Malacologia

J. A. ALLEN

Marine Biological Station
Millport, United Kingdom
jallen @udcf.gla.ac.uk

E. E. BINDER
Museum d’Histoire Naturelle
Geneve, Switzerland

P. BOUCHET

Museum National d'Histoire Naturelle
Paris, France

bouchet @cimrs1.mnhn.fr

P. CALOW
University of Sheffield
United Kingdom

R. CAMERON

Sheffield

United Kingdom

R.Cameron @sheffield.ac.uk

J. G. CARTER
University of North Carolina
Chapel Hill, U.S.A.

MARYVONNE CHARRIER

Universite de Rennes

France
Maryvonne.Charrier@univ-rennes1.fr

В. H. COWIE
University of Hawaii
Honolulu, HI., U.S.A.

A. H. CLARKE, Jr.
Portland, Texas, U.S.A.

B. C. CLARKE
University of Nottingham
United Kingdom

R. DILLON
College of Charleston
SC, U.S.A.

C. J. DUNCAN
University of Liverpool
United Kingdom

D. J. EERNISSE
California State University
Fullerton, U.S.A.

E. GITTENBERGER

Rijksmuseum van Natuurlijke Historie
Leiden, Netherlands

sbu2eg @rulsfb.leidenuniv.de

F. GIUSTI
Universita di Siena, Italy
giustif @ unisi.it

2001
EDITORIAL BOARD

A. N. GOLIKOV
Zoological Institute
St. Petersburg, Russia

S. J. GOULD
Harvard University
Cambridge, Mass., U.S.A.

A. V. GROSSU
Universitatea Bucuresti
Romania

T. HABE
Tokai University
Shimizu, Japan

R. HANLON
Marine Biological Laboratory
Woods Hole, Mass., U.S.A.

G. HASZPRUNAR

Zoologische Staatssammlung Muenchen

Muenchen, Germany

haszi @zi.biologie. uni-muenchen.de

J. M. HEALY

University of Queensland
Australia

jhealy O zoology.uq.edu.au

D. M. HILLIS
University of Texas
Austin, U.S.A.

K. E. HOAGLAND

MCZ
LIBRARY

FEB 14 2002

Council for Undergraduate Research

Washington, DC, U.S.A.
Elaine @cur.org

B. HUBENDICK
Naturhistoriska Museet
Goteborg, Sweden

S. HUNT
Lancashire
United Kingdom

R. JANSSEN
Forschungsinstitut Senckenberg,
Frankfurt am Main, Germany

M. S. JOHNSON

University of Western Australia
Nedlands, WA, Australia

msj @cyllene.uwa.edu.au

R. N. KILBURN
Natal Museum
Pietermaritzburg, South Africa

M. A. KLAPPENBACH

HARVARD

UNIVERSITY

Museo Nacional de Historia Natural

Montevideo, Uruguay

J. KNUDSEN
Zoologisk Institut Museum
Kobenhavn, Denmark

C. LYDEARD

University of Alabama
Tuscaloosa, U.S.A.
clydeard@biology.as.ua.edu

C. MEIER-BROOK
Tropenmedizinisches Institut
Tubingen, Germany

Н. К. MIENIS
Hebrew University of Jerusalem
Israel

J. E. MORTON
The University
Auckland, New Zealand

J. J. MURRAY, Jr.
University of Virginia
Charlottesville, U.S.A.

R. NATARAJAN
Marine Biological Station
Porto Novo, India

DIARMAID O’FOIGHIL
University of Michigan
Ann Arbor, U.S.A.

J. OKLAND
University of Oslo
Norway

T. OKUTANI
University of Fisheries
Tokyo, Japan

W. L. PARAENSE
Instituto Oswaldo Cruz, Rio de Janeiro
Brazil

J. J. PARODIZ
Carnegie Museum
Pittsburgh, U.S.A.

R. PIPE

Plymouth Marine Laboratory
Devon, United Kingdom
RKPI@wpo.nerc.ac.uk

J. P. POINTIER

Ecole Pratique des Hautes Etudes
Perpignan Cedex, France
pointier @ gala.univ-perp.fr

М.Е. PONDER
Australian Museum
Sydney

QIFZ. У.
Academia Sinica
Qingdao, People’s Republic of China

D. G. REID
The Natural History Museum
London, United Kingdom

$. G. SEGERSTRALE
Institute of Marine Research
Helsinki, Finland

A. STANCZYKOWSKA
Siedice, Poland

F. STARMUHLNER
Zoologisches Institut der Universitat
Wien, Austria

Y. |. STAROBOGATOV
Zoological Institute
St. Petersburg, Russia

J. STUARDO
Universidad de Chile
Valparaiso

C. THIRIOT

University P. et M. Curie
Villefranche-sur-Mer, France
thiriot @ obs-vifr.fr

S. TILLIER
Museum National d'Histoire Naturelle
Paris, France

J.A.M. VAN DEN BIGGELAAR
University of Utrecht
The Netherlands

N. Н. VERDONK
Rijksuniversiteit
Utrecht, Netherlands

H. WÄGELE

Ruhr-Universität Bochum

Germany

Heike. Waegele @ruhr-uni-bochum.de

ANDERS WAREN
Swedish Museum of Natural History
Stockholm, Sweden

B. R. WILSON
Dept. Conservation and Land Management
Kallaroo, Western Australia

H. ZEISSLER
Leipzig, Germany

A. ZILCH
Forschungsinstitut Senckenberg
Frankfurt am Main, Germany

MALACOLOGIA, 2002, 44(1): 1-15

SMALL-SCALE MUSSEL SETTLEMENT PATTERNS WITHIN MORPHOLOGICALLY
DISTINCT SUBSTRATA AT NINETY MILE BEACH, NORTHERN NEW ZEALAND

Andrea С. Alfaro! & Andrew G. Jeffs?

ABSTRACT

Microscale settlement patterns of juveniles of the mussel Perna canaliculus were investigated
within drift material at Ninety Mile Beach, northern New Zealand. Size- and site-specific selec-
tivity on various morphologically distinct algal and hydroid species were identified within drift ma-
terial and corroborated in laboratory experiments with similar artificial substrata. Mussel spat
densities were greater within fine-branching natural (-28-57%) and artificial materials
(-13-20%) compared to medium- and coarse-branching natural (-7-8%) and artificial
(-2-3%) materials. Size-frequency distributions of mussel spat within natural and artificial ma-
terials suggested a relationship of increasing mussel size with decreased branching of substrata.
Field and laboratory investigations indicated higher settlement of 1.5-2.0 mm mussel size
classes in coarse-branching substrata, whereas fine-branching substrata had greater settlement
of mussels within the <0.5 mm size class. Mussel settlement comparisons within node and inter-
node areas of all substrata in the field and in the laboratory indicated a strong preference of set-
tlement in node areas over inter-node areas. The microscale settlement patterns observed in this
study are argued to be indicative of a life strategy to maximize juvenile mussel survival within the
dynamic environment of drift material in oceanic currents, before the potential arrival and re-set-
tlement to rocky coastal areas. The present study is the first to elucidate settlement patterns of
Perna canaliculus on drift material that washes up on Ninety Mile Beach, where >70 tonnes/year
of this material is collected and supplied to the New Zealand aquaculture industry to seed mus-
sel farms.

Key words: mussels, small-scale settlement patterns, micro-habitat selection, size-frequency

distribution, drift algae.

INTRODUCTION

The complexity and diversity of planktonic
larval settlement patterns has received a
great deal of attention within various temporal
and spatial scales (Butman & Grassle, 1992;
Bourget & Harvey, 1998). Physical and bio-
logical factors have been investigated as po-
tential contributors to passive and active set-
tlement outcomes (Butman, 1987; Butman &
Grassle, 1992; Grassle et al., 1992). For ma-
rine invertebrates, such as mussels, larval
settlement on filamentous substrata repre-
sents an important intermediate step to the
eventual recruitment to the adult population
(Bayne, 1964; Davies, 1974; Highsmith,
1985; King et al., 1990; Harvey et al., 1993;
Hunt & Scheibling, 1996). However, the dy-
namics of these interactions are not well un-
derstood for most broadcast spawners
(Dame, 1996).

Bayne (1964) first demonstrated that larvae
of the mussel Mytilus edulis tended to settle
on filamentous algae (primary settlement) and
later moved to established adult mussel beds
as secondary settlers (1-2 mm) through a
bysso-pelagic phase. Bysso-pelagic drifting
has since been shown to be a common
means of dispersal among pelagic larvae and
juveniles (Lane et al., 1985; Martel, 1993;
Martel et al., 1994; Buchanan & Babcock,
1997). Buchanan & Babcock (1997) also
found a size-specific settlement pattern for
the New Zealand green-lipped mussel Perna
canaliculus on various intertidal algae, hy-
droids, and adult mussel beds at Piha Beach,
North Island, New Zealand. In their study,
Buchanan & Babcock (1997) observed that
mussels <0.5 mm (primary settlers) were
more abundant in attached filamentous algae
and a hydroid species, whereas secondary
settlers (>0.5 mm) were numerous in coarser-

"Corresponding author. School of Environmental and Marine Sciences, The University of Auckland, Auckland, New Zealand;

a.alfaro@auckland.ac.nz

“National Institute of Water 8 Atmospheric Research Ltd., P. O. Box 109695, Newmarket, Auckland, New Zealand;

a.jeffs O niwa.cri.nz

2 ALFARO & JEFFS

branching algae and the adjacent intertidal
mussel bed. Elsewhere in northern New
Zealand, at Ninety Mile Beach (NMB), early
juveniles or spat of Perna canaliculus also are
found associated with floating algae and hy-
droids. Large quantities (>70 t/y) of spat, at-
tached to this drift material, are used to seed
the extensive mussel farms in New Zealand,
after collection from the surf zone (Jeffs et al.,
2000). Nowhere else in New Zealand but
along the 90 km stretch of NMB, are such vast
amounts of wild spat concentrated in one
place on detached material that can be con-
veniently and economically harvested to sup-
ply the mussel industry (Jeffs et al., 2000).
Distinctive patches of filamentous substrata
that arrive at the beach (wash-up events) may
vary in substratum abundance and density,
and spat size-frequency distribution (Hick-
man, 1982). A single wash-up at NMB may
contain up to 70 tonnes of substratum and
spat material (C. Hensley, personal communi-
cation). These filamentous substrata may de-
rive from intertidal to deep subtidal regions in
particular areas along NMB, and mussel set-
tlement onto these substrata may take place
before or after detachment of the alga or hy-
droid. Regardless of the timing of settlement,
algal and hydroid materials appear to be es-
sential to the transport of mussel spat, as they
may not only provide a means for long-
distance dispersal, but also an alternative
habitat to maintain or increase physiological
processes (Highsmith, 1985; Schneider &
Mann, 1991a). If a size-specific mussel settle-
ment pattern occurs on the filamentous sub-
strata found within a wash-up event at NMB, it
is likely that substratum selection is the pre-
dominant force behind mussel settlement pat-
terns, rather than random physical encoun-
ters along this particularly high energy portion
of the New Zealand coastline. The persis-
tence of size-specific settlement patterns of
mussels on drift material, while exposed to
the energetic hydrodynamic regimes often en-
countered in the ocean, suggests a complex
life history strategy. The aim of the present
work is to elucidate microscale (cm) settle-
ment patterns on the substrata found in wash-
up events that occur along the 90 km length of
NMB. Thus, characterization of substratum
types, and size- and site- specific settlement
patterns upon various drift substrata, may
provide a first step to understanding spat-sub-
stratum interactions at various spatial and
temporal scales.

METHODS AND MATERIALS
Study Site and Terminology

Samples of Perna canaliculus spat and as-
sociated substrata were collected from the
surf zone at various locations along Ninety
Mile Beach (NMB), Northland, New Zealand
(Fig. 1). Each sample was collected from dis-
tinct wash-up events on 17 October 1998, 22
December 1998, and 16 May 1999. Wash-up
samples were collected by net from the surf
along the beach. Once collected, all samples
were immediately frozen for later laboratory
analyses at the University of Auckland, Auck-
land, New Zealand.

Recruitment is an operational word that
does reflect a unique and specific biological
event (Hunt & Scheibling, 1997). There is no
standard size nor age at which mussels are
defined to be settling or recruiting. Bayne
(1964) suggests that primary settlers are
mussels <0.5 mm in shell length, and sec-
ondary settlers are mussels between 0.5-2.0
mm in shell length. Because mussels can,
and often do re-settle even at shell lengths of
up to 25 mm, it is difficult to suggest a cut-off
line between settlement and recruitment. The
mussels that were found attached to algae
and hydroids in our experiments are likely to
have used the drifting substrata as an inter-
mediate settlement step before recruiting to
the adult mussel beds in suitable rocky sub-
strata. We use the term settlement in this
study, because the majority of the mussels we
encountered in the drift samples were <5 mm
in shell length.

Natural Substrata

As afirst step to investigate small-scale set-
tlement patterns of mussel spat on various
drifting natural substrata, two 100-g wet
weight subsamples were taken from each of
three wash-up samples. The original wash-up
samples were between 1-5 t (wet weight) of
very homogeneous substratum and mussel
wash-up material. All distinctive algal and hy-
droid substrata within each subsample were
separated and grouped into four morphologi-
cally distinctive categories, with algal cate-
gories based on the level or degree of branch-
ing. The categories were: coarse-branching
algae (Osmundaria colensoi, Carpophyllum
angustifolium, and Rhodymenia dichotama);
medium-branching algae (Melanthalia ab-

SMALL-SCALE MUSSEL SETTLEMENT 3

Cape Reinga

NEW ZEALAND

Great Exhibition
Bay

Cape Karikari

„Kaitaia

Ahipara

Tonatona Beach

173° 00 E

FIG. 1. Location of study site at Ninety Mile Beach, Northland, New Zealand. Rocky intertidal areas are found
at Tonatona Beach, The Bluff, and Scott Point. Spatfall collection sites on 17 October 1998, 22 December
1998, and 16 May 1999 are marked (x) along the beach.

scissa, Laurencia thyrsifera, Pterocladia lu-
cida, P. capillacea, Gigartina marginifera, G.
alveata, and Pachymenia lusoria); fine-
branching algae (Champia laingii, Plocamium
costatum, Haliptilon roseum, and Corallina of-
ficinalis); and hydroids (Amphisbetia bispi-
nosa, Dictyocladium moniliferum, Craterit-
heca insignis, Aglaophenia acanthocarpa,

and Lytocarpia incisa). The distribution of
these algae and hydroids varies from below
the low water mark of spring tides to deep
subtidal, with the exception of Gigartina sp.
and Pachymenia lusoria, which also can be
found in the low intertidal zone (Morton &
Miller, 1968; Adams, 1994). All algal species
found in the samples were red algae, except

4 ALFARO & JEFFS

Carpophyllum angustifolium, which is a brown
alga (Adams, 1994) and constituted <5% of
each sample. The remaining material (shells,
wood fragments, and other debris) did not
contain attached mussels and was discarded.
Mussels within each algal and hydroid sub-
stratum type were removed by vigorous agita-
tion in water, and the removal of remaining
mussels was achieved with forceps
(Buchanan & Babcock, 1997). Mussels within
five different size-classes (<0.49, 0.5-0.99,
1.0-1.49, 1.5-1.99, >2.0 mm in length) were
separated with a set of sieves of appropriate
sizes. Previous research had verified this
technique as an effective means of reliably
sorting mussel material (A. Alfaro, unpub-
lished data). The mussels were then dried to
constant weight in an oven at 80°C. The num-
ber of mussels within each size class was ini-
tially determined by counting under the micro-
scope. The number of dried mussels of each
size class per unit volume was calculated,
and volume subsequently was used to esti-
mate numbers of mussels. Several test sam-
ples were processed initially to ensure the ac-
curacy of the methodology. The surface area
of flattened algal and hydroid samples was
determined from video-computer images
using NIH Image 1.61 software for the Macin-
tosh (Harvey et al., 1993). All algae and hy-
droids were then dried in an oven at 80°C and
weighed. Percent data sets of natural sub-
stratum abundance and mussel densities

(A)

were arcsine transformed to normalize the
data.

An additional 20 samples of ~25 g wet
weight were taken from each wash-up sample
to determine the number of mussels attached
to nodes and internodes of each of the four
types of substrata (Fig. 2a). Anode area was
defined as a 1 cm? area including at least one
axil, and an inter-node area as a 1 cm? area
without an axil (Fig. 2b). Node and internode
areas were randomly chosen from a large set
of suitable areas. All mussels within node and
internode areas on each substratum sample
were removed with forceps and counted. The
exact area occupied by each substratum
within the 1 cm? region was determined from
image analyses using NIH Image 1.61 soft-
ware. The total number of mussels per unit
area of node and internode was calculated for
each sample of algal and hydroid substrata.

Artificial Substrata

In order to further examine the settlement
dynamics of mussel spat onto morphologically
distinct substrata, settling patterns onto artifi-
cial substrata of different shapes were moni-
tored in the laboratory. Five to six 10-cm-long
plastic aquarium plants of six different types
were placed in separate seawater tanks of 1.5
liters. The artificial substrata were standard
aquatic plant replicas and, for consistency,
also were classified as coarse-branching

< Internode (B)

FIG. 2. (A) Diagram depicting node and internode areas on a filamentous substratum. (B) Close-up of node

and inter-node area (1 cm’).

SMALL-SCALE MUSSEL SETTLEMENT 5

(Myriophylium verticillatum, Ceratophyllum
demersum, and Cabomba aquatica), medium-
branching (Elodea densa, Elodea sp., and Ro-
tala indica), and fine-branching (Vallisneria
americana, V. spiralis, and Ceratopteris thalic-
troides) substrata to facilitate comparisons
with the natural substratum experiments. Al-
though the aquatic plants were not identical to
the algae found in the wash-up samples, the
surface area and degree of branching were
very similar to the natural substrata, and the
degrees of branching were distinctive among
the three experimental groups. Mussel spat
were collected from intertidal habitats at NMB,
detached from their original algal substrata,
and placed in a water tank with seawater on
the same day. The density of mussels of dif-
ferent sizes was adjusted to be similar among
size classes. From this mixture, subsamples
of about 500 mussels, representing all sizes
within 0.5 to 3.0 mm in length, were separated
using a standard plankton splitter. The mussel
subsamples were then placed in each of the
50 tanks containing artificial substrata. Each
tank was aerated to ensure constant resus-
pension of unattached mussels. The water
temperature was maintained similar to the col-
lection site at 15°C, and no food was added to
the tanks during these short-term experi-
ments. The light regime was set to mimic out-
door conditions. After two days, mussel abun-
dance, size-frequency distribution, site of

90
80
70
60
50 -
40

30

Available
Substrate Area (%)

20

Coarse-branching
algae algae

Medium-branching

mussel re-settlement, and substratum area
were determined with the procedures outlined
for natural substrata. Percent mussel spat
densities were arcsine transformed to normal-
ize the data.

RESULTS

Natural Substrata

Substratum characterization in wash-up
samples revealed overall differences in the
total abundance of the four different settle-
ment substrata, possibly due to seasonal
changes in algal and hydroid productivity. The
total amount of algal and hydroid material (+
SE) from the original 100 g subsample for the
three wash-up samples was 96 + 2.3% in Oc-
tober 1998, 31 + 5.2% in December 1998,
and 64 + 3.5% in May 1999. However, the
proportional algal and hydroid substratum
comparisons among replicates and wash-up
samples indicated that coarse-branching
algae were more abundant than any other
substratum for all three wash-up events (Fig.
3). The mean percent area (+ SE) of coarse-
branching algae was 69.1 + 2.7%, followed
by medium-branching algae (14.1 + 1.4%),
hydroids (12.1 + 1.4%), and fine-branching
algae (4.6 + 1.2%). Atwo-way ANOVA of per-
cent substratum among wash-up events (arc-
sine transformed data) showed no statistical

17-Oct-98
0 22-Dec-98
16-May-99

Fine-branching
algae

Hydroids

Substratum Type

FIG. 3. Percent area of four natural substratum types among three spatfall events. Non-significant Tukey

tests for substratum differences are shown (*).

6 ALFARO & JEFFS

significance among wash-up events (ANOVA;
F5 42) = 0.35, р > 0.05), but a significant dif-
ference among substratum types (ANOVA;
F 5 42) = 217.33, р < 0.05). The interaction be-
tween wash-up and substratum type was not
statistically significant (ANOVA; Fi, 12) = 1.91,

р > 0.05). Tukey’s pairwise comparisons of
means failed to find significant differences be-
tween medium-branching algae and hydroids
(Tukey test; p = 0.799).

The total number of mussels differed
greatly among samples of different wash-up
events. The first sample, collected on 17 Oc-
tober 1998, yielded mussel numbers ranging
from 12 to 3,866 mussels/cm? of substratum,
and a mean (+ SE) of 658.7 + 230.7 mus-
sels/cm? of substratum. The second wash-up
sample of 22 December 1998 contained very
few mussels, ranging from 0 to 12
mussels/cm* of substratum and a mean (+
SE) of 2.6 + 0.7 mussels/cm? of substratum.
Finally, the third sample of 16 May 1999 hada
range of 9 to 1,632 mussels/cm“ of substra-
tum and a mean (+ SE) of 255.2 + 123.5
mussels/cm? of substratum. While the ab-
solute number of mussels among the three
wash-up samples was very different, the per-
cent of mussels within each substratum type
was similar for all wash-up samples (Fig. 4).
The mean percent of mussels between repli-

70 -
Ш 19-Oct-98

60 0 22-Dec-98

= 16-May-99

E

о 50

=

&

> 40

==

2)

о 30

Q

= *

®

® 20

5

=

o

Coarse-branching

Algae Algae

MA Er

Medium-branching

cates and among wash-up events indicates
that hydroid substrata consistently accumu-
lated the greatest number of mussels com-
pared to all other substrata (Fig. 4). The mean
percent of mussels/cm? (+ SE) among sub-
strata was 57 + 4, 28 + 2,8 + 2% and 7 +
2% mussels/cm? for hydroids, fine-branching,
medium-branching, and coarse-branching
algae, respectively. Results from a two-way
ANOVA on the arcsine transformed data, with
wash-up event and substratum type as fac-
tors, indicated that there was no statistically
significant ВЕ among wash-up events
tion (ANOVA; Fe = 4.41, р > 0:05), al-
though there was a significant difference
among substratum types (ANOVA; E, ¿> =
419.97, p < 0.05). Tukey’s test comparisons
failed to find significant differences between
coarse- and medium-branching substrata
(Tukey test; p = 0.714).

Results from the size-frequency distribution
of mussels within each substratum type re-
vealed a relationship between increasing
mussel size and decreased degree of branch-
ing of substratum. Coarse-branching algae
had a greater percent of large mussels
(1.5-1.99 mm in length), whereas fine-
branching algae and hydroids had the great-
est percent of small mussels (<0.5 mm in

Fine-branching
Algae

Hydroids

Substratum Type

FIG. 4. Mussel densities within four natural substrate types and three events samples. Non-significant Tukey

tests between substrates are shown (*).

SMALL-SCALE MUSSEL SETTLEMENT

100

Coarse-branching Algae

Medium-branching Algae

60

>
o

№
o

o

an
o
(>)

Fine-branching Algae

Mussel Density (% / cm?)
3

<0.49 0.5-0.99

Hydroids

<0.49 0.5-0.99

1.0-1.49

1.0-1.49

№ 17-Oct-98
О22-Оес-98
2 16-May-99

Ш 17-Oct-98
D22-Dec-98
216-Мау-99

1.5-1.99 >2.0

@ 17-Oct-98
О22-Оес-98
16-May-99

1.5-1.99 >2.0

17-Oct-98
O22-Dec-98
E116-May-99

1.5-1.99 >2.0

Mussel Size Class (mm)

FIG. 5. Size-frequency distribution of mussels within five size classes and three spatfall events for four nat-
ural substratum types. Pairwise comparisons (Tukey test) were performed for substrata that showed non-sig-
nificant interactions (coarse-branching algae, medium-branching algae, and hydroids) in an overall ANOVA

test and are shown (*).

length) (Fig. 5, Table 1). Two-way ANOVA's,
with mussel size class and wash-up event as
treatment, were performed for each substra-
tum (Table 1).

Results from mussel density comparisons
between node and inter-node areas within

each of four natural substrata indicate that
mussels were more abundant in node areas
in all substratum types (Fig. 6). Furthermore,
the relationship between increasing mussel
abundance and decreased degree of substra-
tum branching was generally apparent in

ALFARO &

JEFFS

TABLE 1. Mean (+ SE) percent mussel density within each of five size classes for four natural
substratum types. Two-way ANOVAs are shown for each substratum type. Data were arcsine
transformed for statistical analyses and back transformed to obtain the means.

Mussel Size ANOVA
Class Mean + SE
Substratum Type (mm) %/cm? Source df Е pa=0.05
Coarse-branching <0.49 5.62 + 3.50 Sample 2 0.20 0.819 ns
Algae 0.5-0.99 6.74+ 3.10 Size 4 17.63 0.000
1.0-1.49 23.31 + 6.30 Sample x Size 8 2.33 0:075 15
1.5-1.99 52.63 3:34 Error 15
>2.0 2.75 = 3197
Medium-branching <0.49 15.15 + 4.94 Sample 2 0.10 0.906 ns
Algae 0.5-0.99 14.77 + 3.64 Size 4 10.44 0.000
1.0-1.49 27.35 + 2.37 Sample x Size 8 3.52 0.017
1.5-1.99 31.85 + 4.23 Error 15
>2.0 2.57 = 4:35
Fine-branching <0.49 50.48 + 1.26 Sample 2 1.08 0.365 ns
Algae 0.5-0.99 30.57 + 1.46 Size 4 167.10 0.000
1.0-1.49 15.57 + 1.10 Sample x Size 8 2.23 0.086 ns
1.5-1.99 1576 #228 Error 15
>2.0 0.17 = 1.76
Hydroids <0.49 42.81 + 0.96 Sample 2 0.11 0.894 ns
0.5-0.99 29.27 + 1.15 Size 4 173.34 0.000
1.0-1.49 17.60 + 0.71 Sample x Size 8 1.82 0.150 ns
1.5-1.99 9:50) 2.22 ¡EMO! 15
>2.0 0.05 + 0.48
1200
e =
£ Ш Nodes
2 1000 Internodes
9
©
=
Z 800
>
5
£
+ 600
г
17
= 400
©
Q
©
=. 200
5
=
0

Coarse-branching
Algae

FIG. 6.

Medium-branching

Algae

Fine-branching
Algae

Hydroids

Substratum Type

these samples as well (Fig. 6). However, no
difference between coarse- and medium-
branching algae was observed (Fig. 6). The
mean number of mussels within node areas
were 4.7 + 0.9, 52.4 + 9.5, 557.1 + 33.9, and
1,055.0 + 41.5 individuals/cm?, and within in-

Mussel densities within node and internode areas within four natural substratum types (n = 20).

ternode areas were (0.4 + 0.3, 23.7 + 6.0,
61.8 + 13.9, and 581.2 + 145.6 individuals/
cm?) for coarse-, medium-, and fine-branch-
ing, and hydroid substrata, respectively. Al-
though size classes were not differentiated in
this part of the study, the fact that the pattern

SMALL-SCALE MUSSEL SETTLEMENT 9

is similar for all substratum types (which con-
tained different predominate mussel size
classes) suggests that mussels of all size
classes were more abundant within node
areas. In samples with low total mussel abun-
dances, clumps of mussels within node areas
were often clearly visible. As the total density
of the sample increased, the clumps of mus-
sels extended from internodes along the
branches, and in some cases, the entire algal
or hydroid substratum was covered with mus-
sels. A two-way ANOVA, with site of attach-
ment (node versus internode) and substratum
type, showed statistical Signlucangs for site of
attachment (ANOVA; Fi, 475, = 248.74, р <
0.05) and substratum type (ANOVA; F 3 475) =
562.03, p < 0.05). A statistically significant In-
teraction (ANOVA; Fu 72) = 72.54, p< 0.05)
reflected the inability. to differentiate settle-
ment patterns between coarse- and medium-
branching algae.

Artificial Substrata

While the absolute number of mussels that
settled on the artificial substrata was much
lower than the numbers recorded from the
natural material found in wash-up samples,
the general trend among morphologically dis-
tinct groups was consistent with the natural
substrata (Fig. 7). Mussel settlement within

100 -
90
80
70 -
60 -
50
40

30

Mussel Density (% / cm?)

20

Coarse-branching substrate

Medium-branching substrate

coarse-branching artificial substrata ranged
from O to 2 mussels/cm? of substratum and
had a mean of 0.21 + 0.05 mussels/cm? of
substratum. Medium-branching material had
a settlement range from 0 to 6 mussels/cm*
and a mean of 0.38 + 0.09 mussels/cm? of
substratum. Finally, fine-branching material
had a range of 0 to 25 mussels/cm? of sub-
stratum and had a mean settlement of 3.23 +
0.45 mussels/cm? of substratum. Three one-
way ANOVAs, with artificial plant type as treat-
ment, were run on the arcsine transformed
data to test for settlement differences among
plastic aquarium plants within each of the
three experimental branching groups. The re-
sults of these tests showed no significant dif-
ferences among any of the groups (ANOVA;
F5 = 0.02, р > 0.05, ANOVA; F (2 15) = 0.07,
p > 0.05, and ANOVA; Гель) = 0. 17, P= 0105
respectively for coarse-, medium-, and fine-
branching substrata) (Fig. 7). However, a one-
way ANOVA comparing mussel settlement on
artificial substrata with different branching de-
grees found significant settlement differences
among substrata (ANOVA; F4 51, = 80.17, р <
0.05) (Fig. 7). A one-way ANOVA comparing
all coarse- and medium-branching substrata,
regardless of plant species, resulted in non-
significant substratum effects (ANOVA; Р 34)
— 1106, р> 0:05);

Frequency distribution results from the arti-

Fine-branching substrate

Substratum Type

FIG. 7. Mussel densities within three experimental group containing artificial substrata. A significant ANOVA
test between coarse- and medium-branching algal substrata only are shown (*) (n = 6). See text for further

explanations.

10 ALFARO & JEFFS

Coarse-branching Substrata

<0.49 0.5-0.99 1.0-1.49 1.5-1.99 >2.0

Medium-branching Substrata

Mussel Density (% / cm’)

RR

<0.49 0.5-0.99 1.0-1.49 1.5-1.99 >2.0

Fine-branching Substrata

Mussel Size Class (mm)

FIG. 8. Size-frequency distribution of mussels within five size classes and three experimental groups of arti-
ficial substrata. Pairwise comparisons (Tukey test) were performed for substrates that showed non-signifi-
cant interactions (coarse- and medium-branching substrata) in an overall ANOVA test and are shown (*).

ficial substratum experiments also indicated size class, whereas coarse- and medium-
greater settlement on fine-branching material branching substrata had a similarly high per-
than coarse- and medium-branching material centage of > 1.5 mm mussels, and very little
(Fig. 8). Generally, fine-branching substrata to no settlement of < 1.5 sized mussels (Fig.
had a higher percentage of < 0.99 mm mussel 8). Results from statistical analyses, including

SMALL-SCALE MUSSEL SETTLEMENT ial

two-way ANOVAs for each experimental
group (degree of branching and mussel size
as treatment), are shown in Table 2.
Settlement comparisons between nodes
and internodes areas within artificial substrata
also resulted in greater mussel densities in
node areas than in internode areas (Fig. 9).
Again, no settlement differences between
coarse- and medium-branching substrata
were observed, but fine-branching substrata
did contain greater settlement than the other

two substrata (Fig. 9). Mussels often were ob-
served extruding their foot to test surrounding
substrata. In some cases, mussels were ob-
served moving from the tip of an artificial plant
to the nearest node. This migratory behavior
sometimes resulted in clumping of mussels. A
two-way ANOVA, with site of attachment
(node versus internode) and substratum type,
showed significant differences between node
and internode attachment sites (ANOVA;
Fo 402) = 13.40, p < 0.05) among artificial sub-

TABLE 2. Mean (+ SE) percent mussel density within each of five size classes for three artificial
substratum types. Two-way ANOVAs are shown for each substratum type. Data were arcsine
transformed for statistical analyses and back transformed to obtain the means.

Source df E

Mussel Size

Class Mean + SE

Substratum Type (mm) %/cm?
Coarse-branching <0.49 0.00 + 0.00
Substrata 0.5-0.99 0.00 + 0.00
1.0-1.49 0.00 + 0.00
1.5-1.99 0.29 + 0.06
>2.0 3.66 + 0.09
Medium-branching <0.49 0.01 + 0.01
Substrata 0.5-0.99 0.01 + 0.01
1.0-1.49 0.05 + 0.03
1.5-1.99 0.46 + 0.07
>2.0 5231013
Fine-branching <0.49 47.26 + 0.30
Substrata 0.5-0.99 18.79 + 0.54
1.0-1.49 2165 0:15
1.5-1.99 2.15 028
>2.0 1.44 + 0.09

Ш Nodes
O Internodes

Mussel Density ( # individuals / cm?)

Coarse-branching substratum

Medium-branching substratum

ANOVA

pa=0.05
Substrate 2 0.00 0.998 ns
Size 4 20.61 0.000
Sample x Size 8 0.12 0.998 ns
Error 75
Substrate 2 0.24 0.786 ns
Size 4 15.79 0.000
Sample x Size 8 O83) 01953" ins
Error 75
Substrate 2 3.14 0.049
Size 4 41.75 0.000
Sample x Size 8 5.34 0.000
Error 715

Fine-branching substratum

Mussel Size Class (mm)

FIG. 9. Mussel densities within node and inter-node areas within three artificial substratum types (n = 6).

12 ALFARO & JEFFS

stratum types (ANOVA; F, 192 = 41.60, р <
0.05), and interaction (ANOVA: E

(2,102) —
11.00, p < 0.05).

DISCUSSION

One of the great interests in the field of in-
vertebrate ecology is the question of how ini-
tial settlement patterns affect the distribution
of adult broadcast spawners, such as mus-
sels. While oceanic-current dispersal of lar-
vae and juveniles is a passive means of trans-
port, some degree of habitat choice is
exercised by individuals that encounter and
attach to drift material. This initial settlement
process on various morphologically distinct
drift materials may be of significance to the
survival and successful resettlement of juve-
niles to the adult population. Thus, elucidation
of microscale settlement patterns of mussel
spat on natural drift substrata may enhance
our understanding of the process of spat
transport and arrival to coastal areas.

Substratum characterization within three
wash-up events at NMB indicated that
coarse-branching algal substrata consistently
comprised the majority of settlement sub-
strata found associated with wash-up events.
Medium-branching algae, fine-branching
algae, and hydroids were less abundant. Drift
algal material may originate from rocky inter-
tidal and subtidal areas that may have been
detached from their natural substrata by
storms or strong ocean currents. These mate-
rials may become aggregated by local
oceanographic conditions. However, the ma-
jority of the material (mostly red algae) is sub-
tidal in origin (Osmundaria colensoi, Car-
pophyllum angustifolium, and Rhodymenia
dichotoma; Adams, 1994; Steneck & Dethier,
1994), indicating the importance of subtidal
sources. The large amount of subtidal mater-
ialin NMB wash-up samples also may be a re-
sult of the scarcity of rocky intertidal areas
that could provide intertidal algal sources
along the beach (Fig. 1). Furthermore, none
of the intertidal areas at or near NMB contain
the same species of algae found washed-up
at NMB in enough abundance to indicate a
likely source (A. Alfaro, personal observation).
The often fresh and intact nature of the mate-
rial that arrives at the beach suggests that the
subtidal material comes from rocks situated
just offshore (<35 m water depth) of three
rocky outcrops along the beach (Tauroa Point,
The Bluff, and Scott Point). Indeed, algal beds

have been observed off Tauroa Point (S.
Hooker, unpublished), and extensive rocky
substrata, likely to harbor algal material, have
been noted by local fishers off Scott Point.
Furthermore, spawning peaks of intertidal and
subtidal mussels (< 35 m water depth), as well
as algal seasonal cycles, may strongly influ-
ence the abundance, dispersal and coloniza-
tion potential of spat within temporal and spa-
tial scales (Alfaro et al., in review). However,
more samples collected throughout the year
are necessary to clarify these patterns.
Quantification of mussel spat among algal
and hydroid substrata indicated that a greater
number of spat settle on hydroids, followed by
fine-branching algae; whereas fewer spat set-
tle on medium- and coarse-branching algae.
Laboratory settlement experiments with artifi-
cial substrata also resulted in greater settle-
ment on fine-branching material over coarse-
and medium-branching substrata. Buchanan
& Babcock (1997) found a similar size-
frequency distribution of mussel spat on inter-
tidal algae at Piha, North Island, New
Zealand. In their study, the authors found that
primary settlement of mussels (< 0.4999 mm
in length) was mostly on the hydroid Amphis-
Бена bispinosa, and fine- and medium-
branching algae, whereas larger mussels in
the dispersal (0.5-5.4999 mm in length) and
stable (> 5.0 mm in length) stages of their
life history tended to resettle onto coarser-
branching algae and the adult mussel bed. By
contrast, the present study included consider-
ation of a wider range of free-drifting algal-
branching types (coarser red and brown
algae) that are likely of mostly subtidal origin.
Nonetheless, the size-frequency distribution
of mussel spat on natural (in the field at NMB)
and artificial (in the lab) substrata strongly
corroborates observations at Piha by Bu-
chanan & Babcock (1997). The field experi-
ments at NMB indicated that the percent of
mussel spat among the four algal and hydroid
substrata was similar for all three wash-up
samples and may indicate that settlement pat-
terns are not affected by absolute variations in
substratum abundance. Furthermore, mussel
spat will settle predominantly on less abun-
dant natural material even when existing spat
densities may be quite high (~3,800 individu-
als/cm? on hydroid material). This strong se-
lectivity has been observed elsewhere (But-
man, 1987; Schneider & Mann, 1991 a, b;
Butman & Grassle, 1992; Grassle et al., 1992;
Harvey et al., 1993; Buchanan & Babcock,
1997). Schneider & Mann (1991a) found that

SMALL-SCALE MUSSEL SETTLEMENT 13

there was a strong selectivity of epifaunal in-
vertebrates to macroalgae with varying de-
grees of branching. These authors concluded
that the relationship between invertebrate
species and algal morphology benefited the
associated invertebrates in terms of food
source and living space provisions. It is un-
clear as to which different ecological proper-
ties may be provided by different substrata
(such as coarse-branching algae versus hy-
droids) to the associated mussel spat, but it is
likely that the relationship is based primarily
on attaining a structurally secure place to in-
habit. Indeed, results from the artificial sub-
stratum experiments suggested that mussels
will preferentially settle onto filamentous sub-
strata on the basis of their physical shape.
While these experiments with artificial sub-
strata do not rule out the possibility that chem-
ical cues exuded by natural algae affect mus-
sel settlement patterns, they do support the
idea that substratum morphology alone
strongly influences settlement of different-
sized mussels. The observed movement of
mussels from the tips of artificial aquarium
plants to node areas suggest that attraction
cues among mussels may exist, as well as
substratum selectivity.

The size-frequency distribution of mussel
spat on the various natural substrata within
NMB wash-up samples (Fig. 5), and artificial
substrata (Fig. 8), shows a general inverse re-
lationship between spat size and degree of
substratum branching. Thus, larger mussels
(1.5-2.0 mm) appear to settle predominantly
on coarse-branching substrata, whereas
smaller mussels (< 0.5 mm) preferentially set-
tle on fine-branching material (Figs. 5, 8).
Buchanan & Babcock (1997) suggested that
such size-specific settlement on morphologi-
cally distinct substrata in the intertidal zone
may be largely a result of recolonization of
natural substrata. Our results suggest that the
selection process also may take place in sub-
tidal habitats and in the water column, possi-
bly within accumulated patches of drift algae
and hydroids. Furthermore, this size-specific
selection persists even after the mussels
have been transported large distances by dy-
namic oceanographic conditions evident at
NMB. The possibility exists that the observed
relationship between spat size and substra-
tum type is a result of differential growth rates
that allow the less abundant spat on coarse-
and medium-branching algae to attain a
larger size due to lower crowding effect. How-
ever, the fact that all substratum types and

mussel spat size classes were consistently
represented in all three ММВ wash-up sam-
ples suggests that differential growth rates of
spat within patches of drift material is unlikely.
Furthermore, artificial substratum experi-
ments resulted in similar trends after a period
of only two days, which was not long enough
to note size differences due to growth.

A size-specific settlement pattern may rep-
resent a choice for scaled physical stability,
where, for example, larger mussels may re-
quire larger morphologically stable substrata,
such as the coarse- and medium-branching
algae. However, smaller-scale selectivity also
may contribute to stability requirements of
mussel spat. Comparisons of settlement den-
sities between node and inter-node areas
within four different natural substrata (Fig. 6),
and three artificial plant substrata (Fig. 9), in-
dicate that node settlement is preferred by
mussels settling in all types of substrata.
Micro-scale selectivity has been demon-
strated in laboratory flume experiments (Har-
vey et al, 1993; Harvey & Bourget, 1995;
Bourget & Harvey, 1998). Bourget & Harvey
(1998) found that recruits of nine marine in-
vertebrates, including six bivalve species,
were more abundant in nodes as compared to
internodes, and that the rate of recruitment in-
dicated that passive deposition alone was not
sufficient to obtain such an outcome. The au-
thors ruled out the influence of differential ero-
sion and mortality on higher settlement pat-
terns for nodes over linear areas. Evidence
has been accumulating that points to behav-
ioral responses to small-scale (<1 cm) sub-
stratum irregularities, such as pits, grooves,
and depressions, as determinants in mi-
croscale settlement preferences (Bourget et
al., 1994; Nellis & Bourget, 1996; Bourget &
Harvey, 1998). While chemical cues between
spat and substratum, and among individual
mussels, cannot be excluded as factors in the
site-settlement preference experiments re-
ported here, it is likely that morphology is the
driving force to the strong habitat selectivity
observed in this study.

The large volume of wash-up material (up
to 70,000 t/y; C. Hensley, personal communi-
cation) that can be collected only along NMB
suggests that settlement on drift material may
be an important part of the life history strategy
for Perna canaliculus. Indeed, floating algal
clumps have been found to provide alterna-
tive habitats and transport of invertebrates
(Schneider & Mann, 1991a; Highsmith, 1985;
Bologna & Heck, 1999). Micro-scale selectiv-

14 ALFARO & JEFFS

ity of mussel spat of different sizes onto mor-
phologically distinctive substratum types may
reflect a delicate level of organization within
the transitional environment of drift material.
Transport by drift material may be crucial to
the invasion and retention of mussel spat onto
new intertidal and subtidal sites (Highsmith,
1985). Pulses of new mussel settlements
have been observed at Scott Point, NMB after
large accumulations of drift material on the
rocky shore (C. Hensley, personal communi-
cation). It is possible that entire rocky shore
mussel communities may depend on the peri-
odic arrival of spat from drift material. In the
case of NMB, where rocky habitats constitute
a very small portion of the coastal area, it ap-
pears that most wash-up events may arrive
on the sandy beach where mussels have little
chance of survival, unless collected by spat
collectors and transported to mussel farms.
Additional research on substratum availability
and the dynamics of oceanographic transport
of drift material at NMB is critical to further un-
derstanding of mussel dispersal, colonization,
and maintenance of adult populations at vari-
ous temporal and spatial scales.

AKNOWLEDGMENTS

We thank C. and R. Hensley, K. Campbell,
and D. Tasker for their invaluable assistance
with sample collections in the field. We are
grateful for the technical support of A. Turner,
|. MacDonald, V. Ward, and D. Todd. Algal
identifications were done by W. Nelson, and
S. O’Shea conducted the hydroid identifica-
tions. The Geology Department at the Univer-
sity of Auckland provided lab space and
equipment for the size-frequency analyses.
We thank K. Campbell and two anonymous
reviewers for their comments on the manu-
script.

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Revised ms. accepted 29 March 2001

MALACOLOGIA, 2002, 44(1): 17-22

DETECTION OF PHENOLOXIDASE (PO) IN HEMOCYTES OF THE CLAM
VENUS ANTIQUA

Luis A. Mercado', Sergio H. Marshall & Gloria M. Arenas?

Laboratorio de Genetica e Inmunologia Molecular, Instituto de Biologia, Facultad de Ciencias

Bäsicas y Matemäticas, Universidad Catolica de Valparaiso, Chile

ABSTRACT

The prophenoloxidase (proPO) system, particularly studied in Arthropoda, is one of the im-
portant mechanisms of non-self recognition in the immunological response of some inverte-
brates. Under natural conditions, the activation of the system involves the stimulation of serine
proteases by the presence of LPS and ß-1,3 glycans on the surface of microorganisms. It results
in the triggering of proPO, which in turn generates peptides responsible of opsonizing and Iytic
actions. We report here the preliminary analysis of this type of response in hemocytes of the bi-
valve mollusc Venus antiqua, both in intact cells and in crude lysates. Our strategy was first to
monitor PO activity in whole hemocytes. Second, to confirm its presence in hemocyte Iysate su-
pernatant (HLS) by Dot and Western blot analysis using polyclonal antibodies against tyrosinase,
the commercially available form of PO. Third, to measure its enzymatic activity in HLS by com-
paring the oxidation of the substrate L-DOPA in the presence and absence of known activators
or inhibitors. In this report, we demonstrate PO activity in granular cell subpopulations. In HLS,
we quantitate the specific activity of the enzyme by spectromethomertric methods. Finally, im-
munological characterization of HLS suggests the existence of two PO-like polypeptides, one of

45 kDa and a second one slightly higher.

Key words: mollusc immunity, clam hemocyte, PO activity, immunodetection.

INTRODUCTION

The proPO system, best studied in arthro-
pods, corresponds to a complex of he-
molymph enzymes and associated protein
factors that activate the phenoloxidase (PO)
cascade generating small peptides, which act
as mediators of the host immune response
(Rattcliffe, 1991; Kwon et al., 1997; Perazzolo
& Barracco, 1997). The system has been best
characterized in crustaceans and other se-
lected invertebrates (Johansson & Söderhäll,
1989; Iwanaga et al., 1992; Hoffmann &
Reichhart, 1997). The proPO system has also
been described in such molluscs as Bio-
phalaria glabrata (De Aragao & Bacila, 1976),
Mytilus edulis (Coles & Pipe, 1994; Ren-
wrantz et al., 1996), and Perna viridis (Asokan
et al., 1997). Notwithstanding, the marine bi-
valve Perna perna did not show PO activity
(Barracco et al., 1999).

The proPO system is primarily activated by
polysaccharides from the microorganisms

surface, of which B-1,3-glucans from fungal
cell wall and LPS-peptidoglycan complexes
are considered to be the most classical acti-
vators (Unestam & Söderhäll,) 1977; Char-
alambidis et al., 1996). In arthropods, the
proPO has been reported as a 76 kDa mole-
cule (Aspan & Söderhäll, 1991), which is
turned into an active form (PO) by ppA, a ser-
ine protease of 36 kDa (Aspan et al., 1990).
Other endogenous proteinases activate
proPO in a way that could very well represent
an invertebrate counterpart of the vertebrate
complement system (Hernandez-Lopez et al.
1996). Once PO is formed, a number of meta-
bolic pathways are activated oxidazing L-
DOPA to melanin as the final common end-
product (Nappi et al., 1995; Marmaras et al.,
1996). A great variety of molecules have been
described during these activated intermediate
steps. Among them, aglutinines (lwanaga et
al., 1998; Gollas-Galvan et al., 1999), adhe-
sive proteins (Söderhäll et al., 1990), and
quinones (Ratcliffe, 1991).

"Present Address: Departamento de Bioquimica y Biologia Molecular, Facultad de Veterinaria, Universidad Santiago de

Compostela, Lugo, Spain.

Address correspondence to: Instituto de Biologia, Universidad Catölica de Valparaiso, Avenida Brasil 2950, Casilla 4059,

Valparaiso. Chile; garenas @ucv.cl

18 MERCADO ET AL.

Considering that the proPO system is well
established in arthopods and insects and that
there is some controversy in molluscs, we de-
cided to verify if this relevant innate immune
response could be assayed in another marine
bivalve. In this report using the clam Venus
antiqua as model system, we demonstrate its
activity. Moreover, by introducing immunolog-
ical detection as an innovative tool, we also
demonstrate its presence and potential evolu-
tionary conservation.

MATERIALS AND METHODS

Recovery of Hemolymph

Twelve adult specimens of Venus antiqua
were collected from natural grounds in the
coast of the V Region in Chile. Hemolymph
was drained from the posterior adductor mus-
cle of each individual (2.0 ml) using sterile
plastic seringes (Friebel & Renwrantz, 1995).
Resulting hemolymph was pooled in a
polypropylene tube and kept at 4°C.

Recovery of Hemocytes

The hemolymph was centrifuged in a clini-
cal centrifuge at 300 x g for 5 min at 4°C. The
plasma was stored at 4°C until processed and
the cellular pellet carefully resuspended in ca-
codylate buffer (10 mM sodium cacodylate
buffer (NCB): 0.45 M NaCl, 20 mM CaCl,, 10
mM MgCl, pH 7.4) and intact hemocytes
were washed twice and resuspended in the
same buffer.

Hemocyte Lysate Supernatant (HLS)

Hemocytes in 800 ul of NCB buffer were
fully homogenized in a glass-plungered ho-
mogenator. The homogenate was centrifuged
at 35.000 x g (Sorvall RC-5B centrifuge; HB-4
rotor) for 20 min at 4°C and the pellet dis-
carded. The supernatant constitutes HLS, the
source for PO evaluation.

Cytochemistry

Intact hemolymph diluted 2:1 in NCB buffer
(pH 7.4) buffer were fixed with 5% formalde-
hyde on a polylysine-treated (0.1%) slide.
After 15 min, slides were exposed to L-DOPA

(0.1% in NCB 150 mM NaCl) for 12 h at room
temperature in the dark (Renwrantz et al.,
1996). In control samples, L-DOPA was om-
mitted.

PO Activity in HLS

Triplicates of 50 ul of HLS were incubated
with inducers, either 50 ul of trypsin (1 mg/ml)
or SDS (1 mg/ml), for 5 min at 20°C. Then, 50
ul of L-DOPA (3 mg/ml), with or without 10 mM
CaCl,, a PO cofactor, were added and further
incubated for 30 min. In paralel, samples con-
taining either inducer were supplemented with
50 ul of 0.2 mM phenilthiourea (PTU) as a PO
inhibitor to rule out endogenous peroxidase
activity as well as to determine the real PO
specific activity. Reactions were ended by di-
lutions with deionized water to a final volume
of 1.0 ml. PO activity was determined spec-
trophotometrically by measuring oxidation of
the L-DOPA into a DOPA-chrome detected at
490 nm. The specific activity of the enzyme
(S.A.) is expressed as the ratio of the ab-
sorbance value (Abs) per time per amount of
protein. S.A. = Abs x min ! x mg prot! (Smith
& Söderhäll, 1991; Perazzolo & Barracco,
1997). Final S.A. was expressed as average
values + SD, according to the t-test for inde-
pendent samples (p < 0.05) (Statistica 5.0).

Immunological Tests

Polyclonal monospecific anti-tyrosinase
(commercial mushrom PO, Sigma) were
elicited as previously described (Mercado &
Arenas, 1997) and used in all immunological
detections. Protein concentration was deter-
mined by the method of Bradford (1976) using
Bovine Serum Albumin (BSA) as a standard.

Dot Blot Analysis

5 ug of HLS and plasma; 1 ug of tyrosinase
(Sigma) and horseradish peroxidase (HRP)
(Sigma) were spotted onto nitrocellulose
membranes and incubated for 2 h at room
temperature with anti-tyrosinase antibody
(1:3,000) in TBST buffer (0.05% Tween 20 in
TBS, pH 7.4), in the presence of 3% BSA and
0.2% sodium azide. Second antibody was
rabbit anti mouse IgG-alkaline phosphatase
(PA) (1:30,000). After 1 h incubation the mem-
brane was developed with. BCN-NBT Fast
(Sigma) (Dyson, 1991).

DETECTION OF PHENOLOXIDASE IN HEMOCYTES OF THE CLAM 19

Western Blot Analysis

20 ug of HLS resolved into a 12% SDS-
PAGE (Laemmli, 1970) was electrotransfered
to an immobilon-P membrane (PVDF, Milli-
pore) (Towbin et al., 1979) and processed as
described above. Non-specific sites were
blocked with 3.0% BSA in TBST.

RESULTS

The existence of the proPO activating sys-
tem is demonstrated in hemocytes of the clam
Venus antiqua. 58-60% of intact granular he-
mocytes showed a positive PO reaction by
the conversion of the initial substrate to L-
DOPA into a dark end product (Fig. 1A), as
opposed to the 100% absence in the negative
controls (Fig. 1B). In HLS fractions, pigment
formation was also indicative of PO activity,
although a slight positive reaction is detected
in plasma exposed to L-DOPA. Nontheless,
there is no dark precipitate in the presence of
the enzyme inhibitor PTU (Fig. 2).

In enzymatic activity assays, proPO in HLS
was activated by both inducers trypsin and
SDS, with a slighter higher value for the latter
in the presence of calcium (Fig. 3).

Dot blot analysis demonstrate the speci-
ficity of our antityrosinase antibody (Fig. 4).
The polyclonal antibody showed no cross-re-
activity with either HRP or the negative con-
trol, while an intense reaction is seen in the
HLS sample in contrast to a feeble one in
plasma.

Finally, Western blot shows in HLS, two im-
munoreactive bands: a very intensive one
with a mobility of approximately 45 kDa, anda
less reactive slightly higher molecular weight
band, which correlates with the stained profile
of the corresponding gel (Figs. 5: lanes 3 and
2, respectively).

A

e?

| 2 3 4

FIG. 2. Qualitative assay of PO activity in V. antigua
hemolymph. (1) activated HLS, (2) activated HLS in
the presence of 0.2 mM PTU, (3) non activated
plasma, (4) 10 mM Na* cacodilate buffer.

DISCUSSION

The presence of the PO system among dif-
ferent groups of invertebrates is under dis-
cussion. In arthropods, it has been described
exclusively in the hemocytes of some cray-
fish, such as Penaeus californiensis and P.
paulensis (Hernändez-Löpez et al., 1996;
Perazzolo & Barracco, 1997), or solely in the
plasma of insects Bombyx moriand Manduca
sexta (Ashida et al., 1983; Saul et al., 1987).
In bivalve molluscs, Renwrantz et al. (1996)
described PO activity in hemocytes of Mytilus
edulis, whereas in Perna viridis Asokan et al.
(1997) found it in both plasma and hemo-
cytes.

Our study in clams supports the putative
existence of PO activity in marine inverte-
brates. We demonstrate that a high percent-
age of bulk granular hemocytes display the
activity (Fig. 1). Moreover, statistically signifi-
cant spectrophotometric assays measuring
the oxidation of L-DOPA to DOPA-chrome
seem to be good indicators of PO activity in
HLS from Venus antiqua (Fig. 3). The basal
activity detected in plasma (Fig. 2), might be
interpreted as a reflection of the physiological
stage of the specimens analyzed. Finally, our
novel immunological approach suggest the
existence of two size-specific polypeptides

FIG. 1. A, Hemocytes subpopulations from Venus antiqua with a positive PO oxidizing reaction. Monolayer
incubated with L-DOPA as substrate, 1.000 x. B, Hemocytes incubated in the absence of substrate.

20 MERCADO ET AL.

Control
Trypsin — Ca” *
SDS — Ca” *
Trypsin **

SDS **

Trypsin - 0.2 mM PTU
SDS - 0.2 mM PTU

PO activity in HLS.
Abs 490 nm (min! x mg prot!
0.064 + 0.016
0.133 + 0.033
0.277 + 0.104
0.018 + 0.012
0.037 + 0.009
0.006 + 0.002
0.007 + 0.001

The enzymatic activity values are expressed as average + SD.
* The substrate L-DOPA was added in the presence of 10 mM Call,
** The substrate L DOPA was Ca” free added.

FIG. 3. Differential effect of Trypsin, SDS, Ca?* and PTU over PO activity in HLS. Spectrophotometric quan-

titation of PO specific activity.

MEAN +

| 2 3 À

FIG. 4. PO immunodetection in Dot blot. (1) acti-
vated HLS and (2) non activated plasma, (3) com-
mercial tyrosinase, (4) HRP. Each dot contains 1 ug
of protein.

kDa LS
66—}| 2
4 PA
15 a + <— 45
++

FIG. 5. SDS/PAGE characterization of HLS and
Western blot analysis. (1) HLS, 50 ug (10% gel)
Coomassie blue stained, (2) HLS, 20 ug (12%
stained gel). (3) Western blot of a parallel gel shown
in lane 2. Reactive bands indicated by slim arrows.
Relative molecular weights in kDa indicated on the
left of each lane.

(50 and 45 kDa), although different from those
of crayfish molecule (Aspan & Söderhäll,
1991), are highly conserved and presumably
responsible for the detected PO activity in
Venus antiqua. In Mytilus edulis, the native
enzyme characterized in non denaturant gels

also resulted in two proteins with relative mol-
ecular weights of 381 + 13.7 and 316 + 11.1
kDa, respectively (Renwrantz et al., 1996).
This might very well mean that the two Venus
antiqua polypeptides could represent sub-
units of a larger enzyme.

In spite of the fact that the PO values re-
ported for HLS of Venus antiqua are low com-
pared to other species, we think they are real.
PO activity fully responds to serine proteases,
such as trypsin (Fig. 3), in agreement with
other reports (Nellaiappan & Sugumaran,
1996). Additionally, the same figure summa-
rizes the expected activating effect of SDS
(Moore & Flurkey, 1990). Also, for both cases,
the inducing effect of Са?* as a co-factor, as
well as the fully inhibitory action of PTU (Sug-
umaran et al., 1988). Further studies tending
to optimize the measurement of PO activity in
Venus antiqua or in other mollusc model sys-
tem might contribute to asses the observa-
tions reported here.

The specific recognition of two polypeptides
in hemocytes of Venus antiqua using antibod-
ies elicited agaist a commercial enzyme of
mushrom origin clearly suggests the exis-
tence of common epitopes of evolutionary
conserved enzymes, situation expected for in-
nate immunological responses.

SUMMARY AND CONCLUSIONS

The PO activating system was studied in
hemocytes of the bivalve mollusc Venus anti-
qua. Qualitative assays were used to demon-
strate the formation of dark precipitates in in-
tact hemocytes and quantitative assays of
enzyme activity measured by oxidation of L-

DETECTION OF PHENOLOXIDASE IN HEMOCYTES OF THE CLAM 21

DOPA to DOPA-chrome formation. Immuno-
logical detection was incorporated in order to
characterize the putative polypeptides in-
volved in the activation process in mollusc.
We conclude that the PO system is present
mostly in hemocytes of Venus antiqua. The
putative polypeptides recognized by antibod-
ies elicited to a mushrom enzyme, suggest a
high degree of evolutionary conservation. Al-
though they do not match the expected
molecular weights described for crayfish, they
might correspond to subunits of the native en-
zyme described in Mytillus edulis. In sum-
mary, our results tend to indicate a structured
innate immune response active in a species
of marine bivalve.

ACKNOWLEDGMENTS

The present work was financed by the Di-
recciön de Investigaciön, Vice Rectoria de In-
vestigaciön y Estudios Avanzados, Universi-
dad Catolica de Valparaiso, Chile.

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MALACOLOGIA, 2002, 44(1): 23-31

A SURVEY OF GENETIC VARIATION AT ALLOZYME LOCI AMONG GONIOBASIS

POPULATIONS INHABITING ATLANTIC DRAINAGES OF THE CAROLINAS

Robert T. Dillon, Jr. & Andrew J. Reed

Department of Biology, College of Charleston, Charleston, South Carolina 29424, U.S.A.;
dillonr@cofc.edu

ABSTRACT

We estimated gene frequencies at eight polymorphic enzyme loci in 12 populations of Go-
niobasis from Atlantic drainages of North Carolina, South Carolina, and Georgia. Our sample in-
cluded four populations of G. proxima from the piedmont, five populations of G. catenaria cate-
naria, one population of G. catenaria postellii, and two populations of G. catenaria dislocata from
the coastal plain. The fit to Hardy-Weinberg expectation was good within all populations of both
species (F,, = 0.042 proxima, 0.035 catenaria), while levels of interpopulation divergence were
high (F,, = 0.461 proxima, 0.564 catenaria). In spite of slightly overlapping geographic distribu-
tions, and instances of striking similarity in shell morphology between the two species, G. prox-
ima and G. catenaria were genetically distinct (average values of Nei’s D near 1.0), with no evi-
dence of hybridization. Although their ranges are fragmented into numerous isolated and
genetically distinct populations, both species remain broadly recognizable across states,
drainages, and physiographic regions. The relationship between G. catenaria postelliiand seven
other nominal species and subspecies of Goniobasis sometime recognized from Atlantic

drainages of Georgia is called into question.

Key words: electrophoresis, isozymes,freshwater, Carolina, Georgia, gastropods, pleuroceri-

dae, Elimia.

INTRODUCTION

Freshwater snails of the family Pleuroceri-
dae are abundant, widespread, and important
elements of the grazing and shredding com-
munity in lotic ecosystems throughout the
southeastern United States (Power et al.,
1988; McCormick & Stevenson, 1991; Hill et
al., 1995; Huryn et al., 1995; reviews by Fem-
inella & Hawkins, 1995; Dillon, 2000). Their di-
verse and often isolated populations, easily
sampled year around, have made pleuro-
cerids attractive models for the study of diver-
gence and speciation (Chambers 1980, 1982;
Dillon 1989, 1991; Bianchi et al., 1994; Dillon
& Lydeard, 1998; Lydeard et al., 1997; Holz-
nagel & Lydeard, 2000). Yet the pleurocerid
fauna of much of the southeastern United
States remains obscure and poorly docu-
mented, and their systematic relationships
unclear.

Goodrich (1942) listed three pleurocerid
genera, 15 species, and 26 subspecies in-
habiting Atlantic drainages from New England
to Texas. Four of these taxa will be treated in
the present work: Goniobasis proxima (Say,
1825) from the “highlands of North and South

23

Carolina’; G. catenaria dislocata (Ravenel,
1834) inhabiting “headstreams” in Virginia,
east-central North Carolina, and South Car-
olina; G. catenaria catenaria (Say, 1822) from
“springs of eastern South Carolina”; and G.
catenaria postellii (Lea, 1858) from the “Al-
tamaha, Ogeechee, and Canoochee Rivers”
of Georgia. Goniobasis catenaria and G.
proxima were two of the first four species of
this diverse North American genus to reach
formal description.

Although altering the generic name to
“Elimia,” Burch (1982) and Burch & Totten-
ham (1980) generally adopted Goodrich’s un-
derstanding of the pleurocerid fauna of North
America as outlined above. But the system-
atic relationships among the Georgia repre-
sentatives of this group have also been re-
viewed by Clench & Turner (1956), Krieger
(1977), Chambers (1990), and Mihalchik
(1998). Specific nomina applied to popula-
tions from Georgia Atlantic drainages have in-
cluded Goniobasis bentoniensis (Lea, 1862),
G. boykiniana viennaensis (Lea, 1862), G.
mutabilis (Lea, 1862), G. mutabilis timida
(Goodrich, 1942), G. timida (Goodrich, fide
Mihalchik, 1998), G. postellii (Lea, 1858), and

24 DILLON & REED

G. suturalis (Haldeman, 1840). All these
names would be junior synonyms of G. cate-
naria, to the extent that G. catenaria extends
into Georgia, as suggested by Goodrich.

Between 1986 and 1995, Dillon & Keferl
(2000) surveyed 629 aquatic sites distributed
evenly throughout South Carolina, document-
ing approximately 30 pleurocerid populations
at 44 sites. They found ten populations of G.
proxima in small streams in or near the Ap-
palachian foothills, confirming Goodrich’s
(1942) report. Ten populations of G. catenaria
catenaria were found at 26 sites in streams
and rivers of varying size through the South
Carolina midlands, expanding the known
range of that species substantially from that
suggested by Goodrich. The disjunct distribu-
tion of these populations suggested to Dillon
& Keferl that this species may have been
highly impacted by agricultural siltation and
impoundment. Dillon & Keferl also reported
six G. catenaria dislocata populations at eight
sites in small streams of the coastal plain.

Goniobasis catenaria populations were oc-
casionally found well into the South Carolina
piedmont, closely neighboring populations of
G. proxima in upstate tributaries of the
Broad/Santee and Catawba/Santee. And the
similarity in shell morphology between G.
proxima and G. catenaria dislocata of the
coastal plain was striking. The shells of both
species were identically shaped and marked
by a single spiral carina. Goniobasis catenaria
dislocata shells differed from G. proxima only
by the presence of faint axial costae near the
apex, disappearing after the first 2-3 whorls.
Populations of both species inhabit small,
rapidly flowing streams, G. catenaria dislo-
cata generally being found in those rather un-
usual regions of the coastal plain (often bor-
dering large rivers) with slope and marl
exposure. But whether the gross shell similar-
ity of G. proxima and G. catenaria dislocata
might reflect a genuine genetic relationship,
or might be the result of convergence, Dillon &
Keferl could not determine.

The genetics of North Carolina and Virginia
populations of G. proxima have been the sub-
ject of intensive study for 20 years (Dillon &
Davis, 1980; Dillon, 1984a; Stiven & Kreiser,
1994). Intrapopulation variation seems to be
unusually low, and interpopulation divergence
high, as might be expected from populations
of such a poorly mobile animal isolated in
small streams of the piedmont and mountains
(Dillon, 1984b). Despite the occurrence of
fixed differences at multiple enzyme loci, how-

ever, transplants and artificial introductions
have revealed no evidence of reproductive
isolation among G. proxima populations (Dil-
lon, 1986, 1988a).

The purposes of the present work are
threefold. First we compare the levels of intra-
and interpopulation genetic variance ob-
served in G. catenaria to the better-known G.
proxima. Second, we evaluate the genetic
distinctiveness of G. proxima and G. cate-
naria, paying particular attention to the possi-
bility of hybridization. Third, we examine evi-
dence that populations of G. catenaria
dislocata might be extralimital G. proxima,
rather than G. catenaria with shell morphol-
ogy convergent on G. proxima.

METHODS

We sampled five populations of G. cate-
папа catenaria, two populations of G. cate-
папа dislocata from eastern South Carolina,
and one G. catenaria postellii from Georgia.
The four populations of G. proxima we sam-
pled included Yad from northwestern North
Carolina, which we have previously compared
to other populations of Goniobasis (as Yad in
Dillon & Davis, 1980; Yad7 in Dillon, 1984b,
1988b) and which served as a control for the
present investigation. Our Georgia site was
identical to “Station X” of Krieger & Burbanck
(1976) and YELD of Krieger (1977), inhabited
by a population identified as “G. suturalis” in
the former publication and “G. boykiniana vi-
ennaensis’ in the latter. Population Cirk of G.
catenaria and population Bull of G. proxima
were sampled from tributaries of the Broad
River separated by only about 15 km through
water. The sites at which we collected all 12
populations may be located in Figure 1. Lo-
cality data for all sites are provided in the Ap-
pendix. Voucher specimens have been de-
posited in the Academy of Natural Sciences of
Philadelphia.

At each site we collected at least 30 indi-
vidual snails, which were cracked and stored
in Tris-phosphate 7.4 tissue buffer at -70°C
upon return. Allozyme variation was resolved
from whole-animal homogenates by horizon-
tal starch gel electrophoresis, using equip-
ment and techniques as previously described
(Dillon, 1985, 1992; Dillon & Lydeard, 1998).
We initially screened ten enzyme systems
and six buffers by requiring that polymor-
phism interpretable as the product of codomi-
nant Mendelian alleles be expressed in a

GONIOBASIS POPULATION GENETICS 25

Pee Dee

Santee

Altamaha

FIG. 1. The study area, showing primary drainages and sample sites. Circles indicate G. catenaria, and

squares G. proxima.

comparison of G. proxima population Yad, G.
catenaria postelliipopulation Yel, and G. cate-
naria dislocata population Srp.

Ultimately, we compared all populations on
the basis of eight loci, most resolved on two
buffer systems, as follows. The AP6 buffer of
Clayton & Tretiak (1972) was used to resolve
mannose-phosphate isomerase (MPI, EC
5.3.1.8), 6-phosphogluconate dehydroge-
nase (6PGD, EC 1.1.1.44) and isocitrate de-
hydrogenase (IDHF and IDHS, EC 1.1.1.42 —
cathodal and anodal loci, respectively). A
ТЕВ8 buffer (Shaw & Prasad 1970) was used
for esterases (EST1, ЕС 3.1.1.2 —the strong,
slow locus only), xanthine dehydrogenase
(XDH, EC 1.2.1.37), MPI, and IDHF. The

Poulik (1957) buffer was used for octopine de-
hydrogenase (ODH, EC 1.5.1.11), glucose-
phosphate isomerase (GPI, EC 5.3.1.9) and
EST1.

Chambers (1980) demonstrated that inher-
itance of allozyme phenotype is Mendelian at
the 6PGD locus in G. floridensis. Dillon (1986)
verified Mendelian inheritance at GPI, ODH,
and EST1 by mother-offspring analysis in G.
proxima. The designations of putative alleles
used for population Yad were retained from
the system of Dillon (19846) for GPI, MPI,
ODH, EST1 and XDH. For those loci which
had not previously been examined for popula-
tion Yad (6PGD, ISDHF, ISDHS), the most
common Yad allele was named “100.” Then

26 DILLON & REED

alleles in all other populations were named
according to the mobility of their allozymes in
millimeters relative to the Yad standard.

Data were initially analyzed using BIOSYS-
1 (Release 1.7; Swofford & Selander, 1981).
Genotype frequencies at all polymorphic loci
were tested for conformance to Hardy-Wein-
berg expectation within populations using chi-
square statistics, with Yates correction in 2 x
2 cases, pooling rare classes as required.
Mean F-statistics (Wright, 1978) were calcu-
lated across loci for the four populations of G.
proxima and the eight populations of G. cate-
naria separately. Values of Nei’s (1978) unbi-
ased genetic similarity and distance were cal-
culated pairwise over all populations, as well
as the chord distance of Cavalli-Sforza & Ed-
wards (1967). The matrix of chord distances,
which are Pythagorean in Euclidean space
(Wright, 1978), was input to STATISTICA (Re-
lease 5.0, Statsoft, Inc.) and clustered using
the method of unweighted pair-group averag-
ing (UPGMA).

RESULTS

Example shells are shown in Figure 2. Typ-
ical G. catenaria bore shells with both spiral
costae (or cords) and axial costae (or ribs) in-
tersecting to form nodules. The shells of G.
proxima had no axial costae and only a single
spiral costa (or carina) becoming obsolete
with age. Typical G. catenaria shells also
tended to be wider per unit length than G.
proxima. The shells of the Ye/ population,
nominally G. catenaria postellii, did not differ
noticeably from those of typical G. catenaria

catenaria. But as noted by Dillon & Keferl
(2000), the shells of G. catenaria dislocata
were narrower and lacked both spiral and
axial costae on later whorls, rendering them
more similar to G. proxima than typical G.
catenaria (Dillon & Keferl 2000).

For allozyme analysis, sample sizes aver-
aged across loci ranged from N = 29 at West
to N = 43 at Yad (Appendix). Sample sizes at
all 12 x 8 loci examined were greater than 30,
except N = 29 for Bull (XDH), N = 26 for Bull
(EST1), N = 14 for West (IDH), N = 20 for
Morg, (IDH), and N = 26 for Morg (GPI, XDH).

Allele frequencies are given in Table 1.
Over all observations, a total of 26 loci were
polymorphic by the 95% criterion, a value
somewhat higher than has been observed in
comparable surveys of pleurocerid population
genetics previously published (Chambers,
1980; Dillon & Davis, 1980; Dillon, 1984b; Dil-
lon & Lydeard, 1998). Fits to Hardy-Weinberg
expectation were excellent within popula-
tions. Chi-square tests uncovered no signifi-
cant differences between observed genotypic
frequencies and those expected from Hardy-
Weinberg equilibrium in any case. The values
ofF. for both G. proxima and G. catenaria av-
eraged over eight loci were negligible (Table
2).
As might be expected from their highly iso-
lated population structure, divergence among
the four G. proxima populations was high (F.,
= 0.461; Table 2). Table 1 shows fixed (or
nearly fixed) differences at a minimum of one
locus between all pairs of G. proxima, except
Morg —Bull. Divergence among the eight G.
catenaria was not as striking upon first exam-
ination, the trio of populations from the middle

FIG. 2. Example shells from the four taxa studied. From left, Goniobasis catenaria catenaria (Cirk), G. cate-
naria dislocata (Srp), G. proxima (Bull), and G. catenaria postellii (Yel). The standard length of the G. cate-
naria catenaria shell is 1.75 cm, the remaining shells are figured at the same scale.

GONIOBASIS POPULATION GENETICS 27

TABLE 1. Allele frequencies at 8 enzyme loci т 12 populations of Goniobasis from southern Atlantic
drainages of the United States.

G. catenaria
Locus allele Yad West Morg Bull Сик Uwh Lnch Cola Sant

ES 100; 0.167 0.191 0.513 0.219 0.188
103 0.802 0.794 0.474 0.885 1.000 0.734 0.813 1.000 1.000 0.679 0.789 0.524

а. proxima

Srp MeC Yel

106 0.031 0.015 0.013 0.115 0.047 0.321 0.211 0.012
107 0.463
GPI 98 0.014 0.976
100 1.000 1.000 1.000 1.000 0.986 0.024 0.162 1.000
102 1.000 1.000 1.000 1.000
105 0.784
110 0.054
IDHF 93 0.953
97 0.125
99 0.016 0.319
100 1.000 1.000 0.750 1.000
103 0.050
105 0.075 0.047 0.984 1.000 1.000 0.681 1.000 1.000 1.000
IDHS 100 1.000 1.000 1.000 1.000
104 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000
MPI 95 0.833 0.200 0.750
100 0.167 1.000 0.800 0.250 1.000 1.000 1.000 1.000 1.000 0.989 1.000 1.000
103 0.011
ODH 106 0.738 1.000 0.145
109F 0.125 0.855 1.000
111 0.118 0.431
113F 0.138
114 1.000 0.969 1.000 1.000 1.000 1.000 0.882 0.569
118 0.031
6PGD 100 1.000 0.882 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.522
105 0.118 0.422
110 0.056
XDH 97 1.000 0.731 0.603 0.015 0.875 0.390
98 1.000 0.269 0.397 1.000 1.000 1.000 0.985 0.125 0.610 1.000 0.568
100 0.432

TABLE 2. Values of Wright’s (1978) F-statistics
averaged over eight loci for four populations of G.
proxima and eight populations of G. catenaria.

the eight G. catenaria populations over the
three-state region was 0.564, similar to that
observed for G. proxima.

Nei’s statistics among all populations are

SE Szealenana presented in Table 3, and the result of the
EX 0.042 0.035 UPGMA cluster analysis based on interpopu-
Fr 0.484 0.580 lation chord distances is shown in Figure 3.
== 0.461 0.564 Each G. catenaria dislocata population was

of the study area (Uwh-—Lnch-—Cola) being
genetically indistinguishable. But the south-
ern populations were more divergent. The Yel
population of G. catenaria postellii had un-
usual alleles in high frequency at three loci
(EST1, 6PGD, XDH), and both the Srp popu-
lation of G. catenaria dislocata and the McC
population of G. catenaria catenaria were
strikingly distinct at the GPI locus. Population
Cirk was distinguished by a unique IDHF al-
lele. Consequently, the mean value of F,, for

more similar to its neighboring G. catenaria
catenaria population (| = 0.86, 0.89) than the
pair of dislocata were to each other (| = 0.81).
This tends to support the hypothesis that the
two populations of G. catenaria dislocata
have lost shell sculpture independently.
Reinforcing the impression left from the F-
statistics, Figure 3 suggests that the levels of
interpopulation divergence within G. proxima
and G. catenaria are comparably high. The
four G. proxima populations are nevertheless
quite distinct from the eight G. catenaria pop-
ulations, sharing no alleles at three loci (GPI,

28 DILLON & REED

TABLE 3. Nei’s (1978) statistics among pairs of Goniobasis populations. Unbiased genetic iden-
tity is shown below the diagonal, and unbiased genetic distance above.

Yel McC Srp Sant Cola Lnch Uwh Сик Bull Morg West Yad

Yel == 0.22. 70.29.”.0.21 ¿0:11 0.11” ¿0.107 027 - 1.481.215 124132
McC 0.81 = 015 0:24 0.10 0.10 0.11 0.24 1.08 :0:96 11:06, 0:92
Srp 0.75 0.86 = 0.21 0.17 0.17 0.17 .0.33 1.08 0.91 0.95. 1:10
Sant 0.81 0.78 0.81 ак 0.12 0.13 0.13 0.21, 0.96 0.80 0.73 1.23
Cola 0.90 0.91 0.84 0.89 zu 0.00 0.01 0.12 1.08 0.98 1.05 0.93
Lnch 0.90 0.91 0.84 0.88

1.00 = 0.00 0.13 1.13 096 1.08 0.94
Uwh 0.90 0.90 0.84 0.88 0.99 1.00 = 0.13 1.14 0.95 “1:09 095
Ск 0.76 0.79 0.72 0.81 0.89 0.88 0.88 == 1.07 1:00 1:05 0792
Bull 0.23 0.34 0.34 0.38 0.34 0.32 0.32 0.34 = 0:09" 0:28 70:16
Morg 0.30 0.38 0.40 045 0.38 0.38 0.39 0.37 0.91 = 0:15 70/26
West 0.29 0.35 0.39 0.48 035 0.34 034 0.35 0.76 0.86 Le 0.28

Yad 0.26 0.40 0.33 0.29 0.40 040 0.39 040 0.85 0.77 0.76

0:7 0.6 0.5 0.4 0.3
Cavalli-Sforza & Edwards Chord Distance

G. proxima

0.2

G. catenaria catenaria

| G. catenaria dislocata

| С. catenaria postellii

0.1 0.0

FIG. 3. UPGMA Cluster analysis of the 12 study populations, based on a matrix of genetic distances calcu-
lated using the chord method of Cavalli-Sforza & Edwards (1967).

ODH, IDHS) and almost completely distinct at
IDHF. Table 1 shows no evidence of hy-
bridization between the species, in the
Broad/Santee drainage between Cirk and Bull
or elsewhere.

DISCUSSION

Although inhabiting a more moderate cli-
mate and a less mountainous environment,
the population genetic structure of G. cate-
naria does indeed resemble that of the better-
studied G. proxima. The similarity of the G.
catenaria populations inhabiting the Pee Dee
drainage (Uwh and Lnch) and the (apparently
isolated) Santee populations (particularly
Cola) was unexpectedly high. But the overall
values of Wright’s F-statistics and most val-
ues of Nei’s genetic distances among G. cate-

naria populations were comparable to values
obtained from G. proxima.

Values of genetic similarity and distance
weakly suggest that our two dislocata popula-
tions may have independently lost shell sculp-
turing, Sant deriving from Cola and Srp from
McC. But as indicated in Figure 3 and Table 3,
the two putative source G. catenaria catenaria
populations, Cola and McC, are more similar
to each other than either is to its G. catenaria
dislocata neighbor. Sant and Srp do share a
unique allele (GP198) not found in any other
population, and are also more similar to each
other at the XDH locus than either is to G.
catenaria catenaria. Additional data will be re-
quired before the origin of G. catenaria dislo-
cata can be proposed with any confidence.

It is clear, in any case, that G. catenaria dis-
locata populations are not extralimital G. prox-
ima, nor do they bear more genetic similarity

GONIOBASIS POPULATION GENETICS 29

to G. proxima than to other G. catenaria of
more typical shell morphology. We found no
evidence of hybridization between the two
species, in the upper Santee tributaries where
they are closely neighboring or elsewhere.
Goniobasis proxima and G. catenaria are
quite distinct.

As might be predicted from its geographic
situation on the periphery of the study area,
population Ye/ was the most genetically dis-
tinctive of the populations here identified as
G. catenaria (Fig. 3). But the level of diver-
gence displayed clearly does not warrent the
removal of this population to another specific
name. Population West is more different from
other G. proxima than population Yel is from
other G. catenaria. Goodrich’s (1942) sugges-
tion that Goniobasis populations from this re-
gion of Georgia be referred to G. catenaria as
a subspecies postellii, rather than distin-
guished as a separate species, would seem
to have considerable merit.

Based on a comparison of his freshly col-
lected shells to shells in the University of
Michigan collections, Krieger (1977) identified
Georgia populations of Goniobasis from the
Atlantic drainages as G. postellii in the
Oconee River, G. suturalis (= G. mutabilis) in
the Yellow River above Porterdale, and G.
boykiniana viennaensis in the Apalachee
River and in the Yellow/Ocmulgee River
below Porterdale. Mihalcik (1998) identified a
population of Goniobasis from Snapping
Shoals on the South River, a tributary of the
Ocmulgee 20 km west of Porterdale, as G.
mutabilis. She identified a population from an
Ocmulgee tributary downstream about 350
km from Porterdale as G. timida, and sug-
gested that a third population from the Atlantic
tributaries of Georgia, Rocky Creek of the
Oconee drainage, might be an undescribed
“Species C.” Mihalcik referred Goniobasis
populations from the Savannah River to G.
bentoniensis.

Neither Krieger nor Mihalchik examined
populations from South Carolina, nor did ei-
ther researcher consider the possibility that
Georgia Goniobasis might be referable to the
older name G. catenaria. Resolution of the
longstanding systematic confusion regarding
the Goniobasis of Georgia would seem a fer-
tile direction for future studies.

ACKNOWLEDGMENTS

We thank Mr. Kevin Swift for help with the
field work. This research was supported by

grants from the Biology Department, College
of Charleston.

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APPENDIX

Identification and locality data for study popu-
lations. The sample sizes given are averaged
over the 8 enzyme loci reported in Table 1.

Bull —G. proxima (N = 31). Bullock's Creek at
SC 889 bridge (Boheler Road), 10.9 km
NNW of York, York Co., South Carolina.
81°19’W, 35°05'N. Same as site 20p of
Dillon & Keferl (2000).

Cirk —G. catenaria саепапа (N = 32). Clarks
Fork Creek at SC 55 bridge, 16.1 km NW
of York, York Co., South Carolina.
81°24'W, 35°05’N. Same as site 17c of
Dillon & Keferl (2000).

GONIOBASIS POPULATION GENETICS 31

Cola—G. catenaria catenaria (N = 34). Big
Cedar Creek at SC 59 bridge (Wildflower
Road), 21.3 km N of Forest Acres, Rich-
land Co., South Carolina. 81°06’W,
34°11'N. Same as site 23c of Dillon & Ke-
ferl (2000).

Lnch—G. catenaria catenaria (N = 32).
Lynches River at SC 265 bridge, 3.8 km
SW of Jefferson, Chesterfield/Lancaster
Cos., South Carolina. 80°26’W, 34°38'N.
Same as site 40c of Dillon & Keferl
(2000).

McC—G. catenaria catenaria (N = 38).
Stevens Creek at SC 23 bridge, 24.6 km
WSW of Edgefield, Edgefield/McCormick
Cos., South Carolina. 82°11'W, 33°44'N.
Same as site 4c of Dillon & Keferl (2000).

Morg — G. proxima (N = 33). Small tributary of
Clear Creek at bridge 50 m N of U.S. 64,
5 km S of Morganton, Burke Co., North
Carolina. 81°45'W, 35°40'N.

Sant —G. catenaria dislocata (N = 36). Head
of Chapel Branch, Santee, Orangeburg
Co., South Carolina 80°29'W, 33°30'N.

Same as site 28d of Dillon & Keferl
(2000).

Srp—G. catenaria dislocata (N = 39). Lower
Three-Runs Ck at SC 70 bridge, 9 km $
of Snelling, Barnwell Co., South Carolina.
81°27'W, 33°11'М. Same as site 6d of
Dillon & Keferl (2000).

Uwh-G. catenaria catenaria (N = 32).
Uwharrie River at NC 109 bridge, 1 km
NW of Uwharrie, Montgomery Co., North
Carolina. 80°01’W, 35°26'N.

West -G. proxima (N = 29). Small tributary of
the Chauga River at SC 196 bridge, 1km
W of Mountain Rest, Oconee Co., South
Carolina. 83°10'W, 34°52'N.

Yad—G. proxima (N = 43). Naked Creek at
NC 1154 bridge, 5.2 km N of Ferguson,
Wilkes Co., МС. 81°22’W, 36°09’N.
Same as Yadk of Dillon & Davis (1980),
Yadl of Dillon (1984).

Yel—G. catenaria postellii (N = 40). Yellow
River below dam at Porterdale, Newton
Co., Georgia. 83°53'W, 33°34'N.

MALACOLOGIA, 2002, 44(1): 33-46

SPATIAL DISTRIBUTION, DENSITY AND LIFE HISTORY IN FOUR ALBINARIA

SPECIES (GASTROPODA, PULMONATA, CLAUSILIIDAE)

Sinos Giokas' & Moysis Mylonas?

ABSTRACT

In this study, we analysed field sequential data on density, mortality, and spatial distribution of
four Albinaria species. We also recorded and compared a series of life-history traits, including
the onset and end of aestivation, copulation, oviposition, and hatching. Our aim was to obtain
more information on the ecology of Albinaria, to reveal possible life-history patterns, and to un-
derstand the association of life-history characteristics with both extrinsic (density independent)
and intrinsic (density dependent) factors and microevolutionary processes. The onset of aesti-
vation period occurs always in April and is independent of the end of rainfall, whereas awaken-
ing and copulation is actually synchronous and occurs after the first autumn rains. The duration
ofthe oviposition and hatching period is short, but oviposition can be prolonged under favourable
climatic conditions. Density is high, but its fluctuations were not related to climatic or time factors.
Any density dependent pattern resulting from competition was not detected. Higher mortality did
not coincide with aestivation, and usually relatively high mortality results after hatching. Juveniles
usually exhibit higher mortality than adults. Spatial distribution of these rock-dwelling snails is al-
ways highly contagious. Clustering behaviour was probably influenced by the substratum, mo-
saic or uniform, and the occurrence of crevices. Predictions about the population dynamics of
these iteroparous, long-lived species were not possible because of sampling inadequacies
and/or because fluctuations of population structure, density, mortality and spatial distribution

were random.

Key words: Albinaria, Greece, life history, density, mortality, spatial distribution.

INTRODUCTION

Population dynamics is affected, either di-
rectly or indirectly, by such density-indepen-
dent factors as climate, and/or such density-
dependent factors as predation, disease and
competition. Life-history studies are essential
for understanding the role of these factors and
the action of natural selection (Stearns, 1992).
However, in land snails the influence of these
factors is often abstruse or inadequately un-
derstood. Though regulated life cycles are not
likely to be distinguishable from random den-
sity fluctuations (May, 1975; Cook, 1990), sev-
eral studies indicate the effect of environmen-
tal conditions on abundance (Baur & Raboud,
1988; Heller & Dolev, 1994; Lazaridou-Dimitri-
adou & Sgardelis, 1997), or the influence of
intraspecific crowding on density, via its direct
or indirect affect on individual fecundity, sur-
vival, locomotion, adult size, and growth rate
(Yom-Tov, 1972; Cameron & Carter, 1979;
Dan & Bailey, 1982; Tilling, 1985; B. Baur,
1988; Staikou & Lazaridou-Dimitriadou, 1989;

A. Baur & B. Baur, 1992). Resource or inter-
ference competition is another controversial
subject in land snails (Pearce, 1997).
Albinaria is a pulmonate genus distributed
around the northeastern coasts of the Mediter-
ranean, exhibiting a high degree of morpho-
logical and molecular differentiation. More
than 90, in some cases dubious (Douris et al.,
1995, 1998a, b), nominal species have been
described (Nordsieck, 1999). Ecological stud-
ies of Albinaria populations can be very infor-
mative about evolutionary processes in this re-
gion, which has a complex palaeogeographic
and climatic history (Paepe, 1986; Anasta-
sakis & Dermitzakis, 1990; Westeway, 1994),
because the high species number does not
appear to be associated with proportionate
ecological differentiation (Gittenberger, 1991).
However, although the biogeography and sys-
tematics of Albinaria have been well consid-
ered (for a review, see Giokas, 2000), and A/-
binaria species constitute about 15% of the
total Greek land-snail species, studies of their
life history are lacking. Similarly, very few data

‘Corresponding author — Zoological Museum, Department of Biology, University of Athens, GR-15784, Athens, Greece;

sinosg O biol.uoa.gr

Natural History Museum, Department of Biology, University of Crete, GR-71409, lrakleion, Greece; director @nhmc.uch.gr

34 GIOKAS & MYLONAS

are available (Warburg, 1972; A. Baur, 1990;
B. Baur & A. Baur, 1990; 1991, 1992; Heller &
Dolev, 1994) on the biology and life history of
the Clausiliidae.

Albinaria snails live on limestone substrata
(Schilthuizen, 1994; Giokas, 1996). They feed
on the microflora, that is, lichens and
bryophytes. They are active only during the
wet season, which in southern Greece is from
late October through the end of April. Eggs
are laid in clutches of about 5-7 eggs, gener-
ally shortly after the beginning of the wet sea-
son. During the intervening dry periods, juve-
niles and adults aestivate on the rock
surfaces, in rock crevices, and occasionally
on shrubs, or under stones. Especially during
aestivation, aggregates are often formed,
sometimes including many tens of individuals.
Albinaria live about seven years. The devel-
opment from a juvenile (juveniles do not have
a lip or well-formed internal lamellae) to a fully
grown snail takes two and occasionally three
wet seasons. Shell development precedes
maturation, which occurs during the last dry
season after enlargement of genitalia size.
Copulation then takes place during the first
weeks of autumn rains. Population densities
can sometimes be very high, in spite of mor-
tality caused by desiccation, especially during
aestivation, or of occasionally heavy preda-
tion by larvae of the beetle family Drilidae
(Schilthuizen et al., 1994), or by typical preda-
tors of snails, such as rodents and birds.

This is the first comparative field study of
both qualitative and quantitative life history
characteristics for Albinaria. We analysed se-
quential density, mortality, and spatial distri-
bution data of four well-defined Albinaria
species. We also recorded and compared a
series of life-history traits, including the onset
and end of aestivation, copulation, oviposi-
tion, hatching, and aggregation. Our aim was
to obtain more information on the ecology of
Albinaria, to reveal possible life-history pat-
terns and to understand the association of
life-history characteristics with both extrinsic
(density independent) and intrinsic (density
dependent) factors and microevolutionary
processes.

MATERIALS AND METHODS
Species Studied

Four different Albinaria species, with each
one population per species, were studied

(Fig. 1). These were A. coerulea (Deshayes,
1835)— а{ Vravrona, Attiki, on the Acropolis hill
beside the archaelogical site, A. turrita (Pfeif-
fer, 1850) — Kea island, N. W. Kyklades,
around the Monastery of Panagia Kastriani,
A. discolor (Pfeiffer, 1846) — Aigina Island, Ar-
gosaronikos Gulf, 5 km east from Agios Nek-
tarios Monastery, and A. voithii (Rossmassler,
1836) — at Parori, Lakonia, near Sparti, at the
entrance of the gorge.

Habitat and Bioclimatic Characteristics of the
Studied Sites

Table 1 shows the main bioclimatic
(Mavrommatis, 1978) and habitat characteris-
tics of the studied sites. A/binaria coerulea, A.
turrita and A. discolor face the same biocli-
matic conditions, which generally can be
characterised as arid. However, A. voithii lives
in a more humid habitat and the dominant
vegetation type at Parori is maquis and not
phrygana as in the other areas. Aigina, though
arid, has a mixed vegetation (phrygana and
maquis). Other climatic data with unambigu-
ous seasonality (temperature, precipitation,
humidity) are not presented because they
were uncorrelated with the examined life his-
tory parameters (see Results). All the above-
studied populations are very localized and re-
stricted to the sampled areas, which can be
as small as that of Parori. Though the sub-
Strata at all sites is calcareous, sites are dif-
ferentiated by the presence and density of
crevices and calcareous plates, and the con-
tinuous or mosaic allocation of rock bulks.

Sampling Procedure

We adopted the quadrat random sampling
procedure (Krebs, 1989; Chalmers & Parker,
1986; Williams, 1987). Within each site, sam-
ples were taken every month for A. coerulea
(April 1992-April 1994) and on a seasonal
basis for A. voithii (April 1992-August 1994),
A. turrita (August 1992-April 1994), and A.
discolor (July 1992-April 1994). The size of
the sampling quadrats (50 cm x 50 cm) was
determined after preliminary sampling work
(Krebs, 1989). A 20% confidence limit was
chosen (Elliot, 1971; Hayek & Buzas, 1997) to
set the number of sampling quadrats for each
studied species. The number of quadrats on
each sampling occasion was 15 in Vravrona,
and 20 in Parori, Kea and Aigina. During aes-
tivation we sampled once. All samplings were

LIFE HISTORY IN ALBINARIA 35

a >

TURKEY

FIG. 1. The distribution of the genus Albinaria (shaded area) and the studied sites.

done on non-rainy days, at approximately
9.00 a.m. In each quadrat, all the adult and ju-
venile specimens, both alive and dead, were
recorded; we counted only the dead speci-
mens still attached to the substratum and not
the empty shells fallen to the ground. Dead
specimens were removed after each sam-
pling.

One month before the start and the end of
the aestivation period and one month after
that period we were visiting the sampling sites
more often (every ten days at Vravrona) in
order to record more precisely alterations of
associated life-history traits (aestivation onset
and ending, copulatory activity, oviposition,
hatching).

Statistical Analysis

Besides descriptive statistics, we used
ANOVA and Tukey HSD tests in order to
analyse density fluctuations for the alive and
dead specimens (adults and juveniles). We
used log-transformed values. We examined
ratio (adults/juveniles mortality) differences

with contingency table analysis (G-test,
Fisher exact test) (Zar, 1984). Additionally, we
used time-series analysis (Wilkinson, 1989),
in order to investigate possible autocorrela-
tion and cross-correlation patterns for the
density series of alive and dead specimens
(adults and juveniles).

Spatial distribution was estimated using: (a)
Green’s index of dispersion Gl = (s?/m) — 1/(n
— 1), (m = mean number of individuals, s* =
variance, and n = total number of individuals
in sample), and (b) Taylor’s Power Law (s? =
am?) (a and bare population parameters) (El-
liot, 1971; Ludwig & Reynolds, 1988; Hayek &
Buzas, 1997). Parameter b, in Taylor’s Power
Law, can vary from zero to infinity. When b >
1 the distribution is contagious, when b= 1 the
distribution is random and when b < 1 the dis-
tribution is uniform.

Finally, we used MANOVA and multiple re-
gression analysis in order to examine possi-
ble effects of climatic (precipitation, humidity,
temperature) and time (month, season, year)
factors to density, mortality and spatial distri-
bution.

GIOKAS & MYLONAS

36

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LIFE HISTORY IN ALBINARIA 37

RESULTS

Aestivation, Awakening, Copulation, Oviposi-
tion, Hatching, Activity

Aestivation always began in April. However,
at the more arid sites of Vravrona and Kea
aestivation began earlier (first week of April)
than at Aigina and Parori (last week of April).
Specifically, at Vravrona, on average for the
three sampling years, in the middle of April
77.4% of adults had formed epiphragm, while
at the end of April 100% of them were fully
aestivating. The onset of the aestivation pe-
riod was always independent of the end of the
rainfall period. It is notable that aestivation,
both in the arid springs of 1992 and on the
much more humid and rainy of years 1993
and 1994, started approximately at the same
time (April) for all species.

Awakening and copulation occurred only
after the first autumn rain (in 1992 in the mid-
dle of October and in 1993 in early Novem-
ber). However, destruction of the epiphragm
started in early October. More precisely at
Vravrona, whereas in mid-September we did
not observe any specimens with fragmenting
epiphragm, in the beginning of October, on
average for the two years, 86% of adults had
an epiphragm with some holes, and at the end
of October all specimens had no trace of
epiphragm. Copulation is actually immediate
and synchronous, and it is the main activity of
Albinaria specimens after awakening. Copu-
lation period lasts for about a week on aver-
age.

Oviposition starts about 20 days after cop-
ulation and stops after ten days, but at the
more humid site of Parori continued almost
until the end of the wet period. Eggs were usu-
ally laid in crevices or under stones in
clutches of about five to eight eggs. Hatching
occurred approximately two to three weeks
after oviposition.

During the wet period, on the sampling
days, only a portion of the population was ac-
tive, feeding on the microflora. However, we
did not estimate that portion.

Density

Table 2 shows the mean densities (speci-
mens per 0.25 m?) of the live specimens of the
four studied species, and the significant differ-
ences (between the sequential samples of
each species) are indicated, Figure 2 shows
density fluctuations during sampling. Autocor-

TABLE 2. Mean density of alive specimens (speci-
mens/0.25 m?) of the studied Albinaria species.
Asterisks indicate significant (at o. = 0.05) density
differences between sequential samples of each
population.

total adults juveniles
A»coerulear 14.132417 9.1=24” 5:0) 0:7*
A. turrita 84-15 44-07 4:0 ls
A. discolor OS (6:9 1.2 MIE 105
A. voithii 9.4 = 1.57 5:0 = 0:9* 4.4 =0:8*

relation analysis showed that these were гап-
dom series. It is notable that mean densities
can be quite high. Higher densities were usu-
ally observed during the aestivation period.
However, in neither species, the differences
between sequential samples that were sug-
gested by the Tukey HSD test could be attrib-
uted (MANOVA, Multiple regression) to cli-
matic (temperature, precipitation, humidity)
and time (year, season, month) sources of
variation, and consequently are not pre-
sented.

Significant mean density differences (adults
plus juveniles) were not found among the four
studied populations (ANOVA, p = 0.05). Like-
wise, the mean densities of the adult speci-
mens did not differ significantly (ANOVA, p =
0.09), but mean densities of juveniles differed
significantly (ANOVA, p = 0.003), and the
Tukey HSD test indicated significant differ-
ences between juveniles at Aigina with those
at Vravrona and Parori.

Mortality

Mean densities of dead specimens (speci-
mens per 0.25 m?) of the four studied species,
and significant differences (between the se-
quential samples of each species) are shown
in Table 3; Figure 3 shows density fluctua-
tions during sampling. Adults and juveniles
usually exhibit high mortality after hatching
period; however, differences between se-
quential samples were not caused by climatic
and time sources of variation (MANOVA, Mul-
tiple regression).

Significant differences between the mean
densities of dead individuals (adults plus ju-
veniles) were not found among the four stud-
ied populations (ANOVA, p = 0.05). Mean
densities of adult dead specimens differed
(ANOVA, p = 0.015), and the Tukey HSD test
showed differences between adults at Aigina
and those at Kea and Parori. Densities of

38 GIOKAS & MYLONAS

A соеплеа

45

specimens/0.25m*

specimens/0 25m?

8!

specimens/0 25m с

6}

| | |
4) | >
2} |
|
0 = ==)
Jul Dec Mar Jun Dec Mar

1992 nes

months

FIG. 2. Density fluctuations of alive specimens for the studied Albinaria species (in bars black stands for
adults and white for juveniles).

LIFE HISTORY IN ALBINARIA

A coerulea

specimens/0 25m?

specimens/0 25m°

specimens/0 25m?

specimens/0 25m°

FIG. 3. Density fluctuations of dead specimens for the studied Albinaria species (In bars black stands for
adults and white for juveniles).

39

40 GIOKAS & MYLONAS

dead juveniles (ANOVA: p = 0.016) also dif-
fered significantly, and the Tukey H.S.D. test
indicated significant differences between Kea
and Parori.

Mean mortality ratio (dead specimens/
(dead + living specimens)) can vary signifi-
cantly (p < 0.0001) between sequential sam-
ples (Table 3, Fig. 4). Juvenile mortality (the
ratio of dead juveniles to dead + living juve-
niles) was significantly greater (p < 0.05) than
adult mortality (dead adults/dead + living
adults), across time, for A. coerulea and A.
voithii and greater, however not significantly
for A. discolor. In A. turrita, juvenile and adult
mortality did not differ significantly, even
though juvenile mortality was lower than that
of adults.

Spatial Distribution

Spatial distribution was always contagious
(Table 4), according to the parameters a and
b of Taylor's Power Law and Green’s Index,
except in the case of living adults of A. turrita.
Values of b were (except for A. turrita) high
(above 2). Higher values of b (for total and
adults) were estimated for À. discolor and A.
coerulea. For juveniles, A. coerulea had the
lower b value. Adults were more aggregated
than juveniles, except for A. turrita. For dead
specimens, values of b were more or less of
the same order. However, comparison of
slopes bis not always justified, because bcan
be affected by scale changes (Hayek &
Buzas, 1997).

DISCUSSION

The start of the aestivation period, regard-
less of site, year or species, was not corre-

TABLE 3. Mean density of dead specimens (speci-
mens/0.25 m?) of the studied Albinaria species.
Asterisks indicate significant (at с = 0.05) density
differences between sequential samples of each
species. In parentheses mean mortality ratio (dead
specimens/dead + living specimens).

total adults juveniles
A. coerulea 1.4 = 0.2 07+01 0.6 = 0.1*
(9%) (7.1%) (10.7%)
A. turrita 0.5=0.1 "103: =:0.1/. O12 01
(5.6%) (6.4%) (4.8%)
A. discolor 1.38 015" 1.3 =0:4% 05 +101
(17.3%) (15.8%) (22.7%)
A. voithii 1.2: 0:3* 70:4 0/2" 0.3071”
(11.3%) (7.4%) (15.4%)

lated directly to certain climatic conditions
(rain, temperature and humidity). Even
though it is not possible to separate geo-
graphical, species and population effects,
probably this implies that aestivation onset in
Albinaria is relatively independent from these
fluctuating environmental factors, and the
possible cardinal action of an unknown intrin-
sic physiological mechanism possibly associ-
ated with day-length perception. However, the
end of the aestivation period and the onset of
copulation require rainfall (yet, not before the
end of September). Heller & Dolev (1994) re-
ported similar observations for the clausiliid
Cristataria genezarethana. These life-history
characteristics have been observed for sev-
eral other (32) Albinaria populations belong-
ing to 13 species sampled from 1990 to 1995
throughout Greece (Giokas, 1996). Addition-
ally, laboratory experiments with 25 Albinaria
populations belonging to 12 species (among
them the studied species of A. coerulea, A.
turrita, A. discolor and A. voithil) proved that
awakening is actually synchronous (on aver-
age 90% of snails awake within five hours)
and that copulation is the main activity of Albi-
naria specimens for the first 36 hours after
awakening (Giokas, 1996). According to
Heller & Dolev (1994) and Lazaridou-Dimitri-
adou & Sgardelis (1997), the rapid awakening
response of aestivating snails to autumn rains
is advantageous in seasonally dry habitats,
which prevail in Mediterranean climate condi-
tions. In that way, Snails avoid adverse effects
during unfavourable conditions. Several Albi-
naria species, for example, those belonging to
the grisea group, which aestivate under
stones or deep in crevices and have a thin,
transparent shell, awake later (in late Novem-
ber) (Giokas, 1996), and Giokas et al. (2000),
support that awakening is partly associated
with aestivation behaviour and morphological
features, for example, thin shell.

Oviposition and hatching stopped after the
first month of the wet period at the more arid
sites of Vravrona, Kea and Aigina. However,
at the more humid and inland Parori site,
oviposition and hatching continued through-
out the active period, though mating was re-
stricted to its beginning. Likewise, according
to Lazaridou-Dimitriadou & Sgardelis (1997),
coupling and oviposition are more or less con-
temporaneous within populations of land
snails living in coastal habitats, where the
suitable period is relatively short, whereas
breeding lasts longer for the inland species.
Perhaps egg laying period can be prolonged

41

LIFE HISTORY IN ALBINARIA

A coerulea

Анерош %

months

A. tumta

months

A. discolor

months

A. voithii

Jul

months

FIG. 4. Fluctuations of mortality ratio (dead specimens/(dead + living specimens)) for the studied populations

of Albinaria (black bars stand for total, hatched bars for adults, and white bars for juveniles).

GIOKAS & MYLONAS

42

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LIFE HISTORY IN ALBINARIA 43

under favourable conditions for the survival of
the juveniles. Giokas (1996) reports that A/bi-
naria specimens collected during aestivation
laid eggs during captivity in the laboratory
without copulation (among them A. coerulea
and A. voithii), suggesting that sperm or fer-
tilised eggs can be retained as in other land
snails (Duncan, 1975; Tompa, 1984).

Mean values of density (Table 2) are high
and comparable with those reported for other
clausillids. For example, Vestia elata has 400
specimens/m?, with a maturation period of
five years (Piechocki, 1982), and Balea per-
versa has 70 specimens/m”, with a matura-
tion period of two years (B. Baur & A. Baur,
1992). The Israeli Alopiinae land snail Crista-
taria genezarethana (Heller & Dolev, 1994)
has a mean density of 150-200 individu-
als/m?. Heller & Dolev (1994) attribute those
high densities to the presence of crevices, the
absence of predators and competitive spe-
cies, and to the snails’ small size. Additionally,
they consider that low copulation frequencies,
as a response to lack of food, and growth in-
hibition (11 years to mature), as a response to
high population density through limited activ-
ity (prevention of simultaneous demand for
food, lichens with extremely low growth rate,
in quantities that the rock surface may not be
able to supply) or competitive action of adult
specimens, may regulate population size.
However, studies of resource and interfer-
ence competition in land snails have resulted
to argumentative outcomes (Pearce, 1997).
These particular density-dependent mecha-
nisms suggested by Heller & Dolev (1994)
were not proved to act in Albinaria, in which
(Schilthuizen, 1994; Giokas, 1996): copula-
tion is synchronised, maturation comes after
1.5-2 years, Albinaria specimens are of the
same size (on average 18 mm height) as
Cristataria genezarethana, have the same
diet preferences and similar activity pattern,
and food (lichens and bryophytes) was al-
ways abundant (however only a portion of
specimens was active simultaneously). Addi-
tionally, in neither case, at any time lag, higher
mortality as a consequence of high density
was observed. Therefore, in Albinaria intra-
population competition for food and space or
interference competition that would influence
survival and abundance is still ambiguous
(but of course needs further field and labora-
tory examination).

The abundance of the four studied Albinaria
species fluctuated almost randomly through-
out the sampling period, exhibiting no de-

tectable trend, despite the stable oscillations
of the environmental parameters. Lazaridou-
Dimitriadou & Sgardelis (1997) have reported
a similar pattern for Bradybaena fruticum and
Eobania vermiculata. Though Lazaridou-Dim-
itriadou & Sgardelis (1997) also demonstrated
several examples of predictable phenological
oscillation patterns, they claimed that the lat-
ter are more evident for semelparous and
short-lived species. Albinaria species are
iteroparous and relative long-lived, as are
Bradybaena fruticum and Eobania vermicu-
lata, and population fluctuations could not be
attributed directly to extrinsic environmental
conditions. However, the role of intrinsic fac-
tors remained unclear, in part because regu-
lated life cycles are often difficult to distinguish
from random density fluctuations (May, 1975;
Cook, 1990), or because our sampling tech-
niques failed to estimate with accuracy popu-
lation dynamics. Density estimates of several
Albinaria populations (Giokas, 1996) con-
firmed the established notion (Lazaridou-Dim-
itriadou & Sgardelis 1997) that population
densities of different snail species may differ
considerably, and that even populations of the
same species do not always exhibit a peak
density at the same time of the year.

Mean estimated mortality percentage was
generally low (Table 3) yet higher than the es-
timation of 5% for Cristataria genezarethana
(Heller & Dolev, 1994) and the estimation of
2.5% for marked aestivating specimens of A.
discolor (Giokas et al., 2000). However,
higher mortality did not coincide with aestiva-
tion period (Fig. 4). Besides sampling defi-
ciencies, we can suggest that these rock-
dwelling snails do not suffer considerably
during the adverse hot and dry period, taking
into account the tendency of Albinaria speci-
mens to form clusters near deep and narrow
crevices, the thickness and the white colour of
the shell, and the protective function of the
clausilium. However, high mortality, especially
of adult specimens, was usually reported after
hatching (Fig. 4). Probably this indicates that
a major mortality cause is weakening after
parental investment. Lazaridou-Dimitriadou
(1995) came to a similar conclusion for Heli-
cella pappi. Juveniles also exhibit high mor-
tality after hatching, probably because of low
intolerance.

Albinaria turrita and A. discolor juveniles did
not exhibit significantly higher mortality than
adults, even though A. discolor juveniles
show higher mortality than adults. Heller and
Dolev (1994) state the same for Cristataria

a GIOKAS & MYLONAS

genezarethana, whereas A. Baur (1990) re-
ports higher mortality for the juveniles of
Balea perversa. That was unexpected, be-
cause Albinaria juveniles lack some internal
shell morphological features (lamellae and
door like clausilium) that may prevent desic-
cation (Gittenberger & Schilthuizen, 1996),
even though their significance is disputed
(Arad et al., 1995; Giokas, et al., 2000). Per-
haps aggregation and crevice-dwelling can
be in some cases important for juvenile sur-
vival, even when juvenile mortality is higher
than adult mortality (A. coerulea and A.
voithii).

The contagious spatial distribution of the
four studied Albinaria species conformed to
the general attitude of the xero-thermophilic
species (Lazaridou-Dimitriadou, 1981). This
behaviour seems to be important for rock-
dwelling snails (Arad et al., 1995), and espe-
cially for Clausiliidae (Heller & Dolev, 1994;
Arad et al., 1995), and Albinaria (Warburg,
1972; Kemperman & Gittenberger, 1988;
Schilthuizen & Lombaerts, 1994), as in this
way loss of water and predation is inhibited.
However, the fluctuations and differences of
the contagious spatial distribution are not eas-
ily explainable given the problems that con-
cern the sampling procedure and the indices
used. Nevertheless, higher contagious distri-
bution could possibly be attributed to the frag-
mentation of the biotopes and the presence of
crevices. Parameter b of Taylor's Power Law
had higher values on Aigina and Vravrona.
Rocks on the site of Aigina are scattered, and
on Vravrona crevices are very abundant. On
the contrary, on Kea and Parori the suitable
and favourable rock habitats are united, and
there is a relative lack of crevices.

Unfortunately, the relatively instant picture,
on which we had to rely, as in most short-term
life-history studies, does not allow us to fore-
cast, because on investigating the effect of in-
trinsic and extrinsic factors we have to con-
sider several aspects that prevail or change in
time and space. Consequently, some popula-
tion characteristics, such as density, mortality,
and spatial distribution, cannot be predicted
per se, and we must be skeptical about fore-
casting.

ACKNOWLEDGMENTS

We are grateful to M. Lazaridou-Dimitri-
adou, from the Aristotle University of Thessa-
loniki, for her helpful comments, to the editor

and two anonymous referees for their con-
structive suggestions, and to Stephen
Roberts for grammatical suggestions.

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MALACOLOGIA, 2002, 44(1): 47-105

THE EASTERN PACIFIC RECENT SPECIES OF THE CORBULIDAE (BIVALVIA)

Eugene V. Coan

Department of Invertebrate Zoology & Geology,' California Academy of Sciences, Golden Gate
Park, San Francisco, California 94118-4599, U.S.A.; gene.coan @sierraclub.org

ABSTRACT

There are 18 Recent species of the Corbulidae in the eastern Pacific, of which one has been
introduced from the northwestern Pacific. Division of Corbula into additional genera is premature
without new characters and a formal cladistic analysis. Seven subgenera are utilized, with six
species remaining in Corbula, s. |. Three new species are described: C. (Caryocorbula) otra, C.
(Varocorbula) grovesi, and Corbula ($. |.) colimensis. One neotype and 14 lectotypes are desig-
nated. The distributions and habitats of the species are documented, along with their fossil oc-
currences and the relationships to other Recent and fossil species.

Key words: Corbulidae, Corbula, eastern Pacific, western Atlantic.

INTRODUCTION

Previous Treatments

Deshayes (1845-1848: 212-237, pls. 20,
21) described and illustrated the anatomy
of Corbula gibba (Olivi, 1792) (as “C. stri-
ata” Fleming, 1828, ex Walker ms, one of
its synonyms) and Lentidium mediterraneum
(Costa, 1829). White (1942) described but did
not figure the pericardial area of C. gibba, and
Yonge (1946) discussed the anatomy of C.
gibba (without reference to Deshayes’ earlier
treatment). Morton (1986) discussed the biol-
ogy and functional morphology of C. crassa
Reeve, 1843, at the same time questioning
whether the Corbulidae are properly placed in
the Myoidea. However, the only molecular
phylogenies thus far published that have in-
cluded a corbulid closely ally Corbula to Mya
(Adamkewicz et al., 1997; Steiner & Hammer,
2000).

Important early papers describing a number
of species of Corbula at the same time are
those of G. B. Sowerby | (1833), Hinds
(1843), and C. B. Adams (1852a, b). Reeve
(1843-1844) published the only comprehen-
sive monograph on Corbula. Tryon (1869)
listed the then-known species. Lamy (1926)
discussed the species of Lamarck, and then
gave synonymies for many of the known
species (Lamy, 1941).

Dall (1898, 1900) discussed the genera of

the family in connection with his review of the
fossil species of the eastern United States.
Vokes (1945) and Keen (1969b) also covered
the genera of the family. Zhuang & Cai (1983)
treated the species of China, and Habe (1977:
280-284) those of Japan.

Anderson (1994, 1996) discussed the
species of the Neogene of the Dominican Re-
public, and showed that the eastern Pacific
corbulids now average larger than those of
the western Atlantic (Anderson, 2001).

McLean (1942) discussed sculptural differ-
ences between very inequivalve and less in-
equivalve species of Corbula. Bacesco et al.
(1957) and Gomoiu (1965) treated popula-
tions of Lentidium mediterraneum (Costa,
1829), and Hrs-Brenko (1981) those of Cor-
bula gibba. Lewy & Samtleben (1979), de
Cauwer (1985), Morton (1986), Anderson
(1992), Harper (1994), and Kardon (1998)
discussed predation of corbulids and the role
of the thick layer of conchiolin in combatting it.

A preliminary outline of the results of the
present study is given in Coan & Skoglund
(2001).

Format

In the following treatment, each valid taxon
is followed by a synonymy, information on
type specimens and type localities, notes on
distribution and habitat, and an additional dis-
cussion.

Mailing address: 891 San Jude Avenue, Palo Alto, California, 94306-2640, U.S.A.; also Research Associate, Santa Barbara
Museum of Natural History and Los Angeles County Museum of Natural History.

48 COAN

The synonymies include all major accounts
about the species, but not most minor men-
tions in the literature. The entries are
arranged in chronological order under each
species name, with changes in generic allo-
cation from the previous entry, if any, and
other notes given in brackets.

The distributional information is based on
Recent specimens | have examined, except
as noted. Fossil occurrences are taken from
the literature, except as noted.

References are provided in the Literature
Cited for all works and taxa mentioned.

Abbreviations

The following abbreviations are used in the
text: AMNH, American Museum of Natural
History, New York, New York, USA; ANSP,
Academy of Natural Sciences of Philadelphia,
Philadelphia, Pennsylvania, USA; BMNH,
British Museum (Natural History) collection,
The Natural History Museum, London, Eng-
land; BMSM, Bailey-Matthews Shell Museum,
Sanibel, Florida, USA; CAS, California Acad-
emy of Sciences, San Francisco, California,
USA; ICZN, International Commission on Zo-
ological Nomenclature; LACM, Natural His-
tory Museum of Los Angeles County, Califor-
nia, USA; MCZ, Museum of Comparative
Zoology, Harvard University, Cambridge,
Massachusetts, USA; SBMNH, Santa Bar-
bara Museum of Natural History, Santa Bar-
bara, California, USA; UCMP, University of
California Museum of Paleontology, Berkeley,
California, USA; UMML, University of Miami
Marine Laboratory, Rosenstiel School of Ma-
rine and Atmospheric Sciences, Miami,
Florida, USA; and USNM, United States Na-
tional Museum collection, National Museum
of Natural History, Smithsonian Institution,
Washington, DC, USA.

The eastern Pacific Corbulidae in the pri-
vate collections of Carol C. Skoglund,
Phoenix, Arizona, USA; and Kirstie L. Kaiser,
Puerto Vallarta, Jalisco, Mexico, were also ex-
amined.

Morphological Characters

In spite of the morphological plasticity of
corbulids, there are a number of characters
that are useful in distinguishing among the
species. Some of these characters are sum-
marized in Table 1.

Overall shape is a useful criterion, with
some species oval, some trigonal, and some

other shapes. All corbulids are inequivalve,
with the right valve larger than the left, which
fits into it, overlapping most conspicuously
posteroventrally. Most eastern Pacific species
are only slightly inequivalve, and only three
very inequivalve. Some species may become
thick-shelled as adults, whereas others are
never greatly thickened. The anterior end is
rounded in all species, but more broadly in
some and more sharply in others. The shape
of the posterior end is more characteristic of
each. In some, it may be extended by a short,
shelly “spout” (e.g., Figs. 2, 3), but this may be
present in only some specimens; other
species never have a spout. A useful charac-
ter is the nature of the division between the
central and posterior slopes. In some, it is de-
lineated by a carina, a ridge, a change in
angle, and/or a change in sculpture; in others,
is it scarcely set off. Similarly, a narrow, elon-
gate escutcheon of different degrees of
prominence may be present; it is generally
widest and more evident in the right valve.

Sculpture, while variable in some species,
can nonetheless be of diagnostic value. Com-
marginal sculpture predominates, but radial
ribs are present in some taxa. Color provides
a useful character for several species.

In some taxa, the hinge plate is broad, in
others narrow. In general, the conspicuous
tooth in the right valve is too variable in shape
to be a useful diagnostic tool. The resilifer in
the right valve may be visible on the hinge
plate, or recessed beneath it. The chon-
drophore in the left valve may be very project-
ing or only slightly projecting, and it may be
conspicuously divided into sections in some.
At its posterior margin, a small tooth may be
present, which varies in prominence among
species. (It articulates on the posterior side of
a small tooth in the resilifer of the right valve.)
The pallial line, and its sinus, if present, have
informative characters, as shown in the draw-
ings (Figs. 40-57). A small sinus is evident in
some taxa, not in others; in some, a small
posterior extension is visible at the pos-
teroventral corner of the pallial line (e.g., Fig.
40).

SYSTEMATIC ACCOUNT
Family Corbulidae Lamarck, 1818

Lamarck, 1818: 493, as “corbulidees” (ac-
cepted under ICZN Code, 1999: Art.
11.7.2)

49

EASTERN PACIFIC CORBULIDAE

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50 COAN

Two Recent subfamilies are current recog-
nized, the Corbulinae and the Lentidiinae
Vokes, 1945 (pp. 6, 23-24), the latter con-
taining only the living genus Lentidium, which
is briefly discussed below.

Subfamily Corbulinae Lamarck, 1818
Genus Corbula Bruguiere, 1797

Corbula Bruguiere, 1797: pl. 230, genus with-
out named species (ICZN Code, 1999:
Art. 12.2.7); description by Lamarck
(1799: 89); original list by Lamarck (1801:
237) (ICZN Code, 1999: Art. 67.2.2).
Type species (subsequent designation of
Schmidt, 1818: 77, 177): Corbula sulcata
Lamarck, 1801: 137 (species based on
Bruguiere, 1797: pl. 230, fig. 1a-c). Re-
cent; west Africa.
to be confused with Corbula Röding,
1798: 184-185, the type species of
which (subsequent designation of Winck-
worth, 1930: 15) is Corbula anomala
Röding, 1798, a junior synonym of Venus
deflorata Linnaeus, 1758: 687. Corbula
Röding is thus a synonym of Asaphis
Modeer, 1793 (pp. 176, 182), a member
of the Psammobiidae (Keen, 1969a: 633;
Willan, 1993: 5-6)
Aloides Megerle von Mühlfeld, 1811: 67
Type species (monotypy): A. guineensis
Megerle von Mühlfeld, 1811: 67; = Cor-
bula sulcata Lamarck, 1801, making
Aloides an objective synonym of Corbula.

The genus Corbula Bruguiere was long a
source of nomenclatural confusion, and a
number of authors, lacking key pieces of liter-
ature as well as the modern nomenclatural
rules, attempted to sort out the taxonomic tan-
gle (Vokes, 1945; J. Q. Burch, 1960). The for-
mula given above represents the consensus
under the current rules.

Corbula sulcata, which occurs from Mauri-
tania to Angola, is large, heavy, and sube-
quivalve. It has heavy commarginal sculpture
in the right valve and finer commarginal sculp-
ture in the left valve. Its posterior end has two
radial ridges, one between the central and
posterior slopes and another defining a broad
escutcheon. The hinge is very heavy. None of
the Recent species in the New World is
closely similar to the type species of Corbula.

There are a bewildering array of specific
and generic taxa in this subfamily, with char-
acter sets that do not covary. Many ofthe gen-
era were established based mostly on single
characters, such as Anisocorbula Iredale,

No

—

1930 (p. 404), for an elongate species with a
sharp posterior keel, Solidicorbula Habe,
1949 (p. 2), for a thick-shelled species, and
Minicorbula Habe, 1977 (p. 282), for a small-
sized species. Subsequent authors have at-
tempted to shoehorn every species into vari-
ous subgenera, no matter how uncomfortable
the fit. Efforts to make a meaningful arrange-
ment of genera and subgenera will be fraught
with inconsistencies until additional charac-
ters become available and are rationally eval-
uated. In light of this, elevating subgenera to
genera, as many authors have done (includ-
ing by Coan et al., 2000: 478-480), is prema-
ture.

Here, a conservative effort has been made
to group similar species under some of the
named subgenera and leaving several
species in Corbula, s. 1, but even these
groupings should not be given much weight
until a full-scale revision of the family is un-
dertaken.

Grant & Gale (1931) placed Corbula luteola
and C. porcella in Corbula (Lentidium), and
the first assignment was followed by some
other authors. The type species by monotypy
of Lentidium Cristofori 4 Jan, 1832 (De-
scrizione, p. 9; Disposito, p. [ii]; Conchylia ter-
restria, p. 8; Mantissa, p. 4), is Tellina mediter-
ranea Costa, 1829 (pp. 14, 26-27, 131, pl. 1,
fig. 6), which is small, thin, shiny, translucent,
and shaped like a Tellina. It is sufficiently dif-
ferent from other corbulids that it has been
placed in a separate subfamily, the Lentidi-
inae Vokes, 1945 (p. 23). No eastern Pacific
species is similar.

Lamy (1941) placed Corbula fragilis and C.
luteola in Corbula (Corbulomya). Corbulomya
Nyst, 1845 (p. 59), has as its type species
(subsequent designation of Herrmannsen,
1847: 308) Corbula complanata J. Sowerby,
1822 (p. 86, pl. 362, figs. 7, 8), from the upper
Pliocene of England. This genus is now re-
garded as a synonym of Lentidium (Vokes,
1945: 23, 26; Glibert & van de Poel, 1966: 58;
Keen, 1969b: 696).

The eastern Pacific Corbulidae include the
following two species (or species complexes)
of formidable morphological diversity, which
cannot be fully resolved without new lines of
evidence not available in the current study:

(1) Corbula (Caryocorbula) nasuta is by far
the most common species in collections, and
it occurs in enormous populations on soft-
bottoms in shallow-water. Individuals of this
species can attain a fairly large size, but most
populations contain small individuals. Al-

EASTERN PACIFIC CORBULIDAE 51

though there is a wide range in morphologies,
there is as yet no reliable evidence that more
than one species is present, and specimens
of intermediate morphologies can be found.

(2) Corbula biradiata, while not having as
much morphological plasticity as C. nasuta,
varies considerably with respect to the posi-
tion of its beaks, the strength of its commar-
ginal sculpture, and its color.

Corbulids are not only abundant, but there
are many collection lots in alcohol or with
dried animals. Thus, the group would be a
prime candidate for biochemical genetic stud-
ies, not only to test for possible additional di-
versity at the species level but also to under-
stand phylogenetic relationships in the family
as a whole.

Subgenus (Caryocorbula) Gardner, 1926

Caryocorbula Gardner, 1926: 46

Type species (original designation): Corbula
alabamiensis Lea, 1833 (reference
below, under C. nasuta Discussion).
Eocene, eastern United States.

Serracorbula Olsson, 1961: 433

Type species (original designation): S. tu-
maca Olsson, 1961, = Corbula nasuta
С. В. Sowerby I, 1833 (references below)

Small to moderate in size, subequivalve,
subequilateral, with a strong to weak ridge be-
tween central and posterior slopes. Sculpture
of strong to weak commarginal ribs, similar on
both valves. Specimens of some species with
“marginal” ribs along the ventral and dorsal
margins; these are further discussed below.

| include five eastern Pacific species in this
subgenus. Other authors have placed addi-
tional species here as well.

Lamy (1941) placed Corbula nasuta in Cor-
bula (Cuneocorbula). Cuneocorbula Coss-
mann, 1886 (p. 49 [reprint: 37]), has as its
type species (subsequent designation by Dall,
1898: 836) Corbula biangulata Deshayes,
1857 (p. 231, pl. 13, figs. 19-23).? This
species, from the Paleocene of France, is
thin, elongate, and birostrate.

“Glibert & van de Poel (1966: 55) renamed this
species Cuneocorbula pelseneeri on the grounds
that it was a junior homonym of “Corbula biangulata
Sowerby, 1833,” but there is no such senior
homonym. They must have misread either Corbula
bicarinata G. B. Sowerby I, 1833, or C. biradiata
G. B. Sowerby I, 1833.

Corbula (Caryocorbula) amethystina
(Olsson, 1961)
Figures 1, 39

Caryocorbula (Caryocorbula)
Olsson, 1961
Olsson, 1961: 431, 548, pl. 75, fig. 1-1c;
Keen, 1971: 262, 264, fig. 674 [Corbula
(Caryocorbula)]

amethystina

Type Materials & Localities

ANSP 218902, holotype, pair; length, 27.6
mm; height, 18.1 mm; width, 13.8 mm (Fig. 1).
“Tortutilla” [? = Isla Tortolita, Panamä Pro-
vince], Panama (8.8°N); H. B. Johnson, 1958.
In the plate explanation, Olsson gives the lo-
cality as [Isla] “Taboguilla,” but both the text
and label with the specimen say “Tortutilla”.
UMML 30.11391, paratype, right valve; Puerto
Mensabe, Los Santos Province, Panama.
UMML 30.11368, paratype, left valve; San
Carlos, Panamä Province, Panama. UMML
30.11389, paratypes, right valve, left valve;
San Carlos, Panamä Province, Panama.
UMML 30.11390, paratypes, 3 right valves, 2
left valves; El Lagartillo, Panamä Province,
Panama. UMML 30.11388, paratype, right
valve; Isla Gibraleön, Archipielago de las Per-
las, Panamä. UMML 30.11326, paratype, left
valve; Punta Cocos, Isla del Rey, Archipielago
de las Perlas, Panama. UMML 30.11390,
paratype, left valve; Puerto Callo, Manabi
Province, Ecuador. UMML 30.11388, para-
type, right valve; Santa Elena, Guayas Pro-
vince, Ecuador.

Description

Ovate-trigonal, thick; right valve slightly
larger than left; posterior end longer (beaks
37-43% from anterior end); anterior end
rounded; posterior end pointed, slightly ex-
tended by a spout in some specimens; poste-
rior slope set off from central slope by a mod-
erately sharp ridge that extends to the ventral
margin. Escutcheon defined by a low ridge.

Beaks with fine commarginal sculpture.
Central slope with moderate commarginal ribs
and much finer commarginal striae. Exterior
color light to dark purple; interior color purple
around margins, brown in some. Hinge plate
broad; right valve with a large tooth; left valve
with a broad, slightly projecting chondrophore
and a small tooth. Posterior end of pallial line
with small posterior extension (Fig. 39).

52 COAN

FIG. 1. Corbula amythestina, holotype; ANSP 218902; length, 27.6 mm. FIG. 2. C. nasuta. Lectotype of C.
nasuta; BMNH 1966565/1; length, 17.6 mm.

EASTERN PACIFIC CORBULIDAE 53

Length to 30.8 mm (SBMNH 345487; Playas
[de Villamil], Guayas Province, Ecuador).

Distribution

Mazatlan, Sinaloa (23.2°N) (UCMP E.8424;
CAS 121790, 121793; LACM 152689,
152690, 63-11.78; SBMNH 126540), and La
Paz, Baja California Sur (24.2°N) (CAS
121786; SBMNH 345503 [“Punta Coyote,”
probably one of the two in this vicinity]), Mex-
ico, to Playas [de Villamil], Guayas Province,
Ecuador (2.6°S) (SBMNH 345487). It has
been found living from the intertidal zone to 82
m (mean, 17.2 m; n = 27), on sand bottoms. |
have seen 67 eastern Pacific lots, including
the types. A single lot labeled as having come
„from the western Atlantic at Islas Los Roques
(as “Las Rochas 1$.”), Venezuela (BMSM
15008), seems improbable (R. Cipriani, e-
mail, 30 Nov. 2000) and probably represents
a labeling error.

Discussion

Corbula amythestina merits comparison
with C. dominicensis Gabb, 1873b (p. 247),
from the Miocene of the Dominican Republic
(concerning the latter: Anderson, 1996:
14-15, pl. 1, figs. 9, 12, pl. 2, figs. 1, 2, 4, 5).

Corbula (Caryocorbula) nasuta G. B.
Sowerby I, 1833
Figures 2-7, 40

Corbula nasuta G. B. Sowerby |, 1833
G. B. Sowerby |, 1833: 35; Reeve, 1843:
pl. 1, fig. 1; d’Orbigny, 1845: 571; Car-
penter, 1857a: 183, 228, 300; 1864b: 537
[1872 reprint: 23]; Tryon, 1869: 65; Lamy,
1941: 233 [Corbula (Cuneocorbula)];
Hertlein & Strong, 1950: 240, 252, pl. 2,
fig. 9 [Aloidis (Caryocorbula)]; Hertlein &
Strong, 1955: 205-206; Soot-Ryen,
1957: 11; Keen, 1958: 209, fig. 527 [Cor-
bula (Caryocorbula)]; Olsson, 1961:
429-430, 548, pl. 75, fig. 3-3e [Cary-
ocorbula (Caryocorbula)]; Keen, 1971:
265-266, fig. 677 [Corbula (Caryocor-
bula)]; Gemmell et al., 1987: 59, figs. 72,
73 [as Corbula cf. nasuta]
Corbula nuciformis G. B. Sowerby |, 1833

G. B. Sowerby I, 1833: 35; Reeve, 1843:
pl. 2, fig. 9; Carpenter, 1857a: 183, 300;
1864b: 537, 668 [1872 reprint: 23, 154];

Tryon, 1869: 65; Lamy, 1941: 234 [Cor-
bula (Cuneocorbula)]; Olsson, 1961:
430-431, 548, 549, pl. 75, figs. 7, 8; pl.
76, fig. 7 [Caryocorbula (Caryocorbula)];
Keen, 1971: 265-266, fig. 678 [Corbula
(Caryocorbula)]

First revision herein

[NON Corbula nuciformis, auctt., which = C.
obesa]
Hertlein & Strong, 1950: 241, pl. 3, fig. 1
[Aloidis (Caryocorbula)]; Keen, 1958:
209, fig. 528 [Corbula (Caryocorbula)]

[not to be confused with Corbulolumna nuci-
formis Vokes, 1945: 9-11, pl. 2, figs. 5-8,
from the Cretaceous of Lebanon.]

Corbula fragilis Hinds, 1843
Hinds, 1843: 56; Reeve, 1844: pl. 3, fig.
13; Hinds, 1845: 68, pl. 20, fig. 11; Car-
penter, 1857a: 207, 300; Tryon, 1869:
64; Lamy, 1941: 240 [Corbula (Corbu-
lomya)]; Hertlein 8 Strong, 1950: 243-
244 [Aloidis (Tenuicorbula)]; Keen, 1958:
210, 211, fig. 537; Olsson, 1961: 430 [as
a synonym of Caryocorbula nasuta];
Keen, 1966: 268, pl. 4, fig. 3

Corbula alba Philippi, 1846
Philippi, 1846: 19; Carpenter, 1857a:
224, 244; 1857b: 534, 547 [as asynonym
of Corbula bicarinata]; Tryon, 1869: 63

Corbula pustulosa Carpenter, 1857
Carpenter, 1857a: 244, 300 [nomen
nudum]; 1857b: 22-23; 1864a: 368
[1872 reprint: 204]; 1864b: 553 [1872
reprint: 39]; Tryon, 1869: 65; Lamy, 1941:
143; Hertlein & Strong, 1950: 240 [as a
synonym of Aloidis (Caryocorbula) na-
suta]; Palmer, 1951: 13; Brann, 1962: 29,
pl. 4, fig. 32; Keen, 1968: 402, pl. 56, fig.
25 [as a synonym of Corbula nasuta]

Serracorbula tumaca Olsson, 1961
Olsson, 1961: 433, 549, pl. 76, fig. 4-4d;
Keen, 1971: 268-269, fig. 690 [as Cor-
bula (Serracorbula)]

Type Materials & Localities

Corbula nasuta-BMNH 1966565/1, lecto-
type here designated, open pair, the speci-
men closest to the original length measure-
ment; length, 17.6 mm; height, 11.5 mm;
width, 12.0 mm (Fig. 2). BMNH 1966565/2-4,
paralectotypes: closed pair, length, 18.4 mm;
closed pair labeled “4”, length, 16.0 mm; open
pair, length 15.3 mm, perhaps the specimen
figured by Reeve (1843).”Xipixipi” [Jipijapa;
Puerto de Cayo], Manabi Province, Ecuador

54 COAN

FIGS. 3, 4. Corbula nasuta. FIG. 3. Lectotype of C. nuciformis; BMNH 1966566/1; length, 13.4 mm. FIG. 4a.
Lectotype of C. fragilis; BMNH 1966234/1; length, 7.3 mm. FIG. 4b. Paralectotype of C. fragilis; BMNH

1966234/2; length, 6.4 mm.

(1.3°S); 10 fms. [18 m], sandy mud; Hugh
Cuming. G. B. Sowerby | (1833) also reported
some small specimens, tentatively assigned
to this species, from the Golfo de Nicoya,
Puntarenas Province, Costa Rica.

Corbula nuciformis-BMNH 1966566/1,
lectotype here designated, open pair, la-

beled “6”, probably the specimen figured by
Reeve (1843); length, 13.4 mm; height, 8.4
mm; width, 9.4 mm (Fig. 3). ANSP 50875,
possible paralectotypes, 2 small, closed pairs.
“Real Llejos” [Rio Realejo; Corinto], Chinan-
dega Province, Nicaragua (12.5°N), 6 fms. [11
m], sandy mud; Hugh Cuming. G. B. Sowerby

EASTERN PACIFIC CORBULIDAE 55

| (1833) also cited fossil material from near
Guayaquil, Guayas Province, Ecuador.

Corbula fragilis- 1966234/1, lectotype
here designated, right valve; length, 7.3 mm;
height, 4.3 mm; width, 1.9 mm (Fig. 4a).
BMNH 1966234/2, paralectotype, left valve;
length, 6.4 mm (Fig. 4b). A larger specimen
cited by Hinds (1843) is not in the BMNH. Ve-
ragua[s] Province, Panama (approximately
7.7°N), 18 fms. [33 m]; Edward Belcher.

Corbula alba—Presumably lost. The origi-
nal specimen measured 13.0 mm in length,
8.2 mm in height, and 7.6 mm in width.
Mazatlan, Sinaloa, Mexico (32.2°N); Kinder-
man.

Corbula pustulosa-BMNH 1857.6.4.77,
lectotype (Keen, 1968: 402), closed pair
mounted on a glass slide; length, 4.2 mm;
height, 3.2 mm; width, approximately 2.0 mm
(difficult to measure because of being glued to
the slide) (Fig. 5). USNM 715647, paralecto-
type, left valve (glued to a glass slide); length,
3.4 mm. Mazatlän, Sinaloa, México (32.2°N);
Frederick Reigen.

Serracorbula tumaca—ANSP 218948, lec-
totype here designated, right valve; length,
12.4 mm; height, 8.4 mm; width, 5.4 mm (Fig.
6a). Of the material that had been in the ANSP
lot labeled “holotype,” this valve comes clos-
est to the holotype measurements in Olsson’s
text, but it does not match any of his illustra-
tions labeled “holotype.” Olsson’s fig. 4, 4b,
and 4c corresponds to a paralectotype pair
measuring 12.0 mm in length (ANSP 405291)
(Fig. 6b). His fig. 4d is a paralectotype left
valve measuring 11.8 mm in length (also in
ANSP 405291). The left valve shown in his fig.
4a was not present in the lot. Tumaco, Narino
Province, Colombia (1.8°N). The ANSP lot
also contained a paralectotype right valve
from San Miguel, Isla del Rey, Archipielago de
las Perlas, Panamä (now ANSP 405292). No
type material was located in the Olsson col-
lection in the UMML.

Description

Ovate-elongate, heavy; right valve slightly
larger than left; longer posteriorly to slightly
longer anteriorly (beaks 34-51% from ante-
rior end). Posterior end somewhat pointed
ventrally, sometimes extended by a spout.
Posterior slope set off by a well-defined ridge,
often with a slight radial sulcus just anterior to
it. Ventral margin sometimes growing medially
to form an inflated, flattened ventral surface.
Margins, particularly the ventral margin,

sometimes with “marginal”? ribs, resulting in a
serrate edge. Escutcheon defined by a ridge,
more evident in right valve.

Beaks relatively smooth, with fine, pustules.
Sculpture on central and posterior slopes of
closely spaced moderate commarginal ribs
and fine, radially arrayed pustules.

Periostracum tan. External color white; in-
ternal color white, but yellow or tan in some
specimens. Hinge plate heavy; right valve
with a large tooth; left valve with a broad chon-
drophore, projecting in some, less in others;
tooth small (Fig. 40). Length to 18.4 mm (a
paralectotype of C. nasuta).

Distribution

Isla Natividad, Baja California Sur (27.9°N)
(SBMNH 345488); Bahia Magdalena, Baja
California Sur (24.5°N) (CAS 121538), into
and throughout the Golfo de California to its
head at Puerto Peñasco, Sonora (31.4°N)
(UCMP E8431; CAS 115324, 121917,
121919, 122072; SBMNH 21350, 116514,
119426; USNM 212789, 707996; Skoglund
Collection; and many other lots), Mexico, to
Callao, Lima Province, Peru (12.1°S) (LACM
35-152.1, 35-153.1, 35-177.2); Isla del Coco,
Costa Rica (SBMNH 345489, 345490,
345491; LACM 38-180.2); Isla Santa Cruz,
Islas Galapagos, Ecuador (LACM 38-193.13);
from the intertidal zone to 152 m (mean, 28.7
m; n = 328), on mud or sand. (A single valve
with an indicated depth of 384 m—LACM 35-
177.2—is probably the result of drift from
shallower water.) | have seen 875 eastern Pa-
cific lots, including the types. Two lots labeled
as having come from “Monterey, California”
(USNM 21480, 58346), probably represent la-
beling errors, and one of them may be the
source of the Monterey record of C. fragilis by
Dall (1921: 53) and Oldroyd (1925: 203).

This species has also been recorded in
beds of Pleistocene age at Puerto Penasco,
Sonora (Hertlein & Emerson, 1956: 165), and
at Bahia Magdalena, Baja California Sur (Jor-
dan & Hertlein, 1936: 111, as “C. porcella” and
as “C. fragilis’; CAS Loc. 754), México, in
beds of Pliocene age of the Loreto Basin,

ЗТпезе ribs appear along the outer ventral surface
near the shell margins (Fig. 6a, b). Olsson (1961)
referred only to “marginal serrations”. They are not
radial ribs, because they do not radiate from the
beaks. | settled on the term “marginal ribs” because
they run laterally along the shell margin.

56 COAN

FIGS. 5-7. Corbula nasuta. FIG. 5. Lectotype of C. pustulosa; BMNH 1857.6.4.77; length, 4.2 mm. FIG. 6a.
Lectotype of Serracorbula tumaca; ANSP 218948; length, 12.4 mm. FIG. 6b. Paralectotype of S. tumaca;
ANSP 405291; length, 12.0 mm. FIG. 7. SBMNH 345490; Bahia Chatham, Isla del Coco, Costa Rica; 46-69
m; largest specimen length, 5.0 mm.

EASTERN PACIFIC CORBULIDAE 57

Baja California Sur, Mexico (Piazza & Robba,
1998: 238, 248, 260, as “C. nuciformis”), and
in the middle to late Pliocene Canoa Forma-
tion at Punta Blanca, Manabi Province,
Ecuador (Pilsbry & Olsson, 1941: 11, 75).
G. B. Sowerby | (1833) cited fossil material
of unknown age from Guayaquil, Guayas
Province, Ecuador.

Discussion

Corbula nasuta is the most variable of the
eastern Pacific taxa. When small (< 10 mm),
specimens are thin shelled, with conspicuous
fine pustulose radial sculpture. This is the
morphology named C. fragilis. As specimens
grow, the ventral margin may soon become
flattened, yielding the short, inflated morphol-
ogy that was named C. nuciformis and C. pus-
tulosa. The size at which material thickens
and forms a flattened base varies greatly
among populations, and this can occur in
specimens as small as 5 mm. Corbula nasuta
typically becomes rostrate posteriorly, and a
short spout may be added in some speci-
mens.

In some large specimens, marginal ribs
may be developed at the margins, especially
the ventral margin. This is the form that was
named Serracorbula tumaca. The type lots of
both C. nasuta and C. nuciformis also contain
specimens with such ribs.

| believe that Corbula alba belongs here as
a synonym in that Philippi’s description
speaks of the posterior slope being acute and
subrostrate, and because the two European
Mio-Pliocene species with which he com-
pared C. alba-C. revoluta (Brocchi, 1814:
516, 685, pl. 12, fig. 6 —orginally described as
Tellina) and the closely related C. carinata Du-
jardin, 1837 (p. 257)—are much more similar
to C. nasuta than to C. bicarinata, with which
it was previously synonymized.

Some material from the northwestern coast
of South America is shorter, more rounded,
without much of a posterior sulcus setting off
the posterior end, and it has finer sculpture
than material from further north. A similar mor-
phology is seen in material from Isla del Coco,
Costa Rica (lots cited in the Distribution),
which is uniformly small, rounded, and
smooth (Fig. 7). This might be regarded as a
separable species or subspecies, but given
the variability of this species (or species com-
plex) as a whole, it is premature to bestow ad-
ditional names.

Corbula nasuta is most similar to the later-

named western Atlantic C. swiftiana C. B.
Adams, 1852c (pp. 236-237), which has
been reported from Massachusetts to Ar-
gentina. Given the variability of these two
taxa, | doubt that the two can be told apart on
a morphological basis, and perhaps they
should be synonymized until more evidence is
available. Synonyms in the western Atlantic
may include: C. kjoeriana C. B. Adams, 1852c
(p. 237), C. barrattiana C. B. Adams, 1852c
(pp. 237-238), C. chittyana C. B. Adams,
1852c (p. 238), C. fulva C. B. Adams, 1852c
(pp. 240-241), С. caribaea d’Orbigny, 1853“
(p. 284 [Spanish ed., p. 323], pl. 27, figs. 5-8),
C. lavalleana d’Orbigny, 1853 (p. 284 [Span-
ish ed., p. 323], pl. 27, figs. 9-12), and C.
uruguayensis Marshall, 1928 (p. 5, pl. 4, figs.
7-9). Records of C. nasuta from the western
Atlantic, such as those of Dall (1889b: 70, pl.
2, fig. 6-6c, as С. nasuta “Say”) and Haas
(1953: 204), were presumably based on C.
swiftiana.

Records of C. nasuta from Australia
(Angas, 1867: 913, 1878: 869; Hedley, 1918:
M31) were based on specimens of C. coxi
Pilsbry, 1897 (pp. 363-364, pl. 9, figs. 1-3).

Dautzenberg (1912: 99) reported Corbula
nasuta Sowerby from several localities in
west Africa. This African species was also de-
scribed as С. lyrata Е. A. Smith, 1872 (р. 729,
pl. 75, fig. 2), a junior homonym of С. Iyrata J.
de C. Sowerby, 1840 (expl. to pl. 21), and
Smith’s species was renamed C. dautzenberg
Lamy, 1941 (pp. 235-236), the name cur-
rently in use.

Corbula nasuta Sowerby, 1833 (17 May), is
not to be confused with Corbula nasuta Con-
rad, 1833 (3 Sept.) (p. 38; unpublished pl. 19,
fig. 4 [not “pl. 20, fig. 2,” as stated in text]),
from the Middle Eocene Claiborne Formation
of Alabama, for which the name Corbula al-
abamiensis Lea, 1833 (Dec.) (p. 45, pl. 1, fig.
12), is now used (Palmer & Brann, 1965: 74).
Corbula nasuta Conrad was later reported by
Conrad (1857: 161, pl. 19, fig. 4) from the Ter-

“Dall (1889b: 18), followed by Aguayo (1943: 38),
maintained that the entire Atlas to d’Orbigny’s
monograph on the mollusks of Cuba appeared in
1842, with the French text on the bivalves appear-
ing between 1847 and 1853. Aguayo also thought
that the entire Spanish text might have appeared in
1845; Keen (1971: 1006) dated the Spanish text as
1846. However, there is no evidence that any ofthe
bivalves were published, in text or plates, and in ei-
ther edition, before 1853, when citations to the
species begin to appear in other works.

58 COAN

tiary of western Texas, and this Texas mater-
ial was later named C. conradi by Dall (1898:
842).

Small specimens of this species commonly
occur in shallow water with C. marmorata.
These species may most easily be distin-
guished as follows:

nasuta marmorata
Posterior end tapered, subquadrate,
pointed pointed pos-
posteriorly teroventrally
Exterior color white mottled, with
purplish
blotches near
beaks
Interior color white magenta, espe-
cially around
margins

The complex of nasuta-like taxa has been
vastly overnamed in the Pliocene and
Miocene of the western Atlantic region. A par-
tial list of taxa that belong in this complex in-
cludes: C. (Cuneocorbula) sarda Dall, 1898
(pp. 847-848; 1900: pl. 36, fig. 14); C. (C.)
whitfieldi Dall, 1898 (p. 849; 1900: pl. 36, fig.
18); C. (Caryocorbula) whitfieldi stika Gard-
ner, 1928 (pp. 232-233, pl. 35, figs. 8, 9); and
C. (C.) whitfieldi boyntoni Gardner, 1928 (p.
233, pl. 35, figs. 10-13), from the Miocene of
Florida; C. (Cuneocorbula) sericea Dall, 1898
(pp. 848-849; 1900: pl. 36, fig. 8) [synonyms:
C. (C.) cercadica Maury, 1917: 396-397 [=
232-233], pl. 65 [= 39], figs. 16, 17; C. (C.)
caimitica Maury, 1917: 395 [= 233], pl. 65 [=
39], figs. 18, 19] (concerning: Anderson,
1996: 15-17, pl. 2, figs. 7-21); C. (C.) hele-
nae Maury, 1912 (p. 62, pl. 9, fig. 25) [syn-
onyms: C. (C.) smithiana Maury, 1912: 63, pl.
9, figs. 29, 30; C. (C.) caribaea pergrata
Maury, 1925: 255 [= 103], pl. 20, fig. 8; C. (C.)
daphnis Maury, 1925: 256 [= 104], pl. 20, figs.
10-11] (concerning: Jung, 1969: 407-409, pl.
38, figs. 12, 13, pl. 39, figs. 1-9), from the
Miocene and Pliocene of the Caribbean;
Caryocorbula (Caryocorbula) prenasuta Ols-
son, 1964 (p. 70, pl. 9, fig. 10), from the
Miocene of Ecuador; C. oropendula dolicha
Woodring, 1982 (p. 712, pl. 119, figs. 4-6),
from the Miocene of Panama; Corbula (Cu-
neocorbula) swiftiana harrisii Dall, 1898 (p.

°Corbula conradi Dall is not the renaming of a
homonym, as assumed by Boss et al. (1968: 88),
but rather a new species based on Conrad’s mate-
rial (USNM 9899).

855), from the Miocene of Texas; and C. inae-
qualis Say, 1824 (p. 153, pl. 13, fig. 2), from
the Pliocene of Maryland and Virginia [syn-
onym: C. inaequalis mansfieldi Richards,
1947: 32, pl. 11, figs. 27, 31] (concerning:
Campbell, 1993: 48, pl. 21, fig. 191).

Corbula (Caryocorbula) otra Coan,
new species
Figures 8, 41

Corbula ovulata, auctt., in part, non G. B.
Sowerby |, 1833
G. B. Sowerby |, 1833: 35-36 [material
cited from Mazatlan, México, and Corinto,
Nicaragua]; Hanley, 1843: 47, 4 (pl. expl.),
pl. 10, fig. 52; 1856: 344; Reeve, 1843
[material cited from Mazatlan, México,
and Corinto, Nicaragua]; C. B. Adams,
1852a: 522-523 [1852b: 298-299] [in
part; the large pair cited from Isla Taboga,
Panama]; Carpenter, 1857b: 23 [speci-
men cited from Mazatlan]; Lamy, 1941:
127-128; Hertlein & Strong, 1950: 241,
252, pl. 2, fig. 11 [Aloidis (Caryocorbula)];
Hertlein & Strong, 1955: 206; Keen, 1958:
209, fig. 530 [Corbula (Caryocorbula)];
Olsson, 1961: 428-429, 548, pl. 75, fig.
2c [Caryocorbula (Caryocorbula)]; Keen,
1971: 265, 266, fig. 680 [lower right fig.
only] [Corbula (Caryocorbula)]

Type Material & Locality

SBMNH 345493, holotype, pair; length,
22.8 mm; height, 13.6 mm; width, 12.0 mm,
including portion of dried soft parts (Figs. 8,
41). SBMNH 345494, paratypes; open pair,
length, 23.3 mm; closed pair, length, 21.0
mm; right valve, length, 21.5 mm. Manzanillo,
Colima, México (19.1°N, 104.3°W); 30-45 m;
Carl & Laura Shy; ex Skoglund Collection.

Description

Ovate-elongate; heavy; right valve slightly
larger than left; longer posteriorly (beaks at
40-43% from anterior end). Anterior end
rounded; posterior end tapered, slightly up-
turned posteriorly, often extended by a short
spout. Posterior slope set off by a rounded
ridge, becoming less evident ventrally. Es-
cutcheon weakly defined by a low ridge, most
evident in right valve.

Beaks smooth; most of surface covered by
well-spaced, rounded moderate commarginal

EASTERN PACIFIC CORBULIDAE 59

FIG. 8. Corbula otra, holotype; SBMNH 345493; length, 22.8 mm. FIG. 9. C. ovulata, lectotype; BMNH
1967946/1; length, 26.7 mm.

60 COAN

ribs and very fine commarginal striae. Poste-
rior slope with much less conspicuous ribs.
Ventral and dorsal margins with marginal ribs
in some specimens, such as in the holotype.
Periostracum light tan. Exterior color white
on anterior and posterior ends and ventrally,
purple to pink medially; beaks white. Interior
white to purple. Hinge plate heavy; right valve
with large tooth; left valve with chondrophore
not very extended and a small tooth. Posterior
end of pallial line with short posterior extension
(Fig. 41). Length to 26.1 mm (Bahia Chamela,
Jalisco, México; Skoglund Collection).

Distribution

Isla Carmen, Baja California Sur (26.0°N)
(CAS 121884), and Guaymas, Sonora
(27.9°N) (UCMP E.8425, CAS 121883), Mex-
ico, to La Libertad, Guayas Province, Ecuador
(2.2°S) (SBMNH 109827). A lot in the USNM
21479 labeled as “Monterey, California,” is un-
doubtedly a labeling error. A few valves num-
bered with a locality in the Islas Galapagos,
Ecuador, now isolated as CAS 121890, are
thought to represent numbering errors on a
few shells that had been a large lot from Cor-
into, Nicaragua. Recorded depths of live col-
lected material are from the intertidal zone to
55 m (mean, 17.9 т; п = 31), on mud or sand.
| have seen 107 lots.

Etymology

The species name is the Spanish word for
“other.”

Discussion

This new species is closest to C. ovulata,
and their distributions overlap from Costa
Rica to Ecuador. Corbula ovulata is more
elongate, even as a juvenile, more produced
posteriorly, and it is always white in color. The
radial rib between the central and posterior
slope is more pronounced, and the commar-
ginal sculpture is denser and more evenly dis-
tributed.

Corbula (Carycorbula) ovulata
G. B. Sowerby I, 1833
Figures 9, 42

Corbula ovulata, G. B. Sowerby |, 1833
G. B. Sowerby |, 1833: 35-36; Hanley,
1843: 47 [in part; not the fig.]; Reeve,

1843: pl. 1, fig. 7 [in part]; d’Orbigny, 1845:
571-572; C. B. Adams, 1852a: 522-523
[1852b: 298-299] [in part]; Carpenter,
1857a: 183, 228, 244, 280, 300; 1857b:
23 [in part]; 1864a: 368 [1872 reprint:
204]; 1864b: 537, 668 [1872 reprint: 23,
154]; Tryon, 1869: 65; Lamy, 1941:
127-128 [in part]; Hertlein & Strong,
1950: 241 [Aloidis (Caryocorbula)] [in
part; not the fig.]; Hertlein & Strong, 1955:
206; Keen, 1958: 209 [Corbula (Cary-
ocorbula)] [in part; not the fig.]; Olsson,
1961: 428-429, 548; pl. 75, fig. 2-2b
[Caryocorbula (Caryocorbula)] [in part;
not fig. 2c]; Keen, 1971: 265-266, fig. 680
[Corbula (Caryocorbula)] [in part; not
lower right fig. ]

Type Material & Locality

BMNH 1967946/1, lectotype here desig-
nated, open pair, the largest specimen, that
figured by Reeve (1843); length, 26.7 mm;
height, 15.1 mm; width, 12.1 mm (Fig. 9).
BMNH 1967946/2-3, paralectotypes, two
other pairs: closed pair, length, 24.3 mm;
open pair, length, 24.1 mm. “Caraccas” [Bahía
de Caraques], Manabi Province, Ecuador
(0.6°S). Acollective depth of 7-17 fms. [13-31
m] was given for ail the original material.

Five localities were originally mentioned.
The two northernmost—Mazatlan, Sinaloa,
Mexico, and “Real Llejos” [ = Rio Realejos;
Corinto], Chinendega Province, Nicaragua —
specifically refer to the species [“beautiful pink
color’] here described as Corbula otra and are
not represented by material in the BMNH col-
lection, nor is material present from the Golfo
de Montijo, Veraguas Province, Panama, or
from the other cited Ecuadorian locality —
”Xipixapi” [Jipijapa; Puerto de Cayo], Manabí
Province. Without examining type material,
Hertlein & Strong (1950: 241) “designated”
the latter as the type locality, but this is now
superceded by the locality of the lectotype
designated here (ICZN Code, 1999: Art.
76.2):

Description

Ovate-elongate, heavy; right valve slightly
larger than left; equilateral to longer posteri-
orly (40-50% from anterior end); anterior end
rounded; posterior end produced, extended
by a spout in large specimens. Posterior slope
set off from central slope by a very low ridge

EASTERN PACIFIC CORBULIDAE 61

that disappears ventrally. Escutcheon well de-
fined by a ridge.

Beaks smooth; most of surface with closely
spaced, moderate commarginal ribs and very
fine commarginal striae. Ventral margin with
marginal ribs in some material. Escutcheon
smooth.

Periostracum tan. Exterior white; interior
with brown patches and brown marginal color
in large specimens. Hinge plate heavy; right
valve with a large tooth; left valve with a
broad, not very projecting chondrophore and
a small tooth. Posterior end of pallial line with
posterior extension (Fig. 42). Length to 29.2
mm (Skoglund Collection; Playas, Guayas
Province, Ecuador).

Distribution

Bahia Juanilla, Guanacaste Province,
Costa Rica (10.9°N) (LACM 72-13.30), to
Cabo Blanco, Piura Province, Peru (4.3°S)
(CAS 121777), from the intertidal zone to 55
m (mean, 8.2 т; п = 13), on sand bottoms. |
have seen 58 lots, including the types.

Reported on the Pleistocene third terrace
on the Peninsula de Santa Elena (Hoffstetter,
1948: 81) and, with question, as a subfossil at
Laguna de Salinas (Hoffstetter, 1952: 44),
both in Guayas Province, Ecuador. Also re-
ported in the early Pliocene Jama Formation
at Puerto Jama, Manabi Province, Ecuador
(Pilsbry & Olsson, 1941: 11, 75), in the
Pliocene at Rio la Vaca and Rio Blanca,
Burica Peninsula, Puntarenas Province,
Costa Rica (Olsson, 1942: 171, 172), and in
the Miocene Tumbes Formation at Zorritos,
Tumbes Province, Peru (Olsson, 1932: 140).

Discussion

See under C. otra.

Corbula (Caryocorbula) porcella Dall, 1916
Figures 10, 43

Corbula porcella Dall, 1916
Dall, 1916a: 41 [nomen nudum); Dall,
1916b: 415-416; Oldroyd, 1925: 204;
Grant & Gale, 1931: 422 [Corbula (Len-
tidium)]; Lamy, 1941: 242 [as С. “porcel-
lio”); Hertlein & Strong, 1950: 242, 252, pl.
2, figs. 13, 15 [Aloidis (Caryocorbula)];
Keen, 1958: 210-211, fig. 531 [Corbula
(Caryocorbula)]; Olsson, 1961: 430, 549,
pl. 76, fig. 8 [Caryocorbula (Caryocor-

bula)]; Keen, 1971: 265-266, fig. 681
[Corbula (Caryocorbula)]; Coan et al.,
2000: 478, pl. 102 [Caryocorbula]

Corbula fragilis Hinds, auctt., non Hinds, 1843
Dickerson, 1922: 563; Grant & Gale,
1931: 422 [Corbula (Lentidium)]

Type Material & Locality

USNM 97039, lectotype here designated,
open pair; length, 7.9 mm; height, 5.2 mm;
width, 4.9 mm (Fig. 10). USNM 880652, para-
lectotypes, 1 closed pair, 6 right valves, 8 left
valves, and assorted fragments. USFC Sin.
2838, off the east side of Isla Cedros, Baja
California, México (28.2°N, 115.2°W), 44 fms.
[99 m], green mud; May 5, 1888.

Description

Shell ovate-subquadrate, of moderate
thickness for size; right valve slightly more in-
flated than left; beaks closer to anterior end
(32-34% from anterior end). Anterior end
rounded. Posterior end almost vertically trun-
cate. Posterior slope separated from central
slope by a fairly strong radial ridge. Es-
cutcheon sharply defined by a ridge.

Beaks smooth. Central slope with moder-
ate, irregular commarginal ribs, lamellar on
posterior slope; posterior end also with radial
rows of pustules in many specimens.

Color white exteriorly and interiorly. Hinge
plate broad; right valve with a narrow tooth;
left valve a narrow, non-projecting chon-
drophore and a small tooth (Fig. 43). Length
to 10.2 mm (MCZ 260684; S of Laguna San
Ignacio, Baja California Sur).

Distribution & Habitat

Esteros Bay, San Luis Obispo County, Cal-
ifornia (35.4°N) (USNM 207636), to Punta
Magdalena, Baja California Sur, Mexico
(24.6°N) (LACM 71-13.5), in 27 to 210 m
(mean, 80.3 m; n = 55), on mud and sand. |
have seen 68 lots, including the types.

The record of C. porcella from Bahia Bal-
lena, Puntarenas Province, Costa Rica, by
Hertlein & Strong (1950: 242) was based on
specimens of C. nasuta (CAS 121941).

Also recorded in the late Pleistocene Miller-
ton Formation at Tomales Bay, Marin County,
California (Dickerson, 1922: 563, as “C. frag-
ilis — CAS Loc. 561; Valentine, 1961: 390, as
“C. fragilis’; R. G. Johnson, 1962: 115-120; J.

62 COAN

FIG. 10. Corbula porcella, lectotype; USNM 97039; length, 7.9 mm. FIG. 11. C. esmeralda. FIG. 11a. Lecto-
type; ANSP 218903; length, 20.6 mm. FIG. 11b. Paralectotype; ANSP 403198; length, 21.9 mm.

EASTERN PACIFIC CORBULIDAE 63

E. Johnson, 1987: 116, as both C. porcella
and “C. fragilis’). The record of this species in
the Pleistocene at Bahia Magdalena, Baja
California Sur, México (Loc. 754; Jordan,
1936: 111), was based instead on specimens
of C. nasuta.

Discussion

Corbula procella may be distinguished from
C. luteola, which is sympatric but in shallower
water, in that the latter is more elongate and
longer anteriorly, is frequently colored, has
finer sculpture, is less inflated, has a less pro-
nounced rib separating the central slope from
the posterior slope, and has a less sharply de-
fined escutcheon.

Hertlein & Strong (1950), followed by Soot-
Вуеп (1957:11), Keen (1958, 1971), and Ols-
son (1961), suggested a relationship of C.
porcella to the Panamic C. obesa Hinds,
1843. However, C. obesa is very distinct, at-
taining a larger size, being very inflated, hav-
ing a proportionately more inflated right valve,
having radial ribs on its umbones, in lacking
an escutcheon, in being colored interiorly, and
in having a heavier periostracum.

Specimens of the highly variable C. nasuta
account for Panamic records of C. porcella.
They differ as follows:

C. nasuta

more pointed poster-
oventral corner, often
set off by a shallow
radial sulcus just
anterior to it

radial ridge fairly sharp
all the way to ventral
margin

tinted yellow to brown

escutcheon indistinct

sometime with a poste-
rior spout

C. porcella
even, truncate poste-
rior end

radial ridge becoming
indistinct near ventral
margin

white

eschtcheon defined by
a ridge

never with a posterior
spout

Subgenus (Hexacorbula) Olsson, 1932

Hexacorbula Olsson, 1932: 140

Type species (original designation): Corbula
hexacyma Brown & Pilsbry, 1913, = C.
gatunensis Toula, 1909 (references in

Oddly, Soot-Ryen (1957) made С. obesa Hinds,
1843, a junior synonym of C. porcella Dall, 1916b,
in reporting the latter from Panama. His specimen
was probably one of the variable C. nasuta.

Discussion below). Middle Miocene,
Panama.

Medium-sized, heavy, ovate to ovate-elon-
gate, subequivalve, subequilateral; sculpture
of strong commarginal undulations.

Woodring (1982: 713) and Anderson (1996:
12) suggested that Hexacorbula is very simi-
lar in sculpture to Bothrocorbula Gabb, 1873a
(p. 274, pl. 10, fig. 3, 3a); its type species, by
monotypy, is Corbula viminea Guppy, 1866a
(pp. 293, 295, pl. 18, fig. 11), from the middle
Miocene of Jamaica and the Dominican Re-
public. Bothrocorbula differs from Hexa-
corbula in having a lunular pit.

Corbula (Hexacorbula) esmeralda
(Olsson, 1961)
Figures 11, 44

Caryocorbula (Hexacorbula) esmeralda Ols-
son, 1961
Olsson, 1961: 432-433, 549, pl. 76, fig.
3-3c; Keen, 1971: 266-267, fig. 683
[Corbula (Hexacorbula)]

Type Material & Locality

ANSP 218903, lectotype here desig-
nated, right valve, the specimen shown in
Olsson’s fig. 3a, c; length, 20.6 mm; height,
12.4 mm; width, 5.0 mm (Fig. 11a). ANSP
403198, paralectotypes —left valve, shown in
Olsson’s fig. 3b, length, 21.9 mm; left valve,
shown in Olsson’s fig. 3; length, 20.6 mm
(similar to holotype in size and shape, but
they are not a pair) (Fig. 11b); right valve, not
figured in Olsson (1961), length, 20.3 mm.
UMML 30.11326, paralectotypes, 3 right
valves, 9 left valves. Esmeraldas, Esmeraldas
Province, Ecuador (1.0°N); 20 ft. [5 m]; Axel A.
Olsson, 1958.

Olsson (1961) illustrated an unmatched
pair of valves as the “holotype”, necessitating
a lectotype selection.

Description

Shell ovate-elongate, thin in small speci-
mens to very thick in large specimens; sube-
quivalve; longer posteriorly (beaks at 33-39%
from anterior end). Central slope with a broad
radial sulcus. Posterior slope set off from cen-
tral slope by a relatively sharp ridge. Es-
cutcheon set off by a ridge.

Beaks relatively smooth, with fine commar-
ginal ribs and still finer radial rows of pustules.

64 COAN

Most of surface with broad commarginal un-
dulations and finer commarginal ribs. Poste-
rior slope with only fine commarginal sculp-
ture.

Color white exteriorly and interiorly. Right
valve with a large tooth; left valve with a broad
chondrophore, which is not very projecting,
and a small tooth (Fig. 44). Length to 22.5 mm
(2.5 km S of river at Esmeraldas; Skoglund
Collection).

Distribution

Esmeraldas, Esmeraldas Province (1.0°N)
(type locality), to Chone, Bahia de Caraquez,
Manabi Province (0.6°S) (USNM 709895),
Ecuador, in 5-43 m (mean, 23.8; n = 4); no
bottom types have been recorded. | have
seen only 6 lots, including the types.

Discussion

Corbula esmeralda differs from the middle
Miocene C. (H.) gatunensis Toula, 1909 (p.
733, pl. 27, fig. 12), in being more elongate
and in having its posterior end less set off by
a sulcus. In addition to C. hexacyma Brown 8
Pilsbry, 1913 (p. 518, pl. 26, fig. 4), Woodring
(1982: 714) listed C. (Carycorbula) buenavis-
tana F. Hodson, in F. Hodson & H. Hodson,
1931 (p. 24, pl. 8, fig. 6, pl. 12, figs. 8-13),
from the Miocene of Venezeula as a synonym
of H. gatunensis.

Corbula (H.) cruziana Olsson, 1932 (p. 141,
pl. 3, fig. 5, pl. 4, fig. 9), from the early Mio-
cene of Perú and Panamá is apparently the
oldest member of this subgenus.

Subgenus (Juliacorbula)
Olsson & Harbison, 1953

Juliacorbula Olsson & Harbison, 1953: 148-
149

Type species (original desigation): Corbula
cubaniana d'Orbigny, 1853; = C. knoxi-
anaC. B. Adams, 1852c; = C. aequivalvis
Philippi, 1836 (references in Discussion
below). Recent, western Atlantic.

Shell small to medium in size, heavy, sube-
quivalve; beaks just posterior to midline; pos-
terior end truncate; with a strong ridge be-
tween central and posterior slopes and
another outlining the escutcheon. Sculpture of
strong commarginal ribs in both valves.

After saying that their new genus contained
“several” species, Olsson & Harbison (1953)

referred only three to it: the type species, C.
scutata Gardner, 1944 (reference below), and
the eastern Pacific C. biradiata; the latter was
perhaps an error for C. bicarinata, which is
very similar if not identical to the type species.

Corbula (Juliacorbula) bicarinata
G. B. Sowerby |, 1833
Figures 12, 13, 45

Corbula bicarinata G. B. Sowerby |, 1833

С. В. Sowerby |, 1833: 35; Hanley, 1843:
46, 6, pl. 12, fig. 31; 1856: 344; Reeve,
1844: pl. 3, fig. 23; d’Orbigny, 1845: 571;
C. B. Adams, 1852a: 521 [1852b: 297];
Carpenter, 1857a: 183, 224, 228, 244,
280, 281, 300; 1857b: 21-22, 547:
1864a: 368 [1872 reprint: 204]; 1864b:
537 [1872 reprint: 23]; Tryon, 1869: 63;
Lamy, 1941: 128-129; Hertlein & Strong,
1950: 238 [Aloidis (Caryocorbula)];
Keen, 1958: 208-209, fig. 523 [Corbula
(Caryocorbula)]; Olsson, 1961: 436, 548,
pl. 75, fig. 6-6b [Juliacorbula]; Keen,
1971: 266-268, fig. 684 [Corbula (Julia-
corbula)]; Gemmell et al., 1987: 60

Type Material & Locality

BMNH 1966567/1, lectotype here desig-
nated, closed pair, the specimen figured
by Reeve (1844); length, 10.6 mm; height,
8.5 mm; width, 6.6 mm (Fig. 12). BMNH
1966567/2-3, paralectotypes, 2 open
pairs, lengths, 10.9, 10.5 mm. BMNH
1907.12.30.102, paralectotype, open pair,
length 9.6 mm. Four localities in “Columbiae
Occidentalis” [West Colombia] were given by
С. В. Sowerby |—Panama; “Real Llejos” [=
Rio Realejos; Corinto], Chinendega Province,
Nicaragua; and “Caraccas” [Bahia Carä-
quez], Manabi Province, Ecuador; and Santa
Elena, Guayas Province, Ecuador. Neither of
the two lots in the BMNH collection has a spe-
cific locality, so the type locality is here clar-
ified (ICZN Code, Recommendation 76a) as
being Playa Kobbe, Panama Province,
Panama (8.9°N), where the species is com-
mon (Skoglund Collection). A collective depth
for the original material was given as 7-17
fms. [13-31 m], in sandy mud; Hugh Cuming.

Description

Ovate-subquadrate, moderately heavy;
right valve slightly larger than left; posterior

EASTERN PACIFIC CORBULIDAE 65

FIGS. 12, 13. Corbula bicarinata. FIG. 12. Lectotype; BMNH 1966567/1; length, 10.6 mm. FIG. 13. CAS
120689; San Felipe, Baja California, Mexico; length, 11.2 mm.

end longer (beaks at 38-40% from anterior
end). Anterior end rounded; posterior end
abruptly truncate. Posterior slope set off from
central slope by a sharp ridge that becomes
somewhat more rounded ventrally. Es-
cutcheon set off by a similarly sharp ridge.

Beaks relatively smooth. Most of surface
with moderate, rounded commarginal ribs,
which continue onto posterior slope, and very
fine radial striae. Escutcheon with fine com-
marginal ribs.

Periostracum light tan. White exteriorly; in-
terior white to yellowish. Hinge plate narrow;
right valve with a large tooth; left valve with a
Narrow chondrophore and a very inconspicu-
ous tooth (Figs. 13, 45). Length to 13.0 mm

(Bahia Cholla, Puerto Penasco, Sonora, Mex-
ico; Skoglund Collection).

Distribution

Head of the Golfo de California at Puerto
Penasco, Sonora, México (31.4°N) (Skoglund
Collection; UCMP B.6008, E.8416, E.8431;
LACM 60-11.33, 62-22.28, 63-56.38; SBMNH
113654), south to Zorritos, Tumbes Province,
Peru (3.7°S) (UMML 30.11424); Isla Santa
Maria, Islas Galapagos, Ecuador (LACM 32-
2.4); from the intertidal zone on undersides of
rocks to 110 m, in rubble (mean, 12.4 m; п =
61). | have seen 257 lots, including the types.

This species has been reported in the Pleis-

66 COAN

tocene at Bahia Santa Inez and Isla Carmen
(Hertlein, 1957: 63), and in the Pliocene on
Isla Carmen (Emerson & Hertlein, 1964: 341),
Baja California Sur, Mexico.

Discussion

Carpenter (1857b: 547) and some other au-
thors have synonymized C. alba Philippi,
1846, with this species. However, Philippi’s
description, together with shapes of the Euro-
pean fossil species with which he compared
it, indicate that C. alba should instead be re-
garded as a synonym of C. nasuta (see Dis-
cussion under the latter).

Olsson (1961) suggested that C. ira Dall,
1908, described from Panama, might be a
synonym. Although superficially similar in
shape, C. ira is longer and narrower posteri-
orly, there is no rib defining the escutcheon,
and the commarginal ribs are fewer and much
more prominent.

This species is similar to the western At-
lantic type species of the subgenus, C. ae-
quivalvis Philippi, 1836 (pp. 227-228, pl. 7,
fig. 4), which has been reported from Florida
to Panamä. Synonyms of C. aequivalvis in-
clude C. knoxiana C. B. Adams, 1852c (pp.
238-239), and C. cubaniana d’Orbigny, 1853
(p. 283 [Spanish ed., p. 322], pl. 26, figs.
51-54). Corbula aequivalvis seems to have
finer commarginal sculpture than C. bicari-
nata (based on comparison with CAS 130494;
Salinas Papaya, near Ensenada, southwest
coast, Puerto Rico). Corbula aequivalvis was
discussed by Jung (1969: 410-411, pl. 39,
figs. 11-15) and by Anderson (1996: 17-18,
pl. 2, figs. 22-26), who compared it with
closely related fossil taxa, including C. knoxi-
ana fossilis Pilsbry, 1922 (pp. 427, 435, pl. 46,
fig. 14), from the Miocene of the Dominican
Republic; C. aequivalvis stainforthi Rutsch,
1942 (p. 124-125, pl. 3, figs. 8, 9), from the
Miocene of Trinidad; and C. scutata Gardner,
1944 (pp. 140-141, pl. 23, figs. 16, 30-32),
from the Plio-Pleistocene of Florida and North
Carolina.

Subgenus (Panamicorbula) Pilsbry, 1932

Panamicorbula Pilsbry, 1932: 105
Type species (original designation): Pota-
momya inflata C. B. Adams, 1852a; =
Corbula ventricosa A. Adams & Reeve,
1850 (references below); tropical eastern

Pacific, in mangrove swamps.
Shell large, thin in most material to thick in

largest specimens; subequivalve; beaks just
anterior to midline; left valve with submarginal
marginal ridges; with fine commarginal sculp-
ture; chondrophore broad, conspicuously di-
vided.

C. B. Adams (1852a, b) described his three
synonymous taxa in the genus Potamomya
J. de C. Sowerby, 1835 (p. 241). Its type
species, by subsequent designation of Keen
(1969b: 698), is Mya plana J. Sowerby, 1814
(pp. 173-174, pl. 76, fig. 2), from the Eocene
and Oligocene of Europe. Potamomya is now
considered to be a synonym of Erodona Bosc,
1801, ex Daudin ms (vol. 2:329-330, pl. 6, fig.
2) (Keen, 1969b: 698). Erodona, placed in its
own family, the Erodonidae, within the My-
oidea, is still living in brackish waters on the
east coasts of Central and South America. It
has a projecting chondrophore in the left valve
similar to that of Mya. Tryon (1869) placed
Potamomya aequalis in Corbula (Azara).
Azara d’Orbigny, 1842 (p. 161, pl. 7), is an ob-
jective synonym of Erodona (Vokes, 1945: 26;
Keen, 1969b: 698).

Corbula (Panamicorbula) ventricosa
A. Adams & Reeve, 1850
Figures 14-19, 46

Corbula ventricosa A. Adams & Reeve, 1850
A. Adams & Reeve, 1850: 83, pl. 13, fig.
12; Carpenter, 1857a: 284, 300; Tryon,
1869: 66

NOT C. ventricosa A. Adams & Reeve, auctt.
[= C. colimensis Coan, n. sp.]
Hertlein & Strong, 1950: 242-243, 251,
pl. 2, figs. 3, 4; Keen, 1958: 210, 211, fig.
532; Olsson, 1961: 428, 549, pl. 76, fig.
9; Keen, 1971: 266-267, fig. 682

Potamomya aequalis C. B. Adams, 1852
C. B. Adams, 1852a: 519-520, 547-548
[1852b: 295-296, 323-324]; Carpenter,
1857a: 280, 300; 1864a: 363 [1872
reprint: 204]; Tryon, 1869: 67 [Corbula
(Azara)]; Turner, 1956: 28, 128, pl. 19,
figs. 5, 6 [Potamomya]; Keen, 1958: 210-
211, fig. 533 [Corbula (Panamicorbula)]

Potamomya inflata C. B. Adams, 1852
C. B. Adams, 1852a: 520, 548 [1852b:
296, 324]; Carpenter, 1857a: 280, 300;
1864a: 363 [1872 reprint: 204] [as a ju-
nior synonym of Р aequalis]; Tryon,
1869: 67 [as a junior synonym of C. ae-
qualis]; Pilsbry, 1932: 105 [Corbula
(Panamicorbula)]; Vokes, 1945: 9, 11-
12, pl. 2, figs. 1-4; Turner, 1956: 56, 126,
pl. 17, figs. 12, 13 [Potamomya]; Keen,

EASTERN PACIFIC CORBULIDAE 67

1958: 210-211, fig. 535 [Corbula (Pana-
micorbula);]; Olsson, 1961: 434-435,
549, pl. 76, fig. 1-1c [Panamicorbula];
Keen, 1971: 268-269, fig. 689 [Corbula
(Panamicorbula)]

Potamomya trigonalis C. B. Adams, 1852

C. B. Adams, 1852a: 520, 548 [1852b:

FIGS. 14, 15. Corbula ventricosa. FIG. 14. Lectotype of С. ventricosa; BMNH 1967980/1: length, 22.0 mm.
FIG. 15. Holotype of Potamomya aequalis; MCZ 186325; length, 19.4 mm.

296, 324]; Carpenter, 1857a: 280, 300;
1864b: 363 [1872 reprint: 204] [as proba-
ble synonym of P. aequalis]; Tryon, 1869:
67 [as probable synonym of Corbula ae-
qualis]; Turner, 1956: 93, 128 [Pota-
тотуа; as *triagonalis”], pl. 18, figs. 3, 4;
Hoffstetter, 1952: 44, fig. 10 [Pana-

68 COAN

FIGS. 16, 17. Corbula ventricosa. FIG. 16. Lectotype of Potamomya inflata; MCZ 186315; length, 17.2 mm.
FIG. 17. Lectotype of P trigonalis; MCZ 186314; length, 23.8 mm.

micorbula]; Keen, 1958: 210-211, fig. 214358]; Olsson, 1961: 435 [as a syn-
536 [Corbula (Panamicorbula)] onym of P inflata]

Corbula macdonaldi Dall, 1912 Panamicorbula cylindrica Morrison, 1946
Dall, 1912: 3; Dall, 1925: 15, pl. 17, figs. Morrison, 1946: 47, pl. 1, figs. 15, 17;

1, 3 [catalogue number misquoted as Keen, 1958: 210-211, fig. 534 [Corbula

EASTERN PACIFIC CORBULIDAE 69

FIGS. 18, 19. Corbula ventricosa. FIG. 18. Lectotype of Corbula macdonaldi; USNM 214353; length, 20.7
mm. FIG. 19. Holotype of Panamicorbula cylindrica; USNM 542186; length, 13.3 mm. FIG. 20. Corbula
amurensis; CAS 089104; Martinez, Contra Costa County, California; length, 16.4 mm.

70 COAN

(Panamicorbula)]; Olsson, 1961: 435,
549, pl. 76, fig. 2, 2a [Panamicorbula];
Keen, 1971: 267-268, fig. 688 [Corbula
(Panamicorbula)]

? Corbula ustulata Reeve, auctt., non Reeve,
1844
Menke, 1847: 191; Carpenter, 1857a:
236; 1857b: 539

[non Reeve, 1844 — reference in Discussion
of next species]

Type Materials & Localities

Corbula ventricosa-BMNH 1967980/1,
lectotype here designated, pair, the larger of
two specimens, probably that figured by A.
Adams & Reeve (1850); length, 22.0 mm;
height, 16.8 mm; width, 13.6 mm (Fig. 14).
BMNH 1967980/2, paralectotype, pair;
length, 16.0 mm. “China Sea,” but type local-
ity here clarified as the mangrove swamp at
Paitilla, near Panama City, Panama Province,
Panama (9.0°N), where this species is known
to occur (ANSP 155409).

Potamomya aequalis-MCZ 186325, holo-
type, pair; length, 19.4 mm; height, 16.5 mm;
width, 10.2 mm (Fig. 15). Mangrove thicket,
2.5 miles [6.5 km] E of Panama City, Panama
Province, Panama (9.0°N); soft mud; C. B.
Adams;, 27 Nov. 1850-2 Jan. 1851.

Potamomya inflata-MCZ 186315, lecto-
type (Turner, 1956: 126), pair; length, 17.2
mm; height, 13.8 mm; width, 12.4 mm (Fig.
16). MCZ 186316, paralectotypes, 2 pairs
Same locality as P. aequalis.

Potamomya trigonalis—MCZ 186314, lecto-
type (Turner, 1956: 128), pair; length, 23.8
mm; height, 19.8 mm; width, 13.6 mm (Fig.
17). There are no paralectotypes in the MCZ.
Same locality as P aequalis.

Corbula macdonaldi-USNM 214353, lec-
totype here designated, the specimen fig-
ured by Dall (1925), left valve; length, 20.5
mm; height, 16.5 mm; width, 5.7 mm (Fig. 18).
USNM 517479, paralectotype, right valve;
length, 22.7 mm; and pair; length, 18.6 mm.
“Loc. 5848; Pleistocene muck beds at Colon,”
but label says “Miraflores Locks,” and Olsson
(1961: 435) says “near Panama City,”
Panama Province, Panama (9.0°N).

Panamicorbula cylindrica- USNM 542186,
holotype, pair; length, 13.3 mm; height, 9.3
mm; width, 6.3 mm (Fig. 19). USNM 542187,
paratypes: pair, length, 12.5 mm; right valve,
length, 20.6 mm; broken right valve, length,
approximately 21.2 mm; left valve, length,
19.3 mm. Rio Marina mangrove swamp, Isla

San José, Archipielago de las Perlas, Panamä
(8.3°N); J. P. E. Morrison, 19 February 1944.

Description

Shell variable in shape, from ovate to trigo-
nal, from thin- to thick-shelled; right valve
slightly larger than left. Posterior end slightly
longer (beaks 46% from anterior end). Ante-
rior end rounded; posterior end obliquely sub-
truncate. Posterior slope set off from central
slope by a low ridge, more pronounced in
some specimens than in others. Escutcheon
present in most specimens, set off by a slight
ridge and a change in sculpture.

Anterior and posterior slopes of juvenile
portion of unworn specimens with fine, regu-
lar commarginal ribs. Central and posterior
slopes of larger specimens with fine, irregular
commarginal sculpture and fine radial striae.

Periostracum brown to greenish, generally
eroded away. White exteriorly and interiorly.
Right valve with a large, triangular tooth and
elongate submarginal ridges resembling lat-
eral teeth situated well away from cardinal
tooth; left valve with a large, projecting, di-
vided chondrophore, having a moderate tooth
on its posterior end; anteroventral hinge mar-
gin swollen into a small tooth medially. Pallial
sinus absent (Fig. 46). Length to 35.0 mm
(USNM 612203; Indian kitchen midden at Val-
divia, Guayas Province, Ecuador).

Distribution

Medano Blanco, N of Topolobampo,
Sinaloa, Mexico (25.7°N) (Skoglund Collec-
tion), to Puerto Pizarro, Tumbes Province,
Perú (3.5°S) (SBMNH 127876; UMML
30.11329, 11338). All living material has been
collected from intertidal mudflats, generally in
mangrove swamps. | have seen 78 lots, in-
cluding the types of the various synonyms.

Parker’s (1964: 162) west Mexican offshore
records of C. ventricosa were based on spec-
imens of C. ira (MCZ 260660, 260668), C. na-
suta (MCZ 253667), and C. porcella (MCZ
260013, 260679, 260684).

This species was recorded as a subfossil at
Laguna de Salinas, Guayas Province, Ecua-
dor (Hoffstetter, 1952: 44, fig. 10, as “Pana-
micorbula trigonalis”).

Anderson (1996: 18-19, pl. 3, figs. 1-10)
described C. (P) canae from the upper Mio-
cene Cercado Formation of the Dominican
Republic. It is more rostrate and acuminate
than the Recent species. She also discussed

EASTERN PACIFIC CORBULIDAE 71

one other possible new species from the
same formation.

Discussion

Carpenter (1857a: 284, 300) first sug-
gested that Corbula ventricosa came from the
eastern Pacific. Hertlein & Strong (1950) also
discussed the likelihood that the type material
of C. ventricosa actually came from the
Panamic Province, rather than the “China
Sea,” as originally indicated.” This lot did in-
deed come from the eastern Pacific, but it is
not the species Herilein & Strong thought.

Given the fact that C. B. Adams described
three synonymous taxa from a single station,
subsequent workers might have exercised
more caution in proposing additional names
without a better understanding of the variabil-
ity of this species of Corbula (Panamicorbula).
Although material of this species remains un-
common in most collections, because few
workers have collected in mangrove swamps,
abundant specimens from single stations
at the Instituto Nacional de Biodiversidad
(INBio) in Costa Rica clearly demonstrate that
only a single taxon is present. The largest
specimens tend to become trigonal. Speci-
mens in which the ventral margin turns medi-
ally at a smaller size become inflated and
more cylindrical.

Subgenus (Potamocorbula) Habe, 1955

Potamocorbula Habe, 1955: 272

Type species (original designation): Corbula
amurensis Schrenck, 1861. Recent,
northwestern Pacific.

Small sized, thin, subequivalve, smooth.
Left valve with a more prominent radial ridge
than the right valve. Left valve with a project-
ing chondrophore.

Corbula (Potamocorbula) amurensis
Schrenck, 1861
Figures 20, 47

Corbula amurensis Schrenck, 1861
Schrenck, 1861: columns 412-413;
Schrenck, 1867: 584-586, pl. 25, figs.

"Although the Samarang did not stop in the eastern
Pacific, the earlier Sulphur expedition, on which Ed-
ward Belcher also served, did so and was probably
the source of the eastern Pacific material mixed into
the Samarang collection. This confusion is dis-
cussed by Carpenter (1857a: 224; 1864b: 534) and
Hertlein & Strong (1950: 241).

5-8; Lamy, 1941: 247-248 [as Corbula
(Erodona)]; Oyama, 1980: 116, pl. 55,
figs. 6, 8, 10, 13 [as Potamocorbula];
Scarlato, 1981: 392-393, pl. figs.
415-417, text fig. 14; Zhuang & Cai,
1983: 65, fig. 12; Coan et al., 2000:
479-480, pl. 102.

Corbula amplexa A. Adams, 1862
A. Adams, 1862: 223-224

Corbula frequens Yokoyama, 1922
Yokoyama, 1922: 123, pl. 6, figs. 16, 17

Corbula pustulosa Yokoyama, 1922 [non Car-
penter, 1857]

Yokoyama, 1922: 123-124, pl. 6, fig. 18
[non Carpenter, 1857b: 22-23]

Corbula sematensis Yokoyama, 1922
Yokoyama, 1922: 124-125, pl. 6, fig. 19
[not fig. 20, which is a Poromya (Oyama,
1980: 120)]

Corbula vladivostokensis Bartsch, 1929
Bartsch, 1929: 133, pl. 2, figs. 1-7

Corbula amurensis takatuayamaensis Ando,
1965
Ando, 1965: 209, fig. 28

Type Materials & Localities

Under study in a separate project by an-
other worker.

Description

Ovate, thin; right valve decidedly larger
than left valve; beaks anterior to midline (ap-
proximately 41% from anterior end); anterior
end sharply rounded; posterior end sharply
rounded. Posterior end not set off from central
slope in right valve, but set off by an angle in
left valve. Escutcheon not evident.

Beaks smooth; rest of surface with low, ir-
regular commarginal ribs. Periostracum tan.
Shell white exteriorly and interiorly (Fig. 20).

Hinge plate narrow; right valve with a nar-
row tooth, attached to shell wall below hinge-
line; left valve with a long, projecting chon-
drophore that is conspicuously divided, and
with a very small tooth on its posterior end;
anteroventral hinge margin swollen into a low
tooth medially. Pallial line with a small sinus
(Fig. 47). Length to 19.7 mm (CAS 121534;
Carquinez Strait, San Francisco Bay, Contra
Costa County, California).

Distribution

This introduced species is thus far found
only in San Francisco Bay, California, its ecol-

72 COAN

ogy there discussed by Carlton et al. (1990),
Nichols et al. (1990), and Duda (1994). It oc-
curs from the intertidal zone to 8 m, on mud,
sand or clay.

Discussion

Another worker in a separate project is at-
tempting to understand the taxonomy of the
Asian species attributed to this subgenus.
Corbula laevis Hinds, 1843 (p. 59), and C. us-
tulata Reeve, 1844 (pl. 4, fig. 25), are earlier
names for members of this species complex.
Resolution of this group will require access
not only to the type material of the nominai
species but also suites of specimens from
several Asian localities to fully understand
variability and distributions. Material of the
San Francisco Bay import shows significant
variability in shape and thickness. For exam-
ple, CAS 121534 contains specimens that are
ovate, subtrigonal, and ovate-elongate.

А comparison between С. (Potamocorbula)
amurensis and С. (Panamicorbula) ventri-
cosa is instructive because they both inhabit
brackish waters:

C. ventricosa
subequivalve
subquadrate

C. amurensis
inequivalve
ovate, ovate-elongate,
to subtrigonal
smooth, with fine
posterior radials
in right valve
length to 20 mm

stronger commarginal
sculpture

length to 35 mm

has a small pallial sinus
division between central
and posterior slopes

pallial sinus not evident
division between central
and posterior slopes

evident only in left in both valves

valve
no lateral ridges on lateral ridges in right
hinge valve

tooth in right valve
seated deeply under
hinge plate

chondrophore very
projecting

tooth less deeply seated

less projecting

Subgenus (Tenuicorbula) Olsson, 1932

Tenuicorbula Olsson, 1932: 141
Type species (original designation): Corbula
tenuis G. B. Sowerby |, 1833 (reference
below). Recent, eastern Pacific.
Thin, subequivalve, longer posteriorly. With
a strong keel separating posterior and central
slopes and another keel defining a lunule.
Posterior end truncate. Sculpture of fine,

raised commarginal ribs, strongest on poste-
rior slope.

Corbula (Tenuicorbula) tenuis
G. B. Sowerby I, 1833
Figs. 21, 22, 48

Corbula tenuis G. B. Sowerby |, 1833
G. B. Sowerby I, 1833: 36; Hanley, 1843:
47; Reeve, 1843: pl. 2, fig. 13; C. B.
Adams, 1852a: 523-524 [1852b: 299-
300]; Carpenter, 1857a: 183, 228, 244,
280, 300; 1864a: 363 [1872 reprint: 204];
1864b: 537 [1872 reprint: 23]; Tryon,
1869: 66; Lamy, 1941: 143-144; Vokes,
1945: 9, 14-15, pl. 2, figs. 10, 11; Keen,
1958: 211, fig. 538 [Corbula (Tenui-
corbula)]; Olsson, 1961: 433-434, 550,
pl. 77, fig. 3, За [Tenuicorbula]; Keen,
1971: 268-270, fig. 691 [Corbula (Tenui-
corbula)]

Corbula glypta Li, 1930
Li, 1930: 264, pl. 5, fig. 38, 38a; Pilsbry,
1931: 431 [as a synonym of C. tenuis]

Type Materials & Localities

Corbula tenuis- BMNH 1966563, holotype,
pair; length, 22.8 mm; height, 12.1 mm; width,
10.2 mm (Fig. 21). Bay of [Golfo de] Montijo,
Veraguas Province, Panama (7.7°N); 12 fms.
[22 m]; Hugh Cuming.

Corbula glypta- AMNH 268094 [formerly
Columbia University 22098], holotype, pair;
length, 23.9 mm; height, 13.4 mm; width, 10.6
mm (Fig. 22). Mouth of Rio Grande near La
Boca, Panamä Province, Panamä (8.9°N);
10-40 ft. [3-12 m]; D. F. MacDonald, 1907.
As “Miocene,” but actually Recent (Pilsbry,
1931: 428, 431).

Description

Shell elongate-subquadrate, thin, sube-
quivalve; longer posteriorly (beaks at 40%
from anterior end). Posterior end slightly ta-
pered ventrally, truncate, sharply turned dor-
sally. Posterior slope set off from central slope
by a carina. Escutcheon well defined by a
sharp angle.

Beaks, central and posterior slopes with
fine, dense commarginal ribs, strongest on
posterior slope. Escutcheon much smoother.

Periostracum tan; white exteriorly and inte-
riorly.

Right valve with a long, narrow tooth. Left
valve with a projecting chondrophore; tooth

EASTERN PACIFIC CORBULIDAE

73

FIGS. 21, 22. Corbula tenuis. FIG. 21. Holotype of C. tenuis; BMNH 1966563; length, 22.8 mm. FIG. 22.
Holotype of C. glypta; AMNH 268094; length, 23.9 mm.

not evident (Fig. 48). Length to 24.5 mm (Ve-
nado Beach, Panama Province, Panama;
Skoglund Collection).

Distribution

Southeastern coast of Isla Tiburon, Sonora,
Mexico (28.9°N) (MCZ 319592), to Zorritos,

Tumbes Province, Peru (3.7°S) (UMML
3011808 411858 11359) e11S95a" 11S96:
11397, 11401), from the intertidal zone to 73
m (mean 12.9 m; n = 8), on mud or sand. |
have seen 30 lots, including the types.
Although Olsson (1932) mentioned a spec-
imen from an old collection at Cornell Univer-
sity labeled as having come from Mazatlan,

74 COAN

Sinaloa, México, no specimens from north of
Panama have been collected in recent years
other than the single specimen from the cen-
tral Golfo de California in the MCZ cited here
as the northern record (MCZ 319592).® An-
other specimen in the MCZ (319593) from an
old collection is labeled has having come from
“Margarita Bay, California,” but there is no
such place in California or Baja California,
and this specimen may instead have come
from Panama. The record by DuShane (1962:
44) of С. tenuis from Puertecitos, Baja Cali-
fornia, México, cannot be verified, because
the specimen has not been located in the
AMNH (J. Cordero, e-mail, 17 January 2001),
the present location of the bulk of the
DuShane collection.

This species has also been recorded on the
Pleistocene third terrace, Peninsula de Santa
Elena, Guayas Province, Ecuador (Hoffstet-
ter, 1948: 81).

Discussion

Corbula tenuis lupina Olsson (1932: 143,
pl. 14, figs. 7, 10), described from the Mio-
cene Tumbes Formation at Quebrada Tucillal,
Zorritos, Tumbes Province, Peru, was said to
differ in being heavier, more narrowly elon-
gate, and more coarsely sculptured. Jung
(1965: 477, 620, pl. 62, figs. 8, 9) subse-
quently reported this subspecies from upper
middie Miocene beds on the Paraguana
Peninsula of Venezuela, and he described the
similar Tenuicorbula melajoensis from the late
Miocene of Trinidad (Jung, 1969: 413-414, pl.
40, figs. 7-9); it was said to differ in having
heavier sculpture. These fossil taxa merit
comparison with C. acutirostra Spieker, 1922
(pp. 176-177, pl. 10, figs. 18, 19), described
from the late Miocene Zorritos Formation of
Peru.

Subgenus (Varicorbula) Grant & Gale, 1931

Varicorbula Grant & Gale, 1931: 420, foot-
note 1

Тре voucher collection in the MCZ from Parker's
(1964) study of the benthic fauna of the west Mexi-
can coast was incomplete, and the labels with the
remaining specimens do not always correspond to
the publication. For example, the single specimen
that now constitutes the northern record of this
species was not cited in the report, and the only
other extant Parker voucher lot labeled C. tenuis
(his station 1) contained a mixture of C. nasuta
(MCZ 253549) and C. biradiata (MCZ 320412).

Type species (original designation): Tellina
gibba Olivi, 1792: 101. Recent, Mediter-
ranean.

Shell of medium size, very inequivalve, with
right valve larger, higher, more inflated and
more rostrate. Right valve with pronounced
commarginal sculpture; left valve with sub-
dued commarginal sculpture and often with
sparse radial ribs.

Corbula (Varicorbula) grovesi
Coan, new species
Figures 23, 49

Type Materials & Locality

LACM 2891, holotype, pair; length, 11.0
mm; height, 10.1 mm; width, 6.7 mm (Figs.
23, 50). LACM 2892, paratype, pair; length,
11.0 mm; height, 9.0 mm; width, 6.0 mm. Sal
Si Puedes Basin, S end of Isla San Lorenzo,
Baja California Sur, Mexico (28.7°N,
113.0°W), in 732 m (LACM 67-135).

Description

Trigonal-ovate; right valve much larger than
left; left valve fitting inside right valve, leaving
a wide margin; subequilateral (beaks at 48%
from anterior end). Anterior end rounded; pos-
terior end narrowed, subtruncate, without a
radial rib between the central and posterior
slopes. Escutcheon present, but not defined
by a rib.

Beaks smooth. Right valve with dense,
closely set commarginal ribs. Left valve with
5-6 faint radial ribs; otherwise without sculp-
ture.

Periostracum very thin. White externally; in-
ternally white to yellowish. Hinge plate broad;
right valve with a prominent tooth; left valve
with a large, vertically projecting chon-
drophore and a prominent, elongate tooth
(Fig. 49). Length to 11.0 mm (holotype and

paratype).

Distribution

Thus far known from only the type lot con-
taining two pairs.

Etymology

This species is named for Lindsey T.
Groves of the Natural History Museum of Los

EASTERN PACIFIC CORBULIDAE 75

FIG. 23. Corbula grovesi, holotype; LACM 2891; length, 11.0 mm.

Angeles County, who has helped with this and
many other projects.

Discussion

A similar western Atlantic Recent species is
C. operculata Philippi, 1848 (p. 13), which oc-
curs from North Carolina to Brazil. Synonyms
of С. operculata include С. krebsiana С. В.
Adams, 1852c (p. 234), C. disparilis d’Or-
bigny, 1853 (p. 283 [Spanish ed., p. 322], pl.
27, figs. 1-4), and C. philippii E. A. Smith,
1885 (p. 33, pl. 7, fig. 4-4b [not “pl. 8,” as
stated in text]). Acommensal foraminiferan of

C. operculata was discussed by Bock &
Moore (1968). Corbula caloosae Dall, 1898
(p. 853), 1900 (pl. 36, fig. 16), from the Plio-
Pleistocene Caloossahatchee Formation of
Florida, merits close comparison with C. oper-
culata. The two Recent species differ as fol-
lows (based on a comparison with BMSM
15009; W of Cape Romano, Collier County,
Florida; and BMSM 15011; Boca Grande, Lee
County, Florida):

grovesi operculata
rounded posteriorly truncate posteriorly
fine sculpture in left heavier sculpture in left

valve valve

76 COAN

periostracum inconspic- periostracum shiny, light
uous, transparent tan
right valve smooth, with right valve with moderate
fine radial rays commarginal sculpture
and still finer radial
rays

beaks less prominent beaks prominent

Of Recent eastern Pacific species, C.
grovesiis closest to C. obesa, differing in hav-
ing a thin, transparent periostracum, being
more inequivalve, in lacking radial ribs on the
beaks, in having a smoother left valve with
faint radial ribs, and in having a proportion-
ately broader hinge plate in the left valve with
larger teeth.

Corbula grovesi differs from the European
type species of the subgenus Varicorbula— С.
gibba- in being more triangular, less truncate
posteriorly, and white in color.

There are two Pliocene species of Corbula
(Varicorbula) in western North America. Cor-
bula gibbiformis Grant & Gale, 1931 (pp.
420-421, 920, pl. 19, figs. 4-6), was de-
scribed from the early Pliocene upper
Etchegoin Formation (near lower boundary of
San Joaquin Formation) at Southern Califor-
nia Gas Company Well 1-4 (Sec. 4, T. 28 S.,
R. 23 E.; approximately 35.5°N, 119.5°W),
Kern County, California, at a depth of 3,951-
3,952 feet. Examination of the holotype of C.
gibbiformis (SDMNH 172, right valve; length,
about 13 mm; height, about 13 mm) demon-
strates that it is very different from C. grovesi,
with heavy sculpture, bulbous, projecting
beaks, and more prominent radial sculpture in
the left valve. This Pliocene species is also re-
ported from the late Pliocene San Joaquin For-
mation, Kettleman Hills, Kings County (Grant
& Gale, 1931; Woodring, 1938: 55-56, pl. 6,
figs. 8, 9; Woodring et al., 1941: opposite p.
78), the Niguel Formation, Orange County
(Vedder, 1960: 326), and the San Diego For-
mation, San Diego County (Hertlein & Grant,
1972: 323-324, pl. 57, figs. 3, 4), California,
and northwestern Baja California (Rowland,
1972:29, as С. “gibbiformis (Sowerby, 1833)”),
and from the middle Pliocene Pico [and/or
Saugus] Formations, Ventura County (Watts,
in Grant & Gale, 1931; Woodring et al., in Win-
terer & Durham, 1962: 304-305), the Santa
“Clara” [Clarita] Valley, “Ventura” [Los Ange-
les] County (Grant & Gale, 1931), and the East
Coyote field, USGS Loc. 13873, Los Angeles
Basin, Los Angeles County (Woodring, 1938;
USNM 496103), California.

Corbula (Varicorbula) granti Olsson, 1942
(p. 197 [=45], 238 [=86], pl. 15 [=2], figs. 8, 9),

was described from the Pliocene Charco Azul
Formation, Quebrada Penitas, Burica Penin-
sula, Costa Rica. The type material (PRI
5505, 5506) is now missing (W. Allmon, e-
mail, 11 August 2000). It has a more rostrate
posterior end and heavier sculpture in the
right valve than C. grovesi, and it has no radial
sculpture on the left valve.

The Pliocene species merit further compar-
ison with several other species from New
World Pliocene, Miocene, and Oligocene
strata, including Corbula chowanensis Bailey,
1977 (pp. 129-130), from the Pliocene of
North Carolina, and C. caloosae Dall, 1898 (p.
853; 1900: pl. 36, fig. 16), from the Pliocene of
Florida. Corbula bradleyi Nelson, 1870 (p.
200), from the Miocene Tumbes Formation at
Zorritos, Tumbes Province, Peru, illustrated
and discussed by Spieker (1922: 171-172,
196, pl. 10, figs. 13, 14), was subsequently
tentatively identified from the Pliocene Charco
Azul Formation at Quebrada Melissa, Burica
Peninsula, Panama (Olsson, 1942: 197-
198). Other taxa include: Corbula chipolana
Gardner, 1928 (p. 229, pl. 34, figs. 13-17); C.
chipolana carolina Richards, 1977 (p. 33, pl.
11, figs. 34, 35); C. waltonensis Gardner,
1928 (p. 229, pl. 34, figs. 18-22), and C. wal-
tonensis rubisiniana Mansfield, 1932 (pp.
156-157, pl. 34, figs. 2-4), from the Miocene
of Florida; C. sanctidominici Maury, 1925 (pp.
98-99, pl. 19, fig. 2), from the upper Miocene
of the Dominican Republic; C. vieta Guppy,
1866b (pp. 580-581, 590, pl. 26, fig. 8), and
C. islatrinitalis Maury, 1925 (p. 101, pl. 19,
figs. 8-10), from the Miocene of Trinidad; C.
heterogena Dall, 1898, ex Guppy ms (p. 850;
1900: pl. 36, fig. 15), from the Miocene of
Panama; C. carrizalana F. Hodson, in F. Hod-
son & H. Hodson, 1931 (p. 23-24, pl. 10, figs.
4, 6-9), from the Miocene of Venezuela; C.
prenucia Speiker, 1922 (p. 172, pl. 10, fig. 12),
from the Miocene of Peru, and C. zuliana F.
Hodson, in F. Hodson & H. Hodson, 1931 (pp.
22-23, pl. 10, figs. 1-3, 5), from the upper
Oligocene of Venezeula. (For a discussion of
some of these species: Woodring, 1982:
715-717; Anderson, 1996: 20-22). It it likely
that there are fewer species than there are
names.

Corbula ( Varicorbula) obesa Hinds, 1843
Figures 24, 25, 50

Corbula obesa Hinds, 1843
Hinds, 1843: 57 [1844 reprint: 230];
Reeve, 1844: pl. 5, fig. 38 [in part; text but

EASTERN PACIFIC CORBULIDAE 77

FIGS. 24, 25. Corbula obesa. FIG. 24. Neotype of С. obesa; SBMNH 345495; length, 12.7 mm. FIG. 25. Fig-

ure of C. obesa from Hinds (1845).

not fig., which = C. nasuta]; Hinds, 1845:
68, pl. 20, fig. 12; C. B. Adams, 1852a:
522 [1852b: 298]; Carpenter, 1857a: 207,
280, 300; 1864a: 368 [1872 reprint: 204];
1864b: 537, 668 [1872 reprint: 23, 154];
Tryon, 1869: 65; Oldroyd, 1925: 202 [in
part]; Lamy, 1941: 133; Keen, 1958: 209,
fig. 529 [Corbula (Caryocorbula)]; Keen,
1966: 268; Keen, 1971: 265-266, fig.
679

Corbula nuciformis G. B. Sowerby |, auctt.,
non G. B. Sowerby I, 1833
Hertlein & Strong, 1950: 241, 251, pl. 2,
fig. 1; Keen, 1958: 209, fig. 528

Type Materials & Localities

Corbula obesa- SBMNH 345495, neotype
here designated, pair; length, 12.7 mm;
height, 11.0 mm; width, 9.0 mm, with dried
soft parts (Figs. 24, 50). Mazatlän, Sinaloa,
Mexico (23.2°N, 106.4°W); 91-128 m; Don-
ald R. Shasky, October 3, 1961; ex Skoglund
Collection. (Other material from this station in
the Skoglund and museum collections has no
type status.) Two localities were originally pro-
vided for this species—San Blas, Nayarit,
México (21.5°N), and Veragua[s] Province,
Panama (approximately 7.7°N); the collective

78 COAN

depth distribution originally given was 22-33
fms. [38-60 m], on mud; Edward Belcher. No
material from either station has been located
in the BMNH collection.

Workers have guessed about the identity of
this species. Reeve (1844) illustrated a spec-
imen of C. nasuta as C. obesa. Hertlein &
Strong (1950), followed by Keen (1958), fig-
ured the present species as C. nuciformis,
which is here regarded as a synonym of C.
nasuta. Keen (1958, 1971) speculated on a
possible relationship between C. porcella and
C. obesa, but they are not very similar (see
Discussion under C. porcella). À neotype is
therefore necessary to stabilize nomencla-
ture.

Hinds’ description and subsequent figure
(Fig. 25) are in accord with the neotype here
designated, which is about twice as large as
the originally measured specimen. Mazatlan
is approximately 225 km north of San Blas,
but this species occurs still further north.

Description

Shell ovate, very inflated, rotund; right valve
much more inflated than left; equilateral
(beaks at about 50% from anterior end). Pos-
terior end narrow, subtruncate, only slightly
extended by a spout. Posterior slope set off
from central slope by a broadly rounded ridge.
Escutcheon not evident.

Beaks of both valves with commarginal and
radial ribs. Right valve with heavy commar-
ginal sculpture; left valve with much finer com-
marginal sculpture. Periostracum heavy, light
brown. Exterior surface white; interior surface
white, suffused light brown; adductor muscle
scars and pallial line stained brown in some.
Right valve with a large tooth; left valve with a
narrow, non-projecting chondrophore and a
projecting ridge on its posterior end (Fig. 50).
Length to 12.7 mm (neotype).

Distribution

Isla Espiritu Santo, Baja California Sur
(25.5°N) (CAS 121638; LACM 60-6.27;
SBMNH 996; Skoglund Collection), and
Mazatlan, Sinaloa (23.2°N) (SBMNH 129866,
21358, 21362), México, to near Isla Coiba,
Veraguas Province, Panama (7.4°N) (Kaiser
Collection); from 14-205 m depth (mean,
100.9 m; n = 33), on mud bottoms. There is a
lot in the CAS labeled as having been col-
lected on Isla Cedros, Baja California

(28.2°N) (CAS 121638), presumably in beach
drift. This locality requires further verification.
| have seen 38 lots.

The record of C. obesa from Santa Catalina
Island, California (Dall, 1921: 53), was based
on specimens of C. nasuta (USNM 199001),
and also these were probably from Isla Santa
Catalina in the southern Golfo de California, a
labeling error that has resulted in other mis-
taken Californian records of Panamic taxa.

Discussion

Corbula granti Olsson, 1942, from the
Pliocene of Costa Rica, may be a synonym of
C. obesa (reference in Discussion of C.
grovesi). Olsson’s material was admittedly
worn, and the radial sculpture on the beaks
may not have been visible. Short of rediscov-
ery of the now-missing type material, topo-
typic specimes would be required to make a
convincing case.

This species has some resemblance to C.
patagonica d’Orbigny, 1845 (p. 570; 1847: pl.
82, figs. 18-22), which occurs from Brazil to
Argentina. Corbula patagonica attains a
larger size, has a narrower, more produced
posterior end, and has less bulbous beaks
that lack the prominent radial sculpture pres-
ent on those of C. obesa. Corbula patago-
nica also has sparse radial ribs on its right
valve.

Corbula, $.1.

| am hesitant to place any of the following
species in named subgenera. Much more
work must be done to demonstrate true lin-
eages within this family.

Corbula biradiata G. B. Sowerby |, 1833
Fig. 26-31, 51

Corbula biradiata G. B. Sowerby |, 1833
G. B. Sowerby I, 1833: 35; Hanley, 1843:
47, 4, pl. 10, fig. 51; 1856: 344; Reeve,
1843: pl. 1, fig. 3; d’Orbigny, 1845: 571;
C. B. Adams, 1852a: 521-522 [1852b:
297-298]; Carpenter, 1857a: 183, 244,
280, 300; 1857b: 22; 1864a: 368, 369
[1872 reprint: 204, 205]; 1864b: 534, 537,
553, 637 [1872 reprint: 20, 23, 39, 123];
Tryon, 1869: 63; Lamy, 1941: 134;
Hertlein & Strong, 1950: 238 [Aloidis
(Caryocorbula)]; Keen, 1958: 208-209,

EASTERN PACIFIC CORBULIDAE 79

FIGS. 26-28. Corbula biradiata. FIG. 26. Lectotype of C. biradiata: BMNH 1966564/1; length, 16.2 mm. FIG.

27. Holotype of C. rubra; MCZ 186313; length, 12.5 mm. FIG. 28. Holotype of C. ecuabula; ANSP 14486;
length, 16.3 mm.

80 COAN

FIGS. 29-31. Corbula biradiata. FIG. 29. Holotype of Juliacorbula elenensis; ANSP 218913; length, 17.0
mm. FIG. 30. Paratypes of J. elenensis; UMML 30.11384; lengths, 11.5 mm, 9.4 mm. FIG. 31. LACM 34-
318.2; Isla La Plata, Manabi Province, Ecuador; 13-18 m; length, 12.3 mm.

fig. 524 [poor redrawing of Reeve's fig-
ure] [Corbula (Caryocorbula)]; Olsson,
1961: 437, 548, pl. 75, figs. 6-6b [Juli-
acorbula]; Keen, 1971: 267-268, fig. 685
[Corbula (Juliacorbula)]
Corbula rubra C. B. Adams, 1852

C. B. Adams, 1852a: 523, 548 [1852b:
299, 324]; Carpenter, 1857a: 280, 300;
1864a: 363 [1872 reprint: 204] [as a syn-
onym of C. biradiata]; 1864b: 553 [1872

reprint: 39]; Turner, 1956: 82-83, 126, pl.
17, figs. 8, 9

Corbula polychroma Gould & Carpenter, 1857
Gould 8 Carpenter, 1857: 198-199; Car-
penter, 1857a: 226, 228, 300; 1864a: 31
[1872 reprint: 205] [as a synonym of C.
biradiata]; Carpenter, 1864b: 534, 553
[1872 reprint: 20, 39]; Palmer, 1958: 117;
1963: 318; R. I. Johnson, 1964: 129

Corbula ecuabula Pilsbry & Olsson, 1941

EASTERN PACIFIC CORBULIDAE 81

Pilsbry 8 Olsson, 1941: 75, 78, pl. 12, figs.

3-5; Olsson, 1961: 437 [Juliacorbula]
Juliacorbula elenensis Olsson, 1961

Olsson, 1961: 438, 550, pl. 77, fig. 5;

Keen, 1971: 267, 268, fig. 686 [Corbula

(Juliacorbula)]

Type Materials 4 Localities

Corbula biradiata-BMNH 1966564/1, lec-
totype here designated, closed pair, num-
bered “5” on shell, possibly the originally mea-
sured specimen and that figured by Reeve
(1843); length, 16.2 mm; height, 11.1 mm;
width, 8.7 mm (Fig. 26). BMNH 1966564/2,
paralectotype, open pair, length 15.2 mm.
BMNH 1966564/3, paralectotype, closed pair;
length, 14.7 mm; BMNH 1966564/4, open
pair; length, 14.1 mm. [Golfo de] Chiriquí, Ve-
raguas Province, Panamá (8.0°N); Hugh
Cuming. BMNH 1907.12.30.118, paralecto-
type, open pair, that figured by Hanley (1843);
length, 9.5 mm. “Bay of Caraccas” [Bahia de
Caräquez], Manabi Province, Ecuador; Hugh
Cuming. A collective habitat of 3-6 fms. [5-11
m], on mud and sand, was given for this
species.

Corbula rubra-MCZ 186313, holotype,
pair; length, 12.5 mm; height, 4.6 mm; width,
3.6 mm (Fig. 27). Panamä, presumably near
Panamä City, Panamä Province (about
9.0°N), Panama; C. B. Adams, 27 Nov. 1850-
2 Jan. 1851.

Corbula polychroma-Not located. The
originally cited Gould collection specimens,
stated to have come from Santa Barbara, Cal-
ifornia, might be expected to be either in the
MCZ or the USNM, but they have not been lo-
cated. Carpenter (1864a:31) later said that
these specimens probably instead came from
either Acapulco, México, or Panamä. Speci-
mens were also originally cited from the Cum-
ing Collection obtained from the Golfo de Cal-
ifornia, and these might be expected in the
BMNH, but they have not been located. The
originally stated measurements were: length,
13.4 mm; height, 6.8 mm; width, 9.4 mm
(height and width were probably reversed).

Corbula ecuabula- ANSP 14486, holotype,
right valve; length, 16.3 mm; height, 11.5 mm;
width, 4.2 mm (Fig. 28). ANSP 78998,
paratype, left valve; length, 11.2 mm. Punta
Blanca, Manabi Province, Ecuador (1.1°S);
Canoa Formation, middle to late Pliocene.
UMML 30.11455, paratypes, 1 right valve, 2
left valves; Puerto Callo, Manabi Province,
Ecuador (1.3°S); Recent.

Juliacorbula elenensis-ANSP 218913,
holotype, right valve; length, 17.0 mm; height,
12.0 mm; width, 4.4 mm (Fig. 29). UMML
30.11341, paratypes, 5 right valves, 1 left
valve; [Salinas], Santa Elena Peninsula,
Guayas Province, Ecuador (2.2°S). UMML
30.11384, paratypes, 5 right valves, 1 left
valve (Fig. 30); Puerto Callo, Manabi
Province, Ecuador (1.3°S). UMML 30.11456,
paratype, 1 right valve; Zorritos, Tumbes
Province, Peru (3.7°S)

Description

Ovate-elongate, thin to moderately heavy;
right valve slightly larger than left; posterior
end usually longer (beaks at 41-48% from
anterior end), but anterior end slightly longer
in some specimens (beaks at 54-55% from
anterior end). Anterior end rounded; posterior
end narrowed, obliquely subtruncate, ext-
ended by a small spout in some specimens;
bluntly subtruncate in some specimens. Pos-
terior slope set off from central slope by a
fairly sharp ridge that continues to ventral
margin. Escutcheon well defined by a ridge.

Beaks, central and posterior slopes with
moderate commarginal ribs and very fine ra-
dial striae. Some material with only fine com-
marginal striae. Escutcheon smooth.

Exterior light tan to orange, with light radial
color bands about a third of the way to ends in
small specimens; ends purple. Interior red-
dish-brown to purple, particularly around mar-
gins, but some material nearly colorless.
Hinge plate broad; right valve with a large
tooth; left valve with a narrow chondrophore
and a conspicuous tooth (Fig. 51). Length to
20.8 mm (Bucaro, Los Santos Province,
Panama; UMML 30.11442).

Distribution

El Solita, Laguna Ojo de Liebre [Scam-
mons], Baja California (27.8°N) (SBMNH
31519); Bahia San Luis Gonzaga, Baja Cali-
fornia (29.8°N) (LACM 40-36.5), and Isla San
Jorge, Sonora (31.0°N) (Skoglund Collec-
tion), México, to Punta Pena Mala, Piura
Province, Peru (4.2°S) (UMML 30.11449); Isla
Santa Cruz (LACM 38-193.14) and Isla San
Cristobal (LACM 34-43.22), Islas Galapagos,
Ecuador; from the intertidal zone to 57 m
(mean, 7.3 m; n = 86), on mud or sand. | have
seen 248 lots, including the types of the vari-
ous synonyms.

82 COAN

This species is reported in beds of Pleis-
tocene age at Puerto Libertad, Sonora, Mex-
ico (Stump, 1975: 182, 186, 193, 195), and
from the Pleistocene Armuelles Formation,
Burica Peninsula, Chiriqui Province, Panamä
(Olsson, 1942: 162). It is also recorded from
the middle to late Pliocene Canoa Formation
at Punta Blanca, Manabi Province, Ecuador
(Pilsbry & Olsson, 1941: 11, 75, as both C. bi-
radiata and C. ecuabula; UMML 30.11380).

Rutten (1931: 661) reported but did not fig-
ure this species (as “cf.”) from the Quaternary
of Surinam, based on an unpublished thesis,
a record that requires further verification.

Discussion

| have come to the conclusion that C.
ecuabula is inseparable from C. biradiata.
The key feature of Corbula ecuabula, which
Pilsbry & Olsson (1941) and Olsson (1961)
also reported from the Recent fauna, was that
it is significantly longer anteriorly. Olsson de-
scribed the beaks of C. ecuabula as being in
the “posterior third”, but even in his type ma-
terial, the beaks are only 4-5% behind the
midline. Occasional specimens may be found
throughout the distribution of C. biradiata that
have a slightly longer anterior end.

| then concluded that Juliacorbula elenen-
sis is also a synonym. This species was
based on beach-drift material that is light in
color, rounded, and with even commarginal
sculpture. | have found similar material from
the Golfo de California (LACM 78-30.12, from
Bahia San Carlos, Sonora, and LACM 79-
111.14, from Bahia de los Angeles, Baja Cali-
fornia), and a specimen from Esmeraldas,
Ecuador (UMML 30.11321), came to light with
subdued sculpture in the left valve and “ele-
nensis”-like sculpture in the right valve. An off-
shore specimen from Isla La Plata, Manabi
Province, Ecuador (LACM 34-318.8), is thin,
white, and almost equilateral (Fig. 31).

In the fossil fauna, this Corbula biradiata
merits comparison with C. (Caryocorbula)
urumacoensis F. Hodson, in F. Hodson & H.
Hodson, 1931 (pp. 25-26, pl. 12, figs. 1-7),
and C. (C.) democraciana F. Hodson, in F.
Hodson & H. Hodson, 1931 (pp. 26-27, pl. 11,
figs. 1-6), both described from the middle
Miocene of Venezuela, and perhaps to C. (C.)
retusa Gardner, 1944 (p. 140, pl. 23, figs. 33,
34) [synonym: C. (C.) conradi Gardner, 1944:
139-140, pl. 23, figs. 27, 28, non Dall, 1898),
from the Pliocene of Virginia and North Car-

olina (concerning C. retusa: Campbell, 1993:
47-48, pl. 21, fig. 190).

Corbula colimensis Coan, new species
Figures 32, 52

Corbula ventricosa A. Adams & Reeve, auctt.,
non À. Adams & Reeve, 1850
Hertlein & Strong, 1950: 242-243, pl. 2,
figs. 3, 4 [Aloidis (Caryocorbula)]; Keen,
1958: 210-211, fig. 532 [Corbula (Cary-
ocorbula)}; Olsson, 1961: 428, 549, pl. 76,
fig. 9 [Caryocorbula (Caryocorbula)];
Keen, 1971: 266-267, fig. 682 [Corbula
(Caryocorbula)]

Type Material & Locality

SBMNH 345496, holotype, pair; length,
13.7 mm; height, 9.5 mm; width, 7.7 mm
(Figs. 32, 52). SBMNH 345497, paratypes, 3
closed pairs; lengths, 14.0 mm, 13.4 mm, 3.1
mm. Las Ventanas, Manzanillo, Colima, Méx-
ico (19.0°N, 104.3°W), 42 m; Carl & Laury
Shy, 1969-1970; ex Skoglund Collection

Description

Shell ovate, thin to moderate in thickess;
right valve slightly larger than left; posterior
end longer (beaks at 32% from anterior end).
Posterior end subtruncate, extended by a
short spout in some specimens; posterior
slope set off from central slope by a rounded
ridge. Escutcheon faint, set off by a slight
ridge in right valve.

Beaks with fine commarginal ribs; most of
surface with moderate commarginal ribs. Pe-
riostracum light brown. Shell white exteriorly
and interiorly. Hinge plate of moderate thick-
ness; right valve with a large tooth; left valve
with a chondrophore of medium width, which
is not very projecting, and a moderate tooth.
Pallial sinus with a somewhat sharp bend
posteriorly (Fig. 53). Length to 14.0 mm (a

paratype).
Distribution

Los Corchos, Nayarit (21.7°N) (MCZ
260451), to Bahia Tangola Tangola, Oaxaca
(15.8°N) (LACM 34-240.5), Mexico, in 29-112
m (mean, 55.9 m; n = 7), on mud bottoms.
This species is thus far known from 8 lots. The
lot cited by Hertlein & Strong (1950) from off

EASTERN PACIFIC CORBULIDAE 83

FIG. 32. Corbula colimensis, holotype; SBMNH 345496; length. 13.7 mm. FIG. 33. C. ira, lectotype; USNM
122944; length, 11.4 mm.

84 COAN

Bahia Tangola Tangola has not been located
inthe CAS or AMNH.

Etymology

This species is named for the Mexican state
of Colima.

Discussion

Corbula colimensis is most similar in size,
shape, and color to C. obesa, but the new
species is equivalve, thinner, and less in-
flated, and it has no radial sculpture.

Corbula ira Dall, 1908
Figures 33, 53

Corbula (Cuneocorbula) ira Dall, 1908
Dall, 1908: 423; Lamy, 1941: 233-234;
Keen, 1958: 210 [as a synonym of С. ven-
tricosa]; Olsson, 1961: 436, 549, pl. 76,
fig. 5 [Juliacorbula]; Keen, 1971: 267-
268, fig. 687 [Corbula (Juliacorbula)]

Type Material & Locality

USNM 122944, lectotype here desig-
nated, right valve; length, 11.4 mm; height,
8.6 mm; width, 3.0 mm (Fig. 33). USNM
880651, paralectotypes, 1 left valve, length,
11.8 mm (close in size to lectotype, but they
are not a pair); right valve, length, 11.0 mm.
Albatross Stn. 3355; Dall (1908), followed by
other authors, reported this as “Gulf of
Panama,” but the log and map (Townsend,
1901: 412) place this station at 7.2°N, 80.9°W,
just off Punta Mariato, Peninsula de Azuero,
Veraguas Province, Panama; 182 fms. [333
m], mud; Feb. 23, 1891.

Description

Ovate-subquadrate, moderately heavy;
right valve slightly larger than left; beaks
closer to anterior end (beaks at 30-34% from
anterior end). Ventral surface sometimes flat-
tened. Anterior end sharply rounded. Poste-
rior end tapered, obliquely truncate. Posterior
slope set off from central slope by a sharp
ridge that runs almost to ventral margin. Es-
cutcheon defined by a ridge.

Beaks relatively smooth. Central slope with
strong, rounded, shingle-like commarginal
ribs and very fine radial ribs. Posterior slope
with finer commarginal ribs.

Periostracum tan. Exterior white; interior
white, sometimes suffused with purple or
brown. Hinge plate broad; right valve with a
large tooth; left valve with a very small chon-
drophore and a small tooth (Fig. 53). Length
to 13.6 mm (USNM 810921; Cabo Lobos,
Sonora, México).

Distribution & Habitat

Cabo Lobos, Sonora (29.9°N) (USNM
212797; MCZ 260668), and Bahia San Luis
Gonzaga, Baja California (29.8°N) (LACM 37-
199.10, 40-36.6), México, to Callao, Lima
Province, Peru (12.1°S) (LACM 35-153.2, 35-
177.3); Isla del Coco, Costa Rica (SBMNH
345530), from 15 to 388 m (mean, 98.4 m;
n = 60), on sand and mud. | have seen 85 lots,
including the types.

Discussion

This species can be distinguished from C.
marmorata of similar size in being more
quadrate and elongate, more truncate poste-
riorly, with a stronger radial rib, and commar-
ginal ribs that are higher and narrower. It may
also become ventrally flattened, as in C. na-
suta, which is never true of C. marmorata.

Corbula luteola Carpenter, 1864
Figures 34, 35, 54

Corbula luteola Carpenter, 1864
Carpenter, 1864b: 611, 637 [1872 reprint:
97, 123]; 1865: 207; Tryon, 1869: 65;
Arnold, 1903: 181, pl. 17, fig. 11 [a poor
figure]; Oldroyd, 1925: 203; Grant &
Gale, 1931: 421-422, 920, pl. 19, figs. 2,
7 [Corbula (Lentidium)]; Lamy, 1941:
240-241 [Corbula (Corbuloyma)|;
Hertlein & Strong, 1950: 239 [Aloidis
(Caryocorbula)]; Keen, 1958: 209 [in
part; not fig. 525, which = C. marmorata]
[Corbula (Caryocorbula)]; Palmer, 1958:
117-118, 340, pl. 15, fig. 13-18 [Corbula
(Lentidium)]; Keen, 1971: 264 [in part;
not fig. 675, which = C. marmorata] [Cor-
bula (Caryocorbula)]; Hertlein & Grant,
1972: 324-325, pl. 55, figs. 1, 2, 5, 6, 15
[Corbula (Lentidium)]; Coan et al., 2000:
479, pl. 102 [Juliacorbula]

Corbula luteola rosea Williamson, 1905, non
T. Brown, 1843 (or earlier), non Reeve,
1844
Williamson, 1905: 120; Coan, 1989: 298

EASTERN PACIFIC CORBULIDAE 85

FIGS. 34, 35. Corbula luteola. FIG. 34. Lectotype of С. luteola; USNM 14897; length, 10.2 mm. FIG. 35. Holo-
type of C. luteola rosea; LACM 1421; length, 7.0 mm.

[as a synonym of С. luteola] [non Corbula
rosea T. Brown, 1843 (or earlier): 105, pl.
42, fig. 6; non Corbula rosea Reeve,
1844: pl. 5, fig. 26]

Type Materials & Localities

Corbula luteola-USNM 14897, lectotype
here designated, the largest pair, closest to
Carpenter’s (1865) measurement; length,
10.2 mm; height, 6.8 mm; width, 4.5 mm (Fig.
34). USNM 880650, paralectotypes: pair,
length, 8.4 mm (the specimen figured by
Palmer, 1958); right valve, length, 8.3 mm; left
valve, length 5.9 mm; pair, length, 5.7 mm.

San Pedro, Los Angeles County, California
(33.7°N); James G. Cooper. (Evidently, at
some point in the past, USNM 15668 had
been combined into this lot, because this
number is written on the back of the label.)
USNM 73457, paralectotypes, 1 right valve, 1
left valve; San Diego, San Diego County, Cal-
ifornia (32.7°N); James G. Cooper. Lots in
other collections from Cooper material are not
types, because they were not studied by Car-
penter.

Corbula luteola rosea Williamson —LACM
1421, holotype, pair; length, 7.0 mm; height
4.6 mm; width, 2.3 mm (Fig. 35). Terminal Is-
land, San Pedro, California (33.7°N); on an

86 COAN

anemone in a rock pool on the old breakwa-
ter; Martha Burton Williamson. The original
description specified a single valve, but what
is in the type lot is a matched pair of valves.

Description

Shell ovate, solid; right valve slightly larger
than left; posterior end longer (approximately
42-48% from anterior end). Posterior end
vertically truncate. Posterior slope set off from
central slope by a rounded ridge that be-
comes obsolete ventrally. Escutcheon evident
but not sharply defined.

Beaks, central and posterior slopes with
fine commarginal sculpture.

Exterior white or pink, sometimes with pur-
ple patches on either side of beaks. Interior
white or tan. Hinge plate relatively narrow;
right valve with a triangular tooth; left valve
with relatively broad chondrophore and a
small tooth (Fig. 54). Length to 10V.2 mm
(holotype; CAS 121848; Point Loma, San
Diego County, California).

Distribution

Monterey, Monterey County, California,
U.S.A. (36.7°N) (CAS 121792), possibly a re-
sult of larval settlement in an El Nino year;
Topanga Creek, Los Angeles County, Cali-
fornia (33.7°N) (LACM 68-192.21), south to
Bahia Magdalena, Baja Californ Sur (CAS
121791; USNM 217823; Skoglund Collec-
tion); from the intertidal zone to 80 m (mean,
16.1 т; п = 47), in rubble. | have seen 157 Re-
cent lots, including the types.

Records of this species from the Golfo de
California were based on misidentified C.
marmorata, or, in the case of Parker (1964:
161), C. nasuta (MCZ 253667, 259948,
260263) and C. ira (MCZ 260703).

This species has been widely reported as a
fossil in California and Baja California. In the
late Pleistocene, there are records from ter-
races on Santa Rosa Island, Channel Islands,
Santa Barbara County (A. G. Smith, in Orr,
1960: 1117, A. G. Smith, 1968: 22); Playa del
Rey (Willett, 1937: 391), the Baldwin Hills (B.
L. Burch, 1947: 9), terraces in the Palos
Verdes Hills (Chace, 1966: 169; Marincovich,
1976: 20), San Pedro (DeLong, 1941: oppo-
site p. 244; Valentine, 1961: 375, 376, 377),
Huntington Beach (Valentine, 1959: 54), Los
Angeles County; Newport Bay, Orange
County (Kanakoff & Emerson, 1959: 22); San

Diego, San Diego County (Dall, 1878a: 11,
1878b: 27; Emerson & Chace, 1959: 338;
Valentine, 1961: 357; Valentine & Meade,
1961: 10; Kern et al., 1971: 333), California;
northwestern Baja California (Valentine,
1957: 297; Valentine & Rowland, 1969: 518);
Bahia San Quintin (Dall, in Orcutt, 1921: 24;
Jordan, 1926: 245; Manger, 1934: 293), Baja
California, Laguna San Ignacio (Hertlein,
1934: 64), Bahia San Bartolomé [Turtle Bay]
(Emerson, 1980: 72; Emerson et al., 1981:
111), and Bahia Magdalena (Jordan, 1936:
111), Baja California Sur. Records of this
species from the Pleistocene of the southern
Golfo de California (Durham, 1950) were
based on specimens of C. marmorata (see
under the latter).

It has been recorded in strata of early Pleis-
tocene age in Santa Monica (Woodring, in
Hoots, 1931: 121; Valentine, 1956: 196;
Rodda, 1957: 2484) and San Pedro (DeLong,
1941: opposite p. 244), Los Angeles County,
California.

Cooper, in Watts (1897: 79) and Woodring,
in Hoots (1931: 116) reported this species
from late Pliocene strata in Los Angeles
County, and Hertlein & Grant (1972: see syn-
onymy) reported it from the late Pliocene San
Diego Formation, San Diego County, Cali-
fornia. There is a record in the early Pliocene
Towsley Formation of Los Angeles County,
California (Woodring, in Winterer & Durham,
1962: 304-305).

There are also records from the late
Miocene Santa Margarita Formation (Gale, in
Preston, 1931: 15) of central California, the
late Miocene Castaic Formation of southern
California (Stanton, 1966: 23), from the early
to middle Miocene Temblor Formation (Ade-
goke, 1969: 153) of central California, and
from the Miocene Isidro Formation near Bahia
Magdalena, Baja California Sur (Beal, 1948:
66).

Discussion

This species is most similar to C. mar-
morata, which accounts for Panamic records
of C. luteola. The two differ as follows:

C. luteola C. marmorata

oval pointed posteroventrally

larger (to 10.2 mm) smaller (to 8.2 mm)

beaks closer to midline longer posteriorly

solid color mottled color

without a medial sulcus with a medial sulcus

radial ridge becomes radial ridge strong to
obscure ventrally ventral margin

chondrophore larger chondrophore smaller

EASTERN PACIFIC CORBULIDAE 87

hinge plate narrower at wider hinge plate
same size
finer, sharper sculpture heavier, more undulating
sculpture

beaks sculptured beaks less sculptured

For comparison with the sympatric C. por-
cella, see under the latter.

Of the western Atlantic taxa, С. luteola is
most similar to C. contracta Say, 1822 (p.
312), differing from it in being more inflated,
having a much more prominent ridge between
the central and posterior slopes, and having
more lamellar sculpture, which extends onto
the beaks.

Corbula marmorata Hinds, 1843
Figures 36, 37, 55

Corbula marmorata Hinds, 1843
Hinds, 1843: 58 [1844 reprint: 231];
Reeve, 1844: pl. 5, fig. 39; Hinds, 1845:
69, pl. 20, fig. 13; Carpenter, 1857a: 207,
300; Tryon, 1869: 65; Lamy, 1941: 133-
134; Hertlein & Strong, 1950: 239-240,
252, pl. 2, fig. 17 [Aloidis (Caryocorbula)];
Keen, 1958: 209, fig. 526 [Corbula (Cary-
ocorbula)]; Olsson, 1961: 431-432, 548,
pl. 75, fig. 5 [Caryocorbula (Caryocor-
bula)]; Keen, 1966: 268 [Corbula]; Keen,
1971: 264-265, fig. 676 [Corbula (Cary-
ocorbula)]; Gemmell et al., 1987: 58-59
[Corbula]

Corbula luteola Carpenter, auctt., non Car-
penter, 1864
Durham, 1950: 94, 170, pl. 25, figs. 15,
16; Keen, 1958: 209, fig. 525 [Panamic
records and the fig.]; Keen, 1971: 265,
fig. 675 [Panamic records and the fig.]

Type Material & Locality

Not located in the BMNH collection (Keen,
1966). The description and Hinds’ subsequent
figure, however, are sufficiently clear and un-
ambiguous that a neotype is not required. A
copy of the figure from Hinds (1845) is given
here (Fig. 36). The originally measured spec-
imen was 4.2 mm in length, 2.8 mm in height,
and 2.1 mm in width. Veraguafs] Province,
Panama (approximately 7.7°N); 26 fms. [48
m], mud; Edward Belcher.

Description
Shell ovate-subquadrate, moderately

heavy for size; right valve slightly larger than
left; beaks in anterior third of shell (beaks

28-36% from anterior end). Anterior end
rounded. Shallow medial radial sulcus pres-
ent. Posterior end truncate, pointed pos-
teroventrally. Posterior slope set off from
central slope by a radial ridge that runs to ven-
tral margin. Escutcheon evident, narrow,
strongest in right valve.

Beaks relatively smooth; central slope with
moderate, undulating commarginal ribs.
Sculpture more lamellar on posterior slope.
Exterior white, brown, or red, mottled with ma-
genta or white, and with a magenta patch an-
terior to beaks. Interior magenta, particularly
around margins. Right valve with a triangular
tooth; ligament under valve margin. Left valve
with a narrow chondrophore; tooth small
(Figs. 37, 55). Length to 8.2 mm (Bahia
Cholla, Sonora, México; Skoglund Collec-
tion).

Distribution

Bahia Magdalena, Baja California Sur
(24.6°N) (CAS 121641; LACM 67-70.26), and
throughout the Golfo de California to its head
at Puerto Penasco, Sonora (31.4°N) (UCMP
E.8419; CAS 121646; Skoglund Collection),
México, south to Callao, Lima Province, Perú
(12.195) (LACM 35-153.3). This species also
occurs in the western Atlantic, where speci-
mens are sometimes labeled as C. blandiana
C. B. Adams, 1852, which is a synonym of C.
dietziana C. B. Adams, 1852 (see Discussion
under C. speciosa), as C. barrattiana C. B.
Adams, 1852, a synonym of C. swiftiana C. B.
Adams. 1852 (see Discussion under C. na-
suta), or C. contracta Say, 1822 (p. 312), a
distinct, uncolored, more inflated, more heav-
ily sculptured species that occurs as far north
as New England. Corbula marmorata occurs
from the intertidal zone to 137 m (mean, 19.9
m; n = 115), in rubble and on the undersides
of rocks. | have seen 299 eastern Pacific lots.

The record of this species from the Galapa-
gos Islands (Kaiser, 1997: 25) was based on
a specimen of C. bicarinata (LACM 34-43).

Also present in the Pleistocene at Bahía
Santa Inez, Baja California Sur, México
(Durham, 1950: 94, as “С. luteola”).

Discussion

Corbula marmorata sometimes occurs at
the same stations as C. speciosa. Small spec-
imens of the latter are similar to C. marmorata
in having a mottled color pattern and a purple
patch in front of the beaks. In specimens of

88 COAN

FIGS. 36, 37. Corbula marmorata. FIG. 36. Figure of C. marmorata from Hinds (1845). FIG. 37. SBMNH
345499; Bahia Cholla, Sonora, Mexico; length, 6.8 mm. FIG. 38. C. speciosa, lectotype; BMNH 1967945/1;
length, 19.7 mm.

EASTERN PACIFIC CORBULIDAE 89

similar size, C. speciosa is more elongate;
longer, broader, and more truncate posteri-
orly; has a stronger rib separating the central
from the posterior slope; has a more pro-
nounced medial radial sulcus; and the pos-
terodorsal margin is more elevated and
flange-like.

Species that are possibly ancestral to C.
marmorata include: C. engonata burnsii Dall,
1898 (p. 847), C. (Caryocorbula) franci Gard-
ner, 1928 (pp. 231-232, pl. 35, figs. 1-4), C.
(C.) wakullensis Gardner, 1928 (p. 232, pl. 35,
figs. 5, 6), and C. barrattiana leonensis Mans-
field, 1932 (p. 160, pl. 33, figs. 1, 3), from the
Miocene of Florida, and C. cuneata Say, 1824
(pp. 152-153, pl. 13, fig. 3), from the Pliocene
of Maryland and Virginia.

Corbula speciosa G. B. Sowerby |, 1833
Figures 38, 56

Corbula radiata G. В. Sowerby I, 1833, non
Deshayes, 1824
G. B. Sowerby I, 1833: 36; Hanley, 1843:
47
[non Deshayes, 1824: 58-59, pl. 9, figs.
dientes]

Corbula speciosa Reeve, 1843
Reeve, 1843 (Aug.): pl. 1, fig. 6; Hinds,
1843 (Nov.): 57 [1844 reprint: 230];
Hinds, 1845: 68-69, pl. 20, figs. 7, 8;
Carpenter, 1857a: 207, 300; Tryon, 1869:
66; Lamy, 1941: 133; Hertlein & Strong,
1950: 237 [Aloidis (Aloidis)]; Keen, 1958:
208-209, fig. 522 [Corbula (Corbula));
Olsson, 1961: 438-439, 550, pl. 77, fig.
7-7c [Varicorbula]; Keen, 1971: 269-
270, fig. 692 [Corbula (Varicorbula)]

Type Materials & Localities

Corbula radiata—Not located in the BMNH.
The original measurements were: length, 8.9
mm; height, 6.3 mm; width, 4.3 mm. Acapulco,
Guerrero, México (16.9°N); Hugh Cuming.

Corbula speciosa-BMNH 1967945/1, lec-
totype here designated, the largest speci-
men and closest to Reeve’s figure; length,
19.7 mm; height, 15.4 mm; width, 11.6 mm
(Fig. 38). BMNH 1967945/2, paralectotype,
left valve; length, 18.4 mm. BMNH 1967945/3,
paralectotype, left valve; length, 15.3 mm.
Golfo de Nicoya, Costa Rica (approximately
9.9°N); Edward Belcher. BMNH 1879.2.26.91,

Not in Brocchi (1814), as Reeve (1843) claimed.
Deshayes’ species is a Cardiomya.

pair; length, 17.8 тт. Panama, 6 fms. [11 m],
mud; Edward Belcher. The latter seems to be
the specimen figured in Hinds (1845), but ma-
terial from Panama was not mentioned by
Reeve (1843), who made the name available
three months before Hinds.

Description

Shell subtrigonal as an adult, heavy; right
valve much larger than left, very inflated. Pos-
terior end truncate. Posterior slope set off
from central slope by a rounded ridge. Es-
cutcheon apparent in some specimens only.

Juvenile shell set off by a remarkable
change in growth direction, shape, sculpture,
and color. Subquadrate juvenile shell flat-
tened, with commarginal undulations, a me-
dial radial sulcus, fine radial ribs, and a mot-
tled color pattern.

Right valve of adult with commarginal un-
dulations; left valve with finer, lamellar, some-
what oblique sculpture. Exterior color of red-
dish-brown radial ribs. Periostracum light to
dark brown. Interior white to yellowish, with
red ribs visible ventrally. Right valve with a
large tooth; left valve with a small, non-pro-
jecting chondrophore, with the ligament con-
fined to a small area medial to a large poste-
rior cardinal (Fig. 56). Length to 20.6 mm
(Bahia Tenacatita, Jalisco, Mexico; LACM 34-
146.14).

Distribution

San Felipe, Baja California (31.0°N) (LACM
40-279.3), and Isla Tiburon, Sonora (LACM
36-80.45) (28.7°N), Mexico, to Punta Utria,
Choco Province, Colombia (6.0°N) (LACM
35-63.18, 35-170.7); Isla Socorro, Islas Revil-
lagigedo, Mexico (LACM 34-5.17; Kaiser col-
lection); from the intertidal zone to 125 m
(mean, 40.2 m; n = 98), on sand or gravel, or
in rubble. | have seen 137 lots, including the
types.

This species has been reported in late
Miocene deposits of the Imperial Formation in
Riverside County, California (Powell, 1988:
16).

Discussion

This species is very similar and perhaps
identical to the later-named western Atlantic
C. dietziana C. B. Adams, 1852c (pp. 235-
236), which occurs from North Carolina to
Brazil. Synonyms of C. dietziana include C.

90 COAN

blandiana C. B. Adams, 1852c (pp. 234-235),
and C. cymella Dall, 1881 (p. 115) (based on
comparison with BMSM 15012; Boca Grande,
Lee County, Florida).

Small specimens of Corbula speciosa can
be distinguished from C. ira, with which it
sometimes occurs, by its more quadrate out-
line, longer posterior end, mottled color, less
prominent sculpture, and medial radial sulcus.

Some authors have placed this species in
the subgenus Varicorbula because it is in-
equivalve, but I think that this allocation is pre-
mature. Corbula speciosa differs from other
species of Varicorbula in having a highly dif-
ferentiated juvenile shell. Indeed, in several
respects small specimens of C. speciosa are
closest to C. marmorata (for a comparison,
see Discussion under C. marmorata).

ADDITIONAL NOTES & RECORDS

Corbula altirostris Li, 1930 (pp. 263-264,
pl. 5, fig. 37), supposedly from an offshore
outcrop of the Miocene Gatun Formation in
the Bay of Panamä, was based on a Recent
specimen of the mactrid Mulinia pallida
(Broderip & Sowerby, 1829: 360, as Mactra)
(Pilsbry, 1931: 431).

Carpenter (1857a: 300; 1860: 2) listed a
Corbula boivinei, without authorship, from
Central America. This is a nomen nudum.

The record of “Corbula cf. collazica Maury,”
1920 (pp. 44-45, pl. 6, figs. 10, 11), suppos-
edly from an offshore outcrop of the Gatun
Formation in the Bay of Panama (Li, 1930:
263, pl. 5, fig. 36, 36a), was actually based on
a Recent specimen of Corbula ovulata, ac-
cording to Pilsbry (1931: 431).

Corbula gibbosa Broderip & Sowerby, 1829
(p. 361), from the Arctic coast of Alaska, is a
probable synonym of Mya truncata Linnaeus,
1758 (p. 670) (Coan et al., 2000: 471).

Corbula kelseyi Dall, 1916b (p. 416; Dall,
1916a: 41, nomen nudum), was based on a
worn specimen of Cumingia californica Con-
rad, 1837: 234 (Coan & Scott, 1991; Coan et
al., 2000: 437, 478).

The record of “Corbula cf. swiftiana C. B.
Adams,” 1852c (pp. 236-237), supposedly
from an offshore outcrop of the Gatun Forma-
tion in the Bay of Panama (Li, 1930: 264, pl. 5,
fig. 39), was based on a Recent specimen of
the mactrid Миша pallida (Broderip &
Sowerby, 1829) (Pilsbry, 1931: 431).

Corbula tenuis Moody, 1916 (pp. 59, 62, pl.
2, fig. 4a, 4b), поп ©. В. Sowerby |, 1833, = С.
binominata Hanna, 1924 (p. 163), from the

Pliocene Fernando Formation in downtown
Los Angeles, California [holotype: UCMP
11087; Loc. 3030], is not a corbulid. Although
broken and partially sediment- and glue-filled,
the holotype seems to be a poromyid, closest
and probably identical to the Recent Der-
matomya mactrioides (Dall, 1889a: 448, as
Poromya (Dermatomya)); Coan et al. (2000:
572) treated this Dermatomya. Corbula bi-
nominata was also later recorded from the
Pliocene of the Los Angeles Basin by Soper &
Grant (1932: 1060).

NOTE ADDED IN PROOF

A paper has recently appeared that sheds
new light on the western Atlantic species of
Corbula (Varicorbula) (Mikkelsen & Bieler,
2001). Using Varicorbula as a full genus, they
adopt the traditional early dates of d’Orbigny’s
species of Corbula, with the result that V. dis-
paralis (d’Orbigny, 1842) is regarded as the
valid name for the species closest to Corbula
(Varicorbula) grovesinamed here. Asynonym
that | had not noted, Corbula limatula Conrad,
1846 (p. 25, pl. 1, fig. 2), would be the valid
name if the dates of d’Orbigny are as late as |
now think. They regard Corbula operculata
Philippi, 1848, the name | used for this
species, as a nomen dubium, because of the
absence of type material. Clearly, reaching a
definitive conclusion about the dates of the all-
important d’Orbigny work should be a high pri-
ority for workers on the western Atlantic fauna.

CONRAD, T. A., 1846, Descriptions of new species
of fossil and Recent shells and corals. Proceed-
ings of the Academy of Natural Sciences of
Philadelphia, 3(1): 19-27, pl. 1

MIKKELSEN, P. M. & R. BIELER, 2001, Varicorbula
(Bivalvia: Corbulidae) of the western Atlantic: tax-
onomy, anatomy, life habits, and distribution. The
Veliger, 44(3): 271-293

ACKNOWLEDGMENTS

| appreciated the help of the following cura-
tors, other personnel and their institutions,
who made specimens, literature, and informa-
tion available: Warren D. Allmon, Paleonto-
logical Research Institution, Ithaca, New York,
USA; Laurie C. Anderson, Louisiana State
University, Baton Rouge, Louisiana, USA;
Adam J. Baldinger & Kenneth J. Boss, Mu-
seum of Comparative Zoology, Harvard Uni-
versity, Cambridge, Massachusetts, USA;
Warren Blow, Tyjuana Nickens, and Thomas
Waller, National Museum of Natural History,

EASTERN PACIFIC CORBULIDAE Ji

42

FIGS. 39-42. Diagramatic interior views of eastern Pacific species of Corbula, showing hinge, pallial sinus,
and adductor scars of right and left valves. Stippling in left valve indicates socket for tooth of right valve.
Cross-hatching indicates areas of ligament attachment on chondrophore of left valve and visible areas of at-
tachment on hinge margin of right valve. FIG. 39. C. amethystina; SBMNH 345487: Playas de Villimil,
Guayas, Ecuador; trawled; length, 30.8 mm. FIG. 40. C. nasuta; SBMNH 345492, between Santa Cruz and
Platinitos, Nayarit, Mexico; 6-18 m; length, 15.9 mm. FIG. 41. C. otra, new species; SBMNH 345493, holo-

type; Manazanillo, Colima, Mexico; 30-45 m; length, 22.8 mm. FIG. 42. C. ovulata; SBMNH 345498; Playas
de Villamil, Guayas, Ecuador; trawled; length, 29.2 mm.

92 COAN

FIGS. 43-46. Diagramatic interior views of eastern Pacific species of Corbula, showing hinge, pallial sinus,
and adductor scars of right and left valves. FIG. 43. C. porcella; CAS 142446; E end of Isla Cedros, Baja Cal-
ifornia, Mexico; 82 m; unpaired valves: right valve, 7.4 mm; left valve, 7.2 mm. FIG. 44. C. esmeralda; ANSP
218903, 403198, lectotype and paralectotype; Esmeraldas, Esmeraldas, Ecuador; lengths, 20.6 mm. FIG.
45. C. bicarinata; CAS 120689; San Felipe, Baja California, Mexico; length, 11.2 mm. FIG. 46. C. ventricosa;
CAS 120699; Panama; length, 15.3 mm.

EASTERN PACIFIC CORBULIDAE 93

SS

<
©

SS

FIGS. 47-50. Diagramatic interior views of eastern Pacific species of Corbula, showing hinge, pallial sinus,
and adductor scars of right and left valves. FIG. 47. C. amurensis; CAS 089104: Martinez, Contra Costa
County, California; length, 16.4 mm. FIG. 48. C. tenuis; CAS 120702; Isla Taboga, Panama, Panamá; length,
19.1 mm. FIG. 49. C. grovesi; LACM 2891, holotype; S end of Isla San Lorenzo, Baja California Sur, Mex-
ico; 732 m; length, 11.0 mm. FIG. 50. C. obesa; SBMNH 345495, neotype; Mazatlan, Sinaloa, México;
length, 12.7 mm.

50

94 COAN

se eos
54

FIGS. 51-54. Diagramatic interior views of eastern Pacific species of Corbula, showing hinge, pallial sinus,
and adductor scars of right and left valves. FIG. 51. C. biradiata, SBMNH 345500; Playa Kobbe, Panamá,
Panamá; length, 16.8 mm. FIG. 52. C. colimensis, new species; SBMNH 345496, holotype; Las Ventanas,
Manzanillo, México; length, 13.7 mm. FIG. 53. C. ira; SBMNH 345501; Bahía Carazal, Colima, México; 75

m; length, 11.4 mm. FIG. 54. C. luteola; CAS 120696; Arch Rock, Corona del Mar, Orange County, Califor-
nia; length, 10.4 mm.

EASTERN PACIFIC CORBULIDAE 95

TI

an

II

FIGS. 55, 56. Diagramatic interior views of eastern Pacific species of Corbula, showing hinge, pallial sinus,
and adductor scars of right and left valves. FIG. 55. C. marmorata; CAS 120688; Mazatlan, Sinaloa, México;
length, 5.8 mm. FIG. 56. C. speciosa; CAS 120695; Bahia Santa Inez, Baja California Sur, Mexico; 55-64

m; length, 17.5 mm

Washington, DC, USA; Yolanda Camacho, In-
stituto Nacional de Biodiversidad, Santo
Domingo, Heredia, Costa Rica; James
Cordeiro and Paula Mikkelsen, American Mu-
seum of Natural History, New York, New York,
USA; Thomas A. Deméré and N. S. Rugh,
San Diego Natural History Museum, San
Diego, California, USA; Charlene Fricker,
Mark Kitson, and Gary Rosenberg, of the
Academy of Natural Sciences, Philadelphia,
Pennsylvania, USA; Lindsey T. Groves and
James H. McLean, Natural History Museum
of Los Angeles County, Los Angeles, Califor-
nia, USA; Elizabeth Kools, Department of In-
vertebrate Zoology, California Academy of
Sciences, Golden Gate Park, San Francisco,
California, USA; Rafael La Perna, Universita
degle Studi di Bari, Bari, Italy; José Leal, Bai-
ley-Matthews Shell Museum, Sanibel,
Florida, USA; David R. Lindberg and Karen
Wetmore, Museum of Paleontology, Univer-
sity of California, Berkeley, California, USA;
Joan Pickering and Kathie Way, The Natural
History Museum, London, England, UK; Patri-
cia Sadeghian and Paul Valentich Scott,

Santa Barbara Museum of Natural History,
Santa Barbara, California, USA; Nancy Voss,
University of Miami, Miami, Florida, USA.
Carol C. Skoglund and Kirstie L. Kaiser gen-
erously made available material or informa-
tion from their collections. David Campbell,
Roberto Cipriani, Juan Diaz, Carole M. Hertz,
Alan R. Kabat, and Konstantin Lutaenko pro-
vided some information, and Richard Petit
supplied copies of scarce literature. Laurie C.
Anderson, Lindsey T. Groves, Carol C.
Skoglund, and Paul Valentich Scott made
many helpful comments on the manuscript.
Sharon Williams helped to prepare the plates.

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98 COAN

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EASTERN PACIFIC CORBULIDAE 105

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MALACOLOGIA, 2002, 44(1): 107-134

GENUS PARVITHRACIA (BIVALVIA: THRACIIDAE) WITH DESCRIPTIONS
OF ANEW SUBGENUS AND TWO NEW SPECIES FROM
THE NORTHWESTERN PACIFIC

Gennady M. Kamenev

Institute of Marine Biology, Russian Academy of Sciences, Vladivostok 690041, Russia;
inmarbio @ mail.primorye.ru

ABSTRACT

Anew subgenus, Pseudoasthenothaerus, of the genus Parvithracia Finlay, 1927, and two new
species, Parvithracia (Pseudoasthenothaerus) lukini and P. (Pseudoasthenothaerus) sirenkoi,
are described from the Pacific seas of Russia. Previously representatives of the genus Parvithra-
cia, With the type species Parvithracia suteri Finlay, 1927, were found only off New Zealand.
Study of thraciid genera showed that Japanese species assigned to the genus Asthenothaerus
Carpenter, 1864, should be placed in the genus Parvithracia. For Asthenothaerus sematana
(Yokoyama, 1922) and A. isaotakii Okutani, 1964, new combinations are suggested: Parvithra-
cia (Pseudoasthenothaerus) sematana (Yokoyama, 1922) and Р (Pseudoasthenothaerus) isao-
takii (Okutani, 1964). The main morphological characteristics of the new subgenus are the pres-
ence of conspicuous lateral teeth in both valves and a subumbonal plate for the attachment of
the lithodesma that is supported by pillars. Expanded descriptions of P. suteri, Р (Pseudoas-

thenothaerus) sematana, and P. (Pseudoasthenothaerus) isaotakii are given.
Key words: Parvithracia, Thraciidae, Bivalvia, northwestern Pacific, morphology, anatomy.

INTRODUCTION

The bivalve family Thraciidae Stoliczka,
1870, in the Pacific seas of Russia has long
been represented by a sole genus Thracia
Blainville, 1824 (Scarlato, 1981; Kafanov,
1991). Recently, members of the genus Lam-
peia MacGinitie, 1959, previously recorded
from America (MacGinitie, 1959; Coan, 1990),
were found in this region (Kamenev & Nad-
tochy, 1998). Examination of thraciids col-
lected by expeditions in the shelf and bathyal
zones of the Pacific seas of Russia revealed
two new species. Based on the shell mor-
phology and anatomy, these species were as-
signed to a new subgenus of the genus
Parvithracia Finlay, 1927, previousiy known
only from the South Pacific (Finlay, 1927a, b;
Powell, 1937, 1955, 1979). Study of the rep-
resentatives of other genera of the Thraciidae
showed that Japanese species previously as-
signed to the genus Asthenothaerus Carpen-
ter, 1864, should also be placed in this sub-
genus.

Diagnoses and descriptions of the genus
Parvithracia and description of the type
species given in the literature are brief, some-
times inaccurate, and often illustrated only by
schematic figures of Parvithracia suteri Finlay,

107

1927 (Suter, 1913; Finlay, 1927a, b; Keen,
1969; Powell, 1955, 1979). Thorough study of
shell morphology of P. suteri and of north-
western Pacific species has enabled an ex-
panded and modified diagnosis of the genus
and an improved description of P. suteri to be
made. This paper provides new data on its
shell morphology, and describes a new sub-
genus of Parvithracia and four species, two of
which are new. For Asthenothaerus sematana
(Yokoyama, 1922) and A. isaotakii Okutani,
1964, new combinations are suggested.

MATERIALS AND METHODS

In this study, | have used the material col-
lected by numerous expeditions to the shelf
and bathyal zones of Pacific seas of Russia
from 1931 to 1995. Mollusks were fixed in
70% ethanol or 4% formaldehyde. The mate-
rial was stored in 70% ethanol and in the dry
form in ZIN, IMB, and PRIFO.

For comparison purposes, collections of the
following taxa were used: Asthenothaerus
diegensis (Dall, 1915) (USNM); A. hemphillii
Dall, 1866 (USNM); А. isaotakii Okutani, 1964
(NSMT); A. sematana (Yokoyama, 1922)
(NSMT, CAS); Eximiothracia concinna (Gould,

108 KAMENEV

1861) (NSMT); Lampeia adamsi (MacGinitie,
1959) (UAM; MIMB); L. triangula Kamenev &
Nadtochy, 1998 (MIMB); L. posteroresecta
Kamenev, 1998, in Kamenev & Nadtochy,
1998 (MIMB); Thracia curta Conrad, 1837
(USNM); T. myopsis Moller, 1842 (MIMB,
ZIN); T. kakumana (Yokoyama, 1927) (MIMB,
ZIN); T. seminuda Scarlato, 1981 (ZIN); T.
septentrionalis Jeffreys, 1872 (USNM; ZIN);
Thracidora japonica Habe, 1952 (NSMT);
Trigonothracia pusilla (Gould, 1861) (NSMT);
Parvithracia suteri Finlay, 1927 (MNZ and ma-
terial of R. Asher (CIN)). Asthenothaerus isao-
takiiand Lampeia species from Pacific seas of
Russia were fixed and stored in 70% ethanol.
All other material was stored dry.

The gross anatomy of the new species has
been described from whole mount speci-
mens.

Shell Measurements

Figure 1 shows the position of the shell
morphology measurements. Shell length (L),
height (H), width of each valve (W) (not
shown), anterior end length (A), maximal dis-
tance from the posterior end to the top of pal-
lial sinus (S), and lithodesma length (L1) were
measured for each valve. The ratios of these
parameters to shell length (H/L, W/L, A/L, S/L,
L1/L, respectively) were determined. The

L

FIG. 1. Placement of shell measurements: L — shell
length; H —height; А — anterior end length; L1 —lith-
odesma length; S—maximal distance from poste-
rior shell margin to the top of pallial sinus.

number of pillars (N) supporting the subum-
bonal plate for lithodesma in each valve was
also recorded. Shell measurements were
made using a caliper and an ocular microme-
ter with an accuracy of 0.1 mm.

Statistics

Statistical analysis of the material used a
package of statistical programs STATISTICA
(Borovikov & Borovikov, 1997) and Data
Analysis Module of MS Excel 97.

The calculated indices (H/L; W/L; A/L; S/L
and L1/L) are less susceptible to change as
compared with other measured parameters.
Therefore the statistical analysis was per-
formed using only these characteristics. All
data was tested with a Kolmogorov test for
their fit to a normal distribution. The distribu-
tion of some indices was different from the
norm. Therefore all analyses were performed
on log,, transformations of the original vari-
ables. All indices for pairs of different valves of
new species were compared using the Stu-
dent (t) parametric test and one-way analysis
of variance (ANOVA). A discriminant analysis
was used to test the validity of the hypothesis
of division of all studied specimens into two
groups.

Throughout this study, statistical signifi-
cance was defined as P < 0.05.

Morphological Terminology

Subumbonal plate is a plate on the inner
shell wall to which the internal ligament and
lithodesma are attached. It is located ventral
to the umbo and is partially or completely at-
tached to the shell wall. Various specialists
designate it either as a chondrophore (Ya-
mamoto & Habe, 1959, for the genus Trigo-
nothracia Yamamoto & Habe, 1959; Keen,
1969, for the genus Lampeia; Xu, 1980, for
the genus Trigonothracia), buttressed struc-
ture (Coan, 1990; Kamenev & Nadtochy,
1998, for the genus Lampeia), or resilifer
(Coan et al., 2000, for the genus Lampeia)).

Resilifer is a depression or projection of the
hinge plate not attached to the inner shell wall
that serves for the attachment of the internal
ligament.

Abbreviations

The following abbreviations are used in the
paper: CAS--California Academy of Sci-

THE BIVALVE GENUS PARVITHRACIA 109

ences, San Francisco; CIN —Cawthron Insti-
tute, Nelson; IMB — Institute of Marine Biol-
ogy, Russian Academy of Sciences, Vladi-
vostok; MIMB—Museum of the Institute of
Marine Biology, Vladivostok; MNZ — Museum
of New Zealand, Wellington; NIGNS — Ма-
tional Institute of Geological and Nuclear Sci-
ences, Lower Hutt; NSMT — National Science
Museum, Tokyo; PRIFO—Pacific Research
Institute of Fisheries and Oceanography,
Vladivostok; UAM — University of Alaska Mu-
seum, Fairbanks; USNM--United States
National Museum of Natural History, Smith-
sonian Institute, Washington, D.C.; ZIN—
Zoological Institute, Russian Academy of Sci-
ences, St.-Petersburg.

SYSTEMATICS

Order Pholadomyoida Newell, 1965
Superfamily Thracioidea Stoliczka, 1870
Family Thraciidae Stoliczka, 1870
Genus Parvithracia Finlay, 1927

Type species: Montacuta triquetra Suter,
1913: 915, non Verrill & Bush, 1898: 782, 783,
pl. 91, fig. 3; = Parvithracia suteri Finlay,
1927b: 529

Diagnosis

Shell small (< 11 mm), very thin and fragile
to medium in thickness, ovate-elongate to
subtrigonal, inequivalve; right valve larger and
more inflated. Beaks central or posterior. Pos-
terior end truncate, with a faint radial ridge.
Periostracum thin, adherent, colorless or tan.
Surface with conspicuous growth lines and
very fine granules. Escutcheon present;
lunule present in some. Ligament internal,
supported by a strong lithodesma attached to
elongate-trigonal subumbonal plate that is
sometimes supported by a series of pillars.
Hinge plate weak, with large, short anterior
and more or less conspicuous, elongate pos-
terior lateral teeth in right valve, and long,
lamellate, anterior lateral tooth in left valve.
Pallial line with deep pallial sinus of same
shape and size in both valves, sometimes
reaching midline. Pallial sinus not confluent
with pallial line.

Remarks

Previously this genus included two species:
Р suteri and Parvithracia cuneata Powell,

1937 (Powell, 1937, 1955, 1979). However,
studies of Bruce A. Marshall (MNZ) have
shown that P. cuneata belongs to a different
genus (Bruce A. Marshall, personal communi-
cation).

Subgenus Parvithracia, s.s.
Diagnosis

Shell small (< 5 mm), thin, fragile, subtrigo-
nal, inequivalve; right valve more inflated.
Beaks slightly posterior. Posterior end trun-
cate, with very faint radial ridge. Periostracum
thin, adherent, colorless. Surface with faint
growth lines and very fine granules. Es-
cutcheon and lunule present. Ligament inter-
nal, supported by a strong lithodesma, at-
tached to elongate-trigonal subumbonal
plate. Hinge plate weak, with large, high ante-
rior and more or less conspicuous, elongate,
posterior lateral teeth in right valve and long,
lamellate, anterior lateral tooth in left valve; in-
ternal part of anterodorsal and posterodorsal
margins with long grooves. Pallial line with
deep pallial sinus of same shape and size in
both valves, reaching midline. Pallial sinus not
confluent with pallial line.

Parvithracia (Parvithracia) suteri Finlay, 1927
Figs. 2-10, Table 1

Montacuta triquetra Suter, 1913: 915, pl.
53, fig. 7a, non Verrill & Bush, 1898: 782, 783,
pl. 91, fig. 3; Finlay, 1927a: 461.

Parvithracia suteri Finlay, 1927b: 529; Pow-
ell, 1955: 45; Powell, 1979: 434, fig. 117.

Type Material and Locality

Lectotype (NIGNS TM 399) and 4 paralec-
totypes (NIGNS TM 405-409) (Boreham,
1959; Bruce A. Marshall, personal communi-
cation). Port Pegasus, Stewart Island, New
Zealand, 18 fathoms (approximately 33 m)
(Suter, 1913; Powell, 1955, 1979).

Material Examined

1 lot (MNZ ex M.44790) from North Arm,
Port Pegasus, Stewart Island, New Zealand
(47° 11'S, 167° 41'E), 37-44 m, mud, 22 Feb-
ruary 1972 (R/V “Acheron”) (3 spec.); 1 lot
(CIN) from Tasman Bay, Nelson, New
Zealand (40°35'S, 173°54’E), 60 m,
mud/sand, Coll. Rod Asher, January 1997 (1
damaged spec.).

110 KAMENEV

FIGS. 2-10. Parvithracia (Parvithracia) suteri Finlay, 1927 (MNZ ex M.44790), North Arm, Port Pegasus,
Stewart Island, New Zealand (47°11'S, 167°41’E), 37-44 m. 2, 3: Left and right valves (bar = 1 mm). 4: Gran-
ules on the shell surface (bar = 100 um). 5: Hinge of the right valve (bar = 300 um). 6: Hinge of the left valve
(bar = 300 um). 7: Dorsal view of teeth on the right valve (bar = 100 um). 8: Close-up of a left valve showing
the subumbonal plate and the lateral tooth on the anterodorsal shell margin, ventral view (bar = 300 um). 9:
Close-up of a right valve showing inner part of antero- and posterodorsal shell margins with a shallow groove
(bar = 1 mm). 10: Ventral view of lithodesma (bar = 100 um).

Description (right valve more inflated), inflated (W/L of
right valve 0.281-0.303; W/L of left valve

Expanded and modified from Suter (1913) — 0.235-0.250), slightly inequilateral, thin,
Exterior: Shell small (< 5.0 mm), subtrigonal, solid. Surface with faint growth lines and very

high (H/L = 0.824-0.892), slightly inequivalve dense fine granules. Periostracum dull, thin,

THE BIVALVE GENUS PARVITHRACIA 111

adherent, colorless. Beaks small, high, pro-
jecting considerably above dorsal margin,
slightly posterior to midline (A/L = 0.531-
0.588), somewhat sharp, orthogyrate. Ante-
rior end narrow, rounded. Posterior end de-
cidedly truncate, with a faint radial ridge ex-
tending beaks to junction of posterior end with
ventral margin. Anterodorsal margin straight
or slightly convex, very steeply descending
ventrally, smoothly transitioning to rounded
anterior end. Ventral margin slightly curved
(more curved in right valve). Posterodorsal
margin straight or slightly convex, very
steeply descending ventrally, forming a dis-
tinct angle at transition to posterior end. Pos-
terior end slightly curved, anteriorly directed,
forming a rounded angle at transition to ven-
tral margin. Lunule present only in left valve,
narrow, well expressed along entire an-
terodorsal margin, demarcated by a ridge. Es-
cutcheon narrow, more expressed in left
valve, demarcated by ridges extending along
posterodorsal margin from beaks to posterior
end.

Interior: Right valve with anterior and pos-
terior lateral teeth and long grooves on inner
part of anterodorsal and posterodorsal mar-
gins; left valve with anterior lateral tooth. In
right valve, anterior lateral tooth very large,
high, strongly projecting above inner part of
anterodorsal margin, ventrally directed; pos-
terior lateral tooth small, elongate, somewhat
projecting or not projecting above inner part of
posterodorsal margin, extending along pos-
terodorsal margin; inner part of antero- and
posterodorsal margins thickened, with a long,
shallow groove more expressed in inner part
of posterodorsal margin, running parallel to
anterior and posterior dorsal slopes for almost
their entire length. In left valve, anterior lateral
tooth long, lamellate, noticeably projecting
above inner part of anterodorsal margin; inner
part of anterodorsal margin slightly concave
between beak and tooth; inner part of pos-
terodorsal margin straight. Subumbonal plate
short, slightly elevated above inner shell sur-
face, attached to shell wall, free along its
anteroventral margin. Pillars absent. Litho-
desma large (L1/L = 0.121-0.125), ovate-
trapezoidal. Anterior adductor muscle scar
large, elongate, kidney-shaped. Posterior ad-
ductor scar small, rounded. Pallial sinus dis-
tinct, of the same shape and size in both
valves, deep, broad, rounded anteriorly,
reaching midline (S/L = 0.455-0.5), anterior
limit short of faint vertical line from beaks.
Shell interior with faint radial striae.

Variability

Slight variations occur in shell shape, shell
height, valve width, and position of the beaks
(Table 1). There is also variation in the shape
and size of the teeth, lithodesma shape, and
pallial sinus length.

Distribution and Habitat

Parvithracia (P.) suteri occurs near New
Zealand (Three Kings Islands; Cape Brett;
Hen and Chickens Islands; Mayor Island; Tas-
man Bay; Stewart Island, Port Pegasus;
Bounty Islands) (Suter, 1913; Powell, 1955,
1979), from 33 m (Stewart Island, Port Pega-
sus) to 260 m (Three Kings Islands), on mud
and sandy mud.

Pseudoasthenothaerus Kamenev,
new subgenus

Type species: Pseudoasthenothaerus lukini
Kamenev, new species

Description

Shell small (< 11 mm), ovate-elongate to
ovate-trigonal, inequivalve; right valve larger,
more inflated. Beaks central or posterior. Pos-
terior end truncate, with faint radial ridge. Pe-
riostracum thin, adherent, colorless or tan.
Surface with conspicuous growth lines and
very fine granules. Lunule absent. Es-
cutcheon narrow, well expressed. Ligament
internal, supported by a strong lithodesma, at-
tached to elongate-trigonal subumbonal plate
that extends obliquely posterior from beaks,
free along its anteroventral margin, where it is
supported by a series of pillars separated by
shallow or deep pits. Hinge plate weak, with
large, short anterior and more or less con-
spicuous, elongate posterior lateral teeth in
right valve and long, lamellate anterior lateral
tooth in left valve. Pallial line with deep pallial
sinus, of same shape and size in both valves,
not reaching midline. Mantle lobes fused, with
small pedal and three pallial apertures.
Siphons long, separate. Ctenidia consisting of
two demibranchs; demibranchs subequal to
inner demibranch much larger than outer.
Labial palps broadly triangular. Stomach very
large, to right of visceral mass; much of stom-
ach to right of visceral mass not surrounded
by digestive diverticula. Style sac joined to
mid gut. Intestine passing through heart. Si-

112 KAMENEV

TABLE 1. Parvithracia (Parvithracia) suteri Finlay, 1927. Shell measurements (mm), indices and summary
statistics of all characteristics: L— shell length; H —height; W — width; A— anterior end length; $ — maximal
distance from the posterior shell margin to the top of pallial sinus; L1 —lithodesma length. Numerator indi-
cates shell measurements and indices for the right valve, denominator — for the left valve. ММ — not mea-

sured.

Statistics L H W A S 11

W/L AL SE EVE
NM NM NM NM

Depository
MNZ ex M. 44790

0.303 0.576 0.485 NM MNZ ex M. 44790

0.303 0.576 0.485 0.121 MNZ ex M. 44790

0.281 0.531 0.500 0.125 MNZ ex M. 44790

NM NM NM NM NM NM NM
3:4: 2.8 10:8: 2 00017 0.824
3.3 2.8 1.0 1.9 1.6 ММ 0.848
NM NM NM NM м NM
3:3 2977 1:07 19 16 04. 0:879
3:3, 2.9 20.87 2192 1.5 0.879
Je 28 12 16 09 04 0875
ЗУ Е 5151018 0.844
Меап 3.27 2.83 0.97 1.83 1.60 0.4 0.867
3.30 2.80 0.80 1.87 1.57 0.849
$0 0.06 0.06 0.06 0.12 0 0 0.017
0.10 0.10 0 0:15; (0:12 0.028
ЗЕ 0.03 0.03 0.03 0.07 0 0 0.010
0.06 0.06 0 0.09 0.07 0.016
Min 3212.8 OA 1167 0:4 0848
3:20 02.7 (08 117 51.5 0.824
Мах 3322.95 1.07 1.9 1:6 0:4 0:879
3:4 2:95 10:8: 12:0 117 0.879
n 3 3 3 3 3 20:3
3 3 3 3 3 3

multaneous hermaphrodite. Testes occupying
ventral position in visceral mass above foot.
Paired ovaries occupying posterior position in
visceral mass.

Remarks

The major morphological characters of the
genera recognized within the Thraciidae by
various specialists are presented in Table 2.
The new subgenus has a combination of mor-
phological characteristics not seen in any ex-
isting thraciid genus. Parvithracia (Pseudoas-
thenothaerus) differs from all other genera in
having anterior and posterior lateral teeth in
the right valve and an anterior lateral tooth
in the left valve, and a subumbonal plate sup-
ported by pillars.

On the basis of external and internal shell
morphology, as well as hinge structure, the
new subgenus is most similar to Parvithracia.
This subgenus differs from Parvithracia, s.s.,
in having pillars supporting the subumbonal
plate, in lacking a groove on the inner part of
the dorsal shell margin, and in having a
lunule. In my opinion, these differences are
less substantial compared to the differences
among genera within this family (Table 2).

Therefore, Pseudoasthenothaerus is as-
signed as a subgenus of Parvithracia, with
which it is most similar in shell morphology.

On the basis of internal shell morphology,
hinge structure, and relative size of the
lithodesma, this subgenus is also similar to
Lampeia. Only Lampeia and Parvithracia
(Pseudoasthenothaerus) have a subumbonal
plate supported by pillars. However, unlike
Parvithracia (Pseudoasthenothaerus), Lam-
peia has only an anterior tooth in the right
valve. Moreover, Parvithracia (Pseudoas-
thenothaerus) differs from Lampeia in lacking
a thick brown periostracum, lunule, and exter-
nal ligament, and in having granules on the
shell surface.

In shell form, proportions and morphology,
Parvithracia (Pseudoasthenothaerus) resem-
bles Asthenothaerus. For this reason, Japa-
nese malacologists assigned representa-
tives of Parvithracia (Pseudoasthenothaerus)
found off the coast of Japan to Astheno-
thaerus. However, Asthenothaerus has no lat-
eral teeth, the subumbonal plate is tightly at-
tached to the shell wall, and the lithodesma is
less massive, more angular, with long, sharp
ends. Perhaps, Asthenothaerus huanghaien-
sis Xu, 1989, described from northern Huang-

113

THE BIVALVE GENUS PARVITHRACIA

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114 KAMENEV

hai Sea (China) (Xu, 1989), should also be
placed in Parvithracia (Pseudoastheno-
thaerus).

The subgenus name is derived from the
similarity of external shell morphology of
species of this subgenus to members of the
genus Asthenothaerus.

Parvithracia (Pseudoasthenothaerus) lukini
Kamenev, new species
Figs. 11-34, Table 3

Type Material and Locality

Holotype (MIMB 4047), Evreinova Strait
(Onekotan Island — Makanrushi Island), Kuril
Islands, 100 m, silty sand, Coll. V. I. Lukin and
S. |. Grebelny, 26-VIII-1987 (R/V “Tikhookean-
sky”); paratypes (15): paratype (MIMB 4048)
from the type locality; paratype (MIMB 4049),
Fourth Kuril Strait (Onekotan Island —Рага-
mushir Island), Кий Islands (49°38'5N,
155°22'3E), 500 m, sandy silt, Coll. V. I. Lukin
and S. |. Grebelny, 27-X-1987 (R/V “Tikhooke-
ansky”); paratype (ZIN 1), southern Sea of
Okhotsk (51°35’6N, 151°46E), 418 m, silty
sand, bottom water temperature of +1.53°C,
salinity 33.62%, Coll. Р. V. Ushakov, 10-VII-
1932 (R/V “Gagara”); paratype (ММВ 4050),
west coast of Kamchatka, Sea of Okhotsk
(52°20'N, 155°18’E), 142 m, sand + silt, Coll.
V. A. Nadtochy, 10-V-1989 (R/V “Mys Dalny”);
paratypes (4) (MIMB 4051), Black Cape,
Medny Island, Commander Islands, Bering
Sea (54°41.2'N, 167°59.7’E), 150-200 m, silt,
Coll. V. I. Lukin, 16-IX-1973 (R/V “Rakyt-
noye”); paratype (ZIN 2), Aniva Cape,
Sakhalin Island, Sea of Okhotsk (45°58'N,
143°46.3E), 207 m, sandy silt, bottom water
temperature of —0.4°C, Coll. Z. I. Kobjakova,
24-1Х-1947 (R/V “Toporok”); paratype (ZIN 3),
Svobodnogo Cape, Sakhalin Island, Sea of
Okhotsk (46°47.5'N, 143°52.3’E), 187 m,
sandy silt, bottom water temperature of
—0.4°C, Coll. Z. |. Kobjakova, 4-IX-1947 (R/V
“Toporok”); paratype (ММВ 4052), Morzovaja
Rock, Kunashir Island, Kuril Islands, Sea of
Okhotsk (44°33’8N, 146°28’7E), 101 m, silty
sand + gravel, Coll. V. I. Lukin and V. P.
Pavluchkov, 23-VII-1987 (R/V “Tikhookean-
sky”); paratypes (2) (MIMB 4053), Kunashir Is-
land, Kuril Islands, Pacific Ocean (44°08'N,
146°54’4E), 189 m, silty sand + gravel, Coll. М.
|. Lukin and V. P. Pavluchkov, 19-VII-1987 (R/V
“Tikhookeansky”); paratypes (2) (ZIN 4), Peter
the Great Bay, Sea of Japan (42°32.2’N,
131°13.5’E), 65 m, Coll. Tarasov, 2-IX-1932.

Other Material Examined

One left valve (ZIN 5) from Svobodnogo
Cape, Sakhalin Island, Sea of Okhotsk
(46°47.5'N, 143°52.3’E), 187 т, sandy silt,
bottom water temperature of —0.4°C, Coll. Z.
|. Kobjakova, 4-IX-1947 (R/V “Toporok”); one
left valve (MIMB 4054) from east coast of
Sakhalin Island, Sea of Okhotsk, 280 m, silty
sand, Coll. V. N. Koblikov, 5-VIII-1977 (R/V “8-
452”); one left valve (ZIN 6) from Tatar Strait,
Sakhalin Island, Sea of Japan (49°00.4’N,
141°44.2'E), 99 m, silty sand + gravel + peb-
ble, bottom water temperature of +2.3°C, Coll.
Skalkin, 28-VIII-1949 (R/V “Toporok”); one
right valve (ZIN 7) from Pesherny Cape, Sea
of Japan, 230-238 m, silt + sand, Coll.
Ohromkin, 17-VII-1931 (R/V “Rossinante”);
one specimen (ZIN 8) from Peter the Great
Bay, Sea of Japan (42°32.2’N, 131°13.5’E),
65 m, Coll. Tarasov, 2-IX-1932; one left valve
(MIMB 4055) from Bolshoy Peles Island,
Peter the Great Bay, Sea of Japan (42°31'8N,
131°23'5E), 72 m, large-particle sand, Coll. V.
V. Gulbin, 19-IX-1995 (R/V “Akademik
Oparin”); one specimen and left valve (MIMB
4056) from Black Cape, Medny Island, Com-
mander Islands, Bering Sea (54°41.2’N,
167°59.7'E), 150-200 m, silt, Coll. V. 1. Lukin,
16-1Х-1973 (R/V “Rakytnoye”); one right valve
(MIMB 4057) from Yodny Cape, Iturup Island,
Kuril Islands, Pacific Ocean (44°35'5N,
147°27'5Е), 200 m, sandy silt, Coll. V. 1. Lukin
and S. |. Grebelny (R/V “Tikhookeansky”).
Total of 2 specimens, 5 left, and 2 right valves.

Description

Exterior: Shell small (< 11.0 mm), ovate-an-
gular, high (H/L = 0.785-0.936), slightly in-
equivalve (right valve slightly higher, more in-
flated), moderately inflated (W/L of right valve
0.185-0.273; W/L of left valve 0.185-0.267),
inequilateral, thin, solid. Surface with conspic-
uous growth lines and fine, very dense gran-
ules. Periostracum dull, thin, adherent, color-
less, yellowish, pinkish or light brown, with
dark brown or black patches or crusts near
beaks and on dorsal shell margin, extending
into inner surface. Beaks small, moderately
projecting above dorsal margin, posterior to
midline (A/L = 0.585-0.765), slightly rounded,
opisthogyrate. Anterior end rounded. Poste-
rior end narrow, truncate, with faint radial
ridge from beaks to ventral limit of posterior
end. Anterodorsal margin straight or slightly
convex, gently descending ventrally, smoothly

THE BIVALVE GENUS PARVITHRACIA its

FIGS. 11-24. Parvithracia (Pseudoasthenothaerus) lukini Kamenev, new species. 11-15: Holotype (MIMB
4047), Evreinova Strait (Onekotan Island — Makanrushi Island), Kuril Islands, 100 m, shell length 9.9 mm. 16:
Lithodesma of holotype, ventral view (bar = 1 mm). 17-22: Paratypes (MIMB 4051), Black Cape, Medny Is-
land, Commander Islands, Bering Sea (54°41.2’N, 167°59.7'E), 150-200 m. 17, 18: Left and right valves of
a young specimen (bar = 1 mm). 19: Dorsal view of both valves of a young specimen (bar = 1 mm). 20, 21:
Ventral view of right and left valves showing subumbonal plate and teeth (bar = 1 mm). 22: Ventral view of
lithodesma (bar = 1 mm). 23: Paratype (MIMB 4050), west coast of Kamchatka, Sea of Okhotsk (52°20'N,
155°18’E), 142 m, ventral view of lithodesma (bar = 1 mm). 24: Paratype (MIMB 4053), Kunashir Island, Pa-
cific Ocean (44°08’N, 146°54’4E), 189 m, close-up of right valve showing attachment of lithodesma (ventral
view) to subumbonal plate and the tooth (bar = 1 mm).

116 KAMENEV

FIGS. 25-32. Parvithracia (Pseudoasthenothaerus) lukini Kamenev, new species. FIGS. 25-28, 30.
Paratypes (MIMB 4051), Black Cape, Medny Island, Commander Islands, Bering Sea (54°41.2’N,
167°59.7'E), 150-200 m. 25, 26: Dorsal view of teeth on right and left valves (bar = 1 mm). 27, 28: Hinge of
right and left valves (bar = 1 mm). 29: Paratype (MIMB 4050), west coast of Kamchatka, Sea of Okhotsk
(52°20'N, 155°18'E), 142 m, subumbonal plate with pillars (ventral view), right valve (bar = 1 mm). 30: Sub-
umbonal plate with pillars (ventral view), left valve (bar = 1 mm). 31, 32: Right valve with of specimen with
reversed hinge (ventral view), Black Cape, Medny Island, Commander Islands, Bering Sea (54°41.2’N,
167°59.7'E), 150-200 m (bar = 1 mm).

THE BIVALVE GENUS PARVITHRACIA 117

FIG. 33. Parvithracia (Pseudoasthenothaerus) lukini. Organs of mantle cavity as seen from right side with
right shell valve and mantle removed (shell length, 6.4 mm). AA, anterior adductor muscle; APR, anterior
pedal retractor muscle; DD, digestive diverticula; E, exhalant siphon; EM, edge of mantle; F, foot; HG, hind
gut; I, inhalant siphon; ID, inner demibranch; К, kidney; LP, labial palp; MC, mineralized concretions; OD,
outer demibranch; PA, posterior adductor muscle; PPR, posterior pedal retractor muscle; SM, siphonal mus-
culature.

transitioning to rounded anterior end. Ventral
margin slightly curved (more curved in right
valve), sometimes straight in left valve. Pos-
terodorsal margin slightly concave, rather
steeply descending to form a smooth angle at
posterior end. Posterior end slightly curved,
almost vertical, only slightly turned anteriorly
to form a rounded angle at ventral shell mar-
gin. Escutcheon narrow, well defined, bor-
dered by ridges along entire posterodorsal
margin.

Interior: Right valve with anterior and pos-
terior lateral teeth; left valve with anterior lat-
eral tooth. In right valve, anterior lateral tooth
large, triangular, strongly projecting above
inner part of anterodorsal margin, anteroven-
trally directed; posterior lateral tooth small,
elongate, only slightly projecting or not pro-
jecting above inner part of posterodorsal mar-
gin. In left valve, anterior lateral tooth long,
lamellate, slightly projecting above inner part
of anterodorsal margin; inner part of an-

terodorsal margin straight between beak and
tooth; inner part of posterodorsal margin
straight. Subumbonal plate short, elevated
above inner shell surface. Supporting pillars
high, of varying thickness, generally of the
same thickness throughout their individual
lengths or becoming thinner ventrally; width
and height of pillars, and width of pits between
pillars decreasing posteriorly from anterodor-
sal margin. Number of pillars from 2 to 7,
usually equal in both valves. Lithodesma
large (L1/L = 0.098-0.225), butterfly-shaped,
sometimes trapezoidal, often asymmetrical.
Anterior adductor muscle scar large, elon-
gate, kidney-shaped. Posterior adductor scar
small, rounded. Pallial sinus distinct, of the
same shape and size in both valves, deep,
rounded anteriorly, sometimes reaching mid-
line (S/L = 0.395-0.54), anterior limit short of
faint vertical line from beaks. Shell interior
with faint radial striae.

Anatomy: Gross morphology differs little

118 KAMENEV

MG

FIG. 34. Parvithracia (Pseudoasthenothaerus) lukini. Internal morphology as seen from right side with right
shell valve, mantle, and ctenidium removed (shell length, 6.4 mm). A, anus; AA, anterior adductor muscle;
APR, anterior pedal retractor muscle; DD, digestive diverticula; F, foot; H, heart; HG, hind gut; K, kidney; LP,
labial palp; MC, mineralized concretions; MG, mid-gut; O, ovary; OE, oesophagus; PA, posterior adductor
muscle; PPR, posterior pedal retractor muscle; S, stomach; SS, style sac; T, testis; VG, visceral ganglia.

from previous descriptions of Thracia (Kiener,
1834; Deshayes, 1846). Mantle very thin, ex-
cept at ventral margin, which is slightly thick-
ened, completely fused except at extensive
siphonal and pedal gapes, and small, round
aperture ventral to inhalant siphon. Siphons
long; inhalant siphon longer, larger. Anterior
adductor muscle large, elongate dorsoven-
trally, slightly curved parallel to anterior end.
Posterior adductor muscle smaller, almost
round. Foot small, laterally compressed, pro-
jecting anteroventrally, its shape in preserved
specimens variable depending on degree of
contraction. Anterior pedal retractor muscles
ascending almost vertically from base of foot,
passing dorsally above labial palps, attaching
dorsal to anterior adductor muscle. Posterior
pair of narrow pedal retractor muscles pass-
ing on either side of anus as single muscle
sheets from base of foot, attaching dorsal to

posterior adductor muscle. Ctenidia thin, con-
sisting of two demibranchs; inner demibranch
much larger than outer. Paired labial palps
long, elongate-triangular, of equal length.
Mouth small, situated close to anterior adduc-
tor muscle, at basal junction of inner and outer
palps. Oesophagus long, straight, extending
parallel to anterodorsal side of body, opening
into anterodorsal part of stomach. Stomach
very large, oval, lying along dorsoventral axis,
on right side of visceral mass; stomach wall
very close to dorsal and right sides of visceral
mass; much of stomach on right side not sur-
rounded by digestive diverticula (stomach
contents may be visible through the translu-
cent walls), with a combined style sac and mid
gut. Combined style sac and mid gut leaving
stomach at posteroventral border, passing
ventrally into visceral mass. Mid-gut separate
from style sac, forming large number of short

THE BIVALVE GENUS PARVITHRACIA 119

loops extending anteriorly ventral to stomach
almost as far as mouth, turning back along
dorsal margin of-foot to style sac. Hind gut
thickened, turning dorsally between paired
transparent ovaries, passing through heart
and posteriorly along posterodorsal margin of
body above kidney, partially circling posterior
adductor muscle, terminating near exhalant
siphon as an anal papilla free of attachments.
Digestive diverticula surrounding oesophagus
and greater part of stomach. Kidneys large,
posterodorsal between posterior adductor
muscle and ovaries, containing large number
of mineralized concretions. Cerebral ganglia
consisting of two small commissural bodies
between mouth and anterior adductor muscle.
Pedal ganglia large, Iying at interface of foot
and viscera. Visceral ganglia large, conspicu-
ous, lying ventral to kidney, in contact with in-
testine near anus. This species is a simulta-
neous hermaphrodite. Testes occupying a
ventral position in visceral mass dorsal to foot.
Paired ovaries occupying a posterior position
in visceral mass, appearing as transparent
sacs with large eggs (up to 150-200 um).

Variability

Shell shape and proportions vary markedly
(Table 3). The shell shape varies from ovate-
angular to rounded. Shell height and width,
the position of the beaks, the degree of con-
cavity of posterodorsal margin, and the rela-
tive length of pallial sinus vary. The shape,
length, and width of lithodesma is also
markedly variable, but it is most often butter-
fly-shaped. In young specimens, it is some-
times trapezoidal. The number of pillars
varies and, as a rule, increases with shell
size; some of them bifurcate ventrally or are
fused. The sizes and shapes of the lateral
teeth in both valves, and the inclination of an-
terior lateral tooth in right valve vary little. One
specimen has the hinge reversed (Figs. 31,
32); its left valve has two lateral teeth, the
shape and size of which are identical to the
teeth of right valve of other specimens exam-
ined. The right valve possesses an anterior
lamellate tooth analogous to the tooth of left
valve of normal specimens.

Distribution and Habitat (Fig. 35)

In the Bering Sea, Р (Pseudoastheno-
thaerus) lukini occurs near Medny Island,
Commander Islands; in the Sea of Okhotsk —

near the west coast of Kamchatka, near
Sakhalin Island, and in the southern part of
the sea (51°35’6N, 151°46E); near the Kuril
Islands —in Evreinova and Fourth Kuril
Straits, near Iturup and the Kunashir Islands;
in the Sea of Japan—in Tatar Strait (near
Sakhalin Island), near Pesherny Cape, and in
Peter the Great Bay.

In the Bering Sea, this species was ob-
tained at depth 150-200 m on silt; in the Sea
of Okhotsk — from 142 m (west coast of Kam-
chatka) to 418 m (southern Sea of Okhotsk)
on silty sand and sandy silt at a bottom tem-
perature from —0.4°C (Svobodnogo and Aniva
Capes, Sakhalin Island, depth 187 and 207
m, respectively) to 1.53°C (southern Sea of
Okhotsk, depth 418 m); near the Kuril Is-
lands —from 100 m (Evreinova Strait) to 500
m (Fourth Kuril Strait) on silty sand and sandy
silt sometimes with some admixture of gravel;
in the Sea of Japan—from 65 m (Peter the
Great Bay) to 238 m (Pesherny Cape) on
large-particle sand, silty sand and silt some-
times with admixture of gravel and pebbles at
a bottom temperature 2.3°C (Tatar Strait,
Sakhalin Island, depth 99 m).

Comparisons

Parvithracia (Pseudoasthenothaerus) lukini
is easily distinguished from Parvithracia
(Pseudoasthenothaerus) sematana and P
(Pseudoasthenothaerus) isaotakii in having a
thicker, heavier shell (Table 4). Moreover, in
contrast to P. (Pseudoasthenothaerus) se-
matana, this species has a higher, less elon-
gate shell, with a highly elevated subumbonal
plate and distinct, high pillars. Unlike Р
(Pseudoasthenothaerus) isaotakii, it has a
more oval shell, with a considerably less ex-
panded posterior end, and it has opisthogy-
rous beaks.

This species is most similar to Parvithracia
(Pseudoasthenothaerus) sirenkoi; but differs
in having a more elongate, oval, lower, and
less inflated shell. The shell of Parvithracia
(Pseudoasthenothaerus) lukini is more ex-
panded anteriorly, the beaks are more poste-
rior, the lateral teeth in both valves are
smaller, and there is a deeper pallial sinus.

Etymology

The specific name honors Dr. Vladimir 1.
Lukin, a famous Russian researcher of the
marine fauna of the Kuril Islands, who has col-

120 KAMENEV

TABLE 3. Parvithracia (Pseudoasthenothaerus) lukini Kamenev, new species. Shell measurements (mm),
indices and summary statistics of characters: L— shell length; H— height; W — width; А — anterior end length;
$ — maximal distance from the posterior shell margin to the top of pallial sinus; L1 —lithodesma length; N —
number of pillars under subumbonal plate. Numerator indicates shell measurements and indices for the right
valve, denominator — for the left valve. NM — not measured.

Statistics L H W A SA A W/L A/L S/L L1/L Depository

Holotype
99 80 25 67 46 1.7 6 0808 0.253 0.677 0.465 0.172 MIMB
98 78 22 65 45 6 0.796 0.224 0.663 0.459 0.173 4047

Paratype
97 85 26 70 43 20 4 0.876 0.268 0.722 0.443 0.206 MIMB
97 82 24 70 4.0 4 0.845 0.247 0.722 0.412 0.206 4049

Paratype
81 6.5 1.5 5.7 35 14 Z 0802 0.85 0,704 0432 "0173 ММВ
8.1, 6.4 1:5 5:7 3.4 7 0.790 0.185 0.704 0.420 0.173 4050

Paratype
7.9 6.3 18 54 34 13 5 0.797 0.228 0.84 0.430 0.165 MIMB
79 62 18 55 32 5 0.785 0.228 0.696 0.405 0.165 4053

Paratype
67 58 18 41 28 NM 4 0.866 0.269 0.612 0.418 NM ZIN 2
67157 17 212.8 4 0.851 0.254 0.612 0.418 NM

Paratype
6.5 5.6 13 42 30 10 5 0.862 0.200 0.646 0.462 0.154 ZIN 1
6.5 5.6 1.3 44 3.1 4 0.862 0.200 0.677 0.477 0.154

Paratype
6.4 55 17 40 28 12 3 0.859 0.266 0.625 0.438 0.188 ZIN 3
6.4 55 1.7 40 2.8 3 0.859 0.266 0.625 0.438 0.188

Paratype
6.4 53 15 42 32 12 5 0.828 0.234 0.656 0.500 0.188 ZIN 4
6.3 52 15 43 3.4 5 0.825 0.238 0.683 0.540 0.190

Paratype
6.3 56 15 4.1 28 1.1 4 0.889 0.238 0.651 0.444 0.175 MIMB
6.35.5 15 41 2:8 5 0.873 0.238 0.651 0.444 0.175 4051

Paratype
6.3 54 14 40 29 10 4 0857 0.222 0635 0.460 0.159 MIMB
6.3 53 15 40 29 4 0.841 0.238 0.635 0.460 0.159 4048

Paratype
58 53 13 37 27 09 4 0914 0.224 0.638 0.466 0.155 MIMB
5:88 15.3 ЗАЗ 2. 4 0.914 0.224 0.638 0.466 0.155 4051

Paratype
58 48 12 43 27 10 4 0828 0.207 0.741 0.466 0.172 MIMB
5.8 4.7 1.2 43 27 4 0.810 0.207 0.741 0.466 0.172 4053

Paratype
5.3 48 11 31 24 ММ 2 0.906 0.208 0.585 0.453 NM MIMB
5.34.7 1. 3.1 2.4 3 0.887 0.189 0.585 0.453 NM 4051

Paratype
47 44 10 28 22 08 2 0936 0.213 0.596 0.468 0.170 MIMB
4.7 4A 1.1 29 22 3 0.936 0.234 0.617 0.468 0.170 4051

Paratype
41 34 08 28 1.7 04 3 0.829 0.195 0.683 0.415 0.098 MIMB
410093408228 silt 7 0.829 0.195 0.683 0.415 0.098 4052

Paratype
32, 2.9 07 2.0 16 0.5 2 0906 10:219/ 0.625 0.500’ 10.156 ZIN 4
3:27 02.8: 2077052107 ALG 2 0.875 0.219 0.625 0.500 0.156
11.0 96 3.0 7.0 45 22 7 0.873 0.273 0.636 0.409 0.200 ММВ
NM NM NM NM NM NM NM NM NM NM NM 4057
NM NM NM NM NM NM NM NM NM NM NM NM ZIN5
9.0 7.8 24 54 3.8 6 0.867 0.267 0.600 0.422 NM
NM NM NM NM NM 2.0 NM NM NM NM NM NM MIMB
8:9. 77.8 2.0 26.41 4.0 6 0.876 0.225 0.685 0.449 0.225 4054
86 7.1 21 62 34 15 7 0826 0.244 70.721 (0/3895 0174 ZIN 8
8:5 7.0" (2/0). 6:5 8.7: 7 0.824 0.235 0.765 0.435 0.176

THE BIVALVE GENUS PARVITHRACIA 121

TABLE 3. (Continued)

Le A М А Е М H/L W/L AL S/L L1/L Depository
NM NM NM NM NM NM NM NM NM NM NM NM ZIN 6
MOTOS 1.9: 5:7 3.6 5 0.872 0.244 0.731 0.462 NM
NM NM NM NM NM NM NM NM NM NM NM NM MIMB
PARC TRI 43 3:2 5 0.859 0.239 0.606 0.451 NM 4056
6.0 50 13 4.0 29 ММ 4 0.833 0.217 0.667 0.483 NM ZIN 7
NM NM NM NM NM NM NM NM NM NM NM

Mean 6.77 5.77 1.57 4.49 3.02 1.25

6.87 5.82 1.58 4.59 3.07 0.851 0.228 0.664 0.450 0.171
SD 2.00 1.65 0.64 1.46 0.82 0.51 0.040 0.027 0.043 0.029 0.024
1.78 1.43 0.48 1.35 0.74 0.039 0.023 0.051 0.032 0.027
SE 0.46 0.38 0.15 0.34 0.19 0.12 — 0.009 0.006 0.010 0.007 0.006
0.39 0.31 0.10 0.30 0.16 0.008 0.005 0.011 0.007 0.007
Min 32 72:8) 0:4 20 1.6 0:40’ 2 0797 (08185 0585 0.395 0.098
3:22 52:0) 0 20 16 2 0.785 0.185 0.585 0.405 0.098
Мах ЕО OIG. 308 5720), 4.6; 2.20 их 01936 0:273 074417 0.500) 0.206
OBE 8:2 24 70 45 } 0936’ 0:267 0.765 10.5407 0'225
п 19. 19. 10.180 18 1 10 8 19 19 19 16
PA) PA ah il РЯ 2 21 21 21 16

140°

Russia

Bering Sea

Sea of Okhotsk
y 2
Commander Islands
Sakhalin Is.

Makanrushi Is o Paramushir Is.

A
че Onekotan Is.

9

0
0
0

Tatar Stfait o

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Iturup Is.

JA
Japan e Kunashir ls.

/ Peter the Great Bay Pacific Ocean 40°
Sea of Japan ie LE |

FIG. 35. Distribution of Parvithracia (Pseudoasthenothaerus) lukini (№ type locality).

KAMENEV

122

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THE BIVALVE GENUS PARVITHRACIA 123

lected many of the specimens examined in
this paper.

Parvithracia (Pseudoasthenothaerus)
sirenkoi Kamenev, new species
Figs. 36-54, Table 5

Type Material and Locality

Holotype (MIMB 4058), Iturup Island, Ku-
ril Islands, Pacific Ocean (44°52'N, 149°
27.7'E), 910-920 m, silty sand, Coll. B. I.
Sirenko, 25-\1-1984 (R/V “Odissey”);
paratypes (10): paratypes (2) (MIMB 4059)

from holotype locality; paratypes (2) (ZIN 1),
Кигир Island, Kuril Islands, Pacific Ocean
(44°46.8’N, 149°06.7'E), 880 m, silty sand,
Coll. B. |. Sirenko, 26-VII-1984 (R/V “Odis-
sey”); paratype (ZIN 2), Iturup Island, Kuril Is-
lands, Pacific Ocean (44°02.6'N, 148°11’E),
600 m, silty sand + gravel + pebbles, Coll. B.
|. Sirenko, 22-IX-1984 (R/V “Odissey”);
paratypes (4) (MIMB 4060), Kuril Islands,
Coll. V. I. Lukin, 1987 (R/V “Tikhookeansky”);
paratype (MIMB 4061), Yury Island, Kuril Is-
lands, Pacific Ocean (43°10’N, 146°17’E),
490 m, silty sand + gravel + pebbles, bottom
water temperature of +2.33°C, Coll. V. I. Lukin

FIGS. 36-48. Parvithracia (Pseudoasthenothaerus) sirenkoi Kamenev, new species. 36-40: Holotype
(ММВ 4058), Iturup Island, Kuril Islands, Pacific Ocean (44°52’N, 149°27.7'), 910-920 m, shell length 8.3
mm. 41-45: Paratype (MIMB 4060), Kuril Islands, shell length 8.0 mm. 46-48: Paratypes (MIMB 4059) from
type locality. 46, 47: Right and left valves of a young specimen (bar = 1 mm). 48: Dorsal view of both valves

of a young specimen (bar = 1 mm).

124 KAMENEV

FIGS. 49-54. Parvithracia (Pseudoasthenothaerus) sirenkoi Kamenev, new species. 49, 50: Paratype
(MIMB 4060), Kuril Islands, hinge of the left and right valves (bar = 1 mm). 51, 52: Paratypes (ZIN 2), Iturup
Island, Kuril Islands, Pacific Ocean (44°02.6'N, 148°11’E), 600 m. 51: Subumbonal plate with pillars (ventral
view), left valve (bar = 1 mm). 52: Close-up of right valve showing attachment of lithodesma (ventral view) to
subumbonal plate and teeth (bar = 1 mm). 53: Paratype (MIMB 4060), Kuril Islands, ventral view of lith-
odesma (bar = 1 mm). 54: Paratype (ZIN 1), Iturup Island, Kuril Islands, Pacific Ocean (44°46.8'N,
149°06.7'), 880 m, dorsal view of teeth on right valve (bar = 1 тт).

and V. P. Pavluchkov,
“Tikhookeansky”).

8-VII-1987 (R/V

Other Material Examined

Two right and one left valves (MIMB 4062)
from type locality; one specimen (ZIN 3) from
lturup Island, Kuril Islands, Pacific Ocean
(44°59'N, 148°47'E), 450 m, silty sand + peb-
bles, Coll. B. I. Sirenko, 24-IX-1984 (R/V
“Odissey”); one specimen (ZIN 4) from south-
western Sea of Okhotsk (45°06’N,
143°43.5'E), 188 m, silt, bottom water tem-
perature of +0.5°C, Coll. Yu. I. Galkin, 25-VIII-

1948 (R/V “Toporok”); one left valve (ММВ
4063) from Morzovaja Rock, Kunashir Island,
Kuril Islands, Sea of Okhotsk (44°33’8N,
146°28'7E), 101 m, silty sand + pebbles, Coll.
V. |. Lukin and V. P. Pavluchkov, 23-VII-1987
(R/V “Tikhookeansky”). Total of 2 specimens,
2 left, and 2 right valves.

Description

Exterior: Shell small (<8.9 mm), ovate-trig-
onal, high (H/L = 0.84-0.963), slightly in-
equivalve (right valve slightly higher, more
inflated), inflated (W/L of right valve 0.211-

THE BIVALVE GENUS PARVITHRACIA 125

0.313; W/L of left valve 0.237-0.293), inequi-
lateral, thin, solid. Surface with conspicuous
growth lines and fine, very dense granules.
Periostracum dull, thin, adherent, colorless,
pinkish or light brown, with dark brown or
black patches or crusts near beaks and dorsal
shell margin, extending into inner surface.
Beaks small, high, projecting considerably
above dorsal margin, posterior to midline (A/L
= 0.568-0.72), slightly rounded, opisthogy-
rate. Anterior end sharply rounded. Posterior
end higher than anterior, decidedly truncate,
with a faint radial ridge extending from beaks
to ventral limit of posterior end. Anterodorsal
margin straight or slightly convex, very
steeply descending, smoothly curving to
rounded anterior end. Ventral margin slightly
curved (more curved in right valve). Pos-
terodorsal margin straight or slightly concave,
descending and forming a distinct angle with
posterior end. Posterior end slightly curved,
descending ventrally or slightly anteriorly,
forming a rounded angle with ventral shell
margin. Escutcheon wide, well defined, de-
marcated by ridges extending along pos-
terodorsal shell margin from beaks to poste-
rior end.

Interior: Right valve with anterior and pos-
terior lateral teeth; left valve with anterior lat-
eral tooth. In right valve, anterior lateral tooth
large, high, triangular, projecting considerably
above inner part of anterodorsal margin, ven-
trally (Sometimes anteroventrally) directed;
right posterior lateral tooth small, elongate,
slightly projecting above inner part of pos-
terodorsal margin, extending along pos-
terodorsal margin. In left valve, anterior lateral
tooth long, lamellate, strongly projecting
above inner part of anterodorsal margin; inner
part of anterodorsal margin slightly concave
between beak and tooth; inner part of pos-
terodorsal margin slightly convex. Subum-
bonal plate short, elevated above inner shell
surface. Pillars supporting plate high, varying
in thickness between each other, generally of
same thickness throughout their individual
lengths or becoming thinner ventrally; width
and height of pillars, and width of pits between
pillars decreasing posteriorly from anterodor-
sal margin. Number of pillars from 2 to 6,
usually equal in number in both valves. Lith-
odesma large (L1/L = 0.067-0.225), butterfly-
shaped, sometimes ovate-trapezoidal, often
asymmetrical. Anterior adductor muscle scar
large, elongate, kidney-shaped. Posterior ad-
ductor scar small, rounded. Pallial sinus dis-
tinct, of the same shape and size in both

valves, deep, narrow and rounded anteriorly,
not reaching midline (S/L = 0.2-0.447), ante-
rior limit short of faint vertical line from beaks.
Shell interior with dense, tiny pockmarks and
faint radial striae.

Anatomy: Internal morphology similar to
that of Р (Pseudoasthenothaerus) lukini.
Mantle very thin, except at ventral margin,
which is slightly thickened, completely fused
except for the small pedal and three pallial
apertures. Siphons long; inhalant siphon
longer, larger. Anterior adductor muscle large,
elongate dorsoventrally, slightly curved paral-
lel to anterior shell margin. Posterior adductor
muscle smaller, almost round. Foot small, lat-
erally compressed, projecting anteroventrally.
Anterior pedal retractor muscles almost verti-
cally from base of foot, passing above labial
palps to attach dorsal to anterior adductor
muscle. Posterior pair of narrow pedal retrac-
tor muscles passing on either side of anus as
a single muscle sheets from base of foot to at-
tach dorsal to posterior adductor muscle.
Ctenidia thin, consisting of two subequal
demibranchs. Paired labial palps long, elon-
gate-triangular, of equal length. Mouth small,
close to anterior adductor muscle at basal
junction of inner and outer palps. Oesopha-
gus long, straight, extending parallel to an-
terodorsal side of body, opening to anterodor-
sal part of stomach. Stomach very large, oval,
lying along dorsoventral axis, in right side of
visceral mass; stomach wall located close to
dorsal and right sides of visceral mass, much
of stomach on right side not surrounded by di-
gestive diverticula (stomach contents some-
times visible through the translucent walls).
Combined style sac and mid-gut leaving
stomach at posteroventral border and passing
ventrally into visceral mass. Hind gut thick,
passing dorsally between paired, transparent
ovaries, through heart, posteriorly along pos-
terodorsal margin of body above kidney,
passing over posterior adductor muscle, ter-
minating near exhalant siphon. Anal papilla
free. Digestive diverticula surrounding oe-
sophagus and stomach. Kidney large, oc-
cupying a posterodorsal position between
posterior adductor muscle and ovaries. Vis-
ceral ganglia large, conspicuous, lying below
kidney, touching intestine near anus. This
species is a simultaneous hermaphrodite.
Testes occupying a ventral position in visceral
mass dorsal to foot. Paired ovaries occupying
a posterior position in visceral mass, appear-
ing as transparent sacs with large eggs (up to
150-200 um).

126 KAMENEV

Variability

Shell shape and proportions change little
with age. In young specimens (<6 mm), the
shell is less inflated (Table 5). In adults, shell
height, width, the position of beaks, and rela-
tive length of the pallial sinus vary slightly. The
sizes and shape of the lateral teeth in both
valves, and the inclination of the anterior tooth
in the right valve are fairly variable. The
shape, length, and width of lithodesma can
vary considerably, but it is generally butterfly-
shaped. The number of pillars supporting the
subumbonial plate varies and correlates with
shell size.

Distribution and Habitat (Fig. 55)

South Kuril Islands: Yury Island (43°10’N,
146°17'E), Kunashir Island (44°33’8N, 146°
28'7E), Iturup Island (44°59'N, 148°47'E;
44°52'N, 149°27.7'E; 44°46.8'N, 149°06.7'E;
44°02.6'N, 148°11’E); southwestern Sea of
Okhotsk (45°06'N, 143°43.5’E).

This species was recorded off the South
Kuril Islands at depth from 101 m (Kunashir
Island) to 910-920 m (Iturup Island) on silty
sand, sometimes with some pebbles and
gravel; in the southwestern Sea of Okhotsk, it
was present at a depth of 188 m, on silt, at a
bottom water temperature of +0.5°C.

Comparisons

This species is easily distinguished from
other species of this subgenus by its high,
ovate-triangular shell with a narrow anterior
end (Table 4). Moreover, P. (Pseudoastheno-
thaerus) sirenkoi differs from P. (Pseudoas-
thenothaerus) sematana and P. (Pseudoas-
thenothaerus) isaotakii by its thicker, more
solid shell with a larger lateral tooth. It is clos-
est to Р (Pseudoasthenothaerus) lukini, dif-
fering from it in having a higher, more inflated
shell, higher and less posteriorly placed
beaks, more steeply sloping anterodorsal and
posterodorsal shell margins, a smaller apical
angle, larger lateral teeth in both valves, the
inner part of the posterodorsal margin convex
in left valve, and a shallower pallial sinus.
Mean values and variances of the indices
characterizing the relative height (H/L) and
width (W/L) of the shell, as well as the position
of beaks (A/L), and the relative length of the
pallial sinus (S/L) were significantly different
in these species (Table 6).

Discriminant analysis of all lots containing
P. (Pseudoasthenothaerus) lukini and P.
(Pseudoasthenothaerus) sirenkoi showed
that these species differ significantly in a com-
plex of parameters (Wilks Lambda = 0.3331,
F-value = 15.3134, df = 3, 23, Squared Maha-
lanobis Distance = 8.1063, p < 0.0001; Means
of Cannonical variables: P. (Pseudoastheno-
thaerus) lukini = —1.21764; Р (Pseudoas-
thenothaerus) sirenkoi = 1.52205). Off 30
specimens analysed, 28 were accurately
classified (93.33%), with two specimens mis-
takenly classified as P. (Pseudoastheno-
thaerus) lukini. The most significant indices
for dividing all specimens into two species
were S/L for right valves, and W/L, and A/L for
left valves (Table 7).

Etymology

The specific name honors Dr. Boris |.
Sirenko, Zoological Institute - St. Petersburg,
who has collected almost all ofthe specimens
of this species examined for this paper.

Parvithracia (Pseudoasthenothaerus)
sematana (Yokoyama, 1922)
Figs. 56-60, 65, Table 8

Thracia sematana Yokoyama, 1922: 173,
pl. 14, figs. 17, 18.

Parvithracia sematana (Yokoyama, 1922),
Oyama, 1973: 120, pl. 57, figs. 13, 14.

Asthenothaerus sematensis (Yokoyama,
1922), Ito, 1989: 66, pl. 28, fig. 3.

Asthenothaerus sematanus (Yokoyama,
1922), Tsuchida & Kurozumi, 1996: 15, 16.

Asthenothaerus sematana (Yokoyama,
1922), Habe, 1977: 312; Higo et al., 1999:
524; Okutani, 2000: 1039, but not pl. 517, fig.
6, which is Trigonothracia pusilla

Trigonothracia pusilla (Gould, 1861), Oku-
tani, 2000: pl. 517, fig. 8

Type Material and Locality

Lectotype (CM 21507, left valve) and para-
lectotype (CM 21508, right valve) (Oyama,
1973). Shito, Semata, Ichihara City, Chiba
Prefecture, central Honshu, Japan (Pleis-
tocene) (Yokoyama, 1922; Higo et al., 1999).

Material Examined

1 lot (NSMT Mo 48820) from Jyogashima,
Miura Peninsula, Japan, 73-85 m (3 right and

THE BIVALVE GENUS PARVITHRACIA 127

TABLE 5. Parvithracia (Pseudoasthenothaerus) sirenkoi Kamenev, new species. Shell measurements (mm),
indices and summary statistics of all characteristics: L — shell length; H — height; W — width; А — anterior end
length; S — maximal distance from the posterior shell margin to the top of pallial sinus; L1 —lithodesma
length; N— number of pillars under the subumbonal plate. Numerator indicates shell measurements and
indices for the right valve, denominator — for the left valve. NM — not measured.

Statistics L МА М НЕ W/L AL S/L Li/L Depository

Holotype
SONO 2:5 Desc. 71.37 А 0876 0301 05663 0:398 0:155 ММВ
83172124 53 33:3 4 0.863 0.289 0.639 0.398 0.155 4058

Paratype
80 74 24 53 33 15 5 095 0.300 0.663 0.413 0.188 MIMB
8:07 73 2352 33 5 0.913 0.288 0.650 0.413 0.188 4060

Paratype
Ви 2:5 5.0 32 18 5 0963 0.313’ (01625 0.400 0.225 ММВ
8097745723503 2 NM 0.925 0.288 0.625 0.400 0.225 4060

Paratype
ПИ Э.В 29:5 2577 10948: 107299 00/6231 0377040195 MIMB
ИИ ke 12:27 74:8) 72-6 4 0.935 0.286 0.623 0.338 0.195 4060

Paratype
74 67 20 46 31 13 4 0905 0270 0.622 0.419 0.176 MIMB
74 6.6 2.0 46 2.8 4 0.892 0.270 0.622 0.378 0.176 4060

Paratype
62 5.3 16 39 25 1.0 4 0855 0.258 0.629 0.403 0.161 ZIN 2
629587 1:5 39 2.5 4 0.855 0.242 0.629 0.403 0.161

Paratype
5.4 48 13 33 23 NM 3 0.889 0.241 0.611 0.426 NM ZIN 1
5.4 48 13 33 2.2 3 0.889 0.241 0.611 0.407 NM

Paratype
54 47 13 34 23 08 2 0.870 0.241 0.630 0.426 0.148 MIMB
54-46113 23:47 22:3 2 0.852 0.241 0.630 0.426 0.148 4059

Paratype
0.860 0.240 0.640 0.400 0.160 MIMB
0.840 0.240 0.640 0.400 0.160 4059
Paratype
4.5 40 11 28 09 03 3 0.889 0.244 0.622 0.200 0.067 ZIN 1

5:0) 4.312 32 20 0:8

ND IN

4.5 40 1.1 28 0.9 3 0.889 0.244 0.622 0.200 0.067
Paratype

3.8 36 0.8 22 1.7 06 2 0.947 0.211 0.579 0.447 0.158 ММВ
3.8 36 0.9 22 17 2 0.947 0.237 0.579 0.447 0.158 4061
NM NM NM NM NM NM NM NM NM NM NM NM MIMB
89 80 24 52 3.8 6 0.899 0.270 0.584 0.427 NM 4063
NM NM NM NM NM NM NM NM NM NM NM NM MIMB
SD ON NS ONE Er 2 0.878 0.293 0.720 0.378 NM 4062
6.6 6.0 18 44 25 ММ 4 0.909 0.273 0.667 0.379 NM MIMB
NM NM NM NM NM NM NM NM NM NM NM 4062
58151 14 39 21 0913 0879’ 024100672062 04155 ZIN 3
5.8 51 14 39 2.1 3 0.879 0.241 0.672 0.362 0.155
31811315 08 2:47 1.5 ММ’ 2 101921 0241477 0163270895 NM MIMB
NM NM NM NM NM NM NM NM NM NM NM 4062

Mean 6.27 5.68 1.69 3.95 2.47 1.13 — 0.904 0.262 0.630 0.392 0.168
6.71 5.99 1.79 4.22 2.62 0.890 0.263 0.628 0.387 0.168

SD 1.59 1.53 0.61 1.04 0.75 048 — 0.034 0.033 0.030 0.058 0.041
1.63 1.51 0.56 1.07 0.78 0.031 0.022 0.037 0.059 0.041

SE 0.41 0.39 0.16 0.27 0.19 0.14 — 0.009 0.009 0.008 0.015 0.012
0.42 0.39 0.14 0.28 0.20 0.008 0.006 0.010 0.015 0.012

Min 3:0) 535" 20:87 22 70:9 0°30) 277:0:85577.0211,.0:56877.0:200770!067
3.8 36 09 22 09 2 0.840 0.237 0.568 0.200 0.067

Max 8.3 7.7 25 55 35 180 5 0.963 0.313 0.672 0.447 0.225
8.97 8:0 24 59 38 6 0.947 0.293 0.720 0.447 0.225

п 1 Чо Ша Ша 1 1 Ч 18 15 15 15 12

—^
al
_
a
>
a
an
a
—^
a
=>
al
=>
a
E
a
a
a
A
a
a
№

128 KAMENEV

Russia

Bering Sea

Sea of Okhotsk N
N

Commander Islands
Sakhalin Is.

re
= mm Is.
9 |
Japan CSN Kunashir Is.

; Pacific Ocean

40
| Sea of Japan bes ie 7. |
ae eS)

FIG. 55. Distribution of Parvithracia (Pseudoasthenothaerus) sirenkoi (№ type locality).

TABLE 6. Results of comparison by pairs of mean values (Student (t) test) and variances (ANOVA)
of indices of right and left valves of Parvithracia (Pseudoasthenothaerus) lukini and P.
(Pseudoasthenothaerus) sirenkoi: L— shell length; H —height; W — width; A— anterior end length;
S — maximal distance from posterior margin to the top of pallial sinus; L1 —lithodesma length. P.—
probability that index values in Р (Pseudoasthenothaerus) lukini and P. (Pseudoasthenothaerus)
sirenkoi are drawn from the same population; n— number of valves of P. (Pseudoasthenothaerus)
lukini and P. (Pseudoasthenothaerus) sirenkoi, respectively.

Indices Right valves Left valves
ANOVA ANOVA
t P F (1, 26) P n t P Е (25) P n
H/L -3.59 0.001 9.93 0.004 19,15 -3.09 0.004 9.13 0.006 21, 14
W/L -3.16 0.003 11.36 0.002 19,15 -2.09 0.044 18.48 <0.001 21, 15
АЛ- 2.16 0.038 6.74 0.015 19, 15 2.27 0.030 11.36 0.002 21, 15
S/L 3.82 <0.001 10.93 0.003 19, 15 2.81 0.008 12.86 0.001 21, 15

L1/L 0.13 0.901 0.02 0.900 16,12 -0.76 0.454 <0.01 0.989 15, 12

THE BIVALVE GENUS PARVITHRACIA 129

TABLE 7. Results of discriminant analysis for the
most significant characteristics for dividing all spec-
imens into species of Parvithracia (Pseudoas-
thenothaerus) lukini (17 spec.) and Р (Pseudo-
asthenothaerus) sirenkoi (13 spec.): L—shell
length; W—width; A —anterior end length; S—
maximal distance from the posterior shell margin to
the top of pallial sinus.

Significant Standard-
charac- ized Wilks
Valves teristics coefficient Lambda P
Right S/L —0.7335 0.4930 0.015
Left W/L 0.5985 0.4331 0.003
Left A/L -0.5970 0.4228 0.021

1 left valves); 1 lot (CAS 63354) from gami
Bay, Honshu, Japan (2 right and 1 left valves).
Total of 5 right and 2 left valves.

Description

Expanded from Yokoyama (1922)—Exte-
rior: Shell small (<6.6 mm), elongate, ovate-
elongate, moderately high (H/L = 0.656-
0.776), slightly inequivalve (right valve slightly

higher), moderately inflated (W/L = 0.212-
0.283), inequilateral, thin, fragile, translucent,
white. Surface with faint growth lines and very
fine granules. Periostracum dull, thin, adher-
ent, colorless. Beaks small, moderately pro-
jecting above dorsal margin, posterior to mid-
line (AL = 0.618-0.655), slightly rounded,
opisthogyrate. Anterior end narrowly rounded.
Posterior end truncate, with a faint radial ridge
extending from beaks to where posterior end
meets ventral margin. Anterodorsal margin
straight or slightly convex, smoothly descend-
ing and smoothly transitioning to rounded an-
terior end. Ventral margin slightly curved,
sometimes straight in left valve. Posterodor-
sal margin slightly concave, smoothly de-
scending, forming a smooth angle with poste-
rior end. Posterior end slightly curved, almost
vertical, only slightly turned anteriorly to form
a rounded angle where it meets ventral mar-
gin. Escutcheon narrow, well expressed, de-
marcated by ridges along posterodorsal mar-
gin from beaks to posterior end.

Interior: Right valve with anterior and pos-
terior lateral teeth; left valve with anterior lat-
eral tooth. In right valve, anterior lateral tooth

FIGS. 56-64. Shells of Parvithracia species from Japan. 56-60. Parvithracia (Pseudoasthenothaerus) se-
matana (Yokoyama, 1922) (NSMT Mo 48820), Jyogashima, Miura Peninsula, Japan, 73-85 m. 56, 57: Left
valve, length 6.2 mm. 58, 59: Right valve, length 5.8 mm. 60: Subumbonal plate with pillars (ventral view),
left valve (bar = 1 mm). 61-64. Parvithracia (Pseudoasthenothaerus) isaotakii (Okutani, 1964) (NSMT-Mo
71463), Tosa Bay, Japan, Pacific Ocean (33°05,6'N, 133°41,4’E), 800 m, shell length 7.6 mm.

130 KAMENEV

large, triangular, projecting considerably
above inner part of anterodorsal margin, an-
teroventrally directed; posterior lateral tooth
small, elongate, slightly projecting above
inner part of posterodorsal margin, extending
along posterodorsal margin. In left valve, an-
terior lateral tooth long, lamellate, slightly pro-
jecting above inner part of anterodorsal mar-
gin; inner part of anterodorsal shell margin
slightly concave between beak and tooth;
inner part of posterodorsal margin slightly
convex. Subumbonal plate short, slightly ele-
vated above inner shell surface, almost com-
pletely attached to shell wall, free along its an-
teroventral margin. Pillars very short,
sometimes lying on shell wall, but not sup-
porting subumbonal plate. Number of pillars
from 2 to 4. Anterior adductor muscle scar
large, elongate, kidney-shaped. Posterior ad-
ductor scar small, rounded. Pallial sinus dis-

tinct, of the same shape and size in both
valves, deep, rounded anteriorly, not reaching
to midline (S/L = 0.358-0.385), anterior limit
short of faint vertical line from beaks. Shell in-
terior with faint radial striae, noticeable along
ventral shell margin.

Variability

Shell shape and proportions vary little
(Table 8). Pillars supporting the subumbonal
plate are sometimes difficult to see and count.

Distribution and Habitat

Parvithracia (Pseudoasthenothaerus) se-
matana occurs off Japan: in the Pacific
Ocean — in Otsushi Bay (Honshu) (Tsuchida &
Kurozumi, 1996), near Boso Peninsula and

TABLE 8. Parvithracia (Pseudoasthenothaerus) sematana (Yokoyama, 1922). Shell measurements
(mm), indices and summary statistics of all characteristics: L— shell length; H — height; W — width; A —
anterior end length; $ — maximal distance from the posterior shell margin to the top of pallial sinus; N —
number of pillars under the subumbonal plate. Numerator indicates shell measurements and indices for

the right valve, denominator — for the left valve. NM — not measured.

Statistics L H W A S N H/L W/L AL S/L Depository
Lectotype
NM NM NM NM NM NM NM NM NM NM CM 21507
6.6 48 1.7 NM NM NM 0.27 0.258 NM NM
Paralectotype

61 40 13 NM NM NM 0.656 0.213 NM NM CM 21508
NM NM NM NM NM NM NM NM NM NM
NM NM NM NM NM NM NM NM NM NM NSMT Mo 48820
6.2 42 15 40 2.3 ММ 0.77 0.242 0.639 0.371
58 45 16 38 2.1 NM 0.76 0.276 0.655 0.362 NSMT Mo 48820
NM NM NM NM NM NM NM NM NM NM
5.5 39 14 34 2.1 NM 0.709 0.255 0.618 0.382 NSMT Mo 48820
NM NM NM NM NM NM NM NM NM NM
53 39 14 34 19 NM 0.736 0.283 0.642 0.358 NSMT Mo 48820
NM NM NM NM NM NM NM NM NM NM
52 40 11 34 20 3 0.769 0.212 0.654 0.385 CAS 63354
NM NM NM NM NM NM NM NM NM NM
52 40 11 34 20 3 O769 0212 0654 0385 CAS 63354
NM NM NM NM NM NM NM NM NM NM
NM NM NM NM NM NM NM NM NM NM CAS 63354
52 39 11 34 19 3 0750 0:212 0.654 0.365

Mean 5.52 4.05 1.33 348 2.02 — 0.736 0.242 0.645 0.374
6.00 4.30 1.43 3.70 2.10 0.718 0.237 0.650 0.368

SD 0.37 0.23 0.21 0.18 0.08 — 0.047 0.034 0.016 0.013
0.72 046 0.31 0.42 0.28 0.037 0.023 0.006 0.004

SE 0.15 0.92 0.08 0.08 0.04 — 0.019 0.014 0.007 0.006
0.42 0.26 0.18 0.30 0.20 0.022 0.013 0.005 0.003

Min 52 39 11 34 19 3 0.656 0212 0,618 0.358
5272397 11 84777109537 0677 0,2127 0:645), 0365

Max 6.1 45 16 38 21 3 0.776 0.283 0.655 0.385
6.6 48 17 40 23 3 0.750 0.258 0.654 0.371

n 6 6 6 5 5 5 6 6 5 5

3 3 3 2 2 23 3 2 2

THE BIVALVE GENUS PARVITHRACIA 131

southwards (Honshu) (Higo et al., 1999); in
the Sea of Japan—near Oga Peninsula, off
Honshu (Ito, 1989; Higo et al., 1999); in the
Yellow Sea —off Kyushu (Higo et al., 1999).
This species was recorded at a depth from 20
m to 300 m, on the fine sand (Tsuchida &
Kurozumi, 1996; Habe, 1977; Higo et al.,
1999).

Comparisons

This species is easily distinguished from
other species of the subgenus in having a
small, elongate, very thin translucent shell
with the subumbonal plate almost completely

attached to the shell wall and indistinct, short
pillars (Table 4).

Parvithracia (Pseudoasthenothaerus)
isaotakii (Okutani, 1964)
Figs. 61-64, Table 9

Asthenothaerus isaotakii Okutani, 1964:

84, 85, text fig. 6; Habe, 1977: 312; Higo et al.
1999: 524; Okutani, 2000: 1039, pl. 517, fig. 6

Type Material and Locality

Holotype (shell length 7.9 mm), Sagami
Bay, Japan (35°05.35N, 139°18.65E), 550 m,

FIG. 65. The hinge and subumbonal plate of Parvithracia species from Japan by camera lucida. A, B.
Parvithracia (Pseudoasthenothaerus) sematana (Yokoyama, 1922) (NSMT Mo 48820). A: Hinge of right
valve. B: Hinge of left valve. C-F. Parvithracia (Pseudoasthenothaerus) isaotakii (Okutani, 1964) (NSMT-Mo
71463). C: Hinge of right valve. D: Hinge of the left valve (inner part of anterodorsal shell margin broken). E:
Close-up of right valve showing attachment of lithodesma (ventral view) to subumbonal plate. F: Subumbonal
plate with internal ligament and pillars (ventral view), left valve. Bar = 1 mm.

132 KAMENEV

TABLE 9. Parvithracia (Pseudoasthenothaerus) isaotakii (Okutani, 1964). Shell measurements (mm) and
indices: L— shell length; H — height; W — width; А — anterior end length; $ — maximal distance from the pos-
terior shell margin to the top of pallial sinus; L1 — lithodesma length; N — number of pillars under the subum-
bonal plate. Numerator indicates shell measurements and indices for the right valve, denominator — for the

left valve, ?—ventral margin of the left valve is partly broken. NM — not measured.

pa H W A S L1 N
7.6 6.5 1.6; 03:82, ЗУ 41-0

7-5 6.1? ММ 4.0 3.7

Coll. T. Okutani, 27-IX-1960 (R/V “Soyo-
Maru”) (Okutani, 1964).

Material Examined

1 lot (NSMT-Mo 71463) from Tosa Bay,
Japan (33°05.6'N, 133°41.4'E), 800 m, 12-
IX-1997 (R/V “Kotaka-Maru”) (1 spec.).

Description

Expanded from Okutani (1964)— Exterior:
Shell small (<7.9 mm), subovate, high (H/L =
0.855), slightly inequivalve (right valve slightly
longer and higher), moderately inflated (W/L =
0.211), thin, fragile, ashy white under perio-
stracum. Surface rough, with conspicuous
growth lines and very fine granules. Perio-
stracum dull, thin, adherent, sometimes peel-
ing near beaks, dark grayish, sometimes with
dark brown or black patches or crusts near
beaks and dorsal shell margin. Beaks small,
moderately projecting above dorsal margin,
central or slightly posterior to midline (A/L =
0.5-0.533), slightly rounded, orthogyrate. An-
terior end sharply rounded. Posterior end ex-
panded, vertical, higher than anterior, decid-
edly truncate, with a faint radial ridge
extending from beaks to junction of posterior
end with ventral margin. Anterodorsal margin
Straight, steeply descending, smoothly transi-
tioning to rounded anterior end. Ventral mar-
gin slightly curved. Posterodorsal margin
straight, smoothly descending, forming a
smooth, obtuse angle with posterior end. Pos-
terior end straight or slightly curved, almost
vertical, only slightly turned anteriorly to form
a rounded angle where it meets ventral mar-
gin. Escutcheon very narrow, well developed,
demarcated by ridges extending along pos-
terodorsal margin from beaks to posterior
end.

Interior: Right valve with anterior and pos-
terior lateral teeth; left valve with anterior lat-
eral tooth. In right valve, anterior lateral tooth

W/L AL S/L Li/L Depository

6 0.855 0.211 0.500 0.487 0.132 NSMT
6 0.813? NM 0.533 0.493 0.133 Mo 71463
large, triangular, projecting considerably

above inner part of anterodorsal margin, ven-
trally directed; posterior lateral tooth small,
elongate, slightly projecting above inner part
of posterodorsal margin, extending along pos-
terodorsal margin. In left valve, anterior lateral
tooth long, lamellate, considerably projecting
above inner part of anterodorsal margin; inner
part of anterodorsal margin straight between
beak and tooth; inner part of posterodorsal
margin slightly convex. Subumbonal plate
short, elevated above inner surface. Pillars
supporting plate short, wide, partly fused,
considerably tapering ventrally. Number of pil-
lars in both valves 6. Lithodesma large (L1/L =
0.133), curved. Anterior adductor muscle scar
large, elongate, kidney-shaped. Posterior ad-
ductor scar small, rounded. Pallial sinus dis-
tinct, of the same shape and size in both
valves, deep, narrow and rounded anteriorly,
reaching midline (S/L = 0.487-0.493). Shell
interior polished, with faint radial striae.

Distribution and Habitat

Parvithracia (Pseudoasthenothaerus) isao-
takii occurs on the Pacific coast of Japan:
Tosa Bay (33° 05.6'N, 133°41.4’E), 800 m;
Sagami Bay (35°05.35’N, 139°18.65’E;
35°09.9'N, 139°30.4'E), 550-700 m (Oku-
tani, 1964); Sea of Enshu-Nada (34°25.7'N,
137°58.5'E), 620 m (Okutani, 1964).

Comparisons

In contrast to other species ofthe subgenus,
P. (Pseudoasthenothaerus) isaotakii has a
broader posterior end, and the beak is almost
central and orthogyrate (Table 4). This species
also differs from P. (Pseudoasthenothaerus)
lukiniand P. (Pseudoasthenothaerus) sirenkoi
in having a thin, fragile shell, and a curved lith-
odesma that is not butterfly-shaped. Parvithra-
cia (Pseudoasthenothaerus) isaotakii is dis-
tinguished from P. (Pseudoasthenothaerus)

THE BIVALVE GENUS PARVITHRACIA 133

sematana, which also has a thin and fragile
shell, in having a highly elevated subumbonal
plate, with distinct, high pillars.

ACKNOWLEDGMENTS

| am very grateful to Dr. V. A. Nadtochy
(PRIFO, Vladivostok) and Mrs. N. V.
Kameneva (IMB, Vladivostok) for great help
during work on this manuscript; to Drs. V. B.
Durkina, L. N. Usheva, M. A. Vaschenko, and
|. G. Syasina (IMB, Vladivostok) for consulta-
tions and help during study of anatomy of the
new species; to Dr. H. Saito (NSMT, Tokyo),
Ms. R. N. Germon (USNM, Washington), Dr.
N. R. Foster (UAM, Fairbanks), Mr. R. Asher
(CIN, Cawtron) and Dr. K. A. Lutaenko (IMB,
Vladivostok) for providing at my disposal
specimens of different species of Thraciidae
genera; to Drs. B. |. Sirenko, A. V. Martynov
and all collaborators of the Marine Research
Laboratory (ZIN, St.-Petersburg) for help dur-
ing work with collection of bivalve molluscs at
the ZIN; to Dr. B. A. Marshall (MNZ, Welling-
ton) for consultations and sending material of
the genus Parvithracia and reprints of papers;
to Dr. E. V. Coan (Department of Invertebrate
Zoology, CAS, San Francisco) for consulta-
tions, comments on the manuscript, and
sending reprints of papers; to Drs. T.
Kurozumi and E. Tsuchida (NHMI, Chiba), Dr.
K. Amano (Joetsu University of Education,
Joetsu) for sending reprints of papers; to Mr.
E. V. Jakush (PRIFO, Vladivostok) and D. V.
Fomin (IMB, Vladivostok) for help in work with
the scanning microscope; to Mr. A. A.
Omelyanenko (IMB, Vladivostok) for making
photographs; to Ms. T. N. Kaznova (IMB,
Vladivostok) for translating the manuscript
into English; Dr. George M. Davis for help in
the publication of the manuscript; two anony-
mous reviewers for comments on the manu-
script.

This research was partly supported by
Grants 98-04-48279, 01-04-48010, and 00-
15-97890 from the Russian Foundation for
Basic Research.

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723-735.

ANGAS, G. F., 1868, On a new genus and some

new species of marine Mollusca from Port Jack-
son, New South Wales. Proceedings of the
Zoological Society of London, for 1867, 3: 908-
911.

BOREHAM, A., 1959, Biological type specimens in
the New Zealand Geological Survey. 1. Recent
Mollusca. New Zealand Geological Survey Pale-
ontological Bulletin, 30: 1-87.

BOROVIKOV, V. P. & I. P. BOROVIKOV, 1997,
“STATISTICA”. Statistical analysis and process-
ing of data using “WINDO”. Filin Press, Moscow,
608 pp. [in Russian].

COAN, E. V., 1990, The Recent eastern Pacific
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Revised ms accepted 8 July 2001

MALACOLOGIA, 2002, 44(1): 135-143

LOCALIZATION OF NADPH-DIAPHORASE-POSITIVE ELEMENTS
IN THE INTESTINE OF THE MUSSEL CRENOMYTILUS GRAYANUS
(MOLLUSCA: BIVALVIA)

Anatoly A. Varaksin'*, Eugenia A. Pimenova?, Galina $. Varaksina' & Lydia T. Frolova'

ABSTRACT

By NADPH-diaphorase histochemistry (Hope & Vincent, 1989), localization of nicotinamide
adenine dinucleotide phosphate diaphorase-positive (NADPH-d-positive) elements was studied
in the direct and recurrent loops of the midgut and hindgut of the mussel Crenomytilus grayanus.
Intraepithelial NADPH-d-positive cells were found in the intestinal groove and major typhlosole
of the direct loop and in the recurrent loop of the midgut, as well as in the hindgut. The cells are
fusiform and lie separately or in minor groups. Their apical processes are directed to the gut
lumen, and the basal processes contact with the basiepithelial plexus. The latter, in turn, sends
separate NADPH-d-positive fibers to make contacts with subepithelial plexus. Both plexuses are
well developed in the intestinal groove and major typhlosole of the direct loop and in the recur-
rent loop of the midgut, as well as in the hindgut. In the minor typhlosole and crystalline style sac,
both plexuses are less developed. In the ciliary groove of the hindgut, the basiepithelial plexus
is absent. Possible role of NADPH-d-positive elements in regulation of the digestion in bivalve

mollusks is discussed.

Key words: histochemistry, NADPH-diaphorase, basiepithelial plexus, subepithelial plexus, in-
testine, Crenomytilus grayanus (Dunker), Mollusca.

INTRODUCTION

Nitric oxide (NO) acts as a transcellular
messenger in the mammalian nervous sys-
tem (Garthwaite & Boulton, 1995; Rand & Li,
1995). NO, together with citrulline, is pro-
duced by NO-synthase from L-arginine. For
the reaction to proceed normally, O,, Cas
and nicotinamide adenine dinucleotide phos-
phate (NADPH) are required (Bredt 8 Snyder,
1990). In the course of the histochemical re-
action, exogenous nitro blue tetrazolium is re-
duced by NO-synthase to diformazan precipi-
tate, which thereby marks cells containing the
enzyme. At present, of all NADPH-dependent
oxidoreductases, NADPH-diaphorase is only
considered to retain activity after para-
formaldehyde fixation (Matsumoto et al.,
1993). Histochemical activity of NADPH-di-
aphorase is thus believed to be a topographi-
cal marker of NO-synthase-containing struc-
tures in tissues of both vertebrates (Hope et
al., 1991; Dawson et al., 1991) and inverte-
brates (Moroz & Gillette, 1995, 1996; Moroz
et al., 1996).

To date, NO-ergic element localization and
functional role in the mammalian alimentary

tract have been intensively studied (Schleiffer
8 Raul, 1997; Huber et al., 1998). Among
lower vertebrates, NO-synthase-containing
neurons were found in the alimentary tract of
the toad Bufo marinus (Li et al., 1992, 1993;
Murphy et al., 1993); an agama lizard, Agama
sp. (Knight & Burnstock, 1999); the rainbow
trout, Salmo gairdneri (Li & Furness, 1993);
the Atlantic cod, Gadus morhua; and the spiny
dogfish, Squalus acanthias (Olsson & Karila,
1995). At the same time, NO-ergic element lo-
calization and functional role in the inverte-
brate digestive system are poorly studied.
These elements are revealed in the cardiac
stomach of the starfishes Marthasterias
glacialis (Martinez et al., 1994) and Asterias
rubens (Elphick & Melarange, 1998), and in
the oesophagus of the opisthobranch Pleuro-
branchaea californica (Moroz & Gillette,
1996). To date, no data have been present on
NO-ergic elements in the alimentary tract of
bivalve mollusks.

The present work focused on NADPH-d-
positive element localization in the direct and
recurrent loops of the midgut, and in the
hindgut of the mussel Crenomytilus grayanus
(Dunker).

"Institute of Marine Biology, Russian Academy of Sciences, Vladivostok 690041, Russia; inmarbio@mail.primorie.ru

“Far East State University, Vladivostok 690600, Russia
“corresponding author

135

136 VARAKSIN ET AL.

MATERIALS & METHODS

Samples were obtained from five adult
mussels with shells 10.5-11.0 cm high and
4.5-5.0 cm wide. Mollusks were collected in
Amurskii Bay, Sea of Japan, in November
1998 and kept in aerated aquaria for 2-3
days. During that time, the mollusks were not
fed.

NADPH-d-positive elements in the intestine
were revealed by aNADPH-diaphorase histo-
chemistry method (Hope & Vincent, 1989).
Fragments of the direct and recurrent midgut
loops and of the hindgut, shown in Figure 1,
were fixed in 4% paraformaldehyde in 0.1 M
sodium phosphate buffer (pH 7.2) for 2 h at
4°C. They were then rinsed in 3-4 changes of
15% sucrose prepared in 0.05 M Tris-HCl
buffer (pH 8.0) for 24 h at the same tempera-
ture. Both transverse and longitudinal, 15 um
thick, cryostat sections were prepared and
placed on to slides.

NADPH-Diaphorase Histochemistry

Sections were incubated in a medium con-
taining 0.5 mM B-NADPH, 0.5 mM nitro blue
tetrazolium, and 0.3% Triton X-100 in 0.15 M
Tris-HCl buffer (pH 8.0) for one h at 37°C.
Then, they were rapidly washed twice in
distilled water, dehydrated in an ethanol se-
ries of increasing concentrations, and
mounted in Dammar Resin. Control sections

h hg

were incubated in the following media: (1)
without B-NADPH, (2) without nitro blue tetra-
zolium.

Drugs Used

Nitro blue tetrazolium, B- NADPH were sup-
plied by Sigma Chemical Co, USA.

Statistical Analysis

Sections were observed and photographed
using Olympus BH2-RFCA microscope (BHS
model). NADPH-d-positive cells were drawn
by means of a Carl Zeiss drawing tube ata
total magnification of x400. Cell height and
width were measured using a plotting scale.
For each of the intestine parts studied, a total
number of NADPH-d-positive cells was
counted in ten transverse sections, and a
mean and standard deviation per section was
calculated. Statistical significance was tested
by paired t-tests at the 0.05 confidence level.

RESULTS
Direct Loop of the Midgut

The direct loop of the midgut consists of:
the intestinal groove, the major and minor ty-

rl

sates KR 1
7m

NA

pa

gl

FIG. 1. Anatomy of the digestive system of Crenomytilus grayanus. Abbreviations: aa, anterior adductor; dg,
digestive gland; di, direct loop of the midgut; gi, gills; h, heart; hg, hindgut; ml, mouth lobes; oe, oesophagus;
pa, posterior adductor; rl, recurrent loop of the midgut; s, stomach. Scale bar, 10 mm.

NADPH-DIAPHORASE-POSITIVE ELEMENTS OF THE MUSSEL 137

FIG. 2. Schematic localization of NADPH-d-positive elements in the intestine of Crenomytilus grayanus. A.
Direct loop of the midgut; B. Recurrent loop of the midgut; C. Hindgut. Abbreviations: bep, basiepithelial
NADPH-d-positive plexus; cg, ciliary groove; cs, crystalline style; css, crystalline style sac; ct, connective tis-
sues with separate muscular cells; e, epithelium; gl, gut lumen; ic, intraepithelial NADPH-d-positive cells; ig,
intestinal groove; sep, subepithelial NADPH-d-positive plexus; mat, major typhlosole; mit, minor typhlosole;
rt, the region of the typhlosole, adjacent to the crystalline style sac. Scale bar, 1 mm.

phlosoles, and the crystalline style sac (Fig.
2A). Among these structures, a considerable
difference in intensity and pattern of NADPH-
d-positive elements staining was observed.
In the intestine groove wall, fairly developed
basi- and subepithelial NADPH-d-positive

plexuses were observed (Fig. 3A). The
basiepithelial plexus was dense and inten-
sively stained, with the maximum thickness of
13.7 + 4.6 um. In the intestine groove epithe-
lium, there occur NADPH-d-positive cells av-
eraging 2.6 + 1.9 per section (Table 1). The

138 VARAKSIN ЕТ AL.

FIG. 3. NADPH-d-positive element localization in the direct loop of Crenomytilus grayanus midgut. A. In-
testinal groove. B. Major typhlosole. C. Crystalline style sac. D. Intraepithelial NADPH-d-positive cell in the
major typhlosole. Abbreviations: f, NADPH-d-positive fibers in the connective tissue of the intestinal groove;
for other abbreviations, see caption to Fig. 2. Scale bar, A, C, D, 100 um; B, 150 um.

cells are located mainly in the basal zone of
the epithelium; they are fusiform, 10.9 + 2.0
um high and 3.6 + 0.9 um wide. The subep-
ithelial plexus represents a dense, thick (30.1
+ 8.2 um), interlacement of NADPH-d-posi-
tive fibers (Fig. 3A). Both plexuses are con-
nected to each other with separate thin fiber

bundles. Occasional large, varicose fibers
branch off from the subepithelial plexus to
submerge to the adjacent connective tissue
(Fig. 3A).

In the major typhlosole, the basiepithelial
plexus 6.7 + 2.8 um thick has the appearance
of a rather sparse, slightly branched interlace-

NADPH-DIAPHORASE-POSITIVE ELEMENTS OF THE MUSSEL 139

TABLE 1. The average proportion of NADPH-d-positive cells to the total number of
epithelial cells per section in the Crenomytilus grayanus intestine.

Total NADPH-d- NADPH-d-
epithelial positive cell positive cell
Intestine region cell number number proportion (%)
Direct loop of the midgut
Intestine groove 509 + 152 2.6 + 1.9 0.51
Major typhlosole 1349 + 457 17.3 + 10.4 1.28
Minor typhlosole 1109 + 295 cm =
Crystalline style sac 585 + 154 = 7
Recurrent loop of the midgut 1810 + 452 15.1 + 4.8 0.83
Hindgut 1474 + 244 13 3E 461 0.92

Values represent the mean + the standard error of the mean.

ment of fibers bearing varicosities (Fig. 3B). It
is more prominent in the region of the ty-
phlosole, adjacent to the crystalline style sac.
In the major typhlosole epithelium, NADPH-d-
positive cells also occur averaging 17.3 +
10.4 per section (Table 1). The cells are
fusiform and lie diffusely, close to the gut
lumen (Fig. 3B). They are 14.9 + 2.9 um high
and 5.7 + 1.7 um wide (Fig. 3D). In the minor
typhlosole, the basiepithelial NADPH-d-posi-
tive plexus is formed by thin fibers, most of
which are longitudinally oriented. In the region
of the minor typhlosole, adjacent to the crys-
talline style sac, at the epithelium base, only
rare thin fibers occurred, no NADPH-d-posi-
tive cells being observed. In both typhlosoles,
the subepithelial plexus represents an inter-
lacement of weakly branched NADPH-d-pos-
itive fibers bearing varicosities. The subep-
ithelial layers of the major and minor
typhlosoles are, respectively, 31.9 + 10.7um
and 12.0 + 7.2 um thick. In the adjacent con-
nective tissue, occasional NADPH-d-positive
fibers occur.

Inthe area of the crystalline style sac, the
basiepithelial plexus represents a thin (4.2 +
1.4 um) interlacement of fibers bearing vari-
cosities (Fig. 3C). In the subepithelial plexus,
which is 5.2 + 1.3 um thick, the longitudinal
orientation of NADPH-d-positive fibers pre-
vails. In the adjacent connective tissue, occa-
sional fibers beaded with varicosities occur.

Recurrent Loop of the Midgut

In the recurrent loop of the midgut, NADPH-
d-positive elements were found in the basal
zone of the epithelium and in the underlying
connective tissue (Fig. 2B). The basiepithelial

FIG. 4. NADPH-d-positive element localization in
the recurrent loop of Crenomytilus grayanus
midgut. Abbreviations are the same as in Fig. 2.
Scale bar, 70 um.

NADPH-d-positive plexus represents a
dense, intensively stained nerve fiber inter-
lacement 23.7 + 4.7 um thick (Fig. 4). In the
epithelium of the loop occur NADPH-d-posi-
tive cells averaging 15.1 + 4.8 per section
(Table 1). These lie separately or in groups of

140 VARAKSIN ET AL.

2-3 cells (Fig. 4). The cells are fusiform, 17.3
+ 2.8 um high, and 7.9 + 1.3 um wide. The
subepithelial plexus is 43.2 + 5.9 um thick. It
is formed by densely interlacing NADPH-d-
positive fibers (Fig. 4). Occasional fibers bear-
ing varicosities leave the plexus to enter the
adjacent connective tissue.

Hindgut

In the hindgut, NADPH-d-positive elements
were found in both layers (Fig. 2C). The
basiepithelial plexus represents a dense, in-
tensively stained NADPH-d-positive fibers in-
terlacement 14.3 + 3.7 um thick. (Fig. 5A-D).
The only exception is the ciliary groove re-
gion, which lacks a basiepithelial plexus (Fig.
5D). In the basal zone of the epithelium, sin-
gle fusiform NADPH-d-positive cells are re-
vealed (Fig. 5B, C) averaging 13.3 + 6.1 per
section (Table 1). These are 17.9 + 4.6 um
high and 5.7 + 0.9 um wide. The cells pos-
sess apical processes directed to the gut
lumen, their basal processes contacting the
basiepithelial plexus (Fig. 5B, C).

The subepithelial plexus is formed by an in-
terlacement of NADPH-d-positive _ fibers,
which are mainly longitudinally oriented (Fig.
5A, D). Its thickness reaches 27.2 + 11.2 um
and 106.3 + 11.7 um at the concave and con-
vex sides of the gut, respectively. At the con-
vex side of the gut, the plexus is denser than
at the concave side.

Neither control test revealed NADPH-d-
positive elements in any of the C. grayanus
gut parts studied.

DISCUSSION

We revealed NADPH-d-positive cells lo-
cated in the epithelium of the direct and re-
current loops of the midgut, as well as in the
hindgut of the mussel C. grayanus. In many
invertebrate groups, specialized cells were
found in the epithelial lining of the alimentary
tract that are not directly involved in the
process of digestion and seemingly serve reg-
ulatory functions (Punin, 2000). In bivalve
mollusks, the system regulating gut function-
ing includes both nerve and endocrine cells.
By using methylene blue staining, nerve cells
were revealed in the Anodonta cellensis
midgut epithelium (Gilev, 1952). Electron mi-
croscopy studies of Arctica islandica and
Mytilus edulis gut epithelium revealed intraep-
ithelial cells with a fairly developed rough en-

doplasmic reticulum, the Golgi apparatus of
dictyosomic type, numerous granules, and
first-order processes. The latter formed pro-
nounced basiepithelial plexus. Based on their
morphology, these cells were considered
nerve cells (Punin, 1981, 1989). At the same
time, endocrine cells in the gut epithelium
of bivalve mollusks are characterized by
larger cytoplasmic granules and the lack of
processes (Punin, 2000). From the above
facts, we believe that intraepithelial NADPH-
d-positive cells in the C. grayanus gut are of
nerve type.

We revealed a dense, intensively stained
plexus of NADPH-d-positive fibers in the
basal zone о the mussel intestinal epithelium.
A plexus of similar or even greater density and
complexity was found in the subepithelial
zone of the direct and recurrent midgut loops
and of the hindgut.

The bivalve alimentary tract is known to
possess complicated systems comprised of
the basi- and subepithelial nerve plexuses
(Giusti, 1970; Punin, 1981, 1989). Based on
electron microscopy data, both zones were
shown to be to a certain degree autonomous
(Punin & Konstantinova, 1988; Punin, 1989).
The subepithelial plexus is assumed to be a
peripheral component of the molluscan ner-
vous system (Punin & Konstantinova, 1988).

The basiepithelial plexus was earlier shown
to consist of the processes of intraepithelial
nerve cells (Punin, 1989). Nerve elements of
the subepithelial plexus are also presumed to
contribute to basiepithelial plexus formation.
By light microscope studies of M. edulis
(Punin & Konstantinova, 1988) and of A. is-
landica (Punin, 1981), processes were found
that penetrated into the neighbouring connec-
tive tissue. Terminals of nerve processes can
penetrate into the basal zone of the intestine
epithelium from the surrounding connective
tissue, as in the case in the Tapes waltingii
hindgut (Dougan & McLean, 1970) and in the
M. galloprovincialis midgut (Giusti, 1970). We
repeatedly found processes connecting the
basi- and subepithelial plexuses. This sug-
gests that both plexuses were structurally and
functionally integrated, and the putatively NO-
ergic system of the C. grayanus intestine pos-
sess a complex multilevel structure.

It is worth noting that the basiepithelial
plexus is relatively well developed in all parts
of the gut. However, it contains few intraep-
ithelial cells of putative NO-ergic nature. Nu-
merous NO-ergic fibers were revealed in the
neuropils of the C. grayanus ganglia, whereas

NADPH-DIAPHORASE-POSITIVE ELEMENTS OF THE MUSSEL 141

FIG. 5. NADPH-d-positive element localization in the hindgut of Crenomytilus grayanus. A. Convex side of
the gut. Scale bar, 150 um. В, С. Intraepithelial NADPH-d-positive cells. Scale bar, 50um. D. Concave side
ofthe gut. Scale bar, 250 um. The solid arrow shows the apical process; the dashed arrow, the basal process;
for other abbreviations, see caption to Fig. 2.

the number of specifically stained perikarya
was relatively small there (Annikova et al.,
2000). A similar pattern of NO-positive stain-
ing was observed in other gastropod and bi-
valve mollusks and in echinoderms (Moroz &
Gillette, 1996; Dyuizen et al., 1999). It is pro-
posed that C. grayanus NO-ergic neurons are
not only capable of NO synthesis in the cell
body but also possess a system of local nitric
oxide synthesis within the processes (An-

nikova et al., 2000). The presence of numer-
ous, putatively NO-ergic fibers bearing vari-
cosities in the C. grayanus intestine supports
this proposal.

Thus, in the wall of the C. grayanus intes-
tine, there exists an integrated system com-
prised of intraepithelial, putatively NO-ergic
nerve cells and of basi- and subepithelial
nerve plexuses, too, of putative NO-ergic na-
ture.

142 VARAKSIN ET AL.

To date, the role of NO in regulation of in-
vertebrate digestion is poorly studied. It is
shown that in the oesophagus of Lymnaea
stagnalis, nitric oxide is capable of functioning
as a neuromodulator (Elphick et al., 1994;
Moroz et al., 1993). NO is capable of modu-
lating the frequency of epitheliocyte cilia beat-
ing, for instance, in the ciliary epithelium ofthe
mammalian respiratory system (Jain et al.,
1993). In C. grayanus, NO-synthase activity is
found to be high in the basiepithelial plexus of
the intestine, the fibers of which establish di-
rect contacts with ciliated epitheliocytes. We
suppose that the NO role in controlling ep-
itheliocyte locomotory activity consists in
modulating the transport of water with food
masses kept by epitheliocyte ciliary streaming
and the transport of fecal masses through the
intestine. However, in the epithelium of the
crystalline style sac, where a highly intensive
сша beating is maintained, basiepithelial
plexus is unexpectedly ordinary developed.
Obviously, NO does not play a principal role in
regulating ciliary activity of epitheliocytes in
the C. grayanus intestine.

In the intestine of bivalve mollusks, epithe-
liocytes secrete mucus, digestive enzymes,
and protein-like substances of the crystalline
style matrix (Owen, 1966; Reid, 1966; Giusti,
1970). The secretory activity is especially
prominent in epitheliocytes of the region of the
major typhlosole, adjacent to the crystalline
style sac (Frolova, 1989). In this segment, a
lot of putatively NO-ergic cells (Table 1) were
found and could be related to prominent basi-
and subepithelial plexuses. On the contrary,
putatively NO-ergic cells are absent, and
basi- and subepithelial putatively NO-ergic
plexuses are poorly developed in the minor ty-
phlosole and crystalline style sac, which are
characterized by a low secretory activity.
Then, NO is likely to control epitheliocyte se-
cretory activity in the mollusk intestine.

ACKNOWLEDGMENT

The research described in this publication
was made possible in part by Award No. REC-
003 of the U. S. Civilian Research & Develop-
ment Foundation for the Independent States
of the Former Soviet Union (CRDF).

LITERATURE CITED

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BREDT, D. $. & $. H. SNYDER, 1990, Isolation of
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DAWSON, T. M., D. S. BREDT, M. FOTUHI, P. M.
HWANG & $. H. SNYDER, 1991, Nitric oxide
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Revised ms. accepted 30 May 2001

MALACOLOGIA, 2002, 44(1): 145-151

ORIGIN OF THE EXCRETORY CELLS IN THE DIGESTIVE GLAND OF THE LAND
SNAIL HELIX LUCORUM

Vasilis K. Dimitriadis & Vasiliki Konstantinidou

Department of Genetics, Development and Molecular Biology, School of Biology, Faculty of
Sciences, Arisrotle University Of Thessaloniki, Thessaloniki 54006, Greece;
vdimitr@bio.auth.gr

ABSTRACT

The examination of the digestive gland cells of the land snail Helix lucorum with light and elec-
tron microscopes after starvation and subsequent periods of feeding supports the hypothesis
that the so-called excretory cells are digestive cells in a final step of their cell cycle, rather than
a distinct cell type. Results supporting this hypothesis are: (1) the fact that the so-called diges-
tive cells containing heterolysosomes and residual bodies or the so-called excretory cells con-
taining excretory vacuoles were not found in significant numbers in the digestive gland epithe-
lium at the same time; (2) the fact that the decrease in the number and size of the
heterolysosomes implicated in the endocytotic uptake and digestion of nutrients was combined
with an increase in the number and size of the excretory vacuoles responsible for the excretory
processes, and vice versa; and (3) the fact that starvation caused the appearance of a large
number of excretory vacuoles and the reduction of the heterolysosomes in the digestive gland

epithelium to a minimum.

INTRODUCTION

The digestive gland (or midgut gland, or he-
patopancreas or liver) is the largest organ in
the body of terrestrial gastropods. It consists
of two lobes communicating with the stomach
via large ducts, which branch to form smaller
ducts, ductules, and complex branched blind
tubules. It is an organ dealing with elaboration
of enzymes, absorption of nutrients, endocy-
tosis of food substances, food storage, and
excretion (Runham, 1975).

In spite of the numerous studies on the
morphology and physiology of the digestive
gland cells, there are conflicting reports about
the cell types that constitute the digestive
gland epithelium of terrestrial gastropods.
Even at the ultrastructural level, undoubted in-
terpretation of the results is still difficult be-
cause of the changes in the fine structure as-
sociated with phases of activity. Most of the
findings are consistent with the hypothesis
that the digestive gland epithelium is com-
posed of three cell types: digestive cells, cal-
cium cells, and excretory cells (Fig. 1), sur-
rounded by connective tissues, muscular
layers, and haemocoelic spaces (Sumner,
1965; Dimitriadis & Hondros, 1992). The pres-
ence of a fourth type, the thin cells, is docu-
mented in many instances, but these cells

145

are regarded as undifferentiated precursors of
the other cell types (Sumner, 1965; Walker,
1970).

Digestive cells are the most frequent cell
type found in the digestive gland of snails and
slugs. They are usually columnar in shape,
varied in size, and usually display microvilli.
Digestive cells are characterized by numer-
ous granules usually located in the apical por-
tion of the cells consisting of secondary lyso-
somes, the heterolysosomes, as well as by
vacuoles with dense cores consisting residual
bodies, named green and yellow granules, re-
spectively, after light microscopic observa-
tions in Helix and Agriolimax (Sumner, 1965;
Walker, 1970, 1972), or apical granules and
cisternae with electron-dense cores after
electron microscopic observations in Helix
(Dimitriadis & Hondros, 1992; Dimitriadis &
Liosi, 1992). The second cell type of the di-
gestive gland, the calcium cells, are charac-
terized by their pyramidal shape and the pres-
ence of calcium granules (Sumner, 1965;
Dimitriadis & Hondros, 1992). The third cell
type, the excretory cells are larger in size than
the digestive cells and are characterized by
one or more large excretory vacuoles con-
taining cores or amorphous mass of high
electron density (Sumner, 1965; Dimitriadis &
Hondros, 1992).

146 DIMITRIADIS & KONSTANTINIDOU

FIG. 1. Drawing of the three cell type regarded by
many authors that form the digestive gland epithe-
lium of terrestrial gastropods, based on the original
in Dimitriadis & Hondros (1992). Abbreviations: CC,
calcium cell; DC, digestive cell; EC, excretory cell;
Ev, excretory vacuole; HI, heterolysosome; N, nu-
cleus; Rb, residual body.

In spite of the view that the excretory cells
are a separate celltype, there are also results
supporting the view that these cells are either
digestive cells differentiated towards excre-
tory cells or cells of the final developmental
stage of the digestive cells. The present study
was designed to answer the questions related
to the structure and function of the excretory
cells.

A previous study (Dimitriadis & Hondros,
1992) showed that the digestive gland cells of
Helix lucorum, even after 40 days of starva-
tion or 37 days hibernation, exhibited in-
creased number of large excretory vacuoles,
as well as heterolysosomes compared to con-
trols, and this increase was attributed to the
accumulation of residual indigestible material
inside the tubule cells reflecting decreased di-
gestive and excretory function due to starva-
tion and hibernation. Similar results were also
reported in starved Helix aspersa Muller
(Sumner, 1965; Porcel et al., 1996), in which
prolonged starvation caused a significant de-
crease in the percentage of the digestive cells
and an increase of the excretory and narrow
cells. In addition, the carbohydrate cytochem-
istry of the content of these lysosomal struc-
tures did not change after the same periods of
starvation and hibernation compared to con-
trols (Dimitriadis & Liosi, 1992).

Reports from a variety of molluscs show the
existence of different phases of cell activity,
each with a distinct appearance, the digestive
and fragmentation state being the most fre-

quent (Morton, 1974, 1975, 1979). In the lat-
ter reports, the length of the cell cycle is ap-
proximately 24 hours. Because preliminary
observations in the digestive gland of H. luco-
rum showed a substantially longer cell cycle
and less extensive cell alteration during this
cycle compared to other molluscs, another
purpose of the present study was to present
indications related to the digestive rhythm of
the digestive gland cells of H. lucorum.

MATERIALS & METHODS

The snails used in the experiments were
collected from a natural habitat of Helix luco-
rum (Gastropoda, Pulmonata, Helicidae) in
the Logos region of Edessa, northern Greece.
The shell diameter of the snails varied from 20
to 40 mm, and the body weight was between
14 and 20 q.

The animals were kept in the laboratory
under stable and controlled conditions of pho-
toperiod (L:D 13:11 h), temperature (22 +
2°C), and relative humidity (90 + 5%). They
were fed lettuce, which is the most assimi-
lated food for H. lucorum (Staikou & Lazari-
dou-Dimitriadou, 1989). The controls were
kept in the conditions described above, while
others were starved for 14 days, being in
identical conditions of photoperiod, tempera-
ture, and humidity as the controls. During the
starvation period, the animals developed a
velum of mucus. After starvation, the animals
were fed again with lettuce, and food was kept
in the boxes during the whole experiment,
while each of the animals was observed at
subsequent periods. The snails started to
move at about 1.5 h after feeding. Animals
were examined after 0, 10, 22, 34, 45, 56, 65,
75 h from the beginning of food ingestion. For
each experimental stage, the animals began
feeding at the same time and were actively
feeding for the whole experimental period.
Five actively feeding animals from each ex-
perimental stage were examined under the
light and electron microscope.

For light and electron microscopic observa-
tions, samples were fixed in 3% glutaralde-
hyde, postfixed in 2% osmium tetroxide, de-
hydrated, and embedded in Spurr’s resin.
Ultrathin sections were post-stained with
uranyl acetate and lead citrate and examined
under a JEOL 100B electron microscope op-
erating at 80 KV. For light microscopic obser-
vations, thick sections were stained with 1%
toluidine blue.

DIGESTIVE GLAND OF HELIX LUCORUM 147

RESULTS

In the following paragraphs, the light and
electron microscopic views of the digestive
gland epithelium of snails Helix lucorum,
which were starved for 14 days and then fed
for 10, 22, 34, 45, 56, 65 and 75 h are de-
scribed.

At the beginning of food ingestion (animals
after 14 days of starvation), as well as 10h
from the beginning of food ingestion, exami-
nation of transverse sections of digestive
tubules under the light (Fig. 2A) or the elec-
tron microscope (Fig. 2B) show a large num-
ber of excretory vacuoles in the form of large
vacuoles with dense cores, similar to that de-
scribed in the excretory cells of Helix lucorum.
These large vacuoles, the accumulation of
which in the digestive gland epithelium is at-
tributed to starvation, reach usually 2-8 um,
while in certain cases reach 32 um. However,
cells containing heterolysosomes in the form
of apical granules, which is a main character-
istic of the digestive cells, are very few. The
apical plasma membranes show limited
pinocytotic phenomena at 0 h, expressed by
the formation of a moderate number of coated
vesicles. However, at 10 h these phenomena
become intensive and the formation of coated
pinocytotic vesicles, and a network of micro-
tubules and vesicles in the apical cytoplasm is
apparent (not shown). The calcium cells lo-
cated in the base of the epithelium (Fig. 2A)
exhibit the usual morphology described for
this cell type.

The apical region of the cells show a large
number of excretory vacuoles, as well as in-
tensive pinocytotic phenomena 22 h from the
beginning of food ingestion. Also, a small
number of heterolysosomes is present in the
tubule cells in the form of apical granules of
moderate size (0.5-1, 5 um) filled with mater-
ial with low electron density. After 34 h from
the beginning of food ingestion, these het-
erolysosomes are larger in size and numbers,
reaching 1-2 um and are located near the
apical border of the cells (Fig. 2C). The excre-
tory vacuoles are now located adjacent to the
apical region of the cells, being probably in a
phase just before their excretion, while such
vacuoles are present in the tubule lumen in
certain cases (not shown). The ultrastructural
observations show that micropinocytosis con-
tinues at this stage.

At the 45 h stage, a large number of het-
erolysosomes, about 2 um in size, as well as
an increased number of excretory vacuoles

are obvious in the apical and middle portion of
digestive gland cells under the light micro-
scope. The heterolysosomes 56 h after the
beginning of food ingestion are increased
very much in number, and their size usually
reaches 4 um (Fig. 2D). With the electron mi-
croscope, the presence of recently formed
large vacuoles with dense cores is apparent,
while some of them are observed to fuse with
each other (Fig. 3A). The calcium cells do not
show any obvious alteration in the number
and location, comparing to the previous
stages.

The digestive tubules cells 65 h and 75 h
from the beginning of food ingestion are char-
acterized by an increased number of excre-
tory vacuoles, the size of which being more
evident at 75 h (Fig. 3B). The apical het-
erolysosomes decrease in number, being al-
most absent at 75 h (Fig. 3B). No any visible
change in the number and location of the cal-
cium cells was noted.

Considering the digestive gland cells of the
control animals, that is the snails fed with let-
tuce and kept in identical photoperiod, tem-
perature, and humidity, their functional mor-
phology resemble that already described for
these snails examined under normal condi-
tions.

DISCUSSION

There is a disagreement on the origin and
the function of excretory cells in terrestrial
gastropods. Fretter (1952), Billett & McGee-
Russell (1955), Walker (1970), and Morton
(1979) regarded these cells as differentiated
digestive cells, whereas Thiele (1953) and
Sumner (1965) suggested that they are de-
generate calcium cells. In other studies,
Abolins-Krogis (1961) found mucopolysac-
charides, proteins, lipids, and small quantities
of RNA inside the cisternae of excretory cells
in Helix aspersa and suggested that these
materials are implicated in shell repair.

The results of the present study support the
hypothesis that in the digestive gland, the so-
called excretory cells are, simply, digestive
cells in a final step of their cell cycle rather
than a distinct cell type. The results showed
that the so-called digestive cells containing
heterolysosomes and residual bodies or the
so-called excretory cells containing excretory
vacuoles did not follow independent cell cy-
cles, because they were not found in signifi-
cant numbers at the same time. The increase

148 DIMITRIADIS & KONSTANTINIDOU

FIG. 2A, B. Beginning of food ingestion. Transverse sections of digestive tubules. Under the light (arrows) or
the electron microscope (asterisks), the digestive gland cells of the snail Helix lucorum show absence of het-
erolysosomes, while abundance of large vacuoles with dense cores is noted. A number of calcium cells are
located at the base of the epithelium. C. 34 h from the beginning of food ingestion. Large numbers of het-
erolysosomes (apical granules) are present at the apical portion of the digestive gland cell (asterisks). D. 45
h from the beginning of food ingestion. Transverse sections of a digestive tubule. Under the light microscope,
the cells contain large number of heterolysosomes (small arrows), as well as an increased number of ex-
cretory vacuoles (large arrows). Abbreviations: CC, calcium cell; Lu, lumen; Mv, microvilli. Scale bars —A =
15 um, B= 1.5 um, C = 1 um, D = 16 um.

DIGESTIVE GLAND OF HELIX LUCORUM 149

FIG. 3A. 56 h from the beginning of food ingestion. Under the electron microscope, the digestive gland cells
show abundance of excretory vacuoles in the form of vacuoles containing dense cores (asteriks). The arrows
show possible sites of fusion between adjacent vacuoles. B. 75 h from the beginning of food ingestion. Trans-
verse section of a digestive tubule. The abundance of large vacuoles with dense cores (arrows) and the ab-
sence of heterolysosomes are prominent in the digestive gland cells at this stage. Scale bars: À = 1 um, B =

25 um

in the number and size of the heterolyso-
somes and residual bodies implicated in the
endocytotic uptake and digestion of nutrients
from the gland lumen and their digestion was
combined with a decrease in the number and
size of the excretory vacuoles responsible for
the excretory processes in the epithelium, and
vice versa. The cell changes observed in the
digestive gland epithelium should be re-
garded as different phases of cell activity of
the same cell type, the digestive cells.

Another result that supports the above hy-
pothesis was the fact that in the digestive
gland cells starvation caused the appearance
of a great number of excretory vacuoles and
the reduction of the heterolysosomes to a
minimum (Fig. 2A). It is also supported by re-
sults in starved Helix lucorum (Dimitriadis &
Hondros, 1992) and Helix aspersa (Porcel
et al., 1996), in which prolonged starvation
caused a decrease in the percentage of the
digestive cells compared to the increased per-
centage of the excretory cells.

According to the above approach, the phe-

nomena related to the endocytotic and excre-
tory processes reported in the present study
could exhibit the following sequence: at the
beginning of food ingestion (end of 14 days
starvation period), the digestive gland epithe-
lium consists only of cells with excretory vac-
uoles formed during the previous cell cycle
and were accumulated in the cells during star-
vation (Fig. 4C). In the following periods, the
excretory vacuoles are relocated adjacent
to the apical border of the cells and finally
are extruded from the cells, which could be
conclude by the presence of such vacuoles
in the tubule lumen. The digestive activities
progress and initially increase in number and
size the heterolysosomes, that is, the cell
elements implicated in the function of endocy-
tosis and Iysosomic digestion of the endocy-
tosed material (Fig. 4A) and later (approxi-
mately at the 45 h), the residual bodies
formed by further digestion and condensation
of the content of the heterolysosomes (Fig.
4B). Digestion proceeds, and there is a de-
crease in the number and size of both het-

150 DIMITRIADIS & KONSTANTINIDOU

FIG. 4. Drawing of the various stages that the digestive cells possibly undergo during the endocytotic and
excretory processes in the digestive gland epithelium. A. As the digestive activity progresses, in the diges-
tive cells initially increase in number and size the heterolysosomes (Hl), the secondary Iysosomes implicated
in the function of endocytosis and lysosomic digestion of the endocytosed material. B. Intralysosomal diges-
tion in the heterolysosomes and condensation of their content leads to the formation of large number of resid-
ual bodies (Rb) or heterolysosomes condensed to residual bodies C. Fusion of residual bodies (arrow) leads
to the formation of large excretory vacuoles and to a decrease in the number and size of both heterolyso-
somes and residual bodies. D. Cytoplasmic vessels containing excretory vacuoles, amongst other or-
ganelles, are cut off from the apical region of digestive cells and are extruded to the lumen or the whole cell

is degenerated and is extruded to the lumen.

erolysosomes and residual bodies and an in-
crease in number and size of the excretory
vacuoles, which possibly arise after fusion of
smaller residual bodies (Figs. 3A, 4C). The
excretory vacuoles are finally extruded into
the tubule lumen as described above.

The proposed sequence is supported by
the results of Walker (1970) in Agriolimax
reticulatus, in which the excretory cells were
also regarded as the final step in the develop-
ment of the digestive cells. The excretory cells
in Agriolimax contained the digestive rem-
nants of ingested material, which is absorbed
by the former cells, degraded by Iysosomal
action and subsequently extruded to the
lumen of the digestive gland.

Reports show that in the land pulmonate
Deroceras reticulatum (Walker, 1972; Triebs-
korn & Florschutz, 1993), the food transit
through the digestive tract takes 9-12 h, while
in the freshwater pulmonates Lymnaea stag-
nalis (L.) (Veldhuijzen, 1974) and Biophalaria
grabrata (Say) (Florschutz & Becker, 1999)
the digestive gland is emptied of food every
60-110 min, discharging undigested parti-
cles. In the absence of information, the latter
authors postulated the existence of a rhythmic
activity of the basommatophoran digestive
gland cells. On the other hand, reports froma
variety of different molluscs, from the phy-
tophagous pulmonate Amphibola cerenata

(Morton, 1974) to the zoophagous pulmonate
Deroceras caruanae (Morton, 1979) and the
microphage bivalve Geloina proxima Prime,
1864 (Morton, 1975) showed the existence of
different phases of cell activity, each with a
distinct appearance, the digestive and frag-
mentation state being the most frequent. In
these reports, the length of the cell cycle is es-
timated to be approximately 24 h. Although a
different methodology would be required for
studying cell turnover, the results of the pres-
ent study display indications that in Helix lu-
corum this cycle seems to be longer, reaching
approximately 55 h. This could be concluded
by the fact that at 0-10 h, as well as at 65-75
h from the beginning of food ingestion, identi-
cal morphological features were observed in
the digestive gland cells. In addition, the mor-
phological changes in the cell that accompany
the cell cycle were not as extensive as in other
gastropods and were related to the presence
or absence of certain lysosomic structures,
rather than extensive changes, for example in
the thickness of the epithelium.

By using light and electron microscope ob-
servations the present study provides infor-
mation about the cell types and the physiol-
ogy of the digestive gland cells of H. lucorum.
However, more studies, using for example
histochemistry in cryosections and immuno-
cytochemistry are needed for the better un-

DIGESTIVE GLAND OF HELIX LUCORUM 151

derstanding of the fine structure and function
of these cell types.

LITERATURE CITED

ABOLINS-KROGIS, A., 1961, The histochemistry of
the hepatopancreas of Helix pomatia (L.) in rela-
tion to the regeneration of the shell. Arkiv fur Zo-
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BILLETT, F. & S. M. MCGEE-RUSSELL, 1955, The
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Revised ms. accepted 25 April 2001

MALACOLOGIA, 2002, 44(1): 153-163

INTERPOPULATION VARIATION IN LIFE-HISTORY TRAITS OF POMACEA
CANALICULATA (GASTROPODA: AMPULLARIIDAE) IN SOUTHWESTERN
BUENOS AIRES PROVINCE, ARGENTINA

Pablo R. Martin & Alejandra L. Estebenet

Universidad Nacional del Sur, Departamento de Biologia, Bioquimica y Farmacia, San Juan

670, 8000 Bahia Blanca, Argentina; pmartin@criba.edu.ar

ABSTRACT

The Argentinean apple snail Pomacea canaliculata is a freshwater gastropod with a high inter-
population variation in shell shape, size and thickness. Previous experimental studies have shown
that many life-history traits are highly dependent on rearing conditions. Three natural populations
located in one same drainage basin and climatic regime showed marked differences in birth, ma-
turity and maximum sizes. Most of this variation disappeared when newborns from each popula-
tion were reared under homogeneous conditions in the laboratory, indicating its ecophenotypic ori-
gin. However, a significant variation in reproductive, growth and survival patterns attributable to
genetic differences among the source populations was still discernible among laboratory cohorts.

The three sites studied represent a marked gradient in stability and food availability. Females
from the most unstable and poorer site showed a faster prematurity growth and a higher ovipo-
sition rate than those from the most stable and productive site. This higher oviposition rate was
associated with bigger clutches and a higher mortality rate. The different patterns of survival and
somatic and reproductive allocation in the three populations, being heritable and adaptively cor-
related to different environmental conditions, could be considered as parts of different life-history
strategies.

Since the 1980s, P. canaliculata has become a serious pest of paddy fields in most Southeast
Asian countries. Nearby and recently isolated populations of Р canaliculata from the same basin
showed genetically different life-history strategies, so that different populations of this pest could

require different control programs.

Key words: apple snail, life-history traits, ecophenotypic variation, genetic variation

INTRODUCTION

Pomacea canaliculata (Lamarck, 1822) isa
freshwater prosobranch gastropod with a high
polymorphism in shell shape, size and thick-
ness (Cazzaniga, 1987). Its natural distribu-
tion is basically tropical and subtropical, in-
cluding the Amazonas and La Plata basins
(Ihering, 1919), though in the last two decades
it has invaded most Southeastern Asia coun-
tries, becoming a serious rice pest (Wada,
1997). During an extensive survey in the
southern limit of the native area of this species,
the southernmost apple snail in the world
(Martin et al., 2001), we observed a great in-
terpopulation variation in maximum size, shell
thickness and egg size, even at a small spatial
scale and within the same drainage system.

Experimental trials have shown that growth
rates, longevity and age at maturity are highly
dependent on temperature (Estebenet & Caz-
zaniga, 1992), crowding (Cazzaniga & Es-
tebenet, 1988; Tanaka et al., 1999), and food

153

(Lacanilao, 1990; Estebenet, 1995; Albrecht
et al., 1999; Lach et al., 2000). This evidence
and the strong intersite environmental varia-
tion (Martin et al., 2001) seems to support the
hypothesis of an ecophenotypic origin of the
interpopulation variation. Cazzaniga (1987)
reported that shells from lentic, soft-bottom
water bodies are generally bigger and lighter
than those from hard-bottom running waters.
However, a genetic basis of this interpopula-
tion variation cannot be ruled out.

The aims of this study were to analyze the
variation in birth, maturity and maximum sizes
among populations of P. canaliculata from
three environmentally different sites belonging
to the same drainage basin, and to determine
if they were ecophenotypic or genetic in origin.

STUDY AREA

The study area lay between 36°40’S and
37°35’S and 61°15’W and 63°10’W in south-

154 MARTIN & ESTEBENET

western Buenos Aires Province, Argentina.
The climate of the region is temperate, with a
mean annual temperature and amplitude of
13.8°C and 15.1°C, respectively; mean an-
nual rainfall is 733 mm, mainly concentrated
in fall and spring. The rainfall is highly vari-
able, with marked fluctuations between dry
and wet years (Scian & Donnari, 1997).

The three sites selected belong to the En-
cadenadas del Oeste Basin (Fig. 1), a closed
drainage system composed of a chain of six
shallow lakes of increasing conductivity to-
wards the west, from Alsina Lake (freshwater)
to Epecuen Lake (hypersaline) and de la Sal
Lake (temporary salt lake). Streams and
creeks of pluvial hydrological regime, with
scant and very variable water discharge, run
northwards into the lakes from the Ventania
Mountains.

Alsina Lake is a permanent, eutrophic lake,
with a surface area of 105 km”, maximum and
mean depths of 6.4 m and 2.8 m respectively

Alsina Lake

ween EI Quilcé
del Monte Lake ix x, El Ниазсаг
+

Cochicö Lake

=

del Venado Lake
Epecuén Lake yo
1 Cochicö
; Rivulet

a

de la Bal Lake

Curalmalal
Stréam

`
`
Ventani

mr. y

RT 2 и
E ~~ Mountains
г 1

—

?

and a shoreline length of 62 km. The bottom
is composed of fine sand and mud. The dom-
inant macrophytes during the present study
were the emergent Typha spp. and Schoeno-
plectus californicus and the submerged Pota-
mogeton sp. and Myriophyllum elatinoides,
sometimes heavily colonized by cyanophytes.

Curamalal Stream is a permanent stream
approximately 91 km long that discharges in-
termittently into Alsina Lake. The bottom is
composed of mud with minor areas of lime-
stone bed. The macrophytes were the emer-
gent Cyperus spp. and S. californicus, the
submerged Chara sp. and Potamogeton sp.
and the floating-leaved Ludwigia sp.

Cochico Rivulet is a 20-km-long creek with
intermittent flow along most of its course. It
discharges in the salty Cochicó Lake (con-
ductivity 11.3 mS.cm '), and during high
floods the Curamalal Stream overflows into it.
The dominant substratum is mud with some
areas of limestone bedrock and cobble.

4 .r

Stre

Las Tunas
Stream

kilometers

FIG. 1. Map ofthe Encadenadas del Oeste basin showing the location (®) ofthe three study sites (from north
to south Alsina Lake, Cochicö Rivulet and Curamalal Stream) (Inset: map of Argentina showing the study

zone).

LIFE-HISTORY VARIATION IN POMACEA 155

Emergent macrophytes were absent and only
occasionally was the presence of scarce Lud-
wigia sp., Potamogeton sp. and Enteromor-
pha sp. detected.

MATERIALS & METHODS

The three sites were visited in February,
July and October 1998 and February 1999.
Two or three people searched for apple snails
during up to four hours among the submerged
vegetation, under stones, or buried in the sub-
stratum; all snails larger than 20 mm were col-
lected and their shell length from the apex to
the basal extreme of the aperture was mea-
sured. In October 1998 and February 1999
copulating snails (i.e., couples in which male
penis sheath was inserted into female pallial
cavity) were collected and sized. The pink,
aerial and conspicuous egg masses, de-
posited on riparian or emergent plants, were
obtained in March and October 1998 and
maintained until hatching in the laboratory.
Temperature, conductivity (mS.cm !) and pH
were determined in situ with a multimeter
Horiba U-10. A subsurface water sample was
collected at each site for further analyses. The
concentrations of major ions and other limno-
logical variables were measured as detailed
in Martin et al. (2001).

Progeny from at least five clutches from
each site (March 1998) that hatched in a pe-
riod of 72 hours were pooled and 100 new-
born were randomly selected to generate
three experimental cohorts. They were reared
in sets of ten snails under homogeneous con-
ditions (aereated tap water saturated with
СО.Са, temperature of 25 + 3°C, permanent
illumination and lettuce ad libitum).

During the first two months the sets of ten
snails were kept in 3 | aquaria and then trans-
ferred to 15 | aquaria to avoid crowding (Caz-
zaniga & Estebenet, 1988). When the first egg
mass was observed in each 15 | aquarium,
snails were sexed on the basis of shell shape
(Cazzaniga, 1990; Estebenet, 1998) and
color and appearance of the gonad (Andrews,
1964), visible through the thin shell. One fe-
male and one male were randomly selected,
and reared again in 3 | aquaria. The eight re-
maining snails of each aquarium were sacri-
ficed and stored for later morphometric analy-
sis, which will be published elsewhere.

Water was changed weekly and survivor-
ship, shell length and number of clutches and

eggs were recorded. Dead snails were re-
placed by mature snails of the same sex with
a shell length larger than 30 mm, obtained in
the wild and maintained in the lab under the
same rearing conditions, to keep rearing con-
ditions constant for the survivors, especially
mating possibilities and density. The oviposi-
tion and spawning rates were calculated as
the ratio of life-time production of eggs or egg
masses to the length of the spawning period
(from first to last spawn). Clutch size was es-
timated as the ratio of the life-time production
of eggs to the life-time production of egg
masses.

Field egg masses obtained in March and
October 1998 and one egg mass from each
couple from each cohort were incubated in
the lab. After hatching, groups of 30-50 new-
born were dried (80°C, 48 h), weighed to the
nearest 0.1 mg, and reweighed after ignition
(600°C, 4 h) to obtain total and ash dry
weights. Shell lengths of ten hatchlings from
field spawns and fifteen from laboratory
spawns were measured under a stereoscopic
microscope to the nearest 0.01 mm.

To test for differences among sites in repro-
ductive variables one way Anovas were per-
formed. Two way Anovas were used for length
and age at death, since sexual dimorphism in
these traits has been recorded (Estebenet &
Cazzaniga, 1998). For newborn and imma-
ture snails nested Anovas were performed on
shell length with egg masses or aquaria as
the nested factor.

RESULTS

The values of the physico-chemical vari-
ables measured in Alsina and Curamalal sites
fall within the range of the water bodies of the
Southern Buenos Aires province classified as
“habitable for snails” by Martin et al., (2001).
The Cochico, contrarily, showed marked
fluctuations in conductivity (Fig. 2) and Na*
concentration, reaching values out of the
mentioned range. The variation in water tem-
perature among the three sites was less than
3°C during most part of the year, except in
summer, when temperatures 7°C higher were
measured in Cochico.

Maximum sizes differed markedly among
the three populations. The mean shell length
of the 15 larger snails on each sampling date
is showed in Figure 3. Snails from Cochico
were generally smaller than those from Alsina
and Curamalal. Despite the similarity in maxi-

156 MARTIN & ESTEBENET

& Alsina о

{+ Curamalal
= 41| e Cochicó _
Е
o

—®

>
> 2- 3
$ A
3 РЕН
=
о 17
o

FIG. 2. Seasonal variation in conductivity in the
studied sites of Alsina Lake, Cochicó Rivulet and
Curamalal Stream.

60 =

_ A $$ AS,

A
— à _
Е 50; 5 ti :
Е 1 т
=
= |
2 | №
Е: |
= 40 +
= À
E
” 0 ¢ Feen
] - & Alsina
2 4 {+ Curamalal
| | e Cochicó
30 ————— > > > —
J FMAM J J AS ON D JS FM
1998 date 1999

FIG. 3. Seasonal variation in mean shell length of
the fifteen larger snails in the studied sites of Alsina
Lake, Cochicö Rivulet and Curamalal Stream (bars
are 95% confidence intervals).

mum sizes in spring, the size of the copulating
snails was smaller in the Cochico population
than in the Curamalal population in October
1998, and also in February 1999 (Fig. 4; t,, =
3.07, р < 0.01 and t,., = 6.03, р < 0.001, re-
spectively). Couples were not observed in
Alsina on those dates. Despite the highly sig-
nificant variation in newborn shell length
among clutches of the same site (Fy. 270 =
19.52, Р < 0.0001 and F, jog = 35.33, P <
0.0001, respectively), the intersite variation
was highly significant both in March and Oc-
tober 1998, Cochicö hatchlings being the
smallest in both dates (Table 1). The new-
borns from Cochicö were also lighter than
those from Alsina and Curamalal, though dif-
ferences were significant only in March 1998

50 +

Ger = =]

: {OCuramalal] |
0 Cochic

40 |

30 +

shell length (mm)

October 1998 February 1999
FIG. 4. Mean shell length of copulating snails in the
Cochicö Rivulet and Curamalal Stream sites in Oc-
tober 1998 and February 1999 (bars are 95% con-
fidence intervals).

(Table 1). Egg and clutch sizes were also evi-
dently smaller in Cochico (pers. obs.).

Growth, survivorship and oviposition pat-
terns for the three laboratory cohorts are
shown in Figure 5. The initial size differences
disappeared before the age of two months
(Table 2), suggesting a higher growth rate for
the Cochico cohort. Growth curves were very
similar from two months of age onwards, and
female shell length at first spawn was not sig-
nificantly different (Table 2; one female from
Cochicé and Alsina each died accidentally
and one from Curamalal that never spawned
was excluded from analysis). Length at death
was significantly different between sexes
(males smaller than females) but not among
cohorts (Table 3).

Mortality was null during the pre-reproduc-
tive phase in the three cohorts but after the
beginning of spawning the mortality rate in-
creased steadily. It was highest in the Cochico
cohort, with a maximum longevity of 318
days. The Alsina cohort showed the lowest
mortality, with some snails surviving up to 650
days. Differences in age at death (Table 3)
were significant only between Cochico
(253.53 days) and Alsina cohorts (343.58
days) (critical value for the SNK test = 71.33
days, p < 0.05). The survivorship pattern of
the Curamalal cohort was intermediate, with a
mean survival time of 300.37 days and a max-
imum longevity of 627 days.

Although the age at first spawning and the
duration of the spawning period in the Alsina
cohort were higher than in the Cochico and
Curamalal ones, they were not significantly
different (Table 2), probably due to the high
variation in reproductive behavior in that co-

LIFE-HISTORY VARIATION IN POMACEA

TABLE 1. One-way Anova for length and dry weights of newborn from field egg masses (&:
Nested Anova with four or five spawns (nested factor) per site and ten newborn per spawn).

Underlined means are significantly different to the other two (SNK tests, p < 0.05).

Means

Variable M.S. 9.1. = P Cochico Curamalal Alsina
March 1998

total weight (mg) 0.966 2,1 20.572 0.000 1.053 1.808 1.555
ash weight (mg) 0.411 2,16 9.459 0.002 0.329 0.824 0.648
shell length (mm)& 2.414 2,12 24.738 0.000 2.199 2.628 2.498
October 1998

total weight (mg) 0.181 2,9 2.564 0.131 0.920 1.236 1.356
ash weight (mg) 0.060 2,9 20705404182 0.524 0.735 0.768
shell length (mm)& 0.931 2,9 4.392 0.047 2.156 2.388 2.447

157

hort (Fig. 6). The life-time production of eggs
and clutches, and the spawning rate were
not significantly different among cohorts, al-
though the oviposition rate was significantly
lower in Alsina females. On the other hand,
the clutch size from the Cochicö cohort was
the highest (Table 2).

The total and ash dry weights of newborns
were not significantly different among the lab-
oratory cohorts (Table 2). The variation in
newborn shell length among clutches of the
same cohort was highly significant (F,¿ 25, =
34.27, P < 0.0001) and greater than the inter-
cohort variation (Table 2). Only for the
Cochicö site there was a significant variation
between newborn from egg masses laid in the
field (March 1998) and their own progeny in
the laboratory, both in length (nested Anova
with five spawns —nested factor — per source
and ten newborn per spawn: F, з = 9.62, P <
0.01) and weight (t,, = 3.97, р < 0.001 for total
dry weight and t,, = 8.25, р < 0.0001 for ash
dry weight).

DISCUSSION

Pomacea canaliculata shows great life-his-
tory interpopulation variation, related mainly
to different thermal regimes (Estebenet &
Cazzaniga, 1992). Though the three natural
populations studied here are located in the
same drainage basin and under the same cli-
matic regime, they showed marked differ-
ences in some life-history traits. The variation
in maximum sizes among the sites indicates
different survivorship or growth rates. On the
other hand, at the beginning of the reproduc-
tive season (early spring, Hylton-Scott, 1958)
the size of copulating snails differed between
the two stream populations, though the size
distributions were very similar, suggesting dif-

ferences in size or age at maturity. Interpopu-
lation variation in the size of eggs, clutches
and newborn was also observed.

Most of the interpopulation variation in birth,
maturity and maximum sizes disappeared
when the snails were reared under homoge-
neous conditions in the laboratory, indicating
an ecophenotypic origin of the variation. How-
ever, notwithstanding the elimination of envi-
ronmental differences, some variation in re-
productive, growth and survival patterns was
still discernible and attributable to genetic dif-
ferences among populations. Although the
maternal and environmental effects on the
newborn used to initiate the cohorts cannot be
discarded, their influence seems to be irrele-
vant. The effect of site conditions during em-
bryo development was probably null, since
very fresh clutches (easily recognizable by
their moisture and bright pink color) were
used. On the other hand, the differences in
newborn size, partially attributable to mater-
nal size or condition (see below) soon disap-
peared under the highly favorable rearing
conditions in the laboratory.

The three sites represent a marked gradi-
ent in stability and food availability. The
Cochicö is an intermittent creek, with very
marked seasonal fluctuations in water level,
the lower section being the only one that
never dries completely; however, high con-
ductivity peaks caused by the rise in Cochicö
Lake water levels affect this section. These
peaks exceed the maximum values of con-
ductivity recorded for sites inhabited by Р
canaliculata in this area (2.89 mS.cm !,
Martin et al., 2001). Minimum discharge in
Curamalal Stream is higher than in the
Cochicö Rivulet, and the flow is continuous
even during dry summers. Though the area of
Alsina Lake is quite variable, water level fluc-
tuations are comparatively slow and related

158 MARTIN & ESTEBENET

100 ersrarireraie ea ana - ee 600
fe eS DÍ DABAAAAN =
Alsina SER ¡egg production
8 — = shell length Г 500
\ E survirvorship
da = + 400
60 - Ñ — ТУ
ee ЕН ee at Lalla Г 300
40 II ttt à
+ : AAA RAA AN 200
7 IR a
20 © ee | Il _n | 100
* | | Il | | ol
o Oooo 1 | | HL ВД ола. In. 0 | 0

17 52 87 122 262 297 332 367 402

100 qatar AAA Ahh LA à — 600
Curamalal cg
er \ oo

17 52 87 12 157 192 227 262 297 332 367 402

shell length (mm) or survivorship (%)
egg production (eggs.female ‘.wk")

100 гео —— —— —- 600
— DA
| NES | A
an costed ds |
| +
| \ + 400
| A
4 | À +444
2722257 CH | 300
40 Y \
и ie + 200
$ \
| Ltstytytsh
20 =“ \ г 100
Аа НЙ \
0 | ey u e RETA) 0
17 52 87 157 192 22 262 297 332 . 367 1 1402

age (days)

FIG. 5. Growth, survivorship and oviposition patterns for the three laboratory cohorts from Alsina Lake,
Cochicö Rivulet and Curamalal Stream (bars are 95% confidence intervals for shell length).

LIFE-HISTORY VARIATION IN POMACEA 159

TABLE 2. One-way Anova for growth and reproductive variables from laboratory cohorts. Underlined
means are significantly different to the other two (SNK tests, p < 0.05). (#: Nested Anova with ten aquaria
(nested factor) per cohort and ten snails per aquarium; &: nested Anova with six females (nested factor)
per cohort and fifteen newborn per female; +: data from some females were lost).

Variable M.S. d.f.

Length at 17 d (mm) + 27.883 2,27
Length at 65 d (mm) + 16367 2,27
Length at first spawn (mm) 32.490 2,24
Age at first spawn (days) 2806.48 2,24

Spawning period (days)
Clutch size (eggs.clutch ')
Oviposition rate (е99$.мК`')

Spawning rate (clutches.wk~ ') 0.0191 2,24
Life-time egg production 126115.15 2,24
Life-time clutch production 195.44 2,24
Newborn shell length (mm) & 0.960 2,15
Newborn total weight (mg) 0.179 2, 18+
Newborn ash weight (mg) 0.018 2, 18+

11322.11 2,24
25361.35 2, 24
49543.56 2, 24

TABLE 3. Two-way Anova for growth and survival of males and females from labo-

ratory cohorts.

Variable Source

Length at death (mm) Site
Sex
Site x Sex
Error

Age at death (days) Site
Sex
Site x Sex
Error

— á—]<]á] á]á|l
————

ЕЕ Alsina

oo

——ч
>

—

—.
——
——
4
A,
|

Curamalal

ranked females

Cochicó

m—

—
——

—)
—
——
a
——_—_—

—
-—

т T T = T T T T T T

0 50 100 150 200 250 300 350 400 450 500 550 600
age (days)

FIG. 6. Spawning period duration of females from
the three laboratory cohorts from Alsina Lake,
Cochicö Rivulet and Curamalal Stream ranked by
age at first spawn.

Means
F Pp Cochicó Curamalal Alsina
13.354 0.000 5.07 6.00 Sal
2.052 0.148 23.70 23.99 23.19
1.598 0.223 47.27 47.82 50.80
0.752 0.482 136.78 164.55 169.55
2.655 0.091 90.00 92.78 152.78
6.696 0.005 264.75 204.29 158.94
4.576 0.021 375.10 346.36 234.66
0.748 0.484 1.47 1543 1.48
0.023 0.977 4554.22 4407.22 4641.45
1.261 0.302 19.00 22.45 28.21
3.501 0.056 2.48 2.46 2.65
2.333 07126 1137 1.41 1.67
0.618 0.550 0.78 0.80 0.88
M.S. 9.1. Е Р
52.87 2 2.761 0.073
222.80 1 11.633 0.001
17.56 2 0.917 0.406
19.15 51
38394.78 2 4.495 0.016
10938.61 1 1.281 0.263
1224.78 2 0.143 0.867
8541.13 51
mainly to multiannual cycles of rainfall

(IATASA, 1994; Gonzälez Uriarte & Orioli,
1998). On the other hand, the scarcity of
aquatic macrophytes and riparian vegetation
suggest that trophic constraints are stronger
in the Cochicö Rivulet. In this region, water
bodies inhabited by P. canaliculata generally
are quite shallow, quiet and turbid, with low
Na*/(K* + Mg**) ratios as compared to the un-
inhabited sites (Martin et al., 2001). The
Cochicö Rivulet is a less suitable habitat for
these snails than the other two sites.

The lower maximum and maturity sizes in
Cochicö Rivulet were probably due to lower
growth rates, although the detrimental influ-
ence of recurrent disturbance on longevity
cannot be ruled out. The low food availability,
and not the temperature, was probably the

160 MARTIN & ESTEBENET

growth limiting factor, because laboratory
snails from this source showed the highest
growth rates during most of the pre-reproduc-
tive period, and summer water temperature
was higher at this site. Minimum female re-
productive sizes of approximately 25 mm
have been recorded for P. canaliculata grow-
ing under very different densities, food and
temperature conditions (Martin, 1986; Es-
tebenet & Cazzaniga, 1992; Tanaka et al.,
1999), age at first oviposition being hence
highly dependent on growth rate. In this study,
the first spawn of each cohort was observed
at ages between 80 and 99 days, a very early
age as compared to other cohorts at 25°C
(Estebenet & Cazzaniga, 1992, 1998), but at
39 mm shell length, indicating that maturity
depends not only on size, but that a minimum
age is also required. The slow growth at the
Cochicö site and the lack of genetic interpop-
ulational variation in age and size at maturity
suggest that differences in size of field copu-
lating snails are not due to an earlier matura-
tion in Cochicö. In this population, the mini-
mum age is probably attained before the
minimum size, whereas in the Curamalal the
minimum size is reached before the minimum
age.

Hatching in P. canaliculata occurs by me-
chanical fracture of the calcareous egg shell,
after consuming all the perivitelline egg re-
serve, at which stage the length of the new-
born equals the diameter of the egg (Es-
tebenet & Cazzaniga, 1993; Heras et al.,
1998). Estebenet & Cazzaniga (1993) re-
ported that the egg size variation among
clutches from the same pond was greater
than interpond variation. In the present study,
on the contrary, the intersite variation was
greater than intrasite variation. The size of
newborn was the lowest in Cochicö Rivulet,
probably limited by the small size of females,
because newborns from laboratory reared,
big-sized females from this source were big-
ger and similar in size to those from the other
two sources. Female size in the permanent
ponds studied by Estebenet & Cazzaniga was
greater than in Cochico and probably was not
a constraint for egg size.

Under homogeneous conditions, snails
from the three sites showed variation in re-
productive, growth and survival patterns. Fe-
males from Cochicó showed a genetically
fixed tendency to lay a higher number of eggs
per week and packed in bigger clutches than
those from Alsina. This higher oviposition rate
implies a higher reproductive allocation asso-

ciated with a higher mortality rate, probably
owing to a competence between somatic
maintenance and reproduction only (Jokela,
1997), because growth was almost null during
most of the reproductive period.

The different patterns of survival, somatic
and reproductive allocation in the three popu-
lations could be considered as parts of differ-
ent life-history strategies, because they
showed a genetic component and are corre-
lated in an adaptive way to different environ-
mental conditions (Calow, 1978, 1983). Under
the thermal regime of the study zone, P
canaliculata is iteroparous, with two or more
reproductive periods limited to the warmer
months (Estebenet & Cazzaniga, 1992,
1993). Fast prematurity growth and a higher
reproductive output, despite the detrimental
effect of the latter on survivorship and future
egg production, seems to be adaptive in the
Cochicö Rivulet. At this site, where frequent
and unpredictable disturbance events cause
mass mortality episodes, probably only a few
mature snails survive to reproduce in a sec-
ond season. In contrast, in the Alsina Lake,
where the frequency and magnitude of the
disturbances are lower, a restrained repro-
ductive allocation can led to more reproduc-
tive seasons, thus maximizing the lifetime egg
production.

The adaptive value of early maturation and
high reproductive effort depends on a lower
survival of parents relative to their offspring
(Calow, 1983). Adults of Pomacea spp. from
ephemeral but more predictable water bodies
aestivate (Burky, 1974; Kushlan, 1975; Don-
nay & Beissinger, 1993), but this behavior has
not been described for P. canaliculata. Al-
though water loss and susceptibility are prob-
ably higher in small snails (Turner, 1996), their
chances of fitting in humid microhabitats are
also higher. Pomacea canaliculata mobility
and feeding are highly reduced when water
depth is lower than shell diameter (Wada,
1997), so post-drought survival is probably
negatively related to size. Conductivity peaks
and drought episodes shorter than the lapse
between oviposition and hatching (about 17
days in this region) will affect parent survival
without harmful effects on their aerial eggs. In-
deed, despite their normal nocturnal spawn-
ing behavior (Schnorbach, 1995; Albrecht et
al., 1996), P. canaliculata females exposed to
air for several hours frequently spawn, even
during light hours (Cazzaniga, 1981; pers.
obs.), probably increasing the chances of
leaving progeny. Scouring by floods into the

LIFE-HISTORY VARIATION IN POMACEA 161

salty Cochicö Lake is probably a major source
of catastrophic mortality, specially for big
snails, less able to withstand fast flow and to
find in-stream flow refuges of adequate size
(Calow, 1983; Dussart, 1987; Johnson &
Brown, 1997). On the other hand, clutches
can endure submersion for several days with-
out deleterious effects on the embryos (pers.
obs.).

Apart from local adaptation, genetic drift is
another probable cause of interpopulation ge-
netic variation. Encadenadas del Oeste basin
was once part of an allocthonous river running
through the present Vallimanca River into the
Salado River (Fig. 1). The chain of lakes was
originated in its paleo-valley when discharge
diminished after the end of the last glacial age
and climate became increasingly arid in the re-
gion (Frenguelli, 1945; Malagnino, 1989). Fos-
sil records of P. canaliculata are common in the
Salado basin (Camacho, 1966; Dangavs &
Blasi, 1992), and the species was probably
widespread in it when subtropical conditions
advanced to southern Buenos Aires Province
(8-6 Kyr BP; Iriondo, 1994). Since then, local
populations probably became increasingly
isolated because of increasing habitat discon-
tinuity. The P. canaliculata populations in the
studied sites do not seem to have originated
from independent arrivals from a genetically
heterogeneous source, rendering founder ef-
fects unimportant.

The maintenance of genetic differentiation
among the studied populations is noteworthy,
taking into account that their isolation was not
complete in the recent past. The three study
sites secularly connected each other in most
peaks of the multiyear rainfall cycle, espe-
cially when Alsina and Cochicö lakes merge
and both present freshwater conditions
(IATASA, 1994; Gonzalez Uriarte & Orioli,
1998). However, at present the spread of P
canaliculata in this geographic region seems
to be very slow, even within the same stream
(Martin et al., 2001) and gene flow is probably
strongly unidirectional, mostly due to down-
stream drift. Since 1992, a roadway embank-
ment with sluice gates divided Alsina and
Cochico lakes, probably increasing their iso-
lation. On the other hand, population size in
the Cochicö Rivulet was probably very small,
due to the small size of the habitable section
and low food availability. These factors proba-
bly account for the persistence of the genetic
differentiation provoked by the strong selec-
tive pressures, even in the absence of total
habitat discontinuity.

Asian pest managers have showed deep
concern about the specific identity of apple
snails that invaded paddy fields (Wada, 1997;
Cowie, in press). However, close and recently
isolated populations of P. canaliculata from
the same basin showed genetically different
life-history strategies, so even populations
from the same species could require different
control measures. The situation is compli-
cated by the fact that most Asian countries
suffered repeated introductions of apple
snails (Litsinger & Estano, 1993; Wada, 1997;
Cowie, in press), probably from distant geo-
graphic regions, among which genetic in-
traspecific variation is expected to be even
greater. Moreover, since the early 1980s the
control and culture practices in paddy fields
have been exerting new and strong selective
pressures that are probably already shaping
the major features of Pomacea life history.

ACKNOWLEDGMENTS

We wish to express our gratitude to Natalia
Pizani and Eduardo Albrecht for their assis-
tance in field work. This study was funded with
grants by CONICET (“Consejo Nacional de
Investigaciones Cientificas y Técnicas”) and
“Agencia Nacional de Promociön Cientifica y
Técnica”, Argentina. ALE is a researcher in
CONICET.

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LIFE-HISTORY VARIATION IN POMACEA 163

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Revised ms. accepted 9 August 2001

MALACOLOGIA, 2002, 44(1): 165-168

CHROMOSOMES OF PISIDIUM COREANUM (BIVALVIA: VENEROIDA:
CORBICULOIDEA: PISIDIIDAE), AKOREAN FRESHWATER CLAM

Gab-Man Park,' Tai-Soon Yong,’ Kyung-il Im,* & John В. Burch?

ABSTRACT

Polyploidy is reported as occurring in Pisidiidae, being found in Pisidium coreanum from
Korea. The chromosome number is n = 95 and 2n = 190. Because of the large number of its chro-
mosomes, P. coreanum is obviously a polyploid species. The basic chromosome number for the
superfamily Corbiculoidea is X = 18 and X = 19. The polyploid condition found in P. coreanum
probably originally resulted from a single event. Once the polyploid condition was reached, the

stability of this number has been maintained.

Key words: Pisidium coreanum, chromosome, Pisidiidae, polyploid, Korea.

INTRODUCTION

The bivalve superfamily Corbiculoidea has
two families of special interest, Corbiculidae
and Pisidiidae, both with wide geographical
and ecological distributions (Clarke, 1973;
Burch, 1975; Kuiper, 1987; Kwon & Park,
1993). The Corbiculoidea are represented in
Korea by six species of the Corbiculidae and
two species of the Pisidiidae (Kwon et al.,
1993). The Corbiculidae are Asian in origin,
but in relatively recent times they have been
spread to many other parts of the world
through human commerce. Particularly in
Asia, corbiculid clams are an important food
source, not only for wildlife, including fish, but
also for humans. Sphaeriid clams, on the other
hand, are not economically important, other
than providing an important link in various
aquatic food chains. But from a biological
standpoint, they are significant because of
their peculiar lifestyles. They live in many dif-
ferent habitats and environments, and some
species seem to be nearly cosmopolitan
(Burch, 1975). All the species presumably are
hermaphroditic, with the same individuals pro-
ducing both sperm and ova, although direct
observations have been made for only a few
species. Fertilization is internal, and the young
are brooded in special gill chambers of the par-
ent, in some species until the young are rela-
tively large (Park & Kwon, 1993). However, ex-
actly how fertilization and development take
place, that is, whether by cross- or self-fertil-
ization, or by some type of parthenogenesis, is

not Known, nor is it Known how polyploidy
came about (i.e., are the species autopoly-
ploids or allopolyploids, and in the later case
segmental or genomic?). In the marine bivalve
genus Lasaea, which shares many similarities
with the corbiculoidean clams (O Foighil &
Smith, 1995), polyploidy is also a common oc-
currence, and here, at least in the polyploid
clones, parthenogenetic development, trig-
gered by autosperm (without syngamy) is the
rule (O Foighil & Thiriot-Quiévreux, 1991). Our
preliminary observations on gametogenesis in
sphaeriid clams suggest that it is likely that
some, perhaps many, species of corbicu-
loidean clams have similar reproductive
strategies.

Many species are known to be capable of
propagation by self-fertilization when the
more normal biparental mating behavior is
prevented. In such groups, a large amount of
polyploidy might be expected, but chromo-
some surveys have shown that this is not the
case. Natural polyploidy has also been ob-
served in Argopecten purpuratus (Alvarez &
Lozada, 1992), Sphaerium striatinum (Lee,
1999), in Lasaea spp. (Thiriot-Quiévreux et
al., 1988, 1989; O Foighil & Thiriot-Quiévreux,
1999) and in Corbicula spp. (Park et al.,
2000). The first Corbiculoidea clams in which
polyploidy was detected was Corbicula (Cor-
biculina) leana (Okamoto & Arimoto, 1986).
The chromosome numbers have been deter-
mined for several other species belonging to
the order Corbiculoidea (Table 1). In these,
the chromosome number varies from n = 12 to

‘Department of Parasitology, Yonsei University College of Medicine, Seoul 120-752, Korea; tsyong212@yumc.yonsei.ac.kr
Museum of Zoology and Department of Biology, College of Literature, Science and the Arts, and School of Natural Re-
sources, University of Michigan, Ann Arbor, Michigan 48109, USA

166

TABLE 1. Chromosome numbers in Corbiculoidea.

PARKETAL.

Species Chromosome number References
Corbicula fluminea 54 (3n) Park et al., 2000

C. papyracea 54 (3n) Park et al., 2000

C. leana 54 (3n) Okamoto & Arimoto, 1986
C. colorata 38 (2n) Park et al., 2000

C. japonica 38 (2n) Okamoto & Arimoto, 1986
C. sandai 36 (2n) Okamoto & Arimoto, 1986
“C. leana” 24 Nadamitsu & Kanai, 1978
Musculium securis approx. 247 Burch et al., 1998
Sphaerium corneum 36 (2n) Keyl, 1956

S. occidentale approx. 209 Burch et al., 1998

S. striatinum approx. 68-98 Woods, 1931

S. striatinum approx. 152 Lee, 1999

Pisidium casertanum approx. 150, 180 Barsiene et al., 1996

P. casertanum approx. 190 Burch et al., 1998

P. coreanum 190 (2n) Present study

n = 76. The chromosome number of C. flu-
minea, C. papyracea and C. leana suggests
that these species may be members of a 3n
species that has been established by poly-
ploid evolution. The chromosome numbers of
Sphaeriidae have been reported for five
species (Table 1). The chromosome numbers
obtained in the Sphaeriidae are all very large
(over 150 mitotic chromosomes), except for
the European S. corneum (2n = 36). This sug-
gests that the large numbers of chromosomes
in this family represent polyploids.

This study presents the chromosome
analysis of P. coreanum based on mitotic
metaphase chromosomes.

MATERIALS AND METHODS

Twenty-one specimens of Р coreanum
were collected from a spring pond located in
Bongmung-ri, Chunchon-city, Kangwon-do,
Korea, from June to September 2000, and ex-
amined shortly after collection.

Chromosome preparations were made
from gonadal tissues by the standard air-dry-
ing method. The live specimens were set
aside for one week in a petri-dish containing
10 ml of distilled water with 0.3 ml of 0.05%
colchicine solution. The treated tissues were
dissected and minced with needles in a hypo-
tonic 0.01% NaCl solution. Separated cells
were collected by centrifugation at 930 g for
10 min. These cells were fixed in freshly
mixed modified Carnoy’s fixative (three parts
methyl alcohol and one part glacial acetic
acid). The supernatant was replaced by fresh
fixative. The centrifugation (930 g, 10 min)
was repeated two more times. A drop of the

cell suspension was then pipetted by a micro-
hematocrit capillary tube and dropped onto a
clean slide glass pre-cooled to 4°C. The cells
on the slide were air-dried and then stained
for 10 min with 4% Giemsa (Gurr R66) solu-
tion made up in 0.1 M phosphate buffer, pH
7.0. The prepared slides were observed
under an Olympus (BX50F-3) microscope
with a 100X (n.a. 1.25) oil immersion objective
and a 10X ocular. Voucher specimens of the
shells used in this investigation have been
placed in the Department of Parasitology,
Yonsei University College of Medicine, Korea.

RESULTS

The mean shell size determined in this
study consisted of a measured shell length of
4.2 mm, shell height of 4.1 mm, and shell
breath of 2.0 mm. Twelve individuals were ex-
amined for chromosomes. Eighteen cells
clearly had 190 chromosomes. Chromosome
numbers of n = 95 and 2n = 190 were counted
(Fig. 1). Ninety-five bivalents were observed
during late prophase (diakinesis) (Fig. 1A).
The chromosome types of this species con-
sisted of metacentric, submetacentric and te-
locentric chromosomes as shown (Fig. 1B).
However, this chromosome figure was not
sufficient for analyzing karyotypes, not only
because of the large numbers but also the
small size. The longest dimension of the
largest metaphase bivalent was only 3.6 um.

DISCUSSION

Polyploidy is the multiplication of the normal
chromosome number of an organism. Be-

CHROMOSOME OF PISIDIUM COREANUM 167

A
pp"
tree As
и
% x ? |
eo А ña *
„+ Y AY a
d ’ №,
ARCHE >
< Y e Y @ }
Re de > >» “у
tue tas
& L En JS
wh o/s
ps un
a

FIG. 1. Chromosomes of Pisidium coreanum. A, diakinesis (n = 95). B, metaphase chromosomes (2n = 190)

Scale bars indicate 5 um.

cause of the inability of a newly derived poly-
ploid to breed with its sibling diploid it is usu-
ally chromosomally sterile. Accordingly, poly-
ploidy is a method, and the only clearly
established one, for instantaneous speciation
(Wagner et al., 1993). The occurrence of
triploid chromosomes in three species of the
genus Corbicula has been reported (Okamoto
& Arimoto, 1986; Park et al., 2000) (Table 1).
From the numbers found in the three species
of Corbicula, each of these species is poly-
ploid. The family Pisidiidae contains three sub-
families, the Pisidiinae, Sphaeriinae and Eu-
perinae. In the Sphaeiinae, four species has
been studied, Sphaerium corneum (Keyl,
1956), S. occidentale (Burch et al., 1998), S.
striatinum (Woods, 1931; Lee, 1999) and
Musculium securis (Burch et al., 1998), and
the mitotic chromosome numbers of these
species have been reported ranging from 36 to
approximately 152. In the Pisidiinae, the chro-
mosome numbers of P. casertanumis 2n = ap-
proximately 247 as counted by Burch et al.
(1998). This study, the first report of polyploidy
in Pisidiidae clams in Korea, determined that
the chromosome number of P. coreanumis 2n
= 190. Polyploidy is probably an important
method of evolution in the cosmopolitan and
common genus Pisidium, and may account for
the morphological patterns that have made
taxonomy so difficult. Most molluscan groups
are generally conservative with regard to chro-
mosomal change (Patterson, 1969). However,

the chromosome numbers are very variable
among Mytilidae and Pectinidae (Nakamura,
1985). Also, the chromosome number within
two families of the order Veneroidea, 18 to
95 pairs of chromosomes, is not constant.
The origin of polyploidy in the Pisidiidae is
still speculative. In mollusks, Burch & Huber
(1966) suggested that polyploidy came about
in the African-Near Eastern Bulinus by hy-
bridization, followed by a doubling of the chro-
mosome number, that is, allopolyploidy was
involved. Cytological evidence presented by
Goldman et al. (1983) tends to support this
conclusion. Certainly the ecological conditions
where most of the ploidy states in Bulinus
occur — the Ethiopian highlands, located near
the equator — provide the opportune physical
characteristics in which polyploid events might
be expected to occur (Patterson & Burch,
1988).

Future studies of the chromosomes of other
species of the Pisidiidae family are needed to
document the variability of the chromosome
number within this group.

LITERATURE CITED

ALVAREZ, S. & L. E. LOZADA, 1992, Spontaneous
triploidy in Chilean scallop Argopecten purpura-
tus (Lamark 1819) (Bivalvia, Pectinoidae). /nves-
tigacion Pesqueras, 37: 119-126.

BARSIENE, J., G. TAPIA & D. BARSYTE, 1996,
Chromosomes of mollusks inhabiting some

168 PARKETAL.

mountain springs of eastern Spain. Journal of
Molluscan Study, 62: 539-543.

BURCH, J. B., 1975, Freshwater sphaeriacean
clams (Mollusca: Pelecypoda) of North America.
Malacological Publications, Hamburg, Michigan.
96 pp.

BURCH, J. B. & J. M. HUBER, 1966, Polyploidy in
mollusks. Malacologia, 5: 41-43.

BURCH, J. B., С. M. PARK & Е. У. CHUNG, 1998,
Michigan’s polyploid clams [abstract]. Michigan
Academician, 30: 351-352.

CLARKE, A. H., 1973, The freshwater mollusca of
the Canadian Interior Basin. Malacologia, 13:
1-509.

GOLDMAN, M. A., Р. Т. LOVERDE & С. L. CHIRS-
MAN, 1983, Hybrid origin of polyploidy in fresh-
water snails of the genus Bulinus (Mollusca:
Planorbidae). Evolution, 37: 592-600.

KEYL, H. G., 1956, Boobachtungen über die ©-
meiose der Muschel Sphaerium corneum. Chro-
mosoma, 8: 12-17.

KUIPER, J. ©. J., 1987, Systematic rank, synonymy
and geographical distribution of Pisidium ob-
tusale, P. rotundatum and P. ventricosum. Walk-
erana, 2: 145-158.

KWON, O. K. & J. C. PARK, 1993, Studies of the
development and the spawning season of Pisid-
¡um (Neopisidium) coreanum (Bivalvia: Sphaeri-
idae). The Korean Journal of Malacology, 9:
33-38.

KWON, O. K., G. M. PARK & J. S. LEE, 1993,
Coloured shells of Korea. Academy Publishing
Company, Seoul. 221-224 pp.

LEE, T., 1999, Polyploidy and meiosis in the fresh-
water clam Sphaerium striatinum (Lamark) and
chromosome numbers in the Sphaeriidae (Bi-
valvia, Veneroida). Cytologia Tokyo, 64: 247-
252.

NADAMITSU, S. & T. KANAI, 1978, On the chro-
mosomes of the three species in two families in
freshwater Bivalvia. Bulletin Hiroshima Women’s
University, 10: 1-5.

NAKAMURA, H. K., 1985, A review of molluscan cy-
togenetic information based on the CISMOCH-
Computerized Index System for Molluscan Chro-
mosomes. Bivalvia, Polyplacophora and

_ Cephalopoda. Venus, 44: 193-225.

O FOIGHIL, D. & M. J. SMITH, 1995, Evolution of
asexuality in the cosmopolitan marine clam

_ Lasaea. Evolution, 49: 140-150.

O FOIGHIL, D. & C. THIRIOT-QUIEVREUX, 1991,

Ploidy and pronuclear interaction in northeastern
Pacific Lasaea clones (Mollusca: Bivalvia). Bio-
‚ logical Bulletin, 181: 222-231. _

O FOIGHIL, D. & C. THIRIOT-QUIEVREUX, 1999,
Sympatric Australian Lasaea species (Mollusca,
Bivlavia) differ in their ploidy levels, reproductive
modes and developmental modes. Zoology Jour-
nal and Linnaeous Society, 127: 477-494.

OKAMOTO, A. & B. ARIMOTO, 1986, Chromo-
somes of Corbicula japonica, C. sandai and C.
(Corbiculina) leana (Bivalvia: Corbiculidae).
Venus, 45: 194-202.

PARK, J. C. & O. K. KWON, 1993, Studies on the
development and the spawning season of Pisid-
¡um (Neopisidium) coreanum (Bivalvia: Spheri-
idae). The Korean Journal of Malacology, 9:
33-38.

PARK, G. M., T. S. YONG., K. IM & E. Y. CHUNG,
2000, Karyotypes of three species of Corbicula
(Bivalvia: Veneroida) in Korea. Journal of Shell-
fish Research, 19: 979-982.

PATTERSON, C. M., 1969, Chromosomes of mol-
luscs. Proceedings of the Symposium on Mol-
lusca, Marine Biological Association India, 2:
635-689.

PATTERSON, C. M. & B. BURCH, 1988, Chromo-
somes of pulmonate mollusks. Pp 171-217, in: v.
FRETTER & J. PEAKE, eds., Pulmonates, Vol. 2A,
Systematics, evolution and ecology. Academic
Press, London, New York and San Francisco.
540 pp. |

THIRIOT-QUIEVREUX C., A. M. INSUA POMBO &
P. ALBERT, 1989, Polyploïdie chez un bivalve in-
cubant, Lasaea rubra (Montagu). Comptes Ren-
dus de Séances d’Academie de Science, Paris,
308: 115-120.

THIRIOT-QUIEVREUX C., J. SOYER, F. de
BOVEE & P. ALBERT, 1988, Unusual chromo-
some complement in the brooding bivalve
Lasaea consanguinea. Genetica, 76: 143-151.

WAGNER, РВ. Р. М. P. MAGUIRE & В. L.
STALLINGS, 1993, Variation in chromatin organi-
zation and amount. Pp. 269-272, in: Chromo-
somes: a synthesis. Wiley-Liss. New York.

WOODS, F. H. 1931, History of the germ cells in
Sphaerium striatnum (Lam.). Journal of Morphol-
ogy and Physiology, 51: 545-595.

Revised ms. accepted 15 August 2001

MALACOLOGIA

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VOL. 44, NO. 1 MALACOLOGIA 2002

CONTENTS

ANDREA C. ALFARO & ANDREW G. JEFFS
Small-scale Mussel Settlement Patterns Within Morphologically Distinct

Substrata at Ninety Mile Beach, Northern New Zealand................ 1
EUGENE V. COAN
The Eastern Pacific Recent Species of the Corbulidae (Bivalvia) ......... 47

ROBERT T. DILLON, JR., & ANDREW J. REED
A Survey of Genetic Variation at Allozyme Loci among Goniobasis
Populations Inhabiting Atlantic Drainages of the Carolinas .............. 23
VASILIS K. DIMITRIADIS & VASILIKI KONSTANTINIDOU
Origin of the Excretory Cells in the Digestive Gland of the Land Snail
ESTU Nike tanec Nene М а 145
SINOS GIOKAS & MOYSIS MYLONAS
Spatial Distribution, Density and Life History in Four Albinaria Species
(Gastropoda. Pulmonata, CGlausilidae) 2... 2... ...... 33
GENNADY M. KAMENEV
Genus Parvithracia (Bivalvia: Thraciidae) with Descriptions of a New
Subgenus and Two New Species from the Northwestern Pacific ......... 107
PABLO R. MARTIN & ALEJANDRA L. ESTEBENET
Interpopulation Variation in Life-history Traits of Pomacea canaliculata
(Gastropoda: Ampullariidae) in Southwestern Buenos Aires Province,

ATOME ce RE N See ne ee me: 153
LUIS A. MERCADO, SERGIO H. MARSHALL, & GLORIA M. ARENAS
Detection of Phenoloxidase (PO) in Hemocytes of the Clam Venus antiqua ТИ

GAB-MAN PARK, TAI-SOON YONG, KYUNG-IL IM, & JOHN В. BURCH
Chromosomes of Pisidium coreanum (Bivalvia: Veneroida: Corbiculoidea:
Pisidiidae) a /KoreanFreshwatenClamit ar meer a IR: 165
ANATOLY A. VARAKSIN, EUGENIA А. PIMENOVA, GALINA $. VARAKSINA, &
LYDIA T. FROLOVA
Localization of Nadph-diaphorase-positive Elements in the Intestine of the
Mussel Crenomytilus grayanus (Mollusca: Bivalvia) ................... 135

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VOL. 44, NO. 1 MALACOLOGIA

CONTENTS

ANDREA C. ALFARO & ANDREW G. JEFFS

Small-scale Mussel Settlement Patterns Within Morphologically Distinct

Substrata at Ninety Mile Beach, Northern New Zealand................
LUIS A. MERCADO, SERGIO H. MARSHALL, & GLORIA M. ARENAS

Detection of Phenoloxidase (PO) in Hemocytes of the Clam Venus antiqua
ROBERT T. DILLON, JR., & ANDREW J. REED

A Survey of Genetic Variation at Allozyme Loci among Goniobasis

Populations Inhabiting Atlantic Drainages of the Carolinas ..............
SINOS GIOKAS & MOYSIS MYLONAS

Spatial Distribution, Density and Life History in Four Albinaria Species

(Gastropoda, Pulmonata, Clausiliidae) .. 2... cic. 60 ее, oe Hee
EUGENE V. COAN

The Eastern Pacific Recent Species of the Corbulidae (Bivalvia) .........
GENNADY M. KAMENEV

Genus Parvithracia (Bivalvia: Thraciidae) with Descriptions of a New

Subgenus and Two New Species from the Northwestern Pacific .........
ANATOLY A. VARAKSIN, EUGENIA A. PIMENOVA, GALINA S. VARAKSINA, &
LYDIA Т. FROLOVA

Localization of Nadph-diaphorase-positive Elements in the Intestine of the

Mussel Crenomytilus grayanus (Mollusca: Bivalvia) ...................
VASILIS K. DIMITRIADIS & VASILIKI KONSTANTINIDOU

Origin of the Excretory Cells in the Digestive Gland of the Land Snail

IN A ene de AAA AO
PABLO В. MARTÍN 8 ALEJANDRA L. ESTEBENET

Interpopulation Variation in Life-history Traits of Pomacea canaliculata

(Gastropoda: Ampullariidae) in Southwestern Buenos Aires Province,

ее ee
GAB-MAN PARK, TAI-SOON YONG, KYUNG-IL IM, & JOHN B. BURCH

Chromosomes of Pisidium coreanum (Bivalvia: Veneroida: Corbiculoidea:

Pisidiidae), a Korean Freshwater Clam ....::......:...:.:.,442 02%

2002

17

107

135

145

153

165

J. A. ALLEN

Marine Biological Station
Millport, United Kingdom
jallen @ udcf.gla.ac.uk

Е. Е. BINDER
Museum d’Histoire Naturelle
Geneve, Switzerland

P. BOUCHET

Museum National d’Histoire Naturelle
Paris, France

bouchet @cimrs1.mnhn.fr

P. CALOW
University of Sheffield
United Kingdom

R. CAMERON

Sheffield

United Kingdom

R.Cameron @ sheffield.ac.uk

J. G. CARTER
University of North Carolina
Chapel Hill, U.S.A.

MARYVONNE CHARRIER

Universite de Rennes

France

Maryvonne. Charrier @univ-rennes1.fr

R. H. COWIE
University of Hawaii
Honolulu, HI., U.S.A.

A. H. CLARKE, Jr.
Portland, Texas, U.S.A.

B. C. CLARKE
University of Nottingham
United Kingdom

R. DILLON
College of Charleston
SC, U.S.A.

C. J. DUNCAN
University of Liverpool
United Kingdom

D. J. EERNISSE
California State University
Fullerton, U.S.A.

E. GITTENBERGER

Rijksmuseum van Natuurlijke Historie
Leiden, Netherlands

sbu2eg @rulsfb.leidenuniv.de

F. GIUSTI
Universita di Siena, Italy
giustif @ unisi.it

2002
EDITORIAL BOARD

А. N. GOLIKOV
Zoological Institute
St. Petersburg, Russia

S. J. GOULD

Harvard University
Cambridge, Mass., U.S.A.

A. V. GROSSU
Universitatea Bucuresti
Romania

T. HABE
Tokai University
Shimizu, Japan

R. HANLON
Marine Biological Laboratory
Woods Hole, Mass., U.S.A.

G. HASZPRUNAR

Zoologische Staatssammlung Muenchen

Muenchen, Germany

haszi @zi. biologie. uni-muenchen.de

J. M. HEALY

University of Queensland
Australia

jhealy O zoology.uq.edu.au

D. M. HILLIS
University of Texas
Austin, U.S.A.

K. E. HOAGLAND

Council for Undergraduate Research

Washington, DC, U.S.A.
Elaine Ocur. org

B. HUBENDICK
Naturhistoriska Museet
Goteborg, Sweden

S. HUNT
Lancashire
United Kingdom

R. JANSSEN
Forschungsinstitut Senckenberg,
Frankfurt am Main, Germany

М. $. JOHNSON

University of Western Australia
Nedlands, WA, Australia
msj@cyllene.uwa.edu.au

R. N. KILBURN
Natal Museum
Pietermaritzburg, South Africa

M. A. KLAPPENBACH

Museo Nacional de Historia Natural

Montevideo, Uruguay

MCZ
IBRARY

HATVARD

ıxiVERSITY

J. KNUDSEN
Zoologisk Institut Museum
Kobenhavn, Denmark

C. LYDEARD

University of Alabama
Tuscaloosa, U.S.A.
clydeard @ biology.as.ua.edu

C. MEIER-BROOK
Tropenmedizinisches Institut
Tubingen, Germany

H. K. MIENIS
Hebrew University of Jerusalem
Israel

J. Е. MORTON
The University
Auckland, New Zealand

J. J. MURRAY, Jr.
University of Virginia
Charlottesville, U.S.A.

R. NATARAJAN
Marine Biological Station
Porto Novo, India

DIARMAID O’FOIGHIL
University of Michigan
Ann Arbor, U.S.A.

J. OKLAND
University of Oslo
Norway

T. OKUTANI
University of Fisheries
Tokyo, Japan

W. L. PARAENSE
Instituto Oswaldo Cruz, Rio de Janeiro
Brazil

J. J. PARODIZ
Carnegie Museum
Pittsburgh, U.S.A.

R. PIPE

Plymouth Marine Laboratory
Devon, United Kingdom
RKPI@wpo.nerc.ac.uk

J. P. POINTIER

Ecole Pratique des Hautes Etudes
Perpignan Cedex, France
pointier @ gala.univ-perp.fr

М.Е. PONDER
Australian Museum
Sydney

QUEZY
Academia Sinica
Qingdao, People's Republic of China

D. G. REID
The Natural History Museum
London, United Kingdom

S. G. SEGERSTRÄLE
Institute of Marine Research
Helsinki, Finland

A. STANCZYKOWSKA
Siedice, Poland

F. STARMÜHLNER
Zoologisches Institut der Universitat
Wien, Austria

У. |. STAROBOGATOV
Zoological Institute
St. Petersburg, Russia

J. STUARDO
Universidad de Chile
Valparaiso

C. THIRIOT

University P. et M. Curie
Villefranche-sur-Mer, France
thiriot @ obs-vifr.fr

S. TILLIER
Museum National d'Histoire Naturelle
Paris, France

J.A.M. VAN DEN BIGGELAAR
University of Utrecht
The Netherlands

N. H. VERDONK
Rijksuniversiteit
Utrecht, Netherlands

H. WAGELE

Ruhr-Universitat Bochum

Germany

Heike. Waegele O ruhr-uni-bochum.de

ANDERS WAREN
Swedish Museum of Natural History
Stockholm, Sweden

B. R. WILSON
Dept. Conservation and Land Management
Kallaroo, Western Australia

H. ZEISSLER
Leipzig, Germany

A. ZILCH
Forschungsinstitut Senckenberg
Frankfurt am Main, Germany

MALACOLOGIA, 2002, 44(2): 175-222

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC
(OCTOPODIDAE, CEPHALOPODA)

Bent Muus

Zoological Museum, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen,
Denmark; bbisballe @ zmuc.ku.dk

ABSTRACT

Profound confusion among deep-water octopods of the genera Bathypolypus and Benthocto-
pus stems from misidentifications of the Atlantic Bathypolypus arcticus (Prosch, 1849), which is
here shown to consist of at least two allopatric species—the nominal form is a truly arctic
species; the other, Octopus bairdii Verrill, 1873, is a boreal cold-water form, which is reinstated
as a Bathypolypus. A third species, Bathypolypus pugniger, n. sp., of uncertain systematic posi-
tion, occurs in a narrow belt along the southern limit of B. arcticus.

Synonyms are discussed, and evidence is advanced that all records of Benthoctopus pisca-
torum (Verrill, 1879) are erroneous and that the type specimen is identical with В. bairdii.

Using an amended generic diagnosis, a critical survey of all Bathypolypus species is given. Six
species, all Atlantic, are recognized. Distribution and habitats are given, and a key and tables

provided to facilitate identifications.

Key words: Bathypolypus, Benthoctopus, North Atlantic, Bathypolypodinae (Cephalopoda).

INTRODUCTION

The octopod genera Bathypolypus and
Benthoctopus comprise deep-water species
that have caused much taxonomic confusion
since Victor Prosch described the first species,
Octopus arcticus, from southwest Greenland
waters in 1849.

The two genera were established in a rather
casual way by Grimpe (1921), the respective
type species being Bathypolypus arcticus
(Prosch, 1849) and Benthoctopus piscatorum
(Verrill, 1879). Robson (1924b, 1927) defined
the two genera and then treated all known
species in his monograph on octopods (1932).

From Robson's work on B. arcticus, supple-
mented by findings of Hoyle (1886), Pfeffer
(1908), Joubin (1920), Grimpe (1933), Kon-
dakov (1936), Adam (1939), Bruun (1945),
Jaeckel (1958), Kumpf (1958), Macalaster
(1976), Perez-Gandaras & Guerra (1978),
Nesis (1987), and Voss (1988b), B. arcticus
would appear to be a highly variable species
with a huge geographic range in the North At-
lantic. lt seems in fact to be the most abun-
dant bottom-living octopod on the upper half
of the continental slopes from Florida to West
Greenland, along both sides of the ridge be-
tween Greenland and Scotland and from the
Bay of Biscay to the Barents Sea, Svalbard,
and even the Kara Sea.

175

In a report on the cephalopods from the
Danish Godthaab Expedition 1928 to Davis
Strait and Baffin Bay (Muus, 1962), | dis-
cussed specimens of B. arcticus from near
the type locality. This species was easily iden-
tified by means of the type material т ZMUC.
However, | also described a new, rather abun-
dant species, Bathypolypus proschi, which in
spite of a superficial similarity diverged from
B. arcticus, especially in eye size, ligula,
radula, and spermatophores.

When years later | inspected the Norwegian
collections, | was baffled to find B. arcticus
only in the arctic material and B. proschi,
along the Norwegian West coast, labelled as
В. arcticus. lt dawned upon me that there are
still serious identification problems, not only
with B. arcticus but, as | would learn, also with
other species of the genera Bathypolypus and
Benthoctopus, and that a revision was badly
needed.

Robson (1932) expressed the need for
much larger collections to settle the questions
left by his revision. Fortunately, growing trawl
and dredge activity along the continental
slopes has procured substantial additional
material from most parts of the North Atlantic.
Thus, | have had over 600 specimens at my
disposal and ample opportunity to inspect rel-
evant type material and most of the speci-
mens treated by previous authors to settle

176 MUUS

what seemed to be a case of confusion
among sibling species.

MATERIAL

The main revision concerns North Atlantic
material deposited in various museums as
species placed in one or the other ofthe gen-
era Bathypolypus and Benthoctopus, supple-
mented with recently collected material from
Faroese and Icelandic waters (the BIOFAR
and BIOICE projects respectively) and from
the deep-sea prawn fisheries off southwest-
ern Greenland. The material is listed in Ap-
pendix 1.

METHODS

Measurements, counts and calculation of
indices were performed according to the stan-
dard for descriptive characters of octopods
given by Roper & Voss (1983). The soft body
and variable state of preservation, however,
make many measurements of octopods sub-
jective, and repeated measurements of the
same specimens often gave results deviating
5-10%. If different persons perform the mea-
surements, the deviation may be even
greater, which | noticed when | remeasured
specimens treated by other authors. Meristic
characters, such as number of suckers and
lamellae copulatoriae of the hectocotylus,
were often more reliable. Also the eye lens
was useful, being a solid structure, and sup-
plementing the dorsal mantle length as a
standard of size. The lens was extracted with
tweezers through a slit cut horizontally in the
lower part of the eyeball. Remnants of the
darkish primary cornea suspending the lens
at “equator” was removed, and the diameter
was measured gently with a calipers, or under
a microscope.

Due to the slightly subjective manner in
which many measurements of octopods have
to be conducted, all graphs and figures com-
paring species are based on my own figures
to minimize bias. Only in Figure 9, a few
meristic data from Macalaster (1976) are in-
cluded.

For convenience, the standard measure-
ments used in this work are presented below.
In addition to conventional indices, | have
added some new indices that might be useful
in taxonomic work on other octopod genera.

Note that as a slight deviation from stan-

dards as given by Roper & Voss (1983), ligula
and calamus length are measured from the
center of last sucker, not from its rim. It is ac-
curate and eases the use of calipers. Mantle
length is always dorsal ML (midpoint between
eyes to mantle apex).

Measurements Indices
AL: Arm length ALI: % of ML
CaL: Calamus length Call: % of LigL
ED: Diameter of eye ball EDI: % of ML
HcL: Hectocotylized arm length HcLl: % of ML
HW: Head width HWI: % of ML
LD: Lens diameter LDEDI: LD % of ED (Muus)
LigL: Ligula length LigLl: % of HcL
ML: Mantle length MLTLI: % of TL
MW: Mantle width MWI: % of ML
OAI: HcL % of 3. left AL
SD: Sucker diameter (largest) SDI: % of ML

SDLDI: SD % of LD (Muus)
SDEDI: SD % of ED (Muus)
SpLI: % of ML

SpRl: % of SpL

SpWI: % of SpL

SpL: Spermatophore length
SpRL: Sperm reservoir length
SpW: Spermatophore width
TE: Total length
WD: Web depth

(web sectors: A,B,C,D,E)

WDI: % of longest armpair
WDMI: WD % of ML

Counts
LamC: Laminae copulatoriae LamCl: LamC % of SHcC (Muus)
SHcC: Suckers on hectocotylized arm

Acronyms of museum collections used:
BMNH _ British Museum of Natural History, London, England
IMNH Icelandic Museum of Natural History, Reykjavik, Iceland
IRSNB Institut Royal des Sciences Naturelles de Belgique,
Brussels, Belgium
MNHN Museum National D'Histoire Naturelle, Paris, France
MNHT Museum of Natural History, Torshavn, Faroe Islands,
Denmark
TMDZ Tromso Museum, Department of Zoology, Norway
USNM National Museum of Natural History, Washington, DC,
USA
ZIASP Zoological Institute, Academy of Sciences,
St. Petersburg, Russia
ZMUB Zoological Museum University of Bergen, Norway
ZMUC Zoological Museum University of Copenhagen, Denmark
ZMUO Zoological Museum University of Oslo, Norway

REDESCRIPTION OF BATHYPOLYPUS
ARCTICUS (PROSCH, 1849)

The earliest recognition of octopodids in
Greenland was by Fabricius (1780: 353). His
Sepia octopodia is, however, at most a nomen
dubium, because the meagre description fits
any of the now known species, and no type
material exists. Sepia groenlandica Dewhurst
(1834: 263) is a nomen nudum, because no
description was given, and Octopus granula-

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC

tus Möller (1843: 77) is both a nomen nudum
and a junior homonym of О. granulatus
Lamarck, 1798.

The first direct reference to B. arcticus
(under the name “Octopus granulatus”) was in
Reinhardt & Prosch’s (1846) paper on the
anatomy of Cirroteuthis mülleri, in which
some specimens were used for anatomical
comparison, and it was stated for the first time
that both species are devoid of an ink sac.
The authors mentioned that they had many
specimens of the Greenland species at their
disposal “different in sex, age and develop-
ment” [my translation].

In 1849, Prosch described Octopus arcticus
based on some of the specimens mentioned
in 1846, which were sent from west Green-
land in the early 1840s. He did not designate
a type specimen, and the lectotype, and two
paralectotypes described below were identi-
fied as prosch’s original material and marked
“types” about 1930 by the curator R. Spärck,
among the specimens that were retained in
the ZMUC.

Material Examined

ZMUC CEP-13: Lectotype of Bathypolypus
arcticus (Prosch, 1849), called “holotype” in
Kristensen & Knudsen (1983): Male, ML: 42
mm. Label: Greenland, K. M. Jörgensen, Au-
gust 26, 1841; CEP-14, paralectotype: Fe-
male, ML: about 50 mm. Label: Greenland,
K. M. Jörgensen, July 27, 1840; CEP-15,
paralectotype: The dissected parts of a male
used by Prosch (1849) for his drawings (his
figs. 1-3). Depository: ZMUC.

Further 120 specimens from arctic and sub-
arctic North Atlantic as listed in Appendix 1.

Remarks on the Type Series

The lectotype has been dissected and
some measurements are not reliable. The
funnel organ is lost, but was formerly present

177

and clearly VV-shaped. Measurements and
indices are given in Table 1.

Paralectotype CEP-14: Female, ML about
50 mm. Has been dissected, and most mea-
surements are unreliable. SD: 2-6 mm, ED:
12 mm. Crop diverticulum present containing
polychete bristles and crustacean remains.
Gonads filled with eggs.

Paralectotype CEP-15: The dissected parts
of a male used by Prosch (1849) for his figs.
1-3. Hectocotylus abnormal: only left side of
ligula developed, 14 laminae. Remnants of
typical B. arcticus spermatophores (Fig. 2d).

Material seen by Steenstrup (1856a) and
probably by Prosch (1849): Male, ML: 32 mm,
ED: 9.5 mm, LD: 3.8 mm, SD: 2.7 mm. Ligula
9 mm with 9 laminae. Funnel organ lost. Too
fragile and flabby for measurements. Label:
Holböl and Möller, Julianehab (southwestern
Greenland, probably 1840).

Three females in a jar. They have all been
dissected but agree in all recognizable char-
acters with the lectotypes (sucker size, eye-
balls, crop). | extracted the beak and radula
from one of the specimens. (Fig. 5e) Label:
Octopus arcticus, Greenland (probably 1840).

Synonymy

Octopus arcticus Prosch, 1849: 55, pl. 2, figs.
1-3

Octopus grönlandicus Dewhurst: Steenstrup
1856a: 17, pl. 2, fig. 2; 1856b: 234, pl. 11,
19. 2. 1857297. pl 3-16: 2

Octopus piscatorum Мет: Hoyle, 1886: 91

Polypus piscatorum: Russell, 1922: 7, pl. 2,
fig. 2

Polypus faeroensis Russell, 1909: 446; 1922:
5, pl. 1, fig. 1, pl. 2, figs. 4-6

Bathypolypus arcticus: Robson, 1927: 251,
figs. 1a, 2A; 1932 [in part]: 286, pl. 6, figs.
1, 2, text-figs. 53-60; Kondakov, 1936:
61, figs. 1, 2; Adam, 1939 [in part]: 9, figs.
2-4; Bruun, 1945 [in part]: 6; Muus,
1959: 224, figs. 109A, C, 115; 1962: 10,
figs. 1, 2, 4c

TABLE 1. Measurements (mm) of the B. arcticus Lectotype.

We 165

ML: 42 MLTLI: 25
HW: 27 HWI: 64
MW: (38) MWI: (90)
ED: 9 EDI: 21

LD: 5 ODED: 50
SD: 2) ЗЕ Y

LigL: 19 наи: 20
LamC: 14 SHcC: 42

AL: | Il Ш IV
r 119 112 93 114
| 1207113 112 110

WD:
A B C D E
33.5 332.305 32 29
38 35 35

178 MUUS

Benthoctopus piscatorum: Robson, 1927:
254, figs. 2B, 3; 1932: 224, figs. 31, 34,
35

Benthoctopus sasakii Robson, 1927: 257, fig.
8.

Bathypolypus faeroensis: Toll, 1985: 598, figs.
12

Diagnosis

Body egg-shaped, papillated with minute
warts often in a stellate pattern on small light
spots; head narrower than body; each eye
with a verrucose supraocular cirrus; funnel
organ double, a clear-cut VV; hectocotylus
with about 40 suckers and a deeply exca-
vated ligula with 10-16 laminae. Radula usu-
ally with irregularly multicuspid, seldom ho-
modont rachidians. Esophagus with crop
diverticulum. Total length rarely over 200 mm.

Description

Skin and Colors: In freshly caught speci-
mens, the skin is violet to purple strewn with
lighter yellowish subcircular spots with minute
warts, often surrounding a central slightly big-
ger wart in concentric rings, as observed by
Prosch (1849). Ventral side paler, with few or
no warts. Over each eye is an erectile stout
and verrucose cirrus often with adjacent
smaller protuberances. The cirrus may be
about 10 mm long but often more or less re-
tracted in preserved specimens. Color and
sculpture patterns of the skin vary with state
of preservation. In some specimens, the skin
is smooth with no evident sculpture. This is
often the case with preserved juvenile speci-
mens (ML: < 30 mm.)

Bodily Proportions: The mantle is ovoid in
outline (MWI: 65-90) slightly constricted be-
hind the eyes (Fig. 1). ML rarely over 60-70
mm., TL rarely over 200 mm. The head is nar-
rower than the mantle. Due to allometric
growth, HWI decreases from 60-85 at ML 10,
to 40-70 at ML 60.

The eyeballs are not very prominent. They
decrease in relative size with age, EDI being
28-40 at ML 10, 20-33 at ML 60 (Fig. 4). The
lens measures about 35% of the ED (LDEDI:
30-40).

The mantle aperture is 40-50% of the cir-
cumference of the neck. The funnel is free of
the mantle for about 50%.

The funnel organ is VV-shaped. Typically its
limbs appear as narrow swollen “ropes” that

are easily detached from the funnel wall. They
may vary in form (Fig. 18).

The gills are reduced and have 6-7(8) gill
filaments on each demibranch.

The brachial complex is stout, with the arm
order I. Il. Ш. IV. The ML constitutes 25-35%
of the TL, which leaves 65-75% to the
brachial complex. Arm length Index: | 231, II
218, Ill 202, IV 189 (mean of 12 specimens).

The web extends along the arms almost to
the tips. The web sectors A, B, C and D are
subequal in most specimens (WDI: 33-34 at
an average), E usually slightly shallower
(WDI: mean 30). In a beautifully preserved fe-
male with fully extended umbrella, the WDI
was: A: 38, B: 46/46, C: 46/46, D: 46/43, E: 41
(ML: 24 mm, M/K “Asterias”, Svalbard,
ZMUO).

The suckers are biserial, rather small and
well spaced, SDI: 6.5-7.3-8.4 depending on
degree of expansion. They are of the same
sizes on all arms, and there is no sexual di-
morphism. They number 80-90 on the dorsal
arms, 60-70 on the ventral.

The hectocotylized third right arm is some-
what shorter than the corresponding left arm
(OAl: 75-86-100) and carries about 40 suck-
ers (Fig. 8). The number is individually con-
stant throughout life (Fig. 23). The ligula was
not described by Prosch (1849) probably be-
cause he had only one male with an abnormal
hectocotylus at his disposal when he finally
described B. arcticus (paralectotype CEP-
15). But Steenstrup (1856a, b, 1857) pictured
a male with hectocotylized arm and, based on
five specimens, he stated that the arm carries
41-43 suckers and ligula 13-17 transverse
laminae. The ligula is aspoon-shaped pointed
organ with inrolled curved sides (Fig. 1).
The width is about 50-70% of the length. It
has a central ridge and 11-16 (17) deep, well-
separated laminae (variation shown in Fig. 9).
The number of laminae is individually con-
stant from the onset of maturity (Fig. 22).
LigLl: 9-23, the observed variation also de-
pending on state of sexual maturity (Fig. 3).
Calamus is short and pointed, CaLl: approx.
20. Spermatophoral groove well developed,
the strong membrane curling the arm inwards
in preserved specimens. Already at ML 16
mm the ligula may be discernible as a 1.5
mm-long undifferentiated tip of the third right
arm.

Female Organs: The ovary is large with big
globular and heavily pigmented oviducal
glands (Fig. 2c). Proximally, the united

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 179

FIG. 1. Bathypolypus arcticus (Prosch, 1849). a: hectocotylus, ML: 40 mm; b: upper and lower beak, Q ML
46 mm, “Godthaab” Stn. 87, ZMUC; с: ©’ ML 54 mm, South Greenland, Just & Vibe Stn. 45, ZMUC.

oviducts are partly covered by the outer mem-
brane of the ovary; distally they are short, very
stout and leaving the oviducal glands forming
a right angle. In ripe females, the gonad oc-
cupies 30-40% of the mantle cavity and is
stuffed with 60-80 yellow or brownish eggs.
Ripe eggs measure 16-18 mm in length.
They are smooth, with fine longitudinal lines.
The pointed end of each egg has a short stalk,
and the stalks of all eggs are attached to the
same small area of the ovary wall.

Male Organs: The penis has a well-devel-
oped diverticulum (Fig. 2b). Its size and shape
very much depend on presence or absence of

spermatophores, and it was not measured.
Needham’s sac was often distended by 3-6
spermatophores, as in Figure 2b. The large
brownish spermatophores are very character-
istic (Fig. 2d; Prosch 1849: fig. 2): the sperm
reservoir occupies only about one third of the
total length, and the oral end is a long, stout
horn. The casing is very opaque, and details
of the reservoir and middle piece could not be
obtained. Зри: 105-130, SpWI: approx. 18,
SpRI: 26-32 (data from six males).

Beaks and Radula: The beaks do not have
distinctive features. The radula seems to be
very variable. In most cases, the central teeth

180 MUUS

FiG. 2. Bathypolypus arcticus, a: digestive tract; ant.sal.gl.: anterior salivary glands; cae.: spiral caecum;
dig.gl.: digestive or hepatic gland; post.sal.gl.: posterior salivary glands; stom.: stomach. b: male reproduc-
tive organs; acc.gl.: accessory gland; needh.: Needham’s sac containing ripe spermatophores; pen.div.:
penis diverticulum; sem.ves.: seminal vesicles; test.: testis; vas.def.: vas deferens. c: female reproductive or-
gans; ov.gl.: oviducal gland; d: spermatophore. a,b and а нот $ ML 43 mm, Ymer Island, East Greenland,
1932, ZMUC; c: 2 ML 42 mm, BIOFAR Stn. 274, ZMUC.

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 181

B.arcticus
n=49

10 20 30 40 50 ML mm

FIG. 3. Ligula length versus mantle length in B. arcticus. Open circles: juveniles. Maturity is reached at about
ML 30 mm. Inserted lectotype of В. arcticus (encircled dot) and the : Уре” of В. faeroensis in ZMUC (dot in
square). Juv.: y=-1.90 + 0.225x, г? = 0.81; ad.: у = -3.9 + 0.340x, г? = 0.39.

о B.bairdii n=142

B.arcticus n=92

10 20 30 40 50 60 ML mm

FIG. 4. Eye diameter index versus mantle length in B. bairdii and B. arcticus. No sex „difference was found.
Allometric growth is discernible, but correlation is weak; bairdii: у = 52.68 — 0.2204x, r = 0.249; arcticus: у =
36.7 — 0.199x, r? = 0.321.

are clearly multicuspid (Fig. 5), with a seri- regular rough knots. The ectocones may also
ation of 2-5 symmetrical (Fig. 5c) or asym- be reduced to a certain lateral ruggedness of
metrical teeth (Fig. 5b, d-f). In some speci- the rachis teeth even in presumed unworn
mens, the ectocones are rather delicate, parts of the radula. This may lead to quasi ho-

uniform thorns (Fig. 5b); in others they form ir- modont rachidians, but completely homodont

FIG. 5. Radula and rachidians of В. arcticus; a and b: Y ML 54 тт; с: Y ML 37 mm; d: Y ML 44 mm; e: Y
ML approx. 50 mm; fand 9: ФФ ML 33, 52 тт. Caught at various positions off northern Iceland, except e:
southwestern Greenland, probably 1840. Scale: 100 um.

rachidians are sometimes found. A striking ex-
ample is demonstrated by two otherwise typi-
cal arcticus females (Fig. 5f, g) taken at
BIOICE Stn. 2751. One has multicuspid, the
other one homodont rachidians.

Digestive Tract: The esophagus has a well-
developed crop diverticulum (Fig. 2a) on
which the posterior salivary glands are loosely
attached when in situ. The spiral caecum is
reduced to at most one turn. The liver is large

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 183

and bulbous, about the same size as the
testis. There is no ink sac.

Comparison with Prosch’s Description

The material examined fits well with the
types and Prosch's description, which is sup-
plemented by Steenstrup’s (1856a, b, 1857)
remarks and illustrations of the species. One
serious discrepancy, however, is the state-
ment by Prosch that arcticus has no crop. This
error of Prosch is hard to explain unless one
of the specimens dissected during the study
of the anatomy of Cirroteuthis (Reinhardt &
Prosch, 1846) was B. bairdii, which is very
common in West Greenland. | have however
no evidence for this.

An important point that the present investi-
gation confirms is that the rachidians are usu-
ally heterodont (Muus, 1962: 11, fig. 1A),
though much more variable than | previously
thought.

Junior Synonyms and Supposed Synonyms
of B. arcticus

Three species which have contributed con-
siderably to the prevailing taxonomic confu-
sion are discussed below: Bathypolypus
faeroensis (Russell, 1909), which was re-
moved from the junior synonomy of B. arcticus
by Toll (1985), and the two species that had
been placed in Benthoctopus- sasakii (Rob-
son, 1927) and piscatorum (Verrill, 1879).

Bathypolypus faeroensis (Russell, 1909)

Russell (1909) described Polypus faeroen-
sis based on two males and a female trawled
in the Faeroe Channel by the Fishery Cruiser
“Goldseeker” Stn. 19a, 60°40'N, 4°50’W at
1030 m. August 24, 1908. In 1922, Russell re-
peated his earlier description based on the
same three specimens, adding illustrations of
radula, ligula, a papillary area of the skin, and
a good photograph of the whole animal.

Russell stated that faeroensis is closely al-
lied to the squat form that he knew as *bairdii
= arcticus”, from which it differs in certain well-
defined ways: it has a better marked neck, a
narrow head, and a shorter ligula, with more
numerous laminae. The skin has a character-
istic papillation with tiny warts arranged in a
stellate pattern on light, circular spots.

Bathypolypus faeroensis has been consid-
ered a synonym of B. arcticus (Prosch, 1849)
by Robson (1932, with hesitation), Jaeckel
(1958), Kumpf (1958), Roper et al. (1984),
and Nesis (1987). Toll (1985) redescribed B.
faeroensis and reinstated it as a valid species
based on a female specimen taken by FFS
Walther Herwig in Denmark Strait, 67°21,
5'N, 23°30’W, 480-485 m, September 9,
1973. Because Toll was unable to trace the
types of B. faeroensis, this specimen was
designated neotype.

In ZMUC | found a misplaced jar labelled
“Polypus faeroensis, Type, 61°27'N, 1°47'W,
1240 m, July 25, 1909. Russell det. Received
from A. C. Stephen, the Fishery Board of Scot-
land, March 25, 1924.” The specimen, a male
with ML 34 mm, was caught a year after the
specimens used for the description by Russell
1909 (and later 1922). | am inclined to believe
it was received as a donation to ZMUC, but it
is unknown whether Russell or the ZMUC cu-
rator (R. Spärck) is responsible for the “type”
label. | found in BMNH a female specimen
taken in the same haul as the above-men-
tioned male. Because it had been misplaced,
the ZMUC-type is not recorded in the ZMUC
type list (Kristensen & Knudsen, 1983).

The “type” in ZMUC might have been a bet-
ter choice than the neotype designated by
Toll, because it is a male, caught at the type
locality and identified by the describer. Table 2
shows measurements of the specimen in
ZMUC.

The ZMUC “type” has a VV-funnel organ.
Hectocotylized arm with 39 suckers. The buc-
cal mass has been removed, so the radula
and beaks are missing. The esophagus has a

TABLE 2. Measurements (mm) of the B. faeroensis “type” in ZMUC.

mE 105

ML: 34

HW: 20 HWI: 59
MW: 27 MWI: 79
ED: 12 EDI: 35
LD: 372. SDEDIZ 18:3
SD: 2 SDI: 6.5
LigL: 7 LigLl: 11815
LamC: 11 SHcC: 42

AL: | Il Ш IV
r 67 64 520056
| 70 62 DY

2 25 21

184

crop diverticulum. Needham's sac with four
unripe spermatophores. SpL: 22 mm, SpRi
31, of the characteristic arcticus type (SpRI
approx. 30; Fig. 2d).

In all measurable characters, the ZMUC
specimen agrees with the general descrip-
tions by Russell (1909, 1922) and Toll (1985).

Neither Russell nor Toll examined type ma-
terial of B. arcticus. Both authors assumed
that the “bairdii’-form is the true В. arcticus.
Ironically, however, the species described by
Russell and the faeroensis specimen in
ZMUC is the genuine Bathypolypus arcticus,
and all the data on B. faeroensis fit measure-
ments and indices as indicated in the present
redescription of that species. Toll's careful re-
description of faeroensis might as well be
a redescription of a female arcticus. The ho-
modont rachidians pictured by Toll (his fig. 1b)
are unusual but not unknown in arcticus. Ho-
modont rachidians were found in one of two
females (Fig. 5f, g) caught in the same haul at
68°00'N, 20°40'W (BIOICE Stn. 2751). In all
aspects, Toll's specimen is indistinguishable
from B. arcticus.

Benthoctopus sasakii Robson, 1927

Material examined: Types in BMNH:
89.1.24.33-34. A male and a female labelled:
“Benthoctopus sasakii Robson, HMS ‘Triton’
Stn. 9, Faroe Channel, 60'5'N, 6'21'W, 608
fms. August 23, 1882”.

Male: ML about 32 mm, distorted and dis-
sected. Hectocotylized arm 59 mm with 44
suckers, LigL: 7.8 mm. Ligula with 12-15 in-
distinct lamellae. SD: 2.3 mm, ED: 10 mm,
SpL: 34 mm, SpRL: 9 mm. Funnel organ VV
with the outer limbs somewhat shorter than
the median limbs. Skin smooth.

Female: TL: 100 mm, ML: 27, MW: 24, HW:
20, SD: 2.3 mm, ED: 10 mm. Funnel organ
fragmented.

Robson (1927: 262, fig. 8) depicted the
radula, which shows a more or less irregular
seriation of multicuspid rachidians. Both spec-

MUUS

imens are typical of juvenile B. arcticus, as ev-
idenced by the hectocotylus, the funnel organ
and the indices: SDEDI: 23 and SpRl: 26
(Table 9). They were furthermore trawled in
arctic water masses at the same depth and lo-
cality as B. faeroensis (= B. arcticus). Robson
became doubtful about his sasakii and later
(1932) treated it as a junior synonym of Ben-
thoctopus piscatorum (Verrill, 1879), which
prompted me to reexamination of the type of
piscatorum in USNM.

Benthoctopus piscatorum (Verrill, 1879)

Material examined: Holotype USNM
574641. From western part of Le Have Bank
off Nova Scotia, October 1879, 120 fms. (219
m). Female, ML: 39. Measurements are give
in Table 3. The holotype was a unique speci-
men (Roper & Sweeney, 1978).

Description: The skin is smooth, with no evi-
dent sculpture. A much contracted cirrus de-
tectable over the right eye, no trace over the
left eye. The funnel organ partly missing but of
bairdii type (Fig. 18). Esophagus without crop
diverticulum, only with a slight swelling.
Number of suckers on each arm 65-75.
Radula and jaws not available.

Verrill's description (1879: 470) is not very
exhaustive, as noted by Robson (1932). In
Verrill's opinion, piscatorum was easily distin-
guished from Octopus bairdii “by a more elon-
gated body, longer arms, shorter web, lack of
supraocular cirrus and by its smoothness.”

| believe that the type specimen of Octopus
piscatorum is a somewhat aberrant specimen
of Bathypolypus bairdii (Verrill, 1873), as is
the case with his О. lentus and О. obesus
(see below). None of the bodily proportions
are distinctive. The only index that falls out-
side the natural variation of bairdii is SDEDI:
14 (Table 9), but this is compensated for by
SDLDI: 28, which is a more reliable index,
being based on the firm eye lense as standard
for eye size (Fig. 16).

TABLE 3. Measurements of holotype of Benthoctopus piscatorum.

MESAS

ML: 39

HW: 28 HWI: 71
MW: 33 MWI: 85
ED: 15 EDI: 38
ÉD: 75 ЗОШ 28
$0: 2.1 = SDI: 5.4

AL: | Il ll IV
r 93 75 82+ 78
| 98 102 77+ 75+

WD:

д Ме NID QUE
25 16 27 24 12?
32 32 25

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 185

| believe that the smoothness of the body is
due to contraction of all warts or flaccidity at
the time of death, as seen in varying degrees
in many preserved specimens of both bairdii
and arcticus. Traces of a retracted cirrus over
the right eye suggest that the smoothness
over the left eye hides a potential cirrus.

The identity with bairdii is further substanti-
ated by the lack of a crop diverticulum. This
contrasts with Robson’s statement that pisca-
torum has “a tolerably well-developed crop”
(1932: 225) and his figure 31. However, Rob-
son based this statement on dissections of B.
sasakii (= arcticus).

In 1981, | discussed the Bathypolypus-Ben-
thoctopus problems with Gilbert Voss, who
also examined the type specimen of piscato-
rum. He concurred with my opinion that Verrill
(1879) described a somewhat atypical speci-
men of Bathypolypus and consequently the
genus Benthoctopus Grimpe, 1921, would be
indistinguishable. (Voss & Pearcy 1990).

East Atlantic “piscatorum”

The true identity of piscatorum raises seri-
ous doubts as to the identity of the many
specimens attributed to piscatorum from the
East Atlantic by Hoyle (1886), Appellof (1893),
Pfeffer (1908), Massy (1909), Russell (1922),
Robson (1932), Grieg (1933), and Grimpe
(1933).

The first to record piscatorum from the
Faroe Channel was Hoyle (1886). | examined
his specimens in BMNH, and they are identi-
cal with the specimens later described as
Benthoctopus sasakii by Robson (1927) and
once again as piscatorum by Robson (1932).
As argued above, the type specimens of
sasakii are conspecific with arcticus.

Appellöf (1893), inspired by Hoyle (1886),
identified three very juvenile specimens from
66°41'N, 6°59’E and 78°2’N, 9°25’E as pis-
catorum. He does not give particulars, except
that the body is smooth and that mantle and
head of the male measure 14 mm. One of the
specimens was caught with a juvenile male
“Octopus lentus” (junior synonym of bairdii),
and later identified by Grieg (1933) as pisca-
torum. Because piscatorum specimens from
the Svalbard-Barents Sea area are all caught
in cold water (-0.9°-+0.8°C), | strongly sus-
pect them to be smooth and partly juvenile
specimens of arcticus sensu stricto. | dismiss
the records as unreliable, although | have not
seen the specimens.

Pfeffer (1908) merely repeated the Ameri-

can records of Verrill and the East Atlantic
records of Hoyle (1886) and Appellöf (1893).
He repeated Verrill’s pictures of piscatorum
and for unknown reasons synonymized Ben-
thoctopus ergasticus with piscatorum. As
shown below, В. ergasticus is a distinct
species.

In conclusion, Pfeffer’s records of piscato-
rum are unreliable.

Massy (1907) described Polypus normani,
which she later, having consulted Pfeffer, de-
cided was a piscatorum (Massy, 1909). It is a
male, TL: 206 mm, trawled off Ireland at
51°15'N, 11°47’W, 707-710 fms., September
1907.

| have not examined the type, but Massy’s
measurements and drawings (1909: pl. Il,
figs. 3, 4) allow the conclusion that it is not
conspecific with either the type of Verrill's pis-
catorum nor bairdii or arcticus: the hectocotyl-
ized arm has 64 suckers, far beyond the num-
ber found in bairdii (Fig. 8). OAl is 64.6, which
is off the range of variation in bairdii and arcti-
cus, in which OAI is about 80-90. SDI is 9.5,
which means that the suckers are larger than
in bairdii and arcticus. The smoothness of the
skin and the bodily proportions given by
Massy are not distinctive, but the shortness of
the hectocotylized arm and its relatively high
number of suckers reminiscent of Benth-
octopus ergasticus (P. Fischer & H. Fischer,
1892). Two males and a female of this species
were caught in the same haul as normani.
Still, B. normani may be a valid species.
Massy was advised by Pfeffer who, as men-
tioned above, considered ergasticus synony-
mous with piscatorum. The ligula and cala-
mus of normani is clearly juvenile (Massy
1909: figs. 3, 4). Her figure was erroneously
used by Nesis (1987: 318, fig. 84H) to illus-
trate the calamus and ligula of piscatorum.

Russell (1922) recorded three male and
four female Polypus piscatorum taken in the
Faeroe Channel in the same haul as his Р
faeroensis (“Goldseeker” Stn. 19a). All seven
specimens are juvenile. His measurements
do not allow the species to be even tentatively
identified. But if his measure “Posterior end to
eye” is accepted as expression of the ML, the
SDI of the largest female (ML: 32 mm) and the
largest male (ML: 27 mm) is 9.4 and 7.4 re-
spectively, beyond the sucker size of bairdii,
but compatible with arcticus. The large female
faeroensis (= arcticus) taken in the same haul
(Russell, 1922: 6) has SDI: 8.3. The ligula of
the largest male is depicted and shows a 6.5-
mm-long typical juvenile arcticus ligula with

186

“half a dozen indistinct transverse ridges”
(Russell, 1922: fig. 7).

Like many of the earlier writers, Russell has
attached undue weight to the smoothness of
the skin of his “piscatorum” specimens. | con-
sider it indubitable that they are juvenile arcti-
cus, as all the other specimens | have exam-
ined from the arctic water masses in the depth
of the Faeroe Channel (Fig. 19).

Robson (1932) did not examine the type of
piscatorum. He based his revision on three
specimens, two of which are the specimens
originally described as piscatorum by Hoyle
(1886), later as sasakii by Robson (1927). As
argued above, sasakii is a junior synonym of
arcticus.

The only other specimen seen by Robson is
a female (Robson’s C30) surprisingly caught
in the same haul as the “type” of faeroensis
found in ZMUC: Faroe Channel 61°27'N,
1°47'W, 681 fms., July 25, 1909. | have ex-
amined the specimen at BMNH: the body is
ovoid, the skin smooth with no trace of warts.
The funnel organ is not well preserved but
shows VV, the inner limbs are however
weakly joined anteriorly. Measurements are
given in Table 4.

Robson (1932: 225, fig. 35) shows the
radula of this specimen. The rachidians are
heterodont, showing irregular lateral cusps
and a seriation of about four teeth. The esoph-
agus has a well-developed crop diverticulum.

The multicuspid rachis teeth, the presence
of a crop diverticulum, as well as the mea-
surements and indices in Table 4 show that
Robson’s piscatorum is really the smooth-
skinned specimen of B. arcticus.

In conclusion, Benthoctopus piscatorum
sensu Verrill, 1879, is a junior synonym of Ba-
thypolypus bairdii, whereas В. piscatorum
sensu Hoyle (1886), Russell (1922), and Rob-
son (1932) is smooth-skinned and often juve-
nile Bathypolypus arcticus.

A large female octopod caught 1967 at a
depth of 60 fathoms in Placenta Bay, New-
foundiand, and identified as Benthoctopus
piscatorum by Aldrich & Lu (1968: table 1,

MUUS

figs. 1, 2) is undoubtedly misidentified and
should be reexamined. With a TL of 362 mm,
ML 89 mm and HWI of only 35, the specimen
deviates considerably from both B. bairdii and
B. arcticus. The authors were aware of the in-
consistency with Robson’s description of pis-
catorum but put it down to its poor state of
preservation. Nixon (1991), studied the eggs
of this specimen (as “piscatorum” eggs).

Revision of B. arcticus by Robson (1932)

Robson (1932) was very uncertain about
the large number of forms ascribed to the
arcticus-bairdii complex. Though with hesita-
tion, he concluded that faeroensis with the
ovoid body and narrow head at the one ex-
treme and the bairdii form with the saccular
body and large ligula at the other were one
and the same species, B. arcticus.

Robson presented two excellent photos of
the two forms (1932: pl. VI, figs. 1, 2), one of
which is a “type”-specimen of B. arcticus from
Greenland on loan from ZMUC. | have tried in
vain to identify the specimen in the Copen-
hagen collections, but the photo is easily rec-
ognizable as B. arcticus, sensu stricto. The
other photo depicts a typical B. bairdii, the
square-bodied boreal form, which is rein-
stated as a distinct species below.

Because the two species were confounded,
Robson's text is of limited use. Besides the
photo, he provided (1932: 290-291) three fig-
ures of a B. arcticus, sensu stricto, from the
Barents Sea: outline of mantle (fig. 54), an oc-
ular cirrus (fig. 55), and a funnel organ (fig.
56). The radulae (fig. 58) are from B. bairdii
specimens.

Robson (1932) enforced the prevailing tax-
onomic confusion by his erroneous identifica-
tion of smooth skinned and juvenile B. arcti-
cus as Benthoctopus piscatorum.

Records of B. arcticus by Adam (1939)

Adam (1939: 6, table, figs. 2-4) records
six juvenile specimens of B. arcticus from

TABLE 4. Measurements of Robson’s Benthoctopus piscatorum.

a 185

ML 43

HW: 34 HWI: 79
MW: 48 MWI: 112
ED: 14 EDI: 33
SD: 38: :5Dl: 7.6

SDEDI 24

AL: | Il Ш IV
r 133 130 131 130
| 12911136 % 134

ANSE =
30 33 38 38 30
35 36 39

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 187

the Iceland-Faroe area (about 66°25’М,
12°30'W). | reinspected the material on loan
from IRSNB and found, that four of the speci-
mens (Adam's specimens b and с) were iden-
tical with arcticus as here redescribed. They
all have the typical VV funnel organ and the
diagnostic index SDLDI is: 53, 53, 53 and 59,
respectively, which distinguishes arcticus
from B. bairdii and B. pugniger, n. sp. (with
SDLDI < 40). Two of the specimens are erro-
neously identified as females (specimens b:
ML 14 and 16 mm). They are juvenile male
arcticus with undifferentiated ligulae of 1.5
and 1.9 mm. Adam’s drawing of the radula of
a female arcticus (p. 12, fig. 4, specimen c)
shows a homodont rachidian reminiscent of
the radula from “В. faeroensis” pictured by Toll
(1985: 600, fig. 1) and my Figure 5g. It under-
lines the fact that B. arcticus has a very vari-
able radula.

The remaining two specimens of the mate-
rial treated by Adam (1939: specimens a, figs.
2, 3), | consider to be B. pugniger, n. sp., and
are discussed below.

REINSTATEMENT AND REDESCRIPTION
OF BATHYPOLYPUS BAIRDII
(VERRILL, 1873)

Material examined: about 500 specimens
from boreal parts of the North Atlantic as listed
in Appendix |. Types examined: Octopus
bairdii Verrill, 1873 (syntype, USNM 574638)
Octopus piscatorum Verrill, 1879 (holotype,
USNM 574641) Octopus lentus Verrill, 1880
(holotype, USNM 34223) Octopus obesus
Verrill, 1880 (holotype, USNM 382469).

Synonymy

Octopus bairdii Verrill, 1873a: 5; 1873b: 394,
figs. 76, 77; 1881: 107, pl. 2, fig. 4, pl. 4,
fig. 1; 1882: 368, pl. 33, fig. 1, 1a, pl. 34,
figs. 5, 6, pl. 36, fig. 10, pl. 38, fig. 8, pl.
49, fig. 4, 4a, pl. 51, fig. 1, 1a; Sars, 1878:
339, pl. 17, fig. 8, pl. 33, figs. 1-10;
Kumpf, 1958 (passim)

Octopus piscatorum Verrill, 1879: 470; 1882:
377, pl. 36, figs. 1, 2

Octopus obesus Verrill, 1880: 137; 1882: 379,
pl. 36, figs. 3, 4; Robson, 1932: 299;
Kumpf, 1958 (passim).

Octopus lentus Verrill, 1880: 138; 1881: 108,
ple 4; fig: 2; 1882: 351, 375, pl: 35, figs: 15
ры, Нд. 12; Robson; 1932:=297;
Kumpf, 1958 (passim)

Octopus arcticus: Norman, 1890: 466; Joubin
1920: 32, pl. 7, figs. 4, 5

Polypus arcticus: Pfeffer, 1908: 16, fig. 6

Polypus lentus: Pfeffer, 1908: 17, figs. 7, 8

Bathypolypus arcticus: Robson, 1932 [in
part]: 286, figs. 53-60, pl. 6, figs. 1, 2;
Thiele, 1935: 992, fig. 890; Boone, 1938:
360, pl. 152; Bruun, 1945 [in part]: 6;
Kumpf, 1958: 1-135; Jaeckel, 1958: 563:
Cairns, 1976: 261; Macalaster, 1976;
Perez-Gandaras & Guerra, 1978: 201,
figs. 6-8; O’Dor & Macalaster, 1983: 401,
fig. 24.1, 24.2; Roper et al., 1984: 222;
Nesis, 1987: 315, fig. 83B-E; Guerra,
1992: 251, fig. 89

Bathypolypus proschi Muus, 1962: 13, figs.
2-4

Diagnosis

Square-bodied, with papillated skin; arms
short; head broad, with large eyeballs, each
with a supraocular cirrus; funnel organ dou-
ble, pad-like; hectocotylized arm with about
40 suckers; ligula very large deeply exca-
vated with 8-12 prominent laminae. Radula
with homodont central teeth. Esophagus with-
out crop diverticulum. Total length rarely over
200 mm.

Description

Skin and Colors: In newly caught specimens,
the skin is violet to purple, often without any
conspicuous spots or patterns, but sometimes
speckled with small greyish spots. In pre-
served specimens, the skin may be smooth,
but more often the dorsal surface is papil-
lated, especially in the antero-dorsal region.
Single warts or aggregations occur, the latter
sometimes in a stellate pattern, a number of
small warts encircling a larger one, similar to
B. arcticus. Over each eye is an erectile
pointed cirrus with adjacent smaller protuber-
ances. Erected it measures 5-10 mm.

Bodily Proportions: Square-bodied, with
broad head and huge eyeballs. HWI de-
creases from 80-100 at ML 10 to 60-90 at ML
60 mm. TL rarely over 200 mm.

The eyeballs are very prominent. They de-
crease in relative size from EDI 40-60 at ML
10, to 30-45 at ML 60 (Fig. 4). The lens mea-
sures on an average 41% of the ED (LDEDI:
35-46).

The mantle aperture is 36-42% of the cir-

188 MUUS

FIG. 6. Bathypolypus bairdii (Verrill, 1873). a: habitus (after Vecchione et al., 1989); b: upper and lower beak;
с: hectocotylus of с’ ML 42 mm, Greenl. Fish. Invest. Stn. 5047, ZMUC.

cumference of the mantle. The funnel is free
of the mantle for about 50%.

The funnel organ consists typically of two
pad-like structures with a very variable ap-
pearance (Fig. 18). They are never strictly VV-
shaped, but may have an anterior indentation
like a heart or be broken up into bars, sug-
gesting an evolutionary past as VV-shaped
organs. They are rather loosely connected
with the funnel wall and easily lost.

The gills are reduced and have 6-8 gill fila-
ments on each demibranch.

The arm order is I.Il.IIl.IV. The ML consti-
tutes 28-38% of the TL, which leaves 62-
73% to the brachial complex. ALI: | 201, Il
184, Ill 174, IV 168 (mean of 21 specimens).

The web extends along the arms. The web
sectors B, C and D are subequal. WDI: A 32,

B 35, C 36, D 35, E 27. WDMI: A: 54, B: 59, C:
60, D: 59, E: 46 (mean of 21 specimens).

The suckers are biserial, small and well
spaced. SDI: 2.9-3.9-4.8 depending on de-
gree of expansion. They are similar in size on
all arms, and there is no sexual dimorphism.

The hectocotylized third right arm is shorter
than the left (OAI: mean, 88). Spermatophoral
groove well developed. The number of suck-
ers on the hectocotylized arm shows geo-
graphical variation: the east American popula-
tion (S of 45°N) has 26-40 suckers, whereas
specimens from western Greenland and the
eastern Atlantic have 35-49 (Fig. 8). The
number is individually constant throughout life
(Fig. 23).

The ligula is a large spoon-shaped organ,
which in ripe animals (ML > 30 mm) is often

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 189

FIG. 7. В. bairdii. a: digestive tract; b: reproductive organs; с: spermatophore of с’ ML 41 mm, Dana Stn.
11648, МОС; d: female reproductive organs, ML 41 mm, Greenl. Fish. Invest. Stn. 5112; e: hectocotylus of

juvenile с’ ML 23 mm, Dana Stn. 2346, ZMUC.

rather square, with almost parallel sides dis-
tally ending with rounded flaps and a small
pointed tip (Fig. 6). The width is 50-75% of the
length. It has a central ridge and a number of
deep and well-separated laminae. The num-
ber is individually constant from the onset of
maturity. It has a total range of 7-13, but
shows a geographical variation that is very ap-
parent in the western Atlantic, the Newfound-
land waters being a transitional area between
the American population and the populations

off Labrador and western Greenland (Fig. 9).
Also, the size of the ligula in ripe animals
shows geographical variation: at a ML > 30
mm, LigLI in the eastern American population
is 24-44, whereas in western Greenland and
the rest of the North Atlantic, it is 18-38. The
American population apparently reaches ma-
turity ata ML of 25-35 mm, whereas in the rest
of the Atlantic maturity is generally reached at
ML 30-40 (Fig. 10).

Calamus is short and pointed (CaLl approx.

190 MUUS

per cent

B.arcticus
30 n=40

10- |
РЕ

B.bairdii
301 EAt
n=40

10

B.b.
30 W.Greenl
n=61

10

30

10

27 30 33 36 39 42 45 48 Suckers

FIG. 8. The American population of B. bairdii (< 45°N) deviates in mean number of suckers on the hecto-
cotylus from the western Greenland population (P = 0.95); mean: 32.6, SE 2.6 versus 41.4, SE 3.4. The east-
ern Atlantic population differs only slightly from Greenland (mean: 42.7, SE 3.1). Inserted: arcticus, mean:

39.7, SE 3.1.

20). Already at ML 11 mm the ligula may be
identified as a 1.2-mm-long undifferentiated
tip of the third right arm.

Female Organs: The ovary is large, with con-
spicuous globular blackish oviducal glands
(Fig. 7d). Ripe females with 60-85 yellowish
eggs measuring 15-18 mm in length. The
eggs are smooth with fine longitudinal lines.

Male Organs: The penis with well-developed
diverticulum (Fig. 7b). In Needham's sac was

found up to six spermatophores. The sausage-
shaped sperm reservoir of the spermatophore
occupies about half of the total length, and the
oral end is a slim, rather stiff and curved tube
(Fig. 7c; Verrill, 1882: pl. 36, fig. 10). The sper-
matophore is glossy and opaque; the interior
details are difficult to make out. SpLl: 60-90-
115, SpWI about 19, SpRI 50-60.

Jaws and Radula: The beaks do not present
distinctive features. The radula is homodont,
the rachidians having smooth concave sides

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 191

per cent

Г] Lab.-Greenl.
=> | n=92

Newf.
29 n=65

оз: О

| Am<45°N
40 n=121

65587107512

46 758

628 7077274

N

<= B.bairdii

B.pugniger n.sp
n=17

|
|

B.arcticus
n=36

10 12 14 16LamC

FIG. 9. Number of laminae copulatoriae. B. bairdii shows geographic variation with a highly significant dif-
ference (P: > 0.99, z-test) in mean number between the American (< 45°N) and the western Greenland pop-
ulation (mean: 10.12, SE 0.109 versus 8.38, SE 0.3). The Newfoundland area forms a transitional zone
(mean: 9.56, SE 0.2). East Atlantic (mean: 9.89, SE 0.2) versus western Greenland shows a similar signifi-
cant difference. Variation in B. pugniger, n. sp., and B. arcticus inserted for comparison.

and never showing any sign of ectocones
(Fig. 11; Verrill, 1882: pl. 49, fig. 4).

Digestive Tract: The esophagus has no crop
diverticulum, only a slight swelling where the
posterior salivary glands are fastened when in
situ (Fig. 7a). The spiral caecum is reduced to
less than one turn. The liver is large, about the
same size as the testis.

Junior Synonyms of B. bairdii.

Robson (1932) strongly suspected that Oc-
topus lentus Verrill, 1880, and ©. obesus Ver-
rill, 1880, were conspecific with the typical
bairdii form, because he was not able to find
critical differences. Not having seen the type
specimens, however, he hesitated to place
them in synonomy.

In a master’s thesis, Kumpf (1958) tried to
sort out the Bathypolypus arcticus-bairdii-
lentus-obesus complex. He had a large
amount of material, 178 specimens, caught
off the eastern American coast, including the
types of Verrill’s Octopus bairdii, O. lentus and
O. obesus. In addition, he used measure-
ments of seven “В. arcticus” specimens ex-
amined by Gilbert Voss in the BMNH.

Kumpf applied the standard of measure-
ments of Robson (1927, 1932) and amplified
by Pickford (1945, 1949). He thoroughly com-
pared his material with the types of bairdii,
lentus and obesus and concluded that these
species are conspecific and that in spite of all
variation, only one Bathypolypus species is
represented in his material.

| can confirm this part of his conclusion. The
bulk of Kumpf’s material stems from between

192 MUUS

B.bairdii East America (45°N
n=54

B.bairdii W- Greenland - Europe
n=98

10 20 30 40 50 60 70 ML mm
FIG. 10. Ligula length versus mantle length in B. bairdii. Open circles: juveniles. Lower figure: pooled data
for western Greenland and NE Atlantic (juv.: у = —3.34 + 0.333x, г? = 0.58; ad.: y =-3.20 + 0.505x, г? = 0.66).
Upper figure: Eastern America < 45°N (juv.: у = -0.49 + 0.248x, 1” = 0.39; ad.: у = 4.90 + 0.452x, г? = 0.58).

In eastern America, maturity is often reached already at ML 25 mm and ligula grows larger than in the North
Atlantic. Dot in circle: syntype of bairdii (USNM 574638). Dot in square: holotype of B. obesus (Verrill, 1882)
(USNM 382469).

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 193

FIG. 11. Radula and central teeth of В. bairdii. с’ ML 55 mm., Davis Strait 65°12’N, 56°21’W. Scale: 100 um.

30°N and 45°N, and | examined and remea-
sured most of it, including the types of bairdii,
obesus and lentus, during a visit to USNM in
1975. The data are included in the present re-
vision.

Kumpfs main conclusion, however, that
Verrill's species are junior synonyms of arcti-
cus, is erroneous. The genuine arcticus is a
more northerly species, not found off the east
coast of USA (Fig. 20). Paradoxically, Kumpf
never saw the species he purports to revise.
To understand B. arcticus, he had to rely on
the literature, especially on Robson’s (1932)
revision, with all its ambiguities and errors.
Kumpf focused on the squat “bairdii-type” as
being the typical arcticus, thus missing the
point that Verrill's bairdiiis a separate species.

Kumpf (1958) was incorrect in stating that
Verrill did not know of Prosch's arcticus. In a

footnote describing bairdii, Verrill (1873a: 5)
wrote that his species differed from O. groen-
landicus Dewhurst, which has a smaller hec-
tocotylus “with more numerous folds, and the
basal part bears 41-43 suckers”. This is a ci-
tation from Steenstrup's then well-known
paper (1856a, or the translations of it: 1856b,
1857), in which O. groenlandicus is syn-
onymized with arcticus and in which the
species and its hectocotylized arm are figured
(see also Verrill, 1880: 138, footnote).

The knowledge of West Atlantic Bathypoly-
pus was much extended and elaborated upon
by Macalaster (1976), who studied no less
than 750 Bathypolypus specimens collected
in Canadian waters from Georges Bank to the
Labrador Sea. Macalaster was warned by me
that “arcticus” covered more than one
species, but through morphometric analyses

194 MUUS

she showed convincingly that only one
species occurs in Canadian waters and that it
is identical with Kumpf's “В. arcticus”. Leaning
on КитрР$ revision, she excusably put the
wrong species in the title of her otherwise
valuable contribution, instead of B. bairdii.
This mistake was repeated by O’Dor &
Macalaster (1983) and by Wood et al. (1998),
which are in fact accounts of life cycle and
breeding ecology, respectively, of B. bairdii,
not of B. arcticus, as indicated in the titles.

Macalaster’s primary interest was not tax-
onomy but the opportunity to study the life
cycle, growth, and reproduction of Bathypoly-
pus, including observations of live animals
kept in aquaria. Of principal interest to the
present revision, however, is Macalaster’s
comprehensive measurements, which sup-
plement Kumpf's (1958) and my own Ameri-
can data and which show that B. bairdii is
commonly distributed along the upper part of
the slopes from Florida to the Labrador Sea
and that the genuine B. arcticus seems to be
absent. | included some of her data as a sup-
plement to my own to demonstrate morpho-
logical differences between the southern and
northern populations of В. bairdii.

Octopus piscatorum Verrill, 1879

A full account of this nominal species and
description of the holotype is given above
under the synonymy of B. arcticus, which has
been the species most often mistaken for pis-
catorum. It is here shown beyond reasonable
doubt that O. piscatorum is a junior synonym
of O. bairdii Verrill, 1873. It is also shown that
Robson's concept of piscatorum was based
upon misidentified arcticus specimens from
the Faroe Channel and that consequently
characters of B. arcticus, for example, pres-
ence of crop diverticulum, multicuspid radula,
have crept into his diagnosis of piscatorum.
This of course has repercussions on the con-
cept of the genus Benthoctopus, of which pis-
catorum is the type species.

Bathypolypus proschi Muus, 1962

Holotype: Male TL 115 mm, “Dana” Stn.
10018, 65°02’5”М, 56°00’W, Davis Strait,
730-740 m, trawl, July 19, 1956. Depository:
ZMUC.

The unanimous claim among earlier au-
thors (exception: Sars, 1878) that bairdii is

synonymous with arcticus led me to describe
proschi as a new species, because it was ob-
viously distinct from the type material of B.
arcticus (Muus 1962: 18, table III).

The large amount of material now available
and examination of Verrill’s types of bairdii
made it evident that proschi is identical with
the here-reinstated bairdii and is a junior syn-
onym of that species. It should however be
pointed out that there are (subspecific?) mor-
phological differences between the American
and West Greenland populations (see below).

Bathypolypus Species Recorded from
Northwestern Spain

A paper by Perez-Gandaras & Guerra
(1978) recorded for the first time Bathypoly-
pus from 120-439 m off the Galician coast. Of
22 specimens, 14 were identified as B spon-
Salis, three as B arcticus, another three (type
A) with hesitation as B. proschi, while two de-
fective specimens could not be assigned to
any Known species.

The present revision shows that it is highly
improbable that arcticus, a true arctic species
(Fig. 20), would be found off Spain. The bo-
real bairdii, formerly confused with arcticus, is
a more likely candidate and could be ex-
pected to have its southern East Atlantic limit
somewhere in the Biscayan neighbourhood.

The data for “arcticus” given by Perez-Gan-
daras & Guerra (Table 5) seem to support this
expectation. The specimens concerned are
two males, ML: 27-39 mm, and a female, ML:
37 mm.

With few probably insignificant deviations,
the index values lie within the range of varia-
tion of bairdii (Table 9) and exclude the other
here recognized Bathypolypus species. Such
important meristic characters as number of
laminae copulatoriae (8-10) and suckers on
the hectocotylized arm (31-35) confirm affin-
ity to bairdii. One male specimen figured by
Guerra (1992: 252, fig. 89) shows a typical
bairdii form, apart from the ligula, which has a
pointed tip different from the broad-tipped
norm for bairdii known from eastern America
and the Scotland-Greenland Ridge (Fig. 6).

Some other characters described by Perez-
Gandaras & Guerra (1992) are ambiguous.
The rachidian (their fig. 7) is described as
multicuspid on account of two very small
cusps at the base. This is unusual in the
northern populations of bairdii, which have
smooth bases (Fig. 11). Further the sper-

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 195

TABLE 5. Measurements (mm), indices and counts
of three dubious “arcticus” from the Galican coast
extracted from data of Perez-Gandaras & Guerra
(1978).

Sex male male female
mE 78 102 113
ML: 27 39 42
MLTLI: 35 33 ЗИ
НМ: 85 62 62
EDI: 44 36 31
SDI: 5.2 4.4 Sr
SDEDI: 12 12 18
ALI:
| 178 179 150
Il 174 179 152
Ш 174 179 136
IV 167 167 131
HeLl: 130 115 =
OAI: 74 64 =
наи: 20 18 =
LamC 9-10 8-9 =
SHcC: 31 35 =
Зри: — 144 =
SpRI: = 31 =

matophore (their fig. 8) has a reservoir occu-
pying only 31% of the spermatophore length,
which among the Bathypolypus species is a
value known only from arcticus (Table 9).
Apart from the longer oral end, the sper-
matophore has proportions similar to what is
found in bairdii, for example, the characteris-
tic light swelling seen in the cement gland por-
tion. In arcticus, the oral end of the sper-
matophore is a thick, opaque tube with no
swelling where the cement gland is located
(Fig. 2d).

Unfortunately, funnel organ and digestive
tract were not described. Until further material
can be studied, | think the three specimens
should be assigned to B. bairdii, but with
reservation.

“Type A”, described by Perez-Gandaras &
Guerra (1978: figs. 9-11) includes one female
and two males, ML: 34-40 mm, that could not
be assigned to a known species with any cer-
tainty. The authors suggest B. proschi, but
that species is now shown to be synonymous
with bairdii. The specimens possess a pecu-
liar supraocular cirrus, which is smooth, cylin-
drical and raised at the anterior edge of a
hemispherical wart (their fig. 9). The ligula
(their fig. 10) is a simple spoon-shaped organ
with midrib and 9-12 low laminae and a small
pointed calamus, LigLl: 16-18. Compared
with Багой, it looks juvenile. Proportions of

body, spermatophore and most indices (their
table 4) are compatible with bairdii. The
rachidians have two small denticles at their
bases like the “arcticus” specimens described
above, and one radula is abnormal, having
nine rows of teeth (their fig. 11). One male
(Guerra, 1992: 254, fig. 90) has much smaller
eyeballs (EDI: approx. 27, measured on the
drawing) than indicated by Perez-Gandaras &
Guerra (1978: table 4), in which EDI is given
as 35-41, compatible with bairdii.

| think “type A” has to retain its dubious po-
sition until a larger series of specimens be-
comes available.

Bathypolypus pugniger, n. sp.

Material examined: 31 specimens from the
Iceland-Faroe area as listed in Appendix 1.

Holotype: Male, ML 32 mm, Icelandic Fish-
ery Investigations, haul B5-78-44, 64°58'N,
27°44'W, 860-870 m, March 14, 1978. Mea-
surements: Table 5, specimen 470. Deposi-
tory: IMNH 19990971.

Paratypes: 9 males, 10 females as listed
and measured in Tables 6 and 7.

Synonymy

Bathypolypus arcticus: Adam,
part]: 9, figs. 2, 3

1939 [in

Diagnosis

Ovoid or square-bodied with papillated
skin; arms very short, arm order III:IV:II:1 or of
subequal length; head broad, with large eyes,
each with a supraocular erectile cirrus; hecto-
cotylized arm with about 35 suckers; ligula
globular fleshy and deeply excavated, with
4-6 laminae; funnel organ double and pad-
like. Radula with broad homodont central
teeth; esophagus without crop diverticulum.
Total length rarely over 150 mm.

Description

Skin and Colors: Freshly preserved, the skin
is violet to purple dorsally, ventrally lighter and
yellowish or brownish. The antero-dorsal re-
gion more or less equally strewn with numer-
ous small warts. Over each eye is a 3-7 mm-
long single verrucose cirrus, which may be
bifurcated, the anterior branch being shortest,
or there are two closely set cirri.

196 MUUS

Es =
| ’

1.4,
any

=

“à

FIG. 12. Holotype of Bathypolypus pugniger, п. sp., © ML 32 mm. Depository: IMNH 19990971.

Bodily Proportions: Ovoid (Fig. 12) or more
square-bodied, like bairdii (Fig. 13), with large
eyeballs. MWI: males 67-92-106, females 73-
96-118. HWI: males 73-85-100, females 48-
79-96. TL rarely 150 mm, ML rarely over 50
mm. MLTLI: 33-37.5-42. EDI for both sexes
30-44-50, highest for young specimens. The
lens measures on an average 37% ofthe eye
diameter. The rough diagnostic index SDEDI,
useful to discriminate arcticus and bairdii,
shows pugniger to lie between the two
species (Table 9). The corresponding relation
between SD and LD is supposed to be more
accurate, but still shows some overlap with
young specimens of bairdii (Fig. 16).

The mantle aperture is about 40% of the
mantle circumference, and the funnel is free
for about 50%.

The funnel organ is a couple of pad-like
structures sometimes heart-shaped but vari-
able as in В. bairdii (Fig. 18).

The gills are reduced and have 6-7 gill fila-
ments on each demibranch.

The arm length order is IIl:IV:Il:| or sube-
qual (Tables 6, 7). This is reverse from arm
order of В. arcticus and В. Батай. The ML
constitutes 33-42% of the TL making В. pug-

niger the most short-armed of the three
species. ALI: I 145, Il 150, Ill 155, IV 154
(means in Tables 6, 7).

The web extends along the arms. WDMI: A
53, B56, C 59, D 60, E 51 (mean of 18 spec-
imens). In well-preserved specimens, web
sectors C and D tend to be subequal.

The suckers are biserial, small, and well
spaced. Each arm with 65-75 suckers. They
are of the same order of size on all arms, and
there is no sexual dimorphism. SDI: 4.7-5.
8-7.6.

The hectocotylized third right arm is usually
slightly shorter than the left opposite arm.
OAI: 83-91-106. It has 31-35-45 suckers.
Spermatophoral groove well developed.

The ligula is fleshy, short, and broad, with
firmly inrolled borders, giving it a globular ap-
pearance as a clenched fist (Figs. 12, 13c).
LigLI: 22-27-34 in 11 specimens > 25 mm ML.
The ligula has a tiny pointed tip between the
broadly rounded anterior flaps (Fig. 13a). The
width is 80-100% of the length. The spoon
has a central ridge and 4-6 deep, well-sepa-
rated laminae (Figs. 9, 13a). Calamus is stout,
CLI: 26-38-51. Already at a ML of 6 mm, the
nectocotylized arm may be identified by the

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 197

FIG. 13. Bathypolypus pugniger, п. sp. a: distal hectocotylus of young © ML 18 mm. The “clenched” ligula
was forced open to show the laminae; b: upper and lower beak of с’ ML: 33 mm; с: paratype ZMUC CEP-
17, ML 54 mm. Dana Stn. 16437, southwestern Greenland.

0.8-mm-long, as-yet undifferentiated ligula
(Table 6: no. 77).

Female Organs: The ovary is large with
blackish globular oviducal glands (Fig. 14a,
b). Proximally the oviducts are joined, distally
they are short, stout, leaving the oviducal
glands forming right angles. Supposedly ripe
eggs measure about 10 mm. They are
smooth, with fine longitudinal lines.

Male Organs: The penis with well-developed
diverticulum (Fig. 14f). The spermatophores
are of bairdii type, with sausage-shaped
sperm reservoir occupying about 55% of the
total length. SpLI: 60-100.

Beaks and Radula: The beaks do not present
distinctive features. Only four radulae have
been investigated. The rachidians deviate
considerably from both arcticus and bairdii
(Fig. 15), whereas the lateral teeth show no
distinct features. Basically, the rachidians are
homodont, but they may be very broad with
highly set or drooping shoulders (Fig. 15b, e,
respectively) or just stocky and pointed ho-
modont, with almost straight sides (Fig. 15c,
f), but different from the slim, slightly concave
homodont teeth of bairdii.

Digestive Tract
The esophagus has no crop diverticulum,
but swells to double diameter from the point

198

MUUS

TABLE 6. Measurements (mm) of male Bathypolypus pugniger, n. sp. (Note that in Tables 6 and 7 arm
length is given as average of paired arms, except for males, where AL Ill represent the left arm only. Web
depth for sectors B, C and D are likewise averages.)

No 77 76 75 659 653 463 648 470 453 122
mi 11 30 38 51 67 76 88 90 91 146
ML: 6.2 11 15 18 25 30 33 32 36 54
HW: 5.9 10 12 15 25 22 27 31 29 45
MW: 5.5 9.3 12 19 28 20 34 32 33 45
ED: 2.0 4.5 6.2 10 13 11 13 16 16 22
ED: 1.0 2.0 = 37 5.0 4.2 55 5.8 6.0 7.4
$0: 0.4 0.7 0.8 1,2 1.9 1:5 1.8 2.0 2.0 2.5
AL:
| 8.5 15 17 26 36 39 40 55 50 85
Il 8.5 15 19 26 8% 41 48 55 55 87
Ш 8.5 14 19 29 37 42 45 56 53 89
IV 9.0 = 19 28 40 43 44 55 55 87
HcL: 9.0 18 16 24 34 37 38 52 50 83
LigL: 0.8 2.4 3.1 3.5 10 8.3 13 15 13 18
LamC: 0 4 4 5 6 5 5 6 5 6
SHcC 35 Sil 32 34 31 42 34 40 40 45
WD:
A: 3.8 — = 9.5 14 14 14 20 17 33
B: 3.8 = = 11 14 15 16 19 19 35
C: 3.9 — = 10 15 15 16 20 20 38
D: 3.4 = — 10 16 15 16 20 23 40
= 3.0 — = 7.8 14 14 10 15 18 29
TABLE 7. Measures of females attributed to B. pugniger, n. sp.
No 74 649 71 455 461 466 465 456 457 464
q 38 46 55 83 c 90 100 97 99 103 c115
ML: 15 18 28 27, c 32 33 36 39 40 c 44
HW: 12 16 19 26 = 30 30 33 30 21
MW: 11 17 21 31 a 39 34 35 35 34
ED: 6.6 11 10 13 15 14 14 16 16 13
LD: — 4.0 3.6 GS 5.3 = = 5.8 5.8 5.0
SD: 0.8 1.1 1.1 21 2.0 1.9 1.9 2.0 2.2 2.1
AL:
| 18 26 29 47 53 60 58 50 58 64
Il 19 25 32 52 56 60 66 50 55 69
Ш 22 28 33 55 58 68 62 50 59 64
iv 19 26 32 55 59 60 61 52 56 65
WD:
A: 7 10 12 19 117 19 17 21 14 21
B: 6.5 11 13 16 20 18 18 23 21 21
C: 1.5 10 18 19 22 24 22 26 20 22
D: 8.5 8.5 12 19 23 23 22 22 23 24
Е: 8 8 11 20 19 19 19 Uz 16 20

where the leaf-like second salivary glands are
fastened and down to the stomach (Fig. 14c).
The two ducts from the posterior salivary
glands are separate for more than half of the
distance to the buccal mass (Fig. 14c-e). In
arcticus and bairdii, the united excretory canal
is relatively longer (Figs. 2a, 7a). When in situ,
the very large stomach rests in a deep groove

of the liver, which is almost bilobed. The spiral
caecum is scarcely coiled. There is no ink sac.

Etymology
pugniger, derived from the Latin pugnus, a

fist, alludes to the characteristic boxing glove
appearance of ligula.

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 199

FIG. 14. Bathypolypus pugniger, п. sp. a: reproductive organs of Y ML 40 mm; b: egg from same specimen,
Stn. B5-77-46, IMNH; c-e: digestive tract; f: reproductive organs of с’ ML 38 mm. A spermatophore is at-
tached to the penis. MNHT, haul 25, July 19, 1979, now ZMUC.

FIG. 15. Radula and rachidians of В. pugniger, п. sp. a and b: с’ ML 31 mm., BIOFAR Stn. 269; с: с’ ML 36
mm, Iceland Fish. Invest., haul B5-77-46; d and e: с’ ML 33 mm, BIOFAR Stn. 269; f: Y ML 33 mm, Iceland

Fish. Invest., haul B5-77-48. Scale: 100 um.

Synonyms of B. pugniger, n. sp.

A revision of specimens identified by Adam
(1939) as B. arcticus showed two specimens
to be identical to B. pugniger, n. sp. They are
juvenile, a male (ML: 22 mm) and a female

(ML: 18 mm) caught in 1938 off the east coast
of Iceland (66°23'N, 12°53’W) in 200-250 т.
Adam (1939: 10, specimens a, figs. 2, 3) fig-
ures the reproduction organs of the male and
the globose ligula with five distinct laminae
typical of B. pugniger.

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 201

TABLE 8. Measurements (mm) of a male B. pugniger, n. sp., identified as B. arcticus by Adam
(1939). Adam’s 60-year-old measurements in parenthesis.

We 59 (-) Indices
ML: 22 (25) MLTLI: 37
HW: И (19) HWI: ИТ
MW: 20 (18.5) MWI: 91

ED: 90 (-) EDI: 40
SD: 1 SES) SDI: 5.9
LigL: 62 (6:5) LigLl: 22

Table 8 shows measurements and indices
of the male specimen. Comparison with
Adam’s measurements, which were done
while the material was fresh, show consider-
able preservational shrinkage.

Recognition of B. pugniger as a
New Species

The first specimens seen were in fact iden-
tified as slightly abnormal B. bairdii. The glo-
bose ligula of the males at first believed to be
an accidental deformity or a regeneration phe-
nomenon turned out to be a regularly occur-
ring stable structure with 4-6 laminae, thus
deviating significantly from North and East At-
lantic B. bairdii (Fig. 9). This character, in com-
bination with a reverse arm order as compared
with bairdii and arcticus, deviating rachidians
(Fig. 15), and sucker and eye sizes (Fig. 16)
justified establishment of a new species.

As is usual in octopods, the hectocotylized
males offer the best distinguishing characters.
The females were only recognized after re-
newed inspection of all material from the

Arm Length
right left
) | 32 (33) 34 (36)
) || 30 (33) 35 (36)
Ш 28 (32) 33 (34)
IV = (-) = (28+)

Faroe-Iceland area that | had previously iden-
tified as B. bairdii.

The search for females of pugniger was
based on the assumption that, like in other
Bathypolypus species, there would be no sex-
ual dimorphism. This means that bodily pro-
portions, arm order and size of eyes and
suckers should match pugniger males of sim-
ilar sizes. In this way, 12 females could be at-
tributed to B. pugniger. A circumstantial evi-
dence for the plausibility of the result is the
sex ratio of 39% females, close to the sex
ratio found in bairdii (38% females; n = 235).

Morphologically pugniger is very close to
bairdii. As yet few characters distinguishing
these species have been found. Among juve-
niles and females mistaken identity between
pugniger and bairdii is possible, because the
state of preservation can blur the differences
in arm order and size of suckers and eyes.

The rough diagnostic index SDEDI useful to
discriminate between arcticus and baïirdii,
shows pugniger to lie between the two
species (Table 9). The corresponding relation
between SD and LD should be more accu-

TABLE 9. Range of distinctive indices and counts of the Bathypolypus species based on material examined.
The sparse data for valdiviae are extracted from text and drawings of Chun & Thiele (1915), Robson (1924b),
and Sanchez & Moli (1984). Data for sponsalis from the Mediterranean population.

arcticus bairdii ergasticus pugniger sponsalis valdiviae

Call 15-25 15-25 30-40 26-38-51 33-42 28-38
EDI 20-35 30-55 31-41-47 30-44-50 30-41-47 40
LamC 11-16 7-12 7 (п=4) 4-5-6 4-6-7 4-5
LigLI 9-23 18-44 6-9-12 22-27-32 10-12-15 15-18
MLTLI 24-29-35 27-33-38 13-17-23 33-38-42 16-22-26 30
OAI 83-89-100 71-88-104 53-60-70 83-91-106 47-63-73 75
SDEDI 18-26-34 6-9-13 9-11-13 11-13-16 4-6.5-8.5 13
SDI 6-7-8.4 3-4-5 3.5-4-5.5 4.7-5.1-6 2.1-3-3.7 5
SDLDI 50-70-1100 19-25-36 22-25 (n = 2) 32-35-40 15-19-22 =
SHcC 32-49 36-49 73-77-83 31-35-45 52-57-61 45
SpLI 105-130 60-90-115 110-150 90 (n = 3) 50-70 100
SpRI 26-32 50-60 50-60 50-60 50-60 50
Eye cirri present present absent present absent present
Crop divert. present absent present absent absent ?

202 MUUS

rate, but still shows some overlap with bairdii,
especially with younger specimens (Fig. 16)

In bodily proportions and hectocotylus, B.
pugniger has a striking resemblance to the
South African Bathypolypus valdiviae (Chun &
Thiele, 1915), known from the Agulhas Bank
and off the Namibian coast.

With one exception from West Greenland
(Fig. 13c, Table 6: no. 122), all specimens of
pugniger are caught on the Faroe-lceland
plateau in warm Atlantic water. The zoogeo-
graphical and ecological perspectives are dis-
cussed below.

HISTORICAL REVIEW AND
PRESENT STATUS OF BATHYPOLYPUS
AND BENTHOCTOPUS.

In 1921, Grimpe erected two new genera in
the subfamily Octopodinae to accommodate
some characteristic octopods without ink sac:
Octopus arcticus Prosch, 1849, was desig-
nated type species of Bathypolypus, and O.
piscatorum Verrill, 1879, type species of Ben-
thoctopus. Grimpe just stated that the two
species “erheblich verschieden” [differ con-
siderably], but he did not otherwise define the
genera.

To accommodate two new species (Bath-
ypolypus grimpei and Benthoctopus berryi),
Robson (1924a, b) defined the genera this
way:

“Bathypolypus Grimpe: Deepwater poly-
pods with broad and long hectocotylus and
unicuspidate rhachidian teeth. The skin is
usually covered with warts and may be gelati-
nous. There is no ink sac. Type: B. arcticus.”

“Benthoctopus Grimpe: Abyssal polypods
with small hectocotylus, multicuspid rhachid-
ian teeth and smooth skin. There is no ink sac.
Type: B. piscatorum.”

Without discussing details, Grimpe (1925:
100, footnote) declared himself in complete
agreement with Robson’s generic definitions.

With small modifications these definitions
have persisted (for example, Thiele, 1935;
Mangold & Portmann, 1989). Robson (1927)
amended the generic definitions slightly and
later (1928) erected a new subfamily, Bathy-
polypodinae, comprising the two genera and
simply defined as: “Octopods mainly abyssal
in habitat and devoid of an ink sac”.

In his monograph, however, Robson (1929,
1932) moved Benthoctopus with 14 nominal
species back to the subfamily Octopodinae,
while Bathypolypus with six nominal species

was retained in Bathypolypodinae. With the
characters of the subfamily (Robson, 1932:
286), Bathypolypus was now defined as:
“Abyssal octopodids devoid of an ink sac and
in which the crop is usually reduced and
sometimes absent. Eggs and vaginae are
large and spermatophores large and few in
number. The mantle aperture is very narrow
and the general habit squat and short armed.”

Robson removed Benthoctopus to Octo-
podinae because he felt that some of the
species resembled ordinary forms of Octopus
and that “were it not for the lack of the ink-sac
one would place them in that genus” (Robson,
1932: 51). On the other hand, Robson real-
ized that some species have traits of both
genera.

The latter problem has caused some taxo-
nomic confusion. After a redescription of
Bathypolypus sponsalis, it prompted Wirz
(1955: 146) to declare that the strict distinc-
tion between the genera Benthoctopus and
Bathypolypus made by Robson was unjusti-
fied considering the small number of differ-
ences. Thiele (1935) reunited the two genera
in Bathypolypodinae.

As presented by Robson, the two genera
represent an array of species that in varying
degrees demonstrate traits believed to reflect
adaptation to benthic life in deep water-ab-
sence of ink sac; reduction of gills, radula, and
crop; increasing size of eggs and sper-
matophores; elaboration of ligula; shortening
of the arm complex; and funnel organ tending
to be double.

In general, the least specialized species,
that is, those most similar to Octopus, seem to
be accommodated in Benthoctopus, the most
divergent in Bathypolypus.

The present revision shows that most of the
existing confusion derives from misidentifica-
tions that have led to errors in the concept of
the two type species and subsequent misun-
derstandings in the generic definitions of Ba-
thypolypus and Benthoctopus.

The choice by Grimpe (1921) of Octopus
piscatorum as type for Benthoctopus was es-
pecially unlucky, because an examination of
the type shows it to be identical with the here-
reinstated Bathypolypus bairdii (Verrill, 1873).

Aware of this fact and in connection with the
description of five new species of Benth-
octopus from the Pacific, Voss & Pearcy
(1990) plead for the preservation of the name
Benthoctopus, which now includes 20 species
worldwide (Sweeney & Roper, 1998).

Voss (1988a, b) restricted the subfamily Ba-

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 203

B.arcticus
n=33

B.arcticus

B.pugniger n.sp.

B.bairdii

1 2 3 4 5 6 7 8 9 10 11

FIG. 16. Sucker diameter versus lens diameter in arcticus, pugniger, n. sp., and bairdii. Reduced major axis
regressions show that the relation differs among arcticus and bairdii and among arcticus and pugniger at the
P = 0.01 level of significance. Among pugniger and bairdii at = > 0.1 < 0.05. Regression slopes with 95%
confidence intervals and coefficient of determination: 0.8372 (0.7177-0.9766; 0.9766) for arcticus; 0.4112
(0.3541-0.4774; 0.9025) for pugniger, and 0.2643 (0.2294-9.3045; 0.7921) for bairdii. The regression in-
tercepts are 0.1614, 0.0641, and 0.0592 respectively and not considered different in pairwise comparison
among species.

Inserted in upper figure: data for the type of B. arcticus (crossed square), the “type” of B. faeroensis found in
ZMUC (star) and the holotype of Benthoctopus piscatorum (cross in circle).

204 MUUS

thypolypodinae to benthic octopods with bise-
rial suckers and devoid of ink sac and pro-
posed the following definition of Benthoctopus
Grimpe, 1921:

“Deepwater octopods of normal Octopus-
like appearance with short to long arms, suck-
ers biserial; hectocotylus Octopus-like, ligula
slightly to moderately excavated with indis-
tinct midrib, smooth or bearing low, often in-
distinct rugae, never laminate; crop present,
usually with diverticulum; ink sac absent:
radula with strongly, seldom weakly, multicus-
pid rachidian; body entirely smooth, papillae
or ocular cirri absent. Type species Octopus
piscatorum Verrill, 1879, by original designa-
tion.”

If the genus Benthoctopus is to be pre-
served, it would need a different type species
to be designated by the International Com-
mission on Zoological Nomenclature. A sensi-
ble choice would be Benthoctopus januarii
(Hoyle, 1885). This species is revised and
thoroughly redescribed by Toll (1981) based
on a series of recently caught male and fe-
male specimens. It conforms to the diagnosis
of Benthoctopus suggested by Voss and fur-
thermore has the advantage of being a wide-
spread species in the Gulf of Mexico and off
Brazil.

The final decision and the plea to the Inter-
national Commission on Zoological Nomen-
clature, however, should be made by a reviser
of the Benthoctopus species.

The genus Bathypolypus should under any
circumstances be preserved. Robson, being
the first revisor of the two genera, acknowl-
edged Octopus arcticus as type of a group of
species characterized by large lamellated
ligulae, as opposed to another group of
species characterized by small Octopus-like
ligulae, the present Benthoctopus group. To
uphold the distinction between the two groups
and to match the definition of Benthoctopus
by Voss & Pearcy (1990). | propose the fol-
lowing modification of Robson’s definition of
Bathypolypus in an attempt to make an oper-
ational distinction between the two genera:

Bathypolypus Grimpe, 1921. Type species:
Octopus arcticus Prosch, 1849, by original
designation.

Deepwater octopods of normal Octopus-
like appearance, with stout body and gener-
ally with short arms; suckers biserial; hecto-
cotylus with deeply excavated ligula bearing a
number of well-defined laminae; crop, if pres-
ent, seldom with diverticulum; ink sac absent;
radula with homodont or weakly and irregu-

larly multicuspid rachidians; skin usually with
papillae; supraocular cirri often present.

GENERAL SURVEY OF THE
BATHYPOLYPUS SPECIES

The elaborated hectocotylus, with a promi-
nent, deeply excavated, laminated ligula, is
the most distinctive character in the amended
generic definition of Bathypolypus. In addi-
tion, there are reductions in digestive tract,
gills, radula, relative arm length, and the en-
largement of eyes exhibited by the species in
varying degrees. A tendency to develop some
of these traits is found in Benthoctopus. The
difference between the hectocotyli of the Ba-
thypolypus and the Benthoctopus species
provides sufficient substance to justify pre-
serving the two genera, if only as a prelimi-
nary measure. For the time being, our igno-
rance regarding evolutionary sequence and
weighting of derived characters in Octopodi-
dae precludes serious phylogenetic consider-
ations.

Bathypolypus comprises six species in the
most recent list of accepted cephalopod taxa
(Sweeney & Roper, 1998): arcticus (Prosch,
1849); faeroensis (Russell, 1909); proschi
Muus, 1962; salebrosus (Sasaki, 1920);
sponsalis (P. Fischer & H. Fischer, 1892); and
valdiviae (Chun & Thiele, 1915).

The species proschi and faeroensis are
here shown to be synonyms of the reinstated
bairdii and arcticus respectively, whereas
pugniger is recognized as new. It remains,
however, to reconsider the remaining three of
the hitherto recognized species in the light of
the amended generic definitions of Bathypoly-
pus and Benthoctopus. Also, the position of
Benthoctopus ergasticus (P. Fisher & H.
Fischer, 1892) is reconsidered.

Benthoctopus salebrosus
(Sasaki, 1920), new comb.

Polypus salebrosus Sasaki, 1920: 182; 1929:
99, text-fig. 54, pl. 6, figs. 5, 6.

Bathypolypus salebrosus: Robson, 1929: 41;
1932: 302; Akimushkin, 1965: 134, fig.
34; Nesis, 1987: 315, fig. 83A.

Type and paratype: two females, USNM
332969, TL: 153 mm, ML: 45 mm; USNM
332970, TL: 77 mm, ML: 23 mm (not ex-
amined).

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 205

A characteristic, though not well-known
species from the Okhotsk Sea and off the
Japanese Pacific coast in 212-1070 m.

Based on the female types, Robson (1929,
1932) assigned salebrosus to Bathypolypus
with hesitation, mostly on account of its short
arms, rather deep web (33%), and because it
is sculptured with closely set, well-defined,
roundish warts. He found that the rachidians
were weakly multicuspid, with at most one
denticle on each side, and he established the
absence of an ink sac.

The hectocotylus was later described by
Akimushkin (1965: fig. 34) and Nesis (1987:
fig. 83A). The ligula is narrow, conical and
pointed, LigLi about 14. The central groove is
shallow and transversely striated with numer-
ous indistinct rugae. Calamus is short and
pointed.

Benthoctopus salebrosus has no supraocu-
lar cirri. The funnel organ is figured by Sasaki
(1929: text-fig. 54) as a single W, the outer
legs being the shortest. Gills not much re-
duced, each demibranch with 9-10 filaments,
the mantle aperture correspondingly moder-
ate (B-C in Robson’s terminology).

The slim, striated ligula unknown to Robson
is of a type seen in some Benthoctopus
species but very different from the highly spe-
cialized ligulae of Bathypolypus arcticus and
B. bairdii.

| conclude that salebrosus is best placed in
the genus Benthoctopus due to the simple
non-laminated ligula and the single funnel
organ, irrespective of its warty skin, which is
unusual among other species of that genus.
No other character contradicts this opinion.

Bathypolypus sponsalis
(P. Fischer & H. Fischer, 1892).

Octopus sponsalis P. Fischer & H. Fischer,
1892: 297, fig. A; Fischer & Joubin 1907:
322, pl. 22, figs. 5-11

Bathypolypus sponsalis: Robson, 1927: 252;
1932: 300, figs. 61-63; Wirz, 1954: 139,
figs. 1-5; 1955: 129, figs. 1-12; Adam,
1960: 504, fig. 2; Mangold-Wirz, 1963:
49; Perez-Gandaras & Guerra, 1978:
195, figs. 2-5; Nesis, 1987: 315, fig. 831;
Guerra, 1992: 249, figs. 88, 91.

Material examined: Syntypes of Octopus
sponsalis P. Fischer & H. Fischer, 1892,
MNHN 571097: 4 males ML: 27-41 mm,
Exp. du “Talisman”, 332-1250 m, NW-
Africa; in ZMUC, 8 males, ML: 33-49 mm
and 3 females ML: 27-38 mm, Catalan

Sea, western Mediterranean, June-
September 1954 and June-September
1955, 200-500 m; in BMNH: one female,
ML: 36 mm, Exp. du “Talisman”, July 12,
1885.

This species was originally caught off west-
ern Sahara (22°N, 19°46’E) at the same lo-
cality as В. ergasticus. It has since been re-
ported as a common mesobenthic species in
the western Mediterranean (Wirz, 1954,
1955), the Aegean Sea (D’Onghia et al.,
1993), and off Portugal and Galicia (Perez-
Gandaras & Guerra, 1978).

Good pictures of the whole animal and the
hectocotylus are presented by Fischer &
Joubin (1907: pl. 22), radula and internal or-
gans by Robson (1932: figs. 61-63), and Wirz
(1954: figs. 3-5; 1955: figs. 5-12).

Robson (1927, 1932) placed sponsalis in
Bathypolypus because of its apparent affini-
ties to the arcticus-group: a squat, relatively
short-armed body with huge eyes, small suck-
ers, double funnel organ, a reduced radula
with homodont rhachidians, lack of crop, and
a ligula that is deeply excavated, with 6-7
laminae.

The morphometrics and major features of
the life cycle of the Mediterranean population
of sponsalis are well known (Wirz, 1955;
Mangold-Wirz, 1963) based on about 600
specimens caught at all times of the year.
Table 9 gives the main distinctive indices
based on my own measurements of speci-
mens from the Catalan Sea.

There are, however, some discrepancies
between these data and descriptions of spec-
imens from the type locality (Cape Verde Is-
lands). Examination of the syntypes (MNHN
571097) shows that the Mediterranean popu-
lation deviates in several important traits:

Two spermatophores extracted from one of
the syntypes confirmed Robson’s observation
(1932) that the sperm reservoir is long, SpRI:
65-70 (versus 50-60 in the Mediterranean).
The short oral end has a distinct swelling in
the middle, making it spindle-shaped, unlike
any other spermatophores of the genus (Fig.
17). The apical end is swollen compared with
the smaller and more cylindrical reservoir in
Mediterranean specimens.

Calamus is very large and fleshy (Fischer &
Joubin, 1907: pl. 22, fig. 6). In four syntypes, |
found CaLl: 57-71 (Medit.: 33-42). The dif-
ference was noted by Wirz (1955).

The ligula is larger in the type series, LigLI:
14-22 (n = 5) (Medit.: 10-15), and the num-

206 MUUS

1cm

FIG. 17. Spermatophores of B. sponsalis. Left: syn-
type, ML 41 mm., Cape Verde. Right: specimen
from the Catalan Sea, western Mediterranean, ML
44 mm.

ber of suckers on the hectocotylus is low,
SHcC: 44-50 (n = 4) (Medit.: 57-61).

The type specimens have warts or pustules
over the eyes and often on the antero-dorsal
region. Adam (1960: fig. 2) found supraocular
cirri, as well as a mixture of multifid and sim-
ple warts sprinkled dorsally on two specimens
from the Cape Verde Plateau. In contrast, the
Mediterranean and Galician specimens have
smooth skin without traces of warts.

| conclude that the morphological devia-
tions between populations may be rooted in
subspecific variation or even represent unrec-
ognized sibling species. Not least, the mor-
phology of hectocotylus and spermatophores
speak for the latter possibility. The Mediter-
ranean population is well documented, but
more specimens from the Cape Verde Pla-
teau are needed. Regardless, the specimens
hitherto identified as sponsalis clearly belong
to the genus Bathypolypus.

Bathypolypus valdiviae
(Chun & Thiele, 1915)

Polypus valdiviae Chun & Thiele, 1915: 485,
text-figs 52, 53, pl. 80, figs. 1-5

Bathypolypus grimpei Robson, 1924a: 208;
1924b: 663, text-figs. 37-41, pl. 2, fig. 10

Bathypolypus valdiviae: Massy, 1927: 165;
Robson, 1932: 303, figs. 64-68; San-
chez & Moli, 1984: 19, fig. 18; Nesis,
1987: 315, fig. 83F-H

Type: Z. M. Humboldt Univ. Berlin. Male, ML:
approx. 30 mm (not examined).

This species is known from the South
African Agulhas Bank and off the Namibian
coast.

Bathypolypus valdiviae has a remarkable
resemblance to B. pugniger, n. sp, being a
short-armed, big-eyed, warty species usually
with supraocular cirri and having the same
kind of characteristic hectocotylus. Ligula is
globular and deeply excavated with 4-5 well
developed laminae. LigLl: 15-18. The hecto-
cotylized arm bears about 45 suckers. HcLl:
approx. 75. The arms are of subequal length
(Chun & Thiele, 1915: pl. 1, fig. 4; Robson,
1924b: fig. 37, as “B. grimpei”).

Also in other characters valdiviae shows
affinity to pugniger and bairdii. The funnel
organ is a pair of widely separated V-shaped
pads (Robson, 1932: text-fig. 66); the rhachid-
ians are homodont, but more pointed than the
broad teeth in pugniger; the spermatophore is
a true copy of bairdii and pugniger sper-
matophores (Robson 1924b: text-figs. 39,
41).

| conclude that placement of valdiviae in
Bathypolypus is justified.

Bathypolypus ergasticus
(P. Fischer & H. Fischer, 1892), new comb.

Octopus ergasticus P. Fischer & H. Fischer,
1892: 298, fig. B; Fischer & Joubin, 1907:
325, fig. 2A, pl. 22, figs. 1-4

Octopus profundicola Massy, 1907: 277.

Polypus ergasticus: Massy, 1909: 7, pl. 1,
figs. 1-3, pl. 2, fig. 1; 1913: 1; Robson,
1924b: 668

Benthoctopus ergasticus: Grimpe, 1921: 300;
Robson, 1927: 255, figs. 4-6, 9; 1932:
244, figs. 44, 45; Nesis, 1987: 320, fig.
84E, F.

Material examined: Syntypes of Octopus er-
gasticus P. Fischer & H. Fischer 1892,
MNHN 5811: 3 juv. males ML: approx.
17-35 mm, Exp. du “Talisman” 1883, 830
m, NW-Africa; in BMNH: syntypes of
Polypus profundicola Massy, 1907: 3
males, ML: 50-80 mm; 3 females, ML:
57-61 mm. “Helga” stations 363, 365,
369, 400, 477 and 489, approx. 51°25'N,
11-12°W, 385-720 fath. One male er-

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 207

gasticus, ML: 45 mm, NW-Africa (gift
from Prof. Joubin).

This characteristic species originally caught
off western Sahara (22°24'N, 19°46’E, 860
m) is well described by Fischer & Joubin
(1907: pl. 22) and by Massy (1907, as O. pro-
fundicola; 1909: pls. 1, 2) based on speci-
mens caught off western Ireland. Robson has
described reproductive organs, radula (1927:
figs. 4-6, 9), and the digestive tract (1932:
figs. 44, 45).

The main indices of ergasticus are given in
Table 9 based on my own measurements. Ad-
ditional data in Massy (1909).

Robson found it difficult to accommodate
ergasticus in either Bathypolypus or Benth-
octopus. He decided on the latter genus, how-
ever, mainly because he, in contrast to Massy,
found that the rachidians are multicuspid and
because the esophagus has a crop diverticu-
lum. As mentioned earlier, Robson confused
the type species of the genus, Benthoctopus
piscatorum, with specimens of Bathypolypus
arcticus from the Faroe Channel. Multicuspid
rhachidians and a crop diverticulum are found
in the latter species and are thus not unique to
Benthoctopus.

Robson (1932: 247) admits that the hecto-
cotylus of ergasticus is unlike those of other
Benthoctopus species. The hectocotylized
arm is short (OAI: 60) with a well-developed,
deep, spoon-shaped ligula with about 7
strong laminae (LigLI: 9). Calamus prominent
(CaLl: 30-40). The hectocotylus has a strong
likeness to that of sponsalis. Chun & Thiele
(1915: 485) consider it obvious that ergasti-
cus, sponsalis, and valdiviae are related due
to the form of the hectocotylus.

Another character showing affinities to the
Bathypolypus species is the enormous sper-
matophore. One specimen with a total length
of 111 mm, had a sperm reservoir of 51 mm in
length and 8 mm in width. Apart from the size,
the proportions are very much like the sper-
matophores of bairdii and pugniger (Robson,
1927: 255, fig. 4).

The funnel organ is double, and in the spec-
imens from Ireland consists of two very char-
acteristic subquadratic or pentagonal pads
(Fig. 18), a type unknown in Benthoctopus,
but not far from the structures in bairdii and
pugniger. In the specimen from West Africa,
the funnel organs each had a slight indenta-
tion anteriorly, making them more heart-
shaped.

In the light of the amended generic diag-

noses given, ergasticus is best accommo-
dated in genus Bathypolypus.

The genus Bathypolypus, as here recog-
nized, thus includes only the following six At-
lantic species: arcticus (Prosch, 1849), bairdii
(Verrill, 1873), pugniger, n. sp., sponsalis (P.
Fischer & H. Fischer, 1892), valdiviae (Chun
& Thiele, 1915), and ergasticus (P. Fischer &
H. Fischer, 1892). The species salebrosus
(Sasaki, 1920) is moved to Benthoctopus on
account of the simple, non-laminated hecto-
cotylus and single funnel organ.

DISTRIBUTION OF BATHYPOLYPUS

Bathypolypus arcticus, s. str., are confined
to Norwegian Sea Deep Water (NSW),
whereas the squat, broad-headed species (B.
bairdii, B. pugniger, n. sp.) are found in the
warmer Atlantic water south of, or on top of,
the Greenland-Scotland Ridge.

This is brought out in Figure 19, which is
based on the updated hydrographic and topo-
graphic rewiew papers by Hansen (1985) and
Westerberg (1990) in connection with the
BIOFAR benthic projects. It is seen that sub-
zero NSW fills the trough of the Faroe-Shet-
land Canal 500-700 m under the north-going
warm Atlantic Current. Almost barred in the
south by the Wyville Thomson Ridge, the
main flow of NSW is forced to make a bend
round the southern end of the Faroe Plateau
and flows through the Faroe Bank Canal to
reach, and eventually slide down, the south-
ern slopes of the Iceland-Faroe Ridge mixed
with Atlantic water.

Northern species — В. arcticus, В. bairdli,
B. pugniger, n. Sp.

Bathypolypus arcticus

Horizontal Distribution: Figure 19 clearly
demonstrates the stenothermal arctic affinity
of arcticus. Specimens caught outside NSW
are invariably in places intermittently exposed
to overflow of cold water, which on the Faroe-
Iceland Ridge often is a mixture of NSW and
Arctic Intermediate Water (Al) generated in
the Iceland and Greenland seas.

The arctic affinity of arcticus is confirmed by
its wider distribution in the cold parts of the
Barents Sea, off eastern Greenland, and in
the cold threshold fiords of southwestern

208 MUUS

B.bairdii

CO 0000 00 Uv
JO CI OD Vo 000

B.pugniger

000000 OV MY q pp

B.arcticus

У VVVVYV Уиуму м

B.ergasticus

VO VOUJDOOD OQ

B.sponsalis

VAYAVIVAVAVAVIVA VEUVE,

FIG. 18. Variability of funnel organ in five North Atlantic Bathypolypus species. The left pair in each row is

considered typical of the species.

Greenland, not influenced by the warm
Irminger Current (Fig. 20). According to Kon-
dakov (1936), arcticus occurs in the Kara Sea
(78°01'N, 105°27'W, 175 m, —0.64°C). | have
not had the opportunity to verify this identifi-
cation, but Kondakov’s drawing looks con-
vincing.

Bruun (1945: 8) in his treatise of Icelandic
cephalopods, though following Robson’s

sensu lato concept of arcticus, is the only au-
thor who noticed that the narrow-headed
specimens occur off northern Iceland,
whereas the “bairdii form” is found off the
south and southeast coast of Iceland, that is
south of the North Atlantic Ridge.

The material of adult arcticus are too few to
disclose possible geographic variation in
meristic characters.

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 209

5

4 3 2

== D

ZEN 7 Q
/ Foula 60
4 (SHETLANDS)
р /

AH
72 |
\

+ am
Bee E > N

FIG. 19. Distribution in Faroese waters of B. arcticus (squares), B. bairdii (dots) and B. pugniger, n. sp.
(stars). Arctic deep water with negative temperatures darkly screened. In northwest occasional overflow of

arctic water is indicated.

Bathymetrical Distribution: Based on the
present material, the depth range of arcticus
is 37-565-1210 m. Only in the northernmost
localities is the species caught in less than
100 m. In the south, optimal low temperatures
are found at greater depth, usually over 400
m. Juveniles and sexually mature animals are
evenly scattered at all depths.

Bathypolypus bairdii
Horizontal Distribution: This species seems

to prefer Atlantic water masses with tempera-
tures in the range of 2-8°C (Figs. 19, 21). Due

to the warm Norwegian Current, bairdiiranges
from the northern North Sea and Skagerrak
along the Norwegian Coast to the southern
parts of the Barents Sea. It is common on the
southern slopes of the Iceland-Greenland
Ridge, but is probably barred from southern
Greenland by the eastern Greenland Polar
Current. The southeastern Atlantic limit for
bairdii seems to be northwestern Iberian wa-
ters (Perez-Gandaras & Guerra 1978).

In western Greenland, bairdii is found in
water tempered by the warm Irminger Cur-
rent, and it is common on the prawn trawling
grounds and fishing banks south of the ridge

FIG. 20. Distribution of Bathypolypus arcticus (Prosch, 1849). Open circles: positions not precisely known.

separating Davis Strait and Baffin Bay. There
are fewer records along the Labrador coast,
but the Newfoundland area seems to offer the
species excellent conditions. The Labrador
Current, with admixture of warmer Atlantic
water, fills the deeper parts (> 100-200 m) of
the Laurentian Channel with water that rather
constantly holds 2-5°C (Brunel et al., 1998).
Macalaster (1976, as “B. arcticus’) records an
average temperature of 4.3°C (SD: 1.7°C) for
stations where the species was caught.

The species was reported as far south as
Fowey Rocks, Miami (Boone, 1938). Cairns
(1976) showed bairdii to be the most abun-
dant cephalopod in the Straits of Florida.

Geographic variation: Bathypolypus bairdii is
distributed in a many thousand km long, nar-
row band largely following the upper 180-
1000 m of the continental slopes of the North
Atlantic (Fig. 21). As it probably is a rather sta-
tionary animal and having non-pelagic young,
a certain clinal geographic variation could be
expected. In bodily proportions, there does
not seem to be variation. But characters of

the hectocotylus show statistically significant
geographic variation.

Populations south of 45°N have larger ligu-
lae (Fig. 10), with more laminae (Fig. 9) but
fewer suckers on the hectocotylized arms
(Fig. 8) than populations from Labrador and
western Greenland. As regards the number of
laminae, the Newfoundland area acts as a
transitionary zone (Fig. 9). The clinal variation
may be due to genetic differencies, or it may
be a response to different ecological or hydro-
graphic regimes.

Small but perceptible geographic variation
exists between the population from Labrador
and western Greenland and their eastern At-
lantic fellows. The Cape Farewell area at the
southern tip of Greenland seems to form a
distributional gap (Fig. 21). The occurrence of
B. arcticus in this area (Fig. 20) suggests that
the eastern Greenland Polar Current, which
sweeps the slopes causes the absence of
bairdii. In the eastern Atlantic, the ligula has
more laminae (Fig. 9) and the hectocotylus
slightly fewer suckers than found in Labrador
and western Greenland. The relative size of

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 211

FIG. 21. Distribution of Bathypolypus bairdii (Verrill, 1873). Dots: specimens examined by the author, open
circles: specimens treated by Kumpf (1958) or Macalaster (1976), but not examined by the author.

ligula (LigLl) is the same for both populations
(Fig. 10).

The geographic variation is established for
the males only, and it is most distinct in the
western Atlantic population south of New-
foundland. At the present state of knowledge |
do not think a subspecific division is advis-
able. The two major dividing areas, New-
foundland and Cape Farewell, southern
Greenland, have led to three slightly different
populations.

Bathymetric Distribution: Kumpf (1958) states
the depth range of his large western Atlantic
material of bairdii to be 20-350-1545 m. In
the Strait of Florida, Cairns (1976) found the
depth range to be 190-365-674 m. The
southernmost record (29%45'N, 30°09’W)
was also the deepest. Similar depth ranges
were found off Greenland (maximum 1100 m)
and in the East Atlantic (maximum 910 m).

Juveniles and sexually mature animals are
found at all depths, and there are no evidence
of vertical migrations.

Habitat and Biology: The vast majority of
specimens are caught in trawling grounds on
muddy or sand-mixed bottoms, which seem to

be its natural habitat. The catches suggest a
rather even dispersion. Underwater photos
from the western Greenland prawn grounds
showed bairdiiin its natural surroundings. The
animal was seen resting unprotected in an ev-
idently self-made shallow depression with the
arms neatly curled along its sides. The stom-
ach contents of simultaneously caught speci-
mens showed a variety of polychete bristles
and crustacean remains, including Pandalus.

Based on aquaria observations, O’Dor &
Macalaster (1983) suggest that bairdii prac-
tises a sit-and-wait feeding strategy, and they
list food items demonstrating its omnivorous
nature.

Underwater photos confirm the supposed
feeding strategy. In the prawn trawling
grounds, bairdii is surrounded by a rich food
supply of roaming crustaceans, polychetes,
and molluscs.

Mortality must be very low. O’Dor & Macal-
aster (1983) show that a three-year lifespan,
including one reproduction period, is probable,
but that a longer life cannot be ruled out. They
estimate that due to the few eggs (at most 100)
and even fewer hatchlings, about 4% of the lat-
ter have to survive to replace the parents.

To deposit and guard the eggs, the females

212 MUUS

need a firm substratum. This may explain why
| found a skewed sex ratio on the level trawl-
ing grounds poor in shelters (38% females, n
= 235). Hiding egg-guarding females are less
apt to be caught in a trawl.

Brooding females may be found in rocks
(Macalaster, 1976) or in cans and containers
discharged from ships (my observation) and
even in plastic bags (Bergstrom, fide Macal-
aster, 1976).

Wood et al. (1998) observed mating behav-
iour and brooding of B. bairdii (called “B. arcti-
cus’) from Fundy Bay, Canada. In aquaria,
brooding of eggs lasted over 400 days (at
6-10°C) or roughly one-third of the stipulated
lifetime of bairdii. Hatchlings had a mantle
length of 6 mm.

Bathypolypus pugniger, n. sp.

Horizontal Distribution: While arcticus and
bairdii present a neat picture as distinct al-
lopatric species, B. pugniger, n. sp., brings
new problems through its enigmatic system-
atic position and peculiar distribution. The sin-
gle find from western Greenland (Table 6: no.
122) and the specimens from the Faroe-Ice-
land Ridge are all caught in waters with posi-
tive temperatures but in localities episodically
exposed to overflow of arctic water from the
realm of arcticus. | find this distribution sug-
gestive and propose three different specula-
tive explanations:

(1) В. pugniger is a stunted form of В. bairdii.

Adult bairdii living on the Faroe-Iceland
Ridge may endure periods of overflow with
arctic water. Brooding females probably stay
on. Is it possible that negative temperatures
during embryonic development result in
stunted development? If this is the case, why
are there not transitional stages between
bairdii and pugniger?

(2) В. pugniger is a hybrid between bairdii
and arcticus.

The similarity in lifestyle, habitat, and mor-
phology of the two species suggests that mat-
ing behaviour may also be similar. Is it possi-
ble that male arcticus in places with episodic
overflow of arctic water extend their territory
and seduce (rape?) female bairdii left behind
and perhaps less resistant, chilled as they
are? This and the previous suggestion could
explain the peculiar distribution of pugniger in
a narrow zone bordering the arcticus habitat.

(3) B. pugniger is a valid species.

It has been overlooked and confused with
bairdii. The Bathypolypus species of the East
Atlantic are not yet well Known and more ma-
terial will show a wider distribution of pug-
niger.

The question can best be solved by molec-
ular approaches, such as protein elec-
trophoresis or DNA-sequencing using fresh
material not previously preserved in formalin.

Bathymetric Distribution: The depth range of
20 stations where B. pugniger was caught is
200-610-1000 m.

The Southern Species-B. sponsalis,
B. ergasticus, B. valdiviae

Bathypolypus sponsalis is an East Atlantic
species, which replaces bairdii from the Gali-
cian coast to the Cape Verde Islands. It is
widespread in the Mediterranean. It seems to
prefer the same habitats as bairdii and has a
similar bathymetric distribution: 170-1250 m
(P. Fischer & H. Fischer, 1892; Wirz, 1955;
Perez-Gandaras & Guerra, 1978). Some evi-
dence of upslope ontogenetic migration was
found in the eastern Mediterranean by Vil-
lanueva (1992).

Bathypolypus ergasticus occurs off south-
western Ireland and the Cape Verde Islands
but is not known from the Mediterranean. In
its northern distribution, it overlaps bairdii and
further south sponsalis. However, ergasticus
prefers deeper water than its congeners:
704-1350 m off Ireland (Massy, 1909), 932-
1139 m off Cape Verde Islands (P. Fischer &
H. Fischer, 1892). The three species are para-
patric.

Bathypolypus valdiviae is the only repre-
sentative of the genus known from the south-
ern hemisphere. Off the Namibian coast and
on the Agulhas Bank, the species does not
seem to be rare on soft bottoms. Bathymetric
range: 500-900 m (Chun & Thiele, 1915;
Massy, 1927; Roeleveld, 1974; Sanchez &
Moli, 1984).

REMARKS ON THE
IDENTIFICATION OF SPECIES

The application of bivariate ratios (indices)
in multivariate statistical analysis is inadvis-
able (Atchley et al., 1976), but ratios are use-
ful in taxonomic work, such as in keys. They
are also used here for comparison with earlier

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 213

published data. Ratios based on variables
with isometric or approximately isometric
growth (e.g., SDI, LDI, ОА!) are acceptable
tools for identification of species and of
course so are indices based on size-indepen-
dent meristic characters. Allometric growth,
which may impair the usefulness of indices, is
not prominent in the Bathypolypus species
within the relevant size range of adolescent
and adult animals (ML 20-60 mm). It is mod-
est even where it can best be demonstrated
(EDI, Fig. 8, HWI), and generally intraspecific
inherent variability and artifacts far exceed
and mask variation due to allometric growth in
these phenetically similar species. For pur-
poses of species identification, the allometric
variation was less than overall variation and
was not calculated. Regrettably, interspecific
overlap is often unavoidable (Table 9). The
parameters are normally or approximately
normally distributed, and the application of
several indices tends to obliterate marginal
values and leads to clearer discrimination of
species, supplementing other, more tangible
distinctive characters (funnel organ, crop,
radula, sculpture, meristic characters).

An important point that the present revision
brought out is that the number of suckers on
the hectocotylus is individually constant, even
from the juvenile stage, and that the number
of laminae is constant from the onset of matu-
rity, irrespective of later increase in size (Figs.
22, 23) Counts of suckers and laminae are
good size-independent meristic characters to
use in concert with other characters, even if
cases of overlap exist (Fig. 9, Table 9). The
relative constancy of SHcC was also demon-
strated by Toll (1988), who used differences in

Laminae

о B.arcticus n=35
e B.bairdii n=48

10 20 30

SHcC as additional argument for taxonomic
separation of Atlantic and Pacific Scaeurgus.
In the radula, only the rachidians are dis-
tinctive in the Bathypolypus species. They
may be very uniform (bairdii, sponsalis), in
others very variable (arcticus, pugniger, n.
sp.). A striking example of similar variability is
seen in Graneledone pacifica, in which the
rachidians range from heterodont B8 seriation
to simple homodont or even degenerate con-
dition (Voss & Pearcy, 1990: 87, fig. 19).

KEY TO THE NORTH ATLANTIC
BATHYPOLYPUS SPECIES

The majority of specimens may be identi-
fied by outer distinctive features, supple-
mented by inspection of the funnel organ (Fig.
18). Additional help may be gained from Table
9, which shows ranges of essential indices
and counts. For juveniles, females, aberrant
males, and less well-preserved specimens,
however, inspection of radula, digestive tract,
and spermatophores may be necessary.

1a. Mantle length usually more than 25% of
total length. Arms with fewer than 100
suckers. Length of hectocotylus at least
70% of opposite (3, left) arm and with
fewer than 50 suckers. Skin often warty
and with a pointed cirrus overeach eye .2
1b. Mantle length usually less than 25% of
total length. Arms with over 140 suckers.
Length of hectocotylus at most 70% of op-
posite arm and with over 50 suckers. Skin
smooth, supraocular cirri absent ...... 4
2a. Body egg-shaped, with a constriction be-
hind the head. Diameter of eyes less than
33% of ML. Largest sucker diameter

40 50 60 ML

FIG. 22. In Bathypolypus species the number of laminae on ligula seems to be individually constant from the
onset of maturity. Regress. slopes: < +/- 0.023, 1”: < 0.015 (data for bairdii from western Greenland).

214 MUUS

Suckers hect. arm

B.arcticus

10 20 30

40 50 ML mm

FIG. 23. In Bathypolypus species, the number of suckers on the hectocotylized arm seems to be constant

throughout life. Regress. slopes: < +/— 0.03,

6.5-8.5% of ML and 18-33% of eye di-
ameter. Ligula with 11-16 laminae; funnel
organ a clear-cut VV (Fig. 18); esophagus
with prominent crop diverticulum; radula
usually with heterodont rachidians (Fig.
5); seminal reservoir occupying only 30%
of the spermatophore length .. . .arcticus
2b. Body squat; diameter of eye-balls 30-
45% of ML; largest sucker diameter less
than 6.5% of ML and at most 6-16% of
eye diameter. Funnel organ pad-like,
never a clear-cut VV. Ligula with less than
13 laminae. Esophagus without crop di-
verticulum; radula homodont; seminal
reservoir occupying about 50% of sper-
matophore length
3a. Arm length order usually: 1.2.3.4. Ligula
large, oblong, with 7-12 laminae; cala-
mus 15-25% of ligula length; radula with
long, slim homodont rachidians (Fig. 11)
AM НИЕ bairdii
3b. Arm length order usually 3.4.2.1 or sube-
qual. Ligula globular, fleshy, deeply hol-
lowed, with 4-6 laminae; calamus
26-51% of ligula length; radula variable
but with broad homodont rachidians (Fig.
| =) Pa eee a ere eC ren pugniger, n. sp.
4a. Hectocotylus with 70-85 suckers; other
arms usually with over 200 suckers. Fun-
nel organ almost square pads (Fig. 18).
Esophagus with crop diverticulum. Sper-
matophore very large, longer than ML .er-
gasticus

г = 0.02.

4b. Hectocotylus with 50-65 suckers; other
arms with 140-200 suckers. Funnel organ
VV (Fig. 18); esophagus without crop di-
verticulum. Spermatophore shorter than
MIE ee sponsalis

ACKNOWLEDGEMENTS

| am indebted to the staff of the BIOFAR
and BIOICE projects for access to the valu-
able recent collections from the Greenland-
Scotland ridge and the Faroe-Shetland Canal.
Dr. C. C. Lu kindly supplied me with speci-
mens of B. bairdii from Canadian waters and
Dr. Katharina Mangold blessed me (and
ZMUC) with a collection of B. sponsalis from
the Catalan Sea. With gratitude | have re-
ceived advice and/or valuable information
from Dr. G. Perez-Gandaras, Dr. K. Nesis, Dr.
C.F.E. Roper, and, not least, from the late Dr.
G. Voss, with whom | had fruitful discussions.
Finally, | wish to thank the curators of mol-
luscs at the zoological museums in Bergen,
Berlin, London, Oslo, and Washington for
pleasant stays and help at various phases of
my work. In ZMUC Stine Elle, Tom Schiotte
and Gert Brovad offered me invaluable tech-
nical assistance. My wife Kirsten Muus and
two unknown but meticulous referees have

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 215

improved the first version of the script in nu-
merous ways.

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eastern United States. Mollusca: Cephalopoda.
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Report NMFS, 75: 1-22.

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habiting the coast of New England. American
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the marine fauna of the eastern coast of North
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99-116.

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Revised ms accepted 5 September 2001

APPENDIX |

Collection localities and specimens examined
in this study. Sex and mantel length are
stated.

Bathypolypus bairdii (Verrill) East Atlantic
BMNH:

W of Ireland, May 9, 1896, 610-680 fms., Y
ML: 55.—Off S Ireland, May 21, 1898,
Norman coll., 2 ФФ ML: 47, one dis-
torted.-60°57'N, 05°47’W, June 26,
1909, 348 m, juv. ML: 8—61°16'N,
02°08’W, July 9, 1913, 630 m, juv. ML:
8.—Udsire Hole, 58°58'N, 03°37’E, May
15, 1912. © МЕ; 35.—71°50М№ 282107Е;
Мау 1, 1975, 180 fms., Ф ML: approx.
50.

MNHT:

Haul 46, 61°25’N, 05°13’W, July 7, 1979, 481
т, © ML: 23.—BIOFAR project: Stn. 117,
62°00.7'N, 09°4.63’W, July 25, 1987,
481 m, © ML: 16.—Stn. 124, 62°16.94'N,
09°38.93'W, July 26, 1987, 600 m, ©
МЕ 35-5. 158. 61387 05-38 W,
Мау 7, 1988, 322 m, с’ МЕ: 36.—Stn.
419, 62°25'N, 10°38.17'W, June, 1,
1989, 702 m, 2 oo ML: 40, 42.-Stn.
420, 62°32'81N 10°27.57'W, June 1,
1989, 597 m, © ML: 33.—Stn. 482,
61°01.94’N, 05°13.94'W, July 22, 1989,
509 m, Ф ML: 24.

IMNH:

B5-77-?, 65°42'N, 27°53’W, February 24,
1977, 750-820 m, 2 99 ML: 23, 33.—
B5-77-48, 65°38’N, 29°27’W, March 23,
1977, 450 m, © ML: 41, Q ML: 33.—B5-
77-49, 65°37'N, 29°32’W, March 23,

218 MUUS

1977, 420-430 m, С’ ML: 41, ФФ ML:
21, 50.—B5-77-50, 63°38'N, 29°27'W,
March 23, 1977, 450-460 m, 2 So ML:
35, 45, 2 ФФ ML: 24, 36.—B5-77-73,
63°38'N, 26°14'W, March 27, 1977, 575
m, Ф ML: 35.—B5-78-07, 63°48'N,
27°00'W, March 10, 1978, 1110-1095 т,
O” ML: 63.—B5-78-22, 64°56’N,
27°59'W, March 14, 1978, 970-1030 m,
Q ML: 47.—DALB-1-80-13, 66°39'N,
28°43’W, October 21, 1980, 415 m, ©
ML: approx. 64.—B3-81-34, 65°22’М,
32°37'W, February 25, 1981, 910 m, ©
ML: 75.—BIOICE project: Stn. 2299,
63°00'10N, 22°39'61W, September 10,
1992, 775 m, juv. ML: 28.—Stn. 2346,
63°23'N 12°88'W, May 6, 1993, 501 т,
oO ML: 39, Y ML: 18.

ZIASP:

Nr. 25, near Spitsbergen, 215 m, © ML: 35.

ZMUB:

Norwegian North-Atl. Exp. Stn. 290, 72°27'N,
20°51’E, July 7, 1878, 349 m, © ML: 26,
Q ML: 18.— Outer part of Laksefjord, Fin-
mark (approx. 71°N, 27°E), August 27,
1900, 280 m, © ML: 24, Q ML: 39.—R.V.
“Michael Sars”: Stn. 56, 70°09’N,
31°00’E, May 21, 1901, 200 m, juv. © ML:
23.—Stn. 108, 70°32'N, 18°17’E, June
18, 1909, 300 m, juv. с’ ML: 11.—Stn. 5,
70°07'N, 30°53’E, June 4, 1914, © ML:
approx. 44.—Stn. 28, 70°16'N, 32°20’E,
June 24, 1914, © ML: approx. 30.-MS
“Armauer Hansen” Stn. 2, outer part of
Sognefjord (N of Bergen), March 10,
1917, juv. с’ ML: 15.-Salhus (N of
Bergen), July 17, 1916, 400-500 m, ©
ML: 55.—42837, Salhusfjord (N of Ber-
gen), July 12, 1934, 2 F'T' ML: 40, 42.—
Mangerfjord (N of Bergen), March 18,
1931, 300-400 m, 2 So ML: 36, 38.—
53336, Kvinnheradsfjord, 59°59'50’N,
05°54'E, December 7, 1956, 687-672 m,
3 00 ML: 34-42, 3 ФФ ML: 22-36.—
Hardangerfjord, NE of Varaldsoy, Stn. F
103, November 8, 1957, 690 m, ©’ ML:
40.— 50281, Sognefjord, 61°03'N,
06°25'E, May 3, 1966, 1238-1228 m, ©
ML: 56, 3 ФФ ML: 28-47.-R.V. “G.O.
Sars”, S of Bear Island (Björnöya), August
4, 1974, 500 m, © ML: 74.

ZMUC:

Trondhjemsfjord, Norway, March 4, 1891,

Storm leg., Y ML: 26.- Trondhjemsfjord,
September 19, 1934, 180-220 m, © ML:
16.— Skager-rak, NNW of Skagen, July
28, 1897, 210 Tms., 200 ML: 29;,49.—
Skagerrak, July 28, 1897, C. G. Joh. Pe-
tersen leg., 275 fms., 2 juv. ML: 7, 7.—Sk-
agerrak, about 14 n.m. NW of Hirtshals,
June 21, 1911, 313 т, © ML: 41.— Trond-
hjemsfjord, Norway, N of Tatra, Septem-
ber 19, 1934, 180-220 m, Stephensen
leg, © МЕ: 15.—“Thor: Sin. 167,
63°05'N, 20%07'W, July 14, 1903, 557 m,
Ф м. ML: 9.—Stn. 223, 64°30'N,
12°25'W, March 17, 1904, 535 m, © ML:
60.— Stn. 99, 61°35'N, 9°35’W, May 22,
1904, 900 т, © ML: 38.—Stn. 274, Sk-
agerrak, NW of Hirtshals, October 9,
1904, 660 m, © juv. ML: 23.—Stn. 1074,
Skagerrak, 4.5 n.m. S of Okso light-
house, May 28, 1907, 480 m, © ML: 43,
Ф ML: 45.—Stn. 1570, Skagerrak, 53
n.m. N of Hanstholm, June 23, 1911,
525-550 m, © ML: 58, juv. 11.—“Dana”:
Stn. 6001, 63°33’N, 11°25’W, July 24,
1938, 322 m, с’ ML: 33.—Stn. 6004,
63°06'N, 10°40’W, July 24, 1938, 437 т,
Oo ML: 43.-Stn. 11643, 57°44'N,
07°40'W, April 17, 1961, 440 т, 2 00
ML: 50, 55.-Stn. 13320, 58°10’М,
04°22'E, March 12, 1965, 270 m, с’ ML:
42.—Stn. 15194, 57°35'N, 08°08’E, Sep-
tember 19, 1969, 210 m, 2 SO ML: 27,
29.—Stn. ?, 61°03’N, 05°04'W, April 20,
1988, 800-600 m, © ML: 38.

ZMUO:

Oslofjord, off Filtvet lighthouse, April 26, 1910,

100 fms., juv. ML: 13.—Oslofjord, Filtvet,
Мау 3, 1966, 150-280 m, Ф ML: 61.—
32291, Oslofjord, Torbjornskaer light-
house, с’ ML: 39.—Oslofjord, Drobak, ©
ML: 43, Y ML: 31.- Oslofjord, Vestmedet,
August 14, 1937

Greenland

ZMUC:

“Ingolf”: Stn. 28, 65°14’N, 55°42’W, July 1,

1895, 420 fms., © ML: 37.—Stn. 32,
66°35'N, 56°38’W, July 11, 1895, 2 99
ML: 18, 62 + 4 juv.—Stn. 35, 65°16’N,

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 219

55°05’W, July 18, 1895, 682 m, 2 juv. ML:
8, 9.—“Tjalfe”: Stn. 337, 64°05’N,
55°20'W, Мау 8, 1909, 1100 m, Ф ML:
54.—Stn. 428, 63°54'N, 53°15’W, June
8, 1909, 988 m, © ML: 21.—“Rink”: Stn.
45, Bredefjord, SW Greenl. (approx.
60°49'М, 46°45’W), July 18, 1912,
430-450 m, С’ ML: 52.—“Dana”: Stn.
2346, 66°37'N, 56°37'W, June 22, 1925,
450 m, 6 So ML: 16-68, 2 ФФ ML: 32,
44, 3 juv.— Stn. 2361, 68°08'N, 57°30'W,
June 26, 1925, 398 m, 3 O'O' ML: 46-55,
COMMERS4= Sins 10018. 65°02’N,
56°00’W, July 19, 1957, 730-740 т, ©
ML: 45 (type of B. proschi Muus, 1962).—
Stn. 13662, 64°24'N, 52°57'W, July 27,
1966, 450-510 m, с’ МЕ: 32.—Greenl.
Fish. Invest: Stn. 4539, 64°19’N,
52°55’W, June 4, 1971, 470-570 m, 29
do ML: 26-50, 10 ФФ ML: 25-54.—
Stn. 5043, 63°57'N, 52°21’W, June 18,
1975, 300 m, © ML: 33 + juv.— Stn. 5047,
64°21'N, 52%58'W, June 24, 1975,
550-580 m, 8 So ML: 15-49, 8 ФФ ML:
24-42.-Stn. 5101, 66°34'N, 54°15’W,
August 10, 1975, 335-340 m, © ML:
20.— St. 5112, 64°23’N, 52°58’W, August
21, 1975, 450-510 m, 5 Jo ML: 32-41,
Oe Mizar A = Stns 75206, 63°58:N;
52°21'W, June 3, 1976, 300-310 m, ©
ML: 38.—Stn. 5209, 63°58’N, 52°21'W,
June 8, 1976, 300-308 m, © ML: 34, Y
ML: 46.—Stn. 5215, 64°21'N, 52°59'W,
June 10, 1976, 510-520 m, 4 oo ML:
29-45, 5 ФФ ML: 37-41.—Stn. 5384,
64°21'N, 52°59'W, April 22, 1977, 485-
510 m, © ML: 44.—“Elias Kleist”: Stn.
791010/3, 67°57'N, 57°10'W, October
10, 1979, 310-100 m, Ф ML: 47.

American East Coast

BMNH:

Off Marthas Vineyard, Massachusetts, May

21, 1898, 200-388 fms., © ML: 35 (la-
belled Octopus bairdii).

USNM:

34223, Le Have Bank, Nova Scotia, 120 fms.,

holotype of Octopus lentus Verrill, 1880.—
382469, fishing banks off Massachusetts,
36 miles E of NE Light, Sable Island, from
halibut stomach, holotype of Octopus

obesus Verrill, 1880, © ML: 44.- 39943,
off New Jersey, 39°49'30N, 71°10’W, Au-
gust 3, 1884, 420 fms., Y ML: 51.—52979,
off New Jersey, 39°50'N, 71°43’W, Sep-
tember 18, 1885, 137 fms., © ML: ap-
prox. 41.-574638, Bay of Fundy,
paratype of Octopus bairdii Verrill, 1873,
O ML: 24. 575271, 43°
38'М, 69°13’W, August 2, 1912, 60 fms.,
Ф МЕ20.—575274, Gulf of Maine, August
16) 1878, Ys'ims, 31010) MER2IF26 —
575275, off Саре Cod, August-Septem-
ber 1878, 70-94 fms., 6 G'O ML: 12-26,
4 ФФ ML: 10-18.—575276, off Salem,
41°58'30N, 69°44’W, September 18,
1379: 4 Gio МЕ О-о МЕ 39 =
575277, off Salem, August 1877, 49 fms.,
2.©:@.ME: 27, 3951) 9 МЕ: 10575278,
42°31'N, 70°20’W, September 2, 1878,
98 fms., 3 specimens.—575279, off
Martha’s Vineyard, 39°46’N, 71°05’W,
February 10, 1880, 487 fms., Ф ML: 39,
juv. ML: 9.—575280, 40°02'N, 70°23'6W,
September 4, 1880, 192 fms., © ML: 14,
Q ML: 35.—575281, 37°24'N, 74°17'W,
November 16, 1880, 300 fms., 2 Jo’ ML:
26, 23, 3 ФФ МЕ 17-27.—575282,
39°49'N, 70°54'W, September 13, 1880,
225-252. -imss #2: 10/61 МЕ: -20,-38:—
575285, 37°07'50N, 74°34'20W, Novem-
ber 18, 1884, 167 fms., © ML: 37.—
575288, off Martha’s Vineyard,
39°53’30N, 71°13’30W, August 9, 1881,
319 fms., 2 So ML: 50, one damaged, Y
ML: 50.—575289, 40°04’N, 69°29’
30W, September 28, 1884, 58 fms., ©
ML: 39.—575293, 39°52'20N, 70°58'W,
October 2, 1880, 372 fms., © ML: 28.—
575294, 39°53’N, 70°58’30W, October 2,
1880, 365 fms., 3 oc ML: 38-46.—
575306, off Gay Head, Martha’s Vine-
yard, 39°57'N, 70°31’30W, August 23,
1881, 225-396 fms., © ML: 40,3 Y Q ML:
21-49.-575971, 39°57'N, 70°58’W, Au-
gust 25, 1879, 175-200 fms., O' ML: 44.—
RV “Oregon”: Stn. 6800, 29°48'N,
80°09'05W, July 20, 1967, 183 fms., 2
qo.—Gosnold cruise: Stn. 105, 39°
51'N, 70°56’W, August 10, 1972, 875-
880 m, ©’ ML: 51.—Stn. 120, 39°50'N,
70°32'W, August 16, 1972, 750-775 m,
CO ML: 65.-RV “Chain”: Stn. 243,
39°30'N, 72°20’W, February 28, 1973,
474-529 m, © ML: 21, Q ML: 27.—Stn.
244, 39°28'N, 72°18'W, February 28,
1973, 260-342 m, Ф ML: 44.—Stn. 254,

MUUS

39°51'N, 70°47'W, March 2, 1973,
768-947 m, © ML: 49, Q ML: 56.— Stn.?,
39°31'N, 78°18'W, February 26, 1973,
810-900 m, 2 ML: 41.—Stn.?, 39°31’N,
75°23'W, February 28, 1973, 420-590 m,
Q ML: 26.—RV “Knorr”: Stn. 300, 39°40’N
72°27'W, November 13, 1973, 110-182
m, С’ ML: 26.-Stn. 301, 39°32’N,
72°24'W, November 13, 1973, 475-520
т, 40°C ML: 20-33, Y ML: 28.—GI-75-
08: Stn. 10, 37°53’N, 74°40'08W, Sep-
tember 9, 1975, 290-340 т, 2 сс’ 99%
(distorted).—Stn. 18, 37°04'07N, 74°
34'03W, September 10, 1975, 200-215
m, 3 do, 19.—St. 19, 36°58’04N
74°37'05W, September 11, 1975, 190-
250 т, 2 00,3 9 9 .—St. 22, 36°58’06N,
74°33'08W, September 11, 1975, 880-
920 т, 3 oo ML: 24-42.-Stn. 99,
36°36'05М 74°39'W, September 20,
1975, 850-1000 m, Y ML: 50.

ZMUC:

“Albatros IV”, cruise 76-02: Stn. 221,

40°07'N, 69°04'W, March 28, 1976, 465
m, Q ML: 30.—Stn. 237, 41°44'N,
69°46’W, March 30, 1976, 78 m, © ML:
27.—Stn. 288, 41°53’N, 67°52'W, April 6,
1976, 52 m, с’ ML: 19.-Stn. 300,
42°05'N, 68°15’W, April 7, 1976, 189 m,
Ф ML: 25.—Stn. 305, 42°18'N, 6912'W,
April 8, 1976, 230 m, Ф ML: 38.— Stn. 309,
42°26'М, 70°06'W, April 8, 1976, 84m, ©
МЕ: 42.- Stn. 321, 42°45'N, 70°00'W,
April 14, 1976, 163 m, Ф ML: 41.—Stn.
322, 42°46'N, 69°35'W, April 14, 1976,
163 m, © ML: 25, Y ML: 43.—Stn. 330,
42°59'N, 68°39'W, April 17, 1976, 187 m,
2 99 ML: 20, 22.-Stn. 332, 42°45'N,
67°47'W, April 17, 1976, 200 m, 2 So
ML: 38, 42.- Stn. 333, 42°34’N, 67°58'W,
April 17, 1976, 206 m, 2 ML: 20.—Stn.
339, 42°17'N, 66°49’W, April 18, 1976,
270 m, С’ ML: 26.—Stn. 341, 42°19’N,
66°12'W, April 18, 1976, 262 m, 1 spm.—
Stn. 405, 43°26'N, 67°34’W, April 29,
1976, 218 m, © ML: 37, © ML: 37.—Stn.
406, 43°06'N, 67°48'W, April 29, 1976,
200 m, 2 ML: 29.—Stn. 409, 43°39’N,
68°17'W, April 29, 1976, 190 m, 3 SO
ML: 18-29.—Stn. 426, 43°29'N, 69°
01’W, Мау 6, 1976, 139 m, 2 ML: 50.—
Notre Dame Bay, Newfoundland: October
1975, 3 JO ML: 67-74.—USSR “Belo-
gorsk”, cruise 74-11: Stn. 142, 41°16'N,

68°41'W, October 12, 1974, 80 m, Y
ML: 32.

Bathypolypus arcticus (Prosch) East Atlantic
BMNH:

60°03'N, 05°51'W, August 17, 1880, 540 fms.
(labelled В. faeroensis) S ML: 35, © ML:
30.-HMS “Triton”, Stn. 9, 60°05’М,
06°21'W, August 23, 1882, 608 fms.
(syntypes of Benthoctopus sasakii Rob-
son), © ML: approx. 32, Y ML: 27.—Stn.
40, 57°34'N, 00°01'W, November 27,
1904, Tow net 100 m, © ML: 30.—
61°27'N, 01°47'W, July 25, 1909, 1240
m, © ML: 43 (labelled Benthoctopus pis-
catorum) + Ф ML: 20.—61°42’N,
02°00'W, July 25, 1909, 1236 m, Y ML:
31 (labelled B. faeroensis).—Stn. 77,
75°16'N, 24°46’E, June 1956, 85 m,
Е. Holt, 2 ФФ ML: 28, 29.—Cruise IV,
Stn. 37, 60°25’N, 04°31'W-60°29'N, 04°
22'W, W of Foula, 1973, 940-910 m, 2
ФФ ML: 20, 28.

IRSNB:

(vide Adam, 1939) 66°20’N, 12°28'W, June
21, 1938, 180 —220 m, 2 SO juv. ML: 16,
10, Y ML: 13.—66°20'N, 12°28'W, June
22, 1938, 180-220 m, Ф ML: са. 18.

MNHT:

Haul 96, 62°59'N, 09°54'W, July 23, 1979,
481 m, Y ML: 46.—BIOFAR project: Stn.
15, 62°37'68N, 04%40'37W, July 17,
1987, 683 m, juv. МЕ 12.—Stn. 95,
60%41'51N, 05°18’63W, July 23, 1987,
803 m, Y ML: 21.—Stn. 274, 63°00'79N,
07°49'22W, May 16, 1988, 698 m, Ф ML:
42.—Stn. 294, 60°26'N, 07°28’W, July
17, 1988, 1096 m, © ML: 30.—Stn. 502,
60°30'26’N, 08°04'W, July 25, 1989, 890
m, o МЫ 30.—Stn:~ 589, 60°40'N;
10°00’W, April 9, 1990, 250 m, Ф ML:
15.—Stn. 726, 60°39’N, 06°54'W, Sep-
tember 29, 1990, 400 m, Ф ML: 27.

IMNH:

06-80-50, 67°11’N, 18°30'W, April 20, 1980,
430 m, С’ ML: approx. 41.-B16-80,
66°50'N, 20°26'W, October 29, 1980,
415 m, Y ML: approx. 37.—B3-81-34,

THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 221

62°59’N, 20°23'W, March 7, 1981, 910 m,
Q ML: 95.—B4-81-137, 66°37'N,
12°58’W, April 1, 1981, 320 m, Y ML:
31.-BIOICE project: Stn. 2080,
67°22'79N, 17°20'77W, July 4, 1992, 897
m, © ML: 46.-Stn. 2085, 67°15’67N,
17°26’41W, July 4, 1992, 754 m, Ф ML:
61.—Stn. 2088, 67°02'35N, 13°25’05W,
July 4, 1992, 903 m, с’ ML: 40.—Stn.
2134, 66°44'46N, 18°54'93W, July 8,
1992, 504 m, © МЕ: 17.—Stn. 2135,
66°44'37N, 18°57'32W, July 8, 1992, 418
m, с’ juv. ML: 26.—Stn. 2320, 64°02'N,
09°44'W, May 2, 1993, 758 m, © ML:
49.—Stn. 2322, 63°55'N, 10°04’W, Мау
3, 1993, 627 m, Ф ML: 45.—Stn. 2326,
63°44'N, 10°09'W, May 3, 1993, 563 m,
Q ML: 33.—Stn. 2328, 63°20'N, 10°57'W,
May 3, 1993, 430 m, Y ML: 54.

TMDZ:

M/K “Asterias”: Isfjord, Svalbard, July 18,
1955, 242-228 m, Ф ML: 26.—Isfjord,
Svalbard, August 14, 1958, 230-215 m,
GO juv. ML: 23, Y ML: 23.

ZIASP:

92, Barents Sea, 140-150 m, Kondakov det.,
O” juv. ML: approx. 12.-119, between
Spitsbergen and Frantz Josef Land, 210
m, Kondakov det., с’ ML: 31.— 120, Bar-
ents Sea, 201 m, © ML: 19.—Nr. 607,
Novaya Zemlya, 37 m, 2 ML: 39.

ZMUB:

ESE of Vardo (about 70°N, 32°E), August 10,
1902, © ML: approx. 49.—“Solveig |”,
Stn. 52, Kongsfjord, Svalbard, June 22,
1938, 290-333 m, Ф МЕ: 42.- 42838,
Stn. 64, Svalbard, 78°25'N, 12°06’E,
July 7, 1939, © ML: 44.- 36145, “Sotra”,
Stn. 250, 78°04'N, 14°10'E, September
911930: © МЕ: 38 Ток» эм. 10,
77°44'N, 11°45’E, June 30, 1925,
185-228 m, © juv. ML: 13.-78°03’N,
14°10’E, August 30, 1931, 147 m, 2 ML:
31.-75°00’N 37°20'E, September 1,
1968, 100-150 m, Ф ML: 57.- 18678,
“Michael Sars”, Stn. 37, 62°43’N,
01°26’E, June 29, 1902, 775 m, © ML:
36.

ZMUC:

61°27'N, 01°27'W, July 25, 1909, 1240 m, la-
belled Polypus faeroensis Russell Type.
(leg. A. C. Stephen).

Greenland
ZMUC:

Lectotype of Octopus arcticus Prosch, 1849:
С’ ML: 42, SW Greenland, August 26,
1841, К. M. Jorgensen leg.— Paralecto-
type ©, partly dissected, SW Greenland,
July 27, 1840, К. M. Jorgensen leg.— Dis-
sected male organs pictured by Prosch
(1849: figs. 1-3)—3 ФФ in bad shape
and partly dissected labelled Greenland
and evidently from the 1840s.—Hol-
steinsborg, SW Greenland, 1892, Trau-
stedt leg., Y ML: 64.—“Ingolf”: Stn. 124,
67°40’N, 15°40’W, July 28, 1896, 932 m,
2 do ML: 44, 44.- Near Nanortalik, SW
Greenland, April 15, 1906, © ML: 31.—
Lichtenau fjord, SW Greenland, October
14, 1910, from stomach of Greenland
halibut, Ф ML: 45.—Lichtenau fjord, June
6, 1914, 220 fms., Ф ML: 41.—Lichtenau
fjord, July 3, 1947, Poul Hansen leg., Ф
ML: 51.—“Godthab”: Stn. 81, 75°35’N,
65°41’W, August 1, 1928, 490 m, с’ ML:
54, Q МЕ 23:=Sin. 87, 77°05 №
71°13’W, August 4, 1928, 790 m, 14 00°
ML: 32-58, 2 ФФ ML: 32-46, 1 juv. ML; :
22.—Stn. 90, 77°17'N, 69°59’W, August
5, 1928, 930 m, o ML: 46, Y ML: 38.—
Stn. 116, 76°08’N, 80°53’W, August 17,
1928, 80 m, © juv. ML: 13.—“Godthab’s
summer cruise, Stn. 341, off Kap Hacker,
Jameson Land, E Greenland, August 27,
1933, juv. ML: 15.—Ella Island, Kong
Oscar Fjord, approx. 73°N, 25°W, Octo-
ber 10, 1931, 67-68 m, Y ML: 35.— Ymer
Island, Frantz Joseph Fjord, E Greenland
(approx. 73°20'N), August 8, 1932, ©
ML: 43.—Frantz Joseph Fjord, off Eng-
dalen, August 7, 1931, 45-36 m, © ML:
52.—Lindenow Fjord, 60°30'N, 43°25'W,
July 17, 1935, 100-150 m, Bertelsen
leg., O” ML: 36.—Amerdlok Fjord, near
Holsteinsborg (Sisimiut), July 26, 1938,
approx. 500 m, ® ML: 54.—Skovfjord,
SW Greenland, June 16, 1948, P.
Hansen leg., с’ ML: 58.— Young Sound,
off Daneborg, approx. 74%15'N, 20°W,
July 1947, Ф ML: approx. 58.—Bylot

222 MUUS

Sound, 76°31'N, 69°09’W, August 20,
1968, 240 m, Just & Vibe Stn. 45, ©’ ML:
54.-Kap Farvel Exp.: Stn. 56, 60°13'N,
44°13’W, July 31, 1970, 400-420 m, ©
ML: 35.—Stn. 128, 60°01’М, 43°59'W,
August 21, 1970, 530 m, Y ML: 51.-W
Greenland, 66°21'N, 54°56’W, August 2,
1975, 280-290 m, Max Andersen leg., ©
ML: 12.-Greenland Fish. Invest.: Stn.
3978, 63°53'N, 51°27'W, May 9, 1968,
230-250 m, Ф ML: 35.—Stn. 4360, May
21, 1970, 240-250 m, © ML: 43, © ML:
34.—Stn. 5101, 66°34'N, 54°15’W, Au-
gust 10, 1975, 300 m, 2 So ML: 33 +
juv.

ZMUO:

Hoel’s Greenland Exp.: Stn. 1101, Kong
Oscar Fjord, E Greenland (approx.
72°20'N), August 12, 1930, 55-100 m, ©
juv. ML: 17.-Stn. 1116, Kong Oscar
Fjord, August 15, 1930, 250 m, © ML:
30.—Stn. 1118, Kong Oscar Fjord, August
16, 1930, 120 m, 1 м. ML: 26.—Stn.
1119, Kong Oscar Fjord, Vegasund, Au-
gust 17, 1930, 190-250 m, 4 So ML:
37-44.

American East Coast
No material.
Bathypolypus pugniger n. sp.

IRSNB:

66°23'М 12°53’W, June 14-15, 1938,
200-250 т, o ML: 22, Q ML: 18 (vide
Adam 1939).

MNHT:

Haul 25, 63°16’N, 09°30’W, July 6, 1979, 500
т, 2 °C ML: 27, 28.-BIOFAR project:

Stn. 47, 61°02’31”N, 05°54'W, July 19,
1987, 280 m, Ф ML: 19.—Stn. 80, 60°

38'89N, 08°27'93W, July 22, 1987, 678
m, Ф МЕ: 18.—Stn. 269, 62°49'84N, 08°
15'55W, May 15, 1988, 510 m, © ML:
33.—Stn. 503, 60°38'02N, 08°33’54W,
July 25, 1989, 513 m, © ML: 18.— Sin.
734, 60%10'06N, 07°57'03W, May 8,
1990, 634 m, © ML: 31.—Stn. 738,
62°19’N, 10°13’W, October 1, 1990, 749
mo ML: 25.-Stn: 74076229IN7102
02'W, October 1, 1990, 597 m, o ML:
18.

IMNH:

B5-77-36, 65°40'N, 28°20'W, March 21,
1977, 1000-970 m, Ф ML: 36.—B5-77-
40, 65°36'N 29°17’W, March 22, 1977,
870-910 m, 9 ML: 33.—B5-77-42,
65°34'N, 29°29’W, March 22, 1977,
750-760 m, © ML: approx. 44.—B5-77-
46, 65°29'N, 29°33’W, March 23, 1977,
960 m, 2 SO ML: 36, 30, 3 ФФ ML:
27-40.—B5-77-50, 65°38'N, 29°27’W,
March 23, 1977, 450-460 m, Ф ML: ap-
prox. 32, O' ML: 30.—В5-78-44, 64°58'N,
27°44'W, March 14, 1978, 860-870 m,
holotype 19990971. © ML: 32.

ZMUC:

“Dana” Stn. 5840, 62°44'N, 06°06'W, May 14,
1938, 330 m, © ML: 19.—Stn. 6001,
63°33’N, 11°25’W, July 24, 1938, 322 m,
5 do (3 juv.) ML: 6-13, 2 99 (1 juv.)
МЕ: 15, 23.-Stn. 16437, 64°14’N,
57°26'W, July 24, 1974, 760 m, © ML:
54.—Stn. B5-77-36, 65°40'N, 28°20'W,
March 21, 1977, 1000-970 m, Q ML:
36.— Haul 25, 63°16'N, 09*30'W, July 6,
1979, 500 m, 2 0 ML: 27, 38. BIOFAR
project: Stn. 503, 60°38'N, 08°33’54W,
July 25, 1989, 513 m, с’ ML: 18.—Stn.
738, 62°19'N, 10°13’W, October 1, 1990,
749 т, © ML: 25.—Stn. 734, 60°10’06N
07°57'03W, Мау 8, 1990, 634 m, © ML:
31,

MALACOLOGIA, 2002, 44(2): 223-239

ULTRASTRUCTURE OF THE SUPPORTING CELLS AND SECRETORY CELLS OF
THE ALIMENTARY CANAL OF THE SLUGS, LEHMANNIA MARGINATA AND
BOETTGERILLA PALLENS (PULMONATA: STYLOMMATOPHORA: LIMACOIDEA)'

Ana Maria Leal-Zanchet

Laboratorio de Histologia, Centro de Ciéncias da Saude, Universidade do Vale do Rio dos
Sinos, Av. UNISINOS, 950, 93022-000 Sao Leopoldo- RS, Brasil; ipp @ bios. unisinos.br

ABSTRACT

The supporting cells and the secretory cells of the alimentary canal of two slugs of the super-
family Limacoidea, Lehmannia marginata and Boettgerilla pallens, were studied by electronmi-
croscopy. From the esophagus to the third intestinal region, and in the rectum, ciliated and/or
nonciliated columnar cells occur. In the fourth intestinal region and in the intestinal caecum, the
latter present only in the intestine of L. marginata, there are nonciliated squamous cells. The ul-
trastructure of the columnar cells of the crop, stomach and first and second intestinal regions in-
dicate that these cells are involved in food uptake, digestion and storage of reserve materials.
Ultrastructural features of ciliated cells of the typhlosoles and the leader folds of the alimentary
canal indicate the involvement of these cells in the production of currents, thus aiding food and
faeces transportation. The ultrastructure of the squamous cells of the fourth intestinal regions
and caecum point to one of the roles of these organs in the absorption of water and ions from the
faecal pellets. Eight secretory cell types in the alimentary canal of L. marginata and six secretory
cell types in B. pallens are distinguished by their ultrastructural features, such as diameter and
contents of secretion granules, development and contents of rough endoplasmatic reticulum, as
well as diameter of their cisternae, and development of the agranular endoplasmatic reticulum.

Key words: epithelium, secretion, digestive system, glands cells, intestine, caecum, Limaci-
dae, Boettgerillidae.

RESUMO

As células de revestimento e as células secretoras do tubo digestivo de duas lesmas da su-
perfamilia Limacoidea, Lehmannia marginata e Boettgerilla pallens, sao analisadas ultraestru-
turalmente. Células cilindricas ciliadas e/ou nao ciliadas sao observadas desde o esöfago até a
terceira regiao intestinal bem como no reto. Células pavimentosas nao ciliadas ocorrem na
quarta regiao intestinal e no ceco intestinal, este ultimo presente apenas em L. marginata. As
caracteristicas ultraestruturais das células cilindricas do papo, do est6mago e da primeira e se-
gunda regiôes intestinais indicam que essas células auxiliam na absorcäo, na digestao e no ar-
mazenamento de material de reserva. Caracteres ultraestruturais das células ciliadas presentes
nas tiflossoles e dobras condutoras do tubo digestivo indicam que essas células atuam na pro-
ducäo de correntes, auxiliando no transporte do bolo alimentar e fecal. A ultraestrutura das célu-
las pavimentosas da quarta regiao intestinal e do ceco sugerem que uma das funcöes desses
Orgaos é a absorcao de agua e ions do bolo fecal. Oito tipos de células secretoras ocorrem no
tubo digestivo de L. marginata e seis tipos de células secretoras, em B. pallens, sendo diferen-
ciadas por caracteristicas ultraestruturais, tais como diametro e conteudo dos granulos secre-
tores, forma, diametro e conteudo das cisternas do reticulo endoplasmatico granular, bem como
forma e abundancia relativa das cisternas do reticulo endoplasmatico agranular, dentre outras
caracteristicas.

"Part of a thesis submitted to the Lehrstuhl Spezielle Zoologie of the Eberhard-Karls Univertitat Tubingen, Germany, in par-
tial fulfilment of the requirements for the degree of Doctor of Natural Sciences and supported by a fellowship of the Brazil-
ian Research Council (CNPq).

223

224 LEAL-ZANCHET

INTRODUCTION

Knowledge of the role of the alimentary
canal of pulmonates in digestion and absorp-
tion is still incipient, this related to the paucity
of available information concerning the ultra-
structure its organs. Babula & Wielinska
(1988) described the crop and intestine of De-
roceras reticulatum; Angulo & Moya (1989)
studied the intestine of Arion ater; Boer & Kits
(1990) examined the whole alimentary canal
of Lymnaea stagnalis; and Franchini & Otta-
viani (1992) studied intestinal cell types of
Planorbarius corneus. In addition, Triebskorn
(1989), Triebskorn & Künast (1990), and
Triebskorn & Köhler (1992) analysed ultra-
structural changes in different cell types of the
alimentary canal of D. reticulatum and/or
Arion lusitanicus as a background for obser-
vations on the effects of molluscicides. Dero-
ceras reticulatum and L. stagnalis, among
other species of pulmonates, were also the
object of anatomical and histological investi-
gations of the alimentary canal (reviewed by
Runham, 1975, and Luchtel et al., 1997).

The present work completes a series of
anatomical and histological (Leal-Zanchet,
1998), histochemical (Leal-Zanchet, 1999)
and ultrastructural studies of the alimentary
canal of limacoid slugs. Leal-Zanchet (1998)
analysed comparatively the anatomy and his-
tology ofthe alimentary canal of six species of
the superfamily Limacoidea (Deroceras
laeve, D. rodnae, D. reticulatum, Lehmannia
marginata, Malacolimax tenellus, Boettgerilla
pallens) and the milacid slug Tandonia bu-
dapestensis, and described the following re-
gions in this canal: esophagus, crop, stom-
ach, first, second, third and fourth intestinal
regions and the rectum, besides an intestinal
caecum for D. rodnae, D. reticulatum and L.
marginata (Leal-Zanchet, 1998).

Inthe limacoid slugs cited above, as well as
in 7. budapestensis, the epithelium of the ali-
mentary canal is composed of supporting
cells and secretory cells, the former being cil-
iated and/or nonciliated columnar cells from
the oesophagus to the third intestinal region,
and in the rectum. The supporting cells of the
fourth intestinal region and intestinal caecum
are nonciliated squamous to cuboidal (Leal-
Zanchet, 1998).

Eight secretory cell types could be distin-
guished in the alimentary canal of L. mar-
ginata (Leal-Zanchet, 1998). Two types of
mucous cells occur in the proximal and me-
dian segments of the alimentary tract, type |

(esophagus, crop, stomach and second in-
testinal region) and type Il (stomach and first
intestinal region). Two types of secretory
cells, namely intestinal secreting cells oftypes
| and Il, occur in the second and third intesti-
nal regions, respectively. Besides four other
types of secretory cells, mucous cells of types
Ш, IV and V, and cystic cells, occur in the dis-
tal regions of the tract (third and fourth intesti-
nal regions, intestinal caecum and rectum). In
B. pallens, both mucous cells of type V and
the cystic cells are absent.

Most of the limacoid slugs are herbivorous
(Frömming, 1954), but B. pallens seems to be
zoophagous (Leal-Zanchet, 1995). Because
of the apparently diverse feeding habits, as
well as the differing complexity of the alimen-
tary canal (length of the alimentary canal,
presence/ absence of an intestinal caecum
and number of secretory cell types), Lehman-
nia marginata (Limacidae) and Boettgerilla
pallens (Boettgerillidae) were selected for the
present investigation.

MATERIAL AND METHODS

The animals were collected near the town
of Tübingen, Baden-Württemberg, Germany,

Key to Lettering on Figures

aer: agranular endoplasmic reticulum
ar: apical region

bb: basal bodies

bf: basal feet

ci: cilia

cr: cilia rootlets

cl connective tissue
fe fold

9: Golgi bodies

gl: glycogen
interdigitations

li: lipids

lu: lumen

m: mitochondria

mc: muscle cells

mi: microvilli

n: nucleus

ne: nerve ending

nu: nucleolus

г ribosomes

rb: rootlike basal extensions
rer: rough endoplasmic reticulum
sg: secretory granules
sm: secretion mass
sv: synaptical vesicles
V: vesicles

Va: vacuoles

vi: vacuoles of type |
vil: vacuoles of type Il
za: zonula adhaerens
75: zonula septata

ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 225

and kept in a cool room at 15°C. They were
maintained humid with decanted water in the
laboratory for approximately two weeks in
petri dishes with a certain amount of natural
soil and litter from the collecting site. Lehman-
nia marginata was raised on lettuce, carrot
and cabbage. Boettgerilla pallens consumed
eggs of limacoid slugs and also accepted
carrot. Before preparation, the slugs were
starved for three days. For previous micro-
anatomical, histological and histochemical
studies, eight adult specimens of each L. mar-
ginata and B. pallens were analysed. For
present ultrastructural studies, two adult
specimens of each species were examined.
They were anaesthetised in 5% menthol for
one hour and killed in the fixative solution (2%
paraformaldehyde in 0.05 M phosphate buffer
and 2% glutaraldehyde, pH 7.2) (Plattnert,
1975), in which they were dissected. The
whole alimentary tract was transferred to a
fresh amount of fixative solution, and small
pieces of the various parts of the alimentary
canal were separated, labelled and main-
tained for one hour in a new quantity of fixa-
tive solution. The material was washed in
Sörensen’s phosphate buffer (Ruthmann,
1966), post-fixed in 2% osmium tetroxide in
0.05 M phosphate buffer, washed in 0.025 M
phosphate buffer, dehydrated in a graded se-
ries of ethanol, treated with propylene oxide
and embedded in Epon 812 (Serva). Ultrathin
sections (60-90 nm), obtained with a LKB-UI-
trotome | ultramicrotome, were stained with
Reynolds’ (1963) lead citrate and uranyl ac-
etate. The sections were examined in a
Siemens Elmiskop 102 transmission electron
microscope.

RESULTS

Columnar Cells

Both ciliated (Figs. 1, 12) and nonciliated
(Fig. 13) columnar cells of the alimentary tract
of L. marginata and B. pallens show microvilli.
The apical zone of the cytoplasm (Fig. 18) is
devoid of cell organelles, with the exception of
vesicles and cisternae of the agranular endo-
plasmic reticulum. In the optical microscope,
in sections stained by routine histological
methods, this zone appears as a clear area
(Leal-Zanchet, 1998). In the ciliated columnar
cells, the cilia are anchored to the cytoplasm
by basal bodies from which cross-striated
rootlets extend into the cytoplasm (Table 2).

Below the apical zone, there are numerous
mitochondria (Figs. 17, 18), as well as small
vesicles and multi-vesicular bodies. The mito-
chondria present an oval to oblong form. In
the rest of the cytoplasm, mitochondria are
only sparsely scattered. The supranuclear cy-
toplasm of the columnar cells contains nu-
merous large and slightly electron-dense fat
droplets, the mean diameter of which is 2.2 +
0.4 um in the crop of L. marginata and 3.2 +
0.4 um in the crop of B. pallens, being more
plentiful in the crop and stomach of both
species (Figs. 14, 15). Glycogenrosettes were
often observed in the cytoplasm of the colum-
nar cells of B. pallens (Fig. 17). There are also
membrane-limited vacuoles in the supranu-
clear cytoplasm, which can be categorized
into two types. The vacuoles of type | (Figs.
12, 14, 21) show a electron-dense peripheral
zone and a lesser electron-dense central
core. The mean diameter of the vacuoles is
1.3 + 0.3 and 1.3 + 0.1 umin the crop and 0.7
+ 0.2 and 0.9 + 0.2 um in the intestine of L.
marginata and B. pallens, respectively. In L.
marginata, the peripheral part has mainly het-
erogeneous, granular contents (Fig. 14). The
relative number of vacuoles of type | de-
creases in the second intestinal region of both
species. The vacuoles of type Il (1.2 + 0.3 um
in diameter) were observed only in the colum-
nar cells of the crop, stomach and first and
second intestinal regions of B. pallens. They
contain granular, slightly electron-dense ma-
terial (Fig. 21). In the basal cytoplasm, some
mitochondria, Golgi bodies, rough endoplas-
mic reticulum, and fat droplets are present. In
the optical microscope, in sections stained by
routine histological methods, type | vacuoles
show cromophobe contents and type II vac-
uoles appear acidophillic (Leal-Zanchet,
1998).

Neighbouring cells of the alimentary canal
are interconnected apically by the zonula ad-
haerens and zonula septata (Fig. 18). The
zonula adhaerens is located close to the tran-
sition of the apical to the lateral membranes of
the cells, followed by the zonula septata,
which is partly transversed by interdigitations
(Fig. 18). In the middle and basal zones of the
epithelium, the cells present large intercellular
spaces, wherein occur connections uniting
the epithelial cells.

In the first, second and third intestinal re-
gions the height of the columnar cells gradu-
ally decreases (Leal-Zanchet, 1998), the mi-
crovilli and the cilia rootlets becoming smaller
as well (Tables 1, 2).

226 LEAL-ZANCHET

HE 3 UU UU,

ci

rer

So =

=
poe 7 ГИЯ C= o rer

FIGS. 1-3. Schematic drawings of a ciliated columnar cell of the first intestinal region (Fig. 1), a ciliated
columnar cell of the rectum (Fig. 2) and a squamous cell of the fourth intestinal region (Fig. 3).

In the ciliated columnar cells of the ty-
phlosoles and of the leader groove of the
stomach, the cilia are very numerous and
densely arranged (Figs. 15-17). Between two
neighbouring cilia only three microvilli are
present. The basal bodies of the cilia are in-
terconnected by well-developed basal feet,
and the rootlets are thick and very long (Table
2). In the apical cytoplasm of these cells, the
mitochondria are more numerous than in the
ciliated columnar cells of other regions, being
situated close to the cilia rootlets (Fig. 17).

In the leader folds of the rectum of B. pal-
lens, as well as in the ciliated columnar cells
of the rectum of L. marginata, the microvilli
and the cilia rootlets are long (Tables 1, 2).
The apical cytoplasm consists of vesicles,

channels of the agranular endoplasmic reticu-
lum (Fig. 19) and ribosomes. In the leader
folds, the mitochondria zone occupies most of
the supranuclear cytoplasm, and the mito-
chondria are arranged parallel to the cilia
rootlets. In the rectum of Lehmannia mar-
ginata, where specialized leader folds are ab-
sent, the mitochondria of the ciliated cells do
not show such an arrangement, being less
abundant and scattered in the supranuclear
cytoplasm (Fig. 2). Numerous poliribosomes
and some channels of the agranular endo-
plasmic reticulum are found between the mi-
tochondria. Poliribosomes and mitochondria
in a scattered distribution occur in the basal
cytoplasm. Some vesicles and Golgi bodies
are found in the zone of the nucleus.

ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA PPT

FIGS. 4-7. Schematic drawings of mucous cells of types | (Fig. 4) and Il (Fig. 5) and intestinal secreting cells
of types | (Fig. 6) and II (Fig. 7).

Squamous to Cuboidal Cells the supranuclear zone is narrow. Mitochon-
dria are sparsely scattered in the cytoplasm.

In the squamous, nonciliated cells of the The apical and middle cell zones show chan-
fourth intestinal region and the intestinal cae- nels of the agranular endoplasmic reticulum
cum, the microvilli are short (Fig. 3, Table 1). as well as vesicles. Golgi bodies are found in
An apical mitochondria zone is absent, and the middle zone of the cells. In infranuclear

228 LEAL-ZANCHET

10

hee

JR

/

FIGS. 8-11. Schematic drawings of the cell body of mucous cells of type III (Fig. 8), type IV (Fig. 9), type V

(Fig. 10) and the cystic cell (Fig. 11).

TABLE 1. Length of microvilli in the alimentary canal of Lehmannia mar-
ginata and Boettgerilla pallens (in um).-: absent;--: not measured.

L. marginata
Crop 1.6+0.2
Stomach (typhlosoles and canal) HO O
Stomach (other areas) SEO
First intestinal region 0.7 + 0.1
Second intestinal region O17 0:1
Third intestinal region 0.4 + 0.1
Fourth intestinal region 0.523031
Intestinal caecum 0.5 + 0.06

Rectum 0.7 = 0:07

В. pallens

37-05
SEO
107208
1.028102
0.9 + 0.2

0.6 + 0.08

0.4 + 0.1

ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 229

TABLE 2. Length of cilia rootlets in the alimentary canal of Lehmannia
marginata and Boettgerilla pallens (in шт).-: absent;--: not measured.

Oesophagus

Crop

Stomach (typhlosoles and canal)
Stomach (other areas)

First intestinal region

Second intestinal region

Third intestinal region

Rectum

cytoplasm, there are small vacuoles of type |
(0.5 # 0.1 um in diameter) and ribosomes.
The granular endoplasmic reticulum is poorly
developed. The basal membrane shows nu-
merous folds (107 + 27 nm wide), which form
rootlike basal extensions (Fig. 22), containing
mitochondria, ribosomes and vesicles with
poor electron-dense contents (83 + 16.7 nm
in diameter). These epithelial basal exten-
sions come close to folds of the muscle cells.
The muscle folds are wider (0.25 + 0.06 um)
than the epithelial basal extensions and show
numerous vesicles (83 + 16.7 nm) with elec-
tron-dense contents. The connective tissue
between the folds present collagen fibres,
which are arranged parallel to the epithelial
folds.

Secretory Cells

The mucous cells of type |, the mucous
cells of type Il, the intestinal secreting cells of
type I, and the intestinal secreting cells of type
Il are intraepithelial. The mucous cells of type
Ш, the mucous cells of type IV, the mucous
cells of type V, and the cystic cells show a
subepithelially located cell body and a cell
neck that extends to the surface of the epithe-
lium. Cell extensions emerge from the basal
part of the intraepithelial secretory cells,
which enter into the connective tissue. Nerve
endings, which contain electron-dense as well
as electron-lucent synaptical vesicles, come
close to these cell extensions (Fig. 23). By the
subepithelial secretory cells, nerve endings
make contact with the cell body or with the
subepithelial part of the cell neck (Fig. 30). In
the resting phase, the secretory cells show
microvilli. In the process of secretion, the api-
cal cytoplasm forms a camber and the mi-
crovilli are reabsorbed (Figs. 25, 28).

L. marginata B. pallens
0.9 + 0.2 =
8.7 = 1:3 6.0 + 1.0
2.9 + 0.4 2.0+0.5
1.6 = 0:3 1.7 = 0.4
0.7 = 0:1 5 0:3
0.4 + 0.1 --
0/9 = 0.3 0:6 = 0.1

Mucous Cells of Type |

The mucous cells of type | (Fig. 4) have a
widened basal part that contains the nucleus
and most of the cell organelles (Golgi bodies,
granular endoplasmic reticulum and mito-
chondria) (Fig. 24), as well as some secretion
granules. The middle and apical cell zones
are performed with secretion granules. Vesi-
cles containing electron-dense material occur
between the granules and at the margins of
the cell. Multi-vesicular bodies and numerous
ribosomes are found throughout the cyto-
plasm. Golgi zones are well developed. In L.
marginata, the Golgi lamellae show a width of
40 + 10 nm. Vesicles and recently formed se-
cretion granules are often associated with the
Golgi bodies. In B. pallens, the Golgi bodies
usually consist of minimally widened lamellae
(40 + 10 nm) with electron-dense contents.
The rough endoplasmic reticulum (RER)
shows distended (0.4 + 0.17 um), branched
cisternae that contain tubuli (24 + 4 nm). Usu-
ally mitochondria occur close to the cisternae
(Fig. 24). The secretion granules of L. mar-
ginata (2.1 + 0.3 um in diameter) are slightly
electron-dense, with dense, tube-like scat-
tered structures, containing at the margins a
concentration of strong electron-dense mate-
rial (Fig. 25). In B. pallens, the secretion gran-
ules (1.7 + 0.2 um in diameter) are electron-
lucent contents with speckles of granular,
moderately electron-dense material. The
granules usually form large secretion masses
that are membrane-limited (Fig. 26).

Mucous Cells of Type Il

The mucous cells of type II (Fig. 5) present
an elongated basal part, which contains very
numerous RER cisternae as well as numer-

230 LEAL-ZANCHET

FIG. 12. Ciliated columnar cell of the oesophagus of Lehmannia marginata. Note the numerous vacuoles of
type | in supranuclear cytoplasm. Scale bar, 3 um. FIG. 13. Columnar cell of the crop of Lehmannia mar-
ginata showing long microvilli. Scale bar, 1 um. FIG. 14. Supranuclear zone of the cytoplasm of a columnar
cell of the crop of Boettgerilla pallens. Scale bar, 1 um. FIG. 15. Apical zone of the cytoplasm of a ciliated
columnar cell of the typhlosole of the stomach of Lehmannia marginata. The long cilia rootlets are visible.
Scale bar, 2 um. FIG. 16. Apical zone of the cytoplasm of a ciliated columnar cell of the typhlosole of the
stomach of Lehmannia marginata. Note the basal bodies interconnected by the well-developed basal feet.
Scale bar, 0.3 um. FIG. 17. Apical zone of the cytoplasm of a ciliated columnar cell of the typhlosole of the
stomach of Boettgerilla pallens. Scale bar, 0.7 um. FIG. 18. Apical zone of the cytoplasm of a ciliated colum-
nar cell out of the typhlosoles of the stomach of Lehmannia marginata. Scale bar, 1.3 um.

ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 231

FIG. 19. Apical cytoplasm of a cuboidal cell of the leader fold of the rectum of Boettgerilla pallens. The arrow
shows channels of the agranular endoplasmic reticulum. Scale bar, 0.3 um. FIG. 20. Nonciliated cuboidal cell
of the fourth intestinal region of Lehmannia marginata. Scale bar, 3 um. FIG. 21. Supranuclear cytoplasm of
a columnar cell and part of a mucous cell of type Il of the first intestinal region of Boettgerilla pallens. Scale
bar, 1 um. FIG. 22. Rootlike basal extensions of the basal part of a squamous cell of the intestinal caecum
of Lehmannia marginata and corresponding folds of the muscle cells. Scale bar, 1 um. FIG. 23. Basal cyto-
plasm of a mucous cell of type Il of the stomach of Lehmannia marginata. The arrow shows tubuli of the rough
endoplasmic reticulum. Nerve endings come close to the cell. Scale bar, 0.6 um.

232 LEAL-ZANCHET

FIG. 24. Basal zone of the cytoplasm of a mucous cell of type | of the oesophagus of Lehmannia marginata.
Scale bar, 1 ит. FIG. 25. Apocrine secretion of a mucous cell of type | of the oesophagus of Lehmannia mar-
ginata. Scale bar, 1.3 um. FIG. 26. Supranuclear cytoplasm of a mucous cell of type | of the crop of Boettge-
rilla pallens. Scale bar, 2 um. FIG. 27. Supranuclear cytoplasm of a mucous cell of type II of the typhlosole
of the stomach of Lehmannia marginata. Scale bar, 1 um. FIG. 28. Apical part of a mucous cell of type II of
the stomach of Boettgerilla pallens. The apical cytoplasm forms a camber and the microvilli are reabsorbed
(arrow). Scale bar, 0.5 um.

ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 233

ous Golgi bodies and some mitochondria. The
RER cisternae are dilated (0.4 + 0.1 um) and
also contain tubuli (28 + 4 nm in diameter). In
the basal cytoplasm occur large vacuoles (1.5
+ 0.1 um in diameter) containing finely gran-
ular, electron-dense material (Fig. 27). Occa-
sionally some secretion granules are present
in the infranuclear cytoplasm. In the supranu-
clear cytoplasm, however, the granules are

very numerous. In L. marginata, the granules
are electron-lucent with clumps of osmiophillic
material (Fig. 27). In B. pallens, the granules
are electron-lucent with flocculent contents
(Fig. 28). In both species, the granules are
very small (0.9 + 0.2 and 0.8 + 0.2 um in L.
marginata and B. pallens, respectively). Vesi-
cles and channels of the agranular endoplas-
mic reticulum as well as ribosomes are pres-

FIG. 29. Cell body of a mucous cell of type III of the fourth intestinal region of Boettgerilla pallens. Scale bar,
2.2 ит. FIG. 30. Subepithelial part of a cell neck of a mucous cell of type III of the fourth intestinal region of
Lehmannia marginata. Nerve endings make contact with the cell neck. Scale bar, 0.7 um. FIG. 31. Cell body
of a mucous cell of type IV of the rectum of Lehmannia marginata. Scale bar, 2 um. FIG. 32. Detail of the cy-
toplasm of the cell body of a mucous cell of type IV of the rectum of Boettgerilla pallens. Scale bar, 0.2 ит.
FIG. 33. Detail of the cytoplasm of the cell body of a mucous cell of type V of the third intestinal region of
Lehmannia marginata. Scale bar, 2 um. FIG. 34. Detail of the cytoplasm of the cell body of a cystic cell of the
third intestinal region of Lehmannia marginata. Scale bar, 2.2 um.

234 LEAL-ZANCHET

ent between the granules. Mitochondria occur
in the marginal cytoplasm.

Mucous Cells of Type Ill

The cell body of the mucous cells of type Ill
(Figs. 8, 29) is occupied by secretion granules
(1.2 + 0.1 um in diameter in L. marginata and
1.8 + 0.4 um in diameter in B. pallens). These
granules often fuse to form larger granules. In
L. marginata as well as in B. pallens, their
contents show different electron densities,
slightly to moderately electron-dense (Fig.
29). The granules are so closely located that
seldom are other organelles present. The
RER cisternae occur in the marginal cyto-
plasm and close to the nucleus. The cisternae
are distended (0.5 + 0.2 in L. marginata or
0.25 + 0.1 um in diameter in B. pallens) and
contain tubuli (Fig. 30). Golgi bodies and mi-
tochondria are present at the cell margins.
Very numerous RER cisternae and some mi-
tochondria and channels of the agranular en-
doplasmic reticulum occur in the proximal part
of the cell neck (Fig. 30). In the apical part of
the cell, the secretory granules form a large,
electron-lucent secretory mass.

Mucous Cells of Type IV

The secretion granules are smaller (Figs. 9,
31) than the granules of the mucous cells of
type III (0.6 + 0.1 um in diameter in L. таг-
ginata and 1.3 + 0.3 um in diameter in B. pal-
lens). In both species, the secretion granules
show homogenous, electron-dense contents
(Fig. 31). Usually the granules do not form se-
cretion masses. Numerous RER cisternae,
which contain finely granular, electron-dense
material, occur in the cytoplasm between the
granules (Fig. 32). The cisternae are usually
thin (51 + 14 пт L. marginata and 115 + 54
nm in B. pallens), but in L. marginata some
cisternae reach 0.17 + 0.1 um in diameter.
Golgi bodies are present at the cell margins
and close to the nucleus. The apical part of
the cell neck shows electron-dense secretion
granules that fuse distal to discharging in the
lumen. Vesicles and polyribosomes occur in
the apical cytoplasm.

Mucous Cells of Type V

This cell type does not occur in the alimen-
tary tract of B. pallens. A secretion mass,
formed by the coalescence of the secretion

granules, often occupies most of the cell, so
that the organelles (polyribosomes, mitochon-
dria and RER cisternae) and the nucleus are
found in the basal part of the cell body (Fig.
10). The secretion mass is electron-lucent
and flocculent (Fig. 33). Channels and vesi-
cles of the agranular endoplasmic reticulum
are numerous in the peripheral cytoplasm.
The RER cisternae are thin (75 + 20 nm in di-
ameter) and contain electron-lucent material.
Golgi complexes are well developed (Fig. 33).
Occasionally, recently formed secretion gran-
ules are associated with the Golgi bodies.

Cystic Cells

This cell type is absent in the alimentary
tract of B. pallens. The cell body of the cystic
cell is occupied by a granular, electron-dense
secretion mass surrounded by a membrane
(Figs. 11, 34). Small vacuoles showing a sim-
ilar content as well as vacuoles containing
tubular structures are observed in basal and
marginal cytoplasm (Figs. 34, 35). Granules
(0.5 + 0.4 um in diameter) with a homoge-
neous, stronger electron-dense material also
occur in the cytoplasm (Fig. 35). The RER is
often highly developed and shows thin cister-
nae (113 + 82 nm). Polyribosomes and chan-
nels of the agranular endoplasmic reticulum
are abundant (Fig. 35). Some channels are
connected to the outer cell membrane.

Intestinal Secreting Cells of Type |

The cytoplasm is occupied by the highly de-
veloped RER and abundant free ribosomes
(Figs. 6, 38). Some RER cisternae are thin
(33 + 5 nm) and show a platelike structure.
Most of the RER cisternae are, however, di-
lated (0.22 + 0.02 um in diameter) and occur
scattered throughout the cytoplasm. They
contain a finely granular, slightly electron-
dense material. Golgi bodies occur in the in-
franuclear cytoplasm as well as around the
nucleus (Fig. 36). Their lamellae (0.1 + 0.05
nm) have an electron-dense material. The cy-
toplasm also presents channels of the agran-
ular endoplasmic reticulum, small vesicles
and vacuoles with heterogeneous electron-
dense contents (Fig. 38). The round secretion
granules (2.3 + 0.2 um in diameter in L. mar-
ginata and 1.5 + 0.15 um in diameter in B.
pallens) are usually present in the supranu-
clear cytoplasm. Their content is homoge-

ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 235

FIG. 35. Detail of the cell body of a cystic cell of the third intestinal region of Lehmannia marginata. The arrow
shows a channel of the agranular endoplasmic reticulum. Arrowheads indicate tubular structures in a vac-
uole. Scale bar, 0.3 um. FIG. 36. Detail of the cytoplasm of an intestinal secreting cell of type | of the second
intestinal region of Boettgerilla pallens. Scale bar, 0.7 um. FIG. 37. Apical cytoplasm of an intestinal secret-
ing cell of type | of the second intestinal region of Lehmannia marginata. Scale bar, 0.6 um. FIG. 38. Supranu-
clear cytoplasm of an intestinal secreting cell of type | of the second intestinal region of Boettgerilla pallens.
Scale bar, 1.0 um. FIG. 39. Supranuclear cytoplasm of an intestinal secreting cell of type Il of the third in-
testinal region of Boettgerilla pallens. Scale bar, 1.0 um.

236 LEAL-ZANCHET

neous electron-dense (Figs. 36, 37). Numer-
ous mitochondria occur in the apical cyto-
plasm.

Intestinal Secreting Cells of Type Il

The intestinal secreting cells of type II (Fig.
7) also possess an extensive RER and abun-
dant polyribosomes (Fig. 39). The RER cister-
nae are thinner (119 + 37 nm) compared to
those of the intestinal secreting cells of type I.
They contain finely granular, electron-dense
material. Golgi bodies are found around the
nucleus. They show dilated lamellae (67 + 29
nm in diameter) with electron-transparent ma-
terial (Fig. 39). Vesicles that contain electron-
dense material are often associated with the
Golgi bodies. The secretion granules that
show a distinctive limiting membrane are
larger than those of the intestinal secreting
cells of type | (3.8 + 1.0 um in diameter in L.
marginata and 2.5 + 0.5 um in diameter in B.
pallens) and may form secretion masses.
They contain a flocculent, electron-transpar-
ent material as well as membrane-like struc-
tures (Fig. 39). Mitochondria and polyribo-
somes are often present in the apical
cytoplasm. Channels of the agranular endo-
plasmic reticulum and vesicles may also be
present.

DISCUSSION

According to Boer & Kits (1990) the colum-
nar cells of the alimentary tract of Lymnaea
Stagnalis are involved in the absorption, di-
gestion and storage of reserve material. Mi-
crovilli were demonstrated on the surface of
the columnar and squamous cells of the ali-
mentary canal of L. marginata and B. pallens.
However, the absorption of food particles
should occur mainly through the columnar
cells of the crop, the stomach, and the first
and second intestinal regions, where the mi-
crovilli are long, and numerous mitochondria
are present in the apical cell zones (Pacheco
& Scorza, 1971). This would be consistent
with the presence of alkaline phosphatase in
the epithelial cells of the crop and intestine of
Deroceras reticulatum (Babula & Skowron-
ska-Wendland, 1988) and with the findings of
Walker (1972), who showed in D. reticulatum
an uptake of glucose, glycine and palmitic
acid by the epithelial cells of the crop and in-
testine.

A type of vacuole with electron-dense con-

tents (vacuoles of type 1) occurs in the cyto-
plasm of the columnar cells of the crop, stom-
ach and first and second intestinal regions of
B. pallens and L. marginata. Bowen (1970)
and Babula & Wielinska (1988) also observed
vacuoles with similar contents by ultrastruc-
tural studies of epithelial cells of the crop and
intestine of Arion ater and D. reticulatum, re-
spectively. According to Bowen (1970) and
Angulo et al. (1986), such vacuoles are liso-
somes, presenting a positive reaction to acid
phosphatase. The unavailability of histochem-
ical studies of enzymes in the slugs B. pallens
and L. marginata make it impossible to point
out the functions of type | and type II vacuoles.

The storage of reserve material, such as
glycogen and lipids, was observed in the
columnar cells of B. pallens and L. marginata.
Evidence of the storage of lipids was ob-
served mainly in the crop, stomach and first
intestinal region. The storage of glycogen was
demonstrated by ultrastructural studies, as
well as histochemically (Leal-Zanchet, 1999).
Some columnar cells of the esophagus, crop,
stomach, and intestine of D. reticulatum were
designated by Triebskorn (1989) as storage
cells, because of their high lipid and glycogen
content.

Ciliated cells occur along the entire alimen-
tary tract of B. pallens and L. marginata, ex-
cept for the crop of both species, the fourth in-
testinal region of B. pallens and the intestinal
caecum of L. marginata (Leal-Zanchet, 1998).
In some regions of the stomach and rectum,
such as the typhlosoles and the transversal
fold present in the stomach of limacoid slugs
and the leader folds of the rectum of B. pal-
lens, we observed that the ciliated cells show
distinctive features, namely the cilia are
longer, very numerous and densely arranged
(Leal-Zanchet, 1998). Furthermore, they pre-
sent very long cilia rootlets, being intercon-
nected by well-developed basal feet on the
basal bodies. According to Zylstra (1972),
such a contact between the basal bodies
would be involved in the coordination of ciliary
movements, and this coordination would be
crucial in regions involved in the production of
currents, such as the leader system of the
stomach. As a matter of fact, Walker (1972)
studied the ciliary tracts of the stomach of D.
reticulatum and found three main ciliary path-
ways: a sorting mechanism in the folds sur-
rounding the openings of the digestive glands;
extrusion pathways for material that becomes
bound up into faecal pellets; and a circulation
pathway particularly for fine material. After

ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 237

Walker (1972), the cilia on the typhlosoles and
accessory fold (= transversal fold) beat mainly
towards the gastric channels and intestinal
groove, and cilia in the basis of the gastric
channels (channels between each tiphlosole
and the transversal fold) and anterior intesti-
nal groove (channel between the tiphlosoles)
transport material posteriorly, whereas cilia of
the more posterior region of the intestinal
groove transport material anteriorly. Where
these two opposite directional movements
meet, faecal pellets are formed (Walker,
1972). Physiological studies are not available
for the rectal leader folds of limacoid slugs,
but their similar ultrastructural morphology
may indicate that such folds could be better
suited to aid faeces transportation than are
the usual folds (Leal-Zanchet, 1998).

The squamous cells of the fourth intestinal
region, the intestinal caecum, and the rectum
show microvilli on the apical surface, apart
from numerous folds of the basal membrane,
which form rootlike basal prolongations be-
sides well-developed interconnections be-
tween neighbouring cells. These features are
characteristic of water- and ion-transporting
epithelia and would be consistent with the
findings of Deypup-Olsen & Martin (1987),
who verified that the distal part of the intestine
of Ariolimax columbianus plays a significant
role in osmoregulation. As suggested by Boer
& Kits (1990) for Lymnaea stagnalis, this
would indicate that water and ions are ab-
sorbed from the faecal pellets. In limacoid
slugs that have an intestinal caecum, espe-
cially in L. marginata in which this caecum is
long, this region could provide an additional
site for water and ions absorption.

The occurrence of five mucous cell types
was detected in the alimentary canal of L.
marginata, as well as in such other limacoid
slugs as Malacolimax tenellus, Deroceras
laeve, D. reticulatum, and D. rodnae (Leal-
Zanchet, 1998). By optical microscopical
analysis, these cell types were distinguished
by their distribution along the alimentary tract,
their position related to the epithelium (intra-
or subepithelial location), the mean diameter
of their secretory granules, the morphology of
the basal part or cell body of the cells, and
their appearance after histochemical reac-
tions for protein and mucopolyssaccharides
(Leal-Zanchet, 1998, 1999). Thus, type | and
II mucous cells were distinguished on the
basis of the mean diameter of the secretory
granules and the morphology of the basal part
of the cells, characteristics observed by opti-

cal microscopy and confirmed here. Besides,
type II secretory cells show a restricted distri-
bution in the stomach and first intestinal re-
gion, whereas type | are present in the esoph-
agus, crop and second intestinal region. Both
types secret acid mucopolyssaccharides
(Leal-Zanchet, 1999), but their morphological
distinctive features and distribution in the ali-
mentary canal indicate that a more accurate
histochemical analysis could confirm them as
different types. Types Ш, IV and V mucous
cells, restricted to the distal regions of the ali-
mentary tract (third and fourth intestinal re-
gions, intestinal caecum and rectum) were
distinguished by optical microscopical studies
due to the different mean diameter of their
granules and their reaction to trichromical
stains. Previous histochemical studies
showed that the three types are involved in
the secretion of neutral mucopolysaccha-
rides, but type Ill is also involved in the secre-
tion of acid mucopolysaccharides. Besides, in
B. pallens, where type V mucous cells and
cystic cells are absent, type IV mucous cells
could be histochemically distinguished be-
cause they show positive reaction for protein
and neutral mucopolysaccharides (Leal-
Zanchet, 1999). The present investigation
shows that the granules of types III and IV, in
both species, have a different electron-den-
sity and that their rough endoplasmatic reticu-
lum has a different morphology and distribu-
tion in the cytoplasm. Type V mucous cell is
distinguishable by optical studies through the
elongate morphology of the secretory gran-
ules. The present ultrastructural studies show
that they form a eletron-lucent secretion mass
occupying most of the cell.

The mucous cells of type |, Il and Ill show
tubuli in the cisternae of the rough endoplas-
mic reticulum. The presence of tubuli in the
rough endoplasmic reticulum has been de-
scribed by Wondrak (1967), Moya & Rallo
(1975), Kessel & Beams (1984) and Angulo &
Moya (1989). According to Kessel & Beams
(1984), tubular structures within the cisternae
of the endoplasmatic reticulum appear to be a
feature of more than one cell type. Further-
more, according to these authors, their preva-
lence within secretory cells suggests that
these tubular structures could be secretory
products of the cells.

The secretion of the cystic cells and of the
intestinal secreting cells of type | and Il gave
positive results for protein (Leal-Zanchet,
1999). This would be consistent with the pres-
ence of a highly developed rough endoplas-

238 LEAL-ZANCHET

mic reticulum, observed in the present inves-
tigation. The secreting cells of type |, occur-
ring in the second intestinal region, where the
columnar cells show ultrastructural features
that indicate involvement in the absorption of
food particles, imply that the secretion of this
cell type is probably of an enzymatic nature
and may play a role in digestion (Leal-
Zanchet, 1998). However, additional histo-
chemical studies, including enzyme histo-
chemistry, are needed to clarify the role of
these secretory cells.

Considering that data obtained in labora-
tory experiments assume that B. pallens is
carnivorous, whereas Lehmannia marginata
is herbivorous, this living on a specialised diet
of lichens (Wiktor, 1973), it could be expected
to find more distinguishing morphological fea-
tures in the alimentary canal of the two
species. However, B. pallens shows a mere
shortening of the intestinal regions (Leal-
Zanchet, 1998).

ACKNOWLEDGEMENTS

Dr. Wolfgang Rähle supervised the doctoral
thesis. Prof. Dr. Wolfgang Maier is thanked for
accommodation in his department. Prof. Dr.
Christian F. Bardele gave his permission to
use the Laboratory of Electronmicroscopy.
Ms. Siegrid Schultheiss is thanked for the EM
technical assistance. Mr. Fernando Carbayo
is thanked for the preparation of the drawings.
Mr. Edward Benya and Mr. Ramon A. Clark
corrected the English version of the manu-
script. Thanks are also due to the anonymous
referees for the valuable suggestions.

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TRIEBSKORN, R. 8 H.-R. KOHLER, 1992, Plastic-
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WALKER, G., 1972, The digestive system of the
slug, Agriolimax reticulatus (Múller). Proceedings
of the Malacological Society of London, 40:
33-43.

WIKTOR, A. 1973, Die Nacktschnecken Polens. Ar-
ionidae, Milacidae, Limacidae (Gastropoda, Sty-
lommatophora). Kraköv: Monografie Fauny Pol-
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WONDRAK, G., 1967, Die exoepithelialen Schleim-
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Anatomie, 76: 287-294.

ZYLSTRA, U., 1972, Histochemistry and ultrastruc-
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93-134.

Revised ms. accepted 6 September 2001

MALACOLOGIA, 2002, 44(2): 241-257

SEXUAL MATURATION, SPAWNING, AND DEPOSITION OF THE
EGG CAPSULES OF THE FEMALE PURPLE SHELL, RAPANA VENOSA
(GASTROPODA: MURICIDAE)

Ee-Yung Chung’, Sung-Yeon Kim?, Kwan Ha Park! & Gab-Man Park?

ABSTRACT

Germ cell development, sexual maturation, and fecundity of female Rapana venosa (Valenci-
ennes) have been examined by light and electron microscope observations. The Golgi appara-
tus, vacuoles and mitochondria were involved in the formation of glycogen particles, lipid droplets
and yolk granules in early vitellogenic oocytes. The modified mitochondria and endoplasmic
reticulum in late vitellogenic oocytes were involved in the formation of proteid yolk granules near
the cortical layer. A mature yolk granule was composed of three components: main body (central
core), superficial layer, and limiting membrane. Monthly changes in the gonadosomatic index
were closely associated with ovarian developmental phases. Spawning occurred between May
and early August, when the seawater temperature rose to 18-26°C. The female reproductive
cycle of this species was divided into five successive stages: early active stage (September to
February), late active stage (October to April), ripe stage (March to July), partially spawned stage
(May to August), and recovery stage (June to September). The rate of individuals reaching the
first sexual maturity was 51.6% in females of 7.1-8.0 cm in shell height, and 100% in those >
10.1 cm. The total number of egg capsules per individual and the mean number of eggs in an
egg capsule were 184 to 410 and 976, respectively. Fecundity ranged approximately from
179,000 to 400,000 eggs/individual with two to four broods (spawning frequencies) during the
spawning season. The duration of development in egg capsules was 15-17 days at about 20°C.
Rapana venosa is a species with embryos that hatch as veliger larvae. The sex ratio of female:

male was not significantly different from 1:1.

Key word: Rapana venosa, gametogenesis, reproductive cycle, first sexual maturity, fecundity,

egg capsule, sex ratio.

INTRODUCTION

The purple shell, Rapana venosa (Gas-
tropoda: Muricidae), one of the most impor-
tant edible gastropods in Asia (Yoo, 1976;
Kwon et al., 1993), is abundant along the
coasts of Korea, China and Japan, especially,
in silty sand of the intertidal and subtidal
zones. However, the standing stock of this
species has gradually been decreasing due to
extensive loss of habitats from reclamation
projects and reckless over-harvesting. Thus,
R. venosa has been identified as a target or-
ganism that should be carefully managed.

Prior studies have considered various as-
pects of the biology of R. venosa, including
classification (Kuroda & Habe, 1952; Habe,
1969; Tsi et al., 1983; Wu, 1988; Choe, 1986,
1992; Higo et al., 1999), morphology (Lee &
Kim, 1988), biochemical aspects (Yoon, 1986;
Yoo et al., 1991; Hwang et al., 1991), distribu-

tion (Gomoiu, 1972; Zolotarev, 1996), habitat
and age estimation (Chukhchin, 1984; Hard-
ing & Mann, 1999), reproductive cycle (Chung
et al., 1993; Chung & Kim, 1997), and spawn-
ing and egg capsule (Hirase, 1928; Habe,
1960; Amio, 1963; Smagowicz, 1989; Harding
& Mann, 1999). However, there is still uncer-
tainty in many aspects of reproductive biology
of the purple shell. For example, little informa-
tion is available on ultrastructure of germ cell
differentiation during oogenesis, the repro-
ductive cycle, first sexual maturity and fecun-
dity. Understanding the reproductive cycle
and the spawning period of R. venosa will pro-
vide necessary informations needed for the
determination of age and recruitment period
of natural populations. Additional information
on first sexual maturity, fecundity associated
with egg capsules, sex ratio and reproductive
strategy are needed to facilitate effective
management of this important natural re-

"Faculty of Marine Life Science, Kunsan National University, Kunsan 573-701, Korea; eychung@kunsan.ac.kr
National Fisheries Research and Development Institute, Pusan 619-902, Korea
“Department of Parasitology, Kwandong University College of Medicine, Kanghung 210-710, Korea

241

242 CHUNG ET AL.

source. Therefore, the main aim of the pres-
ent study was to describe vitellogenesis dur-
ing oogenesis, the reproductive cycle and
spawning period, first sexual maturity, fecun-
dity, and sex ratio of R. venosa inhabiting the
west coast of Korea.

MATERIALS AND METHODS
Sampling

Specimens of the purple shell, R. venosa
(Valenciennes 1846), were collected monthly
by subtidal dredging near Biung-do, Chol-
labuk-do Korea, for one year from June 1994
to July 1995 (Fig. 1). Purple shells ranging
from 3.1 cm to 16.9 cm in shell height were
used for the present study. After the purple
shells were transported alive to the laboratory,
shell heights and total body weights were im-
mediately measured.

Gonadosomatic Index
Monthly changes in the mean gonadoso-

matic index (GSI) (Fig. 2) were calculated by
the following equation (Chung et al., 1993):

Br

en

KOREA D 9
>> м
МИ >.
SS er ö
Gogunsangun-Do °V

>

126° 15' 126° 30'

FIG. 1. Map showing the sampling area.

Biung-Do >

Sampling area

GSI = Thickness of gonad x 100
Diameter of posterior appendage
including gonad and digestive gland

Light and Electron Microscope Observations

For light microscopic examination of histo-
logical preparations, gonad tissues were re-
moved from shells and preserved in Bouin’s
fixative for 24 h, and then washed with run-
ning tap water for 24 h. Tissues were then de-
hydrated in alcohol and embedded in paraffin
molds. Embedded tissues were sectioned at
5 — 7 um thickness using a rotary microtome.
Sections were mounted on glass slides and
stained with Hansen’s hematoxylin-0.5%
eosin, Mallory’s triple stain and PAS stain, and
examined using a light microscope.

For electron microscopical observations,
excised pieces of the gonads were cut into
small pieces and immediately prefixed in
2.5% paraformaldehyde-glutaraldehyde in
0.1 M phosphate buffer (pH 7.4) for2hat4°C.
After prefixation, the tissues were washed
several times with the same buffer and then
postfixed in 1% osmium tetroxide dissolved in

Chungcheongnam-Do

36°00'

„. Simje-Gun

; Buan-Gun

35° 45°

126° 45"

REPRODUCTION IN RAPANA 243

FIG. 2. Anatomy of the female purple shell, Rapana
venosa, removed from its shell. A, reproductive
organ of female; B, posterior appendage showing
the gonad and liver. X, Y and Z denote the sections
for measurement of GSI, Three sections are spaced
equally. Abbreviations: A, anus; DG, digestive
gland; F, foot; MA, mantle; MO, mouth; OP, opercu-
lum; OV, ovary; PA, posterior appendage; S, stom-
ach; SC, stomachal caecum.

0.2 M phosphate buffer solution (pH 7.4) for
1 В at 4°C. Tissues were then dehydrated in a
series of increasing concentrations of ethanol,
cleared in propylene oxide and embedded in
Epon-Araldite mixture. Ultrathin sections of
Epon-embedded specimens were cut with
glass knives with a Sorvall MT-2 microtome
and an LKB ultramicrotome at a thickness of
about 800 ~ 1000 A. Tissue sections were
mounted on collodion-coated copper grids,
doubly stained with uranyl acetate followed by
lead citrate, and examined with a JEM 100
CX-2 (80 kv) electron microscope.

First Sexual Maturity

A total of 214 female purple shells
(3.1-17.0 cm in shell height) were used for

the histological study of first sexual maturity.
The percentage of sexual mature was deter-
mined for shell heights that reached maturity
during the breeding season (from May to late
August).

Egg Capsule and Fecundity

To investigate the number of egg capsules
per individual and the number of eggs in an
egg capsule in the laboratory, a total of 25
adult females of 11.2-14.2 cm in shell height
were used for observation of spawning be-
havior in an FRP aquarium (80 cm x 60 cm x
60 cm) bedded with sand and small gravels.
An established filtration and aeration appara-
tus was employed. Purple shells used for this
experiment were those collected in May to
June 1994. Length and width of egg capsules
of the adult purple shells were measured and
their egg developmental processes were
checked under a light microscope.

The purple shells were reared at the spe-
cific gravity of 1.021 and water temperatures
of 18.4-23.8°C. Seawater in the rearing FRP
aquarium was changed every 3 days. Suffi-
cient amounts of bivalves (Ruditapes philip-
pinarum, Meretrix lusoria, and Mactra veneri-
formis) were supplied as food for R. venosa
during the rearing period.

Sex Ratio

Sex ratios in a total of 445 sexually mature
individuals (>7.1 cm in shell height) were in-
vestigated from June 1994 to May 1995. Sex
of individuals was determined by visual ob-
servation of the presence/absence of the
penis. Female individuals do not have any
genital organ (penis) near the tentacles. To
confirm a clear sexuality for each individual,
both sexes were confirmed by light micro-
scopic examination of histological prepara-
tions of the gonads. A Chi square test on
goodness-of-fit was used to test the hypothe-
sis of equal representation of each sex.

RESULTS

Morphology and Internal Structures of Re-
productive Organs

The purple shell Rapana venosa is dioe-
cious, and the ovary is located on the surface
of the digestive gland in the spiral posterior re-
gion of the shell. The ovary is composed of

244

numerous oogenic follicles. With gonad matu-
ration, the external color of the ovary be-
comes pale yellow. Female individuals do not
have the genital organ (penis) near the tenta-
cles (Fig. 2) and can therefore be usually dif-
ferentiated from males.

Changes in the Gonadosomatic Index (GSI)
and Water Temperature

Monthly GSI changes were measured in fe-
male samples collected between June 1994
and May 1995 (Fig. 3). The GSI values slowly
increased from October through February,
when seawater temperatures gradually de-
creased. However, the values rapidly in-
creased from March, when seawater began to
increase again and then reached the maxi-
mum value (mean 17.0) in April, when seawa-
ter temperature rapidly increased. Thereafter,
the GSI showed relatively lower values in May
to August, when relatively higher water tem-
peratures were maintained and spawning oc-
curred. And the value temporarily reached the
minimum level in September, when most of
gonads were completely degenerated or re-
sorbed after spawning.

CHUNG ETAL.

Electron Microscope Observations on Germ
Cell Development during Oogenesis

Ultrastructural observations allow the germ
cell developmental phases during oogenesis
to be divided into four phases: (1) oogonial
phase, (2) previtellogenic phase, (3) vitel-
logenic phase, and (4) mature phase. Char-
acteristic features in each phase during ooge-
nesis were as follows:

Oogonial Phase: Oogonia in the oogonial
phase, which propagated on the follicular
wall, were oval in shape and 15 um in diame-
ter. They were single or formed a cluster in the
oogenic follicle. Each oogonium had a large
nucleus with chromatin, several mitochondria,
and the endoplasmic reticulum in the cyto-
plasm (Fig. 4A). At this stage, the granular
cells and mesenchymal cells were present
around the oogonia (Fig. 4B).

Previtellogenic Phase: With cytoplasmic
growth, several small mitochondria, a well-de-
veloped endoplasmic reticulum and several
vacuoles were concentrated around the nu-
cleus in the cytoplasm of the previtellogenic
oocyte (Fig. 4C, D). The number of Golgi ap-

20 30
AN
©
x 1 5 —1 E —
O Lao dz
À 20 >
= tf <
< Ge
= 10-4 O re
Ф >
O LL]
о 4 HE
> i [= Os ar
oO E LL]
O-5- E Е ==
@
--@-- Water temperature e
—f- Gonadosomatic index

SAS LOAN" DA Me AN

1994

(895

MONTH

FIG. 3. Monthly changes in the gonadosomatic index in Rapana venosa and the mean water temperatures.

Vertical bars represent standard error.

REPRODUCTION IN RAPANA 245

ЕЕ
ER
+

“el

FIG. 4. Electron micrographs of oogenesis of Rapana venosa (A-F). A, An oogonium, with a large nucleus
and several mitochondria in the cytoplasm, scale bar = 2 um; B, the mesenchymal tissue and the granular
cells near the oogonia, scale bar = 2 um; C, a previtellogenic oocyte, with a large nucleus containing chro-
matin, and several mitochondria, a number of vacuoles and well-developed endoplasmic reticula in the cy-
toplasm, scale bar = 2 um; D, E, the previtellogenic oocytes, with the Golgi apparatus and glycogen particles
in the vacuoles, scale bars = 4 um; Е, an early vitellogenic oocyte, with a number of the vacuoles and lipid
droplets and yolk granules near the nucleus, scale bar = 2 um. (Figure continues.)

246 CHUNG ET AL.

FIG. 4. (Continued) G, a late vitellogenic oocyte, with yolk granules and proteid yolk granules, scale bar = 2
um; H, a late vitellogenic oocyte, with a number of proteid yolk granules and immature yolk granules; |, a ma-
ture oocyte, with mature yolk granules near the nucleus, scale bar = 2 um; J, a mature oocyte, with a mature
yolk granule being composed of the main body (central core), superficial layer and a limiting membrane, of
a yolk granule, scale bar = 2 um. Abbreviations: CR, chromatin; ER, endoplasmic reticulum; G, Golgi appa-
ratus; GC, granular cell; GR, granule; IYG, immature yolk granule; LD, lipid droplet; LM, limiting membrane
of mature yolk granule; M, mitochondrion; MBy, main body; MC, mesenchymal cell; MYG, mature yolk gran-
ule; N, nucleus; OC, oocyte; OG, oogonium; PYG, proteid yolk granule; SL, superficial layer; V, vacuole; YG,

yolk granule.

paratus, scattered from the perinuclear region
to the cortical region of the oocyte in the pre-
vitellogenic phase, increased (Fig. 4D). At this
time many vacuoles formed by the Golgi ap-
paratus appeared near the endoplasmic retic-
ulum and some mitochondria (Fig. 4E).

Vitellogenic Phase: In the early vitellogenic
oocyte, especially with the initiation of yolk
formation, glycogen particles, lipid droplets
and a few yolk granules were found in the vac-
uoles formed by the Golgi apparatus in the
perinuclear region. Yolk granules diffused to-
ward the cortical layer, and yolk granules ap-

peared around the well-developed cortical re-
gion of early vitellogenic oocytes (Fig. 4F). In
the late vitellogenic oocyte, accumulation of
yolk granules occurred in the cortical layer.
Particularly, yolk granules, proteid yolk gran-
ules, and glycogen particles were apparent in
the cytoplasm (Fig. 4G). In addition, a number
of modified mitochondria and endoplasmic
reticulum were involved in the formation of
proteid yolk granules. Glycogen particles, yolk
granules and proteid yolk granules were ac-
cumulated in the cytoplasm. Eventually, their
yolk granules containing several different
components were intermingled and became

REPRODUCTION IN RAPANA 247

immature yolk granules in the oocyte in the
vitellogenic phase (Fig. 4H).

Mature Phase: In the oocyte in the mature
phase, small immature yolk granules were
continuousiy mixed and became larger ma-
ture yolk granules in the cytoplasm (Fig. 41). A
mature yolk granule was composed of three
components: (1) main body (central core), (2)
superficial layer; and (3) a limiting membrane
of a mature yolk granule (Fig. 4J).

Occurrence of Germ Cells during Oogenesis
and the Reproductive Cycle

Based on electron microscopic and histo-
logical observations of the germ cells and

60

FREQUENCY (%)
>
о

N
о

1994

Early active

M Ripe
E Recovery

other surrounding cells, the gonadal phases
were classified into five successive stages
(Fig. 5), which show an annual cycle. Occur-
rence of germ cells of various phases at each
stage and their characteristics are as follows:

Early Active Stage: The gonadal volume was
small, and the follicles occupied approxi-
mately 25% of the gonad. The follicular walls
(germinal epithelium) were relatively thick. A
few oogonia of 15 um in diameter were pre-
sent along the follicular walls ofthe ovary, and
the previtellogenic oocytes appeared. Previ-
tellogenic oocytes of 60-70 um in diameter
formed an egg-stalk attached to the follicular
wall (Fig. 6A). The individuals in the early ac-
tive stage were found from September to

ES] Late active
Partially spawned

FIG. 5. Frequency of gonadal phases of Rapana venosa from June 1994 to May 1995.

248 CHUNG ET AL.

FIG. 6. Photomicrographs of the histological gonadal phases of the female purple shell, Rapana venosa. À,
Transverse section of oogenic follicles in the early active stage, scale bar = 60 um; B, C, section of follicles
in the late active stage, scale bars = 60 ит; D, section of ripe oocytes in the пре stage, scale bar = 60 um;
E, section of follicles in the partially spawned stage, scale bar = 60 um; F, section of the follicles in the re-

covery stage, scale bar = 60 um.

February when seawater temperatures were
gradually decreasing.

Late Active Stage: This stage is character-
ized by the presence of developing early vitel-
logenic oocytes. Follicular walls (germinal ep-
ithelium) of oogenic follicles were thin. A
number of early vitellogenic oocytes of
120-140 um in diameter were attached to the
follicular walls through each egg-stalk. With

the initiation of yolk formation, there were nu-
merous yolk granules in the cytoplasm of late
vitellogenic oocytes of 150-190 um. Some
mature oocytes were free in the lumen of the
follicle (Fig. 6B, C). The individuals in the late
active stage appeared from October to April.

Ripe Stage: Follicles occupied over 70% of
the gonad, and follicular walls became very
thin. Mature oocytes growing up to 230-250

REPRODUCTION IN RAPANA

um diameter became polygonal in shape, and
contained a number of mature yolk granules
(Fig. 6D). Mature or ripe ovaries were found in
March through July, when seawater tempera-
tures were gradually increasing.

Partially Spawned Stage: The lumen of the
oogenic follicle became considerably empty
because approximately 50-60% of ripe
oocytes in the lumen were discharged.
Spawned ovaries were characterized by the
presence of a few undischarged vitellogenic
oocytes as well as previtellogenic oocytes in
the follicle (Fig. 6E). This stage appeared in
the individuals collected from May to early Au-
gust, when seawater temperatures were
18-26°C.

Recovery Stage: After spawning, each folli-
cle shrank, and then degeneration or resorp-
tion of undischarged vitellogenic or mature
oocytes occurred. Thereafter, the connective
tissues and a few oogonia and oocytes ap-
peared on the newly formed follicular walls
(Fig. 6F). The individuals in the recovery
stage were found from June to September.

First Sexual Maturity

During the breeding season, a total of 214
individuals (3.1-17.0 cm in shell height) were
histologically examined to check whether they
reached maturity and participated in repro-

249

duction. The rate of shells of different size that
reached the first sexual maturity is summa-
rized in Table 1. The breeding season of R.
venosa was found to be from May to August.
Inthe case of some individuals with gonad de-
velopmental stage in the late active stage in
May through July, it is supposed that they can
reach maturity except for individuals in the
early active stage during the breeding sea-
son. First sexual maturity was 0% in female
purple shells of 3.1 to 5.0 cm high if they were
at the early active stage during the breeding
season. The percentages of first sexual matu-
rity of female snails of 5.1-6.0 cm and
6.1-7.0 cm in shell height were 14.3% and
27.3%, respectively: most of the individuals
were still in the early active stage. Percentage
of first maturity in 7.1 to 8.0 cm in shell height
were over 50%, all of which were at the late
active, ripe or partially spawned stages. Sex-
ual maturity was 100% for shells over 10.1 cm
in height.

Copulatory Behavior and Spawning

In the experimental aquaria in the labora-
tory, copulatory interaction between females
and males could be observed from early April.
During this time, it was confirmed from histo-
logical examinations that most of the ovaries
were filled with a number of late vitellogenic
oocytes or mature oocytes in the follicle rep-

TABLE 1. The shell height and first sexual maturity of female purple shell,
Rapana venosa from early August 1994 to May 1995.

Number of individuals by gonadal stage*

Shell height (cm) EA LA RI
3.1-4.0 6

4.1 ~ 5.0 8

5.1 ~ 6.0 12 2
6.1 ~ 7.0 8 2 1
7.1 ~ 8.0 15 2 11
8.1 - 9.0 5 2 9
9.1 - 10.0 3 16
10.1 ~ 11.0 11
11.1 - 12.0 9
12.1 - 13.0 5
13.1 ~ 14.0 5
14.1 ~ 15.0 5
15.1 ~ 16.0 5
16.1 ~ 17.0 2

Total

PS RE Total Mature (%)
6 0.0
8 0.0
14 14.3
11 2703
iS 31 51.6
4 20 75.0
9 28 89.3
9 4 24 100.0
9 2 20 100.0
4 1 10 100.0
5 2 12 100.0
6 2 13 100.0
6 3 14 100.0
1 3 100.0

214

*Gonadal stage: EA, early active stage; LA, late active stage; RI, ripe stage; PS, partially

spawned stage; RE, recovery stage.

250 CHUNG ET AL.

resenting the late active or ripe stages. In the
peak spawning period (June to July), how-
ever, spawning in female individuals occurred
15-30 days later after copulation. This obser-
vation indicates that copulation occurs one to
two months earlier than the spawning in fe-
male individuals.

Spawning and Number of Spawning
Frequencies (Broods) in Aquarium

Egg capsules and spawning intervals of Я.
venosa were counted by visual observation
(Table 2, Fig. 7). Five out of twenty individuals
successfully spawned in the experimental
aquarium kept in the laboratory from May to
August 1994: a total of 301 egg capsules
were spawned by No. 1 adult female at inter-
vals of 1-4 days (three broods); 410 capsules
by No. 2 at 1-2 days (four broods); 306 cap-
sules by No. 3 at 1-2 days (three broods); 184
capsules by No. 4 at 2 days (two broods); and
No. 5 spawned a total number of 386 egg cap-
sules at intervals of 1-3 days (four broods).

The average time required for a spawning
of this species was 5.5 h (1-6 h). According to
the observation in the laboratory, most
spawning occurred from night to early morn-

ing.

FIG. 7. External feature of deposition of egg cap-
sules on the shell of Rapana venosa.

Fecundity, Egg Size, Hatching Shell Length
and Mode of Development

The total number of egg capsules per indi-
vidual and the mean number of eggs in an egg
capsule of R. venosa were 184 to 410 and
976, respectively (Table 2). Thus, fecundity

TABLE 2. Spawning frequency and the number of egg capsules of Rapana venosa observed from May to

July 1995.
Shell

Ind. height Spawning Spawning

No. (cm) date hours

1 13.5 Мау 23 1994 18:34-24:06
Мау 24 1994 02:36-08:18
Мау 28 1994 01:16-07:08

2 14.6 Мау 24 1994 23:16-05:16
Мау 25 1994 16:31-21:52
Мау 27 1994 16:26-22:10
Мау 28 1994 04:34-10:08

3 13.6 June 15 1994 22:24-03:50
June 16 1994 01:04-06:36
June 18 1994 03:14-08:05

4 14-2 June 20 1994 23:34-04:30
June 22 1994 05:48-18:18

5 14.4 July 24 1994 22:10-03:09
July 25 1994 19:18-23:50
July 28 1994 16:06-20:43
July 29 1994 05:04-09:43

Water No. of Spawning Interval
temp. egg frequency (day)
(°C) capsules (No. of broods)
18.4 113
18.6 96
20.5 92
(301) 3 1-4
19.4 118
20.1 107
20.0 94
20.4 91
(410) 4 1-2
21.6 114
21.8 98
22.3 94
(306) 3 1-2
22.5 96
22.8 88
(184) 2 2
22.9 112
23.0 95
23.6 92
23.8 87

REPRODUCTION IN RAPANA

was estimated to be 179,000 to 400,000 eggs
per individual. The egg size and hatching
shell length of R. venosa in Korea were
230-250 um and 0.41 x 0.30 mm, respec-
tively (Table 3). The duration of development
from fertilized eggs to veliger larvae in egg
capsules just before hatching of R. venosa,
were 15 to 17 days at about 20°C under labo-
ratory conditions (Fig. 8). The mode of devel-

251

opment of this species is a planktotrophic
veliger larval pattern because the embryos

hatch as planktotrophic veliger larvae.

Sex Ratio

Of total 445 purple shells, 228 were fe-
males and 217 males (Table 4). There was no
significant difference in the prevalence of

TABLE 3. Comparison of the sizes of eggs and egg capsules, and larval shells of some species in the

family Muricidae.

Shell length

Number of Egg Duration of of larvae at
Shell length at Egg size eggs in an capsule development haching
hatching (mm) (mm) egg capsule size (mm) (days) (mm) Source
Hatch as veligers:
Rapana venosa 0.26 790 - 1300 30x25 13 0.41 x 0.29 Amio, 1963
R. venosa 0.24 772 - 1190 30x26 15 - 17 0.41 x 0.30 Present study
R. bulbosa 0.28 - 0.32 0.42 Thorson, 1940
Bedevina birileffi 0.19 60 ~ 90 3.0 x 0.85 14 0.30 ~ 0.32 Amio, 1963
Thais clavigera 0.19 160-220 4.5x1.2 0.30 - 0.32 Amio, 1963
T. bronni 0.20 160-360 1.0x1.5 0.32 - 0.34 Amio, 1963
T. tissoti 0.19 75 11 0.32 Middelfart, 1996
T. floridana 0.11 0.13 D’Asaro, 1966
T. carinifera 0.20 - 0.22 0.32 - 0.34 Thorson, 1940
Purpura patula 0.24 0.4 Lewis, 1960

FIG. 8. Morphology of veliger larvae before hatching of Rapana venosa. Scale bar = 0.4 mm.

252

CHUNG ET AL.

TABLE 4. Monthly variations in sex ratio of Rapana venosa.

No. of No. of

Month Females Males
June 1994 24 20
July 1994 20 26
August 1994 23 19
September 1994 22 1174
October 1994 18 24
November 1994 18 15
December 1994 14 18
January 1995 14 19
February 1995 18 13
March 1995 21 18
April 1995 19 15
May 1995 17 13
Total 228 217

Total Sex Ratio x (Chi
Numbers (F/(F + M)) squared)*
44 0.55 0.37
46 0.43 0.78
42 0.55 0.38
39 0.56 0.64
42 0.43 0.86
33 0.55 0.27
32 0.44 0.50
33 0.42 0.76
31 0.58 0.81
39 0.54 0.23
34 0.56 0.47
30 0.57 0.53
445 0.51 0.27

“The critical value for x goodness-of-fit test of equal numbers of females and males (1 df) at 95% sig-

nificance is 3.84.

each sex (not different from a 1:1 sex ratio,
X = 0.27, p > 0.05), and monthly comparisons
showed no statistical differences.

DISCUSSION

Germ Cell Development and Vitellogenesis

Our electron microscope observations of
early vitellogenic oocytes of Rapana venosa
indicate that the Golgi apparatus is involved
in the formation of a number of glycogen
particles filled vacuoles and small vesicles in
the perinuclear region of the cytoplasm. Lipid
droplets and lipid granules are then added to
the vacuoles and vesicles formed by the Golgi
apparatus (referred as autosynthetic), as in
llyanassa obsoleta (Taylor 8 Anderson, 1969),
Biomphalaria glabrata (de Jong-Brink et al.,
1976), Mytilus edulis (Reverberi, 1971), and
Mactra veneriformis (Chung & Ryou, 2000).
Therefore, our observation suggests that the
Golgi apparatus and vacuoles present in the
perinuclear region are involved in the forma-
tion of lipid droplets and lipid granules in the
early vitellogenic oocytes. Some mitochondria
in late vitellogenic oocytes of R. venosa ap-
pear to undergo a process of transformation
during late vitellogenesis, and some evidence
affirms that the yolk precursors are derived
from modified mitochondrial structures found
in the cytoplasm. The endoplasmic reticulum
and modified mitochondria are present near
the proteid yolk granules. However, we could
not find pinocytotic tubules, such as those in-
volved in yolk production in the vitellogenic
oocytes of Agriolimax reticulatus (Hill 8

Bowen, 1976; Dohmen, 1983), or multivesicu-
lar bodies as seen in the opisthobranchs
Hypselodoris tricolor and Godiva banyulensis
(Medina et al., 1986), and another snail, Physa
acuta (Terakado, 1974). Accordingly, it is as-
sumed that the endoplasmic reticulum and
modified mitochondria are involved in the for-
mation of proteid yolk granules (Taylor & An-
derson, 1969), with R. venosa only synthesiz-
ing yolk autosynthetically, as seems to occur in
the majority of gastropods. Exceptions include
gastropod species (Planorbarius corneus,
Lymnaea stagnalis, Hypselodoris tricolor, and
Gordiva banyulensis) that synthesize yolkbya
combination of autosynthetic and heterosyn-
thetic processes (Bottke et al., 1982; Medina
et al., 1986).

Occurrence and Resorption of
Germ Cells during Oogenesis and
the Reproductive Cycle

As in most other marine mollusks (Chung et
al., 1993; Chung & Ryou, 2000), gonad de-
velopmental phases of R. venosa show an
annual periodicity (Chung et al., 1993; Chung
& Kim, 1997), with cyclic change of germ cell
development during the reproductive cycle.

According to occurrence of germ cells with
the gonad developmental stage, the oogonia
and previtellogenic oocytes appeared in the
early active stage, and the early and late vitel-
logenic oocytes occurred in the late active
stage. Numerous fully mature oocytes in the
ripe stage were released in the partially
spawned stage. After spawning, small num-
bers of undischarged vitellogenic and mature

REPRODUCTION IN RAPANA 253

oocytes were degenerated and resorbed.
Newly formed oogonia and previtellogenic
oocytes occurred in the recovery stage.

Regarding reproductive energy allocated to
the production of gametes, in particular, the
continuous production and resorption of germ
cells may be regard as an adaptation to envi-
ronmental temperature and food availability
(Morvan & Ansell, 1988; Paulet, 1990). If the
reproductive energy allocated to the produc-
tion of gametes is too large, nutritive reserves
may not be enough to allow all eggs to reach
the critical size for spawning and fertilization.
In this case, the products of gamete atresia
may be resorbed and the energy reallocated
to still-developing oocytes or used for other
metabolic purpose by marine moliusks (Dor-
ange & LePennec, 1989; Motavkine & Varak-
sine, 1989). Therefore, it is supposed that R.
venosa should have a reproductive mecha-
nism to resorb and utilize the high nutritive
substances rather than releasing hopeless
gametes.

Gonadal Development and Maturation

It is well known that development and mat-
uration of gametes of prosobranchs are gen-
erally influenced by several factors, such as
temperature, food availability, illumination
(day length), and hormones (Boolootian et al.
1962; Fretter, 1984).

We observed that gametogenesis of Я.
venosa initiates at a temperature of about
3.0°C, with maximum gonadal maturation oc-
curring in June, when temperatures were high
and phytoplankton were very abundant. Ac-
cording to Segal (1956) and Sutherland
(1970), in temperate prosobranchs, gonadal
maturation and spawning occur in rapid suc-
cession during the summer. The importance
of food is obvious in the limpet Acmaea spp.:
gametogenic activity of upshore populations
with less available food is more restricted than
those of the same species lower on the shore.
As suggested by Sastry (1963) from a study
with bay scallops, water temperature seems
to be the most important parameter for main-
tenance of gonadal development. In Korean
coastal waters, growth and production of bi-
valves that are predated upon by R. venosa is
relatively high from spring to early summer
seasons (Kim et al., 1977; Chung et al., 1994;
Lee, 1995) due to the abundance in phyto-
plankton. Thus, abundant food supply (e.g.,
bivalves) is available to R. venosa during the
period of gonadal development and matura-

tion. Therefore, it is suggested that gonadal
development and maturation of Korean R.
venosa is closely related to temperature
change and food availability. Fretter (1984)
observed that in temperate zones, the sea-
sonal temperature fluctuation associated with
changing illumination is a controlling factor in
gametogenesis. In consequence, gonadal de-
velopment and maturation of this species may
be retarded under low illumination, due to the
decrease in food (bivalves) availability caused
by diminished primary production of phyto-
plankton.

First Sexual Maturity with the
Gonad Developmental Stage

All specimens of 3.1-5.0 cm high were in
the early active stage although collected dur-
ing breeding seasons, and our gonad histol-
ogy indicates that none of them could fully de-
velop: only small numbers of oogonia and
previtellogenic oocytes were present in the
oogenic follicle. The size of the oocyte indi-
cates that they could not have reached matu-
rity until late August, when spawning was
ended. Accordingly, the percentage of first
sexual maturity of female snails ranging from
3.1-5.0 cm in shell height is 0%. However, in-
dividuals 7.1-8.0 cm in shell height belonged
to one of the early active, late active, ripe and
partially spawned stages during the breeding
season. Sixteen individuals in the late active,
ripe, and partially spawned stage underwent
gonadal development, whereas 15 individuals
in the early active stage did not. It was ob-
served that among snails of 7.1-8.0 cm high
and in the late active stage more than 50%
reached the first sexual maturity. However all
snails, in late active, ripe, or partially spawned
stages, reached it ifthey were larger 10.1 cm.
This means that larger individuals can reach
maturity earlier than smaller individuals. The
results suggests that because catching purple
shells < 7.1 cm can potentially cause a dras-
tic reduction in recruitment, a prohibitory mea-
sure should be taken for adequate natural re-
sources management.

Breeding Pattern

Our histological observations on the purple
shell show that spawning of R. venosa on the
west coast of Korea occurs from late May to
August. Japanese R. venosa have been re-
ported to spawn once a year from June to Au-
gust in the Ariake Sea, Japan (Amio, 1963).

254 CHUNG ET AL.

Our results agree well with those described by
Amio (1963). The slight discrepancy in the
spawning period between these two studies
might be related to geographic differences in
water temperature (Chung et al., 1993). As
the spawning of R. venosa in Korea occurs
from late May to August, this species may be
classified as asummer breeder (Boolootian et
al., 1962).

Morphology of Egg Capsule

The surface of the exit part ofthe egg cap-
sule of R. venosa is flattened and has a chiti-
nous capsular form. The egg capsule in the
genus Rapana have a curved sickle shape,
with a long cylindrical stem, and an albume-
nous substance being contained in the egg
capsules. These findings coincide with Amio’s
observation from R. venosa in Japan (Amio,
1963). Rawlings (1999) described that the
morphology of egg capsules varies within the
marine neogastropods, showing differences
in shape, size, surface texture within families
(D’Asaro, 1970, 1988, 1991, 1993, 1997;
Bandel, 1973). Some species in a genus (Per-
ron & Corpuz, 1982; Palmer et al., 1990; Mid-
delfart, 1996) and sometimes even individuals
of the same species population (Rawlings,
1990, 1994, 1995, 1999) show distinct char-
acteristics. These morphological differences
in neogastropods may be associated with en-
vironmental factors such as physical stresses
or geographical latitude.

Number of Eggs in an Egg Capsule

In the present study, R. venosa in Korea
spawned 184 — 410 egg capsules each, and
the mean number of eggs from an egg cap-
sule was 976. Thus, the fecundity (the total
number of eggs) from each individual ranged
from 179,000 to 400,000 eggs. Amio (1963)
described that the number of eggs in each
egg capsule of R. venosa in Japan ranged
from 790 to 1,300. These results indicate a
close similarity in reproductive output be-
tween the Korean and the Japanese purple
shells.

Egg Size and Hatching Shell Length

The egg sizes of R. venosa collected in
Korea (230-250 um) is slightly smaller than
those of R. venosa (260 um) in Japan, and
shell lengths of veliger larvae at hatching of

the Korean and Japanese purple shells were
0.41 x 0.30 mm and 0.41 x 0.29 mm, respec-
tively. The sizes of eggs and shell length of
veliger larvae at hatching of R. venosa in
Korea were similar to R. venosa in Japan, but
the egg sizes and hatching shell lengths of
veliger larvae of R. venosa were larger than
those of Thais clavigera, T. bronni, and Be-
devina birileffi (Table 3). Spight (1976) and
Middelfart (1996) described that egg size in
the egg capsule and shell length of veliger lar-
vae at hatching are very similar for genera
within Muricidae (Table 3). The factors affect-
ing egg size and hatching shell length of R.
venosa have not been studied yet.

Duration of Development in the Egg Capsule

We observed that the duration of develop-
ment from fertilized eggs to hatching veligers
ranged from 15-17 days at about 20°C in the
laboratory. However, Amio (1963) described
that of R. venosa in Japan took 12 days to de-
velop from trochophore larvae to veliger larvae
(i.e., about 13 days after fertilization). In gen-
eral, our results were similar to those of Amio’s
(1963) in the duration of development in egg
capsules (Table 3). It seems that the duration
of development in egg capsules, from deposi-
tion of an egg mass to hatched larvae, varies
with both internal and external factors, as pro-
posed by Amio (1963). Middelfart (1996) re-
ported that duration of development of Thais
tissotiwas 11 days. Accordingly, it is assumed
that duration of development of this species is
shorter than R. venosa (Table 3).

No Evidence of Nurse Eggs as Larval Food

According to our microscopic observations,
approximately 80% of eggs in a capsule were
normally fertilized, and these embryos
hatched as veliger larvae. The remaining 20%
failed to develop, but they did not serve as
nurse eggs for the normally developing lar-
vae.

Spight (1976) has classified marine gas-
tropods into two groups based on hatching
modes: (1) species with embryos that hatch
as planktotrophic veliger larvae; and (2)
species with embryos that hatch as crawling
young snails (juveniles). Spight (1976) also
differentiated developing embryos into those
that consume nurse eggs and those do not
consume nurse eggs within egg capsules in
the process of embryonic development.

REPRODUCTION IN RAPANA 255

Of Muricidae species, Siratus senegalensis
(Knudsen, 1950), Chicoreus torrefactus (Cer-
nohorsky, 1965), and Thais emarginata (Le
Boeuf, 1971; Middelfart, 1994) have been re-
ported to hatch as young snails. According to
Spight (1976), among Muricidae, 16 species
hatch as snails consuming nurse cells, and 17
species hatch as snails without nurse cells.
Twenty-two species hatch as veligers without
nurse cells, but four hatch as veliger with
nurse cells. These species consume 5.9-91.4
nurse eggs per embryo during development
(Spight, 1976), and the size of newly hatched
young snails is usually large (> 1.15 mm). In
Pisania maculosa, a species with embryos
that do not consume nurse eggs, 90% of the
eggs were not fertilized, 8% became nurse
eggs, and only 2% developed normally
(Staiger, 1950). If the encapsulated embryos
should share a common nutrient of albumen,
developmentally retarded eggs are likely to be
fed by surviving embryos (Staiger, 1951). It
has been suggested that in case of hatching
size of veliger is < 0.5 mm, R. venosa do not
use nurse eggs, whereas young snails > 1.0
mm may use nurse eggs for development.
Shell size of the veliger larvae of R. venosa
was relatively small (< 0.41 x 0.30 mm), indi-
cating that the purple shell hatches as
veligers without utilization of nurse eggs dur-
ing development.

In view of our present findings and those
observed in other prosobranch gastropods
(Spight, 1976), an important postulate can be
made: species that hatch as large (> 1.0 mm)
veligers or juvenile snails consume nurse
eggs during development in the egg capsules,
whereas those with smaller veligers (< 0.5
mm) do not.

ACKNOWLEDGEMENTS

The authors are grateful to Dr. John B.
Burch of the University of Michigan and to the
referees for helpful comments on the manu-
script. The authors are grateful to the Fish-
eries Science Institute of Kunsan National
University for equipment and fund in the pro-
gram year of 2001.

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change related to the introduction of new mollusk
species. Marine Ecology, 17: 227-236.

Revised ms. received 10 September 2001

MALACOLOGIA, 2002, 44(2): 259-272

IMPACT OF SOIL CHEMISTRY ON THE DISTRIBUTION OF
ONCOMELANIA HUPENSIS (GASTROPODA: POMATIOPSIDAE) IN CHINA

Edmund У. W. Seto', Weiping Wu?, Dongchuan Qiu’, Hongyun Ми“, Xueguang Gu’,
Honggen Chen“, Robert С. Spear” & George М. Davis?

ABSTRACT

Oncomelania hupensis subspecies serve as intermediate hosts for the human Schistosoma
parasite in China. In this study we present a multivariate analysis and comparison soil chemistry
data associated with the presence or absence of two subspecies, O. hupensis robertsoni and O.
hupensis hupensis, that are associated with schistosomiasis disease transmission upstream and
downstream, respectively, of the soon to be completed Three Gorges Dam on the Yangtze River.
Soil and water samples were collected and analyzed from the Anning River Valley, an 800 km?
area in the mountainous region of Sichuan Province and from the Poyang Lake floodplain, a
4,647 km? area in Jiangxi Province. A multivariate classification of soil data was able to discrim-
inate between snail habitat and non-habitat in the Anning River Valley with 85.5% accuracy, and
revealed that the lack of critical soil conditions potentially excluded O. hupensis robertsoni from
some sites. The soil classification was also able to correctly identify 72.4% of marginal habitat
sites that were misclassified by field ecologists. We found that O. hupensis hupensis was less
dependent upon soil conditions for sites on the Poyang Lake floodplain, and hypothesize that
changes in water level associated with seasonal flooding may play a larger role than soil in dis-
criminating between snail habitat and non-habitat in this environment.

Key words: classification and regression trees, China, Oncomelania hupensis, schistosomia-
sis, Schistosoma japonica, soil chemistry, snail ecology, Poyang Lake, Anning River, Sichuan,

Jiangxi.

INTRODUCTION

Despite the successful eradication of schis-
tosomiasis in some regions of China, it still re-
mains a serious infectious disease that is en-
demic in 118 counties, infecting nearly one
million Chinese (Chen & Zheng, 1999). It is
estimated that over 40 million are at risk of de-
veloping schistosomiasis in China. Many of
those at risk live along the Yangtze River,
which stretches across China, from the moun-
tains in the west to the coast in the east. Along
this span exist a multitude of terrains and en-
vironments, some of which are ideal habitats
for subspecies of Oncomelania hupensis,
which serve as the intermediate snail hosts
for the Schistosoma japonica parasite that
causes schistosomiasis.

Current research attempts to better charac-
terize the different ecologies related to schis-
tosomiasis transmission (Davis et al., 1999;
Zhou et al., 2001). Such work is of critical im-

portance, as the construction of the new
Three Gorges Dam on the Yangtze will poten-
tially result in the emergence of schistosomia-
sis in entirely new areas (Davis et al., 1999;
Hotez et al., 1997). The Three Gorges Dam
will be the world’s largest dam. The reservoir
created by the dam may impact global cli-
mate, cause dramatic change in the flow pat-
terns of the Yangtze River that will increase
snail habitat, and increase the probability of
snail migration so dramatically that the geo-
graphic distribution of snail habitats may
change. A better understanding of which envi-
ronments can and cannot support snails, and
how these environments are distributed spa-
tially now and in the future can potentially lead
to effective control strategies. This is particu-
larly true in China, where historically, snail
control has been an integral part of success-
ful disease eradication (Chen & Zheng, 1999;
Sleigh et al., 1998).

The need for a better ecological under-

School of Public Health, University of California, Berkeley, California 94720, U.S.A.; seto @uclink.berkeley.edu.

"Institute of Parasitic Diseases, Chinese Academy of Preventive Medicine, 207 Rui Jin Er Liu, Shanghai 200025, P.R.C.
“Sichuan Institute of Parasitic Diseases, Chengdu 610041 Sichuan, P.R.C.

“Jiangxi Provincial Institute of Parasitic Diseases, Nanchang, P.R.C.

School of Public Health, University of California, Berkeley, California 94720, U.S.A.

Department of Microbiology and Tropical Medicine, The George Washington University Medical Center, Washington, D.C.

20037, U.S.A.

260 SETOETAL.

standing of O. hupensis is most evident in cur-
rent research in the use of remote sensing
technologies to identify snail habitats (Spear
et al., 1999; Zhou et al., 2001). Such studies,
motivated in large part by the construction of
the Three Gorges Dam, have been carried out
both upstream above the dam and down-
stream below the dam, in current schistoso-
miasis endemic areas. In such studies it is im-
portant to realize that different subspecies of
O. hupensis exist in different regions, and that
these subspecies have evolved to adapt to
their particular ecological niches and human-
parasite interactions. Oncomelania hupensis
robertsoni exists upstream of the Three
Gorges Dam site, in the mountains of Sichuan
Province where the snails live just above the
waterline in the moist soil and shaded by the
vegetation of irrigation ditch networks feeding
agricultural fields. Oncomelania hupensis hu-
pensis exists downstream of the dam, in the
lower Yangtze basin, where the snails live in
the marshes and grasslands of Poyang Lake,
a low elevation plain, completely enclosed by
an impressive dyke system, and affected by
strong seasonal flooding.

The Anning River Valley in southern Si-
chuan Province characterizes the mountain-
ous environment where O. hupensis robert-
soni live (Figs. 1, 2). The valley, at an
elevation of 1,500 m, is approximately 48 km
in length, 24 km at its widest point, represent-

Three Gorges Area

ing approximately 400 km? of potential snail
habitat. Here, land is predominantly irrigated
agriculture, with two growing seasons and
rice, wheat, corn, tobacco, and various export
vegetables the major crops grown. Domestic
animals play a minor role in the agriculture of
the valley and in schistosomiasis transmis-
sion. Snails do not live within fields, but rather,
along the edges of vegetated irrigation
ditches. Recent prevalence surveys con-
ducted in the summer of 2000 by collabora-
tors from the Xichang County Anti-Schistoso-
miasis Station have shown that the variance
in human schistosomiasis infection is quite
large in the valley, ranging from below 10% in
some areas to above 70% in other areas. On-
comelania hupensis robertsoni that live in this
environment have a smooth shell, which has
no varix on the outer lip and is relatively short
(Davis et al., 1995). The subspecies is re-
stricted to Sichuan and Yunnan provinces
above the Three Gorges.

Conversely, the Poyang Lake environment
where O. hupensis hupensis live, as de-
scribed by Davis et al. (1999), is vastly differ-
ent. Poyang Lake of Jiangxi Province is the
largest lake in China (Figs. 1, 3). During the
flooding season it regularly fills with water and
is held back from towns and villages by a se-
ries of dikes. The time that flooding begins
depends on the onset of the annual monsoon.
Typically the flooding is May through October.

Poyang Lake

Dongting Lake

EN Endemic area
Provincial boundary

FIG. 1. Map of schistosomiasis-endemic areas along the Yangtze River. The Anning River Valley, Poyang

Lake and Three Gorges Dam Area are shown.

SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 261

FIG. 2. Landsat TM satellite image of the Anning
River Valley.

During this period only fisherman can venture
out on the lake when it resembles a vast sea.
The flooding covers all of the marshland used
by cattle for grazing. After the rainy season,
the lake empties, losing as much as 90% of its
water. All snails are found on the flood plains
and on the numerous flat marshy islands in-
side the dikes. All transmission occurs within
the lake basin, where the cattle graze on the
marshlands; there are no snails outside the
dikes. Cattle play a much larger role in trans-
mission in this environment than in the moun-
tains of Sichuan Province. The flooding
drowns adult snails, which cannot withstand
continuous submersion. During drought, the
adults move down into the soil and aestivate.

Oncomelania hupensis hupensis has pri-
marily ribbed shells in habitats regularly
flooded and smooth shells in habitats that are
not flooded (Davis et al., 1995). All popula-
tions have a varix. The length of shell is, on

average, greater than that of O. hupensis
robertsoni. The shell allometry is the same as
that of O. hupensis robertsoni. The life ex-
pectancy for snails in this environment is
about one year (Zhang et al., 1996). On-
comelania hupensis hupensis is restricted to
the Yangtze River drainage below the Three
Gorges; it has spread to Guangxi Province,
presumably by way of the grand canal from
Hunan.

We have studied the characteristic soil con-
ditions for both environments. We apply mul-
tivariate statistics to determine which soil con-
ditions are capable of discriminating between
areas where O. hupensis exist and where
they do not. For the Sichuan environment, we
also look at soil conditions for what we call
“marginal habitat’, which are areas that to the
field ecologist look suitable as snail habitat,
but where snails were not found.

METHODS

Soil samples from sites in the Anning River
Valley were collected as part of a remote
sensing validation study in the summer of
1998 (Spear et al., 1999). Each site was de-
fined by randomly generated geographic co-
ordinates. A global positioning system (GPS)
receiver was used to navigate to each site.
Each site consisted of a 30 x 30 m area,
where a Snail survey was conducted and soil
was collected. A total of 111 sites were visited
(35 snail, 47 non-snail, and 29 marginal sites).
Snail status was determined in two manners.
A snail survey was performed in the sur-
rounding 30 x 30 m area. The snail survey is
a systematic search through the ditches for
the presence of snails (Gu, 1990). The pres-
ence of a single snail in the survey constitutes
a positive snail site. In addition to the snail
survey, a judgment was made by a field ecol-
ogist as to whether the habitat seemed suit-
able for snails. There exists sites where no
snails were found, but where field ecologists
felt the snails could exist.

For each site visited, approximately 1,000
cm? of soil was collected by taking multiple
samples along ditches within the 30 x 30 m
area. Soil samples for each site were com-
bined, dried arid well-mixed to constitute a sin-
gle soil sample for each site. These samples
were later analyzed using Lamotte Company
Electronic Soil Lab, Model DCL-12, Code
1985-01. Measurements were made for pH,
conductivity, nitrate nitrogen, nitrite nitrogen,
ammonia nitrogen, phosphorus, potassium,

262 SETO ET AL.

FIG. 3. Landsat TM satellite image of Poyang Lake at low water, showing TMRC study sites.

sulfur, copper, iron, manganese, zinc, calcium,
magnesium, and chlorides. Soil moisture was
measured using a common garden soil mois-
ture meter. Soil texture was analyzed using
Lamotte Company Texture Kit, Code 1067,
which provided the fractions of sand, silt, and
clay present in the soil.

Water samples were also collected at each
site from the nearest irrigation ditch within the
30 x 30 m area. Water samples were ana-
lyzed in the field using Lamotte Company
GREEN Water Monitoring Kit Code 5848.
Measurements were made for pH, phospho-
rus, turbidity, temperature, dissolved oxygen,
and nitrate.

The Tropical Medical Research Center
based in Shanghai conducted snail surveys at
98 sites in the Poyang Lake environment in
1999 and 2000. Sites were chosen based on a
new randomized snail survey protocol. Ac-
cording to this protocol, buffalo grazing ranges
were mapped, and 10,000 m? “squares” were
located throughout the grazing ranges to pro-
vide adequate coverage of the different areas
of the ranges. Each square is divided into
numbered grids of 10 m x 10 m, of which 20
are selected at random each collecting sea-
son. A frame of 4 m? is placed in the center of

each randomly selected grid-square and all
snails are collected. AGPS record is made of
each grid-square. The percent area with no
snails is calculated from the cells with no
snails. For the area calculated to have snails,
the mean and standard deviation of snails
per m? and the frequency of infections are
recorded. Usable ecological data and soil con-
ditions were determined for 87 ofthe 98 sites.
Snails were found at 50 of the 87 sites. Eco-
logical and soil data measured include ambi-
ent air and soil temperature, humidity, light,
saturation, pH, nitrate nitrogen, phosphorus,
potassium, zinc, ammonia nitrogen, calcium,
chloride, copper, ferric iron, magnesium, man-
ganese, nitrite nitrogen, sulfate, and soil tex-
ture. Soil conditions were measured using the
same Lamotte equipment and methods as that
used in Sichuan Province for the Anning River
Valley.

Univariate statistics were calculated for
each ecological property measured, allowing
for comparison between the Anning River Val-
ley and Poyang Lake environments. To test
for significant univariate differences in the dis-
tribution of ecological properties between the
two environments, Kolmogorov-Smirnov dis-
tance statistics were calculated (Conover,

SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 263

1980). The Kolmogorov-Smirnov statistic is a
non-parametric test of the equality of two dis-
tributions, and was calculated using the Stata
statistical analysis software (Stata Corpora-
tion, 2000).

Multivariate statistics were used to consider
possible interactions between ecological
properties in discriminating between snail
habitat and non-habitat sites. The Classifica-
tion and Regression Trees (CART) method
(Breiman, 1984) was used to create a classi-
fication of habitat versus non-habitat for both
the Anning River Valley and Poyang Lake en-
vironments. CART produces optimal binary
decision trees that describe how different fac-
tors interact to produce particular classifica-
tion outcomes. Trees for the Anning River Val-
ley and Poyang Lake were compared. For
data from the Anning River Valley, information
exists on snail habitat, non-habitat, and mar-
ginal habitat. Only habitat and non-habitat
sites were used in the CART analysis so that
the decision tree could subsequently be used
to predict the outcome for marginal habitat.
CART analyses were performed using the
CART for Windows program from Salford
Systems (Steinberg & Colla, 1997).

RESULTS

Univariate statistics for the ecological vari-
ables related to snail habitats and non-habi-
tats for the Anning River Valley and Poyang
Lake environments are presented in Table 1.
In general, there is greater overall variation in
soil conditions for the Anning River Valley
than in those for Poyang Lake. Snail habitat
within the Anning River Valley is more variable
than that in Poyang Lake, as the standard de-
viations for all but four soil properties (nitrate
nitrogen, potassium, chloride, and nitrite nitro-
gen) are larger for snail habitat in the Anning
River Valley. Similarly, non-habitats within the
Anning River Valley are more variable than
those in Poyang Lake, as the standard devia-
tions for all but four soil properties (potassium,
ammonia nitrogen, chloride, and nitrite nitro-
gen) are larger for non-habitats in the Anning
River Valley. In both regions though, snail
habitat may be complex, as generally higher
variances exist in soil conditions associated
with snail habitat than with non-habitat.

Table 1 shows that there is overlap in the
minimum and maximum ranges of soil proper-
ties for snail habitat and non-habitat, suggest-
ing that there is no clear univariate method for

discriminating between habitat and non-habi-
tat. However, the presence of overlap in the
ranges does not necessarily indicate that the
distributions of soil property for snail habitat
and non-habitat are the same. Kolmogorov-
Smirnov distance statistics are better suited to
determining the difference between two distri-
butions. The distance statistic assesses the
difference in cumulative distributions between
snail habitat and non-habitat sites for a partic-
ular soil property. As an example, the cumula-
tive distributions for sulfate measurements at
snail habitat and non-habitat sites in both the
Anning River Valley and at Poyang Lake are
shown in Figure 4. It is clear from this figure
that there is a significant difference in sulfate
levels for snail habitat versus non-habitat in
the Anning River Valley. This difference in sul-
fate is not apparent for Poyang Lake. Corre-
spondingly, the Kolmogorov-Smirnov statis-
tics presented in Table 2 reveal a relatively
large, statistically significant (P < 0.05) D-sta-
tistic for sulfate in the Anning River Valley ver-
sus a small, statistically insignificant D-statis-
tic for sulfate in Poyang Lake. Overally, for the
Anning River Valley, three soil properties have
statistically significant (P < 0.05) distances
(sulfate, ammonia nitrogen, and chloride),
whereas twice as many distances are statisti-
cally significant for the Poyang Lake environ-
ment (zinc, potassium, nitrite nitrogen, silt,
sand, and calcium). This suggests that a dis-
crimination of snail habitat from non-habitat
will depend upon a different set of soil proper-
ties in the Anning River Valley versus Poyang
Lake.

A graphical illustration of the ranges of the
ecological variables for snail habitat sites in
the Anning River Valley and Poyang Lake is
presented in Figure 5. Comparing the ranges
for snail habitat for the two subspecies, it is
apparent that for most soil properties there is
overlap. However, there are distinct differ-
ences in the range of pH and calcium. More-
over, although there is some overlap, the lev-
els of silt, phosphorus, ferric iron, magnesium,
nitrite nitrogen and sulfate seem different for
the two subspecies. Again, rather than only
looking at ranges, Kolmogorov-Smirnov sta-
tistics were used to compare the distributions
of snail habitat for the two areas (Table 3). All
but one of the distance statistics (chloride)
were statistically significant, reinforcing the
fact that the two snail subspecies live in very
different environments.

To understand how multivariate interactions
work to discriminate between snail habitat and

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SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 265

(A) — + Habitat ——— Non-habitat
ía a m
| Г ;
¿
DE :
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ES P
5 - }
oS р Kolmogorov-Smirnov
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Sulfate (ppm)

FIG. 4. Cumulative distributions and Kolmogorov-Smirnov statistics for snail habitat versus non-habitat for
(A) Anning River Valley and (B) Poyang Lake.

266 SETOETAL.

TABLE 2. Kolmogorov-Smirnov distance between snail habitat and non-habitat for each
soil property. A comparison of Kolmogorov-Smirnov distances for the Anning River
Valley and Poyang Lake suggest that the discrimination of snail habitat and non-habitat
for the two environments depend upon different soil properties. Statistically significant

results (P < 0.05) are shown in bold type.
Anning River Valley

Poyang Lake

Soil Moisture 0.2558
Sand (%) 0.1318
Silt (%) 0.1792
Clay (%) 0.1045
pH 0.2208
Nitrate Nitrogen (Ibs/acre) 0.1695
Phosphorus (Ibs/acre) 0.2000
Potassium (lbs/acre) 0.1604
Zinc (ppm) 0.1091
Ammonia Nitrogen (lbs/acre) 0.3623
Chloride (lbs/acre) 0.3266
Copper (ppm) 0.1257
Ferric Iron (ppm) 0.1435
Magnesium (lbs/acre) 0.2916
Manganese (ppm) 0.1864
Nitrite Nitrogen (lbs/acre) 0.1381
Sulfate (ppm) 0.4013
Calcium (lbs/acre) 0.2942

P value D P value

0.138

0.831 0.3455 0.019
0.483 0.3455 0.018
0.962 0.1480 0.742
0.242 0.2674 0.119
0.554 0.2343 0.225
0.356 0.1534 0.714
0.626 0.4005 0.005
0.945 0.4370 0.001
0.008 0.2020 0.379
0.024 0.0647 1.000
0.874 0.2175 0.298
0.747 0.2470 0.174
0.057 0.2456 0.178
0.436 0.2428 0.192
0.777 0.3695 0.009
0.003 0.2484 0.170
0.052 0.3082 0.048

non-habitat for each subspecies of snail,
CART analyses were performed. The CART
tree forthe Anning River Valley is shown in Fig-
ure 6. The binary decision tree is read from the
top down. All 83 data observations are repre-
sented at the topmost node of the tree, where
42% of the sites are snail habitat. CART fol-
lows a brute force algorithm, where every pos-
sible binary split criterion for every soil prop-
erty is considered. The criterion that produces
the best outcome in terms of splitting snail
habitat from non-habitat is then chosen to split
the top node into two child nodes. For the An-
ning River Valley, magnesium is chosen to be
the best first split. If the level of magnesium at
a particular site is less than or equal to 1671.8,
that site is passed to the left child. Otherwise,
the site is passed to the right child. After this
split, 73 of the original 83 sites are passed to
the left child node, of which 34.2% are snail
habitat sites. The remaining ten sites are
passed to the right child node, where 100% of
the sites are snail habitat. Since all ten sites in
the right child node are snail habitat, there is
no need for further splits. However, for the left
child node with 73 sites, CART determined
that a criterion based on sulfur can further dis-
criminate between snail habitat and non-habi-
tat sites. This criterion leads to the creation of
two additional child nodes. This procedure

continues recursively until child nodes can no
longer be reliably split by a soil criterion. In the
Anning River Valley tree magnesium, sulfate,
phosphorus, and silt are used. Actually, not
displayed in the tree, but still important are
contributions from chloride and potassium, the
values of which were used when values of
the other four soil properties were missing.
The six-parameter tree produces a remark-
ably high 85.5% cross-validated accuracy
level. To independently test the classification,
marginal habitat sites — those sites from the
Anning River Valley where field ecologists felt
snails could exist, however, where snails were
not found — were subsequently passed down
the CART tree. 72.4% of these sites were cor-
rectly classified as non-habitat. Anode of par-
ticular interest is terminal node T1, where a
large number of non-habitat sites fall. This
node represents low magnesium, sulfate,
phosphorus, and silt conditions.

For the Poyang Lake environment, a CART
classification of snail habitat versus non-habi-
tat revealed that a single soil property, zinc
was able to correctly classify 63% (cross-vali-
dated accuracy) of the sites (Fig. 7). The ma-
jority of the snail sites (39 of 50) fall into ter-
minal node T2, where zinc levels are between
2.7 and 6.2 ppm. Recall from the univariate
analyses that zinc was also identified through

SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 267

Sand ARV 3 63
PL | | a em Bene ee ee
Silt ARV 24.7 72
ПИ м
Clay ARV 4 67
PL и а
рН ARV 6 8.5
PL 41M :
Nitrate ARV 5 76
PL ОЕ ek A o оо
P ARV 0.2 693
PL 0: e
K ARV 128.8 647.4

Amm. Nit. ARV 1174

Cl ARV 20 200

411

Cu ARV 0 8
PL RSR CARR SERA ne =

Fe ARV 0 786
PL 0. 4B 28.2

Mg ARV 185.8 9907.2
РЕ 14.4— 248

Мп ARV 0 1002
PL MA A

Nitrite ARV 0.1 3.7
PL A A RS eo) |

Sulfate ARV 7.2 245.5
ее >> +

Ca ARV 2270.4 14035.2
PL BD 288

FIG. 5. Illustration of the approximate ranges of soil properties associated with snail habitat in the Anning
River Valley (ARV) and Poyang Lake (PL) environments.

Kolmogorov-Smirnov statistics to have a large
statistically significant distance between snail
habitat and non-habitat distributions.
Aseparate CART classification (Fig. 8) was
run for the Poyang Lake sites using the same
six soil parameters as those found to be im-
portant in discriminating between snail and
non-snail sites in the Anning River Valley. The
result of this analysis was a considerably
more complex eight-terminal node tree. How-
ever, the accuracy for this complex tree was

only slightly better (65.6%) than that of the
simple three-terminal node zinc tree (63%).
The interactions between the soil parameters
in Poyang Lake seem to be different from
those seen in Sichuan. However, the one fac-
tor that remains important for snails in both re-
gions is the sufficient availability of silt.

Soil moisture measurements were col-
lected for the Anning River Valley. Kol-
mogorov-Smirnov statistics indicate that the
difference in the distribution of soil moisture

268 SETOIETAL:

TABLE 3. Kolmogorov-Smirnov distance between
snail habitat in the Anning River Valley and snail
habitat in Poyang Lake for each soil property. All soil
properties with the exception of chloride are statis-
tically significant to P < 0.05, which suggests that
the Anning River Valley and Poyang Lake environ-
ments are very different.

D P value
Sand (%) 1.0000 0.000
Silt (%) 1.0000 0.000
Clay (%) 1.0000 0.000
pH 1.0000 0.000
Nitrate Nitrogen (Ibs/acre) 0.4327 0.001
Phosphorus (Ibs/acre) 0.5306 0.000
Potassium (Ibs/acre) 0.3333 0.016
Zinc (ppm) 0.4163 0.001
Ammonia Nitrogen (lbs/acre) 0.5102 0.000
Chloride (lbs/acre) 0.2286 0.187
Copper (ppm) 0.4748 0.000
Ferric Iron (ppm) 0.6857 0.000
Magnesium (Ibs/acre) 1.0000 0.000
Manganese (ppm) 0.7959 0.000
Nitrite Nitrogen (lbs/acre) 0.9714 0.000
Sulfate (ppm) 0.5878 0.000
Calcium (lbs/acre) 1.0000 0.000

between snail habitat and non-habitat sites is
not significant (Table 2). However, range of
soil moisture for snail habitat sites is much
narrower than for non-habitat sites, with snails
favoring high moisture environments (Table
1). Corresponding data were not available for
Poyang Lake.

Both univariate and multivariate ap-
proaches were used to analyze water sam-
ples from the Anning River Valley. Univariate
statistics for the water samples are presented
in Table 1. There is considerable overlap in
the ranges of measured water characteristics.
However, Kolmogorov-Smirnov statistics indi-
cate that the distribution of water turbidity for
snail habitat sites is significantly different (P <
0.05) from non-habitat sites (Table 4). Multi-
variate CART analysis produced a decision
tree based on water turbidity and temperature
values. However, there was significant mis-
classification with this tree, suggesting that
the variance in water conditions does not sig-
nificantly affect the existence of snails. Corre-
sponding water data for Poyang Lake were
not collected.

DISCUSSION

Based on the Kolmogorov-Smirnov statis-
tics of Table 3, the Anning River Valley is very

different from Poyang Lake, which is not sur-
prising given the differences in general ecolo-
gies for these two regions. Perhaps the most
important differences between the two envi-
ronments are pH and calcium levels, for which
no overlap exists in the ranges for these soil
factors. Despite these differences, we cannot
conclude that O. hupensis robertsoni would
only be able to survive in the mountains of
Sichuan, and O. hupensis hupensis in Poyang
Lake. It can be argued that even though high
pH and high calcium soils do not exist in
Poyang Lake, O. hupensis hupensis may still
find such environments suitable, and might be
able to survive or adapt to the Anning River
Valley environment.

The CART classification of snail habitat ver-
sus non-habitat relies on six soil properties,
suggesting that the lack of critical levels of
magnesium, sulfate, phosphorus, and silt may
prevent the establishment of snails. However,
one must be careful in interpreting such re-
sults, as it is difficult to say with certainty that
there is biological significance to these partic-
ular soil properties. This is especially true in
light of the number of different variables con-
sidered here, many of which are probably cor-
related. One should also be cautioned that as
a non-parametric discriminant analysis,
CART can only create decision rules based
upon data given. As a consequence, variables
that do not show up in the tree may not nec-
essarily be unimportant to snail survival. Such
variables may indeed be vitally important at
extremely high or low levels. However, CART
does not take these extremes into considera-
tion, as such data do not exist within our
datasets.

Nevertheless, the Anning River Valley clas-
sification tree is remarkably accurate, particu-
larly in light of its performance in classifying
marginal habitat sites. Marginal habitat sites
should be considered as very difficult sites to
classify correctly since field ecologists essen-
tially misclassified them as snail habitat. The
ability of CART to correctly classify 72.4% of
these sites as truly non-habitat suggests that
soil conditions are in fact very important, and
that there is merit to the notion that the lack of
particular soil conditions can make what
would look like great snail habitat, marginal at
best. Such a result is relevant to the changing
ecologies associated with the Three Gorges
Dam, where areas suspected of being snail
habitat may be eliminated as habitat for the O.
hupensis robertsoni subspecies based on the
lack of critical soil conditions. Moreover, exist-

SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 269

Percentage

of snail sites

Number of sites
within node

Mg <= 1671.8

À

PEA

Mg > 1671.8

Е
Е

$ <= 21 SEAS
Number of sites
within terminal node
re) Lea
en P> 168.9 Р <= 54.15 Pe S455
Km г KE
100 x
14
SILT / Ne >62.5 4 7 05 P= 92205

ey SS

ma ва
eo 2 ва

Mg <= => | Mg > 681.1
ni 13
ni 0

6

FIG. 6. CART classification of snail habitat versus non-habitat for the Anning River Valley. Units for silt is per-
cent; magnesium and phosphorus are Ibs/acre; and sulfate is ppm.

ing regional soil maps may be studied in the
future for potential correlation with important
soil factors and snail occurrence, which may
explain historical patterns of disease-endemic
areas.

The comparisons of the variation in soil
properties between the Anning River Valley
and Poyang Lake environments suggest that
the Anning River Valley is amore complex en-
vironment than Poyang Lake, both in general,
as well as with respect to snail habitat. Kol-
mogorov-Smirnov statistics suggest that from

a univariate standpoint, it is easier to discrim-
inate between snail habitat and non-habitat in
the Poyang Lake environment. This is also re-
flected in the multivariate analyses, in which a
more complicated multivariate eight-terminal
node tree was needed to discriminate be-
tween snail habitat and non-habitat in the An-
ning River Valley, whereas a more compli-
cated multivariate eight-terminal node tree
performed just slightly better than the univari-
ate three-terminal node classification tree.
One might expect Sichuan mountain environ-

270

Number of sites
within node

Terminal Node ID

Percentage of snail sites

Number of sites
within terminal node

Zinc <= 2.7

SETO ET AL.

EX

Zinc => 23)

FIG. 7. CART classification of snail habitat versus non-habitat for Poyang Lake. Zinc split criteria are shown

in units of ppm.

TABLE 4. Kolmogorov-Smirnov distance between
snail habitat and non habitat for each water prop-
erty in the Anning River Valley. Only the distance for
turbidity is statistically significant to P < 0.05, which
suggests that turbidity may play a role in discrimi-
nating between snail habitat and non-habitat sites
in the Anning River Valley.

D P value
pH 0.1382 0.826
Dissolved Oxygen 0.0794 1.000
Nitrate 0.1563 0.717
Phosphorus 0.2401 0.221
Temperature 0.2451 0.203
Turbidity 0.3240 0.039

ments to be more complex because of highly
variable geology and earth processes associ-
ated with mountainous terrain. The Poyang
Lake environment is clearly composed of de-
positional sediments from flooding, and thus
might be expected to be more uniform, given
that these sediments have been accumulating
from the same sources and mixed over sev-
eral centuries.

The Anning River Valley is a much more
stable environment for snails than Poyang
Lake, due to the relative lack of seasonal
flooding. This stability may lead to the estab-
lishment of different niche habitats for snails in
the valley. Recent trips to the field reinforce
this hypothesis. Different snail densities were
found at different elevations, where different

land-use and related farming practices soil
conditions existed at different elevations. In
the lowlands, more paddy fields and long lin-
ear irrigation ditches were found, whereas in
the terraced highlands more complex field
and ditch structure and considerably higher
snail density were found. These land-use dif-
ferences within the valley may correspond to
the complexity seen in the snail habitat for this
area.

As a large flood basin, we hypothesize that
there is less spatial variation in soil conditions
for Poyang Lake than for the Anning River Val-
ley due in part to the deposition, accumula-
tion, mixing, and dispersal of soil and nutri-
ents that occurs with flooding over time. It is in
fact the flooding itself that is most likely the
largest determinant of snail habitat versus
non-habitat. Areas that are either too wet or
too dry for too much of the year will exclude
snails. Moreover, strong evidence is provided
by Davis et al. (in press) that the severity of
annual floods can affect whether snails will be
found in an area one year but not another. Soil
conditions only play a small role in discrimina-
tion of snail habitat versus non-habitat for this
area. This is supported by the fact that both
the simple univariate CART tree based on
zinc levels, as well as the complex multivari-
ate CART tree were only able to achieve
65.6% cross-validated accuracy, in compari-
son to the 85.5% accuracy of the tree for the
Anning River Valley.

There are implications of these findings for

SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 271

Percentage

of snail sites

Number of sites
within node

ESE

Number of sites
within terminal node

Mg<=62.4

K <= 85.6

SS

ae

96
K > 85.6

PEN oe pie ee

Serial

Mg>62.4 S<=21.7

S 217

Bee aes

Silt <= 6.8 ne

FIG. 8. CART classification of snail habitat versus non-habitat for Poyang Lake using same six parameters
used in the Anning River Valley classification. Units for silt is percent; magnesium, potassium, and phos-

phorus are Ibs/acre; and sulfate is ppm.

snail control and future research. For O. hu-
pensis robertsoni in the mountains, the strong
relationships between land-use, soil condi-
tions, and snail habitat suggest that the sur-
veillance of disease transmission may be very
much related to the monitoring of changing
land-use and farming practice. If the level or
risk associated with particular types of land-
use is better understood, then land-use
change, such as those associated with envi-
ronmental modification and different agricul-
tural practices, may be effective control

strategies for O. hupensis robertsoni. Remote
sensing studies associated with this sub-
species and environment should focus on
characterizing differences in land-use and
crop types, identifying which land cover types
are most associated with high snail density,
and determining the spatial relationship be-
tween these particular land cover types and
sites of high human exposure. For O. hupen-
sis hupensis in Poyang Lake, the lack of a
strong relationship between soil conditions
and snail habitat suggest that relatively simple

272 SETO ET AL.

remote sensing studies focused on identifying
grasslands and their relationship to variances
in water levels and to human exposure sites
would be most appropriate. Studies of
Poyang Lake must take into account chang-
ing water levels associated with the seasonal
flooding in order to understand the geo-
graphic distribution of snail habitats over time.

ACKNOWLEDGEMENTS

This study was supported by NASA-Ames
Joint Research Interchange (NCC2-5102).
NIEHS Mutagenesis Center at the University
of California, Berkeley (5 P30 ES 01896-20
ZESI), NIH-NIAID (1 ROI-Al43961-01A1),
NSF of China (49825511), the University of
California Pacific Rim Research Program,
and М.1.Н. grant 1 P50 Al3946, the Tropical
Medical Research Center, Shanghai, China (1
P50 AI3946). We acknowledge Ms Zhang
Jing of the Jiangxi Institute of Parasitic Dis-
eases for doing the soil chemistry, and the su-
port of that institute for the intensive GIS field
work. We thank the Xichang County Anti-
Schistosomiasis Station for their field support.

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Revised ms. accepted 1 December 2001

MALACOLOGIA, 2002, 44(2): 273-288

ECOLOGY OF POTENTIAL HOSTS OF SCHISTOSOMIASIS IN
URBAN ENVIRONMENTS OF CHACO, ARGENTINA

A. Rumi', J. A. Bechara’, M. I. Hamann? & M. Ostrowski de Nufez*

ABSTRACT

Some of the Biomphalaria species living in Chaco, such as B. straminea and B. tenagophila,
are natural transmitters of schistosomiasis in Brazil, while those of the genus Drepanotrema are
not intermediate hosts ofthe disease. The aim ofthe present work was to analyze the importance
of a selected set of environmental variables in explaining patterns of distributions and relative
abundance of planorbid gastropod assemblages. The study sites were located in urban areas of
Resistencia City, Chaco Province, and the environmental variables measured were substratum
(macrophytes), water quality (pH, O,, nutrients, among others), as well as other gastropods (An-
cylidae, Hydrobiidae and Ampullaridae). Seasonal samplings were carried out in four distinct en-
vironments. Thirty-one quantitative samples of gastropods and environmental variables were ob-
tained. In canonical correspondence analysis (CCA), seven environmental variables were
retained after a stepwise forward selection, from a total of 26, including [N-NH,*], O,%, and the
macrophytes Eichhornia crassipes, Pistia stratiotes, Panicum elephantipes, Hydrocotyle ranun-
culoides and Canna glauca. They explained 62% of the variation in planorbid association. Canna
glauca was the most significant variable, being positively correlated with all of the species of
Drepanotrema. Axis | separates B. tenagophila from B. straminea, along a gradient related to in-
creasing O,% and P. elephantipes abundance, as well as decreasing [N-NH,"] and P. stratiotes.
Axis Il separates D. lucidum, D. anatinum and D. cimex from the other planorbid species along
a gradient associated with decreasing abundances of H. ranunculoides and C. glauca. Some
common aquatic macrophytes, and to a lesser extent, dissolved oxygen and ammonium in water,
may be useful indicators of favorable environmental conditions for potential intermediate hosts
of schistosomiasis in Chaco Region.

Key words: Gastropoda, Planorbidae, vector-ecology, schistosomiasis, intermediate-hosts,

Chaco Region, Parana River.

INTRODUCTION

The southern expansion of mansonic schis-
tosomiasis in the Neotropical region (Pa-
raense & Corréa, 1987) necessitates devel-
oping preventive strategies in those areas
with major risk of disease penetration. In Ar-
gentina, these zones are related to the
Guyano-Brazilian subregion, mainly occupied
by the Del Plata Basin, that includes geo-
graphic areas such as Chaco, Mesopotamia
and Pampas (Bonetto, 1994). The most prob-
able colonization path is along the sub-basin
of the Parana River, since the most recent in-
festation foci of Schistosoma mansoni Sam-
bon, 1907 (Trematoda: Digenea), discovered
in southern Brazil, were found in localities

within that sub-basin in proximity to Argentina.
One of these foci was located at San Fran-
cisco do Sul, Santa Catarina State, near the
headwaters of the Iguazu River (Bernardini &
Machado, 1981), with Biomphalaria
tenagophila (d’Orbigny, 1835) as intermediate
host. The other was located in the Piquiri
River, a tributary of the Parana River (Pa-
raense, 1986), with B. glabrata (Say, 1818)
the intermediate host. Up to now, the latter
host has not been detected in Argentina. Bio-
mphalaria tenagophila was described with
type locality in “Canton de las Ensenadas”,
Corrientes Province, Argentina (d’Orbigny,
1835), and Biomphalaria straminea from Ca-
racas, Venezuela (Dunker, 1848), was re-
corded here about 30 years ago. These two

"CONICET, Facultad de Ciencias Naturales y Museo, División Zoologia Invertebrados, Universidad Nacional de La Plata,
Paseo del Bosque s/n, 1900, La Plata, Buenos Aires, Argentina; alerumi@museo.fenym.unlp.edu.ar
2CONICET, Instituto de Ictiología del Nordeste, Facultad de Ciencias Veterinarias, Universidad Nacional del Nordeste. S.

Cabral 2139, 3400, Corrientes Argentina.

“CONICET, Centro de Ecología Aplicada del Litoral, Ruta 5, km 2,5, Laguna Brava, 3400 Corrientes, Argentina.
“CONICET, Facultad de Ciencias Exactas y Naturales, Departamento Biología, Universidad Nacional de Buenos Aires, Pa-
bellón 2, Ciudad Universitaria, 1428, Capital Federal, Buenos Aires, Argentina.

274 RUMI ET AL.

latter species are very common in freshwater
habitats of northeastern Argentina.

A detailed knowledge of the biology of pos-
sible hosts of schistosomiasis, as well as the
host-parasite relationships established in
both natural and urban environments, is nec-
essary for any control strategy (Rumi et al.
1997). Several research studies have been
conducted in the Parana and Uruguay river
basins, including species identification, ecol-
ogy, demography, population dynamics, and
spatial distribution of potential intermediate
hosts of S. mansoni (Bonetto et al., 1982,
1990; Olazarri, 1978, 1984; Rumi, 1991,
1993; Rumi & Hamann, 1990, 1992; Rumi &
Tassara, 1985; Tassara & Bechara, 1983).
These potential hosts are always species of
the genus Biomphalaria Preston, 1910 (Mol-
lusca: Gastropoda: Planorbidae), though the
studies also treated non-transmitter planor-
bids of the genus Drepanotrema Preston,
1910. Other studies consider the host-para-
site relationships with autochthonous trema-
todes in natural and urban environments (Os-
trowski de Nunez et al., 1989, 1991; Rumi &
Hamann, 1990, 1992).

Urban environments, like those analyzed in
the present paper, are particularly interesting
because of the high probability of schistoso-
miasis transmission to humans, in case this
parasitism was found in Argentina. In the
humid portion of the Chaco Plains, the Paranä
River and its tributaries frequently generate
an extensive plain of meanders with hundreds
of lakes. Many human settlements are well
established at those places, including small
towns and medium-sized cities. Therefore,
the identification of environmental variables
that would allow a rapid evaluation of the po-
tential of a habitat to support planorbids might
be a useful strategy to prevent proliferation of
the disease.

As a general rule, the environmental alter-
ations produced by human activities modify
the adaptive capacities of host species, as
well as their abilities to transmit the disease.
Consequently, it is necessary to study these
hosts in natural and urban environments in
order to contrast their dynamics in different
conditions. According to ter Braak & Prentice
(1988), most species occupy a limited area of
the available habitat, tending to be more
abundant in their optimal conditions. Conse-
quently, assemblage composition shifts along
gradients of environmental variables, and
species replacements tend to follow the pat-
tern of spatial and temporal variation of those

variables. Gradients are not necessarily phys-
ically continuous in space or time, but it is a
useful abstraction to explain organism distri-
butions (Austin, 1985). The concept of spatial
partition of the life zone also implies the sep-
aration of species along “resource gradients”
(Tilman, 1982), “regulator gradients” or “com-
plex gradients” (Huston, 1994). This scheme
resulted in two classical models: the environ-
mental control, in which environmental vari-
ables regulate the presence and abundance
of organisms (Whittaker, 1956), and biotic
control, in which the relationships among or-
ganisms, such as competition and predation
(Connell, 1983), are considered as the main
factors that structure communities. In an al-
ternative model, Quinn & Dunham (1983) pro-
posed that they are not mutually exclusive,
but both contribute.

This paper aims to identify and analyze, in
different planorbid assemblages found in
urban environments in the city of Resistencia,
Chaco Province, Argentina. The most impor-
tant biotic and abiotic variables that explain
their distribution and patterns of relative abun-
dance.

MATERIAL AND METHODS
Study Area

The sampling area was located between
Resistencia and Barranqueras (59°15'S,
27°44'W), Department of San Fernando,
Chaco Province (Fig. 1). According to Drago
(1990), the environments involved in the pres-
ent study are within in the Parana River Basin,
which encompasses the second most impor-
tant watercourse in South America after the
Amazon. These environments are on the right
margin of the alluvial plain of the Middle
Parana River. A large plain of meanders, with
very low slope (10 to 12 cm km”?), runs across
this sector of the plain. Soils are brown silty-
clay, with abundant calcareous material in
some places, and elevated alkalinity. An im-
portant feature of this alluvial system is the
great density of subcircular, shallow water-
bodies (< 5 m depth), organized in a drainage
arrangement that shows different degrees of
connection between lotic and lentic waterbod-
ies. Commonly, they are oxbow lakes and
other meander-generated lakes and wet-
lands, locally named “madrejones”. Most of
them are rich in floating, submerged or settled

POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 275

о
=
2
5
<

FIG. 1. Geographic location of the study area and sampled biotopes. Scale bar = 1km.

aquatic vegetation, surrounded by hydrophilic
gallery forests. In the plain of meanders, lakes
are found in different states of succession, be-
ginning with the active meandering river chan-
nel, followed by recently generated lakes,
ending in the almost complete coverage of the
pond basin with palustrine vegetation and
sediments. Usually these ponds are shallow
(up to 2 m deep).

The climate of eastern Chaco is subtropi-
cal, subhumid mesothermal, with a mean an-
nual temperature of 23°C, and monthly aver-
ages between 17°C in July and 28°C in
January. The mean annual rainfall ranges be-
tween 950 and 1,100 mm, distributed almost
uniformly around the year, with peaks in
spring (September-October) and fall (March-
April).

276 RUMI ET AL.

Sampling Scheme

Selection of variables, identified a priori as
presumably important for colonization and de-
velopment of planorbid populations, was
based on three criteria:

(1) Relationships with the substratum —
mainly composed of aquatic vegetation,
and described at the scale of their rela-
tive abundances. The pulmonate snail
uses macrophytes as sites for reproduc-
tion, feeding, and shelter. Consequently,
a decrease in abundance of macrophytes
generally results in a decrease of gastro-
pod populations (Thomas & Daldorph,
1994).

(2) Relationships with water quality vari-
ables — including indicators of the trophic
state of the selected habitats (nutrient
concentrations), as well as other vari-
ables related to the snail abundance
along regulator gradients (pH, oxygen,
and calcium concentrations).

(3) Relationships with other members of the
mollusk assemblages — also referred to a
scale of relative abundance, represent-
ing potential competitors or making up a
part of multispecific associations.

To include the four climatic seasons, sam-
plings were carried out quarterly, between
February 2, 1988, and October 23, 1989. This
area was first widely surveyed to inventory
habitats. Four sampling sites were selected
based on their planorbid richness: Negro
River, at the Municipal Beach of Resistencia
City; Paikin, a oxbow-lake related to a suburb
of the same name, used as garbage deposit
by people living in a group of huts without a
sewer system; Rural Society, a pond within
the “Rural Society” of Chaco Province, be-
longing to the Araza River corridor, which was
partially channeled underground; and Golf
Club, a pond near the Resistencia “Golf Club”,
being probably an old oxbow-lake of the
Negro River. The Negro and the Araza rivers
cross the city of Resistencia NE-SW toward
the city of Barranqueras, draining into the
Parana River (Fig. 1). The present human
population inhabiting both cities is around
300,000 people.

Gastropod sampling was carried out with a
mean collection time of 41 min (standard devi-
ation, 18 min) within a range of about 6.5 h
(from 9:30 to 16:00); snail abundance was de-
termined as capture per unit effort (CPUE) in
terms of specimens hour '. Collections were

made with simple meshed, standard samplers
(Thiengo, 1995), locally named “copos” (1 mm
mesh size, and 13.5 cm frame diameter).
Thirty-one samples were obtained from the lit-
toral zone of the habitats (0.5-1.0 m deep).

Collected material was carried alive to the
laboratory and prepared for identification, dis-
section of soft parts, counting and measuring.
The planorbid specimens were relaxed in a
Nembutal solution 0.01% for approximately
24 h, according to the method described by
Paraense (1986). Afterwards, they were sac-
rificed by immersion in water at 65-70°C for
45-55 sec, and submerged in fresh water
equilibrated with air temperature. Finally, the
prepared material was fixed in a Raillet-Henry
solution (Paraense, 1976), to facilitate its
identification by dissection of the soft parts
under a stereoscopic microscope.

At each sampling site, physical and chemi-
cal variables were monitored immediately be-
fore the capture of gastropods. Temperature,
pH and conductivity were measured using
thermistors, electrode pH-meters, and battery
conductivity-meters, respectively. In addition,
water samples were taken with a peristaltic
pump and transported refrigerated to the lab-
oratory, where the following variables were
measured: dissolved oxygen concentration
(Winkler method), oxygen percentage satura-
tion, nutrients (nitrites + nitrates, total soluble
nitrogen, ammonium and total soluble reac-
tive phosphorus), calcium, hardness, and al-
kalinity. Chemical analyses where performed
according to Standard Methods (1980) and
Golterman et al. (1978).

Regarding vegetation, the criterion of rela-
tive dominance was employed, based on vi-
sual inspection of the sampling area. Plants
were coded between 0 and 4 for the data ma-
trix, according to the following scale: 0 = ab-
sent (0%); 1 = rare (1-25%); 2 = frequent
(25%-50%); 3 = co-dominance (50-99%);
and, 4 = complete dominance (100%). The
surveyed macrophytes belonged to free-float-
ing covers: Eichhornia crassipes (Mart.)
Solms. (Pontederiaceae), Pistia stratiotes L.
(Araceae), Spirodela intermedia W. Koch.
(Lemnaceae), Salvinia herzogii de la Sota,
and S. rotundifolia Willd. (Salvinaceae); set-
tled with floating leaves: Victoria cruziana
d’Orbigny (Ninfaceae); settled: Enhydra ana-
gallis Gardn. (Compositae), Hydrocotyle ra-
nunculoides L. (Umbeliferae), and Ludwigia
peploides (H.B.K.) Hara (Onagraceae); emer-
gent: Panicum elephantipes Nees (Grami-
neae), Canna glauca L. (Cannaceae); and

POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 277

unidentified cespitose hydrophilic Gramineae
(Table 3).

Statistical Analyses

Data on gastropod abundance and limno-
logical variables were transformed to decimal
logarithm (log(x+1)) to linearize relationships
among the variables. The multivariate tech-
nique chosen to explore the relationships be-
tween environmental variables (independent
variables) and snails taxocenosis (dependent
variables) was canonical correspondence
analysis (CCA) (ter Braak, 1986; ter Braak &
Prentice, 1988). The program CANOCO 4 (ter
Braak & Smilauer, 1998) was employed to
perform the analyses, including adjustment of
model parameters, goodness of fit tests, and
plotting the results. This canonical ordination
technique is a combination of ordination in re-
duced space and multiple regression. It im-
plies the representation of samples and
species constrained by environmental vari-
ables in the reduced space of orthogonal
axes, which are in turn linear combinations of
species relative abundance and environmen-
tal gradients. This method assume both uni-
modal (Gaussian) or linear relationships be-
tween species and environmental variables,
for long or short environmental gradients, re-
spectively. It shows the major trends of varia-
tion of multidimensional data within a reduced
space of linearly independent canonical axes,
and it is a useful tool to understand commu-
nity composition in terms of species distribu-
tion along different environmental gradients.
Therefore, given the unimodal proprieties of
CCA, linear relationships between a given en-
vironmental variable and species are not re-
quired, such as in most canonical multivariate
techniques.

The unimodal properties of the model allow
the estimation of optimum and tolerances of
the species, both indicators of niche attrib-
utes. Optimum can be approximated by the
CANOCO 4 output named “spaces-by-envi-
ronmental table”, which are weighted aver-
ages of species with respect to standardized
environmental variables. Results are repre-
sented graphically in a reduced space of or-
thogonal axes, displaying the distribution of
species, samples, and/or environmental gra-
dients in the same bidimentional space (bi-
plots and triplots). Since triplots with species
scorers and environmental scorers may be in
error because they contain the main patterns
only, this table is useful to verify that infer-

ences drawn from the plots hold true in the ac-
tual data on weighted averages (ter Braak &
Smilauer, 1998).

Among the 26 environmental variables sur-
veyed, it was necessary to preselect those of
higher contribution to the total variability in
snail relative abundance in order to reduce
the possibility of an artificial increase in the
explained variance by environmental factors,
as well as multicolinearity. Stepwise forward
selection procedure was employed, using
Monte Carlo permutation tests, as imple-
mented in CANOCO 4. A critical rejection
level of P < 0.10 was applied. The amount of
variance explained by the whole model and
the first ordination axes was calculated em-
ploying Monte Carlo permutation tests, with a
critical rejection level of P < 0.05.

RESULTS

Planorbid Species

The following planorbid species were found
(Table 1): Biomphalaria tenagophila (d’Or-
bigny, 1835), B. straminea (Dunker, 1848),
Drepanotrema anatinum (d’Orbigny, 1835), D.
lucidum (Pfeiffer, 1839), D. kermatoides (d’Or-
bigny, 1835), and D. cimex (Moricand, 1937).
In three samples, no planorbids were found;
these were omitted from the subsequent sta-
tistical analysis.

Biomphalaria straminea occurred more fre-
quently than B. tenagophila (48% and 35%,
respectively), but the latter showed much
higher abundances, especially in 1989. Distri-
butional patterns in space were also different,
with B. straminea being more common in
Negro River and Golf Club Pond, whereas B.
tenagophila was more abundant in Rural So-
ciety and Paikin ponds. Both species are in-
termediate hosts of schistosomiasis in Brazil.

Species of Drepanotrema were much less
frequent than Biomphalaria (10-16%), but
abundances were highly variable in time and
space, with values similar in order of magni-
tude to those of B. straminea, and never
reaching the high levels of B. tenagophila.
Drepanotrema cimex and D. anatinum were
found almost exclusively in Paikin Pond, a
biotope where the other species of Drepa-
notrema also showed the highest frequency.
In the remaining environments, this genus
was rarely found, with D. anatinun in Rural
Society Pond (one presence), D. lucidum in
Negro River (two presences), and D. kerma-
toides in Golf Club Pond (two presences).

RUMI ET AL.

278

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POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 279

Environmental Variables

For all sampling periods and sites, physical
and chemical variables are shown in Table 2.
Water temperature varied between 8°C in
June 1988 and 32°C in February 1988. Aver-
ages values were similar among sampling
sites, being only 1-2°C higher in the Rural
Society Pond and Golf Club Pond, respec-
tively. These differences may be partly be-
cause sampling hours were not the same for
all biotopes. The pH ranged from 6.3 to 8.9
(October 1989 and February 1988, respec-
tively), being alkaline in most sampling dates
in Rural Society Pond, and closer to neutral
point in the others waterbodies. Specific con-
ductivity showed marked spatial and temporal
variations within a range of 90 and 1,600 uS
cm | (August 1988 and November 1988, re-
spectively). The highest conductivities were
measured in Golf Club Pond and the lowest in
Negro River. Oxygen concentration [O,] and
oxygen saturation percentage (%O,) were
low or undetectable during the warmest peri-
ods, increasing during winter and spring, with
figures as high as 8.1 mg I" and 90% satura-
tion.

Calcium concentration [Ca*] was also
highly variable (Table 2), ranging from 9.5 to
119.4 mg Г' in Paikin Pond (July 1989 and
February 1989, respectively). According to
Dussart’s (1976) classification of mollusk
habitat regarding calcium concentration, the
highest mollusk abundance generally corre-
sponds to the hardest waters ([Ca*] > 40 mg
Г'), while the highest diversity is usually ob-
served in mid-range waters ([Ca*] between
5-40 mg Г’). Sampled environments of
Chaco generally had waters of medium hard-
ness, although hard waters were observed on
some occasions (Table 2). Calcium concen-
tration was positively correlated with hard-
ness (r = 0.677, n = 31, P < 0.01), a variable
that ranged between 36 and 329 mg Г" (June
1989 and February 1989, respectively). The
range of alkalinity due to [HCO °] was 45-357
mg Г' (October 1989 and August 1988, re-
spectively). Rural Society Pond generally
showed the highest levels of alkalinity, while
Negro River had the lowest.

Concerning nutrient concentrations (Table
2), nitrates and nitrites [N-NO, + N-NO,], were
usually below 0.4 mg Г", reaching exception-
ally 9.6 mg Г' on November 1988 in Paikin
Pond. Ammonium concentration [N-NH,?]
showed maximum levels in Paikin Pond dur-

ing 1989, and minimum levels in Rural Soci-
ety Pond most of the time. This nutrient also
tended to increase in all waterbodies during
the second sampling year. Total soluble nitro-
gen (N-SOL) followed the same pattern of
ammonium. Finally, phosphate concentration
[P-PO,] was generally below 1.0 mg Г",
reaching exceptionally high levels in one sam-
pling date in Paikin Pond and Rural Society
Pond. In Negro River, phosphates had the
lowest concentrations of all waterbodies,
never being over 0.3 mg Г".

Relative abundances of aquatic plants
are shown in Table 3. Among the most impor-
tant emergent macrophytes, Panicum ele-
phantipes was frequent and dominant in Golf
Club Pond and Negro River, but absent in
the other waterbodies. On the other hand,
Canna glauca was always dominant in Paikin
Pond, and completely absent from the other
biotopes. Among other settled macrophytes,
Enhydra anagallis was frequent and important
in Paikin and Rural Society ponds, while Hy-
drocotile ranunculoides was present only on
some dates in Rural Society Pond and Paikin
Pond. Of free-floating plants (Table 3), Pistia
stratiotes was dominant in Rural Society Pond
and during 1989 in Golf Club Pond. Eichhor-
nia crassipes was completely absent from
Negro River, and present in variable abun-
dance and frequency in the remainder of the
waterbodies.

Other gastropods that were present with
planorbids (Table 3), were Heleobias spp. (Hy-
drobiidae), Stenophysa marmorata (Guilding,
1828) (Physidae), Gundlachia moricandi
(d’Orbigny, 1835) (Ancylidae), and species of
the family Ampullariidae, mainly Pomacea
canaliculata (Lamark, 1801) and P. scalaris
(d’Orbigny, 1835). All the gastropods found
with planorbids were eliminated in the step-
wise forward selection of independent vari-
ables. Pomacea canaliculata was the most
frequent gastropod considering all samples.
This species has also a wide distribution in del
Plata Basin, especially in the Argentinean
area.

Canonical Correspondence Analysis

Seven of the 26 environmental variables
used in the canonical correspondence analy-
sis (CCA, Table 4) were retained in the step-
wise forward selection procedure. For all of the
selected variables, the variance inflation factor
(V.I.F.) was below 5, the level suggested for

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282 RUMI ET AL.

TABLE 4. Summary of major results of the canonical correspondence analysis relating planorbid species to

the selected environmental variables.

Canonical Axes

Eigenvalues 0.469 0.401 0.354
Cumulative percentage of variance of species data 23.4 43.4 61.0
Cumulative percentage variance of species-environment relation 37.6 69.8 98.2
Species-environment correlation 0.840 0.837 0.838
Correlation of the environmental variables with the axes

P. elephantipes 0.543 —0.346 —0.190
С. glauca 0.095 0.684 0.413
H. ranunculoides —0.139 0.461 —0.036
E. crassipes —0.221 —0.006 —0.068
P. stratiotes 0.385 —0.274 —0.587
[N-NH47] 0.408 0.133 0.310
%O, —0.291 0.268 —0.237
Total of unconstrained eigenvalues 2.006

Total of canonical eigenvalues

1,247 (62% of explained variance)

avoiding multicolinearity and over-explanation
(ter Braak and Smilauer, 1998). Ammonium
[N-NH,*] and percentage saturation of D.O.
(%0,) were the water quality variables re-
tained. The remaining environmental vari-
ables were all macrophytes, including P. stra-
tiotes, E. crassipes, H. ranunculoides, P.
elephantipes, and C. glauca. The selected en-
vironmental variables explain 62% of the total
variation in planorbid species relative abun-
dance. Moreover, the first three canonical
axes account for 98% of the variance ex-
plained by these variables. The species-envi-
ronment relationship was highly significant,
according to the Monte Carlo permutational
test (F = 4.46; P < 0.001; 999 permutations),
as well as the first canonical axis (F = 5.80; P
< 0.01; 999 permutations). In three samples
from Negro River and one from Rural Society
Pond, planorbids were not found, being ex-
cluded by the CCA program.

The graphic representation of the species
positions along the environmental gradients in
the reduced space of the first three canonical
axes, yielded the following results (Figs. 2, 3):

(1) Axis | separates B. straminea and D. lu-
cidum from B. tenagophila and D. kerma-
toides, along an increasing gradient of
[N-NH,*] and P. stratiotes, as well as a
decreasing gradient of %O, and P. ele-
phantipes. The remaining species of
Drepanotrema occupy intermediate posi-
tions along the first axis gradient.

(2) Axis Il separates D. lucidum, D. anatinum
and D. cimex from the species of Biom-

phalaria and D. kermatoides, mainly
through a decreasing gradient of H. ra-
nunculoides and C. glauca, as well as an
increasing gradient of P. elephantipes.

(3) Finally, along axis Ш, species of Drepa-
notrema appear more clearly distributed,
with D. kermatoides on the top, D. anat-
inum and D. cimex in the middle, finishing
with D. lucidum at the opposite end, close
to B. straminea. Biomphalaria tenago-
phila is placed on the opposite negative
side of the planorbid distribution along
the third axis. This axis is mainly related
to the independent variables C. glauca
and [N-NH,'] (positively) and Р. stratiotes
(negatively).

In addition, employing the environment-by-
species table provided by the program
CANOCO 4 (Table 5), the position of the opti-
mum of planorbid species along gradients of
environmental variables can be better ana-
lyzed, independently of their correlations with
canonical axes. With this alternative analytical
approach, the following results were obtained:

(1) Canna glauca was the most important in-
dependent variable in the stepwise for-
ward selection. All species of Drepan-
otrema appear on the positive side ofthe
gradient, with D. anatinum and D. cimex
placed atthe edge. Biomphalaria species
were on the negative side, with B.
straminea at the outermost end of the
gradient.

(2) Pistia stratiotes, the second most im-

POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 283
an
> O |
Dre ana
Can gla
Dre cim
Dre luc
x
= |
a
<
z =
= ES ARS E ic cra Е Вю ten Y
x
O
2 N-NH4@ 9
© | 2
Biostr # Dre ker* Pis str @
Pan ele
|
a) a a
SES CANONICAL AXIS | +2.0

FIG. 2. Spatial representation of the samples, species and environmental variables in the space defined by
the two first canonical axes. Circles: Paikín pond. Diamonds: Rural Society Pond. Squares: Negro River. Tri-
angles: Golf Club Pond. Empty symbols: 1988. Filled symbols: 1989. Arrows: environmental variables. Stars:
planorbid species. Bio str: Biomphalaria straminea; Bio ten: B. tenagophila; Dre ana: Drepanotrema ana-
tinum; Dre cim: D. cimex; D. ker: D. kermatoides; Dre luc: D. lucidum; Can gla: Canna glauca; Eic cra: Eich-
hornia crassipes; Hyd ran: Hydrocolyle ranunculoides; Pan ele: Panicum elephantipes and Pis str: Pistia

stratiotes.

portant independent variable, had B.
tenagophila at the positive end of the dis-
tribution, whereas all species of Dre-
panotrema were on the negative side.
Biomphalaria straminea was placed
close to the center of the distribution.

(3) Along the gradient of [N-NH,?], both
species of Biomphalaria were clearly
segregated, with B. tenagophila having
its optimum at higher concentrations, to-
gether with D. kermatoides. Moreover, B.
straminea was placed on the opposite
end, close to D. lucidum, while D. cimex
occupied the center of the distribution.

(4) With respect to %O,, planorbid species
responded inversely as [М-МН, "|.

(5) Dense stands of P. elephantipes sup-
ported the highest abundances of B.
straminea, together with D. lucidum.

(6) Hydrocotile ranunculoides had a ten-
dency to house populations of D. ana-
tinum, D. lucidum and D. cimex prefer-
ably, while Biomphalaria species and D.
kermatoides were at the negative side of
the gradient.

(7) Finally, along the E. crassipes gradient,
B. straminea and B. anatinum were on
the positive side, D. cimex, close to the
center, and the remainder on the nega-
tive side.

With respect to sample distribution in the re-
duced space of the first three axes, four
groups were observed, each one character-
ized by a kind of vegetation and a particular
planorbid assemblage (Figs. 2, 3):

(1) Samples in Paikín in 1988, characterized
mainly by C. glauca and H. ranuncu-

284
vw,
A
+
|
A
le
WY
x Dre ana
< Pan ele
a
<
О ET EEE 3
= | xDre luc
= Eic era
5 9,02
|
S
= |

-1.5

CANONICAL AXIS |

RUMI ET AL.

Dre ker
-

Can gla
@
> N-NH4
Dre cim

Bio ten

$80

Pis str
$
$

720

FIG. 3. Spatial representation of the samples, species and environmental variables in the space defined by

canonical axes | апа Ш. Circles: Paikin oxbow lake.

Diamonds: Rural Society Pond. Squares: Negro River.

Triangles: Golf Club Pond. Empty symbols: 1988. Filled symbols: 1989. Arrows: environmental variables.
Stars: planorbid species. Bio str: Biomphalaria straminea; Bio ten: B. tenagophila; Dre ana: Drepanotrema
anatinum; Dre cim: D. cimex; D. ker: D. kermatoides; Dre luc: D. lucidum; Can gla: Canna glauca; Eic cra:
Eichhornia crassipes; Pan ele: Panicum elephantipes and Pis str: Pistia stratiotes.

loides. The most common species were
D. anatinum, D. lucidum and D. cimex.
The genus Biomphalaria was rare.
Samples from Rural Society Pond and
from Paikin Pond in 1989, characterized
by the presence of Р stratiotes and the
highest concentrations of ammonium,
with B. tenagophila and D. kermatoides
as the most common planorbid species.
Samples from Rural Society Pond in
1988, with low-density stands of E. cras-

sipes, and the presence of B. straminea
and B. tenagophila also in low densities.
Samples from Negro River and Golf Club
Pond, characterized by the presence of
P. elephantipes, and the highest concen-
trations of %O,. Biomphalaria straminea
was the most typical species, while the
other planorbids were negatively related
to this kind of vegetation. Within this
group, two separated clusters of samples
can be observed, one of them more

POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 285

TABLE 5. Environment-by-species table showing weighted means and standard deviations of environmen-
tal factors, weighted averages of planorbid species with respect to the seven standardized variables
(“optima”), and back-transformed data to original format as in Tables 2 and 3 (in parentheses). Note that for
limnological variables, original data were transformed to decimal logarithm.

Stand B. B. D. D. D. D.

Mean dev. tenagophila straminea anatinum lucidum kermatoides cimex

C. glauca 1.460 1.881 —0.288 —0.584 1.163 0.536 0.426 0.839
(0.9) (0.4) (3.6) (2.5) (2.3) (3.0)

H. ranunculoides 0.393 0.930 —0.196 —0.158 0.772 0.622 —0.423 0.3826
(0.2) (0.2) (ED (1.0) (0.0) (0.7)

P. elephantipes 0.884 1.648 —0.434 0.688 —0.536 0.393 0.009 —0.5361
(0.2) (2.0) (0.0) (1.5) (0.9) (0.0)

E. crassipes 1055 11.558 —0.185 0.274 0.203 —0.112 —0.301 —0.0004
(0.8) (1:5) (1.4) (0.9) (0.6) (1.1)

Р stratiotes №325 81.775 0.726 —0.068 —0.646 -0.745 —0.745 —0.2038
(2.6) (1.2) (0.2) (0.0) (0.0) (1.0)

[N-NH4*] 0.238 0.276 0.211 —0.328 —0.136 —-0.709 0.884 0.0696
(0.977) (0.403) (0.586) (0.102) (2.034) (0.807)

%0, 1.534 0.493 0.193 0.158 0.435 0.499 0.789 0.2062
(26.5) (39.9) (55.0) (59.2) (13.0) (42.2)

clumped, belonging to 1988, and the
other more dispersed, from 1989.

These results indicate that there is not a
clearly defined seasonal variation in the struc-
ture of planorbid assemblages. The most im-
portant patterns were spatial or inter-annual,
as is shown in the triplot of axes | and Il (Fig.
2). There is a defined tendency of most sam-
ples from all waterbodies to move in a top-left
to bottom-right direction from 1988 to 1989,
following a gradient of increasing ammonium
concentration and decreasing oxygen satura-
tion.

DISCUSSION

The empirical model developed in this paper
explains a relatively high percentage of ob-
served variance compared with other commu-
nity studies (Borcard et al., 1992), and is in ad-
dition highly significant. As a general pattern,
the multivariate model shows that Biom-
phalaria straminea tends to be relatively more
important in well-oxygenated environments
with reophilic vegetation, such as Panicum
elephantipes. Junk (1973) also found that this
planorbid species was abundant in the roots of
“floating meadows” (Paspalum repens Berg,
and Echinochloa polystachya (H.B.K.)) in
várzea lakes ofthe Amazon River. Conversely,
Biomphalaria tenagophila, the other potential
transmitter of schistosomiasis, is more com-

mon in less oxygenated waters and higher am-
monium concentrations, conditions that, to-
gether with pH over 8 and temperatures higher
than 25°C, may be extremely toxic for aquatic
animals due to the high proportion of ammonia
(МН.) (Boyd, 1990). These particular limno-
logical features are generally associated with
continuous floating covers of Pistia stratiotes,
with roots that would function as substratum
and refuge for B. tenagophila. Drepanotrema
kermatoides follows a similar pattern to that of
B. tenagophila, while the association of B.
straminea with species of Drepanotrema was
unclear, except for D. lucidum. This latter
species was also found by Junk (1973) occu-
pying the same habitat as B. straminea. In this
sense, the dominance of Canna glauca and
Hydrocotyle ranunculoides also indicates the
dominance of Drepanotrema species, as well
as lower densities of Biomphalaria species,
particularly B. straminea.

Among the environmental factors taken into
account in the CCA, the regulatory effect of
water temperature on population dynamics
was already known, as was the importance of
calcium concentration related to pH, hardness
and macrophytes, on the distribution and
abundance of mollusks (Dussart, 1976; Pigott
& Dussart, 1995; Thomas, 1982; Rumi &
Hamann, 1992). However, only ammonium
concentration and percentage of oxygen sat-
uration were retained as significant limnologi-
cal variables in the process of forward selec-

286 RUMI ET AL.

tion, and they were negatively correlated with
each other. Biomphalaria tenagophila and D.
kermatoides were distributed along this lim-
nological gradient, mainly at the highest am-
monium concentrations, with optima at 0.98
and 2.00 mg Г', respectively, as well as the
lowest oxygen saturation, with optima at
26.6% and 13.0%, respectively. These results
suggest that the variance explained by the ex-
cluded variables was either unimportant to be
selected in the stepwise procedure, or that
this variation was better explained by oxygen
and ammonium, or even aquatic plants, which
were retained first by the statistical method
employed, due to their larger explained vari-
ance. Therefore, these results do not neces-
sarily mean that variables not retained were
not relevant for planorbids.

The lack of clear seasonal trends in planor-
bid communities may be explained because
the environments in which this study was Car-
ried out are of permanent waters, unlike oth-
ers of tropical areas, with well-defined sea-
sonality in rainfall. These observations agree
with Rumi & Hamann (1992), who compared,
in another permanent pond of eastern Chaco
in Corrientes Province, rainfall pattern with
the relative abundance of Biomphalaria occi-
dentalis Paraense, 1981, finding also a very
low correlation between those variables.
However, in this region of Chaco, inter-annual
variability in water availability can be very im-
portant, particularly the surplus after evapora-
tion, soil infiltration and scouring (Bruniard,
1997). Since environmental variables as well
as planorbid population dynamics are related
to climatic changes, large variations in planor-
bid composition and abundance in samples of
the same biotopes from one year to the next
may be due to different rainy and drought pe-
riods. For example, different amounts of pre-
cipitation can affect total biotope area, pro-
ducing an increase in planorbid density during
dry periods due to retraction in pond area, or
an opposite decrease during wet periods due
to dilution effects. Furthermore, although
ponds are physically isolated most of the time,
extensive superficial interconnections among
wetlands always take place during rainy
episodes or river floods. Thus, migration
events in snail populations would be expected
at those times. These two processes could
provoke rapid changes in abundance, and
could partially explain the marked seasonal
modifications in snail capture observed in the
present paper.

Until not long ago, the urban ponds were
employed for rubbish deposits, which affected
the water quality and planorbid population
size, especially during dry climatic events. In
addition, populations of B. straminea were
much more abundant during the first year,
while B. tenagophila showed an increase in
population densities during the second year.

The non-explained variability in planorbid
relative abundance could be attributed to a
rather extensive list of possibilities, including
the colonization patterns in space and time,
sampling biases, and environmental variables
not included (i.e., fish and invertebrate preda-
tors). First, given the limited capacity of
planorbids to disperse and the time required
for settled populations to grow, it is possible
that even with suitable environmental condi-
tions, populations could not reach maximum
or stable densities within the sampling period.
Second, the observed variation range of envi-
ronmental variables and of planorbid assem-
blages could be related to the specific envi-
ronments selected for sampling, as well as
the sampling technique employed. Thus, in-
cluding more biotopes and making a greater
sampling effort could have resulted in a larger
explained variance or in a somewhat different
distributional pattern. Finally, the role of
predators as controls of planorbid populations
cannot be ignored, given that eastern Chaco
environments usually hold an abundant and
diverse fish fauna (Menni et. al., 1992), with
large proportions of invertebrate-eating, in-
cluding mollusc-eating, species, such as
small to medium-sized silurids, characins, ci-
chlids and gymnotids. Therefore, given similar
environmental conditions, the presence or ab-
sence of predatory fishes could result in dif-
ferent planorbid densities and species com-
position.

From a sanitary standpoint, it is remarkable
that the species B. tenagophila and B.
straminea, living in urban environments in the
Chaco, are natural transmitters of schistoso-
miasis in Brazil. Because the presence and
abundance of these two planorbid species is
explained chiefly by some species of aquatic
macrophytes, these variables may be em-
ployed as indicators for a rapid assessment of
potential sites of proliferation of B. tenago-
phila and B. straminea. However, these re-
sults must be supplemented with new sam-
plings, as well as experimental field and
laboratory tests, to confirm the observed
trends.

POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 287

ACKNOWLEDGEMENTS

This study was made possible by a grant
supplied by the Consejo Nacional de Investi-
gaciones Cientificas y Tecnicas (CONICET)
(AGENCIA PICTO1-03453), in Argentina.
Fieldwork and water analyses were facilitated
by the logistic support of the Centro de
Ecologia Aplicada del Litoral (CECOAL). Lic.
Cecilia Longoni de Meabe supervised water
quality analyses. We also thank Mr. Luis
Benetti by his valuable collaboration in field-
work.

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MALACOLOGIA, 2002, 44(2): 289-305

EGG MASSES OF CHROMODORID NUDIBRANCHS
(MOLLUSCA: GASTROPODA: OPISTHOBRANCHIA)

Nerida G. Wilson

Centre for Marine Studies, University of Queensland, St Lucia 4072, Queensland, Australia;

nwilson O marine.uq.edu.au

ABSTRACT

The egg mass characteristics of 20 species of chromodorid nudibranchs are presented, rep-
resenting the genera Chromodoris, Digidentis, Diversidoris, Glossodoris, Hypselodoris, Noumea
and Pectenodoris. The egg mass details for 14 of the included species appear previously un-
recorded. These results combined with observations from the literature (comprising a total of 67
species from 15 genera) indicate that most genera in the Chromodorididae show only one type
of egg mass. The exception to this is the genus Chromodoris, which includes all three egg mass
types outlined in this study. Based on anatomical evidence, a group of flat-spawning Chro-
modoris species has been suggested to belong to a monophyletic clade, which indicates that egg
mass structure can reflect phylogenetic signal. Genera considered to be more derived among
the Chromodorididae are more likely to lay an egg mass that is outward sloping or crenulated.
Cadlinella shows spawning traits (flat egg mass containing extra-capsular yolk) that indicate it
may be ancestral to the Chromodoris lineage. Developmental data is presented for 11 species
of Chromodoris, seven of which utilized extra-capsular yolk in their egg masses. The presence
of extra-capsular yolk appears to correlate with upright egg masses within this genus. However,
the occurrence of extra-capsular yolk appears to be restricted to Indo-Pacific and Red Sea Chro-

modoris species, while upright spawns from Caribbean species lack this feature.
Key words: nudibranch, egg mass, chromodorid, reproduction, extra-capsular yolk

INTRODUCTION

All doridinean nudibranch egg masses, in-
cluding those of chromodorids, take the form
of a spiral ribbon that is attached to the sub-
stratum and consists of embryos embedded in
a gelatinous matrix. The shape of the egg
mass has been considered to a certain extent
to characterize particular groupings (Eliot,
1910; Ostergaard, 1960; Hurst, 1967; Bandel,
1976; Fernandez-Ovies, 1981; Soliman,
1987), and some authors have equated the
taxonomic value of the structure of egg cap-
sules (and associated coverings) in shelled
gastropods with shell, radular and opercular
characters (Andrews, 1935). In a recent
cladistic analysis, Mikkelsen (1996) confirmed
that the opisthobranch order Anaspidea had a
particular egg mass type (string) common to
all investigated members of the group.

An extensive literature exists on the
anatomy and systematics of chromodorids.
However, comparatively little is known about
chromodorid reproduction aside from the mor-
phology of the reproductive system itself
(Rudman, 1984). In particular, very little is

289

known about the structure of the chromodorid
egg mass in species occurring in Australian
waters, apart from a few brief descriptions, il-
lustrations or unpublished theses (Kenny,
1970; Thompson, 1972; Rose, 1981, 1985;
Avern, 1986; Marshall & Willan, 1999). Often
descriptions of chromodorid egg masses are
brief and include such ambiguous expres-
sions as “tangled coils” or “loosely coiled”
(MacGinitie & MacGinitie, 1949; Boucher,
1983; Johnson & Boucher, 1983). This gives
no indication as to whether these terms per-
tain to the distance between the coils, the reg-
ularity of coiling, or to the actual attachment of
individual whorls of the spiral mass to the sub-
stratum. Numerous papers from Japan have
significantly improved our understanding of
opisthobranch egg masses (Baba & Hama-
tani, 1961; Hamatani, 1960a, b, 1961, 1962).
However, only one account described chro-
modorid nudibranch egg masses (Baba et al.,
1956). Other studies may inadequately iden-
tify the parent species or give conflicting data,
thus rendering the resulting egg mass infor-
mation less useful (Bandel, 1976; Barash &
Zanziper, 1980; Rose, 1985). However, many

290 WILSON

photographic nudibranch identification guides
have helped highlight substantial diversity in
nudibranch egg mass structure (Gosliner,
1987; Coleman, 1989; Behrens, 1991; Wells
& Bryce, 1993; Debelius, 1998). More re-
cently, websites have also contributed signifi-
cantly to our knowledge of egg masses (for
example, www.seaslugforum.net).

Opisthobranch egg masses were classified
according to a scheme introduced by Hurst
(1967). This scheme aimed to describe egg
masses in a format that would facilitate com-
parison between taxa. As it was based on
opisthobranch taxa from the cold temperate
northwest coast of the USA, chromodorids
were not included, although nearly all egg
mass types laid by chromodorids were repre-
sented in the scheme by other dorids. Hurst’s
scheme consists of four categories, which
show some taxonomic correlations, for exam-
ple, Type C (jelly bag attached by string) com-
mon amongst cephalaspideans and Type D
(sac-like structure) typical of very small aeolid
nudibranchs. Doridinean spawns were only
present in one category that Hurst defined as
Type A:

“The egg mass is in the form of a ribbon at-
tached along the length of one edge, capsules
occurring throughout most of it. This is com-
mon amongst dorids, which whilst laying may
grip the mass between foot and mantle edge
tending to flatten it, as mentioned by Fretter &
Graham (1964). This is probably not the sole
cause of the flattened shape” (Hurst, 1967:
256).

Hurst (1967) noted differences in relative
lengths of the free edge compared to the at-
tached edge of the masses, but did not incor-
porate this variation in her classification. Con-
sequently, any descriptions of doridinean
spawn masses that employ Hurst’s categories
define the ribbon as simply being upright. This
does not provide enough information to make
any comparisons at or below the familial level.
Bandel (1976) briefly discussed Hurst’s
scheme and proposed 12 of his own group-
ings. Many of these groupings were based on
a single opisthobranch species, and all dorid-
inean egg masses remained in one group, pro-
viding no improvement over Hurst's original
scheme. Some subcategories were added to
Hurst's classification by Fernandez-Ovies
(1981), who recognised that the scheme did
not adequately describe variation within each
type’. While these subcategories were a sig-
nificant improvement, they still did not account
for all the variation caused by differences in the

length of the free edge of upright egg masses
and did not account for flat egg masses. In
fact, Fernandez-Ovies incorrectly listed Chro-
modoris orientalis (as Glossodoris pallescens)
laying an upright egg mass, whereas it is actu-
ally laid flat (Baba et al., 1956). MacGinitie &
MacGinitie (1949) report that some dorids lay
flat egg masses, but it is not until much later
that this shape is formally recognised in a clas-
sification (Soliman, 1987).

An unusual feature of egg masses that
often goes unnoted is the existence of yolk
reserves external to the capsule, but still con-
tained within the egg mass. Boucher (1983)
termed this “extra-capsular yolk” in a paper
that reported the phenomenon in the saco-
glossan genera Elysia Risso (Elysiidae) and
Bosellia Trinchese (Polybranchiidae) and the
chromodorid genera Chromodoris Alder &
Hancock and Cadlinella Thiele (Chromodori-
didae). Risbec (1928) erroneously interpreted
extra-capsular yolk inthe egg masses of Cad-
linella ornatissima Risbec as crustacean ova.
A few brief descriptions or illustrations have
highlighted the presence of extra-capsular de-
posits in the egg masses of opisthobranchs
(Gohar & Aboul-Ela, 1957; Marcus & Burch,
1965; Gohar & Soliman, 1967a; Kay & Young,
1969), but Thompson (1972) appears to be
the first author to formally recognize these
bodies as being composed of “yolky material”.
The first and only paper to call attention to the
widespread occurrence of extra-capsular yolk
resulted from a taxonomic survey of opistho-
branchs in the Marshall Islands (Boucher,
1983). Since that study, no other literature has
specifically addressed the subject of extra-
capsular yolk in nudibranch egg masses. The
actual deposition of extra-capsular yolk varies
between major taxa, and may take the form of
granules, caps or blobs in chromodorid nudi-
branchs (Boucher, 1983) or yolk strings in
sacoglossans (Clark et al., 1979; Clark &
Jensen, 1981; Boucher, 1983). Goddard
(1991) renamed the phenomenon “extrazy-
gotic” and “extra-embryonic” instead of “extra-
capsular” in order to include his observation of
extra yolky polar bodies inside aeolid nudi-
branch capsules. It is unlikely that all of these
extra yolk sources are homologous.

Although most nudibranchs spawn readily
in captivity, perhaps in response to capture
and handling stress (Hadfield & Switzer-Dun-
lap, 1984), there are few detailed descriptions
of egg masses. The aims of the present study
are to increase awareness of structural varia-
tion in chromodorid nudibranch egg masses,

CHROMODORID EGG MASSES 291

and to highlight the presence of extra-capsu-
lar yolk in certain taxa. Knowledge of egg
mass types and extra-capsular yolk may pro-
vide new data to help evaluate existing phylo-
genies.

MATERIALS AND METHODS

Egg Mass Collection,
Maintenance and Measurement

A total of 20 species were collected for this
study from the east coast of Australia during
1998-2001. The austral summer is repre-
sented by the months December, January,
February; autumn by March, April, May; win-
ter by June, July, August, and spring by Sep-
tember, October, and November. The majority
of specimens were collected using SCUBA,
although some material was collected in the
intertidal zone. Animals were kept in holding
tanks with gentle aeration and flowing seawa-
ter. There is some evidence to suggest that
egg masses produced after a prolonged pe-
riod of stress can be atypical of the species,
that is, when organ systems begin to deterio-
rate (J. Havenhand, pers. comm.). To mini-
mize the likelihood of this effect, only egg
masses that were produced in the first week
after capture were used in the study. A portion
of each egg mass was excised with a scalpel
and preserved in either glutaraldehyde (3%
prepared in 0.1M sodium phosphate buffer
containing 10% w/v sucrose) or neutral-
buffered formalin (10%). The remainder of
each egg mass was maintained in a holding
tank at a water temperature equivalent to that
of the collection locality. Portions of the egg
mass were observed under a compound mi-
croscope at least every second day, and more
frequently closer to hatching. The develop-
mental period was deemed to cease on the
first day that hatching was observed. Un-
cleaved ova were measured either from black
and white photographs taken using an Olym-
pus BH-2 Nomarski contrast compound mi-
croscope fitted with an Olympus OM-2N cam-
era attachment and Kodak T-max 100 ASA
film, or from heat images generated from a
National F10 CCD Video attachment to the
above microscope, printed on a Sony video
graphic printer UP-811. To increase the sam-
ple size for comparisons at generic levels, de-
scriptions and photographs from the literature
were included. However, any data that con-
tained ambiguous terminology or was difficult
to interpret was excluded.

Definition of Egg Mass Types

The egg masses examined in this study
were classified into three types (Fig. 1) based
on whether or not they are attached flat on
the substratum or whether they are attached
along one edge. Type A egg masses are at-
tached to the substratum by the broad side
of the ribbon, and are therefore flat. Type B
egg masses had a free edge that was either
shorter than the attached edge, causing the
ribbon to slope toward the centre of the
spiral or was equal in length to the attached
edge, standing upright. Type C egg masses
had a free edge that was either slightly
longer than the attached edge, causing the
ribbon to slope away from the centre of
the spiral or much longer than the attached
edge, causing undulations or waves along
the ribbon in addition to an outward slope.
In some egg masses, the free edge was so
long that the ribbon showed tight crenula-
tions.

Extra-Capsular Yolk Categories

Boucher (1983) recognised two categories
of extra-capsular yolk in nudibranchs and
these are applied here with some modifica-
tions. Boucher (1983) defined Type 1 as “cap-
like bodies associated with individual cap-
sules”. In this study, caps falling into this
category were further subdivided into (A)
caps that were distributed equally and (B)
caps that were distributed unequally (Fig. 2).
Type 2, as defined by Boucher (1983), was
“discrete yolk bodies strewn throughout the
egg mass”, and were not observed during this
study, although are known to occur within the
genera Cadlinella and Chromodoris (Risbec,
1928; Gohar & Aboul-Ela, 1957; Boucher,
1983).

Developmental Type Definitions

Where possible, larvae were examined by
light microscope (Olympus BH-2 Nomarski
contrast compound microscope) and catego-
rized into developmental types according to
the definitions proposed by Thompson (1967)
and Bonar (1978). A well-developed velum
and larval retractor muscle, small ova and
short embryonic period identified plank-
totrophic larvae, whereas lecithotrophic lar-
vae typically exhibited a less developed but
recognisable velum and larval retractor mus-
cle, prominent propodium, and large ova with

292 WILSON

FIG. 1. Stylized drawing of the three types of chromodorid egg masses. Type A is laid flat on the substratum,
Type B is laid upright and may also slope inwards, and Type C is laid upright, slopes outwards and may be
crenulated.

FIG. 2. Extra-capsular yolk. A, equally distributed. B, unequally distributed. Scale bar = 100 um.

a longer embryonic period (Thompson, 1967). RESULTS

Intracapsular development was determined

to be either metamorphic (undergoing capslar The developmental characteristics of
metamorphosis) or ametamorphic (not under- twenty chromodorid species are described

going a typical veliger stage) (Bonar, 1978). below and summarized in Table 1.

293

CHROMODORID EGG MASSES

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294 WILSON

Chromodorididae
Chromodoris Alder & Hancock
Chromodoris collingwoodi Rudman, 1987

A single egg mass was observed in March
from an animal collected at North Stradbroke
Island, Queensland. The egg mass was up-
right, consisting of two whorls slightly sloping
inward to centre of spiral. Ova were yellow,
and associated with extracapsular yolk of Type
1A. Each capsule contained a single embryo.
The embryonic period was six days at 23°C.
Larvae were planktonic, although the exact
developmental type was not determined.

Chromodoris daphne (Angas, 1864)

A single egg mass was observed in No-
vember from an animal collected at Welling-
ton Point, Moreton Bay, Queensland. The egg
mass was upright, with the broad side of rib-
bon constricted slightly toward centre of spi-
ral, giving it a slightly “hour-glass” shape. Two
whorls were present, containing cream ova
arranged linearly within the spawn mass. Un-
cleaved ova were 123 + 5 um in diameter
(n = 8). Extra-capsular yolk of Type 1A was
present, and each capsule contained a single
embryo. The embryonic period lasted six days
at 25-26°C, and the veligers that were re-
leased were planktotrophic.

Chromodoris elisabethina Bergh, 1877

Four egg masses observed from three indi-
viduals. The adult nudibranchs were all col-
lected from the Gneering Shoals, Mooloolaba,
Queensland, and the egg masses laid in Jan-
uary and May. All egg masses were laid flat,
and while all were spiral in shape, they often
ended askew. The egg masses ranged from
three to five whorls. Ova were cream and
there was no extra-capsular yolk observed.
The rate of deposition of the egg mass for one
individual was measured at 1.58 mm/min. Un-
cleaved ova were 93 + 5 um in diameter (n =
15). Each capsule contained a single embryo.
The embryonic period lasted 5-6 days at ap-
proximately 27°C (n = 1) and 8-10 days at
20.5-22°C (n = 3). Larvae were planktonic, al-
though the exact developmental type was not
determined.

Chromodoris geometrica Risbec, 1928

Two incomplete egg masses were ob-
served in May from two individuals collected

at the Gneering Shoals, Mooloolaba, Queens-
land. Egg masses were upright and consisted
of only a partial whorl (animals tried to lay their
spawn on the air-water interface, which could
not sustain the weight of more than a partial
egg mass). Ova were cream and extra-cap-
sular yolk Type 1B was present in the egg
mass. The ova were 82 + 7 um (n = 8) in di-
ameter. Each capsule contained a single em-
bryo. The embryonic period of one of the
pieces lasted 10 days at 20.5-22°C. Larvae
were planktonic, although the exact develop-
mental type was not determined.

Chromodoris kuiteri Rudman, 1982

Three egg masses were observed from in-
dividuals from Cook Island, New South Wales
(n = 1), and Heron Island, Great Barrier Reef,
Queensland (n = 2). These were laid in De-
cember and March respectively. Egg masses
were laid flat, and consisted of 2-5 whorls.
The regularity of the coiling varied greatly be-
tween egg masses laid by different individu-
als. The ova were pale orange in colour,
arranged linearly, and no extra-capsular yolk
was observed. Each capsule contained a sin-
gle embryo. The embryonic period lasted six
days at 27°C, and also six days at a variable
temperature range of 23-28°C. Larvae were
planktonic, although the exact developmental
type was not determined.

Chromodoris kuniei Pruvot-Fol, 1930

A single egg mass was observed in August
from an individual collected from Heron Is-
land, Great Barrier Reef, Queensland. The
egg mass was upright, consisting of two
whorls. The ova were orange and were asso-
ciated with extra-capsular yolk Type 1B. The
uncleaved ova were 109 + 2 um (n = 7) in di-
ameter. Each capsule contained two em-
bryos. The embryonic period was 15 days at
20-22°C. Larvae were planktonic, although
the exact developmental type remains unde-
termined.

Chromodoris leopardus Rudman, 1987

A single egg mass was observed in Sep-
tember from an individual collected in the
Gneering Shoals, Mooloolaba, Queensland.
The egg mass was upright, consisting of two
whorls. The ova were orange and were asso-
ciated with extra-capsular yolk Type 1B. The
uncleaved ova were 104 + 3 um in diameter

CHROMODORID EGG MASSES 295

(n = 10). Each capsule contained one or two
embryos. The embryonic period lasted 14
days at 20-22°C. Larvae were planktonic, al-
though the exact developmental type was not
determined.

Chromodoris lochi Rudman, 1982

A single egg mass was observed in April
from a specimen collected at Ribbon Reef
Number 10, Great Barrier Reef, Queensland.
The egg mass was laid flat and consisted of
three whorls. The ova were cream in colour,
and no extra-capsular yolk was present. Each
capsule contained a single embryo. The em-
bryonic period lasted nine days at 25°C, and
larvae showed a planktotrophic developmen-
tal pattern.

Chromodoris roboi Gosliner & Behrens, 1998

Two egg masses have been observed from
two individuals collected from Heron Island,
and another egg mass was observed from the
Whitsunday Islands, Great Barrier Reef,
Queensland. The egg masses from Heron Is-
land were laid in March and September, and
were upright and consisted of two whorls. The
ova were orange and were associated with
extra-capsular yolk of Type 1B. The ova were
101 + 8 um in diameter (n = 10). Many cap-
sules contained multiple embryos; typically
they contained two, but up to four were ob-
served. The embryonic period of one egg
mass lasted 11 days at 20-22°C. Larvae were
planktonic, although the exact developmental
type remains undetermined. The egg mass
observed in the Whitsundays was laid in Au-
gust and consisted of one whorl. This egg
mass was upright but laid in an irregular spi-
ral, so that the whorl consisted of short,
straight sections with a distinct kink that joined
to another short, straight section. Thus, the
egg mass appeared to be crenulated, but both
the free edge and attached edge were equal
in length.

Chromodoris strigata Rudman, 1982

A single egg mass was observed in October
from an animal collected on Heron Island,
Great Barrier Reef, Queensland. The egg
mass was flat, and consisted of five whorls.
The ova were pale orange and no extra-cap-
sular yolk was observed. The ova measured
80 + 2 um in diameter (n = 11). Each capsule
contained a single embryo. The embryonic

period lasted eight days at 24°C. Larvae were
planktonic, although the exact developmental
type remains undetermined.

Chromodoris tinctoria
(Rüppell & Leuckart, 1828)

A single egg mass was observed in March
from North Stradbroke Island, Queensland.
The egg mass was upright and consisted of
three whorls. The orange ova were arranged
linearly within the egg mass and were associ-
ated with extra-capsular yolk of Type 1A.
Each capsule contained two to three em-
bryos. The embryos died after 11 days at
22-24°C, and no further details of develop-
ment could be ascertained.

Digidentis Rudman
Digidentis cf. arbutus (Burn, 1961)

A single egg mass was observed in Febru-
ary from Pt Puer, Tasmania. The animal was
disturbed while laying, so the egg mass was
incomplete. However, the spawn was upright
and quite firm. The ova were orange and no
extra-capsular yolk was observed. The ova
measured 491 + 12 um (n = 7) in diameter
and each capsule contained a single embryo.
The developmental type could not be deter-
mined, although the large size of the ova po-
tentially indicates direct development.

Diversidoris Rudman
Diversidoris aurantionodulosa
Rudman, 1987

Four egg masses were observed in April
from Pt. Cartwright, Mooloolaba, Queensland.
The egg masses were upright and consisted of
one to two whorls each. Two egg masses laid
in the laboratory sloped inwards very slightly
while those laid in the field appeared typically
upright. The ova were cream-white in colour,
no extra-capsular yolk was observed and each
capsule contained a single embryo. The em-
bryonic period lasted 8 days at 25°C, with the
larvae showing a lecithotrophic developmen-
tal pattern.

Glossodoris Ehrenberg
Glossodoris vespa Rudman, 1990

A single egg mass was observed in May
from an individual collected in the Gneering
Shoals, Mooloolaba, Queensland. The egg
mass was upright, very firm and consisted of

296 WILSON

two whorls. The cream ova measured 300 +
19 um (n = 10) and no extra-capsular yolk was
observed. The embryonic period lasted for 56
days at 17-22°C, and the development pat-
tern was ametamorphic direct.

Hypselodoris Stimpson
Hypselodoris bullocki (Collingwood, 1881)

Two egg masses were observed from two
individuals during November on Orpheus Is-
land, Great Barrier Reef, Queensland. They
were upright but with the free edge ofthe egg
mass sloping away from the centre of the spi-
ral. Ova were yellow and no extra-capsular
yolk was observed. Each capsule contained a
single embryo, but the developmental type re-
mains unknown.

Hypselodoris obscura (Stimpson, 1855)

Four egg masses were observed in total
from three individuals. Three egg masses
were observed from a pair of nudibranchs col-
lected in April from Amity Point, North Strad-
broke Island, Queensland, and one egg mass
was observed in November from an individual
collected from Wellington Point, Moreton Bay,
Queensland. The egg masses consisted of
2-5 whorls. Allegg masses were upright but
ranged from being slightly outward sloping to
having a crenulated free edge. The ova were
white, arranged linearly in the egg mass and
no extra-capsular yolk was observed. The ova
from one egg mass measured 104 + 5 um
(n = 11). Each capsule contained a single em-
bryo. The embryonic period lasted 9-10 days
at 22°C (п = 2) and 4-5 days at 25-26°C (п =
1). Veligers were planktonic, but the exact de-
velopment type remains undetermined.

Hypselodoris sp. Chromodoris geometrica
Coleman, 1981: 32. Misidentified

Four egg masses were observed in total
from three individuals, all from the Gneering
Shoals, Mooloolaba, Queensland. Three egg
masses were laid in September and one in
January. The egg masses ranged from 1-3
whorls and were upright with the free edge
crenulated. Ova were dark orange and no
extra-capsular yolk was observed. Ova were
146 + 4um in diameter (n = 10). Each capsule
contained a single embryo. The embryonic pe-

riod took 9-11 days at 20-22°C (n = 2), and
the resulting veligers were lecithotrophic.

Hypselodoris zephyra
Gosliner & Johnson, 1999

Two egg masses were observed from a
single animal in December from Cook Island,
New South Wales. The egg masses ranged
from 2-3 whorls and were upright with the
free edge crenulated. Ova were white and the
embryonic period took five days at 27°C.
Veligers were planktonic, although exact de-
velopmental type was not determined.

Noumea Risbec
Noumea norba Marcus & Marcus, 1970

Four egg masses were observed from two
individuals in May, from the Gneering Shoals,
Mooloolaba, Queensland. The egg masses
ranged from 2-3 whorls and ranged from up-
right to having the free edge sloping toward
the centre of the spiral. Ova were cream,
arranged linearly and measured 83 + 3 um in
diameter (n = 12). Each capsule contained a
single embryo. The embryonic period was
12-14 days at 20.5-22°C (n = 2). Veligers
were planktonic, although the exact develop-
mental type was not determined.

Pectenodoris Rudman
Pectenodoris trilineata
(Adams & Reeve, 1850)

One egg mass was observed in August on
Heron Island, Great Barrier Reef, Queens-
land. The egg mass was firm, and consisted
of one whorl. The ova were pale pink in colour
and measured 205 + 11 um (n = 8). Each
capsule contained a single embryo. Develop-
mental details were not recorded.

DISCUSSION

Although only a small fraction of the spawn-
ing details of chromodorid species is known,
there is some evidence to suggest that egg
mass morphology is consistent within genera
and even groups of genera (Table 2). The ob-
vious exception to this is the presence of mul-
tiple egg mass types within Chromodoris, the

TABLE 2. Egg mass types of chromodorid species

CHROMODORID EGG MASSES

297

Species

Cadlina luteomarginata
Cadlina modesta
Cadlina pellucida
Cadlinella ornatissima
Cadlinella sp.

Tyrinna nobilis
Chromodoris aspersa
Chromodoris africana

Chromodoris annulata
Chromodoris aureopurpurea
Chromodoris binza
Chromodoris coi
Chromodoris collingwoodi
Chromodoris clenchi
Chromodoris daphne
Chromodoris elisabethina
Chromodoris geometrica
Chromodoris geometrica
Chromodoris kuniei
Chromodoris kuiteri
Chromodoris leopardus
Chromodoris lineolata
Chromodoris lochi
Chromodoris magnifica
Chromodoris orientalis
Chromodoris perola
Chromodoris roboi
Chromodoris strigata
Chromodoris tinctoria
Chromodoris willani
Chromodoris woodwardae
Glossodoris cincta

Glossodoris pallida
Glossodoris plumbea
Glossodoris sibogae
Glossodoris sp.
Glossodoris sp.
Glossodoris vespa

Noumea decussata
Noumea haliclona

Noumea norba

Noumea simplex

Verconia verconis
Pectenodoris trilineata
Digidentis cf arbutus
Diversidoris aurantionodulosa
Ceratosoma amoena
Ceratosoma brevicaudatum
Ceratosoma magnifica
Mexichromis cf multituberculata
Thorunna australis
Thorunna daniellae
Thorunna florens

Thorunna montrouzieri
Hypselodoris bullocki
Hypselodoris emma

A

B

Egg Mass Type

C

Source

Dehnel & Kong, 1979

Behrens, 1991

Fernandez-Ovies, 1981

Boucher, 1983

Debelius, 1998

Muniain et al., 1996

Gohar & Soliman, 1967b, as C. inornata Pease

Gohar & Aboul-Ela, 1957, as C. quadricolor (Ruppell &
Leuckart)

Gohar & Aboul-Ela, 1957

Baba et al., 1956, as Glossodoris

Ortea et al., 1994

Taylor, 2001

present study

Ortea et al., 1994

present study

Johnson & Boucher, 1983; present study

Johnson & Boucher, 1983; Chuk, 2001; present study

Rose, 1981; Fraser, 2001a

present study; Adams, 2001; Warren, 2001

present study

present study

Kenny, 1970

present study

Klussman-Kolb & Wagele, 2001

Baba et al., 1956, as Glossodoris pallescens Bergh

Bandel, 1976

present study

present study

present study

Gill, 2001

Rudman, 1998a

Gohar & Soliman, 1967c, as C. obseleta (Rüppell &
Leuckart)

Soliman, 1987

Gohar & Aboul-Ela, 1959, as G. atromarginata Cuvier

Baba et al., 1956

Fraser, 2001b

Ostergaard, 1960

present study

Johnson, 2001a

Avern, 1986; present study

present study

Johnson, 2001b

Debelius, 1998

present study

present study

present study

Coleman, 2001

Smith et al., 1989

Jamieson, 1999, as Miamira

Miller, 2001a

S. Johnson, pers. comm.

Miller, 2001b

Coleman, 2001

Rudman, 1998b

present study

Marshall & Willan, 1999

(continues)

298

TABLE 2. (Continued)
Egg Mass Type
Species A B C

WILSON

Source

Hypselodoris festiva .
Hypselodoris kanga .
Hypselodoris maculosa .
Hypselodoris obscura °
Hypselodoris sp. .
Hypselodoris whitei .
Hypselodoris zebra .
Hypselodoris zephyra

Risbecia ghardagana

Risbecia pulchella

Risbecia tryoni

largest genus in the Chromodorididae. Cur-
rently, it is estimated that Chromodoris con-
tains approximately 200 species (Gosliner 8
Draheim, 1996), whereas most other chro-
modorid genera are considerably less spe-
ciose and some are monotypic (eg., Diversi-
doris, Verconia). Although the total percentage
of Chromodoris species sampled within the
present study is very low, all three types of egg
mass structure were detected (Table 2).

The nine Chromodoris species that are
known to exhibit flat egg masses (Type A)
occur in two colour groups. Rudman (1977,
1982, 1983) described these groups in order
to facilitate identification of similarly coloured
species. The first of these groups, the Chro-
modoris quadricolor colour group, contains all
but two of these flat-spawning species. Based
on the distribution of mantle glands and on re-
productive characters, it has been suggested
that this colour group may represent a dis-
crete clade within the genus Chromodoris
(Gosliner & Behrens, 1998). This provides fur-
ther evidence that egg masses can potentially
reflect phylogenetic influence. The remaining
species that lay a flat egg mass, Chromodoris
aspersa (Gould) and C. orientalis Rudman,
both belong to the C. aspersa colour group
(Rudman, 1983). These species have long
been confused, although external colouration
can be used to reliably separate them (Rud-
man, 1983). The notal spots in C. orientalis
are black, whereas in C. aspersa they are
deep purple. The precise nature of the rela-
tionship between the two colour groups re-
mains to be investigated, but it is interesting to
note that most recorded Type A spawners in
Chromodoris share a band of orange around
the mantle. They also typically possess
translucent orange gills and/or rhinophores,
and all but C. aspersa share the presence of

Baba et al., 1956, as Glossodoris

Rudman, 1999
Johnson, 2000
present study
present study

Johnson & Boucher, 1983, as H. mouaci (Risbec)

Geiger, 1999
present study
Gohar & Aboul-Ela, 1957
Gohar & Aboul-Ela, 1957

Johnson & Boucher, 1983, as Chromodoris

black pigment (present as stripes or back-
ground colour in the С. quadricolor colour
group and as spots in C. orientalis).

Upright egg masses (Type B) were present
in at least 13 of the 24 species of Chromodoris
species listed in Table 2. There was some dif-
ficulty in classifying the egg masses of Chro-
modoris coi, C. kuniei and C. roboi. These
species all lay ribbons that in most cases are
upright, but are often attached in short kinks
that cause them to appear outward sloping.
These egg masses may also have grooves on
the broad side of the ribbon running parallel to
attachment, although the significance of this is
unclear. The two reports of aclearly crenulated
egg mass (Type C) occurring in Chromodoris
warrant further attention. Chromodoris mag-
nifica falls into the C. quadricolor colour group
of Rudman and would thus be predicted to lay
a flat egg mass similar to all other known mem-
bers of the group. However, Klussmann-Kolb
& Wagele (2001) report C. magnifica laying an
upright and crenulated egg mass, although
further observations are desirable to confirm
the report. Similarly, conflicting reports occur
regarding the egg mass of Chromodoris geo-
metrica. Boucher (1983) recorded C. geomet-
rica in the Marshall Islands with an upright, or-
ange egg mass containing extra-capsular
yolk. This study confirmed that report for spec-
imens from subtropical eastern Australia, and
an upright orange ribbon was also reported
from Papua New Guinea (Chuk, 2001). How-
ever, Rose (1985) recorded some spawning
details of C. geometrica from temperate east-
ern Australia and reported an absence of
extra-capsular yolk. It is only in his unpub-
lished thesis (1981) that he describes the egg
mass as being fluted. Although it is quite pos-
sible that the single specimen that Rose col-
lected was misidentified, Fraser (2001a) also

CHROMODORID EGG MASSES

shows an egg mass of C. geometrica that is
clearly crenulated. This latter observation from
South Africa also differs from all previous ac-
counts in that the colour of the egg mass is
white. It is likely that this egg mass lacked
extra-capsular yolk as well, as the resulting or-
ange hue is usually visibletothe naked eye. As
both Rose (1981) and Fraser (2001a) made
their observations at similar latitudes in the Pa-
cific and Indian oceans respectively (approxi-
mately the southermost limits for C. geomet-
rica), it is possible that the production of
extra-capsular yolk reserves is related to tem-
perature.

An upright egg mass structure (Type B) was
found in all species of Glossodoris, Noumea,
Verconia, Pectenodoris, Digidentis and Diver-

Ceratosoma

FN
=
Pectenodoris

_ Glossodoris

0) Verconia

Ardeodoris ?

x

Chromodoris ~

Noumea

299

sidoris represented in Table 2. According to
the first phylogeny proposed for the Chro-
modorididae (Rudman, 1984), all these gen-
era are typically considered in the “mid re-
gion” of evolution within the family, neither
basal nor highly derived (Fig. 3). Gosliner &
Johnson's (1999) cladistic analysis found no
resolution between the lineage containing
Chromodoris, Ceratosoma and Glossodoris
and the one containing Noumea, Pecten-
odoris, Verconia, Thorunna and Digidentis
(Fig. 4). However, both phylogenies agree
that the crown group within one lineage con-
sists of Risbecia + Hypselodoris. This crown
group (with the exception of Hypselodoris
zebra) all show egg masses that are either
outwardly sloping or crenulated. This indi-

Hypselodoris

Risbecio uses)
2
Ourvilledoris

Mexichromis =

x
x

\ \

x Thorunna DE

Digidentis

x
{
;
| EZ
:
N
x
‘

Cadlina

Codlinella C= N

re) ))
LAS:

FIG. 3. Egg mass shape mapped onto hypothesized phylogeny of the Chromodorididae (from Rudman,

1984).

300

St
a Pao’

| < У <

> > os RS Se x so Sl

WILSON

Nm) AP a
¿Y x ©

Se
ESOS ES LEN N

RE ER Resigns oe № Qe con с © У ce «3 ce

FIG. 4. Egg mass shape mapped onto hypothesized phylogeny of the Chromodorididae (from Gosliner &
Johnson, 1999). Broken lines indicate that the spawn type illustrated above is also present in the genus.

cates that the most highly derived forms are
more likely to lay egg masses that have the
free edge of the ribbon lengthened, resulting
in outward sloping or crenulated egg masses.
As the structure of an egg mass is said to re-
flect the degree of anatomical complexity of
the reproductive system (Fretter & Ko Bun,
1984), it is likely that more highly derived gen-
era would lay more complex egg masses. The
single Known exception in Hypselodoris (H.
zebra) shows an upright egg mass. This is the
only egg mass known for a member of the At-
lantic/Eastern Pacific clade of the genus.
Hypselodoris is known to consist of two
clades, the above-mentioned clade and an
Indo-Pacific clade (Gosliner & Johnson,
1999), which is represented by all the other
Hypselodoris egg masses observed in this
study.

The genus Thorunna is considered rela-
tively derived in both phylogenies of the Chro-
modorididae, and the egg mass data from
Table 1 appear to sustain this. Thorunna has
been proposed as a sister group to both Digi-
dentis (Rudman, 1984) and Durvilledoris
(Gosliner & Johnson, 1999). Observations on
the egg mass of Digidentis offers no firm evi-
dence in support of this relationship, and the

egg mass structure of Durvilledoris species
remains unknown.

Ceratosoma is closely allied to Chro-
modoris and Glossodoris, according to the
phylogeny of Gosliner & Johnson (1999). This
contrasts with Rudman (1984), who places it
in the “hypselodorid” subgroup (also contain-
ing Digidentis, Thorunna, Durvilledoris, Mexi-
chromis, Risbecia and Hypselodoris), consid-
ering Ceratosoma to have a Chromodoris
ancestor but having diverged early in hypselo-
dorid evolution. Here, the egg masses of
three Ceratosoma species are reported to be
outwardly sloping or crenulated, indicating a
derived condition.

Extra-capsular yolk reserves have so far
been recorded in only two chromodorid gen-
era, Cadlinella and Chromodoris, with five
new records in the present study (Table 3).
While extra-capsular yolk is found in the flat
egg masses of Cadlinella, the flat egg masses
of Chromodoris never contain these reserves.
It is only in the upright egg masses of Indo-Pa-
cific Chromodoris species that extra-capsular
yolk is present. Chromodoris binza and Chro-
modoris clenchi, both found in the Caribbean,
lay upright egg masses but do not incorporate
extra-capsular yolk reserves into the egg

CHROMODORID EGG MASSES 301

TABLE 3. Chromodorids that produce extra-capsular yolk.

Species

Cadlinella ornatissima
Chromodoris albopunctatus

Chromodoris albopustulosa 1A
Chromodoris annulata

Chromodoris collingwoodi 1A
Chromodoris daphne 1A
Chromodoris decora 1A
Chromodoris E-6 1A
Chromodoris fidelis 1A
Chromodoris galactos 1A
Chromodoris geometrica 1B
Chromodoris kuniei 1B
Chromodoris leopardus 1B
Chromodoris marginata 1A
Chromodoris preciosa

Chromodoris горо! 1B
Chromodoris rubrocornuta
Chromodoris E-328
Chromodoris E-48
Chromodoris thompsoni 1A
Chromodoris tinctoria 1A
Chromodoris vibrata

mass (Ortea et al., 1994). It will be of great in-
terest to determine whether extra-capsular
yolk is restricted solely to Indo-Pacific and
Red Sea Chromodoris. While Cadlinella sp.
from the Red Sea does lay flat egg masses,
they have not yet been examined to deter-
mine if they also contain extra-capsular yolk
like Cadlinella ornatissima.

While Cadlina, Tyrinna and Cadlinella are
all currently considered basal within the Chro-
modorididae, there appears to be no indica-
tion that these three genera are themselves
closely related (Rudman, 1984). It is therefore
no surprise that these genera exhibit different
egg mass types. While varying hypotheses
regarding the basal chromodorids have been
proposed or supported (Rudman, 1984; Muni-
ain et al., 1996; Gosliner & Johnson, 1999),
the most recent discussion concludes only
that the phylogeny of these basal groups re-
mains unclear (Schródl & Millen, 2001). Given
that Cadlinella shares a flat egg mass and
extra-capsular yolk with varying Chromodoris
species, it is likely that Cadlinella gave rise to
the Chromodoris lineage.

There is some concern that egg mass
structure may reflect environmental rather
than phylogenetic influences (Wagele &
Willan, 2000), and is therefore not suitable to
be used as a Character in phylogenetic analy-
ses. Observations on spawning in the field
and laboratory have shown some differences,

Yolk type

2, small & pale
2, large & orange

2, large & orange

not determined
not determined

not determined
not determined

not determined

Original source

Risbec, 1928

Boucher, 1983

Kay & Young, 1969
Gohar & Aboul-Ela, 1957
present study

present study

Kay & Young, 1969
Boucher, 1983

Marcus & Burch, 1965
Boucher, 1983, as E-57
Boucher, 1983

present study

present study

Boucher, 1983

S. Johnson, pers. comm.
present study

S. Johnson, pers. comm.
S. Johnson, pers. comm.
S. Johnson, pers. comm.
Thompson, 1972, as C. loringi
Gohar & Soliman, 1967
S. Johnson, pers. comm.

which have been incorporated into the egg
mass classification in this study. Specimens
of Noumea norba and Diversidoris aurantio-
nodulosa laid upright ribbons in the field, while
the same specimens in the laboratory laid egg
masses that sloped inward. Specimens of
Hypselodoris obscura lay egg masses that
range from outward sloping to crenulated.
Some egg masses, particularly those that are
thin and flaccid, can appear slightly fluted
when laid on irregular or uneven substrata.
However, it is possible to differentiate be-
tween these and egg masses that are truly
outward sloping or crenulated by comparing
the length of both the free and attached
edges. The regularity of the coiling, that is, the
space between the whorls of one egg mass,
differed greatly within a species, suggesting
this may be affected by environmental condi-
tions or perhaps the reproductive history of
the parent. It is not yet possible to make any
correlation between egg mass type and the
habitat of the parent nudibranch, since there
is little information regarding movement within
the latter. Many species of nudibranch are
found in both intertidal and subtidal environ-
ments without showing any obvious change in
egg mass structure. However, controlled ex-
periments varying such factors as tempera-
ture, salinity and water flow are desirable to
test this idea.

There are apparently conflicting reports

302 WILSON

where the colour of aegg mass has been re-
ported to differ between localities, even when
extra-capsular yolk is absent. Johnson &
Boucher (1983) reported that Hypselodoris
maculosa lays a pale pink egg mass, whereas
Marshall & Willan (1999) recorded it as white.
While it is possible that a change in prey items
may trigger a corresponding change in ova
colour, differences may also reflect subjective
interpretation of colour. It is also important to
know whether the animal laying the egg mass
is identified correctly. Hypselodoris maculosa
individuals are known to be variable in colour,
and there is the possibility that a complex of
species is currently identified as a single
species. Egg masses may have the potential
to help separate these complexes but need to
be used in conjunction with morphological
data from the parent specimens. Another fac-
tor that can influence the colour of an egg
mass is the amount of time that has elapsed
since it was laid. Gradual colour changes
occur as the developing embryos use up the
available yolk. However, while small changes
in colour may be attributed to such factors,
real disagreement in colour may reflect differ-
ences in the identity of the parent. Risbecia
tryoni has been reported to lay a rose-pink
egg mass with a crenulated free edge (John-
son & Boucher, 1983), whereas Marshall &
Willan (1999) reported the mass as orange
but do not describe its structure. Marshall &
Willan (1999) also incorrectly cited Johnson &
Boucher as attributing extra-capsular yolk to
this species.

While the general form of the egg mass is
usually characteristic of a species or genus,
Rudman & Avern (1989) found both upright
and crenulated egg mass types in the rela-
tively small genus Rostanga (approximately
13 species). This was also the case for Acan-
thodoris (Hurst, 1967), in which both upright
and crenulated egg masses were recorded.
This indicates that some caution may be nec-
essary when interpreting phylogenetic signal
from egg mass structure, as it may be useful
at different taxonomic levels in different
groups. The absence of a fossil record means
there is currently no reliable method of dating
genera. It may only be in more recent genera
that egg mass structure remains conservative
throughout. It is possible that the trend to-
wards crenulation of egg masses in the more
derived Chromodorididae is also seen within
a single “older” genus that has had more time
to evolve.

Soliman (1987) recognized the potential

taxonomic value of egg mass type among
gastropods, but he recommended that when
interpreting phylogenetic relationships, pri-
mary consideration should be given to ana-
tomical, palaeontological or ecological evi-
dence. Because no fossil record exists for the
Nudibranchia, and accurate ecological infor-
mation is still scarce for most groups, alterna-
tive characters may be found in reproductive
data. Egg mass structure may help confirm or
challenge phylogenetic hypotheses based
solely on anatomical data, but much work is
still required to understand the underlying
causes of observed variation.

ACKNOWLEDGEMENTS

Support for this project was obtained from a
University of Queensland Research Grant, a
Mollusc Research Grant from the Malacologi-
cal Society of Australasia, and the Undersea
Explorer, Port Douglas. | would like to thank
many people for assistance in collecting ani-
mals, namely Dan Jackson, Suzie Green,
David Harris, Shane Litherland and Shireen
Fahey. Bill Rudman assisted with identifica-
tions, and Scott Johnson provided valuable
discussion and observations. Maria del Car-
men Gömez-Cabrera and Rosa Garcia Novoa
are thanked for their assistance in translating
the work of Fernandez-Ovies. This manu-
script was improved by comments from John
Healy, Bill Rudman and two anonymous re-
viewers. | would like to acknowledge the
Great Barrier Reef Marine Park Authority
(G98/110), Queensland Parks and Wildlife
Service (QSE99/489) and the Department of
Primary Industries, Water and Environment,
Hobart (P99/00-121) for allowing respective
collection permits. This manuscript forms con-
tribution 2002-01 from the Centre of Marine
Studies, University of Queensland.

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MALACOLOGIA, 2002, 44(2): 307-324

ALLOZYMIC TAXONOMY WITHIN THE GENUS MELANOPSIS
(GASTROPODA: CERITHIACEA) IN ISRAEL:
A CASE IN WHICH SLIGHT DIFFERENCES ARE CONGRUENT

Andrzej Falniowski', Joseph Heller’, Magdalena Szarowska', & Krystyna Mazan-Mamczarz'

ABSTRACT

Molecular differences between the species of Melanopsis distinguished by Heller were stud-
ied by means of cellulose acetate gel allozyme electrophoresis on 26 Melanopsis populations
from Israel: 6 M. buccinoidea Olivier, 1801; 8 M. saulcyi Bourguignat, 1853; 1 M. meiostoma
Heller & Sivan, 2001; and 11 M. costata Olivier, 1804, represented by two subspecies: M. costata
costata Olivier, 1804, and M. costata jordanica Roth, 1839. Fourteen loci (9 polymorphic) were
scorable: Aat, Alp, Est-1, Est-2, Gpi, Hbdh, Idh-1, Idh-2, Iddh, Mdh, Mdhp, Mpi, Pgdh, Pgm. Mean
sample size was 28.983; mean number of alleles per locus: 1.45 (1.1-1.7); polymorphic loci per
population: 36.5% (14.3-57.1); mean observed heterozygosity: 0.085 (0.041-0.121); mean ex-
pected heterozygosity: 0.112 (0.045-0.170). Nei genetic distance (0.000-0.232, mean 0.0761,
no significant association with geographic distance) and Cavalli-Sforza and Edwards arc genetic
distance (0.054-0.441, mean 0.240, significant association with geographic distance) were com-
puted for pairs of populations. Interpopulation differences were analyzed phenetically (corre-
spondence analysis, UPGMA), and phylogenetically (neighbor-joining and Fitch-Margoliash ad-
ditive trees). Despite the slight differentiation of the taxa, the results confirmed the distinctness
of M. costata costata, M. costata jordanica and M. saulcyi although the speciation processes may
not yet be completed. The molecular variability of М. buccinoidea was wider than that of all the
other studied taxa together, and overlapped the variation ranges of each. It may be ancestral
species to the others, which may have speciated as peripheral isolates. The position of M. meios-
toma as a distinct species was not confirmed.

Key words: freshwater snail, species, allozymes, electrophoresis, phenetics, phylogenetics.

INTRODUCTION

The freshwater snail genus Melanopsis
(Ferussac, 1807) (Prosobranchia: Melanopsi-
dae) is one of the most abundant mollusks in
the lakes, rivers and springs of the Middle
East, and is widely distributed throughout the
circum-Mediterranean (Germain, 1921-22;
Bilgin, 1973; Tchernov, 1973, 1975; Schütt &
Bilgin, 1974; Banarescu, 1990-95). Its anat-
omy was described by Bilgin (1973) and Al-
taba (1991), sperm structure by Afzelius et
al. (1989), radula variation by Glaubrecht
(1993), and allozyme variation in the western
Mediterranean by Altaba (1991), who found
considerable genetic differentiation over short
distances. There are numerous studies on
mollusk allozymes, devoted to molecular tax-
onomy (Woodruff et al., 1988; Borsa & Ben-
zie, 1993; Ponder et al., 1993; Davis, 1994;
Haase, 1994; Falniowski et al., 1996), includ-

ing in Melanopsis (Altaba, 1991; Altman &
Ritte, 1996).

In Melanopsis, the shell is extremely vari-
able, with smooth and costate, elongate and
stout, banded and all-black forms found in a
relatively small area. Heller et al. (1999) in-
vestigated the systematics, distribution and
extent of presumed hybridization of Melanop-
sis in the central and northern Jordan Valley.
Basing their study on the shell morphometry
of 700 snails collected at 35 sites, they recog-
nized three species: snails with smooth shells
(M. buccinoidea Olivier, 1801); with straight-
ribbed shells that almost always extend the
entire length of the whorl (M. costata Olivier,
1804); and with tubercle-ribbed shells that do
not extend the entire length (M. saulcyi Bour-
guignat, 1853). Melanopsis buccinoidea is
common in a wide variety of aquatic habitats,
from small trickles to springs and streams; M.
costata occurs in the upper Jordan River,

"Department of Malacology, Institute of Zoology, Jagiellonian University, ul. Ingardena 6, 30-060 Kraköw, Poland;

faln @ zuk.iz.uj.edu.pl

Department of Evolution, Systematics and Ecology, Hebrew University, Jerusalem, Israel

308 FALNIOWSKI ET AL.

Lake Kinneret and lower Jordan River; M.
saulcyi in streams that border swamps.

Heller et al. (1999) also reported the occur-
rence of presumed hybrids characterized by
shells with weak ribs, thus defined based on
shell characters alone. According to those au-
thors, hybrids were found in narrow zones of
contact, both between M. buccinoidea and M.
costata, and between M. buccinoidea and M.
saulcyi. Outside these zones, the species re-
tain their distinct conchological identities. The
narrowness of the hybrid zones suggested to
Heller et al. (1999) that selection was main-
taining the parapatric distribution of the
species involved, with the narrow hybrid
zones acting as partial barriers to gene flow.
Between M. buccinoidea and M. costata, the
hybrid zones were at habitat transition, from
streams to lake.

Within M. costata, Heller et al. (1999) dis-
tinguished three subspecies. Two of them are
dealt with in the present paper: M. costata
costata Olivier, 1804 (shell elongate; the
upper Jordan River, on mud and submerged
vegetation) and M. costata jordanica Roth,
1839 (shell usually stout; Lake Kinneret, on
stones and boulders). The stout shell of M.
costata jordanica seems correlated with a
stormy habitat. Recently a new species, M.
meiostoma, was described from Israel (Heller
& Sivan, 2000) characterized by a small,
slender shell with relatively small mouth and
weak callus.

The aim of the present study is to test
whether the morphological differences be-
tween the species and subspecies distin-
guished by Heller et al. (1999) are accompa-
nied by the molecular differences. The
morphologies are rather finely differentiated:
in fact, the differences are marked in charac-
ters that are prone to environmental factors
(e.g., Gostan, 1966; Palmer, 1985; Falniow-
ski, 1988). Thus, our null hypothesis is that
the entire studied group is a single, concho-
logically variable species, with shell variation
determined environmentally, not genotypi-
cally. The latter is not a rare phenomenon
within the Gastropoda. It is essential in any
discussion on species distinctness to specify
the species concept applied. In this study, we
follow the cohesion species concept, as de-
fined by Templeton (1989), considering not
only genetic but also demographic and eco-
logical factors. In particular, speciation is re-
garded as the development of cohesion
mechanisms, not isolation mechanisms. The
cohesion concept assumes, as cohesion

mechanisms, not only genetic exchangeabil-
ity, but also demographic exchangeability.
The latter means that all the populations of a
species must share the same fundamental
niche, as defined by Hutchinson (1965). Thus,
to demonstrate that the fundamental niches of
two taxa are not the same means to prove
their species distinctness. The cohesion con-
cept covers the full range of biological reali-
ties, from completely closed systems of asex-
ually reproducing taxa to open systems of
syngameons, with species defined practically
based solely on demographic exchangeabil-
ity. It must be stressed that speciation is rather
a process, not an event (Templeton, 1989).
Hence, wherever the process is still going on
and not completed, there obviously will be the
“bad” species undergoing speciation as op-
posed to the “good”, well-defined ones. The
cohesion concept is, therefore, what applies
best to such cases.

MATERIAL AND METHODS

Locality Description

The distribution of the localities is shown in
Figure 1.

Melanopsis buccinoidea

(1) Enot Huga (IG —New Israel Grid—200-
213). Within a recreation site, a small
spring that irrigates by way of a recently
constructed channel into a small pool
consisting of boulders and cobble; snails
collected from the channel, at a depth of
2-3 cm.

(2) En Hanatziv (IG 197-208). A spring
within a large natural pool; snails col-
lected from dead, floating vegetation and
firm boulders.

(3) En Raqgat, spring (IG 199-245). Emerg-
ing 15 m from the shore of Lake Kinneret,
this spring trickles down into the lake
among boulders and sparse, swampy
vegetation; snails collected along the
trickle, at a depth of 2-3 cm.

(4) Dan (IG 212-295). A lowland stream that
forms the principal tributary of the Jordan
River; the physico-chemical aquatic envi-
ronment extremely stable: annual as well
as diurnal water temperatures virtually
constant, at 15.5°C (+ 0.5%), chemical
parameters (NH,, PO,, NO,) vary little,
dissolved oxygen concentrations uni-
formly high at 95% of saturation (Heller et
al., 1997); snails collected from several

ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 309

ha
Y
E
[> 4
£
a
2
Е
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Lake
Kinneret

FIG. 1. Distribution of studied localities. Black arrow
in the small map at the bottom indicates the area
presented in the bigger map. Population numbers:
see text. Symbols of taxa: see Fig. 2.

small brooks near the spring, from boul-
ders at a depth of 5-10 cm.

(6) Banias (IG 214-294). A cool spring form-
ing a major source of the Jordan River;
snails collected from the artificial pool
surrounding the spring, at a depth of
down to 30 cm.

(7) Nahal Tavor (IG 201-223). A narrow,
shallow stream covered by dense vege-
tation; snails collected from within the
stream, on the muddy substratum, at a
depth of 2-3 cm.

Melanopsis meiostoma

(8) Kefar Haruv (IG 211-240). A small,
spring that pours into a small cement
pool; brackishwater (a high, 670 mg/l,
content of chloride anions); snails col-
lected from the walls of the pool, at a
depth of 30 cm.

Melanopsis costata jordanica

Lake Kinneret (21 km x 12 km, 170 km). In
its natural condition, the water level of Lake
Kinneret used to fluctuate annually at an av-
erage of 0.8-1.0 m. In 1964, the lake was
dammed, its level increased by 1 m, and was
left at peak levels for much longer periods
than in its natural condition. Further, in its nat-
ural condition, the lake’s salinity was almost
400 mg/l (chlorinity). Recent diverting of
saline springs from the lake has decreased its
salinity to about 200 mg/l. Daily storms fre-
quent the lake, the shores of which consist of
gravel, cobble, stones, mud, sand, and silt.
There were six localities at Lake Kinneret:

(9) Ginossar, Kinneret (IG 209-250). A nat-
ural shore of boulders and stones; snails
collected along the shore at a depth of
5-10 cm.

(10) 208, Kinneret, (IG 209-253). A natural
shore of boulders and stones; snails col-
lected at a depth of 5-10 cm.

(11) En Raggat, Kinneret (IG 199-245). A nat-
ural shore of boulders and stones; snails
collected along the shore at a depth of
5-10 cm.

(12) En Sheva, Kinneret (IG 201-252). A nat-
ural shore of boulders and stones, snails
collected along the shore at a depth of
3-10 cm.

(13) Ha’on, Kinneret (IG 208-237). A shore
consisting of muddy sand, with a few
submerged dead reeds; snails collected
from the reeds, at water level.

(14) Bet Gavriel, Kinneret (IG 205-234). A re-
cently constructed retaining wall con-

310 FALNIOWSKI ET AL.

sisted of large basalt boulders; snails col-
lected at a depth of 5-10 cm.

Melanopsis costata costata

(15) Gesher Benot Ya'aqov, upper Jordan
River (IG 209-269). Muddy substratum,
willows along the banks; snails collected
from vegetation, a few cm above the
water line.

(16) Gesher Lehavot, upper Jordan River (IG
209-283). Muddy substratum, willows
along the bank; snails collected from veg-
etation a few cm above the water line.

(17) Eastern Canal of the upper Jordan River,
(IG 208-278). Muddy substratum, wil-
lows along the banks; snails collected
from the muddy substratum.

(18) Bet Hillel (IG 207-289). A tributary of the
Jordan River, mainly with mud and cob-
ble along the banks; snails collected from
the cobble.

(19) Sede Nehemia (IG 208-288). A tributary
of the Jordan River; within a recreation
site; snails collected from the mud and
from submerged stems, branches and
tree trunks.

Melanopsis saulcyi

(5) Sede Eliyahu (IG 198-204). A small arti-
ficial pool, with a muddy substratum;
snails collected from dead vegetation
floating in the water.

(20) The same locality as 2.

(21) Sheluhot (IG 196-209). A cement chan-
nel in which water from several springs
flows with a rather strong current; snails
collected from the walls of the channel, at
a depth of 30 cm.

(22) Hamat Gader, 15 m from spring (IG
213-232). A small artificial pool into
which the Hamat Gader spring flows;
snails collected from boulders and stones
along the shores of the pool.

(23) The same locality as 7.

(24) The same locality as 1.

(25) Hamat Gader, spring (IG 213-232). A
pool in which a spring gushes; snails col-
lected off logs and various dead
branches, floating in the water.

(26) En Malkoah (IG 198-198), a small stag-
nant water body; on substratum and
plants.

Electrophoretic Techniques

The snails were transported alive to Poland
and then frozen and stored in a deep freezer,

at —80°С. Unsexed individuals for electro-
phoresis were dissected on ice, and their he-
pato-pancreas tissue was taken for homoge-
nization. The tissue was homogenized on ice,
in a glass homogenizer in 50-100 ul of ho-
mogenizing solution (100 ml distilled water, 10
mg NADP, 10 mg NAD, 100 ul B-mercap-
toethanol), depending on snail size. The ho-
mogenates were centrifuged at 11,000 rpm
for 10 min, then used immediately for cellu-
lose acetate electrophoresis following the pro-
tocol of Richardson et al. (1986). The follow-
ing running buffers were used, depending on
enzyme system: citrate-phosphate pH 6.4
(buffer A), phosphate pH 7.0 (buffer В), or tris-
maleate pH 7.8 (buffer C). The position of ori-
gin was cathode, the duration of run 1.5 h at
220 V. Staining protocols followed Richardson
et al. (1986). The cellogels were from MALTA,
Italy, all other chemicals from SIGMA, USA.
Enzyme names and EC numbers are after
Murphy et al. (1996).

Numerical Techniques

Genetic variability parameters and genetic
distances were computed with BIOSYS-1
(Swofford & Selander, 1981). One of the two
distances we had chosen was Nei unbiased
distance. This is the most commonly used ge-
netic distance, and so it is the best for com-
parisons with the literature. The other one was
Cavalli-Sforza and Edwards arc distance; this
is not affected by intrapopulation polymor-
phism and is the most appropriate when a
group of rather closely related populations is
concerned. GENEPOP (Raymond & Rousset
1995) was used to compute exact tests for
HWE (Guo & Thompson 1992; Rousset &
Raymond 1995), as well as f (equaling F, of
Wright) for each population and for each
locus. Following the notation and computa-
tional procedure introduced by Weir & Cock-
erham (1984) and Weir (1990), F-statistics
was computed over all populations with
Weir’s FSTAT (Weir, 1990; Goudet, 1995), the
confidence intervals estimated with jackknife
and bootstrapping techniques (Weir, 1990).
The same procedure was applied to each of
the taxa represented by more than one popu-
lation (i.e., except М. meiostoma). Mantel
tests, correspondence analysis, nonlinear
multidimensional scaling, minimum-spanning
tree, clustering, and cophenetic distances
were computed with NTSYSpc v.2.0 (Rohlf,
1998). Mantel tests were used for testing the
association between the genetic and geo-

ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 311

graphic distances between the populations,
as well as for evaluation of the phenograms
and testing the association between the origi-
nal and cophenetic distances. Additive neigh-
bor-joining trees (Saitou & Nei, 1987) and
Fitch-Margoliash trees (Edwards & Cavalli-
Sforza, 1964; Fitch & Margoliash, 1967; Swof-
ford & Olsen, 1990; Swofford et al., 1996)
were computed with NEIGHBOR and FITCH,
respectively, of the PHYLIP 3.6 package
(Felsenstein, 1990). From the same package,
the program SEQBOOT was applied to gen-
erate 200 bootstrap pseudosamples. For
each ofthem, Cavalli-Sforza and Edwards arc
distances were computed with GENDIST (the
source code of Felsenstein modified for arc
instead of chord version of that distance), and
the resulted file with 200 pseudosamples of
distance matrices processed with FITCH with
the global search option. Finally, the extended
majority rule consensus tree was computed
with CONSENSE of PHYLIP 3.6 package.

RESULTS

Twelve enzyme systems coded by 14 loci
gave well resolved and always interpretable
results: Aat (aspartate aminotransferase, EC
2.6.1.1., buffer B), Alp (alkaline phosphatase,
EC 3.1.3.1, buffer C), Est-1 (esterase, locus 1,
nonspecific, buffer A), Est-2 (esterase, locus
2, non-specific, buffer A), Gpi (glucose-6-
phosphate isomerase, EC 5.3.1.9, buffers A,
C), Hbdh (3-hydroxybutyrate dehydrogenase,
EC 1.1.1.30, buffer C), Idh-1 (isocitrate dehy-
drogenase, locus 1, EC 1.1.1.42, buffer C),
Idh-2 (isocitrate dehydrogenase, locus 2, EC
1.1.1.42, buffer C), Iddh (L-iditol dehydroge-
nase, EC 1.1.1.14, buffer C), Mdh (malate
dehydrogenase, EC 1.1.1.37, buffer C),
Mdhp [malate dehydrogenase (NADP*), EC
1.1.1.40, buffer C], Mpi (mannose-6-phos-
phate isomerase, EC 5.3.1.8, buffers B, C),
Pgdh (phosphogluconate dehydrogenase,
EC 1.1.1.44, buffers B, C), and Pgm (phos-
phoglucomutase, EC 5.4.2.2, buffer C). Nine
of the loci (64.3%) were polymorphic: Alp, Est-
1, Est-2, Gpi, Hbdh, Idh-1, Idh-2, Pgdh, and
Pgm. Allele frequencies in those loci are pre-
sented in Table 1.

Mean sample size was 28.983 (Table 2).
Mean number of alleles per locus averaged
1.45, varying from 1.10 in population 8 to 1.7
in population 12. Mean values for the taxa
were 1.43 for Melanopsis buccinoidea, 1.10
for M. meiostoma, 1.50 for M. costata jorda-

nica and M. costata costata, and 1.43 for M.
saulcyi. On average there was 36.5% of the
polymorphic loci per population, varying from
14.3% in population 8 to 57.1% in population
11 (Table 2). Mean values for the taxa were:
39.3% for M. buccinoidea, 14.3% for M.
meiostoma, 40.5% for M. costata jordanica,
32.9% for M. costata costata, and 36.6% for
M. saulcyi. Mean observed heterozygosity
(Table 2) varied from 0.041 in population 13 to
0.121 in population 10, mean for all the popu-
lations: 0.085. Mean expected heterozygosity
(Table 2) ranged from 0.045 in population 8 to
0.170 in population 22, mean for all the popu-
lations: 0.112. Departures from Hardy-Wein-
berg equilibrium (HWE), F-statistics, linkage
disequilibrium, estimates of gene flow, and
detailed analysis of the genetic structure of
the studied populations are presented in Fal-
niowski et al. (in press).

The multi-locus (by population) tests per-
formed with GENEPOP showed statistically
significant heterozygote deficits in most popu-
lations. The multi-population (by locus) tests
indicated a statistically significant deficiency
of heterozygotes at all loci, except Idh-1 and
Pgdh. Four populations (8, 17, 18, 25) were at
HWE or had all loci monomorphic. Heterozy-
gote excess was observed at Est-2 in popula-
tion 10, at Gpi in 21, and at Pgdh in 1. All the
other departures from HWE were heterozy-
gote deficits. The ratio of the number of popu-
lations at disequilibrium to the number of pop-
ulations with a given locus polymorphic varied
from 9.1% at Idh-1 to 59.1% at Est-1. Statisti-
cally significant values of f were found in 13
populations for Est-1, in 9 for Gpi, in 8 for
Hbdh, in 7 for Est-2, in 3 for Alp and Pgm, in 2
for Idh-2 and Pgdh, and in 1 for Idh-1 (Fal-
niowski et al., in press). The results of FSTAT
for all populations indicated HWE for Est-2,
Idh-1, and Pgdh: for all those loci f values
were not statistically significant and F approx-
imated 6. The highest values of f were found
for Idh-2, Alp and Est-1. For all the polymor-
phic loci, both Е and 6 differed significantly
from 0. The highest 6 was found for Pgm
(0.770), the lowest for Idh-2, Alp and Gpi. The
values and confidence intervals for particular
loci did not overlap. The highest values of F
were found for Pgm, Est-1 and Idh-2, the low-
est for Pgdh. F was less than 8 and f varied
among loci, but the confidence intervals did
not overlap (Falniowski et al., in press).

For each pair of populations, genetic dis-
tances were computed: Cavalli-Sforza and
Edwards arc distance and unbiased Nei dis-

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ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 313

TABLE 2. Genetic variability at 14 loci in all populations (standard errors in parentheses).

Mean heterozygosity

Mean Mean no. Percentage
sample size of alleles per of loci HdyWbg
Population per locus locus polymorphic* Direct count expected”.
Melanopsis buccinoidea
1. Enot Huga 31-910 1) 1.5102) 42.9 0.116 (+0.053) 0.133 (0.053)
2. En Hanatziv 8.0(+0.0) 1.45 (= 0:2) 28.6 0.089 (+0.062) 0.140 (+0.063)
3. En Rakkat spring 41.0 (+2.4) 1.4 (+0.1) 42.9 0.092 (+0.032) 0.131 (+0.049)
4. Dan 30:9(=0:9)= 1.501) 50.0 0.090 (+0.050) 0.090 (+0.039)
6. Banias 29.4 (+0.3) 1.4 (+0.2) SET 0.068 (+0.033) 0.117 (+0.048)
7. Nahal Tavor AAA 16) - 1.4204) 357 0.053 (+0.023) 0.102 (+0.046)
M. meiostoma
8. Kefar Haruv 19/0119) 1.1 (E05) 14.3 0.042 (+0.029) 0.045 (+0.032)
M. costata jordanica
9. Ginnossar 361111) 1.4(Е0:2) 35.7. 0.074 (+0.030) 0.106 (+0.043)
10. 208 SEO A) 1.202) 35% 0.121 (+0.069) 0.122 (+0.055)
11. En Rakkat Kinneret 38:41 (0.9) 6 (6-04) Hal 0.116 (+0.041) 0.165 (+0.055)
12. En Sheva Sigi E01) Wat. (02) 50.0 0.104 (+0.038) 0.126 (+0.042)
13. Ha’on 3116 (=0.2) 14 (02) 28.6 0.041 (+0.019) 0.101 (+0.049)
14. Bet Gavriel SIMEONE 1.502) 357 0.085 (+0.036) 0.101 (0.042)
M. costata costata
15. Gesher Benot Ya'agov 32.0{+0.0) 1.6 (+0.3) 28.6 0.080 (0.044) 0.093 (+0.044)
16. Gesher Lehavot SOON = 21:57(=02) 3577 0.055 (+0.034) 0.081 (+0.033)
17. Eastern Canal 30:0 (2.5) 1.4.0.2) 28.6 0.104 (+0.049) 0.099 (+0.048)
18. Bet Hillel S2AOMEOO) 717502) 35%. 0.112 (+0.055) 0.102 (+0.050)
19. Sede Nehemia SMED 21.512072) 35.7 0.106 (+0.055) 0.114 (+0.055)
M. saulcyi
5. Sede Eliahu San E NI (EE OA) 35.7 0.059 (=0.031) 0.117 (+0.048)
20. En Hanatziv 3177.(=02) 1-6, (0:2) 42.9 0.091 (=0.040) 0.122 (+0.050)
21. Sheluhot 32.0 (+0.0) 1.4 (+0.2) 35.7 0.063 (=0.040) 0.078 (+0.038)
22. Hamat Gader 15 mf.s. 21.0 (+1.3) 1.4 (+0.1) 42.9 0.106 (+0.042) 0.170 (+0.060)
23. Мапа! Tavor РИ 5 (EE 02) 42.9 0.109 (+0.049) 0.155 (+0.059)
24. En Huga 31:7 (0:2) 5 (02) 42.9 0.054 (+0.022) 0.117 (+0.047)
25. Hamat Gader spring 10:07=0:6) 7 1:24( 01) 21.4 0.113 (+0.074) 0.077 (+0.044)
26. En Malkoah 41.3 (=0.3) 1.4 (+0.2) 28.6 0.067 (+0.033) 0.111 (+0.050)

*A locus is considered polymorphic if more than one allele was detected; ** unbiased estimate (see Nei, 1978)

tance (Table 3). The mean value of Cavalli-
Sforza and Edwards arc distance equaled
0.240, and the Mantel test showed a signifi-
cant association between the matrices of this
distance and the geographic distance (r =
0.52133, t = 8.0717, p = 0.0002). The lowest
four values were found between populations
17 and 18 (0.054), 15 and 16 (0.072), 15 and
18 (0.081), 18 and 19 (0.081), and 15 and 19
(0.091). The highest values (exceeding 0.4)
were between population 2 and the following
populations: 18 (0.441), 19 (0.438), 6 (0.435),
17 (0.433), 14 (0.429), 4, 16, 15 and 13. The
mean value of Nei distance equaled 0.076,
and the Mantel test showed no significant as-
sociation between Nei distance and geo-
graphic distance (r = 0.47674, t = 7.6041, p =
0.3778). The lowest values were found be-
tween populations 24 and 25 (0.000); 15 and
16, 15 and 18, 17 and 18 (0.001); and 18 and

19 (0.002). The highest values, exceeding
0.2, were all found for population 2 and the fol-
lowing: 14 (0.232), 18 (0.229), 16 (0.228), 19
(0.224), 17 (0.219), 4, 15, 12, 13 and 6.

The means and ranges of Cavalli-Sforza
and Edwards arc distance and Nei unbiased
distance, within and between the studied
taxa, are listed in Table 4. In general, the val-
ues are low, and the intraspecific ranges are
not necessarily narrower than the interspecific
ones. The mean values of the distances be-
tween the two subspecies of M. costata are
similar to the mean intraspecific values for the
other species.

The interpopulation allozymic differentiation
was analyzed both phenetically and phyloge-
netically. The phenetic analysis included cor-
respondence analysis of the allele frequen-
cies and clustering based on the genetic
distances. The correspondence analysis re-

FALNIOWSKI ET AL.

314

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ALLOZYMIC TAXONOMY WITHIN MELANOPSIS

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316 FALNIOWSKI ET AL.

sulted in eigenvalues decreasing slowly: the
tenth dimension eigenvalue was 0.0087, ex-
plaining still as much as 2.14% of the total
variability, and the cumulative variability ex-
plained by the first ten dimensions explained
only 93.12% of the total variability. Thus, we
decided to present the grouping in only the
first two dimensions that explained together
51.81% of the total variability. The eigenval-
ues were 0.1583 and 0.0520, and the per-
centages of variability explained were 39.00
and 12.81, respectively. The plot of the col-
umn factors in the first two-dimension space
(Fig. 2) shows distinct groups representing M.
saulcyi (populations 5 and 20-26) and M.
costata (populations 9-19). Within the latter,
M. costata jordanica (populations 9-14) are
not mixed with M. costata costata (popula-
tions 15-19), although they all form a single,
compact group. Melanopsis meiostoma (pop-
ulation 8) lies close to M. saulcyi. On the other
hand, the populations of M. buccinoidea (1-4
and 6-7) are scattered across all the plot,
mixed with both M. saulcyi and M. costata.
Nearly the same picture was observed in all
the ten dimensions analyzed.

The UPGMA clustering was computed on

0 50 025 000

the genetic distances (Fig. 3). For each
UPGMA tree, the cophenetic distances were
calculated, and Mantel tests performed for
association between the matrices of original
versus phenetic distances, the so-called
cophenetic correlation. For Cavalli-Sforza
and Edwards arc distance r = 0.85446; for Nei
unbiased distance r = 0.84413. As the as-
sumptions of the Mantel test were violated
(the matrices were not independent), only the
subjective interpretation of the r-values was
allowed (Rohlf, 1998). The values above 0.8
and below 0.9 were considered a good fit.
Both the two phenograms (Fig. 3) show the
same picture, resembling also the one ob-
tained with correspondence analysis. Mela-
nopsis costata costata always forms a cluster,
and M. costata jordanica forms another clus-
ter; the two clusters are placed together. Also
M. saulcyiis grouped in one cluster, which in-
cludes M. meiostoma. On the other hand, the
populations that represent M. buccinoidea are
scattered in the M. saulcyi as well as M.
costata jordanica clusters. Similar results, al-
though less clear, and with the stress values
belonging to the Ча!” range, gave nonlinear
multidimensional scaling, with the minimum-

0.25 050 075 1.00

FIG. 2. Correspondence analysis, populations projected on 1st and 2nd dimension. Population numbers: see
text and Fig. 1. A— Melanopsis buccinoidea Olivier, 1801, B— M. meiostoma Heller & Sivan, 2001, C—M.
costata jordanica Roth, 1839, D—M. costata costata Olivier, 1804, E and F — M. saulcyi Bourguignat, 1853.

ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 317

0.00 0.03

0.06 0.09 0.12

FIG. 3. UPGMA clustering trees, based on Cavalli-Sforza and Edwards arc genetic distance (above) and Nei
unbiased genetic distance (below). Symbols of taxa: see Fig. 2.

spanning tree superimposed on the plot, so
we do not present it here.

Phylogenetic analysis included distance-
based additive trees, computed with two tech-
niques: Neighbor-joining and Fitch-Margo-
liash (Figs. 4, 5). Surprisingly, the tree
topologies, which means not the branch
lengths but the branching patterns, showed
by neighbor-joining are similar to those re-
vealed by clustering: the populations repre-
senting M. saulcyi form one cluster, whereas
the other includes all the populations of M.
costata. In the latter, one subspecies is not
mixed with the other. Melanopsis meiostoma
falls within the cluster of M. saulcyi, and M.

buccinoidea is scattered in both clusters. The
Fitch-Margoliash additive tree technique
based on Cavalli-Sforza and Edwards arc dis-
tance resulted in a tree (Fig. 5) with an aver-
age percent standard deviation of 9.5672
(6,339 trees analyzed). For unbiased Nei dis-
tance (Fig. 5), the value was much higher:
23.2768 (7,083 trees analyzed). Thus, the
most reliable phylogeny reconstruction is the
one based on Cavalli-Sforza and Edwards arc
distance. The phylogenetic trees are drawn in
the form of a phylogram (Maddison & Maddi-
son, 1992), with the branch lengths made pro-
portional to the amount of change, thus en-
abling one to observe not only the tree

318 FALNIOWSKI ET AL.

FIG. 4. Neighbor-joining additive trees, presented in the form of phylograms, based on Cavalli-Sforza and
Edwards arc genetic distance (above) and Nei unbiased genetic distance (below). Symbols of taxa: see

Big. 2.

topology, but also the amount of anagenetic
evolution. Again, there is a distinct group of
closely related populations that represents M.
costata jordanica and joins with the somewhat
less close M. costata costata group. Another
big cluster includes all the populations of M.
saulcyi, but also the single population that
represents M. meiostoma. Also here M. buc-
cinoidea populations are scattered in both
clusters, some of them terminating long
branches. It must be stressed that allthe trees
are unrooted.

The Fitch-Margoliash tree based on Ca-

valli-Sforza and Edwards arc distance was
evaluated using the bootstrap of the original
frequencies, followed by calculating new dis-
tances, and Fitch-Margoliash technique ap-
plied for each one of the new 200 distance
sets. The extended majority-rule consensus
tree was similar in topology to the original
one; it also showed the M. costata costata, M.
costata jordanica, and M. saulcyi population
groups as distinct from each, and M. bucci-
noidea populations scattered all over the tree.
However, the support of the branches was
low, about 30-40%.

ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 319

a 4
2 A
2 А
7 + 2324
zch
+

20
140
+
+
154
150
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17@
ERS о
135 O
AT
a A EN
14
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110
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ates
=0

FIG. 5. Fitch-Margoliash additive trees, presented in the form of phylograms, based on Cavalli-Sforza
and Edwards arc genetic distance (above) and Nei unbiased genetic distance (below). Symbols of taxa: see

810. 2:
DISCUSSION

The proportion of polymorphic loci in fresh-
water gastropods may approach 62%
(Woodruff et al., 1988); in the freshwater Vi-
viparus, it ranged from 0 to 33.3% (Falniowski
et al., 1996); in Bythinella, 11.1 to 55.6% (Fal-
niowski et al., 1998); from 3.1 to 53.1% in
Dalhousia; and from 0 to 16% in Fluvidona
(Ponder et al., 1996). Hence, the values found
in Melanopsis, although relatively high for
prosobranchs, do not exceed the range of
freshwater gastropods. The value of mean ex-
pected heterozygosity for all the mollusks

studied so far is 0.148 (Lazaridou-Dimitriadou
et al., 1994). In fresh- or brackishwater gas-
tropods, the values are usually lower: 0.002-
0.136 or 0.021-0.049 in Hydrobia (Haase,
1993; Davis et al., 1988, respectively) and
0.04-0.19 on average for various species of
Oncomelania (Woodruff et al., 1988). On the
other hand, in Bythinella mean expected het-
erozygosity ranged from 0.018 to 0.229 (Fal-
niowski et al., 1998). Thus, the mean ex-
pected gene diversity in Melanopsis was
among the highest noted in prosobranchs.
Despite the criticism concerning Nei ge-
netic distance (e.g., Swofford & Olsen, 1990;

320 FALNIOWSKI ET AL.

Davis, 1994; Falniowski et al., 1996; Swofford
et al., 1996), it is the most commonly used
statistic, thus using it is convenient because
one can easily make comparisons with the
rich literature. In this study, contrary to Cavalli-
Sforza and Edwards arc distance, unbiased
Nei distance was not correlated with geo-
graphic distance. This can be explained by
the fact that Nei distance is intended to reflect
the number of codon substitutions, thus muta-
tions, as the main source of variation, and it is
heavily affected by intrapopulation genotypic
polymorphism. Thus, it is certainly not appro-
priate where there is a low level of genetic
drift, and not necessarily realistic as a quan-
tificator of the time and/or amount of evolu-
tionary change after coalescence time. As
concerns mutations, the highest number
should be detectable among species, but the
studied taxa are not geographically distinct:
their distribution does not show any geo-
graphic pattern. On the other hand, the high
level of both genetic drift (shown by the re-
sults of Ohta’s D-statistics and estimates of
gene flow calculated from 0: Falniowski et al.
in press) and polymorphism in the studied
populations has undoubtedly biased the val-
ues of Nei distance.

The values of intraspecific Nei distances
found in Viviparus contectus (Falniowski et al.,
1996) ranged from 0.0014 to 0.0397, mean
0.0166. In his survey of over 7,000 compar-
isons of conspecific populations of plants and
animals, Thorpe (1983) revealed that only 2%
of the intraspecific Nei distance estimates ex-
ceeded 0.10. The highest intraspecific value
for sexually reproducing gastropod (0.63) was
reported in Cepaea nemoralis (Johnson et al.,
1984). Usually the values are lower, for exam-
ple, 0.001-0.131 in Helix (Lazaridou-Dimitri-
adou etal., 1994), 0.001 -0.051 in Trochus and
Tectus (Borsa & Benzie, 1993), 0.000-0.007
in Haliotis (Brown, 1993), 0.000-0.013 in Hy-
drobia (Davis et al., 1988), 0.051-0.191 in On-
comelania (Davis et al., 1994). Thus, the val-
ues found within the presumed species of
Melanopsis are typical intraspecific distances
of a rather weakly to quite considerably genet-
ically differentiated gastropod species.

The Nei distances between the presumed
species were also low. They even did not ex-
ceed in all cases the intraspecific values. In
M. buccinoidea, indeed, the intraspecific dis-
tances were often higher than almost any in-
terspecific distance. The average values were
about 0.08 between M. buccinoidea and all

the other species, about 0.05 between M.
meiostoma and M. saulcyi, about 0.1 between
M. meiostoma and M. costata, and about 0.12
between M. costata and M. saulcyi. The val-
ues of interspecific distances in the European
Viviparus (Falniowski et al., 1996) were be-
tween 0.2306 and 0.9888, mean 0.6871.
Thorpe (1983) reviewed 900 comparisons of
interspecific Nei distance for congeners in
plants and animals and reported a mean dis-
tance of about 0.40 (from 0.03 to more than
1); Davis (1994) listed a number of distances
for mollusks. For gastropods, it may be as
high as 5.383 in Haliotis (Brown, 1993), or
1.726 in Trochus (Borsa & Benzie, 1993), but
also may not exceed 0.199 for Cristilabrum
(Woodruff & Solem, 1990), or 0.077 for Stag-
nicola (Rudolph & Burch, 1989).

The above figures indicate that there is no
general rule as to how high the value of Nei
distance must be to prove species distinct-
ness. However, the values in Melanopsis are
rather of conspecific level. The range of in-
traspecific values, exceeding the ones be-
tween the taxa, does not confirm the species
distinctness of the taxa, either. We can con-
sider them either conspecific, thus not reject-
ing our null hypothesis, or consider them dis-
tinct species but the speciation of which
caused very little change in allozyme pattern.
A similar case was observed in Cerion (Gould
& Woodruff, 1986). Secondly, morphology
may not reflect speciation (Larson, 1989). A
third possibility that by chance the loci we
studied are not representative and do not re-
flect reproductive isolation cannot be ad-
dressed with our data. Thus, we restrict the
discussion to the question: are they finely al-
lozymatically differentiated yet distinct spe-
cies (or at least distinct biological, units of not
necessarily a species level) or else, are the
studied populations conspecific and all their
shell variation is environmentally induced, as
postulated by the null hypothesis?

As it can be seen in Figure 2, the concho-
logical differences between the studied taxa,
although rather well marked, are all ex-
pressed in such characters as the shell di-
mensions, proportions, and sculpture. There
is a vast literature describing high, ecologi-
cally determined variation of shell characters.
For instance, many prosobranchs that live in
stormy habitats, where the water movement is
strong, have ribbed shells (e.g., Wigham,
1975; Verduin, 1976; Palmer, 1985; Fal-
niowski, 1988). There were some fine mor-

ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 321

phometric differences in the radulae of the
studied taxa (Mazan et al., 2001). On the
other hand, one can hardly expect well-
marked differences in radular characters be-
tween so closely related taxa. Finally, the
anatomy of Melanopsis is simplified, the
snails lacking copulatory organs. We did not
find anatomical differences between the stud-
ied taxa. Hence, it is the molecular characters
that are decisive.

As stated above, the cohesion concept of
species assumes that the fundamental niches
of two species cannot be identical. From field
observations of one of us (J. H.), it seems that
the fundamental niches of the studied taxa
are not identical, although the differences are
rather fine. Thus, the demographic exchange-
ability of the taxa may be rejected. On the
other hand, the reasoning may be just re-
versed: slight differences in ecology may
create morphology differences between the
organisms that are basically the same. How-
ever, in such a case, the morphological and
ecological differences would not be accompa-
nied by molecular differences. Although there
are a few well-documented exceptions (Sza-
rowska et al., 1998), the neutral theory holds,
and the vast majority of allozymes seems se-
lectively neutral; thus, one cannot expect eco-
logical differentiation in allozyme pattern.

The results of either phenetic or phyloge-
netic analyses present a surprisingly congru-
ent pattern of distinct but close clusters repre-
senting M. costata costata and M. costata
jordanica, as opposed to another cluster
formed by all the populations of М. saulcyi.
Melanopsis meiostoma invariably adjoins the
cluster of M. saulcyi. On the other hand, M.
buccinoidea is scattered in both big clusters.
Correspondence analysis shows the same
picture; it also shows that the variability range
of M. buccinoidea is wider than the variability
of all the other taxa together. On the other
hand, the support indices provided by boot-
strap were low. This may have been due to
the slight differentiation of the studied popula-
tions, and thus the high sensitivity of the in-
ferred trees to any modification of the data. It
must also be stressed that the number of
characters, that is the number of alleles
found, is far too low for the bootstrap to give
reliable estimates (e.g., Swofford et al., 1996).

It is unusual that clustering and phyloge-
netic techniques of additive trees give such
congruent results. As a rule, when the dis-
tances are short and differentiation in general

is weak, each technique applied will give such
different results that it does not make sense to
compute a consensus tree, because too much
information will then be lost. In Melanopsis,
however, though the differences are small, the
different techniques all yield exactly the same
results. Thus, at least M. costata costata, M.
costata jordanica, and M. saulcyi, each mor-
phologically distinct, appear also allozymati-
cally as distinct biological units, though very
close to each other. The level of molecular dif-
ferences between them suggests that they
speciated not long ago, or even that the speci-
ation process is still not completed. The latter
possibility, as stated above, is a good expla-
nation of those species being not “good”.
Whatever the taxonomic decision, it is evident
that in the studied case, the allozymic differ-
entiation accompanies differentiation in shell
form, thus our null hypothesis of a single, con-
chologically variable species must be re-
jected. The two putative subspecies of M.
costata are indeed allozymatically, most close
to each other. As concerns M. meiostoma, its
species distinctness cannot be confirmed by
the present data: more specimens and more
loci must be studied to confirm this. In all trees,
М. meiostoma falls within M. saulcyi.

The most problematic case is M. bucci-
noidea. Its smooth shell is very characteristic,
but specimens are found in which the shell
has weak costae, suggesting hybridization
with other, ribbed species. Again, it can be in-
terpreted as a proof of the conspecifity of all
the Melanopsis populations studied. How-
ever, conspecifity would hardly result in such
clearly and univocally distinguishable allozy-
matic groups of populations as M. saulcyi and
M. costata. A better explanation of the ob-
served pattern of distribution of the popula-
tions of M. buccinoidea in trees and plots is
that M. buccinoidea is a distinct species, with
perhaps some level of hybridization with other
species, and some admixture of hybrids in the
studied populations (some level of genetic in-
trogression, which nevertheless cannot be
proved without genetic markers). However,
we emphasize again that M. buccinoidea is
notwithstanding far more variable than all the
other taxa together. Its wide variability, includ-
ing all the alleles found in the studied taxa,
indicates rather that it is not a phenotypic,
thus polyphyletic group consisting of smooth,
unribbed forms of both M. costata and M.
saulcyi.

Perhaps the most consistent hypothesis

322 FALNIOWSKI ET AL.

with our data is that M. buccinoidea, with its
high genetic variability, is the ancestral
species from which the other species in this
study eventually speciated: M. costata (with
two subspecies diverging later), and M.
saulcyi. Genetic variation in each of the pre-
sumed daughter species is much narrower
than in M. buccinoidea, which suggests a
founder effect (Barton, 1989) and the al-
lopatric speciation of peripheral isolates. On
the other hand, the founder effect must not
have been too severe, as the high levels of
polymorphism are still maintained in the
daughter species. Later, M. buccinoidea be-
came sympatric with its daughter species.
Speciation in Melanopsis, as in Hydrobia
(Davis, 1993), is perhaps a morphostatic one,
with little differentiation in morphology, as well
as in allozymes and ecological niches. Such
speciation seems not uncommon in gas-
tropods (Falniowski, 1987, 1992; Giusti &
Manganelli, 1992). On the other hand, speci-
ation might have been sympatric, with selec-
tion restricting the range of genotypic, thus
also phenotypic variation, which resulted in
the restricted ranges of environmental vari-
ability experienced by the taxa. As concerns
the potential hybridization, our study has not
been designed adequately to answer the
question. However, even if the hybridization
occurs, it has not resulted in merging the ge-
netic pools of the studied taxa. Thus, its pos-
sible occurrence does not reject the species
distinctness of M. buccinoidea.

ACKNOWLEDGMENTS

The molecular work was supported by a
grant from the Polish Committee of the Scien-
tific Research BW/IZ/UJ/98 to A. Falniowski,
and a travel grant from the Hebrew University
of Israel to J. Heller.

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Revised ms. accepted 5 March 2002

MALACOLOGIA, 2002, 44(2): 325-331

MORPHOLOGICAL AND CYTOCHEMICAL CHARACTERISTICS OF HEMOCYTES

OF MERETRIX LUSORIA (Bivalvia: Veneridae)

Sung-Woo Park, Kang-Soo Lee & Ee-Yung Chung

School of Marine Life Science, College of Ocean Science & Technology,
Kunsan National University, Kunsan 573-701, Korea; eychung@kunsan.ac.kr

ABSTRACT

To help understand the cellular defense mechanism of Meretrix lusoria hemocytes withdrawn
from the posterior adductor muscle were classified on the basis of their morphology, cytochem-
istry and phagocytic characteristics under a light microscope.

Two cell types, granulocytes and hyalinocytes, were found in the hemoctyes. Granulocytes,
13.1 + 2.6 um - 16.2 + 3.3 um in size, contain some large or small eosinophlic granules in their
cytoplasm. Granuloctyes were subclassified into small eosinophilic granulocytes and large
eosinophilic granulocytes based on the size of the granules. Granulocytes were positive to PAS,
acid phosphatase and phenoloxidase but negative to Sudan black B. Granulocytes showed pos-
itive phagocytic activity to zymosan particles.

Hyalinocytes, that is, agranulocytes that did not contain granules in the basophilic small cyto-
plasm, were 11.3 + 2.8 um in diameter and had an oval or irregular nucleus at the center of the
cell. The ratio of nucleus to the cytoplasm was very high. Hyalinocytes were weakly positive to
PAS but negative to Sudan black B and acid phosphatase. Phenoloxidase acivity was not
detected in hyalinocytes. Hyalinocytes phagocytized zymosan particles but the activity was very

low compared with that of granulocytes.

Key words: Meretrix lusoria, hemocytes, morphology, cytochemical characteristics.

INTRODUCTION

Meretrix lusoria (Röding, 1798) (Bivalvia:
Veneridae) is one of the commonly cultured
bivalve species in the western coastal areas
of Korea. It grows well in shallow water, which
may be brackish or full salinity seawater.
Juvenile clams taken from natural beds had
been transferred onto culturing beds
restricted with net fences to protect their
escape and to allow them to grow to commer-
cial size. Because mass mortality occurred
from July to August in 1973 and continued
during the following 2 or 3 years, their com-
mercial production ceased because natural
juvenile clams were not available. Yoo et al.
(1975) reported some possible causes of the
mortality, such as bacterial infections by Vibrio
angillarum, Pseudomoans ichyodermidis and
Acromobacter dermidis, inflow of agricultural
pesticides to the beds, and environmental
deterioration due to high water temperature
and high salinity, but the exact cause still
remains unclear. In recent years, it has not
been difficult to obtain clams larger than 5 cm
in shell size, which indicates a gradual
increase of the natural population in the nat-

325

ural beds. Fishermen have transplanted juve-
nile clams onto new culturing beds where it
has not yet been cultured, but have not gotten
good survival there.

Bivalve hemocytes play various important
roles in defense mechanisms, such as wound
healing and shell repair, nutrient digestion and
transportation, and excretion (Cheng, 1981).
Two types of bivalve hemocytes, granulocytes
and hyanilocytes, have been documented in
several species of bivalve on the basis of mor-
phological, cytochemical, and functional crite-
ria. Most studies on bivalve hemocytes have
been carried out using oysters or mussels
(Foley & Cheng, 1972; Bachere et al., 1991;
Carballal et al., 1997b; Lopez et al., 1977;
Nakayama et al., 1997), but there are a few
studies on the hemocytes and their functions
in Meretrix lusoria. On the basis of staining
and morphological characteristics, the hemo-
cytes of M. lusoria were classified into four
type of cells by Wen et al. (1994) and three
types of cells by Shih et al. (1996), but there
were no studies on the cytochemistry or
phagocytosis of Meretrix lusoria hemocytes.
The phagocytic ability of bivalve hemocytes is
one of the effective cellular defense mecha-

326 PARKETAL.

nisms of killing bacterial invaders, and granu-
locytes have much higher phagocytic ability
than hyalinocytes, which posses few or a few
granules containing bacteriolytic enzymes
(Foley & Cheng, 1975). Phenoloxidase was
detected in many bivalves hemocytes (Coles
& Pipe, 1994; Asokan et al., 1997; Carballal et
al,, 1997a), but its role in defense mecha-
nisms has not been revealed. Paillard et al.
(1994) suggested that this enzyme system
could take part in the defense mechanisms in
Venerupis philippinarum against Vibrio P1.

It is important to understand the roles of
Meretrix hemocytes in defense, and in evalu-
ating the clam’s adaptation to new beds as
well as avoiding damages by epizootics or
from environmental conditions. This study
provides data on the morphological and cyto-
chemical characteristics of Meretrix lusoria
hemocytes as seen under a light microscope.

MATERIALS AND METHODS

Specimens of Meretrix lusoria, 43.8 mm in
mean shell length and 36.1 mm in mean shell
width, were collected from a shellfish farm in
Simpo Bay, Chonbuk, Korea. The clams were
transferred to the laboratory in an ice bag and
washed several times with filtered sea water
(FSW) before using them for hemolymph col-
lection. After making a small hole in the shell
margin near the posterior adductor muscle
with an iron drill, hemolymph was directly
withdrawn from the posterior adductor muscle
with a 2.5-ml plastic syringe containing either
cold Modified Alsever’s Solution (MAS;
Bachere et al., 1991) as anti-aggregant, or
Baker’s formol calcium or MAS containing
neutral formalin at 10% (MAS10) as fixative.

Morphology

Hemolymph collected 1: 3 in MAS were
spread in thin films on glass slides, cover-
slipped, and immediately observed for the
morphology of live hemocytes under a phase
contrast microscope. Hemolymph collected in
MAS10 was transferred into a siliconized
glass tube and then fixed at room temperature
for 10 min. The hemolymph was centrifuged
at 250xg for 10 min, the supernatant
decanted, and the pellet was resuspended in
MAS. The cell suspension was smeared on
glass slides by centrifuging in a cell-collecting
apparatus (Hanil, Korea) at 80xg for 4 min at
room temperature. After staining the smears

with May-Grünwald Giemsa, the cell size of
hemocytes was measured under a light
microscope.

Cytochemistry

Lipids were demonstrated using Sudan
black B stain, as described by Barracco et al.
(1999). Hemocytes smears were air-dried and
fixed with 50% ethanol for 3 min and then
immersed in Sudan black B solution in 70%
ethanol for 30 min. After dipping several times
in 50% ethanol, the smears were counter-
stained with Giemsa solution.

Polysaccharides were detected by the peri-
odic acid-Schiff reaction (PAS). The air-dried
hemocyte smears were fixed with fixative
solution (6 acetone: 3 formalin: 1 acetic acid)
for 10 min and rinsed with running tap water
for 5 min. The fixed smears were treated with
1% periodic acid for 10 min, rinsed with dis-
tilled water for 15 min, and immersed in a
commercial Schiffs reagent (Sigma) for 30
min in a refrigerator. After rinsing for 5 min in
each of three consecutive sulfurous solution,
the smears were kept in running tap water for
30 min, counterstained with Mayer hema-
toxylin for 10 min, and mounted after washing
in distilled water for 10 min. Control smears
were treated twice with saliva (4 ml of saliva in
1 ml of 0.1 M phosphate buffered solution, pH
7.0) for 15 min at 37°C in a wet chamber to
confirm if the positive materials were glyco-
gen before being treated with periodic acid.

Acid phosphatase was revealed using
Gomori’s technique described by Barracco et
al. (1999) or a commercial kit for leucocytes
acid phosphatase (Sigma). Hemolymph was
collected 1:3 in Baker’s formol calcium con-
taining 2% NaCl, allowed to fix for 20 min at
room temperature and washed with distilled
water. Hemocyte smears were prepared by
the methods described above and air-dried.
The smears were incubated for 2 hr in the
Gomoris medium which had preheated over
night at 37°C, washed with distilled water,
stained with 1% ammonium sulfide for 2 min,
and finally counterstained with methylene
blue (Carballal et al., 1997a). Controls were
incubated in a medium without the substrate.

Phenoloxidase was demonstrated by the
method of Coles & Pipe (1994) with a minor
modification. Hemolymph was withdrawn
from the posterior adductor muscles of five
clams, diluted 1:1 in 10% Baker’s formol cal-
cium containing 2% NaCl, and allowed to fix
for 10 min at room temperature. After washing

CHARACTERISTICS OF HEMOCYTES OF MERETRIX LUSORIA 327

three times in 0.01 M phosphate buffered
solution, the hemocytes were smeared on
glass slides by the methods as described
above. The smears were air-dried and incu-
bated in a staining jar with 0.01 M phosphate
buffered solution containing 2 mg/ml of L-3, 4
dihydroxyphenyl-alanine (L-dopa) for 2 hr at
30°C. After being washed with distilled water,
the smears were stained with Giemsa solution
for 10 min at room temperature to differentiate
cell types. Control slides were incubated in
phosphate buffered solution without the sub-
strate.

Phagocytosis

Zymosan particles (Sigma) were sus-
pended in filtered sea water at a concentra-
tion of 1 ug/ml, heated for 30 min at 100°C,
washed and resuspended in MAS. One drop
of the hemocytes pooled from 3-4 individuals
was placed on glass slides and allowed to
adhere on the surface of the glass slides in a
wet chamber for 20 min at 25°C. The glass
slides were then washed with MAS to remove
unattached hemocytes and incubated with
zymosan suspension for 1 hr at 25°C. After
incubation uningested zymosan particles
were removed by several washings of the
glass slides with MAS. The air-dried glass
slides were stained with May-Grünwald
Giemsa and observed for phagocytic ability
under a light microscope.

RESULTS
Morphology of Hemocytes

Two types of hemocytes are easily identi-
fied in the hemolymph of M. lusoria on the
basis of the presence or absence of cytoplas-
mic granules under a phase contrast micro-
scope. Granulocytes possessing some large
or small granules in the cytoplasm were oval
or round large cells and were darkly seen due
to the presence of the granules (Fig. 1a). As
the large granules in granulocytes were round
in shape and were observed brightly, their
presence were easily revealed under a phase
contrast microscope. The small granules in
granulocytes were evenly distributed in the
cytoplasm. The granulocytes with the large
granules were larger in cell size than those
with the small granules. Hyalinocytes with
none or a few granules in the cytoplasm were
round in shape, smaller than granulocytes in

size, and most abundant in number. The cen-
ter of hyalinocytes, containing the nucleus
was seen more darkly than the cytoplasm
(Fig. 1b).

The morphological and cytochemical char-
acteristics, and phagocytic ability of Meretrix
hemocytes, are shown in Table 1 and Figure
2. Meretrix lusoria hemocytes were divided
into two types of cells based on the staining
affinity and the presence of the granules in the
cytoplasm after staining with May-Grünwald
Giemsa (Fig. 1). Granulocytes were round,
ovoid or irregular cells (13-16 um) and the
nucleus was commonly eccentric. They con-
tained numerous large or small eosinophilic
granules in the basophilic cytoplasm.
Granulocytes could be divided into large or
small eosinophilic granulocytes based on the
granule size. The large eosinophilic granulo-
cytes (Lgs) were larger than the small
eosinophilic granulocytes (Sgs) in size (Table
1). As the granules in Lgs were compactly
packed in the cytoplasm, it was difficult to see
the nucleus. The Sgs had basophilic affinity in
the peripheral cytoplasm and their granules
were more plentiful in the peripheral part of
the cells than around the nucleus.

Hyalinocytes possessing none or a few
granules in the cytoplasm were round cells
(11.3 + 2.8 um) and the nucleus was eccen-
tric. But, only a few hyalinocyes had cytoplas-
mic granules. The ratio of nucleus:cytoplasm
of hyalinocyes was higher than that of granu-
locytes, especially much higher in the cells
with a central nucleus than in the cells with
eccentric nucleus. The cytoplasm of hyalino-
cytes was more basophilic than that of the
granulocytes. A few large hyalinocyes had
large cytoplasmic vacuoles.

Cytochemistry and Phagocytosis
of Clam Hemocytes

Both granulocytes and hyalinocytes were
negative in Sudan black B. Only granulocytes
showed PAS-positive reaction in their cyto-
plasm. The PAS-positive materials were
evenly distributed in the cytoplasm but some
strongly PAS-positive granules were also
observed in the peripheral cytoplasm. After
digestion with saliva prior to reacting with
Schiffs reagent, the PAS-positive reaction
was not altered, suggesting the positive mate-
rials in the cytoplasm were different from
glycogen. Any difference in the two types of
granulocytes could not be found in PAS reac-
tion. Most of hyalinocytes were negative in the

328 PARKETAL.

FIG. 1. Photomicrographs of the hemocytes of Meretrix lusoria. a, live granulocytes (Lg and Sg) and
hyalinocyte (H) ovserved with a phase contrast microscope; b, large eosinophilic granulocytes (Lg), small
granulocytes (Sg) and hyalinocyte (H) stained with May-Grünwald Giemsa. Bars indicate 10 um.

TABLE 1. Morphology, cytohemistry and phagocytic ability of hemocytes of Meretrix lusoria

Cytochemistry
Shape

Cell* ==: ¿Sudan Acid Phenol- Phago-
Hemocytes size (um) Cell Nucleus blackB PAS phosphatase oxidase cytosis

Lgs 16.2+3.3 Ovoid, round, Ovoid, = + Fe + à

irregular round
Granulocytes Sgs 13.1 + 2.6 Ovoid, round Round = 4 e = tb
Halinocyte 11.3 + 2.8 Ovoid, round Round = 35 = = +

*Mean + SD of 60 cells from ten individuals.

Lgs, large eosinophilic granulocytes; Sgs, small eosinophilic granulocytes.

PAS reaction, but a few cells showed positive
reaction. In the PAS reaction small hyalino-
cytes were weakly positive but large hyalino-
cyes with a central round nucleus or cytoplas-
mic vaculoes were all negative (Fig. 2a).

Acid phosphatase was observed only in
granules of granulocytes as black deposits
(Fig. 2b). The number of positive cells was
much higher in Lgs than in Sgs.

Phenoloxidase activity was detected as
brown deposits in the peripheral cytoplasm of
Lgs (Fig. 2c). The positive reaction for phe-
noloxidase was seen as a black deposit in the
granules after staining with Giemsa solution.
The activity was not detected in both Sgs and
hyalinocytes.

When Meretrix hemocytes were incubated
with zymosan particles, both granulocytes
and hyalinocytes phagocytized zymosan par-
ticles (Fig. 2d). Granulocytes appeared to be
more phagocytic than hyalinocytes, which
ingested only a few zymosan particles. Just a
few hyalinocytes exhibited phagocytic activity
to zymosan particles.

DISCUSSION

Several authors have classified two types
of hemocytes in bivalves based on morpho-
logical, cytochemical and functional charac-
teristics. These authors (Foley & Cheng,
1974; Löpez et al., 1997; Hunakoshi, 2000)
reported two hemocytes type, granulocytes
and agranulocytes on the basis of staining
characteristics and morphological criteria.
Granulocytes were reclassified into eosino-
philic granulocytes and basophilic granulo-
cytes on the basis of the staining affinity ofthe
cytoplasmic granules. Eosinionophilic granu-
locytes were divided into large and small
eoninophilic guranulocytes depending on the
granular size. Bivalve granulocytes vary in
size or shape of the cytoplasmic granules in
different species (Foley & Cheng, 1972;
Carballal et al., 1997b). Agranulocytes were
classified into hyalinocytes and fibrocytes
based on the cell size and cytoplasmic char-
acteristics. Fibrocytes were described as
larger cells with slightly basophilic cytoplasm

CHARACTERISTICS OF HEMOCYTES OF MERETRIX LUSORIA 329

FIG. 2. Photomicrographs of the hemocytes of Meretrix lusoria, showing cytochemical reactions (a-c) and
phagocytosis (d). a, PAS reaction; b, acid phosphatase; c, phenoloxidase; d, phagocytic activity. Bars indi-
cate 10 um.

that had a few or no granules and in some
cases containing many vacuoles in the cyto-
plasm. Foley & Cheng (1972) also reported
the presence of fibrocytes in Mercenaria mer-
cenaria, but Cheng & Foley (1975) suggested
that fibrocytes were considered to degranu-
lated granulocytes with results of ultrastruc-
tural observation of the cells.

In our study, two types of hemocytes, thatis,
granulocytes and hyalinocytes, occurring in M.
lusoria are distinguishable by the presence or
absence of cytoplasmic granules either under
a phase contrast microscope or a light micro-
scope. Under the phase contrast microscope,
granulocytes fixed with Baker’s formol calcium
revealed visible cytoplasmic granules and
were easily differentiated from hyalinocytes,
which had a few or no cytoplasmic granules.
But when hemocytes were fixed with MAS
which have been commonly used as an anti-
coagulant for bivalve hemocytes, it was diffi-
cult to observe the cytoplamic granules in

granulocytes from preparations of live hemo-
cytes. Baker’s formol calcium appeared to be
an excellent fixative for bivalve hemocytes
even though it could not be used for phago-
cytic assay. To explain granulocytes matura-
tion in bivalve, Cheng (1981) proposed an
hypothesis that ganulocytes with small ba-
sophilic granule would be younger granulo-
cytes, and that the basophilic granules be-
come eosinophilic and larger as they mature.
But it is very interesting that no granulocytes
with basophilic granules could be found in the
present study as in observation of Wen et al.
(1994). Very few large cells with large cyto-
plasmic vaculoles and without any cytoplas-
mic granules were also observed in this study.

Lipid droplets and glycogen have been
found in the hemocytes of Mytilus galloprovin-
ciallis (Cajaraville & Pal, 1995). The presence
of polysaharides in the cytoplasm of hemo-
cytes were reported in Tridacna crocea
(Nakayama et al., 1997) and Perna perna

330 PARKETAL.

(Barracco et al., 1999). In P perna the PAS
reaction was stronger in granulocytes than in
hyalinocytes, and the PAS positive materials
were different from glycogen since its staining
affinity did not weaken with a diastase treat-
ment.

The occurrence of acid phosphatase has
been demonstrated in granulocytes of some
bivalve species, and the cytoplasmic granules
showing acid phosphatase acivity were con-
sidered as a form of lysosomes which partici-
pate in phagocytosis (Carballal et al.,1997a;
Nakayama et al., 1997). Löpez et al. (1997)
reported that acid phosphatase is only found
on the granules of granulocytes and can be
used to distinguish granulocytes from hyalino-
cytes.

Phenoloxidase was detected in hemocytes
of some bivalves (Coles & Pipe, 1994;
Carballal et al., 1997a; Deaton et al., 1999).
This enzyme is known to take part in defense
reactions of arthropod (Söderhäll, 1992), but
the roles of this enzyme in defense mecha-
nisms of bivalve are still unclear. The phe-
noloxidase activity was suggested to partici-
pate in defense reactions against the
etiological agent, Vibrio tapetis, of brown ring
disease in Venerupis philippinarum (Paillard
et al., 1994). Coles & Pipe (1994) reported
that fixation of mussel hemocytes with 10%
Baker’s formol calcium produces the best
preservation ofthe phenoloxidase activity and
the activity is seen in granules as a fine gray
to black deposit.

Both types of hemocytes of M. lusoria did
not contain lipids, but showed positive reac-
tions with Schiff's reagent. Acid phosphatase
activity was detected only in granulocytes,
especially Lgs in the present study.
Phenoloxidase activity in hemocytes of M.
lusoria was first found in the present study.
The activity was detected only in Legs. The
positive activity showing brown color was
seen as black deposits in granules after coun-
terstaining with Giemsa solution. The enzyme
activity was detected in the peripheral cyto-
plasm and a strong reaction was observed in
large cells, whereas small cells showed neg-
ative reaction.

Granulocytes were always more phagocytic
than hyalinocytes in many bivalves (Foley &
Cheng, 1975; Tripp, 1992, Carballal et al.,
1997c; Barracco et al., 1999). Both cell types
in M. lusoria, especially in large eosinophilic
granulocytes could actively pagocytose the
zymosan particles. As most phagocytic hemo-
cytes in М. lusoria were large eosinophilic

granulocytes containing some enzymes such
as acid phosphatase and phenoloxidase, it is
suggested that those ezmymes might be in-
volved in cellular defense mechanisms. And
changes in the number and the enzyme activ-
ities of hemocytes might be used for indicators
to evaluate health condition in cultured or
transplanted bivalves.

ACKNOWLEDGEMENTS

The authors are grateful to Dr. John B.
Burch of the University of Michigan for helpful
comments on the manuscript. This research
was supported in part by the fund from
Fisheries Science Institute, Kunsan National
University.

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Revised ms accepted 2 April 2002

MALACOLOGIA, 2002, 44(2): 333-347

POPULATION GENETICS, MICRO-PHYLOGEOGRAPHY, ECOLOGY,
AND SUSCEPTIBILITY TO SCHISTOSOME INFECTION OF CHINESE
ONCOMELANIA HUPENSIS HUPENSIS (GASTROPODA: RISSOOIDEA:
POMATIOPSIDAE) IN THE MIAO RIVER SYSTEM

Chao-Hui Shi', Thomas Wilke**, George M. Davis’, Ming-Yi Xia? & Chi-Ping Qiu®

ABSTRACT

Chinese Oncomelania h. hupensis from the flood plains of the Yangtze River have ribbed
shells. However, populations living above the effects of annual floods usually are smooth-
shelled. Previous allozyme studies of smooth-shelled populations not affected by flooding, and
ribbed-shelled populations affected by flooding from the Miao River, Hubei Province, showed
that they all belong to O. h. hupensis (Davis et al., 1999a). As the allozyme data were of limited
use for assessing Oncomelania-population genetics, we re-examined the above populations to
answer the following questions using mitochondrial COI sequence data. Will DNA sequences
provide higher resolution for the analysis of population structure than allozyme data? Are
there significant genetic differences among ribbed- and smooth-shelled populations? Do they
differ in their susceptibility to infection with Schistosoma japonicum?

Sequences from 59 individuals revealed four groups of haplotypes. Smooth- and ribbed-
shelled individuals clustered together in two of the groups. The greatest sequence
divergence between a smooth-shelled and ribbed-shelled population was 0.020, indicating
that all populations fall within the range of variation expected for О. h. hupensis. Overall,
the highest genetic diversity was found among downstream ribbed-shelled populations.

The analysis of molecular variance (AMOVA) showed the following distribution of total
variance: 69% (P < 0.0001) within populations, 9% (Р < 0.0025) among populations within
the ribbed- and smooth-shelled groups, and 22% (P < 0.0569) between the ribbed- and
smooth-shelled populations. Mismatch distributions indicated that downstream populations
are aggregates of snails from different populations. Downstream populations also showed
a higher infectivity rate and a higher susceptibility to infection with Schistosoma japonicum.
This is probably due to the importation and mixture of snail and parasite strains in flooded
areas increasing the probability that schistosomes encounter genetically suitable snails, and/
or the possibility of multiple infections by different parasite strains. The low infection rate in
upstream populations is probably due to their relative isolation where there is equilibrium with
low frequencies of infection.

Key words: Oncomelania hupensis, Schistosoma japonicum, China, Red Queen, mtDNA,
population genetics, AMOVA, infectivity.

INTRODUCTION

The dioecious snail genus Oncomelania is
widespread throughout China, with a distribu-
tion extending to Sulawesi, the Philippines, and
Japan. There are three subspecies on the

mainland of China (Davis et al., 1995, 1998).
Oncomelania hupensis hupensis is found
throughout the lower Yangtze River drainage
below the Three Gorges, with an extension into
Guangxi Province. Oncomelania hupensis
robertsoni lives at high elevation on the plateaus

‘Chinese Academy of Medical Sciences, Institute of Laboratory Animal Science, 5 Pan Jia Yuan Nanli, Beijing

100021, China

“The George Washington University Medical Center, Department of Microbiology and Tropical Medicine, 2300 Eye

Street, N.W., Washington, DC 20037, U.S.A.

“Institute of Parasitic Diseases, Chinese National Center of Systematic Medical Malacology, 2007 Rui Jin Er Lu,

Shanghai 200025, China

“To whom correspondence should be addressed; mtmtxw @ gwumc.edu

334 SHI ET AL.

and mountains of Yunnan and Sichuan in
far southwestern China. Oncomelania
hupensis tangi occurs on the seaside of
Fujian Province, removed from the Yangtze
River drainage by a range of tall mountains.
All three subspecies transmit the human
blood fluke, Schistosoma japonicum, argu-
ably the most serious parasitic disease
problem in China now that malaria is highly
controlled.

Historically there has been the question of
how to classify smooth-shelled Oncomelania
living in the same geographic range of typical
ribbed-shelled ©. h. hupensis. On the basis of
allozymes, Davis et al. (1995) classified popu-
lations with both shell types from the lower
Yangtze River drainage as ©. h. hupensis. The
latter study raised the question whether the
character of shell sculpture with its two states
(ribbed, smooth) was influenced by elevation of
habitat above the reach of the annual flood of
the Yangtze River and its tributaries. An experi-
ment to test this hypothesis was provided by
nature in the Miao River of Hubei Province. This
is a short, small river only about 22 km long
with flood waters reaching some 14 km up-
stream. Allozyme data were obtained from four
upstream populations (smooth-shelled) not af-
fected by flooding, and three downstream
populations (ribbed-shelled) affected by flood-
ing each year. These populations were sepa-
rated from each other by discrete stretches of
river with no evidence of snails. The results
showed that there was no genetic basis for dis-
tinguishing between the two groups; they both
belong to О. h. hupensis (Davis et al., 1999a).
However, the results from both allozyme-based
studies (Davis et al., 1995, 1999a) raised three
important issues relative to population genetics
of Oncomelania hupensis. (1) Polymorphism
and heterozygosity are too low to enable use of
marker alleles to track patterns of dispersal or
gene flow. This is apparently the situation
throughout rissooidean snails (reviewed in
Davis et al., 1999a). (2) With increasing num-
ber of populations, it becomes increasingly dif-
ficult to determine homology of presumptive
alleles of polymorphic loci. (3) The few poly-
morphic loci are frequently not in Hardy-
Weinberg equilibrium.

In the present paper, we revisit the Miao
River to re-examine the same populations
studied by Davis et al. (1999a) to answer the
following questions using sequence data of
the mitochondrial cytochrome c oxidase subunit |
(COI) gene:

(1) Has the mitochondrial genome of
Oncomelania hupensis accumulated enough
substitutions to enable differentiation among
populations that are separated by small
geographic distances, such as those found
between populations along the short Miao
River?

(2) Will COI sequence data provide higher
resolution for the analysis of population
structure than allozyme data? Can sequence
data help to explain the departure from Hardy-
Weinberg equilibrium?

(3) Are there significant differences among
ribbed- and smooth-shelled populations relative
to genetic diversity and population structure?

(4) Do ribbed-shelled and smooth-shelled
populations differ in their susceptibility to
infection with Schistosoma japonicum?

METHODS
Locality Data

The distribution of the seven populations
studied is shown in Figure 1 (adapted from
Davis et al. 1999a). Detailed locality data are
given in Davis et al. (1999a). Snails were
collected by C. H. Shi and G. M. Davis in the fall
of 1998 from the same localities (A-G)
previously collected in 1994 for the allozyme
study (Davis et al., 1999a). Sites A, B, and C
are annually flooded by the Yangtze River.
There is a dam between sites Band C. The
sluices of the dam are closed part ofthe year
to retain water at site C in order to drown
snails. Sites D-G are never affected by
flooding. All 28 specimens from sites A-C
had ribbed shells, all 31 specimens from
sites D-G had smooth shells.

DNA Isolation and Sequencing

The methods of Spolsky et al. (1996) and Davis
et al. (1998) were used for isolating DNA from in-
dividual snails.

The primers to amplify a fragment of the COI
gene are LCO1490 and HCO2198 (Folmer et al.
1994). The quality of PCR product was deter-
mined by electrophoresis through a 1% agarose
gel. Amplified DNA products were purified using
Wizard PCR preps (Promega, Madison, Wis-
consin). COI sequences were determined us-
ing the LI-COR (Lincoln, Nebraska) Long
ReadiR 4000 DNA sequencer and the Thermo
Sequenase fluorescent labeled primer cycle se-

GENETIC STRUCTURE AND INFECTIVITY OF ONCOMELANIAH. HUPENSIS 335
A IE a: = pe re = 5 er A à
B
10)
A Wuhan City
=
Е ai Sha Shi City
| Г № eo‘ |
ge
190
| с NE We
e = DES
E er
| U A
N A N > 2
р S
——_—— KT) PA nl 10 20
ee a m

FIG. 1. Localities of Oncomelania h. hupensis populations studied. Sites A-C are affected by the
annual flooding of the Yangtze River. The black bar between sites B and C indicates a dam.

quencing kit (Amersham Pharmacia Biotech,
Piscataway, New Jersey) according to the
manufacturer's protocols.

A total of 59 individuals with an average of
8.4 individuals per population were se-
quenced. Individual codes, DNA sample
numbers and GenBank accession numbers
are provided in the Appendix.

Data Analysis

COI sequences, which do not have base in-
sertions or deletions in Oncomelania, were un-
ambiguously assembled by eye using the
software program ESEE 3.0s (Cabot &
Beckenbach, 1989). The first 2-10 base pairs
behind the 3’ end of each primer are often diffi-
cult to read. We therefore uniformly excluded
the first and last ten bp of each sequence, leav-
ing an alignment of 638 bp without any inser-
tions or deletions.

Mismatch distributions and parameters of
DNA sequence polymorphism including haplo-
type diversity (d,,; Nei, 1987), number of poly-
morphic sites ($), nucleotide-sequence
diversity (п; Nei, 1987), its stochastic variance
(Vst ), and nucleotide-sequence divergence
(Dxy; Nei, 1987), were calculated using the
software program DnaSP 3.0 (Rozas &

Rozas, 1999). The coefficient of correlation
(r) of haplotype diversity and geographic dis-
tance was calculated using the function
CORREL of Microsoft Excel for Windows 95,
version 7.0a.

The genetic structure in our data set was in-
vestigated by analyses of molecular variance
(AMOVA) implemented in the computer pack-
age ARLEQUIN ver. 2.001 (Schneider et al.,
2000). The AMOVA approach is based on gene
(haplotype) frequencies, but takes into account
the number of mutations between haplotypes
(Excoffier et al., 1992). Statistical significance
of variance components was assessed with
20,000 random permutations.

In order to test whether the COI data set as a
whole exhibits phylogenetic signal, the relative
apparent synapomorphy analysis (RASA 3.04Т;
Lyons-Weiler, 2001) was used. The software
compares the observed rate of increase in
pairwise cladistic similarity per unit pairwise phe-
netic similarity (В observed) to a null slope (В
null), where cladistic support and phenetic simi-
larities are randomly distributed among pairs of
taxa (Lyons-Weiler et al., 1996). The data set
showed a significant phylogenetic signal of t.,., =
44.71 (P < 0.05; B observed = 18.71; B null =
6.16) and was therefore considered to be suitable
for further analyses.

336 SHI ET AL.

Prior to the phylogenetic analyses we used
the computer program Modeltest 3.0 (Posada
& Crandall, 1998) in order to test which model
of DNA substitution best fits our data. It per-
forms hierarchical likelihood ratio tests
among 56 possible models. The model se-
lected was HKY85 (Hasegawa et al., 1985)
with a Ti/Tv ratio of 3.942; base frequencies
of А—=0-2270, С = 0.1823; © = 02208) and T=
0.3699; as well as а gamma distribution
shape parameter of 0.0856.

A maximum likelihood tree was then con-
structed from all haplotypes using the computer
program Tree-Puzzle 4.0.2 (Strimmer &
Haeseler, 1999). The analysis was performed
with the parameter of sequence substitution
suggested by Modeltest. In addition, the num-
ber of puzzling steps was set to 100,000 and
the parameter estimates to “exact”. As evolu-
tionary relationships above and below the spe-
cies level are different in nature (Posada &
Crandall, 2001), we also analysed the phyloge-
netic relationships of our seven O. h. hupensis
populations with a method that was specifically
designed to infer intraspecific relationships —
the statistical parsimony (SP) network ap-
proach implemented in the computer program
TCS v. 1.06 (Clement et al., 2000).

Infectivity Study

From each location between 59 and 464
snails (1253 total) were subjected to shed-
ding conditions to determine the degree of
infection (Table 5). Cercariae obtained from
the shedding (populations A-C) were used
to infect mice. Then, snails from upstream
populations (D, F, G) and one downstream
population (C) were infected with miracidia

from the infected mice.

In the infectivity experiment, uninfected
snails were challenged with five miracidia
per snail, and maintained in 10 cm petri
dishes in culture using methods of Davis
(1967). The prepatent period was deter-
mined, using standard shedding tech-
niques, along with the number of cercariae
shed. Mortality was checked for on a daily
basis.

RESULTS

Data on haplotype diversity, number of poly-
morphic sites, nucleotide-sequence diversity
and its variance for each population are pro-
vided in Table 1. A total of 27 different
haplotypes was found. The index of haplo-
type diversity (h,) ranged from a high of 0.978
at site A to a low of 0.356 at site G. There is a
clear trend of decreasing haplotype diversity
from downstream to upstream populations
(r = -0.71). Nucleotide-sequence diversity
was > 0.015 at sites A and B; < 0.0085 at
sites C and G.

A matrix of pairwise comparisons of nucleotide-
sequence divergence (Dxy) between populations
(Table 2) shows that all values are rather similar
with the lowest Dxy of 0.0099 being observed
between the two smooth-shelled populations E
and G and the highest Dxy of 0.0200 between the
ribbed-shell population B and the smooth-shelled
population D.

Among the 28 ribbed-shell specimens (sites
A-C), the total number of haplotypes found was
17 with a haplotype diversity of d, = 0.825; the
total number of polymorphic sites was 41. In
contrast, the total number of polymorphic sites

TABLE 1. Sequence polymorphism in the Miao River populations of Oncomelania h. hupensis.

ribbed smooth total

A B C D E Е G (A-C) (D-G) (A-G)
No. individuals 10 9 5 10 10 28 31 59
sequences
No. haplotypes 9 7 3 5 3 4 2 17 10 27
Haplotype 0.978 0.917 0.417 0.933 0.700 0.644 0.356 0.825 0.692 0.878
diversity (ho)
No. polymorphic 23 30 22 21 16 ТЯ 15 41 26 53

sites (S)
Nucleotide-
sequence
diversity (п)
Variance of x
(Vst(»)

0.0153 0.0159 0.0077 0.0110 0.0144 0.0130 0.0084 0.0131 0.0132 0.0161

0.0301 0.0321 0.0085 0.0156 0.0252 0.0221 0.0099 0.0232 0.0234 0.0344

GENETIC STRUCTURE AND INFECTIVITY OF ONCOMELANIA H. HUPENSIS

337

TABLE 2. Nucleotide-sequence divergence (Dxy) between the populations of Oncomelania h. hupensis
studied. The population from GuiChi (Yangtze River, Anhui Province) was used for comparison.

GuiChi A B
GuiChi -

A 0.0183 =
B 0.0192 0.0152 -
C 0.0182 0.0123 0.0117
D 0.0185 0.0180 0.0200
E 0.0173 0.0158 0.0174
Е 0.0170 0.0154 0.0170
G 0.0159 0.0138 0.0150

C D E 5 G
0.0180 =
0.0141 0.0148 5
0.0152 0.0150 0.0125 à
0.0127 0.0140 0.0099 0.0106 =

TABLE 3. Summary of analysis of molecular variance (AMOVA) for smooth and ribbed-shelled
populations of Oncomelania h. hupensis. Levels of significance are based on 20,000 permutations.

Source of variation df Sum of squares

Among groups 1 3.928
(ribbed vs. smooth)

Among populations 5 3.563
within groups

Within populations 52 17.967

among the 31 smooth-shelled individuals
(sites D-G) was only 26 and the 10 ob-
served haplotypes had a haplotype diversity
of d,= 0.692. However, the nucleotide se-
quence diversity within the ribbed-shell
specimens (x = 0.0131) was almost identi-
cal with the diversity within the smooth-
shelled individuals (x = 0.0132).

The analysis of molecular variance (AMOVA)
showed that most of the variation is within
populations (69%), and the respective fixation
index, although relatively low at 0.114, is highly
significant. The variation among populations
within the ribbed- and smooth-shelled groups
accounts for only 9% of the total variation. How-

Variance components

Absolute % Fixation index E

0.10826 2] 73 Rep = 0:27 < 0.0569
0.04453 8.94 Fsr = 0.307 < 0.0025
0.34551 69.34 Fsc = 0.114 < 0.0001

ever, the fixation index is relatively high and sig-
nificant at 0.307. The differences between the
ribbed- and smooth-shelled populations (22%
of the total variation) resulted in a fixation index
of 0.217. However, the significance limit of 0.050
was slightly exceeded with 0.057 (Table 3).

A pairwise test of differences in population
structure showed that eight out of twelve com-
parisons between smooth- and ribbed shelled
populations were significant, whereas only two
out of twelve pairwise comparisons within the
three ribbed-shelled and the four smooth
shelled populations were significant (popu-
lations D vs. F and D vs. G) (Table 4).

A maximum likelihood tree shows the phy-

TABLE 4. Pairwise test on significant differences between populations of Oncomelania h. hupensis
from the Miao River based on molecular variance (AMOVA). Significance level = 0.05.

A B
A
Ribbed B -
© 2 E
D À 3
Smooth E ь z
5 + +
G + +

C D E F G
+

+ -

+ + -

+ + - -

338 SHI ET AL.

D4C

C7

B1

Ye B6

A6, 8
В5; 8, 9 |
C1, 3, 4, 5, 6, 8, 9

FIG. 2. Phylogenetic tree for seven populations of Oncomelania В. hupensis from the Мао River
based on maximum likelihood analysis. The circle sizes are proportional to the observed number of
individuals with each haplotype. Missing haplotypes are indicated by small circled. Smooth-shelled
individuals are represented by white circles, ribbed-shelled individuals by black circles. Nodes that
have support values between 50 and 80% are marked with a *. All other branches have support
values of > 80%. Main haplotype groups and lineages are marked with roman numerals. Alternative
arrangements of haplotypes, as inferred with statistical parsimony (SP), are indicated by dashed
arrows. Note that in the SP network clades I/II, Ш, and IV could not be connected in a parsimonious
fashion as the three groups exceeded the 95% connection limit of 10 mutational differences.

logenetic relationships of the COl-
haplotypes (Fig. 2). The tree consists of four
main haplotype groups and lineages (I-IV).
Lineage Ill comprises a single individual
(B3), which is separated by twelve nucle-
otide differences (x = 0.0204, = 2.0%) from its

nearest neighbor, A7. Groups 1, Il, and IV con-
tain 19, 20, and 19 individuals, respectively.
The greatest nucleotide-sequence diver-
gence between individuals can be found be-
tween a ribbed-shelled individual (C7) anda
smooth-shelled individual (D4) with x = 0.0454.

GENETIC STRUCTURE AND INFECTIVITY OF ONCOMELANIAH. HUPENSIS 339

) Miao River

Ä Er
& 8
| Population G E D С В А
| №. specimens 10 10 5 6 9 9 10 |
|
| №. haplotypes 2 4 3 5 3 7 9 |

smooth-shelled

no flooding

ribbed-shelled

flooding

FIG. 3. Information on haplotype diversity for the Oncomelania h. hupensis populations studied. Each
site is represented by a circle divided into areas proportional to the number of individuals that belong
to haplotype groups I-IV (see Fig. 2). The black bar between sites Band C indicates a dam.

The greatest nucleotide-sequence divergence
between two ribbed-shelled individuals is
0.0392, and between smooth-shelled indi-
viduals, 0.0376. The smallest difference be-
tween a ribbed- and smooth-shelled individual
is 0.0031.

Two haplotype groups each contain a
common haplotype that is shared by more
than ten individuals. In group II, one haplo-
type is shared by 17 smooth-shelled indi-
viduals (54.8% of all smooth-shelled
individuals); in group |, one haplotype is
shared by 12 ribbed-shelled individuals
(42.9% of all ribbed-shelled individuals). We
consider these to be “core” haplotypes.
Smooth-shelled upstream individuals com-
prise only haplotypes of groups II and IV;
ribbed-shelled downstream individuals
comprise haplotypes of all four groups. Addi-
tional information on haplotype diversity is
given in Figure 3. Sites A and B near the
mouth of the Miao River have the greatest
haplotype diversity. Group IV individuals are
to be found at every site; group II individuals
are to be found at all sites except C and
group | individuals are restricted to the sites
that are affected by flooding. The relatively
low haplotype diversity in upstream popula-
tions is mostly due to the lack of haplotype
group l-specimens in the smooth-shelled
populations (Fig. 3).

The phylogeny obtained with the statistical
parsimony network approach was almost

identical with the maximum likelihood phy-
logeny shown in Figure 2, except for minor
changes in the arrangement of three termi-
nal haplotypes in clade IV (indicated by
dashed arrows in Fig. 2) and that clades ИП,
Ш, and IV could not be connected in a parsi-
monious fashion as the three groups ex-
ceeded the 95% connection limit of ten
mutational differences.

The different haplotype diversities in smooth
vs. ribbed-shell populations are readily re-
flected in the mismatch distributions (Fig. 4).
With the exception of populations C and D
(both are from sites close to the flooding
boundary), there is a trend of decreasing the
number of classes of mutational differences
from downstream to upstream populations. At
site A we find a wide spectrum of classes rang-
ing from 0 to 19 mutational differences (only
with the 5, 11, and 15 bp-classes missing). A
similar spectrum can be found at site B, but
now with a gap between 6 and 14 mutational
differences. The number of classes is further
decreased in site C-specimens, temporarily in-
creases in site D-specimens and then
degreases to two (0 and 15 bp) in the speci-
mens from site G However, mismatch distribu-
tions are sensitive to population size.
Therefore, the results for sites D (only six indi-
viduals studied) and for site E (only five indi-
viduals studied) have to be treated with caution.
Interestingly, the mismatch distribution for the
ten ribbed-shell specimens from site A is very

340

Frequency

FIG. 4. Mismatch distributions of pairwise number of mutational differences between individuals of popu-
lations A to С, between all ribbed-shelled individuals (“ribbed”), between all smooth-shelled individuals

SHI ET AL.

0.7

Inn. ah. tall,

10 12 14 16 18 20 22 24 26

0.6

0.51

04!

0.3

0.2

0.1

0.0

6 8 10 12 14 16 18 20 22 24 26

2

B

thn. In.

8 10 12 14 16 18 20 22 24 26

0.7

0.6

G

0.5

0.4
0.3

0.2

0.1

0.0

072 4 678

10 12 14 16 18 20 22 24 26

с |

0.7

ribbed

0.6}

0.57

04!

0 2 4 6 8 10 12 14 16 18 20 22 24 26

0.3

0.2

0.1
0.0

0 2 4 6 8

10 12 14 16 18 20 22 24 26

D

0.7 —

smooth

0.6}

0.5

blu hu

10 12 14 16 18 20 22 24 26

0.4}

0.3

0.2

0.1

0.0
OZ

4

6 8 10 12 14 16 18 20 22 24 26

0.7

0.6}

ribbed + smooth |

0.5

0.4

0.3!

0274 18

6 8 10 12 14 16 18 20 22 24 26

0.2 +

0.1 =

0
01284568

10 12 14 16 18 20 22 24 26

Pairwise number of mutational differences

(“smooth”), and between all individuals studied (“ribbed + smooth’).

GENETIC STRUCTURE AND INFECTIVITY OF ONCOMELANIA Н. HUPENSIS 341

TABLE 5. Natural infection rates of field-collected Oncomelania h. hupensis from the Miao River.

Total

A B C A-C
No. snails 464 381 408
No. positive 25 49 0 74
% positive 5.38 12.86 0 5.91

1,253

Total

D E F G D-G

212 59 295 278 844
0 0 0 0 0
0 0 0 0 0

TABLE 6. Prepatent period giving the average and standard deviation of the number of days from
infection to shedding of field-collected individual Oncomelania h. hupensis snails.

Average
@ D F G D-G
Prepatent time (days) 81.7 +5.4 92.8 + 1.5 86 82.8 + 3.9 87.6 +5.7
No. cercariae shed 44.2 + 18.7 17.8 + 12.8 21 19.0 + 11.5 18.7 + 10.6
similar to both, the mismatch distribution of the DISCUSSION

combined 28 ribbed-shell specimens and the
mismatch distribution of all 59 specimens
(ribbed and smooth) in our study (Fig. 4).
Infectivity data area given in Tables 5-7.
Only downstream snails were infected, and
these with a prevalence of 5.9% (Table 5).
Infections in these snails were used to test
the prepatent period for cercarial emer-
gence. There was no significant difference
between upstream and downstream groups
with regard to average prepatent period (P >
0.05), that is, 82-83 days. However, there
was a significant difference between groups
regarding numbers of cercariae shed per
snail; the ribbed-shelled downstream snails
shed more (P < 0.05). Also, there was a sig-
nificant difference between groups in per-
centage of snails that could be
experimentally infected (P < 0.01). Site C
snails had 60.9% successful infections
compared to an average 8.1% for the up-
stream D, F, and G site snails. There was no
significant difference in mortality (P > 0.05).

Genetic Diversity

There are a least three possible explana-
tions for the observed cluster patterns of mi-
tochondrial haplotypes in Figure 2: (1)
methodological problems, (2) the presence
of a cryptic species complex, (3) high in-
traspecific variation.

In order to test whether our results are
based on sequencing and/or alignment er-
rors, we reanalyzed the sequence for the
distinct haplotype B3, and the same result
was obtained. Also, the sequences were
free of ambiguities and base insertions/de-
letions, and a translation into protein se-
quences did not result in premature stop
codons. We therefore can likely exclude the
possibility of having nuclear pseudocopies
of the COI gene, though there is a slight
chance of a recent jump to the nucleus.
Other possibilities would be heteroplasmy
or duplications within the mitochondrial ge-

TABLE 7. Prevalence of laboratory infections and death rates of field-collected Oncomelania h.

hupensis in the Miao River.

C
No. snails 48 42
No. dead 2
No. infected 28
Infection (%) 60.9 9.8

Mortality (%) 4.7 2.4

Total

F G D-G
42 42 126
1 0 2
2 4 10
4.9 9.5 8.1
2.4 0 1.6

342 SHI ET AL.

nome (Zhang & Hewitt, 1996).

However, we can safely exclude the possi-
bility of having a cryptic species complex. In
the 59 specimens studied, we have found a
total of 27 haplotypes with a nucleotide-se-
quence diversity of m = 0.0161, a haplotype
diversity of h, = 0.878, and total number of
53 polymorphic sites (Table 1). These num-
bers are well within the known sequence
variation for Oncomelania h. hupensis. Wilke
et al. (2000) studied ten populations with 80
specimens of O. h. hupensis from the lower
Yangtze River. They found a total of 45 COI-
haplotypes with an average haplotype diver-
sity of h, = 0.972, a nucleotide-sequence
diversity of x = 0.0192, and a total of 85 poly-
morphic sites. The highest nucleotide-se-
quence divergence between those ten
populations was Dxy = 0.0207. This is even
slightly higher than the highest divergence
between populations found in the present
study (Dxy = 0.0200). Moreover, Wilke et al.
(2000) showed that the nucleotide-se-
quence divergence between the 80 specimens
of O. h. hupensis and a representative of O. h.
robertsoni (the subspecies that occurs in
southwestern China) ranged from 0.129—
0.132. Using the same specimen of O. h.
robertsoni (GenBank accession AF253074),
the nucleotide-sequence divergence with the 59
specimens of O. h. hupensis studied here is
very similar with Dxy = 0.132. Thus, the diver-
gence between the two subspecies O. h.
hupensis and O. h. robertsoni is more than
eight times higher than the nucleotide se-
quence diversity within specimens of the sub-
species O. h. hupensis studied here.

Phylogenetic trees of O. h. hupensis often
show numerous cluster and lineages that are
separated by two to about ten substitutions.
The respective missing haplotypes are
possibly due to one or more of the following
factors:

(1) Inadequate sampling: Considering the
haplotype diversity in the present study (e.g.,
the fact that we have found nine haplotypes in
ten individuals from site A), it is clear that we
have not reached the end of variation.

(2) Genetic history: Considering the close
coevolved relationships between the snail host
and the schistosome parasite (reviewed in
Davis 1980, 1992), a series of extinction-
events, bottle-necks and strong selection for
certain haplotypes must have occurred. Also, it
is possible that there are multiple substitution
events at the same time, thus some of the

missing haplotypes may never have existed.

(3) Current demographic processes: Human
activities have a profound effect on the
population structure of Oncomelania hupensis.
The massive use of molluscicides and
drowning as measures to control snail
populations and therefore schistosomiasis,
often with only few surviving snails, create
major bottle necks. These measures likely play
a role in the extinction of haplotypes.

The cluster pattern in the phylogenetic tree
(Fig. 2) somewhat resembles the СО! tree
for populations of the rissooidean snail
species Peringia ulvae from the northern
Atlantic (Wilke & Davis, 2000). The brackish-
water P. ulvae also has a similar genetic
diversity (in terms of nucleotide and
haplotype diversities). These patterns are
believed to be the result of a high dispersal
capacity combined with past fragmentation/
secondary contact events caused by
Pleistocene glaciations.

By comparison, while Oncomelania В.
hupensis probably was not directly affected by
Pleistocene glaciations, it also has a very high
dispersal capacity. Oncomelania h. hupensis
has negative rheotropism, that is, snails will,
along a river, gradually move upstream. Snails
can float upside down at the surface of the
water to feed; there is net transport
downstream. Flooding lifts and transports them
downstream, especially during the annual
monsoon floods (reviewed in Davis et al.,
1999b). Snails attached to insects, birds, and
hoofs of water buffalos can be transported
from one locality to another.

Allozyme vs. COI Sequence Data

Allozyme data from the Miao River popula-
tions based on Wright's D were robust enough
to show distinctive differences among popula-
tions, to show that population A was unique due
to the extent of divergence from all other popu-
lations, and to reveal unique alleles at different
sites (Davis et al., 1999a). However, the poly-
morphic loci were not in Hardy-Weinberg equi-
librium. Low heterozygosity and paucity of
unique alleles reduced their usefulness for ana-
lyzing genetic variation within populations, gene
flow, or population structure. Our hypothesis to
account for these facts is that the annual
floods of the Yangtze River transport large
numbers of snails from one locality to the next.
The annual deposition of snails in any area cre-
ates an aggregate that is not a true population,

GENETIC STRUCTURE AND INFECTIVITY OF ONCOMELANIA H. HUPENSIS 343

that is, there is no chance to reach Hardy-
Weinberg equilibrium. Such aggregates have
been called “genetically unstable populations”
(Davis et al., 1999a, b). The cluster patterns of
COl-haplotypes in the phylogenetic tree (Fig.
2), combined with the observed sequence
polymorphism (Table 1) and the inferred mis-
match distribution (Fig. 4) support this hypoth-
esis. The downstream sites А, В, С, and, to a
lesser extent, D are clearly not true populations
but assemblages of diverse genetically distinct
units. These results are concordant with those
in Davis et al. (1999a) and are most likely
due to a combination of the Wahlund (1928)
effect (especially in aggregates A, B, and C)
and intense inbreeding in upstream popula-
tions heavily impacted by man.

In this regard it is interesting that, though
22% of the total AMOVA variation is attributed to
differences between the ribbed- and smooth-
shelled populations, the fixation index (F...) is
only 0.217 and the differences between the two
groups are not quite significant (Table 3).
Though, part of the problem may be the low
sample size, the analysis of population struc-
ture indicates that the differences between
ribbed- and smooth-shelled populations are not
discrete but rather gradually changing from
downstream to upstream populations. Thus, all
pairwise comparisons between the three most-
downstream populations A, B, and C (ribbed-
shelled) and the two most-upstream
populations F and G (smooth-shelled) are sig-
nificant, whereas differences involving popula-
tions C-E may or may not be significant no
matter whether they are ribbed- or smooth-
shelled (Table 4).

The Question of Ribbing

Ribbing in Oncomelania h. hupensis is only
found in snails living along the flood plains of
the Yangtze River and associated tributaries,
where severe flooding is an annual event
driven by the seasonal spring monsoons
(Davis et al., 1995, 1999a, b). In slightly higher
elevation and beyond the effects of flooding, the
shells are smooth. Wilke et al. (2000) studied
the phylogeography of smooth- and ribbed-
shelled populations in eastern China. They
found that smooth and ribbed conditions are
not discrete, but the two extremes of a continu-
ously varying character as specimens from one
site in Anhui Province, which was removed
from the Yangtze River flood plains and prob-
ably only slightly affected by annual flooding,

had only weak ribs. The data also sug-
gested that the smooth-shelled condition
results from relief from flooding, not eleva-
tion per se. Davis (1979) demonstrated that
the primitive conditions historically among
rissooidean snails in general, and Asian
pomatiopsid snails in particular, are shells
both small in size and smooth (Attwood &
Johnston, 2001). Sculpture and increased
size are derived. Oncomelania snails from
Yunnan and Sichuan provinces (Subspecies
O. hupensis robertsoni) have these
plesiomorphic character-states. It is hypoth-
esized that the sculpture and large size of On-
comelania h. hupensis in eastern China are
derived, and apparently evolved under selective
pressure of annual torrential floods to survive
on the flood plains of the lower Yangtze River
(Davis, 1979).

According to Davis & Ruff (1973), ribbing is
controlled by a single gene with ribbing domi-
nant to smooth and with inheritance typically
Mendelian. Ribbing, while a dominant gene,
has multiple alleles controlling size and thick-
ness of the ribs. If there is a founder event with
a ribbed colony established beyond the affects
of flooding, ribbing will persist for a time. We do
not yet know how long it takes for the evolution-
ary process of strong selection against ribbing
to result in a smooth shell, or the converse. As
these snails live and reproduce over a life span
of up to five years, it may take decades for all
traces of ribbing to be lost. In a controlled ex-
periment, Urabe (2000) found for the freshwa-
ter snail Semisulcospira reiniana (family
Pleuroceridae) that phenotypic variation of shell
sculpture (ribbed vs. smooth) is controlled by
environmental factors although it has a genetic
basis and that, after changing the ecological
setting, shell sculpture frequencies already
started to change in the F1 generation.

Rapid morphological changes of shell sculp-
ture based on developmental flexibility or true
adaptation have been reported for various gas-
tropod taxa. The significance of shell sculpture
has been attributed to defense against preda-
tors (e.g., Vermeij, 1976; West & Cohen, 1996;
Quensen & Woodruff, 1997), to thermoregula-
tion (Vermeij, 1973), to prevention from sinking
into the substratum (Hutchinson, 1993), to bur-
rowing habits (Palmer, 1980), or to an in-
creased drag in environments affected by
water currents (Linsley, 1978; Denny, 1988;
Urabe, 2000).

Considering the distinct ecology of On-
comelania hupensis (reviewed in Davis et al.,

344 SHIETAL.

1999b), the above functions of ribbing can
likely be excludes:

(1) Increased drag in aquatic environments.
Not applicable: adult Oncomelania snails are
not aquatic; they do not live in the Yangtze
River. Only the young, newly hatched snails are
aquatic; at about two months of age, the snails
move to an ecotone, a land-water interface.
When swept away by the floods, they can float
upside down at the water surface for a certain
time. They cannot withstand continual sub-
mersion; they will drown.

(2) Defense against predation. Adult On-
comelania snails do not have any major preda-
tors.

(3) Burrowing habits; prevention from sinking
into the substratum. Oncomelania prefers silt-
rich soil supporting an abundance of soil dia-
toms used for food. We have not found
significant differences in ecotone soil quality
(composition, coarseness) from sites with
smooth- and ribbed-shelled individuals. More-
over, we have not observed any differences in
burrowing habits between the two forms; at
temperatures below 10°C both, smooth- and
ribbed-shelled specimens, dig down into soil
and aestivate.

(4) Thermoregulation. We did not study ther-
moregulation in Oncomelania. However, given
the short distance between sites with smooth-
and ribbed-shelled populations, there are no
major differences in micro-climate (soil and air
temperature, humidity, radiation).

In our study, all specimens from sites af-
fected by floods (sites A-C) had ribbed shells,
and all specimens from sites that were isolated
from the ‘flooding (sites D-G) had smooth
shells. We therefore attribute ribbing as a re-
sponse to flooding, as stated by Lou et al.
(1982), and Davis et al. (1999a, b). The two
hypotheses that account for this are (1) ribbing
increases surface area that is important for
floatation; (2) ribbing increases shell strength,
an important factor in surviving the annual
flooding and being swept around in flood wa-
ters. The possibly rapid nature of shell sculp-
ture changes may be responsible for the lack
of intermediate shell types in the Miao River,
which were previously reported from few other
locations in China (see above).

The Question of Susceptibility to Schistosome
Infection

No natural infections were found from site C
to the top of the river. Historically, these popu-

lations were infected (records of the local pub-
lic health offices). In addition to the man-made
flooding of site C (see Methods), there is exten-
sive use of molluscicides to kill snails along the
edges of the river thus controlling schistosome
transmission. Large infection prevalences were
found near the mouth of the river at sites A and
B. This is the hardest area to control against
schistosomes because of annual importation
of adult infected snails swept in by the floods of
the Yangtze River.

It is well Known to Chinese provincial anti-
schistosomiasis control centers that snail
populations with high infection rates, that is, >
1%, are to be found on the flood plains of the
Yangtze River or its tributaries. These popula-
tions have a high genetic diversity. In regionally
isolated areas where populations have a lower
genetic diversity, the infection rates are much
lower, too (i.e., < 0.1%). Accordingly, the infec-
tion rates in popuiations A and B at the mouth
of the Miao River are very high. The site C
population was used in the infection experi-
ments because it is a ribbed-shelled population
affected by flooding but snails were not natu-
rally infected, an essential aspect of the infec-
tivity study. We attribute the lack of natural
infections partly to the snail control measure
(routine drowning) that kills off the adult infected
snails. In the experiment, group C snails were
significantly more susceptible to infection than
were the upstream smooth-shelled populations
(Table 7). However, this difference has more to
do with the genetic isolation of upstream popu-
lations than with the smooth or ribbed nature of
the shells. The argument for this assertion fol-
lows.

There is a coevolved interaction between the
parasite Schistosoma japonicum and its snail
hosts Oncomelania hupensis (reviewed in
Davis, 1980, 1992) that is specific, at this point
in time, at the regional level and frequently at
the population level. The literature on this speci-
ficity is quite large and involves regional differ-
ences in snail infectivity by different geographic
strains of S. japonicum (DeWitt, 1954; Yuan et
al., 1984).

The hypothesis to explain this coevolution is
that infected snails evolve defense mecha-
nisms that act to counter the parasite. The
parasite then counters with an evolved re-
sponse to overcome the host's defenses. This
invokes the Red Queen hypothesis of Van
Valen (1973).

Genetic studies of freshwater snails
(Dybdahl & Lively, 1998) and bumble bees

GENETIC STRUCTURE AND INFECTIVITY OF ONCOMELANIA H. HUPENSIS 345

(Baer & Schmid-Hempel, 1999) showed that
infections with parasites were significantly
lower in populations with higher genetic diver-
sity and that rare genotypes were also less sus-
ceptible to infection. The theory is that
parasites that can successfully infect the most
common host genotype will be favored by natu-
ral selection (Lively, 2001).

The situation in Oncomelania appears to be
different as genetically diverse snail popula-
tions are more susceptible to infection. This
could be due to the high immigration and emi-
gration rates of snails and parasites in the
floodplains of the Yangtze River. The low
temporal stability of host-parasite interac-
tions probably makes it impossible for the
Red Queen to get engaged. Our hypothesis
for the high infection rates in downstream
Miao River population follows from this as-
sumption. Flood-affected populations bring
together snails and the schistosomes they
carry coming from diverse locations
(Wahlund effect). In these heterogeneous
parasites and snails there is a higher prob-
ability that parasite eggs hatching from a lo-
cal definitive host (water buffalo, pig, man)
will encounter genetically suitable snails that
do not have a sufficient defense mechanism
against this particular strain of parasite. More-
over, it is conceivable that snail populations can
effectively fight off only one or few parasite
strains at atime. In cases of multiple infections
by different parasite strains, which likely occur
in the flood-affected areas, the defense system
of the snail host might be inadequate.

In isolated snail populations with low rates of
immigration and emigration, the laws of the
Red Queen probably apply. The outcome may
be equilibrium with low frequencies of infection
(observed for a number of isolated populations
of Oncomelania hupensis with low heterogene-
ity; see also Lively, 2001), fixation in the snails
to resist all attacks of the parasite (seen in
some populations in Taiwan), or snail popula-
tion extinction (of course, not directly detect-
able; but observed for a population of the
rissooidean snail genus Hydrobia caused by
bird trematodes; Davis et al., 1989).

The comparatively low experimental infection
rates obtained in the smooth-shelled Miao
River localities is probably due to the relative
isolation of those populations where there is
equilibrium with low frequencies of infection.
The Miao River is a pleasant stream with great
accessibility for humans and cattle (definitive
hosts). Therefore differential accessibility to

the water for people and animals might not
account for differences in infection rates be-
tween upstream and downstream locations.

ACKNOWLEDGEMENTS

The work was supported by N.I.H. TMRC
grant Al 39461. We are indebted to Drs. X.
Yang and X. Xu of the Hubei Schistosomiasis
Control Institute, Wuhan for helping in the col-
lection of the Miao River snails and S.
japonicum. We thank Professor Feng Zheng,
Principal Investigator of the Shanghai TMRC
grant, for use of facilities in Shanghai and
support of the field studies. We are grateful
to Professor David Blair, James Cook Uni-
versity, Townsville, Australia, for reviewing a
draft of this paper and offering sound advice.

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Revised Ms. accepted 1 March 2002

APPENDIX

TABLE 8. Individual codes, DNA sample numbers and GenBank accession numbers for the ingroup

individuals of Oncomelania h. hupensis studied.

Individualcode DNA sample # GenBank
accession #

Al MG 301 AF306572
A2 MG 302 AF306573
A3 MG 303 AF306574
A4 MG 304 AF306575
A5 MG 305 AF306576
A6 MG 306 AF306577
A7 MG 307 AF306578
A8 MG 308 AF306579
A9 MG 309 AF306580
A10 MG 310 AF306581
B1 MG 341 AF306613
B2 MG 342 AF306614
B3 MG 343 AF306615
B4 MG 344 AF306616
B5 MG 345 AF306617
B6 MG 346 AF306618
B7 MG 347 AF306619
B8 MG 349 AF306620
B9 MG 350 AF306621
C1 MG 259 AF306622
C2 MG 260 AF306623
C3 MG 286 AF306624
C4 MG 287 AF306625
C5 MG 288 AF306626
C6 MG 289 AF306627
C7 MG 290 AF306628
C8 MG 291 AF306629
C9 MG 292 AF306630
D1 MG 335 AF306582
D2 MG 336 AF306583

Individual code DNA sample # GenBank
accession #
D3 MG 337 AF306584
D4 MG 338 AF306585
D5 MG 339 AF306586
D6 MG 340 AF306587
Ei MG 351 AF306598
E2 MG 352 AF306599
ЕЗ MG 353 AF306600
E4 MG 354 AF306601
E5 MG 355 AF306602
FA MG 321 AF306588
F2 MG 322 AF306589
ES MG 323 AF306590
F4 MG 324 AF306591
F5 MG 325 AF306592
F6 MG 326 AF306593
F7 MG 327 AF306594
F8 MG 328 AF306595
F9 MG 329 AF306596
F10 MG 330 AF306597
G1 MG 311 AF306603
G2 MG 312 AF306604
G3 MG 313 AF306605
G4 MG 314 AF306606
G5 MG 315 AF306607
G6 MG 316 AF306608
G7 MG 317 AF306609
G8 MG 318 AF306610
G9 MG 319 AF306611
G10 MG 320 AF306612

4
mA:
¡1

MALACOLOGIA, 2002, 44(2): 349-352

RESEARCH NOTES

SYSTEMATIC IMPLICATIONS OF EXTREME LOSS OR REDUCTION OF
MITOCHONDRIAL LSU rRNA HELICAL-LOOP STRUCTURES IN GASTROPODS

Charles Lydeard', Wallace E. Holznagel', Rei Ueshima? & Atsushi Kurabayashi?

Recent systematic studies on one of the
largest recognized classes of animals —the
Gastropoda —have resulted in the disman-
tling of the traditional classification scheme,
which had been firmly entrenched since the
early 1900s (Haszprunar, 1985, 1988; Bieler,
1992; Ponder & Lindberg, 1997). This revision
was precipitated by the recognition of a new
clade currently referred to as the subclass
Heterobranchia, which includes the pulmo-
nates, opisthobranchs, and several groups of
mostly small marine snails formerly placed in
the now obsolete subclass, Prosobranchia.
Heterobranchs presumably separated from
its sister group the Caenogastropoda, which
includes mostly marine, but some terrestrial
and freshwater taxa from several major, po-
tentially paraphyletic, taxonomic groups (e.g.,
Architaenioglossa, Neotaenioglossa, Pteno-
glossa and Neogastropoda), about 350-400
million years ago (Bieler, 1992; Ponder &
Lindberg, 1997). The monophyly of Hetero-
branchia is strongly supported by many
anatomical features, such as loss of a true
ctenidium, simple esophagus, lack of odon-
tophoral cartilages, and pigmented mantle
organ (see Ponder & Lindberg, 1997, for de-
tailed review). Given that these radical phylo-
genetic hypotheses eliminated such tradi-
tional groups as the Prosobranchia and
Mesogastropoda, which were recognized for
nearly a century (but still erroneously used by
GenBank and many recent textbooks), it is
imperative that they be further tested using in-
dependent data sets. It is well documented
that ribosomal RNA sequences fold into com-
plex secondary structures based largely on in-
tramolecular base pairing. The vast majority
of the rRNA secondary structure models have

been determined with comparative sequence
analyses (Woese et al., 1980; Noller et al.,
1981; Gutell et al., 1994). Based on a detailed
comparative analysis of complete mitochon-
drial (mt) large subunit (LSU) rRNA secondary
structures among 10 molluscan taxa, Lydeard
et al. (2000), discovered three putative struc-
turally based synapomorphies (Lydeard et al.,
2000; 98-99, fig. 7) uniting the stylom-
matophoran gastropods, a group of terrestrial
heterobranchs. Here, we show based on a
more comprehensive analysis of 58 complete
or near-complete mt LSU rDNA sequences
and subsequently derived secondary struc-
tures that the loss or reduction of the three
substantial helical-loop structures described
previously for stylommatophorans are molec-
ular synapomorphies of the Heterobranchia.
We sequenced 50 complete or near-com-
plete mt LSU rDNA sequences and obtained
four gastropod, two bivalves, one cephalo-
pod, and one chiton sequence from GenBank.
Sequencing primers and methods are de-
scribed in detail in Lydeard et al. (2000) and
Kurabayashi & Ueshima (2000). Most of the
taxa (n = 32) that were examined were mem-
bers of Cerithioidea for an examination of the
phylogenetic relationships within the super-
family and are dealt with in more detail else-
where (Lydeard et al., 2002). We generated
secondary structure models for a comparative
analysis based on secondary structure mod-
els described and determined elsewhere for
ten representative mollusks (Lydeard et al.,
2000; available online at http://www.rna.
icmb.utexas.edu), and focused our efforts on
the three relevant regions hypothesized to
be synapomorphies of stylommatophorans
based on the previous study. Exemplar struc-

‘Biodiversity & Systematics, Department of Biological Sciences, University of Alabama, Вох 870345, Tuscaloosa, Alabama

35487 USA; clydeard@ bama.ua.edu

Department of Biological Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; rueshima O biol.s.u-

tokyo.ac.up

Sinstitute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan

350

tures representing the range of variation ob-
served within mollusks for the three relevant
regions of mt LSU rRNA are shown in Figure
1. Figure 2 shows the taxa that were exam-
ined in a phylogenetic framework using a phy-
logenetic hypothesis of the Gastropoda based
on a Cladistic analysis of 117 morphological
characters (Ponder & Lindberg, 1997). To il-
lustrate the evolution of the three secondary
structural characters, we have chosen to map
the characters on the independently derived
morphological-based tree (Fig. 2). The ab-
sence or reduction of three substantial helical-
loop structures was discovered for all hetero-
branch taxa. Within Domain Il, a helical-loop
structure is absent in all heterobranchs, with
the exception of the basal heterobranch taxa
Valvata hokkaidoensis and Psilaxis radiatus.
All other gastropods and outgroup taxa, with
the exception of Nerita atramentosa and
Mytilus edulis, possess a helical-loop struc-
ture that ranges from 42-62 bp in length. Val-
vata, Psilaxis, Nerita, and Mytilus have a re-
duced (15, 15, 19, 16 bp, respectively)
helical-loop structure relative to other mol-
lusks. The most parsimonious hypothesis ex-
plaining the secondary structure variation de-

a. b.

Psilaxis radiatus Littorina saxatilis

LYDEARD ET AL.

picted in Domain II requires four steps. With
the reduction of the helical-loop structure
evolving independently on three separate oc-
casions in Mytilus, Nerita, and the Hetero-
branchia, followed by the complete loss of the
structure in derived heterobranch lineages
(Figs. 1, 2). The second structural-based
character involves the entire Domain Ill. All
outgroup taxa, basal gastropod lineages, and
caenogastropods possess a helical-loop
structure ranging from 30-79 bp; however, all
heterobranchs lack the helical-loop structure
(Figs. 1, 2). The third structural-based molec-
ular character is the presence or absence of a
bulge-stem-loop structure that is 41-43 bp,
whereas all heterobranchs lack the bulge-
stem-loop structure and only have a 20-30 bp
structure. The indel in Domain V has been re-
ported previously in a study of euthyneuran
relationships (Thollesson, 1999). The most
parsimonious hypothesis explaining the varia-
tion found in Domains Ш and Vis that the loss
of the helical-loop structures is the derived
condition uniting all heterobranchs. Interest-
ingly, the extreme reduction or loss of the
three helical-loop structures reveals, in part,
why heterobranchs have among the shortest

С.

Albinaria coerulea

h Ayu - С UUUUAAG
UUUU UUAU P ) СААСАА! А S
DIU ied AS A TUE
AAAAA AAUA - CGAAAUUC
A СС и AGUUUUU U AG
i E AG y À U
A ‚С С U
à Ale Al] U
ñ Uy UL U U=A A
mn ^ У U Ve is
ar hs NA As yA А,
< у А с UA
. А AV “AceG С C : A
Domain И USA ES Amu Domain II A
= UN AT GC ven U—A
>. NU U—A A=U
N U A Ü uV =Ac
D ain II U Sa UN oi Geu ЗА = UY U—A
omaın < ze E Gel y NA ie A À A=Uc © “0
AS с Sie AUA NG AG Realty Uy
s Ba Ar AU NA À = |)
5 U - А J Ù — À
5 | A U Am UV
= J U=A
| Domain Ш Domain ИГ ------- missing
Domain II ------- missing
CAUAA uuuuu® Sg Ne
CA GAAG SAUNA - 4 . . U
LET «I AE la los IT IS GUGUUGAA ¿A
GGU GAGCCUUCA GUGUUGGAAAAAA y ‹ UC С UA
Domain V Domain V Domain V

FIG. 1. Representative gastropod secondary structure models for a, Psilaxis radiatus (Heterobranchia: Ar-
chitectonicoidea); b, Littorina saxatilis (Caenogastropoda: Littorinioidea) and c, Albinaria coerulea (Hetero-
branchia: Euthyneura). Additional secondary structure diagrams for other molluscan taxa can be found in Ly-

deard et al. (2000).

MITOCHONDRIAL LSU rRNA HELICAL-LOOP STRUCTURES IN GASTROPODS 351
a Domain b Domain II e
. .ne
= 5 ee
Ш Ш Ш Katharina ex)
Ш Ш Ш Pectn A ie
о № m Mu ie
Ш Ш Ш Loligo 42 - 62 bp eos
Ш Ш Ш Cellana JPatellogastropoda > DY о
DO MM \eria ]Neritopsina ee e So
eo. 15-19 bp NE
Ш Ш Ш Chpidina ]Vetigastropoda eos oP oe E
Ш Ш m Pomacea/Tulotoma e Ye? 0% N° %
Ш Ш E Coclophorus e o es о
= A ee бо о
Ш Ш Ш Cerithioidea ы
W E E Campaniloidea = A o e
aa
Ш Ш E Apdrobia |Rissooidea 2 o e
ee В = e e
Ш Ш EM Serpulorbis | Vermetoidea = o e
Nodilittorina Sa e o
ини ERW [Littorinioides Le °e—e°
Ш Ш Ш Litorina e—e
Ш Ш Ш Busycotypus ]Neogastropoda Domain Ш 323
e—e 0b
О О О Valvara |Valvatoidea 30-795 о 0000 р
О О DO Psiaxis |Architectonicoidea P =
O O © Cingulina|Pyramidelloidea =
О О а Omalogyra |Omalogyroidea я e—e
e
Siph i 3 A
ооо ue = Domain Y
Albinaria с. | =
O00 8 nan = 5 EYTYYYYYIYIYYT De a ®о
О DD Albinariaı | Е = OTE IN e 41-43bp
о са Cepaea 2 5 0000000000000, 00, e?
О Оо Euhadra |2 00000 особо
P ТЕТЕ III © 20-30bp
ОО 9 pa 00000 00050

Ponder & Lindberg, 1997

FIG. 2. Evidence from mitochondrial LSU secondary structures uniting the Heterobranchia, a group that di-
verged from the Caenogastropoda 350-400 mya. a, Phylogeny of gastropods inferred from a cladistic analy-
sis of 117 morphological characters (Ponder & Lindberg, 1997) showing the taxa included in the present
study. Mitochondrial LSU rRNA sequences for the outgroup taxa (Katharina tunicata, Pecten maximus,
Mytilus edulis, Loligo bleekeri), the heterobranchs Albinaria coerulea, Albinaria turrita, Cepaea nemoralis,
and Euhadra herklotsi, and the caenogastropod Littorina saxatilis (Wilding et al., 1999) were obtained from
GenBank (U09810, X92688, M83756, AB009838, X83390, X71393, X71394, U23045, 271693, AJ132137).
GenBank accession numbers for the remaining taxa are AB028237, AY10505-526, AF101007-08,
AF100991, AYO10314-327, AY081770-772, AY081996-2000. The columns of boxes show the three sec-
ondary structural characters: Domain II (character states = (1) 42-62 nucleotides in length — black box, (2)
15-19 nucleotides in length — gray box, (3) stem-loop structure absent —open box); Domain III (character
states = (1) 30-79 nucleotides in length —black box, (2) stem-loop structure absent — open box); and Do-
main V (character states = (1) 41-43 nucleotides in length — black box, (2) stem-loop structure absent — gray
box). b, Schematic diagrams representing consensus secondary structure models for the three secondary
structural characters showing the character states within Domains Il, Ill, and V (described above) of the ex-
amined taxa (actual secondary structure models for the three Domain regions is available online
(http://www.bama.ua.edu/~clydeard). The shade (black, gray or white) of the helical-loop structure corre-
sponds to the character states shown at the tips of each terminal branch in 2a. The four open dots shown for
Domain Il and III, respectively, indicate no helical-loop structure is found for the taxon.

mitochondrial genomes documented for

metazoans (Kurabayashi & Ueshima, 2000).
The nature of secondary structural variation

exhibited among gastropods is analogous to

mative data for elucidating deep evolutionary
nodes.

conservative variation reported for mitochon-
drial gene order among arthropods (Boore et
al., 1995) and gastropods (Kurabayashi &
Ueshima, 2000). It is possible comparative
analyses of secondary structure of other mol-
luscan taxa will yield phylogenetically infor-

ACKNOWLEDGMENTS

This work was funded, in part, with a grant
from the National Science Foundation to CL
and W. F. Ponder. We thank our many coop-
erative colleagues for help in obtaining mate-

352 LYDEARD ET AL.

rial for study. Don Colgan, Winston Ponder,
and Brian Simison provided helpful comments
on the manuscript.

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Revised ms. accepted 5 March 2002

MALACOLOGIA, 2002, 44(2): 353-361

TWO NEW TROPHONINAE (GASTROPODA: MURICIDAE)
FROM ANTARCTIC WATERS

Guido Pastorino

Museo Argentino de Ciencias Naturales,
Av. Angel Gallardo 470 3° piso lab 57,
C1405DJR Buenos Aires, Argentina; pastorin @ mail.retina.ar

ABSTRACT

Two new species of Trophon from Antarctic waters are described. Trophon emilyae, n.
sp., from the northwestern Amundsen Sea, is characterized by a small, slender, almost
smooth shell and a thin profile. Axial sculpture consists of regular, thin varices, and four
weak cords on the first whorls are the only spiral ornamentation. It is similar to T. declinans
Watson, 1882, but the radula and shell ornamentation are clearly different. Trophon
arnaudi, п. sp., was collected off South Sandwich Island. It is a small species similar to T.
scolopax Watson, 1882, from the Kerguelen Islands. The shell is chalky white, with 10-11
axial lamellae crossed by five spiral cords on the last whorl. Adult specimens of the new
species are illustrated, described and compared with other living species of the same
genus and of similar geographic distribution. Lectotypes of 7. septus Watson, 1882, and 7.
declinans Watson, 1882, are here designated.

Key words: Antarctica, Trophon arnaudi n. sp., Trophon emilyae n. sp., Muricidae,

Trophoninae, systematics, biogeography.

INTRODUCTION

The subfamily Trophoninae is by far the larg-
est group of marine living gastropods in Antarc-
tica. A detailed survey shows more than 100
names published for this group in southern
South America and Antarctica. From this total,
approximately 30 belong to Patagonian species
of the genus Trophon 5. I., 34 to Xymenopsis,
and the rest to Antarctic species. Recently,
Xymenopsis was show to have only four valid
species by Pastorino & Harasewych (2000).

In the last comprehensive revision of
southern species of Trophon, Powell (1951)
described T. cuspidarioides and included
Watson’s species T. scolopax, T. septus, and
T. acanthodes in the same group, because of
their long anterior canals. However, he
considered this feature not of sufficient
taxonomic importance to warrant separation of
this group of species. This is true, because T.
acanthodes Watson, 1882, despite the
similarities in the long anterior canal, has a very
different radula and protoconch (Pastorino, in
prep.).

In this paper, two new living species of the
subfamily Trophoninae are described from

353

Antarctic waters. Adult specimens, operculum,
radula, protoconch, and shell ultrastructure are
described for each. Due to the extraordinary
morphological variation within this genus,
comparison with all similar species from the
same geographic area is mandatory.

MATERIAL AND METHODS

Specimens studied in this work are
housed in the collections of: the National
Museum of Natural History, Smithsonian In-
stitution, Washington, D.C. (USNM); Museo
Argentino de Ciencias Naturales “Bernar-
dino Rivadavia”, Buenos Aires (MACN); The
Natural History Museum, London, (NHM);
and Zoological Institute of the Russian Acad-
emy of Sciences, St. Petersburg (ZIN). Mate-
rial housed at USNM, collected by the ship R/
V Eltanin belongs to the United States Ant-
arctic Program (USAP) and material housed
at ZIN was collected by the R/V Evrica. In ad-
dition, type specimens from the Strebel col-
lection housed in part at the Zoologisches
Institut und Zoologisches Museum der
Universität Hamburg was also studied.

354 PASTORINO

Dissections were performed on ethanol-
preserved specimens to study radulae and
male reproductive system. Radulae were
prepared according to the method described
by Solem (1972) and observed using a LEO
440 scanning electron microscope (SEM) at
the USNM. Radular terminology follows Kool
(1993: fig. 6B). Shell ultrastructure data were
procured from freshly fractured colabral sec-
tions taken from the central portion of the lip
on the last whorl of two individuals per taxon,
whenever sufficient material was available.

Photographs were taken using a digital scan-
ning camera. Several images were scanned
from black and white 35 mm negatives using a
slide scanner. All images were digitally pro-
cessed.

SYSTEMATICS

Class Gastropoda Cuvier, 1797

Order Neogastropoda Wenz, 1938

Family Muricidae Rafinesque, 1815

Subfamily Trophoninae Cossmann, 1903

Genus Trophon Montfort, 1810

Type species: Murex magellanicus Gmelin,
1791, = Trophon geversianus (Pallas, 1774),
by original designation.

Trophon emilyae Pastorino, new species
(Figs. 1-14)

Diagnosis

Shell small, slender, almost smooth; siphonal
canal very long, twisted, narrow. Axial sculpture
of regular, thin, gentle varices; spiral sculpture
of four very weak cords in the first whorls, then
obsolete.

Description

Shell small in size (up to 16 mm), slender,
fusiform, very thin, translucid; protoconch of
one and a half whorls; teleoconch of five mod-
erately convex whorls; spire 1/4 of total shell
length; subsutural shelf faint. Spire angle about
40°, suture abutting; aperture small, its interior
glossy white; anterior siphonal canal very long,
twisted, narrow; umbilicus closed; outer lip
rounded; columellar lip almost indistinct. Axial
ornamentation of regular, thin varices, about 8—
11 on last whorl, slightly developed, running
along whorl surface from adapical suture to
siphonal fasciole, weakly projecting outwards

along keel. Spiral ornamentation of low,
rounded spiral cords, four in the first whorls
then obsolete, the entire shell surface covered
by regular threads. Regular growth lines cover-
ing whorls and lamellae.

Innermost shell layer very thin, comprising
about 5% of shell, composed of aragonite (?),
with the crystal planes oriented perpendicular
to growing edge of shell; second layer arago-
nitic crossed-lamellar, representing 95% of
shell thickness.

Operculum suboval, completely covering
aperture, brownish in color, thin, with terminal
nucleus; growth lines covering external surface
curved at upper ends; attachment area with
two or three horseshoe-shape scars.

Rachiglossan radula with teeth very closely
packed; rachidian two times wider than height;
central cusp very thin; lateral cusps shorter but
of similar thickness to central cusp, denticle
between central and lateral cusp very large.
Base of rachidian teeth curved, large; marginal
area inclined, smooth. Lateral teeth large size,
with a thick attached portion.

Etymology

This species is dedicated to Dr. Emily Vokes,
of Tulane University, New Orleans, Louisiana
(now retired), who helped me with my first
steps on Trophon.

Type Material & Locality

Holotype: USNM 896438; R/V Eltanin Station
1343, 567-604 m, collected with rock dredge, 7
November 1964, 54°50’S/29°50’W.

Paratypes: USNM 1003475, from type local-
ity, 3 specimen (one with animal); USNM
896441, R/V Eltanin Station 1346, 549 m,
54°49’S-129°48’W, 4 specimens; USNM
898917, R/V Eltanin Station 1345, 915-1153 m,
7 November 1964, 54°50’S/129°48’W, 4 shells,
collected with Menzies trawl.

Distribution

Known only from the vicinity of the type locality.
Remarks

The shell morphology of Т. етйуае is com-
parable to some specimens of T. declinans
Watson (Figs. 16, 17) despite the distance be-

tween their distributions on the opposite sides
of Antarctica (Fig. 44). The shell of T. emilyae

NEW ANTARCTIC SPECIES OF TROPHON 355

FIGS. 1-3. Trophon emilyae, new species, three views of holotype, USNM 896438; scale bar = 0.5
cm. FIGS. 4, 5. Two views of a paratype, USNM 896441, uncoated SEM; same scale as in Figs. 1-8.
FIG. 6. Apertural view of a paratype, USNM 896441; same scale as in Figs. 1-3. FIG. 7. External view
of operculum of paratype in Figs. 4, 5; scale bar = 1 cm. FIGS. 8, 9. Two views of protoconch of
paratype in Figs. 4-5; scales = 500 um. FIG. 10. Radula side view; scale bar = 10 um. FIG. 11. Radula
front view; scale bar = 20 um. FIG. 12. Shell ultrastructure, uncoated SEM; scale bar = 30 um. FIG.
13. Trophon declinans Watson, 1882, MACN 34956, collected by P. Arnaud, February 3, 1975, from
NW of Kerguelen Islands in 70 m, radula front view; scale bar = 30 um. FIGS. 14, 15. Two views of
protoconch of same specimen; scale = 500 um. FIGS. 16, 17. Trophon declinans Watson, 1882, two
views of lectotype, NHM 1887.2.9.573; scale Баг = 0.5 cm.

356 PASTORINO

FIGS. 18-20. Trophon arnaudi, new species, three views of holotype, USNM 1003473; scale bar =
0.5 cm. FIG. 21. Apertural view of paratype ZIN 59775; same scale as in Figs. 18-20. FIGS. 22, 23.
Holotype, two views of protoconch, uncoated SEM; scale bar = 400 um. FIG. 24. Radula frontal view;
scale bar = 30 um. FIG. 25. Radula side view; scale bar = 30 um. FIGS. 26, 27. T. scolopax Watson,
1882. FIG. 26. Radula side view, MACN 34955; scale bar = 40 um. FIG. 27 radula frontal view, same
specimen as in Fig. 26, scale bar = 80 um.

NEW ANTARCTIC SPECIES OF TROPHON 357

is more slender, with a longer anterior canal.
The axial lamellae in T. declinans are some-
what protruding from the whorl side, whereas
in T. emilyae they are never detached from the
shell wall. The radulae are also different; the
base of the rachidian in T. emilyae is some-
what curved, with a long, thin intermediate den-
ticle between the central and lateral cusps. The
lateral cusps are thinner and longer in the new
species (Figs. 10, 11). To fix the identity of the
taxon (ICZN, 1999: Art. 74.7), the specimen of
T. declinans illustrated here in Figures 16 and
17 (NHM 1887.2.9.573) is here designated
lectotype. The other three lots with one speci-
men each therefore become paralectotypes
(NHM 1887.2.9.574, 1887.2.9.575, and
1985041).

Cernohorsky (1977) mentioned the Magel-
lanic T. ohlini Strebel, 1904, as being very simi-
lar to T. declinans. The type material of T.
ohlini, housed in the Zoologisches Institut und
Zoologisches Museum der Universitat Ham-
burg, was studied as part of the complete revi-
sion of the genus in progress. Based on the
morphology of its protoconch, it is apparently
not related to any Antarctic species.

Trophon arnaudi Pastorino, new species
(Figs. 18-20)

Diagnosis

Shell small, thin, fusiform; axial ornamenta-
tion of regular low lamellae, projecting out-
wards along periphery; 2-3 spiral rounded
cords on the first whorls become 4—5 on the
last whorl.

Description

Shell small in size (~ 13 mm), thin, fusiform,
chalky; protoconch of 1% whorls; teleoconch of
four and a half, right-angled whorls; spire 1/5 of
total shell length; subsutural shelf short but
clearly defined. Spire angle about 45-50°, su-
ture abutting; aperture small, rounded, its inte-
rior glossy white; anterior siphonal canal long,
curved backwards; umbilicus closed; outer lip
rounded, with lirations from spiral ornament;
columellar lip narrow. Axial ornamentation of
regular, thin lamellae, about 8—11 on last whorl:
lamellae moderately developed, running along
whorl surface from adapical suture to base of
last whorl, projecting outwards along periphery.
Spiral ornamentation of rounded cords, slightly
developed on the first whorls to 4—5 on the last.

Regular growth lines covering whorls, cords
and lamellae.

Operculum and shell ultrastructure unknown.

Radula rachiglossate, rachidian with central
cusp very thin; lateral cusps slightly shorter but
of similar thickness to central cusp, denticle
between central and lateral cusp large, rising
from the base. Base of the rachidian teeth
curved; large, smooth marginal area. Lateral
teeth large, with a thick attached portion.

Etymology

This species is dedicated to Dr. Patrick
Arnaud from Endoume, Marseille, for his many
contributions to Antarctic malacological studies.

Type Material & Locality

Holotype: USNM 1003473; R/V Islas
Orcadas, Cruise 575, Station 53, 26 May 1975,
off South Sandwich Island, 57°41.4’S/
26°22.3’W, 355-468 m; one paratype: ZIN
59775 collected alive by R/V Evrica on 22 Janu-
ary 1987, drag 2, in 370 m, 57°38’S/26°18’W,
volcanic sand bottom.

Distribution
Known only from the vicinity of the type locality.
Remarks

Despite the 100° of longitude separating their
distributions, 7. scolopax Watson, 1882, de-
scribed from off the Kerguelen Islands and
Crozet Island, is comparable, but it has differ-
ent lamellae. In T. scolopax, after a large su-
tural shelf, the lamellae end in vaulted spines
in number of two or three plus the keel. In addi-
tion, the 2-3 rounded spiral threads in T.
scolopax are 4-5 real cords in 7. arnaudi. The
anterior siphonal canal is shorter and curved in
T. arnaudi and always measures less than half
shell height. Trophon scolopax has a straight
siphonal canal that is more than half the shell
length.

Trophon scolopax was described by Watson
from material belonging from the Challenger
Expedition. The holotype (Figs. 28-30) is
housed in the NHM under the number
1887.2.9.580. There were very few literature
citations after the original description. Cantera
& Arnaud (1985) cited living specimens of T.
scolopax between 60 to 620 m off the
Kerguelen Islands.

358 PASTORINO

FIGS. 28-30. Trophon scolopax Watson, 1882; three views of holotype, NHM 1887.2.9.580; FIGS.
31, 32. T. scolopax apertural and adapertural views, MACN 34955, collected by P. Arnaud on April 7,
1974, in 560-525 m, NE of Heard Island; FIGS. 33, 34. MACN 34955 apertural and adapertural view
of a smooth specimen from same lot of specimens in Figs. 31, 32. Figs. 35-37. T. cuspidarioides
Powell, 1951, three views of holotype, NHM 1961547. FIGS. 38, 39. T. septus Watson, 1882,
paralectotype, NHM 1887.2.9.579. FIGS. 40-42, lectotype, NHM 1887.2.9.578; scale bar = 1 cm for
all figs.

NEW ANTARCTIC SPECIES OF TROPHON 359

Trophon cuspidarioides Powell, 1951,
known only from the original material from off
mouth of Cumberland Bay, South Georgia
Island, resembles a somewhat smooth
specimen of 7. arnaudi. However, Т.
cuspidarioides (Holotype: NHM 1961547)
(Figs. 35-37) never develops the spines,
lamellae and the spiral cords that charac-
terize T. arnaudi.

Finally, T. septus Watson, 1882, the last com-
parable Antarctic species, known from off the
Kerguelen Islands, differs in having triangular
upturned spines and smooth whorls. Three
syntypes are in the NHM 1887.2.9.578,
1887.2.9.579 and 1887.2.9.579a, the first two

90°—1

1015 dry are illustrated here in Figures 38-42.
The specimen illustrated in the Figures 40-42
is here selected as lectotype (ICZN. 1999:
Art. 74.7). The remaining syntypes are there-
fore paralectotypes.

Cernohorsky (1977) mentioned some resem-
blance of large specimens of T. septus with T.
coronatus H. Adams & A. Adams, 1864, a bo-
real species not related to the Antarctic groups
and which belongs in Nodulotrophon. Previ-
ously, Powell (1951) pointed out theories of bi-
polarity of some species of gastropods. In fact,
Egorov (1993) in a revision of Russian
Trophoninae illustrated all living Russian
trophonine species, and several of them show

FIG. 43. Type localities of new species of Trophon and other species of the same genus with similar
shell morphology: A = T. emilyae, new species; B = T. cuspidarioides Powell, 1951; C = T. arnaudi,
new species; D = T. declinans Watson; 1882; E = T. scolopax Watson, 1882. The size is not relative.
Arrows point to type localities. Thick line represents the Antarctic Convergence.

360

PASTORINO

TABLE 1. Measurements (in mm), distribution, and depth range (in m) of Trophon emilyae, new
species; T. arnaudi, new species; T. septus Watson, 1882; T. scolopax Watson, 1882; and T.

cuspidarioides Powell, 1951.

Length Width Whorls Depth range

T. emilyae, new species
USNM 896438
holotype
T. arnaudi, new species
USNM 1003473
holotype
ZIN 59775
paratype

T. septus Watson

12:1 3.2 5

13.5 5:9 4

NHM 1887.2.9.578 21:1 10.5 5-6
lectotype
NHM 1887.2.9.579 20.0 9.9 5-6
paralectotype
T. scolopax Watson
NHM 1887.2.9.580 23.4 10.4 6-7

holotype
T. declinans Watson
NHM 1887.2.9.573
lectotype
T. cuspidarioides Powell
NHM 1961547 13 Bo 5
holotype

19.5 8.0 7

a striking similarity with Antarctic species —
for example, T. barvicensis (Johnston, 1825)
with T. arnaudi. However, when these spe-
cies are carefully studied they present suffi-
cient features to ensure a different generic
allocation. Crame (1996) discussed from a
paleontological point of view the evolution of
bipolarity of several groups of mollusks, in-
cluding the genus Trophon as the southern
counterpart of the northern Boreotrophon
and Trophonopsis.

Despite the poor knowledge of fossil repre-
sentatives of the genus Trophon, there is a
good record of Paleogene and Neogene spe-
cies in Patagonian deposits (Griffin 8
Pastorino, in prep.). The oldest record in Ter-
tiary deposits from Patagonia is apparently
early Oligocene in age. However, there is no
formal citation of species of this genus in Ter-
tiary deposits around Antarctica. Nevertheless,
Jonkers (2000), in a non-taxonomic paper, il-
lustrated a shell that apparently belongs to this
genus.

The living species belonging to Trophon
could be easily split based on the morphology

Distribution Source
549-1153 NW Amundsen This paper
Sea
This paper
355-468 Off South
Sandwich Island
370 Off South
Sandwich Island
30-620 Off Kerguelen & Cantera & Arnaud,
Crozet Islands 1985
60-620 Off Kerguelen & Cantera & Arnaud,
Heard Islands 1985
70-274 Marion, Kerguelen Watson, 1882
& Crozet Islands Arnaud (pers. com)
120-204 South Georgia Powell, 1951

Island

of the soft parts into isolated Patagonian and
Antarctic clades (Pastorino, in press). Further
studies will show if the Antarctic clade justi-
fies a new generic placement.

ACKNOWLEDGEMENTS

This work was possible thanks to the mate-
rial of T. scolopax and T. declinans collected
by Dr. Patrick Arnaud from the Kerguelen Is-
lands that he kindly offered during a trip to the
Marine Station of Endoume, Marseille. This
specimens are housed at the MACN. | thank
the following people for access to material in
their institutions: K. Way and J. Pickering
(NHM); P. Bouchet and V. Heros (MNHN); A.
Tablado (MACN), B. Hausdorf (Zoologisches
Institut — Hamburg) and Е. Egorova (ZIN). | am
grateful to M. Griffin and F. Scarabino for thor-
ough reviews and helpful suggestions that im-
proved this paper. Finally R. Bieler, E. Coan
and an anonymous reviewer proposed thought-
ful suggestions that also improved it.

Part of this study was conducted during a

NEW ANTARCTIC SPECIES OF TROPHON 361

Post-Doctoral Fellowship granted by the
Consejo Nacional de Investigaciones
Cientificas y Tecnicas (CONICET), Argentina,
to work at the National Museum of Natural
History, Smithsonian Institution, Washington,
DC. | gratefully appreciate the advise and
friendship of J. Harasewych during my time
at the USNM. It was supported in part by a
Research Award from the NSF-USAP United
States Antarctic Program [Contract No. OPP-
9509761], a grant in aid from the Concholo-
gists of America and the Walter E. Sage
Memorial Award and the project PICT No. 01-
04321 from the National Agency for Scientific
and Technical Promotion, Argentina.

LITERATURE CITED

CANTERA, J. R. & P. M. ARNAUD, 1985, Les
gastéropodes prosobranches des iles
Kerguelen et Crozet (sud de l'Océan Indien)
comparaison écologique et particularités
biologiques. Comité National Français des
Recherches Antarctiques, 56: 1-169.

CERNOHORSKY, W. O., 1977, The taxonomy of
some southern ocean Mollusca (Gastropoda)
mainly Antarctic and subantarctic: Records of the
Aukland Institute and Museum, 14: 105-119.

CRAME, J. A., 1996, Evolution of high-latitude
molluscan faunas. Pp. 119-131, in: J. TAYLOR,
ed., Origin and evolutionary radiation of the
Mollusca. Oxford University Press, Oxford.

EGOROV, R., 1993, Trophoninae (Muricidae) of
Russian and adjacent waters. Ruthenica,
Supplement, 1: 1-49.

ICZN, 1999, International Code of Zoological
Nomenclature: London, The International Trust
for Zoological Nomenclature, 306 pp.

JONKERS, H. A., 2000, Gastropod predation
patterns in Pliocene and Recent pectinid
bivalves from Antarctica and New Zealand.
New Zealand Journal of Geology &
Geophysics, 43: 247-254.

KOOL, S. P., 1993, Phylogenetic analysis of the
Rapaninae (Neogastropoda: Muricidae).
Malacologia 35: 155-260.

PASTORINO, G., in press, Systematics and
phylogeny of the genus Trophon Montfort,
1810 (Gastropoda: Muricidae) from Patagonia
and Antarctica: morphological patterns.
Bollettino Malacologico.

PASTORINO, G. & М. С. HARASEWYCH, 2000, A
revision of the Patagonian genus Xymenopsis
Powell, 1951 (Gastropoda: Muricidae). The
Nautilus, 114: 38-58.

POWELL, A. W. B., 1951, Antarctic and
subantarctic Mollusca: Pelecypoda and
Gastropoda. Discovery Reports, 26: 47-196.

SOLEM, A., 1972, Malacological application of
scanning electron microscopy, Il. Radular
structure and functioning. The Veliger, 14:
327-336.

STREBEL, H., 1904, Beiträge zur Kenntnis der
Molluskenfauna der Magalhaen-Provinz:
Zoologischen Jahrbüchern. Abteilung für
Systematik, Geographie und Biologie der
Tiere, 21: 171-248.

WATSON, В. B., 1882, Mollusca of H. М. S.
‘Challenger Expedition — Part 13. The Journal of
The Linnean Society. Zoology, 16: 358-392.

Revised Ms. accepted 1 May 2002

MALACOLOGIA, 2002, 44(2): 363

CORRECTION TO:
COAN, EUGENE V., 2002, THE EASTERN PACIFIC RECENT SPECIES OF THE
CORBULIDAE (BIVALVIA). MALACOLOGIA 44(1): 47-105

Eugene V. Coan

Department of Invertebrate Zoology & Geology, California Academy of Sciences, Golden
Gate Park, San Francisco, California 94118-4599, U.S.A.; gene.coan Osierraclub.org

It has been pointed out to me that one of the figure captions in my paper on the Corbulidae
(Coan, 2002) was incorrectly labeled. As per my text, the lectotype of Serracorbula tumaca
Olsson is the right valve depicted in Fig. 6b; the left valve in Fig. 6a is a paralectotype, not the
lectotype.

12 May 2002

363

MALACOLOGIA, 2002, 44(1-2): 365-378
INDEX

Taxa in bold are new; pages in italic indicate amurensis, Corbula 49, 69, 71-72, 93

figures of taxa.

abscissa, Melanthalia 2-3
acanthias, Squalus 135
acanthocarpa, Aglaophenia 3
acanthodes, Trophon 353
Acanthodoris 302
Acmaea 253
Acromobacter dermidis 325
acuta, Physa 252
acutirostra, Corbula 74
adamsi, Lampeia 108
aequalis, Corbula 66-67
Potamomya 66-67, 67, 70
aequivalvis, Corbula 64, 66
africana, Chromodoris 297
Agama 135
Aglaophenia acanthocarpa 3
Agriolimax 145, 150
reticulatus 150, 252
alabamiensis, Corbula 51, 57
alba, Corbula 53, 55, 57, 66
Albinaria 33-34, 35, 37, 38-39, 40, 41, 42-44,
351
coerulea 34, 36-37, 40, 42-44, 350-351
discolor 34, 36-37, 40, 42-43
turrita 34, 36-37, 40, 42-43, 351
voithii 34, 36-37, 40, 42-44
albopunctatus, Chromodoris 301
albopustulosa, Chromodoris 301
Aloides 50
guineensis 50
Aloidis (Aloidis) 89
(Aloidis), Aloidis 89
(Caryocorbula) 53, 58, 60-61, 64, 78, 82,
84,87
(Caryocorbula) nasuta 53
(Tenuicorbula) 53
Alopiinae 43
altirostris, Corbula 90
alveata, Gigartina 3
americana, Vallisneria 5
amethystina, Corbula 91
Corbula (Caryocorbula) 51
amoena, Ceratosoma 297
Amphibola cerenata 150
Amphisbetia bispinosa 3, 12
amplexa, Corbula 71
Ampullariidae 153, 273, 279

365

Corbula (Potamocorbula) 71-72
amythestina, Corbula 49, 52, 53
anagallis, Enhydra 276, 279, 281
Anaspidea 289
anatinum, Drepanotrema 273, 277-278, 282-
285
Ancylidae 273, 279
angillarum, Vibrio 325
angustifolium, Carpophyllum 2, 4, 12
Anisocorbula 50
annulata, Chromodoris 297, 301
Anodonta cellensis 140
anomala, Corbula 50
antiqua, Venus 17-21, 19
aquatica, Cabomba 5
Araceae 276
arbutus, Digidentis 293, 295, 297
Architectonicoidea 350-351
Arctica islandica 140
arcticus, Bathypolypus 175, 177-178, 179-
180, 181-187, 182, 190-191, 193-198, 200-
205, 207-210, 208-210, 212-214, 220
Octopus 175, 177, 187, 202, 204, 221
Polypus 187
Ardeadoris 299-300
Argopecten purpuratus 165
Ariolimax columbianus 237
Arion ater224, 236
lusitanicus 224
arnaudi, Trophon 353, 356, 357-360, 359
Asaphis 50
aspersa, Helix 146-147, 149
Chromodoris 297-298
Asterias rubens 135
Asthenothaerus 107, 112-114
diegensis 107
hemphillii 107
huanghaiensis 112
isaotakii 107-108, 131
sematana 107, 126
sematanus 126
sematensis 126
ater, Arion 224, 236
atramentosa, Nerita 350
atromarginata, Glossodoris 297
aurantionodulosa, Diversidoris 293, 295, 297,
301
aureopurpurea, Chromodoris 297
australis, Thorunna 297

366 INDEX

Azara 66
(Azara), Corbula 66

bairdii, Bathypolypus 175, 181, 183-187, 188-
189, 190-198, 193, 201-214, 208-209, 211,
217
Octopus 175, 184-185, 187, 191, 194, 219

Balea perversa 43

banyulensis, Godiva 252

barrattiana, Corbula 57, 87

barvicensis, Trophon 360

Bathypolypodinae 175, 202, 204

Bathypolypus 175-176, 185, 191, 193-195,
201-202, 204-207, 208, 212-214
arcticus 175, 177-178, 179-180, 181-187,
182, 190-191, 193-198, 200-205, 207-210,
208-210, 212-214, 220
bairdii 175, 181, 183-187, 188-189, 190-
198, 193, 201-214, 208-209, 211,217
ergasticus 201, 205-207, 208, 212, 214
faeroensis 178, 181, 183-184, 186-187,
203-204, 220
grimpei 202, 206
lentus 191, 193
obesus 191-193
proschi 175, 187, 194-195, 204
pugniger 175, 187, 191, 195-196, 196-197,
198, 199-200, 200-204, 206-207, 208-209,
212-214, 222
salebrosus 204-205
sponsalis 194, 201-202, 204-207, 206, 208,
212-214
valdiviae 201-202, 204, 206-207, 212

Bedevina birileffi 254

Benthoctopus 175-176, 185, 194, 202, 204-
205, 207
berryi 202
ergasticus 185, 204, 206-207
januarii 204
normani 185
piscatorum 175, 178, 183-186, 202-203,
207,220
salebrosus 204-205, 207
sasakii 178, 183-186, 220

bentoniensis, Goniobasis 23, 29

berryi, Benthoctopus 202

biangulata, Corbula 51

bicarinata, Corbula 49, 51, 53, 57, 64, 65, 66,
87,92
Corbula (Juliacorbula) 64

binominata, Corbula 90

binza, Chromodoris 297, 300

Biomphalaria 273-274, 277, 282-283, 285
glabrata 252, 273
occidentalis 286
straminea 273, 277-278, 282-286
tenagophila 273, 277-278, 282-286

Biophalaria glabrata 17, 150

biradiata, Corbula 49, 51, 64, 74, 78, 79-80,
80-82, 94

birileffi, Bedevina 254

bispinosa, Amphisbetia 3, 12

blandiana, Corbula 87, 89-90

bleekeri, Loligo 351

Boettgerilla pallens 223-225, 228-229, 230-
233, 233-234, 235, 236-238

Boettgerillidae 223-224

boivinei, Corbula 90

Bombyx mori 19

Boreotrophon 360

Bosellia 290

Bothrocorbula 63

boyntoni, Corbula (Cuneocorbula) whitfieldi 58

bradleyi, Corbula 76

Bradybaena fruticum 43

brevicaudatum, Ceratosoma 297

bronni, Thais 254

buccinoidea, Melanopsis 307-308, 311, 313,
315-318, 316, 320-322

budapestensis, Tandonia 224

buenavistana, Corbula (Caryocorbula) 64

Bufo marinus 135

Bulinus 167

bullocki, Hypselodoris 293, 296-297

burnsii, Corbula engonata 89

Bushia 113

Busycotypus 351

Bythinella 319

Cabomba aquatica 5
Cadlina 299-301
luteomarginata 297
modesta 297
pellucida 297
Cadlinella 289-291, 297, 299-301
ornatissima 290, 297, 301
caimitica, Corbula (Cuneocorbula) 58
californica, Cumingia 90
Pleurobranchaea 135
californicus, Schoenoplectus 154
californiensis, Penaeus 19
caloosae, Corbula 75-76
Campaniloidea 351
canaliculata, Pomacea 153, 157, 159-161, 279

canaliculus, Рета 1, 2, 13
Cannaceae 276
Canna glauca 273, 276, 279, 281-285
Cardiomya 89
caribaea, Corbula 57
carinata, Corbula 57
carolina, Corbula chipolana 76
Carpophyllum angustifolium 2, 4, 12
carrizalana, Corbula 76
caruanae, Deroceras 150
Caryocorbula 51, 61
(Caryocorbula) 53, 58, 60-61, 82, 87
(Caryocorbula) amethystina 51
(Caryocorbula) prenasuta 58
(Hexacorbula) esmeralda 63
nasuta 53
(Caryocorbula), Aloidis 53, 58, 60-61, 64, 78,
82, 84, 87
Caryocorbula 53, 58, 60-61, 82, 87
Corbula 51, 53, 58, 60-61, 64, 77, 80, 82,
84, 87
casertanum, Pisidium 166-167
catenaria, Goniobasis 23-24, 25, 26-29
Goniobasis catenaria 23-24, 26, 26-28, 30-
31
Cellana 351
cellensis, Anodonta 140
Cepaea 351
nemoralis 320, 351
Ceratophyllum demersum 5
Ceratopteris thalictroides 5
Ceratosoma 299-300
amoena 297
brevicaudatum 297
magnifica 297
cercadica, Corbula (Cuneocorbula) 58
cerenata, Amphibola 150
Cerion 320
Cerithiacea 307
Cerithioidea 351
Champia laingii 3
Chara 154
Chicoreus torrefactus 255
chipolana, Corbula 76
chittyana, Corbula 57
chowanensis, Corbula 76
Chromodorididae 289-290, 294, 298-302
Chromodoris 289-291, 294, 296, 298-301
africana 297
albopunctatus 301
albopustulosa 301
annulata 297, 301

INDEX 367

aspersa 297-298
aureopurpurea 297
binza 297, 300
clenchi 297, 300
coi 297-298
collingwoodi 293-294, 297, 301
daphne 293-294, 297, 301
decora 301
elisabethina 293-294, 297
fidelis 301
galactos 301
geometrica 293-294, 296-301
inornata 297
kuiteri 293-294, 297
kuniei 293-294, 297-298, 301
leopardus 293-294, 297, 301
lineolata 297
lochi 293,295, 297
loringi 301
magnifica 297-298
marginata 301
obseleta 297
orientalis 290, 297-298
perola 297
preciosa 301
quadricolor 297-298
roboi 293, 295, 297-298, 301
rubrocornuta 301
strigata 293, 295, 297
thompsoni 301
tinctoria 293, 295, 297, 301
vibrata 301
willani297
woodwardae 297
cimex, Drepanotrema 273, 277-278, 282-285
cincta, Glossodoris 297
Cingulina 351
Cirroteuthis 183
mülleri 177
Clausiliidae 33
clavigera, Thais 254
clenchi, Chromodoris 297, 300
Clypidina 351
coerulea, Albinaria 34, 36-37, 40, 42-44, 350-
351
coi, Chromodoris 297-298
colensoi, Osmundaria 2, 12
colimensis, Corbula 47, 49, 66, 82, 83, 84,
94
collazica, Corbula 90
collingwoodi, Chromodoris 293-294, 297, 301
colorata, Corbicula 166

368

columbianus, Ariolimax 237
complanata, Corbula 50
Compositae 276
concinna, Eximiothracia 107
conradi, Corbula 58
Corbula (Caryocorbula) 82
constracta, Corbula 87
contectus, Viviparus 320
Corallina officinalis 3
Corbicula 165, 167
colorata 166
(Corbiculina) leana 165
fluminea 166
japonica 166
leana 166
papyracea 166
sandai 166
Corbiculidae 165
Corbiculoidea 165-166
Corbula 47, 49-50, 87, 90, 91-95
acutirostra 74
aequalis 66-67
aequivalvis 64, 66
aequivalvis stainforthi 66
alabamiensis 51, 57
alba 53, 55, 57, 66
altirostris 90
amethystina 91
amplexa 71
amurensis 49, 69, 71-72, 93
amurensis takatuayamaensis 71
amythestina 49, 52, 53
anomala 50
(Azara) 66
barrattiana 57,87
barrattiana leonensis 89
biangulata 51
bicarinata 49, 51, 53, 57, 64, 65, 66, 87, 92
binominata 90
biradiata 49, 51, 64, 74, 78, 79-80, 80-82,
94
blandiana 87, 89-90
boivinei 90
bradleyi 76
caloosae 75-76
caribaea 57
carinata 57
carrizalana 76
(Caryocorbula) 51, 53, 58, 60-61, 64, 77,
80, 82, 84, 87
(Caryocorbula) amethystina 51
(Caryocorbula) buenavistana 64

(Caryocorbula) conradi 82
(Caryocorbula) democraciana 82
(Caryocorbula) franci 88
(Caryocorbula) nasuta 50, 53
(Caryocorbula) otra 47, 58
(Caryocorbula) ovulata 60
(Caryocorbula) porcella 61
(Caryocorbula) retusa 82
(Caryocorbula) urumacoensis 82
(Caryocorbula) wakullensis 88
chipolana 76

chipolana carolina 76

chittyana 57

chowanensis 76

colimensis 47, 49, 66, 82, 83, 84, 94
collazica 90

complanata 50

conradi58

constracta 87

(Corbula) 89

(Corbulomya) 50, 53, 84

coxi57

crassa 47

cubaniana 64, 66

cuneata 89

(Cuneocorbula) 51, 53
(Cuneocorbula) caimitica 58
(Cuneocorbula) caribaea pergrata 58
(Cuneocorbula) cercadica 58
(Cuneocorbula) daphnis 58
(Cuneocorbula) helenae 58
(Cuneocorbula) ira 84
(Cuneocorbula) sarda 58
(Cuneocorbula) sericea 58
(Cuneocorbula) smithiana 58
(Cuneocorbula) swiftiana harrisii 58
(Cuneocorbula) whitfieldi boyntoni 58
(Cuneocorbula) whitfieldi stika 58
cymella 90

dautzenberg 57

dietziana 87, 89

disparilis 75

dominicensis 53

ecuabula 79, 80-82

engonata burnsii 89

(Erodona) 71

esmeralda 49, 62, 64, 92

fragilis 50, 53, 54, 55, 57, 61, 63
frequens 71

fulva 57

gatunensis 63

gibba 47, 76

INDEX 369

gibbiformis 76 rosea 85

gibbosa 90 rubra 79, 80-81

glypta 72, 73 sanctidominici 76

granti 78 scutata 64, 66

grovesi 75, 75-76, 78, 93 sematensis 71

heterogena 76 (Serracorbula) 53

(Hexacorbula) 63 speciosa 49, 87, 88, 89-90, 95
(Hexacorbula) cruziana 64 striata 47

(Hexacorbula) esmeralda 63 sulcata 50

(Hexacorbula) gatunensis 64 swiftiana 57, 87, 90

hexacyma 63-64 (Tenuicorbula) 72

inaequalis 58 (Tenuicorbula) tenuis 72
inaequalis mansfieldi 58 tenuis 49, 72, 73, 74, 90, 93

ira 49, 66, 70, 83, 84, 86, 90 tenuis lupina 74

islatrinitalis 76 uruguayensis 57

(Juliacorbula) 64, 80-81, 84 ustulata 70, 72

(Juliacorbula) bicarinata 64 (Varicorbula) 76, 89-90

kelseyi 90 (Varicorbula) granti76

kjoeriana 57 (Varicorbula) grovesi 47, 74
knoxiana 64, 66 (Varicorbula) obesa 76

knoxiana fossilis 66 ventricosa 49, 66, 67-69, 70-72, 82, 84, 92
krebsiana 75 vieta 76

laevis 72 viminea 63

lavalleana 57 vladivostokensis 71

(Lentidium) 50, 61, 84 waltonensis 76

limatula 90 waltonensis rubisiniana 76

luteola 49-50, 63, 84-87, 85, 94 zuliana 76

luteola rosea 84-85, 85 (Corbula), Corbula 89

lyrata 57 Corbulidae 47-48, 50, 363
macdonaldi68, 69, 70 Corbulinae 50

marmorata 49, 58, 84, 86-87, 88, 89-90, 95 Corbulomya 50

nasuta 49-51, 52, 53-55, 54, 56, 57-58, 63, (Corbulomya), Corbula 50, 53, 84
66, 74, 77-78, 84, 86-87, 91 coreanum, Pisidium 165-167, 166
nuciformis 53-54, 54, 57, 77-78 corneum, Sphaerium 166-167
obesa 49, 53, 63, 76-78, 77, 84, 93 corneus, Planorbarius 224, 252
operculata 75, 90 coronatus, Trophon 359

oropendula dolicha 58 costata, Melanopsis 307-308, 313, 315-317,
otra 49, 59, 60-61, 91 320-322

ovulata 49, 58, 59, 60, 90, 91 Melanopsis costata 307, 310-311, 313,
(Panamicorbula) 66-68, 70-71 315-316, 376, 3184321
(Panamicorbula) ventricosa 66, 72 costatum, Plocamium 3

patagonica 78 coxi, Corbula 57

philippi 75 crassa, Corbula 47

polychroma 80-81 crassipes, Eichhornia 273, 276, 279, 281-285
porcella 49-50, 55, 61, 62, 63, 70, 78, 87, Crateritheca insignis 3

92 Crenomytilus grayanus 135, 136-139, 139-
porcellio 61 140-142, 141

(Potamocorbula) amurensis 71-72 Cristataria genezarethana 40, 43-44
prenucia 76 Cristilabrum 320

pustulosa 53, 55, 56, 57, 71 crocea, Tridacna 329

radiata 89 cruziana, Corbula (Hexacorbula) 64

revoluta 57 Victoria 276, 281

370

cubaniana, Corbula 64, 66
Cumingia californica 90
cuneata, Corbula 89
Parvithracia 109
Cuneocorbula 51
pelseneeri51
(Cuneocorbula), Corbula 51, 53
curta, Thracia 108
cuspidarioides, Trophon 353, 358-359, 359-
360
Cyathodonta 113
Cyclophorus 351
cylindrica, Panamicorbula 68, 69, 70
cymella, Corbula 90
Cyperus 154

Dalhousia 319
daniellae, Thorunna 297
daphne, Chromodoris 293-294, 297, 301
daphnis, Corbula (Cuneocorbula) 58
dautzenberg, Corbula 57
declinans, Trophon 353-354, 355, 357, 359,
360
decora, Chromodoris 301
decussata, Noumea 297
deflorata, Venus 50
demersum, Ceratophyllum 5
democraciana, Corbula (Caryocorbula) 82
densa, Elodea 5
Dermatomya 90
mactrioides 90
(Dermatomya), Poromya 90
dermidis, Acromobacter 325
Deroceras caruanae 150
laeve 224, 237
reticulatum 150, 224, 236-237
rodnae 224, 237
dichotama, Rhodymenia 2, 12
Dictyocladium moniliferum 3
diegensis, Asthenothaerus 107
dietziana, Corbula 87, 89
Digidentis 289, 295, 299-300
arbutus 293, 295, 297
discolor, Albinaria 34, 36-37, 40, 42-43
dislocata, Goniobasis catenaria 23-28, 26, 31
disparalis, Varicorbula 90
disparilis, Corbula75
Diversidoris 289, 295, 298-299
aurantionodulosa 293, 295, 297, 301
dolicha, Corbula oropendula 58
dominicensis, Corbula 53
Drepanotrema 273-274, 277, 282, 285

INDEX

anatinum 273, 277-278, 282-285
cimex 273, 277-278, 282-285
kermatoides 277-278, 282-286
lucidum 273, 277-278, 282-285
Durvilledoris 299-300

Echinochloa polystachya 285

ecuabula, Corbula 79, 80-82

edulis, Mytilus 1, 17, 19-21, 140, 252, 350-
351

Eichhornia crassipes 273, 276, 279, 281-285

elata, Vestia 43

elatinoides, Myriophyllum 154

elenensis, Juliacorbula 80, 81-82

elephantipes, Panicum 273, 276, 279, 281-
285

Elimia 23

elisabethina, Chromodoris 293-294, 297

Elodea5
densa5

Elysia 290

Elysiidae 290

emarginata, Thais 255

emilyae, Trophon 353-354, 355, 357, 359,
360

emma, Hypselodoris 297

Enhydra anagallis 276, 279, 281

Enteromorpha 155

Eobania vermiculata 43

ergasticus, Bathypolypus 201, 205-207, 208,
212,214
Benthoctopus 185, 204, 206-207
Octopus 206
Polypus 206

Erodona 66

(Erodona), Corbula 71

Erodonidae 66

esmeralda, Caryocorbula (Hexacorbula) 63
Corbula 49, 62, 64, 92
Corbula (Hexacorbula) 63

Euhadra 351
herklotsi 351

Euperinae 167

Euthyneura 350-351

Eximiothracia 113
concinna 107

faeroensis, Bathypolypus 178, 181, 183-184,
186-187, 203-204, 220
Polypus 177,183, 185, 221

festiva, Hypselodoris 298

fidelis, Chromodoris 301

florens, Thorunna 297

floridensis, Goniobasis 25
fluminea, Corbicula 166
Fluvidona 319

fontandraui, Hypselodoris 300
fossilis, Corbula knoxiana 66
fragilis, Corbula 50, 53, 54, 55, 57, 61, 63
franci, Corbula (Caryocorbula) 88
frequens, Corbula 71

fruticum, Bradybaena 43

fulva, Corbula 57

Gadus morhua 135
gairdneri, Salmo 135
galactos, Chromodoris 301
galloprovincialis, Mytilus 140, 329
gatunensis, Corbula 63
Corbula (Hexacorbula) 64
Hexacorbula 64
Geloina proxima 150
genezarethana, Cristataria 40, 43-44
geometrica, Chromodoris 293-294, 296-301
geversianus, Trophon 354
ghardagana, Risbecia 298
gibba, Corbula 47, 76
Tellina 74
gibbiformis, Corbula 76
gibbosa, Corbula 90
Gigartina 3
alveata 3
marginifera 3
glabrata, Biomphalaria 252, 273
Biophalaria 17, 150
glacialis, Marthasterias 135
glauca, Canna 273, 276, 279, 281-285
Glossodoris 289, 295, 297-300
atromarginata 297
cincta 297
pallescens 290, 297
pallida 297
plumbea 297
sibogae 297
vespa 293, 295, 297
glypta, Corbula 72, 73
Godiva banyulensis 252
Goniobasis 23-24, 27-29
bentoniensis 23, 29
boykiniana viennaensis 23-24, 29
catenaria 23-24, 25, 26-29
catenaria catenaria 23-24, 26, 26-28, 30-31
catenaria dislocata 23-28, 26, 31
catenaria postelli 23-28, 26, 31

INDEX 371

floridensis 25

mutabilis 23, 29

mutabilis timida 23

postellii 23, 29

proxima 23-31, 25-26

suturalis 24, 29

timida 23, 29
Gramineae 276-277, 281
Graneledone pacifica 213
granti, Corbula 78

Corbula (Varicorbula) 76
granulatus, Octopus 176-177
grayanus, Crenomytilus 135, 136-139, 139-

142, 141
grimpei, Bathypolypus 202, 206
groenlandica, Sepia 176
groenlandicus, Octopus 177, 193
grovesi, Corbula 49, 75, 75-76, 78, 93

Corbula (Varicorbula) 47, 74
guineensis, Aloides 50
Gundlachia moricandi 279, 281

haliclona, Noumea 297

Haliotis 320

Haliptilon roseum 3

harrisii, Corbula (Cuneocorbula) swiftiana 58

helenae, Corbula (Cuneocorbula) 58

Heleobias 279, 281

Helicella pappi 43

Helicidae 146

Helix 145, 320
aspersa 146-147, 149
lucorum 145-147, 148, 149-150

hemphillii, Asthenothaerus 107

herklotsi, Euhadra 351

herzogii, Salvinia 276

heterogena, Corbula 76

Hexacorbula 63
gatunensis 64

(Hexacorbula), Corbula 63

hexacyma, Corbula 63-64

hokkaidoensis, Valvata 350

huanghaiensis, Asthenothaerus 112

hupensis, Oncomelania 259-261, 333-334,
342-345
Oncomelania hupensis 259-261, 268, 271,
333-334, 334, 336-337, 338-339, 341-343,
347

Hydrobia 319-320, 322, 345, 351

Hydrobiidae 273, 279

Hydrocotyle ranunculoides 273, 276, 279,
281-285

372

Hypselodoris 289, 293, 296, 298-300
bullocki 293, 296-297
emma 297
festiva 298
fontandraui 300
iacula 300
kanga 298
maculosa 298, 302
midatlantica 300
mouaci 298
obscura 293, 296, 298, 301
orsinii 300
purpureomaculosa 300
rudmani 300
tricolor 252
whitei 298
zebra 298-300
zephyra 293, 296, 298

iacula, Hypselodoris 300

ichyodermidis, Pseudomoans 325

llyanassa obsoleta 252

imperialis, Risbecia 300

inaequalis, Corbula 58

incisa, Lytocarpia 3

indica, Rotala 5

inflata, Potamomya 66, 68, 68, 70

inornata, Chromodoris 297

insignis, Crateritheca 3

intermedia, Spirodela 276, 281

isaotakii, Asthenothaerus 107-108, 131
Parvithracia (Pseudoasthenothaerus) 107,
119, 122, 126, 129, 131-132, 131

islandica, Arctica 140

islatrinitalis, Corbula 76

ira, Corbula 49, 66, 70, 83, 84, 86, 90
Corbula (Cuneocorbula) 84

Ixartia 113

januarii, Benthoctopus 204
japonica, Corbicula 166
Schistosoma 259
Thracidora 108
japonicum, Schistosoma 333-334, 344-345
jordanica, Melanopsis costata 307-309, 311,
313, 315-316, 316, 318, 321
Juliacorbula 64, 80-81, 84
elenensis 80, 81-82
(Juliacorbula), Corbula 64, 80-81, 84

kakumana, Thracia 108
kanga, Hypselodoris 298

INDEX

Katharina 351
tunicata 351

kelseyi, Corbula 90

kermatoides, Drepanotrema 277-278, 282-
286

kjoeriana, Corbula 57

knoxiana, Corbula 64, 66

krebsiana, Corbula 75

kuiteri, Chromodoris 293-294, 297

kuniei, Chromodoris 293-294, 297-298, 301

laeve, Deroceras 224, 237

laevis, Corbula 72

laingii, Champia 3

Lampeia 107-108, 112-113
adamsi 108
posteroresecta 108
triangula 108

Lasaea 165

Laurencia thyrsifera 3

lavalleana, Corbula 57

leana, Corbicula 166
Corbicula (Corbiculina) 165

Lehmannia marginata 223-226, 228-229, 230-

233, 233-234, 235, 236-238
Lemnaceae 276
Lentidiinae 50
Lentidium 50
mediterraneum 47
(Lentidium), Corbula 61, 84
lentus, Bathypolypus 191, 193
Octopus 184-185, 187, 191, 219
Polypus 187
leonensis, Corbula barrattiana 89
leopardus, Chromodoris 293-294, 297, 301
Limacidae 223-224
Limacoidea 223-224
limatula, Corbula 90
lineolata, Chromodoris 297
Littorina 351
saxatilis 350-351
Littorinioidea 350-351
lochi, Chromodoris 293, 295, 297
Loligo 351
bleekeri 351
loringi, Chromodoris 301
lucida, Pterocladia 3
lucidum, Drepanotrema 273, 277-278, 282-
285
lucorum, Helix 145-147, 148, 149-150
Ludwigia 154-155
peploides 276, 281

INDEX 373

lukini, Parvithracia (Pseudo- meiostoma 307-311, 313, 315-318, 376,

asthenothaerus) 107, 114, 115-118, 119- 320-321

120, 121, 122, 125-126, 128-129, 132 saulcyi 307-308, 310-311, 313, 315-318,

Pseudoasthenothaerus 111 316, 320-322
lupina, Corbula tenuis 74 Melanthalia abscissa 2-3
lusitanicus, Arion 224 Mercenaria mercenaria 329
lusoria, Pachymenia 3 mercenaria, Mercenaria 329

Meretrix 243, 325-330, 328-329 Meretrix 326-328
luteola, Corbula 49-50, 63, 84-87, 85, 94 lusoria 243, 325-330, 328-329
luteomarginata, Cadlina 297 Mexichromis 299-300
Lymnaea stagnalis 142, 150, 224, 236-237, multituberculata 297

252 Miamira 297
lyrata, Corbula 57 midatlantica, Hypselodoris 300
Lytocarpia incisa 3 Minicorbula 50

modesta, Cadlina 297
macdonaldi, Corbula 68, 69, 70 moniliferum, Dictyocladium 3
Mactra 90 Montacuta triquetra 109

veneriformis 243, 252 montrouzieri, Thorunna 297
mactrioides, Dermatomya 90 morhua, Gadus 135
maculosa, Hypselodoris 298, 302 mori, Bombyx 19

Pisania 255 moricandi, Gundlachia 279, 281
magellanicus, Murex 354 mouaci, Hypselodoris 298
magnifica, Ceratosoma 297 mülleri, Cirroteuthis 177

Chromodoris 297-298 Mulinia pallida 90
Malacolimax tenellus 224, 237 multituberculata, Mexichromis 297
Manduca sexta 19 Murex magellanicus 354
mansfieldi, Corbula inaequalis 58 Muricidae 241, 251, 254-255, 353-354
mansoni, Schistosoma 273-274 Musculium securis 166-167
marginata, Chromodoris 301 mutabilis, Goniobasis 23, 29

Lehmannia 223-226, 228-229, 230-233, Mya 47,66

233-234, 235, 236-238 plana 66
marginifera, Gigartina 3 truncata 90
marinus, Bufo 135 Myoidea 47, 66
marmorata, Corbula 49, 58, 84, 86-87, 88, 89- myopsis, Thracia 108

90, 95 Myriophyllum elatinoides 154

Stenophysa 279, 281 verticillatum 5
Marthasterias glacialis 135 Mytilidae 167
maximus, Pecten 351 Mytilus 350-351
mediterranea, Tellina 50 edulis 1, 17, 19-21, 140, 252, 350-351
mediterraneum, Lentidium 47 galloprovincialis 140, 329
meiostoma, Melanopsis 307-311, 313, 315-

318, 316, 320-321 nasuta, Caryocorbula 53
melajoensis, Tenuicorbula 74 (Caryocorbula) Aloidis 53
Melanopsidae 307 Corbula 49-51, 52, 53-55, 54, 56, 57-58, 63,
Melanopsis 307, 319-322 66, 74, 77-78, 84, 86-87, 91

buccinoidea 307-308, 311, 313, 315-318, Corbula (Caryocorbula) 50, 53

316, 320-322 nemoralis, Cepaea 320, 351

costata 307-308, 313, 315-317, 320-322 Nerita 350-351

costata costata 307, 310-311, 313, 315- atramentosa 350

316, 316, 318, 321 Neritopsina 351

costata jordanica 307-309, 311, 313, 315- Ninfaceae 276

316, 316, 318, 321 nobilis, Tyrinna 297

374 INDEX

Nodilittorina 351

Nodulotrophon 359

norba, Noumea 293, 296-297, 301

normani, Benthoctopus 185
Polypus 185

Noumea 289, 296, 299-300
decussata 297
haliclona 297
norba 293, 296-297, 301
simplex 297

nuciformis, Corbula 53-54, 54, 57, 77-78

obesa, Corbula 49, 53, 63, 76-78, 77, 84, 93
Corbula (Varicorbula) 76
obesus, Bathypolypus 191-193
Octopus 184, 187, 191,219
obscura, Hypselodoris 293, 296, 298, 301
obseleta, Chromodoris 297
obsoleta, Ilyanassa 252
occidentale, Sphaerium 166-167
occidentalis, Biomphalaria 286
octopodia, Sepia 176
Octopodidae 175, 204
Octopodinae 202
Octopus 202, 204
arcticus 175, 177, 187, 202, 204, 221
bairdii 175, 184-185, 187, 191, 194, 219
ergasticus 206
granulatus 176-177
groenlandicus 177,193
lentus 184-185, 187, 191,219
obesus 184, 187, 191, 219
piscatorum 177, 184-185, 187, 194, 202,
204
profundicola 206-207
sponsalis 205
officinalis, Corallina 3
ohlini, Trophon 357
Omalogyra 351
Omalogyroidea 351
Onagraceae 276
Oncomelania 319-320, 333-335, 343-345
hupensis 259-261, 333-334, 342-345
hupensis hupensis 259-261, 268, 271, 333-
334, 334, 336-337, 338-339, 341-343, 347
hupensis robertsoni 259-261, 268, 271,
333, 342-343
hupensis tangi 334
operculata, Corbula 75, 90
orientalis, Chromodoris 290, 297-298
ornatissima, Cadlinella 290, 297, 301
orsinii, Hypselodoris 300

Osmundaria colensoi2, 12

otra, Corbula 49, 59, 60-61, 91
Corbula (Caryocorbula) 47, 58

ovulata, Corbula 49, 58, 59, 60, 90, 91
Corbula (Caryocorbula) 60

Pachymenia lusoria 3

pacifica, Graneledone 213

pallens, Boettgerilla 223-225, 228-229, 230-
233, 233-234, 235, 236-238

pallescens, Glossodoris 290, 297

pallida, Glossodoris 297
Mulinia 90

Panamicorbula 66-68, 70
cylindrica 68, 69, 70
trigonalis 70

(Panamicorbula) 66, 70
Corbula 66-68, 70-71

Pandalus 211

Panicum elephantipes 273, 276, 279, 281-
285

pappi, Helicella 43

papyracea, Corbicula 166

Parvithracia 107, 109, 112, 122, 129, 131,
133
cuneata 109
(Parvithracia) 113
(Parvithracia) suteri 109, 110, 111-112, 122
(Pseudoasthenothaerus) 112-114
(Pseudoasthenothaerus) isaotakii 107, 119,
122, 126, 129, 131-132, 131
(Pseudoasthenothaerus) lukini 107, 114,
115-118, 119-120, 121, 122, 125-126, 128-
129, 132
(Pseudoasthenothaerus) sematana 107,
119, 122, 126, 129, 130, 131, 132-133
(Pseudoasthenothaerus) sirenkoi 107,
119, 122-123, 123-124, 126-129, 128, 132
sematana 126
suteri 107-109

(Parvithracia), Parvithracia 113

Paspalum repens 285

patagonica, Corbula 78

Patellogastropoda 351

paulensis, Penaeus 19

Pecten 351
maximus 351

Pectenodoris 289, 296, 299-300
trilineata 293, 296-297

Pectinidae 167

pellucida, Cadlina 297

pelseneeri, Cuneocorbula 51

INDEX 375

Penaeus californiensis 19
paulensis 19
peploides, Ludwigia 276, 281
pergrata, Corbula (Cuneocorbula) caribaea 58
Peringia ulvae 342
Perna canaliculus 1, 2, 13
perna 17, 329-330
viridis 17, 19
perna, Perna 17, 329-330
perola, Chromodoris 297
perversa, Balea 43
philippii, Corbula 75
philippinarum, Ruditapes 243
Venerupis 326, 330
Pholadomyoida 109
Phragmorisma 113
Physa acuta 252
Physidae 279
Pisania maculosa 255
piscatorum, Benthoctopus 175, 178, 183-186,
202-203, 207, 220
Octopus 177, 184-185, 187, 194, 202, 204
Polypus 177, 185
Pisidiidae 165, 167
Pisidiinae 167
Pisidium 167
casertanum 166-167
coreanum 165-167, 166
Pistia stratiotes 273, 276, 279, 281-285
plana, Mya 66
Planorbarius corneus 224, 252
Planorbidae 273-274
Pleurobranchaea Californica 135
Pleuroceridae 23, 343
Plocamium costatum 3
plumbea, Glossodoris 297
Polybranchiidae 290
polychroma, Corbula 80-81
Polypus arcticus 187
ergasticus 206
faeroensis 177, 183, 185, 221
lentus 187
normani 185
piscatorum 177, 185
profundicola 206
salebrosus 204
polystachya, Echinochloa 285
Pomacea 160-161, 351
canaliculata 153, 157, 159-161, 279, 281
scalaris 281
Pomatiopsidae 259, 333
Pontederiaceae 276

porcella, Corbula 49-50, 55, 61, 62, 63, 70,
7919798
Corbula (Caryocorbula) 61
porcellio, Corbula 61
Poromya 71
(Dermatomya) 90
postellii, Goniobasis 23, 29
Goniobasis catenaria 23-28, 26, 31
posteroresecta, Lampeia 108
Potamocorbula 71
(Potamocorbula) 71
Potamogeton 154-155
Potamomya 66
aequalis 66-67, 67, 70
inflata 66, 68, 68, 70
triagonalis 67
trigonalis 67, 68, 70
preciosa, Chromodoris 301
prenasuta, Caryocorbula (Caryocorbula) 58
prenucia, Corbula 76
profundicola, Octopus 206-207
Polypus 206
proschi, Bathypolypus 175, 187, 194-195, 204
proxima, Geloina 150
Goniobasis 23-31, 25-26
Psammobiidae 50
Pseudoasthenothaerus 107, 111-112
lukini 111
(Pseudoasthenothaerus), Parvithracia 112-114
Pseudomoans ichyodermidis 325
Psilaxis 350-351
radiatus 350
Pterocladia capillacea 3
lucida 3
pugniger, Bathypolypus 175, 187, 191, 195-
196, 196-197, 198, 199-200, 200-204, 206-
207, 208-209, 212-214, 222
pulchella, Risbecia 298, 300
punicea, Thorunna 300
Pupa 351
purpuratus, Argopecten 165
purpureomaculosa, Hypselodoris 300
pusilla, Trigonothracia 108, 126
pustulosa, Corbula 53, 55, 56, 57, 71
Pyramidelloidea 351

quadricolor, Chromodoris 297-298

radiata, Corbula 89

radiatus, Psilaxis 350

ranunculoides, Hydrocotyle 273, 276, 279,
281-285

376

Rapana 254
venosa 241-244, 243, 245-246, 247, 248,
249-255, 250-251
reiniana, Semisulcospira 343
repens, Paspalum 285
reticulatum, Deroceras 150, 224, 236-237
reticulatus, Agriolimax 150, 252
retusa, Corbula (Caryocorbula) 82
revoluta, Corbula 57
Rhodymenia dichotama 2, 12
Risbecia 299-300
ghardagana 298
imperialis 300
pulchella 298, 300
tryoni 298, 300, 302
Rissooidea 333, 351
robertsoni, Oncomelania hupensis 259-261,
268, 271, 333, 342-343
roboi, Chromodoris 293, 295, 297-298, 301
rodnae, Deroceras 224, 237
rosea, Corbula 85
Corbula luteola 84-85, 85
roseum, Haliptilon 3
Rostanga 302
Rotala indica5
rotundifolia, Salvinia 276
rubens, Asterias 135
rubisiniana, Corbula waltonensis 76
rubra, Corbula 79, 80-81
rubrocornuta, Chromodoris 301
Ruditapes philippinarum 243
rudmani, Hypselodoris 300

salebrosus, Bathypolypus 204-205
Benthoctopus 204-205, 207
Polypus 204
Salmo gairdneri 135
Salvinaceae 276
Salvinia herzogii 276
rotundifolia 276
sanctidominici, Corbula 76
sandai, Corbicula 166
sarda, Corbula (Cuneocorbula) 58
sasakii, Benthoctopus 178, 183-186, 220
saulcyi, Melanopsis 307-308, 310-311, 313,
315-318, 316, 320-322
saxatilis, Littorina 350-351
Scaeurgus 213
scalaris, Pomacea 281
Schistosoma 259
Japonica 259
japonicum 333-334, 344-345

INDEX

mansoni273-274

Schoenoplectus californicus 154

scolopax, Trophon 353, 356, 357, 358-359,
360

scutata, Corbula 64, 66

securis, Musculium 166-167

sematana, Asthenothaerus 107, 126
Parvithracia 126
Parvithracia (Pseudoasthenothaerus) 107,
119, 122, 126, 129 130131182485
Thracia 126

sematanus, Asthenothaerus 126

sematensis, Asthenothaerus 126
Corbula 71

seminuda, Thracia 108

Semisulcospira reiniana 343

senegalensis, Siratus 255

Sepia groenlandica 176
octopodia 176

septentrionalis, Thracia 108

septus, Trophon 353, 358, 359-360

sericea, Corbula (Cuneocorbula) 58

Serpulorbis 351

Serracorbula 51
tumaca 51, 53, 55, 56, 57, 363

(Serracorbula), Corbula 53

sexta, Manduca 19

sibogae, Glossodoris 297

simplex, Noumea 297

Siphonaria 351

Siratus senegalensis 255

sirenkoi, Parvithracia (Pseudo-
asthenothaerus) 107, 119, 122-123, 123-
124, 126-129, 128, 132

smithiana, Corbula (Cuneocorbula) 58

Solidicorbula 50

speciosa, Corbula 49, 87, 88, 89-90, 95

Sphaeriidae 166

Sphaeriinae 167

Sphaerium corneum 166-167
occidentale 166-167
striatinum 165-167

spiralis, Vallisneria 5

Spirodela intermedia 276, 281

sponsalis, Bathypolypus 194, 201-202, 204-
207, 206, 208, 212-214
Octopus 205

Squalus acanthias 135

stagnalis, Lymnaea 142, 150, 224, 236-237,
252

Stagnicola 320

stainforthi, Corbula aequivalvis 66

INDEX 377

Stenophysa marmorata 279, 281
stika, Corbula (Cuneocorbula) whitfieldi 58
straminea, Biomphalaria 273, 277-278, 282-286
stratiotes, Pistia 273, 276, 279, 281-285
striata, Corbula 47
striatinum, Sphaerium 165-167
strigata, Chromodoris 293, 295, 297
sulcata, Corbula 50
suteri, Parvithracia 107-109
Parvithracia (Parvithracia) 109, 110, 111-
112122
suturalis, Goniobasis 24, 29
swiftiana, Corbula 57, 87, 90

takatuayamaensis, Corbula amurensis 71
Tandonia budapestensis 224
tangi, Oncomelania hupensis 334
Tapes waltingii 140
tapetis, Vibrio 330
Tectus 320
Tellina 50, 57

gibba 74

mediterranea 50
tenagophila, Biomphalaria 273, 277-278, 282-

286
tenellus, Malacolimax 224, 237
Tenuicorbula 72

melajoensis 74
(Tenuicorbula) 72

Aloidis 53

Corbula 72
tenuis, Corbula 49, 72, 73, 74, 90, 93

Corbula (Tenuicorbula) 72
Thais bronni 254

clavigera 254

emarginata 255

tissoti 254
thalictroides, Ceratopteris 5
thompsoni, Chromodoris 301
Thorunna 299-300

australis 297

daniellae 297

florens 297

montrouzieri 297

punicea 300
Thracia 107, 113, 118

curta 108

kakumana 108

myopsis 108

sematana 126

seminuda 108

septentrionalis 108

Thracidora 113
japonica 108
Thraciidae 107, 109
Thracioidea 109
Thraciopsis 113
thyrsifera, Laurencia 3
timida, Goniobasis 23, 29
Goniobasis mutabilis 23
tinctoria, Chromodoris 293, 295, 297, 301
tissoti, Thais 254
torrefactus, Chicoreus 255
triagonalis, Potamomya 67
triangula, Lampeia 108
tricolor, Hypselodoris 252
Tridacna crocea 329
trigonalis, Panamicorbula 70
Potamomya 67, 68, 70
Trigonothracia 108, 113
pusilla 108, 126
trilineata, Pectenodoris 293, 296-297
triquetra, Montacuta 109
Trochus 320
Trophon 353-354, 359-360
acanthodes 353
arnaudi 353, 356, 357-360, 359
barvicensis 360
coronatus 359
cuspidarioides 353, 358-359, 359-360
declinans 353-354, 355, 357, 359, 360
emilyae 353-354, 355, 357, 359, 360
geversianus 354
ohlini 357
scolopax 353, 356, 357, 358-359, 360
septus 353, 358, 359-360
Trophoninae 353-354
Trophonopsis 360
truncata, Mya 90
tryoni, Risbecia 298, 300, 302
Tulotoma 351
tumaca, Serracorbula 51, 53, 55, 56, 57, 363
tunicata, Katharina 351
turrita, Albinaria 34, 36-37, 40, 42-43, 351
Typha 154
Tyrinna 299-301
nobilis 297

ulvae, Peringia 342

Umbeliferae 276

uruguayensis, Corbula 57

urumacoensis, Corbula (Caryocorbula) 82
ustulata, Corbula 70, 72

378

valdiviae, Bathypolypus 201-202, 204, 206-
207,212
Polypus 206
Vallisneria americana 5
spiralis 5
Valvata 350-351
hokkaidoensis 350
Valvatoidea 351
Varicorbula 74, 76, 89-90
disparalis 90
(Varicorbula) 74
Corbula 76, 89-90
Veneridae 325
veneriformis, Mactra 243, 252
Veneroida 165
Veneroidea 167
Venerupis philippinarum 326, 330

venosa, Rapana 241-244, 243, 245-246, 247,

248, 249-255, 250-251

ventricosa, Corbula 49, 66, 67-69, 70-72, 82,

84, 92

Corbula (Panamicorbula) 66, 72
Venus antiqua 17-21, 19

deflorata 50
Verconia 298-300

verconis 297
verconis, Verconia 297
Vermetoidea 351
vermiculata, Eobania 43
verticillatum, Myriophyllum 5

INDEX

vespa, Glossodoris 293, 295, 297
Vestia elata 43
Vetigastropoda 351
vibrata, Chromodoris 301
Vibrio 326
angillarum 325
tapetis 330
Victoria cruziana 276, 281
viennaensis, Goniobasis boykiniana 23-24,
29
vieta, Corbula 76
viminea, Corbula 63
viridis, Perna 17, 19
Viviparus 319-320
contectus 320
vladivostokensis, Corbula 71
voithii, Albinaria 34, 36-37, 40, 42-44

wakullensis, Corbula (Caryocorbula) 88
waltingii, Tapes 140

waltonensis, Corbula 76

whitei, Hypselodoris 298

willani, Chromodoris 297

woodwardae, Chromodoris 297

Xymenopsis 353
zebra, Hypselodoris 298-300

zephyra, Hypselodoris 293, 296, 298
zuliana, Corbula 76

Vol.
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. 1-2

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2002

VOL. 44, NO. 2 MALACOLOGIA 2002

CONTENTS

EE-YUNG CHUNG, SUNG-YEON KIM, KWAN HA PARK, & GAB-MAN PARK

Sexual Maturation, Spawning, and Deposition of the Egg Capsules of the

Female Purple Shell, Rapana venosa (Gastropoda: Muricidae) .......... 241
EUGENE V. COAN

Correction to: Coan, Eugene V., 2002, The Eastern Pacific Recent Species

of the Corbulidae (bivalvia). Malacologia 44(1): 47-105 ................ 363
ANDRZEJ FALNIOWSKI, JOSEPH HELLER, MAGDALENA SZAROWSKA, &
KRYSTYNA MAZAN-MAMCZARZ

Allozymic Taxonomy within the Genus Melanopsis (Gastropoda: Cerithi-

acea) in Israel: A Case in which Slight Differences Are Congruent ........ 307
ANA MARIA LEAL-ZANCHET

Ultrastructure of the Supporting Cells and Secretory Cells of the Alimentary

Canal of the Slugs, Lehmannia marginata and Boettgerilla pallens (Pul-

топа: Stylommatophora: Limacoidea): =... 0. ee ens ee ee 223
CHARLES LYDEARD, WALLACE E. HOLZNAGEL, REI UESHIMA, &
ATSUSHI KURABAYASHI

Systematic Implications of Extreme Loss or Reduction of Mitochondrial LSU

FRNA Helical-Loop Structures in'Gastropods «05... three eas 332
BENT MUUS

The Bathypolypus-Benthoctopus problem of the North Atlantic

(Octopodidae Cephalopoda) ee nn ae 175

SUNG-WOO PARK, KANG-SOO LEE & EE-YUNG CHUNG
Morphological and Cytochemical Characteristics of Hemocytes of Meretrix

возопа (Е мама менее ав inn. RE ee ee a 325
GUIDO PASTORINO
Two New Trophoninae (Gastropoda: Muricidae) from Antarctic Waters .... 353

A. RUMI, J. A. BECHARA, M. I. HAMANN, & M. OSTROWSKI DE NUNEZ
Ecology of Potential Hosts of Schistosomiasis in Urban Environments of
Ghaco Argentina Arte a ee re me antenne ae 273

EDMUND Y. W. SETO, WEIPING WU, DONGCHUAN QIU, HONGYUN LIU,
XUEGUANG GU, HONGGEN CHEN, ROBERT C. SPEAR,
& GEORGE M. DAVIS
Impact of Soil Chemistry on the Distribution of Oncomelania hupensis
(Gastropoda: Pomatiopsidae):in China "#72... 259
CHAO-HUI SHI, THOMAS WILKE, GEORGE M. DAVIS, MING-YI XIA,
& CHI-PING QIU
Population Genetics, Micro-Phylogeography, Ecology, and Susceptibility to
Schistosome Infection of Chinese Oncomelania hupensis hupensis
(Gastropoda: Rissooidea: Pomatiopsidae) in the Miao River System . ..... 333
NERIDA G. WILSON
Egg Masses of Chromodorid Nudibranchs (Mollusca: Gastropoda: Opistho-
a sare ee Ne ee ee 289

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VOL. 44, NO. 2 MALACOLOGIA

CONTENTS

BENT MUUS
The Bathypolypus-Benthoctopus problem of the North Atlantic
(Setepedidas; Cophalapoda) us ta а wens er de an
ANA MARIA LEAL-ZANCHET
Ultrastructure of the Supporting Cells and Secretory Cells of the Alimentary
Canal of the Slugs, Lehmannia marginata and Boettgerilla pallens (Pul-
monata: Stylommatophora: Limacoidea) ..........................
EE-YUNG CHUNG, SUNG-YEON KIM, KWAN HA PARK, & GAB-MAN PARK
Sexual Maturation, Spawning, and Deposition of the Egg Capsules of the
Female Purple Shell, Rapana venosa (Gastropoda: Muricidae) .........
EDMUND Y. W. SETO, WEIPING WU, DONGCHUAN QIU, HONGYUN LIU,
XUEGUANG GU, HONGGEN CHEN, ROBERT C. SPEAR,
& GEORGE M. DAVIS
Impact of Soil Chemistry on the Distribution of Опсотеата hupensis
(Gastropoda: Pomatiopsidae) in China ............................
A. RUMI, J. A. BECHARA, М. I. HAMANN, & М. OSTROWSKI DE NUNEZ
Ecology of Potential Hosts of Schistosomiasis in Urban Environments of
E A es Soe
NERIDA G. WILSON
Egg Masses of Chromodorid Nudibranchs (Mollusca: Gastropoda: Opistho-
MA nak ee es oo age Hons grees on eta re apes ba ea ER
ANDRZEJ FALNIOWSKI, JOSEPH HELLER, MAGDALENA SZAROWSKA, &
KRYSTYNA MAZAN-MAMCZARZ
Allozymic Taxonomy within the Genus Melanopsis (Gastropoda: Cerithi-
acea) in Israel: A Case in which Slight Differences Are Congruent .......
SUNG-WOO PARK, KANG-SOO LEE & EE-YUNG CHUNG
Morphological and Cytochemical Characteristics of Hemocytes of Meretrix
Lusona (Bivalvia: VONOrdaS) 2.2... csc ca ses sun. dead goss a
CHAO-HUI SHI, THOMAS WILKE, GEORGE M. DAVIS, MING-YI XIA,
& CHI-PING QIU
Population Genetics, Micro-Phylogeography, Ecology, and Susceptibility to
Schistosome Infection of Chinese Oncomelania hupensis hupensis
(Gastropoda: Rissooidea: Pomatiopsidae) in the Miao River System .....
RESEARCH NOTES
CHARLES LYDEARD, WALLACE E. HOLZNAGEL, REI UESHIMA, &
ATSUSHI KURABAYASHI
Systematic Implications of Extreme Loss or Reduction of Mitochondrial LSU
rRNA Helical-Loop Structures in Gastropods ..........................
GUIDO PASTORINO
Two New Trophoninae (Gastropoda: Muricidae) from Antarctic Waters ....
EUGENE V. COAN
Correction to: Coan, Eugene V., 2002, The Eastern Pacific Recent Species
of the Corbulidae (bivalvia). Malacologia 44(1): 47-105 ...............

2002

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333

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