

To describe the reaction of the animals through time,
five temporal categories were created by subdividing the
stimulus category. During each trial, a maximum of 1 1 data
points were collected for each of the following stimulus
categories: 5-s stimulus presentation (5 s stim), 1-5-s post-
stimulus (1-5 s post), 6-10-s post-stimulus (6-10 s post),
11-15-s post-stimulus (11-15 s post), and 16-20-s post-
stimulus (16-20 s post). Categorical averages for each trial,
and subsequently each animal, were obtained and paired-
samples f-tests were used to compare control data to each of
the 5-s post-stimulus categories.

As an additional note, mean ventilation rates varied
greatly between animals so numerical ventilation rates were
converted into percentage change from the control to dem-
onstrate changes in behavior graphically. However, all sta-
tistical tests were performed on the actual ventilation values
as opposed to the percentage values to avoid an artificial

8

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

increase or decrease in probability due to the imposition of
fixed limits (0-100) on the measure.

RESULTS

Overall combined results for all experiments

Twenty trials using 1 1 animals were conducted. A sig-
nificant decrease of 8.23% in ventilation rate/5 s occurred
between control and stimulus treatments across all animals
(Paired-Samples Student’s f-test, N = 1 1, t = 2.61, P - 0.03)
with a mean control VR of 4.06, SD = 1.72 and a mean VR
in the presence of a stimulus of 3.70, SD = 1.45.

Mean ventilation rates for Nautilus remained below
control values for at least 20 s post-stimulus presentation
(Fig. 3). Paired-Samples f-tests revealed that the largest de-
crease of 9.9% was observed during the actual 5-s stimulus
presentation (f-test, N = 11, f = 2.90, P = 0.02) and the
smallest decrease of 6.9% occurred 5 seconds after that (f-
test, N = 1 1, f = 2.37, P = 0.04). The responses of animals in
the remaining three 5-s post-stimulus bins were 8.6% lower
than controls in the 6-10 s post-stimulus bin (f-test, N = 11,
f = 2.80, P = 0.02), 7.4% lower during the 11-15 s post-
stimulus bin (f-test, N = 1 1, f = 2.26, P = 0.048), and lastly
8.2% lower than controls during the 16-20 s post-stimulus
bin (f-test, N - 11, f = 2.26, P = 0.05), respectively.

Data from 15 trials using eight stationary animals were
examined to determine if ventilation rate decreases in Nau-

CC

tilus when animals are closer to a vibrating stimulus. Only
animals that remained stationary throughout the trial were
used so their distance from the source would be constant.
Live of the animals participated in more than one trial and,
unless an animal produced values for both distance catego-
ries, their mean VR values were averaged between trials and
used in the analysis. Six animals <20 cm from the source had
an average of VR 2.83, SD = 1.07 whereas six animals that
were >20 cm demonstrated a slightly higher average VR of
2.87, SD - 0.38. No significant correlation between distance
from the source and VR was found (Spearman’s Rank cor-
relation, AT = 8, ij = 0.22, P = 0.60). Additionally, a subset of
animals was selected for which data existed in both distance
categories for each animal. Means from both categories were
compared to determine if distance from the source caused
significant differences in VR. Although no significant differ-
ences were evident (Paired-Samples f-test, N = 4, f = -2.52,
P = 0.09), animals vented at a rate that was 8.0% lower when
they were closer to the stimulus than when they were >20 cm
from the origin of the vibrations.

When source-displacement increased, animals exhibited
a decrease in their ventilation. Pearson correlations exam-
ined ventilation rates in seven animals from the SSDE and
LSDE (Fig. 4) across nine trials. Three animals were <20 cm
from the source and six animals were >20 cm from the
source. A significant inverse correlation was found between
source-displacement and VR for animals that were <20 cm
from the source (Pearson correlation, N = 6, r = -0.57, P =
0.01). No significant correlation was
found between source-displacement
and VR for animals that were >20 cm
from the source (Pearson correlation,
N - S, r = 0.43, P = 0.06). On average,
animals from the SSDE and LSDE,
when exposed to a vibratory stimulus,
ventilated at a rate that was 11.72%
less than the control VR when they
were <20 cm from the source, com-
pared to a 5.38% decrease for those
that were >20 cm from the source.

When source-velocity increased,
as seen during the FSE, animals also
exhibited a decrease in their ventila-
tion. Mean ventilation rates for five
animals which were used in the FSE
were examined across 12 source-
velocity categories (Fig. 5). Four ani-
mals were <20 cm from the source and
the remaining animal maintained a
distance >20 cm from the source. No
significant relationship was found to
exist between source-velocity and VR

5s Bin

Figure 3. Bar graph depicts the mean percent change in ventilation rate (VR) of the control
when compared to each of the five stimulus and post-stimulus time categories. Each bar
labeled “S” represents a 5-s period of time that begins with the presentation of the stimuli and
continues for a 20-s post-stimuli period. The bar labeled “C” represents a 5-min control
period. Significant decreases between the control and stimuli bins were found for each of the
five time categories but no continual decrease in VR over time was observed. Error bars show
+ 1 SE.

NAUTILUS VIBRATION DETECTION

9

were <20 cm from the source com-
pared to a 0.6% increase for those that
were >20 cm from the source.

Additionally, treatment-order ef-
fects and the possibility of habituation
across trials in nautiluses were exam-
ined. The analysis of presentation
order, control first or stimulus first,
revealed that no treatment-order ef-
fect was evident in the FSE (Indepen-
dent Samples f-test, N = 8, f = 1.55,
P = 1.44).

DISCUSSION

Figure 4. The impact that animal distance from the source and source-displacement has on
ventilation rate. Data shown are from two experiments, the Small Source-Displacement
Experiment (SSDE) and the Large Source-Displacement Experiment (LSDE), and account for
eight animals across nine trials. Three animals were <20 cm and six animals were >20 cm.
Bead displacement refers to the distance traveled by the leading edge of the bead and does not
include bead diameter.

for animals that were <20 cm from the source (Pearson
correlation, iV = 4, r = -0.52, P = 0.08). No statistical cor-
relation could be conducted between source-displacement
and VR for animals that were >20 cm from the source be-
cause of an inadequate sample size {N = 1). Animals from
the ESE, when exposed to a vibratory stimulus, ventilated at
a rate that was 16.3% less than the control VR when they

c

The major finding revealed by
these experiments is that Nautilus
responds to underwater vibrations.
Animals almost always reduce their
ventilation rate in the presence of a
vibratory stimulus: there were signifi-
cant decreases in ventilation rate dur-
ing a majority of trials when the animal was exposed to
vibratory stimuli.

Comparatively speaking, these findings are relevant to
research conducted previously on other invertebrates, such
as Williamson’s (1988) investigation into the vibrational
sensitivity of the statocyst in the northern octopus where a
minimum particle-displacement threshold of 0.12 pm was
determined and the study conducted
by Klages et al. (2002) that noted that
the deep-water amphipod Eurythenes
gryllus produced particle displace-
ments of 0.05-0.3 pm between 70 and
200 Hz when feeding and swimming.
This work has demonstrated that nau-
tiluses are capable of responding well
within these ranges of displacements
and frequencies, so future work should
focus on determining practical appli-
cations of this system in the wild. The
detection of signals in the wild can
benefit Nautilus in many ways. A de-
crease in ventilation rate could possi-
bly serve as a mechanism for predator
avoidance. Similar responses have
been observed across multiple groups
of animals including cephalopods.
King and Adamo (2006) demonstrated
that the cuttlefish Sepia ojficiiialis Lin-
naeus, 1758 reduced ventilation and

Velocity (m/s)

Figure 5. The impact that animal distance from the source and source velocity have on
ventilation rate. Data shown are from the Frequency-Sensitivity Experiment (FSE) and ac-
count for five animals across five trials. Four animals were <20 cm from the source and one
animal was >20 cm from the source. Velocity represents varying source-intensities that were
presented randomly.

10

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

cardiac rates when exposed to sudden visual stimuli, in
preparation for a flight response. Additionally, the authors
identified four hypotheses in the literature that offered ex-
planations for this behavior, one of which was that animals
decrease ventilation to increase crypsis. Although they re-
jected this hypothesis, suggesting that cuttlefish decrease VR
in preparation of a flight response, the hypothesis can be
applied to nautiloids since no movements associated with
the stimulus were observed during experiments.

from a biological standpoint, decreasing respiratory
rates may serve as a defense mechanism. Presumably, ap-
proaching predators emit a range of vibratory stimuli result-
ing from motion, such as the sinusoidal movements of fish.
Therefore, such a mechanism would work most effectively in
concert with cryptic coloration, by reducing overall rocking
movement as the predator nears.

Conversely, decreasing respiration may benefit an ani-
mal’s predatory success. This is not to imply that nautiluses
are formidable hunters — but a sit-and-wait strategy is pos-
sible. These animals spend most of their lives associated with
coral reefs that are teeming with potential prey items. Per-
haps nautiluses, upon detection of certain chemical or vi-
brational cues, decrease respiration to make themselves less
conspicuous to an unsuspecting prey. However, it is improb-
able that a decrease in VR is an offensive strategy since
anecdotal evidence suggests that captive animals increase
respiratory activity when exposed to food sources (Soucier,
pers. obs.).

Nautiluses likely detect vibration with epithelial tactile
receptors on the tentacles, mechanoreceptors below the rhi-
nophore, or some other innervated system. In cuttlefish (Ko-
mak et ah 2005), epidermal lines along the mantle and arms
containing polarized hairs are able to detect local water
movements and subsequently integrate that information
into behavioral responses. The locations of these potential
receptors in Nautilus were, however, not ascertained in our
experiments. Additionally, the role of the gas-filled external
shell acting as a resonating mechanism was not investigated
during our experiments but should not be excluded from
consideration as a contributing factor.

Irrespective of the mechanism, any additional sensory
system that an animal can use, whether it is in conjunction
with alternate systems or serving as a primary system would
be beneficial to the survival of that animal. Based on the
average depth in which these animals live, the nektobenthic
niche that they occupy, and the lack of information regard-
ing their feeding and mating strategies, an evolutionary ar-
gument could be made for possessing a mechano-sensoiy
system capable of detecting hydrodynamic disturbances and/
or substrate-borne vibrations.

In regard to latency of response or time-specific re-
sponses, our experiments revealed no temporal trends

within our time periods because significant decreases in ven-
tilation rate ranged from the stimulus presentation to the
16-20s post-stimulus period. These animals can respond to
the stimulus for up to at least 20 s post-presentation, and the
distance from the source and the components of the signal
should be the focus of future investigations.

The results of these experiments clearly indicate that
Nautilus potnpilius pompilius can detect and respond to vi-
brational stimuli. To what end this sensory system seiwes,
whether it is mate selection, prey acquisition, predator
avoidance, or a combination of multiple evolutionary func-
tions, has yet to be determined. What has been established
is that the recognition of these signals and subsequent be-
havioral response may pose some type of evolutionary
advantage.

ACKNOWLEDGMENTS

We thank Christopher Braun, john Chamberlain, Neil
Landman, Robert “Rocky” Rockwell, and Richard Veit for
discussions, statistical advice, and comments on a previous
version of this manuscript. We are grateful to Kristine
Kuroiwa-Bazzan, David Klein, Robyn Crook, Stephanie
Soucier, Michael Barach, Moses Eeaster, and Daniel Hagler
for invaluable help with experiments and graphics. Robyn
Crook kindly designed Eig. 1 and Stephanie Soucier designed
Fig. 2. Dr. Martin Schreibman and the staff of the Aquatic
Research and Environmental Assessment Center of Brooklyn
College/CUNY kindly allowed us to use their facilities. Louis
Tundis and the BC Machine Shop expertly crafted much of
our apparatus. Funding came from the American Museum
of Natural History (CPS), the Sigma Xi Society for Scientific
Research (CPS), and a PSC-CUNY Grant to JAB.

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Submitted: 1 October 2006; accepted: 30 July 2007; final

corrections received: 17 December 2007

Amer. Maine. Bull. 24: 13-23

Octopus sucker-arm coordination in grasping and manipulation"^

Frank W. Grasso

Department of Psychology, Brooklyn College, City University of New York, 2900 Bedford Avenue, Brooklyn, New York 11210, U.S.A.,
FGrasso@brooklyn.cuny.edu

Abstract: In natural settings octopuses use their arms and suckers in a variety of dexterous manipulation tasks, such as extracting prey from
crevices and burrows, opening bivalve shells, and arranging middens in front of den entrances. Octopuses use multiple suckers on a single
surface for a power grasp that supports their locomotion or permits the animal to carry or move small objects. Similar to squids engaged
in prey capture, octopuses can project an arm from their body, attach a group of distal suckers, and pull an object toward themselves by
shortening the arm. I investigated octopuses’ use of suckers in similar tasks under controlled, reproducible laboratory conditions. Because
larger suckers can generate larger adhesion forces, 1 hypothesized that the larger suckers toward the base of the arm would be preferred in
tasks requiring the arm to employ greater forces. Octopuses did not use the strategy found in squid tentacles; applying suckers of appropriate
force generation to a surface and lifting or pulling the arm. Instead, in many cases they used a variety of arm movements in combination
with different functional groups of suckers. In addition, different arms performed different roles. When animals were restricted to the use
of a single arm, they preferred to use suckers in the middle positions of the arm to support this coordinated arm-sucker activity. Contrary
to a view of suckers as passive agents reflexively reacting to surface contact, these results are consistent with the known neural organization
of the octopus arm and also with complex sucker-arm coordination in the performance of manipulation tasks.

Key words: Octopus, grasping-behavior, suckers, coordination

Octopuses move in a mysterious way. Being flexible,
the movements that they make are often difficult to
specify and correspondingly difficult to investigate.

The literature does not contain a description of octo-
pod walking comparable with descriptions of the six-
legged, tripod gait of insects or the stereotyped loco-
motor patterns of snails or polychaetes. Descriptions
of posture run into very similar difficulties and per-
haps partly because oj this, research on motor control
in cephalopods has proved a less attractive proposition
than research on sensory analysis and learning.
(Wells 1978: 246)

Wells’ claim that studies of motor systems in cephalopods
have lagged behind those of other sensory and learning sys-
tems still rings true today for studies of Octopus Cuvier, 1797
and for the reasons he cites. Progress has been made with
kinematic descriptions of reaching and fetching behavior
that have inspired neural and physiological models of arm
control in these activities (Gutfreund et al. 1996, 1998,
Matzner et al. 2000, Sumbre et al. 2001, 2005, 2006) and a
systematic description of the movements of Octopus arms
has also been developed (Mather 1998). The muscular-
hydrostat mechanisms by which arm movements are ef-
fected have provided a conceptual framework for under-

standing limb movement and manipulation in the absence
of hard parts (Kier and Smith 1985). In addition, researchers
have begun to explore and explain the neurophysiology of
bend generation in the arm (Gutfreund et al. 1998, Matz-
ner et al. 2000, Sumbre et al. 2001). However, since Wells
(Wells and Wells 1957a, 1957b, Wells 1978), little attention
has been directed toward the behavioral repertoire involving
the suckers on the arm which provide the octopus with
contact tactile and chemosensory information and fine local
manipulation.

The arms of octopuses can bring suckers into a position
to sense or grasp a surface of interest to the animal. Though
the flexibility of their arms makes them quite capable of it,
octopuses are rarely observed to wrap their arms to grasp
objects. The method octopuses employ in securing purchase
on objects varies with the object and context; the wrapping
often appears to be a natural continuation of arm momen-
tum following abrupt contact with a fixed object. The suck-
ers are integral to much of the directed behavior of octo-
puses; Yet, apart from some excellent quantitative studies of
their adhesion mechanism (Smith 1991, 1996), their mode
of action has received little attention. This report begins to
fill this gap by analysis of simultaneous observations of arm
movements and the actions of scores of suckers under natu-
ral and experimental conditions.

* From the symposium “Cephalopods: A behavioral perspective” presented at the joint meeting of the American Malacological Society and
Western Society of Malacologists, held 29 luly to 3 August 2006 in Seattle, Washington.

13

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AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

The extent to which sucker and arm movements are
coordinated or independent is currently unknown. Hanlon
and Messenger (1996: 15) reflected that “In fact the nervous
system of the arms, which contains more neurons than the
whole central brain (Young 1971), is in some ways curiously
divorced from the rest the brain and many of the arms
actions are performed without reference to the brain.” The
same comment applies to the relationship of control be-
tween the arms and the suckers: suckers have some degree of
autonomy but must move in ways that
are not in conflict with ongoing arm
activity. Studies have shown that a
single octopus arm detached from the
rest of the animal retains considerable
capability for coherent response to
stimuli (Wells and Wells 1957a, Rowell
1963, 1966, Altmann 1971, Wells 1978,

Gutfreund et al. 2006). Yet, all eight
arms are not completely independent
as is dear because the animal is capable
of coordinating all its arms, and arm
preferences exist (Byrne et al. 2006).

These studies have focused on the ac-
tions of the arms and not the contri-
butions that the suckers, the primary
contact sensing and local action or-
gans, make.

The suckers, too, have the appro-
priate (direct or indirect) neural con-
nections (Fig. lA) to send information
to and receive information from the
brain and the arm on which they are
situated (Graziadei 1971). Each sucker
has a committed local ganglion. This
ganglion receives an enormous num-
ber of afferent fibers: chemosensory
and mechanosensory axons from the
sucker rim as well as proprioreceptors
(muscle sense) from the various
muscles of the sucker. These fibers
pass through the nerve connecting the
sucker and the ganglion of the sucker
(Fig. lA). This nerve also carries motor
neuron axons to control the sucker
muscles. The ganglion also carries on
bidirectional communication with the
main nerve cord of the arm, the chain
of brachial ganglia. This communica-
tion is carried via the nerve connecting
the sucker ganglion to the brachial
ganglion (Fig. 1) and not much is
known about its function. The brachial

ganglion is one of a chain of ganglia which enlarge, increas-
ing their neuron counts and neurpil volume directly over
each sucker. Anatomically, they form a chain of intercom-
municating ganglia along the length of the arm, each of
which appears to be intimately involved in sucker informa-
tion processing. Finally, these ganglia make direct connec-
tions of their own to the sucker, bypassing the sucker gan-
glion, through the nerve connecting the brachial ganglion
and the sucker (Fig. 1). This nerve carries sensory fibers

Figure 1. Schematic diagrams of a typical octopus sucker and arm attachment in cross-
section perpendicular to the long axis of the arm. A, The functional divisions described in the
text for adhesion generation. B, The gross neuroanatomical connectivity of those functional
parts in relation to the arm. Abbreviations follow those used in Young (1971): n.suc.-
gan.suc., nerve connecting the sucker and the ganglion of the sucker; n.gang.br.-suc.gan.,
nerve connecting the sucker ganglion to the brachial ganglion; n.gang.br.-suc., nerve con-
necting the brachial ganglion and the sucker; n.gan.br.-gan.suc., nerve running from the
brachial ganglia to the sucker ganglion.

OCTOPUS SUCKER-ARM COORDINATION

15

from the sucker and possibly motor fibers to the sucker. This
brief sketch of the neuroanatomy demonstrates that the con-
nections exist for rich information exchange between the
suckers and the arm chain ganglia and through the brachial
ganglia indirectly between the suckers and the brain. The func-
tional roles of these identified pathways have yet to be studied.

In squid tentacles the roles of sucker and tentacle have
been studied behaviorally and kinematically and the control
of the suckers appears to be much simpler. From these stud-
ies it appears that the coordination of limb and sucker action
is a passive and not an active one. The squid [Loligo pealei
Lesueur, 1821) combines the actions of its paired tentacles
and suckers in prey capture (Kier 1982, Van Leeuwen and
Kier 1997). The terminal club of the tentacle, covered with
suckers, is ballistically propelled toward the squid’s prey
in <300 ms. The process is too fast for tentacle-sucker co-
ordination, so a local reflex-triggered by mechanical contact,
in turn triggers rapid sucker attachment. It is possible, de-
spite the anatomical connections described above, that the
actions of octopus suckers follows a similar plan where the
movements of the arm bring the sucker into contact with
some surface, and that surface contact in turn triggers a
reflexive sucker attachment. Though there is evidence that
this is not always the case (Wells and Wells 1957a, 1957b,
Rowell 1963, 1966), ihe idea that suckers are triggered to
attach by mechanical contact is a parsimonious explanation
for sucker operation in octopuses that cannot be ruled out in
all situations.

Anatomical organization of octopus suckers, which dif-
fers in sophistication from those of the squid, indicates that
octopus suckers are well suited to support active coordina-
tion. The club suckers on the squid tentacle are composed of
jj a single chamber surmounted by a large internal muscle
which acts like a piston to develop a negative pressure lor
adhesion in a few milliseconds (Van Leuven and Kier 1997).
Octopus suckers are two-chambered, radically-symmetric
structures (infundibulum and acetabulum) suspended from
the oral surface of the octopus arm that incompletely enclose
a volume of the surrounding seawater (Fig. IB). Like squid
suckers, they act on ambient seawater to reversibly attach an
object to the octopus arm or the octopus arm to a fixed
surface with which the sucker makes contact. The mecha-
nisms by which they facilitate grasping in octopuses have
been inferred from anatomy (Kier and Smith 2002). They
have an elegant division of function that is absent in the
squid: the muscles of the infindibulum reshape the sucker
rim to conform to the exterior surface; after a seal is formed
(completing the enclosure of the volume), the muscles of the
acetabulum expand its internal volume to produce negative
pressure (and therefore adhesion force). The major differ-
ence between squid tentacle and octopus-arm suckers resides
with the third functional group of muscles in the sucker. The

extrinsic muscles of each sucker attach at the junction of the
infundibulum and acetabulum and on the arm itself With the
surface held, the extrinsic muscles are arranged to act an-
tagonistically to rotate the sucker rim in virtually any plane
around the long axis of the arm, along with whatever it is
attached to. It is these extrinsic muscles which suggest octopus
suckers evolved to support a numipiilation as well as attach-
ment function.

In addition to this motor function, octopus suckers ap-
pear to play an important sensory role. The surface of the
octopus arm is studded with mechano- and chemoreceptors
but their density is extremely high in the sucker rim: on the
order of 10“* per sucker (Graziadei 1964, Graziadei and
Gagne 1976). As mentioned above, these receptors, along
with anatomically identified proproiceptors in the sucker,
project their axons to make synapses in the small ganglion
that lies over each sucker and in the brachial ganglion of the
axial nerve cord that runs the length of each arm (Fig. IB)
(Graziadei 1965, 1971). Thus, the obseiwed interconnectivity
of the suckers and arm ganglia serve a primarily sensory
function rather than a motor control. The receptor and neu-
ral organization agree with observations of complex motions
made by suckers engaged in apparent sensory exploration.
Suckers in an otherwise stationary arm are occasionally ob-
served to reshape themselves by extension, retraction, and
rotation to follow surface contours and edges with only the
rim in contact and without forming a seal and sucker at-
tachment. These movements occur outside the octopus’s
field of the vision and therefore appear to require local sen-
sory feedback and motor integration.

The studies reported here sought evidence of active
arm-sucker coordination in two forms. First, correlations
between arm and sucker activity during spontaneous behav-
ior of freely moving animals were studied. Patterns of activ-
ity in groups of suckers that varied with the behavior of the
octopus as a whole entity would be consistent with active
coordination. Second, I experimentally manipulated the
force required to complete a task, thus requiring the octopus
employ a different mechanical approach. The adhesive force
of suckers is proportional to their size (Smith 1991, 1996)
and suckers on any given arm become smaller in size distally
(Voight 1993). Thus, if the octopus adjusted the use of its
suckers depending upon the force required for a given task,
this would provide evidence that some feedback about the
appropriate force level was shared between the suckers and
the arm or between the suckers and the brain.

MATERIALS AND METHODS
Natural observations of sucker use

Animals

Four wild-caught adult Octopus bimacidoides Pickford
and McConnaughey, 1949 were filmed in their home tank ad

16

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

libitum to capture examples of their use of suckers on one
vertical glass wall. These animals had arms that were approx.
15-20 cm long at the time of the experiment. I did not
determine the sex of these animals. However, one of them
showed somewhat enlarged suckers toward the base of its
second arms, indicating that this animal was male. The ani-
mals were fed ad libitum on a diet of clam meat, frozen
shrimp, and, occasionally, live crabs. These octopuses were
difterent individuals trom those studied in the following
experiment.

Video acquisition

Animals were filmed at 30 fps using a JVC MiniDV
digital video camera (GR-D250U) positioned outside the
tank to encompass the entire pane (resolution =1 mm per
pixel). The tanks were standard 113.59-L tanks of dimen-
sions 76.2 X 53.34 x 33.02 cm. The animals had lived in
these tanks for at least 3 weeks and were therefore habituated
to the tanks and the conditions in the room where they were
housed. Laboratory personnel were absent from the room or
visually isolated from the animals by a curtain while the
footage was collected. Approx, four hours of this footage was
surveyed for periods during which ( 1 ) single arms could be
observed when (2) 20 or more contiguous suckers were con-
tinuously visible for (3) 10-30 seconds. Several sections of
video were obtained that I refer to here as “continuous video
segments.”

Scoring the video

These sections were scored at 1 -s intervals. When suck-
ers are attached to a glass surface they assume a characteristic
appearance: they are flattened and look like enlarged white
discs compared to their unattached state, and the sphincter
is clearly visible and round. An observer scored each sucker
as “Eree” (F), “In-Contact” with the surface (C), “Partially
Attached” (P), or “Attached” (A) for each second in each
series. Suckers scored as F had no part of the sucker in
contact with the glass; C had less than the whole sucker rim
in contact with the glass; P had the entire rim but not the
sucker sphincter in contact with the glass; and A the entire
rim and sphincter were in contact with the glass - the sucker
was flat and its radius enlarged. These categories were easy to
distinguish. Inter-observer agreement on these categories for
a 30-s section of tape in which 45 suckers were visible was at
least 95% for three practiced observers.

Analysis

The attachment data were a series of “sucker states” in
time. Plots of these data were made to represent the se-
quence of sucker attachment down the arm. The scored
categories of C and P were grouped in a single category while
F and A were retained as separate plot categories. I also com-
puted probabilities that a given sucker and its neighbors

would be co-active. For these analyses I assigned each sucker
a value of 1 each time it was observed to be in state A and a
zero if it were in state F, C, or P. For each time step, I
counted the number of coincident attachments for each
sucker and its neighbors one, two, three, and more suckers
in proximal or distal directions along the arm. With this I
had a description of the coincident activation of each sucker
with all the observed suckers in a coordinate system centered
on the individual sucker. I aligned each individual sucker’s
co-activation pattern to this sucker-centered frame and, by
adding the coincidences for each distance from the sucker,
computed the total number of co-incident attachments for
each neighbor. This total, divided by the total number of
activations observed at that distance, is the proportion of
co-activation, or probability of co-activation, observed in
that particular frame of a continuous video segment. Indi-
vidual traces are shown as time series and averages of the
entire series (Figs. 2A-C).

Object raising experiments

Experimental animals

The animals used in this experiment were six wild-
caught Octopus bimaculoides. I did not determine the sex of
these animals, but two of them showed somewhat enlarged
suckers toward the base of their second arms, indicating that
they were male. They had arms 15-20 cm long. This species
possesses 150-300 suckers, including extremely small suckers
(<1 mm diameter) at the tip, arranged in two staggered rows
(Voight 1993). Octopuses were maintained in individual,
transparent Plexiglas-walled chambers in a recirculating, ar-
tificial seawater system and were fed ad libitum on a diet of
clam meat, frozen shrimp and, occasionally, live crabs. This
did not appear to affect their motivation for capturing live ,

Figure 2. The apparatus used in the dome-raising experiment. The
left side of the figure shows a front view of the apparatus and the
right, a side-view. The position of the mirror permitted the video
capture of a side and bottom view with a single camera. The dia-
gram is schematic and not drawn to scale.

OCTOPUS SUCKER-ARM COORDINATION

17

crabs in the testing tank. Animals could see individuals in
adjacent chambers but could not make physical contact.

Apparatus

Tests were conducted in a glass-bottomed aquarium
(1 14-L), similar to that used in experiment 1, supplied with
continuously refreshed water from the animal’s housing sys-
tem (Fig. 2). A mirror was placed beneath the bottom of the
tank at a 45 degree angle so that the octopuses’ movements
and sucker use could be viewed from below. A single IVC
MiniDV digital video camera (GR-D250U) was placed so
that half the field of view captured this view from below and
half captured the side view of the tank and animal activity.
A transparent, ~5-cm diameter glass dome was placed, rim-
down, on the floor of the tank. The dome was fitted with two
fixed magnets positioned at opposite sides of the rim. These
magnets were held to the dome with a single, long cable tie
and thermal glue. The magnets were aligned with the posi-
tive pole up and the negative down relative to the dome. The
fixed magnets’ flat surfaces were parallel to the rim of the
dome; when the dome was in place, the rim and magnets lay
flush with the floor of the tank. Two electromagnets were
positioned beneath the tank and aligned with the fixed mag-
nets to provide variable force required to complete the task.
Electric current from an Elenco Precision Regulated DC
i power supply (Model XP-603) was adjusted to modify the
I strength of the magnetic field they exerted. The force ol these
I activated electromagnets exceeded the force of gravity so that
j the magnets were held in place. We used a spring scale to
I determine the force required to detach the dome from the
j tank floor. We varied the current through the electro-
magnets over a range of 0 to 1 amperes and recorded the
I force required to detach the dome at a variety of current

I levels. A regression analysis of the current supplied to the

electro-magnets allowed us to estimate the force required
to detach the dome (F = 3.88 C -t 10.48; = 0.54). 1 could

produce a 4 N difference in force due to the action of the
magnets. The weak correlation led us to use the settings as
“weak” or “strong” magnetic force as conditions in our ex-

; periments. The weight of the dome and fixed magnets re-
quired 1 0 N to move when the tank was full of water so the
range of forces an octopus was required to apply to detach
t the dome varied between 10 and 14 N.

1

li Trial procedure

At the start of a trial, a crab (~l-2 cm carapace length)
jj was placed under the dome and the electromagnets were
activated to produce the desired level of force. In early trials,
the octopus was released into the chamber with the dome

II and was free to move about the tank and use all of its
|i appendages. On later trials, the animal was released on the

other side of a partition with a 1.5 cm hole which permitted

the animal to reach the dome but limited its appendage use
to one or at most two arms. The camera recorded the activity
of the animal at 30 fps. My protocol called for the termina-
tion of the trial if the octopus did not raise the dome within
30 minutes. This time limit was never reached, and the oc-
topuses always raised the dome a few minutes alter being
placed in the tank.

Scoring the behavior

We scored the number of suckers attached to the dome
at the time the dome was raised. The scoring method was the
same as that used in the observations reported above (states
F, C, P, A). We also scored the portion of the arm in contact
with the dome when the animal first raised the dome. The
observer judged whether the proximal, middle, or distal
third of the arm was in contact with the dome. The time that
the dome was first raised was judged as the video frame just
before the electromagnets began to fall.

RESULTS

Natural observations of sucker use

Sucker attachments were sparse in the video footage
examined. On average 18.07% ± 13.78 (SD) ot the suckers
observable (20 to 60, depending on the trial) on a given arm
were attached at any given time, and instantaneous values
ranged from 0 to 39%. We observed no occasions on which
all the suckers on an arm were attached — even when the arm
was motionless along its entire extent.

The spatial arrangements and temporal sequences of
sucker attachment varied with the activity in which the
animal and the arm were engaged. Some spatial pat-
terns of simultaneous sucker attachment and certain tem-
poral sequences of attachment were repeated often in these
observations.

Adjacent suckers on opposite sides ot an arm were fre-
quently observed to attach to the surface in anti-phase: al-
ternating attached and unattached states. Groups of six, ten,
or even 15 adjacent suckers would be involved in these co-
ordinated patterns (see Figs. IB, 2A). Sometimes this would
persist as a single alternation; on other occasions it might go
on for several seconds, displaying as many as eight cycles ot
attachment and release. In such an “arm walk”, the arm is
moved along the surface of the glass, held in a fixed orien-
tation for several seconds as the suckers advanced in leading
and trailing pairs across the tank wall.

Suckers could also hold a specific position for periods
up to 25 seconds. Interestingly, these patterns of maintained
attachment often involved suckers from just one side of the
arm (see Figs. 3A-B for examples of this as a horizontal stripe
pattern).

18

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

A.

Ann R1 Sucker Attachment Dynamics

4l‘

3g Distal

31 S

s>

26 B

2,1

16

c/i

Proximal

B.

Ann L2 Sucker Attachment Dynamics

Side

Alternation

Fixed Grip
Kight Side
Suckers

Anchor

Points

RD MagF

Distal

Proximal

While both the “arm walk” and continuous attachments
described above suggest that suckers that were near neigh-
bors often were not simultaneously attached, we also ob-
served occasions when they were. “Anchor points” (Ligs.
3A-B) contrast with the large-scale patterns of coordinated
activity described above in that they involved 3-5 adjacent
suckers. On these occasions, I observed that the arm was
moved by a whip-like motion proceeding from the point of
attachment (anchor point) distally. During these arm mo-
tions, the distal suckers were unattached and the arm was
free to move at all more distal points.

Sometimes the animal would move itself along the tank
wall or floor by extending an arm, attaching some of its
distal suckers, and then shortening the arm to pull the body
toward the attached suckers. When this happened, pat-
terns of coordination involving many and then a few suckers
were evident (Lig. 4C). A period of widespread attach-
ments involving 12-15 adjacent suckers on alternating sides
of the arm preceded the shortening of the arm. This was
followed by a local set of attachments of 2-3 adjacent suckers
that supplied the fixed point toward which the animal’s body
was moved.

Intermediate-sized adjacent groups of four to eight si-
multaneously attached suckers were also observed (Pig. 4B).
These were observed proximal to a portion of the arm that
was extended out from the plane of the tank surface wall.

Figure 3. The attachment state of suckers on the arms of two
octopuses. The horizontal axis is time measured in seconds. The
vertical axis is the sucker number, arranged in sequential order
proximally to distally along the arm. The sucker numbers are rela-
tive; they are not numbered from the first sucker on the arm. They
are simply numbered from the sucker closest the arm base that was
visible during the scored observation period. Observations are con-
tinuous from that first observable sucker: odd numbered suckers
are all on one side of the arm while even numbered suckers are on
the other side. Shades of gray represent attachment state. Corre-
lated activity of the arm is marked along the left margin. A, State of
41 suckers on the right first arm (Rl) observed over a 37-s period.
Dark portions of the plot show attachment, light gray indicates that
the sucker was free or in contact with the glass but not sealed.
During this period the octopus oriented this arm vertically along
the surface of the tank wall as a relatively straight segment. The base
ot the arm was positioned, and the arm was whipped twice from
base to tip in a series of stepped waves. The tip moved freely, its
suckers were not scored, and the base was not visible. The hori-
zontal striped pattern results from only suckers on the leading edge
of the arm attaching to the surface. Two points marked “anchor
points” are groups of attached, adjacent suckers at the start of the
move. B, Attachment patterns of 25 adjacent suckers as the animal
raised the dome discussed in the text. The grey levels progress from
lightest to darkest to indicate “free”, “contact”, “partially attached”,

Object raising experiments

In initial trials, the animals were simply released into the
chamber with the dome. The animals were thus free to ap-
proach the dome in any manner and free to use all of their
arms. The animals invariably draped themselves over the
dome, mouth over the dome apex. In the typical posture, the
web was expanded over the dome and the arms fell around
the sides and made contact with the tank floor. The move-

and “attached” states. The arrows marked RPA, MD, RD, and
MagF point to times when the animal RePositioned the Arm rela-
tive to the dome, the animal Moved the Dome in a sliding motion
along the tank floor, the Raising of the Dome from the floor of the
tank first became visible, and when the Magnets holding the dome
came Free and no longer exerted a force to resist the octopus’s
raising. The zigzag patterns visible at the start and end ot the plot
represent alternate stepping of suckers on opposite sides of the arm.
The horizontal striping marks a period of about 20 seconds during
which the suckers on just one side of the arm were attached to the
dome and during which the octopus was presumably applying a
raising force to the dome. Anchor points toward the base of the
arm are again visible in this figure. Note, from the absence of light
grey, that the majority of the suckers not attached to the dome were
in contact with it during this period.

OCTOPUS SUCKER-ARM COORDINATION

19

' ment and superposition of arms and alteration of suckers
made observations of individual suckers and arm usage dif-
|1| ficult to assess. However, the general pattern at the moment
the dome was raised showed one or more large suckers near
■ the base of the arm(s) attached to the dome, more distal
I suckers on a variety of arms attached to the floor of the tank
I with suckers in between unattached to either the floor or the
; dome. It generally appeared that the lengthening of the arms
between the suckers fixed on the dome and those fixed on
i the tank floor produced the raising of the dome.

Trials with the animal able to reach the dome solely
through a hole in a partition permitted unambiguous ob-
,| servation of the actions of the suckers on one arm on the
dome and tank floor. In these trials, a pattern of dome-

il

t

Figure 4. These plots represent the probability ot attachment pairs
of neighboring suckers during three different types of arm move-
ments. Each line plotted along the vertical axis represents that
probability at one second intervals, with earlier traces lower. The
horizontal axis represents the distance between neighboring suck-
ers; zero is self (probability always equal to one), negative values are
suckers in the proximal direction and positive values are in the
distal direction along the arm. Dashed light grey vertical lines mark
sucker distance and dashed light grey horizontal lines mark the
probability scale (0 to 1) for each trace. A, 41 suckers observed on
arm R1 for 20 seconds. During this “arm walk”, the octopus moved
its arm along the glass with an alternative stepping of the suckers on
either side of the arm. This is reflected in the wavy pattern of traces
between 0 and 6 seconds and again between 9 and 13 seconds.
Between these two periods and after them, the arm was moved by
proximal to distal waves along the arm. The widespread patterns of
coordinated sucker activity during the walk contrast with local
patterns of activity during the wave where anchor points were
formed proximally to enable the movement of the arm. B, 10
seconds of observations from 35 suckers on R3. During this period,
the octopus moved this arm out away from the surface ot the glass
using a proximal set of suckers as an anchor for the arm. At 5
seconds, the local neighborhoods spanned 3-5 attached suckers,
presumably to support the weight of the arm away from the glass.
In the next second, the arm returned to the surface and made
several smaller local points of contact. In the following second, a
widespread pattern of attachment by suckers on just one side of the
arm appeared. This was followed by local groups of suckers in the
next second and the movement of the arm from the surface pre-
sumably supported by suckers on the other arms. C, Observa-
tions of 49 suckers during 16 seconds when the octopus used arm
R1 to lift its entire body up the along the tank wall. The octopus
projected the arm upward from its body, attached it with distal
suckers and then shortened its arm to pull itself upward. The ani-
mal repeated this sequence twice during these 16 seconds. In both
repetitions there is an initial widespread attachment of suckers on
alternative sides of the arms, perhaps probing for a suitable hold,
followed by a narrowing to local neighborhoods of attachment
during the pulls.

raising similar in some respects to that in the unrestrained
animal was observed. Without exception, the arm extended
beyond the partition was draped over the dome. There fol-
lowed a period of adjustment of arm position and repeated
attachment, detachment, and reattachment of individual
suckers. At the time of dome raising, there were always suck-
ers attached to the dome as well as suckers attached to the
floor of the tank, both proximally and distally from those
attached to the dome.

On three single-arm trials, the octopus slid the dome a
short distance across the floor of the tank before the dome
was raised. These slides were distinct from a pull of the arm
toward the animal in that they were made with suckers an-
chored both proximal and distal to the dome as well as on

20

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

the dome itself. They may have been accidentally produced
by forces the animal applied to the dome, but on all three
occasions the animal released its suckers on the dome and
tank floor and repositioned its arm before continuing its
efforts to obtain the crab.

All the animals showed a preference for using suckers
in the middle of the arm for this task over those at the base
or tip. In all six trials with strong and in all six trials with
weak magnetic force, the animal used the suckers on the
middle of its arm to attach to the dome and the adja-
cent tank floor (Fig. 5B). A sign test for six trials indi-
cated that these outcomes were unlikely to be due to chance
(S^ = 6, P <0.05).

The number of suckers used in strong and weak-magnet
trials differed (Fig. 5A). In trials requiring less force, the
animals used a mean of 10.16 ± 1.47 suckers while in trials
requiring more force they used 15.67 ± 2.58 suckers. While

Proximal Middle Distal

Portion of Arm in Contact

Figure 5. Summary ot the results from the dome-raising experi-
ment. A, The average number of suckers used to raise the dome
under conditions of high and low force. There is a trend for the
number of suckers used to increase with the force required. The
means are across animals and the error bars show standard devia-
tion. B, The portion of the arm, divided into rough thirds, used for
the raising task during the trial. In all trials all octopuses used the
middle portion of their arm regardless of the force required to
complete the task.

this difference is in the expected direction, a Student’s f-test
for paired samples did not show that this difference was
significant [fg = 1.99, P < 0.08].

DISCUSSION

Here I report both local and distant coordination
between suckers. Overall, the results of these studies are
consistent with the hypothesis of active arm-sucker co-
ordination and inconsistent with the model of exclusive re-
flexive sucker confrol. This conclusion is in agreement with
observations made in the course of earlier studies of arm
control and tactile discrimination in octopuses (Wells and
Wells 1957a, Rowell 1963, 1966, Altmann 1971, Wells 1978,
Gutfreund et al. 2006).

The patterns of sucker use in freely behaving octopuses
varied with the behavior in which the octopus was engaged.
Octopus, under conditions in which many suckers were in
contact with the surface, attached only a subset of suckers —
often a very small subset. A reflex-based mechanism, in
which suckers attach when stimulated by an available sur-
face, would show much greater proportions of suckers at-
tached on the types of surfaces used in these studies. This
suggests differential control across groups of suckers, at least
in the form of inhibition or excitation of attachment at
selected suckers, based on information about the overall
purpose and state of the ongoing behavior of the animal.

The details of the patterns of sucker use during “arm
walk”, “arm lift”, “octopus lift”, and other patterns not de-
scribed in this report suggest even richer forms of informa-
tion sharing along the arm to determine which suckers will
attach and which will remain free at any given moment. To
walk the arm perpendicular to its long axis, the suckers on
opposite sides of the arm must be differentially attached and
detached in opposing phases. Antagonistic pairs of extrinsic
muscles within individual suckers need to pull and push
with appropriate timing while the sucker is attached to sup-
ply the force necessary to move the arm. This and the other
observed patterns of inactivation in adjacent sucker pairs
demonstrate a side-to-side level of control of sucker attach-
ment within the arm. “Arm walk” would be facilitated with
information about the attachment state of each sucker avail-
able to the coordination centers, although a strictly feed-
forward system can be imagined. The “arm lift” and “octo-
pus lift” examples break the side-to-side coordination
patterns of the “arm walk” by allowing adjacent suckers to
attach simultaneously and suggest a different type of sucker
control. While the arm-walk patterns are widespread, the
sucker attachment patterns in “arm lift” and “octopus lift”
are local, presumably concentrating strong attachment
forces where they are needed to contribute to the ongoing

OCTOPUS SUCKER-ARM COORDINATION

21

behavior. The fact that one of these occurred near the base
of the arm and the other near the tip indicates that this
control is distributed along the arm and not localized, in this
case, to certain portions.

These observations raise the likelihood of many levels of
control and coordination of suckers: locally along the arm in
neighborhoods of many scales as well as potentially each of
those scales in conjunction with the central nervous system.

This absence of localization or, put more succinctly,
coordination of distant suckers, is consistent with the known
neuroanatomy of the arm. The basic unit (Fig. IB) is re-
peated for every sucker down the length of the arm. Above
each sucker the associated brachial ganglia is in a position to
share information supplied by its sucker with the adjacent
ganglia to support such inter-sucker coordination (Graziadei
1971). In a purely reflexive sucker control system, the nerve
running from the brachial ganglia to the sucker ganglion
(Fig. IB) would not be required. Only a local circuit from
the sucker ganglia to the sucker muscles and from the sucker
receptors to the sucker ganglia (Fig. IB) would suffice.
Taken together, our results suggest that the flow of infor-
mation between the brachial ganglia and their correspond-
|i ing suckers is not a one-way sensory channel to inform the
! arm ganglia and possibly the brain about the state ot a given
' sucker. It is instead a two-way channel in which usable in-
li formation flows from the adjacent suckers to each sucker
I, ganglion and/or sucker. The question of whether or not
.1 individual suckers are sometimes activated without intluenc-
: ing other suckers {i.e., through local connections involving a
i sensory motor arc from the sucker through the sucker gan-
I glion to the sucker muscles without involving brachial gan-
glionic connections) remains open,
ijl Specialization of sucker operation, to the extent that it
1 exists, is probably physical in nature, following the proximal
to distal taper of the arm. Given that larger suckers are
capable of supplying greater adhesion forces (Smith 1991)
and that larger suckers are found proximally on the arm
I (Voight 1993), it follows that tasks requiring greater attach-
! ment force will likely call for the use of the suckers toward
the arm base. Alternatively, they might require the use of
more than one sucker since their adhesive force is additive
(Smith 1991 ). In the dome-raising experiment, both of these
■ responses were observed under conditions that varied the
; required force. The results showed that as the force required
ij to raise the dome increased, so did the number of suckers
I used. While this result only approached a traditional signifi-
cance level of 0.05, the trend was in the direction to support
our hypothesis. It is worth noting that variability in force
I generated to hold the dome down was due to the placement
' of the electromagnets and that the change in required force
iwas only 28% above the force required to raise the dome

alone. It is likely that improvements of this method would
reduce experimental error and increase the statistical power
of the experimental design. The use of the portion of the
middle third of the arm in all trials was contrary to my a
priori expectations. The larger suckers of the proximal third
of the arm were able to reach the dome and I had expected
the animal to employ the larger suckers preferentially. The
result suggests that, perhaps, the greater flexibility of the
middle portion of the arm offered an advantage in complet-
ing this task: rather than forming single strong point of
attachment on the dome and pulling, the animal produced
attachments on the dome and the tank floor on both sides.
Thus a trade-off between attachment force and positioning
flexibility may occur. The period of probing, contact, and
varying attachments/detachments and reattachments that
preceded the raising of the dome is consistent with this
idea. Information about the force required to raise the dome
coming from the suckers could inform arm reposition-
ing. Together these results lead me to tentatively conclude
that information about sucker state is available to the arm-
control circuits to inform the guidance ol arm movements.
Given the limitations mentioned above, this conclusion
requires confirmation with a more powerful experimental
design.

A recent study (Byrne et al. 2006) reported that freely
moving individuals of Octopus vulgaris Cuvier, 1797 that
were engaged in visually-guided reaching tasks preferentially
make first contact with a target object using the middle of an
arm. Byrne et al. (2006) were concerned with issues of lat-
erality and arm choice and did not report in detail about the
use of suckers in the tasks. Their results implicate visual
guidance and, therefore, information from the central ner-
vous system, influencing the part of the arm that is applied
to the task and are consistent with the results reported here.
It is likely that vision also contributed to octopus perfor-
mance in the dome raising task. The coincidence of the
preference for the middle of the arm in different tasks and in
different species is interesting and offers a indication for
future studies investigating the relative importance of local
and central control mechanisms in the octopus.

In summary, the results of these studies demonstrate
that arm-sucker coordination exists almost certainly in the
form of descending information influencing sucker activity
and very likely in the reverse direction, with sucker state
influencing arm movement.

As Wells (1978) wrote over 30 years ago, motor prob-
lems in octopuses are rarely studied because of the difficulty
ot working with such flexible systems. Today we say that
these systems are “hyper-redundant” — ottering many routes
of achieving the same end — but we mean the same thing
(Walker et al. 2006). An octopus arm with 40 suckers (the

22

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

number of contiguous suckers typically observable in these
studies) is a subset of the real arm which typically has about
300 suckers (Voight 1993). Even if we limit the actions of the
octopus arm to ( 1 ) suckers that can only be attached or free
and (2) the sections of the arm that link each pair of suckers
to 1 pitch, 1 yaw, and 1 roll, we still find that such an arm
can be in -1.2 x lO'"* states, an enormous number of degrees
of freedom. Such an arm has the potential to interact effec-
tively with surfaces far more complex in shape and texture
than the smooth glass surfaces used in these studies. Indeed,
and the real octopus arm evolved to work on more complex
surfaces (i.e., extracting prey from crevices and burrows,
opening bivalve shells, and arranging middens in front of
den entrances). The studies reported here scratch the surface
of an enormous, unexplored domain of complex control
methods that we might understand if Wells’ challenge of
flexibility were pursued. The octopus proves it is possible.

ACKNOWLEDGMENTS

The research reported here was supported by DARPA
DSO BioDynotics Program (Subcontract 882-7558-203-
2004599 through Clemson University (Ian Walker, PI), Pro-
gram Officer: Morley Stone. I acknowledge the technical
assistance of Cai-Dong Hou, Huma Jahangir, Faiza Arshad,
Michael Kuba, Sasha Sorr, and Fabian Suarez of the BCR lab
in performing and analyzing these studies. I am grateful to
Michael Kuba of the BCR lab; Ruth Byrne and Jean Boal of
Millersville University; Robyn Crook and Jennifer Basil of
Brooklyn College; and Roger Hanlon and Phil Alatalo of the
Marine Biological Laboratory for advice and thoughtful dis-
cussions of octopus behavior and observational methodol-
ogy. I thank Jennifer Basil of Brooklyn College for comments
on the manuscript as well as two anonymous reviewers for
their encouragement and thoughtful comments which im-
proved the manuscript.

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Accepted: 30 luly 2007

Amer. Malac. Bull 24: 25-29

Short-term pain for long-term gain: A hypothetical role for the mantle in coleoid
cephalopod circulation"^

Alison J. King* and Shelley A. Adamo^

* Department of Biology, Life Sciences Centre, Dalhousie University, 1355 Oxford Street, Halifax, Nova Scotia B3H 4J1, Canada,
ajaneking@gmail.com

^Department of Psychology, Life Sciences Centre, Dalhousie University, 1355 Oxford Street, Halifax, Nova Scotia B3H 4J1, Canada,
sadamo@dal.ca

Abstract: Mantle cavity pressures are frequently hypothesized to drive venous return in the high-output circulatoiy systems of coleoid
cephalopods. However, studies using non-invasive, imaging ultrasound on resting cuttlefish {Sepia ofllcimilis Linnaeus, 1758) conclude that
mantle cavity pressures do not drive venous return. Interestingly, data from cuttlefish showing sustained mantle hyperinflation indicate
instead that forces within the mantle’s tissues could aid circulation. We hypothesize that alternating contractions of the radial and circular
mantle muscles create a bellows-like effect on mantle capillaries. This effect could be propulsive during normal ventilation and jetting but
could stop circulation when the cuttlefish is engaged in sustained mantle hyperinflation. Sustained mantle hyperinflation accompanies some
behaviors, for example the Deimatic Display. The metabolic consequences of strangulated circulation might limit the duration ot these
behaviors.

Key words: cardiovascular dynamics, peripheral circulation, mantle cavity pressure. Sepia officinalis, veins

The circulatory system of coleoid cephalopods is closed
and has two separate loops: one though the gills, powered by
the two branchial hearts, and one through the body, pow-
ered by the single systemic heart (Tompsett 1939). During
exercise, increases in systemic heart rate and stroke volume,
combined with increasing arterial pressure (Wells and Smith
1987), result in the work and power output of systemic heart
tissue rivaling or exceeding those of mammals (Shadwick et
al. 1990, O’Dor and Webber 1991). The coleoid hearts are
generally considered insufficient to generate such power out-
put and many authors ascribe an accessory circulatory func-
tion to the contractions of the coleoid mantle. The mantle
encloses most organs (including the hearts, large veins, and
large arteries) in a space called the mantle cavity (Tompsett
1939). At rest, the mantle expands and contracts to move
water through the mantle cavity and over the gills. Maxi-
mum mantle cavity pressures in resting cuttlefish are around
0.16 kPa (King, pers. obs.). During jetting, the muscular
mantle contracts forcefully, increasing maximum mantle
cavity pressures by over an order of magnitude to at least 5.5
kPa in cuttlefish (O’Dor and Webber 1991 ), 8 kPa in octo-
pods (Wells et al. 1987), and 6.6 kPa in squid (O’Dor and
Webber 1991). Could the forces generated by the muscular
mantle help circulate the large amounts of blood needed
during exercise?

One model suggests that the hearts should contract at
the same time as the mantle. The resulting increase in mantle
cavity pressure could augment arterial pressure generated by
the heart, driving blood to the low-pressure periphery out-
side the mantle cavity. Additionally, the slightly negative
mantle cavity pressures created during mantle expansion
could help to pull venous blood back into the mantle cavity
from the head and arms and toward the hearts. However, a
1:1 ratio of heart to mantle contractions is not usually ob-
served in octopods (Wells 1978, Smith 1982), squid (Shad-
wick et al. 1990), or cuttlefish (Chichery 1980, King et al.
2005, King and Adamo 2006), even during jetting. More-
over, the ratio of contractions between the heart and the
mantle can change over time within the same octopus (|o-
hansen and Martin 1962, Wells 1978) or cuttlefish (King et
al. 2005). It would seem that heart contractions are not tied
to mantle contractions in any fixed way.

In a different model, mantle cavity pressures could drive
blood flow in the veins, instead of by helping the heart.
Pressures have been measured in the vena cava cephalica
(probably the lateral vena cava of King et al. 2005) and
efferent gill vessel of the octopus Enteroctopus dolleiui
(Wtilker, 1910) (Johansen and Martin 1962) and the squid
Loligo pcaleii (Lesueur, 1821) (Bourne 1982). In these ves-
sels, there are two overlaid pressure pulses: one that is rela-

From the symposium “Cephalopods: A behavioral perspective” presented at the joint meeting of the American Malacological Society and
Western Society of Malacologists, held 29 July to 3 August 2006 in Seattle, Washington.

25

26

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

lively slow and large and one that is relatively fast and small
(Fig. lA). The slow, large pulse (Fig. IB) is due to ventilatory
movements (Johansen and Martin 1962). The fast, small
pulse (Fig. 1C) is due to venous contraction (King et al
2005). Probably due to the large size of the ventilatory pulse,
many have suggested that the mantle cavity pressure flattens
large, thin -walled veins such as the venae cavae and the
efferent branchial vessels. However, for the veins to flatten,
the compression-resistant blood inside them would have to
move into the adjacent vasculature. To accomplish this,
mantle contractions would have to generate pressure differ-
ences between the anterior and lateral venae cavae or be-
tween the efferent branchial vessel and the systemic heart.
Theoretically, the pressures created in the mantle cavity,
while large at times, are applied equally to all veins within
the cavity, and therefore would not create the pressure dif-

A.

ferences required to move blood between vessels. So, while
the contractions of the mantle create absolute pressure
changes in the vessels, they are unlikely to be propulsive
because they are unlikely to change the relative pressure
between the vessels.

Empirical data do not support the hypothesis that
mantle cavity pressure compresses the veins. If mantle cavity
pressures did compress the veins, we would have two expec-
tations: ( 1 ) the veins would contract at the same rate as the
mantle and (2) the veins would collapse as a unit along their
length as mantle pressure increased. Neither of these occurs
in experimental observations using imaging ultrasound.
First, the lateral venae cavae and efferent branchial vessels do
not contract at the same rate as the mantle and, therefore,
could not be compressed by it (King et al. 2005). Second, the
only large vein that does contract at the same rate as the
mantle in resting cuttlefish, namely the
anterior vena cava, contracts peristal-
tically and not as a unit along its length
(King et al 2005). Furthermore, this
vein’s contractions become unsyn-
chronized with mantle contractions in
a mating female; the vein’s contrac-
tions remain steady and slow during
the rapid and vigorous mantle con-
tractions that accompany the place-
ment ot the male’s arm in her mantle
(King 2005). The anterior vena cava is
evidently able to contract indepen-
dently of the pressures generated in the
mantle cavity. It would seem that pres-
sures in the mantle cavity do not com-
press the large veins.

Not only are contractions of
mantle not propulsive in the veins but
also they could impede venous blood
flow from the head and arm veins into
the anterior vena cava. Increased
mantle cavity pressure would increase
the pressure in the anterior vena cava
without increasing pressure in the ve-
nous spaces of the head and arms, thus
inhibiting the forward flow ot blood
(Wells et al 1987).

We have found no experimental
evidence that supports a role for in-
creasing mantle cavity pressure in ve-
nous return. However, some experi-
mental evidence suggests that mantle
tissue may play a role in circulation. In
this paper, we synthesize this evidence
and present a new hypothetical role for

Figure 1. A, Pressure trace from the efferent branchial vessel measured in vivo by Johansen
and Martin ( 1962). It consists of two superimposed pressure pulses of different frequencies:
a slow and large pulse caused by ventilatory movements (B) and a faster and smaller pulse
caused by venous contractions (C). The base pressure of 1.5 kPa is arbitrarily assigned to the
pressure trace in C.

MANTLE ROLE IN CIRCULATION

27

the mantle in circulation. We also present additional indirect
experimental evidence that supports our hypothesis.

HYPOTHETICAL ROLE FOR MANTLE TISSUE

The mantle may aid circulation by creating pressure
within its own tissues, instead of by creating pressure in the
mantle cavity. This hyp>othesis was spurred by experimental
results from cuttlefish (Sepia ojfidnaUs Linnaeus, 1758).
When cuttlefish are exposed to a sudden visual stimulus,
heart rates drop and mantles hyperinflate for several seconds
(King and Adamo 2006). Mantle hyperinflation is an expan-
sion greater than during normal ventilation (King and
Adamo 2006). Interestingly, a decrease in heart rate occurred
almost simultaneously and proportionally to the mantle’s
hyperinflation (King and Adamo 2006). This coincidence
initiated our interest in the connection between mantle tis-
sue and circulation. To explain the connection we draw, we
first present a summary of mantle tissue structure.

Cuttlefish mantle tissue is composed almost exclusively
of two muscle types, radial and circular muscles (Fig. 2, Bone
et al. 1994). Radial muscles contract to expand the mantle
cavity during ventilation (Bone et al. 1994). Circular muscle
contraction constricts the mantle cavity only during heavy
ventilation and jetting (Bone et al 1994). Both sets of
muscles are partially antagonized by variously arranged col-

Constant length

Figure 2. The bands of radial and circular muscles in the decapod
mantle and the plane along which most capillaries are aligned.
Radial muscles contract to thin mantle tissue and expand the
mantle cavity. Circular muscles contract to thicken mantle tissue
and constrict the mantle cavity. Collagen tunics ensure that the
mantle does not change length during mantle contraction and ex-
pansion. Most capillaries are perpendicular to the radial muscles
and parallel to the circular muscles. After Shadwick ( 1994).

lagen tunics and by each other during all but resting venti-
lation (Bone et al. 1994). The collagen tunics keep the de-
capod mantle the same length so that contractions of the
different muscles translate only into expansion and constric-
tion of the mantle.

Most capillaries in the mantle are oriented perpendicu-
larly to the radial muscles (Fig. 2, Bone et al. 1981). We
suggest that they could be compressed by radial muscle con-
traction (Fig. 3A). Conversely, the capillaries are oriented
parallel to the circular muscles (Bone et al. 1981 ), and there-
fore, we suggest, would not be compressed by circular
muscle contraction, and in fact could be expanded by it (Fig.
3C). The radial muscles are always used to expand the
mantle, albeit minimally, during resting ventilation (Bone et
al. 1994). In our model, the radial muscles always alternate
between creating a gentle force pushing blood out of the
capillaries (radial muscle contraction. Fig. 3A) and creating
a vacuum in the capillaries that draws blood in (radial
muscle relaxation. Fig. 3B). Mantle expansion could thus
drive the flow of blood from the mantle into the veins, while
mantle constriction could aid the flow of blood from the
arteries into the mantle. Even during rest, this would help
power peripheral circulation. We hypothesize that this effect
would be magnified during jetting when radial muscles con-
tract more vigorously, expelling more blood, and when the
circular muscles become active, possibly contributing to cap-
illary dilation (Fig. 3C). The alternating contractions of the
radial and circular muscles could create greater peripheral
pumping forces to help power circulation during exercise.

More experiments are needed to test this hypothesis.
For example, it is unclear whether changes in capillary length
would offset changes in their diameter. Certainly, if capil-
laries were completely occluded by radial muscle contrac-
tion, no change in length would compensate. For anything
other than complete occlusion, the situation is less clear.
Unfortunately, almost no direct observations have been
made on blood flow in the periphery which, considering the
strength and size of the mantle, hinders our understanding
of integrated cardiovascular dynamics (Bourne 1984). Nev-
ertheless, several indirect lines of data support our hypoth-
esis that mantle muscle contraction influences peripheral
circulation.

After exposure to a sudden visual stimulus, cuttlefish
and octopods hyperinflate their mantles for several seconds,
and their hearts slow or stop beating (King and Adamo
2006). Mantle hyperinflation is achieved by forceful contrac-
tion of the radial muscles (Bone et al. 1994). It our hypoth-
esis were correct, we would expect mantle hyperinflation to
dramatically increase peripheral resistance, causing aortic
pressure to remain elevated despite slowing or stopping of
the heart. Elevated aortic pressure is in fact maintained in

28

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

A. B. C.

Constant length

Figure 3. A hypothetical cross-section of mantle tissue during radial muscle contraction (A), relaxation of both muscle sets (B), and circular
muscle contraction (C). This cross-section is along the same plane that shows the circular muscles in Fig. 2. The mantle cavity is above the
blocks, the open water below. The vertical white lines represent the radial muscles and the white circles represent the circular muscles. Active
muscles are represented by heavier lines and circles. The shaded circles represent the capillaries. Capillaries are hypothetically occluded
during radial muscle contraction (A) and hypothetically expanded during circular muscle contraction (C). This could propel blood during
normal ventilation and jetting.

Enteroctopus dofleini during mantle hyperinflation and car-
diac arrest (lohansen and Martin 1962). Furthermore, if our
hypothesis were true, we would expect that the veins would
fill during cardiac slowing and stopping and mantle hyper-
inflation, the blood originating from the compressed capil-
laries in the mantle. The veins and systemic heart in fact do
fill at this time (King 2005).

Further evidence is available from jetting cephalopods.
Brief mantle hyperinflation starts every jetting cycle. The
heartbeat of octopods is interrupted during jetting, although
it was not noted whether this is during mantle hyperinflation
or water expulsion (Johansen and Martin 1962, Wells et al.
1987). During hyperinflation, the radial muscles might com-
press the mantle capillaries, greatly reducing blood flow
through the mantle. The cuttlefish or octopus heart slows or
stops at this time, perhaps to avoid dangerous pressure in-
creases in the head and viscera, where blood can still flow. By
contrast, when Octopus vulgaris Cuvier, 1797 moves using its
arms, cardiac interruption is not seen and in fact heart rate
increases (Wells et al. 1987). It seems that movement does
not affect cardiac function unless the movement is achieved
using mantle hyperinflation. Interestingly, the mean resis-
tance of the peripheral vessels remains constant or even de-
creases during mantle contractions (exercise) in octopods
(Wells et al. 1987) and squid (Shadwick et al. 1990). The
contractions of the circular muscles during exercise might
dilate mantle capillaries, resulting in periods of lowered re-
sistance between the periods of increased resistance associ-
ated with radial muscle contraction. The alternating high
and low resistance could result in no change or a drop in the
mean resistance. Our hypothesis is, thus, consistent with
most existing indirect evidence in the literature.

CONCLUSIONS

Currently, we do not have enough data to understand
the effects of mantle contraction on the circulation of co-
leoid cephalopods. The complete picture should integrate
the effects of fluctuating pressures in the arteries and veins
relative to the periphery during mantle contractions, the
effects of the contractions of circular and radial mantle
muscles on mantle capillaries, and the effects of the con-
tracting veins. Differences in lifestyle and anatomy may
mean this integrated picture differs from one group of co-
leoid cephalopods to the next. Also to be integrated into the
complete circulatory picture are the effects of the accessory
vasoconstricting organs found in both cuttlefish and squid
on the inside surface of the mantle, around the posterior
pallial arteries and veins (Alexandrowicz 1962). Their struc-
ture has been well described, but their function is not clear,
including whether they contract during contractions of the
circular (mantle constriction) or of the radial (mantle ex-
pansion) mantle muscles, why they appear on the posterior
but not the anterior pallial vessels, and why they do not
appear in octopods at all. What is clear is that further re-
search is needed on these accessory vasoconstricting organs
and the other factors affecting peripheral circulation.

With new technology being adopted from medicine, the
area of integrated cephalopod cardiovascular dynamics
promises to be interesting and rewarding in the future. To
spur further research, we present a hypothesis for verifica-
tion— that the mantle could contribute to circulation during
ventilation and jetting by alternately compressing and ex-
panding the capillaries in its own tissues. However, strong,
maintained contractions of the radial muscles (sustained hy-

29

MANTLE ROLE IN CIRCULATION

perinflation) may strangulate blood flow (King and Adamo
2006). What might be useful during normal ventilation and
locomotion might, thus, be non-adaptive in some acute
cases such as the sustained hyperinflation exhibited after a
sudden stimulus. This hypothesis is supported by indirect
evidence from the literature but rec]uires further investiga-
tion. If our hypothesis were supported by direct evidence, it
would have consec]uences for the behaviors that involve sus-
tained mantle hyperinflation, such as the Deimatic Display.
The duration of such behaviors may be limited by how long
a given animal can forgo normal circulation.

ACKNOWLEDGMENTS

This work was supported by the Natural Sciences and
Engineering Research Council ot Canada (NSERC) (S. A.
Adamo and A. J. King) and the Lett fund (A. L King). We
thank Dr. Ron O’Dor for valuable discussions.

LITERATURE CITED

Alexandrowicz, I. S. 1962. An accessory organ of the circulatoiy
system in Sepia and Loligo. Journal of the Marine Biological
Association of the United Kingdom 42: 405-418.

Bone, Q., A. Pulsford, and A. D. Chubb. 1981. Squid mantle
muscle. Journal of the Marine Biological Association of the
United Kingdom 61: 327-342.

Bone, Q., E. Brown, and G. Travers. 1994. On the respiratory flow
in the cuttlefish Sepia officinalis. Journal of Experimental Biol-
ogy 194: 153-165.

Bourne, G. B. 1982. Blood pressure in the squid, Loligo pealei.
Comparative Biochemistry and Physiology 72A: 23-27.

Bourne, G. B. 1984. Pressure-flow relationships in the perfused
post-systemic circulation of the squid, Loligo pealei. Compara-
tive Biochemistry and Physiology 78A: 307-313.

Chichery, R. 1980. Etude du comportement moteur de la seiche Sepia
officinalis I. (Mollusque cephalopode): Approches neurophysi-
ologique et neuropharmacologique. Ph.D. Dissertation,
L’Universite de Caen, Caen, France.

lohansen, K. and A. W. Martin. 1962. Circulation in the cephalo-
pod. Octopus dofJeini. Comparative Biochemistry and Physiol-
ogy 5: 161-176.

King, A. 1. 2005. Coleoid Cephalopod Strategies for Powering Venous
Return, Responding to Sudden Visual Stimuli and Regulating
Male Agonistic Behaviour. Ph.D. Dissertation, Dalhousie Uni-
versity, Halifax, Nova Scotia, Canada.

King, A. J. and S. A. Adamo. 2006. The ventilatory, cardiac and
behavioural responses of resting cuttlefish (Sepia officinalis L. )
to sudden visual stimuli. Journal of Experimental Biology 209:
1101-1111.

King, A. L, S. M. Henderson, M. H. Schmidt, A. G. Cole, and S. A.
Adamo. 2005. Using ultrasound to understand vascular and

mantle contributions to venous return in the cephalopod
Sepia officinalis Linnaeus. Journal of Experimental Biology 208:
2071-2082.

O’Dor, R. K. and D. M. Webber. 1991. Invertebrate athletes: Trade-
offs between transport efficiency and power density in ceph-
alopod evolution. Journal of Experimental Biology 160: 93-1 12.

Shadwick, R. E. 1994. Mechanical organization of the mantle and
circulatory system of cephalopods. In: H. O. Portner, R. K.
O’Dor, and D. L. Macmillan, eds.. Physiology of Cephalopod
Molluscs. Gordon and Breach, Basel, Switzerland. Pp. 69-85.

Shadwick, R. E., R. K. O’Dor, and I. M. Gosline. 1990. Respiratory
and cardiac function during exercise in squid. Canadian Jour-
nal of Zoology 68: 792-798.

Smith, P. I. S. 1982. The contribution of the branchial heart to the
accessory branchial pump in the Octopoda. Journal of Experi-
mental Biology 98: 229-237.

Tompsett, D. H. 1939. Sepia. University Press of Liverpool, Liver-
pool.

Wells, M. |. 1978. Octopus: Physiology and Behaviour of an Ad-
vanced Invertebrate. Chapman and Hall, London.

Wells, M. J. and P. ). S. Smith. 1987. The performance of the
octopus circulatory system: A triumph of engineering over
design. Experientia 43: 487-499.

Wells, M. I., G. G. Duthie, D. F. Houlihan, P. I. S. Smith, and ].
Wells. 1987. Blood flow and pressure changes in exercising
octopuses (Octopus vulgaris). Journal of Experimental Biology
131: 175-187.

Submitted: 10 October 2006; accepted: 27 lune 2007;

final corrections received: 15 November 2007

Amer. Maine. Bull 24: 31-41

A new approach to octopuses’ body pattern analysis: A framework for taxonomy
and behavioral studies'^

Tatiana S. Leite* and Jennifer A. Mather^

' Universidade Federal do Rio Grande do Norte, Departamento de Oceanografia e Limnologia, Via Costeira s/n, Bairro dc Mae Luiza,
Natal/RN, Brazil CEP 59014-100, leite_ts@yahoo.com.br

^ University of Lethbridge, Department of Psychology, 4401 Lhiiversity Drive, Lethbridge, Alberta TIK 3M4, Canada, mather@uleth.ca

Abstract: We systematically analyzed octopus body patterns, based on locations of chromatophore nerve projection, using a proposed new
species in the Octopus vulgaris Cuvier, 1797 complex. Octopus insularis Leite and Haimovici, 2008. Although some taxonomic studies have
used body patterns as characters to describe octopus species, a systematic analysis would provide detailed descriptions to assist reliable
comparisons among species. This approach also links body patterns, behaviors, and underlying physiology of the chromatophore system.
Body patterns were characterized by percent occurrence, areas of skin, and number of components in each. To verify the distribution of
chromatic components, skin patterns, and colors among areas of the body, we ran a cluster analysis on occurrence of the components. We
identified a total of 16 chromatic, 5 texture, 9 skin units, 6 colors, and 9 chronic body patterns. The cluster analysis showed twelve distinct
skin areas of the components’ distribution (expressive fields). Smaller fields were found in areas with complex patterns, especially around
the eyes, while larger ones were found in areas with simple patterns. These findings differentiate between morphological and physiological
units of the display system. The strong degree of similarity among photographs also supports previous taxonomic studies that pointed to
morphological similarity within this species from the oceanic islands of northeastern Brazil.

Key words: Octopus insulaiis, behavior

The complex and changing appearance of cephalopod
molluscs offers a challenge to the biologist, both in descrip-
tion (Packard and Sanders 1969, Hanlon and Messenger
1988) and in linkage of its body display to specific behaviors
(Adamo and Hanlon 1996, Hanlon et al. 1999a, Mather and
Mather 2004, Adamo et al. 2006). Many authors (Packard
and Sanders 1969, Packard and Hochberg 1977, Hanlon and
Hixon 1980, Hanlon and Messenger 1988, Roper and Hoch-
berg 1988, Mather and Mather 1994, Hanlon et al. 1999b)
have constructed a repertoire of the body pattern behavior
either tor one species or to discriminate among species. The
problem of species identity is particularly difficult in the
Octopus vulgaris Cuvier, 1797 species complex (Mangold and
Hochberg 1991, Mangold 1998, Sbller et al. 2000, Warlike et
al. 2004). Morphological and morphometric analyses have
suggested the occurrence of Octopus insularis Leite and Hai-
movici, 2008 (Leite et al, 2008), a cryptic species of this
complex from the northeast of Brazil (Leite and Haimovici
2006, Leite 2007), and cataloging body patterns may be a
useful addition to separate it from other species and to com-
pare conserved characters (Hanlon 1988, Hanlon and Mes-
senger 1996). Such information can be gained from careful
analysis of patterns taken from underwater photographs and

film. Recent analysis of films of Sepia ojftciualis Linnaeus,
1758 has used Bayesian probability to identify pattern
(Crook et al. 2002), Independent Component Analysis to
delineate the basic components (Anderson et al. 2003), and
Principal Components Analysis to look for camouflage units
(Kelman et al. 2007). Multivariate analyses can also be used
to assess symmetry of body pattern expression (Langridge
2006) to understand camouflage (Kelman et al. 2007) or to
aid in species differentiation, as in the present study.

Body pattern is composed of chromatic, textural, and
postural components that combine to produce the final ap-
pearance of the individual (Hanlon 1988). Body patterns are
controlled at several levels, and chromatophores are the
most important elements that define chromatic compo-
nents. The chromatophores, organized on the body surface
into groups designated as “morphological” and “physiologi-
cal” units, are the smallest units (Packard 1974). The mor-
phological unit is a static arrangement of chromatophore
density in the skin, such as patches and grooves, while the
physiological units are a dynamic event, resulting from neu-
ral activation of a particular set of nerves in a specific area
(Messenger 2001). These areas are called “motor fields” or
“chromatophoric fields” (Packard 1974, Messenger 2001)

* From the symposium “Cephalopods: A behavioral perspective” presented at the joint meeting of the American Malacological Society and
Western Society of Malacologists, held 29 July to 3 August 2006 in Seattle, Washington.

31

32

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

and are usually irregular with overlapping boundaries (Pack-
ard 1974). These chromatophoric fields depend both on the
distribution of chromatophores in the skin and organization
of their neuromotor control (Messenger 2001).

Because chromatophores are innervated directly from
the brain, it should be possible to map the projection of the
chromatophore nerves onto the body surface to describe the
larger units that Messenger (2001) calls “chromatomotor
fields.” Froesch ( 1973) found 20 areas of projection of chro-
matophore nerves on the body surface when he made selec-
tive lesions in Octopus vulgaris, and Biihler et al. (1975)
divided the mantle into 23 smaller projection areas, based on
40 nerves leaving the stellate ganglion.

We believe that using photos of living octopus to ana-
lyze body patterns systematically, based on locations of chro-
matophore nerve projection on the body surface would
make it possible to link body pattern components to areas of
nerve projection, as well as linkages to behavior states. Body
patterns were characterized in percentage of occurrence, lo-
cations and numbers of components, and cluster analyses
were used to identify groups of similarities among photo-
graphs and among octopus body surfaces.

MATERIALS AND METHODS

luvenile and adult specimens of Octopus iusularis from
the Fernando de Noronha Islands, a northeastern Brazilian
oceanic archipelago (03°51'S, 32°25'W) located 345 km
northeast of Cape San Roque, Brazil, were photographed
from 1999 to 2005, during walking trips near shore, snor-
keling, and scuba diving. They were found at a depth of 0.1
to 25 m, in areas of rock, rubble, and small sand patches,
with water temperature ranging from 23 to 27°C. We ob-

tained 365 photographs with a digital Canon Power Shot
and Sony S50, both 5.0 megapixel, from 93 octopuses.

Conspicuous characteristics of body patterns, behavior,
and habitat were used to exclude photographs of three other
species of Octopodidae from the analysis: Octopus hum-
melincki Adam, 1936 was identified by the ocellus below the
eyes; Octopus defilippi Verany, 1851, found only on sand and
mud in the Rocas atoll, was identified by the white-cream
color; and Callistoctopus macropus Risso, 1826, a nocturnal
species, could be easily identified by conspicuous white spots
all over the body. All photographs without the characteristics
cited above but with characteristics common to Octopus vul-
garis (Nesis 1987, Voss and Toll 1998) were classified as
Octopus iusularis. A subset of photographs was chosen based
on an a priori assessment of image quality, definition, por-
tion of the body visible, and body pattern. We chose 65
photographs from 23 animals that showed at least three
areas of the body (e.^., mantle, head, and at least one arm),
were of high quality, and were not the same pattern, date,
and individual. We determined different behaviors, body
patterns, and their relationships based on Packard and Sand-
ers (1971), Roper and Hochberg (1988), and Hanlon et al.
(1999a), plus the components (chromatic, textural, colors)
and skin patterns present in each photograph (Tables 1-2).
The components and skin pattern were catalogued based on
Packard and Sanders (1969), Roper and Hochberg (1988),
and Mather and Mather (1994) (Table 2). The colors were
the five cited by Messenger (2001) for O. vulgaris, plus Blue-
Green, derived from the iridophores (Florey 1966, 1969,
Messenger 1974, Cooper et al. 1990).

Presence of components and colors throughout the body
and within each body pattern

We analyzed the photographs for presence or absence of
each chromatic and textural component, color, and skin

Table 1. Behavior states and the chronic body patterns identified in photographs of Octopus iusularis from Fernando de Noronha, Brazil.

Definition

Acronyms

References

Behavior states

Inside Den

D

Outside Den

OUT

Hunting

H

Swimming

S

Mating

M

Body patterns

Blotch-light blotch and spots spread throughout

BL

see chronic Mottle pattern in Hanlon et al. (1999)

more than 50% of the skin surface

Dymantic

D

Packard and Sanders (1971)

Dorsal Light-Ventral Blue-Green

DL-VBG

see counter-shading pattern in Hanlon et al. (1999)

Mottle

M

see Packard (1969)

Uniform Dark

UD

see Flush in Roper and Hochberg (1988)

Flamboyant

F

variation of Packard and Sanders (1971)

OCTOPUS BODY PATTERNS

33

Table 2. Components (chromatic, texture, and skin pattern) de-
termined from photographs of Octopus insularis from Fernando de
Noronha, Brazil (based on Packard and Sanders 1969, 1971,
Mather and Mather 1994). R, restricted to specific body area.

Components Acronym

Chromatic Alternate bands (light/dark) ABA

Alternate light/dark

around the eye (R) ABE

Brown-yellow blotch BB

Blue-green around the eye (R) BCE

Black hood BH

Dark bar across the eye (R) DBE

Dark blotch above eye (R) DBA

Dark spots DS

Longitudinal dark strip LDS

Light blotches LB

Purple around suckers (R) PS

Red bar across the eye (R) RBE

White bar across eye (R) WBE

White spots WS

White dots WD

White V(R) WV

Textural Big papillae (>1 cm) BP

Small papillae (<1 cm) SP

Smooth skin S

Rugose skin R

Textured skin (skin with a large

number of small papillae) T

Skin pattern Alternate bands AB

Bars BR

Blotch BL

Dark smooth DS

Light smooth LS

Reticulate dark DR

Reticulate light LR

Reticulate mixed (dark and light) R

Red/white reticulate RWR

Colors Yellow Y

Red R

Brown B

Black BL

White W

Blue-green BG

pattern on forty-nine parts of the body (Fig. lA). These body
parts were delineated based on the projection of chromato-
phore nerves onto the body surface (Froesch 1973, Btihler et
al. 1975), plus additional divisions in the areas that were
much too large, such as mantle and arms (Fig. lA). Classi-
fication of the arms position followed Mather (1998), with
the right and left arms numbered: D\ 2"*^, 3'^'^, and (cor-
responding to the areas 7, 8, 9, and 10). Within a single arm.

the proximal areas were categorized as 1 and 2 anci the distal
areas, 3 and 4.

To verify occurrence and area of each component and
color throughout the body, we calculated: (the number of
areas in which a component appeared in the photograph
analyzed)/(total areas of body analyzed in the photograph) x
100. For exampile, the Dark bar in the eye (DBE) occurred in
two areas of the body and we analyzed 30 areas in this
photograph, so the occurrence for this component would be
(2/30) X 100 = 6.7% (see Appendix 1 for details). We con-
sidered that components with 80% or more occurrences in
an area could be considered typical for this area, and with
50-80% could be considered common for it.

To verify the distribution of chromatic components,
skin patterns, and colors among the areas of the body, we
ran a cluster analysis based on occurrence of all these com-
ponents, except Brown and White, throughout the areas.
These two colors were not considered because they were
found throughout the body.

Typical and common components for the body patterns
and species

To verify the degree of relationship that each compo-
nent, skin pattern, and color had with each body pattern, we
determined the mean occurrence of each component for the
main body patterns: Mottle, Blotch, Uniform Dark, Dyman-
tic, and Dorsal Light-Ventral Blue-Green. We calculated:
(the times that each component appeared in the photo-
graphs within a distinct body pattern) /(total of photograph
analyzed with this body pattern) x 100. For example the
DBE appeared in 5 of 10 photographs classified as Uniform
Dark, so the occurrence for this component would be 50%
in this body pattern. If a component appeared only in areas
of the body that were not present in the photograph ana-
lyzed, the photograph was not included in the total.

We considered that components with 80% or more oc-
currences in all body patterns could be considered “typical”
for the species and with 50-80% could be considered “com-
mon” for this species (Appendix 2). The components with
>80% or more only in one body pattern were considered
typical for them; those with 50-80% were considered common.

Similarity among photographs

To determine how many groups of animals could be
differentiated from the photographs, a cluster analysis wtis
run, taking into account the presence and degree of expres-
sion of each component throughout the parts of body. A
cluster analysis encompassed a number of different classifi-
cation algorithms to join together objects (photographs and
skin areas) in successively larger clusters, using some mea-
sure of similarity or distance (Statistic Program Contents
2000). A typical result of this type of clustering was a hier-

34

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

Figure 1. Areas of the body of Octopus insularis onto which skin patterns were projected: A,
Areas of protection of chromatophore nerves to the skin (based on Biihler et al. 1975, Froesch
1973). B, Areas of common pattern expression, as determined by cluster analysis of occur-
rence of the pattern components.

archical tree that put together the cases that had similar
indices and separated the ones with different indices.

RESULTS

We found sixteen chromatic components, five textures,
nine skin units, six colors, and six chronic body patterns in
five different behavioral states (Outside den, Inside den. Swim-
ming, Mating, and Hunting) (Tables 1-2). Although many
different body patterns were found in each behavioral state,
some of them were more common than others, for instance.
Mottle (Fig. 2A) was common in Hunting (60.9%) (Table 3).

Presence of components and colors throughout the body
and within each body pattern

Seven of the chromatic components were restricted to
specific areas of the body: ( 1 ) the White V at the proximal
part of the arms IR and IL (area 7D1) (Fig. 2A); (2) blue
green around the eyes (areas 3, 4D, and 6D) (Fig. 2A);

( 3 ) alternate bars on the distal parts of
the dorsal arms (areas 3 and 4) (Fig.
2A); (4) bar across the eye (areas 4D, 5,
and 6D) (Fig. 2B), usually dark but
sometimes red or white; (5) alternate
light/dark around the eye (areas 3, 4D,
and 6D) (Fig. 2B); (6) Dark blotch
above the eye (area 2) (Fig. 2C); and
(7) Purple around suckers (Fig. 2D).
Components described in more than
one area around the eye, such as DBF
and BGE, could vary their location to
one, two, or three of the areas at a
given moment. For example, DBF
could be present in only 4D, in 4D and
5 together, or in three areas (4D, 5,
and 6D) at the same time.

Among textural components, the
Small Papillae (Fig. 2F) were spread
throughout the body, while Big Papil-
lae (Fig. 2F) occurred disproportion-
ately, but not commonly, on dorsal
mantle (ID, 16%) or at proximal-
dorsal area of the arms IR and IF
(7D1, 23%). The skin pattern Light
Smooth was typical of ventral areas of
the mantle, with 93% occurrence, and
Red /White Reticulate on the ventral
arms, with >90%.

The colors Brown and White were
widespread throughout the body,
while all others colors showed some
concentration in different parts of the
body. Blue-Green was typical in ven-
tral mantle (100%); Red was typical to ventral parts of the
arms, the edge of suckers (both with >80%), and common to
eyes (>60%). Yellow was common in areas around the eyes
060%).

Some components could occur in different proportions
throughout the body across distinct Body Patterns. For ex-
ample; Light Blotch (LB) appeared in 40.9% of the body
areas in Blotch, while it appeared in just 4.2% of the areas in
Mottle. White Spot was the most common component of the
areas in Mottle (57.7%), while in Dymantic it appeared just
in 9.9% (see Appendix 1 ).

Looking for clusters among the body areas

Cluster analysis of occurrence of the components
throughout the areas showed twelve distinct groups (Fig. IB,
in Roman numerals and Fig. 3), seven composed of single
nerve projection areas (2, 3, 4D, 5, 6D, 4D, and suckers) and

OCTOPUS BODY PATTERNS

35

Figure 2. Six body patterns, seven chromatic, and two textural components identified in
photographs of Octopus iiisularis from Fernando de Noronha, Brazil: A, Mottle; B, Blotch; C,
Dymantic; D, Dorsal Light-Ventral Blue-Green; E, Uniform dark; and F, Flamboyant. WV,
white V spot in the middle of the dorsal head; BGE, Blue green around the eyes; ABA,
Alternate bars on arm; DBE, Dark bar across the eye; ABE, Alternate bars across the eye; BH,
Black hood on mantle; PS, Purple around suckers; BP, Big papillae; and SP, Small papillae.

dorsal arms, including 7D1, p>lus dor-
sal mantle ID (7D2, 8D1, 8D2, 9D1,
9D2, and ID), and among the ventral
mantle and ventral psarts of D\ 2""\
and arms, plus the dorsal part of
the 4“’ arms (IV, 7V, 8V, 9V, lOD, and
lOV). The areas lODl and 10D2 were
not considered in the analysis because
it was not ptossible to see them in any
photograpih. Dorsal areas showed a
larger number of components (7-10)
than ventral arms and mantle did (4).

Typical components, skin pattern,
and colors for the body patterns
and species

The analysis of occurrence of the
chromatic component, skin pattern,
and colors for the five common Body
Patterns (Mottle, Blotch, Uniform
dark, Dymantic, and Dorsal Light-
Ventral Blue-Green) allowed us to
show that some components were
typical to the species or the body pat-
tern. Typical components of the spe-
cies were Purple Edge on Suckers
(<87.5%), Dark Bar Across the Eye
(85%), and Red/White Reticulate on
ventral arms (100%) (Appendix 2).
Other components considered com-
mon to the species were: paired white
mantle spots (ID) (61%), frontal
white V (7D1), blue green around the
eyes (3, 4D, and 6D), and alternate
arm bars (all >50%).

Only Blotch and Mottle had typi-
cal components (>80%). The typical
chromatic components for Blotch
were Dark Bar across the Eye (DBE),
Light Blotch (LB), Blue-Green around
the Eye (BGE), and Purple Suckers
(PS); for Mottle, they were DBE,
White Spots (WS), White Erontal V
(WV), Alternate Bands on Arms
(ABA), and Purple Suckers (PS) (Ap-
pendix 2 and Fig. 2).

five of more than one. The analysis showed clustering among
all lateral areas of the mantle (ILl, 1L2, 1L3, and 1L4),
among two areas of the head (4V and 6V), among distal
parts of the U\ 2"^, and 3'''* dorsal arms (7D3, 7D4, 8D3,
8D4, 9D3, and 9D4), proximal parts of U*, 2”^', and 3‘‘^'

Similarity among photographs

The cluster analysis indicated that the photographs
formed one large similar group (Fig. 4). This analysis
showed similarity among the pictures, based on occurrence
of the components throughout areas of the body, despite

36

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

Table 3. Occurrence of the six chronic body patterns at the behavior states identified from photographs of Octopus insularis (N - 65) taken
at Fernando de Noronha.

Behavior states/ Outside Inside

body pattern

den

%

den

%

Hunting

%

Swimming

%

DL-VBG

2

15.4

0

0.0

0

0.0

5

45.5

Mottle

3

23.1

4

36.4

14

60.9

0

0.0

Blotch

6

46.2

0

0.0

3

13.0

1

9.1

Dymantic

2

15.4

3

27.3

1

4.3

1

9.1

Uniform dark

0

0.0

4

36.4

4

17.4

4

36.4

Flamboyant

0

0

0

0

1

4.3

0

0

differences among Body Patterns, which probably indicated
that the specimens belonged to the same species. The cluster
analysis just separated three pictures with conspicuous pat-
terns and proportion of components from the larger group:
Flamboyant and two pictures of Uniform Dark during
Swimming.

DISCUSSION

Body patterns are a useful taxonomic characteristic for
identifying cephalopods in the natural environment (Moyni-
han 1975, Hanlon 1988, Hanlon and Messenger 1996). This
study supports this statement using quantitative analyses as
well as qualitative ones to analyze body patterns. Although
components had different areas of occurrence and degrees of
expression, these parameters were uniform enough that al-
most all pictures were considered similar by cluster analysis.
This strong degree of similarity among the pictures classified
as Octopus insularis from Fernando de Noronha supports pre-
vious taxonomic studies that pointed to morphological simi-
larity in this species (Leite and Haimovici 2006, Leite 2007).

Although qualitative analyses are sometimes not enough
to distinguish species or subspecies, they can be used as an
indicator. A comparison of body patterns of Octopus insu-
laris with ones described for Octopus vulgaris from the Medi-
terranean (Packard and Sanders 1969, 1971) and Bermuda
(Mather and Mather 1994) showed that some chromatic and
textural components occurred in both species. These are
frontal white spots (forming a “V” in O. insularis and split
for O. vulgaris from the Mediterranean) (Fig. 2B), mantle
white spots (not described for O. vulgaris from Bermuda),
arm bars, eye bar, black hood, and long papillae on the
mantle and head. Otherwise some components such as the
extended hood and transverse stripes (chevron), eye ring,
head bar mantle shield, and grainy texture were observed for
O. vulgaris only from the Mediterranean. Some components
described in this study for O. insularis, such as blue-green
around the eye (Fig. 2A) and alternate light and dark around

the eye (Fig. 2B), had not been cited for O. vulgaris from
either region.

The quantitative results showed that only a small num-
ber of components can always be observed across different
body patterns. These results make it difficult to do a general
Body Pattern description for this species, such as that of |!
Haplochlaena maculosa (Hoyle, 1883) (Roper and Hochberg i
1988). However, some components were strongly related to
specific body patterns, and this close relationship was useful
to make a solid characterization of the body patterns that ■
will be useful in future research. ■

Simple body patterns are found in cephalopods with i
fewer and larger chromatophores, which could generate j
fewer components, and complex body patterns are found in ;
species with many and small chromatophores, which could ,
generate more components (Messenger 2001), but this may
vary within species. Simple and complex body patterns may,
therefore, depend on the number of components. The com- I
plex ones {e.g., with more components) were observed dur- |
ing Hunting and Outside Den (Blotch and Mottle), while the '
simpler ones (fewer components) were more common dur-
ing Swimming (Dorsal Light-Ventral Blue-Green and Uni-
form Dark). As the octopuses were photographed outside
their den in habitats of different complexity including coral
reef, bed rocks, and rock shores, the high degree of com- I
plexity could be explained if some habitats require complex :
body patterns to match them. That might be true for Octo-
pus insularis, but not for all species: Hanlon et al. (1999a)
found Octopus cyanea Gray, 1849 exhibited little back- |
ground- matching outside its den. The degree of complexity i
that body patterns show within the same species or even
individual in relation to the environment indicates a great, j
sophistication of pattern use (Messenger 2001) that needs to ■
be evaluated in detail for many species. i

Different levels of complexity of distribution can also be j
found throughout different regions of the body in a single
species and may determine the components and patterns ;
that each region can display. During studies of Loligo opal- \
escens (Berry, 1911) chromatophores, Florey (1966, 1969) ;

OCTOPUS BODY PATTERNS

37

60

50

40

0)

u

c

(0

« 30

0)

O)

<0

C 20

10

3 2

11

I- rS

£7

jd

10

fm A h

U^QQ<-OJ^C*)TtfO<Nl^TtfOrOTjfOTt^CNT-'^f^CNlT— TfcOTtrtfOcOr-^^rtcOCNr— CsJ'^CNIt-CNJQC'J

u

3

«

Areas of body

Figure 3. Classification of Octopus insularis body skin areas, using cluster analysis. Similarity
among body areas was calculated by means of the proportion of occurrence of components,
skin patterns, and colors (red, yellow, and blue-green). The line is the cut point, and the
numbers indicate twelve different groups of areas on the skin (see Fig. IB).

found large and sparse chromatophores with single inner-
vation in the ventral mantle, and small and numerous chro-
matophores with multiple innervations in the dorsal mantle.
We also found simple body patterns in ventral areas (mantle
and arms) and complex ones in the dorsal areas (mantle,
head, and arms). Different degrees of complexity of pattern
throughout body areas of an individual can be explained if
more complex areas such as the dorsal areas are more visible
and vulnerable than others, while less visible areas such as
the ventral arms and ventral mantle show simple patterns.
Remember, the skin system is widely believed to have
evolved as camouflage (Packard 1974).

Differential occurrences of components in different skin
areas as defined by Froesch (1973) should have helped us
understand the effective projection area of chromatophore
fields. When we analyzed the spatial extent of components
and patterns, however, we found twelve areas with common
patterns that we call “expressive fields” (Fig. IB). Some ot
Froesch’s (1973) areas, such as the projection of nerve 5 and
3 at and near the eye, do predict the expressive fields that we
found. However, not all of his areas match our findings. For
instance, the mantle divides into three large expressive fields,
not the large number of projections described by Buhler et
al. (1975). This important finding reflects the division into
different morphological and physiological units which is also

seen at the lower level (Packard 1974,
Messenger 2001). Our knowledge of
the projection areas for pattern (Mes-
senger 2001 ) is improved when the ar-
eas used are the expressive fields.

Bringing order to a complex sys-
tem such as expression of body pat-
terns, analyzing them as located in ex-
pressive fields, and linking them to
different situations has many uses. The
first is the possibility of using them as
an additional means of species identi-
fication (Hanlon 1988, Roper and
Hochberg 1988, Hanlon and Messen-
ger 1996) in conjunction with mor-
phological and molecular analyses. Be-
yond this, a systematic analysis of the
locations of components may shed
light on the physiology of the complex,
chromatophore-control system (Mes-
senger 2001) and discriminate the
many levels of motor fields on the
skin. Additionally, linkage of specific
patterns to behavioral states has been
traced only for a few displays such as
those of Sepia officinalis (Adamo et al
2006) and Octopus nibescens Berry,
1853 (Warren et al 1974) during hunting, and the Passing
Cloud of O. cyanea to startle potential prey (Packard and
Sanders 1969, Mather and Mather 2004). With a more sys-
tematic analysis, new linkages of behavior and color pattern
may become clear. This kind of analysis can thus be the key
to accessing the behavioral plasticity and sophisticated neu-
ral control that modern cephalopods have developed (Han-
lon and Messenger 1996) through evolution.

ACKNOWLEDGMENTS

This research had financial support from the Boticario
Foundation of Environmental Protection/Brazil (FBPN).
The first author received a scholarship from CNPq (Nacio-
nal Council of Scientific and Technological Development) at
the Graduate School in Biological Oceanography at the Uni-
versity of Rio Grande and a short term scholarship from
CAPES (Coordenai^xio de Aperfeic^oamento de Pessoal de
Nivel Superior) to stay at the University ot Lethbridge,
Canada. We thank the University of Lethbridge for its hos-
pitality in the academic year 2005-2006. The research at the
National Park of Fernando de Noronha was possible with an
authorization by IBAMA. Professor lorge Lins at the Uni-
versity of Rio Grande do Norte (UFRN) and the adminis-

38

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

Figure 4. Classification of Octopus insularis photographs, using cluster analysis based on the
single-linking method. Similarity among photographs was calculated by means of the pro-
portion of the body patterns components through the body {= number of times that com-
ponents appeared at the areas of the picture/number of areas analyzed).

tration of Lernando de Noronha provided invaluable logistic
support. We would like to thank Atlantis Divers, Aguas
Claras, and Noronha Divers for scuba diving support; Hy-
drosphera Productions and the underwater photographers:
Zaira Matheus, Labio Guilherme (CQ), Dayse Brisola, Li-
zandro Almeida, and Diogo Pagnoncelli for the octopus
photographs and films made available, John Vokey for his
advice on statistical analyses and Ruth Braun for her help
and patience with the manuscript revisions. We also would
like to thank the volunteers that worked at the Cephalopod
Project for their help.

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Submitted: 6 October 2006; accepted: 30 )uly 2007; final

corrections received: 1 1 lanuary 2008

40

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

Appendix 1. Median percentage of occurrence of the components and colors throughout the body of Octopus insularis within the main
body patterns: % occurrence = (occurrence of the component or color in the body areas analyzed)/! total parts of body analyzed in the
picture) X 100. The numbers in boldface type indicate the commonest components observed throughout the body for each body pattern.

Components DL-VBG

Chromatic components CR-WBE 1.6%

CR-DBE 9.0%

CR-RBE 0.0%

CR-WS 15.7%

CR-WD 4.3%

CR-DS 0.0%

CR-LB 14.8%

CR-DBA 0.0%

CR-ABE 1.9%

CR-WV 0.5%

CR-BH 0.0%

CR-ABA 0.0%

CR-PS 2.9%

CR-BB 0.7%

CR-BGE 8.3%

CR-LDS 5.4%

Textural component TE-R 32.9%

TE-SP 9.6%

TE-BP 0.0%

TE-S 63.0%

TE-T 0.0%

C-Y 22.5%

C-R 4.9%

C-BR 73.8%

C-W 63.6%

C-BG 49.2%

Mottle

UD

Blotch

Dymantic

0.0%

0.0%

0.0%

0.0%

12.3%

11.4%

12.8%

18.2%

0.4%

0.0%

0.0%

3.1%

57.7%

28.4%

32.1%

9.9%

1.4%

5.4%

3.5%

0.0%

4.9%

5.3%

0.4%

0.0%

4.2%

0.6%

40.9%

6.2%

2.3%

0.4%

2.9%

4.2%

8.2%

6.9%

2.7%

2.6%

4.1%

3.3%

2.0%

1.3%

0.6%

0.0%

0.0%

18.8%

19.9%

2.3%

12.2%

0.0%

4.0%

4.3%

3.0%

0.6%

11.1%

2.6%

3.4%

2.0%

4.7%

8.0%

9.1%

18.6%

0.0%

0.0%

0.0%

0.0%

47.5%

54.2%

22.1%

29.0%

55.0%

38.3%

12.6%

5.2%

13.3%

9.8%

3.0%

0.0%

36.4%

34.7%

100.1%

62.5%

8.9%

7.1%

0.0%

0.0%

37.2%

39.7%

22.6%

6.7%

26.3%

32.4%

16.6%

4.2%

87.8%

86.8%

91.8%

75.6%

74.7%

53.1%

59.6%

52.7%

49.7%

20.1%

45.1%

56.0%

Colors

OCTOPUS BODY PATTERNS

41

Appendix 2. Relationship of components, skin pattern, and colors of Octopus iiisiilaris with Body Patterns; % occurrence = (the times that
component appeared in the pictures with a distinct body pattern)/(total of pictures analyzed with this body pattern) x 100. The numbers
at the topi of each column indicate the total of pictures analyzed of each body pattern, and the numbers in boldface type indicate the typical
components for the Octopus insiilaris.

Chromatic components

Textural component

Skin pattern

Compionents

Blotch

(11)

Mottle

(22)

UD

(12)

Dymantic

(7)

DL-VBG

(7)

CR-WBE

0.00

0.00

0.00

0.00

42.86

CR-RBE

0.00

4.55

0.00

28.57

0.00

CR-DBE

90.00

100.00

100.00

100.00

85.71

CR-WS

63.64

100.00

83.33

42.86

71.43

CR-WD

9.09

9.09

33.33

42.86

40.00

CR-DS

9.09

36.36

25.00

42.86

0.00

CR-LB

90.91

27.27

8.33

14.29

42.86

CR-DBA

60.00

62.50

8.33

57.14

0.00

CR-ABE

36.36

63.64

33.33

14.29

28.57

CR-WV

36.36

81.82

54.55

0.00

20.00

CR-BH

0.00

4.55

0.00

71.43

0.00

CR-ABA

54.55

81.82

16.67

0.00

0.00

CR-PS

87.50

90.00

100.00

100.00

100.00

CR-BB

18.18

50.00

16.67

14.29

14.29

CR-BGE

90.91

50.00

58.33

100.00

85.71

CR-LDS

0.00

0.00

0.00

0.00

14.29

TE-SP

45.45

95.45

66.67

28.57

42.86

TE-BP

9.09

50.00

41.67

0.00

0.00

TE-T

0.00

31.82

16.67

0.00

0.00

SP-RWR

No photos

100.00

100.00

100.00

100.00

SP-AB

54.55

86.36

16.67

28.57

0.00

SP-DS

0.00

9.09

25.00

14.29

0.00

SP-LS

90.91

45.45

75.00

100.00

100.00

SP-DR

63.64

36.36

100.00

85.71

28.57

SP-LR

27.27

4.55

33.33

85.71

100.00

SP-BL

90.91

27.27

0.00

14.29

28.57

C-Y

81.82

81.82

100.00

42.86

85.71

C-R

72.73

95.45

91.67

42.86

85.71

C-BR

100.00

100.00

100.00

100.00

100.00

C-W

100.00

100.00

100.00

100.00

100.00

C-BG

90.91

100.00

91.67

100.00

100.00

C-BL

9.09

36.36

25.00

42.86

0.00

Colors

Amer. Maine. Bull. 24: 43-50

Observations of deep-sea octopodid behavior from undersea vehicles’^

Janet R. Voight

Department of Zoology, The Field Museum, 1400 Lake Shore Dr., Chicago, Illinois 60605-2496, U.S.A., Jvoight@fmnh.org

Abstract: Despite high octopodid diversity in the deep sea, the few opportunities to observe these animals in situ limit tests of behavioral
predictions made from anatomy. Over the last decade, I have made numerous opportunistic observations of these octopuses using
submersibles and remotely operated vehicles. Most commonly seen were octopuses near hydrothermal vents in the North Pacific Ocean at
greater than 2200 m depth. Despite the potential submersible-created artifact, the observed behaviors of octopuses of the genera Bentlioc-
topiis Grimpe, 1921, Graneledone Joubin, 1918, and Vulcanoctopiis Gonzalez et ai, 1998 are reported here. Benthoctopiis and Graneledone
differ in wariness and in egg-brooding postures, although both genera produce large eggs from which male hatchlings emerge with clearly
developed copulatory arms. In a behavior interpreted as foraging for infauna, octopuses of both genera move the mid-section of their arms
through the upper sediment. Graneledone seems more common, perhaps because individuals are typically larger and move more slowly. The
greater wariness of Bentlioctopus increases the species’ propensity to jet and limits observations and capture. Despite considerable sub-
mersible time spent in their habitat, octopuses of Vulcanoctopiis remain little known; only male specimens are available for study. The few
data available indicate that deep-sea octopuses take small prey, as Voss (1988) predicted based on the lack of the esophageal crop and the
small posterior salivary glands. If deep-sea octopuses rely on small prey, the need for a crop to serve as a food storage organ is minimized,
as is the need for glands that produce venom to subdue large and potentially dangerous prey.

Key words: Bentlioctopus, Graneledone, Vulcanoctopiis, North Pacific Ocean, embryos

Deep-sea octopodids, defined here as occurring at
greater than 2000 m depth, are surprisingly diverse (Voss
1988). Our knowledge of the biology of these animals, how-
ever, is limited by the rigors imposed by the deep-sea habi-
tat. The darkness, cold, and high hydrostatic pressure define
the habitat and prohibit its direct exploration by air-
breathers. In addition to these deep-sea features, the unpre-
dictable distribution of the animals and the expense of
conducting research in the deep sea mean that most obser-
vations of the animals in nature must be opportunistic. Most
type specimens were pulled dead from trawl nets and pre-
served at sea without being seen by specialists. This contri-
bution summarizes our current, minimal knowledge of
deep-sea octopodids and offers a starting point for future
research.

Comparative anatomical studies of the Octopodidae re-
veal apparent convergences among deep-sea benthic octo-
puses (Voss 1988). The ink sac, a symplesiomorphy of the
cephalopods that is thought to be associated with defense
against visual predators, is lost. Morphometric studies find
that deep-sea and high-latitude octopuses tend to have short
arms and small suckers compared to octopuses from shallow
water and low latitudes (Voight 1993). Features of the an-
terior digestive system, including the esophageal crop,
radula, and posterior salivary glands, are small or absent in

deep-sea octopuses, a pattern that Voss (1988) attributed to
their diet of small prey. Although its function was not ex-
plicitly stated by Voss, the crop may primarily serve to store
food rather than digest it, and large posterior salivary glands
produce the venom that immobilizes large, dangerous prey
that deep-sea octopuses rarely encounter.

This report summarizes my observations of deep-sea
octopuses made over the last decade during research cruises,
and images made available by participants in other cruises.
The deep-sea octopodid genera Benthoctopiis Grimpe, 1921,
Graneledone loubin, 1918, and Vulcanoctopiis Gonzalez et
ah, 1998 are the subjects of the observations.

MATERIALS AND METHODS

Observations of seafloor animals such as octopuses in
situ require use of the crewed Deep Submergence Vehicle
Alvin or Remotely Operated Vehicles (ROVs) such as
ROPOS, Jason, or Tibiiron. Towed cameras that are lowered
from a surface ship to a few tens of meters above the bottom
to take images at pre-set intervals also can document benthic
octopuses. Regardless of the technique used, undersea assets
require bright lights and cameras to detect and record the
presence and/or behaviors of any deep-sea animals encoun-
tered. The behaviors observed therefore are presumed to be

* From the symposium “Cephalopods: A behavioral perspective” presented at the joint meeting of the American Malacological Society and
Western Society of Malacologists, held 29 July to 3 August 2006 in Seattle, Washington.

43

44

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

typical, as no observations can be made without the lights
and other disturbances the subsea vehicle creates.

Most dives, and thus most observations, reported here
took place in the North Pacific Ocean, in or near hydrother-
mally active areas of luan de Luca and Gorda Ridges, gen-
erally at depths of 2000 to 2650 m. A few observations were
made near 1550 m depth inside the caldera of Axial Volcano
on luan de Luca Ridge. Because hydrothermally active areas
are of interest to geophysicists, geochemists, biologists, and
others, a disproportionate amount of the available submer-
sible and ROV time is spent there. Observations also derive
from submersible and ROV dives that targeted off-axis rocky
outcrops, such as Baby Bare and Wuzza Bare near 2600 m
depth on the Cascadia Basin and Mendocino Ridge at depths
of 1550 m. Observations of Viilcanoctopus were made on the
East Pacific Rise, between 8.5° and 1 1°N near 2550 m depth.

Observations made during the 1 7 submersible and ROV
cruises in which I participated contribute to this report and
are supplemented by photographic and video images pro-
vided by other researchers and by submersible crews. Several
researchers, notably V. Tunnicliffe and R. Embley, kindly
allowed me access to their compiled seafloor images. In ad-
dition, some researchers provided octopus image(s), without
any comment on the frequency with which the animals were
encountered or the total duration of bottom time. Although
providing vital information, these sources limit attempts to
estimate the frequency with which octopuses were detected,
or with which a given behavior was observed. In addition,
the density of octopus on the deep-sea floor is not uniform
(Voight 2002). In some areas, aggregations of brooding oc-
topuses reached extremely high densities (Voight and Gre-
han 2000, Drazen et nl. 2003), yet in “normal” seafloor areas,
hours of bottom time may be required before a single oc-
topus is seen.

The observations reported here are by their nature ad
hoc, as are the collections. Other than brooding octopuses,
the longest observations were made of individuals that could
not be collected. Gonversely, often only minimal obseiwa-
tions could be made of collected individuals, as they were
collected nearly as soon as they were detected. Octopuses
were collected in two ways. Large octopuses were grabbed by
the submersible’s manipulator arm and placed inside lidded
boxes. Smaller octopuses were more often collected in a
suction sampler. Octopuses were preserved in buffered for-
malin in seawater and are catalogued in the collections of
The Field Museum, Ghicago, Illinois. Catalogue numbers
can be accessed through the online database at http://
emuweb.fieldmuseum.org/iz/mollusks.php. Mantles of pre-
served octopuses were opened; if the stomach or esophagus
appeared full, the organs were also opened and their con-
tents examined under a dissecting scope.

Characters diagnostic of genera (Voss 1988, Gonzalez et

al. 1998) that could be detected without examining the
specimen in hand were used to identify the octopuses to
genus, with the assumption that the ink sac was absent.
Octopuses in the Northeast Pacific with a single row of suck-
ers or with suckers arrayed in a zigzag line, prominent su-
praocular cirri, and textured dorsal mantle were assigned to
Gmneledone Joubin, 1918 (Fig. 1), the only octopodid genus
with a single row of arm suckers known from the North
Pacific (Voss 1988). The identity of some specimens with
zigzag sucker rows was ambiguous after only brief observa-
tion, although with extended observations, the texture of the
dorsal mantle and the presence of supraocular cirri were
nearly always sufficient to resolve any ambiguity. A mantle
fold was seen on some individuals, although this character is
not considered to be typical of this genus. Two species of this
genus have been described from the east and west North
Pacific (Nesis 1982, Voss and Pearcy 1990). Although these
species were synonymized by Hochberg (1998) as Granel-
edone boreopaciftca Nesis, 1982, because cryptic species may
exist, I assigned all individuals to the genus rather than a
given species.

Pigmented octopuses with double sucker rows were as-
signed to Benthoctopiis Grimpe, 1921 (Figs. 2-3), as the only
members of this deep-sea genus have been described from
greater than 1000 m depth north of 40°N in the Pacific. The
dorsal surface of all members of this genus is thought to be
smooth, and none of the available observations indicated
otherwise. Among octopuses attributed to Benthoctopiis,
four apparent species could be distinguished. The largest
species had long arms and strong reverse counter-shading
(Fig. 2); two seemingly moderate-sized, uniformly colored
species were distinguished by differences in eye size. The
fourth species was Benthoctopus canthylus Voss and Pearcy,
1990 which was identifiable by the dramatically enlarged
suckers on the dorsal arms (see photos Norman 2000: 210).

White octopuses with a double sucker row (Fig. 4) ob-
served near hydrothermal vents on the East Pacific Rise
(EPR) from 8.5°N to 13°N were assigned to Viilcanoctopus
hydrothennalis Gonzalez et al, 1998. Their double sucker
rows, the unusually translucent white mantle, long thin arms
with an uneven surface, and small dark eyes that contrasted
strongly with skin color supported the identification. Be-
cause the EPR is the type locality for the only known species
in the genus, referring these octopuses to that species is
conservative.

RESULTS AND DISCUSSION

Seafloor observations of benthic octopuses tend to be
unusual, even rare, except in areas where brooding octo-
puses aggregate (Voight and Grehan 2000, Drazen et al.

OBSERVATIONS OF DEEP-SEA OCTOPODID BEHAVIOR

45

2003). The large size and similarity of deep-sea octopuses to
those of shallow-water, however, mean that deep-sea scien-
tists, regardless of specialty, are often able to recognize the
animals. The comparative rarity of octopuses makes obser-
vations memorable, facilitating information sharing.

Frequent reports of octopuses near hydrothermal vents,
especially those with clams, have been attributed to the avail-
ability of clams as prey {e.g., Mottl et al. 1998) or of hard
substrate as a limited deep-sea resource (Voight 2000a). The
occurrence of octopuses near abundant clams that host
endosymbiotic sulfide-oxidizing bacteria might suggest that
octopuses tolerate exposure to hydrogen sulfide, among the
most common and toxic chemicals at chemosynthetic habi-
tats. Clams that host sulfide-oxidizing endosymbiotic bacter-
ia, however, can live in areas with very little sulfide in the
water (Fisher 1995). Their extensible toot and blood with a
high sulfide affinity allow clams to access sulfide deep in
crevices or in subsurface water (Fisher 1995); potential clam
predators might be exposed to very low sulfide levels.

Graneledone

Size and density: Most frequently observed in the North
Pacific were octopuses of Graneledone (Fig. 1) due to their
large size and comparatively slow movements. These octo-
puses, especially at depths greater than 2000 m, were reluc-
tant to jet away from the vehicle. Among 49 preserved speci-
mens, those from comparatively shallow depths (1300 to
2000 m) were typically larger (up to 59 cm total length) with
longer arms carrying more suckers (up to 1 16 per arm) than
were those from greater (to 2800 m) depths (up to 48.6 cm
total length with up to 86 suckers per arm) (Voight, unpubl.

Figure 1. Octopus of Graneledone photographed by fixed still cam-
era mounted on front of the submersible Alvin during dive 4046
near Wuzza Bare seamount, Cascadia Basin at 2650 m depth.

data). Members of this genus were seen on sedimented
ocean floor, adjacent to hard substrate, brooding eggs in
aggregations on vertical rock surfaces (Voight and Grehan
2000, Drazen et al. 2003) but rarely near areas directly im-
pacted by hydrothermal fluid flow. Voight (2000a) reported
the global distribution of this genus in reference to areas of
chemosynthetic activity.

Behavior: When members of this genus were seen mov-
ing across featureless sediment-covered plains, they often
used the middle part of each arm as a sediment probe, in a
behavior inferred to constitute foraging for infaunal prey. In
this behavior, the proximal halves of the dorsal and dorso-
lateral arms were held nearly straight down to contact the
sediment surface. The arm tips extended radially away from
the octopus, remaining on top of the sediment, with the
middle section under the sediment. As the octopus moved
forward, it continually shifted the middle section of its arms
through the superficial sediment. Several individuals per-
formed this behavior and some preserved specimens even
had a covering of sediment on the distal one-half to two-
thirds of their arms. No direct observations of foraging suc-
cess were possible, as the octopus would have passed any
small infaunal organism it collected to the mouth by the
suckers on the oral arm surface. Although such movements
are very hard to confirm, slight movements of the arms
toward the mouth supported the hypothesis that this behav-
ior constituted foraging.

The guts of octopuses of this genus typically contained
little. One specimen, from the caldera of Axial Volcano, had
eaten snails of Provanna variabilis Waren and Bouchet, 1986
and polychaetes characteristic of the hydrothermal vent
fauna (Voight 2000b), providing evidence that these octopus
feed on small prey, in this case taken from hard substrate
impacted by hydrothermal activity. An octopus collected in
a non-hydrothermally active area had preyed on a small
galatheid crab and a second had an amphipod in its gut.

Octopuses of Graneledone were often seen adjacent to
hard substrate, where this substrate type was present. With
most submersible dives devoted to hydrothermal vents as-
sociated with rocks, observations were biased toward hard
substrate habitats. Regardless, octopuses appeared to prefer-
entially associate with rare hard substrate in non -vent areas,
such as near Ocean Drilling Program (ODP) platforms,
long-term instrument deployments, and rocks. The sex and
reproductive maturity of the individuals near hard substrates
could not be identified.

Color: In situ seafloor observations have limited tability
to identify true color, as the proximity, type, and angle of the
light source can all affect the perceived color. Octopuses of
Graneledone in situ appeared to be maroon, orange, purple,
or red. Preserved specimens are purple, blue-red, or maroon
in color. The skin texture of the dorsal mantle is also quite

46

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

variable among individuals, from very rugose (Eig. 1) to
essentially smooth. The supraocular cirri were always the
most readily seen features in terms of skin texture.

Color change was observed in two octopuses of Granel-
edone. In the better-documented observation, the octopus
was between the ROV and a second octopus of Gmneledone
when half of its body blanched along the mid-sagittal line.
On the other occasion, a lone octopus was approached by
the ROV. It blanched, as did the individual reported above,
with one side of the body colored normally and the other
whitish. The contact between the colors followed the mid-
sagittal line.

Egg brooding: Females of Gmneledone that were seen
brooding eggs had attached them to rocks and most often
aggregated in areas with rock outcrops. Aggregations are
known at 2600 m deep at Baby Bare seamount in Cascadia
Basin (Voight and Grehan 2000) and at 1550 m depth on
Mendocino Ridge (Drazen et al 2003, Voight and Drazen
2004). Voight (2002) reports that densities of brooding oc-
topuses at Baby Bare exceeded triple those of background
areas. Males were present, though rare, among the aggrega-
tions. Brooding females invariably were positioned with
their dorsal mantle facing outward and the oral surface of
their arms touching the rock surface to which their eggs were
attached. The total number of eggs produced is unknown,
although evidence of at least 50 young was seen in one case
at Mendocino Ridge (Voight and Drazen 2004), including
eggs and cement from eggs that had apparently already
hatched. The female that had been brooding these late-stage
eggs showed signs of senescence: slack skin, cloudy eyes, and
dull color. Dissection of the collected female revealed con-
spicuous edema of the tissues, renal organs, and an empty
digestive system.

Eggs and embryos: Voight and Drazen (2004) described
newly hatched embryos from an egg clutch from Mendocino
Ridge that were collected as they hatched. Hatching was
likely mediated by the disturbance created by collecting ac-
tivities, as external yolk sacs were present within the arm
crowns. These premature hatchlings were 55 mm long and
males could be identified by the hectocotylus and ligula
(Voight and Drazen 2004). The single intact egg collected
was 39.6 mm long. Earlier, partially developed, 25 mm long
ovoid eggs had been collected from an apparently conspe-
cific female at Baby Bare at 2660 m depth (Voight and Gre-
han 2000). Attempts to model development time of very
large cephalopod eggs are not considered to be reliable (Lap-
tikhovsky 1999), but the large size of these eggs and the low
temperatures at depth likely mean that their development
occurs over a very long period (J. Drazen and B. Robison,
pers. comm.). There is no indication that the females feed
while brooding.

Benthoctopus

Size and density: Octopuses of Benthoctopus were much
less commonly seen than were those of Graneledone. Whe-
ther the few observations of octopuses of this genus com-
pared to those of Graneledone are a real difference between
the densities of the genera or is, more likely, due to their
smaller size, more rapid movements, and/or greater wariness
cannot be assessed. The total lengths of specimens of Ben-
thoctopus collected during submersible and ROV work were
all under 20 cm. Members of the genus Benthoctopus were
most often seen as single individuals on hydrothermally in-
active rocks on the mid-ocean ridge spreading center (Fig.
2). Brooding aggregations were seen at two locations (be-
low).

Behavior: Octopuses of Benthoctopus were observed for-
aging in the sediment as described above for octopuses of
Graneledone. Individuals of Benthoctopus, including B. can-
thylus, were also observed to extend and hold their dorsal
arms into the water column on multiple occasions. Octo-
puses in open, sedimented areas, and on rocks at mid-ocean
ridges displayed this posture (Fig. 2). This pose was not seen
to result in the acquisition of prey, but small prey may have
been manipulated by suckers unobserved. The behavior
could also increase chemoreception by the suckers. No oc-
topuses of this genus were observed to change skin color or
texture.

Egg brooding: Brooding females of Benthoctopus were
found at Mendocino Ridge (Drazen et al. 2003) and in an

Figure 2. Octopus of Benthoctopus photographed by fixed still cam-
era mounted on front of Alvin during Dive 4044 near GR-14 or Sea
Cliff hydrothermal vent field on Gorda Ridge (North Pacific
Ocean) at 2737 m depth.

OBSERVATIONS OF DEEP-SEA OCTOPODID BEHAVIOR

47

extremely localized aggregation (Fig. 3) near the GR-14, or
Sea Cliff hydrothermal vent field on Gorda Ridge, described
by Rona et nl. (1990). Although located near an area of
hydrothermal activity, the octopuses could not be fully dem-
onstrated to be in direct contact with the diffuse hydrother-
mal fluid in the immediate area. The aggregation was located
in 2002 and in 2005 by the ROV Tiburon but could not be
relocated during an Alvin dive in 2004. The aggregations of
brooding females of both Benthoctopus and Gnmeledone
support the hypothesis (Voight 2000a) that substrate avail-
ability limits the distribution ot brooding females.

A difference between octopuses of Benthoctopus and
Graneledone, based on the estimated 15 females with egg
clutches of Benthoctopus observed, is that when brooding,
these females positioned the oral surface of their arms facing
outward and the arm tips in contact with the rock which
held their eggs, and potentially with the eggs themselves.
Females of Benthoctopus often brooded deeper under over-
hanging rocks than did the larger females of Graneledone,
possibly due to preference or because their smaller body
sizes allowed them to exploit the available niches. However,
near GR-14, octopuses of Benthoctopus were found closely
associated with a nearly vertical wall with their oral, suckered
arm surfaces bent backwards to nearly cover the rest of their
bodies; in one instance, an egg was visible (Fig. 3). Obser-
vations at this site were unfortunately very limited. No col-
lections of late-stage females of this genus have been made.

Embryos: A single embryo of Benthoctopus was collected
from near GR-14 by the ROV Tiburon in 2005. The large
(mantle length 7.5 mm; mantle width 7; head width 5.1)
embryo (FMNH 309724) had 13.1 mm long arms, each car-
rying about 66 suckers crowded into two rows. The hecto-

Figure 3. Two octopuses of Benthoctopus, one with egg visible,
photographed by ROV Tiburon during dive T-456 near GR-14 or
Sea Cliff hydrothermal field on Gorda Ridge (North Pacific Ocean)
at a depth of 2770 m.

cotylus carried 47 suckers and the ligula was clearly visible
on the third right arm tip. The eyes were well developed and
chromatophore organs lay just distal to the mantle openings
on the lateral body, on the dorsal arm tips, and on the arm
base. Few chromatophores, however, were seen on the su-
perficial skin, as those noted on the mantle appear to have
been deeper tegumental chromatophores. How near the em-
bryo was to hatching is unclear. 'Fhe preserved embryo was
received with a ruptured egg capsule; the damage was likely
inflicted during collection. Although there was no external
yolk sac, the torn outer membrane ot a yolk sac was present.

Vulcanoctopiis

Size and density: Vulcanoctopiis hydrothernialis was fairly
common at hydrothermal vents on the EPR and at nearby
non-hydrothermally-active areas. Typically, and most con-
spicuously, a solitaiy octopus was seen moving across bare
basalt up to 500 m away from active hydrothermal vents
(Fig. 4). Octopuses were also seen in active vent fields, often
moving over the surface of giant tube worm (Riftia pachy-
ptila Jones, 1981) mounds. More rarely, octopuses of V.
hydrothernialis were seen at hydrothermal vents in aggrega-
tions (Rocha et al. 2002, Voight 2005). Gonzalez et al. (2002)
reported measurements of 16 individuals of this species;
Field Museum specimens are within this size range.

Behavior: Most observations of octopuses of Vulcanoc-
topiis hydrothernialis were brief, comprising a solitary indi-
vidual moving across the basalt substrate in typical octopo-
did style (Fig. 4). This included random lateral movements
of the arms along the surface of the substrate that may

Figure 4. Octopus of Vulcanoctopiis hydrothernuilis photographed
on the East Pacific Rise by fixed still camera mounted on front of
Alvin during Dive 3927, near 9°N at 2495 m depth.

48

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

expand the area the octopus searches for potential prey. This
species is known only from 27 preserved specimens (the sum
of those reported by Gonzalez et al 2002, and nine Lield
Museum specimens), all of which are male. This severely
biased sex ratio suggests either that the sexes partition habi-
tat or that females are extremely rare. Rocha et al (2002)
reported that five apparently male octopuses swarmed a
sixth octopus, presumed to be a female, in an attempt to
copulate.

Voight (2005) described feeding by a group of these
octopuses in which the interbrachial web was used as a cast
net to secure members of a dense swarm of the amphipod
Malice hesmo?7ectes Martin, Lrance, and Van Dover, 1993. In
that account, at least 12 octopuses were positioned on four
extinct chimneys that nearly ringed a clump of tubeworms.
The amphipod swarm, which formed in warm water over
the tubeworms, was accessible to the octopuses due to the
height advantage the chimneys provided. Such swarms
would appear to be an unlikely standard prey for these oc-
topuses, because swarms typically are high enough over the
substrate that octopuses would be unlikely to reach them
without considerable effort.

Egg brooding and embryos: females of Vnkanoctopus are
unknown, as are the egg-brooding habits of the species and
its embryos.

CONCLUSIONS

Observations of octopuses of Graneledone and Benthoc-
topiis apparently foraging for small infaunal animals and of
octopuses of Vnkanoctopus hydrothennalis foraging on
swimming amphipods (Voight 2005) further document that
these deep-sea octopuses take small prey. They therefore
support Voss’ (1988) interpretation of the apparent evolu-
tionary reductions in the anterior digestive system of deep-
sea octopuses. Voss (1988) did not explicitly state the crop’s
function but identified the posterior salivary glands as the
source of pharmacologically active venoms used by shallow-
water octopus and attributed the reduction of the gland’s
size to the switch to small prey in the deep sea. Predators
ingesting relatively small prey do not require complex ven-
oms, as small prey can be readily subdued. Predators that
take prey encountered at low densities have minimal need
for food storage, as may be supplied by the esophageal crop.
In addition, the radulae of octopuses that feed on bite-sized
prey may serve primarily to push individual prey items into
the esophagus, rather than tear off bits of venom-soaked
flesh, consistent with Voss’ ( 1988) statement that the radula
is simplified in deep-sea octopuses. The comparatively small
suckers of deep-sea octopuses (Voight 1993) would also
function well with small prey (Smith 1996).

Although deep-sea octopuses outwardly are very similar
to those of shallow water, behaviorally they have several
distinctions, first, although several individuals of Granel-
edone were seen adjacent to hard structures, deep-sea octo-
puses were never seen to seek or use shelter, other than for
egg brooding. Second, when encountered on an open sedi-
mented plain, neither octopuses of Graneledone nor Benthoc-
topus made any attempt to camouflage themselves by chang-
ing either their color or skin texture. The blanching seen in
two octopuses of Graneledone may have been a startle reac-
tion, evidence of the presence of the chromatophore system
that may be functional in some of these deep-sea octopuses
not due to its current adaptive value, but to phylogenetic
constraint, freed of visual predators, deep-sea octopuses ap-
parently rely on immobility or jetting away to evade poten-
tial predators.

All octopuses observed and collected have eyes, al-
though those of Vidcanoctopns hydrothennalis are reduced
(Gonzalez et al. 1998). Because eyes are enei'getically expen-
sive organs routinely lost by animals living in perpetual
darkness, such as caves, their retention by deep-sea octo-
puses suggests that they are adaptive. One scenario in which
eyes would be advantageous is if the octopuses sought bio-
luminescent prey. If, for example, agitating the sediment
during foraging stimulates bioluminescence by infaunal ani-
mals, octopuses with eyes could be more likely to capture
those prey than would those without eyes. The dark color-
ation of octopuses of Graneledone and the strong reverse
ventral counter-shading seen in at least one species of Ben-
thoctopns observed here may also be associated with taking
bioluminescent prey as the dark ventral color would mask
any light emitted from within the web. Although the pre-
sence of a light dorsal surface in some species of Benthocto-
piis has been suggested to provide crypsis against a light
colored, sedimented background (Voss and Pearcy 1990),
ambient light at depths of over 2000 m is negligible.

Most observations reported here were made near hy-
drothermal vents but cold-seep habitats also support con-
centrations of biomass that are sustained by chemosynthesis.
Octopuses occur in these habitats (Olu et al. 1996, Sibuet
and Olu 1998, Van Dover et al. 2003). Octopuses have also
been recently photographed on the West florida Escarpment
seeps at near 3000 m (f Zekely, pers. comm.), at seeps near
2330 m depth in the Gulf of Mexico (C. R. fisher and R. S.
Carney, pers. comm.), furthermore, two different species of
Benthoctopiis have been photographed at seeps on Blake
Ridge near 2000 m depth (W. Gilhooly, pers. comm.).

Major questions about deep-sea octopuses clearly re-
main, including life span and reproductive output. Deep-sea
octopuses appear to share the life-history strategy common
to most cephalopods. Octopus eggs were never found with-
out a brooding female (and were often difficult to see due to

OBSERVATIONS OF DEEP-SEA OCTOPODID BEHAVIOR

49

her ministrations). All females of both Graneledone and Ben-
thoctopiis observed with late-stage eggs were gaunt, with dull
color, slack skin, and cloudy eyes; females are therefore con-
cluded to senesce as their eggs mature. The duration of
brooding remains to be determined through long-term ob-
servations. The longevity and growth rates of deep-sea oc-
topuses are also unknown. As the logistics of undertaking
such research remain difficult in shallow waters, doing so in
the deep sea may remain unfeasible. With a robust phylo-
geny in place, comparative study of the brains of deep-sea
octopuses and their color change ability might discover how
the elimination of selection for crypsis has affected the ner-
vous system and energy allocation within these deep-sea ani-
mals. Until we know the evolutionary history of the octopo-
dids, however, discussions of adaptations or modification to
the deep sea remain conjecture.

ACKNOWLEDGMENTS

Many generous scientists contributed images or allowed
precious dive time to be used to observe or to attempt to
collect octopuses to assist this effort, notably, D. Clague and
J. Drazen (Monterey Bay Acjuarium Research Institute); R.
Zierenberg and J. McClain (University of California, Davis);
V. Tunnicliffe (University of Victoria); R. Embley (Pacific
Marine Environmental Laboratory, NOAA); D. Butterfield
(NOAA); K. Becker (University of Miami); H. Sahling (Uni-
versity of Bremen); K. Juniper (Universite de Quebec); J.
Cowen and M. Mottl (University of Hawaii); W. Gilhooly
(University of California, Riverside); J. Zekely (University of
Vienna); C. R. Fisher (Penn State University); and R. S.
Carney (Louisiana State University). The pilots and crews of
the DSV Alvin and the ROVs ROPOS, Jason, and Tilmron
and the captains and crews of the R/V Atlantis, R/V Thomas
G. Thompson, R/V Western Flyer, and the CCGS John P.
Tully made this report possible. Preserved specimens from
California Academy of Science and the Invertebrate Mu-
seum of the Rosenstiel School of Marine and Atmospheric
Sciences, University of Miami contributed data on octopus
sizes. I thank T. Gosliner and N. Voss for having allowed
me access to these specimens. For financial support, in-
cluding ship and vehicle time, I thank NOAA-NURP West-
Coast Polar Programs, and NSF DEB-00072695 and NSF
DEB-0103690.

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Submitted: 10 October 2006; accepted: 27 July 2007; final

corrections received: 14 November 2007

Amer. Maine. Bull. 24; 51-58

To boldly go where no mollusc has gone before: Personality, play, thinking, and
consciousness in cephalopods"^

Jennifer A. Mather

Department of Psychology, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta TIK 3M4, Canada, mather@uleth.ca

Abstract: The study of molluscan behavior offers intriguing possibilities and promising results, although focused mainly on coleoid
cephalopods. Octopuses in particular have enduring individual ditterences in reactions that are strong enough to be called personalities
(Mather and Anderson 1993). Given a floating or manipulable object, octopuses do not always habituate to its presence but may instead
perform simple object play (Mather and Anderson 1999). One can argue they have basic concept formation, both in assessment of complex
sensory information and choice of motor output. Sutherland’s ( 1963) series of tests on octopus shape discrimination revealed that octopuses
had no simple rules but were instead learning what to learn. Anderson and Mather (2007) found that octopuses chose one or more of three
methods to penetrate clam shells. Each method used a different effector and prey orientation, all while the clam was under the arm web
and thus visual information was unavailable. These different aspects of behavior all indicate cephalopods may have a simple ‘primary
consciousness’ (Mather 2007), integrating perception and learned information with motivation to make decisions about complex actions.
Such a conclusion offers new possible directions for the study of molluscs.

Key words: octopus, behavior, cognition

Behavior is often studied in molluscs to understand
some other aspect of their functioning, not to evaluate and
deconstruct the behavior itself. Behavior is, thus, seen as the
consequence of physiology or structure, as in Chase’s (2002)
book on the behavior of gastropods and its neurophysiologi-
cal foundation. Alternately, behavior may be evaluated as the
outcome of evolution and the one best fit to survival and
reproductive success, through foraging strategies (Stephens
and Krebs 1986) or sexual selection (Dugatkin 2004). All
these approaches are useful, but behavior is a valid field of
study on its own, and its study can be based on Tinbergen’s
(1972) four areas of causation, development, evolution, and
function.

Although behavior is emphasized only in the cephalo-
pods— and its complexity revealed — the viewpoint could
spread to all molluscs. Behavior is often studied in the co-
leoid cephalopods because of their learning capacity and
high brain-body size ratio (Packard 1972) that is larger than
in fish and some birds and approaches that of mammals.
They also have excellent visual acuity (Gleadall and Shashar
2004), with eyes convergent to those of higher vertebrates.
Cephalopods are well known for variability of behavior, and
research (reviewed in Wells 1978) established their excellent
learning ability and two storage areas in the brain for visual
and chemo-tactile, learned information. Despite criticism of
this early research (Boal 1996), the basic findings stand.

Hanlon and Messenger (1996) collected information from a
wide variety of areas, but focused on body patterns and
responses to predators, in reviewing cephalopod behavior.

Advances in all four areas of behavior have given mol-
luscan specialists the opportunity to ask new questions. De-
velopment of behavior has been barely touched on, partly
because many molluscs, including cephalopods, are small
and planktonic at hatching. Several authors (Messenger
1968, Chichery and Chichery 1992a, 1992b, Dickel et al.
2000, Darmaillacq et al. 2006) have done developmental re-
search on Sepia officinalis Linnaeus, 1758 and are reviewed in
Mather (2006). Behavioral causation has been studied in
learning (Wells 1978), but evolution and function have been
studied only in the context of sexual selection in squid (Hall
and Hanlon 2001, Jantzen and Havenhand 2003) and for-
aging strategies of octopuses (Ambrose 1984, Hartwick et al.
1978, Mather 1991a, 1991b, Vincent et al. 1998). The study
of animal behavior is becoming both wider and deeper as
researchers learn more, and this should cause malacologists
to see the multiple bases of their animals’ behavior. West-
Eberhard’s (2003) masterful combination of evolution, in-
heritance, environment, and development is one example.
Bekoff et u/.’s (2002) focus on cognition from the animal’s
own perspective and Baars’ ( 1994) theory of a global work-
space as a foundation for simpler consciousness available to
non-human species are others. Some abilities that were pre-

From the symposium “Cephalopods: A behavioral perspective” presented at the joint meeting of the American Malacological Society and
Western Society of Malacologists, held 29 July to 3 August 2006 in Seattle, Washington.

51

52

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

vioLisly thought of as the sole domain of humans, such as
tool use (Beck 1980), play (Burghardt 2004), personality
(Gosling 1999), and consciousness (Edelman et al. 2005) are
being evaluated for non-humans and not just in primates.
Such assessments of cephalopods are the subject of this
paper.

Personalities

Individual differences, similar to behavioral syndromes
(Sih et al. 2004), have recently been rediscovered. The focus
on species or group-specific behavior during the second half
of the 20'*^ century, both from ethological observation and
assessment of learning, was an important and productive
advance. Yet in the process, the individual animal was for-
gotten, and variation among individuals was seen as mostly
noise. Although alternative Evolutionary Stable Strategies
began to be recognized through game theory (Maynard
Smith 1982), this work focused on small groups and not on
the individual. However, psychology has a long tradition of
looking at individual personalities of humans. Ideas devel-
oped in this area, especially theories such as Freudian iden-
tity or conflict with one’s parents, were not easily transferred
to non-human species with quite different experiential bases.
Still, Cattell’s (1965) factor-analytic approach offered a rela-
tively theory-free evaluation method, using the assumption
of individual temperaments. He felt young individuals had a
genetic inheritance which combined with environmental
pressure to shape personality. Gosling (1999, 2001) has ex-
tended this approach to animals, integrated it with research
on humans, and this can be applied to cephalopods.

Personality research uses a different experimental para-
digm than the usual study for differences among groups as a
result of experimental intervention and controls. Instead, we
tested 44 individual Octopus rubescens Berry, 1953 in three
common situations in everyday life: ( 1 ) alerting by our
opening the aquarium lid, (2) threatening by touching the
animal with a test-tube brush, and (3) feeding with a live
shore crab (Mather and Anderson 1993). These interven-
tions resulted in nineteen common behavioral responses that
were then sorted by factor analysis and principal compo-
nents analysis to find common behavioral combinations.
The result was three temperament or personality dimen-
sions, which were labeled activity, reactivity, and avoidance
for their common characteristics. Positions of individuals on
these dimensions were stable across time and surprisingly
similar to dimensions found both in humans (Buss and Plo-
min 1986) and across many animal species (Gosling 1999).

Follow-up studies on the development of temperament
in another species, using 37 individuals of Octopus bimacu-
latus Verrill, 1883, were even more interesting. Sinn et al.
(2001) followed the development of individual differences
through the first nine weeks of octopuses’ lives, a significant

period, as their life span is one year. Fifteen common be-
haviors were used in the analysis and four factors (Arousal/
Readiness, Active Engagement, Aggression and Avoidance/
Disinterest) were isolated. As expected if inheritance had a
significant effect on temperament, the developmental trajec-
tories of octopuses from the same brood (at least 50% re-
lated to one another) were similar. Behavior still showed
clear changes across the study, regardless of brood member-
ship. No experimental manipulation was done to alter the
environment, as the tiny animals were each kept in a small,
barren chamber. A fascinating study would be to see what a
stressful early life would do to form the developing person-
ality of the young octopus.

A third study of cephalopod personalities (Sinn and
Moltschaniwskyj 2005) used the sepiolid squid Eupryrnna
tasmanica Pfeffer, 1884, which has the advantages of having
a short five to eight month life span, small size, and solitary
habit. This species had tour enduring traits: Shy/Boldness,
Activity, Reactivity, and Burying Persistence (bury into the
soft sand substrate to avoid predation). Unlike in the octo-
puses, these traits were situation-specific so that activity in
the threat test, for instance, did not correlate with that in the
feeding one. Also, sex did not affect personality scores al-
though maturity stage did. Fully mature squid were more
Threat Active and more Threat Bold as well as less Feed
Reactive. This change, in a semelparous species, may reflect
a switch from a focus on somatic growth to a short lived
concentration on reproduction (Rocha et al. 2001). Behav-
iors in antipredator situations were heritable, while those in
feeding ones were not (Sinn et al. 2006), and female boldness
in foraging explained a small but significant amount of
variation in brood hatching success. The convergent results
with the studies on octopuses support the idea that relatively
stable dimensions of personality may be a characteristic of
the cephalopod group.

Play

Many animals react to their environment not by sim-
plistic responses to conditioned stimuli behavior but by ex-
ploration, which involves active extraction of information
from the surroundings (Hutt 1966). Given a complex envi-
ronment, many animals will explore and then, as stimuli are
repeatedly presented, will habituate to them (Baldwin and
Baldwin 1986) and cease to respond. Yet sometimes an ani-
mal will instead turn to interactions that are more wide-
ranging; Hutt (1966) suggested that orientation changes
from ‘what does this object do’ to ‘what can I do with this
object?’ Play is often defined as simple behavior having no
immediate benefits, including repetitive or exaggerated in-
teractions out of sequence compared to normal activity
(Burghardt 2004). Play has been considered a human char-
acteristic but is also present in many, though not all, large-

CEPHALOPOD PLAY, PERSONALITIES, AND CONSCIOUSNESS

53

brained mammal species (Iwaniuk et al. 2001) and some-
times in birds (Diamond and Bond 2003) and is useful for
acquisition of adult behavior by altricial young in the pro-
tective care of their parents. So why should octopuses play?
Possibly because they have manipulative, flexible arms ( Kier
and Smith 1985, Mather 1998) and thus researchers can
recognize and categorize their actions and trace brain capac-
ity for learning (Wells 1978) about the complex suhtidal
environment in which many octopus species live.

Object play is thought to be expressed when an animal
is safe from the danger of predation and when items can be
manipulated. We gave Enteroctopus dofleini (Wiilker, 1910)
at the Seattle Aquarium, empty pill bottles weighted to float
just at the air- water interface (Mather and Anderson 1999).
The tank included a slow water current. Eight octopuses
were given ten trials, each lasting until the animal made no
contact with the bottle for 30 minutes. Octopuses habituated
within trials, spending less and less time in contact with the
pill bottle. However, across trials, the situation was quite
different as latency to contact and duration of contact with
the stimulus did not decrease. Possible play with the object
occurred in two of the animals. Each octopus jetted water at
the floating bottle until it passed to the far end of the tank
and waited until the current returned it to repeat the activity,
which resembled bouncing a ball, over 20 circuits in each
case. This behavior was different from repulsing the jar, by
holding it away with 1-2 suckers on an extended arm.

The form this play behavior took was surprising, caus-
ing me to ask why there was not manipulation with the
arms? One possibility is that the arms, with 2/3 of the ani-
mals’ neurons, are under local rather than central control
(Rowell 1963, 1966) and that information about the output
they produce is not centrally monitored. Local control of
pre-programmed responses {e.g., an autotomized arm can
walk) might make it more difficult for the arms to produce
playful behavior. Another possibility is that the control of
the water jet output, which originates from the circulatory
system and is used for respiration, is more available for shifts
in behavior. Octopuses use jet propulsion for swimming
through the water (Wells 1990), though not with the effi-
ciency of the open-ocean squid (O’Dor and Webber 1986).
The octopus jet is also used to clean out potential homes
(Mather 1994), to excavate clams from the sand (High
1976), and to repel scavenging fish from the remains of prey
in the midden outside the home (Mather 1992). This flex-
ibility represents an important characteristic of higher non-
stereotyped behavior, defined as using a behavior in a quite
different situation (Hirschfeld and Gelman 1994). Perhaps
jetting was a behavior that was simply available.

Further investigation of play-like behavior suggested it
is a wider phenomenon in octopuses. Kuba et al. (2006)
tested Octopus vulgaris Cuvier, 1797 with presentations of

plastic blocks, clam prey, and empty clam shells. The octo-
puses ate the dams, ignored the empty shells, and sometimes
engaged in play-like behavior with the blocks. The play be-
havior consisted of passing the block from arm to arm,
extending the arm and pulling it back near the body, and
pulling the block along as the octopus moved. These were
designated only play-like on the basis of numbers of repeti-
tions. The arms were clearly central to the actions, and the
peak of playful behavior came during the sixth ot ten trials,
after which the octopuses habituated again. Interestingly,
Kuba et al. (2006) found that, unlike in mammals (Fagen
1981, Power 2000), young and adult octopuses played the
same amount. For the solitary octopus, play was not the
result of needing to learn the nuances of a social group, nor
was it restricted to the protected environment of the family.
It did not occur frequently but occurred equally at different
times in the lifespan.

Was this an exception, or will playful behavior be con-
fined to the relatively large-brained and exploratory coleoid
cephalopods, perhaps just to the octopuses with their large
repertoire of arm actions (Mather 1998)? Do animals have to
have a large brain to play? Anecdotal evidence of playful
behavior in invertebrates includes snails rising in an
aquarium holding on to bubbles, then sinking to the bottom
and rising again (Burghardt 2004). One reason we were able
to identify play in the octopuses was its similarity to behav-
iors that playful children perform {e.g., bouncing a ball).
Knowledge of the behavioral repertoire of most molluscs is
so limited that out-of-sequence and fragmented behavior
would usually go unrecognized. How could researchers dis-
criminate play of sea hares, scallops, or nautiloids? Perhaps
this is why it has been characterized only in an octopus.

Thinking

Outside of the reflexive behaviors expected of inverte-
brates lies the whole area of learning, including concept
formation, problem solving, and thinking, once thought the
domain of primates but now identified in different species,
contexts, and adaptive situations (Bekoff et al. 2002), includ-
ing for corvid birds (Emery and Clayton 2004). With capac-
ity for learning from visual and chemotactile stimuli (Wells
1978, Mather 1995), octopuses seem to have the potential for
such types of cognitive ability. Yet, in standard indices of
accomplishment like Thomas’ (1980) levels of learning, oc-
topuses have not shown high scores. Using visual discrimi-
nation, octopuses perform reversal learning (Mackintosh
and Mackintosh 1963) and attain Thomas’ (1980) Level 5,
but did not learn the concept of oddity (Boal 1991) to ac-
complish Level 6. However, ecological constraints rather
than learning capacity may be impeding them. Octopuses
have great difficulty sustaining the criterion of eight ot ten
correct responses in continued trials (Papini and Bitterman

54

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

1991 ) used for vertebrates. This may be adaptive since field
studies (Mather 1991a) show that they are “win-switch” for-
agers (Stephens and Krebs 1986), and sampling the unre-
warded stimulus or area is useful rather than maladaptive.
When a crab is removed from under a rock or a clam dug up
from the sand, another will not reappear the next hour or
day, and the octopus needs to explore different areas. More
ecologically meaningful situations, such as using spatial
memory for navigation (Boal et al. 2000) as has been dem-
onstrated in their natural environment (Mather 1991b), may
be a better test for octopod cognitive capacity.

Nevertheless, there are laboratory situations where oc-
topuses show concept formation, if a concept is defined as
abstractions that make it possible for animals to solve novel
choice problems without prior experience of the specific
exemplars offered (Gould 2002: 43). In a series of experi-
ments on visual shape recognition, Sutherland (1963, also
see Wells 1978) hoped to understand what he called the rule
of shape discrimination by the octopus visual system, using
paired stimuli where the octopus was given a reward for
touching one and punishment when touching the other. He
found that octopuses could discriminate vertical vs. hori-
zontal extensions of the shape, as in mammals (Matlin and
Foley 1997), which are also less competent at discriminating
oblique orientations. However, octopuses could also use an-
other rule to discriminate a figure with the same extent on
these dimensions but differing in edge-to-area ratio. They
could tell a square figure from a circular one of the same
dimensions, perhaps by angular changes, and could dis-
criminate the same figure rotated 90 degrees. Sutherland
extended six hypotheses about what stimulus dimension the
octopus was using to evaluate a figure, but Muntz (1970)
produced figures that octopuses could discriminate that did
not differ on any one of these. In short, octopuses did not
have a single, simple rule for encoding visual shapes but
instead chose the correct one for each test. This ability was
also shown in Messenger and Sanders’ (1972) study, where
octopuses trained with two valid cues discriminated faster
than those given only one. Octopuses using one cue took
longer to transfer to a situation where the other was the
relevant one (Mackintosh and Mackintosh 1963). They were
learning what aspects of the stimulus were important.

With a variety of techniques to penetrate dam shells,
octopuses may also show simple concept formation using
chemotactile cues. They use simple trial-and-error learning
for the appropriate penetration method (McQuaid 1994,
Fiorito and Gherardi 1999, Steer and Semmens 2003), trying
to pull the clam valves apart and then switching to the more
time-consuming drilling through the shell if necessary.
Anderson and Mather (2007) noted that Enteroctopiis
dofleiiii used different tactics on different clam species, pull-
ing apart the weaker manila clams, breaking the fragile mus-

sels, and drilling or chipping the valve edge of the stronger
little neck clams. When manila clams were wired shut, oc-
topuses switched to drilling or chipping. Given intact clams,
they ate least of the little neck clams but they preferred this
species on the half shell; the excess effort to open the shell
overrode their prey preference. Different areas of the clam
valves were contacted for each technique, so the octopuses
must have switched shell orientation from umbo-to-the-
moLith for arm pulling to a lateral presentation for salivary
papilla drilling (which was preferentially over the adductor
muscles or the heart) or anterior or posterior-to-beak for
chipping. Three techniques using three different effectors
(arms, salivary papilla, or beak), and three prey positions
were all used in the correct combination without access to
visual information. Without observation inside the arm web,
it is difficult to know it this is simple trial-and-error learn-
ing, but the combinations indicate that it is more.

Are such manipulations of information within the ca-
pacity of only the coleoid cephalopods within the molluscs?
In general, other molluscs have been studied as if such ca-
pacities did not exist, and there is excellent information
about reflexive behavior and its neural control in the sea
hare, scallop and sea slug, for instance. However, Chase
(2002) provides some information about the complexity of
gastropod behavior that would be an excellent place to start.
Owl limpets defend territories on the rocks (Stimson 1970),
and other limpets occupy scars for home sites fitted to their
own shell for up to three years (Hodgson 1999). How is this
behavior controlled? Sea hares migrate long distances (Ham-
ilton 1985), under control of what stimuli? Several gastro-
pods have complex escape responses triggered by the sapo-
nin in seastar tube feet (Bullock 1953) and no extensive
study of this behavior has been carried out. The authors of
chapters in Prete’s (2004) book explore examples of flexible,
adaptive behavior in other invertebrates, such as color vision
of honeybees, prey capture in spiders, and visual recognition
in mantis shrimp. Animals can do a lot with simple nervous
systems, and molluscs are no doubt among them.

Consciousness

The set of behavioral traits described above suggest that
octopuses have a simple form of consciousness. Primary
consciousness can be defined as “a reportable multimodal
scene composed of perceptual and motor events” (Seth et al
2005: 120) and is sought for in non-human animals. Of
course, some theorists argue that no such emergent systems
exist, and Gould and Gould ( 1994) caution against assuming
complex cognition where a chain of simple behaviors might
be the cause. However, others have suggested that if humans
have higher-order consciousness, then homology indicates
that non-human vertebrates might have a simpler form. Evi-
dence might be neural, particularly reentrant connectivity

CEPHALOPOD PLAY, PERSONALITIES, AND CONSCIOUSNESS

55

between brain areas for perception and those involved in
memory (Edelman et al. 2005: 170) and to a lesser extent in
behavior suggesting such connectivity. As cephalopod brain
physiology is still poorly known (but see Williamson and
Chrachri 2004 and Hochner et al. 2006 for suggestions of
such feedback circuits), behavioral evidence must be used
primarily for evidence of simple consciousness in these ani-
mals (Mather 2007).

Such primary consciousness would be the result of an
emergent central representation of the world and oneself
Shepherd (2001) discusses humans’ perceptual representa-
tion of the external world, although he comments it cannot
be a completely accurate one, and Gray (2004) notes that our
perceptual world is largely a construct. In fact, one of the
basic lessons of human perception is that we form constan-
cies (of color, lightness, and shape) which transcend the
immediately available information but make the changing
world intelligible to us (Matlin and Eoley 1997). Suther-
land’s studies suggest that octopuses were building such con-
stancies about visual shapes, though none of the early learn-
ing studies ever used a comparison that might address this
question.

Another important aspect of consciousness is that
awareness is only extended to a small proportion of the
information incoming from perception and outgoing to
muscle control (Gray 2004). Gould and Gould (1994: 149)
comment that “thinking is a potentially dangerous backup
strategy, too slow and error-prone to be applied indiscrimi-
nately.” Merker (2005: 98) comments on thinking and as-
sumes its limited task is “optimizing behavioral choices in
the light of diverse types of information.” One area of in-
formation that researchers have assumed is not centrally
calculated in octopuses and thus omitted from conscious-
ness, as in humans, is arm movement. To control the mus-
cular, hydrostatic skeletal system (Kier and Smith 1985),
octopuses have ganglia all along the arms and above each
sucker, and arm control has long been assumed to be re-
flexive (Rowell 1963, 1966). Recent studies (Sumbre et al.
2001, 2006) have confirmed arm control uses peripheral
motor programs and simple output strategies in octopus
arm extension. Yet Grasso’s (2008) studies of the diversity of
programming of sucker use challenge this view. Is such flex-
ibility a result of the larger, more complex on-board com-
puter represented by the arm neurons, or is central moni-
toring evaluating task demands and planning actions? Our
mammalian heritage leads us to think of central planning as
complex and peripheral as simple, but these divisions may
need re-thinking for octopuses.

Baars’ ( 1994) global workspace model of consciousness,
with a short-term, attentional spotlight lasting less than a
minute, might be a good model for primary consciousness in
cephalopods. Merker (2005) feels that such a capacity might

arise only in mobile animals with centralized brains faced
with decision making in a complex environment. Uie lim-
ited evidence we have suggests that cephalopods do use this
kind of decision making; they are active in exploration of
their environment (Mather 1991a) and ot novel objects in-
troduced to them. For example. Wells (1978) reported that
the ‘life span’ of a floating thermometer introduced into an
octopus tank was 5 minutes, and there is anecdotal evidence
of similar manipulations noted by aquarium keepers. Octo-
puses also quickly habituate to the repeated presence of a
simple object and move the attentional spotlight except
when actions are transformed to play (Mather and Anderson
1999, Kuba et al 2006). Such a central processor could also
make decisions about prey entry techniques of octopuses for
clams, since action, effector, and clam orientation all must
be coordinated (Anderson and Mather 2007)

How might cephalopods’ possession of personalities in-
dicate simple consciousness? Merker (2005: 93) assumes that
consciousness mediates between motivational, sensory, and
motoric functions. The presence of distinct personalities in
cephalopods (Mather and Anderson 1993) suggests both
motivational differences among individuals and a complex
base on which they act. Simple neural structures should
produce more behavior that is stereotyped among individu-
als in the input-output relationships than what is seen in
cephalopods. West-Eberhard’s (2003) detailed evaluation
makes it clear how adaptive plasticity allows combining of
genetic influence with environmental pressure (though she
had no concept of a central controller) to produce individu-
als, each adapted over time to its micro-environment.

The ideas advanced in this paper and a deeper evalua-
tion of behavior per se can indeed take the study of molluscs
in directions it has not traditionally gone. There is much to
be gained by doing so: definitely a better understanding of
the cephalopods, a new respect for the competence of lowly
invertebrates, and new questions to ask, by extension, of all
the mollusc groups.

ACKNOWLEDGMENTS

Studies by the author of this paper were not supported
by a grant from any major funding agency. Thanks are ex-
tended to Dr. Roland Anderson for his years of research
partnership, the Seattle Aquarium for allowing us to carry
out our work, and Ruth Bergen Braun and Jennifer-Laura
Kelly for their assistance with manuscript production.

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Submitted: 1 October 2006; accepted: 7 lune 2007; final

corrections received: 8 lanuary 2008

Amer. Maine. Bull. 24: 59-63

Freshwater snails (Mollusca: Gastropoda) from the Commonwealth of Dominica
with a discussion of their roles in the transmission of parasites

Will K. ReevesS Robert T. Dillon, Jr.^, and Gregory A. Dasch^

' USDA Agricultural Research Service, Arthropod-Borne Diseases Research Laboratory, Agriculture Building, Room 5031, Department
3354, 1000 E University Avenue, Laramie, Wyoming 82071-2000, U.S.A., wreeves@alumni.clemson.edu

^ Department of Biology, College of Charleston, Charleston, South Carolina 29424, U.S.A., dillonr@cofc.edu
^ Centers for Disease Control and Prevention, 1600 Clifton Road NE, MS G-13, Atlanta, Georgia 30333, U.S.A.

Abstract: We collected six species of freshwater snails from Dominica, including Biomphalaria hiliniana (Clessin, 1883), Gundlachin
radiata (Guilding, 1828), Helisoma (=Phworbella) trivolvis (Say, 1817), Melatwides tuberculata (Mtiller, 1774), Neritina puuctidata Lamarck,
1816, and Physa marmorata Guilding, 1828. Our collections indicate that un-reported species such as G. radiata and H. trivolvis are
established on Dominica, West Indies. We tested a limited number of M. tuberculata for rickettsial pathogens, Neorickettsia spp., but did
not identify this agent. Three species of snails previously reported from Dominica, Biomphalaria glabrata (Say, 1818), Biomphalaria
straminea (Dunker, 1848), and Thiara granifera (Lamarck, 1822), were not collected. Our data suggest that B. glabrata has not re-emerged
as a prominent component of the freshwater snail fauna since it disappeared or was locally eradicated. In addition, previous reports of B.
straminea were probably misidentifications of B. kuhniaua, and some abnormally large specimens of M. ttd^erculata from Freshwater Lake
could be misidentified as T. granifera. Our sampling was not adequate to demonstrate that T. granifera was absent from Dominica. We
determined that B. kuhniana was not eradicated by previous molluscan control regimes. Additional studies on the relationships of
freshwater snails in Dominica to helminths of animals and humans are needed to understand the public and veterinary health significance
of these snails.

Key words: Biomphalaria, Gundlachia, Helisoma, Physa, West Indies

The Commonwealth of Dominica is a small (790 km^)
mountainous island nation in the West Indies that receives
over 900 cm of rain per year (Grell 1976). The freshwater
snail fauna of Dominica has been studied in regard to its
significance in the transmission of schistosomiasis {e.g., No-
blet and Damian 1991), but the snail fauna has been largely
ignored in other regards. Freshwater snails are the primary
intermediate hosts for most trematodes, some nematodes,
and some rickettsial pathogens {Neorickettsia spp.). There
have been no reports of autochthonous schistosomiasis on
Dominica, but visitors and immigrants harboring the worm
have been documented (Grell 1976, Prentice 1980, Grell et
al. 1981, Noblet and Damian 1991, Adedayo and Nasiiro
2004). There remains a potential for transmission and es-
tablishment of schistosomiasis as long as susceptible popu-
lations of Biomphalaria Preston, 1910 are established on the
island. Biomphalaria glabrata (Say, 1818) was reported on
Dominica (Prentice 1980) but more recent surveys (Noblet
and Damian 1991) indicate that this snail was replaced by
Biomphalaria straminea (Dunker, 1848). In addition, two
molluscan intermediate hosts of the trematode Paragonimus
westermani were introduced on Dominica (Noblet and
Damian 1991). The potential to establish this trematode is
relatively small because it is not established on neighboring
islands. Prosobranch molluscs are the intermediate hosts for
trematodes that transmit Neorickettsia spp. to humans and

domestic animals throughout the Americas (Pusterla et al.
2000, Headley et al. 2004). Neorickettsia spp. have not been
reported from Dominica. We conducted a survey of fresh-
water ponds, lakes, and rivers to determine the distribution
of freshwater snails on Dominica. We tested selected Mela-
noides tuberculata (Muller, 1774) for the presence of
Neorickettsia by PCR amplification of the 16S rRNA gene of
Neorickettsia.

MATERIALS AND METHODS

Snails were collected (sites listed in Table 1) by remov-
ing them from vegetation and mud using nets and sieves or
by snorkeling in streams and removing them from rocks. All
specimens were preserved in 99% ethanol, which was
changed completely after 24 hours.

All snails were identified with morphological characters.
DNA was extracted from individual specimens in the genus
Biomphalaria and 9 Melanoides tuberculata from each col-
lection locality. The DNA extraction, PCR, PCR cleanup,
and sec]Liencing followed the technic]ues described by Reeves
et al. (2006) with the following modifications. We extracted
DNA from individual snails and amplified the internal tran-
scribed spacer 2 (lTS-2) and a portion of the 28S rRNA gene
from each specimen of Biomphalaria, using the primers de-

59

60

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

Table 1. Collection data and snail species identified from sites in Dominica, West Indies in 2005.

Collection site

Habitat type

Collection date

Species collected

Springfield Estate, Parish
of Sainte Paul

Pond and outflow stream 12-17 May 2005

Biomphalaria kuhniana
Melanoides tiiberculata
Neritinia punctulata
Physa marmorata

Roseau Botanic Garden, Roseau,
Parish of Sainte George

Artificial pond

13 May 2005

B. kuhniana
Helisoma trivolvis
M. tiiberculata
P. marmorata

Roseau, open sewer drains,
Parish of Sainte George

Standing water

13 May 2005

B. kuhniana
P. marmorata

Roseau River, Roseau,

River

13 May 2005

N. punctulata

Parish of Sainte George

Clark Hall River,
Parish of Saint Paul

River

14 May 2005

Giindlachia radiata

M. tiiberculata

N. punctulata

Freshwater Lake,

Parish of Saint George

Lake

15 May 2005

B. kuhniana
M. tiiberculata
P. marmorata

Miranda’s Pond,

Parish of Saint George

Pond

16 May 2005

B. kuhniana
N. punctulata

Cochran, unnamed pond,

Pond

16 May 2005

G. radiata

Parish of Sainte Paul

Middleham Falls,

River

16 May 2005

N. punctulata

Parish of Sainte Paul

scribed by Galdeira et al. (2004).

DNA extracts from M.

ana was described from a

“Ghinese well” in Panama and is

tnberciilata were screened for DNA of Neorickettsia by PCR
using the EHR16SD and EHR16SR PCR primers to amplify
the 16S i RNA gene of Neorickettsia as described by Inokuma
et al. (2000). Positive controls with DNA from Helisoma
trivolvis (Say, 1817) or Wolbachia sp. and a negative control
with distilled water were used. We used positive controls
that could be amplified by PCR but represented organisms
other than those examined in our study.

Voucher specimens of snails were deposited in the
Academy of Natural Sciences of Philadelphia. DNA se-
quences for the ITS-2 and a portion of the 28S rRNA gene of
Biomphalmia kuhiiiana (Clessin, 1883) (GenBank Accession
#DQ1 11952) were deposited in GenBank.

RESULTS AND DISCUSSION

We did not collect Biotnphnlnria glabrata or Biompha-
laria straminea but did collect Bioinphalaria kiilmiana from
isolated ponds and Freshwater Lake (Table 1). The DNA
sequences for the ITS-2 and a portion of the 28S rRNA gene
from our specimens were 100% identical to those of B. kuh-
niarm (GenBank #s AY030380, AY030378, AY030379) from
Columbia, Dominica, and Venezuela. Biomphalaria kiihni-

morphologically similar to B. straminea (Paraense 2003).
The two species can be separated with molecular and mor-
phological characters. Biomphalaria kuhniana is not a com-
petent intermediate host for Schistosoma mansoni (Paraense
2003), which could explain why schistosomiasis has not be-
come established on Dominica even though Biomphalaria
and occasional transient human infections with S. mansoni
have been reported in Dominica. Noblet and Damian ( 1991)
reported populations of B. straminea in artificial ponds on
the island but not from Freshwater Lake. However, we sug-
gest that the previous reports of B. straminea were misiden-
tifications of B. kuhniana, which was not included in the key
by Malek (1985), used to diagnose snails in the previous
surveys. In addition we collected B. kuhniana in Freshwater
Lake, which is a new locality for this snail. Dejong et al.
(2001) had reported a population of B. kuhniana from
Roseau, Dominica. Biomphalaria kuhniana is not known to
serve as the intermediate host of trematodes parasitic to
humans or domestic animals; however, little research has
focused on the possibility that this snail is a host to hel-
minths other than S. mansoni. Other species of Biomphalaria
are intermediate hosts to echinostomatid trematodes and
nematodes in the genus Angiostrongylus (Malek 1980).

We collected ancylid limpets in both streams and ponds.

FRESHWATER SNAILS OF DOMINICA

61

All limpets were morphologically identified as Gniidlachia
radiata (Guilding, 1828), which has not been previously re-
ported from Dominica, but is known from neighboring is-
lands (Starmuhler 1984, Malek 1986). Gimdlachia radiata is
not considered an intermediate host to trematodes ot medi-
cal significance. It does harbor anisakid nematodes, which
are parasitic to fish and some fish-eating mammals (Thiengo
et al. 2000). Ancylidae are small and often go unnoticed by
collectors. These limpets might play important roles in the
natural cycles of helminths in Dominica but are currently
unstudied.

Helisoma trivolvis (Say, 1817), a planorbid snail that
could be mistaken for Biomphalaria by untrained collectors,
was collected in the metal ponds at the Roseau Botanic Gar-
den. Helisoma trivolvis is established in the Dominican Re-
public and possibly Haiti and Cuba (Ayvazian and Mallett
1986, Paraense 2003). A congeneric species, Helisoma diiryi
(Wetherby, 1879) is also established in the Caribbean {e.g..
Pointier 2001). Helisoma trivolvis is naturally resistant
to infection by Schistosoma mansoni (Ayvazian and Mallett
1986), but this snail is an intermediate host to clinostomatid,
cyclocoeliid, echinostomatid, and strigeid trematodes and
is used as a laboratory host to nematodes in the genus
Angiostrongylus (Malek 1980, Ponder and Fried 2004).
Humans and domestic animals can be infected by some
of these worms, including Alaria catiis, which can cause fa-
tal infections in humans (Malek 1980). As with other zoo-
notic trematodes, infections of humans are accidental and
usually involve eating uncooked meat harboring metacer-
cariae.

We did not collect Thiara granifera (Lamarck, 1822),
but the specimens of Melauoides tuhercidata from Freshwa-
ter Lake were abnormally large and were initially misiden-
tified as T. granifera. Melanoides tnbercidata was collected in
both streams and ponds. We did not amplify DNA from
Neorickettsia spp. in any of our collections of M. tuberciilata.
This exotic snail was possibly introduced to Dominica
around 1975 (Pointier and McCullough 1989). Melanoides
tnbercidata is a potential biological control agent for Biom-
phalaria spp. because the two snails appear to compete, and
M. tuberculata might exclude Biomphalaria spp. in some
habitats (Pointier and McCullough 1989). Our data indicate
that exclusion does not occur in Dominica (Table 1). Mela-
noides tnbercidata is not a suitable host for Schistosoma man-
soni but is an intermediate host to Paragonimns westermani,
a lung fluke. There is a possibility that P. westermani or other
Paragonimns spp. will become established on Dominica, be-
cause both M. tnbercidata and the freshwater-crab, second-
intermediate hosts of P. westermani, are present (Noblet and
Damian 1991). Carnivorous mammals are natural hosts for
this fluke so zoonotic cycles of the parasite could become

established without human infections. Melanoides tnbercn-
lata can serve as the intermediate host to other trematodes
that occasionally infect humans, including eye flukes
(Plidophthalmns spp.) of birds (Dimitrov et al. 2000, La-
mothe-Argumendo et al. 2003). Melanoides tnbercidata is
also an intermediate host to Heterophyes heterophyes, a fluke
of fish-eating mammals and birds (Malek 1980). Heterophyes
heterophyes can infect humans.

Physa marniorata Guilding, 1828 was collected from
ponds and water tanks with freshwater plants. Physid snails
are often overlooked as intermediate hosts of helminths, but
P. marniorata is the intermediate host for the echinostoma-
tid trematodes, Echinostoma Inisreyi and Ecliinostoma para-
ensei (Maldonado et al. 2001, 2003). Physa spp. are interme-
diate hosts for diplostomatid, echinostomatid, and strigeid
trematodes and are laboratory hosts for nematodes in the
genus Angiostrongylus (Malek 1980). In addition, Physa
spp. serve as hosts to nematomorph worms and the oligo-
chaete Chaetogaster sp., which are parasites of invertebrates
(Gamble and Fried 1976, Hanelt et al. 2001). The public
health or veterinary significance of populations of P.
marniorata on Dominica are unknown, but further studies
could prove this snail a host to helminths of economic
significance. Noblet and Damian (1991) reported collecting
Physa cnbensis Pfeiffer, 1839 in Dominica. Physa cnbensis
is a junior synonym of Physa acuta Draparnaud, 1805 (Para-
ense and Pointier 2003). However, we collected P. marmo-
rata in the same habitats and localities that Noblet and
Damian (1991) reported P. acuta. These older reports of P.
acuta thus probably represent misidentifications of P. niar-
morata.

The neritid snail Neritina pnnctidata Lamarck, 1816 was
collected from both streams and fish ponds. Neritina pimctn-
lata occurs in streams throughout Dominica (Starmuhlner
1984) and breeds in streams with eggs attached to boulders.
Neritina pnnctidata is possibly the largest freshwater snail on
Dominica and is not known to harbor parasites of humans
or domestic animals. However, this snail is eaten by humans
on Dominica. As long as the snails are adec]uately cooked,
they would pose no threat even if they were intermediate
hosts to helminths or other infectious agents. A detailed
study of the potential helminths or other infectious agents
pathogenic to humans in N. pnnctidata will thus have public
health implications.

Exotic snails continue to be introduced into Dominica.
There is at least one tropical fish store on the island, and
tropical ampullarids, physids, and planorbids are sold by the
tropical fish industry. Freshwater plants such as water lettuce
(Pistia stratiotes) are transported from one Caribbean Island
to another by travelers and are used as ornamental plants in
local water gardens or fishponds. Snails such as Biompha-
laria spp. can be transported on these plants.

62

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

ACKNOWLEDGMENTS

We thank I. Andre for issuing collection and research
permits, P. H. Adler, R. W. Blob, J. A. Korecki, B. A. Powell,
P. D. McMillan, A. G. Wheeler, Jr., and N. G. L. Osier for
logistical and collecting assistance in Dominica, and the
American Society for Microbiology for partially supporting
this research. The findings and conclusions in this report are
those of the authors and do not necessarily represent the
views of the funding agency.

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FRESHWATER SNAILS OF DOMINICA

63

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Accepted: 5 March 2007; final corrections received:

2 November 2007

Amer. Mnlac. Bull. 24: 65-69

Patterns of activity cycles in juvenile California two-spot octopuses
{Octopus bimaculoides)

David L. Sinn

School of Zoology, University of Tasmania, Private Bag 5, Hobart, Tasmania 7001, Australia, david.sinn@utas.edu.au

Abstract: Octopuses function as important prey and predators in many continental-shelf marine ecosystems. Understanding activity cycles
of octopuses should help define their mode of foraging and potential resource utilization and, therefore, their niche within the marine
community. Unfortunately, little is known concerning activity cycles of octopuses, especially during their juvenile life-history stages. Here,
I present observations on juvenile activity in Octopus bimaculoides Pickford and McConnaughey, 1949 over three observational weeks in
a semi-natural laboratory setting. Octopuses on average were nocturnal, but some individuals were active during daylight hours in all three
observational weeks. Nocturnal activity cycles may decrease the risk of predation on juveniles by visual fish predators hunting during
daylight hours. However, inter- and intraspecific competition with other octopuses in different life history stages, including adult O.
bimaculoides and adult and juvenile Octopus bimaculatus Verrill, 1883 is also likely during nighttime hours. Further studies are needed on
the relative influence of predation and competition on octopus activity cycles and the resulting consec]uences for octopus populations.

Key words: cephalopod, octopus activity, juvenile ecology, niche partitioning

Activity cycles play an important role in determining a
species’ niche. At the individual level, an organism’s activity
cycle mediates the ecological trade-off between growth and
mortality (Werner and Anholt 1993). Increased activity can
lead to increased resource acquisition (and therefore,
growth) but may also come at a cost, by increasing suscep-
tibility to predation. At the level of populations, activity is
manifest through levels of intraspecific competition between
life history stages {e.g., between juveniles, sub-adults, and
adults) or through intersp>ecific competition between sym-
patric species competing for similar resources (Werner
1992). For example, two species which overlap in feeding or
habitat niche can avoid direct competition if their popula-
tions exhibit non-overlapping activity cycles.

Octopuses are an important mid-level trophic compo-
1 nent of many shallow continental shelf marine ecosystems,
as both generalist predators on many smaller fish and inver-
tebrate prey (Fawcett 1984, Ambrose 1986) and as important
prey items for larger fish and marine mammals (fianlon and
Messenger 1996, Forsythe and Fianlon 1997). Many of these
predator/prey relationships should influence, and be influ-
enced by, the activity cycles of their constituent organisms
(e.g., Richardson 2001). For example, fish, octopus, and/or
other invertebrate predators could be a major selective force
shaping (but also responding to) the general activity cycles of
fish, octopus, and/or invertebrate prey populations (Daido
2002). Understanding activity cycles of octopuses at multiple
life-history stages (i.e., juvenile, sub-adult, and adult stages)
may therefore contribute to our understanding of the struc-
turing of resource use among populations and ecological
communities in shallow, near-shore marine environments.

Reports of activity cycles in octopuses are scarce, but
several species have been studied and characterized as having
a basic endogenous day, night, or crepuscular activity pat-
tern. At least some populations of several species of octopus
are nocturnal (Enteroctopus dofleiiii (Wiilker, 1910):
Hartwick et al. 1984; Octopus joubtiii Robson, 1929: Mather
1984; Octopus macropus Risso, 1826: Meisel et al 2006),
whereas other species show a tendency towards diurnal pat-
terns of activity {Octopus cyanea Gray, 1849: Forsythe and
Hanlon 1997, Hanlon et al. 1999). Some species (e.g., Octo-
pus vulgaris Cuvier, 1797) may be characterized by popula-
tions or individuals which can be nocturnal, diurnal (Wells
et al. 1983, Meisel et al. 2006), and, under certain conditions,
arrhythmic (Wells et al. 1983, Meisel et al. 2003). Overall,
these previous studies illustrate substantial variation within
and among species and populations of adult octopuses. Un-
fortunately, there is little information regarding activity
cycles of juvenile octopuses of any species, probably due to
their small size and highly cryptic nature in the wild.

Octopus bimaculoides Pickford and McConnaughey,
1949 occurs in shallow continental-shelf regions of central
Calitornia to Baja California, Mexico, where it inhabits rocky
reef, kelp forests, and mudflats (Lang 1997). Currently, most
observations of O. bimaculoides have come from laboratory
studies (Forsythe and Hanlon 1988a, Sinn et al. 2001 ); only
one study has reported limited in situ observations (Lang
1997). Like most shallow-water octopuses, O. bimaculoides
has a short lifespan (Forsythe and Hanlon 1988b). Adult
female O. bimaculoides lay numerous (Forsythe and Hanlon
1988a, 1988b), large teardrop-shaped eggs (3-4 mm long)
from which relatively well-developed benthic hatchlings are

65

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AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

[

produced after an incubation period of 6-8 weeks. Upon
hatching, juveniles disperse by crawling or swimming (as
opposed to solely relying on ocean currents). Little is known
about the foraging ecology of juveniles in this species, but in
laboratory settings juveniles are generalist feeders and will
take small shrimp, crabs, bivalves, fish, and gastropods (Lor-
sythe et al 1984, Sinn 2000). There have been no systematic
reports of activity cycles in any life stage of O. bimacidoides.

The aim of the current study was to determine whether
juvenile Octopus biniaculoides displayed patterns of activity
cycles which could be described as crepuscular, nocturnal, or
diurnal under semi-natural laboratory conditions (i.e., natu-
ral day lengths and ad Ubitwn feeding).

MATERIALS AND METHODS

Brooding female specimens of Octopus bimacidoides
were obtained commercially from the wild (Chuck Winkler,
Long Beach, California) in September 1998 and shipped to
Portland, Oregon, where they were maintained until eggs
hatched. Two broods hatched synchronously during the
week of October 15, 1998, and the majority of individuals
hatched within 3-4 days of this date. Within 2-3 days of
hatching, octopuses from these two broods were assigned
randomly and in equal numbers to two separate holding
tubs where they remained until the end of experiments.
Holding tubs were 76-L fiberglass tubs ( 1 m x Vi m X Vi m)
in-line with a 1900-L closed-seawater system. Salinity (34-36
psLi) and temperature (18 °C) were held constant through-
out experiments. The system had overhead fluorescent light-
ing in addition to natural, direct sunlight from a large bank
of adjacent windows. To allow for diffuse dawn and dusk
periods, the day/night light cycle of the fluorescent lights was
timed to come on one hour after sunrise and turned off one
hour before sunset. Sunrise/sunset times were based on data
for Portland, Oregon from The Astronomical Almanac On-
line (U.S. Naval Observatory and H.M. Nautical Almanac
Office; http://asa.usno.navy.mil/). Low-powered red lights
(25 W), which were never turned off, allowed observations
during nighttime hours. Holding tubs contained crushed
oyster shell substrate and plastic seagrass beds along with
shelter in the form of clay pots, small PVC tubing, and rocks.
Hatchling/juvenile octopuses were fed ad libitum during
non-observation days with littorinid snails (Littorina scutu-
lata Gould, 1849), mysid shrimp (Mysis spp.), and small
shore crabs {Hemigrapsus spp.). Juvenile specimens of O.
bimacidoides have high mortality rates relative to adults
(Lorsythe et al. 1984). Therefore, in order to enhance sur-
vival, an attempt was made to maintain at least one type of
food source in tubs at all times.

A single 24-hr long observation period on both tubs of

octopuses began on October 29, 1998 (N = 98 octopuses),
when animals were approx. 14 days old. Subsequent 24-hr |
long observation periods were made on November 11, 1998 |
(N = 88 octopuses) and November 17, 1998 (N = 87 octo- |
puses). Lor each 24 hr period, counts of ‘active’ octopuses |
were made on each tub once per hour at the beginning of |
each hour, beginning at 5 PM on each date. An octopus was |
considered ‘active’ if it was crawling, swimming, or sitting |
outside of cover (at least 50% of its body). The number of |
‘inactive’ octopuses for each count was calculated by sub- J
tracting the number of active octopuses counted from the /
total number of animals within each tank, which had been
assessed the day previous to observations by removing all
cover and counting total octopuses. The proportion of oc- ^
topuses active across both tubs was then calculated by di- (
viding the total number of active animals by the total num-
ber of active and inactive animals. This method resulted in
twenty-four observations per tub per observation day.

A nocturnal versus diurnal activity cycle was tested by
examining the proportion of active individuals during day-
light hours (7 AM to 5 pm) versus nighttime ones (5 pm to 7
am) for each week. To examine whether octopuses were
crepuscular, mean proportions of active individuals were
compared across two time periods for each week, the first
representing dawn/dusk periods (6 AM to 8 AM and 4 PM to
6 pm) and the second period representing all other hours (8
AM to 4 PM ancJ 6 pm to 6 am). Temporal autocorrelation
between observations within a tub and an unbalanced sta-
tistical design precluded use of hypothesis tests. Lor example,
activity at one observation period was probably not inde-
pendent of activity during another, and 6 observation times
were taken to compute a mean proportion for dawn/dusk
activity while 18 observations were used to compute mean
activity during ‘all other times’. Thus, for statistical com-
parisons, the mean proportion of active octopuses for the
appropriate time period was calculated for each week and
graphed along with 95% confidence intervals. Non-
overlapping 95% confidence intervals of means were used as /;
a conservative estimate of statistically significant differences
because a comparable statistical test would always indicate a
statistically significant difference at P < 0.05 for two non- ,:l
overlapping 95% confidence intervals (Payton et al. 2003). ,;i

RESULTS >

There was little evidence to support a crepuscular ac- |
tivity cycle in juvenile Octopus bimacidoides. The mean pro- |
portion of octopuses active during dawn/dusk periods and I
all other time periods was not different for any of the three ' i
observation weeks (Lig. 1 ). Instead, juveniles on average ex- , ]
hibited nocturnal activity cycles, as the mean proportion of / i

ACTIVITY IN JUVENILE OCTOPUS

67

Figure 1. Mean proportion of active juvenile Octopus binmculoides
during dawn/dusk (6 am to 8 am and 4 pm to 6 pm) versus all other
time periods over three weeks. Error bars represent 95% confidence
intervals.

active juvenile octopuses tvas greater during nighttime hours
than daylight ones in two out of the three weeks (Fig. 2). The
activity cycles could be characterized qualitatively each week
by an increase in activity which coincided with sunset peri-
ods; this activity generally peaked at midnight, after which
time there was a steady decrease in activity until the next
day’s sunset (Fig. 3).

DISCUSSION

This is the first report of activity cycles in juvenile Cali-
fornia two-spot octopuses, Octopus bimaculoides. Under
natural daylight conditions and constant food availability,
juvenile animals tended to be nocturnal in their activity. This
tendency could be potentially adaptive for individuals if pre-
dation risk for juvenile octopuses were greater in daylight
than nighttime. High numbers of visual predators active
during daylight hours [e.g., teleost fish) may favor juvenile
octopuses which are active during nighttime hours (Aronson
1991, Hanlon and Messenger 1996). However, intra- and
interspecific competition with other octopuses through
niche overlap should also influence activity cycles and would
favor temporal spacing of time budgets between populations
(Houck 1982, Meisel et al. 2006). Octopus bimaculoides oc-

^ Daytime
□ Nighttime

Figure 2. Mean proportion of active juvenile Octopus bimaculoides
during nighttime hours (5 pm to 7 am) versus daylight hours (7 am
to 5 pm) over three weeks. Error bars represent 95% confidence
intervals.

Time

Figure 3. Percentage of juvenile octopuses that was active during
each 5-minute monitoring period during each hour. Solid vertical
lines represent average sunrise (mean = 7:01 am, SO = 13 min) and
sunset (mean = 4:48 pm; SD = 12 min). Dashed vertical lines rep-
resent dawn (6 to 8 am) and dusk (4 to 6 pm) periods. Sample sizes
varied by week (week 1: N = 98; week 2: N = 88; week 3: N = 87).

curs sympatrically along its range with Octopus bimaculatus,
and these two sister species most likely occupy similar niches
(Pickford and McConnaughey 1949). Some populations of
adult O. bimaculatus can be nocturnal (Ambrose 1982), and

68

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

nocturnal activity in adult O. bimaculoides in the laboratory
has also been observed (Sinn 2000). Taken together, these
reports suggest that juvenile O. bimaculoides may face mul-
tiple, conflicting selection pressures influencing their activity
patterns. Clearly, further work is needed on the relative in-
fluences of predation risk and niche overlap on the activity
cycles of juvenile octopuses.

Even in daytime periods, at least some octopuses were
active, and during one observation week, there were no dif-
ferences between activity in daytime and nighttime hours.
One explanation could be that metabolic demands for fast-
growing juvenile cephalopods are high (Lee 1994), requiring
animals to feed throughout a 24-hr cycle. Two other envi-
ronmental cues which may have influenced the activity
cycles of juvenile octopuses, namely food availability and
octopus density, are also worth further consideration. For
example, food availability influences adult activity in other
Octopus species, with ad libitum feeding resulting in a lack ot
a discernable 24-hr cycle in adult Octopus vulgaris (Wells et
al. 1983). While constant food availability was chosen here
to maximize juvenile survivorship, in the wild, food avail-
ability may not be limiting for juvenile octopuses (e.g., O.
bimaculatus: Ambrose 1988). Second, octopus densities may
also have influenced individual activity cycles if densities in
tubs were unnaturally high. Increased density may have in-
creased aggressive interactions with conspecifics during peak
activity hours {i.e., nighttime) and resulted in some indi-
viduals becoming active during daylight (e.g., Sinn et al.
2001). Nothing is known concerning natural densities of
juvenile O. bi})mculoides, but adult individuals of O. bimacu-
loides have been reported to spatially group in high densities
in suitable habitat in the wild (Lang 1997).

From a practical standpoint, the environmental circum-
stances experienced by octopuses in the current study were
probably similar to culturing conditions other researchers
employ when studying juvenile octopuses (i.e., constant food
availability to ensure low mortality rates). Understanding
the basic activity cycles of laboratory animals is necessary to
properly perform experimental manipulations and to recog-
nize ‘abnormal’ behavior which could indicate sick or dying
animals (Moltschaniwskyj et al. 2007). Unfortunately, de-
tailed study of cryptic, juvenile octopuses in the wild will
probably remain intractable for some time. Therefore, infer-
ences based on laboratory reports, taken with caution, re-
main the sole information available to understand the inter-
action between circadian rhythms and the juvenile ecology
of many Octopus spp.

This study is a first step toward quantifying activity
cycles in the juvenile life stages of Octopus bimacidoides and
provides a baseline for studying juvenile life stages both in
the laboratory and field. Understanding the activity cycles of
different life-history stages of Octopus spp. under natural or

semi-natural conditions should contribute to our under-
standing of the ecological costs and benefits that arise from
an animal taking a particular activity strategy under a given
set of conditions (Sinn et al. 2001). Further work is clearly
needed on the influences of competition and predation risk
on octopus activity cycles and the resulting population- and
species-specific outcomes (Ambrose 1982, 1986).

ACKNOWLEDGMENTS

Chuck Winkler provided female Octopus bimacidoides
with eggs for this project, and Mark Stapleton helped in data
collection. Leonard Simpson and the Department of Biology
at Portland State University, Oregon, provided essential
laboratory and financial support for this project. Russell
Thomson made helpful suggestions with regards to data
analysis. Roland Anderson and Jennifer Mather provided
constructive criticism on an earlier draft of this manuscript,
and two anonymous reviewers made helpful suggestions on
a later version.

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Submitted: 1 November 2006; accepted: 26 June 2007;
final corrections received: 29 November 2007

p.

I

¥f

*-

’IT-

Amer. Mahic. Bull. 24: 71-77

Discovery of the South African polyplacophoran Stenosemus simplicissimus (Thiele,
1906) (Mollusca, Polyplacophora, Ischnochitonidae) in the Southern Ocean

Enrico Schwabe

Zoologische Staatssammlung Miinchen, Mtinchhausenstrasse 21, D-81247 Mtinchen, Germany, enrico.schwabe@zsm.mwn.de

Abstract: Recent expeditions to the Atlantic sector of the Southern Ocean have yielded valuable collections of shelf and deep water
polyplacophorans. These included several specimens of Stenosemus simplicissimus (Thiele, 1906), a species previously known only by its
I holotype and type locality at the Cape of Good Hope. The new material enabled a thorough morphological redescription of the species hy
studying valve, perinotum, and radula characters with SEM. The new records from Shag Rocks and the eastern Weddell Sea enlarge the
species’ biogeographic distribution from the temperate South African region to the polar South Georgia and Weddell Sea regions. Its
I bathymetric range is extended from 318 m to 285-1064 m. The limited occurrence of deep-water Antarctic polyplacophorans may be caused
j by benthic predators that limit the expansion of non-herbivorous chitons in the Antarctic deep sea.

i Key words: Atlantic Ocean, Antarctica, zoogeography, distribution, new records

: Antarctic waters support a small number of Polyplaco-

phora in contrast to the highly diverse fauna of other marine
molluscs (Thiele 1912, Dell 1990, Numanami 1996, Sirenko
and Schrodl 2001). The species discovered to date are: Lep-
tochiton kergiielensis Haddon, 1886, Callocliiton bouveti
Thiele, 1906, Callochiton gaussae Thiele, 1908, Leloupia hel-
gicae (Pelseneer, 1903), Stenosemus exaratus (G. O. Sars,

, 1878), Stenosemus simplicissimus (Thiele, 1906), Tonicina zs-
; chaui (Pfeffer in von Martens and Pfeffer, 1886), Nuttallo-

i chiton mirandus (E. A. Smith MS, Thiele, 1906), and Hemi-
1 athrum setulosum Carpenter in Dali, 1876. With the

exception of S. simplicissimus, all species are more or less well
described in earlier revisions {e.g., Thiele 1906a, 1906b, 1908,
Dell 1964, Kaas and Van Belle 1985a, 1985b, 1990, Getting
i; 1993). The recent rediscoveries of this species, which was
:: known only from the type material, enable a detailed mor-
jj phological description using scanning electron microscopy

ii (hereafter, SEM). The description will help non-chiton spe-
cialists to separate this species from similar representatives of

; the genus Callochiton Gray, 1847. In addition, deep water
! polyplacophorans from Antarctica are rare and our knowl-
i edge of their biology and habitat preference is limited.
Analysis of abiotic parameters at a certain depth may help
getting a better understanding of how chitons interact with
' their environment. The present paper deals with the un-
known Antarctic deep-water chiton fauna.

MATERIALS AND METHODS

Specimens of Stenosemus sitnplicissinms were collected
during recent expeditions with R/V Polarstern (ANT XIII/3,
ANT XVII/3, ANT XXI/2, and ANT XXII/3) to the Scotia

and eastern Weddell Seas. The material was obtained by
using Agassiz trawls (AGT), bottom trawls (BT), epibenthic
sledges (EBS), and Rauschert dredges (RD). When the catch
reached the deck, the samples were sieved through 500 pm
or 1000 pm mesh, the remainder was fixed in 4% buffered
formaldehyde or 75-96% ethanol and then sorted under ste-
reomicroscopes. Most of the polyplacophorans were found
attached to hard substrates, such as cobbles. Specimens were
stored in ethanol for further morphological examinations.
To confirm the identification of S. simplicissimus, the holo-
type was examined.

The type material is deposited at the Natural History
Museum Berlin, Germany (ZMB). The newly collected ma-
terial is deposited at the British Antarctic Survey, United
Kingdom (BAS), the Zoological Institute St. Petersburg,
Russia (ZISP), and the Bavarian State Collection of Zoology,
Germany (ZSM).

Preparation of the specimens followed Schwabe and Ru-
thensteiner (2001). Specimens used for SEM were partly
disarticulated, enabling examination of valves, perinotum,
and radula. Microsculpture and radular photographs were
made on a LEO 1430VP SEM (at the ZSM). Abiotic factors
were established using a conductivity-temperature-depth
(CTD) data logger, or by visual inspection of the substratum
on board of the research vessel.

SYSTEMATICS

Class Polyplacophora Gray, 1821
Subclass Neoloricata Bergenhayn, 1955
Order Chitonida Thiele, 1910
Family Ischnochitonidae Dali, 1889

72

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

Figure 1. Light micrographs of the holotype of Stenosemus simplicissimiis, ZMB Moll 59908.
A, valves ii to iv in situ, anterior at right; B, detail of fig. lA, showing the dorsal perinotum
scales; C, valves (from left to right) viii, v, i in dorsal view, anterior at right; D, right lateral
view of the tail valve, anterior at right. Scale bars; A, 5 mm; B, D, 1 mm; C, 500 pm.

Genus Stenosemus von Middendorff, 1847
Type species: Chiton albiis Linnaeus, 1767, by subsequent
designation, Winckworth (1926: 15)

Stenosemus simpUcissimus (Thiele, 1906)

(Figs. 1-5)

Ischnochiton (Chondropleura) simpUcissimus Thiele
1906b: 335, pi. 29, figs 21-25.

Additions to the bibliography in Kaas and Van Belle
(1990: 67):

Ischnochiton simpUcissimus; Barnard 1974: 740.
Ischnochiton (Stenosemus) simpUcissimus; Kaas and Van
Belle 1980: 120; 1990: 67, fig. 27; 1998: 171.
Ischnochiton (Chondropleura) simpUcissimus; Kilias
1995: 169.

Stenosemus simpUcissimus; Sirenko 1994: 164; 2005: 36;

Gutt et al. 2000: 40; Linse et al. 2006: 155 (partim).
Type material: ZMB Moll 59908 (partly disarticulated
holotype) (Figs. lA-D).

Type locality: South Africa, Cape of Good Hope, Deut-
sche Tiefsee-Expedition St. 113: 34°33.3'S
18°21.2'E, 318 m.

Additional material examined

ZSM Mol 20050857 ( 1 specimen -
3.4 X 2.2 mm, partly disar-
ticulated) (Figs. 2-3), Antarc-
tica, Weddell Sea: ANT
XXII-3 (ANDEEP III) St. PS
67/074-7: 71°18.60'S
13°59.1 1 'W-71°18.38'S
13°58.17'W, 1047-1064 m, on
rock (quartz-amphibolite
gneiss), laying on a sandy
sediment (only 5 cm thick)
(salinity: 34.7 psu; water tem-
perature: 0.5 °C; O2 concen-
tration: 5.8 ml/1; pressure:
1016.5 dBar; all data from
1004 m, measured by CTD),
AGT, collected by J. M. Bohn
and E. Schwabe, 20 February
2005, preserved in 96%
ethanol.

ZSM Mol 20020914 (1 specimen -
width 3.1 mm [curled]), Shag
Rocks, South Georgia and
South Sandwich Islands: ANT
XIX-5 (LAMPOS) St. PS 61/
169-1: 53°22.94'S 42°41.37'W-
53°22.89'S 42°41.50'W,
284.3 m, RD, collected by Dr. M. Schrodl, 10 April
2002, preserved in 78% ethanol.

ZSM Mol 20008502 ( 1 specimen - 8.7 x 4.2 mm, partly
disarticulated) (Fig. 4), Antarctica, Weddell Sea:
ANT XVII-3 (EASIZ III) St. 97-1: 71°06.27'S
12°50.46'W-71°06.24'S 12°49.92'W, 728-743 m,
EBS, collected by Dr. M. Schrodl, 3 April 2000,
preserved in 78% ethanol.

BAS (Dr. Katrin Linse) 03-802 (1 specimen - width 3.8
mm [curled] ), Antarctica, Weddell Sea: ANT XXI-2
(BENDEX) St. PS 65/324-1: 72°54.52'S
19°47.74'W-72°54.55'S 19°47.30'W, 647.2-693.6
m, RD, collected by Dr. K. Linse, 3 January 2004,
preserved in 96% ethanol.

BAS (Dr. K. Linse) 03-769 (1 specimen - width 3.4 mm
[curled]), Antarctica, Weddell Sea: ANT XXI-2
(BENDEX) St. PS 65/297-1: 72°48.50'S
19°31.66'W-72°48.65'S 19“31.85'W, 630.8-668 m,
RD, collected by Dr. K. Linse, 1 January 2004, pre-
served in 96% ethanol.

Description

Species moderately large, up to 16 mm (the largest
specimen is the holotype). It is elongate, oval, with a cari-

STENOSEMUS SIMPLICISSIMUS IN SOUTHERN OCEAN

73

Figure 2. Stenosemus simplidssvmis (ZSM Mol 20050857), 3.4 X 2.2 mm. A-B, light micro-
graphs, C-F, scanning electron micrographs. A, ciorsal view of the complete specimen, an-
terior at right; B, ventral view of the complete specimen, anterior at right; C, dorsal view ol
the head valve; D, dorsal view of valve ii; E, dorsal view ot the tail valve; F, left lateral view
of the tail valve, anterior at left. Scale bars A-B: 1 mm, C-F: 500 pm.

nated, moderately high-elevated dorsum. Dorsal elevation
quotient (height/width) (of isolated valve v of the holotype):
0.51. Color of tegmentum and perinotum uniform dull
white.

Tegmentum virtually smooth, except for commarginal
growth marks, which occur on all valves (Figs. lA-D, 2C-F,
4A-D) and a micro-perforation. In earlier growth stages, fine
radial striation is visible in apical regions and the middle of
the first valve. The head valve has a wide V-shaped posterior
margin and is clearly notched in the middle (Figs. 1C, 2C,
4A). Intermediate valves (Figs. lA, 1C, 2D, 4B) are trapezoid

(valve ii) to rectangular, with short and
rounded side margins, and straight to
slightly concave posterior margins (on
both sides of the slightly protruding
apex). Anterior valve margin is convex.
Lateral areas are dearly elevated. Tail
valve (Figs. IC-D, 2E-F, 4C-D) is
semicircular with an anteriorly di-
rected, weakly elevated mucro that is
situated in the two anterior thirds of
the valve length. Postmucronal slope is
steep and straight.

Articulamentum is thin and white.
Apophyses (Figs. lA, IC-D, 2D-E, 4B-
D) are well developed, rather short,
wide, and medially connected by a
short smooth jugal lamina. Apophyses
are triangular in intermediate valves,
and rectangular in tail valve. Slit for-
mula varies from 14/1/10 (holotype) to
16/1-2/13 (8.7 mm long specimen,
ZSM Mol 20008502). Slits are wide
and rather long, teeth edges are slightly
thickened and faintly crenulated. Slit
rays are present in all valves. Eaves are
spongy.

Perinotum is rather narrow (Figs.
lA-B), dorsally covered with juxta-
posed, bent, conical, round-topped,
and inwardly directed calcareous
scales, 160-176 pm long, 144-150 pm
in diameter on mid-perinotum, rhom-
boidal at the base, and weakly longitu-
dinally striated (Kaas and Van Belle
1990). Towards the outer margin, the
scales are smaller and measure 80-90 X
50-57 pm (Fig. 3B). Marginal fringe
(Fig. 3C) consists of straight, obtusely-
pointed solid spicules, which are me-
dially keeled and measure about 1 10 x
22 pm. Among them are smaller,
straight, and sharp spicules, 50 pm in length and 10 pm in
width. These spicules are situated distally either on long, veiy
slender shafts ( 140 x 7 pm) or in shorter tubs that may attain
a diameter ot 15 pm (Fig. 4E). Ventrally there are radial rows
of rectangular scales that measure 53 x 27 pm (Fig. 3D).

Radula (Fig. 3D) of a partially disarticulated specimen
(ZSM Mol 20008502) measures 3.3 mm, of which 1.6 mm
were taken up by the radula cartilage, with 44 teeth rows ot
which 31 are mineralized. Central tooth is rectangular, its
distal end a little wider than the base. It measures 71x31 pm
long and has a forward-directed simple, slightly bent cusp.

74

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

Figure 3. Scanning electron micrographs of Stenosemus simplicissimiis (ZSM Mol 20050857),
3.4 X 2.2 mm. A, detail of Fig. 2F, showing the valve microsculpture; B, dorsal perinotum scales
at the margin; C, marginal perinotum fringe; D, ventral perinotum scales. Scale bars: 50 pm.

First lateral tooth is wing-shaped and covers the lower two
thirds of central tooth. It may attain a length of 60 pm and
has a simple inward directed small cusp. Total length of
second lateral tooth is 175 pm; one third of its length is taken
up by the squarish head with the single, sharply pointed, and
inwardly curved elongate denticle. Shaft of second lateral
tooth is sharply keeled on its inner side and slightly curved
in the upper half. Basally the tooth is wing-shaped. First
uncinal tooth is triangular in outline with a straight inner
edge and a steep slightly convex, outer edge. Second uncinal
tooth is very broad and S-shaped. Third uncinal tooth is
extremely slender, measuring 138 x 18 pm, with a spoon-
shaped distal extension, which may attain the double width
of the shaft. First marginal tooth is similar to, but slightly
more slender than, the second uncinal. Second marginal
tooth is arrowhead-shaped, measuring 71 x 31 pm. Third
marginal tooth is rectangular in outline, 75 pm long, and 46
pm wide. Its inner edge is thickened.

Ctenidia are arranged merobranchially with the longest
ctenidium on each side positioned the fourth from the pos-
terior end. Size and number of ctenidia depend on animal
size. The 8.7 mm long specimen has 16 ctenidia on the right
and 17 on the left side; the juvenile (3.4 mm) has 7 ctenidia
on each side of the foot (Fig. 2B).

Since its description, Stenosemus
simplicissimiis was never recollected.
Several expeditions to the Subantarctic
Marion and Prince Edward Islands
failed to locate this species, although |
the congeneric Stenosemus exaratus \
(G. O. Sars, 1878) [reported as Steno-
semus dorsuosus (Haddon, 1886)] was
found together with the following spe-
cies: Leptochiton kerguelensis Haddon,
1886, Hemiathrum setulosum Carpen-
ter in Dali, 1876, and Placiphorella sp.
(Branch et al. 1991). Leptochiton ker-
guelensis and H. setulosum are typical
faunistic elements of the Antarctica; :
the latter is Placiphorella atlantica (Verrill and S. I. Smith in ]
Verrill, 1882) (pers. obs.). \

Stenosemus simplicissimiis was first rediscovered during ■
the ANT XIII-3 (EASIZ I) - Antarctic expedition at St. 01:
71°03.10'S 11°25.50'W, at 462 m (Gutt et al. 2000). During
subsequent expeditions, the species was found again but
only in small numbers.

The Weddell Sea supports a high diversity of mollusc
species and, not surprisingly, their numbers decrease with
increasing depth, along with a significant decrease in total
biomass (Brey and Gerdes 1997, 1998). During the ANDEEP
III expedition (from the Cape Basin to Kapp Norvegia and
across the Weddell Sea), 186 mollusc morphospecies (3801
specimens from 12 EBS and 19 AGT stations) were collected
from depths ranging from 1000 to 4900 m (Linse et al.
2006).

In contrast to former expeditions in this area of com-
parable scope (Sirenko and Schrodl 2001), only two poly-
placophoran specimens were found, the herein mentioned
juvenile of Stenosemus simplicissimiis and Leptochiton ker-
guelensis (ZSM Mol 20060001 ). The latter specimen was col-
lected at station PS67/133-2 (62°46'44"S 53°02'34"W-
62°46'20"S 53°04'08"W) at 1581-1582 m depth. This is the
maximum depth reported for this species, which was for-

Distribution

Known from the Cape of Good
Hope (type locality). Shag Rocks near
South Georgia Island and eastern [
Weddell Sea (this study) (Fig. 5). !i

Bathymetrically the species lives be- h

tween 284-1064 m depth (Thiele i
1906b, Gutt et al. 2000, Sirenko and ^
Schrodl 2001, Linse et al. 2006). '

!

DISCUSSION i

STENOSEMUS SIMPLICISSIMUS IN SOUTHERN OCEAN

75

I

I

I

i

I

Figure 4. Scanning electron micrographs of Stenosermis siiuplicissimus (ZSM Mol 20008502),
8.7 X 4.2 mm. A, dorsal view of the head valve; B, dorsal view of valve ii; C, dorsal view of
the tail valve; D, right lateral view of the tail valve, anterior at right; E, dorsal perinotum
scales, close to the margin; F, rows 3-8 of the radula. Scale bars A-D: 500 pm, E-F; 100 pm.

merly only known from 1335 m in the Ross Sea (Dell 1990).
A bulk of stones was found during the recent expedition, a
substratum that allows settlement of chiton larvae. That they
could be covered by sediment can be excluded because Paul
(1976) has shown that chitons are able to remove sediment
layers. Is the extremely low chiton diversity and density in
the Antarctic merely a function of the great depth or related
abiotic environmental conditions? Temperature is unlikely
to be a problem as it does not change with depth (Brey and
Gerdes 1998). The increase in water pressure can also be
ruled out as diverse deep sea chiton faunas are known from
other regions (Sirenko 2001). Most deep water chitons are

I thank Olaf Klatt (Alfred Wege-
ner Institute for Polar and Marine Re-
search, Bremerhaven, Germany), Dr.
Lawrence W. Carpenter (School of
Marine Science, Virgina Institute of
Marine Science, Virginia, USA), Rob
Middag (Koninklijk Nederlands Insti-
tuut vor Onderzoek der Zee, Den
Brug, The Netherlands), Dr. Michael Thompson (School of
Earth Sciences, University of Leeds, Leeds, UK), and Dr.
John A. Llowe (Scottish Association for Marine Science, Ar-
gyll, UK) for providing physical and chemical data of the
ANDEEP III station. All involved sorting teams and the crew
of R/V Polarstern are warmly thanked for their professional
work. Dr. Katrin Linse (BAS) is especially acknowledged for
providing the material reported herein and for helpful com-
ments on an earlier version of the manuscript. Dr. Douglas
Eernisse (California Sttrte Llniversity, Fullerton, USA) kindly
corrected the English and provided supportive remarks.
Thanks are also due to Dr. Matthias Glaubrecht and Frank

dependent on plant remains (c.^.,
sunken wood) that may not found in
the Antarctic deep sea. The high or-
ganic food input (Brey and Gerdes
1997) in the Weddell Sea, together
with abiotic conditions should favor
colonization by non-herbivorous chi-
tons. Several carnivorous taxa such as
asteroids, ophiuroids, and polychaetes
are highly adapted to the conditions of
the Weddell Sea (Brey and Gerdes
1997, 1998). The chitons may lack the
ability to co-occur with abundant
predators such as the asteroids, which
feed on chitons (Seiff 1975).

In summary, this report gives a
detailed description of Stenosemus siw-
plicissiimis, contributes the first record
of the species’ juvenile stage, and ex-
tends its bathymetric range and that of
Leptochitoit kerguelensis, thus provid-
ing a further piece of the unfinished
puzzle documenting Antarctic deep
sea communities. The material ob-
tained to date for S. siiuplicissimus sug-
gests that it could be geographically re-
stricted to the Southern Atlantic
Ocean.

ACKNOWLEDGMENTS

76

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

Figure 5. The geographical distribution of Stenosemus
simpUcissimus.

Kohler (ZMB) for the loan of type material. I especially
thank two anonymous reviewers for their helpful comments
and constructive criticism. The authors participation to
ANDEEP III was kindly supported by GeoBioCenterLMU.
This is ANDEEP contribution No. 61.

LITERATURE CITED

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Brey, T. and D. Gerdes. 1997. Is Antarctic benthic biomass really
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Dell, R. K. 1964. Antarctic and subantarctic Mollusca: Amphineura,
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Dell, R. K. 1990. Antarctic Mollusca with special reference to the
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Gutt, E., B. I. Sirenko, W. E. Arntz, I. S. Smirnov, and C. De Broyer.
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cies (demersal fish included) sampled during the expedition
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Kaas, P. and R. A. Van Belle. 1980. Catalogue of Living Chitons
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Kaas, P. and R. A. Van Belle. 1985a. Monograph of Living Chitons
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Kaas, P. and R. A. Van Belle. 1985b. Monograph of Living Chitons
(Mollusca: Polyplacophora) 2, Suborder Jschnochitonina, Isch-
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Kaas, P. and R. A. Van Belle. 1990. Monograph of Living Chitons
(Mollusca: Polyplacophora). 4, Suborder Jschnochitonina: Isch-
nochitonidae: Iscluiochitoninae (continued). Additions to vols 1,
2 and 3. E. J. Brill, Leiden, The Netherlands.

Kaas, P. and R. A. Van Belle. 1998. Catalogue of Living Chitons
(Mollusca, Polyplacophora). 2nd, revised edition. Backhyus
Publishers, Leiden, The Netherlands.

Kilias, R. 1995. Polyplacophora-Typen und -Typoide (Mollusca)
im Zoologischen Museum in Berlin. Mitteilungen aiis dem
Zoologischen Museum in Berlin 71: 155-170.

Linse, K., E. Schwabe, and A. Brandt. 2006. Mollusca collected
during ANDEEP III - preliminary results. Berichte zur Polar-
und Meeresforschung 533: 151-160.

Numanami, H. 1996. Taxonomic study on Antarctic gastropods
collected by lapanese Antarctic research expeditions. Memoirs
of the National Jnstitute of Polar Research (E, Biology and
Medical Science) 39: 1-244.

Paul, A. Z. 1976. Deep-sea bottom photographs show that benthic
organisms remove sediment cover from manganese nodules.
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Schwabe, E. and B. Ruthensteiner. 2001. Callochitoti schilfi (Mol-
lusca: Polyplacophora: Ischnochitonidae) a new species from
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Seiff, S. R. 1975. Predation upon subtidal Tonicella lineata of Mus-
sel Point, California (Mollusca: Polyplacophora). The Veliger
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Sirenko, B. I. 1994. Chitons (Polyplacophora) of the continental
slope of the Kurile Islands with a brief review of deep water
species of the Russian Seas. Russian Academy of Sciences Zoo-
logical Jnstitute, Explorations of the Fauna of the Seas 46: 159-
174.

Sirenko, B. I. 2001. Deep-sea chitons (Mollusca, Polyplacophora)
from sunken wood off New Caledonia and Vanuatu. In:
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thos, vol. 22. Memoires dii Musann national d'Histoire naturelle
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(Polyplacophora) in bathyal zone near New Caledonia. In:
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STENOSEMUS SIMPLICISSIMUS IN SOU'l’HERN OCEAN

77

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ANTARKTIS XlII/3 (EASIZ I) of “Polarstern” to the eastern
Weddell Sea in 1996. Berichte znr Polar- and Meeresforschung
402: 85-95.

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Accepted: 24 July 2007; final corrections received:

27 November 2007

Amer. Maine. Bull. 24: 79-89

Imposex level and penis malformation in Hexaplex trunculus from the
Tunisian coast

Youssef Lahbib, Sami Abidli, and Najoua Trigui El Menif

Laboratory of Environment Bio-monitoring (LBE), Department of Biology, University of 7''’ November at Carthage, Faculty of Sciences
of Bizerte, 7021 Zarzouna, Tunisia, lahbibyoussef@yahoo.fr and Youssef.Lahbib@fsb.rnu.tn

Abstract: Hexaplex trunculus (Linnaeus, 1758) is a gonochoric marine gastropod. Previous studies demonstrated that the biocide TBT
(tributyltin) induced a sexual abnormality known as imposex (superimposition of male sexual characters onto females) in this whelk. Our
study showed imposex in 19 stations out of 20 along the Tunisian coast. The frequency of imposex ranged from 0 to 100%. Among the
19 sites where the condition was found, 8 were considered as highly affected by imposex (VDSl > 3.7), 6 were moderately affected
(VDSI > 1.3), and 4 were slightly affected (VDSI > 0). The most affected population was obseiwed in the Bizerta Channel where the highest
boating traffic was recorded; no imposex features were found in the Sea of Zarat where boating traffic was veiy low. Significant differences
in imposex levels were obtained among sites with low, moderate, and high boating traffic. All the imposex indices values (1%, RPSI, RPLl,
VDSI, FPL, and VDL) were significantly more elevated at sites with high boating traffic compared with sites with low and moderate boating
traffic. Malformations of the penis were observed only in five stations and in very low rates, but where imposex rates were high. The
incidence of penis malformation in males was significantly related to the boating traffic, 1%, and VDSI. However, in females, a correlation
was obtained only for the RPLL The present study provides data on imposex level and penis malformations in H. trunculus from the
Tunisian Coast that could be used as a starting point for future monitoring programs and for temporal trend surveillance related to TBT
pollution in Tunisia where the use of TBT is not yet banned.

Key words: Muricidae, TBT-biomarker, marine pollution, Tunisia

Imposex (Smith 1971) or pseudo-hermaphroditism
(Jenner 1979) is the development of male sexual character-
istics (i.e., a penis and/or a sperm-duct) in female proso-
branch gastropods. The active biocide used in anti-fouling
paints, TBT, was suspected to be the major cause of imposex
since a direct correlation with shipping intensities was es-
tablished (Bryan et al. 1986, Gibbs and Bryan 1986, Ten
Hallers-Tjabbes et al. 1994, Axiak et al. 1995, Harino et al.
1998, Rilov et al. 2000, Fernandez et al. 2002). In contrast,
even though there is little doubt that TBT has been the main
cause of imposex in gastropods, it may be not the sole cause.
Nias et al. (1993) reported that exposure to copper induces
imposex. The same condition was also noted by Evans et al.
(2000) in Nucella lapilliis (Linnaeus, 1758) exposed to the
estrogen-mimic nonylphenol. However, Davies et al. ( 1987)
found imposex in 12% of the N. lapilliis from a non-polluted
site and considered it a natural phenomenon. Imposex has
now been observed in about 150 gastropod species (Oehl-
mann et al. 2000).

In Hexaplex trunculus, observations on imposex were
first reported along European coasts by Martoja and Bou-
quegneau (1988) in France, Axiak et al. (1995) in Malta,
Terlizzi et al. (1998) in Italy, Vasconcelos et al. (2006) in
Portugal, Rilov et al. (2000) in Israel, and Lahbib et al.
(2004) in Tunisia. Some authors have indicated the devel-

opment of malformations of the penis in males as well as in
females of some gastropod species, namely Hinia reticulata
(Linnaeus, 1758) (Stroben et al. 1992), H. trunculus (Terlizzi
et al. 1998, Vasconcelos et al. 2006), and Bolinus brandaris
(Linnaeus, 1758) (Ramon et al. 2001 ). However, descriptions
of the penis malformations in H. trunculus were limited. The
aim of the present work was ( 1 ) to provide data on imposex
levels along the Tunisian Coast and (2) to describe malfor-
mations of the penis in H. trunculus.

MATERIALS AND METHODS

Adult Hexaplex trunculus, with shells of 40 to 60 mm in
height and a sample size of 44 to 1 50 specimens per station,
were collected between March and luly 2004 from Tunisian
coastal waters. Twenty locations were chosen according to
the intensity of the marine traffic, from Bizerta to Djerba
(Table 1, Fig. 1). For each zone, both the type of boating
activity and the boat density were recorded. The number ot
working fishing boats in each area was obtained from annual
statistical data of fishing activity in Tunisia in 2004 provided
by the Ministiy of Agriculture, or directly in some sites from
fishermen. In sites with commercial activity, the number and
type of boats were obtained from the annual report of the
Tunisian commercial marine and harbors office in 2003.

79

80

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

Table 1. Collection data at the various stations. BT, boat type (F, fishing boat; P, passenger liner; M, merchant ship; O, oil tanker;
ferry-boat); FD, fishing-boat density expressed in number of working boats in the area in 2004; CD, commercial boat traffic expressed in
number of boats to/from the area in 2003; TC, traffic category (H, high; Md, moderate; L, low; site with low boating density but located
nearby a commercial traffic line or harbor); M, male; F, female; N, number of individuals; SL, average shell length (mm); 1%, imposex
frequency; PL, average penis length (mm); RPSI, relative penis size index; RPLI, relative penis length index; VDSI, vas deferens sequence
index; VDSr, vas deferens sequence range; VDL, mean vas deferens length (mm).

Site

BT

FD

CD

TC

Sex

N

SL

1 %

PL

RPSI

RPLI

VDSI

VDSr

VDL

1. Bizerta Channel

FPMO

505

1014

H

M

63

41.7

12.05

F

37

46.5

100

8.23

33.03

69.13

4.24

4-5

12.17

2. Quarries Bay

F

120

—

H

M

26

41.9

11.81

F

37

49.2

100

7.77

28.47

65.79

4.09

4-4.3

12.29

3. Menzel Abderrahmen

F

150

—

H

M

33

44.9

14.64

F

29

41.7

100

5.18

5.13

37.16

3.73

3-4.7

12.12

4. Menzel Bourguiba

F

30

SA

H

M

29

52.9

12.25

F

27

55.7

100

3.54

2.69

29.96

3.97

3-4.7

12.20

5. Menzel lemil

F

60

—

Md

M

20

44.1

13.56

F

40

43.1

66.6

0.28

0.00

2.05

1.31

0-4.3

3.53

6. El Azib

F

50

—

Md

M

29

43.9

14.33

F

21

46.2

62.0

0.37

0.00

2.52

1.28

0-3

2.64

7. Tunis North Lake

FPM

78

2444

H-'

M

14

48.6

17.41

F

30

49

100

3.60

0.88

20.68

3.97

2-4.3

12.90

8. Small Gulf of Tunis

FPM

0

3001

H*

M

48

55.8

14.32

F

42

56.6

85.7

1.38

0.08

9.63

3.27

0-4.7

11.69

9. Khniss Lagoon

F

64

—

L

M

43

45.9

17.49

F

25

47.1

40

0.17

0.00

0.01

0.75

0-2

2.03

10. NPK Sfax

M

—

SA

H

M

51

42

10.31

F

30

44.1

100

5.63

16.98

55.38

4.00

4-4.3

12.91

11. Sfax Fishing Harbor

F

736

—

H

M

14

51.1

24.72

F

32

52.3

100

8.16

3.62

33.08

4.21

4-4.7

13.19

12. Gargour

F

30

—

H*

M

65

44.8

10.29

F

72

43.1

93.0

0.83

0.05

8.00

3.32

0-4

11.24

13. Skhira

FO

38

236

H

M

38

48.5

15.36

F

32

50.2

84.4

1.06

0.03

6.96

2.53

0-4

8.26

14. Gabes Fishing Harbor

F

108

—

H

M

46

52.5

14.78

F

33

54.2

51.5

1.60

0.02

5.48

1.82

0-4

5.91

15. Sea of Zarat

F

10

—

L

M

70

52.1

13.86

F

80

54.9

0.0

0.00

0.00

0.00

0.00

0

0.00

16. Adjim Channel

Fh

244

4

H

M

18

50.2

18.20

F

37

53.9

100

5.80

2.97

31.02

4.09

3-4.7

13.13

17. Gigthis-Djorf

F

20

—

L

M

62

42.9

9.16

F

81

46.6

6.2

0.02

0.00

0.09

0.13

0-3

1.70

18. Gigthis

F

52

—

Md

M

61

42.8

13.14

F

88

43

19.7

0.07

0 00

0.29

0.60

0-4

2.05

19. Guallala

F

10

—

L

M

26

42.9

14.28

F

27

46.6

3.7

0.00

0.00

0.05

0.04

0-1

0.10

20. Ain Meider

F

8

—

L

M

37

42.5

9.53

F

28

42.9

3.6

0.00

0.00

0.00

0.04

0-1

0.11

In the laboratory, the 1621 collected individuals were
frozen. The shell was broken after thawing, and the soft
tissues carefully removed. The mantles were longitudinally
cut to reveal the pallial oviduct in females. Sexes were de-

termined according to the presence or absence of the capsule
gland and vagina. Normal females were separated from ab-
normal ones using a binocular dissecting microscope. In
males and imposex-affected females, the length of the

IMPOSEX AND MALFORMED PENIS IN TUNISIA

81

Figure 1. Sampling sites of Hexaplex trunculus along the Tunisian coast. 1, Bizerta Channel; 2, Quarries Bay; 3, Menzel Abderrahmen; 4,
Menzel Bourguiba; 5, Menzel lemil; 6, El Azib; 7, Tunis North Lake; 8, small gulf of Tunis; 9, lagoon of Khniss; 10, NPK Sfax; 1 1, fishing
harbor of Sfax; 12, Gargour; 13, Skhira; 14, fishing harbor of Gabes; 15, Sea of Zarat; 16, Adjim Channel; 17, Djorf-Ghigthis; 18, Ghigthis;
19, Guallala; 20, Ain Meidder.

straightened penis (from the base of the penis to the end of
the penial flagellum) and the vas deferens were measured
using an ocular micrometer.

Imposex incidence and levels were quantified by using
the following indices: ( 1 ) the imposex incidence or fre-
quency (1% = percentage of imposex-affected females com-
pared to the total number of females in the sample), (2) the
relative penis size index [RPSI = (average length of female
penises)'"’ x 100/(average length of male penises)^] according
to Bryan et al. (1986), (3) the relative penis length index
[RPLI = (average length of female penises X 100)/(average
length of male penises)] according to Stewart et al. (1992),
(4) the female penis average length (FPL), (5) the female vas
deferens average length (VDL), and (6) the vas deferens
sequence index [VDSl = (sum of imposex stage values of all
females)/(total number of females)] following Gibbs et al
(1987). Some imposex stages and malformations affecting
the penis were photographed under the binocular micro-
scope using a digital camera.

For the statistical comparison of the data, each sampling
station was assigned to 1 of 3 categories in terms of boating
traffic density (Table 1): (1) high shipping density (>100
boats in the area per year), (2) moderate (50-100 boats in the

area per year), and (3) low (<50 boats in the area per year).
Sites with shipyard activities or located near a high traffic
area were classified in the high category. All imposex indices
(1%, RPSI, RPLI, VDSL FPL, and VDL) were calculated for
each category. The significance of differences in imposex
levels between the 3 different categories of boating traffic
were tested using one-way analysis of variance (ANOVA)
and Chi-square test.

With regard to the malformation affecting the penis, the
relationship with boating traffic and some imposex indices
(1%, RPLI, and VDSI), recorded at sites where the condition
was observed, was established, using a regression analysis.

RESULTS

Imposex distribution

Imposex was observed at 19 out of 20 sampling sites.
However, the degree and the intensity of this alteration var-
ied depending on the boating activity at each site (Table 1).
The results indicated that the Bizerta Channel had the high-
est recorded imposex level with an RPLI of 69.08%, a VDSI
of 4.23 and a sterility rate of 8%. High levels of imposex were
also observed for Quarries Bay, Menzel Abderrahmen, Men-

82

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

Low Moderate High
Boating traffic category

60 1 —

T

50 -

5 40 - R 1

q:

c 30 - rn

03

I 20 -

10 -

0 J 1 r-l

Low Moderate High
Boating traffic category

Figure 2. Imposex development in Hexaplex trunciihis in relation to boating traffic. {*), significant difference (P = 0.05, one-way ANOVA
applied to all indices except the 1% index tested by a Chi-square test x^)-

zel Bourguiba, Tunis North Lake, NPK Sfax, Fishing Harbor
of Sfax, and Adjim Channel (VDSI above 3.7, Table 1).
Moderate levels of imposex were recorded for Gargour,
Skhira, fishing harbor of Gabes, Khniss, Small Gulf of Tunis,
El Azib, and Menzel Jemil (VDSI >1.3, Table 1). At the rest
of the stations (Guallala, Ain Meider, Gigthis-Djorf, and
Gigthis), very low levels of imposex were recorded (VDSI

above 0), while no female showing any form of genital dis-
order was observed at the Sea of Zarat (Table 1).

Significant differences were obtained between imposex
indices and categories of boating traffic, indicating that im-
posex development is related to the intensity of marine traf-
fic (Fig. 2). All the imposex indices values were significantly
elevated in the high shipping traffic category of stations

IMPOSEX AND MALFORMED PENIS IN TUNISIA

83

Figure 3. Schematic representation of the imposex pathways ob-
served in Hexaplex truncidiis from Tunisian waters. A, anus; Scg,
split capsule gland; Ip, incipient penis; Ov, occluded vulva; p, penis;
sp, small penis; V, vulva; Vd, vas deferens; Yds, vas deferens
section.

compared to the moderate and low categories. Significant
^ differences were also revealed between the moderate and the
low categories for all imposex indices except RPSI and RPLI
j (Fig- 2).

" Scheme of imposex pathways

^ The sequential stages of the imposex development ob-
J served in this study are illustrated (Fig. 3). The first imposex

characters (Id, 2d, 2d’, and 2d”) were detected only at sites
with low boating activity. Whereas some advanced stages
(3b, 3d, 4d, 4, 4.3, and 4.7) were found at the more affected
areas (Fig. 3). The more advanced stages of imposex re-
corded in this study were the YDS 5b and 5c. These stages,
causing sterility in females, were only revealed in the Bizerta
Channel.

In Hexaplex truncuhis, females start to show evidence of
imposex by the presence of a vas deferens section halfway
between the penis site and the vagina (stage Id). Thereafter,
3 cases are possible: ( 1 ) the vas deferens grows towards the
proximal and ciistal sides (stage 2d), (2) an incipient penis
develops behind the right ocular tentacle (stage 2d’), or (3)
a second portion of the vas deferens appears (stage 2d”). At
stage 3, females could show ( 1 ) a complete vas deferens
without a penis (stage 3b), (2) an incipient penis linked to
the vas deferens section (stage 3d), or (3) a small penis with
penis-duct continuing in a portion ot the vas deferens (stage
3a). At stage 4, the vas deferens reaches the vaginal opening,
passes it (stage 4.3), and runs into the ventral portion of the
capsule gland (stage 4.7). Sterility is reached in stage 5, in
which the vulva is occluded by the development of the vas
deferens tissue at the spawning aperture (stage 5b, Fig. 4A-B)
or the capsule gland is split following the ventral develop-
ment of the vas deferens (stage 5c, Fig. 4C).

Penis malformations in males

The normal male penis in Hexaplex trimciilus is gener-
ally bent (Fig. 5A), it possesses a large base and a long fla-

Figure 4. Imposex stage 5b (A, occluded vulva; B, dark mass of
egg capsules removed from the capsule gland at this stage) and 5c
(C) showing the split capsule gland. Cg, capsule gland; Scg, split
capsule gland; Ov, occluded vulva; Yd, vas deferens (scale bar =
1 mm).

84

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008 ^

Figure 5. Penis malformations in male Hexaplex trunculus. A, normal penis. B-E, abnormal
penises observed in Bizerta Channel ( B), the small Gulf of Tunis (C-D) and the Tunis North
Lake (E). bf, bifurcation; pd, penis duct; ex, excrescence; tl, flagellum; pi, penis 1; p2, penis
2; vd, vas deferens (scale bar = 1mm).

gellum-like tip reaching on fifth of the
total penis length, depending on the re-
productive season. The penis-duct is an
open fissure, but it becomes a closed
tube during reproductive activity. Male
penis malformations were detected in :j
the Bizerta Channel, Tunis North Lake, ;j
and the small gulf of Tunis with 1-2 af-
fected individuals per station (Table 2). j
The malformation varied from the de- |
velopment of a tissue bud in both the |
anterior and the posterior sides of the
penis (Fig. 5B-C) to the biphallic penis
(Fig. 5D) and the bifurcated flagellum
(Fig. 5E). A good correlation was ob-
tained between the incidence of penis ;
malformation, boating traffic, 1%, and |
VDSI (r > 0.85, Fig. 6). Flowever, the cor-
relation with RPLI was moderate (r- 0.49).

Penis malformations in female

Malformations affecting the female
penis were observed in 4 stations with a
number varying from 1 to 3 affected in-
dividuals per station (Table 2). In Gabes
and Bizerta Channel, malformations f
were characterized by the development |
of the bud tissue (excrescence) at the i
base of the penis (Fig. 7A) or half of the ;
total penis length at the posterior side :
(near the penis-duct, Fig. 7B). Biphallic ,
penis (Fig. 7C) and inflated penis tip [|
(Fig. 7D) were revealed in Menzel Jemil i
and Tunis North Lake, respectively. The
regression analysis showed a good rela-
tionship of the malformation rate only
with RPLI. The correlations between ■
boating traffic and 1% and VDSI were j
relatively weak (r < 0.34, Fig. 6). i

I

DISCUSSION !

■i

I

Imposex was found in most of the I
sites. Fligh levels were recorded in sites i
frec]uently used by boats or located near
a source of leaching TBT, such as har-
bors and shipyard activities stations.
Moreover, the highest indices were re-
corded in areas where the predominant
source of TBT involved large boats.
However, the moderate level of imposex 1
recorded in the fishing harbor of Gabes 4

IMPOSEX AND MALFORMED PENIS IN TUNISIA

85

Table 2. Genital malformation in Hexaplex truncidus from Tunisian coast with comparison to data collected from previous studies in the
same snail and other species of gastropod. N, number of specimens; Na, number of affected individuals; VDS, vas deferens sequence; — ,
no data available.

Males Females

Studies

Species

Locations

N

Malformation

Na

N

Malformation

Na

VDS

Present study

Hexaplex tniiiculus

Gabes Fishing Harbor

46

Absent

0

33

Penis excrescence

1

4

Hexaplex trunciilus

Steg Tunis North Lake

14

Bifurcated tip

1

30

Inflated penis tip

1

3

Hexaplex tnwciilus

Small Gulf of Tunis

48

Double penis
Penis excrescence

1

1

42

Absent

0

—

Hexaplex trunciilus

Menzel Jemil

40

Absent

0

40

Double penis

1

2

Hexaplex trunciilus

Bizerte Channel

42

Penis excrescence

2

52

Penis excrescence

3

4.3, 4.7.

and 5

Terlizzi et al. 1998

Hexaplex trunciilus

Italian Coast

—

Bifurcated penis

—

—

Bifurcated penis

—

>4

Branched vas deferens

Branched vas deferens

—

>4

Vasconcelos et al

Hexaplex trunciilus

Ria Formosa (Portugal

621

Penis excrescence

2

562

Rounded penis tip

5

—

2006

Coast)

Ramon et at 2001

Boliniis brandaris

Villanovai (Spain Coast)

59

Penis excrescence

1

75

Absent

0

—

Stroben et at 1992

Hinia reticulata

Brittany and Normandy

2760

Penis and/or vas

4

3562

Bifurcated penis and

10

4

Coast

deferens excrescences

double penis
Coiled vas deferens

7

(51.5%), despite the high boating traffic recorded in the area
( 108 boats), is explained by the heterogeneity of the sampled
population. At this station, many fishermen reject acciden-
tally fished whelks found in fishing nets that come from
locations far from the harbor where no imposex is present.
According to imposex index values and the knowledge that
TBT is the cause factor of imposex induction (Ten Hallers-
Tjabbes et al. 1994, Mensink et al. 2002), the Bizerta Channel
is considered as the most affected site along the Tunisian
coast by TBT pollution, because sterility was recorded. The
absence of imposex in the Sea of Zarat is explained by the
weak intensity of boating traffic recorded at this site. The
imposex level in Hexaplex tnwcidus in the present study, in
comparison to other Mediterranean countries such as Italy
(Terlizzi et al. 1998) and Malta (Axiak et al. 1995), suggests
the Tunisian coast is relatively less polluted by TBT. Sterility
was recorded in only one station among 20 with a frequency
of 8%. Along the Italian coast, Terlizzi et al. ( 1998) reported
the occurrence of sterility in 12 sites out of 15 with a fre-
quency varying from 11.1% at Forio to 100% at SM di
Pagana. In Malta, the most affected site had an RPSI of
98.1% and a VDSI of 4.8 (Axiak et al. 1995), compared to
33.03 and 4.23 in Tunisia. In the lagoon of Venice (Italy),
Pellizatto et al. (2004) recorded a female penis length (FPL)
of 12 mm, a RPSI of 36.02, and a VDSI of 4.9 in S. Nicolo
del Lido, and 8, 8.03 and 4. 1 in S. Maria del Mare. Compared
with our study, FPL and VDSI indicated that imposex level
is relatively similar in Quarries Bay (FPL = 7.77, VDSI =
4.09) and S. Maria del Mare. However, the difference ob-
served in RPSI values (28.47 in Quarries Bay and 8.03 in S.
Maria del Mare) is certainly related to the reproductive sea-
son in which males exhibit penis length variation. For this

reason, we think that using the FPL to asses TBT pollution
is more informative than using RPSI. According to Pellizatto
et al. (2004), the level of TBT found in the entire organism
of H. tniuciiliis from S. Maria del Mare varied between 53
and 60 ng per g dry weight.

The morphological expression of imposex in Tunisian
populations of Hexaplex triinciihis was different at early
stages from the general scheme of vas deferens sequence
proposed for the same species in the Maltese Islands (Axiak
et al. 1995) and Italian coast (Terlizzi et al. 1999). These
authors mentioned that the first sign ot imposex is the pres-
ence ol an incipient penis behind the right ocular tentacle
(stage la). Afterwards, the penis duct appeared (stage 2a)
and the vas deferens developed progressively from the base
of the penis toward the vaginal opening (stage 3a). In Tu-
nisian waters, we observed that the first sign of imposex was
expressed by development of a small portion of vas deferens
halfway between the penis site and the vagina (stage Id).
Thereafter, the penis appeared following the d pathway (at
stage 3d) or the d’ pathway (at stage 2d’). These differences
in imposex developmental stages observed between Tunisia
and Italy and Malta could be explained by factors other than
TBT. These factors could range from exposure to heavy met-
als (Nias et al. 1993), parasites (Gorbushin 1997), and other
androgenic compounds (Cajaraville et al. 2000). Another
hypothesis is genetic differences between Tunisian and Eu-
ropean populations of H. trunciiliis that could also lead to
distinct sequences of imposex induction. The development
of a portion of the vas deferens as a first sign of imposex in
Tunisian H. trwiculus allowed calculating a new index, the
VDL (mean length of the vas deferens), that gives more
information on the level of imposex in the less affected area.

86

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

Boating traffic

M(r: 0 85) F (r: 0.30)

0 20 40 60 80 100 12

1%

M(r: 0.94) F (r: 0.29)

0)

£

c

o

to

E

o

to

M (r: 0.49) F (r: 0.80)

0)

<0

c

o

to

£

o

u—

to

Figure 6. Regression analysis between penis malformation rate and boating traffic and some imposex indices (1%, RPLI, and VDSI) in both
sexes. M, in males; F, in females; r, regression coefficient. Boating traffic was estimated as the number of existing boats and visiting boats
in the area per year.

where penises in females are lacking or less developed, and
RPSI and RPLI are close to 0. These indexes (RPSI and
RPLI) are more informative in populations exhibiting the
imposex (a) pathway (penis developed at first).

Penis morphological alterations in Hexaplex tnmculus
affected by imposex were reported by earlier authors, but no
description nor pictures and schemes were provided. Terlizzi
et al. (1998) observed penis bifurcation in H. tnmculus in
both sexes, but especially in males from the Italian coast. In
the Ria Formosa lagoon (Portugal), Vasconcelos et al. (2006)
found 2 males among 621 examined with penis excrescences
and 5 females among 562 with a rounded penis tip. Com-
pared to our study, the novelty was the observation for the
first time of a penis with bifurcated flagellum in males and
a penis with excrescence in females. No more than one ex-
crescence per penis was observed in the present study, 1 in

males and females, in comparison to male Bolinus brandaris,
in which penises with many excrescences were found
(Ramon et al. 2001). In male Hinia reticulata (Stroben et al.
1992) and in both sexes of Nucella lapillus and Ocenebra
erinacea (Linnaeus, 1758) (Oehlmann et al. 1991, 1992), pe-
nis excrescences were also observed (Table 2). Stroben et al.
(1992) have found females with bifurcated penises and fe-
males with two penises in Hinia reticulata. Contrary to the
present study where no malformations associated to vas def-
erens development were revealed, Terlizzi et al. ( 1998) found
branched vas deferens in H. trunculus, and Stroben et al.
(t992) observed coiled vas deferens in Hinia reticulata.

With regard to the imposex stages in females in which
the malformations were found, the abnormalities were ob-
served since stage 2d’, but especially in the more advanced
stages. The correlation between malformed penises and the

IMPOSEX AND MALFORMED PENIS IN TUNISIA

87

Figure 7. Penis malformations in female Hexaplex tninculiis. A, in Gabes fishing harbor; B, in Bizerta Channel; C, in Menzel lemil; D, in
Tunis North lake; ex, excrescence; fl, flagellum; fsr, fissure of the vas deferens; It, inflated tip; pi, penis 1; p2, penis 2 (scale bar =
1 mm).

boating traffic and imposex indices was significantly better
in males than in females, except for the RPLI where the
converse situation was recorded. These findings indicated
that in males, the development of penis malformation is
related to imposex level. However, in females, the occur-
rence of such conditions is related to the development of the
penis and consequently to the imposex pathway (d or d’).
The causal factor of penis malformation during develop-
ment is unknown; it could be related to the presence of TBT
in sea water, as reported by Mensink et al. (2002). These
authors obtained in the laboratory penis malformations in
juveniles of Buccinum imdatum at 500 ng/L TBT. In our

case, the development of such a condition in males is cer-
tainly linked to TBT pollution, because a good correlation
with 1% and VDSl was obtained. However, this hypothesis
must still be supported by laboratory exposure of H. tnin-
ciilus to TBT. Another hypothesis relates to the natural de-
velopment of malformations, since the rate was low and
relatively similar between males and imposex females. In this
context, Garaventa et al. (2006) have reported the presence
of biphallic males among museum specimens of H. tnincuhis
collected before the use of TBT. Such data suggest the exis-
tence of natural abnormalities of the penis that are not re-
lated to TBT pollution.

88

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

This paper, reporting the imposex level and penis mal-
formations in Hexaplex trunculiis from the Tunisian coast, is
important to assess the environmental consequences of the
boating activity. The high values of VDSI in some popula-
tions suggest the existence of diffuse TBT pollution along the
majority of the Tunisian coast. Further investigation of or-
ganotin in seawater, sediments, and gastropod tissue are in
progress. Such data could be useful in regulation of TBT-
based antifoulants and the legislation to ban the use of TBT-
based paints in Tunisia.

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Submitted: 20 May 2007; accepted: 9 August 2007; final

corrections received: 20 December 2007

X

Amer. Malac. Bull. 24; 91-96

Threatened Bliss Rapids snail’s susceptibility to desiccation: Potential impact from
hydroelectric facilities

David C. Richards and Tristan D. Arrington

EcoAnalysts Inc., Center for Aquatic Studies, 11 E. Main Street, Suite M, Bozeman, Montana 59715, U.S.A., drichards@ecoanalysts.com
and tarrington@ecoanalysts.com

Abstract: Water levels in the regulated Snake River, southern Idaho, U.S.A. can fluctuate daily and seasonally due to hydroelectric
demands. The federally listed threatened Bliss Rapids snail, Taylorconclia serpenticola Hershler et al., 1994 (Family: Hydrobiidae), survives
in and near these fluctuation zones. Remaining T. serpenticola populations occur only in sections of the Snake River that are impacted by
these hydroelectric facilities and associated springs. Because effects of rapid draw-down in fluctuation zones on T. serpenticola are unknown,
we conducted a laboratory experiment to evaluate potential impacts of desiccation. Our experiment compared desiccation resistance at
several air temperatures, on dry and wetted substrates, and for ‘small’ vs. ‘large’ snails. Probit regression-maximum likelihood models
estimated lethal time (LTj,,) values. Suiwival was significantly greater on wetted substrate than on dry substrate and was lowest at
temperatures <0‘’C and at 37'^C on dry substrate. Survival was greatest at 17°C on wetted substrate. There was no significant difference in
survival at temperatures above 0°C on dry substrate other than at 37°C. LTj,, survival ranged from 0.5 hours at -7°C to 157.0 hours at 17°C
on wetted substrate. There were no significant differences in survival relative to snail size in any treatment. Our results suggest that
desiccation could impact T. serpenticola populations if snails become stranded on dry substrates during rapid water-level fluctuations of the
Snake River, particularly during subzero winter or extreme high summer temperatures. The most important factor determining survival
would be the ability to find refuge on the undersides of cobbles, where snails typically occur, or in habitats that remained moist for the
duration ot the draw-down of the river.

Key words: regulated rivers, probit regression, population viability, threatened species. Snake River

The federally listed, threatened Bliss Rapicis snail Tay-
lorconcha serpenticola Hershler et al, 1994 (Family: Hydro-
biidae) (Fig. 1) occurs only in fragmented populations
within approx. 80 river kilometers of un-impounded sec-
tions of the regulated Snake River and in associated cool to
cold-water springs of the Snake River aquifer, south-central
Idaho, U.S.A. (Upper Snake River Basin) (Hershler et al.
1994, Richards 2004, Richards et al. 2006) (Fig. 2). Water
levels in the un-impounded sections of the river fluctuate
daily and seasonally depending on flows, location, geomor-
phology, and weather conditions. Daily fluctuations, mostly
a result of hydroelectric generation from three dams in this
area (Fig. 2), occur for only several hours at a time (Ste-
phenson et al. 2004). Populations of T. serpenticola occur
within fluctuation zones and may be affected by daily fluc-
tuations, whereas spring populations are not subjected to
these same fluctuations. Direct effects of rapid dewatering on
individual T. serpenticola survival are unknown.

Taxonomic history and status of
Taylorconcha serpenticola

Taylorconclia serpenticola was first collected in the Snake
River of south-central Idaho and recognized as a new taxon
by Taylor in 1959 (Taylor 1982). The taxon, although ap-
parently collected and noted as early as 1884, went unde-
scribed until Hershler et al. (1994) placed the snail in the

new genus Taylorconcha and a new species, Taylorconcha
serpenticola. Hershler et al. ( 1994) described the known dis-
tribution of this species as the main stem Snake River and
associated springs of south-central Idaho.

The origins of Taylorconcha serpenticola are distinct in
the molluscan fauna. Taylorconcha can be traced back to the
late Pliocene (Blancan) Glenns Ferry formation in Gooding
County, Idaho; the early Pleistocene Bruneau formation in
Owyhee County, Idaho; and the late Pleistocene and prob-
able Holocene deposits in Gooding County (Smith et al.
1982, Hershler et al. 1994). Of equal significance, Taylorcon-
cha can be identified as a survivor ot the Pliocene Lake
Idaho, geologically dated about 3.5 Ma (Hershler et al.
2006).

Taylorconcha is one ot the tew remaining extant taxa
from ancient Lake Idaho, which once supported a molluscan
fauna of more than 80 endemic taxa (Hershler et al. 1994).
Lake Idaho was thought to have extended from the border
between western Idaho and eastern Oregon upstream of
Hells Canyon eastward to a point near American Falls, Idaho
(Taylor 1985, Hershler et al. 1994). Remnant populations of
T. serpenticola remain in Idaho, inhabiting approx, an 80-km
stretch of the Snake River upstream and downstream of
Hagerman, Idaho in the Thousands Springs reach of the
Snake River. Taylorconcha serpenticola was known histori-
cally from the main stem middle Snake River and associated

91

92

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

Figure 1. Male Taylorconcha serpenticola. Photo courtesy of Dan
Gustafson, Montana State University, and David Richards, Eco-
Analysts Inc., Center for Aquatic Studies, Bozeman, Montana.

springs between Indian Cove Bridge
(Rkm 845.6) and Twin Falls (Rkm
982.5) (Hershler et al. 1994). Taylor
( 1982) believed that prior to dam con-
struction there was probably a single
population throughout this range.

The status of extant Taylorconcha
serpenticola populations is a topic of
concern. Federal action began on the
species primarily in response to peti-
tions submitted in 1980, under section
4(b)(3) of the Endangered Species Act
(ESA) 1973. The snail was a candidate
for category 1 listing from 1984
through December 18, 1990. This 1990
proposed rule listed T. serpenticola as
an endangered species along with four
other aquatic snails: the Snake River
physa Physa natricina (Taylor, 1988),
the Idaho springsnail Pyrgulopsis ida-
lioensis (now Jackson Lake springsnail

Pyrgulopsis robusta (Walker, 1908) (see Hershler and Liu ■
2004), the Utah valvata Valvata utahensis (Call, 1884), and
the Banbury Springs Lanx (Frest, 1988) (limpet). On De-
cember 14, 1992, the U.S. Fish and Wildlife Service classified
T. serpenticola as threatened while still taxonomically an
“undescribed Hydrobiid” (U.S. Fish and Wildlife Service j
1992). ii

Potential susceptibility of Taylorconcha serpenticola ;

to desiccation

Taylorconcha serpenticola is the smallest (2. 0-4.0 mm) of
the Snake River hydrobiids in south-central, Idaho, and of
those that we have observed, it is also the slowest moving
(Richards 2004). Field trials indicated that T. serpenticola
could travel approx. 1 to 10 cm/hour in water, which was |
more than ten times slower than the common pebble snail, ,
Flnminicola (Carpenter 1864), and up to 100 times slower |l
than the invasive New Zealand mudsnail Potarnopyrgus an-
tipodarwn (Gray, 1843) (Richards and Arrington, unpubl. |
data). Thus, T. serpenticola is perhaps the least able of the I
hydrobiid species to actively avoid desiccation in fluctuation !
zones in the Snake River.

Life history and temporal environmental conditions ;
may also affect Taylorconcha serpenticola, as has been shown
for Potanwpyrgns antipodarum survival probability to desic-
cation (Richards et al. 2004). Their results showed that: (1)
smaller size classes had lower survival than larger size classes;

(2) higher temperatures were related to decreased survivabil-
ity; (3) freezing rapidly decreased survival; and (4) survival I
was greater at higher than lower humidity.

Figure 2. Current known distribution (approx. 80 river kilometers) of Taylorconcha serpen-
ticola in the upper Snake River basin, south-central Idaho, U.S. A. Dark lines and dots indicate
current known locations of the species.

TAYLORCONCHA SERPENTICOLA AND DESICCATION

93

Although Tnylorconcha serpeuticola is potentially sus-
ceptible to high rates of desiccation-induced mortality, this
species, like all hydrobiid snails, has an operculum, which
may help it survive. Winterbourn (1970) reported that Pot-
amopyrgus antipodariim from New Zealand was able to sur-
vive desiccation for up to 50 days on a wetted substratum at
20-25°C. Richards et al. (2004) showed that P. antipodarwii,
in the western U.S.A., a parthenogenic clone, can survive
desiccation on wetted substratum at 9°C for at least 48
hours.

The purpose of this study was to evaluate the survival
probability of different sizes of Taylorconcha serpeuticola un-
der different time periods of desiccation at various tempera-
tures and substrates (dry and wetted). Based on results of
effects of desiccation on Potamopyrgiis autipodarwn (Rich-
ards et al. 2004), we hypothesized that T. serpeuticola sur-
vival probability to desiccation was: ( 1 ) positively correlated
with snail size, (2) negatively affected by increased tempera-
ture, (3) greater on wetted substrate than on dry substrate,
and (4) negatively affected by freezing.

MATERIALS AND METHODS

Raising and rearing procedures

Taylorconcha serpeuticola used in our experiments were
from brood stock (250 individuals) collected in 1999 at the
outlet of Banbury Springs, near Hagerman, Idaho. The T.
serpeuticola population at the outlet of Banbury Springs had
the highest densities reported for any T. serpeuticola popu-
lation (>3000/m‘ in summer) (Richards 2004) and was con-
sidered the least likely population to be affected by ‘harvest’
for our experiments. Brood stock was supplemented with
100-200 individuals once to twice per year from the same
source, until 2005. The collected snails and offspring were
reared at EcoAnalysts Inc. Research Laboratory, Bozeman,
Montana under the authority of a U.S. Fish and Wildlife
Service Section 10 permit. Taylorconcha serpeuticola popula-
tions in the lab were maintained in twelve to sixteen, 37.85-L
aquaria at 16-17°C. Varying light: dark regimes were used to
simulate or accelerate natural light conditions and seasons in
an effort to produce more snails. All aquaria had substantial
aeration, moderate flow, and contained various substrates,
including periphyton-covered cobbles (food resource) that
were collected from the outlet of Banbury Springs and the
Snake River. Aquaria also contained native aquatic macro-
phytes including Myriophyllu?n sp., Ceratophyhuu sp., and
Elodea sp. Taylorconcha serpeuticola reproduced slowly in the
laboratory (<5-7 eggs/year) (Richards and Arrington, un-
publ. data); therefore, the number of individuals available
for potential experimental sacrifice restricted experimental
designs.

Experimental design

Twenty ‘small’ (1.50-2.00 mm shell height) and twenty
‘large’ (2.01-2.50 mm) Taylorconcha serpeuticola were ex-
posed to six air temperatures (-7, 0, 7, 17, 27, and 37°C) on
two substrate conditions (dry and wetted) and ten time pe-
riods (2, 4, 8, 16, 24, 48, 72, 96, 120, and 144 hours for 0, 7,
17, and 27°C; 0.5, 1, 2, 4, 8, 16, 24, 48, 72, and 96 hours at
-7 and 37°C). At -7°C only one substrate was used (dry)
and at 7°C only one size class was used (1.5-2.50 mm). A
total of 4360 snails were assayed in this experiment. The
experiment was conducted in September 2004. The six tem-
peratures were selected based on conditions likely to be en-
countered by T. serpeuticola in the Snake River throughout
the year. Based on past experiments (Richards 2004), a water
temperature of 17‘’C appears to be the best temperature to
promote T. serpeuticola growth. The two substrate treat-
ments were chosen to simulate conditions that T. serpenti-
cola would encounter either on the tops of cobbles (dry) or
the bottoms of cobbles (wetted) during dewatering.

Twenty ‘small’ and twenty ‘large’ Taylorconcha serpeu-
ticola were uniformly distributed on either a dry paper towel
or wetted paper towel within a large, covered Petri dish. To
reduce the effect of run order, the sequence of temperature
treatments was randomized. Dishes were then placed in an
environmental chamber and held at the appropriate tem-
perature. Snails were removed from the chamber after the
appropriate treatment interval and were transferred into
new dishes filled with aquaria water at 15 to 17°C. After one
hour in the water-filled dish, snails were observed to see if
they opened their opercula and started crawling or if they
were attached to the dish. If snails were crawling or attached
to the dish, they were classified as ‘alive’. If snails were not
moving or not attached to the dish, they were observed
under a dissection scope at 40x magnification; if no move-
ment was apparent, snails were classified as ‘dead’. Snails
classified as ‘dead’ were then re-obseiwed after 24 hours in
water-filled Petri dishes. It snails were crawling or attached
to dishes they were classified as ‘alive’; if there was no ob-
served movement, they were classified as ‘dead’. Mortalities
were kept as voucher specimens at EcoAnalysts Inc. Research
Laboratory, Bozeman, Montana.

As a control, 20 ‘small’ and 20 ‘large’ Taylorconcha ser-
penticola were placed into two separate Petri dishes that were
filled with aerated aquaria water for 120 hours. Controls
were maintained at ambient lab temperature (16-17°C) and
replenished with aquaria water as needed.

Probit regression was used to develop distribution mod-
els of survival for both ‘small’ and ‘large’ snails at the six
temperatures. This method is widely used to evaluate dose/
response of pesticides on insects and in medical studies
(Finney 1971, Preisler 1988, Preisler and Robertson 1989,
Baker et al. 1995, Peng et al. 2002). Compared with an

94

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

ANOVA, ANOVAs only determine if there is a significant
difference between treatments, but probit analysis deter-
mines significant differences between treatments at any de-
sired percent survival level. Probit analysis also models the
relationship between percent survival and duration of expo-
sure at any given temperature.

The most appropriate probit regression distribution
model (Weibull, normal, lognormal, logistic, etc.) with 95%
confidence intervals (CIs) was selected using maximum like-
lihood methods. Pearson chi-square goodness-of-fit tests
were used for evaluating and selecting models. Goodness-
of-fit tests were also used for comparing slopes of models. In
addition, probabilities of survival, including LTg^ values,
were calculated; these are commonly used metrics for inver-

tebrate bioassays (Dunkel and Richards 1998). A LT50 is the
lethal time (or temperature) at which 50% of individuals
being tested have died.

Treatment effects were considered significant if there
was no overlap in 95% CIs of the probit models. All analyses
were conducted using MINITAB 14.1 (Minitab Inc. 2003)
and S-PLUS 6.1 (Insightful Corp. 2002).

RESULTS

Probit survival distributions were highly variable be-
tween treatments and significantly greater on wetted sub-
strate than on dry substrate for both ‘small’ and ‘large’ snails

Table 1. Probit regression models and LTgj, values of Taylorconcha serpentkola survival probability to desiccation, using best-fit maximum
likelihood estimates. NA, not applicable.

Best-fit probit
regression model
distribution

Pearson goodness-

of-fit test
{df, P- value)

X' test for
equal slopes
(df, P- value)

Log-

likelihood

LTgo hr
(95% Cl)

Temp.

Small

Large

-7°C

Weibull

1.68 (17, 1.00)

0.11 (1, 0.74)

-47.49

0.47

(0.34, 0.60)
Dry
6.82

(2.53,8.66)

0.47

(0.33,0.60)

Dry

8.75

(6.88, 11.08)

0°C

Lognormal

30.21 (35,0.70)

8.39 (3,0.04)

-221.78

Wetted Wetted

28.90 26.98

(23.15,35.85) (21.64,33.41)

7°C’

(Dry)

Normal

0.07 (7, 1.00)

NA

-31.55

4.20“

(2.45, 5.42)

7°C‘’

(Wetted)

Lognormal

7.83 (9,0.55)

NA

-102.15

73.33“

(57.04, 108.37)

17°C

(Dry)

Weibull

5.68 (17, 0.99)

2.23 (1,0.14)

-69.49

2.33

(1.87, 2.97)

3.14

(2.61, 3.88)

17°C

(Wetted)

Logistic

25.25 (17, 0.09)

0.09 (1, 0.76)

-100.07

131.96 156.56

(115.93,154.09) (140.58,181.08)

27°C

(Dry)

Weibull

5.38 (17, 0.99)

1.28 (3,0.26)

-80.98

2.26

(1.82, 2.84)

3.22

(2.57, 4.07)

27°C

(Wetted)

Logistic

13.50 (17, 0.70)

0.01 (3,0.94)

-178.32

89.87 88.59

(75.87,106.36) (74.70,105.91)

37°C

Lognormal

28.90 (35, 0.76)

5.21 (3,0.16)

-289.41

Dry

0.98

(0.64, 1.47)

Wetted

3.08

(2.16,4.34)

Dry

1.52

(1.02, 2.21)

Wetted

3.38

(2.38, 4.73)

At 7°C only one size class was used (1.5-2.50 mm) due to limited number of snails available.

TAYLORCONCHA SERPENTICOLA AND DESICCATION

95

within each temperature treatment (Table 1). Survival was
slightly less for ‘small’ snails than ‘large’ snails within each
temperature treatment at most temperatures on both dry
and wetted substrates but was not statistically significant
(Table 1). Survival consistently decreased with increased
desiccation times. Survival was significantly lower at -7°C
than for any other temperature and almost identical for
‘large’ and ‘small’ snails (Table I ). Snails on wetted substrate
survived >15 times longer than snails on dry substrate at 7°C
(Table 1), and snails on wetted substrate survived >50 times
longer than snails on dry substrate at 17°C (Table 1 ). There
were no mortalities for ‘small’ snails {N - 20) and a single
mortality (5%) for ‘large’ snails {N - 20) in the controls.
Therefore, survivability was considered to be a result of
treatment effects (i.e., desiccation).

DISCUSSION

Our experiments showed that temperature and sub-
strate moisture level could affect survival of Taylorconcha
serpenticola to desiccation under controlled conditions. Our
experiments simulated conditions that would occur on the
tops (dry) and bottoms (wetted) of cobbles during flow fluc-
tuations in the Mid-Snake River. Given that T. serpenticola
moves relatively slowly (Richards 2004), its ability to avoid
rapidly retreating water levels is limited. Therefore, the most
important factor for individual survival is snail location at
the time of receding water levels. Survival may be dictated by
whether snails: ( 1 ) become stranded on the tops or sides of
cobbles, where desiccation to temperature extremes is more
likely, or (2) find refuge on the undersides of cobbles or in
habitat that remains moist for the duration of the draw-
down of the river.

Richards (2004) documented the snail’s preference for
sides or undersides of cobbles at the outlet of Banbury
Springs and that it was only occasionally found on tops of
cobbles. Bowler (2001) reported nocturnal movement of
Taylorconcha serpenticola from the bottom to tops of
cobbles. This preference for undersides of cobbles or ‘pho-
tophobic tendency’ may benefit T. serpenticola during rapid
dewatering of shoreline habitat. Richards et al. (2005) and
Stephenson et al. (2004) reported that densities of Taylor-
concha are often greatest at shallow depths near the shore-
line. Because these habitats are the most intensely affected by
fluctuating river levels, a higher proportion of the river
populations may be subjected to desiccation. Due to differ-
ent shoreline topographies, it is impossible to determine
fluctuation levels at any one location in the river and there-
fore, how much T. serpenticola habitat or what percentage of
the population is affected at any point in time.

No research has been conducted on which stages of its
life cycle are the most critical to population viability. For

example, egg survival may be less important to population
viability than survival of larger, more fecund adults or small
to medium-sized snails that may have greater lifetime repro-
ductive potential (Beissinger and McCullough 2002). It is
also unknown if there is a seasonal effect of exposure on
Taylorconcha serpenticola survival due to density-dependent
interference or exploitative intraspecific competition. There
is ample evidence for density-dependent regulation occur-
ring in river invertebrate populations, including snails
(McAuliffe 1984, Hart 1985, 1987, Lamberti et al. 1987, Os-
enberg 1989, Peckarsky and Cowan 1991, Kohler 1992, An-
holf 1995) and possibly T. serpenticola (Richards and Ar-
rington, Linpubl. data). Exposure may reduce intraspecific
competition of T. serpenticola by reducing densities, but may
increase interspecific competition. For example, Potainopyr-
gus antipodariini and T. serpenticola are often found together
in the Snake River fluctuation zones and have been shown to
compete for limited food resources under in situ experi-
ments (Richards 2004). Because P. antipodariini is better able
to actively avoid exposure than T. serpenticola, flow-
fluctuations may favor P. antipodarnin over T. serpenticola,
thus giving the former species an addifional compefifive ad-
vanfage. Ofher biofic facfors fhat were not evaluated, such as
increased predation or parasite load, may also affect T. ser-
penticola survival during exposure.

ACKNOWLEDGMENTS

We thank Yuliya German for her laboratory assistance
in both raising and rearing Taylorconcha serpenticola and for
collecting experimental data. We thank Dr. Sharlene Sing,
Dr. Webb Van Winkle, and William H. Clark for reviewing
the manuscript and Dr. David K. Weaver for assistance with
probit regression interpretation. We also thank the staff and
crew at EcoAnalysts Inc., Moscow, Idaho and Idaho Power
Company, Boise, Idaho for constructive input from start to
finish of this project and the U.S. Fish and Wildlife Service
for the Section 10 collection permit.

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:i

i

Submitted: 14 February 2007; accepted: 6 November I

2007; final corrections received: 29 November 2007

Amer. Maine. Bull. 24: 97-100

Field observations of the nocturnal mantle-flap lure of Lampsilis teres

Andrew Lee Rypel

Department of Biological Sciences, The University of Alabama, Box 870206, Tuscaloosa, Alabama 35487-0206, U.S.A.,
andrewrypel@yahoo.com

Abstract: Three yellow sandshell mussels, Lampsilis teres (Rafinesque, 1820), were observed in Lake Tuscaloosa, Alabama, and the temporal
display pattern of their mantle-flap lures was investigated in situ. All three gravid females fully displayed their mantle-flap lures after dark
during each nighttime visit {N = 3) but none displayed their lures during daytime (N = 3). An encounter between a mantle-lure and a
largemouth bass was observed. These observations are the first reported of in situ mantle-flap lure displays and fish host encounters for L.
teres, and support previous studies of did display patterns in other mantle-lure displaying mussels. This did lure display may be related to
the ecology of the fish hosts they seek to attract. Future daytime and, especially, nighttime field observations of bivalve mussels with
mantle-flap lures may greatly improve understanding of their reproductive ecology.

Key words: Bivalvia, did, mussel, unionid, largemouth bass

Gravid, mature females of the mussel genus Lampsilis
Rafinesque, 1820 display elaborate mantle-flap lures to at-
tract fish hosts for glochidial larvae (Ortmann 1914, Krae-
mer 1970, Haag et al. 1999). Mantle-flap lure displays vaiy in
response to time of day, light conditions, and presence of
suitable fish hosts, and there aprpear to be interspecific dif-
ferences in when displays begin (Kraemer 1970, Haag and
Warren 2000). These variations may be related to the did
habits of the fish hosts used by each mussel species (Welsh
1933).

Lampsilis teres Rafinesque, 1820 is a unionid bivalve
with a wide distribution from Mexico north to Minnesota
and is found in the Mississippi, Rio Grande, Mobile, and
Gulf drainages (Parmalee and Bogan 1998). The species is
especially common in the southeastern U.S.A., prefers pool
and shallow sandbar habitats (Ortmann 1926, Parmalee and
Bogan 1998), and is often abundant in river impoundments
(C. Lydeard, Smithsonian Institution, pers. comm.). Known
fish hosts of L. teres include alligator gar {Atractosteus
spatula), black crappie [Pomoxis nigromaculatus), white
crappie {Pomoxis annularis), green sunfish (Lepomis cyanel-
lus), largemouth bass {Micropterus salnioides), longnose gar
{Lepisosteus osseus), orangespotted sunfish {Lepomis Innni-
lis), shortnose gar {Lepisosteus platostomus), shovelnose stur-
geon {Scaphirhynclius platorynclnis), and warmouth {Lepo-
mis gulosus) (Watters 1994).

Previous studies (Kraemer 1970, Trdan 1981, Haag and
Warren 1999, Haag and Warren 2000) have documented the
reproductive strategy of displaying mussels and showed, un-
der laboratory conditions, that mantle-lure displays respond
to presence of fish hosts, light conditions, and substrate dis-
turbance. However, no studies have reported in situ field
observations of mantle-flap lure displays, and 1 briefly de-

scribe field observations of the mantle-flap lure of Lampsilis
teres, including morphology, display timing, and physical
encounters with fish.

MATERIALS AND METHODS

In May 2005 in Lake Tuscaloosa (an impoundment of
the North River, Mobile River basin), Tuscaloosa County,
Alabama, three individuals of Lampsilis teres (yellow sand-
shell) were observed displaying mantle-lures after dark be-
neath a boat dock (depth ca. 1. 4-2.0 m). Using a flashlight,
1 observed each specimen from the dock, presumably with-
out affecting the display pattern of their mantle-lures. Sub-
sequent visits to this site were made over the next three days
(11-13 May 2005, 12:00-4:00 pm) and three nights (11-14
May 2005, 9:00 PM- 1:00 AM) to observe diel display behavior.
Observations on the timing, morphological characteristics of
the lure, and any interactions with fish were noted.

RESULTS

Display timing

All three specimens were in full display during each
nocturnal visit, and no lures were displayed during daylight
visits. The mussels were buried in the sediment at a slight
angle (posterior facing up) with their mantle-flap lures fully
extended and pulsating. During one visit just prior to dusk,
none of the mussels were displaying; however, as the sun set,
one specimen began to display. After sunset, the other two
mussels began dispkiying their lures. During this twilight
period, one mussel slowly moved from a horizontal position

97

98

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

adjacent to a rock to soft sediment where it positioned itself
vertically and began to display. The displays were periodic
and occurred in episodes of various lengths. Total palpita-
tions per episode of all 3 mussels ranged from 4 to 26 before
individuals rested. Palpitating episodes lasted for 6 to 177 s
and rest periods ranged in time from 10 to 98 s before
recommencement.

Lure morphology

Mantle-lures were approx. 3-4 cm maximum length and
2 cm in maximum width (Pig. 1 ). The tissue was a dark pink
color in the interior and white and tan on the margins.
When fully displayed, the margins were wavy in appearance
and resembled small fishes. The lures varied slightly in mor-
phology and color depending on the individual, but the
margin of all lures undulated during displays, while the in-
terior of the lures pulsated. As the display was initiated, the
lure would extend slowly from the mantle, motionless at

Figure 1. A, Lateral and B, anterior view of the mantle-flap lure of
Lampsilis teres. Photographs (© 2008 by Paul Frese) reproduced
with the permission of the copyright holder.

first, and slowly begin to palpitate. When only moderately or
minimally displayed, the lure apf>eared to have less motion
and color. ^

Fish encounters

Fish were observed within the immediate vicinity of the
mussels frequently. The fishes included bluegill (Lepomis
macrochirus), longear sunfish {Lepomis megalotis), and large-
mouth bass (Micropterus salmoides). Relatively large num-
bers of small longear and bluegill sunfish were seen during
both day and night visits, but a largemouth bass (N = 1, '

-115 cm total length) was encountered only once, at night.
The bass approached a mantle-lure in full display and struck
it. Following the strike, the bass retreated for 2-5 s and swam
off The mussel continued to display immediately following
the strike and rested only after the bass had left. Although
small bluegill and longear sunfish were present during all
visits, there were no attacks by these fish on the displaying
lures although these fish would often pay close attention to
a lure in full display.

DISCUSSION

Display timing

The observation that Lampsilis teres displays occurred
only at night indicates that daytime is not an effective time
to attract a suitable fish host. These observations provide
further documentation that lure displays vary with time of
day and presence of suitable fish hosts (Haag and Warren |
2000). Many centrarchids and a number of other freshwater ’
fishes are known to exhibit diel movements and generally
move from more midstream or open-water during the day W
to littoral habitats at night (Helfman 1993, Shoup et al. 2004,
Rypel and Mitchell 2007). These movements are coupled
with the movements of their prey, many of which are also
driven by diel cycle (Helfman 1993, Layman and Winemiller
2004). Sunfishes were observed in numbers around the dis-
playing mussels at night and are known to be important prey i
for adult largemouth bass (Cochran and Adelman 1982,
Howick and O’Brien 1983, Gabelhouse 1987). By displaying
lures during times which maximize fish host encounters,
mussels would improve glochidial transmission. The tem-
poral differences in lure display for L. teres at this site were
presumably a product of diel changes in host fish locations. )

Lure morphology

Mussel species that use large predacious fishes as hosts
generally display modified mantle-lures which strongly re- r
semble small prey fishes, insects, and aquatic insect larvae
(Kraemer 1970, Haag and Warren 2000). Considering the
number of small centrarchid fishes consistently present near I

MUSSEL MANTLE-FLAP LURE

99

the mussels and the lure’s color and shape, this mantle- lure
apparently mimics these small sunfishes. Each mussel’s shell
color matched the substrate such that the shells are cryptic in
sand and gravel. Meanwhile, the mantle-lure was pink,
which combined with the palpitating motions, accentuated
the lure’s motion underwater, apparently to attract fishes.

Fish encounters

A largemouth bass biting the LampsUis teres mantle-lure
demonstrates that the lure is effective at attracting a suitable
fish host (Fuller 1974, Watters 1994). In other trips to this
site, I have also collected black crappie and warmouth, both
of which are reported as fish hosts for this mussel (Watters
1994). Channel catfish {Ictaliirns punctatus), flathead catfish
(Pylodictis olivaris), freshwater drum [Aplodinotiis grun-
niens), smallmouth buffalo [Ictiobus biibalus), spotted bass
(Micropterus piinctidatus), and spotted gar [Lepisosteiis ocu-
latus) were also collected, although they are currently not
believed to be hosts for L. teres. Fitness of L. teres, and
possibly other nighttime mantle-lure displaying mussels,
could be tied to diel movements of fishes. Bluegill and
longear sunfish, the other fishes consistently observed near
the vicinity of the lures, have not yet been identified as fish
hosts for L. teres (Watters 1994, Parmalee and Bogan 1998).
However, the fact that they are not listed as hosts does not
preclude their potential as a host under certain environmen-
tal conditions. If one species of Lepomis were a host, another
species within the genus can, at times, also serve as a host
(Haag and Warren 2003). These observations corroborate
previous reports of freshwater unionids utilizing nighttime
displays to attract fish hosts (Haag and Warren 2000,
Toomey et al. 2002) and suggest that nighttime observations
may provide information on display behavior in mussel spe-
cies that have not been encountered displaying during day-
time.

Additional field observations on the diel nature of other
freshwater mussel species are necessary. If night were a criti-
cal display period for other unionids, then such observations
would be critical to future conservation efforts such as cap-
tive breeding programs. Field observations might reveal pri-
mary hosts, especially if the host fishes are nocturnal, cryptic,
or rare, and could generate new data and questions regard-
ing the ecology of mantle-flap lures. Fish host identification
is often based on a “shotgun approach” involving laboratory
infestation tests on a variety of sympatric and common
fishes suspected to be hosts. However, lists of potential fish
hosts may be inadequate, especially if we ignore fish-mussel
encounters occurring at night. The ecology of nocturnal
freshwater fishes is not understood well and the diel move-
ments of even well-studied fishes have gone somewhat un-
derappreciated until only recently (Shoup et al. 2004, Rypel
and Mitchell 2007). Future research on the diel ecology of

freshwater mussels will be similarly necessary to develop a
more robust understanding of unionids.

ACKNOWLEDGMENTS

The author would like to thank Paul Frese who gener-
ously donated the images of the mantle flap lure. Charles
Lydeard and Wendell Haag advised me on aspects of the
biology of this species. I also thank Tim Savidge and two
anonymous reviewers whose constructive comments greatly
improved this manuscript. This research was supported by a
University of Alabama graduate research fellowship.

LITERATURE CITED

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ration and diet of largemouth bass, Micropterus salmoides,
with an evaluation of gastric evacuation rates. Environmental
Biology of Fishes 7; 265-275.

Fuller, S. L. H. 1974. Clams and mussels (Mollusca: Bivalvia). Iti:
C. W. Hart, Jr. and S. I.. H. Fuller, eds.. Pollution Ecology of
Freshwater Invertebrates. Academic Press, New York, New
York. Pp. 215-273.

Gabelhouse, D. W. 1987. Responses of largemouth bass and bluegill
to removal of surplus largemouth bass from a Kansas pond.
North American Journal of Fisheries Management 7; 81-90.
Haag, W. R. and M. L. Warren, Jr. 1999. Mantle displays of fresh-
water mussels elicit attacks from fish. Freshwater Biology 42:
35-40.

Haag, W. R., M. L. Warren, Jr., and M. Shillingsford. 1999. Host
fishes and host-attracting behavior of LampsUis altilis and Vil-
losa vihex (Bivalvia: Unionidae). American Midland Naturalist
141: 149-157.

Haag, W. R. and M. L. Warren, Jr. 2000. Effects of light and pres-
ence of fish on lure display and larval release behaviours in
two species of freshwater mussels. Animal Behaviour 60: 879-
886.

Haag, W. R. and M. L. Warren, Ir. 2003. Host fishes and infection
strategies of freshwater mussels in large Mobile Basin streams,
USA. Journal of the North American Benthological Society 22:
78-91.

Helfman, G. S. 1993. Fish behavior by day, night and twilight. In: T.
J. Pitcher, ed.. Behavior of Teleost Fishes. Vol. 7. Chapman and
Hall, London. Pp. 479-512

Howick, G. L. and W. J. O’Brien. 1983. Piscivorous feeding behav-
ior of largemouth bass: An experimental study. Transactions of
the American Fisheries Society 112: 508-516.

Kraemer, L. IL 1970. The mantle flap in three species of LampsUis
(Pelecypoda: Lhrionidae). Malacologia 10: 225-282.

Layman, C. A. and K. O. Winemiller. 2004. Size-based responses of
prey to piscivore exclusion in a species-rich neotropical river.
Ecology 85: 1311-1320.

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24 • 1/2 • 2008

Ortmann, A. E. 1914. Studies in najades (cont.). The Nautilus 28:
41-47.

Ortmann, A. E. 1926. Unionidae from the Reelfoot Lake region in
West Tennessee. The Nautilus 39: 87-94.

Parmalee, P. W. and A. E. Bogan. 1998. The Freshwater Mussels of
Tennessee. University of Tennessee Press, Knoxville, Tennes-
see.

Rypel, A. L. and |. B. Mitchell. 2007. Summer nocturnal patterns in
freshwater drum. American Midlatid Naturalist 157: 230-234.

Shoup, D. E., R. E. Carlson, and R. T. Heath. 2004. Diel activity
levels of centrarchid fishes in a small Ohio lake. Transactions
of the American Fisheries Society 5: 1264-1269.

Toomey, M. B., D. McCabe, and 1. E. Marsden. 2002. Factors af-
fecting the movement of adult zebra mussels (Dreissena poly-
morpha). Journal of the North American Benthological Society
21: 468-475.

Trdan, R. |. 1981. Reproductive biology of Latnpsilis radiata sili-
quoidea (Pelecypoda: Unionidae). American Midland Natural-
ist 106: 243-248.

Watters, G. T. 1994. An annotated bibliography of the reproduction
and propagation of the Unionoidea (Primarily of North
America). Ohio Biological Survey, Ohio State University, Co-
lumbus, Ohio.

Welsh, I. H. 1933. Photic stimulations and rhythmical contractions
of the mantle flaps of a lamellibranch. Proceedings of the Na-
tional Academy of Science 19: 755-757.

Submitted: 1 March 2007; accepted: 20 December 2007;

final revisions received: 27 December 2007

Amer. Maine. Bull. 24: 101-115

Meta-analysis of the relationship between salinity and molluscs in tidal river
estuaries of southwest Florida, U.S.A.

Paul A. Montagna*, Ernest D. Estevez^, Terry A. Palmer*, and Michael S. Flannery^

* Harte Research Institute for Gulf of Mexico Studies, Texas A&M University - Corpus Christi, 6300 Ocean Drive, Unit 5869,

Corpus Christi, Texas 78412-5869, U.S.A., Paul.Montagna@tamucc.edu
^ Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, Florida 34236, U.S.A.

^ Southwest Florida Water Management District, 2379 Broad Street, Brooksville, Florida 34604, U.S.A.

Abstract: The estuaries and rivers of the western coast of Florida have been under intense study for some time to identify relationships
between inflows, salinity, and natural resources. The molluscs have been shown to be especially sensitive to salinity in other parts of the
world. The current study performed a meta-analysis of existing data sets of southwest Florida mollusc communities to identify salinity-
mollusc relationships at regional scales. The mollusc species are controlled more by water rather than the sediment they live in or on. The
most important variable correlated with mollusc communities was salinity, which is a proxy for freshwater inflow. Although total mollusc
abundance was not a good indicator of inflow effects, certain indicator species characterized salinity zones in southwest Florida rivers.
Corbicula fluminea (Muller, 1774), Rangia cimeata (Sowerby, 1831 ), and Neritina usnea (Roding, 1798) were the only common species that
occurred in the oligohaline zone at salinities below 1 psu. Although C. fluminea was the best indicator of freshwater habitat, it is a
non-native, invasive bivalve species. The bivalve R. cimeata is an indicator of mesohaline salinity zones with an estimated tolerance of up
to 20 psu. The gastropod N. usnea is also common in fresh to brackish-water salinities. Polymesoda caroliniana (Bose, 1801) was present
at salinities between 1 and 20 psu, which span the oligohaline and mesohaline zones. Tagelus plebeius (Lightfoot, 1786), Crassostrea virginica
(Gmelin, 1791), MuHnia lateralis (Say, 1822), Littoraria irrorata (Say, 1822), and Ischadium recurvum (Rafmesque, 1820) are also good
indicators for polyhaline salinity zones. These salinity ranges can be used to predict changes in mollusc assemblages in response to
alterations in salinity that result from actual or simulated changes in freshwater inflow.

Key words: Mollusca, benthos, freshwater inflow, indicator species, water management

Estuaries are among the most productive environments
on Earth (Odum 1959). The mixing of freshwater with sea-
water is the defining characteristic of an estuary, and thus,
there is much interest in how alterations of freshwater inflow
patterns might affect estuarine productivity (Montagna et al.
2002b). Certainly, the increasing size of the human footprint
has had a dramatic effect on altering the courses and char-
acteristics of rivers, streams, and lakes; these watershed-level
changes have had effects on downstream estuaries in the
west (Kimmerer 2002) and Gulf of Mexico coasts (Alber
2002, Powell et al. 2002) of the U.S.A. To identify the effects
of altered flow, ecological indicators must be developed.
Molluscs are ideal organisms to indicate inflow effects be-
cause of their life habits and feeding modes (Estevez 2002).
Molluscs have well-defined relationships between species
distributions and physicochemical variables that are affected
by freshwater inflows, e.g. salinity (Montagna and Kalke
1995). Eilter or suspension-feeding molluscs also depend on
primary productivity in the water column for food, which is
also affected by nutrients carried by freshwater inflow into
estuaries.

The Mote Marine Laboratory (MML) and the South-
west Elorida Water Management District have completed

studies of mollusc distributions for six tidal rivers in south-
west Florida located between the Springs Coast, Charlotte
Harbor, and Tampa Bay (Fig. 1). A consistent methodology
was used in these studies for the Peace River, Alafia River,
Myakka River, Weeki Wachee River, Shell Creek, and the
Shakett Creek Dona/Roberts Bay system (MML 2002, 2003,
Estevez 2004a, 2004b, 2005). Extensive environmental data
also exists for freshwater inflows and physicochemical vari-
ables (e.g., salinity, dissolved oxygen, pH, and sediment
characteristics) in these systems that cover the period of
mollusc data collection. Although there have been studies of
individual river and creek systems in Florida, there has not
been an effort to combine data from many tidal rivers to
quantify factors that affect mollusc distributions at the re-
gional scale. Understanding the relationships between salin-
ity and other environmental parameters that relate to mol-
lusc distributions is important to evaluate the freshwater
flow requirements needed to protect the natural resources in
coastal ecosystems.

The overall goal of the current study was to ( 1 ) identify
indicator species of freshwater inflow effects and (2) to bet-
ter define the physical and chemical requirements ot mollusc
species that inhabit tidal river systems in southwest Florida.

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AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

The purpose was to synthesize existing information on the
relationships between freshwater inflows and the distribu-
tion of mollusc populations among the tidal rivers of south-
west Florida. The approach used in this project was to or-
ganize the mollusc and environmental data from the six tidal
river systems into one database with a common format, to
find the appropriate spatial scales in the data so that the
different tidal rivers could be compared, and to perform a
multivariate analysis on the combined data sets.

MATERIALS AND METHODS

Study area

The study sites were all located on the west coast of
peninsular Florida (Fig. 1 ). They group into four areas ot the
coast: Weeki Wachee River estuary, Alafia River in Tampa
Bay, Curry Creek and Shakett Creek located in the Dona/
Roberts Bay estuary, and Charlotte Harbor estuaiy.

Charlotte Harbor bay and estuary complex contained
six of the 10 sites studied, and four of the six were in the arm
of Charlotte Harbor that is dominated by the Myakka River.
There were three sites that were connected to the Myakka
River: (1) Big Slough is near the 14 km marker, (2) Deer
Prairie Creek is near the 19 km marker, and (3) Blackburn
Canal is near the 32 km marker. The eastern arm of Char-
lotte Harbor is dominated by the Peace River, which is con-
nected to Shell Creek near the 15 km marker. The Peace
River ecosystem has been sampled three times: twice in the
Peace River itself and once just in Shell Creek.

Shakett and Curry Creeks are located in the Dona/
Roberts Bay complex in the region designated as the Venice
Estuary. This area is north of, but adjacent to, the Charlotte
Harbor estuary. Shakett Creek ends in Dona Bay and Curry
Creek ends in Roberts Bay.

The Alafia River is about 80 km long and drains into
Tampa Bay. Further north are the two small tidal rivers: the
Weeki Wachee and the Mud Rivers. The Weeki Wachee
River is a small, spring-fed system in which the penetration
of brackish water is generally less than 2.5 km upstream
from the river mouth. Mud River, which is also spring-fed,
joins the Weeki Wachee about 1.4 km upstream of the river
mouth. While the upstream reaches of the Weeki Wachee
are fresh, the Mud River receives flow from brackish springs
and salinity in the Mud River increases upstream toward the
river head.

Mollusc data

Data for a meta-analysis on molluscs were extracted
from several reports designed and implemented by the Mote
Marine Laboratory (MML) (MML 2002, 2003, Estevez
2004a, 2004b, 2005). The data sets were complex and had to

MOLLUSCS IN SW FLORIDA TIDAL ESTUARIES

103

be concatenated, merged, and formatted prior to analysis.
The initial steps in database creation were to determine the
relationship between site designations in the data set, if there
were any differences in the actual sampling designs in the
different rivers, and if there were aggregation relationships
among the rivers (Table 1).

The sampling design employed by MML consists of
molluscs being sampled along transects within each river
system. The transects run lengthwise originating at the
mouth of each river, heading upstream; hence, distance and
station number names increase with freshwater influence.
The original data sets varied uniquely among river systems;
however, all samples were characterized by distance along
the river transect and the mollusc species composition.
These distances represented the stations within the river site,
and a total of 1 80 such stations were sampled across all sites
(Appendix 1). At each sampling location, molluscs were
sampled systematically across the river channel perpendicu-
lar to the river centerline so that samples were collected from
all major habitats found in mid-channel, shallow subtidal,
and intertidal areas. Most sampling locations were spaced at
half-kilometer intervals.

Eor each sampling event, the variables reported in-
cluded the number of juvenile molluscs, the number of live
molluscs, the number of dead molluscs, the size of shells,
and whether the samples were taken from the subtidal or
intertidal area of the river system. For all statistical analyses
in the current study, mollusc counts from the subtidal and
intertidal zones of each station were combined. Sample area
was 0.464 m", and the raw counts were converted to abun-
dance of individuals per square meter (n m“^) for all univariate
and multivariate analyses. For the current study, meta-
analysis was focused on live molluscs; however, the dead
shells do provide information on historical communities.

Samples from multiple years of sampling were found
only from the Peace River (Table 1). For the purpose of the

current study, the sampling stations at Peace River were
averaged over the two years they were sampled (1999 and
2000). Combining the two years of data was supported in
part by the absence ot evidence for shell drift.

To enable a meta-analysis that simultaneously compares
all rivers using multivariate methods, the distance along each
transect had to be standardized. To do this, the distance
from each river’s mouth of each sampling station was ag-
gregated into 2-km segment bins (Appendix 1). This was
performed by rounding the actual distance from the mouth
of the river (in kilometers) to increments of two. Each seg-
ment was numbered as the midpoint of the actual distance,
thus a segment labeled 2 km would encompass stations
found at 1.0 km to 2.9 km of a transect. Overall, 67 new
stations, or 2-km segments, were created for analysis. Be-
cause more than one sampling station occurred within many
new 2-km segments, species abundances were averaged
across stations within each new 2-km segment prior to
analysis to ensure a balanced sampling design.

The scientific names of all the species were verified and
made consistent across all data sets. In addition, the full
taxonomic description was verified. The convention for spe-
cies names and taxonomy used in the current study is based
on the Species 2000 and Integrated Taxonomic Information
System (ITIS) Catalogue of Life: 2006 Annual Checklist
(Bisby et al. 2006, http://www.sp2000.org).

Hill’s number one (Nl) diversity index was used to
report species diversity (Hill 1973). Hill’s Nl is the expo-
nential form ) of the Shannon-Weaver diversity index
H'. Nl was used because it has units of numbers of domi-
nant species, and it is easier to interpret than most other
diversity indices (Ludwig and Reynolds 1988).

Multivariate analyses

Community structure of mollusc species was analyzed
by non-metric multi-dimensional scaling (MDS). All multi-

Table 1. Location of sites within river systems, sampling year(s), and time period that water hydrography data were collected.

Estuary

River system

Site (or creek)

Molluscs

Hydrography

Tampa Bay

Alafia

Alafia

2001

Jan 1999- Dec 2003

Charlotte Harbor

Myakka

Big Slough

2004

—

Charlotte Harbor

Myakka

Blackburn

2004

—

Charlotte Harbor

Myakka

Deer Prairie

2004

—

Charlotte Harbor

Myakka

Myakka

2004

Feb 1998-Mar 2005

Charlotte Harbor

Peace

Peace

1999 & 2000

Aug 1996- Dec 2004

Charlotte Harbor

Peace

Shell

2004

Feb 1991 -Dec 2004

Venice

Dona/Roberts Bay

Curry

2004

Aug 2003-May 2005

Venice

Dona/ Roberts Bay

Shakett

2004

Aug 2003-May 2005

Weeki Wachee

Weeki Wachee

Mud River

2005

July 2003-May 2005

Weeki Wachee

Weeki Wachee

Weeki Wachee

2005

luly 2003-May 2005

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AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

variate statistical analyses were performed using Primer soft-
ware (Clarke and Warwick 2001). The MDS procedure was
used to compare mean abundances of individuals of each
species for each river-site-segment combination. The MDS
analysis was completed using a Bray-Curtis similarity matrix
on log-transformed In (x -l- 1) data. The distance between
river-site-segment combinations in the MDS plot can be
related to community similarities or differences between riv-
ers, sites, and segments. Differences and similarities among
communities were highlighted based on cluster analysis cal-
culated from the similarity matrix. A subset of species that
represented the spatial pattern in an MDS plot was deter-
mined using the BVSTEP procedure. The BVSTEP proce-
dure employs a step-wise approach to determine the mini-
mum subset of species that can yield the same pattern ot
community structure obtained from the entire data set
(Clarke and Warwick 1998).

Physicochemical variables

Physicochemical data for each tidal river system in-
cluded profiles of temperature, dissolved oxygen, salinity,
and pH taken along all transects. Profiles were measured at
various distances along the transects in each river on mul-
tiple dates over a period of 2-13 years. The length of period
and actual years sampled varied for each river (Table 1). As
with the mollusc data, the distance along each transect was
converted into the same 2-km segments for the physical
data. The four water hydrography parameters measured
(temperature, dissolved oxygen, salinity, and pH) were all
averaged by transect segment and river.

Principle Components Analysis (PCA), a parametric
multivariate method, was used to determine differences for
the environmental measurements among river-segment
combinations. As with MDS, the distance between river-
segment combinations in the PCA plot can be related to
actual similarities or differences in water hydrography be-
tween river-segment combinations.

Sediment

Samples along each transect were also analyzed by MML
for sediment characteristics. Sediment grain size distribu-
tions (median, mean, % sand, % silt, % clay, skewness, kur-
tosis), sediment moisture, and the proportion of organic
material present in the sediment were measured.

Relating molluscs and environmental factors

Relationships between mollusc communities and envi-
ronmental factors were investigated using the Biota-
Environment (BIO-ENV) procedure using Primer software
(Clarke and Warwick 2001). The BIO-ENV procedure is a
multivariate method that matches biotic {i.e., mollusc com-
munity structure) with environmental variables. This is car-

ried out by calculating weighted Spearman rank correlations
(p^^) between sample ordinations of all environmental vari-
ables and biotic variables (Clarke and Ainsworth 1993). Cor-
relations are then compared to determine the best match.
The BIO-ENV procedure uses different numbers of abiotic
variables in calculating correlations to investigate the differ-
ent levels of environmental complexity. Eor this study, the
mollusc species abundance MDS ordination was compared
with all physicochemical and sediment variables. A total of
49 of the 67 river-segment combinations were used in the
multivariate analysis because these stations had all sediment,
physiochemical, and mollusc data necessary for analysis. The
significance of relationships were tested using RELATE, a
non-parametric form of the Mantel test. The BIO-ENV and
RELATE procedures were calculated with Primer software
(Clarke and Warwick 2001).

Salinity was used as a proxy for distance from a fresh-
water source because salinity increases as distance from the
freshwater source increases. Salinity was directly compared
with individual species abundances, total mollusc abun-
dances, and mollusc diversity.

The relationship between mollusc abundance, diversity,
and salinity were examined with a non-linear model, which
was used successfully in Texas estuaries (Montagna et at.
2002a). The assumption behind the model is that there is an
optimal range for salinity and values decline prior to and
after meeting this maximum value. That is, the relationship
resembles a bell-shaped curve. The shape of this curve can be
predicted with a three-parameter, log normal model:

Y = ax exp(-0.5 x (ln(X / c) / b)^)

The model was used to characterize the nonlinear relation-
ship between a biological characteristic (Y, e.g., abundance
or diversity) and salinity (X). The three parameters charac-
terize different attributes of the curve, where a is the peak
abundance value, b is the skewness or rate of change of the
response as a function of salinity, and c the location of the
peak response value on the salinity axis (Montagna et al.
2002a). The model was fit to data using the Regression Wiz-
ard in SigmaPlot (version 10) which uses the Marquardt-
Levenberg algorithm to find coefficients (parameters) of the
independent variables that give the best fit between the equa-
tion and the data (Systat 2006).

RESULTS

Physical environments

With the exception of Mud River, salinity decreased
with distance from the river or creek mouth in all the river
systems (Fig. 2). Because rivers and transects in each river
were different, the length of each transect covered different

MOLLUSCS IN SW FLORIDA TIDAL ESTUARIES

105

Figure 2. Mean salinity along transects at each creek /site system.

salinity ranges; thus, a km segment number in one river did
not correspond to a similar salinity range in another system.
The transects of the Alafia, Myakka, and Peace Rivers were at
least 20 km long and had mean salinity ranges between 20
and 25 psu. Although the Shaken Creek and Week! Wachee
River transects covered less than 8 km, they also covered a
mean salinity range of at least 15 psu. The transects in Curry
Creek, Shakett Creek, and Mud River did not extend to
freshwater, as did the transects on the other river systems. A
salinity barrier on Shakett Creek truncates this river and
structurally isolates a freshwater zone under most flow con-
ditions. As described earlier, the Mud River is an unusual
system that is fed by brackish springs and salinity increases
toward the river head. Only two transect segments were
sampled in each of Curry Creek and the Mud River.

The principal components (PC) analysis reduced the
four environmental variables of salinity, temperature, pH,
and dissolved oxygen (DO) into two PC axes. The first
(PCI) and second (PC2) principal components of the phys-
icochemical data explained 98.7% and 0.7% of the variation
within the data set, respectively (total 99.4%; Fig. 3). PCI
was dominated by salinity and pH differences and PC2 by
temperature (Fig. 3A). Dissolved oxygen differences were
not important because it varied little from the origin. Thus,
PCI represents changes over distance along the transects or
between rivers, and PC2 represents water body and temporal
change, with higher temperatures as higher PC2 values. The
PC analysis demonstrates that Alafia, Weeki Wachee, Sha-

PC2(1%)

Figure 3. Principal Components Analysis (PCA) of water hydrog-
raphy in southwest Florida rivers. A, Principal Component variable
loadings. B, Transect segment-river station scores. Symbol key: Al,
Alafia River; Cu, Curry Creek; Do, Dona/Roberts Bay; My, Myakka
River; Pe, Peace River; Sk, Shakett Creek; Sh, Shell Creek; We,
Weeki Wachee River.

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AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

kett, Curry, and Myakka are all distinct water bodies (Eig.
3B). The differences are primarily a result of separation
along the PC2 axis. Shakett, Cuny, and Myakka had similar
temperature conditions but were distinct from the Alafia
and Weeki Wachee in this regard. Separation along PCI and
PC2 indicates the Peace and Myakka Rivers were very similar
to one another with respect to their physical characteristics.
The Alafia River had a unique pattern where at low salinities
temperatures increased, but at high salinities temperatures
were similar.

Taphonomy

Examining the fossil shells or death-assemblages, i.e.,
taphonomy, is a good technic]ue to understand the deriva-
tion of extant benthic communities because it is an indicator
of the living community prior to sampling and between
sampling occasions (Powell et al. 1986). The total abundance
was similar with a mean of 95 m^“ relict shells compared to
a mean of 82 m“^ live shells. The proportion of dead shells
to live shells was similar overall because a paired-difference
test was not significantly different (P = 0.7822). A total of 56
relict species were found (Appendix 2). However, 22 more
species were found among relict shells than live shells. This
does not mean that species have gone extinct or are no
longer found in the study area. Shells can be transported
after death, and the age of the shells are unknown; therefore,
the remainder of this current report focuses on the living
fauna. However, there was no evidence from field observa-
tions that shells were transported in these low-flow rivers
and creeks.

was found in 27 river-segments and had a mean density of
33 individuals m“^ throughout all 67 river-segments. This
represented 40% of total mean abundance. The next four
most dominant species were Polymesoda caroliniana (Bose,
1801; 11%), Rangia cuneata (Sowerby, 1831; 8%), Tagelus
plebeius (Lightfoot, 1786; 6%), and Amygdalum papyrium
(Conrad, 1846; 5%). These top five most abundant molluscs
were bivalves and comprised 70% of all specimens found.
The dominant gastropod Neritina usnea (Roding, 1798) was
the sixth-ranked species in dominance (4% of total mean
abundance). The second-most dominant species P. carolin-
iann was found in 35 river-segments.

Dominance patterns were different in different rivers
(Appendix 3). Eor example, Corbicida flwninea was domi-
nant only in the Peace and Myakka Rivers. In contrast, P.
caroliniana was dominant in Shell Creek and Big Slough, and
the second-most dominant species in Deer Prairie Creek,
Myakka, and Weeki Wachee Rivers. Rangia cuneata was
dominant in Deer Prairie and was the only organism found
in Blackburn Canal. Tagelus plebeius was co-dominant in
Weeki Wachee and the dominant species in Mud River and
Curry Creek. Geukensia granosissima (Sowerby, 1914) was
dominant in the Alafia River, and Crassostrea virginica
(Gmelin, 1791) was co-dominant in Weeki Wachee and
dominant in Shakett Creek. However, the distribution of C.
virginica in the Weeki Wachee River was largely limited to
individuals located near the river mouth.

Similarity in mollusc communities among the river-
segment sites is generally low (Pig. 4). All of the river-
segment combinations are found in associations of groups of

Mollusc community structure

A total of 33 live species were
found in all of the rivers sampled (Ap-
pendix 2). Of these, 25 species were
bivalves and eight species were gastro-
pods. Two families of bivalves, Tellini-
dae and Mytilidae, were represented by
four species each, and there were three
species of Veneridae. Otherwise, all
families were represented by only one
or two species.

The dominant species was the
Asian Clam Corbicula fluminea
(Muller, 1774) an exotic species intro-
duced to Plorida waters (Appendix 3).
The large number of C. fluminea was
due to very high densities of this spe-
cies in the tidal freshwater reaches of
the Peace River; a lower density was
found in the Myaklca River, and five
rivers had none. Corbicula fluminea

2D Stress:

oil Site

A Alafia
o Curry
■ Shakett

♦ BigSlough

• Blackburn
+ DeerPrairie
X Myakka

=k Peace
A Shell
V MudRiver
□ WeekiWachee

Similarity

10

25

Figure 4. Relationships between mollusc communities from multi-dimensional scaling
(MDS) analysis. Symbols represent the river or creek site with shape and color, and the km
segment number is listed above the river symbol. Segment 16 from the Alafia River is outside
the range of this plot. Similarity is indicated with lines drawn around points.

MOLLUSCS IN SW FLORIDA TIDAL ESTUARIES

107

no more than 10% similarity. At the 10% similarity level
there are three groups, two smaller groups with low station
numbers {i.e., more marine conditions) and one large group.
At the 25% similarity level, the large group splits into 4
smaller groups. Although the pattern of river-segment group-
ings is based on 33 species, it is being driven by just seven
species: Corbicula fliuninea, Crassostrea virgiiiica, Littoraria ir-
rorata (Say, 1822), Neritina itsnea, Polyinesoda caroliuiaita,
Rangia cuneata, and Tagelus plebeius (BVSTEP, rho > 0.95, r =
0.96). These species drive the trend in which downstream seg-
ments close to marine sources (with low 2-km segment num-
bers) tend to group to the left, while higher segment numbers
groups to the right in the MDS plot (Fig. 4).

The four groups at the 25% level within the large central
group at the 10% similarity level (Fig. 4) can be explained
based on the distribution of three species. From left to right
in Fig. 4, the station groups are dominated by Crassostrea
virginica, Polymesoda caroliniana, and Corbicula fluminea.
Two groups fell outside the 10% similarity level. One group
had four Peace River segments (0, 2, 4, and 6). The other
group had just one Shakett Creek 0 segment, and this was
most different from all other segments because it had only
two rare species: Chione cancellata (Linnaeus, 1767) and Cy-
clinella tenuis (Recluz, 1852). The 16-km segment of the
transect in the Alafia River was so different from all others
that it is not included in the MDS plot. This station is 100%
different from all of the other stations sampled, because the
station had only one mollusc present, an unidentified snail
of the family Planorbidae, which was not found in any of the
other river systems.

Mollusc-environment relationships

Two approaches are used to relate molluscs to the en-
vironment, but in all cases salinity is used as the surrogate
for inflow. One approach is to relate (by univariate or mul-
tivariate models) salinity with abundance, diversity, or com-
munity structure. The second approach is to examine the
relationship between abundance and salinity to identify
those species or species groups that might have optimal sa-
linity ranges.

For the first approach, a non-parametric multivariate
analysis procedure (BIO-ENV) was used to identify the com-
binations of environmental variables that could best predict
mollusc abundance. Out of 62 transect-segments sampled
for water hydrography and 67 transect-segments sampled for
molluscs, there were only 45 common transect-segments
that could be analyzed using BIO-ENV because of missing
water hydrography data in the other transect-segments. Sa-
linity, temperature, and pH were the environmental vari-
ables that correlated the highest with the mollusc commu-
nity distributions (p„ = 0.612). The RELATE procedure
indicated that this correlation was significant (P < 0.001).

The single physical variable that correlated the highest with
mollusc communities was salinity (p^^ = 0.566). In fact, sa-
linity was the only variable that fit the community distribu-
tions in all the tests. The water hydrography variables had
higher correlations with the mollusc communities than any
single, or combination of, sediment characteristics. Of the
sediment variables, median and mean grain size fit best, but
all sediment variables were selected after salinity, tempera-
ture, and pH. This indicates that overlying water properties,
especially salinity values, have more control on the mollusc
communities than the sediment characteristics.

In the second approach, total mollusc abundance did
not correlate with salinity among all rivers. The highest
abundances occurred at low salinities, but this is attributed
to the large population of Corbicula fluminea that occurred
in the Peace River at low salinities. Mollusc diversity in-
creased with salinity, particularly as salinity increased from 0
to 2 psLi, but the correlation was weak. Hill’s N1 values were
consistently close to one where mean salinity was close to
one; however, as salinity and overall N1 increased, so too did
the range of N1 values.

Two rivers, the Myakka and Peace, were sampled across
long transects (Fig. 2). Examining distributions along salin-
ity gradients in these two rivers separately avoids bias due to
differences between the systems. In both rivers there were
strong relationships between both diversity and abundance
with salinity where abundance and diversity increased with
increasing salinity, then peaked, before cieclining (Fig. 5).
This response emulates a 3-parameter log-normal distri-
bution, which was found to fit total macrofauna abundance
in a Texas estuary (Montagna et al. 2002a). The nonlinear
relationship between salinity and diversity was stronger in
the Peace River than the Myakka River, based on the prob-
ability levels (P) and goodness of fit parameters (R^)
(Table 2).

The ten dominant species were examined for correla-
tions with salinity (Table 3). Corbicula fluminea was found
only where mean salinities were <7 psu, but it was most
common where mean salinities were <2 psu (Fig. 6A). How-
ever, the maximum salinity value (parameter c in Table 2)
was 0.6 psu. Corbicula fluminea occurred at abundances
higher than 10 m““ only in the Myakka and Peace Rivers.
Polymesoda caroliniana was found in all river systems and
occurred where salinities ranges from 1 to 20 psu (Fig. 6B)
while peaking at 5 psu (Table 2). Both P. caroliniana and C.
fluminea are in the same family (Corbiculidae). Rangia cu-
neata and Tagelus plebeius were found in low to moderate
salinities and occurred at salinity peaks of 4 and 7 psu re-
spectively (Figs. 6C-D). Crassostrea virginica and Geukensia
granosissima were generally found at higher salinities, as in-
dicated by salinity peaks of 24 and 10 psu, respectively. Mu-
linia lateralis ranged from 5 to 15 psu, and the model cal-

108

AMERICAN MALACOLOGICAL BULLETIN

24 • 1/2 • 2008

0 5 10 15 20 0 5 10 15 20

Salinity (psu) Salinity (psu)

Figure 5. Relationship between total mollusc diversity (A) and abundance (B) vs. salinity at Myakka River and diversity (C) and abundance
(D) versus salinity at Peace River. Circles, Hill’s N1 diversity index; squares, abundance.

ciliated a peak at 14 psu. According to the model, Neritina
usnea abundance did not change with salinity (P = 0.43).
Littoraria irrorata and Ischadiuni recurvum were found over
a wide range of salinities, with peak salinities at 14 and 12
psu, respectively. Two other species, Amygdalum papyriiiin
and Tellina versicolor, occurred in less than 9 segments, pre-
cluding an estimation of the salinity range.

DISCUSSION i

The overall purpose of this project was to better define
the biogeography, community structure, and the physical I
and chemical requirements of mollusc species that inhabit
tidal river systems in southwest Florida. To meet this pur-
pose, an inter-river meta-analysis was performed to examine

MOLLUSCS IN SW FLORIDA TIDAL ESTUARIES

109

Table 2. Parameters from nonlinear regressions to predict mollusc characteristics from salinity. These parameters are represented on lines
in Figs. 5 and 6. Probability (P) that model fits the data, percent of variance explained by data (P‘), parameters for maximum biological
value (a), rate of change (b), and salinity in which maximum abundance occurs (c), and standard deviation for parameters in parentheses.
Nl, Hill's diversity index; n, abundance (individuals per m“); all species are n m^~.

Variable

P

a

b

c

Myakka Nl

0.1658

0.26

3.11 (0.36)

2.45 (0.65)

2.15 (0.86)

Myakka n

0.0682

0.36

54.9 (7.9)

2.63 (0.84)

0.59 (0.41)

Peace Nl

0.0098

0.64

7.29 (1.02)

1.61 (0.31)

0.99 (0.28)

Peace n

0.0013

0.77

218 (24.8)

1.44 (0.20)

1.05 (0.20)

Neritina usnea

0.4320

0.03

4.92 (1.71)

2.96 (2.77)

0.45 (1.33)

Corbiciila fluniinea

0.0001

0.31

178 (43.2)

0.78 (0.19)

0.63 (0.18)

Rangia Cuneata

0.0001

0.38

27.3 (4.8)

0.49 (0.08)

3.69 (0.31)

Polymesoda caroliniana

0.0001

0.32

28.8 (5.1)

0.66 (0.13)

4.89 (0.63)

Tageliis pleheiiis

0.0003

0.28

15.4 (3.0)

0.48 (0.12)

7.30 (0.90)

Geukensia granosissima

0.0001

0.77

156 (11.9)

0.006 (3e-7)

10.3 (3e-6)

Ischadiiim recurviim

0.0169

0.16

5.68 (1.81)

0.31 (0.11)

12.3 (1.3)

Mulinia lateralis

0.0001

0.37

324 (53.3)

0.006 (3e-7)

13.6 (8e-6)

Littoraria irrorata

0.0001

0.33

6.43 (1.28)

0.31 (0.07)

13.8 (0.98)

Crassostrea virginica

0.0001

0.33

19.3 (4.2)

0.18 (0.04)

22.4 (1.0)

Table 3. Salinity range of twelve most abundant species.

Species

Salinity range
(psu)

Transect segments
with species present

Corbicula fluniinea

<7 (most <2)

20

Polymesoda caroliniana

1 to 20

32

Rangia cuneata

<16 (most <10)

23

Tagelus plebeius

>2

25

Geukensia granosissima

10 to 24

5

Amygdaliim papyriiim

2 to 20

8 (7 in Peace R.)

Crassostrea virginica

>7

13

Mulinia lateralis

>2

10

Neritinia usnea

<18

20

Tellina versicolor

2 to 18

7 (all in Peace R.)

Littoraria irrorata

>2

17

Ischadium recurvum

>6

1 1

relationships between the distribution of mollusc popula-
tions both within and among tidal river estuaries and tidal
river locations. The meta-analysis combines independent
studies to reach general conclusions (Gurevitch and Hedges
2001). The sampling gear and spatial sampling strategies
were consistent for both water hydrography and mollusc
data, making this meta-analysis a simple task. Although
these data were collected without specific regard to a re-
gional scale design and analysis, the data fit well into a sam-
pling design, even though all samples were not taken in the
same year (Table 1 ). Two exceptions to this lack of synoptic
sampling were the Myakka and Dona/Roberts Bay systems.
However, all the rivers exhibited distinct changes in their

water hydrography characteristics and mollusc community
composition along the estuarine gradient. Therefore, analy-
sis of these data provides meaningful information on how
environmental factors affect the distribution and abundance
ot mollusc populations within these tidal river ecosystems.

River systems were strikingly different. The mollusc
communities among all the river stations shared <25% spe-
cies in common. Although sampling occurred over different
years, there were community similarities at similar transect
segments among rivers. There were upstream clusters,
downstream clusters, and larger clusters of intermediate
range transects. The segments with the most similar mollusc
communities occurred in the most upstream segments of the
Peace, Myakka, and Alafia Rivers. These segments had the
most stable, and lowest mean salinities with minimal tidal
infiuence. Further downstream, freshwater influence de-
creased and salinity was more variable, which allowed dif-
ferent species and communities to persist, compared to
stable upstream waters. Other factors such as tides, waves,
currents, and inshore geomorphology create diverse habitats
both within and between estuarine river systems. This in-
crease in physical diversity from upstream to downstream
can cause the considerable differences found in mollusc
communities along the salinity gradient and among the riv-
ers. The heterogeneity of the salinity regimes is why the river
systems share <25% of species in common.

The highest correlations between physical variables and
mollusc communities were with salinity. Salinity differences
were, thus, more important than sediment differences in
regulating mollusc communities in tidal rivers of southwest
Florida. The physical variables with the highest correlations

no

AMERICAN MALACOLOGICAL BULLETIN

24 • 1/2 • 2008

0 10 20 30

0 10 20 30

0 10 20 30

Salinity (psu) Salinity (psu) j

1:

Figure 6. Relationship between salinity and species abundance. A, Corbicula jluminea; B, Polymesoda caroliniana; C, Rangia ciineata; and 'I
D, Tagehis plebeiiis. Symbol key: Al, Alafia River; Cu, Curry Creek; Do, Dona/Roberts Bay; My, Myakka River; Pe, Peace River; Sk, Shakett 11
Creek; Sh, Shell Creek; We, Weeki Wachee River.

with the macrofaunal community structure almost always
included salinity, temperature and pH. The best single physi-
cal indicator of mollusc communities was salinity. Thus,
freshwater inflow, which is one factor controlling salinity, is
an important factor influencing mollusc community struc-
ture and abundance patterns. Corbicula fluminea, Rangia cu-
neata, and Neritina usnea were the only species common to
rivers, creeks, and canals that occurred at salinities below 1
psu. However, C. fluminea was the best indicator ot fresh-
water habitat and is an introduced bivalve that can survive
salinities up to 13 psu (Morton and Tong 1985) but usually
occurs primarily in freshwater (Batelle 2000). Rangia cuneata

is an indicator of a fresh to brackish -water (Swingle and ji
Bland 1974, Montagna and Kalke 1995). Neritina usnea is 1
also common in fresh to brackish-water salinities (Andrews ;j
1992). Polymesoda caroliniana is a native, brackish-water bi- j'
valve (Gainey and Greenberg 1977) also from the family ;■
Corbiculidae. In the current study, P. caroliniana was pres-
ent at salinities between 1 and 20 psu and was present in all i
creeks/sites. ■

Tagelus plebeius, Crassostrea virginica, Mulinia lateralis, '
Littoraria irrorata, and Ischadium recurvum are also good
indicators for brackish to seawater salinities. The relation- -
ship between C. virginica and salinity is well known (Turner |'

MOLLUSCS IN SW FLORIDA TIDAL ESTUARIES

111

2006). Mulinia lateralis prefers organically-rich muddy sedi-
ments (Grassle et a!. 1992) and has the ability to survive
short periods of anoxia (Shumway et ah 1993). Muliuia lat-
eralis is typically found in euryhaline habitats (Williams
1984). Although these bivalves are most often found in these
brackish to euryhaline salinity zones, they may also be most
susceptible to predation in the same area. For example, the
oyster drill Stramonita liaewashvita (Gray, 1839) can se-
verely crop I. recunnim and Ratigia ciineata at high salinities
and limit these prey to lower salinity areas along the Gulf of
Mexico coast (Brown and Richardson 1988).

Total mollusc abundance and aggregated mollusc spe-
cies diversity did not indicate freshwater inflow across all
rivers, but were useful within rivers. In addition, the trend of
transect numbers increasing from left to right in the MDS
analysis is evidence of seriation (i.e., linearity or spatial as-
sociations) in the mollusc communities.

In summary, from this meta-analysis of southwest
Florida communities, mollusc species appear controlled
more by water column hydrography rather than the sedi-
ment composition. Salinity is the most important environ-
mental variable and is an indicator or proxy for freshwater
inflow. One typical approach to link community responses
inflow changes is to perform long-term studies of inflow
events. The current study used spatial variability at a re-
gional scale to capture a large range of salinity differences,
and hence inflow influences. Certain indicator species have
been identified that characterize salinity ranges in southwest
Florida rivers. These salinity ranges may be useful in pre-
dicting mollusc community reactions to changes in freshwa-
ter inflow.

Although meta-analysis is an emerging and accepted
practice, synoptic sampling over time would greatly improve
the ability to accurately determine the relationships between
inflow and the mollusc communities, relative to those in
other regions. Synchronization of sampling and sample rep-
lication would also improve the ability to accurately corre-
late mollusc communities’ response to freshwater inflows
using the types of data analysis reported here. Nevertheless,
the use of transect-segments in the current meta-analysis
and comparing data from the different surveys has led to
robust conclusions.

The present study clearly demonstrates that estuarine
mollusc species are arrayed along horizontal salinity gradi-
ents within tidal river estuaries, with certain species being
most common in low salinity zones (e.g., <10-15 psu). In
addition to salinity, other factors such as current velocities
or the availability of detrital or planktonic food resources
could contribute to mollusc distribution patterns in tidal
rivers. Low salinity zones are among the habitats that are
most vulnerable to impacts and loss within Gulf Coast es-
tuaries because of proximity to human activities in adjacent

uplands and the sources of pollution from the contributing
watersheds (Lewis and Robison 1995, Beck et al. 2005). Low
salinity zones are also particularly sensitive to shifts and
changes in salinity regimes that could be caused by freshwa-
ter withdrawals or salinity intrusions. Given that distinct
mollusc communities occur within low salinity waters, the
proper management of freshwater inflows and other related
watershed activities are very important for maintaining the
biological integrity of mollusc populations in tidal river
estuaries.

ACKNOWLEDGMENTS

This work is the result of research funded by a contract
from the Southwest Florida Water Management District to
Paul A. Montagna, Principal Investigator. We thank Dean
Rusk, SWFWMD, for drafting Fig. 1.

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Submitted: 24 May 2007; accepted: 21 December 2007;

final corrections received: 8 January 2008.

Appendix 1. Aggregation of Mote Marine Laboratory (MML) sam-
pling data for the current analyses. For each river-site, the new
2-km bin name created and the number of MML stations that were
located within the 2-km bin.

River

Site

2-km bin
name

Number of
stations

Alafia

Alafia

0

2

Alafia

Alafia

2

3

Alafia

Alafia

4

4

Alafia

Alafia

6

4

Alafia

Alafia

8

4

Alafia

Alafia

10

4

Alafia

Alafia

12

3

Alafia

Alafia

16

1

Alafia

Alafia

18

1

Dona/Roberts

Curry

2

3

Dona/Roberts

Curry

4

2

Dona/Roberts

Shakett

0

1

Dona/Roberts

Shaken

2

4

Dona/Roberts

Shakett

4

4

Dona/Roberts

Shakett

6

3

Myakka

Big Slough

2

2

Myakka

Blackburn

0

1

Myakka

Deer Prairie

2

2

Myakka

Deer Prairie

4

1

Myakka

Myakka

-0

2

Myakka

Myakka

2

2

Myakka

Myakka

4

2

Myakka

Myakka

6

2

Myakka

Myakka

8

2

Myakka

Myakka

10

2

MOLLUSCS IN SW FLORIDA TIDAL ESTUARIES

113

Appendix 1. (continued)
River Site

2-km bin
name

Number of
stations

Myakka

Myakka

12

2

Myakka

Myakka

14

3

Myakka

Myakka

16

1

Myakka

Myakka

18

2

Myakka

Myakka

20

3

Myakka

Myakka

22

2

Myakka

Myakka

24

1

Myakka

Myakka

26

3

Myakka

Myakka

28

2

Myakka

Myakka

30

2

Myakka

Myakka

32

2

Myakka

Myakka

36

2

Myakka

Myakka

38

3

Myakka

Myakka

40

1

Peace

Peace

0

1

Peace

Peace

2

1

Peace

Peace

4

1

Peace

Peace

6

1

Peace

Peace

8

4

Peace

Peace

10

4

Peace

Peace

12

4

Peace

Peace

14

4

Peace

Peace

16

5

Peace

Peace

18

5

Peace

Peace

20

4

Peace

Peace

22

5

Peace

Peace

24

4

Peace

Peace

26

5

Peace

Peace

28

4

Peace

Peace

30

4

Peace

Peace

32

4

Peace

Peace

34

3

Peace

Peace

36

1

Shell

Shell

0

2

Shell

Shell

2

4

Shell

Shell

4

4

Shell

Shell

6

3

Shell

Shell

8

4

Weeki Wachee

Mud River

2

2

Weeki Wachee

Mud River

4

1

Weeki Wachee

Weeki Wachee

0

2

Weeki Wachee Weeki Wachee

Total number of segment

2

4

bins and stations

67

180

Appendix 2. Taxonomic list of all live and relict species found.
Abundance of all relict and live individuals found per m^ averaged
over all samples (i.e., river-site-segment combinations). Abbrevia-
tions: CL, Class; OR, Order; and FA, Family.

CL OR FA Species Dead Live

Gastropoda

Pulmonata

Ellobium

Melampus sp.

0.055

0

Basommatophora

Planorbidae

Planorbidae (unidentified)

0.208

0.032

Neotaenioglossa

Littorinidae

Littomria irrorata (Say, 1822)

0.469

1.811

Epitoniidae

Epitonium rupicola (Kurtz, 1860)

0.031

0

Calyptraeidae

Crepidula fortiicala (Linnaeus, 1758)

0.318

0

Naticidae

PoIii:ices duplicatus (Say, 1822)

0.133

0.048

Cerithiidae

Cerithiiim atratum (Born, 1778)

0.495

0

Triphoridae

Triphora melaintra (Adams, 1850)

0.031

0

Cephalaspidea

Bullidae

Bidia striata (Bruguiere, 1792)

0.073

0

Flaminoeidae

Haminoea succinea (Conrad, 1846)

0.851

1.062

Neogastropoda
Conidae
Conus sp.

0.010

0

Nassariidae

Nassarius vibex (Say, 1822)

2.944

1.395

Melongenidae

Melongena corona (Gmelin, 1791)

0.247

0.153

Muricidae
Eupleura sp.

0.021

0

Urosalpinx tampaensis (Conrad, 1846)

0.042

0

Neritopsina

Neritidae

Neritina usnea (Roding, 1798)

5.990

3.028

Bivalvia
Myoida
Pholadidae
Cyrtopleura sp.

0

0.008

Veneroida

Cardiidae

Laevicardiuni mortoni (Conrad, 1830)

0.497

0.131

Corbiculidae

Corbicida fliiminea (Muller, 1774)

23.306

33.107

Polyrnesoda caroliniana (Bose, 1801)

13.281

9.052

114

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

Appendix 2. (continued)

CL OR FA Species

Dead

Live

Dreissenidae

Mytilopsis leucophaeata (Conrad, 1831)

6.093

0.796

Lasaeidae

Mysella phundata (Stimpson, 1851)

0.492

0.137

Lucinidae

Anodontia alba (Link, 1807)

0.062

0

Liicina pectiiiata (Gmelin, 1791)

0.203

0.011

Mactridae

Midii^ia lateralis (Say, 1822)

0.923

1.734

Rangia cuneata (Sowerby, 1831)

11.418

6.619

Spisida solidissima similis (Say, 1822)

0.031

0

Pharidae

Ensis minor (Dali, 1900)

0.031

0

Pisidiidae

Muscidiwn partumeiiiin (Say, 1822)

0.031

0.011

Pisidium sp.

0.008

0

Semelidae

Ahra aequalis (Say, 1822)

0.008

0

Solecurtidae

Tageliis plebeius (Lightfoot, 1786)

5.604

4.553

Solenidae

Soleii viridis (Say, 1821)

0.016

0

Tellinidae

Macoma constricta (Bruguiere, 1792)

0.515

2.662

Macoma tenta (Say, 1834)

0.102

0.056

Tellina versicolor (DeKay, 1843)

0.325

2.741

Tellina sp.

1.265

0.139

Veneridae

Anomalocardia anberiana

(d’Orbigny, 1842)

1.369

0.075

Chione cancellata (Linnaeus, 1767)

2.051

0.348

Cyclinella tenuis (Recluz, 1852)

0.161

0.059

Macrocallista nitnbosa (Lightfoot, 1786)

0.016

0

Mercenaria campechiensis

(Gmelin, 1791)

0.130

0

Veneridae (unidentified)

0.016

0

Arcoida

Arcidae

Anadara transversa (Say, 1822)

0.122

0.064

Noetiidae

Noetia ponderosa (Say, 1822)

0.016

0

Mytiloida

Mytilidae

Amygdalum papyriuni (Conrad, 1846)

0.261

4.268

Brachidontes modiolus (Linnaeus, 1767)

0

0.127

Geukensia granosissima (Sowerby, 1914)

1.201

2.793

Ischadiutn recurvum (Rafmesque, 1820)

1.861

1.780

Ostreoida

Ostreidae

Crassostrea virginica (Gmelin, 1791)

9.923

2.626

Dendostrea frons (Linnaeus, 1758)

0.445

0

Appendix 2. (continued)

CL OR FA Species

Dead

Live

Pectinidae

Argopecten irradians (Lamark, 1819)
Anomiidae

0.224

0

Anomia simplex (d’Orbigny, 1842)

0.916

0

Pterioida

Pinnidae

Atrina serrata (Sowerby, 1825)

0.010

0

Bivalvia (unidentified)

0.062

0.317

Mollusca (unidentified)

0.016

0.023

Total

94.929

81.765

MOLLUSCS IN SW FLORIDA TIDAL ESTUARIES

115

Appendix 3. Domiiiimce of
Species

all species as a percentage of all the mean

number of individuals tound
River or Creek

in each site (river or creek) sampled.

Alafia

Big

Slough

Blackburn Curry

Deer

Prairie

Mud

Myakka

Peace

Shakett

Shell

Weeki

Corbicula fluniinea

1.23

0

0

0

4.65

0

42.12

53.32

0

0.26

1.25

Polymesoda caroliiiiana

19.07

40

0

1.9

44.19

21.74

17.23

3.51

2.13

46.59

21.25

Rangia cuneata

0

24

100

0

51.16

0

8.86

5.79

0

30.9

0

Tagehis plebeiiis

3.69

28

0

34.18

0

30.43

9.54

1.36

24.63

19.31

23.75

Crassostrea virginica

21.88

0

0

5.7

0

26.09

0

1.06

27.59

0

25

Geukensia grauosissima

29.44

0

0

0

0

0

6.22

0.22

0

0

0

Amygdalum papyrium

1.23

0

0

0

0

0

0

8.28

0

0

0

Neritina iisnea

5.89

8

0

0

0

0

0.45

4.95

1.31

0.77

0

Ischadiiim recurviim

0

0

0

1.9

0

0

0.45

2.52

16.26

1.02

15

Littoraria irrorata

4.53

0

0

1.27

0

8.69

7.92

0.47

2.46

0.51

8.75

Macoma constricta

0

0

0

0

0

13.04

0

5.16

0

0

0

Chione cancellata

0

0

0

27.85

0

0

0

0

6.9

0

0

TelUna versicolor

0

0

0

0

0

0

0

5.42

0

0

0

Mulinia lateralis

1.71

0

0

3.8

0

0

2.49

2.44

0

0.13

0

Nassariiis vibex

0

0

0

3.8

0

0

0.11

2.63

0.99

0

0

Mytilopsis leiicophaeata

3.56

0

0

0

0

0

3.85

0

0

0.51

0

Haminoea succinea

0

0

0

0

0

0

0

2.1

0

0

0

Laevicardium mortoni

0

0

0

1 0.76

0

0

0

0

2.46

0

0

TelUna sp.

0

0

0

1.27

0

0

0

0

6.9

0

2.5

Bivalvia (unidentified)

4.35

0

0

0

0

0

0

0.1

0

0

0

Anomalocardia auberiana

0

0

0

1.27

0

0

0

0

3.94

0

0

Anadara transversa

0

0

0

3.8

0

0

0

0.06

0

0

0

Melongena corona

0

0

0

0

0

0

0

0.27

0

0

2.5

Mysella plaimlata

2.24

0

0

0

0

0

0

0

0

0

0

Cyclinella tenuis

0

0

0

1.27

0

0

0.1 1

0

1.97

0

0

Macoma tenta

0.66

0

0

0

0

0

0

0

0.99

0

0

Brachidontes modiolus

0

0

0

0

0

0

0

0.25

0

0

0

Lucitia pectinata

0

0

0

1.27

0

0

0

0

0

0

0

Mollusca (unidentified)

0

0

0

0

0

0

0

0.01

0.99

0

0

Planorbidae (unidentified)

0.53

0

0

0

0

0

0

0

0

0

0

Polinices duplicatus

0

0

0

0

0

0

0.11

0.06

0

0

0

Cyrtopleiira sp.

0

0

0

0

0

0

0

0

0.49

0

0

Musculium partumeium

0

0

0

0

0

0

0.08

0

0

0

0

Ainer. Maine. Bull. 24: 117-119

RESEARCH NOTE

Giant African snail, Achatina fulicay as a snail predator

Wallace M. Meyer III^’ Kenneth A. Hayes*’ and Amanda L. Meyer^

' Center for Conservation Research and Training, University of Hawaii at Manoa, 3050 Maile Way, Gilmore 408, Honolulu, Hawaii
96822, U.S.A., meyerwal@hawaii.edu

^ Department of Zoology, University of Hawaii at Manoa, 2538 McCarthy Mall, Edmonson 152, Honolulu, Hawaii 96822, U.S.A.

Abstract: Individuals oi Achatina fiilica (Bowdich, 1822) were observed preying on veronicellid slugs at two sites on the island of Oahu,
Hawaii. As such, the presence of A. fulica may pose a greater threat to terrestrial mollusc conservation than previously imagined. It is our
hope that this note provides some impetus for other researchers to explore the possible predation impacts of introduced populations ot A.
fulica and to consider the possibility that other introduced snails and slugs may be having as yet unforeseen or unnoticed impacts.

Key words: predation, introduced, invasive, alien species

Invasive species are recognized globally as a major threat
to biodiversity and ecosystem health (Carlton and Geller
1993, Lydeard et at. 2004, Pimentel et al. 2005). Numerous
non-native plants and animals have been implicated in the
extirpaation of native taxa (Vitousek et al. 1997, Gurevitch
and Padilla 2004). However, it is difficult to assign causality
to one factor such as invasive spaecies and ignore others, e.g.,
habitat modification and climate change (Gurevitch and Pa-
dilla 2004). Furthermore, knowledge of life histories and
behavioral characteristics of alien species is usually based on
studies in their native range, making it difficult to predict the
interactions of a particular alien species after introduction to
a new region. As such, many unpredicted interactions may
occur and possibly go unnoticed. Here we report on one
such interaction involving the giant African snail, Achatina
fulica (Bowdich, 1822), in Hawaii that has gone unrepiorted
for more than half a century. We also comment on the
possible implications of this interaction with native species
in Hawaii as well as in other parts of the world where A.
fulica has been introduced.

Achatina fulica is one of the largest land snails in the
world, reaching up to 19 cm in length (Peterson 1957). In
part because of its polyphagous diet, it has become recog-
nized as one of the world’s most damaging pests and is listed
in the Global Invasive Species Database (http://www.issg
.org/database/welcome/) among “One hundred of the
world’s worst invasive alien species” (Lowe et al. 2000). In
addition, this snail is known as a vector of at least two
human disease agents: the rat lung-worm Parastrongyhis
{=Angiostrongyhis) cantonensis (Chen 1935) and a gram-
negative bacterium, Aeronionas hydrophila, which causes a
wide range of symptoms (Mead 1956, 1961, Wallace and

Rosen 1969, Dean et al. 1970, Mead and Palcy 1992). Be-
cause of the snail’s prominence as a pest and disease vector,
a number of researchers have investigated its feeding and
behavioral ecology in both the field and laboratory (Mead
1961, Pawson and Chase 1984, Tomiyama 1994). In addition
to the 500 plant species that A. fulica is known to eat, the
snail will also consume decaying and rotting vegetation,
dung, garbage, wet paper and cardboard, dead animals, and
crushed (i.e., already dead) snails of its own kind (Srivastava
1992). Hence, it is surprising that no one has reported car-
nivorous behavior by this species in which it attacks, sub-
dues, and consumes live prey. This note reports three in-
stances of this predatory behavior on the island of Oahu,
Hawaii. These observations are noteworthy because they in-
dicate an alternative feeding mode for A. fulica, suggesting its
potential to impact other species through predation.

Individuals oi Achatina fulica were observed consuming
veronicellid slugs [Veronicella cubensis (Pfeiffer, 1840)) at
two sites on the island of Oahu (Fig. 1). Both sites were
anthropogenically altered, in close proximity to housing and
dominated by low shrubs. The first two observations were
made in Kaneohe (21°25'02"N, 157°48'51"W) in November
and December 2004. In both instances, an individual A.
fulica (ca. 5 cm in shell length) was observed consuming a
similar sized slug. No detailed observations were made dur-
ing these first two observations.

Slug consumption by Achatina fulica was also observed
in Hawaii Kai (21°16'12"N, 157°44'84"W) in September
2005. However, on this occasion, three smaller ( 10 to 15 mm
in shell length) A. fulica were observed consuming one
veronicellid slug (>5 cm in length) at the same time. In
order to determine if A. fulica kills the slugs or whether it

117

118

AMERICAN MALACOLOGICAL BULLETIN

24 • 1/2 • 2008

Figure 1. The two locations on Oahu (Ka-
neohe and Hawaii Kai) where Achatina fn-
lica were observed consuming veronicellid
slugs.

ti

}\

I't

simply eats slugs that are already dead, a new live slug (2-3
cm in length) was collected and offered to the same three
snails. It was quickly attacked by the three snails (Fig. 2). All
three A. fuUca climbed on top of the slug and proceeded to
consume the integument of the slug. It took over five min-
utes for the snails to kill the slug. During the first three
minutes, the slug crawled and pulled the snails with it as it
moved. In the last two minutes, the slug seemed distressed
and tried to curl up. After the slug stopped moving, the
snails continued to consume the slug for a few minutes. The
remainder of the slug (mostly the integument) was left in the
glass dish for one day after the attack to confirm that it was
actually dead.

In many low lying areas around Oahu, Achatina fiilica
and veronicellid slugs occur sympatrically. The fact that
these observations have been made at different locations on
the island suggests that predation by A. fnlica on veronicel-
lids may be common; yet an extensive literature search failed
to find any previous description of this behavior. Achatina
fnlica, native to east Africa, has been introduced to many
locations throughout the tropics and subtropics (Mead 1961,
Mead and Palcy 1992). Given its anthropogenic distribution,
it seems possible that this behavior might not be restricted to
the Hawaiian Islands and to predation on veronicellid slugs.

As such, the presence of Achatina fnlica may pose a
greater threat to terrestrial mollusc conservation than pre-
viously imagined. This may be especially true because A.
fnlica has become established in areas that harbor a signifi-

cant portion of the world’s molluscan biodiversity and A.
fnlica populations in these areas can attain extremely high
densities. For example, A. fnlica was introduced to Brazil in
1988 and has now been recorded in 23 out of 26 states in

Figure 2. Three Achatina fnlica consuming a slug that was offered (
after the initial observation in Hawaii Kai on September 18, 2005. (

PREDATORY BEHAVIOR OF ACHATINA FULICA

119

that country (Thiengo et al. 2007). A similar rapid spread
took place after its introduction to Hawaii in the 1930s
(Cowie et al. 1995). Both Brazil and Hawaii are known to
have a wealth of land snail diversity. However, in Brazil there
are many native slug species, although there are none in
Hawaii (Cowie et al. 1995, Lewinsohn and Prado 2005).
Kekauoha ( 1966) estimated that there were 537,600 A. fidica
in 6.72 hectares (7.75 snails per m") in Hawaii, demonstrat-
ing the high densities this snail can attain in its introduced
ranges. Although any statement on the impact of A. fulica
predation on species in its newly established areas would be
speculative, it seems possible that A. fulica could deleteri-
ously impact the land snail fauna through competition and
predation in areas where it has become established.

It is our hope that this note provides some impetus for
other researchers to explore the possible predation impacts
of introduced populations of Achatina fulica and to consider
the possibility that other introduced snails and slugs may be
having as yet unforeseen or unnoticed impacts. As it is dif-
ficult to make strong conclusions from only few observa-
tions, we hope that this report motivates further research.
Future experiments should address: ( 1 ) what proportion of
A. fulica have this predatory trait? (2) what proportion of
the A. fulica diet is obtained through predation? (3) how
diverse are the prey? and (4) over what geographical range
does A. fulica display predatory behavior?

ACKNOWLEDGMENTS

We thank Robert Cowie for insightful comments and
review of an early manuscript. We also thank Shyama Pagad
and Gary Barker for help tracking down references and Van-
essa Cooling for responding to our c]uestions. In adciition,
we thank Tim Pearce and an anonymous reviewer for their
excellent comments and review of this manuscript.

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74-77.

Pawson, P. A. and R. Chase. 1984. The life cycle and reproductive
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Peterson, G. D. 1957. Studies on the control of the giant African
snail on Guam. Hilgardia 26; 643-658.

Pimentel, D., R. Zuniga, and D. Morrison. 2005. Llpdate on the
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invasive species in the United States. Ecological Economics 52:
273-288.

Srivastava, P. D. 1992. Problems of Land Snail Pests in Agriculture:
A Study of the Giant A frican Snail. Concept, New Delhi, India.

Thiengo, S. C., F. A. Faraco, N. C. Salgado, R. H. Cowie, and M. A.
Fernandez. 2007. Rapid spread of an invasive snail in South
America: The giant African snail, Achatina fulica, in Brasil.
Biological Invasions 9: 693-702.

Torniyama, K. 1994. Courtship behavior of the giant African snail,
Achatina fulica (Ferussac) (Stylommatophora: Achatinidae) in
the field. Journal of Molluscan Studies 60: 47-54.

VitoLisek, P. M., C. M. D’Antonio, L. L. Loope, M. Rejmanek, and
R. Westbrooks. 1997. Introduced species: A significant com-
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ogy 21: 1-16.

Wallace, G. D. and L. Rosen. 1969. Studies on eosinophilic men-
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Pacific islands. Journal of Tropical Medicine and Hygiene 18;
206-216.

Submitted; December 2006; Accepted: 6 August 2007;

final corrections received: 20 December 2007

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Amer. Maine. Bull. 24: 121-125

RESEARCH NOTE

Life history and host fish identification for Fusconaia burkei and Pleurobema
strodeanum (Bivalvia: Unionidae)

Megan P. White^"'^, Holly N. Blalock-Herod^, and Paul M. Stewart^

' Department of Biological and Environmental Sciences, Troy University, Troy, Alabama 36082, U.S.A., pilamm5@wfu.edu
^ U.S. Fish and Wildlife Service, 300 Ala Moana Blvd., Honolulu, Hawaii 96850, U.S.A., holly_herod@fws.gov
^ Department of Biological and Environmental Sciences, Troy University, Troy, Alabama, 36082, U.S. A., mstewart@troy.edu

Abstract: We documented the period of gravidity, identified the fish host, and described the glochidia for two mussel species, Fusconaia
burkei (Wright, 1898) and Pleurobema strodeanum Walker, 1922, in Eightmile Creek, Walton County, Florida. Populations of both species
were checked monthly from December 2003 to October 2004 and were found to be gravid from the middle of March to late May. The size
and shape of F. burkei and P. strodeanum glochidia were similar. Conglutinates released by F. burkei were pink-colored and cylindrical in
shape, tapering sharply on both ends. Pleurobema strodeanum conglutinates were creamy or peach-colored and wider with a more flattened
appearance than those of F. burkei. Ten potential host fish species were exposed to either F. burkei or P. strodeanum glochidia. We identified
the blacktail shiner (Cyprinella venusta) as a host fish species for both F. burkei and P. strodeanum.

Key words: gravidity, glochidia, juveniles, freshwater mussels, Choctawhatchee River

North America possesses a rich freshwater mussel (Bi-
valvia: Unionidae) fauna, but this group is in rapid decline.
Within the Conecuh-Escambia, Yellow, and Choctawhatch-
ee River basins of Alabama and Florida, freshwater mussel
populations have experienced recent declines in species rich-
ness and relative abundance (Blalock-Herod et al. 2005, Pi-
larczyk et al. 2006), resulting in the recent elevation of eight
mussel species to candidate status under the Endangered
Species Act (U.S. Fish and Wildlife Service 2004). Life his-
tory information is crucial for assessing population viability
and evaluating conservation needs of this declining fauna
(U.S. Fish and Wildlife Service 2003). Flowever, host fish
information has been reported for only one quarter of the
mussel species in North America (Hoggarth 1992), and
other life history information is also lacking for most species.

The tapered pigtoe (Fusconaia burkei (Wright, 1898),
formerly referred to as Quincuncina burkei Wright, 1898) is
a small mussel endemic to the Choctawhatchee River basin
(Williams and Butler 1994). The fuzzy pigtoe (Pleurobetiia
strodeanum Walker, 1922) is native to the Conecuh-
Escambia, Yellow, and Choctawhatchee River drainages in
Alabama and Florida (Clench and Turner 1956). Both of
these species are candidates for protection under the Endan-
gered Species Act, and host fish and life history information

* Current Address: Department of Biology, Wake Forest Univer-
sity, Winston-Salem, North Carolina 27106, U.S. A.

for both species are unknown. The objectives of this study
were to assess the life history and host use of the tapered
pigtoe and the fuzzy pigtoe in order to provide data for
conservation and management decisions.

We studied both species in Eightmile Creek, Walton
County, Florida, -14.5 km east southeast of Florala, Ala-
bama (Fig. 1 ). Eightmile Creek is a tributary of the Pea River
(Choctawhatchee River drainage) and lies within the South-
eastern Plains level III ecoregion (Griffith et al. 2001). At the
study site, Eightmile Creek is moderately to highly sinuous,
with primarily sandy substrates, a wide riparian zone, and
stable, well-vegetated stream banks. Water samples from
Eightmile Creek were taken monthly for 10 months. Stream
water was tea-colored, as a result of natural tannins, with a
mean dissolved oxygen of 7.2 mg/L (SD - 1.6) and mean pH
of 7.0 (SD = 0.7). Mean turbidity was 1.9 NTLJ (SD = 0.9),
and water temperature ranged from 1 1 to 24°C throughout
the year. The creek supports a fish community of at least
twenty species as well as populations of at least eight fresh-
water mussel species, including Fusconaia burkei and Pleu-
robenta strodeanum (Pilarezyk et al. 2006).

Eightmile Creek was checked monthly from December
2003 to October 2004 for gravid mussels, except June 2004
because of high water levels. On each date, we tagged all
Fusconaia burkei or Pleurobema strodeanum using numbered
Floy® shellfish tags. Voucher specimens of both species were
collected, preserved in 70% ethanol, and deposited in the
Troy University collection.

121

122

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

N

100 0 100 200 300 Kilometers

Figure 1. Study site at Eightmile Creek, Walton County, Florida.

Mussels were checked for the presence of embryos by
carefully prying open the shell to inspect the gills for any
inflation or color change that would indicate a gravid female.
Some gravid females were transported to the laboratory to
release glochidia. These mussels were individually bagged in
plastic sandwich bags with stream water and transported in
coolers to reduce stress and abortion of glochidia. Mussels
were held at room temperature (about 22°C) in individual
beakers covered with 105-pm mesh screening in an aerated
aquarium containing filtered water from the study site. After
glochidia were expelled, the adult mussels were returned to
the collection site at Eightmile Creek.

We collected fishes for host trials using a backpack elec-
trofisher in streams within the same watershed as Eightmile
Creek where no Fnsconaia burkei or Pleiirobema strodeanum
were present to reduce the likelihood that the fish had ac-
quired immunity via a previous infection to the glochidia of
these two species (Arey 1923, Rogers and Dimock 2003). All
fishes used in host trials have been found in Eightmile Creek,
with the exception of the blacktip shiner {Lytlmmis ntrnpi-
culiis), a cyprinid that occurs within the native range of the
target mussel species (Mettee et a!. 1996). Fish were trans-
ported to the laboratory in an aerated cooler and held in
tanks for at least one to two weeks prior to conducting
experiments to allow acclimation to the laboratory, and to
slough off any residual glochidial infection.

Methods to infect fish with glochidia followed those
described by O’Brien and Williams (2002). Once congluti-
nates were released in the laboratoiy, they were gently agi-
tated to release the glochidia. Glochidial viability was
checked by exposing a subsample of glochidia to NaCl. Glo-

chidia were considered viable, and used in host fish trials, if ™
more than 50% of the subsample snapped shut in response |
to the NaCl. Fishes from four families and six species were 1
exposed to Fnsconaia burkei glochidia over the course of 10
trials. Over the course of 20 trials, fishes from six families
and 10 species were exposed to Pleiirobema strodeanum
glochidia.

After fishes were infected, each species was held indi-
vidually in covered aerated beakers in a water bath at about
22°C. Every three days, the water from the beakers was
sieved through a 105-pm mesh screen, and filtered material
was examined under a microscope for juvenile mussels, j
Transformed juveniles were identified by foot movement '
and the presence of two adductor muscles, while untrans-
formed glochidia were motionless and had only one adduc- i
tor muscle (Kama and Millemann 1978). Once juveniles
were found, the water was sieved daily. These daily checks
continued for 10 days after the last juvenile was found j
(O’Brien and Williams 2002). If no juveniles were found i
after five weeks, the trial was ended. Host fish were consid- ^
ered those that successfully allowed the transformation of '
glochidia into juvenile mussels. Fish that did not produce '
juveniles were preserved after trials and examined under a '
microscope for evidence of encysted glochidia. j

Several conglutinates from each species were preserved (
in 70% ethanol for description. We measured total length
and maximum width of conglutinates to the nearest 0.01 '

mm for both species, using an ocular micrometer. We also !j
measured valve length, height, and hinge length of glochidia
following Hoggarth (1999). Length was measured as the (j
maximum distance from anterior to posterior margins, and |j
measurements were made parallel to the hinge. Height was jj
measured as the maximum distance from dorsal to ventral
margins, and measurements were made perpendicular to

Table 1. Percent gravid mussels for Fnsconaia burkei and Pleu-
robeina strodeanum out of total number of mussels of each species
checked at Eightmile Creek, Florida (*, very high water level).

Fnsconaia Pleiirobema

Date

Water

temperature (°C)

burkei

%

(N)

strodeanum
% (N)

12/18/2003

not recorded

0.0

(8)

0.0

(21)

1/27/2004

13.4

0.0

(5)

0.0

(21)

2/10/2004*

11.0

—

(0)

0.0

(4)

3/16/2004

17.7

25.0

(4)

53.3

(30)

3/30/2004

17.3

66.7

(9)

22.0

(41)

4/21/2004

18.3

0.0

(3)

23.8

(42)

5/27/2004

20.8

13.6

(22)

26.8

(97)

7/26/2004

23.6

0.0

(4)

0.0

(19)

8/27/2004

24.1

0.0

(8)

0.0

(24)

10/19/2004

19.6

0.0

(6)

0.0

(22)

MUSSEL LIFE HISTORY AND HOST FISHES

123

Table 2. Fish species used, number of individuals used, trial duration (days), reason trial was

ended (I, ten days after the last juvenile was detected; T, trial terminated after no juveniles
were detected for five weeks; X, all fish died), and number of juveniles produced for each ot
the ten host fish trials performed for Fusconaia burkei and Pleurobema strodeanum.

Duration Reason

Fish families and species Individuals (days) trial ended Juveniles

Fusconaia burkei

Centrarchidae

Lepomis macrochirus

2

37

T

0

Lepomis macrochirus

3

10

X

0

Cyprinidae

Cyprinella venusta

4

19

X

0

Cyprinella venusta

3

32

1

35

Notropis texamis

3

35

T

0

Notropis texamis

3

8

X

0

Pteronotropis hypselopterus

I

12

X

0

Fundulidae

Fiindulus olivaceus

3

37

T

0

Fumiulus olivaceus

3

34

T

0

Ictaluridae

Noturus leptacanthus

I

8

X

0

Pleurobema strodeanum

Aphredoderidae

Aphredoderus sayanus

I

36

T

0

Centrarchidae

Lepomis macrochirus

3

7

X

0

Cyprinidae

Cyprinella venusta

3

6

X

0

Cyprinella venusta

I

27

1

84

Cyprinella venusta

I

13

X

17

Cyprinella venusta

I

16

X

71

Cyprinella venusta

I

29

I

56

Lythrurus atrapiculus

2

3

X

0

Notropis texamis

3

28

X

0

Notropis texamis

3

12

X

0

Notropis texamis

4

6

X

0

Notropis texamis

3

2

X

0

Pteronotropis hypselopterus

I

34

T

0

Fundulidae

Fundulus olivaceus

3

4

X

0

Fundulus olivaceus

3

12

X

0

Ictaluridae

Noturus leptacanthus

2

34

T

0

Noturus leptacanthus

1

36

T

0

Percidae

Etheostoma edwini

1

34

T

0

Percina nigrofasciata

2

10

X

0

Percina nigrofasciata

1

11

X

0

length. Hinge length was measured in a straight line from
the points at which the dorsal margins intersect the anterior
and posterior margins. Student’s f-tests were used to com-
pare valve length, height, and hinge length between the two

species. Other characteristics we exam-
ined were valve shape and hook pres-
ence or absence.

We tagged and examined a total of
32 Fusconaia burkei and 161 Pleii-
robeina strodeanum. Gravid specimens
of both species were found at Eight-
mile Creek between March 16 and
May 27, 2004, indicating these species
are short-term brooders, or tachytictic
(Table 1 ). The peak period of gravidity
was late March for F. burkei and mid-
March for P. strodeanum (Table 1)-
Mean water temperature during the
period in which female mussels were
gravid was 18.0°C (range: 15.8-
20.8°C). There was no evidence that
females of either species produced
multiple clutches in a year. Both spe-
cies often released conglutinates within
the first 24 hours following the gravid-
ity check, indicating that handling may
result in premature abortion of glo-
chidia. While many glochidia were vi-
able, unnecessary evaluations of gra-
vidity should be avoided to reduce the
likelihood of abortion, especially for
candidate, threatened, or endangered
species.

Both the inner and outer demi-
branchs of gravid Fusconaia burkei
were slightly inflated and typically had
a pinkish color. In the laboratory, nine
F. burkei released pink-colored conglu-
tinates from March 31 to April 12,
2004. Five of the nine individuals re-
leased conglutinates on only one occa-
sion. However, the other four F. burkei
had two to four conglutinate releases
over the course of two to eight days.

Only the outer demibranchs of the
gills of gravid Pleurobema strodeanum
were inflated and had a creamy-orange
color. Twenty-three P. strodeanum re-
leased cream-colored conglutinates in
the laboratory from March 19 to April
29, 2004. The number of conglutinate
releases per P. strodeanum individual
ranged from one to four, with about half of the individuals
releasing conglutinates only once. Eleven P. strodeanum had
two to four conglutinate releases over the course of three to
12 days.

124

AMERICAN MALACOLOGICAL BULLETIN 24 • 1/2 • 2008

Fiisconaia burkei released slender, pink-colored conglu-
tinates that were cylindrical in shape and tapered to only one
or two glochidia on both ends. Fusconaia burkei congluti-
nates measured about 1 x 6.5 mm. Conglutinates released by
Pleurobeina strodeaniim were creamy or peach-colored, had
a more flattened appearance, and were much wider than
those of F. burkei. Pleurobema strodeaniim conglutinates
were also tapered, though not as narrowly as those of F.
burkei, at both ends. Pleurobema strodeaniim conglutinates
were about 2x8 mm.

The size and shape of both species’ glochidia were simi-
lar and not easily distinguishable. The mean valve height for
Fusconaia burkei glochidia was 160.1 pm {SD - 6.8, n - 5),
mean valve length was 167.3 pm (SD = 4.3, n = 5), and mean
hinge length was 130.6 pm (SD = 5.8, n = 5). For Pleurobema
strodeaniim glochidia, mean valve height was 166.3 pm
(SD = 2.8, n = 5), mean valve length was 176.5 pm (SD - 2.8,
n = 5), and mean hinge length was 126.5 pm (SD - 2.3,
n - 5). While there was no statistically significant difference
in valve height (Student’s f-test, t = -1.85, P = 0.10) and
hinge length (t = 1.46, P = 0.18) between the two species, the
valve length of P. strodeaniim glochidia was significantly
greater than that ot F. burkei (t — -4.02, P = 0.004). The
glochidial hook was absent in both F. burkei and P. strodea-
num, and both species had a subelliptical valve shape.

Of the 10 host fish trials performed for Fusconaia burkei,
only one produced juveniles (Table 2). Twenty-one days
after exposure to glochidia, juveniles were found in the
sieved water of Cyprinella venusta. This trial, the second
attempt to infect C. venusta with F. burkei glochidia, resulted
in the transformation of 35 glochidia into juveniles. Some
juveniles were kept alive in the laboratory for at least six
weeks.

Pleurobema strodeaniim glochidia transformed into ju-
veniles in four of the 20 trials (Table 2), but only on Cy-
prinella venusta individuals. A mean of 14 days from date of
infection elapsed until the first transformed P. strodeaniim
juvenile was collected. The first successful trial produced 84
juveniles: 50 collected on day 15, 32 on day 16, and two on
day 17. The second successful trial produced 17 juveniles on
day 13, but the fish subsequently died, and no additional
juveniles were collected. The third trial produced 71 juve-
niles: 46 collected on day 14 and 25 on day 15. The fish died
on day 15, and no additional juveniles were collected. The
fourth successful trial yielded 56 juveniles: six collected on
day 14, 46 on day 16, and four were collected on day 18.
Some juveniles from these trials were kept alive in the labo-
ratory for over nine weeks. When fishes that did not produce
juveniles were preserved and later examined, no encysted
glochidia were observed on the gills or fins.

The period of gravidity documented in this study for
both species is supported by existing literature. Ortmann

and Walker (1922) reported a gravid female Fusconaia burkei \
at the Choctawhatchee River, Alabama, in May. Gravid Pleii- i
robema strodeaniim have been found at the Choctawhatchee
River, Florida, during April and in the Escambia River drain-
age in July (H. N. Blalock- Herod, unpubl. data). Gravid
females of a closely related species, Pleurobema pyriforme
(Lea, 1857), were reported from March to July at Chick- !
asawhatchee and Kinchafoonee Creeks in the Apalachicola,
Chattahoochee and Flint Rivers Drainage (O’Brien and Wil-
liams 2002).

The successful transformation of Fusconaia burkei and |
Pleurobema strodeaniim glochidia on Cyprinella venusta is |
also consistent with the existing literature on related taxa. |
Fusconaia typically use Cyprinidae as fish hosts. Haag and !l
Warren (2003) reported that schools of C. venusta repeatedly j
interacted with drifting conglutinates from the related taxa 'i
Fusconaia cerina (Conrad, 1838) and Pleurobema decisiim
(Lea, 1831). Host fish trials resulted in Fusconaia cerina glo- ■
chidia transforming on a wide variety of cyprinid species,
including C. venusta; P. decisiim glochidia also transformed :
consistently on C. venusta (Haag and Warren 2003). Addi- •
tional studies confirmed that cyprinids often serve as hosts 1
tor Pleurobema (Haag and Warren 1997, O’Brien and Wil- i
liams 2002, Layzer et al. 2003). The data collected in the i
current study will be useful in determining and implement-
ing conservation actions for the protection of these rare
mussel species.

ACKNOWLEDGMENTS

We thank Will Heath, Jonathan Miller, Kristy Pisani,
Christa Collins, Kelly Huizenga, and Jeffrey Herod for their
field assistance. Thank you to Bonnie Hamiter, Michael
Walters, Kelly Huizenga, Jeffrey Herod, and Vanessa Heath
for their help in the laboratory. This project was made pos-
sible by the financial support provided by the ALFA Fellow-
ship at Troy University and the Alabama Water Environ-
ment Association Graduate Student Scholarship. Jn-kind
support was provided by the USFWS, Fisheries Resources
Office, Panama City, Florida.

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MUSSEL LIFE HISTORY AND HOST FISHES

125

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1-30.

Hoggarth, M. A. 1999. Descriptions of some of the glochidia of the
Unionidae (Mollusca: Bivalvia). Malacologia 41: 1-118.

Kama, D. W. and R. E. Millemann. 1978. Glochidiosis of salmonid
fishes: Comparative susceptibility to natural infection with
Margaritifera margaritifera (L.) (Pelecypoda: Margaritanidae)
and associated histopathology. Journal of Freshwater Ecology 7:
35-44.

Layzer, J. B., B. Adiar, S. Saha, and L. M. Woods. 2003. Glochidial
hosts and other aspects of the life history of the Cumberland
pigtoe (Pleurobema gibberum). Southeastern Naturalist 2: 73-
84.

Mettee, M. F., P. E. O’Neil, and J. M. Pierson. 1996. Fishes of
Alabama and the Mobile Basin. Oxmoor House, Inc., Birming-
ham, Alabama.

O’Brien, C. A. and I. D. Williams. 2002. Reproductive biology of
four freshwater mussels (Bivalvia: Unionidae) endemic to
eastern Gulf Coastal Plain drainages of Alabama, Elorida, and
Georgia. American Malacological Bulletin 17: 147-158.

Ortmann, A. E. and B. Walker. 1922. A new genus and species of
American naiades. The Nautilus 36: 1-6.

Pilarczyk, M. M., P. M. Stewart, D. N. Shelton, H. N. Blalock-
Herod, and |. D. Williams. 2006. Current and recent historical
freshwater mussel assemblages in the Gulf coastal plains.
Southeastern Naturalist 5: 205-226.

Rogers, C. L. and R. V. Dimock, Jr. 2003. Acquired resistance of
bluegill sunfish Lepomis macrochirus to glochidia larvae of the
freshwater mussel Utterbackia imbecillis (Bivalvia: Unionidae)
after multiple infections. Journal of Parasitology 89: 51-56.

U.S. Fish and Wildlife Service. 2003. Recovery plan for endangered
Fat Threeridge (Amblema neislerii), Shinyrayed Pocketbook
(LampsiJis subangulata), Gulf Moccasinshell (Medionidus
penicillatus), Ochlockonee Moccasinshell (Medionidus simpso-
nianus), and Oval Pigtoe (Pleurobema pyriforme); and threat-
ened Chipola Slabshell (Elliptio chipolaensis), and Purple
Bankclimber (Elliptoideus sloatianus). Atlanta, Georgia.

U.S. Fish and Wildlife Service. 2004. Review of species that are
candidates or proposed for listing as endangered or threat-
ened. Federal Register 69: 24875-24904.

Williams, J. D. and R. S. Butler. 1994. Class Bivalvia. In: M. Deyrup
and R. Franz, eds.. Rare and Endangered Biota of Elorida, Vol.
IV, Invertebrates. University Press of Florida, Gainesville,
Florida. Pp. 53-128.

Submitted: October 2006; accepted: 13 November 2007;

final corrections received: 1 1 December 2007

INDEX TO VOLUME 24(1/2)129

AUTHOR INDEX

Abidli, S. 24: 79
Adamo, S. A. 24: 25
Arrington, T. D. 24: 91
Basil, J. A. 24: 3
Blalock-Herod, H. N. 24: 121
Dasch, G. A. 24: 59
Dillon, R. T., Jr. 24: 59
Estevez, E. D. 24: 101
Elannery, M. S. 24: 101
Grasso, E. W. 24: 13

Hayes, K. A. 24: 117
King, A. J. 24: 25
Lahbib, Y. 24: 79
Leite, T. S. 24: 31
Mather, J. A. 24: 1, 31, 51
Meyer, A. L. 24: 117
Meyer, W. M. Ill 24: 117
Montagna, P.A. 24: 101
Palmer, T.A. 24: 101
Reeves, W. K. 24: 59

Richards, D. C. 24: 91
Rypel, A. L. 24: 97
Schwabe, E. 24: 71
Sinn, D. L. 24: 65
Soucier, C. P. 24: 3
Stewart, P. M. 24: 121
Trigui El Menif, N. 24: 79
Voight, J. R. 24: 43
White, M. P. 24: 121

PRIMARY MOLLUSCAN TAXA INDEX

[first occurrence in each paper recorded, new taxa in bold]

Ahdopus 24: 1
acuta, Physa 24: 61
aequalis, Ahra 24: 114
alba, Anodontia 24: 114
alhus, Cliiton 24: 72
Ancylidae 24: 61
Anomiidae 24: 114
antipodanun, Potamopyrgus 24: 92
Arcidae 24: 114
Arcoida 24: 114
atlantica, Placiphorella 24: 74
atratum, Cerithium 24: 113
auberiana, Anomalocardia 24: 114
Basommatophora 24: 113
belgicae, Leloupin 24: 71
Benthoctopiis 24: 43
bimaculatiis, Octopus 24: 52, 65
bimacidoides, Octopus 24: 15, 65
Biomphalaria 24: 59
Bivalvia 24: 97, 113, 121
boreopacifica, Graneledone 24: 44
bouveti, Callochiton 24: 71
brandaris, Bolinus 24: 79
Bullidae 24: 113
burkei, Fusconaia 24: 121
burkei, Quiiicunciua 24: 121
Callochiton 24: 71
Calyptraeidae 24: 113
canipechiensis, Mercenaria 24: 114

cancellata, Chione 24: 107

canthylus, Benthoctopiis 24: 44

Cardiidae 24: 113

caroliniana, Polymesoda 24: 101

Cephalaspidea 24: 113

Cephalopoda 24: 3

cerina, Fusconaia 24: 124

Cerithiidae 24: 113

Chitonida 24: 71

Chondropleiim 24: 72

cirrosa, Eledone 24: 4

Coleoidea 24: 3

Conidae 24: 113

constricta, Maconia 24: 114

Conus 24: 113

Corbiculidae 24: 107

corona, Melongena 24: 113

ciibensis, Physa 24: 61

ciibensis, Veronicella 24: 117

cuneata, Rangia 24: 101

cyanea. Octopus 24: 36, 65

Cyrtopleiira 24: 113

decisum, Pleiirobema 24: 124

defiUppi, Octopus 24: 32

dofleini, Enteroctopus 24: 1, 25, 53, 65

dorsuosiis, Stenosenms 24: 74

Dreissenidae 24: 114

diiplicatiis, Polinices 24: 113

diiryi, Heliosonia 24: 61

Ellobium 24: 113
Enteroctopus 24: 1
Epitoniidae 24: 113
erinacea, Ocenebra 24: 86
Eiipletira 24: 113
exaratus, Stenosemiis 24: 71
fhiniinea, Corbicula 24: 101
Fluniinicola 24: 92
fornicata, Crepidiila 24: 113
frons, Dendostrea 24: 114
fulica, Achatina 24: 117
Fusconaia 24: 124
Gastropoda 24: 59, 113
gaiissae, Callochiton 24: 71
glabrata, Biomphalaria 24: 59
Craneledone 24: 43
granifera, Thiara 24: 59
granosissima, Ceukensia 24: 106
Cundlachia 24: 59
haemastoma, Stramonita 24: 1 1 1
Haminoeidae 24: 113
Elapalochlaena 24: 1
Elelisoma 24: 59
hiitnmelincki, Octopus 24: 32
Hydrobiidae 24: 91
hydrothermalis, Vulcanoctopus 24:
44

idahoensis, Pyrgulopsis 24: 92
insularis, Octopus 24: 31

126

irradians, Argopecten 24: 114
irrorata, Littoraria 24; 101
Ischnochitonidae 24: 71
joubini, Octopus 24: 65
kergiielensis, Leptochitou 24: 71
kiihniana, Biomphalaria 24: 59
Lampsilis 24: 97
Lanx 24: 92
lapillus, Nucella 24; 86
Lasaeidae 24: 1 14
latemlis, Midinia 24: 101
leucophaeata, Mytilopsis 24: 114
Littorinidae 24: 113
Loligo 24: 1
Lucinidae 24: 114
macropus, Callistoctopus 24: 32
macropus, Octopus 24: 65
Mactridae 24: 114
maculosa, Haplochlaena 24: 36
marmorata, Physa 24; 59
Melampus 24: 113
melanura, Triphora 24: 113
Melongenidae 24; 113
minor, Ensis 24: 1 14
mirandus, Nuttallochiton 24; 71
modiolus, Brachidontes 24: 114
mortoni, Laevicardium 24: 113
Muricidae 24: 79, 113
Myoida 24: 113
Mytilidae 24: 106
Mytiloida 24: 114
Nassariidae 24: 113
Natiddae 24: 113
natricina, Physa 24: 92
Nautilus 24: 1, 4
Neogastropoda 24: 113
Neoloricata 24: 71
Neotaenioglossa 24; 113
Neritidae 24: 113
Neritopsina 24: 113
nimbosa, Macrocallista 24: 114

Noetiidae 24: 1 14
Octopodidae 24: 32, 43
Octopus 24: 1, 13, 68
ojftcinalis, Sepia 24: 9, 25, 31, 51
opalescens, Loligo 24: 36
Ostreidae 24: 114
Ostreoida 24: 114
papyrium, Amygdalum 24: 106
partumeium, Musculium 24: 114
pealeii, Loligo 24: 15, 25
pectinata, Lucina 24: 114
Pectinidae 24: 114
Pharidae 24: 114
Pholadidae 24: 113
Physa 24: 59
Pinnidae 24: 114
Pisidiidae 24: 1 14
Pisidium 24: 1 14
Placiphorella 24: 74
Planorbidae 24: 113
plaindata, Mysella 24: 114
plebeius, Tagelus 24: 101
Pleurobema 24: 124
Polyplacophora 24: 71
pompilius. Nautilus 24: 3
ponderosa, Noetia 24: 114
Pterioida 24: 114
Pulmonata 24; 113
piinctulata, Neritina 24: 59
pyriforme, Pleurobema 24: 124
radiata, Gundlachia 24: 59
recurvum, Ischadiiim 24: 101
reticulata, Plitiia 24: 79
robusta, Pyrgulopsis 24: 92
rubescetis. Octopus 24: 37, 52
rupicola, Epitonium 24: 113
scutulata, Littoriua 24: 66
Semelidae 24; 114
Sepia 24: 1

serpenticola, Taylorconcha 24: 91
serrata, Artina 24: 114

setulosum, Heniiathrum 24: 71
simplex, Anotuia 24: 114
simplicissimus, Ischnochitou 24: 72
simplicissimus, Stenosemiis 24; 71
Solecurtidae 24: 114
Solenidae 24: 114
solidissima, Spisula 24: 114
Stenosemiis 24: 72
stramiuea, Biomphalaria 24: 59
striata. Bulla 24: 1 1 3
strodeanum, Pleurobema 24: 121
siiccinea, LLaminoea 24: 113
tampaensis, Urosalpinx 24: 113
tasmanica, Euprymna 24: 52
Taylorconcha 24: 91
Telliua 24: 114
Tellinidae 24: 106
tenta, Macoma 24: 114
tenuis, Cyclinella 24: 107
teres, Lampsilis 24: 97
transversa, Anadara 24: 114
Triphoridae 24: 113
trivolvis, Elelisoma 24: 59
trunculus, Hexaplex 24: 79
tuberculata, Melanoides 24: 59
undatum, Buccimim 24: 87
Unionidae 24: 121
usnea, Neritina 24: 101
utahensis, Valvata 24: 92
variabilis, Provanna 24: 45
Veneridae 24: 106
Veneroida 24: 113
versicolor, Telliua 24: 108
vibex, Nassarius 24: 113
virgiuica, Crassostrea 24: 101
viridis, Solen 24: 114
Vulcanoctopus 24: 43
vulgaris. Octopus 24: 1, 21, 28, 31, 53,
65

zschaui, Tonicina 24: 71

LIST OF REVIEWERS FOR 2006 AND 2007

Adamo, Shelley
Barnhart, Chris
Baur, Bruno

Beese, Kathleen
Bieler, Rudiger
Boal, Jean

127

Brooker, Lesley
Byrne, Maria
Camphell, David

Chase, Ronald
Clark, Roger

Cleveland, Carol (several articles)
Collin, Rachel

Cowie, Robert H. (several articles)
Craze, Paul
Dejong, Randall
Dillon, Jr., Robert T.

Dunn, Heidi

Eckelbarger, Kevin

Eernisse, Doug (several articles)

Eoltz, David

Eried, Bernard

Ghiselin, Michael T.

Goddard, Jeff
Gomez-MoJiner, Benjamin
Haag, Wendell R.

Hamburg, Steven

Harasewych, Jerry (severaJ articles)
Herke, Scott
Hershler, Robert

Hodgson, Alan N. (several articles)
Huffard, Cris
Hughes, Jane
Jyengar, Erika V.

Johnson, PauJ
Jones, Robert
Kenchington, EJJen
Kesler, David H. (severaJ
articles)

Koene, Joris
Komaru, Akira

Leonard, Janet (Guest editor)

Lysne, Steven

Mather, Jennifer (Guest editor)

McMahon, Robert
Minton, Russ
Norman, Mark
O Eoighil, Diarmaid
Okusu, Akiko
Pearce, Timothy
Pearse, John
Perez, Kathryn
Pernet, Bruno
Petit, R. E.

Pojeta, John
Price, Rebecca
Rakocinski, Chet
Reise, Heike
Rilov, Gil L.

Rosenberg, Gary
Sada, Don
Saito, Hiroshi
Schander, Cristoffer
Scheltema, Amelie
Schloesser, Don W.

Schwabe, Enrico (several
articles)

Shaw, Jeremy
Shaw, Paul

Shea, Elizabeth (several articles)
Shively, Steven

Sirenko, Boris (several articles)
Stewart, Timothy
Stickle, William B.

Strack, Hermann
Strong, Ellen
Todd, Chris
Trowbridge, Cynthia D.

Valdes, Angel
Van Der Heyden, Sophie
Vaughn, Caryn
Vendrasco, Michael
Watters, Thomas
Yusa, Yoichi

128

THE AMERICAN MALACOLOGICAL SOCIETY

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The American Malacological Society

74th Annual Meeting • 29 June - 3 July 2008
Southern Illinois University, Carbondale, IL

74th Annual Meeting

American Malacological Society

Carbondale, Illinois
June 29 - July 3, 2008

The American Malacological Society will hold its lA'^ annual meeting in Carbondale, Illinois
from June 29 - July 3, 2008. The venue will be the Southern Illinois University Student Center,
which houses an auditorium, several ballrooms and meeting rooms, and a number of restaurants
and coffee shops. The conference will begin with an icebreaker on Sunday evening. Special
events will include an outdoor reception at Blue Sky Vineyard (www.blueskyvineyard.com) on
Monday night, a poster session and the AMS Auction of molluscan miscellany on Tuesday night,
and a barbecue banquet (with vegetarian options) at the 17'^ Street Bar & Grill Warehouse,
Southern Illinois' most unique banquet facility on Wednesday night. 17‘*^ Street Bar & Grill was
recently recognized by Food Network and Bon Appetit as the home of the best ribs in the
country.

The special sessions and symposia will include:

• a land snail conservation symposium and workshop in honor of the late Leslie Hubricht,
organized by Kathryn Perez (University of North Carolina - Chapel Hill/Duke
University), Jay Cordeiro (NatureServe), Jochen Gerber (Field Museum of Natural
History) and Kevin Roe (Iowa State University)

• a symposium on molluscan taxonomy in the 2U‘ century, organized by Benoit Dayrat
(UC Merced)

• a special session on cephalopod biology organized by Frank Anderson, Christine Huffard
(Monterey Bay Aquarium Research Institute), and Elizabeth Shea (Delaware Museum of
Natural History)

The American Malacological Society

On Thursday, two field trips will introduce meeting participants to wonderful mollusk habitats in
southern Illinois. Participants will be able to take a tour of the Larue Pine Hills/Otter Pond
Research Natural Area, a fantastic area of limestone bluffs and outcrops (and home of
Eiichemotrema hnbrichti, the conference mascot), or a trip to local aquatic habitats to search for
freshwater bivalves and gastropods.

Visitors to Carbondale usually travel through either St. Louis or Chicago, though Memphis is
also an option. There is a convenient shuttle service from St. Louis Lambert International Airport
to Carbondale. From Chicago or Memphis, you can take a train — The City of New Orleans,
which stops in Carbondale on its Chicago-to-New Orleans route. Flights also arrive at the
Williamson County airport several times daily. The airport is located 16 miles east of the SIU
campus.

For meeting registration and abstract submission infonnation, please visit
http://malacological.org/meetings/next.html. We look forward to seeing you in Carbondale,
Illinois in 2008!

For more infonnation, please contact:

Frank E. Anderson
Department of Zoology
Southern Illinois University
Carbondale, IL 62901

Phone: 618-453-4136
E-mail: feander@siu.edu

The American Malacological Society

74th Annual Meeting • 29 June - 3 July 2008
Southern Illinois University, Carbondale, IL

Symposium on the Current State of Land Snail Conservation
& Land Snail Identification Workshop

The American Malacological Society is pleased to announce the Leslie Hubricht
Symposium and Workshop on the taxonomy, distribution and conservation of
terrestrial Gastropods . The land snail symposium & workshop are aimed at
AMS members, state and federal agency employees, and others who are
seeking training in land snail collecting, identification, and ecology. Two of
the major goals of the symposium and associated workshop are ( 1) to provide an
opportunity for networking among established land snail researchers as well
individuals who lack taxonomic experience but are responsible for day-to-day land
snail conservation and (2) to offer an opportunity for non-land snail experts to
receive training in basic aspects of land snail biology and identification. Workshop
attendees are invited to bring their own shells to identify!

Workshop topics covered include:

Introduction to land snail collecting strategies
Introduction to ID tenwinology and literature

The major families of macro and micro land snails of North America
Strategies for the conservation of invertebrates with emphasis on terrestrial
snails/slugs

Introduction to the identification of invasive snails and slugs

For more specific information on workshop registration, symposium topics, and
accommodations in and around Carbondale as it develops, please visit the meeting
website http://www.malacological.org/meetings/next.htiTil.

I

1

I-

-■rpTTsmrrn r ipii r Piai

li

!|

Patterns of activity cycles in juvenile California two-spot octopuses (Octopus bhnaculoides) .

DAVID L. SINN 65

Discovery of the South African polyplacophoran Stenosemus simplicissimus (Thiele, 1906)

(Mollusca, Polyplacophora, Ischnochitonidae) in the Southern Ocean.

ENRICO SCHWABE 71

Imposex level and penis malformation in Hexaplex tmncidus from the Tunisian coast.

YOUSSEF LAHBIB, SAMI ABIDLI, and NAJOUA TRIGUI EL MENIF 79

Threatened Bliss Rapids snail’s susceptibility to desiccation; Potential impact from hydroelectric

facilities. DAVID C. RICHARDS and TRISTAN D. ARRINGTON 91

Field obseiwations of the nocturnal mantle-tlap lure of Lariipsdis teres. ANDREW LEE RYPEL 97

Meta-analysis of the relationship between salinity and molluscs in tidal river estuaries of
southwest Florida, U.S.A. PAUL A. MONTAGNA, ERNEST D. ESTEVEZ,

TERRY A. PALMER, and MICHAEL S. FLANNERY ' 101

Research Note: Giant African snail, Achatina fidica, as a snail predator.

WALLACE M. MEYER III, KENNETH A. HAYES, and AMANDA L. MEYER 117

Research Note: Life history and host fish identification for Fnsconaia burkei and Pleurobetna
strodeanum (Bivalvia: Unionidae). MEGAN P. WHITE, HOLLY N. BLALOCK-HEROD,
and PAUL M. STEWART 121

Index to Vol. 24 126

Membership Form 129

Information for Contributors 131

Meeting Announcement 133

SMITHSONIAN INSTITUTION LIBRARIES

HOI
.A SI 3

rv\ol-i—

AMERICAN
MALACOLOGICA
BULLETIN

Journal of the American Malacological Society

http://www.maIacoIogical.org

VOLUME 25 28 July 2008 number 1/2

Status of freshwater native mussels (Unionidae) in the Oklahoma section of the Verdigris River
after introduction of the zebra mussel [Dreissena polymorpha Pallas, 1771).

after introduction of the zebra mussel [Dreissena polymorpha Pallas, 1771).

CHAD J. BOECKMAN and JOSEPH R. BIDWELL 1

Spatial distribution of soft-bottom molluscs in the Ensenada de San Simon (NW Spain).

EVA CACABELOS, PATRICIA QUINTAS, and JESUS S. TRONCOSO 9

Introduction to the symposium “Advances in Chiton Research”. DOUGLAS J. EERNISSE 21

New information about Echhwchitoii dufoei, the Ordovician spiny chiton.

JOHN POJETA, Jr. and JIMMIE DUFOE 25

Methods of sample preparation of radula epithelial tissue in chitons (Mollusca: Polyplacophora).

JEREMY A. SHAW, DAVID J. MACEY, PETA L. CLODE, LESLEY R. BROOKER,

RICHARD I. WEBB, EDWARD J. STOCKDALE, and RACHEL M. BINKS 35

Gross anatomy and positional homology of gills, gonopores, and nephridiopores in “basal”

living chitons (Polyplacophora: Lepidopleurina). JULIA D. SIGWART 43

Aesthete canal morphology in the Mopaliidae (Polyplacophora). MICHAEL J. VENDRASCO,

CHRISTINE Z. FERNANDEZ, DOUGLAS J. EERNISSE, and BRUCE RUNNEGAR 51

continued on back cover

Cover photo: Fertilization in the chiton Stenosemus alhiis (Linnaeus, 1767) by Buckland-Nicks

AMERICAN MALACOLOGICAL BULLETIN

BOARD OF EDITORS

Kenneth M. Brown, Editor-in-Chief
Department of Biological Sciences
Louisiana State University
Baton Rouge, Louisiana 70803

USA

Cynthia D. Trowbridge, Managing Editor

Oregon State University

P.O. Box 1995

Newport, Oregon 97365

USA

lanice Voltzow
Department of Biology
LIniversity of Scranton
Scranton, Pennsylvania 18510-4625
USA

Robert H. Cowie

Center for Conservation Research and Training

University of Hawaii

3050 Maile Way, Gilmore 408

Honolulu, Hawaii 96822-2231

USA

Carole S. Hickman

University of California Berkeley

Department of Integrative Biology

3060 VLSB #3140

Berkeley, California 94720

USA

Timothy A. Pearce

Carnegie Museum of Natural History
4400 Forbes Avenue
Pittsburgh, Pennsylvania 15213-4007
USA

Paula M. Mikkelsen
Paleontological Research Institution
1259 Trumansburg Road
Ithaca, New York 14850-1313
USA

Alan I. Kohn
Department of Zoology
Box 351800

University of Washington
Seattle, Washington 98195
USA

Dianna Padilla

Department of Ecology and Evolution
State University of New York
Stony Brook, New York 11749-5245
USA

Roland C. Anderson
The Seattle Aquarium
1483 Alaskan Way
Seattle, Washington 98101
USA

The American Malacological Bulletin is the scientific journal of the American Malacological Society, an international society of professional,
student, and amateur rnalacologists. Complete information about the Society and its publications can be found on the Society’s website:
http://www. malacological.org

AMERICAN MALACOLOGICAL SOCIETY MEMBERSHIP

MEMBERSHIP INFORMATION: Individuals are invited to com-
plete the membership application available at the end of this issue.

SUBSCRIPTION INFORMATION: Institutional subscriptions are
available at a cost of $75 plus postage for addresses outside the
USA.

Further information on dues, postage fees (for members outside
the USA), and payment options can be found on the membership
application at the end of this issue.

ALL MEMBERSHIP APPLICATIONS, SUBSCRIPTION ORDERS,
AND PAYMENTS should be sent to the Society Treasurer:

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CHANGE OF ADDRESS INFORMATION should be sent to the
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appears at the end of this issue.

MANUSCRIPT SUBMISSION, CLAIMS, AND PERMISSIONS TO

REPRINT lOURNAL MATERIAL should be sent to the Editor-in-Chief:

Kenneth M. Brown, Editor-in-Chief

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Baton Rouge, Louisiana 70803

USA

Voice: 225-578-1740 • Eax: 225-578-2597
E-mail: kmbrown@lsu.edu

AMERICAN MALACOLOGICAL BULLETIN 25(1/2)
AMER. MALAC. BULL.

ISSN 0740-2783

Copyright © 2008 by the American Malacological Society

AMERICAN MALACOLOGICAL BULLETIN

CONTENTS VOLUME 25 I NUMBER I/2

Status of freshwater native mussels (Unionidae) in the Oklahoma section of the Verdigris River
after introduction of the zebra mussel (Dreissena polymorphn Pallas, 1771).

CHAD J. BOECKMAN and JOSEPH R. BIDWELL 1

Spatial distribution of soft-bottom molluscs in the Ensenada de San Simon (NW Spain).

EVA CACABELOS, PATRICIA QUINTAS, and JESUS S. TRONCOSO 9

Introduction to the symposium “Advances in Chiton Research”. DOUGLAS J. EERNISSE 21

New information about Echinochiton diifoei, the Ordovician spiny chiton.

JOHN POJETA, Jr. and JIMMIE DUFOE 25

Methods of sample preparation of radula epithelial tissue in chitons (Mollusca: Polyplacophora).

JEREMY A. SHAW, DAVID J. MACEY, PETA L. CLODE, LESLEY R. BROOKER,

RICHARD I. WEBB, EDWARD J. STOCKDALE, and RACHEL M. BINKS 35

Gross anatomy and positional homology of gills, gonopores, and nephridiopores in “basal”

living chitons (Polyplacophora: Lepidopleurina). JULIA D. SIGWART 43

Aesthete canal morphology in the Mopaliidae (Polyplacophora). MICHAEL J. VENDRASCO,

CHRISTINE Z. FERNANDEZ, DOUGLAS J. EERNISSE, and BRUCE RUNNEGAR 51

Mopalia kennerleyi Carpenter, 1864, a forgotten species and its southern analogue Mopalia

ciliata (Sowerby, 1840). ROGER N. CLARK 71

Two new chitons of the genus Tripoplax Berry, 1919 from the Monterey Sea Canyon.

ROGER N. CLARK 77

The effect of sampling bias on the fossil record of chitons (Mollusca, Polypilacophora).

STEPHANEY S. PUCHALSKI, DOUGLAS J. EERNISSE, and CLAUDIA C. JOHNSON 87

Fertilization biology and the evolution of chitons. JOHN BUCKLAND-NICKS 97

Chitons (Mollusca: Polyplacophora) associated with hydrothermal vents and methane seeps
around Japan, with descriptions of three new species. HIROSHI SAITO,

KATSUNORI FUJIKURA, and SHINJI TSUCHIDA 113

Index to Vol. 25 125

Membership Form for 2008 128

Information for Contributors 130

1

Amer. Make. Bull 25: 1-8 (2008)

Status of freshwater native mussels (Unionidae) in the Oklahoma section of the
Verdigris River after introduction of the zebra mussel {Dreissena polymorpha
Pallas, 1771)

Chad J. Boeckman and Joseph R. Bidwell

Oklahoma State University, Ecotoxicology and Water Quality Research Laboratory, 430 Life Sciences West, Stillwater, Oklahoma 74078,
U.S.A., chadjb@okstate.edu

Abstract: The Verdigris River in Kansas and Oklahoma, USA once held a diverse native unionid mussel fauna although a number of these
populations have declined in richness and abundance. There is recent evidence that populations of some species of unionid mussels are
increasing in parts of the Verdigris River in Kansas, but no current data exist for Oklahoma. In addition, zebra mussels {Dreissena
polymorpha Pallas, 1771) have been introduced into a major impoundment on the Verdigris River, Oologah Lake, and may further threaten
mussel populations downstream from this reservoir. This study updates the distribution and abundance of native mussels in the Oklahoma
portion of the Verdigris River upstream and downstream of Oologah Lake, and documents the current distribution of zebra mussels in this
region. Thirty-one sites were sampled and a significant increase in species richness and abundance of native mussels was observed as
compared to a 1997 study. Two species of special interest, Cyprogenia aberti (Conrad, 1850) and Quadritla cylindrica (Say, 1817), were
found. Zebra mussels were not found at sites upstream of Oologah Lake but were present at every downstream site. In September 2006,
zebra mussel byssal threads were observed on shells of a number of native mussels downstream from Oologah Lake, but unionid richness
and abundance were not significantly different between sites above and below the reservoir.

Key words: invasive species, Quadnda, field survey, impoundment, semi-quantitative sampling

Freshwater mussels are considered one of the most im-
periled groups of organisms in the world, with 70% of taxa
within the family Unionidae considered of special concern
or endangered (Bogan 1993, Strayer et al. 2004, Warren and
Haag 2005, Jones et al. 2006). As for many other organisms
experiencing population declines, loss of native mussels has
been attributed to habitat alteration (Vaughn and Taylor
1999, Garner and McGregor 2001), point and non-point
source pollution (Richter et al. 1997), and invasive species
(Burlakova et al. 2000, Strayer et al. 2004).

The Verdigris River originates in the Flint Hills of
southeastern Kansas (Schuster 1979) and flows south into
Oklahoma where it joins the Arkansas River near Muskogee,
Oklahoma. Historically, the Oklahoma portion of the Ver-
digris contained a diverse assemblage of native unionid mus-
sels, with Isely (1924) describing this region as among the
richest mussel faunas in the state. Since that time, significant
portions of the river have been altered by impoundments
and the lowest reaches have been dredged to create a navi-
gation channel to the Arkansas River (USAGE 2007). Agri-
cultural activity and urban and industrial development have
led to pollutant and sediment input, degrading water quality.
Surveys conducted in both Kansas and Oklahoma in the
1990s indicated an overall decline in Verdigris River mussel
populations compared to earlier studies (Obermeyer et al.
1997a, Vaughn 1998).

Interestingly, more recent surveys in Kansas have re-

ported increases in the densities of some unionid taxa. For
example. Miller and Lynott (2006) report increases in the
abundance of 10 mussel species in the Kansas portion of the
Verdigris between 1991 and 2003, a change they attributed
to improved habitat quality. The last mussel survey con-
ducted in the Oklahoma portion of the Verdigris was by
Vaughn (1998). In addition, zebra mussels (Dreissena poly-
morpha Pallas, 1771) were discovered in Oologah Lake, an
impoundment of the Verdigris in Oklahoma, in the spring of
2003 (Taney 2005). While the distribution of D. polymorpha
in the Verdigris River was largely unknown, the potential
impact of these invasives on unionid mussels is well estab-
lished (Ricciardi et al. 1996, Baker and Hornbach 1997,
Schloesser et al. 2006). The objectives of this study were to
survey the mussel communities in the Oklahoma portion of
the Verdigris River to determine if increases similar to those
in Kansas were occurring, to characterize the extent to which
zebra mussels occur along this section of the Verdigris, and
to gather preliminary data regarding their potential interac-
tion with native mussels.

MATERIALS AND METHODS

Sampling locations in the Verdigris River were selected
to correspond with sites previously sampled by Vaughn
( 1998). Sample sites were mostly riffle areas and mussel sur-

2

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

veys were conducted in runs immediately downstream of
each riffle. Substrate ranged from loose gravel to cobble.
Physical-chemical characteristics (temperature, dissolved
oxygen, pH, and conductivity) were recorded using a Hy-
drolab Quanta multi-parameter probe (Hydrolab Corpora-
tion, Austin, Texas). Additionally, river water was collected
in acid-washed polyethylene bottles and transported back to
the laboratory on ice for determination of titratable alkalin-
ity and hardness (APHA 1998).

Mussel surveys involved timed snorkel searches by two
individuals. Surveys were conducted for at least 15 min (2
people X 15 min = 30 min total search time), with longer
time intervals devoted to locations that contained greater
areas of stable gravel substrate with good flow and low levels
of fine sediment. Mussels were carefully removed from the
substrate and placed in mesh bags. After each survey, mus-
sels were sorted, measured to the nearest 0.01 mm maxi-
mum length using digital calipers (Lisher Scientific, Pitts-
burgh, Pennsylvania), and identified to species using keys by
Cummings and Mayer (1992), Oesch (1995), and Couch
(1997). Mussels were then carefully returned to the river
throughout the search area. Live voucher specimens were
not collected due to limited representatives of some species;
however, digital photographs and rel-
ict shells from each sample site were
deposited at the Ecotoxicology and
Water Quality Research Laboratory at
Oklahoma State University, Stillwater,

Oklahoma.

Comparisons of mussel richness
and abundance were made using
2-sample paired f-test for means (a =

0.05) performed with SigmaStat ver-
sion 3.1 (Systat Software Inc., San Jose,

California). Regression coefficients
and elevation of richness versus log-
transformed abundance curves were
generated for the present study and
that conducted by Vaughn (1998) and
were compared using f-tests at a =

0.05 (Zar 1999).

km above Oologah Lake. The section of river between the
last upstream site and the reservoir was not surveyed because
of a general lack of riverine mussel habitat as the river wid-
ens and becomes more lake-like. Sites below Oologah Lake
were located 1-25 km below the Oologah Lake dam. Habitat
beyond this point also was considered unsuitable due to
dredging in the McClellan-Kerr Navigation Channel.

Temperature across all sites ranged between 20-35 °C,
dissolved oxygen between 6.0-11.9 mg/L, pH between 6-8
standard units, alkalinity between 120-170 mg/L CaCOj, and
hardness between 112-156 mg/L as CaCOj. Seventeen spe-
cies of mussels were identified from the Verdigris River as a
whole (fig. 1), with Quadrula metanevra (Rafmesque, 1820)
being most abundant and Quadrula nodulata (Rafmesque,
1820) the least. Cyprogenia aberti (Conrad, 1850), Lampsilis
teres (Rafmesque, 1820), and Lasmigona complanata (Barnes,
1823) were found only in sites above Qologah Lake, while
Megalonaias nervosa (Rafmesque, 1820), Quadrula cylindrica
(Say, 1817), and Q. nodulata were found only at sites below
the lake. Generally, abundant species were also the most
widely distributed except for Q. cylindrica which was found
only at three locations but was the sixth most abundant
species (fig. 2). Size-frequency distributions were developed

CD

o

c

ro

T3

c

=J

<

o

350

300

250

200

150 -

100

Upper

Lower

RESULTS

A total of 31 sites (20 above Oo-
logah Lake and 1 1 below) were sur-
veyed for mussels between July and
October 2006. Sites above Oologah
Lake started approx. 0.5 km after the
Verdigris River crossed the Oklahoma-
Kansas border and ended approx. 28

.0® <^\y <>'-

oS

Mussel Species

Figure 1. Abundance of native mussels (total number found) in the Verdigris River, Okla-
homa, above (Upper) and below (Lower) Oologah Lake.

NATIVE MUSSELS OE THE OKLAHOMA VERDIGRIS RIVER

3

Figure 2. Incidence (number of sites at which a species occurred) of native mussels for the
Verdigris River, Oklahoma. Maximum incidence = 31.

Table 1. Mean richness and abundance (#/h, mussels found per
hour sampling effort) for sites within the Verdigris River, Oklaho-
ma. Upper, sites above Oologah Lake; Lower, sites below the lake;
All, all sites combined.

Sites

Mean richness

Mean #/h

N

Upper

5.60

39.31

20

Lower

4.36

35.66

11

All

5.16

38.01

31

for the four most abundant species, Q. metanevra, Tritogonia
verrucosa (Rafmesque, 1820), Obiquaria reflexa (Rafmestiue,
1820), ^nd Amblema plicata (Say, 1817) (Fig. 3). The distri-
butions for all four species were negatively skewed, indicat-
ing fewer small individuals than might be predicted given a
normal distribution, with kurtosis values generally positive
except for O. reflexa with a -0.74 value indicating a slightly
more platykurtic (uniform) distribution.

Live mussels were found at all but 2 sites. Timed abun-
dance estimates for sites with live mussels ranged from 3 to

156 mussels per hour (#/h) with a
mean of 38 for all 31 sites (Table 1).
Taxa richness ranged from 2 to 10 spe-
cies per site with a mean of 5.2 species
for all 31 sites combined.

No evidence of zebra mussels was
found at any site above Oologah Lake;
however, zebra mussels and byssal
threads were found on substrate and
unionid shells at every site below the
lake. While many native mussels col-
lected below the lake had byssal
threads on the valves, few had live ze-
bra mussels attached. In order to assess
the potential impact of zebra mussels
on native mussel community compo-
sition, species richness and abundance
were estimated separately for sites
above and below the lake. Sample lo-
cations above the lake had a mean
richness of 5.6 species per site, and an
abundance of 39.3/h (Table 1). Sites
below Oologah Lake had a mean rich-
ness of 4.4 species per site with an
abundance of 35.7/h (Table 1). There
was no significant difference in overall
mussel species richness or abundance
between upstream and downstream
sites (P - 0.12 and P = 0.41, respec-
tively).

While no differences in mussel richness and abundance
were apparent when sites were combined within upstream
and downstream sections, a downstream longitudinal gradi-
ent in these parameters was apparent. Both mussel abun-
dance (U = 0.697, N = 1 1, P = 0.001 ) and species richness (r
= 0.539, N - 11, P = 0.014) were significantly positively
associated with distance from the dam (Fig. 4). However,
these analyses may be influenced by two downstream sites
that had the greatest abundance of any of the sample loca-
tions. When these two sites are removed from the analyses,
the longitudinal relationship is no longer significant (#/h: r
= 0.31, N = 9, P = 0.120; richness: r = 0.38, N = 9, P =
0.077).

DISCUSSION

Sampling locations in the Verdigris River corresponded
to a previous study (Vaughn 1998) to reassess the status of
the native mussel community since the discovery of zebra
mussels in Oologah Lake in June 2003. Vaughn ( 1998) iden-
tified 16 species of mussels, compared with 17 in this study.

4

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Length (mm)

Figure 3. Size-frequency distributions for the four most abundant mussel species in the
Verdigris River, Oklahoma. N, number of observations for that species.

While species composition was largely similar between the
two surveys, Potamilus ohiensis (Rafmesque, 1820) and Qua-
drula quadrula (Rafmesque, 1820) were not found in 2006.
In contrast, live Cyprogenia aberti, Quadrula cylindrica, and
Quadrula nodidata were found in the 2006 survey while only
relict shells of these species were found in the previous sur-
vey. Of these, C. aberti and Q. cylindrica are of particular
interest because C. aberti was once thought extirpated from
the state (Mather 1990, Serb 2006), and Q. cylindrica is a
federal candidate for listing as an endangered species (David
Martinez, pers. comm., U. S. Fish and Wildlife Service).
Only 2 C. aberti were found, and both individuals were from
sites near the Oklahoma- Kansas border. As abundance of
this species has increased in the Kansas portion of the Ver-
digris (Miller and Lynott 2006), these upstream populations

may be responsible for the re-
introduction into Oklahoma through
mechanisms such as fish host dispersal
or downstream transport of juveniles
after detaching from the fish host
(Morales et al. 2006).

While Quadrula cylindrica was the
sixth most abundant species, it was
found only in a short river section
downstream from Oologah Lake, an
area also infested with zebra mussels.
Lee et al. (1998) and Berg et al. (2007)
suggest host-fish vagility may explain
unionid distribution patterns, with
mussels that utilize fish hosts with
greater home ranges typically showing
greater abundance and distribution.
The known fish hosts for Q. cylindrica
include several species of Notropis
(Yeager and Neves 1986), which have
relatively small home ranges (Goforth
and Foltz 1998). This characteristic of
its fish host may explain the limited
distribution of Q. cylindrica in the Ver-
digris.

Size-frequency distributions of the
4 most commonly encountered mussel
species did not include individuals less
than 30 mm in length (70 mm for Am-
blema plicata) although this may have
been a sampling artifact as timed snor-
kel searches bias against encountering
very small or particularly cryptic indi-
viduals (Hornbach and Deneka 1996,
Obermeyer 1998, Metcalfe-Smith et al.
2000). Furthermore, juveniles fre-
quently bury in the substrate, making
detection difficult using snorkel searches (Neves and Widlak
1987, Amyot and Downing 1991, Yeager et al. 1994, Sparks
and Strayer 1998). Timed snorkel search techniques are,
however, more commonly used for determining mussel rich-
ness and locating rare species (Metcalfe-Smith et al. 2000,
Vaughn and Spooner 2004) and are more cost effective when
surveys of large areas are needed as compared with quadrat
methods.

Vaughn (1998) reported a mean abundance for all 31
sites of 14.4 mussels/h and a mean richness of 3.3 species per
site. Gurrent abundance and richness across all 31 sites were
38.0/h and 5.2 species per site, significantly greater than
reported by Vaughn (P - 0.002 and P = 0.001, respectively).
Mean sampling time between the two surveys was not sig-
nificantly different [P- 0.65) with 46.8 min in this survey vs.

NATIVE MUSSELS OF THE OKLAHOMA VERDIGRIS RIVER

5

km below dam

Figure 4. A, Distance from the dam vs. mussel abundance (#/h). B,
richness in Verdigris River, Oklahoma for sites below Lake Oologah
dam. Coefficients of determination and significance levels are
shown.

49.8 min in Vaughn ( 1998). To evaluate the extent to which
the increase in richness resulted from locating more mussels
overall, abundance vs. richness curves were prepared from
both studies (Fig. 5). These curves were linearized by log-
transforming abundance to facilitate statistical analysis.
There was no significant difference between studies in the
regression coefficient (36.7 for the present study vs. 65.7 for
the Vaughn study, P > 0.05) or elevation (P > 0.05), indi-
cating that mussel abundance explained a similar degree of
variation in taxonomic richness in both studies. Thus the
greater taxa richness in the present study appears to be ex-

plained by our finding a greater number of mussels during
the timed searches.

Given the semi-c]uantitative sampling employed in both
surveys, care should be taken not to over-interpret these
data. Strayer ( 1999) found low statistical power for detecting
population declines when using presence/absence tech-
nic]ues; therefore, these results should at a minimum support
the need for more quantitative techniques throughout the
study area. Miller and Obermeyer (1997) and Miller and
Lynott (2006) reported an increase in 10 different species in
the Kansas portion of the Verdigris River relative to 1991
levels, using quadrat methods. They attribute this increase to
improvements in habitat quality, namely reduction in pol-
lution, increase in fish hosts, and lack of severe drought.
These same factors may be working to improve native mus-
sel populations in the Verdigris River of Oklahoma.

The detrimental effects of zebra mussels on native mus-
sel communities are well documented (Burlakova et al. 2000,
Martel et al. 2001, and Schloesser et al. 2006) although we
found no difference in mussel abundances between sites up-
stream of Oologah Lake and sites downstream of the dam
which are infested with the mussels. However, two relatively
“good” sites greater than 20 km from the dam may have
influenced these analyses. A number of native mussels were
found to have byssal threads on their valves, indicating some
degree of zebra mussel settling. It is possible that zebra mus-
sel colonization of native mussels occurs during the spring,
but the attached Dreissena polymorplm may be eliminated as
water temperatures increase through the summer. The im-
poundment at Oologah Lake was designed for hypolimnetic
releases, meaning if water releases occurred during summer,
downstream habitats would receive cooler water. However,
from August to November of 2006, there were no releases
from the reservoir. Without this influence, it is reasonable
that water temperature in these downstream reaches of the
Verdigris River was similar to that in other streams in the
area and approached 30 °C in mid summer. This tempera-
ture may be lethal to zebra mussels (Karatayev et al. 1998,
Matthews and McMahon 1999, Elderkin and Klerks 2005)
while unionids may be slightly more tolerant (Polhill and
Dimock 1996). Additionally, monitoring of zebra mussel
densities in Oologah Lake indicated a significant die-off be-
ginning in late June 2006. Adult zebra mussel densities de-
clined from nearly 150,000 /m^ to <4,500 /m^ from late June
to September 2006 (Chad Boeckman, unpubl. data). Tem-
perature-induced seasonal die-offs of zebra mussels in the
Verdigris River may mean that zebra mussel fouling of na-
tive mussels does not reach high enough numbers to cause a
significant effect. Other studies have reported mortality of
unionids due to zebra mussel infestation within 2 to 8 years
after initial zebra mussel colonization (Schloesser et al. 1996,
2006, Ricciardi et al. 1998, Schloesser and Masteller 1999).

6

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Abundance

Figure 5. Richness (# of species/site) vs. abundance (total # of mussels found at that site)
curves for the Vaughn (1998) survey (dashed line) and the current survey (solid line). For
statistical analysis, these curves were linearized by log-transforming abundance. The resulting
regression equations were y = 4.332x -I- 0.1135 for the present study and y = 4.284x -I- 0.0257
for Vaughn ( 1998).

Zebra mussels were first reported in Oologah Lake in June
2003 (Laney 2005), with numbers increasing steadily since
that time. Negative effects on unionids due to zebra mussel
colonization may still happen, if a cooler summer were to
occur.

Several studies have shown that zebra mussels and na-
tive mussels can co-exist in habitats with fluctuating water
depth, potential wave action, and substrate soft enough to
allow native mussels to bury (Schloeser et al. 1997, Nichols
and Amberg 1999, Bowers and De Szalay 2004, Strayer and
Malcom 2007). The habitat below Oologah Lake does have
some of these characteristics.

A potentially confounding factor in determining the ef-
fect of zebra mussels on unionid mussels is the influence of
the Oologah Lake dam. The effects of impoundments on
native mussel communities have been well established (Bo-
gan 1993, Vaughn and Taylor 1999, Bednarek 2001, Sethi et
al. 2004). For example, Vaughn and Taylor (1999) described
a reduction in native mussel populations below an im-
poundment and found 20 km was needed for these popu-
lations to recover to pre-impoundment levels. In the current
survey, mussel abundance downstream from the reservoir
was positively correlated with distance from the dam. How-
ever, this relationship may have resulted from two sites

(greater than 20 km from the dam)
that harbored the greatest abundance
of all 31 sites surveyed. Downstream
sites less than 20 km from the dam
generally had poor or less than average
abundance, and two sites immediately
downstream from the dam had no live
mussels at all. Taxonomic richness for
downstream sites was also positively
correlated with distance from the dam
although less distance from the dam
was needed to “recover” as compared
with abundance.

While it appears that zebra mus-
sels are currently having little effect on
native mussel richness and abundance
below Oologah Lake, the consequences
of long-term zebra mussel coloniza-
tion on native mussels in this reach of
the river are still unknown. For this
reason, conservation efforts such as
periodic defouling or propagation and
reintroduction (Hallac and Marsden
2001, Strayer et al. 2004) to sites above
Oologah Lake should be directed at
species of special interest (and native
mussels in general) that are currently
located below Oologah Lake. Qiiadrula
cylindrica should be a high priority for such efforts given its
rather limited distribution in the Verdigris River (Oberm-
eyer et al. 1997a, 1997b), the impoundment upstream and
dredging activity downstream, and the relatively limited dis-
persal of its fish hosts.

ACKNOWLEDGMENTS

Funding for this study was provided by a State Wildlife
Grant (T-40-P) from the Oklahoma Department of Wildlife
Conservation through the Oklahoma Cooperative Fish and
Wildlife Research Unit. The Oklahoma Cooperative Fish and
Wildlife Research Unit is jointly sponsored by the U.S. Geo-
logical Survey, Oklahoma State University, the Oklahoma
Department of Wildlife Conservation, the Wildlife Manage-
ment Institute, and the U.S. Fish and Wildlife Service. Ad-
ditional funds were provided by the Ecotoxicology and Wa-
ter Quality Research Laboratory at Oklahoma State
University. We would like to thank C. Vaughn for providing
maps to previously sampled locations in the Verdigris River
and D. Walter and K. Burgess for their assistance in the
field.

NATIVE MUSSELS OF THE OKLAHOMA VERDIGRIS RIVER

7

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revisions received: 26 February 2008

Amer. Malac. Bull 25: 9-19 (2008)

Spatial distribution of soft-bottom molluscs in the Ensenada de San Simon
(NW Spain)

Eva Cacabelos\ Patricia Quintas^, and Jesiis S. Troncoso^

' Area de Bioloxia Animal, Facultade de Ciencias do Mar, Universidade de Vigo, 36310 Lagoas-Marcosende, Vigo (Pontevedra), Spain,
cacabelos@uvigo.es

^ Biotecnoiogia y Acuicultura, Instituto de Investigaciones Marinas de Vigo, Eduardo Cabello 6, 36208 Vigo, Spain

Abstract: Distribution and abundance of the molluscan fauna was studied in the intertidal and subtidal soft-bottoms of the Ensenada de
San Simon (NW Spain). Depth, grain size, and total organic matter were the most important factors in determining distribution patterns
of molluscs in this inlet. Three major malacological assemblages have been determined in the Ensenada de San Simon, two of them
subdivided in two facies. In the intertidal area of the inlet, one facies (A1 ) was located in areas associated with seagrass meadows of Zostera
spp. and was dominated by Hydrobia idvae (Pennant, 1777) whereas the second facies (A2) had a high dominance of H. idvae, Cerastoderma
edtde (Linnaeus, 1758), and Tapes decussatus (Linnaeus, 1758). An impoverished facies of this community was present in reduced, muddy
bottoms (Group C). In the subtidal bottoms, one group (Bl) was located in the central part of the inlet with H. idvae, Rissoa labiosa
(Montagu, 1803), Turboella radiata (Philippi, 1836), Parvicardium exiguum (Gmelin in Linnaeus, 1791), Loripes lacteus (Linnaeus, 1758),
and Abra nitida (Muller, 1789) as characteristic species. A second facies (B2) was found in outer areas of the inlet, characterized by Thyasira
flexuosa (Montagu, 1803), Mysella bidentata (Montagu, 1803), Abra alba (Wood, 1802), and Niicida nitidosa Winckworth, 1930.

Key words: macrofauna, Macoma community, Abra alba community, multivariate analysis, Atlantic Ocean

Several faunistic and ecological works on the macroben-
thic communities of the Iberian and Galician coasts of Spain
have been carried out in recent years (Anadon 1980, Lopez-
Jamar and Mejuto 1988, Lopez-Jamar and Cal 1990, Tron-
coso and Urgorri 1991, Maze et al. 1993, lunoy 1996). Ben-
thic communities are considered good indicators of marine
bottom conditions (Pearson and Rosenberg 1978, Grail and
Glemarec 1997). The Ensenada de San Simon is located in
the inner part of the Ria de Vigo, the southern-most of the
Galician estuaries. These estuarine systems have been stud-
ied because of their great economic and social importance,
including fisheries, raft mussel cultures, and shellfish
resources.

Benthic communities of the Ria de Vigo have been ana-
lyzed since 1886 when Hidalgo published a list of marine
species of the NW Spanish coast (Hidalgo 1917). Despite the
abundance of studies in the Ensenada de San Simon (Nom-
bela et al. 1995, Fernandez Rodriguez et al. 1997, Alvarez-
Iglesias etal. 2003), few researchers have analyzed patterns of
benthic faunal spatial distribution, and none have quantified
community structure. While the molluscan fauna was stud-
ied in other estuaries (Troncoso et al. 1996, 2005, Olabarria
et al. 1998), the only previous studies in the Ria de Vigo were
by Rolan (1983), Rolan et al. (1989), and Moreira et al.
(2005).

Consequently, there is a need to improve our knowledge
to ensure correct management and conservation in the area,
especially due to Ensenada de San Simon being included in

the Nature 2000 Network as a Special Conservation Zone.
Therefore, the aim of the present study is to describe and
quantify the malacofaunal communities and associations in-
habiting intertidal and subtidal soft substrata throughout the
Ensenada de San Simon. Characteristic and dominant spe-
cies are studied to document their relationship with several
environmental variables.

MATERIALS AND METHODS

Study area

The Ensenada de San Simon is located in the inner part
of the Ria de Vigo (NW Spain), between 42°17' and 42°21'N
and between 8°37' and 8°39'W (Fig. 1 ). The seagrasses Zos-
tera noltii and Zostera marina cover the intertidal and shal-
low subtidal areas. Considerable freshwater input occurs in
the inner-most part of the inlet which results in salinity
fluctuations on a tidal and seasonal basis (Nombela and
Vilas 1991 ). Culture of mussels on rafts is a common prac-
tice in large areas of the mouth of the inlet, and a small
harbor is located in the mouth of the Alvedosa River.

Sampling and sediment laboratory analysis

Samples were collected during November and Decem-
ber 1999 from 29 sites (Fig. 1). Five samples were taken at
each site, by means of a van Veen grab (0.056 m^). Samples
were sieved through 0.5 mm mesh and the retained material
was fixed in 10% buffered formalin. Fauna was sorted from
the sediment and preserved in 70% ethanol. Temperature

9

10

AMERICAN MALACOLOGICAL BULLETIN 25 • 1-2 • 2008

Iberian Peninsula

Figure 1. Study area showing the location of sampling sites in NW
Spain. Gray figures represent mussel raft sites.

and pH were measured in situ from water and sediment
samples taken from each site. An additional sediment sample
was taken at each site for grain-size, calcium carbonate, and
total organic matter content analyses. Granulometric frac-
tions and sediment types were determined. The median
grain size (Q50, mm) and the sorting coefficient (S^) were
also calculated for each sample (Trask 1932). Calcium car-
bonate content (%) was estimated by sample treatment with
hydrochloric acid, and total organic matter content (%) was
estimated from the weight loss after combustion by placing
samples in a furnace for 4 hours at 450 °C (Guitian and
Carballas 1976, Parada et al. 1993).

Data analysis

Abundance (A), species richness (S), Shannon-Wiener’s

diversity index (H', logj) (Shannon and Weaver 1963), and
Pielou’s evenness index (J) (Pielou 1984) were determined
for the five samples pooled in each site (0.28 m^). Domi-
nance was calculated as the percentage of the numbers of
individuals belonging to one species with respect to the total
number of specimens in that sample. Mollusc assemblages
were based on the analysis of the species abundance data
matrix by non-parametric multivariate techniques, using
PRIMER software (Clarke and Warwick 1994). A similarity
matrix was calculated, using the Bray-Curtis coefficient, after
applying the fourth -root transformation to species abun-
dance. from the similarity matrix, classification and ordina-
tion of the sites were analyzed: cluster analysis, algorithm
UPGMA, and non-metrical multidimensional scaling, MDS.
The SIMPER program was used to identify molluscan spe-
cies that contributed to dissimilarity among groups.

Species were classified according to the constancy and
fidelity indexes and to the fidelity-dominance product (Da-
joz 1971). Relationships between abundance of molluscs and
environmental variables were studied by means of the
BIOENV procedure (PRIMER package) and canonical cor-
respondence analysis (CANOCO package; ter Braak 1988).
Environmental variables in percentages were transformed by
logarithm (x-l-1) and all were normalized.

RESULTS

Sedimentary characterization

The soft bottoms of the Ensenada de San Simon were
characterized by a predominance of muddy sediments with
a high total organic matter and low calcium carbonate con-
tent (Appendix 1). Sandy sediments were present in tidal
channels in the inner inlet where low total organic matter
content was also found. Areas around the outer part of the
inlet had muddy sands with a large gravel fraction composed
of the mussel shells cultured there.

Molluscan fauna

A total of 24,605 individuals belonging to 68 species of
molluscs (30 bivalves, 34 gastropods, 3 polyplacophorans,
and 1 scaphopod) was sampled in the study area (Appendix
2). Gastropods were the most abundant group (88.92% of
the total mollusc abundance) due mainly to large numbers
of Hydrobia ulvae (Pennant, 1777). This hydrobiid showed
densities of up to 34,946 individuals/m^ in sandy bottoms of
tidal channels and 14,800 individuals/m^ in sediments colo-
nized by Zostera marina and Z. noltii in the innermost part
of the inlet. Other dominant gastropods were Rissoa labiosa
(Montagu, 1803), Turboella radiata (Philippi, 1836), and
Chrysallida terebellum (Philippi, 1844), mainly in intertidal
and shallow bottoms. Bivalves were numerically the second
most important group (10.99% of total) with the greatest
numbers in the central area and at the mouth of the inlet.

SOUTOXUSTO

42°18’N

REDONDELA

wiiaben-Verdugo

8"36'W

Rio Xunqueira

PONTESAMPAIO

MOLLUSCS OF ENSENADA DE SAN SIMON

11

The most abundant bivalves were Cerastoderma edule (Lin-
naeus, 1758) in sandy intertidal sediments and Thyasira flex-
uosa (Montagu, 1803), Mysella bidentata (Montagu, 1803),
and Ahra alba (Wood, 1802) in subtidal sediments. Polypla-
cophorans and scaphopods were found in small numbers
(0.09%).

The highest abundances of molluscs were recorded at
sites 6, 2, and 3 due to the high abundance of Hydrobia
idvae; the lowest were recorded at sites 24 and 28 (64.3-342.9
individuals/m^). Sites 26, 27, and 22 at the mouth of the inlet
showed the highest species richness (22-31). Only two spe-
cies were found at sites 29 and 24, located in the mouth of
the Alvedosa River. Shannon-Wiener’s diversity index varied
between H' = 0.09 (site 3) and 3.75 (site 27), and evenness
ranged from J = 0.04 (site 3) to 0.97 (site 24). Greatest H'
values were recorded in sites in the mouth of the inlet while
the lowest values were found in intertidal sites with high
numbers of H. idvae.

Spearman’s correlation coefficient indicated that depth
was positively correlated with species richness (r^ = 0.511,
N = 29, P < 0.01 ), diversity {r^= 0.639, N =29, P < 0.01 ), and
evenness (r^ = 0.568, N = 29, P < 0.01), and negatively
correlated with number of individuals (r^ = -0.458, N - 29,
P < 0.05). Species richness was positively correlated with
percent content in gravel (r^ = 0.470, N - 29, P < 0.05) and
was negatively correlated with total organic matter (r^ =
-0.401, N - 29,P < 0.05) and silt/clay content (r^ = -0.376,
N= 29, P < 0.05). The number of individuals was positively
correlated with percent calcium carbonate, median grain size

(ij = 0.596 and = 0.473 respectively, N= 29, P < 0.01) and
sand content (r^ = 0.500, iV = 29, P < 0.01 ) and negatively
correlated with silt/clay content (r^ = -0.511, N = 29, P <
0.01).

Multivariate analysis

The classification diagram based on abundance data
showed three main groups: A, B, and C (Pig. 2). These three
groups were further subdivided into five subgroups. Group
A1 had muddy sediment, sandy mud, and very coarse sand.
Group A2 was composed of coarse sand and muddy sand
bottoms. Group B1 was comprised of muddy sediments
(mud and sandy mud to muddy sand). Group B2 sites had
mud and muddy sand bottoms. Group C sites had muddy
sediments. This classification agreed with ordination of sites
obtained through non-metrical multidimensional analysis
(Pig. 3).

Water depth presented the highest correlation with fau-
nistic data according to the BIOENV procedure (Spearman’s
rank correlation = 0.312). It was followed by the combi-
nation of depth and median grain size (p^ = 0.201), and that
of bottom water temperature, very fine sand, fine silt, clay,
and depth (p^^ = 0.192).

In the canonical correspondence analysis, the first two
axes accounted for 44.4% of the total variance of species-
environment relationships and 34.0% of the species variance
(Table 1). Species-environment correlations close to 1 indi-
cated that abiotic variables were correctly chosen (ter Braak
1988) while the maximum eigenvalue reached in the first
axis was close to the optimal 0.7. Bot-
tom water and sediment temperature,
coarse and fine silts, clay, and calcium
carbonate showed the highest correla-
tions with axis I; however, correlations
with other axes were less significant,
forward selection indicated water
depth as the variable explaining most
of the variance in the species data (F =
3.82, P < 0.01) as well as bottom-water
temperature (F = 1.82, P < 0.05), total
organic matter (F=1.73, P < 0.05), clay
(F= 1.63, P < 0.05), and very fine sand
(F = 1.91, P < 0.05). The scatter dia-
gram showed an ordination of sites
following a gradient of depth and grain
size (Fig. 4). Sites from Group A1 and
A2 were progressively distributed
along the negative part of axis I, in
intertidal or shallow waters with coarse
sediments; sites from group B1 and B2
were deeper with greater content in
finer sedimentary fractions.

B1

B2

A2

20

40

Al

60

9
8
13
28
20
7

25

10

23

17
21
12

18
16
19
27

26
22
\4

5
4
15
3
2

1

6

29
1 1

24

80

100

Bray Curtis Similarity (%)

Figure 2. Dendrogram showing the groups and subgroups considered.

I

12 AMERICAN MALACOLOGICAL BULLETIN 25 • 1-2 • 2008 I

Figure 3. Ordination diagram representing the assemblages con-
sidered in the classification analysis. Numbers refer to sites. Stress
indicates the goodness-of-fit for the model: the smaller the stress,
the better the representation.

The analyses suggested that the distribution of mollus-
can fauna is mainly determined by depth, organic matter
content, and grain size gradients.

Description of assemblages

Classification and ordination analyses differentiated five
different assemblages (Tables 2-3, Fig. 5). Group A located
in the inner-most part of the inlet, in intertidal sediments
close to the mouth of the Oitaben-Verdugo and Xunqueira
Rivers. The most abundant species was Hydrobia ulvae,
mainly in bottoms colonized by Zostern noltii. Subgroup A1
was comprised of four intertidal sites located along the
northern border of the inlet in heterogeneous sediments.
These bottoms exhibited low species richness (S = IT). The
seagrasses Zostera marina and Z. noltii were spread across
most of these bottoms. The most characteristic species ac-
cording to values of fidelity-dominance product (F x D)
were H. ulvae, Chrysallida terebellum, and Cerastoderma ed-
ule. This area had the smallest mean diversity value due to
the high dominance of H. ulvae. SIMPER analysis showed
that H. ulvae and C. edule were the species with a greater
similarity contribution (75%) for this group. Subgroup A2
was located in the intertidal sandy bottoms with the greatest
content in coarse sand (23.2% ± 17.5, mean ± SD) and the
greatest median. A total of 28 species was found, and the
most characteristic, according to F X D, were H. ulvae, C.
edule. Tapes decussatus (Linnaeus, 1758), Ostrea edidis Lin-
naeus, 1758, C. terebellum, and Retusa truncatula (Bruguiere,
1792). In accordance with the SIMPER analysis. Group A2

was mainly defined by H. ulvae, C. edule, R. truncatula, T. f
decussatus, and Lepton nitidum Turton, 1822. J

Group B was present in muddy bottoms, from intertidal f
areas to 28 m depth in the mouth of the inlet. Species rich- "
ness increased from sites in inner areas towards the mouth.
Sediments were mainly composed of silt and clay (>50%). |j
Subgroup B1 was defined in shallow sediments in the center b
of the inlet (0-4.7 m depth). Sites 10 and 20 had Zostera 1]
marina meadows. This subgroup had 27 species and showed
the greatest mean value for evenness. The F x D and domi-
nance values indicated that the most characteristic species
were Hydrobia ulvae, Rissoa labiosa, Turboella radiata, Par-
vicardium exiguum (Gmelin in Linnaeus, 1791), Loripes
lacteus (Linnaeus, 1758), Abra nitida (Muller, 1789), and
Chrysallida terebellum. According to the SIMPER analysis,
Group B1 was characterized by H. idvae, C. terebellum, T.
radiata, and the bivalve P. exiguum. Subgroup B2 was lo-
cated in the external part of the inlet, in subtidal bottoms
(3.7-28.2 m). These bottoms showed the highest mean value
for calcium carbonate. Fifty-nine molluscan species were
found and many of them showed great Fidelity values. '

Among the species with highest values of F x D were Thya- |

sira flexuosa, Mysella bidentata, Calyptraea chinensis (Lin-
naeus, 1758), Abra alba, Nucula nitidosa Winckworth, 1930,
and Hyala vitrea (Montagu, 1803). Other species with high
values of constancy and fidelity were Myrtea spinifera (Mon-
tagu, 1803), Chrysallida fenestrata (Jeffreys, 1848), and Cor-
bula gibba (Olivi, 1792). The bivalves T. flexuosa, M. biden-
tata, A. alba, N. nitidosa, and gastropod C. chinensis defined
group B2, according to SIMPER.

Group C was mainly characterized by Hydrobia ulvae
(86% of cumulative similarity). This group was composed of
muddy sites close to the mouth of several small rivers and '
Redondela harbor. Sediments were predominantly com- |
posed of silt and clay with low carbonate content, and the
greatest values of total organic matter in the inlet. Only 8 j
species were found and densities were less than in other
groups (1202.50 ± 898.21 individuals/m^). The species with '
highest values of F x D were H. ulvae, Turboella radiata, and !
Loripes lacteus, while the group was mainly characterized by |
H. ulvae (86% of cumulative similarity). 1

DISCUSSION I

The distribution of the molluscan fauna seemed to be {
mainly determined by depth, organic matter content, and :
grain-size in the Ensenada de San Simon, NW Spain. Inter- j
tidal and shallow sediments in inner channels were mostly
sandy and then became increasingly muddy towards the ;
deeper bottoms in the center and at mouth of the inlet. The
lack of strong currents in the greater part of the inlet and the \
very common culture of mussels on rafts were responsible |

MOLLUSCS OF ENSENADA DE SAN SIMON

13

Table 1. Canonical correspondence analysis for the Ensenada de San Simon.

Total

Axes

I

11

III

IV

inertia

Eigenvalues

0.680

0.361

0.200

0.170

3.061

Species-environment correlations
Cumulative percentage variance

0.984

0.914

0.976

0.935

of species data

Sum of all unconstrained eigenvalues
Sum of all canonical eigenvalues

22.2

34.0

40.5

46.1

3.061

2.347

for the progressive increase of fine particles and organic
matter content in the sediment. The silt/clay contents found
in intertidal areas were higher than described by Nombela
and Vilas (1986-1987). This situation is not surprising. On
one hand, the presence of Zostera spp. stabilizes the sedi-
ment (Nombela et al. 1995); on the other hand, intense
culture of mussels on rafts is located at the study sites (Fig.
1 ) and in the greater part of Galician estuaries. This culture
is an important human disturbance since it produces large
quantities of fecal pellets that substantially modify sediment

Figure 4. Canonical correspondence analysis ordination of envi-
ronmental variables and sites in relation to axes I and II, repre-
senting the groups and subgroups in the classification analysis. Tb,
temperature of bottom-water; Ts, temperature of surface-water;
CO3, carbonates; OM, organic matter; Qjg, median grain size; S„,
sort coefficient; GR, gravel; VCS, very coarse sand; CS, coarse sand;
MS, medium sand; FS, fine sand; VFS, very fine sand; CSi, coarse
silt, FSi, fine silt; C, clay.

composition, increasing the clay frac-
tion (Nombela et al. 1987, Leon et al.
2004). The granulometric change has
an important impact on the benthos
community and also affects trophic
structure (Abella et al. 1996, Conde
and Dominguez 2004). Moreover, an-
oxic situations can be produced by pel-
let sedimentation with high organic
matter content under the rafts
(Tsuchiya 2002). Since the biodeposits
produced by one raft could reach 190
kg dry weight d ’ (Cabanas et al. 1979) and in San Simon
there are 76 rafts, the effect of this activity reduces the depth
in the inlet between 0.5 and 2 cm y~'. However, this culture
could be considered also as an important depurator because
mussels ingest high quantities of particulate organic matter
(Fernandez Rodriguez et al. 1997).

Species richness, diversity, and evenness were greater on
subtidal bottoms than on intertidal areas. This is a conse-
quence of the stressful conditions that aquatic fauna must
tolerate in intertidal areas: this fauna is subjected to impor-
tant environmental changes such as extreme temperatures,
desiccation, or rough conditions on the floor (Kikuchi
1987). The fauna in these intertidal sediments in the
Ensenada de San Simon must also tolerate changes in salinity
due to the freshwater input from several rivers (Vilas et al.
1995). Salinity fluctuations may greatly influence the species
richness and the species composition of the community
(Planas and Mora 1987), benefiting euryhaline species such
as Hydrobia ulvae. As well as in Ensenada de San Simon,
large densities of H. ulvae are common in inner areas of
other Galician estuaries having organic pollution (Planas et

1984, Curras and Mora 1990, Junoy 1996, Olabarria et al.

1998). This species has a broad range of food sources since
it is a detritivore on organic remains and fecal pellets (Ja-
cobs et al. 1983) or grazes on microalgae (Muus 1967) which
may explain the large numbers in these sediments.

Intertidal sediments colonized by Zostera noltii and Zos-
tera marina showed low diversities of molluscs. Seagrass
meadows provide a complex habitat that may be colonized
by many species (Somersfield et al. 2002), stabilizing the
sediment and providing protection to potential prey. How-
ever, low diversities characterize these meadows in San Si-
mon since they are located in areas subjected to abrupt sa-
linity changes, a major limiting factor for non-euryhaline
species (Planas and Mora 1987, Junoy 1996). Subtidal sedi-
ments show more stable conditions in terms of salinity and
currents (Nombela et al. 1987). However, the sites with the
lowest species richness and abundance were the muddy bot-
toms close to the mouth of freshwater channels and in the
harbor. Fine and homogeneous sediments have been related

14

AMERICAN MALACOLOGICAL BULLETIN 25 • 1-2 • 2008

Table 2. Summaiy of biotic and physical characteristics of the associations. Values: mean ± SD. Depth <2 m is intertidal; Qj;,, median grain '
size; Bt, bottom type (VCS, Very coarse sand; CS, Coarse sand; MS, Muddy sand; M, Mud); OM, percent total organic matter content; CO3, 1

percent carbonate content. Faunistic parameters at each site per m^: S, species richness; A, abundance; I, Pielou’s evenness; H', Shannon- i

Wiener’s diversity index. |

Group

Al

A2

B1

B2

C

Depth (m)

1.60 ±0.00

1.73 ± 0.11

3.10 ±0.88

9.04 ±7.77

3.23 ± 1.10

Q50

0.31 ± 0.56

0.77 ±0.38

0.06 ± 0.08

0.15 ± 0.45

0.01 + 0.00

% Gravel

5.85 ± 10.23

17.08 ± 10.82

3.23 ± 4.12

6.20 ± 11.66

0.03 ± 0.05

% Sand

45.83 ± 26.37

77.58 ± 12.52

36.46 ± 26.40

26.32 ± 10.00

17.72 ± 12.26

% Silt/Clay

48.31 ± 34.88

5.34 ± 2.50

60.30 ± 28.67

67.49 ± 20.35

82.25 ± 12.31

Bt

M-VCS

MS-CS

M-MS

M-MS

M

% OM

17.45 ±11.42

2.69 ± 1.63

17.59 ± 11.61

17.17 ± 4.95

20.75 ± 6.11

% CO3

7.30 ±3.13

7.23 ± 0.96

4.74 ± 1.21

8.04 ± 10.89

4.44 ± 0.37

S

6.75 ± 1.5

15.67 ± 5.03

11.62 ± 3.25

17.73 ± 6.35

3.67 ± 2.89

A

159589.29 ± 139930.00

26916.79 ± 17955.36

8218.57 ± 7489.64

8207.14 ± 6280.71

1202.50 ± 898.21

1

0.06 ± 0.22

0.41 ± 0.17

0.69 ± 0.22

0.62 ± 0.14

0.61 ± 0.33

H'

0.16 ± 0.06

1.56 ± 0.63

2.43 ± 0.82

2.56 ±0.82

0.93 ± 0.59

Table 3. Characteristic species of each group according to SIMPER and F x D values are listed indicating their constancy (Ct, constant; C,
common; VC, Very common) and fidelity (Ex, Exclusive; El, Elective; Pr, Preferential; Ac, Accessory; Oc, Occasional).

Al

A2

B1

B2

C

Hydwbia ulvae (Ct, Oc)

H. ulvae (Ct, Oc)

H. ulvae (Ct, Oc)

Calyptraea cliinensis
(Ct, Ac)

H. ulvae (Ct, Oc)

Cerastoderma edule (Ct, Oc)

Retusa truncatula (Ct, Pr)

Turboella radiata
(VC, Ac)

Nucula nitidosa (Ct, El)

T. radiata (VC, Ac)

Chrysallida terebellum (Ct, Oc)

C edule (Ct, Oc)

Tapes decussatiis (Ct, El)

Lepton nitidum (Ct, El)
Ostrea edulis (C, El)

C. terebellum (VC, Oc)

C. terebellum (Ct, Oc)
Parvicardium exiguurn
(VC, Ac)

Rissoa labiosa (C, Oc)
Loripes lacteus (VC, Oc)
Abra nitida (C, Pr)

Thyasira flexuosa (Ct, Ac)
Mysella bidentata (Ct, Ac)

Abra alba (Ct, Ac)

Hyala vitrea (C, Ex)
Myrtea spinifera (C, Ex)
Chrysallida fenestrata
(C, Ex)

Corbula gibba (C, Ex)

L. lacteus (C, Oc)

to low diversities: as the grain size decreases, there are re-
strictions in interstitial space and oxygen diffusion (Olabar-
ria et al. 1998). In general, diversity values observed in the
Ensenada de San Simon were high (1.92 ± 1.13) in com-
parison to other Galician estuaries with a predominance of
muddy bottoms (Lopez-Jamar and Mejuto 1985). Mean di-
versity values were generally greater in exposed estuaries
with sandy and more heterogeneous sediments, as Ria de
Ares-Betanzos (2.38 ± 0.82, mean ± SD, Troncoso and Ur-
gorri 1991 ), Ensenada de Baiona (2.37 ± 0.74, mean ± SD,
Moreira et al. 2005), and Ria de Aldan (2.77 ± 0.84, mean ±
SD, Lourido et al. 2006).

The assemblages in the Ensenada de San Simon deter-
mined by the different multivariate approaches could be
described as classic communities or facies. The assemblage in

the intertidal areas corresponding to Group A had the typi-
cal fauna of the small Macoma community (community of
Cerastoderma ediile-Scrobicularia plana). The facies located
in areas associated with meadows of Zostera spp. (Al) was
dominated by Hydwbia ulvae\ the second facies (A2) pre-
sented a high dominance of H. idvae, C. edide, and Tapes
decussatiis. Similar faunal assemblages have been reported
from other intertidal and shallow bottoms in the Galician
estuaries (Cadee 1968, Anadon 1980, Mora 1982, Troncoso
and Urgorri 1991, Maze et al. 1993). In estuarine bottoms
with high organic content cited by Junoy (1996) and Ola-
barrla et al. (1998), the assemblage tends to be dominated by
H. ulvae. An impoverished facies of a small Macoma com-
munity was present in reduced muddy bottoms (Group C).
In this case, salinity fluctuations coupled with effects from

MOLLUSCS OF ENSENADA DE SAN SIMON

15

Figure 5. Spatial distribution of molluscan assemblages in the
Ensenada de San Simon, Spain.

human activities, such as organic enrichment and sewage
disposal, may be responsible for the scarce malacological
fauna near shore.

Group B can be ascribed to the Abra alba community
(Petersen 1918) from muddy bottoms (Lastra et al. 1990).
The facies present in subgroup B1 had a transitional fauna
that was between that of the small Macoma community {e.g.,
Hydrobia iilvae, Rissoa labiosa), and of a typical A. alba com-
munity, with species that tend to be more abundant in mud-
dier sediments, such as the bivalves Abra nitida, Mysella
bidentata, and Thyasira flexuosa. The facies in deeper bot-
toms of Subgroup B2 was characterized by the greater domi-
nance of T. flexuosa and M. bidentata. Similar assemblages,
showing transitional faunas between typical “communities”
(as in Thorson 1957) both in composition of species and
numbers for any given species according to gradients in
depth and granulometry, have been reported by Sanchez-
Mata and Mora (1999), Moreira et al. (2005), and Lourido et
al. (2006) in a variety of muddy bottoms of Galicia. T. flex-

uosa has been considered as an opportunist in disturbed
situations (Lopez-Jamar and Mejuto 1988) and prefers mud-
dier sediments (Moreira et al. 2005), as was the case in San
Simon. Lopez-Jamar and Parra (1997) detected high faunal
abundances in similar bottoms of the Galician coasts.

In conclusion, the most important factors in determin-
ing distribution patterns of molluscs in the Ensenada de San
Simon were depth, grain size, and total organic matter con-
tent. The presence of muddy sediments in this inlet is a
consequence both of the hydrodynamic regime, which de-
posits finer fractions in subtidal areas, and mussel bivalve
culture. Similarities both in sediment and faunistic compo-
sition have been reported by Mora et al. (1989) in Ensenada
de Lourizan, Lopez-Jamar and Mejuto (1988) in A Coruna
harbor, Lourido et al. (2006) and Sanchez-Mata and Mora
( 1999) in inner areas of Ria de Aldan and Ares-Betanzos, or
Olabarria et al. ( 1998), Mora ( 1982), and Junoy ( 1996) in the
Zostera meadows in Ensenada de O Baho, O Grove, or Ria de
Eoz, respectively.

ACKNOWLEDGMENTS

We gratefully thank the team at the Adaptations of Ma-
rine Animals laboratory for their invaluable help with
sample collection and statistical methods.

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Maze, R. A., M. Lastra, and J. Mora. 1993. Macrozoobentos del
Estuario del Mino (NO de Espana). Publicaciones Especiales
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Mora, J. 1982. Consideraciones generales sobre la macrofauna
bentonica de la Ria de Arosa. Oecologia Aquatica 6: 41-49 [In
Spanish].

Mora, J., M. Planas, and R. Silva. 1989. Impacto de la contami-
nacion organica en la Ensenada de Lourizan (Proyecto
ESCORP) I. El medio fisico y la macrofauna bentonica.
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Moreira, J., P. Quintas, and J. S. Troncoso. 2005. Distribution of
the molluscan fauna in subtidal soft bottoms of the Ensenada
de Baiona (north-western Spain). American Malacological Bul-
letin 20: 75-86.

Muus, B. 1967. The fauna of Danish estuaries and lagoons. Distri-
bution and ecology of dominating species in the shallow
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Nombela, M. A. and F. Vilas. 1991. Datos hidrograficos en la
desembocadura de los rios Oitaben y Redondela, Ensenada de
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Nombela, M. A., F. Vilas, and G. Evans. 1995. Sedimentation in the
mesotidal Rias Bajas of Galicia (north-western Spain):
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Nombela, M. A., F. Vilas, M. D. Rodriguez, and J. C. Ares. 1987.
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Olabarrla, C., V. Urgorri, and J. S. Troncoso. 1998. An analysis of
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Planas, M. and J. Mora. 1987. Estado de conocimiento actual del

MOLLUSCS OF ENSENADA DE SAN SIMON

bentos en zonas organicamente enriquecidas. Thalassas 5: 125-
134 [In Spanish].

Planas, M., L. Rodriguez Rey, and }. Mora. 1984. Cartografia
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de la Ria de Vigo. II. Poliplacoforos, Bivalvos, Escafopodos y
Cefalopodos. Thalassas 1 (Appendix 2): 1-276 [In Spanish].

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de Ares-Betanzos (NO Peninsula Iberica). 111. Estructura y
tipificacion de las comunidades macrofaunales. Nova Acta
Cienttfica Compostelana (Bioloxia) 9; 219-235 [In Spanish].

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ships between seagrass biodiversity and infaunal communities:
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ter Braak, C. J. F. 1988. CANOCO. A Fortran Program for Canonical
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Thorson, G. 1957. Bottom communities (subtidal or shallow shelf).
Bidletin of the Geological Society of America 67: 461-534.

Trask, P. D. 1932. Origin and Environment of Source Sediments of
Petroleum. Houston Gulf Publications Co., Houston, Texas.

Troncoso, J. S. and V. Urgorri. 1991. Los moluscos intermareales
de la Ria de Ares y Betanzos (Galicia, Espana). Nova Acta
Cientlfica Compostelana (Bioloxia) 2: 83-89 [In Spanish].

Troncoso, J. S., V. Urgorri, and C. Olabarria. 1996. Estructura tro-
fica de los moluscos de substratos duros infralitorales de la Ria
de Ares y Betanzos (Galicia, NO Espana). Iberus 14: 131-141
[In Spanish].

Troncoso, I. S., ]. Moreira, and V. Urgorri. 2005. Soft-bottom mol-
lusc assemblages in the Ria de Ares-Betanzos (Galicia, NW of
Spain). Iberus 23: 25-38.

Tsuchiya, M. 2002. Faunal structures associated with patches of mus-
sels on East Asian coast. Helgoland Marine Research 56: 31-36.

Vilas, F., M. A. Nombela, E. Garda-Gil, S. Garda-Gil, I. Alejo, B.
Rubio, and O. Pazos. 1995. Cartografia de sedirnentos subma-
rinos: Ria de Vigo. Memoria del Dpto. de Recursos Naturales
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Submitted: 17 Sepitember 2007; accepted: 12 March 2008;
final revisions received: 15 April 2008

18

AMERICAN MALACOLOGICAL BULLETIN 25 • 1-2 • 2008

Appendix 1. Depth (<2 m is intertidal) and characteristics of sediments at each site. median grain size; CO3, percent carbonate content;
OM, percent total organic matter content (% based on dry mass).

Site

Depth

(m)

Q50

Gravel

(%)

Sand

(%)

Silt/Clay

(%)

Bottom type

OM

(%)

CO3

(%)

1

1.6

0.01

0.1

16.8

83.1

Mud

26.52

5.52

2

1.6

0.01

2.2

32.9

64.9

Mud

23.30

5.60

3

1.6

0.08

0.0

56.7

43.3

Sandy mud

19.05

6.12

4

1.6

0.32

17.8

74.0

8.2

Muddy sand

2.16

6.00

5

1.8

1.25

30.0

64.3

5.7

Muddy sand

4.90

7.33

6

1.6

1.15

21.1

76.8

2.1

Very coarse sand

0.95

11.98

7

3.4

0.15

0.3

74.3

25.4

Sandy mud

3.95

6.31

8

3.2

0.04

0.6

35.9

63.5

Mud

10.88

5.80

9

2.9

0.01

0.9

27.7

71.4

Mud

18.12

4.28

10

2.9

0.01

0.0

2.3

97.7

Mud

36.93

4.28

11

3.6

0.01

0.0

8.9

91.1

Mud

26.50

4.81

12

3.8

0.01

1.1

19.2

79.7

Mud

19.93

2.12

13

3.5

0.01

3.0

23.0

74.0

Mud

23.00

2.36

14

4.6

0.01

7.1

24.4

68.5

Mud

19.78

2.28

15

1.8

0.74

3.5

94.4

2.1

Coarse sand

1.00

8.35

16

4.2

0.01

1.0

15.5

83.5

Mud

21.47

4.53

17

3.7

0.02

4.7

31.1

64.2

Mud

18.93

5.90

18

4.5

0.01

1.9

20.0

78.1

Mud

15.20

4.52

19

4.7

0.01

0.0

13.9

86.1

Mud

21.05

4.53

20

2.6

0.21

11.8

77.7

10.5

Muddy sand

1.80

4.85

21

18

0.01

0.6

26.3

73.1

Mud

19.50

4.61

22

10.4

0.01

1.0

37.4

61.6

Mud

12.98

5.51

23

5.9

0.01

1.2

25.2

73.6

Mud

22.17

5.40

24

4.1

0.01

0.0

12.6

87.4

Mud

21.42

4.07

25

1.6

0.01

6.8

31.8

61.4

Mud

23.72

5.47

26

28.2

1.50

40.2

48.3

11.5

Muddy sand

7.22

40.46

27

11.5

0.01

9.3

28.2

62.5

Mud

10.60

8.61

28

4.7

0.01

2.4

19.0

78.6

Mud

22.32

4.61

29

2

0.01

0.1

31.7

68.2

Mud

14.33

4.45

MOLLUSCS OF ENSENADA DE SAN SIMON

19

Appendix 2. Faunistic parameters at each site: S, species richness;
A, total abundance (individuals/m^); J, Pielou’s evenness; and H',
Shannon-Wiener’s diversity index.

Site

S

A

I

H'

1

6

2,982.1

0.09

0.24

2

8

15,014.3

0.05

0.14

3

5

10,246.4

0.04

0.09

4

15

821.4

0.61

2.37

5

11

2,139.3

0.42

1.47

6

8

35,592.9

0.05

0.16

7

13

1,385.7

0.48

1.77

8

17

1,189.3

0.62

2.54

9

13

207.1

0.88

3.26

10

6

2,178.6

0.29

0.75

11

7

157.1

0.54

1.51

12

9

157.1

0.60

1.91

13

11

107.1

0.90

3.10

14

21

2,467.9

0.63

2.78

15

21

5,114.3

0.19

0.83

16

13

653.6

0.34

1.25

17

16

925.0

0.47

1.90

18

13

303.6

0.57

2.10

19

12

717.9

0.49

1.75

20

11

396.4

0.83

2.88

21

16

332.1

0.73

2.91

22

31

1,117.9

0.74

3.65

23

18

796.4

0.68

2.84

24

2

17.9

0.97

0.97

25

13

1,014.3

0.64

2.37

26

22

1,046.4

0.74

3.28

27

24

510.7

0.82

3.75

28

9

96.4

0.88

2.79

29

2

185.7

0.32

0.32

Amer. Maine. Bull. 25: 21-24 (2008)

Introduction to the symposium “Advances in Chiton Research”’^

Douglas J. Eernisse

Department of Biological Science, California State University, Fullerton, California 92834, U.S.A., deernisse@fullerton.edu

The present volume features contributions from partici-
pants of the symposium, “Advances in Chiton Research,” in
Seattle, Washington on 31 July 2006. As the organizer for
this symposium, I was impressed with the willingness of
national and international authorities or students whose
diverse research involves chitons to participate in these
meetings. The symposium was a tremendous success and
compared favorably to four previous meetings of interna-
tional scope that were devoted to chitons: (1) 1987 AMS
symposium on “Biology of the Polyplacophora” in Key West,
Florida (see American Malacological Bulletin 6(1), 1988);
(2) C' International Chiton Symposium, 1991, Adelaide,
Australia (see Journal of the Malacological Society of Australia
13, 1992); (3) the 4* **^ International Workshop on Malacology
devoted to Polyplacophora, 2001, Menfi, Sicily, Italy (see
Bollettino Malacologico Supplemento 5: 1-IV, [2003] 2004);
and (4) 2"'^ International Chiton Symposium, 2003,
Tsukuba, Japan (see Venus 65(1-2), 2006). The participants
of the present symposium (Fig. 1) featured 14 speakers, of
whom half were international, and 10 posters devoted to
chitons. Including all co-authors, there were 39 total con-
tributors to the symposium and about a third of these were
students.

Research on chitons is central to many aspects not only
of malacology but also of zoology, paleontology, evolution-
ary biology, molecular systematics, molecular evolution,
physiology, and ecology (reviewed by Schwabe and Wann-
inger 2006, Eernisse 2007, Todt et al. 2008). The present
collection of articles reflects this integrative role for contem-
porary chiton research. Some of the symposium speakers are
not represented here because they have already published
articles related to their talks in other journals, including
Jean-Bernard Caron (Caron et al. 2006a, 2006b), Ifyan Kelly
(Kelly and Eernisse 2007, 2008, Kelly et al. 2007), and Enrico
Schwabe (Schwabe 2008). Lesley Brooker (“Genes and
biomineralization in the radular teeth of chitons”) and Bruce
Runnegar (“Paleontological evidence for the origin of valves
in polyplacophoran molluscs”) gave insightful presenta-
tions and have contributed as co-authors on articles in this
volume. Bernie Lieb and his coauthors have continued to

elucidate the molecular evolution and systematics of mol-
luscan hemocyanin {e.g., Bergmann et al. 2007), and his
forthcoming collaborative studies on chiton hemocyanin
as a promising new phylogenetic marker are eagerly antici-
pated. Those who have contributed articles for the present
volume still represent an impressive cross-section of the di-
verse, ongoing research on chitons.

Pojeta and DuFoe (this volume) have extended what is
known about the earlier described Ordovician spiny chiton,
Echifwchiton dufoei Pojeta, Eernisse, Hoare, and Flenderson,
2003. This fossil has already figured prominently in the on-
going debate on the disparity of Paleozoic chitons, includ-
ing whether the geologically younger multiplacophorans
diverged from within chitons or from an earlier “stem chi-
ton” ancestor, and whether certain Cambrian “problem-
atica” with disputed affinities, such as Wiwaxia Walcott,
1911, halkierids, and Odontogriphus Conway Morris, 1976
could potentially be close relatives of chitons. The four pre-
viously known E. dufoei specimens were already remarkable
for their articulated preservation but details of the anterior
portion of the animal were still unknown. After additional
monumental collecting effort by co-author Jimmie DuFoe,
resulting in the discovery of even better fossil examples that
were also displayed in a special session at the symposium,
Pojeta and DuFoe are now able to provide details of the
anterior portion. They show that the anterior portion has the
same striking hollow girdle spines found surrounding the
rest of the animal. The authors also reconsider the signifi-
cance of E. dufoei in discussions of molluscan and polypla-
cophoran evolution.

Shaw et al. (this volume) have contributed an extremely
useful description of methods they used to analyze radular
tooth formation and biomineralization, ensuring minimum
deformation of the fragile associated tissue layers involved in
biomineralization processes. Based on Jeremy Shaw’s Ph.D.
research, the authors have employed multiple state-of-the-
art electron microscopy approaches to analyzing biominer-
alization processes in chitons, the results of which are being
published elsewhere (e.g., Shaw et al., 2008). The exquisite
results achieved by these authors refiect not only the con-

* From the symposium “Advances in Chiton Research” presented at the joint meeting of the American Malacological Society and Western

Society of Malacologists, held 29 July to 3 August 2006 in Seattle, Washington.

21

22

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 1. Attending participants in the “Advances in Chiton Research” symposium, 31 luly 2006 in Seattle, Washington. Numbers
correspond to the inset key: I. Stephaney Puchalski; 2. Albert Rodriguez; 3. Jeremy Shaw; 4. John Pojeta, Jr.; 5. Roger Clark; 6. Ryan Kelly;
7. Alejandro Herrera-Moreno; 8. Liliana Betancourt; 9. Donald Cadien; 10. Hiroshi Saito; 1 1. Lesley Brooker; 12. Doug Eernisse; 13. Mike
Vendrasco; 14. John Buckland-Nicks; 15. Jimmie DuFoe; 16. Julia Sigwart; 17. Anel Ramirez Torres; 18. Klaus Streit; 19. Bernie Lieb; 20.
Christine Fernandez; 21. Bruce Runnegar; and 22. Jean-Bernard Caron.

siderable contributions by Shaw but also the high quality of
the electron microscope facility at Murdoch University,
Perth, Western Australia, headed by co-author David Macey,
and notably drawing on the considerable expertise of Shaw’s
mentor, co-author Lesley Brooker. Besides a detailed exami-
nation of potential fixation artifacts, with implications for
interpreting electron micrographs, I was especially im-
pressed by the simple method for cleaning a radula using a
high-pressure jet of water. The clever adaptation of a dis-

posable pipet tip not only allows for avoiding artifacts asso-
ciated with applying alkaline treatment but also results in the
most pristine images of a chiton radula that I have ever seen.

Sigwart (this volume) has extended what has long been
recognized as a phylogenetically informative set of traits, the
position of the gill rows relative to the foot, the nephridio-
pores, and the gonopores, and also characteristics and the
number of the gills within each gill row, to reveal unexpected
variation in the most poorly known of all chiton taxa: the

INTRODUCTION TO THE SYMPOSIUM “ADVANCES IN CHITON RESEARCH

23

mostly deep-water Lepidopleurida {sensii Sirenko, 2006; al-
ternatively as Lepidopleurina). Her present contribution and
her ongoing molecular and morphological investigations are
welcome additions to the scant literature on lepidopleurid
chitons.

Vendrasco et al. (this volume) have investigated the
phylogenetic utility of the aesthete (or esthete) canal mor-
phology in Mopaliidae, testing between different expecta-
tions implied by either its conventional classification or the
conflicting arrangement predicted by molecular results
(Kelly and Eernisse 2008, Eernisse, unpubl. data). This is a
significant change because it implies that Mopaliidae, as re-
cently reformulated (e.g., Eernisse et al. 2007), had a rela-
tively recent origin and a dramatic subsequent diversifica-
tion while largely confined to the northern Pacific Ocean.
Based on the pattern of innervation of esthetes, Vendrasco et
al provide independent corroboration generally agreeing
with the molecular arrangement. Moreover, they have fur-
ther demonstrated the phylogenetic utility of considering
esthete innervation patterns across chitons.

Clark (this volume) has contributed two significant
taxonomic articles here, the first clarifying the taxonomic
status a north/south species pair of common, but confusing,
shallow-water chitons found along western North America.
In agreement with recent morphological and molecular
treatments (Eernisse et al 2007, Kelly and Eernisse 2008), he
has formally revived Mopalia kennetieyi Carpenter, 1864
from obscurity for the northern species (Alaska to northern
California) and has restricted Mopalia ciliata (Sowerby,
1840) to the south, occurring no further north than north-
ern California. Clark’s second contribution introduces two
new species discovered by recent exploration of the deep-
water habitat of the Monterey Sea Canyon and also restores
full generic status for members of Tripoplax Berry, 1919, to
which the new species are assigned.

Puchalski et al. (this volume) have assembled a com-
prehensive database of nominal fossil chiton species, made
available on-line (http://biology.fullerton.edu/deernisse/
fossilchitons/) in association with this publication. They
have used this database to investigate potential sampling
biases that have likely affected perceptions of the chiton
fossil record.

Buckland-Nicks (this volume) provides an overview and
new analysis, reviewing phylogenetic inferences that have
been drawn from comparing chiton eggs, egg hull coverings,
sperm morphology, and egg-sperm interactions during fer-
tilization. As he and his colleagues have continued to dem-
onstrate in publications featuring splendid electron micros-
copy (e.g., Buckland-Nicks and Brothers 2008), attention to
chiton gametes and their interaction is highly informative
for chiton phylogenetics.

Saito et al. (this volume) have provided a thorough

description of three newly discovered chiton species found
near hydrothermal vents and cold seeps around Japan. The
authors also consider whether these and other chitons re-
ported from similar habitats are necessarily associated with
these chemosynthetic environments.

Finally, I have contributed (Eernisse, unpubl. data) a
preliminary phylogenetic analysis of worldwide chitons
based on about 350 partial secjuences of the mitochondrial
16S ribosomal DNA gene. While this is planned to be the
first phase before an eventual multi-locus analysis including
these same taxa, the 16S gene appears to be relatively effec-
tive in both separating chiton species and in providing a
higher-level inference of relationships that agrees well with
recent cladistic morphological analyses. The taxon sampling
in this study is much more extensive than in the only pre-
vious DNA-based analysis of chiton phylogeny (Okusu et al.
2003). This has allowed a more complete inference of rela-
tionships across chitons, with important phylogenetic impli-
cations that mostly agree with, but also challenge, certain
aspects of our best available classifications of living chitons
(e.g., Sirenko 2006).

I thank the 2006 AMS/WSM President, Roland Ander-
son (Seattle Aquarium), for enlisting me as organizer for this
symposium. I am grateful to AMS and WSM for helping
with registration costs for the day of the symposium and
travel-cost assistance.

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Caron, f-B., A. H. Scheltema, C. Schander, and D. Rudkin. 2006a.
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and D. R. Lindberg, eds., Phylogeny and Evolution of Mollusca.
University of California Press, Berkeley, California. Pp. 71-96.

Submitted; 6 May 2008; accepted: 6 May 2008; final

revisions received: 6 May 2008

Amer. Maine. Bull. 25: 25-34 (2008)

New information about Echinochiton dufoeU the Ordovician spiny chiton"^

John Pojeta, Jr.^ and Jimmie DuFoe^

‘ U.S. Geological Survey and Department of Paleobiology, Museum of Natural History, Smithsonian Institution, Washington, D.C.
20560, U.S.A., pojetaj@si.edu

^ 417 Grove Street, Rockton, Illinois 61072, U.S. A., jdufoe2001@yahoo.com

Abstract: Echinochiton dufoei Pojeta et ah, 2003 is now known from seven specimens. The new material shows the anterior end and allows
for a full reconstruction of the animal. The hollow spines are circumsomal; they were flexible and perhaps moveable in rotary anterior-
posterior directions. Possible functions for the hollow spines are discussed. The relationships of E. dufoei to other chitons and to other
molluscs and mollusc-like organisms are presented.

Key words: Mollusca, Polyplacophora, Fossil, Wisconsin

Echinochiton dufoei Pojeta et ah, 2003 was based on four
partial specimens, three of which are parts and counterparts.
None of these preserved the anterior hollow spines or fully
preserved the anterior valves, and none indicated that the
spines were flexible. Three new specimens are now known.
Two consist of parts and counterparts (USNM 533989 and
533990; Figs. 1-2); they preserve the anterior valves and
spines (Pojeta and DuFoe 2006) and show that the hollow
spines were flexible. The third specimen (USNM 533991) is
fragmentary; it preserves three valves in oblique cross section
and partial impressions of a pair of spines and is not figured.
Herein, the 2003 reconstruction of E. dufoei is shown, as is
the new reconstruction.

Repositories: The specimens figured herein are repos-
ited at the Department of Paleobiology, United States Na-
tional Museum of Natural History (USNM), Washington,
D.C. or at the Burpee Museum of Natural History (BMNH),
Rockford, Illinois, U.S.A.

MATERIALS AND METHODS

All seven known “crack-out” specimens of Echinoeijiton
dufoei were collected from a 7-15 cm thick mollusc-rich bed
of dolostone near the top of Bauer’s Quarry west of Beloit,
Wisconsin, in the Forreston Member of the Grand Detour
Formation, Platteville Group, of Middle Ordovician (Turin-
ian; Blackriveran) age (Hoare and Pojeta 2006). Catalani and
Frey ( 1998) noted that the Forreston Member was deposited
in a tropical, shallow-water, carbonate platform environ-
ment. Kolata (1975: 11) wrote that studies of the Platteville
Group and the lower part of the overlying Galena Group

“strongly suggest an open platform, shallow to deep subtidal,
normal marine environment. . . .”

Most fossils in the 7-15 cm bed are found parallel to
bedding and occur in “pockets of accumulation.” Cephalo-
pods and pelecypods are the most abundant and diverse
molluscs in the bed; 44% of the 25 known genera of cepha-
lopods in the Forreston Member occur in this thin bed (Du-
Foe et al. 2006). Pelecypods are well represented by several
species of palaeotaxodonts and pteriomorphians. Gastro-
pods and bellerophonts are less abundant. Rostroconchs are
known from a few specimens of Eopteria Billings, 1865. To
date, chitons are the only group that has been studied in
detail (Pojeta et al. 2003, Hoare and Pojeta 2006); this class
is represented by three species, none of which is abundant.
All of the molluscs are preserved as molds and casts.

The non-molluscan fauna includes strophomenoid
brachiopods, bumastid trilobites, ostracodes, and fragmen-
tary bryozoans and corals usually having well-preserved
exoskeletons.

INTERPRETATION OF THE SHELL BED

The 7-15 cm shell bed is a death assemblage or thana-
tocoenosis. The fossils occur in “pockets of accumulation”
separated by areas with few or no shells. The pockets indicate
an irregular sea bottom with the shells accumulating in the
low areas.

That the shells have been moved to their present loca-
tion is likely because most valves of the pelecypods are dis-
articulated, as are the valves of the few brachiopods. Except
Echinochiton dufoei, the chiton remains are disarticulated

From the symposium “Advances in Chiton Research” presented at the joint meeting of the American Malacological Society and Western
Society of Malacologists, held 29 July to 3 August 2006 in Seattle, Washington.

25

26

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 1. Echinochitoti diifoei topotype (LISNM 533989), anterior end up; scale bar 5 mm applies to all three views. A, Part, showing the
anteriorly rounded head valve and valves 2-7 as internal molds of their ventral sides. Oblic^ue arrow points to external mold of the
morphologically right-lateral head valve spine. Horizontal arrows point to holes left by anterior head valve spines; these spine impressions
are vertical to bedding. B, Counterpart filling of the body space below the valves. Arrows point to the same features seen in A; anterior spine
impressions are filled with sediment. C, Counterpart tilted away from observer in order to show the tail valve (horizontal arrow), which
is preserved at right angles to bedding. Vertical arrow marks the right-lateral sediment-filled anterior spine of the head valve.

plates. Most of the cephalopods are preserved as short frag-
ments of phragmocone attached to short sections of the
living chamber. The gastropods have abraded apertures. The
trilobites are usually disarticulated into their constituent
parts, and the corals and bryozoans are fragmented. That the
assemblage was not moved far is indicated by some of the
pelecypods that remained articulated or are preserved with
the two valves splayed open and lying side by side (butter-
tlyed) parallel to bedding and with the umbos touching.

Echiiiodiiton diifoei is an exception to the general pres-
ervation of the other fauna, in that all known specimens are
partially articulated although disarticulated spines are
known.

THE NEW SPECIMENS

As in previous specimens, the new material is preserved
as complex molds. In specimen USNM 533989 (Figs. lA-C)
the counterpart (Figs. IB-C) shows impressions, or seciimen-

tary fillings, of five of the hollow spines on the right side,
including a right-lateral spine on the head valve, and two
spines at the anterior end of the head valve. The sedimentary
filling of the body space does not show the valves well. The
part of the specimen (Fig. lA) shows the undersides of the
anteriorly rounded head valve and valves 2-7 parallel to bed-
ding. The tail valve is at right angles to bedding and is
preserved on the counterpart (Figs. IB-C). The external
mold of the right-lateral spine on the head valve is preserved
as an impression. Anterior to the head valve are the impres-
sions and fillings of two spines (Figs. lA-B).

The length of the eight valves is about 25 mm, the width
of a single intermediate valve is 6 mm, and the length of the
one complete and curved spine preserved parallel to bedding
on the right side of valve 6 (Fig. lA) of the part is 7.5 mm.

It is noteworthy that the impressions of the spines, or
their sedimentary fillings, of this specimen are preserved at
various angles to bedding, ranging from parallel to right
angles (Figs. lA-B), thus indicating that the spines were

ORDOVICIAN SPINY CHITON

27

I A). The scutes are erect structures
that occur in right and left rows be-
tween and parallel to the valves and
between the valves and the hollow
spines (Figs. 3A, 3B). The sediment-
filled hollow spines are best preserved
on the holotype part (BMNH

1996.045.01) and counterpart (BMNH

1996.045.02) and are parallel to bed-
ding (Figs. 3A, 4).

See Figure 5 for the new recon-
struction and the 2003 reconstruction.

Figure 2. Echinochiton dufoei topotype (USNM 533990), anterior end up; scale bar 5 mm
applies to both views. A, Part, showing the anteriorly rounded head valve and valves 2 and
3 as internal molds of their ventral sides. The lateral head valve spines (vertical arrows) are
preserved vertical to bedding as are the spines on the morphologically right side. The spines
on the morphologically left side of valves 2 and 3 are preserved parallel to bedding. The
anterior head valve spines are preserved rotated counterclockwise to the morphologically left
side and have most of their lengths are exposed (horizontal arrows). B, Counterpart filling of
the body space below the valves showing the anteriorly rounded head valve. Arrows mark the
same features as in A above. Left-lateral external molds of spines partially filled with
sediment.

flexible. All previously known specimens of Echinochitoti du-
foei have the hollow spines preserved parallel to bedding
(Pojeta et al 2003, figs. 1-6). As used herein, the word flex-
ible means that at least after death the spines were capable of
being bent. Specimen USNM 533990 (Figs. 2A-B), part and
counterpart, preserves the anteriorly rounded head valve,
valves 2-3 on the part (Fig. 2A), and the head valve and
valves 2-4 on the counterpart (Fig. 2B). On the left side of
the counterpart, the hollow spines attached to valves 2-4 are
preserved parallel to bedding and show some of the sedi-
ment fillings of the bases of the spines. On the right side, the
sediment fillings of the spines of valves 2-4 are preserved at
right angles to bedding (Fig. 2B). The lateral spines of the
head valve are preserved at right angles to bedding. The
anterior spines of the head valve have been rotated counter-
clockwise to the left (Fig. 2B); they are at a low angle to
bedding and much of their lengths are exposed. This speci-
men also shows that the spines were flexible, because they
are preserved at various angles to bedding.

The length of the four preserved valves is 14 mm (valve
4 is incomplete), the width of valve 2 is 6 mm, and the length
of the spine on the left side of valve 4 (Fig. 2B) is about 11
mm (attachment to valve 4 not visible).

Neither of the new specimens described here show well-
preserved scutes or the slots made by scutes in internal
molds; a few slots are on the right side (left side in view. Fig.

MOVEABLE SPINES?

So far as is known, Echinochiton
dufoei is unique among chitons in its
possession of circumsomal hollow
spines that are as long, or longer, than
the valves are wide (Figs. 1-5). Our
search of the literature yielded no
other chitons with the large, hollow
spines of E. dufoei. Eernisse (e-mail
comm., September 2006), comment-
ing on E. dufoei, noted: “There are no
other hollow spines documented [in chitons] that I know of.
We (with Pat Reynolds) cite studies in my chapter on chi-
tons: Eernisse, D. J., and P. D. Reynolds, 1994.”

Scheltema et al. (1994: 20) noted the existence of solid
and hollow spicules in the epidermis of neomenioid aplaco-
phorans. However, these are small structures, about 20-200
microns long “and are secreted extracellularly within an in-
vagination of a single cell.” Thus, it is highly unlikely that the
hollow spicules of aplacophorans are homologous with the
hollow spines of Echinochiton dufoei.

The new specimens show that the spines were flexible.
However, were they moveable in life, or is the flexibility a
post-mortem effect? Examine the color photographs of
the holotype part and counterpart before the specimen was
prepared and before it was whitened with ammonium chlo-
ride sublimate (Fig. 6); compare this with the prepared
specimen (Figs. 3A and 4). It is noteworthy that the impres-
sions of the spines are much darker than the surrounding
rock and the valves; this is also the case with some of the
spines (Fig. 2) and the specimens in Pojeta et al. (2003: figs.
5.2, 6.3, 6.4).

This darker color suggests that the spines contained
more organic matter than did the valves. In some Or-
dovician mytiliform and modioliform pelecypods, the inter-
nal molds and the ligament area are covered with a black
film (Pojeta 1962: 175; Pojeta 1971: pi. 15, figs. 5, 6). This

28

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 3. Echinochiton diifoei holotype (BMNH 1996.045.01 ), anterior end up; scale bar 10 mm applies to both views. A, Part, showing valves
3-8 as internal molds of their ventral sides, mucro of tail valve, external mold impressions of spines parallel to bedding, posterior spines
of tail valve, partial sediment fillings of hollow spines, and slots paralleling the right and left lateral side of the valves (solid black arrow).
Open black arrow points to a remnant impression of the morphologically right side spine of valve 3. White arrow at the tip of the external
mold of the morphologically right side spine of valve 7 shows that the sediment filling of the spines went to their distal ends; thus, the spines
were hollow to their tips. B, Latex cast of A, white arrow points to one of the scutes on the right side. Open black arrow at top right marks
the same structure as in A.

presumed organic film does not extend into the surrounding
sediment; it is limited to the internal mold. The ligament
and the thick periostracum of extant mytiliform and mo-
dioliform pelecypiods contains more organic material than
in other areas of the shell and in other groups of pelecy-
pods. This possible larger organic component of the sp>ines
in Echinochiton diifoei may help explain the flexibility of the
spines.

The part of the holotype (Fig. 3A) preserves external
impressions of the hollow spines, the underside of the valves,
and rows of slots between the valves and the spines. The
latex positive (Fig. 3B) shows that the slots are molds of right
and left rows of raised triangular scutes between the valves
and the spines. The scutes can occur between the spines or
between the spines and the valves.

The spines show growth lines; thus, it seems likely that

mantle extended into them at least to the bases of the spines
(Fig. 7A). If this tissue had sufficient muscle fibers, it
could have moved the spines with the scute acting as a
fulcrum against which to raise and lower them cantilever
style or to move the spines fore and aft in a rotary fashion.
The scutes bend toward the valves and together with the
spine could form a ball-and-socket joint, analogous to what
is seen in regular echinoids in which the solid spine is
mounted on a tubercle (Hyman 1955, fig. 187A). In echi-
noids, the outer cylinder of muscle fibers, by local contrac-
tion, causes the spine to point in the direction of the stimu-
lus (Hyman 1955: 438).

In both our old and new reconstructions (Fig. 5), we
show the spines as being embedded in the mantle girdle. It
is unlikely that they would be below the girdle, because this
would impede the ability of the animal to attach to the

ORDOVICIAN SPINY CHITON

29

Figure 4. Echiuochitoii dufoei holotype (BMNH 1996.045.02), an-
terior end up; scale bar 10 mm. Counterpart filling ol the body
space below the valves of Fig. 3A. White arrow on the left lateral
spine of valve 7 shows that the lumen of the spine decreased in size
distally because the sediment filling decreases in size.

substrate. If the spines were embedded in the girdle, vertical
movements would probably be limited and movement
would be mostly in rotary anterior and posterior directions.
Thus, the spines preserved at right angles to bedding would
be largely a post-mortem effect. It is unlikely the spines were

Figure 5. Echiuochiton dufoei. A, New reconstruction incorporating
the new information from specimens seen in Figs. 1 and 2. Anterior
end up. B, 2003 (Pojeta et al.) reconstruction showing the lack of
information about the anterior end of the species. The lateral spines
on the head valve were postulated, but not seen, in 2003. Size about
2.0X.

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AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

above the girdle, because there seems to be no way that they
could have attached to the shell in this position and have
mantle extend into them. The presence of spicules and scales
in the girdle is indicated by small horizontal markings at
nearly right angles to the lateral edges of the valves (Pojeta et
al. 2003; figs. 6.1 and 7.2; Figs. 7B, 8 herein).

WHY LARGE HOLLOW SPINES?

What function could the large hollow spines serve?
Various reasons can be postulated although none of the
hypotheses stand out as the most likely reason for such
spines:

( 1 ) Somehow the spines were used for protection from
predation. However, all known specimens of Echinochiton
dufoei are small, in the 25-40 mm range. The major preda-
tors in Ordovician time were shelled cephalopods, many of
which were considerably larger than E. dufoei. The cephalo-
pods found in the same bed as E. dufoei range in size from
a few centimeters to over three meters, and E. dufoei would
not even be a small snack for most of the cephalopods except
young juveniles. In addition, hollow spines, even if made
turgid with fluid, would provide little protection from pre-
dation. Thus, it seems unlikely that the spines were used for
protection. Also, as suggested above, because the spines were
probably embedded in the girdle, their vertical movement
would be limited and most movement would be in oar-like
anterior and posterior directions.

K. M. Brown (Louisiana State University) suggested that
“the horizontal spines would make it harder to pry the ani-
mal from the substrate” (pers. comm., June 2007)

Various non-chiton molluscs have spines, including
spondylid and some venerid pelecypods, some muricid gas-
tropods, and a few shelled cephalopods. These spines may or
may not have a lumen, or a longitudinal groove. All are
strongly calcareous and range in shape from sharply pointed
to spatulate. Generally these are regarded as protective or
supportive in function.

The spines of productoid brachiopods are hollow, hard,
and not flexible when fully formed. Muir-Wood and Cooper
(1960: 16) regarded the spines as: “Forming a prominent
part of the ornament and occur to some extent in every
productoid species. The spines were in part protective, but

Figure 6. Echinochiton dufoei, anterior end up; scale bar 10 mm
applies to both images. A, B, Color photographs of part and coun-
terpart of holotype (BMNH 1996.045) as the specimen was found
in the field and before it was prepared. Compare to Fig. 3A and 4.
Note that many of the spine impressions are much darker than the
impressions of the valves and the surrounding sediment.

ORDOVICIAN SPINY CHITON

31

cause of the row of eight bilaterally
symmetrical valves (plates), the mucro
on the tail valve, and the likely pres-
ence of a mantle girdle interpreted
from small impressions lateral to the
valves indicating the presence of spic-
ules and scales. It differs from other
polyplacophorans in the presence of
circumsomal, large, hollow spines, and
the right and left rows of scutes paral-
leling the outer edges of the valves.
Thus, it is placed in the separate family
Echinochitonidae.

Within the Polyplacophora, Echi-
tiochiton diifoei is in the subclass Pale-
oloricata based on the upright valves
with large apical areas (Fig. 8); none of
the known specimens show sutural
laminae and they lack insertion plates.
It is treated as a member of the order
Chelodida because the intermediate
valves are not clearly differentiated into lateral and central
areas (Figs. lA, 2B)

When commenting on Echinochiton dufoei, Sirenko
(2006: 33) misunderstood Pojeta et al. (2003). His thought
that the specimens were external molds is only partially cor-
rect. The specimens are very complex molds; the only part of
the molds that are clearly external are the impressions of the
outside of the spines when piarallel to bedding (Fig. 3A). The
sedimentary fillings of the spines are internal molds (Fig. 4).
The specimens are internal molds of the ventral sides of the
valves (Figs. 1-4).

To date, most of the external molds of the dorsal side of
the valves have been found in a specimen where they cannot
be seen (Fig. 8). An exception to this is the partial impres-
sion of the external surface of the head valve (Fig. lA); it
does not preserve the external features.

Sirenko’s notation that EcJunocJiiton dnfoei has small
apical areas is incorrect as shown herein by the specimen
(Fig. 8) in which the valves are seen in lateral view. The
valves are nearly erect and have large apical areas. The spaces
between the internal and external molds represent a mini-
mum thickness for the valves. In this specimen, the external
molds of the valves could not be exposed.

The valves that are preserved parallel to bedding so that
they cannot be seen in lateral view may have been distorted
by compaction (compare the shapes of the valves in Figs. 1 A,
2A, and 3A). Also when comparing the shapes of the coun-
terparts (Figs. 2B, 3B, 4), there is variation in the shapes of
the valves.

In Echitwchitoii dnfoei, Sirenko suggested the presence
Echinochiton dnfoei is a polyplacophoran mollusc be- of incisura; it is not clear to us what the term incisura means.

Figure 7. Echinochiton dnfoei paratype (USNM 517481). A, Anterior to right; scale bar 10
mm. Enlargement of three lateral spine external molds showing growth lines. B, Anterior
down; scale bar 10 mm. Underside of first three valves seen on the right side of Fig. 8,
showing growth rugae. Lateral to the upper two valves, on the left side ot the image, are
marginal markings interpreted to be the impressions of scales and spicules. Black tailless
arrow marks the same position as the black tailless arrow in Fig. 8.

they also played an important role in the attachment and
support of the shell, and some may have functioned as
strainers.” Grant (1966) showed the support function ot the
multitudinous spines of the productoid Waagenoconcha abi-
chi in soft sediments.

(2) David Pawson, USNM (pers. comm., September
2006) has observed that, among other functions, some regu-
lar echinoids use their spines to define their living space and
form a checkerboard-like pattern on the sea floor. This
seems unlikely for Echinochiton dnfoei because it is such a
rare element in the fauna of the 7-15 cm thick bed; in 15
years of collecting and breaking literally tons of rock from
the 7-15 cm thick bed, only seven specimens have been
found. Of course, E. dnfoei may be found to be abundant
elsewhere. However, the Ordovician fossils of Wisconsin and
Illinois have been studied for at least 140 years, and the first
mention of Echinochiton in the literature is Pojeta et al. (2003).

(3) The function of the spines is as stabilizers —
something akin to outriggers when the animal moved. Al-
ternatively, the spines may have helped to maintain the chi-
ton’s position on a hard substrate in strong waves and
currents. However, extant chitons hug the substrate tena-
ciously using the foot and mantle girdle and are difficult to
dislodge without using an instrument having a blade.

(4) The spines were somehow used as accessory organs
of locomotion, particularly if their motion was largely in the
rotary anterior-posterior directions.

TAXONOMIC PLACEMENT

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AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 8. Echinochiton diifoei paratype (USNM 517481), anterior
end to right; scale bar 10 mm. Lateral view of four valves showing
their nearly erect posture and the filling of the body space below the
valves. Downward facing straight-tailed barbed arrow points to two
sediment fillings of the valve suggesting the presence of two open-
ings into the valve as in Matthevia variabilis Walcott, 1885. Down-
ward facing straight-tailed unbarbed arrow points to markings lat-
eral to the body that are interpreted as having been made by
spicules in the girdle. Wavy arrow at left end points to a piece of a
fifth valve that is only partially preserved. Solid triangular tailless
arrow is at the same position as in Fig. 7B.

Hyman (1967: 74) used the term incisures as follows: “The
insertion plates are commonly cut into teeth by incisures . . .
These incisures are continued ... as slit grooves known as
slit rays.” Echinochiton dufoei does not show insertion plates
and, thus, lacks slit rays separating the insertion teeth from
the rest of the shell (Fig. lA).

PHYLOGENETIC RELATIONSHIPS

Echinochiton shows similarities to mattheviids which are
the oldest known chitons. In Matthevia Walcott, 1885, the
valves are nearly upright and they have large apical areas
(Runnegar et al. 1979, Vendrasco et al. 2004) as does Echi-
nochiton (Fig. 8). The suggestion of a relationship between
Echinochiton diifoei and Matthevia is reinforced by a plate
of E. dufoei which shows the filling of two holes in the
valve (Fig. 8) as is the case in Matthevia variabilis Walcott,
1885, the type species of Matthevia. Hoare (2000) placed
mattheviids at the base of his phylogenetic scheme of
polyplacophorans.

Caron et al. (2006) discussed and redefined the large,
Middle Cambrian, Burgess Shale species Odontogriphus
otnalus Conway Morris, 1976. This animal approximates a
shell-less polyplacophoran. Caron et al. (2006) noted that
the species is up to 125 mm long, is flattened dorso-
ventrally, and has a dorsal stiffened cuticle, a radula, a
straight gut, and simple gills that are present in a groove
running laterally and posteriorly around a muscular foot.
Stratigraphically, Odontogriphus Conway Morris is older
than the first known chitons which occur in Upper Cam-
brian rocks (Vendrasco and Runnegar 2004). At the very
least, an animal closely resembling Odonogriphus would be a
likely stem group for chitons.

Echinochiton increases the known disparity of polypla-
cophorans. Vendrasco et al. (2004) considered Paleozoic chi-
ton disparity to be even greater than that suggested by Echi-
nochiton, after the discovery of a nearly complete Early
Mississippian specimen of the taxon Midtiplacophora Hoare
and Mapes, 1995.

The new species Polysacos vickersianum Vendrasco et al,
2004, is about 25 mm long. The shell has three longitudinal
columns of valves plus head and tail valves, and it is sur-
rounded by many elongate hollow spines. The authors noted
the similarity of this arrangement to Echinochiton dufoei in
which the skeleton has a central column of valves, two flank-
ing columns of small dorsally projecting scutes, and is sur-
rounded by hollow spines that grew by accretion. In both
groups the anterior and posterior valves are morphologically
distinct from the intermediate valves. Vendrasco et al. (2004)
regarded the skeleton of E. dufoei as intermediate in form
between the skeletons of multiplacophorans and typical chi-

ORDOVICIAN SPINY CHITON

33

tons. As in “crown group chitons”, multiplacophorans have
pores in valve surfaces and possess the articulamentum (in-
ner shell layer) that projects from the growing margin of the
shell. Echinochiton and other stem group chitons are not
known to have the articulamentum.

As noted by Vendrasco et al. (2004), there are significant
differences between chitons and multiplacophorans such as
Polysacos. The most striking difference is that in Polysacos
there is a seven-fold, rather than an eight-fold iteration of
the major skeletal elements.

The cladistic analysis performed by Vendrasco et al. (2004:
288) “Strongly supports the placement of multiplacophorans
with the total group Polyplacophora.” They treat the Mul-
tiplacophora as an order within the class Polyplacophora.

Another recently-discovered Paleozoic multi-plated
species is Acaenoplax hayae Sutton et ah, 2001. Sutton et al
(2004) monographed the species. The species is known from
Middle Silurian age rocks. This species is vermiform, up to
40 mm long, and has one ventral and seven dorsal valves.
The dorsal valves are not articulated and most are separated
by a variable number of dorsal ridges. Most of the ridges
bear elongate, thin, rigid, and pointed spines, and although
it is not noted if the spines were hollow, this seems unlikely.
Sutton et al. (2001a) regarded Acaenoplax Sutton et al., 2001
as a mollusc, perhaps allied to the Aplacophora and showing
some polyplacophoran affinities.

Subsequent to Sutton et al. (2001a), there was a debate
about the treatment of Acaenoplax as a mollusc. Steiner and
Salvini-Plawen (2001) argued that Acaenoplax hayae was
best considered to be allied to polychaete annelids; Sutton et
al. (2001b, 2004) defended the molluscan affinities.

ACKNOWLEDGMENTS

We thank John Catalan!, Woodbridge, Illinois, U.S.A.,
for the gift of specimen USNM 533991 for use in this study.
Carl Bauer kindly allowed us to collect in his quarry; access
to his quarry is highly restricted and requires Mr. Bauer’s
permission. M. D. Henderson (BMNH) kindly provided the
color photographs in Fig. 6. We give many thanks to: Mary
Parrish (USNM) who created the reconstructions in Fig. 5;
JoAnn Sanner (USNM) who provided digital images of the
specimens seen in several of the figures so that the images fit
the format of the Bulletin; and Scott Whittaker (USNM)
who took optical digital images of the specimens seen in
Figs. 1 and 2. We thank Robert Purdy (USNM), David Paw-
son (USNM), A. H. Scheltema (Woods Hole Oceanographic
Institution), and D. J. Eernisse (California State University,
Fullerton) for reviewing the manuscript. David Pawson pro-
vided helpful discussions in our understanding of how regu-
lar echinoids function and the construction of their spines.

JP thanks the late Ellis Yochelson for his many helpful dis-
cussions in interpreting Echinochiton diifoei.

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Pojeta, )., Jr., D. J. Eernisse, R. D. Hoare, and M. D. Henderson.
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Runnegar, B., ]. Pojeta, Ir., M. E. Taylor, and D. Collins. 1979. New
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25 • 1/2 • 2008

and Queensland: Evidence for the early history of polyplaco-
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Polyplacophora). Venus 65: 27-50.

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Sutton, M. D., D. E. G. Briggs, D. I. Siveter, and D. 1. Siveter. 2001a.
An exceptionally preseiwed vermiform mollusc from the Si-
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Sutton, M. D., D. E. G. Briggs, D. 1. Siveter, and D. I. Siveter. 2001b.
Invertebrate evolution (communications arising): Acaenoplax-
polychaete or mollusc? Nature 414: 602.

Sutton, M. D., D. E. G Briggs, D. ]. Siveter, and D. J. Siveter. 2004.
Computer reconstruction and analysis of the vermiform mol-
lusc Acaenoplax hayae from the Herefordshire lagerstatte (Si-
lurian, England), and implications for molluscan phylogeny.
Palaeontology 47: 293-318.

Vendrasco, M. I. and B. Runnegar. 2004. Late Gambrian and Early
Ordovician stem group chitons (Mollusca: Polyplacophora)
from Utah and Missouri. Journal of Paleontology 78: 675-689.

Vendrasco, M. E, T. E. Wood, and B. Runnegar. 2004. Articulated
Paleozoic fossil with 17 plates greatly expands disparity of
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Submitted: 6 November 2006; accepted: 6 August 2007;

final revisions received: 13 November 2007

Amer. Malac. Bull. 25: 35-41 (2008)

Methods of sample preparation of radula epithelial tissue in chitons (Mollusca:
Polyplacophora)

Jeremy A. Shaw\ David J. Macey\ Peta L. Clode^, Lesley R. Brooker^, Richard I. Webb‘S,
Edward J. Stockdale^, and Rachel M. Binks*

* School of Biological Sciences and Biotechnology, Murdoch University, Murdoch, Western Australia, 6150,

Australia, jererny.shaw@uwa.edu.au

^ Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, Crawley, Western Australia, 6009,
Australia.

^ Faculty of Science, Health and Education, University of the Sunshine Coast, Maroochydore, District of Cairns, Queensland, 4558,
Australia.

^ Centre for Microscopy and Microanalysis, Department for Microbiology and Parasitology, University of Queensland, St. Lucia,
Queensland, 4072, Australia.

Abstract: A glutaraldehyde fixative developed for preserving the radula superior epithelium of the adult chiton Acanthopleura hirtosa
(Blainville, 1825), was used in conjunction with conventional and microwave-assisted sample processing to produce high c]uality tissue
preservation for light and electron microscopy. In addition, high-pressure freezing (HPF) and cryo-substitution were used to fix the radula
tissue of juvenile specimens. Microwave-assisted fixation was preferred to conventional bench-top techniques due to the superior preser-
vation of fine cell structure together with reduced processing times and chemical exposure. Although restricted to very small (<200 pm)
samples, the quality of juvenile radulae processed by HPF was excellent. The improvements in tissue preservation using microwave and
cryo-preservation techniques are therefore critical for obtaining accurate ultrastructural information on the radula in marine molluscs. In
particular, these findings highlight additional processing options available for the study of cellular structures in biomineralizing tissues.

Key words: microwave, high-pressure freezing, chemical fixation, cryo-fixation, biomineralization

The radula has been the focus of numerous studies over
many decades, with its intricate and varied design used to
elucidate aspects of molluscan ecology, biology, and tax-
onomy (Fretter and Graham 1962, Runham 1963, Steneck
and Watling 1982, Padilla 1985, Scheltema 1988, Salvini-
Plawen 1990). In addition, the radulae of polyplacophoran
and patellid gastropods have received particular attention as
a result of their unique ability to harden their teeth with iron
and other biominerals (Mann et al. 1986, Lowenstam and
Weiner 1989, Webb et al. 1989).

The chiton radula represents an excellent example of
matrix-mediated biomineralization, where minerals are
formed in a highly organized manner within the framework
of an organic matrix (Simkiss and Wilbur 1989, Watabe
1990, Mann 2001, Weiner and Addadi 2002). While consid-
erable progress has been made in elucidating the general
structural organization of minerals deposited within the
tooth matrix and the sequence in which they are deposited
(Lowenstam 1962, Kim et al. 1989, Macey et al. 1994,
Brooker et al. 2006), the mechanisms involved in cellular

transport of ions to the tooth cusps are poorly understood.
The elemental precursors for biomineralization are thought
to be delivered to the teeth by the overlying, superior epi-
thelial tissue, which surrounds the cusps during all stages of
development (Nesson and Lowenstam 1985) (Fig. 1)- The
superior epithelium and teeth are encapsulated within the
radular sheath, the inferior epithelium, and the radula mem-
brane with the whole structure resembling a tube open only
along the dorsal surface (Fig. 1). Histological investigation of
epithelial tissue is difficult due to the complex composition
and structure of the radula, which contains both hard min-
eralized structures and cartilaginous membranes in close
proximity to cellular material.

Chemical fixatives such as glutaraldehyde buffered in
filtered seawater are commonly used for preserving marine
organisms, where the osmotic pressure of the solution acts to
mimic that of the animal, thereby reducing swelling or
shrinkage of the tissues (Dykstra and Reuss 2003). However,
fixation often gives rise to variations in the ultrastructural
morphology of organelles (Hayat 2000), and it is therefore

From the symposium “Advances in Chiton Research” presented at the joint meeting of the American Malacological Society and Western
Society of Malacologists, held 29 luly to 3 August 2006 in Seattle, Washington.

35

36

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 1. Radula apparatus of the chiton Acanthopleura hirtosa (adult) in (A) transverse
section and (B) longitudinal section exhibiting various hard and soft tissue components.
Abbreviations: se, superior epithelium; ie, inferior epithelium; tc, tooth cusp; ts, tooth stylus;
rm, radula membrane; rs, radula sheath. Scale bars = 200 pm.

preferable to utilize several techniques
to acquire comparative information.

Microwave-assisted fixation and cryo-
preservation techniques (McDonald et
al. 2007, Webster 2007), now common
in laboratories, provide two additional
means of obtaining such comparative
information. The main advantage of
microwave protocols is a dramatic re-
duction in sample processing times
(<4 hours), while at least maintaining,
if not improving, fixation quality over
conventional methods (Giberson and
Demaree 1999, Laboux et ah 2004).

Excellent tissue preservation can also
be attained using cryo-preservation
techniques, which achieve vitreous ice
formation in samples and thereby pre-
vent ice crystal damage. However, due
to difficulties associated with heat dissipation, only very
small samples (<200 pm) can be frozen successfully (Wilson
et al. 1998, Sawaguchi et al. 2005).

To improve our current techniques and better under-
stand the detailed cellular structure of the superior epithe-
lium in chiton and limpet teeth, we investigated alternative
fixation methods. Determining the precise structure of these
cells will assist in elucidating their function and the mecha-
nisms involved in the transport of ions into the teeth, a
fundamental obstacle to our wider understanding of the ini-
tial phase of biomineralization. The aim of this study is to
compare three fixation methodologies (conventional chemi-
cal, microwave-assisted, and low temperature), regarding
preservation of the superior epithelial tissues of the chiton
Acanthopleura hirtosa (Blainville, 1825) for both light and
electron microscopy.

MATERIALS AND METHODS

Conventional and microwave-assisted chemical fixation
and cryo-fixation techniques were utilized for the prepara-
tion of epithelial tissues for light microscopy (LM) or trans-
mission electron microscopy (TEM). Adult and juvenile
specimens of the chiton Acanthopleura hirtosa (mean animal
length ~4 cm and -0.8 cm, respectively) were collected from
intertidal limestone at Woodman Point within the Perth
metropolitan area of Western Australia (32°08'S, 115°44'E).
Incisions were made along both pallial grooves from the anus
towards the head, thereby freeing the foot, visceral mass,
and buccal mass as a single entity from the shell plates and
girdle. The visceral mass was then carefully removed to expose
the radula sac. Dissections were performed as quickly as pos-
sible to reduce the deterioration of radula epithelial tissue.

Table 1. Conventional and microwave-assisted processing schedules for the radula epithelial tissues of adult Acanthopleura hirtosa.

Step

Medium

Concentration (%)

Conventional

times

Microwave times

Microwave
wattage (W)

Fixation

glutaraldehyde

2.5*

24

2x(2°U2°fV2°")(min)(v)

80

(buffer A)

Rinse

buffer A

15 min x4

40 sec (v)

250

Post-fixation

OSO4 (buffer B)

U

2h

2x(2“72°^V2°")(min)(v)

80

Rinse

buffer B

15 min x4

40 sec (v)

250

Dehydration

acetone

10, 30, 50*, 75*, 90*, 100*

15 min x2 each

40 sec each (100 x2)

250

Infiltration

Spurr’s resin

5, 10, 20, 40, 50, 60, 75, 80, 90, 100*

8-12 h each

3 min each (100 x2) (v)

250

Polymerization

Spurr’s resin

100*

conventional oven 60 °C overnight

Note: Concentrations in bold represent the microwave schedule, concentrations not in bold represent the conventional schedule, and * represents
concentrations used in both schedules.

(v) = steps undertaken in vacuum

denotes magnetron (irradiation) cycle

SAMPLE PREPARATION OP RADULA EPITHELIUM

37

Table 2. Conventional and microwave-assisted processing schedules for the radula epithelial tissues of juvenile Acauthopleura hirtosa.

Step

Medium

Concentration (%)

Conventional

times

Microwave times

Microwave
wattage (W)

Fixation

glutaraldehyde
(buffer A)

2.5*

24 h

2x{2""/2""/2'’”)
(min) (v)

80

Rinse

buffer A

10 min x4

40 sec (v)

250

Post-fixation

OSO4 (buffer B)

1*

1 h

2x(2"'V2'’'V2"")

(min) (v)

80

Rinse

buffer B

10 min x4

40 sec (v)

250

Dehydration

ethanol/acetone*'*’

50*, 75*, 90*, 100*, 100'‘”, 100‘“’

10 min each (100 x2)

40 sec each

250

Infiltration

Procure Araldite

5, 20, 50, 60, 75, 80, 90, 100*

4-8 h each

3 min each
(100 x2) (v)

250

Polymerization

Procure Araldite

100*

conventional oven 60 °C

overnight

Note: Concentrations in bold represent the microwave shecule, concentrations not in bold represent the conventional schedule, and * represents
concentrations used in both schedules.

(v) = steps undertaken in vacuum
on/off denotes magnetron (irradiation) cycle
= acetone

Figure 2. Light micrograph of a longitudinal section through a
major lateral tooth from adult Acanthopleura hirtosa at row 6 pre-
pared using microwave-assisted protocols. Despite being situated
deep within the tooth stylus (ts), the tissues of the stylus canal (sc)
are well preserved, se, superior epithelium; tc, tooth cusp. Scale
bar = 50 pm.

Preparation of radulae from adult animals

For samples processed using either conventional or mi-
crowave-assisted methods, the dissected tissue mass was im-
mediately immersed in a fixative comprised of 2.5% glutar-
aldehyde buffered in 0.1 M phosphate, with a pH of 7.2 and
an osmolarity of 900 mmol kg"' adjusted using sucrose
(buffer A). The buccal mass and radula were separated from
the remainder of the animal, and the radula was either left
whole or cut transversely into three or four segments (the
buccal mass was discarded). The tissues were then processed
by either conventional bench-top methods or accelerated
microwave-assisted protocols using a Pelco Biowave® fitted
with a cold spot and vacuum chamber, according to the
specific schedules detailed in Table 1. This included fixation
with glutaraldehyde in buffer A, rinsing in buffer A, post
fixation in 1% osmium tetroxide (OSO4) in 0.05 M phos-
phate buffered saline (buffer B) at 4 °C, and a final rinse in
buffer B, prior to dehydration through a graded series of
acetones, then infiltration and embedding in Spurr’s resin.
Preparation of radula tissue by high-pressure freezing was
not possible for adults due to limitations in sample size,
which was again restricted to -200 pm.

Following polymerization, resin blocks were sectioned
for observation at both the LM and TEM level. For LM, 1
pm-thick sections were mounted on glass slides and stained
with ac]ueous 1% Methylene Blue and 1% Azur II (20 sec)
prior to imaging on an Olympus BX51 optical microscope
fitted with an Olympus DP70 digital camera. For TEM, ~60
nm-thick sections were mounted on copper grids and
double stained with uranyl nitrate (single crystal in one drop
of 50% methanol ) ( 10 min) followed by Sato’s modified lead
citrate (10 min) (Hanaichi et al. 1986) prior to imaging on
a lEOL 2000 TEM at 80 kV.

38

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 3. Major lateral tooth rows 12-14 from radulae of adult Acanthopleiira hirtosa pre-
pared using (A) conventional and (B) microwave technic]ues. Note the separation (arrow) of
the superior epithelium from the anterior face (af) of the tooth cusps (tc) in conventionally
fixed material and the poor infiltration of resin into the tooth stylus (ts) and cusp in
microwave prepared material. Scale bars = 200 pm.

Preparation of radulae from juvenile animals

Juvenile specimens of Acanthopleiira hirtosa were pro-
cessed similarly using conventional and microwave-assisted
methods; however, schedules were adjusted in order to ac-
count for the reduction in tissue size, and samples were
embedded in Procure Araldite (formerly Epon Araldite)
(Table 2). Eor cryo-fixation of juvenile radulae, the imma-
ture portion of each radula was removed by cutting trans-
versely approx. 200 pm from the posterior end. Each imma-
ture portion was then placed into a 200 pm membrane filled
with 20% bovine serum albumin in artificial seawater. These
membrane-mounted samples were rapidly frozen in a high-
pressure freezer (Leica EMPACT 2) prior to cryo-
substitution in acetone containing 2% OSO4 at -85 °C for 52
h. Samples were then progressively warmed from -85 °C to
20 °C over 13 h in a Leica automatic freeze substitution unit
prior to being washed in acetone and infiltrated and embed-
ded in Araldite resin. Cryo-prepared sample blocks were
polymerized underwater using a Pelco Biowave® microwave
at 650 W for 90 min. Conventional, microwave-assisted and
cryo-prepared sections were imaged unstained on a JEOL
2100 TEM at 120 kV, using a Gatan Orius SC 1000 digital
camera.

RESULTS AND DISCUSSION

Preservation of adult radulae

While glutaraldehyde fixation of radula epithelium was
satisfactory at the LM level when conventional bench-top

methods were used, microwave-
assisted protocols dramatically re-
duced sample processing times from
six days to one hour (Table 1) and re-
sulted in superior ultrastructural pres-
ervation at the TEM level. In addition,
microwave-assisted protocols in-
creased fixative penetration into the
tooth, improving, for example, tissue
preservation within the stylus canal
(fig. 2), which fixed poorly by conven-
tional methods.

The endothelial radula sheath
layer, recognizable by the presence of
ciliated endothelial cells distributed
over the entire sheath’s surface, re-
mained intact when processed by mi-
crowave methods (Fig. 1), in contrast
to conventional methods where it was
often disrupted. It is likely that the
shorter sample processing and han-
dling times with microwave fixation
reduce the likelihood of damage to this delicate membrane.
Both techniques preserved prominent vesicles that line the
anterior and posterior surfaces of the tooth cusps (Fig. 3).
We have found that these vesicles can be either abundant or
virtually absent at the same stage of tooth development in
different animals. It is currently not known whether these
vesicles are natural features of the adult epithelium or arti-
facts resulting from the fixation process.

Conventional fixation often resulted in the separation of
the superior epithelium from the hard tooth cusps, while in
microwave -fixed material this artifact was rarely observed
(Fig. 3). Despite a number of attempts to improve resin
penetration into the base material using both conventional
and microwave-assisted methods, including lower resin con-
centrations and increased infiltration times, the fibrous ap-
pearance of the major lateral tooth cusps and stylus persisted
and was indeed far more noticeable in microwave-prepared
specimens (Fig. 3). This is indicative of poor infiltration by
the epoxy resin and is a common problem encountered in
mollusc species by many researchers (Nesson and Lowen-
stam 1985, Mackenstedt and Markel 1987).

Microwave-assisted sample processing resulted in far
better preservation of tissue ultrastructure compared to con-
ventional methods. TEM of both conventional and micro-
wave-prepared samples revealed the typical arrangement of
organelles within the superior epithelium near the cusp sur-
face that are common to chitons, including microvilli, mi-
tochondria, rough and smooth endoplasmic reticulum, and
abundant, electron-dense ferritin siderosomes (Fig. 4).
However, at higher magnifications it could be clearly seen

SAMPLE PREPARATION OF RADULA EPITHELIUM

39

Figure 4. Transmission electron micrographs of the radula superior epithelium abutting the
tooth cusp (tc) of (A) conventionally and (B) microwave-processed radulae from adult
Acantliopleiira hirtosa at tooth rows 14 and 13, respectively, f, ferritin siderosome; g, granules;
mt, mitochondria; mv, microvilli. Scale bars = 5 pm.

sue from the tooth cusps and bases,
high-pressure freezing (HPF) resulted
in unsurpassed preservation of juve-
nile AotnOop/enra hirtosa radula tissue
(Fig. 6C). Nuclei (with well defined
chromatin adjacent to the nuclear
membrane), mitochondria (with well
preserved cristae), rough endoplasmic
reticulum, and Golgi apparatus were
clearly represented in the cytoplasm,
together with microvilli and ferritin
siderosomes (Fig. 6C). The ~60 nm
granules observed in microwave-
assisted preparations were also present
in HPF samples (data not shown). The
large vesicles surrounding the tooth
cusps in adult epithelium were also ob-
served in juvenile tissue prepared us-
ing conventional and microwave pro-
tocols but were absent in HPF material
(data not shown). While variations in

that samples fixed using the microwave contained numerous
granules approx. 60 nm in diameter. These granules, which
are likely to be either aggregations of ribosomal material or
glycogen, appeared throughout the cytoplasm, particularly
near the apical poles of the superior epithelium (Fig. 5).
These fine structures are either absent or not well preserved
in conventional preparations and have not been reported by
previous authors (Nesson and Lowenstam 1985, Kim et al.
1989). It is likely that the retention of these structures in
microwave-prepared samples is a result of the dramatic re-
duction in processing time of samples, thereby reducing
chemical exposure and the chance of extracting soluble com-
ponents of the tissue.

Preservation of juvenile radulae

The type and arrangement of organelles within the api-
cal epithelium of juvenile Acanthopleiim hirtosa radulae fol-
lows the same characteristic configuration as that described
for adult tissue. The preservation equality of juvenile radula
epithelium, when using conventional and microwave-
assisted methods, was also very similar to that observed in
adults (Figs. 6A-B). While the reduction in sample size may
improve fixative penetration in juvenile radulae, the com-
parable fixation equality between adult and juvenile tissue
indicates that size is not a limiting factor. In support of this,
the -60 nm granules were absent in conventionally fixed
juvenile tissue but were retained in adult and juvenile tissue
when processed by the microwave protocol (Figs. 5B, 6B).

With the exception of slight separation of epithelial tis-

Figure 5. Transmission electron micrograph of (A) conventional
and (B) microwave-prepared superior epithelium from adult Acan-
thopleura hirtosa showing differences in the preservation of fine
structure. Arrows denote aggregations of ribosomal- or glycogen-
like structures within the cytoplasm, f, ferritin siderosome. Scale
bars = 1 pm.

40

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 6. Transmission electron micrographs comparing the preservation of cell ultrastruc-
ture in radula epithelium from juvenile specimens of the chiton Acmithopleura hirtosa pro-
cessed using (A) conventional, (B) microwave-assisted, and (C) high-pressure freezing pro-
tocols. Black arrows denote aggregations of ribosomes/glycogen, f, ferritin siderosome; mv,
microvilli; mt, mitochondria; n, nucleus; tc, tooth cusp. Note: all images were taken from the
first tooth row after the onset of mineralization in the cusps. Scale bars == 2 pm (images on
left) and 1 pm (images on right).

ultrastructure may arise from differences in the functional
state of the cells at the time of fixation (Hayat 2000), it is
more likely that these vesicles are artifacts resulting from
glutaraldehyde fixation (Bowers and Maser 1988).

Despite a slight improvement in resin infiltration using
the conventional method, microwave-assisted processing of
radula superior epithelium is preferred due to the improved
quality of tissue preservation and the vastly reduced time for
sample processing. While cryo-preservation using HPF re-
sults in excellent ultrastructural preservation, it is limited
with respect to sample size. As such, only the radulae of

juveniles or very small mollusc species
can be prepared using this method. In
addition, the relative portability and
affordability of microwave technology
compared to HPF makes it a more re-
alistic option for many laboratories.
While each of these new techniques
has proven to be suitable for fixation
of tissue at the LM level, the improved
retention of ultrastructural informa-
tion gained by using microwave and
HPF methods highlights the need for a
re-evaluation of fine cell structure in
molluscan radulae.

While the ultrastructure of the
radula epithelium of chitons and lim-
pets has been well documented (Nes-
son and Lowenstam 1985, Mann et al.
1986, Kim et al. 1989, Rinkevich 1993),
no studies have been conducted on the
development of this tissue as teeth
progress from an unmineralized to a
mineralized state, information that is
crucial for resolving the cellular basis
of biomineralization. In addition,
cryo-techniques have recently been
used in conjunction with chemical
fixation to characterize the organic
matrix in limpets, by dramatically
reducing artifacts resulting from stain-
ing, dehydration, and embedding
(Sone et al. 2007). The high-pressure
freezing method outlined in the
current study therefore provides a
valuable first step in preserving
the organic matrix for subsequent
cryo-sectioning in a frozen hydrated
state. The methods of sample prepara-
tion presented here not only will ben-
efit future investigations of the supe-
rior epithelium and organic matrix of
chitons and limpets but also will be of use for taxonomic and
morphological studies of molluscan radulae in general.

ACKNOWLEDGMENTS

The authors would like to thank the assistance of Mr.
Gordon Thompson at Murdoch University for his advice on
histological preparations. This research was carried out using
facilities at The Centre for Microscopy, Characterisation and
Analysis at The University of Western Australia and The
Centre for Microscopy and Microanalysis at The University
of Queensland, both of which are supported by university.

SAMPLE PREPARATION OP RADULA EPITHELIUM

41

state, and federal government funding. Pinancial support
was provided by The Australian Research Council (Grant #
DP0559858). We also acknowledge the technical, scientific,
and financial assistance from the NANO-MNRP. All experi-
ments conducted during the study comply with the Austra-
lian Code of Practice for the Care and Use of Animals for
Scientific Purposes, 7th Edition, 2004. Animals were collected
under Conservation and Land Management permit number
SF005916.

LITERATURE CITED

Bowers, B. and M. Maser. 1988. Artifacts in fixation for transmis-
sion electron microscopy. In: R. F. E. Crang and K. L. Klom-
parens, eds.. Artifacts in Biological Electron Microscopy. Plenum
Press. New York. Pp. 13-42.

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Submitted: 29 January 2007; accepted: 7 August 2007;

final revisions accepted: 12 November 2007

Amer. Make. Bull. 25: 43-49 (2008)

Gross anatomy and positional homology of gills, gonopores, and nephridiopores in
“basal” living chitons (Polyplacophora: Lepidopleurina)’^

Julia D. Sigwart

National Museum of Ireland, Natural History Division, Merrion Street, Dublin 2, Ireland and Queen’s University Belfast, School of
Biological Sciences, University Road, Belfast, BT7 INN, Northern Ireland, UK, julia.sigwart@ucd.ie

Abstract: Traditional shell characters are insufficient to differentiate taxa within the polyplacophoran order Lepidopleurida. Additional
morphological character sets from soft anatomy (e.g., gamete morphology, gill arrangement, and locations of gonopores and nephidiopores)
have previously been described from only a small number of taxa. This study reports for the first time, positions of the gonopores and
nephridiopores for 17 species in the Lepidopleurina. The position of both types of pores on the longitudinal body axis varies within a
generalized range of the posterior third of the body; however, the separation between the pores as a proportion of the specimen’s foot length
varies from 3.7% to 17% in different species. Positions of pores relative to the serial gills are also variable within species, and future studies
may require a new descriptive basis in order to resolve positional homology. The order Lepidopleurida occupies a critical position with
respect to understanding larger-scale patterns in polyplacophoran (and molluscan) evolution.

Key words: Leptochiton, Leptochitonidae, descriptive morphology, molluscan evolution

Recent polyplacophorans are divided into two orders:
the large, diverse order Chitonida, and the putatively basal
Lepidopleurida. All living taxa in this order are grouped into
five families within the suborder Lepidopleurina (Sirenko
2006). The group contains around 120 living species in nine
genera, although more than eighty of these taxa are tradi-
tionally classified in the single genus Leptochiton Gray, 1847.
All species in Lepidopleurina are characteristically small,
without shell insertion plates, rarely with complex shell
sculpture or girdle elements, and often found in deep sea
habitats including hydrothermal vents (Saito and Okutani
1990), sunken-wood deposits (Sirenko 1988, 1997), and
cold-seeps (Kiel and Little 2006). The predominant charac-
teristics of this group could be that they live in the most
inaccessible habitats frequented by chitons and are the most
difficult to examine once captured.

Detailed understanding of these chitons has progressed
slowly; the animals are fundamentally difficult to identify
because of their small, plain appearance, and inconsistent
nomenclature remains a major source of confusion. For clar-
ity, members of the suborder Lepidopleurina are collectively
referred to as “lepidopleurans”. The two genus names “Lepi-
dopleurus” and “Leptochiton" were historically often used
interchangeably. There remains persistent confusion over
the family name Leptochitonidae Dali, 1889 and its pro-
posed replacement Lepidopleuridae Pilsbry, 1892 (Dali 1889,

Pilsbry 1892, Dell’Angelo and Palazzi 1991). Despite the no-
menclatural irregularity with the corresponding subordinal
and ordinal epithets, the family name Leptochitonidae has
clear priority and is the taxonomically correct name for the
group.

Although classical descriptions rely on shell characters
(or lack thereof), lepidopleurans are also universally allied by
their posteriorly arranged gills and apparently simple gam-
etes. Cladistic studies of Polyplacophora have repeatedly re-
covered the major subordinal groups, with Lepidopleurina
as the sister group to other living chitons (Buckland-Nicks
1995, Okusu et al. 2003). These studies have also shown that
gamete morphology corresponds strongly to these major
taxonomic partitions. This corroborates Sirenko’s (1993)
work correlating gamete structures to gill arrangements for
more than 100 species; although Sirenko (1993) reported the
position of nephridiopores and gonopores for 130 species in
the Chitonida, he included no lepidopleurans.

To date, the gamete morphology for seven species of
lepidopleuran chiton have been published: Leptochiton asel-
lus (Gmelin, 1791), Leptochiton rugatiis (Capenter MS, Pils-
bry, 1892), Leptochiton assiniilis (Theile, 1909), Deshayesiella
curvata (Capenter MS, Pilsbry, 1892), Hanleya hanleyi (Bean
in Thorpe, 1844), Hanleyella asiatica Sirenko, 1973 (Sirenko
1993, Hodgson et al. 1988, Buckland-Nicks 2006). Of those,
most have smooth egg hulls, but Hanleyella asiatica has long

From the symposium “Advances in Chiton Research” presented at the joint meeting of the American Malacological Society and Western
Society of Malacologists, held 29 luly to 3 August 2006 in Seattle, Washington.

43

44

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

hair-like chorion processes on the egg hull, similar to other
(unrelated) brooding species. However, work on other
brooding species with reduced chorion processes indicates
that egg hulls show little homoplasy (Sirenko 1993, Eernisse
and Reynolds 1994). The lepidopleuran sperm type is also
considered plesiomorphic as it lacks the relatively complex
acrosome processes characteristic of all described sperm
from non-lepidopleuran chitons (Buckland-Nicks 2006).

Gamete characteristics are of great interest, but exami-
nation is limited in a practical way by fixation and specimen
availability. The gross morphology of the exterior position of
the gonopore, which can be examined on preserved speci-
mens, may also be related to fertilization and reproductive
biology. The gonopore and nephridiopore in all chitons are
located on the roof of the pallial cavity between the gills
(Eig. 1).

More than 50% of lepidopleuran taxa are found at a
minimum depth of at least 100 m, and many recently de-
scribed species in the Leptochitonidae have a maximum

Figure 1. Camera lucida drawing of Leptochiton norfolcensis (Med-
ley and Hull, 1912); scale bar = 5 mm. Note nephridiopore (pos-
terior) between gill 4 and 5, and gonopore between gill 5 and 6 (gill
6 and 7 removed, indicated by dashed outline).

adult length of 5 mm or less. However, it is still not under-
stood how many morphological features may respond to
environmental conditions within the lifespan of the indi-
vidual animal.

This paper presents a series of preliminary descriptions
on the position, number, and distribution of gills, gono-
pores, and nephridiopores across a sample of basal living
chitons. Little previous work of this type has been con-
ducted, especially in these very small animals; it is hoped that
a survey of this kind will initiate additional morphological
(especially microanatomical) and phylogenetic studies
within Lepidopleurina. There is little doubt that this clade of
chitons will prove of critical importance to our understand-
ing of polyplacophoran evolution.

MATERIALS AND METHODS

This study included examination of the alcohol-
preserved material in the former collection of P. Kaas (1915-
1996) held in Naturalis, Leiden, The Netherlands (RMNH)
and that of R. A. Van Belle ( 1920-2005) in the Royal Belgian
Institute of Natural Sciences, Brussels, Belgium (RBINS).
These two collections make up a substantial proportion of
the fluid-preserved polyplacophoran material in their re-
spective institutions. The majority of specimens were his-
torically fixed in formalin before being transferred to meth-
ylated ethyl alcohol (70%); some are presumed to have been
additionally preserved with glycerin. Both collections in-
clude material that was collected by Kaas and Van Belle as
well as other colleagues who contributed material to their
private collections. The specimens vary in collecting age
from the 1970s to the 1990s. As the majority of specimens
were collected by persons knowledgeable about chitons and
were flattened at preservation, they are in excellent condition
for morphological examination.

All specimens that were preserved in a flattened posture
were examined under a dissecting microscope (up to lOOx
power) for observing the number of gills per side. The lo-
cation of the gonopores and nephridiopores relative to gills
was determined without dissection, usually by using 000
gauge insect pins as a probe to separate gills for inspection of
the pallial cavity roof. Use of flexible insect forceps allowed
the specimen to be held in place without damage and while
immersed in fluid. In some cases for specimens in RBINS, a
small strip of tissue was removed from the lateral margin of
the foot at the posterior end, to facilitate viewing the gill
row. In other cases for specimens in RBINS and RMNH,
individual gills were removed (“pinched” off) to facilitate
viewing the pallial cavity roof. Multiple specimens in a spe-
cies were examined where suitable material was available.

ANATOMY AND HOMOLOGY IN BASAL LIVING CHITONS

45

Table 1. Location of nephridiopore relative to closest gills (counted from posterior) and
number of gills per side in specimen.

Species

Nephridiopore

Gonopore

Gills

Leptochitoii algesirensis (Capellini, 1859)

5-6

8-9

15

Lep toclii ton a Igesirei isis

5-6

9-10

16

Leptochiton algesirensis

6-7

9-10

16

Leptochiton algesirensis

7-8

10-11

16

Leptochiton algesirensis

7-8

11-12

16

Leptochiton asellus (Gmelin, 1791)

5-6

7-8

10

Leptochiton asellus

5-6

7-8

10

Leptochiton asellus

5-6

7-8

10

Leptochiton hadius (Hedley and Hull, 1908)

4-5

5-6

8

Leptochiton helknapi Dali, 1878

5-6

7-8

14

Leptochiton cancellatus (Sowerby, 1840)

5-5

6-7

8

Leptochiton cancellatus

5-6

6-7

8

Leptochiton cancellatus

5-6

6-7

8

Leptochiton cancellatus

5-6

7-7

8

Leptochiton cancellatus

5-6

6-7

8

Leptochiton foresti (Leloup, 1981)

7-8

8-9

10

Leptochiton kiirnilatus Kaas, 1985

3-4

6

Leptochiton nhcropustulosns Kaas, 1994

9

10-11

14

Leptochiton inicropustulosus

9

10-11

14

Leptochiton inicropustulosus

9-10

12-13

14

Leptochiton norfolcensis (Hedley and Hull, 1912)

4-5

5-6

8

Leptochiton norfolcensis

4-5

5-6

8

Leptochiton riigatus (Carpenter in Pilsbiy, 1892)

5-6

7-7

10

Leptochiton rugatus

5-6

6-7

10

Leptochiton sarsi Kaas, 1981

5-6

7-8

9

Lepidopleurns cajetanus (Poll, 1791)

7

9-9

14

Lepidopleurus cajetanus

7-8

8-9

14

Parachiton eugenei (Kaas and Van Belle, 1985)

7-8

10

16

Parachiton hylkiae Strack, 1993

7-8

11-12

16

Parachiton hylkiae

8-9

10-11

16

Parachiton politiis Saito, 1996

10-1 1

12-13

16

Nierstraszella lineata (Nierstrasz, 1905)

9-10

10-11

14

Nierstraszella lineata

9-10

10-11

14

Nierstraszella lineata

8-9

13-14

16

Nierstraszella lineata

8-9

13-14

16

Nierstraszella philippina (Leloup, 1981)

5-6

7-8

12

Position of nephridiopores and
gonopores was recorded relative to
the posterior end of the gill row. Gills
were numbered from the one closest
to the anus and counted in series, fol-
lowing Sirenko (1993). Positions of
the pores were recorded as between
the two closest gills, or in some cases,
one gill when the pore was directly in
front of the base of a single gill.

Measurements to the nearest 0.1
mm were taken with calipers for total
body length, foot length, and length
of the gill row. As the number ot gills
may vary between left and right sides
of individual specimens, all pore
placements were confirmed on both
sides; gill counts and measurements
were taken uniformly from the left
side of the animal. These measure-
ments were omitted where specimens
were damaged or too curled to allow
accurate measure. Rapid degradation
of the external gill tissue after death
means that only the best- preserved
specimens were suitable for determi-
nation of the location of the gono-
pores and nephridiopores by this
technique. The separation between
gonopore and nephridiopore was es-
timated based on the number of gills
separating pore locations and the av-
erage inter-gill distance (length of row
divided by number of gills per side).

This is possible with some degree of
accuracy, as pores are consistently
placed in the middle portion of the
gill row, where each individual gill is
of “average” width. All measurements
reported were taken from a mean of
three successive caliper measures.

RESULTS

A total of 49 species from eight lepidopleuran genera
were examined for placement of gonopore and nepridio-
pore; however, pore locations relative to the gills were suc-
cessfully determined in only 17 species (Table 1). The speci-
mens ranged from 4.7 mm to 20.6 mm in body length.
Specimens had between 4 to 21 gills per side, and the gill row
ranged in length from 24% to 68% of foot length (but the

median value of 40% of foot length was characteristic of the
suborder).

Taxa for which pore locations were successfully deter-
mined include the genera Lcptochiton (11 species), Lepido-
pleuriis (one species), Pamcliiton Thiele, 1909 (three species),
and Nierstraszella Sirenko, 1992 (two species), represented
by a total of 36 specimens. These specimens ranged in size
from 5.1 mm to 15.1 mm (Table 2) and had between 6 to 16
gills per side (Table 1 ). The general range of placement of the
gonopore and nephridiopore as a pair was variable through-
out all taxa (Fig. 2). Although the two orifices have no physi-
ological relationship, their placement and relationship with

46

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Table 2. Size of chiton specimens; total body length measured on dorsal side in flattened position; foot length measured ventrally from
mouth to anus; gill row measured on right-hand side from anus or posterior-most gill (ctenidium) to anterior-most gill.

Species

Body length
(mm)

Foog length
(mm)

Gill row length
(nm)

Interpore distance
(mm)

Leptochiton algesirensis (Capellini, 1859)

8

5.1

2.1

0.39

Leptochiton algesirensis

13.5

10

4.6

0.86

Leptochiton aseUiis (Gmelin, 1791)

7.8

5

1.9

0.38

Leptochiton badins (Hedley and Hull, 1908)

6

4.2

1.3

0.16

Leptochiton cancellatiis (Sowerby, 1840)

5.9

3.7

1.1

0.14

Leptochi to n cancellatiis

7.1

4.7

1.5

0.19

Leptochiton foresti (Leloup, 1981)

6.5

4

2

0.20

Leptochiton micropustulosiis Kaas, 1994

12.5

8.8

3.5

0.50

Leptochiton niicropiistulosiis

14.6

9.2

4

0.57

Leptochiton inicropnstulosus

15.1

10.2

3.6

0.77

Leptochiton norfolcensis (Hedley and Hull, 1912)

5.1

3.1

1.1

0.14

Leptochiton rugatus (Carpenter in Pilsbry, 1892)

7.9

5.6

2

0.30

Lcpidopleurns cajetaniis (Poli, 1791)

11.5

8.5

3

0.43

Parachiton engenei (Kaas and Van Belle, 1985)

6.8

5

2

0.31

Parachiton hylkiae Strack, 1993

12.5

7.3

5.0

1.25

Parachiton hylkiae

9.8

6.3

3.7

0.46

Parachiton politns Saito, 1996

10.4

7

3.7

0.46

Nierstraszella lineata (Nierstrasz, 1905)

13.9

9.1

5

1.56

Nierstraszella lineata

13.9

9.1

5

1.56

the respiratory cavity allow us to consider them collectively
for convenience here.

The placement of nephridiopores and gonopores rela-
tive to the posterior-most gill was variable among species
and, in some cases, among presumed conspecifics. The ma-
jority of species in all genera (11 of 17) had a nephridiopore
at the sixth or seventh gill (counted from posterior of the gill
row). Other species with more posterior pores (relative to
the gill row) had fewer gills-six or eight per side. However,
species with more anterior pores were not correlated to
higher gill counts. Although most species retained consistent
pore positions in multiple specimens, some (Leptochiton al-
gesirensisy Nierstmszella lineata) varied between individuals.

The separation between nephridiopore and gonopore
was also highly variable. As an estimated proportion of the
length of the foot, the gap varied between 3.7% to 17% with
the widest separation in Nierstraszella litieata and one speci-
men of Parachiton hylkiae (Table 2). This was radically dif-
ferent from the other specimen of Parachiton hylkiae for
which data was available (with an interpore separation of
only 7.3% of the foot length) and Parachiton politns (6.6%).
The relationships of these proportions were similar whether
considered relative to the gill row or total body length.

All species examined had gills of the adanal condition
[sensii Sirenko 1993), having multiple gill pairs posterior of
the nephridiopore. Some Leptochiton species would be con-
sidered “abanal” (sensu Plate 1899) in that all gills were of

equal length; however, these taxa were adanal (sensu Si-
renko) as are all lepidopleurans.

DISCUSSION

The locations of gonopores and nephridiopores were
considerably variable with respect to gill placements in lepi-
dopleurans, which concurs with variability that has been
reported for other chitons (Sirenko 1993). The position of
the gonopore in the taxa examined was not fixed, neither
positionally relative to the gill row nor in distance to the
nephridiopore. This variability must be constrained by the
internal organ systems but external variability is indicative of
internal variability. The change in pore placement and the
effects on respiration, reproduction, and excretion may
mark physiological differences that impact the daily life of
the animals. Variations in pore placement do not appear to
be consistent with currently recognized genera, as they differ
within as well as among genera.

Sirenko ( 1997: 13) reported pore locations for nine lepi-
dopleuran taxa (but did not comment on these results in the
discussion of his paper). None of these taxa were duplicated
by this survey. However, Sirenko (1997) included two spe-
cies of Parachiton, both of which had more than 20 gill pairs
and found nephridiopore (11-12) and gonopore (12-13)

ANATOMY AND HOMOLOGY IN BASAL LIVING CHITONS

47

Figure 2. Schematic drawing of chiton ventral surfaces and the placements of gonopores
(anterior) and nephridiopores (posterior). Drawings are stylized to emphasize positions of
gills relative to gills only; the position of the nephridiopore is consistently underneath valve
VII of the animal. A, Leptochiton norfolceiisis, inset area illustrated in Fig. 1; B, Leptochiton
foresti (Leloup, 1981); C, Lepidopleuriis cajetamis (Poli, 1791); D, Parachiton eugenei (Kaas
and Van Belle, 1985); E, Nierstraszella Uneata (Nierstrasz, 1905); F, N. lineata.

placements that differ from the species examined in this
study.

Within this morphological survey, species showed dif-
ferent patterns of variability. For instance, most specimens
examined of Leptochiton algesirerisis had the same number of
gills per side, yet there are four different pore arrangements
associated with 16 gills. This contrasts with the other most
sampled species, Leptochiton asellus, which had extremely
consistent pore positions across all specimens. In other cases
there may be morphologies associated with growth patterns
(or potentially cryprtic species): in Nierstraszella lineata there
is one positional morphology associated with 14 gills and
another quite distinct pore arrangement in specimens with
16 gills.

The polyplacophoran nephridiopore is consistently lo-
cated underneath valve VII, and the gonopore is anterior to

the nephridiopore. Non-lepido-
pleurans have a substantial gap be-
tween the anus and the gills, and the
nephridiopore is at the posterior end
of the gill row (between 1 and 2; Si-
renko 1993). In lepidopleurans, how-
ever, the nephridiopore may be central
within or even anterior to the gill row.
This does not necessarily represent
anatomical change in the pore, but
rather shift in the gill arrangement
morphology. Although constrained to
the region under valve VII, the ne-
phridiopore clearly varies in position
relative to the body as well, as illus-
trated by considering the placement
relative to the proportional measure-
ment of the foot length. This should
not be a surprise; for example,
Parachiton has a characteristic en-
larged tail valve, which takes up a
larger proportion ot the animal’s foot
length than in other lepidopleurans.
The nephridiopore in Parachiton is
underneath valve VII, but the ne-
phridiopore is necessarily more ante-
rior relative to the foot (and poten-
tially other organ systems) than in
other genera.

The variability of gill arrange-
ments in chitons was first described by
Plate (1899) by differentiating extent
of the gill row (all lepidopleurans are
approximately “merobranchial” with
posteriorly arranged gills), presence of
small gills posterior of the major gill
row, and the presence of a gap between the posterior- most
gill and the anus. Ontogenetically, the adanal arrangement
means that new gills are added to both ends of the gill row
during growth, whereas abanal gill rows grow in an anterior
direction only (with the oldest and usually longest gills at the
posterior). Contemporary 19th centiny work proposed that
the “adanal” state characterized by extra small gills at the
posterior end of the row, typical of lepidopleurans, repre-
sents the plesiomorphic condition (Pelseneer 1899).

Sirenko (1993) improved Plate’s (1899) classical defini-
tions of “abanal” and “adanal” gill arrangements by reinter-
preting them with reference to the nephridiopore. This usage
effectively segregates the lepidopleuran taxa as an adanal
clade. In the majority of lepidopleuran species, the posterior-
most gills are small and sometimes bud-like processes near
or attached to the anus. These minute gills are visible on

48

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

individual animals of adult size. Ontogenetically, in some
species, these may grow to determinant size, but they may
also be influenced by environmental plasticity, growing to
suit local respiratory requirements. Kaas and Van Belle
( 1985) noted that counts of gills were variable in adult speci-
mens of some species, and that counts of gills on right and
left sides may vary within a single individual. Observation-
ally, it appears that comparisons within taxa are not nega-
tively affected as multiple individuals of the taxa included in
this survey and others do have a consistent number of gills
although the marginal gills may be very different in size.
These structures illustrate that gills within a gill row on an
individual chiton are not equal and interchangeable units.

Sirenko ( 1993) proposed using the posterior- most gill as
a reference point for describing the location of nephridio-
pores and gonopores, as followed in this survey. In abanal
chitons, the gill row grows in the anterior direction, making
posterior-based counting a consistent method regardless of
the animal’s growth state. As adanal gill arrangement is pre-
sumed indicative of bi-directional growth of the gill row,
using the posterior- most gill as a reference point may not be
sufficient in these taxa. Examination of a single specimen
may not give a positional result that can be validated in an
older individual. The question of ontogeny is not fully re-
solved; gill row growth may be bi-directional but examina-
tion of juvenile specimens shows that the primary direction
of growth is still anterior. The gill “buds” at the posterior
end of the gill row appear early in post-settlement individu-
als. These buds are interpreted to be new gills that have not
achieved full length; however, they appear in many adult
sized specimens which brings into question whether some
species have determinate growth in gill numbers. In exami-
nation of the gill row in a chiton of indeterminate age (as
they all are), there is also no way to determine which indi-
vidual gill of full size marks the “beginning” point of growth.

A number of South Pacific lepidopleurans, primarily
those described by Sirenko (2001) and other related taxa,
have only four gills. (Interestingly, these species would be
considered “abanal” sensu Plate because all four gills are
equal in length, but there is no question that the species are
lepidopleurans). Because the entirety of the gill row is lo-
cated underneath the tail valve, both the nephridiopore and
gonopore are probably located on the naked pallial roof.
This is a clear limitation on the present methods for describ-
ing the anatomy of nephridiopores and gonopores exter-
nally: there is no practical way to describe their placement or
compare placements among these species.

Correlation of gonopores and nephridiopores, as well as
internal anatomical structures, to shell morphology requires
further descriptive work. Measurement of position in this
case and in other reports is recorded relative to gills, but it
should additionally be measured relative to dorsal valve and

sutural positions. Some of the variability observed in this
morphological survey is probably an artifact of bi-directional
gill row ontogeny. However, there is clearly a high level of
plasticity that cannot be completely accounted for (particu-
larly in the well-sampled case of Leptochiton algesirensis)
without acknowledging that the pores, and therefore internal
organ anatomy, are variable within and between taxa to a
higher degree than previously recognized.

In this group, as in many groups of invertebrates where
species identification is challenging, new characters that help
differentiate taxa are always welcome. However, the prob-
lems illustrated here demonstrate that either pore placement
relative to gill elements is not positionally homologous or,
alternatively, if pore placements are apomorphic then cur-
rent genera are not monophyletic assemblages. Variability in
this and other soft anatomical features in lepidopleurans
may mask cryptic species not identifiable by traditional shell
and girdle characters.

The inconsistent morphology within recognized genera
in the preliminary results reported in this paper suggest that
these structures may not be positionally homologous. Po-
tential plasticity of elements in the gill row in lepidopleuran
chitons is not sufficiently known and means that the posi-
tions of pores with respect to gills may be homoplasious for
phylogenetic inference. It will be interesting to compare the
results presented here with future hypotheses about internal
relationships within the lepidopleuran clade.

Although these chitons are superficially similar, this an-
cient lineage of molluscs shows a great deal of adaptation to
different marine environments. This is reflected in morpho-
logical differentiation, although not in the traditional shell
and girdle characters used to describe polyplacophoran spe-
cies. The issues raised in this survey indicate that although
the nephridiopores and gonopores of lepidopleuran chitons
are superficially similar in structure, our current means of
identifying and describing their placement is insufficient to
consider them to be positionally homologous across taxa for
the purpose of systematic inference. The patterns of variance
are too clouded by our poor understanding of morphology
to shed more light on internal relationships within this
group; however, the morphological variation observed here
is undoubtedly linked to the high level of evolutionary vari-
ance in this diverse group of chitons. Lepidopleurans lack
the dramatic colors and patterns of their sister-group the
Chitonida, but nonetheless represent a diverse group of ma-
rine organisms.

ACKNOWLEDGMENTS

1 am grateful to the European Commission SYNTH-
ESYS program for funding research visits to Naturalis

ANATOMY AND HOMOLOGY IN BASAL LIVING CHITONS

49

(award NL-TAF-437) and RBINS (award BE-TAF-406). J.
Goud and E. Gittenberger provided access to collections in
Naturalis (RMNH); J. L. Van Goethem and R. Sablon pro-
vided access to collections in RBINS. Thanks also to D.
Eernisse for organizing the AMS Chiton Symposium in Inly
2006 and to the American Malacological Society for financial
support for me to attend the symposium. A. N. Hodgson
and B. I. Sirenko provided reviews that improved this
manuscript.

LITERATURE CITED

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eny. Menioires dii Musann national d’Histoire natnrelk, Paris
166; 129-153.

Buckland-Nicks, L 2006. Fertilization in chitons: Morphological
clues to phytogeny. Venus 65: 51-70.

Dali, W. H. 1889. Report on the results of dredging, under the
supervision of Alexander Agassiz, in the Gulf of Mexico (1877-
78) and in the Caribbean Sea (1879-80), by the United States
Coast Survey Steamer “Blake,” Lieutenant-Commander C.D.
Sigsbee, U.S.N., and Commander I.R. Bartlett, U.S.N., com-
manding. Report on the Mollusca, Pt. 2: Gastropoda and
Scaphopoda. Bulletin of the Museum of Comparative Zoology
18; 1-492.

DelFAngelo, B. and S. Palazzi. 1991. Considerazioni sulla famiglia
“Leptochitonidae” Dali 1889 (Mollusca, Polyplacophora). IV.
Aggiunte e correzioni. Bolletthio Malacologico 27: 35-38 [in
Italian].

Eernisse, D. L and P. D. Reynolds. 1994. Polyplacophora. In: F. W.
Harrison and A. I. Kohn, eds.. Microscopic Anatomy of Inver-
tebrates, vol. 5: Mollusca /, Wiley-Liss, Inc. New York. Pp.
55-110.

Hodgson, A. N., J. M. Baxter, M. G. Sturrock, and R. T. F. Bernard.
1988. Comparative spermatology of 11 species of Polyplaco-
phora (Mollusca) from the suborders Lepidopleurina, Chito-
nina, and Acanthochitonina. Proceedings of the Royal Society,
London (B) 235: 161-177.

Kaas, P. and R. A. Van Belle. 1985. Monograph of Living Chitons.
Vol. 1. Order Neoloricata: Lepidopleurina. E. ]. Brill / Backhuys.
Leiden, The Netherlands.

Kiel, S. and C. T. S. Little. 2006. Cold-seep mollusks are older than
the general marine mollusk fauna. Science 313: 1429-1431.

Okusu, A., E. Schwabe, D. I. Eernisse, and G. Giribet. 2003. To-
wards a phytogeny of chitons (Mollusca, Polyplacophora)
based on combined analysis of five molecular loci. Organisms,
Diversity and Evolution 3: 281-302.

Pelseneer, P. 1899. Sur la mophologie des brachies et des orifices
renaux et genitaux des chitons. Bulletin scientifique de la
France et de la Belgique 31: 23 [in French].

Pilsbry, H. A. 1892. Monograph of the Polyplacophora: Lepido-
pleuridae. In: G. W. Tryon, eds.. Manual of Conchology, Acad-
emy of Natural Sciences, Philadelphia. Pp. 1-128.

Plate, L. H. 1899. Die Anatomie und Phylogenie der Chitonen.
Fauna Chilensis. Zoologische Jahrbuch Systematik 2 (Supple-
ment) 5: 15-216 [in German].

Saito, H. and T. Okutani. 1990. Two new chitons (Mollusca: Poly-
placophora) from a hydrothermal vent site of the Iheya small
ridge, Okinawa Trough, East China Sea. Venus 49: 165-179.

Sirenko, B. I. 1988. A new genus of deep sea chitons Ferreiraella
gen. n. (Lepidopleurida, Leptochitonidae) with a description
of a new ultra-abyssal species. Zoological Journal 67: 1776-
1786.

Sirenko, B. I. 1993. Revision of the system of the order Chitonida
(Mollusca: Polyplacophora) on the basis of correlation be-
tween the type of gills arrangement and the shape of the cho-
rion processes. Ruthenica 3: 93-117.

Sirenko, B. I. 1997. The importance of the development of articu-
lamentum for taxonomy of chitons (Mollusca, Polyplaco-
phora). Ruthenica 7: 1-24.

Sirenko, B. I. 2001. Deep-sea chitons (Mollusca, Polyplacophora)
from sunken wood off New Galedonia and Vanuatu. Memoires
du Museum national d’Histoire naturelle 185: 39-71.

Sirenko, B. I. 2006. New outlook on the system of chitons (Mol-
lusca: Polyplacophora). Venus 65; 27-49.

Submitted: 29 January 2007; accepted: 7 August 2007;

final revisions received: 10 February 2008

Amer. Malac. Bull. 25: 51-69 (2008)

Aesthete canal morphology in the Mopaliidae (Polyplacophora)’^

Michael J. Vendrasco^’*^’^, Christine Z. Fernandez^, Douglas J. Eernisse^, and Bruce Runnegar^

' Institute lor Crustal Studies, 1140 Girvetz Hall, University of California, Santa Barbara, California 93106-1100,

U.S.A., mvendrasco@fullerton.edu

^ 14601 Madris Ave., Norwalk, California 90650, U.S.A.

^ Department of Biological Science (MH-282), California State University, Fullerton, P.O. Box 6850, Fullerton, California 92834-6850,

U.S.A.

“ Department of Earth and Space Sciences, Institute of Geophysics and Planetary Physics and Molecular Biology Institute, 595 Young
Drive East, University of California, Los Angeles, California 90095, U.S.A.

Abstract: The aesthete canals of fourteen chiton species were cast with epoxy, allowing detailed examination and comparison of the entire
canal system that infiltrates their valves (shell plates). Some species in this study have been classified without question in the family
Mopaliidae {Mopalia ciliata (Sowerby, 1840), Mopalia lignosa (Gould, 1846), Mopalia spectabilis Cowan and Cowan, 1977, Mopalia swanii
Carpenter, 1864, Katharina tiinicata (Wood, 1815)), while other species have been placed in that family by some workers but not others
(Dendrochiton flectens (Carpenter, 1864), Dendwchiton limlatus (Berry, 1963), Tonkella insignis (Reeve, 1847), Tomcella lineata (Wood,
1815), Tonicella lokii Clark, 1999, Tonkella marmorea (Fabricius, 1780), Nuttallochiton mirandus (Thiele, 1906), Plaxiphora aurata (Spal-
owski, 1795)), and one has never been placed in the Mopaliidae {Tonicia clnlensis (Frembly, 1827)). The results provide additional evidence
that there is high diversity in aesthete canal morphology but also some striking resemblances interpreted here as homologies, reaffirming
that aesthete canal characters have considerable potential for phylogenetic analyses and for supporting classification ranks ranging from
suborder to species. In this case, the results are broadly consistent with traditional classifications of mopaliids, but Tonkella and Dendro-
cliiton (taxa not always thought not to be mopaliids) share many aesthete canal synapomorphies with undisputed mopaliids, whereas
Plaxiphora (typically thought to be a mopaliid) has an aesthete canal system more similar to non-mopaliid members of the Acanthochi-
tonina. These differences are in line with results of recent phylogenetic analyses of the Mopaliidae.

Key words: Chiton, Mopalia, valve, tegmentum, esthete

The hard layers of chiton valves consist of the upper-
most tegmentum, the articulamentum whose projections
form the sutural laminae and insertion plates, and the un-
derlying hypostracum. The tegmentum, which is the visible
layer of the chiton shell in life, is infiltrated with a complex,
tissue-filled canal system that opens at the dorsal valve sur-
face as sensory or secretory organs known as aesthetes (also
esthetes) (Marshall 1869). The pores on the dorsal surface are
entrances of tiny canals that often pass into bulb-shaped
(aesthete) chambers that then connect to larger horizontal
canals and eventually exit at the valve’s anterior or lateral
margin, or in some regions of the ventral valve surface
(now known to correspond to the nervous innervation of the

* From the symposium “Advances in Chiton Research” presented
at the joint meeting of the American Malacological Society and
Western Society of Malacologists, held 29 luly to 3 August 2006 in
Seattle, Washington.

aesthetes). Knorre (1925) made detailed schematic drawings
of the entire canal system in Lepidochitona cinerea (Linnaeus,
1767) (as Trachydermon cinereus) that revealed this configu-
ration of canals. Prior to Knorre’s (1925) work, Moseley
(1885) noticed two size classes of aesthete pores (termed
micropores and megalopores) on the dorsal valve surface
and coined the term megalaesthete for the organic tissues
within the often bulbous chambers near the valve’s dorsal
surface, and micraesthete for the tissues in the smaller canals
that connect from the bulb of the megalaesthete to the valve
surface. The organs that occupy the upper portion of the
chiton tegmentum include the aesthetes (Blumrich 1891)
and in some cases also the extrapigmental and intrapigmen-
tal ocelli (Nowikoff 1907, 1909). Although aesthetes have
been found in all modern chitons so far examined, ocelli
have so far been found only in some members of the Schizo-
chitonidae (Moseley 1885) and Chitonidae (Boyle 1977).

Although there have been numerous studies of aesthetes
in many chiton species (see the review in Reindl et al. 1997),

** Present address: Department of Biological Science (MH-282), California State University, Fullerton, P. O. Box 6850, Fullerton, California
92834-6850, U.S.A.

51

52

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

their function has been debated. Moseley (1885) docu-
mented the morphology of ocelli in Schizochiton incisus
(Sowerby, 1841) (Chitonina: Schizochitonidae). In this spe-
cies, the ocelli are the largest known for any chiton and are
sparsely distributed in relatively huge chambers —
presumably enlarged megalaesthete cavities. Boyle (1969a,
1969b) confirmed the presence of photoreceptors in ocelli in
Ofiithochiton neglectus Rochebrune, 1861. A photosensory
role for aesthetes had initially been proposed by Blumrich
( 1891 ) and observations that certain chiton species are either
positively or negatively phototactic {e.g., Crozier 1920, Om-
elich 1967, Boyle 1972, Lischer 1988, Currie 1989) have led
many to view photoreception as the primary role of aes-
thetes. Indeed, Crozier and Arey (1918) observed that a
crawling Chiton tiiberculatus Linnaeus, 1758 (which lacks
large conspicuous ocelli) immediately stopped crawling,
temporarily, in response to a shadow from a fly about 2 m
away. However, the function of most of the aesthetes, be-
sides those with ocelli, has been disputed, with suggestions
including mechanoreception (Moseley 1885), chemorecep-
tion (Lischer 1988, Baxter et al 1990), periostracum replen-
ishment and secretion (Boyle 1974, Baxter et al. 1987, 1990),
and secretions for protection, prevention of desiccation, or
fouling by epibionts (Lischer 1988). An electron microscopy
study of aesthete tissues (Omelich 1967), an immunocyto-
chemical study (Reindl et al. 1997), and electrical recordings
(Omelich 1967, Lischer, pers. comm, in Eernisse and Reyn-
olds 1994) have all shown that aesthetes contain neuronal
structures, demonstrating a sensory function in at least some
cases. However, it seems plausible that aesthetes could serve
many roles, or the functions differ in different lineages, as
suggested by Haas and Kriesten (1978), Lischer (1978, 1988),
Sturrock and Baxter (1995), and others.

Because features of the aesthete canal system and the
nature of aesthete caps vary between chiton taxa (e.g., Boyle
1974, Baxter and Jones 1981, 1984, Sturrock and Baxter
1993, 1995, Reindl et al. 1997, Schwabe and Wanninger
2006), they provide a suite of characters that have been
included in phylogenetic and taxonomic studies of chitons
(e.g., Hull and Risbec 1930-1931, Leloup 1940, 1942, 1948,
Bullock 1985, 1988, O’Neill 1985, Watters 1990, Sirenko
1992, 2001, Saito 1996, 2006). Brooker (2004) included ex-
tensive data from the distribution, arrangement, density,
shape, and size of ocelli; aesthete pore area, shape, densities,
and ratios; and size, density, and shape of large pores in the
tegmentum eaves in her cladistic analysis of the Acantho-
pletira Guilding, 1829. Moreover, Schwabe and Wanninger
(2006) documented variation among chiton genera in the
elevation of aesthete pores, pigmentation in the megalaes-
thetes, and the arrangement of micraesthetes around the
megalaesthetes.

Currie (1989), however, cautioned that there can be

much variation in size and density of aesthete pores in dif-
ferent areas of one valve. This point was echoed by Brooker
(2004), who found this to be true in the Acanthopleura and
who emphasized the need to compare data from the same
valve area when describing differences in aesthete patterns
between species.

Recently, using an approach pioneered by Haas and
Kriesten (1978; see Eernisse and Reynolds 1994), Lernandez
et al. ( 2007) made epoxy casts of the aesthete canal system in
twelve chiton species and demonstrated variation among
suborders, families, genera, and species. A cladistic analysis
revealed congruence between relationships inferred from
aesthete canal characters alone and those derived from other
aspects of morphology as well as molecules, suggesting that
aesthete canal characters are useful in helping to infer rela-
tionships between chitons (Lernandez et al. 2007).

This study expands previous work by focusing largely
on internal relationships within one family of chitons, Mo-
paliidae Dali, 1889. This family has conventionally included
at least Mopalia Gray, 1847, Placiphorella Carpenter in Dali,
1879, Amicula Gray, 1847, Katharina Gray, 1847, Plaxiphora
Gray, 1847, and Placiplwrina Kaas and Van Belle, 1994 (e.g.,
Kaas and Van Belle 1994, Sirenko 2006). The placement of
Niittallochiton Plate, 1899 and Dendrochiton Berry, 1911 has
been less consistent. Lor example, Thiele (1931) placed Nut-
tallochiton in the Lepidochitonidae, whereas Van Belle (1983)
and Kaas and Van Belle ( 1987) placed it in the Chaetopleurinae
(Ischnochitonidae) instead. Sirenko (1993, 1997, 2006) later
assigned this genus to the Mopaliidae, although recent molecu-
lar phylogenetic analyses (Okusu et al. 2003, Eernisse, unpubl.
data) suggest that Niittallochiton should be excluded from that
family. Berry (1911) originally proposed Dendrochiton as a
member of Mopaliidae because its members possess girdle se-
tae similar to those of Mopalia, but others have instead con-
sidered it to belong to Lepidochitonidae (e.g., Lerreira 1982,
Van Belle 1983). Van BeUe (1983) and Kaas and Van Belle
(1985) even considered it to be a subgenus of Lepidochitona
Gray, 1821 within Lepidochitonidae.

Eernisse (unpubl. data) used mitochondrial 16S rDNA
data to discover a previously unrecognized association of
some conventional mopaliid genera (Placiphorella, Katha-
rina, Amicula, and Mopalia) with genera normally placed in
other families (Cryptochiton, Tonicella Carpenter, 1873, and
Dendrochiton). Lurthermore, these analyses revealed that the
more southern genera, Plaxiphora and Niittallochiton, are
only distantly related to Mopaliidae. This family was previ-
ously diagnosed by a posterior caudal sinus in the tail valve,
which may instead be interpreted as a convergent trait re-
lated to size increase and the enhancement of respiratory
currents (Eernisse, unpubl. data). In order to provide evi-
dence for or against this proposed rearrangement, we exam-
ined aesthete canal morphology in a number of undisputed

AESTHETE CANAL MORPHOLOGY IN THE MOPALIIDAE

53

mopaliid taxa in addition to representatives newly embraced
into, or excluded from, the Mopaliidae based on this new
classification scheme (Eernisse, unpubl. data).

Thus the goals of this study were to: ( 1 ) determine if
aesthete canal morphology supports this new taxonomic
scheme of the Mopaliidae; (2) determine the degree and
nature of variation in aesthete canal systems in a larger set of
chitons (see figs. 1 and 2) to better assess the hypothesis in
fernandez et al. (2007) that such characters are useful in
chiton phylogeny; and (3) use the new data to refine the
previous attempt (in Fernandez et al. 2007) to define poten-
tial characters and states of the aesthete canal system that
may be useful in future phylogenetic analyses of chitons.

MATERIALS AND METHODS

All chitons used in this study were adults and most were
collected from the Eastern Pacific (collection data: Appendix
1). Valves from at least two individuals from each species
were treated, except for the deep-water Nuttallochiton ini-
randus. Two or three intermediate valves of each individual
were embedded and examined. All epoxy casts and voucher
valves for each species in this analysis have been deposited at
the Santa Barbara Museum of Natural History (SBMNH).

Valves were removed from dried or alcohol-preserved
specimens using a scalpel, tweezers, and scraping tools. Boil-
ing the chitons to remove the valves was not done because
this can break valves into pieces. The isolated intermediate
valves of all species were soaked in household bleach for up
to 24 hours and placed in a sonicating bath for 20-30 min-
utes at room temperature to dislodge remnant organic ma-
terial and other debris. Valves were dehydrated through an
ethanol series and then embedded in epoxy using a method
modified after Golubic et al. (1970). A low viscosity, me-
dium hardness embedding medium was mixed using the
Embed 812 kit from Electron Microscopy Sciences. The Em-
bed 812 kit consisted of Embed 812 embedding resin, Do-
decenyl Succinic Anhydride (DDSA), Nadic Methyl Anhy-
dride (NMA), and Benzyldimethylamine (BDMA). They
were combined in the following proportions: 44.2% Embed
812, 35.4% DDSA, 17.7% NMA, and 2.7% BDMA. The
valves were submerged in resin and placed under a vacuum
in a desiccating chamber for 24 hours and then cured in an
oven at 60 °C for 24 hours. The cured epoxy blocks were
trimmed using a rotary hand tool with a thin-bladed saw.
Cuts were made around the edges of the valves, making sure
to intersect the valve along much of its margin. The epoxy
blocks were placed in 10% HCl for another 24 hours, or
until all of the calcium carbonate in the valves dissolved
away, then rinsed thoroughly with distilled water, cleaned
with bleach, and split apart into a dorsal and ventral cast.

In a few cases (e.g.. Fig. 3F), after the vacuum stage but
prior to curing, valves were drained of most epoxy, with only
a shallow pool extending just above the insertion plates. The
surface of these valves, still with a coating of epoxy, was then
wiped with a Kimwipe dipped in alcohol. This alternative
method was included to better see the meglaesthete bulbs
and associated micraesthetes near the dorsal valve surface (it
allows the aesthete chambers to be visible from the top,
rather than from underneath, where they are largely hidden
by underlying canals).

The casts were gold sputtered for 90 seconds and ex-
amined using a LEO 1430 Scanning Electron Microscope
(SEM) with an accelerating voltage (EHT) of 10-15 kV un-
der high vacuum. Many images were taken using backscatter
electron detectors (two to four quadrants) at high or variable
pressure. The backscatter detectors (QBSD) produced far
less charging than occasionally occurred with the secondary
electron detector (SE). The number and exact backscatter
detectors varied, though the best contrast was usually
achieved with 3 of 4 detector quadrants on. In a few cases,
charging still occurred, so variable pressure (30-40 Pa) and
the Variable Pressure detector (VPSE) were used.

In many species, mopaliids in particular, the dense car-
pet of horizontal canals on the dorsal casts prevented a dear-
view of the overlying (in life; in SEM photographs of the
dorsal casts, they underlie the horizontal canals), near-
surface canal systen-i. In these cases, a thin-tipped needle was
used to pull away soi-ne of the horizontal canals and reveal
the megalaesthete chambers and micraesthete canals that lay
below on the casts. In such cases, re-coating with gold was
necessary.

Epoxy casts made prior to this study and described in
Fernandez et al (2007) were also used in coi-nparative analy-
ses herein. These specimens are: Mopalia miiscosa (Gould,
1846) (SBMNH 83143 and 83144), Mopalia acuta (Carpen-
ter, 1855) (SBMNH 83160 and 369432), Cyatioplax {as Lepi-
dochitona) hartwegii (Carpenter, 1855) (SBMNH 83146 and
83147), Nuttallina californica (Nuttall MS, Reeve, 1847)
(SBMNH 83148, 83149, and 83156), Lepidozona cooperi
(Dali, 1879) (SBMNH 83150 and 83151), Lepidozona
mertensii (Middendorff, 1847) (SBMNH 83145 and 369438),
Lepidozona pectinniata (Carpenter in Pilsbry, 1893)
(SBMNH 83152 and 83153), Placiphorella velata Carpenter
MS, Dali, 1879 (SBMNH 83161 and 369440), Nuttallochiton
hyadesi (de Rochebrune, 1889) (SBMNH 83157), Ischnochi-
ton textilis (Gray, 1828) (SBMNH 83158 and 369435), Is-
chnochiton variegatus (H. Adams and Angas, 1864) (SBMNH
83159 and 369437), and Lepidoplennis cajetanns (Poli, 1791 )
(SBMNH 83154 and 83155).

While most genera thought to belong in the Mopaliidae
(plus a few other families) were included in this study,
Ainicula and Cryptochiton were not. Cryptochiton adults lack

54

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 1. Schematic of the horizontal canal system in different chiton genera. A, Chiton intermediate valve showing dorsal (left side) and
ventral (right side) features and terminology: D, diagonal line; JA, jugal area or jugum; IS, jugal sinus; LA, lateral area; PA, pleural area (also
referred to as median triangle (Baxter and jones 1981) or median area (Baxter and lones 1984)); S, slit; SR, slit ray. B, Schematic of
horizontal canals (lines) and megalaesthetes (filled circles) in Lepidopleurus, based on the pattern seen in L. cajetaniis. C, Lepidozona, based
on L. cooperi, L. pectinidata, and L. mertensii. D, Ischnochiton, based on /. textilis and /. variegatus. E, Mopalia type 1, characterizing M. acuta,
M. nmscosa, and M. lignosa. F, Mopalia type 2, characterizing M. ciliata, M. spectabilis, and M. swanii. G, Tonicella, based on T. lokii, T.
lineata, T. insignis, and T. marmorea. H, Plaxiphora, based on P. aurata. I, Cyamplax, based on C. hartwegii. ], Nuttallina, based on N.
californica. The reconstructions in I and I were based on aesthete casts of Cyatwplax hartwegii (SBMNH 83146, 83147) and Nuttallina
californica (SBMNH 83148, 83149, 83156) shown in Fernandez et al. (2007).

AESTHETE CANAL MORPHOLOGY IN THE MOPALIIDAE

55

A

D

Figure 2. Schematic showing comparative morphology of canals
that extend from the dorsal valve surface to the underlying hori-
zontal canals. A, Form characteristic of Mopalia spp., Tonicella spp.,
Dendrochiton spp., Placiphorella velata, and Katharimi tunkata. B,
Form characteristic of Cyanoplax hartwegii, NuttalUna californica,
Plaxiphora aurata, and Nuttallochiton spp. C, Form characteristic of
Ischnochhon spp., Tonicia chilensis, and Lepidozomi spp. D, Form
characteristic of Lepidopleiirus cajetanus. Reconstructions of C.
hartwegii, N. californica, P. aurata, Nuttallochitofi, Ischnochiton, and
Lepidozona are based on aesthete canal casts described and photo-
graphed in Fernandez et al. (2007).

a tegmentum, and adults of Amicula have only a small rem-
nant of that shell layer, which limits the extent to which their
aesthete canal systems can be compared to those of other
mopaliids. Valves of juvenile Cryptochiton stelleri Midden-
dorff, 1847 have some tegmentum, but we were not able to
obtain juveniles of this species for destructive analysis.

The cladistic analysis using only aesthete canal charac-
ters was constructed with PAUP 4.0bl0 (Swoftord 2002). All
taxa from Fernandez et al. (2007) as well as those herein [N
= 26 total from both studies) were scored for the analysis,
although five taxa had the same exact character states as
another taxon in the analysis, so these “redundant” taxa
were excluded (N = 21 in this analysis) to allow for branch-
and-bound analysis over a reasonable time frame. Specifi-
cally, Mopalia lignosa had the same character states as Mo-
palia ciliata, Mopalia spectabilis had the same as Mopalia
rnuscosa, Tonicella marmorea had the same as Tonicella lin-
eata, Tonicella lokii had the same as Tonicella insignis, and
Lepidozona cooperi had the same as Lepidozona rnertensii. All
characters were un-weighted and all character states un-
ordered (description of characters and their states in Appen-
dix 2). Lepidoplenrus cajetanns was used as the outgroup. A
branch-and-bound search was completed using maximum
parsimony.

All epoxy casts and voucher shell plates from each in-
dividual in this study as well as those in the previous one
(Fernandez et al. 2007) have been deposited at the SBMNH.

RESULTS

Reference to the trend of the canal system in the de-
scriptions to follow is consistent with the flow of sensory

information and the direction of valve growth (see Baxter
and (ones 1981, 1984), such that the pores on the dorsal
tegmentum surface are taken to be the entrance and the sites
where the canals enter the body of the chiton (large pores in
the anterior and lateral tegmentum eaves, slit rays, and un-
derneath the jugum) the exit. The terms anterior, posterior,
dorsal, and ventral refer to the valve in life position. The two
pieces of the aesthete canal cast are termed dorsal and ven-
tral, also defined based on life position.

A nearly complete cast of the aesthete canal system was
achieved in most relatively un-eroded valves. The few eroded
valves {e.g., from one individual of Plaxiphora aurata), in
contrast, had missing canals and a high incidence of tunnels
caused by endolithic organisms. The ventral casts in this
study often had at least a few complete vertical canal ele-
ments (i.e., extending from the dorsal to ventral surface of
the valve), allowing a detailed examination of the megalaes-
thete-micraesthete complex in certain portions of the valve.
The results reveal variation in the horizontal canal system
(Fig. 1) as well as megalaesthete-micraesthete morphology
(Fig. 2).

Data from the aesthete canal casts of Mopalia acuta,
Mopalia rnuscosa, and Nuttallochiton hyadesi, described in
Fernandez et al. (2007), are also incorporated into the fol-
lowing descriptions of the canal system in each genus.

The taxonomic assignments are based on Sirenko ( 1997,
2006) but assignments that differ between Sirenko (2006)
and what is suggested by the phylogeny in Eernisse (unpubl.
data) are indicated with a c]uestion mark. Specifically, Eer-
nisse’s phylogenetic hypothesis suggests Dendrochiton and
Tonicella are in the Mopaliidae and Nuttallochiton is not.

Mopalia ( Acanthochitonina: Mopaliidae) (Figs. 3, 4E-I)

The dorsal casts reveal large (ranging from about 20-75
pm diameter), nearly straight, roughly equal diameter, regu-
larly spaced, primary horizontal canals that run from the
posterior to anterior margin through all valve areas (Figs.
3C, 3E, 3H, 4E, 41; also fig. 2a,b,j in Fernandez et al. (2007)).
The canals are closely spaced (about 10-40 pm between pri-
mary canals) and only rarely do they merge with each other.
At least two vertical levels of primary horizontal canals can
be seen at their exit near the anterior margin of the valve. In
Mopalia ciliata, Mopalia spectabilis, and Mopalia swanii, the
horizontal canals on either side of the jugal area fan out
laterally (Figs. 3H, 4A, 41), whereas in the other species of
Mopalia, the long axes of all horizontal canals in the central
area are consistently straight (Fig. 3C; also fig. 2a, j in Fern-
andez et al. (2007)).

There are also many short, smaller-diameter (about 10-
20 pm) subsidiary horizontal canals, above (in life) and in-
clined relative to primary horizontal canals, that merge with
the primary canals at regular intervals (Figs. 3A-B, 3D, 4G;

56

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 3. SEM images of casts of aesthete canal systems for Mopalia spectahilis (SBMNH 369491 ) (A-B), Mopalia lignosa (C-E), and Mopalia
swaiiii (SBMNH 83329) (F-H). All images are of dorsal casts, except A which is of the ventral cast. A-B, Mopalia spectahilis. A, Close-up
of a complete slit ray canal element on the ventral cast. Scale bar = 200 pm. B, Close-up of lateral area canals along the posterior margin
of a different individual than in A. Scale bar = 200 pm. C-E, Mopalia lignosa. C, Composite image showing view of much of the system
of horizontal canals (SBMNH 83328). Scale bar = 1 mm. D, Close-up of canals in the pleural area (SBMNH 83328). Scale bar = 200 pm.

E, Canals in the pleural area along the anterior margin of a different individual (SBMNH 83327). Scale bar = 200 pm. F-H, Mopalia swanii.

F, Complete canals in the pleural area. Cast was made by draining epoxy off valve prior to curing. Scale bar = 200 pm. G, Close-up of canals
in the pleural area. Scale bar = 100 pm. H, Composite image showing horizontal canal pattern. Scale bar = 1 mm. Key: lAc, jugal area
channel; M, megalaesthete chamber; m, micraesthete canal; SRc, slit ray channel; all other abbreviations as in Fig. lA.

AESTHETE CANAL MORPHOLOGY IN THE MOPALllDAE

57

also fig. 2i in Fernandez et al. (2007)). Gently expanding
megalaesthete chambers connect to these subsidiary canals
(Figs. 3A, 3D, 3G, 4F-H; also fig. 2c, i in Fernandez et al
(2007)). Megalaesthete chambers begin as a short length of
canal with a diameter of about 12-15 pm, before gently
flaring out as they continue down towards the horizontal
canals. At least four micraesthete canals (about 2-4 pm in
diameter) trend in a straight, slightly angled to vertical man-
ner to enter each megalaesthete chamber where it first
reaches maximum diameter (Fig. 4F). At the top of the casts
of the micraesthete canals and megalaesthete chambers are
cup-shaped protuberances (Figs. 3G, 4G) that appear to be
casts of subsidiary and apical caps, respectively.

In the jugal area of dorsal casts, some horizontal canals
have a more flattened appearance and project upward {i.e.,
turn down towards the ventral valve surface in life). The
canals that exit at, or very close to, the jugal sinus, on the
other hand, have a circular cross-section. On the ventral
casts, the corresponding area (referred to as the ventral jugal
triangle in Fernandez et al. (2007)) has short lengths of
similarly flattened canals. These ventral canals occur in rows
that correspond to valve growth lines; about five or more
canals along some growth lines can be seen on the casts of
species such as Mopalia ciliata. Most of the Mopalia spp.
have a large number of canals that exit below the jugum, but
Mopalia acuta has only very few jugal area canals that exit
ventrally.

In Mopalia ciliata, Mopalia spectabilis, and Mopalia
swanii, the horizontal components of the slit ray canals oc-
cur through a large portion of the lateral areas (Figs. 4E, 41),
with canals on either side of the slit ray progressing at a low
angle relative to each other to meet and then turn down-
wards towards the slit ray. In contrast, the other species of
Mopalia have slit ray canals that only extend for a short
distance to either side of the slit ray, with the two horizontal
components meeting at an even lower angle (Fig. 3C; also
Fig. 2a, j in Fernandez et al. (2007)).

Tonicella (Acanthochitonina: Tonicellidae?) (Figs. 4A-D, 5)

This genus has large (ranging from about 40-75 pm
diameter), long, somewhat wavy, wide, very regularly
spaced, main horizontal canals that occur from the anterior
to posterior margin of the valves (Figs. 4A, 5A-B, 5F, 5L).
The spacing of canals is even more regular than in the Mo-
palia spp., giving the aesthete canal system in this genus the
most orderly appearance. The main horizontal canals regu-
larly meet gently-tapering megalaesthete chambers (e.g.. Fig.
4B, 4D, 5E, 5G, 51) that are themselves embedded with nu-
merous micraesthete canals. These megalaesthete chambers
are connected obliquely downward by short canals to the
main horizontal canals. The micraesthetes tend to be rela-
tively short and straight.

The jugal area canals begin as micraesthetes that con-

nect to gently tapering megalaesthete bulbs that connect to
long, occasionally somewhat sprawling (Fig. 4H), canals that
after a short cfistance begin to turn downward. I’hese canals
extend for a fair distance before merging with others into a
larger canal, which then extends for a distance before merg-
ing with an even larger one (Fig. 5J). All the species in this
genus had a high number (>30) of canals exiting ventrally
below the jugum.

The slit ray canals make up the entire lateral area of the
tonicellids, and have a high arc, with a consistent curve
towards each other (Figs. 4A, 4C, 5A, 5C, 5F, 5L). On the
ventral casts, the slit ray exits can be seen to begin as a line
near the apex, but often split up into two or more rows
towards the lateral margins of the valve.

Kathariiia (Acanthochitonina: Mopaliidae) (Figs. 6D-G)

The dorsal casts of Katliarina tunicata reveal straight,
regularly spaced, fairly large (about 30-40 pm diameter),
densely packed horizontal canals that are angled towards the
posterior apex in the lateral and pleural areas. The jugal area
is dominated by canals that exit ventrally (“jugal area chan-
nels” of Baxter and Jones (1981)) (Fig. 6F). The jugal area
canals have a high rate of merging before exiting at the
ventral surface below the jugum. The morphology of the
canals that lead into these horizontal canals is quite variable
within the two individuals observed. In most cases, the mi-
craesthetes connect to gently tapering megalaesthete cham-
bers that then connect through a short canal into a main
horizontal canal. In other cases, a large number of micraes-
thetes connect to long, narrow, often branching canals that
then lead to a main horizontal canal or into a megalaesthete
chamber (Fig. 6D). In the jugal area channels, numerous
long, straight micraesthetes merge into the large canals either
directly or by first merging into short, intermediate sized
canals (Fig. 6E).

The apical area canals are quite noticeable on the ventral
cast (Fig. 6G), perhaps because the apical area is more ex-
tensive in this species than in any other in the study. Many
large horizontal canals occur in the apical area, connecting
to micraesthetes that originate on the ventral or posterior
surface of the apical area. The slit ray canals were not clearly
seen in the SEM images of the casts.

Dendrochiton (Acanthochitonina: Tonicellidae?)

(Figs. 7A-F)

The dorsal casts show large (about 40-50 pm diameter),
long, fairly dense (about 60 pm between adjacent canals),
somewhat wavy main horizontal canals that occur from the
anterior to posterior margin of the valve (Figs. 7A, 7E).
These canals have regular intersections with gently expand-
ing megalaesthete chambers (Figs. 7C-D, 7F). Numerous
straight micraesthetes trend downward and attach at the
megalaesthete chamber all along its extent nearly up to its

58

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 4. SEM images of casts of aesthete canal systems for Tonicella iiisignis (SBMNH 369497) (A-D), Mopalia dliata (SBMNH 369501)
(E-H), and Mopalia spectabiUs (SBMNEl 369501 ) (I). B and C are photos of ventral casts; all others are dorsal casts. A-D, Tonicella insignis. j
A, Composite image showing the whole horizontal canal pattern. Scale bar = 1 mm. B, Close-up of the near-surface portion of a slit ray
canal. Scale bar = 100 pm. C, View of slit ray canals overlying (in this image) apical area canals in a different individual than shown in A-B.
Scale bar = 200 pm. D, Close-up of the lateral area near the posterior margin of the valve. Scale bar = 200 pm. E-H, Mopalia dliata. E,
Composite image showing the entire horizontal canal system (SBMNH 83326). Scale bar = 1 mm. F, View of canals along the posterior j

margin (in between the lateral area and apical area) (SBMNH 83325). Scale bar = 100 pm. G, Close-up of canals in lateral area (SBMNH I

83325). Scale bar - 100 pm. H, Close-up of canals in jugal area (SBMNI I 83325). Scale bar = 100 pm. 1, Mopalia spectabiUs, composite image -|

showing the whole horizontal canal pattern. Scale bar = 1 mm. Key: AA, apical area; others as in Figs. lA and 3. ^

AESTHETE CANAL MORPHOLOGY IN THE MOPALIIDAE

59

Figure 5. SEM images of casts of aesthete canal systems tor Tonicdla inannor'ea (SBMNH 369496) (A-D), Tonicclla liucata (SBMNH
369488) (E-H), and Tonicella lokii (I-L). All images are of dorsal casts, except C, D, G, J, and 1, which are images of ventral casts. A-D,
Tonicella mamiorea. A, Composite image showing complete horizontal canal system, with some remnant shell material in the middle. Scale
bar = 1 mm. B, Close-up of region of pleural area with some horizontal canals scraped aside. Scale bar = 200 pm. C, Close-up of dorsal
portion of slit ray canals. Scale bar = 100 pm. D, Close-up of some canals in the apical area. Scale bar = 100 pm. E-H, Tonicella lineata.
E, Close-up of canals in the pleural area, with some missing adjacent horizontal canals. Scale bar = 200 pm. F, Composite image showing
overall horizontal aesthete canal pattern. Scale bar = 1 mm. G, Close-up of canals in the apical area. Scale bar = 200 pm. H, Close-up of
jugal area canals. Scale bar = 100 pm. I-L, Tonicella lokii. 1, Close-up of dorsal portion of slit ray canals on the ventral cast (SBMNH 83319).
Scale bar = 100 pm. I, Close-up of jugal area canals on the ventral cast (SBMNH 83319). Scale bar = 500 pm. K, Close-up of canals in the
pleural (right) and lateral (upper left) region of the shell, with numerous horizontal canals missing, dorsal cast (SBMNH 83319). Scale bar
= 500 pm. L, Composite image showing the overall horizontal aesthete canal pattern of a different individual (SBMNH 83320). Scale bar
= 1 mm. Key: same as in Figs. lA and 3.

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AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 6. SEM images of casts of aesthete canal systems for Plaxiphora aiirata (SBMNH 83321) (A-C), Katliarina tutiicata (SBMNH 369494)
(D-G), Tomcia chilensis (SBMNH 369486) (H-K), and Cyanoplax hartwegii (SBMNH 83147) (L-M). Photos E, G, and 1 are of ventral casts;
all others are of dorsal casts. A-C, Plaxiphora aurata. A, Composite image showing pattern of the horizontal canal system. Scale bar = 1 mm.
B, Close-up of jugal area canals. Scale bar = 100 pm. C, Close-up of canals in the lateral area, near the posterior margin. Scale bar = 100
pm. D-G, Katharina tiimcata. D, Close-up of canals in the lateral area, along the posterio-lateral margin. Scale bar = 100 pm. E, Close-up
of dorsal portion of jugal area canals. Scale bar = 1 mm. F, Composite image showing horizontal canal system. G, Close-up of canals in
the apical area. Scale bar = 500 pm. H-K, Tonicia chilensis. H, Composite image showing horizontal canal system. Some remnant shell
material visible in middle anterior. Scale bar = 1 mm. 1, Close-up of slit ray canals. Different individual than in H, )-K. Scale bar = 100
pm. I, Close-up of lateral area along the posterior margin. Scale bar = 200 pm. K, Close-up of pleural area showing large chamber (that
presumably contained an ocellus). Scale bar = 50 pm. L-M, Cyanoplax hartwegii. L, Close-up of lateral area canals at the posterio-lateral
corner. Scale bar = 100 pm. M, Composite image showing horizontal canal pattern. Scale bar = 1 mm. Key: GC, giant chamber, presumably
that held an ocellus; others as in Figs. lA, 3, and 4.

AESTHETE CANAL MORPHOLOGY IN THE MOFALIIDAE

61

Figure 7. SEM images of casts of aesthete canal systems for Dendwchitoii linilatits (SBMNH 369493) (A-C), Dendwchitoii jlectciis (SBMNH
369492) (D-F), and Niittallochiton niirandiis (SBMNH 83324) (G-1). All images are of dorsal casts. A-C, Deiidwcliiton lindatus. A,
Composite image showing view of much of the system of horizontal canals, with some remnant shell material in the center. Scale bar =
1 mm. B, Close-up of the lateral area near posterior margin. Scale bar = 200 pm. C, View of pleural area, portion of anterior margin in
upper right. D-F, Dendwchitoii flecteiis. D, View ot pleural area showing a region with horizontal canals pulled aside, showing megalaesthete
and micraesthete canals. Scale bar = 1 mm. E, Composite image showing view oi the complete horizontal canal system. Scale bar = 1 mm.
F, Close-up view of region of D. Scale bar = 100 pm. C-I, Niittallochiton niimndns. C, Composite image showing one hall ol the entire
horizontal canal system. Scale bar = 1 mm. II, View of jugal area surface canals along the broken margin. Scale bar = 100 pm. 1, View ot
another region of jugal area surface canals along the broken margin. Scale bar = 100 pm. Key: same as in Figs. lA and 3.

62

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

intersection with the main horizontal canal (Eigs. 7B, 7D,
7F).

The jugal area channels are more prominent in Dendro-
chiton liridatiis than in Dendrochiton flectens (compare Figs.
7A and 7E). The jugal area channels begin as micraesthetes
that connect to sprawling, sub-cylindrical, horizontal canals
that then connect to cylindrical canals that turn downwards,
merging with others of their kind, before terminating at the
ventral valve surface below the jugum.

In one individual of Dendrochiton flectens, canals exited
at many different places on the ventral surface of the valve,
not just below the jugum and in the slit rays. Such a pattern
has not been seen in any of the twelve species studied by
Fernandez et al. (2007) or in the thirteen other species in this
study, and likely resulted from abnormal growth. The hori-
zontal components of the slit ray canals have a relatively
narrow lateral extent (Figs. 7 A, 7E) and form a single promi-
nent line of pores that make up the slit ray.

Plaxiphom (Acanthochitonina: Mopaliidae?) (Figs. 6A-C)

This species is characterized by micraesthete canals that
enter small narrow canals or gently tapering megalaesthete
chambers (Fig. 6C) that then connect with small horizontal
canals (about 15-20 pm diameter), which may merge a few
times until becoming relatively narrow, widely spaced, wavy
main horizontal canals (Fig. 6B). One individual examined,
whose valves were eroded, had a high density of tunnels
made by endolithic organisms. There are very few jugal area
canals apparent on either the dorsal or ventral casts of either
specimen.

The horizontal components of the slit ray canals extend
for a short width on either side of the slit ray, meeting at a
low angle (Fig. 6A), nearly sub-parallel to the slit ray, before
the merged canal trends upward on the dorsal cast (down-
ward in life) towards pores along the slit ray.

The apical area canals, seen on the ventral cast, show
relatively widely-spaced horizontal canals running most of
the length of the apical area. They originate as micraesthete
canals on the ventral surface of the apical area, near or along
the posterior margin. These small-diameter canals widen
and then, in many cases, merge with another horizontal
canal as they progress toward the anterior margin of the
apical area.

Nnttallochito7i (Acanthochitonina: Mopaliidae?)

(Figs. 7G-I)

The dorsal casts reveal primary horizontal canals (about
30-50 pm in diameter) throughout the entire interface be-
tween the tegmentum and articulamentum (Fig. 7G; also fig.
2g in Fernandez et al (2007)). There is a spacing of about
20-50 pm between canals, with about 18 canals per mm
along the horizontal plane. Micraesthetes ( 1-3 pm diameter)

connect to the elongate, indistinct megalaesthete chambers
that regularly connect, after a short distance, to the primary
horizontal canals (Fig. 7H; also fig. 2h in Fernandez et al
(2007)). The megalaesthete chambers have a diameter of
about 10-12 pm before widening to the same diameter as the
connecting canals (about 17-20 pm).

Nnttallochiton mimndus appears to have no, or very few,
jugal area canals and very few slit ray canals. The latter
condition contrasts with Nnttallochiton hyadesi, which has a
wider lateral extent of the horizontal components of the slit
ray canals (compare Fig. 7G with fig. 2g in Fernandez et al
(2007)).

Tonicia (Chitonina: Chitonidae) (Figs. 6F1-K)

The specimens of Tonicia chilensis show relatively nar-
row (about 30-40 pm diameter), widely spaced (about 50-
100 pm between canals), curving horizontal canals that re-
peatedly intersect canals from megalaesthete chambers. All
the megalaesthete chambers consist of bulbs that are embed-
ded with a relatively small number of micraesthete canals
(Figs. 6I-J). The main horizontal canals on either side of the
jugal area have a very high arc towards the lateral margin. In
a few locations, extremely large aesthete bulbs occur (much
larger than the typical megalaesthete chambers), that are
embedded with an immense number of micraesthete canals
(Fig. 6K).

The jugal area canals are abundant and cover most of
the valve surface (Fig. 6H). Many of these canals originate in
the pleural areas and may even extend to the lateral areas.
Such a wide extent of jugal area canals has not been seen in
any of the twelve species examined in Fernandez et al (2007)
or in any of the other thirteen species in this study. The slit
ray canals take up the entire lateral area (Fig. 6H) and have
a similar degree of convergence of canals as in the jugal area
channels of the central area.

DISCUSSION

Variation in aesthete canal characters

The results provide further evidence for variation in
aesthete canal morphology among chiton suborders, fami-
lies, genera, and often species (Table 1). Building from Fern-
andez et al (2007), the results herein confirm that many
chiton taxa at all taxonomic ranks are unified by synapo-
morphies (whether a character state is primitive or derived,
assessed by the cladistic analysis using Lepidopleurus cajeta-
nus as an outgroup) and that aesthete features have consid-
erable potential as phylogenetic characters at a number of
levels.

The cladistic analysis herein (Fig. 8) yields a phyloge-
netic hypothesis based solely on the broad morphology of

AESTHETE CANAL MORPHOLOGY IN THE MOPALIIDAE

63

the aesthete canal system across a large group of chitons,
mostly within the Suborder Acanthochitonina. The cladistic
analysis is meant to: ( 1 ) show the similarities and differences
in the aesthete canal system between a larger set of chitons;
(2) refine characters and character states from Eernandez et
al (2007), in light of the new information, to make the
characters/states more useful for future phylogenetic analy-
ses of a broader range of taxa; and (3) test a recent view of
mopaliid phylogeny (Eernisse, unpubl data).

Many other aspects of chiton morphology have been
used in phylogenetic studies of chitons, including egg hull
characters (Eernisse 1984, 1988, Sirenko 1993, 1997, 2006),
sperm morphology (Hodgson et al. 1988, Buckland-Nicks
1995, 2006, Buckland-Nicks and Hodgson 2000), radula and
radular tooth biomineralization patterns (Bullock 1985,
Brooker and Macey 2001, Saito 2003), gill placement char-
acters (Eernisse 1984, Sirenko 1993, 1997, 2006), and girdle
and gland characters (Sirenko 2006). Moreover, Okusu et al.
(2003) have been successful in using molecular sequences to
infer chiton phylogeny. Attempts to determine the phyloge-
netic relationships within the Polyplacophora should of
course incorporate as many of these characters as possible
(Sirenko 2006), and we would add aesthete canal morphol-
ogy to this list.

Aesthete canal morphology in the Mopaliidae

Eernandez et al. (2007) described how the mopaliids in
their study (Mopalia muscosa, Mopalia acuta, Placiphorella
velata, and Nuttallochiton hyadesi — but see below) are char-
acterized by wide, straight, closely spaced, primary horizon-
tal canals that exist through much of the valve length, as
well as regular merging of short, subsidiary branches from
the upper tegmentum with these primary canals. The sub-
sidiary branches connect with only slightly expanded mega-
laesthete “chambers” just below the valve surface that are
embedded with a large number of micraesthete canals. This
pattern was also seen in all the Mopalia spp., Katharina
tunicata, and some others (see below) in this analysis,
strengthening the hypothesis that such aesthete canal char-
acters typify mopaliids.

In addition, the results of this study show how Mopalia
ciliata, Mopalia spectahilis, and Mopalia swanii share aesthete
canal characters that are absent in Mopalia acuta, Mopalia
lignosa, and Mopalia tuuscosa, based on the observations that
the former group has a wider range of the slit ray canals and
a more fan-like arrangement of the horizontal canals that
flank the jugal area than the latter group.

Kathariua tunicata shows the typical mopaliid pattern of
long, straight, horizontal canals with gently tapering mega-

Table 1. Aesthete characters used in the PAUP analysis. Descriptions of characters and character states provided in Appendix 2. Key to
abbreviations: jug, number of canals that exit ventrally under jugum; lat, nature of slit ray canals in lateral area; lin, linear arrangement and
orderly spacing of megalaesthete bulbs; mgc, types of megalaesthete chambers; hgc, huge aesthete chambers; deh, density of horizontal
canals; she, connection between surface and main horizontal canals; tlj, divergence of horizontal canals flanking jugal area; hap, straight
horizontal canals; reg, regular merging of short canals into main horizontal ones; lam, lateral merging of main horizontal canals.

Species

Mopalia ciliata
Mopalia muscosa
Mopalia swanii
Mopalia acuta
Tonicella lineata
Tonicella insignis
Tonicia chilensis
Placiphorella velata
Dendrochiton flectens
Dendrochiton linilatus
Katharina tunicata
Nuttallochiton mirandus
Nuttallochiton hyadesi
Plaxiphora aurata
Ischnochiton textilis
Ischnochiton variegatus
Nuttallina californica
Cyanoplax hartwegii
Lepidopleurus cajetanus
Lepidozona mertensii
Lepidozona pectinulata

jug lat lin

1 2 0

1 2 0

1 2 0

1 2 0

1 1 0

1 1 0

1 0 0

1 2 0

? 2 0

1 2 0

1 2 0

0 1 0

0 2 0

0 3 0

1 0 0

1 0 0

1 3 0

1 3 0

1 4 1

1 0 1

1 0 1

mgc hgc deh

0 0 1

0 0 1

0 0 1

0 0 1

0 0 1

0 0 1

2 1 0

0 0 1

0 0 1

0 0 1

0 0 1

1 0 1

1 0 1

1 0 0

2 ? 0

2 0 0

1 0 0

1 0 0

3 0 0

2 1 0

2 1 0

she flj hap

1 2 1

1 0 1

1 2 1

1 0 1

1 2 1

1 2 1

1 0 1

1 0 1

1 0 1

1 0 1

1 1 1

0 0 1

0 0 1

0 0 0

1 0 0

1 0 0

0 0 0

0 0 0

2 0 0

1 0 0

1 0 0

reg lam

1

1

1

1

1

1

0

1

1

1

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

1

1

0

0

0

64

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

-Lepidopleurus caietanus (Lepidopleurma. Lepidoplcundac)
—Tonicia chitensis {Chiroama; Chuomdae)

—Ischnochiton textilis (Clutonina: Ischnochuonidac)
—Ischnochiton vanegatus (Chitonma; Ischnochuomdae)
—Lepidozona mertensii {Chitonma: Ischnochuomdac)
—Lepidozona pectinulata (Chitonma: Ischnochiromdac)
—Cyanoplax hartwegti (Acanthochiconma: Tonicdlidac)
—Nuttallina callfornica (Acanthochiconma: Tomoillidac)
—Plaxiphora aurata (Acandiodutomna: Mopaliidac?)
—Nuttalochiton mirandus (Acanthochitonina: Mopalndacf’)
—Nuttalochiton hyadesi (Acanthochitonina; Mopaliidac?)
—Dendrochiton lirulatus (Acantliochitonma; Tonicdlidac?)
—Dendrochiton flectens (Acanthochitonina Tonicdlidac?)
—Placiphorella ve/afa (Acanthochitonina; Mopaliidac)
—Mopalta acufa (Acanthndutonina; Mopaliidac)

—Mopalia muscosa (Acandiochiconma: Mopaliidac)
—Katharina tunicata (Acanthochitonina; Mopaliidac)
—Mopalia ciliata (Acanthochitonina: Mopaliidac)

—Mopalia swanii (Acanthochitonina; Mopaliidac)

— ToniCBlIa insignia (.\canthochiU)nina: Tonicdlidac?)

— Tonicalla lineata (Acandiod-Utonma: Tonicdlidac?)

Figure 8. Majority-rule consensus tree of the 96 most parsimonious
trees (with 23 steps) that resulted from the dadistic analysis
(PAUP) using only aesthete canal morphology. Numbers indicate
majority rule consensus values. The data matrix for the analysis is
shown in Table 1 and the characters and character states are listed
in Appendix 2. The taxonomic assignments are based on Sirenko
(1997 and 2006) but assignments that differ between Sirenko
(2006) and what is suggested by the phylogeny in Eernisse (unpubl.
data) are indicated with a question mark. Specifically, Eernisse’s
phylogenetic hypothesis suggests Dendrochiton and Tonicella are in
the Mopaliidac and Nuttallochiton is not.

laesthete bulbs, but it also has some long, relatively narrow,
horizontal, occasionally branching, subsidiary canals just be-
low the surface that are embedded with numerous micraes-
thetes along their length, a character also seen in Nuttallina
californicn and Cyanoplax hartwegii (previously Lepidochi-
tona hartwegii) (Fernandez et al. 2007).

Our previous study of chiton aesthete canal casts (Fer-
nandez et al. 2007) revealed that Nuttallochiton bears strong
similarities in the aesthete canal system with other members
of the Mopaliidae, and the results of this study are not in-
consistent with that interpretation. The dadistic analysis
suggests that Nuttallochiton is either a basal group within the
Mopaliidae or is the outgroup to that family (Fig. 8). Re-
gardless of which interpretation is preferred, it is clear that
the aesthete canal system of Nuttallochiton is intermediate
between those of (other) mopaliids and other members of
the Acanthochitonina such as Cyanoplax and Nuttallina.
The Nuttallochiton species in this analysis and the previous
one (Fernandez et al. 2007) share the large, closely spaced
horizontal canals with mopaliids, but also share with Cyano-
plax sprawling megalaesthete super-chambers that connect
to the main horizontal canals via a canal subparallel to the
surface, in addition to regular merging of short horizontal

canals in the posterior half of the valve. Fiowever, Nuttallo-
chiton differs from the members of the Acanthochitonina
so far examined in lacking a large number of micraesthetes,
and it differs from most other chitons so far examined in
having no or very few canals that exit underneath the
jugum. Overall, it shares similar aesthete canal characters
both with the Cyanoplax group and undisputed members of
Mopaliidae, but distinguishing between derived and plesio-
morphic similarities will be best considered in the context of
a more complete analysis of morphological and molecular
evidence.

Plaxiphora had been historically placed in the Mopali-
idae ie.g., Kaas and Van Belle 1987, Sirenko 2006), but
the results of this study suggest, as in Eernisse (unpubl.
data), that this genus belongs outside of this family. Plaxi-
phora has a very high ratio of micraesthetes/ megalaesthete,
and a greater amount of shell material between neighboring
horizontal canals than in the mopaliids. It also shares
sprawling megalaesthete chambers with Cyanoplax and
Nuttallina, other members of the Acanthochitonina, al-
though a broader study incorporating more members of this
suborder and the others is needed to better determine
whether these shared characters are primitive or derived
within this group. Regardless, Plaxiphora lacks the long,
densely packed, straight main horizontal canals that charac-
terize all mopaliids.

Tonicella shares many characters with the mopaliids
[e.g., large, closely-spaced, straight horizontal canals, same
megalaesthete chamber shape, regular merging of short sub-
sidiary canals with the primary horizontal canals). The re-
sults of the dadistic analysis are consistent with those of
Eernisse (unpubl. data), which suggested Tonicella should be
classified within the Mopaliidae. All four members of this
genus analyzed in this study share remarkably similar aes-
thete canal systems (in particular, they have the most orderly
arrangement of canals), and are each more similar to each
other than any is to any of the other species whose aesthete
canals have so far been described in detail.

The two species of Dendrochiton analyzed in this study
share many aesthete characters with other mopaliids, such as
the long, straight horizontal canals, non-descript shape of
the megalaesthete bulbs, and a relatively large number of
micraesthetes per megalaesthete (though not so many as in
Cyanoplax and Nuttallina). Consistent with Kaas and Van
Belle (1987), who listed Dendrochiton as a subgenus of Lepi-
dochitona, the Dendrochiton species in this study share some
characters of the aesthete canal system with Cyanoplax
hartwegii and Nuttallina californica, such as the presence of
long, narrow, horizontal canals that connect with megalaes-
thete chambers with many micraesthetes. Flowever, Dendro-
chiton shares more synapomorphies with the mopaliids than

AESTHETE CANAL MORPHOLOGY IN THE MOPALIIDAE

65

it does with Cyanoplax and Niittallirm, consistent with the
results of Eernisse (unpubl. data).

Dendrochiton and the other members of the Mopaliidae
share many overall similarities in aesthete canal system with
those of fellow members of the Acanthochitonina, Cyano-
plax, and Nuttalliua. These similarities include a high density
of horizontal canals (though higher in mopaliids), high
number of micraesthetes per megalaesthete (though higher
in Cyanoplax/ Nuttallma), and a regular merging of subsid-
iary canals with the main horizontal canal. In fact, the hori-
zontal canal system drawn for Lepidochitona cinerea by
Knorre (1925, fig. 37, as Trachydermon cinereus) is similar to
that of mopaliids (Fig. IF): relatively straight horizontal ca-
nals in the pleural area that extend most of the valve’s length,
with a regular merging of subsidiary canals, and with a rela-
tively wide extent of the slit ray canals. Some of these simi-
larities may provide evidence for grouping Mopaliidae and
Lepidochitonidae [e.g., Lepidochitona, Cyanoplax, Nuttal-
lina) as a subclade within Acanthochitonina, or might be
plesiomorphic features for Acanthochitonina, with differ-
ences noted in Plaxiphora and Niittallochiton best considered
derived features. Future studies of the aesthete canal system
in other members of the Acanthochitonina, and the combi-
nation of these data with a wider range of morphological and
molecular evidence should reveal which interpretation is
more likely.

Conclusions

The results provide further evidence that characters of
the aesthete canal system are phylogenetically informative at
a number of taxonomic levels. This study approximately
doubles the total number of comparisons possible, now 26
species, when these data are combined with those in the
study by Fernandez et al. (2007). This present study is also
significant in providing some of the first morphological evi-
dence corroborating a new proposal based on molecular
evidence. Specifically, variation in aesthete canal morphol-
ogy is largely consistent with the new classification of the
Mopaliidae proposed by Eernisse (unpubl. data), which ex-
cludes the genera Plaxiphora and Niittallochiton from Mo-
paliidae (although the placement of Niittallochiton is uncer-
tain with respect to the Mopaliidae based on aesthete canal
morphology alone) while including Tonicella and Dendro-
chiton within this family. Some characters in common be-
tween members of Mopaliidae and Lepidochitonidae could
reflect synapomorphies for uniting these families within
Acanthochitonina. More resolution is expected with the ad-
dition of other chiton species in future analyses of aesthete
canal morphology, and the combination of these data with
other morphological and molecular evidence will help elu-
cidate relationships within Polyplacophora.

ACKNOWLEDGMENTS

Daniel Geiger provided additional and extensive train-
ing to M)V and CZF on the SEM and indispensable advice
on technic]ues to improve image quality. Hank Chaney,
SBMNH, provided most of the specimens used in this study
and permitted the use of the SBMNH SEM. We also thank
Kevin McKeegan for his generosity in allowing MJV and
CZF to use the SEM at the UCLA Department of Earth and
Space Sciences. Michelle Hopkins provided additional help
with the SEM at UCLA ESS. Roger Clark previously identi-
fied many of the SBMNH chitons used in this analysis. Wil-
liam Hewson helped locate specimens of Niittallochiton and
Plaxiphora. Susanne Lockhart collected the specimens of
Nuttallochiton and Plaxiphora and sent them to DIE. Plaxi-
phora was collected during the ICEFISH (International Col-
laborative Expedition to collect and study Fish Indigenous to
Sub-antarctic Habitats) cruise led by H. William Detrich,
and Niittallochiton was collected during the AMLR (Antarc-
tic Marine Living Resources) cruise led by Dr. Christopher
Jones. Boris Sirenko and an anonymous reviewer provided
suggestions that greatly improved the general focus and
many details of the paper. Comments from the editors Ken
Brown and Cynthia Trowbridge also greatly improved the
clarity and overall quality of the paper.

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Submitted: 30 lanuary 2007; accepted: 24 November

2007; final revisions received: 10 February 2008.

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AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Appendix 1. Collecting and locality information for the chitons used in this study.

Species

Accession

#(s)

Collector(s)

Date

collected

Locality notes

Dendrochiton flectens

SBMNH

369492

George Hanselman

1973

Underside of rocks during a -0.76 m tide.
Cactus Island, Washington

Dendrochiton lindatus

SBMNH

369493

George Hanselman

1971

Intertidal of Ensenada Blanca, Baja
California Norte, Mexico

Katharina tunkata

SBMNH

83323

Michael Vendrasco

1999

Rocky intertidal, Cambria, California

Katharina tunkata

SBMNH

369494

George Hanselman

1972

Vancouver Island, British Columbia, Canada

Mopalia spectabilis

SBMNH

369491

Spencer Thorpe

1965

Morro Bay Harbor breakwater, California

Nuttallochiton mirandus

SBMNH

83324

Susanne Lockhart

2006

235 m depth, about 100 km south of
Penguin Island, Antarctic

Plaxiphora aurata

SBMNH

83321-83322

Susanne Lockhart

2004

Intertidal, Tristan da Cunha, Sub-Antarctic

Tonicella lokii

SBMNH

83319-83320

Christine Fernandez
and Michael Vendrasco

2006

Rocky intertidal, Cambria, California

Tonicella insignis

SBMNH

369497

Roger Clark

2000

Unalaska Island, Aleutian Islands, Alaska,
5-10 m depth

Tonicella lineata

SBMNH

369488

Spencer Thorpe

1965

Anacortes, Washington

Tonicella marmorea

SBMNH

369496

Ron McPeak

1977

Underside of rocks, 5-10 m depth. Seal
Island, Nova Scotia, Canada

Tonicella marmorea

SBMNH

369495

Norm Curran

1964

Newagen, Maine

Tonicia chilensis

SBMNH

369486

Hank Chaney

2004

Under small rocks in tidepools, Cobija, Chile

Mopalia ciliata,

SBMNH

George Hanselman

Unknown

California (additional details unknown)

Mopalia ligtwsa, 83325-83330

and Mopalia swanii

Appendix 2. Description of aesthete characters and
character states used in the cladistic analysis

1. Number of canals that exit ventrally under the jugum
(jug): (0) 0-30, (1) >30.

Comments: area is the same as the “ventral jugal tri-
angle” of Eernandez et al. (2007, fig. 1) and can be seen as
the number of “jugal area channels” as defined in Baxter and
Jones (1981, 1984). The number of canals in this area can be
inferred from the number of pores seen on the ventral sur-
face of valves in this region, the canal pieces in this region of
the ventral cast, and in some cases in the dorsal cast, seen as
upturned, typically flattened canals in the jugal area.

2. Nature of slit ray canals in lateral area (lat): (0) sparse
and highly curved, (1) dense and highly curved, (2) dense
and not highly curved, (3) sparse and not highly curved, (4)
no slit ray canals.

Comments: refers to the extent of the horizontal por-
tions of the slit ray canals that occur at the tegmentum/

articulamentum interface. On the dorsal casts, these canals
can be seen to merge parallel to the slit ray before trending
downwards (in life; upwards on the dorsal cast) to a slit ray
pore. This character is similar to character 8 (hcc: degree of
horizontal canal curvature towards diagonal line) in Eern-
andez et al. (2007), although the divisions between character
states are herein refined to match natural character state
boundaries in the now larger taxon set.

3. Linear arrangement and orderly spacing of megalaes-
thete bulbs (lin): (0) absent, (1) present.

Comments: refers to well-organized anterior-posterior
zones of megalaesthete chambers. This character can best be
seen in some photos of Lepidopleiirus cajetanus in Eernandez
et al (2007), which suggests that this character may be
primitive in the crown group Polyplacophora. This character
is the same as character 9 (apz: canals differentiated into
anterior-posterior columns) in Eernandez et al (2007).

4. Types of megalaesthete chambers (mgc): (0) type A,
(1) type B, (2) type C, (3) type D.

AESTHETE CANAL MORPHOLOGY IN THE MOPALIIDAE

69

Comments: the megalaesthete chamber types are illus-
trated in Pig. 7. Type A is a gently tapering chamber that
only has a subtle bulb shape. Type B is a more sprawling
chamber whose micraesthetes often merge before entering it.
Type C is widest in the middle, with gradual tapering on
both ends. Type D has an elongate form tapered sharply on
both ends, like a sausage. Note this character refers to the
typical shape of the megalaesthete chamber. Most species
have at least some variation in the appearance of these cham-
bers. Character 3 (bib: megalaesthete bulbs in central area)
from Fernandez et al. (2007) makes up a portion of this
newly expanded character.

5. Huge aesthete chambers (hgc): (0) absent, (1) pre-
sent.

Comments: these are much larger than typical mega-
laesthete chambers and may be modified or merged mega-
laesthete chambers. These may contain ocelli. They are
sparsely and apparently randomly distributed in Tonicia and
are regularly distributed in large granules in Lepidozona. This
character is similar to character 7 (hmc: huge aesthete cham-
bers in large granules) in Fernandez et al. (2007), but is more
broadly defined to allow the large chambers in Tonicia and
Lepidozona — which appear homologous — to be coded the
same.

6. Density of horizontal canals (deh): (0) very low
(much visible space between canals), (1) low (some visible
space between canals), (2) high (little visible space between
canals).

Comments: refers to the density of primary horizontal
canals at the tegmentum/articulamentum interface. This
character is the same as that of the same number and code
in Fernandez et al. (2007).

7. Typical connection between surface canals (e.g.,
megalaesthete chambers) and main horizontal canals (she):
(0) long canal that is, in part, parallel to the surface, ( 1 ) short
canal, oblique to surface, (2) each horizontal canal connects
to only one megalaesthete bulb.

Comments: refers to the typical portion of the canal
between the surface chambers and horizontal canals at the
tegmentum/articulamentum interface. In Lepidopleunis
cajetanus, each horizontal canal connects to only one mega-
laesthete bulb, making it difficult to compare with the other
taxa (i.e., it is difficult to say where the connecting canal
“ends” and the horizontal canal “begins”). For this reason, L.
cajetanus was coded as having a unique state (2).

8. Divergence of horizontal canals flanking the jugal
area (flj): (0) absent, (1) tilted towards apex, (2) tilted away
from apex.

Comments: refers to the set of main horizontal canals in
the pleural area immediately adjacent to the jugal area. In
some cases the angle of divergence (from the bisecting line)
is high. Those canals that are tilted away from the apex are

often arced and bend back towards the apex. This character
is a modification/refmement of character 1 1 (doc: direction
of convergence of horizontal canals in lateral area) in Fern-
andez et al. (2007).

9. Straight (or regularly wavy) horizontal canals from
anterior to posterior margins (hap): (0) absent, (1) present.

Comments: this character can also be read as whether
main (or primary) horizontal canals extend nearly the entire
length of the valve.

10. Regular merging of short canals into main horizon-
tal canals (reg): (0) absent, (1) present.

Comments: refers to whether there is a high rate of
merging of obliquely-oriented, connecting canals from the
megalaesthete chambers along the length of the main hori-
zontal canals.

1 1. Lateral merging of main horizontal canals (lam): (0)
absent, ( 1 ) present.

Comments: refers to a high rate of lateral merging of
main horizontal canals, especially in the posterior portion of
the valve.

General comments about characters: many of the char-
acters used in Fernandez et al. (2007) were modified herein
(see above) and some were excluded from this analysis. In
some cases character states were modified to better match
natural boundaries in the now larger taxon set. Character 1
from Fernandez et al. (2007) (age: aesthete/granule correla-
tion) was excluded because granules in many of the species
examined are often indistinct, making it difficult to assess
homology. Character 2 from that paper (are: megalaesthete
canal morphology/pattern differ by valve area) was not used
because it correlates with character 2 (lat) in this analysis
and we decided against indirect weighting of that character.

Amer. Maine. Bull. 25; 71-76 (2008)

Mopalia kennerleyi Carpenter, 1864, a forgotten species and its southern analogue
Mopalia ciliata (Sowerby, 1840)"^

Roger N. Clark*

Department of Invertebrate Zoology, Santa Barbara Museum of Natural History, 2559 Puesta del Sol Road,

Santa Barbara, California 93105-2936, U.S.A., insignis_one@yahoo.com

Abstract: The hairy chiton Mopalia kennerleyi Carpenter, 1864 is distinguished from its congener Mopalia ciliata (Sowerby, 1840), and the
identity of Chiton wosnessenskii von Middendorff, 1847 is clarified. Mopalia kewterleyi and M. ciliata are distinguished by setae structure,
valve sculpture, and radular teeth. Their characteristics are illustrated and discussed, and their distributions defined.

Key words: chiton, sibling species, California, Polyplacophora, mollusc

The examination of several hundred lots of what has
been regarded as Mopalia ciliata (Sowerby, 1840) from
throughout its recorded range of Alaska to Baja California
revealed that two similar but distinctive species could be
distinguished by setae structure: Mopalia ciliata from south-
ern California and Baja, and a northern species ranging from
central California, north to the Aleutian Islands in Alaska,
for which Mopalia kennerleyi Carpenter, 1864 appears to be
the oldest available name. These distinctions in setae and
name for the northern species have already been noted and
illustrated by Eernisse et al. (2007). Recent molecular work
by Kelly et al. (2007) and Kelly and Eernisse (2007) have
clearly verified this conclusion.

It is not new to consider northern specimens as distinct.
Pilsbry (1892; 305) distinguished Mopalia ciliata, from what
he considered its variety M. c. wosnessenskii (von Midden-
dorff, 1847), by the “much fainter sculpture” and by the
latter’s lack of “white thorns or spines (spicules) near the
base of the setae.” As Middendorff s name is currently re-
garded as a synonym of M. ciliata, one would first expect
that this name should be revived for the northern taxon.
However, an examination of the lectotype (fig. 1; ZISP
N834) (designated by Sirenko, pers. comm., October 2007),
the larger of two syntypes of Middendorff s Chiton wosnes-
senskii (Pig. 1) revealed that it was instead a specimen of
Mopalia hindsii (Sowerby MS, Reeve, 1847). The type local-
ity for C. wosnessenskii is given as Atka Island in the Aleu-
tians (52°11'57"N, 174°12'48"W); however, M. hindsii does

' Mailing Address: 3808 E. Pinehurst Drive, Eagle Mountain, Utah
84005-6007, U.S.A.

not occur in the Aleutians. Middendorff s type specimens
were said to have come from both Atka Island and Sitka,
Baranof Island, SE Alaska (57°08'N, 135°55'W), the Russian
Capitol of Alaska during the early 1800s and the type locality
of many of Middendorff s types. Undoubtedly both speci-
mens came from Sitka. The western-most distribution of M.
hindsii is in the vicinity of Kodiak Island, in the Gulf of
Alaska (57°N, 154°W). The question of name priority for
these two nominal taxa is a matter for further investigation.

Pilsbry (1892) considered Mopalia kennerleyi Carpenter,
1864 to be a synonym of Mopalia ciliata, an assignment
followed by Burghardt and Burghardt (1969), Smith ( 1977),
and Kaas and Van Belle (1994).

Although the type of Mopalia kennerleyi is lost (Smith
1977, T. Nickens, USNM, pers. comm. October 2003),
there can be little doubt from Carpenter’s original descrip-
tion and the type locality of “Puget Sound” as to which
species he was referring. Mopalia kennerleyi Carpenter, 1864
is thereby reinstated as the oldest available name for north-
ern species.

MATERIALS AND METHODS

Specimens of ^"Mopalia ciliata” from my own collection,
comprising more than one hundred lots from the Aleutian
Islands to Baja California, were separated into two distinc-
tive types of girdle setae using a dissecting microscope (as in
Clark 1991). Setae and radula from each of these two po-
tential species were prepared and examined with a scanning
electron microscope (SEM) at the Biology Department of
Southern Oregon University, using methods described by

* From the symposium “Advances in Chiton Research” presented at the joint meeting of the American Malacological Society and Western
Society of Malacologists, held 29 July to 3 August 2006 in Seattle, Washington.

71

72

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 1. Chiton wosnessenskii von Middendorff, 1847. Lectotype,
ZISP N834.

Mopalia ciliata wosnessenskii (Middendorff): Abbott
1974: 305.

Mopalia ciliata (Sowerby): Burghardt and Burghardt
1969: 26 (in part); Smith 1977: 250 (in part); Put-
man 1980: 122 (in part); Baxter 1987: 105; Kaas and
Van Belle 1994: 222 (in part).

Diagnosis

Medium sized (to 6.5 cm), oval chitons; valves subcari-
nated to rounded, depressed to moderately elevated, weakly
sculptured. Girdle with strap-like or trough-shaped setae,
bearing two rows of pointed spicules to about 250 pm in
length. Valves variously patterned with green, black, brown,
yellow, red, and white.

Clark (1994). The extensive collections at the Los Angeles
County Museum of Natural History, California Academy of
Sciences, San Diego Natural Histoiy Museum, Santa Barbara
Museum of Natural History, and the Royal British Columbia
Museum were also examined. The higher level systematics
used here follows Sirenko (2006).

Acronyms used in the text are as follows: ZISP, Zoo-
logical Institute, Academy of Sciences, Saint Petersburg;
LACM, Los Angeles County Museum of Natural History;
RNC, Roger N. Clark, personal reference collection.

SYSTEMATICS

Class: Polyplacophora Gray, 1821

Order: Chitonida Thiele, 1910

Suborder: Acanthochitonina Bergenhayn, 1930

Lamily: Mopaliidae Dali, 1889

Genus: Mopalia Gray, 1847

Type Species: Chiton hindsii Sowerby, MS, Reeve, 1847,
by subsequent designation.

Mopalia ciliata (Sowerby, 1840)

Mopalia kennerleyi Carpenter, 1864

Mopalia kennerleyi Carpenter, 1864
(Ligs. 2-6)

Mopalia kennerleyi Carpenter, 1864: 648; Eernisse et al.

2007; Kelly and Eernisse 2007; Kelly et al. 2007.
Mopalia grayii Carpenter, 1864: 603 (noni. niid.).
Chaetoplenra thouarsiana de Rochebrune, 1882: 191.
Mopalia ciliata var. wosnessenskii (Middendorff): Pils-
bry, 1892: 305; Leloup 1942: 49.

Mopalia muscosa kennerleyi Carpenter, 1864; Dali 1921:
195; Oldroyd 1924: 197; Oldroyd 1927: 306.

Description

Chitons of medium size, neotype designated herein (Eig.
2) 49 X 27.5 mm; shell oval, subcarinated to rounded, low to

I

Figure 2. Mopalia kennerleyi Carpenter, 1864. Neotype, LACM
2886. Scale bar = 10 mm.

A FORGOTTEN MOPALIA

73

moderately elevated, beaked. Head valve and lateral areas of
intermediate valves defined with rows of low, weak, fiattened
pustules; interstices of head valve and surface of lateral areas
with radial rows of low, broad pustules, often coalescing into
irregular zigzaging costae; central areas with smooth to
granular, longitudinal (gently, posteriorly curved) ribs, or
rows of low, coalescing, oval pustules. Tail valve small, oval,
about twice as wide or more than long, with distinctive wide
posterior sinus.

Girdle wide, usually more than one half the width of
intermediate valves, notched posteriorly; dorsal surface bear-
ing short (2-3 mm), slender trough-like or flattened (strap-
like) setae (Figs. 3-5) with (normally) two rows of slender,
curved, sharp spicules to about 250 pm in length (Figs. 4-5);
ventral surface of girdle covered with minute, broad, flat-
tened, distally pointed spicules to about 125 x 34 pm; mar-
gin of girdle with similar, but longer spicules, to about
180 pm.

Radula (Fig. 6), typical of a member of Mopaliidae, with
large, robust major lateral teeth, bearing tridentate denticle
caps; central cusp the longest, inner cusp slightly shorter,
and outer cusp only about one third as long as the central;
central tooth subquaudrate, tapering proximally, and in-
dented slightly just below cutting edge.

Ctenidia merobranchial, abanal, extending about 80%
of foot length, from beneath valve two to the suture of valves
seven and eight; about 42 per side in animals 50 mm in
length.

Color: Valves variously patterned (Figs. 7-12) with
green, brown, yellow, black, red, and white, often variegated,
mottled, or suffused. Entire valves or portions one or more
valves often unicolored. Girdle yellowish or brown.

Type material

Type lost (Smith 1977); NEOTYPE, LACM 2886 (leg.
S. R. Thorpe, 1 July 1965) (Fig. 2).

Figure 4. Mopalia kemierkyi. SEM image of seta. Scale bar =
250 pm.

Figure 5. Mopalia kemierleyi. SEM image of seta. Scale bar =
250 pm.

Figure 3. Mopalia kennerleyi. Close-up of girdle. Scale bar = 3 mm.

Type locality

“Puget Sound”, herein restricted to Tacoma Narrows,
Pierce County, Washington, U.S.A. (47°16'N, 122°31'W),
intertidal.

Distribution

Mopalia kennerleyi is a North American boreal species,
occurring from the southern Bering Sea (to about 54°N) and
Aleutian Islands (west to Attu Island, 173°14'E; LACM 79-
72) and Gulf of Alaska (north to 61°N) south to Monterey
Bay, California (36°36'N; RNC 1988), but rare south of San
Mateo County, California (37°10'N), where it begins to be
replaced by the similar Mopalia ciliata. In Monterey Bay it is
always subtidal, at about 10-15 m.

74

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 6. Mopalia kennerleyi. SEM image of radula. Scale bar =
500 |am.

Mopalia ciliata (Sowerby, 1840)

(Eigs. 7-10)

Chiton ciliata Sowerby, 1840: 289
Mopalia ciliata (Sowerby): Pilsbry 1892: 303; Leloup
1942: 49; Burghardt and Burghardt 1969: 26 (in
part); Abbott 1974: 401; Putman 1980: 122 (in
part); Clark 1991: 312; Kaas and Van Belle 1994:
222 (in part); Eernisse et al. 2007; Kelly and Eer-
nisse 2007; Kelly et al. 2007.

Diagnosis

Medium sized (to 5.0 cm), oval to elongate-oval chiton;
valves carinated, moderately elevated, finely sculptured.
Girdle with short, strap-like setae bearing (normally) four
rows of large, white, pointed spicules. Color variable, often
suffused or mottled with pale green, white or dark brown,
sometimes olive, with white, orange, red, and blue markings.

Description

Chitons of medium size, neotype 46 x 28 x 8 mm (Fig.
7). Shell elongate-oval, valves carinated, moderately elevated,
beaked. Head valve and lateral areas of intermediate valves
defined with radial rows of fairly heavy, round or irregular
pustules; interstices of head valve and surface of lateral areas
with large, low, round or irregular pustules, which may be
spaced or touching; posterior edge of lateral areas dentated
by oval pustules; central areas with longitudinal rows of low,
oval pustules or smooth ribs. Girdle moderately wide, about
1/2 to 3/4 the width of valves, notched posteriorly; dorsal
surface spiculose, spicules to about 250 x 25 pm, occurring
(mostly) singularly, or in groups of two to four, as well as

short (2-3 mm), strap-like setae (Figs. 8-9) bearing four rows
or robust, white spicules, to 600 pm in length (often present
only on lower half of seta); ventral surface of girdle with
scattered spicules to 150 x 32 pm; margin of girdle with
similar but longer spicules to about 250 pm. Radula (Fig.
10), with robust major lateral teeth bearing tridentate den-
ticle caps; central cusp longest, inner one slightly shorter,
and outer cusp about 1/3 as long as the inner one. Ctenidia,
merobranchial, abanal, extending about 80% of foot length,
from about the suture of valves two and three, to the suture
of valves seven and eight, 34 per side in neotype.

Color: Variable, neotype green with black-brown mark-
ings. This according to Pilsbry (1892) is the “typical” color-
ation of the species, based presumably on Pilsbry’s exami-

Figure 7. Mopalia ciliata (Sowerby, 1840). Neotype, LACM 2885.

A FORGOTTEN MOPALIA

75

Figure 8. Mopalia ciliata. Close-up of girdle. Scale bar = 3 mm.

Figure 9. Mopalia ciliata. SEM image of setae. Scale bar = 500 pm.

nation of Sowerby’s material or original description. From
the material examined, this is the typical color morph of
southern California (Los Angeles and Orange Counties)
populations.

Type material

Type, BMNFl? Not located (K. Way, in lit., 26 )uly
2000). Presumed lost. NEOTYPE, LACM 2885 [leg. Spencer
R. Thorpe, Jr., low intertidal on rock, 11 December 1958).

Type locality

White Point, Los Angeles County, California, U.S.A.
(33°42.8'N, 118°08.3'W).

Distribution

Mopalia ciliata is a North American, warm-temperate
species, occurring between latitudes 38°40'N (RNC 1308),
Sonoma County, California and 30°20'N (LACM 66-3),
Rancho Soccoro, Baja California. The species is rare north of

Figure 10. Mopalia ciliata. SEM image of radula. Scale bar =
500 pm.

San Mateo County, California (37°10'N), where it is mostly
replaced by Mopalia kennerleyi.

DISCUSSION

Although Pilsbry (1892) separated these two taxa as
subspecies (using von Middendorff s Chiton wosnessenskii
for the taxon presently regarded as Mopalia kennerleyi) based
on the large prominent, white spicules present on the setae
of M. ciliata and absent in “M. wosnessenskii,” recent workers
have had much difficulty separating the two.

The similarities between these two species are remark-
able, as they mimic each other in the form and sculpture ot
the plates, as well as their coloration. Hybrids are unknown
in Mopalia; none of the other eighteen Pacific coast species
exhibit this tendency. However, in central California, a few
specimens have been found that indicate further investiga-
tion into the possibility of hybridization might be warranted.

Mopalia ciliata is particularly puzzling, exhibiting a
myriad of forms, some with broad plates, some rather nar-
row, and the sculpture varying from delicate and nearly
smooth to quite coarse. Additionally, many animals in the
Monterey Bay area often resemble Mopalia spectahilis Cowan
and Cowan, 1977, with greenish (olive to turquoise) plates
marked with brilliant blue zigzag lines and red flecks, but
they are easily distinguished from that species by the gener-
ally broader outline, and the structure of the setae.

The most diagnostic character for distinguishing Mopa-
lia ciliata and Mopalia kennerleyi (as well as all other species
of Mopalia) is the structure of the setae. A comparison of the

76

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

setae (best taken from the central portion of the girdle)
reveals that the setae of Mopalia ciliata are flatter, broader,
and bear (when fully mature) four rows of large, robust,
sharp, curved, white calcareous spicules, to about 600 pm in
length. The setae of M. kennerleyi are more slender, trough-
shaped (often nearly tubular) and bear (when fully mature)
two rows of slender, slightly curved, sharp spicules, similar
to those in M. ciliata, but much smaller, reaching only about
250 pm in length. Another useful distinction is that the
posterior valve margin has denticulations in M. ciliata-, these
are lacking in M. kennerleyi.

The radulae of the two species are very similar, the teeth
are nearly identical in shape and proportion, but those of
Mopalia kennerleyi are proportionally about 20% larger than
those of Mopalia ciliata of the same size. Also the middle
cusp of the denticle cap of the major lateral teeth is slightly
longer in M. kennerleyi.

Taken together, these characters along with their geo-
graphic separation provide ample evidence that Mopalia cili-
ata and Mopalia kennerleyi are both valid species.

ACKNOWLEDGMENTS

I am grateful to Dr. Darlene Southworth, Biology De-
partment, SOU (Ashland, Oregon); Dr. James H. McLean
and Mr. Lindsey Groves, LACM; Dr. Paul Scott and Dr.
Henry Chaney, SBMNH (Santa Barbara, California); Dr.
Boris I. Sirenko, ZIAS; Miss Elizabeth Kools, CAS (San Eran-
cisco, California); Miss Kathie Way, BMNH (London, UK);
Dr. Patricia Beller and Mrs. Carole Hertz, SDMNH (San
Diego); Dr. Philip Lambert and Dr. Kelly Sendall, RBCM
(Victoria, British Columbia); Mrs. Leal Thorpe, El Cerrito,
California (for the gift of the extensive collection of her late
husband Mr. Spencer R. Thorpe); and Dr. Douglas J. Eer-
nisse, CSUF (Eullerton, California) for critically reading the
manuscript.

LITERATURE CITED

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Burghardt G. E. and L. E. Burghardt. 1969. A Collectors Guide to
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Clark, R. N. 1991. A new species of Mopalia (Polyplacophora: Mo-
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Oldroyd, I. S. 1927. The Marine Shells of the West Coast of North
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Stanford, California.

Pilsbry, H. A. 1892. Polyplacophora. In: G. W. Tryon, )r., ed..
Manual of Conchology. Vol. 14. Academy of Natural Sciences,
Philadelphia, Pennsylvania.

Putman, B. F. 1980. Taxonomic Identification Key to the Described
Species of Polyplacophoran Mollusks of the West Coast of North
America (North of Mexico). Report, Pacific Gas and Electric
Company, Department of Engineering Research. 411-79.342.

San Luis Obispo, California.

Sirenko B. 2006. New outlook on the system of chitons (Mollusca:
Polyplacophora). Venus 65: 27-49.

Smith, A. G. 1977. Rectification of west coast chiton nomenclature
(Mollusca: Polyplacophora). The Veliger 19: 215-258.

Submitted: 29 March 2007; accepted: 9 November 2007;

final corrections received: 26 February 2008

Amer. Make. Bull 25: 77-86 (2008)

Two new chitons of the genus Tripoplax Berry, 1919 from the Monterey
Sea Canyon’^

Roger N. Clark^

Department of Invertebrate Zoology, Santa Barbara Museum of Natural History, 2559 Puesta del Sol Road,

Santa Barbara, California 93105-2936, U.S.A., insignis_one@yahoo.com

Abstract: Recent deep-sea trawling in the Monterey Sea Canyon, California has brought to light two previously unknown bathyal chitons.
The new species, members of the family Ischnochitonidae, are placed in the genus Tripoplax Berry, 1919, here in raised to full generic rank
on the basis of morphological and ecological characteristics. Tripoplax calypso spec. nov. and Tripoplax cowam spec. nov. are described,
illustrated, and compared to similar species from the region.

Key words: Polyplacophora, new species, mollusc, Monterey Bay

The Monterey submarine canyon is a gigantic undersea
chasm, starting just offshore in less than 10 m of water, and
plunging to depths of more than 3000 m just 50 km offshore.
The Carmel Canyon is the southern branch of this system.
The fauna of these canyons is extremely rich and diverse,
and is being intensely studied by the Monterey Bay
Aquarium Research Institute and the Moss Landing Marine
Laboratories.

Deep-sea trawling in the canyons by the research vessels
USNS DE Steiguer ( 1975) and the R/V Point Snr (1994-1998)
have procured several specimens of two undescribed chitons
of the genus Tripoplax Berry, 1919. Tripoplax calypso spec,
nov. and Tripoplax cowani spec. nov. were taken at depths ol
650-1044 m on rocks and sponges. The new species are
compared to the similar Lepidozona retiporosa (Carpenter,
1864), Lepidozona scrobiculata (von Middendorff, 1847) and
Lepidozona golischi (Berry, 1919), and Tripoplax abyssicola
(Smith and Cowan, 1966), and Stenoseimis stearnsii (Dali,
1902), respectively.

Both new species are members of the genus Tripoplax
Berry, 1919, herein elevated to full generic status and char-
acterized by fine tegmental sculpturing, relatively small
girdle scales (-300 pm), and multiple slits in the insertion
plates of the intermediate valves, in contrast to the genus
Lepidozona Pilsbry, 1892 which has single-slitted intermedi-
ate valves and usually coarser (often pustulose or tubercu-
lose) sculpturing. Additionally, all members of Tripoplax are

' Mailing Address: 3808 E. Pinehurst Drive, Eagle Mountain, Utah
84005-6007, U.S.A.

cold northern or deep water inhabitants, distributed in the
northern Pacific Ocean between latitudes 36°N (off central
California, U.S.A.) and 37°N (northern Honshu, Japan) and
60°N in the Gull of Alaska and Okhotsk Sea, in cool tem-
perate to sub-arctic and bathy-abyssal waters, restricted to
temperatures below about 9 °C. Tripoplax reaches its greatest
diversity in the Aleutian Islands of Alaska, where seven spe-
cies are presently known. Members of Lepidozona are dis-
tributed nearly worldwide, and they typically inhabit
warmer, temperate to tropical waters, at depths of 400 m or
less. Most inhabit shallow (1-50 m) subtidal waters. Only
four species of Lepidozona are found in the Gulf of Alaska
north of 50°N: Lepidozona mertensii (von Middendorff,
1847), Lepidozona willetti (Berry, 1917), Lepidozona retipo-
rosa, and Lepidozona golischi. The genus is absent in the
Aleutian Islands, and only the species Lepidozona multi-
ganosa Sirenko, 1978 is found in the southern Okhotsk Sea,
near the southern Kurile Islands, north to Urup Island
(46°N). No species of Lepidozona occur in the north Pacific
region between 152°W, east of Kodiak Island, Alaska and
about 150°E, east of Urup Island, Kurile Islands, Russia.

Berry (1919) briefly defined Tripoplax for the Alaskan
species Isclmochito?i (Trachydermon) trifidus Carpenter,
1864, a species that Dali (1871) had erroneously placed in
the subgenus Ischnoradsia Shuttleworth, 1853 with Chiton
australis Sowerby, 1840. Realizing that these two taxa were
only distantly related. Berry separated the two species be-
cause of the rather smooth tegmental sculpturing and rela-
tively small girdle scales (to 315 x 250 pm) possessed by
I. trifidus, in contrast with the coarse sculpture and very

From the symposium “Advances in Chiton Research” presented at the joint meeting of the American Malacological Society and Western
Society of Malacologists, held 29 July to 3 August 2006 in Seattle, Wasliington.

77

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AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

large (to 600 pm in width), subcarinated girdle scales of
I. australis. Ischnoradsia is presently regarded as monotypic
subgenus for Ischuochiton australis (Sowerby) by Kaas and
Van Belle ( 1990). The validity of Trachydermon is somewhat
unclear, at the time of its very brief description and subse-
quent usage by Carpenter; it was an assemblage of several
unrelated species, presently placed in three different families,
without an original type designation. Kaas and Van Belle
(1985) considered it a synonym of Lepidochitona Gray, 1821,
but that may he invalid as well, and a re-evaluation of this
name, its validity, and position is clearly necessary (see
Palmer 1958: 284). Kaas and Van Belle (1987) used Tripo-
plax as a subgenus of Lepidozona Pilsbry, 1892 for members
of that genus with multiple slits in the insertion plates of the
intermediate valves. This combination was followed by Clark
(1991, 2000).

In its morphology and biogeography, Tripoplax appears
to be a natural assemblage. Molecular studies would be valu-
able to test this hypothesis and its relationship to Lepidozoua.

Acronyms used in the text are: CASIZ, California Acad-
emy of Sciences, Invertebrate Zoology; LACM, Los Angeles
County Museum of Natural History; ZIAS, Zoological In-
stitute, Academy of Sciences, Saint Petersburg, Russia; RNC,
Roger N. Clark personal collection.

SYSTEMATICS

Class: POLYPLACOPHORA Gray, 1821

Order: Chitonida Thiele, 1909

Family: Ischnochitonidae Dali, 1889

Genus: Tripoplax Berry, 1919

Type species: Ischuochiton (Trachydermon) trifidus Car-
penter, 1864, by original designation

Ischnoradsia Carpenter MS, Dali, 1871, non Shuttle-
worth, 1853; Gurjanovillia Jakovleva, 1952-, Albrech-
tia I. Taki, 1955

Expanded definition

Small to medium sized chitons (1-6.5 cm), elongate-
oval to broadly oval in outline; central areas with fine pitting,
net-like reticulation of cross threading or finely beaded lon-
gitudinal lirae, crossed by growth lines or very fine trans-
verse lirae. Radial areas with relatively weak or very fine
sculpturing; insertion plates of intermediate valves with two
to four slits. Dorsal girdle scales relatively small (200-320 pm
in length), smooth, or bearing minute riblets or striations,
and often mammillated at apices.

Tripoplax calypso spec. nov.

Tripoplax cowani spec. nov.

Tripoplax calypso spec. nov.

(Figs. 1-7)

Diagnosis

Small (to 17 mm), oval chitons; valves carinated, slopes
convex; lateral areas with three to five low, faint radial ribs
separated by weak sulci; central areas with 26-28 curved,
longitudinal riblets; girdle with imbicating, striated scales to
200 X 160 pm; radula with heavy, bidentate major lateral

Figure 1. Tripoplax calypso Clark, spec. nov. Paratype (RNC 2060),
scale bar = 10 mm.

TWO NEW SPECIES OF TRIPOPLAX

79

teeth. Color: valves and girdle dull reddish-brown with some
white patches.

Description

Holotype (Figs. 2-7) small (13.5 X 8.2 X 2.7 mm), oval,
moderately elevated; valves granular, carinated, side slopes
convex, tegmentum delicately sculptured. Head valve
(Fig. 2) semi-circular, posterior margin widely V-shaped,
bearing 23 low, faint (nearly obsolete), rounded radial ribs.
Intermediate valves (Figs. 3-4) oblong, about four times as
wide as long; lateral areas with three to five low, faint radial
ribs, separated by faint sulci; central areas with about four-
teen curved, longitudinal ribs per side, becoming obsolete at
the jugum; the ribs (when viewed dorsally) are seen to be
made up of faint transverse riblets with numerous raised
posterior extensions, which overlap the next rib in the series,
as if made up of overlapping drips; jugal areas show only
obsolete pitting. Tail valve (Fig. 5) relatively large, almost
diamond shaped; mucro ante-central, slightly raised; post-
mucronal slope concave; ante-mucronal area with obsolete
pitting; terminal area with about 21 low, faint radial ribs.
Articulamentum white, insertion teeth short, blunt; sutural
laminae short, round, connected across the jugal sinus by a
short, concave jugal plate with slits at edges; slit formula
11/2/13. Girdle narrow, about one fifth as wide as interme-
diate valve five; clothed dorsally with imbricating, oval, stri-
ated scales (Fig. 6) to about 200 x 160 pm, and bearing 15-16
rather weak riblets; margin of girdle with minute, pointed
spicules to 60 X 18 pm; ventral surface of girdle with radi-
ating rows of minute, rectangular scales to 100 X 15 pm.
Radula (Fig. 7) 6.0 mm long, bearing 40 mature rows of
teeth; rachidean tooth hourglass shaped, working edge about
50 pm wide; minor lateral teeth wing-shaped, with small,

Figure 2. Tripoplax calypso Clark, spec. nov. Holotype (LACM
2882). Head valve, scale bar = 5 mm.

Figure 3. Tripoplax calypso Clark, spec. nov. Holotype (LACM
2882). Intermediate valve fragments, scale bar = 5 mm.

Figure 4. Tripoplax calypso Clark, spec. nov. Holotype (LACM
2882). Intermediate valve fragments, scale bar = 5 mm. SEM image.

lateral extension near the anterolateral edge; major laterals
relatively large, with bidentate denticle cap, the inner cusp
about twice as long as outer cusp. Ctenidia holobranchial,
adanal about 17 per side. Color: dull reddish-brown with
white patches on terminal valves, and jugal areas of some
intermediate valves. Paratype (Fig. 1 ) agrees with holotype in
all aspects, but is larger (17.0 mm x 10.0 mm x 3.1 mm),
and has 18 ctenidia per side.

Type material: Holotype (LACM 2882); radula, girdle,
and intermediate valve fragment mounted on SEM

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AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 5. Tripoplax calypso Clark, spec. nov. Holotype (LACM
2882). Tail valve, scale bar = 5 mm.

Figure 6. Tripoplax calypso Clark, spec. nov. Holotype (LACM
2882). Dorsal girdle scales, scale bar = 500 pm.

viewing stub; head and tail valves, and intermediate
valve fragments separate, unmounted; body in
ethanol (leg. Roger N. Clark, 28 September 1995;
trawled R/V Point Siir).

Paratype (RNC 2060), whole animal in ethanol [leg. Roger
N. Clark, 6 March 1997; trawled, R/V Point Sur).

Type locality: California, Monterey County, Monterey
Submarine Canyon (36°45.163'N, 122°03.447'W),
650-700 m.

Habitat and ecology: The holotypes and paratypes were

Figure 7. Tripoplax calypso Clark, spec. nov. Holotype (LACM
2882). Radula, scale bar = 200 pm.

found living on large dead chunks of the massive,
ridged, hexactinellid sponges Aphrocallistes vastus
and Heterochone calyx. Another chiton, Stenosemus
stearnsii was also found in this habitat.

Four additional chitons were found on rocks in the
same trawls: Tripoplax cowani new species, Placi-
phorella pacifica Berry, 1919, Leptochiton mesogoniis
(Dali 1902), and Leptochiton sp.

Etymology: The name comes from Greek mythology,
Calypso, the nymph who hid Ulysses.

Remarks: At first sight Tripoplax calypso merely looks
like a deep-water specimen of Lepidozona retiporosa
(Fig. 8) it is only when examined under magnifica-
tion that the unique sculpture of the valves be-
comes evident. Still it might be passed off as a form
of the latter, or perhaps considered to be within the
considerable range of variation attributed to Lepi-
dozona scrobiculata [as Lepidozona siniidentata
(Carpenter in Pilsbry, 1892), Ferreira 1978, Kaas
and Van Belle 1987] (Fig. 9). Although the multiple
slits in the intermediate valves (lacking in both of
the previous species) and the very faint radial sculp-
ture serve to distinguish it. Additionally, the girdle
scales of T. calypso reach about 200 x 160 pm and
have 15-16 weak riblets, while those of L. retiporosa
reach only about 145 X 120 pm and have eight to
ten riblets, and the scales of L. scrobiculata reach
185 X 130 pm and have 10-13 weak riblets. Lepi-
dozona golischi (Berry, 1919) [Lepidozona scabrico-
stata (Carpenter) of Ferreira 1978, Kaas and Van

TWO NEW SPECIES OF TRIPOPLAX

81

Figure 8. Lepidozona retiporosa (Carpenter 1864). Scale bar = 10
mm. Eernisse coll., San luan Island, Washington, depth unknown.

Belle 1987, and Clark 1991, non L. scabricostata
(Carpenter, 1864)] (Fig. 10) might also be confused
with this species but is generally uniformly pale
orange, tan, or white in color; central areas are
ribbed, radial areas are rather flat, sometimes with
sulci, and bear relatively large, often scattered pus-
tules and differently proportioned girdle scales
reaching 130 x 220 pm and bearing 15-16 riblets.

Tripoplax cowani spec. nov.

(Figs. 11-14)

Ischnochiton ahyssicola Smith and Cowan, 1966 (in
part)

Figure 9. Lepidozona scwbiadata (von Middendorff, 1847). (RNC
244). Monterey Bay, California, 15 m, scale bar = 10 mm.

Diagnosis

Medium sized (to 4.5 cm) oval chitons; valves solid,
carinated, moderately elevated; terminal and lateral areas
with radiating rows of oval pustules; central areas with lon-
gitudinal riblets; girdle narrow, less than one sixth the width
of intermediate valve five; dorsal surface clothed with
small, blunt, smooth, rounded, subtriangular scales, to 300 X
275 pm. Color: white, often stained yellowish or black with
deep sea mineral deposits.

Description

Holotype (Fig. 1 1 ) of medium size (38 X 22.5 x 8 mm),
oval, moderately elevated; valves granular, carinated, tin-

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 10. Lepidozona golischi (Berry, 1919). (RNC 616). Cape
Blanco, Oregon, 34 m, scale bar = 10 mm.

beaked, slopes straight to convex; tegmentum strongly sculp-
tured. Head valve (Eig. 12) semi-circular, slope straight; pos-
terior margin widely V-shaped, posterior edge slightly
rounded; surface with 52 low, weak radiating ribs, capped
with a row of oval pustules, and separated by faint sulci;
intermediate valves (Fig. 13, valve V) oblong, about three
times as wide as long, eaves short; lateral areas with six to
eight radiating pustulose ribs, like those of the head valve;
central areas with about 36 longitudinal riblets (18 per
side), becoming obsolete at the jugum; jugum obsoletely
pitted. Tail valve (Fig. 14) slightly convex anteriorly,
rounded posteriorly, mucro ante-central, post-mucronal
slope concave; ante-mucronal area with 32 longitudinal rib-
lets (obsolete at jugum) post-mucronal area with about 43
radiating rows of pustules. Articulamnetum white, insertion

Figure 11. Tripoplax cowani Clark, spec. nov. Holotype (CASIZ
115832). Scale bar = 10 mm.

teeth short, blunt; sutural laminae short, rounded connected
across the moderate jugal sinus by a short, concave jugal
plate with slits at edges; slit formula 16/1-3/14. Girdle
(Fig. 15), narrow, about one sixth as wide as valves; dorsal
surface covered with crowded, juxtaposed, smooth, blunt,
rounded (subtriangular) scales to about 300 x 275 pm; mar-
gin of girdle with pointed spicules to about 250 x 20 pm;
ventral surface covered with radiating rows of minute, rect-
angular scales 200 X 25 pm. Radula (Fig. 16) 13.5 mm long,
bearing 43 mature rows of teeth; rachidean tooth about 150
pm long, broadly dilated anteriorly, working edge about 100
pm wide; minor laterals with small, anterolateral projection;
major laterals large, with bidentate denticle cap, inner cusp
twice as long as outer cusp. Ctenidia holobrachial, adanal,
extending from beneath suture of head and second valves, to
under tail valve, about 35 per side. Color: White, often
stained with yellowish or black deep sea mineral deposits.

TWO NEW SPECIES OF TRIPOPLAX

83

Figure 12. Tripoplax cowaiii Clark, spec. nov. Paratype (Clark 404).
Head valve, scale bar = 10 mm.

Figure 13. Tripoplax cowani Clark, spec. nov. Paratype (Clark 404).
Intermediate valve V, scale bar = 10 mm.

Paratypes agree with the holotype in all aspects, except
for variations in the number of radial ribs, and the number
of slits in the articulamentum, due to the size and age of the
specimens. The number of ribs on the head valves varies
from 42-71, those of the lateral areas, from five to nine, and
those on the tail valve from 32 to 43, slit formula range is
14-17/2-3/13-15. Paratypes range in size from 26.5 mm
(LACM 2883) to ca. 45 mm (CASIZ 001640).

Type material

Holotype, CASIZ 11583, whole animal (curled) in alco-
hol, radula mounted on SEM stub [leg. USNS DE Steiguer
1975); Paratypes, 2, CASIZ 010602 (same data as holotype);
1, RNC 404 (same data as holotype); 1, LACM 2883; 1, ZIAS
1936; 2, RNC 2065 (leg. Chris Mali, 22 October 1994;
trawled, R/V Point Siir, 650-700 m); 2, RNC 2121, west of
San Francisco Bay, California (37°37.464'N, 123°05.43'W)
(leg. R. N. Clark, 8 November 2000; trawled R/V Miller Free-
man, 651-674 m; NMFS 21-0012-212).

Figure 14. Tripoplax cowani Clark, spec. nov. Paratype (Clark 404).
Tail valve, scale bar = 10 mm.

Figure 15. Tripoplax cowani Clark, spec. nov. Paratype (Clark 404).
Dorsal girdle scales, scale bar = 500 pm.

Type locality

California, Monterey County, Carmel submarine can-
yon (36°45.3'N, 122°04.7'W), 954-1044 m.

Additional material

2, CASIZ 103675 and 025503, off Trinidad, Humboldt
County, California (41°05'N) (leg. R. Talmadge, 1972;
trawled, 432-720 m); 1, CASIZ 0198513 Swiftsure Bank,
Washington (leg. I. McTaggart Cowan and D. B. Quayle, 6
September 1964; trawled, 975 m) (Paratype oi Ischnochiton

84

AMERICAN MALACOLOGICAL BULLETIN

25 • 1/2 • 2008

Figure 16. Tripoplax cowani Clark, spec. nov. Paratype (Clark 404).
Radula, scale bar = 200 pm.

ahyssicola Smith and Cowan, 1966); 4, RNC 2108, off Del
Norte County, California (41°41.962'N, 125°00.732'W) {leg.
R. N. Clark, 23 October 2001; trawled R/V Miller Freeman,
855 m; NMFS 21-0112-106).

Distribution

Tripoplax cowani has been collected from the Swiftsure
Bank, Washington (48°30'N) to the type locality, Carmel
Bay, California (36°45'N) at depths of 432-1044 m.

Habitat

luveniles have been found on gravel (Smith and Cowan
1966, as Ischnochiton abyssicola); adults are found on large
rocks and boulders.

Etymology

It is with great pleasure that I name this species after my
friend and colleague. Dr. Ian McTaggart Cowan, of Victoria,
British Columbia, Canada.

Remarks

The similarities between Tripoplax cowani and Tripoplax
abyssicola (Figs. 17-19) in form, color, and sculpturing of the
valves are remarkable, and explain why the present species
has hitherto gone unrecognized. However, despite the simi-
larities in general appearance, the two species may be readily
distinguished by:

( 1 ) Body outline, T. cowani is much broader than T.
abyssicola, length 1.7 times width, compared to 2.3
in T. abyssicola.

Figure 17. Tripoplax abyssicola Smith and Cowan, 1966. Holo-
type. Triangle Island, British Columbia, Canada, 870 m, scale bar =
10 mm.

TWO NEW SPECIES OF TRIPOPLAX

85

Figure 18. Tripoplax abyssicola Smith and Cowan, 1966. Paratype
(Cowan coll., No. 5538). Triangle Island, British Columbia, 870 m,
dorsal girdle scales, scale bar = 500 pm.

Figure 19. Tripoplax abyssicola Smith and Cowan, 1966; (RNC
2106, ex RNC 3106). Farallon Islands, California, 2750 m, radula,
scale bar = 500 pm.

(2) Tegmental sculpturing, in animals of approx, the
same size, T. cowani exhibits fewer and coarser ra-
dial ribs (or rows of pustules/granules). A compari-
son of the largest paratypie of T. cowani (ca. 45 mm)
with the holotype of T. abyssicola (46 mm) illus-
trates this well. The head valve of the T. cowani
paratype has 70 ribs, compared to 85 in the holo-

type of T. abyssicola. The lateral areas of T. cowani
have five to nine ribs, compared to 11-13 in T.
abyssicola. The tail valve of T. cowani has 40 ribs, the
tail valve of T. abyssicola has 65 ribs.

(3) The relative sizes of the pustules/granules on the
radial ribs are t]uite distinct also, in animals of about
the same size (45-46 mm), the pustules of T. cowani
are 200-300 pm at about midpoint of the rihlets,
and form a series ot about 18-19 on the lateral
areas. Those of T. abyssicola are 100-150 pm and
form a series of 28-30 on the lateral areas.

(4) Fewer, coarser rihlets on central areas, 40-42 on T.
cowani, and >60 on T. abyssicola.

(5) The dorsal girdle scales of T. cowani are relatively
large and subtriangular in shape, reaching about
300 X 275 pm, those of T. abyssicola (Fig. 18) are
much smaller and narrower, to about 200 X 125
pm, and are slightly curved at the tip, like diminu-
tive surf boards.

(6) Tripoplax cowani has fewer ctenidia than T. abyssi-
cola, 31 in a specimen 28 mm in length, compared
to 37 in a 22 mm T. abyssicola, 36 compared to 41
in specimens 41 mm in length, and 37 for T. cowani
and 43 for T. abyssicola, respectively in specimens
45 mm in length.

The geographic and bathymetric ranges of Tripoplax
cowani and Tripoplax abyssicola overlap somewhat from
northern California to Washington; however, T. cowani is
generally found much shallower than T. abyssicola, 430-
1050 m compared to 950-2750 m. Tripoplax abyssicola
also has a much broader geographic range than T. cowani,
extending from the western Aleutian Islands, south of
Amchitka Island (51°34.14'N, 178°18.49'E) (RNC 2166;
leg. R. Clark, 16 July 2004, trawled R/V Sea Storm, 478 m) to
near the Farallon Islands, west of San Francisco Ray, Cali-
fornia (38°N; RNC 2106). Range here is extended approx.
900 km west from southwest of Unalaska Island (52°36'N,
169°25'W); CASIZ 129748 (Clark 2000). The range of
T. cowani extends from the Swiftsure Bank, off Washington
(48°30'N) to Carmel Bay, California (36°45'N).

Tripoplax cowani might also be confused with Stenose-
mus stearnsii (Dali, 1902) (Fig. 20), which is smaller (to 25
mm in length) and similar in general appearance, but has
broad, low, somewhat flattened, cobble-stone like sculpture
on the radial areas and large, subcylindrical, curved cor-
puscles to 430 X 160 pm. Stenosemns stearnsii is found from
off Clatsop County, Oregon (45°50'N) to near San Clemente
Island, California (33°N) (Clark 1991), at depths of 400-
700 m.

The additions of Tripoplax cowani and Tripoplax ca-
lypso, along with Tripoplax trifuia (Carpenter, 1864), Tripo-

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AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 20. Stenosemiis stearnsii (Dali, 1902). (RNC, 1583). Mon-
terey Sea Canyon, 650 m, scale bar = 10 mm.

plax abyssicola (Smith and Cowan, 1966), Tripoplax ima
(Sirenko, 1975), Tripoplax aUyui (Eerreira, 1977), Tripoplax
attuensis (Clark, 2000), Tripoplax beringiana (Clark, 2000)
and Tripoplax baxteri (Clark, 2000) brings the number of
known species of Tripoplax along the Pacific coast of North
America to nine. The similar appearing Ischnochitoii regularis
(Carpenter, 1855) from northern California appears to be
genetically distinct at the genus level (D. Eernisse, pers.
comm., September 2007).

preparation of this paper: Elizabeth Kools and Robert Van
Syoc, CAS; lames H. McLean and Lindsey Groves, LACM;
Boris I. Sirenko, ZIAS; Darlene Southworth, Southern Or-
egon University; Philip Lambert, Royal British Columbia
Museum; Douglas J. Eernisse, California State University,
Fullerton; and Chris Mah, USNM, for the gift of several
specimens from his 1994 cruise on the R/V Point Snr. The
comments of two reviewers are also greatly appreciated.

LITERATURE CITED

Berry, S. S. 1919. Notes on West American chitons II. Proceedings
of the California Academy of Sciences 9: 1-36.

Clark, R. N. 1991. Notes on the distribution, taxonomy and natural
history of some North Pacific chitons. The Veliger 34: 91-96.
Clark, R. N. 2000. Three new chitons of the genus Lepidozona
Pilsbry, 1892 (Polyplacophora: Ischnochitonidae) from the
Aleutian Islands. Nemouria, Occasional Papers of the Delaware
Museum of Natural History 42: 1-16.

Dali, W. H. 1871. Descriptions of sixty new forms of mollusks from
the west coast of North America and the North Pacific Ocean,
with notes on others already described. American Journal of
Conchology 7: 93-160.

Ferreira, A. J. 1978. The genus Lepidozona (Mollusc: Polyplaco-
phora) in the temperate eastern Pacific, Baja California to
Alaska, with the description of a new species. The Veliger 21:
19-44.

Kaas, P. and R. A. Van Belle. 1985. Monograph of Living Chitons
(Mollusca: Polyplacophora). Vol. 3, Suborder Ischnochitonina,
Ischnochitonidae: Chaetopleurinae, & Ischnochitoninae (part).
E. J. Brill/Dr. W. Backhuys, Leiden, The Netherlands.

Kaas, P. and R. A. Van Belle. 1987. Monograph of Living Chitons
(Mollusca: Polyplacophora). Vol. 4, Suborder Ischnochitonina,
Ischnochitonidae: Ischnochitoninae (continued). E. Brill,
Leiden, The Netherlands.

Kaas, P. and R. A. Van Belle. 1990. Monograph of Living Chitons
(Mollusca: Polyplacophora). Vol. 5, Suborder Ischnochitonina,
Ischnochitonidae: Ischnochitoninae (concluded) Callistoplacinae;
Mopaliidae. E. J. Brill, Leiden, The Netherlands.

Palmer, K. E. H. V. W. 1958. Type specimens of marine Mollusca
described by P. P. Carpenter from the west coast (San Diego
to British Columbia). Geological Society of America, Memoirs
76: 1-376.

Smith, A. G. and I. McTaggart Cowan. 1966. A new deep-water
chiton from the Northeastern Pacific. Occasional Papers of the
California Academy of Sciences 56: 1-15.

Submitted: 29 March 2007; accepted: 9 November 2007;
final revisions received: 26 February 2008

ACKNOWLEDGMENTS

I am grateful to the following people for their help in the

Amer. Maine. Bull. 25: 87-95 (2008)

The effect of sampling bias on the fossil record of chitons
(Mollusca, Polyplacophora)’^

Stephaney S. Puchalski\ Douglas J. Eernisse^, Claudia C. Johnson'

' Department of Geological Sciences, Indiana University, 1001 E. lO"' St., Bloomington, Indiana 47405, U.S.A., spuchals@indiana.edu
■ Department of Biological Science, California State University Fullerton, Fullerton, California 92834-6850, U.S.A.

Abstract: The chiton fossil record is richer than previously reported in the literature. A newly compiled database comprised of Cambrian
to Pleistocene fossil chitons totals 2594 occurrences of 900 species. Of the 900, 430 are named species known only as fossils, 123 are extant
species that also have a fossil record, and 247 are indeterminate taxa. Most of the database (61%) consists of fossil chiton occurrences
reported from localities other than type localities. A preliminary analysis of the data using the collector curve method suggests that the
chiton fossil record has not been adequately sampled by geographic regions or geologic time. The fossil record of chitons is incomplete,
sporadic, and geographically limited because the sampling record has been incomplete, sporadic, and geographically limited. The current
database comprises enough information to discern diversity patterns throughout geologic time, but whether the patterns are real or artifacts
ot sampling inadequacy remains to be investigated.

Key words: collector curve, database, sampling record, fossil record completeness, sampling adequacy

Data analysis is a fairly recent approach to investigating
and discerning patterns in the fossil record (Raup 1976a,
1976b, Benton 1993, Smith 2001, Alroy et al. 2001, Westrop
and Adrain 2001, Sepkoski 2002, Tarver et al. 2007, and
others). Incompleteness in the data, however, including in-
completeness of the fossil record itself, introduces error in
interpretation of observed patterns. Both taphonomic and
sampling biases cause incompleteness and affect the amount
of data available for analysis (Benton 1998, Benton et al.
2000, Tarver et al. 2007), but taphonomic biases are better
understood than sampling biases. Few researchers have ad-
dressed the latter issue (Tarver et al. 2007 and references
therein). Assessment of sampling bias is essential to evalu-
ating the adequacy of the fossil record. Understanding the
causes of incompleteness allows paleontologists to use sta-
tistical methods to correct for errors in order to differentiate
real patterns from apparent trends.

Despite a Cambrian to Holocene fossil record, chitons
(Polyplacophora) may be less well sampled than other shell-
bearing fossil fauna such as brachiopods, gastropods, bi-
valves, and cephalopods that have similarly long but ‘good’
fossil records. Although significant numbers of chiton valves
(400 or more) have been recorded from some localities (Itoi-
gawa et al. 1976, Bischoff 1981, Baluk 1984, Laghi 1984,
Bellomo and Sabelli 1995, Cleveringa et al. 2000, Hoare and
Pojeta 2006, Sigwart et al. 2007), most extinct chiton species
are represented by relatively few valves that are rare com-
pared to other taxa in an assemblage. Even when character-

ized as ‘exceptionally abundant,’ the valves are still uncom-
mon in comparison to other taxa. For example, chiton valves
were only 7% as common as bivalves found at the same
Silurian localities in Gotland (Cherns 1999). Inadequate
sampling thus may have affected patterns of chiton diversity
and distribution reported in the literature {e.g., Sepkoski
2002, Cherns 2004, Puchalski 2005). Chitons reportedly are
most diverse in the Holocene (Smith 1960, Lindberg 1985,
Benton 1993). About 900 modern chiton species inhabit
mostly shallow coastal waters and are ubiquitous on modern
rocky shores in all oceans and at all latitudes worldwide
(Kaas and Van Belle 1985). In comparison, the reported
chiton fossil record ranges between 256 and 368 fossil species
(Smith 1960, Van Belle 1981, Eernisse 2001, Schwabe 2005),
sporadically distributed through geologic time and geo-
graphically limited mostly to the North American, Euro-
pean, and Australia-New Zealand regions (Van Belle 1981).

This study assesses sampling bias in the chiton fossil
record using the collector curve approach with a database on
fossil chiton occurrences. The occurrence data also were
used to show patterns in chiton diversity from the Cambrian
to Pleistocene.

MATERIALS AND METHODS
Database compilation

The initial database containing 336 fossil chiton species
was compiled by Eernisse (2001). The database included

* From the symposium “Advances in Chiton Research” presented at the joint meeting of the American Malacological Society and Western
Society of Malacologists, held 29 luly to 3 August 2006 in Seattle, Washington.

87

88

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

suborders, families, genera, type locality information, geo-
logic period, year of publication, and museum information
(e.g., type kinds and numbers). The database was expanded
by the first author (S.P.) to include additional occurrences of
fossil chitons from the Cambrian through the Pleistocene at
type and other localities. An occurrence was defined as a
reported difference in geographic location of collection (lati-
tude and longitude) or taxonomy (species). No distinction
was made between geographic and taxonomic occurrences.
Taxonomic differences were determined by cross-referen-
cing the literature to account for synonymies and updating
original taxonomic assignments when systematic relation-
ships were reevaluated in later references. Taxonomic occur-
rences with indeterminate relationships were grouped in the
genus “Indet.” whether originally reported as indeterminate,
unidentified, “polyplacophoran,” or “chiton” regardless of
whether or not the fossils were figured and/or described. The
database was developed further to include modern latitude
and longitude coordinates of the collecting locality, reported
numbers and types of valves, authors’ reasons for publica-
tion, geologic stage, and other geologic information such as
lithology and general fossil associations whenever possible.
The primary data sources were published reports including
descriptions of fossil species, but occurrences obtained from
unpublished sources (e.g., online museum collection data-
bases) also were included.

All taxa regarded as invalid chiton fossils were excluded
from this preliminary analysis of the data. For example,
some Early Cambrian “polyplacophoran” fossils from China
(Yii 1987) may be only superficially similar to chitons (Qian
and Bengtson 1989) or may be valid chiton taxa (Yii 2001,
Schwabe 2005). The fossils have an overlapping series of
plates that are much smaller than other chiton valves but
similarly differentiated into three types with distinct areas on
the dorsal surface and shell layers consisting of ‘articulamen-
tum’ and ‘tegmentum.’ Qian and Bengtson ( 1989) argue that
the poorly preserved ‘plates’ show very few structural details
and are not articulated with one another but rather represent
a series of successively larger growth increments deposited
on the inner side of sclerites. After restudying the specimens,
Yii (2001) maintains that the fossils are indeed polyplaco-
phorans closely related to Gotlaitdochiton Bergenhayn, 1955,
Priscochiton Dali, 1882, Chelodes Davidson and King, 1884,
and c]uestionably Glyptochiton de Koninck, 1883. However,
the first three genera are paleoloricates that are distinguished
from more modern chitons in lacking articulamentum, a
character that does not appear in undoubted chiton fossils
until the Carboniferous (Sirenko 2006). The implication that
chitons with articulamentum “gave rise” to chitons without
articulamentum that then evolved into chitons with articu-
lamentum is problematic. The remaining characters de-
scribed by Yii (1987, 2001) are not necessarily exclusive to

chitons. The Lower Cambrian taxa thus were rejected as !
valid chiton species for the purposes of this study.

The completeness of the sampling record was assessed
using all fossil chiton occurrences including named species,
which represent less than 17% of the entire dataset. Debates
on the validity of some taxa and frequent changes in chiton
systematics made it difficult to directly compare the number
of named species to previous catalogues of fossil chitons
(e.g., Smith 1973, Van Belle 1981, Smith and Hoare 1987,
Schwabe 2005). These catalogues were used in most cases to
determine the validity of taxa for this study, but some cata- |
logues are incomplete and the authors did not always agree
on validity. Some species considered valid for the purposes
of this study thus may not have been considered valid in
previous catalogues. In cases of more recent publications,
omissions from previous catalogs, or where published views
conflicted regarding synonymies or validity of a particular
fossil as a chiton, validity was determined by S. Puchalski
using the primary literature. For example, an exhaustive lit-
erature search revealed multiple species not included in Van
Belle’s (1981) monograph (e.g., Pterochiton tripartitus Ebert,

1889 and Pterochiton silesiacus Ebert, 1889) that were con-
sidered to be valid chiton species. Additionally, Clutonellus
Jmncockianus Kirkby, 1859, Chitonelliis antiqnns (Howse,
1848), and Chitonellns distortus Kirkby, 1859 named and
described by Kirkby (1859) in Permian limestone at Tunstall
Hill, England were listed as “no chiton” by Van Belle ( 1981)
and rejected as polyplacophorans by Smith and Hoare
(1987). The reported occurrence of these taxa was accepted
as valid for this study under the name Dindeloplax antiqua
(Howse 1848) based on Hoare and Mapes (2000), who rec-
ognized the three taxa as a single multiplacophoran species.

The multiplacophorans were accepted as valid chiton taxa
because Vendrasco et al. (2004) referred the multiplacopho-
rans to Class Polyplacophora. The list of valid taxa and as-
sociated geographic and temporal data used in this prelimi-
nary analysis is available at: http://www.biology.fullerton
.edu/deernisse/fossilchitons/. The complete database will be 1
made available after further analysis. |

The data were divided into several different groups for
convenient analysis. Countries of occurrences were grouped
into geographic regions (Table 1 ) that approximate modern
continents (Tarver et al. 2007). Valid chiton taxa were sepa-
rated into seven taxonomic groups consisting of: ( 1 ) named
extinct species known only as fossils (e.g., Lepidopleurus da- 1

void Laghi, 2005), (2) extinct species with names consisting !

of numbers or letters (e.g. Lepidopleurus sp. I Sulc, 1936), (3) |

indeterminate extinct species placed in valid genera (e.g., j
Helmintliochiton sp. Plas, 1972), (4) named extinct species in i
indeterminate genera (e.g., “Chiton” cordiformis Sandberger, j
1845), (5) indeterminate taxa (e.g., “unidentified chiton ;
valves,” Hoover 1981), (6) extant species with a fossil record

EFFECT OF SAMPFING BIAS ON CHITON FOSSIF RECORD

89

Table 1. Countries with fossil chiton occurrences grouped by geographic regions.

Geographic region Countries

Africa

Asia

Australasia

Europe

North America
South America

Algeria, Eritrea, Ethiopia, Morocco, South Africa, Tanzania
China, India, lapan, Malaysia, Russia, Thailand

Australia, Borneo, Eiji, Indonesia, Marshall Islands, New Zealand, Palau

Austria, Belgium, Bulgaria, Czech Republic, Denmark, France, Germany, Greece, Hungary, Ireland, Italy,
The Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Spain, Svalbard, Sweden, Ukraine,
United Kingdom

Bahamas, Canada, Cayman Islands, Jamaica, Mexico, Puerto Rico, United States
Argentina, Brazil, Chile, Columbia, Uruguay, Venezuela

{e.g., Mopalia muscosa (Gould, 1846)), and (7) geographic
occurrences reported from localities other than type locali-
ties. The first reports of extant species were considered
equivalent to type localities of extinct species. Affinities (cf ,
aff ), variations, and subspecies were treated as geographic
occurrences and placed in group seven rather than as taxo-
nomic occurrences in one of the other groups.

Data analysis

The collector curve approach (Weller 1952, Paul 2003,
Fountaine et al. 2005, Tarver et al. 2007) was used to inves-
tigate the sampling completeness of the chiton fossil record.
Assuming no decrease in effort, collector cui-ves are expo-

1750n

1500-

>

0

Sl250-

is

J^IOOO-

(1)

750-

ro

1 500-

o

250-

Taxa Group

Geographic Occurrences
Named extinct species
Valid genus, sp. indet.
Extant species
Indeterminate taxa
Other

1 — ^ ^ ^ — T

1 I T

1 — ^ — T

nential as discovery rates increase and become asymptotic
and sigmoid when virtually every fossil taxon that has been
preserved has been found (Benton 1998, Fountaine et al.
2005, Tarver et al. 2007). Collector curves, thus, are plots
of the cumulative number of discoveries against some mea-
sure of collecting effort. The number of new taxa described
per year and the number of fossil occurrences reported
each year were used as the measure of total collecting effort.
Neither approach assumes that the workers are constant.
The first approach shows the rate at which workers are find-
ing new taxa. The second approach accounts for workers
finding few new taxa. In the latter case, publication history
tends to move away from descriptions and into broader
topics such as preservation potential and biogeography
(Tarver et al. 2007). Publication history, thus, was assessed
by categorizing the authors’ primary reasons for reporting

1400-

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3

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CM

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Geographic Region

— Europe
— North America
■ Australasia
- -Asia
Other

1 — ^ ^ — I — ^ ^ — T

1 — I — \ — T

Figure 1. Collector curves for the seven groups of valid chiton taxa.
The fossil record represented by the database is comprised mostly
of geographic occurrences of previously described taxa discovered
at localities other than type localities. The group of species with
names consisting of numbers or letters and named species of in-
determinate genera have been collapsed into one group labeled
‘other’ for clarity because these groups each represent less than 1%
of the data.

CM

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to

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Year

CM

Figure 2. Collector curves for all fossil chiton occurrences reported
by geographic region (see Table 1 for listing of the countries in each
region). Countries in Africa and South America have been com-
bined into one group labeled “other” for clarity because these two
regions represent less than 1% of the data.

90

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

90-

<u

Q.

(0

a.

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a>

All Occurrences: Asia
N = 100

75-

60-

45-

30-

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O.400-
^ 350H

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All Occurrences:

All Geographic Regions
N=2594

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Year of Publication

240

200

160

120

general faunal papers that did not fit
into another group, biostratigraphic
papers, taphonoinic papers, or pa-
leoecologic papers.

RESULTS AND DISCUSSION

75-

60-

45-

30-

15-

Papers Describing New
Species: All Regions
N = 430

in

CO

Figure 3. Histograms comparing the frequency of published papers reporting occurrences of all
seven groups of fossil chiton taxa from Europe, Australasia, North America, and all geographic
regions as a measure of worker effort. African and South American regions with less than 100
publications total are not shown. The publication frequency of new taxa described from all
geographic regions also is shown. Note: y-axis scale varies among histograms.

the fossils based on the subject area that constituted the bulk
of each publication. For example, the taxonomic group con-
sisted of papers with systematic paleontology sections com-
prising most of the article. The other categories consisted of

At the time of analysis, the data-
base comprised 2594 occurrences of
900 chiton taxa. Of the 900 taxa, 430
are valid fossil species named and de-
scribed from 1802 through 2007, 123
are extant species with a fossil record,
and 247 are indeterminate species. The
900 taxa are placed in 95 genera, 31
families, 9 suborders, and 4 orders of
the Class Polyplacophora. In compari-
son, Van Belle ( 1981 ) listed 250 named
and 49 indeterminate fossil species and
a few extant species with a fossil re-
cord. Smith’s (1960) compilation in-
cluded 293 named and unnamed spe-
cies known only as fossils and 59
extant species with a fossil record.
Smith and Hoare (1987) reported 153
Paleozoic named species and 23 inde-
terminate taxa. Schwabe (2005) re-
ported 368 named species known only
as fossils. Despite the importance of
these previous fossil catalogues, named
fossil and extant species combined
represent only about 21.3% of all 2594
fossil occurrences in the current data-
base. Most of the data (61.0%) consists
of previously identified species that oc-
curred at localities other than type lo-
calities. The large numbers of geo-
graphic occurrences suggest that the
fossil record is richer than previously
indicated in the literature.

The fully exponential pattern of
the collector curves indicates that sam-
pling of fossil chitons has been inad-
equate for all taxonomic groups (Fig.
1 ) and geographic regions (Fig. 2). The
stepped Asian curve and roughly as-
ymptotic Australasian curves poten-
tially suggest that the sampling records
are complete for these regions. The combined African-South
American collector curve also appears relatively flat, but this
is partly due to limited data and partly due to the scale of the
graph required to show the Australasian, European, and

o

o

o

CM

Year of Publication

EFFECT OF SAMPLING BIAS ON CHITON FOSSIL RECORD

91

North American curves that represent the majority of data.
However, flattening of collector curves also may be caused
by decreased collecting effort (Tarver et al. 2007). As a mea-
sure of collecting effort, decreased frequency in the number
of published papers suggests that flattening of the Asian and
Australasian collector curves are due to decreased efforts
rather than failure to find new species (Fig. 3). In compari-
son, the increased frequency of papers on European occur-
rences suggests collecting efforts have risen in the last few
decades. The corresponding European collector curve has
not reached an asymptote (Fig. 2), indicating continued high
rates of discovery of new taxa. In North America, collecting
efforts have remained relatively high from 1 960 to the pres-
ent but the collector curve still is in the exponential phase,
similarly indicating high discovery rates of new taxa. The
incomplete sampling records thus result from heterogeneity
in collecting effort focused on fossil chitons.

The frequency of papers describing new species from all
seven geographic regions shows a fairly steady increase from
1950 to the present (Fig. 3). However, the data also suggest
there is much information in the fossil record of chitons that
has yet to be tapped. Most papers were published for the
purposes of describing new taxa (Fig. 4). Taxonomic reports
comprise most of the reported occurrences (N = 2238,
86.2%), although some occurrences were reported as part
of general fauna (N = 205, 7.9%) or biostratigraphic studies
(N - 95, 3.7%). Few fossil chitons were reported as part of
taphonomic {N - 5, 0.2%) or ecologic (N = 51, 2.0%) stud-
ies. The implication is that the range of research on fossil

Publication Reason

I I Taxonomy

^ General fauna/flora
r~l stratigraphy

II Ecology
H Taphonomy

Figure 4. Pie chart showing the distribution of the reasons for
publication of papers reporting occurrences of fossil chitons. Rea-
sons were determined by the subject areas that constituted the bulk
of each publication; taxonomy, systematic descriptions; biostratig-
raphy, temporal correlations; paleoecology, paleoecological analy-
ses; taphonomy, taphonomic analyses; general fauna/flora, papers
not fitting into previous categories. Numbers shown in each slice
indicate the number of publications in each category.

chitons still is mostly in the discovery phase and has yet to
broaden.

Although the collector curves indicate inadequate sam-
pling of all seven groups of fossil chiton taxa through the
Phanerozoic (Fig. 5), the data may be sufficient for some
geobiological studies depending on the geologic time and/or
geographic region being investigated. Some geologic time
periods and geographic fossil records of chitons are more
complete than others due to the heterogeneity in collecting
effort. For example, most fossil species occur in the Carbon-
iferous and Cenozoic (Eocene and Miocene to Pleistocene,
Fig. 6). Although the shapes of the Cenozoic and Paleozoic
curves are similar among groups of taxa, active research and
focused collecting efforts can be attributed to a limited num-
ber of researchers that have contributed to the increases in
both cases. Richard Hoare, as author or coauthor, has de-
scribed thirty-one new Carboniferous chiton species, result-
ing in a more complete record for the period. The Cenozoic
increase may be attributed to the more complete sampling of
Holocene biota referred to as the ‘pull of the Recent’ (Raup
1979, Foote 2000, Alroy et al. 2001, Peters and Foote 2001 ).
However, Bruno Dell’Angelo, as author or coauthor, ac-
counts for 242 or 1 1.8% of the Cenozoic occurrences, most
in the Mediterranean, suggesting that focused collecting ef-
fort has resulted in a more complete Cenozoic record for the
European region. In comparison, the Mesozoic collector
curves suggest severely inadecjuate sampling of chitons for
that period.

Temporal gaps shown by this analysis do not necessarily
equate to non-existence of chitons in past ecosystems. Al-
though the non-logistic nature of the collector curves indi-
cates that at least some gaps in the current dataset are due to
sampling bias, the generally poor preservation states of most
fossils indicate that taphonomic biases also may have been a
contributing factor. Paleoecologic data show that Paleozoic
chitons mostly inhabited shallow coastal environments simi-
lar to the settings inhabited by most modern chiton species
(Dunlop 1915, 1922, Frederickson 1962, Smith and Toomey
1964, Kues 1978, Yancey and Stevens 1981, Gerk and Levor-
son 1982, Hoare and Smith 1984, Debrock et al. 1984, Farrell
1992, Vendrasco 1999, Cherns 1999, Hanger et al. 2000,
Hoare 2001, Cherns 2004, Vendrasco and Runnegar 2004).
There is no reason to assume that the preservation potential
of chitons in the past differed greatly from that in modern
environments. Most modern chitons tend to live in inter-
tidal or shallow subtidal erosional environments that are
rarely preserved even where fossil deposits are extensive (c.g.,
California Miocene). Modern chitons are more rarely found
living in relatively deep water or on muddy bottoms, but
species diversity in such cases is much lower relative to shal-
low water communities. Fossil chitons interpreted to have
lived on muddy bottoms or in deeper water are rare, but
have been reported (Hoare et al. 1972, Lang and Chlupac

92

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Year of Publication

through geologic time indicate chitons
were affected by mass extinctions (Fig.
6). For example, decreased numbers of
species in the Paleocene imply that
chitons were affected by the end-
CretaceoLis mass extinction. The spe-
cies numbers increased in the Eo-
cene following an apparent slow re-
covery through the Paleocene. The
mean number of species per occur-
rence (= locality) used as a proxy
for diversity suggests that chiton diver-
sity has remained relatively constant
through the Phanerozoic (Fig. 6).
Pleistocene diversity is not signifi-
cantly greater than Eocene or Late Per-
mian diversity, for example. Whether
these patterns are artifacts of the sam-
pling inadequacy or real trends remain
to be investigated. Continued active
collection and study of fossil chitons
should be encouraged because the
non-logistic nature of the collector
curves suggest that many more fossil
chiton species remain to be found and
described. Recent discoveries have
been instrumental in demonstrating
that Paleozoic chitons were more di-
verse in form than modern chitons
{e.g., Pojeta et al. 2003, Vendrasco et
al. 2004). Further analysis will investi-
gate large-scale evolutionary and eco-
logical patterns in the data with the
goal of assessing the fidelity of the chi-
ton fossil record after correcting for
the sampling bias indicated in this pre-
liminary analysis.

ACKNOWLEDGMENTS

Figure 5. Collector curves for each taxa group of fossil chiton occurrences reported by
geologic era and for Phanerozoic. Not all seven groups have been reported from all three eras.

1975, Richardson 1980, Dell’ Angelo and Palazzi 1994, Palaz-
zi and Villari 1994, Goedert and Campbell 1995, Squires and
Goedert 1995, Remia and Taviani 2005, Kiel and Goedert
2006). As with modern settings, diversity appears to be lower
relative to the shallow water assemblages.

Observed changes in chiton diversity through time (Fig.
6) do not correlate to degrees of preservation or skeletal
completeness. In general, changes in species numbers

Financial assistance to S.P. was
provided by grants from the Depart-
ment of Geological Sciences and
Women in Science Fellowship through Indiana University.
D.E. acknowledges a sabbatical fellowship supported by the
National Evolutionary Synthesis Genter (NESCent), NSF
#EF-0423641. Richard Van Belle’s (1981) Catalogue of Fossil
Chitons provided D.E. with the initial inspiration for a com-
puterized version of a fossil chiton species database and,
likewise. Then Engesser provided D.E. with an independent,
unpublished compilation of nominal fossil species. Bruno

EFFECT OF SAMPFING BIAS ON CHITON FOSSIL RECORD

93

Pleistocene
Pliocene
Miocene
Oiigocene
Eocene
Paleocene
L. Cretaceous
E. Cretaceous

L. Jurassic

M. Jurassic
E. Jurassic

L. Triassic

M. Triassic
E. Triassic

L. Permian

M. Permian
E. Permian

L. Pennsylvanian

M. Pennsylvanian
E. Pennsylvanian

L. Mississippian

M. Mississippian
E. Mississippian

L. Devonian

M. Devonian
E. Devonian

L. Silurian
E. Silurian

L. Ordovician

M. Ordovician
E. Ordovician

L. Cambrian
E. Cambrian

Number Number Number Sp.

Genera Occurrences Species Occur.

Figure 6. Diagram showing numbers of genera, occurrences, and species by epoch. Sp./
occur., number of species per occurrence. Bar width of N = 10 shown for scale.

Deir Angelo contributed an extensive list of references on
chitons. R. Hendrickson provided critical proof reading.
Comments by J. Pojeta and an anonymous reviewer greatly
improved the original manuscript.

LITERATURE CITED

Alroy, L, C. R. Marshall, R. K. Bambach, K. Bezusko, M. Foote,
F. T. Fiirsich, T. A. Hansen, S. M. Holland, L. C. Ivany, D.
lablonski, D. K. lacobs, D. C. lones, M. A. Kosnik, S. Lidgard,
S. M. A. I. Low, P. M. Novack-Gottshall, T. D. Olszewski,
M. E. Patzkowsky, D. M. Raup, K. Roy, I. 1. Sepkoski, Ir.,
M. G. Sommers, P. f Wagner, and A. Webber. 2001. Effects of
sampling standardization on estimates of Phanerozoic marine
diversification. Proceedings of the National Academy of Science
98: 6261-6266.

Baluk, W. 1984. Additional data on chitons and cuttlefish from the
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Submitted: 10 April 2007; accepted: 26 November 2007;

final revisions received: 1 February 2008

Amer. Maine. Bull. 25: 97-111 (2008)

Fertilization biology and the evolution of chitons'^

John Buckland-Nicks

St. Francis Xavier University, Antigonish, Nova Scotia B2G 2W5, Canada, jbucklan@stfx.ca

Abstract: Studies of gamete structure and fertilization biology have revealed much about the phylogeny of molluscs. Recent studies of
fertilization in chitons support the view that the basal order of chitons, the Lepidopleurida, fertilize eggs, as most molluscs do, by fusing
the entire sperm with the egg and transferring chromatin, mitochondria, and centrioles into the egg cytoplasm. However, current evidence
suggests that all members of the order Chitonida inject only chromatin into the egg. These chitons, which include the controversial family
Callochitonidae, share a series of synapomorphic characters based on their fertilization biology that makes them unique. Current evidence
suggests that Callochitonidae are basal to this order and sister taxa to the remaining Chitonida, which have been divided into two suborders,
the Chitonina and Acanthochitonina. New evidence indicates that Chitonina have at least two different mechanisms for penetrating the egg.
One group of species has pores in the egg hull [e.g., Chaetoplaira apiculata (Say in Conrad, 1834) and Stenosemus alhus (Linnaeus, 1767),
whereas a second group has a continuous dense layer on the surface of the egg hull that is digested by the sperm (e.g., Rhyssoplax tulipa
(Quoy and Gaimard, 1835) and Stenoplox coiispkua (Pilsbiy, 1892)). However, the genus Ischnochitoii Gray, 1847 appears to be polyphyletic,
as several species have distinctive characters that typify other genera or families. In particular, this genus needs to be re-evaluated using
modern morphological and molecular methods. All of the Chitonina have spiny-hulled eggs with narrow bases and are quite different from
the second suborder, Acanthochitonina, which is characterized by large-hull cupules with wide bases. Within Acanthochitonina, some
species have open-hull cupules, whereas most have closed ones. Open-cupule species lack micropores in the hull for sperm entry, whereas
several closed-cupule species exhibit micropores between hull cupules. These features of the egg are accompanied by alterations in sperm
structure, such as position of the mitochondria and structure of the basal body, acrosome, and flagellum. Knowledge of the gamete structure
of individual species and their fertilization biology, as demonstrated here, provides a different series of characters that can help avoid
mistakes that are inherent during early development of new methods, such as molecular analyses. New details of fertilization biology have
made it possible to revise preliminary analyses and provide an updated phylogeny of chitons, which differs in some important respects from
other recent publications.

Key words: Polyplacophora, egg, sperm, cladistics, phylogeny

We are coming to rely more and more on molecular
analyses to accurately explain phylogenetic relationships
(Peterson and Eernisse 2001, Okusu et al. 2003, Eernisse and
Peterson 2004, Smith et al. 2004). Nevertheless, errors and
inconsistencies do occur, making it useful to have a series of
checks and balances in place based on a different set of
characters. Prior to 1984, chiton taxonomy relied heavily on
the structure of shell valves, spicules, scales, and girdle pro-
cesses in order to distinguish between taxa (Smith 1960, Van
Belle 1983, Kaas and Van Belle 2003), but it became appar-
ent that some of these characters had evolved more than
once by convergent evolution, which introduced some prob-
lems in classification.

Eernisse (1984) first suggested the use of egg structure
and gill placement as additional characters, and Sirenko
(1993) included these and produced a revised classification
that divided chitons into two orders, Lepidopleurida and
Chitonida, with the latter comprising two suborders, Chito-
nina and Acanthochitonina. A cladistic analysis by Buck-

land-Nicks ( 1995), which added characters for gamete biol-
ogy at fertilization, supported this classification, differing
only in positions of certain families within each suborder. In
that analysis the family Callochitonidae came out as basal
within Chitonina or as sister taxon to this suborder within
Chitonida. Cyanoplax Gould, 1849 (= Lepidochitotm Gray,
1821) did not place within Tonicellidae, as suggested by
Sirenko (1993), but rather came out as a sister taxon to
Acanthochitonidae. More recently Okusu et al. (2003) un-
dertook a combined molecular and morphological study
that provided the first detailed analysis of this kind. In gen-
eral, their paper agreed with Sirenko (1993) and Buckland-
Nicks ( 1995), but it differed in some important aspects. For
example, Okusu et al. (2003) placed Callochitoii Gray, 1847
(Callochitonidae) within Lepidopleurida, not in Chitonida.
Furthermore, there were inconsistent results from their dif-
ferent molecular analyses (16sRNA, COI, and histone 143)
for both Schizocliiton Gray, 1847 and Lepidozona Pilsbry,
1892 (Okusu et al. 2003: figs. 2, 3, 4, pp. 288-291). Most

From the symposium “Advances in Chiton Research” presented at tire joint meeting of the American Malacological Society and Western
Society of Malacologists, held 29 July to 3 August 2006 in Seattle, Washington.

97

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AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

previous classifications, including the combined morpho-
logical dataset of Okusu et al. (2003: fig. 8, p. 295), placed
Lcpidozona close to Stefwplax in Ischnochitonidae. The ge-
nus Ischiiochiton Gray, 1847 consistently came out as being
a polyphyletic taxon, with some species of the genus turning
up in different families (Okusu et al. 2003). furthermore,
they did not find support for a monophyletic Acanthochi-
tonina, suggesting instead that it comprised two or more
paraphyletic clades.

A preliminary re-analysis of morphological data (Buck-
land-Nicks 2006) once again placed Callochitonidae outside
the Lepidopleurida, either as sister taxon to Chitonida or
basal within it, as suggested previously (Buckland-Nicks and
Hodgson 2000). More recently scientists have begun looking
at other potential characters for distinguishing taxa, such as
mineral composition of the radula (Brooker et al. 2006) and
the gene sequence coding for hemocyanin protein (Lieb et al.
2006).

This paper seeks to further elucidate the morphology of
sperm and eggs at fertilization in selected key taxa and
thereby provide a focus for discussion of some of these new
ideas. Furthermore, previous morphological data are re-
analyzed and updated with current information, providing
some new insights into chiton phylogeny, which are remark-
ably consistent with those achieved by completely new types
of analysis (Lieb et al. 2006). Ideally, all of these analyses

should be combined to provide the most comprehensive test
of phylogenetic relationships.

MATERIALS AND METHODS

Specimens

Specimens were collected from various sites around the
world (see Table 1). Species of the following families are
represented (terminology following Kaas et al. 2006 and Eer-
nisse et al. 2007): Leptochitonidae {= Lepidopleuridae), Cal-
lochitonidae, Ischnochitonidae, Chitonidae, Acanthopleuri-
dae, Lepidochitonidae, Acanthochitonidae, and Mopaliidae.
Several minor families that have not been included because
insufficient data are available on their reproductive biology
include (terminology following Sirenko 2006): Ferreiraelli-
dae, Nierstraszellidae, Callistoplacidae, Loricidae, Hemiarth-
ridae, Choriplacidae, Schizochitonidae, and Cryptoplacidae.

Light and electron microscopy

Fixation for transmission electron microscopy (TEM)
and scanning electron microscopy (SEM) followed methods
of Buckland-Nicks and Hodgson (2000). Individual chitons
were isolated in petri dishes and induced to spawn by adding
sperm to each dish. Egg-laying females were removed from

Table 1. A list of chiton species collected for the study and arranged in alphabetical order (all coordinates are approximate). Where a new
genus has been assigned, the old genus is shown in brackets; subgenera are not shown.

Species

Acanthopleura gramdata
(Gmelin, 1791)

Acanthocliitona viridis (Pease, 1872)

Callochiton dentatiis (Spengler, 1797)

Chaetopleiira apiculata (Say in Conrad, 1834)

Cryptochiton stellcri (von Middendorff, 1847)
Cyanoplax (Lepidochitona) dentiens (Gould, 1846)
Cyanoplax (Lepidochitona) fernaldi Eernisse, 1986
Deshayesiella ciirvata Carpenter in Pilsbry, 1892
Hanleya hanleyi W. Bean in Thorpe, 1844
Lcptochitou asellus (Gmelin, 1791)

Leptochiton assiniilis Thiele, 1909
Leptochiton nigatus (Pilsbiy, 1892)

Mopalia nniscosa (Gould, 1846)

Nnttallina califoniica (Nuttall MS, Reeve, 1847)
Radsia (Chiton) nigrovirescens de Blainville, 1825
Rhyssoplnx (Chiton) tulipa Quoy and Gaimard, 1835
Stenoplax conspicua (Pilsbry, 1892)

Stenosemus (Ischnochiton) albas (Linnaeus, 1767)

Collection site/year

Latitude, longitude

Collected by

Trinidad 2001

10°40'N, 61°39'W

I. Buckland-Nicks
(J.B.-N.)

Oahu, Hawaii 1987

21°18'N, 158°09'W

J.B.-N.

East London, S. Africa 1999
Florida 2004

33°03'S, 28°03'E

J.B.-N. and A. Hodgson
Gulf Specimen Co.,
Florida

San luan Is., Washington 1988

48°28'N, 122°54'W

J.B.-N.

San Juan Is., Washington 1990

48°28'N, 122°54'W

J.B.-N. and D. Eernisse

San luan Is., Washington 1990

48°28'N, 122°54'W

J.B.-N. and D. Eernisse

Vostok Bay, Russia 1997

42°53'N, 132°44'E

B. Sirenko

Lurcher Bank, Canada 1994

43°15'N, 65°30'W

R. Mayhew

Bergen, Norway 2006

60°20'N, 5°1TE

J.B.-N and C. Schander

Vostok Bay, Russia 1997

42°53'N, 132°44'E

B. Sirenko

Vostok Bay, Russia 1997

42°53'N, 132°44'E

B. Sirenko

San luan Is., Washington 1990
S. California 1993

48°28'N, 122°54'W

J.B.-N. and D. Eernisse
Sea Life Supply, California

Eastern Cape, S. Africa 1999

34°16'S, 18°40'E

J.B.-N.

East London, S. Africa 1999
S. California 1993

33°03'S, 28°03'E

J.B.-N. and A. Hodgson
Sea Life Supply, California

Bergen, Norway 2006

60°20'N, 5°11'E

J.B.-N. and C. Schander

FERTILIZATION AND THE EVOLUTION OF CHITONS

99

dishes, cleaned, and replaced in clean dishes with filtered sea
water. Eggs, and sometimes fertilization events, were ob-
served with Nomarski optics. Overnight primary fixation in
ice cold 2.5% glutaraldehyde in O.IM cacodylate buffer (pH
7.4) in filtered sea water with O.IM sucrose was followed by
secondary fixation with 2% osmium tetroxide for 1 h in the
same buffer. Fixed eggs were rinsed in distilled water and
dehydrated in an ethanol series to 100%. Batches of eggs
were divided into ec^ual alic]uots and processed separately for
TEM and SEM.

For TEM, ethanol was replaced with propylene oxide
and then eggs were infiltrated with either TAAB 812/Araldite
or Spurrs/Epon. Samples in pure resin were left all day be-
fore baking in a 60 °C oven for two days. Thick and thin
sections were cut with a diamond knife (Diatome, Switzer-
land). Thin sections were picked up on naked 150 mesh
copper grids and stained sequentially with aqueous uranyl
acetate (20 min) and lead citrate (5 min) with extensive
washing with degassed distilled water between stains and
after staining. Stained sections were examined in a Philips
TEM 410 operated at 80 kV.

Eggs destined for SEM were aspirated into Teflon fio-
thru vials (Pelco) before critical point drying, mounting on
stubs using double-sided carbon tabs, and coating with gold
in an SPI-Module Sputter Coater. Stubs were examined in a
lEOL ISM 5300 SEM and photographed.

RESULTS

Gamete structure and fertilization in Leptochitonidae
(Order Lepidopleurida)

The sperm of Leptochiton riigatiis (Pilsbry, 1892) and
Leptochiton assimilis Thiele, 1909 are essentially similar in
structure to Leptochiton aselhis (Gmelin, 1791) (Hodgson et
ah 1988), having a bullet-shaped nucleus capped by an elon-
gate acrosome cone about 3 pm in length with subacrosomal
material (Figs. lA, 1C, 2A). The proximal and distal centri-
oles are central and can be distinguished as separate entities
although connected by flocculent material (Fig. IB). They
are surrounded by five or six large, spherical mitochondria
and give rise to a central flagellum (Figs. lA, 2A). Sperm of
the lepidopleurids Deshayesiella curvata Carpenter in Pilsbry,
1892 and Hanleya hanleyi W. Bean in Thorpe, 1844 are
essentially similar in terms of centrioles and mitochondria.
However, in these sperm, as an extension of the nucleus,
there is a short nuclear filament less than 2 pm in length,
which is capped by a smaller acrosome about 1 pm long
(Figs. ID, 2B).

The eggs of Leptocl^iton aselhis have a smooth jelly coat
without pores (Figs. IE, IF, 3A), whereas those of Deshayesi-
ella curvata have been shown to have large pores in the jelly

coat (Pashchenko and Drozdov 1998), similar to those of
Calloclutou ilentatus (Spengler, 1797) (= Callochiton casta-
neus Wood, 1815) (Fig. 3B). Observations of fertilization in
L. aselhis within ten minutes of exposure of eggs to sperm
revealed that the sperm had digested the jelly coat and
breached a large hole in the vitelline layer, coming to rest in
the perivitelline space (Figs. IE, IF). This is the first time in
chitons, and specifically in lepidopleurids, that the entire
sperm has been shown to penetrate below the vitelline layer.

Gamete structure and fertilization in Callochitonidae
(Order Chitonida)

The sperm of Callochiton dentatiis is unlike lepidopleu-
rid sperm because it has a nuclear filament greater than 3 pm
in length, tipped by a highly reduced acrosome (Fig. 2C).
Furthermore, the centrioles are fused into a slightly acentric
basal body and the five mitochondria are not all spherical
(Fig. 2C, 4A). This description would apply to virtually all
sperm of Chitonida but none of Lepidopleurida, so far
described.

The eggs of Callochiton dentatiis are similar to those of
Deshayesiella curvata with open pores in a thick jelly coat
(Fig. 3B). Each pore coincides with a depression in the egg
membrane. At fertilization the sperm nuclear filament
bridges the gap between the vitelline layer and egg mem-
brane, thus replacing the acrosomal process that character-
izes Lepidopleurida and other molluscs. This is significant
because in C. dentatiis, when the acrosome digests the vitel-
line layer, only a tiny pore is made (Eig. 4B), which does not
permit the fertilization cone to raise up and engulf the entire
sperm, and it remains below the vitelline layer (Figs. 4B,
4D). Current evidence suggests that sperm mitochondria
and centrioles are left on the surface of the egg.

Gamete structure and fertilization in Chitonina (Order
Chitonida)

The sperm of all Chitonina have very acentric basal
bodies in which the proximal centriole is positioned perpen-
dicular and lateral (on the mitochondrial side) to the distal
centriole in a fused mass (Figs. 2D, 4E, 5A, 5B). The basal
body is usually in line with one side of the nucleus, with the
mitochondria reduced in number to 3 or 4 on the other side.
As usual, the annulus binds the distal centriole to the plasma
membrane but extending from it, along the plasma mem-
brane, mainly on the side containing the mitochondria, is a
dense thickening (Fig. 2D, 4E, 5A, 5B). In addition to basal
mitochondria, lateral mitochondria are present in sperm of
some Chitonina, such as Ischnochiton, Stenoplax, and Lepi-
dozoncL (Fig. 5B). Glycogen rosettes are visible in the space
between mitochondria and centrioles (Figs. 2D, 4E, 5A, 5B).

All Chitonina have eggs with elaborate hulls raised into

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AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 1. A, Sperm of Leptochitoii aseUiis viewed with SEM. Note anterior elongate acrosome (Ac) and mitochondria (M) posterior to
nucleus. Scale bar = 1 pm. B, TEM of base of sperm of Leptochitoii rugatus showing separate proximal centriole (PC) and distal centriole
(DC), adjacent to spherical mitochondrion and dense nucleus. Scale bar = 0.3 pm. C, Sperm acrosome cone (Ac) and tip of nucleus (N)
of Leptochitoii assiiiiilis, viewed with TEM. Scale bar = 0.5 pm. D, Sperm of Deshayesiella curvata viewed with SEM. Note nuclear filament
(NF) and acrosome (Ac). Scale bar = 1.5 pm. E, Light micrograph of 1 pm section of egg of Leptochitoii aselhis showing that entire sperm
has breached vitelline layer (arrow). Scale bar = 35 pm. F, Part of E magnified to show penetrating sperm (Sp) beneath vitelline layer (VL),
prior to entry into the egg. Scale bar = 7 pm.

a series of spines with narrow bases ranging in size from 5 to
30 pm (Figs. 3C, 3D, 6A-D). Two main types of fertilization
have been observed. The first type involves penetration of
open pores in the hull (Fig. 7B); and the second type in-
volves sperm digestion of a thin dense layer covering the hull
(Figs. 7D, 7E).

Mechanism 1: Fertilization via open pores in hull

Stenosennis albus (= Ischnochiton alhus) and Chae-
toplenra apicnlata have open pores in the egg hull, ranging
in size from 1 to 4 pm, at the base of the spines. Within
about thirty seconds of sperm release into a beaker of
eggs, some sperm locate and penetrate these pores with their

FERTILIZATION AND THE EVOLUTION OF CHITONS

101

crgtj 'Ok

Figure 2. A, Sperm of Leptochiton aselhis; B, sperm of Deshayesiella curvata; C, sperm of
Callochiton dentatiis; and D, sperm of Chiton tubercidatus.

anterior filament (Fig. 7B). The pores provide sperm direct
access to the vitelline layer, which is digested by the
acrosome before fusion occurs between sperm and egg
membranes.

Hull spines of Chaetopleura apiculata eggs are complex
with many branches off each spine (Fig. 6D). Overlapping
branches between spines create complex channels that the
sperm must negotiate in order to reach the base of the
spines, where the pores are located. In C. apiculata the pores
can be large enough to admit entire sperm, which sometimes

gain direct access to the vitelline layer,
before penetrating the egg (Buckland-
Nicks and Brothers 2008).

In Stenosemus albus the hull com-
prises long spines with recurved tips
(Fig. 3C). Pores are arrayed alongside
the junctions of hexagonal bases,
around the perimeter of each base
(Fig. 7B).

Mechanism 2: Fertilization via a dense
layer on hull

Rhyssoplax ttilipa (= CJtiton tulipa
Quoy and Gaimard, 1835) and Steno-
plax conspicua both have a continuous
dense layer overlying the hull. This
dense layer is invariably digested by
the acrosome reaction on contact with
the hull (Figs. 7D-E) and has been ob-
served in numerous thick sections as
well as some thin sections of both spe-
cies. Radsia nigrovirescens (= Chiton
nigrovirescens de Blainville, 1825) is
unusual among other Chitoninae ex-
amined in having pores in the hull
(Fig. 7A) although it is not known if
these are used by the sperm at fertil-
ization. Typically, brooding chitons
have reduced spines or cupules, but
specimens of R. nigrovirescens have
long, simple spines with hooked
tips (Fig. 3D), more like those of Ste-
nosennis albus, than those of other
Chitoninae.

Acanthopleura granulata (Gmelin,
1791) has unusual polymorphic spines
on the egg (Fig. 6C). Some spines are
short and bifurcating at the tip
whereas others are intermediate or
longer in length and have a scaly ap-

pearance (Fig. 6C). This egg does not

appear to have pores in the hull and
the sperm penetrate directly, in the same way as they do in
Stenoplax conspicua and Rhyssoplax tulipa.

Gamete structure and fertilization in Acanthochitonina
(Order Chitonida)

Gamete structure and fertilization in Acanthochitonina
is broadly similar to that in Chitonina. However, all sperm of
Acanthochitonina can be distinguished by having anterior
mitochondria as well as basal and lateral ones, numbering 7
or 8 in total. Below the annulus, a fibrous complex is found

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AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 3. A, Quarter of egg of Leptochitou asellus viewed with SEM, showing smooth surface of coat without pores. Scale bar = 25 pm. B,
Quarter of egg of Callochiton deiitatiis viewed with SEM, showing regularly spaced pores in smooth jelly coat. Scale bar = 25 pm. C, Quarter
of egg of Stenoseiuiis alhiis viewed with SEM, showing simple spines with hooked tips and hexagonal bases measuring 36 pm across. Scale
bar = 50 pm. D, Quarter of egg of the brooding chiton Radsia iiigwvirescens showing long simple spines with hooks which interlock in the
pallial grooves retaining eggs. Scale bar = 50 pm.

Figure 4. A, TEM of sperm of Callochiton dentatiis showing oblong mitochondrion (M) and centriolar basal body (BB) acentric to nucleus.
Scale bar = 1 pm. B, TEM of sperm penetrating egg of C dentatiis, showing long thread of chromatin in egg cortex. Pore in vitelline layer
is too small to admit sperm organelles which, like the elongate mitochondrion, appear to be abandoned on the surface in a bag of
membrane. Scale bar = 1 pm. C, TEM of sperm of Mopalia nuiscosa showing proximal centriole (PC) fused to apex of distal centriole (DC)
in fossa of nucleus (N). Flagellum is reinlorced by a fibrous complex (EC), which characterizes the suborder Acanthochitonina. Scale
bar = 0.2 pm. D, SEM of polyspermic fertilization of Callochiton dentatiis egg, showing multiple sperm (Sp) penetrating vitelline layer (VL)

FERTILIZATION AND THE EVOLUTION OE CHITONS

103

and the induction of several fertilization cones (FC). Note: jelly layer has been completely dissolved. Scale bar = 5 pm. E, TEM of sperm
of Chaetopleura apiailata, showing basal body comprised of proximal centriole (PC) fused laterally to distal centriole (DC), which produces
an acentric flagellum. Thickening of membrane is visible extending posteriorly from annulus (An) on both sides. Mitochondria with
glycogen granules are arranged in posterior extension of mid-piece. Scale bar = 0.5 pm.

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AMERICAN MALACOLOGICAL BULLETIN

25 • 1/2 • 2008

Figure 5. A, Sperm of Chaetopleura apiculata-, B, sperm of Stenoplax conspicua\ C, Sperm of
Acanthochitona viridis; and D, sperm of Mopalia muscosa.

elaborate egg hulls raised into large
cupules with wide bases ranging in size
from 50 to 90 pm (Figs. 8A-D). Two
main types of fertilization have been
observed which are defined also by cu-
pule morphology. The first involves
closed-hull cupules with small pores in
the intercupule area that give sperm
direct access to the vitelline layer (Fig.
7F). The second type involves open-
hull cupules with sperm swimming in-
side and penetrating both hull and vi-
telline layer without access to any
pores (Fig. 7G). In both cases, the
sperm inject the chromatin into the
egg through the narrow nuclear fila-
ment. Other sperm organelles, includ-
ing mitochondria and centrioles, ap-
pear to be excluded and left behind on
the egg surface in a bag of sperm
membrane (Fig. 7C).

Some species with closed cupules
are brooders and in some of these,
such as Cyanoplax fernaldi (= Lepido-
chitona fernaldi Eernisse, 1986) (Fig.
8C), the cupules are reduced but
maintain the same form as non-
brooding species, such as Cyanoplax
dentiens (= Lepidochitona dentiens
(Gould, 1846)) (Fig. 8C). Among
open-cupule species some have a more
complex, folded-cupule structure with
protruding elements inside each cu-
pule (Fig. 7G) (e.g., the genera Toni-
cella. Cryptochiton, and Mopalia of
those studied here). However, Nuttal-
lina californica (Nuttall MS, Reeve,
1847) and related species do not have
these protruding structures (Fig. 8D).

on the side opposite to the mitochondria (Figs. 4C, 5C-D).
More specific differences in the arrangement of centrioles in
the basal body and in the structure of the acrosome may
distinguish among two or more groups in this suborder. For
example, in Cyanoplax and Acanthochitona the proximal
centriole fuses laterally with the distal centriole towards the
axis of the sperm, as was found in Chitonina (Fig. 5C).
However, in the genera Tonicella Carpenter, 1873, Crypto-
chiton Middendorff, 1847, and Mopalia Gray, 1847, the
proximal centriole fuses to the anterior of the distal centriole
and is embedded in a small nuclear fossa (Figs. 4C, 5D).

Acanthochitonina species also are united by having

Cladistic analysis

Sperm and egg characters for all taxa are summarized
(Tables 2-3). The data matrix (Table 2) was run through
branch and bound analysis in PAUP 4.0. The Bootstrap con-
sensus tree (for 500 replicates) resulting from this analysis is
shown with confidence values written above the line for each
node (Fig. 9). Lepidopleurida came out as paraphyletic in
this consensus tree, largely because characters for egg and
sperm shared by Deshayesiella curvata and Hanleya hanleyi
were coded as apomorphic (Table 4). There was 100% sup-
port for a monophyletic Chitonida, which includes Callo-
chitonidae as the sister taxon to Chitonina plus Acanthochi-

FERTILIZATION AND THE EVOLUTION OE CHITONS

105

Figure 6. A, Quarter of egg of Stenoplax conspicna viewed with DIG optics, showing spines with bifurcating tips, which characterize this and
other genera, including Lepidozona. Scale bar = 25 pm. B, Quarter of egg of Rhyssoplax lulipa viewed with SEM, showing spines with
petalloid tips, typical of this genus and of some species of Ischnochitoii. Scale bar = 20 pm. C, Quarter of egg of Acanthopleura gramdata
viewed with SEM, showing unique polymorphic spines. Some spines are elongate with a scale-like outer layer; others are intermediate in
length and a third type is short and bifurcates at the tip. Scale bar = 20 pm. D, Quarter of egg of Cliaetopleura apicidata viewed with SEM,
showing complex branching spines that may be unique to this genus. Scale bar = 30 pm.

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AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 7. A, SEM detail at base of spine on egg of Radsia iiigrovircsceiis showing pores in hull (arrows). Scale bar = 3 pm. B, SEM of egg
oi Steiwseiiuis albiis showing pores at perimeter of hexagonal bases of spines, which allow sperm (arrow) direct access to vitelline layer. Scale
bar = 1 pm. C, LM of 1 pm section of Mopalia muscosa egg fertilized two hours before with dilute sperm suspension. Serial sections revealed
only one membrane bag containing eight or nine particles which correspond to the roughly eight mitochondria and centrioles of this sperm
(Buckland-Nicks and Brothers, unpubl. micrograph). Scale bar = 1 pm. D, SEM of sperm penetrating egg of Stenoplax conspkita showing

FERTILIZATION AND THE EVOLUTION OF CHITONS

107

Figure 8. A, Quarter of egg of Cyanoplax dentiens viewed with SEM, showing dosed hull cupules typical of this genus, as well as
Acanthochitona. Scale bar = 25 pm. B, Quarter of egg of Mopalia innscosu viewed with SEM, showing open cupules typical of this clade. Scale
bar = 25 pm. C, Quarter of egg of brooding chiton Cyanoplax fennildi viewed with SEM, showing reduced hull cupules with typical
hexagonal bases. Micrograph courtesy of D. Eernisse (see also Eernisse 1984). Scale bar = 30 pm. D, Quarter of egg of NuttaUina califomica
viewed with SEM, showing the type of open-hull cupules that lack internal protrusions. Scale bar = 20 pm.

<-

dissolution of outer dense layer around nuclear filament (arrow). Scale bar = 1 pm. E, TEM of sperm penetrating egg of Rhyssoplax tidipa
showing dissolution of outer dense layer around nuclear filament (arrow). Scale bar = 2 pm. F, Fertilized egg of Cyanoplax dentiens showing
penetrating sperm (arrow) between closed cupules. Scale bar = 10 pm. G, Fertilized egg of Mopalia nuiscosa showing protrusions (arrow)
from inside wall of hull cupules that are absent from cupules of NuttaUina califomica. Scale bar = 5 pm.

108

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Table 2. Characters used in cladistic analysis of Polyplacophora and Aplacophora (outgroups) with character state codes in boldface font
(see Table 3 for data matrix).

SPERM AND EGG DATA

(1) Acrosome; 0: Acrosome forms from Golgi body in posterior of spermatid; 1: Acrosome forms by aggregation of small
proacrosome vesicles at filament tip.

(2) Acrosome structure: 0: Cone with subacrosomal granule (SAG), interstitial granule (IG), and subacrosomal plate (SAP);

1: Cone with SAG and SAP; 2: Vesicle with SAP.

(3) Mitochondria position: 0: Sheath around flagellum; 1: Ring around centrioles; 2: Ring around offset basal body; 3: Basal
mitochondria in collar; 4: Lateral mitochondria; 5: Lateral and anterior mitochondria.

(4) Mitochondria number: 0: 1-2; 1: 3-4; 2: 5-6; 3: 7-9.

(5) Mitochondrial shape: 0: Fused spiral; 1: All spherical; 2: Not all spherical.

(6) Nuclear filament: 0: Absent; 1: 1-2 pm; 2: ^3 pm.

(7) Flagellum reinforcement; 0: Absent; 1: Spiral ribbon; 2: Thickened membrane; 3: Unilateral fibrous body.

(8) Chromatin pattern: 0: Granular; 1: Thick short fibers; 2: Fine then coarse fibers; 3: Fine long fibers.

(9) Centrioles: 0: Basal body in deep nuclear fossa; 1: Separate centrioles; 2: Proximal centriole (PC) fused lateral to distal
centriole (DC) and offset; 3: PC fused anterior to DC in nuclear fossa.

(10) Hull projections: 0: Absent; 1: Spines with bases 5-30 pm; 2: Cupules with bases 50-90 pm.

(11) Hull cupules: 0; Absent; 1: Closed; 2: Open.

(12) Jelly coat: 0: Absent; 1: Smooth without pores; 2: Smooth with large pores >5 pm.

(13) Hull structure: 0: Jelly coat; 1: Jelly coat with macropores; 2: Hull with micropores in spines; 3: Hull with dense layer; 4: Hull
with micropores between cupules; 5: Cupules without pores.

(14) Fertilization site; 0: Internal 1: External: anywhere on egg; 2: Sperm enters specific site.

(15) Site-specific sperm entr)^: 0: Absent; 1: Macropores >5 m; 2: Between hull projections; 3: Inside hull projections.

(16) Fertilization cone; 0: Engulfs sperm and organelles; 1: Engulfs only chromatin.

OTHER MORPHOLOGICAL DATA

(17) Gill position: 0; Reduced; 1: Adanal; 2: Abanal.

(18) Gill type; 0: Absent; 1; Merobranchial; 2; Holobranchial.

(19) Insertion plate: 0: Absent; 1: Present; 2: Slitted; 3: Pectinated.

(20) Body covering: 0: Spinous; 1: 8 shell valves.

(21) Foot: 0: Absent; 1: Present.

(22) Shell valves: 0: Absent; 1: Modern valve shape; 2: Terminal valves with fissures.

tonina. There was also strong support (96%) for monophyly
of the rest of Chitonida (= Chitonina -F Acanthochitonina).
However, there was weaker support (65%) for a monophy-
letic Chitonina, even though a number of apomorphic char-
acters are shared by this grouping (Table 4). Also, there was
similar support (62%) for a monophyletic grouping of Acan-
thochitonina, members of which share a different set of apo-
morphic characters (Table 4). However, there was strong
support (97%) for a monophyletic Mopaliidae, which in-
cluded Mopalia, Cryptochiton, and Tonicella. The apomor-
phy hypotheses for each of 8 selected internal nodes are
listed in Table 4.

DISCUSSION

Mechanisms of fertilization

Lepidopleurida: Leptochitonidae

Confirmation of the extrusion of an acrosomal process
(Buckland-Nicks 2006) and breaching of the vitelline layer
by the sperm acrosome indicates that Leptochiton aselhis

penetrates the egg in a manner similar to that of most other
Metazoa, including scaphopods (Dufresne-Dube et al. 1983)
and sea urchins (Longo 1987) although this remains to be
confirmed. Other Lepidopleurida, such as Deshayesiella cur-
vata and Hanleya hanleyi, are likely to do the same as ex-
trusion of an acrosomal process has been observed in D.
curvata (Buckland-Nicks, unpubl. data), and sperm of H.
hanleyi are constructed in the same way. However, this will
require confirmation by direct observation of sperm organ-
elles following fertilization. The current analysis showed
Lepidopleurida to be paraphyletic in agreement with Eer-
nisse et ah (2006). However, this result depends largely on
coding changes in sperm and egg structure in Deshayesiella
and Hanleya as derived, rather than evolving by conver-
gence. Recent molecular analyses (Eernisse, unpubl. data)
have shown that these genera form part of a monophyletic
Leptochitonidae. A combined molecular and morphological
analysis would produce the most robust test of these phylo-
genetic relationships but for the moment we will continue to
regard Lepidopleurida as the basal monophyletic order, as in
Sirenko (2006).

Callochiton dentatus, although retaining the plesiomor-

FERTILIZATION AND THE EVOLUTION OF CHITONS

109

Table 3. Data matrix showing characters and their character states for the species listed. A, Acrosome; As, Acrosome structure; Mp,
Mitochondria position; M#, Mitochondria number; M, Mitochondria shape; Nf, Nuclear filament; Fr, Flagellum reinforcement; Ch,
Chromatin pattern; Ce, Centrioles; Hp, Hull projections; He, Hull cupules; Ic, lelly coat; Hs, Hull structure; Fs, Fertilization site; Se, Site
specific sperm entry; Fc, Fertilization cone; Gp, Gill position; Gt, Gill type; Ip, Insertion plates; Be, Body covering; F, Foot; Sv, Shell valves;
?, unknown state; *, outgroups.

Character typie — >

Ac

As

Mp

M#

M

Nf

Fr

Ch

Ce

Hp

He

Ic

Hs

Fs

Se

Fc

Gp

Gt

IP

Be

F

Sv

J-Genus/species

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

Epimenia australis*

0

0

0

0

0

0

1

?

0

0

0

1

01

0

0

?

0

0

0

0

0

0

Chaetoderma argenteum*

0

2

1

2

1

0

0

0

1

0

0

1

0

1

0

?

0

0

0

0

0

0

Leptochiton asellus

0

1

1

2

1

0

0

1

1

0

0

1

0

1

0

0

1

1

0

1

1

1

Deshayesiella curvata

0

1

1

2

1

1

0

1

1

0

0

1

1

2

1

0

1

1

0

1

1

1

Hanleya hanleyi

0

1

1

2

1

1

0

1

1

0

0

1

1

2

1

0

1

1

1

1

1

1

Callochiton dentatus

1

2

2

2

2

2

0

2

2

0

0

1

1

2

1

1

1

1

2

1

1

1

Chaetopleura apkidata

1

2

3

1

2

2

2

2

2

1

0

0

2

2

2

1

1

2

2

1

1

2

Stenosemus albus

1

2

3

1

2

2

2

2

2

1

0

0

2

2

2

1

1

2

2

1

1

2

Acanthopleura granulata

1

2

3

1

2

2

2

2

2

1

0

0

2

2

2

1

1

2

3

1

1

2

Rhyssoplax tulipa

1

2

3

1

2

2

2

2

2

1

0

0

2

2

2

1

1

2

3

1

1

2

Ischnochiton hakodadensis

1

2

4

1

2

2

2

2

2

1

0

0

2

2

2

1

1

2

2

1

1

2

Stenoplax conspicua

1

2

4

1

2

2

2

2

2

1

0

0

2

2

2

1

1

2

2

1

1

2

Lepidozona retiporosa

1

2

4

1

2

2

2

2

2

1

0

0

2

2

2

1

1

2

2

1

1

2

Cyanoplax dentiens

1

2

5

3

2

2

3

3

2

2

1

0

3

2

2

1

2

1

2

1

1

2

Acanthochitona viridis

1

2

5

3

2

2

3

3

2

2

1

0

3

2

2

1

2

1

2

1

1

2

Tonicella lineata

1

2

5

3

2

2

3

3

3

2

2

0

4

2

3

1

2

2

2

1

1

2

Cryptochiton stelleri

1

2

5

3

2

2

3

3

3

2

2

0

4

2

3

1

2

2

2

1

1

2

Mopalia muscosa

1

2

5

3

2

2

3

3

3

2

2

0

4

2

3

1

2

2

2

1

1

2

Epimenia australis (Aplacophora)
Chaetoderma argenteum (Aplacophora)
Leplochilon asellus (Leptochitonidae)
Deshayesiella curvala (Leptochilonidae)
Hanleya hanleyi (Leptochilonidae)

Callochilon denfalus (Callochitonidae)
Stenosemus albus (Ischnochttonidae)
Chaelopleura apiculala (Chaetopieundae)
Acanthopleura granulata (Acanthopleuridae)
Rhyssoplax tulipa (Chitonidae)

Ischnochilon hakodadensis (Ischnochitomdae)
Slenoplax conspicua (Ischnochitomdae)
Lepidozona retiporosa (Ischnochitomdae)
Cyanoptax denliens (Lepidochitonidae)
Acanthochitona vindis (Acanlhochitomdae)
Mopalia muscosa (Mopaliidae)

Cryplochiton slellen (Mopaliidae)

Tonicella lineata (Mopaliidae)

Acanthochitiiiiiii;)

Figure 9. Consensus tree from branch and bound analysis in PAUP
4.0 of the data matrix in Table 2. Numbers above nodes represent
the bootstrap values for 500 replicates. Numbers below refer to
internal nodes. Apomorphy hypotheses describing these nodes are
listed in Table 4.

phic egg type first discovered in Deshayesiella curvata (Pa-
shchenko and Drozdov 1998), has evolved several apoinor-
phic features in the sperm, which dictate a novel mechanism
of fertilization, and one that characterizes the order Chito-
nida (Buckland-Nicks and Hodgson 2000, Buckland-Nicks
2006). In this mechanism a permanent nuclear filament has
replaced the extrusion of an acrosomal process; after the tiny
acrosome digests a small hole in the vitelline layer, the
nuclear filament fuses with an egg microvillus and delivers

Table 4. Apomorphy hypotheses for selected internal nodes of
bootstrap consensus tree shown in Fig. 9. Unless otherwise stated in
brackets, character changes are from 0 — >1.

Internal node

Character changes

Node 1: Polyplacophora

17, 18, 20, 21, 22

Node 2: Deshayesiella +

6

Hanleya

Node 3: Chitonida

1, 2 (1-2), 3 (1-2), 5 (1-2), 6 (1-2),
8 (1-2), 9(1-2), 16, 19 (1-2)

Node 4: Chitonina +

3 (2-3), 4 (2-1), 10, 12 (1-0), 15

Acanthochitonina

(1-2), 18 (1-2), 22 (1-2)

Node 5: Acanthochitonina

3 (4-5), 4 (1-3), 7 (2-3), 8 (2-3),
10 (1-2), 11, 13 (2-3), 17 (1-2)

Node 6: Chitonina

7 (0-2), 13 (1-2)

Node 7: Cyanoplax +

11, 13 (3-4), 18 (2-1)

Acanthochitona

Node 8: Mopalia +

9 (2-3), 11 (1-2), 13 (4-5), 15 (2-3)

Cryptochiton + Tonicella

the chromatin into the egg. Furthermore, current evidence
suggests that other sperm organelles, including mitochon-
dria and centrioles, do not pass through this minute tube
and are left behind on the surface (Buckland-Nicks and
Hodgson 2000, Buckland-Nicks 2006, Buckland-Nicks and
Brothers 2008). These characteristics place Callochitonidae
firmly within Chitonida and outside the Lepidopleurida

110

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

(Eig. 9). This result is different than that obtained by one
molecular analysis (Okusu et al. 2003) but agrees with pre-
vious results based on morphological data (Buckland-Nicks
1995, 2006, Sirenko 1993, 2006) or DNA sequences encoding
for hemocyanin protein (Lieb et al 2006).

Chitonida: Chitouina

The suborder Chitonina is unified by synapomorphic
gamete characters (Table 4). In the sperm, these include the
very acentric basal bodies with proximal centrioles posi-
tioned perpendicular and lateral to the distal centriole in a
fused mass towards the mitochondrial side (see also Hodg-
son et al 1988) and a dense thickening that extends from the
annulus along the plasma membrane adjacent to the mito-
chondria (Buckland-Nicks 2006). In the egg, synapomorphic
characters include hull spines with narrow bases (Sirenko
1993, 2006, Buckland-Nicks 2006). New data on fertilization
in Chaetopleura apiculata (Buckland-Nicks and Brothers
2008) and Stenosemus albas indicate that they and their close
relatives are more basal among Chitonina, a result which was
found also by Okusu et al. (2003) in their combined mo-
lecular analysis (p. 293, fig. 6) and combined morphological
analysis (p. 295, fig. 8). However, the consensus tree in the
present study found weak support (65%) for a monophyletic
Chitonina and was unable to distinguish between the fami-
lies in this suborder. A combined molecular and morpho-
logical analysis would likely provide a more robust test with
better resolution.

This study supports the finding of Okusu et al (2003)
that the genus Iscluiochiton is polyphyletic. Okusu et al
(2003) showed that Ischnochiton rissoi (Payraudeau, 1826) is
closely related to Chaetopleurinae whereas other Ischnochi-
ton species came out within Ischnochitoninae. Spine form in
general seems to be a good indicator of relationship among
genera such as Stenoplax and Lepidozona (bifurcating spines)
as well as Chiton Linnaeus, 1758 and Rhyssoplax Thiele, 1893
(petalloid spines), whereas the mosaic of spine form found
among different species of Ischnochiton suggests polyphyly
(see diagrams by Sirenko 1993 and 2006). The present study
supports reclassification of Ischnochiton albas (Linnaeus,
1767) as Stenosemas albas (see Sirenko 2006). Only further
studies on the reproductive biology and molecular biology of
other species of Ischnochiton will resolve any further incon-
sistencies in classification.

Radsia nigrovirescens is an anomaly among Chitoninae
as it has pores in the hull and an unusual spine form, more
like that of Stenosemas albas. Furthermore, it is a brooder
and in most brooders the spines are reduced or absent (Eer-
nisse 1984, Buckland-Nicks and Eernisse 1992). Fertiliza-
tion biology has not yet been studied and it remains un-
known if the sperm penetrate these pores, as occurs in
Stenosemas and Chaetopleura, or digest the egg hull to make

an open pathway, as in other Chitoninae (Buckland-Nicks
2006).

Acanthopleara granidata is unique among chitons ex-
amined to date, as it has evolved polymorphic spines, which
include long and intermediate scaly ones, as well as short
bifurcating ones. Previously, eggs of A. granalata were illus-
trated with straight spines ending in points (Sirenko 1993,
2006), which is clearly a simplification. A recent analysis of
a related species, Acanthopleara echinatas Barnes, 1824
(Gaymer et al. 2004), revealed spines with petalloid tips, like
some other genera of Chitoninae, including Chiton and
Rhyssoplax. In keeping with this, the hull does not have pores
like Chaetopleara and Stenosemas, suggesting that the
mechanism of fertilization is more like that in genera of
Chitoninae, such as Chiton, Rhyssoplax, or of Ischnochito-
ninae, such as Stetwplax and Lepidozona. However, a recent
molecular analysis (Eernisse, unpubl. data) indicates that
Acanthopleurinae -t Toniciinae is separate from Chitoninae.

Chitonida: Acanthochitonina

Members of the suborder Acanthochitonina share syn-
apomorphies for sperm, with a fibrous complex on the fla-
gellum and anterior mitochondria, and for eggs, with broad
based cupules. Also this suborder is characterized by abanal
gills (Sirenko 1993, 2006). However, two groups emerge
within this suborder, one united by closed cupules, laterally
fused centrioles and fertilization between cupules {e.g., Cy-
anoplax Pilsbry, 1892 and Acanthochitona Gray, 1821), and
the second united by open cupules, anteriorly fused centri-
oles, and fertilization inside cupules (Mopalia, Tonicella,
Cryptochiton) (see Table 4). Cyanoplax cinerea (= Lepidochi-
tona cinerea Linnaeus, 1767) may represent an exception to
this, as it is reported to have open cupules (Eernisse 1984).
In the present analysis, Cyanoplax places outside Mopaliidae,
as was reported by Buckland-Nicks ( 1995) and Okusu et al.
(2003; figs. 6, 8). This disagrees with Sirenko (2006), who
placed this genus within Tonicellidae. Furthermore, in this
analysis Cryptochiton comes out with Mopalia and Tonicella
within Mopaliidae, a placement which agrees with Okusu et
al (2003: figs. 6, 8) and Eernisse (pers. comm.). However,
Sirenko (2006) placed Cryptochiton in Acanthochitonidae.
Okusu et al. (2003: fig. 7B) suggested that there is a possi-
bility that Acanthochitonina species are paraphyletic based
on one of two consensus trees for combined molecular data.
However, based on morphology alone for the genera in-
cluded here, this study finds some support (62%) for a
monophyletic Acanthochitonina, as was also shown by the
second consensus tree of Okusu et al. (fig. 7 A: 2003).

ACKNOWLEDGMENTS

Thanks are due to Doug Eernisse for advice on dadistic
analysis (mistakes are all mine!), the use of part of a micro-

FERTILIZATION AND THE EVOLUl'ION OF CHITONS

111

graph for Fig. 8C, and help with identifying Stenoplax con-
spicun, NuttaUiim califomica, Cynnoplax fernaldi, and Chae-
topleura apiailntn; to David Garbary for assistance using
MacClade and PAUP; to Boris Sirenko for collecting Lepto-
chiton nigatiis, L. assimilis, and Deshnyesiella ciirvata; to Ross
Mayhew for collecting Hanleya hnnleyr, and to Christoffer
Schander for help collecting and identifying Leptochiton asel-
lus and Stenosemiis albus. Thanks also are due to Haixin Xu
for expert technical assistance and to Norma Mitchell for
encoding plates in Photoshop. This research was supported
by a NSERC of Canada Discovery Grant.

LITERATURE CITED

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Buckland-Nicks, 1. and A. N. Hodgson. 2000. Fertilization in Cal-
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Vehger\7: 141-152.

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1988. Comparative spermatology of 11 species of Polyplaco-
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nina and Acanthochitonina. Proceedings of the Royal Society of
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Kaas, P. and R. A. Van Belle. 2003. Monograph of Living Chitons
(Mollusca: Polyplacophora). Vols. 1-5. Brill Publishers, Leiden,
The Netherlands.

Kaas, P., R. A. Van Belle, and H. Strack. 2006. Monograph of Living
Chitons (Mollusca: Polyplacophora). Vol. 6. Brill Publishers,
Leiden, The Netherlands.

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meets chitons: Phylogeny of polyplacophorans revisited by
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Longo, F. I. 1987. Fertilization. Chapman and Hall, New York.

Okusu, A., E. Schwabe, D. Eernisse, and G. Giribet. 2003. Towards
a phylogeny of chitons (Mollusca, Polyplacophora) based on
combined analysis of five molecular loci. Organisms, Diversity
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metes in five species of Far-Eastern chitons. Invertebrate Re-
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ancestry of bilaterians; Inferences from morphology and 18S
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Sirenko, B. I. 1993. Revision of the system of the order Chitonida
(Mollusca: Polyplacophora) on the basis of correlation be-
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rion processes. Ruthenica 3: 93-117.

Sirenko, B. I. 2006. New outlook on the system of chitons (Mol-
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Submitted: 15 May 2007; accepted; 9 November 2007;

final corrections received; 26 February 2008

Amer. Make. Bull. 25: 113-124 (2008)

Chitons (Mollusca: Polyplacophora) associated with hydrothermal vents and
methane seeps around Japan, with descriptions of three new species'^

Hiroshi Saito\ Katsunori Fujikura^, and Shinji Tsuchida^

' Department of Zoology, National Museum of Nature and Science, Tokyo, 3-23-1 Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073 Japan,
h-saito@kahaku.go.jp

^ Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima, Yokosuka-shi, Kanagawa 237-0061 Japan,
fujikura@jamstec.go.jp

^ Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima, Yokosuka-shi, Kanagawa 237-0061 Japan,
tsuchidas@jamstec.go.jp

Abstract: Three new species of chitons are described from hydrothermal vent sites and methane seep sites around Japan: Deshayesiella
sirenkoi n. sp. from the hydrothermal vent sites on the seamounts in the northern Mariana Islands area, Placiphorella okutanii n. sp. from
Hachijo Depression in the Izu-Ogasawara (Bonin) Islands area where no active vent/seep area has been discovered, but the possibility of
hydrothermal activity has been suggested, and Placiphorella isaotakii n. sp. from methane seep sites on the Kuroshima Knoll off Yaeyama
Islands. Deshayesiella sirenkoi n. sp. as well as two previously known hydrothermal vent species, Leptochiton tenuidontus Saito and Okutani,
1990 and Thermochiton undocostatus Saito and Okutani, 1990, are vent/seep associated species, whereas the two Placiphorella may be guest
species. Additional distributional records are given for the two known species.

Key words: Deshayesiella, Placiphorella, deep-sea, chemosynthetic environment, taxonomy. Pacific Ocean

The number of the molluscan taxa described from che-
mosynthetic environments has rapidly increased in the last
two decades (Sasaki et al. 2005). Most of these taxa are,
however, gastropods and bivalves (Desbruyeres et al. 2006:
520-523). Since Saito and Okutani (1990) described two chi-
ton species from the hydrothermal vent site of Okinawa
Trough, East China Sea, some chiton species have been re-
ported from chemosynthetic environments. Sc]uires and
Goedert (1995) reported Leptochiton alveolus (Loven, 1846)
(sensu Ferreira 1979 and Kaas and Van Belle 1985) from
Eocene and Oligocene cold methane seep limestones, Olym-
pic Peninsula, Washington. Olu et al. ( 1997) reported “Poly-
placophora” from the methane seep of Barbados Prism,
1,000-2,000 m, and Sellanes et al. (2004) reported Leptochi-
ton sp., Stenosemus sp., and Placiphorella sp. from methane
seepage in the bathyal zone off Chile. Schwabe and Sellanes
(2004) have described a new species, Lepidozona balenophila,
from another type of chemosynthetic environment, decom-
posing whale carcasses. However, no vent/seep associated
chiton species, other than the two known species, has been
described anywhere else in the world. Those two known
species that were described from the hydrothermal vent in
the East China Sea are Leptochiton tenuidontus Saito and
Okutani, 1990 and Therinochiton undocostatus Saito and

Okutani, 1990. They were collected by a human-occupied
submersible, Shinkai 2000, belonging to Japan Agency for
Marine-Earth Science and Technology (JAMSTEC). Since
then, some additional chiton specimens have been collected
from Japanese waters by the Deep Sea Research System,
including human-occupied submersibles or ROVs belonging
to JAMSTEC. Here, we describe three new species and fur-
ther describe the morphology and distribution of the two
previously known species.

MATERIALS AND METHODS

Specimens were collected by the Deep-sea Research Sys-
tem of JAMSTEC: human-occupied submersibles Shinkai
2000 (abbreviated as 2K) and Shinkai 6500 (6K) and a re-
motely operated vehicle Hyper-Dolphin (HPD). Sampling
sites are shown in Fig. 1. Preparation for SEM observation
followed Saito (2006). All specimens were deposited in the
molluscan collection of the Department ot Zoology, Na-
tional Museum of Nature and Science (formerly National
Science Museum, Tokyo) (NSMT).

The systematic arrangement used in this paper follows
Sirenko (2006).

Prom the symposium “Advances in Chiton Research” presented at the joint meeting of the American Malacological Society and Western
Society of Malacologists, held 29 July to 3 August 2006 in Seattle, Washington.

113

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AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

SYSTEMATICS

Order Lepidopleurida Thiele, 1909

Suborder Lepidopleurina Thiele, 1909

Eamily Leptochitonidae Dali, 1889

Genus Leptochiton Gray, 1847

Type species

Chiton cinereus Montagu, 1803 [= Leptochiton asellus
(Gmelin, 1791)], by subsequent designation (Gray, 1847).

Leptochiton tenuidontus Saito and Okutani, 1990
(Eig. 2A-B)

Leptochiton tenuidontus Saito and Okutani 1990: 166-
171, figs. 2-12, pi. 1, figs. 1-4; Kaas and Van Belle
1994: 22-23, fig. 7; Kaas and Van Belle 1998: 185;
Saito 2000: 7, pi. 3, fig. 11; Cosel 2006: 81.

Leptochiton sp. Saito and Eujikura 2000: 74-75.

Type material examined

Holotype: NSMT-Mo 69193, body length ca. 16 mm.
Type locality: hydrothermal vent site on the Iheya Ridge,
central Okinawa Trough, East Ghina Sea, 27°32.70'N,
126°58.20'E, 1395 m, 2K, Dive #426, 21 luly 1989.

Additional material examined

NSMT-Mo 73838 (ex. JAMSTEC sample No.: RK4-A-1,
009550-009553), 4 specimens, body length ca. 18-20 mm,
methane seep site otf Kikaijima Island in the Amami Islands
area, 28°26.39'N, 130°19.01'E, 1430 m, 2K, Dive #1020, 24
June 1998; NSMT-Mo 73839 (ex. JAMSTEC sampJe No.:
RK4-A-1, 009548-009549), 2 specimens, body length 22 and
23 mm, methane seep site off Kikaijima Island in the Amami
Islands area, 28°26.42'N, 130°18.98'E, 1442 m, 2K, Dive
#1021, 25 June 1998; NSMT-Mo 73940 (ex. JAMSTEC
sample No.: RK4-A-1, 009544-009547), 3 specimens, body
length ca. 20-22 mm, methane seep site off Kikaijima Island,
in the Amami Islands area, 28°26.45'N, 130°19.1 'E, 2K, Dive
#1022, 1440 m, 26 June 1998. All nine specimens were found
on the shells of Bathyniodiohis platifrons Elashimoto and
Okutani, 1994.

Additional description

Tegmentum sculptured with round granules densely ar-
ranged in quincunx order on head valve, lateral areas of
median valves, and postmucronal area of tail valve, with
elongate granules arranged in quincunx order or, occasion-
ally, in irregular longitudinal rows in central area of median
valves and antemucronal area of tail valve (Fig. 2A). Each
granule with one macraesthete pore and one to four mi-
craesthete pores on anterior slope; size of macraesthete pore
ca. 5-8 pm, that of micraesthete pore slightly smaller than
macraesthete pore (Fig. 2B).

120 E 130° 140° 150°

Figure 1. Sampling sites. Solid circles indicate hydrothermal vent.
Open circles indicate methane seep. -kNo active vent/seep area has
been discovered but the possibility of hydrothermal activity has
been suggested.

Gills merobranchial, adanal, without interspace, 6-8 on
each side.

Distribution and type of habitat

Iheya Ridge and off Kikaijima Island, Nansei Islands,
1395-1442 m; hydrothermal vent and methane seep.

Remarks

This species was described based on a single specimen
with heavily eroded valves missing a large piart of the teg-
mental sculpiture. The remaining small portion of sculptur-
ing and other features, especially the characteristic radula
with elongate “toothpick”-like inner small (third) lateral,
allows the additional specimens to be identified as this
species.

The holotype was collected from undersurface of a rock,
whereas all additional specimens were attached on the shells
of Bathymodiolus platifrons.

Family Protochitonidae Ashby, 1925
Genus Deshayesiella Garpenter in Dali, 1879

Type species

Deshayesiella (Leptochiton) curvatus (Carpenter MS)
Dali, 1879 (nom. nud., = Lepidopleurus (Deshayesiella) cur-
vatus Carpenter in Pilsbry, 1892), by subsequent designation
(Pilsbry, 1892).

Deshayesiella sirenkoi sp. nov.

(Figs. 3, 4, 5A-D)

Type material examined

JTolotypie: NSMT-Mo 73841 (ex. JAMSTEC sample No.:
RK8-B-1, 006983), body length 36.4 mm. Type locality: hy-

CHITONS FROM HYDROTHERMAL VENTS AND METHANE SEEPS

115

Figure 2. Leptochitoii teniddontus Saito and Okutani, 1991 (NSMT-Mo 73940) valve VI. A, sculpture on central area, scale bar = 200 pm;
B, close-up of granules on central area, scale bar = 100 pm.

drothermal vent site on the Kasuga II Seamount in the
northern Mariana Islands area, 21°36.1'N, 143°38.5'E, 400
m, 2K, Dive #986, 23 November 1997; paratypes: NSMT-Mo
73842, I specimen, body length ca. 45 mm, hydrothermal
vent site on the Nikko Seamount in the northern Mariana
Islands area, 23°04.7'N, 142°19.9'E, 460 m, 6K, Dive #144,
19 September 1992; NSMT-Mo 73843 (ex. JAMSTEC sample
No.: EZIO, 061632-061635), 4 specimens, body length ca.
27-31 mm, hydrothermal vent site on the Daikoku Sea-
mount in the northern Mariana Islands area, 21°19.53'N,
144°1 1.51'E, 428 m, on rock, HPD, Dive #498, 1 November
2005.

Diagnosis

Valves thick, low, slightly carinated. Median valves wide,
angulated at antero-lateral corners, weakly protruded at an-
terior margin of jugal area. Tail valve with slightly raised
mucro located anterior to the center, and concave posterior
slope. Pleural areas sculptured with longitudinal, weakly
curving riblets. Girdle with long needles.

Description

Body (Fig. 5A) oval, 36.4 mm in length. Valves (Fig. 5B)
thick, low, slightly carinated. Girdle fleshy, deeply encroach-
ing at sutures.

Head valve semicircular, rounded at postero-lateral cor-
ners. Median valves wide, widest at valves IV-VI, slightly
carinated, beaked, weakly projected at anterior margin of
jugal area. Tail valve more than semicircular, wider than
head valve; mucro slightly raised, located anterior to the
center; posterior slope concave. Tegmentum granulo-
costate. Head valve, lateral areas of median valves, and pos-
terior area of tail valve sculptured with densely packed gran-

ules which are often fused radially, forming larger elongate
granules, marked with concentric growth lines; pleural areas
of median and tail valves sculptured with strong, longitudi-
nal, slightly curving riblets; jugal area densely sculptured
with finer granules. Aesthete pores (Fig. 3A) located on an-
terior portion of each granule. Each group of pores consist-
ing of one macraesthete pore, 10-20 pm in diameter, and
one or two micraesthete pores, 5-8 pm in diameter at both
sides of macraesthete pore. Articulamentum of head valve
thickened, weakly projecting around the anterior margin of
transverse muscle scars. Median valves and tail valve with
widely V-shaped callus. Eaves wide, nearly smooth, scattered
with minute aesthete pores. Tegmentum broadly folded un-
der on posterior margin. Sutural laminae (Fig. 5B) strongly
projected forward, triangular, widely separated from each
other.

Girdle fleshy, thick, brownish. Perinotum (Figs. 3B, 5D)
densely covered with elongate, obtusely pointed, flattened,
distally ribbed spicules (Fig. 4A), 130 pm x 25 pm, inter-
mingled with long, straight, smooth needles (Fig. 4B), up to
680 pm X 55 pm. Girdle margin fringed with long needles
similar to those on perinotum (Fig. 3C). Spicules on hypo-
notum (Figs. 3D, 4C-E) flat with one to three strong riblets,
90 pm X 30 pm.

Gills merobranchial, adanal, without interspace, 16 on
left, 18 on right.

Radula (Fig. 3E-F) long, 15.5 mm in length with 56
transverse rows of mineralized teeth. Central tooth oblong
with narrow cusp at top, weakly expanded laterally, keeled
near base. Centro-lateral (first lateral) teeth with well devel-
oped plate surrounding base of major lateral (second lateral)
teeth, obtusely pointed at antero-dorsal corner. Major lateral
teeth with bicuspid head, of which the larger outer cusp is

116

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 3. Deshayesielln sircnkoi sp. nov., holotype (NSMT-Mo 73841). A, valve II, granules on pleural area near anterior margin of jugal
area, scale bar = 100 pm; B, spicules of perinotum near girdle margin, scale bar = 50 pm; C, marginal spicules, scale bar = 100 pm; D,
spicules of hyponotum, scale bar = 50 pm; E, radula, central part, postero-dorsal view, scale bar = 100 pm; F, radula, central part, oblicjue
antero-dorsal view, scale bar = 100 pm.

pointed and the smaller, inner cusp is rounded. Major un-
cinus (fifth lateral) teeth rounded at top with blade of mod-
erate width. Bolster (radular vesicle and cartilage) length
5.2 mm.

Distribution and type of habitat

Known from the seamounts on Kasuga II, Nikko, and
Daikoku in the northern Mariana Islands area, 400-460 m;
hydrothermal vent.

CHITONS FROM HYDROTHERMAL VENTS AND METHANE SEEPS

117

Figure 4. Deshayesiella sireiikoi sp. nov., holotype (NSMT-Mo 73841). A, spicule of perino-
tum; B, needle on perinotum; C-E, spicules of hyponotum; scale bar = 50 pm.

Etymology

This species is named in honor of
Dr. Boris Sirenko, who has recently
given a new diagnosis for the genus
Deshayesiella.

Remarks

The features of the present species
match the characteristics of Deshayesi-
ella given by Sirenko (1997). These
features include: valves solid, rather
flat, evenly rounded; median valves di-
vided into jugal, two pleural, and two
lateral areas (unlike Leptochiton); teg-
mentum of head valve, lateral area of median valves and
postmucronal area of tail valve sculptured with irregular
granules, strongly marked with concentric lines of growth;
girdle rather wide, dorsally covered with small spicules ( 100-
150 pm) and randomly dispersed large spines (320-550 pm);
radula with bicuspid major lateral teeth. Although there are
some slight differences, such as the slightly carinated valves
and somewhat longer large perinotal spines in the present
species, we think they are insignificant for generic assign-
ment. Sirenko (1997) recognized three known species in
Deshayesiella: D. curvata (Carpenter in Pilsbry, 1892), Ol-
droydia bidentata Is. Taki, 1938, and Hanleya sinica Xu,
1990, as well as two undescribed species (sp. 1 and 2). The
assignment of H. sinica may, however, need reconsideration
because it has rather vaguely regionalized tegmentum with
finer sculpture, and thus is more like members of Leptochi-
ton in this respect. The present species is easily distinguish-
able from all known congeners and one of Sirenko’s unde-
scribed species, sp. 1, in having wider median valves, each
side with more angular antero-lateral corner. The features of
another undescribed species, sp. 2, have not yet been given
in detail; however, the present new species is probably dis-
tinct from Sirenko’s sp. 2 because the latter is distributed in
a different geographic area: the East Pacific off southern
California and in the Gulf of California, Mexico.

Deshayesiella sirenkoi is locally common around the hy-
drothermal vent site on the Daikoku Seamount (see Pig. 5C).
Deshayesiella sirenkoi, as well as two known vent species,
Leptochiton teniiidontus Saito and Okutani, 1990 and Ther-
mochiton undocostatus Saito and Okutani, 1990 coulci be
restricted to hydrothermal vent and/or methane seep habi-
tats because each of these species was found only from those
environments of more than two sites.

Order Chitonida Thiele, 1909

Suborder Chitonina Thiele, 1909

Family Ischnochitonidae Dali, 1889

Genus Thermochiton Saito and Okutani, 1990

Type species

Thermochiton undocostatus Saito and Okutani, 1990, by
original designation.

Thermochiton undocostatus Saito and Okutani, 1990

Therttwchiton undocostatus Saito and Okutani 1990:
171-174, figs. 13-23, pi. 2, figs. 1-4; Kaas and Van
Belle 1994: 36-38, fig. 13; Kaas and Van Belle 1998:
192; Saito 2000: 11, pi. 6, fig. 12; Cosel 2006: 80.

Type material examined

Holotype: NSMT-Mo 69194, body length ca. 13 mm.
Type locality: hydrothermal vent site on the Iheya Ridge,
central Okinawa Trough, East China Sea, 27°32.70'N,
126°58.20'E, 1395 m, 2K, Dive #426, 21 luly 1989.

Additional material examined

NSMT-Mo 73844 (ex. JAMSTEC sample No.: RK4-A-6,
038998-039004), 6 specimens, body length ca. 3-8 mm. Hy-
drothermal vent site on the Hatoma Knoll, 24°51.65'N,
123°50.29E, 1497 m, on small chimney rock, 2K Dive #1277,
29 May 2001; NSMT-Mo 73845, 2 specimens, body length
ca. 7 mm, methane seep site on the Kuroshima Knoll off
Yaeyama Islands area, 24°08.00'N, 124°11.50'E, 686-688 m,
on the shells of Bathynwdiolus hirtus Okutani, Fujikura, and
Sasaki, 2004 or Bathynwdiolus securiformis Okutani, Fujikura
and Sasaki, 2004, and Calyptogena kawamurai (Kuroda,
1943), HPD, Dive #554, 21 May 2002.

Additional description

Gills nearly holobranchial (anterior-most gill located
under the third valve), adanal, with interspace, 19 gills on
each side (NSMT-Mo 73845).

Distribution and type of habitat

Off southern Nansei Islands, 686-1497 m; hydrothermal
vent and methane seep.

Remarks

Characteristic features of the present specimens, such as
undulating sculpture on the valves, granulo-costate dorsal

118

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 5. New species of chitons. A-D, Desliayesiella sirenkoi; E-H, Placiphorella okutanii; I-K, Placiphorella isaotakii. A, E, I, whole animal,
dorsal view, holotypes; B, E, 1, head, median (B: valve II; F, J; valve III), and tail valves, dorsal view, holotypes, scale bar = 5 mm; C, habitat,
Daikoku Seamount, arrow heads indicate position of chitons; D, perinotum, showing long needles, holotype, scale bar = 1 mm; G, tail valve,
paratype, scale bar = 1 mm; H, K, anterior margin of girdle, paratype and holotype, respectively, scale bar = 1 mm.

CHITONS FROM HYDROTHERMAL VENTS AND METHANE SEEPS

119

scales, and head of the major lateral tooth of radula with
basal pointed projection agree well with those of the
holotype.

Suborder Acanthochitonina Bergenhayn, 1930

Family Mopaliidae Dali, 1889

Genus Placiphorella Dali, 1879

Type species

Placiphorella velata (Carpenter MS) Dali, 1879, by origi-
nal designation.

Placiphorella okutanii sp. nov.

(Figs. 5E-H, 6, 7)

Placiphorella stimpsonr. Wu and Okutani 1985: 126-128, figs.

9-18 (not of Gould 1859).

Type material examined

Holotyp^e: NSMT-Mo 73777 (ex. JAMSTEG sample No.:
RK4-B-5, 006454), body length 32 mm. Type locality:
Hachijo Depression in the Izu-Ogasawara (Bonin) Islands
area. 32°48.8'N, 139°27.0'E to 32°51.3'N, 139°31.6'E, 926-
817 m, dredge attached to JAMSTEC Deep Tow Camera,
cruise No.: DK88-3-IZU, 27 August 1988; paratype: NSMT-
Mo 60008, body length ca. 30 mm, off Miyake Island, Izu-
Ogasawara Islands area, 34°03.0'N, 140°02.2'E, 1210-1235
m, R/V Soyo-Maru St. B2, beam trawl, 5 July 1967.

Diagnosis

Valves chalky white, fragile, sculptured with densely
packed, low, rather large, granules. Tail valve with narrow
postmucronal areas separated by shallow sinus behind mu-
cro. Sutural laminae wide, narrowly separated from each
other. Perinotum densely implanted with low spiny tufts.
Bristle implanted around girdle margin.

Description

Body (Fig. 5E) broadly oval, 32 mm in length, light buff
in color.

Valves (Fig. 5F) wide, depressed, subcarinated, chalky
white, fragile. Head valve crescent in outline, anterior slope
concave. Median valves very wide, short, oblong in outline,
weakly projected forward at jugal portion; lateral areas
raised, grooved medially. Tail valve (Fig. 6A) small, inversed
trapezoidal in outline, with narrow postmucronal area sepa-
rated by shallow sinus at posterior end; mucro subterminal,
slightly raised. Tegmentum (Fig. 5F) sculptured with densely
packed, low, somewhat elongate granules on head valve, lat-
eral areas of median valves, and postmucronal area of tail
valve. Remaining tegmental areas with slightly lower, larger
granules. Aesthete pores minute, 3-6 pm in diameter, dis-
tributed both on granules and the tegmental plain (Fig. 6B),
which become denser toward the lateral areas (Fig. 6D). The

difference between macraesthete pore and micraesthete pore
hardly discernible. Articulamentum well developed, white,
heavily calloused anteriorly in head valve, transversely in
median valve, and posteriorly in tail valve; posterior margin
of articulamentum widely covered with folded tegmentum
in head and median valves, narrowly covered in tail valve.
Sutural laminae well developed, narrowly separated from
each other. Insertion teeth short, thick, rugose on anterior
surface, with 12 slits in head valve (Fig. 6C), one on each side
in median valves, none on tail valve. Slit rays represented by
series of minute pores, clearly visible in apical half of head
valve, median valves, inconspicuous in tail valve. Eaves nar-
row, with many minute pores.

Girdle (Fig. 5E) widely expanded anteriorly, becoming
narrower toward posterior end. Perinotum (Fig. 6E) covered
with minute spicules (Fig. 7A-B), mammilated at tip, ca. 150
pm X 30 pm, and densely implanted with low spiny tufts
consist of 5-10 sharply pointed, weakly curved spicules (Fig.
7C-D), ca. 400 pm x 50 pm in width, surrounded by broken
short spicules. Bristle, worn off in holotype, with sharply
pointed spicules similar to spiny tufts. Hyponotum clothed
with obtusely pointed, smooth spicules (Figs. 6F, 7G), 140-
165 pm X 30 pm. Anterior hyponotum with numerous
warts, which are provided with 20-30 pointed spicules (Fig.
7H-I), 150-170 pm X 25 pm. Pallial fold well developed with
9 precephalic tentacles, which are occasionally bifurcated.
Spicules on pallial fold similar to obtusely pointed spicules
on hyponotum, but smaller on precephalic tentacles, 1 10 pm
X 15 pm (Fig. 7J), somewhat narrower on posterior end, 160
pm X 25 pm (Fig. 7K).

Gills holobranchial, abanal, with interspace, 15 on left
side, 16 on right.

Radula (Fig. 6G-H) small, 6.5 mm in length, with 40
transverse rows of mineralized teeth. Central tooth oblong,
with narrow cutting edge, slightly expanded laterally and
bilobed at base. Centro-lateral (first lateral) teeth low, thick-
ened at antero-dorsal corner. Major lateral (second lateral)
teeth with proportionally small tridentate head. Major un-
cjnus (fifth lateral ) teeth with rather long blade of moderate
width. Bolster (radular vesicle and cartilage) length 3.3 mm.

Paratype: Tail valve (Fig. 5G) with narrow posterior
areas separated by shallow sinus at posterior end.

Bristle densely implanted along girdle margin (Fig. 5H).
Thick bristles (Fig. 7E), ca. 300 pm in width, implanted on
girdle margin and apparently thinner bristles (Fig. 7F), at-
taining ca. 1 mm x 100 pm, restricted on dorsal surface
close to girdle margin, and occasionally on other area on
perinotum.

Distribution and type of habitat

Only known from Hachijo Depression and off Miyake
Island in Izu-Ogasawara Islands area, 817-1235 m; un-

120

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 6. Placiphorclln okutaiiii sp. nov., liolotype (NSMT-Mo 73777). A, tail valve, scale bar = 1 mm; B, tegmentum of valve 111, anterior
margin of central area, scale bar = 200 pm; C, insertion teeth of head valve, scale bar = 2 mm; D, tegmentum of valve III, lateral area, scale
bar = 200 pm; E, anterior perinotum, scale bar = 200 pm; F, spicules of hyponotum, scale bar = 100 pm; G, radula, central part (right half),
postero-dorsal view, scale bar = 50 pm; H, radula, lateral part, postero-dorsal view, scale bar = 50 pm.

CHITONS FROM HYDROTHERMAL VENTS AND METHANE SEEPS

121

cause P. pacifica Berry, 1919 (Lecto-
type, SBMNH 34394 designated by
Scott et al. 1990) has almost smooth
surface on the tegmentum, and no
other known species of Phiciphorella
has such an obviously granular teg-
mentum. From Japanese waters, an-
other deep-sea species, Placiphorella
albitestae was described from the Sa-
gami Sea, northern Izu-Ogasawara Is-
lands area. Placiphorella albitestae has
much finer granules on finer, but
sharply raised, growth lines on the
tegmentum, much finer and scarce
spinous tufts on the perinotum, and
shallower bathymetrical range of dis-
tribution, from 80 to 200 m (Saito
2000).

Figure 7. Placiphorella okutanii sp. nov., sclerites, A-D, G-K, holotype (NSMT-Mo 73777); E,
F, paratype (NSMT-Mo 60008). A, B, spicules of perinotum; C, D, spicules of tuft on
perinotum; E, thick bristle (spicules are lost in large part); F, thin bristle; G, spicules of
hyponotum, H, 1, spicules of tuft on hyponotum; J, spicule of precephalic tentacle; K, spicule
of pallial fold near posterior end. Upper scale bar = 100 pm, for A-D, G-K; lower scale
bar = 500 pm, for E and F.

Placiphorella isaotakii n. sp.

(Figs. 5I-K, 8, 9)

known, possibility of hydrothermal activity is suggested in
Hachijo Depression.

Type material examined

Holotype; NSMT-Mo 73778 (ex.
JAMSTEC sample No.: RK4-A-3,

016481), body length ca. 34 mm. Type

locality: methane seep sites on the Ku-
roshima Knoll off Yaeyama Islands area, 24°07.00'N,
124°11.00'E, 691-692 m, 2K, Dive #1100, 22 May 1999.

Etymology

This species is named in honor of Dr. Takashi Okulani,
who has been actively working for deep-sea vent/seep mol-
luscs, and collected this species for the first time.

Remarks

Kaas and Van Belle (1994) synonymized all known
deep-sea Placiphorella species with Placiphorella atlantica
(Verrill and Smith, 1882) and this decision was followed by
Clark (1994). However, at least Placiphorella pacifica Berry,
1919 and Placiphorella albitestae Is. Taki, 1954 are distinc-
tive, and can be separated by the valve shape and sculpture,
girdle element shape and sclerite arrangement, and other
features. Among those deep-sea Placiphorella, the present
new species most closely resembles Placiphorella “pacifica”
reported by Smith and Hanna (1952) from Pioneer Sea-
mount, East Pacific, 500-650 m (CASIZ 064802) by having
granular tegmentum. However, the granules of the former
are irregular in shape and arrangement, especially on the
lateral areas, and the spiny tufts of the perinotum are very
prominent and dense. Placiphorella “pacifica” reported by
Smith and Hanna (1952) can be an undescribed species be-

Diagnosis

Valves solid, sculptured with fine elongate granules. Tail
valve wide triangular, with terminal mucro. Sutural laminae
wide, narrowly separated each other. Insertion teeth low,
hardly separated with slits in head valve. Perinotum densely
implanted with low spinous tufts. Bristle implanted along
girdle margin.

Description

Body (Pig. 51) broadly oval, ca. 34 mm in length, light
buff in color.

Valves (Fig. 5J) wide, depressed, subcarinated, solid.
Head valve crescent in outline; anterior slope concave. Me-
dian valves very wide, short, oblong in outline, weakly pro-
jected forward at jugal portion; lateral areas raised, grooved
medially. Tail valve (Fig. 8A) small, wide triangular in out-
line, with terminal mucro. Tegmentum (Fig. 5J) sculptured
with densely packed, elongate granules on head valve and
lateral areas of median valves. Remaining tegmental areas
with weak, elongated granules, which are occasionally
merged into longitudinal threads. Aesthete pores minute,
3-5 pm in diameter, arranged roughly in concentric patterns

122

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

Figure 8. Placiphorella isaotakii sp. nov., holotype (NSMT-Mo 73778). A, tail valve, scale bar = 1 mm; B, tegmentum of valve III, anterior
margin of central area, scale bar = 200 pm; C, insertion teeth of head valve, scale bar = 1 mm; D, tegmentum of valve III, lateral area, scale
bar = 200 pm; E, perinotum, scale bar = 500 pm; F, spicules of hyponotum, scale bar = 100 pm; G, bristles near anterior margin, scale
bar = 500 pm; H, radula, postero-dorsal view, scale bar = 100 pm.

in pleural areas (Fig. 8B), which are denser and less regularly
arranged on lateral areas (Fig. 8D). Difference between mac-
raesthete pore and micraesthete pore hardly discernible. Ar-
ticulamentum well developed, white, heavily calloused ante-

riorly in head valve, transversely in median valve, and
posteriorly in tail valve; posterior margin of articulamentum
widely covered with folded tegmentum in all valves. Sutural
laminae well developed, narrowly separated from each other.

CHITONS FROM HYDROTHERMAL VENTS AND METHANE SEEPS

123

Insertion teeth low, thick, rugose on outside. Slits incon-
spicuous on head valve (Fig. 8C), one on each side in median
valves, none in tail valve. Slit rays inconspicuous, repre-
sented by minute pores. Eaves narrow, with minute pores.

Girdle (Fig. 51) widely expanded anteriorly, becoming
narrower toward posterior end. Perinotum (Fig. 8E) covered
with minute, thick spicules (Fig. 9A-B), obtuse or weakly
mammilated at tip, 150 pm X 40 pm, and densely implanted
with low spinous tufts consist of 5-10 sharply pointed spic-
ules (Fig. 9C-D), 440 pm x 50 pm surrounded by broken
short spicules. Thick bristle (Figs. 8G, 9E), up to 2.5 mm x
400 pm, implanted along the girdle margin, while thinner
bristle (Fig. 9F) restricted on dorsal surface close to girdle
margin (Fig. 5K) and occasionally on other area of perino-
tum. Spicules on bristle similar to those of tufts, hut less
curved and slightly shorter, 380 pm X 50 pm. Hyponotum
clothed with obtusely pointed, smooth spicules (Figs. 8F,
9G-H), attaining 180 pm x 30 pm. Anterior hyponotum
with numerous warts which are provided with 20-40 pointed
spicules (Fig. 91), 150-170 pm X 25 pm. Pallial fold well
developed with nine precephalic tentacles, which are occa-
sionally bifurcated. Spicules on pallial fold similar to ob-
tusely pointed spicules on hyponotum, but smaller on pre-
cephalic tentacles, 120 pm x 15 pm (Fig. 9J), somewhat
narrower on posterior end, 165 pm x 25 pm (Fig. 9K).

Gills holobranchial, abanal, with interspace, 21 on left
sicie, 22 on right.

Radula (Fig. 8F1) small, 7.5 mm in length, with 41 trans-
verse rows of mineralized teeth. Central tooth oblong, with
narrow cutting edge, slightly expanded laterally and bilobed
at base. Centro-lateral (first lateral) teeth low, thickened at
antero-dorsal corner. Major lateral (second lateral) teeth
with small tridentate head. Major uncinus (fifth lateral) teeth
with rather long blade of moderate width. Bolster (radular
vesicle and cartilage) length 3.4 mm.

Distribution and type of habitat

Known only from the type locality; methane seep.

Etymology

This species is named in honor of the late Dr. Isao Taki,
who described the first deep-sea Placipliorella, P. albitestae
from Japanese waters.

Remarks

This species also resembles Placiphorella “paciftca’ re-
ported by Smith and Hanna (1952) and the preceding new
species, Placiphorella by having a granular tegmentum; how-
ever, the present species differs by having a terminal mucro
on a wider tail valve, and thread-like sculpture of the central
area.

The two new Placiphorella species described here might
be transient species, rather than vent/seep specialists be-
cause Placiphorella species have been shown to be carnivo-
rous, using their anterior expanded
girdle to trap prey (McLean 1962,
Saito and Okutani 1992). They may
be able to live in non-chemosynthetic
environment if enough prey were
available.

ACKNOWLEDGMENTS

The authors extend their sincere
thanks to the operation teams of
the Deep-Sea Research System of
JAMSTEC for sampling the material.
Thanks are also due to Dr. Barry Roth
(formerly at California Academy of
Sciences), Dr. Paul Valentich-Scott
(Santa Barbara Museum of Natural
History) for the loan of the type ma-
terial, Dr. Kenji Okoshi (Ishinomaki
Senshu University) for the iniorma-
tion on specimens. Dr. Takashi Oku-
tani (JAMSTEC), Dr. DougJas Eernisse
(California State University, Fuller-
ton), Mr. Enrico Schwabe (Zoolo-
gische Staatssammlung Miinchen),

Figure 9. Placiphorella isaotakii sp. nov., sclerites, holotype (NSMT-Mo 73778). A, B, spicules
of perinotum; C, D, spicules of tuft on perinotum; E, thick bristle; F, thin bristle (some parts
are not traced due to foreign deposit); G, FI, spicules of hyponotum; I, spicules ot tuft on
hyponotum; |, spicule of precephalic tentacle; K, spicule of pallial fold near posterior end.
Upper scale bar = 100 pm, for A-D, G-K; lower scale bar = 500 pm, for E, F.

124

AMERICAN MALACOLOGICAL BULLETIN 25 • 1/2 • 2008

and Mr. Roger Clark (Eagle Mountain, Utah) for critically

reading the manuscript.

LITERATURE CITED

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Kaas, P. and R. A. Van Belle. 1994. Monograph of Living Chitons
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Saito, H. 2000. Polyplacophora. In: T. Okutani, ed.. Marine Mol-
luscs in fapan. Tokai University Press, Tokyo. Pp. 4-23.

Saito, H. 2006. A new species of Ferreiraella Sirenko, 1988 (Mol-
lusca: Polyplacophora) from the Philippine Basin. Venus 65:
91-96.

Saito, H. and K. Fujikura. 2000. A second species of Leptochiton
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Saito, H. and T. Okutani. 1990. Two new chitons (Mollusca: Poly-
placophora) from a hydrothermal vent site of the Iheya Small
Ridge, Okinawa Trough, East China Sea. Venus 49: 165-170.

Saito, H. and T. Okutani. 1992. Carnivorous habits of two species
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nidae). Journal of the Malacological Society of Australia 13:
55-63.

Sasaki, T., T. Okutani, and K. Fujikura. 2005. Molluscs from hy-
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133.

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Submitted: 17 June 2007; accepted: 3 January 2008; final

corrections received: 1 May 2008

INDEX TO VOLUME 25

AUTHOR INDEX

Bidwell, J. R. 25: 1
Binks, R. M. 25: 35
Boeckman, C. J. 25: 1
Brooker, L. R. 25: 35
Buckland-Nicks, J. 25: 97
Cacabelos, E. 25: 9
Clark, R. N. 25: 71, 77
Clode, P. L. 25: 35
DuFoe, J. 25: 25

Eernisse, D. J. 25: 21, 51, 87
Fernandez, C. Z. 25: 51
Fujikura, K. 25: 113
Johnson, C. C. 25: 87
Macey, D. J. 25: 35
Pojeta, J., Jr. 25: 25
Puchalski, S. P. 25: 87
Quintas, P. 25: 9
Runnegar, B. 25: 51

Salto, Fi. 25: 113
Shaw, J. A. 25: 35
Sigwart, J. D. 25: 43
Stockdale, E. J. 25: 35
Troncoso, J. S. 25: 9
Tsuchida, S. 25: 113
Vendrasco, M. J. 25: 51
Webb, R. I. 25: 35

PRIMARY MOLLUSCAN TAXA INDEX

[first occurrence in each paper recorded, new taxa in bold]

aberti, Cyprogenia 25: 1
abyssicola, Ischnochiton 25: 81
abyssicola, Tripoplnx 25: 77
Acaenoplax 25: 33
Acanthochitona 25: 110
Acanthochitonidae 25: 98
Acanthochitonina 25: 51, 72, 97, 119
Acanthopleura 25: 52, 98
Acanthopleuridae 25: 98
acuta, Mopalia 25: 53
alba, Abra 25: 9
albitestae, Placiphorella 25: 121
Albrechtia 25: 78
albus, Ishnochitou 25: 98
albus, Stenosennis 25: 97
algesirensis, Leptochitou 25: 45
allyni, Tripoplax 25: 86
alveolus, Leptochitou 25: 113
Amicula 25: 52
antiqua, Diadeloplax 25: 88
antiquus, Cliitonellus 25: 88
apiculata, Chaetopleura 25: 97
Aplacophora 25: 33, 108
argenteum, Chaetoderinn 25: 109
asellus, Leptochitou 25: 43, 99, 1 14
asiatica, Lianleyella 25: 43
assimilis, Leptochitou 25: 43, 98
atlantica, Placiphorella 25: 121
attuensis, Tripoplax 25: 86
aurata, Plaxiphora 25: 51

australis. Chiton 25: 77
australis, Epimenia 25: 109
australis, Ischuochitou 25: 78
badius, Leptochitou 25: 45
baleuophila, Lepidozoua 25: 113
baxteri, Tripoplax 25: 86
belhiapi, Leptochitou 25: 45
beriugiaua, Tripoplax 25: 86
bideutata, Mysella 25: 9
bideutata, Oldroydia 25: 117
cajetauus, Lepidopleurus 25: 45, 53
californica, Nuttallifia 25: 53, 98
Callistoplacidae 25: 98
Callochitou 25: 75, 97
Callochitonidae 25: 97
calypso, Tripoplax 25: 77
caucellatus, Leptochitou 25: 45
cardiuui, Lampsilis 25: 2
castaueus, Callochitou 25: 99
Chaetopleura 25: 110
Chaetopleurinae 25: 52, 110
Chelodes 25: 88
Chelodida 25: 31
chileusis, Tonicia 25: 51
chiueusis, Calyptraea 25: 12
Chitonida 25: 43, 97, 117
Chitonidae 25: 51
Chitonina 25: 52, 97, 117
Choriplacidae 25: 98
ciliata, Chitou 25: 74

ciliata, Mopalia 25: 23, 51, 71
ciuerea, Cyauoplax 25: 110
ciuerea, Lepidochitoua 25: 51, 110
ciuereus, Chiton 25: 114
cinereus, Trachydermou 25: 51
cocciueuni, Pleuribema 25: 2
complanata, Lasmigona 25: 2
couspicua, Stenoplax 25: 97
cooperi, Lepidozoua 25: 53
cordifonuis, Chitou 25: 88
cowani, Tripoplax 25: 77
Cryptochitou 25: 52
Cryptoplacidae 25: 98
curvata, Deshayesiella 25: 43, 98, 1 1 7
curvatus, Deshayesiella 25: 114
curvatus, Lepidopleurus 25: 114
Cyauoplax 25: 54, 97
cyliudrica, Quadrula 25: 1
davolii, Lepidopleurus 25: 88
decussatus. Tapes 25: 9
Deudrochiton 25: 51
deutatus, Callochitou 25: 98
deutieus, Cyauoplax 25: 98
deutieus, Lepidochitou 25: 98
Deshayesiella 25: 108, 113
distortus, Chitouellus 25: 88
douacifonuis, Truucilla 25: 2
diifoei, Echiuochitou 25: 21, 25
echiuatus, Acanthopleura 25: 110
Echiuoclutou 25: 31

125

Echinochitonidae 25: 31
ediile, Cemstoderma 25: 9
edidis, Ostrea 25: 12
Eopteria 25: 25
eiigenei, Pamchiton 25: 45
exiguwu, Parvicardhim 25: 9
fenestrata, ChrysaUida 25: 12
fernaldi, Cyanoplax 25: 98
fernaldi, Lepidochitona 25: 98
Ferreiraellidae 25: 98
flava, Fiisconaia 25: 2
flectens, Deudrochiton 25: 51
flexiiosa, Tliyasira 25: 9
foresti, Leptochiton 25: 45
fragilis, Leptodea 25: 2
gibba, Corbula 25: 12
Glyptochitoii 25: 88
goUschi, Lepidozona 25: 77
Gotlandochiton 25: 88
granidatn, Acanthopleura 25: 98
grnyii, Mopalia 25: 72
Giirjanovillia 25: 78
hakodadensis, Ischnochiton 25: 109
hnncockianus, Chitonellus 25: 88
Hanleya 25: 108
haiileyi, Hanleya 25: 98
hartwegii, Cyanoplax 25: 53
hartwegii, Lepidochitona 25: 53
hayae, Acaenoplax 25: 33
Helniinthochiton 25: 88
Hemiarthridae 25: 98
hindsii, Chiton 25: 72
hindsii, Mopalia 25: 71
hirtosa, Acanthopleura 25: 35
hirtus, Bathyfnodiolus 25: 117
hyadesi, Niittallochiton 25: 53
hylkiae, Parachiton 25: 45
ima, Tripoplax 25: 86
incisiis, Schizochiton 25: 52
insignis, Tonicella 25: 51
isaotakii, Placiphorella 25: 113
Ischnochiton 25: 54, 97
Ischnochitonidae 25: 52, 77, 98, 117
Ischnoradsia 25: 77
Katharina 25: 52
kawannirai, Calyptogena 25: 117
kennerleyi, Mopalia 25: 23, 71
kunnlatus, Leptochiton 25: 45
labiosa, Rissoa 25: 9

lactens, Loripes 25: 9
Lepidochitotia 25: 52, 78, 97
Lepidochitonidae 25: 52, 98
Lepidopleurida 25: 23, 43, 97, 114
Lepidopleuridae 25: 43, 98
Lepidopleurina 25: 23, 43, 114
Lepidopleurns 25: 43, 54, 89
Lepidozona 25: 54, 77, 97
Leptochiton 25: 43, 81, 113
Leptochitonidae 25: 43, 98, 114
lignosa, Mopalia 25: 51
lineata, Nierstraszella 25: 45
lineata, Tonicella 25: 51, 109
linilatns, Dendrochiton 25: 51
lokii, Tonicella 25: 51
Loricidae 25: 98
Macoma 25: 9
mannorea, Tonicella 25: 51
Matthevia 25: 32
mertensii, Lepidozona 25: 53, 77
mesogonus, Leptochiton 25: 80
inetanevra, Qiiadrula 25: 2
niicropnstiilosiis, Leptochiton 25: 45
mirandus, Niittallochiton 25: 53
Mopalia 25: 52, 75, 104
Mopaliidae 25: 23, 51, 62, 72, 108
nmltiganosa, Lepidozona 25: 77
Miiltiplacophora 25: 32
Multiplacophora 25: 33
nniscosa, Mopalia 25: 53, 72, 89, 104
neglectns, Onithochiton 25: 52
nervosa, Megalonaias 25: 2
Nierstraszella 25: 45
Nierstraszellidae 25: 98
nigrovirescens, Chiton 25: 98
nigrovirescens, Radsia 25: 98
nitida, Abra 25: 9
nitidosa, Nuciila 25: 9
nitiduni, Lepton 25: 12
nodiilata, Qnadrula 25: 2
norfolcensis, Leptochiton 25: 44
Niittallochiton 25: 52
Odontogriphiis 25: 21, 32
ohiensis, Potamiliis 25: 4
okutanii, Placiphorella 25: 113
onialiis, Odontogriphiis 25: 32
pacifica, Placiphorella 25: 80, 121
Paleoloricata 25: 31
Parachiton 25: 45

pectinnlata, Lepidozona 25: 53
philippina, Nierstraszella 25: 45
Placiphorella 25: 52, 113
Placiphorina 25: 52
plana, Scrobicularia 25: 14
platifrons, Bathymodioliis 25: 114
Plaxiphora 25: 51
plicata, Amblerna 25: 2
politiis, Parachiton 25: 45
polyniorpha, Dreissena 25: 1
Polyplacophora 25: 21, 25, 35, 43, 51,
71, 77, 87, 97, 113
Polysacos 25: 33
Priscochiton 25: 88
Protochitonidae 25: 1 14
purpnratiis, Potamilus 25: 2
pustiilosa, Qnadrula 25: 2
Qiiadnila 25: 1
qnadrula, Qnadrula 25: 4
radiata, Turboella 25: 9
reflexa, Obliquaria 25: 2
regiilaris, Ischnochiton 25: 86
retiporosa, Lepidozona 25: 77, 109
Rhyssoplax 25: 110
rissoi, Ischnochiton 25: 110
riigatiis, Leptochiton 25: 43, 98
sarsi, Leptochiton 25: 45
scabricostata, Lepidozona 25: 80
Schizochiton 25: 97
Schizochitonidae 25: 51, 98
scrobiculata, Lepidozona 25: 77
seciiriforniis, Bathymodioliis 25: 117
silesiacus, Pterochiton 25: 88
sinica, Hanleya 25: 117
sinudentata, Lepidozona 25: 80
sirenkoi, Deshayesiella 25: 113
spectabilis, Mopalia 25: 51, 75
spinifera, Myrtea 25: 12
stearnsii, Stenosemiis 25: 77
stelleri. Cryptochiton 25: 55, 98
Stenoplax 25: 98
Stenosemiis 25: 110, 113
stimpsoni, Placiphorella 25: 119
swanii, Mopalia 25: 51
tenuidontus, Leptochiton 25: 113
terebelliim, ChrysaUida 25: 10
teres, Lampsilis 25: 2
textilis, Ischnochiton 25: 53
Thermochiton 25: 117

126

thoiiarsiaiia, Chaetopleura 25: 72
Tonkella 25: 51, 104
Tonicellidae 25: 57, 97
Tonkin 25: 69
Toniciinae 25: 1 10
Trachydermon 25: 78
trifida, Tripoplax 25: 85
trifidus, Ischnocliiton 25: 77
tripartitus, Pterochiton 25: 88
Tripoplax 25: 23, 77

triincatida, Retnsa 25: 12
tld^ercllIatus, Chiton 25: 52, 101
tiilipa, Chiton 25: 98
tnlipa, Rhyssoplax 25: 97
tunicatn, Katharina 25: 51
idvae, Hydrobia 25: 9
iindocostatns, Thermochiton 25: 113
Unionidae 25: 1
variabilis, Matthevio 25: 32

variegatiis, Ischnochiton 25: 53
velata, Placiphorelln 25: 53, 119
verrucosa, Tritogonia 25: 2
vickersianiim, Polysacos 25: 32
viridis, Acanthochitona 25: 98
vitrea, Hyala 25: 12
willetti, Lepidozona 25: 77
Wiwaxia 25: 21
wosnessenskii, Chiton 25: 71

127

THE AMERICAN MALACOLOGICAL SOCIETY

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Tv . Ti : '

- ^r, ■ • it * I

'i

Mopalia kennerleyi Carpenter, 1864, a forgotten species and its southern analogue Mopalia

ciliata (Sowerby, 1840). ROGER N. CLARK 71

Two new chitons of the genus Tripoplax Berry, 1919 from the Monterey Sea Canyon.

ROGER N. CLARK 77

The effect of sampling bias on the fossil record of chitons (Mollusca, Polyplacophora).

STEPHANEY S. PUCHALSKI, DOUGLAS J. EERNISSE, and CLAUDIA C. JOHNSON 87

Fertilization biology and the evolution of chitons. JOHN BUCKLAND-NICKS 97

Chitons (Mollusca: Polyplacophora) associated with hydrothermal vents and methane seeps
around Japan, with descriptions of three new species. HIROSHI SAITO,

KATSUNORI FUJIKURA, and SHINJI TSUCHIDA 113

Index to Vol. 25 125

Membership Form for 2008 128

Information for Contributors

130

A^I3

yv[ 0

AMERICAN

MALACOLOGICAL

BULLETIN

JUIM 11^
(

Journal of the American Malacological Society

http://www. malacological. org

Introduction to the symposium “Molluscan models: Advancing our understanding of the eye”.

JEANNE M. SERB

Toward developing models to study the disease, ecology, and evolution of the eye in Mollusca.

JEANNE M. SERB 3

Rho signaling mediates cytoskeletal re-arrangements in octopus photoreceptors.

SHAUNTE M. GRAY, SHANNON KELLY, and LAURA J. ROBLES 19

Comparative morphology of the concave mirror eyes of scallops (Pectinoidea).

DANIEL I. SPEISER and SONKE JOHNSEN 27

The evolution of eyes in the Bivalvia: New insights. BRIAN MORTON 35

Understanding the cephalic eyes of pulmonate gastropods: A review.

MARINA V. ZIEGER and VICTOR BENNO MEYER-ROCHOW 47

The genus Buccinanops: A model for eye loss in caenogastropods.

ANDRES AVERBUJ and PABLO E. PENCHASZADEH 67

Evolution of mollusc lens crystallins: Glutathione S-transferase/S-crystallins and aldehyde

dehydrogenase/fl-crystallins. JORAM PIATIGORSKY 73

continued on back cover

Cover photo: Mantle eye of Argopecten irradians from Speiser and Johnsen

AMERICAN MALACOLOGICAL BULLETIN

BOARD OF

Kenneth M. Brown, Editor-in-Chief
Department of Biological Sciences
Louisiana State Lhiiversity
Baton Rouge, Louisiana 70803
USA

Janice Voltzow
Department of Biology
University ot Scranton
Scranton, Pennsylvania 18510-4625
USA

Robert H. Cowie
Center for Conservation Research
and Training
Lhiiversity ot Hawaii
3050 Maile Way, Gilmore 408
Honolulu, Hawaii 96822-2231
USA

Carole S. Hickman

University of California Berkeley

Department of Integrative Biology

3060 VLSB #3140

Berkeley, California 94720

USA

Timothy A. Pearce

Carnegie Museum of Natural History
4400 Forbes Avenue
Pittsburgh, Pennsylvania 15213-4007
USA

EDITORS

Cynthia D. Trowbridge, Managing Editor

Oregon State Lhiiversity

P.O. Box 1995

Newport, Oregon 97365

USA

Paula M. Mikkelsen ;

Paleontological Research Institution i

1259 Truniansburg Road '

Ithaca, New York 14850-1313 |

USA

Alan L Kohn

Department of Zoology i

Box 351800

University of Washington
Seattle, Washington 98195
USA

Dianna Padilla |

Department of Ecology and Evolution

State Llniversity of New York j

Stony Brook, New York 11749-5245 f

USA I

Roland C. Anderson ^

The Seattle Aquarium f

1483 Alaskan Way ;

Seattle, Washington 98101 „

USA

Janet Voight '

The Field Museum
1400 S. Lake Shore Dr.

Chicago, Illinois 60605-2496
USA

The American Malacological Bulletin is the scientific jotirnal of the American Malacological Society, an international society of professional,
student, and amateur malacologists. Complete information about the Society and its publications can be found on the Society’s website:
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AMERICAN MALACOLOGICAL BULLETIN 26(1/2)
AMER. MALAC. BULL.

ISSN 0740-2783

Copyright © 2008 by the American Malacological Society

AMERICAN MALACOLOGICAL BULLETIN CONTENTS VOLUME 26 I NUMBER l/2

Introduction to the symposium “Molluscan models: Advancing our understanding of the eye”.

JEANNE M. SERB 1

Toward developing models to study the disease, ecology, and evolution of the eye in Mollusca.

JEANNE M. SERB 3

Rho signaling mediates cytoskeletal re-arrangements in octopus photoreceptors.

SHAUNTE M. GRAY, SHANNON KELLY, and LAURA J. ROBLES 19

Comparative morphology of the concave mirror eyes of scallops (Pectinoidea).

DANIEL I. SPEISER and SONKE JOHNSEN 27

The evolution of eyes in the Bivalvia: New insights. BRIAN MORTON 35

Understanding the cephalic eyes of pulmonate gastropods: A review.

MARINA V. ZIEGER and VICTOR BENNO MEYER-ROCHOW 47

The genus Biiccimmops: A model for eye loss in caenogastropods.

ANDRES AVERBUJ and PABLO E. PENCHASZADEH 67

Evolution of mollusc lens crystallins: Glutathione S-transferase/S-crystallins and aldehyde

dehydrogenase/1 i-crystallins. JORAM PIATIGORSKY 73

Photoreception and the polyphyletic evolution of photoreceptors (with special reference

to Mollusca). LUITERIED VON SALVINI-PLAWEN 83

Primary inhibition by light: A unique property of bivalve photoreceptors. LON A. WILKENS 101

When a snail dies in the forest, how long will the shell persist? Effect of dissolution

and micro-bioerosion. TIMOTHY A. PEARCE Ill

Bivalve molluscs from the continental shelf of Jalisco and Colima, Mexican Central Pacific.

EDUARDO RIOS-JARA, ERNESTO LOPEZ-URIARTE, and

CRISTIAN M. GALVAN-VILLA 119

A mature female of Bathothauwn Chun, 1906 (Cephalopoda: Cranchiidae) from Hawaii.

JANET R. VOIGHT 133

Conservation of the freshwater gastropods of Indiana: Historic and current distributions.

MARK PYRON, JAYSON BEUGLY, ERIKA MARTIN, and MATTHEW SPILLMAN 137

The feeding behavior and diet of an endemic West Virginia land snail, Triodopsis platysayouies.

DANIEL C. DOURSON 153

Structural community changes in freshwater mussel populations of Little Mahoning

Creek, Pennsylvania. ERIC J. CHAPMAN and TAMARA A. SMITH 161

Diversity and distribution of freshwater gastropods in the Bayou Bartholomew drainage,

Arkansas, U.S.A. RUSSELL L. MINTON, JOHN D. WHITE, DAVID M. HAYES,

M. SEAN CHENOWETH, and ANNA M. HILL 171

Research Note: Conus ligluhourni holotype returned to the Delaware Museum of Natural

History. ELIZABETH K. SHEA and WILLIAM J. EENZAN 179

Index to Vol. 26 181

Membership Form for 2009 185

Information for Contributors 187

Meeting AnnoLincement 189

1

Vendrasco et al. (2008) AMB 25: 5 1-69.
ii

Saito etal. (2008) AMB 25: 1 13-124.

Doursoii (2008) AMB 26: 153-159.

Dear Readers,

I am happy to announce that beginning in January 2009, X\\q American Malacological BuUeiin
will be available on BioOne (www.bioone.org). an essential electronic aggregation of over 140
cutting-edge bioscience research journals. Volumes 22-26 as well as current issues published in
2009 and onward will be available in both PDF and XML formats. The Bulletin will be avail-
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those with any questions should contact BioOne directly. We are also working on developing a
link through our web page to allow subscribers to download pdf files of papers. Our hope is that
this increased electronic visibility of our journal will add to its stature and allow us to recruit
cutting-edge papers on malacology.

Kenneth M. Brown
Eclitor-ln-Cliief

Zieger and Meyer-Rochow
(2008) AMB 26: 47-66.

Pearce (2008)
AMB 26: 111-117.

Amer. Make. Bull. 26: 1-2 (2008)

Introduction to the symposium “Molluscan models: Advancing our understanding
of the eye”’^

Jeanne M. Serb

Department of Ecology, Evolution and Organismal Biology, 253 Bessey Hall, Iowa State University,
Ames, Iowa 5001 1, U.S.A., serb@iastate.edu

Since the time of Darwin, the eye has been a subject of
evolutionary and comparative biologists alike who were
intrigued by the structural complexity and morphologi-
cal diversity of eyes in nature. Much of what we know
about the eye — development, structure, physiology, and
function — has been determined from only a handful of
model organisms, specifically the mouse and the fly. One
major phylum in particular, the Mollusca, has been under-
utilized in investigating the evolution and development of
the eye. This is surprising as molluscs display a myriad of eye
types, such as simple pit eyes without any apparatus to focus
images, compound eyes that superficially resemble the eyes
of flies, camera-type eyes that are similar to vertebrate eyes,
and eyes with mirrors, just to name a few. As a result, mol-
luscan eyes comprise more morphological diversity than
seen even in the largest animal phylum, the Arthropoda.

With all of this incredible diversity, how do we as re-
searchers determine which mollusc species should be devel-
oped as models to study the eye? Serb provides background
for eye research using traditional model organisms and how
using molluscan species would be advantageous to under-
standing the eye. She describes the research potential of mol-
luscan species as model organisms and identifies criteria that
might be used to develop a molluscan model and the ques-
tions molluscan models might address.

One application of molluscan models is to study the
cellular biology of human eye disease. As many degenera-
tive eye diseases, such as macular degeneration, have been
linked to the mis-organization of the cytoskeleton within
retinal cells, understanding the control of cytoskeleton or-
ganization and its influence on photoreceptor cell changes
may lead to prevention and possible cures for some eye
diseases. Gray, Kelly, and Robles utilize Octopus bimoculoides
Pickford and McConnaughey, 1949 as a model organism to
study the molecular controls of cytoskeleton organization
in the retina. Their work identifies a cell signaling path-

way (Rho GTPase) that mediates cytoskeleton rearrange-
ments. Errors in this pathway may prove to be one of the
factors that disrupts cytoskeleton formation, leading to reti-
nal degeneration.

After developing one or several molluscan models of the
eye, how does one set about understanding this great diver-
sity of eyes and place it in an evolutionary context? One way
is to use a comparative approach to identify conserved and
variable components of eye morphology, such as lens com-
position, photoreceptor number and organization, and over-
all eye shape. These morphological features can provide
evidence for functional differences and visual capabilities
among species. Several authors in the symposium take this
approach. Speiser and Johnsen examine eye morphology
in four species of scallop and a closely related spondylid
[Spondyhis americaniis [Hermann, 1781]). They show that
scallop eye structure varies among species, and these struc-
tural differences affect optical resolution and sensitivity. Fur-
ther, they provide evidence that actively swimming species
(e.g; Amiisiuni balloti [Bernardi 1861]) have better optical
resolution than non-swimming species. Speiser and Johnsen
provide several new and exciting hypotheses on how the
scallop eye performs and how visual requirements may differ
between mobile and immobile species. Morton takes a
broader perspective and reviews the diversity of non-
cephalic eye types in the Class Bivalvia. He hypothesizes a
possible evolutionary path to create the double retina system
in Pectinidae and Laternulidae through the duplication of
sensory structures on the pallial folds. Zieger and Meyer-
Rochow review the variation of cephalic gastropod eyes,
concentrating on pulmonate species, which are the best-
studied eyes in gastropods. They discuss eye anatomy, dif-
ferences in retinal design, and the visual capabilities of dif-
ferent optical components. Finally, they describe the
ultrastructure of “additional” or “accessory” eyes associated
with cephalic eyes in several lineages. These data provide

From the symposium “Molluscan models: Advancing our understanding of the eye” presented at the World Congress of Malacology,
held from 15 to 20 July 2007 in Antwerp, Belgium. Co-sponsored by the National Science Foundation and the American Malacological
Society.

2

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

hints of the function of these structures and indicate behav-
ioral experiments to test these hypotheses.

A comparative approach also can be used to examine
changes in development, not just morphological endpoints.
For example, even though most gastropods have eyes, loss of
eyes occurs in some eyed lineages. Often eye loss is associ-
ated with dark environments, such as abyssal depths or
caves, but little is known of when or how eye loss occurs.
Averbuj and Penchaszadeh show that eyes are present in the
“eyeless” genus Biicci)umops (d’Orbigny, 1841) (Caenogas-
tropoda: Neogastropoda) during the encapsulated larval
stage. What happens to these cephalic eyes post-hatching is
unknown, but why have eyes in non-motile larvae? Do other
“eyeless” species have eyes as larvae and lose those eyes after
the veliger stage? Studying these and other “eyeless” taxa
may provide data on the evolutionary constraints of devel-
opment on morphology. This is a promising area for future
research.

Another way to study the eye is to examine differences
among the various components that comprise the organ.
Eyes are not just single, irreducible entities but they contain
levels of biological complexity nested in a hierarchical fash-
ion (e.g., Serb and Oakley 2005). Therefore, the eye can be
subdivided into components, or modules, such as genetic
networks (i.e., Pax6 network), photoreceptor cell types, crys-
tallin proteins that make up the lens, photo-transduction
pathways that convert light into a chemical signal, and the
eye itself as a morphological structure. Several authors focus
on specific eye modules.

One module consists of crystallin proteins, which form
the lens in both vertebrate and invertebrate eyes. Evidence
indicates that these proteins initially performed biochemical
functions unrelated to vision and were later recruited for
optical purposes during the evolution of the eye lens (Cvekl
and Piatigorsky 1996). In the symposium, Piatigorsky dis-
cusses the origin and evolution of lens crystallins in cepha-
lopod and bivalve molluscs via processes of gene recruit-
ment, gene sharing, and gene duplication.

Other eye components are the light-sensitive cells, pho-
toreceptors, which are ubic^uitous in animal eyes. Salvini-
Plawen presents an interesting hypothesis on the evolution
of the major classes of animal photoreceptors. He suggests
that despite the structural differentiation of ciliary versus
rhabdomeric photoreceptor cells, these cells are not distinct
classes, but the result of ontological changes of a single
cell type. Support for his hypothesis includes a comprehen-
sive treatment of molluscan photoreceptor diversity.
Wilkens examines the physiology of photoreceptors in
bivalves — specifically, how do photoreceptor cells respond
to light and how is this information processed outside of the
eye? Based on physiological and behavioral work, he de-
scribes differences between species and among photorecep-

tor cell types within a single eye. finally, he hypothesizes the
functions of bivalve eyes.

In addition to these published papers, other symposium
participants presented work on a range of topics. Eernisse
reviewed the sensory system of chitons (Polyplacophora)
and discussed how the recent appearance of chiton ocelli
may have evolved in parallel in two phylogenetically distant
lineages. Kelly and Robles (Kelly et al. 2008) added to the
work of Gray et al. to identify a translational regula-
tion mechanism for cytoskeleton proteins that have differ-
ential expression in light- versus dark-adapted octopus eyes.
Speiser and lohnsen (2008) experimentally show that scal-
lops use visual cues to adjust feeding behavior relative to the
movement and size of particles suspended in the water.

I would like to thank the participants and the audience
members who made the symposium an interactive experi-
ence and generated much discussion. I would also like to
thank Thierry Backeljau and his team for organizing the
UNITAS Antwerp meeting and Paula Mikkelsen for her sup-
port of the symposium. The symposium was funded by a
grant from the National Science Foundation (NSF) (DEB
0614153) and the American Malacological Society (AMS).

LITERATURE CITED

Cvekl, A. and f Piatigorsky. 1996. Lens development and crystallin
gene expression: Many roles for Pax-6. BioEssays 18: 621-629.
Kelly S., H. Yamamoto, and L. I. Robles. 2008. Analysis ot the 3'
untranslated regions of alpha-tubulin and S-crystallin mRNA
and the identification of CPEB in dark- and light-adapted
octopus retinas. Molecular Vision 14: 1446-1455.

Serb, 1. M. and T. H. Oakley. 2005 Hierarchical phylogenies as a
quantitative analytical framework for evolutionary develop-
mental biology. BioEssays 27: 1158-1166.

Speiser, D. 1. and S. lohnsen. 2008. Scallops visually respond to the
size and speed of virtual particles. Journal of Experimental
Biology 211: 2066-2070.

Submitted: 8 August 2008; accepted: 10 August 2008;
final revisions received: 26 August 2008

Amer. Make. Bull. 26: 3-18 (2008)

Toward developing models to study the disease, ecology, and evolution of the eye
in Mollusca"^

Jeanne M. Serb

Department of Ecology, Evolution and Organismal Biology, 253 Bessey Hall, Iowa State University, Ames, Iowa 50011, U.S.A.,
serb@iastate.edu

Abstract: Several invertebrate systems have been developed to study various aspects of the eye and eye disease including Drosophila,
Plauaria, Platynereis, and most recently, the cubozoan iellyfish Tripcdalia: however, molluscs, the second largest metazoan phylum, so far
have been underrepresented in eye research. This is surprising as mollusc systems offer opportunities to study visual processes that may be
altered by disease, vision physiology, development of the visual system, behavior, and evolution. Malacologists have labored for over a
century as morphologists, systematists, physiologists, and ecologists in order to understand the structural and functional diversity in
molluscs at all levels of biological organization. Yet, malacologists have had little opportunity to interact with researchers whose interests
are restricted to the biology and development of eyes as model systems as they tend not to publish in the same journals or attend the same
meetings. In an effort to highlight the advantages of molluscan eyes as a model system and encourage greater collaboration among
researchers, I provide an overview of molluscan eye research from these two perspectives: eye researchers whose interests involve the
development, physiology, and disease of the eye and malacologists who study the complete organism in its natural environment. 1 discuss
the developmental and genetic information available for molluscan eyes and the need to place this work in an evolutionary perspective.
Finally, I discuss how synergy between these two groups will advance eye research, broaden research in both fields, and aid in developing
new molluscan models for eye research.

Key words: retina, photoreceptor, opsin, Pax6

Traditional model systems to study eyes

There is a great diversity of metazoans, but research on
developmental processes has largely focused on a small
number of “representative” species. The traditional “big six”
model organisms used in developmental biology are the
roundworm Caenorhnbditis elegans, the fly Drosophila inel-
anogaster, the zebrafish Daitio rerio, the African clawed frog
Xenopus laevis, the chicken Gallus gallus, and the mouse Mas
miisculus. These species were developed as model organisms
because they are amenable to experimental and/or genetic
manipulation and possess life history characteristics suitable
for life in the laboratory, i.e., they are easy to obtain, breed
readily, and are fecund. Research focused on these six model
animals has resulted in large-scale genome secjuencing ef-
forts, and complete or near complete inventories of genes
and high-resolution genome maps are now available for all
six species (Waterson et al. 2002).

Of the two traditional invertebrate models, Caenovhab-
ditis elegans and Drosophila nielanogaster, only Drosophila
possesses eyes. The Drosophila compound eye has been an
outstanding model system to study many general develop-
mental processes including cell fate specification, cell divi-
sion, growth, and death (Pappu and Mardon 2004). In ad-

dition to exploring cellular biology, researchers have
determined the molecular basis of eye specification by ge-
netically dissecting the fly eye to understand how it works.
We have discovered how a group of multipotent cells (stem
cells) can be converted to eye primordia during eye organo-
genesis and have identified the set of nuclear genes that
regulate retinal specification. Understanding these genetic
mechanisms involved in eye formation gives researchers cru-
cial information on the origin of eye disease — which is when
the genetic program goes wrong.

The Pax6 paradigm

Comparative work with the Drosophila eye and verte-
brate eye indicates that all eyes may share a similar devel-
opmental pathway in eye formation (Fig. 1 ). This has been
referred to as the eyeless! Pax6 paradigm (Donner and Maas
2004), which states that a single homologous genetic net-
work regulates eye formation, regardless of eye type, across
all metazoans, and the Pax6 gene or its homologs are part of
this regulatory gene network (Fig. lA). There are three lines
of evidence for this conclusion. First, the gene eyeless {ey) in
Drosophila is homologous to the genes Small eye of mice and
Aniridia of humans (Quiring el al. 1994). These two verte-

* From the symposium “Molluscan models: Advancing our understanding of the eye” presented at the World Congress of Malacology, held
from 15 to 20 July 2007 in Antwerp, Belgium. Co-sponsored by the National Science Foundation and the American Malacological Society.

3

4

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

A B

Drosophila

Vertebrate

eyeless / twin
of eyeless

Pax6 (9)

sine oculis

Six (6)

eyes absent

Eya (4)

dachshund

Dach (2)

eye development

Figure 1. A network of regulatory genes involved in eye formation
conserved between Drosophila and vertebrates. A, The network of
genes that regulates eye formation in Drosophila. Proteins from
three genes in circles form biochemical complexes with each other
in vertebrate models. B, List of vertebrate genes homologous to the
Drosophila genes. Multiple vertebrate homologs, due to paralogous
duplication, are indicated by numbers in parentheses (Relaix and
Buckingham 1999, Donner and Maas 2004).

brate genes [Small eye. Aniridia) are collectively referred to
as Pax6. This homology of ey and Pax6 suggests that eye
formation is controlled by a similar genetic mechanism in
insects and vertebrates, despite large differences in eye mor-
phology and development. Second, the eyeless gene has been
shown to initiate eye formation. For example, when the eye-
less gene in fly is mis-expressed (turned on in the wrong
place at the wrong time, developmentally) eyes can be in-
duced to form in wing, antennae, or leg primordia (Haider
et al. 1995). Third, expression of Pax6 gene copies from
other species, including mice, squid, arrow-worm, and pla-
naria, can also induce eye formation in Drosophila (Haider et
al. 1995, Tomarev et al. 1997). The result of this work in
Drosophila and vertebrates illustrates that there is a deep
homology and conservation of eye genes in metazoans. This
has lead some researchers (e.g., Gehring and Ikeo 1999) to
describe Pax6 and its homologs as the “master control” gene
for eye development in metazoans. We now know that this
is an oversimplification of the system, and in fact, a number
of other genes [i.e., eyes absent (Bonini et al. 1997), dachs-
hund (Shen and Mardon 1997), sine oculis (Pignoni et al.
1997) (Fig. IB)] in addition to Pax6, are able to induce
ectopic eye expression. Rather than a single gene, the Pax6
paradigm really refers to a homologous genetic pathway that
controls eye development across metazoans.

general evolutionary processes. Further, studying the eyes in
multiple species expands our understanding of variation
among eye types, how similar visual tasks many be per-
formed under different conditions, how permutations at the
structural level affect performance, and how gene and gene
pathways evolve to create new phenotypes and subsequently,
new functions.

In addition to the “traditional” Drosophila model, sev-
eral other invertebrate organisms have been used to study
the eye and eye disease including the tlatworm Planaria (Salo
and Baguha 2002), the annelid Platynereis (Arendt et al.
2002), and most recently, the cubozoan jellyfish Tripedalia
(Piatigorsky and Kozmik 2004, Nilsson et al. 2005). Work on
planarian worms has provided a better understanding of eye
formation, development, disease, and evolution of genetic
networks (Pineda et al. 2000, Cebria et al. 2002), while Platy-
nereis and Tripedalia have been used primarily as evolution-
ary models. Platynereis and Tripedalia models have broad-
ened the evolutionary perspective of how eyes have evolved
and what the ancestral eye condition may have been for
Urbilateria (Arendt and Wittbrodt 2001, Arendt 2003, Piati-
gorsky 2003). Ultimately, these “non-traditional” models
have given evolutionary depth to eye research by expanding
work from the traditional model organism.

However, the second largest metazoan phylum, the
Mollusca, has been underrepresented in eye research during
the molecular age (post-PuA'6 paradigm) and has been un-
derutilized in the study of developmental processes of the
eye. This is surprising, as molluscan systems have shown
potential for study of basic visual processes, physiology of
vision, development of the visual system, and evolution. For
example, past work (Robles et al. 1995, Torres et al. 1997)
has shown that cytoskeletal organization of photoreceptor
cells is regulated by the state of light- and dark- adaptation
in cephalopod eyes. It is known that some disease states in
the human retina, such as macular degeneration, affect cy-
toskeletal development and organization (Eckmiller 2004).
Therefore, studies on cephalopod photoreceptors could lead
to a better understanding of the role of the cytoskeleton in
photoreceptor function and provide clues that link its orga-
nization to retinal disease. The goals of this paper are to:
( 1 ) provide an overview of the advantages of working with
molluscan eyes; (2) describe the eye types found in molluscs;
and (3) discuss the future directions of the field of eye re-
search using molluscan models.

Molluscs as “non-traditional” model organisms for
studying the eye

Despite the monumental advances in understanding eye
development using traditional model organisms, it is impor-
tant to include non-model systems in eye research. Broad
comparative studies with many animal examples identify

What is an “eye”?

An eye is a structure that can measure the amount of
light (intensity) and compare light intensity from multiple
directions (Land and Nilsson 2002). Therefore, eyes supply
information of light distribution in the environment. Essen-
tially, vision uses the principles of geometry to focus light

USING MOLLUSCAN MODELS TO STUDY THE EYE

5

(optics) and chemistry to transform light energy into chemi-
cal signals. A nerve center, such as the brain or cerebral
ganglia, then interprets these signals. Therefore, the ability of
an organism to ‘see’, referred to as spatial vision, is the
interpretation of the origin and direction of light, intensity,
and contrast in the organism’s environment. These attrib-
utes of light are the basis of pictorial information as resolved
images.

The simplest way to produce spatial vision is to have
series of light sensitive cells (photoreceptors) shielded on
one side by dark pigment cells (Fig. 2). Pigment cells are
often arranged in a cup-shape, which prevents all of the
photoreceptor cells from detecting light from the exact same
angular direction at the exact same time. Adding more pho-
toreceptor cells and increasing the depth of the cup-shaped
eye (Fig. 3A) increases sensitivity to the direction of light and
refines the image (Land and Nilsson 2002).

In metazoans, there are two major types of photorecep-
tor cells, which use two different means of increasing the
cell’s surface area to better capture light (Table 1). Ciliary
photoreceptors have an expansion of the ciliary membrane,
while rhabdomeric (or microvillar) photoreceptors have an
array ofvilli (microvilli) on the cell membrane (Eakin 1979).
Each photoreceptor type is associated with specific families
of photo-pigment molecules, such as opsin (r-opsin in rhab-
domeric vs. c-opsin in ciliary cells), and proteins of the
photo-transduction cascade which convert light energy into
a membrane potential, an electrochemical signal (Arendt
2003, Nilsson 2004) (Table 1). Photoreceptors can either be
excited by light and transmit information on light intensity
and direction, or light may inhibit photoreceptor response
so that neurons are activated only when light is termi-

Figure 2. The simplest eye that produces spatial vision. Pigment
cells (PC) are arranged in a cup-shape, which prevents all of the
photoreceptor cells (RC) from detecting light from the exact same
angular direction at the exact same time. Redrawn from Land and
Nilsson (2006).

Figure 3. Two common eye types in molluscs. A, Open pit eye does
not have a lens or cornea and is open to the environment (opening
to optical cup; OC). B, Closed lenticular (lens) eye has both lens (L)
and cornea (CO). Both eyes have optic nerves (ON), photoreceptor
cells (RC), and pigment cells (PC).

nated — resulting in a response to shadow (Land 1968). The
eye can be specialized further with the addition of lenses
(lenticular eyes; Fig. 3B) and corneas. These structures are
found in some eyes to help focus light and the image onto
the aligned photoreceptors that make up the retina.

Eye performance varies greatly among organisms and
the specific eye structure that they possess. Performance of
the eye can by summarized by two components: resolution
and sensitivity (Land 1981, see Land and Nilsson 2002 for
expanded explanation). Resolution is the precision with

6

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Table 1. Characteristics of two photoreceptor cell types, rhabdomeric and ciliaiy. Information from
Arendt (2003) and Nilsson (2004).

Photoreceptor cell type

Rhabdomeric

Ciliary

Membrane expansion

Microvillar

Ciliary

Photopigment molecule

Gq (G-protein)

Gi or Go’’ (G-protein)

Analogous proteins of

PLC (phospholipase enzyme)

PDE (phosphodiesterase)

phototransduction cascade

Arrestin-P

Arrestin-a

rk 2, 3 (rhodopsin kinase)

rk 1 (rhodopsin kinase)

Membrane potential

Depolarizing

Hyperpolarizing

■' Kojima ct al. (1997).

which the eye can separate light according to its direction of
origin and is directly related to the eye’s ability to discrimi-
nate tine detail. Resolution depends on the number and
spacing ol photoreceptor cells in the retina. Sensitivity is the
ability of the eye to capture enough light for photoreceptors
to produce a usable neural signal, thus fully utilizing the
potential resolution. Sensitivity can be increased by enlarg-
ing the aperture of the optical system, such as increasing the
size of the pupil, or increasing photoreceptor diameter.
However, increasing photoreceptor diameter decreases the
number of photorecepstors in the retina, subsequently reduc-
ing the resolution of the eye. In general, a larger eye, with
more photoreceptors and a large aperture, has both better
resolution and sensitivity.

In molluscs, the placement of eyes is highly variable and
may depend on both the function and the development of
particular regions. In lineages such as gastropods and cepha-
lopods, a pair of eyes is located on a well-developed head
region; these are referred to as cephalic eyes. Other lineages
with reduced head regions, such as polyp>lacop)horans and
bivalves, have many non-cephalic eyes. Some polyplacopho-
ran species have eyes on exposed dorsal regions, while some
lineages of bivalves have eyes on mantle tissue near siphons
or along the valves.

The molluscan eye has many functions. Eyes are used
for visually orienting the animal in its environment. For
example, both bivalves and gastropods use visual cues: in
bivalves, Argopecten irradians (Lamarck, 1819) appears to
orient swimming behavior based on visual information
(Hamilton and Koch 1996) while Littori?in (Linnaeus, 1758)
uses visual cues to discriminate between environments or
objects (Evans 1961, Hamilton and Winter 1982). Eyes are
also used to detect visual motion. For example, behavioral
experiments in ark clams (Arcidae) Area none (Linnaeus,
1758) (Patten 1886), Area zebra (Swainson, 1833), Barbatia
eatieellaria (Lamarck, 1819), and Aaadara notabdis (Roding,
1798) (Nilsson 1994) suggest that the great number of eyes
found on these species are used to detect motion, rather than
responding to shadows. Ability to form an image also varies

among molluscs. C o 1 e o i d
cephalop»ods are probably best
known for their excellent per-
ception of images and ability to
visual discriminate (review in
Messenger 1981); perhaps
lesser known is the wide degree
of visual capabilities found
among gastropods (Messenger
1981, Zieger and Meyer-
Rochow 2008). Finally, it
should be noted that mollusc
eyes may also be important for
migratory behaviors in pelagic and benthic species (Hamil-
ton 1985).

Advantages and limitations to studying the
molluscan eye

There are many advantages to working with a molluscan
model to study the eye. First, molluscs provide an evolu-
tionary perspective in eye research with a diversity of eye
phenotypes within a single lineage rather than a comparison
between the traditional model organisms that belong to dis-
parate animal phyla. Although the Drosophila and vertebrate
models have demonstrated the deep homology in eye genet-
ics, we still lack a detailed understanding of what changes
occur in these genetic networks that create the vast variation
in morphology. With closely related mollusc lineages that
possess different eye morphologies, we can tease apart
changes at the gene level that alter phenotypes. Second, mol-
luscs possess an array of visual adaptations found within a
single species (e.^., Groeger et al 2006) or among closely-
related species [e.g., Kano and Kase 2002). These adaptations
can be experimentally treated as “mutant” phenotypes, dem-
onstrating the vast array of possible morphologies and pro-
viding a study system to examine specific genotypes that
relate to phenotype. Third, molluscs are a powerful example
of multiple, independently derived, image-forming eyes
found across three (possibly four) classes. Multiple origins of
complex structures allow researchers to test questions of
gene or genetic network recruitment, an important mecha-
nism that appears to have wide application to alter develop-
mental processes resulting in novel phenotypies. Fourth, in
molluscs a variety of eye types are expressed at different life
stages within a single individual. This system can be used to
test hypotheses of how duplication of orthologous or paralo-
gous eye structures may have played a role in morphological
and functional diversification of animal eyes (Oakley 2003,
Friedrich 2006). Fifth, molluscs have the ability to regenerate
their eyes, a reactivation of developmental processes in an
adult organism to restore missing tissues (Butcher 1930,

USING MOLLUSCAN MODELS TO STUDY THE EYE

7

Sever and Borgens 1988, Bobkova et al. 2004b). Regenera-
tion occurs across many dilferent animal lineages, including
amphibians, molluscs, crustaceans, planaria, and cnidarians.
Comparative studies to understand how vastly different or-
ganisms are able to regenerate organs will identify both dif-
ferences and similarities in the genetic process, mechanisms,
and elements, such as multipotent progenitor cells or plu-
ripotent “stem cells.” By determining the genetic mecha-
nisms of regeneration across metazoans, we may be able to
apply components of these processes to human medicine.
Molluscs offer a unique system to study how optic nerves are
repaired during regeneration, and may be a useful model to
I develop regeneration therapies. Finally, the large camera-like
I eyes of the coleoid cephalopods are morphologically and
j physiologically similar to a vertebrate eye, but offer unique
I research advantages and opportunities to study eyes without
the disadvantages or constraints of working with a vertebrate
! system.

Despite these advantages to using the molluscan eye to
study eye development and evolution, there are some limi-
tations to our current knowledge of molluscan eyes. First,
most information of eyes in molluscs comes from only a
handful of species (Hamilton 1991 ). Second, there have been
few comparative studies within lineages or within species
(but see Bobkova et al 2004a, Gal et al 2004, Speiser and
Johnsen 2008a), so we may be underestimating the degree of
variation in eye structure and function. Since visual systems
may change during the life time of an organism, due to
metamorphosis or changes in environment/habitat (Groeger
et al 2005, 2006), additional work is needed ter understand
these fine and coarse modifications. Third, although we have
identified eye or eye-like structures in many mollusc lin-
eages, we know little about the function of these structures
(see discussion on sensory structures of the Polyplacophora
below) and how these structures may be important to the life
cycle or ecology of the organism.

Types of molluscan eyes

The number of eye types in the Mollusca mirrors the
incredible diversity in body plans in the phylum. Of the
seven mollusc lineages, four (Polyplacophora, Bivalvia,
Gastropoda, Cephalopoda) contain species with eyes that
minimally consist of photoreceptors (arranged as a
retina), pigment cells, and a lens. These four lineages rep-
resent the greatest biological diversity within the Mol-
lusca, encompassing over 98% of recognized mollusc
species (Ruppert and Barnes 1994). Below is a brief over-
view ot the eye types found in these four molluscan classes.
I chose to focus on only a few examples in each lineage
and refer the reader to many excellent reviews where
appropriate.

Polyplacophora

While most studies on the optics and fine-structure of
the molluscan eye focus on the bivalves, gastropods, and
cephalopods, the polyplacophorans have a unique system of
photoreceptors and eye-like structures. There are no cephal-
ic eyes in polyplacophoran species. Instead, chitons have
developed three types ot photoreceptors in the shell, a con-
dition unique to Mollusca. Shell eyes, or aesthetes, are im-
bedded in the tegmentum that covers the shell plates (Blum-
rich 1891). Aesthetes are found in all chitons and may
function as simple photoreceptors to meditate light-
response behavior (Boyle 1977, see Knorre 1925 for alterna-
tive functions), and most likely do not provide visual infor-
mation. Extra-pigmentary ocelli (Moseley 1885, Nowikoff
1907) and intrapigmentary ocelli (Nowikoff 1909) are re-
stricted to few lineages in the family Chitonidae (Boyle 1977)
and are believed to be photoreceptors capable of determin-
ing direction and intensity of light. Unlike the aesthetes,
both ocelli types have lenses, a vitreous area, and a cup of
retinal cells with microvillous rhabdomes (Boyle 1969b), the
components necessary tor spatial vision. Like other non-
cephalic eyes in molluscs, the ocelli are highly repetitive
structures, where a single individual of Onithocliiton neglec-
tiis (Rochebrune, 1881) can have 411 to 1,472 ocelli in rows
along all shell valves (Boyle 1969b). A detailed account of the
orientation, patterning, and cellular structure of the exter-
nally pigmented eye is provided by Boyle (1969a, 1977).
Reviews on chiton sensory organs can be found in Charles
(1966), Boyle (1977), Messenger (1981), Kaas and van Belle
(1985), and Serb and Eernisse (2008).

There is much work to be done to understand the func-
tion and ability of the different types of sensory organs in
chitons. Optics, physiology, and function of the three sen-
sory organs have not been examined in any detail. Thus, it is
not known if the two ocelli types function as “eyes” with the
capability of spatial vision. Further, there has been no recent
work on visually mediated behavior. Until we have this basic
knowledge ot ocelli in chitons, polyplacophorans cannot
used as effective models.

Bivalvia

There is an incredible amount of morphological varia-
tion in eyes of the Bivalvia (review in Morton 2001). Most
bivalve eyes are not cephalic as bivalves do not have a dis-
tinct head. Instead, the majority of eyes in adults are found
along the edge of the mantle, referred to as pallial eyes. This
position of the eye appears to be a type of ectopic expression,
and many species that possess pallial eyes have a large num-
ber of serially repeated eye structures along the mantle.

Two of the most complex and unusual eye types in
bivalves are found in the ark clams (Arcoida) and the scal-
lops (Pectinidae). The first description of eyes in ark clams

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

was Will (1844). Subsequent work by Patten (1886) and
Nilsson (1994) refined the description of eye structure and
examined eye function with visual behavioral experiments.
Members ol the Arcoida have two eye types: ( 1 ) a multifac-
eted compound eye which is similar in structure to the ar-
thropod compound eye, but appears to be an independent
origin of this eye type (Charles 1966, Nilsson and Kebler
2007) and (2) a simple pigment cup, or invaginate, eye (Fig.
3A). In contrast, Patten (1886, 1887) gives a description of
three eye typies: pseudo-lenticulate (groups of ommatidia,
over which a cuticula is thickened to form a lens-like body),
invaginate, and faceted (compound) eyes. However, recent
treatments recognize only two eye types (Waller 1980, Nils-
son 1994). Both eye types are found on the first outer mantle
fold (Waller 1980), but the anterior-posterior patterning of
the eyes varies across species and has been hypothesized to
be related to the degree of light exposure of that portion of
mantle edge (Waller 1980, Nilsson 1994). The eyes in Ar-
coida are highly repetitive structures, where a single indi-
vidual may possess 200-300 compound eyes. This provides
the animal overlapping visual coverage, which may improve
sensitivity to the visual signal.

Based on measurements of eye performance calculated
by eye anatomy, Nilsson ( 1994) suggests that the pallial eyes
of ark clams function as optical “burglar alarms.” According
to this interpretation, these eyes are used to detect visual
motion, rather than relying on a simple shadow response
that can be accomplished by simple photoreceptors. The
result is that the animal can respond to moving objects that
do not cast a shadow. Although this means that ark clams
have spatial resolution (ability to detect objects), it does not
mean that they have the ability to visually reconstruct their
environment, or spatial vision (Nilsson 1994). To date, the
electrophysiology or neurophysiology of the ark dam eye has
not been examined.

One of the best-known molluscan eye types is the mir-
ror eye of the scallop (Pectinidae) and its close allies (Limi-
dae, Spondylidae) (Patten 1886, Dakin 1910, 1928), where
the image is not formed by the lens, but by reflection from
the hemisphereical tapetum (argentea) that lines the back of
the eye behind a double retina (description of optics in Land
1984). It has been demonstrated mathematically that the
image forms on the distal retina, composed of ciliary pho-
toreceptors (Land 1966a), and both physiological and be-
havioral experiments corroborate this finding (Patten 1886,
Land 1966b). Pectinids respond to ( 1 ) an overall distribution
of brightness in the environment, which determines the di-
rection of swimming behavior via the proximal retina; local
changes in (2) light intensity by shadow or (3) movements in
the optical environment are involved in defensive responses
via the distal retina (original description in Buddenbrock
and Moller-Racke 1953, summary in Land 1968). So while

the distal retina is used for focusing an image and detection
of movement, the proximal retina response is to absolute
levels of light intensity (Land 1966b). The two retinas func-
tion independently from one another with opposing re-
sponses to light (hyperpolarizing in distal retina vs. depo-
larizing in proximal retina) (Hartline 1938, Land 1966b,
Gorman and McReynolds 1969, Gomez and Nasi 1994), are
composed ol different photoreceptor cell types (diary vs.
rhabdomeric) (Miller 1958), and use distinct phototrans-
duction cascades (Kojima et al. 1997) (see Table 1).

Much work has been done on the scallop eye including
recent work on optics (Land 1965, 1966a), comparative
anatomy (Morton 2000, 2001 and references therein, Speiser
and Johnsen 2008a), electrophysiology (Gorman and
McReynolds 1969, Gomez and Nasi 1994), neurophysiology
(Spagnola and Wilkens 1983, Wilkens 2006), visual-
mediated behavior (Wilkens and Ache 1977, Hamilton and
Koch 1996, Wilkens 2006, Speiser and Johnsen 2008b), pho-
totransduction (Kojima et al. 1997), and lens formation and
protein evolution (Carosa et al. 2002, Piatigorsky 2008).

The ark clam and scallop utilize two very different eye
morphologies to obtain spatial information from their en-
vironments. Although the general structure of these eyes is
not comparable, the functions may be quite similar. Yet,
there has been much discussion of why a relatively sedentary
organism, like a bivalve, would need such complex eyes and
so many of them. Regardless of the specific function of bi-
valve pallial eyes, the large number found in scallops and ark
clams strongly suggest that vision or visually mediated be-
haviors are extremely important to these species.

Gastropoda

Except for a few genera, most gastropods have a pair of
cephalic eyes. Eye placement varies among gastropod
groups, and the eye can be located at the base of cephalic
tentacles, on the tips of retractable tentacles that can with-
draw the eye, or on short stalks. Gastropod eyes range from
open pits (Fig. 3A) to closed vesicles with or without lenses.
The majority of gastropod eyes are of the closed lenticular
type (Fig. 3B), composed of cornea, lens, vitreous body, and
a cup-shaped retina (but see heteropods below). The retina
can have multiple photoreceptor types (Table 1); however,
the majority of photoreceptors near the lens are microvillous
R cells that form rhabdomeres. Other photoreceptor cells
(e.g., H cells, basal retinal neurons -BRN) are ciliary (Ghase
2002). Across species, there is considerable variation in reti-
nal composition (number of cells, photoreceptor density,
organization of photoreceptors) (Hamilton 1991, Ghase
2002), but the functional significance of these differences
largely is unknown and unexplored. Generally, gastropod
eyes appear to have several functions including: mediating
phototaxic behavior and locomotion, regulating daily and

USING MOLLUSCAN MODELS TO STUDY THE EYE

9

seasonal activities, and, in some species, visual detection of
forms. However, the extent to which gastropod eyes have
spatial vision is still under investigation (Zieger and Meyer-
Rochow 2008) and will probably vary greatly among species.

There are several unicque structures in the gastropod
sensory system. The “accessory retina” (Smith 1906) is
found in some gastropod lineages {e.g., Limacidae), which
may be involved in infrared detection (Newell and Newell
1968). Dorsal eyes appear in species of the marine slug Oti-
chidiu?7i Buchanan, 1800. These eyes are on papillae pro-
jecting off the dorsum of the animal (Hirasaka 1922) and are
composed of ciliary photoreceptors that may create a “rea-
sonable image” (Land 1968). See detailed descriptions in
Katagiri et al. (2002) and references therein. Probably the
most sophisticated and unique eye in the gastropods is the
scanning lenticular eye of pelagic heteropods. The retina is
not cup-shaped but forms a long strip of 3-6 ceils in width,
resulting in a very narrow field of view and contains several
photoreceptor types that are unlike ciliary or rhahdomeric
receptors found in cephalic eyes of other molluscs (Land
1984). These eyes move in a systematic scanning motion,
which may be used to detect stationary objects (Land 1982).
Further work on the function of these unusual eyes is
needed. For more information on gastropod eye diversity,
there are several excellent and comprehensive reviews
(Charles 1966, Messenger 1981, Chase 2002, Zieger and
Meyer-Rochow 2008).

Cephalopoda

Vision in cephalopods is cjuite different than the visual
information available to most other molluscs as reflected by
the sophisticated eye types found in this lineage. There are
two well-known cephalopod eye types, the pin-hole eye type
of nautiloids and the camera-type eye (Fig. 4) in coleoid
(internal shelled) cephalopods. Eyes of Nautilus Linnaeus,
1758 are unusual as they are open to the environment, have
no cornea or lens, and appear to function like a pin-hole
camera (Messenger 1981). Although it would appear that
this eye is rather unsophisticated, many other features sug-
gest that the eye has an effective visual system. The Nautilus
eye is large, comparable in size to the more elaborate cam-
era-type of the coleoids, and has an adjustable pupil (Hurley
et al. 1978). Having a large eye with a small aperture im-
proves spatial resolution of the eye, until light capture is
limited. In fact, calculations by Land ( 1981) suggest that the
resolving power of a nearly closed pupil of Nautilus is on par
with a typical insect eye. In the retina, density of closely
packed rhabdomeric photoreceptors has been conservatively
estimated at an order of magnitude higher than any other
non-cephalopod mollusc (Barber and Wright 1969, Messen-
ger 1981 ), again implying good capabilities for image reso-
lution (but see Land 1981). In addition, the Nautilus eye

Figure 4. Camera-type eye ot coleoid cephalopods has an iris (I),
nearly circular lens (L), vitreous cavity (VC), and photoreceptor
(RC) and pigment cells that form the retina. Redrawn from Zuker
(1994).

possesses an ocular-motor reflex which compensates for
movement of the animal and allows for the stabilization of
images on the retina. This type of reflex is found only in
animals that are able to detect motion and form, thereby
suggesting Nautilus has these capabilities (Land 1981).

Although these eye properties are important in spatial
vision, the function of the Nautilus eye is still speculative. It
has been suggested that the Nautilus may use its eyes to
stabilize itself under strong oceanic currents, help navigate
during diurnal vertical migrations, or identify potential food
sources (Muntz 1991 ); however, little is known of the Nau-
tilus in its natural condition and these hypotheses remain
untested. Many other questions about the fine structure of
Nautilus eye also remain unanswered.

In contrast to the eye of the Nautilus, considerable in-
formation exists on the eye and visual capabilities of coleoid
cephalopods due to detailed studies of optics, neurophysi-
ology, retinal organization, and extensive behavioral experi-
ments. Since these animals rely on vision for prey capture,
predator avoidance, and intra-specific communication
(Budelmann 1996, Hanlon and Messenger 1996, Muntz
1999), they possess excellent perception and visual acuity
(Messenger 1981).

Coleolid cephalopods have rhabdomeric (microvillous)
photoreceptors in a camera-type eye that optically func-
tions in a similar manner to the vertebrate eye, making the
cephalopod eye a famous example of convergent evolution
(Packard 1972; however, see Serb and Eernisse (2008) and
references within for alternative views). Specifically, the

10

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

cephalopod eye resembles an all-rod elasmobranch eye with
similar optics, speed, sensitivity, and resolution (Packard
1972). Similarly, cephalopods have an iris, nearly circular
lens, vitreous cavity, and photoreceptor cells that form the
retina (Fig. 4); however, the photoreceptors in the cephalo-
pod eye are rhabdomeric, not ciliary as in vertebrate eyes
(Young 1962). Reviews on the similarities and differences of
these eyes can be found in Packard (1972), Messenger
( 1981 ), Land ( 1984), Nicol (1989), Nilsson (1996), and Land
and Nilsson (2002).

There is a large body of literature on the cephalopod
retina. We have a good understanding of the retinal struc-
ture in both Octopus Cuvier, 1797 (for example, see early
work in Babuchin 1864, Hensen 1865, summarized in Young
1962, Yamamoto et al. 1965, Messenger 1981) and squid
(Loligo Lamarck, 1798) (Cohen 1973a, 1973b). Because the
cephalopod retina is structurally simple, comprised of only a
few cell types, it has been a favorite model system for the
study of comparative physiology and photoreceptive mecha-
nisms (Yamamoto et al. 1965). Other work on the cephalo-
pod retina has focused on light/dark adaptation — how the
eye acclimates to changes in light levels in the environment
(Young 1963a). Work on cephalopods has identified impor-
tant sub-cellular alternations to the shape of photoreceptor
cells and movements of cytoskeleton and photopigments
within these cells in response to changes in light intensity
(Robles et al. 1995, Marinez et al. 2000, Gray et al. 2008).
These studies have important application to understanding
human eye disease. Another important question with medi-
cal applications is, how does the retina organize? While
Meister ( 1972) provides a chronology of cell patterning and
differentiation of the squid retina based on light microscopy,
the next step is exploring the developmental regulation ot
the retina and how retinal cell fate is determined. This is a
wide-open area for future research and will provide data to
compliment work in mouse and other vertebrate models
(Livesey and Cepko 2001, Zaghloul et al. 2005). These com-
parative studies of convergent structures will be an impor-
tant contribution to both developmental and evolutionary
biology.

Within coleolid cephalopods, there is both interspecific
and intraspecific variation in their eyes. Some of these dif-
ferences occur in the shape of the eye, which may deviate
from spherical to telescopic (Amphitretus Hoyle, 1885),
stalked (Bathothaiima Chun, 1906), or asymmetrical {His-
tioteutliis Orbigny, 1841) eyes (Chun 1913, Nixon and
Young 2003 and references therein). Composition of the eye
may also vaiy. For example, the eyes of Cirrothauma nwrrayi
(Eschricht, 1836) are simple open cups, lacking lens, iris, or
ciliary body — the muscle and choroid surrounding the eye
typically found in other coleolid cephalopods (Chun 1913).
Patterning, size, and density of the rhabdoms in the retina

vary among species (Young 1963b), and these traits appear
to be correlated with the behavior and pupil shape (discus-
sion in Messenger 1981). Absorbance and transparency of
lenses can differ among species found a different depths
(Denton 1960, Denton and Warren 1968, Sweeney et al.
2007b) as well as within a single species (Denton and Warren
1968). Finally, retinal sensitivity can vary during ontogeny, i
Recent work on the cuttlefish Sepia offteinalis Linnaeus, 1758 I
indicates that aspects of the eye change during growth, in- |
eluding spectral sensitivity, light and contrast sensitivity, and ■
visual acuity (Groeger et al. 2005, 2006). !

The visual abilities in coleolid cephalopods have been |
explored more extensively than any other molluscan group
(reviewed in Messenger 1981). Cephalopods display excel-
lent perception and are able to discriminate between differ- |
ent shapes, but it appears that they are color blind (Messen- i
ger 1981, Mathger et al. 2006). So how do these cephalopods
create and control their camouflage to imitate chromatically
rich environments without color vision (Hanlon 2007)? An |
interesting solution has been suggested by Shashar and Cro-
nin (1996). They propose that polarized vision may provide :
visual information to detect and recognize objects analogous
to color vision systems. Polarized light sensitivity has been
identified in many cephalopods (Moody and Parriss 1960,
lander et al. 1963, Tasaki and Karita 1966), suggesting its
importance in the organism’s ecology (e.g.. Waterman
1981), but the function of this sensitivity needs to be tested |
further.

FUTURE DIRECTIONS

Choosing a molluscan model ^

Considerations j

To further advance eye research in molluscs, a directed
and combined effort to develop one or several model species ,
is needed. Some considerations in choosing a model organ- ^
ism should include its life history traits, the availability of the I
nuclear genome sequence of the target species or a closely ij
related species, and a strong foundation of research in the
eye system of that species (Bolker 1995, Slack 2006, Jenner
and Wills 2007). For experimental work, it will he necessary '■
to maintain the model organism for a period of time in the
laboratory. Development and implementation of a new mol- |
luscan model species will depend on both species character- i
istics and laboratory considerations. For example, to maxi-
mize the number of possible laboratory experiments per
year, it is optimal if both adults as well as embryos are
available year-long by either culturing the species in the
laboratory or collecting samples from wild populations. In j
addition, housing costs for the species must be considered.

USING MOLLLISCAN MODELS TO STUDY THE EYE

1 1

especially if a colony needs to be maintained. Larger species
will rec]uire more laboratory space, and marine species may
be more challenging, especially it the species filter-teeds. For
eye research, placement and size ot the organ is also impor-
tant. Eyes of potential model organisms must be easily ac-
cessible in the adult or at specific development stages, if
experimental manipulation or explants are necessary. For
questions concerning genetic processes, the model organism
would need to be a laboratory-cultured species with quick
embryonic development that reaches sexual maturity in a
short period of time. In these cases, small animals would be
preferred to house many individuals and to keep mainte-
nance cost down. However, it has been pointed out that
species selection based on rapid developmental rate and
small body size may introduce bias such as developmental
and genomic constraints or maternal influence (Bolker 1995,
but see lenner and Wills 2007 tor the opposing view).

For nearly all eye research, an organism with a complete
genome sequence would be advantageous. First, the avail-
ability of the complete nuclear sequence ot a model organ-
ism gives the researcher a complete inventory of all genes in
that organism. Second, identifying and isolating homolo-
gous genes in the new model organism becomes almost
trivial compared to the laborious method of cloning ho-
mologous genes in a new species. Third, all members of a
gene family could be identified in advance of the experiment.
These data are essential to interpretation of gene function
and its manipulation. Currently, Genbank of the National
Center of Biotechnology Information (NCBl; http://
www.ncbi.nlm.nih.gov/) lists three genome projects of mol-
lusc species that are in progress or are being assembled and
annotated. These include the freshwater snail Biowphalaria
glabmta (Say, 1818) (Gastropoda: Basommatophora) — in
progress (Washington University); the sea hare Aplysia caU-
fornica Cooper, 1863 (Gastropoda: Opisthobranchia) (Broad
Institute) — in assembly; and the marine clam Spisiila solid-
issinia Dillwyn, 1817 (Bivalvia: Veneroida) (Marine Biologi-
cal Laboratory) — in progress. Another genome that is cur-
rently being annotated is the limpet Lottia gigantea Sowerby,
1834 (Gastropoda: Patellogastropoda) available at the joint
Genome Institute (JGI; http://genome.jgi-psf.org/Lotgil/
Lotgil.home.html).

Finally, successful development of a molluscan model
organism would benefit from a body of research already
conducted on that species. Previous work on such topics as
optics and visual behavior may direct the types of questions
or direct which organism may be most appropriate for the
project. That being said, development of a new model or-
ganism is a time consuming process as well as a large finan-
cial commitment for genomic resources and laboratory set-
up. Only taxa that have multiple uses can realistically be
considered.

Camiidatc model species

There are several molluscan species that meet several of
the above criteria and make strong model organism candi-
dates. Aplysia californica might be considered the highest
priority for molluscan eye researchers. This species has been
the “workhorse” for both physiology and neurobiology, and
there is a large body of literature on the physiology, neurol-
ogy (neurobiology, neural processes), photoreception, and
visual- mediated behavior (Kandel 1979 and references
therein). Aplysia californica has a pair of small (300-600 pm)
cephalic eyes at the base of the posterior tentacles (rhino-
phores). The eye is a closed chamber with a large spheroid
lens. The retina, which nearly surrounds the lens, appears to
have both rhabdomeric and ciliary photoreceptors that in-
terdigitate to form the rhabdomere (Jacklet et al. 1972).
Based on the close proximity of lens to retina, the A. cali-
foniica eye does not appear to have good spatial vision, but
the eyes respond to light in three different ways (lacklet
1969, 1973). This demonstrates that the two photoreceptor
types respond differently to light, like the scallop, making
this an interesting system. Keeping and culturing Aplysia in
laboratory has been somewhat standardized (Kandel 1979),
and the National Institute of Health (NIH) and University of
Miami run a large-scale mariculture facility, the Aplysia Re-
source Facility, that can provide specimens from known ge-
netic lines for researchers (http://www.rsmas.miami.edu/
groups/sea-hares/), making availability of specimens a non-
issue. In addition, the Aplysia genome has been sequenced
and is being assembled.

A bivalve model for eye research might be a scallop
species. There is a large body of literature on the scallop eye
(see references above). Scallops are commercial species being
cultured in aquaculture facilities for the global market, so it
should be an easy transition to develop a facility for research.
This also presents an opportunity to collaborate with aqua-
culture researchers to develop genomic tools and resources,
such as sequencing the scallop genome.

Unlike Aplysia and scallops, cephalopods have large eyes
making them easy to work with and manipulate. Several
coleoid cephalopod species would make good model organ-
isms to study the eye due to their availability. Three small
species (Sepia officinalis. Sepia pharaonis Ehrenberg, 1831,
and Euprymna scolopes Berry, 1913) are laboratory-cultured
by the National Resource Center for Cephalopods (NRCC),
which is funded by NlH’s National Center for Research Re-
sources and Texas Institute of Oceanography. As squids have
been a popular model organism, many texts and protocols
are available for eye and nervous system work (c.g., Gilbert
et al. 1990, Meinertzhagen 1990, Saibil 1990). Currently, an
EST (expressed sequence tag) library for the eye is available
for a related species. Octopus vulgaris Cuvier, 1797 (Ogura et
al. 2004), which provides a list of genes expressed in a spe-

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AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

cific tissue (the eye) at a particular developmental stage
(adult). This genomic resource could be applied to other
cephalopod species as a list of candidate genes or as a start-
ing point to isolate specific genes or gene families in ceph-
alopod eyes. In addition, one of the transcription factors
initiating eye organogenesis, the Pax6 gene, has been isolated
from the squid E. scolopes and there are developmental data
on the role of Pax6 in eye, brain, and sensory organ devel-
opment (Tomarev et al. 1997). Detailed studies on the de-
velopment of the eye and central nervous system in several
species (Marthy 1973, Shigeno et al. 2001) and development
and structure of the lens (West et al. 1995, Sweeney et al.
2007a) are available. A large body of literature exists for
cephalopod ecology and how these organisms adapt to en-
vironmental conditions (Boyle and Rodhouse 2005 and ref-
erences therein). Finally, cephalopods may be the best mol-
luscan model for medical research because their eye
structure and function are similar to the human eye.

Molluscan models in evolutionary biology

Molluscs are an excellent group for the study of evolu-
tionary biology because, as a group, they possess a diverse set
of eye phenotypes that range in complexity. For example,
within a single lineage like Gastropoda, eye phenotypes
range from simple pit eyes to complex lenticular eyes. Across
Mollusca, nearly every eye type is represented as well as
many unique phenotypes. Among metazoans, molluscan
eyes will provide data for a more comprehensive view of eye
evolution, rather than relying on a few model organisms
found in widespread and distant phyla. In particular, mol-
luscan eyes are a compelling case of multiple, indepenciently
derived, image-forming organs. Within the eye there are
various levels of homology to examine, including the level of
the gene and genetic network (e.g., Pax6 pathway), cell (e.g.,
photoreceptor), or tissue type [lens protein evolution,
(Carosa et al. 2002, Piatigorsky 2008)]. Below are some ex-
amples of evolutionary topics that can be addressed with
molluscan eye models.

Testing the Pax6 paradigni

In a recent paper by Donner and Maas (2004), the ge-
netic pathways used to create an eye were compared in Dro-
sophila and vertebrates. The authors found that while all
genes in the Drosophila Pax6 pathway are expressed in the
vertebrate eye during development, the functions and rela-
tionships of these homologous genes within their respective
pathways have not been strictly conserved. This being the
case, Donner and Maas (2004) argue that Drosophila is still
a valuable study model and may be used to guide research on
vertebrate eye development. They conclude (p. 750) that
when the pathway is not strictly maintained between verte-
brates and invertebrates, this indicates that “the particular

role that the genetic [pathway] ... is either not relevant, or
not sufficient, to meet the complexity of the vertebrate [eye]”
(my italics). One interpretation of these results is that con-
servation of genetic pathways between lineages will decrease
as eye complexity increases, and eye types diverge, in one
lineage. We can test this assertion in two ways using mol-
luscan models. First would be to deal with a major short-
coming with the Donner and Maas’ (2004) hypothesis,
namely that their comparison was between two completely
different eye types: the compound eye of Drosophila and the
camera-type eye in vertebrates. A more appropriate test
might be a comparison of two camera-type eyes — in cepha-
lopods and in vertebrates — that are similar in function but
vary in their degree of retinal complexity. Second, the hy-
pothesis could further be tested by examining changes in the
Pax6 pathways and the resulting phenotype of the eye within
a single molluscan lineage, such as gastropods or bivalves,
that have multiple eye types ranging from simple photore-
ceptor eyespots to more complex lenticular eyes.

Using the molluscan eyes to examine evolution

The eye has long been a target of anti-evolutionists as an
example of “irreducible complexity” (Behe 1996) The idea is
that certain biological systems, such as eyes, are too complex
to have evolved from simpler, less complete, prototypes and
that these structures are too complex to have arisen from
chance mutations (Hall and Hall 1975, Johnson 1991, Oak-
land and Matrisciana 1991, Behe 1996). Anti-evolutionists
often cite a single sentence from Darwin’s Origin of Species to
demonstrate his own doubt in the ability of evolutionary
forces to create the eye:

“To suppose that the eye [in all of its complexity]

. . . could have been formed by natural selection,
seems, I freely confess, absurd in the highest pos-
sible degree” (Darwin 1859: 186).

Less often is Darwin’s (1859: 186) next sentence cited, which
states:

“Yet reason tells me, that if numerous gradations
from a perfect and complex eye to one very imper-
fect and simple, each grade being useful to its pos-
sessor, can be shown to exist; if further, the eye does
vary ever so slightly, and the variations be inherited,
which is certainly the case; and if any variation or
modification in the organ be ever useful to an ani-
mal under changing conditions of life, then the dif-
ficulty of believing that a perfect and complex eye
could be formed by natural selection, though insu-
perable by our imagination, can hardly be consid-
ered real” (my italics).

Molluscs, with their diversity of eye types, offer examples of

USING MOLLUSCAN MODELS TO STUDY THE EYE

13

an “intermediate” eye types and the group is often cited as a
counter-argument to anti-evolutionists. The range of eyes
include the eye spot, pigment cup (Fig. 3A), pin hole camera
eye (Nautilus)., lenticular eye (r.e., Stwmbus Linne, 1758; Fig.
3B), and the camera eye (Octopus; Fig. 4). However, it has
not been demonstrated that a single lineage contains a plau-
sible series of intermediate eye-designs to examine the gra-
dient hypothesis. Gastropods may be a good group to test
the gradient hypothesis as there are several lineages within
the gastropods that contain variation in eye complexity.
Once a species phylogeny has been constructed and eye types
characterized on the tree, ancestral states of eye complexity
can be estimated. In addition, the time to transition from
one eye structure to another could be calculated. This would
be an excellent test of Nilsson and Pelger’s (1994) estimated
time (about 0.5 million years) needed to evolve a lenticular
eye from a simple photoreceptor patch.

Photoreceptor evolution

Both rhabdomeric and ciliary photoreceptors are found
in two (Aplysia and Pecten) of three of potential molluscan
model organisms, which is useful tor testing current views of
photoreceptor evolution. In a recent paper by Plachetzki et
al. (2005), the authors argue for a common origin and sub-
sequent divergence of photoreceptor cells in an early meta-
zoan ancestor based on two lines of evidence. First, both
rhabdomeric and ciliaiy photoreceptors have been found to
coexist in many lineages, including vertebrates (Panda et al
2005) and annelids (Arendt et al. 2004). These data are part
of a growing body of evidence that is in contrast to previous
hypotheses, where rhabdomeric photoreceptors are found
mostly in invertebrates and ciliary photoreceptors generally
occur in vertebrates (Eakin 1979, 1982; however, see Van-
fleteren 1982 for a differing view). Second, rhabdomeric and
ciliary photoreceptors can be identified by specific genetic
signatures, which are gene expression specific to that par-
ticular cell type, such as opsin, rx, and atonal genes. How-
ever, molecular data to support the hypothesis of Plachetzki
et al. (2005) are limited to a few taxa (e.g., the annelid
Platynereis., the cubozoan Tripedalia, and the mouse). Se-
quence data from opsin and phototransduction signaling
proteins expressed in molluscs will provide additional tests
for this new view of photoreceptor evolution.

Conclusions

The range of molluscan eyes provides a diverse array of
structures and functions and represent an excellent system
for investigating developmental processes. In order to reap
the rewards of this system, malacologists will need to take an
interdisciplinary approach. Although tools developed and
used in traditional model systems can be successfully applied
to mollusc eyes, those interested in studying the molluscan

eye must familiarize themselves with much of the existing
eye literature and have an understanding of traditional
model species used to study the eye. Therefore, these re-
searchers must be trained in other biological fields with a
diverse set of methods. In particular, advances in genomic
techniques will make mollusc species more accessible to
studying the genetics of the eye and its processes. Ultimately,
these advances and new husbandry practices will allow the
development of new molluscan species as models to study
the eye.

It is important that molluscs are included in eye re-
search, in part because molluscan eyes have much to offer
developmental and cell biology, physiology, evolution, and
ecology. The phylum provides a diversity of eye structures
that possess a multitude of functions. Many times these
highly diverse eyes are found within a species, providing
opportunities to examine the genetic underpinnings ot phe-
notypic variation. For cell biology, molluscan models have
been successfully used to study cytoskeleton growth, which
could provide clues that link its organization to retinal dis-
ease. In developmental biology, gene expression studies in
molluscan models may offer insight in the mechanisms of
cell differentiation and cell fate. Finally, the addition of mol-
luscan taxa to the study of the eye will fill an evolutionary
gap in understanding eye development and evolution.

ACKNOWLEDGMENTS

I would like to thank all participants of the symposium,
“Molluscan models: Advancing our understanding of the
eye,” held at the World Congress of Malacology in Antwerp,
Belgium, 15-20 luly 2007. The symposium was funded by
the National Science Foundation (DEB 0614153) and the
American Malacological Society (AMS) grants to JMS. NSF
and AMS provided travel support of three graduate students
presenting contributed papers at the symposium.

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Submitted: 27 May 2008; accepted: 29 July 2008; final

revisions received: 14 October 2008

Amer. Maine. Bull. 26: 19-26 (2008)

Rho signaling mediates cytoskeletal re-arrangements in octopus photoreceptors'^

Shaunte M. Gray, Shannon Kelly, and Laura J. Robles

Department of Biology, California State University, Dominguez Hills, 1000 East Victoria Street,

Carson, California 90747, U.S.A., lrobles@csudh.edu

Abstract: Light sensitive rhabdoms in the octopus retina increase in cross-sectional area in the dark and shrink in the light. Growth in the
dark is due to the formation of microvilli in an avillar region of the photoreceptor cell membrane and lengthening of rhabdomere microvilli
already present. Diminution in the light is the result of the disassembly and shortening ot the same microvilli. Each microvillus contains
an actin filament core that must be assembled or disassembled in the dark or light, respectively. To understand the regulation ot the
construction and breakdown of rhabdomere microvilli in the light and dark, we used centrifugation to separate the rhabdom membranes
followed by Western blotting and Rho pull-down assays to investigate the role of Rho GTPases in this process. Western blotting showed
a difference in the distribution of Rho in rhabdom membrane and supernatant fractions. In the light, Rho was mostly present in the
supernatant but in the dark it was found in the fraction enriched with rhabdom membranes. Complementing these results, pull-down assays
showed that Rho is activated in the dark but in the light, Rho is mostly inactive. We believe that in the dark, activated Rho binds to the
rhabdom membrane and initiates signaling pathways, leading to growth of rhabdomere microvilli. In the light, Rho is present in the soluble
fraction, is inactivated, and is likely bound to a Rho GDI. Receptors involved in the activation of Rho in the dark are undetermined and
may involve rhodopsin or another membrane protein.

Key words: cytoskeleton, rhabdoms, Rho pull-down assay

The cytoskeleton, consisting of microtubules, microfila-
ments, and intermediate filaments, is a three-dimensional
infrastructure within cells responsible for a myriad of func-
tions, including cell movement, cytokinesis, and organiza-
tion of the cytoplasm. At any particular point in the cell
cycle, the cytoskeleton has a unique architecture, which can
undergo rapid reorganization in response to environmental
or internal signals. Microtubules and microfilaments, com-
posed of tubulin and actin subunits, respectively, generally
reorganize by the addition or subtraction of their resprective
protein subunits, leading to lengthening or shortening of the
tubules or filaments and consequent changes in cell shape
or movement (Maekawa et nl. 1999). Intricate signaling
cascades activated in response to environmental or internal
cues trigger these changes in cytoskeletal organization and
are regulated by the Ras superfamily of small GTPases, in-
cluding the Rho family GTPases (Ridley 2001, Raftopoulou
and Hall 2004).

The Rho family GTPases, which currently includes 22
members such as the well known Rho, Rac, and Cdc42,
shuttle between an active GTP-bound state and an inactive
GDP-bound state (Takai et al. 1995, 2001, Hall 1998, 2005,

Ridley 2001, 2006, Wheeler and Ridley 2004). In the acti-
vated state, Rho GTPases interact with specific downstream

kinases that affect the state of actin polymerization. Rho and
Rac indirectly affect actin polymerization by targeting
ROCK (Rho-associated serine-threonine protein kinases) or
Pak 1 (p21 -activated kinase) that in turn activate LIM kinase
1 or 2 (LIM motif containing kinase). LIM kinases phos-
phorylate and inactivate actin binding/filament severing
proteins, such as cofilin, which leads to an increase in actin
polymerization (Naruyima et al. 1997, Arber et al. 1998,
Maekawa et al. 1999, Sumi et al. 1999, Ohashi et al. 2000,
Ridley 2006).

Photoreceptors of vertebrate and invertebrate retinas
contain cytoskeletons that reorganize in the light and dark
and may be regulated by the Rho family of GTPases (Miller
et al. 2005). This reorganization is necessary to achieve maxi-
mum absorption of light by the photoreceptors by either
increasing the membrane area containing the photopigment
rhodopsin or a mechanical re-orientation ot the photo-
receptors so the light sensitive area sits more advantageously
in light path, such as occurs in teleosts (Ali 1975). In either
case, the cytoskeleton is involved in membrane growth or
re-orienting the cell but the mechanisms leading to these
changes are not well understood in any photoreceptor.

The octopus retina can serve as a model system to dis-
sect these mechanisms since the photoreceptors of octopus

From the symposium “Molluscan models: Advancing our understanding of the eye” presented at the World Congress of Malacology,
held from 15 to 20 Rily 2007 in Antwerp, Belgium. Co-sponsored by the National Science Foundation and the American Malacological
Society.

19

20

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

species are large and only two cell types are present in the
retina, photoreceptors and supportive cells, facilitating mi-
croscopic and biochemical studies. In Octopus himaculoides
(Pickford and McConnaughey, 1949), distinct changes in
photoreceptor shape occur in the light and dark and may be
directly attributable to changes in the organization of the
cytoskeleton. Torres et al. (1997) compared the relative
cross-sectional area of light- and dark-adapted rhabdoms,
the light sensitive region of the cell, and outer segment core
cytoplasm and found that the rhabdoms of light-adapted
photoreceptors are reduced in cross-sectional area when
compared to those maintained in the dark. Electron micros-
copy showed that the rhabdomeric microvilli in light-
adapted rhabdoms partially disappeared, leaving behind an
avillar membrane connecting the microvilli on opposite
sides of the rhabdoms. In the dark, this avillar membrane
was replaced with additional microvilli making the rhab-
doms larger.

The biochemical mechanisms leading to increased mi-
crovillar formation in the dark and diminution in the light
are not well understood. Miller et al (2005) studied the
presence of Rho GTPases in octopus retinas. Immunoblot
analyses of whole retinal extracts confirmed the presence of
Rho in light- and dark-adapted retinas and confocal micros-
copy localized Rho in the rhabdoms and showed its co-
localization with actin.

The presence of Rho in the rhabdom suggests that it is
involved in a signaling transduction pathway leading to
rhabdom changes in the light and dark. If Rho were activated
in the dark, it could indirectly inactivate the filament sever-
ing protein cofilin leading to rhabdom growth. To test this
hypothesis we have performed detailed immunoblot analyses
on isolated rhabdom compartments and Rho-GTP pull-
down assays on light- and dark-adapted octopus retina tis-
sue. Our work will lead to a better understanding of mecha-
nisms leading to retinal changes in the dark and light in
octopus species which affect their ability to absorb light.
Eurthermore, understanding cytoskeletal dynamics are im-
portant for all species and may lead to understanding spe-
cific types of retinal degeneration in humans attributed to
cytoskeletal protein mutations such as Usher syndrome type
IB (Weil et al 1995).

MATERIALS AND METHODS

Eye structure

Eye tissue was prepared for microscopic observations
according to previously published methods and immuno-
stained with antibodies to rhodopsin (Robles et al. 1995).
Micrographs were made using an Olympus Vanox or BX461
fluorescence microscope.

Rho immunoblots

Adult specimens of Octopus bimaculoides were dark- or
light-adapted for 2-3 hours. Afterwards they were anesthe-
tized on ice, whole eye cups removed, and the sclera and
lenses discarded. To isolate the rhabdom compartment from
the remaining retinal tissue, we used previously described
centrifugation procedures (Robles et al 1984). These centri-
fugation experiments were repeated five times using a total
of 20 octopuses per lighting condition. Results were consis-
tent throughout each experiment.

Equal amounts of total protein supernatant from the
first centrifugation, crude membrane pellet, and final pellet
were diluted 1:1 with Laemmli reducing buffer (Bio-Rad
Laboratories, Inc., Hercules, California), boiled for 5 min-
utes, and electrophoresed on 12% sodium dodecylsulfate
polyacrylamide gels (SDS-PAGE) at lOOV for 2-3 hours
(Laemmli 1970). The proteins were blotted onto polyvinyl-
difluoride (PVDF) or nitrocellulose membranes and incu-
bated overnight in 2.5% blocking solution (non-fat dry milk
and gelatin in phosphate buffer saline [137mM NaCl,
2.7mM KCl, 4.3mM Na2HP04, I.4mM KH2PO4 0.1%
Tween-20] Bio-Rad). The membranes were incubated over-
night with polyclonal rabbit anti-Rho (-A-B-C) (1:1000 and
1:500, Upstate Cell Signaling Solutions) either overnight at
4 °C or for one hour at room temperature, followed by
incubation with AP-GAR secondary antibodies (1:3000,
Bio-Rad) for two hours at room temperature. All antibodies
were diluted in PBST-BSA (Bio-Rad). An Opti-4CN Sub-
strate kit (Bio-Rad) was used for colorimetric band detec-
tion, and the Bio- Rad VersaDoc™ 3000 imaging system
was used for molecular weight analyses. Quantitative
analysis of Rho concentrations in dark- and light-adapted
octopus retinal fractions was obtained via densitometric
analysis using the Quantity Qne 1-D Analysis software on
the VersaDoc™.

Rho activation

At the end of light- or dark-adaptation, the eye cups
from one octopus in each lighting condition were dissected
to use as controls. The remaining octopuses were moved to
the opposite lighting condition and sacrificed at 5, 15, 30, 45,
and 60 minute time points. The preparation of retinal lysates
from the controls and the time points was carried out using
a dry ice-ethanol bath to snap freeze the tissue. Whole eye
cups were removed and placed into a Petri dish in the dry
ice-ethanol bath. Retinal tissue was liberated from the eye
cups and added to 0.5 mL of cell lysis buffer (50mM
Tris-HCL pH 7.4; 1% NP-40; 0.25% sodium deoxycholate;
150 mM NaCl; 1 mM EDTA; 1 mM PMSF; 1 pg/mL each
Aproptinin, Leupeptin, Pepstatin; ImM NajVQ4; 1 mM
NaF) and homogenized. Samples were clarified using cen-
trifugation for 5 minutes, 4 °C at 8,000 rpm and Rho acti-

RHO SIGNALING IN OCTOPUS PHOTORECEPTORS

21

W.

Rhabdoms

Rhabdoms

Pigment/Supportive Cells

# ^ % 4 '§ - . ■- .»! -■

-;\r -ii#

Inner Segments

Figure 1. Retinal structure in Octopus binmctiloides. A, O. binmailoides. B, Dissected eye from
O. biimiculoides. Line through the iris marks the plane of sectioning, after removal of the iris
and lens, to obtain retinal image shown in C. C, Longitudinal section through retina showing
photoreceptor structure. The dotted line indicates plane of sectioning to obtain tangential to
cross-sections through rhabdoms shown in D-E. 1), Tangential section through retina to
show details of rhabdom structure. E, Arrows highlight individual rhabdoms. In the inset, 1-4
denote the cytoplasm of four individual cells. Each side of the cells contributes one rhab-
domere, composed of microvilli, which form the rhabdom.

vation assayed using the Rho Activa-
tion Biochem Kit ( Cytoskeleton,

Inc.). Supernatants were collected and
pellets were discarded. Positive and
negative controls were processed by
adding a 1:10 volume of loading
buffer. A nonhydrolyzable form of
GTP (GTPyS) was added to the mix-
ture in a 1:100 volume to a final con-
centration of 200 pM. This positive
control sample was incubated for 15
minutes at 30 °C. The reaction was
stopped by transferring the tube to
4 °C and adding a 1:10 volume of stop
buffer. The same processing was car-
ried out for the negative control, sub-
stituting GDP for GTPyS.

The supernatants and controls
were added to 60 pi aliquots of GST
tagged Rhotekin-RBD beads. Each re-
action tube was incubated for one
hour at 4 °C with gentle agitation.

Next, the supernatant was removed
and the pellet was rinsed with 500 pL
IX lysis buffer. The beads were pel-
leted at 5,000 g for 3 minutes at 4 °C.

The supernatant was removed again
and the pellet washed in 500 pL IX
wash buffer. Again, the mixture was
centrifuged at 5,000 g for 3 minutes
at 4 °C. The pellet was resuspended
in a 3:1 ratio with Laemmli reduc-
ing buffer, boiled 5 minutes, and
subjected to 15% SDS-PAGE at 120V
for 2 hours.

The proteins were blotted onto ni-
trocellulose membranes and incubated
for 45 minutes to one hour in Super-
Block -I- 0.05% Tween (Pierce) with
gentle agitation. The membranes were
incubated for one hour at room temperature or overnight at
4 °C with RhoA monoclonal antibody ( 1:500, Cytoskeleton)
and for one hour at room temperature with HRP-GAM
secondary antibody ( 1:20,000-1:50.000, Pierce ImmunoPure
Peroxidase Conjugated GAM IgG(H-i-L)) at room tempera-
ture. All antibodies were diluted in TBS-Tween. The Pierce
SuperSignal West Pico Chemiluminescent Substrate Kit was
used for chemiluminescent detection. All blots were devel-
oped and visualized with the Bio-Rad Versal)oc‘“ imaging
system. Quantitative analysis of the RhoA activation blots
was performed using the Quantity One 1-D Analysis soft-
ware previously described.

RESULTS

Eye structure

The octopus eye is similar in its external appearance to
other camera-like eyes. The slit-shaped iris permits light to
pass through the spherical lens and focus on the retina at the
back of the eye (Figs. lA-E). The light sensitive retina of
Octopus biniaculoides, as well as those of other octopus spe-
cies, consists of a layer of photoreceptors and supportive
cells (Fig. 1C). Optic nerves exit the photoreceptors at their
base, form bundles, and extend to the optic ganglia where
they synapse on ganglion cells. The photoreceptors span the

22

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

kDa

210.2

82.5

Std His- LA LA LA DA DA DA
Rho Crude Supnt Final Crude Supnt Final
Pellet Pellet Pellet Pellet

Velasco et at. 1999). As mentioned, the rhabdoms increase in
size in the dark, by the addition and lengthening of the
rhabdomere microvilli, and decrease in the light by the dis-
assembly and shortening of the same microvilli (Torres et al.
1997).

40.5

31.6

17.5

■k k k k k k

1 2 3 4 5 6 7 8

Figure 2. Western blot localizing Rho in purified light- and dark-
adapted rhabdom membrane and supernatant fractions. Rho (*) is
present in dark-adapted (DA) rhabdom membrane fractions (lanes
6 and 8) but not in the supernatant (lane 7). In the light-adapted
(LA) supernatant fraction (lane 4), Rho is present while there is
little or no detection of Rho in the LA rhabdom membrane trac-
tions (lanes 3 and 5). The double asterisk denotes the Ltis-tagged
RhoA control protein (lane 2). The molecular weight sizes corre-
spond to the fragments of the Kaleidoscope pre-stained standard
(lane 1 ).

entire retina and are compartmentalized into the inner seg-
ments, a middle region which passes through the pigment/
supportive cell layer, and the rhabdoms. The inner segments
contain the biosynthetic machinery of the photoreceptor as
well as organelles called myeloid bodies which store a second
photopigment retinochrome. The photoreceptors narrow
above the myeloid body region and pass between the sup-
portive cells giving rise to the outer segments and rhabdoms.
The outer segments consist of a cytoplasmic core and two
sets of microvilli on opposite sides of
the core which run from the base of
the outer segments to their tips. These
microvilli are called rhabdomeres, and
rhabdomeres from four adjacent cells
point toward each other to form the
rhabdoms (Figs. ID-E, see inset on
IE). The membrane of each rhabdo-
mere microvillus contains rhodopsin
and signal transduction proteins nec-
essary to process the visual signal after
light absorption by rhodopsin as well
as a core of actin filaments and acces-
sory proteins (Robles et al. 1995, De

0>

70 n
60 -

£

3

50 -

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30 -

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Lane 3

Rho localization

Rhabdoms are easily separated from the rest of the
retina using centrifugation technic]ues (see Materials and
Methods). The rhabdom membranes can be separated from
the outer segment cytoplasm using sucrose solutions and
higher speed centrifugation. We obtained rhabdom mem-
brane and supernatant (cytoplasm) fractions from light- and
dark-adapted retinal homogenates of Octopus bimaculoides
and performed immunoblot analyses on the rhabdom mem-
brane enriched and supernatant fractions. We identified Rho
GTPase in rhabdom membrane fractions and supernatants
ot light- and dark-adapted animals.

In homogenates obtained from light-adapted animals,
faint Rho bands were visible in the crude and final enriched
rhabdom membrane pellets, but a strong signal was detected
in the supernatant after incubation with polyclonal anti-
Rho-A-B-C (Fig. 2, lanes 3-5). In dark-adapted animals,
bands are present in the crude and final rhabdom membrane
pellet, but only a faint band is visible in the supernatant (Fig.
2, lanes 6-8). Control Rho (Fig. 2, lane 2, double asterisk) is
His-tagged (Cytoskeleton, Inc.) and runs at 25 kDa com-
pared to the endogenous Rho in our samples with a mo-
lecular weight of approximately 21 kDa. These results were
consistent throughout repeated experiments. Background
bands are likely due to non-specific binding and insufficient
blocking of membrane before overnight incubation with
anti-Rho to intensify the bands.

Quantitative analysis of Rho concentrations (adjusted
percent volumes) on the blots reveal that Rho is approx.
10-fold more abundant in the light-adapted supernatant
fractions (Fig. 3, lane 4) than in rhabdom membrane frac-
tions (Fig. 3, lanes 3, 5) of light-adapted retinas. Rho appears

Lane 4 Lane 5

Lane 6

Lane 7

Lane 8

Figure 3. Quantitative
of Rho in the LA super

analysis of membrane and supernatant fractions verifies the presence
natant and DA membrane fi'action. Lanes correspond to lanes in Fig. 2.

RHO SIGNALING IN OCTOPUS PHOTORECEPTORS

23

kDa Std
210.2

His-

Rho

LA

Whole

Cell

Lysate

LA

GTP-

ys

LA

GDP

DA

5'

DA

15'

DA

30'

DA

45'

DA

60'

40.5

31.8

10

Light to Dark

Figure 4. Rho pull-down assay showing Rho activation in light-
adapted retinas that were moved to the dark and sampled at 5, 15,
30, 45, and 60-minute time points after the shift. Activated Rho,
migrating between 20-22 kDa (*) is visible at all time points, most
notably and strongly at 30 and 45 minutes. For comparison, Rho
can be seen in the whole cell lysate (lane 3). There is little or no
activation in the light-adapted GDP control (lane 5), but there is a
strong band below 20 kDa (c]uestion mark) whose identity has not
been determined. His-tagged RhoA control protein (**) migrating
between 25-27 kDa is in lane 2.

most abundant in the rhabdom membrane fractions (Fig. 3,
lanes 6, 8) than in the supernatant fractions (Fig. 3, lane 7)
of dark-adapted retinas.

Rho activation

Rho pull-down assays were performed (Cytoskeleton,
Inc.) to determine if Rho was activated in the light, dark,
or in both lighting conditions and when the activation
reaches its peak. After light- or dark-
adaptation, octopuses were moved to
the opposing lighting condition and
sacrificed at 5, 15, 30, 45, and 60-
minute time points. Pull-down assays
performed at each time point con-
firmed the presence or absence of
endogenous Rho-GTP with a molecu-
lar weight of approximately 20-21 kDa
in dark to light or light to dark ani-
mals (Figs. 4, 6A-B). In addition to
the His-tagged RhoA protein control
(Cytoskeleton, Inc.) having a molecu-
lar weight of approx. 25-27 kDa, posi-
tive and negative controls included
GTPyS and GDP loaded samples.

25

20 -

15

10

0

J

Lane 3

respectively. Light- and dark-adapted GTPyS samples
revealed the presence of activated RhoA while GDP-
loaded samples showed little or no activated RhoA (Fig. 4,
lanes 4, 5; Fig. 6A, lanes 3, 4). We also included whole
cell lysates from light-adapted or dark-adapted animals
to compare the activated RhoA signal with that of the total
native RhoA (active and inactive) signal (Fig. 4, lane 3; Fig.
6, lanes 6, 7).

Light to dark

In animals that were light-adapted and then moved to
the dark. Western blot analysis of pull-down products with
anti- RhoA confirmed the presence of Rho-GTP at the 5, 15,
30, 45, and 60-minute time points, with peak activation at 30
and 45 minutes (Fig. 4, lanes 6-10). Rho is also present in the
whole cell lysates and GTPyS control as expected (Fig. 4,
lanes 3, 4). The GDP control (Fig. 4, lane 5) was negative
except for a band at a very low molecular weight, possibly
resulting from proteolysis of which the identity has not yet
been determined.

Quantitative analysis of the light to dark blot revealed
that the increase in activated RhoA is approx. 2-fold greater
at 30 and 45 minutes after light-adapted retinas are moved
to the dark (Fig. 5, lanes 6-10).

Dark to light

In animals that were dark-adapted and then moved to
the light, Rho-GTP was detected faintly at 15 and 30 minutes
but was more prominent after 45 minutes in the light
(Fig. 6, lanes 9-13). Quantitative analysis of the dark to light
blot revealed that RhoA is activated at much lower levels
after 15, 30, and 45 minutes of light exposure (Fig. 7, lanes
9-13) when compared to retinas that were light-adapted and
moved to the dark (Figs. 4, 5).

The increased background and non-specific binding
seen (Fig. 4) are attributed to overnight incubation with

Lane 4

Lane 5

Lane 6

Lane 7

Lane 8

Lane 9

Lane 10

Figure 5. Quantitative analysis of the blot shown in Fig. 4 verifies the strong presence of
activated Rho with the highest signal at 30 and 45 minutes.

24

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

kDa

Std

RhoA DA

GTPyS

DA

GDP

kDa

std

DA

Whole

Ceil

Lysate

LA

Whole

Cell

Lysate

His- LA LA LA

Rho 5 min 15 nun 30 min

LA LA

45 min 60 min

210-2

82 5

30

31 6

•

20

*

** *

*

* *

5

6

7

8 9 10 11

12 13

A

1

2 3

4

B

Dark to Light

Figure 6. A, Western blot showing controls for the Rho pull-down assay for octopuses
dark-adapted and then moved to the light (lanes 1-4). There is no activation in the dark-
adapted GDP control (lane 4), but there is a strong band in the GTP7S control (lane 3, **).
The strong band migrating below 20 kDa, is also present (*). Lane 2 contains the His-tagged
RhoA control protein and standards are in lane 1. B, Western blot after Rho pull-down assay
showing the detection of weak, residual Rho activation in dark-adapted retinas moved to the
light and sampled at 5, 15, 30, 45, and 60 minutes after the shift. The detection of activated
Rho (*) is visible at 15, 30, and 45 minutes after the shift to the light (lanes 10-12). Rho in
whole cell lysates (activated and inactivated) is shown in lanes 6 and 7 and standards in
lanes 1 and 5.

primary antibody at 4 °C to increase the Rho signal on the
blots. Also, there is an increase in the number of bands
above the targeted Rho protein band in Fig. 6B: the globular
bands above 24-25 kDa are because of dimerization of the
Rho protein with the Rhotekin beads used in the pull-down
assay.

Figure 7. Quantitative analysis of blot in B verifies the weak, re-
sidual Rho in dark-adapted retinas moved to the light.

DISCUSSION

We previously reported the immu-
nocytochemical localization of Rlio in
the rhabdom compartment of photo-
receptors from Octopus bimaculoides
(Miller et al. 2005). Rho, as well as actin,
were present along the length of the
rhabdomere and suggested that the
Rho GTPases were candidates for the
regulation of rhabdomere growth and
diminution in the dark and light, re-
spectively. Rho is present in the rhab-
doms of light- and dark-adapted
O. bimaculoides (Fig. 8 A-E). Using
biochemical methods to localize Rho
in the membrane fraction and pull-
down assays to demonstrate Rho acti-
vation after light-dark-adaptation, the
results reported here expand our ear-
lier studies showing the presence of
Rho in specific regions of the rhabdom
and its activation in the dark or light.

The centrifugation techniques we
used separate the rhabdom mem-
branes from other retinal components
and then further divide the compart-
ment into membrane and soluble fractions (Robles et al.
1984). In tissue obtained from light-adapted animals, West-
ern blotting showed that Rho was enriched in the superna-
tant and veiy little was found associated with the mem-
branes. In the dark, Rho was mostly associated with the
membrane fraction and little was found in the soluble frac-
tion (Figs. 2-3).

Pull-down assays are used to confirm the presence or
absence of a specific protein and are similar to an affinity-
binding column. Specifically, Rho pull-down assays are de-
signed to show the presence or absence of GTP-bound Rho
in the tissue sample. Our assays on retinal homogenates
from light- and dark-adapted Octopus bimaculoides showed
that RhoA is mostly activated in membranes obtained from
animals in the dark, with peak activation at 30-45 minutes,
while RhoA activation was reduced in samples obtained
from animals in the light.

Rhabdom cross-sectional areas increase in size in the
dark and diminish in the light (Torres et al. 1997). This
increase can be explained by either the addition of new
microvilli or increase in size of microvilli already present.
Addition or growth require membrane assembly and assem-
bly of the actin core contained within each microvillus. The
question is what factors initiate rhabdom growth. Our re-
sults are consistent with our hypothesis that in the light,

RHO SIGNALING IN OCTOPUS PHOTORECEPTORS

25

proteins, which are translocated when
cells are activated (Seabra 1998, Kai-
biichi et (il. 1999). Moreover, GDIs are
able to block GDP dissociation, inhibit
protein phosphorylation, and increase
the solubility of the Rac/Rho protein
into the cytosol (Bokoch ct al. 1994).
Since we observed that Rho was mostly
associated with the membrane traction
in the dark and that it is activated, we
believe that Rho could then initiate a
signaling pathway controlling acting
filament assembly. Whether or not the
signal to initiate the Rho signaling
pathway comes from rhodopsin re-
mains undetermined. More work is
needed to better understand Rho sig-
naling pathways governing cytoskeletal
changes in the retina of Octopus bi-
nuiciiloidcs and other species.

ACKNOWLEDGMENTS

The authors thank students in the
NIH MBRS RISE program at Califor-
nia State LIniversity Dominguez Hills
for inspiration and assistance with this
work. Supported by NIH NIGMS/
MBRS GM08156 and GM062252.

Figure 8. Rho localization of the retina of Octopus biiuacidoides. A-C, Rho is present in
the rhabdoms (arrows) of light- and dark-adapted photoreceptors. D-E, Cross-section
through rhabdoms showing that Rho robustly labels some rhabdoms and not others. Scale
bar = 100 pm A-B, 25 pm C-E. Reprinted with permission from Miller et id. (2005) Visual
Neuroscience 22; 295-304.

LITERATURE CITED

RhoA is sequestered by a GDI (Guanine Nucleotide Disso-
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Couchman 2005). We further postulate that rhodopsin in-
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(Bokoch et al. 1994, Boukharov and Cohen 1998, Michael-
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Submitted: 28 May 2008; accepted: 8 July 2008; final

revisions received: 24 September 2008

Amer. Maine. Bull. 26: 27-33 (2008)

Comparative morphology of the concave mirror eyes of scallops (Pectinoidea)'^

Daniel I. Speiser* and Sonke Johnsen^

' Department of Biology, Duke University, Box 90338, Durham, North Carolina 27708, U.S.A., dis4@duke.edu
^ Department of Biology, Duke University, Box 90338, Durham, North Carolina 27708, U.S.A., sjohnsen@duke.edu

Abstract; The unique, double-retina, concave mirror eyes of scallops are abundant along the valve mantle margins. Scallops have the most
acute vision among the bivalve molluscs, but little is known about how eyes vary between scallop species. We examined eye morphology
by immunofluorescent labeling and confocal microscopy and calculated optical resolution and sensitivity for the swimming scallops
Amiisiiim balloti (Bernard!, 1861), Placopecten magellenkus (Gmelin, \79\), Argopecten irnidiam (Lamarck, 1819), Chlaiuys hastata (Sow-
erby, 1842), and Chlamys ritbida (Hinds, 1845) and the sessile scallops Crassadoma gigantea (Gray, 1825) and Spondyhis americamis
(Hermann, 1781). We found that eye morphology varied considerably between scallop species. The eyes ot A. balloti and P. magellenicas
had relatively large lenses and small gaps between the retinas and mirror, making them appear similar to those described previously for
Pecten maximiis (Linnaeus, 1758). In contrast, the other five species we examined had eyes with relatively small lenses and large gaps
between the retinas and mirror. We also found evidence that swimming scallops may have better vision than non-swimmers. Swimming
species had proximal retinas with inter-receptor angles between 1.0 ± 0.1 (A. balloti) and 2.7 ± 0.3° (C. rubida), while sessile species had
proximal retinas with inter-receptor angles between 3.2 ± 0.2 (C. gigantea) and 4.5 ± 0.3° (S. americanus). Distal retina inter-receptor angles
ranged from 1.7 ± 0.1 (A. balloti) to 2.8 ± 0.1° (C. rubida) for swimming species and from 3.0 ± 0.1 (C. gigantea) to 3.6 ± 0.2° (S.
americanus) for sessile species, but did not appear to correlate as strongly with swimming ability as proximal retina inter-receptor angles
did. Finally, we found that optical sensitivity differed between species, measuring from 3 ± 1 (A. balloti) to 21 ± 10 pm^ ■ sr (C. hastata)
for proximal retinas and from 2 ± 1 (C. gigantea) to 8 ± 5 pin^ • sr (C. hastata) for distal retinas. These differences, however, did not appear
to correlate with ecological factors such as a scallop species’ swimming ability, preferred substrate type, or range of habitat depth. In light
of these and previous findings, we hypothesize that scallop distal retinas may perform tasks of similar importance to all species, such as
predator detection, and that proximal retinas may perform tasks more important to swimming species, such as those associated with the
visual detection of preferred habitats.

Key words: vision, visual ecology, comparative morphology, invertebrate biology

Scallops have more acute vision than any other bivalve
mollusc (Warrant and Nilsson 2006), but it has been argued
that their eyes, like those of other bivalves, function merely
as “burglar alarms” that trigger valve closure when large
passing objects are detected (Nilsson 1994). Scallops are also
notable for their ability, in most cases, to swim by a form of
jet-propulsion (Cheng et al. 1996) and there is some indi-
cation that their swimming behavior may be visually in-
fluenced. For example, it appears that scallops are able to
visually detect and swim towards preferred habitats (Bud-
denbrock and Moller-Racke 1953, Hamilton and Koch
1996). Arguments have been put forth, however, that scal-
lops are unable to perform visual tasks of such complexity
due to the limitations of their decentralized nervous sys-
tem (Morton 2000). We suspect that these limitations may
not be as severe as once thought, given recent findings that
other animals with decentralized nervous systems, such as
box jellyfish (Coates 2003) and sea urchins (Blevins and

Johnsen 2004), use image formation to help guide move-
ment. We therefore believe that the relationship between
scallop vision and swimming behavior is one worth contin-
ued study.

If scallops are able to visually detect preferred habitats,
as we hypothesize, it may be expected that swimming species
have more acute vision than non-swimmers. Alternately, if
scallops only use their eyes to detect predators, it is likely
that little difference exists between the eyes of mobile and
immobile species. Little is known about how optical resolu-
tion and sensitivity vaiy among scallop species, but it is
thought that eye morphology is largely conserved within
Pectinoidea (Dakin 1928, Morton 2001), a superfamily
(Waller 2006) containing both scallops and spondylids (for
brevity, we will refer to all members of Pectinoidea as “scal-
lops” in this report). All scallops so far examined have eyes
lined with a concave spherical mirror that reflects focused
light onto a pair of retinas as well as a lens that is believed

* From the symposium “Molluscan models: Advancing our understanding of the eye” presented at the World Congress of Malacology, held
from 15 to 20 luly 2007 in Antwerp, Belgium. Co-sponsored by the National Science Foundation and the American Malacological Society.

27

28

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

to help correct for spherical aberration caused by the mirror
(Land 1965).

To test our hypothesis, we examined eye morphology by
immunofluorescent labeling and confocal microscopy in the
swimming scallops halloti (Bernardi, 1861; Fig. 1),

Plncopecten juagellenicus (Gmelin, \79\), Argopecten irradi-
ans (Lamarck, 1819), Chlamys hastata (Sowerby, 1842), and
Chlamys rubida (Hinds, 1845) and the sessile scallops Cras-
sadonm gigantea (Gray, 1825) and Spondylus americanus (a
spondylid; Hermann, 1781). We calculated inter-receptor
angle (a measure of optical resolution) and optical sensitivity
for each species and explored the relationships between these
calculations and ecological factors such as a scallop’s swim-
ming ability, preferred substrate type, and range of habitat
depth.

MATERIALS AND METHODS

Specimen collection and fixation

Four specimens apiece of Argopecten iiradiaiis and Pln-
copecten niagellenicns were obtained from Beaufort, North
Carolina, U.S.A. and Woods Hole, Massachusetts, U.S.A.,
respectively. Three specimens of Spondylus americanus were
obtained from the Florida Keys (Florida, U.S.A. ), a single
specimen of A)uusiuin halloti was obtained from Australia’s
Great Barrier Reef, and single specimens of Chlamys hastata,

Figure 1. The left valves of the scallop species examined in this
study. Pictured are the swimming scallops Amusiiun halloti (A),
Placopecten niagelkiiiciis (B), Argopecten irradians (C), Chlamys ru-
bida (D), and Chlamys hastata (E) and the sessile scallops Crassa-
doma gigantea (F) and Spondylus americanus (G). The scale bar
represents 1 cm.

Chlamys rubida, and Crassadoma gigantea were obtained
from Friday Harbor, Washington, U.S.A. Animals were
anesthetized in a 3% MgCl, solution prior to dissection.
Excised eyes were fixed in buffered 4% formaldehyde for
between two and twelve hours and then washed three times
in PBTw, a buffer solution containing the mild detergent
Tween 20™. Samples were next rinsed three times in 70%
ethanol and stored in 70% ethanol, except for C. hastata, C.
rubida, and C. gigantea tissue, which was rinsed and stored
in 100% methanol. All samples remained in alcohol for less
than two months before measurements were taken. Except
for C. gigantea, in which all eyes were of nearly equal size, all
examined species had both large and small mantle eyes. Only
large eyes were used for measurements. Eyes from the ven-
tral (middle) section of the left valve mantle margin were
used for measurements whenever possible.

Sample preparation and measurements

For sectioning, fixed scallop eyes were cut in half with a
scalpel blade. Eyes were only used for measurements if a
clean, perpendicular cut was made through the center of the
lens. Sectioned eyes were stained with fluorescently-labeled
antibodies to alpha-tubulin, a microtubule protein, and
Hoescht 33245, a DNA-binding fluorescent dye. Eyes were
incubated in the anti-alpha-tubulin primary at 4 °C over-
night and in an Alexa Flour 488 secondary for 4 hours at
room temperature. Both the primary and secondary anti-
bodies were diluted 1:500 in a blocking buffer which con-
tained BSA powder and goat serum diluted in lx PBS. After
alpha-tubulin staining, 10 mg/mL Hoescht 33245 stock so-
lution, diluted 1:100 in lx PBS, was used to stain the eyes
for five minutes. Stained eye sections were mounted in glyc-
erol on standard microscope slides. Cover-slips were applied
with modeling-clay feet so as not to disturb natural eye
morphology. Eyes were mounted so that pupils and cover-
slips were perpendicular. Images were obtained with the 10
or 20x objective of a Zeiss 510 LSM inverted confocal mi-
croscope housed in the Duke University Light Microscopy
Core Facility. Illumination was provided by 405, 488, and
561 nm lasers. Images were processed on a Zeiss-built Fu-
jitsu Siemens Intel Xeon CPU using Zeiss LSM 510 version
4.2 software.

Eye internal diameter, focal length (/), pupil diameter
(D), photoreceptor spacing for distal (s,,) and proximal (s^)
retinas, and rhabdom length for the photoreceptors of the
distal (/,y) and proximal (Ip) retinas were measured for each
eye section. The image in a scallop eye is formed by the
reflection of light off a concave spherical mirror (Land
1965), making focal length (f) equal to halt the radius of
mirror curvature (Halliday and Resnick 1988). We measured
focal length by manually fitting circles to the mirror layer at
the central section of each eye (the section in which the

SCALLOP EYE MORPHOLOGY

29

apparent curvature of the mirror matches its actual curva-
ture), then calculating half the radius of the circle. Pupil
diameter (D) was estimated from cornea diameter. Image
stacks obtained with the microscope’s 20x objective allowed
us to study the morphology of individual photoreceptors
from each eye’s distal and proximal retina. Photoreceptors
were distinguished from other cells by their strong staining
by alpha-tubulin antibodies. Photoreceptor spacing (5) was
calculated as the distance from the center of one photore-
ceptor’s rhabdom to the center of the rhabdom of its nearest
neighbor.

Calculations for optical resolution and sensitivity

We calculated inter-receptor angle for the distal (Acp,;)
and proximal (Aip^) retinas of each scallop eye section using
the formulas:

= tan

Aipp = tan'

(1.1)

(1.2)

where and Sp correspond to photoreceptor spacing for the
distal and proximal retinas and / is focal length (Land and
Nilsson 2002). Rhabdoms were contiguous in the eyes of all
species examined, letting Atp,; = Ap^ and Atp^, = Ap^, where
Ap^^ and Ap^ are the acceptance angles of the photoreceptors
of the distal and proximal retina, respectively. The optical
sensitivities of distal (S^^) and proximal (Sp) retinas were
calculated using the formulas:

S^=(^) Dh^p,f PJl - P,){\ - Ppf and (2.1)

Sp=(^) D“(App)^Pp(l-P,,)(l-Pp) (2.2)

where D is pupil diameter and the terms ( 1 - P,;)( 1 - Pp)“
and (1 - P^)(l - Pp) account for the light that is absorbed
as it passes through both retinas on the way to the mirror
and through the proximal retina on the way back to the
distal retina. This absorption of unfocused light effectively
lowers sensitivity in the scallop eye. Pp and P^j are the frac-
tions of light absorbed by the photoreceptors during one
pass through the proximal and distal retinas, respectively. Pp
and P^ were calculated using the formula:

700

f /(X)(l

400

^abs - 700 ^ ^ ^

f/(\)d\

400

where /(\) is ambient irradiance (Kirschfeld 1974, Land
1981, Warrant and Nilsson 1998), k (=0.0067) is the absorp-
tion coefficient of the rhabdom, and / is rhabdom length
(measured for distal or proximal photoreceptors where ap-
propriate). For our calculations, we assumed that scallops
live in environments dominated by green light, appropriate
given estimated habitat depths in coastal waters (Table 1).
We also assumed that scallops’ eyes have peak sensitivity at
480 nm, based on evidence by Cronly-Dillon (1966).

Statistical analysis

Measurements ot eye internal diameter, focal length (/),
pupil diameter (D), photoreceptor spacing for the distal (s,,)
and proximal (Sp) retinas, rhabdom length for the photore-
ceptors of the distal (/,) and proximal (Ip) retinas, inter-
receptor angle of the distal (Atp,,) and proximal (Aipp) reti-
nas, and optical sensitivity of the distal (S,,) and proximal
(Sp) retinas were compared between Placopccten niagclleni-
cns, Argopectcti irradicms, and Spondylus anieiicamis us-
ing Tukey-Kramer HSD multiple comparison tests (Zar
1999). Comparisons were not made between measurements
for other scallop species due to insufficient sample sizes
(Table 1).

RESULTS

Scallop eyes were located on the middle mantle fold at
the distill ends of short tentacles. These eye-bearing tentacles
lined the edges ot the right and left valves from one end of
the hinge to the other and were interspersed with longer,
extensible sensory tentacles in all species. The eyes were sur-
rounded by a pigmented epithelium, which was brown in
Aimisium Indloti, blue in Argopecten irradiaiis, and black in
Placopecten luagelleniais, Chlaniys hastata, Chlainys riihtda,
Crassadoma gigatitea, and Spondylus americaniis. The cor-
neas were composed of a monolayer of nucleated cells (Fig.
2). Corneal cells were cuboidal in all species except C. gi-
guutea, in which they were columnar. Lenses were cellular in
all species examined. Fhe lenses of A. balloti and P. inagel-
letucus were the largest observed and had front curvatures
that were approximately hyperbolic, causing them to re-
semble those described for Pecteii ina.ximiis (Linnaeus, 1758)
by Land (1965). In contrast, the lenses of the other five
species were relatively small and had front curvatures that
were relatively spherical (Fig. 2). All scallop eyes contained
the distinctive double retina described in detail in a number
of past reports (Dakin 1910, Barber el al. 1966). Cells com-
pletely negative for alpha-tubulin staining were present in
scallop retinas along with the photoreceptor cells. We sus-
pect that these non-staining cells were glial cells (Barber ct al.
1966), which generally serve to support neural cells and are

30

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Table 1. Morphological measurements and calculations of optical sensitivity and inter-receptor angle, a measure of optical resolution, for
the eyes of the swimming scallops Amiisium balloti, Placopecteu inagelleniciis, Argopecten irradians, Chlamys hastata, and Chia^nys riibida and
the sessile scallops Crassadoma gigantea and Spoiidyliis americanus. Values represent mean ± 2 SE. Measurements and calculations for P.
magelleniciis, A. irradians, and S. americanus (appearing in bold columns) were compared statistically using Tukey-Kramer HSD multiple
comparison tests. Significant differences between one species and the other two are denoted by (if a = 0.05) or ** (if a - 0.01).
Information regarding shell height, substrate type, habitat depth, and attachment type was adapted from Brand (2006), Lauzier and Bourne
(2006), and personal observation (DIS). Shell height refers to the dorsal-ventral length of the valves.

A. balloti P. magellenicus A. irradians C. hastata C. tmbida C. gigantea S. americanus

in = 2) (h=16) (h=16) (n = 2) (n = 2) (n = 3) (h = 16)

Inter-receptor angle, distal

retina (°)

1.7 ± 0.1

2.5 ± 0.2*’<-

2.1 ± O.U'^

2.5 ± 0.5

2.8 ± 0.1

3.0 ± 0.1

3.6 ± 0.2**

Inter-receptor angle.

proximal retina (°)

1.0 ± 0.1

1.3 ± O.U’^

1.9 ± 0.2’^*

2.5 ±0.5

2.7 ± 0.3

3.2 ± 0.2

4.5 ± 0.3**

Optical sensitivity, distal

retina S,, (pm’ • sr)

4 ± 1

8 ± 1**

5 + 1

8± 5

6 ± 1

2 ± 1

5 ± 1

Optical sensitivity, proximal

retina (pm^ • sr)

3 ± 1

4 ± U’*'

11 ± 3’*^’^

21 ± 10

10 ± 6

7 ± 3

19 ± 4**

Eye internal diameter (pm)

570 ± 30

550 ± 40’^’^

670 ± 40’^’^

480 ± 20

480 ± 20

450 ± 20

370 ± 30**

Pupil diameter D (pm)

390 ± 12

350 ± 30><^

400 + 30*

360 ± 40

310± 10

170 ±20

230 ± 20*

Focal length / (pm)

170 ± 10

150 ± lO’"*

180 ± 10**

140 ± 20

110 ± 10

110 ± 10

95 ± 6**

Photoreceptor spacing, distal

retina s,, (pm)

5

6.4 ± 0.3

6.4 + 0.2

6

5.2 ± 0.4

6

5.9 ± 0.3*

Photoreceptor spacing.

proximal retina s^ (pm)

3

3.3 ± 0.2’*-’^

5.8 ± 0.2**

6

5

6.3 ± 0.3

7.4 ± 0.3**

Rhabdom length, distal

retina (pm)

15 ± 2

19 ± 3**

12 ± 2

20

12 ± 1

14 ±4

13 ± 1

Rhabdom length, proximal

retina (pm)

25

33 ±6

30 ± 10

45

20 ± 6

40

25 ± 2

Shell height of specimens

examined (cm)

?

10

6

6

5

10

9

Preferred substrate

sandy

sandy

sandy

rocky

rocky

rocky

rocky

Habitat depth (m)

10-75

20-110

1-12

2-150

1-200

1-80

1-150

Attachment type

unattached unattached

unattached

byssal

byssal

cemented

cemented

not known to act in signal processing. The backs of all eyes
were lined with a concave spherical mirror, again as de-
scribed by Land (1965). Underlying the mirror was a red
pigment layer. Contrary to past reports, we found that a
cavity was present between the mirror and the retinas in all
scallop species examined (Fig. 2). Cavity size varied greatly
between species. Relatively small cavities were found in A.
balloti and P. magellenicus, resulting in eyes that were mor-
phologically similar to those of P. maximus (Land 1965),
while larger cavities were present in the eyes of the other five
species. Dissection and whole-mount microscopy revealed
that the cavity was filled with a clear fluid.

Eye internal diameter, focal length (/), pupil diameter
(D), photoreceptor spacing for distal (s^) and proximal (s^)
retinas, and rhabdom length for the photoreceptors of the
distal (/,,) and proximal (/^) retinas varied between scallop
species (Table 1). Swimming species generally had larger
eyes, larger pupils, longer focal lengths, and proximal retina

photoreceptors that were more closely spaced (Table 1).
Rhabdom length and distal retina photoreceptor spacing did
not appear to correlate with whether a species could swim or
not (Table 1). Our calculations indicated that distal and
proximal retina inter-receptor angle and optical sensitivity
also differed between scallop species (Table 1). Swimming
species tended to have smaller distal and proximal retina
inter-receptor angles than sessile species (Table 1). Optical
sensitivity did not appear to be related to scallop swimming
ability.

DISCUSSION

Our study revealed several new aspects of scallop eye
morphology. First, we found that lens size and shape varied
between scallop species (Fig. 2). The lenses oi Amusiiim bal-
loti and Placopecteu }7mgeUenicus had shapes similar to those

SCALLOP EYE MORPHOLOGY

31

Figure 2. Mantle eye sections from tlie
swimming scallops Placopccteii inagcllcni-
cus (A) and Argopecteii irnidians (C), im-
aged under a lOx confocal objective, and
the sessile scallop Spomlylus americamis
(E), imaged under a 20x objective. Eyes
were stained with Hoescht dye, causing cell
nuclei to appear blue, and alpha-tubulin,
causing microtubules to appear green. The
pigment layer underneath the mirror ap-
pears red both in the images and in vivo.
'I'he diagrams (B, D, and F) correspond
to the confocal images above and are la-
beled accordingly. The scale bars represent
100 pm.

described for Pecteii maximiis (Land 1965). The front of the
P. maximus lens apapears to be curved in such a way as to
correct for spherical aberration caused by the reflection of
light off the mirror (Land 1965), a function we will also
attribute, tentatively, to the lenses of A. balloti and P. magel-
leniais. The lenses of the other five species appeared to have
front curvatures that were relatively spherical, an indication
that they may do little to correct for spherical aberration
caused by the mirror. We are currently exploring the func-
tional consequences of these different lens shapes and the
phylogenetic distribution of lens types among a wide range
of scallop species.

Second, we consistently noted a fluid- filled cavity be-
tween the proximal retina and the mirror in the eyes of all
seven scallop species examined (Fig. 2). This cavity ranged in
size between species. Small cavities were found in the eyes of
Amiishim balloti and Placopecten niagelleiiicus, resulting in
eyes that closely resembled those of Pecten maximus (Land
1965). Conversely, a large cavity was found between the
proximal retina and the mirror in the eyes of the other five
scallop species examined. The optics of the scallop eye are
greatly influenced by the size of the cavity that exists between
the proximal retina and the mirror. Following Land’s analy-
sis (1965) of the optics of P. maximus, which has eyes with
a small cavity, it appears that focused light likely falls on the
distal retina in the morphologically similar eyes of A. balloti
and P. magelleniciis. Alternately, due to the presence of a
large cavity, it appears likely that focused light falls on the
proximal retina in the eyes of the other scallop species we
examined. We would be tempted to conclude that focused
images simply fall on different retinas in different scallop
species, but we have also found that photoreceptor spacing is
tighter in the proximal retinas of A. balloti and P. magcUeni-
cus than it is in their distal retinas (Table 1). This is not
consistent with a model in which the proximal retinas of A.
balloti and P. magellenicus fail to receive focused light. We

also found that A. balloti and P. magelleuiais have the most
tightly packed proximal retina photoreceptors of any ot the
species examined (Table 1), which again suggests that their
proximal retinas may be involved in image formation. As an
explanation for these inconsistencies, we speculate that scal-
lop eyes are optically dynamic structures that can alternately
focus light onto either of the two retinas through slight
changes in shape. We are, at this time, exploring possible
mechanistic bases for such a process.

An analysis of scallop visual capabilities provided evi-
dence that swimming scallops have more acute vision than
non-swimmers and that the best swimmers have the most
acute vision. Among the scallops included in this study,
Amnsium balloti and Placopccteii magellenicus were the
strongest swimmers, capable of moving at speeds of up to
100 cm/s (loll 1989) and 67 cm/s (Brand 2006), respectively.
These scallops had proximal retina inter-receptor angles of
around 1°, the smallest of any we calculated (Table 1).
Weaker swimmers like Argopecteii iiradians, able to swim at
speecis of 40 cm/s (Brand 2006), had proximal retina inter-
receptor angles between 2-3° (Table 1). Our findings in this
case concur with past morphological studies that found that
Pecten maximus, a scallop with swimming abilities compa-
rable to those of A. irradians (Brand 2006), had an optical
resolution of about 2°. Sessile scallops, which cement to their
substrate in a manner similar to oysters (Lauzier and Bourne
2006), had the largest proximal retina inter-receptor angles
observed, at around 3-5° (Table 1). Proximal retina inter-
receptor angle diversity was a product of ditferences in both
focal length and photoreceptor spacing. For example, tighter
photoreceptor packing was largely responsible for A. balloti
having a smaller proximal retina inter-receptor angle than A.
irradians, but longer focal length was responsible for A. ir-
radians having a smaller proximal retina inter-receptor angle
than Chlamys hastata. Factors other than swimming ability
may also help explain why some scallop species have better

32

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

optical resolution than others. Lor example, scallops from
sandy substrates tend to have better vision and be better
swimmers than those from rocky habitats (Table 1 ). Another
important caveat is that our methods have led us to estimate
the theoretical maximum of visual acuity in each scallop
species. Neural processes, like spatial summation, and
optical imperfections, such spherical aberration, may lead
to scallops having actual visual acuities that are below these
estimates (Land and Nilsson 2002). However, behavioral
(Buddenbrock and Moller-Racke 1953) and electro-
physiological (Land 1966) studies on P. maximus provide
evidence that actual scallop visual acuity is close to the the-
oretical maximum derived from focal length and photore-
ceptor spacing. This suggests that our estimates of inter-
receptor angle likely point towards true functional
differences between the eyes of mobile and immobile scallop
species. Linally, interspecific differences in inter-receptor
angle will have little consequence if focused light falls on
different retinas in different scallop species, a possibility that
we address in detail above.

Distal retina inter-receptor angles, ranging from 1.7 ±
0.1° for Awiisiwn balloti to 3.6 ± 0.2° for Spondylus ameri-
canus, only varied two-fold between species, as opposed to
the four-fold difference observed between proximal retina
inter-receptor angles (Table 1). Distal retina inter-receptor
angle also correlated with scallop swimming ability but not
as strongly as proximal retina inter- receptor angle did. Lor
example, proximal retina inter-receptor angle was larger in
Placopecten magellenicus than it was in Argopecten irradians,
despite P. magellenicus being the stronger swimmer (Brand
2006). Perhaps more tellingly, variation in distal retina inter-
receptor angle was largely a product of interspecific differ-
ences in focal length, not photoreceptor spacing. Distal
retina photoreceptor spacing fell between 5 and 6.5 pm in all
species and, unlike proximal retina photoreceptor spacing, a
relationship between this measure and a scallop species’
swimming ability was not indicated by the data (Table 1).

It has been suggested that the two scallop retinas per-
form different visual functions (Land 1966, Wilkens 2006),
in part due to evidence that the retinas operate via different
opsins and signal-transduction pathways (Kojima et nl.
1997) and that the neurons of the distal retina hyperpolarize
in response to light, while those of the proximal retina de-
polarize (Hartline 1938, Land 1966, McReynolds and Gor-
man 1970). This proposal is supported by our evidence that
proximal retina photoreceptor spacing may depend on a
scallop species’ swimming ability, while distal retina photo-
receptor spacing varies little between species (Table 1). This
implies that scallop proximal retinas may be involved in
visual tasks more important to swimming species, such as
those relating to the detection of preferred habitat, and that
the distal retinas are likely involved in tasks of equal impor-

tance to both swimming and sessile species, such as predator
detection.

further support for functional differentiation of this
sort comes from indications that scallop proximal retinas are
better at gathering information about relatively static envi-
ronmental features (Land 1966), like the eelgrass beds to-
wards which Argopecten irradians has been found to swim
(Hamilton and Koch 1996), while the distal retinas are better
at detecting movement, such as that by potential predators.

Unrecognized differences between the eyes of mobile
and immobile species have contributed to arguments that
swimming scallops do not visually detect preferred habitats,
as has the fact that scallops lack a centralized nervous system
(Morton 2000). While it is true that scallops do not process
much visual information in their brain, their visceroparietal
ganglion (VPG) contains optic lobes that likely give these
animals the neural capacity to convert a range of visual
inputs into behavioral output (Wilkens 2006). It has been
noted that information from the proximal retina elicits
greater activity in the VPG’s optic lobes than information
from the distal retina (Wilkens and Ache 1977), a finding |
seemingly at odds with the claim that focused light only falls l
on the scallop distal retina (Land 1965). As a potential so-
lution to this problem, we suggest that focused light may fall 'i
on the proximal retina in at least some scallop species. This
suggests that previously unrecognized interspecific variation
may account for inconsistencies between past reports. It
also suggests that the scallop optic lobes may, at least in
some cases, process visual information from the proximal
retina for the sake of complex behavioral tasks like habitat
detection.

Scallop optical sensitivity, like optical resolution, dif-
fered between retinas and between species (Table 1). How-
ever, unlike optical resolution, optical sensitivity did not
appear to correlate with swimming ability or, as might be
expected, with habitat depth (Table 1 ). Given that irradiance
values in scallop habitats may vary over several orders of
magnitude, depending on tide conditions and time of day,
the differences we observed between optical sensitivities may
have only minor functional consequences for the species
examined in this study.

In conclusion, we found that eye morphology varied
between scallop species anci that swimming scallops tend to
have better vision than sessile scallops. This latter discovery
is consistent with our hypothesis that mobile scallops may
visually detect preferred habitats. We also found evidence
that scallop distal and proximal retinas may be functionally
differentiated. We are currently working to clarify the rela-
tionship between vision and swimming ability in scallops
and to develop new models of scallop optics that focus, in
particular, on the range of lens shapes we have observed in

SCALLOP EYE MORPHOLOGY

33

different scallop species and on the ways that scallops may
utilize both of their retinas for image formation.

ACKNOWLEDGMENTS

We would like to thank Sam lohnson of the Duke Light
Microscopy Core Facility, Hafeez Dhalla, Elizabeth Jenista,
Nina Tang Sherwood, and Eric Warrant for their help on
this project. We would also like to thank Jeanne Serb, Dan
Runcie, and Mark Hooper for their help with specimen col-
lection. SJ was supported, in part, by a grant from the Na-
tional Science Foundation ( lOB-0444674).

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Submitted; 17 March 2008; accepted: 24 April 2008; final

revisions received: 14 October 2008

Amer. Malac. Bull. 26: 35-45 (2008)

The evolution of eyes in the Bivalvia: New insights"*^

Brian Morton

Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, LLK., prol_bmorton@hotmail.co.uk

Abstract: Two types of multi-cellular eyes have been identified in the Bivalvia. Paired cephalic eyes occurring internally above the anterior
end of the ctenidia are seen only in representatives ot the Arcoidea, Limopsoidea, Mytiloidea, Anomioidea, Ostreoidea, and Limoidca. These
eyes, comprising a pit of photo-sensory cells and a simple lens, are thought to represent the earliest method of photoreception. Many
shallow-water marine, estuarine, and freshwater bivalves also possess simple photoreceptive cells in the mantle that enable them to respond
to shadows. In some other marine, shallow-water taxa, however, a second type of more complex photoreceptors has evolved. These
comprise ectopic pallial eyes that can be divided into three broad categories, in terms of their locations on the (i) outer mantle fold in
representatives of the Arcoidea, Limopsoidea, Pterioidea, and Anomioidea, (ii) middle told in the Pectinoidea and Limoidea, and (iii) inner
fold in the Cardioidea, Tridacnoidea, and Laternulidae (Anomalodesmata). Eyes do not occur in deep-sea bivalve taxa. Where ectopic pallial
eyes occur, they measure amounts of light and integrate intensities from different directions, thereby supplying information to the
individual possessing them about the distribution ot light in its immediate environment. This does not mean, however, despite broad,
phylogenetically related advances in pallial eye complexity, that any bivalve can perceive an image. A revised picture ot the independent
evolution of ectopic pallial eyes in the Bivalvia is provided. In bivalves, pallial told duplication has resulted in improvements to the
peripheral visual senses, albeit at different times in different phylogenies and on different components ot the mantle margin. This has been
achieved, it is herein argued, through; (i) selective gene-induced ectopism; (ii) pigment cup evagination in Category 1 eyes; (iii) invagination
in Categories 2 and 3; and (iv) natural selection. The invaginated distal retina in representatives of the Pectinidae and Laternulidae provides
the potential for image formation and the detection of movement. In the absence ot optic lobes capable ot synthesizing such information,
however, these complex eyes must await matching cerebral sophistication.

Key words: cephalic and pallial eyes, evagination, invagination, duplication, mantle folds

A wide range of internal proprioreceptors and external
sense organs has evolved in the Bivalvia. The former includes
muscle proprioreceptors to moderate muscle tonus and,
uniquely, in the watering pot shells (Clavagellidae and Peni-
cillidae), a pair of pericardial proprioreceptors to monitor
body tonus (Morton 2007). The latter sense organs include
paired statocysts (for orientation), osphradia for the recep-
tion of chemical spawning cues and synchronization of ga-
mete emission (Beninger et al. 1995), abdominal (Haszpru-
nar 1985) and pallial sense organs, as in Aulacomya atm
(Molina, 1782) (Zaixso 2003), both for the mechano-
reception of water currents, and light-sensitive cephalic and
pallial eyes (Morton 2001). These are structures that can
measure amounts of light and integrate intensities from dif-
ferent directions, thereby supplying information to the in-
dividual possessing them about the distribution ot light in its
immediate environment (Land and Nilsson 2002). As will be
argued, however, the presence of such light-sensitive struc-
tures does not necessarily mean, despite phylogenetically re-
lated advances in pallial eye complexity, that any bivalve eye.

in the absence of significant integrative optic lobes in the
cerebral ganglia, can perceive an image of its immediate
environment.

When present, cephalic eyes are located internally on
the left and right sides of the body at the base of the anterior-
most filaments of the inner demihranchs of the ctenidia but
have been seen and described only for representatives of the
Arcoidea, Limopsoidea, Mytiloidea, Anomioidea, Ostre-
oidea, and Limoidea (Morton 2001). The eyes, each com-
prising a pit of photo-sensory cells and a simple lens, are
thought to represent the earliest method of photoreception
in the Bivalvia, but their functional efficiency must be con-
strained by the fact that, in life, light would be received by
them after transmission through the thickest part of the shell
in epibenthic taxa and from the posterior in the case ot most
infaunal species. More recent bivalves do not possess such
eyes (Morton 2001).

Kennedy (1960) described the simplest pallial photore-
ceptor known for the Bivalvia. He demonstrated that the
pallial nerves of Spisula solidissima (Dillwyn, 1817) each con-

* From the symposium “Molluscan models: Advancing our understanding of the eye” presented at the World Congress of Malacology,
held from 15 to 20 luly 2007 in Antwerp, Belgium. Co-sponsored by the National Science Foundation and the American Malacological
Society.

35

36

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

tain a single light-sensitive afferent nerve fiber. This re-
sponds directly to illumination and mediates the shadow
response (or reflex) of siphonal retraction. Because the
shadow reflex is so widespread in freshwater and coastal
bivalves, it can be assumed that most of them possess such
sensory fibers. Even, for example, in the absence of any
obvious photoreceptors, Donax vittatus (da Costa, 1778) re-
sponds to changes in incident light intensity by adjusting its
position in the sediment (Ansell et al. 1998). Conversely, the
subterranean freshwater dreissenid Congeria knsceri Bole,
1962 does not possess such a reflex (pers. obs.) and no
multi-cellular pallial eyes have evolved in freshwater, brack-
ish-water, or deep-water bivalves. Only intertidal and shal-
low continental shelf species possess them.

A few other coastal bivalves have, however, evolved
more sophisticated pallial photoreceptors. In a few taxa,
such structures have resulted in the evolution of pallial eyes
of extraordinary (for such sedentary creatures) structural
complexity. Morton (2001 ) reviewed such eyes in the Bival-
via and showed that pallial photoreceptors could be divided
into three categories (typically associated with an increasing
degree of morphological specialization) based upon their
locations on the (i) outer, (ii) middle, or (iii) inner mantle
folds. As will be also discussed, some progress has been made
in understanding how the development of eyes is controlled
genetically, most recently reviewed by Gehring (2001), but
the objectives of this paper are to understand (i) how the
pattern of increasing pallial eye sophistication has come
about and (ii) how, for each category of eye (Morton 2001),
its various components have been assembled.

CEPHALIC EYES

It is believed that molluscan cephalic eyes can be derived
from structures similar to those that occur in planarians,
themselves representative of those that would have been
found in the ancestor(s) of all metazoans. Gehring (2001),
quoting Hesse (1897), believes that the eyes of Planaria torva
are the most likely candidates for and examples of such an
ancestor. Each P. torva eye comprises three photoreceptor
cells surrounding a single pigment cell (Fig. 1 ). The cephalic
eyes of bivalves are only somewhat more complex and the
larvae of oysters, for example, Crassostrea virginica (Gmelin,
1791 ) and Crassostrea gigas (Thiinberg, 1793), develop a pair
of light-sensitive eyespots (Nelson 1926) that gradually start
to degenerate soon after metamorphosis (Baker and Mann
1994). In species of Chlamys Roding, 1798, Pecten Muller,
1776, and Ostrea Linnaeus, 1758, such eyespots consist of a
cup of pigmented cells surrounding an amorphous (unstruc-
tured) lens (Cole 1938, Gragg and Crisp 1991, Hodgson and
Burke 1998). Cole (1938) has described the cephalic eye in

Figure 1. A section through the eye of the flatworm Planaria torva.
Redrawn after Hesse (1897).

the pre-settlement veliger of Ostrea ediilis Linnaeus, 1758
(Fig. 2) and notes that each one is situated on the body wall
just dorsal to where the gill rudiments attach and, oppo-
sitely, to the mantle.

Cephalic eye structure in the Bivalvia appears to be
highly uniform (Morton 2001). This suggests that such eyes
must have a common ancestry and may once have been
much more widespread in the class but have subsequently
either been lost or never developed in the majority of taxa.
Those of Aulacomya atra (Mytilidae) can serve as an example
of cephalic eye structure and have been described by Zaixso

Figure 2. A section through the cephalic eye of the pre-settlement
veliger of Ostrea edulis. Redrawn after Cole (1938).

EVOLUTION OF BIVALVE EYES

37

(2003). One such eye is illustrated in section (Fig. 3). It
comprises a cup ot pigmented and photo-sensory cells en-
closing a simple, amorphous, lens. Nerve fibers arise basally
from the photo-sensory cells to connect up with the cerehro-
pleural ganglia. Such an eye structure is similar to that ot
Philobrya muiiita (Finlay, 1930) and of Pterin brevialnta
(Drinker, 1872) (Morton 1978, 1995).

PALLIAL EYES

Category 1: Pterioidea, Arcoidea, Limopsoidea,
and Anomioidea

Pterioidea: Pteria brevialata

Morton (1995) has described the mantle margin and
pallial eyes of Pteria brevialata (Pteriidae). Although the
mantle margin appears to comprise the usual bivalve three
folds (Yonge 1982), it does not. Instead, the outer mantle
fold is sub-divided into two sub-folds comprising (i) a spe-
cialized inner photo-sensory component on which are lo-
cated the pallial eyes (Category 1: Morton 2001) and (ii)
external to this an outer component that secretes the shell.
Both sub-folds are, therefore, overlain by the shell and two-
layered periostracum when the mantle is retracted and the
pallial eyes thus monitor light even through the periostra-
cum when the mantle margin is extended. The pallial eyes of
P. brevialata are among the simplest multi-cellular optical
structures yet described for the Bivalvia and comprise simple
photo-sensory cells located on the outer epithelium of the
inner component of the outer mantle fold and are backed on
the opposite, inner, epithelium by a patch of pigmented cells
(Fig. 4). Nerve fibers arise from the bases of the photo-
sensory cells and pass towards the pallial nerve.

PIGMENT CUP

Figure 3. A section through a cephalic eye of Aitlacomya atra.
Redrawn after Zaixso (2003).

Figure 4. A section through the simplest known pallial eye ot the
outer mantle fold of Pteria brevialata (Category 1). Redrawn after
Morton (1995).

Arcoidea and Limopsoidea

Representatives of the Arcoidea (and Limopsoidea) pos-
sess two types of pallial eyes, namely multi-faceted com-
pound eyes and simple pigment cups. Both types may occur
in the same species but both develop on the inner sub-fold
of the outer mantle fold and are, as a consequence, covered
by the periostracum even when the mantle margins are ex-
panded, as in Pteria brevialata described above.

Area noae

The mantle margin of Area noae Linnaeus, 1758 is il-
lustrated in section (Figs. 5 and 6B) (after Morton and Pe-
harda 2008). The pallial eyes of A. noae are of the compound
ommatidial type first described by Waller ( 1980, 1981 ), their
fine structure subsequently elucidated by Nilsson (1994).
Each, of many hundreds, compound eye of A. noae is located
on the inner sub-fold of the outer mantle fold and comprises
photosensitive cells surrounded by and intermixed with pig-
ment cells. In A. noae, the plump stalk and mantle beneath
each eye are also pigmented, perhaps enhancing their photo-
sensory efficiency.

Barbatia virescens

The second eye type seen in representatives ot the Arci-
dae (and Limopsoidea) is a pigment cup or invaginate eye.
One of the pallial eyes of Barbatia virescens (Reeve, 1844)
(Fig. 6A), also located on the inner sub-fold of the outer
mantle fold (Morton 1987), comprises a simple cup of in-
termixed photo-sensory cells and pigment cells. The cup

38

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

OPTIC NERVE

Figure 5. A section through the pallial eye of the outer mantle fold
of Area none (Category 1 ). Redrawn after Morton and Peharda
(2008).

mantle beneath the shell (Morton 1976, fig. 2) and, thus,
also the periostracum and have been described by Morton
(2001, figs. 8 and 9). Each adult individual possesses ~22
cups of pigmented retinal cells that have invaginated from
the inner epithelium of the general mantle surface. Above
each cup is a more structured, cellular lens that is formed on
the outer surface of the general mantle. There is also a cili-
ated accessory sense organ. It is unknown how this optical
structure has been assembled from either (i) an invaginated
cephalic eye pigment cup that has moved from its usual
position or (ii) has migrated inwards from the mantle mar-
gin. The occurrence with each eye of an accessory ciliated
sense organ is, however, unusual in that, as will be seen, such
a structure is typical only of Category 3 eyes (those that
occur on the inner mantle fold). Bearing in mind that E.
aenigmatica lies on its right shell valve, it seems possible that
the eyes have, in evolutionary terms, migrated inwards from
the left mantle lobe to lie under the left shell valve. From
which marginal fold, however, is unknown. The fact that the
eyes perceive light through the shell, however, places them in
Category 1 but they do constitute a unique photosensory
structure in the Bivalvia.

Figure 6. Sections through the simple cup-like pallial eye of A,
Barbatia virescens, (Redrawn after Morton 1987) and B, the omma-
tidial pallial eye typical of representatives of the Arcoidea (both
Category 1).

encloses an amorphous lens and the eye is thus similar to the
bivalve cephalic eye.

As will be discussed, the compound ommatidial eye
(Fig. 6B) might have evolved by evagination from the sim-
pler invaginate eye (Fig. 6A). For example, the limopsid
Philobrya munita (Finlay, 1930) has a pallial eye that is pos-
sibly intermediate between the inverted cup and compound
ommatidial eyes (Morton 1978, fig. 4).

Amvnioidea: Enigmonia aenigmatica

The pallial eyes of Enigmonia aenigmatica (Holten,
1803) (Anomiidae) are located, uniquely, on the general

Category 2: Limidae and Pectinidae

Limidae: Ctenoides tloridanus

The pallial eyes of Ctenoides floridamis (Olsson and Har-
bison, 1953) are located on the middle mantle fold and have
been described by Morton (2000a). One is illustrated in
section (Fig. 7A). Each eye comprises a cup of photo-sensory
cells surrounded laterally by pigment cells. Each sensory cell
of Lima scabra (Born, 1778) has been shown by Bell and
Mpitsos (1968) to possess bundles of cilia and basal neural
processes. A large optical nerve arises from the base of the
cup and there is a cellular lens. Interestingly, however, the .

Figure 7. Simplified sections through the pallial eyes (Category 2) |

of A, Ctenoides floridaiius and B, Pecten pusio. Redrawn after Mor- |
ton (2001) and Patten (1886), respectively. '

EVOLUTION OF BIVALVE EYES

39

eye is not closed to the sea, but is open, and an amorphous
plug of tissue occupies the space between retina and lens.

The importance of this invaginated eye is that it illus-
trates, as will be discussed, the manner in which bivalve
ectopic pallial eyes have changed their locations in the Pterio-
morphia from a specialized sub-fold of the outer mantle fold
(and thus beneath the shell and periostracum) to the middle
fold (and thus beyond the shell and periostracum).

Pectinidae: Pecten pusio

The eye of Pccteti pusio (Linnaeus, 1758) (Fig. 7B) has
been described by Patten (1886), and Dakin (1910, 1928)
and Morton (1980, 1993, 1996, 2000b) have described the
eyes of other pectinids, including species of Spondyhis (Lin-
naeus, 1758), Amiisiiim Roding, 1798, Leptopectcn Verrill,
1897, Minnivola Iredale, 1939, and Placopecten Verrill, 1897.
The eyes are located on the middle mantle fold with more on
the mantle margin of the upper right valve than the lower
left [see, for example, the description of Amusiiim pleuro-
nectes (Linnaeus, 1758) by Morton (1980)].

The pectinid eye is the most well-known bivalve optical
structure and has best been described for Pecten maximus
(Linnaeus, 1758) in a series of papers by M. F. Land (see
Morton 2001 for a review). This species and Pecten pusio
uses a lens to focus light onto a parabolic mirror of pig-
mented cells, called the argentea, and reflecting this back
onto the more distal of two, ciliary-based, retinal layers
(Land 1965) where it is capable of defining an image. The
proximal retina, on the other hand, detects changes in light
intensity (Land 1966). Each retina possesses cilia with a 9 -I-
0 structure but the proximal one will be responsible for
stimulating the typical escape response of free-living species
of the Pectinidae, for example, the accomplished swimmer
Amusium pleuronectes (Morton 1980).

Category 3: Myidae, Cardiidae, Tridacnidae,
and Laternulidae

Myidae: Mya arenaria

Light (1930) demonstrated for Mya arenaria Linnaeus,
1758 that photosensitive tissue is located in the inner surface
of the siphons, mostly at their tips, that is, the fused inner
mantle folds. He further identified using silver nitrate stain-
ing and described light sensitive cells each containing an
optic organelle composed of a large hyaline structure — the
lens — surrounded by a network of nerve fibers that he called
the retinella. Two of these cells are illustrated (Fig. 8).

Cardiidae and Tridacnidae: Cerastoderma edule and
Tridacna maxima

The pallial eyes of representatives of the Cardiidae have
been described by numerous authors, but the eyes of Cera-
stoderma edule (Linnaeus, 1758) are the most well-known

PHOTOSENSORY

Figure 8. Mya arenaria. A section through two siphonal photore-
ceptors. Redrawn after Light (1930).

(Barber and Land 1967, Barber and Wright 1968). In an
adult individual, -60 of them occur on the inner folds of
both mantle lobes each atop a siphonal tentacle. Each eye
comprises a cup of pigmented cells enclosing 12-20 receptor
cells. Each photo-sensory receptor cell has about 100 cilia,
each with a 9 -I- 0 filament content (Barber and Land 1967)
and with the optical nerve arising internally and departing
the eye at the apical junction of the cup with the lens. Joining
the optic nerve laterally is a nerve from a ciliated sense organ
of unknown function. It is probably, however, a mechano-
receptor. Weber ( 1909) and Braun ( 1954, fig. 5) showed that
the accessory sense organ of each pallial eye of Cardiuin
oblongum Gmelin, 1791 actually comprises two clusters of
cilia (Fig. 9). This is also true for the pallial eye of C. edule
(Fig. lOA).

Representatives of the Tridacnidae also possess pallial
eyes that are located as in the Cardiidae on the, albeit greatly
expanded, inner mantle folds. An adult Tridacna maxima
( Roding, 1 798) will have thousands of eyespots but they have
the same basic structure as those described (above) for rep-
resentatives of the Cardiidae. As well as fulfilling a visual
function, however, tridacnid eyes may focus light on masses
of zooxanthellae residing around them in the mantle hemo-
coel, prompting Yonge (1936) to refer to them as hyaline
organs (although still derived from true eyes). The light-
receiving properties of the array of tridacnid eyes has been
demonstrated by Stasek ( 1965) who showed that the shadow
reflex results in a giant clam squirting a jet of ‘aimed’ water
at any fish (or human) moving above it. The zooxanthellae
might also, however, function as an accessory light reflecting
pigment cup, enhancing the efficiency of the clearly photo-
sensory eye apparatus and thereby confirming the truly sym-
biotic relationship that exists between the bivalve and its
entrained phytoplankters.

40

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

CILIATED SENSORY NERVE

10 pm

Figure 9. A section through the accessory, ciliated, sense organ of
the pallial eye (Category 3) of Cardiimi ohlongum Gmelin, 1791.
Redrawn after Braun (1954).

image. Morton (1973) showed, however, that the extremely
sedentary infaunal L. tnincata, which is capable of only slow ‘
burrowing, possesses only a shadow reflex that results in
pallial tentacles attempting to flick sand grains over the ex-
posed siphons. Presumably, if the situation is similar to that |
seen in representatives of the Pectinidae, the proximal retina [
is responsible for detecting changes in light intensities and
thus stimulating the shadow reflex. The cells of each retina j
possess cilia that, again as in representatives of the Pectini- ,
dae, have a 9 + 0 structure (Adal and Morton 1973). |

As in Cerastoderma edide, each pallial eye of Laternula [
tnincata has an accessory sense organ except that in this
species the groups of -28 cilia with a 9 X 0 + 2 structure are )
long (50 pm) and contained within an invagination of a j
specialized tentacle (Adal and Morton 1973). Projecting j
from a pore at the apex of the tentacle, each cilium may ,
make contact with the microvilli of the surrounding epithe- i
hum (as the tentacle moves) and is, hence, probably a
mechano-receptor. The eyes of L. tnincata represent a re-
markable example of convergent evolution with those of 1
representatives of the Pectinidae.

CILIATED SENSORY

Figure 10. Simplified sections through the pallial eyes (Categoiy 3)
of Cerastoderma edide (after a number of authors) and Laternula
tnincata. Redrawn after Adal and Morton (1973).

Laterniilidae: Laternula truncata

The Anomalodesmata comprises the most family-rich
subclass of the Bivalvia and yet only representatives of the
Laternulidae, for example Laternula tnincata (Lamarck,
1818), have been shown to possess pallial eyes (Adal and
Morton 1973) (Fig. lOB). As in Cerastoderma edide, each of
the nine eyes sits atop a siphonal tentacle and is hence lo-
cated on the inner mantle fold. As in representatives of the
Pectinidae, for example Pecten maxiimis (Dakin 1910), there
is a cup of pigment cells forming a parabolic mirror, or
argentea, which encloses a double-layered, proximal and dis-
tal retina. The lens focuses light onto the argentea, which
reflects it back onto the distal retina. In such a way, it is
possible, as in representatives of the Pectinidae, to form an

DISCUSSION

Gaten (1998) suggested that superimposition eyes,
which re-direct light from many facets onto the target rhab-
domeres, have evolved only once in the crustacean Deca-
poda, probably in the Devonian (345-395 mya). Morton
(2001) similarly argued that in the Bivalvia, cup-like, cephal-
ic eyes have probably also only evolved once but have been
either retained by or developed in representatives of only a
few (generally older) phylogenies (see above). Morton
(2001) also showed, however, that, conversely, ectopic pallial
eyes have evolved a number of times in various lineages of
the Bivalvia and, typically, in different ways, notably with
regard to their positions on the various folds of the mantle
margin.

The sizes (diameters) of various bivalve pallial eyes are
illustrated (Fig. 11) in relation to their category. What is
obvious is that there is no clear relationship between eye
category and size. That is. Category 1 eyes are all <-800 pm
in diameter whereas Category 2 eyes all range in diameter
between 100 pm and <1,000 pm. Category 3 eyes, however,
are also only -100 pm in diameter. Size does not seem to be
an indicator of structural and, hence, optical complexity
even though the pallial eyes of Pecten maximiis (a large scal-
lop) are among the largest and most sophisticated bivalve
optical structures. Moreover, the pallial eyes of Laternula
tnincata are only 100 pm in diameter but are as morpho-
logically complex as those of P. rnaxinnis (Adal and Morton

I

)

1980).

EVOLUTION OF BIVALVE EYES

41

>

Qi 2
O
O
LU
h-
<

O 1

<•

Latemula

Cerastoderma

• • • •

Ctenoides Patinopeclen

Lima Pecten

cm 9 • •

Glycymeris Enigmonia
Isognomon Area

PhUobrya
Barbatia

100 1,000
EYE DIAMETER (pm)

2,500

Figure 11. The pallial eye diameters of bivalves which are known to
possess them and which have been divided into the three structural
categories of Morton (2001). Note; x-axis scale is not uniform.

Furthermore, the eyes of representatives of the Tridac-
nidae (Fig. 11) are ~2.5 mm in diameter and hence about 25
times the size of those of their nearest relatives, the cardiid
cockles. Although giant clams are the largest extant bivalves,
their pallial eyes also have the same basic structure as rep-
resentatives of the Cardiidae. It is, therefore, possible that
tridacnid eyes are larger because, as well as fulfilling a visual
function, they focus light on masses of zooxanthellae resid-
ing around them in the mantle hemocoel (Fankboner 1981,
Wilkens 1986). The zooxanthellae might also, however,
function as an accessory light-reflecting pigment cup. Ex-
cluding, therefore, as a special case, the hyaline organs/eyes
of the Tridacnidae, it seems (Fig. 11) that all bivalve ectopic
pallial eyes are of about the same size, probably in relation to
the size of the individuals that possess them. I’hompson
(1942: 34-35) noted for a number of vertebrates that:

“A big dog's eye is hardly bigger thau a little dog's; a
squirrel’s is much bigger, proportionately, than an
elephant’s; a robin’s is but little less than a pigeon's or
a crow’s."

Hence, as with Thompson’s vertebrate examples, size alone
is not an indication in the Bivalvia of either structural com-
plexity or visual acuity. Nevertheless, the fact that some bi-
valves, for example representatives of the Arcoidea, Pectini-
dae, and Tridacnidae, possess so many pallial eyes suggests
that they are highly important in another way. First, how-
ever, what controls pallial eye development?

The Sixl/2/so and Six3/6/7/9/optix groups of genes are
implicated in eye and sensory structure development. In
Drosophila, so is associated with the eye marginal disc and
Bolwig’s organ, and the expression of so in the un-patterned
epithelium is required for eye morphogenesis and develop-
ment of the visual system (Cheyette et al. 1994). A mutation
in the so homeobox disrupts the larval visual system of Dro-
sophila and abolishes the larva’s response to light (Flassan el
al. 2000). The expression of so is regulated by eyes absent

{eya) (Bonini et al. 1997) and the expression of both is
required to induce ectopic eyes (Haider et al. 1998). How-
ever, Gehring and Ikeo (1999) considered Pax6 and its ho-
mologs to be the control gene for eye development in meta-
zoans although it is now known that a number of others
(including eya), for example, dachshund (Shen and Mardon
1997) and sine oculis (Pignoni et al. 1997) are also able to
induce ectopic eye expression in Drosophila. Serb (2008, fig.

1 ) provides a model of the network of genes that regulates
eye formation in Drosophila.

In the case of the Bivalvia, however, the recovery of so
sequences from Nutricola tantilla (Gould, 1853) and Cras-
sostrea gigas by Bebenek et al. (2004) suggests that the ability
to develop ectopic eyes is present in both taxa but neither
has produced them, possibly because either eya or the other
genes identified above are not present. Such a generalization
might apply to the vast majority of bivalves (Fig. 12). How-
ever, Light (1930) appears to be the only person who has,
once determining that Mya arenaria does have a shadow
reflex, demonstrated, using silver nitrate staining, the pres-
ence and described the structure of intra-cellular light sen-
sitive bodies mostly in the tips of the siphons. Since it is
known that most shallow-water marine, estuarine, and
freshwater bivalves do possess a shadow reflex, perhaps it
would be beneficial if they too could be examined in more
detail prior to arguing that eya and/or the other genes now
associated with ectopic eye development (in Drosophda) are
not present.

As described by Nilsson (1994), for representatives of
the Arcidae, and reviewed by Morton (2001 ), the pallial eyes
of those bivalves that possess them, regardless of their struc-
tural complexity, are analogous to photoreceptive burglar
alarms that are placed on the outsides of (human) buildings.
That is, although no image is discerned, only simple spatial
movement, the efficiency of this optical sensory system has
been refined in each of the three categories of eyes. How?

In the Arcoida, pallial eyes are restricted to species that
inhabit shallow waters so that the deep-water Bathyarca pec-
tunculoides (Scacchi, 1833) does not possess them (Morton
1982). The pallial eyes of Barbatia virescens and Anadara
notabilis (Roding, 1798) are simple ciliated pits (Morton
1987, Nilsson 1994) whereas the compound ommatidial eyes
of Area noae conform in structure to those of other species
of Area (Waller 1980, 1981) and Barbatia cancellaria (La-
marck, 1819) (Nilsson 1994). Since the pallial eyes of rep-
resentatives of the Arcoida (Category 1) comprise pigment
cups which lack a well-defined lens and the cavity is filled
with rhabdomeric microvilli arising from the receptor cells
(Nilsson 1994), there are no cilia so that there is no base
structure present for the evolution of a ciliary-based retina.
Notwithstanding, an arcid compound ommatidial eye can be
derived by evagination of a pigment cup (as illustrated in

42

AMERICAN MALACOLOGICAL BULLETIN 26 - 1/2 • 2008

{Z

Polyplacophora

Cephalopoda

Scaphopoda

Gastropoda

Solemyidae

Nuculidae

Trigoniidae

Margaritiferidae

Unionidae

Arcoida

Mytilidae

Pterioidea

Ostreoidea

Pinnidae

Ltmoidea

Plicaculoidea

Anomioidea

Pectinidae

Spondylidae

Carditidae

Astartidae

Nuculanoidea

Thyasiridae

Fimbriidae

Lucinidae

Galeommatidae

Lasaeidae

Sportellidae

Pandoroidea

Cuspidariidae

Gastrochaenidae

Pharidae

Hiatellidae

Donacidae

Semelidae

Sphaeriidae

Chamidae

Cardiidae

Petricolidae

Glossidae

Veneridae

Corbiculidae

Vesicomyidae

Arcticidae

Ungulinidae

Mactridae

Pholadidae

Corbulidae

Myidae

Dreissenidae

Teredinidae

Xylophagidae

Palaeoheterodonta

Pteriomorphia

Euheterodonta

Figure 12. A phylogenetic
tree of the Bivalvia based on
Giribet and Distel (2004, fig.
3.6), showing the occurrence
of cephalic eyes (stars) and
ectopic pallial eyes belonging
to Categories 1 (Arcoida,
Mytilidae, Pteriodea, Anomio-
dea), 2 (Limoidea, Pectinidae,
Spondylidae) and 3 (Pando-
roidea, Cardiidae, Myidae).

Fig. 6) to create a structure that (i) has a greater breadth of
view and which could (ii) but only in theory, produce a
‘summed’ image (Dawkins 1997). The limopsid Pljilobrya
niimita has a pallial eye that seems to be intermediate be-
tween the inverted cup and compound ommatidial eyes in
that like the latter it is everted although pigment and photo-
sensory cells are not inter-mixed (Morton 1978).

As Morton (2001 ) pointed out, however, no bivalve has
any kind of brain that could ever recreate within it an image,
regardless of the sensory sophistication of the eye that re-
ceives the enhanced differences in light associated with such
improvements. The paired optic lobes of the cerebral ganglia
of Pecten maximiis are relatively ‘large’ for a bivalve (see
Dakin 1909, plate 6, fig. 28) but nowhere near as large as

EVOLUTION OF BIVALVE EYES

43

those of cephalopods, for example. Octopus vulgaris Cuvier,
1797 (see Wells 1968, fig. 9.7; Hanlon and Messenger 1996,
fig. 2.8) and which possess some 65 million nerve cells
(Young 1971) involved not only in visual analysis but also
act as visual memory stores.

Moreover, the fact that some bivalves, notably represen-
tatives of the Arcoidea and Pectinidae, possess so many pal-
lial eyes along the mantle margin suggests that the eyes are
highly important. In what way? In the case of the Arcidae,
the presence of many compound eyes may allow tor the
detection of movement, as each is stimulated in succession
by a moving object (Nilsson 1994). The same probably ap-
plies to the parallel circlets of eyes along the left and right
mantle margins of scallops.

However, because Category 1 eyes develop on the outer
mantle fold under the shell and periostracum, what changes
in light they detect must be of poor visual c|uality. They,
thus, perceive only a passing shadow. A human eye with a
lens cataract might provide an analogy. This is not so, how-
ever, in those bivalves that developed pallial eyes on either
their middle (Category 2) or inner (Category 3) mantle folds
since they develop beyond the shell and periostracum. The
increased visual acuity associated with such ‘external’ loca-
tions has been enhanced further by increases in structural
complexity. How might this have been achieved?

In the Limidae and Cardiidae, there are ciliated pallial
tentacles or accessory ciliated sense organs, in the former
and latter, respectively, located upon specialized sub-folds of
the middle or inner mantle fold, again respectively. Repre-
sentatives of those two only distantly related bivalve families
with the most sophisticated retina-based eyes, that is, the
Laternulidae and Pectinidae both possess a ciliary-based
proximal retina (Barber and Land 1967, Bell and Mpitsos
1968). It seems possible, however, that invagination of either
an adjacent ciliated sensory tentacle or accessory organ, re-
spectively, and incorporation of such a structure into these
eyes might explain how the ciliary-based distal retina has
been developed in both of them (as illustrated in Figs. 7 and
10, respectively). This would further explain how the double
retina eyes of representatives of both families have evolved.
They are, hence, also, an example of convergent evolution.

It seems possible, therefore, that the duplication of
structures, in these cases either sensory tentacles or accessory
organs, opens up the potential not only for new structures to
evolve but also to alter and enhance functions. Hence, the
original proximal retina was and still is responsible for de-
tecting changes in light intensity and thus stimulating the
shadow reflex. However, the distal retina, derived from an
invaginated ciliated accessory structure, provides the poten-
tial for image formation and the detection of movement in
representatives of the Pectinidae and Laternulidae. In the
absence of optic lobes capable of synthesizing such informa-

tion, however, these complex eyes must await matching ce-
rebral sophistication.

The Arthropoda provides a well-known example of how
the duplication of appendages has facilitated not just ambu-
latory but also mandibular diversification resulting in the
extraordinary adaptive radiation of the phylum’s numerous
representatives and, hence, also of their eyes (Oakley 2003).
In the Bivalvia, pallial fold duplication has resulted in im-
provements to the total array of peripheral pallial senses,
most notably, however, the visual one. Improvements to the
pallial visual senses have, however, occurred at different
times in different phylogenies and on different components
of the mantle margin. This has been achieved through (i)
selective gene-induced ectopism; (ii) pigment-cup evagina-
tion in Category 1 eyes; (iii) invagination of accessory struc-
tures in some eye Categories 2 and 3 to form a distal retina
in representatives of the Pectinidae and Laternulidae; and
(iv) natural selection.

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Submitted: 7 July 2007; accepted: 24 June 2008; final

revisions received: 23 September 2008

Amer. Maine. Bull. 26: 47-66 (2008)

Understanding the cephalic eyes of pulmonate gastropods: A review"^

Marina V. Zieger and Victor Benno Meyer-Rochow

Jacobs University Bremen, School of Engineering and Science, Research II, Camps Ring 6, D-28759 Bremen, Germany,
marinazi@umich.edu

: Abstract: This review showcases one group of gastropod’s ability to perceive light through the eyes. The central c]iiestion is simple: what
are the visual performances and tasks of cephalic eyes in gastropods? That topic in itself is rather broad and is here applied to pulmonate
gastropods, coming from terrestrial and aquatic biomes as well as different habitats and microhabitats, exhibiting different life-styles and
light-tolerances. Therefore, the main objectives have been to analyze (1) anatomical and ultrastructural eye characteristics, (2) optical
systems, (3) image-forming capabilities and possible functional consequences of eye size and design, (4) interactions between gastropods
and their environment mediated by the visual information obtained through the eye, and (5) the specific visual tasks that the eyes serve.
During the course of this study, a range of variations (= adaptations) in both optical and retinal design parameters, including eye size,
aperture size, quality of optical image, retinal shape, sampling density, and optical sensitivity were discovered. All species ot pulmonate
gastropods studied have paired simple camera-type eyes that operate with advanced fixed focal-length optics. However, in terrestrial snails
and slugs as well as freshwater limpets, the optics cannot produce a focused image on their shallow retinas. This seems to indicate that eyes
in these species are not designed to receive a focused image and are likely to measure only the average light intensity or quality over large
angles rather than resolve fine image details. The aquatic snails examined are able to focus a sharp image on the photoreceptive layer of
the retina due to the deepenings of the latter (at least in a localized region). Although there is a significant correlation between specialization
of the eye (e.g., quality of optical image, sensitivity, and resolution) for a particular visual task in a specific habitat that the species
encounters, there is no correlation between cellular composition of the retina and light/dark preferences. Their high optical sensitivity allows
terrestrial snails to perform the necessary visual tasks in both bright and dim light, whereas the eye in aquatic species functions preferentially
under bright light conditions. In conclusion, pulmonate gastropods use their eyes primarily for the following two kinds of visual tasks: ( 1 )
discriminating objects and possible enemies in their environment and (2) monitoring the environmental brightness level to orient towards
dark places. The first type of visual task is characteristic ot the aquatic snails and is served by image-torming eyes; the second is typical of
terrestrial snails and slugs and is best served by a blurred image. Attention is given to visual ecological adaptations, specific visual needs,
and the evolutionary history of gastropods.

Key words: vision, eye optics, visual behavior, retina, molluscs

The literature dealing with invertebrate eyes is extensive
and especially with regard to molluscs, includes excellent
reviews on photoreceptor evolution (Eakin 1968, Salvini-
Plawen and Mayer 1977, Vanfleteren 1982), comparative
anatomy and optics (Land 1981, 1984, Land and Fernald
1992), and phototransduction and physiological mecha-
nisms (Messenger 1981, 1991, Nasi et al. 2000). The most
recent books by Land and Nilsson (2002) and Warrant and
Nilsson (2006) represent a comprehensive account of all
known types of eye and provide an up-to-date synthesis of
our current knowledge of invertebrate vision. However, even
in these latest publications, pulmonate lineages, reviewed
more than 30 years ago by Kerkut and Walker (1975), are
somewhat under-represented. Moreover, most of the con-
clusions on the importance of eyes and vision in pulmonates
stem from just a handful of species, and of them the terres-
trial snails belonging to the genus Cornu (Born, 1778) alone
have attracted the bulk of attention. This, no doubt, was the

result of the sustained, detailed, and elegant anatomical and
ultrastructural examinations of the Cornu aspersum asper-
sum (Muller, 1774) eye by Eakin and Brandenburger and
their colleagues (e.g., Eakin and Brandenburger 1967a,
1967b, 1970, 1975a, 1975b, 1982, Brandenburger and Eakin
1970, Eakin and Eerlatte 1973, Brandenburger 1975, Bran-
denburger ct al. 1976) as well as some early electrophysi-
ological studies by Berg and Schneider (1967), Gillary
(1970), Basic et at. (1974), and Berg (1978). The present
review deals with the cephalic eyes of gastropods generally,
and in pulmonates in particular, focuses on eyes of various
sizes, differences in retinal design, and the extent to which
their optical ec]uipment permits image formation. Some of
our recent observations, together with those of other re-
searchers, will be briefly summarized.

For more than fifteen years we have been studying the
eyes of pulmonate gastropods, and in this paper we shall
review what is known of the morphology and ultrastructure

From the symposium “Molluscan models: Advancing our understanding of the eye” presented at the World Congress of Malacology, held
from 15 to 20 July 2007 in Antwerp, Belgium. Co-sponsored by the National Science Foundation and the American Malacological Society.

47

48

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

of their retinal cells. We shall discuss recent comparative
anatomical and ultrastructural findings and results based on
behavioral tests and investigations that have involved optical
modeling. We will evaluate some of the new ideas that arose
from analyses of morphological and ecophysiological adap-
tations to terrestrial and ac|uatic ways of life and will give
possible reasons for the large variety of retinal designs and
optical systems one encounters in the pulmonate eye. Special
emphasis is given to the research of the authors and their
associates.

ANATOMY

With the exception of a few subterranean stylommato-
phoran forms (e.g., Cedlioides acicula (Muller, 1774) and
Helicoiliscus singleyanus (Pilsbry, 1889) as well as some blind
Ellobiacea (Grasse 1948), all pulmonates have a single pair of
eyes located on the head. The position of the eyes in relation
to the tentacles is frequently employed as a taxonomic cri-
terion. In stylommatophoran land snails and slugs, as well as
in systellommatophoran marine slug-like pulmonates, eyes
are developed at the tips of the mobile, retractile tentacles. In
freshwater basommatophoran snails and limpets, the eyes
are located medially or laterally at the base of a single pair of
mobile tentacles close to the cerebral ganglion. Each cephalic
eye consists of a cornea, a lens, a vitreous body, a non-
inverted retina, the eye capsule, and the optic nerve.

The plane of the bilateral symmetry of the cephalic eyes
coincides with the dorso-ventral plane of the heads in Lym-
iiaea stagnalis (Linnaeus, 1758), Radix peregra (Linnaeus,
1758), Planorharius corneiis (Linnaeus, 1758), and Physa foii-
tiualis (Linnaeus, 1758) and with the ventro-lateral plane in
species with an accessory retina; no preferred plane is de-
monstrable in the remainder of the terrestrial pulmonates.

Integument

In all of the investigated pulmonates the eyes are placed
under a circular area of a specialized region of the integu-
ment, that at this location is thin, unpigmented, dome-
shaped, and translucent. The function of the integument
layer may be a protective one, reducing possible mechanical
injury and desiccation, the latter presumably of importance
for terrestrial and some aquatic molluscs that lead moder-
ately amphibious lifestyles. The integument may also be in-
volved in regulating osmotic and ionic processes, since
aquatic species in particular have to continually osmoregu-
late, while terrestrial gastropods need to minimize water loss.

Perioptic sinus

Willem (1892) described a spacious blood lacuna, the
perioptic sinus, from the region around the eye of Lymnaea
stagnalis. Later, Stoll (1973) confirmed Willem’s observa-

tions in the same species and Bobkova et al (2004a) de-
scribed the perioptic sinus in Radix peregra, Physa fontinalis,
and Planorharius corneas. The observations showed that the
sinus is anatomically isolated from the rest of the hemocoel
and connected to it only through the interstitial tissue.

It is well known that sinuses, located in strategic regions,
may become cavities of the hydrostatic skeleton, with blood
serving as the hydrostatic fluid. In terrestrial species, how-
ever, the situation is different; the blood cavity in the re-
tractable optic tentacles together with the hemocoel may
function as a hydraulic rather than a hydrostatic system. We
suggested (Bobkova et al. 2004a) that freshwater basom-
matophora actively place their eyes into the perioptic sinus,
firstly, in order to fix their rigidly-held eyes in a particular
position with regard to the body, and, secondly, to minimize
the risk of possible mechanical compression when the ani-
mal withdraws its body into the shell. However, limpets like
Latia neritoides (Gray, 1850) and Ancylus fluviatilis (Muller,
1774) apparently do not have the perioptic sinus (Meyer-
Rochow and Bobkova 2001), but at the same time, these
species have cap-like shells and do not have to compress
their body as much as, for example, Lymnaea stagnalis.

Basal lamina and eye capsule

The retinal cells rest on a basal lamina. The lamina
separates the retina and eye capsule as well as the cornea and
interstitial tissue or, as in some aquatic species, cornea and
lumen of the perioptic sinus. The basal lamina is continuous
throughout its length and of constant thickness. Both the
basal lamina and eye capsule continue into branches of the
optic nerve. Along the course of the nerve, the capsule is
then gradually replaced by a sheath of glial cells as, for ex-
ample, in Lymnaea stagnalis (Bobkova 1998).

The eye capsule consists mainly of striated collagen
fibrils and a layer of muscle cells. The muscle cells are em-
bedded circumferentially (except for the area of the cornea)
into an amorphous matrix to which they are attached by
hemidesmosomes [e.g.. Cornu aspersum aspersum: Eakin and
Brandenburger 1972; Lymnaea stagnalis: Bobkova 1998).
The muscles are innervated by fine neurites, containing
cored vesicles. The source of these neurites has still not been
dearly determined. Recently it was demonstrated by immu-
nohistological means that the eye capsule receives serotoner-
gic innervation (Zhukov and Trichina 2008).

Retinal design

As a rule, the eyes of all pulmonates studied to date
possess conventional, cup-shaped retinas (e.g., Cepaea
nemoralis (Linnaeus, 1758) and Trichia hispida (Linnaeus,
1758): Fig. lA-B). The eyes of some freshwater pulmonates
such as Lymnaea stagnalis, Radix peregra, Physa fontinalis,
and Planorharius corneas have retinas that are portioned into

THE PULMONATE EYE

49

!

I

Figure 1. Light micrographs of longitudinal sections of camera-
type eyes of various designs in pulmonate gastropods. (A) Cepaea
nemoralis, (B) Trichia hispida, (C) Lyimmea stagnalis, (D) Deroceras
agreste, (E) Achatina fulica, and (F) section through lens of addi-
tional eye in Deroceras agreste. AL, additional lens; AR, additional
retina; CO, cornea; L, lens; ON optic nerve; R, retina. A, B, and C
from Bobkova et al. 2004a; D and F from Bobkova et al. 2004b with
permission of lohn Wiley and Sons, Inc.

dorsal and ventral depressions (termed “pits”) (Fig. 1C). The
pits are separated by an internal ridge, called a “crest”, and
based on their pigmentation, can be seen in vivo (Bobkova et
al. 2004a). Of all non-pulmonate gastropods, only the opis-
thobranch Navanax if tennis (Cooper, 1863) possesses an eye
with a “bi-lobed” lens and a non-hemispherical retina (Eskin

and Harcombe 1977). However, the c]uestion of whether this
lens can produce an image on the retina has not been ex-
amined. Four distinct retinal layers in the eyes of gastropods
can be distinguished, namely the microvillar, the pigmented,
the somatic, and the plexiform layer.

Additional photoreceptive structures

Some pulmonates have a double eye. The additional
photosensory organ was first described by Henchman ( 1897)
in the slug Umax maximus (Linnaeus, 1758). The terrestrial
snail Achatifia fiilica (Ferussac, 1821) also has an additional
retina (termed “accessory” by Tamamaki and Kawai 1983),
invariably equipped with its own lens and an anatomically
separate retina from that of the main eye (Fig. IE). In Umax
flavus (Linnaeus, 1758), Tamamaki (1989) found traces of a
lens and a discontinuity of the two optic cavities. Therefore,
we suggest calling the structure in question “an additional
eye” rather than an “additional retina” or “accessory retina”.
Moreover, we have demonstrated that the cornea and the
additional eye with its own nerve in the snail A. fulica may
regenerate separately from the main eye (Bobkova et al.
2004b). But what we do not know is if such a regenerated
structure is a functional organ.

The slug Agriolimax reticidatiis (Muller, 1774) (Newell
and Newell 1968) possesses an additional retina but lacks the
additional lens, while Deroceras agreste (Linnaeus, 1758),
also a slug, possesses both an additional retina and an extra
lens (Fig. ID, IF). The additional lens can easily be isolated
from its cavity. The lens is irregularly shaped and the vitre-
ous body of the additional retina is continuous with the
vitreous body of the main eye in Deroceras agreste (Zieger et
al. 2008). Thus, we accept the term “additional retina” for
species of pulmonates in which a vitreous body, associated
with the additional retina, is continuous with vitreous body
of the main eye, but we use the designation of “additional
eye” when a discontinuity ot the two optic cavities is present.

On the basis of the work by Tamamaki and Kawai
(1983) on the eye of Achatina fulica, Newell and Newell
(1968) on Agriolimax reticulatus, Tamamaki (1989) on
Umax flavus, and us on Deroceras agreste (Zieger et al. 2008),
it has become clear that the cellular composition of the
additional retina is similar to that of the main eye. However,
there are no screening pigment granules in the supportive
cells of the additional retina. Neurosecretory cells were ab-
sent as well. The photoreceptor cells in the additional retina
are large and their dome-shaped apices bear well-organized
(regular), long microvilli. Accumulations of photic vesicles
are also present, but the latter are not as tightly packed as
those of the photoreceptors of the main retina. The micro-
villi were observed to be aligned in perpendicular orienta-
tions (Fig. 2), but we could not provide any morphological
evidence for the view that the perpendicularly oriented mi-

50

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Figure 2. Electron micrograph of additional retina in Deroceras
agreste. The inset shows a transverse section through the presum-
ably light-sensitive microvilli. CO, corneal cells; Ph, photoreceptor
cells; Vb, vitreous body. From Zieger et al. 2008 with permission of
lohn Wiley and Sons, Inc.

crovilli might belong to neighboring (different) photorecep-
tor cells (Zieger et al. 2008).

As to the function of the additional retina, two theories
have been propaosed: (1) percejation of changes in the inten-
sity of the ambient light and (2) percejation of infrared ra-
diation. The latter could not be confirmed experimentally,
but it has not been possible either to demonstrate that the
additional eye is a visual organ at all. Moreover, the lack of
screening pigment permits light to reach the presumed pho-
toreceptor cells from any direction, making image formation
impossible. However, Newell and Newel (1968), observing
the behavior oi AgrioUmax reticulatiis in a state with partially
retracted optic tentacles, concluded that the additional eye
would become exposed to light when the aperture of the
main eye becomes fully masked by the pigmented integu-
ment. Therefore, the additional eye may be useful in situa-
tions like the partially retracted tentacle (Tamamaki 1989).

The hypothesis that in Deroceras agreste the additional
retina could play a role as a sensor of polarized light had to
be rejected as a consequence of carefully conducted behav-
ioral tests (Vakoliuk 2005). Although we did not evaluate the
opitical system of the additional retina in D. agreste, we are
nevertheless convinced, for reasons explained above, that it
cannot form an image, even if it should turn out to be a
functional light sensor.

The plexiform layer

The plexiform layer in all pulmonates studied to date is
formed by axons of both photoreceptive and neurosecretory
cell types (e.g., Brandenburger 1975, Eakin and Branden-
burger 1975b, Kataoka 1975, Eakin et al 1980, Katagiri et al.

1995, Bobkova 1998, Meyer-Rochow and Bobkova 2001,
Bobkova et al. 2004a). As shown for Lymnaea stagnalis
(Bobkova 1998), the axons of the photorecepitor cells may be
joined by synapse-like contacts of two morphologictil types.
The first we call “invagination”, for in this typse the mem-
brane of one axon drives a narrow fmger-like protrusion
into the other axon. Such contact seems to be very effective,
as in the case of sensory cells in vertebrate electroreceptors of
the “Lorenzini Ampoule” in skates (Byzov 1994). The sec-
ond inter-axonal contact type in gastropods belongs to the
flat “cH passant" type.

The ultrastructure of synapses in the plexiform layer of
the eye in Lymnaea stagnalis appears similar to the structure
in the gastropod central nervous system, from studies in the
snail Achatina fulica, it appears that there are at least two
morphological types of synapses: (1) asymmetrical, or po-
larized, synapses with vesicle aggregation on one side of the
active zone and (2) symmetrical synapses, with vesicle ag-
gregations and pre- membranous densifications present on
both sides of the synaptic cleft, thus indicating bi-directional
transmission across the junction (cf , review by Chase 2002).

We suggest that contacts of both types can possibly
provide summation of signals from groups of neighboring
photoreceptors. Summation increases the number of pho-
tons received per channel during periods of low luminance.
Such a strategy is employed by several other animals but
only at the expense of spatial resolution (Seyer et al. 1998).

Organelles of great interest in the eye of Lymnaea stag-
nalis are the numerous small, clear, and dense vesicles. They
are abundant close to the photoreceptor axonal membrane
in the extracellular space (Bobkova 1998). Morphologically
identical pictures of them were taken from the extracellular
space in the CNS of Cornu aspersum aspersum (Chalazonitis
1971) where such vesicles were associated with exchanges of
macromolecules between adjacent neuronal axons, axons
and glial cells, and neurons and axons. We presume that the
axons of the photoreceptor and possibly neurosecretory cells
may have a metabolic link at the level of the plexiform layer.
This process might be related to cell respiration because
vesicles have been observed only in the peri-membrane por-
tion of the axoplasm and in the intercellular spaces. The
osmiophilic material in the vesicles can be one of the meta-
bolic products of respiratory pigments like hemocyanin,
which is known to function extracellularly.

Connections to the central nervous system

The axons of the retinal photoreceptors pass out of the
eye by way of the optic nerve and form connections with
second order neurons in the cerebral ganglion of the central
nervous system. As demonstrated for Lymnaea stagnalis and
Planorbarius corneas, afferent information from the eyes is
widely distributed throughout the CNS. Nerve fibers, start-

THE PULMONATE EYE

51

ing in the ipsilateral optic nerve were traced through the
cerebral ganglion to the contralateral optic nerve, suggesting
tight interactions between the two eyes (Zhukov and
Trichina 2008). Optic tract projections were found to end on
the hair cells of the statocyst in Lymuaea stagnalis. The ani-
mal initially responds to light with phototactic behavior and
moves towards the light, but in response to mechanical tur-
bulence it clings to the surface. Such paired visuo-vestibular
conditioning results in conditioned escape behaviors (cf.,
review by Sakakibara 2006).

Efferent innervation of the eyes occurs as well. Efferent
fibers from the CNS form a varicose plexus within the eye
capsule in Lymnaea stagnalis and Planorharins corneas
(Zhukov and Trichina 2008). Serotonergic efferent fibers in-
fluence the amplitude of the electroretinogram and change
absolute sensitivity to light in Lymnaea stagnalis (Zhukov et
al. 2006).

OPTICAL SYSTEM

The optics of the eyes of gastropod molluscs vary greatly
and range from a pigmented pit without a lens to sophisti-
cated eyes with hard spherical lenses and image-forming
capacity (Land 1984). According to Messenger (1981), the
advanced cephalic eyes present in pulmonates can be clas-
sified as a “closed vesicle, camera-type eye”, capable ot form-
ing an image on the retina (Nilsson 1989). However, such a
categorization suggests a certain degree of homogeneity
within the taxon but provides little information on possible
modifications and/or variations of the optical system as well
as the visual adaptations seen in gastropods.

Based on numerous pulmonate species examined by us
( Bobko va et al. 2004a, 2004b), we now conclude that the
positions or shapes of the lenses have evolved to become
optimized in different environments as a response to the
prevailing light conditions. Muscular or other connective
tissues attached to the lens were not seen, and no kind of
membrane or sheath delineating the lens appears to have
developed. For the terrestrial snail Cornu aspersnm aspersam
it has been suggested that the shape of the eye could be
changed through the actions of the musculature of the eye
capsule and associated capsular strands (Mortensen and
Eakin 1974). However, no experimental proof for this view
has ever been presented. Based on our own observations,
Bobkova et al. (2004a) concluded that pulmonates have a
fixed focal-length optical system and a total absence of ac-
commodative ability. The main components of the optical
system in all of the pulmonates studied until now are the
cornea and the lens.

Cornea

The convex-concave cornea represents the most ante-
rior part of the eye vesicle. In terrestrial snails it is much

thicker than that of the aquatic species (e.g., in molluscs with
comparable eye sizes like Trichia hispida and Radix peregra,
the thickness of the cornea at its center is about 1 3 and 3 pm,
respectively) (Bobkova et al. 2004a). Yet, despite the differ-
ence, comparisons of the corneae of the eyes of terrestrial
{e.g., the slug Umax flavns: Kataoka 1977, the snail Achatina
fnlica: Bobkova et al. 2004b) and aquatic pulmonates (e.g.,
the snail Lymnaea stagnalis: Stoll 1973, Bobkova 1998, the
slug Onchidium verrncnlatnm (Cuvier, 1830): Katagiri and
Katagiri 1998, the freshwater limpets Ancylus fluviatilis and
Latia neritoides: Meyer-Rochow and Bobkova 2001) have
revealed that the corneae are structurally similar to each
other (Fig. 3A-B). We believe that the difference in thick-
nesses reflects the degree to which protection of the eye,
for example in terrestrial snails against desiccation, is at a
premium.

The cornea is composed of a monolayer of elongated,
flattened, and iti vivo transparent cells, reaching from the
basal lamina to the optical cavity. The cells in aquatic species
are strongly interdigitating. The inner surface of the corneal
cells forms short irregular microvilli that are oriented toward
the lens. The nuclei are situated in the basal cytoplasm. The
flaky but electron-translucent cytoplasm of the corneal cells
contains a few mitochondria, glycogen particles, rough en-
doplasmic reticulum, free ribosomes, and, directed toward
the vitreous body, secretory vesicles filled with electron-
dense granular material (Fig. 3A-B). The corneal cells are
laterally connected with each other and with the most ante-
rior retinal cells at the end of the cornea by septate junctions
and zonidae adhaerentes. Basally they are anchored to the
basal lamina by hemi-desmosomes. Corneal cells, together
with retinal supportive cells, are known to contain secretory
vesicles. The electron-dense granular content of the vesicles
closely resembles that of the vitreous body and the lens.

Figure 3. Electron micrographs of approximately longitudinal sec-
tions through the cornea: (A) Dcroccras agrcstc, (B) Radix peregra
and section through lens and cornea in (C) Lymnaea stagnalis. CO,
cornea; L, lens; Nco, nucleus of corneal cell; VB, vitreous body;
Arrows, secretory vesicles.

52 AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008 |

I

Therefore, the corneal cells are thought to be involved in the
genesis of lens material {e.g., in Cornu aspersmn aspersum:
Eakin and Brandenburger 1967a; Umax flavus: Kataoka
1977; Lymnaea stagnalis: Stoll 1973, Bobkova 1998; Ariou
riifiis (Linnaeus, 1758); and Deroceras agreste: Zieger et al
2008). Blumer (1995) described the transport of electron-
dense material toward the lumen in juvenile marine non-
pulmonate gastropods.

In an aquatic environment the cornea is not very useful
as a refracting interface, and any necessary refraction there-
fore depends on the properties of the lens alone. Obviously,
we have to be mindful of the fact that the cornea, if in air,
shortens the focal length of the optical system, but even so,
it has earlier been shown that in the terrestrial snails Cepaea
nemoralis and Trichia hispida only the lenses play a signifi-
cant role as the refractory surfaces (Gal et al. 2004). We
therefore assume that the lens is the main element of the
optical system not just in the aquatic, but in terrestrial spe-
cies as well.

Lens

Gastropod eyes possess an “acellular” lens (Eakin 1972)
and a vitreous body that shares certain features with the lens.
Freshwater snails like Lymnaea stagnalis. Radix peregra,
Physa fontinalis, and limpets like Ancylus fliiviatilis and Latia
neritoides have spherical lenses. However, all of the studied
terrestrial snails and slugs (Newell and Newell 1968, Bran-
denburger 1975, Eakin et al. 1980, Meyer-Rochow and
Bobkova 2001, Bobkova et al. 2004a, Zieger et al. 2008) as
well as the marine slug Onchidium verrucidatnni (Katagiri
and Katagiri 1998) have ovoid lenses of lamellar substruc-
ture with optical axes either along or across their ovoid
outlines. Spherical lenses of the aquatic species are usually
hard enough to permit their surgical removal from the eye
for further study. The ovoid lenses of the terrestrial snails
and slugs are also hard (Bobkova et al. 2004a), but some
stylommatophoran snails, like Strophochelius sp. (Pfeiffer,
1842), possess a jelly-like lens that tears easily during dis-
section (Oswaldo-Cruz and Bernardes 1982).

The spherical lenses of most aquatic and terrestrial spe-
cies can easily be isolated from the retinal cavity, but it is
practically impossible to separate the non-spherical lenses
(Zhukov et al. 2002) from the vitreous body of the freshwa-
ter pulmonate snail Planorbarius conieus (Bobkova et al.
2004a), suggesting intimate connections and possibly a com-
mon origin of lens and vitreous body.

To keep the size of the eye sufficiently small, the focal
length needs to be kept short in relation to the size of the
lens. This requires the lens to be more or less spherical.
However, a homogeneous and spherical lens would suffer
from serious spherical abberations, in which rays off axis are
refracted too severely, so that a sharp image cannot be ob-

tained and an undesirably large blurred circle is created in-
stead. Another problem is that the lens would have rather a
long focal length (Land and Nilsson 2002).

Matthiessen (1886) showed that both these problems j
could be overcome if the lenses were optically inhomoge- j
neons with a gradient of higher to lower refractive index ;
from center to periphery. Then, rays entering the lens could
be bent continuously within the lens through refraction and
not just at the lens’s outer and inner surfaces. A lens design
like this would effectively increase the power of the lens and
shorten its focal length, correct spherical aberration, and,
thus, reduce or even abolish the deleterious effects of an
optically homogeneous lens (Land 1984).

As a rule, unstained transverse sections through a snail’s
lens sometimes reveal regular concentric layers that are dis- 1
cernible under both light and electron microscopes (e.g., the
marine slug Onchidium verrnculatunr. Katagiri and Katagiri
1998; freshwater and terrestrial snails and slugs: Bobkova et
al. 2004a, Zieger et al. 2008; and freshwater limpets: Meyer-
Rochow and Bobkova 2001) (Fig. 3C). A heterogeneous
staining pattern can furthermore indicate an optically inho-
mogeneous construction of the lens (Hamilton et al. 1983, '
Bobkova et al. 2004a).

Demian and Yousif (1975) demonstrated that the lens, i
laid down in the lumen of the eye, grows through the addi- j
tion of concentric layers of secretions from the centre out- !
wards. Thus, it seems that lenses with a radial gradient of
refractive index should be relatively easy to build because i
proteins varying in optical density may be synthesized and i
laid down successively. However, the chemical composition
of the acellular gastropod lenses remains enigmatic. The sea '
hare Aplysia californica (Cooper, 1863) is the only gastropod j
that has been examined for crystallins, i.e., proteins known ’
to be responsible for the optical properties of transparent i
lenses and corneae (Tomarev and Piatigorsky 1996). :

Vitreous body

In order to have a potential for image formation, the
lens and the retina need to be separated (Land 1981, Seyer i
1994, Seyer et al. 1998, Gal et al. 2004). In the camera-type
eyes, separation of the lens and retina is achieved by presence !
of a vitreous body. The vitreous body surrounds the lens and
has contact with the receptive and non-receptive parts of the i
retina. The vitreous body and lens both contain grainy or
tubular substructures, but those making up the vitreous ;
body are always less electron-dense than those of the lens
interior (Fig. 3C). The vitreous body is considerably less
extensive in many terrestrial pulmonates, and larger in some ‘
freshwater pulmonates that demonstrate good visual abili-
ties. The near lack of separation between the lens and the |
retina in most terrestrial pulmonates seems to indicate that
their eyes are not built to receive a focused image.

THE PULMONATE EYE

53

The vitreous humor is continuous between the addi-
tional and the main retinae in Agriolimnx rctkulatus (Newell
and Newell 1968) and in Deroceras agreste (Zieger et al. 2008).

ULTRASTRUCTURE OF THE RETINAL CELLS
Photoreceptors

With rare exceptions, e.g., Planorbariiis corneiis (Zhukov
et al. 2002), the majority of the species investigated have two
morphologically distinct kinds of “rhabdomeric photorecep-
tors” (Eakin 1963). The first kind (“type I photoreceptors”)
is characterized by long microvilli and massive aggregations
of so-called “photic vesicles” (Eakin 1990). The second pho-
toreceptor type (“type 11”) is less common. It lacks the dense
packing of photic vesicles and bears shorter microvilli. The
type II photoreceptor has been described from the retinae of
the terrestrial snails Cornu asperswn aspersuin (Branden-
burger 1975), Succinea piitris (Linnaues, 1758) (Zunke
1979), Strophochelius sp. (Oswaldo-Cruz and Bernardes
1982), Tricliia Jiispida (Bobkova et al. 2004a), the terrestrial
slugs Umax rnaximus, ArioUmax californicus (Cooper, 1872)
(Eakin and Brandenhurger 1975b), Athoracophorus biten-
taculatus (Quoy and Gaimard, 1832) (Eakin et al. 1980),
Umax flavus (Kataoka 1975), and Deroceras agreste (Zieger et
al. 2008). The marine slugs Onchidium verniculatiim (Kata-
giri et al. 1995), and the freshwater basommatophoran snails
Lymnaea stagnalis (Stoll 1973, Bobkova 1998), Radix peregra,
and Physa fontinalis (Bobkova et al. 2004a) have the second
photoreceptor type as well.

Photoreceptor cells with long microvilli may have a fin-
ger-like (as in freshwater species (Bobkcwa et al. 2004a) and
the slug Deroceras agreste (Zieger et al. 2008)) (Fig. 4A-C),
flared (as in Arion rufiis (Zieger et al. 2008)) (Fig. 5) or short
and dome-shaped apical portion as in ArioUmax californicus
(Eakin and Brandenhurger 1975b) and Cepaea nemoralis
(Bobkova et al. 2004a). The short microvilli of the photore-
ceptors may be brush-like, whorled (as in the distal depres-
sion of the retina), or regularly arranged (Fig. 4D-E). Elec-
trophysiological results suggest in Umax flavus that type I
photoreceptors operate in dim and type II photoreceptors in
bright light (Suzuki et al. 1979). However, we cannot exclude
the possibility that the cells belong to the same functional
type of photoreceptor and represent stages in the process of
receptor cell renewal (death and replacement) in the mature
retina. Moreover, based on electrophysiological recordings
from a variety of gastropods (e.g., Dennis 1967, Gillary and
Wolbarsht 1967, Hughes 1970, Gillary 1974, Berg 1978, Su-
zuki et al. 1979, Zhukov and Gribakin 1990, Chernorizov et
al. 1994), there is no convincing evidence for more than a
single spectral sensitivity peak between 480 and 505 nm
wavelength, suggesting that at least physiologically there is
only one type of receptor.

Figure 4. Light (A and B) and electron (C, D, and E) micrographs,
featuring two morphological types of photoreceptor cells in pul-
monates. Nearly longitudinal (A and C) and transverse (B) sections
through the eye ot Deroceras agreste, showing fmger-like apices
(asterisks). Photoreceptors with short and regularly arranged mi-
crovilli in Lymnaea stagnalis (D) and whorled microvilli in the
distal depressions of the Tiichia hispida retina (E) are discernible.
CO, cornea: L, lens; Mv, light sensitive layer of microvilli; ON, optic
nerve; Ph, photoreceptor cells; R, retinal cup; Sp, supportive (pig-
mented) cells; Vb, vitreous body. A, B, and C from Zieger et al.
2008; D and E from Bobkova et al. 2004a with permission of lohn
Wiley and Sons, Inc.

As evident from iit vivo observations and sections of
fixed tissues, the retinas of all pulmonate snails are deeply
pigmented. However, in spite of the presence of specialized
supportive (or pigmented) cells, photoreceptor cells also
contain screening pigment granules in freshwater snails
(Bobkova 1998, Bobkova et al. 2004a) and limpets (Meyer-
Rochow and Bobkova 2001 ) as well as in the terrestrial snails
Tricliia hispida (Bobkova et al. 2004a) and Cornu aspersum
aspersum (Eakin and Brandenburger 1982) and the marine
slug Onchidium verruculatum (Katagiri et al. 1995) (Fig. 6A-
B). Photoreceptors in terrestrial slugs (Newell and Newell
1968, Eakin and Brandenburger 1975b, Kataoka 1975) and

54

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Figure 5. Light (A) and electron (B) micrographs of longitudinal
sections through eye and apical portion of the retina of Arion nifus
showing shallow retina (A) and flared apices of photoreceptor cells
with long microvilli (B). CO, cornea; Int, integument; L, lens; Mv,
microvillar layer; Ph, photoreceptor cells; Sp, pigmented supportive
cells. A and B from Zieger et nl. 2008 with permission of lohn Wiley
and Sons, Inc.

Figure 6. Electron micrographs, showing pigment granules in the
supportive cells oi Arion nifus (A) and pigmented granules, affected
by bright light, in the photoreceptor cell of Deroceras agreste (B),
with pigment granules filling the apices of the photoreceptor cells
following deep light adaptation (C) and fewer pigment granules
following deep dark adaptation (D) in the photoreceptor cells of
Lynmaea stagnalis. Mv, microvilli; Ph, apex of photoreceptor cell;
Vb, vitreous body. C and D from Bobkova et al. 2004a with per-
mission of lohn Wiley and Sons, Inc.

the snail Cepaea nemoralis (Bobkova et al. 2004a) contain no
screening pigment granules. A certain vertical light-
dependent pigment granule displacement (often called “mi-
gration”) was revealed in freshwater snails (Bobkova 1998,
Zhukov et al. 2002, Bobkova et al. 2004a). In samples pre-
pared for morphological investigations during periods of
total light adaptation, we were able to observe that the fm-

ger-like apices became filled in with screening pigment gran- j
ules. Conversely, pigment granule dispersion occurred when i
a light-adapted eye was exposed to darkness and, thus, in-
dicates dark adaptation (Bobkova 1998) (Fig. 6C-D). ;

Our ultrastructural observations have shown that pho- '
toreceptor cell types I and II occur in close approximation to :
each other and are jointly present at the level of the retinal
pigment layer in the eye of, for example, Lymnaea stagnalis \
(Bobkova 1998) and Cepaea nemoralis (Bobkova et al.
2004a). However, we never found any specialized mechani- j
cal or functional contacts between the two types of these !
cells. !

Microvilli and photic vesicles

One of the most important selective pressures in the !
evolution of photoreceptors appears to have been the need ,
to increase the area available for visual membranes — in <
other words, to lay the foundation for an increase in pho- [
tosensitivity that depended on the maximization of space for
photoreceptive membranes. Rhabdomeric photoreceptors
achieve this by increasing the total number of microvilli. The
small diameter of an individual microvillus, which is approx.
600 A in the dark-adapted eye irrespective of the type of
photoreceptor, helps in this regard but also poses a lower
limit for miniaturization as the organization and dimen-
sional characteristics of the microvilli appear to be similar in
all of the invertebrate eyes investigated to date.

An axial cytoskeleton is present in the tubular lumen of
the microvilli; it is formed by bundles of fibrils that run
along the length of the microvilli and are connected to the
membrane (Fig. 7 A). Microvilli reach their highest degree of '
order during periods of dark adaptation but are found to
enlarge (“swell up”), following brief exposures to bright [
light, or to become dramatically disorganized when an iso- '
lated eye is exposed to bright light for a longer period of time
(Bobkova 1998) (Fig. 7B). The processes of microvillar dis-
ruptions by light in the snail eye are likely to be similar to the
changes that bright light exposure causes in the compound
eyes of crustaceans (reviewed by Meyer-Rochow 1994, 2001)
and insects (Meyer-Rochow et al. 2002), where bright light is
known to affect the axial cytoskeleton and the lipid fraction
of the microvillar membrane itself (Kashiwagi et al. 1997,
2000).

Photic vesicles are important and characteristic cyto-
plasmic organelles in the eyes of gastropod molluscs (Fig.
7C). Whittle ( 1976) discussed the controversial origin of the
photic vesicles, i.e., of Golgi apparatus origin according to
Eakin and Brandenburger (1967b) but vesiculations of the
smooth endoplasmic reticulum according to Kataoka
(1975), and concluded that the evidence is entirely consis-
tent with reticular specializations. Eakin (1990) has summa-
rized data on structure, origin, fate, and function of the

FHE PULMONATE EYE

55

Figure 7. Electron micrographs, showing transverse section
through microvilli (A) and microvillar layer affected by bright light
in the eye of Lymnnea stagiialis (B) and aggregation of photic
vesicles (C) in the perinuclear area of photoreceptor cells in Cepaea
nemoralis. Arrows, axial cytoskeleton; Nph, nuclei of photoreceptor
cells; Phv, photic vesicles; VB, vitreous body.

photic vesicles as “transporters of a photopigment retino-
chrome and calcium” and likened them to the lamellated
bodies in cephalopod photoreceptors shown by Hara et al.
(1967) to contain retinochrome. Later, fluorescent histo-
chemical technic]ues, using a reducing agent, have shown
that rhodopsin and retinochrome are present in the micro-
villar and somatic layers of the stalks of the eyes of the
marine pulmonate slug Onchidium verriiculatum (Katagiri et
al. 2002) and the terrestrial slug Umax flavus (Ozaki et al.
1983).

Using antibodies against squid retinal proteins, Katagiri
et al. (2001 ) determined the localization of three retinal pro-
teins (rhodopsin, retinochrome, and retinal-binding pro-
tein), and thus demonstrated the presence of a rhodopsin-
retinochrome system in the eyestalk of the marine
pulmonate Onchidium sp. Later, Katagiri et al. (2002) con-
firmed by fluorescence histochemistry the presence of rho-
dopsin and retinochrome in specific regions of the retina.
Accordingly, the visual pigment rhodopsin is present in the
microvilli of the photoreceptive cells, while the photic
vesicles themselves may be regarded as a store for retinalde-
hyde in the form of retinochrome-chromophore.

Rhodopsin and retinochrome function cooperate to
mutually regenerate photopigment (Terakita et al. 1989).
Upon illumination, rhodopsin is converted to metarhodop-
sin, and retinochrome to meta-retinochrome. The 11-cis-

retinal in rhodopsin is photoisomerized to the all-trans
configuration. Conversely, the all-trans-retinal in the retino-
chrome is photoisomerized to its 1 1 -cis-isomer. The 1 1-cis-
retinal is required for rhodopsin formation. In the dark,
rhodopsin and retinochrome are regenerated from these two
isomers by chromophore exchange.

Supportive pigmented cells

Supportive pigmented cells are located between the
photoreceptor cells. Their apical portion bears very short
membranous micrcwilli-like projections into the vitreous
body. The cells form the border of the fixed pupil aperture.
In freshwater snails, limpets, and some terrestrial species,
supportive pigmented cells have column-like apices (e.g.,
Lyimiaea stagnalis: Stoll 1973, Zunke 1979; Onchidium ver-
ruculatunr. Katagiri et al. 1995, Bobkova 1998; Latin neri-
toides and Ancilus jluviatilis: Meyer-Rochow and Bob-
kova 2001; Planorharius corneus: Zhukov et al. 2002; Radix
peregra, Physa fontinalis: Bobkova et al. 2004a). However, in
some terrestrial slugs and snails, the supportive pigmented
cells envelope the photoreceptors, send deep and multiple
projections into the photoreceptor cytoplasm, and are
crowded with innumerous pigment granules (viz.. Helix po-
matia: Schwalbach et al. 1963, Rbhlich and Torok 1963;
Cornu aspersum aspersum: Eakin and Brandenburger 1967a;
Umax flavus: Kataoka 1975; Ariolimax californicus: Eakin
and Brandenburger 1975b; Achatina fulica: Tamamaki and
Kawai 1983; Cepaea nemoralis: Bobkova et al. 2004a; Dero-
ceras agreste and Arion rtifus: Zieger et al. 2008). However,
in species with finger-like apices (cf., section on “Photore-
ceptors”) they never penetrate into the spaces between the
latter. In the nocturnal slug Umax maximiis, lateral branches
along the sides of the photoreceptor cells have been de-
scribed to often lack pigment granules (Eakin and Branden-
burger 1975b).

The nuclei of the supportive (pigmented) cells are lo-
cated in the basal region of the somatic and plexiform layers.
They are much smaller than the nuclei of the photoreceptor
cells and are identifiable by their condensed heterochroma-
tin (Fig. 8A). Multiple rings of rough endoplasmic reticulum
together with large numbers of mitochondria are present in
the perinuclear area, suggesting that the supportive pig-
mented cells are synthetically active retinal cells. The cells
themselves are anchored by hemi-desmosomes to the basal
lamina.

Although the chemical nature of the retinal screening
pigment has not been analyzed in detail, generally it is as-
sumed that the pigment is a form of the melanin (e.g., Lym-
naea stagnalis: Land 1968, Stoll 1973, Bobkova 1996, and
Cornu aspersum aspersum: Eakin and Brandenburger 1967a,
1967b).

56

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Figure 8. Electron micrographs, showing (A) nuclei of pigmented
supportive cells (Nsp) and sites in (B) of gap junctions (arrows)
between adjacent pigmented supportive cells (Sp) in Arion mfiis.
EC, eye capsule; Sc, neurosecretory cell. From Zieger et al. 2008
with permission of lohn Wiley and Sons, Inc.

Cellular contacts between adjacent cells

As a rule, and not restricted to molluscs, adjacent cells
are connected with each other by mechanical cellular contact
specializations like zoniilae adlmeretites, located apically just
above the septate junctions. The latter resemble those exam-
ined in the leech photoreceptors by Aschenbrenner and
Waltz ( 1998) and are thought to contribute to the maintance
of the cell polarity. Contacts of this nature are seen in the
retinae of Arioliinax caUfornicus (Eakin and Brandenburger
1975b), Umax flaviis (Kataoka 1975), Athoracophonis biten-
tacuhitus (Eakin et al. 1980), and Lymnaea stagnalis
(Bobkova 1998). The existence of finger-like folds, project-
ing into adjacent cells, has been reported from the eyes of
Lymnaea stagnalis by Bobkova (1998) and Arioliinax califor-
niciis by Eakin and Brandenburger (1975b). Similar cyto-
plasmic folding is known from vertebrate epithelial cells (i.e.,
bronchial and intestinal kinds) to enable these cells to
change their length. A property such as this, if it held true for
the unusual mechanical contacts with folds, would agree
with observations made by Stoll (1973) on light-dependent
retinomotoric cell extensions and contractions in the retina
of Lymnaea stagnalis.

The supportive cells (with heavily pigmented exten-
sions, but only near their distal ends) form multiple gap-
junctions between each other in Lymnaea stagnalis (Bobkova
1998) and Arion rufiis (Zieger et al. 2008) (Eig. 8B). It ap-
pears as if this type of cell has a kind of syncytium-like
intraretinal net.

Neurosecretory cells

The retinal cells that we termed ‘neurosecretory’ in
Arion rufns (Zieger et al. 2008) are likely to belong to cells

that are known in Limax flavus as “large ganglionic cells of
the nervous system” (Kataoka 1975) and in Cornu aspersum
aspersiini (Brandenburger 1975), Arioliinax reticulatus (New-
ell and Newel 1968), and Lymnaea stagnalis (Stoll 1973,
Bobkova 1998) as ganglion cells.

The cell types listed above are similar in appearance,
contain a very large nucleus, and are endowed with promi-
nent rough endoplasmic reticulum, numerous granulated
osmiophilic dense-core and clear vesicles, and large lipo-
somes or pigment-granule-like bodies, similar to the lipo-
chondria of the Aplysia sp. ganglion cells described by Baur
et al. (1977). Following Strumwasser et al. (1979), we have
accepted the term “neurosecretory cells” for the cells in
c]uestion in Arion riifiis.

These large oval cells were found to form a single cluster
in a delimited region of the eye of Arion rufus (Zieger et al.
2008) (Fig. 9A-C), and in Trichia hispida and Cepaea
nemoralis (Zieger, unpubl. data). In the freshwater snails
Lymnaea stagnalis (Bobkova 1998), Radix peregra, and Pliysa
fontinalis (Zieger, unpubl. data), retinal cells of this type are
diffusely distributed among other retinal cells. No synaptic
contacts were found on the surface of the cell body of these
cells in A. rufus (Zieger et al. 2008) and Lymnaea stagnalis
(Bobkova 1998). However, Brandenburger (1975) demon-

Figure 9. Light micrographs of tangential section (A) through clus-
ter of neurosecretory cells (framed) and (B) enlargement ot the
cluster in the eye of Arion rufus. Electron micrographs of perinu-
clear region (C) and axons (D) containing dense-core vesicles in
Arion rufus. Ax, axons; Int, integument; L, lens; R, retina; Sc, se-
cretory cells; Vb, vitreous body. A and B from Zieger et al. 2008
with permission of )ohn Wiley and Sons, Inc.

THE PULMONATE EYE

57

strated synaptic-like structures on the surface of the “gan-
glion” cells in Cornu aspersum aspcrsiim.

The axons of the neurosecretory cells can be identified
in the plexiform layer of the retina by their contents of dense
core vesicles (Fig. 9D). We cannot completely rule out the
possibility that the neurosecretory cells in the eye of Anon
rufiis are a kind of neuroendocrine cell type that releases its
vesicular content directly into the bociy fluid to reach more
distant effector organs.

As to the function of the retinal neurosecretoiy cells, we
can only speculate that they could be involved in various
aspects of the reproductive cycle (i.e., gonadal development,
sexual maturation, egg laying) or in some aspect of meta-
bolic regulation. It was earlier shown that the retinal neurons
in the marine non-pulmonate gastropods Biilla gouldiana
(Pilsbry, 1895) and Aplysia californica are capable of gener-
ating a circadian periodicity. Actually, the cells are compe-
tent circadian retinal pacemakers and as in circadian pace-
makers, the oscillators entrain to cycles of light and
darkness, especially the 24-h light/dark cycle of the environ-
ment (cf., review by Whitmore and Block 1996).

In behavioural experiments with Avion riifus, it was
shown that these slugs have a circadian rhythm (Lewis
1969). In fact, that light is highly effective in entraining
circadian rhythmicity is also known for other gastropods like
slugs {Linmx flavus: Segal 1960\ Agriolinwx reticniatns: New-
ell and Newel 1968), terrestrial snails like Helix pO)uatia
(Jeppersen 1977), Cornu aspersum aspersum (Bailey 1981),
Helix lucorum lucorum (Linnaeus, 1758) (Flari and Lasari-
dou-Dimitriadou 1995), and Achatina achatina (Hodasi
1982) as well as the slugs Umax pseiuioflavus (Evans, 1978)
(Ford and Cook 1988, 1994), Deroceras reticulatum (Muller,
1774), and Anon distinctus (Mabille, 1868) (Hommary et al.
1998). However, the possession of an internal oscillator,
which is capable of measuring the photoperiod has not been
demonstrated in these pulmonate species. The notion that it
is the retinal neurons, which express circadian rhythmicity
themselves, has yet to be proven.

OPTICAL CALCULATIONS

In 1984 Hamilton and Winter pointed out that the de-
gree of accuracy of the behavioral data on visual abilities can
be checked by carrying out a resolution estimate based on
the optical and structural characteristics of the eye. In other
words, in order to know a snail eye’s functional limitations,
it is imperative to possess some information on the eye’s
optics and structural organization. Resolving power and sen-
sitivity are key parameters in this context, and both can be
approximated from anatomical and optical data. To deter-
mine the limitations of the optical system of the eyes in
pulmonates, we followed methods (with some modifica-
tions: cf., Bobkova et al. 2004a, Gal et al. 2004) that were
used earlier with considerable success in comparisons of eyes
in some caenogastropods (Seyer 1992, 1994, Seyer et al.
1998). The methods described by Nilsson et al. (1988) and
by Seyer ( 1992) were used to determine the focal lengths of
freshly isolated lenses. The same methodological approach
allowed us to compare optical systems in different pulmo-
nate gastropods (Tables 1-3).

All of the pulmonate gastropod eyes investigated to date
operate with fixed focal-lengfh optics, which means that at
least the principal focal length cannot be changed. The fixed
focal-length optics design is described in some vertebrates
(Walls 1942), who in order to accommodate to different
distances adjust the position of the lens relative to the retina.
However, in the absence of accommodation mechanisms in
the eyes of gastropods, it appears that the almost infinite
depth of focus characteristic of the eyes of aquatic gastro-
pods makes accommodation unnecessary (Land 1981).

The shape of the gastropod eye lens was found to be
spherical in aquatic species and elliptic, with an optical axis
either across or along the lenticular ellipse, in terrestrial
species. There are, however, exceptions: the lens of the
aquatic snail Platiorbarius corneus is rather more elliptic than
spherical (Zhukov et al. 2002) and the terrestrial slug Dcro-
ceras agreste has a spherical lens (Zieger et al. 2008). The

Table 1. Comparative eye parameters of terrestrial pulmonates based on light and electron microscopy (in pm). Values are means.

Arion

rufus

Deroceras

agreste

Ccpaea

nemoralis

Trick ia
hispida

Agrioliinax

reticniatns

Size of eyeball

240 X 290

220 X 220

312 X 320

142 X 220

140 X 180

Size of lens

150 X 200

no X 110

152

81

130

Diameter of aperture

100

80

107

92

—

Center-to-center separation of receptors

5.6

17.0

21.0

15.0

6.0

^Receptor length

13.8

27.8

3.4-20.0

3.0-15.0

25.0-30.0

Authors

Zieger et al. (2008)

Bobkova et al. (2004)

Newell and Newell (1968)

* Values based on thickness of microvillar layer.

58

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Table 2. Comparative eye parameters of aquatic pulmonates based on light and electron microscopy (in pm). Values are means.

Ancyhis

fhiviatilis

Latia

neritoides

Lymnaea

stagnalis

Radix

peregra

Pliysa

fontinalis

Planorbarius

Cornells

Size of eyeball

100 X 100

150 X 175

210 X 270

160 X 200

150 X 170

240 X 275

Size of lens

60

90

120

94

80

136

Diameter of aperture

43

63

60

73

65

140

Center-to-center separation of receptors

2.5

6.5

8.3

4.5

8.9

6.2

^Receptor length

7.0

31.0

71.0

18.0

49.1

18.5

Authors

Meyer-Rochow and Bobkova (2001)

Bobkova et al. (2004a)

* Values based on thickness of microvillar layer.

Table 3. Comparative mathematically determined optical parameters in different species of pulmonate gastropods. Values are means; the
symbol ‘ — ’ denotes missing values.

Principal

focal Angular Diameter

Species

length of
the lens
(pm)

Relative

aperture

Matthiessen’s

ratio

receptor

spacing

(degrees)

F-

n umber

of

Airy-disc

(pm)

Resolving
power
(radian^' )

Sensitivity
(pnTsr“‘ )

Authors

Arion rufus

848

0.10

—

0.40

—

—

—

—

Zieger et al. (2008)

Deroceras agreste

207

0.40

3.7

4.70

2.5

3.00

—

—

Zieger et al. (2008)

Cepaea nemoralis

149

0.70

—

8.00

1.3

1.60

4.0

8.00

Gal et al. (2004)

Trichia hispida

122

0.80

—

13.00

1.2

1.50

2.5

4.00

Gal et al. (2004)

Agriolimax reticulatus

100

—

2.8

15.00

—

—

—

—

Newell and Newell (1968)

Lymnaea stagnalis

174

0.30

2.9

2.70-5.20

2.9

3.60

6.3-12.0

0.04-1.80

Gal et al. (2004)

Radix peregra

128

0.60

2.7

2.0-6.50

1.7

2.00

0.2-16.5

0.02-0.20

Gal et al. (2004)

Physa fontinalis

116

0.60

2.9

4.00-5.00

1.8

2.20

7.0-8.0

0.20

Gal et al. (2004)

Planorbarius corneus

241

0.60

3.5

2.50

1.8

2.00

13.0

1.40

Gal et al. (2004)

lenses in all species studied presumably possess a radial gra-
dient of refractive index (i.e., are optically in-homogeneous)
irrespective of lens shape.

It is generally assumed that terrestrial animals cannot
exploit the advantages of a spherical graded index lens. In-
stead, by having a curved outer surface on the cornea, they
must make use of the low refractive index of air (1.0). Be-
cause the cornea then provides much of the refracting
power, an interior lens can be designed specifically, for ex-
ample, to correct spherical aberration (Land 1984, Nilsson
1989).

However, on the basis of our calculations it appears that
the integument-cornea lens complex in the terrestrial snails
Cepaea nemoralis and Trichia hispida is optically much less
important than the refractive power of the lens alone (Gal et
al. 2004). A similar assessment, not based on any calcula-
tions, was made for the slugs Arion riifus and Deroceras
agreste (Zieger et al. 2008 ). Eor accuracy, we have made the

essential calculations, and have included the mean values
obtained into this review (see below).

The focal length of the system of two lenses (/s) can be
calculated as:

l//s ~ ~

where j\ is principal focal length of the lens, is focal
length of the integument-cornea complex and x is the dis-
tance between the two lenses centers.

The focal length of the integument-cornea complex can
be calculated as described by Gal et al. (2004).

Thus, in Cepaea nemoralis the focal length of the lens
alone is 149 pm, and the focal length of the system com-
posed of the two lenses (integument-cornea plus lens) is 144
pm. The corresponding figures are 122 and 113 pm for
Trichia hispida, 207 and 170 pm for Deroceras agreste, and
848 and 450 pm in Arion rufus. As is obvious, in the eyes of
the snails C. netnoralis and T. hispida, the shortening is only

THE PULMONATE EYE

59

5 to 9 i-im, but in the eyes of the slugs it is ca. 400 (A. rufiis)
and 40 jam [D. agreste).

As we see here, the contribution of the first lens, the
integument-cornea complex in the snails’ eyes, is surpris-
ingly small and in slugs, the refractive power of the integu-
ment-cornea complex looks very significant. However, at
this point of our discussion, we still continue to argue that
the lens, but not the integument-cornea, is the principal
optical element of the eyes in terrestrial gastropods, just as in
aquatic species. It is very likely that the optical properties of
the lens in the terrestrial gastropods reflect the lens proper-
ties that their marine ancestors might have had.

The quality of the images produced by the isolated
lenses of both terrestrial and aquatic gastropods demon-
strates that the latter are corrected tor spherical aberration
(Gal et al. 2004). Therefore, we may conclude that lenses in
both aquatic and terrestrial pulmonates, presumably possess
a radial gradient of refractive index are optically inho-
mogeneous), irrespective of lens shape. However, to entirely
eliminate spherical aberration it is not enough to have re-
fractive-index gradients. The lens should be spherically sym-
metrical and have a ratio of focal length to lens radius of
about 2.5 (Warrant and McIntyre 1993). Newell and Newell
(1968) have shown that the lens in the terrestrial slug Ag-
rioUmax reticiilatiis has a focal length of 2.8 radii and, on that
basis, concluded that the lens must have a graded refractive
index.

The ideal spherical geometry, combined with a gradient
of refractive index and an F-number of 2.9, endows Lymimea
stagnalis with a perfect aplanatic lens and superb optical
image quality. Other species, like Radix percgrn, Physa foa-
tinalis, and Phmorbariiis corneiis come close to the optimum,
but imperfections in the optics such as low F-numbers (1.7,
1.8, 1.8, and 1.9, respectively) (Table 3) of the eyes manifest
themselves as degradations in the quality of the image
formed on the retina. Spherical aberration must be worse in
the eyes of the terrestrial snails Cepaea nemoralis and Trichia
hispida because of large, in relation to their focal length,
apertures (0.7 and 0.8 compared to 0.3 in L. stagnalis) and
low F-numbers ( 1.3 in C. nemoralis and 1.2 in T. hispida) as
their eyes are designed to capture as much light as possible
(Table 3) (Gal et al. 2004). The slug Deroceras agreste was
found to have a high F-number equaling 2.5 and a spherical
lens very likely free of aberration (Zieger et al. 2008).

Another source of lens imperfection, namely chromatic
aberration, arises because the transparent material of the
lens is invariably dispersive, that is, light of shorter wave-
length is refracted by the material more strongly than light of
longer wavelengths (Warrant and McIntyre 1993). However,
chromatic aberration becomes important only when the di-
ameter of the aperture exceeds about 500 pm (Land 1981).
We therefore did not take chromatic aberration into con-

sideration because the species studied have apertures in the
range of 40 pm (Ancylns llnviatilis) (Meyer-Rochow and
Bobkova 2001) to 140 pm (Planorbarius corneas) (Zhukov et
al. 2002).

Even if the effects of spherical and chromatic aberration
are negligible, the image produced by the eye can still be
blurred. This limitation in image quality is referred to as
diffraction.

In general, at any given wavelength, lenses with larger
apertures will have narrower Airy discs (the central peak ot
a diffraction blur-circle), and thus suffer least from diffrac-
tion. For instance, aperture and Airy disc are 60 pm and 3.6
pm in Lymnaea stagnalis, 80 and 3.0 pm in Deroceras agreste,
92 and 1.5 pm in Trichia hispida, 107 and 1.6 pm in Cepaea
nemoralis, and 140 and 2.0 pm in Planorbanus corneas
(Tables I and 3). Larger apertures lead to losses in image
quality due to aberration, and therefore for each eye design
there must be some sort of compromise between diffraction
and aberration.

Do the gastropods make use of their definitely advanced
optical designs? It appears that even if eye optics can focus
aberration free and diffraction-limited images, there are still
anatomical limitations within the eye, which can ciestroy the
potential for high resolving power of the gastropod eye.

Retinal geometry

If the position of the retina does not coincide with the
sharp image plane (- position of the focal point within the
microvillar layer), it would lead to wide receptive fields in
individual receptors and thus result in considerable de-
creases of resolution. Such severely under-focused eyes occur
in the terrestrial snails Cepaea nemoralis and Trichia hispida,
where the sharp image falls just below the light-receiving
microvilli within the retina (Gal et al. 2004). The under-
focusing leads to a blurred image and loss of fine visual detail
that the optics of these snails is able to provide.

The situation is even worse in the slugs Anon rafas and
Deroceras agreste (Zieger et al. 2008) as well as the aquatic
limpets Latia neritoides and Ancylns flaviatilis (Meyer-
Rochow and Bobkova 2001 ) where the sharp image lies well
outside the retina. Although refractive power of the integu-
ment-cornea-complex is very significant in slugs, such short-
ening of the focal length ot the two lenses still does not even
allow a blurred image to be formed within the retina in the
eyes of A. rafas and D. agreste.

The optics of the eyes in these pulmonates cannot assist
in focusing an image on their shallow retinae because to
shorten the principal focal length of their lenses to place the
focal point onto light-receiving layer would require a central
refractive index of the lens of approximately 1.9 (Land
1981), which is well beyond that of even the densest crys-
talline proteins (Sweeney et al. 2007).

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AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Such short offset of the lens and the retina seems to
indicate that eyes in terrestrial snails and slugs as well as
some aquatic limpets are not designed to receive a focused
image and are likely to measure the average light intensity
or quality over large angles rather than resolve fine image
details.

Aquatic snails like Lyninnea stagnalis. Radix peregra,
Physa foiitinalis, and Planorbarius corneiis are able to focus a
sharp image on the light-receptive layer of the retina. It is
due to the deepenings of the latter, resulting in the increase
of the vitreous body volume (Bobkova et al. 2004a, Gal et al.
2004).

Finally, it seems that there is no definite demarcation
between lens eyes that form an image and those that do not.
Nevertheless, we argue that it is the inappropriate geometry
of the retina and the size of the eye in general, but not the
optics alone, that cause the biggest differences between the
eye designs in the species examined here.

VISUALLY MEDIATED BEHAVIOR

Visual capabilities and behavior

Numerous generalizations with regard to vision in pul-
monate gastropods were based on observations of Cornu
asperswn aspersum or closely related terrestrial snail species
(Wheeler 1921, Geismer 1935, Zanforlin 1976, Bailey 1981,
Hamilton and Winter 1984). Not surprisingly, a common
assessment was that pulmonates have poor vision, i.e., that
their eyes can detect general light levels and perhaps broad
areas of light and dark regions but not much else. However,
comparisons of the visual capacities of freshwater snails and
limpets, through behavioral tests and estimates based on
analyses of eye structure and optics (Vakoliuk and Zhukov
2000, Meyer-Rochow and Bobkova 2001, Zhukov et al. 2002,
Vakoliuk 2005), with those of terrestrial snails and slugs
(Hermann 1968, Zhukov and Baikova 2001, Bobkova et al.
2004a, Gal et al. 2004, Vakoliuk 2005, Zieger et al. 2008)
suggest a considerable range of visual capabilities exists
within the pulmonate lineage.

Gastropods exhibit two easily distinguishable kinds of
visual behavior. First, they can use light to orient by moving
either toward, or more commonly, away from regions of
high light intensity, and these responses usually take the
form of a phototaxis. Second, gastropods may respond to a
sudden decrease in light intensity by withdrawing into their
shell and adhering tightly to the substratum. The first be-
havior is clearly concerned with habitat selection, while the
second is a defense against a predator that casts a shadow
prior to attack (Land 1968).

In pulmonates the ocular system (= paired cephalic
eyes), but no other sensory system, is involved in the pho-

totactic behavioral response. This conclusion is the result of
behavioral experiments with operated (eyeless) animals that
did not show any sign of phototactic response, while intact
molluscs did. Electrical recordings from the eye of Lymnaea
stagnalis by Stoll and Bijlma (1973) gave only “on-
responses”, confirming that the shadow reflex was mediated
by ( 1 ) dermal receptors, in Nassarius reticulatus (Linnaeus,
1758) according to Crisp (1972), (2) in the skin cind along
the mantle and in L. stagnalis, according to Stoll (1976), and
(3) in the foot, lips, and tentacles as well.

The freshwater pulmonates Lymnaea stagnalis (Stoll
1972, 1973, 1976, Vakoliuk and Zhukov 2000) and Planor-
barins corneas (Zhukov et al. 2002) clearly choose to move
towards illuminated targets, displaying positive phototaxis.
However, terrestrial pulmonate snails like Cornu asperswn
aspersum (Wheeler 1921, Hamilton and Winter 1984), Helix
pomatia (Geismer 1935), Otala lactea (Muller, 1774) (Her-
mann 1968), Euparipha (Helix) pisana (Muller, 1774) (Zan-
forlin 1976), Achatina fulica (Zhukov and Baikova 2001),
Cepaea nemoralis, and Trichia hispida (Vakoliuk 2005) as
well as the slugs Agriolimax reticulatus (Newell and Newell
1968), Arion rufus, and Deroceras agreste (Zieger et al. 2008)
approach the dark regions of an experimental arena and,
thus, display negative phototaxis.

A negative phototactic behavior is fully compatible with
the dim or even dark environments that terrestrial snails and
slugs are most active in (Newell and Newell 1968, Rollo and
Wellington 1981, Hamilton and Winter 1984, Hommay et
al. 1998, Cook 2001, Vakoliuk 2005). In contrast, positive
phototaxis is compatible with the preference for a well-lit
environment as, for example, in Lymnaea stagnalis and Ra-
dix peregra that exhibit moderately amphibious lifestyles and
live on the algal growth of well-illuminated surfaces at the
water/land boundary (Purchon 1977, Vakoliuk 2005).

Counsilman et al. ( 1987) suggested that in the biolumi-
nescent terrestrial snail Dyakia striata (actually Quantula
striata (Gray, 1834): Haneda 1981) the light produced by
this species may have a social function. Although intraspe-
cific communication by light was rejected by Meyer-Rochow
and Moore (1988) for the bioluminescent freshwater pul-
monate limpet Latia neritoides, comparisons of the eyes be-
tween this limpet and the equally large, but non-luminescing
species Ancylus fluviatilis showed that the light-producing
Latia neritoides had a significantly larger eye, larger lens, and
more extensive retina (Meyer-Rochow and Bobkova 2001).

First described by Hamilton and Winter (1982), an ex-
perimental procedure to determine response thresholds for
black stripes of different widths and orientation was then
modified by Zhukov and Baikova (2001) and Vakoliuk
(2005). Zhukov and Baikova (2001) have been able to show
that the terrestrial snail Achatina fulica discriminates vertical
and horizontal black stripes as well as grating patterns of

THE PULMONATE EYE

61

different frequencies. Discrimination tests revealed a strong
preference for vertical bars over diagonal and horizontal bars
of the same width in Otala lactea (Hermann 1968), a signif-
icant preference for horizontal bars over vertical ones in
Cepaea tieuioralis (Vakoliuk 2005), and no preference in
Lymnaea stagnalis, Planorbarius conieus (Vakoliuk 2005),
and Cornu asperswn aspersiim (Hamilton and Winter 1984).

The experimentally determined resolution limits (Va-
koliuk 2005) are well supported by our calculations of re-
ceptor spacings (for comparison in parentheses) (Gtil et al
2004) in Lymnaea stagnalis, Planorbarnis corneas, Cepaea
nemorais, and Trichia hispida: 2.5°-5.7° (2.4°-5.2°),
1.4°-1.9° (1.4°-3.4°), 8.0° (8.0°), and >10.0° (13.0°). Cornu
asperswn aspersiim has a resolution limit of 14.5°-24.6°
(Hamilton and Winter 1984) and Otala lactea, 0.9° for a
single vertical bar and 2.4°-3.7° for a horizontal bar of simi-
lar dimensions (Hermann 1968).

However, we should keep in mind that behavioral re-
sponse threshold estimates do not necessarily approximate
actual sensory thresholds. Thus, our theoretically calculated
performances of slug eyes show that a centrally-placed re-
ceptor subtends about 0.4° in Arion rufus and 4.7° in Dero-
ceras agreste, so that acuity on purely anatomical grounds
should be considered to be quite good in both species. Yet,
behavioral tests show that these slugs do not respond until
resolution thresholds of ca. 26° and ca. 90°, respectively,
are reached (Zieger et al. 2008). The reasons for such dis-
parity between anatomically-determined estimates and be-
haviorally-determined resolution limits are probably to be
sought in features of either the visual processing or the op-
tical system.

Our research has led us to conclude that anatomically
the eyes of the two species of slugs investigated should be
able to form images, but images that would lie in the wrong
plane, well behind any retinal structures. The eyes of the
slugs are designed to transmit spatially averaged intensity
patterns of light and dark to the CNS for orientation and
only one aspect of the visual environment, the overall pat-
tern of light and dark, seems to be important for them. A
sharp image is unnecessary and may even be a hindrance
when the aim is to provide nothing more than orientation.

Newell and Newell (1968) estimated that two adjacent
receptors subtend an angle of 15° at the center of the lens so
that the visual acuity is poor in the eye of Agriolimax reticu-
latus. The authors presumed that the slug’s eyes are adapted
to detect changes in light intensity only and to operate at
night.

Hamilton and Winter (1984) concluded that the poor
vision of Cornu asperswn asperswn may correlate with pri-
marily nocturnal habits. Geismer (1935) reported that in
Helix pomatia a significant orientation response was released
to a 24 X 20 cm black card of angular size extending at least

20° and positioned 25 cm away from the snail. According to
Hermann (1968), another terrestrial snail, Otala lactea, can
orient with respect to a target as small as 22.5°— 30° at 25 cm
distance. We have to conclude that the eye design in Trichia
hispida and Cepaea nemoralis does not prevent image for-
mation, but that resolution of the eyes can certainly not be
great. Nevertheless, the eyes of these two snails are able to
register not just the average light level but the quality of wide
angles, and thus the crude direction, of a light source as well.
This led us to suggest that the eye of terrestrial pulmonates,
inherited from aquatic ancestors, has changed very little.

Secondarily aquatic pulmonates with higher visual
needs are capable of image formation in both air and water.
During their evolution, they have changed the terrestrial eye
into a type that can function under water by deepening their
retinae, so that the latter are capable of handling the increase
in focal length required for vision under water (Bobkova et
al. 2004a, Gal et al. 2004).

CONCLUSION

The terrestrial snails Cepaea nemoralis, Otala lactea.
Cornu asperswn asperswn, Achatina fulica, and Trichia his-
pida are active under twilight conditions (c.g., are crepuscu-
lar) and thus would need to he able to collect some light to
be able to retain the image-forming ability of their eyes.
Although their resolution is very poor, their eyes are able to
register the average ambient light level as well as the quality
of wide angles of light.

The slugs Arion rufus and Deroceras agreste are crepus-
cular but have high F-numbers of 8.5 and 2.5, respectively.
It seems that the eyes of these gastropods have another visual
task: they monitor environmental brightness and assist the
animal in orientating towards dark places. The slugs simply
do not need to perceive sharp images. The eyes of the lim-
pets Latia neritoides and Ancylus fluviatilis also have no im-
age-forming capacity, and the same conclusion as for the
slugs may apply. Elowever we do not know if the eyes of the
limpets assist them in orienting towards dark, or toward
light areas, and whether the somewhat larger eye of L. neri-
toides has something to do with the fact that this species can
produce light when attacked (Meyer-Rochow and Moore
1988).

The eyes of Lymnaea stagnalis and Radix peregra are
well adapted for vision in both watery and terrestrial habi-
tats, and we suggest that the shape of the retina and the
optics of the eyes of these snails match their amphibious
life styles. The eyes of Physa fontinalis and Planorbarius Cor-
nells appear to have been less modified from those of their
ancestors. Probably these snail species have lesser visual

62

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

needs and depend more on other senses like chemo- and
mechanoreception.

Pulmonate gastropods use their eyes primarily for the
following two kinds of visual task: ( 1 ) discriminating objects
and possible enemies in their environment and (2) moni-
toring the environmental brightness level to orient towards
dark places. The first type of visual task is characteristic of
the aquatic snails and is served by image-forming eyes; the
second is typical of terrestrial snails and slugs and is best
served by a blurred image.

The studies on pulmonates can provide a more solid
basis than currently exists for generalizations on the sensory
capabilities of the molluscs in Class Gastropoda. Aside from
the morphological and functional ramifications, this re-
search is important from an evolutionary and taxonomic
standpoint. The eyes are used as identifying characters in
taxonomy or as a basis for comparisons in systematic dis-
cussions. An underlying knowledge of the function of the
eyes, and why they take on a particular shape or design will
obviously enhance their use or disuse as taxonomic or phy-
logenetic characters, finally, an understanding of the various
eye designs can shed light on evolutionary relationships and
serve as a springboard for future research.

ACKNOWLEDGMENTS

Dr. M. Zieger wishes to thank members of UNITAS
Malacologica and the National Science foundation for sup-
port of her travels, and both authors express their gratitude
to Dr. L Serb for much good advice and encouragement. We
are very grateful to our expert reviewers who gave us many
useful suggestions.

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final revisions received: 13 October 2008

Amer. Make. Bull. 26: 67-71 (2008)

The genus Buccinanops: A model for eye loss in caenogastropods"^

Andres Averbuj and Pablo E. Penchaszadeh

Museo Argentino de Ciencias Naturales - CONICET and Facultad de Ciencias Exactas y Naturales - University of Buenos Aires
Avenida Angel Gallardo 470, laboratory 57, C1405D1R, Buenos Aires, Argentina, andresbuj@macn.gov.ar

Abstract: The genus Buccinanops (d’Orbigny, 1841 ) (Caenogastropoda, Nassarriidae) is endemic to the SW Atlantic Ocean, and the name
implies no eyes, due to the lack of visible eyes in adults. We recognize for the first time the occurrence of eyes during several developmental
stages within Buccinanops. Eye spots in Buccinanops cochlidiuin (Dillwyn, 1817) were observed during intracapsular development and in
hatchlings and juveniles. Eyes were histologically confirmed in embiyonic cephalic tentacles; they were comprised of sensory cells, supportive cells,
a lens, and an optic nerve cord. The ontogenetic history of the eyes of B. cochlidiuin is discussed.

Key words: Nassariidae, eye development, embryology, gastropod blindness, ultrastructure

Molluscs represent a group with a diversity of eye types
and range of complexity of eye structure and are becoming
of increasing interest when modeling evolution of eye de-
velopiment (Tomarev et ah 1997, Arendt 2003, Platcheski et
al. 2005). In the caenogastropods, a pair of eyes is usually
located on the outer side of the cephalic tentacles, embedded
in the tentacle or on a small bulge at its base. Originally, a
separate stalk contains the eye in the side of the head im-
mediately posterior to the cephalic tentacle [e.g., Haliotis
Linnaeus, 1758) or partially separated as in the Trochacea
(Hyman 1967, Fretter and Graham 1994).

The interest in eye development in gastropods includes
topics such as the independent development of the eye in
comparison with other classes of molluscs and eye regenera-
tion (Gibson 1984, Bever and Borgens 2005). Even more
interesting is the loss of eyes in eyed lineages. The loss of eyes
has occurred several times in the gastropods. Within them,
some non-related families are cited to have blind represen-
tatives such as the archaeogastropod Pisiilina sp. Neville,
1869 (Neritiliidae) (Kano and Kase 2002), the neogastropods
Buccinanops sp. (d’Orbigny, 1841) and Bnllia sp. (Gray in
Griffith and Pidgeon, 1834, da Silva and Brown 1985) (both
Nassariidae), the opisthobranchs Retusa sp. Brown, 1827
(Retusidae) and Cylichna sp. Loven, 1846 (Scaphandridae)
(Mikkelsen 2002), and the pulmonates Cecilioides sp. de
Ferussac, 1814 (Ferussaciidae) (Heller et ah 1991). A unic]ue
and consistent explanation for eye reduction or loss is not
agreed upon although it is generally associated with habitual
burrowers or living in habitats where light does not reach
such as caves or ocean abysses (Hyman 1967, Fretter and
Graham 1994, Strickler et al. 2001, Kano and Kase 2002).

Comparative studies among eyeless species or lineages could
help our understanding of why eye reduction and loss occur
in nature and complement the modeling of eye development.

The genus Buccinanops (Caenogastropoda, Nassarriidae)
represents a group of seven species, all endemic to the SW
Atlantic Ocean (Pastorino 1993, Rios 1994). The genus name
means ''Biiccinum without eyes” due to the lack of visible eyes
in the adults (d’Orbigny 1841). Within the genus, Bucci-
nanops cochlidiuin (Dillwyn, 1817) is the largest species (Fig.
lA) and ranges from Rio de laneiro, Brazil (23°S) to Pata-
gonia, Argentina (42°S). Animals reach up to 110 mm in
length and are gonochoristic.

A study of the intracapsular embryological development
of Buccinanops cochlidiuin was recently conducted in the
field and conditioned aquaria (Averbuj and Penchaszadeh,
unpubl. ms). Females of the species attach the egg capsules
to the callous region of their own shell. Between 1 and 20
embryos completed their development within the egg cap-
sule after a period of 4 months and the ingestion of thou-
sands of entire nurse eggs. The embryos hatch as 4 mm
shelled, crawling juveniles.

Observations made during this study identified small
dark spots at the base of the cephalic tentacles of the em-
bryos of Buccinanops cochlidiuin while developing inside the
egg capsules. These spots coincided with the description of
eye location in the tentacle and aspect of pigmentation (Fret-
ter and Graham 1994). The structure and location of those
eyes is studied here. Although we tentatively identified these
structures as eyes in the encapsulated embryos, the presence
of these structures in juveniles is not confirmed, and they are
lacking in the adults (Fig. lA).

* From the symposium “Molluscan models: Advancing our understanding of the eye” presented at the World Congress of Malacology, held
from 15 to 20 luly 2007 in Antwerp, Belgium. Co-sponsored by the National Science Foundation and the American Malacological Society.

67

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AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

OCCURRENCE OF EYES IN BUCCINANOPS

69

MATERIALS AND METHODS

Samples were obtained from Villarino Beach, in San
Jose Gulf, Argentina (42°25'S, 64°31'W). Collection was per-
formed by scuba diving over muddy bottoms at depths vary-
ing between 5 and 15 meters. Females with attached egg cap-
sules in different stages of development, juveniles, and adults
were collected and taken to laboratory. The animals were
maintained in seawater conditioned ac]uaria until processed.
Salinity was fixed at 35 PSU and 12 °C, on a 12-12 h light;
dark photoperiod. Individuals’ shell length was measured
with 0.1 mm precision vernier calipers.

Egg capsules representing all stages of embryonic devel-
opment were detached from the shell of gravid females and
dissected. Total shell length (TSL) of the embryos and free
living individuals were measured at different stages of de-
velopment (modified from Bigatti 2005) defined as: cell di-
vision, morulae, “veliger”, late “veliger”, coiling, pre-
hatching, hatchling (all inside the capsules), juveniles, and
adults. All measurements were made under a Zeiss stereo-
scopic microscope with a 0.1 mm precision ocular microm-
eter. Using a microscope, presence or absence of eyes was
recorded at each of the different developmental stages as well
as in hatchlings, juveniles, and adults. Whenever eyes were
found, the cephalic tentacles containing the eye were dis-
sected for histology studies. When the eyes were not visible,
the whole tentacle was fixed and preserved for continuous
sectioning.

Material was pre-fixed in 2.5% glutaraldehyde for two
hours and rinsed in sodium cacodylate buffer, post-fixed
with 2% Osmium for 1 hour, and rinsed again in sodium
cacodylate buffer. Fixed specimens were serially dehydrated
with ethanol in graded steps and embedded in Spur’s epoxy
resin. Sectioning of embedded specimens was done in 1 pm
sections from the base of the tentacle to the apex. Slides were
stained with Methylene Blue for optical microscopy.

When possible, sections of the eye were mounted on
TEM copper grids and stained with 2% uranyl acetate
(Reynolds 1963). This technique enabled the visualization ot
microvilli and/or cilia used to define cell types.

RESULTS

Eye spots were first recognized in all intracapsular em-

bryos at a late “veliger” stage (2.9 ± 0.5 mm of total length;
N = 39) when the cephalic tentacle is developed conspicu-
ously and the foot and shell have already started to develop.
Eyes were also observed at the pre-hatching stage (3.5 ± 0.44
mm; N = 38) and in hatchlings (4.0 ± 0.6 mm of total shell
length; N = 626; Fig. IB).

The eye is located at the basal region of the cephalic
tentacles (Fig. IB-C). In transverse section of the tentacle,
the structure of the eye shows a basal membrane, a retina,
and a lens (Fig. ID-E). Two cellular types (probably photo-
receptor and supportive cells) appear to be present and
forming in the retina. One cell type shows condensed chro-
matin (euchromatin) which is observed as dark nuclei
and corresponds to the photoreceptor cells. The supportive
cells, which occur in a higher frequency than the photore-
ceptor cells, have less condensed chromatin (heterochroma-
tin; Fig. IF).

Pigmentation is present in both cell types in different
degrees of density. The eye’s maximum width in the em-
bryos ranged between 35 and 40 pm (N = 8), in embryos
measuring from 2 to 5 mm of total length. We could not
identify an area for entrance of light to the eye in any section
of the tentacles, as each eye was consistently surrounded by
tentacle tissues (epidermal, muscular, and connective tis-
sues; Fig. ID).

The black spots were also recognized macroscopically in
a single 15 mm crawling juvenile (total shell length), but we
could not find an eye structure microscopically. Tentacles of
an adult (60 mm of TSL) were also studied microscopically
but no eye was found.

DISCUSSION

In this work we recognized for the first time the pres-
ence of eyes in the encapsulated late embryo and confirmed
them histologically in late intracapsular embryo stages. Al-
though there certainly are other species of snails without
eyes in the adult individuals (in Argentina the genus Oliv-
aucillaria d’Orbigny, 1841 (Olividae) and other groups cited
above) but where embryos probably have eyes, to our
knowledge this is the first study on the embryonic eyes of a
blind gastropod species. Loss or reduction ol eyes is usually
associated with living in poorly illuminated environments

f— ^ — : ^ ^ ^ ^ —

Figure 1. Buccinanops cochlidium. A, Adult specimen ot R. cochltdium from Villarino Beach, Patagonia. B, Dark spots at the base ot the

tentacles of a pre-hatching embryo. C, Cross section of the cephalic region of a late “veliger” embryo. The eye on the left tentacle is
pigmented. D, Cross section at the base of the tentacle. E, Detail of the eye with lens and humor and surrounded by tissues (connective,
muscular, and epidermal). The optic nerve is shown. F, Detail of the eye at the base of the tentacle of a pre-hatching embryo, with a unique
lens and different cell types. Abbreviations: e, eye; t, tentacle; on, optic nerve; le, lens; h, humor; pi, pigmentation; phc, photoreceptor cell;
sc, supportive cell; ct, connective tissue; mt, muscular tissue; et, epidermal tissue. Scale bars = 2 cm (A), 1 mm (B), 100 pm (C), and 20
pm (D-F).

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AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

(Hyman 1967, Eretter and Graham 1994). In this case, Bucci-
nauops cochliditim lives in shallow waters in a well-illuminated
environment, yet individuals often are found fo be buried a
few centimeters in the muddy/sandy bottom. An exception
to this observation is when the animals are feeding isolated
or in groups on carrion.

Two cellular types appear to be present in the retina of
Biiccinanops cochlidiu?77. Pigmentation is present in both
types, but in aggregations of different densities. A lens, a
basal membrane, and an optical nerve complete the struc-
ture of the photoreceptor organ. The lens sometimes appears
as one big roundish structure, while in other cases it is
smaller and accompanied by a second similar structure
which is colored darker, resembling a humor (Eig. IE).

Although a pair of dark spots was observed in a 15 mm
(post-hatched) individual, it was not possible to confirm
histologically the presence of eyes. Studying individuals in
this size range would be important to determine whether the
eye is conserved intact or modified, deeply embedded in the
tentacle tissue, or if it degenerates as the animal ages. In
snails measuring more than 20 mm of TSL, eye spots are not
visible macroscopically; thus, the hypothesis that eyes de-
generate in adults is a strong possibility. How does the de-
generation occur? Kano and Kase (2002) discussed possibili-
ties such as reduction in size, loss of retinal pigmentation, or
sinking under the skin.

At the moment, TEM techniques are being used to com-
plete ultrastructure information of the embryonic eyes and
attempt to confirm the photoreceptor cell type, rhabdomeric
or ciliary (Arendt 2003, Plachetzki et u/. 2005). Tentacles of
juvenile and adult individuals of increasing size ranges were
preserved for later studies (continuous serial cut). If eyes
occur in juveniles or adult snails, comparison of the ultra-
structure with that of the embryo will be relevant in order to
know whether it remains equal or if it is modified in type
and number of retinal cells, as happens in other snails
(Blumer 1996, 1998). We found no literature about modi-
fication or loss of embryonic eyes in species with no adult
eyes. Additional work comparing the structure and function
of Buccinanops eyes in related groups, such as the buccinid
Biicdmiw sp. (Linnaeus, 1758) or the nassariid Bidlia sp.
(Brown 1982, Cernohorsky 1984, Allmon 1990) (with and
without eyes, respectively) may also be insightful.

ACKNOWLEDGMENTS

This work was presented at the “Molluscan models:
Advancing our understanding of the eye,” held at the World
Congress of Malacology in Antwerp, Belgium in luly 2007.
A graduate student travel award was given to Liceiiciado
Averbuj by the American Malacological Society. This re-

search was partially supported by Project PICT-10975, GEE
AC-56 (subproject). National Research Council (CONICET)
PIP-5301, and Lie. Averbuj’s PhD Scholarship by CONICET.
Special thanks are given to Isabel Lopez, Eugenia Zavattieri,
and Oscar Wheeler for the technical support in the labora-
tory and field and to Dr. leanne Serb and Dr. Alfredo
Castro- Vazquez for their comments on a preliminary ver-
sion. All research work complies with the current laws of
Argentina.

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Submitted: 18 September 2007; accepted: 24 April 2008;
final revisions received: 1 October 2008

Amer. Maine. Bull. 26: 73-81 (2008)

Evolution of mollusc lens crystallins: Glutathione S-transferase/S-crystallins and
aldehyde dehydrogenase/ fl-crystallins’^

Joram Piatigorsky

Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, 7 Memorial Drive, Building
7, Room 100, Bethesda, Maryland 20892-0704, U.S.A., joramp@nei.nih.gov

Abstract: Diverse crystallins (abundant water-soluble proteins) are responsible for the optical properties of transparent cellular eye lenses
and are multifunctional proteins that have been recruited from stress proteins and enzymes by enhanced lens expression. The major
(S-crystallins) and minor (fi-crystallin) cephalopod crystallins were recruited from glutathione S-transferase (GST) and aldehyde dehy-
drogenase (ALDH), respectively. S-crystallins underwent multiple gene duplications while fi-crystallin appears to be encoded in a single-
copy gene. Except for one S-crystallin (considered a “molecular fossil”), S-crystallins lack enzyme activity due to mutation and insertion
of a variable central peptide by exon shuffling. The li-crystallin is the sole crystallin in scallops. Scallop fi-crystallin does not bind the
co-factor NAD *'/NADH, lacks enzyme activity, and is a tetramer but migrates as a dimer by gel filtration, suggesting structural adaptations
for crystallin function. Similar transcription factors (Pax6 among others) appear to drive high lens expression of crystallin genes in molluscs
and other species consistent with convergent recruitment of the non-homologous crystallin genes.

Key words: cephalopods, scallops, enzymes, lens proteins, eye, gene expression

Cephalopods and scallops have camera-type, lens-
containing eyes that appear grossly like the complex eyes of
vertebrates. Indeed, the similarities in the overall structures
and the common use of opsin family members for photo-
transduction within the photoreceptors have made the cam-
era-type eyes of molluscs and vertebrates prototypical ex-
amples of convergent (independent) evolution of specialized
organs (Packard 1972). However, studies showing that in-
vertebrates and vertebrates employ similar transcription fac-
tors (especially Pax6) for eye development re-opened the
idea that diverse eyes are monophyletic (derived once and
are thus homologous due to common ancestry) rather than
polyphyletic (derived independently multiple times) (Geh-
ring and Ikeo 1999, Gehring 2004). Although the similarities
in their transcriptional networks for eye development fa-
vored a single origin for eyes throughout the animal king-
dom, it is not proof of monophyletic eye evolution for vari-
ous reasons, including the facts that the eye developmental
networks show differences among species, the networks are
not used exclusively for one tissue or organ, and there are
major developmental differences in eye development in dif-
ferent species, especially between invertebrates and verte-
brates. In brief then, the existing data are generally consis-
tent with a great deal of parallel evolution (independent
recruitment of similar networks of genes) among the diverse
eyes of different species.

Analysis of lens crystallins provides a detailed view of
independent processes during eye evolution. Crystallins
comprise 80-90% of the water-soluble proteins of transpar-
ent cellular lenses and are responsible for their optical prop-
erties (Bloemendal and de Jong 1991). The crystallins are
diverse proteins that, despite their common function for lens
refraction, often differ among taxonomic groups, i.e., many
crystallins are taxon-specific (Wistow and Piatigorsky 1988).
In addition to being highly expressed in the lens, crystallins
are often present in lower concentrations in other tissues
where they serve non-refractive roles. Surprisingly, the
taxon-specific crystallins are generally (not exclusively) iden-
tical or related to metabolic enzymes and consequently are
called enzyme-crystallins (Wistow and Piatigorsky 1988, de
Jong et nl. 1989). The use ot the identical protein tor a
refractive role in the lens and for one or more non-refractive
roles in other tissues (as well as in the lens) has been called
gene sharing (Piatigorsky et al. 1988) to convey the situation
of having two or more distinct molecular functions directed
by (i.e., sharing) the identical protein-coding gene sequence
(Piatigorsky and Wistow 1989, Piatigorsky 2007).

Many lens crystallins of invertebrates and vertebrates
display gene sharing since they have non-refractive functions
outside of the lens in addition to their refractive functions in
the lens (Tomarev and Piatigorsky 1996). The present com-
munication reviews the lens crystallins of the camera-type

* From the symposium “Molluscan models: Advancing our understanding of the eye” presented at the World Congress of Malacology,
held from 15 to 20 July 2007 in Antwerp, Belgium. Co-sponsored by the National Science Foundation and the American Malacological
Society.

73

74

AMERICAN MALACOLOGICAL BULLETIN 26- Ml- 2008

eyes of cephalopods and scallops. The existing data are con-
sistent with these molluscan crystallins being recruited from
the metabolic enzymes glutathione S-transferase (GST) and
aldehyde dehydrogenase (ALDH). Many diverse proteins
have been recruited to be crystallins in different species in
vertebrates and invertebrates (Figs. 1 and 2).

S-Crystallins

S-crystallins were named as such because they are the
most abundant proteins in the sc]uid lens (Siezen and Shaw
1982). S-crystallins are a large family of enzyme-crystallins
that are homologous to the metabolic enzyme GST and are
highly specialized for lens tunction (Tomarev and Zinovieva
1988, Tomarev et al. 1991, Tomarev et al. 1993, Chiou et al.
1995). They are differentially expressed in a radial gradient
consistent with their expected role in focusing by creation of
a refractive index gradient (Sweeney et al. 2007). The squid
(Tomarev et al. 1992) and octopus (Tomarev et al. 1991)
S-crystallin mRNAs are expressed strictly in the lens except
that a few are also highly expressed in the squid cornea

(Cuthbertson et al. 1992), consistent with the concept of
corneal crystallins (Piatigorsky 1998, Jester et al. 1999, Pi-
atigorsky 2001). The optical role of corneal crystallins, so
named because of their abundance, remains elusive (Nees et
al. 2002, Jester et al. 2005, Estey et al. 2007).

The cephalopod digestive gland expresses authentic GST
that has high enzymatic activity; this active enzyme is ex-
pressed barely if at all in the lens (Harris et al. 1991, Tomarev
et al. 1993, Tang et al. 1994). The three-dimensional struc-
ture of squid GST (known as GST cr) indicates that it has an
unusually open active site that correlates with its high activ-
ity and has a characteristic dimer interface that differs from
that of the vertebrate GST isoforms (Ji et al. 1995). By
contrast with the digestive gland active GST ct, all but one
(SLll in squid; Lops4 in octopus) of the S-crystallins that
have been examined lack enzyme activity (Tomarev et al.
1995).

The GST-related S-crystallin gene family (>20 mem-
bers) was derived by gene duplications (Tomarev and Zi-
novieva 1988, Tomarev et al. 1992). The S-crystallins lost

/

(Z)

o

/

/

.5*

/

(b

Q

-o

'C-

'tf

a

a

'S

a

t

<Z)

<h

§

is

X q

p

U

51 82

e

T

7t 1 pB

P

Rabbit Elephant

Camel Kangaroo

Platypus

Reptiles

Crocodile

Turtle

Diurnal geckos

Rana pipiens

shrew

Llama

Birds

Duck

Lamprey

(frog)

Guinea pig

Humming

(jawless

bird

fish)

Chimney

/Wo/a mo/a

swift

(fish)

Mammals

Figure 1. Vertebrate taxon-specific lens crystallins. Enzymes in parenthesis lack activity. (Reprinted by permission of the publisher from
Gene Sharing and Evolution: The Diversity of Protein Functions by loram Piatigorsky, p. 62, Harvard University Press, Cambridge, Massa-
chusetts, © 2007 by the President and Fellows of Harvard College.)

MOLLUSC LENS CRYSTALLINS

75

❖

.-S'

Ci-p7 Drosocrystallin SL1 1/Lops4 ; S n/L J1 ; J2 ; J3

Figure 2. Invertebrate taxon-specific lens crystallins. Ci-P^-
crystallin in urochordates may be an ancestor of the vertebrate-P7-
crystallins (Shimeld et al. 2005, Piatigorsky 2006); however, Ckvui
intestinalis does not have a lens in its larval eye (there is no eye in
the adult). Thus, the function of Ci-P^-crystallin is not known.
Enzymes in parenthesis lack activity. (Reprinted by permission of
the publisher from Gene Sharing and Evolution: The Diversity of
Protein Functions by loram Piatigorsky, p. 64, Harvard Llniversity
Press, Cambridge, Massachusetts. © 2007 by the President and
Fellows of Harvard College.)

enzyme activity due to the insertion of an additional exon
encoding peptides of variable length within the genes and to
multiple point mutations sustained throughout the coding
region of the genes. Only the squid SLll gene ancf its or-
thologous gene in the octopus, Lops4, do not contain an
additional central peptide acquired by exon insertion, as in
the other S-crystallin genes; their encoded proteins are thus
more closely related to the enzymatically active GST(t than
are the other S-crystallin proteins. Consec]uently SLl l/Lops4
has residual GST activity (although much lower than GSTcr)
despite being lens-specific. It is interesting, but speculative,
to think of the SLl l/Lops4 gene as a “molecular living fossil”
of the enzyme-crystallin recruitment process during evolu-
tion, representing the original GST duplicate whose expres-
sion was directed to the cephalopod lens (Tomarev ct al.
1995, Tomarev and Piatigorsky 1996).

ll-Crystallin

il-crystallins of molluscs have been recruited from one
or more members of the large group of aldehyde dehydro-
genases (ALDHs) which metabolize a wide number of en-
dogenous and exogenous aldehydes (Perozich et al. 1999,
VasilioLi et al. 1999, Sophos and Vasiliou 2003). ALDHs are

very ancient and found ubiquitously in microbes, plants, and
animals. In eukaryotes alone, the ALDI4s comprise at least
20 gene families. As a general rule, ALDHs with less than
40% identity in amino acid sequence are placed in different
families and those with more than 60% identity belong to
the same subfamily.

Due to the extensive number of ALDH genes, many
showing high sequence identity and extensive gene conver-
sions, their exact relationships to one another and time of
gene duplications during evolution are uncertain. H-
crystallin is a minor crystallin in octopus ( ALDH 1 Cl) and
squid (ALDH1C2) lenses (Chiou 1988, Zinovieva et al. 1993)
and is the only crystallin in the scallop lens (ALDH1A9),
where it comprises —70% of the water-soluble protein (Pi-
atigorsky et al. 2000). It is also the sole crystallin protein
present in the lens of the light organ of the squid Eiiprymna
scolopes where it is known as L-crystallin (Montgomery and
McFall-Ngai 1992). It is noteworthy that aldehyde dehy-
drogenase is the only enzyme known to have been recruited
as a lens crystallin in both vertebrates and invertebrates and
as a corneal crystallin in vertebrates (Tomarev and Piatigor-
sky 1996, Piatigorsky 1998). In vertebrates, the lenses of
elephant shrews contain T|-crystallin (ALDH1A8) (Graham
et al. 1996), and ALDH3A1 and ALDLH Al are the predomi-
nant corneal crystallins of mammals (Abedinia et al. 1990,
Piatigorsky 1998, Jester et al. 2005). Although T|-crystallin
has retinaldehyde dehydrogenase activity (Graham et al.
1996), mollusc (1-crystallins lack enzyme activity (Mont-
gomery and McFall-Ngai 1992, Zinovieva et al. 1993, Piati-
gorsky et al. 2000). A mutation changing a critical cysteine to
arginine in the active site of cephalopod li-crystallins has
occurred, consistent with enzymatic inactivity ot the en-
coded crystallin. Lhiexpectedly however, the active site se-
quence, including the critical cysteine, of the inactive scallop
(l-crystallin is intact. The reason for the absence ot enzy-
matic activity of scallop (l-crystallin appears to be due to the
fact that it does not bind the required co-factor NAD^ or
NADH (Piatigorsky et al. 2000).

In contrast to the S-crystallin mRNAs in cephalopods,
scallop (i-crystallin mRNA is expressed weakly outside of
the lens (gills, muscle) (Piatigorsky et al. 2000). Non-lens
expression of (l-crystallin is surprising because this abun-
dant lens protein appears specialized tor its non-enzymatic
crystallin function. It remains possible that there are two
closely related (l-crystallin genes in the scallop, with the
(l-crystallin gene expressed solely in the lens and its dupli-
cate expressed in other tissues. It is also possible that scallop
(l-crystallin will bind NAD ' and has enzyme activity under
different physiological conditions or can utilize an unknown
substrate, allowing it to act as an enzyme under some cir-
cumstances. ALDHs are known to have wide substrate speci-
ficities as well as having non-enzymatic functions (Sophos

76

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

and Vasiliou 2003, Estey et al. 2007). Thus, further investi-
gations are necessary to reveal the possible enzymatic and/or
other non-refractive function(s) of (l-crystallin.

The squid and octopus fl-crystallins have 78% amino
acid sequence identity to scallop fl-crystallin and 56-58%
amino acid sequence identities to cytoplasmic ALDHIAI
and mitochondrial ALDH2 of vertebrates, while the scallop
H-crystallin is 64% and 67% identical to human cytoplasmic
ALDHIAI and mitochondrial ALDH2, respectively (see
Horwitz et al. 2006). Despite the close similarities of the
cephalopod and scallop ii-crystallins to both cytoplasmic
ALDH 1 and mitochondrial ALDH2 enzymes, phylogenetic
tree analysis shows that the cephalopod Ll-crystallins cluster
with the cytoplasmic ALDHl proteins and the scallop fl-
crystallin clusters with the mitochondrial ALDH2 proteins
(Horwitz et al. 2006).

Despite the sequence homology of scallop H-crystallin
to ALDHl and ALDLI2, the native protein migrates as a
homodimer (100 kD) by gel filtration chromatography
rather than a homotetramer (200 kD) as expected for an
ALDHl or ALDH2 (Piatigorsky et al. 2000). Re-examination
ot scallop fl-crystallin by on-line multi-angle laser light scat-
tering showed, however, that ii-crystallin is a tetrameric
protein despite its chromatographic properties (Horwitz et
al. 2006). It, thus, appears as it the anomalous gel filtration
chromatography behavior of scallop H-crystallin reflects
structural alterations, yet to be discovered, associated with its
specialized crystallin function. In that connection, X-ray
crystallographic studies of elephant shrew Tr)-crystallin pro-
vided direct evidence for numerous structural features indi-
cating that this recruited ALDH-crystallin displays charac-
teristics of the cytoplasmic ALDHl as well as of
mitochondrial ALDH2 (Bateman et al. 2003). Mollusc fi-
crystallins await crystallographic studies.

Finally, mollusc fl-crystallins lack an N-terminal leader
sequence directing the protein to mitochondria (Piatigorsky
et al. 2000, Horwitz et al. 2006). The absence of an N-
terminal leader sequence is unexpected for scallop ii-
crystallin because, in contrast to squid and octopus il-
crystallins, the phylogenetic tree analysis suggests that it was
recruited from a mitochondrial ALDH2-like enzyme. All
known mitochondrial ALDH proteins have a mitochondrial
leader sequence, unlike the cytoplasmic ALDHl proteins
that do not (Piatigorsky et al. 2000, Horwitz et al. 2006). The
absence of a mitochondrial leader peptide allows fi-
crystallins to fill the cytoplasm and serve refractive crystallin
roles rather than be shunted into the mitochondria. Simi-
larly, T|-crystallin of elephant shrews lacks a mitochondrial
signal sequence and is a cytoplasmic, ALDHl protein (Gra-
ham et al. 1996). Cephalopod and scallop H-crystallins also
have numerous sequence differences whose biological sig-

nificances remain to be elucidated, as discussed elsewhere
(Horwitz et al. 2006).

Crystallin gene expression

The hallmark of a water-soluble protein being a crys-
tallin is abundance in the transparent lens. It has been sug-
gested that 5% of the water-soluble protein of the lens be
considered a minimum concentration for a crystallin (de
long et al. 1994). Crystallin genes have complex patterns for
expression in numerous tissues as well as being specialized
for high lens expression consistent with their having been
recruited from proteins with non-crystallin roles presumably
by undergoing a period of gene sharing during evolution.
Moreover, crystallin gene expression in the lens is exquisitely
regulated to create a smooth gradient of protein concentra-
tion that is highest in the center of the lens and lowest at the
periphery. Cephalopod crystallins amount to as much as
70% of the protein in the center of the hard lenses. Scallop
crystallins have a much lower concentration as judged by the
softness of the scallop lenses (personal experience). The gra-
dient of protein concentration in the lens produces a corre-
sponding gradient of refractive index allowing a focused im-
age of incident light upon the retinal photoreceptors. In
addition, the crystallin concentrations within the individual
lens cells must be regulated to minimize refractive index
fluctuations so that the cells are transparent (Benedek 1971,
Bettelheim and Slew 1983, Delaye and Tardieu 1983).

Little is known about crystallin gene regulation in
cephalopods or scallops; therefore, it is useful to apply our
knowledge about the well studied mechanisms of ciystallin
gene regulation in vertebrates. Investigations on vertebrates
have found that lens-specific expression of the diverse, non-
homologous genes is regulated by a similar set ot transcrip-
tion factors that are involved in lens development (Cvekl
and Piatigorsky 1996, Duncan et al. 2004). Foremost among
them is Pax6, which plays a key role in lens development of
all vertebrate lenses (Gehring and Ikeo 1999, Gehring 2005).
Additional factors include members of the Maf family, Sox2
and retinoic acid receptors, among others (Duncan et al.
2004, Piatigorsky 2007). Regulation of lens preferred expres-
sion of non-related crystallin genes by similar transcription
factors implies convergent evolution of crystallin gene regu-
lation. In short, the data indicate that, in vertebrates, the
recruitment of diverse proteins (often with stress protective
or enzymatic functions) to become lens crystallins occurred
by independent modifications of their gene regulatory
motifs (cA-control elements) making them accessible to a
restricted set of transcription factors that direct lens
development.

Although scant, the available data indicate that ciystallin
recruitment in invertebrates has also occurred by indepen-
dent convergent changes in gene regulation. Both S-

MOLLUSC LENS CRYSTALLINS

77

crystallin and ii-crystallin promoters have independently ac-
quired DNA motifs that bind transcription factors from the
same family that are used tor vertebrate crystallin gene ex-
pression. A diagrammatic comparison of transcription factor
binding sites in vertebrate and invertebrate crystallin pro-
moters is shown in Fig. 3. Note that similar transcription
factors (color-coded) bind to or activate the promoters of
the non-homologous crystallin genes. Of particular interest
are the Pax6 (PaxB in jellyfish) and Maf (MARE) binding
sites on the various promoters since these transcription fac-
tors are critical for lens development. Functional studies
showed that minimal squid promoter fragments for the
SL20-1 ciystallin gene and the SLl 1 gene are active in trans-
fected rabbit lens epithelial cells (Tomarev et al. 1992). Pro-
moter activity of these fragments depends upon AP-1 sites.
AP-1 sites are also important for lens activity of vertebrate
crystallin promoters (Piatigorsky and Zelenka 1992). The

Species Crystallin

AP-1 sites in these squid crystallin promoters overlap with
sequences similar to antioxidant and xenobiotic responsive
elements which are involved in rat GST gene and guinea pig
^-crystallin gene expression (Tomarev et al. 1992, 1994).
GST and ^-crystallin (which has quinine reductase activity)
have physiological stress protective functions consistent with
their genes being regulated by stress response elements and
with the stress protective nature of vertebrate crystallin genes
(de long et al. 1989, Horwitz 1992). The putative control
elements of the S-crystallin promoters resemble the PL- 1
and PL-2 (polyomavirus-like enhancer element 1 and poly-
omavirus-like enhancer element 2, respectively) as-control
elements of the lens-specific chicken (3B1 -crystallin pro-
moter (Roth et al 1991).

Although Pax6 appears necessary for establishing the
eye field in cephalopods, it is uncertain at the present time
whether or not it controls S-crystallin gene expression in the

Gene

Promoter

Vertebrates

Mouse

Chicken

aA

aB

yf

PB1

Chaperone/Small heat
shock protein homologue

+44 Small heat shock protein

+45~^

> Ci-Py-crystallin

+30^

Invertebrates

Squid

Scallop

Jellyfish

/ # /

SL11

-93

A-

Q -942 . "f j | t TT

4” 4” ^

-“Czizinn

+93

+50

+ 37

Glutathione S-transferase

Aldehyde dehydrogenase
homologue

Saposin homologue

Figure 3. Diagrammatic representation of ds-control elements in the promoters of crystallin genes of vertebrates (upper) and invertebrates
(lower). Note the similarity in control motifs in the non-homologous crystallin genes. CRE, cyclic AMF-responsive element; MARE, Maf
! regulatory element; RARE, retinoic acid receptor regulatory element. Jellyfish PaxB has a Pax2-like paired domain and a Pax6-like
homeodomain. Ci-Py-crystallin is a urochordate protein and may be ancestral to the vertebrate Py-crystallins (Shimeld cl al. 2005,
[ Piatigorsky 2006). (Reprinted from Piatigorsky (2006) with permission from Nature Publishing Group.)

78

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

lens (Tomarev et al. 1997). A functional Pax6, as judged by
its ability to induce ectopic eyes in Drosophila, is expressed in
the anterior segment of the squid lens, consistent with it
having a role in S-crystallin gene expression. However, nei-
ther the posterior lens cells nor the embryonic squid lenti-
genic cells express Pax6 (Tomarev et al 1997), where S-
crystallin synthesis is active (West et al 1994). Nonetheless,
potential Pax6 binding sites have been identified in the
SL20-1 and SL-1 1 squid ciystallin promoters [A. Cvekl (pers.
comm.) in (Tomarev et u/. 1995)].

Experiments with the scallop Li-crystallin promoter ex-
tend the evidence that lens-preferred crystallin gene expres-
sion in molluscs is controlled by transcription factors that
resemble those regulating lens-preferred crystallin gene ex-
pression in vertebrates (Carosa et al 2002). The scallop il-
crystallin promoter sec]uence contains an abundance of mo-
tifs that are similar to those in vertebrate crystallin
promoters. These include n's-elements for oxidative stress
response factors (CREB/Jun; AP-1 ) and developmental tran-
scription factors ( Pax6; Maf) as well as other gene regulatory
proteins. Moreover, the results of co-transfection and site-
specific mutagenesis experiments are consistent with over-
lapping CREB/lun and Pax6 sites in the fl-crystallin pro-
moter being functional (Carosa et al 2002). Thus, the “lens
nature” of the scallop fl-ciystallin promoter is completely
different from that of other ALDH promoters, which is strik-
ing in view of the fact that the scallop fl-crystallin gene is
homologous to and shares high sequence identity with
ALDHl and ALDH2 proteins (Carosa et al 2002, Horwitz et
al. 2006). Taken together, the data indicate that stress re-
sponse and eye developmental transcription factors regulate
lens-specific expression of mollusc S- and fi-crystallin genes
as they do vertebrate crystallin genes.

Does Pax2 regulate scallop il-ciystallin gene expression?

Cubozoan jellyfish, members of the ancient Cnidarians,
are particularly interesting with respect to the well-
established role of Pax6 in the evolution and development of
eyes throughout animals (Piatigorsky and Kozmik 2004).
Cubozoan jellyfish have camera-type, lens-containing eyes as
do cephalopods, scallops and vertebrates. The complex eyes
of the cubozoan Tripedalia cystophora are situated on four
equally spaced rhopalia nestled in open cavities on the sur-
face bell surrounding the jellyfish (Piatigorsky et al 1989,
Piatigorsky and Kozmik 2004). Each sensory rhopalium con-
tains two of these lens-containing eyes, one larger and one
smaller, situated at right angles to one another as well as four
simpler eyespots comprising photoreceptors that lack lenses.
The rhopalia also have a relatively large statocyst used for
orientation. The complex eyes have a mono-layered cornea
and transparent, cellular lenses which refract incident light
without spherical aberration, indicating the presence of a

gradient of refractive index similar to vertebrate lenses (Nils-
son et al 2005). Three distinct crystallins (J1-, J2-, and J3-
crystallin) (Piatigorsky ef n/. 1989), with J1 comprising three
extremely similar polypeptides (Piatigorsky et al 1993), are
responsible for the optical properties of the lenses of the T.
cystophora.

Despite having sophisticated eyes, jellyfish and other
investigated Cnidaria, lack Pax6 (Sun et al. 1997, 2001,
Groger et al. 2000, Miller et al. 2000). Instead, Cnidaria (as
well as sponges (Hoshiyama et al. 1998)) have PaxB, which
has a Pax2/5/8-like DNA binding paired domain and a Pax6-
like homeodomain. It appears then as if modern Pax2 and
Pax6 genes evolved from a PaxB-like ancestor by duplication
and diversification in higher metazoans (Piatigorsky and
Kozmik 2004). Indeed, PaxB of Tripedalia cystophora is a
functional as well as structural hybrid of Pax2/5/8 and Pax6.
For example, PaxB has the ability to induce small ectopic
eyes in Drosophila as does both Pax2 and Pax6 (Kozmik et al.
2003). With respect to crystallin gene expression, jellyfish
PaxB activates the jellyfish crystallin promoters in co-
transfection tests although its paired domain will not recog-
nize a Pax6 DNA binding site (Kozmik et al. 2003). Pax2, but
not Pax6, is also capable of low-level activation of the jelly-
fish J3-crystallin promoter, consistent with the presence of
Pax2-like ris-control elements that do not recognize a Pax6
paired domain. Crystallin genes of other species are activated
by Pax6 but not by Pax2 since their promoters have Pax6 but
lack Pax2 binding sites.

It is possible, although not known at present, that the
scallop fl-crystallin promoter is responsive to Pax2 as well as
to Pax6. As mentioned above, this promoter has two puta-
tive Pax6 binding sites (Carosa et al. 2002). Inspection of the
sequences of these Pax6 binding motifs reveals that site 2 of
the scallop fl-crystallin promoter has a 3’ C consistent with
preferential binding by Pax2. Moreover, a Pax2 cDNA has
been cloned from scallop eyes (Kozmik and Piatigorsky, un-
publ. data). It remains to be shown that Pax2 is expressed in
the scallop lens and is capable of activating the fl-crystallin
promoter. A Pax2 contribution to the regulation on fl-
crystallin gene expression in scallop lenses would be very
interesting and be consistent with convergent evolution of
crystallin gene recruitment in scallops and jellyfish.

SUMMARY

Cephalopods and scallops have complex camera-type
eyes. Their transparent lenses have recruited crystallins from
the widely distributed families of metabolic GST and ALDH
enzymes. Crystallin recruitment occurred by independent
mutations of the ds-control elements of their genes allowing
their promoters to be activated by transcription factors used

MOLLUSC LENS CRYST ALLINS

79

for lens development. This is the same convergent evolu-
tionary process that has been used in recruitment of the
vertebrate crystallin genes. The GST-derived/S-crystallin
genes of cephalopods are expressed specifically in the lens
and have undergone many gene duplications, insertions and
base changes. Except for one S-crystallin, the cephalopod
S-crystallins are enzymatically inactive proteins.

The cephalopod and scallop ALDH-derived/Il-
crystallins are encoded in single-copy genes. They show high
sequence similarity to vertebrate cytoplasmic ALDHl and
mitochondrial ALDEI2 proteins. Phylogenetic tree analysis
indicates (but does not prove) that cephalopod (l-crystallin
was recruited from a cytoplasmic ALDH 1 -like gene while the
scallop fi-crystallin was recruited from a mitochondrial
ALDH2-like gene. It scallop ii-crystallin was indeed re-
cruited from a mitochondrial ALDH2-like protein, it has lost
its mitochondrial leader to resemble a cytoplasmic ALDHl-
like protein. Neither cephalopod nor scallop fl-crystallins
show enzymatic activity, however for different reasons. The
cephalopod Ll-crystallins have a mutated active site; by con-
trast scallop (i-crystallin has a wild type active site sequence
but the protein is unable to bind the NAD or NADH cofac-
tor required for activity. The scallop (i-crystallin gene is
highly expressed in the lens but, unlike the cephalopod S-
crystallin genes, is also expressed to a lesser extent in tissues
outside of the eye. Scallop fi-crystallin appears to have made
adaptive changes in conformation affecting some of its prop-
erties. Similar to the S-crystallin promoters, the fi-crystallin
promoters have undergone independent sequence modifica-
tions making them responsive to transcription factors used
for lens development in vertebrates, consistent with conver-
gent evolution of crystallin gene recruitment.

ACKNOWLEDGMENTS

I thank Drs. Zbynek Kozmik and Vasilis Vasiliou for
many helpful discussions about various topics in this review.
I thank Iowa State University for support for my attending
the World Congress of Malacology in Antwerp, Belgium
duly 15-20, 2007).

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Submitted: 1 1 September 2007; accepted: 29 July 2008;

final revisions received: 20 September 2008

Amer. Malac. Bull. 26: 83-100 (2008)

Photoreception and the polyphyletic evolution of photoreceptors (with special
reference to Mollusca)"^

Luitfried von Salvini-Plawen

Zoologie, Universitiit Wien, A- 1090 Wien (Vienna), Althanstrasze 14, Austria, Luitfried. Salvini-Plawen@univie.ac. at

Abstract: The general expression of the transcription factor gene Pax-6 homologues and the overall presence of the photopigment opsin
have cast doubt on the polyphyletic evolution of photoreceptors. Herein it is proposed that the evolutionary pathway of photoreceptors
reflects two different, successive processes: (i) the (monophyletic?) differentiation of photoreception as such, mediated by a specific
transcription factor gene (such as Pax-6 or sine ociilis) and (ii) the genetic information of that induction factor (normative unit for
photoreception). The latter stimulates the (polyphyletic) differentiation of the photoreceptors themselves through its multiply convergent
co-options with variable network-modifications (intercalation of different genes). The expression of transcription factor genes does not per
se imply homology of the differentiated photoreceptors (but at most some pattern of homoiology). The differentiation of both receptor
types, ciliary versus rhabdomeric, in one and the same cell during development (of veliger larvae; Blumer 1996) shows them to be
interchangeable structures (mere morphs). Apparently dependent of functional rec]uirements, the structural type of the receptive organelle
has no direct bearing upon the homology identification of the photoreceptors. This obviates the need to propose separate (ciliary and
rhabdomeric) precursor cells in metazoans. A possible primitive “dermal'' receptive cell which was polyphyletically adapted for metazoan
photoreceptors is discussed. The rich morphological diversity of photoreceptors (including the larval organs) in Mollusca appears to
represent in-group differentiations. Their polyphyletic lines are surveyed and the fine structure of eyes of three pteriomorph Bivalvia
species — Lima lima (Linnaeus, 1758) (with a subdivided retina), Clilamys varia (Linnaeus, 1758), and Pseudamiissium peslutre (Linnaeus,
1771) (with incomplete proximal retina) — is reported.

Key words: eye evolution, morphogenesis of photoreceptors, photoreceptor structure, transcription factor gene Pax-6, Lima and Pseii-
damussium (Bivalvia)

Photoreceptors (photoreceptive cells) and more differ-
entiated photoreceptive organs (ocelli, eyes) are crucial sen-
sory equipment for environment information and tor ori-
entation and are, therefore, widely differentiated within the
animal kingdom. Their morphologically diverse organiza-
tion raises questions on evolutionary relationships. New
knowledge provides new views, and these are discussed
herein.

The pioneer in ultrastructure research on photorecep-
tors, R. Eakin (1963, 1965, 1968), advanced the hypothesis
that the evolution of photoreceptors followed two phyloge-
netic lines: the first involved opsin-containing surface en-
largements of the cilia or flagella membrane (ciliary type)
and the second involved enlargements in the form of mi-
crovilli of the distal cell membrane (rhabdomeric type).
Some photoreceptors, however, have cilia as well as micro-
villi (mixed type). In contrast, Salvini-Plawen and Mayr
(1977) proposed a convergent, polyphyletic origin of pho-
toreceptors in about 40-65 independent lines. Later, Salvini-
Plawen (1982) refuted a restriction of the “rhabdomeric

line” to the cerebral photoreceptors of Protostomia or Spi-
ralia (Eakin 1979).

More recently, the widespread findings of the molecular
prerequisites for photoreception have blurred the homology
debate on photoreceptive organs with regard to monophyly
versus polyphyly. Homology implies similar structures or
characters that are based on the same singular (monophy-
letic) evolutionary origin. The debate refers, on the one
hand, to the ubiquitous presence of the (homologous) pho-
topigment opsin and, on the other hand, to the expression of
transcription factor gene paired-box 6 (Pax-6, Pax6) homo-
logues and other genes in photoreceptive cells in Triploblas-
tica. Molecular biologists rapidly postulated that all photo-
receptive cells and organs (ocelli, eyes) — at least of
Triploblastica — are monophyletically homologous (Gehring
and Ikeo 1999, Gehring 2001, 2004, Arendt and Wittbrodt
2001, Arendt 2003). With respect to this new perspective, E.
Mayr (2002: 226) concluded “The origin of eyes in 40
branches of the evolutionaiy tree was always considered to
be an independent convergent development. Molecular biol-

From the symposium “Molluscan models: Advancing our understanding of the eye” presented at the World Congress of Malacology, held
from 15 to 20 fuly 2007 in Antwerp, Belgium. Co-sponsored by the National Science Foundation and the American Malacological Society.

83

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AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

ogy has now shown that this is not entirely correct” [italics
by the present author]. Subsequently Mayr explained (2002:
227) “When survival is favored by the acquisition of a new
structure or other attribute, selection makes use of all avail-
able molecules already present in the genotype” — that also
includes the multiple convergent co-option of a transcrip-
tion factor gene for receptor cell differentiation (see below).

MONOPHYLETIC PHOTORECEPTORS
IN TRIPLOBLASTICA?

According to Arendt anti Wittbrodt (2001: 1546), the
polyphyly of photoreceptors would be restricted to Protista,
Cnidaria, many “aberrant” (“ectoptic/extraocular/extrareti-
nal”) organs (pygidial eyes in Polychaeta, pallial photo-
receptors in Bivalvia, or neural ocelli in Acrania/
Cephalochordata) and the cerebral photoreceptors in “Bila-
teria”. The monophyly of cerebral eyes in Triploblastica (ex-
cluding the bilateral Cnidaria and Ctenophora; cf. Salvini-
Plawen 1978) is based upon the general presence of opsin
and the shared expression of and regulation by the tran-
scription factor gene Pax-6 (Gehring and Ikeo 1999, Gehring
2001, 2004, Arendt and Wittbrodt 2001, Arendt 2003). It is,
however, very difficult to comprehend such a homology in
structurally very different organizations. Examples include
complex eyes in Arthropoda, the inverse eyes of Vertebrata,
or the everse eyes in Cephalopoda. Closer examination leads
to a proposed subdivision of the evolutionary pathway of
photoreceptors to reflect two different processes. The first is
the (monophyletic?) differentiation of photoreception (later
controlled by opsin photopigments) and mediation by a
transcription factor gene that induces receptor cell differen-
tiation. The second is the multiple stimulation by that in-
duction factor to differentiate (polyphyletic) photoreceptors
through multiply convergent co-options with variable net-
work-modifications (intercalation of different genes).

In a strict sense, genetic expression leads to clear ho-
mology of morphological structures or organs only when (in
the simplest case) they correspond by inheritance one-to-
one to a genomic locus or its orthologues (in other species).
More generally, however, genes interact with each other to
effect a specific developmental function and thus form a
hierarchical, developmentally interactive, regulatory net-
work (Fernald 2000, Davidson and Erwin 2006). Transcrip-
tion factor genes act fairly dispersed within such a network,
and the Pax-6 expression is not specific for photoreception.
It is also involved in morphogenesis (structural differentia-
tion) of the cerebral center as well as in chemosensory or-
gans. In squids, besides in the developing eye (but not in the
retina) and cerebral tissues, Pax-6 additionally expresses in

the arms (suckers) and in the mantle (Tomarev et ah 1997, ,

Hartmann et ah 2003). Moreover, Pax-6 generally functions j
in a wide range of processes in further cells, including the j
interactions with other proteins (e.^., Simpson and Price
2002). As underlined by Wagner (2007) with respect to the
transcription factors, the regulatory network within which
Pax-6 expression in Drosophila is embedded includes the
genes eyeless, eyes absent, dachshund, and sine oculis — this
differs from the detailed network in vertebrates as exempli-
fied by Xenopus (Fig. 1). Thus, although they include Pax-6 \
or a homologue, the gene regulatory networks for the de-
velopment of the insect and vertebrate eyes are derived in-
dependently (by intercalation of different genes); in mor-
phological terms, this reflects homoiology at most
(representing convergent structures derived from a homolo-
gOLis character). Therefore, Pax-6 homologues expressing ,
within different regulatory networks do not infer photore-
ceptor homology (see also Zuckerkandl 1994, Fernald 2000, ,

Nielsen and Martinez 2003). Moreover, the sine oculis gene (
of Drosophila (and optix of vertebrates) clearly existed at the
very base of the metazoans (Pichatid and Desplan 2002,
Stierwald et ah 2004). Not the Pax-6, but the sine oculis gene
(or a homologue of it) might be the important ancestral
transcription factor gene for receptor differentiation (see
also Pineda et ah 2002). In any case, only gene regulatory
networks that include an almost identical combination of
transcription factors (called ’character identity networks’ by
Wagner 2007) appear to execute specific developmental pro-
grams that reflect homologous structures and organs.

There is a fundamental difference between potentially
monophyletic, primitive photoreception of cells based upon
a particular gene regulatory network, and polyphyletically
differentiated, distinct photoreceptors or organs (ocelli,
eyes) incorporating independently modified gene regulatory
networks (which in Triploblastica includes Pax-6 homo-
logues). This discrepancy, underlined 30 years ago by Sal-
vini-Plawen and Mayr ( 1977: figs. 2, 9) for dermal light sense
versus photoreceptors, is often neglected (e.g., Vanfleteren
1982, Gehring 2004). Such prerequisite primitive cells with a
transcription factor gene for receptor differentiation possibly
still have “dermal” light sensitivity (sensitive cells without
pigment; cf Millott 1968). This would be similar to the case
in Echinodermata (Yoshida and Takasu 1984), particularly
in the echinoid Paracentrotus (Czerny and Busslinger 1995,
cf also Arendt and Wittbrodt 2001). It would also corre-
spond to the general sensitivity involved in the shadow reflex
(“off response”) in many molluscs and especially of the pal-
lial lobes in Bivalvia (Kennedy 1960, Mpitsos 1973, Messen-
ger 1991, Morton 2001). The particular gene regulatory net-
work of light-sensitive dermal cells became — by selection —
the functionally successful, normative pattern or induction
unit for photoreception. It was then repeatedly co-opted to

PHOTORECEPTION AND POLYPHYLETIC PHOTORECEPTORS

85

Drosophila melanogaster

I

Xenopus laevis

Figure 1. The non-homologous genetic regulatory networks for eye development in insects (Drosophila) and vertebrates (Xenopus)-,
orthologous genes in grey boxes, paralogous genes in white boxes (after Wagner 2007), In Drosophila, the gene twin of eyless (toy) activates
eyeless (ey) which is necessary for eye development; its network includes the genes eyes absent (eya), dachshund (dac), and another
transcription factor sine oculis (so). In Xenopus, the gene retinal hoineohox (Rx) is essential and regulates paired-box 6 (Pax6, a homologue
of eyeless) which includes in its network homologues of eyes absent (Eya 1,2, 3) as well as the sine oadis hoineobox homologue 3 (Six 3) and
its paralogue Optx2. A Rx homologue is also present in Drosophila but not involved in eye development. The non-homology of both
networks is supported in vertebrates by Eya 1,2, 3 which do not regulate the dachshund homologue Dachl, as well as by additional genes
involved.

serve as the foundation for the independent structural dif-
ferentiation (morphogenesis) and evolution of photorecep-
tors. This initial normative induction unit, however, multi-
ply modified its gene regulatory network by intercalation of
different genes (see also Gehring and Ikeo 1999, Gehring
2001, 2002), yielding fhe variously differentialed, polyphy-
lefic phoforecepfors and organs (see Wagner 2007). The ex-
pression of certain specific factors such as Pax-6 of fhe (ofh-
erwise nof idenfical) nefwork has been mistaken for
homology of the photoreceptors themselves. As outlined
elsewhere (Salvini-Plawen 1998a: 139), gene expressions ap-
pear to reflect normative regulators only and do not imply
homology of the induced structures. Moreover, the hypoth-
esis by Arendt and Wittbrodt (2001) of a homology of ce-
rebral photoreceptors throughout all “major bilaterian
branches” suffers from subjecfivify: if claims discrepancies to
be secondary losses and disregards more precise homology
criteria.

Molecular biologists often are not necessarily aware of
the synchronously differentiated organ systems within the
complex “bauplans”. First, the two nervous systems (includ-
ing sense organs) of Triploblastica — gastroneural (at least in
Spiralia) versus epineural — are not homologous and may
simultaneously be present (Salvini-Plawen 2000). Second, it
is functionally consistent that the photoreceptors of direct-
edly moving organisms (“bilaterians”) became differentiated
by selection pressure (stimulation) at the anterior body and

connected to the cerebral ganglia. This condition parallels
the “pygidial” photoreceptors in several retrograde-moving
Polychaeta-Sabellidae (Purschke et al. 2006) as well as the
photoreceptors at the exposed mantle edge in Bivalvia (see
below). Such conditions, however, reflect a general selection
pressure towards convergent formation. Restricted solutions
based on physical laws also play a role (Fernald 2006). Lo-
cation per se is not proof for homology — unless other crite-
ria also provide evidence. Such criteria include relationship,
continuity, structural and/or developmental intermediates,
and an identical gene regulatory network.

Thus, the present analysis focuses not on the origin of
initial light sensitivity (which might be monophyletic) or the
differentiation of phoforeception as such. Rather it deals
with the multiply stimulated “subsequent divergent, parallel
and convergent evolution” of photoreceptors (Gehring 2004:
707 which in fact represents polyphyly). It involves indepen-
dently modified and co-opted gene regulatory networks in
which different genes are intercalated (see also Gehring and
Ikeo 1999, Gehring 2001, 2002). This process leads fo the
convergent, selectively adapted, polyphyletic photoreceptor
cells and organs (ocelli, eyes). Regulator genes are merely
competent for the transcription towards photoreceptive cell
differentiation but not for fhe adaptively convergent-
stimulated, structural differentiation of the multiple cerebral
and “ectoptic/extraocular/extraretinal” photoreceptors
themselves.

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AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

PHOTORECEPTIVE MEMBRANE ENLARGEMENTS

With respect to the distribution ot the two different
types of photoreceptive membrane enlargements, a phylo-
genetically inherited occurrence can be attributed only to a
taxon level ranging between phylum and order (Vanfleteren
and Coomans 1976, Salvini-Plawen and Mayr 1977). This
thoroughly contradicts the separation of photoreceptor evo-
lution into a protostome (rhabdomeric) and a deuterostome
(ciliary) line (Eakin 1963, 1965, 1968).

In addition to the ciliary and rhabdomeric receptor
types, a third type called “diverticular” was recognized
(Salvini-Plawen and Mayr 1977, Salvini-Plawen 1982). It
originates from non-epidermal (sunken, ganglionic), aciliary
cells (neurons) which have an unpleated receptor surface or
show some diverticular membrane enlargements. An ex-
ample is the ganglionic pigment-cup ocelli (Hesse cells) in
the neural tube of Brnnchiostowa; they lack both cilia and
microvilli. Such cells secondarily differentiated their cell
membrane to represent surface-enlarging diverticulae. With-
out knowledge of their genesis, they can easily be confused
with the rhabdomeric type (Salvini-Plawen 1982). Although
later authors (Ruiz and Anadon 1991 ) claimed that this type
was likewise rhabdomeric, Pax-6 is not expressed in these
Hesse cells (Glardon et al 1998). Accordingly, dermal re-
ceptor cell differentiation by that induction factor (norma-
tive unit) is not involved. The seemingly exceptional condi-
tion in Salpa — with neurally derived, aciliary receptor cells
with their hyperpolarizing “off- response” (Gorman et al.
1971; see below) — coincides with the diverticular receptor
type (Salvini-Plawen 1982). This is also valid for the hyper-
polarizing response of two photosensitive neurons of the
abdominal ganglion m Aplysia Linnaeus, 1767 (Krauhs et al.
1979, Andresen and Brown 1982; see below). They also lack
cilia and microvilli (diverticular type).

In view of such a condition, one of the pressing c]ues-
tions is why photoreception involves, on the one hand, cili-
aiy and, on the other hand, microvillar (rhabdomeric, di-
verticular) membrane enlargements. Muhoz-Ctievas (1975)
as well as Vanfleteren and Coomans (1976) presented an
induction theory with a ciliary structure as an organizer
(contradicted by Salvini-Plawen 1982 and Yamamoto 1985).
Eakin (1979) advanced a concept based on mutations (dis-
cussed and contradicted by Salvini-Plawen 1982). Both hy-
potheses are inconsistent. In fact, most ciliary type receptors
represent phylogenetically young organs (Vanfleteren and
Coomans 1976, Salvini-Plawen and Mayi" 1977, Burr 1984).
Based on structural organization, ciliary reception has ap-
parently changed to rhabdomeric reception in certain cases.
These considerations point to ciliary differentiation as the
initial (and/or original) type. This led Salvini-Plawen to pro-
pose a functional dependence and to conclude (1982: 151)

that “the polyphyletic diversity of photoreceptors appears to
be due to different functional requirements at different times
during radiation of evolutionary lines.”

Although functional correlations are still poorly under-
stood, several investigations support such a principal depen-
dence. Some earlier findings in Platyhelminthes-Polydadida
(Ruppert 1978, Eakin and Brandenburger 1981, Lanfranchi
et al. 1981) of ciliary receptors in larvae (adult photorecep-
tors are rhabdomeric) advanced the idea of potential struc-
tural change according to different functional requirements
in larvae and adults.

In Gastropoda, the veliger larvae (but not others, see
below) possess photoreceptors. They represent the organs of
the adult organization precociously advanced into the larval
stage (acceleration; Salvini-Plawen 1980b and below). The
ocelli or eyes of adult gastropods typically show a rhabdo-
meric receptor structure (summarized in Blumer 1996);
some species, however, have ciliated and microvillous recep-
tors (mixed type; see below). Because of acceleration, a rhab-
domeric receptor structure should also be present in the
larval ocelli. This indeed is the case in short-term or actae-
planic veligers (Hughes 1969, Chia and Koss 1978), whose
rhabdomeric photoreceptors might be sufficient for the
short stay in the water column. Based on the argument that
the photoreceptive demands in larvae with a long pelagic life
(long-term or teleplanic larvae) will change accordingly, we
might expect the receptive structure to change. Besides the
evidence in Platyhelminthes-Polydadida (above), the find-
ing of ciliary photoreceptive structures in the long-term ve-
liger larva of llyanassa obsoleta (Say, 1822) (Crowther and
Bonar 1980) supported this point of view.

In accordance with this hypothesis (Salvini-Plawen
1982, 1988), the photoreceptive structures in long-term
(teleplanic) veligers were then demonstrated to correlate
with the ciliary type (Blumer 1994, 1995, 1998, 1999). More-
over, the investigations by Dilly ( 1969) on Pterotrachea mu-
tica (Lesuetir and Peron, 1817), and by Blumer (1999) on
Atlanta peroni Lesueur, 1817, revealed that a permanent pe-
lagic life without a benthic phase (such as in Gastropoda-
Heteropoda) might also be correlated with ciliary photore-
ceptors, even if variously adapted. Other heteropods, such as
Carinaria Lamarck, 1801, however, still differentiate close to
metamorphosis a photoreceptive segment of presumed
rhabdomeric type. Structurally, this represents a mixed type
(Blumer 1998) with a dual function. Thus, short-term (ac-
taeplanic) larvae that spend less than six weeks in the plank-
ton (Scheltema 1989) are correlated with rhabdomeric re-
ceptors whereas long-term pelagic life (larvae/adults)
functionally correlates with ciliary receptors. Finally, Blumer
(1996) demonstrated for teleplanic veliger larvae that the
photoreceptive part of sensory cells themselves is succes-
sively altered during the life cycle: microvilli (for rhabdo-

PHOTORECEPTION AND POLYPHYLETIC PHOTORECEPTORS

87

meric structure) as well as photoreceptive cilia (monociliary
to polyciliary) are added.

These conditions support the idea (Salvini-Plawen
1982) that the ciliary photoreceptive structure is adaptive for
a pelagic existence, i.e., is due to the functional requirements
for this environment. This may refer to the reception of
different wavelengths or to the sensitivity. Mobile benthic
life no doubt demands that photoreceptive organs sense for
orientation and/or vision (correlated to rhabdomeric struc-
ture?). In contrast, larvae within the water column need
information on the direction of the light (correlated to cili-
ary structure?) for optimal feeding close to the surface and
thus for the diurnal migration to that layer (periodicity). In
sessile or semi-sessile animals, photoreceptors must recog-
nize light/shadow (antipredatory) conditions with a hyper-
polarizing “off- response” of (ciliary) receptor potential (cf.
also “dermal” light sensitivity). Accordingly, this is appar-
ently correlated with detecting changes in light intensity (de-
crease or light/shadow reactions) in very slow moving or
sessile animals, with color changes including dorsal/ventral
reactions and paling response in pelagic organisms, and with
rhythmic behavior like diurnal migration of plankton and/or
circadian rhythms (Land 1968, 1984, Salvini-Plawen 1982,
1988). In vertebrates, this appears to be performed by the
dorsal (pineal and parietal) eyes, in direct inheritance of the
(originally paired) ocellus in larvae ot Tunicata-Ascidiacea
(Hamasaki and Eder 1977, Salvini-Plawen and Mayr 1977).
A long-term filter-feeding nourishment — during which the
perception ot light direction was sufficient — may explain
why the ciliary receptor type was genetically firmly an-
chored. Then, when the shift to a directed food uptake
stimulated the differentiation of new, repetitively (itera-
tively) homologous lateral eyes, this receptor type was re-
tained (i.e., not changed to the rhabdomeric morph). Rhab-
domeric photoreceptive organs, however, may be called
upon to correlate yes/no registrations ot light stimuli with a
depolarizing “on-response” (existence and/or increase of
light intensity and direction); this would be more favorable
for orientation.

HOMOLOGY AND THE PRECURSOR
CELL STRUCTURE

Results with veliger larvae (Blumer 1996) show that one
and the same photoreceptive epidermal cell can change its
receptive surface structure according to function. This im-
plies that photoreceptive structure has no direct bearing
upon the identification of homology of the photoreceptive
organs. A case in point is the Gastropoda. The same also
holds true for Placophora, in which the monophyletic aes-
thetes and shell-eyes are ciliary and rhabdomeric structures.

These structures are interchangeable and thus merely func-
tionally conditioned different morphs. In other animal
groups, however, the conditions appear to be different. In
Sipunculida, for example, the inverse cerebral ocelli in the
teleplanic larvae are not continuous with the adult everse
cerebral tubes (Salvini-Plawen and Mayr 1977); the larval
ocelli possess rhabdomeric receptive cells and appear to be
homologous to the inverse pigment cup ocelli with rhabdo-
meric structure in polychaete larvae (Blumer 1997; not con-
sidered by Arendt and Wittbrodt 2001). Similarly, many
members of Polychaeta have different organs for photore-
ception during their life cycle (Purschke et al. 2006): the
larval ocelli may be retained as adult cerebral photoreceptors
but more usually are replaced by adult cerebral organs (e.g.,
Eakin and Westfall 1964, Rhode 1993). In late larvae, there-
fore, both pairs of photoreceptive organs may be visible (e.g.,
in nectochaeta larvae of Polydom sp. (Spionidae) with outer
larval and inner adult ocelli; pers. obs.). Only some ot the
pigmented or unpigmented larval ocelli investigated so tar
possess ciliary receptors. The altered photoreception re-
quirements of larval and adult polychaetes, however, are
reflected in the usual discontinuous differentiation of sepa-
rate, non-homologous organs (versus/in contrast to Barto-
lomaeus 1992). The general predominance of rhabdomeric
structure points to well-established adaptation (directed
movement: cerebral photoreceptors) and, hence, to strong
genetic anchorage. In contrast, ciliar structure appears to be
the initial adaptive answer to photoreceptive requirements
(e.g., light-shadow reaction), with the rhabdomeric structure
as a subsequent differentiation. The ciliary type is, thus,
more frequently (still) represented in phylogenetically rela-
tively young organs (Vanfleteren and Coomans 1976, Sal-
vini-Plawen and Mayr 1977, Burr 1984). Such “new” re-
quirements are evident in the teleplanic veliger larvae versus
the “old” photoreceptors in adult gastropods.

This casts a new perspective on the homology criteria as
related to photoreceptors. The results from long-term ve-
ligers consicierably decrease the value of “compositional cor-
respondence” of Remane’s (1952) three principal homology
criteria (positional correspondence, compositional corre-
spondence, structural, and/or developmental intermediates).
The ability to replace the receptive structure deletes one
prominent morphological character for detailed homology
comparison. Moreover, “positional correspondence” is also
of limited value, as outlined above for cerebral photorecep-
tors. This diminishes the possibility to recognize conver-
gences or parallelisms, unless there is different innervation
or gene expression. At the same time, this contradicts the
arguments of Arendt and Wittbrodt (2001: figs. 8(c), 9) and
Arendt (2003) for separate precursor cells (ciliary versus
rhabdomeric with opsin orthologues) in “Bilateria”: one and
the same cell can alter its photoreceptive part from a ciliary

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

to a rhabdomeric type (Blumer 1996). Thus, the precursor
cells (e.g., “dermal” light sensibility) probably corresponded
to a neutral, “mixed” or combined type with cilium and
microvilli (Arendt and Wittbrodt 2001; fig. 8(b)). This con-
dition was already proposed by Salvini-Plawen and Mayr
(1977: figs. 2, 9) and is supported by recapitulation in cepha-
lopods (Yamamoto 1985).

Gehring’s (2001, 2004) two-cell model of a sensory and
a pigment cell as the original condition in metazoans is no
longer realistic: two types of sensory cells are present. The
first are so-called phaosome photoreceptors, which are
single cells with an invaginated vacuole housing the receptive
organelles (e.g., Clement and Wurdak 1984, Purschke 2003)
which generally show no shading pigment. The second are
receptor cells with intracellular pigment. Although verified
in only in a few photoreceptors, a sensory cell with cilium -I-
microvilli + intracellular pigment probably represents an
early photoreceptive cell associated with shading pigment. In
gastropods, pigmented sensory cells are differentiated in
conservative groups, such as in Patella Linnaeus, 1758
(Docoglossa; Hesse 1902, Marshall and Hodgson 1990) and
in Haliotis Linnaeus, 1758 (Vetigastropoda; Hesse 1902,
Tonosaki 1967, Kataoka and Yamamoto 1981 ). They are also
present in several other representatives (e.g., llyanassa
Stimpson 1865, Murex Linnaeus, 1758, Aplysia, Pleurobrati-
cJnis Cuvier, 1804; Hesse 1902, Hughes 1969, Gibson 1984).
Photoreceptive cells with pigment granules are likewise pres-
ent in the veliger larvae of Smaragdia Issel, 1869 and Strom-
bus Linnaeus, 1758, in Octopus Cuvier, 1798, and olher
cephalopods as well as in addilional animal groups (Eakin
1972, Yamamoto 1985, Blumer 1995).

Going furlher back in evolution, the Cnidaria clearly
point to a single cell type with a mixed/combined or a ciliary
receptor (with opsin) as being the primary precursor struc-
ture for photoreception. Although they lack a cerebral cen-
ter, Cnidaria possess an astonishing range of various
“ectoptic/extraocular/ extraretinal” photoreceptors (Salvini-
Plawen and Mayr 1977), albeit without Pax-6 expression
(Sun et al. 2001). They are generally of the ciliary type,
though microvilli may also be present (e.g., Singla 1974,
Singla and Weber 1982, Blumer et al 1995, Nordstrom et al.
2003). Accordingly, the type of opsins appears to be more
closely related to that of the ciliary receptors or c-opsins
(Plachetzki et al. 2007, Suga et al. 2008). In Leuckartiara
(Anthomedusae) the sensory cells with microvilli, combined
with an unmodified cilium (Singla 1974), correspond fairly
well to the precursor cells (e.g., “dermal” light sensibility)
proposed by Salvini-Plawen and Mayr ( 1977: figs. 2, 9). Also,
the receptor cells in the scyphozoan Cassiopea as well as in
the cubozoans Tamoya and Tripedalia are pigmented (Bouil-
lon and Nielsen 1974, Yamasu and Yoshida 1976, Laska and

Hundgen 1982). All these presumed structural prerequisites
(cilium, microvilli, and pigment granules) may also be com-
bined within one cell. An example is the photoreceptive cell
of cubozoan planula larvae (Nordstrom et al. 2003). It has
no neural connection to any other cells and is structurally
intermediate between a photosensitive dermal cell and a
primitive organ.

The presumed light-sensitive cells in certain sponge lar-
vae (parenchymulae of Ainphimedon qiieenslandica, formerly
Reneira; Porifera-Haplosclerida) each possess a long cilium
and are pigmented. Though neither neurons nor opsins are
present (Plachetzki et al. 2007, Suga et al. 2008), they react to
changes in light intensity (Leys and Degnan 2001).

Thus, the primitive condition is reflected by a single
precursor cell type (also Arendt et al. 2004). As already pro-
posed (Salvini-Plawen and Mayr 1977), cells with a cilium
and with some microvilli (mixed type) probably represent
the basic type for (dermal) photoreception in Metazoa. This
basic type is then convergent-adaptively modified for mor-
phogenesis. Opsins are lacking in poriferans. This supports
the morphological classification of the sponges as Parazoa as
opposed to the Histozoa or Gastrozoa (Salvini-Plawen 1978,
1998b). It also reflects the presence of non-opsin controlled
(“dermal”) photoreception in Metazoa. Accordingly, photo-
reception evolved independently of and prior to the differ-
entiation of opsin photopigment. Later, an opsin-like pro-
tein arose as a visual pigment in basal Histozoa/Gastrozoa
(see also Plachetzki et al. 2007). Due to functional demands/
adaptive “needs”, opsin paralogues (by gene duplication)
were differentiated within the same cells (see Blumer 1994).
A correspondingly adapled opsin type became incorporated
into the membranes of cilia. In the case of mixed receptors,
a paralogue with a different type of transduction cascades
additionally became incorporated into the microvilli (see
also Arendt et al. 2004, Eernald 2006). This implies that two
or more opsin paralogues can be differentiated within the
receptive cells and would explain the expression of the
“wrong” opsin in cells without respective membrane en-
largements (Kumbalasiri and Provencio 2005). Over time,
the cells were provided with intracellular pigment or asso-
ciated with pigment cells.

The photoreceptive organelles in different flagellate Pro-
tista (Gehring 2001, 2004), however, appear to represent one
or several alternative structural answers to the selective profit
of photosensitivity. The most probable ancestors of Metazoa
(based on their primitive monociliar/ flagellate collar cells; cf
Rieger 1976) are the Choanofiagellata or Craspedomonadina
(Salvini-Plawen 1978). They lack photoreceptive organelles
or opsin genes (Plachetzki et al 2007, Suga et al. 2008).
Therefore, the phylogenetic-structural gap between photo-
receptive protists and metazoans additionally points to sepa-

PHOTORECEPTION AND POLYPHYLETIC PHOTORECEPTORS

89

rate structural formations (morphogeneses) of the photore-
ceptive ec]uipment. In contrast, the differentiation of
photoreception itself, it monophyletic, distinctly points to an
earlier process (see above).

POLYPHYLETIC PHOTORECEPTORS IN MOLLUSCA

Our current understanding of the general polyphyletic
structural formation and evolution of photoreceptors re-
mains unchanged: the evolutionary pathway of photorecep-
tors still reflects two different, successive processes. The rec-
ognition that the ciliary and rhabdomeric photoreceptive
structures are interchangeable morphs, however, alters the
premises for detailed homology comparison. Turning to
Mollusca, how often within the phylum was such a tran-
scription factor (as a normative induction unit for photore-
ceptive cell differentiation) convergently co-opted for struc-
tural formation (morphogenesis) of photoreceptors?

The wide range of life-styles in molluscs is reflected in a
considerable variety of photoreceptive differentiations, from
dermal light sensitivity to a rich diversity of ocelli and eyes
(Messenger 1991). Laiwal and/or adult photoreceptors are
present in several dades of Testaria (Placophora and Con-
chifera; cf. Salvini-Plawen 2006). On one hand, there are
cerebrally innervated organs, restricted to larvae and organ-
isms with a free head (Gastropoda, Cephalopoda). On the
other hand, there are quite a number of “extraocular/
ectoptic/extraretinal” organs. In Bivalvia, these are repre-
sented by a rich morphological diversity. Following the
probable evolutionary history of molluscs (Salvini-Plawen
1990, 2006, Salvini-Plawen and Steiner 1996, Haszprunar
2000), however, they originally lacked any photoreceptors
(see Solenogastres, Caudofoveata, Tryblidia, Scaphopoda; cf.
Salvini-Plawen 1972, 1980b, 1982, Salvini-Plawen and Mayr
1977). Nonetheless, scaphopods do show dermal sensitivity
with shadow responses (see Messenger 1991). Thus, all pho-
toreceptive organs of different lines, including larval ocelli,
appear to represent ingroup acquisitions, and no reliable
descendance from differentiations in other spiralians can be
ascertained.

Larval photoreceptors

Mollusc larvae primarily possess no photoreceptors
(Salvini-Plawen 1980b, 1982, in contrast to Bartolomaeus
1992): no ocelli are differentiated in the larvae of Soleno-
gastres, Caudofoveata, Scaphopoda, and protobranch as well
as true lamellibranch Bivalvia. The pseudo-trochophoran
larvae of Placophora, however, have a pair of ocelli which
may persist for some time in the juveniles. Yet, these are
post-trochal and laterally innervated (without a homologous
equivalent throughout metazoans; Salvini-Plawen 1982,
1988). They have a mixed receptor type of microvilli and of

cilia with a somewhat irregular membrane and “a few short
microvilli-like projections” (Fischer 1980: 54; see also Rosen
et al. 1979, Bartolomaeus 1992).

In contrast, the larval ocelli in Bivalvia-Pteriomorpha
are cerebrally innervated and may persist in the adults at
the first branchial filament. Therefore, a homology had been
argued with the cerebral photoreceptors in gastropod
veligers (Rosen et nl. 1978, Bartolomaeus 1992). Four
facts contradict this. (1) The most conservative Bivalvia-
“Protobranchia” as well as the true lamellibranch bivalves
lack ocelli, as mentioned above. (2) Within the Bivalvia (see
Fig. 2), larval ocelli are thus restricted to several families of
Pteriomorpha (Arcidae to Ostreidae; Pelseneer 1908, Hick-
man and Gruffydd 1971, Rosen et al. 1978, Morton 2001).
(3) The larval gastropod photoreceptors are precocious (ac-
celerated) adult organs, not yet differentiated either in the
larvae of basal gastropods or in the plesiomorphically com-
mon organization of bivalves and gastropods. (4) The Bival-
via, whose pre-pedal body is still covered by the shell, clearly
evolved prior to the separation of the free “head” (to which
cerebral eyes are correlated). In the latter organizations — the
Gastropoda and Cephalopoda — the shell is restricted to the
pallio-visceral body. Consec]uently, a common precursor
with “head” and cerebral photoreceptors in Bivalvia (and
thereafter accelerated into the larvae) can be excluded (see
“Protobranchia”). Accordingly, those larval ocelli have in-
dependently evolved only in Pteriomorpha (Pelseneer 1908,
Salvini-Plawen 1982). This reflects an apomorphy for this
dade (see Fig. 2). The sensory cells of these cup-like ocelli
show a microvillous receptor structure with a cilium (Rosen
et al. 1978), and in Mytilus ediilis Linnaeus, 1758 larvae they
react photo-positively (Bayne 1964).

Among the Gastropoda, the lecithotrophic pseudo-
trc^chophores of the most conservative dade, the stereoglos-
sate Docoglossa (patellogastropods), lack photoreceptors.
This is also true for pseudo-trochophores of several Vetigas-
tropoda {Haliotis, Fissurella Bruguiere, 1789) although these
later (“pre-veliger”), however, develop photoreceptors, as do
most true veligers of advanced gastropods. They represent
the precociously advanced (accelerated) organs of the adult
organization (Salvini-Plawen 1980b) rather than primary
larval organs (Bartolomaeus 1992). The Irend lo accelerate
the organs is obvious in Haliotis for example (Grofts 1937,
Buckland-Nicks et al. 2002), and reflects the prolongation of
larval life in the pelagic realm (differentiation of the pro-
totroch to a velum with a second ciliated band, not homolo-
gous to a metatroch; Henry et al. 2007). In short-term ve-
ligers, the photoreceptors are rhabdomeric as in their adults.
In long-term (teleplanic) veligers, however, the receptor
structure is ciliary and changes during metamorphosis to a
rhiibdomeric or mixed type (Blumer 1994, 1995, 1996).

90

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Nuculoidea

Solemyoidea

Nuculanoidea

► "Protobranchia”

d

*Arcoidea ## of
*Limopsoidea U# oj

€L

d

*lMytiloidea
• Pinnoidea
*Ostreoidea
*Pterioidea of

*Anomioidea of
Plicatuloidea

■ Limoidea of-mf(pg)

■ Pectinoidea mf

Trigonoidea

Unionoidea

}

Pteriomorpha

Palaeoheterodonta

Carditoidea

C'rassatelloidea

Lucinoidea

Galeommatoidea

Cyamioidea

Gaimardio\dea

Gastrochaenoidea

Pandoroidea ij

Pholandomyoidea

Thracioidea

Clavagelloidea

Verticordioidea

Poromyoidea

Cuspidarioidea

Hiatelloidea

Solenoidea

Tellinoidea

Sphaerioidea

Arcticoidea

Corbiculoidea

Glossoidea

Veneroidea

Chamoidea

Cardioidea if

Mactroidea
Dreissenoidea
Mvoidea if
Pholadoidea

■o

o

E

o

-g

c

lU

>

C3

"2

'o

s

(Eu-)Heterodonta

Figure 2. Tentative relationships of family groups within Bivalvia (after Steiner and Hammer 2000, Morton 2001, Giribet and Distel 2003),
showing differentiation of photoereceptors. bold, with photoreceptors; underlined, with pallial photoreceptors; *, with larval/cerebral ocelli;
# #, with compound eyes; //, at inner mantle fold; mf, at middle mantle fold; of, at outer mantle fold; pg, in periostracum groove {of-inf.

Organs in adults

Adult Mollusca differentiate several types of photore-
ceptive organs. Most exceptional are the aesthetes and shell-
eyes in Placophora, but most prominent are the cerebral
photoreceptors and eyes in Gastropoda and Cephalopoda. In

autobranch Bivalvia (bivalves except for Protobranchia), dif-
ferent types of photoreoreceptors are present at the exposed
mantle edge, the latter which serves for general orientation.
Such non-cerebral photoreceptive organs are also known in
gastropods and cephalopods (see Table 1).

PHOTORECEPTION AND POLYPHYLETIC PHOTORECEPTORS

91

Placophorn

Adult Placophora possess a particular type of organ, the
aesthete. These are laterally innervated and located dorsally,
embedded within the secondarily calcified outer layer (teg-
mentum) of the eight shell-valves. In addition to other cell
types, they include photoreceptors, which in some taxa have
also given rise to advanced shell-eyes. The photoreceptive
cells of the aesthetes are generally microvillous (rhabdo-
meric; Fischer 1988). \n Acanthochiton fasciciilaris (Linnaeus,
\767), however, the aesthetes of the lateral fields of the valves
differ by including sense cells with ciliary lamellate receptors
(Fischer 1979). The shell-eyes (with lenses) also possess
rhabdomeric receptor structures, whereas their marginal
cells have ciliary receptors (Boyle 1969). In both these cases,
the different receptors probably perform dissimilar func-
tions [e.g., for polarized light?). The extraordinary sensory
organs within the valves of Cryptoplax mysUca Iredale and
Hull, 1905 (see Currie 1992) should also be mentioned al-
though their function is still uncertain. They are surrounded

by pigmented tissue and demonstrate a highly aberrant
structure consistent with a photoreceptive and/or a balance
organ.

Gastropoda

Cerebral photoreceptors in Gastropoda are evcrse but
dilferentiated in various degrees. The structures range from
open eye-pits such as in Patellidae (Docoglossa) or in Neriti-
lidae (Neritopsina; Kano and Kase 2002) to the well-
equipped lens-eyes in higher Caenogastropoda and Euthy-
neura (Salvini-Plawen and Mayr 1977). The receptor
structure is generally rhabdomeric. Some species, however,
show a mixed rhabdomeric and ciliary type: Haliotis (Nor-
dotis) discus Reeve, 1846, Viviparus vivipariis (Linnaeus,
1758), Lacuna viiicta (Montagu, 1803) [-Lacuna divaricata
(Fabricius, 1780)], possibly Fartulunt orcutti (Dali, 1885),
Aporrhais pespelicani (Linnaeus, 1758), Aplysia putrctata
(Cuvier, 1803) (see Howard and Martin 1984, Bartolomaeus
1992, Blumer 1995, 1996, Zhukov et al. 2006). In the ho-

Table 1. Polyphyletic evolution ol photoreceptors, ocelli, and eyes in Mollusca (
Taxon Photoreceptors

13 lines).
Structure

Reference (see also text)

Solenogastres

—

Salvini-Plawen 1982

Caudofoveata

—

Salvini-Plawen 1982

(1) Placophora (larvae)

laterally innervated larval ocelli

mixed

Fischer 1980,

Bartolomaeus 1992

(2) Placophora (adult)

aesthetes in general

rhabdomeric

Fischer 1988

Placophora (adult)

Acanthochiton lateral aesthetes

ciliary

Fischer 1979

Placophora (adult)

extrapigmental shell-eyes

rhabomeric

Boyle 1969

Placophora (adult)

shell-eye marginal cells

ciliary

Boyle 1969

Tryblidia

—

Salvini-Plawen 1982

(3) Gastropoda in general, adult

cerebral photoreceptors

rhabdomeric or mixed

Blumer 1996

Gastropoda-Heteropoda,

adult cerebral eyes

ciliary or mixed

Blumer 1999

Gastropoda, telephanic veligers

cerebral photoreceptors

ciliary

Blumer 1996

(4) Caenogastropoda: Cerithidea

pallial eye

ciliary

Rogge 1987

(5) Gastropoda-Anaspidea: Aplysia

abdominal neurons

diverticular

Messenger 1991

(6) Gastropoda-Onchidiidae

dorsal eyes

ciliary

Katagiri ct al. 1985

Gastropoda-Onchidiidae

lens cells of dorsal eyes

rhabdomeric

Katagiri ct al. 1985

(3?) Siphonopoda (cephalopods)

cerebral photoreceptors

rhabdomeric

Messenger 1991

(7) Siphonopoda

photosensitive vesicles

rhabdomeric

Messenger 1991

Scaphopoda

—

Messenger 1991

(8) Bivalvia-Pteriomorpha

cerebral/iai'val ocelli

rhabdomeric

Rosen et al. 1978

(9) Bivalvia-Arcoida

compound eyes at mantle edge

ciliary

Nilsson 1994

(10) Bivalvia-Arcoida

pigment-cup ocelli at mantle edge

rhabdomeric

Nilsson 1994

Bivalvia-Limoidea

eyes at mantle edge

mixed

Steiner 2001

Bivalvia-Pectinoidea

eyes with two retinae at mantle edge

rhabdomeric ( proximal )
ciliary (distal)

Steiner 2001

(11) Bivalvia-Pandoroidea

eyes with one or two retinae at siphons

both retinae ciliaiy

Adal & Morton 1973,
Prezant 1984

(12) Bivalvia-Cardioidea

eyes at siphons

ciliary or mixed

Salvini-Plawen 1982

(13) Bivalvia-Myidae

phaosomes embedded in siphons

?rhabdomeric?

Light 1930

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AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

lopelagic Heteropoda, the retina of some species is subdi-
vided in adults into an anterior segment with rhabdomeric
receptor cells and a posterior segment with ciliary structure.
Other heteropod species exclusively exhibit a ciliary type of
cerebral photoreceptor, as do all heteropod veligers (Blumer
1998, 1999).

Apart from these cerebral organs, there is a clearly non-
homologous pallial eye in the caenogastropod Cerithidea sca-
lariformis Say, 1825. This has ciliary retinal cells that allow
light/shadow reactions (Rogge 1987).

The photosensitive neurons in the abdominal ganglion
in Aplysia californica J. G. Cooper, 1863 also deserve men-
tion (see Messenger 1991 ). They consist of two receptor cells
of the diverticular type (see above) which respond to light by
hyperpolarization; each possesses intracellular organelles (li-
pochondria) with two orange-red photopigments (Andresen
and Brown 1982).

The Onchidiidae — a family of the Gymnomorpha
(within Aeropneusta the sister-group to Eupulmonata) — are
also noteworthy. Several members possess, apart from cere-
bral eyes (stalked, rhabdomeric), dorsal papillae with up to
seven eyes. These ciorsal mantle eyes show an inverse retina
as well as two lenses above each other (Stantschinsky 1908)
and are pleurally innervated via pallial nerves. The fine
structure reveals particular features: in Onchidiiim verriicu-
latuin Cuvier, 1830, the retina consists of cells with ciliary
receptors which hyperpolarize with an “off-response” (Kata-
giri et al. 1985). The upper, unicellular lens is also photore-
ceptive but with a dense brush of microvilli (rhabdomeric
type) and depolarizes with “on-response”. In addition, On-
chidiiim possesses single dermal photoreceptive cells, iso-
lated or in small clusters, devoid of associated pigment; they
have dense microvilli (rhabdomeric type) and depolarize
with an “on-response”.

Cephalopoda

The typically everse cerebral eyes of Cephalopoda ini-
tially differentiate a rudiment of the retina whose cells have
a root-less cilium and microvilli. This subsequently develops
into a rhabdomeric receptor structure like most Gastropoda
(Yaniiunoto 1985, Muntz and Wentworth 1987, Messenger
1991 ). The free head and other synapomorphic characters in
cephalopods and gastropods (Salvini-Plawen 1990, Salvini-
Plawen and Steiner 1996, Haszprunar 2000) suggest the ce-
rebral eyes of both groups (= Visceroconcha) as being ho-
mologous although evolved to different complexity. Nautdus
Linnaeus, 1758, exhibits simple pin-hole organs, whereas the
Coleoida possess highly differentiated lens-eyes. Additional
photoreceptors are the so-called photosensitive vesicles, viz.
the epistellar bodies in Octobrachia and the parolfactory
vesicles in Decabrachia (Salvini-Plawen and Mayr 1977,

Messenger 1991, Cobb and Williamson 1998, Parry 2000).
These “extraocular/ectoptic/extraretinal” organs are pro-
vided with microvillous receptor cells, contain the visual
pigment rhodopsin, and show a depolarizing reaction to
light.

Bivalvia

In Bivalvia, six different lines of photoreceptive organs
may be recognized (Salvini-Plawen and Mayr 1977, Morton
2001; Pig. 2). One of these (1) includes cerebral ocelli,
whereas five lines (2)-(6) are “extraocular/ectoptic/
extraretinal” photoreceptors that originate at the mantle
rim(s). The border of the mantle, including the siphons,
appears to have acquired a general light sensitivity (see “der-
mal” light sense, above; Kennedy 1960, Mpitsos 1973, Mor-
ton 2001). This gave rise, at different mantle folds and in
various groups (Morton 2001; see Pig. 2), to photoreceptive
organs, all innervated by pallial nerves from the visceral
ganglia.

( 1 ) The cerebrally innervated, cup-like ocelli at the first
branchial filament represent the larval ocelli (see above). In
Striarca lactea (Linnaeus, 1758) they are located at the
posterior labial palp (Thiele 1902: 380 and figs. 145-146).
These organs persist for various times also in the adult ani-
mals. They occur in representatives of several pteriomorph
families (Pelseneer 1908, Rosen et al. 1978, Morton 2001)
and some may be provided with a simple lens (Morton
1978).

(2) A type of compound eye originates at the mantle
edge of arcoidan representatives (cf. Morton 2001). This
includes Arcidae, Glycimeridae (Pectwiadus in Land 1984 =
Glycimeris Da Costa, 1778), Cucullaeidae [Cucidlaea La-
marck, 1801), and Limopsidae (Phdohrya Carpenter, 1872).
Such eyes parallel the compound ocelli of sabellid poly-
chaetes as well as the complex eyes of arthropods (cf. Nilsson
1994, Land and Nilsson 2006). Each “ommatidium” lacks a
real lens and represents an unusually modified ciliary pho-
toreceptive cell accompanied by a supporting pigment cell
(Levi and Levi 1971, Nilsson 1994). Each sensory cell is
covered by a lateral expansion with microvillosities of the
supporting cell and shows an inversed polarity: the nucleus
is positioned distally and the median membrane enlarge-
ments of the laterally located cilia are arranged above each
other in the basal third of the cell. They react to a decrease
in light intensity.

(3) In Pteriomorpha, there are additional photorecep-
tive organs at the mantle edge (see Fig. 2) which may co-exist
with compound eyes or even cerebral ocelli; this underlines
that this clade as adapted to the eulittoral zone. They show
a wide morphological range in configuration and structure.
Examples include the simple cap-shapeci eyespots in Pteri-

PHOTORECEPTION AND POLYPHYLETIC PHOTORECEPTORS

93

oidea (e.g., Isog}iomon Lightfoot, 1786; Morton 2001 ), simple
everse pigment-cup pits in Arcoidea {e.g.. Area Linnaeus,
1758; Nilsson 1994), pinhole-like organs in Limoidea {e.g.,
Lima Bruguiere, 1797; Hesse 1900 and below), everse lens
eyes in Limoidea {e.g., Ctenoides Morch, 1853; Morton 2001 )
as well as on the outer surface of the mantle in Anomioidea
{e.g., Enigmonia Iredale, 1918; Morton 2001), and the un-
usual organs with lens and two retinae in Pectinoidea (Pec-
tinidae, Spondylidae).

With respect to the receptor structure among Pterio-
morpha, except for Pectinoidea, only the pigment-cup pho-
toreceptive organs in Arcidae have been investigated {Area,
Barbatia Gray, 1842, Auadara Gray, 1847; Nilsson 1994).
More ultrastructural studies in other Pteriomorpha are
needed. The organs of Arcidae are rhabdomeric. In the study
by Steiner (2001), three additional species were ultrastruc-
turally investigated: the two pectinids Chlamys varia (Lin-
naeus, 1758) and Pseudat7nissiiim peslutre (Linnaeus, 1771)
as well as the limoidean Lima lima (Linnaeus, 1758) [= Lima
squamosa Lamarck, 1891]. The eye structure of the two pec-
tinids (Fig. 3) is similar to that of Pecten maximns (Linnaeus,
1758) (see Barber et al. 1967), i.e., an inverted retina of
rhabdomeric receptors as well as a distal retina of everse
ciliary receptor cells. The organs possibly function as con-
cave mirror eyes (Land 2000). In both species, the upper,
everted retina shows cells with distally flattened cilia ar-
ranged in whorls (Pig. 4A). Central microtubules and root-
lets are lacking. The two species differ from Pecten in that the
proximal rhabdomeric receptor cells have numerous long
ciliary rootlets (Figs. 4B, 5). The cornea cells are also con-
siderably taller at the center than peripherally (see also Mor-
ton 2001 for Patinopecten yessoensis (Jay, 1857) and Chlamys
pusio (Linnaeus, 1758)). Another major difference is in the
arrangement of the proximal retina. Whereas in Chlamys
varia the rhabdomeric receptive regions of the long sensory
cells form a closed layer below the distal retina, their
nucleus-containing regions are fairly peripherally arranged.
In contrast, the proximal retina of Pseudatnussium peslutre
does not form a continuous layer below the distal retina;
rather, the ciliary retina, which is smaller in diameter, is only
peripherally underlain by the rhabdomeric receptive regions
(Fig. 3). Thus, the proximal retina in Pseudamussium merely
forms an incomplete, ring-like layer centrally without recep-
tors. In the eye axis, only the distal, ciliary retina probably
functions for photoreception.

The distally open photoreceptive organs of Lima lima,
without cornea or lens, are formations of the periostracal
groove between outer and middle mantle folds (Fig. 6). As
already roughly described by Mpitsos ( 1973) for Liiiia scahra
(Born, 1778), rhabdomeric and ciliary sections are separable
in the retina; Nasi (1991) figured isolated rhabdomeric sen-
sory cells with a distal microvillous lobe. Similarly, the retina

CO

Figure 3. Pseudamussium peslutre (Bivalvia). Diagram of a longi-
tudinal section of the eye. a, argentea; cc, ciliary retina cell; co,
cornea; db, distal branch of optic nerve; le, lens; ne, optic nerve;
p, epidermal pigment ring; pb, proximal branch of optic nerve;
rh, rhabdomeric sensory cell; t, pigmented tapetum cells. The
transition from the cell bodies (proximal retina) to the rhabdo-
meric rod is marked by desmosomes (dotted line). Scale bar = 100
pm. (From Steiner 2001).

of L. lima is divided into two regions, the proximal rhabdo-
meric and pigmented cells on the one hand, and distal ciliary
receptor cells on the other hand. The latter also show some
microvilli. In L. lima, however, the transition from rhabdo-
meric to ciliary receptors is gradual. The rhabdomeric cells
show several cilia at the “neck” leading to the bulbous re-
ceptive portion (Fig. 7), thus perhaps representing a mixed
type. The ciliary receptors lack rootlets as well as central
microtubules, and the vast enlargements of their ciliary
membrane surfaces fill the eye cavity (“vitreous mass”, Fig.
8; compare Tridaciia Bruguiere, 1797, below). Part of the
middle mantle fold, above the periostracum-secreting cells,
shows cells with large void spaces and few organelles, thus
representing a “window” for the light (Fig. 6).

No decision can be made as to a homology versus a
homoiology of all these photoreceptive organs at different
mantle folds between the pteriomorph family groups (see
also Morton 2001, 2008). Particularly, no known configura-
tion of photoreceptive organ forms a transition to the ex-

94

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

distal retina. Scale bar 5 |am. B, Rhabdomeric sensory cell of the
proximal retina. Scale bar = 10 pm. a, axon; bb, basal body; c, cilia;
cr, ciliary rootlet; d, desmosomes; g, Golgi stack; m, mitochon-
drion; mv, microvilli; n, nucleus; v, vesicle. (From Steiner 2001).

traordinary eyes of Pectinoidea. The earlier reference (Sal-
vini-Plawen and Mayr 1977, Salvini-Plawen 1982) to a
morphological sequence has been questioned (Morton
2001), ;.e., that an “accessory receptor” of inverse eyes (such
as in Cardiidae, below) could have been modified to the
distal retina in Pectinoidea. The very distant relationship of
the representatives (Veneroidea versus Pteriomorpha; see
Fig. 2) and the independent formation of two retinae in
Laterniila Roeding, 1798 ( Anomalodesrnata, below) renders
the hypothesis speculative. The existence of an “accessory

Figure 5. Pseudainiissium peshitre (Bivalvia). Rhabdomeric sensory
cell of the proximal retina, ax, axon; bb, basal body; cr, ciliary
rootlet; d, desmosome; dmv, double-membraned vesicle; g, Golgi
stack; m, mitochondrion; mv, microvilli; n, nucleus; v, vesicle. Scale
bar = 10 pm. (From Steiner 2001).

organ” associated with the photoreceptors also in some
Pteriomorpha {e.g., Philobryn Carpenter, 1872, Etngmonia-.,
see Morton 2001), however, does not exclude such a struc-
tural sequence as a differentiation model for the eyes of
Pectinoidea. An alternative view (see also Steiner 2001) re-
fers to the more closely related Limoidea (see Fig. 2), whose
retina shows two sections. A major shift and concentration
of their ciliary cells (distal section) towards the light-exposed
side, followed by their separation and medial inversion,
would form a primordial distal retina. This would be fol-
lowed by later reversion to everse cells (compare organogen-
esis in pectinids; Hesse 1908, Kiipfer 1916). In the proximal
section, a concentration of the pigment cells mid-ventrally
(eventually becoming a tapetum) and separation from the
lateral (rhabdomeric) receptive cells would favor a medially
directed inversion of the latter. This would then represent a
peripheral proximal retina such as in Pseiidarniissium
peshitre.

(4) Within Anomalodesmata-Pandoroidea-Pandoridae,
the unusual pallial photoreceptive organs in Laterniila spe-
cies [= Anatina in Pelseneer 1908, footnote] are situated on
tentacles around the siphons. The eyes with lens have two
retinae above each other, both built up (in contrast to Pec-
tinoidea) of everse cells with ciliary receptor structure. They
are surrounded by a cup of pigment cells delimited by a
sclerotic coat (Adal and Morton 1973). Similar to the acces-
sory receptors in Cardiinae and Tridacninae, an eye append-
age associated with the optic nerve is present; this includes a
vertical channel with a bundle of long cilia at its base and
apparently represents a mechanoreceptor (Morton 2001).

Other photoreceptive organs are reported in Pandoroi-

PHOTORECEPTION AND POLYPHYLETIC PHOTORECEPTORS

95

Figure 6. Lima lima (Bivalvia). Diagram of a longitudinal section of
the mantle folds with the eye cup. cc, ciliary sensory cells; imf, inner
mantle fold; mmf, middle mantle fold; mu, muscle; ne, nerve; omt,
outer mantle fold; p, pigment cell; po, periostracum; rh, rhabdo-
meric sensory cell; w, “window”. Scale bar = 100 pm. (From Steiner
2001).

dea-Lyonsiidae by Prezant (1984) for Lyonsia hyalina (Con-
rad, 1831) along the exhalant siphon. These lens-eyes have
pigment cells and they have ciliary sensory cells with the
membrane-enlargements in whorls. Proximally, these whorls
I form concentric rings similar to those in Laternula (above).

(5) Among Veneroida, members of the more closely
related Cardiinae and Tridacninae (Cardioidea) show pho-
toreceptive organs at the siphonal tissues; in Cardiinae a
large number is present at the tips of tentacles. In Cerasto-
derma ediile (Linnaeus, 1758) the eyes are more or less in-
verted without a lens and the receptor cells (ciliary type) are
enclosed in a cup of reflecting cells (Barber and Wright

Figure 7. Lima lima (Bivalvia). Diagram of a rhabdomeric sensory
cell of the proximal eye cup. bb, basal body of cilium, c, cilium;
dmv, double-membraned vesicle; m, mitochondrion; mv, micro-
villi; n, nucleus; v, vesicles. Scale bar = 20 pm (from Steiner 2001 ).

1969). The inner epithelium of the tentacle, basal to the eye
cup, contains pigment granules that presumably also serve as
a reflecting surface (Morton 2001). In addition, an “acces-
sory receptor organ” at the tip of the tentacle is associated
with the optic nerve. Its receptor cells bear numerous un-
modified cilia and microvilli.

In Tridacua, the abundant eyes are everse and the re-
ceptor cells are of the ciliary type. The mass of the micro-
villous membrane-enlargements of the cilia may also func-
tion as a lens (“vitreous mass”; see Lima, above). No cup ot

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AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Figure 8. Lima lima (Bivalvia). Ultrastructural section of a ciliary
sensory cell of the distal area of the retina with tube-like diverticles
of the ciliary membrane filling the eye cup (“vitreous mass”), c,
cilium; cd, tube-like diverticles. Scale bar = 1 pm. (From Steiner
2001).

pigment cells is present, but surrounding layers of olive-
green zooxanthellae may have a reflective function (Erank-
boner 1981). A ciliated “accessory receptor” is associated
with the optic nerve.

(6) Within Myoida, thousands of pear-shaped, pig-
ment-less cells have been reported and described in the si-
phons of Mya arenaria Linnaeus, 1758 (Light 1930). They
are differentiated just beneath the inner epithelium of both
(incurrent and excurrent) siphons. No fine structural inves-
tigation is available, but these cells, which have axons to the
siphonal nerves and an enclosed optic organelle, are devoid
of associated shading pigment. They nonetheless react to an
increase in light intensity with contraction of the joined
siphons. These pear-shaped cells thus parallel the phaosome
cells in Clitellata in both structure and function (see Pur-
schke 2003), the latter representing unicellular photoreceptors.

FINAL CONCLUSION

The proposed evolution of photoreceptors according to
two different, successive processes is compatible with the

general expression of transcription factor genes as well as
with the rich morphological diversity of the photoreceptive
organs throughout the metazoans. Earlier representations
with respect to this polyphyly are now modified: the struc-
tural types of the epidermal photosensitive receptors (ciliary,
rhabdomeric) are now considered to be mere morphs, ob-
viating the need for arguments on homology or non-
homology. The homology identification of photoreceptors
must, therefore, follow other precise criteria (relationship,
continuity, structural and/or developmental intermediates,
identical gene regulatory network). According to such crite-
ria, evolutionarily independent structural differentiation
(morphogenesis) of photoreceptors in Mollusca can be pro-
posed for at least thirteen lines (Table 1), irrespective of
potential multiple homoiology of pallial organs within
pteriomorph Bivalvia (Eig. 2).

ACKNOWLEDGMENTS

The author is very grateful to Prof. Dr. Gunter Wagner
(Yale University) for providing him with the manuscript of
his contribution prior to its publication (2007) and to Mag.
Maria Theresia Steiner (Vienna) for the courtesy of placing
her diploma thesis (2001) at the author’s disposal. Many
thanks also to Dr. Michael Stachowitsch (Vienna) for pol-
ishing the English text.

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Submitted: 3 September 2007; accepted: 2 May 2008;

final revisions received: 4 November 2008

Amer. Maine. Bull. 26: 101-109 (2008)

Primary inhibition by light: A unique property of bivalve photoreceptors’^

Lon A. Wilkens

Center for Neurodynamics and Department of Biology, University ol Missouri-St. Louis, One University Boulevard,

St. Louis, Missouri 63121, U.S.A., wilkensl@umsl.edu

Abstract: Bivalve molluscs are known for shadow responses involving closure or retraction of the siphon and valve adduction. In
representative genera [Spisiila solidissima (Dillwyn, 1918), Mercenaria mercenaria (Linnaeus, 1758), Limn scahrn (Born, 1778)], the pallial
nerves contain photosensitive fibers that exhibit physiological shadow responses. These photoreceptors are inhibited by light but trigger an
excitatory burst of spikes to a shadow, the off-response. Equivalent responses are seen in bivalve eyes, e.g., in optic fibers from the siphon
tentacle eyes of Cardiitm edide (Linnaeus, 1758) and the mantle eyes of scallops (Pectinidae) and hie clams (Limidae). In scallops, they form
a distal retinal layer of ciliary receptors, distinct from a proximal microvillar layer that is excited by light. In otf-receptors (ciliary), light
inhibition is the result of a hypierpolarizing receptor potential with spikes generated on the rebound depolarization at dimming. In contrast,
the proximal on-receptors are excited by light with spikes generated by depolarizing receptor potentials. The inhibitory effect is hrst-order,
i.e., a direct response to light, as is excitation for the proximal receptors. With separate retinas and the absence of synaptic contact, these
are the primary receptors. Aside from Pecten Muller, 1776 and Lima Bruguiere, 1797, the only other bivalve eyes in which receptor potentials
have been investigated are those of the giant clam Tridnam maxima (Rbding, 1798). Here there are two types of hyperpolarizing,
light-inhibited primary receptors, one of which generates spikes at light offset, the other non-spiking. The inhibitory response is universal
in bivalve photoreception, unique among the eyes of invertebrates, but similar in polarity to chordate photoreceptors although the ionic
mechanisms are different. Receptor physiology is discussed relative to image formation in bivalve eyes.

Key words: shadow response, mantle, retina, Pecten, Tridacna

The cast of a shadow onto benthic and/or sessile organ-
isms commonly triggers some form of withdrawal into a
shell or burrow. Such is the case for most shallow, subtidal
bivalve molluscs. In bivalves, this ‘shadow response’ involves
a variety of movements including siphon closure, retraction
of the siphon or mantle, and shell adduction (Wenrich
1916). For most bivalves this response does not require eyes.

Rather, the mantle tissues themselves are sensitive to light as
confirmed in pallial nerve recordings from a number of
species (Kennedy 1960, Mpitsos 1973, Wiederhold et al.

1973). However, a few bivalve families have added func-
tional eyes ectopically around the shell-mantle edge (Morton
2001). These constitute a well-developed visual system for
motion sensitivity that triggers sight reactions independent
of direct shadowing of the animal (Nilsson 1994). Most
prominent are the distinctive blue eyes of the scallop (Fig. 1 ),
visual organs that have attracted the attention of classical
morphologists (Dakin 1910, 1928) to present-day neurosci-
entists. Morphology and optics have been examined in detail
in the eyes of Pecten maxinius (Linnaeus, 1758) (Dakin 1910,

1928, Land 1965, Barber et al. 1967), recently in Aimisium
balloti (Bernardi, 1861), Argopecten irradians irradians (La-
marck, 1819), Chlamys hastate (Sowerby, 1842), Chlaniys
rubida (Hinds, 1845), Spondylns americanus (Herman,

* From the symposium “Molluscan models: Advancing our understanding of the eye” presented at the World Congress of Malacology, held
from 15 to 20 luly 2007 in Antwerp, Belgium. Co-sponsored by the National Science Foundation and the American Malacological Society.

1781), and Crassodoina gigantea (Gray, 1825) (Speiser and
Johnsen 2008), Cardium edide (Linnaeus, 1758), and Tri-
dacna maxima (Roding, 1798) (Barber and Land 1967,
Kawaguti and Mabuchi 1969, Land 2003, Stasek 1966) to
evaluate bivalve vision. Here, physiological properties are
examined with emphasis on the unique inhibitory response
to light in bivalve photoreception and its role in behavior
and visual function.

Light inhibits sensory neurons in the mantle and eyes
Bivalves, alone among the non-chordate invertebrates,
have adopted a near-universal inhibitory response to light, a
physiological mechanism suitably adapted for the shadow
response. Termed ‘primary inhibition’, activity in photosen-
sory neurons is suppressed by the absorption of light inde-
pendent of synaptic interactions at the receptor level and
therefore a first-order response. Fhotoreception in chordates
is also inhibitory, hyperpolarizing by convention, but with
contrasting mechanisms to be considered later. Light inhi-
bition was first observed in optic nerve recordings from
scallop eyes (Hartline 1938). Upon dimming these same fi-
bers produce vigorous bursts of action potentials or spikes,
the ‘off-response’. However, inhibition is also characteristic
of the dermal light response in the bivalve mantle. For ex-

101

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Figure 1. Argopccteu irradians taylome (Fetuch, 1987) in its shal-
low-water, turtle grass habitat, St. Joseph Bay, Florida.

ample, the siphonal nerves of the lamellibranchs Mya are-
naria (Linnaeus, 1758), Mercenaria (=Vemis) tnercenaria
(Linnaeus, 1758), and Spisula solidissima (Dillwyn, 1817)
each contain neurons whose spontaneous activity is inhib-
ited by light. In Spisula this “simple” photoreceptor system
(Kennedy 1960) consists of a single pair of siphonal neurons
leading to the visceral ganglion where the motor response
for siphon retraction is generated. In these neurons, the light
response has been characterized as a dual inhibitory-
excitatory process involving different photopigments. The
response is modeled as the sum of a low-threshold, short-
wavelength inhibition and a long-wavelength excitation re-
sponsible for the off-response burst of impulses (Fig. 2). The
photopigments, as yet unidentified, are assumed to coexist

■111111-^

Figure 2. Model of primary inhibition in Spisula siphonal neuron.
Light (bar) evokes a low-threshold light intensity inhibition (I) and
delayed, long-lasting excitation (E) whose sum is the receptor po-
tential (RC) that accounts for the inhibition of spiking and the
burst of action potentials at light offset. The inhibitoiy effect is
hyperpolarizing and the excitatory effect depolarizing. After
Kennedy ( 1960).

within the neuron since the light response does not originate
from presynaptic receptors. Likewise, photosensory neurons
in the siphon nerves of the hard shell clam M. mercenaria are
inhibited by light, with spikes elicited only in response to
dimming (Wiederhold et al. 1973). Membranous whorls in
the distal portions of these neurons indicate, again, that
these neurons contain photopigments and respond directly
to light. Mantle neurons in the file clam Lima scahra (Born,
1778) also generate off- responses independent of light sen-
sitivity in the eyes (Mpitsos 1973).

Light inhibition in the scallop eye was initially attributed
to synaptic interactions in the retina (Hartline 1938), remi-
niscent of the lateral inhibitory synapses among Limulus
polyphemus ommatidia well known as the basis for contrast
enhancement (Hartline and Ratlliff 1958). This interpreta-
tion grew out of the fact that the scallop retina contains a
dual layer of photoreceptor cells (Fig. 3). Inhibition and the
corresponding excitatory off-response are properties of the
distal layer, whose ciliary photoreceptors and axons form the
distal ramus of the optic nerve. Optic fibers from the proxi-
mal, rhabdomeric layer form the proximal ramus and re-
spond to light with an excitatory ‘on-response’. Although
Hartline favored the interpretation that the proximal recep-
tors were the source of excitation for the off-response in the

Figure 3. Diagrammatic representation of Pecten eye. Modified
from Dakin (1928).

BIVALVE PHOTORECEPTORS INHIBITED BY LIGHT

103

distal receptors, the latter equivalent to vertebrate retinal
ganglion cells, he also considered the possibility thirt distal
receptors represented a new type of cell, “excited only by the
removal of a stimulating agent” (p. 477). The latter inter-
pretation, that the distal receptors are a new type of cell, is
now accepted, both for distal receptors in the eye and for
photosensory fibers in the mantle. In the eye, ultrastructural
examination ot the retina has since revealed no evidence for
synaptic connections either between proximal and distal lay-
ers or among neighboring photoreceptors (Barber et al.
1967), thereby ruling out synaptic interactions as the basis
for inhibition by light. Unecjuivocal segregation of proximal
and distal responses has also been confirmed by cutting the
distal ramus, thereby eliminating off-response activity in the
optic nerve (Hartline 1938, Land 1966). With minor differ-
ences in spontaneous activity the visual responses are
equivalent tor all species ot scallop. As in scallops, the eyes of
Lima scabra have a dual retina and on- and off-responses in
the respective optic fibers of proximal and distal origin
(Mpitsos 1973). Also, ciliary receptors that resemble those in
the scallop distal retina are present in the tentacle eyes of
Cardiiim edule and spiking in their optic fibers occurs only
in response to a decrease in light intensity (Barber and Land
1967).

The inhibitory effect of light in the distal (ciliary) pho-
toreceptors of scallops, along with bivalve siphonal nerves, is
unusual considering that a light stimulus to the eyes ot most
animals is excitatory and triggers bursts ot impulses in optic
nerve fibers, as in the excitatory on-response in the scallop
proximal (rhabdomeric) photoreceptors. An excitatory re-
sponse, to light or any other sensory stimulus is generated by
a depolarizing receptor potential, a reduction in membrane
potential toward spike threshold. Impulses arise in response
to these depolarizing currents, either at the spike initiation
zone of photosensory neurons or in presynaptic non-spiking
retinal cells. Hyperpolarization, in contrast, increases the
membrane potential away from threshold levels exerting a
stabilizing, spike-inhibiting influence. Excitatory, depolariz-
ing receptor potentials in response to light are characteristic
ot all non-chordate invertebrate eyes examined so tar, with
bivalve eyes the only exception. Representative examples,
based on intracellularly recorded receptor potentials, include
annelids (Walther 1965, Eioravanti and Fuortes 1972), in-
sects (Naka 1961), gastropod (Dennis 1967) and cephalopod
molluscs (Tomita 1968), and arthropods, as first demon-
strated in the horseshoe crab Limiilns polypheimis (Hartline
et al 1952, Fuortes 1959). Each of these ‘excitatory’ examples
represents a phylum or taxon (annelid, gastropod, cephalo-
pod, arthropod) with rhabdomeric photoreceptors. Photo-
sensitive neurons in the arthropod central nervous system
are also excitatory, e.g., the crayfish caudal photoreceptor, a
photosensory interneuron in the 6th abdominal ganglion

discovered by Prosser (1934) and later shown to be depo-
larized by light (Wilkens and Larimer 1972). However, ex-
ceptions to the bivalve-only inhibitory effect exist if one
considers extraocular photosensitivity, e.g., gastropod CNS
neurons are inhibited and/or hyperpolarized by light {Aply-
sia calijoriiica (Cooper, 1863), Brown and Brown 1973; On-
chidium verriiciilaliim (Cuvier, 1830), Goto and Nishi 2002;
see review of extraocular photosensitivity by Yoshida 1979).
In most instances these neurons are neither ciliary nor
rhabdomeric.

In bivalves, receptor potentials obtained from intracel-
lular recordings were first observed in photoreceptors from
scallop eyes (Toyoda and Shapely 1967, Gorman and
McReynolds 1969). Receptor potentials in the distal layer are
hyperpolarizing in response to light (Fig. 4B), consistent
with primary inhibition and spike suppression previously
established for these receptors. At light offset the membrane
potential rapidly depolarizes generating the off-response
burst of spikes. In contrast, the proximal photoreceptors are
depolarizing (Fig. 4A), generating spikes at light onset. Simi-
lar hyperpolarizing (inhibitory) and depolarizing (excitato-
ry) responses have been reported for the corresponding reti-
nal receptors in Lima scabra (Mpitsos 1973).

A -

I

Figure 4. Receptor potentials with accompanying action potentials
in excitatory proximal (A) and inhibitory distal (B) photoreceptors
ot Argopecteii (=Pccteii) irradians. Membrane depolarization ex-
ceeds threshold and triggers a spike burst in the proximal receptor
(A). Hyperpolarization inhibits ongoing spontaneous activity in the
distal receptor followed by a burst of spikes with depolarization
following light off. The inhibitory response is similar to the inhibi-
tory model (Fig. 2). From McReynolds and Gorman (1970a).

104

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Receptor physiology has been studied in the eyes of only
a single additional bivalve species, the giant clam Tridacna
maxima (Wilkens 1988). Although no recordings were made
from optic nerve fibers, responses to light recorded intracel-
lularly from retinal cells are hyperpolarizing, consistent with
primary inhibition characteristic of bivalve photorsensitivity.
Unlike the scallop, however, two types of inhibitory photo-
receptors have been described, ancf no excitatory cells were
found. Classified as spiking (S) and non-spiking (NS) recep-
tors (Fig. 5), these cells can be distinguished by the following
criteria. The S-cells, which share most of the characteristics
of scallop distal photoreceptors, are hyperpolarized by light
and spikes are suppressed. Similarly, light has a secondary
excitatory effect seen in the strength of the spike burst aris-

A

I \ f \

i W '■

ing from the off-response depolarization. This is illustrated
(Fig. 5A) where spikes are absent in the second off-response
as a consequence of reduced light adaptation from the initial
dark period, and where the number of spikes increases rela-
tive to increases in duration of the preceding period of illu-
mination (Fig. 5B). Spiking in NS-cells has never been ob-
served. It is possible that the absence of spikes is due to
injury from electrode penetration, as has been reported
commonly for both cell types in scallops (McReynolds and
Gorman 1970a). However, S-cells retain their ability to con-
duct action potentials as long as cell penetrations are main-
tained, for periods up to an hour. Further, T. maxima re-
ceptors are invariably distinguished by differences in
membrane potential and light adaptation. S-cell receptor

V35

B

\

-•-10

■ -30

■ -50
■*-70

Figure 5. Receptor potential in Tridacna photoreceptors. Spiking S-cell off-response beginning when fully light adapted following several
minutes under bright illumination (A) and beginning when fully dark adapted (B). The light-off burst of spikes is greatest for higher degrees
of light adaptation, e.g., the initial dark period in (A) and spike counts in response to relative durations of light exposure in (B). Non-spiking
NS-cell (C) beginning in a fully light-adapted state followed by shadow stimuli of increasing duration. Inhibitory hyperpolarization in
response to light increases following increasing intervals of illumination. Relative light adaptation in S-cells is evident in the spike burst
response but not in receptor potential amplitude, whereas NS-cell responses change dramatically with relative light adaptation. The light
stimulus is monitored in the lower trace of each pair; light on is the upward position. Vertical scale indicates actual membrane potential
in millivolts; horizontal bar = 30 s in (A) and (C), 5 s in (B). Modified from Wilkens (1988).

BIVALVE PHOTORECEPTORS INHIBELED BY LIGHT

105

potentials are seemingly immune to adaptation, either in the
light or in the dark (Pig. 5A-B), whereas the receptor po-
tentials of NS-cells respond variably as a function of light
exposure (Pig. 5C). At the beginning of this record (Fig. 5C),
the cell is fully light adapted and inhibitory-like hyperpolar-
izations increase dramatically following increasing durations
in the dark. On-response hyperpolarizations also adapt rap-
idly, returning to the light-adapted baseline irrespective of
changes in relative dark adaptation as seen throughout this
record. Membrane and receptor potentials also vary consis-
tently, with average resting (dark) potentials of -44 mV in
S-cells and -14 mV in NS-cells and hyperpolarizing off-
responses averaging 14 mV or up to 100 mV in fully light-
adapted S- and NS-cells, respectively. Both cell types have
axons that emerge from the receptor soma, as seen in injec-
tions of fluorescent dyes following recording sessions
(Wilkens 1988). These converge to form the optic nerve
described morphologically by Stasek (1966) and Kawaguti
and Mabuchi (1969). Neither Kawaguti and Mabuchi ( 1969)
nor Fankboner (1981) report synapses in their electron mi-
crographs of the T. maxima retina.

Receptor physiology, consistent in its inhibitory re-
sponse to light, is nevertheless significantly different in the
eyes of scallops and giant clams. Differences, as yet unex-
plained, also exist in the organization of the visual apparatus.
The dual retinas in scallops form a common optic nerve that

projects without branching or synapse formation into the
lateral (optic) lobes of the parietovisceral ganglion (Spagno-
lia and Wilkens 1983, fig. 6). Any mantle reactions to light
are therefore dependent on efferent projections back to the
periphery. In Tridacna maxima small pieces of mantle tissue,
excised and pinned out for recording, exhibit local retrac-
tions to shadows. This requires that optic fibers synapse with
other cells after leaving the eye capsule, or that dermal pho-
tosensitivity exists in the mantle aside from the eyes, as in
Lima scabra (Mpitsos 1973). Receptor organization is also
different, as histological studies in T. maxima do not show
the distinct retinal layers characteristic of scallop eyes. Tri-
dacna maxima receptor cells are described by Fankboner
(1981) as having numerous ciliary processes that give rise to
tangles of microvilli whereas Kawaguti and Mabuchi ( 1969)
distinguish two cell types, one of which is described as rhab-
domeric but having ciliary basal bodies. Both authors may be
describing the same ciliary receptor somewhat diflerently,
but no evidence as yet relates any structural difference to the
physiologically distinct S- and NS-cells.

Contrasting ionic mechanisms of hyperpolarizing
receptor potentials

The receptor potentials and mechanisms of phototrans-
duction in scallops have been studied extensively with re-
spect to their differing polarity. In Argopecten irradians ir-

Figure 6. Diagram of the parietovisceral ganglion of Pecteii. From Dakin (1910). The larger lateral lobe on the right corresponds with the
upper (left) mantle that contains a larger number of eyes than present on the lower (right) mantle. I’he inset (Wilkens, unpublished) shows
autoradiographic labeling of cortical neurons (surface layer) and a spherical glomerulus after injection of tritiated proline into the eye and
axonal transport via the pallial nerves into the lateral lobe.

106

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

radians, both depolarizing and hyperpolarizing receptor
potentials are based on increases in ionic conductance
(McReynolds and Gorman 1970b). The excitatory depolar-
izing potentials of arthropods, and presumably other inver-
tebrate photoreceptors, also arise from increases in mem-
brane conductance (Euortes 1959), whereas in vertebrate
receptors the ‘inhibitory’ hyperpolarization is associated
with a decrease in conductance (Toyoda et al. 1969). There-
fore, receptor polarity and conductance changes must be
explained as a function of ion channel specificity. In scallop
proximal receptors, an increase in inward sodium conduc-
tance drives the excitatory depolarization (Gomez and Nasi
1996), similar to other depolarizing receptors. In the distal
receptors of scallops, which have received the greatest atten-
tion, hyperpolarization is due to an increase in outward
potassium conductance (Gorman and McReynolds 1978),
with similar ion specificity in Tridacna maxima (Wilkens
1988). These experiments are based on ion substitution and
reversal potential measurements, with similar results in scal-
lops and giant clams. However, hyperpolarizing light inhi-
bition, as seen in bivalve photoreceptors and in vertebrate
rods and cones, is based on contrasting mechanisms. In
bivalves, hyperpolarization is due to an increase in conduc-
tance, i.e., outward potassium current. In vertebrates, hyper-
polarization is due to a decrease in conductance shutting off
the leaky, inward sodium currents that depolarize the cell in
the dark. Thus, the inhibitory effects of light are explained
ultimately by ion channel specificity and its activation or
suppression by the photochemical mechanisms following
light absorption. Lor the scallop distal photoreceptors, as in
vertebrates and other animals, much additional work has
been directed towards understanding the cascade of second-
messenger intermediates (Gomez and Nasi 2000), the
evolutionary history of opsin photopigments, and their
control of membrane conductance, and bivalve photorecep-
tors remain of great value in the comparative study of
phototransduction.

Historically, the evolution of the eye has been of im-
mense interest, including the morphology of its light-
sensitive cells. Photoreceptors generally involve an extensive
membrane hypertrophy involving either the cilium or mi-
crovilli of the cell membrane in the formation of a rhabdom.
Aside from the competing theories concerning the phyloge-
netic relationships of photoreceptor morphology, some
trends are in evidence physiologically. Lor example, all de-
polarizing/excitatoiy photoreceptors are exclusively rhabdo-
meric, including the examples mentioned previously, e.g., all
arthropods, annelids, gastropods, cephalopods, and the
proximal receptors in pectinid eyes. In contrast, hyperpolar-
izing photoreceptors are predominantly ciliary although the
list is fairly short, e.g., vertebrate rods and cones, the distal
photoreceptors and siphon/mantle nerves in bivalves, and

the photoreceptors in the larval eyes of the tunicate Ama-
roucium constellatiun (Gorman et al 1971). A lone exception
is found in the eye of another tunicate, Salpa democratica,
where the hyperpolarizing response is associated with a mi-
crovillar-type receptor although these eyes are considered
non-homologous to those of other primitive chordates
(Gorman et al. 1971). In another exceptional instance, a
putative ciliary photoreceptor is found in the brain of the
polychaete annelid Platynereis dumerilii (Arendt et al 2004)
although its response to light is unknown. Ciliary opsin
similar to that of vertebrate rods and cones is associated with
these photoreceptors but not the rhabdomeric receptors of
the eyes.

Despite these trends, information is insufficient for link-
ing physiological sensitivity to the evolution of photorecep-
tor morphology. Hyperpolarization is seemingly a charac-
teristic of all chordate photoreceptors, as it is in bivalve eyes.
However, the ionic and conductance mechanisms are dis-
tinctly different between these two groups. In addition, the
depolarizing excitatory receptors of scallops are anomalous
considering the bivalve emphasis on primary inhibition. Ge-
netic analysis of the various phototransduction mechanisms
is the likely avenue for resolving the evolutionary relation-
ship of photoreception among animals. Nevertheless, further
sampling of the physiological properties of bivalve eyes
would be of great utility in understanding visual function in
this sedentary group, including both cephalic eyes in the
Arcoida and Pterioida and the rich diversity of pallial eyes, as
reviewed by Morton (2001).

Behavioral implications from receptor physiology

The evolution of an inhibitory light response restricted
to just two groups of unrelated animals makes for interesting
comparisons. Inhibition in vertebrates must be viewed in the
context of a complex retina with an extensive set of integra-
tive components underlying image formation and depen-
dent on the functional specificity of synaptic contacts from
rods and cones to their post-synaptic targets. Transmitter
release from receptors in a depolarized state is high while in
the dark. With hyperpolarization transmitter release is re-
duced. However, the ‘inhibitory’ light response can he in-
terpreted as either inhibitory or excitatory postsynaptically,
depending on the specificity of ion channel activation by the
neurotransmitter. For example, light inhibition that sup-
presses the release of an inhibitory transmitter will result in
excitation while suppression of an excitatory neurotransmit-
ter will have a net inhibitory effect. These and other synaptic
interactions are integral to the construction of receptive
fields in elements of the retina that are essential for spatial
imagery, along with focus of the lens.

In bivalve vision, inhibition by light is presented directly
to the central nervous system as an inhibitory signal, i.e., the

BIVALVE PHOTORECEPTORS INHIBITED BY LIGHT

107

cessation of activity from optic (or pallia! ) fibers that would
otherwise be excitatory. Llowever, it is generally accepted
that a photoreceptor inhibited by light at a low-threshold,
but also primed by a latent light-mediated excitation, is
physiologically efficient for responding to a shadow (Land
1966), where spikes are triggered with minimal delay on the
rising phase of the receptor potential (McReynolds and Gor-
man 1970a). Thus, bivalves have encoded response proper-
ties optimized for detecting shadows directly into their sen-
sory receptors. In doing so, they avoid delay intervals
inherent in the synaptic activation of second-ortier neurons
for mediating an off-response, as is the case for the shadow
response in barnacles where the effect of light is excitatory at
the receptor level (Gwilliam 1963).

Light inhibition and the ensuing off-response in distal
receptors is also optimal and potentially most important for
signaling motion sensitivity. The distal retina lies in the im-
age plane of the mirror-like optics of the scallop eye (Land
1965) and the off-response receptors are therefore shadowed
or illuminated sequentially by movements in the visual field.
A moving object whether light or dark is an effective stimu-
lus since movement will invariably darken some part of the
retina (Land 1966). The excitatory receptors in the proximal
retina lie outside the image plane, lack spatial acuity, and
also adapt rapidly to light (McReynolds and Gorman 1970a)
and under lighted conditions would be less responsive to
rapid increases or decreases in light intensity. Thus, the more
phasic off-receptors inhibited by light are optimally designed
and positioned in scallops to detect mcwement, signals that
represent potential predators for a sedentary animal, swim-
ming not withstanding. Off-receptors perform the same
function in the giant clam although without the benefit of an
image-forming mirror in what Stasek (1965) refers to as the
‘sight reaction’. Movements in the environment trigger rapid
mantle retraction and valve adduction without shadowing to
avoid the potential grazing of predatory reef fish. Spatial
resolution is limited to that provided by the aperture of the
invaginated pinhole eye, but Land (2003) has nevertheless
measured acceptance angles for individual receptors that
correspond to the motion-induced behaviors. Primary inhi-
bition in bivalves can therefore be viewed as an evolutionary
adaptation for survival, whether for responding to a shadow
by animals lacking eyes or responding to movement in the
environment. Movement detection has been likened to the
function of a burglar alarm (Nilsson 1994), giving the ani-
mal advance warning not available to animals lacking eyes
and dependent on shadows.

Nevertheless, many aspects of bivalve vision remain
poorly understood, including whether or not eyes are essen-
tial given that the vast majority of species have no eyes
(Morton 2001 ). At the opposite extreme, the ark clams have
hundreds of eyes. Also, there is great diversity in eye mor-

phology among the species where eyes do exist (Nilsson
1994, Morton 2001 ) and physiological information is lacking
except for the handful of species discussed here. Indeed, it is
an open question whether eye diversity is homologous (Nils-
son 1994) so receptor physiology could provide additional
insights concerning the evolution of bivalve vision.

Aside from the burglar alarm theory, what function do
eyes provide? In addition, what is the function of the depo-
larizing on-receptors found so far only in scallops and the
file clam? Despite the evidence for image formation in scal-
lops there is little indication that images are useful, other
than for motion sensitivity, and doubt exists as to whether
the central nervous system has the computational machineiy
required to process an image (Morton 2001).

The functional morphology of eyed bivalves still invites
inquiry into the question of spatial vision. In scallops, for
example, optic tracts entering the parietovisceral ganglion
via the pallial nerves innervate the lateral lobes, the func-
tional equivalent of ‘optic’ lobes (Fig. 6). Optic fibers make
initial synaptic contact with a cortical layer of second-order
neurons that in turn communicate with glomerular struc-
tures deeper into the optic lobes (Spagnolia and Wilkens
1983). Simple methylene blue staining of live tissue shows
that the lateral lobes contain numbers of glomeruli in pro-
portion to the eyes in the mantle, and in the diagram ot the
Pecten tuaxUniis ganglion (Fig. 6), the larger size of the left
lobe reflects the greater number of eyes on the correspond-
ing dorsal mantle. Glomerular structures are characteristic of
high-level sensory integration so it is tempting to speculate
that these structures make use of neural images from the eye.
Curiously, however, in recordings from the cortical surtace
of the lateral lobes in Eitvola (=Pecten) zkzac (Linnaeus,
1758), light stimuli elicit far greater activity from second-
order visual neurons associated with on-responses in the
optic nerves than for off-responses (Wilkens and Ache
1977). This suggests a high degree of input from the depo-
larizing proximal receptors although their ‘unfocused’ role
has been suggested as being limited to detecting only
changes in overall light intensity. New evidence (Speiser and
Johnsen 2008) that both retinal layers receive focused images
from the optical mirror is consistent with the predominance
of excitatory input centrally. Visual function in scallops re-
mains as an interesting avenue for further research.

Vision, when present in bivalves, involves numerous
eyes distributed around the mantle/shell margins of the ani-
mal with numbers ranging up to hundreds in arcaceans. In
the ark clams, pigment-cup and compound eyes are present,
both in high numbers but with poor resolution (Nilsson
1994). The large number of eyes and even larger number of
ommatidia are well designed to function as an alarm system
with a high safety factor estimated at up to 755 receptor
units for any direction in the visual field (Nilsson 1994). The

108

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

visual system of Tridaaia is also comprised of a large num-
ber of eyes, and with a high degree of overlap considering the
scalloped arrangement of the mantle lobes and radial orien-
tation of the eyes plus an acceptance angle for individual
receptors estimated at 16.5° (Land 2003). In essence, all of
these visual systems have the potential to form a mosaic type
of image projecting to the central nervous system. Even ani-
mals without eyes nevertheless are able to form a diffuse
spatial image, as demonstrated in echinoids based on their
orientation and movement toward large contrasting objects
(Blevins and johnsen 2004). Scallops have functional optic
lobes, but very little is known for other bivalves about visual
integration or the tunctional morphology for vision in the
central ganglia. Visual behavior in giant clams suggests at
least the possibility that a coarse visual image of the envi-
ronment can be constructed. Stasek ( 1965) notes anecdotally
that Tridaaia has been observed to aim the exhalent cone at
objects held over the mantle, much as they do when spurting
water in the direction of mantle irritants, e.g., a grazing reef
fish. While this behavior has not been confirmed, valve ad-
duction and mantle retraction responses in these clams have
been shown to habituate to a moving shadow presented
repetitively in one part of the visual field but remaining
responsive to a novel motion stimulus in another area
(Wilkens 1986). In one instance, a giant clam habituated in
this fashion responded vigorously to the shadow of a passing
cloud. These behaviors suggest that some degree of discrimi-
nation for objects or movement in the visual field exists and
that this capacity rec]uires a type of collective image forma-
tion centrally. It remains that bivalves, as a largely sedentary
group of animals, have at best limited needs for a sensory
system with high visual acuity. Nevertheless, the ‘evolution-
ary energy’ in developing functional eyes and a behavioral
repertoire in a few species challenges us to explain the adap-
tive significance of these visual systems. This is especially
true for the scallop and file clam where both inhibitory and
excitatory photoreceptors are present and the animals are
capable of locomotory responses.

ACKNOWLEDGMENTS

The symposium “Molluscan models: Advancing our un-
derstanding of the eye” was funded by grants from the Na-
tional Science Eoundation (NSF) (DEB 0614153) and the
American Malacological Society (AMS) to Jeanne Serb.

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Submitted 19 October 2007; accepted; 30 July 2008; final

revisions received: 2 October 2008

Amer. Maine. Bull. 26: 111-117 (2008)

When a snail dies in the forest, how long will the shell persist? Effect of dissolution
and micro-bioerosion

Timothy A, Pearce

Carnegie Museum of Natural History, 4400 Forbes Ave, Pittsburgh, Pennsylvania 15213, U.S.A., PearceT@CarnegieMNH.org

Abstract: Snail shells persist in the environment after death, but we know little about the rate at which shells decompose. Assumptions
about the rate of shell decomposition are relevant to conservation biologists who find empty shells or biologists using empty shells to make
inferences about assemblages of living individuals. 1 put shells in 1.6 mm mesh litter bags (excluding macro-grazers) in Delaware and
northern Michigan, U.S.A. and monitored shell mass annually for 7 years. Decomposition rates differed among species, but 1 found no
difference in rates at two sites with different habitats. Surprisingly, loss of periostracum had no effect on shell decomposition rate. At the
locations and habitats studied, decomposition rate of snails averaged 6.4% per year, excluding shells that broke during the experiment (shell
half life = 1 1.5 years), or 10.2%, including shell breakage (half life = 7.5 years). Half lives would likely be shorter if macro-grazers had access
to shells. These results caution us to draw conclusions carefully when including empty shells in inferences about assemblages ol living
individuals.

Key words: chemical weathering, death assemblage, decomposition rate, land snail, periostracum

After snails die, their shells persist in the environment.
Although some shells survive as fossils for hundreds of mil-
lions of years, most shells decompose (or effectively disap-
pear) more quickly than that, probably on the order of
months or years. A shell in a dry, protected place such as a
desert or a museum might ptersist for hundreds of years. A
shell on the forest floor might persist more briefly — but how
briefly?

Although we know little about the decomposition rates
of land snail shells in leaf litter, many studies make assump-
tions about the rate of shell decomposition and could benefit
from more information about the decomposition rates (Me-
nez 2002). Management biologists making conservation de-
cisions would find decomposition rates relevant for knowing
how long ago a species was living at a site where an empty
shell was found. Although using data from snails collected
alive would give more reliable results, biologists conducting
biodiversity surveys commonly use empty shells as an expe-
dient way to indicate the presence of species at a site. Fur-
thermore, since methods for recovering snails from leaf litter
{e.g., sieving and picking snails) are labor intensive, includ-
ing information from empty shells is tempting for at least
two reasons. First, empty shells can usually be recovered
along with live specimens with little extra effort, and second,
the only occurrence of rare species in a sample might be
empty shells, so excluding empty shells would discard infor-
mation. Studies in which empty shells and live shells are
counted indiscriminately would, of course, overestimate
population sizes of the living snails. Furthermore, if shell
decomposition rates differ among species, then including

dead shells would overestimate the abundance of robust-
shelled species. Using empty shells to calculate proportions
of species in the assemblage of living individuals requires
assuming that the death assemblage accurately represents the
assemblage of living individuals. This assumption might be
incorrect if different species, robust and fragile-shelled, de-
compose at different rates or if shells at different sites de-
compose at different rates.

Shells of Ovachlamys fiilgens (Glide, 1900) decomposed
in an average of five months in Costa Rica during the dry
season (Barrientos 2000). Aside from that study, most of
what we know about the rate at which snail shells decom-
pose is anecdotal. Welter-Schultes (2000) collected all the
dead shells of Albinaria jaeckeli Wiese, 1989 that he could
find in a particular area once in 1987 and again in 1990. The
number of dead shells he collected was similar in each year,
suggesting that the dead shells had been completely replaced
within three years.

Shells probably disappear by three main processes: dis-
solution, breaking, and bioerosion (shell removal by graz-
ing). Shells that are protected from bioerosion decompose
more slowly than shells that are exposed to this process
(Cadee 1999). Although consumption by larger organisms
such as other snails and decomposition by crushing and
breaking are real processes contributing to disappearance of
shells, in this study 1 excluded macro-grazers larger than 1 .6
mm and breakage (for most analyses) by keeping target
snails in mesh bags. Consequently, the shell decomposition
rates in this study are likely to be slower than in experiments
allowing access by macro-grazers such as other snails. How-

111

112

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

ever, excluding macro-grazers allowed me to focus on the
effects of dissolution and micro-grazers less than 1.6 mm
that might remove shell material by grazing.

In this study, I address 4 questions: ( 1 ) Do shells of
different species decompose at different rates, (2) Do shells
in different habitat types decompose at different rates, (3)
Does periostracum loss influence shell decomposition rate,
and (4) What is the mean half life of a dead snail’s shell?

MATERIALS AND METHODS
Localities and species

To address how long empty shells persist in the forest,
and to test for differences in decomposition rates among
species and among sites, I put individually numbered shells
in mesh litter bags at sites of two different habitats in north-
ern Michigan and at one site in Delaware with three repli-
cates located about 15 meters apart at each locality. The litter
bags were approx. 22 x 22 cm with 1.6 mm screen openings
and held about 1 liter of soil, leaf litter, and the decomposing
shells. The litter bags were placed on the mineral soil surface
and covered with about 5 cm of leaf litter and a small
amount of soil. I monitored shell mass annually for 7 years.

Measuring mass loss from material in mesh bags has
been used in many studies of leaf litter decomposition (Tay-
lor and Parkinson 1988) and is an applicable method to
studying snail shell decomposition. Choice of mesh size in
the bags is a trade-off between retaining fragments and al-
lowing entrance by grazing organisms. If larger animals were
important grazers on shells, then mesh sizes that exclude
them will result in slower shell decomposition rates. For
example, such a decrease in decomposition rate was ob-
served in studies of leaf litter between larger mesh that ad-
mitted and smaller that excluded grazers (Cornelissen 1996).

The two northern Michigan localities included a mixed
pine and hardwood forest on a sandy outwash plain and a
mixed hardwood forest on rich moraine soil. At these lo-
calities, I used locally collected shells from near the sites
where I studied their decomposition. The Michigan outwash
plain site tends to be drier because sandy soil does not hold
water as well. In Delaware, 1 used shells collected from the
Dehnarva Peninsula and put them in a beech-maple forest
on piedmont. Soil pH measured 4.5 at all sites.

In Michigan, I used shells of 7 species: Anguispira alter-
nata (Say, 1816) [n = 48), Discus catskillensis (Pilsbry, 1896)
(n = 18), Euchemotrema fraterinim (Say, 1824) (» = 8), Hap-
lotrema concavum (Say, 1821) {n - 18), Mesodou thyroidiis
(Say, 1816) (n = 18), Neohelix albolabris (Say, 1817) (ii = 6),
and Novisuccineo ovalis (Say, 1817) (n = 2). I chose shells
that ranged in size from 4 to 25 mm diameter and ranged
from the relatively robust and thick-shelled A. alteniata to

the thin and fragile N. ovalis. I individually numbered shells
with India ink and divided specimens of each species evenly
into the 6 replicate bags (3 from each habitat), for example,
8 A. alteniata in each bag. For species that did not divide
evenly by 6 [e.g., N. ovalis and E. fraternum), I put one
remainder in each of the outwash plain and the moraine
localities. The Delaware bags each contained 7 Triodopsis
fallax (Say, 1825).

At the start of the experiment, shells ranged from fresh
to eroded and some had been broken by small mammal
depredation. The fact that some shells were not fresh at the
start is not a problem because I compared relative shell loss
from year to year. Although older shells might be expected to
decompose more rapidly, for example due to periostracum
loss, as will be seen in the results, periostracum loss had no
significant effect on percentage annual shell mass loss. The
Delaware shells were intact, but most were missing some
periostracum at the start of the experiment. In Delaware, I
used only one species, Triodopsis fallax, which has a fairly
robust shell.

The fact that the mesh bags in Michigan and Delaware
had no species in common means that I cannot examine
species-locality effects among all three localities. However,
since I used the same species at both Michigan sites, I can
look for species-locality effects there. Because sample sizes of
some species were very small, caution should be exercised in
interpreting results.

Analyses

To determine shell decomposition, I weighed shells an-
nually after retrieving them from the litter bags, cleaning off
adhering soil, and air-diying them to constant mass. Clean-
ing and drying resulted in little to no observable shell loss
although a few small non-adhering pieces of periostracum
occasionally fell off shells during drying. I did not retrieve
shell fragments less than about 4 mm^, so shells with these
kinds of small fragment losses are interpreted in this study as
shell mass loss through decomposition. Larger shell frag-
ments that could be associated with an individual shell were
included in the mass measurements of that individual. In
addition, in order to examine mass loss by shell breakage
and to examine the effect of periostracum loss on decom-
position rate, I also annually estimated the proportion of the
shell and periostracum that were missing and measured
maximum diameter of the remaining shell. I re-inked iden-
tification numbers onto the shells if the previous numbers
had faded.

I made some adjustments to the data set. In some in-
stances, the apparent mass of a shell increased over a previ-
ous year (perhaps a piece of sand had lodged in the shell).
For instances in which the mass of a shell increased more
than 10%, I removed the increased year from the analysis.

SHELL DECOMPOSITION

113

I excluded the first year of Discus catskilleusis measure-
ments from the analyses. The shells had been collected alive
and dried without removing the bodies. The shells lost much
more mass the first year (mean of 5 mg or 46% of shell mass)
compared to subsequent years (0.5 mg or 15% of shell
mass), suggesting that the soft tissue body masses had been
a significant part of the mass the first year. Indeed, Pearce
and Gaertner (1996) reported dry mass of D. catskilleusis to
be about 2 mg. Because soft part decomposition the first year
seemed to account for a large portion of the mass loss, I
excluded all first-year D. catskilleusis from mass loss analysis.

In most analyses, I was interested in the shell mass loss
due to non-breakage factors rather than shell loss due to
breakage. To focus on effects of dissolution and micro-
grazers, I excluded from statistical analyses shells that suf-
fered catastrophic breakage during a particular year. I in-
cluded shells that were intact for at least 2 contiguous years.
I defined broken shells as those that lost more than 1 5% of
shell (estimated visually and recorded annually) or more
than 5% shell maximum dimension (measured and recorded
annually). Defining shell breakage using the percent shell
present was independent of any changes in shell mass.

Although a control was not used in this experiment, for
example, to assess repeatability of measurements from year
to year, the precision of measurement obtained was much
greater than the variation from year to year.

Statistical tests and comparisons

The primary measure I used to assess shell decomposi-
tion was decrease in mass over time. In order to standardize
so shells of different starting masses could be compared, I
calculated % shell mass loss over time and used this measure
in comparisons. I used this measure for addressing questions
comparing shell decomposition rates among different spe-
cies and different localities. A test for normality of percent
shell mass loss of Angiiispira alteruata showed the data to be
kurtotic; sample sizes of the other species were too small to
allow tests for normality. Consequently, I transformed the
data using Log(x-l-l ) and used ANOVA to compare different
species or localities. I used the Tukey test to examine post-
hoc differences.

In order to evaluate whether shell decomposition rate
increased after periostracum loss, I examined whether shell
mass loss rate correlated with percent periostracum loss.

To calculate the half life of the shell, I extrapolated the
shell decomposition rate to determine when half the mass
would remain.

RESULTS

Examples of shells that had decomposed for 4 and 7
years are shown (Fig. 1 ). An example demonstrating how the

mass changed for individual shells of A. alteruata, for 3 shells
that remained intact, and 3 shells that experienced cata-
strophic breakage at some point in the 7 year experiment, is
shown (Fig. 2).

Shell decomposition rate differed among species in 119
shell specimens that did not break (ANOVA, F = 3.774, P =
0.001 ) (Fig. 3). The Tukey post hoc test showed that decom-
position rate of intact Auguispira alteruata was less than that
of D. catskilleusis, and M. thyroidiis was less than those of D.
catskilleusis, H. coucavuui, and T. fallax. Interestingly, larger
shells had a slower percentage shell mass loss rate than
smaller shells (Pearson correlation, N = 142, - 0.088, P <

0.001, unbroken shells only, not shown). Of the 5 species
having at least 10 unbroken specimens, A. alteruata was the
only one showing a significant within-species correlation of
shell mass loss with shell size (Pearson correlation, N = 48,
R~ = 0.099, P < 0.05), suggesting that it might be the major
contributor to the correlation for all species, although its
pattern is not contradicted by the trends in other species.
When I subjectively classified N. avails and H. coucavuui as
relatively fragile shells and the rest as relatively robust shells,
I saw no striking difference in trends for percent shell mass
loss.

Shell decomposition rate did not differ significantly be-
tween the moraine site and the outwash plain site for 100
unbroken specimens in Michigan (ANOVA, F - 2.536, P =
0.114). Because different species decompose at different
rates, and the shells in Delaware were different species from
those in Michigan, if there were differences between Michi-
gan and Delaware specimens, I would not be able to differ-
entiate species differences anci locality differences. Conse-
quently, I omitted Delaware from the analysis comparing
shell decomposition rates among localities.

Surprisingly, shells that lost more periostracum did not
decompose faster (Pearson correlation, R^ - 0.0004, P > 0.5)
(Fig. 4). Although all values of mass loss per year greater
than 22% had less than 11% periostracum remaining, that
apparent greater variability likely reflects the larger statistical
sample of shells with little or no periostracum remaining.
Furthermore, five species having sample sizes of at least 12
individuals had shell decomposition rates independent of
periostracum loss (separate species P-values > 0.2 to > 0.5).
Although periostracum loss itself did not affect shell decom-
position rate, it varied among species in 113 shells examined
(ANOVA, F = 4.997, P<0.0005) (Fig. 5).

Considering the intact shells only, which decomposed at
an average of 6.4% per year, the half life of an individual
shell (protected from macro-grazers) would be 11.5 years
and after 35.8 years only 10% of the shell would remain.
Considering both intact and broken shells, which decom-
posed at an average of 10.2% per year (Fig. 3), the half life

114

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Figure 1. Appearances of three individual shells after 4 years (left column, A, C, E) and 7 years (right column, B, D, F); Anguispira alternata
No. 03 (13.6 mm diameter; A, B), A. alternata No. 12 (17.6 mm; C, D), Mesodon thyroidiis No. 51 (23.1 mm, E, F).

SHELL DECOMPOSITION

115

Figure 2. Loss of Anguispira altcrnata shell mass over 7 years.
Dashed lines are shells that broke during the experiment; solid lines
are shells that remained intact.

of the shell would be 7.5 years; after 22.4 years, only 10% of
the shell would remain.

DISCUSSION

Because shells of different land snail species decompose
at different rates, these results demonstrate that using shells

from dead snails has potential to bias estimates about as-
semblages of living snails. Admittedly, sample sizes of some
species in this study were very small, so results about those
species must be interpreted with caution; however, being
cautious with those results does not change conclusions
about species with larger sample sizes. Conclusions in stud-
ies using empty shells should indeed be drawn carefully.

Shell decomposition rates are likely influenced by a
plethora of factors. Three of the factors that might influence
shell decomposition rates are surface area to mass ratio, shell
robustness, and physical and chemical environment. Larger
shells, which likely have a smaller surface area to mass ratio,
lost mass more slowly than smaller shells in this study. Me-
nez (2002) also found that larger snail shells degraded more
slowly. Such a size difference might be expected since shells
with a high surface area to mass dissolve more rapidly
(Claassen 1998). Although physical and chemical destruc-
tion has been reported to be faster in thin-shelled than more
robust species (Evans 1972), no effect of shell robustness was
observed on shell decomposition rate in this study although
a better test for an effect of robustness needs to be con-
ducted. Robustness would be influenced by shell thickness as
well as form, such as ridges that add strength; future tests of
robustness should examine crush strength among species.

Shell decomposition rates did not differ significantly at
two habitats in Michigan, despite the habitats differing in
substrate (sandy soil versus poorly
sorted moraine deposits), vegetation
(relatively low oak and pine with
sparser undergrowth versus taller as-
pen forest with denser undergrowth),
and evidently moisture (although the
pH did not differ). This result con-
trasts with leaf litter decomposition
rates, which are slower in sandy soil
having less moisture and less nutrient-
holding capacity (lohnson et al. 2000),
suggesting that processes regulating
leaf litter and shell decomposition
might differ. Because temperature,
moisture, and pH likely play impor-
tant roles in shell decomposition,
shells in environments different from
those I studied are likely to have dif-
ferent decomposition rates. Indeed,
Barrientos (2000) found that shells in
mesh bags (mesh size not stated) in
Costa Rica decomposed in an average
of 5 months.

Surprisingly, periostracum did not
seem to play a protective role in de-
composing shells. Periostracum is usu-

20 -

48

J

n

^ 0

w 20
in

10 -

bl

III

42

intact + broken

18 “ _

i i I i

intact

18

16

/

c?

/

O'

0

/

/

species

19

i * i £ ■ ■ i

1 ^ 1 ^ 1 ^ 1 ^ 1 ^ 1 ^ \ ^ 1

Figure 3. Shell decomposition rate contrasted among species. Lower panel indicates decom-
position rate for unbroken shells, upper panel the rate for both broken and unbroken shells
together. Numbers above bars indicate initial sample size. Bars with different letters within
a horizontal row differed significantly; those without letters did not differ significantly. Data
from broken shells were included in the lower graph only for their unbroken duration, which
explains unequal mass losses in top and bottom graphs for species having same sample sizes.

116

AMERICAN MALACOLOGICAL BULLETIN

26 • 1/2 • 2008

Figure 4. Shells that lost more periostracum did not decompose faster. Points show amount
of periostracum remaining at the end of a year (x-axis) and amount of weight loss since the
preceding year (y-axis). Individual shells can appear more than once for different years.

Figure 5. Periostracum loss rate varied among species. Numbers above bars indicate sample
size. Bars with different letters within a horizontal row of letters differed significantly; those
without letters did not differ significantly.

ally thought to protect shells from boring organisms, ero-
sion, or dissolution by leaf litter acids in terrestrial snails or
acidic water in aquatic molluscs (Solem 1974). While most
living shells have intact periostracum, the apices of some
living shells do erode over time. However, older molluscs
missing large areas of periostracum do not seem to suffer
serious erosion of the shell, suggesting that water and cor-
rosion proofing qualities of the periostracum may be only of
secondary importance (Hunt and Oates 1978). Possibly ero-
sion soon after death starts on the inside surface of the shell,
which is not protected by periostracum. Nevertheless, two
questions remain: in the present study, why did the shell
decomposition rate not increase after the loss of the peri-
ostracum, and why does periostracum apparently become

more pervious after death, after having
stayed intact for years during the
snail’s life? Regarding the second ques-
tion, the living snail might behavior-
ally or chemically maintain a good
bond between the periostracum and
the shell whereas the bond might
weaken after death, allowing ingress of
corrosive solutions.

Living snails would be important
grazers on decomposing snail shells.
Other micro- or meso-organisms that
might graze on decomposing shells are
likely to exist. In a study of shell mass
loss of Helix aspersa Muller, 1774 on a
dune area in the Netherlands, Cadee
(1999) found that shells protected
from bioerosion lost about 8% mass
in a year, similar to the rate found in
this study. However, in that study,
shells exposed to bioerosion by other
land snails lost mass much more rap-
idly, 34% in 70 days, indicating that
shells exposed to bioerosion could dis-
appear in less than one year. If micro-
grazers are important contributors to
shell decomposition, then the soil
conditions [e.g.-, nutrients) would also
be relevant through their effect on the
micro-grazers.

Dissolution and chemical conver-
sion are often the main contributors to
land snail shell decomposition (Claas-
sen 1998). Colder water can dissolve
more calcium carbonate (Claassen
1998) although more rapid dissolution
can be expected at higher tempera-
tures, at least in non-saturated water.
On one hand, presence of moisture and lower pH increase
the speed of shell decomposition (Claassen 1998, Reitz and
Wing 1999). On the other hand, alkaline soils rich in cal-
cium retard the breakdown of empty shells (Claassen 1998,
Schilthuizen and Rutjes 2001, Cameron et al. 2003).

Although influences on decomposition rates of snail
shells on forest floors are poorly known, insights might be
gained from the more numerous studies on decomposition
of leaf litter. Although different processes probably act on
shells and leaves, results from studies finding leaf litter de-
composition differences among habitats and climates are
probably applicable to snail shell decomposition. For ex-
ample, warmer climates would probably increase decompo-
sition rates of shells, as it does in leaf litter (Bell 1974). In

SHELL DECOMPOSITION

117

leaves, factors most important at determining the rate of
decomposition are those that regulate microorganism activ-
ity: temperature, moisture, nutrients, and energy source
(Berg and Ekbohm 1991). It microorganism activity plays a
large role in shell decomposition, then shell decomposition
rates would also be largely affected by processes regulating
microorganism activity.

The result that snail shells of different species have dif-
ferent decomposition rates has important ramifications for
studies of endangered species and community analysis. Find-
ing empty shells of an endangered species in habitats similar
to those studied here would suggest that the species was
I living in the area within the last several decades at most.

; However, the results of this study suggest that for commu-
j nity analysis studies, using empty shells to infer abundances
i of the assemblage of living individuals might violate the
■ assumption that the death assemblage accurately represents
I the assemblage of living individuals. Including empty shells
I could overestimate the abundance of robust species,
j In the geographical locations and habitats I studied, and

with shells protected from macro-grazers, I extrapolate that
i shells will decompose to 10% of their former mass after
I several decades. For practical purposes, e.g., in surveys re-
I covering shells from leaf litter samples, shells missing more
i than 50% of their former mass might not be findable or
I identifiable, so shells in these conditions might effectively
j disappear in 7-12 years, their half life. Half lives would likely
I be shorter if macro-grazers had access to shells.

Future studies might help tease apart the processes in-
volved in shell decomposition. Exploring the decomposition
rates of shells in different environments (and geographic
localities) and noting biotic and abiotic influences would
help to address the importance of different situations (as has
been found in leaf litter decomposition studies) and of
scrapers or chemical weathering. Laboratory experiments
could more directly evaluate the relative importance of the
three decomposition methods and the importance of pH
and temperature.

ACKNOWLEDGMENTS

I am grateful to Amy Cortis, Jeffrey Firestone, and Erika
Martin for collecting most of the shells, to Alice Doolittle for
help with fieldwork, and to Joan McKearnon for advice
about the experimental design. Many thanks to Amanda E.
Zimmerman for revising the figures. Collalaoration with
Marvin C. Fields provided considerable help and encourage-
ment in analyzing and writing up the paper. Philip Myers
photographed Figs. lA, 1C, and IE.

LITERATURE CITED

Barrientos, Z. 2000. Population dynamics and spatial distribution
of the terrestrial snail Ovachlamys fidgens (Stylommatophora:
Helicarionidae) in a tropical environment. Revista de Biologia
Tropical 48: 71-87.

Bell, M. K. 1974. Decomposition of herbaceous litter. In: C. H.
Dickinson and G. I. F. Pugh, eds.. Biology of Plant Litter Decom-
position. Academic Press, London and New York. Pp. yi-Bl .

Berg, B. and G. Ekbohm. 1991. Litter mass-loss rates and decom-
position patterns in some needle and leaf litter types. Long-
term decomposition in a Scots pine forest. VII. Canadian Jour-
nal of Botany 69: 1449-1456.

Cadee, C. G. 1999. Bioerosion of shells by terrestrial gastropods.
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Cameron, R. A. D., M. Mylonas, K. Triantis, A. Parmakelis, and K.
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Cambridge University Press, Cambridge, U.K.

Cornelissen, ). H. C. 1996. An experimental comparison of leaf de-
composition rates in a wide range of temperate plant species
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Evans, J. G. 1972. Land Snails in Archaeology. Seminar Press, London.

Hunt, S. and K. Oates. 1978. Fine structure and molecular organi-
zation of the periostracum in a gastropod mollusc Buccinwn
undatnm L. and its relation to similar structural protein sys-
tems in other invertebrates. Philosophical Transactions of the
Royal Society of London (B, Biological Sciences) 283: 417-459.

Johnson, C. M., D. J. Zarin, and A. H. Johnson. 2000. Post-
disturbance aboveground biomass accumulation in global sec-
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Menez, A. 2002. The degradation ot land snail shells during the annual
dry period in a Mediterranean climate. Iberus 20: 73-79.

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trail following in the carnivorous land snail Haplotrenia con-
cavum (Gastropoda: Pulmonata). Malacological Review 29: 85-
99.

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versity Press, Cambridge, U.K.

Schilthuizen, M. and H. A. Rutjes. 2001. Land snail diversity in a
square kilometre of tropical rainforest in Sabah, Malaysian
Borneo. Journal of Molluscan Studies 67: 417-423.

Solem, G. A. 1974. The Shell Makers, Introducing Mollusks. John
Wiley and Sons, New York.

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Submitted: 23 December 2007; accepted: 20 |une 2008;

final revisions received: 4 September 2008

Amer. Make. Bull. 26: 119-131 (2008)

Bivalve molluscs from the continental shelf of Jalisco and Colima, Mexican
Central Pacific

Eduardo Rios-Jara, Ernesto Lopez-Uriarte, and Cristian M. Galvan-Villa

Laboratorio de Ecosistemas Marinos y Acuicultura, Departamento de Ecologia, Centro Universitario de Ciencias Biologicas y
Agropecuarias, Universidad de Guadalajara, Carretera a Nogales km 15.5, Zapopan 40110, Jalisco, Mexico, edurios@maiz.cucba.udg.mx,
ernlopez@maiz.cucba.udg.mx

Abstract: A survey for bivalves was conducted at 25 sampling stations on the Mexican Central Pacific shelf off Jalisco and Colima, during
the summer of 1988. The bivalves were sampled with a Van Veen grab at 16 stations with medium sand, sandy silt, and silty clay substrata
at depths between 18 and 112 m. A total of 5,196 individuals belonging to 59 genera and 95 species of bivalves were found. A systematic
list is provided with the relative abundance and density (individuals/m') for each species and information on depth, type of substratum,
bottom water temperature, and oxygen concentration for each station. The twelve most common species (>100 individuals/station) in
descending order of abundance were: Nnculana laeviradius (Pilsbry and Lowe, 1932), CrassineUa pacifica (C. B. Adams, 1852), Corhida
nasuta G. B. Sowerby 1, 1833, Anadara adamsi Olsson, 1961, Parvihicma approxhmita (Dali, 1901), Nucula decUvis Hinds, 1843, Corbula ira
Dali, 1908, Radiolucirm amcellaris (Philippi, 1846), Cyclopecteii periioimis (Hertlein, 1935), Nuadana lobida (Dali, 1908), Parviluciim
mazatlanica (Garpenter, 1857), and Goiddia californiai Dali, 1917. The bathymetric patterns in the abundance and species composition of
the bivalve community and their relationship to environmental parameters are discussed. The structure of the assemblages differed with
depth, with peak abundances and species richness (1) between 24 and 40 m with medium sand and sandy silt substrata and (2) at
intermediate depths between 71 and 74 m, with sandy silt and silty clay substrata. The species characterizing shallow, intermediate, and deep
zones were the most abundant or those exclusive of each zone. Diversity, dominance, and evenness decreased at the deeper stations. The
distinctive species composition of these zones may be the result of variation in depth, oxygen concentration, and substratum.

Key words: Bathymetric patterns, diversity, dominance, evenness, benthos

Our understanding of the taxonomic composition of
the bivalve fauna of the Mexican Pacific stems from the
exhaustive survey of Keen ( 1971 ) and the coverage of some
Panamic taxa by Keen and Coan (1974) and Coan et al.
(2000). The literature reviews of Skoglund (1991, 2001) are
also important since they cite new species, redefine taxo-
nomic relationships, and provide new records and range
extensions for many bivalves from the Panamic Province.

Most of the ecological literature on benthic communi-
ties from the Mexican Pacific refers to the Gulf of California.
Parker (1964) reviewed the early biological exploration in
the Gulf of California and provided an extensive description
of macroinvertebrate assemblages and their environments.
Parker provided a list of more than 380 species of bivalves
and assemblages from intertidal and shallow rocky or sandy
shores to the abyssal basin and outer continental slope, to
4,122 m depth. Coan (1968) described the shallow benthic
mollusc community (0-49 m) at Bahia de Los Angeles, lo-
cated on the northeastern coast of Baja Californja, and con-
cluded there was a single assemblage, typical of a silty-sand
substratum in semi-protected bays of tropical and subtropi-
cal areas. Zamorano and Hendrickx (2007) reported a total
of 56 species of deep-water molluscs collected during the
TALUD IV-IX cruises in depths >500 m in the southern
Gulf of California. The most recent biodiversity model of

marine and brackish-water Mollusca from the Gulf of Cali-
fornia, proposed by Hendrickx et al. (2007), analyzed the
latitudinal and bathymetric distribution of 2,194 species, in-
cluding 565 bivalves. Additionally, some catalogues of re-
cords of biological collections include bivalves from the Gulf
of California (Morris 1966, Abbott 1974, Hendrickx and
Toledano-Granados 1994, Hendrickx and Brusca 2002).

Surveys of mollusc communities from the Mexican
Central Pacific are scarce, non-continuous, and have been
carried out mostly in the intertidal and shallow subtidal
zones. In the coast of lalisco and Colima, only a few works
focus mainly on the taxonomic composition and abundance
of gastropods (Rios-Jara et al. 1996, Perez-Peha and Rios-
Jara 1998), scaphopods (Rios-Jara et al 2003a, 2003b), or
both gastropod and bivalve communities (Landa-laime and
Arciniega-Flores 1998, Rios-Jara et al. 2001) with very few
records of bivalves. Holguin-Quinones and Gonzalez-
Pedraza (1994) provide the only catalogue of molluscs for
this region and include 87 species of bivalves mostly from
rocky and sandy beaches and shallow subtidal areas to 39 m
depth.

The Atlas expeditions off the coast of Jalisco and Colima
in 1988 resulted in one of the most important and most
extensive collections to date of benthic marine molluscs
from the Mexican Central Pacific. The R/V El Puma of the

120

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Universidad Nacional Autonoina de Mexico extensively
sampled the shelf of both states, from Puerto Vallarta to the
southern limit of Colima. The specimens were deposited in
the collection of the Laboratorio de Ecosistemas Marinos y
Acuicultura of the Universidad de Guadalajara in the city of
Guadalajara and have resulted in reports mostly related to
the gastropods and the scaphopods (Rios-Jara et al. 1996,
2001, 2003a, 2003b, Perez-Pena and Rios-Jara 1998). The
present study examines the bivalves collected during the
Atlas V expedition along the continental shelf of Jalisco and
Colima. Taxonomic changes are updated, and range exten-
sions reviewed with an analysis of distribution patterns with
respect to depth, oxygen concentration, and substratum.

MATERIALS AND METHODS

Study area

The area of study is the narrow (7-10 km) continental
shelf off the states of Jalisco and Colima, along the Pacific
coast of Mexico (Fig. 1) with an area of approx. 5, 315 km^

(Ruiz-Dura 1985). The area extends ca. 364 km of coastline,
from the mouth of the Rio Ameca, Bahia Banderas
(20°39'N), to the mouth of the Rio Cohuayana (18°39'N).

This region has irregular topography, with foothills and
mountains which form cliffs, bays, estuaries, and beaches of
diverse sizes and shapes. Ca. 70% of the coastline is sandy
beaches; rocky areas are mixed with sands and include vol-
canic rock platforms, boulders, stones, and pebbles. On the
sea bottom, areas of uneven topography intercalate with
relatively flat zones (Galaviz-Solis and Gutierrez-Estrada
1978, Guzman-Arroyo and Flores-Rosas 1988). The compo-
sition of sediments in the continental shelf of the tropical
Mexican Pacific is mostly terrigenous and pelagic clays with
small areas of calcareous and bio-siliceous mud and silts
(McCoy and Sancetta 1985). This coastal region includes
several rivers, coastal lagoons, and estuaries.

Sampling methods

On board the R/V El Puma, topography and depth were
determined continuously with an Edowestern analog echo-

Figure 1. Location of sampling stations
and types of substratum along the
coast of Jalisco and Colima, Mexican
Central Pacific. The stations with a
square indicate those where bivalves
were collected. The isolines at 40, 70,
and 100 m indicate the shallow (18-40
m), intermediate (48-74 m), and deep
(83-129 m) bathymetric zones in
which sampling stations were grouped.

BIVALVE MOLLUSCS FROM JALISCO AND COLIMA, MEXICO

121

sounder system. Tiiirteen transects perpendicular to tlie
coastline were established at intervals of ~10' of latitude (Fig.
1). One to four sampling stations were conducted on each
transect, for a total of 25 samples collected with a Van Veen
grab. The grab collected 20 L of sediment in a surface area of
0.1 m^. At each station, two grab samples were taken. The
depth ranged from 18 to 129 m. Sediments were sieved
through three screens (mesh size = 10, 3, and 1 mm) and
analyzed in the Instituto de Geografia of the Universidad de
Guadalajara, Mexico.

Living bivalves were preserved in 70% ethanol. Indi-
viduals were identified to species using Morris (1966), Keen
(1971), Abbott (1974), and Coan et al. (2000). Only shells
that permitted clear identification were used. Taxonomy and
distributional ranges were updated using Skoglund’s publi-
cations (1991, 2001 ).

There are at least three types of substrata in the area
(Perez-Pena and Rios-Jara 1998): (I) silty clay (most par-
ticles <0.02 mm), (2) sandy silt (0.02-0.015 mm), and
(3) medium sand (>0.2 mm). Greater heterogeneity of the
sediments occurred in shallow sampling stations (18-60 m)
compared to the intermediate (48-74 m) and deeper sta-
tions. Sediments were more homogeneous at deeper stations
than in the intermediate and shallow stations, and decreased
in particle size from medium sand and sandy silt to silty clay.

To analyze the effect of depth and substratum on the
distribution and abundance of the bivalves, sampling sta-
tions were grouped in (1) a shallow zone (SZ) (18-40 m)
with coarse substratum (medium sand and sandy silt), which
includes stations 52, 24, and 47; (2) a transition zone with
intermediate depths (IZ) (48-74 m), sandy silt and silty clay,
including stations 35, 23, 48, 26, 51, 34, 18, 22, and 30; and
(3) a deep zone (DZ) (83-129 m) with homogeneous sub-
stratum made of silty clay, including stations 33, 50, 38, 25,
29, and 37.

Preliminary analysis revealed that abundance values
were asymmetrical (X“ goodness-of-fit statistic, P < 0.01),
and sample variances were not homogeneous ( Hartley’s
test; Byrkit 1987) (Statgraphics plus 5.0 computer program).
Therefore, the non-parametric Kruskal- Wallis test (Sokal
and Rohlf 1989) was used to analyze bathymetric patterns of
bivalve abundance. Significant differences were further ana-
lyzed using the Bonferroni test to identify differences of
bivalve abundance means among sampling stations.

The structure of the bivalve community was analyzed by
estimating ecological indices for the three bathymetric zones
(SZ, IZ, and DZ) using the computer program Species Di-
versity and Richness III (1998). Diversity was estimated by
means of the Shannon- Weaver index (H' ) (Magurran 1988),
species richness with the Margaleff Index (Dj,^g) (Magurran
1988), dominance with the Simpson Index (D') (Simpson
1949), and evenness with the Pielou Index (f) (Pielou 1977).

Differences in these indices among zones were determined
with a Kruskal-Wallis test; significant differences were fur-
ther analyzed using the a posteriori test of Bonferroni.

Pearson correlation coefficients (Minitab computer
program; Sokal and Rohlf 1989) were used to determine the
relationship between environmental (temperature, depth,
and oxygen concentration) and biological parameters (abun-
dance and number of species). The Pearson probability P-
values were used to determine any significant correlations.

RESULTS

Composition and abundance

Bivalves were found at 16 of the 25 sampling stations, at
depths between 18 and 112 m (Fig. 1 ). Most of these stations
had silty clay (8 stations) and sandy silt (6 stations) sub-
strata; only two stations had medium sand. A total of 5,196
individuals belonging to 28 families, 59 genera, and 95 spe-
cies was collected (Appendix I ). The number of species per
family varies considerably (from 1 to 15). Twelve families
(42.85%) contain a single species, while the most diverse
families contain nine or more species: Veneridae (15),
Tellinidae (13), Lucinidae (10), and Arcidae (9).

The density of bivalves obtained with the grab ranged
between 13,015 individuals /m“ in station 47 and 15 indi-
viduals/m^ in station 48 (mean density per station =
1,623.75). The number of species varied between 1 and 63
species/station (mean = 6) (Appendix 1). There was consid-
erable variation in the structure of the bivalve communities
along the continental shelf, with areas of high and low den-
sity of bivalves, and notable differences in the number and
composition of species. In general, the zones with higher
numbers of individuals coincide with those of high species
richness (Fig. 2).

The density of individuals and species decrease at depths
between 48 and 66 m ot the Intermediate Zone (IZ) with
15-210 individuals/m“ and 3-16 species/station, and in the
Deep Zone (DZ) (83-112 m), with 55-2070 individuals/m“
and only 1-11 species/station. Notably, the boundaries
ot these zones present areas with peaks of very high values:
(1) in station 47 (40 m) between SZ and IZ, with total of
13,015 individuals/m^ and 63 species and (2) in station 30
(74 m) between the IZ and DZ, with 5,585 individuals/m"
and 28 species. These two areas include the majority of all
bivalves collected (50.1% and 34.5% of all individuals, re-
spectively) and a large traction of the species (65.6% and
47.9%).

The assemblages of bivalve species across the continen-
tal shelf of Jalisco and Colima thus show a tendency to
decrease in the abundance and number of species from the
shallow to the deeper areas. However, there is not statistical

122

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Surface ^ Deep _ ^ Dissolved

temperature * temperature D Individuals □ Species ------- oxygen

o

30

25

20

15

10

on ^
JU T3
CD

g

-.n ^
20 cn

10

9

8

Y

- 6
- 4

L 0

Depth (m)

Figure 2. Density (individuals per m^) and number of species collected with a Van Veen grab in the continental platform of lalisco and
Colima, Mexico. The differences between surface and bottom temperatures during the sampling period define zones along the continental
shelf with thermal stratification. Variation in dissolved oxygen concentration is also indicated.

difference in the abundance of bivalves between the bathy-
metric zones (Kruskal-Wallis test, N - 3, P - 0.23) (Table 3).

Relationship between environmental and biological
parameters

Highly significant negative correlations occurred be-
tween depth and temperature (Pearson correlation, r =
-0.76, N = 16, P - 0.0003) and depth and oxygen concen-
tration (r = -0.638, N - 16, P = 0.009). Minimal values of
temperature and oxygen occurred in areas deeper than 60 m
although several peaks of both parameters are evident at
depths of 18-24 m, 53-60 m, 84 m. Consequently, bottom
water temperature was significantly correlated with oxygen
concentration (r = 0.874, N - 16, P = 0.00009). Depith was
significantly correlated with the number of species of bi-
valves (r = -0.471, N = \6, P = 0.049). Low oxygen con-
centrations overlap the areas of strong thermal stratification
at 48 m and between 66 and 112 m depth. Oxygen levels
increase considerably between 53 and 60 m where there is no
apparent stratification. Continuous hypoxic conditions oc-
cur in areas deeper than 60 m but become more severe at
station 29 where the oxygen level falls to 0.2 ml/1 and only
one species was collected (Fig. 2).

Because the peaks in the number of individuals and
species closely coincide across tbe continental shelf (Fig. 2),
both variables had a significant positive correlation coeffi-
cient (/■ = 0.740, N = 16, P = 0.0005). However, no signifi-
cant correlation was found between depth and abundance

(r - -0.123, N = 16, P = 0.627) probably because of the large
increment in the number of bivalves registered in the sta-
tion 25 at 99 m, especially the clams Corbula ira (242) and
Nitculana lobiila (149). The correlation coefficients for all
other pairwise comparisons were low and not statistically
significant (P > 0.05).

Twelve species were very common (>100 individuals) in
the samples, representing 78.16% of all individuals (Table 1).
These dominant species had a widespread bathymetric dis-
tribution across the continental shelf, but most individuals
concentrated in high numbers in one or two stations either
in the shallow (SZ) or deeper stations (DZ). This is more
evident in the SZ, where nine dominant species represented
71% of the bivalve abundance and in the DZ where two
species represent 91% of the abundance (Table 2). The ob-
served decrease in the total bivalve density in the IZ is largely
the result of the decline in the populations of the 12 most
abundant species.

The shallow zone (18-40 m) contains an assemblage of
79 species. Eleven species are considered dominant (Table
2). These species are semi-infaunal or infaunal, which can be
attributed to the sediment type, mostly medium sand (67%)
and sandy silt (33%). The only epifaunal species in this
group, Cydopecten pernomiis, was found in great numbers in
medium sand substratum at 40 m. The family with the most
species was Veneridae (3); each of the other eight dominant
species belongs to different families. The stations with this
assemblage of species are in Colima (52 and 47) and in the

BIVALVE MOLLUSCS FROM JALISCO AND COLIMA, MEXICO

123

Table 1. Abundance, habitat, and feeding habits of the most representative bivalve species. MS, medium sand; SS, sandy silt; SC, silty clay;
DF, deposit feeder; FF, filter feeder.

Species

Abundance

(total

individuals)

Relative

abundance

(%)

Cumulative

relative

abundance (%)

Type of
substratum

Depth

range

(m)

Habitat

Feedin:

habit

1. Nucuhma laeviradius

690

13.28

13.28

MS, SS, SC

18-84

Intaunal

DF

2. Crassinella pacifica

680

13.09

26.37

MS, SS, SC

18-74

Infaunal

—

3. Corbula nasuta

535

10.30

36.66

MS, SS, SC

18-74

Infaunal

FF

4. Anadara adamsi

435

8.37

45.03

MS, SS, SC

24-94

Semi-intaunal

FF

5. ParvUucina approxitnata

399

7.68

52.71

MS, SS, SC

18-83

Intaunal

FF

6. Nucula declivis

263

5.06

57.78

MS, SS, SC

18-83

Infaunal

DF

7. Corbula ira

242

4.66

62.43

SC

99

Infaunal

—

8. Radiolucina cancellaris

208

4.00

66.44

MS, SS, SC

40-83

Intaunal

—

9. Cyclopecten pernomus

188

3.62

70.05

MS, SS, SC

24-74

Epifaunal

FF

10. Nucuhma lobula

173

3.33

73.38

MS, SS, SC

18-112

Intaunal

DF

11. ParvUucina mazatlanica

144

2.77

76.15

MS, SS, SC

18-83

Infaunal

DF

12. Gouldia californica

104

2.00

78.16

MS, SS

40-72

Infaunal

FF

middle portion of Jalisco (24), but the assemblage possibly
extends to all shallow areas with similar conditions along the
length of the tropical Mexican Pacific. Many species (32) are
found only in this zone, which indicates this is an optimum
habitat for the bivalve species. The black clam Megapitaria
squalida (G. B. Sowerby I, 1835) was the only dominant
exclusive to this zone and together with other abundant
species [Corbula nasuta, Anadani adamsi, Cydopecten perno-
mus, Gouldia californkn, Chioiie compta (Broderip, 1835),
and Semelinn campbellorwu Coan, 2003] characterizes this
assemblage (Table 2).

The intermediate zone assemblage (48-74 m) is distinct
and is characterized by several species which are dominant
only in this zone: Parvdudna approximata, ParvUucina
mazatlanicn, Ludnisca cetitrifuga (Dali, 1901), and Niiculana
acapulcensis (Pilsbry and Lowe, 1932) (Table 2). The family
with the most dominant species (4) was Lucinidae followed
by Nuculanidae (2). The zone has finer sediments than the
SZ (mostly sandy silt and silty clay), and most of the dom-
inant species are also semi-infaunal or infaunal. Although
five dominant species were also dominant in the shallow
zone, there is also a considerable number of exclusive species
( 10). Some of these species were also found in deeper waters,
until 83 m. The total number of species (55) and individuals
(1,896) decrease with respect to shallower areas although
both species and individuals are considerably higher between
71 and 74 m.

All samples from stations in the outer shelf, 83 to 112
meters, were taken on silty clay bottom. This deeper zone
contained a rather different assemblage of bivalve species
from those found in the shallow and intermediate zones.
This was the zone with the lowest number of bivalve species
(22) although three were exclusive to this zone. Although

only two species, Nuciilcma lobida and Corbtda ira, may be
considered dominant because of their high relative abun-
dance, it is notable that C. ira is also exclusive to this zone.

Species richness, diversity, dominance, and evenness

The shallow and intermediate zones have similar values
tor the ecological indices, but they all decrease toward the
deep zone. However, significant differences were found only
in species richness and diversity among the bathymetric
zones (Kruskal-Wallis test, N = 5, P < 0.05) (Table 3). The
Bonferroni multiple-comparison test revealed that the values
for the SZ and IZ were not statistically different but both
were greater than in the DZ.

DISCUSSION

Although bivalves play a key role in the macroinverte-
brate community of the intertidal zone, there have been few
attempts to relate benthic bivalves to environmental factors
in the continental shelf of Jalisco and Colima. Trawling nets
used in other studies do not collect the smaller semi-infaunal
and infaunal species which comprise this group (Landa-
Jaime and Arciniega-Flores 1998, Godinez-Dominguez and
Gonzalez-Sanson 1999). These studies thus underestimate
their importance in the benthos. During the Atlas V expe-
dition, the fauna from grab samples included many gastro-
pods (Perez-Rena 1989) and some scaphopods (Rios-Jara et
al. 2003a) but bivalves represented the greatest numbers of
individuals and species.

Because most species were tound in more than one
bathymetric zone, specific assemblages were characterized by
the dominant species and by those found in only one zone.

124

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Table 2. Most abundant and exclusive species in the shallow, intermediate, and deep zones.
The most abundant species of each list contain £80% of all individuals within each zone.

Shallow zone

Intermediate zone

18-40 m

41-80 m

Deep zone

67% medium sand.

56% sandy silt.

81-129 m

33% sandy silt

44% silty clay

100% silty clay

Most abundant species

1. Corbula nasiita

1. Parvilucina aproximata 1. Corbula ira

2. Nucidana laeviradiiis

2. Crassinella pacifica 2. Niiculana lobiila

3. Anadara adamsi

3. Niiculana laeviradiiis

4. Crassinella pacifica

4. Radiolucina cancellaris

5. Niicula declivis

5. Parvilucina mazatlanica

6. Cyclopecten pernomus

6. Lucinisca centrifiiga

7. Megapitaria sqiialida

7. Nucidana acapulcensis

8. Goiildia californica

8. Niiciila declivis

9. Chione compta

9. Cyclopecten pernomus

10. Radiohtcina cancellaris

11. Semelina campbellorum

Exclusive species

1. Corbiila ira

2. Kellia suborbicularis

3. Strophocardia megastropha

11. Lncina prolongata

12. Limarca brevifrons

13. Mactrelkma subalata

14. Megapitaria sqiialidn

15. Lirophora kellettii

16. Pitar concimnis

17. PHcatida pendllata

18. Psammotreta aurora

19. Seinele pallida

20. Semele verrucosa

21. Sheldotiella olssoiii

22. Strigilla cicercida

23. Strigilla dichotoina

24. Strigilla sp.

25. Tagelus politiis

26. Tellina pacifica

27. Trachycardium belcheri

28. Trachycardium proceriim

29. Trachycardium senticosum

30. Transeiinella modesta

31. Trigoniocardia granifera

1. Anadara aequatorialis

2. Anadara mix

3. Chione pulicaria

4. Corbula marmorata

5. Crassinella ecuadoriana

6. Doiiax gracilis

7. Dosinia dunkeri

8. Isognomon recognitus

9. Laevicardiiim elenense
10. Lirophora mariae

1. Anadara obesa

2. Barbatia reeveana

3. Conchocele excavata

4. Crassinella varians

5. Lucinisca fenestrata

6. Macoma siliqiia

7. Nucida schenki

8. Pitar aletes

9. Pitar berryi

10. Tellina pristiphora

Considerable changes in species com-
position and dominance occur because
the most abundant species were fre-
quently collected in high numbers
(>100 individuals) but only at one or
two sampling stations. Species with a
wide bathymetric distribution tend to
be found either in the shallow or the
deep zone, forming aggregations at
specific sampling stations. In addition,
even though bivalve abundance can be
highly variable, some predictions of
community structure can be made
based on a few key environmental fac-
tors such as type of substratum and
oxygen concentration.

Thorson (1957) proposed that
parallel bottom communities, charac-
terized by closely related genera, exist
where environmental conditions are
similar. Parker (1964) found similar
species assemblages, including bi-
valves, in areas with similar conditions,
as predicted by Thorson ( 1957). Other
examples of parallel communities have
also been described {e.g., Coull and
Herman 1970, Asakura and Suzuki
1987, Chertoprud et al. 2007). How-
ever, there are also examples that do
not support the parallel communities
hypothesis (Gallardo 2003). A com-
parison of the bivalve assemblage de-
scribed by Parker (1964) from near-
shore (11-26 m) with sand to sand-
mud substrata of the Gulf of California
to the bivalve assemblage of the shal-
low-water zone (18-40 m) with me-
dium sand and sandy silt of Jalisco and
Colima indicates some similarities. In
both regions, the majority of species
live in semi-infaunal and infaunal
habitats, and the most diverse families
are Nuculanidae and Veneridae. In ad-
dition, Megapitaria sqiialida is very
abundant, several species of the genera
Nucidana Link, 1807, Anadara J. E.
Gray, 1847 and Chione Megerle von
Muhlfeld, 1811 are dominant in both
assemblages, and both share the same
three most diverse families (Veneridae,
Tellinidae, and Arcidae) (Hendrickx et
al. 2007). Other comparisons among

BIVALVE MOLLUSCS FROM JALISCO AND COLIMA, MEXICO

125

Table 3. Ecological indices estimated for the bivalve communities from three bathymetric zones of the continental shelf of Jalisco and
Colima, Mexico. H' = Shannon-Weaver’s diversity index, = Margaleff s species richness index, D' = Simpson’s dominance index and
J' = Pielou’s evenness index. Significant differences among zones (*) were found for the Species richness (0^,^,) and Diversity (H') indices
(Kruskal-Wallis Test, P < 0.05) and were further analyzed using the Bonferroni test.

Shallow
zone (SZ)

Intermediate
zone (IZ)

Deep

zone (DZ)

Kruskal-Wallis
test (H value)

Bonferroni test
among zones

Total number of individuals (N)

2834

1896

466

2.41

Total number of species (S)

79

55

22

Species richness (D^.,g)

9.81

7.15

3.41

6.8U

SZ = IZ > DZ

Diversity (H')

2.86

2.63

1.32

6.58*

SZ = IZ > DZ

Dominance (D')

10.28

8.91

2.54

4.16

Evenness (L)

0.62

0.57

0.28

1.61

the bivalve assemblages of Jalisco and Colima and those
described by Parker (1964) show fewer similarities. The
physical characteristics of these environments and the biol-
ogy of the species provide explanations for the presence of
distinct bivalve assemblages in the different zones across the
continental shelf of Jalisco and Colima. A reduction in mac-
rofaunal diversity with depth was also observed in the mol-
luscs from the Gulf of California (Hendrickx et al. 2007).
Other studies, however, showed no significant relationship
between diversity and depth for the demersal invertebrate
communities of the southern continental shelf of Jalisco
(Landa-Jaime and Arciniega-Flores 1998, Godinez-
Dominguez and Gonzalez-Sanson 1999).

The abundance of bivalves depends on their affinity to
type of substratum, temperature, depth, and oxygen concen-
tration (Levin et al. 2001). The structure and composition of
soft-sediment communities are related to sediment charac-
teristics {e.g., Sanders 1968, Gray 1981, Snelgrove and But-
man 1994); the type of substratum is particularly important
since all these species live on or within the sediments. How-
ever, the presence of some species or even the whole assem-
blages may also be determined indirectly by biological fac-
tors, such as feeding mechanisms, competition for food,
predator-prey relationships, etc. In the present study, the
abundance and distribution of bivalves was related to the
type of substratum and feeding habit. Some of the most
abundant species, such as the infaunal, filter feeding Corhula
ira, Nucidana lobiila, and Parvihtcina approxiniata were
more common in finer substrata, while some semi-infaunal
(Anadara adamsi) and epifaunal {Cyclopecten pernomiis) fil-
ter feeding species were characteristic of coarser substrata. In
general, filter feeding was important for most ot the bivalve
species, and was more common in shallow epibenthic and
semi-infaunal habitats.

In the continental shelf of the tropical Mexican Pacific,
the irregular topography of the coastline is closely related to
the different substrata, predominantly of terrigenous origin,
found in this region (McCoy and Sancetta 1985). The spatial

variation in species diversity is correlated with the hetero-
geneity of sediment grain size across the continental shelf
The greater heterogeneity in particle size and texture of the
shallow areas probably offers a greater variety of benthic
habitats. Species composition across the continental shelf
was closely related to the vertical distribution of the sedi-
ments. Most bivalves were collected in the sandy silt and silty
clay substrata of the shallow zone ( 18-40 m). The majority of
these species are filter feeders or deposit feeders that rely on
the infaunal or semi-infaunal habitats of the sea floor. Some
other representative species of this zone are epifaunal filter
feeders including those of the families Arcidae, Chamidae,
Mytilidae, Pectinidae, and Plicatulidae. Among the most im-
portant are the ark shell Aren pacifica (G. B. Sowerby I,
1833), the so called “pata de mula” Anadara spp., the Pacific
chama Chama sordida Broderip, 1835, the scallop Argopecten
ventricosiis (G. B. Sowerliy II, 1842) anci the mother-of-pearl
Pteria sterna (Gould, 1851). These species live attached to
hard substrata, other shells, rocks, or even fixed liy the left
valve (C. sordida).

In the intermediate and deep zones, sediments are finer
and more homogeneous than in the shallow zone and the
number of species decreases. The number of epifaunal spe-
cies also decreases, and most are infaunal deposit or filter
feeders, with a single semi-infaunal species of carrion feeder
(Verticoriidae). In the deep zone, the number of species is
even lower and dominance increases because two infaunal
species, Corbida ira and Nucidana lolnda, represent 31% of
all individuals in this zone.

Bottom water temperatures apparently had little effect
on the abundance and distribution of bivalves; the sampling
stations with the greatest abundance had temperatures
within a wide range of values. However, tlie differences be-
tween surface and bottom water temperatures are probably
important because thermal stratification may control oxygen
and food supply from surface waters. Benthic biomass and
abundance are assumed to reflect the rate of nutrient input
to the seafloor. Other variables related to thermal stratifica-

126

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

tion are the hydrodynamic regime and the water circula-
tion. Bottom water oxygen concentrations in the continental
shelf of the Mexican Tropical Pacific vary from high values
(~5 ml/1) in the shallow areas <100 m when the circulation
is strong enough to maintain high concentrations or little
oxygen consumption in the bottom layer, to low values (0.25
ml/1) toward deeper areas where there is weak circulation
(Pacheco-Sandoval 1991). In the area of study, prolonged
periods of hypoxic conditions may eliminate most macro-
benthos from the seabed (they either emigrate from the area
or die). A hypoxia-stressed benthos is typified by short-lived,
smaller surface deposit-feeding polychaetes and the absence
of bivalves (Rabalais et al. 2002). Thus, peaks in bivalve
abundance occur in the areas of strong stratification where
the availability of dissolved oxygen probably is not a limiting
factor.

In the eastern Pacific Ocean there are extensive mid-
water regions where oxygen is depleted, typically between
100 and 1,200 m depth (Oxygen Minimum Zone, OMZ)
(Kamykowsky and Zentara 1990, Levin et al. 2001). In shal-
low areas, OMZ may be evident at depths lower than 100 m.
Where these low oxygen regions intercept the continental
seabed, the benthos experiences hypoxia (Gallardo 1985,
Levin et al. 1991) and reduced macrofaunal diversity (Parker
1964, Mullins et al. 1985, Levin et al. 1991 ). Our data for the
continental shelf of lalisco and Colima coincide with this
general pattern.

Although oxygen exerts a strong effect on species rich-
ness, organic matter has a greater influence on dominance
(Levin and Cage 1998). Together these factors lower diver-
sity within the OMZs. Significant reduction of macrofaunal
species richness by low oxygen may not occur until concen-
trations fall below 0.4 or 0.3 ml/1; this value may be ever
lower for those species tolerant to hypoxia (Levin et al.
2001). Among the macrofauna, many molluscs appear less
tolerant of hypoxia than other taxa (Diaz and Rosenberg
1995) although there are some exceptions. In the continental
shelf of Jalisco and Colima, the number of species decreases
in the deep zone where oxygen concentrations tall to <0.3
ml/1. However, such species as Corbiila ira and Niiculatia
lolnila are very abundant and cause an overall decrease in
diversity. Members of the family Lucinidae seem particularly
widespread at this zone. The affinity of lucinid bivalves to
numerous OMZ sites within the eastern Pacific has been
documented by Levin et al. (2001).

In summary, the results suggest that substratum vari-
ability, oxygen concentration, and thermal stratitication
have an important influence on the bathymetric distribution
and abundance of bivalves in the continental shelf ot Jalisco
and Colima, and that the bivalve assemblages are not simply
the result of species independently sorting in diverse envi-
ronments. This study also shows that, even under hypoxic

conditions, bivalves may display significant peaks of abun-
dance. During the Atlas V expedition the abundance and the
number of species of gastropods and scaphopods also de-
creased with depth and no live specimens were collected at
stations deeper than 83 m (Perez-Peha and Rios-Jara 1998,
Rios-Jara et al. 2003b). Low dissolved oxygen concentrations
have also been mentioned as limiting the distribution of
benthic molluscs from the Gulf of California (Guerrero-
Pelcastre 1986) and the continental platform of Guerrero
(Lesser-Hiriart 1984). In the present study, the dominant
species were used to characterize the bathymetric zones;
however, the importance of many less common or even rare
species should also be taken into consideration because they
determine the structure of the community.

ACKNOWLEDGMENTS

The oceanographic expedition Atlas V on board of the
R/V El Puma was conducted with the technical and financial
support of the Universidaci de Guadalajara (UdeG) and the
Universidad Nacional Autonoma de Mexico under the su-
pervision of Manuel Guzman Arroyo. All persons in the
Laboratorio de Ecologia Marina at the former Eacultad de
Ciencias, Universidad de Guadalajara (UdeG) offered us
much help during field and laboratory work, especially
Martin Perez Pena, Lucia Lizarraga, and Samuel Renteria.
David Barrera (Instituto de Geografia, UdeG) made the de-
terminations of the types of substrata found in the area of
study. The authors are grateful to Eugene V. Coan for his
critical review of the manuscript and help with the taxo-
nomic identification and validation of the bivalves. Paul Val-
entich Scott of the Invertebrate Zoology Department at the
Santa Barbara Museum of Natural History also validated
some species. Kirstie Kaiser kindly gave us access to her
mollusc collection during the taxonomic revision of the spe-
cies in Puerto Vallarta.

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Submitted: 21 June 2007; accepted: 24 June 2008; final
revisions received: 4 November 2008

BIVALVE MOLLUSCS FROM JALISCO AND COLIMA, MEXICO

129

Appendix 1. Density (individiuils/m^) of bivalve species obtained with a Van Veen grab in the continental platform ot Jalisco and Colima,
Ndexico. MS, medium sand; SS, sandy silt; SC, silty clay.

Sampling station

52

24

47

35

23

48

26

51

34

18

22

30

33

50

25

29

T)'pe of substratum

MS

SS

MS

SS

SS

SC

SC

SC

SS

SS

SS

SC

SC

SC

SC

SC

Depth (m)

18

24

40

48

49

53

57

60

66

71

72

74

83

84

99

112

Temperature (°C)

28

30

24

16

26

29

26

25.5

17

15

14.5

16

16

19

14.5

14.5

Dissolved oxygen (ml/1)

6.3

5.0

3.6

0.6

0.6

6.5

7.4

5.6

0.5

0.2

0.3

0.3

0.4

1.0

0.3

0.2

Family Nuculidae

1. Nucula declivis Hinds, 1843

2. Nucula schenki Hertlein and Strong,

25

895 45

5 5

5

215

115

5

82.18

1940

5

5

80

10

6.25

Family Nuculanidae

3. Nuculana acapulcensis (Pilsbry and Lowe,
1932)

4. Nuculana laeviradius (Pilsbry and Lowe,

5

5

65 20

5 85

125

lit)

5

26.56

1932)

50

15 2235 5

5

10 25

155

940

5 5

215.62

5. Nuculana lobula (Dali, 1908)

5

35

10

15 745 55

50.06

Family Arcidae

6. Acar gradata (Broderip and G. B.

Sowerby I, 1829) 5

7. Anadara adamsi Olsson, 1961 5

8. Anadara aequatorialis (d’Orbigny, 1846)

9. Anadara concinna (G. B. Sowerby I,

1833)

10. Anadara formosa (G. B. Sowerby 1, 1833)

11. Anadara obesa (G. B. Sowerby 1, 1833)

12. Area paeifica (G. B. Sowerby I, 1833) 5

13. Barbatia reeveana (d’Orbigny, 1846)

14. Limarca brevifrons (G. B. Sowerby 1,

1833)

Family Noetiidae

15. Sheldonella delgada (Lowe, 1935)

16. Sheldonella olssoni (Sheldon and Marry,

1922) 30

Family Glycymerididae

17. Tucetona multkostata (G. B. Sowerby 1,

1833) 5

18. Tucetona strigilata (G. B. Sowerby 1,

1833) 5

Family Mytilidae

19. Crenella decussata (Montagu, 1808)

Family Pteriidae

20. Pteria sterna (Gould, 1851)

Family Isognomonidae

21. Isognomon recognitus (Mabille, 1895) 90

Family Pectinidae

22. Argopecten ventricosus (G. B. Sowerby 11,

1842)

23. Leptopecten biolleyi (Hertlein and Strong,

1946)

24. Leptopecten velero (Hertlein, 1935) 5

Family Propeammusiidae

25. Cyclopecten pernomus (Hertlein, 1935) 5

Family Plicatulidae

26. Plicatula pencillata Carpenter, 1857 5 35

Family Crassatellidae

27. Crassinella adamsi Olsson, 1961

28. Crassinella eciiadoriana Olsson, 1961 10

29. Crassinella paeifica (C. B. Adams, 1852) 45

30. Crassinella varians (Carpenter, 1857)

1900

30

1 10
5

5

35

15

225

10

105

20

20

565

25

30

1505

5

90

20

5

10

85 95

40

10

5

10

50

10 45

20

5

90 260

10

15

625 1180

15

15

0.625

135.93

1.87

7.5
0.31

2.5
0.94
0.31

0.31

6.56
1.87

1.25

1.56
18.44

0.625

5.625

7.812

3.125

1.875

58.75

4.06

2.5

0.625

212.5

0.94

130

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Appendix 1. (continued)

Sampling station

52

24

47

Type of substratum

MS

SS

MS

Depth (m)

18

24

40

Temperature (°C)

28

30

24

Dissolved oxygen (ml/l)

6.3

5.0

3.6

Family Carditidae

31. Cardites iaticostata (G. B. Sowerby I,

1833) 55

32. Cydocardia beebei (Hertlein, 1958)

33. Strophocardia megastropha (Gray, 1825)

Family Lucinidae

34. Divalinga pcrparvuln (Dali, 1901) 30

35. Liicina prolongata (Carpenter, 1857) 10

36. Ludnisca ccntrifiiga (Dali, 1901) 10

37. Ludnisca fenestrata (Flinds, 1845)

38. Ludnoma annulatuin (Reeve, 1850) 5

39. Neophysema aphanes Taylor and Glover,

2005

40. Parvilitdna approximata (Dali, 1901) 5 75

41. Parviludna inazatlanica (Carpenter,

1857) 35 5

42. Pegophysema edentnloides (Verrill, 1870)

43. Radioliidna cancellaris (Philippi, 1846) 295

Family Ungulinidae

44. Diplodonta soror (C. B. Adams, 1852) 15

45. Diplodonta suhquadrata (Carpenter,

1856) 5

Family Thyasiridae

46. Thyasira flexuosa (Montagu, 1803)

Family Kellidae

47. Kellia sitborbicularis (Montagu, 1803)

Family Chamidae

48. Chama sordida Broderip, 1835
Family Cardiidae

49. Laevicardium elenense (G. B. Sowerby 11,

1841) 5

50. Trachycardium belcheri (Broderip and

G. B. Sowerby 1, 1829) 5 5

51. Trachycardium procerum (G B. Sowerby

1, 1833) 5 5

52. Trachycardium senticosum (G. B.

Sowerby 1, 1833) 55

53. Trigoniocardia granifera (Broderip and

G. B. Sowerby 1, 1829) 5 85

54. Trigoniocardia obovalis (G. B. Sowerby 1,

1833) 30 85

Family Veneridae

55. Chione compta (Broderip, 1835) 345

56. Chione guatulcoensis Flertlein and

Strong, 1948 15 30 5

57. Chionchione pulicaria (Broderip, 1835) 30

58. Cydinella subquadrata (Hanley, 1844) 5

59. Dosinia dunkeri (Philippi, 1844) 5

60. Dosinia ponderosa (Gray, 1838) 10

61. Gouldia californica Dali, 1917 460

62. Lirophora kellettii (Hinds, 1845) 10

63. Lirophora marine (d’Orbigny, 1846) 5

64. Megapitnria squalida (G. B. Sowerby I,

1835) 5 460

35

23

48

26

51

34

18

22

30

33

50

25

29

SS

SS

SC

SC

SC

SS

SS

SS

SC

SC

SC

SC

SC

Mean

48

49

53

57

60

66

71

72

74

83

84

99

112

number of

16

26

29

26

25.5

17

15

14.5

16

16

19

14.5

14.5

individuals

0.6

0.6

6.5

7.4

5.6

0.5

0.2

0.3

0.3

0.4

1.0

0.3

0.2

per m^

3.44

5 50 3.44

10 0.625

10

2.5

0.625

5

20

190

70

155

5

28.44

10

0.625

5

10

40

10

4.375

45

50

5.93

10

85

1815

5

124.69

10

125

520

25

45

5

5

5

0.94

5

15

70

350

295

10

65

5

10

5

2.19

10 0.94

40 2.5

5 0.31

15 094

0.31

0.625

0.625

3.44

5.63

5 5 7.81

10 5 22.5

5 3.44

1.875

0.31

0.31

5 0.94

60 32.5

0.625

0.31

29.06

BIVALVE MOLLUSCS FROM JALISCO AND COLIMA, MEXICO

131

Appendix 1. (continued)

Sampling station

52

24

47

35

23

48

26

51

34

18

22

30

33

50

25

29

Type of substratum

MS

SS

MS

SS

SS

SC

SC

SC

SS

SS

SS

SC

SC

SC

SC

SC

Mean

Depth (m)

18

24

40

48

49

53

57

60

66

71

72

74

83

84

99

112

number of

Temperature ("C)

28

30

24

16

26

29

26

25.5

17

15

14.5

16

16

19

14.5

14.5

individuals

Dissolved 0X7gen (ml/1)

6.3

5.0

3.6

0.6

0.6

6.5

7.4

5.6

0.5

0.2

0.3

0.3

0.4

1.0

0.3

0.2

per m”

65. Periglypta midticostata (G. B. Sowerby 1,

1835) 5

66. Phar caUicomatus (Dali, 1902)

67. Pitar concinnus (G. B. Sowerby I, 1835) 60

68. Pitar multispinosus (G. B. Sowerby 11,

1851)

69. Transennella modesta (G. B. Sowerby 1,

1835)

Family Mactridae

70. Mactrellona subalata (Morch, 1860) 10

71. Midinia pallida (Broderip and G. B.

Sowerby 1, 1829) 10

Family Tellinidae

72. Cymatoica nndidata (Hanley, 1844)

73. Macoma elytriim Keen, 1958

74. Macoma siliqua (C. B. Adams, 1852)

75. Psammotreta aurora (Hanley, 1844)

76. Strigilla cicercula (Philippi, 1846) 5

77. Strigilla dichotoma (Philippi, 1846) 55

78. Strigilla interrupta Morch, 1860 30

79. Strigilla sp.

80. Tellina carpenteri Dali, 1900 30

81. Tellina coani Keen, 1971

82. Tellina martinicemis d’Orbigny, 1853

83. Tellina paciftca Dali, 1900

84. Tellina pristiphora Dali, 1900
Family Donacidae

85. Donax gracilis Hanley, 1 845
Family Solecurtidae

86. Tagelus politus (Carpenter, 1857)

Family Semelidae

87. Seniele pallida (G. B. Sowerby 1, 1833)

88. Semele verrucosa Morch, 1860

89. Semelina cainpbcUoruni Goan, 2003
Family Corbulidae

90. Corbula ira Dali, 1908

91. Corbula marmorata Hinds, 1843

92. Corbula nasuta G. B. Sowerby 1, 1833 25

93. Corbula ventricosa A. Adams and Reeve,

1850

Family Pandoridae

94. Pandora arcuata G. B. Sowerby 1, 1835 5

Family Verticordiidae

95. Trigonulina novemcostatus (A. Adams
and Reeve, 1850)

5

15

10

5

20

15

15

5

15

5

110

55

200

10

220

75

10

15

15

285

50

2450

25

5

5

10

10

5 10

5

5

5

5

5

10

5

5

5 5

15 10 10

5 5

10

20 5 5

5 5

5 60 90

35 145

10 40

5 10 55

0.94

1.25

4.375

0.625

1.25

0.625

0.625

2.5

1.25

2.19

0.31

1.25

0.31

9.69
3.44
2.81

15

1.25

13.75

0.625

4.69

0.625

0.94

1.56

27.5

1210 75.625

3.125
167.18

3.44

0.625

5.94

Total individuals per station

97

134

2603

23

42

3

24

7

4

146

530

1117

24

17

414 11

Total species per station

22

19

63

12

9

3

16

4

3

21

33

28

11

4

10 1

Density (individuals/m^) per station

485

670

13015

115

210

15

120

35

20

730

2650

5585

120

85

2070 55

Amer. Make. Bull. 26; 133-136 (2008)

A mature female of Bathothauma Chun, 1906 (Cephalopoda: Cranchiidae)
from Hawaii

Janet R. Voight

Department of Zoology, The Field Museum of Natural History, 1400 S. Lake Shore Dr., Chicago, Illinois 60605, U.S.A.,
Ivoight@fieldmuseum.org

Abstract: A gravid female of the cranchiid squid genus Bathothauma Chun, 1906 was collected from 1027 m depth in excellent condition,
other than having a ruptured ovary and tentacles reduced to stubs. The 2 mm diameter eggs, 18 spermatangia embedded in the skin of her
mantle, head, and eye, and the very pronounced nidamental glands indicate full sexual maturity. The eggs are over three times larger than
those previously reported in the genus.

Key words: Oegopsida, egg size, spermatangia, Taoniinae

The deep ocean forms the largest life-supporting area on
earth, yet the animals that inhabit the area remain poorly
known. Bathypelagic cephalopods are fairly abundant
throughout the world’s oceans (Clarke 1966) but continue to
be scarce in collections. “Glass squids” of the Cranchiidae
are a prime example. Many species of this group undergo
ontogenetic vertical descent in which young stages of the life
cycle occur at shallow depths; as the animals mature, they
move deeper (Young 1978). This distributional pattern con-
tributes to our limited knowledge of adults, and especially of
reproductively mature members of this group.

Squids of the cranchiid genus Bathothauma Chun, 1906
have been collected circum-globally, at depths of 100 to
nearly 2000 m (Voss 1980). Perhaps due to ontogenetic de-
scent, the most familiar and frequently collected members of
this genus are the remarkable young with unusually long
stalks supporting eyes that extend well away from the axis of
the body, and a very long brachial pillar that carries the arm
crown and the mouth (Fig. 1). Individuals of up to at least
7 cm mantle length retain this larval morphology (Voss
1980) although the eyes and arms are thought to be become
sessile in both sexes with maturation.

The most complete account of reproductive biology in
Bathothauma is that of Aldred (1974). Of the 87 specimens
considered, 3 specimens were maturing females, as evi-
denced by their detectable nidamental glands (Aldred 1974).
Young (1978) also reported three gravid females trawled
from near Oahu, Flawaiian Islands; he mentioned their en-
larged nidamental glands and large eggs, and described sper-
matangia embedded in one of the females.

To advance our knowledge of the species-level diversity
in the genus, this note reports data concerning a mature
temale collected from near 1000 m depth in Hawaiian wa-
ters. Voss et al. (1992) consider the genus Bathothauma to
include four species and indicate that Bathothauma lyromma

Chun, 1906, the only formally described species, is restricted
to the Atlantic tropical and subtropical regions to about
45°N in the northeast Atlantic. As a result, only the generic
identifier is applied to this specimen.

MATERIALS AND METHODS

The specimen was collected on 7 luly 1996 in a modified
opening/closing Tucker Trawl with a 10 m‘ mouth. Chil-
dress et al. (1978) describe the modifications to the trawl as
including the addition of a 30-L thermally protecting cod-
end to limit mechanical damage and heat shock to which the
animals are exposed at recovery. The collection locality was
off the island of Oahu at 21°35'00"N, 158°35'00"W to
21°20'00"N, 158°20'00"W at a maximum depth of 1027 m.
When the female was removed from the cod-end, eggs began
to spill out of her mantle cavity. She and her eggs were
preserved in 8% formalin in buffered seawater for two
weeks, then transferred to 70% ethanol at The Field Museum
of Natural History (FMNH), Chicago, Illinois where she is
catalogued in the Invertebrate collection as FMNH 286571.

Field Museum collections house six additional speci-
mens of the genus Bathothaiuna that were collected by trawl
from off Bermuda. Voss (1960) reported these specimens,
identifying the largest two, with mantle lengths of 60 and 80
mm as females. These specimens are compared to the com-
paratively newly collected one from Hawaii.

Measurements were performed with electronic calipers.
All eggs, whether loose or remaining in the mantle or rem-
nants of the ovary, were removed and individually counted.

RESULTS

Externally, the female Bathothauma sp. from Hawaii
appears to be in excellent condition (Fig. 2) although her

133

134

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Figure 1. Ventral view of the male specimen of Batlwtliainna lywmma (FMNH 78328, mantle length 73 mm) collected from off Bermuda
showing the larval morphology of the conspicuous stalks (S) carrying the eyes and the brachial pillar (BP). Note, however, the penis (P)
inside the open mantle. The scale carries 1 mm increments. ©2008 The Field Museum, Z94484_01d, Photographer John Weinstein.

tentacles are represented only by stubs. The stubs appear to
have been recently generated as the skin is puckered. Mea-
surements are presented in Table 1. The arm tips are in
excellent condition, with membranes on both sides; brachial
end-organs and photophores are absent. Arm sucker size
transitions fairly abruptly from large to small; the arm tips
clearly carry suckers in two rows. Arm suckers carry only blunt
teeth. The eyes are sessile on the sides of the head (Fig. 2).

Eight spermatangia lie just under the skin of the inner
mantle on its right lateral wall. The ejaculatory apparatus
emerges from the skin of the inner mantle and the skin on
the external mantle is damaged in the area overlying the
spermatangia. A total of six spermatangia are present medi-
ally on the left eye and on the neck posterior to the left eye.
The dorsal mantle posterior to the right eye carries four. The
mean length of four straight spermatangia is -4.5 mm
(range; 4.1 to 5.2).

When opened, in addition to a mass of loose amber-
colored eggs, the female’s mantle cavity contained a decapod
shrimp (carapace length 6 mm) and three other small crus-
taceans. These may have contributed to the apparent rupture
of the ovary. The total number of eggs is at least 1495,
including minimally 747 that had been released prior to
fixation, those removed from the mantle cavity and ovary,
and those found in the jar. Each egg is undifferentiated and
spherical (except where wrinkling of the outer cover implies
preservation-caused shrinkage), with a diameter of 2 mm.
Each egg appears to be identical in size. The outer cover of

the ovary has scattered chromatophore organs; the remain-
ing ovarian tissue carries small white sessile nodules that
conceivably could be eggs or remnants of egg follicles. The
oviducts generally lack any chromatophore organs and, al-
though considerably enlarged, do not contain eggs. The ovi-
ducal glands are covered with a smattering of chromato-
phores and appear veiy similar in size and shape to the
nidamental grants. The extremely large nidamental and ovi-
ducal glands are the most rigid organs in the mantle cavity
(Fig. 2h

The two largest comparative specimens (FMNH 78328,
FMNH 78329) were identified by Voss (1960) as females of
Bathothawna lyromma. Although the specimens have juve-
nile characters of eyestalks and brachial pillar, they also have
developing male reproductive organs. FMNH 78328 carries
a 4.3 mm long penis (Fig. 1), but other male reproductive
organs have been removed. Another large specimen from
Bermuda (FMNH 78329) has a testis with two concentrated
dots of purple pigment and accessory organs in the distal
mantle that are visible to the unaided eye, and the penis was
not removed from the associated tissue. There do not appear
to be stored spermatophores. Neither individual shows clear
evidence ot hectocotylization.

DISCUSSION

The large eggs, presence of spermatangia, and enlarged.

A MATURE FEMALE OF BATHOTHAUMA

135

Figure 2. Ventral view of the female specimen of Batiwthaunw sp. (FMNH 286571, mantle length 93 mm). The funnel (F) is labeled, note
the dark-colored eye (E) which carries two white spermatangia, the light-colored oviducts (OD) exposed in the mantle that emerge from
the remnants of the ovary, and the conspicuous nidamental (N) and oviducal (OG) glands. The scale carries 1 mm increments. ©2008 The
Field Museum, Z94485_01d, Photographer lohn Weinstein.

robust nidamental and oviducal glands (Fig. 2) all demon-
strate that the female Bathothau?}ia sp. examined was fully
gravid. At 2 mm in diameter, the eggs of this female are over
triple the 0.6 mm size of those in the only previous report for
the genus (Aldred 1974). Unfortunately, the crustaceans that
likely entered the mantle cavity of the female during collec-
tion preclude determination of how the eggs were held. The
distribution of spermatangia is very much the same as
Young (1978) reported in a female with very large eggs and
nidamental glands.

The female reproductive anatomy seen here (Fig. 2) is
very similar to that Chun (1910) reported for Leachin
Lesueur, 1821. In Leachia pacificn (Issel, 1908), a member of
the Cranchiinae, Young ( 1975) reported that the nidamental

Table 1. Measurements in mm and counts for Bathothainua sp.
(FMNH 286571).

Mantle length 93

Head width (exclusive of eyes) 10

Head width (including eyes) 25

Arm length I 22.4

Arm length II 29.0

Arm length III 32.0

Number of arm suckers 57, 56

Arm length IV 29.0

Greatest eye length 18.9

Fin length at base 10.5

Maximum fin length 17.5

Nidamental gland length 14.6

glands of near- or perhaps post-spawning females were ge-
latinous. Those of this female are robust with clear lamellae,
with no indication of gelatinous texture. The contrast sug-
gests that the females Young examined were post- rather
than near spawning. The recovery of this female with intact
arm tips also confirms that the brachial end organ is absent
in this genus (Flerring el al. 2002).

Graphically, none of the measurements or counts of this
female distinguish it from the specimens represented on
plots of three characters (Aldred 1974; fig. 1). These plots,
however, rely on mantle length, a measurement that Voss et
al. ( 1992) caution tends to be problematic as a size indicator
in cranchiids due to preservation artifacts. The sexual matu-
ration seen in the “larval” specimens collected off Bermuda
imply that the change to adult morphology is not necessarily
a pre-requisite for sexual development. The female specimen
and data concerning her reproductive status are offered in
hopes they aid futures studies of the genus and its species-
level diversity.

ACKNOWLEDGMENTS

B. Seibel kindly invited my participation in the cruise
that resulted in collection of the specimen; he and A.
Lindgren provided helpful comments on the manuscript, as
did two anonymous reviewers. The Captain and crew of the
R/V New Horizon made the cruise successful. Marshall Field
Fund of the Zoology Department at The Field Museum of
Natural Flistory supported my participation in the cruise.

136

AMERICAN MALACOLOGICAL BULLETIN

26 • 1/2 • 2008

LITERATURE CITED

Aldred, R. G. 1974. Structure, growth and distribution of the squid
Bathothauma lyromma Chun. Journal of the Marine Biological
Association of the United Kingdom 54: 995-1006.

Childress, I. h, A. T. Barnes, L. B. Quetin, and B. H. Robison. 1978.
Thermally protecting cod ends for recovery of living deep-sea
animals. Deep-Sea Research 25: 419-422.

Chun, C. 1910. Die Cephalopoden. Oegopsida. Wissenschaftliche
Ergebnisse der Deutschen Tiefsee-Expedition, “Valdivia”
1898-1899, 18: 1-522, atlas. [Translated by the Israel Program
for Scientific Translations 1975].

Clarke, M. R. 1966. A review of the systematics and ecology of
oceanic squids. Advances in Marine Biology 4: 91-300.

Herring, P. [., P. N. Dilly, and C. Cope. 2002. The photophores of
the squid family Cranchiidae (Cephalopoda: Oegopsida).
Journal of Zoology 258: 73-90.

Voss, G. L. 1960. Bermudan cephalopods. Fieldiana-Zoology 39:
419-446.

Voss, N. A. 1980. A generic revision of the Cranchiidae (Cepha-
lopoda: Oegopsida). Bulletin of Marine Science 30: 365-412.

Voss, N. A., S. I. Stephen, and Zh. Dong. 1992. Family Cranchiidae
Prosch, 1849. In: M. ). Sweeney, C. F. E. Roper, K. M.
Mangold, M. R. Clarke, and S. v. Boletzky, eds., “Larval” and
juvenile cephalopods: A manual for their identification. Smith-
sonian Contributions to Zoology 513. Pp. 187-210.

Young, R. E. 1975. Leachia pacifica (Cephalopoda, Teuthoidea):
Spawning habitat and function of the brachial photophores.
Pacific Science 29: 19-25.

Young, R. E. 1978. Vertical distribution and photosensitive vesicles
ot pelagic cephalopods from Hawaiian waters. Fisheries Bulle-
tin 76: 583-615.

Submitted: 8 April 2008; accepted: 24 July 2008; final

revisions received: 2 October 2008

Amer. Maine. Bull. 26: 137-151 (2008)

Conservation of the freshwater gastropods of Indiana: Historic and
current distributions

Mark Pyron, Jayson Beugly, Erika Martin, and Matthew Spielman

Department of Biology, Ball State University, Muncie, Indiana 47306, U.S.A., mpyron@bsu.edu

Abstract: We surveyed Indiana collections of freshwater gastropods from 220 museum collection lots and found 39 species inhabiting
Indiana historically. Collection dates of museum material ranged from 1900 to 2006, with a median date of 1986. We collected 17,593
gastropods at 123 sites, including 86 sites where museum material was previously collected. Our surveys were combined with recent
literature surveys and indicate a total of 36 species are currently present in Indiana. The Indiana fauna is composed of three species that
are apparently secure globally, and 36 species that are widespread, abundant, and globally secure, including two exotics. However, three
species are locally extinct and many others are locally imperiled or vulnerable. The majority of freshwater gastropod taxa in Indiana are of
local conservation concern. The causes of local gastropod extinctions are unknown but likely include agricultural impacts, hydrologic
alterations from reservoirs, and pollution. We recommend thorough inventory, recognition, and protection of the aquatic gastropods in
Indiana.

Key words: macroinvertebrates, endangered sp>ecies, snails, distributions, biogeography

Freshwater gastropods inhabit all aquatic habitats in
North America. However, relatively little information is
available on species distributions and the ecological require-
ments of this group as a whole (Burch 1982, Thorp and
Covich 2001, Stewart 2006). Although recent distributional
studies exist for several states — e.g., Iowa and New York
(Jokinen 1992, Stewart 2006), large knowledge gaps remain
for geographic distribution and species composition
throughout much of North America. Aquatic gastropods are
a large component of freshwater ecosystems, providing sig-
nificant biomass as herbivores (Brown 2001, Brown et al.
2008). Freshwater gastropods are frequently used in water
quality bioassessments because of the occurrence of several
indicator species or groups that are sensitive to water quality
and habitat alteration (Salanki et al. 2003). In addition,
freshwater organisms are the most imperiled fauna in North
America, and freshwater gastropods are a group that is at
risk (Ricciardi and Rasmussen 1999, Brown et al. 2008).

Indiana’s water resources are at risk because of a com-
bination of agricultural, urban, and other human impacts.
These effects result largely from patterns of human land use
and subsequent effects on aquatic ecosystems (Allan 2004,
Pyron et al. 2006). Although land use patterns are unlikely to
change in the near future, identification and awareness of
existing fauna can provide a baseline for further monitoring
and conservation.

The earliest attempt to produce a guide to the aquatic
gastropods of Indiana was by Goodrich and van der Schalie
(1944), which included an identification key, habitat de-
scriptions for each species, and brief descriptions of spe-
cies distributions. However, most of the taxonomy used in

this guide is out of date, and no other Indiana guides to
aquatic gastropods have been published. Several more recent
studies of Indiana aquatic gastropods provide presence/
absence information for several taxa. Brown (1982) sam-
pled aquatic gastropods of temporary ponds in northeast
Indiana, to test for habitat overlap among species. He found
six species that varied in abundances by pond type, lokinen
(2005) surveyed ponds of the Indiana Dunes National
Park, to compare with a historic survey by Shelford (1913).
Many of the ponds that Shelford (1913) surveyed have
since been destroyed by industrial development. However,
lokinen (2005) found similar overall species richness for
aquatic gastropods, due to a combination of species that
appeared to be extinct and other species that were not found
in Sheltord’s (1913) survey. Greenwood and Thorp (2001)
studied the distributions and substrate selection of two
caenogastropods in the Ohio River, upstream from Louis-
ville, Kentucky. Both species, Lithasia obovata (Say, 1829)
and Pleiirocera canaliculata (Say, 1821), are large river
specialists.

A current survey of Indiana snails is important because
it provides information of local declines and extinctions that
will require action from conservationists. Local extinctions
may suggest problems with water quality or hydrologic al-
terations in the watershed, or other explanations for absence
of gastropods at sites. Information on historical and current
snail distributions throughout Indiana will thus be invalu-
able to future water quality managers and scientists. This
study is such a survey of museum collections from Indiana,
coupled with site visits to assess the current status of aquatic
gastropods in the state.

137

138

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

MATERIALS AND METHODS

Study area

Indiana is in the mid-western United States, with physi-
ography consisting primarily of glacial till plains (Visher
1922) and a total area of 94,000 km“. The majority of the
state is in the Central Lowland province with only local
topographical relief. The southern limit of glaciation is a
boundary line between the Central Lowland and the south-
ern Low Plateau (~Vi of the southern portion of the state).
One fourth of the state along the north is in the Eastern Lake
Section, with many moraine lakes formed from glacial drift.
Two major watersheds drain the state: the Great Lakes are
immediately north, and the remainder of the state is in the
Mississippi River basin. The Illinois River watershed in-
cludes the Kankakee River to the northwest, the southern
section of the state drains directly into the Ohio River, and
the majority of the state is within the Wabash River water-
shed that drains to the Ohio River (Visher 1922).

The human footprint has been large in Indiana. About
98% of land is used for cropland, pasture, or development
(GAP 1996). The northern 24% of the state was predomi-
nately wetland prior to European settlement, and 85% of
these wetlands have been lost, with drainage for agriculture
the primary cause (IDNR 1996). Water quality of Indiana
streams was severely altered by humans (Gammon 1998).
Nearly all Indiana streams that are within the Wabash River
watershed (>70 % of the state) have hydrologic alterations
(significant changes to the natural flow regime) caused pri-
marily by agricultural effects and/or reservoir release (Pyron
and Neumann 2008). The net result is a human-dominated
landscape with habitat fragmentation and degradation,
widespread pollution, and isolated plant and animal
populations.

We used a two-step process for surveying aquatic gas-
tropods of Indiana. The first step was to assess historical
distributions using two natural history collections (Ohio
State University Museum of Biological Diversity and Uni-
versity of Michigan Museum of Zoology) that contain
aquatic gastropod material from Indiana. The second step
was to return to sites where museum collections were taken
and to re-survey the sites to determine if previously recorded
species were still present. The modern surveys would also
reveal any additional species not reported in earlier surveys.
Museum visits included examining specimens and verifying
identifications and recording location information and col-
lection dates. No other Indiana guides to aquatic gastropods
have been published, thus Burch’s (1982) keys and notes are
the primary reference for identification. Unless otherwise
noted, nomenclatural taxonomy was from Turgeon et al.
(1998) or Stewart (2006).

Eield sampling

We visited 123 sites of which 86 were historical sites, in
the summers of 2006-2008 to collect aquatic gastropods (Pig.

1). The additional sites were in locations where historic
samples were sparse. Methods consisted of sampling all
available habitats at each site, primarily by hand collections
in shallow water, on woody debris, on the undersides of
stones, and on aquatic vegetation. Deeper areas and fine
substrates were sampled with a net. Collection durations I
were the equivalent of one individual searching for 60 min. j
For example, two persons searched for 30 min (Brown et al. ;
1998). Gastropods were preserved in 70% ethanol and iden- \i
tified in the laboratory to the lowest possible taxonomic '!
level, using Burch (1982). All specimens will be deposited at j
the Illinois Natural History Survey. We determined global jj
conservation status for species using The Nature Conser- !'
vancy designations (www.natureserve.org), and we described i
the Indiana status based on our collections. j

RESULTS

We found 220 lots of Indiana aquatic gastropods at the
two museums, comprising 39 taxa at 86 sites. Museum ma-

Figure 1. Sites sampled in 2006-2008. Coordinates are available
from authors.

FRESHWATER GASTROPODS OF INDIANA

139

terial was collected between 1900 and 2006 with a median
collection date of 1976. Our current survey of 123 sites
yielded 17,593 individuals in 32 species (Figs. 2-17). Four
additional taxa were included based on the survey by Joki-
nen 2005). Most taxa occurred at few sites — the average
number of sites where an individual taxon occurred was 1 1
(range, 1-75; Figs. 2-17). The mean abundance of individuals
collected at sites was 144 (range, 0-1463). The mean number
of species per site was 3.3 (range, 0-8). Ten of the historic
collection sites and five new sites had no gastropods. Seven-
teen of the 36 taxa we collected, or that were in Jokinen’s
(2005) collections, were not collected in previous surveys.

Mean water hardness was 300 mg CaCO,/L and ranged
from 40 to 1200. Mean water conductivity was 550 pmhos
and ranged from 101 to 1800. Mean pH was 8.2 and ranged
from 6 to 9.6. Although we found variation in mean water
chemistry parameters among species, overall variation
among sites was relatively low. The majority of species had
mean hardness values of 300 mg CaCOj/L, conductivity of
500 pmhos, and pH of 8.0. Only one species occurred at sites
with an exceptional mean water chemistry value: Ferrissia
fragilis (Tryon, 1863) was found at 18 sites with a mean pH
of 7.2. We will examine the influence of environmental vari-
ables on gastropod assemblages in detail in a separate study.

The following list of taxa is organized by family. Distri-
bution maps include historical sites from archival material
and current (2006-2008) collections. Not all museum mate-
rial included specific site details or dates. We did not include
information on maps it collections lacked site information.

Family Valvatidae

Valvatn bicarinatn (Lea, 1841). Goodrich and van der
Schalie (1944) reported the species occurred likely in every
county in Indiana. This species has apparently declined or
disappeared, as in Iowa (Stewart 2006). We consider it to be
extinct in Indiana and secure in the rest of the range.

Valvata lewisi (Currier, 1868). No historical collections
were found. The species was historically present in lakes in
Kosciusko and Marshall County (Goodrich and van der
Schalie 1944). We collected this species only at Clear Lake,
Steuben County. The habitat was silt substrate and sub-
merged vegetation. This species occurs in southern Canada
from Quebec to British Columbia and northern U.S. from
New York to Minnesota (Burch and Tottenham 1980). lo-
kinen (1992) found only one site for this species in New
York, and Stewart (2006) determined the species is extinct in
Iowa. We categorized it as critically imperiled in Indiana but
it is secure in the rest of the range.

Valvata tricarinata (Say, 1817). No historical or recent
collections were found. Jokinen (2005) found this species in
a pond at Indiana Dunes National Lakeshore in 1992-1993.

We categorized it as critically imperiled in Indiana but se-
cure in other parts ot the range.

Valvata sincera (Say, 1824). No historical or recent col-
lections were found. Burch and Tottenham (1980) reported
the range as Maine west to Alberta, and south to South
Dakota and Indiana. We consider it to be extinct in Indiana
but secure in other parts of the range.

Family Viviparidae

Viviparus georgiauus (Lea, 1834). No historical collec-
tions were found. Wright (1932) collected this species in
Indiana at four sites on Maxinkuckee Lake and its outlet, the
Tippecanoe River. The species was historically present in the
Wabcish River and numerous Indiana lakes (Goodrich and
van der Schalie 1944). We collected this species at two sites:
Clear Lake (Steuben County) and Lake Wawasee (Elkart
County). Both lakes had submerged vegetation and either
sand or silt substrates. This species is distributed across the
midwest and eastern U.S. (Burch and Tottenham 1980). lo-
kinen (1992) found many sites with this species in New
York. It appears to be critically imperiled in Indiana but
secure in other parts of the range.

Viviparus subpurpiireiis (Say, 1829). This species was
collected historically at four sites that were large rivers and
one pond (Fig. 2). We did not collect this species. The three
large river sites have reservoirs within their watersheds, and
reservoir releases likely produce hydrologic alterations to
natural flow regimes. This species has a range through out
the Mississippi River watershed to Iowa, Illinois, and Ken-
tucky and south to Louisiana (Burch and Tottenham 1980,
Brown et al. 1989). Goodrich and van der Schalie (1944)
reported the species as confined to larger streams such as the
Mississippi, Ohio, and Wabash rivers. Populations appear to
be possibly extinct in Indiana but secure in other parts of the
range.

Bellamya chiiiensis (Reeve, 1863). No historical collec-
tions were found. We collected this species at four sites that
were lakes and rivers in the northern third of the state (Fig.
2). The sites had submerged macrophytes and various sub-
strates. This Asian snail is an exotic species that has been
introduced and subsequently dispersed across North
America (Stewart 2006).

Bellamya japouica (von Martens, 1861). No historical
collections were found. We collected this species at four sites
that were lakes and rivers in the northern third of the state
(Fig. 2). The sites had submerged macrophytes and various
substrates. This Asian snail is an exotic species that has been
introduced and subsequently dispersed across North
America (lokinen 1992).

Catiipeloina decisum (Say, 1817). We mapped all of the
Campelonia spp. records together, following Stewart (2006).
However, we recognized our current collections as C. de-

140

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Figure 2. Distributions of historic Vivipanis subpttrpureus (circles),
current Bellamya cliinensis (triangles), and current Bellamya ja-
ponica (diamonds).

cisiim. We found 20 historical collections and 11 current
sites (Fig. 3). Habitats included macrophytes, woody debris,
and various substrates of silt, sand, gravel, and cobble. Cam-
peloma spp. occur in the Missouri and Mississippi water-
sheds (Stewart 2006 and references therein). They are com-
mon in lakes and rivers of Indiana and we classified them as
secure.

Family Hydrobiidae

Birgella subglobosns (Say, 1825). No historical collec-
tions were found. We found the species at six sites (Fig. 4).
Habitats included various substrate categories but lacked silt.
The species was historically found throughout Indiana
(Goodrich and van der Schalie 1944) with a range from Ohio
west to Iowa, and from Michigan south to Alabama and
Arkansas (Burch and Tottenham 1980). Jokinen (1992) col-
lected the species in Lake Champlain, St. Lawrence water-
shed. We classified it as imperiled in Indiana but it is ap-
parently secure in other parts of the range.

Figure 3. Distributions of historic (diamonds) and current Cam-
peloma decisiim (circles). Sites where historic and current collec-
tions occurred are triangles.

Cindnnatia Integra (Say, 1821). We found one historical
collection of this species from Lake James and we collected
this species at one site on the Eel River. The habitat at the Eel
River was gravel and sand substrates and submerged vegeta-
tion. Shelford (1913) found this species in ponds at the
Indiana Dunes National Lakeshore. However, Jokinen
(2005) did not find the species at the same ponds in 1992-
1993. This species occurs in the Ohio River and tributaries in
Ohio, Indiana, Kentucky, and southeastern Illinois (Burch
and Tottenham 1980). Jokinen (1992) did not collect this
species in New York, but it was found there historically. The
species was historically common in Iowa (Stewart 2006). We
classified it as critically imperiled in Indiana but secure in
other parts of the range.

Pyrgulopsis histrica (Pilsbry, 1890). Three historical col-
lections were found: Tippecanoe Lake (Elkhart County),
Pine Lake (La Porte County), and Lake Michigan (Lake
County). We found the species in Little Turkey Lake and Big
Turkey Lake (Steuben County). The habitats were sand or

FRESHWATER GASTROPODS OF INDIANA

141

Figure 4. Distributions of current Birgella subglohosus (diamonds),
current Amnkola limosus (circles), and Pomatiopsis cincinnatiensis
(triangles).

silt substrates, woody debris, and emergent vegetation. This
species occurs in southern Quebec and Ontario, and from
Maine and New York west to Iowa and Minnesota (Burch
and Tottenham 1980). Jokinen (1992) found this species
at nine sites in New York. The species was found at several
Iowa locations in 1979 (Stewart 2006). Goodrich and van
der Schalie (1944) found this species was common in lakes,
ponds, and streams that had heavy growths of macro-
phytes and algae. We classified its status as locally imperiled
due to very few populations, but secure in other parts of
the range.

Amnicola limosus (Say, 1817). No historical collections
were found. We found the species at six sites (Fig. 4). Shel-
ford ( 1913) found this species in ponds at the Indiana Dunes
National Lakeshore. lokinen (2005) found the species at one
of the same ponds in 1992-1993. We found the species at six
sites (Fig. 4). The species is imperiled in Indiana but secure
in other parts of the range.

Family Pomatiopsidae

Pomatiopsis ciucimiatiensis (I. Lea, 1850). This species is
amphibious. No historical collections were found. We found
the species at live sites (Fig. 4). The habitats were sand or silt
substrates, and submerged or emergent vegetation. The river
site had sand, gravel, and cobble substrates, and submergent
vegetation. Goodrich and van der Schalie (1944) reported
the historical distribution as Henry and La Porte Counties.
The species is vulnerable in Indiana but it is apparently
secure in other parts of the range.

Family Pleuroceridae

Elimia livescens (Menke, 1830). We included historical
material that was misidentified as Elimia semicarinata (Say,
1829). We found 24 historical sites and 48 current sites (Fig.
5) of which five were lakes. The sites had various substrates
including silt, sand, gravel, cobble, and boulders. This spe-
cies occurs in the St. Lawrence River drainage from the Great

Figure 5. Distributions of historic (diamonds) and current Elimia
livescens (circles). Sites where historic and current collections oc-
curred are triangles.

142

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Lakes to Lake Champlain and Quebec, east of the Scioto
River in Ohio and west to the Illinois River (Burch and
Tottenham 1980). The historic distribution was in the
Wabash River watershed, the Maumee River watershed, the
St. loseph River watershed (Goodrich and van der Schalie
1944). The species was abundant in New York (lokinen
1992). This is a common and iibundant species in large rivers
of Indiana with a secure status.

Pleiirocera acuta (Rafmesque, 1831). We found 12 his-
torical sites and 21 current sites for this species (Fig. 6) of
which four were lakes. Habitat at the sites was varied with
silt, sand, gravel, cobble, or boulder substrates and occasion-
ally included macrophytes and/or woody debris. This species
occurs in the Ohio River headwater streams and tributaries,
the Great Lakes and tributaries, the Mississippi River water-
shed to Nebraska and Kansas, and the Cumberland and
Duck rivers in Tennessee (Burch and Tottenham 1980).
Goodrich and van der Schalie ( 1944) reported the historical

Figure 6. Distributions of historic (diamonds) and current Pleuro-
cera acuta (circles). Sites where historic and current collections
occurred are triangles.

distribution in Indiana as the upper Wabash River, tribu- |
taries, and lakes connected to the river, and the Maumee ;
River and watershed. Stewart (2006) identified numerous
collections in Iowa, lokinen (1992) found the species at
many New York locations. This species is common and
abundant in Indiana with a secure status.

Pleuwcera caiialiculata (Say, 1821). We found 14 his-
torical sites and two current sites on the Ohio River and 1
White River (Fig. 7). Habitat at the Ohio River site was silt, .
sand, and riprap substrates with woody debris. The White I
River site had silt and riprap substrates. This species occurs '
in the Ohio River from Pittsburgh to Illinois, the Wabash ;
River and its tributaries, aberrantly in the Tennessee River )
system, and to Omaha, Nebraska (Burch and Tottenham i
1980). This species was found to be abundant in the Ohio I
River upstream from Louisville, Kentucky (Greenwood and
Thorp 2001). Goodrich and van der Schalie (1944) listed the '
Indiana distribution as in the Wabash River above Lafayette )
downstream to the Ohio River, present in the White River )

Figure 7. Distributions of historic (diamonds) and current Pleuw-
cera canalicidata (circles).

FRESHWATER GASTROPODS OF INDIANA

143

and in the Ohio River. This is a rare species in Indiana but
it is secure in other parts of the range.

Leptoxis praerosa (Say, 1821). No historical collections
were found. We found the species at one site on the Blue
River, Harrison County. The habitat was silt and riprap sub-
strates with woody debris also present. Its range is the Ohio
River below Cincinnati, Ohio to Elizabethtown, Illinois; the
Cumberland River and tributaries; the Duck River, Tennes-
see; and the Tennessee River and tributaries (Burch and
Tottenham 1980). Goodrich and van der Schalie (1944) re-
ported the historical Indiana distribution as the Ohio River
from Scioto County, Ohio to Pope County, Illinois, the
Wabash River at Grand Chains, Posey County, and the Big
Blue River, Crawford County. We categorized its status as
critically imperiled in Indiana but it is secure in other parts
of the range.

Lithasin obovata (Say, 1829). We found one historical
collection from the Blue River in Harrison County, and one
current site on the Eel River in Logansport. The habitat was
silt, sand, and cobble substrates with emergent vegetation.
This species was abundant in the Ohio River upstream from
Louisville, Kentucky (Greenwood and Thorp 2001). This
species occurs in the Ohio River and tributaries, in Penn-
sylvania, Ohio, Indiana, Illinois, Kentucky, and Tennessee
(Burch and Tottenham 1980). Goodrich and van der Schalie
(1944) reported this species present in the Wabash River
downstream from Vincennes, in the Ohio River, the Big Blue
River (Crawford County), and the Kentucky River in Ken-
tucky. We categorized its status as critically imperiled in
Indiana but it is apparently secure in other parts of the
range.

Family Lymnaeidae

Fossaria spp. (Say, 1822). Stewart (2006) attributes
many currently confused taxa to this group. We found seven
historical collections that were in lakes and the Wabash
River, and at 43 of our current sites (Fig. 8) of which live
were lakes. Substrates at the sites varied with silt, sand,
gravel, cobble, or riprap substrates, and occasional woody
debris and vegetation present. Brown (1982) found this
taxon in ponds at the Crooked Lake Field Station at Fort
Wayne. Goodrich and van der Schalie (1944) reported this
taxon present in ponds, lakes, and brooks in Kosciusko,
Starke, Steuben, and La Porte Counties. Shelford (1913)
found this taxon in ponds at the Indiana Dunes National
Lakeshore. lokinen (2005) found the taxon at the same
ponds in 1992-1993. Burch and Tottenham (1980) described
the range of taxa in this group to include eastern North
America west to Vancouver Island. These taxa appear to be
common and abundant in Indiana and thus secure.

Lymnaea stagnalis (Linnaeus, 1758). No historical col-
lections were found. We found the species at Bass Lake,

Figure 8. Distributions of historic (diamonds) and current Fossaria
spp. (circles). Sites where historic and current collections occurred
are triangles.

Starke County. The habitat of the site was silt and sand
substrates and emergent vegetation present. This species
range is the Great Lakes-St. Lawrence River drainage area
northwest to the Mackenzie and Yukon River drainage areas,
west to the Rocky Mountains, south to Colorado, and in
Illinois and Ohio in the Mississippi drainage (Burch and
Tottenham 1980). The historical Indiana distribution was
small lakes and streams of the northern part of the state, and
in Lake Michigan (Goodrich and van der Schalie 1944). We
categorized its status as critically imperiled in Indiana but it
is secure in other parts of the range.

Stagnicola catascopinni (Say, 1867). No historical collec-
tions were found. We found the species at one site. Fish
Creek, Steuben County. The habitat of the site was silt and
sand substrates and woody debris was present. Goodrich and
van der Schalie (1944) reported the species was present in
the Great Lakes and in other bodies of shallow water near
Lake Michigan. The range is eastern Canada and Nova Scotia

144

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

west to North Dakota, Great Slave Lake south to northern
Iowa, northern Ohio, and Maryland (Burch and Tottenham
1980). We categorized its status as critically imperiled in
Indiana but it is secure in other parts of the range.

Stagnicola capernta (Say, 1829). One historical collection
was found from the Maumee River. We did not find this
species. Goodrich and van der Schalie (1944) suggested the
species occurred in every county ot Indiana. The range is
Quebec and Massachusetts west to Galifornia, Yukon Bay,
and lames Bay south to Maryland, Indiana, Colorado, and
California (Burch and Tottenham 1980). We categorized its
status as critically imperiled in Indiana but it is secure in
other parts of the range.

Stagnicola elodes (Say, 1821). No historical collections
were found. We found this species at 17 sites (Lig. 9) of
which four were lakes. The habitats varied with substrates of
silt, sand, gravel, cobble, or hardpan and woody debris and/
or vegetation occasionally present. Goodrich and van der
Schalie (1944) reported this species was expected in ditches,
ponds, and shallow parts of lakes with heavy vegetation.

Brown (1982) found this species was abundant in ponds at
the Crooked Lake field Station at fort Wayne. Shelford
(1913) found this species in ponds at the Indiana Dunes
National Lakeshore. lokinen (2005) found the species at the
same ponds in 1992-1993. It is common and abundant in
Indiana and is secure.

Stagnicola exilis (I. Lea, 1838). No historical collections
were found. We found this species at Brown Ditch, Newton
County. The habitat was silt substrate with submerged mac-
rophytes. Goodrich and van der Schalie (1944) found this
species to occur in temporary aquatic habitats. We catego-
rized its status as critically imperiled in Indiana but it is
secure in other parts of the range.

Psendosnccinea columella (Say, 1817). No historical col-
lections were found. We found 16 current stream sites (Lig.
10) of which seven were lakes or ponds. Habitats varied with
substrates of silt, sand, gravel, or cobble and woody debris
and/or vegetation occasionally present. The range is eastern
North America generally west to Minnesota and eastern
Kansas, south to central Texas and florida (Burch and Tot-
tenham 1980). Goodrich and van der Schalie (1944) re-

FRESHWATER GASTROPODS OF INDIANA

145

ported this species present in northern Indiana counties. The
species is common in New York (Jokinen 1992). It appears
to be common in Indiana and is secure.

Family Physidae

PJiysella gyrimi (Say, 1821 ). We found 22 historical sites
and eight current sites (Fig. 11) of which one was a lake.
Habitats varied with silt, sand, gravel, or cobble substrates
and woody debris and/or vegetation present. Brown (1982)
found this species was abundant in ponds at the Crooked
Lake Field Station at Fort Wayne. Shelford (1913) found this
species in ponds at the Indiana Dunes National Lakeshore.
Jokinen (2005) found the species at the same ponds in 1992-
1993. The species appears to be common and abundant and
is secure.

Physella acuta (Draparnaud, 1805). We found 17 his-
torical sites and 94 current sites (Fig. 12). Habitats varied
with substrates of silt, sand, gravel, riprap, or cobble and

Figure 11. Distributions of historic (diamonds) and current Phy-
sella gyrina (circles). Sites where historic and current collections
occurred are triangles.

Figure 12. Distributions of historic (diamonds) and current Phy-
sella acuta (circles). Sites where historic and current collections
occurred are triangles.

woody debris and/or vegetation occasionally present. This
species occurs throughout North America (Burch and Tot-
tenham 1980) and is abundant in Indiana. We consider it to
be secure in Indiana.

Aplexa ekmgata (Say, 1821). Two historical collections
were found: Tippecanoe Lake (Elkhart County) and the
Elklrart River (Noble County). We did not find this species.
Brown (1982) found this species was abundant in ponds at
the Crooked Lake Field Station at Fort Wayne. Jokinen
(2005) found the species in temporary aquatic habitats in the
Indiana Dunes National Seashore in 1992-1993. Its range is
Ontario to Saskatchewan, Canada, and Alaska (Burch and
Tottenham 1980). We categorized its status as imperiled in
Indiana but it is secure in other parts ot the range.

Family Planorbidae

Gyraiilus circiunstriatus (Tryon, 1866). We found one
historical collection from Rock Creek, Carroll County, and

146

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

none in our collections. Goodrich and van der Schalie
(1944) reported the species present in Lake James, Lake
Maximkuckee, and Webster Lakes in northern Indiana. This
species occurs from Connecticut north to Quebec, rvest to
Alberta, and south in the Rocky Mountains to New Mexico
(Burch and Tottenham 1980). Jokinen (1992) found the
species in New York collections but commented that it ap-
pears to be intolerant to low pH and low calcium, as are
most snails. The species appears to be extinct in Indiana but
secure in other parts of the range.

Gyraulus deflectus (Say, 1824). No historical collections
were found although Shelford (1913) found this species in
ponds at the Indiana Dunes National Lakeshore. Jokinen
(2005) did not find them during 1992-1993 surveys of the
same ponds. We found the species at 10 sites (Fig. 13).
Habitats varied with substrates of silt, sand, gravel, or cobble
and woody debris and/or vegetation occasionally present.
We consider the species to be vulnerable in Indiana but it is
secure in other parts of the range.

Gyraulus parvus (Say, 1817). We found nine historical

sites and nine current sites (Fig. 14). Habitats varied with
substrates of silt, sand, gravel, cobble, or boulder and woody
debris and/or vegetation occasionally present. Shelford
(1913) found this species in ponds at the Indiana Dunes
National Lakeshore. Jokinen (2005) found the species at the
same ponds in 1992-1993. Brown (1982) found this species
was abundant in ponds at the Grooked Lake Field Station at
Fort Wayne. Its range is all of North America (Burch and
Tottenham 1980). Jokinen (1992) found the species at many
sites in New York. Goodrich and van der Schalie (1944)
comment that the species was “doubtless present in every
county.” The species appears to be common and abundant
and is secure.

Helisoina anceps (Menke, 1830). No historical collec-
tions were found. Shelford (1913) found this species in
ponds at the Indiana Dunes National Lakeshore. Jokinen
(2005) did not found the species at the same ponds in 1992-
1993. We found six current sites in lakes and streams (Fig.

Figure 14. Distributions of historic (diamonds) and current Gy-
raulus parvus (circles). Sites where historic and current collections
occurred are triangles.

FRESHWATER GASTROPODS OE INDIANA

147

15). Habitats varied with substrates of silt, sand, gravel, or
cobble and vegetation occasionally present. The range is
throughout North America from lames and Hudson Bays
south to Georgia, Alabama, Texas, and northwestern Mexico,
west to southwestern Northwest Territories (Burch and Tot-
tenham 1980). The species is widespread across New York
(lokinen 1992). Goodrich and van der Schalie (1944) re-
ported the species was probably in every part of Indiana. We
consider the species to be imperiled in Indiana but it is
secure in parts ot the range.

Planorbella awipanuhita (Say, 1821). No historical col-
lections were found. We found one current site in Lake
Wawasee. The habitat was sand and riprap substrate and
submergent macrophytes. Shelford (1913) found this species
in ponds at the Indiana Dunes National Lakeshore. lokinen
(2005) did not find the species at the same ponds in 1992-
1993. The range is Vermont west to North Dakota, south to
Ohio and Illinois, northward to Great Slave Lake ( Burch and
Tottenham 1980). The species was common in New York
(Jokinen 1992). Goodrich and van der Schalie ( 1944) report

that the species is most likely limited to the lakes area of
Indiana, and the species is intolerant of domestic sewage. We
consider the species to be imperiled in Indiana but it is
secure in parts of the range.

PhmorbeUa trivolvis (Say, 1817). Two historical collec-
tions were found in Half Moon Pond and Bass Lake. We
found it at 16 sites (Fig. 16) of which five were lakes. Habi-
tats varied with substrates of silt, sand, gravel, cobble, or
boulder and woody debris and/or vegetation occasionally
present. The species was not found at either historical site.
Shelford (1913) found this species in ponds at the Indiana
Dunes National Lakeshore. Jokinen (2005) found the species
at the same ponds in 1992-1993. Brown (1982) found this
species was abundant in ponds at the Crooked Lake Field
Station at Fort Wayne. The range is Atlantic coast and Mis-
sissippi River drainages, northward to Arctic Canada and
Alaska, and southward to Tennessee and Missouri (Burch
and Tottenham 1980). The species is widespread and abun-
dant in New York (lokinen 1992). The species was histori-

Figure 16. Distributions ot historic (diamonds) and current Pla-
norbella trivolvis (circles).

148

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

cally common in lakes and likely present throughout Indiana
(Goodrich and van der Schalie 1944). We categorized the
status of this species as locally and globally secure.

Planorbula armigera (Say, 1821). No historical collec-
tions were found and we did not find them in our collec-
tions. However, Shelford (1913) found this species in ponds
at the Indiana Dunes National Lakeshore. Jokinen (2005)
found the species at the same ponds in 1992-1993. Historical
locations in Indiana were lakes in Lake, La Porte, Steuben,
Marshall, and Kosciusko Counties (Goodrich and van der
Schalie 1944). We categorized its status as critically imper-
iled in Indiana but it is secure in other parts of the range.

Promenetus exactions (Say, 1821). No historical collec-
tions were found. We found the species at two lakes in
Steuben County, Clear Lake and Pleasant Lake. Habitats
were silt substrates with submerged vegetation at Clear Lake,
and silt and sand substrates with emergent vegetation at
Pleasant Lake. Shelford (1913) found this species in two
ponds at the Indiana Dunes National Lakeshore. Jokinen
(2005) found the species at one of the ponds in 1992-1993.
The species was assumed to occur throughout Indiana by
Goodrich and van der Schalie (1944). We categorized its
status as critically imperiled in Indiana but it is secure in
other parts of the range.

Family Ancylidae

Ferrissia fragilis (Tryon, 1863). No historical collections
were found. We found three current stream sites: Clear
Creek (Huntington County), Coal Creek (Fountan County),
and the Tippecanoe River (Kosciusko County). Habitats var-
ied with substrates of silt, sand, gravel, cobble, or boulder
and woody debris occasionally present. The range is New
York to Michigan, California, and Texas (Burch and Tot-
tenham 1980). Stewart (2006) found that the species has not
been observed in Iowa since 1912. However, he mentioned
that the species is tiny and easily overlooked. In New York,
the species is fairly common (Jokinen 1992). The historical
locations that were published in Indiana were Clear Lake
and a pond in La Porte County (Goodrich and van der
Schalie 1944). We categorized its status as imperiled in In-
diana but it is secure in other parts of the range.

Ferrissia par allelus (Haldeman, 1841). No historical col-
lections were found and we did not collect this species. Shel-
ford (1913) found this species in ponds at the Indiana Dunes
National Lakeshore. Jokinen (2005) also found the species in
ponds at the Indiana Dunes National Lakeshore in 1992-
1993. The only other historical location in Indiana was Lake
Maxinkuckee (Goodrich and van der Schalie 1944). The spe-
cies appears to be extinct in Iowa (Stewart 2006) and rare in
Indiana. We categorized its status as critically imperiled in
Indiana but it is secure in other parts of the range.

Ferrissia rividaris (Say, 1817). No historical collections

were found. We found 18 current sites (Fig. 17) of which
four were lakes. Habitats varied with substrates of silt, sand,
gravel, cobble, or boulder and woody debris and/or vegeta-
tion occasionally present. The range is most of North
America: northward into the Hudson Bay lowlands and
northwestward to Saskatchewan, south to North Carolina
and New Mexico, west to California and Oregon (Burch and
Tottenham 1980). The species is fairly common in New York
(Jokinen 1992). The historical distribution in Indiana was
Lake Knox and Henry Counties (Goodrich and van der
Schalie 1944). The species appears to be widely distributed in
Indiana, except that it is easily overlooked. It is common,
widespread, and secure.

Laevapex fiiscus (C. B. Adams, 1841). No historical col-
lections were found. We found one current site, the Eel River
at Logansport. The habitat was silt and riprap substrates and
woody debris was present. Jokinen (2005) tound the species
in one pond at the Indiana Dunes National Lakeshore in
1992-1993. Historical locations in Indiana were lakes in
Marshall and La Porte Counties, and Grassy Creek, Kosci-

FRESHWATER GASTROPODS OF INDIANA

149

Table 1. Summary of aquatic gastropods ot Indiana by current taxa (from Stewart 2006), synonyms from Goodrich and van der Schalie
(1944), historic sites based on museum material, current sites where we collected the species, and observation status using The Nature
Conservance global ranking system (www.natureserve.org). GS refers to species that are presumed extinct, GH are possibly extinct, Gl are
critically imperiled, G2 are imperiled, G3 are vulnerable, G4 are apparently secure, and G5 are secure.

Historic

Current

Conservation

Current taxa

Synonyms

sites

sites

status

Valvatidae

Valvata hicarinata (Lea, 1841)

0

0

G5

Valvata lewisi (Currier 1868)

Valvata Icwisii

0

1

G5

Valvata trkarinata (Say, 1817)

0

o-*

G5

Valvata sincera (Say, 1824)

0

0

G5

Viviparidae

Viviparus georgianus (Lea, 1834)

Valvata contectoides

0

2

G5

Vivipanis subpurpureus (Say, 1829)

4

0

G5

Bellamya chinensis (Reeve, 1863)

0

4

Exotic

Bellamya japonica (von Martens, 1861)

0

4

Exotic

Campelonia decisum (Say, 1817)

Catnpeloma spp.

20

11

G5

Hydrobiidae

Birgella subglobosus (Say, 1825)

Somatogyrus subglobosus

0

6

G4

Cindnnatia Integra (Say, 1821)

1

1

G5

Pyrogulopsis lustrica (Pilsbry, 1890)

Amnicola lustrica

3

2

G5

Amnkola limosus (Say, 1817)

Ammcola limosa, Amnicola parva

0

6“

G5

Pomatiopsidae

Pomatiopsis cincinnatiensis (I. Lea, 1850)

0

6

G4

Pleuroceridae

Elimia Uvescens (Menke, 1830)

Goniobasis Uvescens

24

48

G5

Pleurocera acuta (Rafinesque, 1831)

12

21

G5

Pleurocera canalkulata (Say, 1821)

14

2

G5

Leptoxis praerosa (Say, 1821)

Anadosa praerosa

0

1

G5

Lithasia obovata (Say, 1820)

1

D

G4

Lymnaeidae

Fossana spp. (Say, 1822)

Lymnaea humilis, Lymnaea dalli,

7

43"

G5

Lymnaea stagnalis (Linnaeus, 1758)

Lymnaea parva

0

1

G5

Stagnkola catascopium (Say, 1867)

Lymnaea catascopium

0

1

G5

Stagnkola caperata (Say, 1829)

Lymnaea caperata

1

0

G5

Stagnkola elodes (Say, 1821)

Lymnaea palustris, Lymnaea reflexa

0

17"

G5

Stagnkola exilis (I. Lea, 1838)

Lymnaea exilis

0

1

G5

Pseudosiicdnea columella (Say, 1821)

Lymnaea columella

0

16

G5

Physidae

Physella gyrina (Say, 1821)

Physella heterostropha, Physella sayii,

22

8"

G5

Physella acuta (Draparnaud, 1805)

Physella andllaria

Physella heterostropha, Physella Integra,

17

94

G5

Aplexa elongata (Say, 1821)

Physella walkeri
Aplexa hypnonim

2

0"

G5

Planorbidae

Gyraulus circumstriatus (Tryon, 1866)

1

0

G5

Gyraulus deflectus (Say, 1824)

Gyraulus hirsutus

0

10

G5

Gyraulus parvus (Say, 1817)

9

9-’

G5

Helisoma anceps (Menke, 1830)

Flelisoma antrosum

0

6

G5

Planorbella campanulata (Say, 1821)

Flelisoma campanulatum

0

1

G5

Planorbclla trivolvis (Say, 1817)

Flelisoma trivolvis

2

16"

G5

Planorbula armigera (Say, 1821)

0

0"

G5

Promcnetus exacuous (Say, 1821)

Menetus exacuous

0

2"

G5

Ancylidae

Ferrissia fragilis (Tryon, 1863)

Gundlachia meekiana

0

3

G5

Ferrissia parallelus (Haldeman, 1841)

Ferrissia parallela

0

0"

G5

Ferrissia rivularis (Say, 1817)

Ferrissia tarda

0

18

G5

Laevapex fuscus (C. B. Adams, 1841)

Ferrissia fusca

0

1"

G5

“ Refers to taxa that were collected in

Indiana by lokinen (2005). '’Refers to taxa that were

collected on

the Ohio River main

stem by Greenwood and

Thorp (2001).

150

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

usko County (Goodrich and van der Schalie 1944). We cat-
egorized its status as critically imperiled in Indiana but it is
secure in other parts of the range.

DISCUSSION

Our historical sui-veys of museum records and literature
search indicated 39 species present historically in Indiana.
Our 2006-2008 survey and literature search resulted in 36
species of ac]uatic gastropods, including two exotics. Three
species are apparently locally extinct in the state (Valvata
bicarinata, Valvata sincera, and Gyraiilus drciimstriatiis) but
globally secure. Our species richness estimates are similar to
species estimates for other states (Stewart 2006). For ex-
ample, states with published aquatic gastropod species rich-
ness values are: Connecticut (35 species; lokinen 1983),
Maine (45 species; Martin 1999), New York (61 species;
lokinen 1992), Virginia (53 species; Stewart and Dillon
2004), Kentucky (29 species; Branson et al. 1987), and Iowa
(49 species; Stewart 2006). The conservation status of Indi-
ana’s gastropods is: three taxa that are apparently secure
globally and 36 taxa that are widespread, abundant, and
globally secure, including two exotics (Table 1). However,
three taxa are locally extinct and many others appear locally
imperiled or vulnerable.

Of three Indiana species presumed to be locally extinct,
only Valvata bicariiiata was collected in Indiana by Good-
rich and van der Schalie ( 1944) and it appears to be declin-
ing elsewhere (Stewart 2006). Whether Valvata sincera was
ever collected in Indiana is an open question. Gyrauhis
circumstriatiis is likely locally extinct due to water quality
degradation, as it is intolerant to low pH and calcium (lo-
kinen 1992).

Only 31 of the sites where the museum material was
collected had the same species present in our collections at
those sites. Our interpretation is that these species are likely
no longer present at the majority of historic sites. Our col-
lection technique may have missed individuals, but the
probability of missing species that were historically abun-
dant seems unlikely. Explanations for local species extinc-
tions include extensive habitat degradation throughout the
state from agricultural impacts, hydrologic alteration by res-
ervoirs, and pollution. The majority of Indiana watersheds
have hydrologic alterations due to reservoir release and/or
channelization for agriculture drainage (Pyron and Neu-
mann 2008). Watersheds that are upstream from reservoirs
are also negatively impacted by the reservoir (Pringle 1997).

Conservation of aquatic gastropods should be consid-
ered as important as conservation of other aquatic organ-
isms, if only for preservation of phylogenetic diversity. In
Alabama, 65% of gill-breathing endemic freshwater snails

are extinct, endangered, threatened, or of special concern
(Lydeard and Mayden 1995). Although the southeastern
U.S. has the highest diversity of freshwater organisms on the
continent, all freshwater fauna of North America are facing
similar losses of biodiversity (Ricciardi and Rasmussen
1999). Ricciardi and Rasmussen (1999) projected future ex-
tinction rates for freshwater organisms in North America at
4% per decade. This is a similar depletion rate as for tropical
forests. Additional protection besides the current approach
to conservation of aquatic invertebrates is obviously
necessary.

A first step toward conservation of aquatic gastropods is
an accurate inventory (Lydeard and Mayden 1995). Efforts
toward inventory of aquatic invertebrates in the U.S. have
lagged behind inventories of vertebrates although some
statewide surveys of aquatic gastropods are appearing (Joki-
nen 1992, Stewart 2006). Accurate inventory of aquatic gas-
tropods will also encourage studies of the taxonomic, eco-
logical, and general biology of the group (Neves et al. 1997).
For example, studies of macro-ecological patterns of fresh-
water gastropods are rare compared with macro-ecological
studies of vertebrates. These endeavors have lagged behind
other taxa largely because of a lack of descriptive and dis-
tributional natural history studies.

Indiana currently lists the conservation status for verte-
brate and invertebrate taxa (www.in.gov/dnr/). However,
there is currently no specific conservation recognition or
protection plan for aquatic gastropods in the state. We rec-
ommend such a thorough inventory, recognition, and pro-
tection plan for the aquatic gastropods in Indiana. A better
understanding of freshwater gastropod ecology demands
conservation and further study to protect this valuable natu-
ral resource.

ACKNOWLEDGMENTS

We are grateful to the Indiana Academy of Science and
Ball State University Office of Research and Sponsored Pro-
grams for financial support. Tim Stewart, Rob Dillon, Jack
Burch, and Randy Bernot assisted with taxonomic difficul-
ties. We are extremely grateful to Rob Dillon for identifica-
tion of specimens and editorial suggestions.

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of Conservation, Division of Geology, Publication No. 21.

1120 pp.

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four species of snails. Ecological Monographs 11; 234-249.

Submitted: 19 December 2007; accepted: 25 July 2008;

final revisions received: 30 September 2008

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Amer. Maine. Bull. 26: 153-159 (2008)

The feeding behavior and diet of an endemic West Virginia land snail,
Triodopsis platysayoides

Daniel C. Dourson'

Belize Foundation for Research and Environmental Education, P.O. Box 129, Punta Gorda, Belize Central America,
bfreemgr@xplornet.com

Abstract: The feeding behavior and diet of the federally threatened land snail Triodopsis platysayoides (Brooks, 1933) are reported. The
species is atypical among eastern North American land snails in that it remains active and feeding during hot, dry summer months while
other land snail species occurring in the region may become motionless or are compelled to estivate. Triodopsis platysayoides has also
coevolved with a rare mammal, the Alleghany wood-rat, Neotoma magister. Clearly, where the wood-rat and T. platysayoides coexist,
wood-rats furnish a nearly constant food supply to the snail, including wood-rat excrement and a host of wood-rat harvested provisions
carried into the snail’s location. Triodopsis platysayoides includes as part of its diet fungi, lichens, flower blossoms of the tulip tree
Liriodendron tulipifera, deceased gray cave crickets Euhadenoecus fragilis, gray cave cricket excrement, yellow birch Betida allegheniensis, and
sweet birch Betula lenta leaves. Senescent leaves of the birch may form a significant pool of foliar calcium available to the snail in an
otherwise acidic environment. Triodopsis platysayoides was witnessed feeding on the vacant shells of Xolotrema denotatum (Ferussac, 1821 ),
Mesomphix cupreus (Rafmesqtie, 1831), and its own kind, presumably for the calcium carbonate content, a critical mineral in regulation of
bodily functions and shell building. Peak activity for the species occurred after nightfall whereas peak feeding occurred when temperatures
were between 18 and 23 °C and relative humidity was between 70% and 85%.

Key words: Cheat threetooth. Cheat River Gorge

The globally rare Cheat threetooth Triodopsis platysay-
oides (Brooks, 1933) is a reclusive snail endemic to the Cheat
River Gorge of northern West Virginia, U.S.A. First collected
by Graham Netting at Coopers Rock and later described by
Stanley Brooks in 1933, the species is associated with inter-
stices of cool boulder talus (Fig. 1), sandstone cliftline fea-
tures, and to a lesser degree limestone caves within a 21 -km
stretch of the gorge (Stihler 1994, ITotopp 2006, Dourson
2007). Listed as federally threatened in 1978 by the U.S. Fish
and Wildlife Service, the snail is also ranked as a G1 species
by NatureServe. A ranking of G1 means that the species is
considered Critically Imperiled — at very high risk of extinc-
tion due to extreme rarity (NatureServe 2008).

The food preferences of Triodopsis platysayoides are
poorly known, owing in part to its enigmatic lifestyle, be-
havior, and feeding preference in the midst of deep rock and
boulder talus. Moreover, the preponderance of work to date
has largely focused on locating new populations (Hotopp
2000) during daytime hours, a time when the snail is gen-
erally less active. Solem (1974) reported that the feeding
niche of T. platysayoides is apparently among seasonal leaf
litter alongside the rocks but made no mention of specific
diet. Hotopp and Grimm (1999) cited lichens as a primary
food source in the wild. Hotopp (2003) reported that rotting

' Present address: P.O. Box 424, Bakersville, North Carolina 28705,
U.S.A.

leaf litter appeared to be a food supply in the wild and that
lichens may also be consumed, but no unambiguous food
taxon was identified. Most reports regarding the diets of land
snails are rather vague, largely describing snail diets only in
generalities.

In this paper, 1 report observations on feeding by Trio-
dopsis platysayoides that I made during field surveys over 2
years. These observations considerably increase the known
foods for this species and, in many cases, provide more
specific identification of food organisms than previous
reports.

STUDY AREA

The Cheat River Canyon is a steep, 26-km winding
stretch along the Cheat River, from Albright in Preston
County, northwest to the upper reaches of Cheat Lake in
Monongalia County. Elevations of the canyon range from
approx. 366 m at the river to approx. 640 m at the upper-
most rim of the gorge. The walls of the canyon expose a
series of Pennsylvanian and Mississippian sedimentary rock
strata deposited 300-350 million years ago. The topmost and
thickest layer is composed of Pottsville Sandstone, which
outcrops along the rim where tributaries enter the canyon,
on periglacial screes and in a variety of other locations. More
than halfway down on the slopes of the canyon is the gray or
whitish Greenbrier Limestone, in which caves occur.

153

154

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Figure 1. Sandstone boulder and rock talus, considered critical
habitat for Triodopsis platysayoides.

Oaks, Quercus, are ubiquitous, sometimes underlain by
dense stands of mountain laurel Knimia latifolia. Oaks domi-
nate upper slopes, while great laurel Rhododendron maxi-
ninni, often found beneath the canopy trees on the shady
slopes, typifies wet acidic ravines and the lower slope.

Tulip poplar often dominates the rich cove and lower
slope habitats. Along the river, sycamore Platanns occiden-
talis becomes a canopy dominant. The herbaceous vegeta-
tion of the canyon is extremely diverse, varied, and depends
largely upon the site.

MATERIALS AND METHODS

Visits were conducted at 81 sites where extant popula-
tions or the shells of Triodopsis platysayoides were docu-
mented, providing a range of conditions and possible food
sources from which to study feeding behavior. These sites
throughout the Cheat River Gorge were studied from May to
September of 2006 and 2007, at various times of day and
night in a variety of weather conditions. Eighty-nine percent
of the sites were visited during daylight hours while eleven
percent of the sites sampled were during nighttime hours,
which equates to 130 daytime and 17 nighttime visits.

Snails were recorded as active when observed in some
form of movement and inactive when the majority of their
bodies were retracted inside the shell and the upper and
lower tentacles were drawn inward. Triodopsis platysayoides
was determined to be feeding when its shell and body pos-
ture remained somewhat stationary; the lower tentacles were
positioned downward and the upper tentacles were short-
ened and curved backward. On occasion, however, the ten-
tacles would remain mostly extended and in an upright po-

sition during feeding behaviors. As T. platysayoides fed,
irregular head movements were observed and monitored to
authenticate actual feeding and not just chemoreception.
After teeding was completed, the food was carefully exam-
ined tor radular abrasions, indicating that the snail had in-
deed removed and consumed portions of the food.

The foods were reported as senescent, fresh, or in the
case of wood-rat guano, both. Food categorized as senescent
was noted to be at any visible stage of decomposition. Food
retaining living tissue or that did not exhibit signs of decay
was classified as fresh. Food sources recognized were iden-
tified to genus and, if possible, to species.

Substrate on which the snail fed, time, and weather
conditions during the feeding episode were recorded. Tem-
peratures outside rock structure and relative humidity were
recorded using a hygrometer. Snails were categorized as ei-
ther juveniles or adults, by the presence of a reflected lip.
Photographs were taken of all feeding behavior and food
sources.

Data analysis

Log-ratio tests (Christensen 1997) were used to ana-
lyze differences in the proportion of observations between
day and night surveys. Due to the disproportionate number
of daytime visits to nighttime visits, no statistical conclu-
sion was made regarding the snail’s diurnal preference for
feeding.

RESULTS

Of the 488 live snails documented in 2006 and 2007, 360
were engaged in some form of activity (mostly in move-
ment). The log-ratio tests clearly demonstrated that there
was an increased activity level seen in Triodopsis platysay-
oides after nightfall when temperatures were 18-24 °C and
relative humidity (RH) was above 70%. Twenty-eight per-
cent more snails were active at night than during the day
(X^ = 20.81, df = 1, P £ 0.0001) (Fig. 2). Results from this
study also indicate that as temperatures increased and hu-
midity (RH) decreased, Triodopsis platysayoides moved
deeper into the screes and clifflines. The following accounts
are for daytime only. In August (historically the hottest
month in West Virginia) of 2006 and 2007, only 7 snails
were found within a meter of the surface of the rock struc-
ture while 43 snails found in August were found from 2-18
meters deep. Inly also showed this trend, though to a lesser
degree. During the survey months of May, feme, and Sep-
tember, the greatest percentage of live snails found (198)
occurred within a meter of the surface while (81) of the live
snails found were 2-18 meters deep. The month of June
found the most live snails (118) within a meter of the surface
whereas (25) snails found in June were 2-18 meters deep.

DIET OF TRIODOPSIS PLATYSAYOIDES

155

Figure 2. Overall activity levels of Triociopsis platysayoides observed
in day {N = 278) and night {N = 74) surveys conducted from May
to September 2006 in Monongalia County, West Virginia.

A total of fifty-nine feeding observations were recorded.
Twenty-eight of the feeding episodes were documented dur-
ing nighttime hours from 8:30 pm to 3:30 am, while thirty-
one feeding episodes were documented in the daylight hours
from 8:00 am to 8:30 pm. The majority of nighttime feeding
events (21 out of 28) occurred in temperatures that ranged
between 18 and 23 °C (recorded outside the rock structure)
and relative humidity (RH) was between 75% and 85% (re-
corded outside the rock structure). During the daytime 18
out of 31 feeding events occurred between temperatures that
ranged between 18 and 23 °C (recorded outside the rock
structure), and relative humidity (RH) was between 70%
and 85% (recorded outside the rock structure). The highest
number of feeding events (35 of 53 or 66%) for both night-
time and daytime hours occurred between 1 8 and 23 °C with
(RH) between 70% and 85%.

The diet of Triociopsis platysayoides was a great deal
more diverse than reported by Solem (1974) and Hotopp
and Grimm ( 1999). The results of my study documented 27
plant and animal food sources (Table 1) for T. platysayoides.
The most common meals included senescent birch leaves,
gilled mushrooms, and wood-rat scat. Less frequently con-
sumed foods were fresh dead cave crickets, flower petals of
great laurel and tulip trees, crustose lichens, and the shells of
several species of terrestrial gastropods. Although two spe-
cies of birch leaves, sweet birch and yellow birch, were the
most frequently consumed, red maple and red oak leaves

were also documented as food for T. platysayoides. The snail
diet included senescent portions of at least two fern species,
wood fern Dryopteris intermedia and hay-scented fern
Dennstaedtia punctiloba. Triodopsis platysayoides also fed on
newly emergent fruiting bodies of mushrooms. Wood-rat
excrement was eaten fresh (Fig. 3) but also in various stages
of decomposition, and the snail also consumed a white mold
that grows from wood- rat scats.

Triodopsis platysayoides habitually fed on decaying veg-
etation. At one location, the species consumed abcissed
flower petals of great laurel (Fig. 4) that were in decompo-
sition. The snail appeared to he interested only in the rankest
ones. When offered newly fallen flowers, the snail declined
to feed on them and instead turned away and headed for the
most putrid. At other feeding stations, only the dead or
dying portions of fern fronds were eaten. While only three
species of Bryophytes (mosses) were documented as food
during the study, other species of bryophytes are likely con-
sumed as well, given their frequency in the snails’ habitat.
Snails fed only on the leafy portions of the moss, intention-
ally avoiding the more fibrous stems altogether.

Triodopsis platysayoides fed on a variety of mushrooms
in the genera Amanita, Tricholonia, Russula, Hygrophorus,
and Marasinius, including several species that had been har-
vested and transported by wood-rats into the boulder talus
habitat. The snail was especially fond of mushroom gills,
often crawling upside down to feed on these portions of the
fungi. One adult T. platysayoides was observed feeding on
Gyroporus castaneus, a mushroom found Iruiting three me-
ters deep in the dark zone of boulder talus. Numerous fungi
species that had feeding evidence from snails were observed
in dark zones of other rock shelters and talus. Although most
were likely fed on by T. platysayoides, it is possible that the
mushrooms were eaten by other common land snail species
such as Neolielix dentifera (A. Binney, 1837) or Triodopsis
tridentata (Say, 1816), sporadically found in talus habitat
with T. platysayoides.

During the study, the vacant shells of Mesoniphix cu-
preus (Rafinesque, 1831) and Xolotrema denotatum (Ferus-
sac, 1821) were offered to Triodopsis platysayoides to deter-
mine if the species would utilize the shells as a calcium
source. Shells were placed 12 cm in front of two actively
crawling T. platysayoides. Both snails found the shells within
several minutes and promptly fed on the surfaces for more
than forty minutes each. After the snails departed, shells on
which they fed were examined and showed signs of radula
abrasions. On another occasion, an adult T. platysayoides
was observed feeding (unsolicited) on the vacant shell of its
own kind for a period of 30 minutes before it moved on.

The length of feeding episodes appeared to correlate to
the solidity of the food source, ranging from 5 minutes to
nearly an hour. While grazing on the vacant shell of Xolo-

156

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Acer ruhrwn

Red maple leaves

Senescent

Amanita flavoconia

a mushroom

Fresh

Betula allegheniensis

Yellow birth leaves

Senescent

Betida lenta

Sweet birch leaves

Senescent

Betula lenta

Sweet birch catkins

Fresh

Dennstaedtia pimctiloba

Hay-scented fern

Senescent

Dryopteris intermedia

Wood fern

Senescent

Euhadenacous fragilis

Gray cave cricket

Fresh

Enhadenacous fragilis

Grave cave cricket guano

Fresh

Gyroporus castaneiis

a mushroom

Fresh

Hygrophoriis sp. (?)

a mushroom

Fresh

Liriodendron tulipifera

Tulip tree flowers

Senescent

Loeskeobryiim brevirostre

a moss

Fresh

Marasmius sp. (?)

a mushroom

Fresh

Mesomphix cuprens

Copper button shell

Senescent

Neotoma magister

Wood rat scat

Both

Plagioinninni cia ra re

a moss

Fresh

Quercus rnbrum

Red oak stems

Senescent

Qnercus rnbrnm

Red oak leaves

Senescent

Rhododendron maximum

Rhododendron flowers

Senescent

Russula foetens

a mushroom

Fresh

Sassafras albidum

Sassafras leaf

Fresh

Thuiditifii delicatnlnm

a moss

Fresh

T riodopsis platysayoides

Cheat threetooth shell

Senescent

Not identified to genus

Crutose lichens

Fresh

Not available to genus

Mold on wood rat scat

Fresh

Xolotrema denotatum

Velvet wedge shell

Senescent

trema denotatum, Triodopsis platysayoides took 48 minutes.
One juvenile T. platysayoides spent 30 minutes feeding on a
freshly dead cave cricket. In contrast, T. platysayoides spent
an average ot 15 minutes or less feeding on the tissues of
mushroom caps and gills and only 5 minutes on the mold
growing from wood-rat scat.

The results of this work found at least a dozen examples
of adult and juvenile Triodopsis platysayoides feeding on the
same foods. For instance, deep within a boulder talus, a fully
adult (22 mm) (Fig. 5) and a juvenile (12 mm) were ob-
served feeding together on a recently deceased grey cave
cricket. luvenile and adult T. platysayoides were also ob-
served feeding on wood-rat scats, birch leaves, and newly
abscised birch catkins. No juveniles of T. platysayoides under
8 mm were documented feeding during this study.

DISCUSSION

One of the most important environmental factors to
land snails in general is moisture (a frequent cause of death

is desiccation). Other factors such as
temperature range, escape from preda-
tors and disease, and a food source are
also important. While most other spe-
cies of snails fulfill their needs without
such a close association with rock, the
needs of Triodopsis platysayoides are sat-
isfied by certain rock structures in ways
that we do not fully understand (Pearce
et al. 2007). For example, it is not en-
tirely clear where or even if, the species
hibernates during winter months. The
snail has been recorded 18 meters deep
in cave-like caverns that form in sand-
stone clifflines and boulder talus (Dour-
son 2007). At this depth, winter tem-
peratures have little effect on the
relatively stable microclimate, which
generally remains around 14 °C with
relative humidity around 70%. During
the study, eight snails were observed in
May actively feeding in these tempera-
ture and ( RH ) ranges. It is entirely pos-
sible that T. platysayoides remains active
and feeding during winter months, par-
ticularly if there are cohabiting wood-
rats (which do not hibernate) in the
same rock features.

Results from the log-ratio x“ tests
established that there were increased ac-
tivity levels seen in Triodopsis platysay-
oides after nightfall, suggesting that perhaps peak overall
feeding activity occurs at night. However, the 59 feeding
events in my study suggest that peak feeding for T. platy-
sayoides may be more a function of temperature and relative
humidity, not time of day. If so, this is likely a consequence
of the snails’ specialized habitat that maintains a relatively
stable microclimate, allowing the snail to feed during the
daytime even if weather conditions outside the rock struc-
ture were less than favorable. Both of these hypotheses await
further testing.

As a food source, rock and boulder talus habitats of the
Cheat River Gorge are comparatively rich, harboring a mul-
tiplicity of bryophyte, lichens, trees, a variety of herbaceous
vegetation, and a few animal species that are known foods of
Triodopsis platysayoides. Talus also accumulates large quan-
tities of forest detritus, an ideal medium for growing an
assortment of fungi and their fruiting bodies. Moreover, a
number of known foods for the species, such as birch leaves
as well as tulip tree and rhododendron blossoms, fall easily
into the interstitial spaces of the open boulder talus. A snail
living in such sites need not travel far for its meal.

Table 1. Confirmed foods of Triodopsis platysayoides.

Scientific name

Common name

# feeding

Stage of material events

1

8

14

1

1

2

2

1

1

1

1

1

1

1

8

1

1

1

1

1

1

1

1

1

2

1

DIET OF TRIODOPSIS PLATYSAYOIDES

157

Figure 3. Sub-adult Triodopsis platysayoides feeding on fresh Alle-
gheny wood-rat scat. Shell 18 mm in diameter.

Figure 5. Adult Triodopsis platysayoides feeding on recently de-
ceased gray cave cricket. Shell 22 mm in diameter.

Triodopsis platysayoides is a dietary generalist, feeding
on a variety of foods largely originating or contained within
the screes. Senescent birch leaves were the most habitually
consumed by the species (with 22 feeding events). At least
ten other senescent materials were included in T. platysay-
oides diet. A number of studies have reported a prevalence of
senescent foods in the natural diet of land snails (Richardson
1975, Hatziioannou et al. 1994, Iglesias and Castillejo 1999).
Many plants contain toxins or refractory compounds such as
tannins, so some gastropods eat senescent plant material that
has lost many of those secondary compounds (Burch and
Pearce 1990). Aging vegetation also concentrates macronu-

Figure 4. Adult Triodopsis platysayoides feeding on aged blossoms
of rhododendron. Shell 23 mm in diameter.

trients in tissues, an example being foliar calcium in birch
leaves (Potter et al. 1987).

Triodopsis platysayoides' most fascinating food relation-
ship is the one it has with the Alleghany wood-rat. In loca-
tions where the two species coexist, this affiliation, coupled
with the protective sanctuary and microclimate of the talus,
has allowed T. platysayoides to remain entirely active and
feeding inside the talus and cliffs during the hot dry months
of summer, particularly in August. As summer temperatures
climb, T. platysayoides simply moves deeper into talus or
cliffline habitats where it remains active and feeding. For
other land gastropods during this time, surviving desiccation
is the priority. This is because most land snails are not
adapted to live in the deep recesses of screes and cliffs. As
summer temperatures climb and conditions dry out, surface
dwelling snails must retain their vital body moisture by be-
coming inactive, followed by aestivation.

Clearly, wood-rats benefit Triodopsis platysayoides in a
number of ways, most importantly as a food supplier.
Wood-rats carry into the talus and rock shelters a plethora of
known and potential T. platysayoides food, including a large
assortment of fungi, freshly cut Dryopteris species (wood
ferns), and scattered, easily accessible deposits of their own
excrements. The snail appeared to he especially fond of a
white mold growing from aged wood-rat scat (Fig. 6).
Wood-rats maintain latrines that are usually sheltered
among these rock features providing the snail a near-
constant food supply in the relative protection of the screes
and cliffs. From a management standpoint, protecting
wood-rats and their habitat in the Cheat River Gorge will no
doubt benefit the snail.

Hotopp and Grimm (1999) reported that lichen was a

158

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Figure 6. Sub-adult Triodopsis platysayoides feeding on mold that
grows on wood-rat scat. Shell 18mm in diameter.

primary food source for Triodopsis platysayoides in the wild,
yet in my study I observed the snail feeding only once on a
crustose variety. Lichens are, in fact, eaten by many species
of terrestrial snails (Peake and lames 1967). The crustose
lichen that T. platysayoides fed upon was frequent on the
surface of outsized sandstone boulders in the Cheat River
Gorge and the number of lichen species occurring in the
gorge appeared particularly diverse. Because lichen was cited
as a primary food source by Hotopp and Grimm in (1999),
further investigation into the use of lichens as food by T.
platysayoides is of special interest.

Diets of juvenile Triodopsis platysayoides may differ
from those of adults (Caldwell et al. 2006). The literature
about the different diets in adults and juvenile land snails is
largely scarce and ambiguous (Iglesias and Castillejo 1999).
luveniles of Cepaea nemoralis (Linnaeus, 1758) are reported
to eat some species of plants that the adult animals avoid and
vice versa (Wolda et al 1971). Other studies have shown no
difference in the diets of juveniles and adults of the same
species (Wolda et al. 1971, Hatziioannou et al 1994, Wil-
liamson and Cameron 1996). I found no evidence of such
life-stage dietary differences in T. platysayoides.

In addition to a food supply, shell-bearing snails require
a calcium source for reproduction, regulation ol bodily
functions, but most importantly shell building. Sandstone
boulder and cliffline habitat and their associated soils are
reported to be largely calcium deficient environments (Ka-
lisz and Powell 2003). The quest for calcium carbonate
sources in these acidic environments are likely more varied
than we know, but several suppliers are speculated on here.
These include but are not limited to, the vacant shells of
gastropods and abscissed leaves of yellow and sweet birch.

Discus macclintocki (P. C. Baker, 1928), known only from
cold rock talus slopes, includes yellow birch leaves in its diet,
which may supply a calcium carbonate source for that spe-
cies. In non-calcareous areas, land snails often appear allied
to a calcium supply (Mason 1974). Gosz et al. (1973) re-
ported that dead birch leaves could form a significant pool of
calcium on the forest floor, since the concentrations of cal-
cium remained high in dead leaves 12 months after abscis-
sion. It is not known if the leaves eaten by T. platysayoides
were consumed for the leaf tissues themselves or perhaps for
something growing on the surface of the leaves such as a
sooty mold or algae. Leaves were always eaten in their en-
tirety, however, and not just surface-grazed by the snails,
suggesting the motive was more for the leaf content.

Besides the 27 documented foods for Triodopsis platy-
sayoides, several other foods are suggested here and deserve
some mention. Numerous unidentified mushrooms that
showed signs of snail feeding, e.g., radula marks and slime
trails, were routinely found in T. platysayoides habitat and
were probably fed on by that species. Fall feeding observa-
tions of T. platysayoides would likely document additional
fungi species, given that fruiting bodies of mushrooms are
more frequent during this time of year (Hartman 2003).
Rock tripe, a common species of foliose lichen growing on
rock surfaces, often exhibited snail-feeding signs close to
sites that harbored live T. platysayoides and although these
lichens were twice offered to the species in the field, the
snails declined to feed.

Additional research on the feeding behavior anci diet of
Triodopsis platysayoides will no doubt yield some interesting
discoveries. Ideally, studies should be conducted after night-
fall, when temperatures are 18-23 °C and relative humidity is
70-85% during late May and early lune when the snail is
most active and potential food sources are plentiful.

ACKNOWLEDGMENTS

I would like to thank the U.S. Fish and Wildlife Service
and West Virginia Division of Natural Resources, Wildlife
Diversity Program for funding the development of a survey
protocol for Triodopsis platysayoides, which allowed me to
observe and document the feeding habits and diet of the
snail. Thanks also to Graig Stihler of West Virginia Division
of Natural Resources for his extensive knowledge of the spe-
cies as well as logistical assistance for this study; Bill Roody
of West Virginia Division of Natural Resources for identifi-
cation of mushrooms consumed by T. platysayoides; Allen
Risk, PhD, of Morehead State University for identification of
mosses; Ken Hotopp, who provided the description of the
study area and who has written several excellent papers on
the species. Thanks also to Timothy Pearce, PhD of Carnegie

DIET OF TRIODOPSIS PLATYSAYOIDES

159

Museum of Natural History for his thorough and construc-
tive review of this manuscript. Special thanks to Robert
Klinger, PhD of USGS for his statistical assistance and Mark
Gumbert of Copperhead Environmental Consulting for con-
ducting literature searches.

LITERATURE CITED

Burch, I. B. and T. A. Pearce. 1990. Terrestrial Gastropoda. In D. L.
Dindal, ed., Soil Biology Guide. John Wiley & Sons, Inc., New
York. Pp. 201-299.

Caldwell, R. S., I. E. Copeland, and G. L. Mears. 2006. Findings for
habitat analysis of Triodopsis platysayoides (Brooks 1933),
Cheat Threetooth. Report submitted to Cheat Lake Environ-
ment and Recreation Association, Morgantown, West Vir-
ginia.

Christensen, R. 1997. Log-Linear Models and Logistic Regression, 2"‘^
Edition. Springer-Verlag. New York.

Dourson, D. D. 2007. Survey Protocol for Cheat threetooth, Trio-
dopsis platysayoides (Brooks) (Revised). Report to United
States Eish and Wildlife Service. Elkins, West Virginia.

Gosz, James R., G. E. Likens, and F. H. Bormann. 1973. Nutrient
release from decomposing leaf and branch litter in the Hub-
bard Brook Forest, New Hampshire. Ecological Monographs
43: 173-191.

Hartman, J. 2003. Wild mushrooms: To eat or not to eat? Univer-
sity of Kentucky Cooperative Extension Newsletter No. 1002, La
Grange, Kentucky.

Hatziioannou, H., N. Eleutheniadis, and M. Lazaridou-
Dimitriadou. 1994. Food preferences and dietary overlap by
terrestrial snails in Logos area (Edessa, Macedonia, Northern
Greece). Journal of Molluscan Studies 62: 495-505.

Hotopp, K. P. 2000. Cheat threetooth (Triodopsis platysayoides
Brooks) Inventory 2000. USFWS in the West Virginia Division
of Natural Resources, Elkins, West Virginia.

Hotopp, K. P. 2003. Cheat threetooth habitat mapping at Snake Hill.
Report to Wildlife Diversity Program. West Virginia Division
of Natural Resources, Elkins, West Virginia.

Hotopp, K. P. 2006. Patera panselenus (Hubricht) on the lower
Cheat River West Virginia (Gastropoda: Pulmonata: Polygy-
ridae). Banisteria 27: 39-42.

Hotopp, K. P. and Grimm, F. W. 1999. Land snails from the mouth
of Cornwell Cave, Preston County, West Virginia. Report to
West Virginia Nature Conservancy, Elkins, West Virginia.

Iglesias, 1. and I. Castillejo. 1999. Field observations on feeding of
the land snail Helix aspersa Muller. Journal of Molluscan Stud-
ies 65: 411-423.

Kalisz, P. J. and I. E. Powell. 2003. Effect of calcareous road dust on
land snails (Gastropoda: Pulmonata) and millipedes (Dip-
lopoda) in acid forest soils of the Daniel Boone National Forest
of Kentucky, USA. Report to US Forest Service Supervisors
Office. Winchester, Kentucky.

Mason, C. F. 1974. Mollusca. In: C. H. Dickinson and F. ]. F. Pugh,

eds.. Biology of Plant Litter Decomposition. Academic Press,
London. Pp. 555-591.

NatureServe. 2008. NatureServe Conservation Status. Available at:
http://www.natureserve.org/explorei7ranking.htm 1 February
2008.

Pearce, T. A., R. S. Caldwell, D. Dourson, and G. T. Watters. 2007.
Criteria for recognizing potential habitat o/ Triodopsis platy-
sayoides (Brooks), the Cheat Threetooth Snail. Report submit-
ted to: Allegheny Wood Products, Inc., Petersburg, West Vir-
ginia.

Peake, I. F. and P. W. lames. 1967. Lichens and Mollusca. The
Lichenologist 3: 425-427.

Potter, C. H„ H. L. Ragsdale, and C. W. Berish. 1987. Resorption
of foliar nutrients in a regenerating southern Appalachian for-
est. Oecologia 73: 268-271.

Richardson, A. M. M. 1975. Food, feeding rates and assimilation in
the land snail, Cepaca nemoralis (Linnaeus). Oecologia 19: 59-
70.

Solem, A. 1974. Information form on file. Office of Endangered
Species, Washington, D.C.

Stihler, C. W. 1994. New records for the land snail I'riodopsis platy-
sayoides, a West Virginia endemic. West Virginia Academy of
Science Proceedings 66: 2-6.

Williamson, P. and R. A. D. Cameron. 1996. Natural diet of the
land snail, Cepaea nemoralis Linnaeus. Oikos 27: 493-500.

Wolda, H., A. Zweep, and K. A. Schuitema. 1971. The role of food
in the dynamics of populations of the landsnail Cepaea
nemoralis Linnaeus. Oecologia 7: 361-381.

Submitted: 13 February 2008; accepted: 29 luly 2008;

final revisions received: 14 October 2008

Amer. Maine. Bull. 26: 161-169 (2008)

Structural community changes in freshwater mussel populations of Little Mahoning
Creek, Pennsylvania

Eric J. Chapman* and Tamara A. Smith^

'Western Pennsylvania Conservancy, Freshwater Conservation Program, Blairsville, Pennsylvania 15717, U.S. A.,
echapman@paconserve.org

^Western Pennsylvania Conservancy, Northwest Field Station, Union City, Pennsylvania 16438, U.S. A., tsmith@paconserve.org

Abstract: As part of a complete biotic survey for Little Mahoning Creek watershed. Western Pennsylvania Conservancy (WPC) used timed
searches to survey for freshwater mussels along the entire mainstem of Little Mahoning Creek (56.8 km). The survey captured all historic
sites sampled as early as 1905. The 15 sites revealed a high diversity of unionids ( 10 species) and several rare and endangered taxa, including
Plewvbemn sintoxia (Rafinesque, 1820), Villosa iris (1. Lea, 1829), and Alasmidonta margiuata (Say, 1818). Present survey results indicate
an established unionid community with several taxa increasing their distributions compared to historical surveys. Upstream dispersal was
halted at the Savan Dam at river kilometer 36.69. No live or dead unionids were found at six sites upstream of the dam. Bray-Curtis
similarity indices were calculated between present survey and historical survey accounts. Species composition in the present survey
compares favorably (86.14%) with collections by Ortmann (1909) and Bogan and Davis (1992). Several locations have relatively high
unionid diversity, despite being located in an area with anthropogenic perturbations.

Key words: Unionids, Bray-Curtis similarity indices, watershed ecology, anthropogenic threats, visual assessment

Little Mahoning Creek, originating near Deckers Point
in Indiana County, Pennsylvania, U.S. A., is home to several
rare and threatened acquatic species. Most notably, the east-
ern hellbender salamander Cryptobnmchiis alleganieiisis and
several rare freshwater mussels and fish are endemic to this
watershed. Three ot the mussels documented in past surveys
of Little Mahoning Creek are presently listed as endangered
in Pennsylvania and have global status of either imperiled or
vulnerable — Epioblasma triqiietra (Rafinesque, 1820), Pleii-
robema sintoxia (Rafinesque, 1820), and Villosa iris (I. Lea,
1829) (NatureServe 2008). Historic records from the greater
Mahoning drainage indicate the presence of the federally
endangered Pleurobenia dava (Lamarck, 1819) and Obovana
subrotunda (Rafinesque, 1820) although neither of those
species has been found in the Little Mahoning Creek (Ort-
mann 1919).

Despite having this diverse unionid fauna, no surveys
for freshwater mussels have occurred since 1991, and past
surveys did not systematically cover the entire stream length
(Ortmann 1909, Bogan and Davis 1992). This study utilized
stream-wide surveys to provide information on the distri-
bution and abundance of freshwater mussels along the entire
length of Little Mahoning Creek, and to document any
changes in species distribution and composition over time.
The survey included all historic sites on Little Mahoning
Creek that were sampled as early as 1905 (a single site and
five in 1991) and filled in gaps by conducting additional
surveys throughout the creek.

There are several methods and sampling designs to es-

timate population sizes of mussels (Strayer and Smith 2003),
and a semi-quantitative sampling method was chosen that
can detect rare species and give accurate estimates of catch
per unit effort (Smith et al. 2001). Timed searches indi-
cated mussel species richness in an area as well as catch per
unit effort (CPUE). In addition, length data were recorded
for each mussel to examine recruitment and age class data.
Bray-Curtis similarity indices were calculated to compare
results to Bogan and Davis (1992) and Ortmann (1909) to
elucidate long-term trends in mussel fauna of this imperiled
watershed.

MATERIALS AND METHODS
Study location

Little Mahoning Creek watershed is located in the Pitts-
burgh Low Plateau Section originating near Deckers Point,
Pennsylvania (Fig. 1 ) and covers an area of 295.61 km‘. Little
Mahoning Creek is a tributary to Mahoning Creek, which
empties into the Allegheny River near Templeton, Pennsyl-
vania. Agriculture and deciduous forests comprise over
86.9% of the available land area in this watershed. The re-
maining 13% of the land is used for light industrial and
residential uses. The watershed is sparsely populated, with
the largest centers being Marion Center Borough (pop.
2,945), Smicksburg Borough (pop. 1,743), and Dayton Bor-
ough (pop. 2,302) in 2000 (United States Census Bureau
2007).

161

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AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Figure 1. Survey map for the
present survey and historical
survey locations.

Site selection

To survey the entire mainstem of Little Mahoning Creek
and all historic sites methodically, Western Pennsylvania
Conservancy (WPC) utilized several Geographic Informa-
tion System (GIS) applications and searched historical col-
lections. First, a visual assessment was completed, using a
modified United States Department of Agriculture (USDA)
protocol, which was used to identify substrate and basic
stream characteristics that were used as predictors for mussel
habitat (/.e., substrate composition, flow, and embedded-
ness) for the entire mainstem of Little Mahoning Creek,
from its confluence with Mahoning Creek upstream to the
headwaters in Deckers Point (Fig. 1). Next, all locations that
were previously sampled were added to the GIS database.
Data from the Bogan and Davis (1992) collections contained
latitude and longitude coordinates for all five sites, so it was
possible to geo-reference those stations before survey work
started in 2007. Museum specimens from the Carnegie Mu-
seum of Natural History were searched to identify historical
collection sites. Only one collection site was recorded by
Ortmann ( 1909), near the mouth of Little Mahoning Creek
at river kilometer (RKM) 0.13 (Fig. 1). Ten additional sites
were chosen throughout the creek that had suitable mussel
habitat (no bedrock slabs or excessive boulders) and private
landowner permission for stream access.

Survey methodology

Freshwater mussel surveys were conducted as in Smith
et al. (2001). Fifteen sites were surveyed in 2007, using a
combination of tactile and visual methods. Although most of
the mussels collected were visible at the surface, observers
periodically brushed away sediment, flipped over non-
embedded rocks, and did some excavation during each
search. Surveyors included two people using masks and
snorkels and two to five persons surveying with glass bottom
buckets. Surveyors collected as many unionid individuals as
possible during a specified amount of time, which was de-
pendent upon the width of each site.

Search area was standardized by the effective sampling
fraction of 0.06 and a target effective search rate of 0.5 m^/
minute (Smith et al 2001 ). Sites were standardized to 100 m
lengths, and areas were calculated based on an average width
at each site. After the area to be surveyed was determined for
each 100 m stretch, the following formula was used to de-
termine total search time. Total search time and search area
was then divided equally among surveyors, with search times
ranging from 35 to 300 minutes.

Search time (min) = Ai'ea (m^) X (Effective sampling fraction)
Effective search rate (m^/min)

LITTLE MAHONING CREEK AND FRESHWATER MUSSELS

163

A

B

0 20 40 60 80 100 120 140 160

Length (mm)

Figure 2. Length-frequency histo-
grams showing size distribution for
common species (N > 10) found in
Little Mahoning Creek.

0 20 <10 60 80 100 120 OO 160

Length (mm)

1)

0 20 40 60 80 100 120 140 160

Length (mm)

E

F

Strophitus u/idulatus

Lampstlis radiata luteola

30 ■

Mean = 54.97 mm

30'

Mean = 78.47 mm

SE= 1.13

SE=Z.52

J 20"

>

3 20

1

2

3

2

10'

10^

0

0

0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160

Length (mm) Length (mm)

G

Elliptio dilatata
N= 137

Mean = 63.98 mm
SF = 1.08

Length (mm)

120 140 160

II

S20

Rychobranchus JascioUins
N= in

Mean = 70.35 mm
SE = 0.96

60 80 100 120 140 160

Length (mm)

Sampling started at the downstream end of the study section,
and observers moved in an upstream direction covering the
entire stream width. Live mussels and dead shells were kept
in submersed mesh bags until the survey was finished at each

site. Mussels were identified, counted, measured, and re-
leased to the study site. Catch per unit effort (CPUE) was
calculated as number of unionid individuals collected di-
vided by person-hours (p-h) spent sampling. Shell length of

164

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

85

90

&

'C

95

100

Transform: Presence/absence
Resemblance: S17 BrayXurtis similarity

Sites

Figure 3. Bray-Curtis similarity dendrogram for the present survey.

each mussel was measured to the nearest 0.1 mm. Length-
frequency histograms were created for each species and were
examined for obvious breaks indicating year classes. A
threshold of 30 mm total length was used to define recent
recruitment based on similar studies (Obermeyer 1998,
Mohler et al. 2006). Mean lengths were calculated for each
species at each site to see if there were any longitudinal
trends in size distribution. Sex ratios of sexually dimorphic
species were also examined.

Statistical analysis

All meristic data collected were analyzed using S-Plus
2000 (1998). Data from all sites were analyzed using
PRIMER version 6 (Clarke and Warwick 2001) to determine
Margalefs index (d), Pielou’s evenness (J’), and Shannon-
Wiener diversity index (H’) calculated on a log, scale. All
sites were then analyzed for similarity using Bray-Curtis
similarity indices. The data were analyzed on two levels.
First, data from 2007 were analyzed to see how sites varied
within the present survey. Second, data were compared to
two historical surveys, Ortmann (1909) and Bogan and
Davis (1992). The Bray-Curtis similarity indices determine
what surveys and groups of species are most similar as de-
termined by the highest coefficient of similarity (0-100%).
Historical survey data were analyzed based on presence-
absence counts, which are not skewed by abundant taxa
(Clarke and Warwick 2001). Rare taxa thus have the iden-
tical weight that dominant species have.

To make comparisons among the
three surveys, data from the historical
surveys were converted to fit present
survey site locations. The 2007 site
closest to both historical surveys is
shown in parentheses. For example,
the author of the survey will appear
first, then the date, and finally, the
closest 2007 survey point in parenthe-
sis, i.e., Bogan and Davis 1992 (16).
Museum specimens from the Carnegie
Museum of Natural History were used
for the Ortmann collection data,
which contained shells from a single
sample site LM 1 (RKM 0.13).

RESULTS

Results from this survey indicate
an established unionid assemblage in
Little Mahoning Creek, with several
taxa increasing their distributions in
comparison to historical surveys. Us-
ing timed searches, we documented 10 species throughout
this third order stream.

Total species summary

A total of 812 live mussels and 10 species was found,
including several threatened and endangered species (Ap-
pendix 1 ). No additional species were detected as dead shells.
All mussels were found at the nine sites locateci downstream
of RKM 31.84; the six sites surveyed between RKM 36.81
and 50.88 were devoid of mussels. Not surprisingly, the least
diverse site — LM 16 (RKM 31.84) — was found closest to
Savan Dam (Fig. 1). Savan Dam, located at RKM 36.69,
appears to be a barrier to upstream dispersal of mussels.

The total number of mussels ranged from 0 to 168 at
each site. Ptycliobranchus fasdolaris (Rafinesque, 1820) was
the most abundant species, found at nine sites and account-
ing for about 28.0% of the total number of mussels found in
the timed searches. Elliptio dilatata (Rafinesque, 1820),
Lampsilis cardimn Rafinesque, 1820, Lasmigona costata
(Rafinesque, 1820), and Lampsilis fasciola Rafinesque, 1820
were also found at all nine sites that had mussels present
(Appendix 1). The aforementioned species accounted for
87.50% of all mussels recovered below Savan Dam. Pleu-
robema sintoxia and Strophitiis luididattis (Say, 1817) were
located at eight of the nine sites, which accounted for an
additional 7.2% of identified adults in Little Mahoning
Creek. CPUE ranged from 0 to 38.8 mussels/p-h. CPUE was
highest at RKM 5.87, with a CPUE of 38.8 mussels/p-h (Ap-

l.HTLE MAHONING CREEK AND FRESHWATER MUSSELS

165

70 -r

75-

Transfomn: Presence/absence
Resemblance: S17 Bray-Curtis similarity

80

£r-

c

85

90

95

100^

Sites (WPC Site #)

Figure 4. Bray-Curtis similarity dendrogram comparing the present survey with historical
surveys.

20.51) compared most Idvorably at
100% similarity. The lowest similarity
among all sites was at 85.98% with LM
16 (RKM 31.84) and all other couplets
combined. The site at LM 16 (RKM
31.84) was tied with the lowest species
count (seven) and had the most un-
even community of all sites with d =
0.66 (Appendix 1).

Historical surveys

The overall similarity was high at
71.30% when comparing all 1 1 sites
(Fig. 3). Highest similarity between
sites was 100%, which occurred three
separate times in this cluster analysis.
The highest similarity among the pres-
ent survey and historical surveys oc-
curs between LM 1 (RKM 0.13) and
Bogan and Davis 1992 (4), at 94.74%,
which best represents the lower
reaches. The single Ortmann survey
compares most favorably with Bogan
and Davis 1992 (12) at 87.5%, but it
still aligns well with all of the lower
river sites at 81.06% (Fig. 4).

pendix 1 ). Except for one 12.5 mm P. fasciolaris found at site
RKM 20.51 and one 27 mm Elliptio dilatata found at RKM
4.38, no evidence of recent recruitment was found (Fig. 2A-
H). No significant longitudinal trend in mean shell length
was found for adult mussels. Observations of sex ratios in
sexually dimorphic species yielded no significant differences
among any of the sites surveyed.

Univariate analysis

Margalef s index values (d) varied from a low of 1.34 at
LM 8 (RKM 12.57) to a high of 1.91 at LM 3 (RKM 4.38)
(Appendix 1 ). Evenness values were often skewed by a highly
successful single taxon, Ptychobranchiis fasciolaris^ which
caused lower scores at RKM 20.51 and 31.84. The most
diverse site for this survey was RKM 8.38, which had a Shan-
non-Wiener index of 2.68, while the lowest diversity value
was 1.85, found at RKM 31.84.

Similarity indices

Present survey

All sites where mussels were collected ( nine sites) during
the 2007 field season were compared using the Bray-Curtis
similarity indices with presence/absence transformed data
(Fig. 2). The sites at LM 8 (RKM 12.57) and LM 12 (RKM

DISCUSSION

Threats to mussel populations in this watershed include
siltation by agriculture and timbering practices (Houp 1993,
Brim Box and Mossa 1999), acid mine drainage (RADER
2007), and hydrologic alterations (Watters 2000). The WRC,
in 2006, documented threats to the watershed by utilizing a
modified (USDA) rapid bioassessment protocol to assess all
265.49 kilometers of first, second, and third order streams as
well as unnamed tributaries in the watershed (Fig. 5). The
most significant problem is excessive sedimentation mainly
from three sources: farming, natural gas extraction, and dirt
and gravel roads (WRC 2007). Farms are contributing sedi-
ment to the watershed through improper stream hulfer en-
croachment and cattle directly entering the stream. The ex-
cessive sedimentation found at sites LM 12 (RKM 20.51 ) and
LM 16 (RKM 31.84), as identified by the visual assessment
and verified by field observation during collections, is effect-
ing mussel diversity, as seen in the depressed evenness values
and correspondingly lower Shannon- Wiener scores (Figs. 1,
5, Appendix 1). A second threat is the active natural gas
extraction that is rapidly expanding in the watershed. There
are currently 2,284 abandoned and/or active gas wells in this
small (295.61 km") watershed. All wells have access roads
that could contribute sediment to the watershed. Third, a

166

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Little M;ihonuig Creek \Vatei*shed
V^isual Assessment

Clearfield

Comity

Caiiibna

County

Friorilj Areas
BH8 Agriculture
m Acid Mine Drainage
mm Ripuniin Zone
■B Channel Alleralion
BBI Oin nod Gravel Ronds
^^mi In stream Habitat
QQ Erosion and Sediment
mu Agriculture and Erosion
IBiB Dm and Gravel and Agncutlun

( { County Bound

I I Little Mtdionii

Figure 5. Visual assessment
results detailing problematic
areas in Little Mahoning
Creek watershed.

large portion, 224.24 km, of the roads are constructed ot dirt
and gravel, with 32% of these roads causing sedimentation
impacts (Fig. 5). WPC is actively working with USDA, In-
diana County Conservation District, several township super-
visors, and the Pennsylvania Game Commission to repair
and mitigate the impacts of these roads to the watershed
(ICCD 2008).

Dams prevent dispersal of mussels by preventing up-
stream movement of their host fish (Watters 1995, 2000). A
diverse mussel population was documented in the river
downstream of Savan Dam, but no mussels were docu-
mented upstream of the dam. Savan Dam has a large head of
about two meters, depending on flow, halting upstream mi-
gration of host fish. If mussels were present upstream prior
to dam construction, it is likely that isolation caused an
eventual die-out of the isolated upstream population. Savan
Dam was built in 1938 to reduce flooding; however, it is
predicted that the stream will eventually meander away from
this structure. The WPC is currently negotiating with the
landowner to remove the structure to restore unionids to the
upper reaches.

Several surveys for unionids completed in this water-
shed indicate similarity of the historical fauna with that in
the present survey. For example, ElUptio dilatata, Lawpsilis
cardium, Lasmigona costata, Pleurohema siritoxio, and Stro-
phitus iindiilatiis were collected by all surveys. Species dis-

tributions within Little Mahoning Creek also appear to be
increasing, because Lampsilis fasciola was also found at LM 1
(RKM 0.08) where it was not recorded by Ortmann (1909).
In addition to L. fasciola, Lampsilis radiata luteola was found
further upstream than it was reported by Bogan and Davis
(1992). These increased ranges could be the result of im-
provements in water quality (PADEP 2007) or the result of
more intensive survey work with additional surveyors in the
present suiwey.

Bogan and Davis ( 1992) documented the presence of 1 1
species of unionids in this system, including all the species
found in the 2007 study. In addition, two freshly dead and
one relict Epioblastna triquetra were found by Bogan but not
found during 2007 surveys or by Ortmann (1909) (Table 1).
Bogan surveyed 1 1 other sites in the Mahoning Creek drain-
age in 1991 (five of which are identical to the present sur-
vey), but no evidence of E. triquetra was recorded at those
sites (Bogan and Davis 1992). Given the small numbers, this
species has questionable viability in the Mahoning system
(Butler 2008). Obovaria subrotunda (Rafinesque, 1820) and
Pleurobema clava (Lamarck, 1819), both historically known
from the Mahoning drainage (Ortmann 1919), were not
detected in this survey. Most species found in these surveys
were relatively large and conspicuous. Two relatively small
and cryptically colored species, Villosa iris and Alasmidonta
marginata, were found infrequently by both surveys, with

LITTLE MAHONING CREEK AND FRESHWATER MUSSELS

167

Table 1. Global and Pennsylvania state ranks for each species found in Little Mahoning Creek. Key to global ranks: G5 = Secure, G4 =
Apparently Secure, G3 = Vulnerable, G2 = Imperiled, G1 = Critically Imperiled. Key to state ranks: S5 = Secure, S4 = Apparently Secure,
S3 = Vulnerable, S2 = Imperiled, SI = Critically Imperiled, SNR = not ranked. Ranks according to NatureServe (accessed 4 February 2008).

Species

Global

rank

State

rank

Ortmann

1909

Bogan

1991

Present

survey

Alasmidonta marginata

G4

S4

X

X

X

ElUptio dilatata

G5

S4

X

X

X

Epioblasma triquetra*

G2 T2

S2

X

Lampsilis cardium

G5

S4

X

X

X

Lampsilis fasciola

G5

S4

X

X

Lampsilis radiata luteola

G5

S4

X

X

X

Lasmigona costata

G5

S4

X

X

X

Pleurohema sintoxia

G4

S2

X

X

X

Ptychobranchus fasciolaris

G4 G5

S4

X

X

X

Strophitus undulatus

G5

S4 S5

X

X

X

Villosa iris

G5

SI

X

X

* Found by Bogan and Davis (1992) as dead shells only.

the majority only in the lower reaches. The only other small,
crypitic mussel was Strophitus imdidatiis, which was found in
numerous sites in the p>resent survey, but was absent in the
Bogan and Davis survey. The ability to detect small and
often overlooked species using timed searches is important
and indicates the present survey methods are more rigorous
than haphazard collections for finding conspricuous species.
In an attempt to classify recent recruitment, excavation is the
only possible method to collect an ample numbers of juve-
niles, and the present survey was not focused on juvenile
abundance as much as a complete assessment of unionid
diversity in Little Mahoning Creek.

Although substrate was not searched, this survey sug-
gests little recent recruitment. The majority of mussels ap-
pear to be older adults, which could however have been a
relic from our sampling protocol. Timed searches are less
likely to find small individuals than quadrat surveys (Horn-
bach and Deneka 1996, Vaughn et al. 1997). To get a better
estimate of recruitment and density and abundance, future
studies in the Little Mahoning Creek should use quantitative
surveys (Smith et al. 2001). Quantitative surveys will also
determine if the sex ratios documented were truly represen-
tative. Despite the anthropogenic perturbations over the past
100 years, the unionid assemblage remains similar to Ort-
mann’s historical collections. However, the watershed is cur-
rently experiencing a level of natural gas exploration that
could severely impact this important and intact resource.
Future studies should include rigorous quantitative surveys
in lower reaches and systematic sampling of excavated quad-
rates for juveniles, definitely documenting active reproduc-
tion or a paucity of juveniles. The WPC is working with local
landowners to ensure conservation stewardship within the

watershed and to protect this under-appreciated resource in
southwestern Pennsylvania.

ACKNOWLEDGMENTS

Funding for this project was provided by Colcom Foun-
dation. Scientific Collecting Permit Number 196 Type 1 was
granted by the Pennsylvania Fish and Boat Commission. We
acknowledge and thank all WPC staff that aided in the field-
work tor this project, including Nick Pinizzotto, Creg
Schaetzle, Kelly Taylor, Emily Warner, Sarah Zeglin, and
leremy Deeds. Dr. Tom Simmons from Indiana Lhiiversity
of Pennsylvania also aided in the collection of unionids in
the lower reaches of Little Mahoning Creek. We would like
to thank Dr. Tim Pearce, Curator of Molluscs, at the Car-
negie Museum of Natural History in Pittsburgh for his as-
sistance with Ortmann’s collections. This manuscript was
dramatically improved by editorial comments from Kylie
Daisley, Dr. Ken Brown, and an anonymous reviewer.

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made during 1991 under Pennsylvania Collecting Permit No. 87
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Brim Box, I. and I. Mossa. 1999. Sediment, land use, and freshwater
mussels: Prospects and problems. Journal of the North Ameri-
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Butler, R. S. 2008. Status assessment report for the stiuffhox, Epio-

168

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

blasma triquetra, a freshwater umsscl occurring in the Missis-
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Ciarke, K. R. and R. M. Warwick. 2001. Change in marine com-
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Hornbach D. I. and T. Deneka. 1996. A comparison of a qualitative
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Houp, R. E. 1993. Observations on long-term effects of sedimen-
tation on freshwater mussels (Mollusca: Unionidae) in the
North Fork of Red River, Kentucky. Transactions of the Ken-
tucky Academy of Science 54: 93-97.

ICCD (Indiana County Conservation District). 2008. Little Mahon-
ing Creek Dirt and Gravel Road Assessment and Implementation
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Mohler, J. W., P. Morrison, and ]. Maas. 2006. The mussels of
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NatureServe. 2008. NatureServe Explorer: An online encyclopedia
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western Pennsylvania. Proceedings of the American Philosoph-
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Ortmann, A. E. 1919. Monograph of the naiades of Pennsylvania.
Part III. Systematic account of the genera and species. Memoirs
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PDEP (Pennsylvania Department of Environmental Protection).
2007. Pennsylvania Nonpoint Source Management Program
FFY 2006 Annual Report. Commonwealth of Pennsylvania.
Bureau of Watershed Management, Division of Watershed
Protection. Available at: http://www.depweb.state.pa.us/
watershed mgmt/lib/watershedmgmt/nonpoint_source/
initiatives/ffy2006_report.pdf 18 April 2008.

Smith, D. R., R. F. Villella, and D. P. Lemarie. 2001. Survey pro-
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S-Plus 2000. 1998. Professional Release 1 c 1998-1999. MathSoft,
Inc., Seattle, Washington.

Strayer, D. L. and D. R. Smith. 2003. A Guide to Sampling Fresh-
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United States Census Bureau. 2007. Smicksburg, Pennsylvania pro-
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Vaughn, C. C., C. M. Taylor, and K. I. Eberhard. 1997. A com-
parison of the effectiveness of timed searches vs. quadrat sam-
pling in mussel surveys. In: K. S. Cummings, A. C. Buchanan,

C. A. Mayer, and T. J. Naimo, eds.. Conservation and Man-
agement of Freshwater Mussels II: Initiatives for the Future.
Proceedings of an Upper Mississippi River Conservation Com-
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servation Committee, Rock Island, Illinois. Pp. 157-162.

Waters, T. F. 1995. Sediment in streams; Sources, biological effects,
and control. American Fisheries Society Monograph 7: 1-251.

Watters, G. T. 1995. Small dams as barriers to freshwater mussels
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Watters, G. T. 2000. Freshwater mussels and water quality: A review
of the effects of hydrologic and instream habitat alterations.
In: R. A. Tankersley, D. I. Warmolts, G. T. Watters, B. J.
Armitage, P. D. lohnson, and R. S. Butler, eds.. Freshwater
Mollusk Symposia Proceedings 1999, Chattanooga, Tennessee.
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Submitted: 27 May 2008; accepted: 1 1 September 2008;

final revisions received: 6 November 2008

LITTLE MAHONING CREEK AND FRESHWATER MUSSELS

169

Appendix 1. Total number, univariate statistics, number of sites, and catch per unit effort (CPUE, numbers per person-hour) for the
present survey.

(Site #) River

(LM 1)

(LM 3)

(LM 4)

(LM 6)

(LM 8)

(LM 12)

(LM 13)

(LM 15)

(LM 16)

(LM 18)

(LM 20)

(LM 21)

(LM 23)

(LM 25)

(LM 27)

Total

Relative

kilometer

0.13

4.38

5.87

8.38

12.57

20.51

22.83

28.17

31.84

36.81

37.86

40.60

42.08

47.39

50.88

numbers

abunddnce

Species

Ahisiiiidonta

1

1

1

3

()AU%

marginata

ElUptio

7

5

16

21

15

1 1

36

12

14

137

16.90'H,

dilatata

Lampsilis

21

8

66

24

23

15

16

6

5

184

22.70'/o

cardium

Lampsilis

3

3

12

11

8

4

8

3

3

55

6.80%

pisciola

Lampsilis

19

5

5

5

1

3

38

4.70'K)

radiata

luteola

Lampsilis

51

2

32

1

4

1

10

4

1

106

13.10%

costata

Pleuwbema

1

1

8

6

5

1

5

2

29

3.60'K)

sintoxia

Ptychohranchiis

18

17

21

36

32

19

34

11

39

227

28.00%

fasciokiris

Strophitus

6

2

6

8

2

1

2

2

29

3.60%

imdulatus

Villosa iris

2

2

4

0.50%

Total numbers

127

39

168

115

89

52

116

39

67

0

0

0

0

0

0

812

(M)

S

9

8

9

10

7

7

8

7

7

0

0

0

0

0

0

d

1.65

1.91

1.56

1.90

1.34

1.52

1.47

1.64

1.43

0

0

0

0

0

0

r

0.77

0.79

0.81

0.81

0.83

0.76

0.83

0.87

0.66

0

0

0

0

0

0

H’ (log,)

2.44

2.37

2.5

2.68

2.34

2.14

2.50

2.43

1.85

0

0

0

0

0

0

Search time

260

260

260

300

300

300

240

260

200

200

132

100

100

35

35

(min)

CPUE (#/p-h) 29.3 9 38.8 23 17.8

Area (nr) 2286 2439 3170 1890 2713

10.4 29 9

1951 21.34

20.1 0
1219 1280

0

1219

0 0 0
1524 762 487

2134

0

335

Amer. Maine. Bull. 26: 171-177 (2008)

Diversity and distribution of freshwater gastropods in the Bayou Bartholomew
drainage, Arkansas, U.S.A.

Russell L. Minton\ John D. White', David M. Hayes'”^, M. Sean Chenoweth^, and Anna M. Hill'

' Department of Biology, University of Louisiana at Monroe, 700 University Avenue,

Monroe, Louisiana 71209-0520, U.S.A., minton@ulm.edu

“ Department of Geosciences, University of Louisiana at Monroe, 700 Lhiiversity Avenue, Monroe, Louisiana 71209-0550, U.S.A.

Abstract: Bayou Bartholomew is a low-gradient river system that drains much of southeastern Arkansas and northeastern Louisiana, U.S.A.
As one of the few southeastern streams remaining un-impounded, the Arkansas reach of the bayou harbors a rich freshwater molluscan
fauna. Collecting efforts have historically focused on documenting freshwater mussel and fish diversity, and there was no prior survey
focusing on freshwater gastropods. I’his survey of the drainage yielded 13 gastropod species representing three orders and seven genera.
Pulmonates were most abundant in low-order reaches of the drainage, while gill-breathing snails dominated higher-order reaches.
Co-occurrence analyses indicated that pulmonates occurred significantly more often with other pulmonates than they did with gill-
breathers; this trend was also observed in gill-breathers. Both stream order and predominant substrate influenced species richness and
abundance. Our findings were consistent with other published studies on freshwater snail distribution but may be confounded by drought
conditions experienced during the survey.

Key words: snails, ecology, drought, co-occurrence

The southern United States harbors one of the most
diverse freshwater mollusc assemblages in the world (Wil-
liams et al. 1993, Neves et al. 1997). Of the over 800 species
of freshwater gastropods in North America (Lysne et al.
2008), up to 40 are reported from Arkansas (Hayes 2008,
NatureServe 2008), depending on source. Like many fresh-
water taxa, snails are experiencing declines due to habitat
degradation, pollution, and anthropogenic effects (Lydearci
et al. 2004, Perez and Minton 2008). Few stream systems in
the United States with abundant snail resources htive re-
mained unaltered or unexploited. Those rare, minimally im-
pacted systems offer a glimpse into the natural condition
that existed prior to widespread impoundment, channeliza-
tion, and other human influences.

Bayou Bartholomew originates in loess hills west of Pine
Bluff, Arkansas and flows 457 km through Jefferson, Lin-
coln, Drew, Desha, and Ashley counties in Arkansas and
Morehouse Parish in Louisiana before its confluence with
the Ouachita River near Sterlington, Louisiana. It is the only
non-channelized river in southeast Arkansas and northeast
Louisiana. Bayou Bartholomew is a low-gradient, Yazoo-
type watershed occupying approx. 20% of the Ouachita
River basin and draining over 400,000 ha in southeast Ar-
kansas and northeast Louisiana (Broom 1973), including

Current Address: Department of Biological Sciences, Arkansas
State University, P.O. Box 599, State University, Arkansas 72467-
0599, U.S.A.

parts of the Arkansas River floodplain (Saucier 1994). Up-
stream reaches of the drainage are subject to frequent drying
and modification due to low water levels; single reaches can
become unconnected chains of pools during summer
months. Most of Bayou Bartholomew occurs within the Mis-
sissippi Alluvial Basin ecoregion characterized by fine tex-
tured and fertile alluvial soils well suited to agricultural de-
velopment (Alley 2005). Agricultural fields, row crops, and
pastures dominate land-use in Bayou Bartholomew’s water-
shed. There is a narrow riparian zone along the river, in
most cases less than 50 m wide, dominated by bottomland
hardwood species such as water tupelo {Nyssa aqiiatka),
bald cypress {Taxodiiim distichiim), and maples (Acer spp.).
Erosion, sedimentation, agriculture and urban nutrient in-
puts, and irrigation water withdrawals have been the main
stressors of the Bayou Bartholomew stream ecosystem for
many years (Alley 2005).

Most research conducted on Bayou Bartholomew has
focused on fishes (Thomas 1976, Hutchins 1988, Pezold et
al. 2002) and mussels (George and Vidrine 1993, Pezold et
al. 2002, Brooks et al. 2005). These surveys found that Bayou
Bartholomew harbors a diverse mussel assemblage in both
Arkansas and Louisiana. However, exact figures for the
number of gastropod taxa in Bayou Bartholomew are lack-
ing. The objectives of this project were ( 1 ) to assess the
current status, diversity, and distribution of snail species in
the Arkansas portion of Bayou Bartholomew and (2) to pro-
vide baseline data for monitoring these species in the future.
We additionally explored potential population structure in

171

172

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

the drainage and assessed any roles that substrate and stream
order had on diversity and distribution.

MATERIALS AND METHODS

The Arkansas portion of the Bayou Bartholomew drain-
age was surveyed from August 2004 to April 2006. Seventy-
four sites were surveyed between the headwaters west of Pine
Bluff, Arkansas and the Arkansas-Louisiana state line (Fig. 1,
Appendix 1). At each site, the predominant substrate en-
countered was classified as mud, silt, sand, clay, gravel, or
rock; dry sites were recorded separately. One person con-
ducted an hour-long visual search at each site, and all live
snails encountered were collected, identified to species, and
returned to the river; dead shells were not included in the
survey. Voucher specimens of all species were preserved in
95% ethanol and are housed at the ULM Museum of Natural
History and await cataloguing. Nomenclature and classifica-
tion followed Turgeon et al. ( 1998). The status of each spe-
cies was determined by comparing our findings to global
heritage ranks given on NatureServe (2008) and state heri-
tage ranks provided by the Arkansas Heritage Program
(2008).

Because the survey was performed during a period of
extreme drought (National Weather Service 2008), only sites
where live snails were present were used for statistical analy-
ses. We felt this was appropriate because we had no way of
establishing if sites without snails indicated a true lack of
gastropods, or if snails had moved out of those areas or died.

The influence of environmental factors on snail diver-
sity and distribution was assessed using one-way analyses of
variances to determine the effects of stream order and pre-
dominant substrate on the numbers of pulmonates and cae-
nogastropods and total richness at each site in JMP 7.0 (SAS
Institute, Cary, North Carolina). To test for potential struc-
turing within the watershed, co-occurrence analyses using
species abundances at each site were performed in EcoSim 7
(Gotelli and Entsminger 2004) using the C-score, checker-
board species pairs, and number of species combinations
settings with fixed row and column sums and all other set-
tings as defaults. C-score calculates co-occurrence values ac-
cording to a model based on Stone and Roberts ( 1990) and
compares observed occurrences with a set of simulated
datasets; if the observed value is significantly larger than the
random simulations, significant co-occurrence exists.
Checkerboard pair analyses calculate how many species
never occur together in the observed and simulated data; if

Figure 1. Map of survey sites
in the Bayou Bartholomew
drainage of Arkansas. Filled
circles indicate sites where
snails were present, open
circles where snails were ab-
sent, and gray squares indi-
cate sites that were dry. Inset
map shows the location of
the drainage in the state.

SNAILS OF BAYOU BARTHOLOMEW, ARKANSAS

173

the observed value is larger, then singleton species that do
not occur with any others may be biasing the analysis toward
significant structuring (Diamond 1975). Number of species
combinations analyses calculate the number of unique spe-
cies that co-occur according to Pielou and Pielou (1968). If
the observed number of species combinations is greater than
random, species co-occur at a significant level.

RESULTS

Of the 74 sites surveyed, 8% were dry, 28% had water
but no snails, and 64% of the sites had snails (Fig. 1 ). A total
of 3,384 individual snails representing 13 species, 12 genera,
seven families, and three orders were found throughout the
drainage (Table 1). These collections represent 35% of the
previously reported state fauna; no new state records or alien
species were tound. Pleuwcera canaliculata (Say, 1821) was
the most abundant species, with 2,230 individuals (66% of
total) surveyed. Snails were most abundant at site 72 in
Ashley County, with 630 individuals representing three spe-
cies. The highest species richness in the drainage was at site
57, Bearhouse Creek in Ashley County, with six species. The
highest species richness for the main stem of Bayou Barthol-
omew was at sites 7 and 65 in Jefferson and Ashley Counties,
each with five species. All species encountered have G4-G5
global heritage rankings, indicating overall stability; how-
ever, these same species hold SU state rankings, meaning
their status in Arkansas is unknown.

Neither stream order ( 1-way ANOVA, F - 1.54, c1f = 45,

Table 1. Gastropod taxa found in the Bayou Bartholomew drainage
of Arkansas. Nomenclature and classification follow Turgeon cl al.
(1998).

ARCHITAENIOGLOSSA (gill-breathing)

Viviparidae Campelonia decisum (Say, 1817)

Vivipariis intertextus (Say, 1829)
Viviparus subpwpureiis (Say, 1829)

BASOMMATOPHORA (pulmonate)

Ancylidae Ferrissia riviilaris (Say, 1817)

Laevapex fitsciis (Adams, 1841)
Lymnaeidae Fossaria hiilimoides {Lea, 1841)

Pseudosucdnea columella (Say, 1817)
Physidae Physella gyrina (Say, 1821)

Planorbidae Flelisoma anceps (Menke, 1830)

Micronienetus dilatatus (Gould, 1841)
Planorbella trivolvis (Say, 1817)

NEOTAENIOGLOSSA (gill-breathing)

Hydrobiidae Cinebmatia Integra (Say, 1829)

Pleuroceridae Pleiirocera canaliculata (Say, 1821)

P > 0.21) nor predominant substrate {F = 0.855, P > 0.52)
had significant effects on total species richness. Stream or-
der, however, had significant effects on the numbers of pul-
monates {F = 3.45, P < 0.03) and caenogastropods (F = 4.19,
P < 0.02); fourth-order reaches had more caenogastropods
and fewer pulmonates than first through third-order
reaches. Predominant substrate showed no effect on the
number of pulmonates (F = 0.72, P > 0.61) but had a sig-
nificant effect on the number of caenogastropods (F == 4.78,
P < 0.002). Caenogastropods were significantly more abun-
dant in areas with sandy substrate than in any other. Co-
occurrence analyses based on C-scores indicated that there
was a significant species structure in the watersheci, with
pulmonates occurring with other pulmonates more fre-
quently than with caenogastropods and vice versa {P <
0.001). Analysis of species combinations additionally sug-
gested significant structuring (P < 0.02), but analysis of
checkerboard species pairs also indicated that singleton
species presence was biasing the data toward co-occurrence
(P < 0.04).

DISCUSSION

The diversity of snails in low gradient, Mississippi allu-
vial and gulf coast rivers remains relatively unstudied. The
few published studies addressing this point {e.g., Gordon
1985, Gordon et al. 1993-1994) indicate these aquatic sys-
tems possess low snail diversity (up to 15 species; Gordon et
al. 1993-1994) due to many factors, including a prevalence
of soft substrate reaches, low flow, and frequent seasonal
drying. The species richness we observed in Bayou Barthol-
omew is consistent with these values, and we speculate the
observed distribution reflects variation in the temporal water
levels in the river. The co-occurrence analyses indicated that
pulmonates were tound with other pulmonates at a signifi-
cantly higher rate than with caenogastropods and vice versa.
Analyses of variance showed these groups were associated
with stream order, with pulmonates occupying lower order
reaches ot the bayou and caenogastropods occupying higher
order reaches.

Bayou Bartholomew is a highly variable system with
periodic drying which would tend to decrease overall species
richness. All ot the species found in Bayou Bartholomew
have broad distributions (Burch and Tottenham 1980, Na-
tureServe 2008) and almost all are from groups of taxa
known to be highly resistant to desiccation and drought
(Pilsbry 1896, Alyakrinskaya 2004) and indicative of tempo-
rary environments (Eckblad 1973). These same taxa fre-
quently characterize lentic and other low flow areas (Brown
et al. 1998). Therefore, the bayou system may be well suited
to species adapted to the high disturbance conditions that

174

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

have characterized Bayou Bartholomew for the last 5,000-
7,000 years (Saucier 1994).

Other longer-term factors may also be influencing snail
diversity in Bayou Bartholomew. The watershed is a rela-
tively young system and, as such, would generally possess a
less diverse fauna than older systems such as the large rivers
of the North American interior (Saucier 1994). The upper
reaches of the bayou were originally connected to the Ar-
kansas River and began to separate into a different drainage
approximately 15,000 years ago following the last glacial
maxima. Subsequent flooding and sediment deposition
caused the Arkansas River to divert into its current channel,
and the abandoned channel connected to the lower portion
of the bayou 5,000 to 7,000 years ago.

The National Weather Service in Little Rock, Arkansas
reported that an extreme drought began in 2005 and ended
late in 2006. By the end of 2005, average precipitation defi-
cits for the state were 35.5 cm below normal; some areas in
the drainage near the Arkansas and Louisiana state line were
over 50 cm below normal (National Weather Service 2008).
These conditions caused many of the low-order tributaries
in the drainage to dry out completely, while many higher
order reaches became isolated pools. Brown et al. (1998)
hypothesized that pulmonate species were more tolerant to
temporary streams and extreme conditions, and would
therefore dominate such areas. We think it is likely that
distribution of snails we observed was strongly influenced by
intense drought conditions during our survey. Our data in-
dicate a similar general pattern in Bayou Bartholomew, with
pulmonate species being more prevalent upstream and in
tributaries, and gill-breathing species occupying higher or-
der downstream areas that are presumably deeper and more
stable. Pulmonates also exhibit greater dispersal abilities and
can re-colonize dry areas more easily (Davis 1982, Brown
1991).

While many natural factors played a role in the observed
diversity and abundance of snails in the bayou, these may
have been confounded by our survey methods. A single man
hour was dedicated to surveying at each site, and this may
have decreased the probability of finding small species like
hydrobiids and freshwater limpets. Most limpets were found
associated with discarded glass beer bottles; visual inspection
found many in bottlenecks. Given their small size and abun-
dance of human-produced refuse at many points in the
drainage, species like limpets could be easily overlooked.
Additionally, some species including viviparids bury them-
selves in soft substrates (Van Cleave and Altringer 1937) and
individuals may have been missed in the survey. Subsequent
surveys during periods of higher water employing more ef-
fort may well provide more accurate estimates of richness
and distribution; however, despite the drought and limited

available effort, we feel our results are consistent with pub-
lished data. ji

Modern assessments of freshwater mollusc populations, f
particularly snail populations serve two important purposes.
First, distribution and status surveys provide baseline data
for tracking population fluctuations and declines (Hartfield
and Rummel 1985, Blalock and Sickel 1996, Lydeard et al. (
1999, Vaughn and Taylor 1999). Second, these studies can ^
reveal biotic and abiotic interactions that may be influencing
snail community structure (Brown et al. 1998) for further f
experimental examination. Since many freshwater molluscs i
are experiencing their highest declines in terms of richness
and abundance (Neves et al. 1997), it is critical that surveys
such as ours are performed to serve as baseline data docu- 1
menting biological diversity. )

i

I

ACKNOWLEDGMENTS

Jeff Brooks and Don White assisted with fieldwork. Ken
Brown, Kathryn Perez, and anonymous reviewers provided
valuable comments for improving the manuscript. RLM was
partially supported by a Howard Hughes Medical Institute
science education grant to ULM.

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revisions received: 14 October 2008

Appendix 1. Survey localities in the Bayou Bartholomew drainage.
Site numbers follow Fig. 1. Sites that were dry are indicated as sueb.

Site Locality

1 Bayou Bartholomew at Hardin Road, Jefferson County

34°15'33"N, 92°09'02"W

2 Unnamed tributaiy at Gorman Itoad, lefferson County

34°16'21"N, 92°08'39"W

176

AMERICAN MALACOLOGICAL BULLETIN 26' Ml- 2008

Site Locality

3 Unnamed tributary, lefferson County

34°15'15"N, 92°10'30"W

4 Bayou Bartholomew at Princeton Pike Road, lefferson County

34°14'09"N, 92°08'00"W

5 Unnamed tributary at Hardin-Reed Road, Jefferson County

34°14'12"N, 92°09'04"W

6 LInnamed tributary at Brantley Road, Jefferson County

34°13'20"N, 92°08'19"W

7 Bayou Bartholomew, Jefferson County

34°12'51"N, 92°06'11"W

8 Nevins Creek at Beechnut Road, Jefferson County

34°11'38"N, 92°07'08"W

9 Nevins Creek at SR 54, Jefferson County

34°11'19"N, 92°05'03"W

10 Boggy Bayou at Brinkley Road, Jefferson County

34°08'55"N, 92°03'02"W

1 1 Boggy Bayou at Middle Warren Road, Jefferson County

34°09'07"N, 92°02'01"W

12 Boggy Bayou at SR 15, Jefferson County

34°07'42"N, 91°59'30"W

13 Bayou Bartholomew at Bohannon Road, Jefferson County

34°08'14"N, 91°58'52"W

14 Bayou Bartholomew at Ohio Street, Jefferson County

34°09'56"N, 91°57'37"W

15 Sandy Bayou at Middle Warren Road, Jefferson County

34°06'30"N, 92°()1'55"W

16 Unnamed tributary at Ty Lane, Jefferson County

34°05'46"N, 92°01'25"W

17 Sandy Bayou at SR 54, Jefferson County

34°06'02"N, 92°00'05"W

18 Sandy Bayou at SR 15, Jefferson County

34°06'34"N, 91°59'36"W

19 Bayou Bartholomew at Gibb- Anderson Road, Jefferson County

34°07'16"N, 91°57'31"W

20 Bayou Bartholomew at Gibb-Anderson Road, Jefferson County

34°05'45"N, 91°56'51"W

21 Bayou Bartholomew at CR 70, Lincoln County

34°04'19"N, 91°54'03"W

22 Bayou Bartholomew at CR 70, Lincoln County

34°04'25"N, 91°52'38"W

23 Melton Creek at CR 10, Lincoln County

34°02'24"N, 91°55'46"W

24 Flat Creek at CR 10, Lincoln County

34“02'06"N, 91°55'36"W

25 Flat Creek at CR 71, Lincoln County

34°03'21"N, 91°53'18"W

26 Flat Creek at CR 11, Lincoln County

34°03'18"N, 91°50'01"W (DRY)

27 Unnamed tributary at CR 8, Lincoln County

34°01'48"N, 91°53'25"W (DRY)

28 Turtle Creek at CR 8, Lincoln County

34°00'34"N, 91°53'32"W

29 Bayou Bartholomew at US 425, Lincoln County

34°01'12"N, 91°48'53"W

Site Locality

30 Bayou Bartholomew at CR 2, Lincoln County

33°59'33"N, 91°44'14"W

31 Bayou Bartholomew at SR 293, Lincoln County

33°55'33"N, 91°42'58"W

32 Abies Creek at SR 54, Lincoln County

33°53'33"N, 9r50'15"W

33 Abies Creek at CR 32, Lincoln County

33°52'33"N, 91°47'54"W

34 Abies Creek at Gentry Road, Lincoln County

33°5T08"N, 91°46'03"W

35 Bayou Bartholomew at SR 54, Lincoln Coirnty

33°51'59"N, 91°39'23"W

36 Chance Creek at CR 32, Lincoln County

33°50'22"N, 91°47'52"W (DRY)

37 Chance Creek at SR 54, Lincoln County

33°49'48"N, 91°45'44"W (DRY)

38 Lyle Creek at CR 32, Lincoln County

33°48'48"N, 91°47'48"W (DRY)

39 Chance Creek at SR 83, Lincoln County

33°49'30"N, 91°44'07"W

40 Bayou Bartholomew at SR 293, Lincoln County

33°50'00"N, 91°36'31"W

41 Bayou Bartholomew at CR 20, Desha County

33°49'27"n, 91°33'07"W

42 Weaver Creek at SR 83, Lincoln County

33°48'03"N, 91°44'51"W (DRY)

43 Prairie Creek at Prairie Creek Road, Drew County

33“46'35"N, 91°40'09"W

44 Bayou Bartholomew at SR 138, Drew County

33°46'24"N, 91°30'17"W

45 Panther Creek at SR 293, Drew County

33°43'56"N, 91°36'16"W

46 Abies Creek at SR 138, Drew County

33°44'10"N, 91°33'41"W

47 Bayou Bartholomew at CR 77, Drew County

33°43'11"N, 91°29'47"W

48 Bayou Bartholomew, Drew County

33°38'52"N, 91°29'09"W

49 Bayou Bartholomew at Fourmile Creek Road, Drew County

33°36'03"N, 91°28'14"W

50 Bayou Bartholomew, Drew County

33°34'32"N, 91°28'41"W

51 Bayou Bartholomew at SR 35, Drew County

33°3T40" N 91°29'51"W

52 Bayou Bartholomew at HiU Community Road, Drew County

33°30'10"N, 91°28'05"W

53 Bayou Bartholomew, Drew County

33°27'17"N, 91°29'30"W

54 Bayou Bartholomew at Panther Break Road, Drew County

33°27'16"N, 91°29'23"W

55 Bayou Bartholomew, Drew County

33°25'22"N, 91°29'41"W

56 Jordan Creek at SR 61, Ashley County

33°20'52"N, 91°39'49"W

Site

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

SNAILS OF BAYOU BARTHOLOMEW, ARKANSAS

177

Locality

Bearhouse Creek at SR 61, Ashley County
33°21'02"N, 91°38'08"V\^

Little Bayou at CR 104, Ashley County
33°20'50"N, 91°33'39"W
Bayou Bartholomew at CR 104, Ashley County
33°20'49"N, 91°31'50"W
Bayou Bartholomew, Asliley County
33°21'44"N, 91°29'51"W
Overflow Creek at US 82, Ashley County
33°17'10"N, 91°35'36"W
Bayou Bartholomew at US 82, Ashley County
33°17'54"N, 91°33'44"W

Bayou Bartholomew at Mount Pleasant Road, Ashley County
33°14'55"N, 91°32'55"W

Bayou Bartholomew below SR 278, Ashley County
33°14'09"N, 91°32'06"W
Beach Creek at SR 8, Ashley County
33°10'28"N, 91°39'14"W
Overflow Creek at SR 8, Ashley County
33°10'27"N, 91°36'31"W
Bayou Bartholomew, Ashley County
33°09'30"N, 91°35'27"W
Bayou Bartholomew at SR 8, Ashley County
33°07T6"N, 91°33'13"W
Bayou Bartholomew, Ashley County
33°04'03"N, 91°34'51"W
Bayou Bartholomew at SR 173, Ashley County
33°04'16"N, 91°34'41"W
Bayou Bartholomew at CR 365, Ashley County
33°01'37"N, 91°39'22"W
Bayou Bartholomew, Ashley County
33°01'47"N, 91°38'19"W
Bayou Bartholomew, Ashley County
33°0T30"N, 91°37'36"W
Bayou Bartholomew, Ashley County
33°01'31"N, 91°37'52"W

Amer. Maine. Bull. 26: 179-180 (2008)

RESEARCH NOTE

Conus lighthourni holotype returned to the Delaware Museum of Natural History

Elizabeth K. Shea* and William J. Fenzan^

’Mollusk Department, Delaware Museum of Natural History, 4840 Kennett Pike, Wilmington,
Delaware 19807, U.S.A., eshea@delmnh.org

^401 Sinclair St., Norfolk, Virginia 23505-4359, U.S.A., bill@fenzan.com
Key words: Bermuda, Conidae

In the early 1970s, the deep water gastropod fauna of
Bermuda was sampled using baited lobster traps (Light-
bourn 1991). This novel approach yielded many unusual
gastropods, many of which were provided to Dr. R. Tucker
Abbott at the Delaware Museum of Natural History
(DMNH) for further study and identification. Several new
species were described based on these specimens, including
Conns (Flomconns) lighthourni Petuch, 1986. The DMNH
was identified as the type repository for C. lighthourni and
catalog numbers were assigned for the holotype (DMNH

134938) and paratype (DMNH 134939) lots; however, the
holotype was never received (Bieler and Bradford 1991).
There was considerable speculation regarding the location of
this specimen for the past 20 years. No neotype was ever
designated.

The original description of Conus lighthourni was based
on 9 specimens examined, all collected south of Castle Is-
land, Bermuda by lack Lightbourn and Arthur Guest. Petuch
(1986) described the holotype as 35 mm long and 16 mm
wide, having a bright orange base color, salmon-pink spire.

Figure 1. The holotype of Conus lightbourni I^etuch, 1986. Dorsal (A) and ventral (B) view of the returned specimen. The shell markings
are an obvious match to the dorsal (C) and ventral (D) view of Petuch ( 1986: fig. 1 ). Three areas of damage to the outer lip are identified
by number for comparison with Fig. 2. Original ® 2008 Biological Society of Washington, from the Proceedings of the Biological Society of
Washington. Reprinted by permission of Allen Press Publishing Services.

179

180

AMERICAN MALACOLOGICAL BULLETIN 26 • 1/2 • 2008

Figure 2. The holotype of Conus lightbourni, lateral view. As in the
original photograph, the outer lip is damaged at three locations.
Ongoing wear at the outer lip makes the damage at location 2
appear diminished.

wide salmon-pink bands, and brown tlammules and dots.
Three paratypaes (26.0-47.7 mm length) were designated as
coming from the same depth and locality as the holotype,
and five additional specimens (22.4-44 mm) were examined
but not included as paratypes.

In 2007, Don Pisor purchased Jack Lightbourn’s private
collection, with one known specimen of Conus lightbourni as
part of the transaction. Upon unpacking, a second shell was
found in a box of miscellaneous Bermuda sprecimens and
other items (Pisor, pers. comm.). One of these two speci-
mens was sold to Bill Fenzan who recognized it as the ho-
lotype. After an indeptendent confirmation of the identifica-
tion, the holotype (Figs. 1-2) was returned to DMNH on 14
March 2008.

The color and breakage piatterns of the returned speci-
men are a convincing match to the original holotypre de-
scription (Figs. 1-2). The shell length and maximum diam-

eter (Kohn and Riggs 1975) of the returned shell were
measured using NIS-Elements D 3.00 digital imagery soft-
ware. The length, measured from the top of the spire to the
base of the columella, is 35.9 mm, within 2.5% of the origi-
nal measurement. The maximum diameter is 16.2 mm,
within 1.2% of the original measurement. Damage at the
edge of the outer lip is consistent in location, but different in
degree (Figs. 1-2). The returned specimen has brown flam-
mules and dots, which match the original description, but
the salmon-pink bands are orange-brown and the base color
aprpears white. These differences are consistent with wear
from improper long-term storage and fading due to expo-
sure to light and do not change the conclusion that the
returned specimen is the holotypie of Conus lightbourni.

ACKNOWLEDGMENTS

We thank Mr. Don Pisor for facilitating the return of
the holotype to DMNH and Dr. Harry Lee for his indepen-
dent evaluation of the identification. Reviews of this research
note by Dr. Rudiger Bieler and Dr. Tom Duda and discus-
sions with Dr. Alan Kohn are greatly appreciated.

LITERATURE CITED

Bieler, R. and A. Bradford. 1991. Annotated catalog of type speci-
mens in the malacological collection of the Delaware Museum
of Natural History. Gastropoda (Prosobranchia and Opistho-
branchia). Nemouria: Occasional Papers of the Delaware Mu-
seum of Natural History 36; 1-48.

Kohn, A. J. and A. C. Riggs. 1975. Morphometry of the Conus shell.
Systematic Zoology 24: 346-359.

Lightbourn, J. R. H. 1991. Dredging and trapping shells in deep
water in Bermuda. American Conchologist 19: 4-7.

Petuch, E. 1986. The Austral-African conid subgenus Floraconus
Iredale, 1930, taken off Bermuda (Gastropoda: Conidae). Pro-
ceedings of the Biological Society of Washington 99: 15-16.

Submitted: 30 May 2008; accepted: 8 September 2008;
final revisions received: 13 October 2008

INDEX TO VOLUME 26

AUTHOR INDEX

Averbuj, A. 26: 67
Beugly, J. 26: 137
Chapman, E. J. 26: 161
Chenoweth, M. S. 26: 171
Dourson, D. C. 26: 153
Fenzan, W. J. 26: 179
Galvan- Villa, C. M. 26: 119
Gray, S. M. 26: 19
Hayes, D. M. 26: 171
Hill, A. M. 26: 171
Johnsen, S. 26: 27

Kelly, S. 26: 19
Lopez-Uriarte, E. 26: 119
Martin, E. 26: 137
Meyer-Rochow, V. B. 26: 47
Minton, R. L. 26: 171
Morton, B. 26: 35
Pearce, T. A. 26: 111
Penchaszadeh, P. E. 26: 67
Piatigorsky, J. 26: 73
Pyron, M. 26: 137
Rios-Jara, E. 26: 119

Robles, L. J. 26: 19
Serb, J. M. 26: 1, 3
Shea, E. K. 26: 1 79
Smith, T. A. 26: 161
Speiser, D. I. 26: 27
Spielman, M. 26: 137
Voight, J. R. 26: 133
Salvini-Plawen, L. V. 26: 83
White, J. D. 26: 171
Wilkens, L. A. 26: 101
Zieger, M. V. 26: 47

PRIMARY MOLLUSCAN TAXA INDEX

[first occurrence in each paper recorded, new taxa in bold]

acopidcensis, Niiciilann 26: 123
achatina, Achatina 26: 49
aciciila, Cedlioides 26: 48
acuta, Physella 26: 145
acuta, Pleurocera 26: 142
adanisi, Anadara 26: 119
aenigmatica, Euigmonia 26: 38
aequatorialis, Anadara 26: 124
Aeropneusta 26: 83
agreste, Deroceras 26: 49
albolabris, Neoljelix 26: 112
aletes, Pitar 26: 124
alternata, Anguispira 26: 112
americamis, Spondylus 26: 1, 27, 101
Amphitretus 26: 10
Amusium 26: 39
Anadara 26: 93, 125
Anatina 26: 94

anceps, Helisoma 26: 146, 173
anciUaria, Physella 26: 149
Ancylidae 26: 148, 173
annulatum, Luclnoma 26: 130
Anomalodesmata 26: 35, 94
Anomiidae 26: 38
Anomioidea 26: 35, 93
antroswn, Helisoma 26: 149
aphanes, Neophysema 26: 130
Aplysia 26: 11, 56, 86

approxiniata, Parvdiicina 26: 119
Area 26: 93

Architaenioglossa 26: 173
Arcidae 26: 6, 37, 89, 121
Arcoida 26: 7, 41, 91, 106
Arcoidea 26: 35, 93
arcuata, Pandora 26: 131
arenaria, Mya 26: 39, 96, 102
armigera, Planorbida 26: 148
aspersa, Helix 26: 116
aspersum, Cornu 26: 47
atra, Aulacomya 26: 35
aurora, Psannnotreta 26: 124
balloti, Amusium 26: 1, 27, 101
Barbatia 26: 93
Basommatophora 26: 11, 173
Bathothauma 26: 10, 133
beebei, Cyclocardia 26: 130
belcheri, Trachycardium 26: 124
berryi, Pitar 26: 124
bicarinata, Valvata 26: 139
bimaculoidcs, Octopus 26: 1, 20
biolleyi, Leptopecten 26: 129
bitentaculatus, Athoracophorus 26: 53
Bivalvia 26: 1, 7, 35, 83
brevialata, Pteria 26: 37
brevifrons, Lunarca 26: 124
Buccinanops 26: 2, 67

Bucciniim 26: 67
bidinwides, Fossaria 26: 173
Bidlia 26: 67

Caenogastropoda 26: 2, 67, 91
califonuca, Aplysia 26: 11, 52, 92, 103
californica, Gouldia 26: 119
califoriucus, Ariolimax 26: 53
callicofnaliis, Pitar 26: 131
canipanidata, Planorbella 26: 147
campamdatum, Helisottia 26: 149
campbellorum, Semelina 26: 123
Campeloma 26: 139
canaliculata, Pleurocera 26: 1 73
cancellaria, Barbatia 26: 6, 41
cancellaris, Radiolucina 26: 119
caperata, Lymnaea 26: 149
caperata, Stagnicola 26: 144
Cardiidae 26: 39, 94, 130
Cardiinae 26: 94
Cardioidea 26: 91
Carditidae 26: 130
cardium, Lampsilis 26: 166
Carinaria 26: 86
carpenteri, Tellina 26: 131
catascopium, Lymnaea 26: 149
catascopium, Stagnicola 26: 143
catskillensis. Discus 26: 112
Caudofoveata 26: 89

181

Cecili aides 26: 67
centrifuga, Lucinisca 26: 123
Cephalopoda 26: 7, 84, 133
Chamidae 26: 125
chiiiensis, Bellamya 26: 139
Chione 26: 125
Chitonidae 26: 7
Chlamys 26: 36
cicercula, Strigilla 26: 124
cincinnatiensis, Poinatiopsis 26: 141
drcumstriatiis, Gyrauhis 26: 145
clava, Pleurobema 26: 161
Clavagellidae 26: 35
coani, Tellitia 26: 131
cochlidiiim, Buccinanops 26: 67
Coleoida 26: 92
cohimelhi, Lymuaea 26: 149
columella, Pseiidosuccinea 26: 144, 173
eompta, Chione 26: 123
concavwn, Haplotrema 26: 112
Conchifera 26: 89
eoncinmi, Anadara 26: 129
concinmis, Pitar 26: 124
contectoides, Valvata 26: 149
Corbulidae 26: 131
Cornells, Planorbariiis 26: 48
Cornu 26: 47

costata, Lasmigona 26: 166
Cranchiidae 26: 133
Crassatellidae 26: 129
Ctenoides 26: 93
Ciicullaea 26: 92
Cucullaeidae 26: 92
cnpreus, Mesoinphix 26: 153
Cylichim 26: 67
dalli, Lymnaea 26: 149
Decabrachia 26: 92
decisnni, Cainpelomci 26: 139, 173
declivis, Nncida 26: 123
decussata, Crenella 26: 129
deflectus, Cyraidus 26: 146
delgada, Sheldonella 26: 129
denotatum, Xolotrenia 26: 153
dentifera, Neolielix 26: 155
dichotoma, Strigilla 26: 124
dilatata, Elliptio 26: 164
dilatatus, Micronienetus 26: 173
discus, Haliotis 26: 91
distinctus, Arion 26: 57
divaricata. Lacuna 26: 91

Docoglossa 26: 88
Donacidae 26: 131
dunkeri, Dosinia 26: 124
ecuadoriana, Crassinella 26: 124
edentuloides, Pegophysema 26: 130
edule, Cardium 26: 101
edu/e, Cerastoderma 26: 39, 95
edulis, Mytilns 26: 89
ediilis, Ostrea 26: 36
elenense, Laevicardium 26: 124
Ellobiacea 26: 48
elodes, Stagnicola 26: 144
elongata, Aplexa 26: 145
elytrum, Maconia 26: 131
Enignionia 26: 93
Eupulmonata 26: 92
Euthyneura 26: 91
exacuous, Menetus 26: 149
exacuous, Pronienetns 26: 148
excavata, Conclwcele 26: 124
exilis, Lymnaea 26: 49
exilis, Stagnicola 26: 144
fallax, Triodopsis 26: 112
fascicularis, Acantlwchiton 26: 91
fasciola, Lampsilis 26: 166
fasciolaris, Ptychobranchus 26: 164
fenestrata, Lucinisca 26: 124
Ferussaciidae 26: 67
Eissurella 26: 89
flavus, Umax 26: 49
flexuosa, Thyasira 26: 130
floridanus, Ctenoides 26: 38
finviatilis, Ancylus 26: 48
fontinalis, Physa 26: 48
formosa, Anadara 26: 129
Eossaria 26: 143
fragilis, Eerrissia 26: 139
fraternum, Euchemotrema 26: 112
fulgens, Ovachlamys 26: 111
fidica, Achatina 26: 49
fuscus, Laevapex 26: 148, 173
Gastropoda 26: 2, 7, 62, 67, 86
georgianus, Viviparns 26: 139
gigantea, Crassodoma 26: 27, 101
gigantea, Lottia 26: 1 1
gigas, Crassostrea 26: 36
glabrata, Biomphalaria 26: 1 1
Glycimeridae 26: 92
Clycimeris 26: 92
Glycymerididae 26: 129

gouldiana. Bulla 26: 57
gracilis, Donax 26: 124
gradata, Acar 26: 129
granifera, Trigoniocardia 26: 124
guatulcoensis, Chione 26: 130
Gymnomorpha 26: 92
gyrina, Physella 26: 145, 173
Haliotis 26: 67, 88
hastata, Chlamys 26: 27
Heteropoda 26: 86
heterostropha, Physella 26: 149
hirsutus, Cyrauliis 26: 149
hispida, Trichia 26: 48
Histioteuthis 26: 10
humilis, Lymnaea 26: 149
hyalina, Lyonsia 26: 95
Hydrobiidae 26: 140, 173
hypnorum, Aplexa 26: 149
Uyanassa 26: 88
inermis, Navanax 26: 49
Integra, Cincinnatia 26: 140, 173
Integra, Physella 26: 149
interriipta, Strigilla 26: 131
intertextus, Viviparns 26: 173
ira, Corbula 26: 119
iris, Villosa 26: 161
irradians, Argopecten 26: 6, 27, 101
Isognomon 26: 93
Isognomonidae 26: 129
jaeckeli, Albinaria 26: 111
japonica, Bellamya 26: 139
kellettii, Lirophora 26: 124
Kellidae 26: 130
kusceri, Congeria 26: 36
lactea, Otala 26: 60
lactea, Striarca 26: 92
laeviradius, Nuculana 26: 119
Laternula 26: 94
Laternulidae 26: 1, 35
laticostata. Cardites 26: 130
Leachia 26: 135
Leptopecten 26: 39
lewisi, Valvata 26: 139
lewisii, Valvata 26: 149
lightbourni. Conus 26: 179
lightbourni, Eloraconus 26: 179
Lima 26: 83, 101
lima, Lima 26: 83
Limaddae 26: 9
Limidae 26: 8

182

Limoidea 26: 35, 91
Limopsidae 26: 92
Limopsoidea 26: 35
limosn, Amnicola 26: 149
limosus, Amnicola 26: 149
Littorina 26: 6
livescens, Eliwia 26: 141
livescens, Goniobasis 26: 149
lobiila, Nuculana 26: 119
Loligo 26: 10
Lucinidae 26: 121
luconimy Helix 26: 57
lustrica, Amnicola 26: 140
lustrica, Pyrgulopsis 26: 149
Lymnaeidae 26: 143, 173
Lyonsiidae 26: 95
lyromina, Bathothaiima 26: 133
maccUntocki, Discus 26: 158
Mactridae 26: 131
tnagellenicus, Placopecten 26: 27
marginata, Alasmidonta 26: 161
mariae, Lirophora 26: 124
mannorata, Corbula 26: 124
martinicensis, Tellijia 26: 131
maxima, Tridacna 26: 39, 101
maximus, Umax 26: 49
maximus, Pecten 26: 27, 39, 93, 101
mazadanica, Parviliicina 26: 119
meekiana, Gimdlachia 26: 149
megastropha, Stroplwcardia 26: 124
mercenaria, Mercenaria 26: 101
Minnivola 26: 39
rnodesta, Transennella 26:
mnlticostata, Periglypta 26:
midticostata, Tiicetona 26:
inultispinosus, Pitar 26:
mimita, Philobrya 26:

Murex 26: 88

murrayi, Cirrothaiima 26: 10
miitica, Pterotrachea 26: 86
Myidae 26: 39, 91
Myoida 26: 96
mystica, Cryptoplax 26: 91
Mytilidae 26: 36, 119
Mytiloidea 26: 35
nasiita, Corbida 26: 119
Nautilus 26: 9, 92
neglectus, Onitliochiton 26: 1
nemoralis, Cepaea 26: 48, 158
Neogastropoda 26: 2

Neotaenioglossa 26: 173
Neritilidae 26: 91
Neritiliidae 26: 67
ueritoides, Latia 26: 48
Neritopsina 26: 91
noae, Area 26: 6, 37
Noetiidae 26: 129
notabilis, Anadava 26: 6, 42
novemcostatus, Trigonulina 26: 131
Nuculana 26: 125
Nuculanidae 26: 123
Nuculidae 26: 129
mix, Anadara 26: 124
obesa, Anadara 26: 124
oblongum, Cardium 26: 39
obovalis, Trigoniocardia 26: 130
obovata, Lithasia 26: 137
obsoleta, Ilyanassa 26: 86
Octobrachia 26: 92
Octopus 26: 88
Oegopsida 26: 133
officinalis. Sepia 26: 10
Olivancillaria 26: 69
Olividae 26: 69
olssoni, Sheldonella 26: 124
Onchidiidae 26: 92
Onchidium 26: 92
Opisthobranchia 26: 1 1
orcutti, Fartulum 26: 91
Ostrea 26: 36
Ostreidae 26: 89
Ostreoidea 26: 35
ovalis, Novisuccinea 26: 112
pacifica. Area 26: 125
paciftca, Crassinclla 26: 119
pacifica, Lcachia 26: 135
pacifica, Tellina 26: 124
pallida, Miilinia 26: 124
pallida, Seinele 26: 131
paliistris, Lymnaea 26: 149
Pandoridae 26: 94, 131
Pandoroidea 26: 42, 91
parallela, Ferrissia 26: 149
parallelus, Ferrissia 26: 149
parva, Ainiiicola 26: 149
parva, Lymnaea 26: 149
parvus, Gy rai lilts 26: 146
Patella 26: 88
Patellidae 26: 91
Patellogastropoda 26: 1 1

Pecten 26: 13

Pectinidae 26: 1, 7, 35, 93, 101, 125
Pectinoidea 26: 27, 35, 91
pectimciiloides, Bathyarca 26: 41
Pectimculus 26: 92
pencillata, Plicatula 26: 124
Penicillidae 26: 35
peregra, Radix 26: 48
pertioinus, Cyclopecteii 26: 119
peroiii, Atlanta 26: 86
perparvula, Divalinga 26: 130
peslutre, Pseudamiissiuin 26: 83
pespelicani, Aporrliais 26: 92
pliaraonis. Sepia 26: 1 1
Philobrya 26: 37, 92
Physidae 26: 145, 173
pisana, Fiipariplia 26: 60
pisana, Helix 26: 60
Pisulina 26: 67
Placopecten 26: 27, 39
Placophora 26: 2, 7, 87
Planorbidae 26: 145, 173
platysayoides, Triodopsis 26: 153
Pleiirobranclius 26: 88
Pleuroceridae 26: 141, 173
pleiiroiiectes, Amusiiim 26: 39
Plicatulidae 26: 125
politiis, Tagelus 26: 124
Polyplacophora 26: 2, 7, 96
poniatia, Helix 26: 55
Pomatiopsidae 26: 141
poiiderosa, Dosiiiia 26: 130
praerosa, Aticulosa 26: 149
praerosa, Leptoxis 26: 143
pristipliora, Tellina 26: 124
proceriim, Trachycardium 26: 124
prolongata, Liiciiia 26: 124
Propeammusiidae 26: 129
Protobranchia 26: 89
Pseudamiissiuin 26: 83
pseiidoflavus, Umax 26: 57
Pteriidae 26: 37, 129
Pterioida 26: 106
Pterioidea 26: 35
Pteriomorpha 26: 89
Pteriomorphia 26: 39
pulicaria, Chione 26: 124
pulicaria, Cliionopsis 26: 130
punctata, Aplysia 26: 92
piisio, Chlaniys 26: 93

183

pusio, Pecten 26: 38
piitris, Sucdnea 26: 53
mdiata, Lawpsilis 26: 166
recognitus, Isognomon 26: 124
reeveana, Barbatia 26: 124
reflexa, Lymnaea 26: 149
reticulatnm, Deroceras 26: 57
reticulatus, Agrioliinax 26: 49
reticulatiis, Nassariiis 26:

Retusa 26: 67
Retusidae 26: 67
vivularis, Ferrissia 26: 173
nibida, Chhvnys 26: 27, 101
rufus, Arion 26: 52
sayii, Physella 26: 149
scabra, Lima 26: 38, 93, 101
scnlarifornus, Cerithiden 26: 92
Scaphandridae 26: 67
Scaphopoda 26: 89
sciwnki, Nucula 26: 124
scolopes, Enprymna 26: 11, 75
Semelidae 26: 131
semicarinata, Elirnia 26: 141
senticosum, Trachycardium 26: 124
siliqna, Macoma 26: 124
sincera, Valvata 26: 139
singleyamis, HeUcodiscus 26: 48
sintoxia, Pleiirobema 26: 161
Smaragdia 26: 88
SolecLirtidae 26: 131
Solenogastres 26: 89
solidissima, Spisida 26: 11, 35, 101
sordida, Chnma 26: 125
soror, Diplodonta 26: 130
Spondylidae 26: 8, 42, 93

Spondyhis 26: 39
squalida, Megapitaria 26: 123
squamosa, Lima 26: 93
stagnalis, Lymnaea 26: 48, 143
sterna, Pteria 26: 125
striata, Dyakia 26: 60
striata, Quantula 26: 60
strigilata, Tucetona 26: 129
St rigid a 26: 124
Strombus 26: 13, 88
Strophochelius 26: 52
subalata, Mactrellona 26: 124
subglobosiis, Birgella 26: 140
subglobosus, Somatogyriis 26: 149
suborbicularis, Kellia 26: 124
subpiirpureus, Valvata 26: 139
subpurpureus, Viviparus 26: 173
subquadrata, Cyclinella 26: 130
subqiiadrata, Diplodonta 26: 130
sidnotunda, Obovaria 26: 161
tantilla, Nutricola 26: 41
Taoniinae 26: 133
tarda, Ferrisia 26: 149
Tellinidae 26: 121
Testaria 26: 90
Thyasiridae 26: 130
thyroidus, Mesodon 26: 112
tricarinata, Valvata 26: 139
Tridacna 26: 93, 101
Tridacnidae 26: 39
Tridacninae 26: 94
Tridacnoidea 26: 35
tridentata, Triodopsis 26: 155
triquetra, Epioblasma 26: 161
trivolvis, Helisoma 26: 149

trivolvis, Planorbella 26: 147, 173
Trochacea 26: 67
truncata, Latermda 26: 40
Tryblidia 26: 89
undulata, Cymatoica 26: 130
undulatus, Strophitus 26: 165
Ungulinidae 26: 130
Valvatidae 26: 139
varia, Chlamys 26: 83
varians, Crassinella 26: 124
velero, Leptopecten 26: 129
Veneridae 26: 121
Veneroida 26: 11, 95
ventricosa, Corbula 26: 131
ventricosiis, Argopecten 26: 125
Venus 26: 102
verrucosa, Seniele 26: 124
verruculatum, Onchidium 26: 51, 93,
103

Verticordiidae 26: 131
Vetigastropoda 26: 88
vincta, Lacuna 26: 91
virescens, Barbatia 26: 37
virginica, Crassostrea 26: 36
Visceroconcha 26: 92
vittatus, Donax 26: 36
Viviparidae 26: 139, 173
viviparous, Viviparus 26: 91
vulgaris. Octopus 26: 11, 43
walked, Physella 26: 149
yessoensis, Patinopecten 26: 93
zebra. Area 26: 6
ziczac, Euvola 26: 107
ziczac, Pecten 26: 107

184

THE AMERICAN MALACOLOGICAL SOCIETY

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Tunison Laboratory of Aquatic Science
3075 Gracie Rd„ Cortland, NY 13045-9357, U.S.A.

Application for new membership in 2009 calendar year: page 1

Please complete both pages of this form and mail it with your dues payment to the treasurer at the address above.

First Name / Middle Initial / Last Name

1. CONTACT OPTIONS

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[H Regular Member - One year dues (2009) S 60.00

D Regular Member - Two years (2009 & 2010) $105.00

n Regular Member - Three years (2009-2011) $145.00

D Each additional family member, per year $ 1.00

D Student Member - One year $ 20.00

D Sustaining Member - Regular dues plus $25.00 $ 85.00

D Affiliate Membership (Shell Clubs & other organizations) $ 60.00

n Membership reinstatement/back issues @ $60 Regular / $20 Student for 2008 (Volumes 24 & 25) $ .

Members outside the US please note that there is a postage and handling fee for the Bulletin.

3. POSTAGE D Canada & Mexico $5.00 per year CH All other non-US addresses $10.00 per year $ .

4. TAX-DEDUCTIBLE GIFT

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TOTAL ENCLOSED: $ . _

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THE AMERICAN MALACOLOGICAL SOCIETY

Application for new membership in 2009 calendar year
Page 2: address and contact details

Please fill in this form and mail it to the treasurer together with the first page. This information will be
included in the annual AMS membership directory. For options, see under [1] on page I

Title (Dr, Mr, Ms, etc)

Name (First/Initial/Last)

Address*

Department
Hall or box #

Institution
Street or PO Box
City

State or Province
Postal/ZIP code
Country
Office phone
Home phone
Cell phone
Fax

E-mail^

Interests^

For official use only

Date r’cd
Paid to
Comments

' Members may provide only a single address, which will be published in the AMS membership directory. Students are advised
to give the address of their institution, to facilitate mail forwarding.

^ Please give a work or institutional e-mail address where possible

^ Please provide some key words outlining your special interests within Malacology. You may also give the URL of your web
site(s) here.

INFORMATION FOR CONTRIBUTORS

Scope. The American Malacological Bulletin is the scientific
publication of the American Malacological Society and
serves as an outlet for reporting notable contributions in
malacological research. Manuscripts concerning any aspect
of original, unpublished research, important short reports,
and detailed reviews dealing with molluscs will be consid-
ered for publication.

Format. Manuscripts and illustrations should be submitted
electronically (in a Word document or pdf file with embed-
ded figures). Text must be typed in 12 pt font on 8.5 x 11
inch (letter-sized) paper, double-spaced, with all pages
numbered consecutively. Leave ample margins on all sides,
and left-justify the text. Final submission of accepted,
revised manuscripts must include the text, tables, etc. as a man-
datory MS Word file on a CD, DVD, or e-mail attach-
ment, along with high resolution EPS or TIFF files of all figures.
Authors should make sure all figures have at least 350 dpi
resolution, and check figure files for acceptability at Sheridan’s
web site (dx.sheridan.com). Please follow current instructions
for authors given at the AMS website or at the back of recent
issues of the Bulletin.

Sections. Text, when appropriate, should be arranged in
sections as follows:

1. Cover page with title, author(s), address(es), e-mail ad-
dress (of corresponding author), and suggested running
title of no more than 50 characters and spaces. Authors
should also supply 5 key words or short phrases, placed at
the base of this page, for indexing purposes. Key words or
phrases should not duplicate terms already in title.

2. Abstract (less than 5% of manuscript length)

3. Text of manuscript starting with a brief introduction fol-
lowed by methodology, results, and discussion.

• Separate main sections of text with centered titles in
bold face, capital letters.

• Subheadings ( D' level) should be left-justified and bold
face; capitalize first letter.

• Subheadings (2"*'^ level) should be in italics with no
bold face. Please refer to example below:

MATERIALS AND METHODS
Taxonomy
Animals

Cultured animals
Wild ani?nals

Behavioral observations

RESULTS

4. Acknowledgments

5. Literature cited

6. Figure legends (together)

7. Tables (each on a separate sheet, headed by a brief
legend)

Taxonomic Authorities. All binomens, excluding non-
molluscan taxa, must include the author and date attrib-
uted to that taxon the first time the name appears in the

manuscript, such as Crassostrea virginica (Gmelin, 1791). A
comma is rec]uired between the authority and date. The full
generic name along with specific epithet should be written
out the first time that taxon is referred to in each paragraph.
The generic name can be abbreviated in the remainder of the
paragraph as follows: C. virgmica. The taxonomic authorities
of generic names must be provided if species names are not
included. Please refer to recent issues for examples.

Text References. Literature citations should be cited within
text as follows: Hillis (1989) or (Hillis 1989). Dual author-
ship should be cited as follows: Yonge and Thompson ( 1976)
or (Yonge and Thompson 1976). Multiple authors of a single
article should be cited as follows: Beattie et al. (1980) or
(Beattie et al. 1980).

Literature Cited Section. References must also be typed
double-spaced. All authors must be fully identified and listed
alphabetically; journal titles must be unabbreviated. When
more than one publication with the same first author is
cited, please arrange citations as follows: (a) single author,
according to publication dates; (b) same author and one
co-author in alphabetical, then chronological order; (c)
same author and more than one co-author.

Citation Format.

Beattie, J. H., K. K. Chew, and W. K. Hershberger. 1980.
Differential survival of selected strains of Pacific oysters
(Crassostrea gigas) during summer mortality. Proceedings
of the National Sliellftsheries Association 70: 184-189.
Hillis, D. M. 1989. Genetic consec]uences of partial self fer-
tilization on populations of Liguus fasciatiis (Mollusca:
Pulmonata: Bulimulidae). American Malacological Bulle-
tin 7: 7-11.

Seed, R. 1980. Shell growth and form in the Bivalvia. In: D. C.
Rhoads and R. A. Lutz, eds.. Skeletal Growth of Aquatic
Organisms. Plenum Press, New York, New York. Pp. 23-67.
Yonge, C. M. and T. E. Thompson. 1976. Living Marine
Molluscs. William Collins Son and Co., Ltd., London.
For more detailed examples of journal series, supplements,
graduate theses, governmental reports, Internet citations,
non-English citations, or other categories of references,
please check the following AMS web page under “Submission”:
http://malacological.org/publications/authors.html

Resources. The manuscript should follow the style rules out-
lined in The CSE Matiual for Authors, Editors, and Publishers
(7‘^ edition, June 2006). This can be purchased from the
CSE at 12100 Sunset Hills Rd., Suite 130, Reston, Virginia
20190, USA or at the following web site: http://www.
councilscienceeditors.org/publications/style.cfm. Spelling
should follow American English as listed in Merriam-
Webster Online (http://www.m-w.com/) or recent hardcopy
editions.

Punctuation. Punctuation should follow that shown in the
most recent issues of AMR. Specific common examples include:

• Italics: e.g., i.e., sensu, per se, et al.

• Non-italicized text: pers. comm., pers. obs., and unpubl. data

• Font: please use Times New Roman 12 (if possible).

Figures. Authors are strongly encouraged to submit elec-
tronic copies of the figures on CDs or DVDs (hardcopy
submissions may incur additional costs). Illustrations should
be clearly detailed and readily reproducible. Fine patterns
and screens do not reproduce well. All figure panels must be
marked with capitalized letters (A, B, C, etc . . .) and ad-
ec]uately labeled with sufficiently large labels to remain read-
able with reduction by one half Magnification bars must
appear on the figure (except for graphs), or the caption must
read horizontal field width = x mm or x pm. All measure-
ments must be in metric units. When creating figures, use
font sizes and line weights that will reproduce clearly and
accurately when figures are sized to the appropriate column
width. Please do not include figure legends in a graphic
file. Explanations of abbreviations used in a figure should
occur in the legend. Please see recent Journal issues for cor-
rect format. Indicate in text margins the appropriate loca-
tion in which figures should appear. Color illustrations can
be included at extra cost to the author (currently about
$650-700/figure).

Figure Format. For final revisions of papers submitted to
the editor, all electronic figure files should be in TIFF or EPS
formats. Each individual figure or graphic must be supplied
as a separate, stand-alone file (not as an embedded object).
Figure files must be named with their respective numbers
and graphic type such as Figl.tif, Figure2.eps, etc.

Figure Resolution. AMB c]uality reproduction will rec]uire
grayscale and color files at resolutions yielding approxi-
mately 350 dpi. Bitmapped line art should be submitted at
resolutions yielding 600-1200 dpi. These resolutions refer
to the output size of the file (85 mm for single column or
170 mm for double column); if you anticipate that your
images will be enlarged or reduced, resolutions should be
adjusted accordingly. Please check your output resolu-
tion prior to manuscript submission. Directions on how to
check are available at the following AMS web page under
“Submission”: http://malacological.org/publications/authors.
html. The production of high-resolution figures is the re-
sponsibility of the author(s).

Mandatory AMB Style. Any manuscript not conforming to
AMB format will be returned to the author for revision
before publication. Manuscripts are accepted contingent
upon authors making final AMB stylistic revisions.

New Taxa. The Bulletin welcomes complete descriptions of
new molluscan taxa. Establishment of new taxa must con-
form with the International Code of Zoological Nomencla-
ture (1999). Descriptions of new species-level taxa must in-
clude the following information in the order as given: higher
taxon designation as needed for clarity; family name with
author and date; generic name with author and date; Genus
species author sp. nov. followed by numeration of all figures

and tables; complete synonymy (if any); listing of type
material with holotype and any other type material clearly
designated along with complete museum catalogue or acces-
sion information; listing of all additional non-type material
also with full museum deposition information; type locality;
diagnosis and full description of material done in telegraphic
style including measurements and zoogeographic distribu-
tion as necessary; discussion; etymology. Descriptions of
new supraspecific taxa should include type species (for new
genus) or type genus (for new family), diagnosis and full
description done in telegraphic style, and list of included
taxa.

Proofs. Page proofs will be sent to the author and must be
checked for printer’s errors and returned to the Managing
Editor within a 1-week period. Changes in text, other than
printer errors, will result in publishing charges that will be
billed to the author.

Charges. There are no mandatory page costs to authors
lacking financial support. Authors with institutional, grant,
or other research support will be billed for page charges. The
current rate is $50 per printed page. Acceptance and ulti-
mate publication is in no way based on ability to pay page
costs. However, changes at the proof stage are mandatory
costs set by the publisher.

Reprints. Order forms and reprint cost information will be
sent with page proofs. The author receiving the order form
is responsible for insuring that orders for any co-authors are
also placed at that time. Electronic e-reprints (with unlim-
ited distribution) are available from the Managing Editor;
the current rate is $20/article.

Submission. Submit all manuscripts to: Dr. Kenneth M.
Brown, Editor-In-Chief, Department of Biological Sciences,
Louisiana State University, Baton Rouge, Louisiana 70803,
USA. Please refer to the AMS web page for more detailed
information prior to submission, http://malacological.org/
publications/authors.html

Subscription Costs. Institutional subscriptions are available
at a cost of $75 per volume. Membership in the American
Malacological Society, which includes personal subscriptions
to the Bulletin, is available for $60 ($20 for students, $60 for
affiliated clubs). All prices quoted are in U.S. funds. Outside
the U.S. postal zones, add $5 airmail per volume (within
North America) or $10 airmail per volume (other locations).
For membership information contact Dr. Dawn Dittman,
Treasurer, Tunison Laboratory of Aquatic Science, 3075
Gracie Rd., Cortland, New York 13045-9357, USA. For in-
stitutional subscription and back-issue information contact
Dr. Kenneth M. Brown, Editor-In-Chief, Department of Bio-
logical Sciences, Louisiana State University, Baton Rouge,
Louisiana 70803, USA. Complete information is also avail-
able at the AMS website: http://www.malacological.org.

The American Malacological Society

The 75th meeting of the American Malacological Society will
be held in Ithaca, New York from July 19th through July 23rd,
2009. The meeting will be held at Cornell University.

Special events will be held all around Ithaca, including an
opening reception at the Museum of the Earth at the
Paleontological Research Institution, a dinner cruise on scenic
Cayuga Lake, the AMS auction on Tuesday at the Holiday Inn
in Downtown Ithaca, and a banquet at the Museum of the
Earth on Wednesday evening.

The large recent and fossil mollusk collections of PRI will be
open throughout the week for scientific work. Please visit the
Collections website at www.pricollectionsdatabase.org.

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Soxciiety y/ytlhi Ainuniuaf
Meeitiuni'g^

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The American Malacological Society

75th Annual Meeting
July 19th - July 23rd, 2009
Ithaca, New York

This meeting's field trips will familiarize meeting participants with
Ithaca's aquatic habitats, both ancient and modern. Chose between
exploring the Devonian marine fossils of the Ithaca Region with
Warren Allmon and the freshwater and land mollusks of the Cayuga
Lake Basin with Eileen Jokinen.

Participants flying to Ithaca can chose among the Ithaca,
Binghamton, Syracuse, or Rochester airports. Binghamton and
Syracuse are both approximately an hour by car from Ithaca, and
Rochester is two hours away. Housing is available either at the
Holiday Inn Downtown, or on campus in residence halls at Cornell
University. Transportation will be provided to all events from each
location.

For registration and abstract submission information, please visit
www.malacological.org

We look forward to seeing you in Ithaca in 2009!

For more information, please contact:

Kelly Cronin
PRI

1259 Trumansburg Rd
Ithaca, Ny 14850

E-mail: cronin@museumoftheearth.org
Phone: 607-273-6623 ext. 10

The American Malacological Society

75th Annual Meeting
July 19th - July 23rd, 2009
Ithaca, New York

Symposium on Speciation in Mollusks

This multi-disciplinary symposium aims to bring together
researchers working on molluscan speciation in a variety of taxa, at
a variety of temporal and geographic scales, and with a variety of
techniques — from fossils to genes.

Confirmed speakers include Warren Allmon (Paleontological
Research Institution, Ithaca, NY), Robert Dillon (College of
Charleston, Charleston, SC), Matthias Glaubrecht (Humboldt
University, Berlin), Patrick Krug (California State University, Los
Angeles), Peter Marko (Clemson University, Clemson, SC), Elinor
Michel (The Natural History Museum, London), Paula Mikkelsen
(Paleontological Research Institution, Ithaca, NY), Rebecca Rundell
(University of British Columbia, Vancouver), and Ursula Smith
(Cornell University, Ithaca, NY).

Please visit the meeting website at www.malacological.org for more
information on symposium topics, accommodations, and things to do
in and around Ithaca.

SMITHSONIAN INSTITUTION LIBRARIES

3 9088 01448 2160

Photoreception and the polyphyletic evolution of photoreceptors (with special reference

to Mollusca). LUITFRIED VON SALVINI-PLAWEN

Primary inhibition by light: A unique property of bivalve photoreceptors. LON A. WILKENS

When a snail dies in the forest, how long will the shell persist? Effect of dissolution

and micro-bioerosion. TIMOTHY A. PEARCE

Bivalve molluscs from the continental shelf of lalisco and Colima, Mexican Central Pacific.

EDUARDO RIOS-JARA, ERNESTO LOPEZ-URIARTE, and

CRISTIAN M. GALVAN-VILLA

A mature female of Bathothanma Chun, 1906 (Cephalopoda: Cranchiidae) from Hawaii.

JANET R. VOIGHT

Conservation of the freshwater gastropods of Inciiana: Historic and current distributions.

MARK PYRON, JAYSON BEUGLY, ERIKA MARTIN, and MATTHEW SPILLMAN

The feeding behavior and diet of an endemic West Virginia land snail, Triodopsis platysay aides.

DANIEL C. DOURSON

Structural community changes in freshwater mussel populations of Little Mahoning

Creek, Pennsylvania. ERIC J. CHAPMAN and TAMARA A. SMITH

Diversity and distribution of freshwater gastropods in the Bayou Bartholomew drainage,
Arkansas, U.S.A. RUSSELL L. MINTON, JOHN D. WHITE, DAVID M. HAYES,

M. SEAN CHENOWETH, and ANNA M. HILL

Research Note: Conus lightboiirni holotype returned to the Delaware Museum of Natural
History. ELIZABETH K. SHEA and WILLIAM J. FENZAN

Index to Vol. 26

Membership Form for 2009

Information for Contributors

Meeting Announcement

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