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Full text of "Malacologia"

HARVARD UNIVERSITY 




LIBRARY 

OF THE 

DEPARTMENT OF MOLLUSKS 

IN THE 

Museum of Comparative Zoology 
Gift of: 



VOL39, NO. 1-2 1998 



MALACOLOGIA 



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



Publication dates 

Vol.28, No. 1-2 19 Jan. 1988 

Vol.29, No. 1 28 Jun. 1988 

Vol.29, No. 2 16 Dec. 1988 

Vol.30, No. 1-2 1 Aug. 1989 

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 



VOL39, NO. 1-2 MALACOLOGIA 1998 

CONTENTS 

R. A. D. CAMERON & L M. COOK 

Forest and Scrub Snail Faunas from Northern Madeira 29 

KATHERINE COSTIL & STUART E. R. BAILEY 

Influence of Water Temperature on the Activity of Planorbarius Corneas (L.) 
(Pulmonata, Planorbidae) 141 

MARIA GABRIELA CUEZZO 

Cladistic Analysis of the Xanthonychidae (= Helminthoglyptidae) (Gastropoda: 
Stylommatophora: Helicoidea) 93 

CHRISTOPHE DESBUQUOIS & LUC MADEC 

Within-Clutch Egg Cannibalism Variability in Hatchlings of the Land Snail Helix 
Aspersa (Pulmonata: Stylommatophora): Influence of Two Proximate Factors 173 

GREGORY R DIETL & RICHARD R. ALEXANDER 

Shell Repair Frequencies in Whelks and Moon Snails from Delaware and Southern 

New Jersey 151 

ROBERT T DILLON, JR. & CHARLES LYDEARD 

Divergence Among Mobile Basin Populations of the Pleurocerid Snail Genus, 
Leptoxis. Estimated by Allozyme Electrophoresis 113 

ALEJANDRA L. ESTEBENET 

Allometric Growth and Insight on Sexual Dimorphism in Pomacea Canaliculata 
(Gastropoda: Ampullariidae) 207 

BENJAMIN J. GOMEZ, ANA M. ZUBIAGA, M.TERESA SERRANO, & EDUARDO ÁNGULO 

Histochemical and Ultrastructural Identification of Biphasic Granules in the Albumen 
Secretory Cells of Arion Subfuscus (Gastropoda, Pulmonata) 77 

SANDRA GORDILLO Y SANDRA N. AMUCHÁSTEGUI 

Estrategias de Depredación del Gastrópodo Perforador Trophon Geversianus (PaWas) 
(Muricoidea: Trophonidae) 83 

MARY ELLEN HARTE 

Translating Trees into Taxonomy within Veneridae (Bivalvia): A Critique of Two Recent 
Papers 215 

J. W. HAWKINS, M. W. LANKESTER, & R. R. A. NELSON 

Sampling Terrestrial Gastropods Using Cardboard Sheets 1 

ALEXANDER I. KAFANOV & ANATOLY L. DROZDOV 

Comparative Sperm Morphology and Phylogenetic Classification of Recent Mytiloidea 
(Bivalvia) 1 29 

MATTY KNIGHT ANDRE N. MILLER, NEIL S. M. GEOGHAGEN, FRED A. LEWIS, 

& ANTHONY R. KERLAVAGE 

Expressed Sequence Tags (ESTs) of Biomphalaria Glabrata, an Intermediate Snail 

Host of Schistosoma Mansoni: Use in the Identification of RFLP Markers 175 

RICHARD M. LEBOVITZ 

The Inheritance of an Embryonic Lethal Mutation in a Self-Reproducing Terrestrial 

Slug, Deroceras Laeve 21 

CHARLES LYDEARD, JOHN H. YODER, WALLACE E. HOLZNAGEL, FRED G.THOMPSON, 

& PAUL HARTFIELD 

Phylogenetic Utility of the 5'-Half of Mitochondrial 16S rDNA Gene Sequences for 
Inferring Relationships of Elimia (Cerithioidea: Pleuroceridae) 183 

CHARLES S. RICHARDS, CAROLYN PATTERSON, FRED A. LEWIS, & MATTY KNIGHT 

Larval Fusion and Development of Conjoined Teratoids in Biompliaiaria Glabrata ... 123 

KEVIN J. ROE & CHARLES LYDEARD 

Molecular Systematics of the Freshwater Mussel Genus Potamilus (Bivalvia: 
Unionidae) 195 

PETER D. ROOPNARINE 

Translating Trees into Taxonomy within Veneridae (Bivalvia): A Reply to Harte 221 

LUIZ RICARDO L.SIMONE 

Morphology of the Western Atlantic Haliotidae (Gastropoda, Vetigastropoda) with 
Description of a New Species from Brazil 59 

LAURA R.WHITE 

Corrections to White et al., 1996, Molecular Genetic Identification Tools for the 
Unionids of French Creek, Pennsylvania Malacologia 38:181-202 225 

J. B. WOOD, E. KENCHINGTON, & R. K. O'DOR 

Reproduction and Embryonic Development Time of Bathypolypus Arcticus, A Deep- 

Sea Octopod (Cephalopoda: Octopoda) 11 

ANA MARIA LEAL-ZANCHET 

Comparative Studies on the Anatomy and Histology of the Alimentary Canal of the 
Limacoidea and Milacidae (Pulmonata: Stylommatophora) 39 



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Address: Malacologia 

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



AWARDS FOR STUDY AT 
The Academy of Natural Sciences of Philadelphia 

The Academy of Natural Sciences of Philadelphia, through its Jessup and 
McHenry funds, makes available each year a limited number of awards to support 
students pursuing natural history studies at the Academy. These awards are primar- 
ily intended to assist predoctoral and immediate postdoctoral students. Awards usu- 
ally include a stipend to help defray living expenses, and support for travel to and 
from the Academy. Application deadlines are 1 March and 1 October each year. 
Further information may be obtained by writing to: Chairman, Jessup-McHenry 
Award Committee, Academy of Natural Sciences of Philadelphia, 1900 Benjamin 
Franklin Parkway, Philadelphia, Pennsylvania 19103-1195, U.S.A. 



MALACOLOGIA, 1998, 39, NO. 1-2 



39 NO. 1-2 



MALACOLGIA 



1998 



INSTRUCTIONS FOR AUTHORS 



1. MALACOLOGIA publishes original re- 
search on the Mollusca that is of high quality 
and of broad international interest. Papers 
combining synthesis with innovation are par- 
ticularly desired. While publishing symposia 
from time to time, MALACOLOGIA encour- 
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diverse topics. Papers of local geographical or 
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where, as should papers whose primary 
thrust is physiology or biochemistry. Nearly all 
branches of malacology are represented on 
the pages of MALACOLOGIA. 

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figures. 



VOL39, NO. 1-2 1998 



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. 



Co-Editors: 



EUGENE COAN 

California Academy of Sciences 

San Francisco, CA 

Associate Editor: 

JOHNB. BURCH 
University of Michigan 
Ann Arbor 



CAROL JONES 
Denver, CO 

Assistant Managing Editor: 

CARYL HESTERMAN 

The Academy of Natural Sciences 

Philadelphia, Pennsylvania 



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



RÜDIGER BIELER 
Field Museum, Chicago 

JOHN BURCH 

MELBOURNE R. CARRIKER, 

President 

University of Delaware, Lewes 

GEORGE M. DAVIS 
Secretary and Treasurer 

CAROLE S. HICKMAN 
University of California, Berkeley 

ERIC HOCHBERG 

Santa Barbara Museum of Natural History 



ALAN KOHN 

University of Washington, Seattle 

JAMES NYBAKKEN 

Moss Landing Marine Laboratory 

California 

CLYDE F E. ROPER 

Smithsonian Institution 
Washington, D.C. 

SHI-KUEIWU 

University of Colorado Museum, 

Boulder 



Participating Members 

EDMUND GITTENBERGER JACKIE L. VAN GOETHEM 

Secretary, UNITAS MALACOLOGICA Treasurer, UNITAS MALACOLOGICA 

Rijksmuseum van Natuurlijke Koninklijk Belgisch Instituut 

Historie voor Natuurwetenschappen 

Leiden, Netherlands Brüssel, Belgium 



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

KENNETH J. BOSS 

Museum of Comparative Zoology 

Cambridge, Massachusetts 



Emeritus Members 

ROBERT ROBERTSON 

The Academy of Natural Sciences 

Philadelphia, Pennsylvania 

W. D. RUSSELL-HUNTER 
Easton, Maryland 



Copyright © 1998 by the Institute of Malacology 



1998 
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. CALOW 

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 

T 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 

Association of Systematics Collections 
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 

Kabenhavn. 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 

Marine Biological Station 

Porto Novo, India 



S.G.SEGERSTRLE 
Institute of Marine Research 
Helsinki, Finland 

A. STAÑCZYKOWSKA 
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.0KLAND 
University of Oslo 
Norway 

TOKUTANI 
University of Fisheries 
Tokyo, Japan 

W. L. PARAENSE 

Instituto Oswaldo Cruz, Rio de Janeiro 

Brazil 

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

J. R POINTIER 

Ecole Pratique des Hautes Etudes 

Perpignan Cedex. France 

W. F PONDER 
Australian Museum 
Sydney 

01 Z.Y 

Academia Sínica 

Qingdao, People's Republic of China 

D. G. REID 

The Natural History Museum 

London, United Kingdom 



J. STUARDO 
Universidad de Chile 
Valparaiso 

S.TILLIER 

Museum National d'Histoire Naturelle 

Paris. France 

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

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

J. A. VAN EEDEN 
Potchefstroom University 
South Africa 

N.H.VERDONK 
Rijksuniversiteit 
Utrecht, Netherlands 

B.R.WILSON 

Dept. Conservation and Land Management 

Kallaroo, Western Australia 

H.ZEISSLER 
Leipzig, Germany 



N.W. RUNHAM 

University College of North Wales 

Bangor, United Kingdom 



A. ZILCH 

Forschungsinstitut Senckenberg 

Frankfurt am Main. Germany 



MALACOLOGIA, 1998, 39(1-2): 1-9 

SAMPLING TERRESTRIAL GASTROPODS USING CARDBOARD SHEETS 
J. W. Hawkins, M. W. Lankester & R. R. A. Nelson 

Department of Biology, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, 

P7B 5E1, Canada 

ABSTRACT 

Cardboard sheets are an efficient way of collecting large numbers of terrestrial gastropods and 
are useful for estimating relative densities and determining species composition of snails and 
slugs active on the surface of the forest floor, provided sampling is conducted under optimal 
weather conditions. Sheets may be less reliable, however, for quantitatively assessing the sub- 
terranean component of gastropod communities. Cardboard sheets placed on the forest floor 
sampled approximately 1/50 the number of gastropods estimated, using soil cores, to be in the 
upper 10 cm of soil beneath the sheets. The numbers collected by the two methods were not cor- 
related (p = 0.96), but cardboard sheets produced up to 30 times as many specimens per unit of 
sampling time. Gastropod numbers beneath cardboard sheets peaked at near-ground tempera- 
tures around 15°C and were augmented by animals moving horizontally over the surface of the 
litter from the surrounding area and vertically from the underlying soil, particularly when condi- 
tions were wet. Although mean ( ± S.E.) gastropod densities determined using sheets allowed 
to weather for a year (27.3 ± 4. 1 m~^) and new sheets (16.2 ± 2.6 m"^) were not significantly dif- 
ferent (p = 0.09), larger samples might confirm a tendency to prefer weathered sheets. In addi- 
tion, 4 of 20 species were collected in greater numbers on weathered sheets, suggesting differ- 
ential use of cardboard sheets by those species. 

Key words: terrestrial gastropods, sampling, cardboard sheets, horizontal movement, temper- 
ature. 



INTRODUCTION 

Corrugated cardboard sheets have fre- 
quently been used in parasitological studies 
to collect terrestrial gastropods that serve as 
intermediate hosts of metastrongyloid nema- 
todes (Lankester & Anderson, 1968; Gleich & 
Gilbert, 1976; Kearney & Gilbert, 1978; Up- 
shall et al., 1986; Beach, 1992; Lankester & 
Peterson, 1996). For this purpose, cardboard 
sheets have provided a convenient, time-effi- 
cient method of collecting large numbers of 
snails and slugs, but whether sample data can 
be used to accurately estimate gastropod 
population densities and species composition 
remains unknown. 

Boag (1982) compared the use of card- 
board sheets and hard masonite boards for 
sampling terrestrial gastropods and con- 
cluded that cardboard sheets were effective 
for repeatedly sampling the same area over 
time but that the technique was subject to po- 
tential limitations. He suspected that card- 
board sheets may not sample all gastropod 
species and life stages equally and that hori- 
zontal movement to the sheets may bias den- 
sity estimates. In addition, Boag (1990) sus- 



pected that sheets may become progressively 
more attractive to gastropods the longer they 
are exposed to the weather. 

Our purpose was to evaluate some con- 
cerns raised about using cardboard sheets 
to estimate population densities and deter- 
mine the species composition of terrestrial 
gastropod communities. We tested the hy- 
pothesis that the number of gastropods col- 
lected using cardboard sheets is correlated 
with that deeper within the underlying litter 
and soil. In addition, we investigated whether 
gastropods migrate horizontally across the 
surface and accumulate beneath cardboard 
sheets, thereby inflating population estimates. 
The relationship between temperature and 
the number of gastropods collected was ex- 
plored, and collections from new and weath- 
ered cardboard sheets were compared. 



MATERIALS AND METHODS 

Two study sites, in recently restocked 
spruce (Picea spp.) plantations, were located 
in Fraleigh Township (48°08'N, 89°49'W), 
about 60 km southwest of Thunder Bay, 



HAWKINS, LANKESTER & NELSON 



Ontario, within the Great Lakes-St. Lawrence 
Forest Region (Rowe, 1972). Site 1 was on a 
south facing slope, had shallow, sandy loam 
soils and deciduous regrowth consisting pri- 
marily of trembling aspen {Populus tremu- 
loides) and mountain maple (Acer spicatum). 
Site 2 was also on a south facing slope but 
had deep, silty soils and regrowth consisting 
of trembling aspen, willow {Salix spp.), alder 
{AInus spp.), fireweed (Epilobium angusti- 
folium), raspberry {Rubus spp.), and white 
birch {Betula papyrifera). Additional informa- 
tion on the study sites can be found in Bell et 
al. (1996). 

Terrestrial gastropods were collected using 
unwaxed, corrugated cardboard sheets 
(80 X 1 07 cm) placed on the surface of the for- 
est floor and weighed down with rocks. 
Gastropods located beneath and on top of 
each cardboard sheet were identified, with the 
aid of Pilsbry (1939-1948), Oughton (1948), 
and Burch (1962), counted and removed. 
Species too small to be identified in the field 
were preserved in glycerin alcohol and identi- 
fied later under a dissecting microscope at 
16x. Voucher specimens were sent to the 
Royal Ontario Museum, Toronto, where iden- 
tifications were confirmed. 

To determine whether the number of gas- 
tropods collected using cardboard sheets was 
correlated with that in the soil beneath them, 
10 cardboard sheets, in place for five days on 
site 1, were examined for gastropods on 
September 14, 1993. Immediately thereafter, 
three soil cores (8x7x10 cm) were taken at 
random locations from beneath each sheet. 
The top and bottom halves (5 cm each) of 
each core were placed in separate bags, la- 
belled, and stored frozen at -5°C for 2-20 
days. Before freezing, an attempt was made 
to collect any slugs present in the soil samples 
by placing fresh lettuce leaves in the bags and 
leaving them overnight in a controlled envi- 
ronment chamber at 20°C. The inner surface 
of the bag and the lettuce were then inspected 
for slugs and snails that moved out of the soil 
sample. 

Thawed soil samples were placed in an 
enamel dissection tray (25 x 45 x 6 cm) half- 
filled with water and visually examined for 
gastropods. Following this preliminary visual 
search, samples were washed through a se- 
ries of sieves (smallest = mesh No. 1 20, 0. 1 25 
mm opening) and the remaining material ex- 
amined for the presence of gastropods under 
a dissecting microscope (6x). Gastropods 
without a visible tissue mass within the shell 



were considered dead at the time soil cores 
were collected and were not counted in den- 
sity estimates. In the case of smaller speci- 
mens, this was determined with the aid of a 
dissecting microscope. 

An experiment was designed to determine 
whether cardboard sheets sample only those 
gastropods residing in the litter and soil di- 
rectly beneath them, or if collections also in- 
clude animals moving laterally from the sur- 
rounding area. Ten pairs of cardboard sheets 
were placed randomly at approximately 10 m 
intervals on the ground vegetation at site 2. 
Each pair consisted of one sheet enclosed 
tightly by a strip of sheet metal (22 gauge, 12 
cm high) penetrating 7 cm into the soil and 
protruding 5 cm above the soil surface, plus a 
control sheet (unenclosed), placed 4 m away. 

The above-ground surface of the sheet 
metal barrier was coated with automotive 
bearing grease and covered with a mixture of 
coarse grain, black pepper and cayenne pep- 
per (2:1) to discourage gastropods from 
crawling over it. All overhanging ground vege- 
tation was removed. Pairs of cardboard 
sheets were sampled simultaneously for gas- 
tropods, between 0800 hrs. and 1000 hrs., 
every 2-3 days from July 22 to August 18, 
1994, for a total of 1 1 sampling days. Gastro- 
pods were removed and each sheet was re- 
turned to its original position. 

The relationship between temperature and 
numbers of gastropods collected beneath 
cardboard sheets was examined. At each of 
30 cardboard sheets on site 2, three type-T 
copper-constanten thermocouple leads were 
positioned, one 2 cm above the sheet to mea- 
sure ambient air temperature, one directly be- 
neath, and one 2 cm deep in the litter beneath 
the sheet. Thermocouple temperatures were 
measured, using a Cole-Parmer digital ther- 
mometer (Model 08500-41), five times 
throughout the summer of 1994 (0700-1100 
hrs.), immediately before collecting all gas- 
tropods on the sheet. 

An experiment designed to determine if 
weathered cardboard sheets sample more 
gastropods than new sheets was initiated in 
early June 1993. Ten cardboard sheets were 
randomly placed on the ground vegetation at 
site 2 and sampled five times over the sum- 
mer of 1 993 as part of a larger gastropod pop- 
ulation study (Hawkins et al., 1996). All were 
left exposed to the weather over winter. On 
May 2, 1994, a new cardboard sheet was po- 
sitioned approximately 4 m from each weath- 
ered sheet. At the same time, the weathered 



SAMPLING TERRESTRIAL GASTROPODS 

TABLE 1 . Gastropods collected on cardboard sheets and from soil cores beneath sheets 





Cardboard sheets* 




Soil cores^ 


Species 


Total 


Density (/m^)* 


Total 


Density (/m^)* 


Zonitoides arbóreas 


71 


8.3 ± 92.1 


103 


613.1 ± 138.6 


Discus cronkhitei 


16 


1.9 ±0.8 


57 


339.3 ±81 .4 


Striatura milium 


16 


1.9 ±0.7 


49 


291.7 ±70.1 


Deroceras laeve 


48 


5.6 ± 1.0 


1 


6.0 ± 6.0 


Vitrina limpida 


29 


3.4 ± 1.0 


19 


113.1 ±39.1 


Strobilops ¡abyrinthica 


14 


1.6 ±0.8 


9 


53.6 ± 24.2 


Vertigo gouldi 


13 


1.5 ± 0.8 


5 


29.8 ± 23.9 


Euconulus fulvus 


13 


1.5 ±0.5 


3 


17.9 ± 12.7 


Cochlicopa lubrica 








14 


83.3 ± 33.4 


Columella edentula 


13 


1.5 ± 0.5 








Zoogenetes harpa 








7 


41.7 ± 19.9 


Striatura exigua 


2 


0.2 ± 0.2 


1 


6.0 ± 6.0 


Vertigo modesta 








1 


6.0 ± 6.0 


Carychium exile canadense 








1 


6.0 ± 6.0 


Total 


235 


27.5 ± 5.2 


270 


1607.1 ±272.3 



*10 sheets of unwaxed corrugated cardboard (80 x 107 cm) 
"^30 soil cores (8 X 7 X 10 cm) 
*mean ± S.E. (/m^ of surface area) 



sheets were moved approximately 1 m from 
their over-winter position and any gastropods 
adhering to the sheets were removed. Each of 
the 10 new sheets was sampled, simultane- 
ously with its corresponding weathered sheet, 
four times during the 1994 field season (May 
20, June 29, July 12, and July 25, 1994). 

Simple linear regression (Neter et al., 1 989) 
was performed to determine whether any re- 
lationship existed between density estimates 
obtained using cardboard sheets and those 
obtained using soil cores. Density estimates 
from soil cores were used as the independent 
variable. The null hypothesis of no difference 
between enclosed cardboard sheets and con- 
trol sheets was tested using the Wilcoxon 
Rank Sum test (Bradley, 1968). A Kruskal- 
Wallis analysis of variance (Bradley, 1968) 
was performed to test for differences in gas- 
tropod densities between the 11 sample peri- 
ods. A repeated measures analysis of vari- 
ance (Gumpertz & Brownie, 1993) was 
performed to detect differences in near- 
ground temperature between the three differ- 
ent thermocouple positions. The dependent 
variable in this analysis was normalized using 
a square root transformation. The Wilcoxon 
Rank Sum test was also used to test the null 
hypothesis of no difference in the use of new 
and weathered cardboard sheets by gas- 
tropods. All statistical differences were con- 
sidered significant at p < 0.05. Statistical pro- 



cedures were performed on SPSS PC-6.1 
(Norusis, 1992a, 1992b). 

RESULTS 
Cardboard Sheets vs. Soil Cores 

The mean (± S.E.) density of gastropods 
active on the surface of the forest floor and es- 
timated using the cardboard sheet method 
was 27.5 ± 5.2 m"^ whereas a mean (± S.E.) 
of 1607.1 ± 272.3 m"^ was estimated using 
the soil core method to be in a 1 m x 1 m x 10 
cm volume of soil (Table 1 ). Fourteen species, 
including 13 snails and one slug (Deroceras 
laeve), were collected. Eighty-eight percent of 
gastropods collected from the soil cores were 
found in the upper 5 cm of the soil and the re- 
mainder in the bottom 5 cm. Linear regression 
analysis revealed no correlation between the 
densities of gastropods estimated using card- 
board sheets and soil cores (F = 0.003; p - 
0.958). Checking a cardboard sheet required 
5-15 minutes with yields ranging from 7-49 
gastropods per sheet; 3-4 hours were re- 
quired to examine each soil core with a range 
of 0-27 gastropods being recovered. 

Barrier-Enclosed Sheets 

Total mean (± S.E.) gastropod density was 
lower on sheets enclosed with a sheet metal 



HAWKINS, LANKESTER & NELSON 

TABLE 2. Mean (± S.E.) densities (/m^) of terrestrial gastropods collected from cardboard sheets 
enclosed with a metal barrier and control sheets (unenclosed) 





Barrier enclosed sheets* 


Control sheets* 


p-value^ 


Deroceras laeve 


1.18 


±0.152 


1.90 


± 0.280 


p = 0.097 


Euconulus fulvus 


0.23 


± 0.060 


0.20 


± 0.058 


p = 0.472 


Columella edentula 


0.12 


± 0.034 


0.27 


± 0.076 


p = 0.328 


Zonitoides arboreus 


0.14 


± 0.047 


0.23 


± 0.056 


p = 0.077 


Striatura milium 


0.12 


± 0.062 


0.04 


± 0.026 


p = 0.462 


Succinea ovalis 


0.06 


± 0.025 


0.09 


± 0.036 


p = 0.974 


Vitrina límpida 


0.03 


±0.018 


0.10 


± 0.034 


p = 0.121 


Vertigo gouldi 


0.02 


±0.015 


0.09 


± 0.033 


p = 0.088 


Strobilops labyrinthica 


0.04 


± 0.021 


0.02 


±0.015 


p = 0.409 


Cochlicopa lubrica 


0.02 


±0.015 


0.04 


± 0.021 


p = 0.409 


Discus cronkhitei 


0.03 


±0.018 


0.02 


±0.015 


p = 0.652 


Pallifera dorsalis 







0.04 


± 0.034 


p = 0.156 


Zoogenetes harpa 


0.04 


± 0.026 







p = 0.082 


Vertigo ovata 


0.02 


±0.015 







p = 0.156 


Anguispira alternata 


0.01 


±0.011 







p = 0.317 


Gastrocopta tappaniana 







0.01 


±0.011 


p = 0.317 


Total 


2.07 


± 0.228 


3.05 


± 0.349 


p = 0.038 



'sampled with removal 1 1 times between July 22 and August 18, 1994 
^p-values based on a Wilcoxon Rank Sum Test 



barrier (2.1 ± 0.2 m ) than on control sheets 
(3.1 ± 0.4 m"2) (p = 0.038), although no dif- 
ference was detected when individual gastro- 
pod species were considered (Table 2). 
Cumulative totals of 195 and 287 gastropods 
were collected from enclosed and control 
cardboard sheets, respectively, over the 
course of the experiment. Pallifera dorsalis 
and Gastrocopta tappaniana were not col- 
lected from the sheets enclosed with a bar- 
rier; Zoogenetes harpa, Vertigo ovata, and 
Anguispira alternata were not collected from 
the control sheets. The mean density of snails 
and slugs, from both enclosed and control 
cardboard sheets, varied over the 1 1 sample 
periods (p < 0.0001 ) (Fig. 1 ). Over the first five 
collection days, mean (± S.E.) gastropod 
density was lower under enclosed (2.0 ± 0.21 



Mean (± S.E.) temperature beneath the 
cardboard sheets (14.4 ± 0.4°C) was slightly 
cooler than that 2 cm above the forest floor 
(15.4 ± 0.6°C) throughout July, while both 
were similar in August. However, interaction in 
temperature among the three thermocouple 
positions was observed over the summer (F = 
4.99; p < 0.05). Throughout July, the tempera- 
tures 2 cm above the forest floor and directly 
beneath (0 cm) the cardboard sheets were 
higher than those measured 2 cm deep in the 
humus layer but, for the first half of August, the 
reverse was true. 

Weathered Sheets 

Total mean (± S.E.) gastropod densities on 
new (16.2 ± 2.6 m"^) and weathered (27.3 ± 



m ) than control sheets (3.9 ± 0.26 m ) 4.1 m ) cardboard sheets were not signifi- 



(p = 0.0029). However, densities increased 
sharply under both, and particularly under 
control sheets, following heavy rains on 
August 3rd and 7th totalling 35.2 mm and on 
August 16 following 3.8 mm of rain (Fig. 1). 

Temperature Beneath Sheets 

Gastropod collections peaked when the 
temperature beneath cardboard sheets was 
approximately 15°C and decreased at lower 
and higher temperatures. Temperature was 
more variable on clear days (4°C to 30°C, x = 
13.5 ± 0.56) than on overcast days (1 1°C to 
22°C, x = 15.8 ±0.33). 



cantly different (p = 0.09). However, of the 
twenty species of terrestrial gastropod col- 
lected (Table 3), Euconulus fulvus. Vertigo 
gouldi, Carychium exile canadense, and 
Striatura milium were present in greater den- 
sities on the weathered sheets than on the 
new sheets. 



DISCUSSION 

Results reported here demonstrate that the 
number of terrestrial gastropods present 
within the litter and underlying 10 cm of soil is 
more than 50 times as great as the number of 



SAMPLING TERRESTRIAL GASTROPODS 



A. Slugs 



Enclosed 
Control 




Sample date 



B. Snails 



5 - 



R 



Enclosed 
Control 



R 




Sample date 



FIG. 1 . Mean (± S.E.) densities of gastropods removed from cardboard sheets enclosed with a metal barrier 
and control sheets (unenclosed), on 1 1 sample days, July 22 to August 18, 1994. [R = rain greater than 3 
mm]. (A) Slugs. (B) Snails. 



HAWKINS, LANKESTER & NELSON 



TABLE 3. Mean (± S.E.) densities {Im'' 
and weathered cardboard sheets 



of terrestrial gastropods collected from beneath new 



Species 


New sheets* 


Weathered sheets* 


p-value^ 


Zonitoides arboreus 


5.12 


± 1.03 


6.53 ± 1.04 


p = 0.187 


Strobilops labyrinthica 


1.85 


±0.46 


4.51 ± 1.08 


p = 0.072 


Euconulus fulvus 


1.71 


±0.42 


4.51 ± 0.85 


p = 0.001 


Deroceras laeve 


2.72 


±0.49 


2.23 ± 0.35 


p = 0.712 


Vitrina ¡impida 


1.30 


±0.49 


1.10 ± 0.45 


p = 0.889 


Discus cronkhitei 


0.40 


± 0.14 


1.30 ±0.38 


p = 0.110 


Vertigo gouldi 


0.35 


±0.18 


1.10 ±0.36 


p = 0.018 


Carycliium exile canadense 


0.09 


±0.09 


1.24 ±0.38 


p< 0.001 


Striatura milium 


0.20 


±0.15 


1.07 ±0.35 


p = 0.008 


Succinea ovalis 


0.75 


±0.28 


0.52 ±0.12 


p = 0.665 


Columella edentula 


0.46 


±0.19 


0.78 ± 0.32 


p = 0.703 


Anguispira alter nata 


0.29 


±0.12 


0.69 ± 0.29 


p = 0.631 


Striatura exigua 


0.23 


±0.12 


0.64 ± 0.24 


p = 0.278 


Cochlicopa lubrica 


0.23 


±0.08 


0.52 ± 0.30 


p = 0.863 


Gastrocopta tappanlana 


0.06 


±0.04 


0.20 ± 0.07 


p = 0.079 


Vertigo ovata 


0.14 


±0.08 


0.12 ±0.09 


p = 0.655 


Vertigo modesta 


0.14 


±0.10 


0.06 ± 0.04 


p = 0.960 


Zoogenetes harpa 


0.06 


±0.04 


0.06 ± 0.04 


p = 1 .000 


Punctum minutissimum 


0.06 


±0.06 


0.06 ± 0.06 


p = 1 .000 


Pallifera dorsalis 


0.03 


±0.03 


0.03 ± 0.03 


p = 1 .000 


Total 


16.19 


±2.61 


27.25 ±4.13 


p = 0.086 



'sampled with removal four times throughout the summer of 1994 (May 20, June 29, July 12, and July 25) 
^p-values based on a Wilcoxon Rank Sum Test 



snails and slugs active on the surface of the 
forest floor and detectable using the card- 
board sheet sampling technique. Although the 
cardboard sheet and soil core sampling meth- 
ods were not correlated, each provides a rel- 
ative density estimate of a particular compo- 
nent of the gastropod community, one active 
on the surface of the forest floor and one pres- 
ent within the litter and underlying soil. Kralka 
(1986), working in the boreal forest of Alberta, 
examined 5 cm deep soil cores and estimated 
a total mean gastropod density of 80 m"^, with 
maximum densities of Discus cronkhitei and 
V. gouldi reaching 340 m"^ and 460 m"^, re- 
spectively. Estimates of gastropod population 
densities in boreal forests using cardboard 
sheets have ranged from 2-38 m"^ (Kearney 
& Gilbert, 1978; Hawkins, 1995; Lankester & 
Peterson, 1996). 

Cardboard sheets provided a more time- 
efficient method of collecting terrestrial gas- 
tropods, yielding up to 30 times as many 
specimens as found in soil cores in similar 
time periods. In addition, the majority of gas- 
tropods found adhering to sheets can be iden- 
tified in the field, with the exception of smaller 
species, such as Vertigo spp. and Columella 
spp., which must be identified beneath a mi- 
croscope. Soil cores, however, not only in- 
volve a greater effort in the field but also re- 



quire considerably more time to extract speci- 
mens from the samples in the laboratory. 

When using cardboard sheets to sample 
terrestrial gastropods, horizontal movement 
across the surface of the litter could affect the 
number of snails and slugs collected. With re- 
peated sampling and removal, the mean num- 
ber of gastropods collected beneath both bar- 
rier enclosed and control sheets would be 
expected to decline if no horizontal movement 
of gastropods towards the sheets were occur- 
ring. However, if individuals were continually 
immigrating from the surrounding area, the 
mean density of individuals found beneath the 
enclosed sheets should fall to a lower level 
than that under control sheets. In fact, this was 
observed over the first five collection days. 
The mean density recovered beneath en- 
closed sheets (2.0 ± 0.21 m"^) was half that 
from unenclosed sheets (3.9 ± 0.26 m"^) sug- 
gesting that horizontal movement does occur. 
Numbers Increased, however, under both en- 
closed and control sheets following separate 
rainfalls totalling 35 mm and 4 mm. Although 
the increase on both occasions was greatest 
beneath the control sheets, indicating that 
some Increase in horizontal movement had 
probably occurred, an increase beneath the 
enclosed sheets suggests that individuals 
also moved vertically in response to the wet- 



SAMPLING TERRESTRIAL GASTROPODS 



ter conditions (Locasciulli & Boag, 1987). A 
more direct demonstration of horizontal move- 
ment was provided by Boag (1990), who 
marked and released snails beneath ma- 
sonite boards and observed that 5% of D. 
cronkhitei and 12% of E. fulvus moved from 
beneath the boards onto the surrounding lit- 
ter. 

The extent to which gastropods might actu- 
ally be attracted to cardboard sheets has not 
been determined, yet there is some empirical 
evidence that they do accumulate beneath 
more permanent sampling structures (Boag, 
1990). Snails and slugs would accumulate be- 
neath cardboard sheets if more individuals 
move under a sheet than leave, implying that 
conditions beneath cardboard sheets are 
generally more favourable than those encoun- 
tered on the surrounding forest floor. These 
animals can be observed moving openly 
across the surface of the litter at night and on 
overcast and rainy days until moisture, tem- 
perature, and/or light conditions become 
unfavourable (Boag, 1985; personal observa- 
tion). Under natural daily conditions of in- 
creasing light intensity and decreasing humid- 
ity, their most direct route to refuge would be 
downward into the litter and soil. Conditions 
that discourage gastropod movement on the 
surface may be delayed in onset and be less 
severe beneath a cardboard sheet. However, 
sheets seldom remain suitable refuge for 
long. They dry readily in the sun, and the veg- 
etation beneath them dies back if left covered 
for more than 2-3 weeks. In some circum- 
stances, the litter beneath a cardboard sheet 
may be drier than its surroundings, for exam- 
ple if a sheet has been put in place before a 
light rain or heavy dew occurs. This expected 
variability can best be minimized by placing 
already dampened sheets in a new location 
during, or immediately after, a rainy period, 
and by checking the sheets within a few days 
and only in the early hours of the morning. 

The temperature beneath cardboard sheets 
influences the number of gastropods that can 
be collected using this method. The mean 
temperature immediately beneath the card- 
board sheets was slightly lower than the air 
temperature 2 cm above them throughout 
July, suggesting that the shading effect of the 
sheets keeps temperatures cooler than those 
on the surrounding forest floor. Collections 
were greatest when the temperature beneath 
the sheets was approximately 15°C. Boag 
(1990) reported the greatest number of gas- 
tropods beneath masonite boards at ambient 
air temperatures between 7.5°C and 17.5°C 



and direct observation of snails in ferrarla in- 
dicated greater activity on the surface of the 
litter at temperatures ranging from 6°C to 
15°C (Boag, 1985). Snails and slugs most 
likely take refuge deeper in the litter and un- 
derlying soil when temperatures reach lower 
or higher extremes. The time of day and 
amount of cloud cover will clearly play a role in 
influencing the temperature beneath the 
sheets. On sunny, hot days, favourable tem- 
peratures beneath the sheets will only prevail 
for a limited period of time early in the morn- 
ing. On overcast days, however, daytime tem- 
peratures will remain cooler and larger num- 
bers of gastropods are likely to be found 
beneath cardboard sheets later into the day. 
The apparent relationship between tempera- 
ture beneath the sheets and the number of 
gastropods collected should be considered 
when using this technique in field studies of 
terrestrial gastropods. 

Overall, mean densities of gastropods col- 
lected from weathered and new cardboard 
sheets were not significantly different. How- 
ever, the low power of the test (n = 10) and a 
reasonably large difference between the mean 
(± S.E.) densities (weathered sheets = 27.3 ± 
4. 1 m"^; new sheets = 1 6.2 ± 2.6 m"^), suggest 
that snails and slugs may in fact favour weath- 
ered sheets. Four species (E. fulvus, V. gouldi, 
C. exile canadense, and S. milium) may pref- 
erentially use weathered sheets. Weathered 
sheets appeared to absorb more moisture and 
retain it longer following a rain. A similar in- 
crease in use of weathered masonite boards 
was seen by Boag & Wishart (1 982) and Boag 
(1 990). It was suggested that boards exposed 
for long periods to the elements may become 
more attractive as a result of having dissipated 
any possible repellant chemicals, or because 
they acquire fungal hyphae and slime trails on 
their lower surface. Boag (1990) also sug- 
gested that various gastropod species may 
use sampling boards differentially, which may 
explain, in part, the greater numbers of four 
species collected from weathered sheets and 
underscores a potential limitation of using 
cardboard sheets to estimate relative numbers 
and densities of terrestrial gastropods. 

We conclude that the cardboard sheet sam- 
pling technique is a time-efficient method of 
collecting large numbers of terrestrial gas- 
tropods that otherwise become difficult to find 
in daylight hours, if sampled under optimal 
weather conditions, cardboard sheets provide 
an acceptable method of quantifying the rela- 
tive abundance and species composition of 
gastropods active on the surface litter. This 



8 



HAWKINS, LANKESTER & NELSON 



method allows comparison between gastro- 
pod communities in different locations and 
habitats provided collections are made under 
similar weather conditions and in the same 
season to account for different reproductive 
life histories (Comfort, 1957; Berry, 1966; 
Uminski & Focht, 1979; Livshits, 1983). Card- 
board sheets are less reliable, however, for 
quantitatively assessing the subterranean 
component of gastropod communities. 



ACKNOWLEDGMENTS 

We gratefully acknowledge funding pro- 
vided for this work by the VMAP (Vegetation 
Management Alternatives Program) under the 
Sustainable Forestry Initiative, Ontario Minis- 
try of Natural Resources, Sault Ste. Marie, 
Ontario. We thank Jackie Hrabok and Cam 
Oomen for assisting with gastropod collection 
and Karen Watt for helping with the prepara- 
tion of the manuscript. 



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MALACOLOGIA, 1998, 39(1-2): 11-19 

REPRODUCTION AND EMBRYONIC DEVELOPMENT TIME OF BATHYPOLYPUS 
ARCTICUS, A DEEP-SEA OCTOPOD (CEPHALOPODA: OCTOPODA). 

J. B. Wood\ E. Kenchington^'2 & R. K. O'Dor^ 



Hobbes 



Worse, in terms of outright scariness, Are the suckers multifarious . . . Bill Watterson, Calvin and 



ABSTRACT 

Mating, brooding, and embryonic development rate of Bathypolypus arcticus, a deep-sea oc- 
topod, are described. Live specimens of B. arcticus were collected in the Bay of Fundy, Canada, 
and kept in a flow-through system in the lab. Two of the octopods laid and brooded viable eggs. 
Brooding and embryological development took over a year at average temperatures of 7.3°C and 
7.8°C. Brooding females ate occasionally and left their eggs shortly before dying. Hatchlings 
weighed 208 ± 17 (SD) mg from the first batch and 283 ± 20 (SD) mg from the second batch. 
There was no evidence of multiple spawning. 

Mating of B. arcticus was also observed. The usually smaller male sits upon the female, en- 
veloping much of the female's mantle in his web, and he inserts his large ligula into her mantle. 
One or two large spermatophores are transferred by a combination of mantle pumping and arm 
groove peristalsis. A filmed mating sequence lasted 140 seconds. 

Key words: cephalopod, Octopoda, Octopodidae, Bathypolypus arcticus, deep-sea, mating, 
embryonic development, brooding. 



INTRODUCTION 

Bathypolypus arcticus (Prosch, 1849) is a 
small octopodid that rarely exceeds 200 g 
(O'Dor & Macalaster, 1983). This species has 
been found to depths of 1,543 m (Voss, 
1988a,b) and is classified as a deep-sea oc- 
topus by Voss (1 988b). Bathypolypus arcticus, 
most common at depths of 200-600 m, is 
widely distributed in the Atlantic Ocean (O'Dor 
& Macalaster, 1 983). Assuming the three-year 
life span estimated by O'Dor & Macalaster, fe- 
males brood eggs for a larger percentage of 
their life than any octopus studied thus far, 
with the possible exception of the iteroparous 
Octopus chierchiae (Jata, 1889) (Rodaniche, 
1984). Like all deep-sea octopods, female B. 
arcticus lay large eggs, from which well-devel- 
oped young hatch. 

Mature male B. arcticus have the largest 
ligula relative to body size of any octopodid. 
The ligula is part of the hectocotylus, the mod- 
ified third right arm that males use to transfer 
their equally large spermatophores while mat- 
ing. Mating has not been previously described 
for B. arcticus — what males do with their hec- 



tocotylus was unknown. Two mating positions 
have been observed in the family Octo- 
podidae: a distant position in which the male 
and female are separated except for the hec- 
tocotylus, and one in which the male mounts 
the female (Mangold, 1987). Mangold notes 
that mating may last from a few minutes to 
several hours. 

As much as there is to be learned from 
cephalopods we can easily obtain, perhaps 
even more is yet to be learned from species 
that dwell in the deep-sea (Forsythe & Van 
Heukelem, 1987). Little laboratory work has 
been done with deep-sea cephalopods due to 
difficulties in collecting undamaged live spec- 
imens and continually providing cold water. 
The only previous laboratory information on 
brooding and embryonic development of 
deep-sea octopods is from O'Dor & Macal- 
aster (1983). They reported that a single fe- 
male B. arcticus laid eggs in August 1 978 and 
brooded them until they hatched in July 1979. 
They state that the temperature varied be- 
tween 3°C and 10°C, but temperatures were 
not recorded. The female was essentially ig- 
nored until the spring of 1979 when the eggs 



^Biology Department, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada 

^Science Branch), Department of Fisheries and Oceans, P.O. Box 550, Halifax, Nova Scotia, Canada 



11 



12 



WOOD, KENCHINGTON & O'DOR 



were discovered to be developing. She only 
had 4 of 40 eggs left in July 1 979 and was not 
offered food while brooding. This female B. 
arcticus died shortly after her eggs hatched. 

Very little is known about the life history of 
deep-sea octopods. This report describes the 
first detailed observations of mating, brood- 
ing, and embryonic development time of Ba- 
thypolypus arcticus. 

METHODS 

Eighteen B. arcticus ^Nete collected from the 
Bay of Fundy off Digby [circa 44.70°N and 
65.90°W) on June 8-21 and below Brier 
Island (circa 43.80°N and 66.30°W) on 
August 22-September 1 , 1 994. The FRV J. L. 
Hart, a 20 m trawler belonging to the 
Department of Fisheries and Oceans (DFO), 
was used. Animals were collected in scallop 
trawls as incidental catch during DFO scallop 
stock surveys. The majority of animals came 
from tows at depths of 75-100+ m. While on 
the boat, specimens were housed in a 
portable cooler for as long as a week and kept 
at temperatures below 10°C. Additional spec- 
imens were collected in the same manner in 
the summer of 1995. 

The B. arcticus collected in 1994 were kept 
together in a flow-through system at DFO's 
Halifax laboratory. The animals were housed 
in a 91 by 91 cm fibreglass tank (internal di- 
mensions). The walls of the tank were 4 cm 
thick and contained chilling coils. Water depth 
was 31 cm. Water temperature was controlled 
by adjusting the amount of ambient and 
heated water that entered the system. 
Temperature varied with the incoming water 
and user demand. 

Initially, temperature was recorded with a 
mercury thermometer (Fig. 1). In late De- 
cember 1994, a min./max. thermometer was 
added to the system to record temperature 
fluctuations. 

The octopoda collected in 1995 were kept 
in two flow-through fiberglass tanks at 
Dalhousie. Most of the females collected laid 
fertile eggs. Temperature was recorded but is 
not presented here. The number of eggs laid 
in these fertile broods was assessed in May 
1996 by removing the females and pho- 
tographing their eggs with an undenwater 
camera. Eggs were much easier to count in 
static photographs. (We knocked eight eggs 
off when removing the females from their 
broods, and these are included in the totals for 
the appropriate females.) 

During the first months after capture, the 



octopodids were hand-fed live crustaceans 
and mollusks removed from their shell, and 
they were offered amphipods ad libitum. Hand 
feeding consisted of bumping the food into 
the octopuses arms. Later, sand shrimp 
(Crangon) and brittle stars (ophiuroids) were 
added in ad libitum quantities to the tank, and 
polychaetes, mussels, crabs, amphipods, and 
other small invertebrates were added as they 
became available. 

Octopodids that laid eggs were observed 
and occasionally offered food by hand. 
However, brooding octopodids were disturbed 
as little as possible. Once the eggs started 
hatching, brooding octopodids were filmed 
with a 24-hour time-lapse VCR under red 
light. 

To measure mantle length (ML), mantle 
width (MW), and interocular width (lOW) of 
day-old individuals, they were filmed and mea- 
surements were made with an Óptimas Video 
Analysis System. This system was used to re- 
duce stress on the animals. Summers (1985) 
used a somewhat similar method, and he 
briefly discussed the reliability of using photo- 
graphic size determination. 

Wet weights ±1 mg of 15 hatchlings from 
each brood were obtained with a Mettler PI 63 
scale. Members of the first batch of hatchlings 
were dried with a tissue to absorb excess 
water before being weighed. Many animals 
weighed with this method died; several of the 
dead hatchlings had tears in their skin. 
Because this method damaged the hatchlings 
and caused mortality, it was abandoned. 
Individuals from the second batch were 
weighed in a weighing tray with a micron 
screen bottom. This tray was placed on a tis- 
sue before being weighed to absorb excess 
water. Preserved and recently dead juvenile B. 
arcticus were weighed with both methods to 
quantify the difference between the two meth- 
ods. The second method produced results that 
were 1 0. 1 % (n = 26) higher. Weights of the first 
batch were converted so that they could be 
compared to those of the second batch. 

Mating and hatching was filmed by Dave 
Gaudet (Halifax Cable) with a housed Sony 
CCD 3-chip DX3 camera using high-8 format. 
The primary author induced several eggs to 
hatch by handling them and/or adding sugar 
to the water. 

RESULTS 

Adult mortality was highest within the first 
few weeks of capture. By September 1 994, 1 2 
of the 18 octopodids collected during the pre- 



BATHYPOLYPUS ARCTICUS 
Brooding and Development Temperatures 



13 



12 



10 - 



2 - 













r 




Г 




1 


r 






■ 




412 


ч 8 

Т 




l- 








¿10 










- 


1 


7 




¿12 




■^ 






1 


no 
















- 






b 


Г 




■ 




Г 


• 


' 




1 






2 


F 


мю 






































2 F MD. 






• 


1 


• 1 


1 




1 




1 . 1 





6 - ^ 



4 - 



60 



120 



180 



240 300 

Time (days) 



360 



420 



480 



540 



FIG. 1. Average and standard deviation of temperatures at which brooding octopuses were kept. The num- 
ber next to the average temperature is the sample size for that 60-day period. The horizontal lines show the 
brooding period for the two females (1 = first inversion, 2 = second inversion, F = first hatchling, M = median 
hatchling, L = last hatchling, D = death). 



vious summer were still alive. Of those, eight 
were still alive in September 1995. Of the 18 
B. arcticus collected in 1994, only one was 
male. However, in 1995 the sex ratio was 
50/50 (n = 32). 

Average temperature during the brooding 
period was 7.3°C for the first female and 
7.8°C for the second female (Fig. 1). During 
the experiment, the min./max. thermometer 
recorded a minimum temperature of -1 .8°C 
on September 28, 1995, caused by a pump 
failure. A similar problem that dropped the 
temperature to 0.0°C occurred in fall 1994. 
A maximum temperature of 16.7°C was re- 
corded by the min./max. thermometer during 
July 17-24, 1995. These extreme tempera- 
tures probably lasted for only a short time. 

On August 16, 1994, a female B. arcticus 
was discovered brooding at least three eggs 
that were laid the previous night. The female 
refused food. There were about ten eggs on 
August 17. The female laid more eggs by 
August 22 and ate a few amphipods. Due to 
our effort to disturb the female as little as pos- 
sible while she was laying eggs, eggs were not 
counted until several months later. Although 



precise observations could not be made, no 
noticeable additions of eggs took place after 
two weeks. To count the eggs, it was neces- 
sary to remove the female for a brief period. 
Fifty-five eggs were counted, but only 54 were 
accounted for at hatching. This discrepancy is 
likely due to difficulties in counting the eggs 
while keeping the female away from them. 
Eggs were glued individually to the side of the 
tank by the female. The glue was initially clear 
but it turned green after a few days. 

While brooding eggs, the female occasion- 
ally ate food offered to her. She took fish pel- 
lets, amphipods, Crangon, and crushed mus- 
sels. Food was offered by hand-feeding as 
described above. 

Brooding females would sit on their eggs 
and cover most of them with their web. They 
often directed their funnel down, which pro- 
vided a water current around the eggs. The 
video tapes of brooding females revealed that, 
although they occasionally moved slightly 
away from the eggs, they would always keep a 
few arms on the eggs while exploring the 
perimeter around the brood with the other 
arms. When a probe was used to try to get a 



14 



WOOD, KENCHINGTON & O'DOR 



better look at the eggs, the female would ac- 
tively defend them, sometimes by blasting jets 
of water at the probe and/or grabbing it. 

An egg was taken on January 20, 1 995, 1 58 
days after the first eggs were laid, to deter- 
mine if the batch was fertilized. Incirrate em- 
bryos, except Argonautidae, flip position in the 
egg twice during development (Boletzky, 
1987). The egg was viable and the embryo 
was in the first inversion. By July 31, 1995 
(day 350), most of the embryos had flipped 
the second time. At least one egg had not 
flipped by August 8, 1995. On August 27, 
1995 (day 377), the first octopus hatched. By 
October 23, 1995 (day 434), all the octopo- 
dids in the first batch had hatched, although 
six of the last seven were induced to hatch so 
they could be filmed (Figs. 2-5). 

Eggs were 1 1 mm long and 6 mm wide be- 
fore hatching. Hatchlings (n = 15) weighed 
208 ± 17 (SD) mg, had a mantle length of 
7.71 ± 0.49 (SD) mm, a mantle width of 7.14 
± 0.39 (SD) mm, and an interocular width of 
2.30 ± 0.20 (SD) mm. The median day of 
hatching was October 14, 1995 (day 425), 
and assuming the median day of egg laying 
was one week after the first egg was laid, the 
average octopus in the first batch took 419 
days at an average temperature of 7.3°C.This 
female died three days after the last egg in her 
brood hatched. 

On October 30, 1994, a slightly larger fe- 
male started laying viable eggs in the lower 
left corner of the same tank. Over 50% of 
these eggs flipped the second time by 
October 23, 1995 (day 359 from first laying of 
eggs). The first egg in the second batch 
hatched on or a few days before November 8, 
1995 (day 375), and the last on December 30, 
1995 (day 427) (Fig. 1). The median day of 
hatching was December 17, 1995 (day 414). 
The average octopus in the second batch took 
407 days at 7.8°C to develop. This batch, 
counted as they emerged, yielded 36 hatch- 
lings with an average weight of 283 ± 20 (SD) 
mg (n = 15), mantle length of 8.91 ± 0.43 
(SD) mm, mantle width of 8.60 ± 0.84 (SD) 
mm, and an interocular width of 2.84 ± 0.23 
(SD) mm. The second female died two days 
before her last egg hatched. Brooding behav- 
iour was as noted for the first female, except a 
few days before the second female died she 
left her eggs, at one point for several hours, 
and then returned to them. She was in very 
bad condition by this time and appeared to be 
having trouble breathing and orienting herself. 
Both females moved away from the eggs 
shortly before dying. Females were preserved 



in formalin after they died. The preserved 
specimens weighed 20.82 g and 17.55 g re- 
spectively. The two females weighed an esti- 
mated 30-40 g prior to laying eggs. 

None of the hatchlings from either batch 
had any of the outer yolk sac remaining upon 
hatching. Video tapes revealed that hatchlings 
are able to hang upside down from the water 
surface (see Marliave, 1981; Van Heukelem, 
1976). A variety of foods were offered to the 
first batch of hatchlings. They were initially 
hand-fed fresh mussel meat, and bits of gam- 
maridean amphipods, mysid shrimp, and 
Crangon septemspinosus. Plankton and live 
gammaridean amphipods were offered to 
some of the hatchlings. Later, they were of- 
fered the small burrowing gammaridean am- 
phipod Corophlum volutator [ad libitum]. 
Hatchlings from the second batch were rarely 
hand-fed and were offered C. volutator ad libi- 
tum within a few days of hatching. 

Several other females that were collected in 
1994 laid eggs on the side of the tank or in a 
clay pipe. These eggs disappeared after a few 
months. An egg was removed from one of 
these females. When this egg was examined 
several months later, it was found to be unfer- 
tilized. Presumably these batches were not vi- 
able and the females ate them. A single male 
was kept in the tank with the females. 

Nine of the females that survived collection 
in 1995 were brooding eggs in May 1996. All 
of these broods were fertile, and seven were 
able to be counted (the eighth and ninth octo- 
pus laid eggs in a plastic pipe). Therefore, the 
nine B. arcticus in this study laid 12, 13, 18, 
19, 36, 48, 54, 89, and 105 fertile eggs. How- 
ever, we estimate that none of these cephalo- 
pods weighed 70 or more grams when they 
laid their eggs. Macalaster (1976) reports that 
70 g is the average size of mature females. 

The following observations were made on 
the 1995 females that laid eggs. Females 
brooding their own eggs can be moved away 
from their eggs and then returned to them. 
Also, females that are brooding eggs can be 
moved to care for another octopuses brood of 
fertile eggs. While females don't differentiate 
between their own fertile eggs and other 
broods of fertile eggs, they seem to be able to 
recognize infertile eggs, which we presume 
they eat. One female was briefly observed to 
be slightly out of arm reach of her eggs but 
was rapidly moving back toward them. This 
occurred shortly after amphipods were added 
to the tank. 

Male B. arcticus uses the mounting position 
to mate. The smaller male initiated mating by 



BATHYPOLYPUS ARCTICUS 



15 




FIGS. 2-5. Hatching. FIG 2. Swollen egg just prior to hatching. FIG. 3. During hatching the pressure in the 
swollen egg pushes out the end of the mantle. FIG. 4. The octopus frees himself from the egg. FIG. 5. The 
fully functional hatchling emerges from the egg. Photographs from high-8 video by D. Gaudet. 



mounting the female and enveloping her man- 
tle in his web (Figs. 7-9). He then inserted his 
hectocotylus into the mantle of the female. Its 
folded shape suggests that it was actually in- 
serted into the oviduct to open it. During mat- 
ing, the male contracts his mantle in a dramatic 
pumping arch motion (Fig. 9); this may help the 
large spermatophore pass to the funnel. We 
presume that the spermatophore is then 
passed from the funnel to the large grove on 
the hectocotylus and that through peristalsis it 
is transferred to the ligula which is in or on the 
oviduct. The female remained motionless dur- 



ing mating, which lasted 1 40 seconds. Bathyp- 
olypus arcticus mates readily — specimens 
collected the following year mated in the cooler 
that they were kept in while still at sea. These 
matings followed the pattern outlined above. 



DISCUSSION 

This study presents the first detailed data 
on the brooding and development time for a 
deep-sea octopodid. Bathypolypus arcticus 
brood eggs for over 400 days, and if they live 



16 



WOOD, KENCHINGTON & O'DOR 








FIGS. 6-1 1 . Mating Bathypolypus arcticus. FIG. 6. The male octopus (foreground) sees the female octopus 
and pounces on her (FIG. 7). FIG 8. He mounts her and inserts his ligula into her mantle cavity. FIG. 9. The 
male stretches his mantle dramatically (this happened twice) presumably to help pump the spermatophore 
to his ligula. FIG 10. The male removes his ligula and departs (FIG. 11). Illustrations by Rebekah McClean. 



BATHYPOLYPUS ARCTICUS 



17 



for three years, as estimated by O'Dor & 
Macalaster (1983), these octopodids spend 
over a third of their life brooding. Additionally, 
this study describes for the first time the mat- 
ing behavior of B. arcticus. Mating in B. arcti- 
cus is of interest because these little cephalo- 
pods have the largest ligula relative to body 
size of any octopod. 

In this experiment, brooding behaviour and 
developmental time were very consistent be- 
tween the two females despite differences in 
egg size, female size, and time of laying. This 
suggests that the length of the brooding pe- 
riod is relatively fixed at a given temperature. 

Brooding ß. arcticus in this experiment were 
kept at average temperatures of 7.3°C and 
7.8°C, which is significantly higher than the 
4 ± 2°C SD that O'Dor & Macalaster (1983) 
reported for wild specimens. Therefore, one 
might expect B. arcticus to brood eggs for 
even longer in nature, because length of em- 
bryonic development has been shown to de- 
pend on temperature in cephalopods (Bolet- 
zky, 1987, 1994). Ken Dnnkwater (personal 
communication) reports that average monthly 
bottom temperatures in the area where the 
octopodids were collected were as warm as 
11-12°C in August and September 1994 and 
that the average annual temperature was 
8.0°C in 1994 — these temperatures are ap- 
proximately 1 .1 °C higher than usual. O'Dor & 
Macalaster stated that laboratory specimens 
experienced mortality with only brief periods 
of temperatures from 10°C to 12°C, whereas 
our specimens survived periods of weekly av- 
erage temperatures in the 11°C range. 
Although O'Dor & Macalaster (1 983) found no 
evidence of migration, we point out that mi- 
gration to warmer water to spawn has not 
been ruled out. Villanueva (1992) found evi- 
dence of up-slope ontogenic migration in 
Battiypolypus sponsalis. 

Brooding Bathypolypus arcticus occasion- 
ally take food to offset the extremely high en- 
ergetic cost of brooding eggs for over a year. 
The octopus reported on by O'Dor & Macal- 
aster (1983) may have eaten her own eggs to 
have sufficient energy to brood the remaining 
four through to hatching. If this is true, it shows 
that B. arcticus can brood for a year by eating 
its own eggs. The cost in such a protracted 
brooding period is a sharp decrease in fecun- 
dity. An alternative explanation is that this fe- 
male only had a few viable sperm from her 
mating, which occurred at least five months 
earlier. Perhaps she ate the other 36 eggs be- 
cause they were not viable. 

The long brooding period in B. arcticus must 



have substantial costs of time, energy, and risk 
of prédation. This period may limit B. arcticus 
to a semelparous strategy, because the costs 
of brooding are not worth the effort unless the 
number of eggs and their chance of survival is 
sufficiently large. Historicity, or phylogenetic 
legacy (Williams, 1992), may also limit many 
octopodids to a semelparous strategy. 

Parental care is necessary for all incirrate 
octopod eggs because they lack a protective 
egg case. Without the mother caring for and 
defending her eggs, they would be rapidly suf- 
focated by fouling organisms (Boletzky, 1 994). 
However, the length of this brooding period 
varies. Battiypolypus arcticus benefit in sev- 
eral ways from their large young and long 
brooding period. Sibling and non-sibling com- 
petition favour smaller broods of larger eggs 
(Stearns, 1992). Longer brooding periods 
may be selected for in B. arcticus because the 
egg stage is likely to have a high survival rate 
compared to hatchling. Stearns (1992) noted 
that "selection should increase the proportion 
of time spent in the safest developmental 
stages." Yampolsky & Scheiner (1996) dis- 
cussed demographic reasons that favour 
large offspring in cold environments for 
exothermic animals. 

Because Battiypolypus arcticus are not 
found in high densities (O'Dor & Macalaster, 
1983) and because they are not very mobile, 
chances to mate may be rare. The female's 
ability to store viable sperm for at least five 
months (often incorrectly cited as ten months; 
O'Dor & Macalaster, 1983) and the male's 
large spermatophores and ligula are likely 
adaptations to maximize fitness under such 
conditions. It is possible that the large ligula 
and spermatophores are selected for in 
sperm competition. Cigliano (1995) noted that 
octopodids meet the criteria for sperm prece- 
dence and that the spoon-shaped ligula could 
be used to scoop out competitors sperm. 
Another (non-exclusive) possibility is that the 
large ligula expands once in the oviduct to an- 
chor the hectocotylus during mating. Further 
investigation will be needed to confirm or re- 
ject these theories. 

The successful manipulation of females 
between broods suggests that experiments 
manipulating brood and egg size may be pos- 
sible. An explanation of the experimental 
significance of manipulation of offspring size 
can be found in Stearns (1992). Such experi- 
ments could answer such questions as 
whether hatchling behaviour (e.g., planktonic 
versus benthic) is a function of hatchling size, 
species, or hatchling age. Also, the effect 



18 



WOOD, KENCHINGTON & O'DOR 



of egg size on development time could be 
tested within a brood, which would minimize 
or eliminate many confounding factors. Work- 
ers should test to see if other octopus species 
can be manipulated in this manner, because 
the long brooding period and cold tempera- 
tures required for keeping B. arcticus make it 
a poor choice for such experiments. 

The fact that a soft-bodied cephalopod can 
survive collection in a scallop trawl, days at 
sea, and acclimation to laboratory conditions, 
indicates the hardiness of B. arcticus. Bolet- 
zky & Hanlon (1983) provided a general re- 
view of low trauma cephalopod collection 
techniques and general culture information. 
Less traumatic collection techniques that 
specifically target deep-sea cephalopods are 
allowing investigators to study more species 
of deep-sea octopods in the lab. Researchers 
at the Monterey Bay Aquarium and Monterey 
Bay Aquarium Research Institute used an 
ROV specifically to capture and film cirrate oc- 
topuses, and they have been able to keep 
these octopuses alive for several months 
(Stein-Hunt and Hochberg, personal commu- 
nication). 

We are just beginning to understand the life 
history of deep-sea octopods. Additional stud- 
ies involving such modern sampling tech- 
niques as video monitoring from submersibles 
and remote operated vehicles (Vecchione & 
Roper, 1 991 ) will continue to complete the pic- 
ture of how deep-sea cephalopods behave in 
nature. However, because deep-sea cephalo- 
pods cannot always be found, workers must 
hope for chance encounters. Telemetry has 
provided more continuous records of the be- 
haviour of Loligo and Nautilus (Carlson et al., 
1 984; O'Dor et al., 1 993, 1 994) and could also 
be employed to study other deep-sea cephalo- 
pods. Cost and logistical problems limit the use 
of these techniques. 

It is widely acknowledged that laboratory 
conditions can bias results. However, studies 
of cephalopods in their natural environment 
are difficult due to their mobility, excellent vi- 
sion, and nocturnal habits (Boletzky & Han- 
lon, 1983). Laboratory studies are currently 
the best, most direct way to look at develop- 
ment time, life span, reproductive strategy, 
growth rates, and other processes that occur 
in individuals over time. Also, the effect of 
such variables as temperature, sex, and diet 
can be analyzed and separated. Laboratory 
conditions allow the experimenter to observe 
and manipulate such behaviour as mating or 
hatching. Further laboratory studies of hardy 



deep-sea species are needed if we want to 
understand how these organisms live in their 
cold, dark world. 



ACKNOWLEDGEMENTS 

This project would not have been possible 
without the help of the Canadian Department 
of Fisheries and Oceans. We acknowledge 
the help of Dale Roddick, Mark Lundy, Brenda 
Bradford, and the crew of the FRV J. L. Hart. 

We thank S. v. Boletzky and J. Voight for 
their helpful comments on this manuscript. A 
thank you is also due to John Cigliano, who 
commented on the mating video. Thanks to 
Joyce Chew for helping collect food for the oc- 
topodids, to Dave Gaudet for filming them, to 
Chris Harvey-Clark for photographing the 
eggs, and to Rebekah McClean for illustrating 
the mating sequence. 

The primary author is supported by a 
Killiam Fellowship, and the work by an 
NSERC Canada grant to RKO. 



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86-100. 

Revised ms. accepted 19 August 1996 



MALACOLOGIA, 1998, 39(1-2): 21-27 

THE INHERITANCE OF AN EMBRYONIC LETHAL MUTATION IN A 
SELF-REPRODUCING TERRESTRIAL SLUG, DEROGERAS LAEVE 

Richard M. Lebovitz 
c/o L.C. Evogen, 3515 Washington Boulevard, #317, Arlington, Virginia 22201, U.S.A. 

ABSTRACT 

A new lethal developmental mutation (EZL-1, embryonic zygotic lethal-1) in Deroceras laeve 
was used as a genetic marker to determine whetiier self-reproduction is by parthenogenesis or 
self-fertilization. An average of 25.7% of the progeny of individuals (N = 38) apparently het- 
erozygous for the mutation exhibited the EZL-1 lethal phenotype, a result that can only be ex- 
plained if reproduction is meiotic. Histological sections of a fertile slug revealed the presence of 
both sperm and eggs in the gonad, consistent with self-fertilization, rather than parthenogene- 
sis. In addition to EZL-1 , other developmental defects were observed in progeny. 

Key words: Deroceras, development, mutation, self-fertilization, slug. 



INTRODUCTION 

Self-reproduction is widespread, particu- 
larly among molluscs (Heller, 1993). Dero- 
ceras laeve, the grey garden slug, a pul- 
monate, reproduces predominantly, if not 
exclusively, by self-reproduction (Foltz et a!., 
1982). It is of particular interest to malacolo- 
gists because it is unsettled whether they self- 
reproduce by self-fertilization or by partheno- 
genesis (Heller, 1993; Hoffmann, 1983; Foltz 
et al., 1982; Nicklas & Hoffmann, 1981). To 
identify the mechanism of reproduction in this 
species, the inheritance of a recessive lethal 
mutation, embryonic zygotic lethal-1 (EZL-1), 
was studied. This is the first report of an em- 
bryonic mutation in a terrestrial slug. 

EZL-1 mutant embryos complete about 
21% of the embryonic stage, arresting after 
the acquisition of most of the rudimentary adult 
structures, including the tentacles, mantle, 
and foot. An average of 25.7% of the progeny 
of apparently heterozygous individuals (N = 
38) for the mutation exhibited the EZL-1 lethal 
phenotype, a result that can only be explained 
if reproduction is meiotic (Asher, 1970). Histo- 
logical sections of a fertile individual revealed 
the presence of both sperm and eggs in 
the gonad, consistent with self-fertilization, 
rather than parthenogenesis (Hoffmann, 
1 983; Asher, 1 970). In addition to EZL-1 , other 
developmental defects were observed in prog- 
eny. 



MATERIALS AND METHODS 

The founder slugs were isolated from the 
wild and maintained on a diet of fresh romaine 
lettuce. Subsequent progeny of the founders 
were isolated after hatching and cultured indi- 
vidually in tupperware dishes containing a 
piece of moistened towel. There were no op- 
portunities for cross-fertilization. Eggs from 
mature, isolated, animals were collected 
every one to two days and transferred to a 
moistened towel in a petri dish, where they 
were stored until hatching. The phenotype of 
unhatched eggs was observed under a dis- 
secting microscope and scored. 

Slugs were fed fresh salad leaves every 
one to two days. Environmental conditions 
were not controlled, although the slugs were 
generally maintained at room or seasonal 
temperatures, between about 16°C to about 
28°C. 

Karotyping was performed on three- to four- 
day-old embryos. After removal from the egg 
case, embryos were incubated for 30 min in 
0.125% colchicine dissolved in 0.45% KCl, 
washed in 0.045% KCl for about 30 min, and 
then fixed in a cold mixture of 3 parts 
methanol to 1 part acetic acid. Fixed embryos 
were placed in a drop of 60% acetic acid, 
squashed under a siliconized coverslip, and 
dried on a hot plate between about 37°C and 
47°C. When the slides were completely dried, 
the coverslip was removed, and the slides 



21 



22 



LEBOVITZ 



were stained with aceto-orcein for micro- 
scopic examination. 

For histology, a fertile slug was fixed in 4% 
formaldehyde, dehydrated, and embedded in 
paraplast. Six fim sections were prepared and 
stained with Harris hematoxylin-phloxine- 
eosin for light microscopic examination. 

The values for lifespan, sexual maturity, and 
egg number were calculated as the mean with 
a standard deviation (±). Statistical analy- 
sis was performed by chi-square, using the 
test for heterogeneity described in Mather 
(1951). 



RESULTS 

Although five different founder lines were 
maintained for a four-year period by self-re- 
production alone, only one line, IF, is de- 
scribed here. This line was selected because 
of its possession of a recessive lethal muta- 
tion, providing an opportunity to characterize 
the mutation and use it as a genetic marker to 
study self-reproduction. 

The animals in this study were identified as 
Deroceras laeve by H. Lee Fairbanks of 
Pennsylvania State University according to 
their description by Pilsbry (1948). In all three 
animals dissected, an inspection of the dorsal 
surface of the internal cavity revealed that the 
ovotestis was hidden under the digestive 
gland, a characteristic of D. laeve but not D. 
reticulatum, a species it closely resembles. In 
addition, the rectal caecum and terminal male 
genitalia were absent. 

Development of the Adult 

Animals collected from eight generations 
were included in this study. The founder (IF) 
was a progeny of an animal captured from the 
wild. Ninety animals, predominantly from the 
sixth and seventh generations, were selected 
for analysis. Only animals that reached matu- 
rity and produced viable offspring were in- 
cluded. For convenience, the ages discussed 
below were calculated from the day of egg de- 
position, rather than the actual day of hatch- 
ing. 

The average lifespan of all 90 animals was 
164 ± 40.7 days (about 5 1/2 months), rang- 
ing from 90 to 250 days. Sexual maturity, 
marked by the first deposition of eggs, was 
reached at about three months (94.8 ± 18.7 
days), with a shortest time of 71 days and a 
longest of 148. Animals produced eggs for an 



average of 57.2 ± 35.2 days. Egg production 
continued until an average of 12 ± 8.8 days 
before death. 

Very few metaphase figures were observed 
in the embryos. Of these, quality of the chro- 
mosomal squashes was low and thus pre- 
cluded detailed karyotyping. However, of 14 
spreads sufficiently clear to count, the chro- 
mosome number averaged 50 (data not 
shown). Patterson & Burch (1978) reported 
that Deroceras are diploid, having a haploid 
chromosomal number of 30. The number here 
is roughly twice that value, consistent with 
diploidy. 

Viability and Phenotypes of Deposited Eggs 

A mean of 66.5 ± 32.5 eggs (N = 90; total 
eggs = 5,981), with a range of 6 to 164, were 
produced per animal. In addition to producing 
eggs with embryos that developed into normal 
adults, two other primary egg phenotypes 
were observed. First, eggs were frequently 
observed that showed very little, if any, em- 
bryonic development. Inside such eggs, a 
small ball-like structure, varying in size, was 
typically observed. These eggs are referred to 
as type A. All animals produced this type of 
egg. Secondly, a category of eggs that un- 
dergo about 20% of normal development and 
then arrest were observed. These are embry- 
onic/zygotic lethal-1 or EZL-1, the mutation 
described here. Not all individuals produced 
EZL-1 eggs. It is assumed that those that did 
were heterozygous for the gene loci and that 
those that did not were homozygous normal 
at the EZL-1 locus. 

Normal Egg Development 

Development of the viable and normal egg 
occurred substantially as described in Carrick 
(1938) for Agriolimax agrestis. There is no 
metamorphosis nor a larval stage. Rather, the 
embryo gradually assumes the adult form, 
progressing through at least five distinct and 
identifiable forms. For the purposes of this 
study, the discussion is focused on the pro- 
gression of the embryo from stage IV to V, be- 
cause this is the stage at which embryonic de- 
velopment is arrested in EZL-1. 

Embryonic development takes about 14 
days. Stage IV, reached at about four days, is 
characterized by the appearance of most of 
the rudimentary structures of the adult organ- 
ism, including the tentacles, mantle, and foot 
(Fig. 1A, reproduced from Carrick, 1938). The 



LETHAL MUTATION IN DEROGERAS 



23 




FIG. 1. Development of Deroceras laeve. Stage IV. 
A, Right aspect, psr, rudiment of posterior sac; eor, 
external opening of right larval nephridia: m, mantle: 
a, anterior sac; h, hepatic mss; rrt, rudiment of right 
tentacles; f, foot. Stage V. Maximum development of 
posterior and anterior sacs. B, Right aspect. C, 
Ventral aspect, ps, posterior sac; po, pulmonary ori- 
fice; ror, optic rudiment of right anterior tentacle; lor, 
optic rudiment of left anterior tentacle; rtl , right an- 
terior tentacle; rt2, right posterior tentacle; rt3, third 
tentacle of right side; Itl, left anterior tentacle; It2, 
left posterior tentacle; It3, third tentacle of left side; 
st, pedal streaks. Reproduced by permission of the 
Royal Society of Edinburgh and R. Carrick from 
Carnck (1938-1939). 

posterior sac, the respiratory organ of the em- 
bryo, and the hepatic mass, another embry- 
onic structure, also become visible at this 
time. The progression from stage IV to V is 
gradual, marked by the enlargement of the 



rudimentary stage IV structures into recogniz- 
able and significantly differentiated forms. 
Stage V is identified by Carrick (1938) when 
the hepatic mass reaches its maximal size 
(Fig. IB). 

A primary characteristic of the transition 
from stage IV to stage V is the differentiation 
of the posterior sac from the embryonic foot. 
The foot and posterior sac originate from the 
same embryonic region. During development 
from stage IV to stage V, the two structures 
gradually assume characteristic forms, ap- 
pearing to comprise different cell types. As the 
foot morphologically separates from the pos- 
terior sac, it thickens, and two streaks (Fig. 
1С, st) appear on its ventral surface, running 
from anterior to posterior. After the appear- 
ance of the pedal streaks, the mantle and ten- 
tacles begin to enlarge, and the embryo grad- 
ually becomes more adult. Concomitantly, the 
hepatic mass begins to shrink, ending stage 
V. The entire sequence takes about seven to 
eight days. 

After about seven days, midway through de- 
velopment, pigmented eye spots appear in 
the first optic tentacles. Subsequently, pig- 
mentation spreads in a wave across the outer 
embryo epithelium. The remainder of develop- 
ment until hatching is characterized in Carrick 
(1938) and is not discussed here. 

Type A Egg Development 

An average of 23 = 13.8% (N = 90) eggs 
from founder 1 F were type A, showing little, in 
any, growth past the blástula stage (Stage I ac- 
cording to Carrick, 1938). A type A egg was 
classified by the appearance in the perivitelline 
egg fluid of a small, round mass and a long, 
twisted, wispy membranous thread. The latter 
was defined by Carrick (1938) as the remains 
of the sperm body, but this conclusion has not 
been generally accepted (South, 1992). The 
type A phenotype cannot be distinguished at 
deposition from a viable egg, except that the 
former does not develop any further over the 
14-day embryonic period. From all appear- 
ances, it is likely that this class represents de- 
velopmentally inactivated or unfertilized eggs. 

A second class of eggs is infrequently iden- 
tified in IF progeny. These eggs, type B, un- 
dergo a small amount of growth, arresting one 
to two days after egg deposition. В exhibits the 
same characteristic ball-like morphology as 
A, but it is about two to three times larger. It is 
uncertain at what development type В arrests, 
but the absence of visible structures suggests 



24 



LEBOVITZ 



TABLE 1 . Percent frequency of egg phenotypes produced by homozygous and heterozygous 
EZL-1 Deroceras laeve. 









% Type A (not fertilized or 




TOTAL EGGS 


% EZL-1 


not activated) 


Group 1, 


1141 


1.3 ±2.1 


20.8 ± 16.4 


homozygotes (N - 22) 




range: 0-7.4 




Group 2, 


2181 


1.0 ± 1.7 


21.4 ± 13.7 


homozygotes (N = 30) 




range: 0-7 




Group 3, 


2659 


25.7 ± 9.6 


25.8 ± 11.8 


heterozygotes (N = 38) 




range: 8.6-61.5 





that it has not, or has just, entered stage III 
(Carrick, 1938) when visible morphological 
differentiation begins. The tissue mass inside 
the perivitelline sac of type В eggs often is 
"fuzzy," rather than a well-defined structure as 
in type A, possibly indicating tissue necrosis. 
It is possible that В defines an early embry- 
onic (or maternal effect) mutation, but this has 
yet to be confirmed. В represents less than 
3% of the total eggs and was included in the 
type A class for all calculations, because it is 
only qualitatively different and therefore diffi- 
cult to score separately. 

EZL-1 Egg Development 

In the line from founder 1F, an embry- 
onic/zygotic lethal mutation (EZL-1) was first 
observed in the fourth generation, when the 
phenotypes of unhatched embryos were first 
inspected for defects. It was observed in all 
subsequent generations. 

The lethal mutation was first distinguished 
on day 4 of the 14-day development period, 
about 21% of the way through its develop- 
ment. Developmental arrest occurs during 
stage V when the embryonic foot and poste- 
rior sac start to become distinguishable struc- 
tures in the rudimentary foot of the zygote 
(Fig. 1С). In the majority of these embryonic 
lethals, although the foot and posterior sac 
became distinguishable from one another, the 
foot is enlarged but arrests before or when the 
pedal streaks would become visible in a nor- 
mal embryo. The posterior sac, on the other 
hand, looks perfectly normal in EZL-1. Its 
characteristic movement throughout the nor- 
mal 14-day period when egg development 
was examined suggests that the EZL-1 zy- 
gote is alive, but that its development halted. 

All development appears to cease in EZL-1 . 
For example, during normal development, the 
optic tentacles begin to enlarge after the ap- 
pearance of the pedal streaks, when the he- 



patic mass reaches its maximum (i.e., stage 
V). In EZL-1, the zygote acquires the rudi- 
ments of the optic tentacles, but they do not 
appear to expand or acquire any visible struc- 
ture, such as the pigmented eyespots ob- 
served in normal embryos. 

Frequency of EZL-1 Phenotype 

Because animals carrying the EZL-1 muta- 
tion could not be distinguished phenotypically 
from non-carriers, genotype was deduced 
from the frequency of the mutation appearing 
in their progeny. To calculate the percentage 
of eggs exhibiting the EZL-1 lethal phenotype, 
the number of type A eggs was subtracted 
from the total eggs and the result was divided 
into the number of eggs scored as EZL-1 . This 
adjustment was done to eliminate type A eggs 
from the analysis, on the assumption that they 
represent a class of eggs that are either un- 
fertilized or unactivated. The results are sum- 
marized in Table 1 . 

Group 1 comprises three generations de- 
scended from (and including) a fifth genera- 
tion animal that apparently did not possess 
the EZL-1 gene mutation. An average of 1 .3% 
of the eggs were scored as EZL-1 . The values 
ranged from 0% to 7.4%. It is assumed that 
the eggs scored as EZL-1 (since all data were 
collected blindly) died for other reasons, but at 
a stage similar to the one at which EZL-1 ar- 
rests. In some of these, it had been noted that 
the phenotype was not characteristic of EZL- 
1 , that is, later developmental arrest or differ- 
ent-sized embryo. 

Groups 2 and 3 were collected from de- 
scendants of three fourth-generation animals. 
These animals were from the same founder 
as Group 1, but from a lineage that diverged 
at the third generation. The animals were clas- 
sified into two groups, using 7% EZL-1 as a 
cut-off, because that was the highest value 
observed in Group 1. Based on this value, 



LETHAL MUTATION IN DEROGERAS 



25 



Group 2 possessed an average of 1% EZL-1 
eggs, similar to Group 1, the animals that do 
not have the EZL-1 mutation. The average 
number of eggs with the EZL-1 phenotype in 
Group 3, however, was 25.7%, with only four 
of the 38 animals being lower than 17% (that 
is, 8.6, 1 2.7, 1 4.3, and 1 5). This result for the 
pooled data (normal = 1,450 eggs; EZL-1 = 
481 eggs) is consistent with the expected 
number for self-fertilization by heterozygotes 
ix^ = 0.027, df = 1, 0.95 > P > 0.90). When 
Group 3 was analyzed as a class, using chi- 
square to test heterogeneity (Mather, 1951), 
the x^ value was high (P < 0.05), suggesting 
that the class was heterogeneous. Homo- 
geneity was established (x^ = 48.2, df = 35, 
0.30 > P > 0.20) by eliminating from Group 3 
the two animals with the highest EZL-1 fre- 
quency (43% and 62%). As discussed below, 
other lethal developmental defects have been 
noticed in slug progeny, albeit at a much lower 
occurrence than EZL-1 .Thus, the appearance 
of EZI-1 in these two slugs (as well as others) 
could have been inflated by scoring other de- 
velopmental defects that resemble but are not 
EZL-1. Figure 2 is a frequency histogram of 
the % EZL-1 per animal. 




10 15 20 25 30 35 40 45 50 55 60 



FIG. 2. Frequency histogram displaying percent 
eggs exhibiting the EZL-1 phenotype from individ- 
ual slugs. Data collected from 38 slugs character- 
ized as heterozygous for the EZL-1 locus. 



clusters, suggesting their origin from individual 
spermatogonia (Fig. ЗА). Mature spermatozoa 
were abundant in the hermaphroditic duct (Fig. 
3B). Because aphallic slugs, lacking most of 
the terminal structures of the male reproduc- 
tive system, were fertile, it is suggested that 
sperm production is dissociated from the de- 
velopment of the reproductive tract required 
for transporting sperm. 



Other Developmental Defects 

The appearance of an EZL-1 like pheno- 
type was observed an average of 1.1% (± 
1 .9%, N = 52) in lines apparently homozygous 
wild-type for the EZL-1 gene locus (Groups 1 
and 2). Other lethal developmental defects 
were noted, as well. These phenotypes in- 
cluded: developmental arrest at earlier stages 
than EZL-1, either with a distinct morphology 
or comprising a bulbous, undifferentiated 
mass; incomplete to fully pigmented embryos; 
and fully developed embryos that did not 
emerge from the egg shell. When added to the 
EZL-1 -like phenotype, the mean value of de- 
velopmental defects for the apparently ho- 
mozygous wild-type animals was 3.4% (± 
3.9%, N = 52). 

Histology of the Gonad 

The simultaneous presence of both sperm 
and egg in slug gonads is well documented 
(South, 1992). In D. reticulatum, for example, 
all individuals are hermaphroditic, and the 
gonad can contain both eggs and sperm at the 
same time (Runham & Laryea, 1968). Both 
sperm and eggs were seen in the gonad of one 
fertile D. ¡aeve. The spermatids are arranged in 



DISCUSSION 

The EZL-1 mutation in D. ¡aeve is clearly a 
recessive mutation at a single gene locus. 
First, it is transmitted through successive gen- 
erations, segregating between carrying and 
non-carrying slugs. Secondly, an apparently 
heterozygous individual produces an average 
of 25.7% eggs exhibiting the EZL-1 pheno- 
type, consistent with either simple Mendelian 
inheritance by self-fertilization or meiotic par- 
thenogenesis with a high frequency of recom- 
bination (Asher, 1970; Hoffmann, 1983). The 
detection of both sperm and egg in the gonad 
favors self-fertilization, in agreement with Foltz 
et al. (1 984), and all other pulmonales (Heller, 
1993), but contrary to the conclusions of 
Nicklas & Hoffmann (1981) and Hoffmann 
(1983). 

EZL-1 is an embryonic lethal mutation that 
arrests during stage V of development 
(Carrick, 1938). Its progression from the blás- 
tula to stage IV is visibly normal, acquiring 
most, if not all, of the rudimentary adult struc- 
tures. However, the progression from stage IV 
to the adult in EZL-1 is arrested. Differen- 
tiation, for example, of the foot rudiment into 
the posterior sac and foot is abnormal in these 



26 



LEBOVITZ 



LwJ\akMÊL** ?*- ••• 






fi? 






> 



FIG. 3. Light micrograph showing the reproductive tract of Deroceras laeve. A, Section through gonad re- 
vealing a cluster of spermatids (arrow head) between egg cells (e). B, Hermaphroditic duct filled with mature 
spermatozoa. Sperm nuclei are darkly stained (box). 



mutants, and the optic tentacles do not signif- 
icantly differentiate from their rudimentary el- 
ements. 

It is likely that the EZL-1 locus is a zygoti- 
cally active gene, rather than a maternally ac- 
tive one. Maternally active genes are con- 
tributed by the maternal chromosome of the 
oocyte, responsible for setting up the spatial 
patterns in the embryo. Once the spatial orga- 
nization is set out, differential expression of 
the zygotic genes is triggered, and embryonic 
development can be completed. In EZL-1 mu- 
tations, development is visibly normal until 
stage IV, when the acquisition of all of the 
adult rudimentary structures is complete. 
Further development, however, is aberrant. 
Thus, the initial spatial patterning of the em- 
bryo is normal, suggesting that the perfor- 
mance of the maternal genes is normal. On 
the other hand, differentiation of the embryo is 
abnormal, arresting about one-quarter into 
development, making it likely that the defect is 
zygotic, a consequence of the malfunction of 
a zygotically active gene. 

The number of eggs in self-fertilizing slugs 
that failed to hatch was high. For example, in 
apparently homozygous individuals, about 
23% of the eggs did not develop at all, and 
3.4% of the developing eggs exhibited a range 
of lethal phenotypes. The failure of eggs to 
hatch into viable offspring has been observed 
with cross-fertilizing snails as well (Doums et 
al., 1994; Jarne & Delay, 1990; Rollinson et 
al., 1989). In one of these studies (Jarne & 
Delay, 1990), a significant difference in egg 



"hatchability" was observed between self- and 
cross-fertilizing Lymnaea peregra snails. It 
was stated that the disparity was due to well- 
formed snails that did not hatch, rather than 
eggs arresting at an early developmental 
stage. This result was interpreted as a de- 
crease in fitness produced by self-fertilization, 
which these authors called self-fertilization 
depression. The studies presented here sug- 
gest the possibility that self-fertilization de- 
pression can be caused by a recessive lethal 
mutation. For example, the number of un- 
hatched eggs produced by EZL-1 heterozy- 
gotes (EZL-1 eggs plus type A) is about two- 
fold greater than for homozygous wild-type 
slugs (type A eggs only) at the EZL-1 gene 
locus. Thus, the presence of a lethal mutation 
decreases egg "hatchability" in self-fertiliza- 
tion, a result that could be avoided by cross- 
fertilization with non-carrying animals. It is in- 
teresting that in the studies reported here with 
self-fertilizing slugs, the largest class of un- 
hatched eggs exhibit no, or little, embryonic 
development. Further studies are needed to 
explain this observation. 



ACKNOWLEDGEMENTS 

Many thanks to Beth Fricano of the Natural 
History Museum of the Smithsonian Insti- 
tution, Washington, D.C., for performing the 
histology; to Dr. Lee Fairbanks of Pennsylvania 
State University, Beaver Campus, for identify- 
ing the slug species; to Dr. Teresa Tansey for 



LETHAL MUTATION IN DEROGERAS 



27 



scientific advice and encouragement; to Hien 
Truong for teclinical assistance; and to Dr. 
Antliony Zelano for encouragement. 

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variability. II. One-locus models for various diploid 
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CARRICK, R., 1938-1939, The life-history and de- 
velopment of Agriolimax agrestis L., the grey field 
slug. Transactions of the Royal Society of 
Edinburgti, 59: 563-597. 

DOUMS, C, B. DELAY & R JARNE, 1994, A prob- 
lem with the estimate of self-fertilization depres- 
sion in the hermaphrodite freshwater snail Buli- 
nus truncatus: the effect of grouping. Evolution, 
48: 498-504. 

FOLTZ, D. W., H. OCHMAN & R. K. SELANDER, 
1984, Genetic diversity and breeding systems in 
terrestrial slugs of the families Limacidae and 
Arionidae. IVIalacologia, 25: 593-605. 

FOLTZ. D., W., B. M. SCHAITKIN & R. K. SE- 
LANDER, 1982, Gametic disequilibrium in the 
self-fertilizing slug Dereceras laeve. Evolution, 
36: 80-85. 

HELLER, J., 1993, Hermaphroditism in molluscs. 
Biological Journal of the Linnean Society, 48: 
19-42. 

HOFFMANN, R. J., 1983, The mating system of the 



terrestrial slug Dereceras laeve. Evolution, 37: 
423-425. 

JARNE, R & B. DELAY, 1990, Inbreeding depres- 
sion and self-fertilization in Lymnaea peregra 
(Gastropoda: Pulmonata). Heredity 64: 169-175. 

MATHER, K., 1951, The measurement of linkage in 
heredity. New York: John Wiley & Sons, Inc. 

NICKLAS, N. L. & R. J. HOFFMANN, 1981, Apo- 
mictic parthenogenesis in a hermaphroditic ter- 
restrial slug. Dereceras laeve (Müller). Biological 
Bulletin, 160: 123-135. 

PATTERSON, C. M. & J. B. BURGH, 1 978, Chromo- 
somes of pulmonate molluscs. Pp. 171-217, in 
Pulmonales, Vol. 2A, Systematics, evolution, and 
ecology, v. fretter & j. peake, eds.. Academic 
Press, London. 

PILSBRY H. A., 1948, Land Mollusca of North 
America. Monograph of the Academy of Natural 
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ROLLINSON, D., R. A. KANE & J. R. L. LINES, 
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RUNHAM, N. W. & A. A. LARYEA, 1 968, Studies on 
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Revised ms. accepted 20 September 1996 



MALACOLOGIA, 1998, 39(1-2): 29-38 

FOREST AND SCRUB SNAIL FAUNAS FROM NORTHERN MADEIRA 
R. A. D. Cameron^ & L. M. Cook^ 

ABSTRACT 

The island of Madeira has two major natural vegetation types, a damp forest association at 
higher altitudes, with below it a dry association of grasses, herbs and low scrub. The open scrub 
is predominantly on the south coast and the eastern peninsula. The land mollusc fauna of the 
high forest and the north coast has been surveyed, and compared with that of other regions. 
Presence or absence of 84 species, 56 of them endemic, has been recorded in 51 samples. The 
forest samples are very clearly separated from those of the other regions; species in the families 
Pupillidae and Vitrinidae have radiated there, whereas radiation of Helicidae is characteristic of 
the other areas. Where non-endemics are present, they increase the species richness and do not 
displace endemics. There is no evidence of subdivisions within the forest fauna. Previous work 
has shown that at lower and drier locations an eastern peninsula fauna is differentiated from that 
of the south coast. The existence of the forest accounts for some of the species richness of the 
Madeiran archipelago, but much of it is attributable to species proliferation, especially in the 
Helicidae, between similar scrub habitats on different islands and on different parts of the same 
island. 

Keywords: land snail, Mollusca, Madeira, competition, endemism. 



INTRODUCTION 

The Madeiran group is in the Atlantic 
Ocean, 900 km southwest of Portugal and 
800 km west of Morocco at 33°N 1 7°W. The is- 
land of Madeira is 58 km at its largest dimen- 
sion and rises to an altitude of 1860 m. There 
are two other clusters of islands, Porto Santo 
with its offshore islets and the three Deserta 
islands, all considerably lower than Madeira it- 
self. The archipelago is exceptionally rich in 
land molluscs, with a high frequency of en- 
demic species. In a count by Waiden (1984) 
there are 261 ±3 taxa, of which 193, or 
73.9%, are endemic. Waldén's (1 983) list con- 
tains 216 species. 

The distribution of the land snail fauna has 
been examined in detail in the southeast and 
eastern part of Madeira (Cameron & Cook, 
1992; Cook et al., 1990). These areas have a 
predominantly dry-zone fauna, but with some 
species found also in the montane forest re- 
gion. Some montane species occur, along 
with those characteristic of the modern east- 
ern fauna, in the fossil deposit on the eastern 
peninsula (Cook et al., 1993; Goodfriend et 
al., 1 994, 1 996). The distribution of species in 
Porto Santo has also been surveyed (Came- 
ron etal., 1996). 

The territory from the peninsula to the west- 



ern extremity of the island in the mountains, 
mostly on the north side of the main dividing 
ridge, has now been investigated. This area 
consists of steeply sloping valleys and moun- 
tainsides, covered in many places by indige- 
nous humid laurel forest (Sjögren, 1972). The 
collections allow comparison with the lower- 
lying and dryer south coast and the eastern 
peninsula of Madeira, and also with Porto 
Santo and the Desertas. In order to under- 
stand how the fauna evolved, it is necessary 
to know the extent to which species are lo- 
calised within islands and whether there is 
any evidence of competition, which might limit 
the diversity at a given locality or lead to se- 
lection for divergence between taxa. 



MATERIALS AND METHODS 

Fifty-one samples were examined, stretch- 
ing from Porto Moniz in the west to the Boca 
do Risco to the east of the north coast, and 
from there along the peninsula and the south- 
east coast, a linear distance of about 70 km. 
Site locations are shown in Figure 1. Each 
site was examined by two people for about 
half an hour, searches being made on rock 
faces, litter and living vegetation. About 5 I of 
soil and litter were collected at each site, tak- 



^ Division of Adult Continuing Education, University of Sheffield, Sheffield SI 4ET, United Kingdom 
^The Manchester Museum, University of Manchester, Manchester M13 9PL, United Kingdom 



29 



30 



CAMERON & COOK 



ing small amounts from favourable patches 
within the sample area. Material so collected 
was air-dried and searched in the laboratory 
after sieving. Material passing through a 0.5 
mm mesh aperture was discarded. Searching 
strategies of this kind give reasonably robust 
and reliable indications of presence and ab- 
sence. Different and more time-consuming 
techniques would be needed to estimate den- 
sities (Cameron, 1973, 1992). For the pur- 
pose of this study, presence and absence 
data are sufficient. A set of specimens of crit- 
ical endemic species has been deposited with 
the Manchester Museum. Nomenclature and 
classification follow Waiden (1983), Cook et 
al. (1990), Groh & Hemmen (1986) and 
Holyoak & Seddon (1986). There were 84 
species recorded, of which 56 are endemics. 
The mean number of species in the 51 sam- 
ples was 14.0, with a standard deviation of 
4.7. The richest sample contained 31 species, 
the poorest 6. 

Salient features of the habitat, including 
dominant vegetation structure, the balance of 
native and alien plant species, and altitude, 
were noted and used to classify the sites into 
the following categories. 

A (Sites 1-5): southern coastal scrub, firmly 
within the Aeonio-Lyntathion alliance (Sjo- 
gren, 1972), usually with introduced Opuntia 
tuna present. All sites subject to some degree 
of past agricultural disturbance, including oc- 
casional burning. 

В (Sites 6-8): sites on the Ponta de Sao 
Lourenço, all open with field layer only present 
and with calcareous sand in the substrate. 
Floristically, they fall into the Biserrulae- 
Scorpiurietum association within the Aenio- 
Lytanthion alliance (Sjögren, 1972; Hamp- 
shire, 1984). These are the richest and least 
disturbed sites on the Ponta as reported by 
Cook et al. (1990). 

С (Sites 9-15): coastal cliff and slope sam- 
ples from the north coast. These also fall in 
the Aeonio-Lytanthlon alliance of Sjögren 
(1972), but with more native scrub elements 
and higher moisture levels than on the south 
coast. 

D (Sites 16-21): coastal cliff and slope 
samples from the north coast, as for С above 
but in which there are significant elements of 
the Clethro-Lauhon alliance, the vegetation of 
typical native laurel forest (Sjögren, 1972), 
which descends to sea level in places along 
the north coast. 

E (Sites 22-33): inland sites (at least 1 km 
from the sea) below 600 m, clearly within the 



Clethro-Laurion alliance, but generally dis- 
turbed and part cleared, with non-native trees 
often dominating the vegetation. 

F (Sites 34-41): native laurisilva above 600 
m but below 900 m above sea level in which 
native species typical of the Clethro-Laurion 
predominate. 

G (Sites 42-51 ): native laurisilva above 900 
m but below 1,400 m, as for F, but with Erica 
species often more important. At and above 
1,300-1,400 m laurisilva is frequently re- 
placed by Erica scrub or by overgrazed mon- 
tane grassland (Sjögren, 1972). 

The topography and land-use pattern of the 
island is such that there is some geographical 
clustering of sites placed in the same cate- 
gory. Categories C, D and E are more hetero- 
geneous than the others, and note should be 
taken of the following sites. 

Site 12 (Group C): extremely arid, very 
heavily grazed and uniquely on Pleistocene 
limestone rather than volcanic rock. 

Site 14 (Group C): very open, with no sur- 
viving laurisilva elements, but with springs 
and permanently moist soil in places. 

Site 20 & 21 (Group D): both with more or 
less complete native forest cover or shaded, 
wet rocks with native Clethro-Laurion vegeta- 
tion, although very close to the sea. 

Sites 22 & 23 (Group E): both clearly within 
the Clethro-Laurion zone altitudinally, but in 
an area of long-term forest clearance with 
much open ground and non-native vegetation 
typical of grassland and scrub. 



RESULTS 
General Patterns 

Table 1 shows the percentage occurrence 
of each species in the samples within each 
sample group as defined above. A full matrix 
of species and sites is available from the au- 
thors, and a copy has been deposited with the 
collections in the Manchester Museum. 

There is evidence of both geographical and 
ecological patterns in the distribution of 
species. Table 2 lists the species found only in 
predominantly Aeonio-Lytanthion habitats 
and their derivatives (groups A, В and C), only 
in predominantly Clethro-Laurion habitats 
(groups D, E, F and G) and also those found 
in combinations of C, D and E, which are to 
some extent intermediate between the ex- 
tremes of A + В and F -t- G. Thirty-five species 
out of 84 show restriction to one or other of the 



MADEIRAN FOREST SNAILS 



31 



Porto Moniz 



Sao Jorge 




Ч-// Boca do Risco 

7 ^2 



Funchal 



FIG. 1 . The island of Madeira showing location of sampling sites. Open circles: sites in groups A and В (south 
coast and peninsula), closed circles: sites in groups С and D (dry and wetter north coastal samples), open 
triangles: group E (inland, wooded or disturbed, below 600 m), closed triangles: groups F and G (woodland, 
6-1 ,400 m). Approximate position of 600 m contour is shown. Maximum distance from west to east is 58 km. 



major vegetation alliances, and a further 12 
are restricted to intermediate habitat types, 
which may represent a third, extensively dis- 
turbed faunal grouping. Helicidae, generally 
the largest species with the thickest shells, 
constitute a far higher proportion of restricted 
species in the open and intermediate habitats 
than they do in the forests (13 out of 30 In the 
former, 2 out of 17 in the latter). Relatively few 
non-endemics are restricted to forest sites. 

To investigate the distinctions between cat- 
egories further, we carried out a cluster analy- 
sis on individual samples. The basis for such 
analyses is a matrix of values of an index of 
similarity in species composition between all 
pairs of samples. The Jaccard Index is one 
such index, calculated by dividing the number 
of species in common between two sites by 
the total number of species present in both. 
When there are different numbers of species 
in the samples being compared, this index 
measures both the taxonomic similarity and 
the difference in number of species, because 
the maximum possible value is number of 
species in the sample with the fewer species 
as a fraction of the total in that with the larger 
number. Cook et al. (1972) normalized the 
Jaccard Index by multiplying it by the ratio of 
the larger species number to the smaller, call- 
ing the result maximum similarity. This would 
be appropriate if all the variation in species 
number was regarded as sampling error, and 
we were concerned exclusively with taxo- 
nomic similarity. However, a measure that is 
affected to some extent by difference in spe- 



cies richness as well as species composition 
would be preferable to either of these indexes, 
because it gives a better idea of the ecologi- 
cal similarity of the sites. When applied to 
presence/absence data, the Nei Index (Nei, 
1987) is the number of species in common di- 
vided by the geometric mean of the number of 
species in each sample of the pair, providing 
the right kind of balance. It is therefore an ap- 
propriate measure, and has been used to ex- 
amine pairwise similarity. Figure 2 shows the 
dendrogram of faunal affinities for all species 
using the Nei Index and UPGMA clustering. It 
confirms the habitat distinctions noted above. 
It is clear that the data fall into two major clus- 
ters (Table 3). All samples of groups A, В and 
С fall into one of these, and all those of groups 
F and G into the other. Samples in groups D 
and E are distributed between the two. There 
are clear separations between groups A and 
В and between these and groups E, F and G. 
Groups C, D and E are more scattered; much 
of the variation being explicable in terms of 
the specific site characteristics noted above. 
We therefore conclude that the groups recog- 
nise differences in ecology and/or geography 
that genuinely influence the faunal composi- 
tion. Intermediate habitats have mixtures of 
species from the two major faunas. 

The Nei index is usually applied to fre- 
quency data, and may be used to examine the 
fraction of sites occupied by each species in 
each group as shown in Table 1 . The result is 
shown in Figure 3. Again, all species were in- 
cluded; when non-endemics are excluded, the 



32 



CAMERON & COOK 



TABLE 1 . Samples grouped into seven geographical and habitat categories, showing fraction of sites 
occupied in each group (to nearest per cent). Endemic species (1 to 56) are listed before non-endemics 
(57 to 84), otherwise species are listed in the systematic order given by Waiden (1 983). Authorities for the 
names are in Waiden (1983), Groh & Hemmen (1986), and Holyoak & Seddon (1986). 



A 


В 


с 


D 


E 


F 


G 


Group 


ENDEMICS 

































66 


42 


83 


91 


100 


100 


1 . Craspedopoma mucronatum 








14 


50 


33 


50 


20 


2. С neritoldes 


20 




















3. C. monizianum 








14 


50 


16 


37 


10 


4. С trochoideum 








14 


33 


33 


62 


80 


5. Columella microspora 


40 


33 


57 


16 











6. Staurodon saxícola 

















37 


50 


7. Leiostyla cheilogona 




















10 


8. L. f i lieu m 











16 











9. L. viñeta 








14 


16 


16 








10. L. irrigua 








14 


33 


8 


50 


30 


^^. L. loweana 




















20 


12. L. eoncinna 

















25 


10 


^3. L. laurínea 








14 


16 


8 


12 





14. L. sphlnetostoma 








14 











10 


15. L. arbórea 








14 


16 


8 








16. L. fusea 


20 





57 


66 











17. L. reeta 


80 


33 


14 














18. L. millegrana 

















25 


10 


19. Lauria fanalensis 

















37 


50 


20. Hemilauria limneana 

















12 





21 . Phenacolimax nitidus 











33 


75 


100 


89 


22. P. mareidus 








14 


33 


58 


62 


80 


23. P ruivensis 











16 


16 


12 





24. P behnil 











16 


8 


12 


30 


25. P albopalliatus 








14 














26. Janulus stephanophora 


80 


33 


42 


50 











27. J. bifrons 








14 


33 


58 


12 


20 


28. Amphorella tornatellina 





100 





16 











29. A. cf. minor 


40 




















30. A. mit ri for mis 





33 

















3^. A. ef irideseens 


80 


33 


28 


33 











32. Pyrgella leacockiana 


40 








16 











33. Boettgeria deltostoma 


60 


66 


14 














34. B. depauperata 





66 


71 


33 











35. B. exigua 











16 





62 


50 


36. В. crispa 


40 


100 


28 


16 











37. HeteroStoma paupereula 








14 











10 


38. Spirorbula latens 








14 














39. S. squalida 


40 


100 

















40. Caseolus compactus 


60 




















41. С leptostictus 


20 




















42. Disculella maderensis 








57 


16 


8 








43. Aetinella lentiginosa 








14 














44. A. aetinophora 


100 




















45. A. areta 








14 


16 


16 





10 


46. A. fausta 











16 


16 








47. А. earinofausta 




















10 


48. A. obserata 


100 


100 


100 


100 


16 








49. A. nitidiuseula 














16 








50. A. giramica 


20 




















51. Discuta tabellata 


100 


100 


85 


50 











52. D. poly mo rp ha 


60 





71 


66 


66 


50 


10 


53. Leptaxis erubescens 











16 








20 


54. L. furva 








14 


33 


58 


87 


100 


55. L. membranácea 


100 


100 


28 





8 








56. L. undata 



MADEIRAN FOREST SNAILS 



33 



TABLE ^ . {Continued) 



Л 


В 


С 


D 


Е 


F 


G 


Group 


NON-ENDEMICS - 
































14 








12 


10 


57. Carychium minimum 








14 


33 


58 


75 


20 


58. C. t ride n tat и m 








57 





16 








59. Cochlicopa lubrica 


80 





57 


100 


66 


75 





60. С. lubricella 


20 





14 


33 


8 


12 





61 . Columella áspera 








14 














62. Truncatellina callicratis 


60 


33 


42 


16 











63. Vertigo pygmaea 


100 





100 


66 


50 








64. Lauria cylindracea 


80 





14 














65. Vallonia costata 


40 


33 


14 














66. V. pu Ichella 




















10 


67. Acanthinula aculeata 














16 


87 


69 


68. Plagyrona placida 











50 


50 


37 


10 


69. Punctum pygmaeum 


100 


33 


85 


66 


75 


87 


69 


70. P. pusillum 











33 


25 


12 


10 


71 . Helicodiscus singleyanus 


20 




















72. Hawaiia miniscula 


60 


33 


85 


83 


91 


87 


30 


73. Vitrea contracta 








14 





16 


12 





74. Nesovitrea hammonis 


80 





100 


83 


8 








75. Oxychilus cellarius 











16 


16 








76. Zonitoides arboreus 














8 








77. Oxychilus alliarius 








14 


83 


58 


87 


40 


78. Euconulus fulvus 


60 


33 

















79. Cecilioides acicula 


20 




















80. Ferrusacia folliculus 


60 


33 

















81 . Caracollina lenticula 





33 

















82. Cochlicella barbara 





33 

















83. Theba pisana 








28 





8 








84. Helix aspersa 


5 


3 


7 


6 


12 


8 


10 


Total sites in group 


19 


14 


30 


31 


21 


19 


23 


Endemic species 


13 


7 


16 


12 


16 


11 


9 


Non-endemic species 


32 


21 


46 


43 


37 


30 


32 


Total species in group 



pattern is almost identical. The impression 
gained from presence/absence data is con- 
firmed. Groups A and B, representing the fau- 
nal composition of the Aeonio-Lytatithion al- 
liance on the southern and eastern parts of 
the island, are similar to each other and form 
a pair distinct from the rest. At a higher level of 
similarity, categories F and G, the higher alti- 
tude Clethro-Lauhon, separate off from C, D 
and E, which contain species characteristic of 
the laurel forest plus others introduced by dis- 
turbance and by natural spreading to those 
coastal areas resembling the eastern sites. 
They include sites 22 and 23 (exceptionally 
dry and open for group E), which resemble the 
open habitat section, and sites 14, 20 and 21 
(in groups С and D), which have above aver- 
age moisture or Clethro-Laurion characteris- 
tics. The faunal separation of the south coast 
and the high laurel forest is due not only to the 
species restricted to them but also to marked 



differences in proportion in several of the 
other, non-restricted species. 

There is also a less obvious correlation be- 
tween similarity and geographical position. 
Sites within groups A and В are close both ge- 
ographically and in terms of affinity, and there 
is a collection of group С and D sites between 
Boca do Risco and Sao Jorge, which also 
show high affinity levels. No geographical pat- 
tern is visible in the forest sites. 

Ratio of Non-endemics to Endemics 

When non-endemics are introduced to an 
endemic fauna, one possible outcome is a 
negative relation between numbers of en- 
demic and non-endemic species in samples. 
This may occur because non-endemics thrive 
in habitats to which endemics are not adapt- 
ed, and vice versa, or because there is direct 
competition between the species. Such a 



34 



CAMERON & COOK 



TABLE 2. Species in the survey which come from (a) Clethro-Lauhon forest or from lower 
altitude habitats of similar type (DEFG), (b) intermediate and disturbed habitats (CDE) or 
(c) from Aeonio-Lytanthion scrub or the damper sites of group С (ABC). 

Groups DEFG CDE ABC 

ENDEMICS 

Cyclophoridae 

Craspedopoma monizianum 

Pupillidae 

Leiostyla cheilogona L. viñeta L. millegrana 

L. filicum L. arbórea 

L. concinna L. fusca 

L laurínea 

Lauria fanalensis 

Hemilauria limneana 

Vitrinidae 

Phenacolimax nitidus 

P. marcidus 

P. behnii 

P. albopalliatus 

Zonitidae 

Janulus stephanophora 

Ferussaciidae 

Amphoreila m i tri form is 
A. cf. iridescens 

Clausiliidae 

Boettgeria crispa В. depauperata 

Helicidae 

Spirorbula squalida 

Caseolus compactus 
С leptostictus 
Disculella maderensis 
Actinella arcta 

A. obserata A. lentiginosa 

A. carinofausta 
A. giramica 

Discula tabéllala 

Leptaxis furva 

NON-ENDEMICS 

Cochlicopidae 

Cochlicopa lubrica 
Valloniidae 

Vallonia costata 
V. pulchiella 

Acanthinuia aculeata 

Plagyrona placida 

Endodontidae 

Punctum pygmaeum 

Helicodiscus singleyanus 

Zonitidae 

Hawai ia miniscula 
Oxychilus alliarius 
Zonitoides arboreus 

Ferussaciidae 

Cecilioides acicula 
Ferrusacia folliculus 

Helicidae 

Caracollina lenticula 
Cochlicella barbara 
Theba pisana 
Helix aspersa 



MADEIRAN FOREST SNAILS 



35 



Sample 



12 
14 
9 
10 
16 
17 
13 
19 
11 
18 
26 
27 
22 
23 
1 
2 
3 
5 
4 
15 
7 
8 
6 
51 
47 
48 
45 
50 
46 
49 
33 
37 
32 
38 
43 
42 
34 
35 
40 
44 
21 
36 
39 
41 
24 
25 
30 
28 
31 
29 
20 



Distance 



20 



40 



60 

"~Г 



80 



100 
1 



J 



V 



и 



H 



FIG. 2. Similarity of the 51 samples in the survey. Scale shows distance as a fraction of the maximum value 
for the analysis. 



36 



CAMERON & COOK 



TABLE 3.The seven defined habitat groups divided between the two main clusters 
of Figure 2. Groups, based on habitat type and reflecting to some extent geo- 
graphical location, are defined in the text. 









Defined groups 








Derived cluster 


A 


В 


С 


D 


E 


F 


G 


Total 


1 


5 


3 


7 


4 


4 








23 


2 











2 


8 


8 


10 


28 


Total 


5 


3 


7 


6 


12 


8 


10 


51 



Category 



Distance 



20 

"T 



40 

"~r 



60 

~~r 



80 

"T 



100 

— I 



ZJ 



FIG. 3. Similarity of the seven categories of samples derived from the frequency of species in the samples 
within categories (Table 1). Scale shows distance as a fraction of the maximum value for the analysis. 



relation occurs, for example, in our study of 
the snails of Porto Santo (Cameron et al., 
1996), where there is a negative relation of 
endemics to non-endemics on a rocky/undis- 
turbed to sandy/disturbed axis. To test for a 
similar effect on Madeira, the association of 
the number of non-endemic species with the 
number of endemic species has been exam- 
ined. The correlation coefficient r is 0.263, for 
which t = 1.906 (P < 0.06). Thus, there is no 
evidence of a negative association; the rela- 
tion is positive, and nearly significant, the 
slope of the reduced major axis being 0.757. 
it could result from the fact that sites vary in 
their suitability for molluscs, good sites being 
favourable to non-endemics and endemics 
alike. The feature most likely to lead to high 
species number may be microhabitat hetero- 
geneity. At any rate, presence or absence of a 
species appears to depend on habitat suit- 
ability, with no evidence of negative associa- 
tion between the two categories. 



DISCUSSION 

Cook et al. (1990) demonstrated differ- 
ences between snail faunas from open habi- 



tats in the south and east of Madeira, which 
suggested a pattern of geographical differen- 
tiation independent of present habitats. 
Similar, indeed more striking differentiation of 
this kind is found in the neighbouring island of 
Porto Santo (Cameron et al., 1996). This 
study extends the survey to the high altitude 
forest and open and intermediate habitats 
along the north coast of Madeira. 

There is a radical difference between the 
fauna of the Clethro-Laurion forest and of the 
open habitats and scrubby areas at lower alti- 
tudes. In the forest, the climate is cooler, rain- 
fall is higher, and soils are less rich in calcium 
(Sjögren, 1972). Table 4 illustrates the differ- 
ent balance of families in Clethro-Laurion 
sites from those of open habitats on Madeira 
and on the other islands of the archipelago, 
which are even more arid. The faunas have 
substantial representation of small, thin- 
shelled species (especially Pupillidae) and 
semi-slugs (Vitrinidae), and are less domi- 
nated by Helicidae species, only six of which 
were found above 600 m altitude. In the open 
sites, there is again evidence of geographical 
differentiation, albeit complicated by distur- 
bance and small-scale mosaics of forest and 
open habitats. Faunas from mid- to high aiti- 



MADEIRAN FOREST SNAILS 



37 



TABLE 4. Species distribution between families in samples collected in different parts of the archi- 
pelago. Madeira north side (Groups D to G): data from this paper. Madeira fossil: samples 45 thou- 
sand years old or older from the sand bed on the eastern peninsula from Cook et a!. (1993). Madeira, 
eastern peninsula and Desertas: contemporary samples from Cook et al. (1990). Porto Santo: con- 
temporary samples from Cameron et al. (1996). 





Madeira 


Madeira 


Madeira 




Porto 


Family 


N side 


fossil 


peninsula 


Desertas 


Santo 


Endemics 












Pupillidae 


13 


7 


4 





5 


Ferussaciidae 


3 


3 


5 


1 


8 


Clausiliidae 


3 


1 


4 


1 


1 


Vithnidae 


5 


2 


2 





1 


Helicidae 


12 


18 


12 


10 


32 


Others 


6 


6 


5 


1 





Non-endemics (all families) 


19 


2 


21 





9 


Total 


61 


39 


53 


13 


56 



tude forests, free from gross disturbance, do 
not show such effects, although distances be- 
tween sites are the same, or greater than 
those involved in open habitat comparisons. 

In drawing the distinction between the rela- 
tively open Aeonio-Lytanthion and Clethro- 
Laurion alliances, Sjögren (1972) noted that 
on the drier south side of the island the 
Aeonio-Lytanthion gives way to forest be- 
tween 300 and 700 m above sea level, 
whereas on the wetter north it is rare for it to 
ascend above 300 m; Clettiro-Laurion or de- 
rivatives frequently approach sea-level. While 
modest changes in altitudinal range of the al- 
liances (for example, in response to Pleisto- 
cene climatic changes) could isolate sections 
of open habitat on the south coast, and extin- 
guish it in the north, the forest area would fluc- 
tuate is size but remain as a largely continu- 
ous block. Extension of forest faunas in the 
past is suggested by the subfossil data in 
Table 4, and confirmed by detailed analysis 
(Cook et al., 1993). 

Although forest faunas are more uniform, 
there is some altitudinal differentiation, with 
species characteristic of low or high altitudes. 
There are some species-rich genera. Of 
these, Leiostyla species are small, rare, cryp- 
tic and apparently patchily distributed. Our 
knowledge of them is as yet too limited to say 
whether they show geographical differentia- 
tion. On the present evidence, however, the 
extensive proliferation of distinct species of 
limited distribution but similar habitat, which 
contributes so much to the species richness 
of the archipelago, is largely a dry-habitat 
phenomenon. Forests, especially at higher al- 
titudes, have few non-endemic species, and 
some of those that do occur may be natives 
rather than introductions. We have no evi- 



dence pointing to adverse effects of non- 
endemics on endemics or to the presence of 
habitats which favour non-endemics over en- 
demics. The greater number and higher den- 
sity of non-endemics in open habitats proba- 
bly reflects the richer conditions there, 
created to some extent by human activity, in 
which both categories can flourish. 



ACKNOWLEDGEMENTS 

We thank Dr. Mary Seddon and the 
National Museum of Wales for essential as- 
sistance in species identification. 



LITERATURE CITED 

CAMERON, R. A. D., 1973, Some woodland mol- 
lusc faunas from southern England. Malacologia, 
14:355-370. 

CAMERON, R. A. D., 1 992, Land snail faunas of the 
Napier and Oscar ranges, V\/estern Australia; di- 
versity, distribution and speciation. Biological 
Journal of the Linnean Society, 45: 271-286. 

CAMERON, R. A. D. & L. M. COOK, 1992, The de- 
velopment of the diversity of the land snail fauna 
of the Madeiran archipelago. BiologicalJournal of 
tfie Linnean Society. 46: 105-1 14. 

CAMERON, R. A. D., L. M. COOK & J. D. HAL- 
LOWS, 1996, Land snails on Porto Santo: adap- 
tive and non-adaptive radiation. Philosophiical 
Transactions of the Royal Society of London, (B) 
351, 309-327. 

COOK, L. M., R. A. D. CAMERON & L. A. LACE, 
1990, Land snails of eastern Madeira: speciation, 
persistence and colonization. Proceedings of the 
Royal Society of London, (B) 239: 35-79. 

COOK, L. M., G. A. GOODFRIEND & R. A. D. 
CAMERON, 1993, Changes in the land snail 
fauna of eastern Madeira during the Quaternary. 



38 



CAMERON & COOK 



Philisophical Transactions of the Royal Society of 
London, (B) 339: 83-103. 

COOK, L. M., T. JACK & С W. A. PETTITT, 1972, 
The distribution of land molluscs in the Madeiran 
archipelago. Boletim do Museu Municipal do 
Fuchal, 26: 5-30. 

GOODFRIEND, G.A., R. A. D. CAMERON & L. M. 
COOK, 1994, Fossil evidence of recent human 
impact on the land snail fauna of Madeira. Journal 
of Biogeography 21: 309-320. 

GOODFRIEND, G. A., R. A. D. CAMERON, L. M. 
COOK, M.-A. COURTY, N. FEDEROFR A. KAUF- 
MAN, E. LIVETT & J. TALLIS, 1996, Quaternary 
eolianite sequence of Madeira: stratigraphy, 
chronology, and paleoenvironmental interpreta- 
tion. Palaeogeography Palaeoclimatology Pal- 
aeoecology 120, 195-234. 

GROH, K. & J. HEMMEN, 1986, Zur Kenntnis der 
Vitriniden des Madeira-Archipels (Pulmonata, 
Vitrinidae). Archiv für l\Aolluskenkunde, 115: 
1-39. 

HAMPSHIRE, R. J., 1984, A study of the vegetation 
of the Ponta de Sao Lourenço in Madeira, llheu 



Chao and Deserta Grande. Boletim do l\Auseu 

Municipal do Funchal, 36: 207-226. 
HOLYOAK, D.T. & M. B. SEDDON, 1986, An unde- 

scribed Leiostyla (Gastropoda: Pupillidae) from 

Madeira. Journal of Conchology 31: 191-193. 
NEI, M., 1987, Molecular evolutionary genetics. 

New York: Columbia University Press. 
SJÖGREN, е., 1972, Vascular plant communities of 

Madeira. Boletim do Museu Municipal do 

Func/ia/, 26:46-125. 
WALDÉN, H. W., 1983, Systematic and biogeo- 

graphical studies in the terrestrial Gastropoda of 

Madeira. With an annotated check-list. Annales 

Zooiogici Fennici, 20: 255-275. 
WALDÉN, H. W., 1984, The land mollusc fauna of 

Madeira in relation to other Atlantic islands and 

the Palaearctic region. Pp. 38-45, In: a. solem & 

A. H. с VAN BRUGGEN, eds., World-wide snails. 

Leiden:E. H.J. Brill. 

Revised ms. accepted 29 October 1996 



MALACOLOGIA, 1998, 39(1-2): 39-57 

COMPARATIVE STUDIES ON THE ANATOMY AND HISTOLOGY 

OF THE ALIMENTARY CANAL OF THE LIMACOIDEA AND MILACIDAE 

(PULMONATA: STYLOMMATOPHORA)^ 

Ana Maria Leal-Zanchet 

Laboratorio de Histología, Centro de Ciencias da Saude, Universidade do Vale do Rio dos 
Sinos, Caixa Postal 275, 93022-000 Sao Leopoldo-RS, Brasil 

ABSTRACT 

The anatomy and histology of the digestive tract of Dereceras laeve, D. reticulatum. D. rodnae, 
Lehmannia marginata, Malacolimax tennelus, Boettgerilla pallens and Tandonia budapestensis 
are described comparatively. The alimentary canal is composed of oesophagus, crop, stomach, 
intestine and rectum. An intestinal caecum is present in D. reticulatum. D. rodnae and L. mar- 
ginata. The intestine is subdivided into four histologically distinct regions. From the oesophagus 
to the third intestinal region, and in the rectum, the alimentary canal is lined by a simple colum- 
nar to cuboidal epithelium. In the fourth intestinal region and in the intestinal caecum, the ep- 
ithelium is simple squamous to cuboidal. Supporting ciliated and nonciliated cells are found in the 
epithelium. Both types carry microvilli. Eight gland cell types can be distinguished: mucous cells 
of types l-V, cystic cells, and intestinal secreting cells of types I and II. The epithelium of the ali- 
mentary canal is surrounded by a layer of connective tissue and two muscle layers, an inner lon- 
gitudinal muscle layer and an outer circular muscle layer. 

Key-words: anatomy, histology, alimentary canal, secretory cells, Limacidae, Agriolimacidae, 
Boettgerillidae, Milacidae. 



INTRODUCTION 

According to Likharev & Wiktor (1980), the 
superfamily Limacoidea is formed by the fam- 
ilies Limacidae, Agriolimacidae and Boett- 
gerillidae, whereas the family Milacidae be- 
longs to the superfamily Zonitoidea. Little 
information exists on the anatomy and histol- 
ogy of the digestive system of the limacids 
and agriolimacids. Simroth (1885). Quick 
(1960) and Wiktor (1973), in their systematic 
studies on the group, only described the 
course of the alimentary canal. Studies on the 
anatomy and histology of the alimentary canal 
of Umax, Arion and Helix have been carried 
out by Gartenauer (1875) and Baecker 
(1 932). Walker (1 972) examined the digestive 
system of Dereceras reticulatum, emphasiz- 
ing the physiology of the crop, stomach and 
intestine. More recently, only a few observa- 
tions have been made on the histology of the 
alimentary canal of a limacoid species (Ba- 
bula & Skowronska-Wendland, 1988). No 
data are found in the literature on the anatomy 
and histology of the digestive system of the 



families Boettgerillidae and Milacidae. The 
purpose of this paper is to report on a com- 
parative anatomical and histological study of 
the alimentary canal of six limacoid species 
and a milacid species. 



MATERIALS AND METHODS 

The following species were studied: 
Malacolimax tenellus (Müller, 1774) and Leh- 
mannia marginata (Müller, 1774) (Limacidae), 
Dereceras laeve (Müller, 1774), D. reticulatum 
(Müller, 1774) and D. rodnae (Grossu & Lupu, 
1965) (Agriolimacidae), Boettgerilla pallens 
(Simroth, 1912) (Boettgerillidae), and Tan- 
donia budapestensis (Hazay, 1881) (Milaci- 
dae). The animals were collected next to the 
city of Tübingen, Baden-Württemberg, Ger- 
many, and kept in a cool room at 15°C (Leal- 
Zanchet, 1995). The species are herbivorous, 
except for B. pallens (Leal-Zanchet, in press). 
In the laboratory, the animals were fed cab- 
bage, lettuce and carrots. For the anatomical 
studies, the slugs were anaesthetized in a 5% 



Vart of a thesis submitted to the Lehrstuhl Spezielle Zoologie of the University of Tübingen, Gernnany, in partial fulfillment 
of the requirements for the degree of Doctor of Natural Sciences. 



39 



40 



LEAL-ZANCHET 



solution of menthol for three to four h, fixed in 
4% formaldehyde for 24 to 48 h and trans- 
ferred to 70% ethanol. The slugs were pro- 
gressively dissected with the aid of a binocu- 
lar microscope. The digestive tract was 
uncovered and drawn under a camera lucida. 
Finally, the alimentary canal was opened lon- 
gitudinally to observe its internal morphology. 
For the light microscopy studies, the slugs 
were anaesthetized in 5% menthol for two and 
fixed in Susa or 4% paraformaldehyde/glu- 
taraldehyde. Tissues fixed with Susa were de- 
hydrated in ethanol, embedded in Paraplast, 
and serially sectioned at 6 um. The sections 
were stained with haematoxylin/eosin, or with 
the triple stain methods of Masson-Goldner 
(MG) and Azan-Heidenhain (AZ) (Romeis, 
1989). This method was used to delimit histo- 
logically the organs and regions of the diges- 
tive system, and also for a first characteriza- 
tion. Small fragments of the various parts of 
the alimentary canal were fixed in 4% 
paraformaldehyde/glutaraldehyde, washed in 
phosphate buffer, dehydrated in ethanol and 
embedded in historesin. The 2 um thin sec- 
tions were stained with methylene blue and 
basic fuchsin (mf) (Bennett et al., 1976). This 
second method gives the best results for de- 
tailed histological studies. 

For measurements of the epithelium and 
the gland cells we used a micrometric glass. 
Four animals of each species were measured. 
All measurements were made on the material 
fixed in paraformaldehyde/glutaraldehyde and 
embedded in historesin. They were made on 
the apices of the folds and between the folds. 
The mean values were given in the tables. 



RESULTS 
Anatomical Features 

The narrow oesophagus (oe) rises from the 
buccal mass (Fig. 2, bcm) and widens poste- 
riorly to form the crop (cr). From this emerges 
the small stomach (s), which is followed by the 
long intestine (Fig. 2, i). The ducts of the di- 
gestive gland open into the stomach (Figs. 3, 
4). The intestines of Lehmannia marginata, 
and Malacolimax tenellus have two forward- 
directed loops, whereas the intestines of 
Deroceras laeve, D. reticulatum, D. rodnae, 
Tandonia budapestensis and Boettgerilla pal- 
lens have only one forward directed loop 
(sensu Quick, 1960). From the second intesti- 
nal loop of Lehmannia and Malacolimax, or 



the single loop of the other species, arises the 
terminal branch of the intestine, which goes 
forward and enters into the body wall and is 
then called rectum (Fig. 1).The rectum goes 
through the palliai complex for a short way 
and unites with the ureter into the anus, which 
is situated in the right anterior part of the body. 
A blind tube, the intestinal caecum (Fig. 2, c), 
is associated with the terminal part of the in- 
testine of L. marginata, D. reticulatum and D. 
rodnae (Fig. 1). 

The oesophagus has longitudinal folds (Fig. 
5, If), and the crop has a smooth wall without 
folds, containing only narrow elevations (Fig. 

KEY TO LETTERING ON FIGURES 

as: apocrine secretion 

bcm: buccal mass 

c: intestinal caecum 

ca: clear area 

cc: ciliated columnar cells 

cet: cells of the connective tissue 

ce: cuboidal epithelium 

ci: cilia 

cia: ciliated area 

cm: circular muscle layer 

en: cell neck 

cr: crop 

ct: connective tissue 

cy: cystic cells 

dg: digestive gland 

dgd: duct of the digestive gland 

dgo: opening of the digestive gland 

e: elevation 

i: intestine 

ic: intestinal secreting cells 

ic I: intestinal secreting cells of type I 

ic II: intestinal secreting cells of type II 

let: leader fold 

If: longitudinal fold 

Ig: leader groove 

Im: longitudinal muscle layer 

lu: lumen 

mc: mucous cells 

mc I: mucous cells of type I 

mc II: mucous cells of type II 

mc III: mucous cells of type III 

mc IV: mucous cells of type IV 

mc V: mucous cells of type V 

mi: microvilli 

n: nucleous 

nc: nonciliated columnar cells 

nu: nucleolus 

oe: oesophagus 

s: stomach 

sac: strong acidophilic cells 

se: squamous epithelium 

sg: salivary glands 

sgd: duct of the salivary gland 

sgr: secretory granules 

si: subepithelial layers 

sm: secretion mass 

sv: supranuclear vacuoles 

t., t^: typhlosoles 

ti: transversal fold 

va: vacuole containing amorphous material 



HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 



41 




FIG. 1. Schematic diagrams comparing the digestive system of (1) Deroceras laeve, (2) Dereceras reticula- 
tum, (3) Deroceras rodnae. (4) Malacolimax tenellus. (5) Lehmannia marginata. (6) Boettgerilla pallens and 
(7) Tandonia budapestensis. A: first intestinal region, B: second intestinal region, C: third intestinal region, D: 
fourth intestinal region, E: rectum, F: intestinal caecum. 



First intestinal region 
Second intestinal region 



Ш Third intestinal region 
'/:' Fourth intestinal region 



Rectum 
Intestinal caecum. 



6, e) due to the irregular height of the epithe- 
lium. The crop of Tandonia budapestensis is 
an exception, with the wall having transverse 
folds (Fig. 28). The stomach contains three 
conspicuous folds, two longitudinal typhlo- 
soles and a transverse fold (Figs. 4, 7). Each 
typhlosole originates close to an opening of 
the duct of the digestive gland and runs pos- 
teriorly as far as the posterior limit of the stom- 
ach (Fig. 4, t^, t2). The transverse fold is 
closely triangular and extends between the 
two openings of the ducts of the digestive 
gland (Fig. 4, f). A leader groove occurs be- 
tween the typhlosoles, as well as between the 
transverse fold and the typhlosoles (Fig. 4, Ig). 
The intestine can be subdivided into four re- 
gions. Figure 1 shows the arrangement of the 
Intestinal regions in relation to the intestinal 
loops. The first and second intestinal regions 
have smooth walls without folds, being differ- 
entiated only by their histological features. 
However, in L. marginata and M. tenellus, only 
small longitudinal folds are present in the first 
intestinal region (Fig. 8, If). The third intestinal 
region has numerous longitudinal folds (Fig. 
10, If), and the fourth region has a smooth 
wall, except for a ciliated area (Fig. 11, cia). 
This occurs in limacids and agriolimacids, but 
not in B. pallens and T. budapestensis. The in- 



testinal caecum has no folds. The rectum has 
a smooth wall in its proximal third and abun- 
dant longitudinal folds in its distal two thirds 
(Figs. 12-16, If). Among these, three to five 
differentiated folds occur in Agriolimacidae, 
Boettgerillidae and Milacidae (Figs. 13-16, 
let). There are five of these folds in B. pallens 
and T. budapestensis, where they are most 
developed (Figs. 13, 14, 43). In Deroceras, 
there are two to three leader folds (Figs. 15, 
16,44). 

Histological Features 

Epithelium: Oesophagus — The oesophagus 
is lined by a columnar simple epithelium. In 
the proximal third of the oesophagus, its sur- 
face is cuticularized, but in most of the oe- 
sophagus the epithelium carries cilia and mi- 
crovilli (Fig. 17). The cilia are especially 
numerous on the crests of folds (Figs. 5, 26). 
The epithelium of the oesophagus consists 
of ciliated columnar cells (cc), nonciliated 
columnar cells (nc), and mucous cells (Fig. 1 7, 
mc I). Both ciliated and nonciliated columnar 
cells bear microvilli. The columnar cells show 
an acidophilic cytoplasm, in which supra- 
nuclear vacuoles (Fig. 17, sv) with strong aci- 
dophilic or cromophobe contents can be seen. 



42 



LEAL-ZANCHET 




FIG. 2. Dorsal view of the digestive system of 
Lehmannia marginata. Scale bar 1 mm. 



The most apical part of the cells is a clear area 
(Fig. 17, ca).The oval nucleus lies in the mid- 
dle or basal third of the cells (Fig. 17, n). 

The columnar cells of the alimentary canal 
show an apocrine secretion (Figs. 17, 18, 22, 
as), and eventually a holocrine secretion in 
which strong acidophilic columnar cells are 
discharged into the lumen (Figs. 30, 31, sac). 

The mucous cells of the oesophagus are 
termed type I (Fig. 17, mcl).They are intraep- 
ithelial and flask shaped, with a broadened 
base and a long neck, and they contain nu- 
merous weakly or strongly basophilic granules 
(MF). The oval or elongated nucleus is basally 
located and surrounded by a strong acidophilic 




FIG. 3. Ventral view of the crop and the stomach of 
Dereceras reticulatum. 

FIG. 4. Internal morphology of the stomach of 
Lehmannia marginata. Scale bar 1 mm. 



cytoplasm. In the paraffin sections, the gran- 
ules and the remaining cytoplasm of the mu- 
cous cells cannot be distinguished; the mu- 
cous cells show a foamy content that stains 
light green with MG and light blue with AZ. 

Crop — The columnar epithelium of the crop 
is higher than that of the oesophagus (Table 
1 ). In the crop, only nonciliated columnar cells 
(nc) and mucous cells of type I (Figs. 18, 27, 
mcl) are present. The columnar cells of the 
crop are clearly distinguishable because of 
the presence of very abundant large vacuoles 
containing amorphous material (Figs. 18, 27, 
va). The contents of the vacuoles can be re- 
moved by histological methods. Supranuclear 
vacuoles (Fig. 18, sv) with strong acidophilic 
or chromophobe contents, similar to those of 
the oesophagus, are also present. The oval or 



HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 



43 




FIG. 5. Cross-section of the oesophagus of 
Lehmannia margínala. 

FIG. 6. Cross-section of the crop of Lehmannia mar- 
gínala. Scale bar 0.1 mm. 




FIG. 7. Cross-section of the stomach of Leiimannia 
margínala. 

FIG. 8. Cross-section of the first intestinal region of 
Lehmannia margínala. 



elongated nucleus lies in the middle or basal 
third of the cells (Fig. 18, n). Type I mucous 
cells are similar to those of the oesophagus 
(Figs. 18, 27, mcl). 

Stomach — The ciliated columnar epithe- 
lium of the stomach usually is lower than that 
of the crop (Table 1). In the stomach, the cili- 
ated columnar cells far outnumber the noncil- 
iated cells. The latter occur only in small areas 
adjoining the typhlosoles (Fig. 7). The cyto- 
plasm of the columnar cells is similar to that of 
the corresponding cell types of the crop, but 
the number of vacuoles containing amor- 
phous material is lower (Fig. 19, va). The 
columnar cells of the leader groove (Fig. 20), 
located between the two typhlosoles, differ 
from the other columnar cells of the stomach 
by their strong acidophilic cytoplasm contain- 
ing rare vacuoles. In addition, the epithelium 
of the leader groove and of the typhlosoles is 



taller than in the rest of the stomach and has 
long cilia (Fig. 20). 

In the stomach, the mucous cells are more 
abundant than in the crop (Table 3). Two types 
of mucous cells can be distinguished. The 
type I mucous cells are found in most of the 
stomach and are similar to those of the crop in 
shape and morphology (Fig. 19, mcl). The 
type II mucous cells occur only in the leader 
groove (Figs. 20, 31 , mcll). However, in B. pal- 
lens and T. budapestensis, type II mucous 
cells are found in the entire stomach. They are 
intraepithelial and have a long base with an 
acidophilic cytoplasm containing many vac- 
uoles. The nucleus lies distal at the base. The 
long neck shows numerous small and strong 
basophilic secretory granules (Table 2, Fig. 
20). 

First Intestinal Region — This region has a 
high columnar epithelium (Table 1) with a 
pseudostratified appearance because the nu- 




LEAL-ZANCHET 




FIG. 9. Cross-section of the second intestinal region 
of Lehmannia marginata. 

FIG. 10. Cross-section of the third intestinal region 
of Lehmannia marginata. Scale bar 0.1 mm. 




FIG. 1 1 . Cross-section of the fourth intestinal region 
of Leiimannia marginata. 

FIG. 12. Cross-section of the rectum of Letimannia 
marginata. Scale bar 0.1 mm. 



ciel tend to be arranged in two rows: the round 
nuclei of the abundant mucous cells form a 
row at the base of the epithelium, and the 
elongated nuclei of the columnar cells lie in 
one or two rows halfway up in the epithelium 
(Figs. 21, 33, n). 

Only ciliated columnar cells occur in the first 
intestinal region (Fig. 8). Their cytoplasm pos- 
sess a small number of vacuoles with amor- 
phous contents (Fig. 21 , va), and is otherwise 
similar to that of the columnar cells of the 
crop. Due to the pressure of the numerous 
mucous cells, the base of the columnar cells 
becomes thinner and their nuclei lie in the 
middle or apical third (Figs. 21 , 33). 

The abundance of mucous cells is the more 
conspicuous feature of this intestinal region 
(Table 3, Fig. 33). The mucous cells are 
mainly of type II (Fig. 21 , mc II), but some mu- 
cous cells of type I may also be present (Table 
4). 

Second Intestinal Region — The second in- 
testinal region is characterized by the pres- 
ence of type I intestinal secreting cells (Fig. 



22, ic I). The columnar epithelium is usually 
lower than in the first intestinal region (Table 
1). Ciliated and nonciliated columnar cells 
occur in the second intestinal region. Both cell 
types contain rare vacuoles with amorphous 
contents. The nucleus of the columnar cells is 
located in the basal or middle third (Fig. 22). 

The number of mucous cells is relatively 
small in the second intestinal region (Table 3). 
Morphologically, the cells are similar to the 
type I mucous cells of the oesophagus and 
crop (Fig. 22, mc I). 

The intestinal secreting cells of type I (Figs. 
22, 34, ic I) have a claviform shape. The large, 
oval nucleous is rich in chromatin and has a 
conspicous nucleolus (Fig. 34, nu).The basal 
cytoplasm stains violet (MF). The numerous 
secretory granules are found in the supranu- 
clear cytoplasm (Fig. 22, sgr). The granules 
stain red with MG and MF. 

Third intestinal region — In the third intesti- 
nal region, type II intestinal secreting cells are 
found (Figs. 23, 35). Ciliated cells occur only 



HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 




45 
15 



FIG. 13. Cross-section of the rectum of Tandonia 
budapestensis. 

FIG. 14. Cross-section of the rectum of Boettgerilla 
pallens. Scale bar 0.1 mm. 



at the crests of the folds (Fig. 10). The re- 
maining cells are nonciliated. The cytoplasm 
of the columnar cells has a small number of 
supranuclear vacuoles (Fig. 23, sv). 

The intestinal secreting cells of type II (Figs. 
23, 35, ic II) have granules, the contents of 
which were not stained by any of the methods 
used. The granules are larger than those of 
the intestinal secreting cells of type I. Other 
features of type II intestinal secreting cells are 
similar to those of type I. 

The gland cells become more abundant 
than in the second intestinal region (Table 3). 
In L. margínala and M. tenellus, four types of 
gland cells (mucous cells of type III, IV and V, 
and cystic cells) occur in the third and fourth 
intestinal regions, in the intestinal caecum, 
and in the rectum. In D. rodnae and D. re- 
ticulatum, all four types are also present, but 
their occurrence is different (Table 4). Only 
three of these gland cell types can be seen in 





FIG. 15. Cross-section of the rectum of Deroceras 
rodnae. 

FIG. 16. Cross-section of the rectum of Deroceras 
laeve. Scale bar 0.1 mm. 



D. laeve, and only two of them in B. pallens 
and T. budapestensis (Table 4). These cell 
types were identified and described with the 
aid of historesin sections. With the exception 
of the cystic cells, the gland cells cannot be 
distinguished on paraffin sections, remaining 
unstained or staining light green with MG and 
light blue with AZ. 

The mucous cells of type III (Figs. 23, 25, 
36, 38, 42, 45, Table 2, mc III) have an aci- 
dophilic cytoplasm and granules staining light 
or dark blue (MF). The cells are usually subep- 
ithelial. Their cell body is located in the subep- 
ithelial connective tissue or external to the 
ring muscle layer (Figs. 25, 38). 

The mucous cells of type IV (Figs. 25, 38, 
39, mc IV) are also usually subepithelial. Their 
cell body has a smaller diameter than that of 
type III mucous cells (Table 2). The nucleus, 
however, is larger than that of type III mucous 
cells. The acidophilic cytoplasm has numer- 
ous small secretory granules that stain red 
(MF). 



46 



LEAL-ZANCHET 




me 



cet — 4it ifet ., --A 




FIG. 17. Semi-schematic drawing of part of a trans- 
verse section of the oesophagus of Lehmannia 
marginata. 

FIG. 18. Semi-schematic drawing of part of a trans- 
verse section of the crop of Lehmannia marginata. 
Scale bar 10 um. 



The mucous cells of type V (Figs. 23, 25, 
47, Table 2, mc V) are always subepithelial 
and are easily distinguishable from the other 
mucous cells by their shape and by their con- 
tents. The cell body has a sacculiform shape, 
so that its transition to the cell neck is very 
gradual, and the cell body is small and located 
close to the epithelium. Most of the cell is filled 
with elongated secretory granules that stain 
blue with MR The granules often coalescens, 
forming a secretion mass. The nucleus is pe- 
ripheral and oval to elongated in shape and is 
surrounded by acidophilic cytoplasm. 

The cystic cells (Figs. 23-25, 46, cy), like 
the mucous cells of type V, are always subep- 
ithelial, but have a very large sacculiform ceil 
body (Table 2). The cystic cells are filled with 
an amorphous secretion that stains pink with 
MP and red with MG. Basophilic cytoplasm 
can be seen in a peripheral zone of the cell 
body. The nucleus is basally located. 

Fourth Intestinal Region — The epithelium 
of the fourth intestinal region is squamous to 
cuboidal, and two to three times lower than in 
the third intestinal region (Table 1, Figs. 25, 
37-39). The cells are nonciliated, except for 
the longitudinal ciliated area (Figs. 11 , 25, 37). 
Both nonciliated and ciliated cells have mi- 
crovilli. The cytoplasm of the cells of the fourth 
region seldom has supranuclear vacuoles 
(Fig. 25). The basal part of the cells is highly 
folded. In the fourth intestinal region of B. pal- 
lens and T. budapestensis, the ciliated area is 
absent. 

The epithelium of the ciliated area is higher 
and has long cilia (Figs. 25, 37). Apical cells 
are more acidophilic than the other cells of the 
region. 

The gland cells (Table 4) are usually more 
abundant than in the third intestinal region 
(Table 3). In the fourth intestinal region, the 
gland cells are always subepithelial (Fig. 25). 
The intestinal secreting cells are absent. The 
cell body of the mucous cells of type III and IV, 
as well as that of the cystic cells, is located ex- 
ternal to the muscle layers. 

Intestinal Caecum — The epithelium of the 
intestinal caecum is squamous and lower 
than that of the fourth intestinal region (Table 
1, Figs. 24, 40, 41). The cytoplasm of the 
squamous cells is similar to that of the fourth 
intestinal region. The number of gland cells is 
small (Tables 3, 4). 

Rectum — The epithelium of the rectum is 
cuboidal to columnar. The epithelial cells of 
the leader folds (Figs. 43, 44, 46) are higher 
and show features similar to those of the 



HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 



47 



TABLE 1 . Epithelial height of the digestive tube of Limacoidea and Miiacidae (цт). 





B. 


L. 


D. 


D. 


D. 


M. 


T. buda- 




pallens 


marginata 


laeve 


reticulatum 


rodnae 


tenellus 


pestensis 


Oesophagus 


49.7 


47.0 


31.5 


40.3 


46.5 


51.3 


29.0 


Crop 


79.8 


65.0 


55.0 


63.0 


58.5 


72.3 


37.8 


Stomach 


52.5 


41.5 


53.8 


49.0 


45.1 


58.5 


56.3 


1. intestinal region 


56.0 


61.0 


58.3 


62.5 


57.3 


61.3 


32.5 


2. intestinal region 


53.8 


54.0 


42.8 


42.0 


57.3 


67.5 


41.8 


3. intestinal region 


34.0 


37.8 


33.3 


21.3 


32.8 


30.3 


17.5 


4. intestinal region 


10.5 


15.5 


15.0 


10.1 


6.8 


9.8 


10.0 


Caecum 


— 


13.8 


— 


6.3 


6.0 


— 


— 


Rectum 


7.1 


10.0 


7.5 


13.4 


11.4 


14.0 


12.1 



leader fold of the fourth intestinal region. The 
rectum of L. marginata and M. tenellus, with- 
out leader folds, has ciliated cells in the crests 
of the folds (Figs. 12, 42, 47). In the rectum of 
B. pallens, T. budapestensis and D. laeve 
(Figs. 13, 14, 16), ciliated cells occur only in 
the leader folds, and all other cells are noncil- 
iated. In D. rodnae and D. reticulatum, ciliated 
cells occur in the leader folds, as well as in the 
crest of other folds (Fig. 1 5). 

In the rectum, the gland cells (Table 2) are 
again fewer than in the fourth intestinal region 
(Table 3). The gland cells are still fewer in the 
leader folds (Figs. 13-16, 43, 44). The cell 
body is located in the subepithelial connective 
tissue or external to the muscle layers (Fig. 
45). 

Subepithelial Layers: The epithelium of the 
alimentary canal is surrounded by a thin layer 
of connective tissue and two muscle layers, 
an inner longitudinal layer, and an outer 
circular layer. The longitudinal muscle layer is 
rather irregular; some longitudinal muscle 
bundles were observed within or externally to 
the circular layer. 

The muscle layers are thicker in the oe- 
sophagus (Fig. 26), in the third intestinal re- 
gion (Fig. 36) and in the rectum (Figs. 42, 44). 
The longitudinal layer is thicker in the oesoph- 
agus and in the third intestinal region, 
whereas in the rectum both muscle layers are 
well developed. 

In the other organs and regions, the muscle 
layers are thin. In the crop (Fig. 27), in the 
stomach (Fig. 32), and in the first and second 
intestinal regions (Figs. 33, 34), the longitudi- 
nal layer is the less developed; in the fourth in- 
testinal region (Figs. 37, 38), the circular layer 
is the least developed. In the intestinal cae- 



cum (Figs. 24, 40, 41), the muscle layers are 
poorly developed and not well demarcated. 



DISCUSSION 

The light microscope observations demon- 
strated that the alimentary canal of the 
Limacoidea and Miiacidae is divisible into five 
morphologically distinct organs; oesophagus, 
crop, stomach, intestine and rectum. In the in- 
testine of the species studied here, I distin- 
guish four histologically different regions, 
whereas other pulmonates have only three in- 
testinal regions (Carriker & Bilstad, 1946; 
Moussa et al., 1983; Roldan & Garcia- 
Corrales, 1988; Boer & Kits, 1990). Walker, in 
Runham (1975) subdivided the intestine of 
Dereceras reticulatum into only three intesti- 
nal regions. He termed our fourth intestinal re- 
gion as rectum without mentioning the actual 
rectum, which is located in the palliai cavity. 
Another peculiar feature of the intestine of 
some Limacidae and Agriolimacidae is the 
presence of an intestinal caecum. This can be 
long, as in Lehmannia marginata, or short, as 
in Dereceras reticulatum and D. rodnae. The 
histological and ultrastructural features of the 
caecum imply the probably function of this 
organ. A simple columnar epithelium is pres- 
ent from the oesophagus to the third intestinal 
region and in the rectum. In the fourth intesti- 
nal region and in the intestinal caecum, how- 
ever, the epithelium is simple squamous, with 
cells showing distinct ultrastructural features 
that are characteristic of water- and ion-trans- 
porting epithelia (Leal-Zanchet, in preparation 
b).This would indicate that water and ions are 
absorbed from the faecal pellets. This was 
also suggested by Boer & Kits (1990) for 



48 



LEAL-ZANCHET 




FIG. 19. Semi-schematic drawing of part of a trans- 
verse section of the stomach (out of the ty- 
phlosoles) of Lehmannia marginata. 

FIG. 20. Semi-schematic drawing of part of a trans- 
verse section of the stomach of Lehmannia mar- 
ginata showing the leader groove. Scale bar 10 iim 




FIG. 21 . Semi-schematic drawing of part of a trans- 
verse section of the first intestinal region of Leli- 
mannia marginata. 

FIG. 22. Semi-schematic drawing of part of a trans- 
verse section of the second intestinal region of 
Lehmannia marginata. Scale bar 10 urn. 



Lymnaea stagnalis and is consistent witfi the 
findings of Deyrup-Olsen (1987), who verified 
that the distal part of the intestine of Ariolimax 
columbianus plays a significant role in os- 
moregulation. 

Most of the epithelium of the alimentary 
canal shows ciliated supporting cells. The cilia 
play a role in the transport of the food bolus 
and the faecal pellets (Roldan & Garcia- 
Corrales, 1988; Boer & Kits, 1990). In the 



stomach and in the rectum, however, there 
are distinct folds with cilia that are longer and 
very numerous. In addition, ultrastructural 
data show that these cilia have very long roots 
and are interconnected by well-developed 
basal feet on the basal bodies (Leal-Zanchet, 
in preparation b). In the stomach, such folds 
are the typhlosoles and the transversal fold, 
and in the rectum they are termed leader 
folds. The cilia of the typhlosoles, the trans- 



HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 



49 



TABLE 2. Length and width of the cell body (CB), of the nucleus (N) and diameter of the secretory cell gran- 
ules (SGR) of gland cells of the digestive tube of Limacoidea and Milacidae (urn). 



B. pallens marginata D. laeve 



D. 
reticulata m 



D. rodnae M. tenellus 



T. buda- 
pestensis 



MCI 


N 


7.5x5.3 


8.2 X 5.8 


8.5x5.7 


6.8x5.1 


8.8 X 5.3 


10.0x7.0 


8.6x5.5 




SGR 


2.0 


2.3 


2.0 


1.8 


1.7 


2.1 


1.7 


MC II 


N 


8.0x6.4 


7.5 X 5.0 


9.5 X 5.0 


8.0x5.5 


11,0x6.5 


8.8 x 7.3 


9.5x5.5 




SGR 


0.5 


0.5 


0.5 


0.5 


0.5 


0.5/2.0 


0.5 


MC III 


CB 


20.9 X 15.9 


19.3 X 15.1 


20.6 X 13.2 


18.0 x 10.9 


21.0 X 15.1 


20.3 X 15.8 


26.5 X 15.7 




N 


7.1 X 5.9 


10.4x6.8 


7.3x4.9 


8.5 X 4.6 


8.0 X 5.0 


8.4 X 6.3 


8.4 X 6.4 




SGR 


1.5 


1.4 


1.4 


1.7 


2.1 


2.3 


1.9 


MC IV 


CB 


20.4 X 13.5 


16.5 x 12.0 


19.8 x 12.8 


16.9 X 10.4 


20.2 X 14.9 


17.6 X 12.0 


19.2 X 14.7 




N 


9.3x7.6 


9.4x6.4 


10.1 x6.8 


7.6 X 5.6 


7.0x6.0 


8.9x7.3 


9.4 X 7.6 




SGR 


1.2 


0.9 


0.9 


0.9 


1.2 


0.9 


1.2 


MCV 


CB 


— 


1 1.4 X 11.9 


— 


11.8x9.5 


14.0x6.5 


14.0x6.5 


— 




N 

SGR 

CB 


— 


6.8x3.7 


— 


5.0 x 3.2 


6.5x3.8 


6.5 X 3.8 


— 


СУ 





27.2 X 17.5 





25.7 X 18.8 


23.0 X 17.5 


17.7 x 13.5 







N 


— 


12.4x6.8 


— 


14.3x6.7 


10.2x5.3 


11.3x4.8 


— 




SGR 


— 


— 


— 


— 


— 


— 


— 



TABLE 3. Frequency of mucous cells in the digestive tube of Limacoidea and Milacidae. 



B. 
pallens 



marginata D. laeve 



D. 

ret icu latum 



D. 
rodnae 



M. T. buda- 

tenellus pestensis 



Oesophagus 


1 5% 


9% 


5% 


3% 


3% 


4% 


10% 


Crop 


8% 


9% 


5% 


9% 


10% 


8% 


8% 


Stomach 


18% 


10% 


11% 


13% 


13% 


10% 


18% 


1. intestinal region 


54% 


41% 


35% 


44% 


46% 


35% 


55% 


2. intestinal region 


15% 


8% 


15% 


4% 


6% 


13% 


4% 


3. intestinal region 


22% 


18% 


19% 


16% 


21% 


11% 


27% 


4. intestinal region 


34% 


17% 


26% 


16% 


21% 


18% 


36% 


Caecum 


— 


4% 


— 


8% 


7% 


— 


— 


Rectum 


27% 


21% 


20% 


25% 


22% 


19% 


44% 



versal fold and the rectal leader folds have a 
distinct function. This would be consistent with 
the studies of Walker (1 972) on the physiology 
of the stomach of Deroceras reticulatum: he 
showed that the typhlosoles, the transversal 
fold, and the leader groove play an important 
role in the transport of fine material. No data 
were found about the physiology of the rec- 
tum. The rectal leader folds seem to be better 
suited to aid faeces transport than the usual 
folds. In the rectum of other pulmonates, the 
presence of leader folds has not yet been de- 
scribed. 

In some regions of the alimentary tract of 
the Limacoidea and Milacidae, the epithelium 
is unciliated, namely in the crop, in the fourth 
intestinal region, and in the intestinal caecum. 
The absence of cilia in the crop was also ob- 
served in other Stylommatophora (Ghose, 



1963; Rigby, 1963, 1965; Roldan & Garcia- 
Corrales, 1988). In the fourth intestinal region 
of Boettgerllla and Tandonia, the epithelium is 
completely unciliated, but in Malacolimax, 
Lehmannia and Deroceras a reduced ciliated 
area is present. According to Runham (1975), 
the variation in the presence or absence of 
cilia in the organs of the alimentary canal may 
reflect the relative importance of cilia and 
muscles for transport and mixing of food ma- 
terial. In the crop, the muscle layers alone are 
responsible for mixing food material with 
the crop juice (Runham, 1975) and also for 
transport of the food material towards the 
stomach. In the fourth intestinal region, the cil- 
iated area present in Malacolimax, Lehman- 
nia and Deroceras seems to aid faeces trans- 
port towards the rectum together with the 
muscle layers. 



50 



LEAL-ZANCHET 



TABLE 4. Distribution of gland cells in the intestine, caecum and rectum of Limacoidea and Milacidae. 





ß. 


L. 




D. reticula- 


D. 


M. 


T. buda- 




pallens 


margínala 


D. laeve 


tum 


rodnae 


tenellus 


pestensis 


1 . intestinal region 


MC II 


MC II 


MC II 


MC II 


MC II 


MC II 


MC II 






MCI 


MCI 


MCI 


MCI 


MCI 




2. intestinal region 


ICI 


ICI 


ICI 


ICI 


ICI 


ICI 


ICI 




MCI 


MCI 


MCI 


MCI 


MCI 


MCI 


MCI 


3. intestinal region 


ICI! 


ICH 


ICH 


ICH 


ICH 


ICH 


ICH 




MC III 


MC III 


MC III 


MC III 


MC III 


MC III 


MC III 




MC IV 


MC IV 

MCV 

CY 


MCIV 


MCIV 


MCIV 


MCIV 

MCV 

CY 


MCIV 


4. intestinal region 


MC III 


MC III 


MC III 


MC III 


MC III 


MC III 


MC III 




MClV 


MCiV 


MCIV 


MCIV 


MCIV 


MCIV 


MCIV 






MCV 




MCV 


CY 


MCV 








CY 




CY 




CY 




Caecum 




MC III 




MC III 


MC III 










MCIV 

MCV 

CY 




MCIV 

MCV 

CY 


MC IV 
CY 






Rectum 


MC III 


MC MI 


MC III 


MC III 


MC III 


MC III 


MC III 




MC IV 


MCIV 


MCIV 


MCIV 


MCIV 


MCIV 


MCIV 






MCV 


MCV 


MCV 


MCV 


MCV 








CY 




CY 


CY 


CY 





The presence of five mucous cell types Is 
now reported for the alimentary canal of li- 
macids and agriolimacids.Type I mucous cells 
are present in the proximal regions of the ali- 
mentary canal, such as the eosophagus, the 
crop, in parts of the stomach, and also in the 
second intestinal region. Type II mucous cells 
are found in the stomach and in the first in- 
testinal region. The mucous cells of type III, IV 
and V occur in distal regions of the canal, such 
as the third and fourth intestinal regions, the 
intestinal caecum, and the rectum. The mu- 
cous cells of type I and II are intraepithelial, 
whereas the mucous cells of type III, IV and V 
are subepithelial. The mucous cells of type V 
are absent in Boettgerilla pallens and Tan- 
donia budapestensis. 

The functional role of the mucus would be 
the lubrification of the lumen, helping in the 
transport of food and faeces, the clumping of 
food particles for the formation of the food 
bolus, the formation of the faecal string, and 
the compaction of the faecas (Carriker & 
Bilstad, 1946; Pereira & Breckenridge, 1981). 
The mucous cells of type I of the Limacoidea 
and Milacidae, the occurrence of which is lim- 
ited to the proximal parts of the tract, must be 
related to the formation of the food bolus. The 
formation of the faecal pellets that takes place 
in the distal part of the stomach (Walker, 
1972) should involve the mucous cells of type 



II. The mucous cells of the distal regions of the 
alimentary cana! — types III, IV and V — should 
be concerned with the compaction of the fae- 
ces. 

Gland cells that are termed intestinal se- 
creting cells have been described for the ali- 
mentary canal of various pulmonates (Haff- 
ner, 1924; Baecker, 1932; Walker, in Runham, 
1975; Roldan & Garcia-Corrales, 1988; Leal- 
Zanchet et al., 1990; Franchini & Ottaviani, 
1992). The intestinal secreting cells described 
by these authors are similar to the type I in- 
testinal secreting cells of the limacoids. We 
observed also another type of gland cells (I.e., 
intestinal secreting cells of type II) that are 
clearly distinguishable from intestinal secret- 
ing cells of type I. The secreting cells of type I 
and II occur in all the species studied in the 
present investigation. The occurrence of the 
intestinal secreting cells of type I and II in 
the second and third intestinal regions, re- 
spectively, and their positive reaction to pro- 
tein (Leal-Zanchet, In preparation a), indicate 
that the secretion of these cells is probably of 
an enzymatic nature and may play a role In di- 
gestion. 

The occurrence of cystic cells In the ali- 
mentary canal of gastropods has not yet been 
described, but similar cells are known in the 
salivary glands of pulmonates (Blain, 1957; 
Bani, 1964, Boer et al., 1967). In LImacldae 



HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 



51 




25 



FIG. 23. Semi-schematic drawing of part of a trans- 
verse section of the third intestinal region of 
Lehmannia marginata. 

FIG. 24. Semi-schematic drawing of part of a trans- 
verse section of the intestinal caecum of Lehmannia 
marginata. Scale bar 10 um. 



and Agriolimacidae, the secretion of the cystic 
cells Is positive to histochemical reactions for 
protein (Leal-Zanchet, in preparation a), but 
the exact role of the cystic cells is still unclear. 
The cystic cells occur in the distal regions of 
the alimentary canal of the limacids and agri- 
olimacids, except in Deroceras laeve, but are 
absent in Boettgerilla and Tandonia. 

The alimentary diet of the species studied 
in the present investigation differs widely. 
Deroceras and Tandonia are herbivorous. 




FIG. 25. Semi-schematic drawing of part of a trans- 
verse section of the fourth intestinal region of 
Leiimannia marginata. Scale bar 10 ¡.im. 



Lehmannia lives on a specialized diet of 
lichens, whereas Malacoiimax feed prefer- 
ently on fungus (Wiktor, 1973). Little is known 
about the diet of Boettgerilla pallens, but 
some data obtained in laboratory experiments 
suggest that this species is carnivorous (Leal- 
Zanchet, in press). Although the animals have 
a distinct diet, only few distinguishing anatom- 
ical and histological features were observed. 
Boettgerilla presents a mere shortening of the 
intestinal regions, a characteristic observed in 
others carnivorous slugs, such as Daude- 
bardia (Zonitidae) and Diplompharus (Rhyti- 
didae) (Wiktor, 1983; Tillier, 1989). If Boett- 
gerilla is carnivorous, the occurrence of a 
powerful protease in its digestive system 
would be expected. At present, having com- 
pleted anatomical, histological, histochemical 
(Leal-Zanchet, in preparation a) and ultra- 
structural (Leal-Zanchet, in preparation b) 
studies, we still cannot relate the different 
diets of the Limacoidea and Milacidae to their 
distinct histological features. An investigation 
of the enzymes of Limacoidea and Milacidae 
would clarify many questions. 




me II 

• \ 



FIG. 26. Cross-section of the oesophagus of Malacolimax tenellus showing a fold. Scale bar 50 iim. 

FIG. 27. Cross-section of the crop of Malacolimax tenellus. Note the abundant vacuoles with amorphous con- 
tents. Scale bar 25 urn. 



FIG. 28. Cross-section of the crop of Tandonia budapestensis showing a fold. Scale bar 25 urn. 

FIG. 29. Cross-section of the distal part of the stomach of Lehmannia margínala demonstrating the ty- 
phlosoles and the leader groove. Scale bar 80 um. 

FIG. 30. Cross-section of the stomach of Lehmannia margínala demonstrating the lining epithelium of a ty- 
phlosole. Note the strong acidophilic columnar cells and the mucous cells of type II. Scale bar 25 iim. 



HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 



53 




mcV 



nc 



cm 



Ч.- 



icil 



/ 






^■ 



mclV rnclll 



••»^ 



FIG. 31. Cross-section of the stomach of Lehmannia marginata showing the lining epithelium of the leader 
groove. The holocrine secretion of a strong acidophilic columnar cell can be seen. Scale bar 25 um. 

FIG. 32. Cross-section of the stomach wall (out of the typhlosoles) of Lehmannia marginata. Note the apoc- 
rine secretion of the columnar cells. Scale bar 25 um. 

FIG. 33. Cross-section of the first intestinal region of Lehmannia marginata showing the numerous mucous 
cells of type II. Scale bar 25 um. 

FIG. 34. Cross-section of the second intestinal region of Malacoiimax tenellus demonstrating the intestinal 
secreting cells of type I. Scale bar 25 urn. 

FIG. 35. Cross-section of the third intestinal region of Malacoiimax tenelius. The cell body of a mucous cell 
of type V can be seen. Scale bar 25 um. 



FIG. 36. Cross-section of the third intestinal region of Boettgerilia pailens. Scale bar 25 um. 




'f.y^- 4I 



mclV 

/ 



Щт* 



». ^»îb 



iu 

. уЛ se «^^^* 



38 



^ 



Г'-^ 



cm 






^*/ 



lu 
mein 



* 



ь 



% ■ / 



, g^ % 







* " Im 
cm 



it, ' , ^ж 



• ^|^l 



Cl 



^1 



42 

lu '% 



Cl 



t'* 



ce 



î*' 









^ 



,<^- % 



mclV 



.1*^5»' 



em 



# 4 



FIG. 37. Cross-section of the fourth intestinal region of Lehmannia marginata. Exceptionally, the circular layer 
lays directly below the epithelium. Note the ciliated area. Scale bar 25 um. 

FIG. 38. Cross-section of the fourth intestinal region of Boettgerilla pallens. A ciliated area is absent. Scale 
bar 25 um. 

FIG. 39. Cross-section of the fourth intestinal region of Deroceras laeve. A ciliated area can be seen. Scale 
bar 25 urn. 

FIG. 40. Cross-section of the intestinal caecum of Lehmannia marginata. Scale bar 25 ^m. 

FIG. 41 . Cross-section of the intestinal caecum of Deroceras reticulatum. Scale bar 25 um. 

FIG. 42. Cross-section of the rectum of Lehmannia marginata showing a fold. Scale bar 25 um. 



HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 



55 







FIG. 43. Cross-section of the rectum of Boettgehlla pallens showing the well developed leader folds. Cilia are 
absent outside the leader folds. Scale bar 25 цт. 

FIG. 44. Cross-section of the recturn of Dereceras laeve. SnnatI leader folds and well-developed muscle lay- 
ers can be seen. Scale bar 25 |.tm. 

FIG. 45. Cross-section of the rectum of Dereceras reticulatum. Note the numerous mucous cells of type III 
and two cell necks of mucous cells of type V. Scale bar 25 цт.[ 

FIG. 46. Cross-section of the rectum of Dereceras reticulatum demonstrating the large cell body of a cystic 
cell. Scale bar 25 цт. 

FIG. 47. Cross-section of the rectum of Malacolimax tenellus. Observe the small cell body of a mucous cells 
of type V. Scale bar 25 |.im. 



56 



LEAL-ZANCHET 



ACKNOWLEDGEMENTS 

The author wishes to thank Dr. Wolfgang 
Rähle for supervising her doctoral thesis and 
Prof. Dr. Wolfgang Maier for providing space in 
his departrлent. Thanks are also due to Dr. 
Dieter Bunke for discussion about the histol- 
ogy and for help with the historesin technique, 
and Dr. Klaus Eisler and Miss Martina 
Hohloch for assistance with the photography. 
The help of Mr. Neuri Zanchet and Miss 
Irmlind Heinze in collecting specimens is also 
gratefully acknowledged. 



RESUMO 

Estudo corлparativo da anatomía e histolo- 
gía do tubo digestivo dos Limacoidea e 
Milacidae (Pulmonata: Stylommatophora) 

Descreve-se comparativamente a anato- 
mía e histología do tubo digestivo de Deroc- 
eras laeve, D. reticulatum, D. rodnae, Leh- 
mannia marginata, Malacolimax tennelus, 
Boettgehlla pallens e Tandonia budapesten- 
sis. O tubo digestivo destes animais é com- 
posto pelo esófago, papo, estómago, intestino 
e reto. Um ceco intestinal está presente em 
D. reticulatum, D. rodnae e L. marginata. O in- 
testino pode ser subdividido em quatro re- 
gióos histológicamente distintas. Do esófago 
à terceira regiáo intestinal, e no reto, o tubo di- 
gestivo é revestido em sua maior parte por 
um epitelio cilindrico a cúbico simples. Na 
quarta regiáo intestinal e no ceco o epitelio 
apresenta-se pavimentóse a cúbico simples. 
As células epiteliais de suporte podem ser ci- 
liadas ou nao, mas apresentam sempre mi- 
crovilos. Distinguem-se também oito tipos 
celulares secretores: células mucosas do tipo 
I, células mucosas do tipo II, células mucosas 
do tipo III, células mucosas do tipo IV, células 
mucosas do tipo V, células císticas, células 
secretoras intestinais do tipo I e células se- 
cretoras intestinais do tipo II. Subepitelial- 
mente, encontram-se uma camada de tecido 
conjuntivo frouxo e duas camadas muscu- 
lares, uma longitudinal interna e outra circular 
externa. 



LITERATURE CITED 

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BAECKER, R., 1932, Die Mikromorphologie von 
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BANI, G., 1964, Osservazione sulle ghiandole sali- 
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BENNETT, H. S., A. D. WYRICK, S. W. LEE & J. H. 
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489-502. 

BOER, H. H., S. E. W. BONGA, & N. ROOYEN, 
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BOER, H. H., & K. S. KITS, 1990, Histochemical and 
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CARRIKER, M. R. & N. M. BILSTAD, 1946, 
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DEYRUP-OLSEN, I. & W.MARTIN, 1987, Osmolyte 
prosessing in the gut and an important role of the 
rectum in the land slug, Ariolimax columbianus 
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FRANCHINI, A. & E. OTTAVIANI, 1992, Intestinal 
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GARTENAUER, H. M,, 1875, Über den Darmkanal 
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GHOSE, K. C, 1963, The alimentary system of 
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HAFFNER, K., 1924, Über den Darmkanal von 
Helix pomatia L. Zeitschrift für Wissenschaftliche 
Zoologie, 121: 126-169. 

LEAL-ZANCHET A. M., J. W. THOME & J. 
HAUSER, 1990, Microanatomia e histología do 
sistema digestivo de Phyllocaulis solelformis 
(Orbigny 1835) (Mollusca: Gastropoda: 
Veronicellidae). III. Tubo digestivo (do estómago 
ao reto). Caatinga, 7: 76-104. 

LEAL-ZANCHET A. M., 1995, Vergleichende 
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Verdauungssystems limacoider und milacoider 



HISTOLOGY OF THE ALIMENTARY CANAL OF LIMACOIDEA 



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limacidae. Limacidae, Boettgerillidae, Milacidae). 
Unpublished D. Nat. Sei. Thesis, Tübingen 
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LEAL-ZANCHET A. M., About the rearing of 
Boettgerilla pallens (SIMROTH, 1912) (PUL- 
MONATA, STYLOMMATOPHORA). Malacolog- 
ical Review, in press. 

LEAL-ZANCHET A. M., Histochemical study of the 
alimentary canal of Limacoidea. In preparation a. 

LEAL-ZANCHET A. M., Ultrastructural aspects of 
the epithelium of the alimentary canal of 
Limacoidea. In preparation b. 

LIKHAREV, I. M. & A. WIKTOR, 1980, The fauna of 
slugs of the USSR and adjacent countries 
(Gastropoda terrestria nuda). Akademia Nauk 
SSSR, Leningrad, 437 pp. 

MOUSSA, T A., A. M. IBRAHIM & A. H. A. SAAD, 
1983, The histology of the alimentary tract of the 
snail Lymnaea caillaudi. Annales Zoologici, 20, 2: 
93-107. 

PEREIRA, C.R.D.&W.R.BRECKENRIDGE, 1981, 
A histophysiological study of the alimentary sys- 
tem of Achatina fúlica. (Gastropoda, Stylom- 
matophora) with particular reference to glands in 
the tract. Ceylon Journal of Science, 14(1-2): 
152-192. 

QUICK, H. E., 1960, British slugs (Pulmonata; 
Testacellidae, Arionidae, Limacidae). Bulletin of 
the British Museum, 6(3); 198-226. 

RIGBY, J. E., 1963, Alimentary and reproductive 
systems of Oxychilus cellarius (Müller) (Stylom- 
matophora). Proceedings of the Zoological 
Society of London, 141:311 -359. 

RIGBY, J. E., 1965, Succinea putris: a terrestrial 
opisthobranch mollusc. Proceedings of the Zoo- 
logical Society of London, 1 44: 445-486. 



ROLDAN, С. & P GARCIA-CORRALES, 1988, 
Anatomy and histology of the alimentary tract of 
the snail Theba pisana (Gastropoda: Pulmonata). 
l\Aalacologia, 28(1-2): 1 19-130. 

ROMEIS, В., 1989, Mikroskopische Technik. Urban 
und Schwarzenberg, München, Wien, Baltimore, 
697 pp. 

RUNHAM, N. W., 1975, Alimentary canal. Pp. 

54-104, in FRETTER V. & J. PEAKE, eds., 

Pulmonates, v. 1 : Functional anatomy and physi- 
ology. Academic Press, London. 

SIMROTH, H., 1885, Versuch einer Naturge- 
schichte der deutschen Nacktschnecken und 
ihrer europäischen Verwandten. Zeitschrift für 
Wissenschaftliche Zoologie, 42: 203-366. 

TILLIER, S., 1989, Comparative morphology, phy- 
logeny and classification of land snails and slugs 
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30 (1-2): 1-303. 

WALKER, G., 1972, The digestive system of the 
slug, Agriolimax reticulatus (MùWer). Proceedings 
of the Malacological Society of London, 40: 
33-43. 

WIKTOR, A., 1973, Die Nacktschnecken Polens. 
Arionidae, Milacidae, Limacidae (Gastropoda, 
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Naukowe, Warzawa, Krakóv, 182 pp. 

WIKTOR, A., 1983, Die Abstammung der holark- 
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Revised ms accepted 30 October 1996 



MALACOLOGIA, 1998, 39(1-2): 59-75 

MORPHOLOGY OF THE WESTERN ATLANTIC HALIOTIDAE (GASTROPODA, 
VETIGASTROPODA) WITH DESCRIPTION OF A NEW SPECIES FROM BRAZIL 

Luiz Ricardo L. Simone 

Seçào de Moluscos, Museu de Zoología da Universidade de Säo Paulo, Caixa Postal 71 72, 
CEP 01064-970, Säo Paulo, SP Brazil 

ABSTRACT 

Haliotis aurantium. new species, is described from the southeastern coast of Brazil and com- 
pared morphologically with Haliotis pourtalesii, which inhabits the Gulf of Mexico and the 
Caribbean Sea. These species differ mainly in characters of the head, epipodium, metapodium 
and digestive system. The species are also compared anatomically with other species of Haliotis 
based on published descriptions and on comparative dissections of H. lamellosa and H. tuber- 
culata. 



INTRODUCTION 

The finding of a haliotid in the western 
Atlantic excited considerable interest (Hender- 
son, 1915; Harry, 1966; Klappenbach, 1968; 
Sarasua, 1968; Merrill & Petit, 1969; Quince, 
1969; Nijssen-Meyer, 1969; Silva & Guerra, 
1981; Martinez & Ruiz, 1994), although until 
now few specimens with soft parts have been 
found (Titgen & Bright, 1985). 

All western Atlantic haliotid specimens 
have been identified as Haliotis pourtalesii 
Dall, 1881, with apparently in two disjunct 
populations, one from North Carolina to Cuba 
and other in Brazil (Rios, 1985; Titgen & 
Bright, 1985). These identifications were 
based only on shell characters, because no 
anatomical information has appeared to date. 
Photos and a brief descnption of the head- 
foot and color pattern of a specimen from the 
Gulf of Mexico were given by Titgen & Bright 
(1985). 

Specimens from the two regions were care- 
fully compared in their morphology, showing 
that, despite the similarity in their shells, the 
specimens from these regions are sufficiently 
distinct to be regarded as separated species. 

Few papers provide anatomical information 
on haliotids (e.g., Freure, 1905; Crofts, 1929, 
1 937, 1 955; Campbell, 1 965; Russell & Evans, 
1989).The present paper provides an anatom- 
ical description, as a basis for comparison be- 
tween the two western Atlantic species, as well 
as for use in a future systematic rearrange- 
ment of the family, which has about 70 species 
in the single genus Haliotis Linné, 1758 
(Abbott & Dance, 1983). 



The complex history of the discovery of H. 
pourtalesii in waters near Florida and the loss 
of its type specimen have been discussed 
elsewhere (e.g., Foster, 1946; Titgen & Bright, 
1985). Also there are misidentifications based 
on young specimens of other species (Abbott, 
1974) and based on similar species occurring 
in Pacific waters (/-/. c/a/// Henderson, 1915; H. 
roberti McLean, 1969), demanding care in lit- 
erature analysis. 



MATERIAL AND METHODS 

The specimens from Brazil are in the col- 
lection of the Museu de Zoología da Univer- 
sidade de Säo Paulo (MZSP), some of them 
collected by Instituto Oceanógrafico da Uni- 
versidade de Säo Paulo (lOUSP) in the pro- 
ject "Monitoramento Ambiental Oceánico da 
Bacia de Campos." The northern specimens 
are in the collection of the National Museum 
of Natural History (USNM) and Marine Invert- 
ebrate Museum, Rosenstiel School of Marine 
and Atmospheric Science, University of Miami 
(UMML). Those with soft parts are preserved 
in 70% ethanol. 

Two Brazilian and five northern specimens 
were available with soft parts for dissections, 
which were made using standard techniques. 
Some organs, such as the buccal mass and 
palliai organs, were dehydrated in ethanol se- 
ries, stained with carmine, fixed and cleared in 
creosote. All drawings were made with the aid 
of a camera lucida. Shells, radulae and jaws 
were also examined using SEM in the Lab- 
oratorio de Microscopía Electrónica do Insti- 



59 



60 



SIMONE 



tuto de Biociências da Universidade de Säo 
Paulo. The shells were not coated with gold. 
Odontophoral muscles were examined by di- 
rect dissection, although the jugal muscles 
were not seen in detail. The nomenclature of 
buccal musculature follows Fretter & Graham 
(1962). 

Anatomical comparison with other Halio- 
tidae is based on the literature (Fleure, 1905; 
Crofts, 1929, 1937, 1955; Fretter & Graham, 
1 962; Campbell, 1 965, digestive system; Rus- 
sell & Evans, 1989, circulatory system) and 
on comparative examination of two lots of the 
MZSP collection: MZSP 13340, Haliotis tuber- 
culata Linné, 1758, 5 specimens in 70% 
ETOH from Trieste, Italy; MZSP 28202, Halio- 
tis lamellosa Lamarck, 1822, 1 specimen in 
70% ETOH from Trieste, Italy. 

In the figures the following abbreviations 
are used: ac: anterior cartilages; af: accessory 
oesophageal fold; al: aperture of left oe- 
sophageal pouch; an: anus; ar: aperture of 
right oesophageal pouch; cm: main (right) col- 
umellar muscle; da: direct anterior radular 
tensors; dg: digestive gland; dr: direct radular 
tensor muscle; ef: efferent gill vessel; ep: epi- 
podium; ff: dorsal epipodial flap; gc: gastric 
caecum; go: gonad; hz: horizontal muscle; if: 
intermediary epipodial flap; im: intertentacular 
membrane; in: intestine; ja: jaws; la: left auri- 
cle; Ic: left columellar muscle; Ig: left gill; Ih: left 
hypobranchial gland; Ik: left kidney; Im: lateral 
protractor muscle; Ip: left oesophageal pouch; 
ma: main epipodial tentacle; mb: mantle bor- 
der; mt: metapodium; nr: nerve ring; oa: outer 
approximator muscle of cartilages; od: odon- 
tophore; oe: oesophagus; om: ommatho- 
phore; os: osphradium; pc: posterior cartilage; 
pr: pigmented region of dorsal epipodial flap; 
pv: posterior ventral radular tensor muscle; ra: 
right auricle; rd: radula; rg: right gill; rh: right 
hypobranchial gland; rk: right kidney; rp: right 
oesophageal pouch; rs: radular sac; rt: rec- 
tum; sa: sorting area; sf: pigmented multipapil- 
late tentacles surrounding ma; si: palliai slit; 
sn: snout; sr: subradular membrane; st: stom- 
ach; tc: metapodial tentacle covered with long 
cilia; te: cephalic tentacle; tm: metapodial ten- 
tacle; ts: slit palliai tentacle; ty: gastric ty- 
phlosole; vp: ventral buccal protractor muscle; 
ve: ventricle; vf: ventral epipodial flap. 

Abbreviations of institutions: MNRJ: Museu 
Nacional do Rio de Janeiro; MORG: Museu 
Oceanógrafico da Fundaçào Universidade de 
Rio Grande; MZSP: Museu de Zoología da 
Universidade de Sao Paulo; UMML: Marine 
Invertebrate Museum, Rosenstiel School of 



Marine and Atmospheric Science, University 
of Miami; USNM: National Museum of Natural 
History, Smithsonian Institution. 

SYSTEMATICS 

Haliotis aurantium, new species (Figs. 3-9, 
11-13, 18-35) 

Haliotis pourtalesii: Klappenbach, 1968: 1-2; 
Ríos, 1970: 16, pi. 1; Silva & Guerra, 
1 971 : 49-50, figs. 1-4; Rios, 1 975: 1 1 , pi. 
1 , fig. 4; Rios, 1 985: 1 0, pi. 5, fig. 35; Rios, 
1994: 22, pi. 5, fig. 39 {non Dali, 1881). 

Types: Holotype, MZSP 28201 , from type lo- 
cality; paratypes: MZSP 18482, 1 shell, 
off Ubatuba, Säo Paulo, 24°07'S 
44°06'W, 150 m depth; MZSP 19569, 2 
shells, 22°27'6"S 40°30'W, off Cabo de 
Sao Tomé, Rio de Janeiro, Brazil, 95 m 
depth (ll/ii/1969); MZSP 28391, 1 spec- 
imen, 21 °05'S 41 °1 9'W, east of Ponta do 
Ubú, Espirito Santo, Brazil, 48 m depth 
(E. С. Oliveira Fo. col., 1986). 

Type Locality: Brazil, Rio de Janeiro, off 
Campos Bay (sta. 21), 22°06'06"S 
40°08'38"W, 95 m depth (R. V. Astro- 
garoupa, 22/vii/1991). 

Diagnosis 

Minute southwest Atlantic species with un- 
pigmented head-foot and mantle; two tenta- 
cles in mantle slit; epipodial tentacles ran- 
domly arranged; pair of large epipodial 
tentacles posteriorly; pair of metapodial tenta- 
cles sometimes present; lobed snout border; 
left pouch of buccal mass covering ventral 
surface of odontophore; several pairs of lat- 
eral radular protractor muscle. 

Description 

Shell (Figs. 3-7). Auriform, fairly thin, subel- 
liptical, up to 15 mm in length, few more than 
three whorls (Fig. 4). Color of exposed areas 
from homogeneous vivid reddish orange in 
living specimens to pale yellow in eroded 
specimens. Protoconch (Fig. 5) of two whorls, 
low, sculptured by several minute, uniform, 
spiral threads. Spire small, low, submarginai, 
situated on posterior fourth of shell (Figs. 4, 
7). Aperture subelliptical, nacreous. Base of 
shell concave with some lateral torsion. 
Columella with a sulcus inside raised parietal 
margin of aperture (Fig. 6). Three to four oval 



WESTERN ATLANTIC HALIOTIDAE 



61 




FIG. 1-7; shells in SEM. (1) and (2) left and dorsal view of Haliotis pourtalesiiüSNM 833627, scales = 2 mm; 

(3) detail of middle-outer region of body whorl of Haliotis aurantium, MZSP 18482, paratype, scale = 0.5 mm; 

(4) to (7) Haliotis aurantium holotype: (4) dorsal view, scale = 2 mm; (5) detail of protoconch, scale = 0.5 mm; 
(6) ventral view, scale = 2 mm; (7) left view, scale = 2 mm. 



shell pores (tremata) open, preceded by sev- 
eral closed, all of them oval, with elevated 
margins (Figs. 4, 6, 7). Spiral sculpture of 
sharp, rather widely shaped cords, between 
which finer threads are occasionally interca- 
lated (Fig. 3). About 30 cords and threads in 
area between suture and outer margin of 



body whorl. Lateral portion of body whorl with 
three cords followed by strong, angular, pe- 
ripheral ridge or carina (Fig. 7). Immediately 
below this, three or four more cords present 
(Figs. 6, 7). Axial sculpture consisting of radi- 
ating lamellae, which roughly correspond to 
pores in their position, considerably variable, 



62 



SIMONE 



sometimes missing. Minute uniform axial 
cords between spiral cords occasionally pre- 
sent. Surface with very fine growth lines. No 
periostracum apparent. 

Head-Foot. Head somewhat protruding 
(Fig. 21). Tentacles stubby, short, broad, cov- 
ered with long cilia, pigmented by regular pale 
brown, successive transverse bands (Fig. 21 ). 
All other structures without pigment. 
Ommatophore well developed, in outer basal 
region of tentacles (Figs. 18, 19, 21), with 
dark, vesicular, opened eyes. Intertentacular 
membrane a semi-transparent, thin flap (Figs. 
21, 26, 27) between the two cephalic tenta- 
cles, covering anterior region of snout and 
inner region of tentacles (Fig. 21). Snout well 
developed (Figs. 21, 25), cylindrical, broad, 
with irregular ventral margin. Foot large, about 
same size as shell aperture (Fig. 19), without 
pigment. Epipodium with many lobed tenta- 
cles (Figs. 19, 22, 23), without pigment, uni- 
form in size, some of them covered with long 
cilia, apparently without special organization 
(Fig. 22); in posterior extremity of epipodium 
two epipodial tentacles larger and longer (Fig. 
23), and a median area without tentacles (Fig. 
23). In the holotype, a pair of long metapodial 
tentacles present on posterior border; dis- 
sected paratype without this structure. Main 
(right) columellar muscle very large, circular in 
section (Fig. 18). Secondary (left) columellar 
muscle very small (Fig. 25: Ic). 

Mantle border. Trifolded and simple, without 
pigment. Slit deep (Figs. 18, 20), with two ten- 
tacles covered with long cilia, one on the left- 
anterior border and other on the right-poste- 
rior border of slit (Figs. 18, 20). 

Palliai cavity. Short, about half of body whorl 
(Fig. 20). Gills short, bipectinate, right gill 
shorter than left (Fig. 20). Afferent gill vessel in 
base of gill's insertion. Efferent vessel be- 
tween two flaps of each gill leaflet, inserting in 
gill sub-terminally, anterior to posterior ex- 
tremity of gill (Fig. 20). Hypobranchial glands 
present, left larger, with several transverse, 
uniform furrows; right much smaller, with three 
oblique furrows. Both hypobranchial glands 
situated at left of slit (Figs. 18, 20, 24). Rectum 
between both hypobranchial glands, slightly 
free in posterior half of palliai cavity; anus 
papillated near posterior extremity of slit (Figs. 
20, 24). 

Circulatory and excretory systems. Kidneys 
and pericardium situated ventrally, in mid-left 
side of animal just behind palliai cavity (Fig. 
24). Left kidney short, broad, with a short 





FIG. 8-13. Jaws and radula in SEM: (8) jaws of 
Haliotis aurantium. scale = 200 цт; (9) detail of 
same, scale = 20 uin (10) detail of central region 
of jaws of Haliotis pourtalesii. scale = 50 um; 

(11) radula of Haliotis aurantium. scale = 100 ¡.im; 

(12) detail of same, central region, scale = 50 цт; 

(13) detail of Fig. 11, marginal region, scale = 
50 um. 



WESTERN ATLANTIC HALIOTIDAE 



63 





ш^^л 


Í 




й 


\ 




i 


5 


^^Ш¥' 




14 



15 



16 



17 



FIGS 14-17. Radulae of Haliotis pourtalesii in SEM: (14) USNM 833627, scale = 100 urn; (15) UMML 
30-8376, scale = 100 um; (16) detail of Fig. 14, marginal region, scale = 100 um; (17) the same, central re- 
gion, scale = 100 ¡.im. 



papillated nephrostome in ventral base of rec- 
tum. Right kidney long, thin, lying right margin 
of palliai cavity (Fig. 24), its nephrostome a 
longitudinal slit in its anterior extremity. 
Ventricle large, surrounding intestine; left auri- 
cle anterior to ventricle and right auricle ven- 
tral to it. 

Digestive system. Mouth in snout, covered 
internally by pavement-shaped papillae (Fig. 
28). Jaws two small plates (Figs. 8, 28), with 
rounded borders, situated in mid-dorsal region 
of mouth; median and anterior region of each 
plate with a small, sharp pointed projection 
(Figs. 8, 9). Buccal mass very large, complex; 
odontophore surrounded by two glandular oe- 
sophageal pouches (Figs. 25-27), both with 
inner surface covered by many tall villiform 
papillae (Fig. 28). Left pouch broad, short, cov- 
ering ventral and lateral-left surfaces of odon- 
tophore. Right pouch narrow, long, beginning 
at right and running obliquely (Figs. 26, 27). 
Both pouches open in ventral-anterior region 
of oesophagus in two separated, tall, ring-like 
folds; a third short accessory fold also at this 
position (Fig. 28: af). Odontophore short, with 
very long radularsac, extending behind buccal 
mass and terminating near stomach (Fig. 25). 
Radula (Figs. 11-13); rachidian teeth broad, 



short, each with a large curved terminal cusp 
and two lateral bolsters (Fig. 12); first lateral 
tooth with triangular base and small rounded 
cusp; second lateral tooth long, with a some- 
what rectangular base and a lateral-terminal, 
hook-like cusp; third lateral tooth the largest, 
with a long, irregular, curved base, and a large, 
long, sharp cusp, of almost the same length as 
base; fourth and fifth lateral teeth similar to 
third, but narrow; fifth narrowest, sharply 
pointed. About 32 pairs of marginal teeth per 
row (Fig. 13), with a long stalk; main cusp 
rounded, curved, spoon-like, flanged on each 
side by two small, sharp secondary cusps; 
marginal teeth gradually decrease in size lat- 
erally. Odontophore muscles (Figs. 31-35) 
consisting of: pair of ventral buccal protractor 
muscles, with their origin in ventral-lateral 
inner surface of peribuccal wall, and their in- 
sertion in ventral lateral region of posterior car- 
tilages (Figs. 31 , 32: vb); pair of direct radular 
tensor muscles, their origin in mid-ventral re- 
gion of posterior cartilages and insertion on 
lateral angles of ventral edge of radula (Figs. 
31, 33, 34: dr); pair of muscles as outer ap- 
proximator of cartilages, their origin in anterior 
surface of posterior cartilages and insertion in 
outer lateral surface of anterior cartilages 



64 



SIMONE 




FIGS. 18 to 20. Haliotis aurantium n. sp. anatomy: (18) topography of the holotype specimen in dorsal view; 
(19) the same in ventral view, scale = 2 mm; (20) palliai organs, mantle deflected, inner-ventral view, scale = 
1 mm. 



(Figs. 33-35: oa); pair of small posterior ven- 
tral radular tensor muscles, their origin in ven- 
tral inner surface of peribuccal wall and inser- 
tion in mid-ventral region of radular sac (Figs. 
31, 32: pv); several pairs of small lateral pro- 
tractor muscles, their origin in dorsal inner sur- 
face of peribuccal wall and insertion in dorsal- 
mid surface of radula (Fig. 32; Im); pair of direct 
anterior radular tensors, their origin in ventral- 
dorsal surface of posterior cartilages and in- 
sertion in lateral borders of sub-radular mem- 
brane and ventral surface of radula up to mid 



line, in a "M" shape (Fig. 33: da); and horizon- 
tal muscle, uniting ventral edge of both anterior 
cartilages (Figs. 31, 35: hz). Anterior odon- 
tophoral cartilages long, flattened, anteriorly 
sharp, posteriorly broad, with rounded borders 
(Fig. 35). Posterior odontophoral cartilages 
very short, elliptic, situated in outer posterior 
extremity of anterior catilages (Fig. 35). 

Oesophagus short, flattened tube (Figs. 25, 
26), with about eight internal longitudinal folds 
(Fig. 28). Stomach very large, U-shaped, near 
mid line in posterior region of animal (Fig. 18). 



WESTERN ATLANTIC HALIOTIDAE 



65 



22 




FIG. 21 to 24. Haliotis aurantium n. sp. anatomy: (21) detail of head, frontal view, mantle removed, scale = 1 
mm; (22) detail of left-posterior fourth of epipodium, scale = 1 mm; (23) detail of posterior extremity of foot, 
ventral view, scale = 1 mm; (24) semi-diagrammatic drawn of cleared pericardial and nearby structures, ven- 
tral-right view, scale = 1 mm. 



Oesophageal branch of stomach ventral, con- 
ical, with a very small caecum (Fig. 26). 
Internally oesophagial branch of stomach with 
mosaic of low, irregular folds near oesophagus 
opening (Fig. 30), where three longitudinal 
folds begin, two of them contouring posterior 
extremity of stomach, becoming weaker pos- 
teriorly; the third fold becoming larger and run- 
ning to intestinal branch of stomach where it 
becomes weaker (Fig. 30: ty). Some radial 
muscle fibers in stomach wall originating be- 
tween oesophageal and intestinal branches of 
stomach (Fig. 30: Ic) and inserting in small left 
columellar muscle (Fig. 25: Ic). Intestinal 
branch of stomach dorsal, conical, larger than 
oesophageal branch (Fig. 26); two typhlosoles 
running alongside gastric intestinal branch 
from caecum into intestine, one of them pre- 
senting in its mid region a series of oblique 
folds, differentiating a small sorting area (Fig. 
29: sa). Other regions of stomach inner surface 



smooth, covered by thin greenish cuticle (Figs. 
29, 30). Intestine long, with thin transparent 
walls, running near right side of head, when it 
twists and returns to posterior region near 
stomach (Figs. 25, 27); in this posterior region, 
it is sigmoid, running through pericardium (Fig. 
24) and exiting into palliai cavity (Figs. 20, 25, 
26). Intestine and stomach full of gravel. 

Digestive glands large, green, with mosaic 
of irregular brown spots on its surface (Figs. 
1 8, 25) and occupying visceral mass ventral to 
gonad, surrounding stomach (Fig. 25). 

Genital system. Very large ovary occupying 
all of spire and part of body whorl (Figs. 18, 
25: go), pale cream in color. Ovary with three 
lobes (Fig. 18), one within spire, one posterior 
to main columellar muscle, the third in left side 
of this muscle (Fig. 18). Oviduct, which proba- 
bly runs within right kidney, not seen. Ventral 
limit of gonad at the digestive gland and stom- 
ach (Fig. 25). Male not examined. 



66 




FIG. 25 to 29. Haliotis aurantium n. sp. anatomy: (25) cephalic organs and visceral mass, ventral view, foot 
and mantle removed; (26) extracted head and digestive ducts, right-dorsal view; (27) detail of anterior region 
of digestive system, dorsal view, head tegument partially removed; (28) snout, buccal mass and esophagus 
opened longitudinally, ventral view; (29) stomach opened longitudinally in intestinal branch. Scales = 1 mm. 



WESTERN ATLANTIC HALIOTIDAE 



67 



Nervous system. Only circum-oesophageal 
region examined, agreeing closely with that 
described by Fleure (1905) and Crofts (1929) 
for Haliotis tuberculata. 

Measurements (respectively length in mm, 
width in mm, number of whorls, of opened 
pores, of closed pores). Holotype, MZSP 
28201: 12.9 by 8.7, 3.2, 4, 14; Paratypes: 
MZSP 18482: 15.0 by 10.5, 3.2, 3, 16; MZSP 
19569: 13.2 by 9.0, 3.0, 4, 13 and 7.2 by 5.2, 
2.1,4, 8. 

Habitat. From 77 to 150 m depth, on gravel, 
generally associated with Laminaria sp. 

Etymology. The specific name refers to the 
orange color of the external shell surface 
(Latin, aurantium). 

Material Examined. Types. 

Material Available. BRAZIL; Amapá: MORG 
1 5000, 1 shell, off Cabo Orange, 1 00 m (R. V. 
Almirante Saldanha, 30/xi/1988). Espirito 
Santo; MNRJ-HSL 6603, 2 specimens, off 
Vitoria (lost); MORG 15930, 1 shell, off 
Vitoria, 87 m (R. V. A. Saldanha, 24/iv/1969); 
MNRJ 3577, 20 shells, 20°37'05"S 
39°59'00"W, 87 m (R. V. A. Saldanha, sta. 
DHN 2027, 24/ÍX/1971). Rio de Janeiro: 
MNRJ 3554, 14 shells, 21°56'05"S 
40°07'00"W, 77 m (R. V. A. Saldanha, sta 
DHN 2012, 1 1/ix/l 969); MNRJ 3578, 4 shells, 
off Cabo Frio, 23°05'00"S 40 05'00"W, 111m 
(R. V. A. Saldanha, sta. DHM 2168); MORG 
15931, 1 shell, off Cabo Frio, 90 m (R. V A. 
Saldanha, 9/ix/1969); MORG 26226, 1 shell, 
off Cabo Frio (R. V. A. Saldanha, x/1986, on 
Laminarla); MORG 15929, 1 shell, off Cabo 
de Sao Tomé, 77 m (R. V. A. Saldanha, 
ll/iv/1969). Rio Grande do Sul; MORG 
17467, 1 Shell, off Conceiçào, 126 m (R. V. 
Mestre Jerónimo, 2/x/1972). 



Haliotis pourtalesHDaW, 1881 (Figs. 1, 2, 10, 
14-17, 36-45) 

Haliotis (Padollus) Pourtalesll Dali, 1881: 79 
[Gulf Stream near Florida Reefs, 180 m 
(31/iii/1869)] (described from memory). 

Haliotis pourtalesll: Dall, 1889: 168 
Henderson, 1911: 81 [neotype]; Cooke 
1914: 103; Henderson, 1915: 660, pis 
45, 46; Smith, 1937: 78, pi. 29, fig. 3 
Foster, 1946: 38-40, pi. 22, figs. 1, 2 
Parker, 1960; Harry, 1966: 207-208, pi 
30; Jung, 1968; Sarasua, 1968: 1-8, figs 
1, 2; Merrill & Petit, 1969: 117; McLean 
1969: 115; Guice, 1969: 140; Abbott 



1974: 18, fig. 30; Titgen & Bright, 1985: 
1 47-1 52 figs. 1 , 2; Abbott & Dance, 1 983: 
19, fig.; Ode, 1986: 69-73; Martinez & 
Ruiz, 1994:63-64, figs. 1-2. 

Type: Neotype USNM 271601 [3 miles off 
Sand Key, Florida, 49 m, 1913] 

Diagnosis 

Minute northwest Atlantic species with pig- 
mented epipodium and metapodium; three 
tentacles in mantle slit; epipodial tentacles 
arranged in three layers around well-devel- 
oped (main) tentacles; without large epipodial 
tentacles posteriorly; without metapodial ten- 
tacles; snout border papillated; ventral surface 
of odontophore free of pouches; only one pair 
of lateral radular protractor muscles. 

Description 

Shell. Figs. 1,2. 

Head-foot. Head somewhat protruding (Fig. 
39). Tentacles long, narrow, covered with long 
cilia; pigmented by regular dark brown, suc- 
cessive, well-spaced transverse bands and a 
mid longitudinal band (Fig. 39). Dark brown 
spots abundant in dorsal and ventral epi- 
podium faces and dorsal face of metapodium, 
scanty in metapodial sole. Ommatophore well 
developed, on outer basal region of tentacles, 
with dark, vesicular, open eyes (Figs. 36, 37, 
39). Intertentacular membrane semitranspar- 
ent, thin, covering anterior region of snout and 
inner region of cephalic tentacles (Fig. 39). 
Snout well developed, cylindrical, broad, with 
regular small, abundant papillae on its ventral 
border (Fig. 39). Foot large, about same size 
as shell aperture (Fig. 37). Epipodium with 
many tentacles (Fig. 37) arranged as follows 
(Fig. 41):(1) a dorsal flap (ff) fringed by flat- 
tened, polytomic tentacles in a uniform zigzag 
pattern; regions nearest foot with dark pig- 
ment in dorsal and ventral faces (pr), other re- 
gions white; (2) intermediary flap (if) with con- 
spicuous, large, white, sharp tentacles (called 
"main" tentacles), covered with long cilia (ma); 
bases of these main tentacles, which are ven- 
tral to pigmented region of superior flap (pr), 
surrounded by two (one on each side) large, 
multipapillated, dark-brown colored tentacles 
(sf); between these structures, many other 
short tentacles, with rounded tips, without pig- 
ment nor evident cilia; (3) ventral flap (vf) ex- 
tremely rich in tentacles, some of them longer 



68 



SIMONE 




FIG. 30 to 35. Haliotis aurantium n. sp. anatomy: (30) oesophageal branch of stomach opened longitudinally; 
(31) ventral view of odontophore; (32) dorsal view of same; (33) ventral view of radular ribbon and subradu- 
lar membrane showing the "M" in shape insertion of "da"; (34) lateral-right view of odontophore, direct ante- 
rior radular tensor muscle (da) partially sectioned; (35) dorsal view of odontophore with part of its muscles 
removed, right anterior cartilage deflected. Scales = 1 mm. 



(tc), sharp, covered with long cilia, sinnilar to 
but smaller than main tentacles; other tenta- 
cles short, without pigment, with rounded tips 
without evident cilia. Epipodium on each side 
begining near snout and ending at posterior 
extremity of foot, where it unites with metapo- 
dial sole (Fig. 40); practically no region without 
tentacles. Number of main epipodium tenta- 
cles In each side from 10 to 1 2. Main columel- 
lar muscle very large, circular in section (Fig. 



36). Secondary (left) columellar muscle very 
small, with some fibers attached to mid wall of 
stomach (Fig. 42: Ic). 

Mantle border. Trifolded, simple, depig- 
mented. Silt deep, with three sharp tentacles 
covered with long cilia, two of them about mid 
region of the slit (one in each side), and the 
third in posterior extremity of slit (Figs. 36, 38). 

Palliai cavity. Short, about half of body whorl 
(Figs. 36, 38). Gills somewhat long, bipecti- 



WESTERN ATLANTIC HALIOTIDAE 



69 




FIG 36 to 38. Haliotis pourtalesii anatomy; (36) topography of the specimen USNM 833627 in dorsal view, 
scale = 2 mm; (37) same in ventral view, scale = 2 mm; (38) pallia! organs, mantle deflected, inner-ventral 
view, scale = 1 mm. 



70 



SIMONE 




FIG. 39 to 42. Haliotis pourtalesii anatomy: (39) detail of head, frontal view, mantle removed, scale = 1 mm; 
(40) detail of posterior extremity of foot, ventral view, scale = 1 mm; (41) detail of left-posterior fourth of 
epipodium, scale = 1 mm; (42) cephalic organs and visceral mass, ventral view, snout opened longitudinally, 
scale = 1 mm. 



nate; right gill somewhat shorter than left (Fig. 
38). Afferent gill vessel in base of gill's inser- 
tion. Efferent vessel between two flaps of each 
gill leaflet. Inserting in gill sub-terminally, an- 
terior to posterior extremity of gill. Hypo- 
branchial glands present, left very large (Fig. 
38), with about 18 transverse, successive, 
uniform folds; right smaller, with two oblique, 
somewhat curved folds (Figs. 36, 38). Both hy- 
pobranchial glands at left of slit (Fig. 38). 



Rectum lying between hypobranchial glands, 
slightly free in posterior half of palliai cavity; 
anus papillated, opening in posterior third of 
slit (Fig. 38). 

Circulatory-excretory systems. Kidneys and 
pericardium in posterior left side of animal, 
just behind palliai cavity. These structures are 
very similar to those of the preceding species 
(Figs. 24, 38). 

Digestive system. Mouth in snout, covered 



WESTERN ATLANTIC HALIOTIDAE 



71 



internally by smooth walls (Fig. 42). Jaws two 
somewhat large plates (Fig. 42), brown, in 
mid-dorsal region of buccal cavity, lateral and 
anterior border with a sharp edge, median-an- 
terior region with a sharp projection (Fig. 10). 
Buccal mass large, complex; odontophore 
(Fig. 42) surrounded, except in its ventral re- 
gion, by two glandular pouches, both with 
inner surface covered by many tall, villiform 
papillae (Fig. 43). Left pouch broad, short, 
covenng left side of odontophore. Right pouch 
narrow, long, running obliquely from right side 
of odontophore. Both pouches opening in ven- 
tral-anterior region of oesophagus in sepa- 
rate, tall, ring-like folds, that of left chamber 
more anteriorly (Fig. 43). Radular sac very 
long, running close to and attached to dorsal 
region of buccal mass and oesophagus (Fig. 
42). Radula (Figs. 14-17): similar to that of 
preceding species, except for the marginal 
teeth, which have a much longer, sharp cen- 
tral cusp (Fig. 16); and in being more abun- 
dant, with about 40 pairs per row (Figs. 14, 
15). Odontophore similar to that of preceding 
species (Figs. 44, 45), including cartilages 
and intrinsic muscles; except lateral protractor 
muscle of radula (Fig. 45: Ip), which in H. pour- 
talesii is a single, larger pair. 

Oesophagus short, flat (Fig. 42), with about 
ten internal longitudinal folds (Fig. 43). 
Stomach very large, U-shaped; walls irregu- 
lar, with two ventral (oesophageal) and three 
dorsal (intestinal) chambers and a well-devel- 
oped, narrow spiral caecum in right dorsal re- 
gion of stomach, with about one whorl (Fig. 
36: gc). Stomach and intestine of studied 
specimen with a large quantity of clear gravel 
and organic materials including foraminiferan 
shells and unidentified bristles. 

Intestine, rectum and digestive gland char- 
acters similar to those of preceding species 
(Fig. 42). 

Genital system. Gonad with only two lobes: 
a lobe within spire and another just posterior 
to main columellar muscle (Fig. 36). Ventrally, 
this gland terminates at the digestive gland 
and stomach (Fig. 42). 

Measurements (respectively length in mm, 
width in mm, number of whorls, of opened 
pores, of closed pores). USNM 833627: 17.8 
by 12.5, 3.2, 6, 1 8. UMML 30-8376: a) 20.1 by 
13.0,3.1,5, 18; b) 17.6by 11.3, 3.0, 5, 17; c) 
9.9 by 7.0, 2.5, 5, 12; d) 21.6 by 15.0, 3.2, 5, 
19; e) 19.6 by 13.0, 3.0, 5, 17; f) 13.7 by 9.9, 
3.0,6, 16; g) 12.9 by 9.1, 2.3, 5, 14. 

Habitat. Depth from 50 to 160 m. In the lit- 
erature, the habitat is referred to as bottoms 



with rocks, stones sand and shell debris, or 
reef (Nijssen-Meyer, 1969). 

Material examined: USNM 833627, 1 spec- 
imen, U.S.A., Gulf of Mexico, off Florida, 
25°16'55"N, 83°37'47"W, 74 m depth 
(15/viii/1984). UMML 30-8376, 4 specimens 
+ 5 shells, off VENEZUELA, 11°03'N 
65°59'W, 69-1 55 m (R. V. Pillsbury sta. P-736, 
22/VÍÍ/1968). 



DISCUSSION 

Haliotis pourtalesii and H. aurantium are 
atypical Haliotidae. Generally, haliotids are 
large gastropods, over 150 mm in length, 
whereas both these species are less than 25 
mm. Haliotids generally are common and 
occur in shallow waters, whereas these 
species are rare and found in deep water 
(slope). However, both have the same general 
anatomical characters of the family, modified 
due to miniaturization. 

Haliotis aurantium differs from H. pourtale- 
sii in having: (1) a smaller size; (2) only two 
tentacles on the mantle slit (not three); (3) 
only transverse bands in the cephalic tenta- 
cles (without a longitudinal band); (4) foot 
without pigment; (5) epipodial tentacles of a 
simpler structure (see below); (6) a pair of 
larger tentacles on the posterior extremity of 
the epipodium and a proportionally large area 
with out tentacles posteriorly; (7) a pair of 
large tentacles on the posterior extremity of 
the metapodium sometimes present (absent 
in paratype) (Fig. 23: tm); (8) right gill much 
shorter than the left (in H. pourtalesii both gills 
are about the same size and are proportion- 
ally longer than in H. aurantium); (9) left hypo- 
branchial gland with three folds (not two); 
(10) right hypobranchial gland proportionally 
smaller and with weaker transverse folds; (11) 
pericardial structures situated more anteri- 
orly; (12) snout bordered by lobes (without 
small papillae); (13) marginal teeth with 
rounded main cusp (those of H. pourtalesii a'ce 
sharp); (14) left pouch of the buccal mass cov- 
ering the ventral surface of the odontophore 
(H. pourtalesii has this region free); (15) sev- 
eral pairs of small lateral protractor muscles of 
the radula (not one large pair); and (16) kid- 
neys and pericardium more ventrally placed. 

Both species show considerable shell vari- 
ation, from specimens with well-developed 
axial ridges to specimens lacking them (e.g., 
the figures of Sarasua, 1968). There is also 
variation in the number of spiral ridges, which 




FIG. 43 to 45. Haliotis pourfa/es/7 anatomy: (43) anterior region of digestive system, odontophore removed, 
left pouch (Ip) and oesophagus opened longitudinally; (44) ventral view of odontophore; (45) same in dorsal 
view. Scales = 1 mm. 



apparently increases with shell growth. Be- 
tween the spiral ridges, shells sometimes 
have delicate axial and uniform threads (Fig. 
3). Shared shell characters of these species 
are small size (up to 25 mm), the reddish-or- 
ange color of the exposed areas, and the 
prominent borders of the tremata. A possible 
difference between the species is the number 
of open pores; in specimens of H. aurantium, 
they varied from 3 to 4, whereas H. pourtale- 
sii had from 5 to 6 open pores. Analysis of 
more specimens is necessary to establish 
whether this is consistent. Comparative exam- 
ination of available shells, as well as those fig- 
ured in the literature, no shell character exclu- 



sive of a species was found. Thus, it is difficult 
to separate them using only the shell, al- 
though, if a specimen is found from North 
Carolina to the Caribbean Sea it is probably 
H. pourtalesii, whereas if collected in Brazil 
(from Espirito Santo to Rio Grande do Sul), it 
is probably H. aurantium. This criterion was 
used in the synonymic list, but the anatomical 
study of specimens from all localities was not 
undertaken and perhaps examination of addi- 
tional specimens could modify this concept. In 
both areas, a considerable number of speci- 
mens were found. Between these areas shells 
have been recorded from three localities: off 
Surinam (Nijssen-Meyer, 1969), off Para 



WESTERN ATLANTIC HALIOTIDAE 



73 



River (Rios, 1975) and off Maranhâo (Kempf 
& Matthews, 1968). Although this could be 
due to transportation, further sampling in 
these areas should clarify the distribution of 
these taxa. Dr. Mello (Museu de Malacologia 
of the University of Pernambuco), has not ob- 
tained haliotids from dredge samples in the 
northeastern coast of Brazil (pers. comm.). 

The epipodial tentacles of H. aurantium 
(Fig. 22) differ from those of H. pourtalesii 
(Fig. 41) in being entirety unpigmented and in 
having no specific arrangement around the 
main tentacles. The characters of the epipo- 
dial tentacles of H. pourtalesii have some sim- 
ilarity to those of the Mediterranean H. lamel- 
losa and H. tuberculata, (pers. obs), but these 
differ from H. pourtalesii in having only two 
epipodial tentaculated flaps, the main tenta- 
cles inserting dorsally in the dorsal flap; the 
ventral flap has a similar organization to the 
dorsal one, but its main tentacles are smaller, 
ventrally inserted, and situated between the 
main tentacles of the dorsal flap. Haliotis tu- 
berculata has a practically straight epipodium, 
with three or four small undulations between 
the main tentacles (Crofts, 1929: pi. I); H. 
lamellosa, in contrast, has two strong undula- 
tions between the main tentacles. Epipodial 
tentacle characters have been used in hali- 
otids by Owen, et al. (1971) for separating 
seven eastern Pacific abalone species, and 
even their hybrids. Using the good figures of 
that paper, it is clear that the species studied 
herein differ considerably from those taxa. 

Haliotis aurantium and H. pourtalesii differ 
anatomically from H. tuberculata (Fleure, 
1905; Crofts, 1927, 1937, 1955; person, obs.) 
and from H. lamellosa (pers. obs.) in several 
characters: the cephalic tentacles possess 
spots (not of uniform color); the intertentacu- 
lar membrane simple and free in its lateral 
margins (H. lamellosa has minute lobes in lat- 
eral margins, and in H. tuberculata the lateral 
regions are attached to omatophores); as well 
as the other characters of epipodial tentacles 
(noted above); the epipodium of Atlantic 
species begins abruptly near the snout (in the 
Mediterranean species, there is a coiled ex- 
pansion in each side, which partially covers 
the snout); gill proportionally shorter and with 
fewer leaflets; hypobranchial glands propor- 
tionally smaller and simpler (those species 
have strong and tall folds, H. pourtalesii has 
low folds and H. aurantium only furrows); rec- 
tum only covered by tegument (both Medi- 
terranean species have the posterior region of 
the rectum covered on both sides by the hy- 



pobranchial glands); anus long and papilliform 
(H. tuberculata and H. lamellosa have a short, 
broad anus); stomach shorter with clear de- 
limitation; and gastric spiral caecum much 
shorter (which also differentiates it from H. 
cracherodii, see Campbell, 1965). With regard 
to the tentacles of the mantle slit, H. pourtale- 
sii, H. tuberculata and H. lamellosa are similar 
in having three tentacles in somewhat the 
same disposition; H. aurantium has only two 
(there is no tentacle situated just in angulated 
posterior extremity of slit). 

The auriform shell with tremata, the com- 
plex tentaculate epipodium and the lack of an 
operculum, are known synapomorphic char- 
acters of the Haliotidae within the Vetigas- 
tropoda. At least three additional synapomor- 
phies are offered here: (1) the insertion in the 
stomach of some fibers of the right retractor 
muscle, (2) the insertion in a "M" shape of the 
direct radular anterior muscle, and (3) the in- 
tertentacular membrane of the head ("head 
pleat" of Crofts, 1927). 

Schremp (1981: 1125, pi., fig. 1) called a 
Pliocene haliotid found in the Imperial 
Formation of California Haliotis pourtalesii. 
Because this identification is based only on 
the shell, this specimen might instead be the 
Pacific Haliotis robe rti McLean, 1969, consid- 
ered a synonym by that author. 



ACKNOWLEDGMENTS 

I thank Dr. Airton S. Pararam and Cyntia 
Miyaji, lOUSR and Dr. Alvaro Migotto, Centro 
de Biología Marinha, USP, for the specimen of 
Haliotis aurantium with soft parts; Tyjuana 
Nickens and Mike Sweeney, USNM, and Dr. 
José H. Leal, Rosenstiel School of Marine and 
Atmospheric Science, University of Miami, for 
the loan of H. pourtalesii specimens. For 
search of haliotids in the collection, I am grate- 
ful to Dr. L. С F Alvarenga, Museu Nacional da 
Universidade Federal do Rio de Janeiro; Dr. 
Rosa L. S. Mello, Museu de Malacologia, 
Universidade Federal Rural de Pernambuco; 
Dr. Eliezer С Rios, Museu Oceanograáfico da 
Fundaçâo Universidade do Rio Grande; Yae R. 
Kim, American Museum of Natural History, 
New York; Dr. Kenneth J. Boss, Museum of 
Comparative Zoology, Cambridge; Edward 
Gilmore, Academy of Natural Sciences, Phila- 
delphia; and John Slapcinsky Field Museum 
of Natural History Chicago. For helping SEM 
exams I thank Marcio V. Cruz and Enio Mattos, 
Instituto de Biociências, USP I specially thank 



74 



SIMONE 



also Dr. Winston F. Ponder, Australia, for valu- 
able revision on manuscript. 



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RIOS, E. C, 1994, Seashells of Brazil, second edi- 
tion. Fundaçâo Universidade do Rio Grande, Rio 
Grande, 368 pp., 113 pis. 

RUSSELL, С W. & B. K. EVANS, 1989, Cardio- 
vascular anatomy and physiology of the black-lip 
abalone, Haliotis ruber Journal of Expehmental 
Zoo/ogfy. 252:105-117 

SARASUA, H., 1968, Primer hallazgo del género 
Haliotis (Mollusca: Gastropoda) en Aguas Cu- 
banas. Poeyana (A), 55:1-8 

SCHREMR L. A., 1981 , Archeagastropoda from the 
Pliocene Imperial Formation of California. Jour- 
nal of Paleontology 55: 1 1 23-1 1 36 

SILVA, S. В. & О. GUERRA, JR., 1971, Uma nova 
localizaçâo geográfica para Haliotis pourtalesii 



WESTERN ATLANTIC HALIOTIDAE 75 

Dali 1881 (Mullusca, Gastropoda, Haliotidae). distribution and ecology of the Western atlantic 

Arquivos do Museu Nacional, 54:49-50, ^p\. abalone, Haliotis pourtalesii Dali 1881 (Mol- 

SMITH, M., 1945, East coast marine stiells. Ed- lusca: Gastropoda). Northeast Gulf Science, 7: 

wards Brothers Inc., Ann Harbor, Michigan, vii + 147-152 

TITGEn'Î' R. H^& T J. BRIGHT 1985, Notes on the Received ms. accepted 3 March 1997 



MALACOLOGIA, 1998, 39(1-2): 77-81 

HISTOCHEMICAL AND ULTRASTRUCTURAL IDENTIFICATION OF BIPHASIC 

GRANULES IN THE ALBUMEN SECRETORY CELLS OF ARION SUBFUSCUS 

(GASTROPODA, PULMONATA) 

BENJAMIN J. GOMEZ\ ANA M. ZUBIAGA^, M. TERESA SERRANO^ & EDUARDO ÁNGULO^ 

ABSTRACT 

Two types of globules, differing in size, structure, and nature, are demonstrated within each se- 
cretory cell of the albumen gland of Arion subfuscus. Large secretory globules, with a homoge- 
neous granular content, are rich in proteins and carbohydrates, with the protein-reacting groups 
probably partially masked by intimate junction with the polysaccharides. Smaller secretory glob- 
ules lie inside the large ones and are more electron-lucent and foam-like in substructure, con- 
taining mostly glycosaminoglycans but also proteins. Lectin-sugar specificities show that the car- 
bohydrates synthesized by the gland are mainly galactogen, but other neutral polysaccharides, 
such as glycogen, are also present in the secretory vesicles. The reactions of each type of glob- 
ule to the lectins are also very different. 

Key words: albumen gland, lectin histochemistry, ultrastructure, Arion subfuscus. 



INTRODUCTION 

The albumen gland is a large female ac- 
cessory gland that surrounds the distal part of 
the ovotestis duct, enclosing the carrefour 
system and the most proximal portion of the 
spermoviduct in stylommatophorans. It is 
composed of a large number of secretory 
branched tubuies, which all open into the slit- 
like central lumen of the gland (Tompa, 1 984). 
This gland synthesizes the perivitelline fluids 
of the molluscan egg, which is composed 
mainly of galactogen (Bayne, 1967; Meenak- 
shi & Scheer, 1968), a polysaccharide of 
galactose (Duncan, 1975). Besides galacto- 
gen, some proteins mineral salts, and mono- 
saccharides are present in the albumen gland 
secretions (Runham, 1988). 

The tubules of the albumen gland in gas- 
tropods contain two cell types — the large 
glandular cells and the small centrotubular 
cells (Jong-Brink, 1969; Breckenridge & Fallil, 
1973; Els, 1978). 

It is the aim of this study to investigate in 
detail the nature of the albumen gland secre- 
tions of the arionid species Arion subfuscus 
(Draparnaud, 1805), by means of general 
and lectin histochemical methods. The ultra- 
structure of secretory granules is also de- 
scribed. 



MATERIALS AND METHODS 

For general and lectin histochemical stud- 
ies the albumen glands were fixed in Carney's 
fluid (Culling, 1974) for six h, dehydrated with 
alcohol, and embedded in paraffin wax. Sec- 
tions, 6-8 um thick, were obtained. 

Neutral carbohydrates were detected by the 
periodic acid Schiff technique (PAS). Acetyl- 
ation followed by saponification, and diastase 
treatment were used as controls and also for 
glycogen demonstration (Martoja & Martoja- 
Pierson, 1970). Alcian Blue staining (AB) was 
used at pH = 0.5 to stain strongly sulphated 
mucosubstances and at pH = 2.5 for carboxy- 
lated and weakly sulphated mucosubstances 
(Bancroft & Stevens, 1982). The combined 
High Iron Diamine with Alcian Blue (HID-AB) 
was used to stain sulphated mucosubstances 
black and non-sulphated acidic mucosub- 
stances blue (Culling, 1974). The Mercuric 
Bromophenol Blue (BB) with deamination 
(Chapman, 1975), as well as Chloramine T- 
Schiff techniques (Martoja & Martoja-Pierson, 
1970), were used for detection of proteins. 
Bock's technique (Bock et al., 1 976) was used 
for demonstrating protein disulphide groups. 
In order to detect lipid material, glands were 
fixed in Baker's formol-calcium and sections 
were stained with Sudan Black B. 



^Departamento de Biología Celular y Ciencias IVIorfológicas. Facultad de Ciencias, Universidad del País Vasco, Apdo. 644- 

48080, Bilbao, Spain 

^Departamento de Biología Animal y Genética. Facultad de Ciencias, Universidad del País Vasco, Apdo. 644-48080, Bilbao, 

Spain 

77 



78 



GOMEZ ET AL. 



TABLE 1. Lectins employed and their major binding specificities: Fuc: fucose; Gal: galactose; 
GalNac: N-acetylgalactosamine; Glc: glucose; GlcNac: N-acetylglucosamine; Man: mannose; 
NeuAc: neuraminic acid or sialic acid. 



Taxonomic name of the source 



Acronym 



Carbohydrate binding specificity 



Ulex europaeus 


UEA-I 


Canavalia ensiformis 


Con A 


Umax flavus 


LFA 


Arachis hypogaea 


PNA 


Ricinus comunis 


RCA-I 


Glycine max 


SBA 



L-Fuc; a-1,2 Gal ß-1,4 GlcNAc, ß-1,6 
(t-D-Man > ft-D-GIc » «-D-GlcNAc 
NeuAc o.-2,3/6Gal; NeuAc a-2,3/6GalNAc 
Gal ß-1,3GalNAc>Gal 
Gal ß-1,4 GlcNAc; Gal 
D-GalNac > D-Gal 



Lectin histochemistry was used according 
to Welsch & Schunnacher (1984). Deparaf- 
fined sections were incubated for 30 min with 
lectins coupled to fluorescein isothiocyanate 
(FITC) : PNA (Peanut agglutinin); SBA (Soy- 
bean agglutinin); LFA {Umax flavus aggl.); 
UEA I {Ulex europeas I aggl.); RCA I {Ricinus 
communis aggl.); Con A (Concanavalin A)(for 
lectin specificities, see Table 1). After incuba- 
tion, the sections were rinsed in phosphate 
buffered saline (pH = 7.4) for 2 h. Preparations 
were studied by fluorescence microscopy. 
Sections not treated with FITC-lectins were 
used as controls to observe the autofluores- 
cence of the tissue. The specificity of the 
staining (Goldstein & Hayes, 1978) was 
tested by preincubating the lectin in a solution 
of the appropriate reactive sugar (Zubiaga et 
al., 1990). 

For transmission electron microscopy, tis- 
sues were fixed for 2 h in 25% Karnovsky in 
0.1 M cacodylate buffer at pH 7.3 (Glauert, 
1981), postfixed for 1 h in 1% osmium tetrox- 
ide in the same buffer and embedded in 
Araldite. Ultrathin sections were stained with 
uranyl acetate and lead citrate. The grids were 
observed in a Zeiss EM 9 and Philips EM 300 
electron microscopes. 



RESULTS 



Ultrastructure 



In the secretory cells of the albumen gland 
of Arion subfuscus, the organelles involved in 
the synthesis of secretory materials are abun- 
dant and include flattened and parallely 
arranged rough endoplasmic reticulum cister- 
nae and Golgi stacks with many vesicles bud- 
ding from them (Fig. 1 ). The middle and apical 
portions of the cells are filled by secretory 
granules (large secretory globules, 2-3 um in 



1* '. 



/ 




FIG. 1 . ТЕМ micrograph showing the matrix of large 
secretory globules (sg) containing small globules 
(*). Rough endoplasmic reticulum cisternae are flat- 
tened and abundant (rer). Arrows indicate Golgi 
system. X 15000. 



diameter) with a matrix of finely granulated 
material, homogeneously distributed (Fig. 1). 
Inside these large globules, there are small 
aggregates of irregular lumps (small secre- 
tory globules, 0.3-0.4 um in diameter), which 
contain a fine granulation (Fig. 1). 

Histochemistry 

The general histochemical tests show that 
the composition of the albumen gland secre- 
tion is complex. The small globules show a re- 
action distinct from that of the general matrix 
of the large globules in which they are in- 
mersed. Within each large globule there are 
one or two small ones. The results of the his- 
tochemical tests carried out are shown in 
Tables 2 and 3. 

The small globules react strongly to the AB 
technique at both pH levels, and they stain 
black with the HID-AB technique indicating 
that they are composed mostly of sulfo- 
mucins. Moreover, these small globules are 



IDENTIFICATION OF BIPHASIC GRANULES 



79 



TABLE 2. Results of the histochemical tests of the albumen gland tubules of Arion subfuscus. Three portions 
of the secretory cells have been distinguished: 1, cytoplasm; 2, large secretory globules; 3, small secretory 
globules; -, negative reaction; +, weak reaction; ++, moderate reaction; +++, strong reaction. 



Secretory cells 



Staining technique 


1 


2 


3 


ABpH = 0.5 


- 


- 


+++ 


ABpH = 2.5 


+ 


- 


+++ 


HID-AB 


+ 


- 


+++ 




blue 




black 


PAS 


++ 


+++ 


- 


PAS-diastase 


+ 


++ 


- 


PAS-acet 


- 


++ 


- 


PAS-acet.-sap 


++ 


+++ 


- 


Deamination-PAS 


++ 


+++ 


- 


Bromophenol blue 


++ 


+ 


+ 


ChloramineT-Schiff 


++ 


++ 


+ 


Bock 


- 


- 


++ 


Sudan Black 


- 


- 


- 



Centrotubular cells 



+ 
++ 

+ 
+ 
+ 



TABLE 3. Lectin specificities of the different portions of the secretory cells of the Arion subfuscus albu- 
men gland. -, negative reaction; +, weak reaction; ++, moderate reaction; +++, strong reaction. 



Lectins 



PNA 



SBA 



LFA 



UEAI 



RCAI 



ConA 



Cytoplasm 


- 


++ 


Large globules 


+++ 


+++ 


Small globules 


+++ 


- 



PAS-negative, whereas exhibiting a strong re- 
action to the Bock technique for protein SH 
groups. 

The large globules show a strong reaction 
to the PAS technique, even after treatment 
with diastase or after acetylation followed by 
saponification. They also show moderate re- 
actions to tests for protein. 

The reactions of each type of globule to the 
lectins are different. The large granules bind 
specifically to PNA, SBA, RCA I, and Con A 
(Fig. 2), whereas the small granules bind to 
PNA, LFA, and UEA I (Fig. 3). 



DISCUSSION 

Previous histochemical and biochemical 
studies have shown that the albumen gland of 
gastropods produces a galactogen and pro- 
tein-rich nutritive fluid for the developing em- 
bryos (Grainger & Shilliote, 1952; Bayne, 
1967; Meenakshi & Scheer, 1968; Okotore et 
al., 1 981 ;Dictus& Jong-Brink, 1987). The pro- 
tein percentage varies from one species to an- 
other. Thus, in the pulmonates Biomphalaria 
glabrata (see Jong-Brink, 1969) and Achatina 



fúlica (see Ramasubramaniam, 1 979) the pro- 
tein content is high, whereas in Dereceras 
laeve only a small amount of protein has been 
demonstrated (Els, 1 978). We have found that 
protein is present in the secretory globules of 
the albumen gland of Arion subfuscus, as well 
as in the cytoplasm of the secretory cells, but 
the reaction to protein tests is only moderate. 
Nevertheless, the rough endoplasmic reticu- 
lum is very abundant. The protein-reacting 
groups could be intimately joined with poly- 
saccharides and thus masked. In this sense, 
Bayne (1967) could not separate the proteins 
from sugars by electrophoretic studies, but he 
found great amounts of free amino-acids in al- 
bumen gland homogenates.This fact, together 
with the near absence of digestion of secretory 
granules by pronase (Kress & Schmekel, 
1992), indicates that there is a very close 
union between both components. 

The presence of galactogen in the albumen 
gland has been shown using different metho- 
dologies (Bolognani-Fantin & Vigo, 1968; 
Varute & Nanaware, 1972). In this work, we 
have used lectin histochemical methods to 
detect galactogen. The secretory granules of 
Arion subfuscus show a strong reaction with 



80 



GOMEZ ET AL. 




FIG. 2. Lectin histochemistry. RCA I binding to large 
secretory globules (arrows). Arrow heads indicate 
the nuclei of cells. x40. 




FIG. 3. Lectin histochemistry. LFA binding to small 
secretory globules (small arrows) which are present 
inside the large secretory globules (arrows). Arrow 
heads indicate the nuclei of cells that show a weak 
reaction to LFA. x65. 



PNA, SBA, and RCA I. These three lectins 
bind specifically to galactose residues (Gold- 
stein & Hayes, 1978; Zubiaga et al., 1990). 
According to Okotore et al. (1981) and 
Okotore & Uhlenbruck (1982), these residues 
are probably D-Gal ß1, 3D-Gal. 

Moreover, the secretory granules of A. sub- 
fuscus as well as the cytoplasm of secretory 
cells also react with Con A. This indicates the 
additional presence of glucose or mannose 
sugar residues (Goldstein & Hayes, 1978) 
free in the cytoplasm as well as inside secre- 
tory vesicles. The strong positive reaction of 
the cytoplasm and secretory granules of the 
albumen gland of this species with the PAS 
technique decreases after acetylation and di- 
astase treatment. Thus, the general histo- 
chemical tests also indicate the presence of 



glycogen and other neutral carbohydrates 
(Culling, 1974; Bancroft & Stevens, 1982) dif- 
ferent from galactogen (Rangarao, 1963; 
Bolognani-Fantin & Vigo, 1968) inside secre- 
tory globules. 

On the other hand, the strong reaction with 
AB at pH 0.5 together with the black-staining 
reaction with HID-AB, indicates the presence 
of sulphated acidic mucosubstances in the 
small granules contained in the large ones 
of the albumen gland secretion of Arion sub- 
fuscus. We conclude that the small and 
foamy-looking globules contain mainly gly- 
cosaminoglycans. Acidic groups have also 
been reported in the albumen gland of other 
gastropods (Rangarao, 1963; Bayne, 1967), 
but the secretory granules have always been 
described as ultrastructurally homogeneous 
without smaller clear granules inside (Nieland 
&Goudsmit, 1969). 

As with other stylommatophorans (Bayne, 
1967; Ramasubramaniam, 1979), no lipid has 
been detected in albumen secretions of Arion 
subfuscus. 



LITERATURE CITED 

BANCROFT J. D. & A. STEVENS, 1982, Theory 
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London. 

BAYNE, C, 1967, Studies on the composition of ex- 
tracts of the reproductive glands of Agrioiimax 
reticulatus, the grey field slug (Pulmonata, 
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BOCK, R., T SALLAND & P. SCWABEDAL, 1976, 
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BOLOGNANI-FANTIN, A. & E. VIGO, 1968, Histo- 
chemistry of the glands associated with the re- 
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chimie. 15:300-311. 

BRECKENRIDGE, W. & S. FALLIL, 1973, Histo- 
logical observations on the reproductive system of 
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118. 

CHAPMAN, D., 1975, Dichromatism of bromophe- 
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CULLING, C, 1974, Handbook of histopathological 
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don. 

DICTUS, W. J. A. G. & M. DE JONG-BRINK, 1987, 
Morphometrical, enzyme cytochemical and bio- 
chemical studies on the secretory activity of a fe- 



IDENTIFICATION OF BIPHASIC GRANULES 



81 



male accessory sex gland (albumen gland) of the 
freshwater snail Lymnaea stagnalis. Proceedings 
of the Koninklijke Nederlandse Akademie van 
Wetenscfiappen, 90: 257-270. 

DUNCAN, С J., 1975, Reproduction. Pp. 309-365, 
in: V. FRETTER & J. PEAKE, eds.. Pulmonales. Vol. 
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ELS, W., 1978, Histochemical studies on the matu- 
ration of the genital system of the slug Dereceras 
laeve (Pulmonata, Limacidae), with special refer- 
ence to the identification of mucosubstances se- 
creted by the genital tract. Annals of University of 
Stellenbosch, 1A:2. 

GLAUERT, A., 1981, Fixation, dehydration and em- 
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GOLDSTEIN, I. & С HAYES, 1978, The lectins: car- 
bohydrate binding proteins from plants and ani- 
mals. Advances in Carbohydrates Chemistry and 
Biochemistry 35: 1 27-240. 

GRAINGER, J. & A. SHILLITOE, 1952, Histochem- 
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JONG-BRINK, M. DE., 1969, Histochemical and 
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ductive tract of Biomphalaria glabrata (Austra- 
lorbis glabratus), intermediate host of Schisto- 
soma mansoni. Zeitschi ft für Zellforschung, 102: 
507-542. 

KRESS, A. & L. SCHMEKEL, 1 992, Structure of the 
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branch mollusc, Runcina. Tissue & Cell, 24: 
95-110. 

MARTOJA, M. & M. MARTOJA-PIERSON, 1970, 
Técnicas de histología animal. Toray-Masson, 
Barcelona. 

MEENAKSHI, V. & В. SCHEER, 1968, Studies on 
the carbohydrates of the slug Agriolimax colum- 
bianus with special reference to their distribution 
in the reproductive system. Comparative Bio- 
chemistry and Physiology 26: 1091-1097. 

NIELAND, M. & E. GOUDSMIT 1 969, Ultrastructure 
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matia. Journal of Ultrastructure Research, 29: 
119-140. 



OKOTORE, R., P KLEIN, M. ORTMANN & G. UH- 
LENBRUCK, 1981, Serological and histochemi- 
cal studies on the Achatina fúlica galactan. 
Comparative Biochemistry and Physiology 70 B: 
469-475. 

OKOTORE, R. & G. UHLENBRUCK, 1982, Addi- 
tional lectin receptors in galactans from the albu- 
min gland of the Achatina fúlica snail. Exper- 
ientia, 38: 507-508. 

RAMASUBRAMANIAM, K., 1979, A histochemical 
study of the secretions of reproductive glands and 
of the egg envelopes of Achatina fúlica (Pul- 
monata: Stylommatophora). International journal 
of Invertebrate Reproduction, 1 : 333-346. 

RANGARAO, K., 1963, The polysaccharides of the 
reproductive system of the land snail Ariophanta 
ligulata in the formation of egg capsules. Journal 
of Animal Morphology and Physiology, 10: 
158-163. 

RUNHAM, N.W., 1988, Mollusca. Pp. 113-188, in: 
K. G. ADiYODi & R. G. ADiYODi, eds., Reproductive 
biology of Invertebrates. Vol. Ill, Accessory sex 
glands. John Wiley & Sons Ltd., Chichester. 

TOMPA, A., 1984, Land snails (Stylommatophora). 
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duction. Academic Press, New York. 

VARUTE, A., & NANAWARE, 1972, Histochemical 
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10:37-46. 

WELSCH, и. & и. SCHUMACHER, 1984, Histo- 
chemical observations on carbohydrates in con- 
nective structures and basement membranes of 
hemi- and cephalochordates. Acta Zoológica, 65: 
105-112. 

ZUBIAGA, A. M., B. J. GOMEZ, J. MOYA & E. ÁN- 
GULO, 1990, Identification and carbohydrate 
content of secretory cell types in the spermovi- 
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Revised ms. accepted 25 February 1997 



MALACOLOGIA, 1998, 39(1-2): 83-91 

ESTRATEGIAS DE DEPREDACIÓN DEL GASTRÓPODO PERFORADOR 
TROPHON GEVERSIANUS (PALLAS) (MURICOIDEA: TROPHONIDAE) 

Sandra Gordillo y Sandra N. Amuchástegul 

Centro Austral de Investigaciones Científicas (CADIC, CONICET). CC 92 (9410) Ushuala, 

Tierra del Fuego, Argentina 

RESUMEN 

Se analizaron las diferentes estrategias de depredación utilizadas por Troplion geversianus 
(Trophonidae) sobre cuatro especies de bivalvos típicos de la región fueguina: Mytilus chilensis. 
Aulacomya atra, IHiatella solida y Tawera gayi. 

Los datos fueron obtenidos utilizando tres metodologías: análisis de valvas perforadas trans- 
portadas sobre la playa actual, estudio de la depredación bajo condiciones de laboratorio, y ob- 
servación de la depredación en poblaciones naturales durante la bajamar. 

Se concluye que (1) no todas las perforaciones producidas por los murícidos sensu lato son 
cilindricas; (2) en las distintas presas hay variación en la forma y sitio de las perforaciones; y (3) 
la intensidad de depredación es baja a moderada. 

Palabras clave: depredación, gastrópodos, perforaciones. Tierra del Fuego. 

ABSTRACT 

Prédation strategies of Trophon geversianus (Trophonidae) on four bivalve species: Mytilus 
chilensis. Aulacomya atra, Hiatella solida and Tawera gayi, from the fuegian region were ana- 
lyzed. 

Data were obtained by three methods: analysis of bored shells transported on modern beachs, 
study of prédation under laboratory conditions, and observation of prédation on populations in 
the field. 

We conclude that (1) not all muricid holes are cylindrical; (2) there is variation among prey 
species in the shapes of holes and in the sites of drilling; and (3) prédation intensity is low to mod- 
erate. 

Key words: prédation, gastropods, drillholes. Tierra del Fuego. 



INTRODUCCIÓN 

Una característica notable de la mayoría de 
los moluscos es su capacidad para secretar 
un exoesqueleto calcáreo de protección. 
Otra, irónicamente, es la habilidad de algunos 
de estos moluscos de excavar o perforar di- 
chos exoesqueletos mediante un proceso 
mecánico-químico, descripto por Carriker & 
Williams (1 978) y Carriker (1 981 ), entre otros. 

Es ampliamente conocido que los princi- 
pales responsables de dichas perforaciones 
son distintas especies que pertenecen a dos 
grupos dentro de Gastropoda. Por un lado, y 
entre los mesogastrópodos, figuran los natíci- 
dos (Naticidae). Por otro lado, y entre los 
neogastrópodos, la superfamilia Muricoidea 
comprende los murícidos sensu stricto (Mu- 
ricidae), táididos (Thaididae) y trofónidos 
(Trophonidae) con especies perforadoras de 
valvas. 



Los bivalvos epifaunales son atacados par- 
ticularmente por los murícidos sensu lato, es 
decir por representantes de las distintas fa- 
milias que integran el grupo Muricoidea 
(Kabat, 1990). Los bivalvos infaunales, en 
cambio, son comúnmente víctimas de los 
nati'cidos (Kabat, 1990; Anderson et al., 
1 991 ), aunque también pueden ser presas de 
algunos Muricoidea como lo mencionan 
Vermeij (1 980) y Gordillo (1 994) para distintas 
especies. 

La morfología de la perforación ha sido uti- 
lizada por distintos autores para diferenciar 
familias. La perforación de los murícidos 
sensu lato es cilindrica con bordes casi rec- 
tos; mientras que la perforación de los nati'ci- 
dos, en contraste, tiene una forma más pa- 
raboloide y bordes biselados (Carriker, 1981; 
Aitken & Risk, 1988; Kabat, 1990; Anderson 
et al., 1991). Un caso atípico dentro de 
Muricoidea lo constituyen los trofónidos, ca- 



83 



84 



GORDILLO & AMUCHASTEGUI 



paces de realizar perforaciones que varían 
entre cilindrica y cono-truncada, según la 
presa (Gordillo, 1994, 1995a). 

Para Tierra del Fuego, trabajos anteriores 
realizados por Gordillo (1994, 1995a) y Annu- 
chástegui (1995) describen la depredación de 
Trophon geversianus en distintas especies 
que viven en el litoral fueguino. El objective de 
este trabajo ha sido reunir y sintetizar toda la 
información recabada sobre la depredación 
de los trofónidos que caracterizan esta región. 



UBICACIÓN Y CARACTERIZACIÓN DEL 
ÁREA DE ESTUDIO 

El Canal Beagle se ubica entre la Isla 
Grande de Tierra del Fuego, la Isla Navarino 
y la Isla Hoste a los 54°LS y 68°LO. Este 
canal es de aguas tranquilas, con una profun- 
didad media de aproximadamente 150-200 
m y un litoral rocoso con playas de rodados. 
El canal posee un régimen de mareas de de- 
sigualdades diurnas con una amplitud media 
de 1 metro. 

La temperatura media anual es de 5.5°C 
con una amplitud térmica moderada. La sali- 
nidad superficial del agua varía entre 27 y 31 
gramos/litro (Iturraspe et al., 1989). La tem- 
peratura estival del agua oscila en 11-12°C, 
mientras que en invierno es de 3-4°C 
(Iturraspe et al., 1989). 



MATERIAL Y MÉTODO 

El principal depredador considerado fue el 
trofónido Trophon geversianus y, en segunda 
instancia, Xymenopsis muriciformis. Las pre- 
sas estudiadas fueron cuatro especies de bi- 
valvos típicos de la región: Mytilus chilensis, 
Aulacomya atra, Hiatella solida y Tawera gayi. 
El Apéndice presenta la posición taxonómica 
de las especies tratadas según Gordillo 
(1995b). El material estudiado procede de 
distintos sitios de las zonas mesolitoral e 
infralitoral del Canal Beagle. Los es- 
pecímenes se obtuvieron por recolección 
manual durante la bajamar o con buceo 
autónomo a profundidades de hasta 3 me- 
tros. 

Los datos sobre los que posteriormente se 
aplicaron los distintos parámetros fueron 
obtenidos utilizando tres metodologías: 

Análisis de valvas perforadas transporta- 
das sobre la playa actual: el material procede 
de distintas playas indicadas en la Figura 1 . 



En cada sitio, y dentro del supralitoral, se 
tomó como unidad de muestreo un cuadrante 
de 0.5 X 0.5 m con un número de réplicas pre- 
determinado. En cada cuadrante se contabi- 
lizaron todas las valvas de las cuatro es- 
pecies que aparecieron en sus respectivas 
superficies. 

Estudio de la depredación bajo condiciones 
de laboratorio: se utilizaron acuarios de vidrio 
de distinta capacidad, según la experiencia, 
variando la cantidad de depredadores y pre- 
sas (Tabla 1). Los ejemplares fueron recolec- 
tados del mesolitoral e infralitoral de distintas 
localidades del Canal Beagle (Fig. 1). Los 
acuarios se mantuvieron en la cámara fría del 
CADIC a una temperatura de 2 a 9°C. Dia- 
riamente se realizaron observaciones sobre 
el comportamiento de los depredadores y se 
retiraron las valvas vacías. 

Observación de la depredación en condi- 
ciones naturales durante la bajamar: se ana- 
lizó la presencia de Trophon geversianus en 
una superficie que varió, según las carac- 
terísticas del sitio elegido, entre 2.5 y 50 m^. 
Estos muéstreos se realizaron mensualmente 
durante la bajamar (con un nivel menor a 0.45 
m) en las localidades indicadas en la Figura 1 . 
Se midió la longitud de los ejemplares halla- 
dos, consignando si depredaban o no, parti- 
cularmente sobre Mytilus chilensis por ser la 
presa más abundante del mesolitoral. 

Los parámetros analizados fueron los si- 
guientes: 

Morfología de la perforación: para analizar 
este parámetro se midieron los diámetros ex- 
ternos e internos (mm) de la perforación, uti- 
lizando una lupa binocular con retículo. 

Selectividad por el sitio a perforar: para de- 
terminar la preferencia con respecto al sitio de 
perforación en las distintas especies, la su- 
perficie de la valva fue dividida en regiones 
según muestra la Figura 2. Para analizar este 
parámetro se sometieron los datos a la prueba 
estadística de Chi-cuadrado (X^). Para este y 
otros parámetros de selectividad se hace re- 
ferencia a "selectivo" para expresar la prefer- 
encia o comportamiento estereotípico y "no 
selectivo" para denotar un comportamiento 
aleatorio. 

Selectividad por la valva derecha o iz- 
quierda: para analizar la preferencia por al- 
guna de las dos valvas opuestas, los valores 
obtenidos también se sometieron a una 
prueba de Chi-cuadrado (X^). 

Selectividad por un tamaño de la presa: en 
valvas transportadas, para determinar la exis- 
tencia de selectividad por un tamaño de valva 



DEPREDACIÓN DETROPHONIDAE 



85 



TABLA 1 . Experiencias de laboratorio. Se incluye el tamaño y el número o peso de las presas y de 
los depredadores y la capacidad de los acuarios. 



ACUARIOS 



PRESAS 



DEPREDADORES 



CAPACIDAD 



Acuario A Mytilus chilensis 

lOOgr. (x <30 mm) 

1 00 gr. (30 mm < x < 50 mm) 

100 gr. (50 mm < x < 70 mm) 

Acuario В igual al Acuario A 

Acuario С Aulacomya atra 

100 gr. (x< 30 mm) 

1 00 gr. (30 mm < x < 50 mm) 

1 00 gr. (50 mm < x < 70 mm) 

Acuario D igual al Acuario С 

Acuario E Mytilus chilensis 

n = 25 (24 mm < x < 65 mm) 

Tawera gay i 

n = 3 (31 mm; 31 .7 mm у 33 mm) 

Acuario F Tawera gayi 

n = 20 (20 mm < x < 36 mm) 

Hiatella solida 

n = 5 (20 mm < x < 45.4 mm) 



6 Trophon geversianus 
(23 mm < X < 45.6 mm) 



6 Trophon geversianus 
(22.7 mm <x < 43.3 mm) 

6 Trophon geversianus 
(18.5 mm < x < 52.3 mm) 



6 Trophon geversianus 
(22.7 mm < x < 45.3 mm) 

3 Trophon geversianus 
(30 mm; 48 mm y 54 mm) 



6 Trophon geversianus 
(33.0 mm < X < 51 .5 mm) 



19 litros 

1 9 litros 
1 9 litros 

19 litros 
4 ^4 litros 

4 ^4 litros 



en particular, se relacionó la distribución de 
frecuencias por tamaño (longitud), consi- 
derando separadamente las valvas no per- 
foradas, y las valvas perforadas. Con los datos 
obtenidos en laboratorio se calculó la relación 
entre la cantidad de presas ofrecidas y las pre- 
sas consumidas según su tamaño. Para 
analizar este parámetro, y en ambos casos, se 
aplicó la prueba de Chi-cuadrado (X^). 

Correlación entre el tamaño del depredador 
y el tamaño de la presa: se calculó en forma 
directa mediante la medición de los espe- 
címenes que fueron observados depredando 
y de sus presas. Se realizó bajo condiciones 
de laboratorio y en poblaciones naturales en 
el caso de Mytilus chilensis. Para expresar la 
correlación se calculó el coeficiente de cor- 
relación (r). 

Éxito-fracaso de la depredación (drilling 
success; Tüll & Bohning-Gaese, 1993): se 
partió del supuesto de que una perforación in- 
completa constituye un evento fallido de 
depredación. Este parámetro expresa el por- 
centaje de éxito y resulta de dividir el número 
de perforaciones completas por el número 
total de intentos, dado por la suma de las per- 
foraciones incompletas y completas. Los por- 
centajes de depredación obtenidos se expre- 



saron según la siguiente escala: 0-20%: muy 
poco exitosa; 20-40%: poco exitosa; 40-60%: 
moderadamente exitosa; 60-80%: exitosa y 
80-100%: muy exitosa. 

índice de depredación (prédation rate, 
Vermeij, 1980): la proporción de depredación 
se calculó tomando la proporción de valvas 
perforadas en relación al número total de val- 
vas. Los valores obtenidos se expresaron 
según la siguiente escala: 0.0-0.25: bajo; 
0.25-0.50: moderado; 0.50-0.75: elevado y 
0.75-1 .0: muy elevado. 

Tiempo de depredación: un ciclo de depre- 
dación está dado por el período de alimen- 
tación y el de descanso hasta que se inicia un 
nuevo periodo de alimentación. Para determi- 
nar el tiempo de depredación, y siempre bajo 
condiciones de laboratorio, se calculó el tiem- 
po transcurrido (en días) desde que el depre- 
dador se apoyó sobre la presa hasta que la 
abandonó. Luego se tomó, también en días, el 
período de descanso transcurrido hasta que 
un mismo depredador atacó a otra presa. El 
tiempo de depredación se expresó arbitraria- 
mente en: "variable" cuando el desvío resultó 
mayor o igual a la media aritmética dividida 2 
y "estable" cuando el desvío fue menor a dicho 
cociente. 



86 



GORDILLO & AMUCHASTEGUI 




О Depredación m s;''j # Esoecimenes para acuario 



Volvos tronsportodos 



FIG. 1 . Mapa de Ubicación. Los símbolos señalan los sitios de procedencia de las muestras. 




A 


r- \ 
II 


[Л 


IV 


V 


.,) 


Ч 


VIII 


vil/ 




FIG. 2. Sectores en que se subdividieron las valvas 
de las presas para analizar la selectividad por el 
sitio a perforar. A, Mytilus chilensis. B, Tawera gayi. 
C, Hiatella solida. 



RESULTADOS 
Morfología de la Perforación 

Las perforaciones producidas por Trophon 
geversianus mostraron algunas diferencias 
morfológicas en las distintas presas (Tabla 2, 
A;Fig.3). 

En Hiatella solida, Gordillo (1994) observó 
que Trophon geversianus realiza perfora- 
ciones cono-truncadas, tal como se esque- 
matiza en la Figura 3, A. 

Las perforaciones excavadas por este 
mismo depredador en valvas de Mytilus chi- 
lensis también son de tipo conotruncado, si- 
milares a las descriptas previamente por Gor- 
dillo (1994, 1995a) para Tawera gayi {F\g.3,B). 

Sobre Aulacomya atra, las perforaciones 
producidas por Trophon geversianus se difer- 
encian de las anteriores en que se asemejan 
más a un cilindro simple (Fig. 3, С). 

En relación al depredador Xymenopsis mu- 



nciformis se constató que bajo condiciones 
de laboratorio, éste produce en Mytilus chilen- 
sis perforaciones que se asemejan más a la 
morfología cilindrica, por las menores diferen- 
cias entre los diámetros externo e interno; i.e. 
sobre un total de 19 ejemplares de esta es- 
pecie, los diámetros externo e interno fueron 
respectivamente 1.79 ± 0.2 mm y 1.22 ± 0.16 
mm. En Tawera gayi y Hiatella solida, bajo 
condiciones de laboratorio, solamente fueron 
observadas perforaciones de este tipo en un 
caso para cada especie. Algunas valvas 
transportadas de Tawera gayi (n = 14) pre- 
sentaron una morfología similar, razón por la 
cual fueron atribuidas a este depredador. En 
estas perforaciones se obtuvo un diámetro 
único de 1.81 ± 0.30 mm, ya que no se notó 
diferencias entre los dos diámetros. 

Finalmente, se observó que las perfora- 
ciones incompletas dejadas por Trophon ge- 
versianus y Xymenopsis muriciformis en las 
distintas presas coincidían en tener el fondo 
plano. 

Sitio de Perforación 

Las zonas elegidas por los depredadores 
variaron según las distintas presas conside- 
radas (Tabla 2, B). 

En las valvas de Mytilus chilensis se ob- 
servó que alrededor del 50% de las valvas 
(53% en el primer caso y 59% en el segundo) 
estaban perforadas en los sectores II y III, que 
corresponden a la zona media ventral y dorsal 
respectivamente, por lo que se consideró que 
hay preferencia por la zona media. 



DEPREDACIÓN DE TROPHONIDAE 87 

A ВС 






VISTA DORSAL: 






2 mm 



FIG. 3. Morfología de la perforaciones producidas por Trophon geversianus en A, Hiatella solida. B, Mytilus 
chilensisy Tawera gayi. C, Aulacomya atra Referencias: Oext, diámetro externo, o,^,, diámetro interno [Tomado 
deGordillo(1994)]. 



Con respecto al mitílido Aulacomya atra, y 
sólo bajo condiciones de laboratorio, se pudo 
constatar que las perforaciones se encon- 
traron en el 100% de los casos en proxinni- 
dades del borde de las valvas, lo que marcó 
una gran diferencia con el sitio que Trophon 
geversianus elige para perforar las valvas de 
Mytilus chilensis. 

En las valvas de Tawera gayi hubo prefe- 
rencia por el sector V o medio central donde 
se concentraron el 50% de las perforaciones 
(Gordillo, 1994). 

En Hiatella solida el 77% de las perfora- 
ciones se encontraron en el sector II o poste- 
rior en torno a una cóstula radial caracterís- 
tica de la especie (línea punteada de la Fig. 2, 
С), descripto con anterioridad por Gordillo 
(1994). Una proporción de las mismas (61%) 
se halló dentro de la zona de inserción del 
músculo posterior. 

Respecto a este parámetro, también se ob- 
tuvo que el segundo depredador considerado, 
Xymenopsis murlciformis, eligió el sector um- 
bonal en valvas de Tawera gayi en el 1 00% de 



los casos (n = 14). Sobre Mytilus chilensis (n 
= 19) no se halló selectividad por un sitio par- 
ticular, estando las perforaciones distribuidas 
en toda la superficie de la valva. En Aula- 
comya atra se produjo un único caso de per- 
foración por Xymenopsis muriciformis en que 
la misma se ubicó en el borde de la valva, 
similar a Trophon geversianus. 

Preferencia por la Valva Derecha o Izquierda 

Para las distintas presas se observó que no 
hubo preferencia por una de las valvas, ya 
sea derecha o izquierda (Tabla 2, C). Este 
parámetro no pudo calcularse para Aulaco- 
mya atra ya que las perforaciones ubicadas 
en el borde abarcaron en la mayoría de los 
casos las dos valvas opuestas. 

Selectividad por un Tamaño 

Hubo variaciones en los resultados obteni- 
dos en relación a este parámetro (Tabla 2, D). 
Con los datos de laboratorio se obtuvo la 



GORDILLO & AMUCHASTEGUI 



proporción de presas ofrecidas y consumidas 
por Mytilus chilensis y Aulacomya atra. 

Para el caso de Mytilus chilensis se ob- 
servó una marcada preferencia de Trophon 
geversianus por las presas más grandes 
(50-70 mm). En Aulacomya atra los ejem- 
plares seleccionados comprendieron un ran- 
go más amplio, ya que el depredador prefirió 
los ejemplares medianos (30-50 mm) y los 
grandes (50-70 mm). 

En valvas transportadas de estas dos es- 
pecies, y en base a la distribución de frecuen- 
cias de valvas perforadas y no perforadas 
también se observó preferencia por las valvas 
más grandes. 

En cambio, con la misma metodología, en 
Tawera gayi y Hiatella solida, no se observó 
selectividad por un tamaño particular. En 
estas dos especies, la mayor proporción de 
perforaciones sobre valvas con un largo 
promedio de 31 mm se relaciona directa- 
mente con la mayor cantidad de valvas de 
dicho tamaño y no con una selección del 
mismo. 

Relación Entre los Tamaños de la Presa y 
del Depredador 

Según la especie y la metodología em- 
pleada, los resultados variaron entre sin cor- 
relación hasta una ligera correlación positiva 
(Tabla 2, E). 

En Mytilus chilensis, y bajo condiciones 
naturales, este parámetro mostró una ligera 
correlación positiva. Sin embargo, los resulta- 
dos de laboratorio indicaron ausencia de cor- 
relación. 

En Aulacomya atra con los datos de labora- 
torio se obtuvo una correlación positiva entre 
los tamaños del depredador y de sus presas. 
Bajo condiciones naturales, solamente en 
cuatro oportunidades se observó a Trophon 
geversianus depredando sobre ejemplares 
chicos (menores a 30 mm) de Aulacomya atra. 

Finalmente, con los datos de laboratorio de 
Tawera gay/ tampoco se encontró correlación 
entre estas variables. 

Perforaciones Incompletas: Éxito-Fracaso de 
la Depredación 

Los resultados mostraron (Tabla 2, F) que 
la depredación de Trophon geversianus varió 
entre muy exitosa en Aulacomya atra (98.7- 
100%); muy exitosa a exitosa en Hiatella so- 
lida (63.7-100%); y exitosa en Tawera gayi 



(64.4-74.1%) y en Mytilus chilensis (79- 
93.3%). 

Para estas especies las valvas presentaron 
una sola perforación, ya sea completa o in- 
completa. Una excepción la constituye la 
depredación en Tawera gayi bajo condiciones 
de laboratorio, en que algunas presas fueron 
atacadas simultáneamente por dos depre- 
dadores distintos, resultando perforadas las 
dos valvas en un 25% de los casos. 

Proporción de Valvas Perforadas: 
Indice de Depredación 

El índice de depredación resultó bajo para 
Mytilus chilensis, Aulacomya atra y Tawera 
gayi, mientras que fue moderado en el caso 
de Hiatella solida (Tabla 2, G). 

Tiempo de Depredación 

Se desconoce aún el tiempo que emplea el 
depredador en excavar la perforación; sin em- 
bargo, los resultados (Tabla 2, H) hacen refe- 
rencia al tiempo promedio transcurrido desde 
que el depredador se apoyó sobre la presa 
hasta que la abandonó. 

Los tiempos de depredación y de descanso 
fueron muy variables en Mytilus chilensis y 
Aulacomya atra, y relativamente más estables 
en Tawera gayi y Hiatella solida, aunque el 
número de observaciones en estas dos es- 
pecies fue menor. 



DISCUSIÓN 

En relación a la morfología de la per- 
foración es importante destacar que la per- 
foración cono-truncada que Trophon gever- 
sianus excava en valvas de Mytilus chilensis, 
Tawera gayi y Hiatella solida se asemeja más 
a la perforación típica de los natícidos que a 
la perforación producida por los murícidos 
sensu lato. 

Sin embargo, una forma de diferenciar 
estas perforaciones de aquellas producidas 
por los natícidos es a través de las perfora- 
ciones incompletas; ya que en los natícidos 
éstas presentan un fondo deprimido con una 
prominencia o giba central (Aitken & Risk, 
1988; Kabat, 1990), mientras que la per- 
foración incompleta realizada por los dos 
trotón idos Trophon geversianus y Xymenop- 
sis muriciformis se caracteriza por tener el 



DEPREDACIÓN DETROPHONIDAE 



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90 



GORDILLO & AMUCHASTEGUI 



fondo plano, y sin ninguna prominencia cen- 
tral. 

En Aulacomya atra la perforación dejada 
por Trophon geversianus es relativamente 
más cilindrica, es decir con menor diferencia 
entre diámetros externo e interno. Este tipo de 
perforación se asemeja más a la perforación 
producida por el otro depredador Xymenopsis 
muriciformis sobre valvas de Mytilus chilen- 
sis, Tawera gay i y Hiatella solida. 

Sólo en una oportunidad se observó una 
perforación producida por Xymenopsis murici- 
formis en una valva de Aulacomya atra, y si se 
compara su morfología con la perforación de 
Trophon geversianus sobre la misma presa, 
no se detectan diferencias morfológicas, in- 
cluso tampoco por su ubicación ya que ambos 
depredadores dejan sus marcas en el borde. 

También cabe mencionar en relación a 
Mytilus chilensis y Aulacomya atra que ejem- 
plares previamente perforados por Trophon 
geversianus fueron capaces de reparar su 
valva en la zona interna exactamente opuesta 
al sitio de la perforación. Este mecanismo de 
reparación también fue observado por Grif- 
fiths & Blaine (1994) en Mytilus galloprovin- 
cialis del hemisferio norte. 

Otro aspecto que merece ser destacado es 
la selectividad por un sitio particular de la 
valva. El registro fósil muestra que en los 
murícidos sensu lato las perforaciones 
pueden estar distribuidas al azar (Aitken & 
Risk, 1988;Tull&Bohning-Gaese, 1993) o ser 
selectivas (Vermeij et al., 1989; Guerrero & 
Reyment, 1988). Las observaciones de este 
trabajo, y las obtenidas previamente por 
Gordillo (1994) y Amuchástegui (1995), tam- 
bién indican selectividad en relación a este 
parámetro, en coincidencia con lo señalado 
por Vermeij et al., (1989) y Guerrero & 
Reyment (1988) para otras especies de este 
grupo. El sitio elegido por Trophon gever- 
sianus para realizar su perforación varía en 
las distintas presas, y estaría condicionado 
por la morfología de la presa en relación a su 
habitat. 

En relación a la selectividad por una valva 
en particular, los resultados muestran que no 
existe selectividad o preferencia por una de 
las valvas ya sea derecha o izquierda, lo que 
se atribuye a que las presas exponen indistin- 
tamente las dos valvas al depredador. 

Las diferencias registradas al analizar la 
selectividad por un tamaño particular de las 
presas quizás se relacione con otros aspec- 
tos no considerados en este trabajo como la 
existencia de otros depredadores, o con los 



tamaños relativos que alcanzan las presas. 
La selectividad observada en Mytilus chilen- 
sis coincide con la selectividad registrada por 
Zaixso & Bala (1995) en poblaciones natu- 
rales de esta especie a una menor latitud, en 
Santa Cruz (Argentina). La ausencia de se- 
lectividad por tamaño en Tawera gayl y 
Hiatella solida concuerda con los resultados 
obtenidos por Aitken & Risk (1 988) para la es- 
pecie antes mencionada del hemisferio norte. 

El éxito de depredación con un rango varia- 
ble entre un 63.7-100% entre las distintas 
presas permiten considerar a Trophon gever- 
sianus como un depredador eficiente. Esta 
característica podría constituir una ventaja 
evolutiva si se considera que una perforación 
incompleta representa un gasto energético 
inútil, tal como lo considera Kabat (1990) para 
los natícidos. 

Los índices de depredación que variaron 
entre bajos a moderados indicarían que Tro- 
phon geversianus es responsable de la mor- 
talidad de una proporción baja a moderada de 
las poblaciones de las distintas presas. 

Finalmente, para interpretar las variaciones 
de los tiempos de depredación de Trophon 
geversianus sobre las distintas especies, 
además del tamaño de la presa habría que 
analizar otros aspectos no considerados en 
este trabajo, como el grosor de la valva y/o su 
naturaleza mineralógica. 

AGRADECIMIENTOS 

Las autoras agradecen al Prof. Geerat J. 
Vermeij y a un arbitro anónimo por sus valio- 
sos comentarios. También al Dr. Nermesio 
San Román por el apoyo brindado como en- 
cargado de la sección de Biología Marina del 
CADIC. 

Este trabajo ha sido subsidiado con fondos 
propios y realizado como parte del trabajo de 
Investigador Asistente del Consejo Nacional 
de Investigaciones Científicas y Técnicas 
(CONICET) de S.G. y de una beca de la 
Provincia de Tierra del Fuego otorgada a S.A. 

LITERATURA CITADA 

AMUCHASTEGUI, S., 1995, Depredación del mejil- 
lón y cholga del Canal Beagle por gastrópodos 
perforadores de valvas. Informe técnico no publi- 
cado de la Secretaría de Planeamiento de la 
Provincia. 58 pp. 

AITKEN, A. E. & M. J. RISK, 1988, Biotic interac- 
tions revealed by macroborings in arctic bivalve 
molluscs. Lethaia, 21: 339-350. 



DEPREDACIÓN DE TROPHONIDAE 



91 



CARRIKER, M. R., 1981, Shell penetration and 
feeding by naticacean and muricacean predatory 
gastropods: a synthesis. Malacologia, 20 (2): 
403-422. 

CARRIKER, M. R. & L. G. WILLIAMS, 1978, The 
chemical mecanism of shell dissolution by preda- 
tory boring gastropods: a review and an hypothe- 
sis. Malacologia, 17 (1): 143-156. 

GORDILLO, S., 1994, Perforaciones en bivalvos 
subfósiles y actuales del Canal Beagle, Tierra del 
Fuego. Ameghiniana (Rev. Asoc. Paleont. Ar- 
gent.), 31 (2): 177-185. Buenos Aires. 

GORDILLO, S., 1995a, Muricid gastropod préda- 
tion on Holocene bivalves from Tierra del Fuego, 
Subantarctic Region. En: International Sympo- 
sium on the Paleobiology and Evolution of the 
Bivalvia, Abstracts, 10. Drumheller, Canada. 

Gordillo, S., 1995b, Moluscos australes. Bivalvos y 
caracoles de las costas del extremo sur de 
América. Zagier & Urruty Publ., 115 pp. Buenos 
Aires. 

GRIFFITHS, С. L. & M. J. BLAINE, 1994, Non-fatal 
cropping of large mussels by drilling whelks, 
Nucella cingulata (Linnaeus, 1771). Journal of 
Molluscan Studies. 60: 346-348. 

GUERRERO, S. & A. REYMENT 1988, Differen- 
tiation between the traces of prédation of muri- 
cids and naticids in Spanish Pliocene Chlamys. 
Estudios Geológicos, 44: 317-328. 

ITURRASPE, R., R. SOTTINI, С SCHROEDER & J. 
ESCOBAR, 1989, Hidrología у variables climáti- 
cas del Territorio de Tierra del Fuego. Información 
básica. Contribución CADIC. 7. Ushuaia. 

KABAT, A. R., 1990, Predatory ecology of naticid 
gastropods with a review of shell boring préda- 
tion. Malacologia. 32 (1): 155-193. 

TULL, D. S. & K. BOHNING-GAESE, 1993, Patterns 
of drilling prédation on gastropods of the family 
Turritellidae in the Gulf of California. Paleobiology, 
19:476-486. 

VERMEiJ, G. J., 1980, Drilling prédation of bivalves 
in Guam: some paleoecological implications. 
Malacologia, 19:329-334. 

VERMEIJ, G. J., E. С DUDLEY & E. ZIPSER, 1989, 
Successful and unsuccessful drilling prédation in 
Recent pelecypods. The Veliger, 32: 266-273. 



ZAIXSO, H. E. & L. O. BALA, 1995, Relaciones de 
tamaño en la predación de Trophon geversianus 
sobre mitilidos. Resúmenes del VI Congreso 
Latinoamericano de Ciencias del Mar, 206. Mar 
del Plata, Argentina. 



Revised ms. accepted 27 May 1997 



APÉNDICE 

Ubicación sistemática de las especies 
consideradas 

Clase Gastropoda 
Subclase Prosobranchia 
Orden Neogastropoda 
Superfamilia Muricoidea 
Familia Trophonidae 

Trophon geversianus (Pallas) 
Xymenopsís muriciformis (King) 

Clase Bivalvia 
Subclase Pteriomorpha 
Orden Mytiloida 
Familia Mytilidae 

Mytilus chilensis (Hupé) 
Aulacomya atra (Molina) 
Subclase Heterodonta 
Orden Veneroida 

Familia Veneridae 

Tawera gay i (Hupé) 
Orden Myoida 
Familia Hiatellidae 

Hiatella solida (Sowerby) 



MALACOLOGIA, 1998, 39(1-2): 93-111 

CLADISTIC ANALYSIS OF THE XANTHONYCHIDAE (= HELMINTHOGLYPTIDAE) 
(GASTROPODA: STYLO M M ATOP HO RA: HELICOIDEA) 

Maria Gabriela Cuezzo 

Facultad de Ciencias Naturales e Instituto Miguel Lillo, Universidad Nacional de Tucumán- 
CONICET, Miguel Lillo 205, 4000 Tucumán, Argentina 

ABSTRACT 

A cladistic analysis of the family Xanthonychidae (= Helminthioglyptidae) was carried out. The 
data set consisted of 34 characters and 25 terminal taxa (including the four outgroups: Oreohelix, 
Neoheiix, Helix, and Bradybaena). Two different analyses were performed using the program 
Hennig86. The preferred hypothesis is represented by one of the two trees obtained after suc- 
cessive weighting in the first analysis, which has the same topology of one of the original set of 
50. The conclusions of this study suggest that: (1) the family Xanthonychidae as defined by 
Pilsbry (1939) and Zilch (1959-1960) is a paraphyletic group, (2) Bradybaenidae, Helicidae and 
Xanthonychidae form a monophyletic group and therefore should be analyzed together as a unit, 
(3) based upon the preferred hypothesis, three monophyletic groups can be defined, although 
taxonomic changes will have to wait until a new analysis of the Bradybaenidae-Xanthonychidae- 
Helicidae group is performed. 

Key Words: Stylommatophora, Helicoidea, Xanthonychidae, cladistics, phylogeny, characters. 



INTRODUCTION 

The Xanthonychidae (= Helminthogly- 
ptidae) are a large group of land snail and 
semislug genera distributed along the Am- 
ericas. Pilsbry (1939), while describing the 
land snail fauna of North America, established 
the family Helminthoglyptidae, composed of 
all the "American dart-bearing helices." In this 
work, he named only five (Helminthoglyp- 
tinae, Sonorellinae, Humboldtianinae, Cepo- 
liinae and Epiphragmophorinae) out of eight 
subfamilies he included in the Helmintho- 
glyptidae. The remaining three subfamilies 
are probably the Central American groups 
(Pilsbry, 1900, 1927): Lysinoinae, Xanthony- 
chinae, and Metostracinae. Zilch (1959-1960) 
gave a complete list of the subfamilies and 
genera included in the Helminthoglyptidae. 
Richardson (1982) catalogued the Helmintho- 
glyptidae at species level. 

Baker (1943, 1959) pointed out that the 
name Xanthonychidae Strebel & Pfeiffer, 
1879, has priority over Helminthoglyptidae 
Pilsbry, 1939. Xanthonyx was also the first 
genus of an American helicoid described 
(Fischer & Crosse, 1 872). However, only a few 
authors adopted Xanthonychidae as the cor- 
rect name for the family (Nordsieck, 1987). 
Helminthoglyptidae continued to be used to 



refer to the North American helicoids (Roth, 
1996), while Xanthonychidae was employed 
for the Central and South American helicoids 
(Emberton, 1991; Miller & Naranjo-Garcia, 
1991; Schileyko, 1991). Other authors, how- 
ever, continued using the name Helmintho- 
glyptidae for all the American helicoids (North, 
Central and South) (Zilch, 1959-1960; Solem, 
1978; Richardson, 1982). For a complete 
chronologic review of previous studies and 
tendencies, see Roth (1996). As a result, 
whenever the names Helminthoglyptidae or 
Xanthonychidae are used, it is not clear to 
which genera the authors are referring, unless 
they state which convention they are follow- 
ing. 

For years many authors (Pilsbry, 1939; 
Nordsieck, 1987; Miller & Naranjo-Garcia, 
1991; Schileyko, 1991) have searched for a 
coherent definition of the family. Pilsbry's 
(1939) was the concept that has lasted 
longest. His definition of the Xanthonychidae 
{- Helminthoglyptidae) is a combination of 
character states, none of which represents 
true synapomorphies for the group. The geo- 
graphic disthbution became important in the 
identification of the genera. Similarly, the ab- 
sence of clearly defined synapomorphies lead 
to poor definitions of the other families in- 
cluded in Helicoidea (Scott, 1996). Recent 



93 



94 



CUEZZO 



studies have appeared reviewing the Heli- 
coidea (Nordsieck, 1987; Schileyko, 1991), 
but they failed to propose their hypotheses in 
a testable way. The only two studies using 
cladistic analysis to formulate phylogenetic 
hypotheses within Helminthoglyptidae are 
Pearce (1990) and Roth (1996). 

The anatomy of several components of the 
Xanthonychidae is still poorly known. Although 
several papers have been published with 
anatomical data on the Central American 
(Binney, 1879; Pilsbry, 1894, 1900; Baker, 
1942; Thompson, 1959; Miller, 1987; Tillier, 
1989; Cuezzo, 1996) and South American 
taxa (Hesse, 1930; Fernandez & Rumi, 1984; 
Tillier, 1989; Cuezzo, 1997), they are still the 
least known components of the Xanthony- 
chidae. 

Three different problems occurred in the 
published literature of the Xanthonychidae: 
(1) Monographic revisions are scarce, and 
most of the existing studies focus on the de- 
scription of single species upon which phylo- 
genetic hypotheses are built (Pilsbry, 1900; 
Pilsbry & Cockerell, 1 937; Hylton Scott, 1 951 , 
1962; Parodiz, 1955; Mass, 1962; Miller, 1970, 
1971, 1976a, b, 1981, 1985). (2) The taxo- 
nomic differences among the genera included 
in the Xanthonychidae are mainly based on 
two organs: mucous glands and dart sac (= 
dart complex), with different combinations of 
their character states (generally shape and 
number). In this way, phylogenetic assump- 
tions and classifications (Miller & Naranjo- 
Garcia, 1991; Schileyko, 1991) had been car- 
ried out on single characters or at least 
single-organ systems (generally the genital 
system) (Bieler, 1992). (3) Phylogenetic rela- 
tionships of the genera are established on ar- 
bitrarily narrative character transformations 
(Nordsieck, 1987; Miller & Naranjo-Garcia, 
1 991 ; Schileyko, 1 991 ). As stated by Nixon & 
Carpenter (1993), there is no clear way to es- 
tablish whether a character is "primitive" or 
"derived" prior to cladistic analysis, and actu- 
ally there is no need for the creation of "evolu- 
tive scenarios" to explain the possible direc- 
tion of character transformation. 

This study was undertaken (1) to test the 
monophyly of the Xanthonychidae (= Hel- 
mintoglyptidae) (2) to carry out a cladistic 
analysis in order to hypothesize the relation- 
ships among the components of the family in 
a testable way, and (3) to review the current 
classification of the Xanthonychidae based on 
the cladograms obtained. 



MATERIALS AND METHODS 

The adult alcohol preserved snails used for 
this study belong to the following Institutions: 

ANSP = Academy of Natural Sciences of 
Philadelphia, Pennsylvania, USA 

INBIO = Instituto de Biodiversidad, Costa 
Rica. 

UF = Florida Museum of Natural Sciences, 
Gainesville, Florida, USA 

FMNH = Field Museum of Natural History, 
Chicago, Illinois, USA 

FML = Fundación Miguel Lillo, Tucumán, 
Argentina 

In the case of the material from ANSP, the 
first number cited after the institution corre- 
sponds to dry lots consulted every time that a 
verification of the species determination was 
necessary. A number preceded by the letter 
"A" (in the case of material from ANSP or 
FML) corresponds to alcohol-preserved ma- 
terial used in this study. All catalogue numbers 
from other institutions correspond to alcohol- 
preserved material. A complete list of the taxa 
studied is documented in Appendix 1. 

The characters used in this study (except 
characters 30 and 31 , which are based on 
published literature) are based on a selection 
made after a study of the anatomy of the pal- 
liai, digestive, reproductive and nervous sys- 
tems, and external morphology of the type 
species of each genus (Appendix 1). Speci- 
mens were dissected under a Wild M3C mi- 
croscope. Illustrations were made with the aid 
of a camera lucida. Illustrations and a brief de- 
scription of the characters are included only 
when there is no agreement with the literature 
or a clarification of a specific character is 
needed. Shell characters have not been con- 
sidered for this analysis under the assumption 
that they are inadequate for reconstructing 
phylogenetic history (Nordsieck, 1986; Em- 
berton, 1995). However, the study of the 
sculpture, apertural barriers and composition 
of the shell could lead in the future to informa- 
tive characters (Solem, 1978; Emberton, 
1995). 

Cladistic analyses were carried out using 
the computer program Henning86 (Farris, 
1988). The program DADA (Nixon, 1992) was 
used for the construction of the data matrix 
and CLADOS (Nixon, 1 992) for the analysis of 
the character distribution on the trees. Jack- 
knife, a statistical test for homoplasy, was per- 
formed with the program NONA (Goloboff, 
1993). FQ, a program provided by P. Goloboff, 



PHYLOGENETICS OF XANTHONYCHIDAE 



95 



integrated jackknife results from 50 replicates. 
FQ reads a "tread" statement (resulting trees 
in parenthetical notation) and calculates the 
majority rule consensus tree showing the fre- 
quencies above 50% (frequencies of 100% 
are not indicated). 
The frequency index is: 



fi 



(T-2) 



where fi is frequency of group i of consensus 
tree, and T is the number of terminals, note 
that these depend on the cutoff value. 

The data set includes 34 characters and 
25 taxa (including the outgroups) (Table 1). 
Thirteen characters are multistate and were 
coded as non-additive so that any state could 
transform into any alternative state at an equal 
cost. Character polarity is derived from the 
analysis rather than being an a priori assump- 
tion (Nixon & Carpenter, 1993). The assign- 
ment of "missing character" ("?") to some taxa 



is not mentioned in the character description 
but indicated in the data matrix. Some apo- 
morphies, although not informative for con- 
structing phylogenies, were included among 
the characters because they are usfull for the 
characterization of certain terminals. Cladistic 
analysis was initially carried out with HennigSO 
using the commands "mh*;bb*;". Successive 
weighting (Farris, 1969; Carpenter, 1988) was 
used after the initial runs. From the cladograms 
obtained after successive weighting, one 
cladogram was selected. When more than one 
tree was obtained, they were sumarized in a 
Nelson consensus tree. 



RESULTS 

Selection of the Ingroup and Outgroup Taxa 

The xanthonychid (= helminthoglyptid) in- 
group taxa considered in this analysis are 
those included in the family by Zilch (1959- 



TABLE 1 . Data set containing thirty four characters and twenty five taxa used 
in analysis #1.Two additional characters (#35-36) were used in analysis #2. 



10 



20 



30 



Outgroups 
Oreohelix 
Neohelix 
Helix 
Bradybaena 

Xanthonychidae 
Cepolis 
Polymita 
Dialeuca 
Helminthoglypta 
Epiphragmophora 
Monadenia 
Sonorella 
Eremarionta 
Micrarionta 
Humboldtiana 
Charodotes 
Plesarionta 
Bunnya 
Tryonigens 
Trichodiscina 
Cryptostrakon 
Lysinoe 
Leptarionta 
Metostracon 
Xanthonyx 
Xerarionta 



123456789 123456789 123456789 1234 56 
-000000100 3011101000 0000000001 01100 00 
-000001002 1011101000 0000000001 70010 00 
-000000001 0011101120 1000000000 10010 11 
-000000011 0000101021 0000000100 10011 24 



-00000001 1 
-00000201 1 
-00000200? 
-00000001 1 
-00000001 1 
-000002012 
-000002012 
-000000010 
-000000010 
-000002012 
-00000001 1 
-00000201 1 
-010010011 
-100000010 
-000000070 
-001110010 
-100001010 
-100007017 
-001110010 
-010010010 
-000002010 



0021131111 
1011131111 
0000130011 
0000111111 
0100101111 
1021111111 
0022101100 
1021101021 
7021101121 
1021101120 
0000121111 
1021101121 
2012101120 
0021001000 
0022101121 
0011201121 
1021101120 
2072101121 
0011101121 
2021101120 
1000101121 



0100001200 
0100001200 
0100001200 
0010002100 
0000002200 
0000003000 
0000000000 
0021000100 
0001000700 
1000000000 
0010002100 
0001000000 
1000113010 
0000000000 
0000000000 
0000000000 
1000000100 
0000000000 
0000023000 
1000017000 
1001000000 



10011 23 
10011 23 
10021 23 
10021 22 
10011 24 
10021 21 
10021 00 
10021 22 
10021 22 
10021 13 
10021 22 
10011 22 

70011 13 

10020 00 

10021 24 

70012 21 

10010 13 
10021 24 
70010 24 
70010 14 

10011 32 



96 



CUEZZO 



1960), with the exception of Averellia Ancey, 
Dinotropis Pilsbry & Cockerell, Sonorelix 
Berry, and Setipelis Pilsbry, for which no alco- 
hol-preserved material was available. For the 
purpose of the analysis, subgenera as well as 
genera (sensu Zilch, 1959-1960) have been 
used as taxonomic units when material was 
available. This is the case for Plesarionta 
Pilsbry, Xerarionta Pilsbry, and Eremarionta 
Pilsbry, which were considered by Zilch to be 
subgenera of Micrarionta Ancey Charodotes 
Pilsbry, considered a subgenus of Helmintho- 
glypta Ancey by Zilch, and Trichodiscina 
Martens, considered a subgenus of Averellia, 
are also treated as separate terminals. 

For each genus, the type species, when 
available, was used as representative of the 
group. The only exceptions are Xanthonyx 
Crosse & Fischer, Eremarionta Pilsbry, Lepta- 
rionta Fischer & Crosse, and Xerarionta 
Forbes. Consequently, other species were se- 
lected as representative of those genera. 

For the selection of outgroups, a previous 
cladistic analysis was used (Nixon & Car- 
penter, 1 993). Based on Emberton (1 991 ), the 
Bradybaenidae, Helicidae, Oreohelicidae, and 
Polygyridae were chosen as outgroups. Al- 
though the use of more than one outgroup is 
not necessary, this option was prefered, with 
the idea that the cladistic inferences might be 
better founded. Outgroups were treated as all 
the other terminals in order to test the mono- 
phyly of the ingroup. The Bradybaenidae were 
traditionally assumed to be the sister group of 
the Helminthoglyptidae (Schileyko, 1978; 
Roth, 1996). In each case, the nominal genus 
was used as representative of its family, except 
in the case of Polygyridae. 

My results are presented in Figures 1-3 
and are discussed below. 

Character Descriptions 

External Morphology: 

Character 1:Ta\\ keeled (Figs. 4, 6): 

Longitudinal row of plaques in mid-dorsal 
tail. Character states: ("0") absent; ("1") pre- 
sent. 
Character 2: TaW horn (Figs. 5, 6): 

Projection on the end of the tail with the ap- 
pearance of a horn. Character states: ("0") ab- 
sent; ("1") present. 

Palliai System: 

Character 3: Lobes of kidney: 

This character is generally associated with 
the reduction in space due to the limacization 



process (Tillier, 1984). Character states: ("0") 
absent; ("1") present. 

Character 4: Position of the heart relative to 
the kidney (Fig. 7): 

The common position of the heart in 
Stylommatophora is to the left of the kidney 
with the roof of the lung observed ventrally. 
However, the heart is partially surrounded by 
the kidney in Cryptostrakon and Metostracon. 
Although this character has usually been as- 
sociated with the morphology of a semislug, in 
the other two semislug genera of the 
Xanthonychidae, the position of the peri- 
cardium is to the left of the kidney, as in most 
of snails. Character states: ("0") to the left of 
the kidney; ("1") partially surrounded by the 
kidney. 

Character 5: Relation of the mantle to the 
shell: 

Character states: ("0") the mantle does not 
enclose the shell; ("1") mantle entirely en- 
closes the shell. 
Character 6: Diaphragm: 

The diaphragm or lung floor forms the base 
of the pulmonary cavity Character states: ("0") 
diaphragm thin, transparent, and membra- 
nous; ("1") diaphragm thick, muscular, not 
transparent; ("2") diaphragm thin but not 
transparent showing some muscular strands. 
Character 7: Anus position (figure and 
description in Emberton, 1991). Character 
states: ("0") near mantle collar; ("1") recessed 
from collar. 

Reproductive System: 

Character 8: Fertilization Pouch-Sperma- 

thecal complex (FPSC): 

Character states: ("0") the FPSC is com- 
pletely free of the albumen gland; ("1") FPSC 
totally or partially embedded in the base of the 
albumen gland. 
Character 9: Penial sheath: 

Character states: ("0") penial sheath ab- 
sent; ("1") sheath thin, membranous; ("2") 
sheath thick, muscular. 
Character 10: Internal penial structure: 

Character states: ("0") with longitudinal 
ridges; ("1") with one or two pilasters; ("2") 
smooth or with wrinkles; ("3") basal portions 
with several pilasters and upper portion with 
pustules. 

Character 11: Penial muscular band: (de- 
scribed and illustrated in Cuezzo, 1997). 
Character states: ("0") absent; ("1") present. 
Character 72.- Verge (Figs. 8, 9): 

Character states: ("0") verge absent; ("1") 
verge present, with thin projections or termi- 



PHYLOGENETICS OF XANTHONYCHIDAE 

Oreohelix 



97 



Neohelix 



Helix 



Monadenia 
— Sonorella 



Eremarionta 



Micrarionta 

Humboldtiana 

Plesarionta 

Tryonigens 



Trichodiscina 
Lysinoe 



Leptarionta 
Xerarionta 

Bunnya 

Xanthonyx 

Cryptostrakon 



80 



86 



r 



78 



Metostracon 

— Bradybaena 

Epiphragmophora 

I Hebninthoglypta 

I Charodotes 

— Cepotis 

Polymita 

Dialeuca 



84! 



66 



FIG. 1 . Consensus tree generated from 50 trees. Nurnbers in the nodes are the frequencies above 50% ob- 
tained with FQ after Jackknife (frequencies of 100% are not indicated). 



7 10 31 32 33 



-Oreo helix 



13 110 
6 9 10 

-Neohelix 



9 20 

f-i 

1 1 



17 18 29 30 



9 13 24 28 34 



10 20 25 26 



12 113 



' — ^Xanthonyx 

34 

Ш -Cryptostrakon 



14 17 18 33 




Lysinoe 



lili 



19 34 
1 1 



9 12 13 27 

-i-i-i-i- 

10 1 



radybaena 



Helminthoglypta 
Charodotes 



11 19 

Щ -^Epiphragmophora 



epolis 



10 12 13 

Г-Ш -^-^Polymita 



X 16 17 33 



-Dialeuca 



,3 I — Trichodiscina 

I 10 



Sonorella 




Humboldtiana 



Xerarionta 
Eremarionta 

o 2 1 

' — Micrarionta 

FIG. la. Prefered phylogenetic hypothesis for Xanthonychidae, generated by Henig86 and constructed using 
CLADOS, from data in Table 1. Filled dash marks represents synapomorphies, gray dash marks represents 
homoplasies. There are no reversals. 



PHYLOGENETICS OF XANTHONYCHIDAE 



99 



13 11 
b 9 lU 33 



-Oreohelix 



1 14 

I — H-Tryo/j/ge/is 



8 12 29 30 33 

-n-H-H 



Sonorella 



I — Trichodiscina 

I 10 

Leptarionta 

17 27 



Bradybaena 



6 9 111 12 13 lb 



2 2 12 13 

„ ,-, 3^ I — Helmiiithoglypta 
И Щ ^ 

■ Щ Ш \^ 

' — m-Charodotes 
-Epiphragmophora 

6 8 16 17 33 

il l Ш -^Dialeu ca 



15 21 26 36 



I — ^Plesarionta 

I 

12 13 35 

ill Xerarionta 



35 36 
-1-t- 



3 

17 22 27 

■^-Щ-Ш -Eremarionta 

2 I 

' — Micrarionta 

Ь 9 

umboldtiana 

1 6 27 



33 34 

-i-i 

1 



10 12 36 
о 1 1 



Helix 



Ч 13 24 28 34 36 



10 25 26 
12 13 



Виппуа 



1 2 I I 1 3 
12 

anthonyx 



Metostracon 



FIG. 2. Cladogram #2 for Xanthonychidae, generated by Henig86 and constructed using CLADOS. 
Alternative hypothesis adding characters 35 and 36. Filled dash marks are synapornorphies, gray dash 
marks are homoplasies. 



100 



CUEZZO 
-Oreohelix 
-Neohelix 

Tryonigens 

-Sonorella 
r-Trichodiscina 
-Leptarionta 

— Bradybaena 



-Monadenia 



—Helminthoglypta 
' — Charodotes 
I — Epiphragmophora 

Dialeu ca 



I — Cepolis 
Polymita 



-Eremaríonta 



—Micrarionta 

Plesarionta 

Xerarionta 



Humboldtiana 
Lysinoe 
— Helix 



Bunnya 

' — Xanthonyx 
— Cryptostrakon 



-Metostracon 



FIG. 3. Consensus of 22 trees, second analysis, generated by Henig86 and constructed by CLADOS. 



PHYLOGENETICS OF XANTHONYCHIDAE 



101 






FIG. 4. Tryonigens: Tail keeled (Tk), character 1(1). Bar = 0.1 cm. 
FIG. 5. Sunnya.- Tail horn (Th), character 2(1). Bar = 3 mm. 

FIG. 6. Epiphragmophora:Jä\\ morphology, character 1(0), 2(0). Td = tail middorsal groove. Bar = 1 cm. 
FIG. 7. Bradybaena: Position of heart (H) respect to the kidney (K), character 4(0). Bar = 3 mm. 
FIG. 8. Polymita: Portion of the penial complex showing small verge (Ve), character 13(1), with terminal thin 
projections (P), character 12(1). Bar = 3 mm. 

FIG. 9. Sonorella: Penial complex showing verge (Ve) without terminal projections, character 12(2), occupy- 
ing half of the penial sac, character 13(2). Bar = 3 mm. 



nal papillae; ("2") verge present, but without 
terminal papillae in which case it is stout. 
Character 13: Relative size of the verge with 
respect to penial sac (Figs. 8, 9): 

Character states: ("0") absent; ("1") present, 
small to medium sized verge, generally lo- 
cated in the upper portion of the penial sac; 



("2") present, large verge, occupying half or 
more of the penial sac. 
Character 14: Atrium: 

Character states: ("0") absent; ("1") present, 
with longitudinal folds; ("2") present, with 
transversal thin folds. 
Character 15: Atrial sac (Figs. 10, 11): 



102 



CUEZZO 



The atrial sac is a projection or prolongation 
of the atrium. In general, both the atriunn and 
the atrial sac presents the same internal 
sculpture. Character states: ("0") absent; ("1") 
present, with internal sculpture consisting of 
thin folds; ("2") present, with a wide pilaster in 
the internal wall; ("3") present, with small ir- 
regular pustules in internal wall. 
Character 16: Epiphallus: 

The epiphallus is the portion of the penial 
complex between the penis and the insertion 
of the vas deferens. Its delimitation is easy 
when the penis bears a verge that clearly 
marks the limit between penis and epiphallus. 
When the verge is absent, the internal struc- 
ture of the epiphallus is an important element 
in determining its limits. The portion of the pe- 
nial complex termed "penis or preputial cham- 
ber" (Gregg & Miller, 1976; Miller, 1981), and 
"double tube of the upper part of penis" (Miller, 
1985) are considered here to be homologous 
to the lower portion of the epiphallus: 
Character states: ("0") absent; ("1") present. 
Character 17: Flagellum: 

In the penial complex, the epiphallus con- 
tinues as a blind duct that can have different 
lengths and that decreases in diameter to- 
ward the tip. In the type species of the genus 
Sonorella, a reduced flagellum is present, 
known in the literature as "epiphallic caecum." 
Because of its position this structure is con- 
sidered here to be homologous to the flagel- 
lum present in the other Xanthonychidae. 
Character states: ("0") absent; ("1") present. 
Character 18: Dart Sac insertion (Figs. 12, 
13): 

The dart sac is a muscular blind sac usually 
containing a calcareous dart, which functions 
in stimulation during copulation. Character 
states: ("0") absent; ("1") present, one dart sac 
inserted in the atrium or in the atrial sac, cylin- 
drical to round; ("2") present, one to four dart 
sacs seated on the vagina. 
Character 19: Mucous glands inserted in dart 
sac (Fig. 14): 

The mucous glands that insert in the dart 
sac are considered homologous because 
they share the same position and probable 
function. Character states: ("0") absent; ("1") 
present, generally bearing one or more ducts. 
Character 20: Mucous glands inserted in 
vagina (Fig. 15): 

The mucous glands inserted in the vagina 
are considered to be homologous because 
they share the same position and probably the 
same function. Character states: ("0") absent; 
("1") present. 



Character 21: Mucous glands inserted in 
atrial sheath (Fig. 12): 

The ducts of these glands are inserted be- 
tween the folds of the atrial sheath. These 
glands are not homologous to others in the re- 
productive system. Character states: ("0") ab- 
sent; ("1") present. 

Character 22: Bulbous reservoirs on mucus 
gland ducts (Figs. 16, 17): 

The reservoirs are swellings in the ducts. 
Character states: ("0") absent; ("1") present, 
with glands ending in a common duct and 
("2") present, with glands ending in separate 
ducts. 

Character 23: Distal portion of mucous glands 
(Fig. 17): 

Character states: ("0") glands not expanded 
in their distal portion; ("1") distal portions of 
mucous glands expanded, flattened and 
spread upon vagina, dart or base of penis. 
Character 24: Vaginal diverticulum: 

The vaginal diverticulum is a round, cecil 
evagination in the ventral side of the vagina 
under the row of dart sacs. There is no possi- 
bility that it is another dart sac, because the 
internal structure is completely different. Also, 
no dart was found in the interior. Whereas the 
dart sacs have two thin dart papillae, the vagi- 
nal diverticulum has only longitudinal folds in 
its interior. Character states: ("0") absent; ("1") 
present. 
Character 25: A\bumen gland: 

Character states: ("0") albumen gland bean 
shaped, located in the visceral mass; ("1") al- 
bumen gland bilobate, located in the visceral 
mass; ("2") albumen gland straight, located in 
the pedal cavity. 
Character 26: Vas deferens: 

Character states: ("0") vas deferens does 
not loop around any structure; ("1") vas defer- 
ens looped around the penial retractor muscle 
close to its insertion in the epiphallus; ("2") 
vas deferens looped around the dart sac; 
("3") vas deferens looped around penis- 
epiphallus. 
Character 27: Basal genital sheath: 

The observation of this character has been 
affected in the past by the tendency to clean 
the genitalia from connective tissue or other 
membranous tissue before any observations 
are made. However, many genera of the 
Xanthonychidae show a conspicuous basal 
genital sheath that overlaps the basal female 
and male terminal genitalia. Character states: 
("0") absent; ("1") present, formed by mem- 
branous tissue; ("2") present, composed of 
thin muscular tissue. 




Ash 



FIG. 10. Charodotes: Lower genitalia showing atrial sac (As) with internal wide pilaster, character 15(2). Bar 

= 3 mm. 

FIG. 1 1 . Dialeuca: Lower genitalia showing atrium (A) and atrial sac (As) with irregularly distributed pustules 

in the internal wall, character 15(3). Bar = 3 mm. 

FIG. 12. Cepo//s.- Terminal genitalia. Dart sac (Ds) cilindrical inserting in the atnal sac (As), character 18(1). 

There are another pair of glands (Mg) inserted in the atrial sheath (Ash), character 21(1). Bar = 0.5 cm. 

FIG. 13. ßunnya.- Terminal genitalia. Dart sacs (Ds) seated on the vagina, character 18(2). Ba = bursa copu- 

latrix appendix; Be = bursa copulatrix. Bar = 2 mm. 



104 



CUEZZO 



Character 28: Bursa copulatrix sac appendix 
(Fig. 13): 

Thick and short appendix in the bursa cop- 
ulatrix sac. Character states: ("0") absent; ("1 ") 
present. 

Character 29: Bursa copulatrix duct swollen at 
the base: 

Character states: ("0") absent; ("1") present. 
Character 30: Copulation modality: 

Character states were selected from the 
published data (Webb, 1947, 1948, 1959, 
1 972; Emberton, 1 985): ("0") copulation is one 
sided; ("1") copulation is reciprocal. 
Character 31: Reproductive modality: 

Character states were selected from pub- 
lished information (Solem, 1978; Tompa, 
1984): ("0") oviparous; ("1") ovoviviparous. 
Character 32: End of penial retractor muscle: 

Character states: ("0") penial retractor in- 
serts without divisions; ("1") penial retractor 
splits in branches. 

Digestive System: 

Character 33: Internal structure of the oe- 
sophageal crop: 

The wall of the oesophageal crop presents 
different kinds of interior sculpture indepen- 
dent of the thickness of the wall. Character 
states: ("0") wall with longitudinal ridges that 
can extend along all the crop length or only 
portions of it length; ("1") wall with pustules or- 
dered as longitudinal cords or irregularly dis- 
tributed. 

Nervous System: 

Character 34: Fusion of the visceral ganglion 

(illustrated in Emberton &Tillier, 1995): 

The visceral ganglion is located in the ven- 
tral chain of the nervous system. Although the 
fusion of the ganglia is traditionally associated 
with the limacization process, surprisingly non 
fusion of the visceral ganglion was observed 
in the case of two semislug genera. This char- 
acter is discussed by Emberton & Tillier 
(1995). Character states: ("0") absent; ("1") 
present, fused with left parietal ganglion; ("2") 
present, fused with both parietal ganglia. 

Characters Added in Analysis #2 

Character 35: Position of mucous glands 

Character states: ("0") absent; ("1") inserted 
in vagina; ("2") inserted in dart sac or close to 
its base; ("3") inserted in vagina and dart sac. 
Character 36: Type and shape of mucous 
glands (according to Miller & Naranjo Garcia, 
1991) 



Character states: ("0") absent; ("1") tubular; 
("2") membranous; ("3") round compact; ("4") 
vesicular club-shaped. 

Cladistic Analysis 

Using the program Hennig86, two different 
analyses were carried out: (1 ) The initial analy- 
sis using the command "mh*; bb*;" produced 
50 trees of 99 steps in length, CI = 52, Rl = 62. 
A consensus tree summarizing the 50 original 
trees obtained is presented in Figure 1. 
Caution should be used to interpret it, because 
consensus trees contain less information, 
being generated from fundamental dado- 
grams instead of original information. After 
performing succesive weighting, two trees 
were retained, each with length 372, CI = 79 
and Rl = 78. One of them (Fig. la) is identical 
to one of the trees of the original set. The other 
differs only in the position of Micrarionta. In 
one of the trees, its position is resolved, but 
there are no characters supporting this resolu- 
tion, whereas in the other, there is no resolu- 
tion on the position of Micrarionta. Conse- 
quently, the Nelsen consensus tree (after 
succesive weighting) has the same topology 
as the one showing the unresolved trichotomy 
for (Plesarionta, Xerarionta) + Eremarionta + 
Micrarionta. There are four synapomorphies 
(characters 17, 18, 29, 30) supporting the 
monophyly of the ingroup (Bradybaenidae- 
Xanthonychidae-Helicidae). Helix '\s the sister 
group of the Bradybaenidae-Xanthonychidae 
complex, supported by character 8. The out- 
groups Oreohelix and Neohelix are clearly 
separated from the other genera. Within 
the ingroup, three monophyletic groups are 
clearly defined: (a) first, the genera Bunnya, 
Xanthonyx, Cryptostrakon and Metostracon 
with character 5 as synapomorphy; (b) sec- 
ond, IHelminthoglypta, Charodotes, Epiphra- 
gmophora, Cepolis, Polymita and Dialeuca, 
supported by character 26; (c) and third, Ere- 
marionta, Micrarionta, Plesarionta and Xerari- 
onta, supported by character 23. 

(2) A second analysis for comparative pur- 
poses has been made by adding to the same 
matrix two characters: (a) type and shape of 
mucous glands (character states as defined 
by Miller & Naranjo-Garcia, 1 991 ; #35 in Table 
I) and (b) mucous glands insertion (assuming 
that all the mucous glands are homologous, 
as it is traditionally considered, with the ex- 
ception of the glands inserted in the atrial 
sheath in the Cepoliinae, #36 in Table I). 
Concurrently, characters 19 and 20 (mucous 



PHYLOGENETICS OF XANTHONYCHIDAE 



105 




FIG. 14. /Wetosiracon.- Terminal genitalia. Mucous glands (Mg) inserted in dart sac (Ds), character 19(1). Bar 

= 0.5 cm. 

FIG. 15. /-/umbo/c/f/ana.- Terminal genitalia. Mucous glands (Mg) inserted in vagina (V), character 20(1). Ds = 

dart sac. Bar = 0.5 cm. 

FIG. 16. /-/e/m/nfA?og'/ypfa.- Terminal genitalia. Bulbous reservoirs (Br) on mucous glands ducts ending in a 

common duct (Mgd), character 22(1 ). Bar = 0.5 cm. 

FIG. 17. Eremar/onfa.- Terminal genitalia. Bulbous reservoirs (Br) on separate mucous glands ducts (Mgd), 

character 22(2). Distal portion of mucous glands (Mg) expanded and spreaded on vagina and base of penis, 

character 23(1)- Bar = 1 mm. 



106 



CUEZZO 



gland insertion in vagina and in dart sac) were 
deactivated. This analysis produced initially 
40 trees, L = 106, CI = 52 and Rl = 62, and 
after succesive weighting the number was re- 
duced to 22 trees, L = 391 , CI = 82, Rl = 84. 
One of them is illustrated in Figure 2. This tree 
has the same topology as one of the original 
set and is very similar to the consensus tree 
(Fig. 3). The differences with the first analysis 
are: (a) Sonorella and Tryonigens, the genera 
that lack a dart complex, are located basally in 
the cladogram, but they do not form a mono- 
phyletic group; (b) excluding Tryonigens, the 
synapomorphy for the rest of the genera is the 
presence of flagellum (character 17); (c) 
Monadenia is the sister group of Helmin- 
thoglypta-Charodotes, this relationship sup- 
ported by character 15 (atrial sac with internal 
thin folds). The other two monophyletic groups 
described in analysis #1 : Bunnya + Xanthonyx 
+ Cryptostrakon + Metostracon (supported by 
character 5) and Plesarionta + Xerahonta + 
Micrarionta + Eremarionta (supported by 
character 23), are also clearly delimited in the 
second analysis; (d) the genus Helix repre- 
senting the Helicidae is internested in the in- 
group. 



DISCUSSION 

Both cladistic analyses suggest that the 
family Xanthonychidae, or Helminthogly- 
ptidae as it was defined by Pilsbry (1939) and 
subsequently treated by various authors 
(Baker, 1959; Zilch, 1959-1960; Schileyko, 
1 991 ), is a paraphyletic group. The results ob- 
tained (Figs. 1, la, 2, 3) support the idea that 
the families Bradybaenidae and Helicidae 
should be jointed with Xanthonychidae, be- 
cause they form a monophyletic group. 

Baker's (1959) concept: "Since the sizes of 
families are matters of convenience or custom, 
we Americans can leave to the wisdom of our 
Old World colleagues the advisability of a sep- 
arate family for the genera of their home lands" 
had prevailed since 1959, and the Xantho- 
nychidae (American Helicoids) had been 
maintained as a separate family from the Bra- 
dybaenidae (mostly Asian Helicoids) and 
Helicidae (mainly European) without much 
justification. Consequently, geographic data 
become important in the identification of cer- 
tain genera of the Bradybaenidae-Xanthony- 
chidae-Helicidae group. 

The cladogram illustrated in Figure la is 
chosen as the preferred hypothesis, because 



is the one that best explained the data: (1) the 
mucous glands are not homologous structures 
as they are traditionally considered in pub- 
lished literature, because they have different 
positions and, therefore, should be treated as 
different characters; (2) this hypothesis (clado- 
gram #1 a) shows closest relationship between 
xanthonychid and bradybaenid snails, with 
Helix, representing the Helicidae, their sister 
group. 

The position of the Helicidae, however, 
should be carefully reconsidered when more 
genera of Helicidae and Bradybaenidae can 
be studied along with the Xanthonychidae. 

Four synapomorphies (Fig. la) support the 
Bradybaenidae-Xanthonychidae-Helicidae 
group (presence of flagellum, dart sac in- 
serted in vagina, bursa copulatrix duct not 
swollen at the base, and copulation recipro- 
cal). One synapomorphy supports the mono- 
phyly of the Bradybaenidae-Xanthonychidae 
group (fertilization pouch-spermathecal com- 
plex embedded in the base of the albumen 
gland). 

Although it is clear that it would be prema- 
ture to translate this phylogenetic hypothesis 
(cladogram #1a) into a new classification (too 
many branches of the cladogram are not 
strongly supported, and the relationships of 
the genera could also change when more taxa 
of the Helicidae and Bradybaenidae are stud- 
ied), three monophyletic groups are well de- 
fined and delimited based upon the analyses 
performed (cladogram #1a). The first is the 
semislug group composed of Cryptostrakon + 
Metostracon + Bunnya and Xanthonyx. The 
monophyly of this group is supported by char- 
acter 5 (mantle entirely concealing the shell). 
Other characters, such as shell reduction, kid- 
ney size reduction and compactation, rotation 
of the longitudinal axis of kidney, pulmonary 
cavity short and reduced, presence of sec- 
ondary respiratory structures (such as alveoli), 
detorsión of digestive tract, position of the 
stomach, oesophageal crop contained in foot 
cavity, digestive gland invading the pulmonary 
cavity, presence of air sacs, have been hy- 
pothesized by Solem (1 978) and Tillier (1 983, 
1984, 1989) as being correlated with the lima- 
cization process. For this reason, none of them 
were used in the final analysis (#1 a) in order to 
avoid redundant characters (in case Solem 
and Tillier are correct). However, when these 
characters were included in preliminary analy- 
ses, they appeared as synapomorphies of the 
semislug group. At this point, it is difficult to test 
whether Solem and Tillier's hypotheses are 



PHYLOGENETICS OF XANTHONYCHIDAE 



107 



simply evolutionary scenarios because, as 
proposed, they appear to be based on circular 
reasoning. Characters 3 and 4, although de- 
scribed also as correlated with the limaciza- 
tion process, were maintained in the analysis, 
because the lobes of the kidney (character 
3) and the position of the heart internested in 
the kidney (character 4) are present only in 
Cryptostrakon and Metostracon but not in the 
other semislug genera. Similarly, character 5 
(mantle entirely enclosing the shell) has been 
used, because it is not possible to correlate 
it with a "semislug state." The reason for this 
is that there are some semislugs that have 
the shell exposed — Vitrina: Vitrinidae; 
Binneya Cooper (Pilsbry, 1939); Pellicula de- 
pressa.- Bulimulidae (Van Mol, 1968, 1971) — 
others with the shell partially enclosed by 
the mantle — Austenia, Parmarion: Heli- 
carionidae (Solem, 1966) — and still in others 
the shell is completely concealed by the man- 
tle — Peltella iheringi: Bulimulidae (Van Mol, 
1968), Malagarion paenelimax: Helicarioni- 
dae (Tillier, 1979) and the xanthonychid 
genera studied here. In other words, it is not 
possible to infer that all semislugs have 
the shell concealed by the mantle due to 
the changes in shape (mainly reduction of 
visceral mass). Also, if the trend towards a 
"slug stage" is a real phenomenon, all slugs 
should have the shell completely concealed 
by the mantle, which is true only in some 
cases (some Arionidae) but not in others, for 
example in Testacella (Testacellidae) and 
Daudebardia (Zonitidae). Solem (1978) and 
Timer's (1983, 1984, 1989) argumentation 
that all the characters mentioned above 
represent compensations for space alter- 
ations in the process of slug evolution must be 
reconsidered with a historical perspective. 
Within the Xanthonychidae, the semislug 
stage has occured only once, and thus the 
monophyly of the semislug genera is sup- 
ported in this study. 

The second monophyletic group delimited 
in the two analyses and also well supported 
by the statistical test of homoplasy (Fig. 1) is 
composed of Helminthoglypta + Charodotes + 
Epiphragmophora + Cepolis + DIaleuca and 
Polymita. This group occurs in all most parsi- 
monious cladograms and thus is "unequivo- 
cally supported" by the data. The monophyly 
of this group is supported by "vas deferens 
looped around the dart sac," a character that 
later changes to "looped around the penial re- 
tractor muscle" in the clade Cepolis + Polymita 
+ DIaleuca. The muscular basal genital 



sheath is the synapomorphy supporting the 
relationship between the Cepoliinae and 
Epiphragmophora. Baker (1943) had pointed 
out that Helminthoglypta and Cepolis, placed 
by Pilsbry (1939) in different subfamilies, 
could be related based on the common pres- 
ence of the "dart sheath" (considered here as 
the basal genital sheath, character 27). Later, 
Baker (1961) proposed that the Cepolinae 
(Cepolis, Polymita, and DIaleuca) should be 
included with Helminthoglypta and Micra- 
rionta in the Helminthoglyptinae as defined by 
Pilsbry (1939). However, both Nordsieck 
(1987) and Roth (1996) denied any relation 
between the two genera because of the "com- 
pletely differently constructed glands" that 
they possess. In this study, the clade com- 
posed by Helminthoglypta + Charodotes + 
Epiphragmophora + Cepolis + Polymita and 
DIaleuca appeared as a monophyletic group 
with the addition of Monadenia in the second 
analysis (cladogram #2). The genus Brady- 
baena (representing the Bradybaenidae) is 
the sister group of this clade in both analyses, 
although its position is not strongly supported 
by any synapomorphy. 

The third monophyletic group is composed 
of Eremarionta + Micrahonta + Plesahonta 
and Xerarionta. The monophyly of this group 
is sustained by character 23 (expansioned 
and spread of distal portion of mucous 
glands). Eremarionta, Plesahonta and Xera- 
rionta were originally proposed as "sections" 
or subgenera of Micrahonta. Later, they were 
elevated to genera based on "major differ- 
ences" that were unfortunately not well de- 
tailed (Bequaert & Miller, 1973; Miller, 1981). 
My results are consistent with Pearce's (1 990) 
hypothesis of the relationships of these gen- 
era (Fig. la). 

Because the type and shape of the mucous 
glands has been traditionally important in the 
various definitions of Xanthonychidae-Hel- 
minthoglyptidae-Helicidae, and often used as 
the only justification for splitting the families 
(Pilsbry, 1939; Miller &Naranjo-Garcia, 1991), 
the second analysis was performed for com- 
parative purposes. In this analysis, there are 
three synapomorphies supporting the in- 
group: bursa copulatrix duct not swollen at 
base, copulation reciprocal, and fertilization 
pouch-spermathecal complex buried in the al- 
bumen gland. Helix is internested in the in- 
group. The hypothesis proposed in Figure 2, 
treating the mucous glands as homologous 
structures, suggests that the dart sac and mu- 
cous glands were originally absent in the 



108 



CUEZZO 



basal clades Tryonigens and Sonorella, and 
secondarily acquired (Figs. 2, 3) in the rest of 
Xanthonychidae. The hypothesis proposed in 
Figure 1,1a, treating the mucous glands as 
non-homologous structures, suggests, how- 
ever, the opposite situation: the dart sac and 
mucous glands inserted in the vagina were 
originally present and secondarily lost in sep- 
arate groups. Comparisons among the dado- 
grams presented (Figs. 1a, 2) are difficult to 
make because a different matrix was used for 
the analysis. Even if we compare the behavior 
of characters 19 and 20 of the first analysis 
and 35 and 36 of the second, which refer to 
the same structure coded differently, the mu- 
cous glands are highly homoplastic and there- 
fore appear to be unreliable as characters for 
phylogenetic reconstruction. In any case, the 
mucous glands and dart sac have been over- 
valued in past studies, and many other non- 
genital characters have not been considered 
in previous classifications of the traditional 
Xanthonychidae. 

The question of whether the genera that 
lack the dart complex form a monophyletic 
unit or whether the absence of these struc- 
tures are products of parallel evolution has 
been discussed several times (Nordsieck, 
1987; Schileyko, 1991). Based on the dado- 
grams obtained in this study, neither analysis 
supports the idea of monophylly of taxa that 
lack dart complex, represented here by Tryo- 
nigens and Sonorella. The taxonomic position 
of Monadenia, originally placed in the 
Helminthoglyptinae by Pilsbry (1939) but later 
moved to Bradybaenidae (Miller & Naranjo- 
Garcia, 1991), remains controversial. In this 
study, the "swollen" in the terminal genitalia 
present in Monadenia was interpreted as an 
athal sac. When the shape of the mucous 
glands are taken into consideration and the 
mucous glands are considered homologous 
structures (Fig. 2), the position of Monadenia 
is similar to the one traditionally accepted 
and proposed by Pilsbry (1939). However, it 
could be concluded that the relationships of 
Monadenia will remain obscure until a phy- 
logeny of the Bradybaenidae-Xanthonychi- 
dae-Helicidae is proposed. 

(a) The family Xanthonychidae (= Helmin- 
thoglyptidae) as defined and used by Pilsbry 
(1939) and Zilch (1959-1960) is paraphyletic. 

(b) Xanthonychidae-Bradybaenidae-Heiici- 
dae conform a monophyletic group. 

(c) The preferred hypothesis (Fig. la) sup- 
ports the delimitation of three monophyletic 



groups within the traditionally named Xan- 
thonychidae. 

(d) Some characters used in previous studies, 
such as the shape and number of mucous 
glands, are of poor value for the reconstruc- 
tion of the phylogeny of the Xanthonychidae. 

Considering the monophyly of the Brady- 
baenidae-Helicidae-Xanthonychidae well es- 
tablished, further cladistic studies are needed 
as a basis for a revised, testable and informa- 
tive classification of its components. 

ACKNOWLEDGMENT 

This study was partially done while I was 
beneficiated with a Jessup Fellowship award- 
ed by The Academy of Natural Science of 
Philadelphia, without which this work would 
not have been possible. I am deeply indebted 
to George Davis for his consistent support 
and encouragement. I am grateful to Quentin 
Wheeler, not only for providing laboratory fa- 
cilities at Cornell University but also for his 
contagious enthusiasm for taxonomy, "the big 
science," and for his unconditional support. 
Thanks are extended to Eduardo Domínguez, 
Pablo Goloboff, Diana Silva, and Quentin 
Wheeler for reviewing the manuscript and to 
P. Goloboff for providing the computer pro- 
gram NONA and FQ. David Robinson facili- 
tated the access to the collection in the 
Academy of Natural Sciences of Philadelphia. 
I also thank the following persons for the loan 
of material of their institutions: George Davis 
(ANSP), Fred Thompson (UF), Zaidett Bar- 
rientes (INBIO), and Rüdiger Bieler and John 
Slapdnsky (FMNH). 

LITERATURE CITED 

BAKER, B. H., 1942, A new genus of Mexican heli- 
cids. The Nautilus, 56(2): 37-41. 

BAKER, B. H., 1943, Some Antillean helicids. The 
/Vauf/Vus, 56(3): 81-91. 

BAKER, B. H., 1959, Xanthonychidae (Pulmonata). 
The Nautilus, 73(1): 25-28. 

BEQUAERT J. С & W. B. MILLER, 1973, The mol- 
lusks of the arid Southwest with an Arizona check 
list. University of Arizona. xvi+ 271 pp. 

BIELER, R. 1992, Gastropod phylogeny and sys- 
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PHYLOGENETICS OF XANTHONYCHIDAE 



109 



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Revised ms. accepted 27 May 1997 



APPENDIX I 



Taxa studied: 



Bradybaena simllaris (Férussac): 

ANSP 434, A8774: West Java, Bogor, Java. 
Feb. ^973. Buiinya bernardinae Baker 

ANSP AI 6728: Ruins of Monastery 20 knn 
southwest of Mexico City, El Desierto de 
Los Leones to La Venta, Distrito Federal, 
Mexico. July 1926. H. В. Baker! 

Cepolis сера (Müller): 

UF 46191: Dept. du Sud, SE slope of Morne 

Formón, Haiti. 1500 m. Thompson! Jan. 

1984. 
UF 235002: Dept. Quest, 3 km S. Kenscoff, 

Haiti. 1470 m. Thompson & Auffenberg! 
UF 48281: Dept. du Sud, Boia Dirant, SW of 

Morne Formón, 1250 m. June 1984, К. A. 

& R.W.P! 

Charodotes traski (Newcomb): 

ANSP AI 4976: 12 miles E. of Las Cruces, 

Baja California, Mexico. 1918. H. N. 

Lowe! 
Cryptostrakon gabbi Binney: 
ANSP 246310, A9639: Costa Rica. Lec- 

totypes selected by Baker (1963). 

Cryptostrakon corcovadensis Cuezzo (in 
press): 

INBIO 468080: R N. Corcovado, Estación 
Sirena, Sendero a Río Los Patos, 10 mt.. 
Madrigal, Puntarenas Province, Costa 
Rica. 18 Aug. 1994. Marianella Segura. 

INBIO 468087: Madngal, Puntarenas Prov- 
ince, P. N. Corcovado, Estación Sirena, 
Sendero a Los Patos, Costa Rica, 10 m. 
14 Aug. 1994. Marianella Segura! 

INBIO 408059: Madrigal, Puntarenas Prov- 
ince, P. N. Corcovado, Estación Sirena, 
Sendero Los Espaveles, 20 m. 12 Aug. 
1994. Marianella Segura! 

Dialeuca nemoraloides (Adams): 
ANSP Al 2685: Mandeville, Manchester 
Parish, Jamaica. June 1933. H. Baker! 

Epiphragmophora hieronymi Doering: 
FML Al 00: Quebrada del Tala, Catamarca, 
Argentina. Jan. 1993. Domínguez! 



PHYLOGENETICS OF XANTHONYCHIDAE 



111 



Eremarionta rowelli (Newcomb): 

ANSP A11327B: Needles Peaks, Topock, Mo- 
have County, Arizona, USA. Ex Ferriss. 

ANSP 164956, A11327G: Rocky Hills, Punta 
Libertad. Feb. 1935. H. N. Lowe! 

Helminthoglypta arrosa (Gould, in Binney): 
ANSP A1 1345: 12 miles from end of Point 

Reyes, Marin County, California, USA. H. 

N. Lowe! 

Helminthoglypta tudiculata (Binney): 
ANSP 94237 A11344F: Oceanside, San Di- 
ego County, California, USA. Sept. 1907. 
ExH. G.Eaton. 

Humboldtiana humboldtiana (Valeciennes, in 

Pfeiffer): 
ANSP AI 3281: El Desierto de Los Leones, 

Mexico. 

Leptarionta quillarmodi (Shuttieworth): 
ANSP Al 6727: 4 km North and slightly east of 
Cordova, foothills east and north of 
Toxpam (Hacienda de San Francisco), 
Cerro de Las Palmas, Vera Cruz, Mexico. 
June 1926. H.B.Baker! 

Lysinoe ghiesbreghti (Nyst): 

FMNH 206294/1 : 2400 m, on trail, 1 km SW of 

Esquipulas Palo Gordo, San Marcos, 

Guatemala. 28 July 1 980. Ken Young! 
UF 190195: Alta Verapaz Prov., Guatemala. 

10.5 Km. SE of Tactic. Feb. 1991. F T. & 

S. P Christman! 

Metostracon mima Pilsbry: 

ANSP 77245, A9636: Morelia, Michoacan, 
Mexico. S. N. Rhoads! Holotype. 

ANSP A9635F: Uruapam del Progresse, Mi- 
choacan, Mexico. S. N. Rhoads! Para- 
type. 

Metostracon mima Pilsbry: 

ANSP A9410D: Near Alvarez at km 53, San 

Luis de Potosi, Mexico. July 1934. H. A. 

Pilsbry! 
ANSP A9411A: Km 42, Potosi and Rio Verde 

Railroad, San Luis de Potosi, Mexico. 

Aug. 1934. H.A. Pilsbry! 

Micrarionta facta (Newcomb): 

ANSP 10789, A1 1342: Santa Barbara Island, 

Santa Barbara County, California, USA. 

Newcomb! 

Micrarionta sp.: 

ANSP 130897, A1 13321: 5 mi W of Leach 

Spring, Granite Mountains, California, 

USA. 1922. Ferris! 



Monadenia fidelis (Gray): 

ANSP 158283, AI 6079: Riverdale, Multno- 
mah County, Oregon, USA. August 1929. 
H. B. Baker! 

ANSP 158278 AI 6078: About 13 mi N of 
Klamath Falls, E side of upper Klamath 
Lake, Ouxy siding, Klamath County, 
Oregon, USA. July 1929. H. B. Baker! 

Plesarionta stearsiana (Gabb): 

ANSP 66091 A1 1336: Coronado Island, San 

Diego County, California, USA. 1895. 

A. W. Anthony! 
ANSP 146098 A11332E: Near San Vicente 

Mission, Baja California Norte, Mexico. 

Dec. 1927. L.G. Ingles! 

Polymita picta Born: 
ANSP A13209: Cuba, 

ANSP 154067, A9341: Mandinga de Yumuri, 
Oriente Province, Cuba. Welch! 

Sonorella hachitana (Dall) 
ANSP AI 0367: Florida Mountains, Luna 
County, New Mexico. 1906. H. A. Pilsbry! 

Trichodiscina cordovana (Pfeiffer): 

ANSP AI 6732: Steep Valley down from sad- 
dle, Twin Peak Valley, Estado Puebla, 
Mexico. July 1926. H. B. Baker! 

ANSP AI 6734: Steep valley down from sad- 
dle. Twin Peak Valley, Estado Puebla, 
Mexico. July 1926. H. B. Baker! 

Tryonigens remondi (Tryon): 
ANSP 166233, A9415A: Hills around Panuco, 
Sinaloa, Mexico. Aug. 1935. H. A. Pilsbry! 

Xanthonyx sp.: 

ANSP AI 6735: 14 km from Cordoba towards 
Orizaba on Mexican railroad, hills south- 
east of town, on opposite side of canyon 
Sumidero, Vera Cruz State, Mexico. June 
1926. H.B.Baker! 

Xerarionta kelletti (Forbes): 

ANSP 138972, A11332A: West of North Bay 
isthmus, hillside west of Isthmus Cove, 
Santa Catalina Island, Los Angeles 
County, California, USA. 1925. H. A. Pils- 
bry! ANSP AI 38973, A11332B: Avalen, 
Santa Catalina Island, Los Angeles 
County, California, USA. 1925. H. A. 
Pilsbry! 



MALACOLOGIA, 1998, 39(1-2): 113-121 

DIVERGENCE AMONG MOBILE BASIN POPULATIONS OF THE PLEUROCERID 
SNAIL GENUS, LEPTOXIS, ESTIMATED BY ALLOZYME ELECTROPHORESIS 

Robert T. Dillon, Jr.^ and Charles Lydeard^ 



ABSTRACT 

Although the Mobile River Basin of Alabama was historically a center of great pleurocerid di- 
versity, populations today are small and scattered. We obtained samples of all four nominal 
species of Mobile Basin Leptoxis currently extant: L. ampia (3 populations), L. picta (1 popula- 
tion), L. plicata (2 populations), and L. taeniata (2 populations). Gene frequencies at nine variable 
enzyme loci were determined for about 30 individuals from each population using horizontal 
starch gel electrophoresis. Samples of about 30 individuals from three populations of the wide- 
spread Leptoxis praerosa were analyzed as controls. Within populations, 1 8 of 99 loci were poly- 
morphic, none showing genotype frequencies significantly different from Hardy-Weinberg ex- 
pectation. Between populations within species, statistically significant divergence was apparent 
at most loci. Comparisons among the nominal species showed L. praerosa and L. plicata to be 
quite distinct from each other, and from all other populations. Much lower levels of divergence 
among populations nominally L. picta, L. ampia, and L. taeniata seem more consistent with a hy- 
pothesis of geographic isolation than reproductive isolation. We refer to these three taxa as the 
"Leptoxis picta group." Our results compare favorably in most respects with previously published 
data on mitochondrial 16S rRNA gene sequence divergence among these taxa, especially in the 
genetic distinction of L. plicata. The relationships within the L. picta group warrant further study 

Key words: genetics, isozymes, speciation, freshwater, gastropods, Alabama, endangered 
species. 



INTRODUCTION 

The rivers and streams of Alabama's 
Mobile River Basin have recently attracted at- 
tention as a center of endemism for a variety 
of aquatic life, including turtles, fish, bivalves 
and prosobranch snails (Lydeard & Mayden, 
1995). Based primarily on the revisions of 
Goodrich (1922, 1924, 1936, 1941), Burch 
(1989) recognized 77 species of pleurocerid 
snails from the region, 95% of which were un- 
known outside the Mobile Basin. Burch's list 
included 6 species of Gyrotoma, 5 species of 
Pleurocera, 52 species of Elimia (synonymiz- 
ing Goniobasis as used by Goodrich), and 14 
Leptoxis (lowering Anculosa, as used by 
Goodrich, to subgeneric level). During the 
present century, however, most of the larger 
rivers of the Mobile Basin have been im- 
pounded for hydroelectric power, channel- 
ized, or otherwise modified for navigation. The 
Mobile Basin pleurocerid fauna has also been 
adversely impacted by changing patterns of 
land use, first from siltation due to intensive 
agriculture, and more recently from pollution. 



Lydeard & Mayden (1995) presumed extinct 
29 species of Mobile Basin pleurocerids, in- 
cluding all six species of the endemic genus 
Gyrotoma. 

The Leptoxis species of the basin have 
been the object of special concern. Of the 1 1 
species of Leptoxis known historically from 
the Coosa River, Bogan & Pierson (1993a) 
found only L. taeniata (Conrad, 1834), appar- 
ently restricted now to but a few small tribu- 
taries. Of the four Leptoxis species docu- 
mented from the Cahaba River, only L. ampia 
(Anthony, 1855) apparently survives, inhabit- 
ing a 30 km reach of the main river and sev- 
eral smaller Cahaba tributaries (Bogan & 
Pierson, 1993b). The only Leptox/s population 
remaining in the Black Warrior drainage is L. 
plicata (Conrad, 1834), restricted to a short 
reach of Locust Fork. Based on these data, as 
well as extensive U. S. Fish & Wildlife Service 
field records, Hartfield (1997) identified L. tae- 
niata, L. ampia, and L. plicata as candidates 
for addition to the U. 8. list of endangered and 
threatened wildlife and plants. The status of 
the only other nominal Leptoxis species 



^Department of Biology, College of Charleston, Charleston, South Carolina, 29424, U.S.A. 

^Aquatic Biology Program, Department of Biological Sciences, University of Alabama, P.O. Box 870344, Tuscaloosa, 

Alabama 35487, U.S.A. 



113 



114 



DILLON AND LYDEARD 



known with certainty to have survived in the 
Mobile Basin, L. picta (Conrad, 1834) of the 
main Alabama River, continues to be moni- 
tored. 

But Hartfield noted that the genetic relation- 
ships among these four nominal species of 
Leptoxis are poorly understood. They are dis- 
tinguished primarily by minor attributes of 
shell shape and size, traits long known for cli- 
nal variability (Goodrich, 1934, 1935). The 
non-genetic component of some aspects of 
pleurocerid shell morphology is well-docu- 
mented (Chambers, 1982; Dillon, 1984a). 

In light of these concerns, Lydeard et al. 
(1997) surveyed 15 pleurocerid populations 
from the Mobile Basin: seven Elimia and four 
Pleurocera, in addition to the four nominal 
Leptoxis species. A molecular phylogeny con- 
structed from mitochondrial 16S rRNA gene 
sequences suggested that Alabama Elimia 
and Pleurocera are sister taxa. The four 
Leptoxis species were quite different from the 
Elimia/Pleurocera group, and depicted as pa- 
raphyletic. Levels of sequence divergence 
were low between L. taeniata and L. ampia. 
with L. picta and L. plicata appearing more 
distinct. 

Allozyme electrophoresis is an older and 
more established technique for evaluating the 
specific status of pleurocerid populations, es- 
pecially the large genus Goniobasis. 
Extensive surveys of variation at allozyme-en- 
coding loci, involving at least 11 species and 
58 populations, have established that 
Goniobasis shows unusually low levels of het- 
erozygosity, high levels of divergence 
between populations within species, and 
very few shared alleles at any locus when 
compared among species (Chambers, 
1978, 1980; Dillon, 1984b, 1988a; Dillon & 
Davis, 1980; Bianchi et al., 1994; Stiven & 
Kreiser, 1994). Recent evidence suggests 
similar trends in Leptoxis, although intrapopu- 
lation variation may be somewhat greater, and 
interpopulation variation less (Dillon & 
Ahlstedt, 1997). 

The purposes of the present work are 
twofold. We survey the allozyme divergence 
displayed by populations representing the 
four nominal species of Mobile River basin 
Leptoxis to gather further evidence regarding 
their genetic distinction. We also compare the 
levels of allozyme divergence estimated here 
to the DNA sequence divergence estimates of 
Lydeard et al. (1997), as a possible guide to 
the future application of the newer technology. 



METHODS 

We analyzed eight populations of Alabama 
Lepfox/s assigned to four species (Appendix). 
Our L. taeniata populations were sampled 
from Buxahatchee and Choccolocco creeks, 
two tributaries of the Coosa River. As no 
Leptoxis inhabit the 50 km reach of the Coosa 
River separating these two creeks, gene flow 
between the populations we designated 
Taebux and Taechc, respectively, would seem 
to be negligible at present. We obtained sam- 
ples of L. ampia from three shoals of the 
Cahaba River separated about 20 river km 
from each other, labeled Ampcahl , Ampcah2, 
and Ampcah3 from upstream down. Our two 
samples of L. plicata are from Locust Fork, 
Plilod about 15 river km upstream from 
Pliloc2. Our single sample of L. picta (Picala) 
was collected by boat from limestone walls 
and outcrops in the lower Alabama River. 

We selected three populations of the well- 
characterized Leptoxis praerosa (Say, 1821) 
to provide calibration for our analysis. 
Populations of this species are common and 
widespread throughout the Ohio, Cumber- 
land, and Tennessee river drainages. Our L. 
praerosa came from three tributaries of the 
Tennessee River, the Elk River (Praelk), the 
Duck River (Praduk), and the Sequatchie 
River (Praseq). Leptoxis from the Sequatchie 
and Duck rivers have been previously ana- 
lyzed by Dillon & Ahlstedt (1997). Analyzing 
all 1 1 populations together, we were able to 
evaluate observed levels of genetic diver- 
gence among nominal Alabama species by 
comparison to divergence among Leptoxis 
populations known to be conspecific, isolated 
at approximately equivalent distances. 

The geographic relationships among the 1 1 
populations analyzed in this work are mapped 
in Figure 1 , and locality data and sample sizes 
are given in the Appendix. Although our sam- 
ple sizes were in most cases greater than 30, 
only 21 individual L. picta were available. The 
Appendix also provides catalog numbers for 
voucher specimens deposited in the Aca- 
demy of Natural Sciences of Philadelphia. 

Our equipment and techniques for hori- 
zontal starch gel electrophoresis of whole an- 
imal homogenates have been previously de- 
scribed (Dillon, 1985, 1992). Samples were 
initially run on gels of five different buffer sys- 
tems and stained to visualize 13 different en- 
zymes. We simultaneously screened these 
gels and stains by requiring that clearly inter- 



LEPTOXIS POPULATION GENETICS 



115 




FIG. 1. The state of Alabama, showing major 
drainages and sample sites. (1) Tombigbee River, 
(2) Black Warrior River, (3) Cahaba River, (4) Coosa 
River, (5) Tallapoosa River, (6) Alabama River, (7) 
Mobile Bay, (8) Tennessee River. 



pretable polymorphism be present in an initial 
comparison of Praduk and Ampcahl, se- 
lected as the most different pair of populations 
in our study. Ultimately we identified the prod- 
ucts of nine putative gene loci for detailed 
analysis over all 1 1 populations. 

The Poulik buffer (Poulik, 1 957) was used to 
resolve glucose phosphate isomerase (GPI, 
EC 5.3.1.9) and octopine dehydrogenase 
(ODH, EC 1.5.1.11). The AP6 buffer (Clayton 
¿Tretiak, 1972) was used to resolve mannose 
phosphate isomerase (MPI, EC 5.3.1.8), 6- 
phosphogluconate dehydrogenase (6PGD, 



EC 1.1.1.44), and isocitrate dehydrogenase 
(IDH, EC 1 .1 .1 .42). The products of two puta- 
tive loci were apparent on the IDH gel, one mi- 
grating cathodally ("IDHF") and the other an- 
odally ("IDHS"). The TEB8 buffer (buffer III of 
Shaw & Prasad, 1970) was also employed for 
IDHF, xanthine dehydrogenase (XDH, EC 
1.2.1.37), and esterase (EST1, EC 3.1.1.2). 
Superoxide dismutase (SOD, EC 1.15.1.1) 
activity was visualized as light bands on TEB8 
gels darkly stained for XDH or IDH. 

Allozyme phenotype has been shown to re- 
sult from simple Mendelian inheritance of 
codominant alleles at the 6PGD locus by 
Chambers (1980), working with Goniobasis 
floridana. Dillon (1986) reported similar find- 
ings for GPI, ODH, and EST1, based on a 
mother-offspring analysis in Goniobasis próx- 
ima. Although the esterase stain employed 
here (c(-napthyl acetate as substrate) yields a 
complex, multi-banded phenotype for each in- 
dividual, only the slowly-migrating, strongly 
staining products of the single locus desig- 
nated EST1 by Dillon (1986) were accorded a 
genetic interpretation in the present work. 

Population Praduk served as the standard 
for allelic designations. We adopted here the 
same designations used for this population by 
Dillon & Ahlstedt (1997) for the four shared 
loci (EST1, GPI, MPI, and ODH), and labeled 
all new alleles accordingly. For the five loci not 
reported by Dillon & Ahlstedt (IDHS, IDHF, 
XDH, 6PGD, and SOD), the most common al- 
lele in Praduk was considered to migrate 100 
mm and all other alleles labeled by their rela- 
tive electrophoretic mobilities in millimeters 
faster or slower. 

Gene frequencies, tests to Hardy-Weinberg 
expectation (by chi-square, with pooling for 
rare genotypes), and Nei's (1978) unbiased 
genetic identities and distances were calcu- 
lated using BIOSYS version 1.7 (Swofford & 
Selander, 1981). Tests for homogeneity be- 
tween populations within nominal species 
were by Fisher's exact method in 2 x 2 cases, 
otherwise by chi-square contingency tests, 
pooling the rarest rows or columns as neces- 
sary. We analyzed the matrix of genetic dis- 
tances using the multidimensional scaling 
module of STATISTICA (Release 5.0, StatSoft, 
Inc.), with a standard Guttman-Lingoes (prin- 
cipal component) starting configuration. The 
distances between any pair of populations 
sharing no alleles at any locus (i.e., similarity = 
0.0) were set to 5.0, a figure greater than any 
value actually observed. 



116 



DILLON AND LYDEARD 



RESULTS 

Example shells from each of the four nomi- 
nal Alabama Leptoxis species are shown in 
Figure 2. Their differences were not striking. 
The shells of L picta tended to be heavier, 
with a higher spire, while those of L. ampia 
were lower and more rounded, and L. taeniata 
intermediate. Apical erosion made spire 
height difficult to evaluate, however, espe- 
cially in the L. ampia population. The shells of 
L. plicata were less eroded, with more shoul- 
dered whorls. They were characterized by low 
folding (or plication) on the whorl periphery, 
barely visible in Figure 2. Although such plica- 
tions have been reported to occur in L. ampia, 
we saw no evidence of them in our samples. 

Gene frequencies are given in Table 1 . 
Levels of intrapopulation variation were low, 
although perhaps not quite as low as in the 
better-studied Goniobasis. Over all 9 x 1 1 = 
99 loci, we found 1 8 polymorphic as judged by 
the 95% criterion. Genotype frequencies at 
none of these 18 loci differed significantly 
from Hardy-Weinberg expectation. 

All nominal species for which more than one 
population was sampled are listed in Table 2, 
along with the loci at which any intraspecific 
polymorphism was observed. Every nominal 
species showed significant interpopulation al- 
lelic frequency difference in at least one locus. 
This was especially striking at the ODH locus 
in L ampia , and at both the ODH and EST1 
loci in L. plicata, where the most common al- 
lele changed over distances as short as 15 
river km. Not only did the three L. praerosa 
populations differ significantly from each other, 
the present Praseq and Praduk populations 
differed from the Sequatchie and Duck sam- 



ples of Dillon & Ahlstedt (1997) located 20-30 
km downstream. 

Figure 3 shows Nei's unbiased genetic 
identities among all pairs of Leptoxis popula- 
tions. The three L. praerosa populations were 
strikingly different from all others, as were the 
two L. plicata populations. The levels of ge- 
netic identity among L. ampia, L. picta, and L. 
taeniata populations were much higher. 
Figure 3 also depicts the Nei's genetic dis- 
tances in two dimensions, from multidimen- 
sional scaling. After 100 iterations, the stress 
for this solution was 0.0015. The six popula- 
tions comprising L. ampia, L. picta, and L. tae- 
niata occupy one end of the long axis of the 
scale, the three L. praerosa the other end, and 
L plicata appears intermediate. 



DISCUSSION 

The species concept under which the pleu- 
rocerid fauna of the Mobile Basin has been 
described and revised differs substantially 
from the biological concept in currency today. 
In his (1922) monograph on the "Anculosae" 
(Leptoxis) of Alabama, Goodrich wrote, "That 
collection of individuals in the Pleuroceridae 
may be called a species whose predominant 
characters are not the predominant charac- 
ters of another collection of individuals. If we 
see only a few specimens of a single species 
its own peculiar characters may often seem to 
be submerged by characters linking it with an- 
other species. But in a long series the individ- 
ual characters stand out, and we are com- 
pelled then to recognize the existence of 
definable differences and to proceed to de- 
scribe them and provide the label of a name." 





5 mm 



FIG. 2. Example shells of four Mobile Basin Leptox/s species. From left, L.p/cia (Picala), L. taeniata (Jaechc) 
L. ampia (Ampcahl), and L. plicata (Pliloc2). 



LEPTOXIS POPULATION GENETICS 117 

TABLE 1 . Gene frequencies at nine allozyme loci tor 1 1 populations of Leptoxis 
Amp- Amp- Amp- 



Locus 


Allele 


cahl 


cah2 


cah3 


Pícala 


Taechc Taebux 


Plilocl 


Pliloc2 


Praduk 


Praelk 


Praseq 


GPI 


108 








0.024 






0.726 


0.726 










104 


1.000 


0.855 


0.625 


0.976 


1.000 


1.000 














97 














0.274 


0.274 


1.000 


1.000 


0.838 




94 




0.145 


0.375 




















90 






















0.162 


MPI 


100 
98 








0.929 


1.000 


1.000 


1.000 


1.000 




0.016 






95 


1.000 


1.000 


1.000 


0.071 










1.000 


0.984 


1.000 


EST1 


106 
105 














0.613 


0.387 


1.000 


1.000 


1.000 




104 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 














99 














0.387 


0.613 








6PGD 


106 

100 

94 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


0.767 
0.233 


0.900 
0.100 


1.000 


1.000 


0.952 
0.048 


ODH 


121 
118 
115 
113 
110 




0.057 


0.682 








0.355 
0.065 
0.323 
0.258 


0.242 
0.048 
0.532 
0,177 


0.855 
0.145 


0.177 
0.823 


0.054 
0.203 




107 


1.000 


0.943 


0.318 


1.000 


1.000 


1.000 










0.743 


IDHF 


103 
100 














1.000 


1.000 


1.000 


1.000 


1.000 




98 


1.000 


1.000 


1.000 


1.000 


0.875 


1.000 














95 










0.125 














IDHS 


103 
102 
100 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


0.177 
0.823 


XDH 


103 
100 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


SOD 


110 
100 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 


1.000 



Goodrich often reported that Mobile Basin 
species overlapped not just in character, but 
in geographic range as well. Populations iden- 
tified by Goodrich (1922) as L. picta histori- 
cally inhabited the lower Coosa River, the 
lower Cahaba River, and the Alabama River 
downstream to Claiborne, Monroe County. 
Goodrich reported the range of L. taeniata as 
substantially identical to that of L. picta, ex- 
cept that L. taeniata extended further up the 
Coosa River and its tributaries. Goodrich 
listed L. ampia from both the Coosa and 
Cahaba rivers and their tributaries, although 



not from the main stem of the Alabama River. 
Goodrich did not consider that the geographic 
range of L. plicata overlapped with those of L. 
picta, L. taeniata, or L ampia. He recorded L. 
plicata as occurring in the Black Warrior River, 
the Tombigbee River, and their tributaries 
only. 

The concept of the species differs today, as 
does the distribution of Leptoxis in the Mobile 
Basin. Under the biological species concept, 
local variation in gene frequencies (and by ex- 
tension, external appearances) is a not-unex- 
pected consequence of limited gene flow in 



118 



DILLON AND LYDEARD 



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LEPTOXIS POPULATION GENETICS 



119 



TABLE 2. The probability of Inomogeneity among nominally conspecific popula- 
tions of Leptoxis (p^ from x^ tests, p, from Fisher's exact tests). A subscript "c" indi- 
cates that rows or columns were combined for the test. The table is blank for loci 
where conspecific populations were not polymorphic. 



GPI 

EST1 

6PGD 

ODH 

IDHF 

IDHS 



L. ampia 



L. taeniata 



L. plicata 



Pxc< 0.001 



p^ < 0.001 



P,= 10 
p, = 0.019 
p, = 0.085 
Pxc = 0.062 



p, = 0.006 



L. praerosa 



p,^ < 0.001 



p^ < 0.001 
p„ < 0.001 



populations of organisms with dispersal capa- 
bilities as low as freshwater snails. For ехагл- 
ple, a culvert placed in a small North Carolina 
stream in the 1950s caused significant diver- 
gence at the ODH locus between upstream 
and downstream populations of G. próxima 
over a distance of just 10 meters (Dillon, 
1 988b). That this did not comprise a speciation 
event became clear when the barrier was re- 
moved, and the genetic difference disap- 
peared. Indeed, geographically isolated popu- 
lations of G. próxima sharing no alleles at as 
many as six allozyme loci have nevertheless 
demonstrated no evidence of reproductive iso- 
lation when transplanted (Dillon, 1986, 
1988a). Evidence of similar interpopulation di- 
vergence is clear in our three samples of L. 
ampia, our two samples of L. taeniata, and our 
two samples of L. plicata. Whether the signifi- 
cant differences highlighted in Table 2 are due 
to some unrecognized barriers to dispersal, or 
whether they may be due to isolation by dis- 
tance alone, cannot be told at present. But it is 
clear that our three populations of L. ampia, for 
example, do not constitute different species. It 
is also clear that, extending the levels of diver- 
gence illustrated within L. ampia down the 1 20 
km length of the Cahaba River as was the sit- 
uation earlier in this century, the Lepfox/s of the 
Alabama River would be expected to show 
striking genetic differences with the Leptoxis oi 
the headwaters, through isolation by distance. 
There is little expectation, however, that repro- 
ductive isolation will evolve in such a circum- 
stance, or that headwaters populations and 
populations from the main river will speciate 
parapatrically. 

The divergence among L. taeniata, L. plata, 
and L. ampia is negligible, given their geo- 
graphic distance. Leptoxis plata has uncom- 
mon alleles at the GPI and MP! loci not de- 



tected in L. taeniata, and one L. taeniata pop- 
ulation has an allele at IDHF not seen in L. 
plata. The levels of divergence appeared 
somewhat greater between L. ampia and L. 
picta/taenlata, due to the results at the MPI 
locus. But although L. ampia is fixed for an al- 
lele not seen in L. taeniata. Table 1 shows that 
both MPI alleles are found in the L. plata pop- 
ulation that may have connected them in the 
main Alabama River. Such small and clinal dif- 
ferences are not comparable to those nor- 
mally displayed by species of pleurocerid 
snails presumed distinct, as illustrated by L. 
praerosa and L. pliaata. We therefore refer to 
all three of these taxa, L. plata, L. taeniata, 
and L. ampia, as the "Leptoxis plata group." 

In most respects, our findings coincide with 
those based on 16S rRNA gene sequence di- 
vergence. Lydeard et al. (1997) also found L. 
plicata to be quite distinct from all other Mobile 
Basin Leptoxis, unique at about 20% of its nu- 
cleotide bases. Lydeard's mtDNA phylogeny 
depicted the three members of the L. plata 
group as a single clade when transversions 
were weighted more than transitions. But while 
very little sequence divergence was apparent 
between L. taeniata and L. ampia (only about 
2%), Lydeard reported about 20% sequence 
divergence between L. taeniata/ampla and L. 
plata. So our finding that L. plata and L. taeni- 
ata are indistinguishable in their allozyme fre- 
quencies at nine loci was quite unexpected. 

A similar discrepancy between allozyme 
and mtDNA divergence in oysters was attri- 
buted to balancing selection at multiple en- 
zyme loci by Karl & Avise (1992), although 
much more data would be required before 
such a suggestion could be made in our case. 
Lydeard et al. (1997) only analyzed a single 
individual for each nominal Leptoxis species. 
There is a clear need for additional surveys of 



120 



DILLON AND LYDEARD 



16S rRNA sequence divergence focused 
below the species level. 

A complete understanding of the genetic re- 
lationships among L. picta, L. taeniata, and L. 
ampia would have required samples from 
populations inhabiting the lower regions of the 
Cahaba and Coosa rivers, where the three 
nominal species were once reported to co- 
occur. All such populations are long extinct. 
Regardless of their specific status, the levels 
of genetic diversity displayed by the small 
populations of the L. picta group that remain 
today, as evidenced by both mtDNA and al- 
lozyme studies, argue strongly for conserva- 
tion measures. The L. plicata population re- 
stricted now to just 20 km of Locust Fork 
(Hartfield, 1 997) is clearly a unique species by 
all measures, and deserves immediate pro- 
tection. 



ACKNOWLEDGMENTS 

We thank Wallace Holznagel, John Yoder 
and J. Malcolm Pierson for assistance in the 
field, Gary Rosenberg for helpful discussions, 
and Paul Hartfield for reading the manuscript. 
This research was supported by a Research 
Grants Committee Award (2-67767) from the 
University of Alabama, a contract with the U. 
S. Department of the Interior (1448-0004-04- 
929), and the National Science Foundation 
(DEB-9527758). 



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Revised ms. accepted 25 July 1997 



APPENDIX 

Locality data, sample sizes, and (where ap- 
plicable) catalog numbers for voucher speci- 
mens deposited in the Academy of Natural 
Sciences of Philadelphia 

Дтрса/7 7— Cahaba River at Co. 52, 1 .5 km W 
of Helen, Shelby County, Alabama. Same 



station as CA-61 of Bogan & Pierson 
(1993b). N = 25 Leptoxis ampia. 

Ampcafi2—Ca\r\aba River at River Road, 3 km 
S of intersection of Co. 1 and Co. 13. 
Shelby County, Alabama. N = 36 Leptoxis 
ampia. 

AmpcahS—Cahaba River at Co. 24, Bibb 
County, Alabama. Just downstream from 
station CA-64 of Bogan & Pierson 
(1993b), and 10 km downstream from the 
L. ampia site of Lydeard et al. (1997). N = 
24 Leptoxis ampia. ANSP 4001 1 1 . 

Pícala — Alabama River about 2 km south of 
U.S. 84 crossing, Monroe County, Ala- 
bama. Same site as the L. picta site of 
Lydeard et al. (1997). N = 21 Leptoxis 
p/ctó. ANSP 400112. 

Plilod — Locust Fork, 0.4 km upstream from 
the Mount Olive Road boat ramp, 
Jefferson County, Alabama. About 20 km 
south of L. plicata site of Lydeard et al. 
(1997). N = 31 Leptoxis plicata. ANSP 
400113. 

P//7oc2^Locust Fork, at shoal 10 km down- 
stream from the Mount Olive Road boat 
ramp, Jefferson County, Alabama. N = 31 
Leptoxis plicata. 

ГаеЬих— Buxahatchee Creek, Shelby County, 
Alabama. N = 34 Leptoxis taeniata. 

Taechc — Choccolocco Creek, Talladega 
County, Alabama. Same site as the L. 
taeniata site of Lydeard et al. (1997). N = 
32 Leptoxis taeniata. ANSP 4001 15. 

Praduk— Duck River at Shelbyville, Bedford 
County, Tennessee. About 30 km up- 
stream from Duck River site of Dillon & 
Ahlstedt (1997). N = 31 Leptoxis prae- 
rosa. 

Praelk—E\k River at Stump Shoals Public 
Access near US 64 bridge, 8 km E of 
Fayetteville, Lincoln County, Tennessee. 
N = 31 Leptoxis praerosa. ANSP 4001 1 4. 

PraseQ— Sequatchie River at Tn 28 bridge, 
Whitwell, Marion County, Tennessee. 
About 20 km upstream from Sequatchie 
site of Dillon & Ahlstedt (1997). N = 37 
Leptoxis praerosa. 



MALACOLOGIA, 1998, 39(1-2): 123-128 

LARVAL FUSION AND DEVELOPMENT OF CONJOINED TERATOIDS IN 
BIOMPHALARIA GLABRATA 

Charles S. Richards, Carolyn Patterson, Fred A. Lewis & Matty Knight 
Biomedical Research Institute, 12111 Parklawn Drive, Hockville, Maryland 20852 

ABSTRACT 

Snails of a genetically isolated laboratory stock of Biomphalaria glabrata, if mated to snails of 
certain ottier stocks, produce polyzygotic egg capsules. When two or more embryos occupy a 
single capsule, some embryos spontaneously fuse during the trochophore larval stage into ter- 
atoids of usually two, but up to seven, conjoined snails. We have observed more than 950 of 
these fused teratoids, with some dizygotic conjoined twins surviving to adulthood. Allophenic 
cell-to-cell adhesions lead to unusual patterns of development, apparently determined by the 
areas of the embryos that are fused. 

Key words: Biomphalaria, Pulmonata, development. 



INTRODUCTION 

Observations of conjoined twinning in mol- 
lusks have been previously reported [re- 
viewed by Bigus (1981) and Mason & 
Copeland (1988)], most often as rare anom- 
alies in otherwise normal populations. The un- 
expected occurrence of large numbers of 
spontaneously conjoining embryos in one of 
our snail stocks and the consistency with 
which we obtain these teratoids have pro- 
vided an abundance of material for study. 
Allophenic cell membrane junctions, the de- 
velopment of conjoined embryos, and the un- 
usual morphological patterns that result are 
areas of interest that lead us to continue the 
culture of these teratoids. 

The pulmonale gastropod Biomphalaria 
glabrata is a simultaneous hermaphrodite and 
is an important intermediate host of the para- 
sitic trematode Schistosoma mansoni. We 
maintain several named stocks of this snail in 
genetic isolation to conserve phenotypic vari- 
ations in pigment and in susceptibility to 
S. mansoni (Richards & Shade, 1987). Pig- 
mentation markers (Newton, 1954; Paraense, 
1955) are useful in elucidating inheritance 
patterns of the susceptibility-resistant pheno- 
types (Richards, 1985; Richards et al., 1992). 



MATERIALS & METHODS 

Snails of one genetic stock, M3 636, pro- 
duced a large number of polyzygotic eggs 
after out-crossing with a different stock (10R2 



was used most often). M3 636 stock snails 
were isolated as juveniles and reared individ- 
ually in 400 ml beakers until the onset of egg 
production by self-fertilization. Each snail was 
then put in a new 400 ml beaker with a part- 
ner of a different snail stock. After 2-7 days, 
during which each snail fertilized the other, 
the snails were re-isolated. A piece of clear 
plastic sheeting was floated in the beaker of 
each isolated snail. Most snails preferentially 
deposit clutches onto this plastic, which can 
then be removed from the beaker for exami- 
nation under a low-power dissecting micro- 
scope. Hybrid pigmentation of offspring from 
the clutches of both parents indicated the two 
snails had each functioned as a male in the 
cross. 

Many (> 200) egg clutches that contained 
polyzygotic egg capsules were sketched at in- 
tervals of their development to record the 
number of zygotes originally deposited in 
each capsule and the timing and pattern of 
embryo fusion. 

Some conjoined teratoids apparently had 
difficulty hatching on their own, in which 
cases we used dissecting needles to break 
the egg capsule. Survival of teratoids after 
hatching was very limited until we began 
feeding the neonate teratoids with cultured 
Nostoc sp. (Liang et al., 1987). This filamen- 
tous cyanobacterium, in addition to being nu- 
tritionally sufficient, reduced the need for 
active foraging and allowed simultaneous 
feeding by component snails fused in a con- 
figuration that made motility and feeding 
problematic. 



123 



124 



RICHARDS ET AL 



RESULTS 
Polyzygotic Egg Capsules 

Because we had not previously observed 
significant polyzygocy in self-fertilizing snails 
or in isogenic interbreeding populations of 
laboratory snail stocks, the production of large 
numbers of polyzygotic egg capsules by an 
out-crossed snail suggested that crossing ge- 
netically distant snails may trigger polyzygocy. 

Most M3 636 snails exhibit a degree of self- 
sterility (Paraense, 1993) and produce, by 
self-fertilization, very few clutches, each of 
which contains few or no viable eggs. After 
mating with a snail from a different stock, M3 
636 snails would generally produce normal 
clutches for several days before producing 
polyzygotic egg capsules. Polyzygocy in- 
creased during the next 4-6 weeks and then 
declined until only a few small clutches were 
produced. A subsequent out-crossing would 
often induce another cycle of polyzygocy. 

Egg capsules that contain multiple zygotes 
are not larger than monozygotic capsules. 
Multiple embryos share the single portion of 
nutritive capsular fluid and are smaller at 
hatching than snails from normal monozy- 
gotic egg capsules. In clutches that contain 
both single embryo and polyzygotic egg cap- 
sules, it is sometimes evident that the egg 
capsules deposited first are those that are 
monozygotic. The egg capsules deposited 
last in the clutch contain increasing numbers 
of zygotes, as if the snail were unable to pro- 
vide enough capsular fluid or other material to 
accommodate available zygotes. Zygotes are 
often observed at one end of the clutch, not 
contained in egg capsules, but loose in the 
fluid that surrounds the egg capsules. 

Fusion of Larval Snails 

Early observations of conjoined twins indi- 
cated that it was unlikely that they resulted 
from incomplete division of a single embryo. 
Conjoined snails of different pigment pheno- 
types (Fig. 1) confirmed that fusion of individ- 
uals occurred. In our many observations of 
new clutches, which were sketched and 
recorded at intervals in their development, we 
saw that the polyzygotic egg capsules con- 
tained excess fertilized oocytes and that divi- 
sion of a single zygote into multiple germs did 
not occur. 

For embryos to fuse they must make con- 
tact. When the clutch is first deposited, the zy- 




FIG. 1 . Conjoined Biomphalaria glabrata twins with 
one albino component and one black-eye (arrow) 
component, (length about 1 mm) 



gotes are non-motile and are spaced through- 
out the viscous capsular fluid. Cleavage con- 
tinues with little change in the size of the em- 
bryo through the blástula stage. During this 
interval, up to about 24 hrs after the clutch is 
deposited, the embryos become more dense, 
and gravity acts to bring the embryos, now 
blastulae, together at the lowest point in the 
spherical egg capsule. Though the embryos 
seem to be in close contact, we have not seen 
fusion at this stage. With gastrulation, the 
vitelline membrane that had surrounded and 
isolated the embryo is lost (Kawano et al., 
1992; Arambasic et al., 1989), the embryo in- 
creases in size, and the prototroch forms. The 
first larval motility is seen in this trochophore 
larva. We see fused embryos only after the 
multiple embryos in a polyzygotic capsule 
begin to move by means of the ciliated pro- 
totroch cells (Fig. 2). However, there may be 
some asynchrony in the development of em- 
bryos within the same egg mass, and the 
growing embryos may crowd each other in a 
polyzygotic egg capsule to the extent that fu- 
sion or the absence of fusion is not discern- 
able. Also, because of the large number of 
polyzygotic egg capsules produced in our lab- 
oratory, we do not follow every clutch through- 
out larval development. Some conjoined twins 
are first observed when the component snails 
are long past the early trochophore stage. 
Fusion of separate embryos may sometimes 
occur in late larval stages. 



DEVELOPMENT OF CONJOINED TERATOIDS 



125 




FIG. 2. Four egg capsules from a clutch laid by a M3 636 snail after crossing with a 10R2 snail: motile tro- 
chophore larvae of which three pairs (arrows) Inave fused. 



Developnnent of Conjoined Embryos 

The differentiation and development of ter- 
atoids subsequent to larval fusion is, of 
course, affected by the regions of the em- 
bryos that are fused. Larval snails fuse in 
many configurations. Most teratoids with more 
than two components do not develop long 
past the trochophore stage (Michelson & 
Schork, 1958;Bigus, 1981). Therefore, the fol- 
lowing generalizations will be discussed only 
in relation to the development of conjoined 
twins. 

Fusion occurs between homologous struc- 
tures. For example, two heads may be fused 
dorsally (Fig. 3) or laterally, occasionally two 
individuals may be joined at the edge of the 
foot, or the shell fields of two embryos may 
fuse and result in a two-headed individual 
under one shell (Fig. 4). Development of a twin 
that would have resulted from fusion of a 
pretrochal area of one component embryo 
with a posttrochal area of the other compo- 
nent embryo (Kawano et al., 1992) has not 
been seen. 

Every conjoined twin that developed and 
survived to the veliger stage had two heads 
(Fig. 5). Mouth parts, including the radula and 
anterior esophagus, were not fused in surviv- 
ing conjoined twins. Occasionally tentacles 
and eyes were partially fused, displaced, or 
distorted, but there was individual develop- 



ment of these structures in both component 
snails. 

For twins that had fused In a configuration 
allowing normal development of two shells — 
for example, fusion of dorsal areas of the 
heads — each component developed a com- 
plete anatomy of basically normal morphol- 
ogy. 

The fusion of the shell fields of two embryos 
often led to the formation of an aberrant shell 
shared by the two components. Often these 
teratoids would initially have two hearts, two 
kidneys, and other separate viscera. As de- 
velopment progressed with the single shell 
sheltering the two-headed teratoid, usually 
only one heart would persist and other inter- 
nal organs would seemingly coalesce into an 
internal anatomy grossly similar to that of a 
normal individual. These individuals, however, 
did not produce eggs, although one lived sev- 
eral weeks (Fig. 4). 

Three sets of conjoined twins fused in con- 
figurations that allowed one component to 
develop fairly normally while the other compo- 
nent, after some early development, degener- 
ated and became a vestigial tumor-like mass 
on the larger component. In these cases, 
head and mouth parts of the smaller compo- 
nent, although initially present, were lost. The 
larger component snails continued to develop 
and two produced offspring. 

Conjoined snails, often with heads oriented 



126 



RICHARDS ET AL. 




e 



^ 




FIG. 3. (a) Teratoid that survived several weeks and produced offspring (each shell --4 mm diameter) and 
(b) a stained section through the separate brains and shared cephalic sinus (arrow) of this teratoid. 





FIG. 4. Conjoined pair sharing one aberrant shell 
(grid = 2 mm). This teratoid initially had two hearts, 
only one of which persisted. 



FIG. 5. A teratoid with one component that initially 
had an everted mouth and was unable to feed. The 
other component fed normally, developed a com- 
plete anatomy, and produced offspring. The mouth 
of the non-feeding component reverted to the nor- 
mal configuration concurrent with the eversión of its 
preputium (shell diameter approximately 5 mm). 



DEVELOPMENT OF CONJOINED TERATOIDS 



127 



in different planes or facing opposite direc- 
tions, are sometimes unable to hatch without 
assistance and do not forage and feed effi- 
ciently. Nearly all teratoids are considerably 
smaller than individual snails of the same age, 
and most do not approach normal adult size. 



DISCUSSION 

Polyzygocy is a necessary condition for the 
formation of the conjoined teratoids described 
in this report. The highly determinate spiral 
cleavage in B. glabrata and other mollusks 
would all but preclude monozygotic twinning 
(Bigus, 1981; Crabb, 1931). One previous re- 
port (Eyster, 1995) of conjoined teratoids in- 
volves one of the many mollusks for which 
polyzygocy is normal. Mason & Copeland 
(1988) report some increase in the frequency 
of double embryo egg capsules in one gener- 
ation of selected breeding of the normally 
mono-embryonic pulmonate slug Lehmannia 
valentiana. Studies of other conjoined mol- 
lusks for which polyzygocy is unusual have 
not demonstrated any heritability of the trait 
(Bigus, 1 981 ; Crabb, 1 931 ; Hall, 1 925). In the 
past, we have seen occasional polyzygotic 
egg capsules in most of our laboratory stocks 
of B. glabrata, but attempts in these cases to 
increase its frequency by selection always 
failed. The fact that snails of one of our genet- 
ically isolated stocks of B. glabrata, when 
mated with snails of certain other stocks, con- 
sistently produce polyzygotic egg capsules in- 
dicates that there is indeed a genetic factor in 
this case of polyzygocy. 

Bigus (1981) reports that polyzygocy in 
Physa acuta increases as reproductive sen- 
escence approaches, concurrent with a re- 
duction in the thickness of the capsule and in 
the amount of capsular fluid. Although we 
have found no age dependence in our study, 
the very small clutch size of most self-fertiliz- 
ing M3 636 snails suggests that polyzygocy 
may serve as a reproductive strategy that 
maximizes number of offspring when re- 
sources for egg capsule or clutch production 
are for some reason limited or declining while 
production of zygotes continues or increases. 

Larval motility does not limit fusion and may 
in some instances be necessary to bring the 
trochophore larvae into contact with each 
other. The embryos of B. glabrata do not move 
until the vitelline membrane is lost. Leh- 
mannia embryos lack a vitelline membrane 
and are reportedly motile as zygotes when 



the polar bodies are visible (Mason & Cope- 
land, 1988). Although these authors observed 
early paired zygotes of Lehmannia in appar- 
ent contact, they do not state when in embry- 
onic development fusion may have occurred, 
only that teratoids survived to hatch. Eyster 
(1 995) increased the number of conjoined ter- 
atoids, first observed as veliger larvae, by 
subjecting the polyzygotic egg capsules of 
Crepidula to acidified seawater, possibly dis- 
rupting or destroying the vitelline membrane. 
We have not been able to demonstrably re- 
move this membrane from B. glabrata em- 
bryos and have not seen larval motility, indi- 
cating loss of the membrane, before the early 
trochophore stage. 

By the trochophore larva stage, the mor- 
phogenetic fields of B. glabrata have been es- 
tablished (Camey & Verdonk, 1970; Kawano 
et al., 1992), and cell membranes on the ex- 
terior of the embryo are evidently primed to 
form cell-to-cell junctions (Serras et al., 1 990). 
Further study of live and fixed, whole-mount 
and sectioned, fused embryos will help deter- 
mine which cells form these junctions, the na- 
ture of the junctions, whether non-homolo- 
gous areas of separate embryos can fuse, 
and the effect on development of communica- 
tion between allophenic cells. 

The morphologies of conjoined teratoids in 
our laboratory are analogous to morphologies 
reported for teratoids of other molluscan 
species, and most closely resemble those re- 
ported by Bigus (1981) for Physa acuta, an- 
other pulmonate snail. The survival to maturity 
of bizarrely configured teratoids of Lehmannia 
(Mason & Copeland, 1988) may be due to the 
lack of shell to interfere with mobility or to limit 
the spatial arrangement of multiple organs. 

From the morphologies we have observed, 
it is apparent that the development of head re- 
gions is not as greatly modified or suppressed 
as the development of other regions often is. 
We cannot readily determine the extent to 
which internal organs are mosaic in a devel- 
oped teratoid, but the head and mouth parts 
are clearly distinct. When separate hearts are 
initially present, in some cases, one will per- 
sist as the other seems to be resorbed. Other 
organ systems, including the kidney, repro- 
ductive tract, and the digestive tract posterior 
to the mouth, develop later than the heart and 
are not as readily visible under the shell as is 
the beating heart. To what degree these and 
other systems are histologically a mosaic of 
genetically different cells or are the result of 
suppression or degeneration of one genetic 



128 



RICHARDS ET AL. 



line of cells while the other cell line develops 
is not known. 



ACKNOWLEDGEMENTS 

We are grateful to Dr. J. A. M. van den 
Biggelaar for comments on an early draft of 
this manuscript. This work was supported by 
grant AI-27777 from the National Institute of 
Allergy and Infectious Diseases. 



LITERATURE CITED 

ARAMBASIC, M., M. PASIC, & L. KOJIC, 1989, 
Embryonic development of Lymnaea stagnalis L. 
2. Partial development in vitro in the absence of 
the egg capsule. Zoologische Jahrbücher 
{Anatomie und Ontogie de Tiere), 119(1): 27-35. 

BIGUS, L., 1 981 , Polyvitellinity and fusion of germs 
in Physa acuta (Pulmonata, Basommatophora). 
Zoologische Jahrbücher (Anatomie und Ontogie 
de Tiere), 105:526-550. 

CAMEY, T & N. H. VERDONK, 1970, The early de- 
velopment of the snail Biomphalaria glabrata 
(Say) and the origin of the head organs. 
Netherlands Journal of Zoology 20: 93-121 . 

CRABB, E. D., 1 931 , The origin of independent and 
of conjoined twins in freshwater snails. Wilhelm 
Roux Archiv fur Entwicklungsmechanik de 
Organismen, 24: 332-356. 

EYSTER, L. S., 1995, Conjoined twins, triplets, and 
quadruplets in the gastropod Crepidula fornicata. 
Invertebrate Biology 1 14(4): 307-323. 

HALL, R. P., 1925, Twinning in a mollusc, Serpu- 
loides vermicularis. Science, 61: 658. 

KAWANO (CAMEY), K.T OKAZAKI & L. RE, 1992, 
Embryonic development of Biomphalaria glabrata 
(Say, 1818) (Mollusca, Gastropoda, Planorbidae): 



a practical guide to the main stages. Malacologia, 
34(1-2): 25-32. 

LIANG, Y S., J. I. BRUCE & D. A. BOYD, 1987, 
Laboratory cultivation of schistosome vector 
snails and maintenance of schistosome life cy- 
cles. Proceedings of the First SIno-American 
Symposium, 1 : 34-48. 

MASON, J. & J. COPELAND, 1988, The incidence 
and variety of Lehmannia valentiana conjoined 
twins: related breeding experiments (Gastro- 
poda, Pulmonata). Malacologia, 28(1-2): 17-27. 

MICHELSON, E. H. & A. R. SCHORK, 1958, Tera- 
togeny in Australorbis glabratus. Nautilus, 72:3-5. 

NEWTON, W. L., 1954, Albinism in Australorbis 
glabratus. Proceedings of the Helminthological 
Society of Washington, 21 : 72-74. 

PARAENSE, W. L., 1955, Self- and cross-fertiliza- 
tion in Australorbis glabratus. Memohas do 
Instituto Oswaldo Cruz, 53: 285-291. 

PARAENSE, W. L., 1993, Egg-laying induced by in- 
semination in Biomphalaria snails. Memorias do 
Instituto Oswaldo Cruz, Rio de Janeiro 88(4): 
551-555. 

RICHARDS, С S., 1985, A new pigmentation mu- 
tation in Biomphalaria glabrata. Malacologia, 
26(1-2): 145-151. 

RICHARDS, С S., M. KNIGHT & F. A. LEWIS, 1992, 
Genetics of Biomphalaria glabrata and its effect 
on the outcome of Schistosoma mansoni infec- 
tion. Parasitology Today 8(5): 171-182. 

RICHARDS, С S. & P С. SHADE, 1987, The ge- 
netic variation in compatibility in Biomphalaria 
glabrata and Schistosoma mansoni. Journal of 
Parasitology 73: 1 1 46-1 151. 

SERRAS, F., W. J. A. G. DICTUS, & J. A. M. VAN 
DEN BIGGELAAR, 1990, Changes in junctional 
communication associated with cell cycle arrest 
and differentiation of trochoblasts in embryos of 
Patella vulgata. Developmental Biology 137(1): 
207-216. 

Revised ms. accepted 27 August 1997 



MALACOLOGIA, 1998, 39(1-2): 129-139 

COMPARATIVE SPERM MORPHOLOGY AND PHYLOGENETIC CLASSIFICATION 
OF RECENT MYTILOIDEA (BIVALVIA) 

Alexander I. Kafanov & Anatoly L. Drozdov 

Institute of Marine Biology, Far East Branch, Russian Academy of Sciences, Vladivostok 

690041, Russia 



ABSTRACT 

In sperm morphology, the genera of Recent Mytiloidea that have been studied thus far — 
Adula, Arcuatula, Aulacomya, Brachidontes, Choromytilus, Crenomytilus, Modiolus, Musculista, 
Musculus, Mytilus, Perna, Semimytilus and Septifer — may be classified into several groups 
based on size of the tip, mode of chromatin packing, number of mitochondria, and presence or 
absence of an axial rod in the acrosomes. Spermatozoa of Modiolus might be regarded as a 
basal or plesiomorphic type within the Mytiloidea. The available sperm morphology data, to- 
gether with conchological characters, is adequate for suggesting a modified classification of 
Recent Mytiloidea, representing a sort of trade-off between the arrangements of Soot-Ryen 
(1969) and Scarlato & Starobogatov (1979, 1984). Only one family (Mytilidae) is conventionally 
acknowledged among Recent Mytiloidea. The family Septiferidae Scarlato & Starobogatov, 1 979, 
has been ranked as a tribe in the subfamily Modiolinae. The subfamily Perninae Scarlato & 
Starobogatov, 1979, поп Zittel, 1895, has been abolished. 

Key works: Mytilidae, sperm morphology classification. 



INTRODUCTION 

It is commonly accepted that, besides the 
Triassic Mysldiellidae Cox, 1964, the super- 
family Mytiloidea includes the large family 
Mytilidae Rafinesque, 1815, incorporating at 
least 57 Recent and fossil taxa of the genus 
group. Numerous Mytilidae are of commercial 
importance in fisheries and aquaculture and 
are of biostratigraphical use. For these rea- 
sons, the systematics of the family is of long- 
standing interest to researchers. 

After the revision by Soot-Ryen (1969) for 
the Treatise on Invertebrate Paleontology, it 
became the usual practice to subdivide 
Mytilidae into four or five separate subfamilies: 
Mytilinae, Crenellinae Gray, 1840, Musculinae 
Iredale, 1939, Lithophaginae H. Adams & A. 
Adams, 1857, and Modiolinae Keen, 1958 
(Kafanov, 1987). Subsequently, these subfam- 
ilies were supplemented by the monotypic 
Dacrydiinae (Ockelmann, 1983: 112). 

A very different scheme was suggested by 
Scarlato & Starobogatov (1979, 1984; Staro- 
bogatov, 1 992), who subdivided the family My- 
tilidae into four separate families — Mytilidae, 
Septiferidae Scarlato & Starobogatov, 1979, 
Crenellidae, and Lithophagidae — and they 
established an additional 13 subfamilies. Un- 
fortunately, their diagnoses of the taxa are ex- 



tremely brief and they did not give arguments 
to support their classification. At the same 
time, a need for new morphological criteria for 
the classification of the Mytiloidea and the tax- 
onomic importance of sperm morphology in 
other groups made us pay attention to specific 
features of spermatozoon morphology. 



TAXONOMIC IMPORTANCE OF 
SPERMATOZOON MORPHOLOGY 

In Recent years, gamete ultrastructure has 
been used extensively for solving various sys- 
tematic and phylogenetic problems in the 
Metazoa (Afzelius, 1979; Jamieson & Rouse, 
1 989; Ferraguti & Gelder, 1 991 ; Justine, 1 991 ; 
Jamieson et al., 1995). Species specificity for 
spermatozoa ultrastructure was initially estab- 
lished for Mammalia (Bishop & Austin, 1957) 
and subsequently confirmed for other animal 
groups (Baccetti & Afzelius, 1976), including 
bivalve molluscs (Drozdov & Reunov, 1986b). 
Structural features of spermatozoa have been 
successfully used for identification of sibling 
species (Meier et al., 1972; Aksenova, 1978). 
Species specificity of spermatozoon morphol- 
ogy, acrosome structure in particular, is 
thought (Popham, 1979) to contribute to re- 
productive isolation of closely related species 



129 



130 



KAFANOV AND DROZDOV 



on a cytological level. In the opinion of a num- 
ber of researchers (Ockelman, 1964, 1965; 
Popham, 1979; Franzen, 1970, 1983; Drozdov 
& Kasyanov, 1985; Pashchenko & Drozdov, 
1991), the spermatozoon structure of bivalve 
molluscs is dependent on the structure of egg 
membrane and specific insemination features. 

A significant amount of data is presently 
available concerning gamete-specific mor- 
phological features for families of various ani- 
mal groups: scleractinias (Steiner, 1991), 
archiannelids (Franzen, 1982; Franzen & 
Sensenbaugh, 1984), and chitons (Hodgson 
et al., 1988; Pashchenko & Drozdov, 1994, 
1997). Such features also are known in the 
Bivalvia (Karpevich, 1961, 1964; Gharago- 
zlou-Van Ginneken & Pochon-Masson, 1971; 
Thompson, 1973; Popham, 1974, 1979; Max- 
well, 1983; Drozdov & Kasyanov, 1985; 
Eckelbarger et al., 1990; Hodgson et al., 
1987, 1990; Healy, 1995, 1996). In particular, 
acrosomal morphology displays characteristic 
features that provide information on phyloge- 
netic relationships (Baccetti, 1970). 

Spermatozoon morphology of Mytiloidea 



has been dealt with in several papers (Table 
1), and sufficient data now exists for a review 
of previous classifications. 



GENERIC FEATURES OF 

SPERMATOZOON MORPHOLOGY 

IN MYTILOIDEA 

All mytiloidean sperm show essentially the 
same organization of the midpiece, that is, 
spherical mitochondria are grouped in a ring 
around the proximal and distal centrioles 
(centrioles arranged at approximately 90° to 
each other), a small rootlet connects the prox- 
imal centriole to the nucleus, and a satellite 
fibre complex of nine terminally forked fibres 
anchors the distal centriole to the plasma 
membrane. Some generic differences in sper- 
matozoon morphology are given below and in 
Table 2. 

Modiolus Lamarck, 1799 (Fig. la). Large- 
sized spermatozoa with flask-shaped head 
and pointed acrosome. Barrel-shaped nu- 
cleus 2.0 um in length and 2.7 um in diameter. 



TABLE 1 . References to the morphology of spermatozoa in Recent Mytiloidea 



Species 



Authors 



Adula falcatoides Habe, 1955 
Arcuatula capensis (Krauss, 1848) 
Aulacomya ater (MoWna, 1782) 

Brachidontes semistriatus (Krauss, 1848) 
Choromytilus chorus (Molina, 1782) 
Choromytilus meridionalis (Krauss, 1848) 
Crenomytilus grayanus (Dunker, 1 853) 

Modiolus americanus (Leach, 1 81 5) 
M. kurilensis Bernard, 1983 

M. modiolus (Linnaeus, 1758) 

Musculista senhousia (Benson, in Cantor, 1842) 

Musculus discors {Linnaeus, 1767), including 

M. laevigatus (Gray 1824) 
Mytilus chilensisHupé, 1854 
M. coruscus Gould, 1861 
Mytilus of the group of M. edulis [M. edulis Linnaeus, 

1758 + M. trossulus Gould, 1850] 

M. galloprovincialis Lamarck, 1819 

Pema pema (Linnaeus, 1758) 

P. viridis (Linnaeus, 1758) 
Semimytilus algosus (Gould, 1850) 
Septifer keenae Nomura, 1936 



Reunov& Drozdov, 1986 

Reunov & Hodgson, 1994 

Hodgson & Bernard, 1986a; Garrido & Gallardo, 

1996 
Reunov & Hodgson, 1994 
Garridos Gallardo, 1996 
Hodgson & Bernard, 1986a 
Drozdov, 1979, 1983; Drozdov & Mashansky 1979; 

Drozdov et al., 1981; Drozdov & Kasyanov, 1985 
Hylander & Summers, 1977 
Drozdov & Kasyanov, 1985; Drozdov & Reunov, 

1986a 
Franzen, 1955 
Drozdov, 1992 
Kaufman, 1977; Franzen, 1983; Drozdov & 

Kasyanov, 1985 
Garndo& Gallardo, 1996 
Reunov & Drozdov 1987 
Niijima & Dan, 1965; Longo & Dornfeld, 1967; 

Endo, 1976; Drozdov & Reunov 1986b; Hodgson 

& Bernard, 1986b 
Hodgson & Bernard, 1986b; Crespo et al., 1990; 

Drozdov 1992 
Boucart et al., 1965; Bernard & Hodgson, 1985; 

Hodgson & Bernard, 1986a 
Drozdov 1992 
Garridos Gallardo, 1996 
Reunov & Drozdov 1986 



MORPHOLOGY AND CLASSIFICATION OF RECENT MYTILOIDEA 



131 





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KAFANOV AND DROZDOV 




FIG. 1 . Structural pattern of spermatozoa of the subfamily Modiolinae: a — Modiolus kurilensis, b — Aulacomya 
ater, с — Adula falcatoides, d — Brachidontes semisthatus, e — Choromytilus meridionalis, f — Musculista sen- 
housia. g — Arcuatula capensis, h — Septifer keenae. A: acrosome; N: nucleus; AR: axial rod; m: mitochondria; 
dc: distal centriole; pc: proximal centriole; AV: acrosomal vesicle; PM: periacrosomal material. Bar = 1 urn. 



No axial rod typically present. Acrosomal 
соглр1ех of two parts: cone-shaped acrosomal 
vesicle and periacrosomal granular material 
arranged between the vesicle and nucleus. 
On side of nucleus, acrosomal vesicle with a 
contraction filled by periacrosomal material. 
Midpiece of spermatozoon formed by two mu- 
tually perpendicular centrioles, surrounded by 
13-14 mitochondria. 

Aulacomya Mörch, 1853 (Fig. 2b). Sperms 
with flask-shaped head, containing a barrel- 
shaped nucleus of about 2.2 um length and a 
large (3.7 um long) cone-shaped acrosomal 
vesicle with a large cavity filled by granular pe- 
riacrosomal material. The midpiece of sper- 
matozoon with five, sometimes six, spherical 
mitochondria. 

Adula H. & A. Adams, 1857 (Fig. 1c). 
Acrosomal complex (1.4 iim long) with a con- 
ical electron-dense acrosomal vesicle with a 
contraction in a distal acrosomal part, filled 
by electron-lucent periacrosomal material. 
Middle part of spermatozoon formed by five 
mitochondria encircling two centrioles. 

Brachidontes Swainson, 1840 (Fig. Id), 
and Arcuatula Lamy, 1919, ex Jousseaume, 
MS (Fig. Ig). Sperm head of B. semistriatus 
4.5 ¡.(m long, containing a barrel-shaped nu- 



cleus (2.0 X 1 .9 um) with minute anterior hole. 
Nucleus adjoining flask-shaped acrosome 
of 2.5 iim length, with a long anterior projec- 
tion. 

Sperm of A. capensis with a relatively long 
head, a barrel-shaped nucleus (length, 1.2 
um, average diameter 1.5 urn) and a small 
conical acrosome, 0.8 um long). 

Cfioromytilus Soot-Ryen, 1952 (Fig. le). 
Sperms of С meridionalis with a compara- 
tively rounded nucleus of 1.6 um diameter 
and a 2 um-long acrosome crowning the 
head. Acrosome with a large-sized conic cav- 
ity filled by granular electronically compact 
material. The middle part of spermatozoon in- 
cludes four (sometimes five) spheric mito- 
chondriae of 6 um diameter. 

/Wuscu//s/a Yamamoto & Habe, 1958 (Fig. 
If). Unlike Modiolus, spermatozoa compara- 
tively small-sized (bullet-shaped head of 
sperm about 2.5 um, middle piece of sperma- 
tozoon 0.4 urn, top head width 1 .2 um). Flask- 
shaped acrosome with two components: acro- 
somal vesicle widened distally and narrowed 
towards the top, possessing a contraction in 
the distal part filled by globular periacrosomal 
material. Proximal and distal centrioles per- 
pendicular. 



MORPHOLOGY AND CLASSIFICATION OF RECENT MYTILOIDEA 



133 



Septifer {Mytilisepta) Habe, 1951 (Fig. 1h). 
Sperms with a bullet-shaped head of 1.2 
length and 1.4 ¡.tm width. Conical acrosome 
0.7 !.im-long with cupola-shaped acrosomal 
vesicle filled with electron-lucent periacroso- 
mal material. Two centrioles surrounded by 
five mitochondriae of 0.4 ¡.im diameter. 

Mytilus Linnaeus, 1758 (Figs. 2a, b, d, e), 
Crenomytilus Soot-Ryen, 1955 (Fig. 2c), and 
Perna Retzius, 1788 (Figs. 2f, g). Sperma- 
tozoa of several species of these genera with 
flask-shaped head between 3.6 um {Perna 
viridis) and 7.0 1.1m {Mytilus coruscus) in 
length. Barrel-shaped nucleus with pointed 
acrosome anteriorly. Posteriorly, nucleus ad- 
jacent to middle part of spermatozoon con- 
taining five spheric mitochondria and two cen- 
trioles. 

Musculus Röding, 1798 (Fig. За, b). 
Sperms with extended cone-shaped head of 
about 8.0 |.im length and 0.8 um base diame- 
ter. Acrosomal complex of two components: 
apical part with an acrosomal vesicle of ap- 
proximately 0.8 um diameter, surrounded by a 



membrane. Periacrosomal material branching 
off from vesicle and consisting of a bundle of 
actine threads entering nucleus channel and 
a small amount of globular material. In some 
specimens, the axial rod reaching midpart of 
spermatozoon {M. discorshom North Pacific), 
whereas in other specimens only reaching 
middle of nucleus (same species from North 
Atlantic and Arctic Ocean). Midportion of 
sperm with four spheric mitochondriae of 0.4 
цт diameter encircling two perpendicular 
centrioles. 



TAXONOMIC ANALYSIS 

Although a thorough taxonomic analysis 
would require the availability of sperm mor- 
phology data for a larger number of genera, 
the available data (Table 1) is adequate for 
some conclusions on alternatives for the sys- 
tematics of the Mytiloidea (Table 3). The more 
so because the taxonomic value of a charac- 
ter depends on its adaptive significance 




FIG. 2. Structural pattern of spermatozoa of the subfamily Mytilinae: a — Mytilus coruscus, b — Mytilus trossu- 
lus. с — Crenomytilus grayanus, d — Mytilus galloprovincialis. e — Mytilus edulis, f — Perna perna, g — Perna 
viridis. Bar = 1 |ят. 



134 



KAFANOV AND DROZDOV 




FIG. 3. Structural pattern of spermatozoa of the 
subfamily Musculinae: a, b — Musculus discors. 
Note that the length of the axial rod varies among 
specimens of one species. Bar = 1 urn. 



(Mayr, 1969), there is no reason to treat sper- 
matozoon morphology as less taxonomically 
important than the conventional conchologi- 
cal characters. 

In terms of the presence or absence of an 
axial rod in acrosomes of intact spermatozoa, 
all the analysed representatives of Mytiloidea 
may be clearly classed into two major groups: 
those possessing no axial rod {Modiolus, Mu- 
sculista, Septlfer, Adula, Choromytilus, Aula- 



comya, Brachldontes, Semimytilus, and Ar- 
cuatula) and those possessing an axial rod 
{Mytilus, Crenomytilus, Perna, and Muscu- 
lus). The axial rod of the acrosome consists of 
a bundle of actine filaments which, in the 
course of acrosomal reaction, form the basis 
for the formation of acrosomal filament (Droz- 
dov & Mashansky, 1 979; Drozdov et al., 1 981 ; 
Drozdov & Podgornaja, 1982; Drozdov, 1992). 
Because the acrosomal reaction has a role in 
fertilization and the onset of ontogenesis, it 
should be assumed that the presence or ab- 
sence of acrosomal the axial rod is of great 
taxonomic importance. The absence of an 
acrosomal axial rod provides evidence con- 
cerning the more primitive spermatozoon 
structure in animals with external fertilization 
(Popham, 1979). 

Spermatozoons of Modiolus are distin- 
guished by an unusually large head with a 
large amount (3.99 pkg) of loosely packed 
(0.42 pkg/um^) DNA (Tuturova, 1989), large, 
variable number (10-14) of mitochondria In 
the middle part of the spermatozoon and, as 
previously mentioned, the absence of an axial 
rod in the acrosome. These characters pre- 
sent strong evidence concerning the apomor- 
phic structure of the spermatozoa of Modio- 
lus, which is confirmed by its long (Devon- 
ian through Recent) geological history com- 
pared to the remaining Mytilidae (Soot-Ryen, 
1969). 

In terms of acrosomic rod presence/ab- 
sence. Recent Mytiloidea form two major 
groups around Modiolus and Mytilus. The 
rank of these taxa is open to debate, because 
there are no objective criteria for establish- 
ment of taxa above the species rank (Mayr, 
1969). Analoguous information about other 
superfamilies provides a perspective on how 
many families should comprise the superfam- 
ily Mytiloidea. In this connection, Cardioidea is 
notable. For instance, the spermatozoa mor- 
phology of Keenocardium californiense (De- 
shayes, 1839) (Clinocardiinae) and various 
representatives of Lymnocardiinae are very 
different from each other (Fig. 4). Never- 
theless, in the most modern classification 
(Schneider, 1992, 1995), the Cardioidea in- 
cludes only one family Cardiidae. Among 
Recent Mytiloidea, the degree of morphologi- 
cal variability of spermatozoa is much less. 

Because all the spermatozoon types of 
Mytiloidea may be derived from that of 
Modiolus, as well as the fact that even the clas- 
sification of Scarlato & Starobogatov (1979, 
1984) imparts only subfamily status to Modio- 



MORPHOLOGY AND CLASSIFICATION OF RECENT MYTILOIDEA 



135 



TABLE 3. Comparison of the classifications of Recent Mytiloidea by Soot-Ryen (1969), Scarlato & 
Starobogatov (1979, 1984) and proposed system 



Soot-Ryen, 1969 



Scarlato & Starobogatov, 
1979, 1984 



Proposed herein 



fam. Mytilidae 

Rafinesque, 1815 
subfam. Mytilinae 

Rafinesque, 1815 



subfam. Modiolinae 
Keen, 1958 



subfam. Crenellinae 
Gray, 1840 



subfam. Lithophaginae 
H. Adams & A. Adams, 1857 



fam. Mytilidae 

Rafinesque, 1815 
subfam. Mytilinae 

Rafinesque, 1815 
subfam. Arcuatulinae 

Scarlato & Starobogatov, 1979^ 
subfam. Musculinae 

Iredale, 1939 
subfam. Modiolinae 

Keen, 1958 

subfam. Brachidontinae 
Scarlato & Starobogatov, 1 979^ 



subfam. Perninae 

Scarlato & Starobogatov, 1979^ 
subfam. Trichomyinae 

Scarlato & Starobogatov, 1979 
fam. Septiferidae 

Scarlato & Starobogatov, 1979 
subfam. Septiferinae 

Scarlato & Starobogatov, 1979 
subfam. Limnoperninae 

Scarlato & Starobogatov, 1979 
fam. Crenellidae 

Gray, 1840 
subfam. Crenellinae 

Gray, 1840 
subfam. Botulinae 

Scarlato & Starobogatov, 1979'^ 
fam. Lithophagidae 

H. Adams & A. Adams, 1857 
subfam. Lithophaginae 

H. Adams & A. Adams, 1857 
subfam. Adulinae 

Scarlato & Starobogatov, 1979 



fam. Mytilidae 

Rafinesque, 1815 
subfam. Mytilinae 

Rafinesque, 1815 



subfam. Musculinae • 

Iredale, 1939 
subfam. Modiolinae 

Keen, 1958 
tribe Modiolini 

Keen, 1958 
tribe Brachidontini 

Scarlato & Starobogatov, 

1979 



tribe Septiferini 
Scarlato & Starobogatov, 
1979 



subfam. Crenellinae 
Gray, 1840 



subfam. Lithophaginae 
H. Adams & A. Adams, 1857 



Notes. ^Type-genus not given in the original publication. Established (according to Sysoev & Kantor, 1992), by name forma- 
tion, on Arcuatula Lamy, 1919, ex Jousseaume MS, non Gugenberger, 1 934, nee Soot-Ryen, 1 955. ^Nom. correct. (Kafanov, 
1987) pro Brachiodontinae Scarlato & Starobogatov, 1979. Invalid because preoccupied repeatedly as family-group name 
based on Perna Bruguiere, 1789, non Retzius, 1788 [Pernahdia Rafinesque, 1815; Pernadae Fleming, 1828, also Guilding, 
1828; Pernidae Zittel, 1895]. "'Dacrydiinae Ockelmann, 1983, may be a junior synonym. 



linae, there is no sound basis for identifying 
any other separate families among Recent 
Mytiloidea. 

The genera grouped around /Woc//o/¿ys differ 
in levels of synplesio- and synapomorphy (in 
the sense of Hennig, 1950, 1966). Morpho- 
logical evolutionary transformations of Sper- 
matozoons of the Modiolus-\ype were then 
accompanied by compactification of DNA, 
diminution of spermatozoon head size, and by 
the development of an acrosomal axial rod. 



The most advanced Spermatozoons appear 
to be those of Septifer, possessing a small- 
sized bullet-shaped head with a negligible 
(1.29 pkg) but very tightly packed (0.72 
pkg/u^) amount of DNA (Tuturova, 1989) and 
a minute acrosome. Spermatozoons of other 
genera characterized by the absence of an 
axial rod in the acrosome {Musculista. Adula, 
Choromytilus, Semimytilus, Arcuatula), being 
variable in details, occupy an intermediary po- 
sition between Modiolus and Septifer. In this 



136 



KAFANOV AND DROZDOV 




FIG. 4. Structural pattern (light microscope) of spermatozoa in cardiid subfamilies Lymnocardiinae Stoliczka, 
1870 (a-f), and Clinocardiinae Kafanov, 1975 (g). Species: a — Hypanis (Monodacna) colórala (Eichwald, 
1829), b — Hypanis (Monodacna) sp. 1, с — Hypanis (Monodacna) sp. 2, d — Hypanis (Adacna) laeviuscula 
(Eichwald, 1829), e~Hypanis (Adacna) vitrea (Eichwald, 1829), ^—Hypanis (Adacna) minima (Ostroumoff, 
1907) (Figs, a-f from: Karpevich, 1964), g — Keenocardium californiense (Deshayes, 1839) (from Drozdov, 
1992). Bar = 8 urn. 



case, the Spermatozoons of Modiolus and 
Musculista are most similar, closely corre- 
sponding to their allocation to the same sub- 
family Modiolinae by Scarlato & Starobogatov 
(1979, 1984) and Scarlato (1981). 

Among spermatozoa of Mytilus-lype, pos- 
sessing an axial rod, the most primitive are 
those of Musculus, which possess a long (ap- 
proximately 8 um), narrow head crowned with 
a minute acrosomal vesicle, from which an 
axial rod almost reaches the middle part of 
spermatozoon and which also possess five mi- 
tochondriae. These features may indicate 
some specialization, because of the large 
(about 600 um) eggs of Muscl//us compared to 
those of Mytilus (about 70 um) and to features 
of their insemination (Drozdov & Kasyanov, 
1985). 

In spermatozoon structure, as well as con- 
chological features (Siddall, 1980), the sub- 
family Perninae Scarlato & Starobogatov, 
1979, appears to be paraphyletic. Although a 
close taxonomic relation among Perna, 
Mytilus, Ciioromytilus, and Aulacomya, was 
proposed by Soot-Ryen (1952), spermatozoa 
of Perna show no morphological differences 
from those of Mytilus and Crenomytilus, 



whereas spermatozoa of Chromytilus and 
Aulacomya prove to be similar to the sperma- 
tozoa of Modiolus. This makes it impossible to 
consider Choromytilus Soot-Ryen, 1 952, to be 
a subgenus of Perna Retzius, 1788, as was 
suggested by Soot-Ryen (1969). In any case, 
however, Perninae Scarlato & Starobogatov, 
1979, Is invalid for nomenclatural reasons 
(Table 2). 

The available spermatozoan data, together 
with conchological characters, is adequate for 
suggesting an slightly modified systematics of 
Recent Mytiloidea, representing a trade-off 
between the classifications of Soot-Ryen 
(1969) and Scarlato & Starobogatov (1979, 
1984) (Table 3).The subfamily status of Litho- 
phaginae in this system is the result of its bor- 
ing habitat, generally uncommon for mytilids, 
resulting in major transformations in shell 
morphology. In any case, the Lithophaglnae Is 
more closely related to Modiolinae than to the 
Mytlllnae. 

ACKNOWLEDGEMENTS 

The manuscript of the present article was 
critically reviewed by Dr. Eugene V. Coan 



MORPHOLOGY AND CLASSIFICATION OF RECENT MYTILOIDEA 



137 



(California Academy of Sciences, San Fran- 
cisco, USA), by three anonimous reviewers, 
and by Dr. Yaroslav I. Starobogatov (Zoo- 
logical Institute, Russian Academy of Sci- 
ences, St. Petersburg, Russia). These per- 
sons made a number of important comments. 
Dr. Klara F. Tuturova (Institute of Marine 
Biology, Far East Branch, Russian Academy 
of Sciences, Vladivostok, Russia) kindly per- 
mitted to make use of the material from her 
unpublished Ph.D. dissertation. Mrs. Elena S. 
Kornienko (Institute of Marine Biology, Vladi- 
vostok, Russia) made the drawings for the 
paper and rendered constant technical assis- 
tance. Initial translation of the Russian text 
into English was made by Mr. Sergei V. 
Solovyev (Research Institute for Nature 
Conservation of the Arctic and North, St. 
Petersburg, Russia). Assistance of all the 
above persons is deeply appreciated. 

The research described in this publication 
was made possible in part by grants #95-04- 
11134 and #96-04-49702 from the Russian 
Foundation for Basic Research and by Grant 
of I NTAS #93-2176. 



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Revised ms. accepted 27 August 1997 



MALACOLOGIA, 1998, 39(1-2): 141-150 

INFLUENCE OF WATER TEMPERATURE ON THE ACTIVITY OF PLANORBARIUS 
CORNEUS{L.) (PULMONATA, PLANORBIDAE) 

Katherine Costil^ & Stuart E. R. Bailey^ 



ABSTRACT 

This study cornpiements our previous study of the influence of temperature on the life-history 
traits of the freshwater snail Planorbarius corneus by examining the effect of temperature on ac- 
tivity The activity of 24 groups of three individuals was recorded over 24 h in a 12 h light: 12 h 
dark photoperiod with six groups at each of four temperatures — 10, 15, 20 and 25°C. 

Despite considerable variations between individuals, activity increased significantly with tem- 
perature, principally due to an increase in time spent feeding. Time spent on non-displacement 
movements declined above 10°C, but time spent on locomotor activity did not alter with temper- 
ature. However, the speed and total distance moved increased significantly with temperature, as 
did the number of contacts made with the water surface and with other individuals. Locomotion, 
feeding and non-displacement movements showed no diurnal rhythms at any temperature. The 
present study shows that a behavioural component contributes to the influence of temperature 
on life-history traits. 

Key words: freshwater snail, behavioural ecology, temperature, light, locomotor and feeding 
activities. 



INTRODUCTION 

Planorbarius corneus is a large basom- 
matophoran snail, common in eutrophic 
ponds of northwest France (Costil & Clément, 
1996). It is often collected in association with 
Lymnaea stagnalis, and both species con- 
tribute to communities showing high species 
richness and diversity (Costil, 1994b). Pla- 
norbarius corneus plays an important part in 
the invertebrate communities of eutrophic 
ponds in this region, where it is eaten by both 
fish and birds. 

Natural populations of freshwater snails 
have to adapt to various abiotic constraints, 
for example, climate, desiccation and water 
chemistry. Temperature is particularly impor- 
tant for freshwater pulmonates, because they 
inhabit shallower waters than most proso- 
branch molluscs, and thus experience a sea- 
sonal range of temperatures almost as great 
as that on land (MacMahon, 1983). We have 
previously studied the life-history traits of 
P. corneus and demonstrated the importance 
of water temperature in the control of these 
snail populations (Costil, 1994a; Costil & 
Daguzan, 1995a, b). To complete this study, 
we have now examined the impact of temper- 
ature on the activity of P. corneus. 



Activity level is an important component of 
adaptive strategy. For example, locomotion in 
most animal species is an essential compo- 
nent of fitness, being necessary for both re- 
production (finding mates and oviposition 
sites) and survival (finding food and avoiding 
predators) (Calow, 1 974). Most studies of mol- 
luscan activity have concerned land pul- 
monates, and concentrated on their diurnal 
activity rhythms (e.g., Rollo, 1982; Dainton & 
Wright, 1 985; Ford & Cook, 1 987; and Wareing 
& Bailey, 1985, on slugs; and Cameron, 
1970a, b; Bailey & Lazaridou-Dimitriadou, 
1986; and Lorvelec, 1988, on helicid snails). 
The fewer studies on the activity of freshwater 
snails have focused on locomotion of tropical 
species (Beeston & Morgan, 1977; Chaudry & 
Morgan, 1983; Pimentel-Souza et a!., 1984), 
the North American planorbid Helisoma trivol- 
vis (Kavaliers, 1981) and the European spe- 
cies Lymnaea stagnalis (MacDonald, 1973), 
Ancylus fluviatilis and Planorbis contortus 
(Calow, 1974). In P. corneus, locomotion in- 
volves mucus, muscles and cilia covering the 
sole of the foot. Deliagina & Orlovsky (1990) 
established the locomotory repertoire of this 
species and the nervous control of locomotion. 

Little attention has been paid to the time 
budget of different activities, and the effect of 



^Laboratoire de Zoologie et d'Ecophysiologie, Campus de Beaulieu, Université de Rennes I, 35042 Rennes Cedex, France. 
^School of Biological Sciences, 3.614 Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdom 

141 



142 



COSTIL AND BAILEY 



temperature on this budget, or on the conse- 
quences of increased speed of movement in- 
duced by elevated temperatures on distances 
travelled and encounters made, for example, 
with other individuals and the water surface. 
This study addresses those aspects. 



METHODS 

The snails were collected from a pond near 
Rennes, northwest France, where the life 
cycle of the population had been studied 
(Costil & Daguzan, 1995b). The snails were 
immature when collected and belonged to the 
same cohort. Brought into the laboratory at 
18°C, they grew to maturity and after nine 
months were grouped into four experimental 
groups of 18 snails, each group having a sim- 
ilar mean diameter (close to 18.8 mm). 

The water temperature of each group was 
gradually adjusted to one of the four experi- 
mental temperatures of 10, 15, 20 or 25°C. 
The experimental temperatures are within the 
range generally recorded in the region. After 
an acclimatization period of at least three 
weeks at the target temperature, the activity of 
the snails was studied by filming them with a 
video-camera for 24 h. The advantages and 
disadvantages of this technique were empha- 
sised by Bailey (1994). The recording appara- 
tus consisted of a black-and-white camera 
sensitive to dim red light (6300 to 7500 A), a 
monitor, and a time-lapse video-recorder. 
Recordings were made at one frame per sec, 
so that 24 h of recording could be played back 
in one hour at normal speed to analyse activ- 
ity. 

A total of 72 snails were tested, 18 at each 
temperature. The snails were filmed in six 
groups of three. Individuals were identified by 
painting spots on one snail, stripes on a sec- 
ond, and leaving the third snail unpainted.The 
snails were fed with rectangular pieces of let- 
tuce (5x7 cm) which were weighted down to 
the bottom of the tank. Each tank (23 x 17 x 9 
cm) was filled with 1800 ml of pond water. To 
aid the recording of distances moved by the 
snails, a grid of lines was drawn on the bottom 
and sides of the tank. There was no artificial 
aeration of the water in the tanks. Recordings 
were made in a photoperiod of 12 h light (from 
08h00 to 20h00) and 12 h dark. As in 
Lorvelec's study (1988), a red light was used 
for filming during the dark phase, since pul- 
monates respond only weakly to long wave- 
lengths (Kerkut & Walker, 1975). When the 



lights went on or off, the snails either did not 
react, or immediately stopped crawling for a 
short period, usually a few seconds. 

Activity was analysed for each individual 
snail, recording its behaviour in each of the 
288 5-min units over the 24 h. Snail activity 
was classified into five categories: 

— inactive (I); 

— locomotion (L); a displacement of 2 cm or 
more in the 5 min; 

— moving without displacement (M), often 
consisting of rocking movements of the shell; 
— feeding (F), with obvious scraping move- 
ments with the head on the lettuce; 
— copulating (C). 

If a snail showed two activity categories 
(e.g., locomotion and feeding) within a five- 
min time unit, both categories were scored as 
a half unit. More than two categories never oc- 
curred within one time unit. 

The following additional variables were also 
calculated for each individual: 

— longest period of inactivity in 24 h (LP!), in 
min; 

— total distance covered in 24 h (TD), in m; 
—mean speed (MNS), the total distance 
moved divided by five times the number of 
five-min units spent locomoting, in cm min"\ 
— maximum speed (MXS), recorded over any 
5-min period; 

— number of contacts with the water surface 
in 24 h (CWS), including contacts with head or 
foot of a snail near the walls of the tank as well 
as contacts when the snail crawls with foot ex- 
tended on the surface; 

— number of contacts with fellow snails in 24 
h (CFS). 

The data on inactivity (I and LP!) were sub- 
jected to non-parametric tests (Mann Whitney 
test and Kruskal Wallis test), because the 
conditions of normality and equivariance re- 
quired for an analysis of variance were not 
met, even after various transformations. 

The data on activity were subjected to mul- 
tivariate analyses of variance (MANOVA), pre- 
ceeded by univariate analyses and Newman 
Keuls tests (Morrison, 1967; Dagnelie, 1977). 
The effects of temperature and light on the dif- 
ferent types of activity were tested using a two- 
way MANOVA with two factors (temperature 
and light), 18 replicates, and three variables 
(times spent locomoting, feeding, and in non- 
displacement movements). The effect of tem- 
perature on activity was tested with a one-way 



ACTIVITY OF PLANORBARIUS CORNEUS 



143 



MANOVA applied to eight variables (L, F, M, 
MNS, MXS, TD, CWS and CFS); M and TD 
were transformed to Log (x + 1 ) to meet the re- 
quirements of the analysis of variance. After 
MANOVA, we used multiple comparisons 
which were in agreement, and thus the results 
of WILKS test and ROY test alone are pre- 
sented here. Calculations were performed with 
STATVIEW (1988) and STAT-ITCF (1988) pro- 
grams. 



RESULTS 

Egg masses were laid at 20 and 25°C, but 
the egg masses of Planorbarius corneus are 
almost colourless, and not sufficiently visible 
to enable us to reliably distinguish ovipositing 
during recording. Mating activity was ob- 
served only at 20°C. Two matings occurred: 
one began at 18h30 and lasted for 5.33 h, the 
other began at 5h40 and lasted 6.50 h. To 
compare the results at 20°C with the others, 
we have excluded those 5.6% of time units 
which were occupied by copulatory activity, 
and brought the percentage occupied by 
other activities back to 100. 

As the high standard deviations given in 
Table 1 indicate, there are considerable differ- 
ences between individuals, and at all temper- 
atures, some snails were continuously active. 
For example, at 10°C, three snails were con- 
tinuously active while four other snails were 
inactive for three quarters of the time. At 1 5°C, 
the percentage of time that individuals spent 
feeding varied from 1 to 75%. At 20°C, the dis- 
tance covered by individual snails varied from 
less than three to more than 32 m. At 25°C, 
some snails made ten times more contacts 
with the surface than other snails. 

Temperature significantly affects both the 
total time spent inactive (H - 28.01, p = 
0.0001, Kruskal Wallis test), and the longest 
period of inactivity (H = 26.89, p = 0.0001, 



Kruskal Wallis test). The time spent inactive 
decreases at higher temperatures, and this 
decrease was most noticeable between 15 
and 20°C (Fig. 1). The mean length of the 
longest period of inactivity (LPI) was nega- 
tively related to temperature (Fig. 2); LPI at 
25°C was 35 min, but at 10°C it was > 21 h. 

The proportion of time spent feeding in- 
creases with temperature, from 17.1% at 
10°C to 56.2% at 25°C. The increase in time 
devoted to feeding accounts for most of the 
decline in inactivity seen with increasing tem- 
perature. Non-displacement movements oc- 
cupy 10.2% of the time at 10°C, but only 3.3% 
to 5.4% of the time at higher temperatures. 
Time spent in locomotion was independent of 
temperature. Figure 3 shows no consistent or 



10°C 



15°C 




a INACTIVITY 

H MOVEMENT 

□ FEEDING 

Ш LOCOMOTION 

■ MATING 




25°C 




FIG. 1 . Mean percentages of inactivity and different 
types of activity in Planorbarius corneus individuals 
at four temperatures (10, 1 5, 20 and 25°C) over 24 
h corresponding to 288 activity units of 5 min. The 
results obtained at 20°C were corrected by exclud- 
ing the activity units corresponding to mating. 



TABLE 1 . Mean numbers (± S.D.) of activity units spent by the individuals of Planorbarius corneus in inactiv- 
ity and different types of activity (locomotion, feeding, movement without displacement and mating) at 10, 15, 
20 and 25°C (n = 18). The results obtained at 20°C were corrected by excluding the activity units corre- 
sponding to mating, thus bringing back the total number of activity units to 288. 



TEMPERATURE 



INACTIVITY LOCOMOTION 



FEEDING 



MOVEMENT 



MATING 



10°C 


97.28 ± 103.89 


112.00 ±71.90 


49.28 ± 45.96 


29.4 ±22.11 





15°C 


79.06 ± 65.63 


87.61 ± 57.32 


107.72 ±64.58 


13.61 ±9.78 





20°C 


9.39 ± 10.36 


114.08 ±43.08 


138.81 ±34.11 


9.61 ±7.27 


16.11 ±31.29 


Corrected 20°C 


10.00 ± 12.02 


118.92 ±41.29 


148.86 ±43.27 


10.22 ± 12.02 





25°C 


5.28 ±4.87 


105.08 ±48.68 


161.97 ±47.30 


15.67 ± 10.13 






144 



COSTIL AND BAILEY 



LPI(ir 


in) 


П Mean 


1400- 
1200- 


Ш 


■ Maximum 


800- 


^1 




600 


" H ^ 




400 


' и r_H 




200- 


. ЩШЩ 


U-^-^— ÍL^^Y ^ 




10 15 


20 25 

Temperature 



FIG. 2. Mean (± S.D.) and maximum values of the 
longest period of inactivity (LPI) in Planobarius 
corneus put at 10, 15, 20 and 25°C. 



Inactivity 




10°C 



1S°C 



Locomotion 



cor ЗОХ 25°C 

Temperature 




10°C 



15°C 



Feeding 



cor 20°C 25°C 

Temperature 




10°C 



15°C 



Movement 



cor 20°C 2S°C 

Temperature 




lO-C 



15°C 



Ш Light period 



cor 20°C 25°C 

Temperature 

И Dark period 



FIG. 3. Mean percentages of inactivity and different 
types of activity occurring during a 1 2h light and 1 2h 
dark period in Planorbarius corneus at 10, 15, 20 
and 25°C. 



light or the light x temperature interaction 
(Table 2). Temperature emerges as the only 
factor influencing activity. As Table 3 shows, 
the time spent feeding changes significantly 
throughout the temperature range, while non- 
displacement movements only alter signifi- 
cantly over the lower part of the range, and 
there are no significant differences in the time 
spent in locomotor activity over the range of 
temperatures employed. 

In general, the higher the temperature, the 
faster the snails moved (Fig. 4). At 25°C, the 
mean speed was on average 3.5 cm min~\ 
with a maximum speed of 7.5 cm min~\ The 
mean distances covered at 25°C were twice 
and four times those recorded at 1 5 and 1 0°C 
respectively, and one snail at 25°C covered 42 
m in 24 h. The number of contacts between 
snails increased with temperature to reach a 
mean of 45.3 encounters in 24 h at 25°C, 
while at 10°C four snails never encountered a 
fellow snail. When two P. corneus snails met, 
one snail generally pushed the other and con- 
tinued on its way. At the three highest temper- 
atures, phoretic behaviour (carrying other in- 
dividuals) was often observed. Individuals at 
25°C has significantly more contacts with fel- 
lows than individuals at 10°C, probably be- 
cause, although they do not move for any 
longer period of time, they move faster, and 
cover more distance. 

The number of contacts with the water sur- 
face in general increased with temperature; 
the mean values are lowest at 15°C and high- 
est at 25°C. However, the differences were not 
significant Although these contacts with sur- 
face included breathing visits, it is very difficult 
to be see breathing from a videorecording, 
and only in a few instances were lung ventila- 
tions with the pneumostome obviously open 
observed. The results of a one-way MANOVA 
applied to eight activity variables (L, F, M, TD, 
MNS, MXS, CWS and CFS) show a signifi- 
cant effect of temperature (Wilks Test, F = 
9.327, p < 0.0001; ROY Test, eigen value = 
5.179, critical value = 0.414, p < 0.001). 
Among these variables, temperature affects 
time spent feeding, total distance, mean and 
maximum speeds, and number of encounters 
with fellow snails (Table 4). 



sizeable differences between the light and 
dark phases in the proportion of time spent in 
any activity category, and applying a two-way 
MANOVA using both Wilks test and Roy test, 
no significant differences were found for either 



DISCUSSION 

The large differences in the level of activity 
of individual snails under identical conditions 
that we observed were also noted by 



ACTIVITY OF PLANORBARIUS CORNEUS 



145 



TABLE 2. Results of a two-way multivariate analysis of variance using two different tests (Wilks 
test and Roy test) and applied to thiree variables (locomotion, feeding, movement without dis- 
placement) of Planorbarius corneus activity. 





WILKS TEST 




ROY TEST 




FACTORS 


F 


P 


Eigen Value 


Critical Value 


P 


TEMPERATURE 
LIGHT 

INTERACTION 
Temp. X Light 


10.807 
0.330 

0.451 


0.0000 
0.8062 

0.9062 


0.723 
0.007 

0.021 


0.102 
0.060 

0.102 


0.0000 
0.8065 

0.9202 



TABLE 3. For each activity, temperatures for which some significant differences were calculated 
with Roy multiple comparison method used after the two-way multivariate analysis of variance. 



LOCOMOTION 



FEEDING 



MOVEMENT 



Temperatures (°C) 
for which some 
differences were 
calculated 



NO 



10-15 
10-20 
20-25 
15-25 



10-20 



MacDonald (1973), who found that the se- 
quences and rates of activity patterns of indi- 
vidual L. stagnalis were so different as to pre- 
clude summing results. Such heterogeneity 
necessitates the use of sufficient numbers of 
individuals, and cautious interpretation of re- 
sults. Video recordings often fail to show 
whether snails and slugs feed all the time that 
they are in contact with food (Bailey, 1994), 
but the use of visible scraping movements 
proves to be an efficient definition of feeding, 
and allows a firm relationship to be estab- 
lished between increasing temperature and 
increase in time spent feeding. 

Despite the inter-individual differences, this 
study has demonstrated that P. corneus 
spends more time active, or, conversely, less 
time inactive, at higher temperatures. This 
may seem an unremarkable conclusion, but it 
does, in fact, contrast with Cameron's (1970b) 
observations on helicid snails, and Dainton & 
Whght's (1985) data on a terrestrial slug. Ford 
& Cook (1987) also showed that Umax pseu- 
doflavus is initially stimulated when the tem- 
perature is lowered from 17°C to 4°C. 
Furthermore, the present study shows that 
the component activities (locomotion, non- 
displacement movement, feeding, mating) 
show different and non-linear relationships to 
temperature. The time spent in locomotion is 
not affected by temperature. The principal 
component of activity which increases with 
temperature is the time spent feeding. This ac- 
tivity levels off between 20 and 25°C, and It is 
possible that at higher temperatures, feeding 



would be depressed, as shown in L. stagnalis 
at 30°C (MacDonald, 1973). Rollo (1982) also 
found a curvilinear relationship between tem- 
perature and activity in the slug Umax máx- 
imas, and no activity was found above 1 9.5°C. 
Non-displacement movement of R corneus is 
not linearly related to temperature, the mean 
value being minimum at 20°C and maximum 
at 10°C (the sole significant difference): more- 
over, this activity showed no significant rela- 
tionship to temperature when the MANOVA 
was applied to a higher number of variables. 

The observations of egg-laying and mating 
at higher temperatures are consistent with our 
previous demonstration that the population of 
P. corneus from which these animals were 
taken reproduces at and above 15°C and is 
most fecund at 20°C, all reproduction para- 
meters being affected by temperature (Costil 
& Daguzan, 1995a). No complex courtship 
was seen, the first contact between partners 
occurring about 1 min before the start of cop- 
ulation. Durations of mating varying from 3 to 
5 h are generally reported for planorbid 
species (Madsen et al., 1983), slightly shorter 
than the times noted here. However, the low 
number of observed matings do not allow us 
to draw any firm conclusions. 

A snail's activity pattern is an energetic com- 
promise between different activities, and pedal 
mucus production accounts for 1 3-32% of the 
energy assimilated (Calow, 1974). According 
to Denny (1980), "it seems likely that the high 
cost of movement has affected the lifestyle of 
these animals, for example by limiting the dis- 



146 



COSTIL AND BAILEY 



MNS ев 

10 IS 20 25 

Temperature 



] MXS ^д 



20 



10 15 20 25 

Temperature 



TD 



10 15 20 25 



Temperaturi- 



UèM 



10 15 20 



25 
Temperature 



CVVS ^^ 



25 
Temperature 



I I Mean jTv] Minimun 



FIG. 4. Mean (± S.D.), minimum and maximum val- 
ues of activity variables calculated in Planorbarius 
corneus put at 10, 15, 20 and 25''C. MNS = mean 
speed for 24 h filmed; MXS = maximum speed; TD 
= total distance covered by snails; CFS = number of 
meetings with the fellow snails; CWS = number of 
contacts with the water surface. 



tance over which it is profitable to crawl in 
search of mates or food." Mouritsen & Jensen 
(1994) found that infection by larval trema- 
todes reduced locomotor activity of Hydrobia 
ulvae, and suggested that the energy for loco- 
motion is probably re-allocated to parasite me- 
tabolism and excessive growth of the host. 

The persistence of locomotion when tem- 
perature decreases is important for species 
that make regular seasonal migrations or bur- 
row into the substratum in winter. Cheatum 
(1934) reported that during autumn, when 
temperatures are declining, some species mi- 
grate from shallow littoral waters to overwinter 
in deeper waters. Several authors have con- 
firmed such migrations, but there is no definite 
information on P. corneus. Caution is required 
when attempting to extend laboratory obser- 
vations on the effect of temperature on speed 
of movement to field conditions: movement 
over a substratum of mud, plants and stones 
could be expected to be slower than on the 
glass of an aquarium tank. Furthermore, 
Calow (1974) showed that the speed of 
Planorbis contortus is affected by starvation, 
food availability and water movement, 
whereas Dimock (1 985) found that after three 
weeks in an aquarium, there was a 50% re- 
duction in speed and a significant decrease in 
overall activity in the marine mud snail 
llyanassa obsoleta. Nonetheless, DeWit 
(1955) reported a maximum speed of 7 cm 
min"^ in P. corneus, close to our highest 
recorded speed, and speeds of 8.5 cm min"^ 
for the slightly larger L. stagnalis and 6.5 cm 
min"^ for the smaller Physa fontlnalls. In our 
experiments, the mean distance travelled in 
24 h ranged from 4.38 m at 10°C to 19.29 m 
at 25°C, with a maximum value of 42 m. Boss 
et al. (1984) recorded much smaller displace- 
ments in 24 h by marked individuals of three 
aquatic species of snail — less than 40% 
moved one metre or more from the release 
point, and the maximum distance moved in 
the speediest species, Physa integra, was 
less than seven m. However, these authors 
observed snail positions only at intervals and 
did not follow the complete tracks of the 
snails. The direction of movement is also im- 
portant, and Deliagina & Orlovsky (1990) re- 
ported that when searching for food, P. 
corneus exhibited very sinuous tracks, turning 
at irregular intervals. In random movement, 
the mean displacement is close to the square 
root of tracklength (Bailey, 1989). 

Freshwater pulmonates renew the air in the 
mantle cavity lung by periodic visits to the sur- 



ACTIVITY OF PLANORBARIUS CORNEUS 



147 



TABLE 4. Temperatures for which some significant differences were calculated with Roy multiple comparison 
method used after the one-way multivariate analysis of variance. L = locomotion, F = feeding, M = movement, 
TD = total distance covered by the snails, MNS = mean speed, MXS = maximum speed, CWS = number of 
contacts with water surface, CFS = number of meetings with fellow snails. 



M 



TD 



MNS 



MXS 



CWS 



CFS 



Temperatures for which 
some differences were 
calculated 



NO 10-20 
10-25 



NO 



10-20 


10-20 


10-20 


10-25 


20-25 


20-25 
15-25 



10-25 



NO 



face. The interval between lung ventilations 
varies considerably between species: in nine 
species studied by Cheatum (1934) the range 
was from 52 min in Lymnaea palustris Xo 1493 
min in Physa sayii crassa. The pseudobranch 
of R corneus enables this species to obtain 
over 50% of its total oxygen uptake by cuta- 
neous exchange at much lower levels than the 
similarly sized L. stagnalis, and the high affin- 
ity of planorbid haemoglobin makes better 
use of pulmonary oxygen, allowing it to re- 
main submerged for extended periods, even 
burrowing into substrate (Jones, 1961, 1964). 
Water contains less dissolved oxygen at 
higher temperatures, and at 11 °C, the aver- 
age interval between breathing periods for 
Cheatum's nine species was nearly 22 times 
that in water at 21 °C. In L. stagnalis, the per- 
centage of time breathing increased with tem- 
perature (MacDonald, 1973). Although snails 
would be expected to visit the surface more 
frequently merely because of the increased 
distances they moved at higher temperatures, 
surface visits increase in response to lowered 
oxygen tensions even at the same tempera- 
ture (Jones, 1961). 

Our conclusion that snail activity was unaf- 
fected by the presence or absence of light, 
might appear surprising, given the well-known 
nocturnality of terrestrial gastropods (e.g., 
Umax pseudoflavus, Ford & Cook, 1987; 
Helix aspersa, Lorvelec, 1988; and Helix luco- 
rum, Bailey & Lazaridou-Dimitriadou, 1986). 
Unlike terrestrial snails and slugs, however, 
freshwater gastropods are not subjected to 
the low humidity that reduces the activity of 
helicids (Cameron, 1970a). Thus, they are not 
constrained to place most of their activities 
during the night. Several studies on the influ- 
ence of light, reviewed by MacMahon (1983), 
suggest that the activity of freshwater gas- 
tropods increases during darkness, or at dusk 
and dawn. In Bulinus tropicus, locomotion, 
feeding, excretion, ovipositing and hatching 
all follow a diurnal pattern (Chaudry & 



Morgan, 1983). Endogenous dawn and dusk 
activity peaks are reported in the prosobranch 
Melanoides tuberculata by Beeston & Morgan 
(1979). Infected individuals of L. stagnalis are 
most active in the first few hours of illumina- 
tion (Anderson et al., 1976). In the absence of 
food, P. corneus exhibited maximum locomo- 
tor activity during the day (Deliagina & 
Orlovsky, 1990). Truscott et al. (1995) found 
maximum activity in Lymnaea stagnalis in the 
morning, and minimum activity around mid- 
night, but the differences were small. Intertidal 
gastropods could constitute an intermediate 
case: the pulmonate limpets Siphonaria 
capensis are active at low tide both by day 
and night in intertidal pools, whereas limpets 
exposed to air at low tide are active only dur- 
ing nocturnal or late evening low tides (Branch 
& Cherry, 1985). Nevertheless, the siphonar- 
ian species S. sirius, inhabiting the low shore, 
is active only during daytime and when awash 
and submerged both at ebb and flood tides 
(Iwasaki, 1995). Barnes (1986) suggested 
that the activity variation in Hydrobia ulvae 
was most likely a direct response to changes 
in light intensity and water cover. 

The tropical planorbid Biomphalaria gla- 
brata exemplifies the great variation in activity 
that can be encountered in a single species 
under the influence of several factors, includ- 
ing parasitization. Moreover, activity, in com- 
mon with many life-history traits, probably 
differs between different populations of a spe- 
cies. Pimentel-Souza et al. (1984) reported a 
dusk and a dawn peak of activity in B. 
glabrata. The levels of locomotion are similar 
in constant darkness and constant light, al- 
though significantly lower than levels shown 
under a natural cycle of illumination. Biom- 
phalaria glabrata shows more locomotor ac- 
tivity by day than by night, with a distinct max- 
imum in the second hour of light, in contrast to 
feeding and egg-laying activities (Hien & 
Disko, 1981). Gerard (1996) also found that 
uninfected B. glabrata move less during the 



148 



COSTIL AND BAILEY 



night than during the day, but the locomotor 
activity of infected individuals is not influenced 
by the time of day or night. 

The importance of light in directly control- 
ling activity or synchronising the endogenous 
rhythm of activity of terrestrial gastropods has 
diverted attention away from the subtle effects 
of temperature in terrestrial species. However, 
Cameron (1970b) found significant effects of 
temperature and time on the activity of three 
species of land snails. All species had their 
maximum daytime activity at 8°C but became 
increasingly nocturnal as temperature in- 
creased. Arianta arbustorum, the least noctur- 
nal species, reached maximum activity at 8°C, 
but Cepaea nemoralis and C. hortensis were 
most active at 22°C. In aquatic snails, also, 
temperature interacts with endogenous activ- 
ity cycles in subtle ways: Kavaliers (1981) 
showed that the planorbid Helisoma trivolvis 
has a circadian rhythm of behavioural ther- 
moregulation, selecting maximum tempera- 
tures of 21-22°C in a thermal gradient during 
the dark phase, and minimum temperatures 
(17-18°C) during the light phase (Kavaliers, 
1981). This rhythm had an endogenous basis, 
and temperature selection continued in con- 
stant darkness. 

Temperature and light influence molluscan 
behaviour in different ways. From the present 
results and those already discussed, we may 
conclude that temperature, light and endo- 
genous circadian rhythm act together to con- 
trol the behaviour of terrestrial gastropods, 
whereas temperature is the most important 
factor controlling the behaviour of freshwater 
species. Interspecific differences probably re- 
flect different environmental constraints to 
which different species have adapted. Basom- 
matophoran snails, including the planorbids, 
are pulmonates that have secondarily re- 
adapted to aquatic life, and different species 
show varying degrees of re-adaptation (Rus- 
sell-Hunter, 1978). It would be of greatest in- 
terest to compare the influence of tempera- 
ture and light on the behaviour of a less 
aquatic species, such as L. truncatula, with 
that of the more aquatic P. corneus. 

In northwest France, P. corneus has a 
spring generation each year, and sometimes 
an autumn one as well (Costil & Daguzan, 
1995b). Compared to Planorbis planorbis, P. 
corneus appears to be more influenced by cli- 
mate, and there are strong differences in the 
growth patterns of the spring and autumn 
generations. Shell growth was fastest in 
Spring, and very slow or nil in Winter. In the 



laboratory, growth increases with temperature 
(Costil, 1994a): this is connected to the en- 
hancement of metabolic processes with a 
temperature coefficient of about two (Ricklefs, 
1990). However, the present study demon- 
strates an additional behavioural explanation 
for enhanced growth. The increased feeding 
activity at higher temperatures is important in 
meeting the increased requirements for 
growth and reproduction. 

This study also provides a behavioural ex- 
planation for the precocious senility at higher 
temperatures. At 10°C, growth is slow, and 
there are many mortalities (Costil, 1994a); 
this is accompanied by inactivity, slow loco- 
motion, and little feeding activity. However, the 
snails reared at the two highest temperatures 
expended energy on fast somatic growth and 
then became senile earlier. Life expectancy 
from hatching falls from 2.64 years at 15°C to 
1.96 y at 20°C and 1.26 y at 25°C (Costil, 
1994a). Snails at the two highest tempera- 
tures were rarely inactive and moved quickly. 

Our wider study demonstrates the impor- 
tance of temperature on the life history of P. 
corneus, and the present study emphasises 
the induction of life history traits by behavioural 
components, as well as physiological ones. 



ACKNOWLEDGEMENTS 

Thanks are due to J. L. Foulon for technical 
help. 



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Revised ms. accepted 27 August 1997 



MALACOLOGIA, 1998, 39(1-2): 151-165 

SHELL REPAIR FREQUENCIES IN WHELKS AND MOON SNAILS 
FROM DELAWARE AND SOUTHERN NEW JERSEY 

Gregory P. DietP & Richard R. Alexander^ 



ABSTRACT 

More than 1 ,500 specimens combined from the extant moon snails Euspira heros (Say) and 
Neverita duplicata (Say), and the whelks Busycon carica (Gmelin) and Busycotypus canalicula- 
tus (Linnaeus) from southern New Jersey tidal flats were examined for breakage-induced shell 
repair. Additionally 500 specimens of E. Iieros and N. duplicata and Busycon scalarispira 
(Conrad) from the Miocene Kirkwood Formation collected from northern Delaware were exam- 
ined. On each specimen, body whorl diameter (WD) and apertural lip thickness (ALT) at three po- 
sitions, namely, anterior-most, mid-length, and posterior-most location on the lip, were measured 
and number of scars per final whorl counted. Although mean number of repairs per specimen dif- 
fered among the five species, averages were comparable for Recent moon snails (1.1 tor N. du- 
plicata and 1 .0 for E. heros) and whelks (5.3 for B. carica and 5.2 for B. canaliculatus). Mean re- 
pairs/specimen were appreciably lower for Miocene naticids (0.4) and the melongenid (0.7). 
Repair frequencies/shell ranged from zero to 12 for Recent whelk species and Recent N. dupli- 
cata, zero to seven for the Recent E. heros, and zero to four for Miocene moon snails and whelks. 
Only four and three percent of shells of B. canaliculatus and B. carica lack repairs, whereas 48% 
and 57% of Recent E. heros and N. duplicata, respectively, lack repairs. The majority of shells in 
every size class of Miocene whelks and moon snails lacked repairs, save for the largest size class 
of B. scalarispira. Repair frequencies accumulated at a greater rate (regression line slope) and 
are more strongly correlated (higher r value) with WD and ALT for thicker lipped Recent whelks 
than either thinner lipped moon snails or Miocene whelks. Presence of an umbilical plug in N. du- 
plicata renders posteriorly located breaks on the apertural lip repairable, unlike the plug-lacking 
E. heros. Greater rate of scar accumulation with increasing shell size among Recent naticids re- 
flects increase in breakage-localizing shell thickness since the Miocene. Greater mean repair fre- 
quencies in Recent whelks relative to Recent moon snails is attributed to the additive effect of 
sublethal prédation on whelks plus prey-induced apertural lip fracture during valve-wedging by 
feeding whelks. The substantially lower frequency of repair/shell in B. scalarispira versus B. car- 
ica and B. canaliculatus suggests that the habit of shell-wedging of prey had not yet evolved, or 
was ineffectually practiced by Miocene whelks. 

Key words: shell repair, moon snails, whelks, durophages, shell-wedging. 



INTRODUCTION 

Shell breakage by durophagous predators 
is potentially a more important agent of an- 
tipredatory adaptive selection with respect to 
gastropod armor today than in pre-Cre- 
taceous time. The most effective shell-break- 
ing predators in Recent gastropod communi- 
ties, brachyuran crabs, elasmobranchs and 
teleost fishes, were rare, absent or had not yet 
achieved durophagy in the Paleozoic and 
early Mesozoic eras. Vermeij et al. (1 981 ) pos- 
tulated that "if all breakage were lethal, there 
would be no selection between weak and 
strong shell variants, and no shells would 
show the scars that record nonlethal injury." 



Therefore, breakage-induced shell repair, al- 
though not a measure of the magnitude of se- 
lection, is a conservative estimate for the im- 
portance of selection in favoring the evolution 
of durophage-resistant gastropod armor. Low 
frequencies of repair implies either that shell- 
breaking predators were rare or that most 
shells were successfully lethally broken, re- 
gardless of predator abundance. High fre- 
quencies of breakage-induced shell repair 
(among other defenses) reveals the effective- 
ness of shell characteristics in protecting the 
snail against contemporaneous durophages. 
Repair frequencies are expected to vary 
within and between snail species both spa- 
tially and temporally. 



Department of Ecology and Evolution, Slate University of New York, Stony Brook, New York 1 1794-5245, U.S.A. 
^Department of Geological and Marine Sciences, Rider University, Lawrenceville, New Jersey 08648-3099, U.S.A. 



151 



152 



DIETL& ALEXANDER 



Several factors influence differences in 
accumulation of repairs of sublethal shell 
breakage on different gastropod species with 
unornamented, oval to tear dropped-shaped 
apertures, among taxonomically closely and 
distantly related species. Shell size, thickness, 
strength, and shape of the gastropod prey is of 
primary importance (Vermeij, 1976, 1977; 
Curry & Kohn, 1976; Palmer, 1979; Vermeij & 
Curry, 1980; Kitchell et al., 1966; Bertness & 
Cunningham, 1981), but other factors, includ- 
ing differences in diversity and abundance of 
shell-breaking predators (Vermeij, 1987), size 
refugia from predators, within-habitat refugia 
(e.g., vegetated vs. barren substrata or dura- 
tion of intertidal exposure) from predators, 
age-structure of different prey species popula- 
tions, difference in locomotion or escape habit 
of prey species, tissue toxicity and palatability, 
as well as differences in mode of subjugation 
and invasion of prey of compared predatory 
snail species influence repair frequencies. In 
order to determine the importance of any one, 
or set of factors, it is desirable to control or 
eliminate many other variables. Studies of co- 
habitating gastropod species afford the oppor- 
tunity to eliminate differences in frequencies of 
shell repair due to disparities in predator 
abundances and within-habitat refugia, and 
focus on the architecture and size of the prey 
species and differences in their escape and/or 
feeding habits. Accordingly, the low intertidal to 
shallow subtidal, vegetation-barren, shifting, 
rippled, fine sandy bottomed community of the 
New Jersey coast offers an opportunity to 
study repair frequencies on shells of four large, 
burrowing, predatory gastropods that are 
prone to durophagous prédation. 

Durophagous predators on snails crush or 
peel their prey. Crushing involves compression 
between two rigid surfaces, such as the claws 
of many crabs (Vermeij, 1976, 1977, 1978; 
Zipser & Vermeij, 1978; Brown et al., 1979). 
Peeling involves breakage of the outer shell lip 
in a spiral direction (Muntz et al., 1965; Shoup, 
1968; Vermeij, 1978; Vermeij et al., 1980). 
Shell characteristics that deter durophagy in- 
clude a low spire, small aperture, thick outer 
lip, tight coiling, external sculpture, and retrac- 
tility (Vermeij, 1 982). In the case of moon snails 
and whelks, deeply embayed fractures (Fig. 2- 
1 , 2-8) and scalloped fractures (Figs. 2-3, 5) in 
the body whorl suggest that peeling and crush- 
ing are employed by crabs preying on whelks 
and moon snails. The four large gastropods 
common to the southern New Jersey coast are 
the globose naticids Euspira heros (Say), 



Neverita duplicata (Say) and the subpyriform 
melongenids Busycon carica (Gmelin) and 
Busycotypus canaliculatus (Linnaeus) (Figs. 
1 , 2). Their common shell-crushing and shell- 
peeling predator is the blue crab Callinectes 
sapidus. Sampled gastropod species from 
New Jersey are compared to the fossil gastro- 
pod assemblage from the Miocene Kirkwood 
Formation of Delaware (Ward, 1 992), which in- 
cludes both E. lieras and N. duplicata, plus the 
whelk Busycon scalarispira (Conrad). 

Objectives of this investigation were deter- 
mination of significant differences, if any, in: (1 ) 
size-dependent accumulation of shell repair 
among the co-existing species; and (2), rela- 
tion between apertural lip thickness at se- 
lected anteroposterior positions and number 
of repairs per shell among the co-existing 
modern and fossil species. Furthermore, this 
study utilizes several perspectives. The evolu- 
tionary perspective deals with whether or not 
repair frequencies for the temperate E. heros 
and N. duplicata are similar to congeneric and 
conspecific fossil populations from the Late 
Cretaceous to Miocene. The latitudinal per- 
spective bears on the issue of different abun- 
dance and diversity of predators on whelks 
and moon snails from different communities. 
Are repair frequencies in these temperate lati- 
tude gastropods higher or lower than those re- 
ported on tropical congeneric Recent assem- 
blages (Vermeij, 1982)? The architectural-size 
perspective focuses on whether or not co- 
habitating, confamilial and congeneric species 
with differing shell thicknesses differ in their 
frequency of breakage-induced repair (i.e., E. 
heros vs. N. duplicata and B. carica vs. B. 
canaliculatus). The trophic habit perspective 
focuses on possible differences in repair fre- 
quencies among co-habitating melongenids 
versus naticids given that whelks risk, and 
moon snails do not risk, fracture of their aper- 
tural lip during invasion of their molluscan prey. 



MATERIALS AND METHODS 

Empty and hermit crab-occupied shells of 
the naticid snails N. duplicata and E. heros 
and the melongenid whelks B. carica and 
B. canaliculatus were collected from Here- 
ford Inlet (HI), Cape May County, and Longs- 
port Beach in Great Egg Harbor (GEH), 
Atlantic County, New Jersey. Collections of E. 
heros, N. duplicata, and B. scalarispira from 
the Miocene Kirkwood Formation of Delaware 
were loaned by Lauck Ward, Virginia Museum 



SHELL REPAIRS IN MOON SNAILS AND WHELKS 



153 





FIG. 1. Apertural view of Busycotypus canaliculatus and Euspira heros showing position of maximum whiorl 
diameter (WD) measured for body whorl and apertural lip thickness (ALT) measured (A) anteriorly near junc- 
tion with penultimate whorl, (B) at lip mid-length, and (C) posteriorly at siphonal canal in whelks and nearest 
umbilicus in moon snails. 



of Natural History. The fauna of the Kirkwood 
Formation correlates with the Calvert For- 
mation of Maryland, which is the lowest of the 
Chesapeake Group (Richards & Harbison, 
1942). Whorl diameter (WD) and apertural lip 
thickness (ALT) at three positions, namely, the 
anterior-most, mid-length, and posterior-most 
location on the lip (Fig. 1 ) were measured with 
Vernier calipers to the nearest 0.05 mm on 
more than 1,500 specimens combined from 
the extant moon snails N. duplicata and E, 
heros, and the whelks B. carica and B. 
canaliculatus. These same measurements 
were made on more than 500 fossil speci- 
mens of E. heros. N. duplicata and B. scalar- 
ispira. Traces of sublethal breakage may be 
seen as scars that cut across the growth lines 
or axial sculpture of the shell (Fig. 2). Number 
of sublethal scars per final whorl were 
counted for each specimen and the repair fre- 
quency determined. Repair frequency was 
calculated as the number of repairs per shell 
divided by the total sample size, following the 
definition by Vermeij et al. (1982), Vermeij & 
Dudley (1982), and Schindel et al. (1982). 

Accordingly, the number of repair scars per 
shell, an index of repair frequency, was re- 



gressed on WD, the index of size, for each 
species in the Recent and Miocene samples 
to determine if repairs were correlated with 
size (age) for either moon snail and/or whelk 
species. Both linear and second-order polyno- 
mial regressions were executed for number of 
repairs regressed on WD. The greater correla- 
tion coefficient generated by these two meth- 
ods was used in statistical comparisons 
(Tukey-HSD test) of r values within and be- 
tween species. Slope of the regression equa- 
tions, beta, were also statistically compared 
(F-test) between and within species in order 
to evaluate if the rate of accumulation of re- 
pairs with increasing size differ within and be- 
tween species. 

Shells of all species were sorted according 
to WD into six size-class divisions, namely, 
class 1 , < 1 9 mm; class 2, 20-39 mm; class 3, 
40-59 mm; class 4, 60-79 mm; class 5, 
80-99 mm; and class 6, 1 00-1 1 9 mm. Repair 
frequencies were calculated for specimens in 
each size class by dividing the total number of 
repairs in the sample by the number of shells 
examined in the size class (Table 1). The pro- 
portion of shells with a given number of scars 
per final whorl, which ranged from zero to 12, 



154 



DIETL & ALEXANDER 




FIG. 2. Specimens of Busycotypus canaliculatus (1-2), Busycon carica (3-5), Neverita duplicata (6, 8), and 
Euspira heros (7) with shell repairs. Multiple repairs in body whorl illustrated in 3, 7. Anteriorly concentrated 
repair in apertural lip shown in 2, mid-length lip repair in 4, 8, and posteriorly concentrated repair near um- 
bilicus of moon snail shown in 6. Repair that extends anteriorly across whorl shoulder to penultimate whorl 
of juvenile whelk shown in 5. Width of bar equals one cm. 



SHELL REPAIRS IN MOON SNAILS AND WHELKS 



155 



TABLE 1. Frequency of shell repair in relation to size class (based on whorl diameter). 
N = number of specimens; f = shell repair frequency. Repair frequencies are calculated 
as total repairs in a sample divided by number of shells examined in that sample. 



Taxon 


Location 


Size Class (mm) 


N 


f 


E. heros 


GEH 


<19 





0.00 


Recent 




20-39 


23 


0.69 






40-59 


29 


1.00 






60-79 


10 


2.10 




HI 


<19 


12 


0.58 






20-39 


79 


0.72 






40-59 


86 


0.96 






60-79 


33 


1.51 






80-99 


13 


1.54 


E. heros 


Kirkwood 


<19 


71 


0.48 


Miocene 


Fm; Delaware 


20-39 


28 


0.18 






40-59 


7 


0.57 


N. duplicata 


GEH 


<19 





0.00 


Recent 




20-39 


64 


0.61 






40-59 


324 


1.05 






60-79 


53 


2.13 




HI 


<19 


17 


0.35 






20-39 


193 


0.73 






40-59 


207 


1.37 






60-79 


132 


2.04 


N. duplicata 


Kirkwood 


<19 


69 


0.26 


Miocene 


Fm; Delaware 


20-39 


82 


0.73 






40-59 


5 


0.80 


B. carica 


Hi 


<19 


12 


1.67 


Recent 




20-39 


49 


3.80 






40-59 


20 


4.55 






60-79 


28 


6.10 






80-99 


40 


7.00 


B. ca nal icu latus 


HI 


<19 


3 


0.33 


Recent 




20-39 


41 


3.00 






40-59 


21 


3.42 






60-79 


28 


5.03 






80-99 


42 


7.78 






100-119 


10 


7.90 


B. scalarispira 


Kirkwood 


<19 


24 


0.13 




Fm; Delaware 


20-39 


28 


0.43 






40-59 


58 


0.59 






60-79 


66 


0.83 






80-99 


25 


1.32 






100-119 


3 


1.67 



Locations: GEH = Great Egg Harbor, Atlantic County, NJ; HI 
Jersey. Kirkwood Fm = Pollac site, Kent County, Delaware. 



Hereford Inlet, Cape May County, New 



was determined for each size class in each 
species. Hereafter this calculation is referred 
to as "proportion of repair" (Table 2). One- 
way ANOVA was used to determine if mean 
number of repairs per specimen was signifi- 
cantly different between the five species. A 



Kolmogorov-Smirnov test was used to deter- 
mine if there were differences in the percent- 
frequency of shells with increasing number of 
repair scars per shell between contemporane- 
ous and noncontemporaneous whelk and 
moon snail species (Table 3). 



156 DIETL& ALEXANDER 

TABLE 2. Proportion of shells of Euspira héros, Neverita duplicata, Busycon carica, В. scalarispira, and 
Busycotypus canaliculatus, with increasing number of repairs per shell (0-12) in relation to size class defined 
by whorl diameter (mm). 



Taxon & 
Geol. Age 




Sizei 
(m 


niqf-n 








Num 


iber of 


repairs per i 


shell 










LOC 


m) 





1 


2 


3 


4 


5 


6 


7 


8 


9 


10 


11 


12 


Eh 


GEH 


<" 


19 




























Recent 


HI 


20- 
40- 
60- 
80- 
<■ 
20- 
40- 
60- 
80- 


-39 

-59 

-79 

-99 

19 

-39 

-59 

-79 

-99 


0.57 

0.44 

0.20 

1.0 

0.70 

0.43 

0.53 

0.30 

0.31 


0.30 
0.30 
0.20 

0.10 
0.24 
0.29 
0.21 
0.23 


0.09 
0.13 
0.30 

0.10 
0.20 
0.11 
0.30 
0.31 


0.13 
0.10 

0.10 
0.07 
0.06 

0.08 


0.10 

0.05 
0.01 
0.09 


0.04 

0.01 
0.03 


0.10 

0.03 
0.08 














Nd 


GEH 


<■ 


19 




























Recent 




20- 


-39 


0.53 


0.38 


0.08 




0.02 






















40- 


-59 


0.44 


0.33 


0.14 


0.04 


0.02 


0.01 


0.01 


0.01 




0.05 






0.05 






60- 


-79 


0.21 


0.34 


0.17 


0.09 


0.06 


0.02 


0.08 




0.02 








0.02 




HI 


<■ 

20- 


19 
-39 


0.61 
0.51 


0.39 
0.38 


0.11 


0.04 


0.01 






















40- 


-59 


0.33 


0.34 


0.17 


0.06 


0.06 


0.02 


0.02 


0.03 
















60- 


-79 


0.16 


0.12 


0.16 


0.23 


0.08 


0.12 


0.04 


0.04 


0.08 










Ben 


HI 


<■ 


19 


0.67 


0.33 
























Recent 




20- 


-39 




0.18 


0.25 


0.25 


0.10 


0.13 


0.03 


0.03 






0.03 




0.03 






40- 


-59 


0.11 


0.16 


0.16 


0.21 


0.11 


0.21 




0.05 
















60- 


-79 


0.04 


0.04 


.04 


0.14 


0.25 


0.14 


0.11 


0.11 


0.04 


0.04 


0.04 




0.04 






80- 


-99 








0.02 


0.07 


0.12 


0.07 


0.17 


0.14 


0.17 


0.12 


0.07 


0.05 






100- 


-119 








0.20 






0.10 


0.20 






0.40 


0.10 




Bcr 


HI 


< 


19 


0.17 


0.08 


0.50 


0.25 




















Recent 




20- 


-39 


0.08 


0.06 


0.12 


0.20 


0.16 


0.10 


0.14 


0.06 


0.04 




0.02 










40- 


-59 


0.11 






0.16 


0.11 


0.37 


0.11 


0.05 




0.11 












60- 


-79 




0.07 


0.07 




0.17 


0.10 


0.10 


0.17 


0.14 


0.10 


0.07 










80- 


-99 


0.03 




0.03 


0.05 


0.05 


0.10 


0.18 


0.10 


0.10 


0.13 


0.13 


0.05 


0.08 






100- 


-119 








0.04 


0.08 


0.25 


0.17 


0.08 


0.17 


0.13 


0.04 


0.04 




Nd 




< 


19 


0.76 


0.21 


0.03 






















Miocene 




20- 


-39 


0.70 


0.21 


0.06 


0.02 




















Kirkwood 




40- 


-59 


0.50 


0.50 
























Eh 




< 


19 


0.66 


0.24 


0.06 


0.04 




















Miocene 




20- 


-39 


0.82 


0.18 
























Kirkwood 




40- 


-59 


0.86 








0.14 


















Bs 




<■ 


19 


0.88 


0.12 
























Miocene 




20- 


-39 


0.71 


0.18 


0.07 


0.04 




















Kirkwood 




40- 

60- 

80- 

100- 


-59 
-79 
-99 
-119 


0.65 
0.50 
0.24 
0.33 


0.21 
0.30 
0.32 


0.09 
0.12 
0.36 
0.33 


0.02 
0.02 
0.04 
0.33 


0.04 
0.06 
0.04 



















Species: Eh - Euspira iieros: Nd - Neverita duplicata: Ben - Busycotypus canaliculatus: Bcr - Busycon carica.: Bs - Busycon 
scalarispira: LOC = Locations; GEH - Great Egg Harbor, Atlantic County, New Jersey; HI - Hereford Inlet, Cape May County, 
New Jersey; Miocene Kirkwood Fm - Pollac site, Kent County, Delaware. 



Vermelj (1982) reported that species with many unsuccessful attacks that leave the lip 

thickened lips have significantly lower fre- unscathed and, therefore, unrepaired. Ac- 

quencies of repair than do thin-lipped species, cordingly, a two-way ANOVA was used for 

because thick-lipped species experience both Recent and Miocene nnoon snails and 



SHELL REPAIRS IN MOON SNAILS AND WHELKS 



157 



TABLE 3. Multiple comparison Fisher PLSD test for equality among mean number of repairs and 
Kolmogorov-Smirnov test for differences in percent-frequency of shells with increasing number of repair 
scars per shell. 



Comparison 



Fisher PLSD K-S Chi-square 



Within naticids 
Neverita duplicata (Recent) vs. Euspira heros (Recent) 
vs. E. heros (Miocene) 
vs. N. duplicata (Miocene) 
E. heros (Recent) vs. E. heros (Miocene) 

vs. N. duplicata (Miocene) 
E. heros (Miocene) vs. N. duplicata (Miocene) 

Within melongenids 
Busycotypus canaliculatus (Recent) vs. Busycon carica (Recent) 

vs. B. scalarispira (Miocene) 
B. carica (Recent) vs. B. scalarispira (Miocene) 



0.225ns 


1.5ns 


0.334* 


35.5** 


0.249* 


67.4*** 


0.379* 


21.8* 


0.296* 


35.8** 


0.394ns 


0.173ns 


0.378ns 


2.1ns 


0.365* 


173.1*** 


0.346* 


220.7*** 



P-values: ns = nonsignificant 
* = < 0.05 
** = <0.01 
*'* = < 0.001 



whelks treated separately to determine if 
mean thickness of the apertural lip for each 
species varies significantly anteriorly to pos- 
teriorly, that is, at lip positions A, B, or С (Fig. 
1). Because repairs are often localized along 
a portion of the apertural margin (Figs. 2-4), 
the number of repair scars per shell was re- 
gressed on thickness of the apertural lip sep- 
arately at positions A, B, or С for each species 
to determine the susceptibility of differing 
parts of apertural lip to sustaining repeated, 
repairable, breakage. As with regressions in- 
volving size (WD), both linear and second 
order polynomial regressions of the number of 
repairs versus apertural lip thickness were 
generated for each sample of each species. 
The greater r value generated by either 
method was used in statistical comparisons of 
correlation coefficients (Tukey-HSD test). 
Similarly, the slopes of the regression lines, 
beta, were statistically compared (F-test) to 
evaluate possible significant differences in the 
rate of accumulation of repairs with increasing 
lip thickness between and within species. 



RESULTS 

Frequency of shell repair is greater in all 
size classes of Recent whelks relative to the 
same size class among Recent moon snails 
(Table 1). However, frequencies of shell repair 
are similar among same size classes of com- 
pared Miocene whelks versus Miocene moon 
snails (Table 1). Number of repair scars per 
specimen ranged form zero to 12 for both 



Recent whelk species, zero to 12 for Recent 
N. duplicata, and zero to seven for Recent E. 
heros (Table 2). Only four and three percent of 
B. carica and B. canaliculatus, respectively, 
lack repairs, whereas 48% and 57% of Recent 
E. heros and N. duplicata, respectively, lack 
repairs. Recent whelks display significantly 
higher proportions of shells with more than six 
repairs relative to moon snails (Table 2). There 
is a significant difference (p < 0.001) between 
noncontemporaneous congeneric melon- 
genids in percent-frequency distributions of 
shells with increasing number of repair scars 
per shell, as well as noncontemporaneous 
confamilial and conspecific Miocene and 
Recent naticids (Table 3). Furthermore, the 
thinner Recent moon snail N. duplicata and 
whelk B. canaliculatus (Table 4) do not have 
percent-frequency distributions of shells with 
increasing number of repair scars per shell 
different from their thicker-lipped Recent rela- 
tives E. heros and B. carica, respectively 
(Table 3). Among Miocene naticid shells, all 
size classes are dominated by shells without 
repairs. The maximum number of repairs per 
shell for any size class is four (Table 2). 
Among Miocene whelks, no shell had more 
than four repairs, and the majority of the spec- 
imens (72%) in the 19-79 mm size classes 
lacked repairs (Table 2). 

The apertural lip of E. heros is thicker on av- 
erage at all three positions (Fig. 1) relative to 
N. duplicata (Table 4), although the shell of the 
latter species is more massive due to an um- 
bilical plug. Average thickness of the apertural 
lip increases by a factor of three at positions A 



158 



DIETL & ALEXANDER 



Q 



0.7 П 



с 0.6 

о 

ел 

•2 0.5 

a 

^ 0.4 



о 

и 

с 

''S 

,2 

"03 

u 
u 

о 

и 



0.3- 



0.2- 



0.1- 



BcnD 



BcrD 



□ Bs 



NdD 



EhD 



П Nd 
□ Eh 



Miocene Recent 

FIG. 3. Distribution of correlation coefficient, r, for 
number of repairs on sfiell regressed on size (in- 
dexed by whorl diameter, WD) for Miocene versus 
Recent samples of Euspira heros (Eh), Neverita du- 
plicata (Nd), Busycon carica (Bcr), Busycotypus 
canaliculatus (Ben), and Busycon scalarispira (Bs). 
Multiple comparison Tukey-HSD test reveal correla- 
tion coefficients are significantly different (p < 0.01) 
among naticids only in comparisons between Nd 
(Recent vs. Miocene). Among melongenids, r val- 
ues are significantly different in comparisons of Bs 
vs. Bcr and Bs vs. Ben (p < 0.05). Greater r value be- 
tween linear or second order polynomial regression 
for a sample used in all comparisons. 



and B, and a factor of four at position С (Fig. 
1) from Miocene to Recent samples of E. 
heros (Table 4). Average thickness values at 
the same three positions on the apertural lip 
more than doubled from Miocene to Recent 
samples of N. duplicata (Table 4). Among 
whelks, shells of B. carica are on average 
twice as thick as those of B. canaliculatus at 
position A, but is no thicker at position С than 
B. canaliculatus or B. scalarispira (Table 4). 

Mean number of repairs per specimen was 
different among the five species (ANOVA, p = 
0.0001), although the average was compara- 
ble for the two moon snails, namely 1.1 and 
1 .0 for Recent confamilial N. duplicata and E. 
heros, respectively (Table 3). Miocene nati- 
cids N. duplicata (0.36) and E. heros (0.41 ), as 
well as the Recent whelks B. carica (5.3) and 



0.Ü8- 




Bcnö 


0.07- 






0.06- 






0.05- 




Bcr D 


0.04- 




Nd ° 


0.03- 






0.02- 






0.01- 


Bsn 

Ndn 


Eh □ 


0- 


ЕЬ° 


1 



о 

с 

О 

¡л 

u 

а 
ей 



а 

с 

*!л 
ел 
а> 

U 
CJD 
О) 



Miocene Recent 

FIG. 4. Distribution of slope of regression lines, 
beta, for number of repairs on shell regressed on 
size (indexed by whorl diameter, WD) for Miocene 
versus Recent samples of Euspira heros (Eh), 
Neverita duplicata (Nd), Busycon carica (Bcr), 
Busycotypus canaliculatus (Ben), and Busycon 
scalarispira (Bs). Multiple comparison F-tests re- 
veal slopes of regression lines are significantly dif- 
ferent (p < 0.001) in comparisons between Nd 
(Recent) and all other naticid samples. Among me- 
longenids, regressions lines for Recent whelks (Ben 
and Bcr) have significantly greater slopes (p < 
0.001) than that for the Miocene whelk (Bs). Slope 
of regression line of Ben is significantly greater (p < 
0.001 ) than that for Bcr. Slopes for regression line of 
Recent whelks are significantly greater (p < 0.001) 
than all naticids. 



B. canaliculatus (5.2), also show comparable 
averages (Table 3). Mean number of repairs 
for Miocene B. scalarispira (0.7) and Miocene 
naticids are significantly different (p < 0.05) 
than Recent congeneric whelks and Recent 
naticids (Table 3). Among naticids, mean val- 
ues are substantially greater for Recent tem- 
perate latitude N. duplicata and E. heros, ver- 
sus Recent tropical naticids and Upper 
Triassic to Miocene naticids (Table 5). Among 
the melongenids. Recent species have a 
seven times greater repair frequency than the 
Miocene species (Table 6). 

Repair frequency is more positively corre- 
lated with size and apertural lip thickness for all 



SHELL REPAIRS IN MOON SNAILS AND WHELKS 



159 



TABLE 4. Two-way ANOVA for moon snails indicates that species (p = 0.0001) and position (p = 
0.0001) are significant factors in mean differences in apertura! lip thiickness (ALT). See Fig. 1 for 
location of position A, B, and С on apertural lip. Factor interaction (species in combination with 
ALT position) is also significant (p = 0.0001). Two-way ANOVA for whelks indicates that species 
(p = 0.0001) and position (p = 0.0001) are significant factors in mean differences in ALT. Factor 
interaction (species in combination with position) is also significant (p = 0.0001); n = sample size. 



Taxon 


Mean ALT (mm) at 
position A 


Mean ALT (mm) at 
position В 


Mean ALT (mm) at 
position С 


E. heros 
Recent 


0.89 
n = 241 


0.76 
n = 241 


2.08 
n = 240 


E. heros 
Miocene 


0.29 
n = 81 


0.22 
n = 80 


0.50 
n = 80 


N. duplicata 
Recent 


0.75 
n = 779 


0.68 
n = 780 


1.62 
n = 780 


N. duplicata 
Miocene 


0.37 
n = 216 


0.26 
n = 222 


0.66 
n = 198 


B. canalicu latus 
Recent 


1.26 
n = 143 


1.36 
n = 143 


1.30 
n = 143 


B. carica 
Recent 


2.11 
n = 173 


1.60 
n = 173 


1.28 
n = 173 


B. scalarispira 
Miocene 


1.99 
n = 194 


1.73 
n = 195 


1.29 
n = 195 



TABLE 5. Comparison of repair frequencies, f (the number of scars per shell), in umbilicate species from the 
present study with extant and fossil populations. 





Geological 








Taxon 


Age/Formation 


Location 


f 


Reference 


Euspira heros 


Recent 


Southern New Jersey 


1.0 


Present study 


Neve rita duplicata 


Recent 


Southern New Jersey 


1.1 


Present study 


Euspira sp. 


Recent 


Tropical 


0-0.05 


Vermeij, 1982 


Polinices tumidus 


Recent 


Aru Islands 


0.10 


Vermeij, 1982 


Ruber 


Recent 


Panama 


0.13 


Vermeij, 1982 


Natica chemnitzii 


Recent 


Panama 


0.50 


Vermeij, 1982 


E. heros 


Miocene (Kirkwood 

Fm.) 
Miocene (Kirkwood 

Fm.) 
Late Cretaceous 


Delaware 


0.41 


Present study 


N. duplicata 


Delaware 


0.36 


Present study 


E. rectilabrum 


Mississippi 


0.71 


Vermeij & Dudley, 




(Ripley Formation) 






1982 


Euspira sp. 


Late Cretaceous 
(Ripley Formation) 


Alabama 


0.64 


Vermeij & Dudley, 
1982 


Amauropsis 


Upper Triassic St. 


Costalaresc, Italy 


0.051 


Vermeij et al., 


paludinaris 


Cassian Gr. 






1982 



three positions (Fig. 1) for Recent nnelon- 
genids versus Recent naticids (Figs. 3, 5, 7, 9). 
Furthermore, rate of accumulation of repairs is 
significantly greater for Recent whelks versus 
moon snails based on slopes of the regression 
lines for repairs regressed on WD and ALT 
(Figs. 4, 6, 8, 10). Repair frequency is more 



positively correlated with size and apertural lip 
thickness in Recent whelks relative to Miocene 
whelks (Figs. 3, 5, 7, 9). In contrast, the only 
significant difference occurs in the r values of 
repair frequency regressed on size for 
Miocene versus Recent samples of N. dupli- 
cata (Flg. 3). No significant differences occur In 



160 



DIETL& ALEXANDER 



TABLE 6. Comparison of repair frequencies, f (the number of scars per shell), in extant and fossil 
whelk populations. 



Taxon 



Geological 
Age/Formation 



Location 



Reference 



Busycon carica 
Busycotypus 

canaliculatus 
Busycon 

scalarispira 



Recent 

Recent 

Miocene (Kirkwood 
Fm.) 



Southern New Jersey 5.3 Present study 

Southern New Jersey 5.2 Present study 

Delaware 0.7 Present study 



< 

С 
О 

ел 

и 

'сЗ 
а 

0^ 



о 

и 

с 

U 
U 

о 
U 



о.ь- 




BcrD 


0.4- 




BcnD 


0.3- 






0.2- 


П Bs 

nNd 




0.1- 




EhD 


А 


D Eh 


NdD 



Miocene Recent 



FIG. 5. Distribution of correlation coefficient, r, for 
number of repairs on shell regressed on apertural 
lip thickness at position A (see Fig. 1) for Miocene 
versus Recent samples of Euspira heros (Eh), 
Neverita duplicata (Nd), Busycon carica (Bcr), 
Busycotypus canaliculatus (Ben), and Busycon 
scalarispira (Bs). Multiple comparison Tukey-HSD 
test reveal correlation coefficients are significantly 
different (p < 0.05) only in comparison between B. 
carica and B. scalarispira. Greater r value between 
linear or second order polynomial regression for a 
sample used in all comparisons of correlation coef- 
ficients. 



H 

< 

с 
© 

"я 

Си 

a 

с 

^o 

*сл 
(Л 
a> 
u 
DD 



1.6-1 

1.5- 

1.4- 

1.3- 

1.2- 

1.1- 

1- 

0.9- 

0.8- 

0.7- 

0.6- 

0.5- 

0.4- 

0.3- 

0.2- 

0.1- 

0- 

-0.1- 

-0.2- 

-0.3 -■■ 



BcnD 



□ Nd 
D Bs 



Ü Eh 



BcrD 



Ndp 
Eh 



Miocene Recent 



FIG. 6. Distribution of slope of regression lines, 
beta, for number of repairs on shell regressed on 
apertural lip thickness at position A (see Fig. 1) for 
Miocene versus Recent samples of Euspira heros 
(Eh), Neverita duplicata (Nd), Busycon carica (Bcr), 
Busycotypus canaliculatus (Ben), and Busycon 
scalarispira (Bs). Multiple comparison F-tests re- 
veal slopes of regression lines are significantly dif- 
ferent only in comparisons involving melongenids. 
Regression lines for Recent whelks (Ben and Bcr) 
have significantly greater slopes (p < 0.001) than 
that for the Miocene whelk (Bs) and Recent naticids 
(Eh and Nd). Slope of regression line of Ben signifi- 
cantly greater (p < 0.001 ) than that for Bcr. 



comparison of r values involving shell thick- 
ness (Figs. 5-10). Furthermore, no significant 
difference occurs between compared correla- 
tion coefficients or slopes of regression lines 
for Miocene and Recent samples of E. heros 
for either size or lip thickness (Figs. 3-10). 



DISCUSSION 

The significant correlation (p < 0.05) be- 
tween the number of repairs per shell and size 
(WD) for Recent whelk and moon snail species 
(Fig. 3) indicates that age-dependent accumu- 



SHELL REPAIRS IN MOON SNAILS AND WHELKS 



161 



0Э 
I 

H 

< 
s 

О 

ел 

u 

a 
pes 



о 

и 

с 

s 

"03 

u 
u 
о 

и 



и.ь- 




Bcr D 


0.4- 




Ben D 


0.3- 






0.2- 


BsD 




0.1- 
0- 


Nd ° 
Eh □ 


Ndp 
Eh° 

1 



Miocene Recent 



FIG. 7. Distribution of correlation coefficient, r, for 
number of repairs on sfiell regressed on apertural 
lip thickness at position В (see Fig. 1) for Miocene 
versus Recent samples of Euspira heros (Eh), 
Neverita duplicata (Nd), Busycon carica (Bcr), 
Busycotypus canaliculatus (Ben), and Busycon 
scalarispira (Bs). Multiple comparison Tukey-HSD 
test reveal correlation coefficients are significantly 
different (P < 0.05) only in comparison between B. 
carica and B. scalarispira. Greater r value between 
linear or second order polynomial regression for a 
sample used in all comparisons of correlation coef- 
ficients. 



S 


1.4 


H 


1.3 


N^ 


1.2 


< 


1.1 


a 


1 


о 


СЛ 


0.9 


и 


0.8 


"a 




a 


0.7 


O) 




Й 


0.6 








0.5 


0) 




a 


0.4 


o^ 


0.3 


^ 




с 


0.2 


0.1 


*!л 





О) 




u 


-0.1 


Ol 




Q¿ 


-0.2 



Ben □ 
Бег □ 



Ndü 



BsD 



Eh D 



NdO 
Eh □ 



Miocene Recent 

FIG. 8. Distribution of slope of regression lines, 
beta, for number of repairs on shell regressed on 
apertural lip thickness at position В (see Fig. 1) for 
Miocene versus Recent samples of Euspira lieras 
(Eh), Neverita duplicata (Nd), Busycon carica (Bcr), 
Busycotypus canaliculatus (Ben), and Busycon 
scalarispira (Bs). Multiple comparison F-tests re- 
veal slopes of regression lines are significantly dif- 
ferent only in comparisons involving melongenids. 
Regression lines for Recent whelks (Ben and Bcr) 
have significantly greater slopes (p < 0.001) than 
that for the Miocene whelk (Bs) and Recent naticids 
(EhandNd). 



lation of scars increased as exposure time to 
potential predators increased over the life 
span of the prey (Fig. 3). In nnost Recent gas- 
tropods, incidence of sublethal shell breakage 
increases as shell length increases (Vermeij et 
al., 1980; Zipser & Vermeij, 1980; Vermeij, 
1982; Vermeij & Dudley, 1982; Vermeij et al., 
1982; Raffaelli, 1978; Dudley, 1980). Similarly, 
Alexander (1989) showed that the frequency 
of repaired valves correlates significantly with 
valve surface area (index of size) for Late 
Ordovician brachiopods. Large size also de- 
creases the probability that a shell-breaking 
predator will lethally fracture the shell (Hughes 
& Einer, 1979; Seed, 1978; Einer & Raffaelli, 
1980; Preston et al., 1996), which may also ex- 
plain why successively larger size classes 
have greater frequencies of shell repair for all 
five gastropod species in this investigation 



(Table 1). However, repaired fractures do not 
continue to accumulate on shells of E. heros 
(maximum = 6) from the penultimate to the 
largest size classes (60-79 vs. 80-99) as they 
do between the two largest size classes of N. 
duplicata (Table 2), suggesting a possible size 
refugia from prédation for E. heros. In contrast, 
the largest individuals of N. duplicata had a 
WD in the 60-79 mm size class (Table 2) and 
had scars near the periphery of the apertural 
lip. 

Failure to continue to accumulate repairs on 
shells of E. heros with increasing size beyond 
the 80 mm WD threshold in the population at 
Hereford Inlet (Table 2) may account for the 
lower correlation coefficient and slope value 
between repair frequency and WD for E. heros 
relative to the slightly smaller N. duplicata 
(Figs. 3-4). Preston et al. (1996) commented 



162 



DIETL& ALEXANDER 






С 

о 

CA 

и 

'я 
а 
а> 

oes 



4> 

О 

и 

с 
"oí 

U 

о 



0.5- 




Всг п 


0.4- 






0.3- 




BcnD 


0.2- 


NdD 






BSD 


NdlH 


0.1- 


EhD 




0- 




Eh п 
1 



Miocene Recent 



FIG. 9. Distribution of correlation coefficient, r, for 
number of repairs on shell regressed on apertural 
lip thickness at position С (see Fig. 1) for Miocene 
versus Recent samples of Euspira heros (Eh), 
Neverita duplicata (Nd), Busycon carica (Bcr), 
Busycotypus canaliculatus (Ben), and Busycon 
scalarispira (Bs). Multiple comparison Tukey-HSD 
test reveal correlation coefficients are significantly 
different (p < 0.01 ) only in comparison of B. carica 
vs. B. scalarispira. Greater r value between linear or 
second order polynomial regression for a sample 
used in all comparisons. 



H 

< 

С 
О 

(Л 

и 
а 



а 

с 
^© 

и 
ОХ) 



1.5- 
1.4- 
1.3- 
1.2- 
1.1- 

1- 
0.9- 
0.8- 
0.7- 
0.6- 
0.5- 
0.4- 
0.3- 
0.2- 
0.1- 

0- 
■0.1- 



BcrD 
Ben D 



Nd 

Bs 

Eh 



В 



Ndü 



Eh D 



Miocene Recent 



FIG. 10. Distribution of slope of regression lines, 
beta, for number of repairs on shell regressed on 
apertural lip thickness at position С (see Fig. 1) for 
Miocene versus Recent samples of Euspira heros 
(Eh), Neverita duplicata (Nd), Busycon carica (Bcr), 
Busycotypus canaliculatus (Ben), and Busycon 
scalarispira (Bs). Multiple comparison F-tests re- 
veal slopes of regression lines are significantly dif- 
ferent (p < 0.001) among naticids only in compar- 
isons between Recent N. duplicata and Recent E. 
heros. Regression lines for Recent whelks (Ben and 
Bcr) have significantly greater slopes (p < 0.001) 
than that for the Miocene whelk (Bs) and Recent 
naticids (Eh and Nd). 



that with increasing shell size, more attacks 
from predators will fail to break the thicker lip. 
Rate of accumulation of repairs is thus ex- 
pected to decline when the predator can no 
longer readily inflict damage to the thicker 
shelled gastropods, a contrast to durophagy 
on skeletonized invertebrates with delicate ap- 
pendages that can be sheared off at any body 
size, such as with crinoid arms (Oji, 1996). 

The significantly greater correlation coeffi- 
cient (p < 0.05) between repair frequency and 
lip thickness at position С (r = 0.16) vs. posi- 
tion A (r = 0.06) or В (r = 0.06) (Fig. 9 vs. Figs. 
5 and 7) for both Recent and Miocene N. du- 
plicata may indicate where the crushing ele- 
ments of the durophage more often exerted 
their peeling force, namely posteriorly near 
the umbilicus (Fig. 1, position C). Peeled 
shells of N. duplicata more often have shell 
material of the body whorl closest to the um- 



bilical plug removed, but retain a wrapped 
around "awning" of shell material anteriorly, 
nearest the suture with the penultimate whorl. 
The aperture is widest posteriorly at position 
C, in N. duplicata, allowing the claw of a crab 
to secure the deepest purchase onto the lip 
and into the body whorl. Hence most sublethal 
shell breakage originates posteriorly on the 
lip. Thus, the thickness of the lip posteriorly 
(position C), where peeling forces are exerted 
on the shell, may be more important than the 
thickness of the lip anteriorly (position A) in 
the determination if a fracture initiated by a 
durophage will be localized by the prey. The 
significantly greater correlation of repair fre- 
quency with lip thickness at position С rather 
than position A is harmonious with that rea- 
soning. 
Among repaired shells the umbilical plug 



SHELL REPAIRS IN MOON SNAILS AND WHELKS 



163 



functioned to stop propagation of fractures 
through this posterior region into the basal 
cavity and thereby render repairable any pos- 
teriorly localized breakage. Vermelj (1987) 
stated that the structural weakness of the um- 
bilicus can be strengthened by the formation 
of the umbilical plug closing the basal cavity. 
Conversely, lack of correlation between ALT at 
any position and number of shell repairs for 
the other Recent moon snail, E. heros (Figs. 5, 
7, 9) may indicate an architectural weakness 
of this unplugged umbilicate design to local- 
ized fractures initiated in the apertural lip. As 
the probability of lethal fractures increased in 
this unplugged moon snail, frequency of sub- 
lethal fractures decrease. The statistical con- 
sequence is diminished correlation between 
ALT and repair frequency in shells of E. heros 
(Vermeij, 1982). 

The better correlations between ALT at any 
lip position for whelks versus moon snails 
(Figs. 5-10) may reflect the fact the whelks 
have a higher probability of accumulating sub- 
lethal fractures on their shell as a conse- 
quence of their feeding habit. They wedge 
apart their bivalve prey (Colton, 1 908; Warren, 
1916; Carriker, 1951), an activity that is not 
likely to induce an unrepairable fracture. 
Indeed, most repairs are scallop-like, shallow 
indentations from the normal axial growth line 
(Figs. 2, 3), a pattern suggestive of breakage 
during valve-wedging of bivalve prey. Only 
deep, irregular embayments from the normal 
axial growth line (Fig. 2-1) indicate repairs of 
shell-breakage more likely to have been in- 
flicted by blue crabs. Thus, the feeding habit of 
the whelk is probably more important than its 
victimization by crabs in accumulation of shell 
repairs with increasing size and apertural 
thickness (Tables 1, 2). 

Vermeij (1997) speculated that predator- 
prey escalation between armored gastropods 
and their shell-breaking enemies can be used 
as a prediction that shell-breakage became 
increasingly important as a selective factor for 
gastropods through Mesozoic and Cenozoic 
time. Interpretations of repair frequencies 
cannot estimate either predator intensity or 
predator efficiency but can reflect the effec- 
tiveness of the predator's crushing ability and 
the prey's resistance to crushing (Schindel & 
Vermeij, 1982). Based on these assumptions, 
Vermeij, after several investigations, con- 
cluded that repair frequencies remained con- 
stant from the Late Carboniferous to the Late 
Triassic (Schindel et al., 1982; Vermeij et al., 
1982), increased from the Late Triassic to the 
Late Cretaceous (Vermeij & Dudley, 1982), 



and then again became relatively constant 
approaching Recent levels (Vermeij, 1982). 
These trends, coupled with an increase in 
power and diversification of shell-breaking 
predators from the Mesozoic to Cenozoic, 
formed the basis of Vermeij's conclusions. 

Vermeij & Dudley (1982) reported that 
Euspira sp. from the Ripley Formation (Late 
Cretaceous) had higher frequencies of repair 
(0.71 , Table 5) than do Recent tropical popula- 
tions of Euspira sp. (0-0.5; Vermeij, 1982). 
Data in this investigation show a frequency of 
repair which is even higher (1 .0) than those of 
Late Cretaceous members (0.71), suggesting 
higher incidence and stronger expression of 
breakage-resistant armor in temperate mem- 
bers of this family. However, repair frequency 
in the Miocene E. heros is lower (0.4) than that 
reported for the Late Cretaceous (Table 5), ev- 
idence in support of Vermeij's contention that 
repair frequencies did not continue to increase 
after the Cretaceous. Disparity in repair fre- 
quencies between Recent tropical (0.05) and 
temperate Euspira sp. (1.0) may reflect differ- 
ences in both the strength and the abundance 
of predators relative to the strength and abun- 
dance of the prey. Similarly, repair frequencies 
for N. duplicata (1.1) are higher than ecologi- 
cally similar Recent tropical populations of 
Polinices tumidus (0.1) and Polinices über 
(0. 1 3)(Vermeij, 1 982) (Table 5). Vermeij (1 983) 
reported that an increase in relative abun- 
dance of the predator would increase the fre- 
quency of repair. 

Size of individual shells is important in com- 
parison of repair frequencies. Vermeij (1982) 
sorted 21 specimens of Natica chemnitzii 
among four size classes, that is, 5-9 mm; 
10-19 mm; 20-29 mm; and 30-39 mm. The 
repair frequency of 0.67 in the 20-29 mm size 
class (n = 9; Vermeij, 1982), is comparable to 
the repair frequency values in the 20-39 mm 
size class in this study, namely, 0.61 (GEH) 
and 0.73 (HI) for N. duplicata and 0.69 (GEH) 
and 0.72 (HI) for E. heros (Table 1). However, 
for all size classes, repair frequency for shells 
of N. chemnitzii was 0.50 compared with 1.1 
and 1 .0 for N. duplicata and E. heros, respec- 
tively (Table 5). This difference may be the re- 
sult of the inclusion of large numbers of shells 
in the 40-99 mm size classes in this study. 

The higher frequency of repair for both B. 
canaliculatus (5.2) and B. carica (5.3) is attri- 
buted to a combination of shell breakage from 
crabs preying on whelks combined with aper- 
tural lip fracture (Fig. 2) during attempts by 
whelks to employ the outer shell lip to wedge 
open the valves of tightly closed bivalve prey. 



164 



DIETL & ALEXANDER 



The rate of accumulation of repairs, reflected 
in the slopes of repair frequencies regressed 
on size and lip thickness (Figs. 4, 6, 8, 10), is 
greater for whelks in comparison to moon 
snails. Because the shell lip of predatory 
whelks is often slightly damaged during at- 
tacks on prey, valve-wedging species fre- 
quently have a high incidence of repaired 
breaks than non-valve-wedging species. The 
frequencies of repair for Recent whelk species 
in this study are higher than that reported for 
any other gastropod in the literature. The fre- 
quency is more than seven times greater than 
that for B. scalarispira from the Miocene. 
Vermeij (1987: 182) suggested that the lip- 
wedging technique may not have evolved until 
the Middle Pliocene on the Atlantic Coastal 
Plain of North America, although no evidence 
was presented. The majority of repaired frac- 
tures in B. scalarispira extend across the 
varices onto the abaxial edge of the sutural 
shelf of the final whorl (Fig. 2-5). In contrast, 
most repairs in Recent whelks were concen- 
trated at the lip mid-length (Fig. 2-4, 2-8). 
Thus, varices appear to be effective in limiting 
the extent of unrepairable damage from lip- 
wedging but not the direct attention to the lip by 
the durophage. This discrepancy suggests 
that Miocene melongenids had not evolved, or 
perfected, valve-wedging, and as a conse- 
quence the frequency of repairs probably re- 
flects the contribution of predators sublethally 
peeling the whelks. 



ACKNOWLEDGMENTS 

We are grateful to Joanne Dietl, Walt Bien, 
and Richard Trub for assistance in collection 
and measuring of specimens, and editing the 
manuscript. We also appreciate the sugges- 
tions of Mel Carriker and Charles Jansen to 
improve the manuscript. We are especially 
grateful to Lauck Ward for the loan of the gas- 
tropod collections from the Miocene Kirkwood 
Fm of Delaware. Critical review of the manu- 
script by Geerat Vermeij and an anonymous 
reviewer is also appreciated. 



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ALEXANDER, R. R., 1989, Influence of valve 
geometry, ornamentation, and microstructure on 
fractures in Late Ordovician brachiopods. 
Lethaia, 22: 133-147. 

BERTNESS, M. D. & С CUNNINGHAM, 1981 , Crab 
shell-crushing prédation and gastropod architec- 



tural defense. Journal of Experimental Marine 
Biology and Ecology, 50: 21 3-230. 

BROWN, S. C, S. R. CASSUTO & R. W. LOOS, 
1979, Biomechanics of chelipeds in some deca- 
pod crustaceans. Journal of Zoology 188: 
153-169. 

CARRIKER, M. R., 1951 , Observations on the pen- 
etration of tightly closing bivalves by Busycon and 
other predators. Ecology, 32: 73-83. 

COLTON, H. S., 1908, How Fulgur ar\d Sycotypus 
eat oysters, mussels and clams. Proceedings of 
the Academy of Natural Sciences of Philadelphia, 
60:3-10. 

CURRY, J. D. & A. J. KOHN, 1976, Fracture in the 
crossed-lamellar structure of Conus shells. 
Journal of Material Science, 11:1615-1 623. 

DUDLEY, E. C, 1980, Crab prédation on two small 
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Revised ms. accepted 12 May 1997 



MALACOLOGIA, 1998, 39(1-2): 167-173 

WITHIN-CLUTCH EGG CANNIBALISM VARIABILITY IN HATCHLINGS OF THE 

LAND SNAIL HELIX ASPERSA (PULMONATA: STYLOMMATOPHORA): 

INFLUENCE OF TWO PROXIMATE FACTORS 

Christophe Desbuquois & Luc Madec 

U.M.R. CNRS 6553, Laboratoire de Zoologie et d'Ecophysiologie (U.A. INRA), Faculté des 
Sciences, Université de Rennes 1, Avenue du Général Ledere, 35042 Rennes CEDEX, France 

ABSTRACT 

This study investigated, under laboratory conditions, the influence of two proximate factors, 
temperature and humidity, on the rate of egg cannibalism in hatchlings of ¡Helix aspersa. Nine 
combinations were tested involving three different temperatures (15, 20, 25°C) and relative hu- 
midities (40, 70, 100% R.H.). Each new-born snail was maintained with a conspecific egg of its 
laying, previously incubated at 15°C instead of 20°C for the tested snails, so that its incubation 
duration was enhanced. Three measurements were carried out, namely the percentage of can- 
nibalism, the snail mortality and the egg desiccation. Temperature, humidity and their interaction 
had a significant influence on egg cannibalism. For every combination of these two factors, the 
percentage of hatchlings having consumed the available egg increased with time. Those snails 
not fed could survive at least four days at high humidity. However, four days after hatching, non- 
cannibalistic hatchlings exhibited a higher mortality than cannibalistic ones, essentially due to 
humidity constraint. Hence, egg cannibalism may represent an adaptive mechanism to resist to 
adverse climatic conditions, such as high temperature and low humidity. In natural conditions, a 
higher rate of egg cannibalism might be expected at low temperature and low humidity in com- 
parison with the one observed in this experiment. Egg cannibalism by juveniles might improve 
survival during adverse conditions (via water ingestion) and would be able to affect other life his- 
tory traits, such as the subsequent growth rate of juveniles having consumed one egg immedi- 
ately after the hatching. It might represent a mean to avoid juvenile size-dependent mortality due 
to the seasonal variations of abiotic conditions. 

Key words: nutrition, intraspecific oophagy, egg cannibalism, proximate factors, IHeiix aspersa. 



INTRODUCTION 

Egg cannibalism in hatchlings of different 
land snail species has been reviewed by Baur 
(1992). In pulmonates, two species have been 
especially investigated, namely Helix pomatia 
Linnaeus (Baur, 1988a,d) and Arianta arbus- 
torum Linnaeus (Baur 1988a-c, 1993; Baur & 
Baur, 1986). However, few studies deal with 
this aspect of juvenile nutrition in Helix as- 
persa Müller, although Elmslie (1988) demon- 
strated its existence in hatchlings of this 
species and Fearnley (1993) gave hypothe- 
ses on its consequences in life history. As in 
H. pomatia (Baur, 1988d), oophagy is re- 
stricted to new-born snails during the hatch- 
ing period. 

Different life history traits may be affected 
by oophagy; for example, subsequent sur- 
vivorship and growth enhancements were ob- 
served in the snail H. aspersa having con- 
sumed one egg immediately after hatching 
(Desbuquois, 1997). Adaptation for terrestrial 



life involves strategies of survival and devel- 
opment under various proximate factors. 
Special forms of behaviour, namely aestiva- 
tion and hibernation, may have evolved in 
land snails to offer some resistance to ad- 
verse abiotic conditions (Riddle, 1983). Such 
dormancies have a diapause value in H. as- 
persa (Bailey, 1981, 1983; Lorvelec 1988) 
and, in the context of life history theory, are 
often related with an adversity selection 
(Greenslade, 1983). In the same way, egg 
cannibalism might be one of these behav- 
ioural tactics with important repercussions on 
survival during winter via the size reached by 
juvenile snails before diapause (Biannic, 
1995). 

Within-clutch egg cannibalism variation can 
be related to microhabitat differences, espe- 
cially in temperature and moisture (Baur, 
1994; Baur & Baur, 1986), which were also 
subject, in Western Europe, to great variation 
during daily and annual cycles. Thus, the aim 
of our study is to test the influence of this vari- 



167 



168 



DESBUQUOIS & MADEC 



ation during hatching, on the extent of egg 
cannibalism in hatchlings of H. aspersa. 



MATERIAL AND METHODS 
Relevant Natural History 

The hermaphroditic land snail H. aspersa 
lays its eggs in batches in a nest excavated in 
the soil. One to three clutches are deposited 
per breeding season, with a mean clutch size 
of around 100 eggs. Newly hatched snails 
might remain in their nest from 3 to 16 days 
after hatching (6 days on average) before 
emergence (Herzberg & Herzberg, 1962); this 
may allow first born hatchlings to feed on un- 
hatched eggs. Juvenile and adult snails are 
herbivorous. 

In the present experiment, adults of H. as- 
persa were collected in August 1994 in a pop- 
ulation living in the salt-pans at Guerande 
(South Brittany, France). They were main- 
tained in hibernation in a room at 4°C for three 
months before breeding. 

Breeding 

The study was carried out on egg-layings 
obtained from 20 January to 23 March 1995 
from adults reared in polythene boxes under 
constant artificial conditions (temperature: 
20 ± 1°C, relative humidity: 80 ± 5%, 18/6 
light/dark cycle) promoting reproduction 
(Daguzan, 1 981 ). They were fed with a cereal 
composed snail food (produced by the com- 
pany Arrivé) supplied ad libitum and renewed 
twice a week. Four laying jars containing moist 
soil were placed in each cage allowing snails 
to deposit their clutches. 

Experimental Conditions 

After each clutch was washed, it was di- 
vided into two equal groups of eggs, which 
were incubated in small Petri dishes (diame- 
ter: 55 mm) lined with moistened filter paper to 
obtain air moisture saturation. Half of the eggs 
were placed at 15°C to slow down the embry- 
onic development, the other at 20°C to obtain 
the snails that will be fed with the 15°C-incu- 
bated eggs. This method, used by Baur 
(1988a, 1993) and Baur & Baur (1986), al- 
lowed to reproduce the natural hatching asyn- 
chronism, that is, a delay between the first 
and the last hatching. According to Le Calvé 
(1987), these conditions lead to a hatching 



delay of about nine days for the 15°C-incu- 
bated eggs. 

Each new-born snail (one day old) was 
placed on a moistened filter paper disc in a 
small aluminium container (diameter: 20 mm; 
height: 6 mm), open at the top, and received a 
conspecific egg incubated at 15°C. 

Animals tested were maintained in the 
shade at different temperatures in hermeti- 
cally closed plexiglass boxes (24 x 18 x 10 
cm). Constant humidities were obtained within 
the boxes by means of NaOH/water mixtures 
(Madge, 1961), which were introduced more 
than three days before starting the experi- 
ment. Three temperatures (15, 20, 25°C) and 
relative humidities (40, 70, 100% R.H.) and 
their eventual interactions were tested. The 
ranges of air temperature and relative humid- 
ity chosen were commonly encountered in 
South Brittany during the breeding season of 
this species. Containers were maintained 
above the NaOH solution with a wired prop 
placed 2 cm above the bottom of the boxes 
and were covered with a polythene net. The 
boxes were opened daily in order to allow ob- 
servations and air renewal; at the same time, 
a drop of water was deposited on each disc of 
paper. 

Measurements Used 

Egg prédation was observed under 12.5x 
magnification; the rate of egg cannibalism 
(percent) was defined as the ratio of hatch- 
lings having consumed the egg divided by the 
number of snails tested per batch. Snail mor- 
tality was equal to the number of hatchlings 
dead divided by the number of snails tested 
(individuals of all clutches were considered to- 
gether). When an egg was dehydrated, his 
weight became very low and his colour white 
and opaque. Egg desiccation was the delay 
(in days) before each egg not consumed in a 
batch was dehydrated. Three or four clutches 
were used for each combination of tempera- 
ture and humidity. A total of 1,555 hatchlings 
was tested for egg cannibalism in the different 
thermohygrometric associations (i.e., from 
130 to 260 hatchlings per combination). 

Statistical Analyses 

First, we calculated regressions of the per- 
centage of egg cannibalism on the age in the 
different thermohygrometric conditions. Then, 
the effects of temperature, relative humidity 
and their eventual interactions on egg canni- 



ENVIRONMENTALLY-INDUCED VARIATION OF EGG CANNIBALISM 



169 



balism were tested using ANOVA on residuals 
of regressions, that is, log(age) was intro- 
duced as a covariable in the analysis. SNK 
multiple comparison tests were carried out 
when means were heterogeneous. The per- 
centages of mortality were compared using 
x2 tests of association. The assumption of 
normality of the residuals was checked using 
Lilliefors test on BIOMECO (1988). Other 
analyses were run with MINITAB (1991). 



with time so they could be consumed more 
than ten days after the beginning of the ex- 
periment. At 40% R.H., all eggs were dehy- 
drated after 2 to 4 days. At 70% R.H., egg des- 
iccation increased with temperature. Hence, 
egg availability duration for potentially canni- 
balistic hatchlings was different according to 
thermohygrometric conditions. 



DISCUSSION AND CONCLUSIONS 



RESULTS 
Egg Cannibalism 

For every combination of temperature and 
humidity, the percentage of hatchlings having 
consumed the available egg increased with 
the logarithm of time (Fig. 1). Temperature 
significantly affected egg cannibalism by 
hatchlings (Table 1; ANOVA, P < 0.001): 
oophagy was highest at 20°C and 25°C and 
significantly lower at 15°C (SNK tests, P < 
0.001). The percentage of egg cannibalism 
was also different according to relative humid- 
ity (Table 1; P < 0.001) and was higher at 
100% R.H. than at the two other humidities 
(SNK tests, P < 0.005). Moreover, a significant 
interaction between the two factors studied 
was found (Table 1; P < 0.001) especially in- 
volving the 20°C-100% R.H. and 25°C-100% 
R.H. modalities (SNK tests, P < 0.05). 

Other Mortality Factors 

Snail survival and egg desiccation were 
highly affected by humidity and, to a lesser ex- 
tent, by temperature. 

Thus, hatchling mortality was higher and 
earlier at 40% R.H., much lower and later at 
100% R.H. After 4 days, mortality was signifi- 
cantly higher in non-cannibalistic hatchlings, 
that is, snails having not consumed the avail- 
able egg, except for 15°C-100% and 20°C- 
1 00% R.H. (x2 test, P < 0.001 ), where no mor- 
tality occurred (Table 2A). In low relative 
humidity (40% R.H.) or high temperature 
(25°C) conditions, lethal water loss was espe- 
cially important in non-cannibalistic hatch- 
lings. After 6 days, results were not different 
but several combinations could not be tested 
because of egg desiccation which prevented 
snails from cannibalism (Table 2B). Obviously, 
humidity drastically affected the time of dehy- 
dration of the eggs (Table 3). At 100% relative 
humidity, eggs did not suffer from desiccation 



The present experiment shows that egg 
cannibalism is influenced by the two environ- 
mental variables studied which might operate 
at two levels: (i) hatchling survival, and proba- 
bly activity and time spent by hatchlings in the 
nest, that is, egg cannibalism opportunity, and 
(ii) egg desiccation and hatching asynchro- 
nism of the clutch, that is, egg availability. 
Obviously, the longer a hatchling is in pres- 
ence of non-dehydrated eggs, the more likely 
it is to eat them. 

As was recorded in other mollusc species 
(Machin, 1975), juveniles of H. aspersa axe 
particularly sensitive to dehydration: at 15°C 
and 20°C, water loss occurs under 90% rela- 
tive humidity; at a temperature of 25°C, three- 
month-old snails lose water even if they are 
maintained at 100% relative humidity (Cha- 
rrier, 1980). Klein-Rollais (1993) showed that 
the rate of water ingestion of juvenile snails 
during the first weeks of life was highest 
above 20°C, and decreased when the relative 
humidity increased from 60% to 100%. Under 
mild climatic conditions, individuals may sur- 
vive more than four days without feeding, 
whereas under harsh conditions of tempera- 
ture and relative humidity, mortality is very 
high in non-cannibalistic hatchlings. Thus, 
snail mortality is greatly influenced by temper- 
ature-humidity interaction through dehydra- 
tion and impossibility of water intake. There- 
fore, egg cannibalism, through water intake, 
might be considered as an adaptive mecha- 
nism which allow some resistance to condi- 
tions which promote water loss. 

In H. aspersa, locomotor activity is influ- 
enced by snail water content (Charrier, 1980; 
Biannic, 1995), which is itself dependent on 
temperature and humidity (Klein-Rollais, 
1 993). Thus, as in other slug and snail species 
(Prior, 1985), activity of H. aspersa is also 
closely related to these two factors (Herzberg 
& Herzberg, 1962; Dan, 1978). Moreover, ju- 
veniles are active in a higher range of temper- 
atures and humidities than adults (Biannic, 



170 



DESBUQUOIS & MADEC 



100 л 




40% RH у = 5.421og(x) + 3.05 г = 0.27 р = 0.554 
70% RH у = 19.581og(x) - 1.96 г = 0.52 р = 0.039 
100% RH у = 38.431og(x) - 10.06 г = 0.67 р = 0.001 



age (days) 



40% RH у = 12.331og(x)- 1.16 г = 0.39 р = 0.268 
70% RH у = 50.771og(x) + 4.97 г = 0.59 р = 0.050 
100% RH у = 105.71Iog(x) - 8.33 г = 0.91 р = 0.001 , 



age (days) 

^ о 40% RH y = 41.341og(x)-5.13 г = 0.83 р = 0.010 

3 70% RH у = 12.401og(x) + 6.20 г = 0.49 р = 0.216 

к 100% RH y = 84.661og(x) + 6.18 г = 0.92 р = 0.001 , 



age (days) 

FIG. 1 . Regressions between the rate of egg cannibalism and the age of hatchlings of /-/e//x aspersa in rela- 
tion to ambient temperature (T) and relative humidity (RH). 



1995). Thus, the two factors tested are indi- 
rectly able to affect the extent of egg canni- 
balism. 

In addition, egg cannibalism was also de- 
pendent on these two abiotic factors via egg 



desiccation which influences egg availability. 
Eggs are particularly sensitive to dehydration 
(Machin, 1 975; Riddle, 1 983). At 25°C, the rate 
of desiccation of isolated eggs of H. aspersa 
placed at 25% R.H. is 6.4 times higher than for 



ENVIRONMENTALLY-INDUCED VARIATION OF EGG CANNIBALISM 



171 



TABLE 1 . Analysis of variance for egg cannibalism by hatchlings of Helix aspersa according to 
ambient temperature and relative humidity 



Source of variation 



Degrees of freedom Sum of squares F-values 



Temperature (T) 
Relative hiumidity (RH) 
Tx RH 


2 
2 

4 


Error 


102 



33.09 


20.87 


<0.001 


18.03 


24.61 


<0.001 


18.67 


11.59 


<0.001 


41.07 







TABLE 2. Mean values of hatchling mortality (%) of Helix aspersa according to ambient tem- 
perature and relative humidity (left value: cannibalistic hatchlings; right value: non-cannibalistic 
hatchlings) (number of clutches in parentheses) 

A. After four days of life. B. After six days of life (When no value appears, the experiment was 
stopped before day six because all the eggs were dehydrated) 





Relative humidity (%) 




Temperature (°C) 




A 


15 


20 


25 




40 
70 

100 


28.6/85.2 (3) 
0.0/6.9 (3) 
0.0/0.0 (4) 


16.7/53.5(3) 
0.0/3.3 (3) 
0.0/0.0 (3) 


0.0/43.8 (3) 
0.0/19.4(3) 
0.0/4.8 (3) 




Relative humidity (%) 




Temperature (°C) 




В 


15 


20 


25 




40 

70 

100 


0.0/8.3 
0.0/0.0 


0.0/3.8 
0.0/20.0 


0.0/4.8 



eggs maintained at 90% R.H. (Bayne, 1968). 
Thus, egg cannibalism decreased when tem- 
perature increased and/or humidity decreased 
because eggs lost water and became uneat- 
able. 

There is also a negative correlation between 
the humidity and the durations of incubation 
and hatching in H. aspersa (Guéméné & 
Daguzan, 1983) and a positive one between 
temperature of incubation and hatching syn- 
chronism (Le Calvé & Daguzan, 1989). Ther- 
mohygrometric conditions can increase hatch- 
ing asynchronism of clutches incubated in 
natural conditions and lead to egg cannibal- 
ism. The occurence of oophagy is also related 
to the time spent by hatchlings in the nest. 
Temperature enhances this period from four 
days at 25°C to 10 days at 15°C (Le Calvé, 
1987). A low soil humidity may also increase 
this delay, because dry soil might prevent 
snails from emerging. 

In natural clutches, as eggs are arranged in 
groups, egg dehydration is significantly lower 
and outer eggs may dry more rapidly (Bayne, 
1969). Therefore, in a dry soil, outer eggs of 
the clutch dehydrate more rapidly and hatch 
later than the inner ones, so that they may be 



TABLE 3. Range of egg dehydration time (in days) 
of all eggs available of several batches of Helix 
aspersa according to ambient temperature and rel- 
ative humidity (number of clutches in parentheses) 







Temperature (°C) 


humidity (%) 


15 


20 25 


40 

70 

100 


2-4 (3) 
>10(3) 
>10(4) 


2-4 (3) 2-4 (3) 
2-5 (3) 2-4 (3) 
>10(3) >10(3) 



consumed by newly hatched snails from the 
internal eggs, as hypothetised by Baur & Baur 
(1986) for A. arbustorum. In A. arbustorum, 
Baur (1988b) noted a preference for wet ovi- 
position sites and assumed that this choice in- 
duced a higher hatching success. Egg canni- 
balism might therefore be influenced by the 
choice of the oviposition site (parental manip- 
ulation), which produces an alteration of the 
hatching asynchronism (Baur, 1992). 

In snail farms, the rates of hatching are 
often around 70% to 90% (Daguzan, 1981); 
although no value on egg cannibalism are 
available in the literature, it seems to be rare. 



172 



DESBUQUOIS & MADEC 



In the wild, numerous causes of egg mortality 
exist in the clutches, but the extent of egg can- 
nibalism is unknown. 

High rates of egg cannibalism observed in 
this experiment compared with farm studies 
and probably wild conditions might be ex- 
plained by two reasons: (i) the incubation time 
was artificially prolonged for half of the hatch; 
natural hatching asynchronism is probably 
lower in field conditions for the reason that the 
eggs of a clutch never undergoes such differ- 
ent environmental conditions, that is a range 
of 5°C in a egg laying site. In the case of high 
temperature and humidity in natural condi- 
tions or in snail farms where incubation condi- 
tions were nearly constant, the hatching syn- 
chronism of the eggs might have prevented 
egg cannibalism, (ii) in snail farms and when 
the conditions were favourable in the wild, the 
time spent by hatchlings in the nest is low. 
Thus, the length of time during which hatch- 
lings may consume eggs is lower than in this 
experiment. On the other hand, egg cannibal- 
ism in natural conditions may increase at low 
temperature and low humidity in comparison 
with our results because hatchings were not 
synchronized and the length of time spent in 
the nest was high. 

The low availability of an alternative food 
tends to favour cannibalism in different spe- 
cies (Elgar & Crespi, 1992). However, prelimi- 
nary experiments demonstrated that new- 
born hatchlings of H. aspersa exhibited similar 
rate of cannibalism in presence and absence 
of humus (unpublished data). Thus, the lack of 
an alternative food can not explain the high 
rates of egg cannibalism observed in this ex- 
periment. 

According to Baur & Baur (1986), egg can- 
nibalism during dry weather might be a sur- 
vival mechanism for the reason that nutritional 
and energetic benefits of egg consumption 
allow an increase in growth and survival of 
hatchlings. In addition, water intake by egg 
feeding leads, in those dry conditions, to a re- 
hydration of hatchlings, which gives them the 
opportunity to wait for humidity and therefore 
increases their survival. Because of a juvenile 
size-dependent mortality based on harsh con- 
ditions encountered in winter for European 
populations of Helix aspersa, a high juvenile 
growth rate might represent an interesting ele- 
ment to avoid mortality due to low tempera- 
tures during winter, because juveniles with 
shell breadth below 19 mm are not able to re- 
ally hibernate and thus, exhibit high mortality 
(Biannic, 1995). The plasticity of egg size and 



the correlation between egg size and hatchling 
size may lead to a seasonal enhancement of 
the juvenile growth rate, for autumnal clutches 
(Madec, 1989; Madec et al., in press). Egg 
cannibalism may be an alternative solution 
favouring higher growth rates. On that ac- 
count, egg cannibalism and its plasticity, via 
their action on juvenile survival and on size at 
maturity (in preparation), may become an im- 
portant component of the fitness in popula- 
tions subject to periods of dryness. 



ACKNOWLEDGEMENTS 

We would like to express our grateful 
thanks to L. Chevalier and R. Spittal for cor- 
recting the English text, and to anonymous 
referees for helpful advices. 



LITERATURE CITED 

BAILEY, S. E. R., 1981, Circannual and circadian 
rhythms in the snail Helix aspersa Müller and the 
photoperiodic control of annual activity and re- 
production. Journal of Comparative Physiology, 
142:89-94. 

BAILEY, S. E. R., 1983, The photoperiodic control of 
hibernation and reproduction in the land snail 
Helix aspersa Müller. Journal of Molluscan 
Studies. SuppL, 12A:2-5. 

BAUR, В., 1988a, Egg-species recognition in canni- 
balistic hatchlings of the land snails Arianta ar- 
bustorum and Helix pomatia. Expehentia, 44: 
276-277. 

BAUR, В., 1988b, Do the risks of egg cannibalism 
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BAUR, В., 1993, Intraclutch egg cannibalism by 
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BAUR, В., 1994, Parental care in terrestrial gas- 
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BAUR, B. & A. BAUR, 1986, Proximate factors influ- 
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ENVIRONMENTALLY-INDUCED VARIATION OF EGG CANNIBALISM 



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Revised ms. accepted 30 September 1997 



MALACOLOGIA, 1998, 39(1-2): 175-182 

EXPRESSED SEQUENCE TAGS (ESTs) OF BIOMPHALARIA GLABRATA, 

AN INTERMEDIATE SNAIL HOST OF SCHISTOSOMA MANSONI: 

USE IN THE IDENTIFICATION OF RFLP MARKERS 

Matty Knight\ Andre N. Miller\ Neil S. M. Geoghagen^, Fred A. Lewis\ 
& Anthony R. Kerlavage^ 

ABSTRACT 

To identify some of the genes expressed in the snail Biomphalaria glabrata, a major interme- 
diate host for the trematode parasite Schistosoma mansoni, we sequenced random cDNA clones 
from either a whole body or a cerebral ganglia cDNA library to generate 1 1 1 expressed sequence 
tags (ESTs). Searches of existing public databases showed that the majority of the snail se- 
quences (54%) showed no significant homology to genes listed in either DNA or protein data- 
bases. Thirty one EST sequences showed significant matches with other genes in the databases. 
These included genes involved in gene expression, such as ribosomal proteins and translation 
factors, and those involved in cell communication, such as acetylcholine receptor and ATP-de- 
pendent transporter. Some ESTs used as probes demonstrated the occurrence of restriction 
fragment length polymorphisms (RFLPs) between parasite-resistant and parasite-susceptible 
snail stocks. Southern hybridization of parasite DNA with the snail EST encoding the acetyl- 
choline receptor as probe showed the presence of a related sequence in the parasite genome, 
with the heterologous probe, indicating that this may be a useful method to identify closely re- 
lated genes between the host and parasite. 

Key words: Biomphalaria glabrata, cDNA libraries, Expressed Sequence Tags (ESTs), RFLP, 
Schistosoma mansoni resistant/susceptible. 



INTRODUCTION 

Considerable progress has been made in 
the analysis of genes of complex organisms 
by partial sequencing of random cDNAs.This 
method of generating expressed sequence 
tags (ESTs), first developed by Adams et al. 
(1991) for the human genome project, has 
rapidly expanded our knowledge of the iden- 
tity and diversity of transcripts in organisms 
for which relatively little genetic information 
previously existed. For the parasitic helminths, 
Schistosoma mansoni and Brugia malayi for 
example, ESTs compiled in recent years have 
greatly expanded the number of cloned tran- 
scripts listed in DNA and protein databases 
from these organisms (Franco et al., 1995; 
Blaxter et al., 1996; Johnston, 1997). The 
identification of previously unknown genes 
from these invertebrates, and homologies 
with those from other organisms, may lead to 
a better understanding of their biology and to 
more effective treatment of the diseases they 
cause. 



Much less is known about the genes of the 
vectors or intermediate hosts that transmit 
these parasites. For the mollusc Biomphalaria 
glabrata, an important intermediate host of 
Schistosoma mansoni, infection by the para- 
site is influenced by both snail and parasite 
genes (Richards, 1973, 1975). In adult snails, 
resistance to parasite infection is controlled by 
a single gene, which is inherited in Mendelian 
fashion, with resistance dominant. In juvenile 
snails, resistance is believed to be a polygenic 
trait, which is based on the complex interac- 
tion of other yet unknown genetic factors 
(Richards & Merrit, 1 972). A more detailed un- 
derstanding of both parasite and snail genes 
involved in this parasite-host interaction may 
lead to new methods for schistosomiasis con- 
trol. 

Compared to some other invertebrates that 
serve as intermediate hosts or vectors for 
major human diseases, relatively little is 
known about the molecular make-up of B. 
glabrata: no genetic or physical maps exist, 
and very few genes have been analyzed. A 



Biomedical Research institute, Rocl<viile, IVlaryiand 20852, USA 
^The Institute for Genomic Research, Gaithersburg, Maryland 20850, USA 



175 



176 



KNIGHT ET AL 



search of sequences in GenBank showed only 
five B. glabrata sequences listed. Relatively 
few molluscs have been karyotyped (Burch, 
1 967; Patterson & Burch, 1 978), and few stud- 
ies have been conducted on the cytological 
analyses of chromosomes from different 
strains of B. glabrata chromosomes (Goldman 
et al., 1984). Based on the genome size of an- 
other closely related freshwater pulmonate 
gastropod, Lymnaea stagnalis, the B. glabrata 
genome is expected to be about 5.0 x 10® bp 
(Boer et al., 1977). 

Using snails of different parasite suscepti- 
bility phenotypes, we previously described the 
identification of restriction fragment length 
polymorphisms (RFLP) and random amplified 
polymorphic DNA (RAPD) markers that can 
be used to distinguish some of our laboratory 
maintained, genetically selected resistant and 
susceptible snails (Knight et al., 1 991 ; Larson 
et al., 1996). In the present study, we have 
used the isolation of ESTs to identify B. 
glabrata genes and to search for polymor- 
phisms within some of these genes. This may 
be useful for conducting genetic linkage stud- 
ies to identify genes associated with either re- 
sistance or susceptibility in B. glabrata. In this 
paper, we describe the identification of new B. 
glabrata ESTs from a survey of whole body 
and cerebral ganglia directional cDNA li- 
braries from a S. manson/'-resistant snail (BS- 
90), and report the occurrence of RFLPs with 
some of these genes. 



MATERIALS AND METHODS 



under liquid nitrogen on dry ice in a chilled 
mortar. RNA was extracted with Rnazol as de- 
scribed by the manufacturer (Sinna Biotech). 
Cerebral ganglia were dissected from 10 indi- 
vidual adult snails (12-14 mm), plunged di- 
rectly into Rnazol on ice, and extracted imme- 
diately. DNA was extracted from adult snails 
(10 mm) by a combination of the methods de- 
scribed by Knight et al. (1 991 ) and Winnepen- 
ninckx et al. (1993). Briefly, snails were 
crushed into a fine powder as described 
above, and the powder was mixed, by inver- 
sion, into 10 ml pre-warmed (60°C) extraction 
buffer containing 2% cetyltrimethylammonium 
bromide (СТАВ) (w/v), 1.4M NaCI, 0.2% (v/v) 
ß-mercaptoethanol, 20 mM EDTA pH 8.0, 100 
mM Tris-HCI pH 8.0 and 100 ^g/ml proteinase 
К (Boehringer Mannheim) and incubated for 1 
h at 60°C. Samples were extracted sequen- 
tially with an equal volume of phenol, phe- 
nol/chloroform (1:1) and chloroform. The 
aqueous phase was digested with RNase A 
(20 ug/ml) for 1 h at 37°C, and extractions 
were repeated as described above. DNA was 
recovered by spooling after the addition of 2.5 
volumes of ethanol (-20°C). Spooled DNA 
was washed in 70% ethanol (-20°C), air 
dried and resuspended in sterile dH20 at a 
concentration of 0.1 mg/ml. Restriction en- 
zyme digestions of DNA samples were per- 
formed as described by Knight et al. (1991), 
except that DNA was heated for 5 min at 65°C 
before enzymatic digestion. Digestions were 
done overnight at 37°C with buffer supplied by 
the manufacturer (New England Biolabs, 
Massachusetts). 



Snails 



Construction of cDNA Libraries 



The BS-90 snail line of B. glabrata is resis- 
tant at any age to S. mansoni infection 
(Paraense & Correa, 1963) and was made 
available to us by Dr. E. S. Loker (University of 
New Mexico). The M-line snail was selected 
for high susceptibility to S. mansoni infection 
by Newton (1955). Both snail lines were main- 
tained as previously described (Miller et al., 
1996). 

RNA and DNA Extraction 

Snails used for nucleic acid extraction were 
cleaned and kept overnight in sterile water 
containing 0.1 mg/ml ampicillin. RNA was ex- 
tracted from either the whole body or cerebral 
ganglia. For whole body extraction, snails 
were crushed with a pestle into a fine powder 



The whole body snail directional cDNA li- 
brary was prepared from 5 |.ig of poly A+ se- 
lected mRNA in the phage vector >^ZAP using 
the >^ZAP-cDNA synthesis kit according to 
manufacturer's instructions (Stratagene, Cal- 
ifornia). Briefly, first strand synthesis with re- 
verse transcriptase was prepared by priming 
with Xho l-oligo-dT primer, followed by second 
strand synthesis with RNase H and DNA poly- 
merase I. The final cDNA product was blunt- 
ended and, after ligation of EcoR I linkers and 
kinase treatment, was size selected on a 
Sephacryl S-400 column. The cDNA recov- 
ered was digested with EcoR I and Xho I and 
cloned directionally {EcoR I at the 5' end 
and Xho I at the 3' end) into the >^Zap vector 
EcoR \IXho I phosphatase treated arms. 
Packaging was performed using packaging 



EXPRESSED SEQUENCE TAGS OF BIOMPHALARIA 



177 



extract (Gigapack gold) from Stratagene and 
plated out on E. со// strain XL1 -blue MRF'.The 
library consisted of 1.1 x 10^ independent re- 
combinants with average insert size of 1000 
bp. 

The cDNA library of cerebral ganglia was 
constructed as described above, with the ex- 
ception that first strand cDNA was prepared 
from total RNA (13.3 цд) extracted from cere- 
bral ganglia from snails exposed for 5 h to 25 
S. mansoni miracidia. The kZap cerebral gan- 
glia directional cDNA library has 1.0 x 10^ re- 
combinants. Phagemids were prepared by 
mass excision of the libraries by co-infection 
with helper phage R408 and plating on E. coli 
(Sure strain) according to the manufacturer's 
instructions (Stratagene). Individual random 
colonies, selected by plating on IPTG/Xgal 
plates, were transferred in ordered array into 
Super broth (100 ¡.il) in a 96-well microtitre 
plate. Phagemid DNA was prepared from 
each well after 37°C overnight incubation, 
with the mini-prep DNA isolation kit (Wizard, 
Promega, Wisconsin). Partial sequencing was 
on double stranded templates in the forward 
and reverse directions with fluorescent M 13 
universal primers and automated sequencers 
(Applied Biosystems 373A) (Adams et al., 
1995). Nucleotide and protein sequence 
searches were conducted as described by 
using the algorithms BLAST (Altschul et al., 
1990) and BLAZE (Brutlag et al., 1993), re- 
spectively. 

Southern Hybridization 

Restriction enzyme digested DNA was 
loaded (10 ug/lane) onto 0.8% TBE agarose 
gels and resolved by horizontal flat bed elec- 
trophoresis. Southern transfer onto nylon 
membranes (Nytran, Schleicher & Schuell) 
was performed using lOx SSC according to 
the standard method (Southern, 1975). DNA 
was immobilized by UV cross-linking and bak- 
ing for 2 h at 80°C. 

Hybridizations were performed overnight at 
42°C in the presence of 50% formamide and 
10% Dextran sulfate in 2x SSPE, 5x 
Denharts, 1% SDS and 100 ug/ml of soni- 
cated salmon testes DNA. Probes were made 
from individual phagemid DNA by labeling 
with ^2p-dCTP (6000 Ci/mmole, Amersham) 
by the random priming method as described 
by Feinberg & Volgelstein (1983). Blots were 
washed at 55°C in 0.2x SSC and 0.1% SDS 
and set up for autoradiography at -70°C, for 
2-5 days, with intensifying screens. 



RESULTS & DISCUSSION 



Sequence Analysis and Identification of 
a glabrata ESTs 

Of the 1 90 clones sequenced (95 from each 
library). 111 provided usable sequences. An 
average of 322 bases was obtained from ei- 
ther the 5' or 3' ends. The standard for elimi- 
nation of unwanted and ambigous sequences 
(vector and poly A tail) was performed as de- 
scribed by Adams et al. (1995). Searches of 
peptide sequences were performed from all 
six possible reading frames. Table 1 shows the 
number of ESTs divided into categories which 
represent (1 ) sequences that show significant 
homology to database sequences, (2) se- 
quences that show no significant matches, 
and (3) sequences of mitochondrial DNA. As 
indicated, significant matches were detected 
with 28% of the snail sequences, 54% 
showed no homology to existing database se- 
quences, and 18% of the sequences corre- 
sponded to mitochondrial DNA. Because the 
RNA utilized to construct the cerebral ganglia 
library was not poly-A selected, we expected 
to identify large numbers of ribosomal RNA 
sequences, but none were detected among 
the clones we sequenced. On the other hand, 
the higher number of EST sequences corre- 
sponding to mitochondrial-related sequences 
in the cerebral ganglia library (14), compared 
to the whole adult library (5), may be the result 
of not using poly-A RNA as starting material in 
the construction of the former library. 

The 31 EST sequences that showed signifi- 
cant matches with other genes in the data- 
bases are listed in Table 2. Several sequences 
identified corresponded to genes involved in 
protein/gene expression (ribosomal protein, 
translation elongation factor 1) or to genes in- 
volved in cell communication (acetylcholine re- 
ceptor, ATP-dependent transporter). From 
the neural tissue library, we identified se- 
quences with significant homologies (57%- 



TABLE 1 . Summary of B. glabrata ESTs 







Cerebral 




Libraries 


Whole Snail 


Ganglia 


Total 


Database Match 


15 


16 


31 


Unknown 


31 


29 


60 


Mitochondrial 


5 


15 


20 


Total 


51 


60 


111 



178 



KNIGHT ET AL. 



TABLE 2. Database similarities of B. glabrata ESTs. ESTs witin database matches are listed with their puta- 
tive identification, the length, percent identity, and percent similarity of the match, and the accession number 
of the sequence matched. Match lengths are in nucleotides. In addition to those listed above, EST1 88741 
had 83% nucleotide identity with GB:M69023, which is misidentified as a human globin gene. We counted 
this EST as unknown. 



est# 


putative ID 


len 


%id 


%sim 


acc# 


Adult library 












EST1 88651 


acetylcholine receptor 


182 


30.6 


51.6 


SP:P22770 


EST1 88652 


possible glycoprotein 


233 


34.2 


51.9 


GP:912490 


EST1 88653 


possible glycoprotein 


233 


34.2 


51.9 


GP:912490 


EST1 88654 


cystatin 


155 


36.5 


57.7 


PIR:S12913 


EST1 88655 


cystatin 


239 


30.9 


48.2 


PIR:A29632 


EST1 88656 


endo-1 ,3-beta-glucanase 


296 


45.5 


58.6 


GP; 144808 


EST1 88657 


endo-1 ,3-beta-glucanase 


242 


47.6 


59.8 


GP: 144808 


EST1 88658 


major secreted protein MPB70 


119 


42.5 


60.0 


PIR:A37195 


EST1 88659 


major secreted protein MPB70 


119 


45.0 


62.5 


PiR:A37195 


EST1 88660 


major secreted protein MPB70 


119 


45.0 


62.5 


PIR:A37195 


EST1 88661 


moesin 


182 


59.7 


88.7 


GP:623040 


EST1 88662 


ribosomal protein L13 


119 


50.0 


65.0 


SP:P26373 


EST1 88663 


ribosomal protein LI 7 


344 


93.0 


95.6 


PIR:JC1253 


EST1 88664 


ribosomal protein LI 7 


242 


91.4 


95.1 


PIR:JC1253 


EST1 88665 


ribosomal protein S20 


218 


93.2 


95.9 


GP:292443 


Cerebral ganglia 


library 










EST1 88671 


antigen HuD, neuronal 


281 


41.5 


54.3 


GP:1 79537 


EST1 88672 


ATP-dependent transporter 


296 


53.0 


70.0 


SP;P40024 


EST1 88674 


DNA topoisomerase II 


104 


42.9 


60.0 


SP:Q01320 


EST1 88675 


DNA topoisomerase II 


104 


42.9 


60.0 


SP:Q01320 


EST1 88676 


FMRFamide precursor 


233 


43.8 


61.2 


SP:P42565 


EST1 88677 


FMRFamide precursor 


262 


53.3 


60.0 


SP:P42565 


EST1 88678 


FMRFamide precursor 


143 


37.3 


56.9 


SP:P42565 


EST1 88679 


globin 


389 


30.8 


49.2 


SP:P02215 


EST1 88680 


heat shock protein 90 


374 


88.8 


97.6 


GP:256089 


EST1 88681 


heat shock protein 90 


338 


81.6 


90.4 


GP:256089 


EST1 88682 


proclotting enzyme precursor 


242 


35.7 


54.8 


SP:P21902 


EST1 88683 


ribosomal protein L41 


74 


92.0 


92.0 


GP:36136 


EST1 88684 


ribosomal protein SI 7 


164 


78.2 


89.1 


GP:337501 


EST1 88685 


ribosomal protein SI 7 


101 


82.4 


82.4 


GP:337501 


EST1 88686 


serine protease 


500 


29.2 


47.0 


GP:868212 


EST1 88687 


translation elongation factor 1, alpha 


293 


79.6 


85.7 


GP:214111 



61% similarity) to the neuropeptide FMRF- 
amide precursor. A number of sequences from 
both libraries were highly redundant. For ex- 
ample, the EST sequences for ribosomal pro- 
teins occurred frequently as did sequences for 
housekeeping genes, hsp90 and DNA topo- 
isomerase. The high frequency of certain 
clones may either reflect the metabolic state of 
the tissue samples when RNA was isolated 
(abundant transcripts) or a bias created during 
the manipulation (amplification and mass ex- 
cision) of the libraries. Using the partial EST 
sequence for globin, the corresponding full- 
length cDNA has been isolated and se- 
quenced from the cerebral ganglia library 
(Dewilde et al., manuscript submitted). The 



1116. glabrata EST sequences discussed in 
this manuscript have been deposited in the 
dbEST database of the National Center for 
Biotechnology Information with the follow- 
ing accession number: dbEST: 1 193734 to 
1 1 93844; GenBank: AA547685 to AA547795. 

Identification of Polymorphic ESTs 

Hybridization patterns were compared be- 
tween genomic DNA from parasite-resistant 
(BS-90) and -susceptible (M-line) snails, 
using the ESTs as probes. As shown in Table 
3, RFLPs were detected with some ESTs. 
Most polymorphisms were observed with re- 
striction enzymes Hind \\\ and Hinf\\\. RFLP 



EXPRESSED SEQUENCE TAGS OF BIOMPHALARIA 



179 





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180 



KNIGHT ET AL. 



analysis was conducted on either individual or 
pooled snail DNA samples. Linkage studies, 
using these EST RFLPs as probes, will be 
performed on progeny snail DNA derived from 
a cross between the resistant and susceptible 
snails as part of an ongoing study to identify 
sequences associated with either the resis- 
tant or susceptibility loci in B. glabrata. 

Hybridization of parasite DNA using heterol- 
ogous snail ESTs as probe, in some cases, 
demonstrated the occurrence of related se- 
quences in the parasite genome. For example. 
Figure 1 shows the hybridization of the 
snail EST encoding acetylcholine receptor 
(EST1 88651 ) to Hae III digested DNA from the 
resistant snail (Lane 1), susceptible snail 
(Lane 2) and S. mansoni (Lane 3). As indi- 
cated, the snail probe detects a major frag- 
ment (590bp) in both the snail and parasite 
genomes. Considerable sequence homolo- 
gies have previously been reported between 
parasite and snail genes (Dissous et al., 1 990; 
Weston et al., 1 994). Cross hybridization stud- 
ies, using these snail probes, may be a useful 
strategy to identify and clone corresponding S. 
mansoni genes for which no sequence infor- 
mation currently exists. Conversely, ESTs that 
are currently being generated from the para- 
site (Franco et al., 1995; Neto et al., 1997), 
may serve as useful heterologous probes for 
genome studies of the intermediate host. 
Unlike the parasite, which shows an amino 
acid codon preference for A/T in the third base 
position (Meadows & Simpson, 1989), analy- 
sis of the partial amino acid sequences gener- 
ated from the B. glabrata ESTs does not reflect 
a similar bias (data not shown). Full-length se- 
quences will, however, be required in order to 
assess relative structural similarities and di- 
vergences between snail ESTs and related 
parasite sequences. 

This study shows that the generation of B. 
glabrata ESTs is a useful approach that 
should quickly expand our knowledge on the 
molecular biology of this organism. Although 
most of the sequences identified in this study 
showed no homology to sequences listed in 
existing public data bases, indicating they rep- 
resent novel snail-related sequences, the ac- 
cumulation of such sequences will help in our 
collective efforts in this field towards the iden- 
tification of genes involved in the genetic con- 
trol of parasite infection in this snail host. The 
ability to identify RFLP EST sequences pro- 
vides a useful method to limit the search of 
probes to known genes for which a biological 
function can be ascribed. These polymorphic 



Kbp 

1.4 - 

1.1 — 

0.87 - 

0.60 - 

0.31 - 
0.28 - 






Ш ш 



FIG. 1 Southern blot of Нае III digested DNA frorn 
resistant (1) and susceptible (2) B. glabrata snail 
lines and S. mansoni (3) hybridized with EST probe 
EST1 88651 (acetylcholine receptor). 



EST markers can form the basis to begin to 
create a physical map of the B. glabrata 
genome. 

The comparative analysis of the profile of 
transcripts obtained from the two libraries 
screened in this study suggests that for B. 
glabrata, the generation of ESTs from various 
tissues, rather than the whole snail, may be a 
more meaningful strategy to adopt in the iden- 
tification of transcripts that may be relevant to 
a particular biological phenomenon. In this 
context, work being conducted in our labora- 
tory is employing an EST strategy to compare 
the profile of transcripts, as described by Lee 
et al. (1995), between hemocytes of resistant 
B. glabrata snails, with and without parasite 
exposure. 

In summary, ESTs have been generated by 
partial sequencing of clones from B. glabrata 
cDNA libraries. Most sequences showed no 
homology to sequences in existing data 
bases. Some snail ESTs may serve as useful 
probes to identify homologous genes in the 
parasite. RFLPs identified with ESTs provides 
useful markers to conduct genetic linkage 



EXPRESSED SEQUENCE TAGS OF BIOMPHALARIA 



181 



studies for the identification of the parasite re- 
sistance/susceptibility loci in this snail host. 



ACKNOWLEDGMENT 

This work was supported by NIH grant A! 
27777. 



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Revised ms. accepted 8 October 1997 



MALACOLOGIA, 1998, 39(1-2): 183-193 

PHYLOGENETIC UTILITY OF THE 5'-HALF OF MITOCHONDRIAL 16S rDNA 

GENE SEQUENCES FOR INFERRING RELATIONSHIPS OF ELIMIA 

(CERITHIOIDEA: PLEUROCERIDAE) 

Charles Lydeard\ John H.Yoder\ Wallace E. HolznageP, Fred G.Thompson^ & Paul Hartfield^ 

ABSTRACT 

Mitochondrial 16S rDNA sequences have proven useful for mesolevel phylogenetic questions. 
To date, most published studies have used primers that amplify the conservative 3'-half of the 
gene. We recently developed primers that amplify an approximately 550 bp portion of the more 
variable 5'-half of the gene. The primers work well for a wide range of gastropods tested. Because 
the 5'-half of the gene exhibits greater variation than the 3 -half, we wanted to determine whether 
there is sufficient phylogenetic signal for resolving relationships among closely related taxa. We 
examined the utility of the 5' portion by assessing relationships within the pleurocerid genus 
Elimia of the Mobile River basin. Although the 433 bp data matrix possessed significant phylo- 
genetic signal, and the resultant 320 most parsimonious trees had some aspects that were well 
resolved, most of the phylogenetic signal seemed to be partitioned among genera. Only 36 phy- 
logenetically informative sites were found within Elimia, which is too few to resolve all nodes for 
such a diverse assemblage. We recommend the continued use of the 16S rDNA gene for stud- 
ies examining intergeneric relationships of molluscs, and suggest researchers employ mito- 
chondrial protein coding genes for interspecific studies. 

Key words: mollusks, Cerithioidea, Pleuroceridae, Elimia, mitochondrial DNA, 16s rDNA. 



INTRODUCTION 

The Introduction of conserved "universal" 
primers (Kocher et al., 1989), which permit 
amplification of specific regions of homolo- 
gous DNA via the polymerase chain reaction 
(PCR) (Saiki et al., 1 985), has offered tremen- 
dous opportunities for systematic studies. 
Many of the earlier molecular phylogenetic 
studies of molluscs have employed 18S or 
28S nuclear ribosomal sequence data in an 
attempt to resolve relationships among dis- 
tantly related taxa. For example, the relation- 
ships of molluscs to other metazoans (e.g., 
Ghiselin, 1988; Winnepenninckx et al., 1994), 
gastropod relationships (e.g., Emberton et al., 
1990; Tillier et al., 1992; Rosenberg et al., 
1994), and unionoidean bivalve relationships 
(e.g., Rosenberg et al., 1994). Although these 
genes appear useful for resolving some as- 



pects of higher-level relationships among mol- 
luscs, they are too conservative to be of much 
use for studying relationships among more 
closely related genera or species. 

Mitochondrial DNA has proven to be quite 
useful for studying evolutionary relationships 
of animals (Brown, 1985; Avise et al., 1987; 
Moritz et al., 1987). Sequencing the complete 
genome, although relatively labor intensive, 
has provided data for studying molluscan rela- 
tionships based both on nucleotide homology 
and on mitochondrial gene order (Boore & 
Brown, 1994). In contrast, restriction fragment 
or site analysis of the mtDNA genome pro- 
vides information for intraspecific population 
structure (e.g., Reeb & Avise, 1990; Liu et al., 
1996). On the other hand, molecular phyloge- 
netic studies employing mitochondrial DNA 
sequence data are only recently becoming 
more commonly conducted in malacology. The 



^Aquatic Biology Program, Department of Biological Sciences, University of Alabama, Box 870345, Tuscaloosa, Alabama, 

35487 USA 

2Florida Museum of Natural History, Museum Road, University of Florida, Gainesville, Florida, 3261 1 USA 

3U.S. Fish and Wildlife Service Endangered Species Office, Dogwood View Parkway, Suite A, Jackson, Mississippi, 39213 

USA 



183 



184 



LYDEARD ET AL. 



delay is most likely due to the fact that many of 
the first universal primers reported in the liter- 
ature do not work well if at all for molluscs 
(Spolsky et al., 1996). Thus, more effort is re- 
quired on the part of the investigator to design 
primers that will work efficiently and yield suf- 
ficient variation for the question in hand. 
Folmer et al. (1994) developed primers that 
amplify a portion of the protein coding gene, 
cytochrome с oxidase subunit I from a wide 
range of invertebrate taxa, including molluscs. 
These primers have since been used to exam- 
ine the evolution of gender-associated mito- 
chondrial lineages in bivalves (Hoeh et al., 
1996), and are now being used in a variety of 
other systematic studies of freshwater unionid 
mussels (Roe & Lydeard, 1998; Hoeh, pers. 
comm.; Liu & Mulvey, pers. comm.) and gas- 
tropods (Harasewych, pers. comm.). Primers 
for another protein coding gene, cytochrome b, 
have also been developed, which have proven 
useful for studying relationships within the 
gastropod genus Nucella (Collins et al., 1996) 
and for intraspecific relationships within the 
gastropod genus Oncomelania (Spolsky et a!., 
1996). Mitochondrial protein coding genes ap- 
pear to be useful for a wide range of system- 
atic questions. This can be attributed to rate 
variation among codon positions. For exam- 
ple, third codon positions are under fewer se- 
lective constraints and hence evolve faster 
than first and second codon positions. Thus, 
sufficient variation may be found to estimate 
relationships even among closely related taxa. 
In contrast, for deeper phylogenetic questions, 
substitutions in the third codon position can be 
downweighted or dropped (e.g., Lydeard & 
Roe, 1997). 

Mitochondrial ribosomal genes have been 
of considerable use for systematics studies 
(Hillis & Dixon, 1991). The genes are thought 
to be more conservative than protein coding 
genes and are therefore more useful for 
meso-level phylogenetic questions. Recently 
the mitochondrial 16S rDNA gene has been 
used successfully for estimating relationships 
among genera of North American freshwater 
unionids (Lydeard et al., 1996). The primers 
used by Lydeard et al. (1996) were designed 
by modifying the universal primers of Palumbi 
et al. (1991), which amplify a 550 bp fragment 
of the 3' half of the gene. In another study 
Lydeard et al. (1997) developed primers that 
extended into the 5' half of the gene, which is 
rarely sequenced in phylogenetic studies. The 
combined data matrix of nearly 900 bp was 
used to study relationships among three gen- 



era of pleurocerid gastropods of the Mobile 
River basin. Interestingly the 5' half of the 
gene exhibits more variation than the 3' half 
(Gutell et al., 1992; Lydeard et al., unpubl.), 
which lead us to believe the gene might be 
useful for assessing relationships among 
more closely related taxa. Here, we present 
our analysis of the utility of the 5' half of the 
16S rDNA gene for examining relationships 
within the gastropod genus Elimia. The pri- 
mers used to amplify this region work well on 
a wide range of gastropod species, so our 
findings will be of interest to other malacolo- 
gists interested in examining systematic rela- 
tionships among similarly divergent taxa. 



MATERIALS AND METHODS 
Specimens Studied 

Elimia were collected from various localities 
throughout the Mobile River basin, but with a 
particular emphasis on Coosa River species 
(Appendix). Twenty-three Elimia specimens 
representing eleven nominal species were in- 
cluded in the analysis, along with three 
Pleurocera prasinatum and one Leptoxis tae- 
niata as outgroup taxa (Table 1 ). 

Sequence Procurement, Alignment, 
and Analysis 

Genomic DNA was isolated from frozen or 
80% ethanol-preserved specimens (typically 
the proboscis or entire head of the snail; less 
tissue seems to yield better quality DNA) by 
standard phenol-chloroform extractions. Mito- 
chondrial DNA sequences were obtained for 
an amplified segment of the mitochondrial 
16S rDNA gene using primers SNL002 (5'- 
aaatgattatgctacctttgc-3' and SNL-448 (5'- 
gaaatttcattcgcactag-3'). These primers were 
designed by Lydeard et al. (1997) specifically 
for pleurocerids and related gastropods, and 
amplify an approximately 550 bp fragment at 
the 5' end. 

Approximately 50 - 500 ng of genomic DNA 
provided template for double-stranded ampli- 
fications via PCR in 25 ul of Tris (67 mM, pH 
8.8) containing 2 mM MgCI2, 1 mM of each 
dNTR 1 uM of each primer, and Taq poly- 
merase (1.25 units, Perkin-Elmer-Cetus).The 
amplification regime consisted of 30 cycles of 
denaturation at 92°C for 40 s, annealing at 
52°C for 60 s, and extension at 72°C for 90 s. 
Single-stranded DNA was produced for se- 



16S rDNA GENE SEQUENCES IN ELIMIA 



185 



TABLE I.Taxa and number of specimens included 
in the present study. 

Pleuroceridae 
Elimia catenaria group 

E. crenatella (2) 
Elimia carinocostata group 

E. carinocostata (9) 
Elimia gerhardtii group 

E. gerhardtii (3) 
Elimia haysiana group 

E. alabamensis {^) 

E. haysiana (1) 
Elimia hydei group 

E.hydei(^) 
Elimia olivula group 

E. cylindracea (1) 

E. olivula (1) 

E. showalteh (^) 
Elimia vanuxemiana group 

E. fascinans (2) 

E. caelatura infúscala (2) 
Pleurocera prasinatum (3) 
Leptoxis taeniata (2) 



quencing via asymmetric amplification 
(Gyllensten & Eriich, 1988) using low-melt 
agarose (FMC BioProducts) purified double- 
stranded PCR products as templates. 
Reaction conditions for asymmetric PCR 
were as above with the exceptions that one 
primer was held limiting, and the final volume 
of the reaction cocktail was increased to 50 
uL. Asymmetric reactions were conducted for 
each specimen using both amplification 
primers in limited quantity in separate reac- 
tions. Thermal cycling was performed in a pro- 
grammable heating block (Perkin-Elmer- 
Cetus) with negative (-DNA) controls included 
with each reaction set. 

Following purification by centrifugal filtra- 
tion (Millipore Ultra-free-MC 30,000), single- 
stranded DNA was sequenced by dideoxy 
chain termination using Sequenase Version 
2.0 (United States Biochemical) and instruc- 
tions supplied by the manufacturer. Both 
strands were sequenced using the appropri- 
ate amplification primer as a sequencing 
primer. The sequencing reaction products, 
which included S to permit autoradiographic 
visualization, were run on 6% Polyacrylamide 
gels (Long Ranger, FMC BioProducts) from 2 
to 4.5 h. Following electrophoresis, all gels 
were vacuum-dried and exposed to X-ray film 
for 48- 120 h. 

Sequences were initially entered in the soft- 
ware program XESEE version 3.0 (Cabot & 
Beckenbach, 1 989) and subsequently aligned 



using CLUSTALW version 1 .5 (Thompson et 
al., 1994) and visual inspection. In addition, a 
consensus sequence of the ingroup taxa was 
superimposed over the hypothesized sec- 
ondary structure of the fruitflies Drosophila 
yakuba and D. melanogaster (Gutell & Fox, 
1988; Gutell et al., 1992) in an attempt to fur- 
ther refine the alignment and identify regions 
corresponding to hypothesized loops and 
stems. Pairwise percent sequence differences 
were calculated using Kimura's two-parame- 
ter model (Kimura, 1980), which corrects for 
multiple hits using the software package 
MEGA (Kumar et al., 1993). 

The phylogenetic analyses were conducted 
using maximum parsimony of the orthologous 
sequences using the heuristic search option 
(10 replicates) of PAUP (version 3.1.1; 
Swofford, 1993). We employed the following 
options in PAUP: uninformative characters 
were ignored, only minimal trees were kept, 
gaps were treated as missing data, and zero- 
length branches were collapsed. A bootstrap 
analysis (Felsenstein, 1985) with 100 itera- 
tions was conducted to estimate the internal 
stability of the data matrix. Although we usu- 
ally prefer to run more iterations, this particu- 
lar data set tested the limits of memory for 
more than 100 replicates. A skewness test 
statistic (gl) was calculated based on the dis- 
tribution of tree lengths of a random sample of 
10,000 topologies. Data matrices with a 
strong phylogenetic signal are significantly 
more structured than random data (Hillis & 
Huelsenbeck, 1992). 



RESULTS 

Multiple sequence alignment of the ampli- 
fied region from our selected taxa resulted in 
a data matrix consisting of 433 nucleotide po- 
sitions (Fig. 1) including insertions and dele- 
tions. Several DNA sequences from conspe- 
cific specimens were identical, and therefore 
combined in subsequent analyses: Elimia cre- 
natella (cren18 = 41A-2), E. carinocostata 
(5A-2 = 1 1 A-1), E. caelatura infúscala (16-1 = 
16-2), and Leptoxis taeniata. (2 individuals, 1 
locality). Thus, 25 unique sequences from 28 
individuals. Of the 433 nucleotide positions 
examined, 135 (31%) are variable among all 
taxa, including the outgroups, and 71 (16%) 
are potentially phylogenetically informative. 
Within the genus Elimia, 75 (17%>) are vari- 
able and 36 (8%) are potentially phylogeneti- 
cally informative. 



carino llA-l,5A-2 
carino llA-2 
carino llB-1 
carino llB-2 
carino 4 7 A- 2 
gerhard 10-1 
gerhard 10-2 
gerhard 42-1 
alabamensis 
haysiana 
olivula 
cylindracea 
hydei 

carino 46A-2 
carino 4 9 A- 2 
carino 49B-2 
fascin 7-1 
fascin 7-2 
cael inf (2) 
crenatella (2) 
shovalteri 
pras 12A-1 
pras 12B-1 
pras 12Б-2 
taen (2) 



carino llA-l,5A-2 
carino 11 A- 2 
carino llB-1 
carino llB-2 
carino 47A-2 
gerhard 10-1 
gerhard 10-2 
gerhard 42-1 
alabamensis 
haysiana 
olivula 
cylindracea 
hydei 

carino 46A-2 
carino 49A-2 
carino 49B-2 
fascin 7-1 
fascin 7-2 
cael inf (2) 
crenatella (2) 
showalteri 
pras 12A-1 
pras 12B-1 
pras 12B-2 
taen (2) 



carino llA-l,5A-2 
carino 11 A- 2 
carino llB-1 
carino llB-2 
carino 47A-2 
gerhard 10-1 
gerhard 10-2 
gerhard 42-1 
alabamensis 
haysiana 
olivula 
cylindracea 
hydei 

carino 46A-2 
carino 4 9 A- 2 
carino 49B-2 
fascin 7-1 
fascin 7-2 
cael inf (2) 
crenatelia (2) 
showalteri 
pras 12A-1 
pras 12B-1 
pras 12B-2 
taen (2) 



1 ..... 60 
GACGAGAAAATAATTATAAAATATTAATTA-TTTCATAAAATATTTCTCGATTAATTTTT 





. .T. . . . 


















. .T. . . . 




































. .T. . . . 




































. .T. . . . 




































. .T. . . . 


















. .T. . . . 












. .T. . . . 






. .T.G. . 


. .G. . . . 


. . .С 


Г 






. . TG . . . 


...G 


. . .A 














. .T. . . . 


. . .A 


. .T. . . . 












.CT 

.CT 

. .T. . . . 


...G 

. ..G 

. . .G. . . 


, . .A 














. . .A 


. .T. . . . 




G. , 














G. . 








. .T. . . . 


. . .G. . . 




T . 




G. . 




-NN. 




. .T. . . . 


. . .G. . . 




. .T. . . . 


. . .С. . , 










. . TG . . . 


С 

. . .G. . . 


. . .A 








г 






. .T. . . . 






G 










. .T. . . . 




. .T 


TA 


G. 










. .T. . . . 




. .T 


..TA... 


..G 








__, 


. .T 




..T 



CGGCT.G. ..C.C С A. ..T. С. G CTTGC . 



61 . . . . .120 
TTGAGGATAAGCTCGAAAAAAAGTTAAGAAATTTTACTAATTTAGGTT ATTATGTGG 



.T GAAG G 

.T GT.G G 

.T GT.G G 

.T — . .G. .A.AA. .TGTG. .GC. 



121 . . . . . 180 

GCTTAAAATTGGCCATCATAAGAGTTTGTTATAAAACAATAATCTTAATATTTAAGATAA 



.GC. 
..C. 
..C. 
. .C. 



.ce. 
.ce. 



FIG. 1 . An aligned data matrix of 433 nucleotide positions of nnitochondrial 16S rDNA sequences for 25 pleu- 
rocerid specimens. Dashes correspond to gaps and N's are missing data. E. carino = Elimia cahnocostata; 
E. gerhard = E. gerhardtii; E. fascin = E. fascinans: E. cael inf = Elimia caelatura infúscala; P. pras = 
Pleurocera prasinatum; L. taen = Leptoxis taeniata. Numbers following the name of the species is the spec- 
imen number. Locality information of the specimens can be found in the Appendix. 



E. 


carino llA-l,5A-2 


E. 


carino llA-2 


E. 


carino llB-1 


E. 


carino 11 В- 2 


E. 


carino 4 7 A- 2 


E. 


gerhard 10-1 


E. 


gerhard 10-2 


E. 


gerhard 42-1 


E. 


alabamensis 


E. 


haysiana 


E. 


olivula 


E. 


cylindracea 


E. 


hydei 


E. 


carino 46A-2 


E. 


carino 49A-2 


E. 


carino 49B-2 


E. 


fascin 7-1 


E. 


fascin 7-2 


E. 


cael inf (2) 


E. 


crenatella (2) 


E. 


showalteri 


P. 


pras 12A-1 


P. 


pras 12B-1 


P. 


pras 12B-2 


L. 


taen (2) 



181 . . . . . 240 

ATATATTTTATTCTAATTTTTT-ACAGAAATAAAGACCTCAATTAATAAATGCCTTATAC 



.ACT GTGCTG. 



.TG. 
. .G. 



.G. . 
.G.. 
.GG. 



.TT. 
.ТТ. 



.GA. 
.GA. 
.GA. 



.-.T. 



TAT.. 




.С. ..С. ТТ. 


. . .G, 


TAT.. 




.С. .С. ТТ. 


. ..G, 


TAT.. 




. с . . . с . TT . 


...G 


. .T.. 


.Г. 


.. .т. ..т.. 


.GC. 



241 . . . . . 300 
carino 1 1А- 1 , 5А-2 CTATGCTAGGATGAGTATTAAAACTTTTTATATTCTAAGAAAGTTTTATGTATTTTTCTT 
carino llA-2 N. . 



E. 


carino llB-1 


E. 


carino llB-2 


E. 


carino 47A-2 


E. 


gerhard 10-1 


E. 


gerhard 10-2 


E. 


gerhard 42-1 


E. 


alabamensis 


E. 


haysiana 


E. 


oliwla 


E. 


cylindracea 


E. 


hydei 


E. 


carino 46A-2 


E. 


carino 4 9 A- 2 


E. 


carino 49B-2 


E. 


fascin 7-1 


E. 


fascin 7-2 


E. 


cael inf (2) 


E. 


crenatella (2) 


E. 


showalteri 


P. 


pras 12A-1 


P. 


pras 12B-1 


P. 


pras 12B-2 


L. 


taen (2) 


E. 


carino IIA- 1,5. 


E. 


carino llA-2 


E. 


carino llB-1 


E. 


carino llB-2 


E. 


carino 4 7 A- 2 


E. 


gerhard 10-1 


E. 


gerhard 10-2 


E. 


gerhard 42-1 


E. 


alabamensis 


E. 


haysiana 


E. 


olivula 


E. 


cylindracea 


E. 


hydei 


E. 


carino 46A-2 


E. 


carino 49A-2 


E. 


carino 49B-2 


E. 


fascin 7-1 


E. 


fascin 7-2 


E. 


cael inf (2) 


E. 


crenatella (2) 


E. 


showalteri 


P. 


pras 12A-1 


P. 


pras 12B-1 


P. 


pras 12B-2 


L. 


taen (2) 



T 

A CT 

A T 

Г С . .CGT 

Г С. . .CGT 

Г С. . .CGT 

.GCA GGT.G. . 



.G. A. С AA.C 



301 . . . . . 360 

CAAAAAAAATATTGATTGAATTAAATTAGTTAAAAGGAACTCGGCAAAATTAATGCTTCG 



.T. 



.CGGG. 
.CGGG. 
.GGGG. 
.T. G. . 



. .CA. 
. .CA. 
.NCA. 
. .CA. 
..CA. 



FIG. 1. [Continued) 



188 



LYDEARD ET AL 



carino llA-l,5A-2 
carino IIA- 2 
carino llB-1 
carino llB-2 
carino 47A-2 
gerhard 10-1 
gerhard 10-2 
gerhard 42-1 
alabamensis 
haysiana 
olivuLa 
cylindracea 
hydei 

carino 46A-2 
carino 49A-2 
carino 49B-2 
fase in 7-1 
fascin 7-2 
cael iní (2) 
crenatella (2) 
showalteri 
pras 12A-1 
pras 12B-1 
pras 12B-2 
taen (2) 



361 . . . . . 420 

CCTGTTT-ATCAAAAACATGGCTCTCTGAATTCATTTTTATAGAGAGTCAGGCCTGCCCA 



421 .433 
carino llA-l,5A-2 GTGAATAATATTT 

carino 1 lA-2 

carino 1 IB- 1 

carino 1 lB-2 

carino 47A-2 

gerhard 10-1 NNNN 

gerhard 10-2 

gerhard 42-1 

alabamensis 

haysiana 

olivTjla 

cylindracea 

hydei 

carino 46A-2 ... 

carino 49A-2 ... 

carino 49B-2 

fascin 7-1 

fascin 7-2 

cael inf (2 ) ... 

crenatella (2) ... 

showalteri 

pras 12A-1 

pras 12B-1 

pras 12B-2 

taen ( 2 ) 



. .NNN 
.NNNN 



FIG. ^ . (Continued) 



Pairwise percent sequence differences cor- 
rected for multiple hits by the two-parameter 
method of Kimura (1980) were from 0% to 
3.93% within Elimia species, and 0.3% to 
1 1 .08% among Elimia species. Intergeneric 
values ranged from 10.64 to 15.33% for com- 
parisons of Elimia and Pleurocera and 
17.75% to 23.29% for comparisons of Elimia 
and Leptoxis. All painA/ise genetic distances 
are shown in Table 2. 

Maximum parsimony analysis was con- 
ducted treating all base substitutions as 
equally weighted based on a previous analy- 
sis of nucleotide substitutions within pleuro- 



cerids (Lydeard et al., 1997). Maximum parsi- 
mony analysis resulted in 320 equally most 
parsimonious trees (tree length = 207; consis- 
tency index = 0.619, excluding uninformative 
characters). The gl statistical analyses 
showed the data were significantly skewed (p 
= 0.01), suggesting the data contain signifi- 
cant phylogenetic signal. Phylogenetic signal 
was stronger when outgroup taxa were in- 
cluded in the gl statistical analysis (gl = 
-1.84) than when they were excluded (gl = 
-0.583). A strict consensus tree of the 320 
equally most parsimonious trees and a phylo- 
gram of one of the 320 equally most parsimo- 



16S rDNA GENE SEQUENCES IN ELIMIA 



189 



TABLE 2. Estimated percentage nucleotide sequence difference (Kimura's 2-parameter) among pairwise 
comparisons of taxa based on mitochiondrial 16S rDNA sequences 





eren 


earn AI car11A2 


carllBI car11B2 


car46A2 


: car47A2 


car49A2 ear49B2 


E. eren 





6.99 


7.27 


6.99 


6.72 




6.67 


6.74 


7.33 




7.02 


E. carl 1 AI 




- 


- 


0.24 


0.23 


0.47 




1.98 


0.71 


2.93 




3.67 


E.car11A2 








— 


0.47 


0.71 




2.23 


0.95 


3.19 




3.93 


E. carllBI 










— 


0.23 




1.98 


0.95 


2.93 




3.67 


E. car11B2 












— 




1.98 


0.71 


2.68 




3.42 


E. car46A2 
















— 


1.98 


1.23 




1.98 


E. car47A2 


















— 


2.68 




3.42 


E. car49A2 




















— 




1.20 


E. car49B2 
























— 


E. ger10-1 


























E.ger10-2 


























E. ger42-1 


























E.ala 


























E. hay 


























E. hydei 


























E.cyl 


























E.oli 


























E. sho 


























E.fas7-1 


























E. fas7-2 


























E. cael 


























P. pra12A-1 


























P. pra12B-1 


























Ppra12B-2 




























ger10-1 


ger10-2 


ger42-1 


ala 


hay 


hyd 


cyl 


oil 


sho 


fas7-1 


fas7-2 


cael 


E.cren 


7.10 


7.26 


7.06 


6.99 


7.26 


11.08 


6.99 


7.52 


4.16 


7.29 


7.02 


7.04 


E. carl 1 AI 


0.72 


0.71 


0.71 


0.71 


0.71 


10.20 


1.43 


0.95 


4.63 


3.64 


3.90 


3.15 


E.car11A2 


0.96 


0.95 


0.72 


0.95 


0.95 


10.50 


1.67 


1.19 


4.90 


3.90 


4.16 


3.41 


E.carllBI 


0.96 


0.95 


0.96 


0.95 


0.95 


10.48 


1.67 


1.18 


4.63 


3.90 


4.15 


3.40 


E.car11B2 


0.96 


0.71 


0.96 


0.71 


0.71 


10.20 


1.43 


0.95 


4.38 


3.64 


3.90 


3.15 


E. car46A2 


2.74 


2.48 


2.74 


2.48 


2.48 


9.99 


2.23 


2.74 


4.78 


3.51 


3.77 


3.00 


E. car47A2 


1.21 


0.95 


1.20 


0.95 


0.95 


10.22 


1.67 


1.19 


4.39 


3.40 


3.65 


2.91 


E. car49A2 


3.45 


3.18 


3.47 


3.18 


3.18 


9.20 


2.93 


3.43 


4.42 


3.18 


3.43 


2.68 


E. car49B2 


3.71 


3.92 


3.71 


3.92 


3.92 


9.99 


3.67 


4.18 


4.15 


3.42 


3.16 


2.92 


E.ger10-1 


— 


1.20 


0.96 


1.20 


1.20 


10.94 


1.94 


1.45 


4.71 


3.70 


3.44 


3.71 


E.ger10-2 




— 


1.20 


0.95 


0.95 


10.20 


1.67 


1.18 


4.89 


3.90 


4.15 


3.40 


E. ger42-1 






— 


1.20 


1.20 


10.88 


1.93 


1.44 


4.68 


4.19 


3.93 


3.69 


E.ala 








— 


0.95 


9.91 


1.67 


1.18 


4.63 


3.39 


3.64 


3.40 


E. hay 










— 


9.33 


1.66 


0.23 


4.37 


3.90 


4.15 


3.40 


E. hydei 












— 


10.17 


9.64 


8.81 


8.30 


8.57 


8.87 


E.cyl 














— 


1.43 


4.62 


3.64 


3.90 


3.15 


E.oll 
















— 


4.63 


4.15 


4.40 


3.65 


E.sho 


















— 


3.89 


3.64 


3.65 


E. fas7-1 




















— 


0.24 


1.92 


E. fas7-2 






















— 


2.17 


E. cael 
























— 








Ppra12A- 


•1 


P 


pra12B-1 




Rpra12B-2 




Ltae 




E. eren 






15.33 






14.96 






14.93 




23.29 




E. car11A1 






12.03 






11.70 






11.72 




19.72 




E.car11A2 






12.36 






12.02 






12.05 




20.02 




E.carllBI 






12.03 






11.70 






11.72 




20.06 




E.car11B2 






11.74 






11.40 






11.43 




20.06 




E. car46A2 






11.78 






11.50 






11.53 




17.75 




E. car47A2 






11.77 






11.43 






11.46 




19.78 




E. car49A2 






12.10 






11.82 






11.85 




19.44 




E. car49B2 






12.33 






12.05 






12.08 




21.14 




E. ger10-1 






12.17 






11.89 






11.91 




19.25 




E. ger10-2 






11.74 






11.40 






11.43 




20.40 




E.ger42-1 






12.77 






12.42 






12.45 




19.02 




E.ala 






12.33 






11.99 






12.02 




19.06 




E. hay 






12.33 






11.99 






12.02 




19.72 




E. hydei 






15.27 






14.59 






14.66 




23.09 




E.cyl 






10.87 






10.64 






10.66 




19.39 




E.oli 






12.03 






11.70 






11.72 




20.06 




E.sho 






13.47 






13.12 






13.09 




21.11 




E. fas7-1 






11.50 






11.17 






11.14 




19.47 




E. fas7-2 






11.79 






11.46 






11.43 




19.81 




E. cael 






12.12 






11.78 






11.76 




20.21 




Ppra12A-1 






— 






0.47 






0.71 




24.48 




Ppra12B-1 












— 






0.24 




24.09 




Ppra12B-1 


















— 




24.04 




L. tae 






















— 





190 



LYDEARD ET AL. 



E. crenatella (2) 
E. showaiteri 
E. carino11A-1,5A-2 
E. carino11A-2 



car¡no11B-1 
carinol 1 B-2 
carino47A-2 
gerhard10-1 
gerhard42-1 
E. gerhard10-2 
E. alabamensis 
E. haysiana 
E. olivula 
E. cylindracea 
E. carino46A-2 
E. car¡no49A-2 
E. carino49B-2 
E. fascin7-1 
E. fascin7-2 
E. cael inf (2) 
E. hydei 
P. pras12A-1 
P. pras12B-1 
P. pras12B-2 
L. taen (2) 



FIG. 2. A strict consensus tree of 320 most parsi- 
monious trees obtained in the maximum parsimony 
analysis of thie mtDNA sequence data using equal 
weighting for all substitutions. Bootstrap values are 
noted to the left of the corresponding node. Tree 
length = 207; consistency index = 0.619, excluding 
uninformative characters. 



Ч 



E. crenatella (2) 



E. showaiteri 
E. carino11A-1,5A-2 
E. carlno11A-2 
E. carino11B-1 
E. carlno11B-2 
E. carino47A-2 
E. gerhard10-1 
E. gerhard42-1 
E. gerhard10-2 
E. alabamensis 
E. haysiana 
E. ollvula 
■- E. cylindracea 
г E. carino46A-2 
Xr E. carino49A-2 
•- E. carino49B-2 
>— E. cael Inf (2) 
E. fascin7-1 
E. fascin7-2 
— E. hydei 
г P. pras12A-1 
P. pras12B-1 
P. pras12B-2 
L. taen (2) 



С 



Ц. 



FIG. 3. A phylogram representing one of the 320 
most parsimonious trees obtained in the maximum 
parsimonious analysis of the mtDNA sequence 
data using equal weighting for all substitutions. Tree 
length = 207; consistency index = 0.619, excluding 
uninformative characters. Branch lengths reflect 
total number of substitutions. 



nious trees is shown in Figures 2 and 3, re- 
spectively Bootstrap values are reported for 
each node of the strict consensus tree. Nodes 
without nunnbers have bootstrap scores of 
50% or less. 



DISCUSSION 

The molecular phylogeny obtained here 
shows strong support for the monophyly of 
Elimia of the Mobile River basin. The basal- 
most species is Elimia hydei, which is sister to 
a large unresolved clade of remaining Elimia 
species. The unresolved polytomy is com- 
prised of four clades: (1) E. caelatura infús- 
cala; (2) E, fascinans; (3) E. crenatella + E. 
showaiteri; and (4) E. cannocostata + E. ger- 
hardtii + E. alabamensis + E. haysiana + E. 
olivula + E. cylindracea. There appears to be 
little congruence between the molecular phy- 
logeny and the current classification scheme 
of pleurocerids (Burch, 1980). For example, 



the E. olivula group (E. cylindracea, E. olivula, 
and E. showaiteri) and the E. haysiana group 
(E alabamensis and E haysiana) are para- 
phyletic. However, some of the most parsimo- 
nious trees depict E fascinans + E. caelatura 
infúscala of the E. vanuxemiana group as sis- 
ter taxa. Of course, it is worth noting that the 
classification scheme of pleurocerids is pre- 
Hennigian, and many of the groups are no 
doubt recognized by shared plesiomorphic 
characters. Within the largest of the four afore- 
mentioned clades, E cannocostata (speci- 
mens 46A-2, 49A-2, and 49B-2) is depicted 
as being sister to a clade that contains 
E. cylindracea + E. olivula + E. haysiana + 
E alabamensis + E gerhardtii + E. can- 
nocostata (specimens 5A-2, 11A-1, 11A-2, 
11B-1, 11 B-2 and 47A-1), rendering E ca- 
nnocostata paraphyletic. Elimia cylindracea is 
the next most-basal member of the largest of 
the four clades, and it is sister to the remain- 
ing Elimia species. The only resolved relation- 



16S rDNA GENE SEQUENCES IN ELIMIA 



191 



ships within this large clade are E. haysiana 
+ E. olivula and E. gerhardtii (10-1 + 42-1). 
The relationship of the remaining E. gerhardtii 
(10-2) remains uncertain. The phylogeny ob- 
tained in the present study is consistent with 
the findings presented by Lydeard et al. 
(1997). 

Of the five EHmia species that had more 
than one specimen sequenced, E. crenatella 
(identical sequences), E. caelatura infuscata 
(identical sequences), and E. fascinans are 
depicted as monophyletic. However, the 
monophyly of the aforementioned species 
should be tested with additional specimens 
and sequence data. Three EHmia gerhardtii 
specimens were sequenced from two sepa- 
rate locales. Interestingly, E. gerhardtii speci- 
mens from different locales are sister taxa, but 
this clade does not include the third E. ger- 
hardtii specimen. Eight E. carinocostata spec- 
imens were sequenced. The three most-basal 
E. carinocostata specimens (46A-2, 49A-2, 
49B-2) were collected in headwater streams 
of the Coosa River, whereas the remaining E. 
carinocostata specimens were obtained in 
sites located further downstream. 

Genetic differentiation among EHmia 
species was generally low. This was under- 
scored particularly in the E. carinocostata (in 
part) + E. gerhardtii + E. alabamensis + E. 
haysiana + E. olivula + E. cylindracea clade. 
Most species of this large clade differed by no 
more than 1 .8%. Confounding this problem, is 
the presence of intraspecific variation that is 
equal to or exceeds the amount of variation 
present among several of the species. The low 
genetic variation detected among the afore- 
mentioned species can be interpreted in two 
ways. First, they represent a single evolution- 
ary entity, and therefore should be syn- 
onymized or second, they represent valid 
species, but the gene is simply too conserva- 
tive to detect any significant differences. We 
recommend a more detailed investigation of 
the genus using other more potentially useful 
genetic markers before any formal taxonomic 
decisions be made. 

EHmia is the second most diverse genus of 
freshwater gastropods in North America. 
Burch (1988) lists 83 species within the 
genus, but this number is likely to change fol- 
lowing more detailed studies. Support for this 
claim comes from Hershler's (1994, 1998) re- 
view of the hydrobiid genus Pyrgulopsis, 
which is now considered the most diverse 
genus of North American gastropods. Prior to 
Hershler's reviews, EHmia would have ranked 



first, and the hydrobiid genus Somatogyrus 
would have ranked second, with 35 species 
(Burch, 1988). This dramatic increase in the 
number of hydrobiid species underscores the 
need for detailed monographic studies of all 
freshwater gastropods (Hershler, 1996). 

The 5'-half of the mitochondrial 16S rDNA 
gene seems to be of limited utility for assess- 
ing relationships among closely related EHmia 
species. Although it is evident that there is sig- 
nificant variation among more distantly re- 
lated EHmia, there were only 36 phylogeneti- 
cally informative sites, which is not very many 
when examining relationships among 20+ 
taxa. Despite exhibiting more variation than 
the 3'-half of the gene, most phylogenetic sig- 
nal seems to be partitioned among genera. 
Although we recognize that evolutionary rate 
differences exist among taxa, and that pilot 
studies should be carried out before under- 
taking any major sequencing project, the 5'- 
half of the ribosomal gene is likely to be of use 
to investigators interested in resolving rela- 
tionships among molluscan genera. 

ACKNOWLEDGMENTS 

We thank Rob Dillon, Jr., K. Roe, L. 
Thompson, R J. West, and an anonymous re- 
viewer for helping to improve the quality of the 
manuscript. This research was supported by a 
Research Grants Committee Award (2-67767) 
from the University of Alabama, a contract with 
the U.S. Department of the Interior (#1448- 
0004-95-938), and the National Science 
Foundation (DEB-9527758, DEB-9707623) 
to CL. John Yoder was a participant of a 
NSF Undergraduate Research Supplement 
Award. GenBank accession numbers for se- 
quences are U73761 to U73767, U73771 , and 
AF050037 to AF050053. Vouchers of speci- 
mens have been deposited at the Florida 
Museum of Natural History. 

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APPENDIX 

Elimia alabamensis - 

Coosa River just below Mitchell Dam, down- 
stream of fishing platform (east bank), 

Coosa Co., Alabama. 
Elimia caelatura infuscata-Kahatchee Creek, 

on Co. Rd. 008 (Childersburg Parkway), 

Talladega Co., Alabama. 
Elimia cahnocostata - 
(5A-2) Camp Branch Creek, 3 miles W of 

Shelby on Co. Rd. 42, Shelby Co., 

Alabama. 
(11A-1, 11A-2, 11B-1, 11B-2) Waxahatchee 

Creek, 2.7 miles W of Shelby, Shelby Co., 

Alabama. 



(46A-2) Beaver Creek, Co. Rd. 26 bridge, St. 

Clair Co., Alabama. 
(47A-2) Shoal Creek, at Co. Rd. 21 bridge, 4.7 

miles NW of Ragland, St. Clair Co., 

Alabama. 
(49A-2, 49B-2) Little Canoe Creek, Etowah 

Co. line on St. Clair Co. Rd. 44, St. Clair 

Co., Alabama. 
Elimia crenatella - 
(ere 18) Cheaha Creek at Co. Hwy 005, 5.1 

mile SSW of Eastaboga, Talladega Co., 

Alabama. 
(41 A2) Yellow Leaf Creek, 2 mile S of 

Westover on Co. rd. 51, Shelby Co., 

Alabama 
Elimia cylindracea - 
Noxubee River, 6 river miles above Alabama 

state line, Noxubee Co., Mississippi. 
Elimia fascinans - 
Shoal Creek, Pine Glen Recreation Area, 

Cleburne Co., Alabama. 
Elimia gerhardtii - 
(10-1, 10-2) Weogufka Creek, 0.8 miles NW 

of Moriah on Co., Rd. 15, Coosa Co., 

Alabama. 
(42-1 ) Cheaha Creek, Co. Hwy 005, Talladega 

Co., Alabama. 
Elimia haysiana - 
Coosa River, main channel about 2.6 miles 

downstream of Jordan Dam, 4.0 miles 

NW of Wetumpka, Elmore Co., Alabama. 
Elimia hydei - 
Locust Fork of Black Warrior River at Warrior, 

0.3 miles E of U.S. Hwy 31 on unnum- 
bered Co. rd., Jefferson Co., Alabama. 
Elimia olivula - 
Alabama River ca. 1.5 miles downstream of 

US Hwy 84, ca. 300 m upstream from 

grain elevator, Monroe Co., Alabama. 
Elimia showalteri - 
Cahaba River at Booth's Ford, 4.7 mile NW of 

Pea Ridge, Shelby Co., Alabama. 
Pleurocera prasinatum - 
Coosa River, public boat ramp, just S of hwy 

22 intersection with Coosa River S of 

Mitchell Dam, Chilton Co., Alabama. 
Leptoxis taenlata - 
Buxahatchee Creek, 5 miles ESE of Calera, 

Hiawatha Rd. off Co. Rd. 86, Shelby Co., 

Alabama. 



MALACOLOGIA, 1998, 39(1-2): 195-205 

MOLECULAR SYSTEMATICS OF THE FRESHWATER MUSSEL GENUS 
POTAMILUS (BIVALVIA: UNIONIDAE) 

Kevin J. Roe & Charles Lydeard 

Aquatic Biology Program, Department of Biological Sciences University of Alabama, Box 
870345, Tuscaloosa, AL 35487-0345, USA 

ABSTRACT 

Few explicit hypotheses for the relationships of unionid mussels exist. The absence of explicit 
phylogenetic hypotheses is problematic and is in part responsible for the lack of taxonomic sta- 
bility seen in this group. In this paper we examine the relationships of mussels in the genus 
Potamilus, based upon the DNA sequences of a 600 base pair portion of the first subunit of the 
mitochondrial cytochrome с oxidase (COI) gene. We also examine the genetic distinctiveness of 
populations of the inflated heelsplitter P. inflatus. The molecular phylogeny indicates that 
Potamilus is paraphyletic with Leptodea fragilis and Lampsilis ornata nested between P. capax 
and the remaining Potamilus species. With the exception of P. capax. the remaining Potamilus 
species are depicted as monophyletic and form three distinct clades: (1) a reciprocally mono- 
phyletic P. inflatus clade; (2) a P. ohlensis/P amphichaenus clade; and (3) a P. purpuratus/P. p. col- 
oradoensis/P alatus clade. While bootstrap values indicate a high degree of support for these 
three clades, relationships among these three clades are not as strongly supported. 

The genetic distinctiveness of two populations of the inflated heelsplitter exceeds that seen be- 
tween some other species in the genus. These populations represent geographically isolated, 
genetically distinct entities, and we therefore recommend the recognition of both the Amite and 
the Black Warrior populations of P. inflatus as separate species. 

Key words: Unionidae, Potamilus, cytochrome с oxidase subunit I. 



INTRODUCTION 

The freshwater mussel genus Potamilus 
Rafinesque, 1818 (Bivalvia: Unionidae), cur- 
rently contains six species: P. alatus (Say, 
1817), P amphichaenus (Frierson, 1898), P 
capax {Green, 1832), P inflatus {I.Lea, 1831), 
P. ohiensis (Rafinesque, 1820), and P. purpu- 
ratus (Lamarck, 1819) (Turgeon et al., 1988; 
Williams et al., 1992). In addition to these 
taxa, Simpson (1914) included P. {Lampsilis) 
coloradoensis (I. Lea, 1856), which is now 
generally considered a western form of Ppur- 
puratus. Potamilus is distributed in the St. 
Lawrence and Mississippi drainages and in 
Gulf drainages from Alabama to Texas 
(Valentine & Stansbery, 1971; Burch, 1975; 
Clarke, 1 981 ). The type species for the genus 
was designated as Unio alatus Say, 1817, by 
Morrison (1969). 

The genus Potamilus in its current form was 
first recognized as a natural assemblage of 
species by Frierson (1927) in the synonymous 
genus Proptera Rafinesque, 1819. Several re- 
searchers have proposed classifications that 
render the genus paraphyletic (Simpson, 



1914; Hoggarth, 1988; Burch, 1975) and have 
placed mussels currently assigned to Pot- 
amilus in the genus Lampsilis {P. capax) 
(Simpson, 1914), the genus Leptodea {P. lae- 
vissima [= ohiensis], P. amphichaenus) 
(Burch, 1975) or the resurrected genus Las- 
tena (Hoggarth, 1988). Whereas Potamilus is 
generally perceived as a natural group by 
freshwater malacologists, it has not yet 
achieved taxonomic and nomenclatura! stabil- 
ity, as evidenced by the continual change in 
generic assignments over the last 170 years. 
Even after successful petitioning by Bogan et 
al. (1990) of the International Commission on 
Zoological Nomenclature for the retention of 
Potamilus (BZN, 1992), Proptera, a junior 
synonym of Potamilus, appears in publica- 
tions as late as 1993 (e.g., McMahon, 1993). 
While many descriptions of the genus include 
the presence of a posterior wing as diagnos- 
tic, this character alone does not discriminate 
members of Potamilus from their putative sis- 
ter genus Leptodea (Ortmann, 1912; Valen- 
tine & Stansbery, 1971). Ortmann's (1912) 
statement that "this genus {Potamilus) stands 
in all characters except the glochidia, by that 



195 



196 



ROE & LYDEARD 



of Paraptera [= Leptodea]" supports the simi- 
larity of tliese two genera. Valentine & 
Stansbery (1971) stated that the only unique 
feature that defines Potamilus is the posses- 
sion of axe-head shaped or ligulate glochid- 
ium (Fig. 1), and Utterback (1915) noted that 
with the exception of the unique glochidia and 
the more developed hinge, "this genus 
(Potamilus) stands with Lasmonos [= Lep- 
todea]" A phonetic analysis by Hoggarth 
(1988) of the utility of glochidia morphology 
for deducing the relationships among North 
American freshwater mussels indicated that 
Potamilus is not a monophyletic group and 
that P. ohiensis and P. amphichaenus are 
more closely related to mussels in the genus 
Leptodea than to other members of Po- 
tamilus. Hoggarth's analysis indicated two 
distinct groups of mussels within Potamilus: 
those with lateral hooks on the ventral valve 
edges [alatus, capax, purpuratus) and those 
without such hooks {ohiensis, amphichaenus. 
inflatus). He concluded that the glochidia bore 
only a superficial resemblance to each other, 
and implied that the axe-head shaped 
glochidia were not homologs. 

The historic lack of taxonomic stability of 
Potamilus reflects the fact that no detailed or 
comprehensive cladistically based study has 
been conducted on this genus. Despite in- 
creasing interest in freshwater mussels, only 
a few cladistically based analyses have been 
published to date (Hoeh, 1990; Hoeh et al., 
1996; Lydeard et al., 1996; Mulvey et al., 
1 997). The primary objective of this study is to 
test the monophyly of Potamilus using a mol- 
ecular data set composed of the DNA se- 
quences of a portion of the first subunit of the 
mitochondrial cytochrome с oxidase (COI) 
gene, and develop hypotheses for relation- 
ships within the genus. 

Additionally, we wish to examine the level of 
intraspecific genetic variation in the inflated 
heelsplitter, P. inflatus. Potamilus inflatus was 
known from the Amite and Tangipahoa rivers 
in Louisiana, the Pearl River in Mississippi, 
and the Black Warrior, Coosa, and Tombigbee 
rivers in Alabama. Presently, it is limited to the 
lower and middle reaches of the Amite River, 
and a portion of the Black Warrior River. In 
1990, the U.S. Fish and Wildlife Service listed 
P. inflatus as a threatened species, because of 
its diminished range and potential threats to 
its continued survival in those rivers where it 
still occurs (USFWS, 1992). Knowledge of 
how genetic variation is partitioned in P. infla- 



tus will aid in making management decisions 
concerning this species. 



MATERIALS AND METHODS 

Twenty-four specimens representing ten 
species and five genera were included in the 
analysis (Table 1 ). Genomic DNA was isolated 
from fresh frozen or ethanol preserved tissues 
using the OlAamp Tissue Kit (QIAGEN 
#29304) following manufacturers instructions. 
Care was taken to use only somatic tissues as 
unionid mussels exhibit bi-parental inheri- 
tance of mitochondria (Hoeh et al., 1 996; Liu et 
al., 1996b). Double-stranded and single- 
stranded DNA was generated via the poly- 
merase chain reaction (PCR) using the 
primers LCO1490 and HC02198 (Folmer 
et al., 1994). Approximately 100 ng of geno- 
mic DNA provided the template for double 
stranded reactions performed in a 25 ul solu- 
tion containing each dNTP at 0.1 mM, each 
primer at 1 .0 uM, 40 mM MgCl2, 2.5ul 1 0X Taq 
buffer, and 0.6 units of AmpliTaq polymerase. 
Reactions were amplified for 32 cycles at 94° 
for 40 sec, 55° for 60 sec, and 72° for 90 sec. 
The amplified DNA was gel purified and then 
used as template for single-stranded amplifi- 
cation (Gyllensten & Eriich, 1988) using the 
same conditions and primer pair, with the H- 
primer used in limited quantity. Single stranded 
DNA was concentrated on Millipore Ultrafree 
MC filters, and sequenced using the Se- 
C|uenase version 2.0 kit (U.S. Biochemical) and 
*S-labeled dATP following the manufacturers 
instructions. The heavy strand was sequenced 
using overlapping primers: HC02198 (5'- 
taaacttcagggtgaccaaaaaatca-3'), UNICOIH 
(5'-tcagcaaccaacccaggag-3'), and HUNI- 
COIC (5'-aacaacactctctaccaaag-3'). 

DNA sequences were visualized via autora- 
diography, and aligned by eye using the soft- 
ware package XESEE (Cabot & Beckenbach, 
1989). P-distances (uncorrected for multiple 
hits) and Kimura's "two parameter" distances 
(Kimura, 1 980) were calculated using the soft- 
ware package MEGA (Kumar et al., 1993). 
Prior to phylogenetic analysis, the DNA se- 
quences were examined for evidence of satu- 
ration by plotting the number of transversions 
and transitions at each codon position vs. p- 
distance. Trees were generated under maxi- 
mum parsimony using PAUP version 3.1.1 
(Swofford, 1993). Trees were rooted using 
Fusconaia cerina (Conrad, 1838) and 



MOLECULAR SYSTEMATICS OF POTAMILUS 



197 





В 



FIG. 1. (А) Glochidia of Potamilus purpuratus, 
showing the axe-head shape and lateral hooks. 
Redrawn from Surber (1915). (B) Glochidia of 
Lampsilis cardium for comparison. Redrawn from 
Surber (1912). Bar = 100 ¡.im. 



Obliquaria reflexa (Rafinesque, 1820). 
Bootstrapping (Felsentein, 1985) was em- 
ployed to measure the internal stability of the 
topologies generated using 200 iterations. 
Skewness of tree-length distributions as a 
measure of phylogenetic signal (Hillis & 
Huelsenbeck, 1992) was estimated by gener- 
ating 10,000 random trees. 



RESULTS 

Sequence Variation 

DNA sequencing procedures yielded -600 
base pairs of CO! sequence for 24 taxa for a 
total of 14,400 nucleotides (Genbank acces- 
sion numbers AFO 49499-AFO 49522). Pre- 
liminary analysis of the sequence data re- 
vealed 182 variable sites, 151 of which were 
phylogenetically informative. Of those sites 
that were phylogenetically informative 16 
were at the first position, 10 were at the sec- 
ond position, and 125 were at the third. 
Translation of codons into amino acids indi- 
cates 23 variable residues. Pairwise percent 
sequence differences corrected for multiple 
hits using the "two parameter" model (Kimura, 
1980) ranged from to 2.6% for intraspecific 
comparisons. Values for interspecific compar- 
isons within Potamilus were between 1.2% 



and 14.5%. PainA/ise comparisons for all taxa 
are presented in Table 2. 

Scatterplots of pairwise genetic sequence 
differences versus the absolute number of 
transitions and transversions are presented 
for each codon position in Figure 2. Trends re- 
vealed by the scatterplots are typical for those 
seen in other protein coding genes (Roe et al., 
1997a; Lydeard & Roe, 1997), transversions 
were relatively rare at first and second posi- 
tions, not exceeding four and two substitu- 
tions respectively for any comparison. Trans- 
versions were considerably more common at 
the third codon position. A slight decrease in 
the number of transitions relative to the num- 
ber of transversions at the third position pro- 
vides evidence that some saturation is pre- 
sent. Saturation has the potential to affect 
phylogenetic analyses, therefore differential 
weighting of substitutions in the third codon 
position was employed. 



Phylogenetic Analyses 

Based on the analysis of nucleotide substi- 
tution patterns, phylogenetic analyses were 
performed under maximum parsimony using 
equal weighting and weighting transversions 
2x transitions at the third codon position. 
The g^ values (-0.362894, -0.625367) for 
weighted and equal weight analyses indicate 
the presence of significant phylogenetic signal 
(p = 0.01). Parsimony analysis of the data 
using equal weighting of transitions and trans- 
versions resulted in five equally parsimonious 
trees (01 = 0.636, RC = 0.517, 352 steps), the 
strict consensus of which is presented in 
Figure 3. Analysis of the data weighting trans- 
versions 2x transitions resulted in two equally 
parsimonious trees, which are presented in 
Figure 4. With the exception of the equivocal 
placement of Pp. coloradoensis, the two trees 
from the weighted analysis represent a single 
topology, identical to two of the five trees from 
the equal weight analysis. Whereas differ- 
ences exist between the trees generated using 
transversion weighted and equal weighted 
parsimony analysis, all topologies depict 
Potamilus as paraphyletic. In addition, all 
topologies support the monophyly of all 
species with the exception of the P. purpuratus 
clade. All topologies also support the sister 
relationships of P. ohiensis and P. am- 
phichaenus, and the reciprocal monophyly of 
the Amite and Black Warrior populations of P. 



198 



ROE & LYDEARD 



TABLE 1. Localities and number of specimens included in this study. 



SPECIES 


# INDIVIDUALS 


Potamilus alatus^ 

P. alatus^ 

P. amphichaenus^ 


1 
1 
1 


P. amphichaenu^ 


1 


P. capax 


2 


P. ohiensis'^ 


1 


P. ohiensis^ 


1 


P. purpuratus^ 


2 


P. purpuratu^ 


1 


P. p. coloradoensis 


1 


P. in flatus 


4 


P. inflatus 


4 


Leptodea fragilis^ 


1 


L. fragilis^ 


1 


Lampsilis ornata 


1 


Obliquaria reflexa 


1 


Fusconaia cerina 


1 



LOCALITY 



Elk River, Limestone Co., AL, 29 September 1994. 
Clinch River, Hancock Co., TN., 12 August 1994 
B.A. Steinhagen Resevoir, Noches River Dr., Tyler 

Co.,TX., 28 January 1996. 
Sabine River, at US Highway 59, Panola Co., TX., 5 

July 1995. 
Iron Mines Ck., -1.25 mi.W. of AR. Highway. 140 

and Red Oak Baptist Church, Poinsett Co., AR., 

26 October 1994. 
St. Francis floodway, near Wittsburg, Cross Co., AR., 

16 July 1995. 
Lake Arrowhead, Little Wichita River, Red River Dr., 

Clay Co., TX., 12 July 1994. 
Cahaba River, below Cooper Island, Bibb Co., AL., 

15 September 1994. 
Cahaba River, - 1 mi. downstream of Hwy. 24, Bibb 

Co., AL., 30 June 1993. 
Twin Buttes Resevoir, Concho River Dr., Tom Green 

Co.,TX., 30 August 1993. 
Amite River, above Port Vincent, Baton Rouge Pa., 

LA., 3-4 August 1994. 
Black Warrior River, (river mile 327.3), Tuscaloosa 

Co., AL., 15 October 1994. 
Cahaba, River, above AL. Highway 58, Centreville, 

Bibb Co., AL., 14 November 1994. 
Elk River, upstream of AL Highway 127, Limestone 

Co., AL., 14 October 1996. 
Cahaba, River, above AL. Highway 58, Centreville, 

Bibb Co., AL., 14 November 1994. 
Cahaba, River, above AL. Highway 58, Centreville, 

Bibb Co., AL., 14 November 1994. 
Cahaba River, -1 mi. downstream of Hwy. 24, Bibb 

Co., AL., 30, June 1993. 



inflatus. Weaker support was found for some 
deeper nodes as evidenced by the low boot- 
strap values. 



DISCUSSION 
Phylogenetic Analysis 

The CO! data do not support the recogni- 
tion of Potamilus as a monophyletic group. 
Whereas the majority of the species of 
Potamilus form a natural assemblage, the 
placement of Lampsilis ornata and Leptodea 
fragilis nested between P. capax and the re- 
maining members of Potamilus renders the 
genus paraphyletic. The single morphological 
character that serves to unite members of 
Potamilus is the possession of axe-head 
shaped glochidia. Hoggarth (1988) suggested 
only a "superficial resemblance" between the 
glochidia of P. ampfiictiaenus, P. ohiensis and 
those of P. alatus, P. purpuratus and P. capax, 



and recommended that mussels with axe- 
head shaped glochidia possessing hooks 
{alatus, capax ano purpuratus) should remain 
in Potamilus, while those that lacked hooks 
{amphichaenus, inflatus and ohiensis) should 
be placed in the resurrected genus Lastena 
Rafinesque, 1820. Hoggarth had not exam- 
ined the glochidia of P. inflatus and placed it in 
Lastena on the basis of the morphology of 
adult shells. His phonetic analysis indicated 
that Lastena was more closely allied to 
Leptodea than to Potamilus. Within Lastena, 
Hoggarth placed P. ohiensis and P. am- 
phichaenus as sister to P. inflatus. However, 
recent examination of the glochidia of P. infla- 
tus revealed the presence of large supernu- 
merary hooks (Roe et al., 1997b). Based on 
Hoggarth's criteria, P. inflatus should have 
been placed in a group containing P. alatus, P. 
purpuratus and P. capax, all of which have 
glochidia that possess hooks. The molecular 
phylogeny (Fig. 4) agrees with the classifica- 
tion of Hoggarth (1988) in the recognition of P 



MOLECULAR SYSTEMATICS OF POTAMILUS 



199 



TABLE 2. Pairwise genetic distances based on Kimura's "two parameter" model. Values are percentages. 





R 


R 


P 


R 


R 


P 


P 


R 


R 


R 


R 


R 




inf.wl 


inf.w2 


inf.w3 


inf.w4 


inf.al 


inf.a2 


inf.aS 


int.a4 


purpi 


purp.2 


purp.c. 


alatusl 


p. inf.wl 




0.00 


0.00 


0.34 


2.46 


2.44 


2.62 


2.08 


9.68 


10.16 


10.55 


10.16 


p. inf.w2 






0.00 


0.34 


2.12 


2.29 


2.47 


1.93 


9.55 


9.51 


9.70 


9.88 


P inf.w3 








0.34 


2.29 


2.45 


2.62 


2.09 


9.49 


9.82 


10.39 


10.00 


R inf.w4 










2.46 


2.08 


2.26 


2.07 


9.26 


9.72 


10.10 


9.72 


R inf.al 












0.35 


0.35 


0.17 


9.36 


9.32 


9.49 


9.10 


R inf.a2 














0.17 


0.17 


9.13 


9.58 


10.17 


9.18 


R inf.a3 
















0.34 


9.49 


9.82 


10.19 


9.39 


R inf.a4 


















9.09 


9.53 


10.32 


9.34 


P. purp.1 




















0.00 


1.40 


1.22 


R purp.2 






















1.55 


1.38 


P purp. col. 
























1.20 


R alatusl 


























R alatus2 


























P. capaxi 


























R capax2 


























P. ohien.1 


























R ohien.2 


























P. amph.1 


























P amph.2 


























L. frag.1 


























L. frag. 2 


























L. ornata 


























0. refiexa 


























R cerina 




























R 


R 


R 


R 


R 


R 


R 


L. 


L. 


L. 


0. 


R 




alatus2 


capaxi 


capax2 


ohieni 


ohien.2 


amph.1 


amph2 


frag.1 


frag. 2 


ornata 


reflexa 


cerina 


R inf.wl 


10.11 


14.40 


14.48 


12.40 


13.02 


12.88 


12.80 


9.95 


9.61 


14.48 


16.43 


14.92 


R inf.w2 


9,83 


14.42 


14.48 


12.39 


13.00 


12.89 


12.81 


9.90 


9.55 


14.70 


16.69 


14.93 


P inf.w3 


9.92 


14.28 


14.31 


12.47 


13.09 


12.96 


12.88 


9.81 


9.46 


14.34 


16.53 


14.79 


R inf.w4 


9.66 


13.91 


14.02 


12.16 


12.76 


12.64 


12.57 


9.51 


9.18 


13.98 


16.15 


14.65 


R inf.al 


9.02 


14.24 


13.88 


11.13 


11.75 


11.59 


11.54 


9.09 


8.74 


12.95 


16.26 


14.95 


R inf.a2 


9.11 


14.25 


14.11 


11.20 


11.82 


11.66 


11.61 


8.98 


8.64 


12.98 


16.02 


15.17 


R inf.aS 


9.31 


14.08 


13.89 


11.02 


11.63 


11.47 


11.42 


9.40 


9.06 


12.82 


15.86 


15.45 


R inf.a4 


9.27 


13.99 


13.88 


10.98 


11.56 


11.42 


11.38 


9.13 


8.79 


12.94 


15.55 


15.11 


P. purp.1 


1.23 


13.79 


13.62 


10.18 


10.56 


11.64 


11.61 


7.16 


7.21 


11.74 


14.51 


14.37 


R purp.2 


1.40 


14.19 


13.88 


10.82 


11.20 


12.28 


12.24 


7.62 


7.67 


12.16 


14.91 


14.55 


R purp. с. 


1.22 


13.52 


13.18 


10.19 


10.56 


11.66 


11.61 


8.20 


8.25 


12.35 


16.84 


16.27 


R aiatüsl 


0.00 


13.12 


12.99 


9.98 


10.37 


11.27 


11.22 


7.24 


7,29 


11.33 


15.75 


14.79 


P alatüs2 




13.33 


13.16 


9.92 


10.32 


11.22 


11.18 


7.36 


7.41 


11.32 


16.03 


14,83 


P. capaxi 






0.00 


13.54 


14.15 


14.28 


14.17 


11.42 


11,29 


13.87 


16.77 


17,44 


R capax2 








13.40 


14.04 


14.14 


14.04 


11.30 


11.16 


13.98 


16.89 


17,57 


P. ofiien.1 










0.34 


4.68 


4.39 


9.76 


9.42 


14.49 


17.10 


16.96 


R ofiien.2 












5.24 


4.94 


10.34 


10.00 


14.66 


17.52 


17.59 


P. amph.1 














0.17 


10.78 


10.44 


16.65 


17.84 


17.37 


P ampli. 2 
















10.77 


10.43 


16.26 


17.63 


17.19 


L. frag. 1 


















1,03 


9.87 


13.41 


13.64 


L. frag. 2 




















9.33 


13.49 


12.67 


L. ornata 






















15.02 


16.62 


0. reflexa 
























13.44 


F. cerina 



























Note Taxen abbreviations: P inf.wl -4, Potamilus inflatus-B\ack Warrior River; R inf.al -4, Potamilus inflatus-Amite River; P. 
purp. 1-2, Potamilus purpuratus: P. purp, col., Potamilus purpuratus coloradoensis: P. alatusl -2, Potamilus alatus; P. 
capax1-2, Potamilus capax: P. chien. 1-2, Potamilus ohiensis: P. amph.1-2, Potamilus amphichaenus; L. frag. 1-2, Leptodea 
fragilis: L. ornata, Lampsilis ornata: O. reflexa, Obliquaria reflexa: F. cerina, Fusconaia cerina. 



amphichaenus. R inflatus, and R ohiensis as 
a natural group; however, it is not due to the 
shared absence of hooks. Clearly, given the 
homoplastic nature of hook development this 
character appears to be of limited phylogenet- 
ically utility. 



The phylogenetic position of P. capax is 
problematic. In analyses of the molecular 
data, P. capax is depicted as the most basal 
member of the in-group in the weighted analy- 
sis, and is the most basal or second most 
basal member in the equal weight analysis. 



OO eso QO 



(3D CD OS О О 



О 03 0.04 

P-d ¡Stance 



5 x 

4.5 ■• 
4 ■• 

3.5 ■■ 

> 

Ь 3 •■ 

« 2.5 ■■ 

"5 2 -■ 

1 5 ■• 

1 ■• 

0.5 ■• 

i- 



<nni> OO 



0.005 0.01 



02 
P-distance 



0.025 0.03 035 0.04 



60 ■ 


■ 










50 ■ 


■ 










40 ■ 


■ 








s ,Л * °<í'o 


> 










:.;.;^^^" ^5?, ^ 


И 30 - 


- 








o " . _ • • 


о 










* -fc« .11 


« 










• '.% .1* 


20 ■ 








o-° 


' -.--*^ 


10 ■ 


■ 


0° 


^° 






- 


Ú- 


-ss«H 


•M«. 


— 1 


1 1 1 1 1 1 



0.2 0.25 

P-distance 



FIG. 2. Scatter plots of number of nucleotide substitutions (transitions (TS) = open circles, transversion.s (TV) 
= filled circles) versus genetic difference (p-distance) at (A) first, (B) second and (C) third codon positions. 



MOLECULAR SYSTEMATICS OF POTAMILUS 



201 



О. reflexa 
P. inf.wl 
P. inf.w2 
P. inf.w3 
P. inf.w4 
P. inf.al 
P. inf.a4 
P inf.a2 
P inf.a3 
P. purp.1 
p. риф.2 
P. риф. col. 
p. alatusl 
P. alatus2 
p. ohien.1 
P. ohien.2 
P. amph.1 
P. amph.2 
L. frag.1 
L. frag.2 
P. capaxi 
P. capax2 
L. ornata 
F. cerina 



FIG. 3. Strict consensus tree for five equally parsi- 
monious cladograms based on maximum parsi- 
mony analysis using equal weighting of all substitu- 
tions. Numbers correspond to the percentage of 
bootstrap replicates where the clade was found 
(200 total replications). Only values greater than 
50% are shown. Taxon labels follow Table 2. 



The placement of P. capax outside the re- 
maining members of Potamilus indicates pos- 
sible affinities with other genera. Potamilus 
capax had been placed in Lampsilis by 
Simpson (1914) based on similarities of the 
adult shells, particularly L. satura (I. Lea, 
1852) (Valentine & Stansbery, 1971). Based 
on glochidia morphology, Coker & Surber 
(1911) indicated that capax was not a 
Lampsilis but a Potamilus. The molecular evi- 
dence presented here indicate no support for 
the placement of P. capax in Potamilus; for the 
present, we withhold a formal recommenda- 
tion concerning the generic affinity of P. capax 
until a more inclusive analysis can be per- 
formed, including the type species of both 
Leptodea and Lampsilis. 

Our analyses suggest that P. p. coloradoen- 
sis may represent a species distinct from P. 
purpuraíüs (Fig. 4B). Simpson (1914) also rec- 
ognized P. coloradoensis (I. Lea, 1856) as a 
distinct species, although he admitted he v\/as 
doubtful of its validity. The placement of the 
specimen referable to P. coloradoensis in our 



analysis is equivocal, either being sister to P. 
purpuratus or P. alatus. Examination of adult 
shells reveals differences in periostracum and 
nacre color between P. p. coloradoensis anä P. 
purpuratus shells from east of the Mississippi 
River. Specimens of Pa/afusare generally dis- 
tinguishable from those of P. purpuratus, but 
examination of the glochidia of representa- 
tives of these taxa reveals no detectable dif- 
ferences. Based upon genetic distances P. p. 
coloradoensis\s phenetically more similar to P. 
alatus (1.2%) than to P purpuratus (1.5%). 
Genetic distances between these taxa exceed 
the intraspecific variation observed in all other 
species included in the study, with the excep- 
tion of P. inflatus. Further research involving 
representatives of P. purpuratus and P. alatus 
from throughout their respective ranges is nec- 
essary to resolve the relationships of this 
clade. For the present, we recommend caution 
in treating P. p. coloradoensis and P. purpura- 
tus as the same evolutionary entity. 

Both P. ohiensis and P. amphichaenus were 
placed in the genus Leptodea by Burch 
(1 975), however no support for the sister rela- 
tionships of Leptodea and these taxa is found 
in this analysis. The molecular data do provide 
strong support for the sister relationships of P. 
ohiensis and P. amphichaenus, and indicate 
they represent distinct evolutionary entities, 
more closely related to other members of 
Potamilus than to L. fragilis. 

The paraphyletic nature of Potamilus raises 
questions about the monophyly of other 
closely related unionid genera, such as 
Leptodea. Leptodea contains three species: 
L. fragilis, L. ochracea and L. leptodon. Of 
these, L. ochracea was assigned to Lampsilis 
by several authors (Simpson, 1914; Johnson, 
1970; Burch, 1975) because of similarities in 
appearance of adult shells, particularly to 
Lampsilis cariosa. Morrison (1 975) placed it in 
Leptodea because it lacked the mantle flaps 
often seen in species of Lampsilis. Hoggarth 
(1988) found the glochidia of L. ochracea to 
be more similar to L. fragilis and recom- 
mended retaining it in Leptodea. The type 
species, Leptodea leptodon, was originally 
assigned to Leptodea by Rafinesque (1820). 
It was also placed in Lampsilis by Simpson 
(1914). This species has always been consid- 
ered rare (Oesch, 1984) and has become 
very difficult to find recently. Ultimately, any 
taxonomic revision of these taxa must include 
type species. Future phylogenetic analyses 
including these and other allied taxa are 
needed in order to more fully resolve relation- 
ships among these genera. 



202 



ROE & LYDEARD 




FIG. 4. (A, В). Two equally parsimonious cladograms based on maximum parsimony analysis weighting trans- 
versions 2x transitions at the third codon position. Numbers above the branches correspond to the percent- 
age of bootstrap replicates where the clade was found (200 total replications). Only values greater than 50% 
are shown. Boldface numbers below the branches correspond to the number of nucleotide substitutions at 
those nodes. Taxon labels follow Table 2. 



Conservation Genetics of Potamilus inflatus 

DNA sequence data have been used to 
clarify relationships both between and within 
species for a large variety of organisms from 
whales (Milinkovitch et al., 1993) to hermit 
crabs (Cunningham et al., 1992). However, 
very few intraspecific comparisons of DNA se- 
quences exist for studies involving unionids 
(Liu et al., 1996a; Mulvey et al., 1997). 

Intraspecific studies are necessary for wise 
management decisions concerning endan- 
gered and threatened species. Phylogenetic 
analysis of sequence data of the COI gene in- 
dicates that populations of Я inflatus from the 
Amite River, Louisiana, and the Black Warrior 
River, Alabama, are reciprocally monophyletic 
(Figs. 3, 4) and represent distinct evolutionary 
entities (Moritz, 1994; Mayden & Wood, 
1995). Genetic distances and the number of 
nucleotide substitutions that separate these 
two populations were compared with the num- 
ber of substitutions that separate well-estab- 
lished species. Examination of genetic dis- 



tances reveals that the two populations of P. 
inflatus are more distinct genetically than P. 
purpuratus is from P. alatus (Table 2). 
Examination of nucleotide substitution pat- 
terns reveals that a total of 12 diagnostic sub- 
stitutions separate the two populations of P. in- 
flatus, whereas P. alatus and P. purpuratus are 
separated from each other by eight substitu- 
tions. In another comparison of congenerics, 
P. ohiensis and P. amphichaenus are sepa- 
rated by 26 substitutions. 

Nucleotide substitutions are considered by 
some researchers to accumulate at a similar 
rate for closely related taxa (Wilson et al., 
1 987; Vigilant et al., 1 991 ; Wayne et al., 1 991 ; 
Li, 1993). If this is true for Potamilus, it would 
indicate a more distant divergence time for the 
two populations of P. inflatus than that for 
some conspecifics. Alternatively, the differ- 
ences observed could indicate an increased 
rate in nucleotide substitutions for the inflatus 
clade. In either case, based on these data, a 
strong argument can be made for the recogni- 
tion of the Black Warrior and Amite popula- 



MOLECULAR SYSTEMATICS OF POTAMILUS 



203 



tions of P. inflatus as distinct species. To date 
no conchological characters have been found 
that support the molecular data, and discrimi- 
nation between these two species is based 
solely upon DNA sequence data. The recog- 
nition of cryptic unionid species is not without 
precedent. Davis (1983) identified allozymic 
differences for two phenotypically similar 
species of Uniomerus. The degree of genetic 
differentiation observed between populations 
of P. inflatus was greater than that seen in a 
comparison of two other morphologically dis- 
tinct species of Potamilus and exceeded in- 
traspecific values for all other species. The 
current geographic isolation of these two pop- 
ulations can only lead to further genetic differ- 
entiation of these entities and has serious im- 
plications for any plans to reintroduce P. 
inflatus in areas where it once occurred. Other 
studies involving mitochondrial DNA variation 
in unionids have come to similar conclusions 
regarding the protection of genetically distinct 
forms. For example, in a study of the conser- 
vation genetics of two unionid genera, Mulvey 
et al. (1997) confirmed the distinctiveness of 
Amblema neislerii{\. Lea, 1858) and A. plicata 
(Say, 1817) using allozyme and DNA se- 
quence data. Mulvey et al. (1997) recom- 
mended additional protection for A. neislerii 
because of its restricted range and particular 
habitat requirements. In another study, Liu et 
al. (1996b) urged caution regarding any ef- 
forts aimed at re-establishing populations of 
the giant floater, Pyganodon grandis, in 
Colorado, because of observed mitochondrial 
DNA differentiation between different river 
drainages. Given the unique genetic status of 
the Amite and Black Warrior forms of P. infla- 
tus, we recommend that each should be man- 
aged as a distinct evolutionary entity. 

The utility of the COI gene for elucidating 
relationships at the species level in our study 
is based primarily on the relatively high num- 
ber of substitutions at the third codon position. 
The relative lack of support, as measured by 
bootstrapping, for deeper nodes in the phy- 
logeny is due in part to the smaller number of 
variable sites at the first and second positions. 
It is possible that sequencing a larger portion 
of the COI gene would result in higher support 
for these internal nodes. Lydeard & Roe 
(1997) found that the complete cytochrome b 
gene proved useful for diagnosing relation- 
ships of representative actinopterygian fishes, 
contrary to previous studies based on only a 
portion of the gene. These studies questioned 
the usefulness of this particular gene for re- 



solving deeper phylogenetic relationships 
(Hillis & Huelsenbeck, 1992; Graybeal, 1993), 
but merely lacked sufficient data to address 
the question at hand. 

Historically, much of the uncertainty sur- 
rounding the placement of particular unionid 
species in one genus or another can be at- 
tributed to the use of characters of unknown 
phylogenetic utility and the absence of any 
objective analysis. In the case of Potamilus, 
the phylogenetic analysis of an independent 
molecular data set indicates that such charac- 
ters as glochidia shape and spines on 
glochidia may be homoplastic and thus not 
useful in diagnosing natural groups of mus- 
sels. Further investigations involving Po- 
tamilus and other genera are warranted and 
should include morphological as well as mol- 
ecular characters. Davis (1983) recom- 
mended the use of multiple data sets for re- 
solving relationships between unionid taxa. 
The use of multiple data sets, such as mor- 
phological and molecular characters, both in- 
dependently and in a total evidence approach 
(Kluge, 1989) would provide a more accurate 
test of the phylogenetic utility of molecular and 
traditional morphological characters in an 
evolutionary context and provide much 
needed insight into the evolution of these 
traits. 

ACKNOWLEDGMENTS 

We wish to thank the following individuals 
for their assistance in procuring specimens: 
R. Howells, Texas Parks and Wildlife; J. Harris 
and R. Doster, Arkansas State Highway and 
Transportation Department, and R Hartfield, 
USFWS. A special thanks to R Hartfield for 
bringing the P. inflatus question to our atten- 
tion. This research was supported by the 
Conchologists of America, the Hawaiian 
Malacological Society (to K. J. R.) and USFW 
Contract #43910-5-0098, National Science 
Foundation DEB-9527758 and 9707623 (to 
С L.). We also thank David Neely for the 
glochidium illustration and A. M. Simons, A. 
Bogan, P. Harris and two anonymous review- 
ers for their comments on drafts of this manu- 
script. 

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MALACOLOGIA, 1998, 39(1-2): 207-213 

ALLOMETRIC GROWTH AND INSIGHT ON SEXUAL DIMORPHISM IN POMACEA 
CANALICULATA (GASTROPODA: AMPULLARIIDAE) 

Alejandra L. Estebenet 

Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 

670 - 8000, Bahía Blanca, Argentina 

ABSTRACT 

The shape changes associated with increase in size and sexual dimorphism in Pomacea 
canaliculata are described using bivariate and multivariate statistical analysis. Allometric growth 
was found in the studied population, the shell becoming relatively more globose and both aper- 
ture and operculum becoming rounder as shell height increases. Related to this ontogenetic 
change in shell shape is a relative increase in body dry weight. Adult snails show sexual dimor- 
phism, males having both aperture and operculum rounder than females. Because juvenile snails 
do not exhibit these differences in aperture form, shell dimorphism seems to be associated with 
sexual maturity; that is, it is possibly related to the development of the penial complex. In sum- 
mer, adult females of any given shell height weigh significantly more than males of equivalent 
size; this being possibly due to the remarkable development of the albumen gland in this period. 

Shell dry weight shows great variability in similarly sized snails, this fact being partially ascrib- 
able to the seasonal growth pattern of P. canaliculata in waters showing thermal seasonality. 

Key words; Sexual dimorphism, Pomacea, growth. 



INTRODUCTION 

Freshwater snails in the family Ampu- 
llariidae have a tropical and subtropical distri- 
bution. The literature describing shell shape in 
adult snails is extensive. Many species have 
been described according to external shell 
characters. However, shell shape changes as- 
sociated with increase in size have not been 
explored. Allometric relationships between 
some linear shell measurements and snail 
weight have been investigated for adult 
Pomacea canaliculata (Lamarck 1822) (Que- 
des et al., 1981; Cazzaniga, 1990), but the 
values of proportionality constants cannot be 
related to specific or relative growth unless we 
are dealing with ontogenetic data (Shea, 
1985). 

Because young P. canaliculata have no de- 
veloped sexual structures (penial complex 
and albumen gland) (Hylton Scott, 1957), 
their sex can be determined only by the mi- 
croscopical analysis of the gonad. Cazzaniga 
(1990) reported external differences between 
sexes in adult P. canaliculata; however, shell 



shape in undifferentiated juvenile snails was 
not analyzed. 

In this paper, ontogenetic changes in shell 
form and sexual dimorphism in P. canaliculata 
are analyzed using bivariate and multivariate 
statistics. 



MATERIALS AND METHODS 

Live P. canaliculata were collected from 
standing waters in an artificial pond at Paseo 
del Bosque, La Plata city, Argentina, in 
January and February 1 989. Their shells were 
cleaned of adhering matter (mostly algae), 
and they were then starved in aquaria with tap 
water for 18 h to empty their guts. The snails 
were killed by immersion into warm water (75 
°C) for 3-5 min. 

The sex of each snail was determined by 
the presence of the penial complex or the al- 
bumen gland; the snails were classed as un- 
differentiated (or juveniles) when these sexual 
structures could not be clearly identified. 

The shells, soft parts and opercula were 



207 



208 



ESTEBENET 



dried separately at 80 °C for 48 h and weighed 
on a scale to the nearest 0.1 mg. 

The following seven measurements were 
taken on each specimen: shell height (SH), 
spire height (SpH), operculum height (OH), 
aperture height (AH), shell width (SW), aper- 
ture width (AW) and operculum width (OW). 
All measurements were made along straight 
lines either parallel or perpendicular to the 
imaginary axis shown on Figure 1 . Shells <25 
mm high were measured using a stereoscopic 
microscope fitted with a camera lucida. Some 
shell reference points were projected on a 
graph paper ruled in squared millimeters. 
From lines that joined these points, I obtained 
the linear measurements for each snail to the 
nearest 0.5 mm. Shells >25 mm high were 
measured also from a plane projection but ob- 
tained from X-ray images. 

A total of 363 shells were measured (171 ju- 
veniles, 89 females, 103 males). Because 
some parts were spoiled while separating tis- 
sue from shell and operculum, only 226 snails 
were used to weight operculum and body (57 
juveniles, 80 females, 89 males) and 233 
snails to weigh shell (64 juveniles, 80 females, 
89 males). 

The power function y = ax'^ was used to de- 
scribe the relationships among the linear and 
weight measurements. Least squares regres- 
sion analyses were carried out for the whole 
population, and for males and females sepa- 
rately. The logarithm of SH was used as inde- 
pendent variable in all regressions. All the 
slopes of the regressions involving whole pop- 
ulation were compared with isometry (b = 1 for 
linear variables or b = 3 for weight variables) 
using t statistics (Sokal & Rohlf, 1979). 
Analysis of covariance was used to test the 
equality of regression coefficients and inter- 
cepts between sexes (BMDP1V, Statistical 
Software, UCLA, 1982). Residual analysis 
was performed in all cases to test the basic 
assumptions of the linear regression analysis 
and to assess the adequacy of the linear 
model. 

Stepwise discriminant function and cano- 
nical variate analysis were performed 
(BMDP7M, Statistical Software, UCLA, 1982) 
in order to reveal which variables contributed 
to discriminating the sexes. Only the linear, 
not weight, variables were used. Allometric 
growth exists in all characters measured (see 
results); all variables were therefore log-trans- 
formed before the analyses were carried out. 

Twenty eight newly hatched snails from a 



single egg-mass were reared in the labora- 
tory. They were placed individually in cylindri- 
cal plastic tubes submerged in an aquarium 
with aerated, warm (25 ± 1°C), tap water 
(hardness 90 to 96 ppm CaCOg). The snails 
were fed with lettuce, and the water was 
changed periodically at the time the linear 
shell dimensions were measured. When the 
snails were approximately 25 mm high, their 
sexes were determined (14 males and 14 fe- 
males). 



RESULTS 

Table 1 is a statistical summary of the 10 
measured variables. 

Analyses of residual plots showed no de- 
partures from the assumptions of the regres- 
sion model; the linear log-log regression ac- 
curately describes the relationships between 
the pairs of selected variables, the only ex- 
ception being log shell weight (SWt) - log shell 
height (SH) regression. 

Table 2 shows the different allomethc rela- 
tionships calculated for the whole population. 
The null hypothesis that b = 1 was rejected in 
all regressions involving linear variables. The 
ratios of shell width, aperture width, opercu- 
lum width and spire height to shell height 
(SW/SH, AW/SH, OW/SH and SpH/SH re- 
spectively) increase as the shell increases in 
size. The dry weight of the soft parts (BWt) 
and operculum (OWt) also increase relative to 
SH (the null hypothesis that b = 3 was re- 
jected). 

Table 3 shows the results of the ANCOVA 
on the two sexes of P. canaliculata. Except for 
log SW and log SpH, the slopes or intercepts 
of the regression lines were significantly dif- 
ferent between the sexes. The values of b for 
aperture and operculum variables were signif- 
icantly larger for males. The slopes of the log 
BWt-log SH and log OWt-log SH regressions 
did not differ between sexes, but the inter- 
cepts were larger for the females. 

The linear log SWt - log SH regression was 
not appropriate to describe the relationship 
between both variables, because the ratio of 
specific growth rates changes during the on- 
togeny. Residual plot showed that residual 
variance increases along the independent 
variable. 

In this study, the best discriminating model 
for separating sexes uses only three of the 
seven available measurements (SW, OH, 



SEXUAL DIMORPHISM IN POMACEA 



209 




FIG. 1 . Variables measured on the shell and the op- 
erculum of Pomacea canaliculata (SH; shell height, 
SpH: spire height, AH: aperture height, SW: shell 
width, AW: aperture width, OH: operculum height 
and OW: operculum width). 



OW). The other four variables were not in- 
cluded, because they contribute no additional 
dischnninating information. A discriminant 
score was computed for each snail by multi- 
plying each of three characters by the corre- 
sponding coefficient and adding together 
these products (Fig. 2). There was a signifi- 
cant difference between the mean discrimi- 
nant scores of the two sexes; that is, the func- 
tion will discriminate, significantly, between 
the two groups (Wilk's lambda = 0.534 or x^ = 
118, d.f = 3, p < 0.0001). The percentage of 
correctly allocated individuals by sex using 
this function was greater than 78% for the 
males and 85% for the females. The results of 
canonical variate analysis showed that OW 
had the greatest importance in separating 
sexes (Table 4). 



The growth rate (measured as increase in 
SH) was similar in males and females reared 
in the laboratory, at least until sexual maturity 
was reached (Fig. 3). The form of the aperture 
(AW/AH) was compared between sexes twice 
during growth. The first comparison was made 
when the snails were 7.42 ± 0.707 mm high 
(mean ± SD); there was no significant differ- 
ence (male mean ratio: 0.7605 ± 0.0255 SD, 
female mean ratio: 0.7636 ± 0.0269 SD; t = 
0.313, p > 0.10, d.f. = 26). However, when the 
snails were 23.51 ± 2.996 mm high (mean ± 
SD), the aperture was significantly rounder in 
males than in females (male mean ratio: 
0.8042 ± 0.0302 SD; female mean ratio: 
0.7698 ± 0.0427 SD; t = 2.461 , p < 0.05, d.f. = 
26). 



DISCUSSION 

Allometric growth occurs in Pomacea 
canaliculata, the shell becoming relatively 
more globose, and both aperture and opercu- 
lum becoming rounder as the shell increases 
in size. Related to this ontogenetic change in 
shell shape, there is a relative increase in the 
dry weight of the soft parts. The positive al- 
lometry of AW and SW relative to SH may be 
related to a positive allometric growth of the 
foot. 

Though SpH shows positive allometric 
growth with respect to SH, it is a widely vari- 
able character within populations. The shell of 
P. canaliculata usually has a short spire. 
However, in our material, snails range from 
those whose shell apex does not exceed the 
plane that delimits the superior edge of the 
last whorl, to snails with a much higher spire. 
This variation has been noted by earlier au- 
thors (d'Orbigny, 1847; Barattini, 1939; Hylton 
Scott, 1957) and prevents SpH be used as a 
diagnostic character. 

Adults of P. canaliculata from Paseo del 
Bosque show sexual dimorphism, as shown 
by Gazzaniga (1 990) for another population of 
the same species. The external differences 
between males and females are manifested 
by both the form of the aperture and the oper- 
culum. The AW/SH ratio increases with in- 
crease in size. However, in females AW in- 
creases less rapidly than in males. Another 
ampullariid, Marisa cornuarietis (L.), also ex- 
hibits positive allometric growth of AW 
(Demian & Ibrahim, 1972), and the differ- 



210 



ESTEBENET 



TABLE 1. Statistic summary of the variables measured on Pomacea canaliculata. Linear measurements are 
in mm; weights are in mg. 



Variable 


Mean 


SD 


Minimum 


Maximum 


N 


Shell height 


35.21 


15.61 


8.84 


80.00 


363 


Shell width 


29.38 


13.65 


7.12 


70.50 


363 


Spire height 


4.10 


2.10 


0.88 


10.00 


363 


Aperture height 


24.69 


10.45 


6.28 


54.00 


363 


Aperture width 


19.01 


8.79 


4.80 


42.50 


363 


Operculum height 


21.88 


9.53 


5.64 


49.00 


363 


Operculum width 


15.03 


6.95 


3.64 


33.50 


363 


Body weight 


1480 


1250 


110 


6130 


226 


Shell weight 


3865 


2795 


253.8 


14946 


233 


Operculum weight 


90 


70 


6 


370 


226 



TABLE 2. Values for different linear regressions for a population of Pomacea canaliculata. with tests of sig- 
nificance of deviation from the values of isometry (values of test t) 









Isometry t 








Regression 


N 


b±SE 


b = 1 


P 


a 


r2 


log SW/log SH 


363 


1.042 ±0.005 


8.13 


<0.0001 


-0.147 


0.99 


log SpH/log SH 


363 


1.070 ±0.020 


3.47 


<0.001 


-1.053 


0.88 


log AH/log SH 


363 


0.945 ± 0.004 


12.08 


<0.0001 


-0.067 


0.99 


log AW/log SH 


363 


1.025 ±0.006 


4.17 


<0.0001 


-0.308 


0.99 


log OH/log SH 


363 


0.967 ± 0.005 


5.61 


<0.0001 


-0.156 


0.98 


log OW/log SH 


363 


1.015 ±0.006 


2.5 

b = 3 

7.36 


<0.02 


-0.395 


0.97 


log BWt/log SH 


226 


3.456 ± 0.062 


<0.0001 


-2.590 


0.93 


log OWt/log SH 


226 


3.253 ± 0.068 


3.68 


<0.001 


-3.470 


0.91 



TABLE 3. Values for different linear regressions in males and females Pomacea canaliculata. 



Regression 


Sex 


N 


b±SE 


a 


R2 


F** 


log SW/log SH 


females 


89 


1.020 ±0.021 


-0.104 


0.96 


FbNS 




males 


103 


0.984 ± 0.022 


-0.051 


0.95 


FaNS 


log SpH/log SH 


females 


89 


1.084 ±0.103 


-1.083 


0.56 


FbNS 




males 


103 


0.987 ±0.103 


-0.912 


0.48 


FaNS 


log AH/log SH 


females 


89 


0.886 ±0.019 


-0.032 


0.96 


Fb 13.70** 




males 


103 


0.983 ±0.018 


-0.127 


0.97 




log AW/log SH 


females 


89 


1.048 ±0,021 


-0.353 


0.96 


Fb 3.69* 




males 


103 


1.114±0.027 


-0.445 


0.94 




log OH/log SH 


females 


89 


0.924 ± 0.023 


-0.083 


0.95 


Fb 5.44* 




males 


103 


1.003 ±0.024 


-0.209 


0.94 




log OW/log SH 


females 


89 


0.975 ± 0.030 


-0.342 


0.92 


Fb8.51** 




males 


103 


1.123 ±0.040 


-0.545 


0.88 




log BWt/log SH 


females 


80 


3.229 ±0.154 


-2.145 


0.86 


FbNS 




males 


89 


3.081 ±0.112 


-2.015 


0.90 






females 




common slope 


-2.034 




Fg 59.63** 




males 




3.164 ±0.086 


-2.151 






log OWt/log SH 


females 


80 


3.076 ±0.149 


-3.230 


0.85 


FbNS 




males 


89 


3.365 ±0.172 


-3.590 


0.82 






females 




common slope 


-3.326 




F^ 34.77** 




males 




3.204 ±0.1 13 


-3.443 







#F test for null hypothesis that b^^^^^ = b,eniales ^ъ °^ a^g.^s = a,en,ales Fa 
NS: not significant (p > 0.05) *p < 0.05 **p < 0.01 



SEXUAL DIMORPHISM IN POMACEA 



211 



3.562 - 


М= 


=103 ■ 


1 


■ 


Males 
Females 


2.149 - 






0.736 - 












1 












1 


676- 








1 






1 






1 1 












1 




1 


2 089 - 


1 


1 


F=89 

1 




1 


3.620 - 


1 



15 



10 



5 5 

RELATIVE FREQUENCY (%) 



10 



15 



FIG. 2. Frequency histograms of discriminant scores (DS) for male and female Pomacea canaliculata. DS 
-35.42(logOW) + 22.91 (logOH) + 19.31(logSW) - 18.07 



enees between the sexes in this species are 
greater than in P. canaliculata. 

Multivariate analysis showed OW is a better 
discriminant between sexes than AW, be- 
cause OW grows with positive allometry re- 
spective to SH in males, whereas in the fe- 
males the growth is isometric. 

In P. canaliculata, the penial complex, which 
arises as a ventral outgrowth from the mantle 
edge, appears to develop at the same rate in 
both sexes until the gonad becomes active, 
when its growth is arrested in females. This 
occurs when the females are 8 mm in diame- 
ter (Andrews, 1964). Around this point, the 
aperture form is still similar between sexes. 
The sexual dimorphism in the aperture form 
seem therefore to be associated with the sex- 
ual maturity, possibly with the posterior devel- 
opment of the penial sheath in the males. 

About 50% of the males of P. canaliculata 
greater than 40 mm in SH show a slight re- 



TABLE 4. Results of canonical varíate analysis for 
shape differences between sexes. 



Standardized coefficients 


logSW 




1.487 


logOW 




-3.655 


log OH 




2.188 


Eigenvalue 




0.871 


Canonical correlation 


0.682 



flection of the free edge of the peristomal lip. 
This feature was only present in three of the 
89 females analyzed. This other differential 
shell feature was present in some males of M. 
cornuarletis (Demian & Ibrahim, 1972). 

Sexually dimorphic growth and/or survivor- 
ship patterns resulting in sexually dimorphic 
sizes are known for many freshwater proso- 
branchs (Browne, 1978; Aldrige, 1982; 



212 



ESTEBENET 




-о Females 
-"- Males 



60 
DAYS 



FIG. 3. Pattern of growth of Pomacea canaliculata 
reared under laboratory conditions (mean ± 95% 
confidence interval). 



A linear model did not accurately describe 
the SWt-SH relationship. Great variability in 
SWt exists among similar sized snails that can 
be partly ascribed to seasonal pattern of 
growth showed by P. canaliculata in waters 
with marked thermal seasonality (Estebenet & 
Cazzaniga, 1992). Snails hatching at the be- 
ginning of the breeding season grow rapidly; 
they generally have a thin shell and are often 
similar sized as snails hatched at the end of the 
previous breeding season, the later generally 
having a heavier shell. Another source of vari- 
ation in the SWt could be the sex of the snails, 
because Cazzaniga (1990) determined that 
shells of P. canaliculata males are significantly 
heavier than the shells of females. 



ACKNOWLEDGMENTS 



Jokinen, 1982; Jokinen et al., 1982; Brown & 
Richardson, 1992). Females reach sizes 
greater than males in several species of 
Ampullariidae: Pila spp. (Keawjam, 1987), 
Pomacea urceus (Müller) (Lum Kong & Kenny, 
1989) and Marisa cornuarletis (Demian & 
Ibrahim, 1972). Burky (1974) reported that P 
urceus males can attain a maximal size simi- 
lar to that of females, but usually the proportion 
of males in the largest size classes is less than 
those of females. In my material, the maximal 
size attained by one male was 62.5 mm (SH) 
whereas around 12% of the females were 
larger, up to 80 mm. This size difference could 
be attributed to a sexually dimorphic growth 
pattern (Estebenet & Cazzaniga, 1997). 

Females of any given size weigh signifi- 
cantly more than males. Because there was 
not marked shell form differences between 
sexes, this could be attributed to the sampling 
date (summer) that coincided with reproduc- 
tive season (from late spring to late summer). 
The albumen gland grows up to represent 
68% of the dry body mass in a reproductive 
active female, this being why Guedes et al. 

(1981) discarded reproductive active females 
to obtain reliable regressions for biomass es- 
timation in P. canaliculata. Bourne & Berlin 

(1982) determined a similar weight difference 
between sexes of Pomacea dolioides 
(Reeve). It is therefore probable that the re- 
gressions involving soft parts weight are sea- 
sonally variable. This fact could be extended 
to other temperate ampullariid populations 
with seasonal reproductive patterns. 



This work was funded with grants by CON- 
ICET (Consejo Nacional de Investigaciones 
Científicas y Técnicas, Argentina: P.I.D. # 
3368-80092), CIC (Comisión de Investiga- 
ciones Científicas de la Provincia de Buenos 
Aires, Argentina), and UNS (Universidad 
Nacional del Sur). 

I am grateful to Dr. Néstor J. Cazzaniga for 
his critical reading of the manuscript and en- 
couragement and to Lie. Pablo R. Martin for 
his valuable comments along the course of 
this work. 



LITERATURE CITED 

ALDRIDGE, D.W., 1982, Reproductive tactics in re- 
lation to life-cycle bioenergetics in three natural 
populations of the freshwater snail, Leptoxis ca- 
rinata. Ecology, 63: 196-208. 

ANDREWS, E. В., 1964, The functional anatomy 
and histology of the reproductive system of some 
pilid gastropod molluscs. Proceedings of the 
Malacological Society of London, 36: 121-1 40. 

BARATTINI, L. R, 1939, Alteraciones con cierto 
carácter constante observadas en algunas es- 
pecies uruguayas. Revista Chilena de Historia 
Natural, 43: 47. 

BOURNE, G. R. & J. A. BERLIN, 1982, Predicting 
Pomacea dolioides (Reeve) (Prosobranchia: 
Ampullariidae) weights from linear measure- 
ments of their shells. The Veliger 24: 367-370. 

BROWN, K. M. & T D. RICHARDSON, 1992, 
Phenotypic plasticity in the life-histories and pro- 
duction of two warm-temperate viviparid proso- 
branchs. The Veliger 35: 1-11. 

BROWNE, R. A., 1978, Growth, mortality, fecundity, 
biomass and productivity of four lake populations 



SEXUAL DIMORPHISM IN POMACEA 



213 



of the prosobranch snail, Viviparus georgianas. 
Ecology, 59: 742-750. 

BURKY, A., 1974, Growth and biomass production 
of an amphibious snail, Pomacea urceus (Müller), 
from the Venezuelan Savannah. Proceedings of 
the Malacological Society of London, 41: 
127-143. 

CAZZANIGA, N. J., 1990, Sexual dimorphism in 
Pomacea canaliculata (Gastropoda: Ampulla- 
riidae). Thie Veligen 33: 390-394. 

DEMIAN, E. S. & A. M. IBRAHIM, 1972, Sexual di- 
morphism and sex ratio in the snail Marisa cor- 
nuarietls (L.). Bulletin of tfie Zoological Society of 
Egypt. 24: 52-63. 

D'ORBIGNY, A. D., 1847, Voyage dans lAmérique 
Méridionale. 5. Mollusques. Paris. 

ESTEBENET, A. L. & N. J. CAZZANIGA, 1992, 
Growth and demography of Pomacea canalicu- 
lata (Gastropoda: Ampullariidae) under labora- 
tory conditions. Malacological Review, 25: 1-12. 

ESTEBENET, A. L. & N. J. CAZZANIGA, 1997, Sex- 
related differential growth in Pomacea canalicu- 
lata (Gastropoda: Ampullariidae). Journal of 
Molluscan Studies, 63(4): 292-297. 

GUEDES, L. M., A. M. FIORI & С О. DIEFENBACH, 
1981, Biomass estimation from weight and linear 
parameters in the apple snail Ampullaria canali- 
culata (Gastropoda: Prosobranchia). Compara- 
tive Biochemistry and Physiology 68(A): 285- 
288. 



HYLTON SCOTT, I., 1957, Estudio morfológico у 
taxonómico de los Ampulláridos de la República 
Argentina. ¡Revista del Museo Argentino de 
Ciencias Naturales "Bernardino Rivadavia", 
(Serie Zoología) 3: 233-333. 

JOKINEN, E. H., 1982, Cipangopaludina chinensis 
(Gastropoda: Viviparidae) in North America, re- 
view and update. The Nautilus, 96: 89-95. 

JOKINEN, E. H., J. GUERETTE & R. W. KORT- 
MANN. 1982, The natural history of an ovovivipa- 
rous snail, Viviparus georgianus (Lea), in a soft- 
water eutrophic lake. Freshwater Invertebrate 
Biology 5:2-17. 

KEAWJAM, R. S., 1987, The apple snails of 
Thailand: Aspects of comparative anatomy. 
Malacological Review, 20: 69-90. 

LUM KONG, A. & J. S. KENNY 1989, The repro- 
ductive biology of the ampullariid snail Pomacea 
urceus (Müller). Journal of Molluscan Studies, 55: 
53-66. 

SHEA, B.T, 1985, Bivariate and multivariate growth 
allometry: statistical and biological considera- 
tions. Journal of Zoology 206: 367-390. 

SOKAL, R. R. & F. J. ROHLF 1979, Biometn'a. 
Omega Ed. 832 pp. 

Revised ms. accepted 1 April 1997 



MALACOLOGIA, 1998, 39(1-2): 215-219 



LETTERS TO THE EDITOR 



TRANSLATING TREES INTO TAXONOMY WITHIN VENERIDAE (BIVALVIA): 
A CRITIQUE OF TWO RECENT PAPERS 

Mary Ellen Harte 
1180 Cragmont Ave., Berkeley, California 94708, USA 



Cladistic analysis has been developed over 
the past two decades into a scientifically rig- 
orous nnethod for determining probable evo- 
lutionary relationships among taxa, and it is 
increasingly used throughout all biological dis- 
ciplines concerned with evolution. In each 
case, the end result is a rooted or unrooted 
tree of taxa — commonly referred to as a dado- 
gram — from which phylogenetic inferences 
are made. At the same time, various phenetic 
algorithms have been developed to create 
rooted or unrooted trees of taxa based on bio- 
molecular data (e.g., Neighbor-Joining; Saitou 
& Nei, 1 987), and these have been used to val- 
idate or refute existing taxonomies of organ- 
isms (e.g., Sibley & Ahlquist, 1990; Albert et 
al., 1992; Simon et al., 1993; Wainright et al., 
1993;HalnychetaL, 1995). 

Two recent papers, Roopnarine (1996) in 
Malacologia and Canapa et al. (1996) pub- 
lished elsewhere, utilize such algorithms to 
study the bivalve family Veneridae and attempt 
to apply the resulting inferences to current tax- 
onomy. An examination of these papers indi- 
cates some taxonomic and methodological 
misapplications to an already taxonomically 
controversial group. 



A STUDY OF THE CHIONINAE 

Roopnarine (1996) focuses on the venerid 
subfamily Chioninae, which represents a pro- 
lific evolutionary radiation centered in North 
America, judging by the number of current 
and extinct taxa. He collected data on 25 con- 
chological characters from six extinct and ten 
Recent American chionine species, most re- 
cently classified into four genera and 1 1 sub- 
genera (Keen, 1969; Fischer-Piette & Vuka- 
dinovic, 1977). Two of the four genera are 



represented by their type species; nine of the 
1 1 subgenera are represented by their type 
species. 

Character Definitions 

Of the 25 characters, inadequately defined 
character states are present among the fol- 
lowing seven: palliai sinus depth, spacing of 
commarginal sculptural elements, anterior de- 
velopment of commarginal elements, ventral 
margin crenulation, width of anterior cardinal 
tooth, posterior cardinal tooth shape, and 
width of central cardinal tooth. For example, 
the character of palliai sinus depth is defined 
as greatly reduced to absent for one state, 
and present but short for another. It is not 
clear where the demarcation is between these 
two states. Similarly, I disagree with the as- 
signment of a character state in one of the 
taxa for spacing of commarginal elements, but 
the ambiguous definition of states ("close, 
widely spaced, widely spaced but narrowing 
later") allows independent workers to reach 
different conclusions. Such ambiguities exist 
in the other six characters, allowing for a sig- 
nificant amount of subjectivity in the analysis, 
and preventing other workers from replicating 
the results. 

A second source of ambiguity arises be- 
cause Roopnarine fails to define what per- 
centage of examined specimens must have a 
character state in order for it to be considered 
present in the species. For example, in defin- 
ing types of sculpture present, he asserts that 
"radiais are absent in Mercenaria" (p. 140; 
presumably M. mercenaria, the only species 
included in his study), countering the obser- 
vations of previous workers (Jones, 1979; 
Harte, 1992), who note the characteristic 
presence of radial sculpture in that species. 



215 



216 



HARTE 



Representation of Genera 

Inadequate representation of some of the 
genera weakens his analysis. The taxonomic 
placement of Chione (Puberella) based on the 
consensus tree is problematic, but its very 
presence is weakened because it was not 
represented by its type species. Two other 
taxa in the analysis, Protothaca (Leukoma) 
and Timoclea (Glycydonta), are parts of much 
larger groups that would require more analy- 
sis of related taxa to adequately assess their 
taxonomic status. If Roopnarine represented 
all of the Chione subgenera solely by their 
subgeneric names on his cladograms, how- 
ever, he should have treated Leukoma and 
Glycydonta in the same manner. Using their 
generic classifications implies relationships 
that might not be taxonomically valid to those 
much larger groups. To justify inclusion of 
Protothaca and Timoclea in the cladograms, 
type species of the taxa should have been 
used. 

Not using type species of taxa could possi- 
bly influence the results. For example, in his 
resulting consensus tree, Roopnarine (1996: 
fig. 20) shows Timoclea grouped with Proto- 
thaca in a clade separate from that of Chione 
s.S. In the study, the species representing 
Protothaca and Timoclea were P. {Leukoma) 
asperrima (Sowerby) of the east Pacific, and 
T. {Glycydonta) marica (Linnaeus), of the west 
Pacific, respectively. The type species of 
Timoclea s.S., Timoclea ovata (Pennant), an 
eastern Atlantic species, has different charac- 
ter states for at least some of the characters. 
Whether these different character states 
might make it more closely related to the type 
species of Chione s.S., Chione cancellata 
(Linnaeus), a western Atlantic species, than 
to P. asperrima, remains untested. 

Roopnarine said that specimens of some of 
the type species were not available to him, but 
these species are not rare. For example, 
Timoclea ovata is present in the UCMP col- 
lection, one of several he used in his study, 
and availability of specimens is adequate 
through the current museum loan system of 
the major collections in North America. 



translation into the taxonomy? Here, I believe 
he is only partly successful. 

Reducing taxonomic ambiguity is a major 
consideration for creating hierarchical, supra- 
specific taxa. Thus, the introduction of sub- 
genera into a classification clearly aligns 
some generic groups with one genus and not 
another, reducing ambiguity in intergeneric 
relationships. While taxonomic hierarchy does 
not have the flexibility for incorporating pre- 
cisely the hierarchical information offered in a 
cladistic network, major cladistic hierarchical 
elements can be incorporated into the taxo- 
nomic framework, which can significantly im- 
prove its information content and reduce its 
taxonomic ambiguity. By demonstrating that 
Chione, as previously defined, was para- 
phyletic, Roopnarine justifies its breakup, but 
not, on the basis of his results and the stan- 
dard of minimizing taxonomic ambiguity, his 
creation of genera from almost all former sub- 
genera of Chione. For example, Roopnarine 
(1996) (Fig. 1) illustrates that the clade con- 
taining Chione subdivides into two clades. If 
he had incorporated this information into 
current chionine taxonomy following the taxo- 
nomic seniority rule, he could improve chion- 
ine taxonomy by proposing that Anomalo- 
cardla, the senior taxon of one clade, remain 
a genus under which the other members of 
that clade — lliochione, Lirophora, and Panchi- 
one — are subsumed as subgenera. He did 
propose that the senior taxon of the second 
clade, Chione. remain a genus, under which 
the other supraspecific taxon of that clade, 
Chionista, was subsumed as a subgenus. He 
also proposed, justifiably, that Chionopsis 
stand as a genus based on its solitary loca- 
tion in his cladograms. Taken in combination, 
these steps reduce ambiguity in chionine 
taxonomy, thereby justifying the revision, and 
they incorporate much, but admittedly not all, 
plausible evolutionary information derived 
from the analysis. But elevation of all subgen- 
era except Chionista to generic rank indicates 
an underutilization of cladistic hierarchical in- 
formation, creating unnecessary taxonomic 
ambiguity by decreasing the hierarchical infor- 
mation within the existing taxonomy. 



Classification 



A STUDY OF VENERID PHYLOGENY 



Assuming these two sources of ambiguity 
do not seriously interfere with the cladistic re- 
sults, can one justify his proposed taxonomic 
revision based on his analysis and its minimal 



The second paper, Canapa et al. (1 996), uti- 
lized biomolecular data from a length of 16s 
rRNA to construct two Neighbor Joining trees 
for nine Mediterranean venerid species from 



TRANSLATING TREES INTO TAXONOMY 



217 




Mercenaria 
Puberella 

Chionopsis 
Protothaca 

Timoclea 
Chionista 

Chione 

AnomaJocardia 
Iliochione 
Lirophora 

Panchione 



FIG. 1 . Consensus tree after Roopnarine (1 996: fig. 
20, lower figure). 



the five largest presently recognized subfami- 
lies: Chamelea gallina (Linnaeus) [Chioninae], 
Dosinia lupinus (Linnaeus) [Dosiniinae], 
Callista chione (Linnaeus) and Pitar rudis 
(Poli) [Pitarinae], Tapes decussatus (Lin- 
naeus), Tapes philippinarum (Adams & 
Reeve), and Venerupis aurea (Gmelin) [Tape- 
tinae], and Venus verrucosa Linnaeus [Ve- 
nerinae]. Three of the species, Chamelea gal- 
lina, Callista chione and Venus verrucosa are 
the type species for these genera, and the last 
is the nominal genus of the subfamily 
Venerinae and the Veneridae. The authors 
concluded that the resulting trees (Canapa et 
al., 1 997) (Figs. 2, 3) support the results of tra- 
ditional classification at the subfamily level but 
do not support the concept of a genus Tapes. 
Indeed, Chioninae and Venerinae, long con- 
sidered to be closely related, fall within the 
same clade on both trees, and each of the 
other subfamilies forms a distinct clade, al- 
though the relationships among those clades 
differ substantially between their two trees. 

Contrary to the paper's assertions, how- 
ever, the data do not disprove the concept of 
the genus Tapes. The tapetine species used 
in this study do not represent true Tapes, but 
rather belong to the subgenus Ruditapes 
(Fischer-Piette & Métivier, 1971). In order to 
prove or disprove the concept of Tapes, both 
the species representing the genus Tapes 
s.S. — T literatus (Linnaeus), an Asian 
species — and at least one of the 3-4 addi- 
tional Asian species considered to be mem- 



bers of Tapes s.S., should have been included 
in the study. If the tapetine species included in 
the study had fallen among these Asian 
species in the resulting trees, then this would 
indeed disprove the concept of Tapes. While 
the choice of species in this study reflects the 
accessability of material (local Mediterranean 
venerid species), this also limits what one can 
interpret, taxonomically, from the analyses. 

As it is, a more accurate taxonomic inter- 
pretation of the analyses indicates some 
equally interesting insights and illustrates 
some of the limitations presented by the 
species used. For example, Ruditapes decus- 
satus is the type species of Ruditapes. How- 
ever, what malacologists identify as Rudi- 
tapes decussatus in the Mediterranean might 
not be the same species as from the type lo- 
cality in the Indian Ocean, according to 
Fischer-Piette & Métivier (1971: 28). 

Venerupis aurea is not the type species of 
Venerupis; that honor belongs to the British 
Venerupis perforans Montagu, 1803, which in 
turn is a synonym of Venerupis corrúgala 
Gmelin, 1791 (Fischer-Piette & Métivier, 
1971). Fischer-Piette & Métivier (1971) 
lumped several nominal taxa under V. corrú- 
gala, resulting in a geographic distribution 
from Norway to Natal, Mozambique, and be- 
yond, and thus presenting the possibility that 
this is not one wide-spread species but a se- 
ries of similar taxa. This, then, limits what can 
be inferred about the integrity of Venerupis. 

What can be inferred about these taxa? 
Since Venerupis aurea falls among the 
Ruditapes, it calls into question the placement 
of V. aurea within Venerupis, the correct 
placement of the Mediterranean Ruditapes 
decussatus within Ruditapes, OR, if one as- 
sumes that the Mediterranean Ruditapes de- 
cussatus is indeed a valid representative of 
that taxon, the integrity of Ruditapes as a 
taxon. With respect to the last possibility, 
Venerupis Montagu, 1803, was established 
long before Ruditapes Ch\amen\\, 1900. Thus, 
if species of Venerupis are found to fall within 
Ruditapes, then Ruditapes would be a junior 
synonym of Venerupis. The data presented do 
not indicate which of the three possibilities 
might be correct, so no such inference can be 
made. 



CONCLUSION 

Careful study of these papers illustrates 
that the species chosen for analyses will limit 



218 



HARTE 
58 



99 



54 



100 



100 



96 



Crassostrea gigas 



Ruditapes decussatus 
Venerupis aurea 
Ruditapes philippinarum 
Dosinia lupinus 
Chamelea gallina 

Venus verrucosa 

Pitar rudis 
Callista chione 



98 



35 



100 



37 



96 



81 



73 



Crassostrea gigas 



Venus verrucosa 

Chamelea gallina 
Dosinia lupinus 
Pitar rudis 

Callista chione 
Ruditapes philippinarum 
Venerupis aurea 

Ruditapes decussatus 



FIG. 2. Neighbor-Joining trees constructed with pairwise distances calculated following an application of two- 
parameter correction for multiple substitutions (upper), and calculated on the basis of the sole transversions 
(lower), modified after Canapa et al. (1996; figs. 2, 3). The numbers represent the percentage of 100 boot- 
strap replications in which a given node appeared. 



what can be interpreted about higher taxa. In 
order to make significant inferences about 
generic and subgeneric taxonomy from the 
trees of cladistic or other types of analyses, 
studies must include at least the type species 
of those taxa, and preferably other species 
belonging to the same taxon or, in the case of 



larger taxa, that represent adequately the di- 
versity of the taxon. In morphological cladis- 
tics, character states must be carefully de- 
fined. An initial review of the taxonomy of the 
group to be analyzed should be conducted, so 
as to optimize choice of species for insights 
into the taxonomy. To ensure that the material 



TRANSLATING TREES INTO TAXONOMY 



219 



is indeed the originally described species, the 
specimens should come from the same geo- 
graphic locale as the originally described type 
specimens for the species. Conversely, if 
availability limits choice, this must be reflected 
in the limits of the resulting discussion of the 
taxa and presentation of cladograms. 

LITERATURE CITED 

ALBERT V. Д., S. E. WILLIAMS & M. W. CHASE, 
1992, Carnivorous plants: phylogeny and struc- 
tural evolution. Science, 257: 1491-1495. 

CANAPA, A., I. MAROTA, F. ROLLO & E. OLMO, 
1996, Phylogenetic analysis of Veneridae 
(Bivalvia): comparison of molecular and palaeon- 
tological data. Journal of Molecular Evolution, 43: 
517-522. 

FISCHER-PIETTE, E. & B. MÉTIVIER, 1971, Re- 
vision des Tapetinae (mollusques bivalves). Mé- 
moires du Muséum National d'Histoire Naturelle, 
(A-Zoologie) (n.s.) 71: 106 pp., 16 pis. 

FISCHER, E. & D. VUKADINOVIC, 1 977, Suite des 
révisions des Veneridae (Mol!. Lamellib.) Chion- 
inae, samaranginae et complément aux Vénus. 
Mémoires du Muséum National d'Histoire Natu- 
relle, (A-Zoologie) (n.s.) 106: 186 pp., 22 pis. 

HALANYCH, K. M., J. D. BACHELLER, A. M. A. 
AGUINALDO, S. M. LIVA, D. M. HILLIS & J. A. 
LAKE, 1995, Evidence from 18s ribosomal DNA 
that the lophphorate are protostome animals. 
Science, 267: 1641-1643. 



HARTE, M. E, 1992, An eastern Pacific Mercenaria 
and notes on other chionine genera (Bivalvia: 
Veneridae). The Veliger, 35: 137-140. 

JONES, C, 1979, Anatomy of Chione cancellata 
and some other chionines (Bivalvia: Veneridae). 
Malacologie, 19: 157-199. 

KEEN, A. M., 1969, Veneracea. Pp. 670-690, in 
Treatise on invertebrate paleontology. Part N. 
Mollusca 6. Bivalvia, vol. 2. R. с mugre, ed. The 
Geological Society of America and The University 
of Kansas. 

ROOPNARINE, P D, 1996, Systematics, biogeogra- 
phy and extinction of chionine bivalves (Bivalvia: 
Veneridae) in tropical America: Early Oligocène — 
Recent. Malacologia. 38: 103-142. 

SAITOU, N. & M. NEI, 1987, The Neighbor-Joining 
method: a new method for reconstructing phylo- 
genetic trees. Molecular Biological Evolution, 4: 
406-425. 

SIBLEY, С G. & J. E, AHLQUIST 1990, Phylogeny 
and classification of birds. Yale University Press, 
Hartford. 

SIMON, L., J. BOUSQUET R. С LEVESUE & MAU- 
RICE LALONDE, 1993, Origin and diversification 
of endomycorrhizal fungi and coincidence with 
vascular land plants. Nature, 363: 67-69. 

WAINRIGHT P O., G HINKLE, M. L. SOGIN, & S. K. 
STICKEL, 1993, Monophyletic origins of the 
Metazoa: an evolutionary link with fungi. Science, 
260: 340-342. 

Revised ms. accepted 22 April 1997 



MALACOLOGIA, 1998, 39(1-2): 221-224 

TRANSLATING TREES INTO TAXONOMY WITHIN VENERIDAE (BIVALVIA): 

A REPLY TO HARTE 

Peter D. Roopnarine 

Department of Biology, Southeast Missouri State University, Cape Girardeau, 
Missouri 63701 , U.S.A. 



Harte (this volume) has initiated, in com- 
ments on two papers (Capana et al., 1996; 
Roopnarine, 1996), an interesting and much 
needed discussion of the status of venerid 
systematics. The major focus of her commen- 
tary on Roopnarine (1996) is the selection 
and utilization of morphological characters for 
a phylogenetic analysis of the Chioninae, and 
the subsequent revision of the taxonomy of 
that subfamily on the basis of the phylogenetic 
analysis. In recognition of the fact that modern 
phylogenetic analysis is a dynamic process of 
hypothesis construction, testing and restruc- 
turing, I welcome her comments on my paper 
and below will admit shortcomings of my 
study but will also defend what I believe are 
valid and valuable contributions. 

Character Definitions 

Harte's first source of objection is the de- 
scriptive nature of several conchological char- 
acters employed in the phylogenetic analysis, 
for example palliai sinus depth. It should be 
made clear that, while the explanations of 
character states (for example, "greatly re- 
duced to absent" versus "present but short") 
may be verbally ambiguous, morphologically 
they are not. Close examination of taxa in- 
volved, such as Cliione (von Mühlfeld) and 
Lirophora Conrad, reveals that these charac- 
ter states are consistently identifiable and 
separable. Certainly a more robust method of 
presentation that would permit precise repli- 
cation by other workers, besides productive 
intenworker communication, would be the 
quantitative description of such characters. 
Attempts have been made to incorporate 
quantitative description of continuously vary- 
ing characters into phylogenetic analyses, 
such as gap coding (Archie, 1985) and more 
recently thin plate spline decomposition of 
landmark data (Naylor, 1996; Fink & Zelditch, 
1 995; Zelditch et al., 1 995), but much remains 
to be done in this area. In fact, salient argu- 
ments have been presented against the feasi- 



bility of such approaches (e.g., Bookstein, 
1994). 

One other reason for apparent character 
ambiguity is a less fundamental one. While 
many characters can be coded as dichoto- 
mously discrete states, several have addi- 
tional states that represent single taxa. An ex- 
ample is ventral margin crenulation, which is 
normally either very regular and fine, or alter- 
natively consists of significantly larger, 
coarser and more variable subunits (for ex- 
ample, Liropfiora versus Cliione). All taxa in- 
cluded in my analysis could be coded as one 
state or the other with the single exception of 
Chionista Keen, which has marginal crenula- 
tions intermediate in relative size and transi- 
tions posteriorly from regular to coarse and ir- 
regular. The trichotomous nature of this 
character then is based on one taxon out of 
sixteen. However, the potential ecomorpho- 
logical importance of this character (Vermeij, 
1987, 1993) justifies its inclusion in the analy- 
sis. 

Another cited source of ambiguity is the fail- 
ure to report the relative occurrence of a char- 
acter state in a species. Only character states 
that were recognized as invariable with re- 
spect to relevant taxa were analyzed. Char- 
acter conditions resulting from ontogenetic or 
environmental variation were purposefully ex- 
cluded. Such an example would be the radiais 
of Mercenaria mercenaria (L.) cited by Harte. 
These are typically obvious on the smoothed 
central portions of adult valves, and have even 
been used in the past as support of subspe- 
cific recognition (for example, M. mercenaria 
subradiata Palmer). The appearance of radial 
lines on the surfaces of smoothed, worn or 
eroded venerid valves that normally possess 
only concentric or commarginal sculpture is 
very common, but these radiais should not 
necessarily be recognized as primary sculp- 
ture. They are perhaps indeed of phylogenetic 
importance, an issue not explored in my 
paper, but until they are examined in that con- 
text should not be considered homologous 



221 



222 



ROOPNARINE 



with the prominent and primary radial sculp- 
ture of taxa such as Chione, Chionopsis 
Olsson and Puberella Fischer-Piette. Harte 
(1992) makes such an assumption (in addi- 
tion to the possession of a rugose nymph) 
when arguing for the assignment of the 
species Lirophora kellettii (Hinds) to genus 
Mercenaria. As discussed in Roopnarine 
(1 996), this species is more properly assigned 
to Panchione Olsson (Early Miocene-Recent) 
because of its obvious and overwhelming sim- 
ilarity to the numerous described extinct 
species also assigned to the genus, for exam- 
ple P. mactropsis (Conrad) and P. ulocyma 
(Dalí). Panchione kellettii bears faint radial 
lines on its surface, but this is characteristic of 
all species of Panchione, but certainly not 
Mercenaria. 



Representation of Genera 

The absence of type species for some of 
the genera used in the systematic analysis 
does introduce a certain amount of ambiguity 
in the results if they are viewed at a level of 
phylogenetic universality beyond mere con- 
sideration of former genus Chione. The extent 
and effect of the ambiguity of course remains 
itself ambiguous until the data are reanalyzed 
with the inclusion of the type species. I do not 
believe that inclusion of the type or additional 
species of Puberella will alter the resulting 
cladograms in any way, because the species 
are morphologically very similar, being sepa- 
rated primarily by time, geography, and such 
labile characteristics as shell size (e.g., 
Stanley & Yang, 1987). Increasing the cover- 
age of Protothaca Dall and Timoclea Brown in 
the analysis was unnecessary, because the 
major focus was to analyze the evolutionary 
histories of the tropical American Chione sub- 
genera, and to place their histories in a 
testable phylogenetic framework. The phylo- 
genetic relationships of these two genera, 
which range far beyond tropical American wa- 
ters, to the former Chione subgenera is un- 
doubtedly of great interest. That analysis 
would definitely require the examination of 
generic type species, as well as subgeneric 
types, a process that I am currently undertak- 
ing as an analysis of subfamily Chioninae. 
Moreover, varying degrees of relationship of 
say Timoclea {Timoclea) and T (Glycydonta) 
to Chione would lead to much more than a 
simple change of Timocieäs position on the 
current consensus tree. It would instead imply 



paraphyly of that genus and would require its 
entire taxonomic reconstruction. 

Classification 

In converting the results of my phylogenetic 
analyses to a revised hierarchical taxonomy 
of Chione, I relied upon two criteria: (1) 
changes to the existing taxonomy should be 
minimized, and (2) the revised taxonomy 
should be logically consistent with the under- 
lying hypothesis of phylogeny (Wiley et al., 
1991; deQueiroz & Gauthier, 1992). As Harte 
points out, the revised taxonomy is consistent 
with the phylogenetic results, but alternative 
revisions are available. Her major emphasis 
and point of contention seems to be the ele- 
vation of subgenera in the Lirophora clade 
{Anomalocardia Schumacher, lliochione 
Olsson, Lirophora and Panchione) to generic 
status. She suggests instead that the hierar- 
chical information implied by the topology of 
this subclade could be retained in a revised 
taxonomy by subsuming all subgenera under 
the historically senior taxon, in this case 
Anomalocardia. This alternative, while simpler 
and almost as informative, would unfortu- 
nately be inconsistent with the cladistic re- 
sults. Anomalocardia and lliochione spring 
from an unresolved polytomy, along with a 
branch bearing Lirophora ano Panchione.The 
placement of lliochione with respect to the 
other two branches is therefore unknown, and 
additional information may well place it out- 
side of Harte's Anomalocardia (Fig. 1 ). The re- 
sulting Anomalocardia, while monophyletic, 
would conflict with the hierarchical structure 
implied by the phylogeny. Interestingly, the re- 
cent description of an extinct chionine genus 
(Roopnarine, in press) (Fig. 2) also from trop- 
ical America partially supports Harte's sug- 
gestion. It may now be reasonable to consider 
lliochione a subgenus of Anomalocardia. 

I would like to reiterate that the taxonomic 
revision of Chione suggested in Roopnarine 
(1996) merely gives phylogenetic and paleon- 
tological support to revisions implemented by 
previous workers. For example, Olsson (1 961 ) 
regarded Chionopsis as a genus distinct from 
Chione: Keen (1 969) treats Panchione as dis- 
tinct from Lirophora: and Woodring (1982) 
and Ward (1992) treat Lirophora as a generic 
rank taxon. Finally, at the risk of portraying 
myself as an unrepentant cladist, I must point 
out that the maintenance of an artificial sys- 
tem of hierarchical categorization and classifi- 
cation is one of convenience (albeit a very in- 



A REPLY TO HARTE 



223 




Mercenaria 

Puberella 

Chionopsis 

Protothaca 

Timoclea 

Chionista 

Chione 

Anomalocardia 

I lio chione 

Lirophora 

Panchione 

Mercenaria 

Puberella 

Chionopsis 

Protothaca 

Timoclea 

Chionista 

Chione 

Anomalocardia 

Iliochione 

Lirophora 

Panchione 

Mercenaria 

Puberella 

Chionopsis 

Protothaca 

Timoclea 

Chionista 

Chione 

Iliochione 

Anomalocardia 

Lirophora 

Panchione 



FIG. 1 .Three possible solutions to the polytomy pre- 
sented in Roopnarine (1996: tigs. 4, 20). The upper 
two cladograms would support the inclusion of 
Iliochione within a new genus Anomalocardia. but 
the lowest cladogram would be incompatible with 
the hierarchical structure implied by such an inclu- 
sion (that is, genus Anomalocardia could then also 
include genera Chione, Protothaca, Timoclea, etc.). 






FIG. 2. Strict consensus tree of four equally most 
parsimonious cladograms illustrating the relation- 
ship of Liromissus Roopnarine (a recently described 
genus endemic to the late Neogene of Venezuela) to 
other chionine genera (consistency index = 0.426) 
(Roopnarine, in press). This solution supports the in- 
clusion of Iliochione m\h\n Anomalocardia as a sub- 
genus. Note the loss of resolution, with respect to 
Chione. Chionopsis. Protothaca. Puberella and 
Timoclea in the other portion of the tree. The corre- 
spondence between addition of a taxon and loss of 
resolution suggests the need for more characters, 
possibly non-conchological (although this would be 
difficult for the extinct Liromissus). 



valuable one). The Linnean system of taxo- 
nomic classification does not necessarily lend 
itself to descriptions of history, and it was 
never intended to, nor will it ever be capable of 
capturing fully the depth of phylogenetic rela- 
tionships implied by cladistic hypotheses. 

LITERATURE CITED 

ARCHIE, J. W., 1985, Methods for coding variable 
morphological features for numerical taxonomic 
analysis. Systematic Zoology. 34(3): 326-345. 

BOOKSTEIN, F L., 1994, Can biometrical shape be 
a homologous character? Pp. 220-222, in: в. к. 
HALL, ed., Homology: the hierarchical basis of 
comparative biology. Academic Press, London. 

CAPANA, A., I. MAROTA, F ROLLO & E. OLMO, 
1996, Phylogenetic analysis of Veneridae 
(Bivalvia): comparison of molecular and palaeon- 
tological data. Journal of Molecular Evolution, 43: 
517-522. 

DEOUEIROZ, K. & J. GAUTHIER, 1992, Phylo- 
genetic taxonomy. Annual Review of Ecology and 
Systematics, 23: 449-480. 



224 



ROOPNARINE 



FINK, W. L & M. L. ZELDITCH, 1995, Phylogenetic 
analysis of ontogenetic shape transformations: A 
reassessment of the piranha genus Pygocentrus 
(Teleostei). Systematic Zoology, 44(3): 343-360. 

HARTE, M. е., 1992, An eastern Pacific Mercenaria 
and notes on other chionine genera (Bivalvia: 
Veneridae). The Veliger, 35(2): 137-140. 

KEEN, A. M., 1969, Veneracea. Pp. 670-690, in 
Treatise on invertebrate paleontology Part N. 
Mollusca 6. Bivalvia, Vol. 2. R. с moore, ed. The 
Geological Society of America and The University 
of Kansas. 

NAYLOR, G. J. P, 1996, Can partial warp scores be 
used as cladistic characters? Pp. 519-530. in: 

L. F. MARCUS, M. CORTI, A. LOY, G. J. P. NAYLOR & 

D. E. SLICE, eds., Advances in Morphometries. 
Plenum Press. New York and London. 

OLSSON, A. A., 1 961 , Mollusks of the tropical east- 
ern Pacific. Panamic-Pacific Pelecypoda. Pale- 
ontológica! Research Institution. Ithaca, New 
York, 256pp. 

ROOPNARINE, P D., 1996, Systematics, biogeog- 
raphy and extinction of chionine bivalves (Bivalvia: 
Veneridae) in tropical America: Early-Oligocene- 
Recent. Malacologia, 38(1-2): 103-142. 

ROOPNARINE, P. D., in press, Endemism and ex- 
tinction of a new genus of chionine (Veneridae: 
Chioninae) bivalve from the late Neogene of 
Venezuela. Journal of Paleontology, in press. 



STANLEY S. M. & X. YANG, 1987, Approximate 
evolutionary stasis for bivalve morphology over 
millions of years: a multivariate multilineage 
study. Paleobiology 13(2): 113-139. 

VERMEIJ, G. J., 1987, Evolution and escalation, an 
ecological history of life. Princeton University 
Press. Princeton, New Jersey, xiii + 332 pp. 

VERMEIJ, G. J., 1993, A natural history of shells. 
Princeton University Press. Princeton, New 
Jersey, viii + 207 pp. 

WARD, L. W., 1992, Molluscan biostratigraphy of 
the Miocene, Middle Atlantic Coastal Plain of 
North America. Virginia Museum of Natural 
History Memoir, 2: 159 pp. 

WILEY E. O., D. SIEGEL-CAUSEY D. R. BROOKS 
& V A. FUNK, 1991, The compleat cladist: a 
phmer of phylogenetic procedures. The University 
of Kansas Museum of Natural History, Special 
Publication 19. 

WOODRING, W. P, 1982, Geology and paleontol- 
ogy of Canal Zone and adjoining parts of Panama. 
United States Geological Survey Professional 
Paper, 306-F: 757 pp. 

ZELDITCH, M. L., W. L. FINK & D. L. SWIDERSKI, 
1995, Morphometries, homology, and phyloge- 
netics: quantified characters as synapomorphies. 
Systematic Zoology 44(2): 179-189. 

Revised ms. accepted 22 April 1997 



MALACOLOGIA, 1998, 39(1-2): 225 

CORRECTIONS TO WHITE ETAL, 1996, MOLECULAR GENETIC 

IDENTIFICATION TOOLS FOR THE UNIONIDS OF FRENCH CREEK, 

PENNSYLVANIA MALACOLOGIA 38:181-202 

Laura R.White 

School of Forest Resources, The Pennsylvania State University, University Park, 
Pennsylvania 16802, U.S.A. 



It has recently come to my attention that several errors appeared in the "Laboratory 
Techniques" section of the article "Molecular genetic identification tools for the unionids of French 
Creek, Pennsylvania" [White et al., Malacologia, 1996, 38(1-2): 181-202]. Specifically, the con- 
centration of proteinase К in the standard phenol-chloroform nucleic acid extraction protocol 
should be 0.05 цд /^il (instead of 5 ug/i.11); the concentration of MgCl2 in the manufacturer-supplied 
amplification buffer at 1x final concentration should be 1.5 mM (instead of 15 mM); and the con- 
centration of gelatin should be 0.001% (instead of 0.01%). I regret any inconvenience or confu- 
sion that these errors might have caused. 



7 May 1997 



The editor-in-chief of Malacologia welcomes let- 
ters that comment on vital issues of general im- 
portance to the field of Malacology, or that com- 
ment on the content of the journal. Publication is 
dependent on discretion, space available and, in 
some cases, review. Address letters to: Letter to 
the Editor, Malacologia, care of the Department of 
Malacology, Academy of Natural Sciences, 1900 
Benjamin Franklin Parkway, Philadelphia, PA 
19103-1195, U.S.A. 



225 



MALACOLOGIA, 1998, 39(1-2): 227-234 



INDEX 



Page numbers in italics indicate figures of 
taxa. No new taxa appear in this number of 
Malacologia. 

Achatina fúlica 79 
Acer spicatum 2 
acicula, Cecilioides 33, 34 
Actinella actinophora 32 

arcta 32, 34 

carinofausta 32, 34 

fausta 32 

giramica 32, 34 

lentiginosa 32, 34 

nitidiuscula 32 

obserata 32, 34 
actinophora, Actinella 32 
aculeata, Acanthi nula 33, 34 
acuta, Physa 1 27 
Adacna 136 
Adula ^2S, 131, 132, 134, 135 

falcatoides ^ 30, 132 
Adulinae 135 
Aenio-Lytanthion 30 
Aeonio-Lyntathion 30, 33, 34, 37 
agrestis, Agriolimax 22 
Aghoiimacidae 39, 41 , 47, 51 
Agriolimax, agrestis 22 
alabamensis, Elimia 185-191 
alatus, Potamilus 195, 196, 198-202 
alatus, Unio 1 95 

albopalliatus, Phenacolimax 32, 34 
algosus, Semimytilus 1 30 
alliarius, Oxychilus 33, 34 
AInus 2 

minor 32 

mitriformis 32, 34 

tornatellina 32 
alternata, Anguispira4, 6 
Amauropsis paludinaris 1 59 
Amblema neislerii 203 

p//caia 203 
americanus. Modiolus 1 30 
amphichaenus, Potamilus 1 95-202 
Amphorella iridescens 32, 34 
amp/a, Leptoxis 113-121, 116 
Ampullariidae207, 212 
Anculosa 1 1 3 
Anculosae 116 
Ancylus fluviatilis 141 
Anguispira alternata 4, 6 
angustifolium, Epilobium 2 



Anomalocardia 216, 217, 222, 223 
arbórea, Leiostyla 32, 34 
arboreus, Zon ¡toldes 3, 4, 6, 34 
arcta, Actinella 32, 34 
arcticus, Bathypolypus 11-19 
Arcuatula ^29, 131, 132-135 

capens/s 130, 132, 132 
Arcuatulinae 135 
Argonautidae 14 

Arianta arbustorum 148, 167, 171 
Ariolimax columbianus 48 
Arion 39 

subfuscus 77-81 
Arionidae 107 
áspera, Columella 33 
áspera, Helix 33, 34, 145, 167-173 
asperrima, Protothaca 216 
ater, Aulacomya 130, 732 
atra, Aulacomya 83-91 , 87 
Aulacomya :29, 131, 132, 134, 136 

ater 130, 132 

atra 83-91 , 87 
aurantium, Haliotis 59-75, 61, 62, 64-66, 68 
au rea, Venerup/s 21 7, 218 
Austenia 1 07 
Averellia 96 

barbara, Cochlicella 33, 34 
Bathypolypus arcticus 11-19 

sponsalis 1 7 
be/7 л /7, Phenacolimax 32, 34 
Seíü/a papyrifera 2 
bifrons, Janulus 32 
Binneya 1 07 
Biomphalariaglabrata 79, 123, Г24, 127, 147, 

175-182, 700 
Biserrulae-Scorpiurietum 30 
Boettgeria crispa 32, 34 

deltostoma 32 

depaupérala 32, 34 

exigua 32 
Boettgerilla pallens 39-57, 41,45, 53-55 
Boettgerillidae 39 
Botulinae 135 
Brachidontes 129, 131, 132, 134 

semistriatus 130, 132, 732 
Brachidontinae 135 
Bradybaena 93, 95, 97-100, 707, 107 
Bradybaenidae 93, 96, 104, 106-108 
Brugia malayi 1 75 

budapestensis, Tandonia 39-57 , 41,45, 52 
Bulimulidae 107 



227 



228 



INDEX 



Bulinus tropicus 1 47 

Виппуа95, 97-100, 101, 103, 104, 106 

Busy con carica 1 51 -1 65, 154 

scalarispira 1 51 -1 65 
Busycotypus canaliculatus 151 -^ 65, 153, 154 
caelatura infúscala, Elimia 185-191 
californiense, Keenocardium "[ЗА, 136 
callicratis, Truncatellina 33 
Callinectes sapidus 1 52 
Ca///síac/7/one 21 7, 218 
canadense, Carycíiium exile 3,6,7 
canaliculata, Pomacea 207-213 
canaliculatus, Busycotypus 151-165, 153, 154 
cancellata, Chione 216 
capax, Potamilus 195, 196, 198-202 
capensis, Arcuatula 130, 132, 132 
capensis, Siphonaria 1 47 
Caracola na lenticula 33, 34 
Cardiidae 134 
Cardioidea 134 
carica, Busy con 151-165, 154 
carinocostata, Elimia 185-191 
carinofausta, Actinella 32, 34 
cariosa, Lampsilis 201 
Carychium canadense exile 3, 6, 7 

minimum 33 

tridentatum 33 
Caseolus compactus 32, 34 

leptostictus 32, 34 
catenaria, Elimia 1 85 
Cecilioides acicula 33, 34 
Cepaea hortensis 1 48 

nemoralis 1 48 
Cepoliinae93, 104, 107 
Cepolis95, 97-100, 103, 104, 107 
cerina, Fusconaia 196, 198-202 
Cerithioidea 183 
Chamelea gallina 21 7, 21 8 
Charodotes 95-100, 103, 104, 106, 107 
cheilogona, Leiostyla 32, 34 
chemnitzii, Natica 159, 163 
chierchiae, Octopus 1 1 
chilensis, Mytilus 83-91 , 86, 87, 130 
chione. Callista 21 7, 21 8 
C/7/one216, 217, 221-223 

cancellata 216 
Chioninae215, 217, 221 
Chionista 21 6, 21 7, 221 , 223 
Chionopsis 216, 217, 222, 223 
Choromytilus 129, 131, 132, 134-136 

chorus 1 30 

grayanus 1 30 

meridionalis 130, 132, 732 



chorus, Choromytilus 1 30 
Clausiliidae 34, 37 
Clethro-Laurion 30, 33, 34, 36, 37 
Clinocardiinae 134, Г36 
Cochlicella barbara 33, 34 
Cochlicopidae 34 
Cochlicopa lubrica 3, 4, 6, 33, 34 

lubricella 33 
colórala, Hypanis 136 
columbianus, Ariolimax 48 
Columella áspera 33 

edentula 3, 4, 6 

microspora 32 
compactus, Caseolus 32, 34 
concinna, Leiostyla 32, 34 
contortus, Planorbis 141, 146 
contracta. Vitrea 33 
corneus, Planorbarius 141-150 
cornuarietis, Marisa 209, 21 1 , 212 
Corophium volutator 1 4 
corrúgala, Venerupis 217 
coruscus, Mytilus 130, 133, 733 
costóte, l/a//on/a 33, 34 
cracherodii, Haliotis 73 
Crangon 12, 13 

mucronatum 32 

neritoides 32 

septemspiosus 1 4 

trochoideum 32 
Craspedopoma monizianum 32, 34 
crenatella, Elimia 185-191 
Crenellinae 129, 135 
Crenomytilus 131, 133, 134, 136 

grayanus 133 
Crepidula 127 
crispa, Boettgeria 32, 34 
cronkhitei. Discus 3,4,6,7 
C/yptosíra/con 95-1 00, 104, 106, 107 
Cyclophoridae 34 
cylindracea, Elimia 185-191 
cylindracea, Lauria 33 
Dacrydiinae 129, 135 
dalli, Haliotis 59 
Daudebardia 51 , 107 
decussatus, Ruditapes 217 , 218 
decussatus. Tapes 217 
deltostoma, Boettgeria 32 
depaupérala, Boettgeria 32, 34 
depressa. Pellicula 1 07 
Deroceras 21 -27 

/aei^e 3, 4, 6, 21-27, 39-57, 41, 45, 54, 55, 

79 

reticulatum 22, 25, 39-57, 47, 42, 54, 55 



INDEX 



229 



rodnae 39-57, 41,45 
Dialeuca 93, 95-98, 705, 104, 107 
Dinotropis 96 
Diplompharus 51 
discors, Musculus 130, 133, 134 
Discuta polymorpha 32 

tabellata 32, 34 
Disculella maderensis 32, 34 
Discus cronl<hitei 3, 4, 6, 7 
dolioides, Pomacea 2^2 
dorsal is, Pallifera 4, 6 
Dosinia lupinus 21 7, 21 8 
Dosiniinae217 

duplicata, Neverita 151-165, 154 
edentula. Columella 3,4,6 
edulis, Mytilus 130, 133 
Elimia^^3, 114, 183-193 

alabamensis 185-191 

caelatura infúscala 185-191 

carinocostata 185-191 

catenaria 1 85 

crenaíe//a 185-191 

cylindracea 185-191 

fascinans 185-191 

ger/7ardi/'/' 185-191 

haysiana 185-191 

/7yde/ 185-191 

o//Vu/a 185-191 

showalteri ^85-^9^ 

vanuxemiana 1 85 
Endodontidae 34 

Epiphragmophora 95, 97-100, 707,104, 107 
Epiphragmophorinae 93 
Epilobium angustifolium 2 
Eremarionta 95-'i 00, 104, 705, 106, 107 
Erica 30 

erubescens, Leptaxis 32 
Euconulus fulvus 3, 4, 6, 7, 33 
Euspira ^59, 163 

Ьелоз 151-165, 153, 154 

rectilabrum 1 59 
exigua, Boettgeria 32 
exigua, Striatura 3, 6 
falcatoides, Adula 130, 132 
fanalensis, Lauria 32, 34 
fascinans, Elimia 185-191 
fausta, Actinella 32 
Ferrusacia folliculus 33, 34 
Ferussaciidae 34, 37 
filicum, Leiostyla 32, 34 
f leridana, Goniobasis 1 1 5 
fluviatilis, Ancylus 141 
folliculus, Ferrusacia 33, 34 



fragilis, Leptodea 195, 198-202 

fúlica, Achatina 79 

fulvus, Euconulus 3, 4, 6, 7, 33 

fun/a, Leptaxis 32, 34 

fusca, Leiostyla 32, 34 

Fusconaia cerina 196, 198-202 

ga///na, Cfiamelea 217, 218 

galloprovincialis, f^ytilus 90, 130, 733 

Gastrocopta tappaniana 4, 6 

gay/, Tavvera 83-91, Ö6, 37 

gerhardtii, Elimia 185-191 

geversianus, Troption 83-91 , 87 

giramica, Actinella 32, 34 

glabrata, Biomphalaria 79, 123, 724, 127, 

147, 175-182, 730 
G/ycydon/a 21 6, 222 
Gon/obas/s 11 3-1 16 

floridana 1 1 5 

próxima 115, 119 
gouldi, Vertigo 3, 6, 7 
grandis, Pyganodon 203 
grayanus, Cfioromytilus 130 
grayanus, Crenomytilus 133 
Gyrotoma 1 1 3 
Haliotidae 59-75 
Haliotis 59, 67 

auranîium 59-75, 61, 62, 64-66, 68 

cracherodii 73 

da/// 59 

pourtalesii 59-75, 61-63, 69, 70, 72 

lamellosa 59, 60, 73 

roberti 59, 73 

tuberculata 59, 60, 67, 73 
hammonis, Nesovitrea 33 
harpa, Zoogenetes 3,4,6 
Hawaiia miniscula 33, 34 
haysiana, Elimia 185-191 
Helicahonidae 105 

Helicidae 34, 36, 37, 93, 96, 106, 107 
Helicodiscus singleyanus 33, 34 
Helicoidea91, 106 
Helisoma trivolvis 141 , 148 
Helix 39, 93, 95, 97-100, 104, 106, 107 

aspersa 33, 34, 147, 167-173 

lucorum 1 47 

pomatia 167 
Helminthoglypta 95-100, 104, 705, 106, 107 
Helminthoglyptidae 93-1 1 1 
Hemilauria limneana 32, 34 
héros, Euspira 151-165, 153, 154 
Heterodonta 91 
HeteroStoma paupercula 32 
Hiatella solida 83-91 , 86, 87 



230 



INDEX 



Hiatellidae 91 
hortensis, Cepaea 148 
НитЬоШала 95, 97-100, 105 
Humboldtianae 93 
Hydrobia ulvae 146, 147 
hydei, E//m/a 185-191 
Hypanis colorata 136 

¡aeviuscula 136 

minima 136 

vitrea 136 
iheringi, Peltella 1 07 
///oc/7/one216, 217, 222, 223 
llyanassa obsoleta 1 46 
inflatus, Potamilus 1 95-203 
integra, Physa 1 46 
iridescens, Amptiorella 32, 34 
irrigua, Leiostyla 32 
Janulus bifrons 32 

steptianoptiora 32, 34 
keenae, Sepi/fer 130, 132 
Keenocardium californiense 134, 136 
l<ellettii, Lirophora 222 
kellettii, Panchione 222 
l<urilensis, Modiolus ^30, 132 
labyrinthica, Strobilops 3, 4, 6 
laeve, Deroceras3, 4, 6, 21-27, 39-57, 41, 45, 

54, 55, 79 
laevigatus, Musculus 1 30 
laevissima [= ohiensis], Potamilus ^95 
laeviuscula, Hypanis 136 
lamellosa, Haliotis 59, 60, 73 
Laminaria 67 
Lamps/7/s 195, 201 

cariosa 201 

omaía 195, 198-202 

satura 201 

ventricosa 197 
Lasmonos 1 96 
Lasiena 195, 198 
latens, Spirorbula 32 
Lauria cylindracea 33 

fanalensis 32, 34 
laurínea, Leiostyla 32, 34 
leacockiana, Pyrgella 32 
Lehmannia 1 27 

marginata 39-57, 41-44, 46, 48, 50-54 

valentiana 1 27 
Leiostyla 37 

arbórea 32, 34 

cheilogona 32, 34 

concinna 32, 34 

W/cum 32, 34 

fusca 32, 34 



/mgfua 32 

laurínea 32, 34 

loweana 32 

millegrana 32, 34 

recia 32 

sphinctostoma 32 

wncia 32, 34 
lentiginosa, Actinella 32, 34 
lenticula, Caracollina 33, 34 
Lepiar/onte 95-1 00 
Leptaxis erubescens 32 

fL//va 32, 34 

membranácea 32 

undafa 32 
Leptodea 195, 196, 198, 201 

fragilis ^95, 198-202 

leptodon 201 

ochracea 201 
leptodon, Leptodea 201 
leptostictus, Caseolus 32, 34 
Leptox/s 11 3-1 21, И6 

amp/a 113-121, 7 76 

p/cfâ 11 3-1 21, 7 76 

p//caia 113-121, 776 

praerosa 113-121 

7aen/aia 11 3-1 21, 7 76,184-191 
/.eu/coma 21 6 
Limacidae 39, 47 
Limacoidea 39-57 
/./max 39 

maximus 1 45 

pseudoflavus 145, 147 
limneana, Hemi lauria 32, 34 
Limnoperninae 135 
I impida. Vitrina 3,4,6 
Liromissus 223 
/./лорЛога216, 217, 221-223 

/íe/tem/ 222 
literatus, Tapes 2М 
Lithophagidae 135 
Lithophaginae129, 135, 138 
Loligo 1 8 

loweana, Leiostyla 32 
/иЬл/са, Cochlicopa 3, 4, 6, 33, 34 
lubricella, Cochlicopa 33 
lucorum. Helix 1 47 
lu pi nus, Dosinia 2\7, 218 
Lymnaea palustris 1 47 

peregra 26 

sfagna/Zs 141, 145, 146, 175 

truncatula 1 48 
Lymnocardiinae 134, 736 
Lys/noe 95, 97-100 



INDEX 



231 



Lysinoinae 93 

mactropsis, Panchione 222 

maderensis, Disculella 32, 34 

Malacolimax tennelus 39-57, 41, 52, 53, 55 

Malagarion paenelimax 1 07 

malayi, Brugia 1 75 

mansoni, Schistosoma 123, 175-177, 180- 

182, 180 
marcidus, Phenacolimax 32, 34 
margínala, Lehmannia 39-57, 41-44, 46, 48, 

50-54 
marica, Timoclea2^6 
Marisa cornuarietis 209, 211,212 
maximus, Umax 1 45 
Melanoides tuberculata 1 47 
membranácea, Leptaxis 32 
Mercenaria 21 5, 21 7, 222, 223 

mercenaria 21 5, 221 

mercenaria subradiata 221 
meridionalis, Clioromytilus^^Q, 132, 132 
Metostracinae 93 

A/íetosíracon 95-100, 104, 105, 106, 107 
/W/crar/onfâ 95-100, 104, 106, 107 
microspora. Columella 32 
Milacidae39, 41,47, 49-51 
milium, Striatura 3, 4, 6, 7 
millegrana, Leiostyla 32, 34 
minima, H y pan i s 136 
minimum, Carychium 33 
miniscula, Hawaiia 33, 34 
minor, AInus 32 
minutissimum. Punctum 6 
mitriformis, AInus 32, 34 
modesta, Vertigo 3,4,6 
Modiolinae 129, 732, 135, 136 
modiolus. Modiolus 1 30 
Wod/o/ivs 129-132, 134, 135 

americanus 1 30 

kurilensis -\30, 132 

modiolus 1 30 
Monadenia 95, 97-100, 106-108 
monizianum, Craspedopoma 32, 34 
Monodacna 136 
mucronatum, Crangon 32 
Muricidae 84 

muriciformis, Xymenopsis 84, 86-91 
Muricoidea 83, 84, 91 
Musculinae 129, 134 
Musculinae 135 
Musculista ^29, 131, 132, 134-136 

senhousia 130, 132 
Musculus Л29, 131, 133, 134, 136 

d/scors 130, 133, 134 



laevigatus 1 30 
Myoida 91 
Mysidiellidae 129 
Mytilidae91, 129, 131, 134, 135 
Mytilinae129, 133, 135, 136 
Mytilisepta ^33 
Mytiloida 91 
Mytilus ^29, 131, 133, 134, 136 

chilensis 83-9^ , 86, 87, 130 

coruscus^ 30, 133, 133 

edulis Л30, 133 

galloprovincialis 90, 130, 133 

trossulus ^ 30, 133 
Mytiloidea 129-139 
Natica chemnitzii 1 59, 1 63 
Naticidae 84 
Nautilus ЛЗ 

neislerii, Amblema 203 
nemoralis, Cepaea 1 48 
Neogastropoda 91 
Neohelix 93, 95, 97-100, 104 
neritoides, Crangon 32 
Nesovitrea hammonis 33 
Neverita duplicata 151-165, 154 
nitidiuscula, Actinella 32 
nitidus, Phenacolimax 32, 34 
Nostoc 1 23 
Nucella^84 

Obliquaria reflexa 197-202 
obserata, Actinella 32, 34 
obsoleta, llyanassa 1 46 
ochracea, Leptodea 201 
Octopoda 1 1 
Octopodidae 11, 12 
Octopus 11 -18, 15, 16 

chierchiae 1 1 
ohiensis, Potamilus 1 95-202 
olivula, E/zm/a 185-191 
Oncomelania 1 84 
Opuntia tuna 30 
Oreohelicidae 96 
Oreohelix 93, 95, 97-100, 103 
ornata, Lampsilis 195, 198-202 
ovalis, Succinea 4, 6 
ovata. Vertigo 4, 6 
ovata, Timoclea 216 
Oxychilus alliarius 33, 34 
Padollus 67 

paenelimax, Malagarion 1 07 
pallens, Boettgerilla 39-57, 41, 45, 53-55 
Pallifera dorsal is 4, 6 
paludinaris, Amauropsis 1 59 
palustris, Lymnaea 1 47 



232 



INDEX 



Panchione 216, 217, 222, 223 

kellettii 222 

mactropsis 222 

ulocyma 222 
Paraptera ^96 
Parmahon 1 07 
papyrifera, Betula 2 
paupercula, Heterostoma 32 
Pellicula depressa 1 07 
Peltella iheringi 1 07 
peregra, Lymnaea 26 
perforans, Venerupis2M 
perna, Peгna^ 30, 133 
Perna ^29, 131, 133-136 

perna 1 30, 133 

viridis -\30, 133, 133 
Pernadae 135 
Pernidae 135 
Perninae 129, 135, 136 
Phenacolima ruivensis 32 
Phenacolimax albopalliatus 32, 34 

behnii 32, 34 

nitidus 32, 34 

marcidus 32, 34 

ruivensis 32 
philippinarum, Ruditapes 218 
philippinarum, Tapes 2M 
Physa acuta 1 27 

integra 1 46 

sayii 1 47 
Picea 1 

picta, Leptoxis 113-121, 116 
Pila 212 

pisana, Theba 33, 34 
Pitar rudis 2M, 218 
Pitarinae217 
placida, Plagyrona 33, 34 
Plagyrona placida 33, 34 
Planorbarius corneus 141-150 
planorbis, Planorbis 1 48 
Planorbis contortus 141 , 146 

planorbis 1 48 
Planorbidae 141 

Plesarionta 95-100, 104, 106, 107 
Pleurocera 1 1 4 

prasinatum 184-191 
Pleuroceridae 116, 183 
plicata, Amblema 203 
plicata, Leptoxis 113-121, 116 
Polinices tumidus 159, 163 

über 159, 163 
Polygyridae 96 
Polymita 95, 97-100, 101, 104, 107 



polymorpha, Discuta 32 
Pomacea 207 

canaliculata 207-213, 209 

dolioides 212 

urceus 212 
pomatia, Helix 167 
Populus tremuloides 2 
Poiam//L/s 195-205 

alatus 195, 196, 198-202 

amphichaenus 1 95-202 

capax 195, 196, 198-202 

inflatus 195-203 

laevissima 1 95 

o/7/ens/s 195-202 

purpuratus 195-202, 197 

purpuratus coloradoensis 1 95, 1 97-202 
pourtalesii, Haliotis 59-75, 61-63, 69, 70, 72 
praerosa, Leptoxis 113-121 
prasinatum, Pleurocera 184-191 
Proptera 195 
Protothaca 216, 217, 222, 223 

asperrima 216 
próxima, Goniobasis 1 1 5, 1 1 9 
pseudoflavus, Limax 145, 147 
Pteriomorpha 91 
Puberella 216, 217, 222, 223 
pulchella, Vallonia 33, 34 
Pulmonata 77, 123, 141, 167 
Punctum minutissimum 6 

pusillum 33 

pygmaeum 33, 34 
Pupillidae 34, 36, 37 
purpuratus, Potamilus 195-202, 197 
purpuratus coloradoensis, Potamilus 195, 

197-202 
pusillum, Punctum 33 
Pyganodon grandis 203 
pygmaea, Vertig 33 
pygmaeum, Punctum 33, 34 
Pyrgella leacockiana 32 
Pyrgulopsis 191 
recta, Leiostyla 32 
rectilabrum, Euspira 1 59 
reflexa, Obliquaria 197-202 
reticulatum, Dereceras 22, 25, 39-57, 41, 42, 

54,55 
Rhytididae 51 
roberti, Haliotis 59, 73 
rodnae, Dereceras 39-57, 41, 45 
Rubus 2 

rudis, Pitar 217, 218 
Ruditapes 217 
decussatus 217, 218 



INDEX 



233 



philippinarum 2^Ъ 
ruivensis, Phenacolima 32 
Salix 2 

sapidus, Callinectes 1 52 
satura, Lampsilis 201 
Saxicavidae 90 
saxícola, Staurodon 32 
sayii, Physa 1 47 
scalarispira, Busycon 151-165 
Schistosoma тап50пП23, 175-177, 180-182, 

750 
Semimytilus^2Q, 131, 133, 135 

algosus 1 30 
semistriatus, Brachidontes 130, 132, 132 
senhousia, Musculista 1 30, 132 
septemspiosus, Crangon 1 4 
Septiferidae 129, 135 
Septiferinae 135 
Septifer^29, 131, 133-135 

keenae ^30, 132 
Setipelis 96 

singleyanus, Helicodiscus 33, 34 
Sirius, Siphonaria 1 47 
Siphonaria capensis 1 47 

Sirius 1 47 
solida, Hiatella 83-9^ , 86, 87 
Somatogyrus 191 

Sonorella95, 97-100, 101, 102, 106, 108 
Sonorelix 96 
Sonorellinae 93 
sphinctostoma, Leiostyla 32 
spicatum, Acer 2 
Spirorbula latens 32 

squalida 32, 34 
sponsalis, Bathypolypus 1 7 
squalida, Spirorbula 32, 34 
stagnalis, Lymnaea 141, 145, 146, 175 
Staurodon saxícola 32 
stephanophora, Janulus 32, 34 
Stri atura exigua 3, 6 

m/7/L/m 3, 4, 6, 7 
Strobilops labyrinthica 3, 4, 6 
Stylommatophora 49, 93, 96, 167 
subfuscus, Arion 77-81 
subradiata. Mercenaria mercenaria 221 
Succinea ovalis 4, 6 
tabellata. Discuta 32, 34 
taeniata, Leptoxis 113-121, / 76, 1 84-1 91 
Tandonia budapestensis 39-57, 41,45, 52 
Tapes 217 

decussatus 2M 

Hteratus2M 

philippinarum 2M 



Tapetinae217 

tappaniana, Gastrocopta 4, 6 

Tawera gayi 83-91 , Ö6, 87 

tennelus, Malacolimax 39-57 , 41, 52, 53, 55 

Thaididae 84 

Theba pisana 33, 34 

Teratiodae 123-128 

Testacella 1 07 

Testacellidae 107 

Timoclea 216, 217, 222, 223 

marica 216 

ovata 216 
tornatellina, AInus 32 
tremuloides, Populus 2 
Trichodiscina 95-1 00 
Trichomyinae 135 
tridentatum, Carychium 33 
trivolvis, Helisoma 141, 148 
trochoideum, Crangon 32 
Trophon geversianus 83-91 , 87 
Trophonidae 83-91 
tropicus, Bulinus 1 47 
trossulus, Mytilus 130, 733 
Truncatellina callicratis 33 
truncatula, Lymnaea 1 48 
Tryonigens 95, 97-100, 707, 106, 108 
tuberculata, Haliotis 59, 60, 67, 73 
tuberculata, Melanoides 1 47 
tumidus. Polinices 1 59, 1 63 
tuna, Opuntia 30 
über, Polinices 159, 163 
ulocyma, Panchione 222 
ly/vae, Hydrobia 1 46, 1 47 
undata, Leptaxis 32 
l/n/o a/aiL/s 1 95 
Uniomerus 203 
Unionidae 195,225 
urceus, Pomacea 21 2 
valentiana, Lehmannia 1 27 
Valloniidae 34 
Vallonia costata 33, 34 

pulchella 33, 34 
vanuxemiana, Elimia 1 85 
Veneridae91,215, 217, 221 
Venerinae217 
Veneroida 91 
l/enert/p/s217 

аалеа 21 7, 218 

corrugata 2M 

perforans 2M 
ventricosa, Lampsilis 197 
Venus verrucosa 21 7, 21 8 
verrucosa, Venus 217, 218 



234 INDEX 

Vertigo gouldi 3, 6, 7 Vitrinidae 34, 36, 37, 1 07 

modesta 3, 4, 6 volutator, Corophium 14 

ovata 4, 6 Xanthonychidae 93-1 1 1 

pygmaea 33 Xanthonyx 93, 95-1 00, 1 04, 1 06 

Vetigastropoda 59, 73 Xerarionta 95-1 00, 1 04, 1 06, 1 07 

viñeta, Leiostyla 32, 34 Xymenopsis muriciformis 84, 86-91 

viridis, Perna 1 30, 1 33, 133 Zonitidae 34, 51 , 1 07 

Vitrea contracta 33 Zonitoidea 39 

vitrea, 14 y pan is 136 Zonitoides arboreus 3, 4, 6, 34 

Vitrina ¡impida 3, 4, 6 Zoogenetes harpa 3, 4, 6 



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VOL 39, NO. 1-2 MALACOLOGIA 1998 

CONTENTS 

J. W. HAWKINS, M. W. LANKESTER, & R. R. A. NELSON 

Sampling Terrestrial Gastropods Using Cardboard Sheets 1 

J. B. WOOD, E. KENCHINGTON, & R. K. O'DOR 

Reproduction and Embryonic Development Time of Bathypolypus Arcticus, A Deep- 

Sea Octopod (Cephalopoda: Octopoda) 11 

RICHARD M. LEBOVITZ 

The Inheritance of an Embryonic Lethal Mutation in a Self-Reproducing Terrestnal 

Slug, Dereceras Laeve 21 

R. A. D. CAMERON & L. M. COOK 

Forest and Scrub Snail Faunas from Northern Madeira 29 

ANA MARIA LEAL-ZANCHET 

Comparative Studies on the Anatomy and Histology of the Alimentary Canal of the 
Limacoidea and Milacidae (Pulmonata: Stylommatophora) 39 

LUIZ RICARDO L. SIMONE 

Morphology of the Western Atlantic Haliotidae (Gastropoda, Vetigastropoda) with 
Description of a New Species from Brazil 59 

BENJAMIN J. GOMEZ, ANA M. ZUBIAGA, M. TERESA SERRANO, & EDUARDO ÁNGULO 

Histochemical and Ultrastructural Identification of Biphasic Granules in the Albumen 
Secretory Cells of Arion Subfuscus (Gastropoda, Pulmonata) 77 

SANDRA GORDILLO Y SANDRA N. AMUCHÁSTEGUI 

Estrategias de Depredación del Gastrópodo Perforador Trophon Geversianus (Pallas) 
(Muricoidea: Trophonidae) 83 

MARIA GABRIELA CUEZZO 

Ciadistic Analysis of the Xanthonychidae (= Helminthoglyptidae) (Gastropoda: 
Stylommatophora: Helicoidea) 93 

ROBERT T DILLON, JR. & CHARLES LYDEARD 

Divergence Among Mobile Basin Populations of the Pleurocerid Snail Genus, 
Leptoxis, Estimated by Allozyme Electrophoresis 113 

CHARLES S. RICHARDS, CAROLYN PATTERSON, FRED A. LEWIS, & MATTY KNIGHT 

Larval Fusion and Development of Conjoined Teratoids in Biomphalaria Glabrata ... 1 23 

ALEXANDER I. KAFANOV & ANATOLY L. DROZDOV 

Comparative Sperm Morphology and Phylogenetic Classification of Recent Mytiloidea 
(Bivalvia) ''29 

KATHERINE COSTIL & STUART E. R. BAILEY 

Influence of Water Temperature on the Activity of Planorbarius Corneas (L.) 
(Pulmonata, Planorbidae) I^l 

GREGORY R DIETL & RICHARD R. ALEXANDER 

Shell Repair Frequencies in Whelks and Moon Snails from Delaware and Southern 

New Jersey ^^^ 

CHRISTOPHE DESBUOUOIS & LUC MADEC 

Within-Clutch Egg Cannibalism Variability in Hatchlings of the Land Snail Helix 
Asperea (Pulmonata: Stylommatophora): Influence of Two Proximate Factors 173 

MATTY KNIGHT ANDRE N. MILLER, NEIL S. M. GEOGHAGEN, FRED A. LEWIS, 

& ANTHONY R. KERLAVAGE 

Expressed Sequence Tags (ESTs) of Biomphalaria Glabrata, an Intermediate Snail 

Host of Schistosoma Mansoni: Use in the Identification of RFLP Markers 175 

CHARLES LYDEARD, JOHN H.YODER, WALLACE E. HOLZNAGEL, FRED G.THOMPSON, 

& PAUL HARTFIELD 

Phylogenetic Utility of the 5'-Half of Mitochondrial 16S rDNA Gene Sequences for 
Inferring Relationships of Elimia (Cerithioidea: Pleuroceridae) T ; 183 

KEVIN J. ROE & CHARLES LYDEARD 

Molecular Systematics of the Freshwater Mussel Genus Potamilus (Bivalvia: 
Unionidae) ^9^ 

ALEJANDRA L. ESTEBENET 

Allometric Growth and Insight on Sexual Dimorphism in Pomacea Canaliculata 

(Gastropoda: Ampullariidae) 207 

LETTERS TO THE EDITOR 

MARY ELLEN HARTE 

Translating Trees into Taxonomy within Veneridae (Bivalvia): A Critique of Two Recent 
Papers 215 

PETER D. ROOPNARINE 

Translating Trees into Taxonomy within Veneridae (Bivalvia): A Reply to Harte 221 

LAURA R.WHITE 

Corrections to White et al., 1996, Molecular Genetic Identification Tools for the 
Unionids of French Creek, Pennsylvania Malacologia 38:181-202 225