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Full text of "Malacologia"

HARVARD UNIVERSITY 




LIBRARY 

OF THE 

DEPARTMENT OF MOLLUSKS 

IN THE 

Museum of Comparative Zoology 
Gift of: 



VOL 41, NO. 2 1999 



MALACOLOGIA 



International Journal of Malacology 



INTERACTIONS BETWEEN MAN AND MOLLUSCS 



UNITAS MALACOLOGICA-AMERICAN MALACOLOGIGAL SOCIETY SYMPOSIUM 

WASHINGTON, D.C. 
26-30 JULY 1998 



Publication dates 

Vol.31, No. 1 29 Dec. 1989 

Vol. 31, No. 2 28 May 1990 

Vol.32, No. 1 30 Nov. 1990 

Vol.32, No. 2 7 Jun. 1991 

Vol. 33, No. 1-2 6 Sep. 1991 

Vol. 34, No. 1-2 9 Sep. 1992 

Vol. 35, No. 1 14 Jul. 1993 

Vol. 35, No. 2 2 Dec. 1993 

Vol. 36, No. 1-2 8 Jan. 1995 

Vol. 37, No. 1 13 Nov. 1995 

Vol.37, No. 2 8 Mar. 1996 

Vol.38, No. 1-2 17 Dec. 1996 

Vol.39, No. 1-2 13 May 1998 

Vol. 40, No. 1-2 17 Dec. 1998 

Vol.41, No. 1 22 Sep. 1999 



VOL. 41, NO. 2 MALACOLOGIA 1999 

CONTENTS 

DANIEL L. ALKON, PLENARY ADDRESS 

Molecular Principles of Associative Memory that are Conserved During the 
Evolution of Species 321 

A. T ABD ALLAH, S. N. THOMPSON, D. B. BORCHARDT & M. 0. A. WANAS 

Biomphalaria glabrata: A Laboratory Model Illustrating the Potential of 
Pulmonate Gastropods as Freshwater Biomonitors of Heavy Metal 
Pollutants 345 

ROLAND С ANDERSON, PAUL D. HUGHES, JENNIFER A. MATHER & 

CRAIG W. STEELE 

Determination of the Diet of Octopus rebescens Berry, 1953 (Cephalopoda: 
Octopodidae), Through Examination of Its Beer Bottle Dens in Puget Sound 455 

THERESE D. BRACKENBURY & С С. APPLETON 

Structural Damage to the Foot-sole Epithelium of Bulinus africanus 
Following Exposure to a Plant Molluscicide 393 

JAMES T CARLTON 

Molluscan Invasions in Marine and Estuarine Communities 439 

GEORGE M. DAVIS 

Introduction 319 

GEORGE M. DAVIS, THOMAS WILKE, Yl ZHANG, XING-JIANG XU, 

CHI-PING OIU, CHRISTINA SPOLSKY DONG-CHUAN OIU, YUESHENG LI, 

MING-YI XIA & ZHENG FENG 

SnaW- Shistosoma, Paragonimus Interactions in China: Population Ecology, 
Genetic Diversity, Coevolution and Emerging Diseases 355 

THOMAS K. KRISTENSEN & DAVID S. BROWN 

Control of Intermediate Host Snails for Parasitic Diseases— A Threat to 
Biodiversity in African Freshwaters? 379 

MARIA CHRISTINA DREHER MANSUR & MARIA DA GRAÇA OLIVEIRA DA SILVA 

Description of Glochidia of Five Species of Freshwater Mussels (Hyriidae: 
Unionoidea) from South America 475 

RICHARD NEVES 

Conservation and Commerce: Management of Freshwater Mussel (Bivalvia: 
Unionoidea) Resources in the United States 461 

J. P. POINTIER 

Invading Freshwater Gastropods: Some Conflicting Aspects for Public 
Health 403 

DAVID G. ROBINSON 

Alien Invasions: The Effects of the Global Economy on Non-marine 
Gastropod Introductions into the United States 413 

TIMOTHY P YOSHINO, CHRISTINE COUSTAU, SYLVAIN MODAT & 

MARIA G.CASTILLO 

The Biomphalaria glabrata Embryonic (BGE) Molluscan Cell Line: 
Establishment of an In Vitro Cellular Model for the Study of Snail Host- 
Parasite Interactions 331 



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Address: Malacologia 

Department of Malacology 
Academy of Natural Sciences 
1900 Benjamin Franklin Parkway 
Philadelphia, PA 19103-1195, U.S.A. 



VOL 41, NO. 1 1999 



MALACOLOGIA 



International Journal of Malacology 
Revista Internacional de Malacologia 
Journal International de Malacologie 
Международный Журнал Малакологии 
Internationale Malakologische Zeitschrift 



MALACOLOGIA 

EDITOR-IN-CHIEF: 
GEORGE M. DAVIS 

Editorial and Subscription Offices: 

Department of Malacology 

The Academy of Natural Sciences of Philadelphia 

1900 Benjamin Franklin Parkway 

Philadelphia, Pennsylvania 19103-1195, U.S.A. 



EUGENE GOAN 

California Academy of Sciences 

San Francisco, CA 



Co-Editors: 



Assistant Managing Editor: 

CARYL HESTERMAN 

Associate Editor: 

JOHN B. BURGH 

University of Michigan 

Ann Arbor 



CAROL JONES 
Denver, CO 



MALACOLOGIA is published by the INSTITUTE OF MALACOLOGY, the Sponsor Members of 
which (also serving as editors) are: 



RÜDIGER BIELER 

President 

Field Museum, Chicago 

JOHN BURGH 

MELBOURNE R. CARRIKER 
University of Delaware, Lewes 

GEORGE M. DAVIS 
Secretary and Treasurer 

CAROLE S. HICKMAN 

President Elect 

University of California, Berkeley 



ALAN KOHN 

University of Washington, Seattle 

JAMES NYBAKKEN 

Vice President 

Moss Landing Marine Laboratory, California 

CLYDE F. E. ROPER 

Smithsonian Institution, Washington, D.C. 

SHI-KUEI WU 

University of Colorado Museum, Boulder 



Participating Members 



EDMUND GITTENBERGER 

Secretary, UNITAS MALACOLOGICA 

Rijksmuseum van Natuurlijke 

Historie 

Leiden, Netherlands 



JACKIE L. VAN GOETHEM 
Treasurer, UNITAS MALACOLOGICA 
Koninklijk Belgisch Instituut 
voor NatuuHA/etenschappen 
Brüssel, Belgium 



Emeritus Members 



J. FRANCES ALLEN, Emérita 
Environmental Protection Agency 
Washington, D.C. 

KENNETH J. BOSS 

Museum of Comparative Zoology 

Cambridge, Massachusetts 



ROBERT ROBERTSON 

The Academy of Natural Sciences 

Philadelphia, Pennsylvania 

W. D. RUSSELL-HUNTER 
Easton, Maryland 



Copyright © 1999 by the Institute of Malacology 
ISSN: 0076-2997 



1999 
EDITORIAL BOARD 



J.A.ALLEN 

Marine Biological Station 

Millport, United Kingdom 

E. E. BINDER 

Museum d'Histoire Naturelle 

Geneve. Switzerland 

A.J.CAIN 

University of Liverpool 
United Kingdom 

P. CA LOW 

University of Sheffield 
United Kingdom 

J. G. CARTER 

University of North Carolina 

Chapel Hill. U.S.A. 

R. COWIE 
Bishop Museum 
Honolulu, HI.. U.S.A. 

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

B. С 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 

F. GIUSTI 

Universita di Siena. Italy 

A. N. GOLIKOV 
Zoological Institute 
St Petersburg. Russia 



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

A. V. GROSSU 
Universitatea Bucuresti 
Romania 

J. HABE 

Tokai University 

Shimizu. Japan 

R. HANLON 

Marine Biological Laboratory 

Woods Hole. Mass., U.S.A. 

J. A. HENDRICKSON, Jr. 
Academy of Natural Sciences 
Philadelphia, PA, U.S.A. 

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

K. E. HOAGLAND 

Council for Undergraduate Research 

Washington, DC. U.S.A. 

B. HUBENDICK 
Naturhistoriska Museet 
Göteborg, Sweden 

S. HUNT 
Lancashire 
United Kingdom 

R. JANSSEN 

Forschungsinstitut Senckenberg. 
Frankfurt am Main, Germany 

R. N. KILBURN 
Natal Museum 
Pietermaritzburg, South Africa 

M. A. KLAPPENBACH 

Museo Nacional de Historia Natural 

Montevideo. Uruguay 

J. KNUDSEN 

Zoologisk Institut Museum 

Kobenhavn, Denmark 

A. LUCAS 

Faculte des Sciences 

Brest, France 



с. MEIER-BROOK 
Tropenmedizinisches Institut 
Tubingen. Germany 

H. 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 
Mahne Biological Station 
Porto Novo. India 

DIARMAIDO'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 Oswalde Cruz. Rio de Janeiro 

Brazil 

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

J. P. POINTIER 

Ecole Pratique des Hautes Etudes 

Perpignan Cedex. France 

W. F. PONDER 
Australian Museum 
Sydney 

Ol Z. Y. 

Academia Sínica 

Qingdao. People s Republic of China 

D. G. REID 

The Natural History Museum 

London. United Kingdom 



S. G. SEGERSTRALE 
Institute of Mahne Research 
Helsinki. Finland 

A. STANCZYKOWSKA 
Siedlce. Poland 

F. STARMÜHLNER 

Zoologisches Institut der Universität 

Wien. Austria 

Y I. STAROBOGATOV 
Zoological Institute 
St. Petersburg. Russia 

W. STREIFE 
Universite de Caen 
France 

J. STUARDO 
Universidad de Chile 
Valparaiso 

S. TILLIER 

Museum National d'Histoire Naturelle 

Pahs. France 

R. D.TURNER 
Harvard University 
Cambridge. Mass.. U.S.A. 

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

N. H. VERDONK 

Rijksuniversiteit 
Utrecht, Netherlands 

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, 1999, 41(1): 1-118 

DESCRIPTIONS OF SOME OF THE GLOCHIDIA OF THE UNIONIDAE 
(MOLLUSCA:BIVALVIA) 

Michael A. Hoggarth^ 

Museum of Zoology. The Ohio State University, 1813 North High Street, Columbus, Ohio 
43210, U.S. A: mhoggarth@otterbein.edu 



ABSTRACT 

The primary objective of this study was to describe glochidia of the family Unionidae. Glochidia 
from 82 nominal taxa representing 30 genera in four subfamilies of the LJnionidae were exam- 
ined. The glochidium of the European Unio elongatulus glaucinuswas found to share many char- 
acters with those of the Anodontinae. Both have triangular valves and styliform hooks. Further- 
more, the glochidium of Simpsonaias ambigua and U. e. glaucinus share exterior valve sculpture 
and styliform hook fine structure. 

The glochidia of some members of the Alasmidontini (subfamily Anodontinae), including some 
species in the genera Lasmigona and Alasmidonta. and that of Pegias fabula are depressed- 
pyriform, with large adductor muscles, looped exterior valve sculpture, and a double row of mi- 
crostylets on the hook. These glochidia are very similar to those of Stroptiitus. The glochidia of 
Arcidens and the remaining members of Alasmidonta and Lasmigona are high-pyriform, with 
small adductor muscles, beaded to rosette exterior valve sculpture, and complex hooks with at 
least four rows of microstylets. 

The glochidia of the Ambleminae demonstrate structures also found in the primitive lampsiline 
genera. The Lampsilinae are divided into four groups with the main lineage including Ptycho- 
branchus. Actinonaias. Obovaria. Ligumia. Venustaconcha. Villosa and Lampsilis. Branches 
from this lineage include: (1) Obliquaria. Cyprogenia and Dromus: (2) Ellipsaria. Leptodea and 
Potamilus: and (3) Epioblasma. 

Key words: Unionidae, glochidia, glochidial morphology, electron microscopy. 



INTRODUCTION 

Leeuwenhoek made the first substantial 
contribution to the study of glochidia (Leeu- 
wenhoek, 1695). He correctly interpreted 
these tiny bivalves as young molluscs. He 
also observed limited development of the lar- 
vae and saw the characteristic snapping be- 
havior they display when mature. It does not 
appear that he ever doubted that these tiny 
molluscs, which had developed within the gills 
of a female, were her young. However, like so 
many others during the century and a half to 
follow, he was unable to provide an environ- 
ment outside of the female where develop- 
ment could continue. This, combined with the 
observations that these tiny molluscs were al- 
most identical in both Anodonta and Unio. 
numbered within the gills of the larger mollusc 
by the thousands, and had structures, either 
real or imagined, that were quite different from 
those of the larger mollusc, led some to be- 
lieve that the small shelled animals were not 
larvae but parasites. The tiny bivalve para- 

^Also: Department of Life and Earth Science, Otterbein College, Westerville, Ohio 43081, U.S.A. 

1 



sites were given the name Glochidium para- 
siticumby Rathke (1797). 

The next major advance in the understand- 
ing of the life history of the Unionacea came 
with the observations of Carus and Leydig. 
Carus (1 832) watched the brightly colored ova 
of Potamida littoralis (Cuvier, 1 797) pass from 
the oviduct to the outer gills of a female mol- 
lusc. Continued observation demonstrated 
that the animal known as G. parasiticum was 
not a species separate from the larger mollusc 
but its larval stage. Leydig's (1866) discovery 
of glochidia embedded in the fins of a fish 
solved the primary developmental mystery 
and led the way for life history investigation. 
One of the primary objectives of these early life 
history investigations was to document the 
changes that occur during parasitism, when 
the glochidium transforms into a juvenile 
(Braun, 1878; Schmidt, 1885; Schierholz, 
1878, 1888; Harms, 1907a, b, c, 1908, 1909). 
These studies also demonstrated that artificial 
infection could be used to indicate the suscep- 
tibility of fish to glochidia. Fueled by the com- 



HOGGARTH 




FIG. 1 . Typical life histories of two species of Unionidae. (Drawing of A. rupestris after Trautman, 1 981 , and 
drawings of adult shells of P. g. grandis and L. r. luteola after Burch, 1975.) 



mercial importance of North American unionid 
shells in the button industry, and the dwindling 
supply of those shells, the staff of the U. S. Bu- 
reau of Fisheries began to use artificial infec- 
tion to determine the hosts for commercially 
important species (Lefevre & Curtis, 1910, 
1912; Coker & Surber, 1911; Surber, 1912, 
1913, 1915; Howard, 1912, 1914a, b, c). Dia- 
grams of the unionid life cycle, were soon to 
follow. 

Figure 1 demonstrates that there are es- 
sentially two very different life histories. Not 
only are the larvae themselves morphologi- 
cally different (triangulate, ligulate, etc.), but 
so are their sites of eventual parasitism and 
the way in which they gain access to their 
host. The relatively large, subtriangular 
glochidium of the Anodontinae bears a hook 
on the ventral margin of each valve. If this 
glochidium clamps down on the fin of a pass- 
ing fish and pierces the fin epithelium, it may 
become encapsulated by host tissue. The 
smaller, subelliptical glochidium of the Lamp- 
silinae lacks hooks. If this glochidium is taken 
in through the mouth of its host, travels to the 
gills and clamps down on a gill filament, irri- 
tating the gill epithelium, it too may become 



encapsulated by host tissue. Attraction de- 
vices, such as the mantle flaps of Lampsilis, 
facilitate the exchange of glochidium from 
unionid to fish (Morrison, 1973). 

The dissimilarities in these life histories re- 
veal three periods when selection might be 
acting upon the glochidium: (1 ) during release 
from the female, (2) during initial contact with 
the host, and (3) during encapsulation. The 
selective advantage of glochidial structures 
might be understood, therefore as they facili- 
tate attraction of the host, attachment to the 
host, or induction of encapsulation (Hoggarth 
& Gaunt, 1988). Encapsulation provides a 
stable environment where transformation can 
occur, a ready source of nutrients (whether 
used or not), and protection from predators 
and from being washed or tumbled down- 
stream (Coker & Surber, 1911; Howard & 
Anson, 1922; Arey, 1932a, b; Kat, 1984). Fur- 
thermore, the host functions as a dispersal 
mechanism, and releases the newly trans- 
formed juvenile into suitable habitats. With the 
use of scanning electron microscopy, minute 
glochidial structures can be examined and 
used to interpret relationships among unionid 
species. It is the objective of this paper to de- 



GLOCHIDIAOF UNIONIDAE 



scribe the glochidia of a large portion of tine 
North American fauna to begin this endeavor. 



MATERIALS AND METHODS 
Specimens Examined 

Glochidia were removed from the marsupia 
of 150 female unionids representing 82 nomi- 
nal taxa from 30 genera. Forty-three lots of 
material were processed from specimens col- 
lected or received during this study. These 
specimens were deposited in the collection of 
The Ohio State University Museum of Zool- 
ogy (OSUM), but glochidia samples from 
each specimen were retained by the author 
and have MAH catalog numbers. The remain- 
ing specimens were located in collections of 
unionids at The Ohio State University Mu- 
seum of Zoology, University of Michigan Mu- 
seum of Zoology (UMMZ), University of Wis- 
consin Zoological Museum (UWZY), and The 
Illinois Natural History Survey (INHS). 

Glochidia removed from the marsupia of fe- 
male unionids were preserved (for freshly col- 
lected specimens) and stored in a solution of 
80% alcohol, 5% glycerin, and 15% water. 
Each vial of glochidia was labeled with the 
catalog number of the female from which the 
sample came. Subsamples of glochidia taken 
from a vial for dehydration in acetone, and 
subsamples of these that were placed on a 
stub for viewing with scanning electron mi- 
croscopy (SEM) were also labeled with the 
catalog number of the female mollusc. There- 
fore, each glochidium examined can be 
traced back to its maternal parent. 

Procedures for Scanning 
Electron Microscopy 

Preserved and freshly collected glochidia 
were cleaned by the removal of the glochidial 
soft parts. Only glochidia that had valves gap- 
ing apart (in the case of the preserved mater- 
ial) or that were actively snapping their valves 
(in the case of freshly collected glochidia) 
were processed for the SEM. 

The initial step in cleaning was to wash the 
glochidia in three changes of distilled water. 
Each sample was suspended in distilled water 
and then allowed to settle, after which the su- 
pernatant was removed by using a Pasteur 
pipette. Generally, the glochidia settled to the 
bottom of the vial within 10-15 sec, whereas 



small pieces of the marsupium and the matrix, 
within which the glochidia may be found within 
the marsupium, were still suspended. These 
impurities were removed with the super- 
natant. 

Freshly collected glochidia were cleaned 
according to the method of Calloway & Turner 
(1979). These glochidia initially were washed 
as above, with two drops of 1 N NaOH added 
to the final wash (10 ml). The glochidia were 
allowed to stand in this slightly basic solution 
for ten minutes and then washed in three 
changes of distilled water. 

Previous studies using SEM to examine 
glochidia employed only freshly collected ma- 
terial from which the soft parts were removed 
as described above, or preserved material 
from which the soft parts had not been re- 
moved (Giusti, 1973; Giusti et al., 1975; 
Clarke, 1981a, 1985; Rand & Wiles, 1982). 
No practical method for the removal of the 
preserved soft parts had been developed. 
However, the procedure outlined below pre- 
sents a method that was found to produce ad- 
equate specimens for SEM examination. This 
procedure is sufficiently flexible to allow for 
differences in preservation, initial valve gape, 
and amount of extraneous material in the 
sample. 

Following the final rinse in distilled water, 
specimens were placed in 10 ml of 1% aque- 
ous trypsin solution (Sorensen's Phosphate 
Buffer, pH 7.00) and thoroughly mixed. This 
was accomplished by drawing and expelling 
the liquid in the vial into a pipette 10-15 times. 
The action of the rapidly moving liquid often 
dislodged the soft parts from the valves, 
thereby reducing the time required in the 
trypsin solution. The sample was placed in an 
incubator at 37-C, visually inspected every 1 5 
min, and removed when the valves began to 
gape at an angle approaching 180°. The di- 
gestion process was discontinued as soon as 
the first valves began to open, rather than 
after all valves had opened. The supernatant 
was carefully removed from the sample and 
the sample was washed in three changes of 
distilled water. Any remaining soft parts were 
mechanically dislodged by drawing a large 
number of glochidia into a pipette and ex- 
pelling them 20-30 times per wash. 

Cleaned glochidia, whether freshly col- 
lected or preserved, then were dehydrated in 
an ascending gradation of acetone (10%, 
30%, 50%, 70%, 90%, 95%, 100%, 100%) 
and stored in Borosilicate Glass Scintillation 
Vials (VWR Scientific) in the final acetone 



HOGGARTH 



wash. Subsamples of these glochidia were 
processed for the SEM. Glochidia were air 
dried on a clean glass slide and mounted on 
double stick tape (3M) on 13 mm aluminum 
stubs. The tape was rimmed with silver paint 
and then the entire stub was placed in a vac- 
uum desiccator for 24 h. Critical point drying 
was found to be unnecessary since the soft 
parts, which would tend to resist desiccation, 
were removed. Following desiccation, the 
specimens were coated with 30 nm of gold- 
palladium in a Hummer VI Sputter Coater, and 
then viewed in a Cambridge Stereoscan S4- 
10 SEM or a Hitachi S-500 SEM at an accel- 
eration voltage of 20kV. 

Orientation of Specimens on Figures 

The orientation of the glochidial valve, 
demonstrated by the electron micrographs on 
each plate, follows Hoggarth (1987). The dor- 
sal aspect of the valve has been oriented to- 
ward the top of the page, ventral is down. The 
anterior margin is toward the right hand mar- 
gin of the page and posterior is to the left. 

Characters Examined 

Each character examined was defined in 
terms of two or more states or expressions of 
that character. An index to glochidial valve 
characters is found in Table 1 . Measurements 
were made directly from SEM micrographs 
with care taken when glochidia were placed 
on the stub to ensure that some were flat and 
not tilted in respect to the stub surface. Micro- 
graphs of these glochidia were taken at 0- tilt. 

Length - Glochidial valve length was mea- 
sured as the greatest distance from anterior to 
posterior margins. This measurement was 
made parallel to the hinge (Fig. 2). Table 2 
contains the morphometric data for each 
specimen of glochidium examined. 

Height - Glochidial valve height was mea- 
sured as the greatest distance from dorsal to 
ventral margins. This measurement was 
made perpendicular to length (Fig. 2). 

Hinge length - The hinge was measured in 
a straight line from the points were the dorsal 
margins intersect the anterior and posterior 
margins, regardless of whether the hinge was 
curved or straight (Fig. 2). 

Hinge ligament length - In the glochidium 
the hinge ligament extends the entire length 
of the hinge. A portion of the ligament can be 



seen when viewing the valve externally and 
another portion can be viewed internally. 
Herein, central ligament refers to the ex- 
panded central portion of the hinge ligament 
or that portion that can be viewed internally. 
Posterior ligament refers to the posterior por- 
tion of the hinge ligament, measured from the 
posterior margin of the hinge to the posterior 
margin of the central ligament. Anterior liga- 
ment is the portion of the hinge ligament from 
the anterior margin of the hinge to the anterior 
margin of the central ligament. 

Central ligament position - Central liga- 
ment position was found by adding one half 
the central ligament length to the length of the 
posterior ligament and then dividing by the 
length of the hinge. The mid-point of the cen- 
tral ligament was expressed as % length of 
the hinge from posterior to anterior. 

Valve shape - Valve shape refers to the 
outline of the shell when viewed sagittally (the 
plane bisecting anterior and posterior). The 
terms used to describe valve shape have 
been loosely defined in the past. Valve shape 
descriptions and representative electron mi- 
crographs are indexed in Table 1 . 

Lateral valve gape - With the valves fully 
adducted some species of glochidia were 
found to possess anterior and posterior valve 
gape. This character was expressed as either 
absent or present (Table 1 provides an index 
to these and all subsequent glochidial valve 
characters). 

Dorsal alae - This structure was found at 
the dorsal-anterior and dorsal-posterior mar- 
gins of most lampsiline glochidia. They can 
best be described as arch-like extensions of 
the glochidial valve. This character was ex- 
pressed as absent, short or long. 

Microstylets - Clarke (1 981 a) proposed the 
term microstylet for the larger (> 1 .0 цт long) 
points on the ventral margin of the glochidial 
valve. Microstylets were, (1) absent, (2) many 
and unorganized, (3) arranged in one distal 
row on the hook, (4) arranged in two distal 
rows on the hook, or (5) arranged in many dis- 
tal rows on the hook. 

Micropoints -Clarke (1981a) used the term 
micropoints for the small (< 1.0 |.im long) 
points on the ventral margin of the glochidial 
valve. Micropoints were, (1) lanceolate, aris- 
ing as single attenuate points from the ventral 
margin of the valve, (2) lamellate, arising as 
single plate-like points, or (3) coronal, with 
three to seven attenuate points arising from a 
common base. 



GLOCHIDIAOF UNIONIDAE 



TABLE 1. Glochidial shell characters used in analysis of relationships between the species 
of Unionidae. Following each character state is a reference to a figure illustrating the char- 
acter state. 



1. Length 

a. short (46A) 

b. moderate (57A) 
с long (17A) 

2. Height 

a. short (32A) 

b. moderate (58A) 
с high (24A) 

3. Hinge length 

a. short (48C) 

b. moderate (64G) 
с long (4F) 

4. Central ligament length 

a. short (47E) 

b. long (9C) 

5. Central ligament position 

a. posterior (7F) 

b. central (50F) 

6. Valve shape 

a. subelliptical (36B) 

b. depressed subelliptical (71 B) 
с subrotund (38B) 

d. subspatulate (63B) 

e. fabelliform (40B) 

f. subligulate (45B) 

g. ligulate (51 B) 

h. subtriangulate (5B) 

i. lachrimiform (11 B) 

j. pyriform (27B) 

k. depressed pyriform (20B) 

I. quadrate (23B) 

7. Lateral valve gape 

a. absent (64C) 

b. present (50C) 



8. Dorsal alae 

a. absent (36A) 

b. short (47A) 
с long (62A) 

9. Microstylets 

a. absent (61 C) 

b. many unorganized (15D) 
с one distal row (13D) 

d. two distal rows (16D) 

e. many distal rows (28D) 

10. Micropoints 

a. lanceolate (29E) 

b. lamellate (49E) 
с coronal (33E) 

11. Micropoint organization 

a. unorganized (36H) 

b. horizontal rows (71 C) 

с broken vertical rows (59E) 
d. complete vertical rows (45D) 

12. Hook 

a. absent (55D) 

b. styliform (25E) 

с supernumerary (70C) 
d. lanceolate (50D) 

13. Exterior surface sculpture 

a. rough (68E) 

b. beaded (15E) 
с rosette (26E) 

d. loose looped (9D) 

e. ribbed loose looped (5C) 

f. tight looped (23E) 

g. vermiculate (51 E) 



Micropoint organization - Micropoints were, 
(1) unorganized, (2) arranged in horizontal 
rows, (3) arranged in broken vertical rows, or 
(4) arranged in complete vertical rows. 

Hook - As pointed out by Clarke (1981a), 
this term has been used indiscriminately to 
apply to independently derived structures that 
serve a similar function. He proposed using 
the term stylet to refer to the complex hook of 
the anodontine glochidia. In this study, the 
glochidial hook was either, (1) absent, (2) 
styliform, a V-shaped extension of the ventral 
margin of the glochidium, (3) lanceolate, a re- 



curved, attenuate extension at each corner of 
the ventral margin of the glochidia of the 
genus Potamilus, or (4) supernumerary, the 
straight and sharply pointed hook found in an 
area along the ventral margin of the genus 
Epioblasma. 

Exterior surface sculpturing - This refers to 
the fine structure of the exterior surface of the 
glochidial valve (viewed between 10,000 to 
20,000 x). Exterior surface sculpture was, (1) 
rough, (2) beaded, (3) rosette, (4) loose- 
looped, (5) ribbed, loose-looped, (6) tight- 
looped, or (7) vermiculate. 



HOGGARTH 




FIG. 2. Subelliptical glochidium of the Lampsilinae 
demonstrating length (L) and height (H). The dorsal 
margin of the valve is toward the top of the page, 
ventral down, anterior to the right, and posterior to 
the left. The adductor muscle (AM) is located in the 
dorsal-anterior quadrant of the valve. 



DESCRIPTIONS OF GLOCHIDIA 
Subfamily Unioninae 

Unio elongatulus glaucinus Porro, 1838 
(Fig.3A-F) 

Material Examined 

MAH 2055-Rivanazzano, Pavia, Italy, ex 
Coll. F. Giusti. 

Description 

Glochidium subtriangular, length 21 8 to 232 
(am, (227 ± 6.45 ¡.im, n = 4), height 21 to 21 8 
um (216 ± 4.08 цт, n = 4). Anterior and pos- 
terior margins arcuate, meeting at base of 
hook to form a broadly rounded ventral termi- 
nus. Valve outline only slightly asymmetric, 
with anterior margin slightly more produced 
than posterior margin. Exterior surface of 
valve malleated (large dimple-like depres- 
sions) and pitted (smaller depressions gener- 
ally within malleated valve surface), except 
along smooth valve border. Fine sculpture of 
exterior valve surface beaded (Fig. 3E). Cen- 
tral ligament (seen in internal views of valve) 
55 to 58 |jm (56 ± 1 .73 |im, n = 3) in length, 
located about 40% from posterior to antehor. 
Posterior ligament 41 to 44 цт (42 ± 1 .53 



).im, n = 3) in length; anterior ligament 68 to 75 
\xvr\ (72 ± 3.60 цт, n = 3) long. Dorsal margin 
straight, 164 to 175 цт (171 ± 4.36 ¡xm, n = 
4) in length. Styliform hook extending from 
ventral terminus of each valve as a broad tri- 
angular plate. Hook gradually and uniformly 
tapered to a broad point distally, located about 
50% from both lateral margins. Surface of 
hook covered by microstylets (> 1.0 ¡am long 
points on hook) and micropoints (< 1.0 |.im 
long points on the hook). Most microstylets 
(about 35) bluntly pointed, with some of more 
proximal microstylets multifaceted, sharply 
pointed. Micropoints extending from lateral 
and proximal margins of styliform hook cover- 
ing most of its lateral surface, except within 
narrow band distally. 



Remarks 

The glochidium of UnIo has been described 
as subtriangular with a ventral hook (Ort- 
mann, 1912). Giusti (1973) stated that other 
than its smaller size, this glochidium resem- 
bles that of Anodonta. Ortmann (1918), how- 
ever, reported the absence of a hook in Unio 
caffer, but Heard & Guckert (1970) suggested 
that this was the result of examining immature 
specimens. They noted that Giusti (1973) 
demonstrated hooks in U. e. glaucinus and 
that McMichael & Hiscock (1 958) found hooks 
on mature glochidia of Velesunio ambiguus, 
even though the species was earlier de- 
scribed as having bookless glochidia (His- 
cock, 1951). 

Ortmann (1912) and Heard & Guckert 
(1970) placed this genus near the North 
American genus Pleurobema (Ambleminae) 
on the basis of similar shell morphology and 
anatomy. However, Ortmann (1912) recog- 
nized the similarity between the glochidia of 
the Unioninae and the Anodontinae and sug- 
gested that the former may have given rise to 
the latter. Morhson (1955) agreed with Ort- 
mann's conclusion regarding the origin of the 
Anodontinae. He chose to stress develop- 
mental characters (the similarity in glochidia) 
over adult characters and therefore placed 
the subfamily Unioninae near the Anodonti- 
nae. Harms (1908, 1909) and Haas (1910) 
gave length and height measurements of 290 
цт for the glochidium of U. pictorum. Giusti 
(1973) noted that the glochidium of U. e. 
glaucinus is smaller than that of Anodonta. 
but gave no measurements. 



GLOCHIDIAOF UNIONIDAE 



TABLE 2. Morphometric data for glochidia examined during this study. All measurements in ¡.im. 



Species 



Catalog # 



Length 





Hinge 
Length 


Ligament Length 


ieight 


Ante. 


Cent. 


Post. 


218 


172 


75 


56 


41 


216 


175 










168 


68 


57 


43 


210 


164 










173 


74 


55 


44 


218 


175 








352 


268 


112 


79 


77 


348 


273 








345 


268 










279 


115 


85 


79 


356 


277 








356 


279 


115 


88 


76 


352 


279 


118 


83 


78 


344 


272 








346 


270 










268 


115 


85 


68 


357 


279 








361 


273 








354 


267 








352 


262 








361 


264 


116 


91 


57 


350 


260 


114 


86 


60 


358 


274 








361 


280 


125 


96 


59 


289 


212 








293 


214 








288 


211 








290 


210 


118 


58 


34 


284 


209 








340 


264 


111 


88 


65 




257 


110 


88 


59 


345 


260 


111 


89 


60 




267 


114 


89 


64 




250 


106 


85 


59 


350 


253 


114 


84 


55 


345 


163 








350 


166 


52 


77 


37 




160 


48 


74 


38 


328 


237 


80 


101 


56 


324 


228 


74 


100 


54 


323 


233 


81 


98 


54 


320 


232 


78 


102 


52 


322 


231 








360 


250 


104 


92 


54 


355 


250 








360 


257 


111 


94 


52 


352 


250 








355 


259 


107 


90 


62 


353 


250 








350 


252 








367 


260 








368 


275 








363 


263 


100 


93 


70 


368 


288 


102 


115 


71 


366 


277 








370 


277 








358 


291 








351 


289 


98 


116 


75 



U. e. glaucinus 



A. cygnea 



A. anatina 



A. bering iana 
A. kenneriyi 

A. i m plicata 
A. suborbiculata 

P. g. grandis 



P. g. corpulenta 



P. с Cataracta 



MAH 2055 
MAH 2055 
MAH 2055 
MAH 2055 
MAH 2055 
MAH 2055 
OSUM 20911. 
OSUM 20911. 
OSUM 20911. 
OSUM 20911. 
OSUM 20911. 
OSUM 20911, 
OSUM 20911, 
OSUM 20911, 
OSUM 20911, 
OSUM 20911, 
OSUM 20911, 
OSUM 20912. 
OSUM 20912. 
OSUM 20912. 
OSUM 20912. 
OSUM 20912. 
OSUM 20912.2 
OSUM 20912.2 
OSUM 3711.1 
OSUM 3711.1 
OSUM 3711.1 
OSUM 3711.1 
OSUM 3711.1 
OSUM 52882.2 
OSUM 52882.2 
OSUM 52882.2 
OSUM 52882.2 
OSUM 52882.3 
OSUM 52882.3 
OSUM 52463.7 
OSUM 52463.7 
OSUM 52463.7 
OSUM 13634 
OSUM 13634 
OSUM 13634 
OSUM 13634 
OSUM 13634 
OSUM 38467.10 
OSUM 38467.10 
OSUM 38467.10 
OSUM 38467.10 
INHS2247 
INHS2247 
INHS2247 
OSUM 47890 
OSUM 47890 
OSUM 53653 
OSUM 52462.27 
OSUM 52462.27 
OSUM 52462.27 
OSUM 52462.35 
OSUM 52462.35 



225 
218 

231 

232 
358 
351 
345 

347 
351 
351 
357 
346 

350 
348 
355 
352 
353 
353 
356 
361 
289 
292 
288 
286 
288 
354 

352 



350 
342 
343 

328 
326 
326 
323 
325 
358 
355 
355 
365 
353 
350 
358 
350 
350 
343 
380 
374 
375 
375 
378 



{continued) 



HOGGARTH 



TABLE 2. (Continued) 












Species 


Catalog # 


Length 


Height 


Hinge 
Length 


Ligament Length 
Ante. Cent. Post. 



P. с margínala 
P. doliaris 
U. imbecillis 

U. imbecillis 



A. ferussacianus 



S. ambigua 



S. и. und и latus 

S. и. tennesseensis 
S. subvexus 

A. viridis 



A. heterodon 



A. und и lata 



OSUM 38962.6 
OSUM 38962.6 
OSUM 26405 
OSUM 26405 
MAH 435 
MAH 435 
OSUM 9436.2 
OSUM 9436.2 
OSUM 9436.2 
OSUM 9436.2 
OSUM 9436.2 
OSUM 9436.2 
OSUM 9436.2 
OSUM 9436.2 
UWZY 24971.1 
UWZY 24971.1 
UWZY 24971.1 
OSUM 18275.2 
OSUM 18275.2 
OSUM 18275.2 
MAH 989.4 
MAH 989.4 
MAH 989.4 
MAH 989.4 
UMZY 22658 
UMZY 22658 
UMZY 22658 
UMZY 22662 
UMZY 22662 
UMZY 22672 
OSUM 55995 
OSUM 49443 
OSUM 52458.4 
OSUM 52458.4 
OSUM 52458.4 
OSUM 52458.4 
OSUM 52458.4 
OSUM 33381 .2 
OSUM 55449 
OSUM 36240 
OSUM 36240 
OSUM 36240 
OSUM 36240 
OSUM 47518 
OSUM 47518 
OSUM 47518 
OSUM 47518 
OSUM 47518 
OSUM 47518 
OSUM 47518 
OSUM 25106.2 
OSUM 25106.2 
OSUM 25106.2 
OSUM 25106.2 
OSUM 52434.4 
OSUM 52434.4 
OSUM 52434.4 
OSUM 52434.4 
OSUM 52434.4 



358 
360 

317 
405 
291 
310 
310 
306 
299 
300 
299 
310 
309 
300 
313 

303 
323 
325 
326 
322 
325 
321 
319 
253 
251 
255 
258 
257 

255 
369 
360 
360 



346 
354 
348 

359 
300 
313 
313 
300 
319 
303 
300 
330 
338 
332 
335 
358 
356 
355 
354 
343 



364 
369 
317 
368 
297 
297 
300 
295 
292 
289 
303 
304 
303 
303 
306 

305 
325 
326 
327 
322 
325 
320 
322 
256 
257 
265 
264 
264 

259 
299 
289 
296 



298 
288 
292 

289 
245 
250 
246 
253 
258 
250 
250 
267 
258 
268 
258 
378 
365 
375 
368 
369 



276 
277 
240 
305 
246 
242 
245 
251 
243 
242 
240 
243 
249 
256 
252 
255 
250 
235 
234 
238 
231 
238 
231 
233 
168 
168 
167 
170 
170 
166 
164 
281 
271 
280 
272 
281 
280 
268 
268 
271 
275 
277 
273 
245 
260 
250 
250 
258 
250 
245 
253 
266 
265 
255 
258 
247 
260 
257 
247 



102 



95 



98 



103 
103 

83 

85 
89 
65 
61 
63 

101 

109 

108 

110 

96 

123 

100 
108 

109 

110 

105 
103 

105 



110 



90 



93 



93 
89 

92 

91 
88 
58 
63 
54 

97 

87 

103 

92 

94 

84 

80 
81 



64 



61 



60 



60 
63 

59 

62 
56 
45 
49 
49 

73 

76 
70 
78 
78 

70 

65 

71 



73 68 



80 

98 
89 



89 



75 

55 
55 



53 



GLOCHIDIAOF UNIONIDAE 



TABLE 2. (Continued) 











Hinge 


Ligament Length 


Species 


Catalog # 


Length 


Height 


Length 


Ante. 


Cent. 


Post. 


A. m arg i nata 


MAH 277.1 


340 


360 


233 










MAH 277.1 


341 


362 


235 


107 


67 


61 




MAH 277.1 


335 


372 


232 


100 


73 


59 




MAH 724.1 


341 


367 


230 








P. fabula 


OSUM 41308.3 


386 


325 


210 


74 


73 


63 




OSUM 41309.1 


388 


319 


200 


69 


68 


62 




OSUM 41309.1 


385 


323 


208 


79 


73 


56 


A. confragosus 


OSUM 52015 


357 


353 


240 


100 


70 


70 




OSUM 52015 


352 


354 


252 


108 


74 


70 




OSUM 52015 


363 


355 


237 










OSUM 52015 


360 


355 


252 


115 


78 


59 




OSUM 52015 






250 


115 


63 


72 


L. compressa 


OSUM 23179.1 


322 


285 


234 










OSUM 23179.1 


327 


287 


233 


82 


84 


67 




MAH 702 


320 


286 


230 










MAH 702 


317 


283 


239 










MAH 727 


327 


288 


232 


81 


83 


68 


L. subviridis 


OSUM 27131.66 


370 


309 


253 


100 


88 


65 




OSUM 27131.66 


383 


318 


259 










OSUM 27131 .66 


380 


311 


244 










OSUM 27131.68 


368 


310 


260 


105 


87 


68 




OSUM 27131.68 


381 


312 


253 


107 


89 


57 


L. holstonia 


OSUM 55826.6 


286 


294 


234 


109 


73 


52 




OSUM 55826.6 


291 


276 


235 










OSUM 55826.6 


281 


275 


221 










OSUM 55826.7 


290 


275 


228 


110 


63 


55 




OSUM 55826.7 


284 


291 


221 








L. costata 


MAH 585 


343 


365 


240 


97 


79 


64 




MAH 585 


347 


375 


239 










MAH 585 


343 


373 


239 


104 


71 


64 




MAH 585 


345 


364 


248 


105 


82 


61 




MAH 882.1 


343 


368 


241 


90 


83 


68 




MAH 882.1 


348 


377 


244 


99 


84 


61 




MAH 882.1 


340 


363 


239 








L. complánate 


MAH 278.2 






193 


75 


68 


50 




MAH 278.2 


290 


295 


197 


79 


65 


53 




MAH 278.2 


296 


310 


201 


88 


60 


53 




MAH 278.2 


289 


293 


195 


82 


62 


51 




MAH 278.2 


294 


298 


199 










MAH 278.2 


293 


297 


208 










MAH 278.2 


295 


308 


206 








M. nervosa 


OSUM 13032.66 


268 


347 


155 










OSUM 541 78 


254 


348 


145 










OSUM 54178 






153 


60 


49 


44 




OSUM 541 78 


262 


350 


147 










OSUM 1986:22 


260 


340 


145 










OSUM 1986:22 






155 


60 


52 


43 




OSUM 1986:22 






155 


63 


49 


43 


M. boykiniana 


OSUM 51107.5 


245 


350 


150 








P. dombeyana 


OSUM 53273.2 


223 


238 


132 


55 


46 


31 




OSUM 53273.2 


223 


240 


134 










OSUM 53273.2 


228 


259 


135 










OSUM 53273.2 






133 


58 


43 


32 




OSUM 53273.3 


224 


252 


134 










OSUM 53273.3 


230 


251 


132 


59 


42 


31 




OSUM 53273.3 


225 


243 


133 










OSUM 43011 


223 


250 


136 










OSUM 43011 


226 


243 


132 










OSUM 43011 


231 


241 


134 










OSUM 43011 


223 


243 


133 


51 


47 


35 



(continued) 



10 




HOGGARTH 










TABLE 2, (Continued 
























Hinge 


Ligament Length 


Species 


Catalog # 


Length 


Height 


Length 


Ante. 


Cent. 


Post. 


T. verrucosa 


МАИ 654.1 


92 


101 


45 










MAH 654.1 






46 


9 


32 


5 




MAH 654.1 


88 


100 


40 


6 


31 


3 




MAH 654.1 






43 


6 


33 


4 




MAH 654.1 


90 


100 


46 


8 


33 


5 




MAH 654.1 


85 


97 


44 










MAH 654.1 


94 


101 


45 










MAH 654.1 






44 


9 


30 


5 




MAH 654.1 


93 


99 


44 


6 


34 


4 


Q. infucata 


OSUM 48537.1 






104 


28 


54 


22 




OSUM 48537.1 


234 


275 


104 


26 


58 


20 




OSUM 48537.2 


240 


287 


105 










OSUM 48537.2 


242 


285 


102 










OSUM 48537.2 


242 


285 


102 








E. dilatata 


MAH 946.9 


217 


225 


140 


56 


45 


39 




MAH 946.9 


210 


219 


142 


56 


50 


36 




MAH 946.9 


219 


219 


142 










MAH 946.9 


219 


219 


147 








P. fasciolaris 


MAH 641 


170 


195 


89 










MAH 641 


174 


184 


85 


26 


40 


19 




MAH 651 


175 


182 


80 


22 


36 


22 


P. occidentalis 


OSUM 45361.14 


197 


241 


101 


30 


48 


23 




OSUM 45361.17 


203 


234 


102 










OSUM 45361.17 






104 


34 


44 


26 


P. greeni 


OSUM 19025.2 


189 


227 


96 


26 


49 


21 




OSUM 19025.2 


190 


228 


93 










OSUM 19025.2 


183 


226 


90 








P. subtentum 


OSUM 43156.5 


195 


251 


85 


25 


36 


24 




OSUM 43156.5 


181 


239 


90 










OSUM 43156.5 


191 


251 


85 










OSUM 43156.5 






83 


26 


36 


21 




OSUM 43156.5 


190 


236 


82 










OSUM 43156.5 


194 


239 


83 


25 


38 


20 


0. reflexa 


OSUM 54361.1 


219 


221 


119 










OSUM 54361.1 


213 


219 


127 


44 


52 


31 




OSUM 54361.1 


219 


206 


121 


37 


54 


30 


С stegaria 


OSUM 6298.21 


204 


164 


113 










OSUM 6298.21 


206 


167 


116 


46 


40 


30 




OSUM 6298.21 


208 


170 


120 










OSUM 6298.21 






115 


43 


39 


33 




OSUM 6298.21 






114 


47 


35 


32 


С aberti 


OSUM 48067 


200 


145 


128 


50 


29 


49 




OSUM 48067 


207 


155 


136 










OSUM 48067 


200 


143 


125 


48 


31 


46 




OSUM 48067 


211 


156 


128 


51 


36 


41 




OSUM 48067 


218 


161 


132 










OSUM 48067 


211 


161 


131 


60 


28 


43 




OSUM 48067 






136 


61 


32 


43 


D. dramas 


OSUM 23209 






171 


76 


39 


56 




OSUM 20407.1 


230 


120 


182 










OSUM 20407.1 


230 


114 


182 


82 


39 


61 




OSUM 20407.1 


222 


118 


169 


78 


40 


51 




OSUM 20407.1 


221 


120 


160 










OSUM 20407.1 


219 


120 


182 








A. pectorosa 


OSUM 24337 


253 


270 


139 


46 


62 


31 




OSUM 48748.3 


248 


270 


151 


52 


62 


37 




OSUM 48748.3 


244 


260 


144 


47 


58 


39 


A. 1. cari nata 


MAH 842.1 


220 


243 


125 


38 


57 


30 


0. retusa 


UUMZ UNCAT. 


233 


278 


115 










UUMZ UNCAT. 


218 


272 


117 










UUMZ UNCAT. 






119 


38 


45 


36 



GLOCHIDIAOF UNIONIDAE 



11 



TABLE 2. {Continued) 












Species 


Catalog # 


Length 


Height 


Hinge 
Length 


Ligament Length 
Ante. Cent. Post. 



O. olivaria 



O. subrotunda 



O. jacksoniana 



O. unicolor 



E. lineolata 



L. frag i I is 
L. ochracea 

P. otiiensis 



P. amphiciiaena 
P. alatus 



P. purpuratus 



L. recta 



OSUM 51282.2 
OSUM 51282.2 
OSUM 51282.2 
OSUM 51282.2 
OSUM 51282.2 
MAN 659.2 
MAH 659.2 
MAH 659.2 
MAH 805.1 
MAH 805.1 
MAH 805.1 
OSUM 50233.8 
OSUM 50233.8 
OSUM 50233.8 
OSUM 50233.8 
OSUM 33158 
OSUM 33158 
OSUM 33158 
OSUM 47696.6 
OSUM 47696.6 
OSUM 47696.6 



1984 
1984 
1984 
1984 
1984 
1984 
1984 



OSUM: 
OSUM: 
OSUM: 
OSUM: 
OSUM: 
OSUM: 
OSUM: 
MAH 626.1 
MAH 626.1 
MAH 626.1 
MAH 896 
MAH 896 
MAH 896 
MAH 896 
MAH 896 
MAH 896 
OSUM 54521 
OSUM 54521 
OSUM 54521 
OSUM 54521 
OSUM 54521 
OSUM 33163.13 
OSUM 33163.13 
OSUM 55465 
OSUM 55465 
OSUM 55465 
OSUM 55465 
OSUM 55465 
OSUM 15738.2 
OSUM 15738.2 
OSUM 15738.2 
OSUM 15738.2 



OSUM 
OSUM 
OSUM 
OSUM 
OSUM 
OSUM 



1984:2 
1984:2 
1984:2 
1984:2 
1984:2 
1984:2 



202 
198 



206 

174 
180 
178 
180 
174 
175 
183 
184 
187 
172 
174 
180 
168 
168 

240 

229 

240 

235 

245 

233 

241 

73 

72 

72 

246 

246 
241 
241 
241 

126 

122 

120 
113 
111 
206 
208 
227 
223 
217 
200 

195 
190 
205 
219 



207 
211 



254 
261 



258 

197 
210 
205 
210 
199 
243 
230 
230 
240 
222 
224 
229 
218 
234 

324 

319 

325 

310 

325 

319 

325 

82 

80 

83 

289 

293 
289 
291 
291 

182 

187 
175 
171 
170 
371 
371 
386 
386 
375 
356 

347 
348 
257 
263 



257 
265 



107 

106 

105 

101 

110 

94 

89 

95 

92 

88 

85 

89 

95 

100 

97 

85 

88 

96 

93 

89 

88 

93 

88 

91 

87 

96 

91 

89 

35 

35 

30 

109 

110 

102 

105 

105 

110 

42 

45 

50 

42 

47 

41 

40 

104 

96 

109 

96 

106 

106 

103 

100 

109 

105 

112 

115 

110 

105 

110 



32 


44 


29 


41 


34 


26 


43 


40 


27 


29 


40 


25 


24 


43 


21 


26 


42 


21 



31 



14 



15 



11 



41 
45 



41 



11 



21 



20 



40 
35 



25 



41 


32 


23 


32 


33 


28 


32 


32 


24 


23 


50 


20 


27 


37 


24 


29 


35 


23 


23 


51 


22 



10 



11 


11 


8 


32 


47 


30 


36 


42 


32 


34 


43 


28 


37 


39 


34 


13 


16 


13 



14 



10 



35 


42 


19 


38 


42 


29 


37 


41 


28 


37 


37 


29 



34 
30 



[continued) 



12 




HOGGA 


RTH 










TABLE 2. (Continued) 






















Hinge 


Ligament Length 


Species 


Catalog # 


Length 


Height 


Length 


Ante. 


Cent. 


Post. 


V. ellipsiformis 


MAH 947.2 


230 


280 


110 


39 


40 


31 




MAH 947.2 






102 


34 


40 


28 




MAH 947.2 


226 


287 


105 










MAH 947.2 


226 


287 


103 










MAH 947.2 






104 


34 


38 


32 




MAH 947.2 


223 


284 


102 








V. trabalis 


OSUM 9516.49 


213 


278 


94 










OSUM 9516.49 


211 


277 


95 


36 


31 


28 




OSUM 9516.49 






99 


36 


36 


27 




OSUM 9516.49 


214 


279 


96 


40 


31 


25 




OSUM 9516.49 


216 


280 


97 








V. perpurpurea 


OSUM 16262 


165 


241 


88 








V. villosa 


OSUM 45943 


246 


300 


114 










OSUM 45943 


245 


298 


105 










OSUM 45943 


246 


296 


110 


35 


42 


33 




OSUM 45947 


250 


308 


116 










OSUM 45947 


248 


304 


108 










OSUM 45947 


240 


307 


110 










OSUM 45947 


241 


308 


113 


43 


37 


33 


V. vibex 


OSUM 54631 


230 


298 


117 










OSUM 24124 


231 


302 


120 










OSUM 24124 


232 


304 


102 


29 


44 


29 




OSUM 24124 


231 


302 


117 










OSUM 24124 


239 


299 


105 


32 


45 


28 




OSUM 48628 


231 


300 


109 










OSUM 48628 


225 


297 


105 










OSUM 48628 


224 


299 


103 








V. i. iris 


MAH 641.1 


230 


289 


112 










MAH 641.1 


217 


289 


113 


38 


42 


33 




MAH 641.1 


230 


300 


112 










MAH 641.1 


232 


303 


115 


40 


41 


34 




MAH 641.1 


228 


305 


115 










OSUM 55828.3 


220 


293 


113 










OSUM 55828.3 


227 


300 


114 


39 


42 


33 




OSUM 55828.3 


217 


290 


107 








L t. teres 


OSUM 36409 


189 


256 


103 










OSUM 36409 






113 


54 


27 


32 




OSUM 36409 


189 


255 


100 










OSUM 36409 


190 


255 


107 










OSUM 36409 


191 


256 


108 










OSUM 36409 






115 


49 


34 


32 




OSUM 36409 






106 


42 


34 


30 




OSUM 36531.1 


190 


258 


106 










OSUM 36531.1 






107 


42 


33 


32 




OSUM 51669.2 


190 


259 


115 










OSUM 51669.2 






116 


49 


37 


30 




OSUM 51669.2 


192 


261 


115 










OSUM 51669.2 


193 


265 


112 


41 


39 


32 




OSUM 51669.2 


194 


260 


112 








L. t. anodontoides 


OSUM 35612 


197 


255 


105 










OSUM 35612 


206 


249 


115 


46 


36 


33 




OSUM 35612 


207 


254 


110 










OSUM 35612 






107 


39 


36 


32 




OSUM 41762.2 






115 


47 


38 


30 




OSUM 41762.2 


187 


249 


105 










OSUM 41762.2 


199 


254 


110 










OSUM 41 762.2 


204 


249 


118 


50 


37 


31 




OSUM 41762.2 


194 


249 


118 


46 


42 


30 



GLOCHIDIA OF UNIONIDAE 



13 



TABLE 2. {Continued) 












Species 


Catalog # 


Length 


Height 


Hinge 
Length 


Ligament Length 
Ante. Cent. Post. 



L. r. radíala 



L. r. luteals 



L. r. luteola 



L. abrupta 



L. higginsi 



L. ovata 



L. ornata 



L. satura 



L. ventricosa 



MAH 897,1 
MAH 897.1 
MAH 897.1 
MAH 897.1 
MAH 321.6 
MAH 321 .6 
MAH 587.1 
MAH 587.1 
MAH 587.1 
MAH 587.1 
MAH 587.1 
MAH 628.1 
MAH 628.1 
MAH 727.2 
MAH 727.2 
MAH 727.2 
OSUM 13303 
OSUM 13303 
OSUM 13303 
OSUM 13303 
OSUM 13303 
OSUM 38841 
OSUM 38841 
OSUM 49024.1 
OSUM 49024.1 
OSUM 49024.1 
OSUM 49024.1 
OSUM 43164.1 
OSUM 43164.1 
OSUM 43164.1 
OSUM 43164.1 
OSUM 54661 
OSUM 54661 
OSUM 54661 
OSUM 38973.51 
OSUM 38973.51 
OSUM 38973.51 
OSUM 38973.52 
MAH 846.1 
MAH 846.1 
MAH 846.1 
MAH 846.1 
MAH 954.1 
MAH 954.1 
MAH 954.1 
MAH 954.2 



255 
260 
250 
255 
230 
232 
227 
235 
230 



232 

232 

230 
208 
214 
207 

210 
214 
208 
214 
217 



232 
232 



203 
204 
198 

220 

223 
255 
253 

254 
245 
250 
240 
255 



311 
295 
304 

288 
295 
280 
290 
280 



280 

280 

285 
251 
252 

257 

259 
252 
251 
257 
254 



271 
276 



258 
257 
260 

269 

268 
292 
287 

291 
275 
274 
274 
286 



128 
129 
122 
121 
110 
112 
109 
118 
107 
120 
119 
113 
115 
110 
120 
123 

96 
112 

98 
111 
106 

96 

97 
108 
111 
108 
118 
113 
116 
116 
119 

98 
100 

96 
117 
115 
116 
113 
111 
109 
118 
111 
117 
108 
106 
105 



50 



39 



46 



46 



46 



38 



32 



46 


47 


29 


49 


45 


27 


37 


40 


33 



33 



40 


40 


40 


42 


45 


32 


37 


42 


36 


40 


41 


39 



27 



38 


39 


31 


40 


47 


31 


39 


43 


34 


40 


44 


35 


38 


33 


29 


42 


40 


35 


43 


40 


33 


45 


41 


33 


38 


40 


33 


45 


39 


33 



Subfamily Anodontinae 

Anodonta cygnea (Linnaeus, 1758) 

(Fig. 4A-F) 

Material Examined 

OSUM 20911.1 -"Maubroux (Genval) petit 
etang près du lac", Belgium [étang = pool, 
pond], 24 January 1949, W. Adam. 



Description 

Glochidium subtriangular, length 345 to 358 
цт (351 ± 4.33 цт, n = 9), height 344 to 357 
\xm (351 ± 4.41 [im, n = 9). Anterior margin 
rounded, slightly more produced than poste- 
rior margin, with maximum inflation at approx- 
imately 50-60% from dorsal to ventral. Poste- 
rior margin gently curved throughout its length, 



14 



HOGGARTH 




FIG. 3. Glochidium of Unió elongatulus glaucinus. MAH 2055: A. exterior valve, bar length = 30 цт; В. inte- 
rior valve, bar length = 30 |im; С styliform hook, bar length = 10 цт: D. styliform hook, bar length = 10 |дт; 
E. exterior valve sculpture, bar length = 1 цт; F. hinge, bar length = 25 |.im. 



producing a moderately asymmetric valve out- 
line. Ventral terminus narrowly pointed, lo- 
cated about 40% from posterior to anterior. Ex- 
terior surface of valve malleated and pitted, 
except in a narrow band along lateral margins. 
Pit density reduced in the area of adductor 
muscle scar (Fig. 4B, C). Loose-looped sculp- 



turing covering exterior surface of valves (Fig. 
4E). Central ligament 79 to 88 |.im (84 ± 3.27 
j.im, n = 5) in length, centered about 43% from 
posterior to anterior. Posterior ligament 68 to 
79 \.ivn (73 ± 5.22 |.im, n = 5) in length; anterior 
ligament 112 to 118|.im(115 ± 2.12мт,п = 5) 
long. Hinge straight, 268 to 279 цт (274 ± 



GLOCHIDIAOF UNIONIDAE 



15 




|HHK ' 'ЖУК 


^- ■ 


■ *, '..jk 


i *- 


.^.„-я^,^^ , ,„.„^_.^,, ^„^ 


^ ' *. 


•>. fc - * 


s 





FIG. 4. Glochidium of Anodonta cygnea. OSUM 20911.1; A. exterior valve, bar length = 55 jam; B. interior 
valve, bar length = 50 |am; С interior valve pitting, bar length = 5 (im; D. styliform hook, bar length = 20 цт; 
E. exterior valve sculpture, bar length = 1 цт; F. hinge, bar length = 40 \xm. 



3.35 ).im, n = 1 1 ) in length. A styliform hook ex- 
tends from ventral terminus as a broad trian- 
gular plate. Lateral hook margins concave, 
rapidly narrowed, producing a sharp point. 
Microstylets lanceolate, sharply pointed, ar- 



ranged in four row/s near ventral terminus, re- 
duced to single row distally. Micropoints cov- 
ering ventral terminus and lateral surfaces of 
the hook, leaving narrow unsculptured band 
along distal edge of the hook. 



16 



HOGGARTH 



Remarks 

The glochidium of A. cygnea can be distin- 
guished from that of Unio by its shape, hook 
structure, exterior valve sculpturing and size. 
Length and height measurements of 350 дт 
have been given for this glochidium by Harms 
(1909), Haas (1910), and Ortmann (1912). It 
lias been figured by Ortmann (1912: pi. 19, 
fig. 2) and Wood (1974: figs. 3, 4). 

Anodonta anatina (Linnaeus, 1758) 
(Fig. 5A-G) 

Material Examined 

OSUM 20912.1, 20912.2 -"lxelles,etang", 
Belgium [etang = pool, pond], 16 March 1950, 
W. Adam. 

Description 

Glochidium subtriangular, moderately 
asymmetric, length 350 to 361 |дт (357 ± 
3.91 |.im, n = 7), height 348 to 361 |im (354 ± 
4.46 цт, n == 7). Anterior margin broadly 
curved near the dorsal margin, more moder- 
ately curved ventrally. Posterior margin gently 
and evenly curved throughout its length. Mal- 
leations and pits covering exterior surface of 
valve, except along the valve margin. Interior 
surface uniformly pitted, without a noticeable 
adductor muscle scar. Exterior valve sculptur- 
ing ribbed loose-looped, consisting of raised 
loops resthcted to bands that run parallel to 
the dorsoventral axis of valve, separated by 
unsculptured bands (Fig. 5C). Larval thread 
present (Fig. 5B, E, F). Central ligament 86 to 
96 |im (91 ± 5.00 |.im, n = 3) in length, cen- 
tered about 43% from posterior to anterior. 
Posterior ligament 57 to 60 (.im (59 ± 5.51 
|im, n = 3) long; anterior ligament 114 to 125 
|im (118 ± 5.86 |im, n = 3) long. Hinge 
straight, with a length of 260 to 280 yim (269 
± 6.37 цт, n = 7). Styliform hook with bicon- 
cave lateral margins, forming sharp distal 
point. Microstylets (about 15) lanceolate (Fig. 
5G), sharply pointed, reduced to a single row 
distally. Micropoints numerous, covering ven- 
tral terminus and lateral surface of hook. Un- 
sculptured distal hook margin narrow. Hook 
located about 40% from posterior to anterior. 

Remarks 

This glochidium is distinguished from that of 
A. cygnea by its unique exterior valve sculp- 



turing. Giusti (1973) and Giusti et al. (1975) 
were the first to demonstrate this unusual 
sculptuhng. They refer to their species as A. 
cygnea {Anodonta piscinalis). Anodonta pisci- 
nalis was placed in the synonymy of A. 
cygnea by Simpson (1900, 1914) and Ort- 
mann (1912). Anodonta anatina has also 
been synonymized with A. cygnea by Ort- 
mann (1912), although it was treated as a 
variant of A. cygnea by Simpson (1900, 
1914). 

There is little question that there are two 
species of European Anodonta based on 
glochidial characters. These species have 
many characters in common, but their exterior 
valve sculpturing is sufficiently different to 
separate them. Glochidia of A. anatina were 
figured by Flemming (1875: pi. 3, fig. 11), 
Schierholz (1888: pi. 2, fig. 29), Ortmann 
(1 91 2: pi . 1 9, fig. 3, as A. complanata), Giusti 
(1973: figs. 13-25, as A. cygnea), and Giusti 
et al. (1 975: figs. 10-21, as A. cygnea). 

Anodonta beringiana Middendorff, 1851 
(Fig. 6A-F) 

Material Examined 

OSUM 3711.1 - Outlet of Peper Lake, 
Kenai Peninsula, Alaska, 13 August 1957, R. 
Rausch. 

Description 

Glochidium subtriangular, moderately 
asymmetric, length 286 to 292 ¡.im (289 ± 
2.28 |.im, n - 5), height 284 to 293 |.im (290 ± 
3.42 |.im, n = 5). Anterior and posterior mar- 
gins about equal, more or less gently curved, 
with anterior margin slightly more produced, 
especially in the dorsal half of valve. Valve 
outline only slightly asymmetric. Exterior sur- 
face of valve malleated and pitted. Pits uni- 
formly distributed, except along the valve 
margin, where they are absent. Loose-looped 
sculpture covering extehor surface of valve. 
Larval thread present (Fig. 6A, C). Hinge 
straight, length 209 to 214 цт (211 ± 2.35 
(.im, n = 5). Posterior, central and anterior lig- 
aments of a single specimen, 34 цт, 58 цт 
and 1 1 8 |jm, respectively. Central ligament far 
posterior, at 31% from posterior to anterior. 
Styliform hook ahsing as a broad biconcave 
triangular plate. Microstylets robust, multifac- 
eted few in number (about 15). Micropoints 
bluntly lanceolate, on ventral rim of valve, 
along lateral edges of the microstylets and for 



GLOCHIDIAOF UNIONIDAE 



17 




FIG. 5. Glochldium of Anodonta anatina: A. exterior valve, OSUM 20912.2, bar length = 55 цгл; В. interior 
valve, OSUM 20912.2, bar length = 50 |am; С exterior valve sculpture, OSUM 20912.1, bar length = 1 цт; 
D. styliform hook, OSUM 20912.2, bar length = 20 цт; E. larval thread, OSUM 20912.2, bar length = 25 цгл; 
F. larval thread, OSUM 20912.2, bar length = 15 ¡am; G. microstylets, OSUM 20912.2, bar length = 5 цт. 



HOGGARTH 




FIG. 6. Glochidium of Anodonta beringiana, OSUM 3711.1: A. exterior valve, bar length = 45 цт; В. interior 
valve, bar length = 50 |.im; С ventral valve view, bar length = 40 |.im; D. styliform hook, bar length = 10 |.im; 
E. styliform hook, bar length = 10 \хт; F. styliform hook, bar length = 10 pm. 



GLOCHIDIAOF UNIONIDAE 



19 



a short distance on lateral, surfaces of hook. 
Unsculptured distal hook margin, wide. Hook 
located approximately 41% from posterior to 
anterior. 

Remarks 

This species was allied, on the basis of 
adult shell morphology, to A. cygnea by Simp- 
son (1914). The shape of this glochidium also 
allies A. beringiana with A. cygnea. The glo- 
chidium of A. beringiana can be distinguished 
from that of A. cygnea by its smaller overall 
size and hook structure. Inaba (1941) gave 
296 |.im for the length and height of this glo- 
chidium, and Cope (1959) reported length 
and height measurement of 275 |im x 300 [im. 

Anodonta lienneriyi Lea, 1 860 
(Fig. 7A-F) 

Material Examined 

OSUM 52882.2, 52882.3 - "Griffin Creek," 
Snohomish Co., Washington, January 1972, 
L. Gilbertson. 

Description 

Glochidium subtriangular, moderately 
asymmetric, length 350 to 354 ).im (352 ± 
2.08 |Lim, n - 3), height 340 to 350 |.im (344 ± 
5.13 цт, n = 3). Anterior margin rounded, ta- 
pering to meet a slightly curved posterior mar- 
gin at ventral terminus. Ventral terminus nar- 
rowly pointed, located about 40% from 
posterior to anterior. Malleations and pits uni- 
formly distributed on valve, except along 
valve margin, where dorsally converging lon- 
gitudinal ridges occur (Fig. 7C). Exterior valve 
sculpturing intermediate between beaded and 
loose-looped (Fig. 7E), resembling short 
lengths of strung beads closely packed on 
surface of valve. Hinge straight, 250 to 267 
|.im (259 ± 7.31 ¡am, n = 6) in length. Central 
ligament 84 to 89 \xvc\ (87 ± 2.07 цт, n = 6) 
long, centered about 40% from posterior mar- 
gin. Posterior ligament 55 to 65 цт (60 ± 3.25 
цт, n = 6) in length; anterior ligament 106 to 
114 |.im (111 ± 2.94 lam, n = 6) long. Styliform 
hook sharply pointed, with fewer than 20 mi- 
crostylets (Fig. 7B, D). Microstylets lanceo- 
late, sharply pointed, arranged in four proxi- 
mal rows, reduced to a single row distally. Two 
to three microstylets, and as many as seven 
micropoints, forming a cluster near tip of 



hook. Additional micropoints covering lateral 
surfaces of hook and along lateral borders of 
microstylets, leaving narrow unsculptured dis- 
tal hook edge. 

Remarks 

This species was also allied to A. cygnea by 
Simpson (1914) due to similarity in adult shell 
morphology. The shape of this glochidium is 
also similar to that of A. cygnea, although this 
species can be distinguished by its exterior 
surface sculpturing and the cluster of mi- 
crostylets and micropoints near the point of 
the hook. This aspect of the styliform hook re- 
sembles that of Pyganodon Cataracta and re- 
lated species east of the Rocky Mountains. 

Anodonta implicata Say, 1 829 
(Fig. 8A-F) 

Material Examined 

OSUM 52463.7 - Great Herring Pond, S 
shore by the Herring River, 0.8 mi. N of 
Bournedale, 1.6 mi. WNW of Sagamore, 
Barnstable Co., Massachusetts, 4 October 
1982, D. H. Stansbery & K. E. Wright. 

Description 

Glochidium lachrimiform, asymmetric, 
length 342 to 343 цт (343 ± 0.71 цт, n = 2), 
height 345 to 350 |.im (348 ± 3.54 цт, n = 2). 
Posterior margin gently curved, anterior mar- 
gin subrotund. Maximum inflation of anterior 
margin at about 50% from dorsal to ventral, 
broadly rounded ventral terminus occuring 
about 35% from posterior to anterior. Exterior 
valve surface finely malleated and pitted, ex- 
cept at valve margin. Pits uniformly distributed 
in the malleated surface, exterior valve sur- 
face with sculpturing intermediate between 
beaded and loose-looped (Fig. 8F). Hinge 
straight, 160 to 166 цт (163 ± 3.06 цт, n - 
3) long. Central ligament 74 to 77 \лт (76 ± 
2.12 |im, n = 2) in length, centered about 46% 
from posterior border of hinge. Posterior liga- 
ment 37 to 38 цт (38 ± 0.71 цт, n = 2) long; 
anterior ligament 48 to 52 цт (50 ± 2.36 ¡лт, 
n = 2) long. Hook styliform, broadly triangular, 
gradually tapered to blunt point (Fig. 8C). Mi- 
crostylets lanceolate, gradually increasing in 
size toward center, arranged in four rows near 
proximal border of hook. Micropoints numer- 
ous, covering lateral surfaces of hook. Un- 
sculptured distal hook margin, narrow. 



20 



HOGGARTH 




FIG. 7. Glochidium of Anodonta kennerlyi:A. exterior valve, OSUM 52882.2, bar length = 50 цт; В. interior 
valve, OSUM 52882.2, bar length = 50 |.im; С lateral view, OSUM 52882.3, bar length = 55 цт: D. styliform 
hook, OSUM 52882.2, bar length = 1 5 цгл; E. exterior valve sculpture. OSUM 52882.3. bar length = Uim; F. 
hinge, OSUM 52882.3, bar length = 35 цт. 



GLOCHIDIAOF UNIONIDAE 



21 




FIG. 8. Glochidium of Anodonta implicata. OSUM 52463.7; A. exterior valve, bar length = 50 |im; B. interior 
valve, bar length = 45 |im; С styliform hook, bar length = 1 ¡xm; D. valve pitting, bar length = 1 5 |.im; E. hinge, 
bar length = 25 цт; F. exterior valve sculpture, bar length = 2 цгл. 



22 



HOGGARTH 



Remarks 

Johnson (1946) describes this glochidium 
as, "typical of the genus Anodonta," even 
though his figure shows the short hinge line 
and greatly inflated anterior margin, both char- 
acters far from typical for this genus. Rand & 
Wiles (1 982) have also failed to recognize that 
this species can be distinguished from all other 
glochidia simply by the tear-drop outline of the 
valve. This glochidium is further distinguished 
by its hook structure and exterior valve sculp- 
ture. The glochidium of this species is figured 
by Johnson (1 946: pi. 1 6, fig. 3), Wiles (1 975: 
figs. 1 , 2, as A. ¡mplicataand A. Cataracta), and 
Rand & Wiles (1982: figs. 5-8). Rand & Wiles 
gave length and height measurements of 345 
|im X 345 |.im. 

Anodonta suborbiculata Say, 1831 
(Fig. 9A-F) 

Material Examined 

OSUM 13634 - Black River at U.S. Rt. 67 
bridge W of Hendrickson, Butler Co., Mis- 
souri, 14 October 1964, С В. Stein. 

Description 

Glochidium subtriangular, length 323 to 328 
|im (325 ± 2.08 \хт, n = 5), height 320 to 328 
).im (323 ± 3.46 [ivn, n = 5). Dorsal margin 
straight, 231 to 237 ¡.im (232 ± 2.41 цт, n = 
5) long. Posterior margin gently curved; an- 
terior margin, broadly curved. Maximum an- 
terior inflation at about 50% from dorsal to 
ventral. Ventral terminus, narrowly pointed, lo- 
cated about 42% from posterior to anterior. 
Exterior surface malleated, pitted (Fig. 9E), 
except along the edge of valve (Fig. 9C); fine 
structure of exterior surface consisting of fine 
non-overlapping lines referred to here as ver- 
miculate sculpturing (Fig. 9F). Central liga- 
ment 98 to 1 02 цт (1 00 ± 1 .71 цт, n = 4) in 
length, centered about 45% from posterior to 
anterior (Fig. 9B). Posterior ligament 52 to 56 
|im (54 ± 1.83 [xvr\, n = 4) long; anterior liga- 
ment, 74 to 81 fim (78 ± 2.99 дт, n = 4) long. 
Hook styliform, with about 40 lanceolate mi- 
crostylets and many micropoints. Unsculp- 
tured distal margin of hook narrow. 

Remarks 

This glochidium can be distinguished by its 
exterior valve sculpturing and hook. It resem- 



bles A. cygnea in shape but it has no other 
characters to tie it to that species. Surber 
(1915) stated that in, "general outline [the 
glochidium of] suborbiculata closely resem- 
bles Anodonta grandis but may be distin- 
guished by its smaller size". He gave mea- 
surements of 325 цт X 320 ¡am for length and 
height. However, the glochidia of these spe- 
cies are only superficially similar, and a close 
relationship between this species and any 
other member of the genus is not supported by 
glochidial characters. The glochidium of A. 
suborbiculata is figured by Surber (1 91 5: pi. 1 , 
fig. 1 ) and Utterback (191 5-1 91 6: fig. 7). 

Pyganodon grandis grandis (Say, 1 829) 
(Fig. 10A-F) 

Material Examined 

P. g. grandis: OSUM 38467.10 - Miami 
River, R.Mi. 82.4, at 1-75 bridge at Dayton, just 
above mouth of Mad River, Harrison Twp., 
Montgomery Co., Ohio, 6 February 1976, D. 
H. Stansbery. MAH 668-Olentangy River 
below Fifth Ave. bridge near Ohio State Uni- 
versity main campus, Columbus, Franklin Co., 
Ohio, 30 September 1984, K. Wright & K. Gal- 
lant. INHS 2247-Kankakee River at Kanka- 
kee, below hydroelectric plant, Kankakee Co., 
Illinois. 11 October 1985, J. M. Kasprowicz. P. 
g. corpulenta: OSUM 47890-Stonelick Creek 
at Stonelick Reservoir, 1.1 mi. SW of Edenton, 
2.8 mi. N of Newtonville, Wayne Twp., Cler- 
mont Co., Ohio, 1 October 1978, D. H. Stans- 
bery & K. G. Borror. OSUM 53653-Ohio River 
bank, R.Mi. 442.8-443.0, 0.3-0.4 mi. NW of 
Moscow, 2.2-2.4 mi. S of Point Pleasant, 6.9 
mi. SE of New Richmond, Clermont Co., Ohio, 
22 October 1984, K. E. Wright et al. 

Description 

Glochidium subtriangular, asymmetric, 
length 350 to 365 f.im (356 ± 5.56 цт, n = 7), 
height 350 to 360 |.im (355 ± 3.10 цт, n = 7). 
Posterior margin gently curved throughout its 
length. Anterior margin broadly curved to its 
point of maximum inflation at about 70% from 
dorsal to ventral. Ventral terminus broadly 
rounded, located about 40% from posterior to 
anterior. Valve surface malleated and pitted, 
except along its margin, where dorsally con- 
verging longitudinal ridges are found (Fig. 
IOC). Adductor muscle scar not evident. 
Coarse loose-looped exterior valve sculpture 
covering surface of valve (Fig. 10F). Hinge 



GLOCHIDIAOF UNIONIDAE 



23 




FIG. 9. Glochidium of Anodonta suborbiculata. OSUM 13634; A. exterior valve, bar length = 40 цт; В. inte- 
rior valve, bar length = 40 |дт; С. lateral view, bar length = 40 |am: D. styliform hook, bar length = 10 |im; E. 
valve pitting, bar length = 10 |am; F. exterior valve sculpture, bar length = 2 ¡am. 



24 



HOGGARTH 




FIG. 10. Glochidium of Pyganodon д. grandis. OSUM 38467.10: A. exterior valve, bar length = 55 (.im: B. in- 
terior valve, bar length = 55 |.im; С lateral view, bar length = 55 |.im: D. styliform hook, bar length = 15 |.im; 
E. interior valve pitting, bar length = 15 цт; F. exterior valve sculpture, bar length = 1 цт. 



GLOCHIDIAOF UNIONIDAE 



25 



straight, 250 to 259 цт (253 ± 3.20 цт, n = 
7) in length. Central ligament 90 to 94 ¡.im (92 
± 2.08 |.im, n = 3) long, centered between 
41 -42% from posterior margin. Posterior liga- 
ment 54 to 62 цт (57 ± 4.04 |.im, n = 3) long; 
anterior ligament 104 to 111 (.im (107 ± 3.61 
j.im, n = 3) in length. Hook styliform, with about 
20 microstylets. Microstylets arranged in four 
proximal rows, reduced to a single row distally 
(Fig. 10B, D). Five to six sharply pointed mi- 
crostylets forming a cluster near tip of hook. 
Micropoints few in number, found only along 
ventral rim of valve and along borders of mi- 
crostylets. Unsculptured distal hook margin 
very wide. 

The glochidium of P. g. corpulenta is nearly 
identical to that of P. g. grandis, with a length 
of 343 to 350 цт (348 ± 5.13 цт, n - 3), a 
height of 363 to 368 цт (366 ± 3.54 цт, n = 
3), and a hinge length of 260 to 275 цт (266 
± 7.94 j.im, n = 3). The posterior, central and 
anterior ligaments of a single specimen were 
70 i-im, 93 цт and 100 (.im in length, respec- 
tively. Surber figured this glochidium (1912: 
fig. 4; 1913: fig. 1 ) and gave length and height 
measurements of 350 цт. 

Remarks 

Ortmann (1912) gave 360 цт x 370 цт for 
length and height of the glochidium of P. g. 
grandis, and Tucker (1928) reported the fol- 
lowing ranges: length, 350-398 цт; height, 
343-390 цт. However, Surber gave measure- 
ments of 410 |im X 420 |.im. This extremely 
large range in size led Tucker (1928) to sug- 
gest that this was the result of examining the 
glochidia of a far ranging, variable species. 
Her material, Ortmann's and mine came from 
small streams, whereas Surber's material 
probably came from the Mississippi River and 
probably represents P. g. gigantia. The glo- 
chidium of P. g. grandis was figured by Lea 
(1858: pi. 5, figs. 32, 33, 34, as A. lewisii. A. 
ovata and A. decora = P. g. grandis, fide Ort- 
mann, 1919) and Surber (1912: pi. 3, fig. 45). 
Lea's figures are slightly different from each 
other, and none show the correct outline. 
Surber's figure demonstrates the asymmetri- 
cal valves of this glochidium. 

Pyganodon Cataracta Cataracta (Say, 1817) 
(Fig. 11A-E) 

Material Examined 

P с Cataracta; OSUU 52462.27, 52462.35 
- Great Herring Pond, S Shore by the Herring 



River, 0.8 mi. N of Bournedale [1.6 mi. WNW 
of Sagamore], Barnstable Co., Massachu- 
setts, 4 October 1984, D. H. Stansbery & K. 
Wright. P с marginata - OS UM 38962.6 
"Black Moshannon Lake" [near or part of 
Black Moshannon Creek?], Snowshoe Twp., 
Centre Co., Pennsylvania, September 1975, 
D. S. Gussman. 

Description 

Glochidium subtriangular, asymmetric, 
length 374 to 380 цт (376 ± 2.61 |im, n = 5), 
height 351 to 370 цт (363 ± 7.40 цт, n = 5). 
Dorsal margin straight; posterior margin gen- 
tly, evenly curved; anterior margin broadly 
curved to its maximum inflation at about 70% 
from dorsal to ventral. Ventral terminus 
broadly pointed, about 43% from posterior to 
anterior. Malleations and pits uniformly dis- 
tributed on the valve, except along valve mar- 
gin. Coarse loose-looped sculpture covering 
exterior surface of valve (Fig. 11 D). Hinge 
straight, 277 to 291 |im (284 ± 5.92 цт, n = 
5) long. Central ligament 98 to 1 02 |.im (1 00 ± 
2.83 }.im, n = 2) in length, centered about 42% 
from posterior to anterior. Posterior ligament 
71 to 75 |im (73 ± 2.83 (лт, n = 2) in length; 
anterior ligament, 115 to 116 цт (116 ± 0.71 
|im, n = 2) long. Hook styliform, with about 20 
lanceolate microstylets (Fig. 11 E). Micro- 
stylets arranged in four rows near ventral ter- 
minus, reduced to a single row distally, clus- 
tered near tip of hook (four to six per hook). 
Micropoints limited to proximal half of hook, 
except along lateral margins of microstylets, 
where they form a single row, leaving wide un- 
sculptured distal hook margin. 

The glochidium of P c. marginata had a 
length of 358 to 360 цт (359 ± 1 .41 цт, n = 
2), a height of 364 to 369 цт (367 ± 3.54 |im, 
n = 2), and a hinge length of 276 to 277 ¡im 
(277 ± 0.71 цт, n = 2). The posterior, central 
and anterior ligaments of a single specimen 
were 64 цт, 1 1 цт and 1 02 цт in length, re- 
spectively. 

Remarks 

Ortmann (1912) believed this species was 
the eastern representative of the P grandis 
complex. The similarities between the glo- 
chidia of these two species support the view 
of close relationship; however, the glochidium 
of P. Cataracta can be distinguished from 
that of P grandis by its longer central liga- 
ment. The glochidia of both P с Cataracta and 
P. с marginata have longer anterior ligaments 



26 



HOGGARTH 




FIG. 11. Glochidium of Pyganodon с Cataracta: A. exterior valve, OSUM 52462.27, bar length = 55 |.im; B. 
interior valve, OSUM 52462.35, bar length = 55 цгл: С. hinge, OSUM 52462.27, bar length = 40 |.im; D. ex- 
terior valve sculpture, OSUM 52462.27, bar length = 2 |.im; E. styliform hook, OSUM 52462.27, bar length = 
20|am. 



than any species in the Anodontinae. Rand & 
Wiles (1982) gave length and height mea- 
surement of 382 цт X 383 |.im, whereas Ort- 
mann (1912) reported 360 |.im x 370 цт 
(identical to his figures for P. g. grandis). This 
glochidium is figured by Lefevre & Curtis 
(1910: fig. C; 1912: fig. 1С), Calloway & 
Turner (1979: pi. 3, figs. 1, 3, 5, 7, 8), Wiles 
(1975: figs. 3, 7), and Rand & Wiles (1982: 
figs. 1-4). 



Pyganodon doliaris (Lea, 1863) 
(Fig. 12A-D) 

Material Examined 

OSUM 26405 - Potomac River along W 
shore Theodore Roosevelt Island, W. edge 
Washington D.C., just E of Arlington, Va., Dis- 
thct of Columbia. 22 February 1970, K. Hef- 
felfinger Olson. 



GLOCHIDIAOF UNIONIDAE 



27 




FIG. 12. Glochidium of Pyganodon doliaris. OSUM 26405; A. exterior valve, bar length = 55 jam; B. interior 
valve, bar length = 55 |.im; С exterior valve sculpture, bar length = 1 цт; D. styliform hook, bar length = 20 
|im. 



Description 

The glochidium of this species is essentially 
identical to that of P. g. grandis and P. с 
Cataracta. Its shape and hinge structure, ex- 
terior valve sculpture (Fig. 12C) and hook 
structure (Fig. 12D) place it firmly with these 
species. One glochidium measured 31 7 цт x 
317 цт X 240 |.im (length x height x hinge 
length) (Fig. 12B), while another measured 
405 |im x 368 urn x ЯП.Я nm (F\a. 12A). Both 



glochidia were removed from the marsupium 
of the same female. 

Remarks 

With such a limited amount of material and 
such great variability in size, it would not be 
appropriate to suggest limits. However, the 
larger measurements seem out of range for 
glochidia in this group, with the exception of P. 
g. gigantia. 



28 



HOGGARTH 



Utterbackia imbecillis (Say, 1 829) 
(Fig. 13A-F) 

Material Examined 

OSUM 9436.2 - Lake Erie off East Sister Is- 
land, Ontario, Canada, 13 July 1960, D. 
Mount. OSUM 34463 - Clear Fork Mohican 
River at Clear Fork State Park Campground 
B, 5 mi. NNW of Jelloway, Hanover Twp., Ash- 
land Co., Ohio, 13 July 1973, E. W. Tittsler. 
MAH 435 - Hellbranch Run at Bausch Rd. 
(Co. Rt. 1 1 ) bridge, 0.7 mi. S of Galloway, 1 0.0 
mi. SW of Columbus, Prairie Twp., Franklin 
Co., Ohio, 15 June 1983, M. A. Hoggarth. 
UWZY 24971.1 -Baraboo River, Til N, R6E, 
Sec. 1, NW 1/4 of NE 1/4, 25 meters down- 
stream of dam at Baraboo Sauk Co., Wiscon- 
sin, 12 October 1984, D. J. Heath. 

Description 

Glochidium subtriangular, length 291 to 313 
).im (304 ± 6.41 |.im, n = 1 2), height 289 to 306 
цт (300 ± 6.11 |.im, n = 12). Dorsal margin 
straight 240 to 256 цт (246 ± 5.97 )im, n = 
13) long. Anterior and posterior margins 
slightly and evenly curved. Anterior margin 
only slightly more produced than posterior 
margin, producing a slightly asymmetric valve 
outline. Surface of valve weakly malleated 
and pitted, except along smooth valve margin. 
Surface of valve covered with a uniform 
looped pattern, referred to here as tight- 
looped sculpture (Fig. 13F). Central ligament 
85 to 93 (.im (91 ± 3.50 цт, n = 4) long, cen- 
tered about 42% from posterior border of 
hinge. Anterior ligament 95 to 103 цт (100 ± 
3.79 jim, n = 4) in length; posterior ligament 
60 to 63 |.im (61 ± 1 .50 |.im, n = 4) long. Hook 
styliform, arising as a broad triangular plate 
from ventral terminus. Microstylets lanceolate 
near distal margin of hook, multifaceted near 
proximal border (Fig. 13C, D), arranged in 
three to four rows near ventral terminus, re- 
duced to a single row distally. A cluster of mi- 
crostylets and micropoints near point of hook 
present in some material (Fig. 13C); however, 
most micropoints cover proximal half of lateral 
surface of hook, leaving wide unsculptured 
distal margin. Ventral terminus located about 
45% from posterior to anterior. 

Remarks 

This glochidium can be distinguished from 
others in the subfamily by its exterior valve 



sculpture, its rather small size, and its rela- 
tively long central ligament. It resembles A. 
suborbiculata in these last two characters, but 
differs in valve sculpture and hook structure. 
The outline of this glochidium is like that of A. 
cygnea, whereas the hook allies this species 
to P. grandis. Ortmann (1912) gave length 
and height measurements of 300 цт x 310 
).im (for A. imbecillis) and 290 ¡.im x 300 цт 
(for /A. henryana. = U. imbecillis, fide Johnson, 
1 970). Surber (1912) gave 31 ¡im x 290 |im, 
and Tucker (1 927) reported 290 |am x 300 ¡im. 
This glochidium was figured by Lea (1858: pi. 
5, fig. 36), Ortmann (1911: pi. 89, fig. 13), 
Surber (1912: pi. 1 , fig. 2), and Tucker (1927: 
pi. 10, figs. 1,2). 

Anodontoides ferussacianus (Lea, 1834) 
(Fig. 14A-E) 

Material Examined 

OSUM 18275.2 - South Branch Phelps 
Creek at Rt. 322 bridge, 1.5 mi. E of Hunts- 
burg, 5.5 mi. NE of Middlefield, Huntsburg 
Twp., Geauga Co., Ohio, 23 March 1966, R. 
E. Jezerinac. MAH 989.4 - Silver Creek, R.M. 
4.6, at St. Rt. 15 bridge, 1.5 mi. N of Pioneer, 
8.4 mi. NNE of Montpelier, T9S, R2W, Sec. 
8/9, Madison Twp., Williams Co., Ohio, 2 Oc- 
tober 1986, M. A. Hoggarth. 

Description 

Glochidium subtriangular, only slightly 
asymmetric, length 319 to 326 цт (323 ± 
3.20 цт, n = 7), height 320 to 327 цт (324 ± 
2.64 ).im, n = 7). Hinge 231 to 238 цт (234 
± 2.93 |.im, n = 7) in length. Dorsal margin 
straight. Posterior and anterior margins about 
equally curved to a bluntly rounded ventral 
terminus. Exterior surface malleated and pit- 
ted, except along lateral margins and within a 
circular dorsomedial area or umbo (Fig. 14A, 
C). Exterior surface of valve covered by 
loose-looped sculpture (Fig. 14E). Central lig- 
ament 88 to 92|.im (91 ± 2.31 |.im, n = 3) long, 
centered about 42% from posterior to anterior. 
Anterior ligament 83 to 89 pm (86 ± 3.06 цт, 
n = 3) in length; posterior ligament 56 to 62 
(.im (59 ± 3.06 ^m, n = 3) long. Hook styliform, 
with two rows of microstylets extending dis- 
tally for at least three quarters of its length, lo- 
cated about 50% from posterior to anterior. A 
cluster of microstylets and micropoints occur- 
ring at distal end of hook. Micropoints located 



GLOCHIDIAOF UNIONIDAE 



29 




FIG. 13. Glochidium of Utterbackia imbecillis: A. exterior valve, UWZY 24971 .1 , bar length = 50 ¡.im; B. inte- 
rior valve, OSUM 9436.2, bar length = 60 |am; С styliform hook, UWZY 24971 .1 , bar length = 1 5 |am; D. styli- 
form hook, MAH 435, bar length = 1 5 ).im; E. hinge, UWZY 24971 .1 , bar length = 2 |.im; F. exterior valve sculp- 
ture, UWZY 24971 ,1 , bar length = 2 цт. 



30 



HOGGARTH 




FIG. 14. Glochidium of Anodontoides ferussacianus:A. exterior valve. MAH 989.4, bar length = 50 цт; В. in- 
terior valve, MAH 989.4, bar length = 50).im;C. hinge ligament. OSUM 18275.2, bar length = 30цт: D. styli- 
form hook, MAH 989.4, bar length = 25 |дт; E. exterior valve sculpture, MAH 989.4, bar length = 1 цт. 



GLOCHIDIAOF UNIONIDAE 



31 



on ventral rim of valve and on proximal sur- 
faces of hook, leaving wide unsculptured dis- 
tal hook margin. 

Remarks 

The glochidium of Anodontoides ferussa- 
cianus can be distinguished from above-de- 
scribed members of the Anodontinae by its 
double row of microstylets distally and its 
symmetry. The hook of this glochidium re- 
sembles that of Strophitus, but the outline of 
its valve is essentially like that of Anodonta. 
Ortmann (1912) and Surber (1912) gave the 
following size range for the glochidium of A. 
ferussacianus: 320-330)дт x 320-330 цт. 
This glochidium has been figured by Lea 
(1858: pi. 5, fig. 35), Ortmann (1911: pi. 89, 
fig. 12), and Surber (1912: pi. 13, fig. 43). Lea 
figured this glochidium without hooks, but Ort- 
mann (1919) suggests that Lea's specimens 
were immature. 

Simpsonaias ambigua (Say, 1 825) 
(Fig. 15A-F) 

Material Examined 

UWZY 22658, 22662, 22672 - Wisconsin 
River, T8N, RIB, Sec. 5, 9.5 mi. S. of Rich- 
land Center, Richland Co., Wisconsin, 1 6 July 
1984, D. J. Heath. OSUM 55995 - Wisconsin 
River, T8N, RÍE, Sec. 5, N side of river, 100 
meters upstream of public boat landing, 9.5 
mi. S. of Richland Center, Richland Co., Wis- 
consin, 20 April 1 985, D. J. Heath. 

Description 

Glochidium ovate subtriangular, slightly 
asymmetric, length 250 to 258 |im (255 ± 
3.39 (im, n = 6), height 256 to 265 (im (261 ± 
3.43 (im, n = 6). Dorsal margin straight, 164 
to 170 (im (168 ± 2.22 (im, n = 7) in length. 
Posterior margin gently and evenly curved. 
Anterior margin slightly more rounded than 
posterior margin. Maximum -anterior inflation 
occurring near 60% from dorsal to ventral. 
Ventral terminus bluntly rounded, about 45% 
from posterior to anterior. Exterior surface of 
valve finely malleated, uniformly pitted, ex- 
cept along smooth valve margin and umbo. 
Exterior surface sculpture beaded (Fig. 15E). 
Central ligament 54 to 63 (.im (58 ± 4.51 (im, 
n = 3) long, centered about 45% from poste- 



rior to anterior. Posterior ligament 45 to 49 (im 
(48 ± 2.31 (.im, n = 3) long; anterior ligament 
61 to 65 (im (63 ± 2.00 (Lim, n = 3) in length. 
Hook styliform, arising as a broad biconcave 
triangular plate from ventral terminus, taper- 
ing to a sharp point. Micropoints grading more 
centrally located microstylets. Proximal mi- 
crostylets multifaceted, sharply pointed, ar- 
ranged four to five abreast. Distal microstylets 
and micropoints lanceolate. Micropoints lo- 
cated on ventral terminus and lateral surfaces 
of hook, leaving narrow unsculptured distal 
hook margin. 

Remarks 

The glochidium of S. ambigua has only 
rarely been available for description. Lea 
(1858) described the glochidium of Margari- 
tanaliildrettiiana{^ S. ambigua, f /сУе Simpson, 
1900) as subrotund, with a straight or slightly 
incurved dorsal line and lacking hooks. He 
suggested, however, that hooks might be 
present in more mature specimens. Howard 
(1915, 1951) described the glochidium as 
triangular, with well-developed hooks, and 
Clarke (1985) added that the glochidium is 
slightly asymmetric with malleated surfaces. 
Clarke's description, however, is of Howard's 
figure rather than of glochidia he examined. 

This glochidium can be distinguished from 
most others by its shape, and from that of the 
other Anodontinae by its size, hook structure 
and exterior valve sculpturing. In regards to 
valve symmetry, size, hook structure, hinge 
structure and exterior valve sculpture, this 
glochidium is reminiscent of Unio. The 
glochidium of S. ambigua was figured by Lea 
(1 858: pi. 5, fig. 31 ), and Howard (1 951 : figs, 
4a, b). Lea's figure is much too round and 
lacks hooks. Howard's figures show the out- 
line of this glochidium correctly, but the draw- 
ings lack microstylets on the styliform hook. 
Howard's figures are reprinted in Clarke 
(1985: fig. 20d). 

Strophitus undulatus undulatus (Say, 1817) 
(Fig. 16A-F) 

Material Examined 

S. u. undulatus -OSUM 49443 Mississippi 
River, R.Mi. 635.0, East Channel across from 
Prairie du Chien, N tip of island above U.S. Rt. 
1 8 bridge, Crawford Co., Wisconsin, 21 March 



32 



HOGGARTH 




FIG. 15. Glochidium of Simpsonaias ambigua: A. exterior valve, UWZY 22658, bar length = 40 |.im; B. inte- 
rior valve, UWZY 22662, bar length = 40 цт; С, lateral view, UWZY 22662, bar length = 45 цт: D. styliform 
hook, UWZY 22658, bar length = 10 цт; E. exterior valve sculpture, OSUM 55995, bar length = 2 цт; F. 
hinge, UWZY 22672, bar length = 20 pm. 



GLOCHIDIAOF UNIONIDAE 



33 




FIG. 16. Glochidium of Strophitus u. undulatus:A. exterior valve, OSUM 49443, bar length = 50 цт; В. inte- 
rior valve, OSUM 52458.4, bar length = 50 urn; С lateral view, OSUM 52458.4, bar length = 70 |im; D. styli- 
form hook, OSUM 52458.4, bar length = 20 urn: E. exterior valve sculpture, OSUM 49443, bar length = 1 |дт; 
F. microstylets, OSUM 52458.4, bar length = 10 цт. 



34 



HOGGARTH 



1981, M. E. Havlik et al.. OSUM 52458.4 - 
Ashuelot River 0.4 mi. S of Surrey Mountain 
Dam, 4.3 mi. NNW of Keene, Cheshire Co., 
New Hampshire, 22 August 1 982, K. Wright & 
J. LeBlanc. MAH 792.1 - Fish Creek 0.4 mi. 
above its mouth at Co. Rt. 49 bridge, 1.1 mi. N 
of Edgerton, 10.4 mi. W of Bryan, St. Joseph 
Twp., Williams Co., Ohio, 29 October 1 986, D. 
H. Stansbery et al. S. и. tennesseensis - 
OSUM 33381.2 Laurel Creek 0.4 mi. N of 
Bradford along Va. Rt. 91, 6.3 mi. NE of 
Saltville, Rich Valley District, Smyth Co., Vir- 
ginia, 29 September 1971, D. H. Stansbery & 
W. J. Clench. OSUM 55449 - Clinch River, 
R.Mi. 270.6-270.9, 0.7-1 .0 mi. SW of Cleve- 
land, 1.5-1.8 mi. NE of Carbo, Russell Co., 
Virginia, 3 October 1985, G. T. Waiters. 

Description 

Glochidium depressed pyriform, asymmet- 
ric, length 360 to 369|.im (363 ± 5.20 ,um, n - 
3), height 289 to 299 |.im (295 ± 5.29 цт, n = 
3). Dorsal margin straight, 271 to 281 цт (278 
± 3.78 |jm, n = 6) in length. Posterior margin 
broadly arcuate. Anterior margin almost 
round, meeting posterior margin at a slightly 
rounded and ventrally produced, nipple-like, 
ventral terminus. Ventral terminus located 
about 44% from posterior to anterior. Exterior 
valve surface malleated and pitted. Pits uni- 
formly distributed throughout valve, except 
along valve margin and at umbo. Coarse 
loose-looped sculpture covering exterior sur- 
face of valves (Fig. 16E). Central ligament 87 
to 103 цт (95 ± 6.55 цт, n = 4) in length, lo- 
cated about 44% from posterior margin. Pos- 
terior ligament 70 to 78 |.im (74 ± 3.30 цт, n 
= 4) long; anterior ligament 101 to 110 цт 
(107 ± 4.08 |дт, n = 4) long. Hook styliform, 
covered with about 30 microstylets and nu- 
merous micropoints. Proximal microstylets 
bluntly pointed. Distal microstylets, multifac- 
eted, sharply pointed (Fig. 16F). Two rows of 
microstylets extend distally, and a few mi- 
crostylets and micropoints forming a cluster 
near sharply pointed terminus of hook. Un- 
sculptured distal hook margin narrow. 

The glochidium of S. u. tennesseensis is 
identical to that described above. One glo- 
chidium had the following dimensions: length, 
346 |.im; height, 298 (.im; hinge length, 268 
jjm; central ligament length, 94 |дт; posterior 
ligament length, 78 ¡im; anterior ligament 
length, 96 jjm. 



Remarks 

The glochidium of Strophitus is depressed- 
pyriform and possesses a styliform hook at 
the ventral terminus of each valve. Lea (1 858) 
described the glochidia of Strophitus edentula 
(= S. undulatus, ^/de Johnson, 1970) and S. 
undulata as subthangular, with a long straight 
dorsal margin, inflated side margins (Lea er- 
roneously viewed all glochidia as symmetrical 
about the dorsoventral axis), and a large hook 
with four rows of "granules" proximally, re- 
duced to two rows distally. Other than shape. 
Lea's description is surprisingly accurate. 

The glochidium of S. u. undulatus is unlike 
any so far described. Its shape, hook structure 
and coarse looped sculpture will distinguish it 
from other species examined. Glochidia of S. 
u. undulatus have been figured by Lea (1858: 
pi. 5, fig. 37, as S. edentula, pi. 5, fig. 38, as S. 
undulatus), and Surber (1912: pi. 1, fig. 3, as 
S. edentula). Surber gave length and height 
measurements of 350 цт x 285 |jm, and Ort- 
mann (1 91 2) gave 360 цт x 300 |Lim. 

Strophitus subvexus (Conrad, 1834) 
(Fig. 17A-F) 

Material Examined 

OSUM 36240 - Buttahatchie River about 
0.5 mi. above its mouth, 12 mi. NNW of 
Columbus, T16S, R19W, Lowndes Co., Mis- 
sissippi, 4 October 1974, R. Grace et al. 

Description 

Glochidium depressed pyriform, asymmet- 
ric, length 348 to 359 цт (354 ± 5.51 цт, n = 
3), height 288 to 292 ,um (290 ± 2.08 цт, n = 
3). Dorsal margin straight, 271 to 277 цт (274 
± 2.50 jam, n = 4) in length. Posterior margin 
broadly curved; anterior margin rounded. Lat- 
eral margins meeting at a narrowly rounded, 
nipple-like ventral terminus located about 
50% from posterior to anterior. Surface of 
valve coarsely malleated, uniformly pitted, ex- 
cept along valve margin and at umbo (Fig. 
17B, C). Coarse loose-looped sculpture cov- 
ering the exterior surface of valves (Fig. 1 7E). 
Central ligament about 84 |Lim in length, cen- 
tered about 40% from posterior to anterior. 
Posterior ligament about 70 цт long; anterior 
ligament about 123 цт long. Hook styliform, 
with proximal microstylets bluntly pointed dis- 



GLOCHIDIAOF UNIONIDAE 



35 




FIG. 17. Glochidium of Strophitus subvexus. OSUM 36240: A. exterior valve, bar length = 50 |.im; B. interior 
valve, bar length = 50 |.im; С exterior valve, bar length = 50 цт; D. styliform hook, bar length = 20 цт; E. ex- 
terior valve sculpture, bar length = 2 |.im; F. hinge, bar length = 35 ).im. 



36 



HOGGARTH 



tal microstylets lanceolate, multifaceted and 
sharply pointed. Microstylets arranged in a 
double row distally, forming a cluster near 
point of hook. Micropoints located on ventral 
valve rim and on lateral surfaces of hook, 
leaving narrow unsculptured distal hook mar- 
gin. 

Remarks 

The glochidium of S. subvexus resembles 
that of S. u. undulatus, except the former has 
a roundish outline and a more centrally posi- 
tioned ventral terminus. The near symmetrical 
outline of this glochidium is overemphasized 
slightly by Lea (1874: PI. 21, fig. 15, as Mar- 
gan tana spillmanii. = S. subvexus, f/de John- 
son, 1967), but his figure correctly shows the 
broadly curving margins. 

Alasmidonta wr/d/s (Rafinesque, 1820) 
(Fig. 18A-F) 

Material Examined 

OSUM 47518 - Horse Lick Creek, 0.3 mi. 
below mouth of Raccoon Creek at Dango, 7.6 
mi. SW of Mckee, Jackson Co., Kentucky, 28 
February 1980, S. Call et al. 

Description 

Glochidium depressed pyriform, only 
slightly asymmetric, length of 300 to 319 \.^m 
(307 ± 7.59 |im, n - 7), height 245 to 260 цт 
(251 ± 4.35 ¡am, n = 7). Dorsal margin 
straight, 245 to 258 |.im (250 ± 3.93 ,um, n = 
7) long. Posterior margin gently and evenly 
curved; anterior margin only slightly more pro- 
duced than posterior margin. Ventral terminus 
roundly pedicellate, about 45% from posterior 
to anterior. Exterior surface of valve finely 
malleated and pitted, except along valve mar- 
gin (Fig. 18C) and at umbo. Pit density re- 
duced in area of adductor muscle scar (Fig. 
1 8F), and loose-looped sculpture covering the 
exterior surface of valve (Fig. 18E). Central 
ligament 73 to 81 цт (76 ± 4.1 6 |.im, n = 3) in 
length, centered about 42% from posterior to 
anterior. Postehor ligament 65 to 71 цт (68 ± 
4.04 цт, n = 3) long; anterior ligament 100 to 
109|.im(105 ± 4.93 |.im,n = 3) in length. Hook 
styliform, broadly connected to ventral termi- 
nus and covered with about 30 microstylets 
and numerous micropoints. Microstylets 
lanceolate, arranged in three proximal rows 



and reduced to two distal rows. Six to eight 
microstylets and micropoints forming a cluster 
near point of hook. Micropoints extend over 
nipple-like ventral terminus beyond edge of 
ventral margin of valve, leaving wide unsculp- 
tured distal hook margin. 

Remarks 

Clarke (1981a) described many of the 
glochidia of the Alasmidontini using SEM, in- 
cluding this species. Our descriptions of this 
glochidium are almost identical, except for the 
location of the hook. Actually, we agree here 
as well, but Clarke confused the anterior-pos- 
terior orientation of the glochidium. He stated, 
"apices are located slightly anterior of center 
(about 47%)." Actually the apices (= ventral 
termini) are located about that distance from 
the posterior margin. 

Ortmann (1912) reported length and height 
measurements of 300 цт x 250 |am, and 
Surber (1912) gave 300 |am x 255 цт for this 
glochidium. Clarke (1 981 a) gave the following 
ranges; length 286-292 цт, height 232-235 
|лт, and hinge length 205 (.im. The figure for 
hinge length is probably a typographical error 
and should read 250 цт (approximate hinge 
length taken from his fig. 6b, c). 

This glochidium is distinguished from that of 
Strophitus by its finer exterior valve sculpture 
and its broadly connected hook. This glochid- 
ium is figured by Lea (1858; pi. 5, fig. 30, as 
Margaritana deltoidea. = A. viridis, fide Simp- 
son, 1900), Surber (1912: pi. 1,fig. 1, as Л /as- 
midonta calceola. = A. viridis, fide Clarke, 
1 981 a), Ortmann (1 91 2: pi. 1 9, fig. 4, as Alas- 
midonta minor = A. viridis, fide Clarke, 
1981a), Clarke (1981a: fig. 6), and Zaie & 
Neves (1982: fig. 1 , as A. minor). 

Alasmidonta heterodon (Lea, 1829) 
(Fig. 19A-F) 

Material Examined 

OSUM 25106.2 - Canoe River at old New- 
land St. bridge, 2.45 mi. NNE of Norton, Bris- 
tol Co., Massachusetts, 2 June 1 969, H. D. At- 
hearn. 

Description 

Glochidium depressed-pyrlform, symmetric 
when immature (Fig. 18A). becoming asym- 
metric with the development of hook (Fig. 



GLOCHIDIAOF UNIONIDAE 



37 





— «-««-^ШЩ^ 


1Ш^^ 




eWnîlffraSRI«» 




w 


rW^ 


^ЩЯЁ^Ш- 


i 


'M 


J 




^ 


D 




FIG. 18. Glochidium of Alasmidonta viridis, OSUM 47518; A. exterior valve, bar length = 45 цт; В. interior 
valve, bar length = 45 lam; С lateral view, bar length = 45 ¡.im; D. styliform hook, bar length = 15 |дт; E. ex- 
terior valve sculpture, bar length = 2 (.im: F, interior valve pitting, bar length = 10 цт. 




FIG. 19. Glochidium of Alasmidonta heterodon. OSUM 25106.2; A. exterior valve of immature glochidium. 
bar lengthi = 45 |am; B. interior valve, bar length = 70 цт; С. exterior valve, bar length = 45 цт; D. interior 
valve, bar lengtfi = 60 цт; E. styliform hook, bar length = 10 |.im: F. exterior valve sculpture, bar length = 
1 \im. 



GLOCHIDIAOF UNIONIDAE 



39 



18B, С, D), length 330 to 338 цт (334 ± 3.65 
цт, n = 4), height 258 to 268 цт (265 ± 4.86 
|am, n = 4). Dorsal margin straight, 253 to 
266 цт (261 ± 5.94 цт, n = 4) in length. Pos- 
terior margin slightly and evenly curved; ante- 
rior margin more broadly curved, especially 
near dorsal margin. Point of maximum lateral 
inflation at between 40-50% from dorsal to 
ventral. Ventral terminus curved, slightly out- 
wardly produced. Exterior valve surface finely 
malleated and pitted, except along its margins 
and at umbo. Interior pitting not uniform; pit 
density reduced in adductor muscle scar. 
Loose-looped sculpture occurring on exterior 
surface of valves (Fig. 19F). Central ligament 
about 80 |.im long, centered about 42% from 
posterior to anterior. Posterior ligament about 
75 цт long; anterior ligament about 110 цт 
long. A styliform hook extending dorsally from 
ventral terminus of each valve. Hook covered 
with about 40 microstylets and many micro- 
points, located about 42% from posterior to 
anterior. Microstylets arranged in four to six 
proximal rows and reduced to two rows dis- 
tally. Micropoints extending over edge of ven- 
tral terminus, along ventral margin of valve, 
and for a short distance on lateral surfaces of 
hook, leaving wide unsculptured distal hook 
margin. 

Remarks 

Clarke's (1981a) and my descriptions of 
this glochidium are, not surprisingly, very sim- 
ilar. We both examined glochidia removed the 
same adult female. He reported length, 
height, and hinge length measurements of 
325 ,um X 255 цт x 267 цт, respectively. All 
of these are within the ranges found during 
this study. This glochidium is very similar to 
that of A. viridis, but differs from that species 
in being more inflated toward the anterior end 
and in having a less broadly attached styli- 
form hook. This glochidium has been figured 
by Clarke (1981a; fig. 9). 

Alasmidonta undulata (Say, 1817) 
(Fig. 20A-F) 

Material Examined 

OSUM 52434.4 - Merrimack River just 
below Sewalls Fall's Dam, 2.7 mi. SE of Pe- 
nacook, 3.7 mi. NNW of Concord, Concord 
Twp., Merrimack Co.. New Hampshire, 31 Oc- 
tober 1982, K. E.Wright. 



Description 

Glochidium pyriform, much higher than 
long, asymmetric, length 343 to 358 ¡.im (353 
± 5.89 |.im, n = 5), height 365 to 378 |am (371 
± 5.13 цт, n ^ 5). Dorsal margin straight, 247 
to 260 |.im (254 ± 5.59 (.im, n = 5) in length. 
Posterior margin outwardly curved dorsally, 
becoming straight to slightly incurved before 
ventral terminus. Anterior margin broadly 
curved. Ventral terminus slightly incurved. 
Maximum anterior and posterior inflation at 
about 30% from dorsal to ventral, with ventral 
terminus rounded, located about 40% from 
posterior to anterior. Valve surface coarsely 
malleated, uniformly pitted, except at the 
umbo and along valve margin, where pits and 
malleations are absent. Exterior surface cov- 
ered with sparse rosette sculpture, separated 
by areas of beaded sculpture (Fig. 20E). Lar- 
val thread present (Fig. 20D). Central liga- 
ment 89 to 98 |.im (92 ± 5.19 цт, n = 3) in 
length, centered about 40% from posterior to 
anterior. Posterior ligament 53 to 55 цт (54 ± 
1.15 jjm, n = 3) in length; anterior ligament 
103 to 105 |im (104 ± 1.00 цт, n = 3) long. 
Hook styliform, covered with numerous (about 
120) microstylets and micropoints. Micro- 
stylets pyramidal, multifaceted, arranged in 
about six proximal rows, reduced to about 
four rows distally. Micropoints present along 
ventral valve margin, at lateral margins of mi- 
crostylets and for a short distance down lat- 
eral surfaces of hook, leaving wide unsculp- 
tured distal hook margin. 

Remarks 

The glochidium of A. undulata can be dis- 
tinguished by its large size, pyriform valve 
shape, complex hook and unique exterior 
valve sculpture. Ortmann (1912) described 
this glochidium as, "moderately large, higher 
than long, with strong hooks. Length 0.34 
mm; height 0.36 mm." Clarke (1981a) re- 
ported length and height measurements of 
310 i-im X 370 i^m and figured the glochidium 
(his fig. 13). This glochidium also is figured by 
Wiles (1975; fig. 6, as Anodonta Cataracta). 

Alasmidonta marginata Say, 1818 
(Fig.21A-l) 

Material Examined 

M AH 277.1 - Big Darby Creek at and above 
McLean Mill Rd. bridge, 0.2 mi. SW of Fox, 4.7 



40 



HOGGARTH 














FIG. 20. Glochidium of Alasmidonta undulata. OSUM 52434.4; A. exterior valve, bar length = 60 цт: В. in- 
terior valve, bar length = 55 цт; С. styliform hook, bar length = 20 ¡.im; D. interior valve, bar length = 50 цт; 
E. exterior valve sculpture, bar length = 1 цт; F. micropoints, bar length = 10 |.im. 



GLOCHIDIAOF UNIONIDAE 



41 




FIG. 21 . Glochidium of Alasmidonta marginata: A. exterior valve, MAH 724.1 , bar length = 55 цт; В. interior 
valve, MAH 724.1 , bar length = 45 (.im; С exterior valve sculpture, MAH 724.1 , bar length = 1 |im; D. exte- 
rior valve sculpture, MAH 277.1 , bar length = 1 цт; E. styliform hook, MAH 277.1 , bar length = 25 |am; F. hair 
cell, MAH 724.1 , bar length = 5 ¡am; G. adductor rnuscle, MAH 724.1 , bar length = 5 ¡jm: H. mantle cells. MAH 
724.1, bar length = 10 |im; I. larval thread, MAH 724.1, bar length = 10 цт. 



42 



HOGGARTH 



mi. NW of Circleville, Jackson Twp., Pickaway 
Co., Ohio, 1 October 1982, IVl. A. Hoggartln et 
a!.. MAH 724.1 - Fisti Creek above and below 
Edon Rd. bridge, 1 .9 mi. NW of Edgerton, St. 
Josepfi Twp., Williams Co., Ohio, 2 October 
1985, M. A. Hoggarth & D. Rice. 

Description 

Glochidium pyriform, higher than long, 
asymmetric, length 335 to 341 ).im (339 ± 
2.87 (.im, n = 4), height 360 to 372 (im (365 ± 
5.74 [im, n = 4). Dorsal margin straight, 230 to 
235 |.im (233 ± 0.96 |im, n - 4) in length. Pos- 
terior margin broadly curved (especially dor- 
sally), with maximum inflation at about 30% 
from dorsal to ventral. Anterior margin more 
broadly curved than posterior margin. Maxi- 
mum anterior inflation at about 40% from dor- 
sal margin. Lateral margins slightly incurved 
ventrally, producing a narrowly rounded ven- 
tral terminus. Ventral terminus about 40% 
from posterior to anterior. Exterior surface of 
valve coarsely malleated and densely pitted, 
except at valve margins and at umbo. Exterior 
surface covered with beaded sculpture (Fig. 
21 D), although fine loose-looped sculpture 
present near umbo of one glochidium (Fig. 
21 C). Larval thread present (Fig. 21 1). Central 
ligament 67 to 73 |лт (70 ± 3.54 цт, n = 2) in 
length, centered about 40% from posterior to 
anterior. Posterior ligament 59 to 61 цт (60 ± 
1 .41 цт, n = 2) long; anterior ligament 1 00 to 
1 07 |.im (1 04 ± 4.95 цт, n - 2) in length. Hook 
styliform, with many microstylets (about 120) 
and many micropoints. As in A. undulata, dis- 
tal margin of hook parallel to ventral margin, 
except at its center, where it becomes 
strongly curved to a sharp distal point. Mi- 
crostylets pyramidal, multifaceted, arranged 
in six to seven proximal rows, reduced to four 
rows distally. Micropoints occur along rim of 
ventral terminus, at lateral margins of mi- 
crostylets and for a short distance down lat- 
eral surfaces of hook. Distal hook margin with 
wide unsculptured band. 

Sensory hair cells (Fig. 21 F) have been de- 
scribed using light microscopy (Lillie, 1895; 
Wood, 1974), SEM (Giusti et al., 1975; Rand 
& Wiles, 1982) and transmission electron mi- 
croscopy (Zs.-Nagy & Labos, 1969). It is gen- 
erally thought that during attachment to the 
host, the tissues of the host push down on the 
hair cells and that the response to this stimu- 
lus is prolonged muscle contraction. It ap- 
pears that when the hairs are bent, their 
movement stimulates a ring of tissue, possibly 



composed of nerve tissue that encompass the 
base of the cell (at arrow). 

The adductor muscle (Fig. 21 G) is com- 
posed of long cells of contractile elements 
that attach to the crystalline matrix of the 
valves. The adductor muscle scar is often vis- 
ible as a rough elliptical area or by its reduced 
number of pits. There is no evidence that the 
muscle cells actually insert within these pits. 
The larval mantle cells are five to seven sided 
(Fig. 21 H) and do not appear to be pitted, as 
suggested by Rand & Wiles (1982). They 
suggested that the larval mantle was pitted to 
correspond to the pits in the valve and that 
this might facilitate gas exchange, nutrient up- 
take, or waste elimination. This was not ob- 
served, and therefore it is suggested that the 
pits are simply a result of the absence of 
stress traversing the body of the shell. Where 
stress is transferred from ventral margin to 
hinge (the lateral margins), pitting is absent 
and ridges (providing additional strength) 
occur. The pits may serve no function and 
may simply be a consequence of glochidial 
valve morphology (Hoggarth & Gaunt, 1988). 

Remarks 

This glochidium can be distinguished from 
that of A. undulata by differences in exterior 
valve sculpturing. Otherwise they are very 
similar. Lea (1858) described this glochidium 
as subthangular, with a long, straight dorsal 
line and inflated side margins. He described 
the hook as terminating in an arrowhead 
point. Clarke (1 981 a) also described the point 
of the hook as arrowhead-like. The arrowhead 
effect is probably due to the collapse of the 
lateral surfaces of the hook and is therefore 
an artifact of drying. This glochidium has a 
wide size range: (length x height) 330 цт x 
360 |Lim (Ortmann, 1912); 350 |im x 380 |im 
(Surber, 1912); 300 цт x 350 |nm (Utterback, 
1915-1916); 341 |.im x 346 |.im (Clarke, 
1981a) and is figured by Lea (1858:pl. 5, fig. 
27), Surber (1912: pi. 3, fig. 42, as A. truncata. 
= A. marginata, fide Stansbery et al., 1985), 
and Clarke (1981a: fig. 20). 

Pegias fabula (Lea, 1838) 
(Fig. 22A-F) 

Material Examined 

OSUM 41 308.3 - Little South Fork Cumber- 
land River at Freedom Church Ford. 2.0 mi. 
ENE of Ritner, 14.3 mi. E of Monticello, Wayne 




FIG. 22. Glochidium of Pegias fabula: A. exterior vaive, OSUM 41 308.3, bar length = 55 цт; В. interior valve, 
OSUM 41 309.1 . bar length = 55 \xm; С exterior valve. OSUM 41 308.3, bar length = 55 |дт; D. lateral view, 
OSUM 41308.3, bar length = 55 цгл; E. exterior valve sculpture, OSUM 41308.3, bar length = 2 |im; F. styii- 
form hook, OSUM 41309.1, bar length = 20 urn. 



44 



HOGGARTH 



Co., Kentucky, 22 October 1977, W. & L. 
Starnes. OSUM 41309.1 - Little South Fork 
Cumberland River at Freedom Church Ford, 
2.0 mi. ENE of Ritner, 14.3 mi. E of Monticello, 
Wayne Co., Kentucky, 22 October 1977, A. 
Bogan. 

Description 

Glochidium oval quadrate to roundly trape- 
zoidal, length 385 to 388 цт (386 ± 1 .53 |am, 
n = 3), height 319 to 325 цт (322 ± 3.06 |am, 
n = 3). Dorsal margin straight, 200 to 210 |am 
(205 ± 5.03 цт, n = 3) long. Posterior margin 
roundly arcuate. Anterior margin inflated dor- 
sally, slightly incurved near ventral terminus. 
Ventral terminus slightly rounded, about 35% 
from posterior to anterior. Exterior surface pit- 
ted but not malleated, smooth in area of ad- 
ductor muscle scar and at margin of valve. 
Adductor muscle scar very large (Fig. 22B-D). 
Exterior sculpturing tight-looped (Fig. 22E). 
Central ligament 68 to 73 цт (71 ± 2.89 |jm, 
n = 3) long, centered about 45% from poste- 
rior to anterior. Posterior ligament 56 to 63 цт 
(60 ± 3.51 цт, n = 3) in length; anterior liga- 
ment 69 to 79 ).im (74 ± 5.03 |.im, n = 3) long. 
Hook, styliform, sharply pointed, broadly con- 
nected to ventral valve margin. Hook with 
about 75 lanceolate microstylets arranged 
three abreast near proximal end of hook and 
in a double row distally. About 15 microstylets 
forming a cluster near distal end of hook. Mi- 
cropoints limited to ventral rim of valve and 
along margins of microstylets, leaving very 
wide unsculptured distal hook margin. 

Remarks 

The glochidium of P. fabula cannot be con- 
fused with that of any other species. Its 
quadrate shape, broadly connected styliform 
hook, tight looped exterior valve sculpturing 
and extremely large adductor muscle scar 
distinguish it. Clarke (1981a) figured this 
glochidium (his fig. 3) and provided length and 
height measurements for two specimens: 354 
jim X 309 цт and 380 цт x 310 yim. 

Arcidens confragosus (Say, 1 829) 
(Fig. 23A-G) 

Material Examined 

OSUM 52015 - Green River at Glenmore 
below lock 5 dam, 12 mi. N of Bowling Green, 



Warren Co., Kentucky, 2 November 1977, 
D. H. Stansbery et al. 



Description 

Glochidium pyriform, about as long as high, 
asymmetric, length 352 to 363 цт (359 ± 
5.32 |.im, n - 4), height 353 to 355 |am (354 ± 
1 .41 |im, n = 2). Dorsal margin straight, 237 to 
252 |.im (246 ± 7. 1 6 цт, n = 5) in length. Pos- 
terior margin produced dorsally, incurved ven- 
trally, with its maximum inflation between 
30-40% from dorsal to ventral. Anterior mar- 
gin broadly rounded dorsally, incurved just be- 
fore ventral terminus, maximum inflation at 
about 50% from dorsal margin. Ventral termi- 
nus narrowly rounded, located about 40% 
from posterior to anterior. Exterior surface 
coarsely malleated and densely pitted, except 
at umbo and along valve margin. Dense 
rosette sculpturing covering exterior surface 
of valve (Fig. 23E). Central ligament 63 to 
78 |im (71 ± 6.40 |лт, n = 4) long, centered 
about 42% from posterior to anterior. Poste- 
rior ligament 59 to 72 |.im (68 ± 5.91 ¡am, n = 
4) long; anterior ligament 100 to 115 цт (110 
± 7.14 цт, n = 4) long. Styliform hook ex- 
tending from ventral terminus as a very 
strongly biconcave triangular plate with about 
80 pyramidal microstylets arranged in about 
five rows (Fig. 23D, F). Number of rows of 
microstylets same from proximal to distal 
ends of hook. Micropoints occur on rim of 
valve, at ventral terminus and along mi- 
crostylet border, leaving wide unsculptured 
distal hook margin. 

Remarks 

The glochidium of A. confragosus is pyri- 
form and about as long as high. Anterior and 
posterior margins are greatly inflated dorsally 
and slightly incurved ventrally. The ventral ter- 
minus is narrowly rounded, and the number of 
rows of microstylets remain constant from 
proximal to distal ends of the styliform hook. 

This glochidium is similar to that of some 
members of the genera Alasmidonta and Las- 
migona but can be distinguished by its equal 
height and length, its exterior valve sculpture, 
and the arrangement of microstylets on the 
hook. Length and height measurements are 
given by Surber (1912: pi. 1, fig. 5) 355 цт x 
350 цт, and Clarke (1981a: fig. 31) 359 pm x 
360 i.im, both of whom also figure the glochid- 
ium. 



GLOCHIDIAOF UNIONIDAE 



45 





i 



FIG. 23. Glochidium of Arcidens confragosus. OSUM 52015; A. exterior valve, bar length = 65 цт; В. inte- 
rior valve, bar length = 65 (.im; С styliform hook, bar length = 25 i-im; D. styliform hook, bar length = 5 цт; E. 
exterior valve sculpture, bar length = 1 цт; F. microstylets, bar length = 5 |.im; g. hinge, bar length = 40 |im. 



Lasmigona compressa (Lea, 1829) 
(Fig. 24A-F) 

Material Examined 

OSUM 23179.1 - Little Darby Creek above 



Rosedale-Plain City Rd. bridge, 2.8 mi. E of 
Rosedale, Pike Twp., Madison Co., Ohio, 20 
October 1969, С В. Stein et at.. MAH 702 - 
Big Darby Creek below access point within 
Battelle-Darby Metro Park, 0.6 mi. S of 
Georgesville, 3.5 mi. SW of Galloway, Pleas- 



46 



HOGGARTH 







Ki 



FIG. 24. Glochidium of Lasmigona compressa: A. exterior valve. OSUM 23179.1, bar length = 45|.im: B. in- 
terior valve, OSUM 23179.1, bar length = 45 цт; С lateral view, MAH 702, bar length = 70цт; D. styliform 
hook, MAH 702, bar length = 30 цт; E. extehor valve sculpture, MAH 702, bar length = 1 |.im; F. microstylets, 
MAH 702, bar length = 10 цт. 



GLOCHIDIAOF UNIONIDAE 



47 



ant Twp., Franklin Co., Ohio, 9 September 
1985, H. T. Albin. MAH 727 - Fish Creek 
above and below Edon Rd. bridge, 1.9 гл1. 
NW of Edgerton, St. Joseph Twp., Williams 
Co., Ohio, 2 October 1985, M. A. Hoggarth & 
D. Rice. 



Description 

Glochidium depressed pyriform, longer 
than high, strongly asymmetric, length 317 to 
327 |iim (323 ± 4.39 цт, n = 5), height 283 to 
288 цт (286 ± 2.51 цт, n = 5). Dorsal mar- 
gin straight, 230 to 239 (.im (234 ± 3.36 цт, n 
= 5) long. Posterior margin strongly curved; 
anterior margin greatly inflated dorsally, be- 
coming more gently curved ventrally. Poste- 
rior and anterior margins joining at a gently 
rounded, nipple-like ventral terminus. Exterior 
valve surface coarsely malleated and pitted 
throughout, except at umbo and at margin of 
valve (Fig. 24C). Loose-looped sculpture cov- 
ering exterior surface of valve (Fig. 24E). 
Central ligament 83 to 84 цт (84 ± 0.71 ¡um, 
n = 2) in length, centered about 46% from 
posterior margin. Posterior ligament 67 to 68 
|nm (68 ± 0.71 |im, n = 2) long; anterior liga- 
ment 81 to 82 )im (82 ± 0.71 ¡.im, n - 2) in 
length. Styliform hook armed with about 25 
stout microstylets arrange in three proximal 
rows reduced to two widely off-set distal rows, 
extending from ventral terminus and located 
approximately 45% from posterior to anterior. 
Micropoints extend over edge of valve at ven- 
tral terminus and along valve margin, leaving 
very wide unsculptured distal hook margin. 



Remarks 

Surber (1912), Ortmann (1912), Tompa 
(1979), and Clarke (1985) gave length and 
height measurements for this glochidium; 353 
|im X 31 3 цт, 340 цт x 280 ^im, 320 цт x 260 
|im, and 344 ¡.im x 275 |.im. This glochidium 
can be distinguished by its widely offset dou- 
ble row of microstylets and its wide unsculp- 
tured distal hook margin. Clarke (1985) de- 
scribed the hook of this species as having a 
single distal row of microstylets; however, his 
micrographs show only collapsed hooks that 
are very difficult to interpret. This glochidium 
was figured by Lea (1 858: pi. 5, fig. 23, as Unio 
pressus, = L. compressa, f/de Simpson 1900), 
Ortmann (1911: pi. 89, fig. 10), Surber (1912: 
pi. 3, fig. 44), and Clarke (1985: fig. 13). 



Lasmigona subviridis (Conrad, 1835) 
(Fig. 25A-D) 

Material Examined 

OSUM 271 31 .66, 271 31 .68 - Little River at 
U.S. Rt. 221 bridge, at Woods Store, 5.3 mi. 
NE of Floyd, 33.7 mi. SW of Roanoke, Floyd 
Co., Virginia, 3 October 1 970, D. H. Stansbery 
& W.J. Clench. 

Description 

Glochidium depressed pyriform, 368 to 383 
цт (376 ± 5.81 цт, n = 5) in length, 309 to 
31 8 |.im (31 2 ± 3.71 цт, n = 5) in height. Pos- 
terior margin, strongly curved; anterior margin 
broadly rounded. Point of maximum posterior 
inflation at about 30% from dorsal to ventral; 
point of maximum anterior inflation at about 
40-50% from dorsal margin. Dorsal margin 
straight, 245 to 264 |.im (254 ± 6.82 цт, n = 
5) in length. Exterior valve surface coarsely 
malleated and uniformly pitted, except within 
a narrow marginal band. Ventral terminus 
broadly rounded, not produced or nipple-like 
as in the other members of the genus, located 
about 40% from posterior to anterior. Exterior 
surface sculpturing loose-looped (Fig. 25C). 
Central ligament 87 to 89 цт (88 ± 1 .00 цт, 
n = 3) in length, centered about 40% from 
posterior to anterior. Posterior ligament 57 to 
68 цт (63 ± 5.51 |im, n= 3) long; anterior lig- 
ament 100 to 107 |im (104 ± 3.61 |.im, n = 3) 
long. Styliform hook armed with about 25 mi- 
crostylets and numerous micropoints. Mi- 
crostylets lanceolate, about five to six abreast 
proximally, arranged in a double off-set row 
distally. Micropoints located on ventral margin 
of valve and on the lateral surfaces of hook 
but not over the edge of valve at the ventral 
terminus (Clarke 1 985, Fig. 1 6b), leaving wide 
unsculptured distal hook margin. 

Remarks 

This glochidium can be distinguished by its 
more broadly curved lateral margins and the 
absence of micropoints extending onto the 
valve at the ventral terminus. Ortmann (1912) 
gave 360 |im x 300 цт for the length and 
height of this glochidium, and Clarke (1985) 
gave 350-372 |am x 285-303 цт. This 
glochidium was figured by Lea (1874: pi. 21, 
fig. 14, as Unio tappanianus, = L. subviridis, 
fide Ortmann & Walker, 1922) and Clarke 



48 



HOGGARTH 




FIG. 25. Glochidium of Lasmigona subviridis: A. exterior valve. OSUM 27131 .68, bar length = 55 цт: В. in- 
terior valve, OSUM 27131 .68, bar length = 80 цт: С. extehor valve sculpture, OSUM 27131 .66, bar length 
= 1 jam; D. styliform hook, OSUM 271 31 .68, bar length = 1 5 цт. 



(1 985: fig. 1 6). Lea's figure does not show the 
morphologically depression of the glochidium. 



Tazewell, Tazewell Co., Virginia, 13 October 
1985, D. H. Stansbery. 



Lasmigona holstonia (Lea, 1838) 
(Fig. 26A-D) 

Material Examined 

OSUM 55826.6, 55826.7 - South Fork 
Clinch River at St. Rt. 61 bridge, E edge of 



Description 

Glochidium subtriangular, length and height 
about equal. 281 to 291 |.im (286 ± 3.65 |.im, n 
= 5) in length, 275 to 294 |.im (282 ± 7.76 pm. 
n = 5) in height. Dorsal margin straight, 221 to 
235 цт (228 ± 5.32 |.im, n = 5) long. Posterior 



GLOCHIDIAOF UNIONIDAE 



49 




FIG. 26. Glochidium of Lasmigona holstonia. OSUM 55826.6; A. exterior valve, bar length = 45 цт; В. inte- 
rior valve, bar length = 40 цт; С. exterior valve sculpture, bar length = 1 ¡.im; D. styliform hook, bar length = 
10 цт. 



margin slightly and evenly curved, with its 
point of maximum inflation about 20-30% 
from dorsal margin. Anterior margin broadly 
curved, with a tendency to be incurved before 
ventral terminus, with its maximum inflation at 
about 40-50% from dorsal to ventral. Ventral 
terminus narrowly rounded, located about 
40% from posterior to anterior. Exterior sur- 
face finely malleated, uniformly pitted, except 



along valve margin. Tight-looped exterior 
valve sculpture covering surface of valve (Fig. 
26C). Clarke (1985) reported a central liga- 
ment length of 65 |.im centered 35% from the 
anterior margin (actually the posterior margin). 
A central ligament length of 63 to 73 |jm (68 ± 
7.07 |лт, n = 2) was found during this study, 
with a midpoint about 38% form posterior to 
anterior. Posterior and anterior ligaments 52 to 



50 



HOGGARTH 



55 |.im (54 ± 2.12 |.im, n = 2), and 109 to 110 
цт (110 ± 0.71 |.im, n = 2) long, respectively. 
Hook styliform, with about 20 microstylets and 
many micropoints. Micropoints located on 
ventral rim and lateral surfaces of hook, leav- 
ing narrow unsculptured distal hook margin. 
Microstylets lanceolate, arranged in a double 
offset rows distally. 

Remarks 

The shape of this glochidium resembles 
that of Anodonta. except for the slightly in- 
curved margins prior to ventral terminus. The 
hook structure also differs from that of An- 
odonta. These two characters, as well as the 
far posterior position of the hook, the broadly 
rounded anterior margin and looped exterior 
valve sculpture ally this glochidium with some 
members of the genera Alasmidonta and Las- 
migona. It is easily distinguished from these, 
however, by its shape and the tightness of its 
looped sculpture. 

Lasmigona costata (Rafinesque, 1820) 
(Fig. 27A-E) 

Material Examined 

MAH 279.1 - Big Darby Creek at and above 
McLean Mill Rd. bridge, 0.2 mi. SW of Fox, 
4.7 mi. NW of Circleville, Jackson Twp., Pick- 
away Co., Ohio, 1 October 1982, M. A. Hog- 
garth et al. MAH 585 - Big Darby Creek at ac- 
cess, 0.9 mi. N of Harrisburg, 1.7 mi. NW of 
Orient, Pleasant Twp., Franklin Co., Ohio, 27 
September 1983, M. A. Hoggarth. MAH 882.1 
- Fish Creek at bridge 0.7 mi. W of Arctic, 3.8 
mi. NE of Butler, Sec. 20/29, Troy Twp., 
Dekalb Co., Indiana, 30 October 1985, D. H. 
Stansbery et al. 

Description 

Glochidium pyriform, asymmetric, length 
340 to 348 |im (344 ± 2.73 |im, n = 7), height 
363 to 377 ¡im (369 ± 5.68 |im, n = 7). Dorsal 
margin 239 to 245 цт (241 ± 2.23 цт, n - 7) 
long. Posterior and anterior margins broadly 
rounded dorsally, slightly incurved ventrally. 
Maximum inflation of posterior margin at 
about 30% from dorsal to ventral; maximum 
inflation of anterior margin at about 40% from 
dorsal margin. Exterior valve surface coarsely 
malleated and densely pitted, except at valve 
margins and at umbo. Exterior surface cov- 



ered with densely beaded sculpture (Fig. 
27C). Central ligament 78 to 84 ¡.im (80 ± 
2.49 Mm, n = 5) long, centered at about 45% 
from posterior to anterior (Fig. 27B). Posterior 
ligament 61 to 68 цт (64 ± 2.77 |.im, n = 5) in 
length; anterior ligament 90 to 105 цт (99 ± 
7.05 |.im, n = 5) long. Hook styliform, arising 
from ventral terminus as a broadly incurved 
triangular plate. Microstylets lanceolate, mul- 
tifaceted, arranged in about seven proximal 
rows, reduced to five distal rows and number- 
ing about 100. Micropoints on proximal border 
of hook but ending abruptly at ventral rim of 
valve, not extending onto exterior valve sur- 
face (Fig. 27D) nor very far onto lateral sur- 
face of the hook, leaving wide unsculptured 
distal hook margin. 

Remarks 

The glochidium of L. costata can be distin- 
guished by its distinctly pear-shaped outline, 
its exterior valve sculpture, and hook struc- 
ture. Glochidia of this species were figured by 
Lea (1858: pi. 5, fig. 26, as Margaritana ru- 
gosa, = L. costata. fide Ortmann & Walker, 
1922), Lefevre& Curtis (1910: fig. B, 1912:fig. 
1 B), Surber (1912: pi. 1 , fig. 7), Arey (1 924: pi. 
1, fig. 2), and Clarke (1985: fig. 5). This glo- 
chidium varies greatly in size: (length x height) 
Lea, 368 цт x 400 цт; Surber, 385 jam x 390 
jjm; Lefevre & Curtis, 350 ¡am x 390 |jm; Ort- 
mann, 340 |im X 370 цт ; Clarke, 333 цт x 
364 цт. 

Lasmigona complanata (Barnes, 1823) 
(Fig. 28A-F) 

Material Examined 

MAH 278.2 - Big Darby Creek at and above 
McLean Mill Rd. bridge, 0.2 mi. SW of Fox, 
4.7 mi. NW of Circleville, Jackson Twp., Pick- 
away Co., Ohio, 1 October 1982, M. A. Hog- 
garth et al. 

Description 

Glochidium pyriform. almost symmetrical, 
length 289 to 296 |.im (293 ± 2.90 цт, n = 6), 
height 293 to 310 |.im (300 ± 6.90 цт, n = 6). 
Dorsal margin straight, 193 to 208 i^im (200 ± 
4.69 цт, n = 7) in length. Maximum inflation 
of anterior and posterior margins at about 
40% from dorsal to ventral. Exterior surface 
coarsely malleated, densely pitted except 
along valve margin. Umbo malleated but not 



GLOCHIDIAOF UNIONIDAE 



51 




FIG. 27. Glochidium of Lasmigona costata:A. exterior valve, MAH 585, bar length = 50 |am; B. interior valve, 
MAH 279.1 , bar length = 50 цт; С. exterior valve sculpture, MAH 585, bar length = 1 ¡im; D. styliform hook, 
MAH 585, bar length = 20 цт; E. styliform hook, MAH 585, bar length = 20 цт. 



pitted. Dense rosette sculpture covering exte- 
rior surface of valves (Fig. 28E, F). Central lig- 
ament 60 to 68 (.im (64 ± 3.32 ¡.im, n = 4) long, 
centered about 42% from posterior to anterior. 
Posterior ligament 50 to 53 цт (52 ± 1 .29 |лт, 
n = 4) in length; anterior ligament 75 to 88 цт 
(81 ± 5.48|.im,n = 4)long. Hook styliform, very 
similar to that of L. costata. Microstylets (about 
100) lanceolate, multifaceted, arranged in six 
proximal rows reduced to four rows distally. Mi- 
cropoints restricted to the proximal margin of 



hook, leaving wide unsculptured distal hook 
margin. Ventral terminus located about 40% 
from posterior to anterior. 

Remarks 

This glochidium can be distinguished by its 
nearly equal length and height, few micro- 
points and exterior valve sculpture. It is figured 
by Lea (1858: pi. 5, fig. 29), Lefevre & Curtis 
(1910:fig.A, 1912:fig. 1A), Ortmann (1911 :pl. 



52 



HOGGARTH 




FIG. 28. Glochidium of Lasmigona complanata, MAH 278.2; A. exterior valve, bar length = 45 |лт; В. interior 
valve, bar length = 45 цт; С. styliform hook, bar length = 15 |um; D. styliform hook, bar length = 20 цгл; E. 
exterior valve sculpture, bar length = 1 цт; F. exterior valve sculpture, bar length = 2 цт. 



GLOCHIDIAOF UNIONIDAE 



53 



89, fig. 11), Surber (1912: pi. 1, fig. 6), Arey 
(1 921 : pi. 1 , fig. 1,2), and Clarke (1 985: fig. 8). 
Measurements given for this glochidium are: 
290 цгл X 300 |.im (Lefevre & Curtis, 1 91 0), 340 
(im X 340 ц m (Ortmann, 1911), 310 цт x 320 
|лт (Surber, 1912), and 337 j.im x 337 (.im 
(Clarke, 1985). This glochidium appears to 
vary a great deal in size, but its relative di- 
mensions remain fairly constant (i.e., length = 
height). 

Subfamily Ambleminae 

Magalonaias nervosa (Rafinesque, 1820) 

(Fig. 29A-G) 

Material Examined 

OSUM - 13032.66 St. Francis River Bay 
halfway between Wynne and Perkin, 1 .0 mi. S 
of Rt. 64 bridge, Cross Co., Arkansas, 25 Oc- 
tober 1964, С В. Stein. OSUM 54178 - Mis- 
sissippi River, R.MI. 299.8-301 .1 , 7.8-9.0 mi. 
SE of Hannibal (MO), Pike/Ralls Co., Illi- 
nois/Missouri, 16 October 1979, R. B. Lewis 
et al. OSUM 178-Licking River, 1.6 mi. E of 
Butler, immediately above mouth of Flour 
Creek, 22 mi. SE of Cincinnati, Pendleton 
Co., Kentucky, 31 October 1986, D. H. Stans- 
bery et al. OSUM - 1986:22 Green River at 
Glenmore, below Lock 5 Dam, 12.0 mi. N of 
Bowling Green, Warren Co., Kentucky, 1 No- 
vember 1986, D. H. Stansbery et al. 

Description 

Glochidium subelliptical, length 254 to 268 
¡.im (261 ± 6.58 (.im, n = 4), height 340 to 350 
(.im (346 ± 4.78 (дт, n = 4). Dorsal margin 
straight, 145 to 155 (.im (150 ± 3.27 (.im, n = 
6) in length. Lateral margins gently curved but 
unequal. Maximum inflation of posterior mar- 
gin at about 70% from dorsal to ventral; max- 
imum inflation of anterior margin at about 40% 
from dorsal margin. Ventral margin narrowly 
rounded. Tight-looped sculpture covering ex- 
terior surface of valve (Fig. 29F). Coiled larval 
thread present (Figs. 29C, D). Central liga- 
ment 49 to 52 ¡.im (50 ± 1 .53 (.im, n - 3) long, 
centered about 44% from posterior to anterior. 
Anterior ligament 60 to 63 ¡.im (61 ± 2.00 (.im, 
n = 3) long, posterior ligament 43 to 44 (.tm (43 
± 0.58 (.im, n = 3) in length. Lanceolate mi- 
cropoints occurring in broken vertical rows on 
a narrow ventral flange, and along rim of ven- 
tral margin of valve, covering most of ventral 
flange, leaving very narrow unsculptured dis- 
tal flange margin. 



Remarks 

Surber (1915) stated, "notwithstanding its 
great variation in size, and even outline, this 
species cannot be readily confused with any 
other, even though the larval gland may have 
been absorbed. . ." Surber (1912), Howard 
(1914c), and Surber (1915) gave length and 
height measurements of 260 ¡.im x 340 (.im, 
250-260 (.im X 316-340 (.im, and 250-280 
(.im X 300-380 jLim (as Quadrula heros = M. 
nervosa, fide Stansbery et al., 1985). This 
glochidium has been figured by Lea (1858: pi. 
5, fig. 3, as Unio multiplicatus. = M. nervosa 
fide, Stansbery et al., 1985), Surber (1912: pi. 
2, fig. 32), Howard (1914c: pi. 3, fig. 21; pi. 5, 
fig. 35), Surber (1915: pi. 1, fig. 10), and Ut- 
terback (1915-1916: fig. 3a, b). Surber's, 
Howard's, and Utterback's figures agree with 
mine, whereas Lea's does not. 



Megalonaias boykiniana (Lea, 1840) 
(Fig. 30A-D) 

Material Examined 

OSUM 511 07.5 - Apalachicola River below 
U.S. Rt. 90 bridge, 1.0 mi. W of Chatta- 
hoochee, 17.8 mi. WNW of Quincy, T4N, 
R6W, Sec. 32, Gadsden Co., Florida, 29 Oc- 
tober 1981, D. H. Stansbery et al. 



Description 

Glochidium subelliptical, with a straight dor- 
sal margin, a narrowly rounded ventral mar- 
gin, and gently but unequally curved lateral 
margins. A single specimen gave the following 
measurements for length, height, and hinge 
length: 245 (im x 350 jim x 150 (.im. Tight- 
looped sculpture covering exterior surface 
of valve (Fig. 30C), lanceolate micropoints 
cover a narrow ventral flange (Fig. 30D), and 
a larval thread is coiled around adductor mus- 
cle, not supercoiled as in /W. nervosa (Fig. 
ЗОВ) 



Remarks 

The glochidium of this species can be dis- 
tinguished from that of M. nervosa by its larval 
thread and from all other glochidia by its di- 
mensions and outline. No published figure of 
this glochidium was found. 



54 



HOGGARTH 




FIG. 29. Glochidium of Megalonalas nervosa: A. exterior valve, OSUM 54178, bar length = 50 цт; В. inte- 
rior valve, OSUM 178, bar length = 50 цт; С. larval thread, OSUM 54178, bar length = 25 цт; D. larval 
thread, OSUM 54178, bar length = 5 цт; E. micropoints, OSUM 54178, bar length = 5 ¡.im: F. exterior valve 
sculpture, OSUM: 1986:22, bar length = 2 |дт; G. hinge, OSUM 178, bar length = 20 цт. 



Plectomerus dombeyana 

(Valenciennes, 1827) 

(Fig. 31A-H) 

Material Examined 

OSUM 42011 - Black Warrior River at Hall 
Shoals, below Eutaw Dam, 5.8 mi. SE of 
Eutaw, Sec. 25, T21N, R2E, Green Co., Al- 
abama, 28-30 July 1 975, J. D. Williams et al.. 
OSUM 53273.2, 53273.3 - Calcasieu River at 



Novel's Bluff, 2.0 mi. NE of Indian Village, 6.9 
mi. WSW of Kinder, 22.0 mi. NE of Lake 
Charles, Allen Pahsh, Louisiana, 25 July 
1982, D. H. Stansbery & M. A. Hoggarth. 

Description 

Glochidium subelliptical, length 223 to 231 
|.im (226 ± 2.99 цт. n = 1 0). height 238 to 259 
цт (246 ± 7.07 цт, n = 10). Dorsal margin 
straight, 130 to 135 |.im (133 ± 1.36 цт, n = 



GLOCHIDIAOF UNIONIDAE 



55 




FIG. 30. Glochidium of Megalonaias boykiniana, OSUM 51107.5; A. exterior valve, bar length = 50 |.im; B. 
larval thread, bar length = 25 jam; C. exterior valve sculpture, bar length = 1 |im; D. micropoints, bar length 
= 5 [ivn. 



11) long. Lateral nnargins gently and equally 
curved, symmetrical. Ventral margin semicir- 
cular. Valve surface finely malleated, with 
many pits (Fig. 31 E). Loose-looped sculpture 
covers exterior valve surface (Fig. 31C,H). 
Figure 31 С demonstrates that the larval valve 
has an exterior membrane that is at least 
analogous, if not homologous, with the pe- 
riostracum of the adult. It is also evident from 
this micrograph that the exterior valve sculp- 
ture occurs within this membrane. Central lig- 
ament 42 to 47 |im (44 ± 2.06 fim, n = 4) long, 
centered about 40% from posterior to anterior. 
Anterior ligament 51 to 59 |лт (56 ± 3.59 ¡am. 



n = 4) long; posterior ligament 31 to 35 ¡am (32 
± 1.89 цт, n = 4) in length. Lanceolate mi- 
cropoints in broken rows on rim of ventral 
margin of valve and on a narrow ventral 
flange. Surface of ventral flange mostly cov- 
ered with micropoints, leaving narrow un- 
sculptured distal flange margin. 

Remarks 

This glochidium is distinguished from oth- 
ers examined by its shape and dimensions. 
No published figure of this glochidium was 
found. 



56 



HOGGARTH 




FIG. 31. Glochidium of Plectomerus dombeyana: A. exterior valve, OSUM 53273.3, bar length = 35 цт: В, 
interior valve, OSUM 53273.3, bar length = 35 цт; С. exterior valve sculpture and torn exterior valve mem- 
brane, OSUM 53273.3, bar length = 2цт; D. hinge, OSUM 53273.2, bar length = 25 мт; E. interior valve 
pitting, OSUM 42011, bar length = 10|.im; F. micropoints, OSUM 53273.3, bar length = 5 ^m: G. micropoints, 
OSUM 42011 , bar length = 3 цт; H, exterior valve sculpture, OSUM 42011 , bar length = 1 pm. 



GLOCHIDIAOF UNIONIDAE 



57 




FIG. 32. Glochidium of Tritogonia verrucosa, MAH 654.1; A. exterior valve, bar length = 15 цт; В. interior 
valve, bar length = 15 цгл; С. micropoints, bar length = 5 цт; D. hinge, bar length = 7 \im; E. exterior valve 
sculpture, bar length = 2 цт. 



Tritogonia verrucosa (Rafinesque, 1820) 
(Fig. 32A-E) 

Material Examined 

MAH 654.1 - Big Darby Creek at Scioto- 
Darby (Mt. 31ег11пд-Сотглегс1а1 Pt.) Rd. 
bridge, 3.4 mi. S of Orient, 1 5.3 mi. NW of Cir- 
cleville, Scioto/Darby Twp., Pickaway Co., 
Ohio, 18 May 1984, M. A. Hoggarth & G. T. 
Watte rs. 



Description 

Glochidium subelliptical to subrotund, 
length 85 to 94 ).im (90 ± 3.38 |jm, n = 6), 
height 97 to 101 цт (100 ± 1.75 цт, n = 6). 
Dorsal margin slightly curved, 43 to 46 |im (44 
±1.11 jim, n = 7) long. Lateral margins gen- 
tly and equally curving throughout their 
lengths. Ventral margin semicircular. Exterior 
surface rough, sparsely pitted (Fig. 32E). 
Central ligament 30 to 34 |am (32 ± 1 .47 цт, 



58 



HOGGARTH 



n = 6) long, centered about 45% from poste- 
rior to anterior. Anterior ligament 6 to 9 |.im (8 
± 1 .51 |.im, n = 6) long; posterior ligament 3 to 
5 цт (4 ± 0.82 цт, n = 6) in length; central lig- 
ament comprising about 75% of total hiinge 
length. Micropoints extremely small, almost 
undetected even at high magnification, unor- 
ganized (Fig. 32C), on rim of ventral valve 
margin and on exterior surface of valve. Ven- 
tral flange not observed. 

Remarks 

This glochidium is figured by Surber (1912; 
pi. 2, fig. 31 , as T. tuberculata, = T. verrucosa, 
fide Ortmann, 1919), who gave length and 
height measurements of 85 цт x 90 цт. It can 
be distinguished from all other glochidia by its 
very small size and the outline of its valve. 

Quincuncina ¡nfucata (Conrad, 1834) 
(Fig. 33A-G) 

Material Examined 

OSUM 48537.1, 48537.2 - Suwannee 
River at Fl. Rt. 51 bridge, 8.4 mi. SSE of 
Jasper, Sec. 17, Hamilton/Suwannee Co., 
Florida, 14 May 1978, W. J. Clench et al. 

Description 

Glochidium subelliptical, with a short hinge 
line, equally curved lateral margins, a broadly 
curved ventral margin. Glochidium 234 to 242 
|im (240 ± 4.00 цт, n = 4) in length, 275 to 287 
[im (283 ± 5.68 цт, n - 4) in height. Dorsal 
margin straight, 102 to 105 цт (103 ± 1.34 
|im, n = 5) in length. Valve surface densely pit- 
ted, except along valve margin, and rough 
sculpture covering exterior valve surface (Fig. 
33F). Central ligament 54 to 58 цт (56 ± 2.83 
|im, n = 2) long, centered about 48% from pos- 
terior to anterior. Anterior ligament 27 to 28 ¡.im 
(28 ± 0.71 jam, n = 2) long, posterior ligament 
20 to 22 f.im (21 ± 1 .54 |.im, n - 2) in length. Mi- 
cropoints coronal, with fused bases and lance- 
olate points, not extending onto ventral rim or 
on ventral margin of valve, covering about 
90% of ventral flange, leaving narrow un- 
sculptured distal flange margin. 

Remarks 

This glochidium will not be confused with 
that of any other species examined. The most 
striking feature is its unusual micropoint struc- 



ture. The more proximal micropoints resem- 
ble crowns. The bases of the micropoints are 
fused with their points extending outward. The 
number of points in each "crown" range from 
seven along the proximal margin of the flange 
to two points distally. The furthest micropoints 
on the flange are simple lanceolate points. No 
published figure of this glochidium was found. 

Elliptio dilatata (Rafinesque, 1820) 
(Fig. 34A-F) 

Material Examined 

MAH 946.9 - Kalamazoo River above St. 
Rt. 60 bridge, 3.0 mi. WSW of Spring Arbor, 
12.0 mi. WSW of Jackson, Jackson Co., 
Michigan, 13 May 1986, M. A. Hoggarth. 

Description 

Glochidium subelliptical, length 210 to 219 
цт (216 ± 4.27 цт, n = 4), height 219 to 225 
(.im (221 ± 3.00 (im, n = 4). Dorsal margin 
straight, 140 to 147 [xm (143 ± 2.99 }im, n = 
4) in length. Ventral margin semicircular. Lat- 
eral margins subequal, with anterior margin 
slightly more produced than posterior margin. 
Valve pitting eliminated in region of adductor 
muscle scar (Fig. 34D) and sparse throughout 
remainder of valve. Loose-looped sculpture 
covering exterior surface of valve (Fig. 34C). 
Central ligament 45 to 50 цт (48 ± 3.54 цт, 
n = 2) long, centered about 43% from poste- 
rior to anterior. Anterior ligament about 56 цт 
long; posterior ligament 36 to 39 цт (38 ± 
2.12 цт, n = 2) in length. Micropoints lanceo- 
late, located on ventral rim of valve and 
on narrow ventral flange, arranged in broken 
vertical rows, covering most of the area of 
flange. Unsculptured distal flange margin nar- 
row. 

Remarks 

This glochidium is figured by Lea (1 874: pi. 
21, fig. 10, as Unio gibbosus, = E. dilatata. 
We Ortmann & Walker, 1922), Lefevre & Cur- 
tis (1910; fig. N; 191 2; fig. 10, as U. gibbosus), 
Ortmann (1911 ; pi. 89, fig. 7, as E. gibbosus), 
and Surber (1912: pi. 2, fig. 38, as U. gibbo- 
sus). Lefevre & Curtis (1910, 1912) gave 
length and height measurements of 220 цт x 
190 МГП for this glochidium, Surber (1912) 
gave 200 цт x 21 5 ¡.im, Ortmann (1912) gave 
200 цт x 220 yivn, and Ortmann (1919, as E. 
cuprous. = E. dilatata, fide Johnson, 1970) 
gave 200 |.im x 200 ).im. 



GLOCHIDIAOF UNIONIDAE 



59 




FIG. 33. Glochidium of Quincuncina infucata:A. exterior valve. OSUM 48537.2, bar length = 40 .urn; B. inte- 
rior valve, OSUM 48537.2, bar length = 40 цт; С. micropoints, OSUM 48537.2, bar length = 5 цт; D. mi- 
cropoints. OSUM 48537.2, bar length = 5 цт; E. micropoints, OSUM 48537.1 , bar length = 2 цт; F. exterior 
valve sculpture. OSUM 48537.1, bar length = 2|лт; G. hinge, OSUM 48537.1, bar length = 15 цт. 



Subfamily Lampsilinae 

Ptychobranchus fasciolaris 

(Rafinesque. 1820) 

(Fig. 35A-E) 

Material Examined 

MAH 640.1 - Little Darby Creek at Co. 
Rt. 131 bridge (Grewell Rd.), 1.8 mi. E of 



Plumwood, 7.7 mi. NW of West Jefferson, 
Monroe Twp., Madison Co., Ohio, 11 May 
1984, M. A. Hoggartli & G. T. Watters. MAH 
651 - Little Darby Creek at Little Darby Rd. 
access, 2.7 mi. SE of Plumwood, 6.4 mi. NW 
of West Jefferson, Monroe Twp., Madison 
Co., Ohio, 17 May 1984, M. A. Hoggarth & G. 
T. Watters. 



HOGGARTH 





FIG. 34. Glochidium of Elliptio dilatata. MAH 946.9: A. exterior valve, bar length = 35 ,um: B. interior valve, 
bar length = 35 цт; С. exterior valve, bar length = 1 \.ivr\: D. interior valve, bar length = 1 |.im; E. micropoints, 
bar length = 5 |jm; F. hinge, bar length = 20 цт. 



Description 

Glochidium subelliptical, higlier than long, 
length 1 70 to 1 75 цт (1 73 ± 2.89 цт, n = 3), 
height 182 to 195 цт (187 ± 6.81 цт, n = 3). 
Dorsal margin slightly curved, 80 to 89 )Lim (83 
± 4.93 |лт, n = 3) in length. Anterior and pos- 



terior margins equally curved to a maximum 
inflation at about 60% from dorsal to ventral; 
ventral margin broadly rounded. Surface of 
the valve smooth, with only a few pits. Dorsal 
alae absent. Loose-looped sculpture covering 
exterior surface of valve (Fig. 35E). Central 
ligament 36 to 40 мт (38 ± 2.83 цт, n = 2) 



GLOCHIDIAOF UNIONIDAE 



61 




FIG. 35. Glochidium of Ptychobranchus fasciolaris: A. exterior valve, MAH 640.1 , bar length = 25 цгл; В. in- 
terior valve, MAH 640.1 , bar length = 25 цгл; С. lateral view, MAH 640.1 , bar length = 35 |am; D. ventral valve 
edge, MAH 651 , bar length = 5 цт; E. exterior valve sculpture, MAH 651 , bar length = 1 |am. 



long, centered about 49% from posterior to 
anterior. Posterior ligament 1 9 to 22 цт (21 ± 
2.12 цт, n = 2) long; anterior ligament 22 to 
26 цт (24 ± 2.83 |.im, n = 2) in length. Micro- 
points very small, few in number, located on 
ventral margin of valve rather than on broad 
ventral flange. 



Remarks 

This glochidium can be distinguished by its 
small size, central ligament position, and its 
very simple ventral margin. It was figured by 
Lea (1858: pi. 5, fig. 12, as Unio phaseolus, = 
P. fasciolaris, fide Ortmann & Walker, 1922) 



62 



HOGGARTH 



and Ortmann (1911: pi. 89, fig. 14, as P. 
phaseolus). Ortmann (1912) gave 170 ).im x 
190 |.im for length) and height of this glochid- 
ium and noted that Lea's figure does not rep- 
resent its shape or size accurately. The 
glochidium of P. fasciolaris is much smaller 
than that of Ligumia recta rather than, as pic- 
tured by Lea (1858), larger than that species. 

Ptychobranchus occidentalis (Conrad, 1836) 
(Fig. 36A-D) 

Material Examined 

OSUM - 45361 .14, 45361 .17 Current River 
"between Mo. Rt. 106 and Van Buren," about 
19 mi. of stream, Shannon/Carter Co., Mis- 
souri, 24 October 1977, R. D. Oesch. 

Description 

Glochidium subelliptical, higher than long, 
length 197 to 203 цт (200 ± 4.24 ¡am, n = 2), 
height 234 to 241 |im (238 ± 4.95 |.im, n = 2). 
Dorsal margin slightly curved, 101 to 104 |im 
(1 02 ± 1 .53 |jm, n = 3) in length. Anterior and 
posterior margins equally and gently curving 
to their maximum inflation at about 60% from 
dorsal to ventral; ventral margin broadly 
curved. Dorsal alae absent; valve surface 
sparsely pitted. Loose-looped sculpture cov- 
ering exterior valve surface (Fig. 36C). Cen- 
tral ligament 44 to 48 цт (46 ± 2.83 цт, n = 
2) in length, centered about 47% from the 
posterior margin. Anterior ligament 30 to 34 
|jm (32 ± 2.83 |iim, n = 2) in length; posterior 
ligament 23 to 26 ¡am (25 ± 2.12 цт, n = 2) 
long. Ventral flange poorly developed, very 
narrow, covered with micropoints. Micropoints 
lanceolate and unorganized. 

Remarks 

This glochidium is slightly larger than that of 
P. fasciolaris and is further distinguished from 
that species by its larger and more numerous 
micropoints. No published figure of this glo- 
chidium was found. 

Ptycliobranctius greeni (Conrad, 1 834) 
(Fig. 36E-H) 

Material Examined 

OSUM 19025.2 - Conasauga River above 
lower Kings Bridge, 5 mi. WNW of Eton, Mur- 



ray/Whitfield Co., Georgia, 25 September 
1966, D. H. Stansbery & H. D. Athearn. 



Description 

Glochidium subelliptical, higher than long, 
symmetrical, length 183 to 190 ¡.im (187 ± 
3.61 (jm, n = 3), height 226 to 228 |im (227 ± 
1.15 |.im, n = 3). Dorsal margin sligfitly curved, 
90 to 96 |im (93 ± 3.06 цт, n = 3) in length. 
Anterior and posterior margins equal and 
slightly curved to their point of maximum infla- 
tion at about 70% from dorsal to ventral. Ven- 
tral margin broadly rounded. Surface of valve 
sparsely pitted. Loose-looped sculpture cov- 
ering exterior surface of valve (Fig. 36G). Dor- 
sal alae present but very small, oriented al- 
most perpendicular to hinge. Central ligament 
about 49 )im long centered about 48% from 
posterior to anterior. Anterior ligament about 
26 |jm long; posterior ligament 21 |im in 
length. Micropoints occur on ventral rim of 
valve, small, lanceolate, unorganized. Ventral 
flange not observed. 

Remarks 

This glochidium is about the same size as 
that of P. fasciolaris; however, it can be distin- 
guished from that species by its dorsal alae 
and micropoints. This glochidium is figured by 
Lea (1858: pi. 5, fig. 16, as Unio woodwar- 
dianus, - P. greeni. fide Ortmann, 1923- 
1924). His figure is essentially correct except 
for size. Glochidia of Lampsilis ovata and L. 
fasciola are larger than those of P. greeni 
rather than smaller as figured by Lea. 

Ptychobranchus subtentum (Say, 1 825) 
(Fig. 37A-F) 

Material Examined 

OSUM 43156.5 - Clinch River at mouth of 
Copper Creek, 1 .3 mi. S of Clinchport, 9.3 mi. 
W of Gate City, Scott Co., Virginia, 21 October 
1978, D. H. Stansbery et al. 



Description 

Glochidium subelliptical, higher than long, 
symmetrical, length 181 to 195 цт (190 ± 
5.54 |.im, n = 5), height 236 to 251 цт (241 ± 




FIG. 36. Glochidium of Ptychobranchus occidentalis ( A-D) and Ptychobranchus greeni (E-H); A. exterior 
valve OSUM 45361.17. bar length = 35|im: B. interior valve, OSUM 45361.14. bar length = 35 цт; С. exte- 
rior valve sculpture, OSUM 45361 .17, bar length = 1 цт; D. hinge, OSUM 45361 .17. bar length = 1 5 |am; E. 
exterior valve, OSUM 19025.2, bar length = 35 цт; F. interior valve, OSUM 19025.2, bar length = 35 |лт; 
G. exterior valve sculpture, OSUM 19025.2, bar length = 1 |am; H. micropoints, OSUM 19025.2, bar length 
= 2|im. 



64 



HOGGARTH 




FIG. 37. Glochidium of Ptychobranchus subtentum OSUM 431 56.5: A. exterior valve, bar length = 35 ^m; B. 
interior valve, bar length = 35 |.im; С exterior valve sculpture, bar length = 1 ¡.im; D. hinge, bar length = 
20 |.im; E. micropoints, bar length = 5 (.im; F. interior valve pitting, bar length = 10 цт. 



5.76 |.im, n = 5). Dorsal margin slightly curved, 
82 to 90 |.im (85 ± 2.88 |im, n = 6) in length. 
Anterior and posterior margins about straight 
to their maximum inflation at about 60% from 
dorsal to ventral, where they begin to curve to 
form a broadly rounded ventral margin. Valve 
surface sparsely pitted, loose-looped sculp- 



ture covering exterior surface of valve (Fig. 
37C). Adductor muscle scar present (Fig. 
36B,D,F). Dorsal alae short, poorly devel- 
oped. Central ligament 36 to 38 цт (37 ± 
1.15 цт, n = 3) in length, centered about 48% 
from posterior to anterior. Anterior ligament 25 
to 26 |.im (25 ± 0.58 |.irn, n = 3) long; posterior 



GLOCHIDIA OF UNIONIDAE 



65 



ligament 20 to 24 цт (22 ± 2.08 ¡im, n = 3) in 
iengtli. Micropoints small, numerous, unorga- 
nized, covering a large portion of ventral rim, 
leaving very narrow unsculptured distal mar- 
gin. 

Remarks 

This glochidium can be distinguished by its 
relatively short hinge line, simple micropoints 
and small dorsal alae. Ortmann (1912) noted 
that the glochidium of P. subtentum is larger 
than that of P. fasciolaris. He gave 180 цт x 
220 |im for the length and height of P. subten- 
tum glochidia. This glochidium is figured by 
Ortmann (1912: pi. 29, fig. 5). 

Obliquaria reflexa Rafinesque, 1820 
(Fig. 38A-D) 

Material Examined 

OSUM 54361.1 - Mississippi River, R.Mi. 
564.5-566.1 , about 3.5 mi. SW of Galena (IL), 
Jo Daviess/Jackson Co., Illinois/Iowa, 7-8 
August 1979, R. B. Lewis et al. 

Description 

Glochidium subrotund, symmetrical, length 
213 to 219 |Lim (217 ± 3.46 |jm, n = 3), height 
206 to 221 [im (215 ± 8.14 цт, n = 3). Dorsal 
margin slightly curved outward, 1 1 9 to 1 27 ¡am 
(122 ± 4.16 цт, n = 3) in length. Lateral and 
ventral valve margins more or less round in 
outline, with maximum inflation of both side 
margins at about 50% from dorsal to ventral. 
Exterior surface malleated and pitted, except 
along valve margin, where shell fairly smooth. 
Within this smooth marginal area, longitudinal 
ridges present (Fig. 38C). Dorsal alae absent. 
Central ligament 52 to 54 ¡.im (53 ± 1.41 ¡am, 
n = 2) in length, centered about 46% from 
posterior to anterior. Anterior ligament 37 to 
44 jam (41 ± 4.95 \xm, n = 2) long; posterior 
ligament 30 to 31 цт (31 ± 0.71 |im, n = 2) in 
length. Large, bluntly pyramidal micropoints 
occurring on ventral rim and on narrow ventral 
flange, decreasing in size distally, arranged in 
broken vertical rows that extend two thirds 
length of flange, leaving a narrow unsculp- 
tured distal flange edge. Micropoints extend- 
ing laterally to about point of maximum lateral 
inflation of valves. 



Remarks 

Lefevre & Curtis (1910, 1912), Surber 
(1912), and Ortmann (1912) gave the follow- 
ing measurements for this glochidium; 230 
цт X 225 |im, 225 |im x 235 цт, and 220 цт 
X 220 |im. Ortmann (1919) noted that the 
shape of this glochidium, "may best be com- 
pared with a circle a small section of which is 
cut off." This glochidium can be distinguished 
by its shape and micropoints. It was figured by 
Lefevre & Curtis (1910; fig. M, 1912; fig. IN), 
Surber (1912; pi. 2, fig. 39), and Ortmann 
(1912; pi. 20, fig. 1). 

Cyprogenia stegaria (Rafinesque, 1820) 
(Fig. 39A-D) 

Material Examined 

OSUM 6298.21 - Green River at Munford- 
ville. Hart Co., Kentucky, 22 October 1 961 , D. 
H. Stansbery. 

Description 

Glochidium elongate-oval, subrotund, 
length 204 to 208 |.im (206 ± 2.08 цт, n = 3), 
height 1 64 to 1 70 |.im (1 67 ± 3.06 цт, n = 3). 
Dorsal margin straight, long, with a length of 
113to120|jm(116 ± 3.00 |.im,n = 5). Anterior 
and posterior margins curving greatly to create 
mirror images of each other, with maximum lat- 
eral inflation at about 50% from dorsal to ven- 
tral. Exterior surface smooth, lacking mal- 
leations, with only a few pits. As pointed out 
by Sterki (1 898), concentric ridges occur near 
margins of valve. Valve disc smooth, except 
for loose-looped exterior valve sculpture (Fig. 
39C). Dorsal alae absent. Central ligament 35 
to 40 |лт (38 ± 2.65 \im, n = 3) in length, cen- 
tered about 45% from posterior to anterior. 
Posterior ligament 30 to 33 \im (32 ± 1 .53 ¡am, 
n = 3) in length; anterior ligament 43 to 47 jam 
(45 ± 2.08 |im, n = 3) long. Micropoints lance- 
olate, bluntly pointed, located on the ventral 
rim of valve. Ventral flange not observed. 

Remarks 

This glochidium can be distinguished from 
other species by its more broadly rounded 
valve outline. Sterki (1898), Surber (1912), 
and Ortmann (1912) gave the following mea- 



66 



HOGGARTH 




FIG. 38. Glochidium of Obliquaria reflexa, OSUM 54361 .1 ; A. exterior valve, bar length = 35 |am; B. interior 
valve, bar length = 35 ¡.im; С ventral view, bar length = 25 urn; D. micropoints, bar length = 7 ¡.im. 



surements for length and height; 210 цт x 
170).im,210|.imx 185|.im, and 180дтх 150 
цт. This glochidium was figured by Ortmann 
(1911: pi. 19, fig. 6, as С /rroratà) and Surber 
(1912: pi. 1, fig. 11, as С ¡rrorata). 

Cyprogenia aberti (Conrad, 1 850) 
(Fig.39E-l) 

Material Examined 

OSUM - 48067 St. Francis River along Mo. 
Rt. E, 2.5 mi. NNE of French Mills, 11.5 mi. 
SW of Fredericktown, SE 1/4, Sec. 3, T32N, 
R5E, Madison Co., Missouri, 5 October 1977, 
W. L. Pflieger & T. Grace. 



Description 

Glochidium elongate oval, subrotund, 
length 200 to 218 (.im (208 ± 7.03 |.im, n = 6), 
height 143 to 161 цт (154 ± 7.79 цт, n = 6). 
Dorsal margin straight, 125 to 136 цт (131 ± 
4.18 |.im, n = 7) long. Anterior and posterior 
margins equally curved to their maximum lat- 
eral inflation at about 50% from dorsal to ven- 
tral. Ventral margin flatly curved, dorsal alae 
absent. Central ligament 28 to 36 цт (31 ± 
3.11 i-im, n = 5) long, centered about 46% 
from posterior to anterior. Anterior ligament 48 
to 61 \.[m (54 ± 6.04 \.im. n = 5) in length; pos- 
terior ligament 41 to 49 ¡.im (44 ± 3.13 цт. n 
= 5) long. Micropoints lanceolate, bluntly 




FIG. 39. Glochidium of Cyprogenia stegaria (A-E) OSUM 6298.21, and Cyprogenia aberíi (F-l) OSUM 
48067: A. exterior valve, bar length = 30 цт: В. interior valve, bar length = 30 цт; С. exterior valve sculp- 
ture, bar length = 2 цт: D. hinge, bar length = 20 ).im: E. exterior valve, bar length = 30 |агл; F. interior valve, 
bar length = 30 цт; G. micropoints, bar length = 5 |.im; H. micropoints, bar length = 5 цт; I. hinge, bar length 
= 20|im. 



68 



HOGGARTH 



pointed, unorganized on ventral margin. Ven- 
tral flange only poorly developed. 



Remarks 

This glochidium is essentially like that of С 
stegaria. although it is slightly more de- 
pressed than that species. The only other 
glochidium that approaches this shape is that 
of Dromus dramas. However, the glochidium 
of D. dramas would not be confused with ei- 
ther of these because of its extreme valve de- 
pression. No published figure of the glochid- 
ium of С aberti\Nas found. 



Dromus dramas (Lea, 1 834) 
(Fig. 40A-E) 

Material Examined 

OSUM 20407.1 - Powell River 2.5 mi. ENE 
of Hoop, 11.5 mi. NE of Tazewell, Clairborne 
Co., Tennessee. 20 October 1968, G. F. 
Ahrens. OSUM 23200.9 - Powell River at 
Hoop ("Brooks Bridge"), 9.5 mi. NE of 
Tazewell, Clairborne Co.. Tennessee, 19 Oc- 
tober 1969, D. H. Stansbery et al. 



Description 

Glochidium fabelliform or bean-shaped, 
much longer than high, with a straight dorsal 
margin, narrowly rounded anterior and poste- 
rior margins, broadly curved ventral margin. 
Glochidium 219 to 230 |.im (224 ± 5.09 цт, 
n = 5) long, 114 to 120 |.im (118 ± 2.49 цт, 
n = 5) in height. Dorsal margin 160 to 182 
цт (174 ± 8.65 цт, n = 6) long. Anterior and 
posterior margins equal, with their points of 
maximum inflation occurring about 30% 
from dorsal to ventral. Exterior valve surface 
smooth, with pits only evident in internal view 
(Figs. 40B-D). Pits absent at valve mar- 
gins, but present in central portion of valve 
(Fig. 40C). Dorsal alae absent. Central liga- 
ment 39 to 40 |.im (39 ± 0.58 ,um, n = 3), 
centered at about 45% from posterior to ante- 
rior. Anterior ligament 76 to 82 um (79 ± 3.06 
|im, n = 3) in length; posterior ligament 51 to 
61 |im (56 ± 4.58 |.im, n = 3) in length. Nu- 
merous bluntly pointed micropoints present 
on valve margin. Ventral flange only poorly 
developed. 



Remarks 

This glochidium is easily distinguished by 
its shape. It represents the end of an evolu- 
tionary line from the nearly round glochidium 
of O. reflexa. through the progressively more 
depressed valve of С stegaria and C. aberti, 
to the extremely depressed valves of this 
species. Surber (1912), Lefevre & Curtis 
(1912), and Ortmann (1912) gave 190 [im x 
1 00 i^m for length and height. This glochidium 
was figured by Surber (1912: pi. 1, fig. 13), 
Lefevre & Curtis (1912: fig. IM) and Ortmann 
(1912: pi. 29, fig. 7). 

Actinanaias pectarasa (Conrad, 1834) 
(Fig. 41A-G) 

Material Examined 

A. pectarasa: OSUM 24337 - Middle Fork 
Holston River, 3.7 mi. S of Glade Spring at Va. 
Rt. 91 bridge (Craigs Bridge), Washington Co., 
Virginia, 16 September 1968, D. H. Stansbery 
& W. J. Clench. OSUM 48748.3 - Clinch River 
below footbridge at Slant. 7.1 mi. NNW of Gate 
City, 8.1 mi. ENE of Clinchport, Dekalb Twp., 
Scott Co., Virginia, 22 December 1980, С С. 
Coney. A. I. cahnata: МАИ 842.1 - Fish Creek 
at bridge 2.0 mi. NW of Edgerton, 11.9 mi. W 
of Bryan, Sec. 20, T6N, R1 E, St. Joseph Twp., 
Williams Co., Ohio, 29 October 1985, D. H. 
Stansbery et al. 

Description 

Glochidium subelliptical, nearly symmetri- 
cal, length 244 to 253 цт (248 ± 4.58 цт, 
n = 3), height 260 to 270 f.im (267 ± 5.77 цт, 
n = 3). Dorsal margin straight, 139 to 151 \xm 
(144 ± 5.20 |.im, n = 3) in length. Anterior and 
posterior margins gently and evenly curving: 
ventral margin semicircular. Exterior surface 
of valve malleated and pitted, except at its 
margin, where longitudinal ridges occur (Fig. 
41 С D). Dorsal alae well developed, about 38 
|.im in length (Fig. 41 A, C). Beaded exterior 
valve sculpture covering surface of valve (Fig. 
41 G). Central ligament 58 to 62 ,um (60 ± 
3.21 j.im, n = 3) in length, centered about 46% 
from posterior to anterior. Anterior ligament 46 
to 52 цт (48 ± 3.21 цт, n = 3) long: posterior 
ligament 31 to 39 .um (35 ± 3.61 urn, n = 3) in 
length. Micropoints, lanceolate, located on 
ventral margin and on a narrow ventral flange. 



GLOCHIDIA OF UNIONIDAE 



69 




FIG. 40. Glochidium of Dromus dramas: A. exterior valve, OSUM 20407.1, bar length = 30 цт; В. interior 
valve, OSUM 20407.1 , bar length = 30 |агл: С. interior valve pitting. OSUM 23200.9. bar length = 5 \xm; D. in- 
terior valve pitting, OSUM 20407.1 , bar length = 3 цт: E. micropoints, OSUM 23200.9, bar length = 2 ¡am. 



Ventral flange slightly produced centrally to 
give the abducted valve a beak-like appear- 
ance (Fig. 41 C, D); this region of flange 
densely covered with micropoints and proba- 
bly facilitates attachment by digging deeply 
into host tissue. 

Remarks 

The glochidium of A. I. carinata is nearly 
identical to that of A. pectorosa. except it is 
smaller. A single glochidium of A. I. carinata 
gave the following measurements: length, 220 
цт; height, 243 (.im; hinge length, 125|.im; an- 
terior ligament length, 38 цт; central ligament 
length, 57 цт; and posterior ligament length, 
30 um. Ortmann (1912) gave 220 цт x 240 |.im 



for length and height measurements for A. li- 
gamentina and 250 цт x 290 \xrr\ for Nephro- 
najas pendix (= A. pectorosa. fide Ortmann & 
Walker, 1922). The glochidium of A. liga- 
mentina was figured by Lea (1858: pi. 5, fig. 
18), Ortmann (1911: p1. 89, fig. 16), and 
Surber (1 91 2: pi . 2, fig. 1 8). The glochidium of 
A. pectorosa was figured by Ortmann (1912: 
pi . 1 9, fig. 1 2, as N. pendix). 

Obovaria retusa (Lamarck, 1819) 
(Fig. 42A-D) 

Material Examined 

UMMZ Uncataloged Tennessee River (Ken- 
tucky Lake), U.S. Rt. 70 bridge. New John- 



70 



HOGGARTH 








FIG. 41 . Glochidium of Actinonaias pectorosa;A. exterior valve, OSUM 48748.3, bar length = 35 цт; В. in- 
terior valve, OSUM 48748.3, bar length = 40 мт; С. lateral view, OSUM 24337, bar length = 60 цт: D. mi- 
cropoints, OSUM 24337, bar length = 5 pm; E. micropoints, OSUM 24337, bar length = 2 цт; F. hinge, OSUM 
48748.3, bar length = 20 цт; G. exterior valve sculpture, OSUM 48748.3, bar length = 1 цт. 




FIG. 42. Glochidium of Obovaria retusa UMMZ Uncataloged (A-D) and Obovaria olivaria OSUM 51282.2 
(E-H): A. exterior valve, bar length = 40 ¡im; В. interior valve, bar length = 40 цт; С. exterior valve sculp- 
ture, bar length = 1 um; D. hinge, bar length = 20 цт; E. exterior valve, bar length = 40 цт; F. interior valve, 
bar length = 40 цт; G. exterior valve sculpture, bar length = 1 ¡лт; H. hinge, bar length = 1 5 цт. 



72 



HOGGARTH 



sonville, Humphreys Co., Tennessee, October 
1958. J. Bates. 

Description 

Glochidium subelliptical, slightly asymmet- 
ric, length 21 8 to 223 цт (221 ± 3.54 |лт, n - 
2), height 272 to 278 цт (275 ± 4.24 |ит, n = 
2). Dorsal margin slightly curved, 115 to 119 
|am (1 1 7 ± 2.08 ¡.im, n = 3) in length. Dorsal half 
of posterior margin straight, oblique to the dor- 
sal line. Ventral half of posterior margin gently 
curved initially, then straight to run more or less 
perpendicular to dorsal line. Anterior margin 
gently curved throughout its length, with max- 
imum lateral inflation occurring at about 70- 
80% from dorsal to ventral. Ventral margin 
semicircular in outline. Exterior valve surface 
finely malleated and evenly pitted, except at 
valve margin, where surface is smooth. Dorsal 
alae about 39 \xvr\ in length. Beaded to loose- 
looped sculpture covering exterior surface of 
valve (Fig. 42C). Central ligament about 45 |im 
in length, centered at about 45% from poste- 
rior to anterior. Posterior ligament about 36 цт 
long; anterior ligament about 38 lam long. Mi- 
cropoints lanceolate, arranged in broken verti- 
cal rows on ventral rim of valve and on narrow 
ventral flange. 

Remarks 

Surber (1912) gave measurements for 
length and height of 240 цт x 295 |im, 
whereas Ortmann (1912) gave 220 цт x 270 
jam. This rather wide discrepancy is not ad- 
dressed by either author and demonstrates 
the difficulty of trying to determine the species 
of a glochidium based solely on size. In the 
case of O. retusa, the moderately sized dorsal 
alae and valve shape distinguish the glochid- 
ium from species other than those of Obo- 
varia. Its beaded to loose-looped sculpture 
distinguish this species from the other mem- 
bers of the genus. This glochidium was fig- 
ured by Lea (1858: pi. 5, fig. 7), Surber (1912: 
pi. 3, fig. 47), and Ortmann (1912: pi. 19, fig. 
9). Lea's figure shows the anterior and poste- 
rior margins evenly curved, whereas Surber's 
figure shows the correct outline. 

Obovaria olivaría (Rafinesque, 1820) 
(Fig. 42E-H) 

Material Examined 

OSUM 51282.2 Mississippi River, R.Mi. 
634.7, at U.S. Rt. 18 bridge, main channel. 



1.5 mi. W of Prairie du Chien, Crawford Co., 
Wisconsin, 1 5 May 1 981 , M. E. Havlik et al. 



Description 

Glochidium subelliptical, slightly asymmet- 
ric, length 1 98 to 206 цт (202 ± 4.00 |im, n = 
3), height 254 to 261 цт (258 ± 3.51 цт, n = 
3). Dorsal margin slightly curved, 101 to 118 
|.im (109 ± 5.81 \лт, n = 5) long. As in O. re- 
tusa, posterior margin is mostly straight sided, 
with a bend about half the distance from dor- 
sal to ventral. Anterior margin gently curved; 
ventral margin semicircular. Dorsal alae well 
developed, about 37 цт in length. Loose- 
looped sculpture covering exterior surface of 
valve (Fig. 42G). Central ligament 34 to 44 ¡am 
(39 ± 5.03 |im, n = 3) in length, centered 
about 45% from posterior to anterior. Poste- 
rior ligament 26 to 29 цт (27 ± 1 .53 |im, n = 
3) long; anterior ligament 32 to 43 цт (39 ± 
5.86 |.im, n = 3) long. Micropoints lanceolate, 
located on ventral rim and on narrow ventral 
flange. 



Remarks 

This glochidium is very similar to that of O. 
retusa. However, it is smaller and it has a dif- 
ferent exterior valve sculpture. This glochid- 
ium was figured by Surber (1912: pi. 2, fig. 25, 
as O. ellipsis, = O. olivaría, fide Ortmann & 
Walker, 1922) and Ortmann (1912: pi. 19, fig. 
11). Surber gave length and height measure- 
ments of 210 цт X 265 цт and Ortmann 
(1912) recorded length and height measure- 
ments of 1 90 |jm X 220 |лт. Ortmann's figures 
are considerably smaller than Surber's or 
mine. 



Obovaria subrotunda (Rafinesque, 1820) 
(Fig. 43A-E) 

Material Examined 

MAH 659.2 - Big Darby Creek at Scioto- 
Darby (Mt. Sterling-Commercial Pt.) Rd. 
bridge, 3.4 mi. S of Orient, 1 5.3 mi. NW of Cir- 
cleville, Scioto/Darby Twp., Pickaway Co., 
Ohio, 18 May 1984, M. A. Hoggarth & G. T 
Watters. MAH 805.1 - Fish Creek at Co. Rt. 
49 bridge, 0.4 mi. above its mouth, 1.1 mi. N 
of Edgerton, 10.4 mi. W of Bryan, St. Joseph 
Twp., Williams Co., Ohio, 29 October 1985, D. 
H. Stansbery et al. 



GLOCHIDIAOF UNIONIDAE 



73 




FIG. 43. Glochidium of Obovaria subroturda; A. exterior valve, MAH 805.1, bar length = 30 цт; В. interior 
valve, MAH 805.1 , bar length = 35 цт: С. micropoints, MAH 805.1 , bar length = 3 цт; D. hinge, MAH 659.2, 
bar length = 1 5 цт; E. exterior valve sculpture, MAH 805.1 , bar length = 1 ¡jm. 



Description 

Glochidium subelliptical, length 174 to 180 
|im (177 ± 2.70 j.im, n = 5), height 197 to 210 
|im (204 ± 5.63 jim, n = 5). Dorsal margin 
slightly curved, 85 to 95 |дт (91 ± 3.50 ¡am, n 
= 6) in length. Posterior margin straight, 
oblique dorsally; ventral portion of posterior 
margin curved. Anterior margin gently curved 
throughout its length, with maximum lateral in- 
flation at about 60% from dorsal to ventral. 
Ventral margin broadly rounded. Dorsal alae 
about 29 ¡ivn in length, and loose-looped valve 
sculpture covering exterior surface of the 



valve (Fig. 43E). Central ligament 40 to 43 цт 
(42 ± 2.12 |дт, n = 2) in length, centered 
about 48% from posterior to anterior. Anterior 
ligament 24 to 29 |дт (27 ± 3.54 цт, n = 2) 
long; posterior ligament 21 to 25 цт (23 ± 
2.83 |дт, n = 2) in length. Micropoints lanceo- 
late, arranged in broken vertical rows on ven- 
tral rim and on short ventral flange. Unsculp- 
tured distal flange margin narrow. 

Remarks 

Surber (1915) described this glochidium as, 
"semielliptical in shape; ventral margin 



74 



HOGGARTH 



rounded; hinge line long and slightly de- 
pressed near center; size medium." He sug- 
gested that this glochidium is intermediate be- 
tween those of O. retusa and O. olivaria but 
differs from both by its smaller size. This study 
supports Surber's observations. This glochid- 
ium shares moderately long dorsal alae with 
O. retusa. and moderately dense loose- 
looped sculpture with O. olivaria. Ortmann 
(1912) gave 200 |.im x 230 цт for length and 
height, whereas Surber (1915) gave 1 70 |Lim x 
215 |.im. This glochidium was figured by Ort- 
mann (1911: pi. 89, fig. 15, as O. circulus, = O. 
subrotunda. fide Ortmann & Walker, 1922), 
and Surber (1915; pi. 1 , fig. 8, as O. circulus). 

Obovaria jacksoniana (Frierson, 1912) 
(Fig. 44A-D,G) 

Material Examined 

OSUM 50233.8 - Sipsey River below Rt. 21 
bridge, 1.3 mi. W of Brownville, 16.8 mi. NW 
of Tuscaloosa, Tuscaloosa Co., Alabama, 10 
October 1981, L. M. Koch. 

Description 

Glochidium subelliptical, almost symmetri- 
cal, length 1 75 to 1 87 |im (1 82 ± 5.74 цт, n = 
4), height 230 to 243 цт (236 ± 6.56 ¡.im, n = 
4). Dorsal margin slightly curved, 89 to 1 00 цт 
(95 ± 4.57 jjm, n = 4) long. Posterior and an- 
terior margins about equal, except posterior 
margin slightly more produced as a result of a 
larger angle of divergence. Posterior margin 
curving ventrally at about 50% from dorsal to 
ventral, becoming almost perpendicular to 
dorsal margin, anterior margin gently rounded. 
Exterior surface smooth with just a few pits 
and very little malleation, except at the umbo. 
Loose-looped sculpture covering exterior sur- 
face (Fig. 44C). Dorsal alae with a length of 
about 34 |.im. Central ligament 41 to 42 ¡.im (42 
± 0.71 jim, n = 2) in length, centered 46% from 
posterior to anterior. Anterior ligament 26 to 31 
цт (29 ± 3.54 pm, n = 2) long; posterior liga- 
ment 21 to 25 |.im (23 ± 2.83 |.im, n = 2) long. 
Micropoints lanceolate, located on ventral rim 
of valve and on short ventral flange. 

Remarks 

This glochidium is very similar to the other 
members of the genus. It is intermediate in 
size between O. olivarla and O. subrotunda. 
and much smaller than O. retusa. No pub- 
lished figure of this glochidium was found. 



Obovaria unicolor (Lea, 1845) 
(Fig. 44E, F, H) 

Material Examined 

OSUM 33158 - Sabine River at La. Rt. 8 
and Tex. Rt. 3 bridge, 1 .3 mi. W of Burr Ferry, 
Vernon Parish/Newton Co., Louisiana/Texas, 
14 July 1968, D. H. Stansbery et al. OSUM 
47696. 6-Pearl River, 0.8 mi. SSE of Drysdale, 
6.7 mi. SW of Carthage, Sec. 25/36, T10N, 
R6E, Lake Co., Mississippi, 5 October 1979, 
R. G. Rummel & P Hartfield. 

Description 

Glochidium subelliptical, almost symmetri- 
cal, length 1 68 to 1 80 цт (1 72 ± 4.36 (im, n = 
5), height 218 to 234 (.im (225 ± 5.86 цт, n = 
5). Dorsal line slightly curved, 85 to 96 цт (90 
± 4.22 j.im, n = 6) long. Anterior and posterior 
margins about equal, straight sided dorsally, 
slightly curved ventrally, as in O. jacksoniana. 
Ventral margin broadly rounded. Exterior sur- 
face mostly smooth, with malleations at umbo 
and only a few pits. Loose-looped sculpture 
covering exterior surface of valve Dorsal alae 
about 32 j.im in length. Central ligament 32 to 
33 цт (32 ± 0.58 цт, n = 3) in length, centered 
about 45% from posterior to anterior. Anterior 
ligament 32 to 41 ,um (35 ± 4.62 (.im, n = 3) 
long; posterior ligament 23 to 28 ¡am (25 ± 
2.65 um, n = 3) long. Micropoints lanceolate, 
arranged in incomplete vertical rows, located 
on ventral rim of valve and on short ventral 
flange. 

Remarks 

This glochidium is essentially identical to 
that of O. jacksoniana. My measurements 
would indicate that the glochidium of O. uni- 
color is smaller than that of Q. jacksoniana. 
but even here there is overlap. This glochid- 
ium is figured by Ortmann (1912; pi. 19, fig. 
10), who gave length and height measure- 
ments of 160 цт X 210 |im. 



Ellipsaria lineolata (Rafinesque, 1820) 
(Fig. 45A-F) 

Material Examined 

OSUM 1984; 14 - Ohio River, R.Mi. 443.0- 
445.0, from 0.4 mi. NW of Moscow, Ohio, to 
Point Pleasant, Ohio, 5.0-6.8 mi. SE of New 
Richland, Ohio, Pendleton/Campbell Co., 



GLOCHIDIAOF UNIONIDAE 



75 




FIG. 44. Glochidium of Obovaria jacksoniana (A-D, G) OSUM 50233.8 and Obovaria unicolor (E, F, H); A. 
exterior valve, bar length = 30 \.im; B. interior valve, bar length = 30 |.im; С exterior valve sculpture, bar length 
= 1 цт; D. hinge, bar length = 15 |am; E. exterior valve, OSUM 33158, bar length = 30 |лт; F. interior valve, 
OSUM 331 58, bar length = 30|im;G. micropoints, OSUM 33158, bar length = 2 цт; H. micropoints, OSUM 
47696.6, bar length = 3 |.im. 



76 



HOGGARTH 



Kentucky, 16 November 1984, D. H. Stans- 
bery et al. 



Description 

Glochidium subiiguiate, much higher than 
long, length 229 to 245 (.im (237 ± 5.06 |im, 
n = 7), height 310 to 325 цт (321 ± 5.15 цт, 
n = 7). Dorsal margin slightly curved, 87 to 96 
lam (91 ± 3.55 цт, n = 7) long. Anterior and 
posterior margins more or less straight to 
slightly incurved dorsally. At about 40% from 
dorsal to ventral, margins again becoming 
straight, slightly divergent. Point of maximum 
lateral inflation at about 80% from dorsal to 
ventral. Ventral margin broad, over twice 
length of hinge margin, but more narrowly 
curved than found in Obovaria, Actinonaias ox 
Ptychobranchus. External valve surface 
finely malleated and evenly pitted, except 
along valve margins, where pits and mal- 
leations are absent (Fig. 45C). Vermiculate 
sculpture covering exterior surface of valve 
(Fig. 45F); dorsal alae very small and ori- 
ented at a 45° angle to hinge. Central liga- 
ment 35 to 51 |im (43 ± 8.42 \im, n = 4) long, 
centered about 48% from posterior to ante- 
rior. Posterior ligament 20 to 24 |.im (22 ± 
1.63 |im, n = 4) long; anterior ligament 23 to 
29 |jm (26 ± 2.75 цт, n = 4) in length. Mi- 
cropoints lanceolate, arranged in complete 
vertical rows on ventral valve margin and on 
wide ventral flange, decreasing in size dis- 
tally, covering proximal two thirds of ventral 
flange, leaving a narrow unsculptured distal 
flange margin. The ventral flange becomes 
folded laterally so that it forms small hook-like 
points (Fig. 45C). 



Leptodea fragilis (Rafinesque, 1820) 
(Fig. 46A-E) 

Material Examined 

MAH 626.1 - Big Darby Creek at Scioto- 
Darby (Mt. Sterling-Commercial Pt.) Rd. 
bridge, 3.4 mi. S of Orient, 15.3 mi. SW of 
Columbus, Scioto/Darby Twp., Pickaway Co., 
Ohio, 13 April 1984, M. A. Hoggarth & G. T. 
Watte rs. 

Description 

Glochidium subelliptical, very small, length 
72 to 73 |im (72 ± 0.58 |im, n = 3), height 80 
to 83 |im (83 ± 1.53 цт, n = 3). Dorsal mar- 
gin straight, 30 to 35 цт (33 ± 2.89 |im, n = 
3) long. Anterior and posterior margins about 
equally divergent dorsally, ventrally curved. 
Ventral margin broadly rounded, joining lat- 
eral margins at their point of maximum infla- 
tion, near 70% from dorsal to ventral. Exterior 
surface only malleated near umbo; however, 
valve surface otherwise densely pitted, ex- 
cept along valve margin (Fig. 46A, C) and at 
adductor muscle insertion (Fig. 46A, B). Exte- 
rior surface lightly covered with loose-looped 
valve sculpture (Fig. 46E); dorsal alae very 
small. When fully adducted, valves gape in 
lateral view (Fig. 46C). Central ligament about 
11 |im long, centered about 45% from poste- 
rior to anterior. Posterior ligament 8 to 10 )ит 
(9 ± 1.41 цт, n = 2) in length; anterior liga- 
ment 11 to 14 (.im (13 ± 2.12 цт, n = 2) long. 
Micropoints wide, lamellate plates arranged in 
complete vertical rows on ventral valve mar- 
gin and on narrow ventral flange. Unsculp- 
tured distal flange margin narrow. 



Remarks 

Lefevre & Curtis (1910), Surber (1 912), and 
Ortmann (1912) gave the following measure- 
ments for length and height: 230 цт x 31 цт, 
230 |jm X 330 |im, and 260 цт x 350 цт. This 
glochidium can be distinguished from all oth- 
ers by its fan-shaped outline, small dorsal 
alae, exterior valve sculpture and its distinct 
hook-like folding of the ventral flange. This 
glochidium is figured by Lea (1 858: pi. 5, fig. 6, 
as Uniosecuris, = E. lineolata. f/de Ortmann & 
Walker, 1922), Lefevre & Curtis (1910: fig. H, 
1912: fig. 1H, as Plagióla securis), Ortmann 
(1 91 1 : pi. 89, fig. 1 7, as P. securis), and Surber 
(1 912: pi. 2, fig. 1 4, as P securis). 



Remarks 

This glochidium will not be confused with 
any other. Its size, round subelliptical shape, 
small dorsal alae, and lamellate micropoints 
distinguish it from all others. Lefevre & Curtis 
(1910, 1912), Surber (1912), and Ortmann 
(1912) gave the following measurements for 
length and height: 75 цт x 85 yivn, 70 |.im x 95 
|лт, and 80 цт x 90 |.im. This glochidium is fig- 
ured by Lefevre & Curtis (1910: fig. K; 1912: 
fig. IK, as Lampsilis gracilis. = L. fragilis. fide 
Ortmann & Walker, 1922), Ortmann (1911: pi. 
89, fig. 19, as Paraptera gracilis), Coker & 
Surber (1911: pi. 1, fig. 2, 2a, as L. gracilis). 
and Surber (1912: pi. 2, fig. 28, as L. gracilis). 



GLOCHIDIAOF UNIONIDAE 



77 




FIG. 45. Glochidium of Ellipsaha lineolata, OSUM:1984:14; A. exterior valve, bar length = 45 |am; B. interior 
valve, bar length = 45 |am; С lateral view, bar length = 35 jam; d. micropoints, bar length = 5 ¡am; E. hinge, 
bar length = 15 jam; F. exterior valve sculpture, bar length = 1 цт. 



Leptodea ochracea (Say, 1817) 
(Fig.47A-E) 

Material Examined 

MAH 896 - Great Herring Pond at Carter's 
Brook Rd., 4.0 mi. NNE of Buzzards Bay, 3.0 
mi. NW of Sagamore, Plymouth Co., Massa- 
chusetts, 31 July 1985, G.T. Watters et al. 



Description 

Glochidium subelliptical to subligulate, 
length 241 to 246 |.im (243 ± 1 .95 |im, n = 5), 
height 289 to 294 |.im (291 ± 1 .92 |.im, n = 5). 
Dorsal margin straight, short, 102 to 110 ¡im 
(1 07 ± 3.90 |am, n = 6) in length. Anterior and 
posterior margins equal, more or less straight 
to slightly incurved dorsally, bending ventrally 



78 



HOGGARTH 



^m^g_¿^j\__^ 


а 


1 


^Яе^^^ЯШЁш* 


^^^^ь, 


1 




' ^ 


^^ЖЯ 





FIG. 46. Glochidium of Leptodea fragilis, MAH 626.1 ; A. exterior valve, bar length = 10 цт: B. interior valve, 
bar length = 1 цт; С. lateral view, bar length = 1 цт: D. micropoints, bar length = 2 |.im: E. exterior valve 
sculpture, bar length = 1 ¡.im. 



about 50% from dorsal margin, slightly curved 
to their maximum inflation at about 80% from 
dorsal to ventral. Ventral margin gently and 
evenly curved throughout its length. Exterior 
surface malleated and pitted, except along 
valve margins where longitudinal ridges occur 
(Fig. 47A, C). Small dorsal alae occur at dor- 
sal lateral margin of valve. Lateral valve gape 
moderate. Central ligament 39 to 47 цт (43 ± 
3.65 jim, n = 4) in length, centered about 48% 
from posterior to anterior. Anterior ligament 32 
to 37 |.im (35 ± 2.22 |.im, n = 4) long; postehor 
ligament 28 to 34 }.im (31 ± 2.58 цт, n = 4) in 
length. Micropoints narrow lamellate plates 



arranged in complete vertical rows on ventral 
flange and ventral valve rim, becoming 
smaller, more lanceolate distally covering 
about four fifths of the surface of flange. Un- 
sculptured distal flange edge very narrow. 

Remarks 

The adult shell of L. ochracea is so similar 
to that of Lampsilis cariosa that the adults are 
often confused. The glochidia are very dis- 
similar, however, and are easily distinguished 
by valve outline, development of dorsal alae, 
presence of lateral valve gape, and micro- 



GLOCHIDIAOF UNIONIDAE 



79 




.) 
V 

1 


Ы \ 


с 




FIG. 47. Glochidium of Leptodea ochracea, MAH 896; A. exterior valve, bar length = 35 цгл; В. interior valve, 
bar length = 35 цт; С. lateral view, bar length = 40 j.im; D. hinge, bar length = 15 цт; E. micropoints, bar 
length = 5 |дт. 



point Structure (see glochidium of L. cariosa 
below). The glochidium of L. ochracea was 
figured by Porter & Horn (1981 : fig. 7). 

This species is often included in the genus 
Lampsilis. primarily because the adult shell re- 
sembles that of other members of that genus 
and because of the pronounced sexual dimor- 
phism of the shell (Simpson, 1900, 1914; 
Johnson, 1947, 1970; Burch, 1975; Clarke, 
1 981 b). However, Morrison (1 975) was unable 



to find mantle flaps in female L. ochracea. 
Nonetheless, Fuller & Bereza (1975) sug- 
gested that this was not sufficient evidence to 
re-classify L. ochracea. and Porter & Horn 
(1 981 ) concluded that because the shape and 
size of the glochidium of ochracea is similar to 
that of cariosa, the species should remain in 
Lampsilis. In fact, the glochidium of this 
species does not ally ochracea with any 
species of Lampsilis examined (see below). 



80 



HOGGARTH 



Because the anatomy of this species is not 
that of Lampsilis. and the glochidium, although 
larger, is only closely allied to L. fragilis, I agree 
with Morrison (1 975) that this species belongs 
in Leptodea. 

Potamilus ohiensis (Rafinesque, 1820) 
(Fig. 48A-E) 

Material Examined 

OSUM 54520.1 - Big Blue River below 
bridge, 3.0 mi. NNW of Barneston, 4.0 mi. SB 
of Wymore, T2N, R7E, Gage Co., Nebraska, 
7 September 1981, F. E. Hoke. 

Description 

Glochidium ax-head shaped, dorsally ligu- 
late, becoming very broad ventrally (Fig. 
47A), length 120 to 126 цт (123 ± 3.05 |im, 
n = 3), height 175 to 187 |im (181 ± 6.03 |im, 
n = 3). Dorsal margin straight, very short, 42 
to 50 |im (45 ± 3.42 |im, n = 5) in length. An- 
terior and posterior margins equal, straight 
sided dorsally, crescent shaped ventrally, out- 
wardly curved to their maximum inflation at 
about 90% from dorsal to ventral. Exterior sur- 
face smooth, without malleations or pits, and 
loose-looped exterior valve sculpture cover- 
ing the valve (Fig. 48D). Dorsal alae small, lat- 
eral valve gape very large (Fig. 48B). Central 
ligament 1 6 to 21 цт (1 9 ± 3.54 ^im, n = 2) in 
length, centered about 50% from both lateral 
margins. Anterior ligament, 1 3 to 1 5 |лт (1 4 ± 
1 .41 |am, n = 2) long; posterior ligament 1 3 to 
14 [im (14 ± 0.71 цт, n = 2) long. Lamellate 
micropoints present on ventral rim and on nar- 
row ventral flange. Lanceolate hooks absent. 

Remarks 

Lea (1 858, 1 863) described this glochidium 
as wedge shaped, with a hook-like process at 
each corner of the ventral margin. However, 
Coker & Surber (1 91 1 ) reported that the spines 
or hooks were absent, and Surber (1912) re- 
ported, "Glochidium without spines (?)." This 
glochidium can be distinguished from that of 
most other members of the genus Potamilus 
by the absence of spines. They are further dis- 
tinguished by micropoint structure, exterior 
valve sculpture and shape, especially the 
roundness of the ventral margin. This glochid- 
ium shares these characters with Leptodea 
fragilis, but can be distinguished from that 
species by its ax-head shape. The glochidium 



of P. ohiensis \Nas figured by Lea (1858: pi. 5, 
fig.24),Coker&Surber(1911:pl. 1,fig. 1, la), 
Surber (1 91 2: pi. 1 , fig. 1 0), and Arey (1 921 : pi. 
2, figs. 5, 6;1924: pi. 1, fig. 3). Lea incorrectly 
figured the glochidium with hooks, whereas 
Coker & Surber (1911), Surber (1912), and 
Arey (1 921 ) did not. The shape of the glochid- 
ium is correctly drawn in each figure. 

Potamilus amphichaena (Frierson, 1898) 
(Fig. 49A-E) 

Material Examined 

OSUM 33163.13 - Sabine River at La. Rt. 
8 and Tx. Rt. 9 bridge, 1 .3 mi. W of Burr Ferry, 
Vernon Parish / Newton Co., LouisianaЯexas, 
14 July 1968, D. H. Stansbery et a!. 

Description 

Glochidium ax-head shaped, length 111 to 
113 цт (112 ± 1.41 |im, n = 2), height 170 to 
171 цт (1 71 ± 0.71 |am, n = 2). Dorsal margin 
short, 40 to 41 цт (41 ± 0.71 цт, n = 2) in 
length; anterior and posterior margins lie par- 
allel dorsally, becoming strongly curved ven- 
trally. Anterior and posterior margins meeting 
broadly rounded ventral margin at point of 
maximum lateral inflation at about 90% from 
dorsal to ventral. Dorsal alae small. Exterior 
surface of valve with loose-looped sculpture 
(Fig. 49D). The hinge of a single specimen 
gave the following measurements: anterior lig- 
ament, 11 |.im; central ligament, 20 ¡am; poste- 
rior ligament, 10 ¡am. The central ligament is 
centered about 49% from posterior to anterior. 
Micropoints lamellate, as in L. fragilis and P. 
ohiensis. arranged in vertical rows on ventral 
valve margin and on short ventral flange. 
Lanceolate hooks absent. 

Remarks 

This glochidium is almost identical with that 
of P. ohiensis but can be distinguished by its 
slightly more broadly rounded ventral margin. 
No previously published figure of this glochid- 
ium was found. 

Potamilus alatus (Say, 1817) 
(Fig. 50A-F) 

Material Examined 

OSUM: 1983:58 - Muskingum River, R.Mi. 
31 .8-33.4, 1 .4 mi. N of Luke Chute Lock and 



GLOCHIDIAOF UNIONIDAE 



81 




FIG. 48. Glochidium of Lastena ohiensis, OSUM 54520.1; A. exterior valve, bar length = 25 |дгл; В. lateral 
view, bar length = 30 |am; C. hinge, bar length = 1 цт; D. exterior valve sculpture, bar length = 2 jam; E. mi- 
cropoints, bar length = 5 |лт. 



Dam, 4.3 mi. W of Beverly, Windsor/Waterford 
Twp., Morgan/Washington Co., Ohio, 25 Sep- 
tember 1983, W. N. Kasson & K. Perkins. 
OSUM 55465 - Clinch River, R.Mi. 213.0 at 
Clinchport, just above swinging bridge, Scott 
Co., Virginia, 5 October 1985, G. T. Walters. 



Description 

Glochidium ligulate, ax-head shaped, much 
higher than long, length 206 to 227 цт (21 6 ± 
9.15 |im, n = 5), height 371 to 386 (.im (378 ± 
7.66 цт, П = 5). Dorsal margin straight, al- 



though appearing slightly curved in exterior 
view due to deep umbo cavity (Fig. 50C), 96 to 
109 |im (102 ± 5.93 |im, n = 5) in length, or 
less than half total length of valve. Anterior and 
posterior margins equal, parallel sided for 
about 70% their length, gently outwardly 
curved to their point of maximum inflation at 
about 95% from dorsal to ventral. Ventral mar- 
gin slightly curved. Adductor muscle scar very 
rough, with numerous ridges; however, pit 
density not appearing to be reduced. Dorsal 
alae small (Fig. 50A); lateral valve gape pro- 
nounced (Fig. 50C); exterior surface covered 
with vermiculate sculpture (Fig. 50E). Central 



82 



HOGGARTH 




FIG. 49. Glochidium of Lastena amphichaena. OSUM 331 63.1 3; A, exterior valve, bar length = 25 цт; В. mi- 
cropoints, bar length = 5 j.im; С hinge, bar length = 1 цт; D. exterior valve sculpture, bar length = 2 цт; E. 
rлicropoints, bar length = 2 ).im. 



ligament 42 [xm in the two specimens mea- 
sured, centered about 44% from posterior to 
anterior. Anterior ligament 35 to 38 цт (37 ± 
2.1 2 |лт, n = 2) in length; posterior ligament 1 9 
to 29 |im (24 ± 7.07 ¡am, n = 2) long. Lanceo- 
late hooks present at lateral margins of ventral 
flange, long, slightly inwardly curved, attenu- 
ate. Micropoints arranged in complete vertical 
rows on the ventral rim of valve and on rather 
wide ventral flange, bluntly pointed, becoming 
gradually smaller toward distal edge of flange. 



covering most of flange, leaving narrow to ab- 
sent unsculptured distal flange edge. 

Remarks 

This glochidium was figured by Lea (1858: 
pi. 5, fig. 25), Lefevre & Curtis (1910: fig. D; 
1912: fig. ID), Ortmann (1911 : pi. 89, fig. 18), 
Coker & Surber (1911: pi. 1, fig. 3). Surber 
(1912: pi. 1, fig. 8). and Utterback (1915-6: 
figs. 9a, b). Length and height measurements 



GLOCHIDIAOF UNIONIDAE 



83 




FIG. 50. Glochidium of Potamilus alatus; A. exterior valve, OSUM 55465, bar length = 50 цт; В. interior 
valve, OSUM 55465, bar length = 50 цгп; С. lateral view, OSUM 55465, bar length = 50 цгл; D. lanceolate 
hook, 0SUM:1 983:58, bar length = 5 |.im; E. exterior valve sculpture, OSUM 55465, bar length = 1 цт; F. 
hinge, OSUM 55465, bar length = 15 цт. 



84 



HOGGARTH 



were given by Lefevre & Curtis (1910, 1912), 
Surber (1912), and Ortmann (1912), 230 цт 
X 410 цт, 220 цт x 380 ¡.im, and 200 }.im x 
380 |.im. This glocliidium can be distinguished 
from other species by its size, shape, and mi- 
cropoint structure. 

The glochidium of Potamilus capax is also 
similar to that of P. alatus (Cummings et al., 
1 990). The glochidium of P. capax is about the 
same size as that of P. ohiensis (105 |лт in 
length and 185 цт in height, Coker & Surber, 
1911). This glochidium possess lanceolate 
hooks, vermiculate exterior valve sculpture, 
and a narrowly curved ventral margin. The 
glochidium of P. capax is figured by Cum- 
mings et al. (1990: fig. 6). 

Potamilus purpuratus (Lamarck, 1819) 
(Fig. 51A-F) 

Material Examined 

OSUM 15738.2 - Brazos River at foot of 
Whitney Dam, about 20 mi. NW of Waco, 
Bosque/Hill Co., Texas, 20 March 1966, С В. 
Stein. 

Description 

Glochidium ligulate, ax-head shaped, much 
higher than long, length 1 90 to 200 цт (1 95 ± 
5.00 |im, n = 3), height 347 to 356 цт (350 ± 
4.93 |im, n = 3). Dorsal margin short, straight, 
100 to 109 |im (105 ± 3.87 |.im, n = 4) long. 
Anterior and posterior margins parallel to 
about 80% from dorsal to ventral, becoming 
evenly curved to point of maximum lateral in- 
flation at about 95% from the dorsal margin. 
Ventral margin only slightly curved. Exterior 
surface of valve malleated, pitted dorsally, 
ventrally smooth, with sparse pitting in area of 
adductor muscle scar compared to remainder 
of valve. Dorsal alae small (Fig. 51 A, C), lat- 
eral valve gape wide, and exterior surface 
covered with vermiculate sculpture (Fig. 51 E). 
Central ligament 37 to 41 цт (39 ± 2.83 цт, 
n = 2) long, centered about 46% from poste- 
rior to anterior. Anterior ligament 37 i^m in 
length; posterior ligament 28 to 29 ¡am (29 ± 
0.71 |im, n = 2) long. Lanceolate hooks lo- 
cated at lateral margins of ventral flange. Mi- 
cropoints arranged in complete vertical rows 
on ventral rim of valve and on wide ventral 
flange. Micropoints bluntly pointed (Fig. 51 D), 
becoming gradually smaller toward distal 
edge of flange, covering a large portion of 



flange, leaving narrow unsculptured distal 
edge. 

Remarks 

This glochidium can be distinguished from 
that of P. alatus by its micropoint structure and 
its size. This glochidium was figured by Lea 
(1874: pi. 21, fig. 13) and Surber (1915: pi. 1, 
fig. 5). 

Ligumia recta (Lamarck, 1819) 
(Fig. 52A-E) 

Material Examined 

0SUM:1 984:2 - Mississippi River, R.Mi. 
635.6, E channel, 1.1 mi. NW of Prairie du 
Chien, Crawford Co., Wisconsin, 29 April 
1984, D. H. Stansbery et al. 

Description 

Glochidium subelliptical, length 205 to 219 
j^m (211 ± 6.02 |лт, n - 5), height 257 to 265 
fim (260 ± 3.08 }im, n = 5). Dorsal margin 
slightly curved, 105 to 115 |im (109 ± 3.24 
)im, n = 7) in length. Anterior and posterior 
margins equally curved, valve outline sym- 
metrical. Exterior surface of valve malleated 
and pitted, except along valve margin, where 
shell is smooth. Dorsal alae long, well devel- 
oped, about 52 цт in length. Rough exterior 
surface sculpture covering valve (Fig. 52E). 
Central ligament 35 to 40 ¡am (38 ± 3.54 цт, 
n = 2) long, centered about 45% from poste- 
rior to anterior. Anterior ligament 41 to 45 |лт 
(43 ± 2.83 |im, n = 2) in length; posterior liga- 
ment 30 to 34 lam (32 ± 2.83 |am, n = 2) long. 
Micropoints numerous, lanceolate, located on 
ventral margin of valve and on rather wide 
ventral flange (not figured). Center of ventral 
margin almost beak-like, similar to that of A. 
pectorosa (Fig. 41 C). 

Remarks 

This glochidium has the same valve outline 
as that of A. pectorosa. However, the glochid- 
ium of L. recta can be distinguished from that 
glochidium by its larger size, broader ventral 
flange, and longer dorsal alae. The glochid- 
ium of L. rec/a was figured by Lea (1858: pi. 5, 
fig. 11), Lefevre & Curtis (1910: fig. L, 1912: 
fig. 1 L), Ortmann (1 91 1 : pi. 89, fig. 21 ), Surber 
(1912: pi. 2, fig. 17), Isom & Hudson (1982: 
fig. 1), and Isom (1983: figs, la, b). 



GLOCHIDIAOF UNIONIDAE 



85 




FIG. 51 . Glochidium of Potamilus purpuratus, OSUM 1 5738.2; A. exterior valve, bar length = 50 lam; B. inte- 
rior valve, bar length = 50 |jm; С lateral view, bar length = 50 lam; D. micropoints, bar length = 5 цт; E. ex- 
terior valve sculpture, bar length = 1 цт; F. hinge, bar length = 15 цпп. 



86 



HOGGARTH 




FIG. 52. Glochidium of Ligumia recta. 0SUM:1 984:2; A. exterior valve, bar length = 30 |.im: B. exterior valve, 
bar length = 35 jam; С lateral view, bar length = 35 |.im: D. micropoints, bar length = 3 |um: E. micropoints, 
bar length = 5 pm. 



Venustaconcha ellipsiformis ellipsiformis 

(Conrad, 1834) 

(Fig. 53A-G) 

Material Examined 

MAH 947.2 - Kalamazoo River above St. 
Rt. 60 bridge, 3.0 mi. WSW of Spring Arbor, 



12.0 mi. WSW of Jackson, Jackson Co., 
Michigan, 13 May 1986, M. A. Hoggarth. 

Description 

Glochidium subeiliptical, symmetrical, 
length 223 to 230 |.im (226 ± 2.94 цт, n = 4), 
height 280 to 287 цт (285 ± 3.32 pm, n = 4). 



GLOCHIDIAOF UNIONIDAE 



87 




FIG. 53. Glochidium of Venustaconcha e. ellipsiformis. MAH 947.2; A. exterior valve, bar length = 35 |am; B. 
exterior valve, bar length = 35 |.im; С micropoints, bar length = 2 |.im; D. micropoints, bar length = 2 |am; E. 
exterior valve sculpture, bar length = 1 ¡.im; F. interior valve pitting and adductor muscle scar, bar length = 
10 |лт; G. hinge, bar length = 15 \xm. 



Dorsal margin straight, 102 to 1 1 цт (1 04 ± 
2.48 цт, П = 6) in length. Anterior and poste- 
rior margins equally and gently curving to their 
maximum inflation at about 70% from dorsal 
to ventral. Exterior surface of valve finely mal- 



leated near umbo, with fine loose-looped ex- 
terior valve sculpture (Fig. 53E). Adductor 
muscle scar indicated by numerous small 
ridges and reduced pitting (Fig. 53F). Central 
ligament 38 to 40 |am (40 ± 3.21 [im, n = 3) 



88 



HOGGARTH 



long, centered 47% from posterior to anterior. 
Posterior ligament 28 to 32 цт (30 ± 2.08 
|дт, n = 3) long; anterior ligament 34 to 39 |.im 
(36 ± 2.65 цт, n = 3) in length. Micropoints 
lanceolate, located on ventral rim of valve and 
on moderately wide ventral flange, arranged 
in broken vertical rows that extend about half 
distance of flange, leaving a wide unsculp- 
tured distal flange margin. 

Remarks 

This glochidium is identical to that of L. 
recta, except it is larger. It was figured by Lea 
(1858: pi. 5, fig. 9, as Unio spatulatus, = V. el- 
lipsiformis, fide Simpson, 1900) and van der 
Schalle (1963: pi. 1 , center, left). 



Villosa trabalis (Conrad, 1834) 
(Fig. 54A, B, D, E) 

Material Examined 

V. trabalis: OSUM 9516.49 - Rockcastle 
River, Rt. 80 W of London, Rockcastle/Laurel 
Co., Kentucky, 26 October 1 963, С В. Stein & 
D. H. Stansbery. V. perpurpurea (Fig. 53C): 
OSUM 16262 - Clinch River at St. Rt. 460 
bridge at Richlands, Tazewell Co., Virginia, 6 
October 1965, D. H. Stansbery & J. J. Jenkin- 
son. 



Description 

Glochidium subelliptical, higher than long, 
with a short, straight dorsal margin, gently 
curved, almost equal lateral margins and 
broadly rounded ventral margin. Glochidium 
21 1 to 21 6 цт (214 ± 2.36 |im, n = 4) in length, 
277 to 280 цт (278 ± 1 .50 }.im, n = 4) in height. 
Dorsal margin 94 to 99 цт (96 ± 2.1 7 ¡.im, n = 
5) in length. Exterior valve surface mostly 
smooth, with relief occurring at pits, which are 
surrounded by smooth, circular discs (Fig. 
54E). Dorsal alae moderate in length. Central 
ligament 31 to 36 |.im (33 ± 2.89 |.im, n = 3) 
long , centered about 45% from posterior to an- 
terior. Posterior ligament 25 to 28 ).im (27 ± 
1 .53 |.im, n = 3) long; anterior ligament 36 to 40 
jim (37 ± 2.31 цт, n = 3) in length. Micropoints 
lanceolate, numerous, unorganized, found on 
ventral rim of valve and on distal half of wide 
ventral flange, leaving wide unsculptured dis- 
tal flange margin. 



Remarks 

Surber (1912) gave 193 цт x 255 |im for 
the length and height of this glochidium, and 
Ortmann (1912) gave 220 цт x 270 цт. This 
glochidium is similar in shape, size and dorsal 
alae structure to Obovaria, but it can be dis- 
tinguished by its rather wide ventral flange. 
The glochidium of Villosa perpurpurea (Lea, 
1861 ) is virtually identical to that of V. trabalis, 
although smaller: length, 165 |.im; height, 241 
|.im; hinge length, 88 |лт. The glochidium of V. 
iraba//s was figured by Surber (1912: pi. 3,fig. 
40) and Ortmann (1912: pi. 20, fig. 4). 



Villosa villosa (Wright, 1898) 
(Fig. 55A-G) 

Material Examined 

OSUM 45940.3, 45940.7 - Santa Fe River 
at U.S. Rt. 41/441 bridge, 2.0 mi. NNW of 
High Spring, 27.3 mi. NW of Gainsville, Sec. 
27/28, Alachua/Columbia Co., Florida, 4 Au- 
gust 1975, J. M. Condit & E. P. Keferl. 

Description 

Glochidium subelliptical (Fig. 54C) to sub- 
spatulate (Fig. 55A, B, D), length 240 to 250 
|jm (245 ± 3.89 цт, n = 7), height 296 to 308 
|im (303 ± 4.26 |.im, n = 7). Dorsal margin 
straight, 105 to 116 цт (111 ± 3.65цт,п = 7) 
in length. Glochidium usually characterized by 
a gently curved ventral margin and lateral 
margins that are oblique to the dorsal margin 
dorsally and perpendicular ventrally. Dorsal 
alae long, extending about half distance of 
dorsal oblique section of lateral margins. 
Valve finely malleated, densely pitted, except 
along margins; exterior sculpturing rough but 
not quite beaded, best described as very fine 
pustules (Fig. 55G). Central ligament 37 to 42 
|.im (40 ± 3.54 цт, n = 2) long, centered about 
49% from posterior to anterior. Anterior liga- 
ment 35 to 43 цт (38 ± 4.24 (.im, n = 2) long; 
posterior ligament about 33 цт long (both 
specimens had identical posterior ligament 
lengths). Micropoints lanceolate, arranged in 
more or less complete vertical rows on ventral 
flange and on the rim of ventral margin of 
valve. Micropoints becoming smaller from 
proximal to distal portion of flange, covering 
about 75% of flange surface, leaving narrow 
unsculptured distal flange margin. 



GLOCHIDIA OF UNIONIDAE 



89 




FIG. 54. Glochidium of Villosa trabalis (A, B, D, E) OSUM 9516.49, and Villosa perpurpurea (C) OSUM 
1 6262; A. exterior valve, bar length = 35 |jin; B. interior valve, bar length = 35 pm; С exterior valve, bar length 
= 40 |im; D. micropoints, bar length = 2 |im; E. exterior valve sculpture, bar length = 1 цт. 



Remarks 

The outline of the valve of this glochidium 
distinguish it from the glochidia previously de- 
scribed, but not from other members of this 
genus (see Villosa vibex and Villosa iris iris 
below) and all members of the genus Lamp- 
silis. This appears to be the final change in 
glochidial shape within this lineage. This 
glochidium is figured here for the first time. 



Villosa vibex {Conrad, 1834) 
(Fig. 56A-F) 

Material Examined 

OSUM 54631 - Yellow River at U.S. Rt. 84 
bridge, 3.0 mi. SE of Sanford, 5.0 mi. W of 
Opp, Sec. 33, T4N, R17E, Covington Co., Al- 
abama, 2 June 1969, H. Harima & B. Wall. 
OSUM 241 24 - Santa Fe River at U.S. Rt. 27 



90 



HOGGARTH 




FIG. 55. Glochidium of Villosa villosa:A. exterior valve, OSUM 45940.7, bar length = 40 |.im; B. interior valve, 
OSUM 45940.3, bar length = 40 цт; С. exterior valve, OSUM 45940.7, bar length = 40 ¡.im: D. interior valve. 
OSUM 45940.7, bar length = 40 цт: E. micropoints, OSUM 45940.3. bar length = 5 [im: F. micropoints. 
OSUM 45940.3, bar length = 2 цт; G. exterior valve sculpture, OSUM 45940.7, bar length = 1 urn. 



GLOCHIDIAOF UNIONIDAE 



91 




FIG. 56. Glochidium of Villosa vibex: A. exterior valve, OSUM 24124, bar length = 40 цт: В. interior valve, 
OSUM 24124, bar length = 40 |.im; С adductor гли5с1е insertion, OSUM 54631, bar length = 5 |am; D. mi- 
cropoints, OSUM 54631 , bar length = 5 |im; E. exterior valve sculpture, OSUM 54631 , bar length = 2 |лт; F. 
exterior valve sculpture, OSUM 24124, bar length = 2 |.im. 



bridge, 2.5 mi. NW of High Springs, 7.5 mi. SE 
of Fort White, Columbia/Alachua Co., Florida, 
24 February 1970, J. J. Jenkinson. OSUM 
48628 - "Nichols Cr." Monroe Co., Missis- 
sippi, in the Tombigbee River drainage (Mo- 
bile River), 18 March 1972, collector un- 
known, ex J. D. Williams. 



Description 

Glochidium subspatulate, length 224 to 239 
|im (230 ± 5.42 |im, n - 8), height 295 to 304 
цт (300 ± 4.17 |jm, n = 8). Dorsal margin 
straight, 1 00 to 1 20 цт (11 ± 7.79 цт, n = 8) 
long. Lateral margins straight, divergent dor- 



92 



HOGGARTH 



sally, ventrally parallel; ventral margin gently 
curved. Malleations and pits more or less uni- 
form over surface of valve; fine sculpture of 
exterior surface rough (Fig. 56E, F). Mem- 
brane covering exterior surface torn in Figure 
56F, intact in Figure 56E. No adductor muscle 
scar evident, but just as in Alasmidonta mar- 
ginata, adductor muscle inserting on interior 
surface of valve, rather than on walls of pits 
(Fig. 56C). Long dorsal alae like those in V. 
villosa. Central ligament 44 to 45 |.im (45 ± 
0.71 цт, n = 2) long, centered about 48% 
from posterior to anterior. Anterior ligament 29 
to 32 |Lim (31 ± 2.1 2 цт, n = 2) in length; pos- 
terior ligament 28 to 29 ¡im (29 ± 0.71 цт, n 
= 2) long. Micropoints pyramidal, arranged in 
broken vertical rows on wide ventral flange 
and on ventral rim of valve, covering proximal 
half of ventral flange and leaving wide un- 
sculptured distal flange margin. 

Remarks 

This glochidium was figured by Lea (1858: 
pi. 5, fig. 4, as Unio rutilans, = V. vibex, fide 
Johnson, 1970; 1874: pi. 21, fig. 7, as Unio 
sudus, = V. vibex, fide Johnson, 1970). The 
1874 figure is closer to my figure of this 
glochidium than the 1858 figure, except that 
the ventral half of the figure is much too round. 
This glochidium is very close to that of V. vil- 
losa, but it can be distinguished by its bluntly 
pyramidal micropoints and its wide unsculp- 
tured distal flange margin. 

Villosa iris iris (Lea, 1 829) 
(Fig.57A-F) 



qua!, dorsally divergent, ventrally parallel. An- 
terior margin may be slightly more rounded 
ventrally than the posterior margin; ventral 
margin gently curved. Dorsal alae long, but 
only about half as long as the dorsal portion of 
lateral margins. Valve malleated and pitted; 
umbo deeply folded (Fig. 57A, C, D). Concen- 
tric ridges extending from umbo ventrally for a 
short distance; loose-looped sculpture cover- 
ing exterior surface of valve (Fig. 57E). Central 
ligament 41 to 42 [xm (42 ± 0.58 цт, n = 3) 
long, centered 49% from posterior to anterior. 
Anterior ligament 38 to 40 ^im (39 ± 1 .00 |am, 
n = 3) long; posterior ligament 33 to 34 цт (33 
± 0.58 цт, n = 3) long. Micropoints lanceolate, 
arranged in broken rows on ventral rim of valve 
and on wide ventral flange, covering proximal 
half of flange, leaving wide unsculptured distal 
flange margin. 

Remarks 

This glochidium can be distinguished from 
all other subspatulate glochidia examined by 
its loose-looped exterior valve sculpture, its 
wide unsculptured distal flange border, and 
smaller dorsal alae. Surber (1912) gave 
length and height measurement of 240 ¡am x 
300 цт for this glochidium, and Ortmann 
(1912) gave 220 цт x 280 |.im. This glochid- 
ium was figured by Lea (1 858: pi. 5, fig. 1 4, as 
Unio novi-eboraci, = V. iris, fide Simpson, 
1900), Ortmann (1911: pi. 89, fig. 20), and 
Surber(1912:pl. 3, fig.46). 

Lampsilis teres teres (Rafinesque, 1820) 
(Fig. 58A-E) 



Material Examined 



Material Examined 



MAH 641.1 - Little Darby Creek at Co. Rt. 
131 bridge (Grewell Rd.), 1.8 mi. E of Plum- 
wood, 7.7 mi. NW of West Jefferson, Monroe 
Twp., Madison Co., Ohio, 11 May 1984, M. A. 
Hoggarth & G. T Watters. OSUM 55828.3 
55828.5 - South Fork Clinch River at Four- 
way, at E edge of Tazewell, at U.S. Rt. 19 
bridge, Tazewell Co., Virginia, 13 October 
1985, D. H. Stansbery. 

Description 

Glochidium subspatulate, length 21 7 to 232 
}im (225 ± 5.84 [im, n = 8), height 289 to 305 
|дт (296 ± 6.64 fim, n = 8). Dorsal margin 
straight, 107to 115|.im (113 ± 2.51 цт, n = 8) 
in length. Anterior and posterior margins sube- 



OSUM 36409 - Tombigbee River 0.5 mi. 
below mouth of Luxaplilla Creek, 3.2 mi. S of 
Columbus, Sec. 32/5, T18S, R18W, Lowndes 
Co., Mississippi, 5 October 1974, R. Grace & 
T Whitfields. OSUM 36531.1 - Whitewater 
River at Bollinger Mill Dam, at Burfordville, 
T31N, R11E, Cape Girardeau Co., Missouri, 
17 October 1974, F. Schilling & H. Kemper. 
OSUM 51669.2 - Mississippi River, R.Mi. 
632.1, E. Shore main channel, 2.9 mi. SW of 
Prairie du Chien, Craford Co., Wisconsin, 10 
June 1 981 , D. J. Heath & M. С Weisensei. 

Description 

Glochidium subspatulate, length 1 89 to 1 94 
|im (191 ± 1.87 цт, n = 9), height 255 to 265 



GLOCHIDIAOF UNIONIDAE 



93 




FIG. 57. Glochidium of Villosa i. iris: A. exterior valve, OSUM 55828.4, bar length = 50 цт; В. interior valve, 
MAH 641 .1 , bar length = 40 цт; С. exterior valve, MAH 641 .1 , bar length = 40 iim; D. umbo, MAH 641 .1 , bar 
length = 15 |.im; E. exterior valve sculpture, MAH 641.1, bar length = 1 ¡.im; F. micropoints, MAH 641.1, bar 
length = 5 цт. 



94 



HOGGARTH 




FIG. 58. Glochidium of Lampsilis t. teres; A. exterior valve, OSUM 51669.2, bar length = 35 |im; B. interior 
valve, OSUM 51669.2, bar length = 40 |am; С hinge, OSUM 36409, bar length = 15 |.im; D. exterior surface 
sculpture, OSUM 51669.2, bar length = 1 цт; E. micropoints, OSUM 51669.2, bar length = 5 |im. 



¡am (258 ± 3.71 |im, n = 9). Dorsal margin 
straight, 100 to 116 |jm (110 ± 3.64 цт, n = 
14) long. Anterior and posterior margins 
equal, dorsally divergent, parallel to slightly 
convergent ventrally. Ventral margin gently 
curved. Exterior valve surface malleated and 
pitted; exterior surface rough (Fig. 58D). Dor- 
sal alae about half as long as divergent por- 
tion of lateral margins. Central ligament 27 to 
39 |дт (34 ± 4.09 цт, n = 6) long, centered 
about 45% from posterior to anterior. Anterior 
ligament 41 to 54 цт (46 ± 5.08 цт, n = 6) 
long; posterior ligament 30 to 32 цт (31 ± 
0.82 )im, n = 6) in length. Micropoints lanceo- 



late (deformed in Fig. 58E), sharply pointed, 
restricted to proximal half of ventral flange 
and ventral rim of valve. This leaves a wide 
unsculptured distal flange margin. 

Remarks 

Surber figured this glochidium (1912: pi. 2, 
fig. 22, as L. fallaciosa. = L. teres, fide John- 
son, 1972) and gave length and height mea- 
surements of 200 цт X 240 ).im. This glochid- 
ium is much smaller than any other recorded 
for Lampsilis. except L. t. anodontoides (see 
below). 



GLOCHIDIAOF UNIONIDAE 



95 




FIG. 59. Glochidium of Lampsilis t. anodontoides: A. exterior valve, OSUM 35612, bar length = 40 ¡.im; B. in- 
terior valve, OSUM 35612, bar length = 35 ¡.im; С hinge, OSUM 41762.2, bar length = 15 ¡am; D. exterior 
valve sculpture, OSUM 41762.2, bar length = 1 |дт; E. micropoints, OSUM 41762.2, bar length = 5 |im. 



Lampsilis teres anodontoides (Lea, 1 831 ) 
(Fig. 59A-E) 

Material Examined 

OSUIVI 35612 - Uphapee Creek at 1-85 
bridge, 3.4 mi. N of Tuskegee, 6.3 mi. S of No- 
tasulga. Sec. 7, T17N, R24E, Macon Co., Al- 
abama, 4 April 1968, J. S. Ramsey et al. 
OSUM 41762.2 - St. Francis River 2.2 mi. N 
of Parkin, 33.0 mi. WNW of Memphis (TN), 



Sec. 21, T8N, R5E, Cross Co., Arkansas, 14 
March 1978, D. H. Stansbery et al. 

Description 

Glochidium subspatulate, length 1 87 to 207 
цт (199 ± 7.19 (im, n = 7), height 249 to 255 
\.im (251 ± 2.87 цт, n = 7). Dorsal margin 
straight, 1 05 to 1 1 8 цт (1 1 1 ± 4.42 цт, n = 9) 
long. Valve outline like that of L. t. teres; lat- 
eral margins dorsally divergent, becoming 



96 



HOGGARTH 



parallel, convergent ventrally; ventral margin 
gently curved. Exterior surface of valve mal- 
leated, especially near umbo; pits numerous. 
As in L. t. teres, dorsal alae about half as long 
as dorsal portion of lateral margins. Exterior 
surface rough under high magnification (Fig. 
59D). Central ligament 36 to 42 цт (38 ± 
2.45 цт, n = 5) long, centered about 45% 
from posterior margin. Anterior ligament 44 to 
50 цт (46 ± 2.88 |.im, n = 5) long; posterioR 
ligament 30 to 33 ).im (31 ± 1.14 цт, n = 5) 
long. Micropoints lanceolate, located on ven- 
tral rim of valve and on wide ventral flange, 
arranged in incomplete vertical rows; un- 
sculptured distal flange margin moderate in 
length. 

Remarks 

This glochidium is identical to that of L. t. 
teres. Surber (1912) gave length and height 
measurements of 185 |.im x 210 цт for this 
glochidium, and Ortmann (1912) gave 200 |.im 
X 260 i-im. Surber's measurements seem too 
small, even for this glochidium, and his figure 
(1912: pi. 2, fig. 21) shows the ventral margin 
as semicircular rather than gently curved. 
These discrepancies suggest that his material 
was not mature. This glochidium was also fig- 
ured by Lea (1858: pi. 5, fig. 2) and Ortmann 
(1912: pi. 20, fig. 9). Lea's figure is much 
closer to Ortmann's and mine than to Surber's 
figure. 

Lampsilis radiata radiata (Gmelin, 1791) 
(Fig. 60A-E) 

Material Examined 

L. r. radiata: MAH 897.1 - Great Herring 
Pond at Carter's Brook Rd. 4.0 mi. NNE of 
Buzzards Bay, 3.0 mi. NW of Sagamore, Ply- 
mouth Co., Massachusetts, 31 July 1985, G. 
T Watters et al. L. г. tuteóla: MAH 321 .6 - Big 
Darby Creek at Scioto-Darby (Mt. Sterling- 
Commercial Pt.) Rd. bridge, 3.4 mi. S of Ori- 
ent, 15.3 mi. SW of Columbus, Scioto/Darby 
Twp., Pickaway Co., Ohio, 7 January 1983, 
M. A. Hoggarth & G. T Watters. MAH 587. 
1 - Big Darby Creek at access 0.9 mi. N of 
Harrisburg, 1.7 mi. NW of Orient, Pleasant 
Twp., Franklin Co., Ohio, 27 September 1 983, 
M. A. Hoggarth. MAH 628.1 - Big Darby 
Creek at Scioto-Darby (Mt. Sterling-Commer- 
cial Pt.) Rd. bridge, 3.4 mi. S of Orient, 15.3 
mi. SW of Columbus, Scioto/Darby Twp., 



Pickaway Co., Ohio, 13 April 1984, M.A. Hog- 
garth & G. T Watters. MAH 652 - Little Darby 
Creek at Little Darby Rd access, 2.7 mi. SE of 
Plumwood, 6.4 mi. NW of West Jefferson, 
Monroe Twp., Madison Co., Ohio, 17 May 
1984, M. A. Hoggarth & G. T Watters. MAH 
721 .2 - Fish Creek at St. Rt. 49 bridge, 0.4 mi. 
above its mouth, 1.1 mi. N of Edgerton, 10.4 
mi. W of Bryan, St. Joseph Twp., Williams 
Co., Ohio, 1 October 1985, M. A. Hoggarth & 
D. Rice. 

Description 

Glochidium subspatulate, length 250 to 260 
|im (255 ± 4.43 цт, n = 4), height 295 to 311 
}.im (303 ± 6.58 |im, n = 4). Dorsal margin 
straight, 121 to 129 ¡.im (125 ± 3.78 ¡jm, n = 
4) in length. Lateral margins dorsally diver- 
gent, ventrally parallel, ventral margin gently 
curved. Dorsal alae long; fine structure of ex- 
terior surface of valve rough (Fig. 60D). Exte- 
rior surface finely malleated. Valve at umbo 
deeply folded. Central ligament 45 to 47 цт 
(46 ± 1.00 |Lim, n = 3) long, centered about 
42% from posterior to anterior. Anterior liga- 
ment 46 to 50 цт (48 ± 2.08 цт, n = 3) long; 
posterior ligament 27 to 32 цт (29 ± 2.52 цт, 
n = 3) long. Micropoints lanceolate, located on 
rim of ventral margin and on wide ventral 
flange, arranged in broken vertical rows and 
covering proximal half of ventral flange. Un- 
sculptured distal flange margin wide. 

Morphometries from the subspecies L. r. lu- 
teola are: length, 227 to 235 цт (231 ± 2.39 
цт, n = 8); height, 280 to 295 цт (285 ± 6.63 
|.im,n = 8); hinge length 1 07 to 123 (.im (115 ± 
5.23 |im, n = 12); anterior ligament length 37 
to 42 |дт (39 ± 1.94 цт, n = 6); central liga- 
ment length 40 to 46 [im (42 ± 2.58 цт, n = 
6); posterior ligament length 32 to 40 цт (36 
± 3.39 |.im, n = 6). 

Remarks 

Surber (1912) and Ortmann (1912) gave 
length and height measurements for the 
glochidium of L. r. luteola: Ortmann's figures 
are 230 цт x 280 ,um (almost identical to 
mine), and Surber gave 250 ¡.im x 290 цт (al- 
most the same as my measurements for L. r. 
radiata). This species is as far ranging as 
Pyganodon grandis and appears to have just 
as wide a range in glochidial size. The 
glochidium of L. r. radiata was figured by Lea 
(1858: pi. 5, fig. 20), Wiles (1975: fig. 5), and 



GLOCHIDIAOF UNIONIDAE 



97 




FIG. 60. Glochidium of Lampsilis r. radiata, MAH 897.1 ; A. exterior valve, bar length = 45 ¡am; B. interior valve, 
bar length = 45 |am; С hinge, bar length = 20 цт; D. exterior valve sculpture, bar length = 1 цт; E. micro- 
points, bar length = 10 цт. 



Calloway & Turner (1 979: pi. 3, figs. 2, 4, 6, 9). 
The glochidium of L. r. luteola was figured by 
Lea (1958; pi. 5, fig. 10), Surber (1912: pi. 2, 
fig. 1 5), and Arey (1 921 : pi. 2, fig. 3, 4; 1 924: 
pl. 1,fig. 1). 

Lampsilis abrupta (Say, 1 831 ) 
(Fig. 61A-F) 

Material Examined 

OSUM 1 3303 - Gasconade River 3 mi. N of 
Mount Sterling, Gasconade Co., Missouri, 17 



September 1964, D. H. Stansbery & J. J. 
Jenkinson. OSUM 38841 - Kanawha River 
immediately below Kanawha Falls, 0.3 mi. 
SSE of Glen Ferris, 1.4 mi. SW of Gauley 
Bridge, Falls Twp., Fayette Co., West Virginia, 
27 November 1976, K. G. Borror et al. 

Description 

Glochidium subspatulate, length 207 to 214 
jim (210 ± 2.66 |im, n = 6), height 251 to 259 
¡am (254 ± 3.13 |.im, n = 6). Dorsal margin 
straight, 96 to 112 цт (102 ± 5.62 цт, n - 7) 



98 



HOGGARTH 




FIG. 61. Glochidium of Lampsilis abrupta: A. exterior valve, OSUM 13303. bar length = 40 цт: В. interior 
valve, OSUM 38841, bar length = 40 цт: С. micropoints, OSUM 13303. bar length = 2 цт: D. micropoints, 
озим 38841, bar length = 2 |.im; E. exterior valve sculpture. OSUM 13303. bar length = 1 цт: F. hinge, 
OSUM 13303, bar length = 15 |.im. 



in length. Exterior surface finely malleated, 
wrinkled near umbo. Fine structure of exterior 
surface of valve rough (Fig. 61 E). Pits few in 
number, more or less evenly distributed 
throughout valve. Dorsal alae long, extending 
about one fifth height of valve. Hinge of a sin- 



gle specimen with the following ligament 
lengths: anterior ligament, 46 ¡.im: central lig- 
ament, 38 j.im; posterior ligament, 27 ¡.im. 
Central ligament centered about 44% from 
posterior to anterior. Micropoints lanceolate, 
arranged in broken vertical rows on wide ven- 



GLOCHIDIAOF UNIONIDAE 



99 



tral flange and on rim of ventral margin, cov- 
ering proximal half of ventral flange, leaving 
wide unsculptured distal flange margin. 

Remarks 

Lea (1863) stated that this glochidium is, 
"almost exactly the same with multiradlatus" 
[= Lampsilis fasciola. fide Ortmann & Walker, 
1922]; however, the glochidium of L. abrupta 
is much smaller than that of L. fasciola. Ort- 
mann (1912) gave length and height mea- 
surements of 1 90 (.im X 21 i-im and 200 цт x 
250 i-im for the glochidium of L. orbiculata {= 
L. abrupta, fide Stansbery et al., 1985). My 
figures are nearer his second set of measure- 
ments, and the glochidia were all about the 
same size. There is often differential matura- 
tion of glochidia and Ortmann's smaller indi- 
viduals may have been immature. This glo- 
chidium was figured by Ortmann (1 91 1 : pi. 89, 
fig. 22, as L. orbiculata). 

Lampsilis higginsi {Lea, 1857) 
(Fig. 62A-F) 

Material Examined 

OSUM 49024.1 Mississippi River, R.Mi. 
633.3, E channel, N end of Indian Isle, 2.1 mi. 
SW of Prairie du Chien, Sec. 1/2, T6N, R7W, 
Craford Co., Wisconsin, 11 November 1980, 
A. Reed, donor: M. E. Havlik. 



Description 

Glochidium subspatulate, length 21 4 to 21 7 
lam (216 ± 2.12 |im, n = 2), height 254 to 257 
|дт (256 ± 2.12 цт, n = 2). Dorsal margin 
straight, 1 08 to 1 1 8 ¡jm (1 1 1 ± 4.72 цт, n = 4) 
in length. Anterior and posterior margins dor- 
sally divergent, subparallel ventrally. Ventral 
margin gently curved. Surface of valve finely 
malleated, with concentric ridges near umbo. 
Dorsal alae strongly curved, extending about 
half distance of dorsally divergent portions of 
lateral margins. Fine structure of exterior sur- 
face rough (Fig. 62E). Central ligament 39 to 
47 |am (43 ± 5.66 |im, n = 2) long, centered 
about 47% from posterior to anterior. Anterior 
ligament 38 to 40 цт (39 ± 1 .41 цт, n = 2) in 
length; posterior ligament about 31 \.im long. 
Micropoints lanceolate, arranged in incom- 
plete rows on ventral flange and on rim of ven- 
tral margin. Micropoints decreasing in size dis- 
tally, covering about three quarters of flange 



surface. Unsculptured distal flange margin 
moderate in width. 



Remarks 

Waller et al. (1988) gave almost identical 
morphometric data for this glochidium. They 
also found that the glochidium of L. recta had 
similar morphometries. They suggest, how- 
ever, that the glochidia are also the same 
shape and can be distinguished only by the ex- 
tent of the development of dorsal alae and 
placement of the central hinge ligament. Their 
conclusion regarding shape was based on 
their inability to distinguish any differences 
in outline upon overlaying light microscopy 
transparencies. I found that the glochidium of 
L. recta can best be described as subelliptical, 
with a rounded ventral border and equally 
curved lateral margins (similar to that of A. 
pectorosa), whereas the glochidium of L. hig- 
ginsi is subspatulate, with a gently curved ven- 
tral margin and lateral margins that are dor- 
sally divergent and ventrally parallel. The 
anterior margin of L. higginsi is slightly more 
rounded than the posterior margin, and the 
margins are clearly not equal. These addi- 
tional differences help distinguish these glo- 
chidia. This glochidium was figured by Waller 
et al. (1 988: figs. 2, 4), who gave length, height 
and hinge length measurements of 215 jam x 
259|.imx 110 (.im. 

Lampsilis ovata (Say, 1817) 
(Fig. 63A-F) 

Material Examined 

L. ovata: OSUM 43164.1 - Clinch River 
above mouth of Copper Creek, 1.3 mi. S of 
Clinchport, 9.3 mi. W of Gate City, Scoot Co., 
Virginia, 21 October 1978, D. H. Stansbery et 
al. L. ornata: OSUM 54661 -Amite River 1 mi. 
NNW of Denham Springs, about 13 mi. WNW 
of Livingston, T6S, R3E, Ward 2, Livingston 
Parish, Louisiana, 1 October 1967, E. N. Lam- 
bremont et al. L. satura: OSUM 38973.51, 
38973.52 - West Fork San Jacinto River 
below 1-45 bridge, 4.5 mi. S of Conroe, 36 mi. 
N of Houston, Montgomery Co., Texas, 20 
July 1968, D. H. Stansbery et al. 

Description 

Glochidium broadly subspatulate, length 
about 232 (.im, height 271 to 276 цт (274 ± 



100 



HOGGARTH 




FIG. 62. Glochidium of Lampsilis higginsi, OSUM 49024.1 ; A. exterior valve, bar length = 35 ит; B^interior 
valve, bar length = 35 цт; С. lateral valve view, bar length = 35 um; D. micropoints, bar length = 2 цт, t. ex- 
terior valve sculpture, bar length = 1 цт; F. hinge, bar length = 1 5 (.im. 



GLOCHIDIAOF UNIONIDAE 



101 




FIG. 63. Glochidium of Lampsilis ovata. OSUM 43164,1; A. exterior valve, bar length = 40 цт; В. interior 
valve, bar length = 40 цт; С. micropoints, bar length = 5 |im; D. exterior valve sculpture, bar length = 1 ¡ivn; 
E. hinge, bar length = 20 |.im; F. hinge, bar length = 20 (.im. 



3.54 |.im, n = 2). Dorsal margin straight, 11 3 to 
119 lam (116 ± 2.58 )im, n = 4) in length. Dor- 
sal portions of lateral margins strongly diver- 
gent; ventral portions of these margins sub- 
parallel; anterior margin slightly curved. 
Dorsal alae extending about three quarters 
length of dorsal divergent portion of lateral 



margins, ventral margin gently curved. Fine 
structure of exterior surface of valve rough 
(Fig. 63E). Central ligament 43 to 44 цт (44 
± 0.71 |.im, n = 2) long, centered about 49% 
from posterior to anterior. Anterior ligament 39 
to 40 |дт (40 ± 0.71 цт, n = 2) long; posterior 
ligament 34 to 35 |im (35 ± 0.71 |im, n = 2) in 



102 



HOGGARTH 



length. Micropoints bluntly pyramidal to lance- 
olate, arranged in broken vertical rows on 
ventral flange and on rim of ventral margin, 
covering tfiree quarters of ventral flange, leav- 
ing moderately wide unsculptured distal 
flange margin. 

Remarks 

The glochidium of L. ovata is distinguished 
from those previously described by its broadly 
subspatulate shape. However, this shape is 
also found in the glochidia of the other mem- 
bers of the L. ovata complex (i.e., L. ventri- 
cosa, L. satura and L. ornata). Relative size 
may be the only way to distinguish these 
species. Morphometric data for Lampsilis 
satura is: length, 220 to 223 (.im (222 ±2.12 
|im, n = 2); height, 268 to 269 цт (269 ± 0.71 
|im, n = 2); hinge length, 113 to 117 цт (115 
± 1 .82 |im, n = 4); anterior ligament length, 42 
to 43 |im (43 ± 0.71 цт, n = 2); central liga- 
ment length, 40 ¡am; and posterior ligament 
length, 33 to 35 цт (34 ± 1 .41 |im, n = 2). The 
position of the central ligament is about 48% 
from posterior to anterior. Morphometric data 
for Lampsilis ornata: length, 198 to 204 |.im 
(202 ± 3.21 lam, n - 3); height, 257 to 260 |.im 
(258 ± 1.53 )im, n = 3); hinge length, 96 to 
100 |im (98 ± 2.08 jam, n = 3). A single spec- 
imen gave the following ligament lengths: an- 
terior ligament, 38 цт; central ligament, 33 
|im; posterior ligament, 29 ¡am. The midpoint 
of the central ligament is about 46% from pos- 
terior to anterior. The glochidium of L. ovata 
was figured by Lea (1858: p1.5, fig. 15) and 
Isom (1983: fig. 1c). 

Lampsilis ventricosa (Barnes, 1823) 
(Fig. 64A-G) 

Material Examined 

OSUM 44619 - Green River below Lock 5 
dam at Glenmore, 1 2 mi. N of Bowling Green, 
Warren Co., Kentucky, 21 October 1979, D. 
H. Stansbery et al. МАИ 846.1 - Fish Creek at 
bridge 2.0 mi. NW of Edgerton, 1 1 .9 mi. W of 
Bryan, Sec. 20, T6N, RÍE, St. Joseph Twp., 
Williams Co., Ohio, 29 October 1985, D. H. 
Stansbery et. al. MAH 954.1, 954.2, 954.3, 
954.4, 954.5 & 954.6-Sugar River 300 m 
downstream of Lake Belle View Dam, 150 m 
upstream of St. Rt. 69 bridge, at Belleville, 
Sec. 34, T5N, R8E, Dane Co., Wisconsin, 15 
May 1986, M. A. Hoggarth & D. J. Heath. 



Description 

Glochidium broadly subspatulate, length of 
240 to 255 цт (249 ± 4.46 цт, n = 1 8), height 
274 to 293 [xm (283 ± 6.30 |.im, n - 18). Dor- 
sal margin straight, 1 04 to 1 1 8 ¡am (1 1 1 ± 4.06 
).im, n = 21) in length. Shape very much like 
that of L. ovata. Straight dorsal portion of lat- 
eral margin strongly divergent; ventral portion 
of lateral margin almost parallel; ventral mar- 
gin gently curved. Dorsal alae long, strongly 
arched (Figs. 64A, C). Fine structure of exte- 
rior valve sculpture rough (Fig. 64F). Central 
ligament 37 to 42 цт (39 ± 1.98 цт, n = 8) 
in length, centered about 47% from posterior 
to anterior. One female produced glochidia 
with a more posterior central ligament (43%) 
whereas another produced glochidia with a 
more anterior central ligament (50%). Anterior 
ligament 38 to 50 цт (44 ± 4.82 цт, n = 8) 
long; posterior ligament 29 to 33 \лт (32 ± 
1.41 \im, n = 8) in length. Micropoints lanceo- 
late to bluntly pyramidal, arranged in broken 
vertical rows on wide ventral flange and on 
rim of ventral margin, covering about 75% of 
flange and leaving moderately wide unsculp- 
tured distal flange margin. 

Remarks 

The glochidium of L. ventricosa is longer 
and higher than that of L. ovata, but both 
species have about the same hinge length. 
This glochidium was figured by Lea (1858: 
pi .5, fig. 1 3, as Unio occidens, = L. ventricosa, 
f/de Johnson, 1970), Ortmann (1911: p1.89, 
fig. 23), Surber (1912: p1. 2, fig. 24), and 
Waller et al. (1988: figs. 5, 6). Surber gave 
length and height measurements of 200 ¡am x 
250 цт for this glochidium; Ortmann (1912) 
gave 250 |лт x 290 цт; and Waller et al. gave 
21 6 í-im X 257 [im. My measurements are 249 
|jm X 283 цт. Waller et al. and Surber col- 
lected their material from the Mississippi River, 
whereas Ortmann's and my material came 
from smaller streams. 

Lampsilis reeviana brevicula (Call, 1 887) 
(Fig. 65A-E) 

Material Examined 

OSUM 45363.20, 45363.22, 45363.35 & 
45363.50 - Current River "between Mo. Rt. 
106 and Van Buren," about 9 mi. of stream. 
Shannon/Carter Co., Missouri, 24 October 
1981, R. D. Oesch. 



GLOCHIDIAOF UNIONIDAE 



103 




FIG. 64. Glochidium of Lampsilis ventricosa; A. exterior valve, MAH 954.6, bar length = 40 цт: В. interior 
valve, MAH 954.3, bar length = 40 цт; С. lateral view, OSUM 44619. bar length = 40 цт; D. rnicropoints, 
MAH 954.3, bar length = 2 ¡.im; E. micropoints, MAH 954.4, bar length = 2 |im; F. exterior valve sculpture, 
MAH 954.1, bar length = 1 цт; G. hinge, MAH 954.6, bar length = 15 цт. 



104 



HOGGARTH 




FIG. 65. Glochidium of Lampsilis breviculata: i\. exterior valve, OSUM 45363.35, bar length = 40 цт; В. inte- 
rior valve, OSUM 45363.20, bar length = 40 |jm; С hinge, OSUM 45363.50, bar length = 15 цт; D. exterior 
valve sculpture. OSUM 45363.50, bar length = 1 цш; E. micropoints, OSUM 45363.35, bar length = 5 цт. 



Description 

Glochidium broadly subspatulate length 
230 to 245 цт (235 ± 6.05 )im, n = 8), height 
286 to 297 |jm (290 ± 4.37 |im, n - 8). Dorsal 
margin straight, 113 to 127 цт (119 ± 4.50 
|am, n = 9) long. Lateral margins straight, 
strongly divergent dorsally, subparallel ven- 
trally. Anterior margin slightly more curved 
ventrally than the posterior margin; ventral 
margin gently curved throughout its length. 
Dorsal alae about as long as the divergent 
dorsal portion of lateral margins. Fine struc- 
ture of the exterior surface rough (Fig. 65D). 
Central ligament 37 to 48 |im (43 ± 4.39 |im, 



n = 5) in length, centered about 47% from 
posterior to anterior. Anterior ligament 38 to 
50 цт (43 ± 5.45 |.im, n = 5) long; posterior 
ligament 32 to 36 цт (34 ± 2.1 9 цт, n = 5) in 
length. Micropoints lanceolate, arranged in 
broken vertical rows on ventral rim of valve 
and on a wide ventral flange, covering proxi- 
mal half of ventral flange, leaving a wide un- 
sculptured distal flange margin. 

Remarks 

This glochidium was figured by Surber 
(1915: pi. 1, fig. 14), who gave length and 
height measurements of 230 |im x 290 цт. 



GLOCHIDIAOF UNIONIDAE 



105 



Lampsilis crocata (Lea, 1841 ) 
(Fig. 66A-F) 

Material Examined 

OSUM 54485.1, 54485.2 - Tar River at 
U.S. Rt. 64 bridge, 2.4 mi. SW of Spring Hope, 
10.8 mi. mi. WSW of Nashville, Nash Co., 
Tennessee, 1973, M. Imlay. OSUM 42060.1 - 
Waccamaw River just below Lake Waccamaw 
Dam, 6.2 mi. SE of Hallsboro, 11 .7 mi. ESE of 
Whiteville, Columbus Co., North Carolina, 21 
July 1978, D. H. Stansbery et al. 

Description 

Glochidium narrowly subspatulate, length 
235 to 249 |.im (242 ± 6.23 (im, n - 8), height 
287 to 303 [im (293 ± 5.03 цт, n = 8). Dorsal 
margin straight, 110 to 125 цт (118 ± 4.81 
|im, n = 8) in length. Lateral margins weakly di- 
vergent dorsally, ventrally subparallel. Ventral 
margin evenly and broadly curved; dorsal alae 
long, strongly arched (Fig. 66A, C) Fine struc- 
ture of exterior surface of valve rough (Fig. 
66E). Central ligament 52 to 56 |jm (54 ± 2.83 
)im, n = 2) long, centered about 46% from pos- 
terior to anterior. Anterior ligament 36 to 39 цт 
(38 ± 2.12 |im, n = 2) long; posterior ligament 
about 32 |im long. Micropoints bluntly lanceo- 
late, covering about 75% of ventral flange, 
arranged in broken vertical rows on ventral 
flange and on rim of ventral margin, remaining 
about equal in length from proximal to distal. 
Unsculptured distal flange margin narrow. 

Remarks 

The shape of this glochidium is like that of 
L. teres, but it is much larger. No published 
figure of this glochidium was found. 

Lampsilis cariosa (Say, 1817) 
(Fig. 67A-D) 

Material Examined 

OSUM 54500 - Tar River at U.S. Rt. 15 
bridge, 2.7 mi. SW of Clay, 6.7 mi. SSW of Ox- 
ford, Granville Co., North Carolina, 1973, M. 
Imlay. 

Description 

Glochidium narrowly subspatulate, length 
about 241 |nm, height about 314 ,um, hinge 
length about 109 |am. Dorsal margin straight; 



ventral margin evenly and broadly curved; lat- 
eral margins about equally divergent dorsally 
and ventrally parallel. Dorsal alae long. Fine 
structure of exterior surface of valve rough 
(Fig. 67C). Valve pitted, except along mar- 
gins; pitting reduced in adductor muscle scar 
(Fig. 67B). Umbo deeply folded and the cen- 
ter of numerous concentric ridges. Hinge liga- 
ments not seen. Micropoints lanceolate, re- 
stricted to proximal half of ventral flange and 
on rim of ventral valve margin, arranged in 
broken vertical rows. Unsculptured distal 
edge of ventral flange wide. 

Remarks 

It is clear that this glochidium is very differ- 
ent from that of Leptodea ochracea. The long 
dorsal alae, lanceolate micropoints, and rough 
exterior valve sculpture place the glochidium 
of L. cariosa firmly in Lampsilis, whereas the 
small dorsal alae, lamellate micropoints, 
loose-looped exterior sculpture, and lateral 
valve gape separate the glochidium of Lep- 
todea ochracea from Lampsilis. No published 
figure of this glochidium was found. 

Lampsilis fasciola Rafinesque, 1820 
(Fig. 68A-F) 

Material Examined 

OSUM 25467 - Kanawha River immedi- 
ately below Kanawha Falls, 1.2 mi. below 
Gauley Bridge, Falls Twp., Fayette Co., West 
Virginia, 1 October 1970, D. H. Stansbery & 
W. J. Clench. MAH 562.3 - Big Darby Creek 
at and above Scioto-Darby (Mt. Sterling-Com- 
mercial Pt.) Rd. bridge, 3.4 mi. S of Orient, 
1 5.3 mi. SW of Columbus, Scioto/Darby Twp., 
Pickaway Co., Ohio, 19 August 1983, M. A. 
Hoggarth. OSUM 55977 - Olentangy River 
just above Powell Rd. bridge, 1.6 mi. E of 
Powell, 9.8 mi. S of Delaware, T3N, R19W, 
Delaware Co., Ohio, 29 September 1983, С 
В. Stein et al. OSUM 55033.2 - Clinch River, 
R.Mi. 269.7, 1 .0 mi. above Carbo, 1 .8 mi. W of 
Cleveland, 6.2 mi. NW of Lebanon, Castle- 
wood Twp., Russell Co., Virginia, 26 July 
1985, D. H. Stansbery & С В. Stein. 



Description 

Glochidium narrowly subspatulate, length 
240 to 250 цт (247 ± 4.47 ¡am, n = 5) height 
287 to 295 \хг(\ (290 ± 3.08 цт, n = 5). Dorsal 



106 



HOGGARTH 




FIG. 66. Glochidium of Lampsilis crocata;fK. exterior valve. OSUM 42060.1 , bar length = 40 цт; В. interior 
valve, OSUM 42060.1, bar length = 50 цт; С. lateral view. OSUM 54485.1, bar length = 50 |.im; D. micro- 
points, OSUM 54485.1 , bar length = 2 pm; E. exterior valve sculpture, OSUM 54485.1 , bar length = 1 цгл; F. 
micropoints, OSUM 42060.1, bar length = 5 pm. 



\ 



GLOCHIDIA OF UNIONIDAE 



107 




FIG. 67. Glochidium of Lampsilis cariosa, OSUM 54500; A. exterior valve, bar length = 45 цт; В. interior 
valve pitting and adductor muscle scar, bar length = 15 |дт; С. exterior valve sculpture, bar length = 1 |im; 
D. micropoints, bar length = 5 |.im. 



margin straight, 107 to 115 цт (110 ± 2.95 
цт, П = 5) in length. Posterior margin strongly 
divergent from dorsal margin for about half its 
length. Near its midpoint, posterior margin 
bending ventrally, straightening out to run 
more or less perpendicular to hinge. Anterior 
margin diverging from dorsal margin at a 
lesser angle, straight for about three quarters 
of its length, curving continuously to ventral 
margin. Ventral margin gently and evenly 
curved. Dorsal alae long, with a strong arch 
(Fig. 68A, C). Fine structure of exterior sur- 
face of the valve rough (Fig. 68E). Central 
ligament about 44 цт long, centered about 



48% from posterior margin. Anterior ligament 
about 38 цт long; posterior ligament about 33 
цт long. Micropoints lanceolate, arranged in 
broken vertical rows on ventral rim of valve 
and on a wide ventral flange. Micropoints de- 
creasing in size distally on flange covering 
about three quarters of its proximal surface, 
leaving narrow unsculptured distal flange 
margin. 

Remarks 

The glochidium of L. fasciola was figured by 
Lea (1858; pi. 5, fig. 17, as Unio multiradia- 



108 



HOGGARTH 





^_ ' ^^.^1 




/ 


\ 




' 


% ^ 


A 




FIG. 68. Glochidium of Lampsilis fasciola; A. exterior valve, OSUM 55033.2, bar length = 45 цт: В. interior 
valve, OSUM 55033.2, bar length = 45 цт; С. lateral view, OSUM 25467, bar length = 40 цт; D. micropoints, 
OSUM 55033.2, bar length = 5 |.im; E. extehor valve sculpture, OSUM 55033.2, bar length = 1 цт; F. hinge, 
OSUM 55033.2, bar length = 15 цт. 



tus, = L. fasciola, fide Ortmann & Walker, 
1922) and Surber (1915: p1. 1, fig. 2, as L. 
multiradiatus). This glochidium is like other 
narrowly subspatulate glochidia, except that 
the anterior margin is slightly more rounded. 
In this regard, this glochidium resembles that 



of Obovaria, but it will not be confused with 
Obovaria because of it posterior margin, wide 
ventral flange, and rough exterior valve sculp- 
ture. The glochidium of Obovaria has a curved 
posterior margin, narrow ventral flange, and 
loose-looped exterior valve sculpture. 



GLOCHIDIAOF UNIONIDAE 



109 




FIG. 69. Glochidium of Epioblasma triquetra, MAH 588.1; A. exterior valve, bar length = 35 |jm; B. interior 
valve, bar length = 40 цт; С. interior valve, bar length = 40 цт; D. supernumerary hook and micropoints, bar 
length = 2 |дт; E. micropoints, bar length = 5 цт. 



Epioblasma triquetra (Rafinesque, 1820) 
(Fig. 69A-E) 

Material Examined 

IVIAH 588.1 - Big Darby Creek at access 
0.9 mi. N of Harrisburg, 1 .7 mi. NW of Orient, 
Pleasant Twp., Franklin Co., Ohiio, 27 Sep- 
tember 1983, M. A. Hoggarth. MAH 631.1 - 
Big Darby Creek at Scioto-Darby (Mt. Ster- 
ling-Commercial Pt.) Rd. bridge, 3.4 mi. S of 
Orient, 15.3 mi. SW of Columbus, Scioto/ 



Darby Twp., Pickaway Co., Ofiio, 13 April 
1984, M. A. Hoggarth & G. T. Watters. 

Description 

Glochidium depressed subelliptical, length 
208 to 217 |im (214 ± 3.54 }лт, n = 5), height 
205 to 21 4 цт (21 1 ± 4.09 цт, n = 5). Dorsal 
margin straight, 149 to 156 \im (152 ± 3.05 
цт, n = 5) in length. Lateral margins equally 
and gently curved; ventral margin broadly 
curved. Exterior surface sparsely malleated, 



110 



HOGGARTH 



pits few. Exterior valve sculpture loose- 
looped. Dorsal alae absent (Fig. 69A). Central 
ligament 48 to 53 |.im (50 ± 2.89 |.im, n = 3) in 
length, centered about 44% from posterior to 
anterior. Anterior ligament 56 to 65 ¡.im (60 ± 
4.51 |im, n = 3) long; posterior ligament 39 to 
48 |.im (43 ± 4.73 |.im, n =; 3) in length. Micro- 
points blunt, irregular in shape. Ventral flange 
narrow; distal unsculptured flange edge wide. 
Supernumerary hooks (Fig. 69D) about 2 ¡.im 
in length, on unsculptured portion of flange. 

Remarks 

This glochidium was figured by Lea (1858: 
pi. 5, fig. 19, as Unio triangularis, = E. trique- 
tra. fide Ortmann & Walker, 1922) and Ort- 
mann (1911: pi. 89, fig. 24). Ortmann (1919) 
note that Lea's figure was incorrect and gave 
length and height measurements of 210 |.im x 
210 \xn\ (Ortmann, 1912). Lea's figure does 
not show the morphological depression of the 
valve. 



Epioblasma brevidens (Lea, 1 831 ) 
(Fig. 70A-F) 

Material Examined 

OSUM 16173 - Cedar Creek about 1 mi. 
SE of Mingo at bridge, Tishomingo Co., Mis- 
sissippi, 5 November 1965, R Yokley & B. G. 
Isom. 



Description 

Glochidium depressed subelliptical, length 
213to220|.im (216 ± 3.11 |im, n = 4), height 
205 to 21 4 (.im (21 ± 4.24 цт, n = 4). Dorsal 
margin straight, 141 to 153 |im (147 ± 5.06 
\im, n = 4) long. Lateral margins equally and 
evenly curved; ventral margin broadly curved. 
Exterior surface sparsely malleated, pits few. 
Valve pitting reduced in adductor muscle scar; 
however, irregular ridges, probably to in- 
crease surface area for attachment of large 
adductor muscle, found within muscle scar 
(Fig. 70B, D). Fine structure of exterior sur- 
face of valve rough (Fig. 70E), Dorsal alae ab- 
sent. Central ligament 48 to 49 yim (49 ± 0.71 
|im, n = 2) in length, centered about 46% from 
posterior margin. Anterior ligament 49 to 52 
цт (51 ± 2.12 )Lim, n = 2) in length; posterior 
ligament 41 to 47 цт (44 ± 4.24 |.im, n = 2) 
long. Micropoints small, triangular, arranged 
in horizontal rows on ventral flange and on 



ventral rim of valve. Supernumerary hooks tri- 
angular (Fig. 70B) to lanceolate (Fig. 70C). 
Unsculptured distal margin of ventral flange 
narrow. 

Remarks 

This glochidium is similar to that of E. tri- 
quetra. They are of about the same shape and 
size and have similar micropoints and central 
ligament positions. However, the glochidium 
of E. brevidens has rough exterior valve 
sculpture, whereas that of E. triquetra has 
loose-looped exterior valve sculpture. 

Epioblasma capsaeformis (Lea, 1834) 
(Fig. 71A-G) 

Material Examined 

OSUM 42007 - Clinch River at Clinchport, 
above swinging bridge, 2.3 mi. N of Speer's 
Ferry, Scoot Co., Virginia, 2 July 1978, С R. 
Ciola & K. L. Ciola. 

Description 

Glochidium depressed subelliptical, length 
240 to 252 цт (246 ± 3.88 цт, n = 8), height 
226 to 238 цт (234 ± 4.34 цт, n = 8). Dorsal 
margin straight, 154 to 173 цт (162 ± 6.33 
jim, n = 8) long. Lateral margins about equal 
and gently curved (Fig. 71 A, B) to more 
broadly curved (Fig. 71 E). Ventral margin 
broadly curved. Dorsal alae absent. Loose- 
looped sculpture covering valve exterior (Fig. 
71 F). Adductor muscle scar large, with only a 
few pits and with numerous small ridges (Fig. 
71 B, E). Central ligament 51 to 60 цт (57 ± 
3.43 jim, n = 6) in length, centered about 46% 
from posterior to anterior. Anterior ligament 52 
to 64 j.im (59 ± 4.45 |дт, n = 6) long; posterior 
ligament 40 to 57 цт (47 ± 5.89 |im, n = 6) in 
length. Micropoints blunt, irregular (Fig. 71 C), 
arranged in broken horizontal rows on a nar- 
row ventral flange. Near proximal border, mi- 
cropoints coalescing to form broken ridges. 
Distal unsculptured margin narrow. Attenuate 
supernumerary hooks present on otherwise 
unsculptured distal flange margin (Fig. 71 D). 

Remarks 

This glochidium is slightly larger than that of 
E triquetra or E brevidens. but it is otherwise 
very similar. No published figure of this glo- 
chidium was found. 



1 



GLOCHIDIAOF UNIONIDAE 



111 




FIG. 70. Glochidium of Epioblasma brevidens. OSUM 16173; A. exterior valve, bar length = 40 цт; В. inte- 
rior valve, bar length = 40 цт; С. supernumerary hook and micropoints, bar length = 5 |im: D. adductor mus- 
cle scar, bar length = 20 (.im: E. exterior valve sculpture, bar length = 1 ¡.im: F. hinge, bar length = 25 |.im. 



Epioblasma rangiana (Lea, 1839) 
(Fig. 72A-F) 

Material Examined 

MAH 632.1 - Big Darby Creek at Scioto- 



Darby (Mt. Sterling-Commercial Pt.) Rd. 
bridge, 3.4 mi. S of Orient, 15.3 mi. SW of 
Columbus, Scioto/Darby Twp., Pickaway Co., 
Ohio, 13 April 1983, M. A. Hoggarth & G. T. 
Waiters. MAH 701 - Big Darby Creek at 
Scioto-Darby (Mt. Sterling-Commercial Pt.) 



112 



HOGGARTH 




FIG. 71 . Glochidium of Epioblasma capsaeformls. OSUM 42007; A. exterior valve, bar length = 35 цт; В. in- 
terior valve, bar length = 35 цт: С. micropoints, bar length = 5 цт: D. зирегпиглегагу hook, bar length = 
5 цт; E. interior valve, bar length = 35 цт; F. exterior valve sculpture, bar length = 1 ).im; G. hinge, bar length 
= 25цт. 



Rd. bridge, 3.4 mi. S of Orient, 1 5.3 mi. SW of 
Columbus, Scioto/Darby Twp., Pickaway Co., 
Ohio, 6 September 1985, M. A. Hoggartli. 

Description 

Glochidium depressed subelliptical, length 
238 to 258 |im (249 ± 7.34 \xm, n = 8), height 
21 to 238 um (224 ± 9.34 ¡jm, n = 8). Dorsal 
margin straight, 160 to 188 |дт (170 ± 6.30 
цт, n = 8) in length. Lateral margins and ven- 
tral margin are about equally rounded. Shape 
of this glochidium approaching that of Obli- 



quaria reflexa (subrotund). Dorsal alae ab- 
sent. Surface sculpturing loose-looped (Fig. 
72F). Central ligament 53 to 59 ¡.im (57 ± 3.21 
|.im, n = 3) long, centered about 46% from 
posterior to anterior. Anterior ligament 60 to 
77 |лт (68 ± 8.62 ¡.tm, n = 3) long; posterior 
ligament 50 to 55 |.im (52 ± 5.21 (.im, n = 3) in 
length. Micropoints blunt, located on a narrow 
ventral flange and on ventral rim of valve (Fig. 
72E), covering about 50% of flange, leaving a 
wide unsculptured distal flange margin. Su- 
pernumerary hooks present as triangular ex- 
tensions of this unsculptured area of flange. 



GLOCHIDIAOF UNIONIDAE 



113 




FIG. 72. Glochidium of Epioblasma torulosa rangiana;A. exterior valve, MAH 632.1 , bar length = 40 цт; В. 
interior valve, MAH 632.1 , bar length = 35 |.im; С interior valve, MAH 632.1 , bar length = 35 цт; D. hinge, 
MAH 701 , bar length = 25 цт; E. micropoints and supernumerary hooks, MAH 701 , bar length = 5 ¡im; F. ex- 
terior valve sculpture, MAH 632.1 , bar length = 1 |im. 



Remarks 

This glochidium can be distinguished by its 
extreme morphological depression. It was fig- 
ured by Lea (1858: pi. 5, fig. 21, as Unio per- 
plexus, = E. rangiana, fide Ortmann & Walker, 
1922), who provided length and height mea- 
surements of 224 |im X 240 |im. Lea's figure 
is correctly drawn, so it must be assumed that 
his valve length equals my valve height and 
that his valve height equals my valve length. 



His measurements would then agree with 
mine and Ortmann's (1912) 260 цт x 230 |nm. 



DISCUSSION 

Anyone interested in the literature dealing 
with the glochidium of a particular species is 
referred to the remarks section above for that 
species, and this information will not be re- 
peated here. Instead, this discussion will re- 



114 



HOGGARTH 



late glochidial structure to an understanding 
of relationships within the Unionidae and pro- 
vide some suggestions for future investiga- 
tions. Larval stages have been instrumental in 
our understanding of relationships among 
higher taxa of invertebrates (phyla, classes 
and orders) and have played increasing roles 
in lower level taxonomic decisions, even 
within the Unionidae (Sterki, 1903; Ortmann, 
1912: Morrison, 1955). 

Hoeh (1990) proposed a radically new 
arrangement of the genus Anodonta, which 
has resulted in the splitting of the that genus 
into Anodonta s.S.. Utterbackia, and Pygan- 
odon in North America. The genus Anodonta 
was restricted to A. cygena and related 
species found in Europe and along the east 
coast of North America. Glochidial characters, 
support this reclassification of the genus An- 
odonta. with one exception. Hoeh (1990) in- 
cludes the species implicata in the genus An- 
odonta. but glochidial valve shape, fine 
structure of the styliform hook, and exterior 
valve sculpture appear to separate this 
species from from all other species of An- 
odonta, Utterbackia and Pyganodon and sug- 
gests that additional work to refine the rela- 
tionship between this species and the other 
members of this group is warranted. Also, it 
might be noted that glochidial morphology 
confirms the close relationship between the 
members of these genera and A. ferrusa- 
cianus. 

Glochidial characters assist in confirming 
the relationship between the Ambleminae and 
the Lampsilinae. Davis & Fuller (1981) con- 
clude that the Ambleminae and Lampsilinae 
are monophyletic, and the lack of a clean mor- 
phological break in glochidial structure ap- 
pears to agree with that conclusion. Glochidial 
morphology of the more primitive lampsiline 
species, such as P. fasciolaris. agree almost 
completely with that of some members of the 
amblemine species, such as E. dilatata ana P. 
dombeyana. It is suggested that an examina- 
tion of more of the glochidia of the amblemine 
species would be helpful to confirm this con- 
clusion. 

Perhaps, however, the most powerful dem- 
onstration of the use of glochidial characters 
in the determination of relationship is the 
confirmation of ochracea in the genus Lep- 
todea. The species has been assigned to the 
genus Lampsilis because of adult shell mor- 
phology, including pronounced sexual dimor- 
phism (Simpson, 1900, 1914; Johnson. 1947, 
1970; Burch, 1975; Fuller & Bereza, 1975; 



Clarke, 1 981 b; Porter & Horn 1 981 ). Morrison 
(1975), on the other hand, was unable to lo- 
cate mantle flaps on gravid females and 
placed the species in Leptodea. The size and 
shape of the glochidium of this species con- 
firm Morrison's conclusion, which is also in 
agreement with most modern arrangements 
of unionid taxa (e.g., Turgeon et al. 1988). 

The glochidia described above suggest 
other taxonomic relationships within the Un- 
ionidae which have not been proposed pre- 
viously. Within the tribe Alasmidontini, two 
types of glochidia occur. One has a de- 
pressed, pyriform shape, with two row of mi- 
crstylets on the styliform hook and looped ex- 
terior valve sculpture. Furthermore, these 
glochidia posses exceptionally large adductor 
muscles in cross-sectional area and dorsal 
placement of the adductor muscle (Hoggarth 
& Gaunt, 1988). Species with this type of 
glochidium included all members of the genus 
Strophitus examined, A. viridis. A. heterodon, 
L. compressa. L. subviridis. L. iiolstonia and P. 
fabula. The other type of glochidium is high, 
pyriform, with at least four rows of micro- 
styletes on the hook and beaded to rosette ex- 
terior valve sculpture. These species, which 
include A. undulata. A. marginata. A. confrigo- 
sus. L. costata and L. complanata. have a 
smaller adductor muscle in cross-sectional 
area and the adductor muscle is placed further 
from the dorsal shell margin (Hoggarth & 
Gaunt, 1988). Additional study using anatomi- 
cal or molecular methods might confirm closer 
relationships among the members of these 
two groups and thereby support a different tax- 
onomy of the tribe. 

Glochidial structures suggest a much differ- 
ent arraignment of the species of Potamilus 
and Leptodea than currently accepted (e.g., 
Turgeon at al., 1988). Furthermore, E. lineo- 
lata is shown to belong to this group of taxa 
as it posses lateral valve gape and small, 
obliquely oriented dorsal alae. Ortmann 
(1912) was the first to suggest this associa- 
tion based on the presence of lateral valve 
gape in the glochidia of Potamilus and Ellip- 
saria. 

The genus Potamilus is currently defined 
on the basis of a single character; the pres- 
ence of a ligulate glochidium. Ortmann (1912) 
stated, ". . .this genus [Potamilus] stands in 
all characters except the glochidia, by the side 
of Paraptera [= Leptodea]." The current study 
suggests that there are many other, more 
subtle, glochidial characters that can be used 
to define the genus. If the genus is restricted 



GLOCHIDIA OF UNIONIDAE 



115 



to alatus. purpuratus. and capax (the glochid- 
¡um of capax was described by Cummings et 
al., 1990), then it can be defined as having 
ligulate glochidia with lanceolate hooks at the 
lateral margins of the ventral flange. The 
glochidia of these species are generally large 
(210 ,um X 380 ,um in length and height) (ex- 
cept capax). and they posses vermiculate ex- 
terior valve sculpture. Two species currently 
in the genus, P. ohiensis and P. amphichaena. 
lack these characters. The glochidia of these 
two species, although possessing lateral 
valve gape, lack lanceolate hooks at the ven- 
tral margin of the valve and posses looped 
rather than vermiculate exterior valve sculp- 
ture. They are much more rounded at the ven- 
tral margin than any other member of the 
genus: they are smaller (about the same size 
as capax): and they have lamellate rather 
than lanceolate micropoints on the ventral 
flange. These two species share these glo- 
chidial characters with L. fragilis and L. ochra- 
cea. I believe a study of adult anatomical 
characters could provide useful data that 
would support a different relationship among 
these taxa. Roe & Lydeard (1998) found mol- 
ecular genetic characters support the separa- 
tion of ohiensis and amphichaena from alatus 
and purpuratus. 

Finally, it is suggested that an examination 
of the glochidia of additional species would 
suggest other taxonomic questions and/or 
provide support for currently accepted views 
of unionid taxonomy. An examination of the 
glochidia of the Ambleminae would be partic- 
ularly helpful. Fewer amblemine glochidia 
have been examined with SEM than any other 
group primarily, because it is more difficult to 
tell when amblemine females are gravid and 
because they hold mature glochidia for a 
shorter period of time. Still, such structures as 
the coronal micropoints of Q. infucata and the 
exaggerated larval threads of /W. nervosa and 
M. boyiiiniana suggest a fruitful area of inves- 
tigation. 



ACKNOWLEDGEMENTS 

This work formed part of a Ph.D. disserta- 
tion submitted to The Ohio State University. I 
wish to thank my advisor, Dr. David H. Stans- 
bery for his support, for use of the collection at 
OSU and for the many discussions he has 
had with me concerning glochidia. I wish to 
thank the National Science Foundation (grant 



BSR-8401209) for financial support and Dr. 
George Davis and two anonymous reviewers 
for helpful criticism of a previous draft. I thank 
Dr. J. B. Burch. David Heath, and Kevin Cum- 
mings for providing access to specimens, and 
I must thank all those who have contributed 
specimens to this study. Their names, too nu- 
merous to list here, are found in the material 
examined sections of each species account. 



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Revised ms. accepted 10 January 1999 



MALACOLOGIA, 1999, 41(1): 119-137 

GROWTH PATTERN AND DYNAMICS OF A SOUTHERN PERIPHERAL 

POPULATION OF PISIDIUM AMNICUM {MÖLLER, 1774) 

(BIVALVIA: SPHAERIIDAE) IN SPAIN 

R. Araujo\ M. A. Ramos^ & R. Molinet^ 



ABSTRACT 

The dynamics of a Spanish population of P. amnicum. one of the southernmost populations of 
the species in the world, is presented. P. amnicum is the only living European species of the sub- 
genus Pisidium s. s. The study is based on monthly samples, from June 1 990 to May 1 991 , from 
the Miño River, Galicia, northwestern Spain. Specimens from all size classes were dissected and 
all embryo/larval stages were counted and measured. Studies were conducted to find out the 
growth pattern of this population using the von Bertalanffy model. Pisidium amnicum in Spain is 
semelparous and univoltine. Only two cohorts coexisted from late spring to late summer. The life 
span of the species is about 15 months. The parental generation disappear in August. Juvenile 
recruitment occurs in April-May when water temperature ranges between 15-20°C. The mini- 
mum observed size (shell length) of a gravid specimen was 3-4 mm. Fertilized eggs were 
brooded for approximately nine months in the inner demibranches until they reached up to 2 mm. 
The bigger clams had, in the same month, more and bigger embryos (or larvae), than the smaller 
ones. Not all the initial embryos completed their development. Nevertheless, this phenomenon 
of intramarsupial suppression does not seem to be very important in the Spanish population. A 
main feature of the Spanish population of P. amnicum is the high number of larvae incubated dur- 
ing the months immediately before birth. 

Key words: Pisidium amnicum. population dynamics, growth, Spain, peripheral population. 



INTRODUCTION 

The natural species range is the area in 
which it is well adapted both morphologically 
as ecologically. However, it is known that en- 
vironmental factors may be extreme at the 
edge of a species distribution and may pre- 
vent it from extending its range. Under such 
conditions peripheral populations may show 
restriction to particular biotopes (Ford, 1964), 
tendency to isolation (Mayr, 1 963), changes in 
the genetic structure of the populations 
(Ramos, 1985), as well as in other physiolog- 
ical aspects, such as development, fecundity 
or life span (Möller & Swaddle, 1997). 

All known species of the family Spaheriidae 
are hermaphroditic, and incubate fertilized 
eggs in brood sacs developed in the inner gill. 
Therefore, they are an excellent material for 
studying reproductive biology. Taking into ac- 
count that some reproductive aspects have 
been mentioned as very important not only in 
the subgeneric characterization (Heard, 1965) 
but also at the specific level, it seems impor- 



tant to know the specific variation in these 
characters along a geographical range. 

Pisidium amnicum is the only European 
species of the subgenus Pisidium s. s. Only a 
few papers include data on the biology and re- 
production of this species (Odhner, 1929; 
Meier-Brook, 1970; Holopainen, 1979; Holo- 
painen & Hanski, 1986) or deal specifically 
with its population dinamics and growth (Da- 
neel & Hinz, 1976; Bass, 1979, Vincent et al., 
1981), but they all are referred to northern or 
central paleartic populations and do not in- 
clude data on larval stages prior to shell for- 
mation. 

This paper describes the dynamics of a 
Spanish isolated population, the southern- 
most known of the species in Europe and in 
the world, with the exception of the North 
African population cited by Kuiper (1972). 
Therefore, its reproductive characteristics can 
also be used as a sensitive measure to as- 
certain the extent to which this peripheral pop- 
ulation has adapted to local conditions. Data 
such as life span, birth period, size at sexual 



^Museo Nacional de Ciencias Naturales С. S. I. С. José Gutiérrez Abascal 2. 28006 Ivladrid. Spain; rafael@mncn.csic.es 
^Programa de Cooperación Técnica para la Pesca CEE-VECEP ALA 92/43. Centro Profesional Santa Paula, Torre A. 
Avenida Circunvalación, Santa Paula, Caracas, Venezuela. 



119 



120 



ARAUJOETAL. 




FIG. 1 . Live Pisidium amnicum specimen from the Miño River, Galicia, Spain. 



maturity, and percentage and size of incu- 
bated larvae, when compared with the avail- 
able data from central and northern popula- 
tions, should allow us to determine if the 
geographical position of this population at the 
edge of the species range represents margin- 
ality in the ecological and/or physiological 
sense, or whether the response remains es- 
sentially similar in all the populations. Studies 
were conducted for the first time to discover 
and describe the growth pattern of a popula- 
tion of P. amnicum. dissecting specimens 
from all size classes, and counting and mea- 
suring all embryo/larval stages. 



MATERIAL AND METHODS 

Specimens of Pisidium amnicum (Fig. 1) 
were collected monthly between June 1990 
and May 1991 in the Miño River in north- 
western Iberian Peninsula (Fig. 2). From 
June to August the sample locality was the 
Miño River near Goian, Pontevedra, close to 
the Spain-Portugal ferryboat docks. As no 
more live specimens of the species were 
found after September (Araujo, et al., 1993). 
the site was changed to 15 km upstream on 
the same river (Fig. 2). The two sites are 
on the same shore of the river, which is 400 m 
wide, and are exposed to tidal influences as 



they are 10 and 25 km, respectively, from the 
Atlantic Ocean. 

The sampling method consisted of drag- 
ging the bottom with a dredge in which sand 
and mud were retained so that specimens of 
all size classes were collected. Animals were 
sorted from the sediment using a 1-mm mesh 
sieve. They were carried alive on ice to the 
laboratory in plastic jars containing water from 
the sample site. Artificial aeration was pro- 
vided for five sec every eight h. In the labora- 
tory, specimens of each monthly sample were 
measured (maximum shell length) and sorted 
into 1-mm size classes. 

To discover the ratio of gravid animals in 
each size class and month, ten animals (when 
possible) from each class and month were 
dissected. To obtain data on number and size 
of incubated embryos (or larvae), ten speci- 
mens from each size class per month, from 
June to December, were prepared in the field 
using the method developed by Eggleton 
(Heard, 1965), which consists of isolating 
each animal in a glass tube filled with water. 
When the tisúes decayed, the larval shells 
were counted and measured. Once it was re- 
alized that this technique does not permit the 
recovery of embryonic phases prior to shell 
formation, data for all months were obtained 
directly from the animals. After dissection, 
embryos and larvae present in each gill were 



POPULATION DYNAMICS OF PISIDIUM AMNICUM 



121 




-^ PORTUGAL 



5 Km 



FIG. 2. Map of sample sites. 



counted and measured (maximum length). 
The sizes of the embryos and larvae were ob- 
tained by recording the largest and smallest in 
each gill. Observations, dissections, counting 
and measuring were carried out under a 
Bausch and Lomb stereomicroscope with 1 0x 
oculars, 1x to 7x zoom and micrometric ocu- 
lar. Due to the extremely small size of the in- 
cubated phases after fertilization, values for 
June to September are less reliable than 
those for the rest of the year. 

The frequency of gravid animals was calcu- 
lated from the data of dissected animals, and 
the number and mean size of the incubated 
embryos and larvae refer to the number of 
gravid animals. In order to check if there was 
lateralization, that is differences in the number 
and sizes of the incubated phases between 
gills, and as these variables are not normally 
distributed, a repeated measures ANOVA test 



was performed using SuperAnova. Descrip- 
tive statistics were carried out using StatView 
4.1 . Both software packages are from Abacus 
Concepts by Macintosh. The possible influ- 
ence on the specimens' gravidity, coded as 
gravid (1) and non-gravid (0), of both month 
and the specimen size, as well as the interac- 
tion between these two factors, was explored 
by means of a logistic regression analysis 
using the SPSS 6.0 statistical package. The 
effect of these factors (specimen size and 
month) on the number of incubated embryos 
for each of the gravid specimens was tested 
by an ANCOVA performed with the Statistica 
4.1 package from Statsoft. The variances of 
the dependent variable (number of embryos) 
were not homogeneous, so the squared-root 
transformation was used in the analysis. In 
the analysis, data from June to August were 
excluded due to very low sample size (n < 1 0). 



122 



ARAUJOETAL 



The same statistical procedure was used to 
study the relation of these factors to the size 
of the incubated embryos. 

The von Bertalanffy model (Bertalanffy, 
1934) was used for analysis of growth. Al- 
though this model was designed specifically 
for fisheries, it has been applied to freshwater 
bivalves (Morton, 1969. 1977). This mathe- 
matical model expresses length (L) as a func- 
tion of age (t) as follows: Lt = L,^, (1-е '^""'o*), 
where Ц^, = the mean length of an "infinitely" 
old animal, К = the curvature parameter 
showing the speed at which the animal be- 
comes L|^, and tQ = the parameter of initial 
condition, is the time at which the animal has 
zero length. It does not have biological signif- 
icance. 

As no data about age were available, for- 
mula parameter values were estimated from 
size class composition of the monthly sam- 
ples. With these data, the Bhattacharya 
(1967) method was used to identify both the 
different cohorts alive during the year and the 
mean of the normal curve adjusted to each 
generational group. These values were the 
basis to calculate the parameters of the von 
Bertalanffy formula. They were estimated 
using ELEFAN (Gayanilo et al., 1988) soft- 
ware. As the time between samples was con- 
stant (one month) the value of Ц^, was esti- 
mated using the graphic method of Ford 
(1933) and Walford (1946), as discussed in 
Sparreetal., (1989). 

While collecting specimens, physical and 
chemical characteristics of the water were 
recorded at the sample site. Temperature (± 
0.5°C), dissolved oxygen (± 1 .0 ppm) and pH 
(± 0.1 pH) were monitored using an Horiba U- 
7 water checker. Conductivity (|.is ± 0.3%) 
was measured using a Crison 523 conduc- 
tivimeter. Alkalinity, calcium, total water hard- 
ness and carbonate hardness values were 
obtained in situ with Merck Aquamerck kits. 
The influence of the measured water physical 
parameters on gravidity was explored using a 
logistic regression analysis. 

RESULTS 
Population Dynamics and Growth 

The raw data on collected, dissected and 
gravid specimens, and the number and size of 
the incubated phases for each gravid animal 
from each size class and month are in Araujo 
(1995). Appendix 1 summarizes this informa- 
tion. 



Monthly histograms of the different size 
classes (Fig. 3) showed that there was only 
one reproductive period per year. Juveniles 
1 -2 mm long only appeared in May, when the 
next size class (2-3 mm) reaches its highest 
annual frequency. This suggests that birth oc- 
curs between April and May. Between June 
and July, there was notable mortality in the old- 
est classes (Fig. 3). The largest individuals 
(10-11 mm) represent a minimum ratio in June 
and August, and were not present the rest of 
the year. There is hardly any growth in winter. 
In fact, the Bhattacharya (1967) method iden- 
tified only two cohorts that coexisted from late 
spring to late summer (Table 1). Recruitment 
occurred in May, and no members of the 
parental generation appeared in August, indi- 
cating that the life span of each generation is 
about 15 months. Values in Table 1 show that 
animals were born proportionally very big, and 
that their growth was fast, reaching a mean 
size between 3.3 and 4.5 mm in summer when 
the reproductive period began. This growth 
continued until December delaying until Feb- 
ruary, when it started again but more slowly. 
Animals were largest at the time of juvenile re- 
lease, and stabilized at around 8 mm until the 
disappearance of the adults. 

The growth curve of this population was 
tested using a Ford-Walford plot (Table 2). 
The corresponding regression analysis (Fig. 
4) gave the formula: L(t -(- 1) = 1.499 + 
0.81 8L(t), which approaches the 45° line as 
expected if growth fits the von Bertalanffy for- 
mula. Therefore, the value of the asymptotic 
length (Ц^, = 8.245 mm) was employed to cal- 
culate the dependent variable of regression in 
the von Bertalanffy method, the data from 
which appear in Table 2. Values for the inter- 
cept and slope of this regression are a = 0. 1 83 
and b = 0.201 , respectively. Parameters of the 
growth formula of von Bertalanffy are: L,^, = 
8.254: К = 0.201 : to = 0.913 years. 

Figure 5 shows the monthly frequencies of 
gravid animals in each size class. No gravid 
animals under 3-4 mm have ever been found. 
Therefore, the minimum size to be gravid ap- 
pears to be 3-4 mm, although this size was 
only recorded in October (50% of collected 
specimens). From June (just after juvenile re- 
cruitment) to September, some gravid ani- 
mals in classes up to 7 mm were found, but 
from October to May practically the whole 
adult population was gravid. The logistic re- 
gression to test the influence of the size and 
the period of the year on gravidity revealed 
a significant (X^,2 = 182.66: p < 10"^) model 



POPULATION DYNAMICS OF PISIDIUM AMNICUM 

January n = 164 •'"'У 



50 
40 
30 
20 
10 





120 
100 
80 
60 
40 
20 




n=260 



1 



I 



123 



February 

60 
50 
40 
30 
20 
10 



n = 163 



80 
60 
40 
20 




Jh 



n = 170 



ll 



L 



August 

25 
20 

15 I 
10 

5 





n = 93 



JdiL 



^ in <© h. 

A 4 Л ¿ 



(¿ к ¿ Т 



о» о ^ 



September 

150 

100 

50 

О 



n=350 



Jl 



April 

100 
60 
60 
40 
20 




n=258 



October 




10 



n=215 



.lliü 



May 



n=285 




November 



80 
60 
40 
20 




n = 202 



^M 



I 



It 



5 Î s 



n = 404 



JJL 



70 
60 
50 
40 
30 
20 
10 



n = 173 



j| 



1 



FIG. 3. Size histograms of the population structure of P. amnicum in the Miño River between June 1990 and 
May 1991. 



124 



ARAUJOETAL. 



TABLE 1. Mean length (mm) of the cohorts identi- 
fied by the Bhattacharya (1967) method on the size 
distribution of P. amnicum. 





Cohort 1 (mm) 


Cohort 2 (mm) 


Months 


X 


X 


January 




7.00 


February 




7.27 


March 




7.47 


April 




7.66 


May 


2.42 


7.83 


June 


3.27 


7.90 


July 


4.21 


7.72 


August 


4.57 


7.92 


September 


5.77 




October 


6.12 




November 


7.11 




December 


7.22 





TABLE 2. Variables used for the Ford-Walford and 
von Bertalanffy regression analyses. *t expressed 
in months. 





Ford-Walford Plot 


von Bertalanffy Plot 


Time (t)* 


Lt 


L(t + 1) 


Ln(1-(Lt/LJ 


1 




2.42 


3.27 


0.3470 


2 




3.27 


4.21 


0.5045 


3 




4.21 


4.57 


0.7135 


4 




4.57 


5.77 


0.8067 


5 




5.77 


6.12 


1.2008 


6 




6.12 


7.11 


1.3527 


7 




7.11 


7.22 


1.9761 


8 




7.22 


7.00 


2.0772 


9 




7.00 


7.27 


1 .8843 


10 




7.27 


7.47 


2.1267 


11 




7.47 


7.66 


2.3539 


12 




7.66 


7.83 


2.6314 


13 




7.83 


7.90 


2.9685 


14 




7.90 


7.73 


3.1489 


15 




7.73 


7.92 


2.7568 


16 




7.92 







that correctly classified 87.2% of individuals. 
The model accuracy was higher for "gravids" 
(90.6%) than "non-gravids" (72.3%). The sea- 
sonal variation observed in Figure 5 is signifi- 
cant (Wald = 52.33; df = 11 ; p < 10^^), time 
being the main factor influencing gravidity in 
this species as can be deduced from the par- 
tial correlation (Грд^ = 0.302). The effect of in- 
dividual size on gravidity, although weaker 
than that of seasonal variation (Wald = 10.42; 
df = 1 ; p = 0.001 ; r^^^ - 0.1 59), is positive, and 
the probability of a clam being gravid is higher 
among the biggest specimens throughout the 
year. The effect of size on gravidity does not 



change during the year (interaction between 
month and size; Wald = 1.914; df = 11; p > 
0.95). 

The repeated measures ANOVA test gave 
no significant differences either in number 
(F^ 263 = 0-009, p = 0.922) or size (F^ 2бз = 
0.182, p = 0.67) of incubated phases between 
the two gills. Thus, the corresponding values 
for the two gills of each individual were added 
for analysis. 

The ANCOVA test showed that both, the 
month (F(8 254^ = 10.27; p < 10"^) and the 
mother size (F(i 254) = 324.50; p < 1 0"^) highly 
influence the number of incubated embryos 
(Table 3, Fig. 6). However, in this case the ef- 
fect of time is not homogeneous within all age 
classes, because the interaction between 
these two factors is highly significant (F(8 246) 
= 10.686; p < 10"^). From December to May, 
we found a progressive decrease in the total 
number of embryos (larvae), especially in the 
smaller classes. It is minimum in July and Au- 
gust, and thereafter the number of embryos 
increased following the oocyte fertilization pe- 
riod, which occurs from June to mid-autumn 
(Araujo & Ramos, 1999). Figure 7 illustrates 
how the seasonal variation is lower among 
the smaller gravid specimens. The influence 
of mother size is positive (r = 0.749). In other 
words, in the same month, as a whole, lar- 
ger clams contained more embryos (or lar- 
vae) than smaller clams. To show this temp- 
oral variation, the standardized regression 
coefficients were calculated for all the 
months. (The period June-August was ex- 
cluded because of the lack of enough gravid 
specimens.) Their values were positive and 
similar for all the months except May when 
negative values (Table 4, Fig. 8) were re- 
corded. 

Regarding the size of the incubated phases 
(Table 5, Fig. 9), the ANCOVA analysis re- 
sults, although weaker than the ones regard- 
ing number of embryos, suggest that both 



month (F, 



= 1 73.45; p< 10" 

v5 , 



and mother 
0.51) have 



(8,255) 
(1,255) 

an effect. The effect of the latter is not homo- 
geneous, as it changes monthly (F^g247) = 
1.99; p = 0.05); in a given month, larger 
classes incubated larger embryos (or larvae) 
than smaller ones. The monthly variation of 
this regression can be observed in Figure 10. 
The progressive increase after August was 
proportional to each size class, with a maxi- 
mum in April-May just before birth. In the last 
month of incubation, larval growth was high- 
est. 



POPULATION DYNAMICS OF PISIDIUM AMNICUM 



125 




FIG. 4. Ford-Walford plot of P. amnicum from the 
Miño River in which the mean length of each age 
grouping (Lt) has been plotted against the suc- 
ceeding age grouping (Lt + 1 ). 



Influence of Abiotic Factors 

Monthly physico-chemical water values are 
shown in Table 6. There were significant sea- 
sonal variations in conductivity that may be 
due to tidal influences, total hardness, alkalin- 
ity, calcium values, and water temperature. 
The latter increased progressively from April- 
May (15-20"C) to July (27X). Conductivity 
values are low, as occurs in rivers on granitic 
soils poor in soluble salts. This parameter re- 
mained constant between 60 and 70 mScm"^ 
at 25°C during the year, increasing in June to 
reach 225 mScm"' in August, and decreasing 
to 112 mScm"^ in September. A summer in- 
crease also occurs in total hardness and al- 
kalinity. Calcium values change in summer, 
reaching a maximum in September. 

The influence of the physical water param- 
eters on the reproductive cycle of P. amnicum 
was analyzed using logistic regression (dis- 
solved oxygen was excluded, because of the 
lack of measurements for two months). The 
stepwise procedure (backward) obtained a 
highly significant model (X^^ = 168.41; p = 
10'"), accounting for 88.30% of the observed 
variability in gravidity. The accuracy was 
higher when explaining "gravids" (91.37%) 
than "non-gravids" (75%). The model identi- 
fied four water variables as significant (Table 
7), water temperature being the most impor- 
tant, as shown by the partial correlation coef- 
ficients, although calcium, pH and alkalinity 
were also significant. 

To test to what extent the reproductive cycle 
of P. amnicum (measured by the frequency of 
gravid specimens during the year), is influ- 
enced by abiotic factors, a multiple regression 



analysis was performed using the residuals 
obtained from the logistic model (gravidity = f 
(mother size, month)) as the dependent vari- 
able. The result of the regression analysis 
was not significant (r = 0.028; F^^ 33^, = 0.039; 
p = 0.99), which means that none' of tne tested 
water parameters were directly influencing 
the reproduction of the species after removing 
the effect of mother size and month of the 
year, the previous significances being a sec- 
ondary effect of the seasonal variation of 
these parameters and their influence on other 
intrinsic biotic factors. 



DISCUSSION 

Reproductive Strategies 

In the Spanish study population, P. amnicum 
had only one annual reproductive cycle with 
births and juvenile recruitment in April-May, 
when water temperature ranged between 
1 5°C and 20°C. Danneel & Hinz's (1 976) find- 
ings for a German population were similar, al- 
though the new generation appeared in May- 
June, a little later than the Spanish one. The 
apparent absence of gravid animals in August 
in the German population, and in June and 
July in the English one (Bass, 1 979) was prob- 
ably a consequence of the method used, as 
larval stages prior to shell formation were ig- 
nored. With very few P. amnicum specimens 
from two different localities in Germany, Meier- 
Brook (1970) cited a total of eight gravid indi- 
viduals containing embryos between 0.25 and 
0.4 mm long in September, and concluded that 
the reproductive period was synchronous and 
began in autumn; also data for six individuals 
collected at the beginning of spring in England 
agree with this, "they had big embryos be- 
tween 0.5 and 1." Similar data were reported 
by Thiel (1928) in Germany and Odhner 
(1929) in Sweden, whereas in a Finnish lake 
juveniles were born later on in summer (July) 
(Holopainen, 1979). Table 8 provides a com- 
parative summary of the data on the five stud- 
ied populations of P. amnicum. 

The species in Europe seems to be very 
conservative in reproductive cycle with syn- 
chrony among populations: it begins in sum- 
mer, the newborn appearing in spring, with 
some differences depending on the country. 
Such variation seems to follow a latitudinal 
dine, which might be related to water temper- 
ature. One cycle follows as soon as the for- 
mer has finished. Bass (1 979) reported that in 



126 



ARAUJOETAL. 



% gravid animals 



JANUARY 



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FEBRUARY 



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MARCH 



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APRIL 



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MAY 



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JUNE 



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JULY 



AUGUST 



SEPTEMBER 



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OCTŒER 



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NOVEMBER 



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DECEMBER 



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Ш 


m, 


= 


111 


""] 


H 


■ 


■ 


D 


■ 


_l 


Ю 


00 


-vl 


en 


СЛ 


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CO 


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cp 


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00 


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M 



FIG. 5. Monthly variation, by size class, in the frequencies of gravid animals in relation to the number of an- 
imals dissected. 



POPULATION DYNAMICS OF PISIDIUM AMNICUM 



127 



TABLE 3. Mean number and standard deviation of the embryos (larvae) occurring in the gravid animals. 



CLASS 














(mm) 


JANUARY 


FEBRUARY 


MARCH 


APRIL 


MAY 


JUNE 


1-2 
2-3 


— 


— 


— 





— 


— 


3-4 


— 


— 


— 


— 


— 


— 


4-5 


7.00 


— 


— 


— 


— 


— 


5-6 


7.9 ± 2.28 


10.5 ± 3.94 


3.67 ± 3.21 


11.5 ± 0.71 


11.00 


— 


6-7 


16 ± 4.92 


16.2 ± 5.29 


15.5 ±4.95 


15.5 ± 5.27 


19 ±7.07 


— 


7-8 


34.33 ± 11.18 


36.1 ± 14.43 


30.3 ± 12.41 


29.3 ± 9.43 


16.17 ± 16.13 


10.00 


8-9 


44.1 ± 10.94 


48.3 ± 14.69 


44.6 ± 15.85 


43.1 ± 9.37 


11.67 ± 5.03 


6 ± 4.24 


9-10 


73.00 


56 ± 1.41 


63 ± 5.66 


42.2 ± 11.61 


5.5 ± 6.36 


11.00 


10-11 


— 


— 


— 


— 


— 




TOTAL 


26.07 ± 18.09 


31.08 ± 19 


29.74 ± 18.11 


30.08 ± 14.56 


13.71 ± 11.4 


8.25 ± 3.59 


CLASS 














(mm) 


JULY AUGUST SEPTEMBER 


OCTOBER 


NOVEMBER 


DECEMBER 


TOTAL 


1-2 
2-3 


- - 


I 


: 


I 


— 


: 


3-4 


— — 


— 


6.5 ± 7.78 


— 


— 


6.5 ± 7.78 


4-5 


— — 


13.00 


13.33 ± 2,07 


15 ±2.83 


— 


13 ± 2.87 


5-6 


— — 


12 ± 4.63 


18.5 ± 5.95 


15.33 ± 2.52 


14.5 ± 3.69 


11.89 ± 5.6 


6-7 


— — 


18.33 ± 6.11 


28.83 ± 6.97 


28.33 ± 4.84 


23.11 ± 9.95 


19.33 ± 7.73 


7-8 


— — 


25.6 ± 2.89 


31.83 ± 6.49 


37.5 ± 6.56 


42.17 ± 6.62 


31.45 ± 12.33 


8-9 


3 ± 2 2.33 ± 


1.53 25.67 ± 4.93 


35.67 ± 7.5 


41.17 ± 11.65 


43.33 ± 9.65 


36.97 ± 17.61 


9-10 


— — 


42.00 


— 


40.00 


44.33 ± 16.62 


42.17 ± 20.36 


10-11 


— — 


— 


— 


— 


— 


— 


TOTAL 


3 ± 2 2.33 ± 


1.53 21.94 ±8.64 


24.44 ± 11.06 


31.58 ±11.92 


32.57 ± 14.71 


— 



England a small proportion of the largest 
adults initiated a second reproductive cycle, 
but died before the brood was fully developed. 
None of the European populations show a de- 
cline in fertility in relation to age, as occurs in 
the related genus Sphaerium (Meier-Brook, 
1970). 

Avery close similarity can also be found be- 
tween the reproductive strategies of P. am- 
nicum and the vicahant North American 
species P. dubium. Both species are of similar 
size, share the same incubation and birth 
months, and, in both cases, the mean number 
and size of larvae increased with the size of 
the parents, although the number of incu- 
bated larvae was considerably higher in the 
Spanish population. The strategy of the Cana- 
dian population of P. amnicum (Vincent et al., 
1981) also fits perfectly with the one of P. du- 
bium, with only two exceptions: P. dubium at- 
tains its sexual maturity in its first year of life, 
and its number of larvae is considerably 
higher, suggesting that these two populations 
might belong to the same taxon. 

Meier-Brook (1970) suggested that, with 
the exception of P. amnicum for which no data 
were available, sexual maturity in populations 
of Pisidium occurs when individuals reach 50 
or 60% of maximum size. According to this 
rule, as the maximum size of the Spanish 



population ranges from 10-11 mm, it follows 
that sexual maturation would occur in a size 
class near 5-6 mm, which is higher than the 
4-5 mm that we found to be the minimum 
needed to be gravid (with the exception of two 
gravid specimens of 3-4 mm collected in Oc- 
tober). However, it applies perfectly to the 
maximum theoretical length (8.25 mm) esti- 
mated by the von Bertalanffy method. More 
surprising are the proportions reported by Da- 
neel & Hinz (1976) (Table 8). Thus, although 
most of the available data (Table 8) seem to 
follow Meier-Brook's rule, in some popula- 
tions maturity is reached before specimens 
are 40-50% of maximum size. Then, if any 
rule were applicable to P. amnicum, we would 
expect a positive correlation between mini- 
mum gravid length and maximum adult size 
throughout the species range (Table 8), which 
was not found. 

The main feature of the Spanish population 
of P. amnicum seems to be the high number 
of larvae incubated during the months imme- 
diately before birth. The difference with all the 
other previous data may be explained by dif- 
ferences related to temperature and/or lati- 
tude. In warmer climates, the same species 
becomes bigger, incubates a greater number 
of larvae (maximum of 73 in Spain and 12 in 
Finland), and the larvae are also bigger. Such 



128 ARAUJOETAL 

mean number of embryos 

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SEPTEMBER 



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NOVEMBER 



DECEMBER 



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II 


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FIG. 6. Monthly variation, by size class, in the mean number of embryos (larvae) occurring in gravid animals. 



POPULATION DYNAMICS OF PISIDIUM AMNICUM 



129 



80 














• 


/U 














• 


bU 














• 


50 


- 










• 




40 
30 


• 






• 

• 
• 


• 

1 

• 


1 

• 
• 


i 


10 


• 


• 

• 


• 

1 

• 


t 


• 
• 


• 

t 


• 
• 



















Size classes (mm) 



FIG. 7. Variation in the mean number of embryos 
(larvae) in each size class with parental size. 



TABLE 4. Monthly standardized regression coeffi- 
cients of the variation in the number of incubated 
embryos in relation with the mother size. 



Month 


BETA 


St. Err. of BETA 


January 


0.898 


0.070 


February 


0.837 


0.091 


March 


0.842 


0.094 


April 


0.800 


0.101 


May 


-0.325 


0.272 


September 


0.824 


0.157 


October 


0.846 


0.097 


November 


0.794 


0.129 


December 


0.733 


0.133 



north-south clinal reproductive behaviour was 
apparently not found in preliminary studies 
with the related North American species P. 
idahoense Roper, in which the northern popu- 
lations were smaller but had more progeny 
(Heard, 1965). On the other hand, as the life 
span of P. amnlcum in Spain appears to be 
about 15 months, our results do not confirm 
Bass's idea (1 979) of an extended life span or 
a successful second brood in southern areas. 
The scarce data on the Finnish population, 
one of the northermost in the species range, 
indicate a reduction in fertility in relation to the 
central European populations. This reduction 
may be accompanied by a long life span, al- 
lowing iteropahty, as reported by Holopainen & 
Hanski (1 986) and Vincent et al., (1 981 ) in Fin- 
land and Canada, respectively. Given that the 
Spanish population broods the maximum 
number of larvae in the species range, and 
that the maximum embryo length is similar to 
all other reported populations, we may state 
that in P. amnicum there is no trade-off be- 
tween litter size and embryo size as was pro- 



posed by Holopainen & Hanski (1986) for the 
genus Pisidlum. However, the characteristics 
of the Spanish population as a whole reflect its 
breeding success. The species seems to be so 
well adapted to local conditions in this range 
margin that it is possible to speculate that such 
attributes might allow the species to expand its 
range if the opportunity presented itself. In 
fact, the Goian (Fig. 2) colony, which experi- 
enced a drastic reduction after colonization by 
Corbicula fluminea (Müller, 1774) (Araujo et 
al., 1993), has re-established itself, sharing its 
habitat with the latter species as we have ob- 
served recently (unpublished data). 

Study of the phenomenon that Meier-Brook 
(1977) called intramarsupial suppression of 
fetal development has deserved special atten- 
tion. This author cited 50% of the brood dying 
before birth in P obtusale and P. lilljeborgii, 
whereas it is very variable in P amnicum 
(0-54%) according to Danneel & Hinz (1976). 
Our results show a progressive increase in 
embryos from June to December, which 
means that settlement of eggs or zygotes may 
occur over several months. On the other hand, 
we observed that from December, and in sev- 
eral size classes, the embryo number may 
decrease, suggesting that not all the initial em- 
bryos complete their development. Neverthe- 
less, this phenomenon does not seem to be 
very important in the Spanish population. The 
idea suggested by Araujo & Ramos ( 1 997) that 
ova fertilization occurs in the gills and not in the 
spermoviduct as was proposed (Okada, 1 935; 
Odhner, 1929; Meier-Brook, 1970), may pro- 
vide a new perspective, suggesting that the so 
called "embryos" present in the gill during the 
first months of the reproductive period may be 
eggs not yet impregnated. This may explain 
the differences found by other authors when 
comparing the initial and final number of larval 
stages in the gills. 

Although there is the same high mortality 
following the birth of juveniles, the corre- 
sponding ratio of the size classes over 6 mm 
in the Spanish population is higher than in the 
others during the year. 



Influence of Environmental Factors 

None of the water measured parameters in 
itself seems to directly influence the reproduc- 
tive cycle of the Spanish population of P am- 
nlcum as measured by specimen gravidity, 
although seasonal variation in water tempera- 
ture, calcium, pH, and alkalinity might be im- 



130 



ARAUJOETAL. 




December 



FIG. 8. Monthly variation in the relation between the mean number of embryos and parental size. 
TABLE 5. Mean lenght (mm) and standard deviation of the embryos (larvae) occurring in the gravid animals 



CLASS 






















(mm) 




JANUARY 


FEBRUARY 


MARCH 


APRIL 




MAY 


JUNE 


1-2 
2-3 
3-4 
4-5 




- 




— 




- 


— 




— 


— 




0.5 




— 




— 


— 




— 


_ 


5-6 




0.52 ± 0.12 


0.55 


±0.1 


0.45 


± 0.39 


0.76 ± 0.04 




1.3 


— 


6-7 




0.66 ± 0.19 


0.73 


± 0.88 


0.84 


± 0.09 


0.88 ± 0.16 




1.37 ± 0.19 


— 


7-8 




0.73 ± 0.14 


0.87 


± 0.11 


0.92 


± 0.09 


1.23 ± 0.25 




1.61 ± 0.4 


0.08 


8-9 




0.79 ± 0.17 


0.9 


± 0.1 


0.94 


± 0.13 


1.2 ± 0.22 




2.02 ± 0.08 


0.09 ± 0.04 


9-10 




0.85 


1 


± 0.14 


1.09 


± 0.13 


1.08 ± 0.15 




2.26 ± 0.09 


0.1 


10-11 




— 




— 




— 


— 




— 


— 


TOTAL 




0.67 ± 0.17 


0.8 


± 0.16 


0.87 


± 0.2 


1.08 ± 0.25 




1.77 ± 0.4 


0.09 ± 0.02 


CLASS 






















(mm) 


JULY AUGUST 


SEPTEMBER 


OCTOBER NOVEMBER 


DECEMBER 


TOTAL 


1-2 
2-3 




— 


- 


- 


- 


- 


- 




- 


- 


3-4 




- 


- 




- 


0.08 ± 







- 


0.08 ± 


4-5 




— 


— 


0.08 


0.12 ± 


0.04 0.13 ± 


0.07 


— 


0.15 ± 0.13 


5-6 




— 


— 


0.09 ± 0.01 


0.18 ± 


0.10 0.25 ± 


0.09 


0.42 ± 0.07 


0.46 ± 0.24 


6-7 




— 


— 


0.12 Í 


t 0.02 


0.22 ± 


0.02 0.37 ± 


0.07 


0.42 ± 0.14 


0.62 ± 0.29 


7-8 




— 


— 


0.09 = 


: 0.01 


0.22 ± 


0.04 0.34 ± 


0.03 


0.55 ± 0.15 


0.75 ± 0.47 


8-9 


0.08 


± 0.01 0.08 


±0.02 


0.14 d 


: 0.12 


0.25 ± 


0.03 0.34 ± 


0.05 


0.6 ± 0.08 


0.75 ± 0.47 


9-10 




— 


— 


0.18 


- 


0.35 


0.45 ± 0.15 


0.84 ± 0.54 


10-11 




— 


— 




- 


- 


— 




— 


— 


TOTAL 


0.08 


± 0.01 0.08 


± 0.02 


0.11 ± 0.05 


0.19 ± 


0.07 0.32 ± 


0.09 


0.49 ± 0.14 


— 



portant for their influence on other species 
intrinsic factors (i.e., mother size, previous epi- 
sodes of reproduction). Moreover, since P. am- 
nicum incubates its larvae within a ctenidial 
marsupium, larvae release and subsequent 
growth could be induced, both directly and in- 
directly, by these factors. 

Temperature increases progressively over 
April-May, when births occur, to July, when fer- 
tilization begins. The 27'C maximum is more 
or less maintained in August and September, 



and it is followed by a sudden decrease of 
about 9 С in October, precisely when the pe- 
riod of maximum population growth ends. It 
could be argued that temperature has so im- 
portant relation with birth that larvae big 
enough to be born in April did not because 
temperature was below 15'C. A relationship 
between temperature and growth in freshwa- 
ter bivalves has also been described in the Asi- 
atic clam Corbicula fluminea (Morton, 1977; 
Eng, 1979; Joy, 1985; Ituarte, 1985). 



POPULATION DYNAMICS OF PISIDIUM AMNICUM 131 

mean size of the incubated embryos 



JAN'UAR^ 



FEBRUAR'» 



ÎTTiiiirinmirTiiiiiirTrnii 



ппитпитпигтгтпттлт 



MARCH 



В 



rniiiiiTimiriiiiiiimTTrmnmii 

APRIL iggiiiigggggggi^^ 

[тппптпттттп i тттттптгтп i ii i 

MAY 



AUGUST 



SEPTEMBER 



OCTOBER 



NOVEMBER 



DECEMBER 



h 



Ii 




+ 



lllllllllini 



IIIIIIIIIIIIIITT 



JUNE L_ 



JULY 

im 



M п г m m 

ЧО 00 -J о '-П -t>- OJ 

>— чО oc ~J C> '-Л -t^ 



FIG. 9. Monthly variation, by size class, in mean embryos (larvae) size in gravid animals. 



132 



ARAUJOETAL 




Size classes 

FIG. 10. Monthly variation in the relation between mean embryo size and parental size. 

TABLE 6. Montly and mean values of the chemical and physical properties of the water at the sample site 









Dissolved 


Conductivity 






Total 


Carbonate 




Temperature 




oxygen 


(las cm-1) 


Calcium 


Alkalinity 


hardness 


hardness 


Month 


(^C) 


ph 


(ppm) 




(mg/l) 


(mmol/l) 


(°dh) 


(°dh) 


January 


8.13 


6.33 


12.23 


53.00 


8.00 


0.50 


1.20 


1.00 


February 


9.17 


7.13 


— 


60.00 


9.00 


0.50 


1.75 


1.40 


March 


10.80 


6.00 


14.77 


60.00 


10.00 


0.35 


1.30 


1.40 


April 


13.97 


6.07 


13.97 


68.00 


11.00 


0.50 


1.30 


1.40 


May 


18.83 


5.97 


9.77 


78.00 


7.00 


0.60 


1.50 


1.70 


June 


22.73 


5.87 


11.23 


84.80 


8.00 


0.40 


1.20 


1.50 


July 


26.97 


8.17 


8.43 


149.00 


7.00 


0.45 


1.60 


1.50 


August 


26.17 


7.33 


— 


225.00 


13.00 


0.80 


2.00 


1.50 


September 


26.27 


7.37 


9.93 


112.00 


20.00 


0.80 


1.90 


1.70 


October 


17.67 


8.27 


8.43 


92.50 


10.00 


0.75 


1.55 


1.50 


November 


14.80 


7.23 


9.77 


82.00 


10.00 


0.65 


1.85 


1.60 


December 


11.63 


7.20 


9.93 


76.00 


13.00 


0.50 


1.70 


1.40 


Mean 


17.26 


6.91 


10.85 


95.03 


10.50 


0.57 


1.57 


1.47 


sd 


6.62 


0.81 


2.07 


46.53 


3.45 


0.15 


0.27 


0.17 



TABLE 7. Water parameters selected by the logistic 
regression analysis as the most important influenc- 
ing the gravidity of P. amnicum 



Variable 



Wald 



df 



sig. 



Alkalinity 


3.97 


1 


0.05 


-0.077 


Calcium 


16.44 


1 


0.001 


0.209 


pH 


10.27 


1 


0.001 


0.158 


Temperature 


53.25 


1 


10-^ 


-0.40 



Calcium, alkalinity and pH increased in 
summer suggesting that a relation with the 
higher growth speed in these months may 
also exist. Our results show a certain corre- 
spondence between the increase in the val- 
ues of the abovementioned parameters and 
the disappearance of the adults. 

Finally, Hornbach & Cox (1987) suggested 
that in Pisidium casertanum (Poli) there exists 



a possitive relation between high calcium and 
alkalinity values and adult shell length, and 
probably also embryo number and size. If we 
compare the values of these two parameters 
for the Spanish P. amnicum (alkalinity: mean 
= 0.57 mmol/l; calcium: mean := 10.50 mg/l) 
with those of the Finnish population (alkalinity: 
0.17-0.19 mmol/l; calcium = 6.1 mg/l) (Holo- 
painen, 1979), the only available ones, we 
could explain the biggest population parame- 
ters found in Spain (Table 6) as a function of 
the relatively "high" calcium and alkalinity val- 
ues. 

AKNOWLEDGEMENTS 

We are very indebted to Dr. Emilio Rolan 
and his wife for the facilities provided during 
the monthly sampling field trips, and to Diego 
Moreno for his help in the field. We are also 



POPULATION DYNAMICS OF PISIDIUM AMNICUM 



133 



TABLE 8. Reproductive and growth features of several populations of P. amnicum.{^) Several specimens live 
longer but do not breed. (2) Recorded two months before birth in adults bigger than 4 mm. (3) Not all months 
recorded. (4) Recorded in the month immediately after birth. (5) Recorded in the first year of life.(*) Values 
dependant on parents' size and date. 





Germany 


England 


Finland 


Canada 






Danneell & 


Bass 


Holopainen 


Vincent et al. 


Spain This 




Hinz (1976) 


(1979) 


(1979) 


(1981) 


study 


Maximum adult length (mm) 


8.9 


8.5 


8.8 


9 


10-11 


Life span (months) 


12 


12(1) 


— 


36 


15 


Birth period 


May-June 


May 


July 


June 


April- May 


Minimum gravid length (mm) 


2-3 


4 


4.3 


4 


3-4 


% of gravid animals (2) 


80-100% 


1 00% 


— 


42-62 


100% 


Maximum number of 


34 


37 


12 


18 


73 


embryos/larvae* 












Maximum length of larvae (mm)* 


2 


1.4 


0.6(3) 


— 


2.4 


Lateralization 


no 


— 


— 


— 


no 


% of new born in relation to 


< 50% 


< 50% 


— 


62% 


< 50% 


whole population (4) 












Premature births 


yes 


probably 


— 


— 


yes 


Annual mean growth of adults 


3 


4 


— 


2-3 (5) 


5-6 


(mm) 
Winter interruption of larval 


yes 


no 


— 


- 


no 


growth 













very grateful to Dr. Luis M. Carrascal for his in- 
valuable help and advice as regards the sta- 
tistical analysis of the data. Two anonymous 
reviewers made interesting comments which 
improved the manuscript. Thanks also to Dr. 
Pablo Penchaszadeh for his comments and 
help in the preparation of the manuscript and 
to Lesley Ashcroft for the revision of the Eng- 
lish version. This work received financial sup- 
port from the Project "Fauna Ibérica И" (SEUI, 
DGICYTPB89 0081). 



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FORD, E., 1933, An account of the herring investi- 
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Revised ms. accepted 10 August 1998 



POPULATION DYNAMICS OF PISIDIUM AMNICUM 



135 



APPENDIX 1. Summarized information on the specimens collected, dissected and gravid, and of the total 
mean number and mean size of the incubated embryos (or larvae). Frequency of gravid animals at 1% 









freq. 








total 


mean 


mean 








collected 


dis- 




freq. 


of 


number of 


length of 


month 


class 


collected 


% 


sected 


gravid 


gravid 


embryos 


embryos 


embryos 


January 


1-2 




























2-3 




























3-4 


1 


0.61 






















4-5 


4 


2.44 


1 


1 


1 


7 


7 


0.5 




5-6 


34 


20.73 


10 


10 


1 


79 


7.9 


0.52 




6-7 


44 


26.83 


10 


10 


1 


160 


16 


0.66 




7-8 


41 


25.00 


10 


9 


0.9 


309 


34.33 


0.73 




8-9 


37 


22.56 


10 


10 


1 


441 


44.1 


0.79 




9-10 


3 


1.83 


1 


1 


1 


73 


73 


0.85 




10-11 


























Total January 




164 


100 


42 


41 


0.98 


1069 


26,07 


0.67 


February 


1-2 




























2-3 




























3-4 




























4-5 


1 


0.61 






















5-6 


18 


11.04 


7 


6 


0.86 


63 


10.5 


0.55 




6-7 


44 


26.99 


10 


10 


1 


162 


16.2 


0.73 




7-8 


60 


36.81 


10 


10 


1 


361 


36.1 


0.87 




8-9 


35 


21.47 


10 


10 


1 


483 


48.3 


0.9 




9-10 


5 


3.07 


2 


2 


1 


112 


56 


1 




10-11 


























Total February 




163 


100 


39 


38 


0.97 


1181 


31.08 


0.8 


March 


1-2 




























2-3 




























3-4 




























4-5 


1 


0.59 






















5-6 


11 


6.47 


3 


2 


0.66 


11 


3.67 


0.45 




6-7 


35 


20.59 


10 


10 


1 


155 


15.5 


0.84 




7-8 


75 


44.12 


10 


10 


1 


303 


30.3 


0.92 




8-9 


41 


24.12 


10 


10 


1 


446 


44.6 


0.94 




9-10 


7 


4.12 


2 


2 


1 


126 


63 


1.09 




10-11 


























Total March 




170 


100 


35 


34 


0.97 


1041 


29.74 


0.87 


April 


1-2 




























2-3 




























3-4 




























4-5 


1 


0.39 






















5-6 


5 


1.94 


2 


2 




23 


11.5 


0.76 




6-7 


62 


24.03 


10 


10 




155 


15.5 


0.88 




7-8 


91 


35.27 


10 


10 




293 


29.3 


1.23 




8-9 


82 


31.78 


10 


10 




431 


43.1 


1.2 




9-10 


17 


6.59 


5 


5 




211 


42.2 


1.08 




10-11 


























Total April 




258 


100 


37 


37 


1 


1113 


30.08 


1.08 


May 


1-2 


52 


18.25 


10 



















2-3 


106 


37.19 


11 



















3-4 


36 


12.63 


10 



















4-5 




























5-6 


3 


1.05 


1 


1 


1 


11 


11 


1.3 




6-7 


12 


4.21 


3 


1 


0.33 


38 


38 


2.73 




7-8 


35 


12.28 


10 


6 


0.6 


97 


16.17 


1.61 




8-9 


34 


11.93 


10 


3 


0.3 


35 


11.67 


2.02 




9-10 


7 


2.46 


2 


2 


1 


11 


5.5 


1.72 




10-11 


























Total May 




285 


100 


57 


13 


0.23 


192 


14.77 


1.78 
{continued) 



136 



ARAUJOETAL 



APPENDIX 1, (Continued) 









freq. 








total 


mean 


mean 








collected 


dis- 




freq. 


of 


number of 


length of 


month 


class 


collected 


% 


sected 


gravid 


gravid 


embryos 


embryos 


embryos 


June 


1-2 




























2-3 


32 


7.92 






















3-4 


87 


21.53 






















4-5 


4 


0.99 






















5-6 


8 


1.98 


2 



















6-7 


35 


8.66 


5 



















7-8 


106 


26.24 


5 


1 


0.2 


10 


10 


0.06 




8-9 


101 


25 


5 


2 


0.4 


12 


6 


0.07 




9-10 


30 


7.43 


5 


1 


0.2 


11 


11 


0.08 




10-11 


1 


0.25 




















Total June 




404 


100 


22 


4 


0.18 


33 


8.25 


0.07 


July 


1-2 




























2-3 




























3-4 


66 


25.38 


4 



















4-5 


120 


46.15 


4 



















5-6 


10 


3.85 






















6-7 


18 


6.92 


3 



















7-8 


24 


9.23 


5 



















8-9 


12 


4.62 


4 


3 


0.75 


9 


3 


0.06 




9-10 


10 


3.85 






















10-11m 


























Total July 




260 


100 


20 


3 


0.15 


9 


3 


0.06 


August 


1-2 




























2-3 




























3-4 


4 


4.3 






















4-5 


17 


18.28 


5 



















5-6 


6 


6.45 


1 



















6-7 


11 


11.83 


5 



















7-8 


25 


26.88 


4 



















8-9 


22 


23.66 


4 


3 


0.75 


7 


2.33 


0.08 




9-10 


7 


7.53 


2 



















10-11 


1 


1.08 




















Total August 




93 


100 


21 


3 


0.14 


7 


2.33 


0.08 


September 


1-2 




























2-3 




























3-4 


13 


3.71 


2 



















4-5 


37 


10.57 


8 


1 


0.125 


13 


13 


0.08 




5-6 


67 


19.14 


10 


3 


0.3 


26 


4.33 


0.03 




6-7 


143 


40.86 


6 


3 


0.5 


55 


18.33 


0.12 




7-8 


65 


18.57 


6 


5 


0.83 


128 


25.6 


0.09 




8-9 


19 


5.43 


5 


3 


0.6 


77 


25.67 


0.14 




9-10 


6 


1.71 


2 


1 


0.5 


42 


42 


0.18 




10-11 


























Total Septembe 


r 


350 


100 


39 


16 


0.41 


341 


17.95 


0.09 


October 


1-2 




























2-3 




























3-4 


12 


5.58 


4 


2 


0.5 


13 


6.5 


0.06 




4-5 


50 


23.26 


6 


6 




80 


13.33 


0.12 




5-6 


52 


24.19 


6 


6 




111 


18.5 


0.18 




6-7 


28 


13.02 


6 


6 




173 


28.83 


0.22 




7-8 


40 


18.6 


6 


6 




191 


31.83 


0.22 




8-9 


32 


14.88 


6 


6 




214 


35.67 


0.25 




9-10 


1 


0.47 






















10-11 


























Total October 




215 


100 


34 


32 


0.94 


782 


24.44 


0.19 



POPULATION DYNAMICS OF PISIDIUM AMNICUM 



137 



APPENDIX 1, (Continued) 









freq. 








total 


mean num- 


mean 








collected 


dis- 




freq. 


of 


ber of 


length of 


month 


class 


collected 


% 


sected 


gravid 


gravid 


embryos 


embryos 


embryos 


November 


1-2 




























2-3 




























3-4 


2 


0.99 






















4-5 


9 


4.46 


2 


2 




30 


15 


0.13 




5-6 


19 


9.41 


3 


3 




46 


15.33 


0.25 




6-7 


49 


24.26 


6 


6 




170 


28.33 


0.37 




7-8 


85 


42.08 


6 


6 




225 


37.5 


0.34 




8-9 


33 


16.34 


6 


6 




247 


41.17 


0.34 




9-10 


5 


2.48 


1 


1 




40 


40 


0.35 




10-11 


























Total November 




202 


100 


24 


24 


1 


758 


31.58 


0.32 


December 


1-2 




























2-3 




























3-4 


1 


0.58 






















4-5 


5 


2.89 






















5-6 


15 


8.67 


4 


4 




58 


14.5 


0.42 




6-7 


43 


24.86 


9 


9 




208 


23.11 


0.42 




7-8 


69 


39.88 


6 


6 




253 


42.17 


0.55 




8-9 


36 


20.81 


6 


6 




260 


43.33 


0.6 




9-10 


4 


2.31 


3 


3 




133 


44.33 


0.45 




10-11 

























Total December 




173 


100 


28 


28 


1 


912 


32.57 


0.49 


TOTAL 




2737 




398 


276 


0.69 


7438 


26.95 


0.66 



MALACOLOGIA, 1999, 41(1): 139-145 

RELATIONSHIPS BETWEEN LENGTH OF PREY/PREDATOR FOR THE MOST 

IMPORTANT PREY OF THE CUTTLEFISH SEPIA OFFICINALIS L. 

(MOLLUSCA: CEPHALOPODA) 

Alexia Blanc, Guy Pinczon du Sel & Jacques Daguzan 

Laboratoire de Zoologie et d'Ecophysiologie, Bat 13, Campus de Beaulieu, Université de 
Rennes I, 35042 Rennes Cedex, France; alexia.blanc@univ-rennes1.fr 

ABSTRACT 

As is typical of cepinalopods, the cuttlefish Sepia officinalis L. is an opportunist predator. This 
study shows that Sepia selects the length of its prey taking into account its own size. In the nat- 
ural environment, the most important prey are fishes and crabs. For each group, we have linked 
the prey remains (found regularly in the stomach contents of the cuttlefish) to total prey length. 
Prey remains consist of the merus of pereiopods 2 to 5 for crabs and otoliths for fishes. These 
pieces are not attacked by enzymes during their transit through the digestive tract. 

Fishes become prey when they measure between 25-80% of the DML (Dorsal Mantle Length) 
of the cephalopod. The cuttlefish attack crabs when they measure between 20-40% of their own 
DML. Cuttlefish attack crabs by jumping or pouncing without using their tentacles. 



INTRODUCTION 

Throughout its geographical distribution, 
the diet of the cuttlefish Sepia officinalis L. 
consists of fishes and crustaceans (Brachy- 
ura and Macrura): in the English Channel 
(Richard, 1 971 ), in the Bay of Biscay (Pinczon 
du Sel, 1 996), on the Spanish coast (Castro & 
Guerra, 1989), on the Italian coast (Scalera 
Liaci & Piscitelli, 1982), and on the Tunisian 
coast (Najai & Ktari, 1 979). In the Ria de Vigo, 
Guerra (1 985) showed that the composition of 
the diet of Sepia officinalis changes with 
growth (Amphipoda and Caridea for individu- 
als with a DML less than 45 mm, and Porcel- 
lanidae, Brachyura, and fishes when the DML 
is above 55 mm). Castro & Guerra (1990) re- 
marked that in the diet of Sepia officinalis the 
proportion of crustaceans decreases whereas 
that of fishes increases with cuttlefish growth. 
However, the decrease of crustaceans in gen- 
eral did not modify the importance of Brachy- 
ura for larger cuttlefish. 

Only a few studies have been carried out to 
determine the relationships between the 
length of the prey and the length of the cuttle- 
fish. Moreover, these studies have been car- 
ried out only in the laboratory (Boulet, 1964; 
Duval etal., 1984). 

Fishes and Brachyura were identified re- 
spectively by the otoliths and principally by 
the periopods or walking legs. We next linked 
these to the size of the prey. Thus, we can 



begin to elucidate the relationship between 
length or size of the predator to its prey. 



MATERIALS AND METHODS 

All cuttlefish examined for this study were 
collected by 2 trawls (10 x 3 x 0.6 m with 20 
mm mesh size and 3.5 x 1.2 x 0.4 m with 5 
mm mesh size to capture young) in the Bay of 
Biscay from June 1 990 to July 1 992, thus tak- 
ing into account all the stages between hatch- 
ing and death. Only two Sepia were under 10 
cm, 19 between 10-14 cm, 12 between 15- 
19 cm, 15 between 20-24 cm, and 12 Sepia 
greater than 25 cm. The prey items was iden- 
tified under binocular microscope. The stom- 
ach contents were examined in all 60 cuttle- 
fish. 

Crabs parts were present in 28 and fishes 
in 32 of the cuttlefish studied. For fishes and 
crabs, when the weight of the stomach con- 
tents is significant there is only one type of 
prey present in the stomach (Pinczon du Sel, 
1996). In five cuttlefish, there were two prey 
items in the stomach (two fishes or fishes with 
Brachyura not Identified). 

Carcinus maenas L. (Crustacea: Brachyura) 

In the Bay of Biscay, the most important 
Brachyura taken by Sepia officinalis is Carci- 
nus maenas L. (Pinczon du Sel, 1996). To ap- 



139 



140 



BLANC, PINCZON DU SEL & DAGUZAN 



preciate the size range of the captured crabs, 
a total of 100 crabs were measured and 
weighed. References to size are based on the 
maximum length and width of the carapace 
and maximum width of the merus of perei- 
opods 2 to 5 (Fig. 1 ). This portion of the walk- 
ing legs was often found in the stomach con- 
tents. In a laboratory study, cuttlefish eat only 
52.5% of the crabs and do not consume the 
legs (Pinczon du Sel, 1996). All the measure- 
ments were made to the nearest 0.1 mm, and 
weights to the nearest 0.1 g. 

The merus of the walking legs identified in 
the stomach contents were measured. Then, 
the width of the crab was estimated from the 
allometric relation as well as the surface area 
of the céphalothorax. When several merus 
were present in the same gut content, the 
pairs were reconstituted by minimizing the 
variations. The size used to calculate crab 
length was the mean width of each pair. Next, 
the width of the céphalothorax was linked to 
the size of the merus (Fig. 2) and to the sur- 
face area of the céphalothorax (surface area 
was calculated by length times width) (Fig. 3). 

Duval et al. (1 984) studied the method used 
by cuttlefish to capture crabs in relation to the 
surface area of the céphalothorax. The com- 
parison index is R = weight of the cuttlefish 
(g) / surface of the céphalothorax of the crab 
(mm^). Duval et al. (1984) noted that when R 
was greater than 1 (i.e., the crab was "small"), 
the crab was captured using the tentacules. 
However, when R was less than 1 (i.e., the 
crab was large), capture was done by jumping 
or pouncing without using the tentacules (data 
not submitted). 

Fishes 

A diversity of fishes constitute the second 
most important prey for cuttlefish. The re- 
mains of fish prey are identified in stomach 
contents by the presence of otoliths, bones of 
the inner ear, shape. Moreover, as is typical of 
bones, there is an allomethc relationship be- 
tween the length of the otolith and the total 
length (Lt: from head to caudal fork) and 
weight of the fish. 

This study was carried out on seven 
species of fishes, which represented the most 
important elements found in the stomach con- 
tents (Table 1, Fig. 4). We compare otoliths 
found in stomach contents with a collection of 
otoliths made for several fish species of the 
Bay of Biscay. 

Before using the allometric relationship be- 




FIG. 1. Measurements made on the crab Carcinus 
maenas L. (scale bar = 2 cm). LC, length of the 
céphalothorax; WC, width of the céphalothorax; 
WM, width of the merus of P3 (pereiopods 3) (draw- 
ing from Christiansen, 1969) 




0.2 0.3 0.4 0.5 0.6 

width of crab merus (cm) 

FIG. 2. Carcinus maenas L. Allometric relationship 
between width of the merus of pereiopods 2 to 5 
and width of the céphalothorax. Y = 13.082 x + 
1.040; r = 0.965 



tween length of otolith and length of fish, we 
had to be sure that the otoliths were not 
eroded by digestive enzymes during their 
passage through the cuttlefish digestive tract. 
As a control, fishes were weighed, measured 
and the length of the otolith estimated and fed 
to cuttlefish. When otoliths were recovered in 
the faeces, the two lengths could be com- 
pared. 

All otoliths found in the cuttlefish stomach 
content were identified and measured to the 
nearest 0.01 mm. When several otoliths were 
present in the same stomach, they were all 
measured and the pairs were reconstituted by 
minimizing the variations. Then, the allometric 



CUTTLEFISH PREY/PREDATOR-LENGTH RELATIONSHIPS 



141 




width (cm) 

FIG. 3. Carcinus maenas L, Allometric relationship 
between width of the céphalothorax and its surface 
area. Y = 0.739 x 2.003; r = 0.995 

relations allowed us to estimate the length 
and weight of the fishes caught by Sepia. 

RESULTS 

Of the 60 cuttlefish examined, 28 contained 
crabs parts in the stomach. The cuttlefish that 
contained crab pereiopods ranged in size 
1 1 -29 cm DML and 200-2200 g in total body 
net weight. 

The ratios of prey to predator sizes (width of 
the céphalothorax of prey to DML of predator 
= WC/DML) ranged from 20% about 35% of 
the predator DML (Fig. 5). Thus, the lengths of 
captured crabs increase as the length of the 
predator increases. Therefore, there was a 
slight trend for crab size to decline as cuttle- 
fish size increases. The size range of wild 
crabs was between 30-80 mm in carapace 
width. As the cuttlefish grow, if they do not 
have enough large crabs, they eat smaller 
ones. The ratios of weights range are be- 
tween 0.1 -4.0% of the weight of the cuttlefish, 
with a greater inter-individual variation. Some 
adult cuttlefish (w = 1 .875 kg) eat small crabs 
(w = 38.04 g) and vice versa (Fig. 6). 

The ratio R (weight of cuttlefish to surface 
area of crab céphalothorax) is given in the 
Table 2. The majority of the ratios are less 
than 1 and the crabs are considered to be 
"large". 

The size range of cuttlefish with otoliths in 
their gut was between 4 cm to 29 cm, with a 
weight between 1 8 g to 2025 g. The results of 
our study on the digestion of otoliths indicates 
that they are not attacked by the cuttlefishes' 



digestive enzymes (Table 3). Thus, we can 
use the allometric relationship between length 
of otolith and length of fish to estimate the size 
of the captured fish. 

The majority of the size ratios (length of fish 
to DML of cuttlefish) are between 25-80% 
(Fig. 7) in Morbihan Bay (breeding area) as 
well as in the Morbraz (growth area). These 
are both areas of the Bay of Biscay. Only 
three cuttlefish had otoliths in their stomach 
contents, indicating fishes with length ratios 
greater than 80% of the DML. Moreover, we 
can see that among these three cases, two 
show a ratio greater than 100% (116 and 
120%). These three fish belong to the same 
species with a greatly elongate shape, the eel 
Anguilla anguilla L. 

The majority of cuttlefish (73%) eat fish with 
an estimated weight of less than 15 g (Fig. 8). 
Sepia seems to be more selective of fishes 
than crabs. Anguillidae, Gadidae and Labri- 
dae eaten were greater than 15 g, Gobiidae 
and Atherinidae less than 1 5 g. 



DISCUSSION 

Although the cuttlefish. Sepia officinalis is 
opportunist in its choice of prey-species (Najai 
& Ktari, 1979), it seems to be more selective 
when it comes to prey size. Size ratios for 
fishes ranges from 25-80% of the DML of the 
predator and between 20-40% for the crabs. 
Sepia officinalis L. can also seize its prey with 
its arms by jumping on it (Wilson, 1946; Mes- 
senger, 1968). 

The prey's capacity to defend or to escape 
capture increases with prey size; larger prey 
are also more difficult for the cuttlefish to at- 
tack and capture successfully. In Sepia ele- 
gans, there was a relationship between tenta- 
cle club length and body size. Individual 
cuttlefish equipped with comparatively longer 
clubs can capture larger prey (Bello, 1991). 

Boulet (1964) observed that the ratio be- 
tween the size of the crab and of the cuttlefish 
may inhibit predatory behaviour of the cepha- 
lopod; if the crab is too big the cuttlefish will 
not attempt to capture it. 

A study on the energy expended in prey cap- 
ture and the energy gained from ingestion and 
assimilation could perhaps explain the larger 
size limit. In the case of Octopus vulgaris, the 
stimulus for prédation is controlled by the 
repletion ratio of the crop (number of full stom- 
achs of the octopus to number of total number 
of stomach contents examined (Young, 1964; 



142 



BLANC, PINCZON DU SEL& DAGUZAN 



TABLE 1 . Allometric relationship between otolith length and the length and weight of several species of fish 
prey. 



Family 



Species 



L(mm) = f(Lo(mm)) 



r 


n 


References 


0.94 


25 


Monaix, 1992 


0.92 


58 


Pinczon du Sel, 1996 


0.99 


30 


La Мао, 1985 


0.88 


28 


Pinczon du Sel, 1996 


0.99 


32 


Pinczon du Sel, 1996 



Anguillidae 

Gadidae 

Atherinidae 

Callonymidae 

Gobiidae 



Anguilla anguilla L. 
Trisopterus lu se и s L. 
Atherina presbyter Cuvier 
Callyonymus lyra L. 
G obi и s s p./ 
Pomatochistus sp. 



L= 186.73LO- 85.35 
L = 31 .877LO- 89.924 
L = 31.91Lo-0.35 
L = 87.323Lo- 72.783 
L = 29.461 Lo- 9.238 



Lo = length of the otolith (see also Fig. 4) 



CaLLIONWIIDAE'S OTOLITH 



Atherimdaf's otolith 




о I о 




FIG. 4. Measurements made on fish otoliths. Lo, length of otolith. 



40%- 










■ 


■ 






30%- 


■ 


■ 


■ 
■ 




■ ■ 
Ш 


■ 


20%- 


Ш 


1 ■ 

■ 


■ 


Ш 

»-- 


..Л. 












Ш 




10%- 
















1 


1 




1 


1 






10 


15 




20 


25 


3( 



200- 



cuttlefish DML (cm) 

FIG. 5. Ratio of the "width of the céphalothorax of 
the captured crab ^ DML of the cuttlefish" plotted 
against cuttlefish (predator) DML. 



g, 150-1 
0) 

g 100- 

"О 

Е 50- 



IxfS-? 



.# 



cuttlefish weigth (g) 



.^ 



.# 



FIG. 6. Relationship between cuttlefish weight and 
estimated crab weight. 



CUTTLEFISH PREY/PREDATOR-LENGTH RELATIONSHIPS 



143 



TABLE 2. Estimates of the surface area of the céphalothorax of crabs captured by cuttlefish in their natural 
environment and the calculation of the ratio R. 



Sepia 


officinalis 




Carcinus maenas 






DML 


Weight 


Width of the 


Carapace surface 


Crab 


Ratio 


(cm) 


g) 


carapace (mm) 


area (mm^) 


weight (g) 


R 


11 


200 


29.8 


632.9 


1.74 


0.32 


13 


250 


33.3 


796.3 


3.26 


0.31 


14 


372 


40.6 


1178.2 


8.86 


0.32 


15 


325 


51.6 


1888.3 


27.11 


0.17 


17 


400 


62.1 


2716.6 


61.33 


0.15 


18 


525 


43.8 


1364.3 


12.66 


0.38 


20 


1078 


73.2 


3758.5 


123.84 


0.29 


21 


840 


56.2 


2232.5 


39.65 


0.38 


22 


1100 


69.3 


3371.8 


98.13 


0.33 


23 


1246 


36.6 


957.7 


5.27 


1.30 


24 


1100 


55.0 


2181.6 


37.64 


0.50 


24 


1413 


58.8 


2441.9 


48.44 


0.58 


25 


1220 


52.9 


1983.7 


30.35 


0.62 


25 


1265 


78.6 


4320.4 


166.43 


0.29 


26 


1560 


65.3 


3005.8 


76.52 


0.52 


10 


145 


30.4 


666.3 


2.01 


0.22 


13 


225 


43.8 


1364.3 


12.66 


0.16 


13 


254 


34.9 


871.9 


4.16 


0.29 


14 


325 


30.9 


689.1 


2.20 


0.47 


15 


325 


40.4 


1163.3 


8.59 


0.28 


16 


450 


53.3 


2012.8 


31.37 


0.22 


18 


600 


50.8 


1832.1 


25.30 


0.33 


23 


1157 


67.3 


3186.2 


86.84 


0.36 


24 


1375 


64.2 


2900.1 


70.77 


0.47 


25 


1344 


46.4 


1529.6 


16.62 


0.88 


25 


1550 


62.3 


2739.3 


62.45 


0.57 


28 


2200 


81.4 


3409.6 


193.20 


0.64 


29 


1875 


55.7 


2191.7 


38.04 


0.86 



R = weight of the cuttlefish (g). surface of the céphalothorax (mm ) (Duval et al., 1984). 



Nixon, 1965, 1966). Mather (1980) obtained 
the same results with a second species of oc- 
topus, Octopus joubinii. 

There is another hypothesis that must also 
be taken into account in the attack and cap- 
ture of larger prey. This relates to the effi- 
ciency of the toxic secretions from the poste- 
rior salivary glands of Sepia on larger fishes 
or crabs. The injection of a toxic saliva pro- 
vokes the rapid paralysis of the crab (Chich- 
ery & Chichery, 1992). 

For each type of prey (crabs and fishes), the 
length ratios remain constant in relation to the 
DML of the predator. The anatomical charac- 
teristics of the digestive tract constrains the 
ability of the cuttlefish to break down prey tis- 
sue into particles that can pass through the oe- 
sophagus where it penetrates the central ner- 
vous system. Guerra et al. (1 988) showed that 
the antero-postehor length of the buccal mass 
increases in size as the oesophagus grows, 
until the cuttlefish reaches a DML of 150 mm. 



This implies that the size of ingested particles 
is limited by these two parameters. In other 
words, the larger the prey is, the more the buc- 
cal mass -the beaks and radula-must work 
to reduce the prey before ingestion take place. 

Fishes appear to be preferentially captured 
by the tentacle strategy, whereas crabs are 
captured by both strategies (Messenger, 
1968). The tentacle strategy was often used 
for smaller crabs (Chichery & Chichery, 1 991 ). 

Messenger (1968) demonstrated in a labo- 
ratory study that crabs can be attacked by two 
methods -a jump or tentacular strike. Duval 
et al. (1984) indicated that the attack behav- 
iour is dependent on the ratio R (weight of the 
cuttlefish/surface area of the crabs' céphalo- 
thorax). Estimates based on stomach con- 
tents show that the majority of crabs are cap- 
tured by the jump method. Control of the two 
first parts of the attack (attention and position- 
ing) is a visually controlled loop system (Mes- 
senger, 1968). The prey and its movements 



144 



BLANC, PINCZON DU SEL & DAGUZAN 



TABLE 3. Comparison of estimated otolith lengtlns after digestion of thie fisfi prey by tfie cuttlefisfi (n = 14) 









Total length 


Otolith length 


Otolith length 


Teleostan 






of the 


estimated 


following 


family 


Species 




fish (mm) 


(mm) 


digestion (mm) 


Gadidae 


Trisopterus luscus 




200 


9.29 


8.70 


Gadidae 


Trisopterus luscus 




180 


8.47 


8.60* 


Gadidae 


Trisopterus luscus 




215 


9.57 


9.80* 


Gadidae 


Trisopterus luscus 




230 


10.04 


10.60* 


Gadidae 


Trisopterus luscus 




94 


5.77 


5.30 


Callionymidae 


Callionymus lyra 




205 


3.18 


3.55* 


Callionymidae 


Callionymus lyra 




230 


3.47 


3.41 


Callionymidae 


Callionymus lyra 




185 


2.95 


3.02* 


Callionymidae 


Callionymus lyra 




255 


3.75 


3.55 


Callionymidae 


Callionymus lyra 




275 


3.98 


3.58 


Gobiidae 


Gobius sp. or Pomatoschistus 


sp. 


91 


3.40 


3.55* 


Gobiidae 


Gobius sp. or Pomatoschistus 


sp. 


73 


2.79 


2.78 


Gobiidae 


Gobius sp. or Pomatoschistus 


sp. 


103 


3.81 


3.71 


Gobiidae 


Gobius sp. or Pomatoschistus 


sp. 


54 


2.15 


2.14 


Gobiidae 


Gobius sp. or Pomatoschistus 


sp. 


89 


3.33 


3.30 


Gobiidae 


Gobius sp. or Pomatoschistus 


sp. 


102 


3.78 


3.30 


Mean ± sd 








4.98 ± 2.72 


4.96 ± 2.77 



*Some data on otolith length following digestion were greater than these of estimated otolith length. The latter came from the 
equations given in Table 1 . There were also variations. 




10 15 20 

cuttlefish DML (cm) 

FIG. 7. Ratio of the "length of the captured fish ^ 
DML of the cuttlefish" plotted against DML cuttlefish 
(predator) DML. 

are identified by the cuttlefish before begin- 
ning an attack. The possibility of identification 
of the prey in terms of type and size, possibil 
with an accurate spatial location, increases 
the rate of attack success. The tentacle strat- 
egy is the best adapted for prey that possess 
a rapid escape response (Duval et al., 1984; 
Chichery & Chichery, 1992). 

Hurley (1976) noted that the young squid 
Loligo opalescens showed an absence of se- 
lectivity for prey length. But this author also 
noticed that, although success of the hunt de- 





60 П 




B, 










Ш 






3 

Où 

1 


50- 
40- 




В 

■ 

Ш 


E 






30- 
20- 


BW 

■ 


■ E 


шЕ 




10- 










и 


Л 










OH 


■^ ■ 


— ^— 


V- 


— 1 



s^ Ф* г^ 
cuttlefish weight (g) 



FIG. 8. Relationship between cuttlefish weight and 

estimated fish weight. 

E: eels G: gobies 

B; bibs S: sand smelts 

BW: bailan wrasses 



pended on prey size. Other factors have to be 
taken into account to explain the variations in 
behaviour ^the prey species hunted, and the 
age, experience, and motivation of the preda- 
tor. 

Otoliths are routenely used to identify fishes 
and to estimate their sizes. Digestive en- 
zymes do not attack these hard structures. 



CUTTLEFISH PREY/PREDATOR-LENGTH RELATIONSHIPS 



145 



Jobling & Breiby (1986) noticed the same re- 
sult with the squid Todarodes sagittatus. They 
estimated that the pH (5.2 to 6.3) of the di- 
gestive tract of oceanic squid was not acidic 
enough to dissolve these structures. 

The only fish that is attacked even if the 
length ratio is unfavourable to the predator is 
the eel. Our observations in the laboratory re- 
vealed that the tentacular method undergoes 
some modifications. In these case, the head 
of the eel is not targeted first. The first bite is 
made on the spinal column. Subsequently, 
manipulation to bring the head of the fish to 
the mouth can be done because risks of flight 
have been minimized. 

ACKNOWLEDGEMENTS 

We are grateful to Dr. A. Leroux for his ad- 
vice and L. Allano for his technical assistance. 
We also thank the two referees for their com- 
ments and advice. 



LITERATURE CITED 

BELLO G., 1991, Relationship between tentacle 
club length and body size in Sepia elegans. pp. 
92-97, in: E. boucaud-camou, ed., Acta of the 
first international symposium on the cuttlefish 
Sepia, Centre de Publications de l'Université de 
Caen. 

BOULET, P. С, 1964, La prédation chez la seiche. 
Actualités l\/lannes. 8(2): 26-32. 

CASTRO, B. G. & A. GUERRA, 1989, Feeding pat- 
tern of Sepia officinalis (Cephalopoda: Sepi- 
oidea) in the Ria de Vigo (NW Spain). Journal of 
Marine Biological Association of the United King- 
dom. 69: 545-553. 

CASTRO, B. G. & A. GUERRA, 1990, The diet of 
Sepia officinalis L. and Sepia elegans Blainville 
1827 (Cephalopoda, Sepioidea) from the Ria de 
Vigo (NW Spain). Sciencia Marina. 54(4): 375- 
388. 

CHICHERY, R. & M. P CHICHERY, 1991, The 
predatory behaviour of Sepia officinalis, pp. 233- 
239, in: E. BOUCAUD-CAMOU, ed.. Acta of the first 
international symposium on the cuttlefish Sepia. 
Centre de Publications de l'Université de Caen. 

CHICHERY R. & M. P CHICHERY, 1992, Learning 
performances and aging in cuttlefish {Sepia offic- 
inalis). Experimental Gerontology. 27: 233-239. 

CHRISTIANSEN, M. E., 1969. Decapoda Brachy- 
ura. Marine Invertebrates of Scandinavia n'2, 
Scandinavia University Books, Universtets for- 
laget, Oslo, 143 pp. 

DUVAL, P, M. P CHICHERY & R. CHICHERY, 
1984, Prey capture by the cuttlefish (Sepia offici- 
nalis): an experimental study of two strategies. 
Behavioural Processes, 9: 13-21. 



GUERRA, A. 1985. Food of the cuttlefish Sepia of- 
ficinalis and Sepia elegans in the Ria de Vigo 
(NW Spain) (Mollusca: Cephalopoda). Journal of 
Zoo/ogfy, 207:511-519. 

GUERRA, A., M. NIXON & B. G. CASTRO, 1988, 
Initial stages of food ingestion by Sepia officinalis 
(Mollusca: Cephalopoda). Journal of Zoology 
214: 189-197. 

HURLEY, A. C, 1976, Feeding behavior, food con- 
sumption, growth and respiration of the squid 
Loligo opalescens raised in the laboratory. Fish- 
ery Bulletin, 74{^)■. 176-182. 

JOBLING, M. & A. BREIBY 1986, The use and 
abuse of fish otoliths in studies of feeding habits 
of marine piscivores. Scarcia, 71 : 265-274. 

LA MAO, P., 1985, Peuplements piscicole et 
teuthologique du bassin maritime de la Ranee: 
Impact de l'aménagement marémoteur Thèse de 
Doctorat de 3ème cycle. ENSA Rennes I. 1 25 pp. 

MATHER, J. A., 1 980, Some aspects of food intake 
in Octopus joubini. The Veliger 22(3): 286-290. 

MESSENGER, J. В., 1968, The visual attack of the 
cuttlefish Sepia officinalis. Animal Behaviour, 16: 
342-357. 

MOUNAIX, В., 1992, Intercalibration et validation 
des méthodes d'estimations de l'âge de l'anguille 
européenne (Anguilla anguilla L.). Application au 
bassin versant de la Vilaine, Bretagne. Publica- 
tions du Département d'Halieutique, ENSA 
Rennes, 14: 1-150. 

NAJAI, S. & M. H. KTARI, 1 979, Etude du régime al- 
imentaire de la seiche commune Sepia officinalis 
L. du Golfe de Tunis. Bulletin Institut National Sci- 
ences Techniques Océanographique Pêche, 
Salammbô, Tunisie, 6(1 -4): 53-61 . 

NIXON, M., 1965, Some observation on food intake 
and learning in Octopus vulgaris. Journal of Zool- 
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NIXON, M., 1966, Changes in body weight and in- 
take of food by Octopus vulgaris. Journal of Zool- 
ogy 150: 1-9. 

PINCZON DU SEL, G., 1996. Régime alimentaire 
de la seiche Sepia officinalis L. (Mollusque, 
Céphalopode, Sepiidae) dans le secteur Bre- 
tagne Sud. Thèse d'Université Rennes I, 270p. 

RICHARD, A., 1971, Contribution à l'étude expéri- 
mentale de la croissance et de la maturation sex- 
uelle chez le céphalopode Sepia officinalis L. 
(Mollusque, Céphalopode), Thèse Doctorat d'E- 
tat, Univ Lille, 264 p. 

SCALERA LIACI, L. & G. PISCITELLI, 1982, Ali- 
mentazione di Sepia officinalis L. nella laguna di 
Lesina. Bollettino del Musei Istiti Biologici dell' 
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Revised ms. accepted 14 October 1998 



MALACOLOGIA, 1999, 41(1): 147-150 

THE ACCUMULATION OF TAXONOMIC KNOWLEDGE: THE HISTORY OF 
SPECIES DESCRIPTIONS OF SOME PREDATORY GASTROPODS 

Geerat J. Vermeij 

Department of Geology and Center for Population Biology. University of California at Davis 
One Sfiields Avenue Davis, California, 95616 USA; vermeij(g)geology ucdavis.edu 

ABSTRACT 

In order to assess how perceived temporal and spatial patterns of distribution of species de- 
pend on taxonomic knowledge, I compiled dates of description of all 558 known gastropod 
species with shells having a labral tooth. Peaks in the number of described species were reached 
in the intervals of 1 820- 1 859 and 1 980- 1 999 for Recent species, and 1 920- 1 939 for fossil taxa. 
Distributional patterns were already evident by 1859, when about half of the living and 13% of 
the fossil species had been described; but geographical and temporal ranges are very sensitive 
to accumulated taxonomic knowledge, and have changed substantially as more species were 
described. An asymptote in the number of species has not yet been approached either in these 
gastropods or in molluscs and other organisms generally. This fact underscores the importance 
of continuing support for taxonomic research. 

Key words: taxonomy, gastropods, labral tooth, history. 



INTRODUCTION 

For some years I have been studying the 
functional morphology, taxonomy, phylogeny, 
and distribution in time and space of predatory 
gastropods with a labral tooth, a sharp, ven- 
trally and sometimes anteriorly directed pro- 
trusion of the abapical or medial sector of the 
shell's outer lip. In all studies of this kind, ques- 
tions arise about the completeness of taxo- 
nomic data, and about the robustness of the 
patterns that those data reveal. One approach 
to tackling these questions is to document the 
history of description of species since the be- 
ginnings of formal zoological taxonomy in 
1758. If the number of species described per 
time interval falls off, we may infer that an as- 
ymptotic number is being approached, which 
would mean either that we are close to dis- 
covering all existing or knowable species, or 
that taxonomic interest is waning. This latter 
possibility is potentially a matter of concern, 
because it affects directly the amount and 
quality of fundamental data on which most 
studies of ecology, evolution, biogeography, 
stratigraphy, and many other fields depend. 

Here I report the results of my compilation 
of the times of description of all fossil and 
living gastropod species with a labral tooth 
known to me. Although these gastropods rep- 
resent a tiny and potentially unrepresentative 



fraction of gastropods as a whole, to say noth- 
ing of living things generally, they reveal pat- 
terns of description that are both surprising 
and informative. 



METHODS 

I have assembled a list of all fossil and liv- 
ing gastropod species with a labral tooth. A 
species was included if I observed a labral 
tooth on specimens, or if authors describing 
or discussing the species in question clearly 
indicated the presence of a labral tooth. In a 
large number of cases, the original describers 
were unaware of, or failed to record, the pres- 
ence or characteristics of the labral tooth. My 
own examination of material in museum col- 
lections therefore revealed many cases that 
would not have come to light from a search of 
the literature alone. 

For each species, I used the earliest pub- 
lished name, even if that name is not the cur- 
rently accepted one because of homonymy. 
Names that on the basis of my studies or tax- 
onomic revisions by other authors appear to 
be synonyms were not included. 

I grouped dates of publication into 20-year 
time intervals. For each interval, I compiled 
separately the number of living and fossil 
species. 



147 



148 



VERMEIJ 



RESULTS AND DISCUSSION 

My current tally shows that 558 species (21 9 
Recent and 339 fossil) have a labral tooth. 
These species range in age from Late Creta- 
ceous (Campanian) to Recent, and belong to 
ten families. These include the tonnoidean 
Ranellidae, and the neogastropod families 
Muhcidae, Buccinidae, Melongenidae, Nas- 
sariidae, Fasciolariidae, Pseudolividae, Tur- 
binellidae, Lividae, and Marginellidae. 

The temporal pattern of description of Re- 
cent species differs from that of fossil ones 
(Fig. 1). For Recent species, there was a 
broad peak from 1820 to 1859, and a very 
conspicuous maximum in the present 20-year 
interval, 1980-1999. The first half of the 20th 
century (1900-1959) represents a broad 
trough in the number of described Recent 
species. The fossil data reveal a general rise 
in the number of described species per inter- 
val to a peak in the years 1920-1939, fol- 
lowed by more than half a century of stability 
at a high level (about 30 species per 20-year 
Interval) of taxonomic activity. 

A striking result of this study is that the most 
recent time interval (1980-1999) has con- 
tributed a sizable fraction of our accumulated 
taxonomic knowledge. Of the 219 Recent 
species, 48 (21.9%) have been described 
during this interval. Among the 339 fossil spe- 
cies, the corresponding number is 34 (9.4%). 
For Recent species, taxonomic knowledge 
has doubled since 1 860; for fossil species, the 
number has doubled since 1920. 

As Sepkoski et al. (1981) and Sepkoski 
(1993) have pointed out in their studies of 
the number of families and genera of marine 
fossil invertebrates, increases in taxonomic 
knowledge do not necessarily require revision 
in our perception of temporal and spatial pat- 
terns of distribution of species. A general in- 
crease in the number of taxa over geological 
time, and the existence of a latitudinal in- 
crease in diversity from the poles to the equa- 
tor, are robust patterns that were discernible 
by 1859, the date of publication of Charles 
Darwin's Origin of Species. Similarly, the 
broad outlines of distribution of gastropods 
with a labral tooth were already apparent by 
this date when, according to my data, about 
half the living species with a labral tooth and 
13% of the presently known fossil taxa had 
been named. By the middle of the 19th cen- 
tury, observers able to examine specimens 
could already have concluded that the inci- 



dence of labral teeth is higher among living 
predatory gastropods on the Pacific than on 
the Atlantic side of the Americas, and that the 
incidence in Europe declined from the Mio- 
cene to the Recent. 

Other details of distribution, especially first 
appearances and unusual geographical 
records, are very sensitive to accumulated 
knowledge. For example, only one of 30 Cre- 
taceous gastropods with a labral tooth -Sl/c- 
cinopsis parryi Conrad, 1 857 -was described 
before 1859, and this species was based on 
material so poorly preserved that no contem- 
porary scholar could have inferred the pres- 
ence of a labral tooth. Muricids with a labral 
tooth from the Oligocène, the earliest epoch 
from which this structure is known in this fam- 
ily, were not described until 1 91 8. In that year, 
Clark described Thais pacl<i, but its labral 
tooth was not recognized until 1993 (Amano 
et al., 1 993). A second Oligocène muricid with 
a labral tooth was described by Vokes (1963), 
but its age was originally thought to be early 
Miocene. Until about 1970, therefore, taxono- 
mists might legitimately have concluded that 
muricids with a labral tooth did not originate 
until early Miocene time. 

The continuing high rate of discovery of 
species with a labral tooth mirrors a general 
pattern for molluscs (Bouchet, 1997) and for 
organisms generally (Winston & Metzger, 
1 998). This is surprising in view of the fact that 
most labral-tooth-bearing gastropods are rel- 
atively large (greater than 2 cm in height) and 
thus more easily collected than are micromol- 
luscs. 

The fossil data in Figure 1 are likely to be 
less representative for the description of fossil 
taxa in general than are the data for Recent 
species. Large parts of the world remained 
essentially unexplored paleontologically until 
the present century. This is especially true for 
older rocks and for small fossils. Despite this, 
the vigor of descriptive paleontological activity 
in the latest 20-year interval as indicated in 
Figure 1 is noteworthy. 

The early peak (1820-1859) in taxonomic 
description of Recent gastropods with a labral 
tooth probably reflects the collective efforts of 
major scientific expeditions throughout the 
world. Many of the common shallow-water 
taxa became known during this time. The 
1980-1999 maximum, preceded by a rise in 
taxonomic activity that began in the 1960s, 
likely represents the efforts of divers and of 
expanded trawling and dredging operations. It 



TAXONOMIC DESCRIPTION THROUGH TIME 



149 



1740-1759 



17604779 



17804799 



18004819 




19804999 



10 20 30 40 50 60 70 
Number of species described 



FIG. 1 . Number of gastropod species with a labra! tooth described per 20-year interval, 1 740-present. 



150 



VERMEIJ 



also demonstrates unequivocally that interest 
in molluscan taxonomy is as strong as ever. 

The pattern of description of fossil species 
probably reflects exploratory activities by ge- 
ologists and paleontologists working in the oil 
industry or for other commercial interests. It is 
noteworthy that, whereas the number of de- 
scribed Recent species reached a broad low 
in the decades during and between the great 
world wars, the fossil data show no such ap- 
parent effect of worldwide conflict. There is no 
dramatic upsurge in the number of described 
fossil species in the most recent time interval 
as there is for Recent species, but neither is 
there the decline that might be expected if tax- 
onomic expertise and the number of working 
taxonomists were declining. 

In the future, the rate of species description 
is likely to dwindle for either or both of two rea- 
sons. The first, and most worrying, is the long 
feared reduction in funding for, and interest in, 
taxonomy. This problem may be mitigated 
somewhat, at least for shell-bearing molluscs, 
by the increased participation of highly knowl- 
edgeable amateurs. According to my data, 
amateurs account for about one-quarter of the 
authors of labral-tooth-beahng gastropod 
species since 1980. The second reason is 
thiat fewer species remain to be discovered. 
Potentially counteracting this trend is the find- 
ing that taxa previously interpreted as repre- 
senting a single species in fact comprise two 
or more genetically distinct species. In any 
case, the limited data in Figure 1 give no indi- 
cation that either taxonomic activity or the rate 
of discovery of species id declining or reach- 
ing asymptotic values. 

The fact that taxonomic data are funda- 
mental to all branches of comparative and his- 
torical biology and that large numbers of taxa 
remain to be described provides a strong ar- 
gument for continued support of what many 
observers disparage as mundane, descriptive 
science. Phylogenetic and ecological studies 
are only as good as their underlying data and 
assumptions. Continued research and publi- 
cation on taxonomy should remain an impor- 



tant component of the study of the history and 
distribution of living things. 

ACKNOWLEDGMENTS 

This research was funded by Grant 97- 
06749 from the National Science Foundation. 
I thank Janice Cooper, Janice Fong, and Mary 
Graziöse for technical assistance. 

LITERATURE CITED 

AMANO, К., G. J. VERMEIJ & К. MARITA, 1993, 
Early evolution and distribution of the gastropod 
genus Nucella, with special reference to Miocene 
species from Japan. Transactions and Proceed- 
ings of the Palaeontological Society of Japan, 
(n.s.)171:237-248. 

BOUCHET P., 1 997, Inventorying the molluscan di- 
versity of the world: what is our rate of progress? 
Ttie Veligen 40Л -^^ . 

CLARK, B. L., 1918, The San Lorenzo Series of 
middle California: a stratigraphie and palaeonto- 
logic study of the San Lorenzo Oligocène series 
of the general region of Mount Diablo, California. 
University of California Publications. Bulletin of 
ttie Department of Geology. 1 1 :45-234. 

CONRAD, T. A., 1857, Descriptions of Cretaceous 
and Tertiary fossils. Pp. 141-174, in w. н. emory, 
ed., Reports on the United States and Mexican 
boundary survey, made under the direction of the 
Secretary of the Interior. Volume 1 , Part 2. Wash- 
ington, D.C. 

SEPKOSKI, J. J., JR., 1993, Ten years in the li- 
brary: new data confirm paleontological patterns. 
Paleobiology 19:43-51. 

SEPKOSKI, J. J. JR., R. K. BAMBACH, D. M. 
RAUP & J. W. VALENTINE. 1981, Phanerozoic 
marine diversity and the fossil record. Nature. 
293:435-437. 

VOKES, E. H., 1963, Notes on Cenozoic Muricidae 
from the western Atlantic region, with descrip- 
tions of new taxa. Tulane Studies in Geology, 1 : 
151-163. 

WINSTON, J. E. & K. L. METZGER, 1998, Trends 
in taxonomy revealed by the published literature. 
BioScience. 28: 125-128. 

Revised ms. accepted 16 December 1998 



MALACOLOGIA, 1999, 41(1): 151-162 

THE ROLE OF SUBSTRATUM STABILITY IN DETERMINING ZEBRA MUSSEL 

LOADONUNIONIDS 

Susan A. Toczylowski, R. Douglas Hunter & Lisa M. Armes 

Department of Biological Sciences, Oakland University. Rochester Michigan 48309 - 4476 

USA; hunter@oakland.edu 

ABSTRACT 

Data reported herein do not support the existence of preference for or attraction to unionids by 
settling or migrating zebra mussels compared to alternative hard substrata. Despite claims and 
inferences in the Dreissena literature suggesting that unionids are preferred substrata, higher 
Dreissena loads on unionids compared to alternative hard substrata can be explained by mech- 
anisms other than preference. Our data indicate that substratum conditions are often critical in 
determining the relative zebra mussel loads that accrue on unionids. On stable and relatively 
hard lake/river bottoms, zebra mussel loads on unionids tend to be similar to those on other hard 
substrata. However, on bottoms mostly composed of very soft or unstable substrata, discreet 
hard objects become silted-over and/or buried, hence sub-optimal for zebra mussels. Under 
such conditions, unionids develop a higher load of zebra mussels due to their ability to maintain 
position in relation to the sediment/water interface. We conclude that high Dreissena loads on 
unionids relative to other substrata are not a matter of preference for or attraction to the union- 
ids, but are the outcome of differential survival/emigration of the Dreissena due to unstable or 
changing bottom conditions. 

Key words: Dreissena. zebra mussel, substratum, preference, unionids, stability. 



INTRODUCTION 

Among the many ecological changes attrib- 
uted to the zebra mussel invasion of North 
American lakes, none is more readily appar- 
ent than the virtual elimination of entire 
unionid communities (Schloesser & Kovalak, 
1991: Schloesser & Nalepa, 1994: Nalepa, 
1994). This consequence is especially unfor- 
tunate due to the already precarious status of 
the majority of unionid taxa (Williams et al., 
1993: Steins Flack, 1997). 

The common impression from examination 
of unionids in lakes recently invaded by zebra 
mussels is that they are highly suitable as 
substrata for attachment. This often gives the 
appearance that unionids are being singled 
out by zebra mussels by some mechanism 
of preference (Lewandowski, 1976; Mackie, 
1 993: Ricciardi et al., 1 995, 1 996). One of the 
difficulties in using the word preference in the 
context of settling zebra mussels is that it 
specifies active choice, a choice based on the 
ability to discriminate among alternatives. The 
underlying mechanism would of necessity in- 
volve an ability to detect positive or negative 
stimuli from amongst alternatives (e.g., differ- 
ent kinds of substrata) and to choose based 



on the perception of those qualities. With the 
example of settling larvae in marine ecosys- 
tems, preference is typically based on detec- 
tion of chemical or textural cues originating 
from the biofilm or from components of the 
biofouling community (Morse, 1991; Ro- 
driguez et al., 1993). However, it is possible 
for a different mechanism, not involving pref- 
erence, to result in clumped or aggregated 
distribution of a species. In such cases, larvae 
settling on both suitable and non-suitable sub- 
strata would experience post-settlement dif- 
ferential mortality with higher mortality among 
the latter group. The resulting greater aggre- 
gation on suitable substrata might give the im- 
pression of preference, but in the absence of 
active choice the resulting distribution would 
simply be the outcome of differential mortality 
on dissimilar substrata. 

Although it is often taken for granted that 
zebra mussels "prefer" hard substrata, they 
have been reported living on soft substrata 
and can achieve surprisingly high densities 
(Hunter & Bailey, 1992; Dermott & Munawar, 
1993; Coakley et al., 1997). Despite these re- 
ports, the highest densities are on hard sub- 
strata, and it is likely that they can colonize 
soft substrata only from an initial seed object 



151 



152 



TOCZYLOWSKI ETAL. 



and if the bottom is undisturbed by current. 
Zebra mussels are clearly epifaunal and the 
morphological adaptations for this mode of 
existence are obvious: presence of a byssus 
and heteromyarian shell morphology (Morton 
& Yonge, 1964; Yonge & Campbell, 1968). 

One form of preference that has been doc- 
umented for zebra mussels is settlement on 
or near conspecifics. This was first suggested 
by Lewandowski (1976) and supported by the 
field studies of Wainman et al. (1996), Chase 
& Bailey (1996), and Toczylowski & Hunter 
(1 997). Although none of these studies exper- 
imentally addressed the release of chemical 
cues by established adults, the results of 
Wainman et al. (1996) suggest that it is more 
likely that conspecific shell surface chemistry 
was the basis for veliger preference. 

Some of the recent literature largely rein- 
forces the perception of unionid preference by 
D. polymorpha, without specifically identifying 
the mechanism by which preferred substrata 
are recognized (Ricciardi et al., 1 995, 1 996). A 
previous study (Toczylowski & Hunter, 1997) 
found no evidence to support zebra mussel at- 
traction to unionids based on field studies of 
Dreissena seWWng on unionid and non-unionid 
test surfaces. The present work extends and 
refines those findings and examines the ef- 
fects of substratum stability on differential mor- 
tality of zebra mussels attached to stones as 
compared to unionids. 

We hypothesize that zebra mussels do not 
actively choose unionids over other hard sub- 
strata. Higher Dreissena density on unionids 
in relation to other hard substrata, where it oc- 
curs, is the outcome of differential mortality re- 
sulting from bottom instability. 

EXPERIMENTAL DESIGN AND METHODS 
Site Descriptions 

All of the field sites were in the upper Clin- 
ton River or in Loon Lake, a lake connected to 
the Clinton River located in Oakland County in 
southeastern Michigan (Fig. 1). The upper 
mainstem of the Clinton River between Loon 
Lake and Dawson's Millpond Outlet (DM0) is 
mostly less than 10 m in width, with discharge 
rates averaging 1.42 and 1.75 m^/s for the 
1995 and 1996 calendar years respectively 
(Blumer et al., 1997). The range in 1996 was 
0.28 - 15.01 m^/s. The dominant unionid 
species in this region of the river, Ptycho- 
branchus fasciolaris (23%), Elliptio dilatata 
(21%), and Strophitus undulatus (15%), are 



indicative of a hydrological tendency toward a 
stable river as opposed to an event river 
(Hunter et al., 1 997; DiMaio & Corkum, 1 995). 
The water is relatively clear with the silt load 
sufficiently low that one can see the bottom in 
most places. Mean seston load for 1995 from 
12 monthly measurements was 8.7 mg/L dry 
mass (range = 0.7 - 37.4 mg/L) and values for 
1 996 were similar (Hunter et al., 1 997). In this 
stretch of the river, the watershed is mostly 
residential with a few small towns and no 
heavy industry. The river bottom is mostly 
hard consisting of cobble and/or sand with 
soft silt elsewhere. 

Loon Lake is a mesotrophic lake with an 
area of 0.95 km^ and maximum depth of 21 m. 
The bottom is mostly soft mud except near 
shore where it is mostly cobble and sand ex- 
cept for a few marshy areas. Macrophyte 
growth along the shore is moderate. 

Natural Density of Zebra Mussels on 
Unionids and Stones 

To determine the field density of zebra mus- 
sels on unionids and stones, samples of nat- 
ural substrata were collected at DM0 on 16 
October 1996 (Fig. 1). Unionids and stones 
were from an area where the water was about 
0.5 m deep and the bottom was partially cob- 
ble and partially sand. Bottom sediments in 
this area were subject to considerable move- 
ment during periods of high water flow; conse- 
quently hard objects, such as stones, were al- 
ternately buried in sand or exposed as cobble. 
At this site, the most abundant three unionids 
were ElUptio dilatata. Lampsilis siliquoidea. 
and Ptyctiobranchus fasciolaris. 

The density measured in this instance, 
refers to number or biomass of zebra mussels 
per unit of unionid or stone surface area, not 
per unit total bottom area. Although the latter 
meaning is the standard one, our modified 
density (= suitable substrate density) allows a 
more direct comparison between different 
types of discreet hard surfaces. Ten unionids 
along with ten stones of approximately the 
same exposed surface area as the unionids 
were selected. Both the unionids and the 
stones were carefully transported to the lab 
where all attached zebra mussels were re- 
moved with a scalpel under a dissecting mi- 
croscope to ensure that small individuals 
were included. After removal, the zebra mus- 
sels were counted and their length was mea- 
sured using digital calipers. For each of the 
two surface types, area above the sedi- 



ZEBRA MUSSEL LOAD ON UNIONIDS 



153 




Dawson's Mlllpond 
Outlet 
(DM0) 



Sylvan 
Lake 



^Clinton 
River 



FIG. 1 . Map of the upper mainstem of the Clinton River in southeastern Michigan with the sample and study 
sites indicated. 



154 



TOCZYLOWSKI ETAL. 



ment/water interface was obtained using a foil 
method. This method is essentially the same 
as that used in Mackie (1993). Because 
buried areas on the test surfaces became 
darkened, this made the sediment/water in- 
terface clearly visible, so the area exposed to 
zebra mussel settling was easy to see. The 
method involved removing attached zebra 
mussels, covering the exposed area with alu- 
minum foil, removing the foil, and trimming 
and flattening it. After placing the foil on met- 
ric graph paper, the outline was traced and 
the surface area counted. Data from this 
study were compared using an unpaired t test 
to determine if there were significant differ- 
ences between zebra mussel density on 
stones and unionids. Mean values given in 
the text are accompanied by standard error. 

Settling and Migration Preference Study 

An experimental test of preference utilized 
test quadrats placed in Loon Lake and in the 
Clinton River at Drayton Plains Nature Center 
(DPNC; asterisks mark these locations in Fig. 
1). Quadrats at Loon Lake were placed in 
water of 1.2 - 1.4 m deep and ~10 m from 
shore, where the bottom sediment was soft 
and silty, lacking in cobble or stones. The 
DPNC quadrats were placed at a depth of 
about 1 .0 m at the center of a slow-flowing 
stretch of the river where the bottom was also 
mostly silt. Quadrats were circular and en- 
closed an area of about 0.75 m^. Each 
quadrat was an open-topped enclosure using 
plastic garden edging as a boundary, with 
about 3 cm above and about 9 cm below the 
sediment, to facilitate recovery of unionids 
later in the summer. Each quadrat contained 
four replicates of each of the following test 
surfaces; unionids with no attached zebra 
mussels (= control), unionids with marked 
zebra mussels, control stones, and stones 
with marked zebra mussels. Although they dif- 
fered slightly in size, the surface area of each 
object was measured at the end of the exper- 
iment and the density of attached zebra mus- 
sels expressed per unit area. Average total 
exposed unionid surface area was 31 cm^ 
and that of stones was 43 cm^. 

The species composition of test unionids at 
Loon Lake was Pyganodon grandis. Lampsilis 
siliquoidea, and Elliptio dilatata in a ratio of 
4:3:1 and at a density of 8/quadrat. Unionids 
placed in Clinton River quadrats were col- 
lected from the river nearby and included 
Strophitus undulatus, Ptychobranchus fascio- 
laris, and Pyganodon grandis in a ratio of ap- 



proximately 4:2:1, also at a density of 
8/quadrat. 

The unionids and stones with zebra mus- 
sels each had about 5 - 30 zebra mussels at 
the outset of the experiment. Each of these 
zebra mussels was marked with a dot of 
enamel model paint; those on unionids were 
marked with red paint and those on stones 
were marked with blue. All unionids and 
stones had an identification number painted 
on them at the start of the experiment. By 
marking the zebra mussels attached to the 
test surfaces at the beginning (= residents), it 
was possible to identify non-residents that im- 
migrated onto the test surfaces during the ex- 
periment. 

Test surfaces were put into Loon Lake on 6 
June 1996 and remained in situior 103 days. 
At the Clinton River site they were put in on 1 
June 1996 and were in situ for 99 days. This 
period coincided with the major period of the 
veliger presence and spat settling for Loon 
Lake. After completion of the experiment, all 
attached zebra mussels were removed and 
counted. Area of the test surfaces above the 
sediment/water interface was measured 
using the foil method. For unionids with at- 
tached zebra mussels, the surface area of the 
original resident zebra mussels was included 
in the total surface area. However, for the 
density and biomass data, the ohginal at- 
tached zebra mussels were not included. 

The mean of four replicates of each of the 
four test surfaces was statistically analyzed 
using two-way ANOVA, with treatment and 
site as the factors. To evaluate the relation- 
ship between number of attached zebra mus- 
sels at the start and number of immigrating 
zebra mussels that attached during the study, 
a regression ANOVA was used that calculated 
an F-statistic using sum of squares and mean 
square. This indicated how important the in- 
dependent variable was in explaining the be- 
havior of the dependent variable. Emigration 
data was expressed as % decrease of resi- 
dent mussels for plotting, but two-factor 
ANOVA was done on arcsin transformed data 
(p' = arcsin [sqrt p]), the results of which are 
reported in Table IB. 

Zebra Mussel Survival and Migration on 
Unstable Substrata 

To determine survival and migration of 
zebra mussels on unionids and stones lo- 
cated on shifting river bottom, we performed a 
field experiment in the Clinton River at the 
CLR site (Cooley Lake Road Bridge. Fig. 1). 



ZEBRA MUSSEL LOAD ON UNIONIDS 



155 



Flow rate at this site is moderately fast for the 
upper Clinton River, and the bottom is rela- 
tively unstable. At a given point in time, most 
of the bottom is sand with a few areas of cob- 
ble, the boundaries of which change over the 
year. Depressions in the sand are common 
here, and their size and location also shift. 
The test surfaces were located on the up- 
stream slopes of such depressions. Test sur- 
faces consisting of unionids and stones of ap- 
proximately the same size were collected and 
given an identification mark. The test unionids 
were collected on site and included Villosa 
iris, Elliptio dilátala, Pyganodon grandis, 
Strophitus undulatus, Lampsilis siliquoidea, 
and Ptychobranchus fasclolaris in ratios of 
20:3:3:2:2:1 . Unionids were allocated to repli- 
cates in approximate proportion to their nat- 
ural abundance. Zebra mussels on both test 
surfaces were counted and marked at the 
start of the experiment, so that any unmarked 
zebra mussels found at the end of the experi- 
ment could be recognized as immigrants. To 
ensure recovery at the conclusion of the ex- 
periment, unionids were tethered to a stake in 
the river bottom using nylon string attached to 
one valve with cyanoacrylate glue. Stones 
were not tethered, and were simply placed on 
the sand bottom. Ten unionids and ten stones 
were placed around each of the three repli- 
cate stakes on 23 June 1 997 and remained in 
situ for 78 days, after which they were re- 
trieved and brought to the laboratory. In the 
lab, the number of resident and non-resident 
zebra mussels was recorded for each test 
surface. Surface area was not measured due 
to the inability to determine the exact amount 
of the test surface that was above the sedi- 
ment-water interface (i.e., the sediment was 
not chemically reducing at this site). The data 
were analyzed with an unpaired t test to de- 
tect significant differences using mean num- 
ber of zebra mussels per test surface at each 
stake, hence N = 6 (i.e., 3 replicates x 2 treat- 
ments/replicate). When the total number of 
zebra mussels (residents + immigrants) was 
expressed as a % change on unionids and 
stones, the decimal equivalents were arcsin 
transformed before using the t test; p' = arcsin 
(sqrt p). 

RESULTS 

Natural Density of Zebra Mussels on Union- 
ids and Stones 

Unionids at DM0 had a zebra mussel den- 
sity of 1.40/cm^, which was significantly 



3.5-, 

3.0 

^g 2.5 

"^ 2.0 

0) 

J3 

E 1.5, 

^ 1.0- 

0.5- 





Loon Lake 



a 

T 



Unionid 



Stone 



3.5 

3.0- 

^c 2.5- 

О 

"i: 2.0- 
0) 

E 1.5^ 
^ 1.0. 
0.5- 




В 



Clinton River at DMO 



У///////////////////////////Л 



Unionid 



Stone 



FIG. 2. Natural density of zebra rnussels (mean ± 
SE) on unionids and on stones from (A) Loon Lake 
and (B) Clinton River at DMO (Dawson's Millpond 
Outlet). Histograms with the same letter are not sig- 
nificantly different from each other. 



greater than the zebra mussel density ob- 
served on stones, 0.14/cm^ (Fig. 2B; t^^g^ = 
5.332; P = 0.0001). There was also a signifi- 
cant difference in wet biomass; unionids had 
600.3 mg zebra mussels/cm^, stones had 
78.0 mg/cm^ (t^^g, = 4.568; P - 0.0002). 
Hence, the river results did not agree with 
those from the Lake. DMO is a river site that 
is frequently exposed to high current due to 
the proximity of a dam used to maintain 
nearby lake levels. As a result, it is a high-en- 
ergy location with an unstable bottom. The re- 
sults we obtained at this site contrasted with 
those obtained from Loon Lake a year earlier 
(Toczylowski & Hunter, 1997). 

Settling Preference Experiment 

All test surfaces in Loon Lake, whether 
stones, unionids, with or without resident 
zebra mussels, attracted a significantly 
greater density of unmarked (= immigrant) 



156 



TOCZYLOWSKI ETAL. 



(Л 

3 2 

E 

¿ 1 

0) 
N 




Unionid with zm 



Stone with zm 



-- ^ 



250 



^ 200 

E 

OÎ 150 

to 

^ 100 

E 
о 

CÛ 50 



Lake 
В 



River 



t^ Unionid control 



Unionid with zm 




11,2^ 



Lake 



River 



FIG. 3. Interaction plots for the settling preference 
study. (A) Numerical density of zebra mussels in 
Loon Lake and Clinton River treatments. (B) Bio- 
mass density of zebra mussels in Loon Lake and 
Clinton River treatments. The four treatments used 
at each site are identified on the plots. 



zebra mussels/cm^ than those in the Clinton 
River (Fig. ЗА). The lake test surfaces aver- 
aged five times as many newly recruited 
zebra mussels as their river counterparts. As 
used here, immigrants denotes both spat 
zebra mussels that have recently settled out 
of the plankton directly onto the test surface, 
as well as later stage individuals that settled 
elsewhere and subsequently relocated onto 
the test surfaces. This latter group consists of 
young-of-the-year juveniles as well as adults 
that settled in previous years. No attempt was 
made to separate these groups in this data 
set; spat and later stages were lumped as "im- 
migrants". This higher rate of accumulation of 
zebra mussels on lake compared to river sub- 
strata is simply the result of higher settling 
rates due to higher veliger density in the lake 
(Hunter et al., 1997; unpublished data for 
1996). 



There were also significant differences in 
immigrant zebra mussel density among the 
test surface types (Table 1 A). Unionids in the 
lake had about a 4x higher density of immi- 
grant zebra mussels than did the stones. 
However, there was no such difference at the 
river site; that is, both unionids and stones ap- 
peared to attract similar numbers of immi- 
grants. It also appeared to be of little impor- 
tance whether the test surface had resident 
zebra mussels or not. That is, immigration of 
zebra mussels was not enhanced by the pres- 
ence of previously settled (resident) con- 
specifics. There was also a significant interac- 
tion between the main effects (Table 1A). 
Hence, zebra mussel density was responsive 
to surface type at the lake site, but not at the 
river site. 

The biomass data showed a similar pattern 
of significance as was seen in the numerical 
density data above (Fig. 3B). There were 
strong site effects; unionids had higher zebra 
mussel loads than did stones, and presence 
of resident zebra mussels had little effect 
(two-factor ANOVA F 



(1, 112) 



= 29.6; P < 
0.0001). Unionid test surfaces had a signifi- 
cantly greater biomass of immigrants (ap- 
proximately five times greater) than the river 
unionid surfaces (F^g ^^^, = 7.3; P = 0.0002). 
There was also a significant interaction be- 
tween main effects (F^g ,^2) = 6.5; P = 0.0005). 
Density differences between lake and river 
are great for unionids but small for stones. 

In order to evaluate the effects of emigra- 
tion of resident zebra mussels and whether it 
differed by test surface type, the percent de- 
crease of marked individuals was calculated. 
There was a significant difference in percent 
decrease by site and surface type (Table 1 B). 
Loon Lake had a larger percent decrease of 
resident zebra mussels on both unionid and 
stone test surfaces compared to those in the 
river (Fig. 4). Stone surfaces had a signifi- 
cantly greater overall percent decrease in res- 
ident mussels regardless of site (Table 1 B). 
However, in contrast to number and biomass 
data, the interaction of the two factors was not 
significant. 

The number of resident zebra mussels at 
the start of the study was not a good predictor 
of the number of mussels that subsequently 
immigrated to that surface at the lake site; 
however, the river site showed the opposite 
result. At Loon Lake, neither unionids (F 3^^ = 
0.391 ; P = 0.537) nor stones {F^^z) = 2-"'Û5; P 
= 0.162) attracted more immigrants if the 
number of resident mussels at the start was 



ZEBRA MUSSEL LOAD ON UNIONIDS 



157 



TABLE 1 . Analysis of variance tables based on settling preference data. (A) Results of a three- 
factor ANOVA on number of immigrant zebra mussels per cm^. (B) Results of a two-factor 
ANOVA on percent decrease (emigration) of resident zebra mussels from test settling sur- 
faces. 







A. Immigration: no./cm^ 






Source of variation 




Deg 


rees of freedom 


Mean square 


F 


Main effects 












site 






1 


122.6 


29.2" 


surface 






1 


90.4 


21.6** 


res/non-res 






1 


0.3 


0.8 ns 


Two-way interaction 












site X surface 






1 


54.3 


12.9* 


surf X res/non-res 






1 


1.5 


0.3 ns 


res/non-res x site 






1 


14.1 


3.4 ns 


Three-way interaction 












site x surface x res/non 


-res 




1 


8.1 


1.9 ns 


Error 






104 


4.2 




Total 






111 


6.8 




*P = 0.0005; **P< 0.0001 




B. 


Emigration: 


% decrease of residents 




Source of variation 




Deg 


rees of freedom 


Mean square 


F 


Main effects 












site 






1 


1.758 


14.99* 


surface type 






1 


1.160 


9.89** 


Two-Way interaction 












site X surface type 






1 


0.243 


2.07 ns 


Error 






56 


0.117 




Total 






59 


3.278 




*P = 0.0003; **P = 0.003; ns = 


p> 


0.05 









80- 



Í 60- 



O 40- 

(U 
CO 
CO 

03 
T3 



Stone 




Unionid 



Lake 



River 



FIG. 4. Emigration of zebra mussels as % decrease 
(mean ± SE) of residents from stones and unionids 
by the end of the settling preference study. See 
Table 1 В for statistical analysis. 

greater (Fig. 5A). When unionids and stones 
at the river site were analyzed, there was a 
significant correlation between the two vari- 
ables (unionids, F,2g) - 528.6; P = 0.0001; 
stones, F(28, = 210.1 ; P = 0.0001 ; Fig. 5B). In 
other words, at the river site, having more res- 



ident mussels at the start significantly in- 
creased the number of zebra mussels that re- 
cruited to the test surfaces. However, at the 
lake site, where recruitment was about an 
order of magnitude higher, there was no such 
relationship. The mean length of recruited 
zebra mussels did not differ between surface 
type (t(g) - -2.205; P = 0.696). 

Zebra Mussel Survival and Migration on 
Unionids and Stones on Unstable Substrata 

Test unionids and stones at the start of this 
experiment had virtually identical mean num- 
bers of attached zebra mussels by object: 
unionids, 6.48 ± 0.435 zebra mussels, and 
stones, 6.45 ± 0.449 zebra mussels (t^gg) = 
0.041 ; P = 0.967). After 78 days on the unsta- 
ble river bottom at CLR, unionids had a signif- 
icantly higher number of zebra mussels (t^^j == 
4.855; P = 0.008), averaging 8.4 ± 0.91 zebra 
mussels, whereas stones averaged 1.9 ± 
0.50 zebra mussels (Fig. 6A). This gain of 
zebra mussels by unionids occurred for two 



158 



TOCZYLOWSKI ET AL. 



О) 

E 
о 
ó 



О) 
(О 

со 

Е 

со 

к_ 

JD 
Ф 
N 

■«— • 

С 

СО 

1_ 

Е 

«4— 

О 

ó 



600 

500- 

400- 



_ço 

О) 
ел 

СО 

13 

Е 

со 

1_ 

JD 
0) 

i^ 300 H 

С 
СО 



Я^ 200- 



Loon Lake 

Unionid: Y = 127.46- 1.323Х; г = 0.113 
Stone: Y = 29.38 + 2.092X; г = 0.302 




5 10 15 20 25 

No. of zebra mussels at start 



30 



В 



зо-п Clinton River 



Unionid: у = -0.111 +0.764X; r = 0.975 о о 



XX 




су^ Stone: 

^ Y = -0.438 + 0.71 5X; r = 0.941 



10 15 20 25 30 35 

No. of zebra mussels at start 

FIG. 5. Scatter plot of the number of zebra mussels immigrating to unionids and to stones at Loon Lake (A) 
and the Clinton River (B) in relation to the number of resident zebra mussels on these test objects at the start 
of the settling preference study. Lines are fitted by least squares regression. 



ZEBRA MUSSEL LOAD ON UNIONIDS 



159 



10 -1 



t =4.855; P = 0.008 




Unionid 



В 



3.0 



i2 2.5 

с 

^ 2.0- 

E 



Z 1.5- 



1.0- 



0.5- 



Stone 



t =3.326; P = 0.0292 




Unionid 



Stone 



FIG. 6. (A) Total number of zebra mussels (mean ± 
SE) on unionid and stone test objects at the end of 
the experiment at CLR (Cooley Lake Road; unsta- 
ble substratum site). (B) Number of immigrant 
zebra mussels (mean ± SE) that were present on 
test objects at the end of the experiment. 



reasons. First, resident zebra mussels on 
unionids (those marked at the start) showed 
little tendency to emigrate, so that the mean 
number of residents on unionids at the end of 
the experiment was 6.1 compared to 6.5 at 
the start. In contrast, the number of resident 
zebra mussels on stones at the end of the ex- 
periment averaged 1.8, a mean decrease of 
4.7 zebra mussels on each stone. Second, 
there was greater immigration of zebra mus- 
sels to unionids than there was to stones, so 
that at the end of the experiment, the unionids 
averaged 2.3 ± 0.48 immigrants per unionid 
compared to 0.04 ± 0.037 immigrants for 
stones (Fig. 6B). These values were signifi- 
cantly different: t^^, = 3.326, P = 0.029. 

It is not known if the decrease in resident 
zebra mussels was due to mortality or emi- 
gration. Evidence for the former came from 



field observations that many of the stones 
were entirely buried, and these often had 
empty shells of resident zebra mussels still at- 
tached. These differences between unionids 
and stones in terms of survival and behavior 
of attached zebra mussels led to large per- 
centage changes by the end of the experi- 
ment (Fig. 7). Average percent change in 
zebra mussels attached to unionids was both 
positive and significantly higher (24.5 ± 
8.66%) than that for stones, the latter de- 
creasing by 73.1 ± 7.36% (t,^. = 5.14; P = 
0.007). 



DISCUSSION 

Our data show that unionids in both lake 
and river conditions are not preferentially set- 
tled on or colonized by zebra mussels com- 
pared to other hard substrata. We found no 
evidence to suggest that unionids are more 
attractive to either migrating zebra mussels or 
to settling veligers than are alternative hard 
substrata nearby. This finding agrees with 
Toczylowski & Hunter (1997), who examined 
a variety of substrata, including natural (live 
unionids, unionid shells, wood) and artificial 
surfaces (tiles, plastic mesh) and reached the 
same conclusion. The density of zebra mus- 
sels on unionids is approximately the same as 
it is on other natural hard substrata located in 
the same area, the outcome that would be 
predicted if there were no preference. These 
data were obtained for lake and river bottoms 
that are relatively firm and in which inanimate 
hard substrata do not become buried. 

However, under different bottom conditions, 
we obtained different results. When the bot- 
tom is soft enough for hard substrata to be- 
come buried, or if the bottom sediment is un- 
stable due to a strong current, as in a river, 
then any hard surfaces that are buried or 
silted-over become sub-optimal for zebra 
mussels. In these conditions, unionids main- 
tain their position with respect to the sedi- 
ment-water interface, and the zebra mussels 
attached to the posterior end are carried 
above the sediment. Zebra mussels on 
stones or other kinds of inanimate substrata 
become buried and either die or are forced to 
emigrate. Therefore, under conditions of un- 
stable substrata, greater loads of zebra mus- 
sels will develop on unionids than on inani- 
mate hard substrata due to differential 
survival of the immigrant zebra mussels. 

This study provides one observation and 



160 



TOCZYLOWSKI ETAL. 



чи- 




"Г 


t^^=3.60; Р = 0.023 


20- 








n 




^^^^^^; 




и - 




^^^^^^^ 




-20- 




^^^^^ 




-40^ 




^^^^ 




-60- 




^^^Щ 




-80- 




J- 




1 пп 




i ии- 




Unionid 


1 


Stone 





FIG. 7. Percent change (mean ± SE) in the total 
number of zebra mussels (residents -i- immigrants) 
on unionids and on stones at CLR (unstable sub- 
stratum site) over the course of the experiment. 



two experiments that support our hypothesis. 
The observation is from measurements of 
natural zebra mussel density on unionids and 
on stones from a river site with an unstable 
bottom (DM0). Test stones and unionids from 
this site were located on patchy sand and 
cobble where sand movement alternately 
buried and exposed the cobble, rendering the 
stones less suitable for zebra mussels. The 
result was that unionids carried significantly 
higher zebra mussel loads per unit area of ex- 
posed surface than did stones from the same 
site. This result differs from that reported in an 
identical study in Loon Lake in 1995 (Toczy- 
lowski & Hunter, 1997). 

We believe the different outcomes of these 
two studies can be explained by different sub- 
stratum stability. The lake work was done on a 
predominantly hard substratum (cobble), on 
which test stones had little tendency to sink 
and maintained their position relative to the 
sediment/water interface. Unionids, which 
were similarly positioned, had loads that were 
basically the same as those on the stones. At 
the river site, with its unstable bottom, zebra 
mussels located on stones were buried and 
died, whereas those on unionids survived by 
remaining above the sediment. 

Our hypothesis was further tested by ex- 
periments conducted at both lake and river 
sites. Pre-marked test substrata placed on an 
unstable bottom in the Clinton River at CLR 
confirmed that zebra mussels previously at- 
tached to unionids were not only more likely to 
remain attached over the summer than their 
counterparts attached to stones, but were 
also more likely to be joined by immigrants. 



On stones, not only were there negligible 
numbers of immigrants, but previously at- 
tached zebra mussels died or emigrated due 
to burial under bottom sediments. 

A further test was provided by experiments 
on a soft bottom site in Loon Lake and a sim- 
ilar site in the Clinton River (DPNC). Under 
such conditions, we would predict that union- 
ids should accumulate a higher zebra mussel 
load than stones, due to siltation of the stone 
surfaces that occurred during the course of 
the experiment. Our data supported the hy- 
pothesis in both in lake and river conditions; 
however, the results were more pronounced 
under lake conditions despite the similarity of 
substratum type. We believe this was due to 
the presence of low current at the river site. 
Current may have reduced silt accumulation 
on upper stone surfaces and prevented re- 
duced O2 levels from occurring at the sedi- 
ment/water interface. These improved bottom 
conditions would reduce the negative effects 
to which zebra mussels on the river bottom 
were exposed, reducing mortality. These 
same subtle lake/river bottom differences 
would also explain our finding that emigra- 
tion/mortality of previously marked resident 
zebra mussels from lake test surfaces was 
higher than that from river test surfaces. 

More recently, studies by Ricciardi et al. 
(1995, 1996) have purported to demonstrate 
that unionids are preferred substrata, and 
consequently zebra mussels reach greater 
densities on unionids than on the bottom in 
general. A model presented in Ricciardi et al. 
(1995) in support of preference was tested 
using data from sites that were mostly soft 
substrata. Under these conditions, the prefer- 
ence by Dreissena for unionids is simply a 
choice between hard and soft substrata. This 
model has tested the obvious: that zebra 
mussels reach higher density on hard, com- 
pared to soft substrata. The control that was 
not done would have examined zebra mussel 
load at the same sites on stones, bricks, or 
other hard substrata of comparable size and 
surface texture. Although their model is useful 
in extrapolating from general zebra mussel 
density to zebra mussel loads on unionids, it 
does not provide any useful insight into the 
real issue of preference: do zebra mussels 
select unionids over other natural hard sur- 
faces? 

In a further examination of the impact of 
Dreissena on unionids, Ricciardi et al. (1996) 
offered more evidence in support of prefer- 
ence by showing that there was higher zebra 



ZEBRA MUSSEL LOAD ON UNIONIDS 



161 



mussel density on unionids (in the mud bot- 
tom of a canal) than on the concrete walls of 
the canal. The problem with these data is that 
Dreissena density on the wall is based on the 
total wall area, whereas the density of Dreis- 
sena on unionids is based only on the area of 
unionid shell, not on total area of the bottom 
(unionids plus mud). Using the terminology of 
Bailey et al. (1995), what Ricciardi et al. 
(1996) have done is to express zebra mussel 
density on unionids in terms of number per 
unit unionid surface (BMI/RSA) and zebra 
mussel density on the concrete wall in terms 
of number per unit wall area (BMI/BA). It is not 
surprising that the former density is greater. 
Because unionid shells are discreet optimal 
surfaces for zebra mussel settling and sur- 
vival and are dispersed in a habitat that is 
generally sub-optimal, the result is intense 
crowding. In contrast, the entire wall is suit- 
able substratum. 

We have shown the absence of preference 
where the substratum is relatively firm and an 
appearance of preference where the substra- 
tum is soft and/or unstable. In this latter case, 
it is likely that higher zebra mussel densities 
on unionids are simply the outcome of the 
ability of unionids to actively maintain their 
posterior shell surface in an exposed position 
on the bottom. Unionids are a relatively stable 
surface due to their responsiveness to chang- 
ing bottom conditions. Therefore, the devel- 
opment of higher Dreissena densities on 
unionids is not an outcome of preference in- 
volving substratum choice using sensory per- 
ception and subsequent taxes or directed 
movement. Instead, our data indicate that 
these density differences have arisen from 
lower mortality and emigration rates on a re- 
sponsive live surface compared to that on un- 
responsive, inanimate hard surfaces. 

Although it is well known that unionids move 
both horizontally and vertically (extent of pro- 
trusion from the sediment), the conditions 
stimulating these responses and the adaptive 
value of specific movements are largely un- 
clear and have received scant attention in 
most species (Balfour & Smock, 1995; Amyot 
& Downing, 1997). Vertical movement involv- 
ing shifts from epibenthic to endobenthic posi- 
tion are known to occur in some species. The 
best studied species is Elliptio complánala, 
which showed seasonal movement resulting 
in most of the population becoming endoben- 
thic in winter and epibenthic in summer (Amyot 
& Downing, 1997). In terms of the present 
study, should any of the species that were test 



unionids become endobenthic in winter it 
could be a means of eliminating or reducing 
their load of zebra mussels. The authors have 
observed unionids in the field that are almost 
entirely buried, with only the siphons visible. 
Yet adjacent to the siphons are a few zebra 
mussels attached to the extreme posterior 
margins of the unionid shell, where they have 
avoided burial but remain at the sediment/ 
water interface. Because they are epifaunal, 
zebra mussels are likely to be less tolerant of 
burial than unionids, hence might be partially 
or entirely eliminated over winter when much 
of the unionid community is endobenthic (un- 
published observations). Although as yet we 
have no overwinter survival data for zebra 
mussels attached to unionids, we do have ev- 
idence from zebra mussels loads in fall and the 
following spring that indicate there is no signif- 
icant reduction of loads over winter (Hunter et 
al., 1997). It is entirely possible that once a sig- 
nificant load of zebra mussels has accumu- 
lated, it may act as a kind of stop, limiting the 
ability of the unionid to bury itself in the sub- 
stratum. If this occurs, it would prevent fresh- 
water mussels that vertically migrate from be- 
coming endobenthic. 



ACKNOWLEDGMENTS 

We acknowledge the Drayton Plains Nature 
Center for allowing access to the Clinton 
River from their property. Nicole Rudolph pro- 
vided help with recovery of test surfaces from 
lake and river. Jon Guilliat and Mike Armes 
provided field assistance; Deborah Bishop 
and Michael Attan provided laboratory assis- 
tance. Thanks to the Michigan Department of 
Natural Resources Wildlife Division for sup- 
porting this work through Natural Heritage 
Small Grants in 1996 and 1997, and to the 
MDNR Fisheries Division for loaning us a boat 
and trailer in 1996 and 1997. Thanks are also 
due to the MDNR Parks and Recreation Divi- 
sion for providing free access to the Loon 
Lake and Cass Lake boat ramps. 



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Revised ms. accepted 16 December 1998 



MALACOLOGIA, 1999, 41(1): 163-174 

GANULA GADIRANA N. SR, A NEW HYGROMIIDAE FROM SOUTHERN SPAIN 

(PULMONATA: HELICOIDEA) 

Benito Muñoz, ^ Arturo Almodovar,^ & José R. Arrebola^ 

ABSTRACT 

Ganula gadirana Muñoz, Almodovar & Arrebola, is proposed as a new species from the south- 
ernmost corner in the Iberian Peninsula. This species, sometimes erroneously recorded as Helix 
(= Ganula) lanuginosa Boissy, 1835, is characterized by a globose conical-depressed shell, with 
ovate aperture, small umbilicus, and periostracum with long, persistent hairs; it has a right om- 
matophore retractor between the penis and the vagina, a penial nerve arising from the right cere- 
bral ganglion, ring-shaped glandular area on the distal penis wall, a fenestrate and elongate pe- 
nial papilla, and a circumvaginal tuft of long, annulated digitiform glands. A dart-sac complex is 
on one side of the vagina and is formed by an outer dart-bearing stylophore and inner apically 
bilobed dartless stylophore; each open separately into a canaliculate deep groove that divides 
two large pleats, distally detached from the vaginal wall. Comparisons with Ichnusotricha and 
Ganula suggest that the new species may be closely related to G. lanuginosa (Boissy, 1835). 
The bilobate distal portion of the inner stylophore, the larger number of tufts in the digitiform 
glands, the fenestrate penial papilla, the penial glandular area, and the radular formula to distin- 
guish G. gadirana from G. lanuginosa. 

Key words: Ganula gadirana. Gastropoda, Stylommatophora, Hygromiidae, Spain. 



INTRODUCTION 

Many new generic taxa have been recently 
described by the Giusti-Manganelli's team for 
the western Mediterranean area, most of 
them being monotypical: Helicotricha carusoi 
from the Aeolian Islands (Giusti et al., 1992), 
Cilliellopsis oglasae from Montecristo Island 
(Giusti & Manganelli, 1990), Schileykiella ior 
Helix parlatoris Bivona, 1839, and Helix 
reinae Pfeiffer, 1856, from Sicily (Manganelli 
et al., 1989), Ichnusotricha berninii anä Nien- 
huisiella antonellae from Sardinia (Giusti & 
Manganelli, 1987), and Tyrrheniella josephi 
from Sardinia/Capraia Islands (Giusti & Man- 
ganelli, 1989). The number of new morpho- 
logical patterns is striking, and more new 
generic taxa can be expected. 

Ganula lanuginosa, according to biblio- 
graphical records, seems to be distributed in 
four areas: eastern Balearic Islands (type lo- 
cality of Helix lanuginosa Boissy, 1835) Sar- 
dinia (Giusti & Manganelli, 1987), southern 
Spain (Servain, 1880), and northwestern 
Africa (Terver, 1839; Bourguignat, 1864; Le- 
tourneux & Bourguignat, 1887). This species 
could be of great zoogeographical value if its 
presence in these areas is anatomically con- 
firmed (Giusti & Manganelli, 1984), although 



Balearic records could be the result of an in- 
troduction from northwestern Africa during the 
Middle Ages (Gasull, 1963). 

Nevertheless, the anatomical confirmation 
has been carried out only for the Balearic Is- 
lands (Hesse, 1931; Gittenberger, 1968) and 
Sardinia (Giusti & Manganelli, 1987). Search- 
ing to confirm its presence in southern Spain 
where it was repeatedly cited (Servain, 1880 
Sacchi, 1956, 1957; Sacchi & Nos, 1958 
Gasull, 1963), was unsuccessfull. In nearly 
identical shells, we have found snails with 
genitalia similar to those in Ganula lanugi- 
nosa, but differing in the configuration of the 
dart-sac complex and in a larger number of 
tufts in the digitiform glands. These charac- 
ters support introduction of a new species 
from southern Spain: Ganula gadirana. 



MATERIALSAND METHODS 

Living specimens were drowned in tap- 
water and preserved in 70% ethanol. Re- 
moved bodies were dissected and studied by 
optical stereomicroscopes (Zeiss and Nikon). 
Genital system and other structures were 
drawn using a camera lucida (Zeiss and 
Nikon). Nerve rings were removed from the 



'Dpto. Biología Animal I (Zoología), Universidad Complutense de Madrid, Avda. Complutense s/n, 28040-Madrid, Spain; 

titomu@eucmax.sim.ucm.es 

^Dpto. Fisiología Animal y Biología, Universidad de Sevilla, Avda. Reina f^ercedes, 6,4101 2-Sevilla, Spain 

163 



164 



MUÑOZ ETAL 



connective tissue with a pointed watchmaker 
forceps prior to drawings. 

Serial microscopical sections were coloured 
with Mallory's stain and photographed with a 
Zeiss Stemi SV6 photostereomicroscope. 
Radulae were removed from buccal bulbs by 
hot digestion in KOH and then washed in pure 
ethanol. Some radulae were mounted on 
metallic blocks with electron-conductive glue, 
coated with gold, and photographed in a JEOL 
820 SEM, and others were stained by a modi- 
fied hemalumpicroindigocarmine method and 
observed using a Zeiss stereomicroscope. 
Shells were photographed with the same SEM 
or with a Pentax P30N camera. 

Shell parameters were measured in adult 
specimens using a Zeiss stereomicroscope 
with millimethc lens. Features of the central 
nervous system were described according to 
the character states proposed by Tillier (1 989: 
40-42). 

The nomenclature for the genital system 
(that used by other authors in brackets) is the 
following: outer stylophore (= dart sac or lower 
stylophore), inner dartless stylophore (= ac- 
cessory sac or upper stylophore), penial 
papilla (= verge), digitiform glands (= mucous 
glands). 



SYSTEMATIC DESCRIPTION 

Ganula gadirana Muñoz, Almodovar & 
Arrebola, п. sp. 

Helix lanuginosa. Servain, 1880: 51 {non 
Boissy) ["Environs d'Algesiras"]. 

Fruticicola lanuginosa. Sacchi, 1956: 17 
{non Boissy) [only, "nelle regioni tra Malaga e 
Algesiras"]. 

Fruticicola lanuginosa. Sacchi, 1957: 19 
{non Boissy) [only: "presse Malaga e presso 
Algesiras"]. 

Fruticola lanuginosa, Sacchi & Nos, 1958: 
93 {non Boissy) [only: "region d'Algesiras"]. 

Shell (Figs. 1, 2). Shell medium sized, glo- 
bose-depressed, conical-convex above, in- 
flated below, uniform light brown translucent 
(Fig. 1A-B). Spire low conical, consisting of 
5-5 1/2 convex, regularly increasing whorls 
separated by deep sutures. Apex small, pro- 
truding apex; protoconch with 1 1/4-1 1/2 
whorls, striated, with hair scars (Fig. 2A). Last 
whorl large, one and one half times broader 
than the penultimate whorl, rounded at pe- 
riphery, and variably descending near aper- 
ture. Umbilicus very small (0.6-0.8 mm), 
deep, partly covered by the reflected margin 



of peristome. Aperture oblique, oval or nearly 
circular, without internal lip, but with a whitish 
band (reddish in external view). Peristome in- 
terrupted straight, smooth, not thickened, with 
very separated, non-convergent marginal 
edges; columellar edge gently curved, wid- 
ened at origin, very reflected over umbilicus. 
External teleoconch surface with long (0.4 
mm), erect, persistent hairs in transverse 
rows 0.4 mm far apart, with a density of 1 4- 1 7 
hairs/mm^ (Fig. 2B); microsculpture of teleo- 
conch formed by fine, small crests among 
hairs; hair scars curved. 

Dimensions (n = 16): Shell diameter, 11.0- 
14.0 mm (holotype, 13.0 mm); shell height, 
8.0-10.9 mm (holotype, 9.8 mm); aperture 
maximum diameter, 6.8-8.2 mm (holotype, 
7.9 mm). 

Foot (Fig. 2C). Sole with a tripartite appear- 
ance (also visible in preserved specimens): 
central zone light, lateral zones darker. 

Lung roof (Fig. ЗА) with an irregular pattern 
of small, blackish, irregular spots, which are 
larger over kidney borders. 

Kidney (Fig. 3B) with numerous, long, 
raised internal pleats, more numerous on 
ureteric side; primary ureter broad, mostly sit- 
uated on the kidney; secondary ureter, closed 
at first, but open 1 mm from the ureteric angle 
(in front of heart ventricle) to form a broad 
ureteric groove. 

Heart oval, somewhat longer than half 
length of kidney. Slender primary pulmonary 
vein with inconspicuous secondary veins. 

Mantle collar (Fig. 3C). Left lateral lobe, 
thin, small, with lobed upper border and 
straight lower border. Right lateral lobe long, 
thick, with an upper corner forming an anal 
lobe. Both left and right dorsal lobes with free 
marginal borders. Subpneumostomal lobe 
separating anal and pneumostomal orifices. 

Central nervous system (Fig. 3D-F). Cere- 
bral commissure shorter (CC3) and right cere- 
bropedal connective somewhat longer 
(CPD2) than right cerebral ganglion width; 
both right and left cerebropedal connectives 
of similar length (CPR2), somewhat longer 
than cerebropleural connectives; both right 
and left pleural ganglions closer to pedal gan- 
glions than cerebral ganglions (PLD1 , PLG1 ); 
visceral ganglion in median plane of pedal 
ganglions (VG2); right parietal ganglion in 
contact with both visceral and right pleural 
ganglions (PAD2), apparently fused with the 
latter; left parietal ganglion only in contact with 
visceral ganglion (PAG3), both ganglia appar- 
ently fused (FG3). 

Genital system (Figs. 4A-B, 5A-E, 6A-E). 



GANULA GADIRANA N. SP 



165 




FIG. 1. Shells of Ganula gadirana. n. sp., from the type locality. A. Holotype; B. Two paratypes (upper col- 
lected on 30 October 1992, and lower on 14 May 1992). 



Multilobate gonad and hermaphrodite duct 
without special features (not figured). Albu- 
men gland relatively short. FPSC (Fig. 5A), 
with both seminal receptacle and fertilization 
pouch simple, sac-like, the former shorter, 
slender. Ovispermiduct (Fig. 4A), wide, cir- 
cumvoluted, with prostatic and uterine parts 
well differentiated. Vas deferens slender. 

Reniai complex (Figs. 4A-B, 5D) consisting 
of flagellum, epiphallus (i.e., from end of vas 
deferens to attachment point of penial retrac- 
tor muscle), and penis (i.e., from point of 
attachment of penial retractor muscle to geni- 
tal atrium); penial retractor muscle attached to 
penial complex proximal to point in which 
base of penial papilla is contained; a strong 
muscular band (Fig. 4B) extends from outer 
penial wall of proximal penis to wall of genital 
atrium, bending the penial complex; flagellum 
short, 1/3 of epiphallus length, conical in 
shape, with thick walls; epiphallus cylindrical, 
somewhat longer than penis, its inner wall 
pleated; penis with distal yellowish, ring- 
shaped, glandular area (Fig. 5C-D); penial 



papilla long, cylindrical (Fig. 5B), with three 
fenestrations through which wide distal lacu- 
nae inside penial papilla walls communi- 
cate with penial cavity; a nearly isolated 
central canal traverses the penial papilla, its 
walls fixed by radial septa to walls of penial 
papilla. 

Female part (Fig. 4A-B) consisting of a 
short free oviduct (half length of bursa copu- 
latrix duct); bursa copulathx large, shoe- 
shaped, with short pedunculus; vagina short, 
with digitiform glands and dart-sac complex, 
the latter located on one side. Digitiform 
glands (Fig. 5E) composed of many (14-20), 
long, unbranched or basally branched tubes 
disposed all around vagina; tubes with an an- 
nulate appearance, the inner secretory ep- 
ithelium forming small ridges (Fig. 7A, G). 
Dart-sac complex (Figs. 6A-B, D-G, 7A-F) 
short, broad, composed of two stylophores; 
outer stylophore basally surrounded by 
groove of vaginal wall (Fig. 6A-B); inner sty- 
lophore broader, dartless, with thick walls and 
narrow inner cavity, extended into two apical 



166 



MUÑOZ ETAL 




; _.:^r\- 




1 


^шл 


^ 


^ * \ 


1 

^ 


^Pçy^l^ 


SIbé 


Ш 


f 


^_1Щ^ 


п^НШвн 


j^^^ 


ià 


ik N v%r%lîf'*^ 




FIG. 2. Shell (SEM micrographs) and pedal sole of Ganula gadirana, n. sp. A. Protoconch of a paratype from 
Cortijo de Ahojiz; В. Microsculpture and one hair of teleoconch (same locality); С Ventral view of pedal sole 
showing the tripartite sole (from type locality). 



lobes (Fig. 6D, 7A). Inner stylophore opening 
into vagina through a wide orifice, far from 
outer stylophore aperture (Fig. 6B, 7C). Dart 
(Fig. 6C) smooth, straight, circular in section, 
but with flattened, keeled tip, elongate, ex- 
tending out of outer stylophore. Inner vagina 
(Fig. 6B, F-G, 7C-F) with two thick, large 
pleats, which distally fuse to form a tongue- 
like structure, U-shaped in traverse section, 
its tip detached from vaginal walls; outer sty- 
lophore and inner stylophore open far apart 
inside groove of tongue-like structure. 

Juvenile specimens (Fig. 6E-G) with dart- 
sac complex placed at half of vagina length, 
with outer stylophore shorter than bilobed 
inner stylophore and tongue-like structure 
present inside vagina. 

Jaw (Fig. 3H) odontognathous, with 24 ribs, 
central larger. 

Radula (Fig. 8) consisting of many rows of 
teeth each with a formula of 35-37 -i- С -i- 
35-37, with the lateral/marginal transition to- 
wards the 15-1 6th tooth. Central tooth with 
wide basal plate, pointed vertices and body 
with large mesocone and two small ectocones 
nearly fused to base of mesocone. Lateral 
teeth with wide basal plates and body with en- 
docone, large, pointed mesocone and short 



ectocone; both mesocone and ectocone pro- 
gressively slender towards radular margin, 
mesocones with little lateral protuberance and 
pointed ectocones split into two points. 

Other anatomical characters. Right om- 
matophore retractor running between penis 
and vagina. Penial nerve apparently arising 
from nght cerebral ganglion. 

Type Locality 

Arroyo de la Cabañuela, Puerto de Bolonia, 
Tarifa (Cádiz, Spain, UTM: 30STE552990). 

Type Material 

Holotype (shell and dissected specimen) - 
30 October 1992, A. Almodovar, В. Muñoz 
and P. Refoyo leg. Museo Nacional de Cien- 
cias Naturales, Madrid, Spain. 

Paratypes: 

-Type locality, 15 paratypes: 11 specimens 
(10 dissected), 30 October 1992, A. Almo- 
dovar, В. Muñoz and P. Refoyo leg., 1 
shell, 9 May 1994, A. Almodovar leg.; 3 



GANULA GADIRANA N. SP 



167 




FIG. 3. Some body parts of Ganula gadirana. n. sp., from Los Barrios, 16 May 1993 (A-G) and the type lo- 
cality (H). A. Pigmentary patches on the pulmonary cover (external view); B. Kidney, ureters and pericardium; 
С Mantle collar; D-E. Left and right views of central nervous system; F. Posterodorsal view of central ner- 
vous system from another specimen; G. Position of salivary glands in respect to buccal bulb; H. Jaw. Abré- 
viations: an, anal lobe; bb, buccal bulb; ce, cerebral ganglion; dsg, ducts of salivary glands; in, intestine; ki, 
kidney; Id, left dorsal lobe; II, left lateral; oe, oesophagus; pa, parietal ganglion; pc, pericardium; pe, pedal 
ganglion; pi, pleural ganglion; pu, primary ureter; pv, pulmonary vein; rd, right dorsal lobe; re, rectum; ri, right 
lateral lobe; sg, salivary glands; sp, supneumostomal lobe; su, secondary ureter; vi, visceral ganglion. Scale 
bar, 1mm. 



168 



MUÑOZ ETAL 




FIG. 4. Genital system of Ganula gadirana. n. sp. A. Specimen from type locality (gonad excluded); B. Spec- 
imen from Los Barrios (distal ducts), 3 November 1991. Abréviations: ag, albumen gland; apb. atrio-penial 
muscular band; is, inner stylophore; be, bursa copulatrix; dbc, duct of bursa copulatrix; dg, digitiform glands; 
OS, outer stylophore; dsc, dart sac complex; e, epiphallus; f, flagellum; to, free oviduct; ga, genital atrium; hd, 
hermaphrodite duct; p, penis; po, prostatic part of ovispermiduct; pr, penial retractor muscle; uo, uterine part 
of ovispermiduct; v, vagina; vd, vas deferens. Scale bar, 1 mm. 



GANULA GADIRANA N. SP 



169 




FIG. 5. Some genital parts of one specimen of Ganula gadirana. n. sp., from Los Barrios, 16 May 1993. A. 
Fertilization pouch and seminal receptacle complex (external view) and four transversal sections: B. Reniai 
papilla within penis and transversal sections at different levels (1 .33 times enlarged): С Glandular area on 
distal part of penis (internal view): D. Penial complex (contracted) and transversal sections (1.5 times en- 
larged) of epiphallus and flagellum: E. Shape and disposition of digitiform glands on vagina. Abréviations: 
apb, atho-penial muscular band: dl, distal lacuna: e, epiphallus: f, flagellum: fp, fertilization pouch: gr, glan- 
dular ring: hd. hermaphrodite duct: os, ovispermiduct: p, penis: pf, papillär fenestration: sd, seminal duct: sr, 
seminal receptacle. Scale bar, 1 mm. 



170 



MUÑOZ ETAL 




FIG. 6. Some genital parts of Ganula gadirana. n. sp. A. External views of dart-sac complex in a contracted 
specimen from Los Barrios, 16 May 1993; B. Same dart-sac complex in longitudinal section: С Dart of a 
specimen from type locality; D. Inner vaginal view of dart-sac complex and cavity of bilobed inner stylophore 
from a specimen from Los Barrios, 5 February 1990; E. Genital system (gonad excluded) of a juvenile spec- 
imen from Los Barrios, 1 6 May 1 993. Note position of dart-sac complex on vagina and bilobation of inner sty- 
lopfiore; F Inner vaginal view; G. Longitudinal section of dart-sac complex of same juvenile specimen. Ab- 
breviations as in Fig. 4. Scale bar, 1mm. 



GANULA GADÍRANA N. SP 



171 



os ]s dg 



A e 



/' 



OS 



'^•^^Щ^£^ 



в 



D 






\ 



/ 



■/ ; 



dbc 



Л' 







I 



ga 

/ 










G 










FIG. 7. Outline of dart-sac complex (digitiform glands not figured) and transversal sections at different lev- 
els. A. Bilobed cavity of inner stylophore and inner ridges of digitiform glands; B. Monoluminar cavity of inner 
stylophore and opening of a digitiform gland inside vagina; C. Opening of inner stylophore inside groove 
formed by vaginal pleats; D. Groove between inner and outer stylophiores forming a functional channel; E. 
Opening of outer stylophore inside groove and fused pleats partially detached from vaginal wall; G. Enlarged 
transversal section of digitiform glands showing inner ridges and secreted mucus. 



specimens (dissected), 14 May 1994, A. 
Almodovar leg. 

-Cortijo de Ahojiz, between km 90-91 of 
C-440 road to Los Barrios (Cádiz, UTM: 
30STF701 0), 1 6 paratypes: 3 specimens (one 
dissected), 5 February 1990; 6 specimens (2 
dissected), 18 May 1991; 1 specimen (dis- 
sected), 24 March 1991 ; 6 specimens (3 dis- 
sected), 3 November 1991. All, J. Arrebola 
leg. 

-'Campo de Gibraltar' sawmill, Los Barrios 



(Cádiz, UTM: 30STF7507), 30 paratypes: 21 
specimens (mostly juveniles, 5 dissected) and 
9 shells, 16 May 1993, E. Unamuno and J. С 
Ruiz leg. 

-Algeciras, near a shelter (Cádiz, UTM: 
30STE7394), 1 paratype (damaged shell), 16 
May 1993, E. Unamuno and J. С Ruiz leg. 

-2 km towards Punta Paloma from N-340 
road. Tarifa (Cádiz, UTM: 30STE5694), 10 
paratypes (3 dissected), 6 December 1993, J. 
Arrebola leg. 



172 



MUÑOZ ETAL 




FIG. 8. SEM micrographs of the radula of Ganula gadirana. n. sp. A. Central tooth and lateral teeth; B. Last 
lateral and marginal teeth. 



Dehvatio Nominis 

From the name of the Fenician colony 
Gadir, which gave origin to Cádiz, in the 
southernmost province of Spain, where the 
described snails were collected. 

Ecology 

Ganula gadirana has been found under 
stones, half buried in the ground or on herba- 
ceous vegetation associated to mediter- 
ranean bushes {Chamaerops humilis and Ne- 
hum oleander) and close to periodically river 
flows. The associated malacological fauna is 
composed by Rumina decollata (Linnaeus, 
1758), Ferussacia follicula (Gmelin, 1791), 
Helix aspersa Müller, 1774, Cochlicella acuta 
(Müller, 1774), Cochlicella barbara {Linnaeus, 
1758), Gasulliella simplicula (Morelet, 1845), 
Caracollina lenticula (Michaud, 1831), Xe- 
rotricha apicina (Lamarck, 1822), Cernuella 
virgata (Da Costa, 1 778), Theba pisana (Mül- 
ler, 1774), Ótala láctea (Müller, 1774), Can- 
didula д1дахИ{Р^еШг, 1848), Oestophora bar- 
bula (Rossmässler, 1838), and Oestophora 
íam /ел/ (Morelet, 1854). Specimens from Los 
Barrios, collected on 1 6 July 1 993, were inten- 
sively parasitized by small nematodes, which 
were found in all growth stages inside the pul- 
monary cavity. 



all records from southern Spain referred to 
this species have been assigned to Ganula 
gadirana. 

There are older conchological records for 
Helix lanuginosa from northern Africa - from 
Morocco (Hidalgo, 1909: "Muluya") to north- 
western Algeria (Terver, 1839; Bourguignat, 
1864), but with one record from Tunisia (Le- 
tourneux & Bourguignat, 1887). Other two 
nominal species are considered to be related 
to G. lanuginosa - Helix flava Terver, 1839, 
and Helix roseotlncta Forbes, 1838 (Clessin, 
1881; Richardson, 1980). Helix flava from 
"Gourayah," near Bougie (Terver, 1839), and 
H. roseotinctawas cited from northeastern Al- 
geria (Bourguignat, 1864) to northern Tunisia 
(Letourneux & Bourguignat, 1887) (Bourguig- 
nat, 1 864, considered the two species as syn- 
onymous). Taking into account that Ganula 
gadirana has been an overlooked species 
identified as H. lanuginosa, there is a possi- 
bility that some of the African records belong 
to Ganula gadirana. Further studies must be 
carried out to test this. Nevertheless, Giusti & 
Manganelli (1 987) stated that H. flava is an Al- 
gerian species clearly distinct from G. lanugi- 
nosa and also from G. gadirana (Giusti, pers. 
com.). 



DISCUSSION 



Geographical Distribution (Fig. 9) 

All known localities are in the province of 
Cádiz in the southernmost corner of Spain, 
concentrated on a small region around the 
northern side of the Gibraltar Strait. Because 
the previously recorded presence of Ganula 
lanuginosa has not been confirmed for that re- 
gion after many searches (see Introduction), 



The new species has similar features to 
Ichnusotricha berninii Giusti & Manganelli, 
1987, and Ganula lanuginosa (Boissy, 1835). 
Ichnusotricha berninii has a similar shell 
shape, but the sutures are more superficial, 
the aperture is descendent, the umbilicus is 
nearly closed, and the periostracal hairs are 
very short. Anatomically, it differs from the 
new species, because /. berninii has a long, 



GANULA GADIRANA N. SP 



173 





— 5 


5 


— 7 


5 






2 












1 






I 






^ 






■ 


О 


IP 


\ 
9 


■ 


^ 


■^ 


5 


tE 


8 


















X 




3¿5 


''\r-- 



FIG. 9. Geographical distribution of Ganuia gadirana, n. sp. (square and upper left inset) and other endemic 
Hygromiinae from the western Mediterranean area (larger letters): C, Cernuellopsis ghisottii Manganelli & 
Giusti, 1987; G, Ganuia lanuginosa (Boissy, 1835); I, Ichnusotricha bern I ni i GlusW & Manganelli, 1987; N, 
Nienhuisiella antonellae Giusti & Manganelli, 1987; Y, Cyrnotheba Corsica (Shuttleworth, 1843); X, Xero- 
plana lacosteana (Bourguignat, 1864). Smaller letters from northern Africa show old conchologica! records 
for Helix flava Terver, 1 839 (F), Helix lanuginosa Boissy, 1 835 (L), and Helix roseotincta Forbes, 1 838 (R). 



non-fenestrated penial papilla; small, narrow 
dart-sac complex placed far apart; shorter 
digitiform glands: and long cylindrical vagina, 
with two long, slender pleats fused distally to 
form an apical tap or dart gun (Giusti & Man- 
ganelli, 1987). 

Ganuia lanuginosa has a nearly identical 
shell, although it is convex-depressed above, 
with more superficial sutures and less convex 
whorls, shorter (length, 0.3 mm; in shells from 
Mallorca), less spaced hairs (25-30/mm^), 
wider umbilicus (1/10 of shell diameter), and 
descending aperture. Anatomically, G. lanugi- 
nosa and G. gadirana are very different, be- 
cause G. lanuginosa has four bifurcated digi- 
tiform glands, standard inner stylophore, and 
non-fenestrated penial papilla (Gittenberger, 
1968; Giusti & Manganelli, 1987). Ganuia 
gadirana has 1 4-20 digitiform glands, and the 
tubes have an annulate appearance, apically 
bilobed inner stylophore, fenestrate penial 
papilla, and penial glandular area. 

On the other hand, in the pedal sole G. 
gadirana appears tripartite, and the left pari- 
etal ganglion is in contact with the visceral 
ganglion; these features are unknown in G. 
lanuginosa, but are present in a few species 
of the family Hygromiidae. The tripartite ap- 
pearance of the pedal sole appears frequently 



among helicoids (Schileyko, 1978), and is il- 
lustrated in a drawing of Leucozonella rubens, 
although we do not know other similar cases. 
The genera of Hygromiidae studied by Tillier 
(1 989) have a more anterior position of the left 
parietal ganglion, being in contact with both 
visceral and left pleural ganglions. 

Ganuia lanuginosa has radular formula of 
32 + С + 32 (Giusti & Manganelli, 1987), 
whereas G. gadirana has a formula of 35- 
37 -b С -b 35-37; both species have a similar 
central tooth, but G. lanuginosa has lateral 
teeth with an apex formed by a wide, robust, 
pointed mesocone and a short, sharp, robust 
ectocone, whereas the new species has lat- 
eral teeth with a large endocone, pointed 
mesocone, and short ectocone. Two points on 
the mesocone apex of the extreme marginal 
teeth appear occasionally in G. lanuginosa, 
whereas there is a little lateral protuberance in 
G. gadirana. 



ACKNOWLEDGEMENTS 

Special thanks for comments on a previous 
version and precise observations on the de- 
scription of the genital system to Dr. Foico 
Giusti (Siena, Italy). The authors wishes to 



174 



MUÑOZ ETAL 



thank to Dr. С E. Prieto (Bilbao, Spain) for his 
helpful comments on the description genital 
system and for his drawing (Figs. 3, 4B, 5 and 
6). 



LITERATURE CITED 

BOURGUIGNAT J. R.. 1864, Malacologie de l'Al- 
gérie. 2 vols. Paris. 686 pp. + 58 pis. 

CLESSIN, S., 1881, Nomenclátor Heliceorum 
viventium quo continetur nomina omnium hujus 
familiae generum et specierum hodie cognitarum 
disposita ex affinitate naturali. Opus postum- 
mum. Ed. Fischer, Kassel. 617 pp. 

EMBERTON, K. С & S. TILLER, 1995, Letter: to 
the editor. Clarification and evaluation of Tillier's 
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3-80. 

GITTENBERGER, E., 1968, Zur Systematischen 
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Revised ms. accepted 17 December 1998 



MALACOLOGIA, 1999, 41(1): 175-186 



COMPARATIVE KARYOLOGICAL STUDY OF CUPPED OYSTER SPECIES 

Alexandra Leitao,^ Pierre Boudry,^ Jean-Philippe Labat^ & 
Catherine Thiriot-Quiévreux^* 



ABSTRACT 

Chromosomes of six cupped oyster species were studied using karyometric analysis after 
conventional Giemsa staining, and silver staining. Karyotypes of Crassotrea angulata (nine 
metacentric and one submetacentric chromosome pairs), C. sikamea (nine metacentric and one 
submetacentric chromosome pairs), С virginica (eight metacentric and two submetacentric 
chromosome pairs), С ariakensis (eight metacentric and two submetacentric chromosome 
pairs), C. grasar (six metacentric and four submetacentric chromosome pairs), and Saccostrea 
commercialls (eight metacentric and two submetacentric chromosome pairs) are distinguishable 
by the number and position of the submetacentric chromosome pair and by the location of nu- 
cleolus organizer regions. Comparative karyological analysis of these six cupped oysters and of 
С gigas was made using a Principal Component Analysis and a Hierarchical Clustering Analy- 
sis. Crassostrea gasar appears isolated from the other oyster species. Then, two clusters are 
separated. The first one groups С gigas. С angulata and С sikamea, in which С gigas is ple- 
siomorphic. The second one consists of C. ariakensis. С virginica and S. commercialis. Results 
are discussed with regards to oyster species relationships based on other genetic characters and 
to hybridization possibilities. 

Key words; cupped oyster, chromosome, karyotype, NORs, cytotaxonomy, Bivalvia. 



INTRODUCTION 

Chromosomes of Ostreidae have been 
studied in 22 species (Nakamura, 1985; Vit- 
turi et al., 1985; leyama, 1990). Cupped oys- 
ter species of the genera Crassostrea and 
Saccostrea show a common diploid chromo- 
some number of 2n = 20, and their karyotypes 
include only metacentric and submetacentric 
chromosomes (Table 1). Interspecific differ- 
ences consist of the occurrence and differing 
proportions of these morphological types, 
identified either by observation or after chro- 
mosome measurements. Karyotype differ- 
ences may be seen within a species (e.g., С 
rhizophorae; Table 1 ) which could be due ei- 
ther to intraspecific polymorphism or to the 
different techniques used. 

Oyster species might have become differ- 
entiated through pericentric inversions of cen- 
tric shifts. However, cytotaxonomic compari- 
son needs to be based on karyological 
analysis carried out by the same technique 
and the same worker. For example, the con- 
centration and time of incubation in the 
colchicine and in the hypotonic treatment, re- 
sulting in differential condensation or elonga- 



tion of chromosomes (Sharma & Sharma, 
1980), vary from one author to another. Kary- 
ometric analysis brings a more quantitative 
method to assess chromosome morphology, 
but still depends on the condensation or elon- 
gation of chromosomes. 

Banding techniques have been found to be 
very useful for the identification of individual 
chromosomes and also of particular regions 
of chromosomes. Few studies have looked at 
banding patterns in the chromosomes of oys- 
ters (Rodriguez-Romero et al., 1 979c; Insua & 
Thiriot-Quiévreux, 1991; Li & Havenhand, 
1997). Fluorescence in situ hybridization has 
been tested in Crassostrea gigas (Clabby et 
al., 1 996; Quo & Allen, 1 997). Selective stain- 
ing of the nucleolus organiser regions (NORs) 
has been shown to have potential as a cyto- 
taxonomic tool (e.g., Amemiya & Gold, 1990). 
Patterns of specific NORs have been de- 
scribed in five species of oysters (Thiriot- 
Quiévreux & Insua, 1992; Insua & Thiriot- 
Quiévreux, 1993; Ladron de Guevara et al., 
1994). Identification of structural chromo- 
some features is useful in hybrid breeding 
programs and in oyster stock conservation. 

In the present study, karyotypes and NORs 



'corresponding author 

ЮЬ5егуа1о1ге Océanologique, LIRA 2077, BP 28, 06230 Villefranche-sur-Mer, France 

^Station IFREMER, Laboratoire de Génétique, Aquaculture et Pathologie, BP 1 33, 1 7390 La Tremblade, France 



175 



176 




LEITAOETAL 




TABLE 1 . Chromosome data 


in cupped oysters. 






Species 


2n 


karyotype 


Origin 


Authors 


Crassostrea 










С amasa (Iredale) 


20 




Australia 


Menzel, 1968 


С. angulata (Lamarck) 


20 




Portugal 


Menzel, 1968 




20 


10m* 


France (Barfleur) 


Thiriot-Quiévreux, 1984 


С. belcheria (Sowerby) 


20 


10 m-sm 


Japan 


leyama & Inaba, 1974 


С. corîeziensis (Hertiein) 


20 


7m-3sm* 


Mexico 


Rodriguez-Romero et a!, 1979a 


С. gigas (Th un berg) 


20 


8m-2sm 


USA 


Ahmed & Sparks. 1967 




20 


10m* 


France (Barfleur) 


Thiriot-Quiévreux, 1984 


С. glomerata (Gould) 


20 




West Pakistan 


Ahmed, 1973 


С. gryphoides (Scholteim) 


20 




West Pakistan 


Ahmed, 1973 


С. /reda/e/ (Faustino) 


20 




Philippines 


Menzel, 1968 


С. rhizophorae (Guilding) 


20 


5m-5sm* 


Mexico 


Rodriguez-Romero et al., 1979b 




20 


8m-2sm* 


Venezuela 


Marquez, 1992 


С. 'Tivularis (Gould)" 


20 




West Pakistan 


Ahmed, 1973 


Syn. C. ariakensis (Fujita) 


20 


lOm-sm 


Japan 


leyama, 1975 


С sikamea (Amemiya) 


20 




West Pakistan 


Ahmed, 1973 


(Kumamoto variety of 










С gigas) 










С virginica (Gmeiin) 


20 


6m-4sm* 


East coast USA 


Longwell et al., 1967 




20 


6m-4sm* 


Mexico 


Rodriguez-Romero et al., 1978 




20 


6m-4sm* 


Venezuela 


Marquez, 1992 


Saccostrea 










S. commercialis (Iredale & 


20 




Australia 


Menzel, 1968 


Roughley) 










S. cucullata (Born) 


20 


10m* 


India 


Goswami, 1992 


S. echinata (Quoy & 


20 


lOm-sm 


Japan 


leyama & Inaba, 1974 


Gaimard) 










S. mordax (Gould) 


20 


lOm-sm 


Japan 


leyama & Inaba, 1974 



2n; diploid chromosome number 
*: after chromosome measurements 
m: metacentric; sm: submetacentric 



were studied in six species of cupped oysters: 
Crassostrea angulata, С sikamea. C. vir- 
ginica, С ariakensis, С gasar, and Sac- 
costrea commercialis. These species originat- 
ing from different areas were imported and 
reared in common quarantine facilities. Com- 
parative karyologicai analysis was made with 
reference to C. gigas (Thiriot-Quiévreux, 
1984, and unpublished data, 1997). 



MATERIALS AND METHODS 
Species Studied 

Five cupped oyster species of the genus 
Crassostrea and one of the genus Saccostrea 
were studied, none native to Europe. Cras- 
sostrea gigas and С angulata have been in- 
troduced into the natural environment for 
decades (Grizel & Héral, 1991) or centuries 
(Boudry et al., 1998) respectively. The other 
species were recently imported into France as 
part of a genetic resources research program. 
They have been strictly confined to the quar- 



antine facilities of the IFREMER hatchery in 
La Tremblade, Charente-Maritime, France, 
according to international recommendations. 
All the oysters studied were reared in the 
same environmental conditions for at least 
three months before sampling. The С angu- 
lata oysters studied originate from the Rio 
Sado estuary, Setubal, Portugal. The taxo- 
nomic status of these oysters was assessed 
using mitochondrial DNA markers as de- 
scribed in Boudry et al. (1998). Crassostrea 
sikamea were imported from Bodega Marine 
Laboratory, University of California, USA. 
Their taxonomic status was confirmed using 
mitochondrial DNA markers as described in 
Banks et al. (1993). Crassostrea virginica 
were imported from a wild stock located in 
Shippagan, New Brunswick, Canada. Cras- 
sostrea ariakensis ("C. rivularis." auctt.) were 
imported from the Shellfish Research Labora- 
tory, Rutgers State University, New Jersey. 
USA. This species was introduced from 
Japan into the Northwest waters of the USA. 
and its aquaculture potential has been re- 
cently reviewed by Langdon & Robinson 



KARYOLOGY OF CUPPED OYSTERS 



177 



(1996). Mangrove oysters, С gasar, were im- 
ported from a wild stock located in Kafountine, 
Casamance, Senegal. Saccostrea commer- 
cialis were collected from the wild at Port 
Stephens, New South Wales, Australia. 

Because of the low number of animals 
available, only two animals from each species 
(except three of C. sikamea) were used for 
this study. 

Chromosome Preparations 

Oysters were incubated for 7 h with 0.005% 
colchicine in sea water. The gills were then dis- 
sected out and treated for 30 min in 0.9% 
sodium citrate in distilled water. The material 
was then fixed in a freshly prepared solution of 
absolute ethanol and acetic acid (3:1), with 
three changes of 20 min duration each. Slide 
preparation was made using an air-drying 
technique (Thiriot-Quiévreux & Ayraud, 1 982). 
For conventional karyotypes, slides were 
stained directly with Giemsa (4%, pH 6.8) for 
10 min. Photographs of suitable mitotic meta- 
phases were taken with a Zeiss III photomi- 
croscope, and after karyotyping, chromosome 
measurements of ten metaphases in each 
species were made with a digitizer table 
(Summa Sketch II) interfaced with a Macin- 
tosh. Data analysis was performed with an 
Excel macro program. Terminology relating to 
centromere position follows that of Levan et al. 
(1964). NORs were silver-stained directly on 
unstained slides using the technique of How- 
ell & Black (1980), modified by Gold & Ellison 
(1982). 

Statistical Analysis 

In order to evaluate the relationships be- 
tween the six species studied here and С 
gigas, a principal component analysis (PCA) 
was carried out. The data set is a matrix of 70 
objects, that is, ten metaphases in seven 
species described by the centromeric index 
values of ten chromosome pairs. Means of 
centromeric index values for each species 
were considered as supplementary objects 
and were projected in the PCA space. The po- 
sition (i.e., component score) of the ten 
metaphases around this mean point gives in- 
formation of the scattering of each species. 
Their correlations give a criterion of their ex- 
planation by the PCA axes considered. As a 
second step, a hierarchical clustering analysis 
(HCA) was performed between the species 
described by their component scores on the 
first four axes of the PCA, using the Ward ag- 



glomeration method (Ward, 1963). This clus- 
tering offers the possibility of representing the 
distances between species by a dendogram. 
PCA and HCA were computed with the SPAD 
software (CESIA) (Lebart et al., 1995). 



RESULTS 

The results obtained for each species are 
summarised in Table 2. 

Crassostrea angulata 

The karyotype (Fig. 1 A, Table 3) consists of 
nine metacentric and one submetacentric (no. 
8) chromosome pairs. Ag-NORs were found 
terminally on the metacentric pair 10. The two 
homologous chromosomes showed hetero- 
morphism involving apparent NOR activity. 
The most frequent case (69%) was one silver- 
stained NOR chromosome (Fig. 2A). 

Crassostrea sikamea 

The karyotype (Fig. 1 B, Table 3) shows nine 
metacentric and one submetacentric (no. 6) 
chromosome pairs. Ag-NORs were found ter- 
minally on the metacentric pairs nos. 9 and 1 
(Fig. 2B). A variable number of one to three 
Ag-NORs was observed. 54% of the silver 
stained metaphases only showed NORs on 
pair 10. The most frequent case (61%) was 
one silver-stained NOR chromosome in pair 
10. 

Crassostrea virginica 

The karyotype (Fig. 1С, Table 3) has eight 
metacentric and two submetacentric (nos. 4 
and 8) chromosome pairs. Ag-NORs were 
found terminally on the short arms of meta- 
centric pairs nos. 1 and 5 (Fig. 2C). A variable 
number of one to three Ag-NORs was ob- 
served. The most frequent case (52%) was 
one silver-stained NOR chromosome in pair 1 
and in pair 5. 

Crassostrea ariakensis 

The karyotype (Fig. 1 D, Table 3) consists of 
eight metacentric and two submetacentric 
(nos. 4 and 8) chromosome pairs. Ag-NORs 
were found terminally on the metacentric 
pairs 9 and 10. A variable number of one to 
three Ag-NORs was observed (Fig. 2D). 68% 
of the silver stained metaphases showed Ag- 
NORs only on pair 10. 

Crassostrea gasar 

The karyotype (Fig. IE, Table 3) includes 
six metacentric and four submetacentric (nos. 



178 



LEITAOETAL. 



TABLE 2. Summary of karyological data of the six cupped oysters studied. 



Species 



No. metaphases 
studied 



No. karyotypes 
studied 



Giemsa 



NOR 



Giemsa 



NOR 



2n 



Chromosome 

type (no. 
chromo. pairs) 

m sm 



No. of (haploid) 

NOR-chromo- 

somes 



С angulata 
C. sikamea 
C. virginica 
С ahakensis 
С gasar 
S. commercialis 



42 
32 
29 
30 
33 
34 



31 
62 
57 
46 
55 
35 



13 
15 
10 
17 
13 
13 



12 
8 

7 



20 
20 
20 
20 
20 
20 



1 (pair 10) 

2 (pairs 9 and 10) 
2 (pairs 1 and 5) 
2 (pairs 9 and 10) 

1 (pair 2) 

2 (pairs 9 and 10) 



*after chromosome measurements of 10 metaphases 
m: metacentric; sm: submetacentric 



2, 8, 9, and 10) chromosome pairs. Ag-NORs 
were found terminally on the short arms of two 
homologous chromosomes of the metacentric 
pair 2 (Fig. 2E). Heteromorphism involving 
NOR-size occurred in 49% of the metaphases 
examined. 

Saccostrea commercialis 

The karyotype (Fig. IF, Table 3) shows 
eight metacentric and two submetacentric 
(nos. 4 and 7) chromosome pairs. Ag-NORs 
were found terminally on the metacentric 
pairs 9 and 10. A variable number of one to 
three NORs were observed. 77% of the silver 
stained metaphases showed Ag-NORs only 
on pair 10 (Fig. 2F). 



Comparative Karyological Analysis 

Figure 3 shows ideograms constructed from 
relative length and centromeric index values 
(Table 3) of the six oyster species studied here 
and of Crassostrea gigas. Chromosome mea- 
surements of this later species were taken 
from ten metaphases of animals collected at 
La Tremblade in 1 997. Mean values of relative 
length and centromeric index are similar to 
those found in C. gigas from Barfleur (Thiriot- 
Quiévreux, 1984). Crassosíreagfasar is distin- 
guishable from the other species first, due to 
the occurrence of four submetacentric chro- 
mosome pairs. Crassostrea angulata and С 
sikamea showed only one submetacentric 
chromosome pair, whereas С virginica, С ari- 
akensis and S. commercialis have two sub- 
metacentric chromosome pairs. Crassostrea 
angulata and С sikamea may be differentiated 
by the different positions of the submetacentric 
chromosome pair and by the Ag-NORs which 
appear on pair 10 and on pairs 9 and 10 re- 
spectively. Crassostrea virginica and С ariak- 



ensis share a similar karyotype, but Ag-NORs 
are observed in different locations (pairs 1 and 
5, and pairs 9 and 10, respectively). Sac- 
costrea commercialis is close to С ariakensis. 
Their karyotypes differ by the position of the 
second submetacentric pair and by the fre- 
quencies of Ag-NORs observed on pair 10. 
Crassostrea gigas has the most symmetrical 
karyotype, with only metacentric chromosome 
pairs. 

Principal component analysis of the data 
set of 70 objects (ten metaphases for seven 
species described by centromeric index val- 
ues of ten chromosome pairs) gives percent- 
ages of variance for the first five axes of 
31.74, 20.06, 12.61, 11.17 and 7.77 respec- 
tively. The variance decreases progressively 
from 5th axis. We have thus only considered 
the information provided by the first four axes 
as relevant. The 1/2 plan (Fig. 4) explains 
51.80% of the variance. It shows the sepa- 
rated position of С pasar (correlation with 1/2 
plan of 0.98) without continuity with the other 
species. The six other species overlap along 
a continuum. Crassostrea gigas (correlation 
of 0.87) is the most distant from this contin- 
uum. Then, С ariakensis and С virginica are 
very close and overlap a part of S. commer- 
cialis (correlations of 0.57, 0.43 and 0.52, re- 
spectively). Crassostrea sikamea (correlation 
of 0.38) shows a larger scattering. Cras- 
sostrea angulata is unexplained by this plan, 
as shown by its correlation of 0.01. The 3/4 
plan explained less of the total variance: 
23.78%. There is a trend of separation be- 
tween С virginica. С ariakensis and S. com- 
mercialis (correlations of 0.40, 0.17 and 0.55 
respectively). Crassostrea angulata and С 
sikamea remain together (correlations of 
0.48 and 0.55 respectively). Figure 5 shows 
the dendogram of a Hierarchical Clustering 
Analysis made using the information from the 



в 



KARYOLOGY OF CUPPED OYSTERS 1 79 

1 2 3 4 5 

6 7 Я 8 9 10 

;^K M )!>t ÍS U 

>X XX KU At KI 



9( 

С 



II a:i fiA *,, ¿ai 

^ix st ¿л ï» 

» 

JIK >1х p%jr^ /^êy. .-^ 

'>;к .'^'■(^ лх г./- 

FIG. 1 . Giemsa-stained karyotypes of six cupped oysters. A: Crassostrea angulata. B: Crassostrea sikamea. 
C: Crassostrea virginica. D: Crassostrea ariakensis. E: Crassostrea gasar F: Saccostrea commercialis. Ar- 
rows show submetacentric chromosome pairs. Scale bar = 5 цт. 



180 



LEITAOETAL. 



TABLE 3. Chromosome measurements and classification in ten cells of six cupped oyster species. 



Chromosome 
pair No. 


Relative 


length 


Arm ral 


tio 


Centromei 


ric index 


Chromosome 
Type 


Mean 


SD 


Mean 


SD 


Mean 


SD 


С angulata 
1 


12.81 


0.91 


0.79 


0.08 


43.79 


2.50 


m 


2 


11.34 


0.41 


0.84 


0.09 


45.22 


2.45 


m 


3 


10.75 


0.41 


0.83 


0.09 


45.02 


2.65 


m 


4 


10.33 


0.63 


0.64 


0.09 


38.46 


2.89 


m 


5 


10.12 


0.74 


0.82 


0.06 


44.79 


1.86 


m 


6 


9.82 


0.60 


0.62 


0.07 


38.01 


2.56 


m 


7 


9.53 


0.82 


0.88 


0.07 


46.47 


1.94 


m 


8 


9.25 


0.68 


0.59 


0.07 


36.84 


3.01 


sm 


9 


8.98 


0.66 


0.68 


0.12 


40.07 


3.83 


m 


10 


7.08 


0.67 


0.75 


0.10 


42.36 


3.44 


m 


C. sikamea 
















1 


12.40 


1.17 


0.85 


0.17 


45.24 


4.35 


m 


2 


11.28 


0.59 


0.77 


0.10 


43.08 


2.99 


m 


3 


11.20 


0.64 


0.87 


0.12 


46.03 


3.38 


m 


4 


10.39 


0.93 


0.86 


0.12 


45.92 


3.14 


m 


5 


10.38 


0.78 


0.85 


0.13 


45.41 


3.89 


m 


6 


9.87 


0.89 


0.53 


0.13 


34.20 


5.27 


sm 


7 


9.50 


0.76 


0.79 


0.13 


43.72 


4.36 


m 


8 


9.27 


1.19 


0.79 


0.08 


43.52 


2.48 


m 


9 


8.61 


0.89 


0.82 


0.20 


44.28 


6.39 


m 


10 


7.10 


1.05 


0.95 


0.18 


47.75 


4.98 


m 


С virginica 
1 


12.71 


0.67 


0.88 


0.08 


46.46 


2.51 


m 


2 


11.50 


0.56 


0.87 


0.11 


46.27 


3.03 


m 


3 


10.89 


0.90 


0.79 


0.11 


43.61 


3.62 


m 


4 


10.57 


0.71 


0.46 


0.08 


30.97 


3.49 


sm 


5 


10.33 


0.47 


0.82 


0.08 


44.63 


2.34 


m 


6 


9.62 


0.61 


0.74 


0.09 


42.18 


3.19 


m 


7 


9.38 


0.92 


0.79 


0.16 


43.52 


5.43 


m 


8 


9.16 


0.69 


0.43 


0.10 


29.34 


4.57 


sm 


9 


8.64 


0.51 


0.73 


0.11 


41.81 


3.78 


m 


10 


7.19 


0.54 


0.86 


0.12 


45.63 


3.49 


m 


С ariakensis 
















1 


12.10 


0.63 


0.82 


0.07 


44.65 


2,09 


m 


2 


11.34 


0.43 


0.77 


0.10 


42.98 


3.47 


m 


3 


10.67 


0.46 


0.78 


0.07 


43.71 


2.10 


m 


4 


10.54 


0.59 


0.51 


0.05 


33.77 


2,32 


sm 


5 


10.21 


0.79 


0.81 


0.09 


44.44 


2,55 


m 


6 


9.91 


0.39 


0.81 


0.12 


44.30 


3,78 


m 


7 


9.64 


0.57 


0.74 


0.07 


42.12 


2,03 


m 


8 


9.44 


0.78 


0.53 


0.08 


34.25 


3,08 


sm 


9 


9.09 


0.90 


0.79 


0.17 


43.41 


5,00 


m 


10 


7.07 


0.43 


0.76 


0.12 


42.64 


3.86 


m 


С gfasar 

1 


11.36 


0.63 


0.79 


0.10 


43.64 


3.11 


m 


2 


11.19 


0.74 


0.38 


0.05 


27.52 


2.34 


sm 


3 


11.04 


0.41 


0.85 


0.08 


45.62 


2.47 


m 


4 


10.62 


0.76 


0.62 


0.07 


37.80 


2.31 


m-sm 


5 


10.54 


0.51 


0.90 


0.09 


46.95 


2.45 


m 


6 


9.97 


0.43 


0.86 


0.08 


45.77 


2.33 


m 


7 


9.64 


0.54 


0.84 


0.12 


45.21 


3.54 


m 


8 


9.54 


0.64 


0.45 


0.07 


30.78 


3.43 


sm 


9 


8.82 


0.60 


0.41 


0.06 


28.65 


3.01 


sm 


10 


7.28 


0.56 


0.39 


0.08 


27.89 


3.78 


sm 



KARYOLOGY OF CUPPED OYSTERS 



181 



TABLES. (Continued) 





Relative 


length 


Arm ratio 


Centromeri 


С index 




ChromosomG 














Ciiromosome 


pair No. 


Mean 


SD 


Mean 


SD 


Mean 


SD 


Type 


S. commercialis 
















1 


13.41 


1.07 


0.81 


0.08 


44.41 


2.45 


m 


2 


12.39 


1.04 


0.78 


0.06 


43.58 


1.77 


m 


3 


10.84 


0.56 


0.81 


0.09 


44.50 


2.68 


m 


4 


10.36 


0.70 


0.44 


0.05 


30.48 


2.29 


sm 


5 


9.89 


0.74 


0.80 


0.08 


44.07 


2.58 


m 


6 


9.82 


0.66 


0.81 


0.11 


44.32 


3.48 


m 


7 


9.13 


0.98 


0.48 


0.07 


32.24 


2.98 


sm 


8 


9.11 


0.63 


0.78 


0.11 


43.42 


3.56 


m 


9 


8.44 


0.59 


0.81 


0.10 


44.34 


3.01 


m 


10 


6.60 


0.63 


0.79 


0.12 


43.19 


3.76 


m 



m: rnetacentric; sm: submetacentric 



first four axes of the PCA. Crassostrea gasar 
appears clearly separated from the other 
species. Then, two clusters are differentiated, 
one with the grouping of С virginica, С ariak- 
ensls and S. commercialis. and the other with 
two close species С angulata, C. sikamea 
and C. gigas at a higher distance. 



DISCUSSION 

Our chromosome study of these six cupped 
oyster species confirms the diploid chromo- 
some number of 2n = 20 found up to now in all 
cupped oysters examined (Table 1). 

The karyotype of C. angulata differs from 
that described in the animals reared at 
Barfleur (Thiriot-Quiévreux, 1984). This differ- 
ence could be due to the origin of animals. 
Samples in this study came from the Bay of 
Setubal, Portugal, and are considered as pure 
C. angulata (Boudry et al., 1 998). The origin of 
samples in the Barfleur study in unknown. 
The karyotype of С virginica. showing two 
submetacentric chromosome pairs, differs 
from those with four submetacentric chromo- 
some pairs described by Longwell et al. 
(1967), Rodriguez-Romero et al. (1978), and 
Marquez (1992). However, the position of 
these submetacentric chromosome pairs is 
different between these authors. Genetic dis- 
continuity has been observed in this American 
oyster along the Atlantic coast and the Gulf of 
Mexico (Buroker, 1983; Reeb & Avise, 1990; 
Hare & Avise, 1 996). The origin of our animals 
is close to those studied by Longwell et al. 
(1967). Therefore, the karyological variation 
observed could be due either to the effect of 
acclimation or to differences in the technique 
(e.g., different concentrations of colchicine: 



0.02% in the Longwell et a!. 1967 study and 
0.005% in this study). Karyotypes made by 
the same scientist, with the same techniques 
carried out within a short period of time give a 
more valid comparison than karyotypes made 
by different authors. 

Karyotypes of С sikamea, С ariakensis. C. 
gasar 3.^0, S. commercialis are first described 
here. 

Our observations on Ag-NORs are original 
in the six species studied. In С virginica, 
Longwell & Stiles (1996) suggested that NOR 
sites could be located on the secondary con- 
striction observed on the longest metacentric 
chromosome pair. Our results confirm the lo- 
cation of Ag-NORs on this pair 1 , but another 
Ag-NOR was observed on pair 5. Heteromor- 
phism involving apparent NOR activity and 
NOR-size is a common phenomenon in bi- 
valves (Thiriot-Quiévreux & Insua, 1992; 
Insua et al., 1994; Martinez-Exposito et al., 
1994). However, the number of Ag-NORs, 
their chromosomal location and their position 
within karyotypes are considered as species- 
specific characters (Sumner, 1990). In this 
study, the majority of species showed Ag- 
NORs on pair 9 or pairs 9 and 10, in a fre- 
quency that varies according to the species 
considered. The position of NORs was differ- 
ent in С virginica and С gasar. Ag-NORs al- 
lowed the separation of C. angulata and C. 
sikamea. and of С virginica and C. ariakensis 
which have similar karyotypes. 

Comparative karyological analysis (Figs. 
3-5) highlights the isolation of C. gasar. Then 
two clusters are separated. The first cluster 
consists of C. gigas. С angulata and C. 
sikamea. in which C. gigas, with the most sym- 
metrical karyotype, could be considered as 
plesiomorphic. Crassostrea gigas and C. an- 



182 




LEITÄO ET AL. 






A 

Il ъ 


■■•'i 


Щ 


.^^!^ 


,^%^ 




''"'' ^■ 


-i^'^ 




.:■> Ö 

tt 


в 




i* i^ 


« ' V 


«% 


f f 


^' «- 


1^ 


1^ 


Ha 


С *| . 


i'^.' 


>iït 


|l^^^ 
/% .»-% 






aÍÍk.» 


^Ik 












Л^ 


A^ 




¿1^ 




» » 


ПЛ 

^ 


m- 




i' 


« 


nt 


*« cf 


•y 


^í 


/t 


Л Л 


'1? 


f ♦ 


■1 


u 




1С 


II 


if. 


31 


91 

-9^ ^ 



FIG 2 Silver-stained karyotypes of six cupped oysters. A: Crassostrea angulata. B; Crassostrea sikamea. 
C: Crassostrea virginica. D: Crassostrea ariakensis. E: Crassostrea gasar. F: Saccostrea commercialis. Ar- 
rows show Ag-NORs. Scale bar = 5 цт. 



KARYOLOGY OF CUPPED OYSTERS 



183 



C. gigas 




С virginica 



Xy 



Шт 




X 


с. gasar 



s. commercialis 



X 



X 



X 



Шт 



gulata are often considered as a same species 
(Menzel, 1974), as are С. gigas and С. 
sikamea, of which the latter has sometimes 
been considered as the "Kumamoto variety" 
(Ahmed, 1973). Recent molecular genetic 
studies have displayed differences between 
C. gigas and С sikamea (Banks et al., 1994), 
and between С gigas and C. angulata 
(Boudry et al., 1998; Ó Foighil et al., 1998). 
Our karyological study confirms these genetic 
differences. The second cluster put together 
C. ariakensis. С virginica and S. commer- 
cialis. Molecular phylogenetics of cupped oys- 
ters (Littlewood, 1994) distinguished two lin- 
eages: (1) С gigas. С belcheri. "C. rivularis" 
(= С ariakensis) and (2) С virginica, C. rhi- 
zophorae and S. commercialis. Ó Foighil et 
al. (1995) using mitochondrial 16S ribosomal 
gene sequences confirm a genetic divergence 
between С wrg /'л/са and two Asian congeners 
С gigas and С ariakensis. Ladrón de Gue- 
vara et al. (1996) suggested that С. virginica 
showed the most primitive karyological fea- 
tures when compared with C. rhizophorae and 
С corteziensis. Our study is in agreement with 
the relationship between С virginica and S. 
commercialis, but does not agree on the posi- 
tion of C. ariakensis. Multidisciplinary ap- 
proaches would help in understanding evolu- 
tionary relationships of oyster taxa. 

Interspecific hybridizations have been pro- 
duced in cupped oyster species (Gaffney & 
Allen, 1993, provide a review). Crassostrea 
gigas is known to hybridize with С angulata, 
С sikamea and, rather less successfully, with 
С ariakensis. These observations are in 
agreement with our study. Looking at kary- 
ological features, С ariakensis and S. com- 
mercialis would be good candidates for hy- 
bhdization, although this cross has apparently 
never been thed. Ag-NORs observed in С vir- 
ginica isolate this species from the others. 
This could explain inviability of hybrids of C. 
gigas and С ariakensis with С virginica 
(Allen & Gaffney, 1 993). In the future, it will be 
of great interest to study the mitotic and mei- 
otic chromosomes of interspecific hybrids 
such as С gigas x С sikamea or С gigas x 
С ariakensis and their backcross offspring. 



ACKNOWLEDGMENTS 



Chromosome pair 

FIG. 3. Ideograms of seven cupped oysters con- 
structed from relative length and centromeric index 
values. White chromosome: metacentric, grey chro- 
mosome: submetacentric. Circles indicate Ag- 
NORs, dark circles the most frequent case. 



This work was supported by the CNRS 
(URA 2077), IFREMER, and by the Région 
Poitou-Charentes (Convention RPC-R-57), 
the French-Portuguese cooperation (no. 158 
CI ), and a research training project (FAIR GT 
97-3599). We are very grateful to S. K. Allen, 



184 



LEITAOETAL 



3 . о 



2.0 - 



1.0 - 



О 

о 



о 0.0 



-1.0- 



2 .0 




3.0 -2.0 -1.0 0.0 1.0 

Axis 1 (31.74%) 



2 . о 



3 . о 



4. о 



FIG. 4. 1/2 plan determined by Principal Component Analysis of chromosome data. Small characters repre- 
sent active objects, large characters indicate the mean for each species. A line is drawn around each species 
to show the dispersion within species. AN: Crassotrea angulata. CO: Saccostrea commercialis. GA: Cras- 
sostrea gasar. Gl: Crassostrea gigas. Afí: Crassostrea ariakensis. SI: Crassostrea sikamea. VI: Crassostrea 
virginica. 



VI 



AR 



CO 



GI 



AN 



SI 



GA 



FIG. 5. Hierarchical Clustering Analysis showing the distances between the seven species from the first four 
axes of the PCA. AN: Crassostrea angulata. CO: Saccostrea commercialis. GA: Crassostrea gasar. Gl: Cras- 
sostrea gigas, AR: Crassostrea ariakensis. SI: Crassostrea sikamea. VI: Crassostrea virginica. 



KARYOLOGY OF CUPPED OYSTERS 



185 



A. Mallet, W. Borgeson, F. Noble, J. Mazurié 
for supplying live oysters. We thank S. Heur- 
tebise and S. Sabini for excellent technical as- 
sistance, V. Thiriot for collaboration in Figure 
3 and H. McCombie for advice on the English. 



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40. 

VITTURI, R., R CARBONE & E. CATALANO, 1985, 
The chromosomes of Pycnodonta cochlear (PoW) 
(Mollusca, Pelecypoda). Biologisches Zentral- 
blatt. 104: 177-182. 

WARD, J. H., 1963, Hierarchical grouping to opti- 
mize an objective function. Journal of the Ameh- 
can Statistics Association. 58: 236-244. 



Revised ms. accepted 8 June 1998 



MALACOLOGIA, 1999, 41(1): 187-195 

ULTRASTRUCTURAL AND CYTOCHEMICAL STUDY OF THE KIDNEY AND 

NEPHRIDIAL GLAND CELLS OF THE MARINE PROSOBRANCH MOLLUSC 

NUCELLA LAPILLUS{L.) IN RELATION TO FUNCTION 

Vasilis K. Dimitriadis^ & Elizabeth B. Andrews^ 

ABSTRACT 

There are three epithelial cell types over the dorsal wall folds of kidney of Nucella lapillus (L.). 
The most numerous, excretory cells, are unciliated columnar cells, characterised by the pres- 
ence of a variety of large membrane-bound cytoplasmic vacuoles, some containing granular ma- 
terial that gives a positive reaction for periodate-reactive carbohydrates. The second type, re- 
sorptive cells, are usually cone-shaped ciliated cells, with a well-developed endocytotic canal 
system and numerous vesicles in their apical cytoplasm. The apical cytoplasm contains large 
clusters of periodate-reactive granules, possibly glycogen and occasional large vacuoles con- 
taining some granular material. The third type, "small mucous cells," are carbohydrate-contain- 
ing cells with many small mucous granules, which react positively for sulphated and carboxylated 
carbohydrates showing a reticulate positive reaction. 

In the "secondary" folds, which are interspersed with the primary ones, the epithelium is 
cuboidal and in some places almost squamous, and most of the cells display some cilia, well- 
recognizable microvilli, many pinocytotic vesicles, phagosomes and elements of the canal sys- 
tem on their apices. Many mitochondria and deposits of glycogen are also observed. 

The nephridial gland epithelium is composed of ciliated resorptive cells and "small mucous 
cells," very similar to those in the dorsal wall folds. The significance of the intracellular presence 
of carbohydrates in relation to the ability of the tissues to absorb and store sugars is discussed. 



INTRODUCTION 

The questions addressed by Fretter & Gra- 
ham (1962) regarding the complex kidney of 
the carnivorous mesogastropods and neogas- 
tropods are still not adequately answered. A 
series of papers by Delhaye (1974-1976) 
gives a comparative systematic, primarily his- 
tological, survey of a range of archaeogas- 
tropods and mesogastropods, while other in- 
vestigations deal with the excretory system of 
neogastropods, archaeogastropods, proso- 
branchs or molluscs in general (Little & An- 
drews, 1977; Andrews, 1981; 1985; Taylor & 
Andrews, 1987; Andrews, 1988). However, 
there is little information on kidney of neogas- 
tropods at the ultrastructural level. 

Andrews (1981) describes the cell types 
composing the kidney epithelium of the car- 
nivorous neogastropod Nucella lapillus (L) 
and other monotocardians, such as Littorina, 
Viviparus. Assiminea. Turritella and Lunatia. 
According to this study, three main types of 
epithelial cell have been identified in the two 



regions of the kidney folds and nephridial 
gland of Nucella lapillus. only one, the ciliated 
resorptive cells, being common to both and 
possibly implicated in reabsorption of organic 
solutes on the basis of well-developed apical 
microvilli with coated vesicles at their bases. 
The vacuolated excretory cells appear to 
have prominent microvilli, although coated 
vesicles are rarely observed. 

Data for Littorina indicate that the nephridial 
gland is the major resorptive site for glucose 
(Andrews & Taylor, 1990), which is consistent 
with ultrastructural observations showing that 
the dorsal wall is also involved in uptake of 
solutes. The latter is believed to be responsi- 
ble for the resorption of residual glucose and 
for nitrogenous excretion (Taylor & Andrews, 
1987; Andrews & Taylor, 1990). Thus, one of 
the goals of the present study is the investi- 
gation of the intracellular presence of carbo- 
hydrates in the kidney and nephridial gland 
epithelium of Nucella lapillus and the compar- 
ison of the results with those obtained from 
other gastropods. 



^Department of Genetics. Development and Molecular Biology, School of Biology, Aristotle Univ of Thessaloniki, Thessa- 
loniki, 54006, Greece: vdimitr@bio.auth.gr 

^Biology Division, School of Biological Sciences, Royal Holloway and Bedford New College, University of London, Egham, 
Surrey, TW20 OEX 

187 



188 



DIMITRIADIS& ANDREWS 



In the present study, the ultrastructure of 
the kidney and nephridial gland cells of Nu- 
cella laplllus is considered and compared with 
analogous ones of other carnivorous or her- 
bivorous molluscs. In addition, cytochemical 
tests for carbohydrate presence and acid 
phosphatase activity were applied. 



MATERIALSAND METHODS 

Specimens of Nucella lapillus from the Uni- 
versity Marine Station, Millport, Scotland, 
were kept in an aquarium with circulating arti- 
ficial sea water at 10°C. Some of the speci- 
mens were fixed for electron microscopy and 
histochemistry on arrival and others were 
used within a few days. 

Electron Microscopy 

Samples were fixed in 3% glutaraldehyde in 
Sorensen's phosphate buffer, ph 7.2 adjusted 
to 1100 mOsM with 14% sucrose. Material 
was embedded in TAAB resin, and thin sec- 
tions were stained in 1% aqueous uranyl ac- 
etate and Reynolds' lead citrate. 

Cytochemistry 

For carbohydrate cytochemistry, finely 
minced pieces of tissues were incubated 
overnight after fixation in high iron diamine 
(HID) (Spicer et al., 1 978; Sannes et al., 1 979) 
using a medium containing 36 mg N,N-di- 
methyl-m-phenylenediamine, 6 mg of the para 
isomer and 0.45 ml ferric chloride 40%, in 15 
ml d water. Other pieces were incubated in low 
iron diamine (LID) (Takagi et al., 1982), using 
a medium containing 27 mg N,N-dimethyl-m- 
phenylenediamine, 4.5 mg of the para isomer, 
and 0.45 ml ferric chloride 40% in 45 ml d 
water. After incubation, the tissues were post- 
fixed with 2% aqueous osmium tetroxide and 
embedded in Spurr's resin. Thin sections of 
these specimens were stained with the thio- 
carbohydrazide-silver proteinate (TCH-SP) 
sequence, using a medium containing 2% 
thiocarbohydrazine in 20% acetic acid, to re- 
veal periodate-reactive substances. Control 
tissues were incubated in 1 M MgClj in place of 
LID or HID. Specimens without osmium tetrox- 
ide treatment were used for the postembed- 
ding pehodate-thiocarbohydrazide-silver pro- 
teinate (PA-TCH-SP) method (Thiéry, 1967). 
Control sections were stained without the pe- 
riodate treatment. 



For acid phosphatase demonstration, a 
modification (Lewis & Knight, 1992) of the 
method proposed by Barka & Anderson 
(1 962) was applied. Glutaraldehyde was used 
as a fixative, and the tissues were incubated 
in a medium containing 0.2M tris/maleate as a 
buffer and 0.1 M ß-glycerophosphate as sub- 
strate at 37° for 15-30 min. Control sections 
were taken through an identical sequence ex- 
cept that the substrate is omitted from the in- 
cubation medium or had 0.01 M sodium fluo- 
ride added. 



RESULTS 



Kidney 



The dorsal wall of kidney of Nucella lapillus 
(L.) (Fig. 1 ) consists of two sets of folds, "pri- 
mary" folds, which receive blood from a 
branch of the afferent renal vein running over 
the ventral wall of the kidney sac, and smaller 
"secondary" folds, which receive blood from a 
branch running over its dorsal wall (Fretter & 
Graham, 1962). 

Three different cell types comprise the ep- 
ithelium of the "primary" folds. The most com- 
mon, excretory cells (Fig. 2), are columnar 
cells, which present a variety of excretory vac- 
uoles in their cytoplasm (Figs. 4, 8). 

The vacuolated excretory cells have promi- 
nent microvilli (Figs. 4, 8), which are lost in 
later stages when apical blebs are formed. 
The most conspicuous organelles of these 
cells are a series of large excretory vacuoles 
containing in certain cases a concentration of 
finely fibrous excretory material (Fig. 4). Due 
to the presence of the large vacuoles in the 
apical and mid cytoplasmic region, the ovoid 
nuclei are usually displaced in their base (Fig. 
4). The fibrous material of the excretory vac- 
uoles in most cases gives a positive reaction 
for periodate-reactive carbohydrates (Fig. 8). 
In certain cases, this material reacts positively 
for acid phosphatase activity (Fig. 11). Other 
clusters of fibrous and/or granular periodate- 
reactive material is also present in the cyto- 
plasm of these cells (Fig. 8). Small vesicles 
are seen in the process of fusing with excre- 
tory vacuoles of varying size and may appear 
as evaginations of the vacuolar membrane. 

The rough endoplasmic reticulum of the ex- 
cretory cells is usually restricted to a narrow 
zone of apical cytoplasm, often associated 
with clusters of glycogen. The Golgi com- 
plexes lie close to the nuclei and are small, 



ULTRASTRUCTURAL AND CYTOCHEMICAL STUDY 



189 



pfk skf 




varv 



FIG. 1. Drawing of the kidney sac of Nucella lapil- 
lus, opened to show double series of folds on the 
wall, based on the original in Fretter & Graham 
(1962). darv, dorsal branch of afferent renal vein; 
hg, hypobranchial gland; ko, kidney opening; ng, 
nephridial gland; pa, posterior aorta; pfk, primary 
kidney folds; skf, secondary kidney folds; te, testis; 
varv, ventral branch of afferent renal nein; ve, ven- 
tricle. Bar = 1 mm. 

conspicuous and rarely observed, whereas 
the mitochondria are positioned parallel with 
the infoldings of the basal plasma membrane. 
In the basal cytoplasm, the excretory cells 
display long slender basal processes, which 
invaginate the basal lamina and extend into 
underlying blood spaces (Fig. 9). Near the 
basal membrane, in the narrow cytoplasmic 
spaces between the adjacent infoldings and 
very close to the blood spaces clusters of per- 
iodate-reactive granules, mostly glycogen are 
present (Fig. 9). 

The second cell type in the "primary" dorsal 
folds, the ciliated resorptive cells (Fig. 5), bear 
apical microvilli and cilia. Usually, they appear 
to be cone-shaped, with their distended 
apices bulging into the lumen in umbrella-like 
fashion over the neighbouring cells (Fig. 5). 

The dense cytoplasm of the resorptive cells 
possesses an endocytotic canal system and 
many small vesicles (Fig. 10), some near the 
apical membrane being coated. Pinocytosis 
occurs at the base of the microvilli and the 
pinocytic vesicles are coated (Fig. 10). Pino- 
cytotic vesicles are coated on the cytoplasmic 
face by closely spaced, bead-like particles. 
The apical membrane itself bears a fine gly- 



cocalyx and microvilli 0.7-1 .2 ¡am long are in- 
terspersed amongst groups of long cilia 4.5- 
5 }.im long. 

In the apical cytoplasm there are some 
lysosomes, usually in the form of residual 
bodies and occasional large vacuoles, some 
of them containing a little granular material. 
Also in the apical cytoplasm are prominent 
large clusters of periodate-reactive particles, 
mostly glycogen (Fig. 10). Similar particles 
are also present throughout the cytoplasm. 

The third cell type, the "small mucous 
cell" (Fig. 6), is very rare and consists of small 
cells with small periodate-reactive secretory 
granules, or cells having discharged their se- 
cretion. The granules of these cells reacted 
positively for sulphated and carboxylated car- 
bohydrates showing a reticulate positive reac- 
tion (not shown). 

The "secondary" folds of dorsal wall of kid- 
ney of N. lapillus are present in troughs be- 
tween "primary" folds. In the "secondary" folds 
the epithelium is cuboidal and in some places 
almost squamous (Fig. 7), while the presence 
of fewer cilia comparing to those of the pri- 
mary kidney folds makes the recognition of 
the cell limits under the scanning microscope 
obscure, comparing to the "primary folds" 
(Figs. 2, 3). In the apical portion, the cells 
show many pinocytotic vesicles, phagosomes 
and elements of the canal system. Many mi- 
tochondria, lysosomes and deposits of perio- 
date-reactive particles are also observed. In 
the "secondary" folds, the presence of the 
"small mucous cells" is less obvious than in 
the "primary" ones. 

Nephridial Gland 

The nephridial gland of N. lapillus receives 
post branchial blood from the auricle. In the 
epithelium of this gland, the same type of cili- 
ated resorptive cell covers the surface and the 
more superficial parts of the tubules (Figs. 12, 
13). Unlike the resorptive cells of the dorsal 
wall folds, cone-shaped forms in the nephridial 
gland are very rare, the cells consisting mostly 
of cuboidal form. The resorptive cells show 
apical microvilli and cilia and a well-developed 
endocytotic canal system with small coated 
vesicles. In the apical portion, the presence of 
small, empty vacuoles is usually apparent 
(Fig. 13). Periodate-reactive particles, mostly 
glycogen, are also located in clusters usually 
in the apical cytoplasm (Fig. 14). 

Dense bodies frequently lie in the apical 
portion of the cells and usually exhibit an elec- 



190 



DIMITRIADIS& ANDREWS 




FIGS. 2 & 3. Scanning electron micrographs of the apical surface of "рг1глагу" (Fig. 2) and "secondary" folds 
(Fig. 3) of the kidney dorsal wall. In the "secondary" folds, the decreased presence of cilia (arrow), compared 
to the "primary" folds, makes the recognition of the cell limits not well recognizable. EC, excretory cell; RC, 
resorptive cell. Fig. 2 Bar = 2.5 |.im. Fig. 3 Bar = 8 цт. 



tron-dense matrix. The material of most 
dense bodies gives a positive reaction for per- 
iodate-reactive carbohydrates, as well as for 
suiphated (Fig. 15) and carboxylated carbo- 
hydrates. In certain cases, membranous rem- 
nants of the dense bodies display a positive 
acid phosphatase reaction (not shown). 

Like the dorsal wall epithelium of the kidney, 
in the nephridial gland there is a second cell 
type, the "small mucous cells" consisting of 
small cells with small periodate-reactive se- 
cretory granules giving also a reticulate posi- 
tive reaction for suiphated and carboxylated 
carbohydrates (not shown). 

Control sections of all cytochemical tech- 
niques constantly lacked reaction product. 



DISCUSSION 

The kidney of N. lapillus is histologically 
similar to that of other monotocardians proso- 
branchs (Andrews, 1981) in that the epithe- 
lium is composed of distinct vacuolated ex- 
cretory and ciliated resorptive cells. The 
morphological and functional parameters of 
the excretory and ciliated cells of Nucella 
lapillus documented by the results of the pres- 
ent study, such as the structure of their excre- 
tory vacuoles, canal system compartments, 
and basal processes, are very similar to those 
of the herbivorous mesogastropod Littorina lit- 
torea (Andrews, 1981; Taylor & Andrews, 
1987; Andrews, 1988), albeit that Nucella is 
carnivorous. A differentiation in the density of 
the excretory vacuoles contents was reported 
by Andrews (1981) between Nucella and her- 
bivores species, being denser in Nucella than 
in the other species, as well as a differentia- 
tion in the colour of the lysosomes of the cili- 



ated cells, being greenish in the herbivores 
species studied. 

The vacuolated excretory cells of the dorsal 
wall folds of the kidney of Nucella, like the cor- 
responding cells in Littorina (Andrews, 1988), 
contain excretory vacuoles with finely fibrous 
excretory material, unlike those of archaeo- 
gastropods and amphibious and terrestrial 
species, which contain layered concentra- 
tions. It is generally accepted that the excre- 
tory vacuoles are formed and increase in size 
after fusion of smaller vesicles and finally are 
ultimately shed in blebs of apical cytoplasm 
that are nipped off from the cell membrane 
(Andrews, 1981, 1985). Delhaye (1976) dem- 
onstrated pinocytosis at the bases of the ex- 
cretory cells of Monodonta by which the cells 
abstracted ferritin injected into the blood. A 
second indication for this function is the clearly 
well-recognisable long slender processes that 
invaginate the basal lamina of the excretory 
cells of Nucella, and as in Littorina (Andrews, 
1988) and Monodonta (Andrews, 1985), per- 
meate the underlying blood spaces, which 
suggest transport of molecules across the 
lamina. 

The ciliated resorptive cells comprising the 
epithelium of N. lapillus display many similari- 
ties to those of the ciliated resorptive cells of 
the dorsal wall folds. The ciliated cells in both 
epithelia were regarded as cells of the same 
cell type (Andrews, 1981; Andrews & Taylor, 
1990). The results of the present study show 
that the ciliated cells in the kidney folds differ 
from those in the nephridial gland in certain 
features, for example their cone shape, where 
they are interspersed with excretory cells. 
However, other differences, such as the num- 
ber of their apical vesicles and lysosomes, 
should be attributed to the different phases of 



ULTRASTRUCTURAL AND CYTOCHEMICAL STUDY 1 91 

Lu 



i^ 



''^'- .f^"'''''^k' 


Lu 


EV 




•A 






N 


Lf 




'jtopH 






^ - 



Lu 



EV * 




Lu 

V 






â^-'à^h:'- ■ 'j.l# -1 k^< 



N 




FIG. 4. Apical portion of excretory cells in a "primary" fold of dorsal wall of kidney showing large excretory 
vacuoles (EV). Lu, lumen; N, nucleus. Bar = 4 ¡am. 

FIG. 5. The apex of a ciliated resorptive cell displaying microvilli and cilia, as well as apical vesicles (aster- 
isk), is protruded into the lumen. Bar = 3.5 дт. 

FIG. 6. A "small mucous cell" (asterisk) is positioned in the apical portion of the epithelium, possibly just be- 
fore the secretion of the cell product. EV, excretory vacuole; Lu, lumen; N, nucleus. Bar = 3 [xm. 

FIG. 7. In a "secondary" fold of the kidney dorsal wall, there is a transition in the form of the cells from colum- 
nar-cuboidal towards cuboidal-squamous. Db, dense body; Lu, lumen; N, nucleus; V, apical vesicle. Bar = 
5 um. 



192 



DIMITRIADIS & ANDREWS 




FIG. 8. Cytochemistry for periodate-reactive carbohydrates. Excretory cells show periodate-reactive fibrous 
material in large excretory vacuoles (EV). Other "primary" fold cells show large clusters of other periodate- 
reactive material in their cytoplasm (arrow). Lu. lumen. Bar = 3 цт. 

FIG. 9. Cytochemistry for periodate-reactive carbohydrates. Basal cytoplasm of excretory cell. The basal 
membrane infoldings form long slender cytoplasmic processes, which permeate the blood spaces (BS) and 
contain periodate-reactive carbohydrates (arrow). Bar = 0.6 |.im. 

FIG. 10. Cytochemistry for periodate-reactive carbohydrate. Apical cytoplasm of a ciliated resorptive cell in 
the kidney of Nucella lapillus. Clusters of periodate-reactive particles, mostly glycogen, are located in the cy- 
toplasm. Note the coated vesicle (arrow) and the well-stained vesicles and tubules of the canal system (small 
arrows). Bar = 0.8 цт. 

FIG. 1 1 . Cytochemistry for acid phosphatase. Materials in an excretory vacuole of an excretory cell react pos- 
itively for enzyme presence (arrow). Mv, microvilli. Bar = 1 ¡.im. 



ULTRASTRUCTURAL AND CYTOCHEMICAL STUDY 



193 



* 



•чЧ 






h2 У 



■л .,_^ ^^ 



vr- 



Db 



Lu 



Db 



•> 



,.^ N 



. ^ Ы 



Ы 



^ 13 



tv 



'MV 



\ 






Lu 



Lu 



V 



14 



'.Й^Р"^ 



'4' 






,.■/*■ 



■■ . ... '. i. .t ' 



49P*M' 



rf'Db 



15 



FIG. 12. Ciliated resorptive cells and a "small mucous cell" (asterisk) in the nephridial gland of Nucella lapil- 
lus. Db, dense body: Lu, lumen. Bar = 3.5 ,um. 

FIG. 13. Ciliated resorptive cells in the nephridial gland of Nucella /ap/7/us display many mitochondria, some 
iysosomes and many apical small vesicles (arrow). Ci, cilium; Db, dense body; Mv, microvilli. Bar = 2 ¡.im. 

FIG. 14. Carbohydrate cytochemistry. The cytoplasm of a ciliated resorptive cell shows abundance of perio- 
date-reactive particles, mostly glycogen. Lu, lumen; V, apical vesicle. Bar = 1 .5 |im. 

FIG. 15. In the nephridial gland, a dense body (Db) in the apical region of a ciliated resorptive cell react pos- 
itively for sulphated carbohydrates. Lu, lumen. Bar = 4 цт. 



cell activity rather, than to differences be- 
tween cell types. 

The apical canal system observed in the re- 
sorptive cells of both kidney dorsal wall and 
nephridial gland cells of Nucella lapillus is 
also observed in the pericardial and kidney 



cells of Scrobicularia plana (Andrews & Jen- 
nings, 1993) and the left kidney of archaeo- 
gastropods (Andrews, 1985), as well as in 
the digestive gland of Nucella (Dimitriadis & 
Andrews, submitted for publication), Lasaea 
(McQuiston, 1969), Cardium (Owen, 1970), 



194 



DIMITRIADIS& ANDREWS 



Mytilus (Owen, 1972), and Rissoa (Wigham, 
1976), where it was regarded as structure re- 
lated to the delivery of nutrients to the endo- 
cytotic system. In the light of the recent data, 
the elements of the canal system should be 
regarded as endosomes, that is, the cell com- 
partments, where the endocytosed material 
enters the lysosomal pathway (Alberts et al., 
1994). 

In the excretory cells of N. lapillus, perio- 
date-reactive fibrous material is located inside 
the excretory vacuoles, while in certain cases 
this intravacuolar material reacts positively for 
acid phosphatase. Carbohydrates, among 
other substances, have been identified in the 
excretory vacuoles in the right kidney of dioto- 
cardians (Delhaye, 1976). The presence of a 
significant amount of carbohydrates and hy- 
drolases inside the excretory vacuoles proba- 
bly indicates that the role of these vacuoles 
should be regarded as multiple and that 
they are implicated in more functions than 
the transport and release of the excretory ma- 
terial. 

In the kidney dorsal wall of N. lapillus. clus- 
ters of periodate-reactive particles, mostly 
glycogen, are present in the cytoplasm of the 
resorptive cells. It is well known that carbohy- 
drates are the major source of energy in gas- 
tropods molluscs (Livingston & De Zwaan, 
1983) and that carbohydrates are largely 
stored as glycogen, especially in connective 
tissue. The excretory system of prosobranch 
gastropods, which forms urine by a filtration 
mechanism, has the capacity to resorb valu- 
able organic solutes, such as glucose, from 
the primary urine, limiting markedly the 
amount that is lost to the exterior (Andrews & 
Taylor, 1990). Glucose resorption has been 
demonstrated in the abalone Haliotis rufes- 
cens (Harrison, 1962) and the mesogastro- 
pod Littorina littorea (Taylor & Andrews, 
1987), while glucose influx to both dorsal wall 
and nephridial gland of the kidney appeared 
to reflect net inward transport by a saturable 
Na'^-dependent carrier mechanism believed 
to be responsible for reabsorption in vivo 
(Taylor & Andrews, 1 987). On the other hand, 
in the herbivorous mesogastropod Littorina lit- 
torea, the nephridial gland cells were re- 
garded as sites implicated in resorption of or- 
ganic solutes on the basis of well-developed 
apical microvilli with coated vesicles at their 
bases (Andrews & Taylor, 1990). Also in Litto- 
rina. Delhaye (1974b) demonstrated the ca- 
pacity of the nephridial gland cells to remove 
Indian ink, iron saccharide and ferritin from 



the blood, like the resorptive cells of the dor- 
sal wall, while their ability to accumulate pH] 
glucose has been shown in vitro (Taylor & An- 
drews, 1990). In N. lapillus. where the mor- 
phological features of the nephridial gland 
cells are very similar to those in Littorina, an 
equivalent absorptive function of the cells is 
very possible. Thus, the presence of large 
amounts of periodate-reactive particles in the 
resorptive cells of kidney folds and nephridial 
gland is a strong indication that these cells ab- 
sorb carbohydrates and store, at least a part 
of them, in their cytoplasm. 

The "small mucous cells" observed in the 
examined tissues of N. lapillus exist also in 
the digestive gland of the same gastropod, 
where they also reacted positively for com- 
plex carbohydrates (Dimithadis and Andrews, 
submitted for publication). Probably this cell 
type is related to the mucoid cells described in 
the nephridial gland of other monotocardian 
prosobranch gastropods (Andrews, 1981), as 
well as in the kidneys of freshwater mesogas- 
tropods (Andrews, 1988), where they were 
specialised as cells related to the resorptive 
mechanism of the epithelium. The secretion 
product of these mucoid cells is not stained by 
alcian blue and is probably a mucoprotein or 
neutral mucopolysaccharide. 

In conclusion, by using light and electron mi- 
croscopic observations in combination with 
cytochemical characterization, the present 
study provides information about the structure 
and function of the kidney and nephridial gland 
cells. Additional information, especially by the 
use of histochemistry in cryosections and im- 
munocytochemistry, is necessary for the bet- 
ter understanding of the fine structure and 
physiology of the studied interesting cell types. 

ACKNOWLEDGEMENTS 

This work was undertaken during the sab- 
batical leave of the first of the authors at the 
Royal Holloway and Bedford New College. 
The authors gratefully thank the staff of the 
Electron Microscope Unit of the College for 
their help and invaluable assistance in the 
preparation of some of the material. 



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Revised ms. accepted 1 February 1999 



MALACOLOGIA, 1999, 41(1): 197-208 

ENERGETICS OF THE RED SLUG ARION RUFUS (GASTROPODA) AND OF THE 
GASTROPOD COMMUNITY IN A BEECH FOREST ON LIMESTONE 

Anne Theenhaus & Matthias Schaefer 

Institut für Zoologie and Anthropologie, Abteilung Ökologie. Berliner Strasse 28, 
D-37073 Göttingen, Germany; theenhau@mail.uni-mainz.de 

ABSTRACT 

The energy budget of the red slug Arion rufus (L.) in a mull beech forest on limestone in Lower 
Saxony, Germany, was calculated. Somatic growth, reproduction, secretion of mucus, respira- 
tion, excretion of faecal materials, and assimilation efficiency were measured experimentally. 
The consumption was calculated from the excretion of faecal materials, using the assimilation ef- 
ficiency. 

Mean biomass of the population of A. rufus in the forest studied was 0.2 g d wt m~^. The av- 
erage density of slugs that grew to maturity and reproduced was one slug per 3 m^. The annual 
consumption of the population of A. rufus in the beech forest studied was equivalent to 361 .9 kJ 
m"^, the investment into somatic growth to 52.5 kJ m"^, the reproduction to 3.6 kJ m"^, the res- 
piration to 27,6 kJ m^^, and the excretion of faecal materials to 79.7 kJ m"^. About 105.3 kJ m"^ 
were invested into the secretion of mucus, which was 65% of the production. The considerable 
investment of gastropods in locomotion probably limits the range of conditions under which pop- 
ulations can survive. Ecosystem characteristics and slug qualities that allow the high slug bio- 
mass in the beech forest ecosystem were discussed. 

The population of A. rufus and the whole gastropod community consumed 0. 1 5% and 2.4% of 
the annual leaf litter input, 3.3% and 26% of the annual production of green plant material, and 
0.24% and 1.7% of the annual net primary production (NPP), respectively. The direct contribu- 
tion of the gastropod community to decomposition processes therefore was small. The con- 
sumption of green plant material by the gastropod community was similar to that annually in- 
gested by phytophages (Curculionidae and lepidopteran larvae) in the forest studied. 

Key words: Arion rufus. gastropod community, energy budget, mucus, beech forest. 



INTRODUCTION 

In the present investigation, we calculate 
the energy budget of the red slug Arion rufus 
(L.) in order to understand life history strate- 
gies of this slug species in the forest ecosys- 
tem. We are especially interested in the role of 
mucus in the energy flux of A. rufus. In previ- 
ous studies, the energy budget of A. rufus and 
A. ater\r\ beech forests was calculated (Stern, 
1 969; Jensen, 1 975), however the authors did 
not quantify the secretion of mucus. Since 
mucus is a major component of gastropod 
production (Denny, 1980; Davieset al., 1990), 
the omission of the mucus term caused a con- 
siderable underestimation of production. 

Arion rufus feeds on dead organic material, 
living plants, fungi and carcasses and there- 
fore contributes directly to decomposition 
processes (Jennings & Barkham, 1976; 
1979a). In the present investigation, special 



attention was given to the consumption data 
of the population of A. rufus, because with 
these data the impact of A. rufus on its food 
and the direct contribution to decomposition 
processes could be evaluated. Furthermore, 
we extrapolated the consumption data of A. 
rufus to the gastropod community in order to 
assess the role of gastropods in the forest 
ecosystem. Schaefer (1990) constructed the 
energy budget of the heterotrophic subsystem 
of the forest investigated in the present study. 
He found that the contribution of the gastro- 
pod community to the energy and material 
turnover was low. However, the calculations 
were made without data on A. rufus, because 
biomass data of A. rufus were not available. 
Presumably, this omission resulted in a dras- 
tic underestimation of the role of the gastro- 
pod community in nutrient cycling in the forest 
ecosystem studied. Arion rufus has a lifespan 
of little more than a year (Laviolette, 1950; 



197 



198 



THEENHAUS & SCHAEFER 



Smith, 1966), and since the body weight of 
mature specimens is high, the population of 
A. rufus builds up a high biomass during a 
short time period. Therefore, we assume that 
the energy flow through the population of A. 
rufus is high and that A. rufus makes a signif- 
icant direct contribution to plant decomposi- 
tion processes. 

As a result of the high production of bio- 
mass of the population of A. rufus in the forest 
ecosystem studied, we hypothesize that this 
slug species turns high amounts of living 
plants and dead organic material into faecal 
material, which is then colonised by a rich 
community of microfauna (Theenhaus & 
Scheu, 1996a) and mesofauna (Theenhaus, 
1997). Furthermore, it is assumed that in the 
forest ecosystem studied the population of A. 
rufus secretes high amounts of slug mucus. 
Since slug mucus contains high nutrient con- 
centrations, A. rufus directly modulates the 
availability of resources for microorganisms 
through the secretion of mucus (Theenhaus & 
Scheu, 1996b). We assume that the creation 
of habitats for other species through the ex- 
cretion of faecal material and the secretion of 
slug mucus is of quantitative importance in 
the forest ecosystem studied. Arion rufus 
might therefore be called an ecosystem engi- 
neer (Lawton, 1994). 



MATERIALSAND METHODS 
Study Site 

The Göttinger Wald beech forest is situated 
on a plateau of Muschelkalk at about 420 m 
above sea level in southern Lower Saxony, 
Germany. The forest, which is approximately 
110-125 years old, has a uniform canopy 
layer, which consists almost exclusively of 
beech trees {Fagus sylvatica). A shrub layer is 
not developed. The herb layer is dominated 
by Allium ursinum and Mercurlalls perennis. 
In the years 1995 and 1996, in which the ex- 
periments were conducted, mean annual tem- 
perature in the litter layer was 9.0°C and 
7.1 °C, respectively, and mean annual precipi- 
tation was 700 mm and 567 mm, respectively. 



Energy Budget of A. rufus 

In the present study, we first calculated the 
energy budget of a "model specimen" of A. 
rufus, which is an animal that grows to matu- 



rity, deposits one egg clutch, and then dies. 
Based on this model specimen, the energy 
budget of the population of A. rufus in the for- 
est studied was estimated. The energy bud- 
get was calculated using the equation given 
by Petrusewicz (1967), which was modified 
to include a mucus term (e.g., Edwards & 
Welsh, 1982; Davies et al., 1990): С - Pg + P^ 
-f- P^ + R + F, where С = consumption, P = 
somatic growth, P^ = reproduction, P^ = 
secretion of mucus, R = respiration, and F 
= faecal materials. 

Defined-area traps similar to those of Fer- 
guson et al. (1989) were constructed in coop- 
eration with С Döring to estimate slug popu- 
lations in the beech forest. The traps 
consisted of a square PVC frame (1 m^ area, 
30 cm high). This frame was placed onto the 
forest soil to enclose slugs, which were sup- 
posed to be caught during the following trap- 
ping pehod. A PVC lid could be screwed 
tightly on a metal rim, which was attached to 
the inner edge of the frame. Twelve round 
holes in the lid, which were sealed with gauze 
(meshsize 1 mm) created a light regime (twi- 
light) inside the traps being favourable for 
slugs. To prevent slug movements in or out of 
the traps, outside the traps soil was piled up 
(20 cm high) and pressed tightly to the frame. 
A glass jar (diameter 5 cm, 11 cm high; Bar- 
ber, 1931) was imbedded into the soil inside 
the traps and filled with beer (3 cm high), 
which served as baiting agent. Duhng periods 
of drought, the ground inside the traps was 
moistened regularly. In the years 1995 and 
1996, the population of A. rufus was esti- 
mated using 12 traps, which were arranged in 
four groups (distance 10 m) of three traps 
each (distance 3-5 m; block-design; Sokal & 
Rohlf, 1 995). Every two weeks, the glass jars 
were emptied and refilled with fresh beer. 
Trapped slugs were identified, and their ash- 
free dry weight was determined. When no 
slugs were captured in the traps for four 
weeks, it was assumed that all slugs inside 
the traps were caught, and therefore the trap- 
ping period was finished. Traps were re- 
arranged, and a new trapping period started. 

To quantify the production of eggs, in au- 
tumn 1994 and 1995 mature specimens of A. 
rufus were collected in the beech forest and 
transferred into the laboratory. Each egg 
clutch was divided into three portions and in- 
cubated at 5, 10 and 20""C, respectively. The 
time between egg deposition and hatching of 
juveniles was measured. 

The COg production of slugs was measured 



ENERGY BUDGET OF ARION RUFUS 



199 



titrimetrically (1 N KOH as a CO2 absorbent) at 
5, 10 and 15'C for 24 h (day/night cycle of 
12/12 h). Measurements were done in exper- 
imental chambers, which consisted of per- 
spex tubes (diameter 6 cm, 15 cm high). 
Slugs were acclimated at the respective tem- 
perature for 14 days prior to measurements. 

To quantify the excretion of faecal materi- 
als, specimens of A. rufus were collected in 
the beech forest in summer following periods 
of drought, in summer following wet periods, 
and in autumn (three parallel measurements). 
Still in the forest, each slug was placed into a 
separate round, perspex vessel (12 cm diam- 
eter, 9 cm height), which contained food 
(leaves of Allium ursinum, Mercurialis peren- 
nis. and Sambucus nigra). The vessels were 
then transported into a temperature controlled 
room, in which temperature and day/night 
cycle were close to those of the respective 
time of year. The fresh weight of each slug 
and the dry weight of faecal materials, which 
were excreted during the incubation by each 
slug, were determined 24 h after the collection 
of slugs. 

To quantify the secretion of mucus, speci- 
mens of A. rufus (fresh weight between 0.3 
and 15.6 g) were incubated in pre-weighed 
perspex vessels (see above) at defined tem- 
perature and humidity conditions (5, 10, 15 
and 20°C in combination with 100% RH and 
10'C in combination with 75% and 55% RH, 
respectively) for 2 h. The fresh weight of 
mucus, which was secreted during the time of 
incubation, was determined by reweighing the 
vessels after the incubation and calculating 
the difference in weight. Slugs were accli- 
mated at the respective experimental condi- 
tions (temperature, RH) for 12 h prior to use. 

The assimilation efficiency of juvenile A. 
rufus (fresh weight between 0.8 g and 2.2 g) 
feeding on leaves of M. perennis and A. 
ursinum and of mature A. rufus (fresh weight 
between 7 g and 1 5 g) feeding on leaves of M. 
perennis and Sambucus nigra was deter- 
mined gravimetrically. Before measurement, 
slugs were fed with one of the respective herbs 
for three days. Then the slugs were transferred 
to perspex vessels (see above), which con- 
tained 7 g f wt of one of the respective herbs of 
known water content, five juvenile slugs or one 
mature slug per vessel (10°C, day/night cycle 
of 14/10; juvenile slugs: six parallel measure- 
ments; mature slugs: 15 parallel measure- 
ments). After three days of incubation, the 
dry weight of faecal materials, which was ex- 
creted during the incubation, and the dry 



weight of herb materials were determined. The 
dry weight of herbs, which were eaten by slugs 
during the three days of incubation, was cal- 
culated as the difference before and after feed- 
ing. Carbon content of faecal materials and 
herbs was determined using an elemental 
analyser (Carlo Erba Co., Milan). In order to 
determine the change in weight of herbs with- 
out the influence of slugs, nine vessels without 
slugs but with herbs (three vessels with M. 
perennis, A. ursinum and S. nigra, respec- 
tively) were incubated for three days. The as- 
similation efficiency of slugs was calculated 
with the formula: 



d wt food - cc food - d wt faecal materials 
- cc faecal materials 

d wt food ' cc food 



100, 



where a = assimilation efficiency, cc = carbon 
content. 

The conversion of data into calorific values 
was accomplished with conversion factors 
from the literature: 1 g d wt tissue of A. rufus 
and of egg mass was equated with 20.1 kJ 
(Jensen, 1975; Bless, 1978); 1 g d wt mucus 
was equated with 18.8 kJ (Calow, 1974; 
Richardson, 1975); 1 I CO2 was equated with 
21 .8 kJ. This conversion factor applies to ani- 
mals with a respiratory quotient of 0.95 
(Southwood, 1991), which was found for the 
slug Ariolimax columblanus by Denny (1980). 
One g d wt faecal materials was equated with 
21.4 kJ, since 1 g carbon corresponds to 46 
kJ (Humphreys, 1979), and the mean carbon 
content of slug faecal materials is 46.5%. One 
g d wt green plant material was equated with 
18.8 kJ (Schaefer, 1990, 1991). 



RESULTS 

In both years (1995 and 1996), traps were 
moved three times (four catching periods in 
each year; Table 1 ). To get a conservative es- 
timate of the mean yearly biomass of the pop- 
ulation of A. rufus in the forest studied, we as- 
sumed that during the time when no trapping 
occurred the dry weight of A. rufus was zero. 
As a result, the mean dry weight of the pop- 
ulation of A. rufus was 0.2 g m"^ (0.15 and 
0.25 g d wt m"^ in 1995 and 1996, respec- 
tively). 

Mean fresh weight of A. rufus after the de- 
position of eggs was 9.4 g and 8.4 g in the year 
1994 and 1995, respectively (Table 2). Juve- 
nile slugs hatched 1 34.0 (n - 39, SD = 8.7) and 



200 



THEENHAUS & SCHAEFER 



TABLE 1 . Ash-free d wt [mg] of specimens of Arion rufus caught with 1 2 defined-area traps in 
the Göttinger Wald in the years 1995 and 1996. 



Catching period 



Ash-free d wt per specimen 



Mean 



(SD) 



23.03.-19.05.95 14, 25, 214, 227, 235, 241, 316, 475, 491, 827, 2095 

20.05.-28.06.95 66, 91 , 94, 1 55. 742, 975, 1 030, 1 083 

29.06.-11.08.95 960 

12.08.-05.10.95 407,1267,1762 

07.05.-02.07.96 1 37, 300, 41 2, 581 , 790, 1 466, 1 690 

03.07.-12.08.96 231, 939, 1100, 1990 

13.08.-10.10.96 812, 1280, 1560, 1650, 1916, 1920, 2009 

11.10.-03.12.96 1,2,6,17 



*306 


(228) 


529 


(438) 


960 


(0) 


1145 


(559) 


768 


(550) 


1065 


(626) 


1562 


(396) 


7 


(6) 



"without the mature specimen of 2095 mg 



TABLE 2. Mean fresh weight (f wt) of Arion rufus and egg clutches after the deposition of eggs 
in the year 1994 and 1995, number of slugs and egg clutches examined (n), standard devia- 
tion (SD), minimum and maximum values and mean number of eggs per clutch. 

















number of 




year 


f wt (g) 


n 


SD 


minimum 


maximum 


eggs per clutch 


A. rufus 


1994 


9.40 


11 


3.7 


4.6 


17.7 






1995 


8.40 


32 


2.8 


4.5 


19.3 




egg clutch 


1994 


2.57 


11 


1.2 


1.31 


5.13 


58 




1995 


2.26 


32 


1.0 


0.68 


5.94 


63 



TABLE 3. Regression lines between dry weight of specimens of Arion rufus and COg pro 
ductionat5, 10 and 15'C. R: COj production [ml COg h ^]: W:dwt of /4. rufus 
ber of slugs examined: inter.: intercept: exp.: exponent. 



2 

n: num- 



Temperature 


Regression equation 


n 


r2 


p inter. 


SD inter. 


SD exp. 


5°C 
10°C 
1 5=C 


R = 0.17xW^°^ 
R = 0.36 X W° ^2 
R = 0.49xW^°^ 


14 
15 
15 


0.59 
0.99 
0.89 


< 0.001 

< 0.001 

< 0.001 


0.041 
0.019 
0.016 


0.246 
0.026 
0.101 



TABLE 4. Regression lines between the dry weight of specimens of Arion rufus and the excretion in 
summer following dry and wet periods and in autumn (three parallel measurements in each season). 
F: d wt cast materials (mg 24 h ^): W: d wt A. rufus (g): n: number of slugs collected. 



Regression equation range of n range of R"" 



P (Slope) 



Summer, following dry periods F = 10.6 x W 

Summer, following wet periods F = 29.2 x W° 

Autumn F = 19.4xW° 



52-73 


0.47-0.65 


< 0.001 


68-79 


0.68-0.79 


< 0.001 


58-77 


0.36-0.64 


< 0.001 



66.8 days following the deposition of eggs (n = 
69, SD = 6.1 ) at 5°C and 1 0"C, respectively. At 
20°C slugs did not hatch. 

The COg production of A. rufus increased 
with increasing temperature. The relationship 
between the dry weight of specimens and the 
respiration rate was almost linear (Table 3). 

There was a positive correlation between 
the log dry weight of A. rufus and the log ex- 



cretion of faecal materials (Table 4). In sum- 
mer following periods of drought, the faecal 
weight excreted per unit weight of slug de- 
creased with increasing weight of slugs. In 
summer following wet periods, and in autumn, 
the exponent of the regression equations 
ranged between 0.88 and 1.00 indicating an 
almost linear relationship between the excre- 
tion and slug weight. 



ENERGY BUDGET OF ARION RUFUS 



201 



The weight of mucus secreted increased 
with the weight of the slug following the equa- 
tion S = a X W'^, where S = d wt mucus, W = d 
vj\A. rufus. < b< 1 (Fig. 1). 

The assimilation efficiency of juvenile slugs 
was 78.7% (n = 6; SD = 8.1) and 88.5% (n = 
6; SD = 5.6) when feeding on M. perennis and 
A. ursinum, respectively. The assimilation effi- 
ciency of mature slugs was lower than that of 
juvenile slugs, being 73.1% (n = 15; SD = 5.9) 
and 71 .8% (n = 1 5; SD = 7.2) when feeding on 
M. perennis and S. nigra, respectively. 



ENERGY BUDGETS 

The Energy Budget of Arion rufus 

Monthly weight classes of A. rufus were cal- 
culated using literature data (Kunkel, 1916; 
Abeloos, 1944; Laviolette, 1950; Frömming, 
1 954; Smith, 1 966) and present data. As a re- 
sult, the model specimen deposits one egg 
clutch at the beginning of September (Table 
5). Mean temperature in the litter layer in 
September and October is 10"C, and there- 
fore it is assumed that the juveniles hatch 
two months following the deposition of egg 
clutches (beginning of November). The model 
specimen weighs 9 g in July, deposits one egg 
clutch in September, and then dies. Based on 
these data, the energy budget of the model 
specimen was calculated as follows; mean 
dry weight of slugs, which deposited egg 
clutches in the laboratory was 1.35 g (27.2 
kJ). In the laboratory, slugs never deposited 
more than one egg clutch, and therefore it 
was assumed that the model specimen de- 
posits only one egg clutch. Average d wt of 
egg clutches that were deposited in the labo- 
ratory 1 994 and 1 995 was 0.54 g (1 0.9 kJ). To 
calculate the amount of mucus secreted, it 
was assumed that during a period of 24 h 
slugs are active for 6 h (Newell, 1971) and 
that during slug activity relative humidity is 
100%. Therefore, the model specimen se- 
cretes 4.58 g d wt mucus (85.9 kJ) during its 
life (Table 5). Considering the monthly aver- 
age temperature in the litter layer, the model 
specimen respires 2.0 I COj (43.5 kJ) during 
its life (Table 5). The regression line between 
dry weight of A. rufus and weight of faecal ma- 
terials excreted was similar in summer follow- 
ing wet periods and in autumn (Table 2). 
Therefore, data of both seasons were pooled, 
resulting in the regression equation F = 19.3 x 
W° ^^. For June, July and August the excretion 



of faecal materials was calculated from the re- 
gression equation of data from summer fol- 
lowing periods of drought, which is F = 10.6 x 
W°^\ As a result, during its life the model 
specimen excretes 3.2 g d wt faecal materials 
(68.5 kJ; Table 5). The consumption rate of 
the model specimen was calculated from the 
excretion of faecal materials using the assim- 
ilation efficiency of 75%. This value is closer 
to that of mature slugs (72%) than to that of ju- 
veniles (84%), because the contribution of the 
model specimen to total consumption rate is 
higher in the mature than in the juvenile stage. 
As a result, the model specimen consumes 
5.96 g carbon during its life, which is equiva- 
lent to 1 3.2 g dry plant material and 248.7 kJ. 
Summing up, the energy budget of the 
model specimen in the Göttinger Wald is; 

248.7 = 27.2 + 1 0.9 + 85.9 + 43.5 + 68.5 [kJ] 
(C = Pg + P. + P, + R + F) 

The Energy Budget of the 
Population of Arion rufus 

Using data of Table 1 , the number of mature 
A. rufus per m^ was determined. Slugs with a 
dry weight of 0.75 g and more were regarded 
as mature, and only those catching periods 
were considered in which slugs deposit eggs 
(August-October). As a result, the number of 
mature specimens per 12 m^ was two and 
seven in the year 1995 and 1996, respec- 
tively, the average number being one mature 
slug per 3 m^. Based on this abundance, the 
energy budget of the population of A. rufus 
was calculated. It is assumed that each ma- 
ture slug lays one egg clutch in autumn, which 
consists of 61 eggs. In November, 61 juve- 
niles hatch, of which one specimen survives 
until July. In September, this specimen de- 
posits an egg clutch and then dies. Based on 
these data, the monthly abundance of A. rufus 
in the Göttinger Wald was calculated using a 
survivorship curve. Survivorship curves of 
slug populations in forest ecosystems are not 
known, but those of laboratory cultures 
(Szabó & Szabó, 1929) and of slug popula- 
tions on permanent pasture (South, 1989) re- 
vealed a more or less constant mortality rate 
throughout life. Therefore, a survivorship 
curve with a constant mortality rate was used, 
which is N, = N^e""^, where Nq = number of ju- 
veniles hatched, N, = number of slugs at time 
t, r = death rate, and t = time (Slobodkin, 
1962). Using the abundance data, monthly 
secretion of mucus, respiration and excretion 



202 



THEENHAUS & SCHAEFER 




— 5°C, 100% RH 
•■-10°C, 100% RH 
--15°C, 100% RH 

— 20°C, 100% RH 
-•10°C, 75% RH 

■ ■■10°C, 55% RH 



0.5 1 1.5 2 2.5 3 

dry weight Arion rufus (g) 

FIG. 1 . Weight of mucus secreted by A. rufus at different temperatures and relative fiumidities as a function 
of animal dry weight. Regression equations are for 5'C and 100% RH: S = 3.55 x W°^^ (n = 49, R^ = 0.81, 
p (slope) < 0.001 ), for 1 0X and 1 00% RH: S = 3.08 x W° ^^ (n = 53, R^ = 0.83, p (slope) < 0.001 ), for 1 5°C 
and 1 00% RH: S = 3.47 x W° ^^ (n = 45, R^ = 0.52, p (slope) < 0.001 ), for 20°C and 1 00% RH: 1 .58 x W° ^^ 
(n = 34, R^ = 0.54, p (slope) < 0.001 ), for 1 0°C and 75% RH: S = 0.97 x W° ^^ (n = 60, R^ = 0.79, p (slope) < 
0.001), and for 1 0X and 55% RH: S = 0.81 x W°^^ (n = 54, R^ = 0.66, p (slope) < 0.001), where RH = rela- 
tive humidity, S = d wt mucus [mg h" ^], W = d wt Л. rufus [g], n = number of slugs examined. 



TABLE 5. Mean monthly temperature in the litter layer and relative humidity (RH) 2 m above ground 
(Göttinger Wald, mean data of the years 1991-1995). Mean monthly fresh weight (F wt) and dry weight (D 
wt) of the model specimen of Arion rufus. Monthly secretion of mucus (Mucus), respiration and excretion of 
faecal materials (F) of the model specimen. 





Temp 


Temp. 


RH 














[°C] 


[°C] 


[%] 




















Fwt 


D wt 


Mucus 


Respiration 


F 


Month 




rounded off 




[g] 


[g] 


[mg d wt] 


[ml COJ 


[mg d wt] 


September 


11.2 


10 


85 






с^слгл ctano 










Cyy OLdLJC 






Optnhpr 


8.2 


10 


89 






(^c\r\ ctanp 






\_/V-il\JUd 






cyy oidyc? 






November 


4.4 


5 


92 


0.05 


0.01 


20 


0.7 


7 


December 


2.0 


5 


92 


0.05 


0.01 


21 


0.7 


7 


January 


1.0 


5 


87 


1 


0.15 


160 


17.1 


97 


February 


0.3 


5 


85 


1 


0.15 


144 


15.5 


87 


March 


2.7 


5 


78 


3 


0.46 


368 


55.8 


284 


April 


5.7 


5 


72 


3 


0.46 


356 


54.0 


275 


May 


9.1 


10 


71 


6 


0.92 


528 


247.7 


552 


June 


11.4 


10 


81 


6 


0.92 


511 


239.8 


300 


July 


14.3 


15 


78 


9 


1.38 


857 


514.8 


412 


August 


14.7 


15 


75 


9 


1.38 


857 


514.8 


412 


September 


11.2 


10 


85 


9 


1.38 


756 


348.5 


789 


Sum 












4578 


2009.4 


3222 


Sum [kJ] 












85.9 


43.5 


68.5 



ENERGY BUDGET OF AfílON RUFUS 



203 



TABLE 6. Number of Arion rufus. monthly secretion of mucus, respiration and excretion per 3 m 



Month 



A. rufus 
[Number] 



Mucus 
[mg d wt] 



Respiration 
[ml COg] 



Faecal Material 
[mg d wt] 



September 





egg stage 





October 




P^nn QtPriP 






cyy oiayC 




November 


61 


1220 43 


427 


December 


36.5 


767 26 


256 


January 


21.9 


3504 374 


2124 


February 


13.1 


1886 203 


1140 


March 


7.8 


2870 435 


2215 


April 


4.7 


1 673 254 


1293 


May 


2.8 


1478 694 


1546 


June 


1.7 


869 408 


510 


July 


1 


857 515 


412 


August 


1 


857 515 


412 


September 


1 


756 349 


789 


Sum 




16737 3816 


11124 


Sum [kJ] 




315.8 82.8 


239.2 



was calculated (Table 6). The consumption 
rate of the population was calculated using an 
assimilation efficiency of 80%. As a result, the 
annual consumption rate of the population is 
8.7 g carbon m"^, which is equivalent to 19.3 
g dry plant material and 361 .9 kJ. The annual 
investment of the population of A. rufus into 
the somatic growth was calculated as sum of 
dry weight of slugs, that die each month, 
which is 2.6 g d wt m"^ (52.5 kJ). 

Summing up, the energy budget of the pop- 
ulation of A. rufus in the Göttinger Wald ¡s: 

361.9 = 52.5 + 3.6 + 105.3 + 27.6 + 79.7 
[kJ m-2 yr-^j 



DISCUSSION 

Slug populations are difficult to estimate, 
because, particularly during periods of 
drought, slugs hide underground, where they 
can stay for up to three months (Kunkel, 
1916). For the determination of slugs in the 
field, Ferguson et al. (1989) constructed de- 
fined-area traps, which consisted of an iron 
ring covering 0.1 m^. A wet sacking was 
placed into the traps to encourage the slugs to 
remain above ground. In the present investi- 
gation, defined-area traps similar to those of 
Ferguson et al. (1989) were constructed. 
These traps cover a larger area (1 m^) than 
those of Ferguson et al. (1989), and a glass 
jar with beer is imbedded into the soil inside 
the traps, because beer is supposed to be a 
good baiting medium for slugs. However, dur- 
ing periods of drought, only low number of 



slugs were caught by the traps, presumably 
because the slugs hid underground. Obvi- 
ously, during dry seasons the slug number is 
underestimated by the defined-area trap 
method. Furthermore, the defined-area traps 
are not suitable for the determination of the 
age distribution of slug populations, because 
in the present investigation the number of ju- 
venile slugs was much lower than one would 
expect from the number of adult specimens 
(Table 1). Presumably juvenile slugs are not 
baited by the beer as efficiently as mature 
slugs. As a consequence of these shortcom- 
ings of traps, in the present investigation the 
energy budget of the population of A. rufus 
was calculated using data on the abundance 
of mature slugs only, and the monthly popula- 
tion structure of A. rufus derived from a sur- 
vivorship curve. However, the abundance of 
mature specimens was calculated from the 
average value of two abundance values only, 
which were strongly dissimilar (two and seven 
mature A. rufus per 12 m^ in the year 1995 
and 1996, respectively). Furthermore, repro- 
duction does occur at various times of the 
year and therefore our model is too simple. Fi- 
nally, it was assumed that the mortality rate of 
A. rufus remains constant throughout life. This 
assumption was based on observations on 
other slug species made in the laboratory and 
on permanent pasture (Szabó & Szabó, 1929; 
South, 1989). However, respective data on A. 
rufus in the forest ecosystem are not avail- 
able, and therefore the survivorship curve 
used in the present investigation is just a 
broad estimate. Therefore, when evaluating 
the energy budget of the population of A. 
rufus it should be kept in mind that this budget 



204 



THEENHAUS & SCHAEFER 



is based on a broad estimate of slug abun- 
dance. 

Mean biomass of tine population of A. rufus 
was 0.2 g d wt m~^. Corsmann (1990) deter- 
mined the biomass of A. rufus in the forest in- 
vestigated in the present study by hand sort- 
ing and found a very similar biomass (0.19 g 
d wt m"^). This accordance shows that the de- 
fined-area trap of the present study is a suit- 
able tool for the determination of slug biomass 
in forest ecosystems. Biomass data of A. ater 
in different beech forests are comparable to 
data of A. rufus found in the present investi- 
gation. Jensen (1975) estimated 0.27 g d wt 
m""^ in a forest in Denmark, and Jennings & 
Barkham (1 976) calculated 1 .5 g fresh weight 
m"^ in Great Britain, which is equivalent to a 
dry weight of 0.2 g m"^. The similar biomass 
of A. rufus and A. ater in different beech 
forests suggests that slug biomass is limited 
by the same factor in different forests. Jen- 
nings & Barkham (1979b) suggested that on 
windy nights and during drought or sub-zero 
temperatures, intraspecific competition for 
food may occur, because slugs remain low on 
the ground or beneath the litter layer where 
green food is unavailable. However, another 
limiting factor of slug biomass may be the 
number of hiding places in the forest soil. Hid- 
ing places are essential for the survival during 
drought or harsh weather conditions. In differ- 
ent forest ecosystems, the number of hiding 
places might be similar, and therefore the reg- 
ulation of slug density through the number of 
hiding places would result in a similar slug 
biomass in different beech forests. This hy- 
pothesis is supported by the fact, that slugs 
usually keep the same hiding place for a long 
pehod of time ("homing"; Frömming, 1954; 
Cook, 1979). 

McNeill & Lawton (1970) calculated a re- 
gression line between annual production and 
respiration of short-lived poikiiotherms being 
log R = 1.17 log P -i- 0.14 [kcal]. The insertion 
of the production of A. rufus (P = P^ -i- P^ -i- P^) 
into this equation results in a respiration value 
which is 60 times of that actually calculated in 
the present investigation. However, in their 
calculations, McNeill & Lawton (1970) explic- 
itly omitted data on animals characterized by 
high mucus secretion, and when P^ was ex- 
cluded from our calculations the respiration 
value of A. rufus would be one fourth of that 
expected by McNeill and Lawton's equation. 
The very high net population production effi- 
ciency may be a consequence of life history 
characteristics of A. rufus. This slug species 



builds up a very high biomass within just one 
year, reproduces and then dies. Therefore, no 
standing crop of older individuals has to be 
maintained. However, in the present investi- 
gation the respiration of A. rufus was extrapo- 
lated from laboratory measurements at three 
temperatures to the field. This extrapolation of 
course is too simple, because in the field 
slugs are exposed to a complex environment 
with fluctuating biotic and abiotic conditions. 
In a further experiment, the respiration rate of 
A. rufus should be measured in the field. 

In the present investigation, the secretion of 
mucus constitutes an important component of 
the energy budget; however, the determina- 
tion of this parameter implies some inaccura- 
cies. First, handling of slugs in the laboratory 
might have stimulated mucus production dur- 
ing incubation, causing an artificial high value 
of P^. However, preceeding experiments 
showed that the amount of mucus secreted 
during the first hour of incubation in the labo- 
ratory was not significantly different from that 
secreted during the second and third hour. 
Second, the secretion of mucus during copu- 
lation of slugs was not considered in the pres- 
ent calculation. Since copulation is very ex- 
pensive in mucus and since slugs may 
copulate several times, the amount of mucus 
lost due to copulation might cause a signifi- 
cant increase in P^. Third, when determining 
the energy content of mucus of A. rufus in the 
present investigation, the mean of calorific 
values of the mucus of two snail species were 
used (23.9 and 1 3.9 kJ g"^ dw mucus for Lym- 
naea stagnalis and Cepaea nemoralis, re- 
spectively). Obviously, such calorific values 
vary widely interspecifically, and had another 
value been used the outcome of the energy 
budget would have been different. Fourth, it 
was assumed that during a period of 24 h 
slugs are active for 6 h. However, during cold 
seasons, slug activity may be very low, result- 
ing in low mucus secretion. If between No- 
vember and April (months of an average tem- 
perature of 5"C; Table 5) zero mucus 
secretion is assumed, the term P^ of the en- 
ergy budget of the population of A. rufus 
would decrease by 35%. 

The exponent b in the relationship between 
the weight of mucus secreted by A. rufus and 
animal dry weight was about 1 at favourable 
(100% RH, 5-1 5"C), and about 2/3 at un- 
favourable conditions (20"C or low RH, Fig. 
1). This dichotomy may reflect the different 
shapes of slugs during moving, being more or 
less flat at favourable and more or less spher- 



ENERGY BUDGET OF ARION RUFUS 



205 



ical (rather elliptic) at unfavourable abiotic 
conditions. In general, doubling in weight of a 
flat body results in doubling of the surface (b 
= 1 ), whereas the surface of a spherical body 
doubles when its weight increases by a factor 
of 3 (b = 2/3). 

Denny (1980) stated that gastropod crawl- 
ing is the most costly form of locomotion in the 
animal kingdom. Actually, in the present inves- 
tigation, the secretion of mucus of the model 
specimen (85.9 kJ) comprises 69% of total 
production and therefore is an important com- 
ponent of the energy budget. In many calcula- 
tions of the energy flow through molluscs, the 
secretion of mucus is ignored (Paine, 1965; 
Stern, 1969; Hughes, 1970; Mason, 1971; 
Jensen, 1975; Jennings & Barkham, 1976; 
Streit, 1976; Andreassen, 1981; Phillipson & 
Abel, 1 983; Workman, 1 983). The secretion of 
mucus is considered in the calculations of 
Carefoot (1 967), Paine (1 971 ), Kofoed (1 975), 
Richardson (1975), Otto (1976), Edwards & 
Welsh (1982), Horn (1986), Davies et al. 
(1 990), and Santini et al. (1 995). Calow (1 974) 
calculated for freswater gastropods that 
13-23% of the assimilated energy is lost via 
mucus. The respective value for Cepaea 
nemoralis is 12% (Richardson, 1975), for 
llyanassa obsoleta 80% (Edwards & Welsh, 
1982), and for Patella vulgata 52% (Davies et 
al., 1990). In the present investigation, the 
model specimen of A. rufus invested 51% of 
the assimilated energy into the secretion of 
mucus. Most obviously, the considerable in- 
vestment of gastropods in locomotion limits 
the range of conditions under which popula- 
tions can survive. In the light of this high in- 
vestment of energy into mucus, the question 
arises as to which ecosystem characteristics 
and slug qualities allow the relative high slug 
biomass in the beech forest ecosystem? First, 
the dense herb layer of the beech forest pro- 
vides plenty of food for population growth, at 
least during favourable weather conditions. 
Second, since slugs are omnivorous, their 
food intake is independent of single food 
items, and therefore food limitation becomes 
rare. Third, the high assimilation efficiency of 
gastropods allows an exceptionally high effi- 
ciency in food utilization. These factors might 
contribute to the high biomass of gastropods in 
the beech forest ecosystem and make up for 
the high cost of gastropod crawling. 

In this and the following section, data ob- 
tained for A. rufus are used to draw broad 
conclusions about the energetics of the gas- 
tropod community in the forest ecosystem 



studied. For example, it is assumed that the 
biomass/excretion ratio of all gastropod spe- 
cies in the forest ecosystem studied is identi- 
cal to that of A. rufus. This of course is a broad 
generalization, because there may be marked 
differences in the ecological efficiencies of dif- 
ferent pulmonales (Lamotte & Stern, 1987). 
Therefore, the calculated values of excretion 
and consumption of the gastropod community 
should be regarded as a broad estimate. The 
production of faecal materials of the popula- 
tion of A. rufus in the forest ecosystem stud- 
ied is equivalent to 1.7 g carbon m"^ уг\ 
which is 3.7 g d wt faecal materials m"^ yr"\ 
The biomass of A. rufus makes up 19% of 
total biomass of gastropods in the forest stud- 
ied (Corsmann, 1 990). Assuming that the bio- 
mass/excretion ratio of all gastropod species 
in the forest studied is identical to that of A. 
rufus, the gastropod community deposits 1 9 g 
m~^ yr~^ as faeces. In comparison, the excre- 
tion of faecal materials of lumbhcids (more 
than 10 kg m"^ yr"^ Scheu, 1987) is much 
higher, which shows that the relative contribu- 
tion of the gastropod community to the biotur- 
bation in the forest soil is negligible. 

The population of A. rufus consumes 1 9.3 g 
d wt food m"^ yr~^ in the forest studied. The 
input of canopy litter fall in this forest is 331 g 
d wt m"^ yr"^ (leaves and bud scales; Schae- 
fer, 1990). About 2.6% of food material of A. 
rufus is leaf litter (Corsmann, 1990), and 
therefore the population of A. rufus has an 
annual consumption of 0.50 g d wt leaf litter 
m"^, which represents 0.15% of the leaf litter 
input per year. Corsmann (1990) determined 
the biomass of the most abundant gastropod 
species of the beech forest studied. Further- 
more, the author examined the food material 
in the gut of these gastropod species and de- 
termined the relative amount of certain food 
material (e.g., leaf litter, green plant material, 
fungi) in their gut. Considering these data and 
assuming that the biomass/excretion ratio of 
all gastropod species in the forest studied is 
identical to that of A. rufus, the gastropod 
community consumes 8.1 g d wt leaf litter m'^ 
yr"\ which is 2.4% of the annual leaf litter 
input. This value compares with those ob- 
tained by Phillipson (1983) and Phillipson & 
Abel (1983) in a beech forest. The authors 
calculated that the gastropod community con- 
sumes 1.8-2.9% of leaf litter input per year. 
Much higher values were calculated by Jen- 
nings & Barkham (1976, 1979a). These au- 
thors found that the population of A. rufus 
consumes 1.54% and that the slug commu- 



206 



THEENHAUS & SCHAEFER 



nity consumes 8.4% of the annual leaf litter 
input of a beech forest, and therefore makes 
a significant direct contribution to plant de- 
composition processes. In contrast, data of 
the present investigation suggest that the di- 
rect contribution of the gastropod community 
to decomposition processes is low. 

Mean above- and below-ground net pri- 
mary production (NPP) of the herb layer in the 
forest studied is 100 g d wt m"^ yr"'' (Schaefer, 
1990). Green herb material makes up 17% of 
the food material of A. rufus (Corsmann, 
1 990), and therefore the population of A. rufus 
consumes 3.3 g d wt green herb material m"^ 
yr"\ which represent 3.3% of the annual pro- 
duction of green plant material. The gastro- 
pod community has a consumption of 26.1 g 
d wt green herb material m"^ yr"\ which rep- 
resent 26% of the annual production of green 
plant material. This phytomass compares well 
with that which is annually ingested by phy- 
tophages (Curculionidae and lepidopteran 
larvae) in the forest studied (Schaefer, 1990), 
and therefore the relative consumption of 
green plant material by the gastropod com- 
munity is high. 

The NPP of higher plants in the forest stud- 
ied is 2078 g d wt m"^ year"^ (Schaefer, 1 990), 
and the population of A. rufus consumes 
0.24% of the annual NPP. The gastropod 
community consumes 34.5 g d wt higher 
plants m year"\ which is 1.7% of the NPP of 
higher plants. Obviously, the gastropod com- 
munity consumes a very small part of the phy- 
tomass in the forest ecosystem and therefore 
most probably the feeding pressure of gas- 
tropods on plants is low. This is partly due to 
the fact that gastropods are omnivorous and 
that therefore the strength of prédation dif- 
fuses to different trophic unities (Polis, 1994). 
It is stated by many authors (e.g., Hairston et 
al., 1960; Fretwell, 1987; Oksanen et al., 
1981) that herbivores of forest ecosystems 
consume only a small part of the phytomass, 
because their biomass is controlled by preda- 
tors (top-down control). The low feeding pres- 
sure of gastropods on higher plants, however, 
most probably is not caused by top-down 
forces, because gastropod biomass is limited 
by abiotic factors (e.g., drought; Jennings & 
Barkham, 1979b). 

The energy budget of the present investi- 
gation is based on a series of assumptions, 
which are strongly subjective. For example, 
the population structure of A. rufus was calcu- 
lated using data on the abundance of mature 



slugs and on the mean number of eggs per 
clutch and a survivorship curve. "Mature 
slugs" were defined as specimens with a dry 
weight of 0.75 g and more, and had another 
weight been taken the abundance of mature 
specimens would have been different. The 
survivorship curve used is a broad estimate 
taken from the literature, and it therefore rep- 
resents another shortcoming of the present 
investigation. Furthermore, the conversion of 
data into calorific values was accomplished 
with conversion factors from the literature, 
and therefore the calorific energy budget 
should be evaluated with caution. In addition, 
the monthly amount of respiration, secretion 
of mucus and excretion of faecal materials of 
A. rufus was calculated using mean monthly 
temperature and air humidity values of the for- 
est under study. Of course, the abiotic char- 
acteristics of the forest ecosystem are incom- 
pletely simulated by this approach. However, 
the energy budgets of populations in general 
embody many assumptions about population 
structure and generally lack true seasonal 
changes in budget terms. The energy budget 
of the present investigation should be re- 
garded as a broad estimate. 



ACKNOWLEDGEMENTS 

We are grateful to Thomas Theenhaus, 
Claus Döring, Klaus Dornieden, Alexander 
Sührig. and Ulrich Strothmann for collecting 
large numbers of slugs and for their help in 
setting up the defined-area traps. We are also 
indebted to Sonja Migge for her help in raising 
slugs. We thank two anonymous reviewers for 
helpful comments on the manuscript. This 
work was supported by a grant of the 
Friedrich-Ebert-Stiftung (Anne Theenhaus). 



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Revised ms accepted 1 February 1999 



MALACOLOGIA, 1999, 41(1): 209-230 

SPECIES OF MACOMA (BIVALVIA: TELLINIDAE) FROM THE PACIFIC COAST OF 
RUSSIA, PREVIOUSLY DESCRIBED AS ABRINA (BIVALVIA: SEMELIDAE) 

Gennady M. Kamenev^ & Victor A. Nadtochy^ 



ABSTRACT 

Abrina cuneipyga Scarlato, 1981 ; A. sachalinica Scarlato, 1981 ; A. shiashkotanika Scarlato, 
1981 ; and A. tatarica Scarlato, 1981, described from the Pacific coast of Russia belong instead 
in the genus Macoma Leach, 1 81 9. For one species, a new combination is suggested, Macoma 
cuneipyga (Scarlato, 1 981 ). Abrina sachalinica. A. tatarica and A. stiiastikotanika are synonyms 
of Macoma /oi/en/ (Jensen, 1905), Macoma calcárea (Gmelin, 1791), and Macoma sp., respec- 
tively. The main morphological characteristic on the basis of which these species were previously 
included in Abrina Habe, 1952, was the presence of an internal ligament in an oblique resilifer 
posterior to the cardinal teeth. Studies of the common northwestern Pacific M. loveni. Macoma 
balthica (Linné, 1758), Macoma crassula (Deshayes. 1855), Macoma lama Bartsch, 1921, Ma- 
coma incongrua (Martens, 1865), and M. calcárea (Gmelin, 1791) show the presence of a simi- 
lar morphological characteristic in young specimens. In Macoma. this characteristic is reduced 
as the molluscs grow, but it is preserved in adult Abrina. The results of a comparative analysis of 
species of genera Abrina and Macoma, and expanded descriptions of M. cuneipyga. M. loveni, 
and Macoma sp.. are presented. 

Key words: Macoma, Abrina, northwestern Pacific, systematics, morphology, distribution. 



INTRODUCTION 

Scarlato (1981), In his monograph on fauna 
and distribution of bivalve molluscs of the 
northwestern Pacific, gave a description with 
very schematic pictures of the type specimens 
of four new species of the genus Abrina Habe, 
1952 (Fam. Semelidae Stoliczka, 1870): Ab- 
rina cuneipyga Scarlato, 1981 ; A. sachalinica 
Scarlato, 1981; A. shiashkotanika Scarlato, 
1981; and A. tatarica Scarlato, 1981. Previ- 
ously, representatives of this genus were not 
mentioned in lists of the Russian molluscan 
fauna and were found only in subtropical lati- 
tudes of the western Pacific (Gould, 1861; 
Kuroda, 1951; Habe, 1952, 1958, 1977, 
1981). 

A study of the type specimens of Abrina 
species from the Pacific coast of Russia, as 
well as material of Macoma species from the 
Arctic seas and the northwestern Pacific, and 
of Abrina species from Japan, have shown 
that the species described by Scarlato (1 981 ) 
should instead be included in the genus Ma- 
coma Leach, 1819 (Fam. Tellinidae Blainville, 
1814). 

For one of the four species described by 
Scarlato (1 981 ) as Abrina, a new combination 
is suggested, Macoma cuneipyga (Scarlato, 



1981). Abrina sachalinica, A. tatarica, and A. 
shiashkotanika are considered to be syn- 
onyms of M. loveni, M. calcárea and Macoma 
sp., respectively. The goal of this paper is to 
present the results of a comparative analysis 
of species of genera Abrina and Macoma and 
to give an expanded description of M. 
cuneipyga, M. loveni and Macoma sp., sup- 
plemented by new data on their shell morphol- 
ogy, ecology, and geographical distribution. 



MATERIALSAND METHODS 

In this study we have used the material col- 
lected by expeditions of the following re- 
search institutes: 

PRIFO expeditions on the shelf zone of the 
northern Sea of Okhotsk (R/V "8-452", 1977; 
R/V "8-461", 1979), eastern coast of Sakhalin 
Island, Sea of Okhotsk (R/V "8-452", 1977), 
western coast of Kamchatka, Sea of Okhotsk 
(R/V "Professor Levanidov", 1996), eastern 
coast of Kamchatka, Pacific Ocean (R/V "Mys 
Tikhiy", 1985), and Bering Sea (R/V "Mys 
Tikhiy", 1984). A joint IMB-PRIFO expedition 
on the shelf and bathyal zones of the Kuril Is- 
lands (R/V "Tikhookeansky", 1987). 

A joint FERHI-SakhRIFO expedition under 



Institute of Marine Biology, Russian Academy of Sciences. Vladivostok 690041 , Russia; inmarbio@mail.primorye.ru 
^Pacific Research Institute of Fistiehes and Oceanography, Vladivostok 690600. Russia 



209 



210 



KAMENEV & NADTOCHY 



the supervision of ENL Company on the shelf 
zone of the northeastern Sakhalin Island. Sea 
of Okhotsk, in the vicinity of an oil-drilling site 
(R/V "Pavel Gardienko", 1997). 

Expeditions of 1MB on the shelf zone of the 
Tatar Strait. Sea of Japan (R/V "Atna", 1974; 
R/V "Berill", 1977) and Peter the Great Bay, 
Sea of Japan (R/V "Ametist", 1983). A joint 
IMB-PIBOC expedition on the shelf zone of 
the Sea of Japan (R/V "Akademik Oparin", 
1995). 

The material from the western Kamchatka. 
Sea of Okhotsk, southeastern Kamchatka, 
Pacific Ocean, and the Bering Sea was fixed 
and stored in 4% formaldehyde in PRIFO. All 
the other material was fixed in 70% ethanol 
and stored dry in 1MB. 

We have also used collections of the follow- 
ing taxa: M. balthica from the Bering Sea 
(MIMB); M. calcárea, M. crassula and M. in- 
congrua from the Sea of Japan (MIMB); M. 
lama from the Sea of Okhotsk (MIMB); M. 
/oi /ел/ from the Arctic seas, the Sea of Japan, 
the Sea of Okhotsk, and the North Atlantic 
(USNM, ZIN); Abrina lunella (Gould, 1861) 
and A. kinoshitai {Kuroda & Habe, 1958) from 
the coastal waters of Japan (Drs. T. Kurozumi 
and E. Tsuchida, NHMI, NSMT); /4. cuneipyga. 
A. sachalinica. A. shiashkotanlka. and A. tatar- 
/ca from the Pacific seas of Russia (ZIN). Ma- 
coma from the Arctic and Pacific seas of Rus- 
sia were fixed and stored in 70% ethanol. All 
other material was stored dry. 

For the collection material stored in ZIN, in- 
ventory numbers are given for holotypes; for 
other specimens, their catalogue numbers are 
given. 

Shell Measurements 

For measurements, we have chosen those 
parameters most often used in diagnosis, de- 
scription, and comparative analysis of species 
of the genus Macoma (Dunnill & Ellis, 1969; 
Coan, 1971; Scarlato, 1981; Kamenev. 1989, 
1990; Kafanov et al., 1997). 

Figure 1 shows the position of our shell 
morphology measurements. Shell length (L), 
height (H), width of each valve (W) (not 
shown), anterior end length (A), maximal dis- 
tance from posterior shell margin to top of pal- 
liai sinus (LI), and minimal distance from top 
of palliai sinus to anterior adductor muscle 
scar (L2) were measured for each valve. The 
ratios of these parameters to shell length 
(H/L, W/L, A/L, L1/L, L2/L, respectively) were 
determined. Shell measurements were made 




FIG. 1 . Placement of shell measurements: L — shell 
length; H — height; A — anterior end length; LI — 
maximal distance from posterior shell margin to top 
of palliai sinus; L2 — minimal distance from top of 
palliai sinus to anterior adductor muscle scar. 



using a calipers and an ocular micrometer 
with an accuracy of 0.1 mm. 
We made measurements of: 

(1) 51 specimens and 2 left valves of M. 
loveni from Spitzbergen (USNM 
108789, 2 spec); Laptev Sea (ZIN 
106, 3 spec); Gulf of St. Laurence, 
North Atlantic (USNM 95638, 1 spec, 
1 left valve); Gulf of Maine, North At- 
lantic (USNM 159769, 1 spec); Bering 
Sea (our material, 1 spec); Kronotsky 
Bay, eastern Kamchatka, Pacific 
Ocean (our material, 4 spec); Sea of 
Okhotsk (ZIN 9853. holotype of A. 
sachalinica: ZIN 57, 2 spec; USNM 
204814, 1 spec; our material, 5 spec); 
Kuril Islands (our material, 19 spec); 
Sea of Japan (ZIN 65, 3 spec: ZIN 67, 

1 spec: USNM 2048015. 2 spec: our 
material 5 spec, 1 left valve). 

(2) 22 specimens, 2 hght, 7 left valves of 
M. cuneipyga from Kuril Islands (ZIN 
9800, holotype of A. cuneipyga: ZIN 
10, 1 left valve; our material, 18 spec, 

2 right, 6 left valves); Sea of Okhotsk 
(our material, 3 spec). 

(3) 3 specimens of Macoma sp. from Shi- 
ashkotan Island, Middle Kuril Islands 
(ZIN 9867, holotype of A. shi- 
ashkotanlka: 2 spec. (ZIN 1) from the 
type locality). 

(4) 3 specimens and one right valve of M. 
calcárea from the Sea of Japan (ZIN 
9900, holotype of A. tatarica: A. tatar- 



STATUS OF ABRINA SPECIES FROM RUSSIA 



211 



ica (ZIN 5), 1 spec); Sea of Okhotsk 
(A. tatahca (ZIN 6), 1 spec, 1 left 
valve). 

(5) 2 specimens, 5 right, 2 left valves of A. 
lunella from Sagami Bay, Japan 
(NSMT 45451). 

(6) 1 specimen, 1 right, 7 left valves of A. 
kinoshitai from Esu-zaki, Wakayama 
Prefecture, Japan (NSMT 51909, 1 
spec); Funakashi Bay, Iwate Prefec- 
ture, Japan (material of Drs. T Kuro- 
zumi and E. Tsuchida (NHMI, no num- 
ber); 1 left, 1 right valves), Otsuchi Bay, 
Iwate Prefecture, Japan (material of 
Drs. T Kurozumi and E. Tsuchida 
(NHMI, no number), 6 left valves). 

Statistics 

Statistical analysis of the material used a 
package of statistical programs for Windows 
STADIA 6.0 (Kulaichev, 1996) and STAT- 
GRAPHICS. 

The calculated indices (H/L; W/L; A/L; L1/L 
and L2/L) are less susceptible to change com- 
pared to other measured parameters. A com- 
parison by pairs of different parameters of the 
samples using parametric and nonparametric 
tests was conducted using these indices. All 
data was tested with a Kolmogorov test for 
their fit to a normal distribution. For the left 
valves of M. loveni and M. cuneipyga, the 
original data on L2/L and the log^Q of H/L cor- 
responded to the norm; for the right valves of 
these species, original data on H/L, A/L and 
L2/L also corresponded to the norm. For this 
reason, we used the Student (T) parametric 
test for a comparison by pairs of different 
valves of M. loveni and M. cuneipyga for 
these indices. 

The distribution of the original data by other 
indices was different from the norm. Fifteen 
transformations were used in an attempt to 
bring the obtained data to the norm; however, 
none were successful. Therefore, in the com- 
parative analyses of the samples of other 
indices (W/L, A/L, and L1/L for left valves 
and W/L and A/L for right valves), we used 
the nonparametric criterion of Kolmogorov- 
Smirnov (two sample test). 

We used a discriminant analysis to test the 
validity of the hypothesis of division of all stud- 
ied specimens into two groups (M /oi/en/and 
M. cuneipyga). 

In linear and stepwise multiple discriminant 
analyses of the samples of M. loveni and M. 
cuneipyga. we used original data on the 
parameters H, L, W, LI, L2. Data on these 



parameters corresponded to the norm; 51 
specimens of M. loveni and 22 specimens of 
M. cuneipyga were used for the analysis. 

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

The following abbreviations are used in the 
paper: ENL -commercial company "Exon 
Neftegas Limited"; FERHI-Far East Re- 
search Hydrometeorological Institute, Vladi- 
vostok; 1MB -Institute of Marine Biology, 
Russian Academy of Sciences, Vladivostok; 
MIMB- Museum of the Institute of Marine 
Biology, Vladivostok; NHMI -Natural His- 
tory Museum and Institute, Chiba; NSMT- 
National Science Museum, Tokyo; PIBOC- 
Pacific Institute of Bio-organic Chemistry, 
Russian Academy of Sciences, Vladivo- 
stok; PRIFO- Pacific Research Institute of 
Fisheries and Oceanography, Vladivostok; 
SakhRIFO -Sakhalin Research Institute of 
Fisheries and Oceanography, Yuzhno-Sakha- 
linsk; USNM- United States National Mu- 
seum of Natural History, Smithsonian In- 
stitute, Washington, D. C.; ZIN -Zoological 
Institute, Russian Academy of Sciences, St. 
Petersburg. 



COMPARATIVE ANALYSIS 

In Tables 1 and 2 the results of a morpho- 
metric analysis of the common Japanese 
spec. Abrina lunella and A. kinoshitai are pre- 
sented (Figs. 2-11). On the basis of the re- 
sults of these studies, as well as descriptions, 
pictures and photos of Abrina (Gould, 1861; 
Kuroda, 1 951 ; Habe, 1 952, 1 958, 1 961 , 1 964, 
1 977, 1 981 ; Ito, 1 967; Kuroda et al., 1 971 ; Ito 
et al., 1986; Ito, 1989; Tsuchida & Kurozumi, 
1995) and Macoma species (Golikov & Scar- 
lato, 1967; Keen, 1969; Coan, 1971 ; Bernard, 
1979; Lubinsky, 1980; Scarlato, 1981), Table 
3 has been constructed giving the main diag- 
nostic characteristics of Abrina and Macoma. 

Table 3 shows that the genera Abrina and 
Macoma are similar in most morphological 
characteristics. The main distinguishing char- 
acteristic of Abrina is the presence of a well- 
developed internal ligament in a resilifer, a 
narrow groove posteria to the cardinal teeth. 
In Macoma. an internal ligament is absent. 

The results of studies different age groups 
of the well-identified and widely distributed 
northwestern Pacific M. loveni, M. balthica. M. 
calcárea. M. crassula. M. incongrua and M. 
lama have shown that young individuals of 
these species have a rather large, well-devel- 



212 



KAMENEV & NADTOCHY 




FIGS. 2-11. Shells of Abrina species. 2-8. Abrina lunella (Gould, 1 861 ) (NSMT 45451 ). Sagami Bay, Japan. 
2-6: Largest specimen, shell length 16.1 mm. 7, 8: Hinge of the right valve, valve length 13.0 mm (bar = 1 
mm). 9-11. Abrina kinoshitai (Kuroda & Habe, 1958) (NSMT 51909), Esu-zaki, Wakayama Prefecture, 
Japan, shell length 14.8 mm. 



oped internal ligament (Figs. 12-21). In its 
form, position, and size it is similar to the in- 
ternal ligament of the genus Abrina. It is situ- 
ated in an oblique resilifer posteria to the car- 
dinal teeth and almost reaches the ventral 
edge of the hinge plate (Figs. 12, 13). With 
age, its relative size is reduced (Figs. 14, 21), 
and in adult specimens of M. crassula. M. 
balthica, M. calcárea, M. incongrua and M. 
lama, it disappears. In adult specimens of 
M. loveni, it can just be noticed on the hinge 
plate ventral to the beaks. 

Also, the form of the resilifer changes with 
age. In young specimens of M. crassula, M. 



balthica, M. calcárea, M. incongrua, and M. 
lama (shell length, <5-6 mm) and M. loveni (< 
10 mm), the form and size of the resilifer rela- 
tive to the width of the hinge plate (Figs. 15, 
16, 18, 20) is similar to those of adult Abrina 
lunella and A. kinoshitai. In larger specimens 
(> 8-10 mm), the resilifer is much shorter than 
the width of the hinge plate and ovate (Fig. 
19). With the exception of M. loveni. in adult 
specimens of all investigated species it is ab- 
sent. In adult specimens of M. loveni, it is pre- 
served in the form of ovate pit on hinge plate 
ventral to the beaks posterior to the cardinal 
teeth (Fig. 17). 



STATUS OF ABRINA SPECIES FROM RUSSIA 



213 



TABLE 1 . Abrina lunella (Gould, 1 861 ). Shell measurements (mm), indices and summary statistics of all char- 
acteristics (NSMT 45451): L — shell length; H — height; W — width; A — anterior end length; LI —maximal dis- 
tance from posterior margin to the top of palliai sinus; L2 — minimal distance from the top of palliai sinus to 
anterior adductor muscle scar. Numerator indicates shell measurements and indices for the left valve, 
denominator — for the right valve. NM — not measured. 



Statistics 


L 


H 


W 


A 


L1 


L2 


H/L 


W/L 


A/L 


L1/L 


L2/L 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 




7.8 


6.0 


1.7 


4.8 


6.2 


0.8 


0.77 


0.22 


0.62 


0.79 


0.10 




10.8 


7.7 


2.1 


6.6 


7.2 


1.5 


0.71 


0.19 


0.61 


0.67 


0.14 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 




11.1 


7.9 


2.4 


7.0 


7.6 


1.8 


0.71 


0.22 


0.63 


0.68 


0.16 




11.4 


8.0 


2.1 


7.2 


8.0 


1.3 


0.70 


0.18 


0.63 


0.70 


0.11 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 




11.9 


8.2 


2.5 


7.6 


7.7 


2.3 


0.69 


0.21 


0.64 


0.65 


0.19 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 




13.0 


10.0 


3.0 


8.0 


8.3 


2.3 


0.69 


0.21 


0.64 


0.65 


0.19 




14.1 


10.2 


2.8 


9.1 


9.5 


2.4 


0.72 


0.20 


0.65 


0.67 


0.17 




14.0 


10.3 


3.2 


9.1 


9.0 


3.0 


0.74 


0.23 


0.65 


0.64 


0.21 




NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 




15.0 


10.5 


3.3 


9.0 


10.0 


2.5 


0.70 


0.22 


0.60 


0.67 


0.17 




16.1 


12.5 


3.5 


9.8 


10.7 


3.0 


0.78 


0.22 


0.61 


0.66 


0.19 




16.1 


12.5 


4.0 


9.8 


11.4 


2.4 


0.78 


0.25 


0.61 


0.71 


0.15 


Mean 


13.10 


9.60 


2.63 


8.18 


8.85 


2.05 


0.728 


0.198 


0.625 


0.675 


0.153 




12.70 


9.34 


2.87 


7.90 


8.60 


2.17 


0.737 


0.226 


0.624 


0.683 


0.166 


SD 


2.46 


2.23 


0.67 


1.52 


1.56 


0.79 


0.036 


0.017 


0.019 


0.017 


0.035 




2.76 


2.13 


0.74 


1.67 


1.71 


0.70 


0.037 


0.013 


0.017 


0.053 


0.035 


SE 


1.23 


1.12 


0.34 


0.76 


0.78 


0.40 


0.018 


0.009 


0.010 


0.009 


0.018 




1.05 


0.81 


0.28 


0.63 


0.65 


0.26 


0.010 


0.005 


0.006 


0.020 


0.013 


Min 


10.8 


7.7 


2.1 


6.6 


7.2 


1.3 


0.70 


0.18 


0.61 


0.66 


0.11 




7.8 


6.0 


1.7 


4.8 


6.2 


0.8 


0.69 


0.21 


0.60 


0.64 


0.10 


Max 


16.1 


12.5 


3.5 


9.8 


10.7 


3.0 


0.78 


0.22 


0.65 


0.70 


0.19 




16.1 


12.5 


4.0 


9.8 


11.4 


3.0 


0.78 


0.25 


0.65 


0.79 


0.21 



Thus, a well-developed internal ligament 
lodged in oblique resilifer in representatives of 
the genus Macoma is a juvenile characteristic 
that is preserved in Abrina during its entire 
life. 

A study of the type material of Abrina 
species from the seas of Russia has shown 
that the main character for their description as 
new species of Abrina is the presence of an 
internal ligament posterior to the cardinal 
teeth (Fig. 22) and having different degrees of 
development. It is most distinct in A. sfii- 
ashl<otanil<a and A. cuneipyga. However, the 
type specimens of all the species of Abrina 
described by Scarlato (1981) have a less de- 
veloped internal ligament compared with that 
of Abrina species inhabiting the coastal wa- 
ters of Japan. The size of internal ligament of 
the holotypes of these species corresponds to 
their shell size and species, similar to the 
species of Macoma studied. In other taxo- 
nomic characteristics, they are also identical 
to the genus Macoma. All the new species de- 
scribed by Scarlato (1981) are of a relatively 



small size. Therefore, we consider that Scar- 
lato (1981) described new species of Abrina 
on the basis of a study of young specimens of 
Macoma that had internal ligaments. We con- 
sider that all species of Abrina described from 
the Pacific coast of Russia should be included 
in the genus Macoma. 



SYSTEMATICS 

Family Tellinidae Blainville, 1814 
Genus Macoma Leach, 1819 

Type species (by monotype): Macoma te- 
ñera Leach, 1819; = Teilina calcárea Gmelin, 
1791 

Diagnosis 

Shell small to large in size (20 to 100 mm), 
medium in thickness to heavy, moderately in- 
flated, ovate-triangular, rounded-triangular, 
ovate or round, white, chalky, smooth with 



214 



KAMENEV & NADTOCHY 



TABLE 2. Abrina kinoshitai {Kuroäa & Habe, 1958). Shell measurements (mm), indexes and summary sta- 
tistics of all characteristics: L — shell length; H — height; W — width; A — anterior end length; L1 —maximal 
distance from posterior margin to the top of palliai sinus; L2 — minimal distance from the top of palliai sinus 
to anterior adductor muscle scar. Numerator indicates shell measurements and indices for the left valve, 
denominator — for the right valve. NM — not measured. 



Statistics L 


H 


W 


A 


LI 


L2 


H/L 


W/L 


A/L 


L1/L 


L2/L 


Otsuchi Bay (39°22'N, 


142°00'E), Iwate Prefecture, Japan, depth 75 m. 


sandy 


mud 






7.5 


4.7 


1.1 


4.8 


5.3 


0.6 


0.63 


0.15 


0.64 


0.71 


0.08 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


8Л 


5Л 


L3 


5.2 


5.8 


0.9 


0.61 


0.16 


0.63 


0.70 


0.11 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


8Л 


5^3 


13 


5.4 


6.2 


0.9 


0.62 


0.15 


0.64 


0.73 


0.11 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


93 


5.9 


1.5 


5.9 


7.0 


1.0 


0.62 


0.16 


0.62 


0.74 


0.11 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


10.0 


6.3 


1.6 


6.5 


7.2 


1.1 


0.63 


0.16 


0.65 


0.72 


0.11 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


12.8 


8.0 


2.2 


8.3 


9.3 


1.6 


0.63 


0.17 


0.65 


0.73 


0.13 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


Funakochi Bay (39 23'N, 142° 


OO'E), Iwate Prefecture. 


Japan, depth 70 


m, mud and sand 






NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


12.0 


7.7 


1.9 


8.0 


8.1 


2.1 


0.64 


0.16 


0.67 


0.68 


0.18 


12.7 


8.0 


2.2 


8.3 


9.3 


1.5 


0.63 


0.17 


0.65 


0.73 


0.12 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


Esu-Zaki, Wakayama Prefectu 


re, Japan (NSMT 51 909) 












14.8 


9.3 


2.4 


9.8 


11.1 


2.1 


0.63 


0.16 


0.66 


0.75 


0.14 


14.8 


9.3 


2.4 


9.8 


10.2 


3.0 


0.63 


0.16 


0.66 


0.69 


0.20 


Mean 10.51 


6.58 


1.70 


6.78 


7.65 


1.21 


0.625 


0.160 


0.643 


0.726 


0.114 


13.40 


8.50 


2.15 


8.90 


9.15 


2.55 


0.635 


0.160 


0.665 


0.685 


0.190 


SD 2.61 


1.66 


0.50 


1.81 


2.04 


0.49 


0.008 


0.008 


0.013 


0.016 


0.018 


1.98 


1.13 


0.35 


1.27 


1.49 


0.64 


0.007 





0.007 


0.007 


0.014 


SE 0.92 


0.59 


0.18 


0.64 


0.72 


0.17 


0.003 


0.003 


0.005 


0.006 


0.006 


1.40 


0.80 


0.25 


0.90 


1.05 


0.45 


0.005 





0.005 


0.005 


0.010 


Min 7.5 


4.7 


1.1 


4.8 


5.3 


0.6 


0.61 


0.15 


0.62 


0.70 


0.08 


12.0 


7.7 


1.9 


8.0 


8.1 


2.1 


0.63 


0.16 


0.67 


0.69 


0.2 


Max 14.8 


9^ 


2.4 


9.8 


11.1 


2.1 


0.63 


0.17 


0.66 


0.75 


0.14 


14.8 


9.3 


2.4 


9.8 


10.2 


3.0 


0.64 


0.16 


0.67 


0.69 


0.20 



TABLE 3. Diagnostic characteristics of the genera Abrina and Macoma. 



Abrina Habe, 1952. Type species; Abra 

kanamarui Kuroúa. 1951 ; = Macoma 

lunella Gou\ä, 1861 



Macoma Leach. 1819. Type species: 

Macoma teñera Leach, 1819; = Tellina 

calcárea Gmelin, 1 791 



Shell form and 
proportions 



Shell surface 
Periostracum 
Hinge 

Ligament 



Palliai sinuses 



Ovate or triangular-ovate, moderately 
inflated; anterior end longer than poste- 
rior; posterior end with radial ridge 
along posterodorsal margin, twisted to 
the right 

Smooth, with faint growth checkmarks 

Thin, colorless, silky or shiny 

Weak, two cardinal teeth in each valve, 
lateral teeth absent 

Both external and internal; external — 
seated on a nymph not projecting 
above dorsal margin; internal — lodged 
in oblique groove behind cardinal teeth 

Long, often of different length and form in 
right and left valves, partly confluent 
with palliai line 



Ovate to subtrigonal, moderately inflated; 
equivalve or with left valve some what 
larger; equilateral to longer anteriorly; 
posterior end with radial ridge along pos- 
terodorsal margin, twisted to the right 
Smooth, with faint growth checkmarks 
Thin, dark to colorless, silky or shiny 
Weak, two cardinal teeth in each valve, lat- 
eral teeth absent 
External, seated on a nymph not projecting 
above dorsal margin 



Long, often of different length and form in 
right and left valves, partly confluent with 
palliai line 



STATUS OF ABRINA SPECIES FROM RUSSIA 



215 




FIGS. 12-21. Hinge of /Wacoma species. 12-17. Macoma loveni (Jensen, 1905). 12: Cardinal teeth andin- 
ternal ligament of right valve (ZIN 106), Laptev Sea (76'"38'N, 118°20'E), 46 m, valve length 7.1 mm (bar = 
1 mm). 13: Cardinal teeth and internal ligament of left valve, Iturup Island (44°23'4"N, 147"^37'8"E), 230 m, 
valve length 8.7 mm (bar = 1 mm). 14: Cardinal teeth, external and internal ligament of right valve (ZIN 65), 
western coast of Sakhalin Island, Tatar Strait, Sea of Japan, 84-86 m, valve length 13.4 mm (bar = 1 mm). 
15: Cardinal teeth and resilifer of right valve, Iturup Island (44°23'4"N, 147'^'37'8"E), 230 m, valve length 8.6 
mm (bar = 100 цт). 16: Cardinal teeth and resilifer of right valve (USNM 159769), Gulf of fVlaine, North At- 
lantic, valve length 5.0 mm (bar = 1 00 |.im). 1 7: Cardinal teeth and ligament pit of right valve, Shikotan Island 
(43"45'N, 146 55'E), 100 m, valve length 15.6 mm (bar = 1 mm). 18, 19. Macoma balthica (Linné, 1758) 
(MIMB 12/14742), Signalny Island, Olyutorskiy Bay, Bering Sea, intertidal zone. 18: Cardinal teeth and re- 
silifer of left valve, valve length 4.5 mm (bar = 100 (.im). 19: Cardinal teeth and ligament pit of right valve, 
valve length 8.2 mm (bar = 1 00 |im). 20. Macoma calcárea (Gmelin, 1 791 ), cardinal teeth and resilifer of right 
valve. Peter the Great Bay, Sea of Japan, 16 m, valve length 4.2 mm (bar = 1 mm). 21 . Macoma nipponica 
(Tokunaga, 1906), cardinal teeth and ligament of right valve, Peter the Great Bay, Sea of Japan, 12 m, length 
6.3 mm (bar = 100 |jm). 



216 



KAMENEV & NADTOCHY 



TABLE 4. Macoma cuneipyga (Scarlato, 1981). Summary statistics of the shell measure- 
ments (mm) and indexes: L — shell length; H — height: W — width; A — anterior end length; 
LI —maximal distance from posterior margin to the top of palliai sinus; L2 — minimal dis- 
tance from the top of palliai sinus to anterior adductor muscle scar. Numerator indicates the 
summary statistics for the left valve, denominator — for the right valve. 



Characteristics 


Mean 


SD 


SE 


Min 


Max 


п 


L 


11.29 


5.52 


1.03 


3.1 


20.0 


29 




10.43 


5.66 


1.16 


3.1 


19.7 


24 


H 


7.93 


3.75 


0.70 


2.4 


14.0 


29 




7.50 


3.96 


0.81 


2.4 


14.0 


24 


W 


2.28 


1.13 


0.21 


03. 


4.1 


29 




2.05 


1.09 


0.22 


0.6 


3.8 


24 


A 


8.29 


4.19 


0.78 


1Л 


15.3 


29 




7.73 


4.35 


0.89 


1.8 


15.3 


24 


LI 


7.22 


3.41 


0.63 


2.2 


12.9 


29 




5.80 


3.07 


0.63 


1.9 


11.0 


24 


L2 


1.70 


0.94 


0.17 


QA 


3¿ 


29 




2.50 


1.54 


0.31 


0.7 


5.5 


24 


H/L 


0.714 


0.038 


0.007 


0.65 


0.80 


29 




0.730 


0.031 


0.006 


0.69 


0.80 


24 


W/L 


0.201 


0.015 


0.003 


0.17 


0.24 


29 




0.197 


0.013 


0.003 


0.17 


0.22 


24 


A/L 


0.721 


0.010 


0.056 


0.58 


0.82 


29 




0.723 


0.013 


0.061 


0.58 


0.82 


24 


L1/L 


0.653 


0.040 


0.007 


0.59 


0.75 


29 




0.565 


0.037 


0.008 


0.46 


0.65 


24 


L2/L 


0.146 


0.027 


0.005 


0.09 


0.19 


29 




0.233 


0.043 


0.009 


0.18 


0.30 


24 



faint growth lines, equivalve or with left valve 
somewhat larger, equilateral to longer anteri- 
orly. Posterior end with a radial ridge along 
posterodorsal margin, twisted to the right, 
usually slightly gaping. Periostracum thin, 
dark or colorless, silky or shiny. Bevelled es- 
cutcheon present in some species. Hinge 
weak, two cardinal teeth in each valve, lateral 
teeth absent. Ligament external, seated on a 
nymph that does not project above dorsal 
margin. In young specimens, ligament both 
external and internal; internal ligament small, 
lodged in lanceolate resilifer posterior to car- 
dinal teeth. Palliai sinus long, often of different 
length and form in each valve, partly confluent 
with palliai line. 

/Wacoma силе/руда (Scarlato, 1981) 
(Figs. 22-37, Table 4) 

Abrina cuneipyga Scarlato, 1 981 : 371 -372, 
figs. 192, 193; Kafanov, 1991: 79. 

Type material and Locality 

Holotype (ZIN 9800), Onekotan Island, Mid- 
dle Kuril Islands, 150 m, silty sand, bottom 
water temperature of 0.7°C, Coll. N. S. Spi- 
rina, 18-VII-1954 (R/V "Lebed"). 



Other Material Examined 

1 lot (ZIN 6) from Green Island (1 spec); 1 
lot from Shikotan Island (5 spec); 5 lots from 
Kunashir Island (5 spec); 5 lots from Iturup Is- 
land (ZIN 2, 1 spec; 4 lots of our material, 7 
spec); 3 lots from Onekotan Island (ZIN 1 0, 1 
spec; 2 lots of our material, 4 spec); 1 lot 
(ZIN 11) from Makanrushi Island (1 spec); 2 
lots from Fourth Kuril Strait (5 spec); 1 lot 
from Paramushir Island (1 spec); 3 lots from 
the western coast of Kamchatka, Sea of 
Okhotsk (3 spec); 1 lot from northeastern 
coast of Sakhalin Island, Sea of Okhotsk (1 
spec). Total of 35 specimens. 

Description 

(Expanded from that of Scarlato, 1981)- 
Exterior: Shell small (to 20.0 mm in length, 
west coast of Kamchatka, Sea of Okhotsk), 
ovate, of moderate height (H/L = 0.65-0.80), 
inequilateral, moderately inflated (W/L of left 
valve 0.17-0.24; W/L of right valve 0.17- 
0.22), thin, white under periostracum, smooth 
with faint growth lines, inequivalve (left valve 
slightly longer, more inflated, less twisted to 
right than right valve); posterior end distinctly 
twisted, slightly gaping. Periostracum non- 



STATUS OF ABRINA SPECIES FROM RUSSIA 



217 





EL IL PCT ACT 





FIG. 22. The hinge plate of holotypes of Abrina species from Pacific seas of Russia aided by camera lucida: 
A — Abrina sachalinica (left valve, length 15.0 mm); B — Abrina cuneipyga (left valve, 13.0 mm); C — Abrina 
tatarica (right valve, posterior cardinal tooth is broken, 9.4 mm); D — Abrina sliiaslil<otanika (right valve, 11 .8 
mm). ACT; anterior cardinal tooth; EL: external ligament; IL; internal ligament; PCT: posterior cardinal tooth. 
Bar = 1 mm. 



polished, thin, adherent, sometimes peeling 
off near beaks, gray, yellowish or pink-brown, 
thrown into numerous small wrinkles, more 
conspicuous at shell margins. Beaks small, 
somewhat projecting above dorsal margin, 
rounded, not opisthogyrate, well posterior to 
midline (A/L = 0.58-0.82). Anterior end ob- 
tusely rounded, expanded vertically. Posterior 
end pointed, angular, with a faint radial ridge 
extending from posterior portion of beaks to 
transition of posterior margin to ventral mar- 
gin. Anterodorsal margin near beaks slightly 
convex, forming small smooth obtuse angle, 
extending almost horizontally, smoothly tran- 
sitioning to rounded anterior margin. Ventral 
margin slightly curved. Posterodorsal margin 
very short, straight, steeply extending ven- 
trally, forming a hardly noticeable angle at 
transition to posterior margin, better ex- 
pressed in left valve. Posterior margin 
straight, abruptly transitioning to ventral mar- 
gin, forming slightly smoothed acute angle in 
left valve, more distinctly rounded in right 
valve. External ligament short (1/2-2/3 of 
posterodorsal margin length). 

Interior: Hinge plate very narrow, not arcu- 
ate in area of cardinal teeth. Hinge weak, with 
two cardinal teeth in each valve. In left valve, 
anterior tooth wide, deeply bifid, long, reach- 



ing edge of hinge plate; posterior tooth nar- 
row, lamellate, shorter, not reaching edge of 
hinge plate. In right valve, anterior and poste- 
rior teeth of almost same length and width 
(anterior slightly longer), reaching edge of 
hinge plate; posterior tooth sometimes slightly 
bifid. Internal ligament in adult specimens 
(> 15 mm) weakly developed, short (< 1/2 of 
hinge plate width), lodged in a small pit on 
hinge plate under beaks posterior to cardinal 
teeth; internal ligament in young specimens 
(< 8 mm) well developed, almost reaching 
edge of hinge plate, lodged in lanceolate re- 
silifer, which extends obliquely posterior to 
beaks. Anterior adductor muscle scar large, 
ovate, angular, vertically extended; posterior 
adductor scar large, rounded, shorter and 
wider than anterior scar. Palliai sinuses dis- 
tinct, of different length and shape in each 
valve. In left valve, palliai sinus long, with wide 
rounded top, reaching past midline (L1/L = 
0.59-0.75); ventral sinus branch confluent 
with palliai line for more than 1/2 of its length. 
In right valve, palliai sinus shorter (L1/L = 
0.46-0.65) with a narrow, rounded top; ven- 
tral sinus branch confluent with palliai line for 
more than 1/2 of its length. Shell polished, 
within sometimes with faint radial striae espe- 
cially noticeable along ventral margin. 



218 



KAMENEV & NADTOCHY 




FIGS. 23-37. Macoma cuneipyga (Scarlato, 1981). 23-27: Holotype of Abrina cuneipyga (ZIN 9800), 
Onekotan Island, 1 50 m, shell length 1 3.0 mm. 28: Cardinal teeth and resilifer of left valve of specimen from 
the type locality (ZIN 1 0), length 1 3.5 mm (bar = 1 mm). 29: Cardinal teeth and ligament of right valve. South 
Kuril Strait (44' 1 2'N, 1 46' 32'E), 1 03 m, valve length 3.6 mm (bar = 1 mm). 30: Cardinal teeth and resilifer of 
right valve, Iturup Island (44' 55'1 "N, 1 48''02'4"E), 1 97 m, valve length 6.6 mm (bar = 1 00 |.im). 31 : Cardinal 
teeth and ligament of right valve, Iturup Island (44 55'1"N, 148 02'4"E), 197 m, valve length 11.1 mm (bar = 
100 |.tm). 32-35: Fourth Kuril Strait, 200 m, left valve length 17.7 mm, right valve length 17.5 mm. 36, 37: 
Kunashirsky Strait, Kunashir Island (43'58'5"N, 145 27'2"E), 200 m, left valve length 15.0 mm. 



Variability 

Shell shape and proportions change little 
with age. In young specimens (< 8 mm) in con- 



trast to adults, the shell is more rounded, 
higher (H/L = 0.73-0.80) and angular: the an- 
terodorsal margin is horizontal, then bends 
(forming a smooth obtuse angle) and is 



STATUS OF ABRINA SPECIES FROM RUSSIA 



219 




Sakhalin Is. .^ ^ ^ Paramushir Is. 
#^ 



о 



Onekotan Is. 



Ogvlturupb 



Sea of ps 
Japan 1^^ 



Kimashirls. Pacific ОсвЕП 



FIG. 38. Distribution of Macoma cuneipyga (♦ — type locality). 



Steeply turned ventrally; the posterodorsal 
margin at the transition to posterior margin 
forms a distinct smooth angle; the posterior 
margin at the transition to ventral margin forms 
a pointed acute angle; the beaks one placed 
less posteriorly and only slightly rounded; the 
ridge on the posterior end of the shell is better 
expressed; in left valve, the anterior tooth is 
non-bifid and only slightly sulcate; the internal 
ligament is very well developed, reaching 
edge of hinge plate and lodged in a lanceolate 
resilifer; the palliai sinus in left valve is longer 
and reaches closer to the anterior adductor 
muscle scar (L1/L - 0.65-0.75; L2/L = 0.11- 
0.16). In adult specimens, the degree of con- 
fluence of the palliai sinus with palliai line 
varies. In left valve, ventral sinus branch con- 
fluent for 1 /4 to 1 /2 of its length with palliai line; 
in right valve, sometimes completely conflu- 
ent. 

Distribution and habitat (Figure 38) 

Green Island (43°19'3"N, 146^23'7"E); Shi- 
kotan Island (43°39'N, 147°08'E); South Kuril 



Strait, Kunashir Island (from 43°48'1"N, 
147°30'5"E to 44°12'N, 146°32'E); Blizky Is- 
land, Kunashirsky Strait, Kunashir Island (43° 
58'5"N, 145°27'2"E); Rok Bay, Iturup Island 
(44"00'N, 147°44'E); Kasatka Bay, Iturup Is- 
land (44'=527"N, 147°46.1'E); Iturup Island, 
coast of the Sea of Okhotsk (45°2176"N, 
148°187E); Drakon Cape, Iturup Island (44° 
55'N, 148°02'4"E); Onekotan Island (49°16'N, 
155°387"E); Makanrushi Island, Fourth Kuril 
Strait (from 49°32'3"N, 155°47'2"E to 49°46' 
6"N, 155°46'E); Dym Island, Paramushir Is- 
land (49°36'1"N, 156°06'E); west coast of 
Kamchatka, Sea of Okhotsk (52°45'N, 155° 
42'E; 54°00"N, 155°30'E); northeastern coast 
of Sakhalin Island, Sea of Okhotsk (143°45' 
15"N,52°29'27"E). 

This species was registered off the South 
Kuril Islands at a depth from 53 m (South Kuril 
Strait (43°51'3"N, 146°05'5"E)) to 300 m (Rok 
Bay, Iturup Island, and Kunashir Island) on 
sand and silty sand, sometimes with some ad- 
mixture of gravel and small stones; near the 
North Kuril Islands from 1 50 to 590 m on sand 
with a admixture of gravel, small stones and 



220 



KAMENEV & NADTOCHY 



TABLE 5. Macoma loveni (Jensen, 1905). Summary statistics of the shell measurements 
(mm) and indexes: L — shell length; H — height; W — width; A — anterior end length; L1 — 
maximal distance from posterior margin to the top of palliai sinus; L2 — minimal distance 
from the top of palliai sinus to anterior adductor muscle scar. Numerator indicates the sum- 
mary statistics for the left valve, denominator — for the right valve. 



Characteristics 


Mean 


SD 


SE 


Min 


Max 


n 


L 


15.56 


5.95 


0.82 


3.9 


32.5 


53 




15.36 


5.86 


0.81 


3.9 


32.0 


52 


H 


10.90 


4.04 


0.56 


3.0 


23.0 


53 




10.88 


3.97 


0.56 


3.0 


23.0 


51 


W 


3.12 


1.30 


0.18 


0.8 


7.0 


53 




2.89 


1.12 


0.16 


0.8 


6.1 


51 


A 


10.52 


3.96 


0.54 


2.4 


21.5 


53 




10.48 


3.90 


0.55 


2.4 


21.5 


51 


L1 


9.76 


3.47 


0.48 


2.8 


18.0 


53 




8.11 


3.00 


0.42 


2.2 


15.6 


52 


L2 


2.24 


1.06 


0.15 


0.4 


5.0 


53 




3.77 


1.60 


0.22 


1.0 


7.0 


52 


H/L 


0.705 


0.024 


0.003 


0.65 


0.77 


53 




0.706 


0.025 


0.003 


0.66 


0.77 


51 


W/L 


0.199 


0.016 


0.002 


0.17 


0.23 


53 




0.187 


0.016 


0.002 


0.15 


0.22 


51 


A/L 


0.675 


0.035 


0.005 


0.62 


0.80 


53 




0.667 


0.073 


0.010 


0.22 


0.80 


51 


L1/L 


0.639 


0.047 


0.006 


0.55 


0.88 


53 




0.530 


0.034 


0.005 


0.46 


0.61 


52 


L2/L 


0.139 


0.025 


0.003 


0.08 


0.20 


53 




0.243 


0.031 


0.004 


0.18 


0.32 


52 



debris; near the western coast of Kamchatka 
at a depth of 50-53 m, on silty sand at a bot- 
tom water temperature of 0.83° to 1.42°C. 
Near the Kuril Islands, it was observed at a 
bottom water temperature of 0.7-5.4°C (Scar- 
lato, 1981). 

Comparison 

M. cuneipyga is distinguished from all other 
species of Macoma by its short, pointed pos- 
terior end. in shape and proportions, this 
species is closest to M. loveni, from which it is 
distinguished in having a very narrow, pointed 
gaping posterior end, beaks less projecting 
above the dorsal margin and less posteriorly 
placed, and a narrower hinge plate that is not 
arcuate in the area of the cardinal teeth. 

Remarks 

Scarlato (1 981 ) described this species from 
of 22 specimens (10 samples) (Scarlato by 
mistake mentioned 12 samples: specimens in 
ZIN 1 and 3, 7 and 9 were taken from 2 sam- 
ples, not from 4), of which the holotype is the 
largest (shell length, 1 3.0 mm). We have stud- 
ied all of this material. In addition to the holo- 



type, only 4 specimens (samples 2, 6, 10 and 
11) could be surely identified as M. cunei- 
pyga. All others are young specimens of the 
genus Macoma that are not easily identified 
and are different from the holotype. 

In our material, we have found 31 speci- 
mens of different ages. The studies show that 
the typical characteristics of this species are 
not much prone to age or individual variation. 
A well-developed internal ligament is a juve- 
nile characteristic. We consider that A. 
cuneipyga is a separate species of Macoma. 
for which a new combination of M. cuneipyga 
is suggested. 

Macoma loveni {Jensen, 1905) 
(Figs. 12-17, 22, 39-51, Table 5) 

Tellina (Macoma) loveni Jensen, 1905: 45, 
pi. 1, figs. 5a-h (Steenstrup MS) (cited from 
Coan, 1971); Coan, 1971: 31-32, fig. 19, pi. 
8, figs. 42, 43, synonymy. 

Macoma loveni (Jensen, 1905), Coan, 
1971: 31-32, fig. 19. pi. 8, figs. 42, 43. syn- 
onymy; Bernard, 1979: 49, fig. 81; Lubinsky 
1 980: 42, pi. 9, figs. 2,5,8,11; Scarlato, 1 981 
362-363, fig. 362, synonymy; Bernard, 1983 
45; Romejko & Kamenev, 1985: 94; Baxter 



STATUS OF ABRINA SPECIES FROM RUSSIA 



221 




FIGS. 39-53. Macoma loveni {Jensen, 1905). 39-43; Holotype of Abrina sachalinica (ZIN 9853), Terpeniya 
Bay, Sakhalin Island, Sea of Okhotsk (48'22'N, 145^1 7'E), 220 m, shell length 15.0 mm. 44, 45: ZIN 106, 
Laptev Sea (76'38'N, 118"20'E), 46 m, left valve length 11.2 mm. 46, 47: Yury Island (43''11'N, 146°14'E), 
300 m, left valve length 22.0 mm. 48, 49: Paramushir Island (49'45'9"N, 1 55"58'5"E), 1 35 m, left valve length 
17.5 mm. 50, 51: Northern Sea of Okhotsk (53°34'N, 139°34'E), 130 m, left valve length 13.3 mm. 52, 53: 
Northern Sea of Okhotsk (58 •16'5"N, 142°54'3"E), 134 m, left valve length 26.1 mm. 



222 



KAMENEV & NADTOCHY 



1987: 25; Kafanov, 1991: 72; Feder et al., 
1994: 160; Kamenev, 1995: 6. 

Abrina sachalinica Scarlato, 1981 : 372, fig. 
194; Kafanov, 1991:79. 

Material Examined 

Abrina sachalinica— holotype (ZIN 9853) 
and 3 specimens of this species from the type 
locality, Terpeniya Bay, Sakhalin Island, Sea of 
Okhotsk (48°22'N, 145°17'E), 220 m, silty 
sand. Coll. L. G. Nazvich, 30-VI-1951 (R/V 
"Vityaz"); 1 lot (ZIN 4) from Hokkaido Island, 
southern Sea of Okhotsk (1 spec); 1 lot (ZIN 
5) from west coast of Kamchatka, Sea of 
Okhotsk (1 spec); /W. loveni - 1 lot (USNM 
1 08789) from Spitzbergen (2 spec); 1 lot (ZIN 
94) from Barents Sea (3 spec); 1 lot (ZIN 1 01 ) 
from Franz-Joseph Land, Barents Sea (4 
spec); 3 lots (ZIN 107) from Novaya Zemlya, 
Kara Sea (17 spec); 1 lot (ZIN 106) from the 
Laptev Sea (4 spec); 1 lot (ZIN 98) from the 
East Siberian Sea (7 spec); 2 lots from 
Vrangel Island, Chukchi Sea (2 spec); 1 lot 
(USNM 95638) from Murray Bay, Gulf of St. 
Lawrence, Atlantic Ocean (2 spec); 1 lot 
(USNM 159769) from Gulf of Maine, Atlantic 
Ocean (1 spec); 1 lot (ZIN 57) from Terpeniya 
Bay, Sakhalin Island, Sea of Okhotsk (2 
spec); 3 lots from Shikotan Island (ZIN 82, 1 
spec; 2 lots of our material, 3spec.); 1 lot (ZIN 
67) from Tatar Strait, Sea of Japan (1 spec); 1 
lot (ZIN 65) from the western coast of Sakhalin 
Island, Tatar Strait, Sea of Japan (5 spec); 1 
lot (USNH 204814) from Aniva Bay (1 spec); 

1 lot (USNM 204815) from coast of Japan 
("coast Yesso", 37°9N), Sea of Japan (2 spec); 

2 lots from Peter the Great Bay, Sea of Japan 
(2 spec); 1 lot from east coast of Sakhalin Is- 
land, Sea of Okhotsk (1 spec); 3 lots from the 
northern Sea of Japan (5 spec); 1 lot from 
Blizky Island, Kunashirsky Strait (3 spec); 2 
lots from Yury Island (2 spec); 4 lots from Itu- 
rup Island (10 spec); 1 lot from Paramushir Is- 
land (1 spec); 1 lot from Kronotsky Bay, east- 
ern coast of Kamchatka, Pacific Ocean (4 
spec);1 lot from Bering Sea (1 spec). Total of 
92 specimens. 

Description 

(Expanded from that of Coan, 1971; 
Bernard, 1979; and Scarlato, 1981) - Exte- 
rior: Shell small (to 37.0 mm, Terpeniya Bay, 
Sakhalin Island, Sea of Okhotsk), ovate, of 



moderate height (H/L = 0.65-0.77), inequilat- 
eral, moderately inflated (W/L of left valve 
0.1 7-0.23; W/L of right valve 0.1 5-0.22), thin, 
white under periostracum, smooth, with faint 
growth lines, sometimes inequivalve (left 
valve sometimes slightly longer, more in- 
flated than right valve); posterior end slightly 
twisted to right, without a gape. Periostracum 
thin, gray, grayish-olive and from light to 
bright brown with an iridescent sheen, dehis- 
cent, non-polished, easily peeled off near 
beaks, thrown into small wrinkles, better 
expressed at shell margins. Beaks small, 
somewhat projecting above dorsal margin, 
rounded, not opisthogyrate, well posterior to 
midline (A/L = 0.62-0.80). Anterior end ob- 
tusely rounded, expanded vertically. Posterior 
end slightly truncate, with faint radial ridge ex- 
tending from posterior portion of beaks to 
transition of posterior margin to ventral mar- 
gin. Anterodorsal margin slightly convex, ex- 
tending almost horizontally, smoothly transi- 
tioning to rounded anterior margin. Ventral 
margin slightly curved. Posterodorsal margin 
slightly concave, smoothly extending ven- 
trally, forming a very smooth angle at tran- 
sition to posterior margin. Posterior margin 
slightly convex, vertically extending ventrally, 
forming a smooth angle at transition to ventral 
margin. External ligament short (2/3 of pos- 
terodorsal margin length). 

Interior: Hinge plate narrow, arcuate in area 
of cardinal teeth. Hinge weak, with two cardi- 
nal teeth in each valve. In left valve, anterior 
tooth wide, bifid, long, reaching edge of hinge 
plate; posterior tooth narrow, lamellate, 
shorter, not reaching edge of hinge plate. In 
right valve, anterior and posterior teeth of al- 
most same length and width (posterior slightly 
longer), almost reaching edge of hinge plate; 
posterior tooth bifid. Internal ligament in adult 
specimens (> 1 5 mm) weakly developed, short 
(< 1/2 width of hinge plate), lodged in a small 
pit on hinge plate under beaks behind cardinal 
teeth; internal ligament in young specimens 
(to 5-10 mm) well developed, reaching almost 
to edge of hinge plate, lodged in a lanceolate 
resilifer, which extends obliquely posterior to 
beaks. Anterior adductor muscle scar large, 
ovate-angular, vertically extended; posterior 
adductor scar large, rounded, shorter and 
wider than anterior scar. Palliai sinus distinct, 
of different length and shape in each valve. In 
left valve, palliai sinus long, with wide, rounded 
top, reaching past midline (L1/L = 0.55-0.88), 
approaching anterior adductor muscle scar 



STATUS OF ABRINA SPECIES FROM RUSSIA 



223 



(L2/L = 0.08-0.20): ventral sinus branch con- 
fluent with palliai line for more than 1/3 of its 
length. In right valve, pallia! sinus shorter (L1/L 
= 0.46-0.61), with narrow, rounded top stop- 
ping just short of midline; ventral sinus branch 
confluent with palliai line for more than 1/3 of 
its length. Shell interior polished, with faint ra- 
dial striae especially noticeable along ventral 
margin. 

Variability 

Shell size, shape and proportions, as well 
as the inner shell morphology of M. loveni 
from the Arctic Ocean and the Pacific Ocean 
differ slightly. This species is much larger 
(maximal shell size: L = 37.0; H = 25.5; W = 
25.5 mm, Terpeniya Bay, Sakhalin Island, Sea 
of Okhotsk; Scarlato, 1 981 ) in the Pacific than 
in other parts of its distribution. In the Arctic 
and the North Atlantic oceans, M. loveni at- 
tains 20 mm (Coan, 1971; Bernard, 1979). 
Specimens (especially young) from the Pa- 
cific Ocean also often have a more elongate 
shell, with the beaks placed less posteriorly. 

The shell shape of M. loveni from the Pa- 
cific varies from ovate to elongate-ovate. 
Specimens from the Bering Sea and the 
northern Sea of Okhotsk have a more ovate 
shell compared to specimens from the Kuril 
Islands and the Sea of Japan (Figs. 46-51). 
The shape of the posterior end varies from 
angular and distinctly truncate to almost 
rounded. The length and width of the posterior 
end also varies. 

The degree of confluence of the palliai 
sinus with the palliai line varies in both valves. 
In some specimens, the ventral branch of the 
palliai sinus in left valve is not confluent with 
palliai line. Usually the ventral sinus branch in 
the left valve is confluent with the palliai line 
for 1/4-1/5 of its length. In the right valve, the 
ventral sinus branch is frequently confluent for 
1/3 of its length. 

In specimens from the Pacific Ocean with a 
shell length up to 10-12 mm, the resilium is 
large, reaching the edge of hinge plate and 
lodged in a lanceolate resilifer (Figs. 13, 15). 
In M. loveni from the Arctic Ocean and the 
North Atlantic Ocean, a similar internal liga- 
ment was observed only in specimens up to 
7-8 mm (Figs. 12, 16). Moreover, in speci- 
mens from the Arctic and Atlantic oceans, the 
hinge plate is relatively wider and more arcu- 
ate in the area of the cardinal teeth. In some 
specimens from throughout the distribution. 



the anterior tooth of the left valve is slightly 
bifid. 

Distribution and Habitat in the Northwestern 
Pacific (Fig. 54) 

In the Pacific Ocean, M. loveni occurs in the 
Sea of Japan -near Japan (37°9N), Possyet 
Bay, Peter the Great Bay (42°33'N, 
131 °22'7"E) ; from Olga Bay to Syurkum Cape, 
near Moneron Island, near Sakhalin Island 
(from Kholmsk City to Kitoushi Cape 
(50^=1 0'0"N, 140°57'1"E)); in the Sea of 
Okhotsk -near Sakhalin Island (Aniva Bay, 
Terpeniya Bay, east coast) and in the northern 
part of the sea (58^^16'5"N, 142°54'3"E), near 
western Kamchatka (53°35'N, 155°11'5"E); 
near the Kuril Islands -in Kunashir Strait 
(Blizky Island, 43"58'5"N, 145°27'2"E), near 
Yury Island (43''07'N, 1 46°1 2'E; 43°1 1 'N, 1 46° 
14'E), near Shikotan Island (43°29'N, 147° 
OO'E), near Iturup Island (Pacific and Sea of 
Okhotsk coasts), near Paramushir Island 
(Dym Island, 49°45'9"N, 155°58'5"E); near 
eastern Kamchatka in Kronotsky Bay, Pacific 
Ocean; in the Bering Sea (61°34'8"N, 179° 
59'7"E): near Bering Island, the Commander 
Islands. 

In the Sea of Japan, this species was ob- 
tained at a depth from 40 m (western coast of 
Sakhalin Island, Krasnogorsk Village (48° 
277"N, 1 41 °56'6"E) to 850 m (coast of Japan, 
USNM 20481 5) on sand, silty sand, sandy silt, 
often with admixture of gravel and shell debris 
at a bottom temperature of 0.4-5.7°C; in the 
Sea of Okhotsk from 16 m (Aniva Bay, 
Sakhalin Island) to 220 m (Terpeniya Bay, 
Sakhalin Island) on silt and silty sand with 
some admixture of gravel at a bottom temper- 
ature from -1.7°C(55°34'N, 139°34'E, depth 
130 m) to 3.6°C (Aniva Bay, Sakhalin Island, 
depth 16 m); near the Kuril Islands from 75 m 
(Pacific coast of Iturup Island) to 1,000 m 
(Yury Island, 43°07'N, 146°12'E) on sand, 
silty sand, sandy silt and silt often with admix- 
ture of gravel at a bottom temperature from 
1 .3°C (Shikotan Island, depth 1 00 m) to 6.4°C 
(Pacific coast of Iturup Island, 75 m); in Kro- 
notsky Bay (eastern cost of Kamchatka, Pa- 
cific Ocean) -from 134 to 150 m on silt and 
sandy silt with admixture of gravel and small 
stones at a bottom temperature of 1 .0°C 
(53°33'N, 160°09'E, depth 150 m); near the 
Commander Islands at a depth of 60-80 m on 
sand with some admixture of gravel; in the 
Bering Sea at 134 m on sandy silt with some 



224 



KAMENEV & NADTOCHY 




■ 60 



Bering Sea 

Bering Is. 



Kronotslo' Bay t^ , ^. 
o** 



ф ^ Paramushir Is. 



>\Ä\^Iturup_Is. 

Kunashiris. Pacific Ocean 



FIG. 54. Distribution of Macoma loveniin the northwestern Pacific (♦ — sampling site of holotype of Abrina 
sachalinica). 



admixture of gravel at a bottom temperature 
of 2.56°C. 

Comparison 

In shell shape and proportions, as well as in 
palliai sinus size and form, this species is 
closest to M. cuneipyga. However, M. lovenl 
differs in having a wide, truncate posterior end 
without a gape, with more projecting beaks, 
and a wider hinge plate that is arcuate in the 
area of the cardinal teeth. A comparison of 
these species with reference to other charac- 
teristics (Table 6) shows that the in left valves 
differ significantly in A/L and L1/L indices, and 
their right valves in all indices with the excep- 
tion of L2/L. Thus, in addition to the charac- 
teristics mentioned above, the beaks of M. 
loveni compared to M. cuneipyga are placed 
less posteriorly, the palliai sinus in the left 
valve is more elongate, and in the right valve 



is shorter proportional to shell length. More- 
over, as compared with M. loveni. the right 
valve of /W. cuneipyga is shorter and more in- 
flated than the left valve. This is because M. 
cuneipyga is more gaping posteriorly and be- 
cause the posterior end of right valve is more 
twisted to the right and more rounded. In M. 
loveni, the shape of posterior end of right and 
left valves is similar. 

Discriminant analysis of all lots containing 
M. loveni and M. cuneipyga showed that 
these species differ significantly (Table 7) in a 
complex of parameters (group centroids for 
M. cuneipyga and M. loveni 1.60979 and 
-0.694421, respectively). Of 73 specimens 
analysed, 65 were accurately classified 
(89.04%) (1 specimen was mistakenly classi- 
fied as M. cuneipyga and 7 as M. loveni). The 
most significant characteristics for dividing all 
specimens into two species were shell length 
(L) and shell anterior end length (A) for right 



STATUS OF ABRINA SPECIES FROM RUSSIA 



225 



TABLE 6. Results of comparison by pairs of mean values of indices of left and righit valves of Macoma 
cuneipyga and Macoma loveni using Student (T) and Kolmogorov-Smirnov (S) tests: L — shell lengthi; 
H — height; W — width: A — anterior end length; LI —maximal distance from posterior margin to the 
top of palliai sinus: L2 — minimal distance from the top of palliai sinus to anterior adductor muscle 
scar. P — probability that the index values in M. cuneipyga and M. loveni are drawn from the same 
population. 







Left valves 






Right valves 




Indices 


Test value 


P 


Test 


Test value 


P 


Test 


H/L 


1.10 


0.272 


T 


3.30 


0.002 


T 


W/L 


0.259 


0.162 


S 


0.471 


0.001 


S 


A/L 


0.581 


<0.001 


s 


0.593 


<0.001 


s 


L1/L 


0.344 


0.024 


s 


3.84 


<0.001 


T 


L2/L 


1.16 


0.246 


T 


0.94 


0.649 


T 



TABLE 7. Results of discriminant analysis for M. cuneipyga (22 spec.) and M. loveni (5^ spec): L — shell 
length: H — height: A-anterior end length: LI —maximal distance from posterior margin to the top of pal- 
liai sinus. P — probability that characteristic values in M cuneipyga ano M. /oi/en/are drawn from the same 
population. 





Discriminant 


Canonical 


Wilks 




Significant 


Standardized 


Valves 


Function 


Correlation 


Lambda 


P 


characteristics 


coefficient 


Both 


1 


0.73126 


0.465254 


<0.001 






Right 


1 


0.62539 


0.608883 


<0.001 


L 
A 


-1.20894 
1.612 


Left 


1 


0.63291 


0.599421 


<0.001 


H 
A 
L1 


4.80796 
-6.96378 
2.562 



valves, and height (H), anterior end length 
(A), and distance from the shell posterior mar- 
gin to the top of palliai sinus (LI) for left 
valves. 

Remarks 



is short in contrast to the width of the hinge 
plate (Fig. 41 ). We have found resilium of sim- 
ilar shape and size in the specimens of M. 
loveni of the same size as the holotype of A. 
sachalinica (Fig. 14). Therefore, we consider 
that A. sachalinica is a synonym of /W. loveni. 



Scarlato (1 981 ) described A. sachalinica on 
the basis of a study of 13 specimens (7 sam- 
ples), from which the holotype is the largest 
(shell length, 1 5.0 mm). We have studied all of 
this material. In addition to the holotype, only 
5 specimens (samples ZIN 1 , 4 and 5) could 
be included in this species. Other specimens 
are young specimens (< 8 mm) of Macoma of 
uncertain identity. The studies of M. loveni 
from the Arctic Ocean and the northwestern 
Pacific, as well as its descriptions, photos and 
pictures (Golikov & Scarlato, 1967; Coan, 
1971; Bernard, 1979; Lubinsky, 1980; Scar- 
lato, 1 981 ), show that in shell morphology the 
holotype of A. sachalinica is identical to spec- 
imens of M. loveni. The holotype of A. 
sachalinica has a small internal ligament that 



Macoma calcárea (Gmelin, 1791) 
(Figs. 20, 22, 55-59, Table 8) 

For complete synonymy, see Coan, 1971: 
20-21; Scarlato, 1981:356. 

Abrina tatarica Scarlato, 1981: 373-374, 
fig. 197; Kafanov, 1991:79. 

Material Examined 

A. /atenea -holotype (ZIN 9900), southern 
coast of Sakhalin Island near Kholmsk town 
(47°02'N, 142"00'5E), Sea of Japan, 46 m, 
sand with some admixture of shell debris, bot- 
tom water temperature of 3.9°C, Coll. V. A. 
Skalkin, 8-X-1949 (R/V "Toporok"); 1 lot (ZIN 



226 



KAMENEV & NADTOCHY 



TABLE 8. Macoma calcárea (Gmelin, 1791). Shell measurements (mm) and indices of specimens identi- 
fied as Abrina tatarica (ZIN 1. 5, 6): L-shell lengtfi; H -height; W- width; A — anterior end length; LI — 
maximal distance from posterior margin to the top of palliai sinus; L2 — minimal distance from the top of 
palliai sinus to anterior adductor muscle scar. Numerator indicates shell measurements and indices for the 
left valve, denominator — for the right valve. NM — not measured. Holotype of Abrina tatarica in italics. 



L 


H 


W 


A 


L1 


L2 


H/L 


W/L 


A/L 


L1/L 


L2/L 


7.0 


4.6 


1.0 


4.7 


5.1 


0.4 


0.66 


0.14 


0.67 


0.73 


0.057 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


NM 


9.4 


6.2 


1.4 


6.2 


6.8 


0.7 


0.66 


0.15 


0.66 


0.72 


0.074 


9.4 


6.2 


1.4 


6.2 


5.8 


1.6 


0.66 


0.15 


0.66 


0.62 


0.17 


9.7 


6.3 


1.6 


6.0 


7.0 


0.5 


0.65 


0.16 


0.62 


0.72 


0.052 


9.7 


6.3 


1.1 


6.0 


6.0 


1.5 


0.65 


0.11 


0.62 


0.62 


0.15 


10.5 


6.8 


1.6 


6.6 


8.0 


0.7 


0.65 


0.15 


0.63 


0.76 


0.067 


10.5 


6.8 


1.6 


6.6 


6.5 


1.5 


0.65 


0.15 


0.63 


0.62 


0.14 




FIGS. 55-63. Shells of Macoma calcárea (Gmelin, 1791) and Масогла sp. 55-58. Holotype of Abrina tatar- 
ica (ZIN 9900). southern coast of Sakhalin Island, Sea of Japan (47"02'N, 142 00'5"E), 46 m, shell length 
9.4 mm. 59. Cardinal teeth and ligament of left valve of specimen identified as Abnna tatarica (ZIN 6), Ter- 
peniya Bay, Sakhalin Island, Sea of Okhotsk (49 03'1 "N, 1 14 1 7'3"E), 1 6 m, valve length 1 0.5 mm (bar = 1 
mm). 60-63. Holotype of Abrina shiashkotanika (ZIN 9867), Shiashkotan Island, 2270 m, left valve length 
1 1 .9 mm, right valve length 1 1 .8 mm. 



5) from Kievka Bay, Sea of Japan (1 spec); 1 
lot (ZIN 6) from Terpeniya Bay, Sakhalin Is- 
land, Sea of Okhiotsk (1 spec, 1 left valve); M. 
calcárea -^0 lots from Peter the Great Bay, 
Sea of Japan (64 spec). Total of 67 speci- 
mens, 1 left valve. 



Remarks 

Abrina tatarica was described from a study 
of 16 specimens (6 samples) (Scarlato, 
1 981 ). We have studied this material and con- 
sider that in addition to the holotype only 2 



STATUS OF ABRINA SPECIES FROM RUSSIA 



227 



TABLE 9. Macoma sp. Shell measurements (mm) and indices: L — shell length; H — height; W — width; 
A — anterior end length; LI —maximal distance from posterior margin to the top of palliai sinus; L2 — min- 
imal distance from the top of palliai sinus to anterior adductor muscle scar. Numerator indicates shell 
measurements and indices for the left valve, denominator — for the right valve. NM — not measured. 
Holotype of Abrina shishkotanika in italics. 



L 


H 


W 


A 


L1 


L2 


H/L 


W/L 


A/L 


L1/L 


L2/L 


9.5 


6.1 


L5 


5^ 


5.6 


L6 


0.64 


0.16 


0.62 


0.59 


0.17 


9.5 


6.1 


1.5 


5.9 


4.9 


2.0 


0.64 


0.16 


0.62 


0.52 


0.21 


10.0 


6.9 


1.5 


6.2 


6.5 


1.5 


0.69 


0.15 


0.62 


0.65 


0.15 


10.0 


6.8 


1.5 


6.2 


NM 


NM 


0.68 


0.15 


0.62 


NM 


NM 


11.9 


7.6 


1.8 


7.4 


8.0 


2.0 


0.64 


0.15 


0.62 


0.67 


0.17 


11.8 


7.6 


1.6 


7.4 


6.8 


3.0 


0.64 


0.14 


0.63 


0.58 


0.25 



specimens (samples ZIN 5 and 6) can be re- 
ferred to this species. The other specimens 
differ significantly from holotype and are 
young specimens of different species of Ma- 
coma. Scarlato (1981) stated that the holo- 
type is the largest specimen (shell length, 9.4 
mm). However, the largest is a specimen from 
a sample ZIN 6 (shell length, 10.5 mm). 

On the basis shell morphology, the holotype 
of A. tatarica is identical to young specimens 
of M. calcárea from Peter the Great Bay, Sea 
of Japan. The internal ligament of the holo- 
type is small, very short (about 1/3 of hinge 
plate width) and lodged in a small pit on the 
hinge plate ventral to the beaks posterior to 
the cardinal teeth (Fig. 22). We have found ré- 
silia of similar shape and size in specimens of 
M. calcárea of the same size as the holotype 
of A. tatarica. Kafanov et al. (1997) presented 
the results of a morphometric analysis of a 
large sample (150 spec.) of specimens of dif- 
ferent ages (L = 14.3-39.8 mm) of M. cal- 
carea from the Bering Sea. A comparative 
analysis of these data with ours shows that 
the values of all the indices of A. tatarica are 
within the range of variation of similar indices 
(H/L = 0.657-0.773; W/L - 0.125-0.192; A/L 
= 0.503-0.662; L1/L = 0.566-0.801 ) of M. cal- 
carea from the Bering Sea. Therefore, we 
consider that A. tatarica is a synonym of M. 
calcárea. 

Macoma sp. 
(Figs. 22, 60-63, Table 9) 

Abrina shiashkotanika Scarlato, 1981 : 373, 
figs. 1 95, 1 96; Kafanov, 1 991 : 79. 

Type material and locality 

Holotype (ZIN 9867), Shiashkotan Island, 
Middle Kuril Islands, 2,270 m, sand. Coll. L. 
G. Nazvich, 7-8-VII-1951 (R/V "Vityaz"). 



Other Material Examined 



Two specimens (ZIN 1) from the type local- 



ity. 



Description 

(Expanded from that of Scarlato, 1981)- 
Exterior: Shell small (to 11.9 mm, holotype), 
elongate-ovate, of moderate height (H/L = 
0.64-0.69), inequilateral, flattened (W/L of left 
valve 0.15-0.16; W/L of right valve 0.14- 
0.16), thin, smooth, with faint growth lines, in- 
equivalve (left valve slightly longer, more in- 
flated than right valve); posterior end distinctly 
twisted to right. Periostracum thin, adherent, 
grey, thrown into small wrinkles more con- 
spicuous at shell margins. Beaks small, not 
very prominent above dorsal margin, slightly 
rounded, not opisthogyrate, posterior to mid- 
line (A/L = 0.62-0.63). Anterior end obtusely 
rounded, not expanded vertically. Posterior 
end slightly truncate, with faint (better ex- 
pressed in right valve) radial ridge extending 
posteriorly from beaks to transition of poste- 
rior margin to ventral margin. Anterodorsal 
margin straight, extending almost horizontally 
parallel to ventral margin, smoothly transi- 
tioning to rounded anterior margin. Ventral 
margin slightly curved. Posterodorsal margin 
slightly concave, smoothly extending ven- 
trally, forming a smooth angle at transition to 
posterior margin. Posterior margin slightly 
convex, almost vertical, extending ventrally 
and forming a strongly smooth angle at tran- 
sition to ventral margin. External ligament 
short (approximately 1/2 of posterodorsal 
margin length.) 

Interior: Hinge plate narrow, not arcuate in 
area of cardinal teeth. Hinge weak, with two 
cardinal teeth in each valve. In left valve, an- 
terior tooth bifid, wide, long, reaching edge of 
hinge plate; posterior tooth narrow, lamellate, 



228 



KAMENEV & NADTOCHY 



shorter, not reaching edge of hinge plate. In 
right valve, anterior and posterior teeth of al- 
most same length and width (posterior slightly 
longer); posterior tooth sometimes bifid. Inter- 
nal ligament large, reaching edge of hinge 
plate, lodged in lanceolate resilifer, which ex- 
tends obliquely posterior to beaks. Anterior 
adductor muscle large, ovate, extended verti- 
cally; posterior adductor scar large, rounded, 
shorter and wider than anterior scar. Palliai si- 
nuses indistinct, of different length and shape 
in each valve. In left valve, palliai sinus long, 
with narrow rounded top, reaching past mid- 
line (L1/L = 0.59-0.67), approaching anterior 
adductor muscle scar (L2/L = 0.15-0.17); 
ventral sinus branch confluent for 1/2 of its 
length with palliai line. In right valve, palliai 
sinus shorter (L1/L = 0.52-0.58; L2/L = 
0.21 -0.25), with a narrower rounded top; ven- 
tral sinus branch half confluent with palliai 
line. Shell inside polished, with faint radial stri- 
aes. 

Distribution and Habitat 
Known only from type locality. 

Comparison 

In shell form and proportions, this species is 
similar to M. loveni and Macoma moesta (De- 
shayes, 1855). However, it has a narrower, 
more elongate shell, with beaks placed less 
posteriorly, and a narrower posterior end 
compared to M. loveni and M. moesta of the 
same size. 

Remarks 

Abrina shiashkotanika was described 
based on the study of only three specimens (1 
sample) (Scarlato, 1981), and of which the 
holotype is the largest (shell length, 1 1 .9 mm). 
We have studied these specimens and con- 
sider them to belong to one species. In the 
description of this species, Scarlato (1981) 
mentioned that the anterior tooth of the right 
valve is deeply bifid. Our studies have shown 
that in the right valve of the holotype both 
teeth were non-bifid, but in the other two 
specimens the posterior tooth is deeply bifid. 
Probably, Scarlato (1981) made a mistake 
and described the anterior tooth of the left 
valve. Of the three specimens studied, only 
the smallest had a deeply bifid anterior tooth 
in the left valve. 



In describing this species, Scarlato (1981) 
had only a small amount of material, with all 
specimens mainly smaller than 10 mm and in 
poor condition. We think that these are young 
specimens of a Macoma, for which it is difficult 
to make an accurate species identification. 
Possibly, as a result of further studies, addi- 
tional material will be obtained representing 
all age groups of this species. It is not incon- 
ceivable that this species will be considered 
synonymous with a known species of Ma- 
coma. At present, taking into consideration 
the peculiarities of shell morphology and the 
depth from which this species was collected 
(2,270 m), as well as the lack of any additional 
material, we assume it to be a separate 
species of Macoma. 



ACKNOWLEDGMENTS 

We are very grateful to Ms. M. B. Ivanova 
(1MB, Vladivostok) for help and consultation 
during our work; to Mrs. N. V. Kameneva 
(1MB, Vladivostok) for great help during work 
on this manuscript; to Professor O. G. Kus- 
sakin. Academician of the Russian Academy 
of Sciences (1MB, Vladivostok) for consulta- 
tion during our work and comments on the 
manuscript; to Mr. K. A. Lutaenko (1MB, Vladi- 
vostok), Ms. R. N. Germon (USNM, Washing- 
ton) and T. N. Belan (FERHI, Vladivostok) for 
providing at our disposal the specimens of M. 
loveni and M. cunelpyga: \o Dr. B. I. Sirenko, 
Mr. A. V. Martynov and all collaborators of Ma- 
rine Research Laboratory (ZIN, St. Peters- 
burg) for sending to us the specimens of 
species of Abrina and help during work with 
collection of bivalve molluscs of ZIN; to Mr. A. 
Yu. Voronkov (ZIN, St. -Petersburg) for pro- 
viding the additional information on the dis- 
tribution of M. loveni: to Dr. E. V. Coan (De- 
partment of Invertebrate Zoology, California 
Academy of Sciences, San Francisco) for the 
consultations and comments on the manu- 
script; to Dr. H. Saito (NSMT, Tokyo) for send- 
ing to us the specimens of A. lunella and A. ki- 
noshltalAo Drs. T Kurozumi and E. Tsuchida 
(NHMI, Chiba) for sending to us the speci- 
mens of A. kinoshital and reprints of neces- 
sary papers; to Dr. K. Amano (Joetsu Univer- 
sity of Education, Joetsu) for sending to us the 
reprints of scientific papers necessary for our 
work; to Mr. E. V. Jakush (PRIFO, Vladivos- 
tok) for help in work with scanning micro- 
scope; to Mr. A. A. Omelyanenko (1MB, Vladi- 
vostok) for making photographs; to Mrs. A. V. 



STATUS OF ABRINA SPECIES FROM RUSSIA 



229 



Vysotskaya and Ms. T. N. Koznova (1MB, 
Vladivostok) for translating the manuscript 
into English. 

This research was supported by Grant 98- 
04-48279 from the Russian Foundation for 
Basic Research. 



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BERNARD, F R., 1983, Catalogue of the living Bi- 
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ITO, K., Y. MATANO, Y. YAMADA, & S. IGARASHI, 
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KAFANOV, A. I., D. D. DANILIN & A. V. MO- 
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613-643, in R. с MOORE, ed.. Treatise on inver- 
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valvia. Geological Society of America and Uni- 
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molluscs of the sublittoral zone of Moneron Is- 
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TSUCHIDA, E. & T. KUROZUMI, 1995, Fauna of 
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Revised ms. accepted 19 January 1999 



MALACOLOGIA, 1999, 41(1): 231-252 

TERRESTRIAL GASTROPOD RICHNESS OF CARBONATE CLIFF AND 
ASSOCIATED HABITATS IN THE GREAT LAKES REGION OF NORTH AMERICA 

Jeffrey С Nekola 

Department of Natural and Applied Sciences. University of Wisconsin - Green Bay. 
Green Bay. Wisconsin 54311, U.S.A.; nekolaj@gbms01.uwgb.edu 



ABSTRACT 

The richness of terrestrial gastropod communities in 19 different habitat types in a 1,300 x 
1 ,000 km region in the Great Lakes region of North America was analyzed using 349 0.01 -0.1 
ha samples. Sites supporting high-richness faunas (24 or more taxa) were limited in the study 
region to areas south of 45' N. Only weakly significant longitudinal gradients in richness were ob- 
served, while a significant latitudinal gradient was present. When only wooded carbonate out- 
crops were analyzed, a significant negative correlation between richness and latitude was pres- 
ent only between 44'N and 45'N. Highly significant differences in richness between habitats 
were also observed. Carbonate cliffs harbored the richest faunas, possessing a mean greater 
than 20. Approximately 25% of these sites contained 24 or more taxa, with a maximum richness 
of 34 being recorded. Algific talus slopes and lakeshore carbonate ledges were also found to 
commonly harbor faunas of 1 7 or more taxa. All of these sites are characterized by shaded, ver- 
tical exposures of carbonate bedrock. Only two of the habitats (old fields and open dunes) were 
found to never support a dozen or more co-occurring taxa. Based on these analyses, carbonate 
cliffs and related habitats in the Great Lakes region should be included among the most impor- 
tant habitats on a global scale for molluscan biodiversity 

Keywords: terrestrial gastropods, community ecology biodiversity, conservation, North Amer- 
ica, Niagaran Escarpment, cliff ecology. 



INTRODUCTION 

One of the more important components of 
community structure is richness, or the num- 
ber of co-occurring species (Peet, 1974). 
While Solem & Climo (1 985) suggest that land 
snail community richness rarely exceeds 1 2, a 
number of other studies have documented 
much higher rates of sympatry. Tropical forest 
ecosystems have the highest reported rich- 
nesses at various sample grains, with up to 40 
taxa being reported from individual sites in the 
Greater Antilles (Solem, 1984), 45 species 
from a 400 m^ area in southwestern Came- 
roon (de Winter & Gittenberger, 1998), 50 
species from a <4 ha site near Amboni Cave in 
eastern Tanzania (Emberton et al., 1997), 52 
species from a 4 ha area near Manombo, 
Madagascar (Emberton, 1995), and 56 spe- 
cies from the 4.2 ha Waipipi Scenic Reserve in 
New Zealand (Solem et al., 1 981 ). Communi- 
ties with high land snail richness have also 
been reported from the temperate zone. Up to 
24 species have been recorded from 0.01 ha 
areas in the Italian Alps (Bishop, 1980), 26 
species 0.09 ha regions in British Columbia 



coniferous forests (Cameron, 1986), 27 
species from 9.1 ha Ekholmen Island in Swe- 
den (Nilsson et al., 1 988), 39 species from ap- 
proximately 1 ha samples in SW Sweden 
(Waiden, 1981), and 44 species from an ap- 
proximately 4 ha site on Pine Mountain in Har- 
lan County, Kentucky (Emberton, 1995). How- 
ever, such sites are uncommon enough that 
Tattersfield (1996) concludes, based upon his 
review of the international literature, that sites 
with 24 or more sympatric terrestrial gastropod 
taxa in small to moderate sample sizes (ap- 
prox. <10 ha) are of global conservation im- 
portance. 

Reconnaissance of a dozen eastern Wis- 
consin limestone and dolomite cliff sites for 
glacial relict snails (Nekola et al., 1996) docu- 
mented three sites that possessed 24 or more 
co-occurring species. Previous surveys made 
from similar habitats in northeastern Iowa 
(Frest, 1982, 1987; Prest & Pay, 1981) docu- 
mented at least five additional sites that also 
equalled or exceeded this level of sympatry. If 
these surveys are reflective of such sites as a 
whole, carbonate cliffs and associated habi- 
tats in central North America could be consid- 



231 



232 



NEKOLA 




о = sites with 23 or fewer taxa 
• = sites with 24 or more taxa 

FIG. 1. Distribution of high-richness terrestrial gastropod communities within Great Lakes region. 



ered among the richest global terrestrial gas- 
tropod communities, particularly at small 
scales. Unfortunately, given the preliminary 
nature of these investigations, it was not pos- 
sible to determine whether these sites were 
simply outliers, or whether other habitats in 
this landscape harbored similarly high num- 
bers of species. 

The purpose of this paper is to investigate 
the terrestrial gastropod richness of carbon- 
ate cliffs and other habitats in a 1 ,300 x 1 ,000 
km region centered on the North American 
Great Lakes. Through this it will be possible to 
better estimate the frequency of high diversity 
assemblages from various habitats, to identify 
potential geographic gradients in species rich- 
ness over this extent, and to compare the 
richness of carbonate cliff habitats to others in 
this landscape. 



MATERIALS AND METHODS 
Study Sites 

A total of 349 areas were surveyed for their 
terresthal gastropod faunas (Fig. 1, Appendix 
I). Sites were chosen for survey if they repre- 
sented typical examples of their respective 



habitat and (except for anthropogenic habi- 
tats) were undisturbed. They ranged from 
north-central Iowa through Wisconsin, the 
Upper Peninsula of Michigan, and southern 
Ontario (including Manitoulin Island and the 
Bruce Peninsula), to central New York State 
(an extent of approximately 1,300 km), and 
from northeastern Minnesota and the Keewe- 
naw Peninsula in Michigan to southern Illinois 
(an extent of approximately 1 ,000 km). The 
bulk of collections were made along or adja- 
cent to the Niagaran Escarpment, a narrow 
zone of exposed Siluhan-age carbonates ex- 
tending from Rochester, New York, to West 
Union, Iowa. Sampling was most intensive 
(252 sites) from Drummond Island, Michigan, 
through northeastern Iowa, where an effort 
was made to sample along the Escarpment 
from all areas supporting carbonate bedrock 
outcrops. 

Collections were made from nineteen dis- 
tinct habitat types: carbonate cliffs (114 sites), 
igneous cliffs (72), rocky woodlands (21), 
lakeshore carbonate ledges (19), fens (19), 
algific talus slopes (16), tamarack wetlands 
(16), lakeshore alluvial banks (12), upland 
woods (8), lowland woods (8), white cedar 
wetlands (8), calcareous meadows (7), cob- 
ble beaches (7), alvars (6), carbonate glades 



TERRESTRIAL GASTROPOD RICHNESS IN THE GREAT LAKES 



233 



(5), igneous shorelines (4), old fields (3), 
shale cliffs (3), and open dunes (1). 

Carbonate cliffs represent 3-30 m tall, 
wooded limestone or dolomite outcrops that 
typically support moss or fern-covered 
ledges. Igneous cliffs are wooded, 2-20 m tall 
basalt, serpentine, or granite outcrops and as- 
sociated open talus slopes located on the 
Precambrian Shield of northern Wisconsin, 
the Upper Peninsula of Michigan, and north- 
eastern Minnesota. Rocky woodlands are up- 
land tracts with abundant bedrock or glacial- 
erratic boulders. Lakeshore carbonate ledges 
are <3 m tall, wooded limestone or dolomite 
outcrops that are within 3 km of the Lake 
Michigan or Lake Huron shore. Fens are peat- 
land areas formed at locations of ground- 
water discharge that maintain higher soil 
moisture and a cooler soil temperatures than 
is otherwise found in the surrounding land- 
scape (Nekola, 1994). Sampling was only 
conducted from sites in which Sphagnum 
mosses were either uncommon or lacking. Al- 
gific talus slopes are associated with me- 
chanical karst systems harboring year-round 
ice reservoirs. Air and water drainage from 
these ice caves through loose carbonate talus 
has created an unique buffered microclimate 
where soil temperatures rarely range lower 
than -10°C in winter or exceed 10°C in the 
summer, and have a more constant soil mois- 
ture as compared to surrounding forest soils. 
Such sites have been shown (Frest, 1991) to 
support populations of the glacial relict snails 
Catinella gélida (F. С. Baker, 1927), Discus 
macclintockii {F. С. Baker, 1928), Henderso- 
nia occulta (Say, 1831), and Vertigo hubrichti 
Pilsbry, 1934. Tamarack wetlands represent 
almost pure Larix laricina (DuRoi) K. Koch, 
stands that are open and support abundant 
AInus rugosa (DuRoi) Spreng, and Carex 
growth. Collections were limited to areas that 
lacked Sphagnum cover. Such sites appear 
restricted to regions with thin soils over car- 
bonate bedrock. Lakeshore alluvial banks 
represent steep wooded banks along the 
Lake Michigan shore that are developed 
into unconsolidated lacustrine material. Up- 
land woods represent wooded tracts devel- 
oped on soils lacking large rocky debris. Low- 
land woods represent deciduous forests 
found in floodplains or depressions. White 
cedar wetlands represent forested peatlands, 
dominated by Thuja occidentalis L., that 
are associated with groundwater seepage. 
Surficial soil chemistry can vary from acidic 
(where Sphagnum moss is abundant) to neu- 



tral or alkaline (where Sphagnum is largely 
absent). Litter collections were limited to the 
latter class of sites. Calcareous meadows are 
open or very sparsely forested wet meadows 
found on carbonate-rich mineral (rather than 
organic) substrate. Cobble beaches are con- 
stantly wet shoreline grassland habitats de- 
veloped on flat limestone or dolomite pave- 
ment with little or no soil development except 
in bedrock fracture planes. Alvars are similar 
to cobble beaches except that they are found 
in upland locations and become xeric by mid- 
summer. Carbonate glades are xeric grass- 
land communities with thin soils overlying 
limestone, dolomite, or calcareous shales. Ig- 
neous shoreline sites occur along the Lake 
Superior coast in the Keewenaw Peninsula 
where basalts or basalt-derived conglomerate 
sequences are exposed. They are largely 
treeless, have only limited soil development, 
and support a number of western and arctic 
disjunct vascular plants. Old fields represent 
early successional grasslands that develop 
following agricultural abandonment. Shale 
cliffs represent wooded cliffs or banks devel- 
oped into shale exposures that are often kept 
wet through constant groundwater seepage. 
Open dunes are xeric grasslands found in 
sandy soils along the Great Lakes shore. 

Field Sampling 

Documentation of the terrestrial gastropod 
communities from each site was accom- 
plished by hand collection of larger shells and 
litter sampling for smaller taxa from represen- 
tative 100-1,000 m^ regions within sites. As 
suggested by Emberton et al. (1996), sample 
collection was concentrated at places of high 
micro-mollusc density, with a constant volume 
of soil litter (approximately 4 liters) being col- 
lected from each site. For woodland sites, lit- 
ter collection was concentrated: (1) at places 
with an abundance of larger shells; (2) along 
the base of rocks or trees; (3) on soil covered 
ledges; and/or (4) at cold air vents on the cliff 
face or in the associated talus. For open sites, 
collections consisted of: (1) small blocks (ap- 
prox. 1 25 cm^) of turf; and/or (2) loose soil and 
leaf litter accumulations under or adjacent to 
shrubs, cobbles and/or boulders. 

The location of each sample was marked 
on USGS (or equivalent) 7.5 minute topo- 
graphic maps. The latitude-longitude coordi- 
nates for each was then determined through 
digitization of these maps using the ATLAS 
DRAW software package. Conversion of loca- 



234 



NEKOLA 



tions into UTM Zone 1 6 coordinates was com- 
pleted using ARCINFO. 

Laboratory Procedures 

Samples were slowly and completely dried 
in either a low-temperature soil oven (approx. 
бО-Эб^С) or in full sun in a greenhouse. Dried 
samples were then soaked in water for 3-24 
hours, and subjected to careful but vigorous 
water disaggregation through a standard 
sieve series (ASTME 3/8" (9.5 mm), 10 (2.0 
mm), 20 (0.85), and 40 (0.425 mm) mesh 
screens). Sieved sample fractions were then 
dried and passed again through the same 
sieve series. These dry, resorted fractions 
were then hand picked against a neutral- 
brown background. All shells and shell frag- 
ments were removed. 

All recovered, identifiable shells were as- 
signed to species (or subspecies) using the 
author's reference collection and the Hubricht 
Collection at the Field Museum of Natural His- 
tory. From this, the total number of taxa per 
site was determined. All specimens have 
been catalogued and are housed in collec- 
tions maintained at the University of Wiscon- 
sin-Green Bay. 

Statistical Analyses 

The frequency of high richness (24 or more 
taxa) sites was calculated across all habitats, 
and for wooded carbonate outcrops (carbon- 
ate cliffs, algific talus slopes, lakeshore car- 
bonate ledges) only, within each of the in- 
cluded states or provinces (Illinois, Iowa, 
Minnesota, Michigan, southern Ontario, and 
New York). Testing for statistical differences in 
the ratio of high vs. normal or low richness 
sites was conducted via the Pearson chi- 
square and likelihood ratio tests. The likeli- 
hood ratio test was calculated as some of the 
predicted values were sparse (< 5), complicat- 
ing interpretation of Pearson's chi-square sta- 
tistic. The asymptotic distribution of the likeli- 
hood ratio test, however, is trustworthy when 
the number of observations (349 and 149, re- 
spectively) equal or exceed the number of 
cells (1 4) by a factor of ten (Zar, 1 984). Based 
on apparent differences in the ratio of high-di- 
versity sites between northern and southern 
sections of the study area, these tests were 
repeated following exclusion of sites from Min- 
nesota, Michigan and Manitoulin Island. 

The relationship between geographic posi- 
tion and richness was graphically represented 



by plotting site richness vs. UTM N-S or UTM 
E-W coordinates for (1) all habitats, and (2) 
for wooded carbonate outcrop (carbonate 
cliff, algific slope, and carbonate lakeshore 
ledge) sites only. The central tendencies in 
these relationships were indicated though lo- 
cally weighted scatterplot smoothing (Cleve- 
land, 1979). The statistical significance of 
these relationships, and amount of variance in 
richness accounted for by geographic posi- 
tion, was estimated using least-squares re- 
gression. Cartesian UTM coordinates were 
analyzed to preclude biases originating from 
use of polar-coordinate latitude and longitude 
coordinates. 

For the N-S relationships, locally weighted 
scatterplot smoothing indicated that the re- 
sponse of richness might not be constant. 
Tests for such differences in response were 
conducted by splitting the data sets into dif- 
ferent N-S position regions, and repeating 
regression analyses separately for each. The 
p-values and r^ for each of these models were 
recorded. 

The central tendency in site richness 
among habitat types was graphically repre- 
sented via a box plot with habitats being 
sorted along the horizontal axis from the high- 
est to lowest means. In box plots the central 
line represents the median of the sample, the 
margins of the box represent the interquartile 
distances, and the fences represent 1 .5 times 
the interquartile distances. For data having a 
Gaussian distribution, approximately 99.3% 
of the data will fall inside of the fences (Velle- 
man & Hoaglin, 1 981 ). Outliers falling outside 
of the fences are shown with asterisks. Test- 
ing for significant differences in the average 
richness between habitats was conducted 
using ANOVA. 



RESULTS 

Regional Patterns 

Forty of 349 sites harbored 24 or more ter- 
restrial gastropod taxa within 0.01-0.1 ha 
samples (Fig. 1). Seven sites (4 Iowa, 1 Illi- 
nois, 1 Ontario, 1 Wisconsin) harbored 30 or 
more taxa, with a maximum richness of 34 
being observed from a Brown County, Wis- 
consin, site. Eighty-five percent of high rich- 
ness sites were found on carbonate cliff habi- 
tats. The only non-carbonate cliff habitats that 
possessed high terrestrial gastropod richness 
were three algific talus slopes (all with imbed- 
ded carbonate cliffs) and single white cedar 



TERRESTRIAL GASTROPOD RICHNESS IN THE GREAT LAKES 



235 



TABLE 1. Ratio of high richness (24 or more taxa) to medium and low richness (23 or fewer taxa) sites in 
states and provinces within study region. 







All sites 


Wooded с 
#high 


arbonate outcrops 


State or Province 


#high 


# medium-low 


# medium-low 


Illinois 

Iowa 

Michigan 

Minnesota 

New York 

Ontario 

Wisconsin 


4 
8 

1 

1 
5 
21 


5 
25 

74 

39 

5 

17 

144 


4 

7 
1 

1 
5 
19 


5 

16 
18 

2 
14 
57 






All states and provinces 


- 






All sites 


Wooded carbonate outcropi 




Pearson Statistic 

p-value 

Likelihood Ratio Statistic 

p-value 


30.763 

< 0.00005 
34.2086 

< 0.00005 


6.2791 
0.2800 
7.3618 
0.1951 




Minnesota, Michigan and Manitoulin Island s 


ites excluded 








Ail sites 




Pearson Statistic 

p-value: 

Likelihood Ratio Statistic 

p-value: 




12.2529 
0.0156 

10.4536 
0.0334 





wetland, tamarack wetland, and rocky wood- 
land sites. 

Approximately 11% of all sampled sites had 
24 or more taxa (Table 1). The frequency of 
these high richness sites in the seven states 
or provinces varied between 0% and 44% of 
all sites. These differences were significant 
(Pearson chi-square and likelihood ratio p < 
0.00005). It appeared possible that this differ- 
ence may be attributed to the much lower fre- 
quency of high-richness sites in Minnesota, 
Upper Peninsula of Michigan, and Manitoulin 
Island. However, differences in the frequency 
of high-richness sites was found to remain 
marginally significant (Pearson chi-square p = 
0.01 56; likelihood ratio p = 0.0334), even after 
removal of the most northern regions from 
analysis. This marginal significance is appar- 
ently related to a lowered frequency of high- 
richness sites in Wisconsin. 

Approximately 25% of all wooded carbon- 
ate outcrop sites harbored high richness com- 
munities. The frequency of these in the five 
states or provinces ranged between 5% and 
44% (Table 1), and occurred over the entire 
extent of the sample region (Fig. 1). While 
carbonate cliff sites of high-richness ap- 
peared scarce in the Upper Peninsula of 
Michigan and Manitoulin Island, Pearson's 
chi-square (p = 0.2800) and the likelihood 



ratio (p = 0.1951) tests demonstrated that at 
the state or province scale, these differences 
were non-significant. 

Geographic Gradients 

Only a marginally significant (Fig. 2; p = 
0.031 ) and weak {r^ = 0.01 3) trend was found 
between richness and E-W UTM position 
across all habitats. This relationship was 
found to not be significant when only wooded 
carbonate outcrops were analyzed (Fig. 3; p = 
0.106). The relationship between richness 
and N-S UTM location, however, was found to 
be stronger and more significant both for all 
habitats (p < 0.0005; л^ - 0. 1 88) as well as for 
wooded carbonate outcrop sites only (p = 
0.003; r^ - 0.059), with northerly sites pos- 
sessing lower richness than southerly sites. 

The shape of the locally weighted scatter- 
plot smoothing lines for the N-S relationships, 
in conjunction with additional regression 
analyses, demonstrate that this pattern is not 
constant over the study region. Across all 
habitats, only a weak {r^ = 0.063) but statisti- 
cally significant (p = 0.001) relationship was 
observed south of 5,000 km while north of this 
position this same relationship was more sig- 
nificant (p < 0.0005) and over 4V^ times 
stronger {r^ = 0.289; Fig. 2). When only 



236 



NEKOLA 



E-W Variation 




35 
30 

25 \- 
20 
15 
10 
5 



N-S Variation 

"T 1 r 



■ 


All sites 




r2 - 0.188 




p < 0.0005 


.."...■ 


Sites <5000km N: 


'..'..11 .".■ 


n = 181 


- • 


r2 - 0063 


""fl^v •.;••■ " 


p - 0,001 


.-•■VT^ . 


Srtes >5000km N 


... •.•.■-• .;' 


n = 168 




1-2 - 0.289 




p < 0.0005 



500 1000 1500 

E-W UTM Coofdrate (1000 meter uits) 



4000 4200 4400 4600 4800 5000 5200 6400 
N-S 1ЛМ Coordinate (1000 meter uiüs) 



FIG. 2. Relationship of terrestrial gastropod richness to E-W an(j N-S UTM location across all 19 habitat 
types. A locally weighted scatterplot smoothing line has been fitted to each relationship. 



E-W Variation 




35 
30 

25 h 
20 



N- 


-s 


Variation 










All sites 
r2 - 0059 
p - 0003 


' ~7 


-— 


-^^Щ,- 


Sites <49(X)km N: 
n = 58 
r2 - 0,010 
p - 0.456 


- 






Sites from 4900-5000 km N 
n = 44 
r2 - 0,211 
p - 0002 


- 






Sites >5000km N. 
n = 47 
r2 - 0002 
p - 0742 



500 1000 1500 

E-W UTM Cocrdirete (1000 meter uils) 



N-S UTM Coordinate (1000 meter irits) 



FIG. 3. Relationship of terrestrial gastropod richness to E-W and N-S UTM location for wooded carbonate 
outcrop sites (carbonate cliffs, algific talus slopes, and lakeshore carbonate ledges). A locally weighted scat- 
terplot smoothing line has been fitted to each relationship. 



wooded carbonate outcrops were considered, 
no relationship was apparent south of 4900 
km (roughly 44" N; p = 0.456) and north of 
5,000 km (approx. 45" N; p = 0.742). How- 
ever, a significant (p = 0.002) and moderately 
strong (r = 0.221) relationship was apparent 
between 4,900 and 5,000 km (Fig. 3). 

Habitat Patterns 

Comparison of site richness values demon- 
strate striking differences among the 19 sam- 
pled habitat types (Fig. 4). Carbonate cliffs 
were the richest habitats sampled, possess- 
ing a mean score approaching 21 . Algific talus 
slopes and lakeshore carbonate ledges fol- 
lowed, having mean richness scores exceed- 
ing 1 7. Both carbonate cliffs and algific slopes 
had upper data fences that exceeded 30 
species per site. Rocky woodlands, carbonate 



glades, calcareous meadows, white cedar 
wetlands and fens had mean richness scores 
ranging from 1 5.3 to 1 3.9. Igneous shorelines, 
tamarack wetlands, lakeshore alluvial banks, 
lowland woods and cobble beaches had 
mean richness scores ranging from 12 to 
1 0.6. Igneous cliffs, alvars, shale cliffs, upland 
woods, old fields, and open dunes all had 
mean richness scores of less than 1 0. ANOVA 
showed these differences to be highly signifi- 
cant (p < 0.0005), with almost 50% of ob- 
served variance in richness being accounted 
for by habitat type. 

DISCUSSION 

Regional Species Richness Patterns 

Although Solem & Climo (1985) stated that 
land snail community richness rarely exceeds 



TERRESTRIAL GASTROPOD RICHNESS IN THE GREAT LAKES 

П — \ — \ — I I \ I — \ — I — I — \ — I — г 



237 




г2 = 0.473 
p < 0.0005 



Habitat Type 

FIG. 4. Box-plot diagrams of terrestrial gastropod richness across 1 9 habitat types. Habitats are sorted along 
the horizontal axis from highest to lowest mean scores. 



1 2 taxa, fully 232 of the sites inventoried (66% 
of the total) equalled or exceeded this level. 
Sites with 1 2 or more taxa were found in 1 7 of 
the 19 sampled habitats. Only old field and 
open dune habitats never equalled or ex- 
ceeded this richness level. It is not clear 
whether Solem & Climo were unnecessarily 
pessimistic about terrestrial gastropod com- 
munity richness, or if the Niagaran Escarp- 
ment in the Great Lakes region possesses 
uniquely rich community assemblages. While 
it seems likely that the former is true, it should 
be mentioned that sites with a dozen or more 
co-occurring terrestrial gastropod taxa may 
be less frequent in other landscapes. For ex- 
ample, in this study only 34% of northern Wis- 
consin, western Upper Peninsula and north- 
eastern Minnesota sites had 12 or more 
co-occurring taxa. Burch (1956) reported 
maximum mean richness of nine taxa per site 
in the eastern piedmont and coastal plain of 
Virginia. Clarke et al. (1968) found no more 
than nine co-occurring taxa in New Brunswick 
forests. In their survey of 1 89 sites (many with 
carbonate substrates) in the Black Hills of 
South Dakota, Prest & Johannes (1993) re- 
port only seven (less than 4%) that harbor a 
dozen or more taxa. Cowie et al. (1 995) found 



that no more than 1 2 taxa coexisted within ap- 
proximately 100 m^ samples on Hawaiian 
vegetated lava flows. It will be necessary to 
expand these analyses to additional land- 
scapes with a greater diversity of geological 
substrates and ecological histories to deter- 
mine whether the terrestrial gastropod com- 
munities of the Great Lakes are uniquely rich, 
or if our definition of what constitutes a 
species-rich community must be expanded. 

Little or no variation in richness was re- 
corded over most of the region. Only a very 
weak longitudinal trends were identified, and 
only north of UTM 5,000 were strong latitudi- 
nal trends observed. When only wooded car- 
bonate outcrops (carbonate cliffs, algific talus 
slopes, and lakeshore carbonate ledges) 
were considered, the significant latitudinal 
trend in richness was restricted to sites falling 
between UTM 4,900 and 5,000 km N (or 
roughly 44- N to 45" N). Similarly, the occur- 
rence frequency of high richness sites (using 
Tattersfield's criteria of 24 or more taxa) was 
found to only weakly differ between Illinois, 
Iowa, southern Ontario, New York, and Wis- 
consin. However, in Minnesota, the Upper 
Peninsula of Michigan, and Manitoulin Island 
this ratio was over ten times lower across all 



238 



NEKOLA 



habitats, and at least five times lower on 
wooded carbonate outcrops. 

A number of factors could be responsible 
for the significantly lower richness levels ob- 
served in the northern reaches of the study 
area. At least some of this decrease in rich- 
ness may be due to lower-Ca and pH soils as- 
sociated with igneous (rather than carbonate) 
bedrock on northern sites. However, this can 
not explain the significant reductions in rich- 
ness observed on the northern-most wooded 
carbonate outcrop sites in the Upper Penin- 
sula and Manitoulin Island. Perhaps the low 
richness values are related to the greater iso- 
lation of these sites, because they are sepa- 
rated from other carbonate outcrops by the 
waters of Green Bay and Georgian Bay as 
well as the acidic soils of the Precambrian 
Shield. Additional research will be necessary 
to tease apart the differential roles played by 
contemporaneous and historical processes in 
determining regional terrestrial gastropod 
richness patterns. 

Habitat-Specific Species Richness Patterns 

Significant differences were observed 
among the 16 sampled habitats, with carbon- 
ate cliffs possessing the highest average num- 
ber of taxa per site. Over one-half of such sites 
harbored 21 or more species. Other habitats 
found to harbor rich assemblages of species 
included algific talus slopes, lakeshore car- 
bonate ledges, rocky woodlands, carbonate 
glades, calcareous open meadows, white 
cedar wetlands, and fens. All of these habitats 
are associated with calcareous substrata, ei- 
ther in the form of exposed bedrock, boulders, 
talus, wet marl, calcareous alluvium or nutri- 
ent-rich peat. The lowest richness habitats 
were, in general, associated with more acidic 
substrata such as igneous outcrops, sand 
dunes, or exposed alluvium. However, this 
pattern is not without exception as low-rich- 
ness cobble beach and alvar faunas are de- 
veloped on carbonate outcrops. 

Carbonate Cliffs as Terrestrial Gastropod 
Diversity Hot Spots 

Wooded carbonate cliffs, on average, sup- 
port the highest number of terrestrial gastro- 
pod taxa within any habitat in the study re- 
gion. The richest 5% of these support 29 or 
more taxa, with a maximum of 34 taxa being 
recorded. Such sites appear to be among the 
richest reported globally from 1 ha or smaller 



quadrats. Waiden (1981) observed up to 39 
taxa from 1 ha quadrats in wooded talus 
slopes in Sweden, while Tattersfield (1996) 
identified up to 33 taxa per one-sixth hectare 
samples from Kenyan rain forest. Other pub- 
lished reports of terrestrial gastropod richness 
from 0.1 ha or less quadrats (e.g., Schmid, 
1966; Bishop, 1980;Nilssonetal., 1988;Getz 
& Uetz, 1994; Cowie et al., 1995; de Winter & 
Gittenberger, 1998) have reported no more 
than 45 co-occurring taxa. Maximum richness 
in Great Lakes carbonate cliff sites is also 
within 25% of the richest known North Ameri- 
can site (at Pine Mountain, Kentucky; Ember- 
ton, 1995). 

Further research will be necessary to deter- 
mine if the richness levels of carbonate cliffs 
in the Great Lakes region are unique, or if sim- 
ilar levels are present in other landscapes. 
Research from other regions (e.g.. New South 
Wales, Australia; Stanisic, 1997; Germany: 
Schmid, 1966; Scotland; Cameron & Green- 
wood, 1 991 ; Sweden: Waiden 1 981 ) indicates 
that maximum terrestrial gastropod richness 
frequently occurs on wooded carbonate out- 
crops. Based on this current and previous re- 
search it seems likely that carbonate cliffs will 
be found to be among the most important 
habitats for molluscan biodiversity on a global 
scale. 



ACKNOWLEDGEMENTS 

Useful comments on earlier versions of this 
manuscript were provided by Douglas Larson, 
John Slapcinsky, George Davis, and two 
anonymous reviewers. Donna Boelk, Patrick 
Comer, Gary Fewless, John Gerrath, Mike 
Grimm, Doug Larson, and Mary Standish 
helped identify potential survey sites, and as- 
sisted in field collection. Matt Barthel, Candice 
Kasprzak, Pete Massart, Chela Moore, Eric 
North, and Tamara Smith processed many 
soil litter samples, and assisted in field collec- 
tion. Assistance in litter sample processing 
was also provided by students participating in 
the Land Snail Ecology Practicum at the Uni- 
versity of Wisconsin -Green Bay. Funding 
was provided by the Door County Office of the 
Wisconsin Chapter of The Nature Conser- 
vancy, a Louis Almon grant (administered by 
the Wisconsin Academy of Sciences, Arts, 
and Letters), three Cofrin Arboretum grants 
(administered by the Cofrin Arboretum Com- 
mittee at the University of Wisconsin -Green 
Bay), the U.S. Fish and Wildlife Service, and 



TERRESTRIAL GASTROPOD RICHNESS IN THE GREAT LAKES 



239 



the Small Grants Program of the Michigan 
Department of Natural Resources. Funding 
for the survey of Minnesota sites was re- 
ceived from the Minnesota Nongame Wildlife 
Tax Checkoff and Minnesota State Park Na- 
ture Store Sales through the Minnesota De- 
partment of Natural Resources Natural Her- 
itage and Nongame Research Program. 

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Revised ms. accepted 18 March 1999 



APPENDIX I 

Name, location, habitat type, and terrestrial gastropod richness of sample sites 

Site Name Location Habitat Type Richness 





ILLINOIS 






Calhoun County 








Franklin Hill 


90°36'38"W, 39°3'57"N 


Carbonate Cliff 


28 


Jackson County 








Kings Ferry Bluff Base 


89°26'16"W, 37°36'36"N 


Carbonate Cliff 


27 


Kings Ferry Bluff Crest 


89°26'15"W, 37°36'36"N 


Carbonate Cliff 


19 


Madison County 








Cliffton Terrace 


90°13'36"W, 38"54'51"N 


Carbonate Cliff 


20 


Monroe County 








Fults Reserve 


90''11'15"W, 38=9'19"N 


Carbonate Cliff 


20 


Fountain Gap 


90°15'33"W, 38°22'36"N 


Carbonate Cliff 


30 


Pike County 








Shewhart Bluff 


9Г6'48"\Л/, 39"39'N 


Carbonate Cliff 


21 


Randolph County 








Prairie du Rocher 


90°11'56"W, 38 6'28"N 


Carbonate Cliff 


20 


Chester 


89°53'6"W, 37"56'42"N 


Carbonate Cliff 


25 



Bremer County 

Brayton-Horsley 
Buchanan County 

Rowley West 
Chickasaw County 

Stapelton Church 
Clayton County 

Bixby East 

Buck Creek 4 

Buck Creek Tributary 1 

Buck Creek Tributary 2 

Buck Creek Tributary 3 

Buck Creek Tributary 4 

Buck Creek Tributary 5 

Elkader South 

South Cedar 2 

South Cedar 3 
Delaware County 

Backbone West 

Elk River East 
Des Moines County 

Iowa Ammunition Plant 
Dubuque County 

Roosevelt Road 



IOWA 

92°6'28"W, 42°48'35"N 

91°54'39"W, 42°22'15"N 

92"6'14"W, 43°1'35"N 

91°23'56"W, 42-40'28"N 
91°11'23"W, 42'51'35"N 
91°10'55"W, 42 51'47"N 
91°10'55"W, 42= 51'56"N 
91°10'55"W, 42 52'5"N 
91°10'58"W, 42 52'1"N 
91°11'2"W, 42 51'52"N 
91°23'45"W, 42 50'23"N 
91°14'38"W, 42 49'58"N 
91°14'23"W, 42M9'51"N 

91°33'43"W, 42^37'5"N 
91°17'27"W, 42°37'42"N 

91°17'16"W, 40"46'27"N 

90"44'30"W. 42' 32'55"N 



Fen 



Fen 



Fen 



14 



20 



17 



Algific Talus Slope 


16 


Algific Talus Slope 


13 


Algific Talus Slope 


21 


Algific Talus Slope 


10 


Algific Talus Slope 


25 


Algific Talus Slope 


31 


Algific Talus Slope 


23 


Algific Talus Slope 


11 


Algific Talus Slope 


20 


Carbonate Cliff 


30 


Carbonate Cliff 


26 


Algific Talus Slope 


32 


Rocky Woods 


26 


Carbonate Glade 


17 



TERRESTRIAL GASTROPOD RICHNESS IN THE GREAT LAKES 



241 



Fayette County 








Brush Creek Canyon 1 


91°41'27"W, 42°47'2"N 


Algific Talus Slope 


11 


Brush Creek Canyon 2 


91°4r20"W, 42°46'46"N 


Carbonate Cliff 


27 


Floyd County 








Juniper Hill Shale Glade 


92"59'2"W, 43°3'10"N 


Carbonate Glade 


12 


Franklin County 








Hampton East Glade 


93'^8'13'Ж 42°43'41"N 


Carbonate Glade 


14 


Howard County 








Larkin Bridge East 


92°5'8"W, 43- 29'32"N 


Algific Talus Slope 


21 


Jackson County 








Hamilton Glade 


90°34'8"W, 42"4'23"N 


Carbonate Glade 


14 


Lytle Creek 6 


90°45'21"W, 42 = 15'34"N 


Algific Talus Slope 


20 


Lytle Creek 16 


90°45'28"W, 42^-1 5'40"N 


Algific Talus Slope 


16 


Maquoketa Caves 


90°46'22"W, 42°7'3"N 


Carbonate Cliff 


23 


Pine Creek 


90°50'41"W, 42"8'27"N 


Carbonate Cliff 


31 


Jones County 








Canton Glade 


90°59'52"W, 42' 10'46"N 


Carbonate Glade 


18 


Pictured Rocks 


91 °6' 18"W, 42^12 '28"N 


Carbonate Cliff 


23 


Linn County 








Dark Hollow 


91°30'W, 4r53'54"N 


Carbonate Cliff 


20 


Paris Fen 


9r35'41"W, 42°13'39"N 


Fen 


11 


Winneshiek County 








Bluffton West 2 


91°55'16"W, 43°24'10"N 


Algific Talus Slope 


17 


Heritage Farm 


91°47'59"W, 43"22'55"N 
MICHIGAN 


Algific Talus Slope 


20 


Chippewa County 








Bass Cove Cobble Beach 


83"32'45"W, 45°55'10"N 


Cobble Beach 


11 


Bass Cove Upland 


83"32'46"W, 45"55'24"N 


Rocky Woods 


11 


Hill Lake East 


84°30'51"W, 46°7'48"N 


Carbonate Cliff 


19 


Huron Bay 


83°45'17"W, 45°57'12"N 


Cobble Beach 


11 


Maple Hill 


84°46'55"W, 46"9'34"N 


Carbonate Cliff 


25 


Marble Head Center 


83°28'28"W, 45°59'3"N 


Carbonate Cliff 


18 


Marble Head North 


83=28'30"W, 45°59'17"N 


Carbonate Cliff 


19 


Marble Head South 


83=28 '35"W, 45°58'46"N 


Carbonate Cliff 


22 


Maxton Plains Center 1 


83°39'48"W, 46°4'26"N 


Alvar 


8 


Maxton Plains Center 2 


83"39'24"W, 46°4'44"N 


Tamarack Wetland 


8 


Poe Point 


83°38'30"W, 46°6'10"N 


Lakeshore Carbonate 
Ledge 


14 


Prentiss Bay 


84 13'49"W, 45°59'25"N 


Tamarack Wetland 


13 


Scott Bay 


83°40'1"W, 46°3'31"N 


Fen 


12 


Scott Quarry 


84°50'4"W, 46°10'43"N 


Carbonate Cliff 


23 


Seastone Point 


83°45'34"W, 46°1'21"N 


Carbonate Cliff 


22 


Spencers Mountain Lower 


84 56'39"W, 46"11'33"N 


Rocky Woods 


19 


Spencers Mountain Upper 


84"56'43"W, 46°11'29"N 


Carbonate Cliff 


16 


Tourist Road 


83°43'48"W, 46°1'44"N 


Carbonate Cliff 


18 


Delta County 








Ansel's Point 


86°34'26"W, 45°48'12"N 


Carbonate Cliff 


23 


Burnt Bluff 


86°42'39"W, 45°41'11"N 


Carbonate Cliff 


19 


Cooks Ridge 


86"29'27"W, 45"57'59"N 


Rocky Woods 


20 


Fayette St. Park 


86"39'46"W, 45°43'40"N 


Carbonate Cliff 


18 


Garden Bluff 


86^37 '48"W, 45°46'47"N 


Carbonate Cliff 


17 


Garden Corners 


86"32'4"W, 45 53'23"N 


Tamarack Wetland 


11 


Garden Peninsula Alvar 


86°38'38"W, 45°39'48"N 


Alvar 


5 


Goully Harbor 


86°36'47"W, 45°46'42"N 


Carbonate Cliff 


21 



242 



NEKOLA 



Jacks Bluff 

Kregg Bay Northwest 1 
Kregg Bay Northwest 2 
016 West 
Pt. Detour 1 
Pt. Detour 2 
Pt. Detour 3 

South River Bay 
Gogebic County 

Bessemer NE 

Copper Mountain 

IVlt. Zion Park 
Keewenaw County 

Agate Point 

Brockway Mountain 

Cliff Range North 1 

Cliff Range North 2 

Cliff Range South 

Cliffton 

Copper Harbor Shore 

Copper Harbor Marina 

Dans Point 

Delaware Gap 

Eagle Harbor 

Grand Marais Harbor 

Mt. Bohemia 
Luce County 

McLeod Hill 
Mackinac County 
Dinkey Line Road 

Gamble Road 
Greene Cedar 
Kenneth Road 
Martineau Creek 
McCann High School 
Point St. Ignace 
Round Lake 1 
Round Lake 2 
Summerby Swamp 
Townhall Road 
Ontonagon County 
Adventure Mountain 
Cloud Peak 
Cranberry River Hill 
Cuyahoga Peak 
Miscowawbic Peak 
Norwich Mountain 
Rodgers Cedar Swamp 
Summit Peak Lower 
Summit Peak Upper 
School Craft County 
Manistique North 
Merwin Creek 
Seul Choix Point 1 
Seul Choix Point 2 



86°3r33"W, 45 5riO"N 


Lakeshore Alluvial 
Bank 


21 


86°32'10"W, 45°43'13"N 


Alvar 


9 


86°31'59"W, 45°42'39"N 


Cobble Beach 


1 


86'=43'1"W, 45'40'17"N 


Rocky Woods 


14 


86^37 '47"W, 45^38 '3"N 


Alvar 


9 


86°36'21"W, 45°36'25"N 


Cobble Beach 


15 


86°36'25"W, 45°36'16"N 


Lakeshore Carbonate 
Ledge 


19 


86°37'19"W, 45°45'22"N 


Carbonate Cliff 


20 


90°2'24"W, 46°29'31"N 


Igneous Cliff 


16 


90°5'16"W, 46°36'5"N 


Igneous Cliff 


11 


90°10'8"W, 46°28'37"N 


Igneous Cliff 


2 


88°1 '4"W, 47°28'38"N 


Igneous Lakeshore 


14 


87°58'15"W, 47°27'48"N 


Igneous Cliff 


6 


88°15'11"W, 47°23'55"N 


Igneous Cliff 


11 


88°15'9"W, 47°23'55"N 


Igneous Cliff 


13 


88°19'50"W, 47°2r36"N 


Igneous Cliff 


12 


88°19'23"W, 47°21'54"N 


White Cedar Wetland 


12 


87°54'15"W, 47°28'36"N 


Igneous Lakeshore 


10 


87°54'14"W, 47°28'19"N 


White Cedar Wetland 


11 


87°58'37"W, 47°28'47"N 


Igneous Lakeshore 


15 


88°6'57"W, 47 25'29"N 


Igneous Cliff 


13 


88°8'54"W, 47°27'39"N 


Igneous Lakeshore 


9 


88°7'9"W, 47°27'37"N 


Igneous Cliff 


15 


88°0'51"W, 47°23'34"N 


Igneous Cliff 


12 


85°15'37"W, 46°15'17"N 


Rocky Woods 


23 


85°14'16"W, 46°10'49"N 


White Cedar Wetland 


7 


84°45'W, 46°7'42"N 


Rocky Woods 


21 


84°51'51"W, 46°1'41"N 


White Cedar Wetland 


14 


84°50'34"W, 46°5'50"N 


Carbonate Cliff 


19 


84°43'11"W. 45°59'8"N 


Fen 


14 


84°43'36"W, 45=51 '48"N 


Rocky Woods 


12 


84°42'34"W, 45°57'8"N 


Rocky Woods 


18 


84°52'31"W, 45^57' 18"N 


Fen 


3 


84°52'31"W, 45"57'11"N 


White Cedar Wetland 


13 


84°47'43"W, 45^58' 15"N 


Fen 


19 


85°10'28"W, 46"8'19"N 


White Cedar Wetland 


22 


89°4'51"W, 46^46' 17"N 


Igneous Cliff 


18 


89°43'58"W, 46°48'51"N 


Igneous Cliff 


16 


89°26'28"W, 46=42'47"N 


Igneous Cliff 


8 


89°41'59"W, 46°48'56"N 


Igneous Cliff 


17 


89°48'47"W, 46°47'N 


Igneous Cliff 


16 


89°22'54"W, 46°39'37"N 


Igneous Cliff 


12 


89°44'3"W. 46°33'7"N 


White Cedar Wetland 


7 


8946'27"W, 46°44'55"N 


Igneous Cliff 


3 


89°46'25"W, 46°44'48"N 


Igneous Cliff 


5 


86°16'W, 46°1'37"N 


Carbonate Cliff 


17 


86°5'40"W, 46°2'30"N 


Rocky Woods 


13 


85°54'50"W, 45=55' 12"N 


Cobble Beach 


19 


85°54'53"W, 45°55'14"N 


Rocky Woods 


11 



TERRESTRIAL GASTROPOD RICHNESS IN THE GREAT LAKES 
MINNESOTA 



243 



Cook County 








Caribou Lake E 


90°40'1"W, 47M2'23"N 


Igneous Cliff 


9 


Caribou Lake N 


90°40'40"W, 47 42'46"N 


Igneous Cliff 


6 


Carlton Peak 


90°51'22"W, 47''35'9"N 


Igneous Cliff 


2 


Cascade River Cedars 


90°31 '32"W, 47 '44'36"N 


Igneous Cliff 


14 


Cascade River Cliff 


90°32'15"W, 47M3'28"N 


Igneous Cliff 


12 


Iceland Fen 


90°7'3"W, 47^47'44"N 


Tamarack Wetland 


11 


John Lake 


90°3'29"W, 48^3'56"N 


Igneous Cliff 


9 


Lake Cliff 


90°58'23"W, 47 29'40"N 


Igneous Cliff 


5 


Lutsen Mountains 


90°41'54"W, 47 '40'49"N 


Igneous Cliff 


4 


McFarland Lake Cliff 


90°5'25"W, 48 ■3'34"N 


Igneous Cliff 


6 


McFarland Lake Talus 


90°5'18"W, 48°3'36"N 


Igneous Cliff 


6 


Moose Mt. Cliff 


90°44'9"W, 47^'39'7"N 


Igneous Cliff 


11 


Mt. Josephine Cliff 


89°39'14"W, 47°58'58"N 


Igneous Cliff 


14 


Mt. Josephine Talus 


89°39'14"W, 47"58'50"N 


Igneous Cliff 


11 


Oberg Mountain 


90°46'38"W, 47^37'44"N 


Igneous Cliff 


5 


Pine Lake 


90°6'7"W, 48°3'3"N 


Igneous Cliff 


12 


Pine River Road 


90°18'4"W, 47°54'59"N 


Igneous Cliff 


10 


Poplar River 


90°43'15"W, 47°39'55"N 


Igneous Cliff 


13 


Poplar River Overlook 


90°43'34"W, 47°40'13"N 


Igneous Cliff 


6 


Port of Entry Cliff 


89^37' 12"W,47°59'51"N 


Igneous Cliff 


9 


Port of Entry Talus 


89°37'6"W, 47=59'47"N 


Igneous Cliff 


9 


Portage Brook 


90°1 '24"W, 48°0'2"N 


Igneous Cliff 


6 


Sawbill Road N Cliff 


90°51 '37"W, 47''35'50"N 


Igneous Cliff 


10 


South Fowl Lake 


90°0'28"W, 48^2'27"N 


Igneous Cliff 


11 


Sugarloaf Cove 


90°58'50"W, 47^29' 12"N 


Igneous Cliff 


7 


Temperance River Road 


90°53'3"W, 47°34'6"N 


Igneous Cliff 


8 


Temperance River Upland 


90^52 '22"W, 47^34 '22"N 


Igneous Cliff 


14 


Timber Creek 


90°15'19"W, 47^'53'53"N 


Igneous Cliff 


10 


LaKe County 








Day Hill 


91°22'59"W, 47=11'36"N 


Igneous Cliff 


10 


Finland Forest 


91 °5'25"W, 47^33' 13"N 


Igneous Cliff 


7 


Goldeneye Lake Cliff 


91°3'55"W, 47°35'53"N 


Igneous Cliff 


6 


Goldeneye Lake Talus 


91°3'48"W, 47°35'50"N 


Igneous Cliff 


7 


Manitou River Falls 


9r4'27"W, 47"26'44"N 


Igneous Cliff 


12 


Sawmill Creek 


91 °9'43"W, 47^24' 15"N 


Igneous Cliff 


3 


Water Tanks 


91°17'45"W, 47°17'42"N 


Igneous Cliff 


7 


St. Louis County 








Chester Bowl 


92°5'55"W, 46°48'48"N 


Igneous Cliff 


10 


Hawk Ridge Sanctuary 


92°1 '55"W, 46°50'49"N 


Igneous Cliff 


13 


Skyline Drive West Cliff 


92°10'6"W, 46°45'47"N 


Igneous Cliff 


5 


Skyline Drive West Talus 


92"10'6"W, 46°45'43"N 
NEW YORK 


Igneous Cliff 


14 


Cayuga County 








Fillmore Glen State Park 


76°23'52"W, 42°41 '45"N 


Shale Cliff 


3 


Madison County 








Cazenova Gorge 1 


75°50'41"W, 42°58'49"N 


Carbonate Cliff 


25 


Cazenova Gorge 2 


75°50'49"W, 42°58'49"N 


Carbonate Cliff 


20 


Niagara County 








Gasport Ravine 


78°35'2"W, 43"10'52"N 


Carbonate Cliff 


9 


Schuyler County 








Watkins Glen State Park 


76=53'24"W, 42°22'16"N 


Shale Cliff 


14 



244 


NEKOLA 






Wyoming County 








Letchworth State Park 


78"1'26"W, 42''35'41"N 
ONTARIO 


Shale Cliff 


10 


Bruce County 








Grotto Trail 


81°31'19"W, 45°14'38"N 


Carbonate Cliff 


19 


Lions Head 


81°13'4"W, 45 0'28"N 


Carbonate Cliff 


20 


Overhanging Point 


81°31'51"W, 45°14'40"N 


Carbonate Cliff 


17 


Grey County 








Inglis Fails 


80°56'2"W, 44°3r50"N 


Carbonate Cliff 


28 


Metcalfe Rock 


80°26'31"W, 44°25'3"N 


Carbonate Cliff 


22 


Skinners Bluff 


80°59'31"W, 44°47'36"N 


Carbonate Cliff 


30 


Halten County 








Crawford Lake 


79°56'27"W, 43°28'27"N 


Carbonate Cliff 


29 


Royal Municipality of Hamilton-Wentworth 






Dundas Park 


79°58'48"W, 43°14'18"N 


Carbonate Cliff 


13 


Manitoulin District 








Burnt Island 


82°56'9"W, 45°49'26"N 


Cobble Beach 


7 


Cooks Dock Lower 


82°47'20"W, 45°52'50"N 


Carbonate Cliff 


12 


Cooks Dock Upper 


82"47'20"W, 45°52'48"N 


Carbonate Cliff 


15 


Cup & Saucer East 


82°6'10"W, 45°51'21"N 


Carbonate Cliff 


18 


Cup & Saucer North 


82°5'59"W, 45°51'9"N 


Carbonate Cliff 


18 


Janet Head 


82°29'16"W, 45 56'43"N 


Carbonate Cliff 


22 


McLean Park Lowland 


81°54'28"W, 45"41'31"N 


Lowland Woods 


8 


McLean Park Upland 


81°54'25"W, 45"41'44"N 


Rocky Woods 


12 


Mississagi Lighthouse 


83°13'19"W, 45°53'36"N 


Lakeshore Carbonate 
Ledge 


13 


Niagara County 








Beamers Falls 


79°34'1"W, 43°11'16"N 


Carbonate Cliff 


20 


Niagara Whorlpool 


79°4'1"W, 43°7'24"N 


Carbonate Cliff 


20 


Peel County 








Devils Pulpit 


79°59'27"W, 43"48'4"N 


Carbonate Cliff 


26 


Simcoe County 








Glen Huron 


80°11 '41 "W, 44^21' 1"N 


Carbonate Cliff 


24 


Wellington County 








Guelph Jail 


80°10'44"W, 43 32'51"N 
WISCONSIN 


Carbonate Cliff 


8 


Ashland County 








Beaverdam Lake 


90°48'33"W, 46°19'26"N 


Igneous Cliff 


3 


Loon Lake Bluff 


90-38'49"W, 46 20'52"N 


Igneous Cliff 


12 


St. Peter's Dome 


90°54'39"W, 46 21 '5"N 


Igneous Cliff 


9 


Bayfield County 








Rainbow Lake Wilderness 


9r20'13"W. 46°26'35"N 


Upland Woods 


7 


Brown County 








Bayshore Park 


87°47'59"W, 44°38'12"N 


Carbonate Cliff 


17 


Benderville Wayside 


87°50'31"W, 44^36'47"N 


Carbonate Cliff 


27 


Blueberry Marsh 


87°53'35"W, 44 31'46"N 


Tamarack Wetland 


1 


Celtis Site 


87°50'52"W, 44 36'35"N 


Carbonate Cliff 


34 


Edgewater Villas 


87-^49 '8"W, 44 37'42"N 


Carbonate Cliff 


14 


Escarpment Glade 


87^^49 '4"W, 44 37'9"N 


Alvar 


12 


Fonferik Glen 


87°58'15"W, 44 25'34"N 


Carbonate Cliff 


25 


Gibson Alvar 


87°50'52"W, 44°35'26"N 


Alvar 


12 



TERRESTRIAL GASTROPOD RICHNESS IN THE GREAT LAKES 



245 



Gravel Pit Road 
Greenleaf Cliff 
Greenleaf Talus 
Hilly Haven 
Iron Fence Wayside 
Lily Lake County Park 1 
Lily Lake County Park 2 
Neshota County Park 1 
Neshota County Park 2 
Scray's Hill 

UWGB Upland Woods 
UWGB Cedar Swamp 
UWGB Escarpment 

Calumet County 
Calumet County Park 
Charlesburg Ledge 
East River Road 
High Cliff State Park 1 
High Cliff State Park 2 
High Cliff State Park 3 
High Cliff State Park 4 
Kiel Marsh 
Stockbridge 

Dodge County 
Ledge County Park 
Mayville South 
Messner Ledge South 
Neda Mine 

Door County 
Bjorklunden 

Boyer Bluff 1 
Boyer Bluff 2 
Brussels Hill North 
Brussels Hill Radio Tower 
Carlsville Road 
Cave Point Shore 

Cave Point Uplands 
Cherry Escarpment 
Door Bluff Park 
Ellison Bay Park 
Fifield Tract 

Prey Tract 

Glidden Drive 

Hemlock Road 1 

Hemlock Road 2 
H utter Tract 
Kangaroo Lake 
Kuchar Fen 
LaSalle Park 

Little Harbor 
McKnight Escarpment 



87°47'59"W, 44 36'7"N 
88°4'1"W, 44"20'20"N 
88°44"W, 44'20'16"N 
88°2'43"W, 44°20'59"N 
87^49 '40"W, 44=37' 14"N 
87°51'3"W, 44^25 '19"N 
87^51 '3"W,44"25'22"N 
87^48'22"W, 44°24'5"N 
87°48'17"W, 44°24'6"N 
88"1'37"W, 44°23'N 
87°55'22"W, 44'31'33"N 
87°55'29"W, 44°31'33"N 
87°54'21"W, 44°31'48"N 

88°19'11"W, 44 6'45"N 
88°12'3"W, 43°58'10"N 
88=3'42"W, 44°8'21"N 
88°17'56"W, 44°9'11"N 
88°16'44"W. 44°10'13"N 
88°17'52"W, 44°9'18"N 
88^17'49"W, 44"9'14"N 
88°3'34"W, 43°53'52"N 
88°17'17"W, 44°3'10"N 

88°35'2"W, 43°28'11"N 
88"32'24"W, 43°27'27"N 
88°35'41"W, 43"37'55"N 
88°32'5"W, 43°25'23"N 

87°7'58"W, 45°1 '54"N 

86°56'2"W, 45°25'10"N 
86"55'55"W, 45"25'6"N 
87°35'45"W, 44"46'13"N 
87^35 '27"W, 4444 '47"N 
87^22' 1"W, 44^57 '4"N 
87°10'33"W, 44"55'38"N 

87°10'44"W, 44°55'51"N 
87°9'25"W, 45°11'11"N 
87°3'53"W, 45°17'48"N 
87°5'38"W, 45^15'20"N 
87°3'36"W, 45°6'N 

87°3'45"W, 45"14'34"N 

87°12'36"W, 44°52'53"N 

86°52'12"W, 45°20'50"N 

86°52'4"W, 45^20 '42"N 
87°22'55"W, 44"56'5"N 
87°10'4"W, 45"3'12"N 
87°10'51"W, 45°5'12"N 
87^21 '50"W,44°41'23"N 

87°24'7"W, 44"54'45"N 
87°19'47"W, 45°0'43"N 



Calcareous Meadow 


19 


Carbonate Cliff 


29 


Carbonate Cliff 


21 


Carbonate Cliff 


26 


Carbonate Cliff 


20 


Tamarack Wetland 


13 


Lowland Woods 


20 


Fen 


20 


Rocky Woods 
Carbonate Cliff 


18 
23 


Upland Woods 
White Cedar Wetland 


3 

26 


Carbonate Cliff 


25 


Carbonate Cliff 


20 


Carbonate Cliff 


26 


Tamarack Wetland 


12 


Carbonate Cliff 


24 


Carbonate Cliff 


26 


Rocky Woods 
Upland Woods 
Tamarack Wetland 


14 

7 
24 


Carbonate Cliff 


28 


Carbonate Cliff 


22 


Carbonate Cliff 


23 


Carbonate Cliff 


25 


Carbonate Cliff 


19 


Lakeshore Carbonate 


21 


Ledge 
Carbonate Cliff 


17 


Carbonate Cliff 


16 


Carbonate Cliff 


20 


Carbonate Cliff 


19 


Carbonate Cliff 


12 


Lakeshore Carbonate 


9 


Ledge 
Rocky Woods 
Carbonate Cliff 


12 
17 


Carbonate Cliff 


18 


Carbonate Cliff 


26 


Lakeshore Carbonate 


17 


Ledge 
Lakeshore Carbonate 


15 


Ledge 
Lakeshore Carbonate 


13 


Ledge 
Lakeshore Carbonate 


23 


Ledge 
Rocky Woods 
Carbonate Cliff 


20 
24 


Rocky Woods 
Fen 


13 
11 


Lakeshore Alluvial 


15 


Bank 




Carbonate Cliff 


18 


Carbonate Cliff 


15 



246 



NEKOLA 



Meridian Park 


87 9'57"W, 45°0'22"N 


Lakeshore Carbonate 
Ledge 


15 


Monument Point South 


87°21 '35"W, 44°58'35"N 


Carbonate Cliff 


15 


Moonlight Bay 


87°4'1"W, 45°4'54"N 


Lakeshore Alluvial 
Bank 


4 


Mountain Park 


86^54 '10"W, 45^23 '16"N 


Carbonate Cliff 


24 


Mud Lake 


87^4'44"W, 45°5'44"N 


Lakeshore Carbonate 
Ledge 


13 


Newport State Park 1 


86°59'13"W, 45°14'52"N 


Lakeshore Carbonate 
Ledge 


20 


Newport State Park 2 


86°59'23"W, 45°15'3"N 


Lakeshore Carbonate 
Ledge 


21 


Newport State Park 3 


86°59'49"W, 45°15'13"N 


Lakeshore Carbonate 
Ledge 


21 


Peninsula State Park 1 


87°13'8"W, 45°9'42"N 


Carbonate Cliff 


18 


Peninsula State Park 2 


87°12'7"W, 45°9'45"N 


Carbonate Cliff 


10 


Port de Mort 


86°59'31"W, 45°17'50"N 


Carbonate Cliff 


22 


Potawatomie State Park 1 


87°25'29"W, 44°52'38"N 


Carbonate Cliff 


20 


Potawatomie State Park 2 


87°24'46"W, 44°51 '40"N 


Carbonate Cliff 


18 


Potawatomie SW 


87°26'31"W, 44°51'45"N 


Carbonate Cliff 


21 


Red River Bluff 


87°41'6"W, 44°45'43"N 


Carbonate Cliff 


12 


Ridges Sand Swale 


87°7'8"W, 45°4'12"N 


Calcareous Meadow 


10 


Rock Island 1 


86°49'44"W, 45°25'20"N 


Carbonate Cliff 


26 


Rock Island 2 


86°49'40"W, 45"24'53"N 


Upland Woods 


15 


Rock Island 3 


86°49'4"W, 45°25'37"N 


Carbonate Cliff 


19 


Rock Island 4 


86°49'8"W, 45°25'31"N 


Carbonate Cliff 


18 


Rock Island 5 


86°49'37"W, 45°25'19"N 


Carbonate Cliff 


28 


Rock Island 6 


86°49'44"W, 45"25'6"N 


Carbonate Cliff 


11 


Rocky Point 


87°19'11"W, 44°46'23"N 


Lakeshore Alluvial 
Bank 


13 


Sand Dune Park 


86°53'52"W, 45°20'14"N 


Lakeshore Alluvial 
Bank 


14 


Shivering Sands 


87°15'57"W, 44°52'30"N 


Lakeshore Carbonate 
Ledge 


22 


Ski Slope, E-facing 


87°14'5"W, 45°6'10"N 


Carbonate Cliff 


23 


Ski Slope, N-facing 


87°14'16"W, 45"6'18"N 


Carbonate Cliff 


15 


South Shore Road 


86"53'6"W, 45"20'10"N 


Lakeshore Carbonate 
Ledge 


21 


Standish 


87°21'57"W, 45°57'36"N 


Carbonate Cliff 


22 


Thorpe Pond 


87°13'40"W, 45°4'20"N 


Fen 


10 


Toft Point 1 


87°5'20"W, 45"3'23"N 


Upland Woods 


13 


Toft Point 2 


87°6'3"W, 45°4'50"N 


Fen 


12 


Toft Point 3 


87°5'52"W, 45°4'43"N 


Tamarack Wetland 


11 


Toft Point 4 


87°5'59"W, 45"4'58"N 


Lowland Woods 


13 


Toft Point 5 


87°5'52"W, 45"4'22"N 


Lakeshore Carbonate 
Ledge 


14 


Toft Point 6 


87°5'5"W, 45°4'40"N 


Lakeshore Carbonate 
Ledge 


19 


Toft Point 7 


87°5'2"W, 45°3'59"N 


Cobble Beach 


10 


Toft Point 8 


87'^5'45"W, 45'4'14"N 


Rocky Woods 


6 


Ulman Woods 


87°32'3"W, 44"44'58"N 


Rocky Woods 


11 


Werkheiser Escarpment 


87°20'27"W, 44°59'39"N 


Carbonate Cliff 


15 


Wilson Escarpment 


87°15'43"W, 45°5'3"N 


Carbonate Cliff 


5 


ouglas County 








Flanagan Lookout 1 


9r56'9"W, 46"35'8"N 


Igneous Cliff 


5 


Flanagan Lookout 2 


9r56'6"W, 46^^35'7"N 


Igneous Cliff 


7 


Pattison State Park 1 


92°7'26"W, 46°32'14"N 


Igneous Cliff 


9 



TERRESTRIAL GASTROPOD RICHNESS IN THE GREAT LAKES 



247 



Pattison State Park 2 


92°7'26"W, 46°32'15"N 


Igneous Cliff 


8 


Pattison State Park 3 


92°7'26"W, 46^32' 16"N 


Igneous Cliff 


7 


South Range 


91°58'26"W, 46°36'15"N 


Old Field 


4 


Fond du Lac County 








Ledge Bar 


88°21'3"W, 43°52'4"N 


Carbonate Cliff 


27 


Messner Ledge 


88"35'31"W, 43'38'13"N 


Carbonate Cliff 


23 


Messner Ledge North 


88°35'24"W, 43 38'25"N 


Carbonate Cliff 


23 


Oakfield Brick Yard 


88°33'10"W, 43°40'27"N 


Carbonate Cliff 


26 


Oakfield Ledge 


88°34'55"W, 43^38 '55"N 


Carbonate Cliff 


23 


Peebles 


88°22'40"W, 43"48'32"N 


Carbonate Cliff 


21 


ShIey Pond 


88°33'39"W, 43°40'8"N 


Carbonate Cliff 


25 


Iron County 








Hurley Visitor Center 


90°12'14"W, 46"28'13"N 


Igneous Cliff 


11 


Lake Lavina Bluff 1 


90°10'55"W, 46"26'6"N 


Igneous Cliff 


11 


Lake Lavina Bluff 2 


90°10'55"W, 46°26'6"N 


Igneous Cliff 


8 


Whitecap Mountain 


90°23'49"W, 46^24' 15"N 


Igneous Cliff 


15 


Kewaunee County 








Kewaunee Fish Hatchery 


87'=33'50"W, 44^^28'45"N 


Lakeshore Carbonate 
Ledge 


19 


Lipsky Swamp 1 


87°37'15"W, 44°28'57"N 


Tamarack Wetland 


11 


Lipsky Swamp 2 


87°37'13"W, 44°28'50"N 


Lowland Woods 


7 


Little Scarboro Creek 1 


87°37'26"W. 44°30'39"N 


Rocky Woods 


18 


Little Scarboro Creek 2 


87°37'22"W, 44''30'45"N 


Cacareous Meadow 


16 


Mud Lake 


87°39'45"W, 44"39'37"N 


Tamarack Wetland 


19 


Stony Creek Woods 


87°22'51"W, 44''39'51"N 


Lakeshore Alluvial 
Bank 


16 


Thiry Daems 


87°42'14"W, 44'='36'8"N 


Calcareous Meadow 


19 


Tisch Mills 


87°38'21"W, 44°20'50"N 


Tamarack Wetland 


12 


Manitowoc County 








Cato Falls County Park 1 


87°50'34"W, 44"5'31"N 


Carbonate Cliff 


16 


Cato Falls County Park 2 


87°50'49"W, 44°5'29"N 


Lakeshore Alluvial 
Bank 


9 


Cooperstown Swamp 


87°53'12"W, 44°16'16"N 


Tamarack Wetland 


11 


Frelich Road Swamp 


87°52'8"W, 44°17'40"N 


Lowland Woods 


21 


Kingfisher Farm 1 


87°42'3"W, 43°57'43"N 


Lakeshore Alluvial 
Bank 


20 


Kingfisher Farm 2 


87°42'17"W, 43°57'47"N 


Lakeshore Alluvial 
Bank 


6 


Kingfisher Farm 3 


87°42'3"W, 43°57'51"N 


Lakeshore Alluvial 
Bank 


6 


Kingfisher Farm 4 


87°42'14"W, 43°57'46"N 


Upland Woods 


2 


Kingfisher Farm 5 


87°42'10"W, 43°57'51"N 


Lowland Woods 


3 


Kingfisher Farm 6 


87°42'21"W, 43°57'48"N 


Lowland Woods 


9 


Kingfisher Farm 7 


87°42'25"W, 43°57'49"N 


Fen 


10 


Maribel Caves 


87°46'11"W, 44°17'9"N 


Carbonate Cliff 


17 


Point Beach 1 


87°31'11"W, 44°12'5"N 


Upland Woods 


11 


Point Beach 2 


87°30'39"W, 44°11'52"N 


Open Dune 


4 


SLC Bog 1 


87°54'5"W, 43°59'31"N 


Calcareous Meadow 


7 


SLC Bog 2 


87°54'3"W, 43°59'21"N 


Lowland Woods 


5 


SLC Bog 3 


87°53'50"W, 43^59 '3r'N 


Old Field 


9 


Tamarack Road 


88°0'55"W, 44°12'12"N 


Tamarack Wetland 


2 


Marinette County 








Kimlark Lake 


87°50'56"W, 45°39'37"N 


Calcareous Meadow 


11 


Niagara East Bluff 1 


87°56'41"W, 45°45'24"N 


Igneous Cliff 


10 


Niagara East Bluff 2 


87°56'41"W, 45°45'23"N 


Igneous Cliff 


15 


Pound Roadside 


88°1'1"W, 45°7'29"N 


Calcareous Meadow 


21 


Spur Lake 1 


88°14'5"W, 45°43'5"N 


Fen 


15 



248 


NEKOLA 






Spur Lake 2 


88 13'58"W, 45°42'59"N 


Fen 


12 


Ozaukee County 








Cedarburg Bog 1 


88°0'34"W, 43°23'9"N 


Fen 


10 


Cedarburg Bog 2 


88°1'4"W, 43°22'59"N 


Tamarack Wetland 


16 


Harrington Beach 1 


87"48'10"W, 43"29'26"N 


Old Field 


6 


Harrington Beach 2 


87°47'56"W, 43^29' 16"N 


Upland Woods 


2 


Harrington Beach 3 


87=4741 "W,43"29'42"N 


Lakeshore Carbonate 
Ledge 


10 


Sauk County 








Devils Lake 


89°43'57"W, 43°24'30"N 


Igneous Cliff 


17 


Sawyer County 








Pipestone Falls 


91°14'12"W, 45°51'19"N 


Igneous Cliff 


11 


Shawano County 








Porter Road 


88°30'10"W, 44°44'16"N 


Carbonate Cliff 


19 


Sheboygan County 








Evergreen Park 


87°44'29"W, 43°46'56"N 


Lakeshore Alluvial 
Bank 


4 


Waters Edge 


87°46'46"W, 43"34'53"N 


Lakeshore Alluvial 
Bank 


7 


Mehles Springs 


88"1'16"W, 43°51'38"N 


Tamarack Wetland 


9 


Walworth County 








Bluff Creek Fen 


88°40'54"W, 42°48'2"N 


Fen 


19 


Washington County 








Allenton Fen 


88°18'25"W, 43°22'41"N 


Fen 


19 


Waushara County 








Bass Lake 


89"16'58"W, 44°0'15"N 


Fen 


17 



TERRESTRIAL GASTROPOD RICHNESS IN THE GREAT LAKES 



249 



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MALACOLOGIA, 1999, 41(1): 253-269 

TERRESTRIAL GASTROPOD RICHNESS PATTERNS IN WISCONSIN 
CARBONATE CLIFF COMMUNITIES 

Jeffrey С Nekola^ & Tamara M. Smith^ 



ABSTRACT 

The patterns of terrestrial gastropod richness within two species-rich carbonate cliff habitats in 
eastern Wisconsin were analyzed at two differing sample scales. Up to 23 taxa were found in 
1 m^ quadrats, and 21 taxa in 0.04 m^ quadrats. These observations are among the highest re- 
ported globally for 1 ha or smaller samples. At the 1 m^ scale, samples collected within 5 m of 
bedrock outcrops had higher richness than more distant sites. At this scale, only soil pH (not Ca, 
Mg, N, P, K, percent organic matter, vascular plant species richness, or surface and 20 cm depth 
soil temperatures) was found to significantly correlate with species richness. At the 0.04 m^ 
scale, the richest sites were restricted to areas within 0.5 m of cliff bases. Comparison of maxi- 
mum richness levels across varying spatial scales demonstrate that up to a third of the total 
fauna may co-exist in <0.04 m^ regions (alpha diversity), up to half of the fauna may coexist in 
<100 m^ regions (beta diversity), while the remainder of the taxa (gamma diversity) occurs be- 
tween regions separated by at least 10 km. 

Key words: terrestrial gastropods, species richness, diversity patterns, conservation, North 
America, Niagaran Escarpment. 



INTRODUCTION 

While a number of studies have docu- 
mented richness in terrestrial gastropod 
communities at relatively large (> 100 m^) 
sample scales (e.g., Paul, 1975; Solem et al., 
1 981 ; Waiden, 1 981 ; Cameron, 1 986; Nilsson 
et al., 1988; Emberton, 1995; Tattersfield, 
1996; Emberton et al., 1997), fewer have ana- 
lyzed terrestrial gastropod community struc- 
ture at smaller scales within sites. The re- 
search that has been conducted at this scale 
has demonstrated terrestrial gastropod com- 
munity structure can change markedly over 
limited (e.g., <100 m) spatial extents. For in- 
stance. Berry (1966) demonstrated significant 
changes in faunal composition between moss- 
covered and moss-free segments on a single 
limestone cliff. Agócsy (1968) reported sub- 
stantial differences in species composition 
and abundance between adjacent limestone 
and sandstone outcrops. Cameron (1978) re- 
ported significant shifts in the faunas found on 
adjacent vertical and horizontal surfaces. 
Kralka (1986) demonstrated that over 60% of 
terrestrial gastropod species clustered signifi- 
cantly within individual boreal forest stands. 
Small-scale patterns in terrestrial gastropod 
distribution are also suggested by the control 



of soil chemistry on community structure (Out- 
eiro et al., 1993; Hermida et al., 1995), as soil 
chemistry is known to be highly variable over 
1 -2 cm distances (Burrough, 1986). 

Unfortunately, little is known about small- 
scale community composition and diversity 
patterns within the richest known global sites. 
At the Waipipi Scenic Reserve in New 
Zealand, Solem et al. (1 981 ) did not quantita- 
tively subsample the fauna, but rather docu- 
mented total gastropod diversity over the 
entire 4.2-ha reserve. Emberton (1995) docu- 
mented total species richness within a 4- 
hectare region near Manombo, Madagascar 
without measuring diversity from subsamples 
within this site. Similarly, Emberton et al. 
(1997) only documented total richness from 
entire 4-hectare regions in eastern Tanzania. 
Diversity gradients at Pine Mountain, Ken- 
tucky, the richest North American terrestrial 
gastropod site, have not been documented 
(Emberton 1 995). The smallest quadrats sam- 
pled by Tattersfield (1 996) from the Kakamega 
Forest Reserve in Kenya were 40 x 40 meters 
in size, while the minimum quadrat size 
sampled by deWinter & Gittenberger (1998) 
in southwestern Cameroon was 20 x 20 me- 
ters. While Schmid (1 966) did measure terres- 
trial gastropod richness from individual 1 m^ 



^Department of Natural and Applied Sciences, University of Wisconsin-Green Bay, Green Bay, Wisconsin 54311, U.S.A.; 

nekolaj@gbms01 .uwgb.edu 

^Department of Natural Resources, Cornell University. Ithiaca. New Yorl< 14853, U.S.A. 

253 



254 



NEKOLA& SMITH 




ОгееЯ Bay-, 




Hwy.54. 
New Franken 




FIG. 1 . Location of the Celtis and UWGB Escarpment sites in northeastern Brown County, Wisconsin, USA. 



quadrats near Tübingen, Germany, these 
were not part of a larger, systematic sampling 
regime and were not used to document diver- 
sity gradients within habitats. 

Previous analyses (Nekola, 1999) have 
shown that carbonate cliffs from the Great 
Lakes region in North America are among 
the richest terrestrial gastropod communi- 
ties reported from less than 1 ha scales. How- 
ever, like other high diversity communities, 
nothing was known of: (1) diversity gradients 
within these sites; (2) the environmental fac- 
tors within sites that correlate with high rich- 
ness microsites, and (3) the scales of organi- 
zation for faunal diversity within and between 
sites. This paper attempts to address these 
questions by analyzing terrestrial gastropod 
diversity patterns at two sampling scales (1 
m^ and 0.04 m^) within two high-richness car- 
bonate cliff sites in northeastern Wisconsin, 
U.S.A. 



MATERIALSAND METHODS 
Study Sites 

Two wooded carbonate cliffs in Brown 
County, Wisconsin, with high levels of terres- 



trial gastropod species richness were chosen 
for study (Fig. 1). Both occur along the Nia- 
garan Escarpment, a 1 ,300 km band of out- 
cropping Silurian-age limestones and dolo- 
mites that can be roughly divided into five 
200-300 km long regions (northeastern Iowa; 
eastern Wisconsin through the Garden Penin- 
sula of Michigan; eastern Upper Peninsula of 
Michigan though Manitoulin Island; Bruce 
Peninsula though south-central Ontario; and 
southeastern Ontario to western New York 
State) which are separated by low areas with 
little or no bedrock exposure. Within the east- 
ern Wisconsin region, the Niagaran Escarp- 
ment is naturally divided into 31 isolated 2-8 
km sections of exposed bedrock that emerge 
above Pleistocene tills and alluvium. 

The Celtis site (87^^50'52"W, 44°36'35"N) is 
situated within a 7 km-long Niagaran Escarp- 
ment section near the settlement of Ben- 
derville. Its canopy is dominated by old-growth 
sugar maple {Acer saccharum Marsh.), white 
cedar ( Thuja occidentalis L.), paper birch {Be- 
tula papyrifera Marsh.) and hackberry {Celtis 
occidentalis L.). The bedrock outcrop at the 
Celtis site is divided into a 3-6 m primary up- 
land cliff and a 2-5 m secondary cliff associ- 
ated with a large bedrock block displaced 
downslope approximately 10 m (Fig. 2). Cool 



SMALL-SCALE RICHNESS PATTERNS WITHIN CARBONATE CLIFFS 

4 



255 




35 meters 

FIG. 2. Schematic profile of tine bedrock outcrop at the Celtis site, with location of 1 m^ quadrats along each 
transect. Vertical exaggeration is approximately 4x the linear extent. 



air seepage from bedrock fissures and talus 
occurs throughout the growing season. The 
presence of large displaced talus blocks, ex- 
panded bedrock joints, and an extensive sub- 
tending talus indicate that this area was sub- 
jected to intense periglacial erosion during the 
late Pleistocene (Stieglitz et al., 1 980). This lo- 
cation harbors the single most diverse terres- 
trial gastropod assemblage known from the 
Great Lakes region, with 34 taxa (Nekola, 
1 999). Included in this fauna are the glacial re- 
licts Catinella gélida (F С. Baker, 1927), Hen- 
derson ia occulta (Say, 1831), Vallon ia gracili- 
costa albula (Sterki, 1893), and Vertigo 
hubrichti PWsbry, 1934 (Nekola et al., 1996) 

The University of Wisconsin -Green Bay 
(UWGB) Escarpment site (87"54'21"W, 44° 
31'48"N) is located within a 4-km Niagaran 
Escarpment section located near the Bay Set- 
tlement community. This site consists of a sin- 
gle 2-5 m tall upland cliff within a white cedar 
and box elder (Acer negundo L.) canopy. 
Large displaced talus blocks are absent. This 
site was extensively modified by small-scale 
quarrying for lime and building material ap- 
proximately a century ago (Stieglitz et al., 
1980). As these activities were carried out 
with hand tools and were spatially limited, this 
site consists of a mixture of modified and un- 
modified cliff segments. Unmodified seg- 
ments appear essentially identical to the up- 
land outcrop at the Celtis site. A total of 25 
terrestrial gastropod taxa have been located 
here, including the glacial relicts Vallonia gra- 



cilicosta albula, Vertigo hubrichti, and Vertigo 
n. sp. {'V. iowaensis" o\ Frest, 1991). 

Data Sets 

A representative and relatively undisturbed 
section of exposed cliff was identified at each 
site. From a random starting point within 
these sections, five transects were laid out 
perpendicular to the cliff face at 5-m intervals. 
Along each transect both 1 m^ and 0.04 m^ 
samples were sampled: 

1 ruf Quadrats: At the Celtis Site, seven 
quadrats, each separated by 5-m distances, 
were collected along each transect (Fig. 2) for 
a total of 35. At the UWGB Escarpment, three 
quadrats, separated by 5-m distances, were 
collected from each transect for a total of 15. 
Only three samples per transect were 
gathered from the UWGB Escarpment site to 
avoid highly disturbed forest and recreational 
trails occurring at greater distances from the 
cliff base. 

The vascular plants growing on or over 
each quadrat were recorded and their species 
richness calculated. Soil temperatures at the 
ground surface and at 20 cm depth were mea- 
sured using a thermocouple thermometer. A 
100 gm dry weight soil sample was collected 
by subsampling the corners and center of 
each quadrat. These samples were sent to 
the Wisconsin State Soils Lab at the Univer- 
sity of Wisconsin -Madison for analysis of 



256 



NEKOLA& SMITH 



percent organic matter, N, P, K, S, Ca, Mg, 
and pH, using methods outlined in Dahnke 
(1988). 

Terrestrial gastropod assemblages were 
determined by collecting a total of 4-5 
deciliters of soil litter from tfie corners and 
center of each quadrat. Samples were slowly 
and completely dried in either a low-tempera- 
ture soil oven (approx. 80-95°C) or in full sun 
in a greenhouse. Dried samples were then 
soaked in water for 3-24 h, and subjected to 
careful but vigorous washing through a stan- 
dard sieve series (ASTME 3/8" (9.5 mm), 10 
(2.0 mm), 20 (0.85), and 40 (0.425 mm) mesh 
screens). The washed fractions were re-dried 
and then re-sifted through the original sieve 
series. The dry, resorted fractions were hand 
picked against a neutral-brown background 
using a small sable brush. All shells and shell 
fragments were removed. 

Recovered, identifiable shells were as- 
signed to species (or subspecies) using the 
author's reference collection and the Hubricht 
Collection at the Field Museum of Natural His- 
tory. From this, species composition and rich- 
ness per quadrat was calculated. All speci- 
mens are housed in collections maintained at 
the University of Wisconsin -Green Bay. 

0.04 nf Quadrats: 20 x 20 cm quadrats were 
collected adjacent to 1 m^ quadrats along 
each of the five established transect lines. 
Quadrats were sampled at distances of 0, 0.5, 
1 .0, 1 .5, and 2.0 meters from cliff bases. The 
primary and secondary cliffs at the Celtis site 
(located at positions 1 and 5, respectively, on 
Fig. 2), and the primary cliff at the UWGB 
Escarpment were analyzed in this fashion for 
a total of 75 observations. For each quadrat, 
transect position and distance from the cliff 
base were recorded, and a 2-3 deciliter soil 
litter sample collected. These litter samples 
were subjected to the same laboratory 
procedures described for the 1 m^ samples to 
determine terrestrial gastropod composition 
and richness. 

Comparison of Maximum Richness Levels: 
The maximum richness of 0.04 m^ and 1 m^ 
samples from each site were compared with 
known richness values from a series of nested 
samples of increasing sample grain. These 
grains include each site (observed from a 100 
m^ quadrat), escarpment section, escarpment 
region. Brown County, and the state of Wis- 
consin. Richness values at these increasing 
scales of observation are based upon species 
lists from other sites (summarized in Nekola, 



1999), augmented by other published records 
(Levi & Levi, 1 950; Teskey, 1 954; Jass, 1 986). 
Richness estimations were limited to carbon- 
ate cliff habitats for Escarpment sections 
and regions, while those for Brown County and 
the state of Wisconsin included all habitat 
types. 

To compare the maximum richness values 
from this study with other reported maximum 
richness values, a survey was made of the 
published literature to identify other datasets 
in which both terrestrial gastropod richness 
and sample grain were reported. If multiple 
examples of such data were found from a sin- 
gle paper, only the richest were entered for a 
given sample size. Through this process, a 
total of 35 records from four continents 
(Africa, Australia, Eurasia, and North Amer- 
ica) were recorded (Burch, 1956; Schmid, 
1966; Agócsy, 1968; Mason, 1970; Berry, 
1973; Paul, 1975; Uminski & Focht, 1979; 
Bishop, 1980; Solem et al., 1981; Van Es & 
Boag, 1981; Waiden, 1981; Nilsson et al., 
1 988; Cameron & Greenwood 1 991 ; Young & 
Evans, 1991; Cameron, 1992; Outeiro et al., 
1993; Getz & Uetz, 1994; Cowie et al., 1995; 
Emberton, 1995; Wardhaugh, 1995; Tatters- 
field, 1996; Emberton et al., 1997; de Winter 
& Gittenberger, 1998). 

Statistical Analyses 

1 nf Quadrats: Analysis of the effect of 
quadrat position and site on richness was con- 
ducted via ANOVA. These data were graphi- 
cally represented using box plots (Velleman & 
Hoaglin, 1981). In box plots, the central line 
represents the median of the sample, the mar- 
gins of the box represent the interquartile dis- 
tances, and the fences represent 1 .5 times the 
interquartile distances. For data having a 
Gaussian distribution, approximately 99.3% of 
the data will fall inside of the fences. Outliers 
falling outside of the fences are shown with as- 
terisks. Identification of the environmental 
variables that best predict observed richness 
was accomplished through multiple linear re- 
gression using a backwards stepwise selec- 
tion procedure. Beginning with the most non- 
significant, variables were removed from the 
model until all remaining p-values fell below 
the 0.05 level. Analysis of residuals and indi- 
vidual variable distributions indicated that data 
transformations were not necessary. 

0.04 nf Quadrats: Analysis of the effect of 
quadrat position, cliff position, and site 
location on richness was conducted via 



SMALL-SCALE RICHNESS PATTERNS WITHIN CARBONATE CLIFFS 



257 



ANOVA. These data were graphically repre- 
sented for each of the three sampled cliffs 
(Celtis Site primary and secondary, and 
UWGB Escarpment primary) using box plots. 
A full ANOVA with all interaction terms was not 
conducted as a secondary cliff was not 
present at the UWGB Escarpment site. 

Comparison of Maximum Richness Levels: 
The percent of total richness from the five 
nested larger sample grains overlying each 
maximally-rich 0.04 m^ and 1 m^ quadrat was 
calculated for each site. The richness of these 
different sample areas was natural-log trans- 
formed and regressed against natural log- 
transformed estimates of habitat area. Habitat 
size estimates for Niagara Escarpment sec- 
tions and regions were generated by multiply- 
ing average cliff-base habitat width (approx. 5 
m) by habitat length. The natural log of maxi- 
mum richness vs. the natural log of sample 
size was also plotted for the 35 literature rich- 
ness records and for the maximum richness 
0.04, 1, and 100 m^ quadrats from the Celtis 
and UWGB Escarpment sites. While the lim- 
ited number of samples prevented use of in- 
ferential statistics to test for significant differ- 
ences between the maximum species area 
curves for carbonate cliffs (n = 3 for each site) 
and other habitats, a qualitative assessment 
was made. 



RESULTS 



1 m^ Quadrats 



Richness of terrestrial gastropods at the 
two sites ranged between and 23 taxa (Ta- 
bles 1 , 2), with a mean of 6.6 taxa/ quadrat. 
Across both sites, mean richness was 9.9 
from quadrats collected at transect position 1 , 
7.1 at position 2, 7.2 at position 3, 6.8 at posi- 
tion 4, 5.8 at position 5, 2.4 at both positions 6 
and 7 (Fig. 3). ANOVA of these data demon- 
strated that this variation was weakly signifi- 
cant (p = 0.04; Table 3). Further ANOVA tests 
demonstrated that no significant differences 
were present between the mean richness of 
transect positions 1 -5 (p = 0.326), or between 
the Celtis and UWGB Escarpment sites (p = 
0.646). No interaction between transect posi- 
tion and site on richness was observed (p = 
0.947). Backwards stepwise linear regression 
of ten environmental variables on richness 
demonstrated that only pH (p < 0.0005) and P 
(p = 0.04) were significant predictors (Table 




3. 4 5^ 6, 7. 

Transect Position 



FIG. 3. Box-plot diagram of terrestrial gastropod 
richness at 1 m^ scales with increasing distance 
from the primary upland cliff. 



25 



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FIG. 4. Scatterplot of terrestrial gastropod richness 
in 1 m^ quadrats vs. soil pH, with best-fit line. 
Twelve of the data points are not apparent in this 
graph as they overlap previously plotted values. 



4), accounting for almost 30% of observed 
richness variation. However, this level of sig- 
nificance of P appears to based upon a single 
outlier. When this observation was removed 
from analysis, the p value for P in a multiple 
linear regression of pH and P on richness 
dropped to 0.266. The amount of variation in 
richness accounted for by pH alone was 
found to exceed 28% (Fig. 4). 



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SMALL-SCALE RICHNESS PATTERNS WITHIN CARBONATE CLIFFS 



259 



TABLE 2. Abundance and richness of terrestrial molluscs in 1 5 1 x 1 m quadrats at the UWGB Escarpment 
site 



Transect 


A 


A 


A 


В 


В 


В 


С 


С 


С 


D 


D 


D 


Е 


Е 


Е 


Quadrat 


1 


2 


3 


1 


2 


3 


1 


2 


3 


1 


2 


3 


1 


2 


3 



Auguispira altérnala (Say, 1 81 7) 
Carychium exile H. С. Lea, 1842 
Catinella avara (Say. 1 824) 
Cochlicopa lubrica (Müller, 1774) 
Cochlicopa lubricella (Porro, 1838) 
Discus cronkhitei {Nev\/comb, 1865) 
Gastrocopta armífera (Say, 1 821 ) 
Gastrocopta holzingeri {S\ei\<\. 1889) 
Hawaiia miniscula (A. Binney, 1 840) 
Helicodiscus shimeki Hubricht, 1962 
Strobilops labyrinthica (Say, 1 81 7) 
Succinea ovalis Say, 1817 
Triodopsis multilineata (Say, 1821 ) 
Valíanla costata (Müller, 1 774) 
Valíanla gracilicosta Reinhardt, 1883 
Valíanla pulchella (Müller, 1774) 
Vertiga gauldi [к. Binney, 1843) 
Vertiga hubrichtl (Pllsbry. 1934) 
Vertiga "lawaensis" 
Vertiga pygmaea (Draparnaud, 1 801 ) 
Immature Individuals 

Total Individuals 
Richness 



2 






6 






1 










3 


1 


3 


2 


2 


6 


11 


8 


15 


8 


18 


10 


3 


2 




6 


2 


6 


5 


1 


2 

1 


1 
2 


3 

1 
1 
1 

1 


8 
3 


7 
1 


5 


5 

1 


4 
3 

1 


3 
3 


11 
3 

4 


1 
1 

1 


2 

1 
1 


3 

1 

1 
1 


1 
2 


2 




1 

4 


1 
8 


2 




1 




3 


2 


2 


1 


1 


1 
1 


2 




1 




6 


19 

1 

1 


14 


5 


16 


7 
1 


8 
3 


30 
1 


10 


20 

1 

2 
5 


2 




4 




2 


2 


1 


3 


2 


9 


1 


1 


4 


1 


7 






1 


2 




4 


2 


3 


5 


3 




2 




2 


13 


3 





22 12 21 42 52 36 37 45 23 25 55 26 57 22 12 
546 11 85668878 12 95 



TABLE 3. Summary statistics for ANOVA of terrestrial gastropod species richness 
in 1 m^ quadrats vs. quadrat distance from base of primary cliff at both the Celtis 
and UWGB Escarpment sites. 



Source 


Sum-of-Squares 


df 


F-Ratio 


P 


Distance from base 

Error 

Squared multiple r: 


313.48 
835.90 

0.273 


6 
43 


2.688 


0.026 



TABLE 4. Results of backwards stepwise linear 
regression of 10 environmental variables on terres- 
trial gastropod species richness at 1 m^ grains. 
Variables are listed in the order in which they were 
removed from the model. The p-values reported are 
those immediately prior to removal of that variable 
from the model. 



Variable 


p-value 


Mg 


0.968 


Percent Organic Matter 


0.783 


Soil Temperature at 20 cm Depth 


0.406 


К 


0.314 


Vascular Plant Species Richness 


0.293 


Ca 


0.283 


N 


0.167 


Surface Soil Temperature 


0.121 


P 


0.040 


PH 


< 0.0005 



0.04 m^ Quadrats 

Terrestrial gastropod richness varied from 
to 21 taxa on all three sampled cliffs (Tables 
5-7). At the primary upland cliff at the Celtis 
site, average species richness was 1 6.8 at the 
cliff base, 9.4 at 0.5 m, 3.2 at 1 m, 2.8 at 1.5 
m, and 2.2 at 2 m distances from the cliff. At 
the secondary cliff at the Celtis site, average 
species richness was 14.4 at the cliff base, 
1 1 .2 at 0.5 m, 5.2 at 1 m, 2.8 at 1 .5 m, and 2.8 
at 2 m distances from the cliff. At the UWGB 
Escarpment site, average species richness 
was 8.0 at the cliff base, 8.0 at 0.5 m, 6.0 at 1 
m, 5.8 at 1 .5 m, and 6.4 at 2 m distances from 
the cliff (Fig. 5). ANOVA of these data (Table 
8) demonstrated that distance from the cliff 
base and the interaction between this variable 



260 



NEKOLA& SMITH 



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SMALL-SCALE RICHNESS PATTERNS WITHIN CARBONATE CLIFFS 



263 



TABLE 8. Summary statistics for ANOVA of terrestrial gastropod species rich- 
ness in 0.04 m^ quadrats vs. quadrat distance from cliff base, cliff position, and 
site location. 



Source 


Sum-of-Squares 


df 


F-Ratio 


P 


Site 


0.08 


1 


0.008 


0.928 


Cliff position 


2.88 


1 


0.296 


0.589 


Distance from base 


406.08 


4 


10.423 


< 0.0005 


Distance * Site 


271.72 


4 


6.974 


< 0.0005 


Distance * Cliff 


25.12 


4 


0.645 


0.633 


Error 


584.04 


60 







Squared multiple л; 0.699 



Primary Cliff. Celtis Site 



Secondary Cliff. Celtis Site 



Primary Cliff. Campus Site 



• 1 




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0.0 0.5 1.0 15 20 
I frc5m Clitf (m) 



00 05 1,0 15 20 
Distance from Cliff (m) 




0.5 1,0 15 20 
Distance from Clrtf (m) 



FIG. 5. Box-plot diagram of terrestrial gastropod richness from 0.04 m scales at increasing distances from 
three cliff exposures at the Celtis and UWGB Escarpment sites. 



and site were both highly significant (p < 
0.0005). In all, distance from cliff base and 
site accounted for 70% of the observed varia- 
tion in richness. Mean richness was not af- 
fected by cliff type (phmary or secondary; p = 
0.649) and cliff type did not interact with the 
rate of richness decrease from the cliff base (p 
-0.518). 

Comparison of Maximum Richness Levels 

At the UWGB Escarpment, a maximum of 
1 2 taxa were observed from single 0.04 and 1 
m^ quadrats. As this site represents the lone 
surviving carbonate cliff community within the 
6 km Bay Settlement section, the total rich- 
ness of this Escarpment section equals site 
richness (25 taxa). For the Celtis site, all 
small-scale richness values were higher, with 
a maximum richness of 21 taxa occurhng in a 
single 0.04 m^ sample, and 23 taxa in a single 
1 m^ sample. Carbonate outcrops along the 8 
km Benderville Escarpment Section (within 
which the Celtis site occurs) support 38 taxa. 
All carbonate cliffs along the 350 km Eastern 
Wiscosin-Garden Peninsula Escarpment Re- 



gion support 62 taxa. The total richness of 
Brown County terrestrial gastropods, across 
all habitat types, is 65 taxa, while a total of 95 
taxa have been documented across all habi- 
tats in the state of Wisconsin (Table 9). 

The faunas of maximum richness 0.04 m^ 
quadrats at the Celtis site thus account for up 
to 91% of maximum 1 m^ richness, 62% of 
site richness, 55% of Escarpment section 
richness, and 34% of Escarpment region rich- 
ness. Individual 0.04-m^ quadrats also har- 
bored up to 32% of the entire county fauna, 
and 22% of the entire state fauna. Given their 
similar maximum richness, almost identical 
results are present for maximum richness 1 
m^ quadrats, which can harbor up to 68% of 
the entire site fauna, 61% of the Escarpment 
section fauna, and 37% of the entire Escarp- 
ment region fauna. Individual 1-m^ quadrats 
can also harbor up to 35% of the entire county 
fauna, and 24% of the entire state fauna. Be- 
cause of the lower richness levels at the 
UWGB Escarpment Site, these numbers 
tended to be lower by almost 30-50% from 
Celtis site levels. Regression analysis demon- 
strates that a high correlation (p < 0.0005; /^ = 



264 



NEKOLA& SMITH 



TABLE 9. Percent of site, escarpment section, escarpment region, county, and 
state terrestrial gastropod faunas contained withiin maximum diversity 0.04 and 
1 m^ quadrats. 



UWGB Escarpment Site 








Percent Overlap 


Sample Grain 


Richness 


0.04 m^ 1 m^ 


0.04 m^ 


12 




1m2 


12 


100 


Site (100 m^) 


25 


48 48 


Niagara Escarpment 






Bay Settlement Section 


25 


48 48 


Eastern Wisconsin Region 


62 


19 19 


Brown County 


65 


18 18 


Wisconsin 


95 


13 13 




Celtis Site 


Percent Overlap 


Sample Grain 


Richness 


0.04 m^ 1 m^ 


0.04 m^ 


21 




1m2 


23 


91 


Site (100 m^) 


34 


62 68 


Niagara Escarpment 






Benderville Section 


38 


55 61 


Eastern Wisconsin Region 


62 


34 37 


Brown County 


65 


32 35 


Wisconsin 


95 


22 24 



0.839) exists between natural log-trans- 
formed richness and sample area, with the 
best-fit line having an intercept of 2.93 and a 
slope of 0.063 (Fig. 6). This translates to an 
average richness of 18.7 taxa per 1 m^ and 
33.4 taxa per hectare. 

Comparison of maximum richness from 
0.04 and 1 m^ quadrats to other reported 
maximum richness values demonstrates the 
Celtis site site is among the richest reported 
globally from small observational scales (Fig. 
7). Maximum richness levels at the Celtis site 
compare favorably with the richest reported 
1 -400 m^ samples in Germany, Sweden, and 
Scotland (Schmid, 1966; Waiden, 1981; 
Cameron & Greenwood 1991). The richest 
global terrestrial gastropod faunas, collected 
over larger areas (400-40,000 m^) in New 
Zealand, Madagascar, Tanzania, and Came- 
roon appear to fall along the same maximum 
species-area curve defined from the Celtis 
Site. While maximum richness at the UWGB 
Escarpment is lower, these observations still 
lie within the upper half of previously reported 
maximum richness levels for the given range 
of sample scales. 



г2 = 0.839 
p < 0.0005 



у = 293 + ООбЗх 



-10 О 10 20 

Natural Log Area (Square Meters) 



30 



FIG. 6. Scatterplot of natural log-transformed ter- 
restrial gastropod richness vs. natural log-trans- 
formed sample area for Wisconsin carbonate cliff 
land snail faunas. The scales of observation in- 
clude: maximum-richness 0.04, 1, and 100 m^ 
quadrats from the Celtis and UWGB Escarpment 
sites: the respective escarpment sections for each: 
escarpment region: county: and state. 



SMALL-SCALE RICHNESS PATTERNS WITHIN CARBONATE CLIFFS 



265 



3.6 



3.0 - 



2.4 - 



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о 




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Global Localities 

FIG. 7. Species-area plot of maximum-richness 
0.04, 1 and 100 m^ quadrats from the Celtis and 
UWGB Escarpment sites compared to other maxi- 
mum richness levels reported from other global 
sites. 



DISCUSSION 

Diversity Patterns Within Carbonate 
Cliff Communities 

The high richness of terrestrial gastropods 
within carbonate cliff habitats occurs down to 
very limited spatial scales, with up to 62% of 
site richness (and up to 22% of total state rich- 
ness) being found within single 0.04 m^ areas 
along cliff bases. The limitation of high-diver- 
sity assemblages to the immediate vicinity of 
vertical bedrock outcrops is striking, with rich- 
ness decreasing by almost six-fold from to 1 
m from cliff bases. This rapid and drastic re- 
duction in richness helps explain why, on av- 
erage, 0.04 m^ quadrats at cliff bases harbor 
more species than adjacent 1 m^ quadrats. As 
the 1 m^ samples consisted of pooled sub- 
samples taken from the corners and center of 
each quadrat, only two subsamples per 
quadrat were thus collected from mollusc-rich 
microsites. This had the unintended effect of 
diluting snail density and richness. However, 
the 0.04 m^ cliff-base quadrats, which con- 
sisted to a similar total volume of soil litter, 
were collected entirely from the richest mi- 
crosites, so that no dilution in snail density or 
richness occurred. 

While diversity at the 0.04 m^ scale was 
markedly higher adjacent to cliffs at the undis- 



turbed Celtis site, such spatial limitation of 
richness was not observed at the UWGB Es- 
carpment. In addition, maximum (but not 
mean) richness at both 0.04 and 1 m^ scales 
at the UWGB Escarpment site was roughly 
one-half that recorded at the Celtis Site. It is 
probable that these lower maximum diversity 
levels, and lack of strong micro-scale diversity 
gradients, are related to this site's past quar- 
rying history, which may have simplified the 
range of microhabitats present along the cliff 
base. 

The exact mechanisms that lead to high 
levels of terrestrial gastropod richness at 
micro-scales within these carbonate cliff sites 
have not been documented. First, it is not 
known how many of these shells originate 
from individuals living in the quadrat versus 
shells that have been deposited from nearby 
areas, such as adjacent vertical rock faces. If 
this latter process is important, levels of 
micro-scale sympatry could be substantially 
lower than the observed shell richness sug- 
gests. However, preliminary observations of 
living individuals on cliff bases and adjacent 
vertical faces suggest that the majority of 
shells originate from within quadrats. Addi- 
tionally, most species observed from cliff- 
base quadrats are represented by at least one 
living snail or recently dead shell, while spe- 
cies reported from more distant quadrats are 
almost always represented by long-dead shell 
fragments. 

Second, even if shells from high-richness 
microsites are locally derived, observed rich- 
ness may be exaggerated if shells persist in 
the soil for long periods. In this case, species 
lists will represent an integration over the per- 
sistence-time of shells. This could lead to an 
overestimate of microsympatry if local faunas 
are in a state of constant flux. Use of radio- 
isotope dating on shells could provide a pos- 
sible test for shell half-life, which would help 
set the temporal scales of integration for such 
shell-banks in carbonate cliff soils. However, 
the fact that most taxa are represented in 
samples by at least one live or recently dead 
shell suggests that high levels of sympatry are 
likely maintained at both limited temporal and 
spatial extents. 

The existence of diverse terrestrial gastro- 
pod assemblages at very small scales on or 
adjacent to carbonate outcrops is also likely 
not unique to the two sites chosen for analy- 
sis. Oualitative observations of other carbon- 
ate cliffs in Illinois, Iowa, New York, Ontario, 
Wisconsin, and southwestern England sug- 



266 



NEKOLA& SMITH 



gest that the co-occurrence of 20 or more taxa 
at 1 m^ or smaller grains may be typical in 
undisturbed sites. 

The limitation of high richness terrestrial 
gastropod assemblages to the immediate 
proximity of cliffs also indicates that these 
microhabitats must be afforded special pro- 
tection if their biodiversity is to be protected. 
Unfortunately, planned and spontaneous 
recreational trails in reserves are often routed 
through these exact areas as they are aes- 
thetically pleasing and provide access to 
charismatic natural features such as caves, 
fissures, and rock walls. Such trails may place 
any high-richness terrestrial gastropod as- 
semblages in serious jeopardy. For instance, 
the cliff base at Bayshore County Park, 6 km 
north of the Celtis Site, has been turned into a 
graveled trail which now lacks a terrestrial 
gastropod fauna (Nekola, unpublished data). 

Environmental Controls on Small Scale 
Richness Patterns 

The literature regarding the environmental 
controls of terrestrial gastropod richness and 
abundance is very conflicting. Burch (1955) 
suggested that in eastern Virginia snail abun- 
dance (and presumably diversity) was related 
to soil organic matter, Ca, Mg, and К, but not 
pH. Lack of correspondence between terres- 
trial gastropod distribution and pH has also 
been demonstrated in southwestern Ireland 
(Bishop, 1977) and the Italian Alps (Bishop, 
1980). However, Gleich & Gilbert (1976), 
stated that in central Maine soil moisture, but 
not soil Ca, was the most important determi- 
nant of snail abundance. Outeiro et al. (1993) 
demonstrated that soil texture and pH were 
the most important factors effecting terrestrial 
gastropod distribution in central Spain. Soil 
pH was also identified as an important deter- 
minant of terrestrial gastropod density and di- 
versity by Waiden (1981) and Gärdenfors 
(1992) in southern Sweden, and by Bishop 
(1976) in Somerset, England. Getz (1974) 
and Getz & Uetz (1994) identified soil mois- 
ture, tree diversity, and leaf litter diversity as 
major determinants of diversity in the Great 
Smoky Mountains. However, Locasciulli & 
Boag (1987) demonstrated in Alberta forests 
that terrestrial gastropod abundance was not 
related to vegetation. 

Within eastern Wisconsin carbonate cliff 
habitats, only soil pH (and not soil Ca, Mg, N, 
P, K, percent organic matter, soil temperature, 
or vascular plant richness) was found to cor- 



relate significantly with terrestrial gastropod 
richness at the 1 m^ scale. This result is con- 
sistent with the analysis of Bishop (1980), 
who stated that soil pH will only be an impor- 
tant environmental correlate of terrestrial gas- 
tropod assemblages when soil Ca levels are 
high. Interactions between environmental 
variables may explain why such a diversity of 
factors exist that have (and have not) been 
shown to influence terrestrial gastropod rich- 
ness across various habitats and regions. 
Thus, extrapolation of these results to other 
habitats or regions may be risky. These re- 
sults do, however, provide insight into the en- 
vironmental correlates of terrestrial gastropod 
richness within Ca-rich carbonate cliffs. 

Spatial Scales of Terrestrial 
Gastropod Coexistence 

A complete assessment of the scales over 
which terrestrial gastropod coexistence is me- 
diated is not possible because the current 
data are restricted to five discrete spatial 
scales (0.04 m^, 1 m^, 100 m^, approx. 3.5 ha, 
and approx. 1750 ha). However, given the 
wide total range covered, some preliminary 
insights into this issue can be made. 

Three of the measured scales appear to 
harbor the bulk of terrestrial gastropods. Up 
to one-third of the total regional fauna may 
occur within individual 0.04 m^ regions. Over 
50% of the total regional fauna may occur 
within 100 m^ regions on individual sites. The 
remainder of the fauna (almost 50% of the re- 
gional total) is largely found between sites on 
different Escarpment Sections (e.g., sites 10 
km or more apart, corresponding to 3.5 -i- ha 
of cliff base habitat). However, little increase 
in richness was observed between 0.04 m^ 
and 1 m^ extents within sites, and between 
sites within the same Escarpment Section 
(100 m^-3.5 km of cliff base habitat). 

One tentative conclusion that can be drawn 
from these results is that alpha diversity 
{sensu Whitaker, 1975) in these sites is best 
measured at scales no larger than 0.04 m^, 
beta diversity {sensu Whitaker, 1975) is best 
measured at scales no larger than 100 m^, 
and gamma diversity {sensu Cody, 1986) is 
best measured between sites at least 10 km 
distant from one another. Although Emberton 
(1995) states that distinctions between alpha, 
beta, and gamma diversity are hazy for ter- 
restrial gastropod communities, these data 
suggest that such problems in resolution may 
be due to poorly chosen observational scales. 



SMALL-SCALE RICHNESS PATTERNS WITHIN CARBONATE CLIFFS 



267 



For instance, if the Celtis and UWGB Escarp- 
ment sites had been sampled at typical mala- 
cologial sampling scales of 0.1 ha or larger, 
distinctions between alpha and beta diversity 
would be impossible to make, as sample res- 
olution would be at least 1000 times greater 
than the scale at which alpha diversity likely 
exists. 

Additional research will be necessary to 
document the mechanisms that allow for 
species coexistence at these differing scales. 
Ecological theory suggests that alpha diversity 
levels may be related to levels of niche parti- 
tioning between species (Auerbach & Shmida, 
1987). However, competition and prédation 
have only rarely been shown to influence ter- 
restrial gastropod distribution and abundance 
(Cain, 1 983; Cowie & Jones, 1 987; Smallhdge 
& Kirby, 1988). The high density of shells (up 
to 330 per 0.04 m^ quadrat) further suggests 
that resource levels are also high, cautioning 
against use of resource-ratio models (e.g., 
Tilman, 1988). Identification of the small-scale 
coexistence mechanisms in these habitats 
may be of broad ecological importance, as it is 
rare for alpha diversity to constitute such a 
high proportion of regional diversity, and thus 
for the rate of species accumulation with in- 
creasing sample size to be so low (Rosen- 
zweig, 1995). 

At larger scales of observation, habitat het- 
erogeneity may be important in determining 
levels of species richness (Auerbach & 
Shmida, 1987; Rosenzweig 1995). The few 
additional species added between site and 
escarpment section scales suggests that the 
universe of microenvironments found within a 
given carbonate cliff site may be very similar 
to those present within an entire Escarpment 
section. At the largest scales, coexistence of 
terrestrial gastropods will likely be mediated 
by large environmental gradients (including 
climate), differential colonization histories of 
habitats (Ricklefs & Schlüter, 1993; Nekola, in 
press), and the incomplete dispersal of 
species between sites (Auerbach & Shmida, 
1987). 

It is important to note that the spatial scales 
of coexistence for terrestrial gastropod com- 
munities, and hence the optimal scales of ob- 
servation, may differ between systems and 
landscapes. For instance, it is not clear that 
alpha diversity will always be confined to such 
small scales in habitats that support substan- 
tially lower densities of individuals and taxa. 
Gamma diversity will also likely vary between 
landscapes, as preliminary analyses have 



documented for Niagaran Escarpment car- 
bonate cliffs in which rates of community 
turnover may vary by an order-of-magnitude 
(Nekola, unpublished data). Such observa- 
tions suggest that additional research will be 
necessary within and between a diversity of 
habitats and landscapes to determine if any 
general rules exist to guide the ecological 
sampling of terrestrial gastropod communi- 
ties. 



ACKNOWLEDGEMENTS 

Robert Cameron, Douglas Larson, John 
Slapcinsky and an anonymous reviewer pro- 
vided useful comments on earlier versions of 
this manuscript. Assistance in sample pro- 
cessing was provided by Catherine Steele 
and students enrolled in the spring 1998 Ter- 
restrial Gastropod Ecology Practicum at the 
University of Wisconsin -Green Bay. Work at 
the Leslie Hubricht collection was made pos- 
sible through a Prince Visiting Scholar grant 
from the Field Museum of Natural History. 
Field research was funded by the Louis Almon 
Fund (administered through the Wisconsin 
Academy of Sciences, Arts, and Letters) and 
a Cofrin Arboretum Grant (administered 
through the Cofrin Arboretum committee of 
the University of Wisconsin —Green Bay). 



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Revised ms. accepted 2 March 1999 



MALACOLOGIA, 1999, 41(1): 271 -281 

SAMPLING TERRESTRIAL GASTROPOD COMMUNITIES: USING ESTIMATES OF 
SPECIES RICHNESS AND DIVERSITY TO COMPARE TWO METHODS 

Karen D. McCoy^ 
Department of Zoology. University of Guelpti, Ontario, N1G 2Wb Canada 

ABSTRACT 

Terrestrial gastropods are sometimes sampled for ecological and parasitológica! studies using 
simple traps of cardboard placed on the ground. Tfiis method enables the collection of large 
numbers of individuals with relatively little effort. However, the violation of assumptions of the trap 
method may mean that samples are biased. I examined the reliability of this method by compar- 
ing species composition, richness and diversity of gastropod collections from traps to paired 
hand-searched plots in Algonquin Park, Ontario, Canada. Ten samples for each method were 
made from five replicate sites in three habitat types, white birch, hardwood and logged forest 
stands. Of the 18 species found overall, 17 and 16 species were found using hand search and 
trap methods respectively. Some species, especially Arion circumscriptus. were consistently 
over-represented using traps, while most other species had greater representation in hand- 
search collections. In general, hand-search collections had significantly greater species richness 
and diversity than trap collections. Further, the similarity in species composition of the two col- 
lections seemed to depend, at least in white birch and hardwood habitats, on the level of site di- 
versity; as site diversity increased the percent similarity decreased. However, estimated rich- 
ness, calculated using a jackknife estimator that accounts for heterogeneity in species detection, 
was not different for the two collection methods. There was no difference in average species' de- 
tection per sample between collections; the probability of sampling a species using a single sam- 
ple within a site was low for both methods (16-34% of species). However, overall detection of 
species on a site (average across all samples within a site) tended to be higher for hand- 
searched plots, especially in more complex habitats. Thus, as trap sampling does not provide 
representative gastropod collections, this method may not be appropriate to use when physical 
collections or detailed information on species abundances are required. Conversely, as there is 
no difference between methods in estimated richness, traps may be suitable to estimate gastro- 
pod community metrics, provided appropriate estimation models are used. 

Key words: Gastropoda, land Mollusca, community ecology, species richness, diversity, sam- 
pling techniques, richness estimation. 



INTRODUCTION 

Terrestrial gastropods are often sampled 
using simple traps of moistened cardboard, 
masonite or tile, particularly for parasitological 
studies (e.g.. South, 1965; Gleich & Gilbert, 
1976; Kearney & Gilbert, 1978; Boag, 1982; 
Samuel et al., 1 985; Strayer et al., 1 986). This 
method is relatively simple and time efficient, 
and has been considered an effective means 
of sampling to estimate relative population 
density and compositional changes of gastro- 
pod communities, both spatially and tempo- 
rally (Boag. 1 982). This method assumes that 
(1 ) all species and age classes are equally at- 
tracted to the traps at the times when the traps 
are typically examined, dawn or dusk, and (2) 



that individuals are only attracted vertically 
from the litter layers beneath and not horizon- 
tally to the trap. However, there are interspe- 
cific differences in gastropod activity (Blinn, 
1963; Cameron, 1970, 1978; Cain & Cowie, 
1978; McCoy & Nudds, 1997), and in the use 
of these traps (Boag, 1990). Further, some 
gastropod species are thought to have large 
home ranges (Blinn, 1963; Thomas, 1944; 
Cook, 1979) such that individuals may be at- 
tracted to traps for shelter or food from an 
area of unknown size; there is now evidence 
that such horizontal movement to traps exists 
(Boag, 1990; Hawkins et al., 1997). Thus, the 
violation of the assumptions of trap sampling 
could result in biased gastropod collections 
both in terms of the number of individuals per 



Vresent address: Laboratoire d'Ecologie, Université Pierre et Marie Curie, C.N.R.S. - UMR 7625, 7 quai Saint Bernard, 
75252 Paris, FRANCE: kmccoy@snv.jussieu.fr 

271 



272 



McCOY 



species collected and the number of species 
represented (species richness). 

The extent to which any collection tech- 
nique accurately represents the gastropod 
community in a given area is difficult to deter- 
mine as there are limitations associated with 
all techniques (e.g., Bishop, 1977; Emberton 
et al., 1996). Bishop (1977) suggested that 
the least biased way to sample terrestrial gas- 
tropods would use stratified random quadrats 
in which litter is either searched, or removed 
and searched in the laboratory. Likewise, 
Newell (1971) reviewed numerous direct and 
indirect methods and found that hand sorting 
soil samples, while slow and laborious, pro- 
vided more accurate estimates of abundance 
of some species than did surface counts 
alone. He recommended the use of indirect 
methods (such as trap sampling) only for 
those animals that could not be reliably esti- 
mated by a direct method. Conversely, Van Es 
& Boag (1 981 ) compared hand-searched field 
samples to samples sorted in the laboratory 
after litter was dried and sieved and found that 
hand-searched collections contained fewer 
individuals than did litter samples and were bi- 
ased towards larger specimens. Emberton et 
al. (1996) suggested that combining direct, 
timed searches with litter-plus-soil samples 
was the most efficient means of quantifying 
microgastropods (< 5 mm) in tropical rain- 
forests. Others have recommended using in- 
direct trap sampling, within certain climatic 
limits, due to its efficiency in collecting 
(Hawkins et al., 1997). Thus, depending on 
the nature of ecological question under con- 
sideration and the degree of sampling bias in 
the technique, a less laborious method such 
as traps could provide a viable alternative to 
labour-intensive methods such as hand 
searching individual quadrats. 

Bias in gastropod sampling can be prob- 
lematic for numerous reasons. First, terres- 
trial gastropod fauna have been collected for 
various ecological studies: examining pat- 
terns of species distributions and biogeo- 
graphical relationships (e.g., Burch, 1956; 
Roth & Lindberg, 1 981 ; Van Es & Boag, 1 981 ; 
Cameron, 1986; Gascoigne, 1994; Tatters- 
field, 1996), evaluating biodiversity and com- 
munity changes (e.g., Nilsson et al., 1988; 
Niemelä, 1997), and studying population dy- 
namics (e.g., Williamson et al., 1977; Uminski 
& Focht, 1979). Bias in sampling methods 
may mean that ecological inferences drawn 
from collected gastropods are incorrect. Like- 
wise, terrestrial gastropods are commonly 



sampled to estimate the prevalence of para- 
sites for whom they act as intermediate hosts. 
These collections are typically used to infer 
the relative importance of different species in 
transmission (e.g., Lankester & Anderson, 
1968; Kearney & Gilbert, 1978) and to predict 
the risk of infection to vertebrate hosts (e.g., 
Boag, 1985). Nonrepresentative samples of 
gastropods can lead to inaccurate assess- 
ments of parasite prevalence and misconcep- 
tions about transmission dynamics. If surveys 
are to provide useful ecological information, a 
rigorous approach to sampling is required, 
and evaluations of existing procedures need 
to be performed to improve sampling protocol. 
In this study, I used a stratified, paired-plot 
design to directly compare trap and hand- 
search collection techniques for sampling ter- 
restrial gastropod communities in three differ- 
ent habitat types to determine whether traps 
provide representative samples of gastropod 
species composition, richness and diversity. In 
order to partially overcome the size bias inher- 
ent in hand-searched collections and to pro- 
vide a more representative collection for com- 
parison, I combined hand searches in the field 
with litter-plus-soil subsamples hand sorted in 
the laboratory. Further, to evaluate the effec- 
tiveness of trap sampling for different types of 
ecological studies, I compared estimates of 
species richness calculated using both direct 
counts and the jackknife estimator of Burnham 
& Overton (1979). The jackknife estimator of 
species richness specifically takes into ac- 
count heterogeneity in species detectability, 
that is, differences among species in their 
probability of being sampled given that they 
are present at a given sampling location. 



MATERIALS AND METHODS 
Field Methods 

The study took place in Algonquin Park, 
Ontario, Canada (45^'35'N, 78°30'W), in June 
and July 1 996. As this work was carried out in 
connection with a broader study of the trans- 
mission of a nematode parasite {Pare- 
laphostrongylus tenuis) of white-tailed deer 
{Odocoileus virginianus), three habitat types 
commonly used by white-tailed deer were 
chosen to compare collection methods: white 
birch {Betula papyrifera), mixed hardwood 
and selectively logged sites (Kohn & Mooty, 
1 971 ; Kearney & Gilbert, 1 978). Five sites for 
each habitat type were randomly selected 



SAMPLING TERRESTRIAL GASTROPOD COMMUNITIES 



273 



from a list of suitable sites. Suitable sites were 
determined by stand content (greater than 
60% white birch or hardwood content), acces- 
sibility and, for the logged sites, time since 
disturbance (logged between 1990 and 
1994). The order in which the 15 sites were 
examined was determined by lottery. 

At each site, two transects, each with five 
plot markers, were set up. The plot markers 
were spaced 10 m apart and there was 20 m 
between the two transect lines. On either side 
of the transects, 10 m from each plot marker, 
a 1 X 1-m piece of moist cardboard was set 
out. Therefore, at each site, a total area of 40 
X 40 m was examined using ten plots and ten 
paired traps. As traps are typically left out for 
a period of time before snails are collected 
from them (e.g.. Gleich & Gilbert, 1976; Kear- 
ney & Gilbert, 1978), the boards were set out 
seven days before the site was scheduled to 
be searched. Pieces of woody debris were 
placed on the top of the boards to keep them 
in place and to help maintain the moisture be- 
neath. 

All sites were examined between 0600h 
and 0900h. At each plot marker, a 1 x 1-m 
area was outlined using a wooden frame such 
that the marker was in the center of the plot. 
Two random subsamples were taken from 
within each plot using a corer of 15 cm diam- 
eter. These subsamples included leaf litter, 
soil to at least a 5 cm depth, and any plants 
and debris from within the subsample area. 
Each subsample was later handsorted in the 
laboratory. Once subsamples were removed, 
the remainder of the plot was searched for 1 
min for gastropods; all vegetation and possi- 
ble shelters were examined and leaf litter and 
soil were sifted by hand. Although it is clear 
that not all gastropod individuals were found 
using this method, the 10-min period was suf- 
ficient to thoroughly examine the entire plot 
once. After a plot was searched, its corre- 
sponding cardboard trap was examined for 
gastropods and all specimens on and under 
the board, and on the debris on top of the 
board were collected. 

The subsamples removed from each plot 
were examined the same day they were col- 
lected. To examine for gastropods, a subsam- 
ple was emptied into a large, light-coloured 
sorting pan. During a 10-min period, each leaf 
or piece of debris was individually examined, 
soil was spread and sifted by hand and all 
gastropods found were removed. Because 
the volume of each subsample was relatively 
small (1.0-1.5 L), the 10-min period allowed 



an exhaustive examination of all material. 
Nevertheless, to ensure greater representa- 
tion, particularly of microgastropods, the two 
subsamples from a single plot were sorted by 
different observers. 

All collected gastropods were counted and 
identified in the lab. Species identifications 
were made using keys by Pilsbry (1946) and 
Burch (1962). Identifications were aided by a 
zoogeographical study by Oughton (1948) 
and were confirmed using voucher specimens 
at the Royal Ontario Museum, Canada. 

Statistical Methods 

Richness for each collection method was 
calculated two ways: (1) as a direct count of 
the number of species found at a site and, (2) 
because it was unlikely that all species pres- 
ent at a site were sampled, as estimated by 
the jackknife estimator of Burnham & Overton 
(1979) using the program CAPTURE (species 
richness values can also be calculated using 
subprogram SPECRICH from software COM- 
DYN available on the Internet -http ;//www. 
mbr-pwrc.usgs.gov/comdyn.html). 

The jackknife estimator for closed popula- 
tions calculates species richness using the 
pattern of observed species occurrences 
(presence/absence) across replicated sam- 
ples within a site (Nichols & Conroy, 1996) 
and is based on a capture-recapture model 
(model M^) that takes into account hetero- 
geneity in the probability of capture among 
species. The program CAPTURE has a built- 
in selection procedure that chooses the ap- 
propriate capture-recapture model for the pre- 
sented data. There are several different 
models possible, each with different assump- 
tions regarding the source of variation in de- 
tection probabilities (Boulinier et al., 1998, 
provides an overview). Nonetheless, here I 
used only the richness estimates provided by 
model M^ as this model has been found to be 
relatively robust to other factors affecting de- 
tection probabilities (e.g., observer influence, 
quadrat heterogeneity) (Otis et al., 1978) and 
as heterogeneity in species detection is likely 
to be the main source of variation in detection 
probability when estimating species richness 
(Boulinier et al., 1998). Detectability refers to 
the probability of sampling at least one indi- 
vidual of a given species in a plot (the sam- 
pling unit) given that the species is present in 
that plot (Boulinier et al., 1998). 

It is well recognized that terrestrial gas- 
tropods tend to be heterogeneously distrib- 



274 



McCOY 



uted in space (e.g., Cameron, 1986; Ember- 
ton et al., 1996). Thus, for estimating species 
richness, the assumption of closure (i.e., that 
the community being sampled does not 
change among replicated sample locations) 
(Burnham & Overton, 1979) is likely to be vio- 
lated at some spatial scale. For this study, 
sampling methods equally covered a small 
(40 X 40 m), uniform area of forest. As terres- 
trial gastropods should be relatively mobile at 
this scale, I felt it was safe to assume that, at 
each plot or trap within a site, I sampled from 
the same gastropod community. 

The program CAPTURE also provides an 
estimate of the average instantaneous de- 
tectability of species for a given site (or the av- 
erage probability of sampling species on a 
plot) based on the number of species cap- 
tured on each plot within the site. From the 
values of estimated species richness, the 
overall detectability of species (observed # 
species/estimated # species) on a site can 
also be calculated. Thus, the average instan- 
taneous detectability gives an indication of the 
probability of capturing all species at a single 
sample location within a site (average proba- 
bility per plot), and the overall detectability de- 
scribes the probability of capturing all species 
across all sampling locations within a site 
(probability across ten plots). The jackknife 
estimator can be biased if there is a low prob- 
ability of detecting species but regardless is 
considered to provide a better estimate of 
richness than counts alone (Nichols & Con- 
roy, 1996). 

The diversity at each site was calculated 
separately for each collection method using 
DIVERS (Krebs, 1989). The Shannon-Wiener 
diversity index was considered the most ap- 
propriate diversity index to use for gastropod 
communities, as it makes no assumptions 
about the shape of the underlying distribution 
of species-abundance and is relatively insen- 
sitive to changes in the dominant species 
(Krebs, 1989). This index will likely be biased 
to different degrees depending on the species 
present in different sites, but it is still appropri- 
ate here as I compare the relative diversity of 
the two collection methods for the same loca- 
tions where the same species are available to 
be sampled. As microgastropods from hand- 
searched subsamples in the laboratory were 
directly included in the list of species for a 
given plot without extrapolating the numbers 
to the entire area of the plot, I considered tests 
for differences in diversity between methods 
to be conservative. 



If the two collection methods sample differ- 
ent species at a given location, we might not 
find differences between methods using such 
measures as richness or diversity. Thus, I cal- 
culated the average similarity in species com- 
position of methods on a site using the 
% similarity (Renkonen) coefficient for each 
plot pair (a hand-searched plot and its cor- 
responding cardboard trap). This coefficient 
uses the abundance of each species on a plot 
and can be calculated using the program SIM- 
ILAR (Krebs, 1 989). It can be biased if sample 
sizes are small and diversity is high (Wolda, 
1 981 ), but should still perform well in this case 
as gastropod diversity is relatively low in the 
study area considered and sample sizes are 
equal. Further, because the effectiveness of 
the two sampling methods could change with 
varying levels of diversity/richness at different 
sampling sites, I regressed the similarity in 
species composition of the two methods at a 
site against diversity and estimated species 
richness. As there were two estimates of di- 
versity/richness for a given site (one for each 
method), the diversity/richness estimate used 
for regressions was the higher of the two esti- 
mates. 

Calculated estimates for the two collection 
methods were statistically compared using 
analysis of variance and paired t-tests (Zar, 
1984). All tests were performed using SAS 
(SAS Institute, 1996) and were considered 
significant at the 0.05 level. 



RESULTS 

Across all three habitat types a total of 18 
gastropod species were sampled; 17 and 16 
species were found using hand-search and 
trap methods, respectively. The average num- 
ber of individuals of each species collected 
using each method is summarized in Table 1. 
One slug species, Arlon circumscriptus, was 
consistently collected in greater numbers by 
the trap method in all habitats. Six other spe- 
cies {Anguispira alternata. Discus cronkhitei. 
Zonitoides arboreus. Deroceras laeve. Meso- 
don sayanus and Stenotrema fraternum) also 
tended to be found in higher numbers in trap 
collections, although the difference between 
methods changed depending on habitat type; 
all other species had similar or greater repre- 
sentation using hand searches (Table 1). Two 
unique species (Paravitrea multidentata and 
Columella edentula) were found only by hand 
searching; individuals of С edeníu/a were only 



SAMPLING TERRESTRIAL GASTROPOD COMMUNITIES 



275 



TABLE 1. Number of individuals of each gastropod species collected in white birch, hardwood and logged 
sites using the hand-search collection method (Hand) and the trap-collection method (Trap). 



Family 


White Birch 


Hardwood 


Logged 


Total 




Species 


Hand 


Trap 


Hand 


Trap 


Hand 


Trap 


Hand 


Trap 


Arionidae 


















Arion circumscriptus (Johnston) 
Endodontidae 


222 


349 


104 


307 


57 


261 


383 


917 


Discus cronkhitei (Newcomb) 
Anguispira altérnala (Say) 
Helicodiscus parallelus (Say) 
Punctum minutissimum (Lea) 
Limacidae 


82 


42 
38 


88 
3 




16 

9 

20 


78 

1 
1 


27 

1 

26 

45 


97 

26 



1 


125 

1 

77 

103 


263 

29 

1 

2 


Dereceras laeve (Müller) 


8 


12 


3 


13 


2 


10 


13 


35 


Philomycidae 
Palifera dorsalis (Binney) 

Polygyridae 
Mesodon sayanus (Pilsbry) 
Stenotrema fraternum (Say) 
Triodopsis dentifera (Binney) 

Pupillidae 


9 

1 




3 

2 

1 



11 

1 





11 

4 
6 



17 

7 
3 



1 

28 

11 
3 


37 

9 
3 




15 

34 

18 

3 


Columella edentula (Draparnaud) 
Strobiiopsidae 

Strobilops labyrinthica (Say) 
Succineidae 


4 
77 



5 



14 



1 



2 




1 


4 
93 



7 


Succinea avails (Say) 
Vallonidae 


6 


5 


11 


18 


9 


10 


26 


33 


Zoogenetes harpa (Say) 
Zonitidae 


10 


2 


1 











11 


2 


Retlnella binneyana (Morse) 
Euconulus fulvus (Müller) 
Paravltrea multidentata (Binney) 
Zonitoides arboreus (Say) 


22 
9 
6 
4 



2 

9 


3 
15 


12 


3 
5 


24 


13 

18 

3 

39 


1 
1 


160 


38 

42 

9 

55 


4 

8 



193 


TOTAL 


540 


481 


220 


472 


269 


611 


1,029 


1,564 



detected in white birch stands. On logged 
sites, one unique species (Triodopsis denti- 
fera) was collected by traps and was not found 
during hand searches (Table 1 ). 

Overall, for both richness and diversity, 
hand-searched samples had significantly 
greater estimates than did samples from traps 
(Fi^4 = 4.75, P - 0.047; F^^^ = 7.04, P = 
0.019; Tables 2,3). Richness on a site varied 
from 4 to 13 species and diversity from 0.28 
to 2.38 for hand searches. For trap collec- 
tions, from 1 to 12 species were found on a 
site and diversity levels varied from to 
1.73. By habitat type, white birch and logged 
stands showed greater differences between 
methods in both components than hardwood 
stands, which showed very little (Table 2). 
Overall, there was no difference among habi- 
tat types in either richness or diversity, but 
there was significant variation in both mea- 
sures among different sites within the same 
habitat (Table 3). 

Unlike for observed richness, there was no 



significant difference in species richness of 
the two collections as estimated by the jack- 
knife (F^^4 - 0.27, P = 0.61; Tables 2,3). 
There was also no difference in average in- 
stantaneous detectability between the two 
methods (hand: 0.28 ± 0.03, trap: 0.23 ± 
0.03; t = 1 .1 1 , P - 0.28). Over all habitat types 
and methods, estimates of average instanta- 
neous detectability were relatively low (be- 
tween 16 and 34%), suggesting only a small 
proportion of the community is sampled using 
one plot or trap on a site. However, the over- 
all detectability at a site was higher, ranging 
between 10% and 100%, with averages of 
80% and 70% for hand and trap methods re- 
spectively (Table 2). As there was a significant 
interaction in overall detectability between 
habitat and collection technique (Table 3), the 
effect of technique for this variable was exam- 
ined for each habitat type independently. The 
overall detectability of species was higher for 
hand searches than for traps for two of the 
three habitats (Table 2); however, this differ- 



276 



McCOY 



TABLE 2. Summary of community estimates for two gastropod collection methods across three habitat 
types. Hand refers to the average estimate value (± standard error) based on collections sampled by 
quadrat based plot searches and trap refers to the average estimate value {± standard error) based on 
collections sampled using cardboard traps. 







White birch 


Hardwood 


Logged 


All sites 


Richness 


Hand 


8.8 ± 1.83 


8.0 ± 1.05 


10 ± 0.63 


8.9 ± 0.71 




Trap 


6.4 ± 1.03 


7.8 ± 1.07 


7.4 ± 1.89 


7.2 ± 0.76 


Diversity 


Hand 


1.32 ± 0.40 


0.99 ± 0.15 


1.29 ± 0.20 


1.20 ±0.15 




Trap 


0.60 ± 0.14 


0.86 ± 0.17 


0.95 ± 0.30 


0.80 ± 0.12 


Est. Richness 


Hand 


9.2 = 1.74 


15.4 ± 4.45 


11 ± 0.84 


11.87 ± 1.65 




Trap 


8.4 = 1.69 


8.4 ± 1.03 


15 ± 5.55 


10.6 ± 2.0 


Overall Detectability 


Hand 


0.86 ± 0.12 


0.67 ± 0.34 


0.87 X 0.09 


0.80 ± 0.22 




Trap 


0.76 ± 0.18 


0.88 ± 0.10 


0.47 ± 0.32 


0.70 ± 0.27 



TABLE 3. Summary of ANOVAs for gastropod community estimates from trap and hand 
search methods across three habitat types. Sources were considered significant at 
P<0.05. 





Source 


df 


Type III 


F value 


P value 


Richness 


Habitat 


2 


6.47 


0.68 


0.522 




Site(habitat) 


12 


154.40 


2.71 


0.039 




Technique 


1 


22.53 


4.75 


0.047 


Diversity 


Habitat 


2 


0.22 


0.65 


0.537 




Site(habitat) 


12 


5.28 


2.57 


0.048 




Technique 


1 


1.20 


7.04 


0.019 


Est. Richness 


Habitat 


2 


94.87 


1.07 


0.369 




Site(habitat) 


12 


697.00 


58.08 


0.310 




Technique 


1 


12.03 


0.27 


0.610 


Overall Detectability 


Habitat 


2 


0.10 


1.44 


0.275 




Site(habitat) 


12 


0.69 


1.60 


0.215 




Technique 


1 


0.071 


1.96 


0.187 




Habitat*technique 


2 


0.46 


6.34 


0.013 



ence was significant only on logged sites (t = 
3.621, P = 0.022). 

Sinnilarity in species composition of the two 
collections varied from to 86% across sites. 
White birch and hardwood sites showed re- 
lated levels of similarity [53.30% (± 21.28) 
and 52.56% (± 17.75) respectively], but aver- 
age similarity was much lower on logged sites 
[25.37% (± 25.00)]. Regressions of diversity 
and similarity, both across all habitat types 
and within each habitat type, were not signifi- 
cant at the 0.05 level. However, data from 
white birch and hardwood showed similar 
trends. When I combined the data from only 
these two habitat types, I found that as site di- 
versity increased, the two methods had more 
dissimilar compositions (Fig. 1 ; % similarity = 
(-18.93)diversity + 75.65, F. „ = 6.52, P = 



0.034, R^ = 0.449). The regressions of esti- 
mated richness and similarity showed similar 
trends, but for this measure the regression 
using only white birch and hardwood sites 
was not significant (F^ g = 2.80, P = 0.133). 
For all regressions performed, however, the 
power of significance tests were low (between 
6% and 30%), indicating a high potential to 
commit Type II errors. Further, when the com- 
position of collections were highly dissimilar at 
a site, hand searches always had higher di- 
versity than did trap collections. 



DISCUSSION 

If we assume that the hand-search collec- 
tion method provides a relatively accurate 



SAMPLING TERRESTRIAL GASTROPOD COMMUNITIES 



277 




FIG. 1. Regression of diversity of terrestrial gas- 
tropods versus percent similarity in species compo- 
sition of hand search and trap collections. Regres- 
sion line is based on data from white birch (solid 
circles) and mixed hardwood (solid squares) sites. 



representation of the gastropod community, 
then the trap collection method using card- 
board appears to be generally biased in terms 
of both observed species richness and diver- 
sity. There were evident differences between 
methods in observed richness and diversity 
for white birch and logged sites, but very little 
for hardwood stands. Further, there was a 
much lower similarity in species composition 
between the two collections in logged sites 
compared to the other two habitats. Regres- 
sions of diversity and similarity in collection 
methods, while only significant when data 
from logged sites were removed, implied that 
the methods yield relatively similar lists of 
species when community diversity is low and 
become increasingly dissimilar as diversity in- 
creases. Because of high variation in esti- 
mates among sites, even within the same 
habitat, there was a high probability of com- 
mitting a Type II error in all regressions per- 
formed; further replicates would be required 
to examine the relationship between similarity 
in collections and diversity more completely. 
Nonetheless, traps seemed to perform best, 
in terms of consistency, in hardwood stands 
and worst in logged sites. 

The observed differences among habitats 
in the degree of bias in trap collections could 
be related to either direct differences in the 
species present in the three habitats or, more 
indirectly, could be associated with differ- 



ences in habitat spatial complexity. For exam- 
ple, there tends to be less understory vegeta- 
tion and debris in hardwood stands compared 
to white birch or logged sites (pers. obs.). 
Where there are fewer potential refuge sites, 
gastropods may be more readily attracted to 
traps. Conversely, where there are erratic 
areas of potential refuge (as in recently 
logged sites), the ability to collect using traps 
will depend on the placement of the trap and 
the performance will be less predictable. 

The distance across which gastropods 
might be attracted to cardboard traps is 
largely unknown. In a study of dispersal of 
Agriolimax reticulatus (= Dereceras reticula- 
tum) on a fallow field. South (1 965) found that 
slugs travelled a mean distance of 1.13 m in 
seven days. However, Umax maximus has 
been known to move directly towards a food 
source from up to 7.5 m away (Cook, 1979). 
The slugs Arion circumscriptus and Dero- 
ceras laeve. and five snail species (family 
Polygyridae: Angulspria alternata. Mesodon 
sayanus. Stenotrema fraternum: family En- 
dodontidae: Discus cronkhitei: family Zonti- 
dae: Zontitoides arboreus), tended to be col- 
lected in higher numbers from cardboard 
traps. All of these species were, with the ex- 
ception of A. alternata, consistently found dur- 
ing hand searches and most are relatively 
large, obvious gastropods that can be easily 
spotted during searches. Such aspects sug- 
gest that these species are over-represented 
in trap collections. Because most of these 
species are robust and all are active in the leaf 
litter (McCoy & Nudds, 1997), they may "find" 
cardboard traps from greater distances and 
aggregate there. Other species, such as Stro- 
bilops labyrinthica (family Strobilopsidae), 
and some members of families Endodontidae 
{Helicodiscus paral lelus. Punctum minutlssi- 
mum) and Zonitidae (Paravitrea multidentata. 
Euconulus fulvus) appear to be underrepre- 
sented in cardboard trap collections. All of 
these species are relatively small (< 3.5 mm), 
and while some (such as H. parallelus) are 
more subterranean in nature, at least one has 
been found on tree trunks (pers. obs.) and is, 
therefore, relatively mobile. 

It is not clear why some species would be 
attracted to cardboard traps and others would 
not. It was previously shown that different 
species show different affinities for traps. For 
example, Boag (1990) demonstrated that Dis- 
cus cronkhitei used artificial masonite shelters 
consistently less often than did Euconulus ful- 



278 



McCOY 



vus. I found the opposite: D. cronkhitei was 
sampled relatively more frequently than E. ful- 
vus by cardboard traps. This further suggests 
that the affinity of different species for traps 
can change depending on the trap material 
used, in addition, considerable variation has 
been found in the frequency of snails adher- 
ing to shelters over time and depending on 
ambient temperature and moisture conditions 
(Boag, 1990; Hawkins et al., 1997). Likewise, 
there are substantial differences in activity 
and use of different micro-sites by species 
found at the same location (Cain & Cowie, 
1978: Cameron, 1978). These aspects imply 
that the quantitative characteristics of gastro- 
pod populations can vary greatly depending 
on both the sampling technique and material 
used for collection, and on the weather condi- 
tions at the time collections are performed. 

I estimated species richness using a cap- 
ture-recapture model (model M^) that incorpo- 
rated variation in detection probabilities 
among species in the community. This model 
was most frequently selected in bird commu- 
nity studies, emphasizing the potential impor- 
tance of heterogeneity in species' detection 
(Boulinier et al., 1998). While the model se- 
lection procedure of CAPTURE did not select 
model Mf., more frequently than other capture- 
recapture models for the gastropod collection 
data, this model did fit more than 80% of the 
time for both methods, suggesting that het- 
erogeneity in species detection exists among 
gastropod species. 

Overall species detection at a site (i.e., 
probability of capturing a species at least 
once across all sampling locations) tended to 
be higher for hand-searched plots compared 
to traps, but was typically between 70% and 
80%, meaning that most species were seen 
by both methods when ten replicates within a 
site were used. At the scale of the plot, how- 
ever, the average instantaneous detectability 
of gastropod species was low. A low probabil- 
ity of detecting species on plots means that 
estimated richness could be slightly biased. 
Nonetheless, previous work suggests that it 
should still be closer to the actual number of 
species than simple counts alone (Nichols & 
Conroy, 1996). For this study, the estimated 
richness values found match well with the 
number of species recorded for the area. For 
example, I observed an average of nine 
species per site, but calculated a estimated 
average richness of 12 species. As 18 spe- 
cies were found overall and 27 species have 
been previously reported in the Algonquin re- 



gion (Oughton, 1948), I expect that a few 
species were never sampled on a site. Thus, 
the low probability of detecting species on a 
plot underlines the potential problems associ- 
ated with using simple counts for determining 
species richness, regardless of the sampling 
technique used and demonstrates the useful- 
ness of species richness estimators. These 
types of estimators are now being more fre- 
quently employed in community-level studies 
(e.g., Derleth et al., 1989; Morrison, 1996; 
Boulinier et al., 1998; Nichols et al., 1998a). 

The differences I found between the collec- 
tion methods can have important implications 
for studies that require representative sam- 
ples of the gastropod community. For exam- 
ple, this study was performed as part of a 
larger project examining the transmission of 
Parelaphostrongylus tenuis, a common nema- 
tode parasite of white-tailed deer {Odocoileus 
virginianus) in northeastern North America. 
Terrestrial gastropods act as intermediate 
hosts for this and other such parasites (Ander- 
son & Prestwood, 1 981 ). To assess the poten- 
tial risk of transmission of these parasites and 
to study the transmission ecology in general, 
gastropods are commonly collected using 
traps (Kearney & Gilbert, 1 978; Samuel et al., 
1985; Upshall et al., 1986: Robb & Samuel, 
1990). If certain species are missed or mis- 
represented in samples, calculated estimates 
of prevalence may be incorrect. For example, 
many of the species I found to be over-repre- 
sented in trap collections (e.g., D. cronkhitei, 
Z. arboreus anä D. laeve) are typically consid- 
ered to be important intermediate hosts of P. 
tenuis based on their relative abundance and 
prevalence of infection (e.g., Lankester & An- 
derson, 1968: Piatt, 1989). Further, the role in 
parasite transmission played by many of the 
species I found to be commonly underrepre- 
sented in trap collections has never been as- 
sessed. In addition, the bias in the trap method 
appears to be stronger in high diversity sites, 
such as white birch stands, and these stands 
have been considered to be potential trans- 
mission foci (Kearney & Gilbert, 1978). Thus, 
due to sampling technique, estimates of 
prevalence and our understanding of trans- 
mission of parasites such as P. tenuis could be 
inaccurate. 

Richness, based on simple counts, and di- 
versity were significantly different for the two 
collection methods, but there was no differ- 
ence between methods in estimated richness. 
This suggests that for the examination of gas- 
tropod community richness alone, collections 



SAMPLING TERRESTRIAL GASTROPOD COMMUNITIES 



279 



from cardboard traps can be as representa- 
tive as collections based on hand searches. 
Estimates of richness are essential in many 
ecological studies, and invertebrates are only 
now being considered more in studies of bio- 
diversity (e.g., Dobyns, 1997; Niemelä, 1997). 
Therefore, while cardboard traps are not reli- 
able for physically obtaining a representative 
gastropod collection, they may be sufficient 
for establishing the pattern of species occur- 
rences such that estimates of richness are 
similar to what they would be if a more labori- 
ous and time consuming method of searching 
individual quadrats was performed. Other 
studies on invertebrates have also found that 
certain species were missed by different col- 
lection methods and that estimation tech- 
niques were useful for determining overall 
richness (Morrison, 1996; Dobyns, 1997). 
Nevertheless, estimation techniques are still 
limited in that they can not necessarily provide 
information about specific species changes 
within the community as some species are 
never actually detected. However, methods to 
make inferences about such community at- 
tributes are now being developed (Mines et 
al., 1999; Nichols et al., 1998a, b). 

Various methods have been developed and 
used to sample terrestrial gastropods. How- 
ever, as there are problems and biases asso- 
ciated with all sampling techniques, the most 
appropriate method will depend on the ques- 
tion being asked. If the goal is to estimate the 
richness of a gastropod community, it is ap- 
propriate to use simple cardboard traps to 
sample as long as the estimation model used 
takes into account potential heterogeneities 
inherent in the system. However, if the goal is, 
for example, to determine the relative abun- 
dance of species over space and time and/or 
to estimate the prevalence of a parasite (such 
as P. tenuis), traps may not be sufficient; po- 
tentially important components of the commu- 
nity may be missed or mis-represented in in- 
consistent manners. I recommend collecting 
gastropods by hand searching quadrats, 
using sorted subsamples, whenever this 
method can practically be undertaken. 



ACKNOWLEDGMENTS 

Many thanks to Stephanie Edwards, 
Thomas Nudds, Alison Stuart, Lisa Enright, 
Heather Hager, Elizabeth Boulding, Ronald 
Brooks and Nils Chr. Stenseth for comments 
and assistance at various stages of this work. 



Thanks also to Thierry Boulinier for running 
my data through CAPTURE and for critical 
comments. Helpful comments and sugges- 
tions for improving this manuscript were also 
provided by two anonymous reviewers. The 
Ontario Ministry of Natural Resources, Parks 
Ontario kindly permitted this work to be car- 
ried out in Algonquin Park and the staff at the 
Wildlife Research Station provided a wonder- 
ful place to work. This work was supported by 
a Natural Sciences and Engineering Re- 
search Grant to TD. Nudds and by the Envi- 
ronmental Youth Corp (EYC). 



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MALACOLOGIA, 1999, 41(1): 283-296 

POPULATION STRUCTURE IN A SNAIL SPECIES FROM ISOLATED MALAYSIAN 
LIMESTONE HILLS, INFERRED FROM RIBOSOMAL DNA SEQUENCES 

M. Schilthuizeп^*^ J. J. Vermeulen^ G. W. H. Davison^ & E. Gittenberger2'^ 

ABSTRACT 

We sequenced the first internal transcribed spacer (ITS-1) of the ribosomal DNA in nine pop- 
ulations of the vertiginid Gyliotrachela hungerfordiana. which lives on isolated (and threatened) 
limestone hills in the Malaysian peninsula. Current data suggest that the species is an obligate 
calcicole. The application of a tentative molecular clock suggests a Quaternary divergence for 
the G. hungerfordiana populations. A strong positive correlation between genetic and geographic 
distance was observed, which, combined with geological data, suggests that the hill populations 
may be interconnected by as yet unsampled populations. 

Key words: internal transcribed spacer, ITS-1, Gastropoda, Pulmonata, Vertiginidae, 
Gyliotrachela, gene flow, Southeast Asia. 



INTRODUCTION 

Land snails have proverbially poor abilities 
for dispersal (e.g., Cowie, 1984; Schilthuizen 
& Lombaerts, 1994), which causes them to 
show evolutionary patterns at much smaller 
spatial scales than many other organisms of 
similar size. As a result, strong geographic 
structunng of populations is common in snails 
(e.g., in Liguus: Hillis et al., 1987). Another 
consequence is endemism, which is seen, for 
example, in the Mediterranean clausiliid 
genus Albinaria, of which almost 30 species 
are endemic to the island of Crete, with distri- 
bution areas of sometimes only one kilometer 
across (Gittenberger, 1991; Welter-Schultes, 
1998). 

An impressive situation of high endemism 
and geographic structuring of land snails in a 
strongly fragmented habitat exists in peninsu- 
lar Malaysia. Here, limestone is exposed in 
the form of "tower karst" and other karstifica- 
tions, limited to about three hundred hills, 
scattered over the peninsula. These hills are 
often very small, the largest with a diameter of 
a few kilometers, but most measuring only a 
few hundred meters across. In spite of their 
small size, the hills are a prominent feature of 
the landscape, because they usually stand 
isolated, are riddled with caves and are 
bounded by precipitous cliffs. 



For more than a century, malacologists 
have been interested in the rich malacofauna 
that the hills support (de Morgan, 1885). High 
numbers of species are found, and the mor- 
phologies of some Diplommatinidae fore- 
shadow the bizarre and extravagant forms 
found in this group in Borneo (Vermeulen, 
1993, 1994; Gittenberger, 1995). But espe- 
cially fascinating is the staggering degree of 
endemism in these calcicolous snails. 
Tweedie (1961) gave an overview of six taxa 
containing many obligate caldcóles {Diplom- 
matina, Opisthostoma. Vertiginidae, Discarte- 
mon, Oophana, and Sinoennea). He listed the 
presence of 106 species on 28 hills or hill- 
clusters, of which 70 are endemic to only one 
locality. Some calcicolous species, however, 
are widespread and occur on almost all hills 
without a trace of morphological differentia- 
tion (e.g. Gyliotrachela hungerfordiana and 
some Alycaeus species). 

Geologically, the hills form the exposed 
parts of a number of larger paleozoic lime- 
stone deposits, which are elsewhere overlain 
by non-calciferous alluvial deposits (Gale, 
1986; Crowther, 1986). Some hills may thus 
have been connected in the past, while others 
have always been separate. Consequently, 
the hills form virtual "islands" for obligately 
calcicolous land snails, which they may reach 
by incidental dispersal. Alternatively, the pop- 



^ Department of Genetics. Wageningen Agricultural University. Dreijenlaan 2. 6703 HA Wageningen, Ttie Netherlands; 

Menno.Schilthuizen@fungen.el.wau.nl 

^National Museum of Natural History "Naturalis". P.O. Box 9517. 2300 RA Leiden, The Netherlands 

%WF Malaysia. Locked Bag No. 911. Jalan Sultan P.O.. 46990 Petaling Jaya. Selangor, Malaysia 

''institute of Evolutionary and Ecological Sciences, Leiden University, Leiden, The Netherlands 

'correspondence 



283 



284 



SCHILTHUIZENETAL. 



ulations on the hills may be relicts from a time 
when the hills were part of large continuous 
plateaus, which were subsequently frag- 
mented. 

In this paper, we examine a relatively wide- 
spread representative of the peninsular 
Malaysian hill malacofauna, using molecular 
and geological data, to answer the following 
questions; (1) what pattern of phylogeo- 
graphic relationships exists among the popu- 
lations of this widespread species, and (2) 
how has the population structure been 
shaped, that is, what are the relative influ- 
ences of dispersal and habitat fragmentation 
over geological time? 

By analyzing the variance in a noncoding 
nuclear DNA marker, we attempt to differenti- 
ate between various alternative population 
structures. In the case of ancient vicariance, 
we expect to find genetic distances that reflect 
the age of fragmentation of the limestone hills, 
while dispersal would result in genetic dis- 
tances more or less related to geographic dis- 
tance. Under the latter hypothesis (dispersal), 
indications of the type and frequency of dis- 
persal may be gleaned from the degree of 
correlation between genetic and geographic 
distance; if dispersal is randomly oriented 
(i.e., corresponding to an island model of pop- 
ulation structure; Wright, 1931), stochasticity 
would result in a poor fit, while dispersal 
occurring mainly among neighboring hills (i.e., 
corresponding to a stepping-stone model; 
Kimura, 1953) would be revealed by a strong 
correlation (Kimura & Weiss, 1964). 

Sadly, there are other motives for working 
on this fauna. The hills of peninsular Malaysia 
are disappearing and becoming depauperate 
at an alarming rate. Forest clearing has de- 
stroyed the vegetation on some hills; and in 
the densely populated areas near Ipoh and 
Kuantan, many hills are being removed by 
quarrying. The true rate of species loss can 
only be guessed at, but the extinction of at 
least one endemic snail species, Opisthos- 
toma sciaphilum, from Bukit Ranching, has 
been documented (Schilthuizen et al., un- 
publ.). 




FIG. 1. Gyliotrachela hungerfordiana (von Möilen- 
dorff). Scale bar = 1 mm. 




FIG. 2. Gyliotrachela frequens van Benthem 
Jutting. Scale bar = 1 mm. 



species G. frequens (Fig. 2) was selected to 
serve as an outgroup in the phylogenetic 
analysis. 



MATERIAL AND METHODS 
Selection of Taxa 

We selected the widespread and morpho- 
logically uniform vertiginid Gyliotrachela hun- 
gerfordiana for study (Fig. 1). The related 



Collecting 

In July 1 997, the first author visited 22 lime- 
stone hills in the West-Malaysian states of Pa- 
hang, Kelantan, Perakand Perils. Living snails 
were discovered by eye using two strategies: 
(a) close inspection of limestone rock faces, 



POPULATION STRUCTURE OF A MALAYSIAN LAND SNAIL 



285 



either damp or dry, bare or covered in algae, 
mosses and lichens; and (b) sifting through 
damp and decaying leaf litter on limestone 
rocks or at the base of the limestone cliffs. All 
snails were put in 100% ethanol on the spot 
and kept at ambient temperatures until arrival 
in the laboratory for further processing. Identi- 
fication of the material was carried out by the 
second author while the material remained in 
alcohol. Gyliotrachela hungerfordiana was 
collected from nine of the 22 localities (Fig. 3): 
loc. 5, State of Pahang: Qua Bama (ca. 10 km 
W of Kuala Lipis); loc. 8, State of Kelantan; 
Gua Musang, southern of the two hills that the 
road to Kuala Kerai passes between; loc. 9, 
State of Kelantan: rocks 59 km in the direction 
of Gua Musang, measured along the road 
from Kuala Krai; loc. 16, State of Perak; Bukit 
Tambun (ca. 6 km E of Ipoh); loc. 22, State of 
Perak; hill directly east of Sungai Siput Ufara 
hospital; loc. 23, State of Perlis; hill ca. 1 km S 
of Kangar; loc. 24, State of Perlis; 9 km along 
the road from Kangar to Kaki Bukit; loc. 25, 
State of Perlis; Gua Kelam at Kaki Bukit; loc. 
26, State of Perlis, Timah Tasoh (ca. 1 6 km NE 
of Kangar). All samples were taken between 
27. vi. 1997 and 17.vii.1997. Gyliotrachela fre- 
quens was taken only from locality 8. Voucher 
specimens have been deposited in the collec- 
tion of the National Museum of Natural History 
"Naturalis", Leiden. 

Molecular Techniques 

DNA was isolated from pools of between 
one and five complete snails with their shells, 
using either a phenol/chloroform extraction as 
described previously (Schilthuizen et al., 
1998a) or a sucrose-based protocol (van 
Moorsel & van Nes, unpublished), which can 
be briefly summarized as follows. Snails were 
ground in 200 ц1 of sucrose-buffer (0.1 M Tris; 
0.02 M NaCI; 0.2 M sucrose; 0.05 M EDTA) 
and centhfuged. The pellet was incubated at 
65°C for 60 min in 200 ).il SDS-buffer (0.02 M 
Tris; 0.01 M EDTA, 1 .25% SDS), 1 5 |.il of cold 
KAc was added, and the mixture was incu- 
bated on ice for 60 min and centrifuged. The 
DNA was precipitated from the supernatant by 
the addition of two volumes of 100% ethanol 
and incubation at -20°C for 30 min. The DNA 
was dried and treated with 200 ng of RNase. 
Full details can be obtained from M.S. on re- 
quest. Homogenization was always done with 
a sterile, disposable plastic pestle. The DNA 
was dissolved in 50 |.il of Tris-EDTA buffer 
(phenol protocol) or 30 )il of ddH20 (sucrose 



protocol) and stored at -20''C. The first inter- 
nal transcribed spacer of the nuclear riboso- 
mal DNA was amplified with the SuperTaq 
enzyme (HT Biotechnology, Cambridge, Eng- 
land) as described previously (Schilthuizen et 
al., 1995) and isolated using the "freeze- 
squeeze" technique (Tautz & Renz, 1 983). Be- 
cause PCR-amplification was at times too 
weak for direct sequencing, we resorted to 
cloning (PCR-based error is usually not a con- 
cern with this methodology; Schilthuizen et al., 
1 998b). After isolation, the fragments were lig- 
ated into Promega or Invitrogen T-tailed vec- 
tors, following the manufacturer's instructions. 
Colonies were screened for the presence of 
the correct insert by PCR. Plasmid DNA was 
isolated from the bacteria using QIAPrep spin 
columns (QIAGEN). One or two clones per 
sample were sequenced in both directions on 
an ABI automated sequencer. 

Alignment 

Before alignment, all chromatograms were 
checked and reading errors were corrected 
blindly where necessary (this never amounted 
to more than three corrections in a single 
sequence). Vector and primer sequences 
were removed. Sequences in the ingroup 
were sufficiently similar to allow manual align- 
ment. Wherever alignment with the outgroup 
was ambiguous, missing data were intro- 
duced into the outgroup sequence. 

Phylogenetic Analysis 

Phylogenetic analyses of the data set were 
performed in PAUP3.1 (Swofford, 1993). Gaps 
were treated as missing data. Searches for the 
most parsimonious trees were carried out with 
the branch-and-bound option. Bootstrap repli- 
cates were carried out 1 00 times, using heuris- 
tic searches. In addition, Bremer (1988) sup- 
port was determined. Kimura's 2-parameter 
genetic distances (Kimura, 1980) were calcu- 
lated with the DNADIST program of the 
PHYLIP package (Felsenstein, 1995). 



RESULTS 

PCR-products ranged in length from 755 to 
772 bp, including primers (52 bp), and the 
flanking regions of 1 8S (1 46 bp) and 5.8 S (87 
bp). These lengths correspond well with other 
ITS-1 lengths reported in mollusks (Anderson 



286 



SCHILTHUIZENETAL. 



50 km 



Penang 




N 



Kuan tan 



FIG. 3. A map of the northern part of Peninsular Malaysia, with the limestone hills drawn in black (modified 
after Gobbett, 1965). The numbers refer to localities where Gyliotrachela hungerfordiana and G. frequens 
were collected (see text for further details). 



& Adiard, 1994; Schilthuizen et al., 1995; 
Armbruster et al., unpubl.). 

We obtained sixteen sequences from G. 
hungerfordiana and one sequence for the out- 
group, G. frequens (Appendix, Table 1). They 
have been deposited in GenBank under 
accession numbers AF118000-AF118016. 
Only small genetic distances were found 
among the G. hunderfordiana sequences, the 
largest being 0.048 between sequence a from 
locality 5 and sequence b from locality 23. A 
comparison between pairwise genetic dis- 
tances and pairwise geographic distances 
between sequences revealed a strongly sig- 
nificant (p < 0.005) positive correlation (Fig. 4, 
Appendix, Table 2). The phylogenetic analysis 
produced 18 most parsimonious trees (length 
= 89 steps, Rl = 0.95), which showed two 



alternative topologies for three monophyletic 
groups of sequences, and otherwise only 
minor differences in topology within each of 
these three monophyletic groups (Figs. 5, 6). 
The fact that duplicate sequences from a sin- 
gle locality always formed monophyletic 
groups might justify the small sample sizes. 
Geographic structuring is apparent in the 
trees also, as these show monophyly for the 
sequences derived from populations in Perils, 
Pahang -i- Kelantan, and Perak. 



DISCUSSION 

Unfortunately, it is difficult to estimate reli- 
ably from the molecular data the time since 
divergence. Unlike the situation for mitochon- 



POPULATION STRUCTURE OF A MALAYSIAN LAND SNAIL 



287 



0.05 -[- 

0.045 -- 

0.04 -- 

0.035 -- 

0.03 -- 

0.025 -- 

0.02 -- 

■ 

0.015 --. ■ 

■ ■ 

0.01 I"' 

0.005 ¿-„ 

I 

■ ■■ 

Á 



100 200 300 400 

geographic distance (km) 

FIG. 4. The relationship between geographic dis- 
tance and Kimura's 2-parameter distance for the 
sequences of Gyliotrachela hungerfordiana. 

drial DNA, corroborated molecular clocks for 
the ITS regions are hardly available yet, and 
where they are, they differ by orders of mag- 
nitude among taxonomic groups. In the 
angiosperm families Cucurbitaceae and 
Winteraceae, substitution rates of 3.62 x 10"^ 
and 3.4 X 10"^ per site per million years (MY) 
were calculated, respectively (Jobst et al., 
1998; Suh et al., 1993), while in Chlorophyta, 
a rate of 0.8 - 2.0 x 10"^ was estimated 
(Bakker et al., 1 995). In animals, rates of sub- 
stitution in ITS appear to be somewhat higher. 
Schlötterer et al. (1994) give a figure of 1 .2 x 
10~^ for Drosophila, and preliminary data for 
clausiliid land snails from Greek islands indi- 
cate a similar rate (van Moorsel, unpublished 
data). 

Here, we will adopt a substitution rate of 1 x 
10~^ per site per MY as a very rough molecu- 
lar clock. Applying this rate to the average 
genetic distance between sequences on 
either side of the node basal to all G. hunger- 
fordiana sequences in the trees, we obtained 
an estimated divergence time of 1.8 MYA for 
the populations of G. hungerfordiana. It 
should be stressed that, given the lack of 
agreement in the few calibrated molecular 
clocks available, not too much confidence 
should be placed on this date. However, it 
may be safe to assume a Late Tertiary or 
Quaternary origin for G. hungerfordiana. 



Given the low degree of genetic divergence 
among the G. hungerfordiana populations, it 
seems unlikely that vicahance has played an 
important role; hills which have been studied 
geologically are thought to be older than Late 
Tertiary/Quaternary (Gale, 1986). However, in 
view of the uncertainty about the calibration of 
the ITS-1 molecular clock, this reasoning may 
be little meaningful. More importantly, geolog- 
ical data indicate that most of the hills from 
which the species was sampled have never 
been part of one continuous plateau (Paton, 
1961). It is for this reason not likely that vic- 
ahance events have been important in its dis- 
tribution pattern. Rather, the limestone hills on 
which it lives now must have been colonized 
after dispersal. 

Several mechanisms for passive dispersal 
in small snails have been suggested, includ- 
ing wind and water mediated dispersal. In ref- 
erence to Gyliotrachela and similar snails, 
Tweedie (1961) has suggested that flooding 
may be important in producing dispersal 
among hills that are situated close together. 
However, the drainage patterns in the penin- 
sula preclude any long-range dispersal by this 
mechanism. Stagnant water may also provide 
means of dispersal, and geological data 
(Gale, 1986; Crowther, 1986) indicate that 
lacustrine conditions have prevailed around 
several limestone hills in the past. But here, 
too, dispersal would be across very small dis- 
tances. Another possibility is wind-dispersal. 
Kirchner et al. (1997) demonstrate how 
Truncatellina, a vertiginid very similar in size 
to G. hungerfordiana, could be blown over 
distances of several kilometers during storms. 

Some additional characteristics of dispersal 
may be gleaned from Figure 4, which suggests 
a linear relationship between geographic and 
genetic distance. If dispersal from one hill to 
another were infrequent and undirected (i.e., a 
population structure corresponding to Wright's 
[1 931 ] island model, where all possible pairs of 
subpopulations are equally likely to exchange 
migrants), such a clear relation would not be 
expected. The fact that genetic distance is re- 
liably predicted (r^ = 0.77) by geographic dis- 
tance, suggests that a structured network of 
dispersal connects the hills. This corresponds 
to a stepping-stone model (Kimura, 1953). 
Under such a model, genetic similarities drop 
steeply with increasing numbers of intervening 
populations (Kimura & Weiss, 1964). The fact 
that we observe a strong relationship with ge- 
ographic distance, suggests that the hill popu- 



96 



hungerfordiana.lod 6#a 



32 — hungerfordiana.loc22#a 
''^ — hungerfordiana.lod 6#b 

hungerfordiana.loc22#b 

hungerfordiana.loc23#a 
hungerfordiana.loc23#b 



dO I 



d2 



hungerfordiana.loc24#a 
hungerfordiana.loc24#b 
hungerfordiana.loc25#a 
hungerfordiana.loc25#b 
hungerfordiana.loc26#a 
hungerfordiana.loc5#a 



611- hi 
M 



d2 L 
83 



— hungerfordiana.loc5#b 
hungerfordiana.loc8#a 

hungerfordiana.loc8#b 

hungerfordiana.loc9#a 



frequens 



FIG. 5. A representative most parsimonious tree of the Gyliotrachela sequences. Bootstrap percentages and 
decay indices have been indicated on the branches. 



hungerfordiana.lod 6#a 

hungerfordiana.loc22#a 

hungerfordiana.lod 6#b 

hungerfordiana.loc22#b 

hungerfordiana.loc23#a 

hungerfordlana.loc24#a 

hungerfordiana.loc24#b 

hungerfordiana.loc25#a 

hungerfordiana.loc25#b 

hungerfordiana.loc26#a 

hungerfordiana.loc23#b 

hungerfordiana.loc5#a 

hungerfordiana.loc5#b 

hungerfordlana.loc8#a 

hungerfordiana.loc8#b 

hungerfordiana.loc9#a 

frequens 



FIG. 6. Strict consensus over all 18 most parsimonious trees. 



POPULATION STRUCTURE OF A MALAYSIAN LAND SNAIL 



289 



lations cannot represent directly adjacent pop- 
ulations in a two-dimensional stepping-stone 
lattice. Rather, to obtain this result, it is neces- 
sary to postulate unsampled populations in 
between. Unfortunately, the population genet- 
ics of ribosomal DNA are as yet far from clear 
(Hillis et al., 1991; Rich et al., 1997), which 
makes a quantitative analysis of dispersal pa- 
rameters and spatial details of the population 
structure impossible. Therefore, it is not possi- 
ble to tell whether the hills that separate our 
sample sites (e.g., the six or more hills be- 
tween sites 8 and 9) will suffice as additional 
stepping stones. This might be tested, for in- 
stance, by exhaustively sampling the hills in a 
given subregion. 

ACKNOWLEDGEMENTS 

The authors wish to thank Sook-Peng 
Phoon, Fenna Schilthuizen, Jan Schilthuizen, 
and Kai Foon Phoon for help in the field. 
Stephen Gale (University of Sydney, Aus- 
tralia), Ian Metcalfe (University of New Eng- 
land, Armidale, Australia), and the Geological 
Survey (Kuala Lumpur, Malaysia) provided 
geological data and references. Tony van 
Kampen operated the automated DNA se- 
quencer, and the Department of Entomology 
(Wageningen Agricultural University) provided 
a camera lucida. Coline van Moorsel and Ton 
de Winter participated in fruitful discussions 
and kindly checked the manuscript. M.S. ac- 
knowledges financial support from the Nether- 
lands Organisation of Scientific Research. 

LITERATURE CITED 

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BAKKER, F. T, J. L. OLSEN & W. T STAM, 1995, 
Evolution of nuclear rDNA ITS sequences in the 
Cladophora albida sericea clade (Chlorophyta). 
Journal of l^olecular Evolution. 40: 640-651 . 

BREMER, K., 1988, The limits of amino acid 
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COWIE, R. H., 1984, Density dispersal, and neigh- 
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Revised ms. accepted 29 April 1999 



POPULATION STRUCTURE OF A MALAYSIAN LAND SNAIL 
APPENDIX 



291 



TABLE 1 . Aligned sequences for Gyliotrachela hungerfordiana and G. frequens. The 5' end of the 1 8S region 
is at position 146, the 3' end of the 5.8S region is at position 694. 





10 

1 


20 
1 


30 

1 


40 
1 


50 
1 


iiungerfordiana 


.loc5#a 


AGCGGTTCAG 


TGAGGGCCTC 


GGATTGGTCT 


CGGTCTGGTG 


CGCAAGTGCC 


nungerfordiana 


.loc5#b 


AGCGGTTCAG 


TGAGGGCCTC 


GGATTGGTCT 


CGGTCTGGTG 


CGCAAGTGCC 


lunger fordiana 


. loc8#a 


AGCGGTTCAG 


TGAGGGCCTC 


GGATTGGTCT 


CGGTCTGGTG 


CGCAAGTGCC 


lunger fordiana 


.loc8#b 


AGCGGTTCAG 


TGAGGGCCTC 


GGATTGGTCT 


CGGTCTGGTG 


CGCAAGTGCC 


lunger fordiana 


.loc9#a 


AGCGGTTCAG 


TGAGGGCCTC 


GGATTGGTCT 


CGGTCTGGTG 


CGCAAGTGCC 


lunger fordiana 


locl6#a 


AGCGGTTCAG 


TGAGGGCCTC 


GGATTGGTCT 


CGGTCTGGTG 


CGCAAGTGCC 


lunger fordiana 


locl6#b 


AGCGGTTCAG 


TGAGGGCCTC 


GGATTGGTCT 


CGGTCTGGTG 


CGCAAGTGCC 


lunger fordiana 


loc22#a 


AGCGGTTCAG 


TGAGGGCCTC 


GGATTGGTCT 


CGGTCTGGTG 


CGCAAGTGCC 


lunger fordiana 


loc22#b 


AGCGGTTCAG 


TGAGGGCCTC 


GGATTGGTCT 


CGGTCTGGTG 


CGCAAGTGCC 


lunger fordiana 


loc23#a 


AGCGGTTCAG 


TGAGGGCCTC 


GGATTGGTCT 


CGGTCTGGTG 


CGCAAGTGCC 


rtunger fordiana 


loc23#b 


AGCGGTTCAG 


TGAGGGCCTC 


GGATTGGTCT 


CGGTCTGGTG 


CGCAAGTGCC 


lunger fordiana 


loc24#a 


AGCGGCTCAG 


TGAGGGCCTC 


GGATTGGTCT 


CGGTCTGGTG 


CGCAAGTGCC 


lunger fordiana 


loc24#b 


AGCGGTTCAG 


TGAGGGCCTC 


GGATTGGTCT 


CGGTCTGGTG 


CGCAAGTGCC 


lunger fordiana 


loc25#a 


AGCGGTTCAG 


TGAGGGCCTC 


GGATTGGTCT 


CGGTCTGGTG 


CGCAAGTGCC 


lunger fordiana 


loc25#b 


AGCGGTTCAG 


TGAGGGCCTC 


GGATTGGTCT 


CGGTCTGGTG 


CGCAAGTGCC 


lunger fordiana 


loc26#a 


AGCGGTTCAG 
AGCGGTTCAG 

60 

1 


TGAGGGCCTC 
TGAGGGCCTC 

70 
1 


GGATTGGTCT 
GGATTGGTCT 

80 

1 


CGGTCTGGTG 
CGGTCTGGTG 

90 
1 


CGCAAGTGCC 
CGCAAGTGCC 

100 

1 


frequens 




lunger fordiana 


loc5#a 


GGCACCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


lunger fordiana 


loc5#b 


GGCACCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


lunger fordiana 


loc8#a 


GGCACCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


lunger fordiana 


loc8#b 


GGCACCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


lunger fordiana 


loc9#a 


GGCACCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


lunger fordiana 


locl6#a 


GGCGCCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


lunger fordiana 


locl6#b 


GGCACCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


lunger fordiana 


loc22#a 


GGCACCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


lunger fordiana 


loc22#b 


GGCATCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


lunger fordiana 


loc23#a 


GGCACCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


lunger fordiana 


loc23#b 


GGCACCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


lunger fordiana 


loc24#a 


GGCACCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


lunger fordiana 


loc24#b 


GGCACCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


lunger fordiana 


loc25#a 


GGCACCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


lunger fordiana 


loc25#b 


GGCACCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


lunger fordiana 


loç26#a 


GGCACCGCTG 


GCCGAGAAGA 


AGCTCGAACT 


CGATCGCTTG 


GAGAAAGTAA 


frequens 




GGCACCGCTG 

110 
1 


GCCGAGAAGA 

120 
1 


AGCCCGAACT 

130 

1 


CGATCGCTTG 

140 
1 


GAGAAAGTAA 

150 

1 




lunger fordiana 


loc5#a 


AAGTCGTAAC 


AAGGTTTCCG 


TAGGTGAACC 


TGCGGAAGGA 


TCATTAACGG 


lunger fordiana 


loc5#b 


AAGTCGTAAC 


AAGGTTTCCG 


TAGGTGAACC 


TGCGGAAGGA 


TCATTAACGG 


lunger fordiana 


loc8#a 


AAGTCGTAAC 


AAGGTTTCCG 


TAGGTGAACC 


TGCGGAAGGA 


TCATTAACGG 


lunger fordiana 


loc8#b 


AAGTCGTAAC 


AAGGTTTCCG 


TAGGTGAACC 


TGCGGAAGGA 


TCATTAACGG 


lunger fordiana 


loc9#a 


AAGTCGTAAC 


AAGGTTTCCG 


TAGGTGAACC 


TGCGGAAGGA 


TCATTAACGG 


lunger fordiana 


locl6#a 


AAGTCGTAAC 


AAGGTTTCCG 


TAGGTGAACC 


TGCGGAAGGA 


TCATTATCGG 


lunger fordiana 


locl6#b 


AAGTCGTAAC 


AAGGTTTCCG 


TAGGTGAACC 


TGCGGAAGGA 


TCATTATCGG 


lunger fordiana 


loc22#a 


AAGTCGTAAC 


AAGGTTTCCG 


TAGGTGAACC 


TGCGGAAGGA 


TCATTATCGG 


lunger fordiana 


loc22#b 


AAGTCGTAAC 


AAGGTTTCCG 


TAGGTGAACC 


TGCGGAAGGA 


TCATTATCGG 


lunger fordiana 


loc23#a 


AAGTCGTAAC 


AGGGTTTCCG 


TAGGTGAACC 


TGCGGAAGGA 


TCATTATCGG 


lunger fordiana 


loc2 3#b 


AAGTCGTAAC 


AAGGTTTCCG 


TAGGTGAACC 


TGCGGAAGGA 


TCATTATCGG 


iiunger fordiana 


loc24#a 


AAGTCGTAAC 


AAGGTTTCCG 


TAGGTGAACC 


TGCGGAAGGA 


TCATTATCGG 


tiunger fordiana 


loc24#b 


AAGTCGTAAC 


AAGGTTTCCG 


TAGGTGAACC 


TGCGGAAGGA 


TCATTATCGG 


lunger fordiana 


loc25#a 


AAGTCGTAAC 


AAGGTTTCCG 


TAGGTGAACC 


TGCGGAAGGA 


TCATTATCGG 


lunger fordiana 


loc25#b 


AAGTCGTAAC 


AAGGTTTCCG 


TAGGTGAACC 


TGCGGAAGGA 


TCATTATCGG 


lunger fordiana 


loc26#a 


AAGTCGTAAC 
AAGTCGTAAC 


AAGGTTTCCG 
AAGGTTTCCG 


TAGGTGAACC 
TAGGTGAACC 


TGCGGAAGGA 
TGCGGAAGGA 


TCATTATCGG 
TCATTATCGG 


frequens 





292 



SCHILTHUIZEN ETAL. 



TABLE 1. Continued. 



160 170 

\ L 

TATAAT CATCA 

TATAAT CATCA 

TATAAT CATCA 

TATAAT CATCA 

TATAAT CATCA 

TATAAT CATCA 

TATAAT CATCA 

TATAAT CATCA 

TATAAT CATCA 

TATAAT CATCA 

TATAAT CATCA 

TACAAT CATCA 

TACAAT CATCA 

TATAAT CATCA 

TATAAT CATCA 

TATAAT CATCA 

TATTATTACA AAATACGTCA 

210 220 

I L 

TGAAATTA — TGCTGAT 

TGAAATTA — TGCTGAT 

TGAAATTA — TGCTGAT 

TAAAATTA — TGCTGGT 

TGAAATTA — TGCTGAT 

TGAAAATA — TGCTGAT 

TGAAAATA — TGCTGAT 

TGAAAATA — TGCTGAT 

TGAAAATA — TGCTGAT 

TGAAAATA — TGCTGAT 

TGAAAATA — TGCTGAT 

TGAAAATA — TGCTGAT 

TGAAAATA — TACTGAT 

TGAAAATA — TGCTGAT 

TGAAAATA — TGCTGAT 

TGAAAATA — TGCTGAT 

TG — TATAGA TAATGCTGAT 

260 270 

I L 

TCCCGTT GCCGATCGGG 

TCCCGTT GCCAATCGGG 

TCCCGTT GCCGATCGGG 

TCCCGTT GCCGATCGGG 

TCCCGTT GCCGATCGGG 

TCCCGTT GCCGATCGGG 

TCCCGTT GCCGATCGGG 

TCCCGTT GCCGATCGGG 

TCCCGTT GCCGATCGGG 

TCCCGTT GCCGATCGGG 

TCCCGTT GCCGATCGGG 

TCCCGTT GCCGATCGGG 

TCCCGTT GCCGATCGGG 

TCCCGTT GCCGATCGGG 

TCCCGTT GCCGATCGGG 

TCCCGTT GCCGATCGGG 

TCGTCTCATT GCCGATCGGG 



180 190 200 

\ I L 

GGCTGCAGCG GGGCGCGCAG CGGCTTATGA 
GGCAGCAGCG GGGCGCGCAG CGGCTTATGA 
GGCAGCAGCG GGGCGCGCAG CGGCTTATGA 
GGCAGCAGCG GGGCGCGCAG CGGCTTATGA 
GGCAGCAGCG GGGCGCGCAG CGGCTTATGA 
GGCAGCAGCG GGGCGCGCAG CAGCTTATGA 
GGCAGCAGCG GGGCGCGCAG CGGCTTATGA 
GGCAGCAGCG GGGCGCGCAG CGGCTTATGA 
GGCATCAGCG GGGCGCGCAG CGGCTTATGA 
GGCAGCAGCG GGGCACGCAG CGGCTTGTGA 
GGCAGCAGCG GGGCGCGCAG CGGCTTGTGA 
GGCAGCAGCG GGGCGCGCAG CGGCTTGTGA 
GGCAGCAGCG GGGCGCGCAG CGGCTTGTGA 
GGCAGCAGCG GGGCGCGCAG CGGCTTGTGA 
GGCAGCAGCG GGGCGCGCAG CGGCTTGTGA 

GGCAGC -GGCTTGTGA 

GGC ATGA 

230 240 250 

I I L 

TGAACGTCTG TC 

TGAACGTCTG TC 

TGAACGTCTG TC 

TGAACGTCTG TC 

TGAACGTCTG TC 

TGAACGTCTG TC 

TGAACGTCTG TC 

TGAACGTCTG TC 

TGAACGTCTG TC 

TGAACGCCTG TC 

TGAACGCCTG TC 

TGAACGCCTG TC 

TGAACGCCTG TC 

TGAACGCCTG TC 

TGAACGCCTG TC 

TGAACGCCTG TC 

GGAACGTGTC TCGTCTCGTC TCGTCTCGTC 

280 290 300 

I I L_ 

GACCGCAAGA AGCGCCGCCC CGGTCGGTTG 
GACCGCAAGA AGCGCCGCCC CGGTCGGTTG 
GACCGCAAGA AGCGCCGCCC CGGTCGGTCG 
GACCGCAAGA AGCGCCGCCC CGGTCGGTCG 
GACCGCAAGA AGCGCCGCCC CGGTCGGTTG 
GACCGCAAGA AGCGCCGCCC CGGTCGGTTG 
GACCGCAAGA AGCGCCGCCC CGGTCGGTTG 
GACCGCGAGA AGCGCCGCCC CGGTCGGTTG 
GACCGCAAGA AGCGCCGCCC CGGTCGGTTG 
GACCGCAAGA AGCGCCGCCC CGGTCGGTTG 
GACCGCAAGA AGCGCCGCCC CGGTCGGTTG 
GACCGCAAGA AGCGCCGCCC CGGTCGGTTG 
GACCGCAAGA AGCGCCGCCC CGGTCGGTTG 
GACCGCAAGA AGCGCCGCCC CGGTCGGTTG 
GACCGCAAGG AGCGCCGCCC CGGTCGGTTG 
GACCGCAAGA AGCGCCGCCC CGGTCGGTTG 
GACCGCAAGA AGCGCCGCCC CGGTCGGTTG 



tiungerf ordiana . 
bungerf ordiana . 
fiunger f ordiana . 
hungerf ordiana . 
hungerf ordiana . 
[lungerf ordiana . 
nungerf ordiana . 
lungerf ordiana . 
hiungerf ordiana . 
nungerf ordiana . 
fiungerf ordiana . 
hungerf ordiana . 
[lungerf ordiana . 
lunger f ordiana . 
lungerf ordiana . 
lunqerf ordiana . 



loc5#a 

loc5#b 

loc8#a 

loc8#b 

loc9#a 

locl6#a 

locl6#b 

loc22#a 

loc22#b 

loc23#a 

loc2 3#b 

loc24#a 

loc24#b 

loc25#a 

loc25#b 

loc26#a 



hungerf ordiana , 
hiungerf ordiana . 
[lungerf ordiana . 
[lungerf ordiana , 
lungerf ordiana . 
hungerf ordiana . 
[lungerf ordiana . 
[lungerf ordiana . 
[lungerf ordiana , 
[lungerf ordiana . 
[lungerf ordiana , 
lungerf ordiana . 
lungerf ordiana . 
lungerf ordiana . 
[lunger f ordiana . 
[lungerf ordiana . 



loc5#a 

loc5#b 

loc8#a 

loc8#b 

loc9#a 

locl6#a 

locl6#b 

loc22#a 

loc22#b 

loc23#a 

loc2 3#b 

loc24#a 

loc24#b 

loc25#a 

loc25#b 

loc26#a 



[lungerf ordiana . 
lungerf ordiana . 
[lungerf ordiana , 
[lungerf ordiana . 
[lungerf ordiana . 
[lungerf ordiana , 
hungerf ordiana . 
[lungerf ordiana , 
[lungerf ordiana . 
[lungerf ordiana . 
[lungerf ordiana . 
[lungerf ordiana . 
[lungerf ordiana , 
[lungerf ordiana . 
[lungerf ordiana , 
[lungerf ordiana . 



f requens 



loc5#a 

loc5#b 

loc8#a 

loc8#b 

loc9#a 

locl6#a 

locl6#b 

loc22#a 

loc22#b 

loc2 3#a 

loc23#b 

loc24#a 

loc24#b 

loc25#a 

loc25#b 

loc26#a 



POPULATION STRUCTURE OF A MALAYSIAN LAND SNAIL 



293 



TABLE 1. Continued. 



310 



320 



330 



340 

_1_ 



350 



tiungerfordiana 
rtungerfordiana 
fiungerfordiana 
hiungerfordiana 
fiungerfordiana 
riungerfordiana 
hunger fordiana 
riungerfordiana 
lunger fordiana 
lunger fordiana 
lunger fordiana 
lunger fordiana 
lunger fordiana 
lunger fordiana 
lunger fordiana 
lunger fordiana 



frequens 



. loc5#a 
. loc5#b 
, loc8#a 
. loc8#b 
.loc9#a 
. locl6#a 
,locl6#b 
,loc22#a 
.loc22#b 
,loc2 3#a 
.loc2 3#b 
,loc24#a 
,loc24#b 
, loc25#a 
,loc25#b 
. loc26#a 



ACCGCTCCCC 
ACCGCTCCCC 
ACCGCTCCCC 
ACCGCTCCCC 
ACCGCTCCCC 
ACCGCTCCCC 
ACCGCTCCCC 
ACCGCTCCCC 
ACCGCTCCCC 
ACCGCTCCCC 
ACCGCTCCCC 
ACCGCTCCCC 
ACCGCTCCCC 
ACCGCTCCCC 
ACCGCTCCCC 
ACCGCTCCCC 
ACCGCTCCCC 

360 

L_ 



TGTTTCGGGG 
TGTTTCGGGG 
TGTTTCGGGG 
TGTTTCGGGG 
TGTTTCGGGG 
TGTTTCGGGG 
TGTTTCGGGG 
TGTTTCGGGG 
TGTTTCGGGG 
TGTTTCGGGG 
TGTTTCGGGG 
TGTTTCGGGG 
TGTTTCGGGG 
TGTTTCGGGG 
TGTTTCGGGG 
TGTTTCGGGG 
TGTTTCGGGG 

370 
L_ 



TACCTAGTCT 
TACCTAGTCT 
TACCTAGTCT 
TACCTAGTCT 
TACCTAGTCT 
TACCTAGTCT 
TACCTAGTCT 
TACCTAGTCT 
TACCTAGTCT 
TACCTAGTCT 
TACCTAGTCT 
TACCTAGTCT 
TACCTAGTCT 
TACCTAGTCT 
TACCTAGTCT 
TACCTAGTCT 
TACCTAGTCT 

380 



TATCTCGCAC 
TATCTCGCAC 
TATCTCGCAC 
TATCTCGCAC 
TATCTCGCAC 
TATCTCGCAC 
TATCTCGCAC 
TATCTCGCAC 
TATCTCGCAC 
TATCTCGCAC 
TATCTCGCAC 
TATCTCGCAC 
TATCTCGCAC 
TATCTCGCAC 
TATCTCGCAC 
TATCTCGCAC 
CGTCTCGCAC 

390 



TCAATACGGC 
TCAATACGGC 
TCAATACGGC 
TCAATACGGC 
TCAATACGGC 
TCAATACGGC 
TCAATACGGC 
TCAATACGGC 
TCAATACGGC 
TCAATACGGC 
TCAATACGGC 
TCAATACGGC 
TCAATACGGC 
TCAATACGGC 
TCAATACGGC 
TCAATACGGC 
TCAATACGGC 

400 



(lungerf ordiana , 
hungerf ordiana . 
tiungerf ordiana . 
hungerf ordiana . 
Iiungerf ordiana . 
tiungerf ordiana , 
tiungerf ordiana , 
hungerf ordiana . 
hungerf ordiana . 
hungerf ordiana . 
Riungerfordiana . 
hungerf ordiana , 
Riungerfordiana . 
Riungerfordiana . 
Riungerfordiana . 
Riungerfordiana . 



loc5#a 

loc5#b 

loc8#a 

loc8#b 

loc9#a 

locl6#a 

locl6#b 

loc22#a 

loc22#b 

loc23#a 

loc23#b 

loc24#a 

loc24#b 

loc25#a 

loc25#b 

loc26#a 



CCACGGTGAC 
CCACGGTGAC 
CCACGGTGAC 
CCACGGTGAC 
CCACGGTGAC 
CCACGGTGAC 
CCACGGTGAC 
CCACGGTGAC 
CCACGGTGAC 
CCACGGTGAC 
CCACGGTGAC 
CCACGGTGAC 
CCACGGTGAC 
CCACGGTGAC 
CCACGGTGAC 
CCACGGTGAC 
CCACGGTGAC 

410 

L 



GGCAAGAGCT 
GGCAAGAGCT 
GGCAAGAGCT 
GGCAAGAGCT 
GGCAAGAGCT 
GGCAAGAGCT 
GGCAAGAGCT 
GGCAAGAGCT 
GGCAAGAGCT 
GGCAAGAGCT 
GGCAAGAGCT 
GGCAAGAGCT 
GGCAAGAGCT 
GGCAAGAGCT 
GGCAAGAGCT 
GGCAAGAGCT 
GGCATGAGCT 

420 



TTCAGCTCGC 
TTCAGCTCGC 
TTCAGCTCGC 
TTCAGCTCGC 
TTCAGCTCGC 
TTCAGCTCGC 
TTCAGCTCGC 
TTCAGCTCGC 
TTCAGCTCGC 
CTCAGCTCGC 
TTCAGCTCGC 
TTCAGCTCGC 
TTCAGCTCGC 
TTCAGCTCGC 
TTCAGCTCGC 
TTCAGCTCGC 
CTCAGCTCGC 

430 



CGGGTCGTCA 
CGGGTCGTCA 
CGGGTCGTCA 
CGGGTCGTCA 
CGGGTCGTCA 
CGGGTCGTCA 
CGGGTCGTCA 
CGGGTCGTCA 
CGGGTCGTCA 
CGGGTCGTCA 
CGGGTCGTCA 
CGGGTCGTCA 
CGGGTCGTCA 
CGGGTCGTCA 
CGGGTCGTCA 
CGGGTCGTCA 
CGGGTCGTCA 

440 



GGTCTAAGGA 
GGTCTAAGGA 
GGTCTAAGGA 
GGTCTAAGGA 
GGTCTAAGGA 
GGTCTAAGGA 
GGTCTAAGGA 
GGTCTAAGGA 
GGTCTAAGGA 
GGTCTAAGGA 
GGTCTAAGGA 
GGTCTAAGGA 
GGTCTAAGGA 
GGTCTAAGGA 
GGTCTAAGGA 
GGTCTAAGGA 
GGTCTAAGGA 

450 



Riungerfordiana 
Riungerfordiana 
Riungerfordiana 
Riungerfordiana 
Riungerfordiana 
Riungerfordiana 
Riungerfordiana 
Riungerfordiana 
Riungerfordiana 
Riungerfordiana 
Riungerfordiana 
Riungerfordiana 
Riungerfordiana 
lunger fordiana 
Riungerfordiana 
Riungerfordiana 



,loc5#a 
loc5#b 
loc8#a 
loc8#b 
, loc9#a 
,locl6#a 
,locl6#b 
, loc22#a 
,loc22#b 
, loc23#a 
,loc2 3#b 
,loc24#a 
.loc24#b 
. loc25#a 
.loc25#b 
loc26#a 



GCGCTGCTCT 
GCGCTGCTCT 
GCGCTGCTCT 
GCGCTGCTCT 
GCGCTGCTCT 
GCGCTGCTCC 
GCGCTGCTCC 
GCGCTGCTCC 
GCGCTGCTCC 
GCGCTGCTCT 
GCGCTGCTCT 
GCGCTGCTCT 
GCGCTGCTCT 
GCGCTGCTCT 
GCGCTGCTCT 
GCGCTGCTCT 
GCGCCGCTCT 



GATTGCTCTG 
GATTGCTCTG 
GATTGCTCTG 
GATTGCTCTG 
GATTGCTCTG 
GACTGCTCTG 
GATTGCTCTG 
GATTGCTCTG 
GATTGCTCTG 
GATTGCCCTG 
GATTGCTCTG 
GATTGCTCTG 
GATTGCTCTG 
GATTGCTCTG 
GATTGCTCTG 
GATTGCTCTG 
GACTGCTCTA 



TGAGCGGCGC 
TGAGCGGCGC 
TGAGCGGCGC 
TGAGCGGCGC 
TGAGCGGCGC 
TGAGCGGCGC 
TGAGCGGCGC 
TGAGCGGCGC 
TGAGCGGCGC 
TGAGCGGCGC 
TGAGCGGCGC 
TGAGCGGCGC 
TGAGCGGCGC 
TGAGCGGCGC 
TGAGCGGCGC 
TGAGCGGCGC 
TGAGCGGCGC 



CGCCCCGGTG 
CGCCCCGGTG 
CGCCCCGGTG 
CGCCCCGGTG 
CGCCCCGGTG 
CGCCCCGGTG 
CGCCCCGGTG 
CGCCCCGGTG 
CGCCCCGGTG 
CGCCCCGGTG 
CGCCCCGGTG 
CGCCCCGGTG 
CGCCCCGGTG 
CGCCCCGGTG 
CGCCCCGGTG 
CGCCCCGGTG 
CGCCCCGGTA 



GTTG-TGAGG 
GTTG-TGAGG 
GTTG-TGTGG 
GTTG-TGTGG 
GTTG-TGTGG 
ATTG-CGTGG 
ATTG-CGTGG 
ATTG-CGTGG 
ATTG-CGTGG 
ATTG-TGTGG 
ATTG-TGTGG 
ATTG-TGTGG 
ATTG-TGTGG 
ATTG-TGTGG 
ATTG-TGTGG 
ATTG-TGTGG 
GTTGGTGTGG 



294 



SCHILTHUIZENETAL. 



TABLE 1. Continued. 



460 470 480 

L I L 

-ATAATGGAG G GTACCTG 

-ATÄATGGAG G GTACCTG 

-ATAATGGAG G GTACCTG 

-ATAATGGAG G GTACCTG 

-ATAATGGAG G GTACCTG 

GATAATGGAG G GTACCTG 

GATAATGGAG G GTACCTG 

GATAATGGAG G GTACCTG 

GATAATGGAG G GTACCTG 

-ATAACGGAG G GTACCTG 

-ATAACGGAG G GTACCTG 

-ATAACGGAG G GTACCTG 

-ATGACGGAG G GTACCTG 

-ATAACGGAG G GTACCTG 

-ATAACGGAG G GTACCTG 

-ATAACGGAG G GTACCTG 

-ATCAAGGAG GCAAGGCCGG AGGGTACCTG 

510 520 530 

I \ L_ 

TCGGCGGATC CGGGTGCGAT AGCTCCTGCG 

TCGGCGGATC CGGGTGCGAT AGCTCCTGCG 

TCGGCGGATC CGGGTGCGAT AGCTCCTGCG 

TCGGCGGATC CGGGTGCGAT AGCTCCTGCG 

TCGGCGGATC CGGGTGCGAT AGCTCCTGCG 

TCGGCGGGTC CGGGTGCGAT AGCTCCTGCG 

TCGGCGGGTC CGGGTGCGAT AGCTCCTGCG 

TCGGCGGGTC CGGGTGCGAT AGCTCCTGCG 

TCGGCGGGTC CGGGTGCGAT AGCTCCTGCG 

TCGGCGGGTC CGGGCGCGAG AGCTCCTGCG 

TCGGCGGGTC CGGGTGCGAG AGCTCCTGCG 

TCGGCGGGTT CGGGTGCGAG AGCTCCTGCG 

TCGGCGGGTT CGGGTGCGAG AGCTCCTGCG 

TCGGCGGGTT CGGGTGCGAG AGCTCCTGCG 

TCGGCGGGTT CGGGTGCGAG AGCTCCTGCG 

TCGGCGGGTT CGGGTGCGAG AGCTCCTGCG 

TCGGCGGGTC TGGGTGCGAC AGCTCCTGCG 

560 570 580 

1 I L 

GCTTAAAGA? GTCGGCC-GT A TGCT 

GCTTAAAGA? GTCGGCC-GT A TGCT 

GCTTAAAGA? GTCGGCC-AT A-TATATGCT 
GCTTAAAGA? GTCGGCC-AT A-TATATGCT 

GCTTAAAGA? GTCGGCCCAT A TGCT 

GCTTAAAGA? GTCGGCC-AT G TGCT 

GCTTGAAGA? GTCGGCC-AT G TGCT 

GCTTAAAGA? GTCGGCC-AT G TGCT 

GCTTAAAGA? GTCGGCC-AT G TGCT 

GCTTAAAGA? GTCGGCC-AC G TGCT 

GCTTAAAGA? GTCGGCC-AT G TGCT 

GCTTAAAGA? GTCGGCC-AT G TGCT 

GCTTAAAGA? GTCGGCC-AT G TGCT 

GCTTAAAGA? GTCGGCC-AA G TGCT 

GCTTAAAGA? GTCGGCC-AA G TGCT 

GCTTAAAGA? GTCGGCC-AT G TGCT 

GCTTAAAGA? GTCGGCC-AT GCTCGCGGCT 



490 



500 



hungerf ordiana . 
Iiungerf ordiana , 
bungerf ordiana , 
hungerf ordiana . 
hungerf ordiana . 
iiungerf ordiana . 
Iiungerf ordiana . 
lungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 
nungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 
iiunqerf ordiana . 



loc5#a 

loc5#b 

loc8#a 

loc8#b 

loc9#a 

locl6#a 

locl6#b 

loc22#a 

loc22#b 

loc23#a 

loc2 3#b 

loc24#a 

loc24#b 

loc25#a 

loc25#b 

loc26#a 



TGCGCTCGAC 
TGCGCTCGAC 
TGCGCTCGAC 
TGCGCTCGAC 
TGCGCTCGAC 
TGCGCTCGAC 
TGCGCTCGAC 
TGCGCTCGAC 
TGCGCTCGAC 
TGCGCTCGAC 
TGCGCTCGAC 
TGCGCTCGAC 
TGCGCTCGAC 
TGCGCTCGAC 
TGCGCTCGAC 
TGCGCTCGAC 
TGCGCTCGAC 

540 



CCGCT-CTGC 
CCGCT-CTGC 
CCGCT-CTGC 
CCGCT-CTGC 
CCGCT-CTGC 
CCGCT-CTGC 
CCGCT-CTGC 
CCGCT-CTGC 
CCGCT-CTGC 
CCGCTGCTGC 
CCGCTGCTGC 
CCGCTGCTGC 
CCGCTGCTGC 
CCGCTGCTGC 
CCGCTGCTGC 
CCGCTGCTGC 
CG-CT-CTGC 

550 



iiungerf ordiana . 
iiungerf ordiana , 
iiungerf ordiana . 
hungerf ordiana . 
hungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 
bungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 
nungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 



loc5#a 

loc5#b 

loc8#a 

loc8#b 

loc9#a 

locl6#a 

locl6#b 

loc22#a 

loc22#b 

loc23#a 

loc23#b 

loc24#a 

loc24#b 

loc25#a 

loc25#b 

loc26#a 



GTGCAAACGC 
GTGCAAACGC 
GTGCAAACGC 
GTGCAAACGC 
GTGCAAACGC 
GTGCAAACGC 
GTGCAAACGC 
GTGCAAACGC 
GTGCAAACGC 
GTGCAAACGC 
GTGCAAACGC 
GTGCAAACGC 
GTGCAAACGC 
GTGCAAACGC 
GTGCAAACGC 
GTGCAAACGC 
GTGCAAACGC 

590 



AGGCCGCGA? 
AGGCCGCGA? 
AGGCCGCGA? 
AGGCCGCGA? 
AGGCCGCGA? 
AGGCCGCGA? 
AGGCCGCGA? 
AGGCCGCGA? 
AGGCCGCGA? 
AGGCCGCGA? 
AGGCCGCGA? 
AGGCCGCGA? 
AGGCCGCGA? 
AGGCCGCGA? 
AGGCCGCGA? 
AGGCCGCGA? 
AGGCCGCGA? 

600 



iiungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 
hungerf ordiana . 
iiungerf ordiana . 
Iiungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 
hungerf ordiana . 
iiungerf ordiana . 
bungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 
iiungerf ordiana . 



loc5#a 

loc5#b 

loc8#a 

loc8#b 

loc9#a 

locl6#a 

locl6#b 

loc22#a 

loc22#b 

loc23#a 

loc23#b 

loc24#a 

loc24#b 

loc25#a 

loc25#b 

loc26#a 



CGAGCA?ACC 
CGAGCA?ACC 
CG?GCA?ACC 
CG?GCA?ACC 
CGAGCG?ACC 
CGAGCA?ACC 
CGAGCA?ACC 
CGAGCA?ACC 
CGAGCA?ACC 
CGAGCA?ACC 
CGAGCA?A-C 
CGAGCA?ACC 
CGAGCA?ACC 
CGAGCA?ACC 
CGAGCA?ACC 
CGAGCA?ACC 
-GACCC-GCC 



CGCCCGCTCC 
CGCCCGCTCC 
CGCCCGCTCC 
CGCCCGCTCC 
CGCCCGCTCC 
CGCCCGCTCC 
CGCCCGCTCC 
CGCCCGCTCC 
CGCCCGCTCC 
CGCCCGCTCC 
CGCCCGCTCC 
CGCCCGCTCC 
CGCCCGCTCC 
CGCCCGCTCC 
CGCCCGCTCC 
CGCCCGCTCC 
CGCCC — T — 



POPULATION STRUCTURE OF A MALAYSIAN LAND SNAIL 



295 



TABLE 1. Continued. 





610 620 630 640 650 
1 1 1 1 1 


lungerf ordiana . loc5#a 
-lungerf ordiana . loc5#b 
lungerf ordiana . loc8#a 
lungerf ordiana . loc8#b 
lungerf ordiana. loc9#a 
lunger f ordiana . loc 1 6#a 
lunger f ordiana . loc 1 6#b 
lungerf ordiana . loc22#a 
lungerf ordiana. loc22#b 
lungerf ordiana. loc23#a 
lungerf ordiana . loc2 3#b 
lungerf ordiana. loc24#a 
lungerf ordiana. loc24#b 
lunger f ordiana . loc2 5#a 
lungerf ordiana. loc25#b 
lungerf ordiana. loc26#a 


GTCTTCCT — TATTT-AATT TGTTACGCTT GGTGGCTCGT CTGTCTTATT 
GTCTTCCT — TATTT-AATT TGTTACGCTT GGTGGCTCGT CTGTCTTATT 
GTCTTCCT — TATTT-AATT TGTTACGCTT GGTGGCTCGT CTGTCTTATT 
GTCTTCCT — TATTT-AATT TGTTACGCTT GGTGGCTCGT CTGTCTTATT 
GTCTTCCT — TATTT-AATT TGTTACGCTT GGTGGCTCGT CTGTCTTATT 
GTCTTCCT — TATTT-AATT TGTTACGCTT GGTGGCTTGT CTGTCTTATT 
GTCTTCCT— TATTT-AATT TGTTACGCTT GGTGGCTCGT CTGTCTTATT 
GTCTTCCT— TATTT-AATT TGTTACGCTT GGTGGCTCGT CTGTCTTATT 
GTCTTCCT— TATTT-AATT TGTTACGCTT GGTGGCTCGT CTGTCTTATT 
GCCTTCCT— TATTT-AATT TGTTACGCTT GGTGGCTCGT CTGTCCTATT 
GCCTTCCT — CATTT-AATT TGTTACGCTT GGTGGCTCGT CTGTCCTATT 
GCCTTCCT — TATTT-AATT TGTTACGCTT GGTGGCTCGT CTGTCCTATT 
GCCTTCCT — TATTT-AATT TGTTACGCTT GGTGGCTCGT CTGTCCTATT 
(3CCTTCCT — TATTT-AATT TGTTACGCTT GGTGGCTCGT CTGTCCTATT 
GCCTTCCT — TATTT-AATT TGTTACGCTT GGTGGCTCGT CTGTCCTATT 
GCCTTCCT — TATTT-AATT TGTTACGCTT GGTGGCTCGT CTGTCCTATT 


trequens 


660 670 680 690 700 
1 1 1 I 1 




hungerfordiana. loc5#a 
hungerfordiana. loc5#b 
nungerf ordiana . loc8#a 
lungerf ordiana. loc8#b 
lungerf ordiana . loc9#a 
nungerf ordiana . loc 1 6#a 
lunger f ordiana . loc 1 6#b 
lungerf ordiana . loc22#a 
lungerf ordiana . loc22#b 
lungerf ordiana. loc23#a 
lunger f ordiana. loc23#b 
lungerf ordiana. loc24#a 
lungerf ordiana . loc24#b 
lungerf ordiana . loc25#a 
lungerf ordiana . loc25#b 
lunqerf ordiana. loc26#a 


TGTCAGTTAC CGAAAAA С AAGATTGCTT GTCGTACAAC 

TGTCGGTTAT CGAAAAA С AAGATTGCTT GTCGTACAAC 

TGTCCGTTAT CGAAAAA С AAGATTGCTT GTCGTACAAC 

TGTCGGTTAT CGAAAAG С AAGATTGCTT GTCGTACAAC 

TGTCGGTTAT CGAAAAA С AAGATTGCTT GTCGTACGAC 

TGTCGGTTAT CGAAAAA С AAGATTGCTT GTCGTACAAC 

TGTCGGTTAT CGAAAAA С AAGATTGCTT GTCGTACAAC 

TGTCGGTTAT CGAAAAA С AAGATTGCTT GTCGTACAAC 

TGTCGGTTAT CGAAAAA С AAGATTGCTT GTCGTACAAC 

TGTCGGTTAT CGAAAAA — A AAAACAAA-C AAGATTGCTT GTCGTACAAC 
TGTCGGTTAT CPAAAAAAAA AAAAC7AAAC AAPATTGCTT GTCGT7CAAC 

TGTCGGTTAT CGAAAAAAAA AAAA С AAGATTGCTT GTCGTACAAC 

TGTCGGTTAT CGAAAAAAAA AAA С AAGATTGCTT GTCGTACAAC 

TGTCGGTTAT CGAAAAAAAA A С AAGATTGCTT GTCGTACAAC 

TGTCGGTTAT CGAAAAAAAA A С AAGATTGCTT GTCGTACAAC 

TGTCGGTTAT CGAAAAA -AAACAAAAC AAGATTGCTT GTCGTACAAC 

GGTTAT CGAAAAA-CC AAAACAAAA- TGCTT GTCGTACAAC 

710 720 730 740 750 
1 1 1 1 1 


rfrequens 




lungerf ordiana . loc5#a 
lungerf ordiana . loc5#b 
lungerf ordiana. loc8#a 
lungerf ordiana. loc8#b 
lungerf ordiana. loc9#a 
lunger f ordiana . loc 1 6#a 
lungerf ordiana . loc 1 6#b 
lungerf ordiana. loc22#a 
lungerf ordiana. loc22#b 
lungerf ordiana . loc2 3#a 
lungerf ordiana. loc23#b 
lungerf ordiana. loc24#a 
lungerf ordiana. loc24#b 
lungerf ordiana. loc25#a 
lungerf ordiana. loc25#b 
lungerf ordiana . loc26#a 


TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCAACCAGC 
TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCAGCCAGC 
TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCAGCCAGC 
TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCAGCCAGC 
TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCAGCCAGC 
TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCAGCCAGC 
TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCAGCCAGC 
TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCAGCCAGC 
TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCAGCCAGC 
TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCGGCCGGC 
TTT ??????? ?????????? ?????????? ?????????? ?????????? 
TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCAGCCAGC 
TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCAGCCAGC 
TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCAGCCAGC 
TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCAGCCAGC 
TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCAGCCAGC 
TTTGAGCGGT GGATCACTCG GCTCGTGCGT CGATGAAGAG CGCAGCCAGC 


freauens 





296 



SCHILTHUIZENETAL. 



TABLE 1. Continued. 





76C 
1 


77C 
1 


780 790 800 
1 1 1 


lungerf ordiana . loc5#a 


TGCGTGAATT 


AATGTGAATT 


GCAGAACACA 




lungerfordiana. loc5#b 


TGCGTGAATT 


AATGTGAATT 


GCAGAACACA 




lungerf ordiana. loc8#a 


TGCGTGAATT 


AATGTGAATT 


GCAGAACACA 




lungerf ordiana. loc8#b 


TGCGTGAATT 


AATGTGAATT 


GCAGAACACA 




-lungerf ordiana. loc9#a 


TGCGTGAATT 


AATGTGAATT 


GCAGAACACA 




lungerf ordiana. locl6#a 


TACGTGAATT 


AATGTGAATT 


GCAGAACACA 




lunger f ordiana . loc 16#b 


TGCGTGAATT 


AATGTGAATT 


GCAGAACACA 




lungerf ordiana . loc22#a 


TGCGTGAATT 


AATGTGAATT 


GCAGAACACA 




lungerf ordiana. loc22#b 


TGCGTGAATT 


AATGTGAATT 


GCAGAACACA 




lungerf ordiana. loc23#a 


TGCGTGAATT 


AATGTGAATT 


GCAGAACACA 




lungerf ordiana . loc2 3#b 
lungerf ordiana . loc24#a 


■>'>'>■>■>■>'>'>■>■> 


■>■>■>■>■>■>■>■>■>■> 


■?->■?'>'>'}'>'>'>'> 




TGCGTGAATT 


AATGTGAATT 


<X.KQ.M^Q.KQ.K 




lungerf ordiana . loc24#b 


TGCGTGAATT 


AATGTGAATT 


<X.KGM^Q.KCK 




lungerf ordiana. loc25#a 


TGCGTGAATT 


AATGTGAATT 


GCAGAACACA 




lungerf ordiana. loc25#b 


TGCGTGAATT 


AATGTGAATT 


GCAGAACACA 




lungerf ordiana. loc26#a 


TGCGTGAATT 


AATGTGAATT 


GCAGAACACA 






TGCGTGAATT 


AATGTGAATT 


GCAGAACACA 







TABLE 2. Pairwise Kimura's 2-parameter distances among the G. hungerfordiana sequences. 



hunger f ordiana 


loe5#a 


0.0000 


































hungerfordiana 


loc5#b 


0.0085 


0.0000 
































hungerfordiana 


loo8#a 


0.0129 


0.0107 


0.0000 






























hungerfordiana 


loc8#b 


0.0193 


0.0150 


0.0085 


0.0000 




























hungerfordiana 


loo9#a 


0.0128 


0.0085 


0.0064 


0.0107 


0.0000 


























hungerfordiana 


lool6#a 


0.0324 


0.0230 


0.0259 


0.0302 


0.0236 


0.0000 
























hungerfordiana 


lool6#b 


0.0280 


0.0236 


0.0215 


0.0258 


0.0193 


0.0085 


0.0000 






















hungerfordiana 


loc22#a 


0.0280 


0.0236 


0.0215 


0.0258 


0.0193 


0.0085 


0.0042 


0.0000 




















hungerfordiana 


loc22#b 


0.0281 


0.0237 


0.0216 


0.0259 


0.0193 


0.0085 


0.0042 


0.0042 


O.OOOO 


















hungerfordiana 


loc23#a 


0.0394 


0.0349 


0.0328 


0.0372 


0.0304 


0.0282 


0.0238 


0.0238 


0.0238 


0.0000 
















hungerfordiana 


loc23#b 


0.0481 


0.0436 


0.0414 


0.0458 


0.0391 


0.0368 


0.0324 


0.0324 


0.0324 


0.0126 


0.0000 














hungerfordiana 


loc24#a 


0.0414 


0.0369 


0.0348 


0.0392 


0.0324 


0.0302 


0.0258 


0.0253 


0.0258 


0.0063 


0.0147 


0.0000 












hungerfordiana 


loc24#b 


0.0459 


0.0413 


0.0392 


0.0436 


0.0369 


0.0345 


0.0302 


0.0302 


0.0302 


0.0105 


0.0190 


0.0042 





0000 








hungerfordiana 


loc25#a 


0.0415 


0.0370 


0.0348 


0.0392 


0.0325 


0.0302 


0.0258 


0.0258 


0.0259 


0.0064 


0.0127 


0.0042 





0084 


0.0000 






hungerfordiana 


loo25#b 


0.0437 


0.0392 


0.0370 


0.0414 


0.0347 


0.0324 


0.0280 


0.0280 


0.0280 


0.0085 


0.0148 


0.0063 





0105 


0.0021 


0.0000 




hungerfordiana 


loo26#a 


0.0405 


0.0359 


0.0337 


0.0382 


0.0313 


0.0289 


0.0244 


0.0244 


0.0244 


0.0043 


0.0108 


0.0022 





0065 


0.0022 


0.0044 


0.0000 



MALACOLOGIA, 1999, 41(1): 297-317 

ALLOZYME ANALYSES TEST THE TAXONOMIC RELEVANCE OF RIBBING IN 
CHINESE ONCOMELANIA (GASTROPODA: RISSOACEA: POMATIOPSIDAE) 

George M. Davis\ Yi Zhang^, Xingjiang Xu^ & Xianxiang Yang^ 

ABSTRACT 

The Tropical Medical Research Center of the Institute of Parasitic Diseases, Shanghai, China, 
is studying the genetics of Oncomelania snail populations throughout China relative to the trans- 
mission of the human blood parasite Schistosoma japonicum by genetically competent popula- 
tions of Oncomelania. On the basis of allozymes and C01 gene sequence data, Oncomelania 
has diverged significantly on a regional basis. There are three groups of populations we classify 
as subspecies: O. h. robertsoniin Yunnan and Sichuan above the Yangtze River Three Gorges 
(small, smooth and varix-less shells); O. h. tangio\ Fujien Province, removed from the Yangtze 
River drainage (small, smooth squat snails with a double thick varix); and O. /7. hupensis in the 
Yangtze River drainage below the Three Gorges (large shells, always with varix, primarily 
ribbed). These three clusters of populations differ from each other by Nei's "D" (allozymes) of 
0.267 to 0.405. Thus, there is considerable genetic divergence among the subspecies. 

There is the residual problem of what, genetically and taxonomically, are large smooth shelled 
Oncomelania. with varix, found below the Three Gorges in the Yangtze drainage? It is well known 
that in the floodplains of the Yangtze, Oncomelania populations are ribbed; above the effects of 
the annual flooding, the snails are smooth. Some taxonomists have called these smooth-shelled 
populations with varix O. fausti. The Miao River of Hubei Province offers a natural experiment to 
resolve this problem. In a small river there are four population centers that are affected by flood- 
ing; they have ribbed shells (RSP); above the flood zone there are several population centers 
where the shells are smooth but with pronounced varix (SSP). 

Allozyme data (35 loci, 72 alleles; mean samples per locus, 44 to 122) resolve the problem. 
Mean Nei's D for RSP = 0.045 ± 0.036; for SSP = 0.024 ± 0.016; comparing RSP and SSP = 
0.038 ± 0.035. There are no significant differences among these populations; they are all O. h. 
hupensis. Ribbing is genetically controlled by a single gene with multiple alleles (Davis & Ruff, 
1973). It thus appears that ribbing is a genetically controlled adaptation for dealing with annual 
flooding and survival by water transport. It also appears that terminal varix formation is controlled 
by a second gene. We predict that there will be no difference among all populations in the Miao 
River for the capacity to transmit the same genetic strain of S. japonicum. The role of unique al- 
leles and gene flow are addressed. 

Key words: systematics, Oncomelania, China, allozymes, population genetics, ribbing, 
Yangtze River, flooding, Hardy-Weinberg equilibrium. 



INTRODUCTION 

Oncomelania is one of eight Gondwanian 
genera of the rissoacean family Pomatiopsi- 
dae. The genus is comprised of a morphosta- 
tic radiation (defined in Davis, 1994) with only 
two species, O. hupensis Gredler, 1881, and 
O. minima Bartsch, 1936. The former, an am- 
phibious species, is distributed from Burma 
(fossil) throughout southern China, Taiwan, 
the Philippines, Sulawesi and Japan, with a 
series of geographically isolated subspecies; 
the latter, an aquatic species, is found in 



northern Japan. Oncomelania hupensis is of 
particular importance to humans as it is the in- 
termediate host for the human blood fluke 
Schistosoma japonicum. The Tropical Med- 
ical Research Center of the Institute of Para- 
sitic Diseases, Shanghai, China, is studying 
the coevolution of Oncomelania snail popula- 
tions with Schistosoma japonicum. The hy- 
pothesis is: "as snail populations diverge 
genetically, so must the associated schisto- 
somes; genetic divergence of schistosomes 
may have an important impact on human 
pathology and vaccine development for anti- 



^ Academy of Natural Sciences of Philadelphia. 1900 Benjamin Franklin Parkway. Philadelphia, Pennsylvania 19103 U.S.A.; 
Davis@acnatsci.org 

Chinese National Center of Systematic Medical Malacology, Institute of Parasitic Diseases. Shanghai, China; 
Zhangyi@hotmail.com 

Hubei Institute of Schistosomiasis Control, Zhuodaoquan, Wuchang, Wuhan. Hubei Province, China 



297 



298 



DAVIS ETAL. 




FIG. 1 . General position of the Miao River with respect to The Yangtze River and Wuhan City. Locations for 
control populations HB 22 and HB 6 are shown. 



Schistosoma vaccines". We have used al- 
lozyme electrophoresis and gene sequencing 
of the mt C01 and CYTb genes to begin to ad- 
dress the hypothesis. On the basis of al- 
lozymes and C01 data, Oncomelania has di- 
verged significantly on a regional basis. There 
are three primary groups we call subspecies: 
O. h. robertsoni in Yunnan and Sichuan, 
above the Yangtze River Three Gorges, with 
small, smooth and varix-less shells; O. h. 
tangi of Fujian Province, removed from the 
Yangtze River drainage, with small, smooth 
squat snails with a double thick varix; O. h. hu- 
pensis in the Yangtze River drainage below 
the Three Gorges with large shells, always 
with varix, primarily ribbed (Davis et al., 
1 995). These three clusters of populations dif- 
fer from each other by Nei's "D" (allozymes) of 
0.267 to 0.405; by 0.28 (28%) C01 sequence 
divergence (over 578 nucleotide positions). 
There is great genetic divergence among the 
subspecies! 

There is the residual problem of what, ge- 
netically and taxonomically, are large smooth- 
shelled Oncomelania, with varix, found below 
the Three Gorges in the Yangtze drainage? It 
is well known that in the floodplains of 
the Yangtze, Oncomelania populations are 
ribbed; above the effects of the annual flood- 
ing, the snails are smooth. Some taxonomists 
have called these smooth-shelled populations 
with varix O. fausti (reviewed in Davis et al., 
1995). The Miao River of Hubei Province 
(Figs. 1, 2) offers a natural experiment to re- 




FIG. 2. Localities along the Miao River and the re- 
lationship of the Miao River to the Yangtze river. 
A-C populations have ribbed shells and are below 
the ti)ridge. Populations D-G have smooth shells 
and are above the bridge. 



solve this problem. In this short river, there are 
four population centers that are affected by 
flooding; they have ribbed shells (RSP); 
above the flood zone there are several popu- 
lation centers where the shells are smooth but 
with pronounced varix (SSP). The purposes 
of this paper are to determine the following; 
(1) Are the RSP and SSP populations signifi- 



ALLOZYME ANALYSES TEST THE TAXONOMIC RELEVANCE OF RIBBING 



299 



cantly different genetically? (2) Are there indi- 
cations of gene flow or allele gradients? (3) 
Are there populations with unique alleles? (4) 
Are the populations subject to annual flooding 
genetically unstable, that is, with polymorphic 
loci in considerable Hardy-Weinberg disequi- 
librium due to immigration of snails floated 
into the river by the annual flooding of the 
Yangtze from various other population cen- 
ters (in contrast to genetically stable popula- 
tions above the effects of the flooding)? (5) 
Are the Miao River populations significantly 
different from control populations from other 
parts of Hubei Province? 



MATERIALSAND METHODS 
Locality Data 

All populations collected in 1994 are from 
Hubei Province, except the universal control, 
GC, from Anhui Province (Figs. 1, 2). GC, 
HB6 and HB22 are control populations. GC is 
the universal control population used with all 
populations of Oncomelania studied from 
throughout China. HB6 is the control popula- 
tion for Hubei Province. HB22 is a population 
outside the Miao River basin but not distant 
from the Miao River. Shells of these popula- 
tions are shown in Figure 3. 

HB6 (Figs. 1, 31): 28 Sept. 1994; Jian Li 
County; Mao Shi Township; Su Hu Canal; Su 
Jia Yuan Section of the canal. The snails were 
collected from the banks of the Grand Canal 
about 1 .5 m from the edge of the water under 
dense grass. The grass indicated that the 
area was not often flooded; at the edge of the 
water there were very narrow beaches of 
sand-shell perhaps a foot to 2 ft. wide in 
places. The greatest density of snails was in a 
narrow band. Above the band the soil was 
drier and snails correspondingly fewer. The 
slope was about 1 5"; at the upper edge about 
1 5 ft. from the water it was about 25". With the 
Oncomelania^ere Parafossarulus, and some 
dead shells of Semisulcospira. There were 
the same millipedes as in previous localities 
with Oncomelania. High humidity under the 
grass. Snails were in pockets of two to ten per 
pocket. We collected about 1,200 snails. The 
snails were numerous in this locality; all snails 
came from a 1 5.2 m-long stretch of bank. The 
canal had high density of Viviparidae [Bel- 
lamya quadrata type]; shells of Anodonta 
were abundant. 

The canal was built in 1959 to 1962; ac- 



cording to the Hubei Institute of Schistosomi- 
asis Control, in 1962 there were no Oncome- 
lania in the canal; they came down from the 
three lakes region at the head of the canal. 
The shells are longer and more slender than 
those from Gui Chi (contrast Fig. 3 I and H), 
and they are not as heavily ribbed. 

HB11 (A, Figs. 2,3): 1 Oct.; Song Zi County; 
Xin Jiang Kou Township; De Shun Village. 
Miao River about 0.4 km above the mouth of 
the river. Snails were collected from the moist 
to wet mud under grasses of small ponds in the 
narrow flood plain of the river. The snails were 
ribbed and with a thick varix. Snail density was 
great. All snails were heavily ribbed. 

HB12 (not on Fig. 2): 1 Oct.; Song Zi 
County; Cheng Dian Township; Ten Zi Qiao 
Village; Miao River 13 km upstream from 
HB1 1 . Snails, very few, were collected from a 
small stream flowing over a small dam into the 
Miao River some 50 m above the bridge 
crossing the Miao. Snails came from the wet 
mud under thick grass at the edges of the 
stream near the dam and rapidly flowing 
water. No snails were found on the banks of 
the main river that had a rapid flow over cob- 
bles. There were too few snails for elec- 
trophoresis. Snails had smooth shells. 

The bridge (Fig. 2, bridge) has great signif- 
icance. For some 60 m up- and downstream 
the snails are reputed to have a mixture of 
phenotypes from ribbed to smooth. One km 
downstream one begins to get ribbed snails 
that transmit Schistosoma japonicum. There 
is apparently no transmission, at this time, 
by smooth snails at the bridge or above it. 
The bridge marks the uppermost limit of the 
Yangtze flood during the rainy season. The 
bridge is about 13 km from the mouth of 
the river. 

HB1 3 (D, Figs. 2, 3): 1 Oct.; Song Zi County; 
Cheng Dian Township; Ten Zi Qiao Village; 
Miao River 1 .0 km above the bridge and HB1 2. 
Snail population density high; snails came 
from behind a natural dike or bank between 
the river and the true bank of the river. This 
backwater has water flowing through it cutting 
through the secondary bank into the river. A lit- 
tle upstream, the river bed widens and this 
ridge separates the river from the natural river 
bank by some 40 m with filled in land with 
crops. The snails came from mud under thick 
grasses at the edge of the water filling in the 
backwater. The snails were smooth. 

HB14 (F, Figs. 2, 3): 1 Oct.; Song Zi County; 
Cheng Diam Township; Ten Zi Qiao Village; 
Miao River 1 .5 km above bridge. At this local- 



300 



DAVIS ET AL. 




FIG. 3. Representative shells from the populations of this study. Figs. A-G are from populations A-G in Fig. 
2. Fig. H is a shell from GuiChi; I is from Jian Li; J is from Jing Men. 



ALLOZYME ANALYSES TEST THE TAXONOMIC RELEVANCE OF RIBBING 



301 



Ity two branches of the river bend around and 
are separated from each other by only the 
road's width of some 14 m. On the south side 
is a dam and spillway. Behind the spillway 
there is no discernable current. The water is 
choked with aquatic plants. Extensive marshy 
areas are at the margins. Snails are dense on 
the mud under the grass at the water's edge. 
HB1 4 is on the exact opposite side of the road 
from the north branch. Snails are smooth. 

HB15 (E, Figs. 2, 3): 1 Oct.; Song Zi 
County: Miao River 1 .5 km above bridge. Op- 
posite HB1 4 from the north branch of the river. 
Wide marshy areas on each side of the slow 
flowing steam harbor dense snail populations. 
Snails come from the wet mud under dense 
grass cover. Snails are smooth. 

HB1 6 (G, Figs. 2, 3): 1 Oct.; Song Zi County; 
Cheng Diam Township; Long Tan He Village; 
Miao River; 7 km above the bridge and 5 km 
from the top of the river; elevation about 47 m. 
The bridge elevation is 42 m. Snails numerous 
on mud under dense grass cover in the 
marshy margin of the river. Marshy border 
some 4.6 m wide. Stream banks some 15 m 
apart. Free flowing water about 4.6 m wide and 
choked with aquatic vegetation. Current very 
slow flow. Riverbanks 1 .8 m high on S side; 1 .2 
m high on N side. Snails are smooth. 

HB1 7 (B, Figs. 2, 3); collected by the schis- 
tosomiasis control station about 30 Oct. Song 
Zi County; Xinjiang Kou Township; De Shun 
Village; Miao River main branch; Lower Miao 
River 3 km upstream from HB11. Snails with 
ribbed shells. 

HB18 (C, Figs. 2, 3); 26 Sept.; Song Zi 
County; Lao Cheng Township; Yu Jia Du Vil- 
lage. Miao River mainstream. Collected by 
Schistosmiasis Control Unit. 2.0 km down- 
stream from the bridge. 8.5 km upstream from 
HB17. Snails with ribbed shells. 

HB22 (Figs. 1 , 3J; Control; not Miao River); 
6 Oct.; Jing Men City; 50 km SE of the city; 
Sha Yang Agricultural Management; Mia 
Liang State Farm; group 1 3. Snails came from 
a drainage canal bounded by cotton fields on 
one side and a road on the other with rice 
fields beyond that. The ditch was about 5.2 m 
wide from bank top to bank top; about 1 .2 m 
to 1.5 m deep with sides about 40". Snails 
were on the mud at the water level of the ditch 
up about 0.3 m under grass cover. Density 
was high. Shells smooth. 

GC (Control) (Fig. 3H): Anhui Province; Gui 
Chi City; 117° 20.6' E; 30° 30' N. collected in 
1994. Control population. The snails came 
from the flood planes of the Yangtze River. 



Electrophoresis 

Horizontal starch gel electrophoresis of tis- 
sue proteins from ten populations was fol- 
lowed by staining for the following 35 loci; 
AAT-1, AAT-2 (aspartate aminotransferase, 
2.6.1.1); AK-1 and 2 (adenosine kinase, 
2.7.1.20); АО (aldehyde oxidase, 1.2.3.1); 
ACPH-1 and 2 (acid phosphatase, 3.1.3.2); 
APH (alkaline phosphatase, 3.1.3.1); CK-1 
and 2 (creatine kinase, 2.7.3.2); EST-1, EST- 
2, EST-3 (esterase, 3.1.1.1); GDH (glutamate 
dehydrogenase, 1.4.1.2); G6PD (glucose-6- 
phosphate dehydrogenase, 1.1.1.49); GPI 
(glucose-6-phosphate isomerase, 5.3.1.9); 
HBD (hydroxybutyrate dehydrogenase, 
1.1.1.30); ISDH-1, ISDH-2 (isocitrate dehy- 
drogenase, 1.1.1.42); LDH (L-lactate dehy- 
drogenase, 1.1.1.27); MDH (malic dehydroge- 
nase, 1.1.1.37); ME-1, ME-2 (malic enzyme, 
1.1.1.40); MP! (mannose-6-phosphate iso- 
merase, 5.3.1.9); NADD-1 and 2 (NADH de- 
hydrogenase, 1 .6.99.3); 6PGD-1 and 2 (phos- 
phogluconate dehydrogenase, 1.1.1.44); 
PGM-1, PGM-2 (phosphoglucomutase, 
5.4.2.2); OCT (octopine dehydrogenase, 
1 .5.1 .11 ); SDH-1 ; SDH-2 (sorbitol dehydroge- 
nase, 1.15.1.1); SOD (SOD (superoxidismu- 
tase, 1.15.1.1); XDH (xanthine dehydroge- 
nase, 1.1.1.204). Procedures are those of 
Ayala et al. (1973) as modified by Dillon & 
Davis (1980), Davis (1983), Davis & Fuller 
(1981), Davis et al. (1981, 1988), and most re- 
cently for Oncomelania, by Davis et al. (1994, 
1995). 

Genetic parameters were calculated using 
BIOSYS-1 (Swofford & Selander, 1981). 
Hardy-Weinberg equilibrium was analyzed for 
all polymorphic loci. Nei's (1978) genetic dis- 
tance and Wright's (1978) modified Rogers 
distance were calculated and phenograms 
constructed using the UPGMA method. An 
unrooted tree based on Wright's D was also 
constructed using the FITCH program of 
PHYLIP version 3.4 (Felsenstein, 1989). This 
phylogentic analysis program does not as- 
sume equal rates of evolution. Some 50 repe- 
titions of FITCH were run with randomized 
input order and optimization by global branch 
rearrangement. We used both Nei's D and 
Wright's modified Rogers D as explained in 
Davis (1994). The former is traditional and 
widely used, and as such sets a standard for 
comparisons. However, it is not metric, and 
closely related populations are rather com- 
pacted towards the 0.01 end of the range. 
With Wright's D, a metric, the closely com- 



302 



DAVIS ETAL. 



TABLE 1. Shell length and smooth/ribbed characteristics. N = 5 unless stated otherwise. 



Population 


No. 
Whorls 


Shell 
Length 


Length Body 
Whorl 


No. Ribs 


CONTROLS 

GUICHI 

JINGMEN 

JIAN LI 
MIAO RIVER 

BELOW BRIDGE 


6-7 eroded 

8.5-9.0 

9.0-9.5 


9.6 ± 0.3 
8.4 ± 0.6 
9.1 ± 0.3 


5.3 ± 0.2 
4.0 ± 0.2 
4.3 ± 0.1 


14 ± 1 
SMOOTH 
18 ±2 


A 
В 
С 

ABOVE BRIDGE 


8.5-9.0 
8.5-9.0 
9.0-10.0 


8.2 ± 0.7 
8.1 ± 0.4 
9.4 ± 0.2 


4.2 ± 0.3 
4.1 ± 0.2 
4.6 ± 0.3 


19±2 
18 ± 2 

21 ± 4 


D 
E 
F 
G 


8.5 
8.5 

8.5-9.0 
8.5 


7.0 ± 0.5 
6.8 (N = 2) 

7.0 ± 0.5 
7.4 ± 0.5 


3.7 ± 0.2 
3.6 

3.6 ± 0.1 

3.8 ± 0.3 


SMOOTH 
SMOOTH 
SMOOTH 
SMOOTH 



pacted Nei's values are more spread out and 
better reflect the actual differences among 
populations in terms of a finite limit. 



RESULTS 

Table 1 provides basic shell data. Shells 
above the bridge (Fig. 3, D-G) are of the 
same whorl number (possibly excepting pop- 
ulation C) as populations below the bridge 
(Fig. 3, A-C) but are significantly smaller. The 
Gui Chi controls (Fig. 3, H) are significantly 
larger than the other ribbed populations 
(based on length of body whorl, because shell 
length cannot be calculated due to the eroded 
condition of the apices of all shells). The Gui 
Chi population also has significantly fewer 
ribs per whorl than do the other ribbed popu- 
lations. 

Table 2 lists the genotype frequencies for 
the ten populations listing the 16 of 35 loci 
(46%) that are either polymorphic or have al- 
ternative alleles; these involve 71 alleles. The 
Miao River populations are arranged in the 
table from downstream (left side) to upstream 
(right side). Indices of genetic variability are 
given in Table 3. Mean sample sizes per locus 
ranged from 44.4 to 122.3. The percentage of 
polymorphic loci ranged from 11 .4 to 22.9, the 
value not correlated with position along the 
river. The highest values were from terminal 
populations at the head and mouth of the 
river. Heterozygosity was low, ranging from 
0.014 to 0.059 (direct count). The mean num- 
ber of alleles per locus was low with the aver- 
age for all populations = 1 .36 ± 0.09. 



Populations are scored for the number of 
polymorphic loci (out of 12 such loci) and 
number of loci considered to differ signifi- 
cantly from Hardy-Weinberg equilibrium 
(Hwe) (Table 4). All but population A had some 
loci significantly different from HWe (range of 
0-4), with the highest number occurring in the 
Anhui, Gui Chi population, the middle popula- 
tion below the bridge (B), and the uppermost 
population above the bridge (G). The actual 
loci involved are scored in Table 5, where it is 
seen that the EST1, AAT1, ME1, and PGM1 
were the loci most often out of HWe (from 4 to 
7 populations). The ME1 locus was only out of 
HWe in populations above the bridge. Proba- 
bilities and fixation indices (F) and direction 
and index of deviation (D) are given for all rel- 
evant loci for all populations in Table 6. Het- 
erozygote deficiency was the predominant 
phenomenon. We examined the populations 
to see if there were discernible patterns of 
gene flow. Table 7 lists cases of alternative al- 
leles (in monomorphic loci where populations 
have different alleles), unique alleles to On- 
comelania. and alleles unique to the Miao 
River. Pie diagrams showing the frequencies 
of alleles (alternative alleles or polymor- 
phisms) are given in Figure 4, with columns 
arranged in order from downstream (right 
end) to upstream (to the left). There are alter- 
native alleles or unique alleles in all but one 
Miao River population. Two alternative alleles 
are found in population A at the mouth of the 
river, while one alternative allele is found in 
population D just above the bridge. Of partic- 
ular interest are the unique alleles found in 
populations E and F, found only some 20 m 



ALLOZYME ANALYSES TEST THE TAXONOMIC RELEVANCE OF RIBBING 



303 



TABLE 2. Allele frequencies for the 16 loci (46%) that are polymorphic or with alternative alleles among the 
1 populations. Populations GC, HB6 and HB22 are not in the Miao River drainage. There are 71 alleles total. 
For A-G, see Fig 2. 











HB11 


HB17 


HB18 


HB13 


HB14 


HB15 


HB16 


Locus 


GC 


HB6 


HB22 


A 


В 


С 


D 


F 


E 


G 


AAT1 






















(N) 


100 


80 


50 


50 


50 


50 


50 


70 


50 


160 


A 


0.985 


0.938 


1.000 


1.000 


0.980 


1.000 


1.000 


1.000 


1.000 


0.972 


В 


0.015 


0.044 











0.020 











0.022 


D 





0.019 























0.006 


AAT2 






















(N) 


100 


80 


50 


50 


50 


50 


50 


70 


50 


160 


A 


1.000 


0.906 


1.000 


1.000 


0.970 


1.000 


1.000 


1.000 


1.000 


1.000 


В 





0.013 








0.020 

















С 





0.081 








0.010 

















ACPH2 






















(N) 


60 


65 


50 


50 


50 


70 


50 


60 


50 


110 


A 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 




1.000 


1.000 


1.000 


D 














1.000 








AK1 






















(N) 


60 


65 


50 


50 


50 


70 


50 


60 


50 


110 


A 


1.000 


0.923 


1.000 





1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


В 











1.000 




















E 





0.077 


























APH 






















(N) 


100 


80 


50 


50 


50 


60 


50 


70 


50 


160 


A 


1.000 


1.000 


1.000 


1.000 


0.980 


1.000 


1.000 


1.000 


1.000 


1.000 


В 














0.020 

















CK1 






















(N) 






















A 


1.000 


1.000 


1.000 





1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


D 











1.000 




















EST1 






















(N) 


100 


80 


50 


50 


44 


50 


50 


70 


50 


159 


A 


0.175 


0.788 


0.930 


0.450 


0.602 


0.610 


0.780 


0.679 


0.940 


0.755 


В 


0.115 


0.087 


0.070 


0.310 


0.205 


0.190 


0.070 


0.050 


0.030 


0.097 


С 


0.020 








0.240 


0.080 








0.007 


0.020 


0.038 


D 


0.150 


0.125 








0.114 


0.200 


0.150 


0.257 





0.110 


E 























0.007 








F 


























0.010 





EST2 






















(N) 


100 


80 


50 


50 


50 


50 


50 


50 


100 


153 


A 


0.995 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


0.990 


С 


0.005 


























0.010 


GPI 






















(N) 


80 


30 


50 


50 


50 


50 


50 


70 


50 


160 


A 


0.656 


0.883 


1.000 


0.760 


0.740 


0.770 


0.930 


0.871 


0.980 


0.962 


В 


0.262 


0.113 





0.060 


0.220 


0.190 


0.060 


0.036 





0.031 


С 


0.044 








0.100 








0.010 


0.093 


0.020 





E 


0.038 


0.033 





0.040 


0.040 


0.040 











0.006 


H 











0.040 




















HBD1 






















(N) 


50 


6 


50 


50 


50 


60 


50 


10 


50 


90 


A 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


0.444 


С 





























0.556 


ME1 






















(N) 


80 


30 


50 


50 


50 


50 


50 


70 


50 


160 


A 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


0.700 


0.957 


0.570 


0.956 


В 




















0.020 








0.006 


С 




















0.140 


0.029 


0.290 


0.019 


D 




















0.100 


0.014 


0.140 


0.019 


E 




















0.040 









continued) 



304 








DAVIS ETAL. 












TABLE 2. 


(Continued) 




























HB11 


HB17 


HB18 


HB13 


HB14 


HB15 


HB16 


Locus 


GC 


HB6 


HB22 


A 


В 


С 


1 


D 


F 


E 


G 


OCT 
























(N) 


86 


80 


50 


47 


50 


50 


50 




70 


50 


152 


A 


0.547 


0.444 


0.620 


0.649 


0.680 


0.530 


0.940 


0.557 


0.550 


0.651 


В 


0.064 


0.019 





0.021 


0.010 
















0.010 


С 


0.314 


0.525 


0.230 


0.277 


0.260 


0.360 


0.030 


0.279 


0.120 


0.204 


D 


0.076 





0.150 


0.053 


0.050 


0.110 


0.030 


0.136 


0.300 


0.135 


Е 





0.013 




























F 

























0.029 


0.030 





PGM1 
























(N) 


70 


20 


50 


50 


50 


50 


50 




70 


50 


143 


A 


0.679 


1.000 


0.530 


0.610 


0.810 


0.530 


0.930 


0.740 


0.790 


0.745 


В 


0.007 





0.030 


0.010 





0.030 













0.003 


E 


0.071 








0.080 


0.130 


0.020 


0.060 


0.214 


0.180 


0.105 


F 


0.229 





0.440 


0.300 


0.060 


0.420 


0.010 


0.043 


0.030 


0.147 


G 


0.014 































PGM2 
























(N) 


80 


30 


50 


50 


50 


50 


50 




70 


50 


160 


A 


0.975 


0.983 


0.980 


1.000 


0.940 


1.000 


1.( 


300 


0.993 


1.000 


1.000 


В 


0.025 


0.017 








0.060 










0.007 








D 








0.020 

























XDH 
























(N) 


60 


65 


50 


50 


50 


60 


50 




60 


50 


110 


A 


1.000 





1.000 


1.000 


1.000 


1.000 


1.( 


DOO 


1.000 


1.000 


1.000 


D 





1.000 




























TABLE 3. 


Populatioi 


n genetic 


indices. 






















Mean 




Mpon no 










Mean heterozygosity 


Populations 


sample No. 
per locus 


1 VIC^Cll 1 1 IVJ. 

alleles 
per locus 


% loci 
polymorphic 




Direct count 


Hdywbg 
expected 


CONTROLS 






















GC1-Gui Chi 
НВ-6 -Jian Li 
НВ-22 Jing 


66.7 (3.3) 
54.7 (3.9) 


1.5(0.2) 
1.4(0.1) 




20 
20 




0.039 (0.020) 
0.030(0.017) 


0.061 (0.028) 
0.047 (0.020) 


Men City 
MIAO RIVER 


50.7(0.7) 


1.2(0.1) 




11.4 




0.034(0.021) 


0.036 (0.022) 


HB-11 = 
HB-17: 
HB-18: 
BRIDGE 


= A 
= B 
= C 


47.1 (1.9) 
49.8 (0.20) 
56.6(1.30) 


1.3(0.02) 
1.4(0.10) 
1.3(0.10) 




11.4 
20 

14.3 




0.059 (0.029) 
0.034(0.016) 
0.046 (0.022) 


0.060 (0.029) 
0.057 (0.025) 
0.060 (0.028) 


HB-13: 

HB-14: 
HB-15: 

HB-16: 


= D 
= F 
= E 
= G 


48.6(1.7) 

55.8(3.1) 

44.4 (2.7) 

122.3(4.6) 


1.3(0.20) 
1.4(0.2) 
1.3(0.1) 
1.5(0.2) 




14.3 
17.1 
14.3 
22.9 




0.014(0.008) 
0.038(0.019) 
0.023(0.015) 
0.027(0.014) 


0.035(0.018) 
0.052 (0.024) 
0.048 (0.025) 
0.059 (0.025) 



from each other but in different branches of 
the river (Fig. 2). With perhaps the exception 
of allele В in EST-1, there is no discernable 
pattern of gene flow or allele frequency gra- 
dation among populations. Allele В in the case 
cited decreases from the mouth of the river to 
in HB15; however, it is present in low fre- 
quency in HB-4 and 16. 

Nei's and Wright's modified Rogers' genetic 
distances are given in Table 8; the relevant 



phenograms are given in Figures 5 and 6. The 
respective cophenetic values are 0.995 and 
0.978. The overall genetic distances involved 
(clustering at <0.08, Nei's D) clearly shows 
conspecificity (for species concepts involving 
Oncomelania: Davis 1994; Davis et al., 1995). 
There is no separate cluster of ribbed snails 
or separate cluster for smooth snails. The 
ribbed control from Anhui Province (GC) clus- 
ters most closely with hbbed populations B. С 



ALLOZYME ANALYSES TEST THE TAXONOMIC RELEVANCE OF RIBBING 



305 



TABLE 4. Populations scored for number of polymorphic loci and number of loci significantly 
differing from Hardy-Weinberg equilibrium where significance using exact probabilities is set 
at 0.06. There are 12 polymorphic loci total. 





No. Polymorphic 


No. Significantly 


% Significantly 


Population 


loci 




different 


different 


Controls 










Gui Chi 


7 




3 


43 


HB-6 


7 




4 


57 


HB-22 


4 




1 


25 


Miao River 










Below Bridge 










A=HB-11 


4 










B = HB-17 


7 




4 


57 


C = HB-18 


5 




2 


40 


Above Bridge 










D = HB-13 


5 




2 


40 


E = HB-15 


5 




2 


40 


F = HB-14 


6 




3 


50 


G = HB-16 


7 




4 


57 



TABLE 5. Chi-square tests for deviation from Hardy-Weinberg Equilibrium; N = not polymorphic, S significance is 
derived from using exact probabilities; S = P < .06. [ ] = number of loci differing significantly from H-W. P values 
are given. 



Locus: 



AAT1 AAT2 AKI APH EST1 EST2 GPI HBD1 ME1 OCT PGM1 PGM2 



Population 

GuiChi [4] S N 

HB-6 [4] S S 

HB-22 [1] N N 

MIAO RIVER BELOW BRIDGE 

A=HB-11 [0] N N 

В = HB-17 [4] N S 

C = HB-18[2] S N 

MIAO RIVER ABOVE BRIDGE 

D = HB-13[3] N N 

E = HB-15[2] N N 

F = HB-14[3] N N 

G = HB-16[4] S N 



N 


N 


S 


1.000 


0.218 


N 


N 


0.831 


S 


S 


S 


N 


S 


N 


1.000 


N 


N 


1.000 


N 


1.000 


N 


N 


1.000 


N 


N 


N 


N 


0.559 


0.579 


s 


N 


N 


0.774 


N 


0.249 


N 


N 


0.754 


0.338 


N 


N 


S 


S 


N 


0.269 


N 


N 


0.210 


S 


1.000 


N 


N 


0.388 


N 


0.254 


N 


N 


0.267 


S 


N 


N 


N 


S 


N 


1.000 


N 


s 


1.000 


S 


N 


N 


N 


1.000 


N 


1.000 


N 


S 


0.097 


S 


N 


N 


N 


s 


N 


0.588 


N 


S 


0.628 


S 


1.000 


N 


N 


s 


1.000 


1.000 


N 


S 


0.110 


S 


N 



and smooth population F. Population A at the 
mouth of the river stands slightly apart from all 
others. Table 9 provides inter-population com- 
parisons of values of Nei's D. There is no sig- 
nificant difference between the average D for 
all Miao River populations (0.038 ± 0.035) 
and the separate average D for populations 
above the bridge (0.024 ± 0.01 6) or below the 
bridge (0.045 ± 0.036). 

The unrooted FITCH tree is given in Figure 
7, freed from the constraints of phenogram 
construction and based on a metric distance 
measure. There are no two distinct clusters of 
populations representing smooth and ribbed 
shells. Populations A, Jian Li, and D indicate 
their unique genetic structure and divergence 
from all the other populations. Note that A dif- 
fers from D by a Nei's D of 0.1 06 (Table 8), the 



greatest divergence among Miao River popu- 
lations. 



DISCUSSION 

Do Smooth and Ribbed Snails Deserve 
Separate Taxonomic Rank? 

The question appears resolved. Based on 
this natural experiment, populations of On- 
comelania within the Yangtze River drainage 
below the Three Gorges of the Yangtze River 
that are smooth (but with varix), and with the 
same allometry as snails of ribbed populations 
in the lower Yangtze drainage, are one sub- 
species, O. hupensis hupensis. Wang et al. 
(1998) support this concept on the basis of a 



306 



DAVIS ETAL. 



TABLE 6. Deviation from Hardy-Weinberg and heterozygote deficiency for all popula- 
tions. P is based on exact probabilities and significance is set at 0.06. Population A is 
at the mouth of the Miao River; G is farthest upstream. 



Population 


locus 


P 


Fixation index (F) 


D 


Controls 










Gui Chi 


AAT1 


0.02 


0.662 


-0.663 




EST1 


0.00 


0.713 


-0.714 




GPI 


0.22 


0.220 


-0.225 




OCT 


0.83 


0.137 


-0.142 




PGM1 


0.05 


0.318 


-0.323 




PGM2 


0.00 


1.000 


-1.000 


Jian Li 


AAT1 


0.00 


0.790 


-0.791 




AAT2 


0.00 


0.927 


-0.928 




AK 


0.00 


1.000 


-1.000 




EST1 


0.00 


0.369 


-0.373 


Jing Men 


PGM2 


0.01 


1.000 


-1.000 


MIAO RIVER 










Below Bridge 










A(HB-11) 


GPI 


0.25 


-0.134 


0.123 




PGM1 


0.39 


0.172 


-0.180 


B(HB-17) 


AAT2 


0.03 


0.659 


-0.662 




APH 


0.01 


1.000 


-1.000 




EST1 


0.00 


0.645 


-0.649 




GPI 


0.27 


0.205 


-0.213 




OCT 


0.21 


0.144 


-0.153 




PGM1 


0.00 


0.567 


-0.571 


C(HB-18) 


AAT1 


0.01 


1.000 


-1.000 




EST1 


0.39 


0.166 


-0.175 




GPI 


0.25 


0.188 


-0.196 




OCT 


0.27 


0.169 


-0.177 




PGM1 


0.01 


0.335 


-0.342 


Above bridge 










D(HB-13 


EST1 


0.00 


0.451 


-0.456 




ME1 


0.00 


1.000 


-1.000 




PGM1 


0.00 


0.848 


-0.849 


E(HB-15) 


ME1 


0.00 


0.965 


-0.965 




OCT 


0.10 


0.122 


-0.131 




PGM1 


0.00 


0.708 


-0.711 


F(HB-14) 


EST1 


0.00 


0.393 


-0.660 




GPI 


0.59 


-0.115 


0.107 




ME1 


0.00 


1.000 


-1.000 




OCT 


0.63 


0.108 


-0.115 




PGM1 


0,010 


0.358 


-0.362 


G(HB-16) 


AAT1 


0.00 


0.659 


-0.660 




EST1 


0.00 


0.444 


-0.446 




HBD1 


0.00 


1.000 


-1.000 




ME1 


0.00 


1.000 


-1.000 




OCT 


0.11 


0.235 


-0.237 




PGM1 


0.00 


0.458 


-0.460 



few enzymes (esterases and MDH, slab PAG), 
and a few specimens of ribbed and smooth- 
shelled Oncome/an/a from three counties (five 
populations) in Hube! Province. They found 
very little difference among populations (data 
not sufficient or scoreable for population ge- 
netic analysis). 

Ribbing is associated with annual flooding 
of the Yangtze River and its tributaries. As is 



well known to Chinese field workers attempt- 
ing to monitor and control Oncomelania snails 
(Liu et al., 1 981 ), snails from any elevation, or 
a man-made situation that removes a popula- 
tion from the annual floods, attain a smooth 
shell but still retain the varix. Molecular ge- 
netic data do not support the concept of dif- 
ferent taxonomic status for these two shell 
types. Accordingly, Katayama fausti Bartsch, 



ALLOZYME ANALYSES TEST THE TAXONOMIC RELEVANCE OF RIBBING 



307 



TABLE 7. Distribution of unique and alternative alleles among the 1 populations. 





Alternative 




Alleles unique to 


Population 


alleles 


Unique alleles 


the Miao River 


CONTROLS 








Gui Chi 


none 


none 




Jian Li 


XDH/d 


AAT1/d (0.019) 
АК-1/е (0.077) 




Jing Men 


none 


none 




MIAO RIVER 








Below Bridge 








A 


AK1/b 
CKI/d 


none 


GPI/h (0.040) 


В 


none 


none 


AAT2/b (0.020) 
AAT2/C (0.010) 
APH/b (0.020) 


С 


none 


none 


none 


Above Bridge 








D 


ACPH2/d 


none 


MEI/e (0.040) 


E 


none 


none 


EST1/f (0.010) 


F 


none 


none 


ESTI/e (0.007) 
PGM2/b (0.007) 


G 


none 


HBD/c (0.556) 


AATI/d (0.006) 
EST2/C (0.010) 



1 925, is a synonynn of O. hupensis hupen- 
sis Gredler, 1 881 . {Katayama used to be used 
as a genus to include all smooth forms of On- 
comelania hupensis.) 

Davis & Ruff (1973) employed breeding ex- 
periments and showed that ribbing in On- 
comelania hupensis from mainland China is 
controlled by a single gene with multiple alle- 
les. Davis (1979) considered the smooth 
shelled condition to be primitive and that rib- 
bing is the derived condition. We argue that the 
earliest ecology and shell morphology of On- 
comelania hupensis is that seen in Yunnan 
and Sichuan, areas into which pomatiopsine 
snails were introduced through a trajectory 
from the Indian Plate into northern Burma and 
Yunnan, China, in the Miocene (Davis, 1979). 
The habitat involves marshy ecotones in hilly 
regions not subjected to annual flooding. It is 
noteworthy that these Yunnan and Sichuan 
snails, O. hupensis robertsoni, are smooth 
and have no varix. The natural course of evo- 
lution would have been through dispersal 
down the evolving Yangtze River. With the ge- 
ological formation of the Three Gorges section 
of the Yangtze creating an effective barrier to 
passive movement of snails up or down the 
Yangtze River, snails living in the flood planes 
and on the islands of the Yangtze River, 
through mutation, developed ribs and a varix. 
The hypothesis is that ribbing contributes 
greatly to survival by significantly increasing 
shell surface area, facilitating floatation and 



dispersal during flooding. Additionally, ribbing 
may also increase shell strength as well as 
surface area. Greater strength would abet sur- 
vival during flotation and vagaries of being 
swept into solid objects. The loss of ribbing but 
not the varix indicates that there is a different 
gene(s) governing varix formation. 

Floatation during Yangtze River flooding is 
a major source of dispersion for Oncomelania 
hupensis hupensis and the schistosomes 
they transmit. This phenomenon is apparently 
not known outside China. During the floods, 
snails are lifted off the islands in the Yangtze 
and floodplains and floated by the millions 
down the river to be deposited on down- 
stream floodplains or swept into canals when 
the flood gates are opened. The impact on im- 
portation into the canals of Hubei Province 
has been documented by Xu & Fang (1988) 
and Xu et al. (1 989, 1 993), Yang et al. (1 992). 
While snails on the islands either float off or 
drown, snails on the floodplains often escape 
flooding by climbing tree trunks, often to 
heights of more than three meters. With refer- 
ence to drowning, Oncomelania hupensis is 
an amphibious species. The young stay sub- 
merged during their early stages of develop- 
ment, often floating upside down, feeding on 
the surface of quiet water. As adults, the 
snails are found out of but near water on the 
banks of irrigation ditches and swamps, on 
shaded, moist soil. During drought, the adults 
move down into the soil and aestlvate. Adults 



308 DAVIS ETAL. 

HB16 HB14 HB15 HB13 HB18 HB17 HBll 



©■ 



DA 

■ в 

DC 

■ D 

■ E 

■ F 

DH 



О 







AAT-1 



ААГ-2 



ACPH-2 



- AK-1 



APH 




О-Э-О-0-Э-С1-О- 








■(3-е- 







(3-(3-(3C Э 0-Э 



■o- 



■o- 



CK-1 



EST-1 



EST-2 



GPI 



HBD-1 



ME-1 



OCT 



PGM-1 



PGM-2 



FIG. 4. Pie diagrams showing allele frequencies and alternative alleles for each population. Populations are 
in rows with HB 11 (=A) at the mouth of the river and HB 1 6 (=G) at the head of the river. The colors are coded 
A, B, etc. for alleles A, B, etc. listed in Table 2. For example, solid orange in Fig. 4, HB11 indicates an alter- 
native allele B. The dominant allele is A. 



ALLOZYME ANALYSES TEST THE TAXONOMIC RELEVANCE OF RIBBING 



309 



TABLE 8. Matrices of Nei's 1978 genetic distance (above tine diagonal) and Wright's modified 1978 Rogers 
genetic distance (below the diagonal). 







JIAN 
















JING 


Population 


GC 


LI 


A 


D 


F 


E 


G 


В 


С 


MIN 


GC 




0.035 


0.066 


0.039 


0.003 


0.010 


0.012 


0.001 


0.001 


0.005 


JIAN LI 


0.182 




0.103 


0.072 


0.035 


0.042 


0.045 


0.035 


0.038 


0.040 


A 


0.247 


0.305 




0.106 


0.068 


0.077 


0.077 


0.066 


0.066 


0.069 


D 


0.193 


0.258 


0.310 




0.036 


0.035 


0.044 


0.036 


0.043 


0.040 


F 


0.052 


0.181 


0.250 


0.185 




0.006 


0.010 


0.002 


0.004 


0.006 


E 


0.100 


0.198 


0.265 


0.184 


0.080 




0.014 


0.010 


0.013 


0.009 


G 


0.108 


0.205 


0.265 


0.203 


0.100 


0.118 




0.011 


0.013 


0.009 


В 


0.041 


0.181 


0.246 


0.185 


0.048 


0.098 


0.105 




0.004 


0.007 


С 


0.040 


0.190 


0.245 


0.201 


0.067 


0.113 


0.113 


0.064 




0.004 


JING MIN 


0.071 


0.195 


0.253 


0.196 


0.077 


0.093 


0.108 


0.087 


0.063 





г GCl -Anhui-Ribs 

- HB18 -С -Ribs 

г- HB 14 -F-Smooth 

- HB17 -В -Ribs 

— HB22 - Control. Jing Men - Smooth 

— HB 15 -Е- Smooth 

— HB16 -G -Smooth 

— HB6 - Control. Jian Li - Ribs 

— HB 13 -D-Smooth 

— HBll -A-Ribs 



H 1 1- 



H 1 1 1 1 1 1 1- 



.10 .09 .08 .07 .06 .05 .04 .03 

FIG. 5. Phenogram based on Nei's genetic distance. 

cannot withstand continual submersion; they 
will drown. Because of this, drowning is a 
method used to control schistosomiasis is in 
some areas of China, including the Miao 
River. During the dry season, a control dam is 
closed at times, flooding a large area of graz- 
ing land with numerous marsh-edged pools 
harboring large populations of Oncomelania. 
Our site В is in this section of river. The flood- 
ing does reduce the sizes of populations in- 
undated (personnel communication; Dr. Yang, 
Xian-Xiang, Director, Hubei Institute of Schis- 
tosomiasis Control). 

After revisiting and collecting snails on Lao 
Zhou Island in the Yangtze River in Tong Ling 
County, Anhui Province, we now understand 



.02 .01 



.00 



why three populations, including the Lao 
Zhou, snails did not group with the clusters we 
classified as O. h. hupensis, O. h. tangi, and O. 
h. robertsoni (Davis et al., 1995) in the Fitch 
tree based on allozyme data from 14 On- 
comelania populations. The other populations 
were Gui Chi (one of our control populations) 
from Anhui, and Jian Li from Hubei Province. 
These three localities are heavily flooded and 
swept during the annual floods. Snails found in 
these locations are not populations, really, but 
aggregates of snails imported from diverse 
areas and deposited with the receding flood- 
waters. In Davis et al. (1 995), these three pop- 
ulations were considered as possible "hybrid" 
populations, because of alleles shared with 



310 



DAVIS ETAL. 



CK'I - Anhui - Ribs 

HB 18 -С -Ribs 

HB 14 -F -Smooth 

UBI 7 -В -Ribs 

HB22 - Control. Jing Men - Smooth 

HB 1 5 - E - Smooth 

HB 16 -G -Smooth 

HB6 - Control. Jian Li - Ribs 

HB 13 -D -Smooth 

HBll -A- Ribs 



40 .36 .32 .28 .24 .20 .16 .12 .08 .04 .00 

FIG. 6. Phenogram based on Wright's modified Rogers genetic distance. 



TABLE 9. Comparison of Nei's D among popula- 
tions. 



Populations 


Mean and S.D. 


No. 


Above bridge x Below 

Bridge 
Below Bridge 
Above Bridge 
Controls y Below Bridge 
Controls X Above Bridge 


0.038 (±0.035) 
0.045 (±0.036) 
0.024 (±0.016) 
0.041 (±0.039) 
0.032 (±0.023) 


12 
3 
6 
6 
8 



Other populations and subspecies, especially 
with the upstream robertsoni subspecies. We 
here call such populations "genetically unsta- 
ble" populations where true population struc- 
ture is not reached and HWe is not attained in 
most polymorphic loci. 

Genetically stable populations, theoreti- 
cally, would be defined as those with normal 
panmixia, little or no immigration, and all loci 
in HWe, that is, populations that are large and 
have had stable population structure for many 
years. There are three populations that could 
arguably fit these criteria; HB-11, HB-22 with 
one locus not in HWe due to very low fre- 
quency of one allele, HB-18, and HB-15 with 
2 of 5 polymorphic loci not in HWe. The popu- 
lation at the head of the river, HB-1 6 should be 
genetically stable but equals GuiChi in having 
57% polymorphic loci not in Hwe. 

Theoretically, population HB-11 at the 
mouth of the Miao River should be "unstable" 
due to annual flooding, but on the basis of H- 
W, this is not the case. The mouth of the Miao 
River is not on the Yangtze, but on a loop that 
branched off the main channel of the Yangtze. 



While the lower Miao River is flooded annu- 
ally, it may not receive many introductions of 
snails from upstream localities. However, as 
clearly seen in Figures 5-7, population A is 
clearly divergent from all the others. 

The Question of Heterozygote Deficiency 

Four reasons could account for heterozy- 
gote deficiency such as seen here: (1 ) organ- 
isms came from two or more populations with 
different allele frequencies (Wahlund effect), 
(2) natural selection acts against heterozy- 
gotes at some loci, (3) there is inbreeding, or 
(4) our scoring is in error and is biased against 
heterozygotes (Ayala et al.. 1973). Error in 
scoring may result in one, or perhaps two loci 
being considered not in HWe, especially 
where EST is involved and gels can be difficult 
to score. However, we consider that 1 and 3 
above are probably mostly responsible, al- 
though we cannot discount selection against 
heterozygotes. 

How often is heterozygote deficiency found 
in molluscs overall, and such rissoacean 
snails as Oncomelania. in particular? It is im- 
portant to place Oncomelania. a rissoacean 
snail, in context with other molluscs with re- 
gard to what one might expect in terms of het- 
erozygosity and heterozygote deficiency. Het- 
erozygote deficiency is common among 
molluscs, especially bivalves where heterozy- 
gosity mostly exceeds 0.250 (see references 
below). Numerous authors have discussed 
this phenomenon in the Bivalva (for example, 
Ayala et al.. 1973; Mitton & Koehn, 1973; 
Lassen & Turano, 1978; Wilkins, 1978; 



ALLOZYME ANALYSES TEST THE TAXONOMIC RELEVANCE OF RIBBING 31 1 

В 
F 

Jing Men 




Jian Li 



FIG. 7. An unrooted FITCH tree based on Wright's D. Line lengths are proportional to branch lengths. 



Gosling & Wilkins, 1981; Mallet & Haley, 
1983; Skibinski et al., 1983; Adamkewicz et 
al., 1984; Hedgecock & Okazaki, 1984; Singh 
& Green, 1984; Koehn & Gaffney, 1984; 
Zouros & Foltz, 1984; Diehl & Koehn, 1985; 
Mallet et al., 1985; Hoagland, 1986; Volckaert 
& Zouros, 1989; Gaffney et al., 1990; Beau- 
mont, 1991; Nirchio et al., 1991; Bricelj & 
Krause, 1992; Gaffney et al., 1992; Liu et al., 
1995; Gardner et al.. 1996; Marsden et al., 
1996; Michinina & Rebordinos, 1997). Signifi- 
cant deviation from HWe in bivalves has been 
attributed to Wahlund effect in limited cases, 
and natural selection on young just post set- 
tlement in many instances. 

In bivalves generally, heterozygosity is 
much greater than that found in gastropods. 
Selander & Ochman (1983) summarized al- 
lozyme data relating to the genetic structure 
of about 100 species of gastropods. Het- 
erozygosity of amphimictic species averaged 
about 0.12 and tanged from 0.004 to 0.198. 
Issues of selfing and parthenogenesis involve 
both aquatic and terrestrial pulmonates, es- 
pecially certain slugs in which there is little or 



no heterozygosity, or in which there is consid- 
erable heterozygote deficiency do not pertain 
to Oncomelania. Foltz et al. (1982 a, b, 1984) 
provided data on slugs. There are also strong 
indications of self fertilization in certain ba- 
sommatophoran freshwater pulmonates of 
the genera Biomphalaria and Bulinus. taxa in- 
volved in the transmission of schistosomes in- 
fecting humans in Africa and South America 
(Biomphalaria) (Bandoni et al., 1990; Mimp- 
foundi & Greer, 1990a, b; Bandoni et al., 
1995; Doumas et al., 1996; Mukaratirwaetal., 
1996). In stylommatophoran pulmonate land 
snails, selection seems to operate on some 
loci. Emberton (1993) found over-representa- 
tion of some rare alleles in the young of wide- 
spread populations of some polygyhds. Fal- 
niowski et al. (1998) found only one of many 
loci of Bradybaena fructicum not to be in 
HWe. 

Among the "prosobranch" grade, non-hy- 
drobioid gastropods, populations of some 
species of some genera have been found to be 
in HWe (all loci in HWe); Nassarius (Gooch et 
al., 1972; Sanjuan et al., 1997), Patella 



312 



DAVIS ETAL 



(Wilkins, 1977), /We/ano/des (Livshits& Fishel- 
son, 1983), and some populations of Littorina 
(Rolan-Alvarez et al., 1 995). Some have been 
out of HWe. including some Littorina (Rolan- 
Alvarez et al., 1995) and Sipiionaria (Johnson 
& Black, 1 984), both attributed to the Wahland 
effect. 

Papers involving hydrobioid snails and al- 
lozymes have been few, and most of these 
have been concerned primarily with genetic 
distances and taxon relationships. Heterozy- 
gosity in hydrobioid populations is lower than 
the average for snails overall; in Oncomelania 
it was 0.052 ± 0.030 (N = 14) (Davis et al., 
1994, 1995). Heterozygosities reported for 
two Australian genera were 0.038 ± 0.01 6 (N 
= 26 populations) and 0.043 ± 0.018 (N = 48 
populations) (Ponder et al., 1996; also see 
Ponder et al., 1994, 1995). Heterozygosity for 
IHydrobia was equally low, reported as 0.008 
- 0.074 (Davis et al., 1988, 1989). Haase 
(1993) studied one population each of three 
species of ¡Hydrobia sensu lato and found that 
for one there were heterozygote deficiencies 
at virtually all polymorphic loci, whereas in the 
other two populations there was low or no 
variability. Haase attributed the heterozygote 
deficiencies to selection due to parasite pres- 
sure; the cases of low to no heterozygosity to 
genetic drift and bottlenecks. 

To what do we attribute heterozygote defi- 
ciencies in this study? There are two probable 
explanations. (1) There appears to be a 
Wahland effect in "unstable" populations such 
as Gui Chi and Jian Li, localities swept by the 
Yangtze River annual flooding. There is a mix- 
ture of alleles from snails of diverse localities. 
Because there are no diagnostic loci that 
serve to separate the subspecies, the use of 
multidimensional scaling and a Prim network, 
or a FITCH unrooted tree aides one to identify 
unstable populations. Such trees are freed 
from the constraints imposed in a UPGMA 
phenogram. For example, Davis et al. (1995: 
fig. 9) present a FITCH tree in which there are 
three clusters of populations attributed to 
three subspecies, but three populations did 
not fit into the clusters; on further investiga- 
tion, it was apparent that these three acted as 
hybrid populations but were actually geneti- 
cally unstable populations. 

(2) There have been extensive efforts along 
the Miao River to control snails using mollus- 
caciding and by flooding to drown snails. 
However, the snails have not been eradi- 
cated, and we have been able to collect snails 
from these same localities over several years. 



The life span of Oncomelania hupensis is 
three to four years (Davis, 1967; van der 
Schalle & Davis, 1968), and a female, fertil- 
ized just once, can store viable sperm over 
the coarse of her lifetime. Accordingly, as fe- 
males produce quantities of eggs it is possible 
for one or two females to totally repopulate a 
locality. Accordingly, we attribute the het- 
erozygote deficiencies in the upper Miao 
River, in localities where there should be "sta- 
ble" populations, to extensive inbreeding. As 
Oncomelania hupensis is under continual as- 
sault in efforts to control schistosomiasis, and 
habitats for these snails are ever changing 
due to land-use changes in China, it may be 
difficult to locate genetically stable popula- 
tions using allozymes. 

We are now testing an alternative to al- 
lozymes, COI gene sequences. They serve 
very well to clearly demonstrate the diver- 
gence of the subspecies of Oncomelania 
(Davis et al., 1998) but are much more con- 
servative within populations. We have come 
to expect variation of to 2 base substitutions 
in a sequence length of 648 base pairs (0.00 
to 0.31%) in isolated and genetically stable 
populations, but 4 to 8 (0.62 to 1.23%) or 
more base substitutions in unstable popula- 
tions, indicating the import of differing geno- 
types with flooding. A study on this phenome- 
non is in progress. 

The Subspecies Question in Mainland China 

As discussed above, smooth shelled snails 
in the Yangtze River drainage previously clas- 
sified as Oncomelania fausti belong to On- 
comelania hupensis hupensis. The genetic 
relationships among the three subspecies 
found on the mainland are graphically por- 
trayed in Figure 8 (data from Davis et al., 
1995) (justification for using the polytypic 
species designation given in Davis, 1994, and 
Davis et al., 1995). All the populations of this 
study fall within the variance around the mean 
value for O. hupensis hupensis. 

There is one more name that requires some 
discussion, Oncomelania hupensis guang- 
xiensis. named recently by Liu et al. (1981) 
from the Xun Jiang (Xun River) drainage, 
which becomes the Xi Jiang flowing into 
Guangdong Province from Guangxi Province. 
The Xi Jiang does not flow to the Yangtze 
River, but to the South China Sea near Macao. 
Snails classified as guangxiensis are gener- 
ally smooth but with sporadic, irregular ap- 
pearance of low ribs (Davis et al.. 1 995: figs. 2, 



ALLOZYME ANALYSES TEST THE TAXONOMIC RELEVANCE OF RIBBING 313 
O. h. robertsoni 




O. h. tangi 



O. h. hiipensis 

FIG. 8. Graphic relationships, in scale, among the three subspecies of Oncomelania hupensis on the main- 
land of China. The standard deviations are marked (curved lines). 



4A). The shells have a strong vahx. We place 
this nominal subspecies in the synonymy of O. 
hupensis hupensis because snails studied 
electrophoretically by Davis et al. (1995) are 
within the vahance ofO. h. hupensis (Davis et 
al., 1995: fig. 9) We hypothesize that these 
snails reached Guangxi Autonomous Region 
via a canal dug some centuries ago connect- 
ing the Xiang Jiang River in Hunan Province to 
Guangxi, thus connecting the snail-rich Tong 
Ting Lake district of Hunnan to the southern 
coast. On the basis of allozymes, the Gui Ping 
County Guangxi snails from near the conflu- 
ence of the Qian Jiang and Yu Jiang that forms 
the Xun Jiang are most closely related to a 
population from Hunan (Nei's mD of 0.150 
from Yue Yang, Hunan). 

There are still some unresolved problems 
involving smooth shells with a strong varix. 
While it seems clear that Oncomelania hupen- 
sis hupensis living along the Yangtze and up 
into the lower stretches of rivers flowing into 
the Yangtze belong to the same subspecies, 
whether they have ribbed or smooth shells, 
there are some populations in the hills in east- 
ern China of coastal provinces, Jiangsu and 
Zhejiang, isolated from the Yangtze River 
drainage, that are problematic. 

Zhou et al. (1995) studied the allozymes of 



34 populations from nine provinces They used 
16 loci, of which 5 were esterase loci. In 
UPGMA clustering of Nei's (1978) "D", they 
also found that Sichuan and Yunnan snails 
clustered apart from O. hupensis {sensu Davis 
et al., 1995), as did the one population they 
had from Fujian (O. hupensis tangi). One pop- 
ulation with a smooth shell and varix, from 
Anhui) clustered with the ribbed-shelled popu- 
lations. Using Fitch-Margoliash least-squared 
cluster analysis, all smooth-shelled Oncome- 
lania clustered together apart from ribbed- 
shelled populations, but the Anhui smooth- 
shelled population was basal and distinctly 
apart, rather intermediate between the smooth 
and ribbed-shelled populations. Zhou et al. 
(1995) concluded that there were two taxo- 
nomically distinct groups: ribbed and smooth- 
shelled types. Aside from the Sichuan, Yun- 
nan and Fujian populations, they examined 
only three other smooth-shelled populations, 
the one from Anhui and two from Jiangsu 
provinces. They did not considerthe shell mor- 
phological and biogeographical differences 
that separate hupensis, robertsoni and tangi. 
They did consider that smooth-shelled popu- 
lation groups might be separated into sub- 
species, that is, those from Fujian, Yunnan, 
Sichuan, and the hilly region of Jiangsu 



314 



DAVIS ETAL. 



Province at the extreme eastern edge of 
China. 

Davis et al. (1994) had studied three popu- 
lations of Oncomelania from Zhejiang Prov- 
ince, like Jiangsu, a coastal province. Two 
populations were ribbed and one smooth. 
One ribbed and the smooth population were 
very close to each other geographically, 
whereas the second ribbed population was a 
considerable distance from the others. On the 
basis of Nei's (1978) "D", the smooth-shelled 
population was highly divergent from the 
ribbed snails, a result similar to that later 
found by Zhou et al. (1995) for the two 
smooth-shelled from hilly areas of Jiangsu 
Province. All these smooth-shelled popula- 
tions had a strong varix. Davis et al. (1995) 
found that one smooth-shelled population 
from Zhejiang Province clustered closely with 
the Yunnan and Sichuan O. h. robertsoni: it 
had the least genetic distance with the 
Sichuan populations considering all pair-wise 
comparisons among populations. Davis et al. 
(1995) classified this Zhejiang population as 
roberstoni. 

It has yet to be determined how these east- 
ern hill-dwelling populations relate to smooth- 
shelled Miao River snails studied here. These 
few special populations require intense study. 
Are tfiey truly genetically divergent from O. h. 
hupensis? Are they part of the robertsoni 
complex but have independently evolved a 
varix? Are they a distinct subspecies? 

ACKNOWLEDGEMENTS 

Figure 3 was made by Zhang Yi from SEM 
photographs made by Shen Bing-Gui. The re- 
maining figures were produced by Dr. 
Thomas Wilke of ANSP who also did the 
PHYLIP analysis. We thank Drs. Walter R. 
Hoeh, Kent State University, and David. O. F. 
Skibinski of the University College of Swan- 
sea for reviewing and criticizing this paper. 
The work was funded by U.S.A., N.I.H. grants 
All 1373 (Davis), and AI 39461 (Shanghai 
T.M.R.C). 



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SINGH, S. M. & R. H. GREEN, 1984, Excess of al- 
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SKIBINSKI, D. O. F., J. A. BREADMORE & T. F. 
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XU. X., T FANG, X. YANG, С YU, С XIAO, F. LIU 
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317 



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Revised ms. accepted 1 May 1999 



. VOL. 41, NO. 1 1999 



MALACOLOGIA 



Infernational Journal of Malacology 
Revista Internacional de Malacologia 
Journal International de Malacologie 
Международный Журнал Малакологии 
Internationale Malakologische Zeitschrift 



Publication dates 

Vol. 31, No. 1 29 Dec. 1989 

Vol. 31, No. 2 28 May 1990 

Vol. 32, No. 1 30 Nov. 1990 

Vol. 32, No. 2 7 Jun. 1991 

Vol. 33, No. 1-2 6 Sep. 1991 

Vol. 34, No. 1-2 9 Sep. 1992 

Vol. 35, No. 1 14 Jul. 1993 

Vol. 35, No. 2 2 Dec. 1993 

Vol. 36, No. 1-2 8 Jan. 1995 

Vol. 37, No. 1 13 Nov. 1995 

Vol. 37, No. 2 8 Mar. 1996 

Vol.38, No. 1-2 17 Dec. 1996 

Vol. 39, No. 1-2 13 May 1998 

Vol.40. No. 1-2 17 Dec. 1998 



VOL. 41, NO. 1 MALACOLOGIA 1999 

CONTENTS 

R. ARAUJO, M. A. RAMOS, & R. MOLINET 

Growth Pattern and Dynamics of a Southern Peripheral Population of 
Pisidium amnicum (Müller, 1774) Bivalvia: Sphaehidae) in Spain 119 

ALEXIA BLANC, GUY PINCZON DU SEL, & JACOUES DAGUZAN 

Relationships Between Length of Prey/Predator for the Most Important Prey 

of the Cuttlefish Sepia officinalis L. (Mollusca: Cephalopoda) 139 

GEORGE M. DAVIS, Yl ZHANG, XINGJIANG XU, & XIANXIANG YANG 

Allozyme Analyses Test the Taxonomic Relevance of Ribbing in Chinese 
Oncomelania (Gastropoda: Rissoacea: Pomatiopsidae) 297 

VASILIS K. DIMITRIADIS & ELIZABETH B. ANDREWS 

Ultrastructural and Cytochemical Study of the Kidney and Nephridial Gland 
Cells of the Marine Prosobranch Mollusc Nucella lapillus (L.) in Relation 
to Function 1 87 

MICHAELA. HOGGARTH 

Descriptions of Some of the Glochidia of the Unionidae (Mollusca:Bivalvia) 1 

GENNADY M. KAMENEV & VICTOR A. NADTOCHY 

Species of Macoma (Bivalvia: Tellinidae) from the Pacific Coast of Russia, 
Previously Described as Abrina (Bivalvia: Semelidae) 209 

ALEXANDRA LEITÄO, PIERRE BOUDRY JEAN-PHILIPPE LABAT, & 

CATHERINE THIRIOT-QUIÉVREUX 

Comparative Karyological Study of Cupped Oyster Species 175 

KAREN D. MCCOY 

Sampling Terrestrial Gastropod Communities: Using Estimates of Species 
Richness and Diversity to Compare Two Methods 271 

BENITO MUÑOZ, ARTURO ALMODOVAR , & JOSÉ R. ARREBOLA 

Ganula gadirana n. sp., A New Hygromiidae from Southern Spain 
(Pulmonata: Helicoidea) 163 

JEFFREY С NEKOLA 

Terrestrial Gastropod Richness of Carbonate Cliff and Associated Habitats in 

the Great Lakes Region of North America 231 

JEFFREY С NEKOLA & TAMARA M. SMITH 

Terrestrial Gastropod Richness Patterns in Wisconsin Carbonate Cliff 
Communities 253 

M. SCHILTHUIZEN, J. J. VERNEULEN, G. W. H. DAVISON, & E. GITTENBERGER 

Population Structure in a Snail Species from Isolated Malaysian Limestone 

Hills, Inferred from DNA Sequences 283 

ANNE THEENHAUS & MATTHIAS SCHAEFER 

Energetics of the Red Slug Arion rufus (Gastropoda) and of the Gastropod 
Community in a Beech Forest on Limestone 197 

SUSAN A. TOCZYLOWSKI, R. DOUGLAS HUNTER, & LISA M. ARMES 

The Role of Substratum Stability in Determining Zebra Mussel Load 

on Unionids 151 

GEERATJ. VERMEIJ 

The Accumulation of Taxonomic Knowledge: The History of Species 
Descriptions of Some Predatory Gastropods 147 



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VOL. 41, NO. 1 MALACOLOGIA 1999 

CONTENTS 

MICHAELA. HOGGARTH 

Descriptions of Some of tlie Glochidia of the Unionidae (Mollusca:Bivalvia) 1 

R. ARAUJO, M. A. RAfVlOS, & R. MOLINET 

Growth Pattern and Dynarnicsjr of a Southern Peripheral Population of 
Pisidium amnicum (Müller, 1774|) Bivalvia: Sphaehidae) in Spain 119 

ALEXIA BLANC, GUY PINCZON DU SEL, & JACQUES DAGUZAN 

Relationships Between Length of Prey/Predator for the Most Important Prey 

of the Cuttlefish Sepia officinalis L. (Mollusca: Cephalopoda) 139 

GEERATJ. VERMEIJ 

The Accumulation of Taxonomic Knowledge: The History of Species 
Descriptions of Some Predatory Gastropods 147 

SUSAN A. TOCZYLOWSKI, R. DOUGLAS HUNTER, & LISA M. ARMES 

The Role of Substratum Stability in Determining Zebra Mussel Load 

on Unionids 151 

BENITO MUÑOZ, ARTURO ALMODOVAR , & JOSÉ R. ARREBOLA 

Ganula gadirana n. sp., A New Hygromiidae from Southern Spain 
(Pulmonata: Helicoidea) 163 

ALEXANDRA LEITÄO, PIERRE BOUDRY JEAN-PHILIPPE LABAT, & 

CATHERINE THIRIOT-QUIÉVREUX 

Comparative Karyological Study of Cupped Oyster Species 175 

VASILIS K. DIMITRIADIS & ELIZABETH B. ANDREWS 

Ultrastructural and Cytochemical Study of the Kidney and Nephridial Gland 
Cells of the Marine Prosobranch Mollusc Nucella lapillus (L.) in Relation 
to Function 1 87 

ANNE THEENHAUS & MATTHIAS SCHAEFER 

Energetics of the Red Slug Arion rufus (Gastropoda) and of the Gastropod 
Community in a Beech Forest on Limestone 197 

GENNADY M. KAMENEV & VICTOR A. NADTOCHY 

Species of l\/lacoma (Bivalvia: Tellinidae) from the Pacific Coast of Russia, 
Previously Described as Abrina (Bivalvia: Semelidae) 209 

JEFFREY С NEKOLA 

Terrestrial Gastropod Richness of Carbonate Cliff and Associated Habitats in 

the Great Lakes Region of North America 231 

JEFFREY С NEKOLA & TAMARA M. SMITH 

Terrestrial Gastropod Richness Patterns in Wisconsin Carbonate Cliff 
Communities 253 

KAREN D. MCCOY 

Sampling Terrestrial Gastropod Communities: Using Estimates of Species 
Richness and Diversity to Compare Two Methods 271 

M. SCHILTHUIZEN, J. J. VERNEULEN, G. W. H. DAVISON, & E. GITTENBERGER 

Population Structure in a Snail Species from Isolated Malaysian Limestone 

Hills, Inferred from DNA Sequences 283 

GEORGE M. DAVIS, Yl ZHANG, XINGJIANG XU, & XIANXIANG YANG 

Allozyme Analyses Test the Taxonomic Relevance of Ribbing in Chinese 
Oncomelania (Gastropoda: Rissoacea: Pomatiopsidae) 297 



VOL. 41, NO. 2 1999 



MALACOLOGIA 



International Journal of Malacology 



INTERACTIONS BETWEEN MAN AND MOLLUSCS 



UNITAS MALACOLOGICA-AMERICAN MALACOLOGIGAL SOCIETY SYMPOSIUM 

WASHINGTON, D.C. 
26-30 JULY 1998 



MALACOLOGIA 

EDITOR-IN-CHIEF: 
GEORGE M. DAVIS 

Editorial and Subscription Offices: 

Department of Malacology 

The Academy of Natural Sciences of Philadelphia 

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Philadelphia, Pennsylvania 19103-1195, U.S.A. 



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California Academy of Sciences 

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Co-Editors: 



Assistant Managing Editor: 

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Associate Editor: 

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University of Michigan 

Ann Arbor 



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Denver, CO 



MALACOLOGIA is published by the INSTITUTE OF MALACOLOGY, the Sponsor Members of 
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Field Museum, Chicago 

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University of Delaware, Lewes 

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University of Washington, Seattle 

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University of Colorado Museum, Boulder 



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Rijksmuseum van Natuurlijke 

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Environmental Protection Agency 
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Copyright © 1999 by the Institute of Malacology 
ISSN: 0076-2997 



1999 
EDITORIAL BOARD 



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Marine Biological Station 

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Museum d'Histoire Naturelle 

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University of Liverpool 
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University of Sheffield 
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Bishop Museum 
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University of Liverpool 
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California State University 
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Rijksmuseum van Natuurlijke Historie 
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Carnegie Museum 
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Ecole Pratique des Hautes Etudes 

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Australian Museum 
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Harvard University 
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Rijksuniversiteit 
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MALACOLOGIA, 1999, 41(2): 319 

UMITAS MALACOLOGICA-AMERICAN MALACOLOGICAL SOCIETY SYMPOSIUM 
INTERACTIONS BETWEEN MAN AND MOLLUSCS 

26-30 JULY 1998 

WASHINGTON, D.C. 

Organizing Committee 

Philippe Bouchet 
George M. Davis 
Gerardo R. Vasta 



INTRODUCTION TO THE SYMPOSIUM ON INTERACTIONS 
BETWEEN MAN AND MOLLUSCS 

GEORGE M. DAVIS 

Department of Malacology, Academy of Natural Sciences of Philadelphia, 1900 Benjamin 
Franklin Parkway, Philadelphia, Pennsylvania 19103, USA; davis@acnatsci.org 



The 1998 World Malacological Congress symposium Interactions Between Mol- 
luscs and Humans was one of three symposia offered during the five-day congress. 
As molluscs have an enormous impact on man, it is not surprising that many persons 
from around the world wished to participate in the symposium and that the symposium 
was well attended. Thirty-seven papers were presented, of which 14 were invited and 
the others submitted for presentation. The papers may be grouped into four broad cat- 
egories: advanced biotechnology (4 papers); medical malacology and disease trans- 
mission (15 papers); economics, including diseases of molluscs, impact of introduced 
species, and molluscan food production (8 papers); conservation and mariculture (10 
papers). 

Twelve of the papers are published here, including the elegant opening plenary ad- 
dress given by Dr. Dan Alkon of the United States National Institutes of Health, who 
reviewed the use of an opistobranch snail for working out the molecular basis of learn- 
ing and memory, and presented the implications of the findings to date for under- 
standing Alzheimer's disease. The papers presented by Alkon and Yoshino are in the 
biotechnology category, but Yoshino's work also groups with papers by Davis et al., 
Kristensen & Brown, Brackenbery & Appleton, and Abd Allah in the medical malacol- 
ogy category. The papers by Anderson, Carlton, Pointier and Robinson involved eco- 
nomics and introduced species, although Pointier's paper also had implications for 
control of schistosomiasis (medical malacology category). Finally, Neves and Mansur. 
presented papers that involve conservation. The Kristensen & Brown paper has im- 
plications for conservation of freshwater snails in Africa. 

I would make special comment on Robinson's paper involving commercial transport 
of molluscs all over the world in ever increasing numbers and involving an incredible 
spectrum of species. It was an eye-opener to learn that over 4,900 interceptions of 
molluscs aboard cargo inbound to the United States from throughout the world had 
been intercepted by the United States Department of Agriculture. These interceptions, 
made in a five-year period up to 1998, involved 71 families, 197 genera and 369 
species. In reading both Robinson's and Carlton's papers, one becomes immediately 
concerned about the impact of introduced species into all environments on ecology, 
survival of endemic species, and implications for plant pathogens and human disease. 



319 



MALACOLOGIA, 1999, 41(2): 321 -329 

MOLECULAR PRINCIPLES OF ASSOCIATIVE MEMORY THAT ARE CONSERVED 
DURING THE EVOLUTION OF SPECIES 

Daniel L. Alkon 

Laboratory of Adaptive Systems, National Institutes of Neurological Disorders and Strokes, 
Building 36, Room 4A24, MSC4007, Bethesda, Maryland 20892-4007; dalkon@codon.nih.gov 

ABSTRACT 

Time domains of homeostatic adaptation encompass: 1.) the lifetime of the species (an evo- 
lutionary domain) in which predictive information with survival valve is incorporated into the 
genome, and 2.) the lifetime of organisms in which predictive information is stored within brain 
networks. Experiments over many years in our NIH lab have confirmed that behavioral, bio- 
physical, and molecular mechanisms for storing learned information have been conserved in 
species as diverse as the nudibranch mollusc Hermissenda and mammals such as rat and rab- 
bit. Further studies now indicate that these conserved molecular memory steps (e.g. K^ channel 
inactivation, PKC and calexcitin activation, and intracellular calcium release) are also consistent 
targets of the human syndrome known as Alzheimer's disease. These findings highlight the ele- 
gant economy of natural selection and the utility of animal models for investigating complex brain 
disorders such as Alzheimer's disease. 

Key words: molecular mechanisms, Hermissenda, memory, Alzheimer's disease 



Adaptation of earth's organisms may be 
considered to occur over at least two distinct 
time domains. The first, one that may be 
called the "evolutionary" time domain, often 
occurs over many thousands, even millions of 
years. During this evolutionary time domain, a 
species survives by incorporating genetic 
programs that encode for adaptive structures 
and functions. The species acquires adaptive 
information that is then stored within the ge- 
nome. 

A second time domain for organisms' adap- 
tation occurs during the lifetime of individual 
members of the species. Environmental stim- 
uli in real time impact on an animal's develop- 
ment during critical periods of the animal's 
early life cycle. Environmental stimuli, particu- 
larly when they occur in temporal patterns that 
reoccur repeatedly within a brief time Interval, 
also impact on mature, fully differentiated an- 
imals, and thus impact on an animal's subse- 
quent behaviors. For the latter, information is 
acquired by the mature species member dur- 
ing a learning experience and stored within 
brain networks as "long-term" memory. 

Given these two distinct time domains of 
adaptation, "evolutionary" and "learning", the 
cellular mechanisms responsible for informa- 
tion acquisition and storage are also quite dis- 
tinct. Mutation-induced variations of genomic 



information provide for the "evolutionary" time 
domain of adaptation. Second-messenger in- 
duced signaling cascades within neurons and 
their synaptic specializations provide for the 
time domains of learning adaptation. 

A third, even longer time domain concerns 
the evolution of progressively more adaptive 
species. Thus, not only do individual species 
members adapt, and the species itself adapts, 
but progressively different successive species 
adapt. In this longest time domain, "life" on 
earth itself adapts to the environment by as- 
suming different forms as distinct but still re- 
lated species. 

All of this adaptation and transformation 
notwithstanding, certain biological entities 
and their underlying functional principles may 
remain essentially unchanged, even over the 
course of many millions of years of evolution. 
One clear example of such conservation in- 
volves membrane channels or "pores" that 
gate the flux of ions across cell walls. One 
such channel, called 1^, is a calcium-depen- 
dent pore for potassium flux. When calcium 
rises inside a cell to a critical threshold level, 
it triggers the opening of channels that allow 
K"^ ions to flow down their chemical electrical 
gradient. This almost invariably means that K"*^ 
ions flow from the inside to the outside of the 
cell, thereby making the electric potential 



321 



322 



ALKON 




Out 



FIG. 1 . A molecular model of a generic K+ channel formed from an octomeric protein (after Scott et al. 
provided courtesy of Noam Meiri). 



1994, 



across the cell wall negative inside the cell 
with respect to the outside environment. Such 
calcium-dependent K"^ channels (of 1^ chan- 
nels) occur within the walls of single-cell or- 
ganisms, such as Paramecium. Remarkably, 
very similar 1^, channels also occur within the 
walls of large pyramidal-shaped cells in the 
human brain area known as the hippocam- 
pus. Once nature designed (through evolu- 
tionary time domains) these marvels of mole- 
cular configurations in single cell organisms it 
essentially maintained the channel design 
(Scott et al., 1994), even within our own, 
human species (Fig. 1). 

Some years ago, I wondered if such exam- 
ples of biological conservation -that is, across 
evolution -might also subserve adaptation it- 
self in the time domain of individual human life- 
times. Specifically, were there mechanisms of 
cellular and molecular adaptation within the 
"learning" time domain that were conserved 
across evolutionary time? 

To press this question of conserved cellular 
mechanisms of learning, I set out in search of 
what I thought of as a possible "evolutionary 
compromise". To survive, a species would 
have evolved by making a compromise be- 



tween a central nervous system that was not 
very complex, but just complex enough to 
achieve the capacity to learn crucial stimulus 
relationships. The nudibranch mollusc Her- 
missenda crassicomis can, after being sub- 
jected to many years of experiments, now be 
considered as such a compromise (Alkon, 
1983). A similar sea snail, the gastropod mol- 
lusc Aplysia califomica, had previously been 
found to be a favorable electrophysiologic 
preparation as well as to show behavioral 
changes, such as habitualization and sensiti- 
zation (Bailey & Kandel, 1993). 

Most psychologists agree that human 
memory is diverse and requires diverse brain 
structures. Memories can be explicit or de- 
clarative, procedural or motoric, sensory or 
emotional. Despite its diversity, however, 
human memory invariably involves relation- 
ships of stimuli in time and/or space. This 
seemingly universal relational aspect, often 
called associational, can even be encoun- 
tered in the context of introspective reporting, 
including that of therapeutic settings. No be- 
havioral model of associative learning has 
been more precisely formulated or more pre- 
cisely controlled than Pavlovian conditioning. 



MOLECULAR PRINCIPLES OF ASSOCIATIVE MEMORY 



323 



When a neutral stimulus, such as the sound 
of a bell, precedes a well-valued or reflexive 
stimulus, such as the smell of meat, by a pre- 
cise temporal interval 350-450 msec, a dog 
will learn to salivate in response to the bell 
alone. The meaning (or response) of the smell 
is associated in the dog's brain with a previ- 
ously undefined or "neutral" sound of the bell. 

From the point of view of evolutionary con- 
servation, it is most remarkable that virtually 
the same quantifiable characteristics of Pav- 
lovian conditioning have been identified for 
dogs, rabbits, rats, and humans (Figs. 2, 3), 
but also for Hermissenda (Fig. 4) as well 
(Gormezano, 1966; Gormezano & Kehoe, 
1 981 ; Gormezano et al., 1 983; Gormezano et 
al., 1962; Schreurs, 1993). These characteris- 
tics (e.g., CS-UCS transfer, temporal speci- 
ficity, pairing specificity, extinction, savings) 
are themselves conserved across evolution. 
This remarkable behavioral conservation 
would seem to require that there be consider- 
able conservation of underlying principles of 
neuronal network functions. Conserved be- 




FIG. 2. Human conditioned eyeblink training. A puff 
of air (from the white outlet) on the subject's corneal 
surface is preceded by a discrete tone received 
through the earphone system. Courtesy of Bernard 
G. Schreurs. 



Purr 



^ 

^-/, 



^' 









^ 


—TONE ON 


■' . .' 


TONF AND PUFF OFF 


8 


- 


PUFF ON — 


/ 


■Ч. 


6 






r 


N. 


4 
2 

П 






1 


^^-"^^■■^ 


1 


1 , . , 1 




1 



< 



z. 

U- 

O 
z 
о 



100 300 500 700 

TIMF (MIILISFCONDS) 



900 



- 


— TONE ON 

PUFF ON — 

/ 


^ 


! 

-TONF AND PUFF OFf 




1, 1 




1 



00 300 500 700 900 

TIME (MILIISFCONOS) 



FIG. 3. Transfer of behavioral response in the rabbit occurs as a result of associative, or Pavlovian, condi- 
tioning. In this case the animal is taught to associate an auditory tone with a puff of air to its eye. The be- 
havioral response — extension of the nictating membrane (left panel)— is transferred from the unconditioned 
stimulus (the puff of air) to the conditioned stimulus (the tone). The graphs show that before conditioning the 
membrane extends after the puff of air (top right); about 70 trials later, the animal learns to extend the mem- 
brane when it heats the tone (bottom right). Bernard G. Scheurs supplied the data for this figure. 



324 



ALKON 




FIG. 4. Classical conditioning in the sea slug Hermissenda crassicornis leads to simple visual-vestibular as- 
sociations. Excitation of sensory hair cells in the paired vestibular organs (statocysts) during rotation of this 
slug leads reflexively to contraction of the foot. In contrast, stimulation of photoreceptors in the paired eyes 
of the slug by flash of light does not cause the foot to contract but to weakly orient toward the light gradient 
(left). However, after a series of paired rotation and light flash stimuli, in which the rotation stimulus quickly 
follows a light stimulus, subsequent light stimulation of photoreceptors alone now causes the foot to contract 
(right). 



havioral principles of association employ con- 
served network mechanisms that, in turn, 
imply crucial conserved biophysical and mol- 
ecular mechanisms. 

To test these conservatlon(s), we first 
turned to our evolutionary compromise. Ap- 
plying a variety of electrophysiologic and bio- 
physical recording techniques (e.g., current 
clamp, two microelectrode voltage clamp, 
patch clamp) to the neural pathways that me- 
diate the Pavlovian training stimuli, we were 
able to construct a wiring diagram (Alkon, 
1983) for the visual and vestibular pathways 
and their synaptic interactions (Fig. 5). With 
this circuit diagram, we traced precisely how 
the associated stimuli (CS: light, DCS: rota- 
tion) traveled throughout the visual-vestibular 
circuits to produce a learned conditioned re- 
sponse. Using this "blueprint" of synaptically 
connected neurons, we then reconstructed 
how synaptic strengths were changed during 



learning by long-term alterations of voltage- 
dependent K"" currents within post-synaptic 
membranes. Subsequently, it was possible to 
implicate an elaborate molecular cascade that 
regulates these channels and possibly struc- 
tural aspects of the synaptic apparatus itself 
during learning and memory. The critical mol- 
ecular cascade showed clear analogy to the 
molecular cascade (Fig. 6) responsible for 
muscle contraction and long-term changes of 
muscle structure (Alkon, 1989; Alkon et al., 
1998). Among the crucial events in these cas- 
cades are enhanced mobilization of intracel- 
lular calcium, activation of the signaling en- 
zyme protein kinase С (PKC), activation of the 
PKC substrate, the signaling protein calex- 
citin, activation of the calcium releasing chan- 
nel, the ryanodine receptor, and calcium-de- 
pendent regulation of m-RNA turnover for 
specific proteins (Fig. 7). 
Many years of further studies on mam- 



MOLECULAR PRINCIPLES OF ASSOCIATIVE MEMORY 



325 




FIG. 5. Neural responses to stimulus pairing. Neural system (schematic and partial diagram) responsive to 
light and rotation. Each eye has two Type A and three Type В photoreceptors; each optic ganglion has 13 
second-order visual neurons; each statocyst has 12 hair cells. The neural interactions (intersection of verti- 
cal and horizontal processes) identified to be reproducible from preparation to preparation are based on in- 
tracellular recordings from hundreds of pre- and post- synaptic neuron pairs. 



Swimming control 



Water-maze trained 




-PGK1- 
-RyR2- 




M 



6 12 24 



6 12 24 M 



FIG. 6. Hippocampal changes in RYR2-mRNA levels after water-maze training. (A) RT-PCR analysis of 
RYR2-mRNA in control swimming and water-maze-trained rats at 2, 6, 12 and 24 h after training. No change 
in RYR2-mRNA levels was observed between control swimming and naive animals (data not shown). {Fig- 
ure continues.) 



глаПап learning nnodels, such as rat nnaze 
learning, rabbit eyeblink conditioning, and ol- 
factory discrimination learning, provided 
strong confirmation that the molecular cas- 
cades found in Hermissenda were also criti- 
cally involved in mammalian learning and 
memory. Thus, conserved associative learn- 



ing behaviors from mollusc to mammal were 
correlated with conserved cellular and subcel- 
lular mechanisms responsible for those learn- 
ing and memory behaviors. This conservation 
of learning behavior and its molecular basis 
received still further confirmation through 
studies of Alzheimer's disease and its molec- 



в 



(Л с: 

^ 8 

ОС от" 
E-g 
см =5 
СП О) 

>>> 

cu 



350.00 



30000 - 



250 00 



200.00 



150.00 




S 12 

Hours after water-maze training 



24 



С 











Water-maze trainee 



255 






í^íísíi'-^*' 



D 



20 



16 



10- 



I Swimming control 
I Water-maze trained 




FIG 



CA1 CA3 DG 

6. (Continued) (B) Relative RYR2-mRNA levels in water-maze-trained rats at 2, 6, 12 and 24 h after 



training. To control for tine integrity of RNA and for differences attributable to errors in experimental manipu- 
lation from tube to tube, primers for rat phosphoglycerate kinase 1 (PGK1) were included in the RT-PCR re- 
actions. (C) Localization by in situ hybridization of RYR2-mRNAs in hippocampal subfields of control, swim- 
ming, and water-maze-trained rats 6 h after training. The color spectrum on the right side of the figure 
represents the pixel value of gray levels. (D) Relative RYR2-mRNA levels in different hippocampal subfields 
of control swimming and water trained-rats. Quantification of induction increase is achieved by comparison 
of pixel values of an area of interest in four sections from each of four pairs of rats. Changes in mRNA lev- 
els are expressed as density ratio of trained to control animals. 



MOLECULAR PRINCIPLES OF ASSOCIATIVE MEMORY 



327 



2+ . 



Periods of Ca signaling 



I. Depolarization 

Ca Influx 

PKC auto- 
phosphorylates 

PKC translocates 

CE phosphorylated 

CE inactivates K'^ channels 



II. Calexcitin 
Binding to RyR 

CE translocates 
to membrane 
and ER 

CE activates RyR 



III. Ca reuptake 

CE activates 
Ca-ATPase 



IV. DNA synthesis 



Transcriptional 
factors activate 
DNA synthesis 



Late genes 



V. Protein Syntliesis 

Axonal transport 
Structural changes 

Increased RyR expression 



VI. Ion Channels 



^2+ 1^ Periods of 

Receptor Channel Channel aSSOCiative ШеГТЮГу 



INDUCTION 

I. msecs^ 
seconds 




II. seconds- 
minutes 



CONSOLIDATION 



III. mjnutes-»^ 
hours 



IV. hours-* 
days 



STORAGE 

V. days^ 
weeks 



Vi. weeks ^ 
months 



FIG. 7. Schematic diagram illustrating time domains of calcium signaling and associative memory. (Legend 
continues.) 



328 



ALKON 



FIG. 7. {Continued) Stage I: The neuron depolarizes as a result of a convergence of synaptic inputs, which 
activates G-protein coupled receptors (i.e., for acetylcholine, GABA, glutamate). Membrane depolarization 
also opens Ca^"^ channels, causing an influx of Ca^^. Diacylglycerol (DAG), arachidonic acid (AA), and inos- 
itol triphosphate (IP3) are released by phospholipases A2 and С (PLAg and PLC) and, along with Ca^*, acti- 
vate protein kinase С (PKC), which is thereby translocated to the plasma membrane. Ca also activates 
CaM kinase. The kinases undergo autophosphorylation which renders their activity independent of Ca^"^. 
PKC and CaM kinase may also inhibit K"^ and other channels by direct phosphorylation. 
Stage II: Elevated [Ca^"^], activates the Ca^'"-binding protein calexcitin (CE). Phosphorylation of CE by PKC 
promotes its translocation to membrane compartments, where it inhibits K"" channels, making the membrane 
more excitable to further depolarizing stimuli. CE also elicits Ca^* release from ryanodine receptors on the 
membrane of the endoplasmic reticulum (ER) and possibly synaptic membranes, resulting in amplification of 
Ca^* signals. IP3 also releases Ca^"^ by activating the IP3 receptor (IP3R). 

Stage III: CE, after phosphorylation by PKC, no longer activates the RyR, and no longer inhibits Ca^'"-AT- 
Pase at the ER membrane, facilitating the removal of excess Ca^"". 

Stage IV: CE and/or Ca^"", probably acting indirectly through transcriptional activators, induce new DNA tran- 
scription. CE may also indirectly increase mRNA turnover. 

Stage V: Late genes are transcribed, resulting in increased synthesis of at least 21 different proteins, in- 
cluding RyR. At this stage, through an as-yet undetermined mechanism, retrograde axonal transport is also 
inhibited by CE; this may underlie the structural changes in branch morphology that were observed after Her- 
missenda associative learning. 

Stage VI: New RyR receptors and ion channels are synthesized and transported to their respective mem- 
branes. These may be related to enhanced Purkinje cell dendritic excitability with rabbit conditioning. 



с 
с 
пз 

x: 

и 1Л 
Ê ^ 

(Л ™ 
1Л Cl- 

4_) It- 

о о 
û. 

СЛ 
Q. 



100 
90 
80 
70 
60 
50 
40 
30 
20 
10 




- 




113 pS 


- 




ШШ^^Ш 1 \ 1 



Untreated 
Controls 



B-Amyloid 
Controls 



Alzheimer's 
Disease 



FIG. 8. Bar graph of the percentage change (from resting Ca^"' concentrations) shows the virtual elimination 
of the TEA response in treated control cells (eight cell lines, 194 cells) similar to observations for AD fibrob- 
lasts (four cell lines, 285 cells measured). Preparations of the various cell lines were tested for response to 
TEA in the absence (left) or presence (center) of ßAP. The responses of AD fibroblasts shown here are in 
agreement with our previous report showing no responses in 13 different AD lines (> 700 cells measured). 



ular correlates. To test the possibility of con- 
served molecular events responsible for hu- 
man memory, we turned to that clinical entity 
most specific for human memory loss: Alz- 
heimer's disease. This disease early in its 
progression rather specifically impairs human 
memory. Beginning with the hypothesis that 



Alzheimer's disease is systemic (i.e., involving 
multiple tissue types through out the body) but 
causes clinical symptoms only through effects 
on the brain, we analyzed peripheral cell 
types, such as human skin fibroblasts. After 
many studies, we could conclude that, indeed, 
a succession of molecular steps in the con- 



MOLECULAR PRINCIPLES OF ASSOCIATIVE MEMORY 



329 



served memory cascade were in fact consis- 
tently targets of dysfunction in Alzheimer's dis- 
ease. Diagnostic clianges of K"" channels (Fig. 
8), PKC intracellular calcium release, and 
calexcitin were consistently found in the skin fi- 
broblasts (Etcheberrigaray et al., 1994) of 
Alzheimer's patients, but not for a variety of 
age-matched controls. Furthermore, low lev- 
els of soluble ß-amyloid (nanomolar-compara- 
ble to endogenous levels found throughout the 
body) induced these diagnostic phenotypic 
abnormalities in the normal fibroblasts. Here, 
then, was evidence that early Alzheimer's dis- 
ease involves several of the same molecular 
steps implicated as mechanism for animal 
learning. 

Thus, based on these studies of species 
that range from the nudibranch Hermissenda 
to humans, we may infer that adaptation in the 
"learning" time domain uses functional princi- 
ples that have been conserved across the 
"evolutionary" time domain. This conservation 
deserves our future attention not only be- 
cause it illustrates and elucidates the elegant 
economy of natural selection. It also provides 
a strategy whereby we can use molecular 
cascades in primitive species as accessible 
models to guide our investigations of daunt- 
ingly complex and often inaccessible myster- 
ies of human physiology and disease. 



LITERATURE CITED 

ALKON, D. L., 1 983, Learning in a marine snail. Sci- 
entific American, 249: 70-84. 

ALKON, D. L., 1989, Memory storage and neural 
systems. Scientific American, 260: 42-50. 

ALKON, D. L., T J. NELSON, W. Q. ZHAO & S. 



CAVALLARO, 1998, Time domains of neuronal 
calcium signaling and associative memory: steps 
through a calexcitin, ryanodine receptor, K^ chan- 
nel cascade. Trends in Neuroscience, 21: 
529-537. 

BAILEY, С H. & E. R. KANDEL, 1993, Structural 
changes accompanying memory storage. Annual 
Review of Phiysiology, 55: 397-426. 

ETCHEBERRIGARAY R-, E. ITO, S. KIM & D. L. 
ALKON, 1994, Soluble ß-amyloid induction of 
Alzheimer's pheontype for human fibroblast K* 
channels. Science, 264: 276-279. 

GORMEZANO, I., 1966, Classical conditioning. Pp. 
385-420, in J. B. siDOWSKi, ed.. Experimental 
methods and instrumentation in psychology. New 
York, McGraw-Hill. 

GORMEZANO, I. & E. J. KEHOE, 1981, Classical 
conditioning and the law of contiguity. Pp. 1-45, 
in p. HARZEM & M. D. ZEiLER, Advances in analysis 
of behavior, vol. 2, Predictability, correlation, and 
contiguity. Sussex, England, Wiley & Sons. 

GORMEZANO, I., E. J. KEHOE & B. S. MAR- 
SHALL, 1983, Twenty years of classical condi- 
tioning research with the rabbit. Progress in Psy- 
chobiology and Physiological Psychology, 10: 
197-275. 

GORMEZANO, I., N. SCHNEIDERMAN, E. G. 
DEAUX & I. FUENTES, 1962, Nictitating mem- 
brane: classical conditioning and extinction in the 
albino rabbit. Science, 138: 33-34. 

SCHREURS, B. G., 1993, Long-term memory and 
extinction of the classically conditioned rabbit nic- 
titating membrane response. Learning & Motiva- 
tion, 24: 93-302. 

SCOTT V. E. S., J. RETTIG, D. N. PARCEJ, J. N. 
KEEN, J. B. С FINDLAY O. PONGS & J. O. 
DOLLY, 1994, Primary structure of a ß subunit of 
a-dendrotoxin-sensitive K"^ channels from bovine 
brain. Proceedings of the National Academy of 
Sciences, USA, 19: 1637-1641. 

Revised ms accepted 30 April 1999 



MALACOLOGIA, 1999, 41(2): 331-343 

THE BIOMPHALARIA GLABRATA EMBRYONIC (BGE) MOLLUSCAN CELL LINE: 

ESTABLISHMENT OF AN IN VITRO CELLULAR MODEL FOR THE STUDY OF 

SNAIL HOST-PARASITE INTERACTIONS 

Timothy P. Yoshino^ ^, Christine Coustau^, Sylvain Modat^ & Maria G. Castillo^ 

ABSTRACT 

Cell lines of invertebrates, especially those of arthropod origin, have played crucial roles in ad- 
dressing fundamental questions related to cell signaling and differentiation, gene expression, 
cell-pathogen interactions, and the like. They also have been instrumental in the development of 
genetic transformation systems and the development and testing of microbial insecticides. Re- 
cently, we have utilized a cell line originally derived from embryos of the freshwater snail Biom- 
phalaria glabrata (Bge cell line; Hansen, 1976a) to investigate the complex cellular, biochemical 
and molecular interactions between snails and their trematode parasites. Because this cell line 
was derived from B. glabrata, possesses a fibroblast-like appearance similar to circulating he- 
mocytes, and shares in common several hemocyte functions (substrate adhesion, phagocytosis, 
encapsulation, enzyme content), the Bge cell line is proposed as a cellular model for B. glabrata 
hemocyte structure and function. In support of this proposal, a hemocyte ß-integrin cell adhesion 
receptor homologue recently was identified and cloned based on information from a previously 
acquired Bge cell ß integrin subunit cDNA sequence. As a general approach, it is anticipated that 
Bge cells can be evaluated for genes associated with immune recognition/adhesion, and sub- 
sequently employed to generate molecular or immunological probes for use in hemocyte stud- 
ies. Other applications of Bge cells to the study of parasite-snail host interactions include their 
use in the in vitro cultivation of intramolluscan stages of diverse trematode species, and in the 
development of genetic transformation systems for molluscan cells. Research in this latter area 
has focused on the identification of suitable Bge cell promoters and testing their abilities to drive 
expression of reporter gene constructs. It is concluded that the Bge cell line offers a diversity of 
valuable experimental approaches when applied to the study of molluscan cellular immune 
mechanisms or snail-trematode physiological interactions. 

Key words: Biomphalaria glabrata, Mollusca, embryonic cell line, Bge cell line, hemocyte. 
Schistosoma mansoni, in vitro culture, transgenic. 



INTRODUCTION 

The use of cell lines as tools for addressing 
fundamental questions regarding molecular 
structure-function relationships is well estab- 
lished in those animal species for which such 
lines are available. For coelomate inverte- 
brates, the vast majority of cell lines are of in- 
sect or arachnid origin (Bayne, 1998) and 
have been extensively employed in studies of 
gene regulation and protein expression 
(Berger & Morganelli, 1984; Jones et al., 
1996), cellular shape-change and motility 
(Kosik & McConlogue, 1994), adhesion 
(Bleber, 1994), induction of immune peptides 
(Hultmark, 1994; Hoffmann & Reichart, 1997), 
and pathogen-host cell interactions (Stellar, 
1993; Kopecky & Stankova, 1998; Lawrence, 



1 997). Moreover, there has been a long-stand- 
ing interest in the use of insect cell lines in the 
production of recombinant proteins through 
baculovirus expression systems (Thomsen et 
al., 1993; McCarroll & King, 1997; Merhngton 
et al., 1997), the selection and testing of mi- 
crobial pesticides (Maramorosch & Mit- 
suhashi, 1997), and the development of new 
genetic transformation technologies (Fallon, 
1 991 ; Cherbas et al., 1 994; O'Brachta & Atkin- 
son, 1996). However, for reasons that are not 
yet understood, there exist very few cell lines 
outside of the arthropod classes Insecta and 
Arachnida, despite a critical need and strong 
interest in cell lines of diverse invertebrate or- 
ganisms (Bayne, 1998). 

Of the non-arthropod cell lines currently 
available, only one, the Biomphalaria glabrata 



^ Department of Pathobiological Sciences, 201 5 Linden Dr. W, University of Wisconsin-Madison, Madison, Wisconsin 53706, 

U.S.A.; yoshinot@svrn. vetmed.wisc.edu 

^Corresponding author 

^Centre de Biologie et d'Ecologie Tropicale et Méditerranéenne, Université de Perpignan, France 

331 



332 



YOSHINOETAL 



embryonic (Bge) cell line, is of molluscan ori- 
gin (ATCC# CL1494; American Type Culture 
Collection, Rockville, Maryland). It was de- 
rived from embryos of the freshwater pul- 
monate snail B. glabrata by Hansen (1976a), 
and has been stored/maintained in various 
laboratories over the past 20 years. The sig- 
nificance of the Bge cell line, in addition to its 
unique molluscan origin, also lies in the fact 
that the snail species from which this line was 
derived represents a primary intermediate 
host for transmission of the blood fluke. Schis- 
tosoma mansoni (Platyhelminth; Digenea), 
causative agent of human intestinal schisto- 
somiasis in the New World and Africa (Basch, 
1991). Schistosoma spp. are estimated to in- 
fect 200 million people in over 74 endemic 
countries, and, among parasitic diseases, is 
considered second in importance only to mos- 
quito-transmitted malaria in its public health 
impact (World Health Organization, 1998). 

Because molluscan and arthropod interme- 
diate hosts of infectious diseases (broadly re- 
ferred to as "vectors") are essential to human 
transmission, they have long been recognized 
as potentially vulnerable targets in disease 
control programs. However, because tradi- 
tional methods of vector control, including pes- 
ticide usage and habitat destruction, have not 
provided sustainable solutions to disease con- 
trol or prevention, new approaches aimed at 
disrupting parasite development within the 
host through genetic and/or molecular manip- 
ulation of vector competence are now being 
explored. This field has advanced most rapidly 
in insects where inducible antimicrobial and 
antiparasitic peptides (Faye & Hultmark, 1 993; 
Hoffman & Reichhart, 1997; Lowenberger et 
a!., 1 999) and enzymes (Ashida & Brey, 1 998) 
have been identified, their genes cloned, and 
the molecular pathways/factors regulating an- 
timicrobial responses elucidated. In addition, 
methods for introducing and expressing im- 
mune peptides or function-disrupting anti- 
sense sequences into whole organisms 
through genetic transformation technologies 
are being developed (Carlson et al., 1997; 
O'Brachta & Atkinson, 1996). It is envisioned 
that the basic knowledge of anti-pathogen 
immune mechanisms and parasite develop- 
mental pathways will lead to molecularly- 
based strategies for interrupting pathogen sur- 
vival in the vector host. Significantly, insect cell 
lines have played important roles in advancing 
the general area of host-pathogen relation- 
ships. 

In contrast to the insects, little progress has 



been made in the development of molecular 
vector control approaches for molluscs of 
medical and veterinary importance. For ex- 
ample, in the freshwater gastropods, few 
genes have been cloned and/or identified as 
being of potential significance in regulating 
vector competence (Dissous et al., 1990; 
Ноек et al., 1996; Adema et al., 1998; Knight 
et al., 1998), and there are still no genetic or 
physical maps available for vector snails 
(Knight et al. 1998). Recently, however, appli- 
cation of the B. glabrata embryonic (Bge) cell 
line to the in vitro cultivation of larval schisto- 
somes (Yoshino & Laursen, 1995; Coustau et 
al., 1997) has provided significant opportuni- 
ties to investigate larval blood fluke-host cell 
interactions at the molecular level. Work utiliz- 
ing this cell line has already led to the identifi- 
cation of molecules potentially involved in 
host cell signal transduction (Lardans et al., 
1998), immune adhesion (Davids et al., 
1999), trematode development (Laursen & 
Yoshino, 1999) and to the establishment of 
protocols for DNA-mediated gene transfer 
into molluscan cells (Lardans et al., 1996; 
Yoshino et al., 1998). The present review fo- 
cuses on these recent advances in the use of 
the Bge cell line as a tool for studying chemi- 
cal communication mechanisms between 
trematode parasites and cells of their mollus- 
can host. 

Bge Cells as a B. glabrata Hemocyte Model 

Two important criteria for establishing a 
comparative cellular model system include 
the demonstration that the cells being com- 
pared have a similar origin, both in terms of 
species and ontogeny, and that they share 
structural and functional similarities under in 
vitro conditions. In terms of species origin, it is 
fortunate that the Bge cell line was derived 
from B. glabrata (Hansen, 1976a), a major in- 
termediate host of S. mansoni and an impor- 
tant experimental snail host for the study of 
schistosome-snail immune interactions (Fryer 
& Bayne, 1996a; Yoshino & Vasta, 1996; 
Adema & Loker, 1997). The precise ontologi- 
cal origin of the Bge cell line is not known 
since it was originally obtained from develop- 
ing five-day old B. glabrata embryos (Hansen, 
1976a). However, it has been speculated, 
based on morphology (Fig. 1) and Bge cell's 
ability to synthesize collagen-like molecules 
(Hansen, 1976b; Stein & Basch, 1977), that 
these cells probably are of fibroblast or fibro- 
blast-like origin. A similar ontological origin 



THE BIOMPHALARIA GLABRATA EMBRYONIC MOLLUSCAN CELL LINE 



333 



111 



%j 



<í¿^ 



B.glahraía hemocytes 



Bge cells 



FIG. 1. Phase-contrast photomicrographs of Biomphalaria glabrata hemocytes (left) and cells of the B. 
glabrata embryonic (Bge) cell line (right) as they appear attached to and spread on a glass slide substrate. It 
has been noted that, as depicted here, a subset of Bge cells assume a more hemocyte-like appearance when 
they are washed of medium and permitted to reattach under serum-free conditions. This differs from more typ- 
ical spindle-shaped morphology of Bge cells as they appear under normal culture conditions (Lardans & Dis- 
sous, 1 998). Whether these differences in cell morphology reflect the possible presence of differentiating Bge 
cell subpopulations or a behavioral response to manipulation is presently unknown. Scale bar = 10 (am. 



also has been suggested for circulating he- 
mocytes (Pan, 1958), although their exact 
embryological source in B. glabrata is still 
controversial (Jeong et al., 1983). 

The second criterion for Bge cells to serve 
as an effective model for B. glabrata hemo- 
cytes is that the two cell-types must share im- 
portant structural and functional characteris- 
tics in their interactions with foreign materials. 
Here we assume that shared function is 
based on similarities in biochemical and/or 
molecular mechanisms being used by these 
snail cell-types. Therefore, recently we have 
initiated experiments to begin evaluating the 
"immune" functions of phagocytosis and en- 
capsulation in the Bge cell line. Phagocytic 
and encapsulation responses represent "hall- 
mark" behaviors for hemocytes since these 
activities, in large part, define their immune ef- 
fector capabilities (Ratcliffe et al., 1985; 
Bayne, 1990). 

Phagocytosis: It is well documented that B. 
glabrata hemocytes are capable of phagocy- 
tosing a variety of foreign particles including 
bacteria (Cheng et al., 1 978), mammalian ery- 
throcytes (Abdul-Salam & Michelson, 1980; 
Noda & Loker, 1989; Zeick & Becker, 1992), 
yeast/zymosan (Fryer & Bayne, 1989; 
Connors & Yoshino, 1990), and latex beads 
(Uchikawa & Loker, 1992; Fryer & Bayne, 
1996b). Moreover, excretory-secretory prod- 
ucts (ESP) released by developing S. man- 
soni sporocysts exert inhibitory effects on 
phagocytosis by host hemocytes (Connors & 
Yoshino, 1990; Fryer & Bayne, 1990), sug- 
gesting the presence of ES factors capable of 



binding to hemocytes and interferring with he- 
mocyte-particle interactions. 

However, to date, only ancedotal evidence 
is available that Bge cells possess similar 
phagocytic activity. Therefore, this function 
was tested more rigorously using an adhe- 
sion/phagocytosis assay modified from 
Uchikawa & Loker (1992). In this assay, un- 
charged latex beads (6.4 ¡am in diameter; 
Sigma Chemical Co., St. Louis, Missouri;) 
were rinsed 2x in Chernin's balanced salt so- 
lution (CBSS; Chernin, 1963) and resus- 
pended in CBSS to a final concentration of 
0.1%. To determine the effect of S. mansoni 
ESP (Connors & Yoshino, 1990) on phagocy- 
tosis, beads were treated with ESP (10 ¡iL 
beads in 1mL ESP) for 4 h at 22°C, followed 
by two washes in CBSS and resuspension in 
CBSS at a concentration of 0. 1 %. Beads were 
then presented to monolayered Bge cells in a 
ratio of 2 beads/cell. Because of the difficulty 
in discerning between surface membrane ad- 
herent and endocytosed beads (Fig. 2) 
(Uchikawa & Loker, 1992), an adhesion index 
(percentage of cells with adherent/phagocy- 
tosed beads) was determined for each treat- 
ment and control group at 1, 3, 6 and 24 h 
post-bead innoculation. As shown in Figure 3, 
untreated latex beads readily attached to Bge 
cells by 1 h (45%), and by 6 h had reached 
near maximum association (adherence/pha- 
gocytosis) with cells (approx. 80%). This rate 
of bead association was considerably higher 
when compared to B. glabrata hemocytes, 
which attained between 10-15% bead adher- 
ence (Uchikawa & Loker, 1992) at 1 h of incu- 
bation. In a similar study (Fryer & Bayne, 



334 



YOSHINOETAL 




FIG. 2. Photomicrographs of В. glabrata embryonic 
(Bge) cells demonstrating surface attached and 
phagocytosed latex beads following and 24 h in- 
cubations in snail saline (CBSS). 



1996b), however, it is noted that hemocyte at- 
tained a phagocytic rate of 70% using smaller 
(2.5 |im) charged latex beads. Regardless of 
discrepancies in association rates, clearly 
Bge cells, like hemocytes, possess the ca- 
pacity to phagocytose foreign particles. 

Pretreatment of beads with ESP prior to in- 
cubation with Bge cells resulted in a signifi- 
cant increase in bead adhesion at all time 
points (Fig. 3), suggesting that ESP contains 
molecule(s) that mediate enhanced bead ad- 
hesion through interaction with putative ESP 
receptors on the Bge cell surface. The fact 
that untreated beads did not associate with 
cells above control levels when incubated in 
the presence of ESP supports this hypothe- 
sis; in this case, free ESP molecules saturate 
Bge cell receptors, thereby blocking en- 
hanced adhesion due to bead-bound ESP. 
Recent flow cytometric analyses using fluo- 
rescein-labeled ESP (fESP) as a "probe"con- 
firm that polypeptide(s) contained in ESP di- 



100 -| 
90- 
80- 
70 
60 
50 
40- 
30- 
20- 
10 








10 15 

Time (hour) 



FIG. 3. Percentage of B. glabrata embryonic (Bge) 
cells with attached/phagocytosed beads acquired 
during 24 h of incubation in snail saline. Control: 
beads pretreated with CBSS prior to addition to Bge 
cells in CBSS (-□-); ESP treatment #1 : beads pre- 
treated with S. manson/ sporocyst excretory-secre- 
tory products (ESP) before addition to Bge cells in 
CBSS (-O-); ESP treatment #2: beads pretreated 
with CBSS prior to addition to Bge cells in CBSS 
containing ESP (-0-). 'designates values that dif- 
fer significantly (p < 0.05) from the control. 



rectly bind to Bge cells, and that such binding 
is inhibited by the sulfated poly-fucose, fu- 
coidan. Of particular relevance to this review, 
B. glabrata hemocytes also have been found 
to bind fESP via a fucoidan-inhibitable recep- 
tor (Johnston et al., in preparation), and it is 
hypothesized that these, or related, receptors 
are responsible for the ESP-mediated modu- 
lation of hemocyte function (e.g., Yoshino & 
Lodes, 1988; Lodes & Yoshino, 1990; Con- 
nors & Yoshino, 1990; Loker et al., 1992; 
Adema & Loker, 1 997). In this case, Bge cells 
may serve as a unique model for investigating 
parasite ESP-binding hemocyte receptors. 

Encapsulation: Encapsulation, or the multiple 
layering of cells around large foreign objects, 
is another function shared in common be- 
tween B. glabrata hemocytes and Bge cells. 
Capsule formation represents the first line of 
cellular defense against larval helminths in- 
fecting molluscs, and is typified in the B. 
glabrata/S. mansoni system where hemo- 
cytes of resistant snail strains encapsulate 
and kill schistosome sporocysts under both in 
vivo (Sullivan & Richards, 1981; Loker et al., 
1982) and in vitro (Bayne et al., 1980) condi- 
tions. At present, however, little is known 



THE BIOMPHALARIA GLABRATA EMBRYONIC MOLLUSCAN CELL LINE 



335 












"'»* к / 



' -^"¡^'^'^ ' . 






•■/^■' 



CBSS 



Fucoidan 



FIG. 5. Bright-field photomicrographs of B. glabrata embryonic (Bge) cells binding to the surface of S. man- 
soni mother sporocysts in the absence (left; CBSS only) and presence of an inhibiting polysaccharide, fu- 
coidan (right; 1 mg/ml CBSS). 



about the hemocyte molecules mediating par- 
asite adhesion/recognition or how the encap- 
sulation process in regulated. In order to ad- 
dress these questions, it may also be possible 
to use Bge cells as a encapsulation model. 
Bge cells co-cultured with S. mansoni sporo- 
cysts avidly bind to the larval surface form- 
ing multicellular capsules (Fig. 4) (Hansen, 
1976b; Yoshino & Laursen, 1995), reminicent 
of encapsulations by resistant snail hemo- 
cytes (Boehmler et al., 1996). In the case of 
Bge cells, however, encapsulation does not 
lead to parasite destruction, but instead are 
more comparable to in vitro capsules formed 
around sporocysts by hemocytes of suscepti- 
ble snail strains (Bayne et al., 1980; Fryer & 
Bayne, 1995). 

Due to this shared sporocyst adhesive be- 
havior, Bge cells are currently being used to 
identify and characterize the surface recep- 
tor(s) responsible for initiating parasite adher- 
ence and for mediating subsequent cell-to- 
cell binding required for capsule formation. 
For example, using an in vitro cell adhesion 
assay, in which S. mansoni sporocysts were 
placed into 1 .5-mL microcentrifuge tubes (200 
larvae/tube) followed by addition of 2 x 10^ 
Bge cells in a final volume of 200 |iL of CBSS, 
we have determined that Bge cells in suspen- 
sion were capable of binding to the sporocyst 
tegument after incubation for 24 h at 26°C 
(Fig. 5). Qualitative and quantitative assess- 
ment of cell adherence in the presence or ab- 
sence of various chemical inhibitors further 
demonstrated that Bge cell binding was medi- 
ated through a carbohydrate-inhibitable 
mechanism, possible involving a lectin-like re- 
ceptor(s) expressed on the Bge cell surface 
(Fig. 5). Whether or not B. glabrata hemo- 
cytes possess similar sporocyst binding sites 



^ ^7 /" V Л. '! ^},\ 



'»if 



FIG. 4. Bright-field photomicrograph of an in vitro 
cultured S. mansoni mother sporocyst encapsu- 
lated by numerous B. glabrata embryonic (Bge) 
cells. Within Bge cell capsules sporocysts continue 
to develop, eventually giving rise to daughter sporo- 
cyst stages under in vitro conditions. 



is at present unknown. However, it is antici- 
pated that in-depth biochemical/molecular 
studies on the parasite-reactive receptors of 
Bge cell will lead to the development of valu- 
able molecular and/or chemical probes for in- 
vestigating similar membrane receptors asso- 
ciated with snail hemocytes. 

Lysosomal Enzyme Content: Finally, since B. 
glabrata hemocytes previously have been 
shown to possess a variety of lysosomal en- 
zymes (mainly hydrolases), we sought to de- 
termine whether or not Bge cells exhibited a 
similar enzyme repertoire. Acid and alkaline 
phosphatase, lysozyme, ß-glucuronidase, li- 
pase, nonspecific esterase, peroxidase, and 
aminopeptidase activities (Rodrick & Cheng, 
1974; Cheng et al., 1978; Granath & Yoshino, 
1983a; McKerrow et al., 1985; Cheng, 1985; 



336 



YOSHINOETAL 



TABLE 1 . Hydrolytic enzyme activity observed in Bge cell lysates (cell) or 2 hr-cultured supernatants (sn). C: 
Bge cells incubated for 2 hr in CBSS. B: Bge cells in the process of phagocytosing latex beads. ES: Bge cells 
in the presence of ES products. ES total: Control for enzymatic activity contributed by ES products alone. 
Semi-quantitative scores according to enzyme kit standards (APIzym^i^ Galery; bioMèrieux): - , nanomole 
of enzyme; +, 5 nanomoles; ++, 10 nanomoles; +++ 20 nanomoles; ++++, 30 nanomoles. 



Enzymes tested 


С cell 


Csn 


Beeil 


Bsn 


ES cell 


ESsn 


ES total 


alkaline phosphatase 


+ 


— 


+ 


— 


++ 


-H-l- 


— 


esterase 


++ 


+++ 


+++ 


Ч-++ 


+++ 


-H-l- 


-1- 


esterase lipase 


++ 


+ 


+-1-1- 


+ 


-H-l- 


+ 


— 


leucine arylamidase 


++++ 


+ 


-H-l-l- 


+ 


++++ 


+ 


— 


valine arylamidase 


+ 


- 


-1- 


- 


+ 


- 


— 


cystine arylamidase 


+ 


- 


-1- 


- 


+ 


- 


— 


acid phosphatase 


++++ 


+ 


++++ 


-1- 


-i~i~i~i- 


+ 


-1- 


naphtol-AS-BI-phosphohy- 


+-I-I- 


+++ 


+++ 


-l"H- 


-l-l-H 


+++ 


++ 


drolase 
















ß galactosidase 


++++ 


++ 


-I--I--1-I- 


-l-l- 


++++ 


-n- 


- 


N-acetyl-ß glucosaminidase 


++ 


- 


-ы- 


- 


-l-l- 


- 


- 



and others) have been detected in B. glabrata 
hemocytes using various enzyme cytochemi- 
cal and biochemical assays. Our assumption, 
as previously suggested, was that similarities 
in enzyme profiles may reflect shared func- 
tional and biochemical "backgrounds". Deter- 
minations of hydrolase activities in Bge cells 
were carried out using the APIzym™ Galery 
semi-quantitative enzyme assay kit (bio- 
Mèrieux Vitek, Hazelwood, Missouri). In these 
experiments, Bge cell monolayers initially 
were washed with CBSS and cultured for 24 h 
in CBSS at 26°C prior to assay. Cells were 
then hypotonically lysed, cellular debris pel- 
leted by centrifugation, and enzyme activity 
measured in the supernatants. 

As shown in Table 1 , the general profile of 
Bge cell hydrolase activities is comparable to 
that of snail hemocytes. Specifically, cells 
contained significant amounts (>10 nano- 
moles) of nonspecific (simple) esterase, li- 
pase, leucine arylamidase, carbohydrases, 
phosphohydrolase and the "classical" lysoso- 
mal marker, acid phosphatase. Several en- 
zymes (e.g., esterase, phosphohydrolase and 
ß galactosidase) appeared to be secreted into 
the medium during a 2 h incubation in CBSS. 
Also of interest was the observation that 
phagocytic stimulation or treatment of Bge 
cells with S. mansoni ESP had little effect on 
the hydrolase activity profile (Table 1). Excep- 
tions included alkaline phosphatase, in which 
exposure to ESP Induced an increase in both 
cellular and secreted enzyme activity, and 
simple esterase and lipase, in which phago- 
cytosis appeared to increase their cellular en- 
zyme content. Although selective, these types 
of inductive responses are similar to those re- 



ported in snail hemocytes exposed to bacteria 
in vitro or from hosts with larval infection 
(Cheng, 1983, 1985; Granath & Yoshino, 
1 983b). Overall, the above functional and bio- 
chemical similarities exhibited by Bge cells 
and hemocytes reinforce the notion that Bge 
cells may serve as a legitimate model for B. 
glabrata hemocytes in investigations of para- 
site-snail immune interactions. In the follow- 
ing section, several examples are described 
as to how Bge cells are being used as tools to 
investigate schistosome-snail relationships 
under in vitro conditions. 

Applications of Bge Cells to the Study of 
Snail Host-Parasite Interactions 

Structure and Function of Hemocyte Adhe- 
sion Molecules: Since both Bge cells and he- 
mocytes are capable of adhering to and 
spreading on glass or plastic substrates via 
pseudopodial extensions of the cytoplasm 
(Fig. 1), it was hypothesized that these cells 
employ similar adhesion molecules to medi- 
ate substrate adherence or spreading. In a 
test of this hypothesis, we recently found that 
cell spreading on glass surfaces by both he- 
mocytes (Davids & Yoshino, 1998) and Bge 
cells (Davids et al., 1 999) was inhibited by the 
tetrapeptide, arg-gly-asp-ser (RODS), but not 
the glu-substituted peptide, RGES (Fig. 6). 
Because the RGD peptide sequence is well 
known as a specific ligand for cellular recep- 
tors of the integrin family (Sonnenberg, 1 993), 
our findings suggested that hemocytes and 
Bge cells share a common cell adhesion/ 
spreading mechanism mediated by integrin- 
like receptors. In order to further investigate 



THE BIOMPHALARIA GLABRATA EMBRYONIC MOLLUSCAN CELL LINE 337 



% 






RGES 




FIG. 6. Phase-contrast photomicrographs of B. glabrata embryonic (Bge) cells incubated in presence of the 
tetrapeptides RGES (nonspecific control peptide) or RGDS (specific test peptide). Note that RGDS strongly 
inhibits cell spreading on the glass substrate (arrow), whereas no such inhibition is seen in the presence of 
RGES (arrow). Scale bar = 10 цт. Reprinted with modification from Gene, Davids, B. J., X. J. Wu & T. P. 
Yoshino, Cloning of a ß integrin subunit cDNA from an embryonic cell line derived from the freshwater mol- 
lusc, Biomphalaria glabrata, 1999, with permission from Elsevier Science. 



this possibility, we took advantage of the 
ready availability and ease of producing large 
numbers of Bge cells to identify and clone a 
molluscan integrin homologue that may be 
serving as a putative RGD-binding receptor. 
This was accomplished by PCR amplifica- 
tion of a 137 base pair Bge cell cDNA se- 
quence corresponding to the highly con- 
served ß integrin ligand binding domain (LBD) 
using degenerate primers to the LBD of sev- 
eral known ß integrin subunits (Gettner et al., 
1995), followed by PCR amplification of the 3' 
region of the Bge ß integrin cDNA using an 
exact 5' primer contained within the 137 bp 
Bge LBD and an oligo-dT 3' primer. Sequence 
of the 5' end of the integrin cDNA was ob- 
tained by 5' RACE (random-amplification of 
cDNA ends) methods. Based on its predicted 
molecular mass of 87.6 kDa and characteris- 
tic domain structure (Davids et al., 1999), this 
molecule was identified as an authentic ß in- 
tegrin subunit homologue (designated ßBGE) 
and the first to be cloned from the phylum Mol- 
lusca. 

We then asked the question, do B. glabrata 
hemocytes also express a ß integrin subunit 
cDNA similar to that of Bge cells? Based on 
Bge cell sequence data, RT-PCR was per- 
formed on snail hemocyte cDNA using exact 
primers synthesized from the ßBGE integrin 
LBD. Amplified products from snail hemo- 
cytes shared 99% nucleic acid similarity and 
100% amino acid identity to the Bge cell ß in- 
tegrin subunit, and from 49-71% amino acid 
identity with LBDs of other known invertebrate 
and human ß subunits (Fig. 7). Cloning and 
sequencing of the complete B. glabrata he- 
mocyte cDNA currently is in progress, but to 



date, comparison of a 1900-nucleotide hemo- 
cyte sequence with ßBGE has revealed 97% 
and 96% identities at the nucleic acid and 
amino acid levels, respectively (unpublished 
data). Thus, it appears that hemocytes also 
express a ß integrin homologue, although like 
the ßBGE subunit, it still remains to be deter- 
mined whether this integrin subunit, in con- 
junction with an as yet unidentified alpha 
subunit, is responsible for the observed RGD- 
dependent cell spreading response (Davis & 
Yoshino, 1998). Moreover, it is important to 
recognize that the integrin gene sequence in- 
formation acquired from Bge cells greatly fa- 
cilitated our ability to generate comparable 
hemocyte data, and it is envisioned that this 
research strategy will continue to contribute 
critical molecular tools for investigating a vari- 
ety of molecules and their functions in hemo- 
cytes. 

In Vitro Cultivation of Larval Trematodes: A 
major technical hurdle in investigating the 
molecular mechanisms underlying intramol- 
luscan schistosome development has been 
the lack of an in vitro culture system capable 
of supporting continous growth and differenti- 
ation through all larval stages. Soon after the 
successful isolation of the Bge cell line from 
B. glabrata embryonic tissues (Hansen, 
1976a), attempts were made to develop a 
Bge cell-based in vitro culture system capable 
of supporting the larval development of S. 
mansoni. Co-cultivation of Bge cells with in 
vivo-dehved daughter sporocysts resulted in 
the production of a second daughter sporo- 
cyst generation (Hansen, 1976b), while in 
more recent experiments, S. mansoni mother 



338 



YOSHINOETAL 



в. glabra ta &Bge 

B. glabra ta Hemocyte 
D. melanogaster ßPS 
S. purpura tus ßG 

C. elegans ßpat-3 
P. leniusculus ß 
A. millepora ßCnl 

D. melanogaster ßv 
0. tenuis ßPol 

H. sapiens ßl 

H. sapiens ß3 
Consensvis 



ENYPVDLYYFMDLSNSMEDDKEKLALLGNKIAEQMSAITKNFRLGFGSFVDKWSPYV . STVPQK 

DLYYFMDLSNSMEDDKEKIJU.IX3№<IAEQMSATTKNFRLGFGSFVDKVVSPYV. STVPQK 

-G L К А— ST— D-LS-T-KR— N~H LM .--I-K- 

_D V К LS— MD— DIL-SE-KN— S T-M . E- 

VD L Y — К Q — SE— DLL--R-RTV 1— KLM-FI . DPRIE- 

RD L S KQ — A — SEL-KL-KGL-SQ-T LM— A.D-S 

L~M-S— K— LGN-RS-AGQM-TT-KE— S— К А F-RT E 

R-N-L VL TWT-R KT-EE— AQLSQTLKN— G-Y A— PTL-MIL. . . — H 

— F-I LL Y — R — LDN-KQ — ADL-ASIVGLST 1 E A-ETTLD-RPQ 

-D — I L Y — К — L-NVKS--TDIWNE-RR— SD— I E-T-M— I . — T-A- 

-D 1 — L Y — К— LWSIQN— T-L-T— RK~S-L-I А P MYISP-EA 

EDYPVDLYYI^DLSYSMKDD — KL — LGDKLAETMK-ITSNFRLGFGSFVDKWMPYV-STVP-K 



FIG. 7. Multiple amino acid (AA) alignment and consensus sequence of ß integhn ligand binding domains 
(LBD). The sequence from B. glabrata embryonic (Bge) cells and hemocytes are aligned with the LBD of all 
known invertebrate species and two human LBD GenBank Accession Nos.: В. glabrata Bge cell [AF060203], 
D. melanogaster ^PS [J03251], S. purpuratus [U77584], C. elegans [U19744], P. leniusculus [X98852], A. 
millepora [AF005356], D. melanogaster ^v [L13305], O. tenuis [AF005357], H. sapiens ß1 [X07979], and H. 
sapiens ß3 [J02703]. The LBD consensus sequence was derived from the nine LBD listed in the figure and 
is shown in the bottom line. The periods (.) represent gaps in the sequence introduced by PILEUP (GCG) to 
align each sequence. AA residues identical to those of the Bge/hemocyte sequence are indicated by dashes. 
Reprinted with modification from Gene, Davids, B. J., X. J. Wu & T P. Yoshino, Cloning of a ß integrin sub- 
unit cDNA from an embryonic cell line derived from the freshwater mollusc, Biomphalaria glabrata, 1 999, with 
permission from Elsevier Science. 



sporocysts, derived from in vitro transformed 
miracidia, were found to produce daughter 
sporocysts (Yoshino & Laursen, 1995). This 
latter study represented the first direct linkage 
of miracidium-through-daughter sporocyst 
development under in vitro conditions. In an 
important breakthrough, Barnes, Bayne and 
colleagues have now succeeded in complet- 
ing the the entire intramolluscan cycle of S. 
mansoni development (miracidium-to-cer- 
caria) under in vitro conditions by co-cultiva- 
tion of larvae in the presence of Bge cells and 
employing a combination of several media 
formulations (Ivanchenko et al., 1999). These 
studies now provide an established in vitro 
culture system that can be used to identify 
and characterize host (Bge) cell factors criti- 
cal to the regulation of larval differentiation, or 
that can be chemically modulated to screen 
various agents (e.g., growth factors, cy- 
tokines, snail plasma factors, etc.) for their ef- 
fects on parasite development (Lardans & 
Dissous, 1998). 

In followup experiments, we were surprised 
to find that, like S. mansoni, Bge cells also 
supported significant parasite growth and dif- 
ferentiation when other trematode species 
were used in this culture system. For exam- 
ple, miracidia of S. japonicum (Coustau et al., 
1997) and the deer liver fluke, Fascioloides 
magna (Laursen & Yoshino, 1999), trans- 
formed to mother sporocysts, which in turn 
produced daughter sporocysts and mother re- 
diae, respectively. In these studies, of particu- 



lar interest was the observation that Bge cells 
did not form large cellular encapsulations 
around mother sporocysts of either species 
as was the case with S. mansoni. In order to 
confirm this observation, a direct comparison 
of S. mansoni and S. japonicum sporocysts 
was made using the Bge cell adhesion assay 
described previously. In contrast to 94% of S. 
mansoni sporocysts exhibiting Bge cell bind- 
ing, only 51 % of S. japonicum sporocysts pos- 
sessed adherent cells. Moreover, using a 
semi-quantitative measure of adhesion inten- 
sity (1 = no binding, 2 = <10 cells/sporocyst, 3 
= >10 cells but <50% of sporocyst surface 
with cell binding, and 4 = >50% of sporocyst 
surface with bound cells), a cell adhesion 
index (CAI) was calculated according to the 
following formula: total adhesion intensity 
score/# sporocysts evaluated. Preliminary re- 
sults of this analysis indicate that Bge cell ad- 
herence to S. japonicum sporocysts was sig- 
nificantly less (CAI = 1.51) than their ability to 
adhere to sporocysts of S. mansoni (CAI = 
2.48). Two possible explanations for this out- 
come may be (1) the tegumental surface lig- 
ands responsible for Bge cell adhesion differ, 
either quantitatively or qualitatively, between 
Schistosoma spp. or (2) S. japonicum larvae 
may be secreting a factor(s) that interferes 
with Bge cell-sporocyst binding interaction 
(Adema & Loker, 1997). However, regardless 
of the mechanisms involved, these molecular 
differences exhibited between Bge cells and 
different schistosome species may be related 



THE BIOMPHALARIA GLABRATA EMBRYONIC MOLLUSCAN CELL LINE 



339 



to the snail host specificity normally ex- 
pressed by these parasites. It is anticipated 
that continued efforts to identify and charac- 
terize these Bge cell adhesion receptors will 
facilitate ongoing nnolecular studies on com- 
parable hemocyte receptors, and eventually 
shed light on fundamental questions related 
to host-parasite compatibility. 

Bge Cell Genetic Transformation-a Tool for 
the Future: The ability to transfect Bge cells 
with foreign DNA and effect its stable in vitro 
expression represents another important step 
in fully developing the research potential of 
this cell line. Successful application of this 
technology to Bge cells would provide a num- 
ber of extremely useful tools: (1) a homolo- 
gous genetic expression system in which re- 
combinant snail proteins can be produced in 
"native" form; (2) in conjunction with S. man- 
soni (or other schistosome species), an in 
vitro culture system for evaluating the effects 
of snail host products (e.g., hemocyte cyto- 
toxic peptides, growth factors, etc.) on larval 
survival; and (3) a system for testing gene 
transfer methodologies or approaches in ad- 
vance of attempts to transfect whole organ- 
isms. However, as alluded to in the introduc- 
tion of this paper, in comparison to the 
arthropods, the field of molluscan transgenic 
technology is only in its infancy. 

The first gene transfer attempts into mollus- 
can cells were reported in 1996 in which tran- 
sient expression of luciferase reporter gene 
constructs driven by heterologous Drosophila 
heat-shock protein (HSP70) or human cy- 
tomegalovirus (CMV, early) promoters was 
achieved in the Bge cell line (Lardans et al., 
1996) and oyster heart primary culture cells 
(Boulo et al., 1996). In efforts to develop a ho- 
mologous promoter system for DNA transfers 
into Bge cells, Laursen et al. (1997) cloned a 
Bge cell HSP70 cDNA, which was then used 
as a probe to isolate and characterize the en- 
tire HSP70 gene, including a putative pro- 
moter region (Yoshino et al., 1998). The pro- 
moter function of this region was confirmed by 
demonstrating the heat-inducible expression 
of luciferase activity (reporter enzyme) in Bge 
cells transfected with HSP70 promoter-lu- 
ciferase reporter constructs (Fig. 8) (Yoshino 
et al. 1998). These results, although prelimi- 
nary in nature, demonstrate the feasibility of 
employing the Bge cell line as model mollus- 
can system for developing new or adapting 
previously successful approaches to the 
eventual establishment of efficient, stable 




□ 26° с 
И 40° с 



FIG. 8. Mean ±SD of specific luciferase activity (in 
relative light units/mg protein) for В. glabrata em- 
bryonic (Bge) cells transfected with various test and 
control vector constructs, and subjected to heat- 
shock (40°C) or no heat-shock (26°C). A: Bge 
HSPgg^ pronnoter-Luc construct; B: Bge HSP^ q^ 
promoter-Luc construct; C: Bge HSPgsk promoter 
only (no /.uc control); D: Luc only (no promoter con- 
trol); E: DOTAR only (lipofectin control), n = 3 inde- 
pendent relicates. 



DNA gene transfers into snail cells and whole 
organisms. Whether or not advancement of 
this technology in molluscs will lead to practi- 
cal applications in the control or prevention of 
human schistosomiasis is presently unknown. 
However, it is anticipated that in continuing to 
strive towards this goal, a great deal of valu- 
able information on various molluscan genes, 
their regulation and the consequences of their 
expression on snail host-parasite interactions 
will be generated. 



SUMMARY 

Biomphalaria glabrata embryonic (Bge) 
cells, currently the only available molluscan 
cell line, is proposed as cellular model for cir- 
culating hemocytes of B. glabrata, a major 
snail intermediate host of the human blood 
fluke. Schistosoma mansoni. In addition to 
originating from the same snail species and 
possibly sharing a similar ontological origin, 
Bge ceils and B. glabrata hemocytes also 
share important functional and biochemical 



340 



YOSHINOETAL 



characteristics including substrate adhesive 
properties, phagocytic activities, encapsula- 
tion responses, and similar lysosomal en- 
zyme content. Investigations of these proper- 
ties in Bge cells should lead to development 
of useful molecular tools (e.g., DNA probes, 
antibodies) that, in turn, will facilitate similar 
studies on snail hemocytes. Such an ap- 
proach currently is being applied to the identi- 
fication and characterization of cellular adhe- 
sion proteins in B. glabrata hemocytes. In 
addition, Bge cells are further being exploited 
in the in vitro cultivation of larval trematodes 
of medical and veterinary importance and in 
the development of molluscan genetic trans- 
formation systems. To date the Bge cell line 
has proved to be an invaluable tool in its ap- 
plication to molluscan biotechnology, and will 
play an increasingly critical role in future stud- 
ies on the molecular basis of snail-trematode 
compatibility. 



ACKNOWLEDGEMENTS 

The authors thank Laura Johnston for as- 
sistance in reproducing the figures presented 
in this paper. The original and previously pub- 
lished work was supported in part by NIH 
grant AM 5503 (TPY) and NIH-NIAID schisto- 
some supply contract N01-AI-55270. 



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THE BIOMPHALARIA GLABRATA EMBRYONIC MOLLUSCAN CELL LINE 343 

line. American Journal of Tropical Medicine and 
Hygiene, 59: 414-420. 
ZELCK, U. & W. BECKER, 1 992, Biomphalaria gla- Revised ms. accepted 30 April 1 999 

brata: influence of calcium, lectins, and plasma 



MALACOLOGIA, 1999, 41(2): 345-353 

BIOMPHALARIA GLABRATA: A LABORATORY MODEL ILLUSTRATING THE 
POTENTIAL OF PULMONATE GASTROPODS AS FRESHWATER BIOMONITORS 

OF HEAVY METAL POLLUTANTS 

A. T Abd Ailah\ S. N. Thompson^^ D. B. Borchardt^ & M. Q. A. Wanas^ 

ABSTRACT 

The potential value of pulmonate gastropod snails as biomonitors of pollution in freshwater en- 
vironments is discussed, with the laboratory M line strain of Biomphalaria glabrata used to illus- 
trate bioaccumulation of heavy metals. Adult B. glabrata exposed at 28°C to 0.25 |iM chloride 
salts of lead, cadmium or mercury accumulated these heavy metals in the soft tissues within four 
weeks exposure. The mean tissue lead concentration increased approximately three fold, cad- 
mium ten fold, and mercury 25 fold over the levels of these metals in snails not exposed to the 
dissolved chloride salts. Exposure to any of the three metal salts caused snail mortality. The 
mean LC25 values for lead, cadmium and mercury at two weeks exposure were 82, 0.22 and 0.94 
цМ, respectively. Although survival was reduced in exposed snails, surviving individuals were vi- 
able as indicated by the relative levels of high energy phosphorus metabolites in the in vivo ^^P 
NMR spectrum. The results suggest that pulmonate gastropods snails display potential for bio- 
monitoring heavy metal pollution in freshwater environments. Surveys of the natural molluscan 
populations in waterways of lower Egypt are currently underway in an effort to identify potential 
molluscs, including pulmonates, as biomonitors in polluted areas. 

Key words: Biomphalaria, freshwater, biomonitor, heavy metals. 



INTRODUCTION 

Direct methods of chemical analysis have 
long been employed for identifying and quan- 
tifying environmental pollutants in air, water 
and soil. Although improvements in the sensi- 
tivity of such analytical methods will ensure 
their continued role as a means for monitoring 
pollution, the low concentration range of many 
environmental contaminants is often a serious 
obstacle, with analyses requiring special-pur- 
pose ultraclean laboratories and lengthly pre- 
concentration techniques. For many years, 
laboratory and field studies have indicated 
that analysis of pollutants in tissues of biolog- 
ical organisms can prove highly beneficial as 
an adjunct to traditional approaches of sam- 
pling and examination (Martin & Coughtrey, 
1982). In aquatic environments, many plants 
and animals absorb and accumulate trace or- 
ganic and inorganic pollutants, frequently 
concentrating these pollutants many-fold over 
the levels occurring in the natural abiotic envi- 
ronment. 

Biomonitoring can provide significant ad- 
vantages over direct chemical analysis. Be- 



cause the pollutants are concentrated by the 
biomonitor, additional concentration prior to 
analysis is seldom required, thus simplifying 
the analytical procedure. In addition, biomoni- 
tors provide infomation on the "bioavailability" 
of a pollutant rather than the total abundance 
of a pollutant in the aquatic environment at 
large (Phillips & Segar, 1986). Exhaustive an- 
alytical studies to Identify all chemical species 
of a pollutant are often unnecessary and In- 
vestigations on the biological effects of each 
and every form may be avoided. Among the 
principal characteristics of a potential biomon- 
itor are: (1) tolerance to the pollutant, (2) dis- 
tinctive morphological changes associated 
with exposure to the pollutant, (3) accumula- 
tion of the pollutant within the organism, and 
(4) accumulation of the pollutant in a manner 
dependent on its concentration in the environ- 
ment (Butteret al., 1971). 

Although biomonitors have seldom been 
used as the sole means for routine detection 
and monitoring of aquatic pollutants, they 
have been employed to some degree for 
about three decades (Phillips & Rainbow, 
1993). The "Mussel Watch Program" employ- 



Department of Zoology, Al-Azhar University, Cairo, Egypt 

Analytical Chemistry Instrumentation Facility 

Department of Entomology, University of California, Riverside, California, U.S.A.; nelsont@mail.ucr.edu 

345 



346 



ABDALLAH ETAL 



ing Mytilis spp. for monitoring organic and 
heavy metal pollution is a well-known exam- 
ple demonstrating the considerable success 
achieved in monitoring contaminated marine 
and estuarine ecosystems (Goldberg et al., 
1983; Cessa, 1989). While numerous bio- 
monitors have also been identified for various 
pollutants in freshwater habitats, only a few 
studies have been conducted on molluscs 
(Phillips & Rainbow, 1993). Most investigation 
has focused on fish, plants and algae, al- 
though a few investigations have appeared on 
bivalves (Graney et al., 1984; Bias & Karbe, 
1985), and Fantin et al. (1982) reported the 
accumulation of lead in Viviparus viviparus, a 
freshwater gastropod. 

The present report summarizes the results 
of several investigations on a laboratory strain 
of Biomphalaria glabrata, a pulmonate gastro- 
pod, suggesting the potential of freshwater 
pulmonates as biomonitors of three heavy 
metals, cadmium and mercury, two List 1 sub- 
stances (European Economic Community, 
1976), and lead, a List 2 contaminant. The 
study was conducted to demonstrate the ac- 
cumulation of heavy metals in the soft tissues 
and to examine the effects of metal exposure 
on snail viability and mortality. The results sug- 
gest that the metals do accumulate in snails 
and exposure to concentrations in excess of 
those generally encountered in contaminated 
environments a limited effect on snail survival. 
Thus, this pulmonate gastropod and related 
species may prove useful as biomonitors. A 
preliminary observation demonstrating that 
lead is accumulated within the digestive gland 
is also described. 



MATERIAL AND METHODS 
Snail Culture 



Metal Treatments and Mortality 

Groups of 1 adult snails measuring 1 1 .5 to 
12.5 mm were placed in glass beakers con- 
taining 500 ml of an artificial spring water 
(Maclnnis & Voge, 1970) prepared in pure 
water (< 15 mega-ohm/cm^). A population 
density of 1 snail/50 ml water replaced every 
three days was previously reported as that re- 
sulting in optimal growth (Thomas & Benjamin, 
1 974). Water was purified with a Millipore Milli- 
Q" water system. "Ultra-pure" (> 99.99%) 
reagent grade chloride salts of cadmium, (Cd), 
lead (Pb), and mercury (Hg 2""), purchased 
from Aldrich Chemical Co. (Milwaukee, Wis- 
consin, USA), were dissolved to various con- 
centrations. Preliminary trials were conducted 
to establish