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HARVARD UNIVERSITY 




LIBRARY 

OF THE 

DEPARTMENT OF MOLLUSKS 

IN THE 

Museum of Comparative Zoology 
Gift of: 



MALACOLOGIA 



International Journal of Malacology 



Vol. 42(1-2) 2000 



Publication dates 

Vol. 32, No. 1 30 Nov. 1990 

Vol.32, No. 2 7 Jun. 1991 

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

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

Vol. 35, No. 1 14 Jul. 1993 

Vol. 35, No. 2 2 Dec. 1993 

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

Vol.37, No. 1 13 Nov. 1995 

Vol. 37, No. 2 8 Mar. 1996 

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

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

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

Vol.41, No. 1 22 Sep. 1999 

Vol.41, No. 2 31 Dec. 1999 



VOL. 42, NO. 1-2 MALACOLOGIA 2000 

CONTENTS 

J.A.ALLEN 

A New Deep-Sea Species of the Genus Neolepton (Bivalvia; Cyamioidea; 
Neoleptonidae) from the Argentine Basin 123 

MARÍA R. ALONSO, MANUEL J. VALIDO, KLAUS GROH & MIGUEL IBÁÑEZ 

Plutonia (Canarivitrina), New Subgenus, from the Canary Islands, and the 
Phylogenetic Relationships of the Subfamily Plutoniinae (Gastropoda: 
Limacoidea: Vitrinidae) 39 

ROBERT E. BENNETTS, STEVEN A. SPARKS & DEBORAH JANSEN 

Factors Influencing Movement Probabilities of Florida Tree Snails Liguus 
fasciatus (Müller) in Big Cypress National Preserve Following Hurricane 
Andrew 31 

EE-YUNG CHUNG & DONG-KI RYOU 

Gametogenesis and Sexual Maturation of the Surf Clam Mactra veneriformis 

on the West Coast of Korea 1 49 

EUGENE V COAN 

The Eastern Pacific Recent Species of the Bivalve Genus Gari (Tellinoidea: 
Psammobiidae), with Notes on Western Atlantic and Fossil Taxa 1 

EUGENE V COAN 

A New Species of Panacea from Chile (Bivalvia: Pholadomyoidea: 
Paralimyidae) 165 

MARTIN HAASE 

A Revision of the Genus Belgrandia, with the Description of a New Species 

from France (Caenogastropoda: Hydrobiidae) 171 

VASILIS K. DIMITRIADIS & ELIZABETH B. ANDREWS 

Ultrastructural and Cytochemical Study of the Digestive Gland Cells of the 
Marine Prosobranch Mollusc Nucella lapillus (L.) in Relation to Function ... 103 

KRISTEN M. LEWIS, JEFFREY L. FEDER, THOMAS G. HORVATH & 

GARY A. LAMBERTI 

Heterozygosity and Fitness: No Strong Association in Great Lakes 
Populations of Zebra Mussel, Dreissena polymorpha (Pallas) 113 

MARGHERITARAINERI 

Early Neurogenesis Pattern in Patella coerulea (Patellogastropoda) and its 
Possible Phylogenetic Implications 131 

M. A. RAMOS, B. ARCONADA, E. ROLAN & D. MORENO 

A New Genus and a New Species of Hydrobiid Snail (Mollusca: Gastropoda: 
Hydrobiidae) from Eastern Spain 75 

COLINE H. M. VAN MOORSEL, W. JOHAN VAN NES & HENDRIK-JAN MEGENS 

Letters to the Editor 203 

JANET R. VOIGHT 

The Distribution of Octopuses of Graneledone (Cephalopoda: Octopodidae) 

in Reference to Deep-Sea Features 63 



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MALACOLOGIA 



/" 



International Journal of Malacology 









A 












Vol. 42(1-2) 




^ 



2000 



MALACOLOGIA 

EDITOR-IN-CHIEF: 
GEORGE M. DAVIS 



Editorial Office 

Malacologia 

P.O. Box 1 222 

West Falmouth, MA 02574-1222 



Business & Subscription Office 

Malacologia 

P.O. Box 385 

Haddonfield, NJ 08033-0309 



Co-Editors: 
EUGENE COAN 
California Academy of Sciences 
San Francisco, CA 



CAROL JONES 
Denver, CO 



Managing Editor: 

CARYL HESTERMAN 
Haddonfield, NJ 



Associate Managing Editor 

TIMOTHY A. PEARCE 

Delaware Museum of 

Natural History 

Wilmington, DE 

Associate Editor: 

JOHN B. BURCH 

University of Michigan 

Ann Arbor 



Graphics Editor 

THOMAS WILKE 
The Academy of 
Natural Sciences 
Philadelphia, PA 



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



RUDIGER BIELER 

President 

Field Museum, Chicago 

JOHN BURCH 

MELBOURNE R. CARRIKER 
University of Delaware, Lewes 

GEORGE M. DAVIS 
Secretary and Treasurer 

CAROLE S. HICKMAN 

President Elect 

University of California, Berkeley 



ALAN KOHN 

University of Washington, Seattle 

JAMES NYBAKKEN 

Vice President 

Moss Landing Marine Laboratory, California 

CLYDE F. E. ROPER 

Smithsonian Institution, Washington, D.C. 

SHI-KUEIWU 

University of Colorado Museum, Boulder 



Participating Members 



EDMUND GITTENBERGER 

Secretary, UNITAS MALACOLOGICA 

Rijksmuseum van Natuurlijke 

Historie 

Leiden, Netherlands 



JACKIE L. VAN GOETHEM 
Treasurer, UNITAS MALACOLOGICA 
Koninklijk Belgisch Instituut 
voor Natuurwetenschappen 
Brüssel, Belgium 



Emeritus Members 



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

KENNETH J. BOSS 

Museum of Comparative Zoology 

Cambridge, Massachusetts 



ROBERT ROBERTSON 

The Academy of Natural Sciences 

Philadelphia, Pennsylvania 

W. D. RUSSELL-HUNTER 
Easton, Maryland 



Copyright © 2000 by the Institute of Malacology 
ISSN: 0076-2997 






2000 
EDITORIAL BOARD 



J.A.ALLEN 

Marine Biological Station 

Millport, United Kingdom 

E. E. BINDER 

Museum d'Histoire Naturelle 

Geneve, Switzerland 

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 

Council for Undergraduate Research 

Washington. DC. U.S.A. 

B. HUBENDICK 
Naturhistoriska Museet 
Göteborg. Sweden 

S HUNT 
Lancashire 
United Kingdom 

R.JANSSEN 

Forschungsinstitut Senckenberg, 
Frankfurt am Main. Germany 

R. N. KILBURN 
Natal Museum 
Pietermaritzburg, South Africa 

MA. KLAPPENBACH 

Museo Nacional de Historia Natural 

Montevideo, Uruguay 

J. KNUDSEN 

Zoologisk Institut Museum 

Kobenhavn, Denmark 

A. LUCAS 

Faculte des Sciences 

Brest, France 

C. 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 

DIARMAIDO'FOIGHIL 
University of Michigan 
Ann Arbor, U.S.A. 

J. OKLAND 
University of Oslo 
Norway 

T. OKUTANI 
University of Fisheries 
Tokyo, Japan 

W. L. PARAENSE 

Instituto Oswaldo Cruz, Rio de Janeiro 

Brazil 



A. STANCZYKOWSKA 
Siedlce, Poland 

F. STARMÜHLNER 

Zoologisches Institut der Universität 

Wien. Austria 

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

W. STREIFF 
Universite de Caen 
France 

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

J. P. POINTIER 

Ecole Pratique des Hautes Etudes 

Perpignan Cedex, France 

W. F. PONDER 
Australian Museum 
Sydney 

Ql Z. Y 

Academia Sínica 

Qingdao. People's Republic of China 

D. G. REID 

The Natural History Museum 

London, United Kingdom 

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



N. H. VERDONK 
Rijksuniversiteit 
Utrecht, Netherlands 

ANDERS WAREN 

Swedish Museum of Natural History 

Stockholm, Sweden 

B. R. WILSON 

Dept. Conservation and Land Management 

Kallaroo. Western Australia 

H.ZEISSLER 
Leipzig, Germany 

A. ZILCH 

Forschungsinstitut Senckenberg 

Frankfurt am Main, Germany 



MALACOLOGIA, 2000, 42(1-2): 1-29 

THE EASTERN PACIFIC RECENT SPECIES OF THE BIVALVE GENUS GARI 
(TELLINOIDEA: PSAMMOBIIDAE), WITH NOTES ON WESTERN ATLANTIC 

AND FOSSIL TAXA 

Eugene V. Coan 1 

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

ABSTRACT 



A study has been conducted of the type and other material of the Recent eastern Pacific 
species of the bivalve genus Gari Schumacher, 1817. There are seven species of Gari (Gob- 
raeus): (1) Gari (G.) californica (Conrad, 1849) (synonyms: Psammobia rubroradiata Carpenter, 
1864: P. lilacina Wilkins, in Palmer, 1958 [in synonymy]) occurs from Kachemak Bay, Alaska, to 
Bahía Magdalena, Baja California Sur, Mexico, but with a gap between Puget Sound and Men- 
docino County, California. Based on the material currently available in the United States, it can- 
not be distinguished from the northwestern Pacific G. kazusensis (Yokoyama, 1922), which is 
also regarded as a synonym, along with G. k. atsumiensis Hayasaka, 1961. (2) Gari (G.) fucata 
(Hinds, 1845) (synonym: Siliquaria edentula Gabb, 1869), occurs from Ventura County, Califor- 
nia, to Punta Eugenia, Baja California Sur, Mexico, and perhaps as far south as Bahía Mag- 
dalena. (3) Gari (G.) lata (Deshayes, 1855) (synonym: Psammobia regularis Carpenter, 1864), 
occurs from Bahía Magdalena, Baja California Sur, Mexico, throughout the Gulf of California, 
south to Santa Elena, Ecuador. Records of Gari regularis from the Pacific coast of northern Baja 
California are based on misidentified small, elongate, inflated specimens of G. californica. (4) 
Gari (G.) maxima (Deshayes, 1855) occurs from Mazatlán, Mexico, to Panama. (5) Gari (G.) 
panamensis Olsson, 1 961 , occurs from the central Gulf of California to Playas, Ecuador. (6) Gari 
(G.) solida (Gray, 1828) (synonyms: Psammobia solida Philippi, 1844; P. crassa Hupé, 1854), oc- 
curs from Arica to Rio Inio, Chile. (7) A probable new species of Gari (G.) occurs in the Galapa- 
gos Islands, thus far represented by only a single, small, broken specimen. An eighth species, 
Gari (Dysmea) helenae Olsson, 1961, occurs from Laguna Ojo de Liebre, Baja California Sur, 
Mexico, throughout the Gulf of California, south to Isla Salango, and the Galapagos Islands, 
Ecuador. Its relationship to the western Atlantic Gari circe (Mörch, 1 876) and G. linhares Simone, 
1998, remains to be resolved. Several lectotype designations are made, and a list is provided of 
New World Recent and fossil taxa that have been placed in Gari. 

Key words: Psammobiidae, Bivalvia, eastern Pacific, western Atlantic, Gari, Gobraeus, Dys- 
mea. 



INTRODUCTION 
Previous Treatments 

Some years ago, I published a paper cov- 
ering the members of the Psammobiidae and 
Solecurtidae of the northeastern Pacific 
(Coan, 1973). Some key problems with re- 
gard to the genus Gari had to be left unre- 
solved, particularly concerning the relation- 
ships of these northern species to those of the 
Panamic Province. Since then, much more 
material has become available, and other 
studies on this genus have been published, 
allowing additional comparisons and an im- 
proved context for classification. 



Important papers on the anatomy and biol- 
ogy of members of the genus Gari include 
Ansell (1967), Bloomer (1911), and Yonge 
(1949). The anatomy of the psammobiid Nut- 
tallia was discussed by Pohlo (1972) and that 
of Heterodonax was treated by Narchi & Do- 
maneschi (1993). 

Key taxonomic reviews covering Gari in- 
clude the short treatment of Philippi (1844- 
1845) of eight species and the more compre- 
hensive monographs of Reeve (1856-1857) 
and Bertin (1880). Deshayes (1855) de- 
scribed many new species in a single, unillus- 
trated paper, and Tryon (1869) provided a list 
of taxa. Discussions of the genera in the 
Psammobiidae were given by Dall (1898a, 



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



COAN 



1900: 970-978), Stoliczka (1870: 113-115), 
and Keen (1969: 629-634). Dautzenberg & 
Fischer (1913, 1914) and Lamy (1914) dis- 
cussed Lamarck's species. Lamy (1918) cov- 
ered the species occurring in the Red Sea. 
Matsukuma (1989) treated some Japanese 
species, and Cosel (1 989) dealt with the West 
African species. 

The most important monograph in recent 
years is that of Willan (1993) on the Psam- 
mobiidae of the Indo-Pacific. He recognized 
the genera Asaphis, Heteroglypta, Gari, and 
Soleotellina. The genus Gari, was divided 
into nine subgenera, Gari, s.S., Crassulobia, 
Dysmea, Gobraeus, Kermadysmea, Psam- 
mobella, Psammobia, Psammodonax, and 
Psammotaena. Of these subgenera, only Dys- 
mea and Gobraeus are present in the Recent 
fauna of the New World. 

Format 

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

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

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

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

Abbreviations 

The following abbreviations are used in the 
text: AMNH, American Museum of Natural 
History, New York, New York, USA; ANSP, 
Academy of Natural Sciences of Philadelphia, 
Philadelphia, Pennsylvania, USA; BMNH, 
British Museum (Natural History) collection, 
The Natural History Museum, London, Eng- 
land; CAS, California Academy of Sciences, 
San Francisco, California, USA; ICZN, Inter- 
national Commission on Zoological Nomen- 
clature; LACM, Natural History Museum of 
Los Angeles County, California, USA; MCZ, 
Museum of Comparative Zoology, Harvard 



University, Cambridge, Massachusetts, USA; 
MNH-U, Museum für Naturkunde der Hum- 
boldt-Universität zu Berlin, Germany; MNHN, 
Muséum National d'Histoire Naturelle, Paris, 
France; MNHN-UC, Museo Nacional de His- 
toria Natural, Universidad de Chile, Santiago, 
Chile; MZSP, Museu de Zoología, Universi- 
dade de Säo Paulo; SBMNH, Santa Barbara 
Museum of Natural History, Santa Barbara, 
California, USA; UCMP, University of Califor- 
nia Museum of Paleontology, Berkeley, Cali- 
fornia, USA; UF, Division of Invertebrate Pale- 
ontology, Florida Museum of Natural History, 
University of Florida, Gainesville, Florida, 
USA; UMML, University of Miami Marine Lab- 
oratory [Rosenstiel School of Marine and At- 
mospheric Sciences], Miami, Florida, USA; 
USNM, United States National Museum col- 
lection, National Museum of Natural History, 
Smithsonian Institution, Washington, DC, 
USA; Hickman Collection, collection of Cleve- 
land P. Hickman, Jr., Lexington, Virginia, USA; 
Kaiser Collection, collection of Kirstie L. 
Kaiser, Puerto Villarta, Jalisco, Mexico; Sko- 
glund Collection, collection of Carol C. Sko- 
glund, Phoenix, Arizona, USA. 

Morphological Characters 

Overall shell shape is characteristic of each 
species; for example, G. solida is ovate (1/h = 
1 .4-1 .5), whereas G. fucata is the most elon- 
gate (1/h = 2.1-2.2). Shell width is more vari- 
able and more difficult to quantify and is there- 
fore less useful. The anterior end may be 
produced or rounded; the posterior end may 
be subtruncate to truncate. The position of the 
beaks relative to the midline, although vari- 
able, is a reliable character, as, for example, 
differentiating G. ¡ata (beaks 40-42% from 
anterior end) from G. maxima (31 -36%). Two 
taxa have conspicuous shell sculptural char- 
acters, but most have only fine commarginal 
ribs. Several taxa can be distinguished by 
shell color. The length of the palliai sinus, its 
height, and the extent to which its ventral mar- 
gin overlaps the palliai line are useful charac- 
ters (Figs. 1 9-26). Finally, some details of the 
hinge teeth are of value, although the teeth 
may be broken off in large specimens. The 
scars of the cruciform muscles -intersecting 
muscles associated with the bases of the 
siphons in the Tellinoidea are very difficult to 
see in the Psammobiidae. Where a scar could 
be seen, I have included it in my drawings, but 
these scars are insufficiently known to provide 
useful characters. 



THE EASTERN PACIFIC RECENT SPECIES OF THE BIVALVE GENUS GARI 



Important differentiating characters are 
summarized in Table 1 . 

Psammobiidae Fleming, 1828: 437 

Genus Gar/ Schumacher, 1817: 44, 

131-132, pi. 9 

Type species (subsequent designation in 
ICZN Opinion 910, 1970): Gari vulgaris Schu- 
macher, 1817: 131, = Tellina gari Linnaeus, 
1758: 674 (suppressed ICZN Opinion 910); = 
Tellina trúncala Linnaeus, 1767: 1118 (ICZN 
Opinion 910). Recent, Indo-Pacific. 

In Gari, s.s, there are oblique cords on the 
anterior and central slopes. All ten Recent 
members of this subgenus are restricted to 
the Indo-Pacific Province. 

Subgenus {Gobraeus) Brown, 1844: 102, 
ex Leach ms 

Type species (monotypy): Solen vespertinus 
Gmelin, 1791: 3228, = Tellina depressa Pen- 
nant, 1777: 87 (octovo ed.); 73 (quarto ed.), 
pi. 47, fig. 27. Recent, Europe. 

In the subgenus Gobraeus, the shells have 
only commarginal sculpture. They lack the ra- 
dial ridge between the posterior and central 
slopes present in the subgenus Psammobia 
Lamarck, 1818. Gobraeus occurs in Europe, 
west Africa, the eastern Pacific, and Australa- 
sia and contains 11 species. 

While Willan (1993) has made consider- 
able progress in sharpening the definitions 
among the subgenera of Gari, the characters 
amongst them overlap to such an extent that 
additional characters are clearly needed, as 
well as a more formal cladistic analysis. 

Gari (Gobraeus) californica (Conrad, 1849) 
Figures 1-4, 19 

Psammobia californica Conrad, 1849 

Conrad, 1837: pi. 19, fig. 3 [fig. only; no 
name or description]; 1849: 121; Carpen- 
ter, 1864: 633 [1872 reprint: 119]; Dall, 
1898a: 58 [Psammobia (Gobraeus)]; I. S. 
Oldroyd, 1924: 57, 215, pi. 46, fig. 3; 
1925: 185, pi. 43, fig. 5 [Psammobia]; 
Grant & Gale, 1931: 382 [Gari (Gob- 
raeus)]; Nomura & Hatai, 1935: 117-118; 
J. Q. Burch, 1 945: 21 -22, 26 (fig.); Coan, 
1973:42-43, figs. 1,20 

Psammobia rubroradiata Carpenter, 1864, ex 
Nuttall MS 

Carpenter, 1857a: 212; 1857b: 195, 301; 
1860: 1 [as Sanguinolaria rubroradiata; 



nom. nud.]; 1864: 540, 563, 602, 638 
[1872 reprint: 26, 49, 88, 124] [Psammo- 
bia]; 1865: 55; Tryon, 1869: 77 [as Gari 
(Psammacola) "rubrolineata"]; Bertin, 
1880: 123 [Gari]; Keep, 1881: 56, pi. 15, 
fig. 4; 1887: 198-199, fig. 170; Dall, 
1898a: 61 [as = Psammobia californica]; 
Palmer, 1958: 17, 20, 112-113, 341; pi. 
16, figs. 2-5, 7. 

Psammobia lilacina Wilkins, in Palmer, 1958, 
ex Carpenter MS [in synonymy] 
Wilkins, in Palmer, 1958: 113 [in syn- 
onymy] 

Psammobia kazusensis Yokoyama, 1922 
Yokoyama, 1922: 136-137, pi. 9, fig. 4; 
Yamamoto & Habe, 1959: 100, 120, pi. 
1 0, figs. 3, 4; Scarlato, 1 981 : 367, fig. 1 90 

Psammocola kazusensis atsumiensis 
Hayasaka, 1961 
Hayasaka, 1961: 54-55, pi. 7, figs. 1, 2 

Gari regularis, auctt., non (Carpenter, 1864) 
Coan, 1973: 45 [in part], figs. 7, 22 

Gari fucata, auctt., non (Hinds, 1845) 
Hertz, 1994: 85, fig. 3 

Type Materials & Localities 

P. californica & P. rubroradiata-BMNH 
1861.5.20.88, pair; length 41.2 mm; 
height, 25.5 mm; thickness, 13.8 mm 
(Fig. 1). This specimen is the holotype of 
P. californica and the lectotype (Palmer, 
1958: 113) of P. rubroradiata. USNM 
15636, paralectotype of P. rubroradiata; 
Neah Bay, Clallam County, Washington 
(48.4°N), pair; length 79.1 mm. USNM 
15492, paralectotype of P. rubroradiata; 
San Pedro, Los Angeles County, Califor- 
nia (33.7°N), pair, length 109.5 mm (one 
valve broken). USNM 3364, paralecto- 
type of P. rubroradiata; Puget Sound, 
Washington (47.5°N); length 69.0 mm. 

P. kazusensis- University Museum, Univer- 
sity of Tokyo, CM21262, syntype, left 
valve, length, 80 mm; height, 45 mm; 
thickness, 13.5 mm (originally stated di- 
mensions) (Fig. 2). Upper Musashino 
Formation at Shito, Ichihara-gun, Chiba 
Prefecture, Japan; Pleistocene. Yoko- 
yama (1972) mentions two left valves, 
only one of which remains in the collec- 
tion. 

P. kazusensis atsumiensis- Institute of Geol- 
ogy and Paleontology, Tohoku University, 
IGPS 78419, holotype, left valve; length, 
57.4 mm; height, approx. 33 mm; thick- 
ness, 7.8 mm (originally stated dimen- 



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THE EASTERN PACIFIC RECENT SPECIES OF THE BIVALVE GENUS GARI 




FIGS. 1-4. Gari californica. FIG. 1. Holotype of Psammobia californica, right and left valves; BMNH 
1861.5.20.88; length, 41.2 mm. FIG. 2. Syntype of Psammocola kazusensis, left valve; University of Tokyo 
CM21262; length, 80 mm. FIG. 3. CAS 115849, right valve, False Narrows, Vancouver Island, British Co- 
lumbia; 18-37 m; length, 71.8 mm. FIG. 4. CAS 113109, right valve, Catalina Island, California; 27-82 m; 
length, 37.0 mm. 



COAN 



sions). IGPS 78420, paratype, left valve; 
length, approx. 41 mm. "Tonna Bed" 
(holotype) and "Mya Bed" (paratype) at 
Takamatsu, Atsumi Peninsula, Aichi Pre- 
fecture, Japan; Pleistocene. 

Description 

Shell elliptical (1/h = 1.5-1.8), moderately 
inflated (h/th = 1.7-2.0), equivalve, with nar- 
row gapes anteroventrally and posteriorly. 
Valves moderately heavy. Beaks at 36-43% 
from anterior end. Anterior end sharply 
rounded. Posterior end vertically subtruncate. 
Surface with moderate to heavy commarginal 
growth lines. Periostracum light to dark tan, 
often present only as marginal remnants. Sur- 
face light yellowish-tan, with narrow purple ra- 
dial bands in most specimens. Small speci- 
mens light in color. 

Interior radial thickenings medial to adduc- 
tor scars heavy. Palliai sinus broad, reaching 
to under beaks or slightly beyond them, its 
ventral margin merged with palliai line for 
70-82% of sinus length. Palliai line not turned 
ventrally at its posterior end (Fig. 19). 

Nymphs heavy, 21-25% of shell length. 
Right valve with a moderate, projecting ante- 
rior cardinal and a narrow posterior cardinal. 
Left valve with a moderate, nearly vertical an- 
terior cardinal and a very narrow posterior 
cardinal. Length to 141.0 mm [LACM 67-44; 
Arbolito, Baja California (Norte)]. 

Distribution 

Kachemak Bay, Kenai Peninsula (59.5°N) 
[LACM 85-139.6, 86-289.1 ], 1 northwest to 
Hanning Bay, Montagu Island, Alaska 
(60.0°N) [CAS 116956], south to the Strait of 
Juan de Fuca, Washington [UCMP R2398] 
(48.1°N); without any records or vouchered 
specimens from the Pacific coasts of Wash- 
ington or Oregon; Portugese Beach, Mendo- 
cino County, California [R. White, 1 993: 1 8; R. 
White, personal communication, 1993; UCMP 
3094, "Mendocino Co."] (39.3°), to the Punta 



1 A record previously thought to be from further west 
in Alaska -USNM 207429, "Shelikov Strait"- prob- 
ably represents a labeling error; whereas the label 
specifies an appropriate Albatross station for this lo- 
cality [stn. 4295; 92 fms.], a different station number 
is written in pencil on one valve of the specimen 
[stn. 4209], corresponding to a more likely locality 
and depth— off Admiralty Head, Port Townsend, 
Jefferson County, Washington; 24-25 fms. 



Entrada, Bahía Magdalena, Baja California 
Sur, Mexico [LACM 71-14.56] (24.5°N); from 
the intertidal zone to 55 m (mean, 14 m; n = 
136). I have seen 321 Recent eastern Pacific 
lots, including the type. Also present in the 
western Pacific from Petropavlovsk, Kam- 
chatka (53.0°N), south to Vladovostok, Sea of 
Japan (43.2°N), and to Tokyo, Honshu, Japan 
(35.7°N). 

Gari californica has been reported from late 
Pleistocene terraces on San Nicolas Island, 
Channel Islands, Ventura County (Vedder, in 
Vedder & Norris, 1 963: 46); on the second ter- 
race, Palos Verdes Hills, Los Angeles County 
(Chace, 1966: 169); terraces at Newport Bay, 
Orange County (Kanakoff & Emerson, 1959: 
22); and Nestor Terrace, San Diego, San 
Diego County (Bishop & Bishop, 1972: 6; 
Valentine, 1961: 357), California. It has been 
recorded in strata of early Pleistocene age in 
Santa Monica (Potrero Canyon terrace - 
Woodring, in Hoots, 1931: 120; Anchor Silt — 
Rodela, 1957: 2484), and San Pedro (San 
Pedro Sand-B. L. Burch, 1947: 8; Clark, in 
Natland, 1957: opp. p. 548; T S. Oldroyd, 
1 925: 7; Valentine & Meade, 1 961 : 1 9; Lomita 
Marl -Clark, in Natland, 1957: opp. p. 548), 
Los Angeles County, California. In the late 
Pliocene, there are records from the Careaga 
Formation, Santa Barbara County (Woodring 
& Bramlette, 1951: 66, as "Gari? cf. G. califor- 
nica'), and the Pico Formation of Los Angeles 
County (Soper & Grant, 1932: 1060), Califor- 
nia. In the early Pliocene, it is known from the 
Towsley Formation of Los Angeles County 
(Kern, 1973: 79). 

Also recorded from the Pliocene (Nomura & 
Hatai, 1935: 117-118) and the Pleistocene 
(type localities of Asian synonyms cited 
above) of Japan. A possible record of this 
species from the lower Miocene or upper 
Oligocène of Japan (Makiyama, 1934: 154- 
155, pi. 7, fig. 53) seems improbable, be- 
cause the specimen has undulating commar- 
ginal sculpture. 

Discussion 

In southern California, typical Gari califor- 
nica is ovate-subquadrate, flattened, and con- 
spicuously rayed with color, and the shells are 
heavy. In Washington and British Columbia, 
specimens are thin-shelled, ovate-elongate, 
inflated, and lack conspicuous radial color 
bands (Fig. 3). On the Channel Islands of 
southern California and northern Baja Califor- 
nia, thin, elongate specimens are also en- 



THE EASTERN PACIFIC RECENT SPECIES OF THE BIVALVE GENUS GARI 



7 



countered (Fig. 4), but this material is scarce 
and represented by only a few lots. Such ma- 
terial accounts for my record of Gari regularis 
from the Islas Coronados, Baja California 
(Coan, 1973: 45; USNM 207676), and proba- 
bly that of G. fucata from San Nicolas Island, 
California, by Hertz (1994). 

In the original description of the Pleistocene 
Psammobia kazusensis, Yokoyama (1922) 
compared it only to the Panamic G. maxima 
(Deshayes, 1855). Recent Japanese material 
was allocated by various authors to G. califor- 
nica. Yamamoto & Habe (1959) first recog- 
nized that Yokoyama's species was the same 
as that identified as G. californica in the Re- 
cent fauna. Based on comparison with speci- 
mens of Gari californica probably from main- 
land southern California, they concluded that 
western Pacific material differed from eastern 
Pacific specimens in its lack of color rays, 
more elongate shape, and more rounded an- 
terior margin. I have long had doubts about 
these supposed differences (Coan, 1973: 43) 
and have still more now. The known variation 
of eastern Pacific material completely encom- 
passes the supposed differences between G. 
californica and G. kazusensis. I have endeav- 
ored to find additional characters to differenti- 
ate the seven Asian lots available to me from 
abundant eastern Pacific material, including 
such features as degree of inflation, beak po- 
sition, shape of the anterior and posterior 
margins, hinge teeth morphology, nymph 
length, and palliai sinus shape and extent of 
its overlap with the palliai line. I was not able 
to find any conclusive patterns. Until a more 
thorough, statistical study is possible with 
many more lots, there is no choice at present 
but to consider these taxa as disjunct popula- 
tions of the same species. 

Such a statistical study might also reach a 
conclusion that the taxonomic situation on the 
coast of western North America is more com- 
plex than is represented by a single taxon. 
Perhaps populations in the Pacific Northwest 
and/or on the offshore islands of southern 
California may come to be regarded as dis- 
tinct taxa. Such conclusions should also be 
supported by additional characters, ideally 
both anatomical and biochemical. 

Gari (Gobraeus) fucata (Hinds, 1845) 
Figures 5-7, 20 

Tellina fucata Hinds, 1845 

Hinds, 1845: 67, pi. 21, fig. 4; Carpenter, 
1857b: 207; Bertin, 1880: 113 [Gari]; Dall, 



1898a: 58, 62 [Psammobia (Gobraeus)); 
Keen, 1958: 190 [Gari]; Olsson, 1961: 
357 [possibly senior synonym of Gari reg- 
ularise Palmer, 1963: 313; Keen, 1966: 
268, pi. 46, fig. 4; Keen, 1971: 240, 241, 
fig. 602; Coan, 1973: 43-44 [as a possi- 
ble senior synonym of Gari edentula] 

u ?Siliquaria" edentula Gabb, 1 869 

Gabb, 1869: 53, 89, pi. 15, fig. 11; Dall, 
1898a: 58 [Psammobia {Gobraeus)]; 
Arnold, 1903: 168 [Psammobia (Psam- 
mobia)]; Dall, 1925: 23, 37, pi. 19, fig. 1 
[Psammobia (Gobraeus)]; I. S. Oldroyd, 
1 925: 1 85, pi. 57, fig. 1 ; Stewart, 1 930: 7, 
281-282, pi. 13, fig. 3; Grant & Gale, 
1931: 382-383, 924, pi. 21, fig. 5; J. Q. 
Burch, 1945: 22; Hertlein & Grant, 1972: 
305, pi. 48, figs. 13, 15 [as Gari H enden- 
tulus"]; Coan, 1973: 43-44, figs. 2-5, 21. 

Gari "fuscata," auctt., nom. null. 
Simone, 1998: 37 

not Gari fucata, auctt. 

Hertz, 1994: 85, fig. 3 [= Gari californica] 

Type Materials & Localities 

T. fucata- BMNH 1879.2.26.192, holotype, 
pair; length, 41.2 mm; height, 20.4 mm; 
thickness, 7.5 mm (Fig. 5). Bahía Mag- 
dalena, Baja California Sur, Mexico 
(24.6°N); R. B. Hinds. It is possible that 
this specimen was actually from further 
north, because no specimens of this 
species have yet been obtained from 
south of Punta Eugenia, Baja California 
Sur. 

S. edentula-MCZ (Paleontology) 15035, 
holotype, left valve, length, 66 mm (Fig. 
6). San Fernando, Los Angeles County, 
California; "San Fernando" [? = Pico] For- 
mation; late Pliocene. 

Description 

Shell elongate (l/h = 2.1-2.2), moderately 
inflated (h/th = 2.2), subequivalve, with nar- 
row anteroventral gape and moderate poste- 
rior gape. Valves thin. Beaks at approximately 
38-43% from anterior end. Anterior end 
sharply rounded. Posterior end obliquely sub- 
truncate. Surface with weak commarginal 
striae, strongest on ends. Periostracum dark 
tan, eroded near beaks. Surface yellowish- 
tan, with a few brownish radial rays. 

Interior radial thickenings medial to adduc- 
tor muscle scars conspicuous. Palliai sinus 
reaching to just under beaks, its ventral mar- 




FIGS 5-7. Garifucata. FIG. 5. Holotype of Tellina fucata, right and left valves; BMNH 1879.2.26.192; length, 
41 .2 mm. FIG. 6. Holotype of Siliquaria edentula, left valve; MCZ (Paleontology) 15035; length 66 mm. FIG. 
7. CAS 113143, right and left valves, San Pedro, California; 46 m; length, 122.8 mm. 



THE EASTERN PACIFIC RECENT SPECIES OF THE BIVALVE GENUS GARI 



gin merged with palliai line for 85-95% of its 
length (Fig. 20). 

Nymphs of moderate thickness, approxi- 
mately 21 -23% of shell length. Right valve 
with narrow, projecting anterior and posterior 
cardinal teeth. Left valve with a narrow, very 
projecting, slightly posteriorly directed ante- 
rior cardinal and a very narrow posterior 
cardinal tooth. Length to 139.8 mm [CAS 
116475; San Pedro, California]. 

An additional Recent specimen is illustrated 
in Figure 7. 

Distribution 

From Ventura, Ventura Co., California 
(34.3°N) [SBMNH 126388; LACM 59-59.11], 
to Punta Eugenia, Baja California Sur (27.9°N) 
[UCMP E2323], Mexico, possibly as far south 
as Bahía Magdalena, Baja California Sur 
(24.6°N) (type locality of Tellina fucata), but no 
other collections contain material from this far 
south; 15-110 m (mean, 36 m; n = 31). I have 
seen 48 Recent lots, including the type of T. fu- 
cata. 

This species has been recorded from the 
late Pleistocene Point Dume terrace (Addicott, 
1964: 146), the Palos Verdes Sand at Pacific 
Palisades (Valentine, 1956: 196), Playa del 
Rey (Willett, 1937: 391), and San Pedro (De- 
Long, 1941: 238), Los Angeles County, and 
the Newport Bay terraces (Kanakoff & Emer- 
son, 1959: 22), Orange County, California. 
From strata of early Pleistocene age, it has 
been recorded from the Anchor Silt, northwest 
Los Angeles Basin (Rodda, 1957: 2484), and 
the San Pedro Sand of San Pedro (Arnold, 
1903: 168; B. L. Burch, 1947: 8; Valentine & 
Meade, 1961: 17), Los Angeles County. It is 
here reported from the early to middle Pleis- 
tocene Santa Barbara Formation at Bath- 
house Beach, Santa Barbara, Santa Barbara 
County (SBMNH 125792), California. In the 
late Pliocene, it is recorded from the Pico For- 
mation (probable type locality of Siliquaria 
edentula; English, 1914: 210; Kew, 1924: 78), 
the Saugus Formation (Groves, 1991 : 16-17), 
and the Fernando Formation (Pressler, 1929: 
336), Ventura County, and the Pico Formation 
(Woodring et al., in Winterer & Durham, 1962: 
304, as "? edentula") and the Fernando For- 
mation, Los Angeles County (Willett, 1946: 
29), and the San Diego Formation, San Diego 
County (Hertlein & Grant, 1972: 305), Califor- 
nia, and northern Baja California (Rowland, 
1972: 29). It has been recorded in the early 



Pliocene Etchegoin Formation, central Califor- 
nia (Adegoke, 1969: 124; Nomland, 1917: 
212, 220, opp, p. 230), and the Towsley For- 
mation, Los Angeles County (Kern, 1 973: 79). 
There are two records in strata of late Miocene 
age in central California (Santa Margarita For- 
mation -Adegoke, 1969: 124; Gale, in Pre- 
ston, 1 931 : 1 5). There is one middle Miocene 
record from the Temblor Formation (Stewart, 
1 946: Table 2, as "? edentula"), and a record in 
the early to middle Miocene Astoria Formation 
of Oregon (Moore, 1 964: 82, pi. 31 , fig. 1 0, as 
"?aff. G. edentula"). There is also one doubtful 
Miocene or Oligocène record from the central 
California San Ramon Formation (Clark, 
1918: 305, as "aff. edentula"). 

Discussion 

When I reviewed this group several years 
ago, I had seen very few juvenile specimens 
of this species, and no material was present in 
collections from the Pacific coast of Baja Cal- 
ifornia Sur, Mexico. As a result, I remained 
tentative about the synonymy of Gari fucata 
with G. edentula. While neither of these lacks 
has been completely filled, because this 
species is rare, I have now examined the type 
specimen of G. fucata, other small specimens 
that match it, and a new southern record of 
the species has come to light in the UCMP. As 
a result, I am now fully convinced that Tellina 
fucata represents a small specimen of what 
was long known as G. edentula. 

Gari (Gobraeus) lata (Deshayes, 1855) 
Figures 8, 9, 21 

Psammobia lata Deshayes, 1855 

Deshayes, 1 855: 31 8; Reeve, 1 857: pi. 1 , 
fig. 7; Tryon, 1869: 76 [Gari (Psam- 
macola-sic)]; Bertin, 1880: 124 [Gari]; 
Hertlein & Strong, 1950: 218 [Gari 
(Psammocola)]; Keen, 1958: 190, 191, 
fig. 464; Olsson, 1961: 356, 536, pi. 63, 
fig. 9 [Gari (Gobraeus)]; Keen, 1971: 242, 
243, fig. 605. 

Psammobia (? Amphichaena) regularis Car- 
penter, 1864 

Carpenter, 1864a: 312 [1872 reprint 
210], 1864b: 618 [1872 reprint: 104] 
Tryon, 1869: 78 [Gari (Amphichaena)] 
Bertin, 1880: 128 [Gari]; Dall, 1898a: 57 
[Psammobia (Gobraeus)]; I. S. Oldroyd, 
1925: 184 [in part]; Palmer, 1958: 
113-114, 340, pi. 15, figs. 1-6 [Gari (Go- 



10 



COAN 




FIGS. 8-9. Gari lata. FIG. 8. Lectotype of Psammobia lata, right and left valves; BMNH 1966549/1; length, 
61.2 mm. FIG. 9. Holotype of Psammobia regularis, right and left valves; USNM 19407; length, 26.6 mm. 



braeus)]; Olsson, 1 961 : 356-357 [in part; 
as a possible synonym of Gari fucata]; 
Palmer, 1963: 313; Coan, 1973: 45, fig. 5 
[in part; figures 7, 22 = Gari cali fornica]; 
Keen, 1971: 242, 243, fig. 608; Myers et 
al., 1984:22-23, figs. 9-11. 
not Gari regularis, auctt. 

Hertlein & Strong, 1950: 218-219, pi. 2, 
fig. 10; Keen, 1958: 190, 191 [ = Gari 
(Dysmea) helenae] 



Gari maxima Deshayes, auctt., non 
Deshayes, 1855 
Olsson, 1 961 : 356 [in part], pi. 63, fig. 7a, b 

Type Materials and Localities 

P. /aia-BMNH 1966549/1, lectotype herein, 
the largest pair, closest to Reeve's figure 
(1857:pl. 1, fig. 7); length, 61.2 mm; 
height, 38.5 mm; thickness, 19.3 mm 



THE EASTERN PACIFIC RECENT SPECIES OF THE BIVALVE GENUS GARI 



11 



(Fig. 7). BMNH 1966549/2-3, paralecto- 
types, pairs, lengths, 55.7 mm, 52.5 mm. 
"Central America; Sancta Elena"; Hugh 
Cuming. BMNH 1984279, probable para- 
lectotype, pair, labeled as "var.," length 
57.7 mm. Both lots labeled as having 
come from Santa Elena, Guayas Prov- 
ince, Ecuador (2.2°S). No Cuming speci- 
mens from Central America have come to 
light. 
P. regularis-USNM 19407, holotype, pair, 
length, 26.5 mm; height, 14.5 mm; thick- 
ness, 5.2 mm (Fig. 8). Cabo San Lucas, 
Baja California Sur, Mexico (22.9°N); 
John Xantus. 

Description 

Shell ovate (l/h = 1.5-1.7), moderately in- 
flated (h/th = 1 .9-2.1 ), subequivalve, with nar- 
row anteroventral and posterior gapes. Valves 
moderately heavy. Beaks at approximately 
40-42% from anterior end. Anterior end 
sharply rounded. Posterior end subtruncate. 
Surface with light to moderate commarginal 
growth lines, somewhat stronger on ends. Pe- 
riostracum light tan to dark brown, sometimes 
present only as marginal remnants, often less 
eroded than in other species. Surface suf- 
fused with purple, sometimes accentuated in 
commarginal bands, and with tan radial 
bands. Small specimens purple. 

Interior radial thickenings medial to adduc- 
tors present but not conspicuous, most evi- 
dent at anterior end. Palliai sinus of moderate 
height, reaching to or slightly beyond beaks, 
its ventral margin closely paralleling palliai 
line and fused with it for about 70-83% of 
sinus length. Palliai line turned ventrally near 
its posterior end (Fig. 21 ). 

Nymphs of moderate thickness, 20-24% of 
shell length. Right valve with a moderate, pro- 
jecting anterior cardinal and a narrow project- 
ing posterior cardinal, slightly bifid on end. 
Left valve with a narrow, vertical, projecting 
anterior cardinal, slightly bifid on end, and a 
narrow posterior cardinal. Length to 65.5 mm 
[SBMNH 145078; Isla Taboga, Panama]. 

Distribution 

Punta Entrada [north entrance], Bahía 
Magdalena, Baja California Sur, Mexico 
(24.5°N) [LACM 71.14.57], into the Golfo de 
California as far north as Isla San Luis, Baja 
California Norte (29.9°N) [SBMNH 12984], 



and Puerto Lobos, Cabo Tepoca, Sonora 
(29.3°N) [Skoglund Coll.], Mexico, south to 
Santa Elena, Guayas Province, Ecuador 
(2.2°S) [type locality; CAS 113126; UMML 
30.10883]; intertidal zone to 110 m (mean, 20 
m; n = 37). I have examined 62 lots, including 
the types. 

It is likely that the record of Gari maxima 
from the Pleistocene Third Terrace at Santa 
Elena, Guayas Province, Ecuador (Hoffstet- 
ter, 1948: 80), was instead based on Gari lata. 

Discussion 

This species overlaps the distribution of 
Gari californica at Bahía Magdalena, Baja 
California Sur, Mexico, where they have been 
collected at the same station [LACM 71-14]. 
Gari lata can most easily be distinguished by 
its purple color, more ovate outline, narrower 
palliai sinus, and a palliai line that is turned 
ventrally at its posterior end. 

The specimens tentatively identified by Dall 
(1898a: 57, 1900: 976) as Psammobia (Gob- 
raeus) "vaginatus" [sic for vaginoides] Reeve, 
1857, supposedly from Charlotte Harbor, 
Charlotte County, Florida [USNM 36167], 
prove to be based on mislocalized specimens 
of Gari lata (see also Other Records and 
Notes below). 

Gari (Gobraeus) maxima (Deshayes, 1855) 
Figures 10, 22 

Psammobia maxima Deshayes, 1855 

Deshayes, 1 855: 317; Reeve, 1 857: pi. 1 , 
fig. 4; Tryon, 1869: 77 [Gari (Psam- 
macola-sic)]; Bertin, 1880: 123 [Gari;] 
Dall, 1898a: 57 [Psammobia (Gob- 
raeus)]; Hertlein & Strong, 1950: 218 
[Gari (Psammocola)]; Keen, 1958: 190, 
191, fig. 465; Olsson, 1961: 356, 536, pi. 
63, fig. 7 [Gari (Gobraeus); in part; figs. 
7a, b = Gari lata]; Keen, 1971: 242, 243, 
fig. 606. 

Type Material & Locality 

P. maxima- BMNH 1966552/1, lectotype 
herein, the smaller of two pairs, figured 
by Reeve (1857); length, 118.4 mm; 
height, 68.9 mm; thickness, 37.0 mm 
(Fig. 10). BMNH 1966552/2, paralecto- 
type, pair, length, 125.1 mm; Hugh Cum- 
ing. Originally described without a local- 



12 



COAN 




FIG. 10. Gari maxima. Lectotype of Psammobia maxima, right and left valves; BMNH 1966552/1; length 
118.4 mm. 



ity; attributed to Panama by Reeve 
(1857) (approximately 9°N). 



specimens. Length to 125.1 mm (the paralec- 
totype). 



Description 

Shell ovate (l/h = 1.6-1.7), moderately in- 
flated (h/th = 1 .8-2.5), subequivalve, with nar- 
row anteroventral and posterior gapes. Valves 
moderately heavy. Beaks at approximately 
31 -36% from anterior end. Anterior end 
sharply rounded; posterior end subtruncate. 
Surface with weak commarginal growth lines, 
strongest at margins. Periostracum moderate 
to dark tan, frequently present only as mar- 
ginal remnants. Shell exterior white to yellow- 
ish-tan, with conspicuous reddish-brown ra- 
dial bands. Small specimens mottled with 
purple near beaks. 

Interiorly with radial shell thickenings me- 
dial to adductor muscle scars, most conspic- 
uous anteriorly. Palliai sinus of moderate 
height, not reaching past beaks, its ventral 
margin paralleling palliai line and merged with 
it for 50-70% of sinus length; palliai line 
turned ventrally near its posterior end (Fig. 
22). 

Nymphs heavy in large specimens, 22- 
36% of shell length. Right valve with a moder- 
ate, projecting anterior cardinal and a very 
narrow posterior cardinal. Left valve with a 
narrow, projecting, slightly posteriorly directed 
anterior cardinal and a very narrow posterior 
cardinal. Teeth frequently broken off in large 



Distribution 

Mazatlán, Sinaloa, Mexico (23.2°N) [CAS 
113118], to Panama (no specific stations; ap- 
proximately 9°N) [type locality; UCMP 
R2416]; 3-15 m (mean, 9 m; n = 10). I have 
seen only 13 lots, including the types. 
Records of this species from Ecuador (Ols- 
son, 1961) were based on specimens of Gari 
lata (UMML 30.10881 -10883; PRI 25826), or 
probably were (Hoffstetter, 1952: 43). A 
record of Gari maxima from the Pleistocene 
Third Terrace at Santa Elena, Guayas Prov- 
ince, Ecuador (Hoffstetter, 1948: 80), was 
likely instead to have been based on speci- 
mens of Gari lata. 



Discussion 

Almost all known specimens are large. Until 
small specimens came to light [e.g., SBMNH 
145079; Bahía Santiago, Colima, Mexico], I 
was not completely convinced that this 
species was separable from Gari lata. How- 
ever, Gari maxima differs from G. lata in being 
light tan rather than purple in color and in hav- 
ing a heavier hinge plate at a similar size. It 
differs from Gari panamensis in being more 
oval. It differs from Gari californica in having 



THE EASTERN PACIFIC RECENT SPECIES OF THE BIVALVE GENUS GARI 



13 



its beaks closer to the anterior end, the lower 
limb of its palliai sinus parallels the palliai line 
for a longer distance before merging with it, 
and the anterior cardinal in the right valve is 
more projecting. 

Gari (Gobraeus) panamensis Olsson, 1961 
Figures 11, 23 

Gari (Gobraeus) panamensis Olsson, 1961 
Olsson, 1961: 357, 536, pi. 63, fig. 11; 
Keen, 1971: 242, 243, fig. 607; Myers et 
al., 1984:21-22, figs. 6-8. 



Type Material & Locality 

G. panamensis- ANSP 282396, holotype, 
pair; length, 55.2 mm; height, 31.7 mm; 
thickness, 16.3 mm (Fig. 11). UMML 
30.10880, four paratypes, lengths: 51.2 
mm (pair), 49.0 mm (right valve), 48.9 
mm (left valve), 43.5 mm (right valve, 
broken and repaired). Palo Seco, 
Panama Province, Panama (8.9°N). 



Description 

Shell ovate-elongate (l/h = 1.7-1.8), aver- 
age in inflation (h/th = 1.9-2.1), subequivalve, 
left valve slight more inflated, with narrow an- 
teroventral and posterior gapes. Valves thin. 
Beaks at approximately 40-42% from anterior 
end. Anterior end sharply rounded. Posterior 
end obliquely subtruncate. Surface with mod- 
erate commarginal striae on central slope, 
more raised on anterior slope, especially 
raised, lamellar on posterior slope and in left 
valve. Periostracum light to moderate tan, 
eroded in some specimens and present only 
as marginal remnants. Surface light tan, with 
dark tan to purple radial color bands. Juvenile 
shell mottled with purple. 

Interior radial thickenings medial to adduc- 
tors weak, most prominent anteriorly. Palliai 
sinus reaching to just below beaks, narrow, its 
ventral margin closely paralleling palliai line, 
merged for about 70-78% of sinus length. 
Palliai line turned ventrally at its posterior end 
(Fig. 23). 

Nymphs of moderate thickness, approxi- 
mately 17-18% of shell length. Right valve 
with a moderate, projecting anterior cardinal 
and a narrow, projecting posterior cardinal, 
slightly bifid at end. Left valve with a narrow, 
nearly vertical, projecting anterior cardinal 



and a very narrow posterior cardinal. Length 
to 55.2 mm (holotype). 

Distribution 

Off west Isla Danzante, Baja California Sur 
(25.8°N) [Skoglund Coll.], Mexico, to Playas 
[de Villamil], Guayas Province, Ecuador 
(2-6°S) [Skoglund Coll.]; 2-59 m (mean, 31 
m; n = 5). This is the least common eastern 
Pacific Gari, known from only 9 Recent lots, 
including the types. 

Discussion 

Olsson differentiated Gari panamensis from 
"G. maxima" on several grounds. (The only 
specimens in his collection labeled as G. 
maxima are here reidentified as G. lata- lots 
listed in the Discussion under G. maxima.) He 
said that G. panamensis was (1) longer and 
lower, (2) the beaks were less central, (3) the 
posterodorsal margin was longer and more 
descending, and (4) the posterior end was 
longer and more attenuate. Gari panamensis 
is indeed more elongate than G. lata, but the 
beaks are in approximately the same position. 
Whereas the holotype of Gari panamensis 
has a very tapered posterior end, three of the 
four paratypes are not as tapered, and the 
other shape differences he noted are not use- 
ful criteria as expressed. On the other hand, 
Gari lata is more ovate than G. panamensis, 
has a more rounded anterior end, a more 
truncate posterior end, is always purple in 
color, and lacks conspicuous commarginal 
sculpture on the anterior and posterior ends, 
particularly the lamellar sculpture present on 
the posterodorsal slope of G. panamensis. 

Gari panamensis is similar to Gari (Gob- 
raeus) wagneri (Dall, 1898a), described from 
the Late Pliocene-early Pleistocene Caloosa- 
hatchee Formation of Florida (Dall, 1 892: 211, 
nomen nudum; 1898b: 920, pi. 25, fig. 10; 
1900: 977) (Fig. 12). This fossil species at- 
tained a larger size; the holotype, a set of 
paired valves [USNM 156871], measures 
76.6 mm in length, 39.8 mm in height, and 
19.0 mm in thickness. Examination of several 
lots from the Pliocene and Plio-Pleistocene of 
Florida [UF 9569, 16105, 26063, 35848, 
56578, 56578] reveals that this fossil species 
is also more pointed anteriorly and has more 
prominent commarginal sculpture, which is 
especially lamellar on the left valve in a radial 
band between the central and posterior 
slopes. Additionally, the ventral margin of the 




FIGS. 11-12. Gari panamensis. Holotype of Gari panamensis, right and left valves; ANSP 282396; length 
55.2 mm. FIG. 12. Gari wagneri (Dall, 1898), right and left valves. UF 16105, Macasphalt shell pit 01 (origi- 
nal pit) (SO009), Sarasota Co., Florida; Pincrest Beds; Pliocene; length, 72.2 mm. 



palliai sinus is coincident with the palliai line 
for only about 50% of the sinus length. It is 
possible that G. wagneri was ancestral to G. 
panamensis. 



Gari {Gobraeus) solida (Gray, 1828) 
Figures 13, 14, 24 

Solen (Solenocurtus) sol id us Gray, 1828 
Gray, 1828: 7, pi. 3, fig. 12, 12a [plate 



prepared but never published]; d'Or 
bigny, 1845: 539 [Arcopagia]; Hupé 
1854: 364-365; 1858: pi. 7, fig. 4 [Psam 
mobia]; Reeve, 1856: pi. 3, fig. 18; Berlin 
1880: 124-125 [Gari]; Stempel, 1899 
239 [Psammobia]; Lamy, 1908: 51; Dall 
1 909: 273; Carcelles & Williamson, 1 951 
344; Marincovich, 1973: 17, fig. 20 [Gari] 
Soot-Ryen, 1959:61;Osorioetal., 1979 
31 -32, fig. 36; Santa Maria, 1982: 26, pi 
10, fig. 10 



THE EASTERN PACIFIC RECENT SPECIES OF THE BIVALVE GENUS GARI 1 5 




FIGS. 13-15. Gari solida. FIG. 13. Lectotype of Solen solidus Gray, right and left valves; BMNH 1985180/1; 
length, 62.2 mm. FIG. 14. Original figure from Philippi (1844) of Psammobia solida Philippi, right valve; 
length, 67.3 mm. 

FIG. 1 5. Garin. sp.?, right and left valves; LACM 34-283.8, Isla Española, Galapagos Islands; 55 m; length, 
20.3 mm. 



16 



COAN 



Psammobia solida Philippi, 1844 
Philippi, 1844: pi. 1, fig. 1 

not Solecurtus solidus (Gray, 1 828), auctt. 
G. B. Sowerby II, 1874: pi. 1, fig. 3; 
Küster & Clessin, 1888: 95, pi. 24, fig. 1 
[= Tagelus peruanus (Dunker, 1862: 426, 
as Siliquaria)] 

Psammobia crassa Hupé, 1854, 1858 [in syn- 
onymy] 

Hupé, 1854: 365 [in synonymy], 1858: pi. 
7, fig. 4 [on pi. expl.] 

Type Materials & Localities 

S. solidus Gray BMNH 1985180/1, lecto- 
type herein, the larger of two pairs and 
closest to the original dimensions; length, 
62.2 mm; height, 41.6 mm; thickness, 
24.8 mm (Fig. 13). BMNH 1985180/2, 
paralectotype, length, 56.5 mm. Arica, 
Tarapacá Province, Chile (18.5°S); Rev. 
W. Hannah [label]. 

P. solida Philippi -Presumably lost. Isla 
Chiloe, Chiloe Province, Chile (approx. 
42°S); E. B. Philippi [brother of R. A. 
Philippi]; originally stated dimensions: 
length, 76.3 mm; height, 56.7 mm; thick- 
ness, 32.7 mm (Fig. 14). A specimen in 
the MNH-U [ZMB 3145] is too small 
(length, 50.8 mm) to be the originally fig- 
ured specimen, and its label says only 
"Chile", with no mention of Isla Chiloe. 
Not in the Museo Nacional de Historia 
Natural, Santiago, Chile (D. Frassinetti, 
e-mail, 22 January 1999). 

Description 

Shell ovate (1/h = 1.4-1.5), inflated (h/th = 
1.7-1.8), equivalve, with very narrow gapes 
anteroventrally and posteriorly. Valves heavy. 
Beaks at 34-46% from anterior end, with pos- 
terior end proportionately longer in large 
specimens. Anterior end rounded. Posterior 
end rounded to subtruncate, slightly set off by 
a very shallow radial sulcus. Surface with 
heavy commarginal growth lines. Perios- 
tracum tan to dark brown, often eroded and 
present only along margins. Surface white to 
yellowish-tan; some specimens with faint, 
narrow tan radial bands. Small specimens are 
proportionately more elongate and more cov- 
ered with periostracum. 

Interiorly with heavy radial thickenings me- 
dial to adductor muscle scars. Palliai sinus 
narrow, rounded anteriorly, not reaching past 



beaks, its ventral margin merged with palliai 
line from 30-50% of sinus length; palliai line 
only slightly turned ventrally near its posterior 
end (Fig. 24). 

Nymphs very heavy, 25-32% of shell 
length. Right valve with a moderate, project- 
ing anterior cardinal and a very narrow poste- 
rior cardinal. Left valve with a narrow, project- 
ing, posteriorly directed, slightly bifid anterior 
cardinal and a very narrow posterior cardinal. 
Length to 93.0 mm [MNHN-UC 500-37; Los 
Molles, Aconcagua Province, Chile]. 

Distribution 

Arica, Tarapacá Province (18.5°S) (type lo- 
cality), to Fiordo Reñihué, Chiloe Province 
(42.5°S) [CAS 113130], and to Rio Inio, 
Chiloe Province, Chile (43.4°S) [MNHN-UC 
500-44]; 3-46 m (mean, 17 m; n = 3). I have 
seen 17 lots, including the types. 

Discussion 

In Philippi's account of Psammobia solida, 
there is no mention of Gray's publication, and 
Philippi quotes the solida as being of his own 
authorship. His treatment must therefore be 
taken as the proposal of a new, homonymous, 
synonymous taxon. Fortunately, there is no 
doubt as to what his species is, because the 
type material has evidently been lost. 

The plates to Hupé's work were completed 
years before they were issued. On his plate 7, 
this species was illustrated as new, Psammo- 
bia crassa. However, the text appeared be- 
fore the plates, and the name P. crassa first 
appears this 1854 text in the synonymy of P. 
solida Gray, with mention of the plate expla- 
nation; the eventual publication of the actual 
plate in 1858 is thus part of this same appear- 
ance as a junior synonym (ICZN Code Art. 
11e) and not the proposal of a new taxon. 

This species is of commercial importance in 
Chile. For example, some 26 tons were har- 
vested in 1992 (C. Osorio, e-mail, 28 Jan. 
1999). 

Gari (Gobraeus) new species? 
Figures 15, 25 

There is a single small, slightly broken pair 
of valves from 55 m near Isla Española, Gala- 
pagos Islands, in the LACM (34-283.8) that 
probably represents an undescribed species 
of Gari (Gobraeus). This 20.3-mm long speci- 



THE EASTERN PACIFIC RECENT SPECIES OF THE BIVALVE GENUS GAFtl 



17 



men is very elongate (1/h = 2.0), thin (h/th = 
2.3), fragile, and light tan, with a few light, in- 
distinct brown spots. The beaks are at 47% 
from the anterior end. The left valve has a 
slightly projecting lunule, and the posterior 
slopes of both valves lack radial ribs. The pal- 
liai sinus is narrow and reaches to just under 
the beaks, its ventral margin overlapping the 
palliai line by 68% in the left valve and by 88% 
in the right valve; it posterior end is not turned 
ventrally at its posterior end (Fig. 25). The 
right valve has narrow, projecting anterior and 
posterior cardinal teeth. The left valve has a 
narrow, projecting anterior cardinal and a very 
thin posterior cardinal. 

With only a single, small, broken specimen 
in hand, it cannot be described. It is typical of 
young specimens of Garito be more elongate 
than adults. I expect that an adult of this 
species will eventually come to light from the 
Galapagos Islands. 



Subgenus (Dysmea) Dali, Bartsch & Rehder, 
1939: 174 

Type species (original designation): Solen oc- 
cidens Gmelin, 1791: 3228. Recent, 
Indo-Pacific. 

This subgenus is as yet not well differenti- 
ated from Gobraeus, some members of which 
also have a lunular projection on the left valve, 
albeit less prominent (Willan, 1993: 56). In- 
deed, such a structure is present in small 
specimens of G. {Gobraeus) californica. The 
only other characteristics cited for this sub- 
genus are subdued sculpture and a flared pos- 
terior end. The latter feature is not true of the 
eastern Pacific Gari helenae, but this species 
is clearly allied to G. occidens on the basis of 
its color, overall shape, and the lunular projec- 
tion. There are four Recent species in the sub- 
genus, which occurs in the Indo-Pacific, east- 
ern Pacific, and western Atlantic. 

The lunular projection in the left valve acts 
as a third cardinal tooth. Although there is pe- 
riostracum on its surface, there is no evidence 
that it acts as a chondrophore, as suggested 
by Myers et al. (1 984), because there is no lig- 
ament attached to its surface. This structure 
was identified as a cardinal tooth by Simone 
(1998) in describing G. Unbares. 

There is a single specimen of Gari occidens 
from an old collection in the ANSP, labeled as 
having been collected in Panama [ANSP 
51669; length, 83 mm]. Without additional ev- 
idence, it cannot be assumed that this wide- 



spread Indo-Pacific species reaches the west 
coast of the New World. 

Gari {Dysmea) helenae Olsson, 1961 
Figures 16, 26 

Gari (Gobraeus) helenae Olsson, 1961 

Olsson, 1961: 357-358, 536, pi. 63, fig. 
12, 12a; Keen, 1971: 240, 241, fig. 603; 
Myers et al., 1984: 20-25, figs. 1-4 
12-14; Willan, 1993: 58 [Gari {Dysmea)}; 
Hickman & Finet, 1999: 34, fig. 53 

Gari regularis Carpenter, auctt., non Carpen- 
ter, 1864 

Hertlein & Strong, 1950: 218-219, pi. 2, 
fig. 10 [Gari {Psammocola)]; Keen, 1958: 
190, 191, fig. 466. 

Gari spp., auctt. 

Olsson, 1 961 : 356-357, 536, pi. 63, fig. 1 
[as the "Gari regularis o\ Hertlein & Strong, 
1950"] Keen, 1971: 240, 243, fig. 604 [an 
unusual specimen allied to Gari helenae] 

Type Material & Locality 

G. helenae- ANSP 282395, holotype; length, 

52.3 mm; height, 27.5 mm; thickness, 

14.4 mm (Fig. 16). Archipelago de las 
Perlas, Panama (approx. 8.3°N); Helen 
L. Beil. 

Description 

Shell ovate to ovate-elongate (1/h = 1.7- 
1.9), moderately inflated (h/th = 1.8-2.3), 
subequivalve, with narrow posterior and very 
narrow anterior gapes. Beaks at approxi- 
mately 44-50% from anterior end. Anterior 
end sharply rounded. Posterior end obliquely 
subtruncate. Surface with weak commarginal 
striae; posterior end with very fine radial ribs, 
which show along interior posterior margin as 
fine granules. Periostracum tan to dark 
brown, eroded over dorsal and central slopes. 
Surface tan to purple mottled with white, with 
conspicuous brownish radial rays in many 
specimens; rarely with brownish zigzag lines. 

Interior radial thickenings medial to adduc- 
tors scars inconspicuous. Palliai sinus reach- 
ing nearly to beaks, broad, its ventral margin 
paralleling palliai line and merged with it for 
45-50% of sinus length (Fig. 26). 

Nymphs moderately thick, 15-17% of shell 
length. Right valve with a moderate, project- 
ing anterior cardinal and a moderate, project- 
ing, slightly bifid posterior cardinal; with an an- 



18 



COAN 




FIGS. 16-18. Gari helenae. FIG. 16. Holotype of Gari helenae, right and left valves; ANSP 282395; length, 
52.3 mm. FIG. 17. Gari ?circe (Mörch, 1876). ANSP 368524, right valve; Grand Bahama Island; length, 29.2 
mm. FIG. 18. Gari ' linhares Simone, 1998. Paratype, right and left valves; MZSP 28786; length, 41.6 mm. 



THE EASTERN PACIFIC RECENT SPECIES OF THE BIVALVE GENUS GARI 



19 



terior recess for lunular projection of left valve. 
Left valve with a moderate, projecting, nearly 
vertical, slightly bifid anterior cardinal and a 
narrow posterior cardinal; and with a lunular 
projection that functions as an anterior cardi- 
nal tooth. Length to 61.3 mm (Kaiser Coll.; 
Isla San Pedro Mártir, Gulf of California, Mex- 
ico). 

Distribution 

Laguna Ojo de Liebre [Scammons], Baja 
California Sur (27.8°N) [SBMNH 1 45062], into 
the Golfo de California where it occurs as far 
north as Puertecitos, Baja California (30.3°N) 
[CAS 113135], and Cabo Tepoca, Sonora 
(29.3°N) [Skoglund Coll.], Mexico, south to 
Isla Salango, Manabi Province, Ecuador 
(1 .6°S) [SBMNH 1 45064]; also on Isla Isabela 
in the Galapagos Islands on (Hickman & 
Finet, 1999; Hickman Coll.) (0.2°N), intertidal 
zone to 155 m (mean, 22 m; n = 58). I have 
seen 98 lots. It may also occur in the Galapa- 
gos Islands on Isla Rábida (Charles Darwin 
Research Station 654) (Finet, 1994: 116; 
Finet, e-mail, 19 April 1999). 

Discussion 

This species differs from Gari (Dysmea) oc- 
cidens (Gmelin, 1791), the Indo-Pacific type 
species of the subgenus, in not attaining as 
large a size (G. occidens attains 120 mm), in 
having fine radial sculpture on the posterior 
slope, a less flaring nymph, and a more 
rounded, less truncate, less flaring posterior 
end. In addition, the ventral margin palliai 
sinus of G. occidens is detached from the pal- 
liai line for a greater distance. Gari occidens 
was discussed at length by Willan (1993: 56- 
58, 116-117, 126, figs. 223-236, 397). 

The relationship of G. helenae to members 
of this subgenus occurring in the western At- 
lantic is far from clear. All specimens of Gari 
from the western Atlantic I have had an op- 
portunity to examine belong in Gari (Dysmea). 

The first-named taxon is Psammobia circe 
Mörch, 1876 (p. 373) (originally stated dimen- 
sions: length, 40 mm; height, 22 mm; Tórtola, 
U. S. Virgin Islands; R. Swift) (see also Dall, 
1898: 57, 1900: 976). Unfortunately, the type 
material seems to be lost. It is not at the Zoo- 
logical Museum, University of Copenhagen, 
Mörch's home institution (T Schiotte, e-mail, 
12 January 17, 1999); nor in the Muséum Na- 
tional d'Historire Naturelle in Paris, where 



many types of species described in the Jour- 
nal de Conchyliologie are held (P. Maestrati, 
e-mail, 24 November 1998); nor in the Acad- 
emy of Natural Sciences in Philadelphia, 
where the Swift collection was eventually de- 
posited (G. Rosenberg, 30 November 1998); 
nor in the BMNH collection (J. Pickering, e- 
mail, 16 Feb. 1999); nor in the MNH-U (L. 
Maitas, e-mail, 4 March 1999). This species 
was figured from St. Thomas, U. S. Virgin Is- 
lands, by R. McLean (1951: 102, 166, pi. 21, 
fig. 2), but I have been unable to locate the de- 
pository of this material, which is not in the 
AMNH, ANSP, MCZ, nor USNM. This species 
may also have been what was reported as 
"Psammobia squamosa Lamarck, 1818," by 
Krebs (1864: 104[1948 reprint: 21], as "sqva- 
mosá') (see also, Dall, 1898a: 57). This is not 
Lamarck, 1818: 514, an Indo-Pacific Gari 
(Gari). 

There are three lots in public collections 
that may be G. (D.) c/rce-two lots of small, 
worn specimens from Grand Bahama Island 
(26.7°N) in the ANSP [368542, 369982] (Fig. 
17), and one lot from off Palm Beach Inlet, 
Palm Beach County, Florida (27.5°N) [USNM 
890240]. 

Sutty & Abbott (1986: 108-109, fig. 121) 
figure what appears to be similar material 
from Martinique, (as Gari sp.), and they report 
the same species from Mayreau (as 
"Mayero"). I have not located these speci- 
mens. I have seen the specimen, now broken, 
of the Gari sp. illustrated by Diaz & Puyana 
(1994: 94) from Parque Natural Tayrona, near 
Santa Marta, Magdalena Department, Colom- 
bia. They correctly concluded that it is very 
close to Gari helenae. 

Finally, Simone (1998: 35-38) has named 
Gari linhares from Barra Beach, Salvador, 
Bahia Province, Brazil, in 5 m [holotype: 
MZSP 28786, pair; length, 44.5 mm; height, 
25.0 mm; paratype: MZSP 28786; length, 
41.6 mm] (Fig. 18). It differs from eastern Pa- 
cific specimens of Gari helenae in lacking 
color, having a narrower hinge plate, and in 
having an anterior cardinal in the left valve 
that is anteriorly directed, whereas that of G. 
helenae is vertical. 

These relationships cannot be resolved 
until more abundant material is available from 
the western Atlantic. There are a range of pos- 
sibilities: (1) that Gari helenae occurs in both 
oceans, but G. circe being restricted to the 
eastern Caribbean; (2) that there is a single, 
variable species, the earliest name for which 
is G. circe. 








19 



20 



21 








22 



23 



24 






25 




FIGS. 19-26. Drawings of the interior features of eastern Pacific species of Gari. FIG. 19. Gari californica; 
CAS 113117; San Pedro, California; intertidal zone; length, 98.3 mm. FIG. 20. Gari fucata; CAS 113119; San 
Pedro, California; 46 m; length, 122 mm. FIG. 21. Gari lata; SBMNH 145082, Guaymas. Sonora, Mexico; 15 
m; length, 45.7 mm. FIG. 22. Gari maxima; В M N H 1966552/2, paralectotype; length, 125.1 mm. FIG. 23. Gari 
panamensis; LACM 72334; Isla San Pedro Gonzales, Panama; 1 -3 m. FIG. 24. Gari solida; CAS 113140; 
Coquimbo, Chile; 46 m; length, 80.8 mm. FIG. 25. Gar/n. sp?; LACM 34-283.8, Isla Española, Galapagos 
Islands; 55 m. FIG. 26. Gari helenae; CAS 113113; Bahía de los Muertos, Baja California Sur; 3-9 m; length, 
53.7 mm. 



THE EASTERN PACIFIC RECENT SPECIES OF THE BIVALVE GENUS GARI 



21 



OTHER RECORDS AND NOTES 
Recent Taxa -Eastern Pacific 

Psammobia californica (Conrad, 1837) was a 
temporary combination by Conrad (1 849: 
120) for Sanguinolaria california Conrad, 
1837 (p. 231, pi. 17, fig. 7), a synonym of 
Macoma balthica (Linnaeus, 1758: 677) 
(Coan, 1971:44). 

Psammobia casta Reeve, 1857 (pi. 8, fig. 55), 
is a synonym of Temnoconcha cognata 
(C. B. Adams, 1852a: 279; 1852b: 503) 
[Tellinidae] (Keen, 1971: 234). 

Psammobia decora Hinds, 1842 (p. 81, pi. 6, 
fig. 1), is a synonym of Nuttallia nuttallii 
(Conrad, 1837: 230-231, pi. 17, fig. 6) 
(Coan, 1973:48). 

Psammobia kindermanni (Philippi, 1847) was 
a temporary combination by Carpenter 
(1857b: 297, 301) for Amphichaena kin- 
dermanni Philippi, 1847 (pp. 63-64, pi. 3, 
fig. 7), which is now placed as Donax 
(Amphichaena) kindermanni (Coan, 
1983:287). 

Psammobia pacifica Conrad, 1 837 (p. 241 , pi. 
18, fig. 13), is the original combination for 
Heterodonax pacificus (Conrad, 1837) 
(Coan, 1973:46). 

Psammobia sp. of Li (1930: 262-263, pi. 5, 
fig. 34) was based on a specimen of Le- 
porimetis dombei (Hanley, 1844: 144) 
[Tellinidae] (Pilsbry, 1931: 431). 

-Western Atlantic 

Psammobia affinis С. В. Adams, 1845: 10, is 
a synonym of Heterodonax bimaculatus 
(Linnaeus, 1758: 677) (Clench & Turner, 
1950:250). 

Psammobia biradiata С. B. Adams, 1845: 10, 
is a synonym of Heterodonax bimacula- 
tus (Linnaeus, 1758) (Clench & Turner, 
1950:250,261). 

Psammobia cayennensis Lamarck, 1818: 
514, is a synonym of Macoma constricta 
(Bruguière, 1792: 126) (Dali, 1900: 
1050). 

Psammobia cerina С. В. Adams, 1845: 9, is a 
synonym of Heterodonax bimaculatus 
(Linnaeus, 1758) (Clench & Turner, 1950: 
250, 265). 

Psammobia declivis (Turton, 1819) was a 
temporary combination by Turton (1822: 
91) for Solen declivis Turton, 1819 (p. 
164, fig. 80), a synonym of Tagelus gib- 
bus (Spengler, 1794: 304) (Dali, 1900: 
983). 



Psammobia? lusoria Say, 1822: 304, from the 
"southern states," was most probably a 
senior synonym of Macoma tenta (Say, 
1834: pi. 65, fig. 3, as Tellina) (Dall, 1900: 
1048-1049), but it is now regarded as a 
nomen dubium. 

Psammobia purpureomaculata С. В. Adams, 
1845: 10, is a synonym of Heterodonax 
bimaculatus (Linnaeus, 1758: 1120) 
(Clench & Turner, 1950: 250, 335). 

The record by Krebs (1864: 104; 1948 reprint 
p. 21) of Psammobia sqvamosa [sic, for 
squamosa] Lamarck, 1818, from the Vir- 
gin Islands is undoubtedly in error, be- 
cause this species is restricted to the 
Indo-Pacific (Willan, 1993: 33-35, 111, 
126). This record, repeated by Dall 
(1898a: 57), was probably based on the 
only Gari present in the Virgin Islands, 
Gari circe. 

The specimens cited by Dall (1898a: 57) from 
Charlotte Harbor, Florida, tentatively 
identified as Gari (Gobraeus) u vaginatus" 
(Reeve, 1857), are based on mislocalized 
specimens of the eastern Pacific Gari lata 
(see above). The name was an error for 
Psammobia vaginoides Reeve, 1857: pi. 
8, fig. 57, described without a locality. The 
type material of this species cannot now 
be located in the BMNH collection (J. 
Pickering, e-mail, 16 Feb. 1999), and it is 
best regarded as a nomen dubium. 

The "worn fragment" reported by Dall (1898a: 
57) of Psammobia (Psammobia) from 
880 fms. east of Tobago could not be lo- 
cated in the USNM. 



Fossil Taxa [formations are of the 

type localities and do not include 

known distributions] 

-Western North America 

Gari (Psammacola: sic for Psammocola) 
alata Gabb, 1866: 21, pi. 5, fig. 36, from the 
Miocene San Pablo Group of Contra Costa 
County, California, is now regarded as a 
species of Nuttallia (Roth & Guruswami- 
Naidu, 1978: 65). 

There are ten western North American 
species of Gari described from the Eocene; 
the relationships among them remain unclear. 
Psammobia martini Dickerson, 1917: 168, 
184, pi. 27, fig. 7a, b, from the uppermost 
Eocene "Gries Ranch beds" of the Lincoln 
Creek Formation, in Washington, is based on 
a fragmentary right valve. It merits close com- 



22 



COAN 



parison with Gari fucata. Other taxa are: Tel- 
lina hornii Gabb, 1864: 160-161, 231; 1865: 
pi. 30, fig. 244, and Gari (Psammobia) texta 
Gabb, 1864: 155, 230, pi. 22, fig. 130, from 
the lower to middle Eocene Tejon Formation 
of south-central California; Psammobia 
columbiana Weaver & Palmer, 1922: 22, pi. 
10, fig. 18; P. cowlitzensis Weaver & Palmer, 
1922: 22-23, pi. 9, fig. 18; and P. olequahen- 
sis Weaver & Palmer, 1 922: 23, pi. 9, figs. 1 1 , 
12, from the upper middle Eocene Cowlitz 
Formation of Washington; P. obscura С. A. 
White, 1889: 61, pi. 10, figs. 4-6, from the 
middle to upper Eocene Puget Group of 
Washington; Gari eoundulata Vokes, 1939: 
93, 94, 215, pi. 14, figs. 23, 24, from the lower 
middle Eocene Domengine Formation of cen- 
tral California; Psammobia diegoensis 
Hanna, 1927: 293, 368, pi. 42, fig. 3, from the 
lower middle Eocene Ardath Shale of south- 
ern California; and Gari hornii umpquaensis 
Turner, 1938: 62, 102, pi. 7, fig. 11, from the 
lower Eocene White Trail Ridge formation of 
Oregon. 

Psammobia cylindrica Dickerson, 1914: 
139, 168, pi. 12, fig. 2a, b, from the Upper Pa- 
leocene Martinez Formation of north-central 
California. 

-Eastern North America 



Harris, in Harris & Palmer, 1946: 97-98, pi. 
21, figs. 12-14, from the upper Eocene 
Moodys Branch Formation of Louisiana; Sole- 
curtus blainvillii Lea, 1833: 39, pi. 1, fig. 7 
[sometimes misspelled as blainvillei]; Psam- 
mobia eborea Conrad, 1833: 42; P. filosa 
Conrad, 1833: 42; and P. {Graum) claibor- 
nense Dall, 1900: 978, from the middle 
Eocene Claiborne Formation of Alabama; P. 
harrisi Aldrich, 1921: 24, pi. 3, figs. 19, 20, 
from the lower Eocene Tuscahoma Formation 
of Alabama; P. ozarkana Harris, 1897: 65, pi. 
1 2, fig. 1 4, pi. 1 3, fig. 8, from the lower Eocene 
Hatchetigbee Formation of Alabama; and P. 
smithi Aldrich, 1921: 23, pi. 3, figs. 17, 18, 
from the lower Eocene Tuscahoma Formation 
of Alabama. 

-Central America 

Woodring (1982) described Gari (Gob- 
raeus) listrota listrota (p. 672, pi. 90, figs. 7, 
13), from unnamed late Eocene beds, and G. 
(G.) listrota hadratera (p. 672-673, pi. 91, 
figs. 7, 12), from the late Oligocène Bohio For- 
mation, of Panama. These taxa do not appear 
to be closely related to Recent forms, al- 
though Woodring suggested a relationship of 
the latter to Gari panamensis. 



Psammobia gubernatoria dalli Olsson, 
1916: 25, pi. 2, fig. 2, was described from the 
lower Pliocene Chowan River Formation at 
Chocowinity, North Carolina. However, the 
record of Gari dalli Olsson, 1916, from the 
Pliocene of Virginia Campbell, 1993: 41-42, 
pi. 17, fig. 153) instead represents a new 
species (L. Campbell, e-mail, 2 January 
1999). 

Miocene taxa include Psammobia guberna- 
toria Glenn, 1904: 292-293, pi. 71, fig. 7a, b, 
from the Choptank Formation of Maryland 
(see also, Vokes, 1 957: 21 , 45, pi. 1 8, figs. 1 , 
2); and P. bo wdichi Gardner, 1928: 214, pi. 32, 
figs. 23-26, from the early Miocene Chipóla 
Formation of Florida. 

The only lower Oligocène taxon is Psam- 
mobia papyria Conrad, 1848a: 291, from the 
Vicksburg Group, Mississippi. Psammobia 
mississippiensis Conrad, 1848b: 122, pi. 12, 
fig. 26, from the same formation is a nomen 
dubium (Dockery, 1982: 80). Psammobia 
linea Conrad, 1848a: 291, also from this for- 
mation, is a Tellina (Dockery, 1982: 77). 

There are eight named Eocene taxa in 
eastern North America: Gari jacksonensis 



-South America 

Psammobia tehuelcha von Ihering, 1907: 
417, pi. 17, fig. 112a, b, from the Pliocene 
"Terraza de Cerro Laziar" at Sierra Laziar, be- 
tween San Jorge and Puerto Deseado, Santa 
Cruz Province, Argentina, is similar, and is 
perhaps identical or ancestral to Gari solida. 
Their relationship merits additional investi- 
gation by those with access to adequate 
material. The strata involved may instead 
be Pleistocene (M. Griffin, e-mail, 11 Feb. 
1999). (Psammobia tehuelcha von Iherling, in 
Ameghino, 1902: 301 [1903 repr., p. 190], is a 
nomen nudum.). See also Feruglio (1933: 96, 
138, 140), Parodiz (1996: 272-273). 

Solenocurtus hanetianus d'Orbigny, 1842 
(pp. 124-125, pi. 15, figs. 1, 2), from the Ter- 
tiary of Coquimbo, Chile, is based on an inde- 
terminate mold (record repeated in Hupé, 
1854: 368; Philippi, 1887: 137, pi. 32, fig. 1). 

Psammobia darwini 'Philippi, 1887 (p. 136, 
pi. 26 [not pi. 16, as stated on p. 136], fig. 16 
[in legend as "Tellina"]), from the lower to mid- 
dle Miocene of central Chile, also remains in- 
determinate. 



THE EASTERN PACIFIC RECENT SPECIES OF THE BIVALVE GENUS GARI 



23 



Psammobia patagónica Philippi, 1887 (p. 
137, pi. 26, fig. 17 [one of two figs, numbered 
17 on pi., the one without an explanation]), 
probably from the early Miocene Monte En- 
trada member of the Monte León Formation at 
Cañadón de los Misioneros near Santa Cruz, 
Santa Cruz Province, Argentina, is a mactrid 
(Steinmann & Wilckens, 1908: 47-49, pi. 5, 
fig. 3), perhaps a Scalpomactra (M. Griffin, e- 
mail, 11 Feb. 1999). 

Psammobia burmeisteri von Ihering, 1907: 
312, pi. 12, fig. 81a, b, from the Late Oligo- 
cene-early Miocene Camarones Formation at 
Bahía Camarones, Chubut Province, Ar- 
gentina (Parodiz, 1996: 207), was based on a 
poorly preserved interior mold (M. Griffin, e- 
mail, 11 Feb. 1999). 

Psammobia guassus von Ihering, 1907: 
312-313, pi. 14, fig. 95, from the late Eocene 
to Oligocène San Julián Formation at Gran 
Bajo de San Julián, Santa Cruz Province, Ar- 
gentina (Parodiz, 1996: 229) {guassu, subse- 
quent emendation, probably unjustified). It is 
known only from the holotype, a mold. 



ACKNOWLEDGMENTS 

I appreciated the help of the following cura- 
tors, other personnel and their institutions, 
who made specimens, literature, and informa- 
tion available: Kenneth J. Boss, Museum of 
Comparative Zoology, Harvard University, 
Cambridge, Massachusetts, USA; James 
Cordeiro and Paula Mikkelsen, American Mu- 
seum of Natural History, New York, New York, 
USA; Juan M. Díaz M., Instituto de Investiga- 
ciones Marinas y Costeras, Santa Marta, 
Colombia; Jon Fajans and Roger Portell, 
Florida Museum of Natural History, Gaines- 
ville, Florida, USA; Yves Finet, Muséum d'His- 
toire Naturelle, Genève, Switzerland; Nora R. 
Foster, University of Alaska Museum, Fair- 
banks, Alaska, USA; Daniel Frassinetti O, 
Museo Nacional de Historia Natural, Santi- 
ago, Chile; Raye Germon and Thomas 
Waller, National Museum of Natural History, 
Washington, DC, USA; Ned Gilmore and Gary 
Rosenberg, of the Academy of Natural Sci- 
ences, Philadelphia, Pennsylvania, USA; 
Matthias Glaubrecht and Lothar Maitas, Mu- 
seum für Naturkunde, Humboldt-Universität, 
Berlin, Germany; Michael Griffin, Museum of 
La Plata, La Plata, Argentina; Lindsey Groves 
and James H. McLean, Natural History Mu- 
seum of Los Angeles County, Los Angeles, 



California, USA; Elizabeth Kools, Department 
of Invertebrate Zoology, California Academy 
of Sciences, Golden Gate Park, San Fran- 
cisco, California, USA; Konstantin Lutaenko, 
Institute of Marine Biology, Vladivostok, Rus- 
sia; Philippe Maestrati, Muséum National 
d'Histoire Naturelle, Paris, France; Akihiko 
Matsukuma, Kyushu University, Hakozaki, 
Japan; Cecilia Osorio R., Departmento de 
Ciencias Ecológicas, Universidad de Chile, 
Santiago, Chile; Guido Pastorino, Museo de 
Ciencias Naturales, Buenos Aires, Argentina; 
Joan Pickering and Kathie Way, The Natural 
History Museum, London, England, UK; Patri- 
cia Sadeghian and Paul Valentich Scott, 
Santa Barbara Museum of Natural History, 
Santa Barbara, California, USA; Takenori 
Sasaki and Kazushige Tanabe, Geological In- 
stitute, University of Tokyo, Japan; Tom 
Schiotte, Zoological Museum, University of 
Copenhagen, Denmark; Luiz R. L. de Si- 
mone, Museu de Zoología, Sao Paulo, Brazil; 
Judith Smith, Stanford University, Stanford, 
California, USA; Richard L. Squires, Califor- 
nia State University, Northridge, California, 
USA; Nancy Voss, University of Miami, Miami, 
Florida, USA; Karen Wetmore, Museum of 
Paleontology, University of California, Berke- 
ley, California, USA; and Richard Willan, Mu- 
seum & Art Gallery of the Northern Territory, 
Darwin, Australia. Cleveland P. Hickman, Jr., 
Kirstie L. Kaiser, Richard McClincy, Charlotte 
Norrid, and Carol С Skoglund generously 
made available material or information from 
their collections, and Lyle Campbell provided 
two fossil species from his collection. Lyle 
Campbell, Lindsey Groves, and Carol С 
Skoglund made useful comments on the man- 
uscript. Carole M. Hertz provided some infor- 
mation. Sharon Williams helped to prepare 
the plates. I acknowledge valuable comments 
on the manuscript by James H. McLean and 
an anonymous reviewer. 



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Revised ms. accepted 22 August 1999 



MALACOLOGIA, 2000, 42(1-2): 31-37 

FACTORS INFLUENCING MOVEMENT PROBABILITIES OF FLORIDA TREE 

SNAILS LIGUUS FASCIATUS (MÜLLER) IN BIG CYPRESS NATIONAL PRESERVE 

FOLLOWING HURRICANE ANDREW 

Robert E. Bennetts, 1,3 Steven A. Sparks 2 & Deborah Jansen 2 

ABSTRACT 

Beginning in 1993, approximately one year after Hurricane Andrew, we marked 2,547 Florida 
tree snails (Liguus fasciatus) during six sampling (mark-resighting) occasions twice per year over 
a three-year period. During each sampling occasion, we conducted a search for all tree snails 
within 16 sample plots on eight hammocks (tree islands) and for an extended radius of approxi- 
mately 20 m around each plot. We individually marked all trees > 5 cm dbh within each plot and 
all trees within the extended radius in which a marked snail was found. Because the host tree for 
each marked animal was known, inter-tree movements could be directly measured as the pro- 
portion of animals found on different host trees at times t and t + 1. We observed 533 marked 
snails during two or more consecutive sampling occasions. Of these, 414 (77.7%) snails had 
moved from one host tree to another between sampling occasions. Based on a conditional lo- 
gistic regression model, movement probabilities were influenced by whether or not the snail's 
host tree had been Lysiloma at time t, the diameter of the host tree at time t, and interactions 
among these and with year. Overall, snails had a lower probability of moving from one tree to an- 
other if their host tree had been Lysiloma and if they were on larger host trees. We found no ev- 
idence that movement probabilities were influenced by the snail's subspecies, age, hammock 
size class, or by the season. An effect of latitudinal gradient or proportion of host-trees damaged 
was not retained in our final model. However, a preliminary univariate analysis did indicate a dif- 
ference in movement probabilities between our northernmost hammocks, which had relatively lit- 
tle hurricane damage, and the southern hammocks, which had extensive damage. 

Key words: Florida tree snail, hammock, host tree, Liguus fasciatus, logistic regression, 
movement. 



INTRODUCTION 

The Florida tree snail Liguus fasciatus 
(Müller), as other species of this genus, is 
likely of Antillean origin, with a distribution in- 
cluding Cuba, Haiti, Isle of Pines, and Florida 
(Pilsbry, 1946; Voss, 1976). Throughout the 
range of Liguus, hurricanes are a natural cli- 
matic event that occurs at periodic, and often 
frequent, intervals (Duever et al., 1994). It is 
now widely accepted that the variability of 
Liguus fasciatus resulted from repeated immi- 
grations from Cuba (Brown, 1978), and hurri- 
canes have been hypothesized to play a role 
in those immigrations. Simpson (1929) sug- 
gested that adult snails on trees or eggs laid in 
decaying logs could float as rafts along cur- 
rents during hurricanes. Thone (1 937) also hy- 
pothesized that snails cemented to branches 
could have been carried by hurricane winds 



from Cuba to Florida. However, more recent 
evidence and scrutiny leads to considerable 
doubt about these hypotheses (Tuskes 1 981 ). 
Hurricanes also occasionally result in the de- 
struction of local tree snail colonies, although 
negative effects on the population as a whole 
are believed to be minimal (Young, 1 951 ). Re- 
gardless of how hurricanes affect the distribu- 
tion, variation, or population dynamics of 
Liguus snails, there can be little doubt that hur- 
ricanes play a major role in shaping the land- 
scape of this region (Duever et al., 1994), in- 
cluding habitat for tree snails. Despite this 
important role of hurricanes, little is known 
about the relationship of Liguus to its environ- 
ment (Brown, 1978), which includes hurri- 
canes. On 24 August 1992, Hurricane Andrew 
hit southern Florida and its path crossed di- 
rectly over much of the remaining peninsular 
habitat for Liguus. Here we explore the post- 



Florida Cooperative Fish and Wildlife Research Unit, P.O. Box 110450 University of Florida, Gainesville, Florida 32611- 
0450, USA. 

2 Big Cypress National Preserve, HCR61, Box 110, Ochopee, Florida 34141, USA 
3 Present address: Station Biologique de la Tour du Valat Le Sambuc, 13200 Arles, France; bennetts@tour-du-valat.com 



31 



32 



BENNETTS. SPARKS & JANSEN 





Path of Hurricane Eye 



FIG. 1 . Hammocks within Big Cypress National Pre- 
serve reported to have populations of Liguus tree 
snails in relation to the path of the eye of Hurricane 
Andrew. The eight hammocks sampled (also 
shown) were intended to reflect a gradient of po- 
tential hurricane damage. 



hurricane movements of tree snails in relation 
to their habitat characteristics along a gradient 
of hurricane damage. 



METHODS 

Our study population consisted of tree 
snails within Big Cypress National Preserve. 
Within the preserve, 136 hammocks have 
been individually identified and mapped (Pils- 
bry, 1946). These hammocks extend from the 
Dade-Collier Training and Transition Airport 
(25°51 'N, 80°53'E) south to boundary of Ever- 
glades National Park (Fig. 1). From this initial 
pool of 136 hammocks, we selected a sample 
of eight. We believed that eight hammocks 
would provide reasonable representation of 



the potential variability and would be a logisti- 
cally feasible number of sites. Because we 
were interested in the potential effects of Hur- 
ricane Andrew, we wanted our study sites to 
reflect the gradient of hurricane impacts that 
occurred latitudinally. Conseguently, we di- 
vided the pool of hammocks into four latitude 
zones that reflected clusters of hammocks 
that were positioned along this gradient. 
There also is considerable variability in the 
size of hammocks, which we believed could 
have influenced their susceptibility to damage 
by Hurricane Andrew. Consequently, we digi- 
tized each hammock from U.S.G.S. 7.5- 
minute ortho photos and used Geographic In- 
formation System (GIS) to estimate the 
approximate size of each hammock. We then 
used a stratified random sampling to select 
one hammock of greater than the median size 
and one hammock of lesser size from each of 
the four latitude zones. Thus, our sample re- 
flected the latitudinal gradient and was bal- 
anced among larger and smaller hammocks. 
Because it was logistically infeasible to 
sample snails or vegetation from each entire 
hammock, we established two 80 m 2 study 
plots (sub-samples) within each hammock 
from which we individually marked both snails 
and their host trees. These plots were ran- 
domly located within portions of the hammock 
where tree snails occurred. 

Snail Sampling Protocol 

Beginning in 1993, approximately one year 
after Hurricane Andrew, we marked snails 
during six sampling (mark-resighting) occa- 
sions twice per year over a three-year period. 
Our sampling was intended to reflect the biol- 
ogy of the animal. Our spring sampling was 
conducted as soon as possible after emer- 
gence of the snails from aestivation. This co- 
incided with the onset of the annual rainy sea- 
son. At this time, snails were active, but had 
not yet put on new growth; the shell was frag- 
ile and had to be handled with care. Our fall 
sampling coincided with the onset of the dry 
season just prior to aestivation. At this time, 
the summer's growth had been terminated, 
the shells had thickened, but the animals had 
not yet entered aestivation. 

During each sampling occasion we con- 
ducted a search for all tree snails within each 
plot and for an extended radius of approxi- 
mately 20 m around the plot in order to detect 
animals that may have moved outside of the 
plot. Each snail found was individually marked 



MOVEMENTS OF TREE SNAILS 



33 



by engraving unique number on the outer 
shell near the apex. Care was taken not to 
penetrate the inner shell, which could directly 
injure the animal or cause moisture loss and 
subsequently influence survival. Snails in 
their first year were not marked because of 
the potential for damaging their relatively frag- 
ile shell. For each snail, we recorded sub- 
species in accordance with Pilsbry (1946), 
age (Jones, 1954; Tuskes, 1981), and host 
tree. We then returned snails to their host tree 
by placing them in a conical paper cup at- 
tached to the trunk of the tree. Thus, snails 
would exit the cups directly on to the host tree 
from which they were found. We observed 
snails on several occasions as they exited 
these cups onto their original host tree and 
never observed a snail leave their host tree 
after being returned. Thus, we are confident 
that the inter-tree movements we observed 
were not a post-handling artifact. 

Tree Sampling Protocol 

We individually marked all trees > 5 cm dbh 
within each plot and all trees within the ex- 
tended radius in which a marked snail was 
found. We also measured the extent (i.e., pro- 
portion of trees) of severe damage to host 
trees by Hurricane Andrew. We considered a 
tree damaged if it was: (1) uprooted to the 
point of root exposure, (2) broken at the main 
trunk and/or a primary fork (i.e., broken sec- 
ondary branches were not considered), and 
(3) leaning at least 45°. 

Movements 

Because the host tree for each animal was 
known, inter-tree movements could be di- 
rectly measured as the proportion of animals 
found on different host trees at times t and t + 
1. This resulted in a minimal estimate, since 
additional movements could have occurred 
between our sampling occasions and be- 
cause any snails that might have moved out- 
side of our extended radius would not have 
been detected. We used a conditional logistic 
regression model to evaluate the influences 
on snails having moved from one host tree to 
another. Thus, we assessed the probability 
that a snail moved to a different tree between 
times t and t + 1 , conditional on snails having 
been alive and their location known at both 
times. Model selection was based on Akaike's 
Information Criterion (AIC)(Akaike, 1973; Shi- 
bata, 1989). Our model selection philosophy 



have been described in detail elsewhere 
(Burnham & Anderson, 1992, 1998). 

As potential explanatory variables for 
movement probabilities, we considered host 
tree species, the proportion of trees dam- 
aged, latitudinal gradient, two-class damage 
zone (see results of tree damage), hammock 
size class (< or > than the median size), snail 
age, snail subspecies, year, and season. 
Throughout the peninsular habitats, Lysiloma 
latisiliqua is the most widely used host tree for 
Florida tree snails (Voss, 1976; Brown, 1978), 
accounting for 67% of the snail locations (in- 
cluding non-consecutive locations) on live 
trees within our study plots (Bennetts et al., 
submitted). Use of Lysiloma also greatly ex- 
ceeds its expected use based on its availabil- 
ity (Brown, 1978; Bennetts et al., submitted). 
Consequently, we used whether or not the 
host tree at time t was Lysiloma as a potential 
explanatory variable in our logistic model. 



RESULTS 

A complete evaluation of host-tree damage 
as a result of Hurricane Andrew is well beyond 
the scope of this paper and will be reported 
elsewhere (D. Jansen, Big Cypress National 
Preserve, unpubl. data). However, prelimi- 
nary results from our assessment indicated 
that the extent of total damage (i.e., cumula- 
tive percentage of trees broken, uprooted, 
and/or substantially leaning) was reasonably 
well modeled (using an arcsin transformation) 
in relation to a latitudinal gradient (R 2 = 0.74, 
P = 0.006), but was better modeled as reflect- 
ing two zones: the northern two hammocks 
being relatively unaffected and the remaining 
six hammocks having been heavily damaged 
(R 2 = 0.83, P = 0.002)(Fig. 2). Thus, we con- 
sidered both a linear gradient and a two-class 
characterization of hurricane damage as po- 
tential explanatory variables for movement 
probabilities. 

We marked a total of 2,547 snails (Table 1) 
from which we observed 533 marked snails 
during two or more consecutive sampling oc- 
casions. Of these, 414 (77.7%) had moved 
from one host tree to another between sam- 
pling occasions. Our final logistic regression 
model indicated that movement probabilities 
were influenced by whether or not the snail's 
host tree had been Lysiloma at time t, the di- 
ameter of the host tree at time t, and interac- 
tions among these and with year (Table 2). 
Overall, snails had a lower probability of mov- 



34 



BENNETTS. SPARKS & JANSEN 




4 5 6 

Hammock 

FIG. 2. Percentage of trees damaged by Hurricane 
Andrew on each of the study Hammocks. 

TABLE 1 . Number of Liguus snails marked on each 
hammock. Also shown is the number of consecutive 
locations and the percentage of those locations that 
were on different host trees. 









Number 








(percentage) 






No. of 


of consecu- 




Total No. 


consecu- 


tive locations 




of snails 


tive loca- 


on different 


Hammock 


marked 


tions 


host trees 


1 


311 


56 


40(71%) 


2 


246 


41 


28 (68%) 


3 


266 


59 


49 (83%) 


4 


326 


50 


40 (80%) 


5 


294 


89 


68 (76%) 


6 


332 


57 


41 (72%) 


7 


426 


94 


79 (84%) 


8 


346 


87 


69 (79%) 



TABLE 2. Likelihood-ratio analysis of variance table 
for our final logistic regression model for the proba- 
bility that a snail moved from one tree to another 
between sampling occasions t and t + 1 . 



Source 




x 2 


df 


P 


LYSILOMA 3 




7.57 




0.006 


DBH b 




5.62 




0.018 


YEAR C 




0.34 




0.558 


LYSILOMA* 


YEAR 


10.92 




< 0.001 


LYSILOMA* 


DBH 


7.32 




0.007 



a Whether or not the host tree at time t was Lysiloma. 
"Diameter at breast height of the host tree at time t. 
c Year was not significant as a main effect, but was retained 
in the model because of its interaction effect. 



¡ng from one tree to another between times t 
and t + 1 if their host tree had been Lysiloma 
at time t and if they were on larger host trees 
(Fig 3). We found no evidence that overall 
movement probabilities differed among years. 
However, we did find evidence (i.e., a signifi- 



cant interaction term) that movement proba- 
bilities of snails showed a different pattern 
over time depending on what host tree they 
were using. Movement probabilities of snails 
using Lysiloma decreased over time, whereas 
the movement probabilities of snails on other 
host trees increased over time (Fig. 4). We 
also found a significant interaction between 
diameter and species of the host trees. Snails 
that were in smaller host trees (e.g., < 20 cm 
dbh) had similar movement probabilities, re- 
gardless of the species of host tree (Fig. 5). In 
contrast, snails using larger Lysiloma as a 
host tree had a lower probability of movement 
compared to snails using other host-tree 
species of similar size. 

We found no evidence that movement 
probabilities were influenced by the snail's 
subspecies (x 2 = 1.71, 3 df, P = 0.635), age 
(X 2 = 2.90, 4 df, P = 0.575), hammock size 
class (x 2 = 0.18, 1 df, P = 0.671), or by the 
season (x 2 = 0.018, 1 df, P = 0.894). We did 
not find an effect of latitudinal gradient on 
movement probabilities (x 2 = 199, 1 df, P = 
0.158) or an effect of proportion of host-trees 
damaged (x 2 = 3.48, 1 df, P = 0.062) at the a 
= 0.05 level, although host-tree damage was 
nearly significant at this level. However, a uni- 
variate analysis did indicate a significant ef- 
fect of a two-class hurricane zone (x 2 = 3.87, 
1 df, P = 0.049), with snails in the two north- 
ernmost hammocks (i.e., with less hurricane 
damage) having a lower movement probabil- 
ity (0.70) than those in the southern ham- 
mocks (0.79). However, our model selection 
procedure indicated that this relationship was 
better modeled using the terms included in 
our final model; thus, hurricane zone was not 
retained. 



DISCUSSION 

Our data indicate that Liguus move from 
one host tree to another quite frequently. 
Given that snails in the northern-most ham- 
mock, which was virtually unaffected by the 
hurricane, also had a high movement proba- 
bility (0.71 ), we suggest that overall high rates 
of movement are part of the natural history of 
animals in this region. Our data also showed 
that even three years after the hurricane, the 
overall probability of movement remained 
high (0.75). Similarly, Brown (1978) observed 
marked Liguus in adjacent Everglades Na- 
tional Park during a period that was not im- 



MOVEMENTS OF TREE SNAILS 



35 



03 

с 
00 

о 
с 
о 
tr 
о 

Q. 
О 



1 .и 

0.9 




+ 


0.8- 


+ 




0.7 






0.6 







Lysiloma Other 

Host Tree Species 







A Л 




Host Tree Species 

Lysiloma 

Other 













1994 
Year 



FIG. 4. The proportion of snails that moved from 
one tree to another between times t and t + 1 for 
each year and depending on whether or not their 
host tree at time t was a Lysiloma. 




< 20 cm > 20 cm 

Diameter of Host Tree 

FIG. 3. The proportion of snails that moved from 
one tree to another between times t and t + 1 de- 
pending on whether or not their host tree at time t 
was a Lysiloma (top) and whether their host tree at 
time t was greater than or less than 20 cm DBH 
(bottom). 



mediately following a hurricane and observed 
similarly high rates of movement (0.65). 

Although generally high movement proba- 
bilities per se were not likely a result of Hurri- 
cane Andrew, our data indicate that move- 
ment probabilities may have been indirectly 
affected by habitat changes induced by Hurri- 
cane Andrew. Snails were less likely to move 
from one host tree to another if they were 
using Lysiloma, particularly if the host tree 
was large. Lysiloma was one of the species 
dramatically impacted by Hurricane Andrew, 
particularly if they were large, hence more 



09 










0.8- 


Î 


+ 






0.7 - 






+ 




06 











Lysiloma Other 
< 20 cm 



Lysiloma Other 
> 20 cm 



FIG. 5. The proportion of snails that moved from 
one tree to another between times t and t + 1 de- 
pending on whether or not their host tree at time t 
was a Lysiloma and whether their host tree at time 
t was greater than or less than 20 cm DBH. 



susceptible to wind damage (Loop et al., 
1994; D. Jansen, Big Cypress National Pre- 
serve, unpubl. data). 

It is not entirely clear why movement of 
snails on Lysiloma would have decreased 
over time, while movements increased for 
other host species. Host trees that were dam- 
aged by Hurricane Andrew, but survived, may 
have had a temporary reduction in quality if 
the damage was severe enough to affect its 
food resources (i.e., algae) for snails. During 
such a reduction, snails may have sought al- 
ternative trees. This could explain why move- 
ments on Lysiloma, the preferred host tree, 
were initially high and decreased over time 
(i.e., the interaction term between species 



36 



BENNETTS. SPARKS & JANSEN 



and year). By 1995, movement rates in more 
affected southern zone (in Lysiloma) were 
similar to rates in northern zone. 

One likely pathway for snails to move from 
one tree to another is via intertwined foliage 
(Jones, 1954). The damage from Hurricane 
Andrew included uprooting, breaking of trunks 
and branches, and defoliation (Loope et al., 
1994; D. Jansen, Big Cypress National Pre- 
serve, unpubl. data), all of which would have 
decreased the connectivity of foliage between 
adjacent trees. Thus, the potential for inter- 
tree movement through foliage in areas influ- 
enced by Hurricane Andrew would have been 
reduced by the hurricane. This too would indi- 
cate that the high rates of movement we ob- 
served may have been conservative. 

The other likely pathway is movement over 
ground, particularly when tree spacing pre- 
cludes intertwined branches (Brown, 1978). 
Movement over ground would seem most 
likely following egg laying, which occurs on 
the ground near the base of their host tree. 
However, if egg-laying were a strong catalyst 
of inter-tree movements, we would have ex- 
pected to see a strong seasonal effect, be- 
cause our fall sampling occurred just after the 
primary egg-laying period. In contrast to this 
expectation, we found no evidence of a sea- 
sonal effect. Nor did we find an age effect for 
snails < 2 years (i.e., largely non-breeding) 
compared to snails > 2, which were more 
likely to have been breeding. Further, the 
physical structure of the ground in the vicinity 
of a host-tree base is usually a gentle slope 
toward the tree's trunk, making it quite easy 
for snails to return to the same host tree. 

Our assessment of the influence of Hurri- 
cane Andrew was likely conservative. First, 
our study was not initiated until a year follow- 
ing the hurricane. Although severe damage to 
trees certainly was still prevalent, there likely 
had already been some adjustment. The re- 
sponse along a latitudinal gradient of hurri- 
cane damage also may have reflected two op- 
posing factors regarding movement. In the 
southern hammocks that were strongly ef- 
fected by the hurricane, food resources may 
have been temporarily reduced, which would 
have encouraged snails to move in search of 
better food resources. However, the potential 
to move among trees was also reduced in 
these hammocks because of the loss of inter- 
twining foliage. Likewise, the northernmost 
hammocks retained the intertwining foliage, 
and thus a higher potential for movement, but 
may have had less reason for snails to move 



if food resources also had been retained. Re- 
gardless of the specific effect of Hurricane An- 
drew on movement probabilities, our data 
clearly indicate that Liguus are a highly mobile 
snail species, which may give them a capac- 
ity to move in response to localized damage 
resulting from many types of disturbance 
events. 



ACKNOWLEDGMENTS 

Funding was provided by the National Park 
Service. Big Cypress National Preserve pro- 
vided additional logistic support. We very 
much appreciate the field assistance of 
Jimmy Conner, Phil Darby, Sue Davis, Vicky 
Dreitz, Guy Fischer, Katie Golden, Paul 
Hinchcliff, Teresa Johnson, Amy Kazmier, 
Steve McGehee, Jeff Ripple, Tim Towles, 
Patty Valentine-Darby. We are grateful to 
George Francioni for the safe helicopter 
flights to otherwise inaccessible hammocks. 
We appreciate the helpful comments of Gaé- 
tan Lefebvre, Brigitte Poulin, and two anony- 
mous reviewers. 



LITERATURE CITED 

AKAIKE, H., 1973, Information theory and an ex- 
tension of the maximum likelihood principle. Pp. 
267-281, in в. PETROv & F. czakil, eds., Pro- 
ceedings of the 2nd International Symposium on 
Information Theory. Akademiai Kiado Budapest. 

BENNETTS, R. E., S. A. SPARKS & D. JANSEN, 
Host-tree selection by Florida tree snails (Liguus 
fasciatus) in Big Cypress National Preserve. 
Nautilus (submitted). 

BROWN, С. A., 1978, Demography dispersal, and 
microdistribution of a population of the Florida 
tree snail Liguus fasciatus. M.S. Thesis, Univ. 
Florida, Gainesville. 

BURNHAM K. P. & D. R. ANDERSON, 1992, Data- 
based selection of an appropriate biological 
model: the key to modern data analysis. Pp. 

16-30 in D. R. MCCULLOUGH & R. H. BARRETT. 

wildlife 2001: populations. Elsevier Applied Sci- 
ence, New York. 

BURNHAM K. P. &D. R.ANDERSON, 1998, Model 
selection and inference: a practical-theoretic ap- 
proach. Springer-Verlag, New York. 

DUEVER, M. J., J. F MEEDER, L. О MEEDER & 
J. M. MCOLLOM, 1994, The climate of South 
Florida and its role in shaping the Everglades 
ecosystem. Pp. 255-248, in s. Davis & J. ogden, 
eds., Everglades: the ecosystem and its restora- 
tion. St. Lucie Press. 

JONES, A. L, 1954, How Florida tree snails live. 
Everglades Natural History Magazine. 2: 59-62. 



MOVEMENTS OF TREE SNAILS 



37 



LOOPE, L, M. DUEVER, A. HERNDON, J. SNY- 
DER & D. JANSEN, 1994, Hurricane impact on 
uplands and freshwater swamp forest. Bio- 
science, 44: 238-246. 

PILSBRY, H. A., 1946, Land Mollusca of North 
America. Academy of Natural Sciences Philadel- 
phia, Monograph, 3(2): 1-520. 

SHIBATA, R. 1989. Statistical aspects of model se- 
lection. Pp. 215-240, in j. с Williams, ed., From 
data to model. Springer-Verlag, New York. 

SIMPSON, С. T., 1929, The Florida tree snails of 
the genus Liguus. Proceedings U.S. Natural Mu- 
seum, 73: 1 -44. 

THONE, R, 1937, Cuban land shells in Florida. Sci- 
ence, 86: 8. 



TUSKES, P. M., 1981, Population structure and bi- 
ology of Liguus tree snails on Lignumvitae Key, 
Florida. Nautilus, 95: 162-169. 

VOSS, R. S., 1976, Observations of the biology of 
the Florida tree snail, Liguus fasciatus (Müller). 
Nautilus, 90: 65-69. 

YOUNG, F. N., 1951, Vanishing and extinct colonies 
of tree snails, Liguus faciatus, in the vicinity of 
Miami, Florida. Occasional Papers, Museum Zo- 
ology, University of Michigan, 531: 

Revised ms. accepted 18 August 1999 



MALACOLOGIA, 2000, 42(1-2): 39-62 

PLUTONIA {CANARIVITRINA), NEW SUBGENUS, FROM THE CANARY ISLANDS, 

AND THE PHYLOGENETIC RELATIONSHIPS OF THE SUBFAMILY PLUTONIINAE 

(GASTROPODA: LIMACOIDEA: VITRINIDAE)* 

Maria R. Alonso 1 , Manuel J. Valido 1 , Klaus Groh 2 & Miguel Ibáñez 1 ** 

ABSTRACT 

Plutonia (Canarivitrina), n. subgen., and four new species of this subgenus are described from 
three islands of the mid-Atlantic Canarian archipelago. The genus Plutonia Morelet, in Stabile, 
1864 (of which Insulivithna Hesse, 1923, is a new synonym), restricted to the mid-Atlantic is- 
lands, is characterized by the autapomorphy of the special course of the penial retractor muscle, 
rounding the right optic nerve. Canarivitrina, n. subgen., has a penis with two easily distinguish- 
able portions: a distal one, short and slightly widened, containing a globular to cylindrical and per- 
forated penial papilla level with the opening of the vas deferens (far from the penial apex), and a 
proximal one, long and slender, with apical insertion of the penial retractor muscle and two par- 
allel longitudinal inner structures: a torus (thick fold, with roundish cross section) and, opposite, 
a laminar crest (velum). Torus and penial papilla are coated by glandular tissue, the penial gland. 
The autapomorphic character state of this subgenus is the proximal portion of the penis, long and 
equipped with internal torus and velum. 

New data are given about the sheath that surrounds the spout of the glándula amatoria in Ca- 
narivitrina, n. subgen., and in other supraspecific Plutoniinae taxa demonstrating the homology 
of the glándula amatoria and its spout sheath with the sarcobelum of the genus Semilimax, a 
basal branch of the Vitrinidae: the subfamily Plutoniinae has the "sarcobelum" fused with the 
proximal part of the vagina to form the glándula amatoria. 

A phylogenetic scenario of the supraspecific taxa of the subfamily Plutoniinae is presented. 
The cladistic analysis shows the Plutoniinae as the sister group of Semilimax, and Phenacolimax 
as the sister group of the remaining Plutoniinae: the genera Phenacolimax, Gallandia (which is 
almost certainly a junior synonym of Oligolimax), Arabivitrina and Plutonia; but this analysis does 
not resolve for the other taxa because the data are still too few to apply to a cladistic study of the 
group. 

The genus Plutonia includes the subgenera Plutonia, s. str. , Guerrina, Insulivitrina, Madeirovit- 
rina and Canarivitrina, n. subgen. The presence of this genus in the archipelagoes of the Azores, 
the Madeiras and the Canary Islands is probably as old as the humid "laurisilva" laurel-forest, a 
Tertiary relict that colonized these archipelagoes before the impact of the Pleistocene glacia- 
tions. 

Key words: Gastropoda, Vitrinidae, Plutoniinae, Canarivitrina, phylogeny, biogeography. 



INTRODUCTION 

The Canarian Vitrinidae were studied only 
superficially between 1821-1954; eight spe- 
cies were described -one doubtful: Vitrina 
fasciolataA. Férussac, 1832; one of Guerrina 
Odhner, 1954: Helix cutícula Shuttleworth, 
1852; and six grouped in Insulivitrina: Helico- 
limax lamarckii A. Férussac, 1821; V blauneri 
Shuttleworth, 1852; V latebasis Mousson, 
1872; V canariensis Mousson, 1872; V retic- 
ulata Mousson, 1872; and V parryi Gude, 
1896. In the last years, ten additional species 



were described: G. christinae Groh, 1993; /. 
eceroensis Alonso & Ibáñez, 1987; /. tubercu- 
lata Ibáñez & Alonso, 1987; /. gomerensis 
Alonso & Ibáñez, 1988; /. emmersoni Morales, 
1988; /. oromii Ibáñez & Alonso, 1988; /. 
machadoi Ibáñez & Alonso, 1990; /. nogalesi 
Alonso & Ibáñez, 1990; /. tamaranensis 
Valido, 1990; and /. mascaensis Morales, 
1 987 (Alonso et al., 1 987; Ibáñez et al., 1 987; 
Morales et al., 1 988; Valido et al., 1 990, 1 993). 
The last species, from Tenerife Island, has a 
peculiar penis, with a very long and slender 
proximal portion having a remarkable distance 



Department of Animal Biology, University of La Laguna, E-38206 Tenerife, Canary Islands, Spain 
2 Mainzer Strasse 25, D-55546 Hackenheim, Germany 
"Notes on the Malacofauna of the Canary Islands, 38 
"To whom correspondence should be addressed; mibanez@ull.es 

39 



40 



ALONSO ETAL. 



between the vas deferens entrance to the 
penis and the insertion of the penial retractor. 
We have recently found four additional new 
species with the same penial characteristics: 
three from La Gomera and one from La Palma 
(Fig. 5); these five species are assigned to a 
new subgenus, Canarivitrina, n. subgen. In 
this paper, we describe these taxa and at- 
tempt a phylogenetic study of the entire sub- 
family Plutoniinae. 



METHODS 

The introduction of Canarivitrina, n. sub- 
gen., is the consequence of our anatomical 
studies of the European Phenacolimax major 
(A. Férussac, 1807), Arabivitrina jansseni 
Neubert, 1988 (from the Asir province, Saudi 
Arabia), Madeirovitrina nitida (Gould, 1848) 
and M. marcida (Gould, 1848) from the 
Madeira Archipelago, and the Canarian 
species above mentioned. Moreover, biblio- 
graphic data on the Plutoniinae have been ob- 
tained from Forcart (1 959), Hubendick (1 953), 
Mermod (1930), Zilch (1979), and several ad- 
ditional papers (Table 1). 

Calculation of number of shell whorls fol- 
lows Kerney et al. (1 979: 1 3). The terms "shell" 
and "specimen" in the enumerations of the ma- 
terial studied refer to empty shells and live 
specimens respectively, and "proximal" and 
"distal" refer to the position in relation to the 
gonad. The term "semislugs" refers to the clas- 
sification of Tillier (1984), that is, the animal 
usually cannot withdraw completely into the 
shell, the posterior edge of the foot cavity is 
lower and further forward than the most poste- 
rior part of the digestive tract, and the stomach 
is retained in the upper visceral cavity. 

The cladistic analysis involves the eight 
genus-group taxa of the Plutoniinae and was 
performed using PAUP 4.0 Beta 2 for Windows 
(Swofford, 1 999) and the Treeview application 
(Page, 1996). Tree searches were performed 
using the exhaustive search command. Zero- 
legth branches were collapsed, MulTrees was 
activated and the accelerated transformation 
option (Ace Tran) was used. The characters 
and character states used are listed in Table 2 
and the data matrix in Table 3 mainly based on 
the type species of the single supraspecific 
taxa. Character polarity was determined by 
outgroup comparison using Semilimax as out- 
group for the reasons indicated in the com- 
ments after the family diagnosis. All characters 
have equal weight. Two out of nine characters 



Abbreviations 



a 


atrium 


A-A 5 


stimulator portions; terminology based in 




Hausdorf (1988) and Schileyko (1984: 39, 




fig. 18) 


ag 


albumen gland 


AIT 


Alonso & Ibáñez collection, Department of 




Animal Biology, University of La Laguna, 




Tenerife, Canary Islands, Spain 


ANSP 


Academy of Natural Sciences. Philadelphia, 




Pennsylvania, U.S.A. 


ar 


atrial retractor muscles 


Ara 


Arabivitrina 


bb 


buccal bulb 


be 


bursa copulatrix 


Can 


Canarivitrina, n. subgen. 


CGH 


K. Groh private collection, Hackenheim, 




Germany 


CRD 


T. Ripken private collection, Delft, The Nether- 




lands 


CRT 


W. Rähle private collection, Tübingen, Ger- 




many 


CGS 


F. Giusti private collection. Siena, Italy 


FMNH 


Field Museum of Natural History, Chicago, 




Illinois, U.S.A. 


ga 


glándula amatoria inside proximal vagina 


Gal 


Gallandia 


Gue 


Guerrina 


Ins 


Insulivitrina 


Ic 


laminar crest (velum) 


Mad 


Madeirovitrina 


MHNG 


Muséum d'Histoire Naturelle, Genève, 




Switzerland 


MNHN 


Muséum National d'Histoire Naturelle, Paris, 




France 


NHM 


The Natural History Museum, London, 




England 


NNM 


Nationaal Natuurhistorisch Museum, Leiden. 




The Netherlands 





free oviduct 


P 


penis 


pg 


penial gland 


Phe 


Phenacolimax 


Pi 


pilaster 


Plu 


Plutonia 


PP 


penial papilla 


pr 


penial retractor muscle 


ps 


penial sheath 


ror 


right ommatophore retractor muscle 


s 


sarcobelum 


Sem 


Semilimax 


SMF 


Natur-Museum Senckenberg, Frankfurt/Main, 




Germany 


sp 


spout 


St 


soft tissue 


ST 


supraspecific taxon grouping the five species 




herein described 


t 


torus 


TFMC 


Museo de Ciencias Naturales de Tenerife, 




Canary Islands, Spain 


V 


distal vagina 


vd 


vas deferens 


vf 


vaginal fold 


ZMH 


Zoologisches Museum der Universität, 




Hamburg, Germany 



CANARIVITRINA AND THE PLUTONIINAE PHYLOGENY 



41 



TABLE 1. Genus-group taxa, type species and bibliographical sources of anatomical data. 



Genus-group taxa 



Type species 



Sources 



Arabivitrina Thiele, 1931 
Canarivitrina, n. subgen. 

Oligolimax Fischer, in Paulucci, 

1878 
Gallandia Bourguignat, 1880 

(= Trochovitrina O. Boettger, 

1Í 



Guerrina Odhner. 1954 



Insulivitrina Hesse, 1923 



Madeirovithna Groh & Hemmen, 

1986 
Phenacolimax Stabile, 1859 



Plutonia Morelet, in Stabile, 1864 
Semilimax Agassiz, 1845 



Vitrina arabica Thiele, 1910 

P. (Canarivitrina) taburientensisn. 
sp. 

Vitrina paulucciae Fischer, in 
Paulucci, 1878 

Vitrina conoidea, sensu 
Bourguignat, 1880 [non 
Martens, 1874] = Gallandia 
o/ymp/ca Hausdorf, 1995 

Helix cutícula Shuttleworth, 1852 



Helicolimax lamarckii A. 
Férussac, 1821 



Vitrina nitida Gould, 1848 

Helicolimax major A. Férussac, 
1807 

Viquesnelia atlántica Morelet, 

1860 
Helix semilimax J. Férussac, 

1802 



Thiele, 1931; Forcart, 1957; Zilch, 

1959; Neubert, 1998 
this paper 

Manganelli et al., 1995 

Schileyko, 1986; Hausdorf, 1995, 
and personal communication 



Odhner, 1954; Ibáñez et al., 1987; 

Valido et al., 1993; personal 

unpublished data 
Hesse, 1923; Odhner, 1937; 

Forcart, 1957; Ibáñez et al., 

1987; Schileyko, 1986; personal 

unpublished data 
Groh & Hemmen, 1986 

Forçait, 1944, 1949, 1957; 
Hausdorf, personal communica- 
tion; personal unpublished data 

Wiktor & Backeljau, 1995; Mordan, 
personal communication 

Eckhardt, 1914; Forcart, 1944; 
Kerney et al., 1979; Grossu, 
1983; Schileyko, 1986; Hausdorf, 
personal communication 



(1,7) had three character states that were 
treated as nonadditive; character 1 was 
treated as unordered, and 7 as ordered for the 
reasons indicated in the explanation of the 
cladistic analysis; four characters (3, 5, 8, 9) 
were autapomorphies with an additional au- 
tapomorphy in each multi-state character. 



RESULTS 
TAXONOMIC DESCRIPTIONS 

Family Vitrinidae Fitzinger, 1833 
Type genus: Vitrina 
Draparnaud, 1801 

Diagnosis 

Limacoidea, sensu stricto (semislugs and 
slugs with vas deferens running along the in- 
side of the penial sheath; without sper- 
matophore, epiphallus and flagellum), with 
shell very thin, glossy, pale and translucent, 
usually with fewer than three rapidly expand- 



ing whorls. Protoconch generally spirally dot- 
ted with numerous pits, which sometimes are 
also present on the teleoconch. Animal with 
penial gland and without penial nerve passing 
through the cerebral ganglion (see Hausdorf, 
1998). 



Comments 

Schileyko (1986) divided the Vitrinidae into 
three subfamilies -Vitrininae Fitzinger, 1833; 
Phenacolimacinae Schileyko, 1986; and 
Semilimacinae Schileyko, 1986. Vitrina Dra- 
parnaud, 1 801 , and Calidivitrina Pilsbry, 1 91 9, 
were included in the subfamily Vitrininae (sar- 
cobelum and glándula amatoria absent; 
vagina very reduced). Semilimacinae, charac- 
terized by the short vagina and by the pres- 
ence of a distinct, atrial sarcobelum, included 
Semilimax Agassiz, 1845, Oligolimax Fischer, 
in Paulucci, 1878, Vitrinobrachium Kunkel, 
1929, and dubitatively Eucobresia Baker, 
1 929. This last genus lacks a sarcobelum and 
has a vaginal papilla near the atrium possibly 



42 



ALONSO ETAL. 



TABLE 2. Characters used on the phylogenetic 
analysis. The numeration refers to the explanations 
in the text. 

(1) Shell 

External, the animal cannot withdraw into it = 
External, the animal can withdraw into it = 1 
Internal, reduced = 2 

(2) Teleoconch ornamentation 
Glossy = 

Entirely and very densely ribbed = 1 

(3) Radular type 
Dichoglossan = 
Beloglossan = 1 

(4) Location of the glándula amatoria — stimulator 
organ 

Distinct from vagina (Sarcobelum) = 
Fused with the proximal part of vagina = 1 

(5) Proximal portion of penis long, equipped with 
torus and velum 

Absent = 
Present = 1 

(6) Penial sheath 
Strong = 
Rudimentary or absent = 1 

(7) Arrangement of the penial retractor muscle 
Free of the right optic nerve = 

Passing rounding the right optic nerve (Fig. 3G) 

= 1 
Penial retractor absent = 2 

(8) Vaginal retractor muscle 
Absent = 

Present = 1 

(9) Oviducal retractor muscle 
Absent = 

Present = 1 



homologous to the spout of the sarcobelum 
(Schileyko 1 986: figs. 11,12). Phenacolimaci- 
nae, characterized mainly by the long vagina 
and the presence of a glándula amatoria 
on the proximal vagina, includes Guerrina 



Odhner, 1954; Arabivitrina Thiele, 1931; In- 
sulivitrina Hesse, 1923; Phenacolimax Sta- 
bile, 1859; Trochovitrina O. Boettger, 1880; 
and Plutonia Morelet, in Stabile, 1864. 

Hausdorf (1998) regarded this system as 
unacceptable, the Vitrininae being poly- 
phyletic, the Semilimacinae paraphyletic, and 
only the Phenacolimacinae monophyletic. 
Moreover, the name Phenacolimacinae is a 
junior synonym of Plutoniinae Cockerell 
(1893), established for Plutonia. 

According to Schileyko (1986), the Vitrini- 
nae and the Phenacolimacinae evolved inde- 
pendently from an unknown common ances- 
tor, while the Semilimacinae derived from the 
Phenacolimacinae by the transformation of 
the glándula amatoria ("vaginal gland") into a 
sarcobelum. We assume the contrasting the- 
ory, sketched by Simroth (1889) and sup- 
ported by Hausdorf (1997, 1998), is the most 
probable: the Phenacolimacinae derived from 
a Semilimax-Uke ancestor by the fusion of the 
sarcobelum with the proximal vagina so as to 
give the glándula amatoria. 

The sarcobelum of the type species of 
Semilimax (Fig. 1) consists of a voluminous 
oblong oval or club-shaped stimulator not 
covered by soft tissue, with a thick glandular 
layer wrapping a longitudinal duct, which 
opens distally into the atrium through a spout, 
the duct being upholstered (internally with re- 
spect to the glandular layer) by a thin layer of 
circular muscles and a horny tube, the latter 
missing in all the other Vitrinidae. The wall of 
the distal portion of the sarcobelum invagi- 
nates sheathing the spout; at the invagination 
edge, the sarcobelum wall is connected with 
the base of the spout by a very thin layer of 
connective tissue. 

As Hausdorf (1998) has shown in his hypo- 



TABLE 3. Original data matrix used for cladistic analysis. All characters with three character states are 
treated as nonadditive; the character T is treated as unordered and the character '7' as ordered. The 
keys and numeration refer to the explanations in the text. The variable or unapplicable character states 
were coded as a '?'. 



Key 


Taxa 


1 


2 


3 


4 


5 


6 


7 


8 


9 


A 


Sem 





























В 


Ara 





? 





















С 


'ST 













1 




1 








D 


Gal 


1 


? 





















E 


Gue 


1 


1 












1 








F 


Ins 


















1 








G 


Mad 


















1 


1 





H 


Phe 




























I 


Plu 


2 


? 


1 







1 


2 





1 



CANARIVITRINA AND THE PLUTONIINAE PHYLOGENY 



43 




FIG. 1. Genital system of Semilimax showing the sarcobelum without the inner horny tube; redrawn from 
Schileyko (1986: figs. 6-7) without scale, as the original drawings. 



thetical explanation of stimulator evolution in 
the Stylommatophora, the genus Semilimax 
may be seen as a basal branch of the Vit- 
rinidae, the atrial sarcobelum possibly being 
homologous to the penial appendix of the Or- 
thurethra, with portions A 3 and A 4 absent and 
A 2 and A 5 fused. But another explanation is 



also possible: the sarcobelum could be origi- 
nated by the ancestral loss of portions A 2 and 
A 3 , the shortening of the A 4 portion (the 
spout), and the invagination of A 4 into the A 1 
portion (Giusti, pers. comm.). We assume the 
last theory because it explains favourably the 
presence of the spout sheath. 



44 



ALONSO ET AL. 



Subfamily Plutoniinae 
Cockerell, 1893 

(junior synonym: Phenacolimacinae Schi- 

leyko, 1986) 
Type genus: Plutonia Morelet, in Stabile, 1864 



Diagnosis 

Vitrinidae with a thin shell variable in shape, 
normally with a narrow basal periostracal 
fringe. Distinct atrial stimulator (sarcobelum) 
absent. Vagina long, its proximal portion 
housing a muscular glándula amatoria with a 
distal spout (see Schileyko, 1986). 

The autapomorphic character state of this 
subfamily is the fusion of the sarcobelum with 
the proximal vagina to give the glándula ama- 
toria (Hausdorf, 1995). 



Comments 

The name Plutoniinae was established by 
Cockerell (1893) for Viquesnelia atlántica 
Morelet, 1860, an Azorean carnivorous slug 
feeding on such animals as earthworms and 
slugs, and with an almost subterranean way 
of life, sheltered in moist places, in moss or 
under stones and detritus; due to its very spe- 
cialized features for a predatory life style, Wik- 
tor & Backeljau (1995) proposed to remove it 
from the family Vitrinidae and assign it to a 
separate family. 

Cockerell (1893) mentioned the possibility 
that Plutonia had also been used for a genus 
of trilobites, and Collinge (in Cockerell, 1893) 
proposed an eventual change of the name 
Plutoniinae to Vitriplutoniinae. However, as 
Backhuys (1975) indicated, the name Pluto- 
nia for a genus of the Trilobita was published 
later: Plutonia Hicks, 1871, Invalid Generic 
Name by homonymy (ICZN, 1997, Opinion 
1880). 

Shelley & Backeljau (1995) recognized a 
case of homonymy between Plutoniinae Boll- 
man, 1893 (Arthropoda, Chilopoda, type- 
genus: Plutonium Cavanna, 1881) and Plu- 
toniinae Cockerell, 1893, and proposed the 
emendation to Plutoniainae Cockerell, 1893. 
However, Backeljau & Shelley (1996) modi- 
fied their original application and proposed to 
amend the myriapod name to Plutoniuminae 
and to retain the molluscan name unchanged 
as Plutoniinae; this proposition has been ac- 
cepted by the ICZN (1997, Opinion 1880). 



Taxa Belonging to Plutoniinae 

Schileyko (1986: 147) referred Guerrina, 
Arabivitrina, Insulivitrina, Phenacolimax, Tro- 
chovitrina, and PlutoniaXo his Phenacolimaci- 
nae. The type species of Trochovitrina, T. led- 
eri (O. Boettger, 1878) and Phenacolimax 
annularis (Studer, 1820), studied by Schileyko 
(1986) as examples of the respective genera, 
were later grouped with Gallandia olympica 
Hausdorf, 1995, in the genus Gallandia Bour- 
guignat, 1880, by Hausdorf (1995). Hausdorf 
recognized this genus to be characterized by 
the following autapomorphies: shell conoid- 
globose, shell and mantle lobes reduced, free 
oviduct short, pedunculus [of the bursa copu- 
latrix] short and penis short. 

Ibáñez et al. (1987) grouped the taxa in- 
cluded by Forcart (1957) in Phenacolimax, s. 
I. (Phenacolimax, s. str., Arabivitrina, Oligoli- 
max, Insulivitrina, Plutonia, and Guerrina) and 
Madeirovitrina in the Phenacolimacinae, 
mainly based on the same characters as Schi- 
leyko (1986). 

The type species of Oligolimax, Vitrina 
paulucciae Fischer, in Paulucci, 1878, was 
considered by Forcart (1965) and Giusti 
(1971) to be a synonym of V. bonelli Targioni 
Tozzetti, 1873, which belongs to the genus 
Semilimacella Sobs, 1917, anatomically not 
far from Semilimax. Recently, Manganelli et 
al. (1995) indicated that V. paulucciae is not a 
synonym of Semilimacella bonelli and that its 
genital system anatomy is so similar to that of 
the species grouped in Insulivitrina and Gal- 
landia, that these last two names could be 
seen as junior synonyms of Oligolimax. 

Gallandia is almost certainly a junior syn- 
onym of Oligolimax, but the complete descrip- 
tion of O. paulucciae has not yet been pub- 
lished (Giusti et al., in prep.). We therefore 
continue to utilize the name Gallandia. On the 
other hand, Insulivitrina is clearly distinguish- 
able from Gallandia (and Oligolimax) because 
of the peculiar course of its penial retractor. 

Genus Plutonia Morelet, in Stabile, 1864 

Type species by monotypy: Viquesnelia at- 
lántica Morelet, 1860 

(Junior synonym: Insulivitrina Hesse, 1923, 
n. syn.) 

Diagnosis 

Plutoniinae with the autapomorphic charac- 
ter state "penial retractor muscle (starting 



CANARIVITRINA AND THE PLUTONIINAE PHYLOGENY 



45 



from the penis) moving forward, forming loop 
around the optic nerve, passing below the 
right ommatophore retractor, then running 
backwards (left of the same right om- 
matophore retractor) to end on left side of di- 
aphragm" (Fig. 3G). 

Description 

Slugs (Plutonia atlántica), semislugs (the 
"Insulivitrina group") or Vithna-Wke helicoid 
snails (the "Guerrina group"). Mantle border 
with well-developed mantle and shell lobes 
(the "Insulivitrina group"), with lobes small to 
very small (the "Guerrina group"), or with shell 
lobes absent {Plutonia atlántica, with a vesti- 
gial shell entirely hidden in the mantle cavity). 
Right side of mantle generally with distinct 
darker coloured "lateral band" above pneu- 
mostome. Foot aulacopod. Sole tripartite, with 
distinct sole furrows, the lateral fused at short 
distance from tail end. Foot dorsally flattened 
beneath the shell, bordered by a pair of dis- 
tinct lateral crests extending for about half the 
tail length; tail tip dorsally keeled. 

Shell thin, greenish, translucent, glossy, 
with 2-4 whorls. Shell variable in shape, from 
slightly depressed, keeled, conical above and 
with small aperture, to auricular with lower 
side very little developed, very large aperture 
and with narrow periostracal fringe. Proto- 
conch pitted, sometimes with radial ribs. 

Jaw oxygnathous, smooth, with median rib 
and central denticle. Radula mainly di- 
choglossan (Jungbluth et al., 1985); central 
tooth little smaller than first lateral teeth, with 
long, slender mesocone having sharp pointed 
tip and two basal ectocones reaching half the 
mesocone length; lateral teeth with long, wide 
mesocone, one basal ectocone and one small 
laterodistal endocone; marginal teeth variable 
in shape. 

System of retractor muscles basically as 
described by Odhner (1937) for Insulivitrina: 
right ommatophore retractor free of penis and 
vagina. Penial retractor, long and slender; 
soon after it leaves the penis wall, it moves 
forward passing below some nerves arising 
from the right pedal ganglion, then above the 
nerve and the retractor of the lower right ten- 
tacle; soon after, it forms loop around the optic 
nerve and, passing below the right om- 
matophore retractor, moves backwards (left of 
the same right ommatophore retractor) to end 
on left of diaphragm. This situation is secun- 
daria lost in Plutonia atlántica and in at least 



one of the Azorean "Insulivitrina" due to re- 
duction of the penial retractor (see Com- 
ments). 

Nervous system as described by Tillier 
(1989: 64) for the family. Palliai complex as 
described by Wiktor & Backeljau (1995) in 
Plutonia atlántica; lung small, kidney sig- 
murethrous, bean-shaped, secondary ureter 
closed like a tube, renal opening near anus 
and pneumostome. 

Genital atrium, penis and distal vagina be- 
come completely evaginated when protruded 
(in some preserved specimens and possibly 
also at mating; Fig. 2J). Genital atrium vari- 
able in length, connected with adjacent part of 
animal integument by short atrial muscles. 
Penial complex without epiphallus and flagel- 
lum. Penial sheath thin, inconspicuous, rudi- 
mentary or absent. 

Bursa copulatrix duct and free oviduct in- 
sert side by side at proximal end of glándula 
amatoria. Glandula amatoria variable in di- 
mensions, wider than distal vagina in which it 
opens through a spout. It can be partially to 
completely coated by a layer of soft tissue that 
can easily be stripped off and it is formed by 
thick outer muscular layer and inner glandular 
layer wrapping the longitudinal duct. 

Distal vagina variably long, its wall invagi- 
nated to envelop the spout of the glándula 
amatoria, to its tip where it fuses with the 
same spout tip. Invaginated vaginal wall and 
spout wall entirely connected by thin connec- 
tive fibres easily removable without destroy- 
ing the vaginal wall (allowing the invagination 
to be eliminated in the laboratory); at the in- 
vagination edge, the vaginal wall is also con- 
nected with the base of the spout by a very 
thin layer of connective tissue (as found in the 
sarcobelum of Semilimax; Fig. 1). 

Spermoviduct long, about one-quarter of 
total genital system length. Prostate poorly 
distinguishable. Albumen gland relatively 
small and oval in shape. Hermaphrodite duct 
long. Gonad follicular, located at apex of vis- 
ceral sac. 



Comments 

The autapomorphic character state of the 
diagnosis was firstly evidenced in Plutonia 
(Insulivitrina) lamarcki, two other Plutonia (In- 
sulivitrina) species and Plutonia (Guerrina) 
cutícula by Hoffmann (1929) and Odhner 
(1937, 1954). It is shared by all the Canahan 
and Madeiran Plutoniinae and was also evi- 



46 



ALONSO ETAL. 



denced in the Azorean Plutoniinae (those 
which retain a penial retractor; Mordan, pers. 
comm.). It is interesting to note that one 
Azorean species (different from Plutonia at- 
lántica) has been recently discovered to have 
a very vestigial or perhaps missing retractor 
(Mordan & Frias Martins, in prep.), which 
hence has been interpreted as a possible 
synapomorphy of this species and P. at- 
lántica. Thus, all the Macaronesian Plutoni- 
inae would belong to the same genus, Pluto- 
nia Morelet, in Stabile, 1864, of which 
Insulivitrina Hesse, 1923, will become a sub- 
genus. The relations of P. atlántica with the 
other Azorean Plutoniinae taxa is supported 
by the cladistic analysis given below. 

The adult animal (with the exception of the 
Guerrina species), cannot withdraw com- 
pletely into the shell. This may happen only 
after a long period of desiccation and (or) 
without feeding, a situation that has been ver- 
ified in some probably senile adult specimens. 

Forcart (1957) attributed a glandular func- 
tion to the soft tissue coating the glándula am- 
atoria. Other authors believe that despite it 
glandular appearance, it has a different func- 
tion, as yet undetermined (Schileyko, 1986). 
The glandular function is feasible, but un- 
proven since this tissue wraps the thick mus- 
cular layer of the glándula amatoria externally, 
and there is no evident communication with 
the central lumen of the glándula amatoria. 

The Plutonia species are endemic to three 
mid-Atlantic archipelagoes located at the 
western fringe of the Palaearctic: Azores, 
Madeira and Canary Islands. The alpine Vit- 
rina glacialis Forbes, 1837, once assigned to 
Insulivitrina (Forcart, 1944: 654), has been re- 
cently presumed as belonging to Eucobresia 
Baker, 1929 (H. Nordsieck, in Falkner, 1991: 
103); data on the course of its penial retractor 
are nevertheless still pending. 

Plutonia taburientensis 

Groh & Valido, n. sp. 

Diagnosis 

Plutonia with penis as long as vagina (in- 
cluding its glándula amatoria portion), divided 
into two portions: proximal penis long, slen- 
der, with penial retractor muscle inserted at 
apex and two internal parallel longitudinal 
structures, a torus and, opposite, a wide 
velum. Distal penis short, slightly widened, 
containing a globular to cylindrical penial 
papilla level with the entrance of the vas def- 



erens into the same penis (far from the penial 
apex). Penial papilla perforated, its central 
channel continuous with vas deferens. Penial 
papilla and torus coated by glandular tissue, 
the "penial gland". Distal portion of penis with 
two very thin penial folds, which join level with 
the penial papilla, a small pit remaining be- 
tween them and the papilla itself. Thin vaginal 
fold sometimes present, ending before reach- 
ing genital atrium. 

Type Material 

Holotype (Fig. 2A): collected by K. Groh 
and M. Ibáñez (8-1-1988) in the Caldera de 
Taburiente (La Palma Island; UTM: 
28RBS1879), at 750 m altitude; deposited in 
AIT. Paratypes: 144 specimens and 22 shells 
collected between January 1988 and Febru- 
ary 1 997, in various localities of the island and 
deposited in ANSP (A 18852/2), CGH (32 
paratypes, 29 specimens and 3 shells), CRT 
(8 paratypes), CGS (1 paratype), SMF 
(311875/5), TFMC (MT 0314/2), ZMH 
(2745/2) and AIT. 

Type Locality 

Caldera de Taburiente, La Palma Island. 

Etymology 

The specific name refers to the Caldera de 
Taburiente, a geological feature of La Palma. 

Habitat and Distribution 

A species endemic to La Palma, occurring 
in wide areas climatologically different, with 
pinewood and sometimes with lowland vege- 
tation, at an altitude between 300 and 1,800 
m (Fig. 5). 

Animal 

Body whitish-beige, with numerous small 
pale-beige spots, more dense in number at 
head. Mantle and tail with darker grey spots 
and a short blackish-grey lateral band. Foot 
sole sometimes with marginal grey spots. 

Shell 

Oblong, with about 2.5 whorls. Protoconch 
pitted; starting from beginning of second 
whorl, the pits are less deep and more scat- 




FIG. 2. A-D. Holotype shells. A. P. taburientensis, n. sp. В. P. ripkeni, n. sp. С P. dianae, n. sp. D. P. falcifera, 
n. sp. E. Protoconch of P. taburientensis, n. sp. F-G. Protoconch and pits detail of P. ripkeni, n. sp. H-l. 
Radula of P. ripkeni. n. sp. H, central tooth and lateral teeth; I, marginal teeth. J. A specimen of P. mascaen- 
sis with the distal part of the genital system everted, showing the penis with the penial papilla and the vagina 
with the glándula amatoria (by transparency). Scale: A-D, J, 5 mm; E, F, 200 цт; G, 100 \ivn; H, I, 20 цт. 



48 



ALONSO ETAL. 




FIGS. 3, 4. Genital system and anatomical details. A. P. taburientensis. n. sp., paratype; Caldera de Taburi- 
ente. B. P. ripkeni, n. sp., paratype; Jerduñe. С. P. dianae, n. sp.. paratype; Montaña Bejira. D. P. falcifera. 
n. sp., paratype; Tamolde. E. P. mascaensis (redrawn from Ibáñez et al., 1987, fig. 14). F. Internal penial de- 
tails of P. taburientensis, n. sp. G. Arrangement of the Insulivitrina penial retractor muscle. Scale: A-E, 1 mm; 
F-G, without scale. 



CANARIVITRINA AND THE PLUTONIINAE PHYLOGENY 



49 




FIG. 4. See figure 3 for legend. 



50 



ALONSO ETAL. 



tered, disappearing after first half of second 
whorl (Fig. 2A, E). 

Radula 

Formula: 31 -33M + 11-12L + C; marginal 
teeth with only one cusp. 

Genital System 

Shown in Figures ЗА, F, 4A; 12 specimens 
dissected. Penis as long as vagina (including 
its glándula amatoria portion). Laminar crest 
inside the proximal portion of penis wide and 
same length as torus, the latter distally 
smaller and thinner, as a laminar fold, joins 
the penial papilla; this fold partially surrounds 
the penial papilla and extends into the distal 
portion of penis; opposite the papilla, another 
small laminar fold present inside distal penis; 
the two folds join level with the penial papilla, 
a small pit remaining between them and the 
papilla itself; finally, the folds extend sepa- 
rately into the atrium. 

Distal vagina twice as long as the glándula 
amatoria portion, nearly one-third shorter than 
the free oviduct and 2-2.5 times longer than 
the bursa copulatrix duct; sometimes with one 
thin internal longitudinal fold ending before 
the atrium. Glandula amatoria with a protrud- 
ing spout. 

Comments 

Of all the described Plutonia species, P. 
taburientensis, n. sp., shares only with P. 
mascaensis the presence of a proximal penis 
with the penial retractor muscle inserted at 
apex and internal torus and velum; the main 
differences between them are listed in the Di- 
agnosis and Comments on the latter. The 
proximal penis is eversible (Fig. 2J) and it is 
not a flagellum. There is no spermatophore. 

Plutonia ripkeni 

Alonso & Ibáñez, n. sp. 

Diagnosis 

As for P. taburientensis, n. sp., but the vagi- 
nal fold, always present, extends to fuse with 
a penial fold into the atrium. 

Type Material 

Holotype (Fig. 2B): collected by K. Groh 
and M. Ibáñez (3-1 -1 988) from Tagamiche (La 



Gomera Island; UTM: 28RBS85 10), at 950 m 
altitude; deposited in AIT. Paratypes: 71 spec- 
imens collected between December 1 985 and 
March 1997 in various localities of the island 
and deposited in ANSP (A 18853/2 and 
400849/2), CGH (2 paratypes), CRD (2 
paratypes), CRT (6 paratypes, 3 specimens + 
3 shells), NNM (55868/1), SMF (311874/5 
and 311876/2), TFMC (MT 0313/2), ZMH 
(2746/2) and AIT. 

Type Locality 

Tagamiche, La Gomera Island. 

Etymology 

The specific name derives from the family 
name of Mr. Theodor "Theo" E. J. Ripken, 
Delft, The Netherlands, to whom the species 
is dedicated. 

Habitat and Distribution 

A species endemic to La Gomera, occurring 
in wide areas with lowland vegetation, border- 
ing the evergreen "lauhsilva" forest of the Na- 
tional Park of Garajonay, at an altitude be- 
tween 200 and 1 ,025 m (Fig. 5). 

Animal 

Body whitish-beige-grey, with numerous 
small darker grey spots, more dense on man- 
tle and tail areas. Lateral band blackish-grey. 

Shell 

Oblong, with 2.25 whorls. Pits in spiral rows 
on protoconch and teleoconch; from begin- 
ning of second whorl, the pits of some rows or 
of adjacent rows frequently fuse forming an ir- 
regular drawing. Pits disappear after first half 
of second whorl (Fig. 2B, F, G). 

Radula 

Formula: 29M + 12L + C; first marginal 
teeth with very small ectocone and last mar- 
ginal teeth with only one cusp (Fig. 2H, I). 

Genital System 

Shown in Figures 3B and 4B; 1 5 specimens 
dissected. Penis similar or little longer than 
vagina (including its glándula amatoria por- 
tion). Laminar crest inside proximal penis 



CANARIVITRINA AND THE PLUTONIINAE PHYLOGENY 



51 



wide and same length as torus, the latter dis- 
tally thinner, as a fold, joins the penial papilla; 
this fold partially surrounds the penial papilla 
and extends into the distal portion of penis; 
opposite the papilla, another fold is present in- 
side distal penis; the two folds join level with 
the penial papilla, a small pit remaining be- 
tween them and the papilla itself; finally, the 
folds extend separately into the atrium, the 
fold deriving from torus fuses with the vaginal 
longitudinal fold. 

Distal vagina nearly three times longer than 
the glándula amatoria portion, as long as free 
oviduct and 1 .5-2 times as long as bursa cop- 
ulatrix duct; with one internal longitudinal fold 
which fuses with the penial fold inside the 
atrium. Glandula amatoria with small spout. 

Comments 

Other than the characters listed in the diag- 
nosis, P. ripkeni, n. sp., differs from P. taburi- 
entensis, n. sp., in vaginal folds more devel- 
oped; distal portion of penis, penis and vagina 
longer; glándula amatoria smaller; and spout 
less protruding. 

Plutonia dianae 

Valido & Alonso, n. sp. 

Diagnosis 

As for P. tabuhentensis, n. sp., but the penis 
is approximately half as long as the vagina 
(including its glándula amatoria portion). 

Type Material 

Holotype (Fig. 2C): collected by R. Hutterer 
and M. Ibáñez (3-11-1994) from Montaña Be- 
jira (La Gomera Island; UTM: 28RBS7320), at 
530 m altitude; deposited in AIT. Paratypes: 
1 specimens and 8 shells collected the same 
day in the type locality and the nearby Argua- 
mul and deposited in ANSP (A 1 8854/1 ), CGH 
(1 paratype), TFMC (MT 0312/1) ZMH 
(2747/1, shell) and AIT. 

Type Locality 

Montaña Bejira, La Gomera Island. 

Etymology 

The specific name derives from that of the 
young daughter of the junior author, Diana 



Valido de Armas, to whom this species is ded- 
icated. 

Habitat and Distribution 

A species endemic to northwest La 
Gomera, occurring in a small area with low- 
land vegetation, at an altitude between 350 
and 530 m (Fig. 5). 

Animal 

Body whitish-beige, with numerous small 
pale-beige spots, more dense at head. Mantle 
and tail with darker grey spots. Lateral band 
short, blackish-grey, interrupted level with 
pneumostome. Foot sole sometimes paler 
than body. 

Shell 

Very oblong, with slightly more than 2.75 
whorls. Pits in spiral rows on protoconch and 
teleoconch, disappearing at beginning of sec- 
ond whorl (Fig. 2C). 

Genital System 

Shown in Figures 3C and 4C; five speci- 
mens dissected. Penis approximately half as 
long as vagina (including its glándula amato- 
ria portion), flattened, tongue-shaped. Proxi- 
mal penis finger-like, with rounded tip. Lami- 
nar crest inside proximal penis wide and 
same length as torus; distal torus slightly 
smaller, partially surrounding the penial 
papilla and extending thicker into distal por- 
tion of penis; opposite the papilla, another 
similar fold present inside distal penis; the two 
folds join level with penial papilla, a deep pit 
remaining between them and papilla. 

Distal vagina very long, up to four times 
longer than the glándula amatoria portion, lit- 
tle longer than free oviduct and 1.5-2 times 
longer than bursa copulatrix duct, with internal 
longitudinal fold that ends before reaching 
atrium. Glandula amatoria very short, with 
small spout. 

Comments 

Other than the characters listed in the diag- 
nosis, P. dianae, n. sp., differs from P. taburi- 
entensis, n. sp., and P. ripkeni, n. sp., in the 
distal penis with deeper pit between penial 
papilla and the two thicker penial folds. Pluto- 
nia dianae, n. sp., differs from P. taburienten- 



52 



ALONSO ETAL. 



sis, n. sp., also in the spout less protruding 
and from P. ripkeni, n. sp., in the vaginal fold, 
which ends before genital atrium. 



Plutonia falcifera 

Ibáñez & Groh, n. sp. 



Diagnosis 



As for P. taburientensis, n. sp., but the lam- 
inar crest of the proximal portion of penis is 
narrow or even vestigial; distal portion of 
penis without pit near penial papilla; the pit is 
substituted by a pilaster connected proximally 
with the papilla and ending distally in a small 
slope with transversal furrows, reminiscent of 
a fingerprint. 



Type Material 

Holotype (Fig. 2D): collected by R. Hutterer 
and M. Ibáñez (31-1-1994) from Tamolde (La 
Gomera Island; UTM: 28RBS9112), at 200 m 
altitude; deposited in AIT. Paratypes: 23 spec- 
imens collected between January 1988 and 
March 1997 in various localities from the is- 
land and deposited in ANSP (A 18855/1), 
CGH (7 paratypes), SMF (311979/1) TFMC 
(MT 0311/2), ZMH (2748/1) and AIT. 



Type Locality 
Tamolde, La Gomera Island. 



Etymology 

The specific name derives from the Latin 
falcifer ("sickle carrier"), due to the shape of 
penis. 



blackish-grey. Foot sole sometimes with mar- 
ginal grey spots. 

Shell 

Oblong, with 2.75 whorls. Pits in spiral rows 
on protoconch and up to the end of second 
whorl where they become smaller to disap- 
pear at the beginning of third whorl (Fig. 2D). 

Genital System 

Shown in Figure 4D; eight specimens dis- 
sected. Penis as long as vagina (including its 
glándula amatoria portion), with proximal por- 
tion curved, entire penis being sickle shaped 
when extended after dissection; proximal 
penis with an empty zone at its beginning, 
which is narrower than the rest. Laminar crest 
narrow, vestigial in some specimens. Torus 
big, starting after the empty zone at the be- 
ginning of proximal penis. Distal torus thin, 
partially surrounding the penial papilla and ex- 
tending into the distal penis as a thick pilaster, 
which ends in a small slope with transversal 
furrows, reminiscent of a fingerprint. 

Distal vagina with thin internal longitudinal 
fold, nearly twice as long as glándula amato- 
ria portion and little longer than both free 
oviduct and bursa copulatrix duct. Glandula 
amatoria with spout well protruding. 

Comments 

Other than the characters listed in the diag- 
nosis, P. falcifera, n. sp., differs from all the 
other species herein described for: penis 
sickle shaped when extended after dissec- 
tion; at its beginning the proximal penis has 
an empty zone. The vagina has a thin internal 
longitudinal fold. 



Habitat and Distribution 

A species endemic to east La Gomera, oc- 
curring in a small area with lowland vegetation 
and pinewood, at an altitude between 200 and 
600 m (Fig. 5). 



Plutonia mascaensis 
(Morales, 1987) 

Insulivitrina mascaensis Morales, 1987, in 
Ibáñez et al.: 133-135, 139, figs. 7, 14, 
26-28, 36, 47, 48, 51 [loe. typ.: Barranco 
de Masca, Tenerife]. 



Animal 

Body whitish-beige, with numerous small 
beige spots, more dense at head. Mantle and 
tail with darker grey spots. Lateral band short, 



Diagnosis 

As for P. falcifera, n. sp., but the proximal 
penis ends pointed and lacking an empty 
zone. 



CANARIVITRINA AND THE PLUTONNNAE PHYLOGENY 



53 



28R UTM zones 




CANARY ISLANDS 
UTM grid: 100 km 
18°00': meridian 



Lanzarote^J^ 




Fuerteventura 



Gran Canaria 




31 



Africa 




FIG. 5. Geographical distribution of the Canarivitrina. n. subgen, species; the symbols represent 1 x 1 km 
squares. 



54 



ALONSO ETAL. 



Type Material 

Holotype (AIT) and 114 paratypes, de- 
posited in ANSP (A1 8856/2), CGH (2 
paratypes), FMNH (205917/1), MHNG 
(984647), MNHN, NHM (1986130/2), NNM 
(55862/1), SMF (305945/2), TFMC (MT 
0310/2), ZMH (2749/2) and AIT. 

Habitat and Distribution 

A species endemic to west Tenerife, occur- 
ring in areas with lowland vegetation or 
pinewood, at an altitude between 90 and 
1,100 m (Fig. 5). 

Animal 

Body whitish-grey, with numerous small 
darker grey spots, more dense on mantle, 
head and tail areas. Lateral band grey, 
blurred. Some specimens have foot sole uni- 
formly whitish. 

Shell 

Slightly oblong, with 2.5 whorls. Pits in spi- 
ral rows on protoconch and teleoconch, be- 
coming smaller after first whorl to disappear at 
end of second whorl. 

Radula 

Formula: 29M + 10L + С (Ibáñez et al., 
1987: figs. 26-28); marginal teeth with very 
small ectocone. 

Genital System 

Shown in Figures 2J, 4E; 18 specimens dis- 
sected. Penis longer than vagina (including its 
glándula amatoria portion), generally coiled in 
natural position, fringed on one side. Proximal 
penis very long, proximally pointed. Distal 
penis short, slightly widened. Laminar crest 
narrow, sometimes vestigial. Distal torus thin, 
partially surrounding penial papilla and ex- 
tending into distal penis as a thick pilaster, 
which ends in a small distal slope with trans- 
versal furrows, reminiscent of a fingerprint. 

Vagina twice as long as the glándula ama- 
toria portion, one-third longer than free 
oviduct and little longer than bursa copulatrix 
duct, with two thick internal longitudinal folds 
which converge near the atrium. Glandula 
amatoria with small spout, almost blunt. 



Comments 

Other than the characters listed in the diag- 
nosis, P. mascaensis differs from the other 
species described herein because the penis, 
coiled in natural position, is fringed on one 
side; from P. falcifera, n. sp., it also differs in 
not having a sickle-shaped penis, its penis in- 
stead having a shorter distal portion and a 
longer proximal portion. 



DISCUSSION 

The five species are cohesive and probably 
represent a distinct supraspecific taxon, ST, 
due to the following synapomorphies: genital 
system with long penis divided into two por- 
tions; proximal penis long and slender, with 
penial retractor muscle inserted at apex and 
with two internal parallel longitudinal struc- 
tures, a torus and, opposite, a velum; distal 
penis short and slightly widened, containing a 
globular to cylindrical and perforated penial 
papilla level with the entrance of the vas def- 
erens into the same penis (far from the penial 
apex); penial papilla and torus coated by glan- 
dular tissue (the "penial gland"). 

ST differs from all the other Plutoniinae in 
the following autapomorphy: proximal penis 
long and equipped with two internal parallel 
longitudinal structures, torus and velum. Fol- 
lowing the praxis of traditional taxonomy, this 
autapomorphy is sufficient to support inclu- 
sion of ST in a distinct supraspecific taxon 
closely related to the other Macaronesian Plu- 
toniinae. 

The structure of the glándula amatoria in ST 
and in the other Plutoniinae supports the hy- 
pothesis of a homology with the sarcobelum of 
Semilimax, even if the latter is distinct from the 
proximal vagina. The homology was advanced 
by Simroth (1886, 1889), rejected by Wieg- 
mann (1886), and later accepted by several 
authors, such as Hesse (1923), Schileyko 
(1986), and Hausdorf (1995). 

To verify the status (genus/subgenus) of 
ST, that of the other Macaronesian Plutoni- 
inae and their relationships we applied a 
cladistic approach. 

Plutoniinae Phylogenetic Relationships 

Explanations of the characters used in the 
phylogenetic analysis; the numeration refers 
to Table 2 and 3 and the cladogram (Fig. 6). In 



CANARIVITRINA AND THE PLUTONIINAE PHYLOGENY 



55 



these explanations and Table 3, we utilize the 
name Plutonia in the old sense, that is, only 
for P. atlántica. 

(1) Shell. Semilimax, as well as ST, Arabi- 
vitrina, Insulivitrina, Phenacolimax, and Ma- 
deirovitrina have an external shell with more 
than 1 .5 whorls, into which the animal cannot 
withdraw. 

Gallandia and Guerhna have a conical-de- 
pressed shell, keeled in Guerhna and Gallan- 
dia lederi, with a small aperture through which 
the animal can withdraw entirely. 

Plutonia has a reduced, capuliform, internal 
oval shell, with about half a whorl. 

(2) Teleoconch ornamentation. ST, Insulivi- 
trina, Madeirovitrina, and Phenacolimax have 
a glossy teleoconch, like Semilimax. 

The Arabivithna teleoconch ornamentation 
is variable (see Appendix). 

Gallandia lederi and G. olympica have an 
entirely ribbed teleoconch, with the ribs rami- 
fied and interconnected in G. olympica; G. an- 
nularis has a glossy teleoconch. Therefore 
the character state is variable within the 
genus. 

Guerhna has an entirely and very densely 
ribbed teleoconch, with a pattern different 
from that of Gallandia lederi and Gallandia 
olympica. 

Plutonia has the teleoconch with concentric 
growth lines but the shell is internal and its or- 
namentation is not comparable with that of the 
other Plutoniinae. 

(3) Radula. Semilimax, as well as almost all 
the Vithnidae, has the radula of the dichoglos- 
san type. 

The radula of Plutonia belongs to the bel- 
oglossan type. 

(4) Location of the glándula amatoria. In 
Semilimax, this is a club-like atrial diverticu- 
lum distinct from vagina. 

All the Plutoniinae have a glándula amato- 
ria fused with proximal vagina. 

(5) Penis. Semilimax, as well as Arabivit- 
hna, Gallandia, Guerhna, Insulivitrina, Ma- 
deirovitrina, Phenacolimax, and Plutonia, 
have a penis without a long proximal portion 
equipped with torus and velum. 

ST has a penis with a long proximal portion 
equipped with torus and velum. 

(6) Penial sheath. Semilimax and 
Phenacolimax have a strong penial sheath. 

In the other Plutoniinae, the penial sheath is 
rudimentary or absent. 

(7) Penial retractor muscle. Semilimax, as 
well as Arabivithna, Gallandia, and Phenacol- 



imax, have the penial retractor muscle with a 
normal course between its insertions, free 
from the right optic nerve. 

In ST, Guerhna, Insulivitrina, and Madei- 
rovitrina the penial retractor muscle has a pe- 
culiar course, rounding the right optic nerve 
(Fig. 3G). 

Plutonia has no penial retractor muscle, 
probably due to a secondary reduction origi- 
nating independently from that of S. semili- 
max. 

(8) Vaginal retractor muscle. ST, Arabivi- 
thna, Gallandia, Guerhna, Insulivitrina, Phe- 
nacolimax, and Plutonia have no vaginal re- 
tractor muscle, like Semilimax. 

Madeirovitrina, with the exception of M. al- 
bopalliatus (see above), has a vaginal retrac- 
tor muscle. 

(9) Oviducal retractor muscle. ST, Arabivi- 
thna, Gallandia, Guerhna, Insulivitrina, Ma- 
deirovitrina, and Phenacolimax have no ovid- 
ucal retractor muscle, like Semilimax. 

Plutonia has a short oviducal retractor with 
a branch inserting at the bursa copulatrix 
duct. 

Cladistic Analysis 

The Plutoniinae phylogenetic relationships 
are not easy to establish, because it is not yet 
possible to determine the state of several 
characters which initially appear to be taxo- 
nomically interesting, due to insufficient 
knowledge, intrageneric variability or redun- 
dancy (see Appendix). 

Character 7 was treated as ordered by the 
possible synapomorphy of one Azorean In- 
sulivitrina species and Plutonia atlántica, re- 
ferred to the absence of the penial retractor 
muscle (as indicated in the Comments after 
the description of the genus Plutonia); a simi- 
lar scenario was found also in the outgroup. 
{Semilimax semilimax does not have a penial 
retractor muscle but one is present in S. kotu- 
lae, free from the right optic nerve: see Ap- 
pendix.) 

Character 4 justifies the monophyly of the 
Plutoniinae with respect to the outgroup. Un- 
fortunately, only three other characters (1, 6 
and 7) are phylogenetically informative; the 
remaining characters (2, 3, 5, 8, and 9) are 
only found in one taxon. 

Eight informative (but not resolutive) trees 
from the matrix (Table 3) were found. How- 
ever, the strict consensus of these trees 
shows the Plutoniinae as the sister group of 



56 



ALONSO ETAL. 



Semilimax (character 4) and Phenacolimax 
as the sister group of the remaining Plutoni- 
inae (character 6). Moreover, the eight trees 
show ST (key C), Insulivitrina (key F), and 
Madeirovitrina (key G) grouped together at 
the same level. The trees are as follows: 

tree 1 = (A((B(CFG)(D(EI)))H)) 

tree2 = (A((B(C(DEI)FG))H)) 

tree3 = (A((B(CDEFGI))H)) 

tree4 = (A((B(CEFGI)D)H)) 

tree5 = (A((B(CFGI)(DE)H)) 

tree6 = (A((BC(DEI)FG)H)) 

tree 7 = (A((B(C(DE)FGI))H)) 

tree8 = (A((BC(DE)FGI)H)) 

Strict consensus tree = (A((BCDEFGI)H)) 

Figure 6 represents tree number 4 of the 
analysis, with a consistency index (CI) of 
0.917, a retention index (Rl) of 0.800 and a 
rescaled CI index (RC) of 0.733. This tree 
shows a probable phylogenetic scenario with 
the Macaronesian taxa included in the genus 
Plutonia (of which Insulivitrina is a new syn- 
onymy), with a single conflict: the character 
state 1.1 ('shell external, the animal can with- 
draw into it') appears in Gallandia and Guer- 
rina, but it could be homoplasic. This supposi- 
tion is based on: 

(A) Guerrina clearly differs from Gallandia 
and belongs to the genus Plutonia. This is 
demonstrated by the fact that it shares the 
same synapomorphy (the peculiar course of 
the penial retractor) which links all the other 
taxa of the Plutonia group. 

(B) The capacity of the animal to withdraw 
into the shell is related to the respective di- 
mensions and the shell form; the suitable re- 
lation could be acquired independently in Gal- 
landia and Guerrina. 

(C) The independent action of the Vitrinidae 
limacization process, found in Semilimax, In- 
sulivitrina, and Madeirovitrina, as well as in 
other Vitrinidae; these taxa have some 
species with the ventral side of the shell well 
developed and other species with the shell 
auricular with very wide aperture and very re- 
duced ventral side (see Appendix). The Guer- 
rina shell form could have originated as a 
phase opposite to the auricular form in the li- 
macization process of the genus Plutonia. 

(D) Gallandia and Guerrina live at opposite 
ends of the geographical range of the Plutoni- 
inae. 

After excluding character 1 of the analysis, 
only one tree was found: the same one repre- 
sented in Figure 6, with a consistency index 
(CI) of 1.0000. 



1-1 



1-1 



1-2 



Semilimax 

Phenacolimax 

Arabivitrina 

Gallandia 

P. (Canarivitrina) 
n. subgen. 

P. (Guerrina) 
P. (Insulivitrina) 

P. (Madeirovitrina) 
P. (Plutonia) 



FIG. 6. Hypothesis of phylogeny of Plutoniinae. 
Numbers refer to characters listed within the 
Table 2. 



CONCLUSIONS 

(1) The subfamily Plutoniinae includes the 
genera Phenacolimax, Gallandia (which is al- 
most certainly a junior synonym of Oligoli- 
max,) Arabivitrina, and Plutonia. 

(2) The genus Phenacolimax is the sister 
group of the remaining Plutoniinae. 

(3) The supraspecific taxon ST has a sub- 
generic category in the genus Plutonia, which 
also includes the subgenera Plutonia, s. str., 
Guerrina, Insulivitrina, and Madeirovitrina. 

(4) We describe the supraspecific taxon ST 
as follows: 

Subgenus Canarivitrina 

Valido & Alonso, n. subgen. 

Type species: P. (C.) taburientensis Groh & 
Valido, п. sp. 

Diagnosis 

Plutonia with a long penis divided into two 
portions. Proximal portion long, slender, with 
penial retractor muscle inserted at apex and 
two internal parallel longitudinal structures: 
torus and, opposite, velum. Distal portion 
short, slightly widened, containing a globular 
to cylindrical and perforated penial papilla 
level with entrance of the vas deferens into 
penis (far from proximal apex). Penial papilla 



CANARIVITRINA AND THE PLUTONIINAE PHYLOGENY 



57 



and torus coated by glandular tissue, the "pe- 
nial gland". 



BIOGEOGRAPHY 

The monophyly of the Macaronesian Plu- 
toniinae has biogeographical implications. 
The three archipelagoes housing these Plu- 
toniinae, plus the Cape Verde Islands (which 
also house a vitrinid, probably a Plutoniinae: 
Groh, 1983) are often considered to form a 
biogeographic unit called Macaronesia (= 
"fortunate islands"), located in the north-east- 
ern Atlantic at the western fringe of the 
Palaearctic. The Azores are the most remote 
from continental land, nearly in the centre of 
the Atlantic, and are situated at about 850 km 
NW of Madeira, the latter lying about 650 km 
from Morocco. 

The Canary Islands lie about 450 km south 
of Madeira, about 500 km north of the Tropic 
of Cancer, and their easternmost island is 
only 100 km distant from Morocco. Land 
bridges with Morocco have been hypothe- 
sized in the past but today the oceanic origin 
of all the Canary Islands is currently attributed 
to the activity of a hotspot in the Earth's man- 
tle (Holik et al., 1991; Carracedoetal., 1998). 

The oldest island of the Azores dates back 
nearly 5.5 Ma (Nunn, 1994), the oldest part of 
the Madeiran archipelago has an age be- 
tween 13-20 Ma (Mitchell-Thomé, 1976) and 
that of the Canaries, 22.5 Ma (Ancochea et 
al., 1993). 

The present subtropical flora of the three 
northern archipelagoes, which includes the 
evergreen laurel-forest or "laurisilva," is 
closely related and also with that of the 
Miocene and Pliocene of the Mediterranean 
region. Nevertheless, the fauna is different, 
although it is predominantly of a Palaeartic 
origin (the Azores have also Neartic affinities; 
Eason & Ashmole, 1 992). The flora and fauna 
of the Cape Verde Islands show a remarkably 
strong Ethiopian influence, especially of the 
region called Saharo-Sindian, (Lobin, 1982), 
but it also include some relicts of the laurel- 
forest. With respect to the malacofauna there 
is a mixture of Palaeartic, Ethiopian and Mac- 
aronesian elements in equal portions (Groh, 
1983). As Baez (1993) indicated, most of the 
Madeiran faunal groups present more affini- 
ties with those of Central Europe than with 
those of the Mediterranean area. The contrary 
is true for the neighbouring Canary Islands. 



Waiden (1984) concluded that "the most con- 
spicuous difference between the mollusc fau- 
nas of Madeira and the Canary Islands is the 
complete absence of taxa with NW African 
affinities on Madeira". 

The different faunistic composition of these 
archipelagoes is probably due, at least par- 
tially, to the different origin of the colonizers 
that landed on them after the Pleistocene 
glaciations. The Azorean fauna and flora are 
much less diverse than those of the other 
archipelagoes (Ashmole et al., 1996). 

A conspicuous example of the different fau- 
nistic composition is offered by the Bulim- 
inidae, a taxon present with different subfam- 
ilies on the Azores and the Canaries (Hesse, 
1933; Henriquez et al., 1993). The Madeiran 
archipelago lacks Buliminidae, their niches 
occupied by the Clausiliidae, a group absent 
from the Azores and the Canaries (Neubert & 
Groh, 1998). 

Eason & Ashmole (1992) evidenced isola- 
tion of the Azores to be severe and that it de- 
pends not only on their distance from conti- 
nents, but also on meteorological and 
océanographie conditions, which are not con- 
ducive to the arrival of animals or plants by 
sea from western Europe or Africa. Surface 
currents are, in fact, basically from the west 
throughout the year. The same authors also 
pointed out that water circulation in the North 
Atlantic was in a highly dynamic state around 
the end of the last glaciation and earlier in the 
Pleistocene, and that colonization by the an- 
cestors of the modern Azorean forms might 
have occurred during a relatively short phase 
in one of the glaciation cycles during which 
oceanic currents were more conducive to col- 
onization than they are at present. 

As for the Madeiran fauna, Baez (1993) 
suggested it must have been originated 
mainly by anemochore or idrochore trans- 
oceanic colonization. Nevertheless, the sea- 
mounts along the Madeira-Iberian crest may 
have provided stepping stone islands in early 
times. 

Finally, the Canary Islands colonization has 
probably taken place from northwest Africa, 
which in the past was covered by dense 
forests and had mighty rivers (Baez et al., 
1983). In the Miocene-Pliocene, the Atlantic 
Moroccan rivers would drag vegetation islets, 
floating rafts, that would be transported to the 
nearby coasts of the Canarian archipelago by 
the trade winds and the marine streams. The 
wadi Drâa was the most probable colonizing 
source because its mouth is located opposite 



58 



ALONSO ETAL. 



to Lanzarote and Fuerteventura, the eastern- 
most Cañarían Islands. 

We consider that the presence of Plutonia 
on the three (or four) archipelagoes is not 
recent, Put probably as old as the humid lau- 
rel-forest, that is, existing long before the 
Pleistocene glaciations, as Waiden (1984) 
suggested, and that the evolution of the 
genus took place in all archipelagoes in a pe- 
riod preceding the Pleistocene glaciations. 
The Plutonia species survived the glaciations 
probably associated to the Tertiary relict forest 
and later on they conquered some others of 
their present biotopes, whereas their extinc- 
tion in the continental lands may also be as- 
sociated to the extinction of the laurel-forest. 

ACKNOWLEDGEMENTS 

Our special thanks go to Dr. B. Hausdorf 
(Hamburg), Dr. P. Mordan (London), and Dr. 
E. Neubert (Frankfurt), as well for the critical 
review of the earlier versions of the manu- 
script and their very valuable informations and 
suggestions as for disposing us some of their 
very important unpublished data; to Dr. F. 
Giusti (Siena) and an anonymous referee for 
the exhaustive review of the paper; addition- 
ally to Dr. Giusti, Dr. G. Manganelli (Siena), 
and Dr. Jorge Crisci (La Plata) for their com- 
puterized cladistic analysis of the data matrix; 
to Dr. Neubert for the gift of specimens of Ara- 
bivitrina jansseni; to Dr. W. Rähle (Tübingen) 
for contributing his data on P. taburientensis, 
n. sp., and P. ripkeni, n. sp.; to Mr. T. E. J. Rip- 
ken (Delft), for his collaboration in the collect- 
ing of the P. ripkeni, n. sp., material; and to 
Mrs. P. Agnew for linguistic revision. 

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Revised ms. accepted 1 June 1999 



APPENDIX. TAXONOMIC COMPARISON 
OF THE GENUS-GROUP TAXA 

Semilimax (anatomical data taken mainly 
from Schileyko, 1986). Shell variable in 
shape: auricular with very wide aperture 
and reduced ventral side (S. semilimax) 
or with ventral side well developed (Vit- 
rina carniolica O. Boettger, 1 884: Grossu, 
1983). Animal cannot withdraw into shell. 
Teleoconch glossy. Right shell lobe 
rather narrow, twisted backwards to 
cover shell apex. Radula dichoglossan 
(Jungbluth et al., 1985). Genital atrium 
short (Fig. 1). Penis short, without long 
proximal portion (torus and velum conse- 
quently absent). The presence of a perfo- 
rated penial papilla needs verification. 
Semilimax semilimax does not have a 
penial retractor muscle, but one is pres- 
ent in S. kotulae (Weste rl und, 1883), free 
from the right optic nerve. "Glandula am- 
atoria" voluminous, oblong oval or club- 
shaped, not covered by soft tissue, lo- 
cated in atrial sarcobelum, which wall 
invaginates sheathing the spout (no data 
available about connection between in- 
vaginated sarcobelum wall and spout 
wall). Vagina short. 

Arabivitrina (anatomical data taken mainly 
from Neubert, 1998). Shell medium to 
large, auricular to subglobose, with 3-3.5 
whorls. Animal cannot withdraw com- 
pletely into shell (at least in A. jansseni). 
Teleoconch ornamentation variable, al- 
most smooth, sometimes with blunt axial 
riblets in several species (as in type 
species, Vitrina arabica Thiele, 1910), or 
without blunt axial riblets. Aperture bor- 
dered by a small periostracal fringe. 
Right shell lobe rather narrow, twisted 
backwards to cover shell apex. Radula 
dichoglossan. Genital atrium long. Penis 
as a broad tube, about as long as vagina, 
without long proximal portion (torus and 
velum are consequently absent); with a 
longitudinal pilaster consisting at least of 
an apical glandular part followed by a 
narrow to broadened part with transverse 
lamellae. Typical penial papilla missing. 



CANARIVITRINA AND THE PLUTONIINAE PHYLOGENY 



61 



Penial retractor free from right optic 
nerve. Vas deferens entering penis sub- 
apically, then turning towards retractor in- 
sertion area to run inside glandular tissue 
of apical organ and open at apex. Distal 
vagina and glándula amatoria similar in 
length. Glandula amatoria covered with 
soft tissue, large, club-shaped to oblong 
oval, but A. darnaudi has a short glándula 
amatoria. Invaginated vaginal wall and 
spout wall entirely connected by strong 
connective fibres in A. jansseni. 

Gallandia (anatomical data taken mainly from 
Hausdorf, 1995). Shell conoid-globose, 
with 2.5-3.25 whorls and small aperture. 
Teleoconch entirely ribbed or glossy. Ani- 
mal can withdraw more-or-less entirely 
into shell. Shell and mantle lobes re- 
duced (as Hausdorf, 1995, stated, this re- 
duction is connected with the fact speci- 
mens can withdraw completely into the 
shell). Radula dichoglossan. Genital 
atrium long. Penis short, without long 
proximal portion (torus and velum conse- 
quently absent). Vas deferens entering 
subapically at penis, under penial gland. 
Penial retractor free from right optic 
nerve. Glandula amatoria small, club- 
shaped to oblong-oval, sometimes par- 
tially covered by soft tissue, arranged like 
a flower calyx (no data available about 
connection between invaginated vaginal 
wall and spout wall). 

Guerrina (anatomical data taken mainly from 
Ibáñez et al., 1987, and Valido et al., 
1993). Shell slightly depressed, keeled, 
conical above, with 3.25-3.75 whorls and 
small aperture. Teleoconch entirely and 
densely ribbed. Animal can withdraw en- 
tirely into shell. Shell lobes and right body 
lobe of mantle very small, not evident in 
live specimens; left body lobe small. 
Radula dichoglossan. Penis short, with 
perforated penial papilla and two small 
portions, the homologies of which are un- 
clear because of different location of in- 
sertion of penial retractor in G. cutícula 
and G. christinae; without long proximal 
portion (torus and velum consequently 
absent). Vas deferens entering penis lat- 
erally, between the two portions. Penial 
retractor rounding right optic nerve. Glan- 
dula amatoria small, spheroidal, with 
proximal end covered with soft tissue, 
arranged like a flower calyx. Distal vagina 
very long, up to four times longer than 
glándula amatoria portion. Invaginated 



vaginal wall and spout wall entirely con- 
nected by thin connective fibres. 

Insulivitrina (anatomical data taken mainly 
from Ibáñez et al., 1 987). Shell variable in 
shape, as in Semilimax. Teleoconch 
glossy. Animal cannot withdraw into shell. 
Right shell lobe well developed, covering 
almost entire shell in undisturbed condi- 
tions, but disturbed specimens (Ibáñez et 
al., 1987: figs. 6, 8) show this lobe similar 
to those published for Semilimax semili- 
max, S. pyrenaicus (A. Férussac, 1821), 
and Phenacolimax major (Falkner, 1989: 
1 73; 1 992: 270-271 ; Kerney et al., 1 979, 
pis. 6, 7). Radula dichoglossan with vari- 
able marginal teeth (finger-shaped with 
or without ectocones, or multicuspid). 
Genital atrium variable in length. Penis 
short, without long proximal portion (torus 
and velum consequently absent) and 
with perforated penial papilla. Vas defer- 
ens entering penis apically to subapically. 
Penial retractor rounding right optic 
nerve. Glandula amatoria covered with 
soft tissue, usually voluminous, club- 
shaped to oblong-oval, but Insulivitrina 
canariensis (Mousson, 1872) has a small 
glándula amatoria. Distal vagina similar 
in length or even shorter than glándula 
amatoria portion. Invaginated vaginal 
wall and spout wall entirely connected by 
thin connective fibres. 

Madeirovitrina (anatomical data taken mainly 
from Groh & Hemmen, 1986). Shell vari- 
able in shape, as in Semilimax. Teleo- 
conch glossy. Animal cannot withdraw 
into shell. Right shell lobe well developed, 
covering almost entire shell in undis- 
turbed conditions. Radula dichoglossan. 
Genital atrium medium to long. Penis 
large and proximally globe-shaped, with 
perforated penial papilla subapical and 
without long proximal portion (torus and 
velum consequently absent). Vas defer- 
ens entering penis subapically. Penial re- 
tractor rounding right optic nerve. Glan- 
dula amatoria voluminous, club-shaped 
to oblong-oval, covered with soft tissue. 
Invaginated vaginal wall and spout wall 
entirely connected by thin connective fi- 
bres. Distal vagina wide and shorter than 
glándula amatoria portion, with a strong 
vaginal retractor muscle. (Madeirovitrina 
albopalliatus Groh & Hemmen, 1986, dif- 
fers from the other species in its tubular 
penis and the absence of a vaginal re- 
tractor muscle; this species should there- 



62 



ALONSO ETAL. 



fore be referred to another genus, possi- 
bly Insulivitrina). 
Phenacolimax (anatomical data taken mainly 
from Forcart, 1957; Hausdorf, pers. 
comm., and our own unpublished data). 
Shell globose-depressed, with 2.5-3.25 
whorls and small aperture. Teleoconch 
glossy. Animal cannot withdraw into shell. 
Right shell lobe rather narrow, twisted 
backwards to cover shell apex. Radula 
dichoglossan. Genital atrium very short. 
Penis long, tubular, with a clear constric- 
tion wrapped by a short but strong penial 
sheath (similar to those shown by Schi- 
leyko, 1986: figs. 11-12, in Eucobresia); 
without long proximal portion (torus and 
velum consequently absent) and with 
perforated penial papilla. Vas deferens 
running inside penial sheath and entering 
penis subapically. Penial retractor free 
from right optic nerve. Glandula amatoria 
small, not completely covered by soft tis- 
sue (no data available about connection 



between invaginated vaginal wall and 
spout wall). Distal vagina and glándula 
amatoria similar in length. 
Plutonia (referred to P. atlántica; anatomical 
data taken mainly from Wiktor & Backel- 
jau, 1995). Shell entirely hidden by the 
mantle (shell lobes absent), oval, vesti- 
gial, whitish, transparent, with about half 
a whorl and a wide aperture. Teleoconch 
with growth lines. Radula beloglossan, 
with dagger-shaped teeth. Vas deferens 
entering penis apically. Penis short, with- 
out long proximal portion (torus and 
velum consequently absent), penial 
papilla and penial retractor. Glandula am- 
atoria voluminous, club-shaped to ob- 
long-oval (no data available about con- 
nection between invaginated vaginal wall 
and spout wall). Distal vagina similar in 
length or even longer than glándula ama- 
toria portion. Short oviducal retractor with 
one branch inserted into bursa copulatrix 
duct. 



MALACOLOGIA, 2000, 42(1-2): 63-74 

THE DISTRIBUTION OF OCTOPUSES OF GRANELEDONE 
(CEPHALOPODA: OCTOPODIDAE) IN REFERENCE TO DEEP-SEA FEATURES 

Janet R. Voight 

Department of Zoology, Field Museum of Natural History, Roosevelt Road at Lake Shore Drive, 
Chicago, Illinois 60605 USA; jvoight@fmnh.org 

ABSTRACT 

Although deep-sea hydrothermal vents are renowned for having many apparently endemic 
taxa, normal deep-sea taxa also occur at vents. These opportunistic taxa have been hypothe- 
sized to tolerate these physiologically stressful habitats due to pre-adaptation to low oxygen con- 
ditions. Octopuses of the deep-sea genus Graneledone, likely a monophyletic group, appear to 
be one such taxon as they occur at several East Pacific hydrothermal vents. To further our knowl- 
edge of this genus and of other taxa opportunistic at chemosynthetic habitats, collection locali- 
ties of these octopuses north of 40°S were verified and whether features such as vents, cold 
seeps, tectonic activity or hydrocarbon deposits were present near the collection localities was 
determined. 

Hydrothermal vents, cold seeps or hydrocarbon deposits lie within one degree of 17 of the 24 
localities confirmed north of 40°S. This distribution suggests that the octopuses routinely occur 
in low oxygen habitats, potentially due to a pre-adaptation, a hypothesis that will require a 
species-level phylogeny for the genus. Low oxygen habitats offer opportunists increased prey 
biomass and possibly reduced prédation rates. In addition, several of these areas offer hard sub- 
strata essential for egg attachment and brooding by females of Graneledone. 

Key words: Graneledone, deep sea, habitat use, Octopodidae, macrofaunal predators. 



INTRODUCTION 

Benthic octopuses of the family Octopodi- 
dae occur from the intertidal zone to depths 
as great as 3,931 m (Voss, 1988), but what 
determines the distribution and abundance of 
deep-sea octopuses and of other mobile 
deep-sea macrofauna remains unknown. 
Trawled specimens provide few details on 
habitat use and few observations of deep-sea 
octopuses from submersibles have been re- 
ported (e.g., Vecchione & Roper 1991). Com- 
pounding the lack of taxon-specific data is our 
meager knowledge of what constitutes dis- 
tinct sea floor habitats. If we are unable to 
distinguish what the animals recognize as dis- 
tinct habitats, we cannot distinguish preferen- 
tial habitat use. 

Hydrothermal vents, characterized by ele- 
vated temperatures, anoxic, metal-rich fluids 
and extremely high biological productivity, 
offer one distinctly recognizable deep-sea 
habitat. Although the vent fauna appears to be 
highly endemic, members of the "normal" 
deep-sea fauna are observed occasionally in 
apparent association with these habitats. 
Whether these are chance sightings of the an- 



imals near the vent habitat (Tunnicliffe et al., 
1998), or whether these taxa actively exploit 
vent resources is unresolved. The ability to 
exploit vent resources is thought to be possi- 
ble only if these non-vent specialized animals 
are unusually tolerant of low oxygen availabil- 
ity (Tunnicliffe, 1991). 

This paper explores the global distribution 
of the octopodid genus Graneledone, "nor- 
mal" deep-sea predators that occur on three 
different ridge systems and prey on vent 
fauna (Voight, in review). If some members of 
the genus exploit hydrothermal vent habitats 
because tolerance of low oxygen availability 
has evolved in the group, then other members 
are predicted to occur in such areas. To test 
this hypothesis, documented occurrences of 
members of the genus are compiled. For each 
locality, geological indicators of reduced oxy- 
gen availability are noted. Hypotheses of pre- 
adaptation to low oxygen cannot be assessed 
until the phylogeny of the genus is resolved, 
but recognition of a such a distributional pat- 
tern could motivate such an effort and help us 
distinguish deep-sea habitats. Global exami- 
nations of the distribution of other taxa that 
appear to be opportunists at vents may aid in 



63 



64 



VOIGHT 



determining the generality of the pattern seen 
here. 

MATERIALS AND METHODS 
The Genus 

The genus Graneledone is argued to be 
monophyletic because several character 
states that are derived among the Octopodi- 
dae (Voight, 1997) characterize the genus in 
a unique combination. The nearly equal-sized 
papillae (also called warts or tubercles) on the 
skin of the dorsal mantle and head, stout 
papillae over each eye, comparatively few gill 
lamellae (6-9), the absence of the plesiomor- 
phic ink sac and crop diverticulum, and the 
presence of small tubular bodies at the junc- 
tion of the tube and cement gland, as illus- 
trated for spermatophores of G. pacifica (Voss 
& Pearcy, 1990: fig. 20e, f), support the hy- 
pothesis of monophyly. Six species are cur- 
rently recognized in the genus, although 
species distinctions are poorly defined (Voss 
& Pearcy, 1 990; Villaneuva & Sánchez, 1 993). 
Subtle morphological variation among North 
Pacific specimens collected from different 
depths has been detected and suggests the 
presence of cryptic species (Voight, 1998). 
With few specimens available from other 
areas, the potential existence of other cryptic 
species cannot be assessed. As a result, 
species names are applied here only in refer- 
ence to type specimens or type localities. 

Octopuses of the genus Graneledone can 
reach weights of nearly 1 kg and exceed 50 
cm in total length. They have been recorded 
from depths as shallow as 512 m (O'Shea & 
Kubodera, 1996), although most range be- 
tween 850 (Voss, 1988) and 2,755 m (Voss & 
Pearcy, 1990). Their biology remains poorly 
known. Reported gut contents include cope- 
pods, amphipods (Voss & Pearcy, 1990) and 
crabs, vent gastropods and polychaetes 
(Voight, in review). Reproduction among 
these species likely follows that typical of 
cephalopods, being limited to the extreme ter- 
minal portion of the life span (Boyle 1983, 
1987). Females brood eggs attached to hard 
substrate (Voight & Grehan, in review). 

Documentation of Distribution 

Collection localities north of 40°S of speci- 
mens of Graneledone documented by mu- 
seum holdings, literature reports and/or by 
submersible-based photographs or video- 



tapes (Appendix 1) were compiled. Literature 
reports were also evaluated as a data source, 
although when possible, museum specimens 
were examined to verify generic identification. 
Well-illustrated taxonomic accounts by estab- 
lished workers (e.g., Berry, 1 917; Villanueva & 
Sánchez, 1993) were accepted, as were re- 
cent re-determinations at the South Africa Mu- 
seum (M. Roeleveld, pers. comm.) and Boyle 
et al. 's (1998) report of three members of 
Graneledone from the Hebrides Shelf. Two 
reports, however, had to be rejected. Bruun's 
(1945) report of the genus from Iceland re- 
mains unverified. The report lacks illustra- 
tions, and the specimen cannot be located in 
the collections of the Zoologisk Museum of 
Copenhagen (T Schiotte, pers. comm.). 
Grieg's (1933) report of the genus from Nor- 
way, cited uncritically by Stephen (1944), was 
also rejected. The photographs of the speci- 
mens and the measurements Grieg (1933) re- 
ported do not support the identification of 
these specimens as members of Granele- 
done based on criteria discussed by Thomsen 
(1931). 

Submersible-based photographs and vid- 
eotapes that showed key characters were 
also used to document the presence of mem- 
bers of the genus. If the photographs and 
videotapes of octopuses in situ clearly 
showed the single row of arm suckers and 
dorsal skin papillae that among octopodids 
north of 40°S at over 800 m depths are unique 
to members of Graneledone (Voss, 1 988), the 
records were accepted. Examination of seven 
submersible-collected specimens from the 
North Pacific (Appendix 1 ) that were identified 
in videotapes and photographs as members 
of Graneledone supported their generic deter- 
mination. 

Habitat Characterization 

A description of the sea floor geology of 
every documented collection locality of the 
genus was sought. Pertinent data, reported in 
Appendix 1, include the presence of hy- 
drothermal vents or vent deposits, plate mar- 
gins (where either subduction or transform 
movement occurs), turbidite flows (massive 
submarine landslides), and hydrocarbon (gas 
hydrate and/or petroleum) deposits. Under at 
least some conditions, these geological fea- 
tures are associated with environments rich in 
sulfides or methane. Bacteria mediate the ox- 
idation of these reduced compounds and can 
form the basis of chemosynthetic species as- 



DISTRIBUTION OF DEEP-SEA GRANELEDONE 



65 




FIG. 1 . The non-Antarctic collection localities of octopuses in the genus Graneledone documented are indi- 
cated by stars. Dotted lines indicate the position of spreading centers; solid lines indicate subduction zones. 
Map adapted from Baker et al. (1995). 

TABLE 1 . Summary of the habitat types from which octopuses of Graneledone 
have been documented. Localities are considered different if they are sepa- 
rated by at least one degree latitude or longitude. Of the 24 localities north of 
40°S, 17 are unusual in being near a hydrothermal vent, cold seep or hydro- 
carbon deposit. Of the seven seemingly normal northern habitats, five are in 
the Atlantic Ocean (Appendix 1). 



Number of different localities 



Habitat type 



Hydrothermal vents 
Cold seeps at tectonically active areas 
Hydrocarbon (petroleum/gas) deposits 
Seemingly normal areas north of 40°S 
Apparently unknown areas south of 40 c 



semblages in areas with these features (Kulm 
et al., 1986; Kennicutt et al., 1988; Mayer et 
al., 1988). 

Collection records of such taxa as vesi- 
comyid bivalves that appear entirely depen- 
dent on endosymbiotic chemosynthetic bac- 
teria (Fisher, 1995) have been used to 
hypothesize the existence of chemosynthesis 
in unknown areas (e.g., Kennicutt et al., 
1989). The underlying assumption, however, 
is that members of these taxa do not vary in 
habitat use. Because variation in habitat use 
within the taxon Graneledone is being investi- 
gated here, these reports cannot contribute 



non-circular data to this study. Marine geology 
is poorly known south of 40°S, so records 
of the genus from this area could not be 
considered. As a result, several records, in- 
cluding the type localities of G. antárctica, G. 
macrotyla, although listed in Appendix 1, 
could not be detailed. 



RESULTS 

Verified localities of Graneledone (Fig. 1) 
and pertinent geological features that charac- 
terize the localities are summarized in Table 1 ; 



66 



VOIGHT 



supporting data are detailed in Appendix 1. 
Below is a brief review of the areas and the 
pertinent geological data. 

Occurrences at Hydrothermal Vents 

Benthic Graneledone are documented at 
eight hydrothermal vent fields on Explorer and 
Juan de Fuca Ridges, and Guaymas Basin 
and Galapagos Rift on the East Pacific Rise. 
A Graneledone was photographed on the 
Galapagos Rift (Corliss & Ballard, 1977: 448) 
among shells of chemosynthetic clams and 
live mussels. A specimen of Graneledone cf. 
boreopacifica collected near chemosynthetic 
clams at Axial Volcano on Juan de Fuca 
Ridge had preyed on vent gastropods and 
polychaete worms (Voight, in review). 

On-going marine geological studies at Ker- 
madec Ridge are documenting active hy- 
drothermal vents (Matsumoto et al., 1997; 
Stoffers et al., 1999). This ridge is the type lo- 
cality of Graneledone challengeri Hoyle, 
1886. 

Occurrences at or Near Cold Seeps at 
Plate Boundaries 

Heat and pressure created by tectonic ac- 
tivity such as the subduction of oceanic plate 
can release reduced fluids that sustain 
chemosynthetic species assemblages at cold 
seeps (e.g., Kulm et al., 1986). The distribu- 
tion and fauna of cold seeps are poorly docu- 
mented compared to hydrothermal vents, but 
a published photograph shows a Granele- 
done at a tube worm cluster (Moore et al., 
1990: fig. 6e) on the Oregon Subduction 
Zone. 

Known cold seeps in Monterey Bay occur 
near 3,000 m depth on the Monterey Fan Val- 
ley system (Embley et al., 1990) and at three 
localities near 36°N, 122°W between 580- 
1,010 m (Barry et al., 1997). Octopuses have 
not been collected or reported from any of 
these sites. Specimens of Graneledone, how- 
ever, are numerous between 1 , 1 00 and 1 ,660 
m depth near a fault seaward of the Sur High 
at the mouth of Monterey Bay (Greene, 1 990). 
The collection area, a zone of transform fault- 
ing (Greene, 1990), is likely to have addi- 
tional, undiscovered cold seeps (Kennicutt et 
al., 1989) and oxygen tensions below 1.0 ml/l 
due to heavy upwelling (Kamykowski & Zen- 
tara, 1990) and the presence of a dysaerobic 
zone (Mullinsetal., 1985). 

Members of Graneledone are also known 



from the northwest Pacific where subduction 
also is linked to the presence of cold seeps. 
As at the Oregon margin, subduction at the 
Japan and the extreme southern Kuril-Kam- 
chatka Trenches is documented to support 
chemosynthesis at depths greater than 3,000 
m (Métivier et al., 1986; Le Pichón et al., 
1987). In addition to what may be subduction- 
linked cold seeps, gas hydrates (frozen 
methane deposits) occur off Sakhalin Island 
in the Sea of Okhotsk (Ginsburg et al., 1993). 
Without citing precise localities, Nesis (1982) 
reports that G. boreopacifica occurs in the 
Okhotsk Sea and the Pacific Ocean northeast 
of Honshu. Unfortunately, there are no further 
data for this area. 

The collection locality of a member of the 
genus off New Zealand (O'Shea & Kubodera, 
1996) lies within two minutes longitude of a 
sea floor plume, interpreted as methane-rich 
water released in response to plate boundary 
processes (Lewis & Marshall, 1996). 

Occurrences Near Hydrocarbon Deposits 

Assemblages of chemosynthetic species, 
characteristic of the most extreme habitat dis- 
cussed in this paper, also occur on passive 
continental margins (Sibuet & Olu, 1998). On 
the Northwest Atlantic margin, release of flu- 
ids from a 1929 slide of organic-rich sediment 
created extreme reducing conditions at 3,000 
m depth (Mayer et al., 1988). In addition, bac- 
terial production linked to hydrocarbon seep- 
age on the continental shelf has been sug- 
gested to increase surface water productivity 
(Levy & Lee, 1988; Fader, 1991; D. Piper, 
pers. comm., cited by Fader, 1991). Hydro- 
carbon deposits occur in the Northwest At- 
lantic, as evidenced by: (1) bottom simulating 
reflectors, indicative of gas hydrates (e. g., 
Pauli et al., 1995) that lie seaward south of 
Massachusetts (Fig. 2; Dillon et al., 1986); (2) 
salt domes and salt-cored anticlines that can 
be associated with petroleum deposits and 
cold seeps from Nova Scotia to Massachu- 
setts (Fig. 2; Uchupi & Austin, 1979; Dillon et 
al., 1986; Swift, 1987); and (3) the immense 
Hibemia Oil Field (Fig. 2; Brown et al., 1989). 
In a seemingly similar geological setting, 
physical and chemical indicators of hydrocar- 
bon seepage occur over broad areas of the 
Gulf of Mexico (Kennicutt et al., 1 988; Sassen 
et al., 1998). The relationship between the 
collection localities of G. verrucosa and these 
three subsurface geological features is shown 
in Figure 2. 



DISTRIBUTION OF DEEP-SEA GRANELEDONE 



67 



80° 




/~N regions of salt domes and sail-cored 
anticlines of the continental margins 



region of Bottom Simulating Reflector 
( BSR ) probably formed at base of gas 
hydrate-cemented sedimentary layer 



FIG. 2. The Northwest Atlantic collection localities of 
Graneledone verrucosa (Appendix 1 ) are indicated 
by stars. HOF indicates the position of the Hibernia 
Oil Field; shading represents regions of salt domes 
and salt-cored anticlines; cross-hatching repre- 
sents Bottom Simulating Reflectors. Map adapted 
from Dillon etal. (1986). 



Off Walvis Bay, Namibia, near the collection 
locality of a Graneledone (Villanueva & 
Sánchez, 1993), is the site of a turbidite flow 
in which about 90 km 3 of sediment created a 
debris flow that extends 250 km downslope 
(Summerhayes et al., 1979). The slide may 
have been initiated due to unstable gas hy- 
drates in the sediment column (Summer- 
hayes et al., 1979) which may contribute to 
vertical hydrocarbon expulsion (Hyndman & 
Davis, 1992). Sedimentary studies show that 
organic carbon is abundant locally (Embley & 
Morley, 1980). 

Occurrences in Seemingly Normal Areas 

Seven collection localities of Graneledone 
north of 40°S cannot be said to have unusual 
sea floor features. Some of these areas ap- 
pear to be normal, such as Norfolk Canyon 
(Malahoff et al. 1982). Others, such as the 
area south of Iceland and the Gulf of Panama, 
appear to be virtually unexplored. Baby Bare 
has been closely explored. This outcrop of 
basement basalt is where tens of octopuses 
of Graneledone brood eggs (Mottl et al., 1 998; 
Voight & Grehan, in review). Although warm 
water springs with detectable sulfide concen- 
trations exist at Baby Bare (Mottl et al., 1998), 
in the immediate area of brooding neither ele- 
vated sulfide concentrations or temperatures 
have been detected (G. J. Massoth, pers. 
comm.). 

South of 40°S, specimens of the genus 
have been collected from seven areas that 



appear to have received little study by marine 
geologists. Perhaps notable is that three 
specimens are from two localities directly sea- 
ward of the San Jorge Basin, a major petro- 
leum deposit (Fitzgerald et al., 1990). In addi- 
tion, upwelling off the southwest coast of 
Africa, may lower oxygen availability or create 
anoxic conditions in the area (Kamykowski & 
Zentara, 1990). 



DISCUSSION 

Octopuses of the genus Graneledone are 
documented at 31 localities around the world 
(Table 1 , Fig. 1 ). Of the 24 collection localities 
north of 40°S, 12 are within one degree of 
chemosynthetic communities at hydrothermal 
vents or cold seeps associated with tectonic 
activity and five are near hydrocarbon de- 
posits (Table 1, Appendix 1). No available 
data contradict the classification of seven of 
these localities as normal deep-sea habitats, 
including Baby Bare, a basalt outcrop that re- 
leases warm water with measurable concen- 
trations of hydrogen sulfide from its summit 
(Mottl et al., 1998). This distribution suggests 
that at least some members of the deep-sea 
octopodid genus Graneledone are able to tol- 
erate low oxygen availability. This ability may 
allow them to exploit resources unavailable to 
other taxa. 

These data are robust to suggestions the 
submersible observations provide significant 
bias. Submersible-based observations are 
the sole source of distributional data for all hy- 
drothermal vents, Baby Bare and Norfolk 
Canyon. Trawls document the presence of the 
octopuses in all other habitats. When locali- 
ties based solely on submersible data north of 
40°S are eliminated from consideration, the 
octopuses are still documented at ten low- 
oxygen areas and five normal areas. These 
totals are not significantly different from 17 
versus 7, the totals from Table 1 (G-test, G = 
0.302). 

The large size of these octopuses is not ex- 
pected to bias these results. On average, 
members of Graneledone pacifica are mid- 
sized among the five species of northeast Pa- 
cific deep-sea octopodids described by Voss 
& Pearcy (1990). Octopuses of the genus 
Graneledone, however, appear to move more 
slowly than do those of Benthoctopus and 
could therefore be more readily collected by 
trawl and submersible. 

Evidence that the octopuses actually enter 



68 



VOIGHT 



vent areas and therefore experience low oxy- 
gen environments at vents is three-fold. The 
gut of a specimen of G. cf. boreopacifica con- 
tained two species of vent-restricted gas- 
tropods and three taxa of vent polychaetes 
that the octopus ingested prior to its collection 
from Axial Volcano (Voight, in review). Sec- 
ond, a photograph from Galapagos Rift 
(Corliss & Ballard, 1977) shows a Granele- 
done among empty Calyptogena valves and 
live chemosynthetic mussels, the presence of 
which correlates with elevated hydrogen sul- 
fide concentrations (Lutz & Vrijenhoek, 1997). 
The third line of evidence that these octo- 
puses enter areas of active vents is circum- 
stantial. Disarticulated valves of chemosyn- 
thetic clams are present up to one meter away 
from clam beds at North Pacific vents. Be- 
cause such predators as spider crabs, 
galatheids and fishes are unlikely to either 
pick up or to prey on clams, octopuses are 
most likely to have entered the clam bed and 
removed the clams. 

Even in non-low oxygen areas, their hy- 
pothesized foraging behavior may expose oc- 
topuses of the genus Graneledone to sulfide, 
a small molecule that is readily absorbed by 
tissues, in anoxic layers of deep-sea sedi- 
ment. The distal half to two-thirds of the arms 
of a Graneledone photographed at Axial Vol- 
cano (V Tunnicliffe, pers. comm.) and of one 
near Galapagos Rift (R. W. Embley, pers. 
comm.) were so covered with sediment that 
their color matched the sea floor. The octo- 
puses may sometimes forage by probing un- 
consolidated sediment with their arms, which 
emerge covered in sediment. This presumed 
prey search behavior could dramatically in- 
crease the octopus' exposure to sulfide, a 
toxin that readily permeates cells, because it 
increases in concentration dramatically just 
under the sediment surface, especially at cold 
seeps and sedimented vents (Grehan & Ju- 
niper, 1996). Whether exposure of their arms 
to anoxic sediment through this behavior may 
have contributed to the evolution of reduced 
oxygen availability by members of this genus 
remains speculation. 

Low oxygen environments may offer both 
reduced prédation pressure (Fischer & Bot- 
tjer, 1995) and increased prey availability 
(Chevaldonné & Olu, 1996). Because hard- 
shelled molluscs constitute about 34% of the 
taxa at hydrothermal vents (Tunnicliffe et al., 
1998), and octopuses of the genus Granele- 
done may be among the few deep-sea preda- 
tors able to prey on them, possible advan- 



tages of entering the habitat are clear. In ad- 
dition, vent and seep habitats also tend to 
offer exposed hard substrata. Although mem- 
bers of this genus occur on soft substrata, as 
documented by the many trawl-collected 
specimens and the photographs of individuals 
with sediment-covered arms (R. W. Embley, 
V. Tunnicliffe, pers. comm.), females of 
Graneledone attach and brood their eggs on 
hard substrata (Mottl et al., 1998; Voight & 
Grehan, in review). The availability of basalt at 
mid-ocean ridges and carbonates produced 
by cold seeps (e.g., Moore et al., 1990) may 
attract many mature octopuses (Voight & Gre- 
han, in review). 

Pre-adaptation to tolerate low-oxygen avail- 
ability has been suggested to be necessary to 
allow "normal" deep-sea animals to enter vent 
habitats (Tunnicliffe, 1991). Whether octo- 
puses of the genus Graneledone are pre- 
adapted to tolerate low oxygen environment is 
unknown, but these data suggest that in sev- 
eral geographically distinct areas, members of 
the genus Graneledone enter low oxygen 
habitats produced by diverse geological 
mechanisms. Resolution of octopodid phy- 
logeny is required to assess the hypothesis of 
pre-adaptation (Voight, 1997). If, when a 
species-level phylogeny of the genus be- 
comes available, taxa that occur in low oxygen 
habitats are monophyletic, than tolerance to 
low oxygen availability can be concluded to 
have evolved only once in the group. 

More knowledge of the distribution of these 
deep-sea octopuses and other macrofauna 
that are opportunistic predators at vents may 
help identify the level of biological isolation of 
hydrothermal vents, the degree to which 
chemosynthetic-produced biomass is distrib- 
uted to the resource-poor ocean floor and the 
impact of vent habitats on biota of surrounding 
deep-sea areas. In addition, investigations of 
the seemingly anomalous distributions of little- 
known deep-sea animals such as Granele- 
done can increase our biological knowledge of 
the group and help identify behaviors such as 
sediment-probing that may relate to evolution- 
ary changes in their physiology. 



ACKNOWLEDGMENTS 

Critically insightful comments by R. A. 
Zierenberg and the collection of specimens 
from hydrothermal vents by R. A. Lutz, A. J. 
Grehan, and M. Mottl (NSF grant OCE93- 
14632), K. Becker, H. P. Johnson and R. A. 



DISTRIBUTION OF DEEP-SEA GRANELEDONE 



69 



Zierenberg stimulated this research and 
earned my gratitude. Becker, Lutz, P. Cheval- 
donné, J. Delaney, R. W. Embley, S. K. Ju- 
niper, J. О Moore, V. Robigou, P. Rona and V. 
Tunnicliffe generously shared photographs 
and videotapes that were vital to this re- 
search. Specimens made available by W. W. 
Wakefield, J. Moore, J. Wood, and E. Dawe 
enhanced the project's completeness. I thank 
F. E. Anderson, M. E. Anderson, P. Bertrand, 
P. Chevaldonné, J. T P. Copley, J. J. Flynn, L. 
Kulm, M. А. С. Roeleveld, J. Rogers, С D. 
Trowbridge, and S. E. Walker for assistance 
and discussion. C. Simpson prepared the fig- 
ures. Comments by M. J. Brooks, R. S. Car- 
ney, A. C. Driskell, R. W. Embley and two 
anonymous reviews improved earlier manu- 
script drafts. Curatorial staffs of the Academy 
of Natural Sciences of Philadelphia, California 
Academy of Sciences, Canada Museum of 
Nature, Muséum National d'Histoire Na- 
turelle, Paris, University of Miami Marine Lab- 
oratory, and the Zoological Museum, Univer- 
sity of Copenhagen allowed access to 
specimens that were critical to this research. 
This research was partially supported by the 
West Coast and Polar Regions Undersea Re- 
search Center at the University of Alaska Fair- 
banks and NOAA's National Undersea Re- 
search Program. 

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Revised ms. accepted 18 August 1999 



APPENDIX 1 

Occurrences of members of Graneledone 
are listed by their habitat type, with data that 
characterize the habitat and how the octo- 
puses' presence was determined. All FMNH 
specimens were examined. Localities sepa- 
rated by one degree are considered distinct. 
For museum specimens, the depository and 
catalogue number for each are reported if the 
data are available. (ANSP = Academy of Nat- 
ural Sciences at Philadelphia; CAS = Califor- 
nia Academy of Sciences; CMN = Canada 
Museum of Nature; FMNH = Field Museum of 
Natural History; ICM = Instituto de Ciencias 
del Mar, Barcelona, Spain; MNHN = Muséum 
National D'Histoire Naturelle, Paris; MOM = 
Musée Océanographique de Monaco; NSMT 
= National Science Museum, Tokyo; SAM = 
South African Museum; UMML = University of 
Miami Marine Laboratory; USNM = United 
States National Museum; ZMUC = Zoologisk 
Museum of Copenhagen). 

Documented Occurrences at Hydrothermal 
vents, n = 8. 

(1) EXPLORER RIDGE. 49°46'N, 130°16'W, 
1,870 m. Reported by Tunnicliffe et al. 
(1986) as octopus identified from photo- 
graph (V. Tunnicliffe pers. comm.). 

(2) JUAN DE FUCA RIDGE: MIDDLE VAL- 
LEY. 48°30'N, 128°41'W, 2,400 m; FMNH 
278061; 278062; 278063; 281001; 
281002. 

(3) JUAN DE FUCA: ENDEAVOUR SEG- 
MENT. 47°56-58'N, 129°08'W, 2,250 m 
(including Main Field, High Rise and Clam 
Bed vent fields). Identified from video- 



tapes and photographs (V. Tunnicliffe, 
S. K. Juniper, J. Delaney, pers. comm.). 

(4) JUAN DE FUCA: CO-AXIAL SEGMENT. 
46°19.4'N, 129°42'W, 2,200 m. AXIAL 
SEAMOUNT: 45°57'N 130°01'W, 1,560 
m. Identified from photographs and video- 
tapes (V. Tunnicliffe, R. W. Embley pers. 
comm.); FMNH 282751. 

(5) JUAN DE FUCA: CLEFT SEGMENT. 
44°38-58'N, 130°15-30'W, 2,210-2,280 
m. Identified from photograph (V. Tunni- 
cliffe, pers. comm.). 

(6) GUAYMAS BASIN. 27°01'N, 111°24'W, 
1,996-2,000 m. Identified from pho- 
tographs (V. Robigou, P. Chevaldonée, 
pers. comm.). 

(7) GALAPAGOS RIFT: CLAM BAKE. 
0°47-48'N, 86°01-14'W, 2,480 m. Identi- 
fied from published photograph Corliss & 
Ballard (1977). 

(8) KERMADEC RIDGE. Active vents occur 
on this ridge (Wright et al., 1998; Stoffers 
et al., 1 999) but the region with the type lo- 
cality of Graneledone challenger! remains 
a critical gap in our knowledge (Stoffers et 
al., 1999). G. challenger! Hoyle, 1886. 
Type locality: 29°45'S 178 Û 11'W, 630 fm 
(1,175 m), Challenger Station 170A, spec- 
imen illustrated by Berry (1917). Not ex- 
amined 

Occurrences at or Near Cold Seeps at 
Tectonically Active Areas, n = 4. 

(1) OREGON SUBDUCTION ZONE. Over 
600 trawls systematically sampled the 
Cascadia and Tufts Abyssal Plains and 
Oregon's continental slope between 
125°23.6'W and 135°22.7'W (Carney & 
Carey, 1982). All 29 type specimens of 
Graneledone pacifica were collected by 
17 trawls between 125°13.4'W and 
125°52.9'W at 1,427 to 2,755 m depth 
(Voss & Pearcy, 1990), a narrow area that 
includes the documented cold seeps 
(Kulm et al., 1986), areas of upwelling and 
the Astoria Fan (Kulm & Scheidegger, 
1979). A photograph taken at 44°40'N, 
125°18'W, 2,100 m shows an octopus of 
Graneledone with vestimentiferan worms 
(Moore et al., 1990: fig. 6e and J. О 
Moore, pers. comm.). Upwelling produces 
large amounts of biomass which deplete 
oxygen through decomposition on the sea 
floor. The topographically complex Fan 
may support broader areas of chemosyn- 
thesis based on three-D perspective ba- 
thymetry map and a slope map made 
using SeaBeam swath bathymetry (L. 
Kulm, pers. comm.). Seven paratypes and 
20 specimens from same area in April 
1997 (FMNH 278303, 278304, 278305, 
278306, 278312, 278313, 278316; 



DISTRIBUTION OF DEEP-SEA GRANELEDONE 



73 



278317; 278318; 278319; 278320) were 
examined. 

(2) MONTEREY BAY. Cold seeps are docu- 
mented at 36°47.1'N, 122°2.6'W, 580 
700 m; 36°44.0'N, 122°2.0'W, 875-920 
m; 36°44.7'N, 1 22°1 6.6'W, 1 ,004 m (Barry 
et al., 1997); the oxygen minimum and 
dysaerobic zones reach these depths off 
Point Sur (Broenkow & Greene, 1981, 
cited by Mullins et al., 1985). Specimens 
of Graneledone examined: off Bodega, 
4-5 mi. N on 600 fm. line (estimated 
38°20'N, 123°40'W), 1,116 m, Dec. 1976; 
CAS 003318. 10 miles SE of N. Farallón 
Island, (estimated 37°45'N, 123°20'W), 
1,414 m, Nov. 1950; CAS 035044. 
36°40.7-41.0'N, 122°17.6-19.4'W, 1,341 
m, Nov. 1975; CAS 031500. 36°29.9- 
30.5'N, 122°18.3'W, 1,415 m, Feb. 1985; 
CAS 084669. 36°29.2'N, 122°18.6'W, 
1,400-1,420 m, March 1985; FMNH 
286436. 36°28.3-29.3'N, 122°17.4- 
20.0'W, 4,409-1,384 m, Nov. 1975; CAS 
031474 and 084666. 36°28.3-29.3'N, 
122°1 7.8-03. 8'W, 1,400 m, May 1985; 
CAS 084667. 36°15.1-15.6'N, 122°25.5- 
26.1'W, 1,600 m, June 1984; CAS 
084665. 

(3) JAPAN/KURIL TRENCH. In addition to 
subduction-related cold seeps associated 
with the Trench (Métivier et al., 1986; Le 
Pichón et al., 1987), ten gas hydrate de- 
posits occur between 53°20.634'N, 
144°29.206'E and 54°26.782'N, 
144°04.858'E at depths of 620 and 1,040 
m (Ginsburg et al. 1993). Graneledone 
boreopacifica Nesis, 1982. Type locality. 
50°04'N, 151°35'E, 1,350 m, also re- 
ported from Sea of Okhotsk and the Pa- 
cific Ocean northeast of Honshu. Not ex- 
amined 

(4) NEW ZEALAND. Seafloor plume of 
methane-rich water rising over seabed at 
39°58.58'S, 178°14.18'E, est. 900 m 
(Lewis & Marshall, 1996). 39°59'S, 
178°13'E, 940-1,070 m, April 1994; 
NMNZ M. 118324 Graneledone sp. 
(O'Shea & Kubodera, 1996). Not exam- 
ined 

Occurrences Associated with hydrocarbon 
deposits, n = 5. 

(1) NORTHWEST ATLANTIC: BOTTOM- 
SIMULATING REFLECTOR (Fig. 2). Fish 
Hawk, Blake and Albatross specimen 
records from Verrill (1884); station locali- 
ties from Tanner (1885). 39°59'45"N, 
68°54'W, 787 fm (1 ,464 m), 26 Aug. 1 882; 
Fish Hawk Sta. 1123. 39°53'N, 70°35'W, 
588-832 m deter. M. Vecchione (J. Moore 
pers. comrm.); Feb. 1999. 39°50'45"N, 
70°11 'W, 466 fm (853 m), 1 880; Blake Sta. 



312. Paratype. 39°44'N, 70°28'W, 1,830- 
1,912 m, July 1975; USNM 730996 (re- 
ported by Villanueva & Sánchez, 1993). 
39°42'50"N, 69°21'20"W, 1,050 fm (1,953 
m), 1 Aug. 1883; Albatross Sta. 2050. 
39°41'00"N, 69°20'20"W, 1,106 fm (2,057 
m), 1 Aug. 1883; Albatross Sta. 2051. 
39°34.45'N, 71°31.30'W, 466-1,255 fm 
(853-2,334 m), Albatross Sta. 2209, 
ANSP A6934. 38°44'00"N, 72°38'00"W, 
1,209 fm (2,249 m), 5 Nov. 1883; Alba- 
tross Sta. 2102. 38°23'N, 73°45'W, 100- 
800 fm (186-1,488 m), March 1996; 
FMNH 281721. 

(2) NORTHWEST ATLANTIC: SALT DOME/ 
SALT-CORED ANTICLINES (Fig. 2). 
Blake and Albatross specimen records 
from Verrill (1884); station localities from 
Tanner (1885). 42°40'N, 62°29' W, 1,458 
m, reported by Joubin (1924); Villanueva 
& Sánchez (1993) likely examined and 
cited the same specimen as MOM 
295353, collected 1913. 42°38-40'N, 
63°20-43'W, 1,116-1,488 m, August 
1995; FMNH 286457 n = 20. 41°33'15"N, 
65°51'25"W, 810 fm (1,483 m), 1880; 
Blake Sta. 305. Holotype. 41°09'40"N, 
66°02'20"W, 1,255 fm (2,334 m), 4 Sept. 
1883; Albatross Sta. 2077. Not examined 

(3) NORTHWEST ATLANTIC: HIBERNIAN 
OIL FIELD (Fig. 2). Collection depths of 
the examined specimens, below, not re- 
corded. 45°41'N, 47°37'W, Sept. 1982; 
CMN 92735. 45°24'N, 48°35'W, June 
1980; CMN 92734. 45°18'N, 48°35'W, 
Sept. 1982; CMN 92736. 45°18'N, 48° 
36'W, June 1980; CMN 92522 and 92732. 
45°18'N, 47°51'W, Sept. 1982; CMN 
92737. 

(4) NORTHWEST ATLANTIC (same petro- 
leum reserve). 53°11.82'N, 51°56.82'W, 
1,145 m, Sept. 1995; FMNH 286459. 

(5) CONTINENTAL MARGIN SOUTH OF 
WALVIS RIDGE. 22°30'S, 12°20'E, 1,900 
m. Walvis Bay Slide Scrap and high or- 
ganic material in sediment (Summerhayes 
et al., 1979; Embley & Morley, 1980). 
23°05'S, 12°41'E, 1,193 m; August 1984. 
Graneledone sp. ICM-SA 179 reported & 
illustrated by Villanueva & Sánchez 
(1993), not examined. 

Specimens from seemingly normal areas 
north of 40°S, n = 7. 

(1 ) SOUTH OF ICELAND. Water column sur- 
veys found no indication of active hy- 
drothermal vents near 63°05'N, 24°25- 
35'W (German et al. 1994). Graneledone: 
62°10.8'N, 19°36'W, 2,150 m, July 1903; 
ZMUC 20-11-1903. Examined. 

(2) HEBRIDES SHELF. Large petroleum de- 
posits at 60°N (Parnell et al. 1998). Three 



74 



VOIGHT 



specimens of Graneledone 55-59°N (ca. 
8-1 5°W) depths of 900 to 1,400 m re- 
ported by Boyle et al. (1998) but not ex- 
amined. Boyle et al. (1998) perpetuate 
Griegg's (1933) statement that members 
of the genus are "not uncommon" off the 
coasts of Norway by citing Stephen's 
(1944) uncritical mention of the account. 

(3) NORFOLK CANYON. 37°N, 74°30'W 
(Upper Norfolk Canyon and Continental 
slope north of Norfolk Canyon) ca. 1100 
m. Photographs from ALVIN dives 807 
and 808 R. W. Embley, pers. comm.). No 
evidence of unusual biological activity 
(Malahoff etal. 1982). 

(4) CAPE VERDE BASIN. Area of mass sed- 
iment movements (Kidd et al. 1987) that 
are apparently of unknown origin. Gra- 
neledone: 20°33'N, 18°35'W, 2,003 m, 
Feb. 1991; MNHN 2013. Examined. 

(5) SOUTHEAST ATLANTIC. 34°S, 17°E, 
1,211 m, 22 July 1903; SAM 2734. 34°S, 
17°E, 1,027-1,284 m, 17 Sept. 1903; 
SAM 2735. 34°21'S, 17°47'E, 900-1,500 
m, 17 April 1982; SAM S807. 34°16'S, 
17°29'E, 1,150 m, 11 Jan 1995; SAM 
S3569. (Determinations relayed by M. 
Roelveld, pers. comm.). 

(6) BABY BARE BASEMENT BASALT OUT 
CROP. 47°42.64'N, 127°47.15'W, 2,591 
m (Mottl et al., 1998). Although warm 
water with detectable sulfide concentra- 
tions is released from Baby Bare, areas in 
which octopuses occur have normal tem- 
peratures and sulfide has not been de- 
tected (G. J. Massoth, pers. comm.). 
FMNH 282750. 

(7) GULF OF PANAMA. Reports of speci- 
mens of Graneledone from the Gulf of 
Panama had been undocumented be- 
cause specimens were severely damaged 
and/or poorly preserved (Hoyle, 1904; 
Voss, 1988). 07°15'N, 79°36'W, 1,020 fm 
(1,890 m), March 1891; Albatross Sta. 
3393: Graneledone sp. reported by Hoyle 
(1904), not examined. 07°11 -12.5'N, 
79°16-18.5'W, 1,463-1,984 m, Jan. 
1972; UMML 31.2484. Examination of a 



spermatophore in this lot supported the 
late G. L. Voss' identification of the mater- 
ial as Graneledone n. sp. 

Records from south of 40°S which lack 
marine geological data and are unconfirmed, 
n = 7. 

NEW ZEALAND. 

(1)43°9'S, 177°49'E, 780 m. Reported by 
Kubodera & Okutani 1994. 

(2) 45°21.1'S, 173°35.8'E, 1,386 m, Oct. 
1979; NZOI Stn. S153. Reported by 
O'Shea & Kubodera (1996). 

(3) 48°15.5'S, 179°48.5'E, 512 m, Nov. 1975; 
FSFRL El 259. 48°27.30'S, 179°27.56'E, 
565-599 m, Nov. 1993; NMNZ M. 117874. 
Both reported by O'Shea & Kubodera 
(1996). 48°40'S, 179°51'E, 732 m, Nov. 
1983 (also reported as 48°40'S, 179°1'E); 
NSMT M067835 as G. sp. b. 48°48'S, 
179°16'E, 725 m, Nov. 1983 (also re- 
ported as 48°48'S, 179°6'E); NSMT 
Mo67836 as G. sp. b. Both reported by 
Kubodera & Okutani (1994). 

SOUTH AMERICA- both South American 
records of the genus are reported by 
Kubodera & Okutani (1994) from directly 
seaward of the petroleum-rich San Jorge 
Basin, positioned between 45°S, 71 °W and 
47°S, 65.5°W (Fitzgerald et al. 1990). Off- 
shore geology apparently unknown. 

(4) 45°44'S, 59°46'W, 858 m, Oct. 1984; 
NSMT Mo67838 as G. macrotyla and 
NSMT M067837 as G. sp. A. 

(5) 47°00'S, 59°37'W, 972 m, Oct. 1984; 
NSMT Mo67839 as G. macrotyla. 

ANTARCTICA. 

(6) 54°43'S, 55°30'W 1,647-2,044 m, 14 
March 1 966. Graneledone macrotyla type 
locality (Voss 1976). 

(7) 74°05.6'S, 175°05.2'W, 2,341 m, 8 Feb. 
1 968. Graneledone antárctica type locality 
(Voss 1976). 



MALACOLOGIA, 2000, 42(1-2): 75-101 

A NEW GENUS AND A NEW SPECIES OF HYDROBIID SNAIL (MOLLUSCA: 
GASTROPODA: HYDROBIIDAE) FROM EASTERN SPAIN 

M. A. Ramos 1 , B. Arconada 1 , E. Rolan 2 & D. Moreno 3 



ABSTRACT 

The identity of Hauffenia (Neohoratia) gasulli Boeters, in Gasull, 1981 , type material, with shell 
topotypes and live specimens collected in neighbouring localities, has been established on the 
basis of shell characters (statistical analysis DFA and Anova). Anatomical studies confirmed its 
allocation to a new genus, Tarraconia, which is only known from the Iberian Peninsula. We pre- 
sent histological data to demonstrate the lack of a seminal receptacle, its function having being 
transferred to a wide part of the proximal renal oviduct. This character, together with a well-de- 
fined data set, including lack of a suboesophageal connective and a gastric caecum, penis size 
and shape, and a well-developed bursa copulatrix fully protruding from the albumen gland char- 
acterise the new genus. Tarraconia rolani, n. sp., is also described. 

The function of the seminal receptacle having moved to a different part of the genital system, 
found for the first time among Iberian hydrobiids, has occurred several times in the Hydrobioidea 
evolution in different families, such as Hydrobiidae, Pyrgulidae, Pomatiopsidae, showing it not to 
be a useful character to justify suprageneric-level taxa. Detailed anatomical studies are neces- 
sary to unravel the obscure phylogenetic relationships of this group of gastropods, which, due to 
their small size, yield very few diagnostic characters and a high degree of convergence. 

Key words: Prosobranchia, Hydrobiidae, Tarraconia, anatomy, taxonomy, Iberian Peninsula. 



INTRODUCTION 

Many of the initial descriptions of hydrobiid 
species were based only on shell features, 
mainly because often only empty shells were 
found. Since shell characters are highly con- 
vergent, most early supraspecific assess- 
ments have to be reviewed to clear up the ex- 
isting confusion and to establish a better 
taxonomic classification (Falniowski & Szar- 
owska, 1995). 

Boeter's work (1988) on hydrobiid gas- 
tropods from the Iberian Peninsula shows that 
the problem is still greater among the minute 
valvatoid Hydrobiidae species, the shells of 
which are very similar and their anatomy un- 
known or poorly known. Preliminary studies on 
Iberian Peninsular hydrobiid snails (Ramos et 
al., 1992, 1995: Arconada et al., 1996) showed 
considerable morphological diversity and high 
endemicity. 

The case of Hauffenia (Neohoratia) gasulli 
Boeters, in Gasull, 1 981 , clearly illustrates the 
problem. It was described by Boeters in a 
paper by Gasull (1981) reviewing the terres- 
trial and freshwater mollusc fauna of Castel- 



lón de la Plana Province, eastern Spain. The 
description and illustrations were based on 
shell characters, because Gasull only found 
empty shells. The genus Hauffenia Pollonera, 
1898, in which species here placed in Tarra- 
conia were first included, is widely distributed 
in freshwater springs on the Balkan Penin- 
sula, Italy, France, Austria, Switzerland, and 
Slovenia (Bole, 1967, 1970; Boeters, 1973; 
Bernasconi, 1984; Haase, 1992, 1993; Bodon 
& Giovanelli, 1994). Described as a subgenus 
of Horatia Bourguignat, 1887, it was treated 
as a full genus by Bole (1970) after studying 
the anatomy of the type species, Hauffenia 
tellini Pollonera, 1898. Neohoratia Schutt, 
1961, was first described as a subgenus of 
Horatia, then ranked as a subgenus of Hauf- 
fenia by Boeters (1974) and raised to a full 
genus by Bole & Velkovrh (1986). It is distrib- 
uted in the Balkans, being represented on the 
Iberian Peninsula. 

In his 1988 paper, Boeters recognised the 
existence of two genera of valvatoid hydro- 
biids in Iberia: Horatia and Neohoratia, and in- 
cluded the latter species as Neohoratia (?) 
gasulli. He described differences in the shape 



'Museo Nacional de Ciencias Naturales (CSIC). José Gutiérrez Abascal, 2. 28006 Madrid. Spain, m.ramos@mncn.csic.es; 

mcna313@mncn.csic.es 

2 Cánovas del Castillo, 22. 36202 Vigo, Spain; 0208378g01 ©abonados. cplus.es 

3 Araña, apartamentos Las Dunas 2, 04150 Cabo de Gata, Almería, Spain; dmoreno@telebase.es 



75 



76 



RAMOS ETAL. 



of both the aperture and the shell in general 
compared with the other Neohoratia species, 
and pointed out the need for anatomical stud- 
ies in order to confirm its generic allocation. 

Within the framework of the macroproject 
Fauna Ibérica, only empty shells of the 
species were found at El Bañador, Altura, 
Castellón, the type locality and thus far the 
only known locality. However, many live spec- 
imens, with shell characters agreeing with 
those described by Boeters, have been col- 
lected in several springs 3.5 km from the type 
locality. Once the similarity in shell characters 
with type material and newly collected topo- 
types was established using both morpho- 
metrical and microsculptural characters, study 
of the anatomy, confirmed by histological evi- 
dence, demonstrated that the combination of 
character-states in that species does not fit 
any known genus. 

This paper aims to give a detailed account 
of the species' morphology, describing a new 
genus and a new species included in the sub- 
family Hydrobiinae (sensu Giusti & Pezzoli, 
1984, and Ponder & Waren, 1988). 



MATERIALAND METHODS 
Material Studied 

Snails collected for this study came from 
the area marked in Figure 1 in the eastern- 
most part of the Iberian Peninsula. They were 
collected by hand, either by picking them off 
the substratum, by washing stones, aquatic 
vegetation, and dead leaves, or by sieving 
mud and vegetation. 

The stations are listed below consecutively 
numbered and are referred to in the text by 
this number. The spring name is followed by 
biotope type, municipality, province, UTM co- 
ordinates, collection date, collector's initials 
(abbreviations given below). Locality names 
and UTM are from the official map of the Army 
Geographical Service. Below each taxon in 
the following section, locality number, locality 
name, museum catalogue number, and 
preservation of specimens is given, except for 
type material. 

01 El Bañador (irrigation ditch), Altura, 
Castellón, UTM: 30SYK145133. 7/3/1990, 
Diego Moreno (= D.M.), Jose Miguel Remón 
(= J.M.R.), Rafael Araujo (= R.A.), and 
17/10/1992, D.M. and Nuria Martín (= N.M.). 

02 Manantial La Esperanza (spring), 
Navajas, Castellón, UTM: 30SYK132163, 



Tarragona 




/ Castellón 




Valencia 






• T. gasulli 
A T. rolani 


100 Km 



FIG. 1. Map of Tarraconia gasulli and T. rolani lo- 
calities. 



7/3/1990, R.A., D.M., J.M.R., 15/6/1994, Glo- 
ria Tapia (= G.T.), 25/5/1998, Beatriz Ar- 
conada (= B.A.). 

03 Acequia (irrigation ditch), Navajas, 
Castellón, UTM: 30SYK133161, 7/3/1990, 
R.A., D.M. and J.M.R. 

04 Fuente de Las Provincias (piped 
spring), Segorbe, Castellón, UTM: 
30SYK1 51 151, 6/10/1990, E.R. 

05 Ermita N a Sra. de la Esperanza (piped 
spring), Segorbe, Castellón, UTM: 
30SYK134162 , 6/10/1990, E.R. 

06 Font Nova (spring), Benifaió, Valencia, 
UTM: 30SYJ513218, 17/3/1994, G.T 

07 Ullal Baltasar (spring), Amposta, Tar- 
ragona, UTM: 31TBF969054, 30/3/1990, 
Jose Bedoya (= J.B.), D.M., R.A., 25/7/1996, 
Carolina Noreña (= C.N.) and 13/11/1999, 
B.A. 

08 Irrigation ditch on the road from San 
Carlos de la Rápita to Tortosa, La Carroba, 
Tarragona UTM: 31TBF943134, 29/3/1990, 
R.A., D.M. and J. В., 30/9/1990, E.R., 13/3/ 
1999, B.A. 



Anatomy 

To avoid losing characters through fixation 
in the newly collected material, such external 
features as penis shape and colour, snout pig- 
mentation, and presence of calcareous gran- 
ules at the base of the tentacles, were studied 
and drawn in the field. To get the best results 
for anatomical studies, they were relaxed 
using different narcotic agents and methods 
(Araujo et al., 1995). On the whole, the best 
results were obtained by adding a few men- 
thol crystals to the water surface in the con- 



A NEW GENUS AND A NEW SPECIES OF HYDROBIID SNAIL 



77 



tainer, followed by hot water (60°C, five sec), 
and then fixing and preserving them in 70% 
ethanol. 

For anatomical studies, specimens were 
dissected in water on a Petri dish with a black 
layer of paraffin, wax and coal (Davis, 1967). 
A Stemi SV8 Zeiss stereomicroscope was 
used. Anatomical illustrations were based on 
camera-lucida drawings. Since all anatomical 
measures were taken from specimens fixed in 
ethanol, a slight variation in relation to fresh 
material values was expected, although the 
effect should be similar in all the study popu- 
lations. 

Radula, protoconchs and opercula were ex- 
amined with a Philips XL20 Scanning Electron 
Microscope (SEM). Before the shells were 
coated with a thin (10-15 nm) gold layer in a 
Bio-Rad SC515 sputter coating unit, the shell 
periostracum was removed by immersion in 
5% commercial Chlorox (sodium hypochlo- 
rite), the time depending on the thickness of 
the sediment layer covering it. If necessary, a 
fine paint brush was used to remove any per- 
sistent material. The holotype was studied 
without coating it. Radulae were extracted by 
digestion of the head with diluted KOH at 
room temperature. The same method was 
used to clean the opercula. Both kinds of 
structures were then rinsed in distilled water 
and air-dried before mounting on stubs. 

Histology 

The shells of specimens used for dissec- 
tions and histological studies were decalcified 
in 5% agueous dilution of ethylenediaminete- 
tracetic acid (EDTA). For histology, a male 
and five females preserved in 70% ethanol 
were dehydrated in different ethanol series 
(10 min at both 80%, 90%, and 1 h at both 
95%, 100%), transferred to benzyl benzoate, 
and then embedded in 100% Paraplast Plus. 
After 14 h in a heater at 59 °C, serial thin sec- 
tions (4-6 (im) were made with a Leica RM 
2045 microtome and hand-stained. We used 
the Carazzi's hematoxylin-eosin and azan 
staining methods. 

Morphometric Analysis 

Eleven shell parameters and six operculum 
parameters, as shown in Figure 2, were mea- 
sured. The number of protoconch whorls was 
counted, beginning at the initial suture 
(Solem, 1976; Burch, 1982). 



Radular central tooth width indicates the 
distance between the outer cusps of the cen- 
tral tooth. 

Bursa copulatrix length does not include its 
duct. Penis width excludes the penial lobe. 
Maximum length of prostate, palliai oviduct 
and ctenidium maximum length were mea- 
sured without taking curvature into account. 
Stomach width includes the oesophagus. For 
the nervous system, RPG ratio was calcu- 
lated following Davis et al. (1976). 

We also calculated the following indices: 
SL/SW, SUM, SW/AW, AL/AW and OL/OW, 
which seem to describe better the shape of 
the shell and operculum (abbreviations given 
below). 

All variables (mean value, standard devia- 
tion and coefficient of variation) were stan- 
dardized in order to avoid the effect of the 
measurement scale. For shell variables, a 
one-way analysis of variance (ANOVA) was 
computed for each character to evaluate their 
contribution to species differences and the 
post hoc Scheffe's F test was used to exam- 
ine the statistical significance of the differ- 
ences between all pairs of means for each 
level of the factor (population) with a probabil- 
ity level of 5% (a = 0.05). The opercular and 
anatomical variables were compared with a 
t-test of independent samples. 

In order to identify the most important mor- 
phological attributes to distinguish directly be- 
tween species when no anatomical data were 
available, a discriminant function analysis 
(DFA) was performed on shell measurements 
(not ratios). For missing data, casewise option 
was selected. The significance of the overall 
discriminatory power of the analysis was 
tested using the Wilks' lambda standard sta- 
tistic. Standardized coefficients for canonical 
variables and the canonical correlation (R) 
were studied to examine each character's 
contribution in the discriminant function and to 
analyze the percentage of variance attributed 
to interspecific variation, respectively. Classi- 
fication functions were computed for each 
group (population) to determine, with the 
highest probability, to which population each 
case most likely belonged. Cases were as- 
signed to the group having the highest classi- 
fication score. 

Descriptive statistics were obtained using 
the Statview 4.1 package for Macintosh. DFA 
was performed using the Statistica 4.1 for 
Macintosh, Statsoft, Inc., 1984-1994, and 
Statistica for Windows, Statsoft Inc., 1995, 
packages. 



78 



RAMOS ETAL. 




FIG. 2. Shell and operculum measurements. А, В, D. Shell measurements. С. Method employed for count- 
ing protoconch whorls. The count begins at initial suture (Solem, 1976; Burch, 1982). E-F. Operculum mea- 
surements (see abbreviations in text). 



Abbreviations 

Shell and Operculum Measurements 

AH Aperture height 

AL Aperture length 

AW Aperture width 

LBW Length of body whorl 

NL Nucleus length of operculum 

NW Nucleus width of operculum 

NSW Number of spire whorls 

OL Operculum length 

OLWL Length of last whorl of operculum 

OLWW Width of last whorl of operculum 

OW Operculum width 

PNW Protoconch nucleus width 

PW Protoconch width 

SL Shell length 



SW Shell width 

WBW Width of body whorl 

WAW Width of the antepenultimate whorl 

WPW Width of the penultimate whorl 

Anatomical Measurements 

Ac Anterior chamber of the stomach 

Ag Albumen gland 

Be Bursa copulatrix 

Ca Egg capsule 

Cc Cerebral commisure 

Cg Capsule gland 

Cgl Left cerebral ganglion 

CI Columellar muscle 

Co Coiled part of the oviduct 

Ct Ctenidium 

Dbc Duct of the Bursa copulatrix 



A NEW GENUS AND A NEW SPECIES OF HYDROBIID SNAIL 



79 



Dgo 


Digestive gland opening 


Di 


Digestive gland 


Int 


Intestine 


К 


Kidney 


Lp 


Left pleural ganglion 


Od 


Oviduct 


Oes 


Oesophagus 


Os 


Osphradium 


Ov 


Ovary 


P 


Penis 


Pe 


Posterior chamber of the stomach 


Po 


Palliai oviduct 


PI 


Penial lobe 


Pr 


Prostate 


Psc 


Pleurosupraoesophageal 




connective 


Pvd 


Posterior vas deferens 


R 


Rectum 


Sbo 


Suboesophageal ganglion 


Spo 


Supraoesophageal ganglion 


Ss 


Style sac 


St 


Stomach 


Sv 


Seminal vesicle 


T 


Testis 


Ve 


Ventral channel 


Collections 



The material collected has been deposited 
in the MNCN collections (catalogue numbers 
included). One male and one female from 
Navajas, Manantial La Esperanza have also 
been deposited in each of the following col- 
lections: SMF (9452, 9453) and NNM 
(312215, 312216). SMF: Senckenberg Mu- 
seum, Frankfurt; NNM: Natural History Mu- 
seum in Leiden; MNCN: Museo Nacional de 
Ciencias Naturales, Madrid; ВОЕ: Boeters' 
private collection. 



SYSTEMATIC DESCRIPTION 

Tarraconia Ramos & Arconada, new genus 

Type Species 

Hauff en ia (Neohoratia) gasulli Boeters, in 
Gasull, 1981 

Etymology 

Tarraconia is derived from Hispania Tarra- 
conensis, an ancient Iberian community that 
became one of the three provinces into which 
the Roman Empire divided the Iberian Penin- 
sula in the second century A.D. and which in- 



cluded the current provinces of Tarragona, 
Castellón and Valencia. 



Diagnosis 

Shell valvatoid, very small (approx. 1 .5 mm 
in length) and depressed, with 3.5-4 whorls, 
deep sutures and a wide umbilicus usually 
carrying an egg capsule; rounded and straight 
aperture with a characteristic varix behind the 
outer lip. Operculum has a subcentral nucleus 
and lacks any outgrowth on the inner surface. 
Radula typically hydrobiid with a single cusp 
on each of the basal angles of the central 
tooth. Right pleural ganglion connected with 
the supraesophageal ganglion by a long con- 
nective; left pleural ganglion and subesoph- 
ageal ganglion fused. Palliai tentacle absent. 
Ctenidium well developed, with long lamellae; 
large osphradium opposite middle of ctenid- 
ium. Stomach without caecum; style sac pro- 
truding from the intestinal loop that makes a 
simple coil around the style sac. Rectum U- 
shaped. Faecal pellets are oval and yellowish. 
Bursa copulatrix large, fully protruding at the 
end of the palliai oviduct; renal oviduct very 
wide near the ventral channel, without semi- 
nal receptacles. Penis has a lobe approxi- 
mately in the middle. 



Tarraconia gasulli (Boeters, 1 981 ) 

Hauffenia (Neohoratia) gasulli Boeters, in 
Gasull, 1981 Neohoratia gasulli (Boeters): 
Bole & Velkovrh, 1986 Neohoratia (?) gasulli 
(Boeters): Boeters, 1988 



Type Material 

Holotype: SMF 256390 one shell by original 
designation (Figs. 3A-C, 4A, B). Shell mea- 
surements in Table 1; paratypes: ВОЕ 952 
Gas., SMF 305476 (one shell) and NNM 
55554 (one shell). (01) MNCN 15.05/32057 
(dried material) (7/3/1990) (Figs. 3D-F) and 
MNCN 15.05/32059 (dried material) (17/10/ 
1992). 

Localities: Manantial La Esperanza (02) 
MNCN 15.05/32058, 15.05/32060 and 
1 5.05/32066 (dried, ethylic alcohol and frozen 
material and golden SEM preparation) (Figs. 
3G-I, 4C-H); Acequia in Navajas (03) MNCN 
15.05/32062 (dried and ethyl alcohol mater- 
ial); Fuente de Las Provincias (04) MNCN 
15.05/32064 (ethyl alcohol material and 



80 



RAMOS ETAL. 




FIG. 3. SEM microphotographs of Tarraconia gasulli shells. A-C: Holotype; D-F: from the type locality, El 
Bañador, Altura, Castellón; G-l: from Manantial La Esperanza, Navajas, Castellón. 



golden SEM preparation); Ermita N a Sra. de 
la Esperanza (05) MNCN 15.05/32065 (ethyl 
alcohol material and golden SEM prepara- 
tion); Font Nova (06) MNCN 15.05/32063 
(ethyl alcohol material). 

Type Locality 

The type locality is the only place described 
in Gasull's paper (1981): El Bañador (01), Al- 
tura (Castellón). 



Morphology 

Shell (Figs. 3, 4A-F; Table 1). The shell is 
dextral, valvatoid, very small (SL: 1.24-1.92; 
SW: 1 .1 8-1 .84), almost as high as wide, thin, 
whitish, and translucent when fresh, with 
3.5-4 whorls. The whorls are rounded, the su- 
tures are deep, and the body whorl occupies 
5/6 of total shell length. The aperture adhering 
to the last whorl is rounded, with slight angu- 
larity close to the penultimate whorl. Its outer 



A NEW GENUS AND A NEW SPECIES OF HYDROBIID SNAIL 




FIG. 4. SEM photomicrographs of Tarraconia gasulli shell and opercula. A, B. Holotype. C-H. From Manan- 
tial La Esperanza, Navajas, Castellón. А, С. Shell from below (note wide umbilicus and typical varix behind 
the outer lip); B, D. Protoconch; E. Protoconch sculpture. F. Part of the sculpture of the teleconch. G, H. Outer 
and inner side of the opercula. 



82 



RAMOS ETAL. 



TABLE 1. Shell measurements (in mm) of Tarraconia gasulli and T. rolani. L = Length, W = Width, SD = 
Standard Deviation, CV = Coeff. of Variation, * n = 31 . 





Holotype 






T.< 


gasulli 








T. rolani 






El Bañador, Altura 


La Espe 


ranza, Navajas 




La Carroba 










(n = 31) 




I 


[n = 33) 






(n = 13) 




Variables 


Mean 


S.D. 


C.V. 


Mean 


S.D. 


C.V. 


Mean 


S.D. 


C.V. 


SL 


1.80 


1.58 


0.08 


0.05 


1.61 


0.15 


0.09 


1.38 


0.18 


0.13 


SW 


1.80 


1.59 


0.07 


0.04 


1.51 


0.14 


0.09 


1.73 


0.13 


0.08 


LBW 


1.50 


1.34 


0.06 


0.04 


1.32 


0.12 


0.09 


1.11 


0.15 


0.13 


WBW 


1.35 


1.23 


0.05 


0.04 


1.21 


0.09 


0.07 


1.17 


0.12 


0.10 


AH 


1.09 


0.89 


0.04 


0.04 


0.88 


0.07 


0.08 


0.88 


0.07 


0.08 


AL 


0.83 


0.81 


0.05 


0.06 


0.81 


0.05 


0.07 


0.78 


0.06 


0.08 


AW 


0.74 


0.70 


0.03 


0.04 


0.69 


0.05 


0.07 


0.72 


0.07 


0.10 


NSW 


3.75 


3.75 


0.18 


0.05 


3.84* 


0.22* 


0.06* 


3.62 


0.19 


0.05 


SL/SW 


1.00 


1.00 


0.04 


0.04 


1.06 


0.06 


0.06 


0.80 


0.08 


0.10 


SL/AL 


2.16 


1.96 


0.12 


0.06 


2.00 


0.17 


0.08 


1.77 


0.17 


0.09 


SW/AW 


2.43 


2.25 


0.08 


0.04 


2.20 


0.09 


0.04 


2.43 


0.25 


0.10 


A U AW 


1.12 


1.15 


0.04 


0.04 


1.17 


0.08 


0.07 


1.09 


0.05 


0.05 



lip is straight, parallel to the columella or very 
slightly inclined backwards, with a character- 
istic and marked varix behind the outer lip. 
The umbilicus is very wide, around 0.20 mm, 
about 1/7 of shell diameter. 

The protoconch (Fig. 4B, D, E) is clearly dif- 
ferentiated from the teleoconch (Fig. 4F). The 
external surface of the protoconch is wrinkled 
with smoothly pitted sculpture. It has 1.5 
whorls. Total width (PW) is approximately 
0.32 mm and nucleous width (NPW) approxi- 
mately 0.15 mm. The teleoconch is smooth, 
with fine growth lines. 

Shell measurements are given in Table 1 
both for type locality specimens and those 
used for anatomical studies (Manantial La Es- 
peranza). 

External Body Features. The head is pig- 
mented with black melanin, but the pigment 
fades towards the snout, which is totally un- 
pigmented, allowing observation of the radu- 
lar sac due to transparency of the epithelium. 
There are characteristic orange spots around 
the eyes (Fig. 8C). The tentacles are dark, 
mainly at the base, and have a central white 
band. Tentacle tips are unpigmented. The 
mantle edge is also dark. On the sole of the 
foot there are some white granules. 

Nervous System (Fig. 5B). The two cere- 
bral ganglia are connected by a cerebral com- 
misure and lie over the oesophagus. The right 
and left pleural ganglia are about the same 
size and rise out of the curvature of the cere- 
bral ganglia. Seen from above, it is not possi- 
ble to discern the left pleural ganglion nor the 
subesophageal ganglion, because at that 



point the oesophagus forms a curve that pre- 
vents direct observation. Once it is removed, 
the left pleural ganglion and the suboesoph- 
ageal ganglion appear fused without any con- 
nective. The right pleural ganglion is joined to 
the supraoesophageal ganglion by a long 
connective. Nervous system measurements 
are: length of the right cerebral ganglia: 0.24 
mm; length of the cerebral commisure: 0.08 
mm; length of the right pleural ganglion: 
0.08 mm; length of the left pleural ganglion: 
0.10 mm; length of the supraesophageal gan- 
glion: 0.07 mm; length of the subesophageal 
ganglion: 0.06 mm; length of the pleuro- 
supraesophageal connective: 0.15 mm. RPG 
ratio = 0.48. 

Mantle Cavity. The mantle epithelium is pig- 
mented uniformly black, except on the body 
whorl, which is less pigmented making it pos- 
sible to follow the position of the intestine. The 
ctenidium occupies nearly the entire lenght of 
the palliai cavity with a variable number of 
well-developed lamellae. There are usually 
about 14, but may vary from 11 to 18 (Fig. 
5C). At the base of the gill, near the neck, 
there is a well developed ovoid-annular os- 
phradium. 

Operculum (Fig. 4G, H, Table 2). It is 
corneus, paucispiral and thin. The pale yel- 
lowish nucleus is subcentral. Measurements 
are shown in Table 2. 

Digestive System (Table 4). In the palliai 
cavity, the rectum forms a more or less 
markedly U-shaped bend beside the prostate 
or palliai oviduct (Figs. 5C, D, 7). It ends near 
the distal corner of the mantle cavity. The di- 




Int Oes 




FIG. 5. Shell, nervous system, palliai organs and digestive system of Tarraconia gasulli from Manantial La 
Esperanza, Navajas, Castellón. A. Shell with an egg capsule in the umbilicus; B. Partial nervous system; C. 
Osphradium and ctenidium; D. Lateral view of the shell. The U-shaped intestine can be seen by trans- 
parency; E. Stomach. Scales bars: A, D: 1 mm; B: 250 цт; С, E: 500 ¡im. Abbreviations: Ac. anterior cham- 
ber of the stomach; Ca. capsule; Cc. cerebral commisure; Cgl. left cerebral ganglion; Ct. ctenidium; Dgo. di- 
gestive gland opening; Int. intestine; Lp. left pleural ganglion; Oes. oesophagus; Os. osphradium; Pc. 
posterior chamber of the stomach; Psc. pleurosupraoesophageal connective; R. rectum; Sbo. suboe- 
sophageal ganglion; Ss. style sac; Spo. supraoesophageal ganglion. 



84 



RAMOS ETAL. 



TABLE 2. Operculum measurements (in mm) of Tarraconia gasulli (La Esperanza, Navajas) and 
T. rolani. (La Carroba). SD = Standard Deviation, CV = Coeff. of Variation. 





T. 


gasulli 




T. rolani 






La Esperanza, Navajas 




La Carroba 




Variables 


Mean 


S.D. 


C.V. 


Mean 


S.D. 


C.V. 


OL 


0.74 (n = 7) 


0.11 


0.14 


0.73 (n = 4) 


0.03 


0.04 


OW 


0.60 (n = 7) 


0.08 


0.14 


0.62 (n = 5) 


0.03 


0.04 


OLWL 


0.21 (n = 3) 


0.05 


0.22 


0.22 (n = 5) 


0.03 


0.14 


OLWW 


0.18 (n = 3) 


0.03 


0.18 


0.19 (n = 5) 


0.03 


0.15 


NL 


0.40 (n = 4) 


0.09 


0.23 


0.35 (n = 4) 


0.03 


0.08 


NW 


0.43 (n = 4) 


0.12 


0.27 


0.43 (n = 5) 


0.01 


0.02 


OL/OW 


1.23 (n = 7) 


0.04 


0.03 


1.16(n = 4) 


0.02 


0.01 



gestive gland is continuously distributed from 
the posterior chamber of the stomach to the 
end of the body. The posterior stomach has 
no caecum (Fig 5E). The style sac is formed 
by a monostratified ciliated epithelium, with 
cubical cells filled with a rather dense cyto- 
plasm in which a large rounded nucleus is 
centrally placed. The distal surface is densely 
clothed with long, close-set cilia (Fig. 9A). The 
stomach has two different types of epithelium. 
The first is a high, columnar, ciliated, mono- 
stratified epithelium with basally situated nu- 
clei. The second is a pseudostratified ciliated 
epithelium. These cells are sometimes ob- 
served to be full of vesicles. 

The typically taenioglossate radula has a 
central tooth with the usual butterfly-shaped 
hydrobiid structure (Fig. 6A-E). The cutting 
edge is markedly excavated. The apex is V- 
shaped, with a long middle cusp and five 
smaller cusps on each side that decrease in 
size towards the edge. The lateral wings have 
one cusp on each of the basal angles. The 
lateral teeth have slender lateral wings with 
one pointed basal cusp on each side. They 
have a large wide central cusp pointing 
toward the central teeth, and 4 or 5 other 
cusps at each side, decreasing in size. The 
two pairs of marginal teeth are similar in 
shape, the apex of the outer one being small. 
Both have a long row of denticles of similar 
size. Radular formula and measurements ap- 
pear in Table 3. 

Female Reproductive System. An uncoiled 
female snail is shown in Figure 7A. Other de- 
tails in Figure 8A, B. Measurements in Table 4. 

The palliai oviduct is divided into an albu- 
men gland and a capsule gland, the border of 
which is not straight, but slightly lobulated. It is 
slightly lengthened in its anterior part. The 
capsule gland comprises more than half the 
palliai oviduct. 



The bursa copulatrix clearly protrudes at 
the end of the palliai oviduct. It is well devel- 
oped, pyriform and not ciliated. It has a long, 
slender anterodorsal duct and constitutes al- 
most half the length of the palliai oviduct. 

There is no seminal receptacle. The renal 
oviduct is unpigmented and forms a loop lean- 
ing against the palliai oviduct. The part of the 
coiled oviduct proximal to the bursa copulatrix 
is greatly thickened because of its strong 
columnar epithelium that makes longitudinal 
folds and leaves a stretched lumen (Fig. 9C). 
This epithelium is highly ciliated, and some of 
spermatozoa are arranged with their heads 
between the cilia and their tails projecting to- 
wards the lumen (Fig. 9D). The epithelium of 
the rest of the oviduct is much thinner and un- 
ciliated (Fig. 9E), very similar to that of the 
bursa copulatrix (Fig. 9B). In the breeding pe- 
riod, the lumina of both are filled with unori- 
ented sperm. Externally, the swollen area is 
clearly recognisable by its refractivity, irides- 
cent colour, and its position with regard to the 
oviduct loop. The mature female gonad con- 
sists of vitellogenic oocytes that have a very 
well-developed cytoplasm full of yolk, each 
measuring 120-200 urn. These yolk granules 
are iridescent yellow. Previtellogenic oocytes 
are roundish and measure approximately 
30-60 urn in diameter (Fig. 9F). The sex cells 
are bordered by a germinal epithelium. The 
gonad is separated from the digestive gland 
by a thin layer of connective tissue. The ven- 
tral channel is ciliated. 

Egg Capsule. In some specimens, an egg 
capsule was found inside the umbilicus with a 
single egg (Fig. 5A). Embryos have been 
found at different stages of maturation (Fig. 
6F). The capsule is approximately 200 urn 
and can be seen in both males and females. 

Male Reproductive System. An uncoiled 
male snail is shown in Figure 7B. Other de- 



A NEW GENUS AND A NEW SPECIES OF HYDROBIID SNAIL 



85 




FIG. 6. A-E. SEM photomicrographs of Tarraconia gasulli radula from Manantial La Esperanza, Navajas, 
Castellón. А, В. Transverse rows; С. Central teeth; D. Central, lateral and inner marginal teeth; E. Lateral 
view of the central, inner and outer marginal teeth; F Embryo before hatching after partially removing the 
capsule. 



tails are shown in Figure 8C, D. Penis and 
prostate measurements are shown in Table 4. 
The anterior part of the testis does not over- 
lap the posterior chamber of the stomach. The 
male gonad is very well developed, and con- 
sists of multiple lobes that extend towards the 
end of the body whorl (Fig. 7B). Inside these 
lobes, maturation of the sex cells goes from 
the outer part to the inner part so spermato- 
gonia! cells can be found on the periphery and 



spermatozoids in the centre (Fig. 9G). The an- 
terior coils of the seminal vesicle overlap the 
posterior chamber of the stomach. The poste- 
rior vas deferens enters the prostate gland 
approximately in the middle part. The vas ef- 
ferens and seminal vesicle can clearly be 
seen because of the iridescent pink sperm. 
The prostate is typically bean-shaped and the 
lumen U-shaped. The anterior deferens vas 
can be seen near its anterior tip. 



86 



RAMOS ETAL. 



TABLE 3. Radula of Tarraconia gasulli (La 
Esperanza, Navajas) and T. rolani (La Carroba). 
Formula and measurements (in urn). 



T. gasulli 



Variables 



La Esperanza, 
Navajas 



T. rolani 

La 
Carroba 



Central teeth (4)-5+C+(4)-5/1-1 5+C+5/1-1 
Cutting edge 

width =7.05 =8.3 

Lateral teeth 4-5+C+4 6+C+4 

Inner marginal 

teeth 21-25 25-28 

Outer marginal 

teeth 11-18 14 



The penis is inserted in the right middle part 
of the neck. It is cylindrical in the transverse 
section, very well developed, and has a 
medium-sized roundish lobe on the concave 
side in its central part. On histological slides, 
no glandular structures were seen in this lobe 
(Fig. 9H). The penis is slightly pigmented in its 
distal part below the lobe, where it narrows 
and finishes in a blunt tip. The narrow penis 
duct is straight and lies towards the right 
edge. 

Remarks 

The Segorbe population shows slight varia- 
tions in shell proportions, but has the typical 
shell shape and marked varix behind the 
outer lip. The female genital system follows 
the pattern described for T. gasulli, and the 
penis seems to be more slender, having a 
smaller lobe. Its geographical location be- 
tween the type locality and Navajas led us to 
include it as T. gasulli until new records are 
available. 



well preserved. In Benifaió, Valencia, only 
three adult specimens, one juvenile and one 
shell were collected. The female anatomy and 
shell features suggest tentatively including 
the Benifaió population as T. gasulli. 

Tarraconia rolani Ramos, Arconada & 
Moreno, new species 

Type Material 

Holotype (Figs. 10A, E, F) and paratypes 
(Figs. 10C, G, I, 11B) (09) MNCN 15.05/ 

33131 (ethylic alcohol material and golden 
SEM preparation) (30/9/1990), MNCN 15.05/ 

33132 (in ethylic alcohol material and golden 
SEM preparation) (29/3/1990) (Figs. 10B, D, 
H, 11 A, MNCN 15.05/33136 (ethylic alcohol 
and frozen material) (13/3/1999). 

Localities 

(07), MNCN 15.05/33133 (dried and ethylic 
alcohol material) (30/3/1990), MNCN 15.05/ 
33134 (ethylic alcohol material) (25/7/1996) 
and MNCN 15.05/33137 (ethylic alcohol ma- 
terial) (13/11/1999). 

Type Locality 

Irrigation ditch on the road from San Carlos 
de la Rápita to Tortosa (08), La Carroba, Tar- 
ragona. 

Etymology 

This new species is dedicated to Dr. Emilio 
Rolan in recognition of his important contribu- 
tion to Spanish freshwater malacological 
fauna. 



Habitat and Distribution 

The species seems to be restricted to a few 
localities in a small area in Castellón Province 
in the easternmost part of Spain. It lives in 
springs and irrigation ditches with clean water 
and much aquatic vegetation. In these envi- 
ronments, specimens can be found on the 
vegetation, stones or even in the mud, to- 
gether with a wide variety of other freshwater 
molluscs, the most common being species of 
the genera Pisidium, Melanopsis, Theodoxus, 
Belgrandia, Pseudamnicola and Lymnaea. 
Live specimens were found in all the locali- 
ties, except the type locality, most being very 



Morphology 

Shell (Figs. 10, 11 A, B; Table 1). The shell 
is dextral, valvatoid, very small, wider (1 .52-2 
mm) than it is tall (1.17-1.80 mm), thin, 
whitish and translucent, with 3.5-4 whorls. 
Periostracum is yellowish. The whorls are 
rounded, the sutures are deep, and the body 
whorl occupies 5/6 parts of total shell length. 
The aperture close but not adhering to the last 
whorl, rounded, with a slight angularity close 
to the penultimate whorl. Its outer lip is 
straight, parallel to the columella or very 
slightly inclined backwards, with a slight varix 
behind the outer lip. The umbilicus is very 



A NEW GENUS AND A NEW SPECIES OF HYDROBIID SNAIL 



87 



TABLE 4. Anatomical measurements (in mm) of Tarraconia gasulli (La Esperanza, Navajas) and 
T. rolani (La Carroba). SD = Standard Deviation, CV = Coefficient of Variation. 





T. 


gasulli 




T. rolani 






La Esperanza, Navajas 




La Carroba 




Variables 


Mean 


S.D. 


C.V. 


Mean 


S.D. 


C.V. 


BcL 


0.27 (n = 4) 


0.04 


0.16 


0.32 (n = 3) 


0.03 


0.08 


BcW 


0.19 (n = 4) 


0.04 


0.18 


0.32 (n = 3) 


0.03 


0.09 


DBcL 


0.24 (n = 4) 


0.07 


0.28 


0.08 (n = 3) 


0.04 


0.50 


PoL 


0.71 (n = 3) 


0.05 


0.07 


0.66 (n = 3) 


0.03 


0.05 


Po W 


0.43 (n = 3) 


0.01 


0.02 


0.44 (n = 3) 


0.06 


0.13 


AgL 


0.31 (n = 3) 


0.14 


0.45 


0.37 (n = 2) 


0.02 


0.06 


CgL 


0.39 (n = 3) 


0.09 


0.24 


0.27 (n = 2) 


0.04 


0.14 


PL 


1.00 (n = 6) 


0.24 


0.24 


1.61 (n = 4) 


0.45 


0.28 


PW 


0.14 (n = 6) 


0.04 


0.30 


0.14 (n = 4) 


0.03 


0.19 


PrL 


0.52 (n = 2) 


0.08 


0.15 


0.70 (n = 3) 


0.07 


0.62 


PrW 


0.25 (n = 2) 


0.10 


0.41 


0.38 (n = 3) 


0.05 


0.14 


PI L 


0.17 (n = 4) 


0.03 


0.20 


0.15 (n = 4) 


0.03 


0.22 


PI W 


0.22 (n = 6) 


0.05 


0.22 


0.20 (n = 4) 


0.04 


0.22 


P L/Head L 


1.31 (n = 5) 


0.43 


0.33 


2.13 (n = 4) 


0.75 


0.35 


OsL 


0.21 (n = 5) 


0.09 


0.42 


0.29 (n = 5) 


0.05 


0.18 


OsW 


0.12 (n = 5) 


0.03 


0.29 


0.14 (n = 5) 


0.03 


0.23 


CtL 


0.64 (n = 5) 


0.26 


0.41 


0.62 (n = 5) 


0.14 


0.23 


ctw 


0.15 (n = 5) 


0.04 


0.29 


0.16 (n = 5) 


0.02 


0.12 


Ss L 


0.33 (n = 5) 


0.04 


0.13 


0.45 (n = 5) 


0.07 


0.15 


SsW 


0.20 (n = 5) 


0.03 


0.16 


0.32 (n = 5) 


0.05 


0.15 


StL 


0.41 (n = 5) 


0.09 


0.23 


0.59 (n = 4) 


0.17 


0.28 


StW 


0.32 (n = 5) 


0.03 


0.11 


0.45 (n = 4) 


0.09 


0.20 



wide, around 0.33 mm, about 1/5 of shell di- 
ameter. 

The protoconch is clearly differentiated 
from the teleoconch (Fig. 11 A, B). The exter- 
nal surface of the protoconch is wrinkled, with 
smoothly pitted sculpture. It has 1.5 whorls. 
Total width (PW) is about 0.34 mm and the 
width of the nucleous (PNW) approximately 
0.12 mm. The teleoconch is smooth, with fine 
and regular growth lines. 

External Body Features. The head is pig- 
mented brown, but the pigment fades towards 
the snout, which is totally unpigmented, al- 
lowing observation of the radular sac. The 
tentacles are dark, mainly at the base, and 
have a central white band (Fig 12C). Tentacle 
tips are unpigmented. The mantle edge is 
also dark. There are a few white granules on 
the sole of the foot. 

Nervous System (as in T. gasulli). The two 
cerebral ganglia are connected by a cerebral 
commisure and lie over the oesophagus. The 
right and left pleural ganglia are about the 
same size and rise out of the curvature of 
the cerebral ganglia. The right pleural gan- 
glion is connected with the supraoesophageal 
ganglion by a long connective. Nervous sys- 
tem measurements are: length of the right 



cerebral ganglia: 0.17 mm; length of the cere- 
bral commisure: 0.05 mm; length of the right 
pleural ganglion: 0.09 mm; length of the left 
pleural ganglion: 0.10 mm; length of the 
supraesophageal ganglion: 0.09 mm; length 
of the subesophageal ganglion: 0.08 mm; 
length of the pleurosupraesophageal connec- 
tive: 0.10 mm. RPG ratio = 0.39. 

Mantle Cavity. The mantle epithelium is pig- 
mented uniformly brown, except on the body 
whorl, which is less pigmented, making it pos- 
sible to follow the position of the intestine. 
There is a well-developed ctenidium, with the 
number of lamellae varying from 11 to 16, but 
usually about 14. At the base of the gill, near 
the neck, there is a well-developed ovoid an- 
nular osphradium. 

Operculum (Fig. 11C-E, Table 2). Opercu- 
lum corneus, paucispiral, thin. The pale yel- 
lowish nucleus is in a subcentral position on 
the operculum. 

Digestive System. The stomach and style 
sac, although similar in overall shape, differ in 
size and proportion from those of T. gasulli 
(Table 4). The rectum forms a markedly ll- 
shaped bend, sometimes directed towards 
the mantle border. It ends near the distal cor- 
ner of the mantle cavity. 



88 



RAMOS ETAL. 




500 M" 1 



FIG. 7. A. Uncoiled bodies without head of A. female and B. male of Tarraconia gasulli. Abbreviations: Be. 
bursa copulatrix; CI. columellar muscle; Di. digestive gland; K. kidney; Int. intestine; Oes. oesophagus; Ov: 
ovary; Pg: paleal gland; Pr. prostate; St. stomach; Sv. seminal vesicle; T. testis; Pvd. posterior vas deferens. 



A NEW GENUS AND A NEW SPECIES OF HYDROBIID SNAIL 



89 




5 oo Mm 





FIG. 8. Female and male genitalia of Tarraconia gasulli from Manantial La Esperanza, Navajas, Castellón. 
А, В. Anterior female genitalia (note the enlargement of the oviduct); C, D. Head of a male and penis. Ab- 
breviations: Ag. albumen gland; Be. bursa copulatrix; Co. coiled part of the oviduct; Cg. capsule gland; Od. 
oviduct; PI. penial lobe; Vc. ventral channel. 



90 

A 



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RAMOS ETAL. 








В 


■ s ■ ■ 

sp 


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% % 




4* 



sp 



ч 




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sp 




. 



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FIG. 9. Histological sections of Tarraconia gasulli from Manantial La Esperanza, Navajas, Castellón. A. Ep- 
ithelium of the style sac; B. Bursa copulatrix and duct; С Thickened part of the oviduct (note narrow lumen); 

D. Heads of the spermatozoids attached to the ciliated epithelial cells of the thickened oviduct; E. Oviduct 
not enlarged (note spermatozoids in lumen); F. Previtellogenic oocytes; G. Male gonadal lobes; H. Penis and 
lobe. Abbreviations: с cilia; ge. germinal epithelium; sp. spermatozoids; PI. penial lobe. Scale bar: A, C, D, 

E, F: 25 urn; B, G, H: 0.1 mm. 



A NEW GENUS AND A NEW SPECIES OF HYDROBIID SNAIL 



91 




FIG. 10. SEM microphotographs of Tarraconia го/ал/ shells from the type locality (La Carroba, Tarragona). A, 
E, F Holotype. B, C, D, G, H, I. Paratypes. The arrow in Fig. F indicates the slight varix behind the outer lip. 
(note the embryo inside the umbilicus in Fig. I). 



The radula is typically taenioglossate. The 
total size of radular ribbon is 0.45 urn and is 
medium-sized relative to mean shell dimen- 
sion (Fig. 12A) (Hershler & Ponder, 1998). 
The central tooth, with the usual butterfly- 
shaped hydrobiid structure, has a slightly ex- 
cavated cutting edge (Fig. 12B-D). The apex 
is V-shaped with a long middle cusp and five 
smaller cusps on each side that decrease in 
size towards the edge (Fig. 12C). The lateral 
wings have one cusp on each of the basal an- 
gles. The lateral teeth have slender lateral 
wings with one pointed basal cusp on each 



side. They have a large wide central cusp that 
points toward the central teeth, and 4 to 6 
other cusps at each side, decreasing in size 
(Fig. 12C-E). The two pairs of marginal teeth 
have a long row of denticles of similar size 
(Fig. 12E-F). Radular formula and measure- 
ments are given in Table 3. 

Female Reproductive System (Fig. 13A, B, 
Table 4). The palliai oviduct is sometimes 
slightly lengthened in its anterior part. The 
capsule gland is about 1/3 the palliai oviduct. 
The bursa copulatrix protrudes at the end of 
the palliai oviduct. It is well developed, 



92 



RAMOS ETAL. 




FIG. 11 . SEM photomicrographs of Tarraconia rolani protoconch and opercula from the type locality (La Car- 
roba, Tarragona). A, B. Protoconch; C, D, E. Outer and inner side of the opercula. 



roundish, has a short duct and constitutes half 
the length of the palliai oviduct. The renal 
oviduct is unpigmented and forms a wide loop 
leaning against the palliai oviduct. 

Egg Capsule. Several specimens were ob- 
served to be carrying a single egg capsule in- 
side the umbilicus with an embryo at different 
stages of maturation (Fig. 101). 

Male Reproductive System (Fig. 13C, D, 
Table 4). The posterior vas deferens enters 
the prostate gland approximately in the mid- 
dle part. The vas efferens and seminal vesicle 
can clearly be seen because of the iridescent 
pink sperm. The prostate is oval or bean- 
shaped and the lumen U-shaped. The ante- 
rior deferens vas can be seen near its anterior 
tip. 

The penis is inserted in the right middle part 
of the neck. It is cylindrical in the transverse 
section, very long and slender, and has a 
small lobe on the concave side in its central 
part that looks like a tip and forms a 90° angle 
to the central axis of the penis. The penis is 
not pigmented in its distal part below the lobe, 
where it narrows and tapers at the end. The 



narrow penis duct is straight and lies towards 
the right edge. 

Remarks 

This species differs from T. gasulli in a set 
of character states as follows: shell shape 
(wider than taller), a much slighter varix be- 
hind the outer lip, the shape of the cutting 
edge of the central teeth, overall shape of the 
bursa copulatrix, shorter duct of the bursa 
copulatrix, penis size, overall shape of the 
penis lobe, tapered shape of penis tip, lack of 
pigmentation on the penis, and size and 
shape of the anterior digestive system. Exter- 
nal pigmentation appears lighter than in T. 
gasulli. 

Habitat and Distribution 

The species seems to be restricted to a few 
localities in a small area in Tarragona prov- 
ince. It lives in springs and irrigation ditches 
with clean water and much aquatic vegeta- 
tion. In these environments, it can be found on 



A NEW GENUS AND A NEW SPECIES OF HYDROBIID SNAIL 



93 







..■."'■'""* 4 win| 


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FIG. 12. A-E. SEM photomicrographs of Tarraconia rolani radula from the type locality. A. Complete radula. 
B. Transverse rows. С Central and lateral teeth. D, E. Central, lateral and inner marginal teeth. F Outer mar- 
ginal teeth. 



the roots of the vegetation, stones or even in 
sand and other artificial substrata with a wide 
variety of other freshwater molluscs, the most 
common being species of the genera Pisid- 
ium, Melanopsis, Theodoxus, Belgrandia, 
Pseudamnicola, Potamopyrgus, Ferrisia, 
Lymnaea and Physa. Live specimens were 
found in all localities except the type locality, 
most being very well preserved. 



STATISTICAL ANALYSIS AND 

MORPHOLOGICAL DIFFERENTIATION 

BETWEEN SPECIES 

Variation in shell measurements in the 
three study populations can be seen in Tables 
1 and 5 and Figures 14 and 15. The t-values 
for the anatomical data and the significance 
level are shown in Table 7. 



94 



RAMOS ET AL. 




FIG. 13. Female and male Tarraconia ло/ап/ genitalia. A, B. Anterior female genitalia (note the enlargement 
of the oviduct); C, D. Head of a male and penis. 



On the basis of shell characters (the only 
characters available both from type material 
and topotypes collected at the type locality of 
T. gasulli), the ANO VA (Table 6) demonstrated 
that the live specimens collected in the neigh- 
bouring locality, Manantial La Esperanza, 
clearly belong to T. gasulli. The only differ- 



ences between both populations are the num- 
ber of spire whorls (NSW) and the width of the 
antepenultimate whorl (WAW). In addition. 
ANOVA showed significant differences be- 
tween T. gasulli and I rolani for some of the 
most important conchological variables stud- 
ied. These differences are related to shell 



A NEW GENUS AND A NEW SPECIES OF HYDROBIID SNAIL 



95 



TABLE 5. F-values of the Anova statistical analysis 
for shell variables and differences between popula- 
tions resulting from the Scheffé Test. POP. 1: T. 
gasulli (La Esperanza, Navajas). POP. 2: T. gasulli 
(El Bañador, Altura). POP. 3: T. rolani (La Carroba). 



Variables 


F (2.74) 


POP. 1 


POP. 2 


POP. 3 


SL 


14.550"* 


3 


3 


1.2 


SW 


16.296*** 


3 


3 


1,2 


AH 


0.583* 








LBW 


24.572*** 


3 


3 


1,2 


WBW 


2.639* 








AmL 


1.505* 








AmW 


2.341* 








WPW 


4.597** 


3 




1 


WAW 


8.777*** 


2.3 


1 


1 


NSW 


5.611** 


2,3 


1 


1 



length (SL) and width (SW), length of body 
whorl (LBW), and width of the penultimate 
whorl (WPW). Mean WPW values only differ 
between Altura (type locality of T. gasulli) and 
La Carroba (type locality of T. rolani) popula- 
tions, but not between both T. gasulli popula- 
tions. Two variables, the width of the ante- 
penultimate whorl (WAW) and the number of 
spire whorls (NSW), differ between T. gasulli 
(type locality) and the rest. Aperture height 
(AH), width of body whorl (WBW) and aper- 
ture length and width (AmL, AmW) are not 
useful variables to investigate differences be- 
tween the two species. All the above suggests 
that the variables that most contribute to shell 
shape are those that are most different be- 
tween T. gasulli ano T. rolani п. sp. 

Differences between species were also con- 
firmed by the DFA. Two highly significant dis- 
criminant functions were found (Wilk's lambda 
= 0.102, F (10,134) = 28.458, p < 0.001) for 
shell characters. The variables included in 
both functions were: LBW, SW, SL, AmW and 
WAW. For the first function, which accounted 
for 82% of explained variance, the characters 
that contribute (highest weight) were (in 
order): LBW, AmW, SW, WAW and SL. For the 
second function, the order was: LBW, SL, SW, 
WAW, AmW. Both discriminant functions were 
highly significant (p < 0.001). The classifica- 
tion functions being: El Bañador, Altura: [f1 = 
-117.179 + (95.665 x LBW) + (49.806 x SW) 
+ (67.485 x SL) + (1 85.587 x AmW) + (39.565 
x WAW)]. La Esperanza, Navajas: [f2 = 
- 128.328 + (147.123 x LBW) + (66.343 x SW) 
+ (1 04.885 x SL) + (81 65.244 x AmW) + (6.51 5 
x WAW)]. LaCarroba: [f3 = -142.381 + 
(21.809 x LBW) + (91.157 xSW) + (-77.679 
x SL) + (261 .688 x AmW) + (71 .541 x WAW)]. 



Seventy nine percent of variance from the 
statistical data was due to interpopulational 
variation. On the scatterplot, three clusters 
can be observed, two of them overlapping 
and corresponding to the two T. gasulli study 
populations. The third, which is separated and 
does not overlap, corresponds to T. rolani. 
From the total data, 75% of cases were cor- 
rectly assigned to T. gasulli {type locality). The 
percentage increased to 96% for Manantial 
La Esperanza (Navajas) and to 92% for the T. 
rolani type locality. 

The opercula variables did not present sig- 
nificant differences among the three study 
populations, not being, therefore, informative 
in differentiating species. 

Study of the anatomical characters statisti- 
cally confirmed the aforementioned differ- 
ences between both species (Table 7). Two 
female genitalia variables presented signifi- 
cant differences: bursa copulatrix width (BcW) 
and length of the bursa copulatrix duct (DBc 
L). Among male genitalia variables studied, 
penis length (PL) was significantly longer in T. 
rolani n. sp. Differences in non-genital system 
variables were mainly related to the size and 
shape of the digestive system: style sac 
length (Ss L), style sac width (Ss W) and 
stomach width (St W). 



GENERIC DIFFERENTIATION 

Boeters (in Gasull, 1981) described Hauffe- 
nia (Neohoratia) gasulli as: "Concha valvati- 
forme, cónica, con aprox. 3.5 vueltas de es- 
pira. La boca en la penúltima vuelta 
permanece en la misma linea, ni más alta ni 
más baja. Peristoma cortante, no ensan- 
chado, algo circular, solamente algo hinchado 
por encima, y situado directamente en la 
penúltima vuelta, por lo que el ombligo queda 
totalmente abierto. El tuberculito, bien visible, 
cae detrás del borde del peristoma. Altura de 
la concha aprox. 1.5 mm y diámetro aprox. 
106 mm)" (shell diameter was surely a typo- 
graphical mistake, which should have read 
1.6 mm). These characters, the lack of infor- 
mation about soft parts and some differences 
regarding the other Neohoratia species led 
him to doubt the allocation of the species to 
the genus Neohoratia (Boeters, 1988). The 
set of character/character-states resulting 
from the addition of the anatomical data con- 
firms that the species studied does not fit the 
description of any of the valvatoid genera de- 



96 



RAMOS ET AL. 



i в\\ 



1.34 
1.30 
1.26 
1.22 
1.18 
1.14 
1.10 
1.06 
1.02 



0.80 
0.78 
0.76 
0.74 
0.72 
0.70 
0.68 
0.66 
0.64 
0.62 



ш 



POP 



WBW 









































































POP 



AMW 
























■ 






■ | 


■ 

























3 POP 



0.98 
0.94 
0.90 
0.86 
0.82 
0.78 



1.7 
1.6 
1.5 
1.4 
1.3 

0.80 
0.76 
0.72 
0.68 
0.64 
0.60 
0.56 
0.52 



AH 


























Ш Ï 















3 POP 



sw 



+ 






POP 



VVPW 




3 POP 



35 



0.88 
0.86 
0.84 
0.82 
0.80 
0.78 
0.76 
0.74 
0.72 
0.70 



0.36 
0.34 
0.32 
0.30 
0.28 
0.26 
0.24 
0.22 
0.20 






Í 



3 POP 



AML 






POP 



WAVY 



T ++ 



1 2 3 POP 



FIG. 14. Box-Whisker plots showing variation in shell measurements for the three populations considered. 
Pop. 1: type locality (T. gasulli). Pop. 2: La Esperanza, Navajas (T. gasulli). Pop. 3: La Carroba {T. rolani). 
Mean/SE/SD are represented. 



scribed in Europe, and justifies the introduc- 
tion of a new genus (Table 7). 

Hauffenia is a controversial genus as can 
be deduced from the description of the "Hauf- 
fenia group" from the Balkan Peninsula 
(Radoman, 1978): "a group of minute subter- 
ranean forms (white or colourless) that is ho- 
mogenous in shell shape, but very heteroge- 
neous in anatomical structure". After 
Radoman (1983) Hauffenia is characterised 
by having a valvatiform shell, a peg-bearing 
operculum, two basal cusps in the central 
teeth, absence of RS1, but presence of a 
RS2, a small bursa copulatrix not protruding 
from the albumen gland, anterior origin of the 
bursal duct, and a broad penis. All these char- 
acters, except shell shape, differ from those of 
the new taxon. 



Six species of Neohoratia have been de- 
scribed on the Iberian Peninsula (Boeters, 
1988), two of them, N. (?) gasulli and N. (?) 
fezi (Altimira, 1960), are known only from 
shell characters. The anatomically known 
Neohoratia species differ from the new taxon 
in the lack of a bursa copulatrix, the existence 
of two well-developed seminal receptacles, 
their oviduct not being wide and the penis 
lobe being larger and situated in a terminal 
position. 

Finally, in relation to the genus Horatia 
Bourguignant, 1887, which also has species 
on the Iberian Peninsula, the new taxon dif- 
fers in shell characters, but mainly in anatomy. 
Species of this genus usually have two semi- 
nal receptacles, an anteroventral origin of the 
bursal duct, a very extended simple penis with 



A NEW GENUS AND A NEW SPECIES OF HYDROBIID SNAIL 



97 



2 



* 



-1 



О T. gasulli (type loci 

D T. gasulli (Navajas) 

7. rolani (type lot.) 





-10 



-2 
Root 1 



FIG. 1 5. Plot of discriminant scores on the two canonical axes, obtained from DFA of shell measurements for 
T. gasulli and T. rolani for the three populations considered. Confidence interval for ellipses: 0.95. 

TABLE 6. t-values of anatomical variables for Tarraconia gasulli (La Esperanza, Navajas) and 
T. rolani (La Carroba). * n.s.; ** p < 0.05; *** p < 0.001. 



t-value 



(df) 



t-value 



(df) 



t-value 



(df) 



OsL 


1.399* 


(8) 


BcL 


1.555* 


(5) 


PL 


2.776** 


(8) 


Os W 


0.639* 


(8) 


BcW 


5.149** 


(5) 


PW 


-0.134* 


(8) 


CtL 


-0.187* 


(8) 


DBcL 


-3.433** 


(5) 


PrL 


2.566* 


(3) 


Ct W 


0.438* 


(8) 


PoL 


-1.532* 


(4) 


PrW 


1.845* 


(3) 


SsL 


3.101** 


(8) 


Po W 


0.261* 


(4) 


PIL 


-1.266* 


(6) 


Ss 


4.447** 


(8) 


AgL 


0.512* 


(3) 


PIW 


-0.935* 


(8) 


StL 


1.962* 


(7) 


CgL 


-1.697* 


(3) 


HeadL 


-0.534* 


(7) 


stw 


2.852** 


(7) 















one or two small excrescences on the left 
side, more than one basal cusp in the central 
teeth and a long suboesophageal connective. 
The new genus, Tarraconia, is character- 
ised by a female genital tract with a well-de- 
veloped and protruding bursa copulatrix, but 
lacks a distinct sac-like seminal receptacle, its 
function being undertaken by a greatly thick- 
ened portion of the oviduct, proximal to the 
bursa copulatrix duct. This is suggested by 
the columnar ciliated epithelium, which differs 
from the rest of the oviduct and the bursa cop- 
ulatrix, and by the orientated sperm not found 



in the other structures, which is the typical 
method of sperm storage in the seminal re- 
ceptacle (Fretter & Graham, 1994: 303-306). 
The presence of an egg capsule in the umbili- 
cus of T. gasulli is described for the first time 
in a hydrobiid valvatoid species in Spain. This 
character has been observed in other popula- 
tions of wide-umbilicated valvatoid hydrobiid 
species from the Iberian Peninsula (unpub- 
lished data). 

The lack of a seminal receptacle has been 
reported in some other genera of Hydrobiidae 
that present different anatomical strategies to 



98 



RAMOS ET AL. 



TABLE 7. Character and character-state scores for 13 characters and 6 genera. Tarraconia, n.g., is com- 
pared with the valvatoid-hydrobid genera mentioned for Spain, and with other genera having a similar female 
genital feature for Character 2. Character states have no phylogenetical significance. 



Character and character-states 



."3 

с 


■S 


■2 


с 


.пз 

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TD 


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о 




С/1 


is 


э 


2 


-с 
о 


£ 


•2 


■С 
О 


5 


о 


О) 


iS 


о 
5 


о 
к: 


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2 





2 


2 


2 





1 


2 


1 


1 








1 


4 


2 


3 


? 


1 


0,2 


2 


3 


3 


3,4 





1,2 


1 


1 


0,1 








1 


1 


1 


0,2 






Female Reproductive System 

1 . Bursa copulatrix: absent (0), small (1 ), well developed (2) 

2. Position of the bursa copulatrix relative to the albumen 
gland: not protruding (0), protruding (1), absent (2) 

3. Origin of the bursal duct: anterior (0), anteroventral (1), 
anterodorsal (2), posterior (3), absent (4) 

4. Seminal receptacle: RS1 (0), RS2 (1), RS1 + RS2 (2), lost 
with function moved within coiled oviduct (3), lost with func- 
tion moved to a swollen part of posterior ventral channel of 
capsule gland (4) 

Male Reproductive System 

5. Penis: simple (0), unilobed (1), bilobed (2) 

6. Penis with lobe (s): no lobes (0), concave edge (1), convex 
edge (2) 

Radula 

7. Central tooth lateral wings: with one basal cusp (0), one to 
three basal cusps (1) 

Ctenidium 

8. Gill filaments: slightly developed (0), well developed (1 ), 
absent (2) 

Rectum and Stomach 

9. Without making any loop (0), slightly sinuous (1), strongly 
sinuous (2) 

Nervous system 

10. Suboesophageal connective: short or absent (0), long (1) 
Shell 

11. Shape: elongated cylindro-conical (0), valvatoid (1) 

12. Umbilicus: narrow (0), wide (1) 
Operculum 

13. Operculum: simple (0), peg-bearing (1) 
Sources (*) 



1,2 



1.2 



2 


1 


1,2 


1 


1,2 


1 


? 


1 


0,1 





1 





1 
1 


1 
1 


1 
1 


1 
1 






1 
1 


0,1 
ABC 
DEF 



GHI 



DIN 



L 




JM 




к 



(*) Sources: (A) Haase, 1992: (B) Bemasconi, 1975; (C) Bole, 1970; (D) Bole, 1967; (E) Haase, 1993; (F) Giusti & Pezzoli, 
1 980; (G) Radoman, 1 966; (H) Radoman, 1 983; (I) Boeters, 1 988; (J) Bodon & Giusti. 1 991 ; (K) Ponder et al., 1 989; (L) This 
paper; (M) Boeters, 1973; (N) authors' unpublished data. 



fulfil its function. In the genus Moitessieha 
Bourguignat, 1863 (Hydrobiinae) (Bodon & 
Giusti, 1991), sperm storage seems to be lo- 
cated in several portions of the oviduct in- 
stead of being concentrated in only one por- 
tion, as histological evidence demonstrates 
for T. gasulli. Moitessieria is a well-defined 
genus with characters that as a whole differ 
widely from Tarraconia, n. g. (Table 7). In 
short, it has elongated cylindro-conical shells 
with characteristic net-like teleoconch mi- 
crosculpture, a narrow umbilicus, a posterior 
origin of the bursal duct, a conical, smooth, 
point-tipped penis with, sometimes, small 



glandular excrescences that are apical on the 
right side, and a long suboesophageal con- 
nective. 

Trochydrobia Ponder, Hersler & Jenkins, 
1 989, described from Australia, is another val- 
vatoid genus of Hydrobiidae lacking a seminal 
receptacle. In Trochydrobia, the role of the 
seminal receptacle is played by a swollen part 
of the posterior ventral channel of the capsule 
gland or in the coiled oviduct. Moreover, Tro- 
chydrobia punicea Ponder, Hersler & Jenkins, 
1 989, has a thickened coiled oviduct similar to 
that described for Tarraconia, and egg cap- 
sules in the umbilicus, as in I gasulli. How- 



A NEW GENUS AND A NEW SPECIES OF HYDROBIID SNAIL 



99 



ever, according to Ponder et al. (1989), in 
spite of the similarities between this species 
group and the European Horatia-Pseudamni- 
cola complex, they have several distinct fea- 
tures (Table 7), such as a simple penis, a very 
well-developed albumen gland with a bursa 
copulatrix not protruding from it, up to two 
pairs of basal cusps in the central tooth of the 
radula, and a rectum that runs close to the 
palliai oviduct and prostate without making 
any loop. 

Other groups of species without the semi- 
nal receptacle are included in the family Pyr- 
gulidae, in which glandular structures, such 
as an oviduct with a blunt beak-shaped dilata- 
tion or a pouch-like dilatation or simply a glan- 
dulous oviduct loop without dilatation, can be 
found instead of the seminal receptacle 
(Radoman, 1983). The species belonging to 
this family differ from all the others described 
here in the glandular origin of this structures. 
In addition, all the genera in this clade differ in 
the most important characters, that is, they 
have an oesophageal caecal chamber and a 
radular central tooth without basal cusps and 
without the typical lateral wings of the Hydro- 
biinae. 

The genus Gammatricula Davis, Liu & 
Chen, 1990, was introduced to isolate two 
species of Pachydrobiini (Triculinae, Poma- 
tiopsidae) from China that have this female 
character with the same shape and position in 
the oviduct as described for Tarraconia, n. g. 

In an investigation of the phylogenetic rela- 
tionships of the new genus Tarraconia with 
the aforementioned genera of Hydrobiidae 
using PAUP 3.1 (Swofford, 1993), only 8 of 
the 13 characters (all characters treated as 
unordered and unweighted) for which enough 
bibliographical data were available proved to 
be informative (characters 2,4-10). Cladistic 
analysis yielded three not fully resolved trees 
(22 steps, C.I. = 0.66). It is well known that 
cladistic methodology, mainly based on es- 
tablishing phylogenetic relationships through 
synapomorphies, is hard to achieve when few 
characters are available or when there is a 
high degree of homoplasy. Two main reasons 
account for the lack of available morphologi- 
cal characters in the case of hydrobiids: (1) 
the minute size results in a very few diagnos- 
tic characters and a high degree of conver- 
gence between different taxa, and (2) most 
European valvatoid hydrobiid genera have 
scarcely been studied and very few charac- 
ters are available for phylogenetic inferences. 
In this case, the lack of data particularly af- 



fects the genera Hauffenia and Horatia. More- 
over, most of the characters compared in 
suprageneric classifications in hydrobiids 
(Ponder & Waren 1988; Davis et al., 1982; 
Hershler, 1985) cannot be used when com- 
paring genera or species. The few phyloge- 
nies done in this sense show a high degree of 
homoplasy in some of the most currently used 
characters of shell and genital systems (Pon- 
der et al., 1993). 

The preliminary phylogenetic analysis 
showed hardly any conflict among the ob- 
served characters, although its validity is un- 
certain due to the low number of available 
data sets. The absence of a seminal recep- 
taculum, its function being transferred to the 
renal oviduct, is one of the most important 
characters of this new genus. Nevertheless, 
the fact that the function of a lost evolutionary 
character has been taken up by similar struc- 
tures in different and separated taxa, even be- 
longing to different families (Hydrobiidae, Pyr- 
gulidae and Pomatiopsidae), suggests that it 
could be another case of parallel derived 
change. This argument seems to support 
Bodon & Giusti's (1991) hypothesis that the 
family Moitessieridae, whose isolation as a 
family was mostly based on this female 
anatomical character (Boeters, 1973), should 
only be considered as a junior synonym of Hy- 
drobiidae. Nevertheless, while the obscure 
phylogenetic relationships of this family are 
being unravelled on the basis of well-founded 
data sets, it is clear that the description of new 
taxa might be based not only on one charac- 
ter, but on a set of characters, with as much 
detail as possible. Progressively thorough 
knowledge of the European hydrobiid fauna 
will help in tracing the evolution of characters. 



ACKNOWLEDGEMENTS 

We are indebted to Dr. G. M. Davis for his 
teaching and advice at the beginning of the 
project and for his useful suggestions con- 
cerning the manuscript. Dr. M. Haase and an 
anonymous reviewer made very useful and 
constructive comments. We thank Dr. R. 
Araujo, J. Bedoya, J. M. Remón, Dr. G. Tapia, 
Dr. O Noreña and N. Martín for providing us 
with field samples. Dr. J. Lobo provided sta- 
tistical advice. Histological sections were 
made by J. Cabanillas. SEM photomicro- 
graphs were made by J. Bedoya at the 
MNCN. Drawings were re-done by I. Diaz- 
Cortaberria. Advice on nomenclature was 



100 



RAMOS ET AL. 



provided by Dr. M. A. Alonso-Zarazaga. The 
English was corrected by L. Ashcroft. This 
work was supported by the "Fauna Ibérica" 
Project (DGES PB95-0235). 



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A NEW GENUS AND A NEW SPECIES OF HYDROBIID SNAIL 



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Revised ms. accepted 22 June 1999 



MALACOLOGIA, 2000, 42(1-2): 103-112 

ULTRASTRUCTURAL AND CYTOCHEMICAL STUDY OF THE DIGESTIVE GLAND 
CELLS OF THE MARINE PROSOBRANCH MOLLUSC NUCELLA LAPILLUS (L.) IN 

RELATION TO FUNCTION 

Vasilis K. Dimitriadis 1 & Elizabeth B. Andrews 2 

ABSTRACT 

The functional morphology of the digestive gland of the carnivorous prosobranch mollusc Nu- 
cella lapillus (L.) is examined and compared to with that of other molluscs. Three distinct types 
cell types compose the tubule epithelium. The first type, digestive cells, are numerous columnar 
unciliated cells displaying large membrane-bounded structures, the heterolysosomes and resid- 
ual bodies, as well as a prominent endocytotic canal system of microvesicles and tubules. The 
material of most residual bodies indicates a positive reaction for periodate-reactive and/or sul- 
phated and carboxylated carbohydrates. In certain cases, the material of heterolysosomes and 
membrane remnants of the residual bodies shows a positive acid phosphatase reaction. 

The second type comprises basophilic secretory cells, which are located into crypts of the 
tubules and are distinguished by their strong basophilia, their pyramidal shape, and their well- 
developed tubular rough endoplasmic reticulum. 

The third cell type is very rare and consists of "small secretory cells" with small periodate-re- 
active secretory granules. These granules react positively for sulphated and carboxylated car- 
bohydrates, the positive reaction located mainly on their periphery. 

Key Words: digestive gland, Nucella, Gastropoda. 



INTRODUCTION 

The light and electron microscopic results 
concerning the functional morphology of di- 
gestive glands of marine prosobranchs are 
few (Graham, 1932; Owen, 1958, 1972; 
Campbell, 1965; Pugh, 1963; Merdsoy & Far- 
ley, 1973; Bush, 1986), in spite the important 
role of these organisms in environmental 
studies. The above-mentioned studies have 
revealed that the digestive gland tubules are 
lined by at least two mature cell types, the di- 
gestive cells dealing with the absorption and 
digestion of food substances, and the ba- 
sophilic cells to which various functions have 
been attributed. The existence of a well-de- 
veloped heterophagic-lysosomal system in 
the digestive cells indicating intracellular di- 
gestion of nutrients is also documented in 
these mentioned studies. 

Nucella lapillus is one of the prosobranchs 
feeding on animals, the bodies of which are 
enclosed within shells. It feeds largely on 
mussels and limpets, and on barnacles; it is 
able to force barnacle shells apart by muscu- 
lar action of the proboscis without boring 



(Fretter & Graham, 1962). A detailed ultra- 
structural study dealing with the functional 
morphology of the digestive gland of a carniv- 
orous prosobranch is missing, in spite of the 
attention that this organ and these organisms 
have received from other points of view. Thus, 
the consideration of the functional morphol- 
ogy of the digestive gland of a carnivorous 
prosobranch compared with that of other her- 
bivorous, carnivorous or deposit-feeder mol- 
luscs might be interesting. 

In the present study, the ultrastructure of 
the digestive gland cells of Nucella lapillus is 
examined. In addition, cytochemical tests for 
carbohydrate characterization and acid phos- 
phatase localization were applied. 



MATERIALSAND METHODS 

Specimens of adult Nucella lapillus ob- 
tained from the University Marine Station, 
Millport, Scotland, were kept in an aquarium 
with circulating artificial sea water at 1 0°C and 
fed with common mussels. During the experi- 
ments their physiological state was good. 



1 Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, Thes- 
saloniki. 54006, Greece; vdimitr@bio.auth.gr. 

2 Biology Division, School of Biological Sciences, Royal Holloway and Bedford New College, University of London, Egham, 
Surrey, TW200EX, England 

103 



104 



DIMITRIADIS & ANDREWS 



Some of the specimens were fixed for elec- 
tron microscopy and histochemistry on arrival 
and others were used within a few days. 

Electron microscopy 

Samples were fixed in 3% glutaraldehyde in 
Sorensen's phosphate buffer, ph 7.2 adjusted 
to 1100 mOsM with 14% sucrose and post- 
fixed with 1% aqueous osmium tetroxide. Ma- 
terial was embedded in TAAB resins (TAAB 
Laboratories Equipment Ltd., Reading, U.K.) 
and thin sections were stained in 1 % aqueous 
uranyl acetate and Reynolds' lead citrate. 

Cytochemistry 

For carbohydrate cytochemistry, finely 
minced pieces of tissues were incubated 
overnight after fixation in high iron diamine 
(HID) (Spicer et al., 1 978; Sannes et al., 1 979) 
using a medium containing 36 mg N,N-di- 
methyl-m-phenylenediamine, 6 mg of the para 
isomer and 0.45 ml ferric chloride 40%, in 15 
ml d water. Other pieces were incubated in low 
iron diamine (LID) (Takagi et al., 1982), using 
a medium containing 27 mg N,N-dimethyl-m- 
phenylenediamine, 4.5 mg of the para isomer 
and 0.45 ml ferric chloride 40%, in 45 ml d 
water. After incubation, the tissues were post- 
fixed with 2% aqueous osmium tetroxide and 
embedded in Spurr's resin. Thin sections of 
these specimens were stained with the thio- 
carbohydrazide-silver proteinate (TCH-SP) 
sequence, using a medium containing 2% 
thiocarbohydrazine in 20% acetic acid, to re- 
veal periodate-reactive substances. Control 
tissues were incubated in 1 M MgCI 2 in place of 
LID or HID. Specimens without osmium tetrox- 
ide treatment were used for the postembed- 
ding periodate-thiocarbohydrazide-silver pro- 
teinate (PA-TCH-SP) method (Thiéry, 1967). 
Control sections were stained without the pe- 
riodate treatment. 

For acid phosphatase demonstration, a 
modification (Lewis & Knight, 1992) of the se- 
quence proposed by Barka & Anderson 
(1 962) was applied. Glutaraldehyde was used 
as a fixative and the tissues were incubated in 
a medium containing 0.2M tris/maleate as a 
buffer and 0.1 M ß-glycerophosphate as sub- 
strate at 37°C for 15-30 min. Control sections 
were taken through an identical sequence ex- 
cept that the substrate was omitted from the 
incubation medium or had 0.01 sodium fluo- 
ride added. 



RESULTS 

The epithelial cells of the tubules of the di- 
gestive gland of Nucella exhibit different 
phases, each with a distinct appearance, the 
digestive and fragmentation state being the 
most frequently encountered. The results pre- 
sented here are a static representation of cell 
types of the digestive gland, rather than a de- 
scription of cell types in a distinct phase. 

Three different cell types compose the 
tubule epithelium. The most numerous, diges- 
tive cells, are columnar unciliated cells, the 
main characteristic of which is the presence of 
a variety of membrane-bound vesicles in their 
cytoplasm. The second type, basophilic se- 
cretory cells are located in crypts of the tubules 
and are distinguished from the digestive cells 
by their strong basophilia, pyramidal shape 
and well-developed tubular rough endoplas- 
mic reticulum. The third cell type, "small mu- 
cous cells", are carbohydrate-containing cells 
with many small mucous granules. 

A very thin connective tissue layer sur- 
rounds each tubule, and poorly developed 
muscle layers of circular, longitudinal and 
oblique fibres surround each tubule. Amoebo- 
cytes do not cluster around the tubules. 

Digestive cells are columnar and their api- 
cal surface bears short microvilli (Fig. 1A). 
The most conspicuous organelles of these 
cells are a series of membrane-bound vesi- 
cles composing the heterophagic-lysosomal 
system. This system consists of microvesicles 
and large or very large membrane-bound 
structures, the heterolysosomes, appearing 
as electron-opaque and homogeneous or 
moderately opaque and containing floculant 
material (Figs. 1A, 2C). Microvesicles include 
pinocytotic vesicles (pinosomes) and profiles 
of the endocytotic canal system (Fig. 1С). 
Pinocytosis occurs at the base of the microvilli 
(Fig. 2A), and the pinocytotic vesicles are 
coated on the cytoplasmic face and usually 
display a size at about 0.1-0.3 urn. 

The endocytotic canal system branches 
through the cytoplasm and in section has the 
appearance of short, tubular profiles and 
vesicles. The bounding membrane and gran- 
ular content stain intensely, making it promi- 
nent even at low magnification (Fig. 1С). Cer- 
tain elements of the canal system are also 
stained for periodate-reactive carbohydrates 
(Fig. 2B). 

Heterolysosomes are frequently situated in 
the mid-region of the cell and are homoge- 
neous granules with a usually moderate elec- 



DIGESTIVE GLAND CELLS OF NUCELLA 



105 



Lu 


; 


* ',, 


m 


i» 


SG 

'% , ЦК 




ч 

*г г. 


* 


Í2 f 

■ „ ) 


""г 


г* . 

• ч 


■ * • 






'Ф***. 




« 


" ЙВ- - 

• 




щ 


A 








Яр 



.О 



HI 



RB 



Li 



N 






в 



' / 



Lu 









>^ 



V 



/ 



Mi 



/ 



Go 

I 



С 



D 



FIG. 1 . Digestive cells in the digestive gland of Nucella lapillus. A. Apical portion of a digestive cell showing 
electron dense secretory granules (SG) and large pale heterolysosomes (HI). Scale bar = 3 |.im. B. A large 
residual body (RB) with a granular clumped matrix is located in the cytoplasm amongst lipid inclusions (Li). 
Scale bar = 1 .9 (.im. C. Elements of the canal system comprising of vesicles and tubules (arrows) are promi- 
nent in the apical portion of a digestive cell. Scale bar = 0.8 um. D. A Golgi complex (Go) producing small 
electron dense granules (arrows). Scale bar = 0.6 urn. Abbreviations: Lu, lumen; Mi, mitochondria; N, nu- 
cleus 



106 




FIG. 2. Digestive cells in the digestive gland of Nucella lapillus. A-D. Cytochemistry for periodate-reactive 
carbohydrates. A. Coated vesicles in the apex of a digestive cell (arrows). Scale bar = 1 (.im. B. In the apical 
portion of a digestive cell, certain elements of the canal system are stained for periodate-reactive carbohy- 
drates (arrows). Scale bar = 0.8 ¡am. С A heterolysosome (HI) indicates a negative reaction for periodate-re- 
active carbohydrates. Scale bar = 0.8 ¡am. D. Residual body (RB) showing moderately positive reaction for 
periodate reactive carbohydrates. Scale bar = 0.8 ,iim. E. The matrix of a heterolysosome (HI) shows a pos- 
itive acid phoshatase reaction. Scale bar = 0.7 ¡j.m. F. Membrane remnants of a residual body (RB) display 
a positive acid phosphatase reaction. Scale bar = 0.7 pm. Abbreviations: Lu, lumen; Mv, microvilli. 



DIGESTIVE GLAND CELLS OF NUCELLA 



107 



tron density. They usually have a size at about 
1 -2 }im, while some of them reach 1 0- 1 2 (.im. 
In certain cases, the material of the het- 
erolysosomes reacts positively for acid phos- 
phatases (Fig. 2E), while these structures 
react negatively to periodate-reactive sugars 
(Fig. 2C). Residual bodies or heterolyso- 
somes condensed towards residual bodies, 
frequently lie in the mid portion of the cells and 
usually exhibit a granular matrix clumped in 
the centre of the granular structure, while oth- 
ers are filled with a similar matrix (Fig. 1B). 
They usually have a size of about 1 -2 urn, 
while in certain cases these structures pre- 
sent larger sizes (Fig. 2B). The material of 
most residual bodies indicates a positive re- 
action for periodate carbohydrates (Fig. 2D), 
as well as for sulphated and carboxylated 
sugars (not shown). In certain cases, mem- 
brane remnants of the residual bodies display 
a positive acid phosphatase reaction (Fig. 2F) 

Lipid droplets (Fig. 1B) are common in the 
basal half of cells of recently collected ani- 
mals. Mitochondria are numerous in the mid- 
region amongst the heterolysosomes. The 
oval nuclei are located basally, and the short, 
thread-like cisternae of the rough endoplas- 
mic reticulum are present in the basal third of 
the cell, around the nucleus or against the 
basal and lateral membranes. The Golgi com- 
plexes are conspicuous and rarely observed. 
They are small in size and their trans face pro- 
duces small or medium electron-dense gran- 
ules (Fig. 1D). Granules displaying the same 
electron density, but larger size, which are 
possibly produced after fusion of the smaller 
one, are apparent in the apical region of the 
cells (Fig. 1A). These granules, which are 
possibly secretory, reacted negatively to both 
acid phosphatase and carbohydrate pres- 
ence reactions. Golgi complex cisternae in 
both digestive and basophilic cells give in cer- 
tain cases a distinct acid phosphatase posi- 
tive reaction (Fig. 3D). 

The basophilic cells (Fig. ЗА) display a 
broad base which upon the thin, flat basal 
lamina and the rest of the cell tapers forming 
a neck, the apical surface of which bears mi- 
crovilli bordering the lumen of the tubule. In 
the apical cytoplasm, the endocytotic canal 
system is not very well developed as in the di- 
gestive cells. A highly developed rough endo- 
plasmic reticulum branches through the cyto- 
plasm showing a tubular and dilated structure 
(Fig. ЗА). The cisternae are very often filled 
with a very pale granular material, while the 
great amount of rough endoplasmic reticulum 



and free ribosomes in the cytoplasm are re- 
sponsible for the strong basophilia of the 
cells. The Golgi complexes are usually posi- 
tioned in the supranuclear cytoplasm and are 
larger in size than the analogous complexes 
of the digestive cells (Fig. ЗА, В). The mito- 
chondria are large and some times hyper- 
trophic, indicating high activity in these cells. 

The third cell type, the "small secretory 
cell", is very rare and consists of small cells 
with small periodate-reactive secretory gran- 
ules (Fig. 3C). These granules reacted posi- 
tively for sulphated (Fig. 3D) and carboxylated 
carbohydrates (not shown), the positive reac- 
tion located mainly on their periphery. 

Control sections of all cytochemical tech- 
niques constantly lacked reaction product. 



DISCUSSION 

The digestive gland of Nucella lapillus is 
histologically similar to those of other proso- 
branchs in that the epithelium of the tubules 
are composed of distinct digestive and ba- 
sophilic cells (Graham, 1932; Pugh, 1963; 
Owen, 1958, 1972; Campbell, 1965; Mersoy 
& Farley, 1973; Bush, 1986). The results of 
the present study clearly indicate the exis- 
tence of a third cell type in the digestive gland 
epithelium, the "small secretory cell". These 
cells produce secretory granules containing 
periodate-reactive, as well as sulphated and 
carboxylated carbohydrates. Similar carbohy- 
drate content was detected with a light micro- 
scope in the snail Achatina fúlica (Pereira & 
Breckenridge, 1981), in the intestinal gland 
cells of the slug Arion ater (Ángulo et al., 
1986), in the intestinal mucocytes of the sty- 
lommatophoran Semperula maculata (Varute 
& Patil, 1971), and in the salivary gland cells 
of the snail Helix lucorum (Dimithadis & Do- 
mouhtsidou, 1995) and of Helix aspersa 
(Moreno et al., 1982; Charrier, 1989). The 
main function of the "small secretory cells" in 
Nucella digestive gland is possibly analogous 
to that of the mucous cells existing in the di- 
gestive tissues of other organisms, that is, the 
lubrication and protection of the tubule sur- 
face (Powell, 1984). 

To our knowledge a detailed electron mi- 
croscopic study on digestive gland cells of 
marine prosobranch molluscs has been con- 
ducted only on the prosobranch archaeogas- 
tropod Patella vulgata (Bush, 1986), which is 
a deposit-feeder that scratches mineral parti- 
cles, phytoplancton and zooplancton, on the 



108 



DIMITRIADIS & ANDREWS 







N 



\ 



\ 



л 



■'■■. 



В 



• 
• # 



* 



DC 



■ ■ 







FIG. 3. A. A basophilic cell in the digestive gland of Nucella lapillus displays a well-developed rough endo- 
plasmic reticulum. Scale bar = 2.5 urn. B. The cisternae of a Golgi complex (arrows) of a basophilic cell show 
a positive acid phosphatase reaction (arrows). Scale bar = 0.5 (.im. С The secretory granules of a "small se- 
cretory cell" display a positive reaction for periodate-reactive carbohydrates. Scale bar = 1.2 \im. D. The 
granules of a "small secretory cell" show a peripheral positive reaction for sulphated carbohydrates (arrows). 
Scale bar = 0.3 urn. Abbreviation: DC, digestive cell; Go, Golgi complex; N, Nucleus. 



DIGESTIVE GLAND CELLS OF NUCELLA 



109 



rocky shores and on algae. The morphologi- 
cal and functional parameters of the digestive 
and basophilic cells, such as the structure of 
heterolysosomes, residual bodies, secretory 
granules, of the latter mollusc are very similar 
to those in Nucella lapillus, albeit the fact that 
the latter is a carnivorous neogastropod 
prosobranch. 

The observations reported here on the di- 
gestive cells of Nucella lapillus support the 
view of most authors that this part of the mol- 
luscan gut is the main site of nutrient absorp- 
tion via endocytotic processes (for example, 
Owen, 1966; Morton, 1979; Oxford & Fish, 
1979). The results support the hypothesis of 
intracellular digestion via lysosomal activity of 
material taken up by pinocytosis. Morphologi- 
cal, histochemical and cytochemical results 
confirm this procedure in various molluscs 
(Sumner, 1965; McQuiston, 1969; Owen, 
1970; Walker, 1970; Owen, 1972; Pal, 1972; 
Bowen & Davies, 1971; Wigham, 1976; Ox- 
ford & Fish, 1979; Dimitriadis & Hondros, 
1992; Dimitriadis & Liosi, 1992). In the Nu- 
cella digestive gland, two main lysosomal 
structures implicated in this procedure domi- 
nate in the digestive cells examined. The first 
one is similar to the "phagolysosomes" (or 
"heterolysosomes", or "type 2 microvesicles", 
or "digestive vacuoles", or "green" granules, 
or "apical" granules) reported in other mol- 
luscs and are structures produced by fusion of 
pinocytotic vesicles and enzyme-rich vesicles 
derived from the Golgi complexes (references 
above). The second one, the residual bodies 
(or "yellow" granules, or "cisternae with elec- 
tron-dense cores") (references above) are 
produced by digestion and release of soluble 
material from the "phagolysosomes", as well 
as by accumulation of undigested material. 
Continuous intracellular digestion results in 
large accumulation of residual bodies, which 
are probably pinched off and fall into the gland 
lumen, together with the apical cytoplasm of 
the cells. Walker (1970) proposed that in Agri- 
olimax reticulatus the final step in the devel- 
opment of the digestive cells are the excretory 
cells containing the digestive remnants of in- 
gested material, which is absorbed by the for- 
mer cells, degraded by lysosomal action, and 
subsequently extruded to the lumen of the di- 
gestive gland. In the same slug, some of the 
digestive remnants probably leave the cells 
basally, as amoebocytes containing them are 
found in the underlying connective tissues 
(Walker, 1970). 



The results of the present study, as well as 
those on other molluscs (Sumner, 1965; 
Walker, 1970; Bush, 1986; Dimitriadis & Hon- 
dros, 1 992) did not demonstrate the existence 
of phagocytosis of solid food material by the 
digestive cells in the midgut gland of any of 
these gastropods. Thus, the occurrence of 
pinocytotic, rather than phagocytic processes 
implicating in the absorption of nutrients seem 
to be the case for these cells (Owen, 1966). 

The canal system observed in the digestive 
gland cells of Nucella lapillus is also reported 
in Lasaea (McQuiston, 1969), Cardium 
(Owen, 1970), Mytilus (Owen, 1972), and Ris- 
soa (Wigham, 1976). This system is also re- 
ported in the pericardial cells and the kidneys 
of Scrobicularia plana (Andrews & Jennings, 
1993) and the right kidney of archaeogas- 
tropods (Andrews, 1 985). In the light of the re- 
cent data, a number of the vesicles and 
tubules observed in the canal system should 
be regarded as endosomes, that is, the cell 
compartments where the endocytosed mater- 
ial enters the lysosomal pathway (Alberts et 
al., 1994). 

In Nucella, as well as in the above-men- 
tioned molluscs, there were no connections of 
the canal system with the apical membrane. 
In Patela vulgata (Bush, 1986), on the other 
hand, connections of the tubules of the canal 
system with heterolysosomes were reported. 
There are suggestions that pinosomes empty 
their contents into the canal system, thereby 
filling the heterolysosomes indirectly (Owen, 
1972), or that the canals lead from the lumen 
directly to the heterophagosomes (McQuis- 
ton, 1969; Wigham, 1976). Bush (1986) re- 
garded as possible a transient connection of 
the system to the lumen persisting long 
enough to allow the diffusion of nutrients into 
the canals as separate pulsations, thus es- 
tablishing a concentration gradient along the 
canal-heterophagosome complex. 

In the absence of reports of secretory activ- 
ities in molluscan digestive gland cells, Run- 
ham (1975) proposed the existence of a par- 
ticular secretory sequence into the digestive 
gland producing the digestive enzymes. The 
proposed sequence includes production of di- 
gestive enzymes, their concentration into 
phagolysosomes and residual bodies, and 
their release via exocytosis of the latter struc- 
tures into the digestive tubule lumen. The re- 
sults of the present study demonstrated a 
well-definable secretory production by the 
Golgi complexes of the digestive gland cells 



110 



DIMITRIADIS & ANDREWS 



of Nucella, which possibly release digestive 
enzymes in the digestive tubules and the 
stomach. Similar secretory granules have 
been reported in only a few other molluscs 
(Bush, 1986). 

In lysosomal structures of molluscs, a posi- 
tive acid phosphatase reaction was detected 
by light and electron microscope (Bowen & 
Davies, 1971; Oxford & Fish, 1979; Bush, 
1986; Pipe, 1986; Dimitriadis & Liosi, 1992). 
In Nucella, a weak acid phosphatase activity 
was demonstrated in the heterolysosomes 
and a stronger one in membrane remnants in 
the residual bodies. The latter reactions pos- 
sibly reflect the enzymatic activity inside these 
lysosomal compartments during the intracel- 
lular digestion of the endocytosed exogenous 
material. 

In Nucella digestive cells, lysosomes such 
as residual bodies showed a positive reaction 
for periodate reactive, as well sulphated and 
carboxylated carbohydrates. Similar reac- 
tions were reported for analogous lysosomal 
structures in Helix lucorum digestive gland 
and crop epithelial cells (Dimitriadis & Liosi, 
1993; Dimitriadis et al., 1992). The interesting 
relation between lysosomes and periodate or 
acid sugars presence remains to be verified 
by further studies. 

The basophilic cells observed in the diges- 
tive gland of Nucella lapillus resemble ultra- 
structurally the homologous cells in a variety 
of molluscs (Sumner, 1966; McQuiston, 1969; 
Owen, 1970; Pal, 1971; Owen, 1973; Boghen 
& Farley, 1974; Wigham, 1976). Concerning 
their role, Graham (1932) proposed that these 
cells produce a mucus-like secretion that ce- 
ments pieces of plant cell walls and diatoms 
into a liver string. Supporting the latter hy- 
pothesis on the basis of histochemical and ul- 
trastructural studies, Bush (1986) suggested 
that they are protein-secreting cells, the se- 
cretion of which probably act as a cement in 
the fragmentation phase of the digestive 
tubule. 

In conclusion, by using light and electron 
microscopic observations in combination with 
cytochemical techniques, the present study 
provides information about the structure and 
function of the digestive gland cells of Nucella 
lapillus. However, additional information, es- 
pecially by the use of histochemistry in 
cryosections and immunocytochemistry, is 
needed for the better understanding of the 
structure and mainly of the function of these 
interesting cell types. 



ACKNOWLEDGEMENTS 

This work was undertaken during the sab- 
batical leave of the first of the authors at the 
Royal Holloway and Bedford New College. 
The authors gratefully thank the staff of the 
Electron Microscope Unit of the College for 
their help and invaluable assistance in the 
preparation of some of the material. 



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MALACOLOGIA, 2000, 42(1-2): 113-122 

HETEROZYGOSITY AND FITNESS: NO STRONG ASSOCIATION 

IN GREAT LAKES POPULATIONS OF THE ZEBRA MUSSEL, 

DR E ISS ENA POLYMORPH A (PALLAS) 

Kristin M. Lewis 1 , Jeffrey L. Feder 1 , Thomas G. Horvath 1,2 & Gary A. Lamberti 1 

ABSTRACT 

A number of studies have found positive associations between allozyme heterozygosity and 
fitness surrogates (e.g., body size and growth rate) for marine molluscs. We investigated 
whether similar relationships exist for freshwater populations of the zebra mussel, Dreissena 
polymorpha. Only one significant correlation between multi-locus heterozygosity and shell length 
was observed for a total of 22 D. polymorpha populations surveyed from midwestern U.S.A. 
lakes and streams, and the result was not significant on a table-wide basis. Meta-analysis re- 
vealed a significant common correlation coefficient (effect magnitude) between multi-locus het- 
erozygosity and shell length across all 22 sites (r c = 0.052, P = 0.019, 1557 df). However, the 
variance in shell length explained by multi-locus heterozygosity was small (r c 2 = 0.0027), imply- 
ing a weak causal relationship if any. Also, we saw no relationship between heterozygosity and 
growth rate in a one-year field enclosure experiment. A significant heterozygosity-shell length 
correlation previously reported for a zebra mussel population at Put-in-Bay, Lake Erie, Ohio, may 
have been the product of unique population dynamics, rather than natural selection. Similar de- 
mographic considerations may contribute to inconsistencies in heterozygosity-fitness correla- 
tions seen for other molluscs. 

Key Words: Allozymes, fitness, shell length growth rate, bivalves. 



INTRODUCTION 

Previous studies on a variety of organisms 
ranging from pine trees to humans have re- 
ported positive associations between al- 
lozyme heterozygosity and fitness-related 
traits (reviewed in Mitton & Grant, 1984; 
Zouros & Foltz, 1987; Zouros & Pogson, 
1994; Mitton, 1997). Such relationships ap- 
pear to be particularly common in populations 
of marine molluscs (reviewed in Zouros, 
1987; Britten, 1996). For instance, multi-locus 
heterozygosity has been correlated with 
growth rate, body size, metabolic rate, protein 
metabolism, fecundity, or resistance to envi- 
ronmental stress in the blue mussel, Mytilus 
edulis (Koehn & Gaffney, 1984; Diehl et al., 
1986; Zouros et al., 1988; Gentili & Beau- 
mont, 1988; Gosling, 1989), the American 
oyster, Crassostrea virginica (Singh & Zouros, 
1 978; Koehn & Shumway, 1 982; Zouros et al., 
1983), the Pacific oyster, Crassostrea gigas 
(Fujio, 1982), the edible oyster, Ostrea edulis 
(Alvarez et al., 1989), the sea scallop, Pla- 
copecten magellanicus (Foltz & Zouros, 1 984; 



Pogson & Zouros, 1994), the dwarf surfclam 
clam, Mulinia lateralis (Gaffney & Scott, 1 984; 
Gallon et al., 1984; Koehn et al., 1988), and 
the surf clam Spisula ovalis (David et al., 
1995). 

The relationship between heterozygosity 
and fitness has two general explanations. The 
first is that the allozymes themselves are the 
causative selective agents for the relationship 
(the so-called "direct effect" or "overdomi- 
nace" hypothesis; Mitton & Grant, 1984; 
Zouros, 1987). In this case, heterozygotes for 
specific allozymes grow faster or survive bet- 
ter than homozygotes. The second explana- 
tion is that the allozymes are neutral markers 
that predict the level of homozygosity of an or- 
ganism for recessive deleterious genes (the 
"indirect" or "associative overdominance" hy- 
pothesis; Mitton & Grant, 1984; Zouros, 
1987). This correlation can occur because 
specific allozyme alleles are in linkage dise- 
quilibrium with deleterious genes or because 
the allozymes are general indicators of in- 
breeding (Mitton & Grant, 1984; Houle, 1994; 
David et al., 1995). Both of these hypotheses 



1 Department of Biological Sciences, University of Notre Dame, P.O. Box 369, Notre Dame, Indiana 46556-0369 U.S.A.; 
lewis. 32@nd.edv 

2 Current address: U.S. Geological Survey, Lake Michigan Ecological Research Station, 1100 N. Mineral Spring Road, 
Porter, Indiana 46304 U.S.A. 



113 



114 



LEWIS ETAL. 



assume a causal link between genetic het- 
erozygosity and individual performance in 
some form. 

In North America, the exotic zebra mussel, 
Dreissena polymorpha, is a common pest in 
lakes and rivers (Ludyanskiy et al., 1993). 
Zebra mussels are unique among freshwater 
mussels in that they share several biological 
attributes with marine molluscs, including the 
presence of free-swimming planktonic veliger 
larvae and the retention of byssal threads into 
the adult stage (Ludyanskiy et al., 1993). 
Zebra mussels also have a similar population 
genetic structure as marine bivalves (Boileau 
& Hebert, 1993; Marsden, 1995; Lewis et al., 
2000). Although zebra mussel populations are 
fairly homogeneous in allozyme frequencies 
across freshwater systems in the midwestern 
United States, significant differentiation exists 
on a local level among sites within lakes 
(Boileau & Hebert, 1993; Lewis et al., 2000). 
In addition, heterozygote deficiencies from 
Hardy-Weinberg equilibrium are common in 
zebra mussel populations (Lewis et al., 2000). 
These results suggest that local population 
subdivision and inbreeding can occur in D. 
polymorpha, despite the apparent capacity of 
veligers for long-range dispersal. 

We asked whether heterozygosity-fitness 
relationships exist in zebra mussels as are 
found for many marine molluscs with similar 
demographics and genetics as D. polymor- 
pha. Previous studies have examined het- 
erozygosity-fitness relationships in North 
American populations of D. polymorpha with 
mixed results. Garton & Haag (1991) ob- 
served a significant positive relationship be- 
tween multi-locus heterozygosity and shell 
length for a zebra mussel population from Put- 
in-Bay, Lake Erie, Ohio. They also reported 
significant single-locus effects for phospho- 
glucose isomerase (PGI) and peptidase-2 
(PEP-2). Mass-specific oxygen consumption, 
however, was not significantly negatively cor- 
related with multi-locus heterozygosity for 
these mussels (Garton & Haag, 1991)-a rela- 
tionship hypothesized to account for faster 
growth rates of heterozygotes (reviewed in 
Mitton, 1997). Furthermore, Boileau & Hebert 
(1993) failed to detect any effect of allozyme 
heterozygosity on growth rates of zebra mus- 
sels within field enclosures. The question of 
whether heterozygosity is an important deter- 
minant of fitness in zebra mussels therefore 
remains unresolved. 

We used observational and experimental 
approaches to investigate whether allozyme 



heterozygosity was correlated with fitness 
surrogates in zebra mussels. First, we con- 
ducted a large-scale survey of 22 mussel pop- 
ulations from lakes and streams in the mid- 
western United States and tested for genetic 
relationships with shell length. Second, we 
conducted a one-year growth experiment in 
field enclosures to test for genetic correlates 
with growth rate. Our results indicate that al- 
lozyme heterozygosity is generally unrelated 
to shell length and growth rate in D. polymor- 
pha. We hypothesize that the occasional ob- 
servation of significant correlations is most 
likely due to a chance synergism of demo- 
graphic factors, rather than to natural selec- 
tion (i.e., overdominance or associative over- 
dominance). 



MATERIALS AND METHODS 
Surveys of Natural Populations 

To test for relationships between shell 
length and single and multi-locus genetic het- 
erozygosity, zebra mussels were analyzed 
from a total of 22 different sites in the Great 
Lakes region of the United States (Fig. 1), in- 
cluding both lakes and streams. Mussels 
were collected from within a 10 m 2 area of 
hard substrata at a depth of 0.5-1 .5 m at each 
site. Individuals were transported back to the 
laboratory on ice and then subjected to mor- 
phological and genetic analyses. The maxi- 
mum shell length of each mussel was mea- 
sured to the nearest 0.1 mm using Manostat 
6" dial calipers. Soft tissue was then removed 
from the mussels for electrophoretic analysis, 
and shells marked and catalogued for future 
reference. 

Growth Experiment 

To test for relationships between growth 
rate and single and multi-locus genetic het- 
erozygosity, zebra mussels were reared for a 
one-year period in field enclosures in Chris- 
tiana Lake and its outflow, Christiana Creek, 
in southwestern Michigan. Mussels were col- 
lected from connected Eagle Lake, Michigan 
(Fig. 1b), on June 17, 1994, and their shell 
lengths measured to the nearest millimeter. 
Individuals ranging in size from 12-15 mm 
were selected and individually marked with 
numbered plastic tags. Fifteen tagged mus- 
sels were placed into a plastic mesh bag 
(mesh size = 2 mm). Each bag was closed 



HETEROZYGOSITY AND FITNESS IN ZEBRA MUSSELS 



115 



(A) 



50 km 









02 



Lake Ontario 



i.<>3 



LOI 



Lake Michigan 



Mil 1-4* 



I.M3* 



• I. Ml 



• im: 



Lake Erie 



i 1.3 



I i ! 



LE2 




(В) 



1 km 



Eagle Lake 

KL3. * 



í : 1 , 2 



• ( L3 

<i> . Christiana Lake 

( I |V"< ! 

• CC2 

• ( C4 



Christiana Creek 



FIG. 1 . (A) Collection sites and sample sizes (n = #mussels genetically scored) within the Great Lakes. Lake 
Michigan samples were from Douglas, Michigan (LM1, n = 50), New Buffalo, Michigan (LM2, n = 49), 
Chicago, Illinois (LM3, n = 50) and Milwaukee, Wisconsin (MH1, n = 50; MH2, n = 50; MH3, n = 50; MH4, n 
= 50). Lake Erie samples were from Maumee, Ohio (LE1 ; n = 77), Huron, Ohio (LE2; n = 50), and Headlands 
Beach, Ohio (LE3; n = 48). Lake Ontario samples were from Sodus Point, New York (L01 ; n = 50), Cape Vin- 
cent, New York (L02; n = 50), and 30 Mile Point, New York (L03; n = 49). (B) Collection sites within the Eagle 
Lake Complex, Michigan. Zebra mussels were collected from 3 sites each in Eagle Lake (EL1 , n = 432; EL2, 
n = 142; EL3, n = 142), Christiana Lake (CL1, n = 127; CL2, n = 128; CL3, n = 124), and Christiana Creek 
(CC1, n = 128; CC2, n = 142; CC4, n = 138). Original Eagle Lake collecting site for mussels used in the 
growth bag experiment is indicated by the asterisk (*). 



116 



LEWIS ETAL. 



and put into the upper hole of a cement cinder 
block. Twenty-one blocks were then placed at 
five sites in Christiana Creek. These sites 
were located at the outflow of Christiana Lake 
into Christiana Creek and then 100 m, 400 m, 
4 km, and 10 km downstream. Each site re- 
ceived four blocks, except for the lake outflow 
site which received five. Four blocks were 
also placed within Christiana Lake near the 
outflow. We monitored the growth of 375 mus- 
sels during the experiment (25 bags x 15 
mussels per bag). All bags were collected 
from the lake and stream on June 17, 1995. 
Tagged mussels were recovered from the 
bags (n = 293) and remeasured prior to elec- 
trophoretic analysis. Yearly growth rate was 
calculated for each mussel by subtracting the 
initial length from the final length. Additional 
details concerning the experimental design 
are given in Horvath & Lamberti (1999). 

Genetic Techniques 

Soft tissue was removed from mussel shells 
and a portion was homogenized in approxi- 
mately 15 mL of grinding buffer for genetic 
analysis. Mussels from the Eagle Lake com- 
plex and a majority of the Great Lakes were 
scored for nine different enzyme systems 
using standard starch gel electrophoresis 
(Murphy et al., 1990). We resolved a total of 
13 different polymorphic loci for these nine 
systems. Five of these allozymes, ACON, 
IDH, MDH1, ODH, and PGI, were resolved 
using a Tris-citrate electrode buffer system, 
pH = 6.3. The remaining loci, EST1, EST2, 
ME, MDH2, and the peptidases (PEPG1, 
PEPG2, PEPG3, and PEPD), were resolved 
using a Tris-citrate electrode buffer system, 
pH = 8.0. Stain recipes were those of Murphy 
et al. (1990). Individuals from four sites col- 
lected from Lake Michigan near Milwaukee, 
Wisconsin, were scored for only a subset of 
five loci: ACON, IDH, MDH1 , ODH, and PGI. 

Data Analysis 

Single-locus relationships with shell length 
and growth rate were analyzed using non- 
parametric Spearman rank correlations (r s ), 
with mussels grouped into homozygote or het- 
erozygote classes depending upon whether 
they possessed the same or different alleles at 
the locus. This pooling procedure was the 
same as that used by Garton & Haag (1 991 ) in 
their earlier study of zebra mussels. In addi- 



tion, for those loci with more than two common 
alleles (allele frequency > 0.05), single locus 
comparisons for shell length were carried out 
for specific allele pairs, that is, heterozygotes 
for alleles 1 00/1 1 were compared to homozy- 
gotes for the 1 00 and 1 1 allele. Significance 
was evaluated using Spearman rank correla- 
tions. 

Relationships between multi-locus het- 
erozygosity and shell length or growth rate 
were evaluated using Spearman rank correla- 
tion, with the total number of heterozygous loci 
possessed by a mussel serving as the genetic 
score. Separate tests were performed for each 
of the 22 natural populations surveyed for both 
the single and multi-locus analyses, because 
shell length distributions were not identical 
among sites. Mussels from the growth bag ex- 
periments were grouped into four different 
size-classes based on their initial shell lengths 
(12, 13, 14, and 15 mm). This was necessary 
due to significant variation in growth rates 
among these size-classes. Correlation coeffi- 
cients were combined across sites or size- 
classes to estimate a common coefficient (r c = 
effect magnitude) by meta-analysis, using 
methods outlined by Hedges & Olkin (1985). 
Correlation coefficients were z-transformed 
into normal variâtes to test for significance. 
One-tailed tests were performed under the al- 
ternate hypothesis that heterozygotes should 
be significantly larger or grow faster than ho- 
mozygotes. Table-wide significance was de- 
termined using a sequential Bonferroni proce- 
dure (Holm, 1979; Rice, 1989). 



RESULTS 

Relationship Between Shell Length and Het- 
erozygosity in Natural Populations 

Shell length generally was not correlated 
with single-locus genetic heterozygosity 
within zebra mussel populations. The mean 
shell length of heterozygotes was significantly 
larger than that of homozygotes for 16 of 254 
single-locus tests conducted within sites 
(Table 1). However, none of these 16 tests 
was significant on a table-wide basis (a 
needed for 5% table-wide significance of at 
least one test = 0.05/254 = 0.0002). An addi- 
tional ten significant tests was obtained using 
specific pairwise allele comparisons (Table 2), 
but again none of these tests were significant 
on a table-wide basis (o needed for 5°o table- 
wide significance of at least one test = 



HETEROZYGOSITY AND FITNESS IN ZEBRA MUSSELS 



117 



TABLE 1. Significant Spearman rank correlations (r s ) between single locus heterozy- 
gosity and mussel shell length at sites. Significance was determined by z-transformed 
correlation coefficients under the one-tailed alternate hypothesis that heterozygotes 
have larger shell lengths than homozygotes. No single locus test was significant on a 
table-wide basis, as determined by a sequential Bonferroni procedure. N = sample size. 













Mean shell 


Mean shell 












length of 


length of het- 












homozygotes 


erozygotes 


Enzyme locus 


Site 


N 


r s 


P-value 


(mm) 


(mm) 


ACON 


LM3 


45 


0.299 


0.024 


11.05 


12.38 


ACON 


L02 


50 


0.367 


0.005 


13.15 


15.32 


EST1 


LE3 


48 


0.327 


0.013 


14.89 


18.49 


IDH 


CC1 


125 


0.160 


0.038 


13.96 


16.10 


IDH 


EL1 


427 


0.085 


0.040 


10.99 


11.87 


IDH 


LE1 


76 


0.241 


0.019 


13.30 


15.72 


MDH2 


LE1 


76 


0.238 


0.020 


13.74 


16.41 


MDH2 


LM2 


49 


0.268 


0.032 


14.57 


15.50 


ODH 


L03 


47 


0.259 


0.040 


15.37 


16.88 


PEPD 


CC1 


119 


0.193 


0.018 


14.11 


16.73 


PEPD 


CC2 


136 


0.177 


0.020 


13.46 


16.18 


PEPD 


LM3 


45 


0.343 


0.012 


10.41 


12.07 


PEPG1 


CL2 


126 


0.269 


0.002 


14.36 


18.85 


PGI 


CL3 


139 


0.145 


0.045 


18.74 


20.23 


PGI 


EL1 


427 


0.097 


0.023 


10.79 


11.62 


PGI 


LM3 


50 


0.282 


0.024 


10.54 


11.97 



TABLE 2. Significant Spearman rank correlations (r s ) between single locus heterozygosity and 
mussel shell length at sites by specific allele pair. Significance was determined by z-trans- 
formed correlation coefficients under the one-tailed alternate hypothesis that heterozygotes 
have larger shell lengths than homozygotes. No single locus test was significant on a table- 
wide basis, as determined by a sequential Bonferroni procedure. N = sample size. 















Mean shell 


Mean shell 














length of 


length of 


Enzyme 


Allele 










homozygotes 


heterozy- 


locus 


pair 


Site 


N 


r s 


P-value 


(mm) 


gotes (mm) 


ACON 


87/1 00 


CC2 


91 


0.187 


0.038 


14.95 


19.36 


PEPD 


80/100 


EL3 


27 


0.357 


0.034 


12.35 


16.58 


PEPG2 


100/113 


L02 


32 


0.392 


0.013 


13.31 


16.04 


PEPG3 


100/110 


CC2 


82 


0.266 


0.008 


14.84 


20.02 


PEPG3 


100/110 


L03 


24 


0.428 


0.018 


15.17 


19.63 


PEPG3 


110/114 


CL3 


9 


0.606 


0.043 


14.20 


20.75 


PGI 


146/172 


CC3 


23 


0.493 


0.008 


20.79 


24.58 


PGI 


172/196 


CL1 


9 


0.730 


0.012 


10.88 


24.70 


PGI 


172/196 


EL3 


19 


0.404 


0.043 


11.78 


17.32 


PGI 


172/196 


LE3 


12 


0.750 


0.002 


14.40 


21.58 



0.05/301 = 0.00016). Three allozymes (EST- 
1, IDH, and PEPD) displayed significant rela- 
tionships when correlation coefficients for the 
22 sites were combined into a single effect 
magnitude by meta-analysis (r c [EST-1] = 
0.051, P = 0.011, 1863 df; r c [IDH] = 0.049, P 
= 0.012, 2102 df; r c [PEPD] - 0.046, P = 
0.026, 1814 df). As before, none of the three 
results was significant on a table-wide basis 
(a needed for 5% table-wide significance = 
0.05/13 = 0.0038), and the variance in shell 



length explained by heterozygosity for each 
allozyme was small. 

Multi-locus heterozygosity also was only 
weakly related to shell length within popula- 
tions. Only one site (Eagle Lake # 1 ) out of the 
22 analyzed exhibited a significant positive 
correlation between overall heterozygosity 
and shell length (r s = 0.103, P = 0.035, 309 
df), but this correlation was not significant on 
a table-wide basis (a needed for 5% table- 
wide significance = 0.05/22 = 0.0023). Never- 



118 



LEWIS ETAL. 



TABLE 3. Significant Spearman rank correlations (r s ) between single locus heterozygosity and mussel 
growth rate (over one year) in the enclosure experiment. Individuals were grouped by initial length for 
analysis. Significance was determined by z-transformed correlation coefficients under the one-tailed 
alternate hypothesis that heterozygotes have larger shell lengths than homozygotes. No single locus 
test was significant on a table-wide basis, as determined by a sequential Bonferroni procedure. N = 
sample size. 













Mean growth of 


Mean growth of 




Initial length 








homozygotes 


heterozygotes 


Enzyme Locus 


(mm) 


N 


r s 


P-value 


(mm) 


(mm) 


EST1 


12 


10 


0.223 


0.026 


14.33 


15.49 


EST1 


13 


83 


0.192 


0.049 


13.48 


14.18 


IDH 


14 


62 


0.283 


0.014 


12.21 


13.87 


IDH 


15 


45 


0.318 


0.018 


12.13 


13.57 


ODH 


12 


10 


0.226 


0.017 


14.29 


15.58 


PEPG1 


13 


83 


0.212 


0.028 


13.38 


14.49 



theless, the effect magnitude for multi-locus 
heterozygosity calculated by meta-analysis 
for all 22 populations combined was signi- 
ficant (r c = 0.052, P = 0.019, 1557 df). How- 
ever, the variance in shell length explained by 
multi-locus heterozygosity was small (r c 2 = 
0.0027, 95% confidence interval = 0.0103 to 
0.000004), suggesting a very weak relation- 
ship. 

Relationship Between Growth Rate and 
Genetic Heterozygosity 

Of the original 375 mussels marked in the 
field enclosure experiment, 46 lost their tags 
and 36 died during the study, leaving 293 
mussels for which we measured allozymes 
and growth rate. The annual growth rate for 
mussels in enclosures was 13.6 ± 0.15 mm 
(x ± s.e.). Yearly growth rate did not differ sig- 
nificantly among sites (F 6 19 = 0.62, P = 0.71), 
and so the six sites were pooled for all subse- 
guent analyses. However, growth rate varied 
significantly among groups of mussels with 
different initial shell lengths (F 4290 = 8.21 , P = 
0.0001). Tests for genetic correlates with 
growth rate were therefore performed sepa- 
rately for mussels having initial shell lengths 
of 12, 13, 14, and 15 mm, and then combined 
by meta-analysis. 

Growth rate generally was not related to ge- 
netic heterozygosity. Heterozygotes dis- 
played significantly higher growth rates than 
homozygotes in six of 52 single-locus tests 
(Table 3). EST1 was significant in both the 12 
and 13 mm size-classes, and IDH in the 14 
and 15 mm classes (Table 3). None of these 
single-locus tests was significant on a table- 
wide basis (a needed for 5% table-wide sig- 
nificance = 0.05/52 = 0.00096). Multi-locus 



heterozygosity also was not significantly cor- 
related with growth rate for any initial size- 
class in the enclosure experiment (r [12 mm] 
= 0.043, P = 0.342, 93 df ; r [1 3 mm] = - 0.075, 
P = 0.745, 76 df; r [14 mm] = 0.094, P = 0.242, 
55 df; r [15 mm] = - 0.147, P = 0.821, 39 df). 
The effect magnitude for multi-locus heterozy- 
gosity across size-classes was not signifi- 
cantly different from (r c = - 0.008, P = 0.548, 
254 df). 



DISCUSSION 

We found only weak evidence, if any, for a 
relationship between allozyme heterozygosity 
and fitness surrogates in zebra mussels. Only 
one site (Eagle Lake, Michigan, site 1) out of 
22 populations tested displayed a significant 
correlation between multi-locus heterozygos- 
ity and shell length. However, this result was 
not significant when considered on a table- 
wide basis. The effect magnitude for multi- 
locus heterozygosity calculated for all 22 sites 
combined by meta-analysis was significant (r c 
= 0.052, P = 0.019, 1557 df). But multi-locus 
heterozygosity explained only a small fraction 
( 0.27%) of the total variation in shell length. 
Similarly, our field enclosure experiment pro- 
vided no convincing evidence for a relation- 
ship between heterozygosity and growth rate, 
consistent with the results of Boileau & Hebert 
(1993), but contrary to reports for many ma- 
rine molluscs (Britten, 1996). 

Garton & Haag (1991) found a significant 
multi-locus correlation with shell length for a 
zebra mussel population at Put-in-Bay, Lake 
Erie (r p = 0.265, P < 0.002 for 1 -tailed test, 1 1 9 
df). When combined with our 22 populations, 
the Put-in-Bay site is significant on a table- 



HETEROZYGOSITY AND FITNESS IN ZEBRA MUSSELS 



119 



wide basis (i.e., one-tailed P < 0.002 for Put- 
in-Bay result is less than the P = 0.0022 
needed for 5% table-wide significance). Gar- 
ton & Haag's (1991) result therefore cannot 
be dismissed simply as statistical artifact. In 
this regard, low repeatability of findings is a 
common problem plaguing heterozygosity 
studies for molluscs. It is not unusual for cer- 
tain populations of a species to display signif- 
icant genotype-fitness relationships, while 
others do not (Pogson & Fevolden, 1998). Re- 
sults can even vary when the same popula- 
tion is sampled over consecutive years 
(Gaffney, 1990; Pecon-Slattery et al., 1993; 
David & Jame, 1997). 

Several hypotheses have been advanced 
to explain why heterozygosity relationships 
are inconsistent within and among mollusc 
populations. One argument suggests that het- 
erozygosity-fitness relationships are primarily 
manifest during times of environmental stress 
(Samollow & Soule, 1983; Gentili & Beau- 
mont, 1988; Scott & Koehn, 1990; Borsa et 
al., 1992). A second hypothesis is that het- 
erozygosity relationships are age- or size- 
specific and, conseguently, can change de- 
pending upon whether certain classes are 
included or excluded in the analysis. For ex- 
ample, studies have shown that heterozygos- 
ity-growth rate relationships tend to dissipate 
with increasing age or the onset of sexual ma- 
turity (Diehl & Koehn, 1985; Rodhouse et al., 
1986). Finally, non-genetic factors such as 
heterogeneous settling times or physical loca- 
tion within clumps of mussels (Koehn & 
Gaffney, 1984) could also dampen allozyme 
correlations with body size or growth rate. 

None of the above hypotheses seems ade- 
quate to reconcile the difference between our 
results and those of Garton & Haag (1 991 ). All 
three of the above scenarios require that an 
underlying relationship exists between al- 
lozyme heterozygosity and fitness, with medi- 
ating factors influencing the magnitude of the 
relationship on a case-by-case basis. Our 
data suggest that only a weak relationship (if 
any) exists between heterozygosity and shell 
length or growth in zebra mussels. A causal 
relationship for Put-in-Bay mussels would 
therefore require that: (1) environmental con- 
ditions at Put-in-Bay were somehow uniquely 
stressful compared to other locations, (2) a 
critical size-class sampled at Put-in-Bay was 
missed at all other sites, (3) epigenetic noise 
present elsewhere, was absent at Put-in-Bay, 
or (4) unique loci scored at Put-in-Bay hap- 
pened to be under selection. 



In our study, mussels under the most envi- 
ronmental stress likely came from the two in- 
dustrial harbor sites (MH1 and MH2) at Mil- 
waukee, Wisconsin (Fig. 1), where water 
temperatures were well above (> 5°C) those 
at other sites (Lewis et al., 2000). Fetisov et 
al. (1992) reported that allozyme frequencies 
for D. polymorpha were related to water tem- 
perature in basins adjacent to the Chernobyl 
atomic power station. The Milwaukee popula- 
tions did not show a significant single or multi- 
locus correlation with shell length (multi-locus 
r c for sites MH1 and MH2 combined = 0.062, 
P = 0.291, 78 df). In addition, allele frequen- 
cies at sites MH1 and MH2 were not signifi- 
cantly different from those of other Lake 
Michigan populations (Lewis et al., 2000). Our 
population surveys included the entire range 
of genetically scorable mussels at sites 
(2.5-30 mm), spanning the size distribution 
analyzed at Put-in-Bay (5-21 mm). We there- 
fore did not miss a critical portion of the 
age/size distribution present in Garton & 
Haag's (1 991 ) study. Our survey also included 
three Lake Erie sites, although not Put-in-Bay. 
Given the overlap in size distributions, it 
would be odd if epigenetic factors present at 
all 22 of our sites were absent at Put-in-Bay. 
Finally, the seven allozymes analyzed by Gar- 
ton & Haag (1991) formed a subset of the 13 
we scored in the current study. 

Fluctuations in the breeding structure and 
recruitment pattern of local zebra mussel pop- 
ulations could explain the Put-in-Bay relation- 
ship. Under this scenario, population demog- 
raphy would enhance a normally weak or 
non-existent correlation between heterozy- 
gosity and shell length. For example, environ- 
mental conditions for an initially large, pan- 
mictic population may sometimes change and 
limit mating to a relatively few number of indi- 
viduals, subdivided into local demes. If most 
juveniles settle and breed in their natal 
demes, then a positive correlation could be 
produced between multi-locus heterozygosity 
and shell length across the metapopulation, 
even if heterozygotes do not grow faster or 
survive better than homozygotes. Of course, 
this correlation would be ephemeral. Once the 
metapopulation became panmictic again the 
relationship would disappear-or could even 
reverse -when subsequent cohorts settled. 

Our hypothesis presumes that zebra mus- 
sels have a dynamic population structure. 
This seems inconsistent with the biology of an 
organism that has large population numbers, 
mass external fertilization, and a lengthy, 



120 



LEWIS ETAL. 



planktonic larval phase (Ludyanskiy et al., 
1993). However, genetic studies of D. poly- 
morpha have revealed significant allozyme 
frequency heterogeneity among local popula- 
tions within lakes (Boileau & Hebert, 1993; 
Lewis et al., 2000). Genetic heterogeneity has 
even been detected among samples collected 
from within a 10 m 2 area at one site (Lewis 
et al., 2000). Zebra mussel larvae can have 
different PGI genotype frequencies than 
parental populations (Haag & Garton, 1995), 
similar to the case for mtDNA haplotypes 
in Pacific oysters (Li & Hedgecock, 1998). 
Furthermore, heterozygote deficiencies from 
Hardy-Weinberg equilibrium are common in 
adult zebra mussel populations (Lewis et 
al., 2000), similar to other molluscs (David 
et al., 1997). These results suggest that 
fine-scale population subdivision, inbreeding, 
and uneven mixing of larval cohorts occur in 
zebra mussels, despite their capacity for long- 
range dispersal as veligers. Furthermore, 
Garton & Haag (1993) have reported yearly 
variation in spawning synchrony in zebra 
mussel populations, suggesting that neigh- 
borhood sizes can change over time. All of the 
ingredients necessary for a fluctuating de- 
mography to generate heterozygosity-shell 
length correlations are therefore present in D. 
polymorpha. 

In conclusion, we saw no compelling evi- 
dence for a heterozygosity-fitness relation- 
ship in 22 populations of D. polymorpha. At 
most, multi-locus heterozygosity was only 
very weakly correlated with shell length (r c [22 
sites] = 0.052). We propose that the popula- 
tion dynamics of zebra mussels may dictate 
the strength of the relationship rather than 
natural selection per se. If our hypothesis is 
correct, then the sequence of demographic 
events necessary to produce a robust and 
positive heterozygosity-shell length correla- 
tion will likely happen infrequently in D. poly- 
morpha. The phenomenon we describe may 
also contribute to the inconsistency of het- 
erozygosity-fitness correlations observed for 
many marine molluscs. 



ACKNOWLEDGMENTS 

We thank the following people who assisted 
in the completion of this project. Gerry 
Bouchard, Brian Bouchard, Bill Perry, Steve 
Beaty, Melanie Vile, Bill Webb and Eileen 
Smith aided in various mussel collections. 
Randy Owens provided mussels from 30 Mile 



Point. Marty Berg provided mussels from 
Chicago, Illinois. Jerry Castor and Art Brooks 
at the University of Wisconsin-Milwaukee 
helped arrange the Milwaukee Harbor survey. 
Becky Bruckert and Sheila Meehan assisted 
with processing and electrophoresis of mus- 
sels. This research was funded through a co- 
operative agreement with the U.S. Environ- 
mental Protection Agency as part of the EPA 
Introduced Species Research Program (CR 
820290-02). 



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KOEHN, R. K.. W. J. DIEHL & T. M. SCOTT, 1988, 
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122 



LEWIS ETAL. 



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Revised ms. accepted 10 January 2000 



MALACOLOGIA, 2000, 42(1-2): 123-129 

A NEW DEEP-SEA SPECIES OF THE GENUS NEOLEPTON (BIVALVIA; 
CYAMIOIDEA; NEOLEPTONIDAE) FROM THE ARGENTINE BASIN 

J.A.Allen 
University Marine Biological Station, Millport, Isle of Cumbrae, Scotland, KA28 OEG 1 

ABSTRACT 

A new species of Neolepton is described from mid to lower slope depths of the Argentine 
Basin. This, the first deep-water species of the Neoleptonidae to be described, differs little from 
the shallow-water species of the genus. It has features in common with species of the family Kel- 
liellidae, but it is concluded that these reflect convergence due to similar life styles, rather than a 
particularly close relationship. Neolepton also has features in common with the venerid bivalves, 
and as has been suggested by other authors, it is possible that this is an example of paedomor- 
phism. 

Key Words: Bivalvia, Neoleptonidae, deep-sea 



INTRODUCTION 

In studies of the deep-sea bivalves of 
the Atlantic, a small, white, ovate species, 
with a heart-shaped, lunule, was provisionally 
placed in the family Kelliellidae (Allen, in ms). 
When specimens were opened and the hinge 
and internal morphology examined, it was 
found that the species belonged to the Ne- 
oleptonidae and as such is the first recorded 
deep-sea species of the family. 

This new species was taken from the Ar- 
gentine Basin as part of studies of the abyssal 
fauna of the Atlantic led by Dr. Howard 
Sanders of the Woods Hole Océanographie 
Institution. It was taken with an epibenthic 
sledge, preserved first in 4% formal saline, 
and then transferred to 95% ethanol. The in- 
ternal morphology was studied using whole 
mounts lightly stained in Erlich's haemo- 
toxylin. 



SYSTEMATICS 

Although Neolepton sulcatulum (Jeffreys, 
1859), type species (Crosse, 1885) of the 
genus Neolepton Monterosato, 1875 (type 
genus of the family Neoleptonidae Thiele, 
1935), is European, most of the described 
species of Neolepton are found south of the 
equator (Soot-Ryen, 1960; Ponder, 1969; 
Boss, 1982; Salas & Gofas, 1998), although 



Salas & Gofas (1998) concluded that Halo- 
draka from the eastern Pacific and north of 
the equator that had been placed in the 
Bemardinidae (Coan, 1984) is synonymous 
with Neolepton. Until now the described 
species have all been found in the intertidal or 
shelf seas. 

The species described here has two poste- 
rior mantle apertures, and prior to the work of 
Salas & Gofas (1998) this fact would have 
placed it in the genus Notolepton Finlay, 1927 
(Ponder 1 969). Salas & Gofas (1 998) showed 
that Neolepton sulcatulum, which, before their 
elegant research, had been thought to have 
only one posterior mantle aperture, has two, 
and thus they synonymized the two genera. 

Genus Neolepton Monterosato, 1875 

Type species: Lepton sulcatulum Jeffreys, 
1859, subsequent designation by Crosse 
(1885). 

For further details of the taxonomy and diag- 
nosis of the genus, see Ponder (1969) 
and Salas & Gofas (1998). 

Neolepton profundorum, new species. 

Type Locality: Argentine Basin, 36°53.4'S, 
53°10.2'W, 2,323 m. 

Type Material: Holotype, The Natural History 
Museum, London, 1998186; 3 paratypes, 



1 Address for correspondence; also Woods Hole Océanographie Institution. Massachusetts 02543, USA. 

123 



124 



ALLEN 



Museum of Comparative Zoology, Harvard 
University, Cambridge, Massachusetts, 
318093. 

Material: Atlantis II, sta.237, 36°32.6'S, 
53°23.0'W, 1,011 m, 173 spec; sta. 240, 
36°53.4'S, 53°10.2'W, 2,323 m, 8 spec. 

Although station 237 yielded many more 
specimens, they had been much eroded 
through poor preservation and thus station 
240 with the smaller number of specimens, 
but in good condition, was chosen for type 
material and type locality. 

Distribution: Found at lower slope depths in 
the Argentine Basin. Depth range: 1,011- 
2,323 m. 

Shell Description (Figs. 1 -3): The shell is 
very small (largest specimen 2.5 mm total 
length), somewhat elongate, becoming more 
so with increasing size (length/height ratio 
0.81-0.75; length/width ratio 0.49-0.50); it is 
semitransluscent, white, with very fine, faint, 
irregular concentric lines and very faint dis- 
continuous radial plicae. The umbo is not very 
prominant and is directed slightly anterior and 
inturned. The lunule is elongate, lozenge- 
shaped, and the margin is well defined by a 
fine line. There is no escutcheon. The antero- 
ventral and ventral shell margins are smoothly 
curved, the postero-ventral and posterior mar- 
gins are broadly concave, and the postero- 
dorsal margin is straight, angled downwards 
to meet posterior margin posterior to adductor 
in a rounded angle. The antero-dorsal lunular 
margin is more or less straight. The ligament 
has internal and external components; the ex- 
ternal, which is formed from fusion layer, is 
elongate, the posterior part being twice as 
long as the anterior. It is attached to dorsal 
edge of hinge plate with little visible externally. 
The internal ligament (resilium) forms an 
arched wedge ventral to umbo that extends to 
the ventral edge of hinge plate. It is formed by 
inner and outer layers with no obvious miner- 
alization. The hinge plate is moderately 
broad. In the right valve, cardinal (1a) forms a 
broad triangular shelf at the ventral edge of 
the plate anterior to umbo. A slender ridge 
(3a) dorsal and parallel to cardinal 1a lies 
close to the dorsal margin of plate. In larger 
specimens, this ridge may end anteriorly as 
an incurved scroll. Immediately anterior to the 
resilium, a small cardinal (3b) extends verti- 
cally across hinge plate. Posterior to the resil- 
ium, a slender posterior lateral tooth (PI) 
forms a diagonal ridge posterior to resilifer. 



List of Abbreviations in Figures 



AA 


anterior adductor muscle 


AL 


anterior outer ligament (fusion layer) 


AN 


anus 


AR 


anterior pedal retractor 


CG 


cerebral ganglion 


DG 


digestive diverticula 


DL 


dorsal lip 


DP 


dorsal palp 


EA 


exhalant aperture 


EG 


epithelial glands 


ES 


exhalant siphon 


FM 


first gill filament 


FT 


foot 


GA 


gill axis 


GE 


attachment of ascending lamella of inner 




demibranch 


HG 


hindgut 


IA 


inhalant aperture 


ID 


inner demibranch 


IL 


inner layer 


KD 


kidney 


LV 


left valve 


MO 


mouth 


OD 


outer demibranch 


OE 


oesophagus 


OL 


outer layer 


OG 


proximal oral groove 


OV 


ovary 


PA 


posterior adductor muscle 


PG 


pedal ganglion 


PL 


posterior outer ligament (fusion layer) 


PR 


posterior pedal retractor muscle 


RS 


resilium 


RV 


right valve 


ST 


stomach 


VG 


visceral ganglion 


VP 


ventral palp 


VT 


ventricle 


1a 


right anterior cardinal 


2a 


second left anterior cardinal 


2b 


first left anterior cardinal 


3a 


first right anterior cardinal 


3b 


second right anterior cardinal 


4b 


possible rudimentary left posterior cardinal 


PI 


right anterior lateral 


PH 


left posterior lateral 



The hinge plate merges with antero-dorsal 
and postero-dorsal shell margins. The left 
hinge plate has inverted spoon-shaped ante- 
rior cardinals (2a and 2b), which interlock dor- 
sally with cardinal (1a). Anterior to cardinal 
(2a) and ventral to the lunule, the plate is rel- 
atively broad and ventrally raised. In some 
specimens, immediately posterior to the resil- 
ifer, there is a small incipient ridge aligned 
with posterior edge of the resilifer and possi- 
bly equivalent to cardinal (4b). A broad, elon- 
gate posterior lateral tooth (Pll) lies close to 
and slightly oblique to the dorsal margin. The 
prodissoconch (length 364 ¡.im) is almost cir- 
cular, with a well-marked peripheral rim. 



A NEW SPECIES OF NEOLEPTON 



125 




ANT 



ч 



FIG. 1. Dorsal and left lateral views of paratype specimens from the type locality showing variation in shell 
outline (scale = 1 .0 mm), and an enlarged dorsal view of a prodissoconch showing the neoleptonid outer rim 
(scale = 0.2 mm). 



There is some variation in the outline of the 
shell (Fig. 1) and more particularly in the 
hinge plates, in the latter (Fig. 2), there is vari- 
ation in the size and shape of the cardinal 
teeth, although the basic form remains the 
same. The fine raised posterior edge of the 
resilifer of the left hinge plate present in some 
specimens, may be homologous with the car- 
dinal 4b of veneraceans (see Discussion). 

Internal Morphology (Figs. 4-6): The mantle 
edge is very broad and particularly so in the 
region of the posterior mantle apertures. As in 
other neoleptonids (Salas & Gofas, 1998), it 
has four folds, the additional fourth fold being 
formed as a subdivision of the middle sensory 
fold. Internal to the inner fold, extending from 



the ventral margin of the anterior adductor 
muscle to the junction of the gill axis with the 
mantle, there is a broad band of radial palliai 
muscles overlain by a very glandular inner 
mantle epithelium. The latter extends from the 
anterior adductor to the posterior limit of the 
pedal gape. The gland cells close to the in- 
halent aperture, at the point where pseudo- 
faeces might be expected to collect before ex- 
pulsion, appear to be larger and more diffuse 
than those more anterior. 

The anterior adductor muscle is large and 
elongate. The posterior adductor muscle is 
somewhat larger and much more rounded in 
cross-section. Fusion of the inner mantle folds 
separates both the inhalant aperture from the 
pedal gape and the short exhalant siphon. 



126 



ALLEN 




RV 



LV 




« 2a 




RV 



LV 



FIG. 2. Lateral views of the left and right hinge plates of three specimens from the type locality to show indi- 
vidual differences. For list of abbreviations, see p. 124. Scale = 0.5 mm. 



A NEW SPECIES OF NEOLEPTON 



127 




H 



h 



FIG. 3. Ventral view of a ligament of a specimen 
from the type locality. For list of abbreviations, see 
p. 124. Scale = 0.5 mm. 



The latter in the preserved specimens lies 
fliped within the suprabranchial chamber. The 
inhalant aperture is surrounded by numerous 
short sensory tentacles developed from the 
inner of the two sensory folds. The number 
varies from specimen to specimen, with 9-12 
on each side, and the tentacles are arranged 
in two rings (Fig. 5). Those of the inner ring 
are slightly more slender than those of the 
outer and tend to point inwards. The base of 
the exhalant apertures is surrounded by a 
similar number of small, circular papillae in a 
single ring. These papillae stain much more 
darkly in haematoxylin than do the inhalant 
tentacles. 

The gills comprise both demibranchs, 
which have non-plicate, homorhabdic fila- 
ments. There are no interlamellar connectives 
and few interfilamentar connectives. The lat- 
ter are present at the free margins, and in the 
case of the inner demibranch there are two or 
three rows of interfilamentar connectives be- 
tween margin and axis. The outer demibranch 
is shorter than the inner, only extending along 
the posterior half of the gill axis and reflected 
dorsally to cover the kidney and heart. 

The foot is a broad blade, laterally com- 
pressed and with a well-defined heel. No 
byssus was observed. The palps are small 
(Fig. 6). The anterior (dorsal) are finger-like 
processes at the outer limits of the lips, each 
with three internal ridges. The posterior (ven- 
tral) palps form small, semicircular, lateral 
pads and also have three internal ridges. The 
gut is simple. The oesophagus opens into a 
relatively voluminous stomach. The style sac 
and midgut are combined. The hind gut 
passes posterior to the stomach to the mid- 
dorsal margin, where it turns posteriorly to 
pass through the ventricle of the heart and 



dorsal to the kidney and posterior adductor 
muscle to the anus. Digestive diverticula lie, 
for the most part, lateral and anterior to the 
stomach. 

The sexes are separate. The gonads lie 
posterior to the stomach. A mature female ap- 
proximately 2.5 mm total length had 12-14 
large eggs. The largest eggs measured 200 
urn longest dimension. No brooding larvae 
were observed within the mantle cavity. 

Neolepton profundorum most closely re- 
sembles N. antipodum in shell and body mor- 
phology, but differing in shell outline, with N. 
profundorum being the slightly more elongate 
and with the concentric sculpture very much 
less obvious (Ponder, 1969). Neolepton pro- 
fundorum possesses a protoconch with a 
clearly marked, smooth marginal rim, similar 
to that described by Salas & Gofas (1998). It 
also possesses a lunule defined by a fine line 
that is reminiscent of the kelliellids. 



DISCUSSION 

Although the concentric ridging is much less 
marked, the shell of N. profundorum, including 
the prodissoconch with its smooth marginal 
rim, is similar to that of other species described 
by Ponder (1969) and Salas & Gofas (1998). 
This also applies to the body morphology. 
Judging by the size of the foot N. profundorum, 
like N. antipodum, can be highly mobile. The 
latter species can fix itself to the substratum by 
mucus and extend the mantle margins some- 
what beyond the shell margins (Ponder, 
1 969). Whether N. profundorum has the same 
habit is unknown, but from the similarity in mor- 
phology, it might be expected. Both species 
have extensive mucus glands in the mantle in- 
ternal to the inner mantle folds. While other 
functions have been suggested, for example, 
Soot-Ryen (1960) found eggs adhering to the 
glandular epithelium, Ponder (1968) thought 
that they, like other bivalve mantle glands, 
were associated with the transport of particles 
and the formation of pseudofaeces. It is likely 
that this is the case for the posterior gland cells 
in N. profundorum; unfortunately, there is no 
evidence from the present samples to say 
whether the anterior mantle glands are asso- 
ciated with egg brooding. Neolepton antipo- 
dum occurs, mucus bound, among the thalli of 
coralline algae and also in soft sediments, 
where it lies partially buried at the surface ei- 



128 




FIG. 4. Lateral semidiagrammatic view of the internal anatomy from the left side of a specimen from the type 
locality with the shell removed. For list of abbreviations, see p. 124. Scale = 0.5mm. 



ther obliquely upside down or vertically (Pon- 
der, 1969). Again, it seems likely that N. pro- 
fundorum may have the same habit in soft sed- 
iment. 

Although they belong to separate super- 
families (Cyamioidea and Glossoidea), the 
shell and internal morphology of N. abysso- 
rum shares similarities with species of the 
family Kelliellidae. These include the form of 
the gill and palps, and the structure of the pos- 
terior apertures and their associated sensory 
papillae. In addition, the general shape of the 
shell and the form of the lunule could well be 
mistaken for that of a kelliellid. Points of major 
difference include the hinge teeth and liga- 
ment and the form of the foot. The similarities 
are likely to be a matter of convergence, with 
Kelliella and Neolepton probably having simi- 
lar life styles. 

Salas & Gofas (1998) debated the relation- 



ship of the neoleptonids with the Veneridae 
and came to the conclusion that there were 
similarities between the hinge teeth and the 
internal anatomies of the two. The signifi- 
cance of these is debateable. Salas & Gofas 
(1998) pointed out that differences in the 
hinge include the lack of cardinal 4b and pos- 
terior lateral teeth in Neolepton as compared 
with venerids. To these differences must be 
added the great differences in the size of the 
palps and the fact that the outer demibranchs 
of the gill in the venerids is not reflected. 
These latter might well be a reflection of dif- 
ference in size of the adults. In the case of the 
hinge of some specimens of N. profundorum, 
there is what appears to be a tiny cardinal 4b 
situated close to the margin of the resilium, 
and in some of the photographs in the paper 
by Salas & Gofas (1998) (e.g., N. cancella- 
tum. p. 46, fig. 28), there is an indication of a 



A NEW SPECIES OF NEOLEPTON 



129 



•• 




FIG. 5. Diagram of the arrangement of tentacles 
around the inhalant and exhalant apertures. 




)- 



similar structure. It is possible that the occur- 
rence of cardinal (4b) and the well-developed 
posterior lateral tooth of the venerids is but a 
reflection of the structural need of the stouter 
shell, and as such Neolepton may well be a 
paedomorphic veneracean as Salas & Gofas 
(1998) suggest. 



LITERATURE CITED 

BOSS, K. J., 1982, Mollusca. Pp. 945-1166, in: S. 
p. Parker, ed., Synopsis and classification of liv- 
ing organisms, 1. McGraw-Hill, New York. 

COAN, E., 1984, The Bemardinidae of the eastern 
Pacific (Mollusca: Bivalvia). Veliger, 27: 227- 
237. 

CROSSE, H., 1885, [Review of] Nomenclatura 
genérica e specifica di alcune Conchiglie 
Méditerranée, pel márchese di Monterosato. 
Journal de Conchyliologie, 33: 139-143 

FINDLAY, H. J., 1927, A further commentary on 
New Zealand molluscan systematics. Transac- 
tions of the New Zealand Institute, 57: 320-485. 

JEFFREYS, J. G., 1859, Further gleanings in 
British conchology. Annals and Magazine of Nat- 
ural History, (3)3:30-43. 

MONTEROSATO, T. A. di, 1875, Nuova revista 
delle conchiglie Méditerranée. Atti delta Accade- 
mia di Scienze, Lettere ed Arti di Palermo, (n.s) 
13: 1-50. 

PONDER, W. F, 1969, Notes on two neoleptonid 
bivalve molluscs. New Zealand Journal of Marine 
and Freshwater Research, 3: 262-272. 

SALAS, С. & GOFAS, S., 1998, Description of four 
new species of Neolepton Monterosato, 1875 
(Mollusca: Bivalvia: Neoleptonidae), with com- 
ments on the genus and on its affinity with the 
Veneracea. Ophelia, 48, 35-70. 

SOOT-RYEN, T, 1960, Pelecypods from Tristan da 
Cunha. Results of the Norwegian Scientific Ex- 
pedition to Tristan da Cuhna 1937- 1938, 49: 47 

PP- 
THIELE, J., 1935, Handbuch der systematischen 
Weichtierkunde, 2. Gustav Fischer, Jena. 



FIG. 6. Internal view of the region of the mouth of a 
specimen from the type locality. For list of abbrevia- 
tions, see p. 124. Scale = 0.5 mm. 



Revised ms. accepted 28 October 1999 



MALACOLOGIA, 2000, 42(1-2): 131-148 

EARLY NEUROGENESIS PATTERN IN PATELLA COERULEA 
(PATELLOGASTROPODA) AND ITS POSSIBLE PHYLOGENETIC IMPLICATIONS 

Margherita Raineri 

Department of Experimental, Environmental and Applied Biology, (DIBISAA), University of 
Genoa, V. le Benedetto XV, 5, 16132 Genova, ITALY; tgfgra@unige.it 



ABSTRACT 

In Patella coerulea (Mollusca: Gastropoda), as shown by acetylcholinesterase (AChE) histo- 
chemistry, the primordia of the cerebral ganglia started to differentiate in the young trochophore. 
Approximately at the same stage, two bilaterally symmetrical neural cells, each with an axon-like 
fibre running parallel to the midline towards the anterior, were detected in the perspective foot 
near the posterior pole. Soon later, other ectodermal cells in their vicinity and running parallel to 
their fibres, became AChE-positive, and then, gave origin, respectively, to the primordia of the 
pedal ganglia, and to the rudiments of the visceral, intestinal and pleural ganglia. 

As shown by previous studies, in a bivalve, Mytilus, and in a polychaete, just as in Patella, the 
initial primordia of the post-trochal nerve cords differentiated parallel to the fibres of two poste- 
rior pioneer neural cells. In the gastropod and in the bivalve, these cells were located in the rudi- 
ment of the foot, which was shifted towards the oral side during ontogeny (anopedial flexure). 
However, in Patella this occurred at an earlier stage than in Mytilus; then, the primordia of the 
pedal ganglia started to differentiate in an anterior position, closer to those of the cerebral and 
pleural ganglia. This may be related to the development of cerebro-pedal and pleuro-pedal con- 
nectives, which are lacking in Mytilus and other bivalves. 

The present findings support an initial differentiation of pre- and post-trochal nervous system 
from bilaterally symmetrical neurogenesis centres located near the opposite poles of the tro- 
chophore. They may also suggest an origin of molluscs from the same ancestor as polychaetes, 
and a role of the anopedial flexure in their evolutionary radiation. 

Key words: acetylcholinesterase histochemistry, posttrochal nervous system, anopedial flex- 
ure, molluscs, polychaetes. 



INTRODUCTION 

In recent times, different hypotheses on the 
origin of molluscs have been elaborated on 
morphological grounds. In the opinion of 
some authors (e.g., Willmer, 1990), molluscs 
should be linked to acoelomate spiralians, 
since they share a number of characters, in- 
cluding tetraneury, with platyhelminthes. Al- 
ternatively, they could be closer related to 
polychaetes (e.g., Eernisse et al., 1992), 
sipunculans (Scheltema, 1993, 1996), or en- 
toprocts (Haszprunar, 1996). 

A phylogenetic relationship between mol- 
luscs and polychaetes is supported by embry- 
ological and larval similarities (Dawydoff, 
1928; Beklemishev, 1969; van den Biggelaar 
et al., 1997). Moreover, in a bivalve, Mytilus 
(Raineri & Ospovat, 1994), just as in a poly- 
chaete, Platynereis (Dorresteijn et al., 1993), 
the primordia of the post-trochal nerve cords 
differentiated parallel to the fibres of two pos- 
terior pioneer neural cells. In the bivalve, 



these cells were located in the rudiment of the 
foot. As it was shifted towards the oral side 
during ontogeny (anopedial flexure; Raven, 
1966; Verdonk & van den Biggelaar, 1983), 
the post-trochal nerve cords, unlike in the 
polychaete, were bent while differentiating. 
This may suggest an origin of molluscs from 
the same ancestor as polychaetes through a 
dorso-ventral developmental bending of the 
body rudiment (Raineri, 1995). In view of a 
comparable flexure that occurs in the brachio- 
pod Crania during ontogeny (Nielsen, 1991), 
the fossil record may indicate a similar origin 
of brachiopods (Conway Morris & Peel, 1995; 
Conway Morris, 1998). This possibility may be 
controversial, as other data contradict a close 
relationship with spiralians (e.g. Willmer, 
1990; Nielsen, 1995). However, analyses of 
gene sequences have firmly grouped mol- 
luscs as well as brachiopods within the coelo- 
mate spiralians (lophotrochozoans; Halanych 
et al., 1995; Mackey et al., 1996; Aguinaldo et 
al., 1997; de Rosa et al., 1999). 



131 



132 



RAINERI 



Broader biomolecular and embryological 
data sets may help to elucidate the affinities 
between these different taxa. For instance, 
making reference to Mytilus and Crania, as 
mentioned above, one could ask whether 
their bending, which has such significant ef- 
fects on morphogenesis, is a basic ancestral 
feature, or an independently evolved sec- 
ondary adaptation to sessile life, as probably 
are the reduction of the cerebral ganglia and 
of the whole head in the brachiopod as well as 
in the bivalve. In the latter, in turn, the "from- 
backwards-to-forwards" post-trochal neuro- 
genesis may be ascribed to the diminished 
importance of these anterior organizing cen- 
tres, since it is generally believed that the 
cerebral ganglia are the first to develop, then, 
neurogenesis should proceed from anterior to 
posterior. If so, the absence of tetraneury in 
Mytilus could be secondarily derived. 

However, free-swimming polychaetes show 
an initial differentiation of the post-trochal ner- 
vous system in a posterior position (Dor- 
resteijn et al., 1993), as well as a dorso-ven- 
tral developmental bending of the body 
rudiment, though, less pronounced than the 
anopedial flexure (Beklemishev, 1969). 

A comparison with the ontogeny of creeping 
molluscs could shed some light on this prob- 
lem. Aplacophorans and polyplacophorans 
may be good candidates, but only occasion- 
ally a few specimens are available to our lab- 
oratory. On the other hand, a better knowl- 
edge of early neurogenesis in gastropods 
would be important, as they appear to be 
more closely related to the bivalves (Run- 
negar, 1996; Kim et al., 1996). The present 
light microscopic study uses histochemically 
detected acetylcholinesterase (AChE) activity 
as a marker of neural cells (Dorresteijn et al., 
1993; Raineri, 1995) to investigate the initial 
phases of neurogenesis in the archaeogas- 
tropod Patella coerulea L, which is believed 
to be a rather primitive gastropod (van den 
Biggelaar, 1996). Since acetylcholine as well 
as biogenic monoamines are often implicated 
in the initial differentiation of neurogenic cells 
(Buznikov et al., 1996), the distribution of 
AChE positivity in embryos and young tro- 
chophores was compared to that of serotonin- 
like immunoreactivity. 

MATERIALS AND METHODS 

Specimens of Patella coerulea were col- 
lected on rocky shores in the Ligurian Gulf 



from February to June. Mature gonads were 
dissected out. Eggs were washed into Petri 
dishes and kept for 30 min at 22-25°C in fil- 
tered pasteurized seawater, alkalinized to pH 
8.6 by adding a few drops of KOH or NaOH 
0.5M (Wilson, 1904b), before artificial fertil- 
ization. 

Undiluted sperm from 3-4 different males 
were mixed and added to the eggs, 200-300 
ul for 50 ml of seawater. After the extrusion of 
the polar bodies, the fertilized eggs were 
washed thoroughly with filtered, pasteurized 
seawater and transferred into fingerbowls. 

The alkalinizing pre-treatment of eggs was 
routinely used, since when it was omitted they 
could not be fertilized successfully to a signif- 
icant percentage. It led, however, to many ab- 
normal embryos and larvae, which often 
showed a fairly normal trochal, yet, a com- 
pletely disorganized posttrochal pattern, but 
not the opposite. 

Histochemical Procedure 

Normally developing embryos, young tro- 
chophores 18-20 h after extrusion of the 
polar bodies, and more advanced larvae up to 
80 h old, were fixed at 4°C for 15-30 min in 
1.5% glutaraldehyde in 0.1 M Na cacodylate 
buffer, pH 7.0, containing 2mM histochemical 
substrate. Then, they were incubated in toto 
for 6-8 h at room temperature, or 14-18 h at 
4°C, in the histochemical medium of Kar- 
novsky & Roots (1964) to localize sites of 
Cholinesterase activities. 

The iodides of acetyl-ß-methylthiocholine 
(AcMThCh), or propionylthiocholine (PrThCh) 
and butyrylthiocholine (BuThCh), purchased 
from the Sigma Chemical Co., U.S.A., were 
used as substrates for acetylcholinesterase 
(AChE), or pseudoCholinesterase (BuChE) 
activities, respectively. 

Cholinesterase inhibitors, eserine and di- 
isopropylfluorophosphate (DFP) for AChE, 
and tetraisopropylpyrophosphoramide (iso- 
OMPA) for BuChE activity, were added at 
10~ 5 M final concentration to the histochemical 
media of controls, previously fixed without the 
substrate. 

After staining, some specimens were 
mounted in toto in Eukitt (Kindler GmbH & 
Co.), others were embedded in methacrylate 
(JB-4, Polysciences) and serial sections were 
made at 2 or 5 urn thickness. Some sections 
were post-stained with haematoxylin or tolui- 
dine blue. 



NEUROGENESIS IN A GASTROPOD 



133 



Immunocytochemical Procedure 

Advanced gastrulae and young tro- 
chophores 1 8-20 h old were fixed for 2-4 h in 
4% paraformaldehyde in 0.1 M phosphate 
buffer, pH 7.2, washed thoroughly in phos- 
phate buffered saline (PBS) 0.2 M, pH 7.4, de- 
hydrated and embedded in Paraplast (Sher- 
wood Medical, U.S.A.). Serial sections were 
made at 4 urn thickness. Serotonin-like im- 
munoreactivity was detected by the indirect 
immunoperoxidase method. Dewaxed sec- 
tions were incubated overnight at 4°C in a 
moist chamber with the serotonin antiserum 
(from a rabbit) that was diluted 1 :500 in PBS. 
Then, sections were washed in PBS and in- 
cubated for 1 h at room temperature in the 
second antiserum (from a sheep) that was di- 
luted 1:100 in PBS. Peroxidase activity was 
revealed by treating the sections for 15-20 
min with a solution of 3,3'diaminobenzidine 
tetrahydrochloride (DAB), 50 mg in 100 ml 
PBS, containing 33 ul 30% H 2 2 . Finally, sec- 
tions were dehydrated and mounted in Eukitt. 

Controls were made by substitution of the 
primary antiserum with either nonimmune 
rabbit serum or PBS. The antibodies were 
purchased from the Immuno Nuclear Corpo- 
ration, U.S.A. 



RESULTS 

AcMThCh and PrThCh gave comparable 
localizations in developing nerve and muscle 
cells, as well as in ciliated cells, like those of 
the prototroch. Moreover, histochemical stain- 
ing that was stronger with PrThCh as a sub- 
strate was found transiently in the mesoder- 
mal (Figs. 7, 17, 19) and endodermal cells, in 
the shell gland and the stomodaeum (Fig. 29). 
No significant enzyme reactions were ob- 
served with BuThCh. Eserine and DFP almost 
completely inhibited the histochemical stain- 
ing, while iso-OMPA had no visible effect. 



The same cell region also showed serotonin- 
like immunoreactivity. 

Young Trochophore 

In young trochophores 18-20 h old, the dif- 
ferentiating apical organ showed a moderate 
AChE positivity that was more evident in the 
medial cells, seemingly two, which gave origin 
to the cilia of the apical tuft (Fig. 2). In tro- 
chophores 24-28 h old, this medial area in- 
cluded prismatic cells with centrally located 
nuclei and flask-shaped cells with basal nuclei 
and stronger AChE activity which supported 
the cilia of the apical tuft (Figs. 3, 4). The un- 
derlying basal membrane was in contact with 
intensely AChE-positive fibres, which ran 
across the pretrochal region from two cells lo- 
cated just above the mouth (Fig. 5). On the 
sides of the apical organ, two cells covered by 
a jelly-like coat ("refractive bodies", Smith, 
1935; Figs. 3-5) showed hair-like processes 
shorter than the apical tuft. Basally, they were 
in contact with a branched AChE-positive cell 
of the pretrochal area that was connected with 
the prototroch (Fig. 6). Here a few stained 
cells with either a single or two processes 
were detected near the basal membrane (Fig. 
6). Other larger, superficial cells located just in 
front and behind the trochoblasts showed 
strong AChE positivity (Figs. 28, 29). 

In the pretrochal ectoderm beneath the re- 
fractive bodies (cephalic plates), mitoses 
could be often observed. Antero-ventrally on 
each side, a few cells became AChE-positive 
(Figs. 3, 28). 

In young trochophores 18-20 h old, 
one- two AChE-positive cells were detected in 
the pedal plates (presumptive foot) near the 
posterior pole on each side, adjacent to the 
mesoteloblasts, which showed a moderate 
AChE activity (Fig. 13). In the same region, 
serotonin immunocytochemistry labelled two 
bilaterally symmetrical nerve-like cells, each 



Embryos 

The distribution of AChE activity could not 
be observed in detail in cleaving embryos and 
gastrulae, as the pigmented yolk interfered 
with the histochemical staining. The enzyme 
reaction was more evident in 2d, 3D and 4d 
blastomeres, then, increased in the primary 
and accessory trochoblasts, mostly at the 
base of the differentiating ciliary band (Fig. 1 ). 



Abbreviations for All Figures 

A: apical organ; AN: anal cells; AT: apical tuft; C: cerebral 
gangliar rudiment; CM: cerebral commissure; CP: cerebral 
pit; E: endodermal cells; F: foot rudiment; FG: foregut; FN: 
nerve-like cells of the foot; IN: intestinal gangliar rudiment; 
M: mouth; MF: muscle fibre; MS: mesodermal cells; MC: 
mantle cavity; MT: mesodermal teloblast; OS: osphradia; 
P: pedal gangliar rudiment; PL: pleural gangliar rudiment; 
R: refractive body; S: statocyst; SG: shell gland; ST: sto- 
modaeum; T: prototrochal cell: TN: tentacle rudiment; V: 
visceral gangliar rudiment. 



134 



RAINERI 




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FIGS. 1 -6. Legends on facing page. 



NEUROGENESIS IN A GASTROPOD 



135 



with an axon-like fibre growing out towards the 
anterior (Figs. 15, 16). Soon after, two lateral 
bands of ectodermal cells running parallel to 
these fibres became AChE-positive (Fig. 14). 
In trochophores 20-24 h old, the shell field 
invaginated on the dorsal side and gave origin 
to the shell gland (Fig. 17). In this stage, cor- 
responding approximately to the "conchos- 
toma larva" of Mytilus (Malakhov & 
Medvedeva, 1985), three, then, more numer- 
ous ectodermal cells became AChE-positive 
on each side of the perspective foot (Figs. 7, 
17). However, they were pushed from poste- 
rior to anterior while differentiating, as the 
rudiment of the foot was shifted towards the 
oral side when the residual opening of the 
blastopore lined by moderately AChE-positive 
stomatoblasts (mouth primordium) rotated 
from the vegetal pole to the future ventral sur- 
face (anopedial flexure). Then, the lateral 
bands of histochemically stained cells were 
bent. When the rudiment of the foot started to 
bulge out on the ventral side, they were left 
progressively aside in a more dorsal position. 



FIGS. 1 -6. AChE activity revealed with AcMThCh 
as substrate. Figs. 1, 3, 5, 6, whole mounts. Figs. 2 
and 4, 2 |im sections through apical organ post- 
stained with toluidine blue. Figs. 1, 5, 6, scale bar 
50 (im. Figs. 2, 3, 4, scale bar 10 цт. 1 —Advanced 
gastrula viewed in Nomarski optics. Arrow: AChE- 
like activity is localized at base of developing ciliary 
band of prototroch. 2 — Trochophore 20 h old; arrow 
indicates medial cells of apical organ which support 
cilia of apical tuft. 3 — Trochophore 24 h old; tuft 
cells (small arrows) show stronger AChE activity 
than adjacent cells (large arrows). Arrows indicate a 
few AChE-positive cells localized antero-ventrally 
beneath refractive bodies (compare with Figs. 5 
and 28). 4 — Trochophore 24 h old: cytoplasmic stri- 
ation seen in a tuft cell (arrow), as well as in a cell 
of a refractive body (arrow), may indicate an intra- 
cellular origin of the cilia. 5 — Trochophore 28 h old, 
frontal view of pretrochal region: a few superficial 
(large arrow heads) and deeper (small arrows) 
AChE-positive cells localized antero-ventrally on 
each side, are first visible sign of differentiation of 
cerebral ganglia. Two rows of stained cells (fo- 
cussed on one side, large arrow) run from these 
rudiments posteriorly. Two fibres (small arrow) run 
from upper side of mouth towards apical organ. 6 — 
Trochophore 24 h old, fronto-lateral view of 
pretrochal region: an AChE-positive branched cell 
(large arrow head) is connected with refractive bod- 
ies and, seemingly, with another stained cell (small 
arrow) adjacent to prototroch. 



Trochophore 

In 28-35 h old trochophores, the AChE- 
positive areas of the cephalic plates invagi- 
nated slightly (Fig. 29) and a few cells lost 
contact with the surface. These cell clusters 
were the initial primordia of the cerebral gan- 
glia. Soon they became connected with two 
rows of AChE-positive cells, which ran to- 
wards the posterior (Fig. 5). However, it was 
unclear if they had a pre- or a posttrochal ori- 
gin. 

In the rudiment of the foot, two-three AChE- 
positive cells dropped inside, maintaining 
contact with the surface by short ciliary 
processes (Fig. 18). Soon after, a few histo- 
chemically stained cells were distinguished 
more in depth on each side (Fig. 19). Then, in 
trochophores 40-45 h old, more numerous 
nerve-like cells formed two bilaterally sym- 
metrical clusters, the rudiments of the pedal 
ganglia. They appeared to be connected to 
the anterior surface of the foot by a few den- 
dritic-like processes with intensely AChE-pos- 
itive ciliary endings (Figs. 8, 20). Almost si- 
multaneously with the pedal ganglia, other 
gangliar primordia, each first consisting of 
two-three nerve-like cells, differentiated from 
the lateral bands of AChE-positive ectodermal 
cells. One gangliar pair was located above the 
mouth in a latero-dorsal position (Fig. 8). It 
was interpreted as pleural, but was difficult to 
distinguish in toto, as its cells were masked by 
the intense AChE staining of the prototroch. 
Two gangliar rudiments that were interpreted 
as intestinal (also called oesophageal or pari- 
etal), developed near the foregut lying one 
slightly above the other on a plane oblique to 
the plane with the pedal ganglia. Another gan- 
glion that was interpreted as visceral differen- 
tiated near the midgut as a bilaterally sym- 
metrical fused primordium characterized by 
two large nerve cells. In 40 h old tro- 
chophores, the lateral bands of histochemi- 
cally stained cells were no longer detected, 
and the gangliar rudiments were connected to 
one another by longitudinal nerve fibres asso- 
ciated with small AChE-active glial-like cells. 

Advanced Trochophores 

In 55-65 h old trochophores, a few nerve fi- 
bres formed the primordium of the pedal com- 
missure. Each pedal ganglion was connected 
to the homolateral pleural (Fig. 12) and cere- 
bral ganglion, and associated antero-laterally 



136 



RAINERI 







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10 




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12 



11 



FIGS. 7-12. Legends on facing page. 



NEUROGENESIS IN A GASTROPOD 



137 



with a ciliary sensory-like organ, as described 
above (Figs. 21 , 23), dorsally, with a statocyst 
(Fig. 21), and ventrally, with a row of AChE- 
positive nerve-like cells running to the poste- 
rior surface of the foot (Fig. 22). 

As the visceral hump increased rapidly in 
size, the rudiment of the visceral ganglion was 
pushed towards the ventral side (Figs. 9, 10, 
12). The viscero-intestinal connectives were 
of different length, did not run parallel to one 
another, and showed an initial twist in tro- 
chophores 65-70 h old (Fig. 11). 

The intestinal gangliar rudiments appeared 
to be connected to the homolateral pedal gan- 
glia, which were lying at a short distance in an 
antero-ventral position (Figs. 10, 12). They 
were also linked to one another by a few 
nerve fibres that ran beneath the foregut. 
These connections are not reported in the 
adult, then, probably are lost in more ad- 
vanced stages. The intestinal gangliar rudi- 
ments were also connected, dorsally, to the 
homolateral pleural ganglia, and laterally, to a 
few AChE-positive ectodermal cells covered 
by a mucous-like coat, which became local- 
ized at the inner surface of the mantle cavity 
when the mantle fold spread downward. 
These cell clusters were pushed towards the 
posterior during the growth of the visceral 
area. In 70-80 h old trochophores, they sup- 



ported AChE-positive hair-like processes pro- 
truding in the mantle cavity and were linked to 
the homolateral intestinal ganglia by nerve fi- 
bres associated with AChE-positive glial-like 
cells and with a few larger, less intensely 
stained cells (Figs. 24-27). 

In the developing gangliar rudiments of the 
same advanced trochophores, AChE positiv- 
ity of many nerve cell bodies was restricted to 
the plasma membrane and the nuclear enve- 
lope. The nerve fibres and the glial-like cells 
showed a more evident histochemical stain- 
ing (Figs. 21, 23-26). 

In this stage, the proliferation centres of the 
cerebral gangliar rudiments invaginated 
("cerebral pits", Smith, 1935). These cells 
were stained more intensely than adjacent, 
more differentiated neural cells (Fig. 31). Two 
prominences, the rudiments of the tentacles 
(Fig. 32), and two pigmented eyes, developed 
in the close vicinity of the cerebral ganglia. 
Possibly due to the contraction of muscle fi- 
bres connected to the basal membrane be- 
neath the apical tuft, the apical organ sank 
inside above the developing cerebral com- 
missure. Both structures showed a strong 
AChE positivity (Figs. 30-32). 

The findings are summarized in Figures 
33-38. 



DISCUSSION 



FIGS. 7-12. whole mounts stained with AcMThCh 
as substrate. Figs. 7-9, scale bar 25 jam. Figs. 
10-12, scale bar 10 um. 7 — Trochophore 24 h old, 
viewed from the ventral side in Nomarski optics: 
arrow indicates a few AChE-positive ectodermal 
cells (those on opposite side are not in focus) local- 
ized in the rudiment of foot. Arrows indicate the 
mesodermal teloblasts. Compare with Fig. 13. 8 — 
Trochophore 40 h old, frontal view; AChE-positive 
ciliary tufts (arrows) formed by cells of pedal gan- 
gliar rudiments (see Fig. 20) appear as two sen- 
sory-like organs on anterior surface of the rudiment 
of foot. 9, 10, 12— Trochophore 55 h old, focussed 
on different planes in ventrolateral view. 9, large 
arrow indicates position of left sensory-like organ 
shown in Fig. 8; small arrow head indicates left vis- 
cero-intestinal connective. 10, arrow indicates 
same connective as in Fig. 9 and its glial-like 
sheath. 12, nerve fibres associated with glial-like 
cells (large arrow) run from pedal gangliar rudiment 
towards a pleural ganglion (not shown). Small ar- 
rows indicate other fibres which run towards pro- 
totroch (see Fig. 22). Arrow indicates emergence of 
a nerve fibre that is directed to visceral mass. 11 — 
Trochophore 70 h old, lateral view of viscero-in- 
testinal connectives; initial twist is evident. 



The results show that in Patella the devel- 
oping nervous system can be investigated 
conveniently by comparing the cell morphol- 
ogy to the distribution of AChE histochemical 
positivity. In early stages, as in many other an- 
imals (e.g., Raineri, 1989, 1995; Dorresteijn & 
Graffy, 1993), AChE activity could play a role 
in neural induction, cell differentiation and 
control of directional axon growth. However, 
in advanced stages a great percentage of 
neurons probably are not cholinergic, as their 
cell bodies showed no significant AChE posi- 
tivity. A more evident staining was associated 
with the nerve fibres, but also with the glial- 
like and muscular cells, as well as with other 
non-neural cells. In the trochoblasts, particu- 
larly, AChE positivity was co-localized with 
serotonin-like immunoreactivity, and with an 
actin band that appears to be crucial for the 
ciliary beating (Serras & Speksnijder, 1991) 
before any larval or adult nerve cell differenti- 
ation. This distribution could be referred to a 
non-nervous, more general role of acetyl- 
choline and biogenic monoamines in ion-me- 



138 



RAINERI 



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FIGS. 1 3- 1 7. 2 |дт sections stained with AcMThCh as substrate, scale bar 25 цт. Figs. 1 5 and 1 6, sections 
4 |дт thick treated by immunocytochemical method for serotonin, scale bar 5 ¡дт. Figs. 13 and 14-Tro- 
chophore 20 h old, consecutive parafrontal sections. 13, arrows indicate two AChE-positive cells with short 
ciliary-like processes localized at surface of perspective foot. 14, section through a plane dorsal to that of Fig. 
13; arrow indicates lateral stripe of AChE-positive ectodermal cells, partly cut tangentially. 15 and 16-Tro- 
chophore 18 h old, consecutive parafrontal sections; serotonin-like immunoreactivity is detected in nerve-like 
cell (large arrow) localized posteriorly in perspective foot; small arrow indicates emergence of its axon-like 
fibre (arrow) that runs in an anterior direction. 17-Trochophore 20 h old, parasagittal section; arrow indi- 
cates three AChE-positive cells in same position as those shown in Figs. 7 and 13. 



NEUROGENESIS IN A GASTROPOD 



139 






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FIGS. 18, 19,21 -23. 5 um sections stained with AcMThCh (1 8, 1 9, 21 , 22) and PrThCh (23) as substrate. 
Fig. 20, whole mount stained with PrThCh as substrate. Figs. 18, 19, 21, scale bar 10 um. Figs. 20, 22, 23, 
scale bar 25 urn. 18-Trochophore 28 h old; in rudiment of foot a cell (arrow) localized basal to ectodermal 
layer keeps contact with surface by means of an AChE-positive process (arrow). 19 — Trochophore 30 h old: 
arrows indicate two AChE-positive cells localized beneath ectodermal layer. 20 — Trochophore 40 h old, lat- 
eral view of rudiment of foot; arrow indicates one of the dendritic-like processes that run from pedal nerve 
cells to foot surface, where they form AChE-positive ciliary tufts (arrows). 21 —Trochophore 65 h old, section 
through pedal gangliar rudiment; arrow indicates statolith inside statocyst, arrow, AChE-positive ciliary tufts 
as in Fig. 20. 22, 23 — Trochophore 65 h old, oblique sections showing position of intestinal, pedal and vis- 
ceral gangliar rudiments; large arrow, AChE-positive ciliary tufts at foot surface; small arrows, AChE-positive 
cells localized near basal membrane of prototroch; one of them is reached by a fibre (arrows) that takes its 
origin near intestinal gangliar rudiment. 






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FIGS. 24-27.2 |лт sections stained with PrThCh (24 and 25) and AcMThCh (26 and 27) as substrate, scale 
bar 10 |дт. 24, 25 — Trochophore 55 h old, consecutive sections, 24 is viewed in Nomarsky optics; arrows in- 
dicate intensely AChE-positive hair-like processes that protrude in mantle cavity. Large arrows indicate nerve 
fibres; small arrows, AChE-positive cells (rudiment of an osphradial ganglion?). 26 and 27 — Trochophore 80 
h old, consecutive sections; large arrows indicate same sensory-like structures (osphradia?) seen in Figs. 24 
and 25; small arrow heads: glial-like cells; arrow: nerve-like cell. 



NEUROGENESIS IN A GASTROPOD 



141 



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FIGS. 28-32. 2 цт sections stained with AcMThCh (28, 31 , 32) and PrThCh (29, 30) as substrate. Figs. 28 
and 29, scale bar 25 \xm. Figs. 30-32, scale bar 10 цт. 28 — Trochophore 24 h old, frontal section cut nearly 
tangential through pretrochal region; arrows indicate AChE positivity localized in two anterolateral ectoder- 
mal regions that will give origin to rudiments of cerebral ganglia; arrows, AChE-positive cells localized above 
trochoblasts. 29 — Trochophore 28 h old, oblique section through pretrochal region; arrow indicates one of 
two ectodermal areas shown in Fig. 28, that invaginates slightly; arrow, AChE-positive cell above prototroch. 
30-32 — Trochophore 80 h old, consecutive sections through apical region, 30 and 32 are viewed in No- 
marski optics. 30, apical organ sunken inside, and tuft cells seen adjacent to rudiment of cerebral ganglion 
localized near a cerebral pit. 31 , muscle fibres, seemingly two, insert in thick, intensely AChE-positive basal 
membrane beneath tuft cells. 32, fibres of developing cerebral commissure, as well as those directed to rudi- 
ments of tentacles, show very evident AChE activity. 



142 



RAINERI 



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FIGS. 33-38. Legends on facing page. 






NEUROGENESIS IN A GASTROPOD 



143 



diated processes (Falugi, 1993), including a 
local regulation of the ciliary motility (Buznikov 
et al., 1996) and assembly-disassembly of 
actin filaments (Shmukler et al., 1999). Due to 
this lack of specificity, the nerves of the tro- 
chophore should be investigated by methods 
that allow a better discrimination between 
nerve and muscle cells, and preceding cyto- 
logical differentiation neural cells could be de- 
tected by the combined use of different mark- 
ers. 

A Possible Basic Pattern of 
Neurogenesis in Molluscs 

As described above, in Patella during post- 
trochal neurogenesis two bilaterally symmet- 
rical neural cells differentiated initially on each 
side of the perspective foot near the posterior 
pole. A pair of comparable nerve cells has 
been reported previously in the bivalve 



FIGS. 33-38. schematic drawings of whole 
mounts. 33-35, lightly stippled, dotted nuclei: 
mesodermal cells; densely stippled, heavily out- 
lined: neural cells. 33 — Young trochophore 18 h 
old, frontal view; blastopore closes from back to 
front and rotates contemporarily from vegetal pole 
to ventral side of body. Its residual opening gives 
origin to mouth, which eventually becomes local- 
ized beneath prototroch. The first posttrochal neural 
cells are detected posteriorly in two ventro-lateral 
ectodermal areas (pedal plates). Here oriented mi- 
toses with spindle axes parallel to midline can be 
observed. 34 and 35 — Young trochophore 20 and 
24 h old, lateral and frontal views; two bands of 
neural cells differentiate parallel to mesodermal 
bands, running at first nearly parallel to main axis of 
trochophore. However, they change their growth di- 
rection while differentiating, as perspective foot with 
first-arising neural cells is shifted from posterior to 
anterior on the oral side (anopedial flexure). Then, 
neuroectodermal bands become oriented obliquely 
from antero-ventral to postero-dorsal. 36-38, 
densely stippled, heavily outlined: cells of develop- 
ing adult nervous system. 36— Trochophore 35 h 
old, ventrolateral view. 37— Trochophore 55 h old, 
frontal view; rudiment of visceral ganglion is not de- 
tected in this orientation, but is located in a more 
ventral position than in trochophores 35 h old (Fig. 
36); rudiments of osphradia are localized in devel- 
oping mantle cavity in a postero-lateral position. 
38 — Trochophore 65 h old, ventro-lateral view; dur- 
ing development, rudiments of osphradia, as well 
as, to a lesser extent, those of intestinal ganglia, are 
shifted posteriorly. Viscero-intestinal connectives 
are of different lengths; in lateral view (Fig. 1 1 ), they 
show an initial twist 



Mytilus (Raineri & Ospovat, 1994). In the gas- 
tropod as well as in the bivalve, they grew out 
a pioneering fibre towards the apical pole; 
then, other cells lying in their vicinity and par- 
allel to their fibres, became AChE-positive. In 
trochophores 28-40 h old, the rudiments of 
the pedal ganglia, and those of the visceral, 
intestinal and pleural ganglia, took origin 
through cell ingression from these ectodermal 
areas. 

In Patella, the posterior nerve cells were 
detected in 18-20 h old trochophores, when 
the initial primordia of the cerebral ganglia just 
started to differentiate. In Mytilus, these rudi- 
ments developed relatively later in the ad- 
vanced trochophore, but the post-trochal pio- 
neer neural cells could be distinguished near 
the posterior pole as early as the end of gas- 
trulation, also due to their close association 
with two AChE-positive presumptive byssus 
gland cells, which are lacking in gastropods 
(Raineri, 1995). 

We may conclude that in Patella, a rather 
primitive gastropod (van den Biggelaar, 
1996), post-trochal neurogenesis starts rela- 
tively early, just as in Mytilus, in spite of the 
different developmental rate of the cerebral 
ganglia. Future studies should verify if other 
molluscs, such as aplacophorans and poly- 
placophorans, show this "from-backwards-to- 
forwards" early neurogenesis. On the other 
hand, a pair of posterior nerve cell progenitors 
was found in a polychaete (Dorresteijn et al., 
1993), and polychaetes are believed to be 
phylogenetically related to molluscs on the 
basis of other embryological similarities 
(Dawydoff, 1928; Beklemishev, 1969; van den 
Biggelaar et al., 1997). This pattern, then, 
may be an ancestral one. 

However, in Mytilus and Patella, unlike in 
the polychaete, these early-arising nerve 
cells, which were coincident with the pedal 
gangliar primordia, were pushed to an antero- 
ventral position when the rudiment of the foot 
was shifted from posterior towards the oral 
side (anopedial flexure; Raven, 1966; Ver- 
donk & van den Biggelaar, 1983). In Patella, 
this occurred in the young trochophore 18-24 
h old, but in Mytilus, in the veliger larva 40-48 
h old. This appears to be a general difference 
between gastropods and bivalves (Beklemi- 
shev, 1969). As a conseguence, in the gastro- 
pod cerebral, pedal and pleural gangliar pri- 
mordia started to differentiate anteriorly rather 
close to one another, but in the bivalve the 
cerebral gangliar primordia started to difieren- 



144 



RAINERI 



tiate when the rudiments of the pleural ganglia 
were anterior, but those of the pedal, posterior 
(Raineri, 1995). 

As a rule, basic nerve pathways must be 
established when embryonic distances are 
shorter in early stages of ontogeny (Klose & 
Bentley, 1989). Then, these differences might 
explain why pleuro-pedal and cerebro-pedal 
connectives form in gastropods, but are lack- 
ing in Mytilus and other bivalves. 

In contrast, previous studies (e.g., Smith, 
1935; Moor, 1983; Page, 1992a, b; Lin & 
Leise, 1996) reported that in Patella and other 
gastropods during neurogenesis the nerve 
connectives of the visceral loop grow out from 
the cerebral ganglia, the different post-trochal 
ganglia derive from separate cell proliferation 
centres and the visceral ganglia only appear 
in advanced larvae. Partly, at least, the dis- 
agreement may derive from the non-specific 
stainings employed in these investigations, as 
the gangliar primordia seen in the present 
study consist initially of two or three cells that 
cannot be detected easily even by AChE his- 
tochemistry. 

Sense Organs and their Relationships with 
the Developing Nervous System 

According to Lacalli (1 981 , 1 984, 1 994), the 
larval apical organ could be an ancestral pho- 
toreceptor, which first acts as a target for 
growing axons of the cerebral commissure, 
then, is incorporated in the commissure itself. 
On the contrary, Lin & Leise (1996) reported 
that in gastropod larvae the apical organ be- 
comes associated with an apical ganglion in 
advanced stages, may act as a chemorecep- 
tor implicated in metamorphosis, and eventu- 
ally undergoes regression. In Patella, the api- 
cal organ shows apparent morphological 
changes from young to advanced larvae. 
However, the present study was focused on 
its structural relationships in the young tro- 
chophore. Here the apical organ may include 
two sensory structures, the medial cells of the 
apical tuft and the two lateral "refractive bod- 
ies", which could be transitory larval receptors 
(Smith, 1935). They appeared to be con- 
nected, respectively, to the mouth and to the 
prototroch by the processes of a few AChE- 
active cells, which may correspond, partly at 
least, to the "primitive nervous net" observed 
by Smith (1935). However, in other tro- 
chophores this "larval" nervous system is as- 
sociated with muscle cells (Lacalli, 1981, 
1984), which also can show AChE positivity. 



Different markers, then, should be used to in- 
vestigate these cells, as well as the larger 
cells in front and behind the trochoblasts. 
They may be coincident with the anterior and 
posterior "supporting cells" (Smith, 1935; Dic- 
tus & Damen, 1997), but their strong AChE 
positivity and regular pattern of actin filaments 
(Serras & Speksnijder, 1991) suggest they 
could play some active role in the prototroch. 
Anyhow, all these cells arose separately from, 
and coexisted with, the rudiment of the adult 
nervous system. Thus, it seems unlikely that 
the cerebral ganglia and the longitudinal 
nerve cords of gastroneuralians evolved in di- 
rect relationship with the apical organ and the 
innervation of ancestral larval-like ciliary 
bands, as suggested by the trochaea theory 
(Nielsen, 1985, 1995). 

This conclusion is in agreement with the hy- 
pothesis that larval and adult patterns may be 
regulated by different sets of genes (Davidson 
et al., 1995), and with experimental evidence 
that a radially symmetrical embryo with tro- 
choblasts, but without adult structures, is ob- 
tained after deletion of the D blastomere or the 
polar lobe in Patella or Dentalium, respectively 
(Wilson, 1904a, b). On the other hand, the 
same embryological studies suggest that api- 
cal organ and adult body rudiment share some 
common developmental mechanism. These 
relationships are unclear (Sweet, 1998), but 
may be pertinent to the bilateral symmetry 
shown by the first-differentiating pre- and post- 
trochal neural cells of Patella prior to the es- 
tablishment of nerve connections. 

On the contrary, a link between sensory and 
gangliar rudiments was more evident in the 
post-trochal region. Justas in Mytilus (Raineri, 
1995), in Patella the pedal ganglia started to 
differentiate in connection with ciliary organs, 
tentatively, chemo- and/or mechanoreceptors. 
The gangliar rudiments interpreted as intesti- 
nal were associated with other sensory-like 
structures, which probably are the chemore- 
ceptor osphradia (Haszprunar, 1992). In 
agreement with Pelseneer and Bouvier 
(quoted by Lang, 1898), they were bilaterally 
symmetrical. Then, the single osphradium of 
other gastropod larvae (Lin & Leise, 1 996) can 
be the result of a reduction determined by tor- 
sion. 

Early Neurogenesis Pattern and its Possible 
Phylogenetic Implications 

The present observations support a com- 
bined origin of pre- and post-trochal nervous 



NEUROGENESIS IN A GASTROPOD 



145 



system from bilaterally symmetrical, but sepa- 
rate, neurogenesis centres (Moor, 1983). 
However, they conflict with the opinion that 
molluscs are typically tetraneural, like platy- 
helminthes (e.g., Willmer, 1990; Salvini- 
Plawen, 1991), due to a hypothetical, ances- 
tral fourfold symmetry of the brain, where 
each quadrant should originate a nerve cord 
(Lacalli, 1983). 

According to the same authors, a kinship 
between molluscs and platyhelminthes is sup- 
ported also by the foot, which should derive 
from the muscular ventral sole of a turbellar- 
ian-like ancestor (see also Haszprunar, 
1992). However, as discussed in detail by 
Beklemishev (1969), the presumptive foot is 
not the homologue of the future ventral side of 
platyhelminthes, but can be compared to the 
adult body rudiment of a polychaete tro- 
chophore. Indeed, they have a similar origin 
referred to the quadrants of the egg, are at 
first postero-dorsal, then, are shifted towards 
the oral side during ontogeny. In apla- 
cophorans and polyplacophorans, just as in 
polychaetes, the longitudinal body axis be- 
comes nearly aligned with the axis of the 
upper hemisphere of the trochophore (larval 
and adult axis regulation). However, in 
scaphopods, gastropods, bivalves to a lesser 
extent, and cephalopods in a modified way, 
the body axis is bent by the more pronounced 
anopedial flexure, which produces dorsally a 
visceral hump and anteroventrally a muscular 
organ, the foot. In a bivalve and in a gastro- 
pod, as discussed above, the latter contains 
initially a pair of posterior pioneer neural cells, 
just as those found at the rear of the body 
rudiment in a polychaete trochophore (Dor- 
resteijn et al., 1993), then, may be compared 
to the adult body rudiment of a polychaete 
bent on the ventral side (Raineri, 1995). 

These observations support a closer rela- 
tionship of molluscs to polychaetes rather 
than to platyhelminthes (e.g., Eernisse et al., 
1992). They may also corroborate Schel- 
tema's view (1993, 1996) that aculiferans 
(aplacophorans + polyplacophorans) are the 
sister-group of the conchiferans. The same 
author suggested a sister-group relationship 
between molluscs and sipunculans based, 
however, on controversial structural homolo- 
gies (Rouse & Fauchald, 1995). On the other 
hand, the hypothesis that molluscs (Raineri, 
1995), and also brachiopods (Conway Morris 
& Peel, 1995; Conway Morris, 1998), evolved 
from the same ancestor as polychaetes 
through a dorso-ventral developmental bend- 



ing of the body rudiment, would be in agree- 
ment with the embryological as well as the 
biomolecular data. Indeed, analyses of gene 
sequences showed that molluscs are closer 
related to polychaetes and to a brachiopods + 
phoronids clade (Halanych et al., 1995; 
Mackey et al., 1996; Aguinaldo et al., 1997). 

A comparison of neurogenesis in Patella 
and Mytilus may have some bearing on these 
relationships. As discussed above, it could 
suggest an origin of the gastropod tetra- 
neural-like pattern related to the compaction 
of the perspective nervous system in earlier 
stages and to the development of foot nerve 
cords depending on the larger size of the foot. 
Since bivalves are modified in relation to ses- 
sile life and, in comparison with other mol- 
luscs, their embryology is more similar to that 
of polychaetes (Dawydoff, 1928), the bending 
of the body rudiment, as observed in Patella, 
could be the primitive condition. If so, it be- 
came less and less pronounced, while seg- 
mentation evolved, on the phyletic line which 
led to the annelids. Alternatively, these differ- 
ent taxa could derive from a straight common 
ancestor, but diverged in evolution also due to 
a different degree of their adult body rudiment 
rotation in ventral direction during ontogeny 
(Raineri, 1995). If so, segmentation, or serial- 
ly, may have been lost in the phylogeny of 
molluscs (and possibly, brachiopods). 

This problem could be investigated by a 
comparative embryological approach, includ- 
ing developmental studies of the foot nerve 
cords in molluscs. This could also lead to a 
deeper insight into gastropod torsion. 

As shown in the present study by the posi- 
tion of the intestinal gangliar primordia, in 
Patella torsion starts in 28-35 h old tro- 
chophores. Smith (1935) suggested the twist 
of the visceral loop was predetermined in the 
neuroectoderm, but described the first signs 
of torsion at the end of the third day of devel- 
opment. However, according to Hasseigaku 
(1968) and Collier (1997), they appear at the 
end of gastrulation, when the anopedial flex- 
ure also takes place. Indeed, these processes 
may be related to one another at the mechan- 
ical (Pelseneer, quoted by Dawydoff, 1928; 
Haszprunar, 1989) and functional level (Lind- 
berg & Ponder, 1996). Moreover, it appears 
that the biomolecular regulation of the left- 
right body axis and its asymmetries is linked 
to the regulation of the antero-posterior and 
dorso-ventral body axes (Danos & Yost, 1 995; 
Vogan & Tabin, 1999). On one hand, these 
mechanisms have been partly conserved in 



146 



RAINERI 



the evolution of bilaterians (Levin, 1997); on 
the other hand, a bending of the body rudi- 
ment occurs also in brachiopods and 
phoronids during ontogeny (Nielsen, 1991). 
Phoronids show, in addition, left-right asym- 
metries, and sipunculans, anterior anus and 
coiled gut (Hyman, 1959). This may suggest 
that perturbations of axial patterning based on 
some common molecular mechanism have 
contributed to both the diversity of the body 
forms of molluscs and the evolutionary radia- 
tion of lophotrochozoans. 



ACKNOWLEDGEMENTS 

I am indebted to Mario Mori, who collected 
the Patella. The helpful comments of one of 
the referees are aknowledged with thanks. 



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Experimental Zoology, 1: 197-268. 

Revised ms. accepted 8 December 1999 



MALACOLOGIA, 2000, 42(1-2): 149-163 

GAMETOGENESIS AND SEXUAL MATURATION OF THE SURF CLAM MACTRA 
VENERIFORMIS ON THE WEST COAST OF KOREA 

Ee-Yung Chung 1 & Dong-Ki Ryou 2 



ABSTRACT 

Gametogenesis and sexual maturation of the surf clam Mactra veneriformis Reeve, 1854, 
were studied by histological and cytological observations. In the early vitellogenic oocyte, the 
Golgi apparatus and mitochondria present in the perinuclear region are involved in the formation 
of lipid droplets and in lipid granule formation. In the late vitellogenic oocyte, the endoplasmic 
reticulum, mitochondria in the cytoplasm are involved in the formation of proteid yolk granules. 
At this time, exogenous lipid granular substances and glycogen particles in the germinal epithe- 
lium are passed into the ooplasm of the oocyte through the microvilli of the vitelline envelope. 
Ripe oocytes are about 60-70 urn (65.35 ± 4.82 urn) in diameter. The head of the spermato- 
zoon is approximately 2.5 urn in length, and its tail is about 58.76 ± 7.20 urn The axoneme of 
the tail flagellum consists of nine pairs of microtubules at the periphery and a pair at the center. 
The spawning period was from early June to September, with the main spawning occurring 
between July and August when the water temperatures are greater than 22°C. The reproductive 
cycle of this species can be divided into five stages: early active, late active, ripe, partially 
spawned, and spent/inactive. 

Percentages of first sexual maturity of female and male clams ranging in length from 26.0 to 
30.0 mm are over 50%, and 100% for clams over 31.0 mm in shell length. 

The sex ratios of females to males over 26.0 mm in shell length were not significantly different 
from a 1 :1 sex ratio. No evidence of hermaphroditism was found in histological sections of any 
surf clam examined. 

Key words: Mactra veneriformis, gametogenesis, reproductive cycle, first sexual maturation, 
sex ratio. 



INTRODUCTION 

The surf clam Mactra veneriformis Reeve, 
1854 (Pelecypoda: Mactridae), is distributed 
along the coasts of Korea, China and Japan. 
More specifically for Korea, this clam is found 
in silty sand in the intertidal zone of the south 
and west coasts of Korea (Yoo, 1976; Kwon 
et al., 1 993), and it is one of the important edi- 
ble bivalves. However, on account of the 
recent sharp reduction in the standing stock 
by reclamation works, marine pollution, and 
reckless overcatching of this clam, it has been 
noted as a target organism and fisheries 
resource that should be managed by a more 
reasonable fishing regime. Therefore, it is 
important to understand some population 
characteristics in regard to gametogenesis 
and sexual maturation of this species. Pre- 
viously, there has been some work on the 
artificial discharge of reproductive substances 
of this species (Iwata, 1948), classification 
(Lee, 1956), aspects of ecology (Choi, 1969; 



Ryou, 1995), and sexual maturation (Chung 
et al., 1988). There are still gaps in our knowl- 
edge regarding reproductive biology. There- 
fore, the main purpose of the present study 
was to understand the gonadal develop- 
ment and germ cell development, the re- 
productive cycle, and the first sexual maturity 
and sex ratio based on histological, cytolo- 
gical examinations and some morphometric 
data in order to get important information 
for natural resources management of this 
clam. 



MATERIALSAND METHODS 
Sampling 

Specimens of Mactra veneriformis Reeve, 
1854, were collected monthly in the intertidal 
zone of Naechodo, Kunsan, Korea, from 
March 1 993 to February 1 994 (Fig. 1 ). A total 
of 669 clams ranging from 15.1 mm to 45.8 



department of Marine Living Resources, Kunsan National University. Kunsan 573-701, Korea; eychung@kunsan.ac.kr 
department of Aquaculture, Kunsan National University, Kunsan 573-702, Korea 

149 



150 



CHUNG & RYOU 




Sampling area 



^O 3 



*<№*> 



V 




- 36°00' 



- 35°00- 



126° 30" 

FIG. 1. Map showing the sampling area. 



126°45' 



mm in shell length were used for the histolog- 
ical and cytological studies. After the clams 
were transported alive to the laboratory, shell 
lengths and heights were measured by a 
Vernier caliper, and their total weights were 
determined using a chemical balance. 

Histological and Cytological Analysis 

Histological preparations were made for 
analysis of the gonadal phases by light 
microscopy. The gonad tissues were removed 
from shells and preserved in Bouin's fixative 
for 24 h and then washed with running tap 
water for 12 h. Tissues were then dehydrated 
in alcohol and embedded in paraffin and sec- 
tioned at 5-7 urn using a rotary microtome. 
Sections were mounted on glass slides, 
stained with either Hansen's hematoxylin- 
0.5% eosin, Mallory's triple stain or PAS stain, 
and examined. 

For electron microscopical observations, 
excised pieces of the gonads were cut into 
small pieces and fixed immediately in 2.5% 



paraformaldehyde-glutaraldehyde in 0.1 M 
phosphate buffer solution (pH 7.4) for 2 h at 
4°C. After prefixation, the specimens were 
washed several times in the buffer solution 
and then postfixed in a 1% osmium tetroxide 
solution in 0.2 M phosphate buffer (pH 7.4) for 
1 h at 4°C. Specimens then were dehydrated 
in increasing concentrations of ethanol, 
cleared in propylene oxide and embedded in 
an Epon-Araldite mixture. Ultrathin sections of 
Epon-embedded specimens were cut with 
glass knives on a Sorvall MT-2 microtome and 
LKB ultramicrotome at a thickness of about 
800-1 ,000 A°. Tissue sections were mounted 
on collodion-coated copper grids, doubly 
stained with uranyl acetate followed by lead 
citrate, and observed with a JEM 100 CX-II 
(80-KV) electron microscope. 

Frequency Distributions of Egg Diameters 

In order to investigate monthly relative fre- 
quency distributions of egg diameters, some 
one thousand eggs a month were centrally 



GAMETOGENESIS AND SEXUAL MATURATION OF THE SURF CLAM 



151 



cut and measured, and then graphed by the 
frequency curve method of Pearse (1965). 



First Sexual Maturity and Sexually 
Mature Length 

The percentages of first sexual maturity 
were investigated histologically in order to 
certify shell lengths of specimens that partici- 
pated in reproduction from April to October 
1993. First sexual maturity was estimated by 
the rate of mature individual to number of 
females. 

To calculate sexually mature length, after 
fitting the rate of group maturity (first sexual 
maturity) to an exponential equation, the 
size equivalent to 50% of first sexual maturity 
was estimated to be the sexually mature 
length. 




FIG. 2. Anatomy of Mactra veneriformis. 
Abbreviations: A, anus; AM, anterior adductor mus- 
cle; ES, exhalent siphon; F, foot; G, gonad; H, 
hepatopancreas; IG, inner gill; IS, inhalent siphon; 
LV, labial valve; M, mantle; OG, outer gill; PM, pos- 
terior adductor muscle; U, umbo. 



Sex Ratio 

The sex ratios of the surf clams were esti- 
mated by collecting monthly samples of 592 
sexually mature individuals (shell length > 26 
mm) from February to December 1993. The 
clams were sexed by microscopic examina- 
tion of histological slides. A Chi square, good- 
ness-of-fit test was used to test the hypothe- 
sis of equal representation of females and 
males. 



RESULTS 
Position and Structure of the Gonads 

This species is dioecious. The gonads are 
located between the subregion of the mid- 
intestinal glands in the visceral cavity and the 
reticular connective tissues of the foot. The 
ovary and testis are composed of a number of 
ovogenic follicles, and the testis comprises 
several spermatogenic follicles (Fig. 2). 

As maturation progresses, an external view 
of the ovary is light pink in colour, and the 
mature testis is yellowish-white. At this time, if 
they are slightly scratched, ripe eggs and 
milky-white sperms readily flow out. 
Therefore, the sex of the clams could be dis- 
tinguished easily by external features. But 
after spawning, the gonads degenerate and it 
becomes difficult to distinguish the sex of the 
clams. 



Monthly Changes in Relative Frequency 
Distributions of Egg Diameters 

Monthly changes in relative frequency dis- 
tributions of the ovarian egg diameters from 
March 1993 to February 1994 are shown in 
Figure 3. In January and February 1993, rela- 
tive frequencies ranging from 10 urn to 20 цт 
in diameter were over 90%. In March, the 
number of clams with large egg diameters 
began to increase, and in April 9% had eggs 
over 40-50 u.m (mature oocyte) in diameter. 
Percentages of eggs over 50-65 u.m (ripe 
oocyte) in diameter were about 35% in May. In 
June, when the spawning period began, ripe 
oocytes measuring about 65-70 ¡.im began to 
decrease considerably in number because of 
their discharge. A few large remaining oocytes 
degenerated after July. From September to 
February, a few oogonia and oocytes measur- 
ing 10-20 u.m in diameter remained in the 
ovogenic follicles. 

Gametogenesis — Ovary 

Oogenesis occurs in the ovogenic follicles 
of the ovary and can be divided into five 
stages: (1) oogonium, (2) previtellogenic 
oocyte, (3) vitellogenic oocyte, and (4) mature 
oocyte stages. 

Oogonial Stage: Oogonia are small, oval in 
shape, and 9-10 urn in diameter. They con- 
tain a large nucleus with a nucleolus, and 



152 



CHUNG & RYOU 



FREQUENCY(%) 



E 
3. 


0—1 


^v 




ОС 




Ш 


1 1—20 


H 




Ш 


21—30 


s 




< 


31—40 


IB 




Û 


41—50 


(D 




(3 


51 — 60 


ш 






61 — 70 




FIG. 3. 

formis. 



M. A. M. J. J. A. S. 0. N. D. J. F. 
1993 1994 

MONTH 

Monthly changes in relative frequency distribution of the ovarian egg diameter of Mactra veneri- 



have several mitochondria in the cytoplasm 
(Fig. 4A). 

Previtellogenic Oocyte Stage: The oogonia 
develop into the previtellogenic oocytes. At 
the beginning of cytoplasmic growth, several 
small mitochondria and vacuoles are concen- 
trated around the nucleus (Fig. 4B). 

Vitellogenic Oocyte Stage: In the early vitel- 
logenic oocyte, the Golgi apparatus and mito- 
chondria are usually present in the perinu- 
clear region, and the vesicles are scattered 
from the perinuclear region to the vitelline en- 
velope of the oocyte. Lipid droplets are accu- 
mulated in the small vacuoles, which are 
formed by the Golgi apparatus near the nu- 
clear envelope and are dispersed toward the 
cortical layer near the vitelline envelope (Fig. 
4C, D). At this time, microvilli on the vitelline 
envelope begin to appear, and the contours of 
the microvilli are round or oval in shape (Fig. 
4E). With the initation of the formation of lipid 
granules and proteid yolk formation (vitelloge- 
nesis), the cortical granules, small vesicles 
and lipid granules and mitochondria are lo- 
cated around the cortical region (Fig. 4F). In 
the late vitellogenic oocyte, lipid granules, 
which occupy the area around the nuclear en- 



velope, dispersed toward the cortical layer, 
and an accumulation of cortical granules 
occur in the cortical layer autosynthetically. 
However, proteid yolk granules, which were 
formed by the cortical granules and endoplas- 
mic retícula in the cytoplasm, are dispersed 
from the cortical layer near the vitelline enve- 
lope to the perinuclear cytoplasm (Fig. 4G). At 
this time, especially, exogenous electron- 
dense granular substances and lots of glyco- 
gen particles in the germinal epithelium or the 
nurse cells are passed into the ooplasm of the 
oocyte through the microvilli of the vitelline 
envelope. The tips of the microvilli, some of 
which bifurcate, protrude and extend just be- 
yond the outer border of the vitelline envelope 
(Fig.4H). 

Mature Oocyte Stage: In the mature oocyte, 
mature proteid yolk globules appear in the 
cytoplasm (Fig. 4I). At this time many small 
yolk globules are fused to each other and 
become larger mature yolk globules (Fig. 4J). 



Gametogenesis —Testis 

Based on the gonadal development and 
morphological characteristics of germ cells, 



GAMETOGENESIS AND SEXUAL MATURATION OF THE SURF CLAM 



153 




M VE 



ft 



CG 






LG* 



irf 



ш 
m 




ш 



FIG. 4. Electron micrographs of oogenesis of Mactra veneriformis (A-J). A, section of an oogonium, with a 
large nucleus and several mitochondria in the cytoplasm; B, a previtellogenic oocyte, with a large nucleus 
with a few nucleolus and several mitochondria in the cytoplasm; C, an early vitellogenic oocyte, with a num- 
ber of vacuoles near the Golgi apparatus in the perinuclear region; D, an early vitellogenic oocyte, with the 
Golgi apparatus, a number of the vacuoles and lipid droplets near the nucleus; E, an early vitellogenic oocyte, 
with formation of the vitelline envelope with the round microvilli; F, an early vitellogenic oocyte, with several 
cortical granules between lipid granules and the vitelline envelope; (continues on next page) 




CHUNG & RYOU 



- I*' Щ ' *r* ф G 0. 5/лл 





FIG. 4. (Continued) G, a late vitellogenic oocyte, with several cortical granules near the vitelline envelope; H, 
a late vitellogenic oocyte, with a number of granular substances and glycogen particles from germinal epithe- 
lium passing into the ooplasm in the oocyte through the microvilli of the vitelline envelope; I, a mature oocyte, 
with several of proteid yolk globules and mitochondria; J, a mature oocyte, with a number of mature yolk glob- 
ules. Abbreviations: CG, cortical granule; G, golgi apparatus; GE, germinal epithelium; GP, glycogen parti- 
cle; GR, granule; LD, lipid droplet; LG, lipid granule; LGRS, lipid granular substance; M, mitochondria; MV, 
microvilli; MYG, mature yolk globule; N, nucleus; NE. nuclear envelope; NO, nucleolus; ОС, oocyte; OG, 
oogonium; PYG, proteid yolk granule, PYGL, proteid yolk globule. V, vacuole; VE, vitelline envelope. 



spermatogenesis can be divided into five 
stages: (1) spermatogonia!, (2) primary sper- 
matocyte, (3) secondary spermatocyte, (4) 
spermatid and (5) spermatozoon stages. 

Spermatogonia! Stage: Spermatogonia, each 
containing a large oval nucleus, are located in 
the follicle wall (germinal epithelium) of the 
spermatogenic follicles. The nucleus contains 
dense, unevenly distributed heterochromatin. 
Several mitochondria are distributed in the cy- 



toplasm (Fig. 5A). Undifferentiated mesenchy- 
mal cells locates near the spermatogonia and 
spermatocytes (Fig. 5B). 

Primary Spermatocyte Stage: Spermato- 
gonia develop into primary spermatocytes. 
The primary spermatocytes have a large nu- 
cleus with aggregated chromatin. During the 
prophase of the primary maturation division, 
synaptonemal complexes appear in the nu- 
cleus of the primary spermatocytes (Fig. 5C). 



GAMETOGENESIS AND SEXUAL MATURATION OF THE SURF CLAM 



155 






* Лг л~ * a ..Ja 



SG 



; ■ * «r ¿va 

, .% t. ♦* л •■;> 






2 ¿от 



M 

5"^ 




N ' 

«A 




0. 5/яп 



^5/лГ р^^^и 



FIG. 5. Electron micrographs of spermatogenesis of Mactra veneriformis (A-l). 

A, section of spermatogonia, with a large nucleus with chromatin; B, mesenchymal cells near the multipli- 
cating spermatogonia, with irregular nucleus of the mesenchymal cells; C, the primary spermatocyte during 
the meiosis, with synaptonemal complex in the nucleus and several mitochondria during the prophase of the 
primary maturation dividion; D, the secondary spermatocytes, with small secondary spermatocytes with 
gradually condensed nucleus; E, a spermatid in the early stage of differentiation during spermiogenesis, with 
a large nucleus with electron dense heterochromatin, the Golgi apparatus and mitochondria in the cytoplasm 
of the spermatid; F, the spermatid in the early stage of the differentiation, with centrosome and mitochondria 
beneath the nucleus; G, acrosome formation in the middle stage of differentiation during spermiogenesis, 
with horn-like acrosomal vesicle and nucleus; H, transformations of the acrosome in the late stage of differ- 
entiation, with an acrosomal vesicle in rectangular type; I, longitudinal section of a spermatozoon after 
spermiogenesis, with acrosome, spermhead, proximal centriole, distal centriole and the axial filament; J, 
cross sectioned tail of a spermatozoon, with a pair of longitudinal fibers along its middle and nine pairs of lon- 
gitudinal fibers surrounding it. Abbreviations: AC, acrosome; AV, acrosomal vesicle; AX, axial filament; C, 
centosome; G, Golgi apparatus; M, mitochondria; MT, mesenchymal tissue; N, nucleus; PSC, primary sper- 
matocyte; SG, spermatogonium; SSC, secondary spermatocyte; ST, spermatid. 



156 



CHUNG & RYOU 



Secondary Spermatocyte Stage: The pri- 
mary spermatocytes develop into secondary 
spermatocytes by the first meiotic division. At 
this time, dense heterogeneous chromatin 
increases in the nucleus (Fig. 5D). 

Spermatid Stage: The secondary spermato- 
cytes develop into the spermatids as a result 
of the second meiotic division. At this time, the 
spermatid nucleus has the typical structure of 
the nucleus with aggregated chromatin (Fig. 
5D). 

At the early differentiation of the spermatid, 
the shape of the nucleus changes gradually, 
becoming slightly elongated and narrow. The 
Golgi apparatus and small acrosomal granule 
in the cytoplasm move to a position just 
before the nucleus, while the mitochondria 
move to a position just behind the nucleus 
(Fig. 5E). 

During the late spermiogenesis (transfor- 
mation of the spermatid into the spermato- 
zoon), the Golgi vacuole becomes closely 
applied to the tip of the elongating nucleus. 
The mass of mitochondria locate in the mid- 
piece of the spermatozoon, with two or more 
spherical mitochondria forming the paranu- 
cleus around the centrosome (Fig. 5F). Three 
horn-like acrosomal vesicles make contact 
with the nucleus (Fig. 5G), and then, the acro- 
some is formed by way of morphological 
change of the acrosomal vesicle (Fig. 5H, I). 
Of the two centrioles lying in the middle piece 
of the spermatozoon, the distal centriole takes 
up a position behind the proximal centriole, 
and the proximal centriole gives rise to the 
axial filament of the flagellum of the sperma- 
tozoon (Fig. 5I). 

The axial filament of the spermatozoon has 
a pair of longitudinal fibers along its middle 
and nine pairs of longitudinal fibers surround- 
ing it. When spermiogenesis is completed, the 
head of a ripe spermatozoon is approximately 
2.5 urn in length, and its tail is 58.76 ± 7.20 
fim in length. The axoneme of the tail flagel- 
lum consists of nine pairs of peripheral micro- 
tubules at the periphery and one pair of cen- 
tral microtubules at the center (Fig. 5J). 

Reproductive Cycle with Gonad 
Developmental Phases 

Based on the morphological features and 
sizes of the germ cells and the tissue cells 
around them, the reproductive cycle with go- 



nadal phases can be classified into five suc- 
cessive stages (Fig. 6). The stages and the cri- 
teria used in defining them are as follows: 

Early Active Stage: In females, oogonia and 
previtellogenic oocytes propagate along the 
oogenic follicle wall and the mesenchymal tis- 
sues of the ovary. The oogonia and previtel- 
logenic oocytes are about 9-11 urn and 
12-15 цт in size, respectively. At this time, 
the total volume of the ovary is small, and the 
follicle wall is thick (Fig. 7A). 

In males, spermatogenesis occurs in the 
spermatogenic follicles of the testis. The sper- 
matogonia and spermatocytes are 7-8 |im 
and 5-7 urn in diameter, respectively, and 
appear in a layer along the spermatogenic fol- 
licle wall (Fig. 8A). Compared with the visceral 
mass, the volume of the testis is small. 

The individuals in the early active stage 
appear from January to March when sea 
water temperatures are very low. 

Late Active Stage: In females, there is a large 
amount of ovogenic activity. A number of the 
early vitellogenic oocytes, ranging from 16- 
20 urn in diameter, appear in the oogenic fol- 
licles. When the late vitellogenic oocytes grow 
to 30-50 urn in diameter, each oocyte has an 
egg-stalk attached to the follicle wall (germi- 
nal epithelium). At this time, the follicle wall is 
thin (Fig. 7B). 

In males, spermatocytes develop into sper- 
matids. The spermatids move toward the cen- 
ter of the lumen, measure 3-4 urn in diameter, 
and show layers. As the testis develops, a 
number of spermatocytes, spermatids and 
small number of spermatozoa occupy approx- 
imately one-third to one-half of the lumina in 
the spermatogenic follicles (Fig. 8B, C). 

Individuals in the late active stage are found 
from March to May when seawater tempera- 
tures increase gradually. 

Ripe Stage: In females, the majority of 
oocytes grow to 50-60 цт in diameter, be- 
coming round or oval in shape, and are lo- 
cated in the center of the lumen. There is an 
increase in the ratio of cytoplasm to the nu- 
cleus. At this time, the follicle wall becomes 
very thin, the ripe eggs measure about 60-70 
urn (65.35 ± 4.82 urn) in diameter, and they 
are surrounded by the gelatinous mem- 
branes. The cytoplasm of the eggs contains a 
large number of yolk granules, while the folli- 
cle wall is very thin (Fig. 7C, D). 



GAMETOGENESIS AND SEXUAL MATURATION OF THE SURF CLAM 157 

40 



P 

^ 20 -I 


80 - 



> 


60 


О 




2 




LU 




D 
О 


40 


ш 




ce 




Li. 






20 



о 




MAMJJASONDJF 

1993 months 1994 



;Шз Early active HI Late active SSS Ripe 

im Partially spawned Y/A Spent / Inactive I I Water Temp 



FIG. 6. Frequency of gonadal phases of Mactra veneriformis from March 1993 to February 1994. 



In males, a number of spermatids undergo 
transformation into differentiated spermato- 
zoa in the center of the lumen. The ripe testis 
is characterized by the formation of a number 
of spermatozoa (Fig. 8D). 

Mature and ripe gonads are found from 
April to August when seawater temperatures 
are relatively high (13-26.4°C). 

Partially Spawned Stage: In females, since 
about 50-70% of the oocytes in the oogenic 
follicles are discharged, the lumen of the 



oogenic follicles becomes considerably 
empty. Spawned ovaries are characterized by 
the presence of a few ripe undischarged 
oocytes and very young oocytes in the lumen 
(Fig. 7E). 

In males, a large number of spermatozoa in 
the spermatogenic follicles are discharged 
into the surrounding water, and the lumen 
becomes empty. But a number of spermato- 
zoa, as well as spermatids and spermato- 
cytes, still remain in the lumen (Fig. 8E). 

The spawning period occurs once a year 



158 



CHUNG & RYOU 




50 [ж 



t£\ 






С 50 ¡m 







vi 

: 4 
*26 




50 ¡m 



FIG. 7. Gonadal phases of female surf clam Mactra veneriformis as seen by light microscopy. A, transverse 
section of the oogenic follicles in the early active stage; B, Section of the follicles in the late active stage; C, 
section of a mature ovary in the ripe stage; D, section of fully mature oocytes in the ripe stage; E, section of 
the ovary in the partially spawned stage; F, section of the follicles in the spent and inactive stage. 



from early June to September, and the main 
spawning occurs between July and August 
when seawater temperatures are greater than 
22°C. 

Spent/Inactive Stage: In females, after 
spawning, the undischarged oocytes in the 



lumen of the ovogenic follicle undergo cytoly- 
sis, and each follicle is contracted and degen- 
erated. The products of gamete atresia are re- 
sorbed. Thereafter, the rearrangement of 
newly formed connective tissues occur in the 
follicles (Fig. 7F). 
In males, the few remaining spermatozoa 



GAMETOGENESIS AND SEXUAL MATURATION OF THE SURF CLAM 



159 



50 ¡m I 




'if не 








FIG. 8. Gonadal phases of male surf clam Mactra veneriformis as seen by light microscopy. A, transverse 
section of the spermatogenic follicles in the early active stage; B, section of the follicles in the late active 
stage; C, section of the follicles in the late active stage; D, section of a fully mature testis in the ripe stage; 
E, section of the follicles in the partially spawned stage; F, section of the testis in the spent and inactive stage. 



and spermatids are degenerated. Thereafter, 
the rearrangement of a few newly formed con- 
nective tissues occur in the spermatogenic fol- 
licles in this stage (Fig. 8F). The individuals in 
this stage appear from September to February 
when seawater temperatures decrease grad- 
ually. 



First Sexual Maturity and Sexually 
Matured Length 

First Sexual Maturity: The first sexual maturi- 
ties of a total of 272 (1 33 females, 1 39 males) 
individuals of M. veneriformis were investi- 
gated histologically in order to certify the shell 



160 



CHUNG & RYOU 



TABLE 1 . Shell length of first sexual maturity of M. veneriforms 







Female 




Male 




Shell length (mm) 


Number 


Mature (%) 


Number 




Mature (%) 


15.0-20.0 


12 


0.0 


16 




0.0 


21.0-25.0 


24 


25.0 


25 




36.0 


26.0-30.0 


36 


58.3 


38 




65.8 


31.0-35.0 


30 


100.0 


25 




100.0 


36.0-40.0 


16 


100.0 


22 




100.0 


41.0-45.0 


15 


100.0 


13 




100.0 


Total 


133 




139 







lengths of clams participating in reproduction 
from March (before spawning) to late Septem- 
ber (after spawning). As shown in Table 1 , per- 
centages of first sexual maturity of female and 
male clams ranging from 26.0 to 30.0 mm are 
over 58%, and it is 100% for clams over 31 .0 
mm in shell length. 

Shell length of Sexually Mature Clams: Shell 
length of sexually mature clams (50% of rate 
of group maturity) that fitted to an exponential 
equation was 26.08 mm in shell length (Fig. 9). 

Sex Ratio 

As shown in Table 2, of 592 surf clams ex- 
amined, 294 were females and 281 males. 
The remaining 17 could not be distinguished 
by sex because of trematode parasites or be- 
cause of having only a few indistinguishable 
sex cells in various stages. There was no sig- 
nificant difference in the numbers of females 
and males present (x 2 = 0.29, p > 0.05), and 
monthly comparisons showed no statistical 
differences in the numbers of female and male 
clams. In five out of eleven months there were 
more females than males, and for the remain- 
ing six months there were more male than fe- 
male clams. The sex ratios of individuals over 
26.0 mm in shell length were not significantly 
different from a 1 :1 sex ratio. All the surf clams 
sampled were dioecious, and no evidence of 
hermaphroditism was found in histological 
sections of any surf clam examined. 



DISCUSSION 

Germ Cell Development and 
Gonadal Development 

Reverberi (1971) reported that in Mytilus 
edulis the Golgi apparatus present in the peri- 
nuclear region is involved in lipid droplet for- 
mation. In the present study, a similar result 



was observed, although we assumed that the 
Golgi apparatus, and mitochondria present in 
the perinuclear region are involved in lipid 
droplet formation, and that the Golgi appara- 
tus, endoplasmic reticulum and mitochondria 
are involved in the formation of yolk granules 
in the cytoplasm autosynthetically. Light 
microscope observations with the PAS stain 
showed a strong positive reaction at the site 
of the egg-stalk of the late active oocyte con- 
nected to the follicle wall (germinal epithe- 
lium). At this time, according to the results 
by electron microscope observation, lipid 
granular substances and glycogen particles in 
the germinal epithelium are passed into the 
ooplasm of the oocyte through the microvilli of 
the vitelline envelope. Therefore, we assume 
that some vitellogenic substances in the late 
vitellogenic oocyte are originated from exoge- 
nous substances in the germinal epithelium 
during vitellogenesis. 

The continuous production and resorption 
of gametes may be regarded as an adaptation 
to environmental temperature and food avail- 
ability (Morvan &Ansell, 1988; Paulet, 1990). 
If the reproductive energy allocated to the pro- 
duction of gametes is too large, nutritive 
reserves may not be enough to allow all eggs 
to reach the critical size for spawning and fer- 
tilization. In this case, the products of gemete 
atresia may be resorbed and the energy real- 
located to still-developing oocytes or used for 
other metabolic purpose by the bivalves 
(Dorange & LePennec, 1989; Motavkine & 
Varaksine, 1989). Therefore, it is assumed 
that the bivalve should have a reproductive 
mechanism to resorb and utilize the high 
nutritive substances rather than releasing 
hopeless gametes. 

Most of the bivalves have a primitive type of 
spermatozoa with a small head and cap- 
shaped acrosome, and a short mid-piece with 
four to five mitochondria surrounded the cen- 
trioles (Longo & Dornfield, 1967; Chung et al., 
1991). In this study, we found that the mor- 



GAMETOGENESIS AND SEXUAL MATURATION OF THE SURF CLAM 161 



100 



^■^ 


80 


>5 

o 4 




> 


70- 






k. 




3 




Ш 


60- 


2 

Q. 




50- 


3 




О 




ь_ 




О 


4U- 


«^ 




о 


30- 


О 




«^ 




Ш 
CE 


20- 




20 30 40 

SHELL LENGTH(mm) 

FIG. 9. Relationship between the rate of group maturity (%) and shell length (mm) of Mactra veneriformis. 



phology of spermatozoon is similar to that of 
other bivalve spermatozoa in having a short 
mid-piece with over two mitochondria sur- 
rounding the centrioles. However, compared 
with the formation of the acrosomal vesicles in 
species of other families, one of the morpho- 
logical or phylogenetical characteristics of 
Mactra species is the three horn-like acroso- 
mal vesicles during spermatogenesis. There- 
fore, we assume that the presence of a special 
acrosomal vesicle during spermatogenesis 
could be used as a key characteristic for iden- 
tification of species of the genus Mactra. 

Spawning 

Iwata (1948) and Oshima et al. (1965) 
found that the spawning period of M. veneri- 



formis in Tokyo Bay occurred twice a year 
from April to early July and from September to 
early October. But in our study, the spawning 
period occurred only once a year from early 
June to September, and the main spawning 
occurred between July and August. 

Regarding food and the spawning period, 
Griffiths (1977) and Jaramillo & Navarro 
(1995) found that the abundance of food is 
generally associated with the breeding period 
of marine bivalves, probably because clams 
do not store large reserves in the mantle and 
have a immediate gonadic regeneration with 
high food availability. Bayne (1976) reported 
that "bivalves tend to spawn during periods 
when food is available to ensure adequate 
nutrition for the planktotrophic larvae and for 
replenishing the energy the adults spend in 



162 



CHUNG & RYOU 



TABLE 2. Monthly variations in sex ratios of the adult surf clam Mactra veneriformis (>26.0 mm in 
shell length) 





Female 


Male 


Indeterminate 


Total 


Sex ratio 


Chi 


Date 


(inds.) 


(inds.) 


(inds.) 


(inds.) 


(F/(F+M) 


Squared 


Feb. 1993 


15 


17 


4 


36 


0.47 


0.13 


Mar. 1993 


23 


21 





44 


0.52 


0.09 


Apr. 1993 


18 


17 





35 


0.51 


0.03 


May. 1 993 


31 


32 





63 


0.49 


0.02 


Jun. 1993 


67 


62 





129 


0.52 


0.19 


Jul. 1993 


25 


24 





49 


0.51 


0.02 


Aug. 1993 


14 


13 





27 


0.52 


0.04 


Sep. 1993 


25 


27 


2 


54 


0.48 


0.08 


Oct. 1993 


19 


17 


1 


37 


0.53 


0.11 


Nov. 1 993 


31 


29 


5 


65 


0.52 


0.07 


Dec. 1993 


26 


22 


5 


53 


0.54 


0.33 


Total 


294 


281 


17 


592 


0.51 


0.29 



The critical value for x goodness of the test of equal numbers of females and males. (1 df) at 95% significance 

is 3.84. 

Ind means number of individuals. 



spawning". Thus, we can accept that "spawn- 
ing is induced by a combination of internal 
and environmental factors and that their inter- 
action may vary seasonally, producing annual 
variations in onset and intensity of spawning" 
(Paulet, 1990). 

Studies of the reproductive cycle or spawn- 
ing period of the clams are very important for 
elucidating their life histories. If they can be 
determined, their age determinations and 
recruitment period will be figured out. On the 
one hand, the inhibition period of harvesting 
clams during the spawning period of the year 
also will be determined. Therefore, data on 
the reproductive cycle or the spawning period 
are very useful for natural resources manage- 
ment of this species. 

First Sexual Maturity and Sexually 
Mature Length 

Ryou (1995) found that M. veneriformis 
reaches a shell length of 22.0 mm after one 
year and that the percentage of first sexual 
maturity of clams over 26.0 mm in length was 
over 50%, and in those over 32.0 mm in shell 
length was 100%. In the present study, per- 
centages of first sexual maturity of female and 
male clams were over 58% among those indi- 
viduals ranging from 26.0 mm to 30.0 mm in 
shell length, and 1 00% in those over 31 .0 mm. 

Sexually mature shell length of this popula- 
tion (50% of rate of group maturity) was 26.08 
mm. According to the growth curves for the 
mean shell length fitted to von Bertalanffy's 



equation by Kim & Ryou (1991), ages and 
shell lengths are as follows: 



Age (years) 


Mean shell length (mm) 


0.75 


17.7 


1.75 


32.3 


2.75 


39.7 


3.75 


43.9 


4.75 


47.2 


5.75 


49.5 



Therefore, individuals ranging from 26.0- 
30.0 mm (26.08 mm) in shell length are con- 
sidered to be one year old. We assume that 
both sexes of this population begin reproduc- 
tion at one year of age. In the aspect of nat- 
ural resources management, the present 
study suggests that catching surf clams <25 
mm in shell length or <1 year old causes a 
drastic reduction in its recruitment. 



ACKNOWLEDGEMENTS 

The authors are grateful to Dr. John B. 
Burch of the University of Michigan for helpful 
comments on the manuscript. Thanks are due 
also to Mr. Ye-Kyu Lee of the Electron Micro- 
scope Laboratory, Korea University, for his as- 
sistance with the transmission electron mi- 
croscopy, and to Mr. Fritz Paper, Mollusk 
Division of Museum of Zoology, University of 
Michigan, for computer assistance. This re- 
search was supported in part by the funds from 
the Environmental Research Projects ('92- 
'94) of the Coastal Research Center, Kunsan 



GAMETOGENESIS AND SEXUAL MATURATION OF THE SURF CLAM 



163 



National University and the Saemankeum 
Development Corporation of Chollabuk-Do, 
Korea. We appreciate this support. 

LITERATURE CITED 

BAYNE, B. L., 1976, Marine mussels: their ecology 
and physiology. Cambridge University Press. 
Cambridge. 506 pp. 

CHOI, К. C, 1969, Studies on the structure of tidal 
flat ecosystem for increasing commercial clam 
yield. The Korean Journal of Limnology, 2(3-4): 
1-21 (in Korean). 

CHUNG, E. Y.,S. Y. KIM & T. Y LEE, 1988, A study 
on sexual maturation of Mactra veneriformis 
Reeve. The Korean Journal of Malacology, 4(1): 
30-40. 

CHUNG, E. Y,T. Y LEE & С. M. AN, 1991, Sexual 
maturation of the venus clam, Cyclina sinensis, 
on the west coast of Korea. Journal of Medical 
and Applied Malacology, 3: 1 25 - 1 36. 

DORANGE, G. & M. LEPENNEC, 1989, Ultra- 
structural study of oogenesis and oocytic degen- 
eration in Pectén maximus from the bay of St. 
Brieuc. Marine Biology (Berlin), 103: 339-348. 

GRIFFITHS, R. J., 1977, Reproductive cycles in lit- 
toral populations of marine Choromytilus meri- 
dionalis (Kr) and Aulacomya ater (Molina) with a 
quantitative assessment of gamete production in 
the former. Journal of Experimental Marine 
Biology and Ecology 30: 53-71 . 

IWATA, K. S., 1 948, Artificial discharge of reproduc- 
tive substances by k-salts injection in Mactra 
veneriformis (Bivalves). Bulletin of the Japanese 
Society of Scientific Fisheries, 1 3(6): 1 88- 1 92 (in 
Japanese). 

JARAMILLO, R. & J. NAVARRO, 1995, Repro- 
ductive cycle of the Chiean ribbed mussel 
Aulacomya ater (Molina, 1782), Journal of Shell- 
fish Research, 14(1): 165-171. 

KIM, Y H. & D. К. RYOU, 1991, Study on the growth 
of Mactra veneriformis Reeve in the coast of 
Kunsan. Bulletin of Gunsan National Fisheries 
Junior College, 25(2): 41-47 (in Korean). 

KWON, O. K., G. M. PARK & J. S. LEE, 1993. 



Coloured shells of Korea. 288 pp. Academy 
Publishing Corporation, Seoul (in Korean). 

LEE, B. D., 1 956, The catalogue of molluscan shells 
of Korea. Bulletin of Pusan Fisheries College, 
1(1): 1-48 (in Korean). 

LONGO, F. J. & E. J. DORNFIELD, 1967, The fine 
structure of spermatid differentiation in the mus- 
sel, Mytilus edulis. Journal of Ultrastructure 
Research, 20: 462-480. 

MORVAN, О & A. D. ANSELL, 1988, Stereological 
methods applied to the reproductive cycle of 
Tapes rhomboïdes. Marine Biology (Berlin), 97: 
355-364. 

MORTAVKINE, P. A. & A. A. VARAKSINE, 1989, La 
reproduction chez les mollusques bivalves: rôle 
du système nerveux et régulation. Reports 
Scienti figues et Techniques de l'IFREMER, 10: 
250 pp. 

OSHIMA, Y, T. SUDO, J. HANAOKA & S. INO, 
1965, Shallow sea aquaculture. Daisei Publishing 
Corporation, Limited, pp. 252-253. 

PAU LET, Y. M., 1990, Role de la reproduction dans 
la déterminisme du recrutement chez Pectén 
maximus (L.) de la Baie de St. Brieuc. Thèse de 
Doctorat, Université de Bretagne Occidentale, 
France. 

PEARSE, J. S., 1965, Reproductive periodicities in 
several contrasting populations of Odontaster 
validus (Koechler), a common Antarctic asteroid. 
Biology of the Antarctic Sea, 2: 39-85. 

REVERBERI.G., 1971, Mytilus in experimental em- 
bryology of marine and freshwater invertebrates. 
Pp. 175-187, in G. REVERBERi, ed., Experimental 
embryology of marine and fresh-water inverte- 
brates. North-Holland, Amsterdam, London, xxiii 
+ 587 pp. 

RYOU, D. K., 1995, Ecological studies on the pop- 
ulation of surf clam, Mactra veneriformis Reeve. 
Thesis for Degree of the Ph. D. of Graduate 
school of Cheju National University. 110pp. (in 
Korean). 

YOO, J. S., 1976, Korean shells in colour, llgisa, 
Seoul. 129 pp. (in Korean). 

Revised ms accepted 23 February 2000 



MALACOLOGIA, 2000, 42(1-2): 165-170 



A NEW SPECIES OF PANACCA FROM CHILE 
(BIVALVIA: PHOLADOMYOIDEA: PARALIMYIDAE) 

Eugene V. Coan 

Department of Invertebrate Zoology, California Academy of Sciences, Golden Gate Park, San 
Francisco, California 94117-4599, U.S.A.; gene.coan@sierraclub.org 

ABSTRACT 

Panacea chilensis is described from off the Islas Juan Fernández, Chile, in 130-180 m, and 
is compared to other members of the family. All Recent species of the Pholadomyoidea are also 
listed to facilitate access to the relevant literature. 

Key words: Pholadomyoidea, Parilimyidae, Pholadomyidae, Panacea, Chile. 



INTRODUCTION 

In allocating a backlog of miscellaneous 
eastern Pacific bivalves in the Natural History 
Museum of Los Angeles County, a single right 
valve of a previously undescribed species of 
Panacea was encountered. This represents 
the first Recent record of the Pholadomyoidea 
from the eastern Pacific. This paper provides 
a name and description for this species and a 
list with references for the 14 named and one 
unnamed Recent members of the Pho- 
ladomyoidea. 

All members of the Pholadomyoidea are 
rare, fragile and represented in collections by 
only a few specimens. Thus far, the anatomy 
of only two taxa have been studied, as indi- 
cated below. 



SYSTEMATIC ACCOUNT 
Superfamily Pholadomyoidea Gray, 1847 
Family Parilimyidae Morton, 1982 

Anatomically this family is differs from the 
Pholadomyidae in having a smaller palliai 
sinus, in having large taenioid muscles, lack- 
ing pedal retractors, in having radial mantle 
glands, reduced labial palps, an extensively 
muscularized gut, having a type II rather than 
a type VI stomach, with a large left pouch, and 
in lacking a opisthopodium (Morton, 1982: 
205, table I). 

These differences are based on the anat- 
omy of a single species, Parilimya fragilis 
(Greig, 1920), and additional species must be 



examined before relationships are completely 
clear. 

Genus Panacea Dall, 1905 

Panacea Dall, 1905: 143, new name for 
Aporema Dall, 1903: 1532, non Scudder, 
1890: 369-370, pi. 20 [Insecta]. Type species 
(original designation): Pholadomya arata Ver- 
rill & Smith, in Verrill, 1881 . Recent, New Eng- 
land. 

Notomya Cotton, 1931: 342, non M'Coy, 
1847: 303 [Bivalvia: Megadesmidae]. Type 
species (original designation): Pholadomya 
tasmanica Hedley & May, 1914. Recent, Tas- 
mania. 

Shell cunéate, anterior end much shorter, 
with conspicuous radial sculpture. 

Runnegar (1974: 928) suggested that the 
Cretaceous genus Procardia Meek, 1871: 
184, might prove to be a senior synonym of 
Panacea (type species, by monotypy, Isocar- 
dia? hodgei Meek, 1871: 183-184); this 
clearly merits additional investigation. 

Panacea chilensis, new species 
(Figures 1, 2) 

Type Material & Locality 

LACM 2876, holotype, a right valve; length, 
21.0 mm; height, 11.3 mm; width, 6.5 mm. 
LACM 65-1 01 , off west side of "Juan Fernán- 
dez Island" [Isla Robinson Crusoe], Islas Juan 
Fernández, Chile (33°34-41'S, 78°45-55'W); 
130-180 m; Anton Bruun Cruise 12; 13-15 
December 1965. 



165 



166 



COAN 



Description 

Shell cunéate, much longer posteriorly 
(beaks at 25% from anterior end). Entire exte- 
rior surface covered with minute granules, 
most coarse ventrally and posteriorly, and 
moderate commarginal growth lines. Anterior 
slope rounded, slightly sinuous, with commar- 
ginal growth lines only; central slope with 11 
conspicuous radial ribs, smallest posteriorly, 
lower dorsally, more elevated ventrally, scal- 
loping ventral margin; with three much finer 
radial ribs posterior to these; posterior end 
produced; posterior slope with commarginal 
growth lines only. 

Ligament external, approximately 2.8 mm 
long, seated on a low nymph. Anterior adduc- 
tor muscle scar elongate; palliai line conspic- 
uous, with a small sinus; anterior adductor 
muscle scar larger, rounded. Taenioid muscle 
scar conspicuous (Fig. 2). 



DISCUSSION, COMPARISONS AND 
LIST OF SPECIES 

The genera within the Parilimyidae are 
much in need of further evaluation. Morton 
(1982) hesitated to synonymize Panacea with 
the earlier-named Parilimya, because it had 
not then been shown that species placed in 
Panacea have large taenioid muscles. The 
large scar in the middle of the holotype of 
Panacea chilensis shows that such a muscle 
is indeed present (Fig. 2). This leaves only the 
cunéate shape and sinuous anterior margin of 
the six species of Panacea to distinguish them 
from those placed Parilimya. 

No characters seem to separate Nip- 
ponopanacca from Parilimya, and they are 
here regarded as synonyms. Although Mat- 
sukuma (1989) cited Morton's (1982) detailed 
anatomical study of Parilimya, he relied on 
shell characters for his arrangement of gen- 
era, with Nipponopanacca placed as a sub- 
genus of Pholadomya. 

The situation is further complicated by the 
substantial fossil record of seemingly related 
forms (Cox & Newell, 1969: 828-838; Run- 
negar, 1974: 927-928). Indeed, Runnegar 
suggests that the Cretaceous genus Procar- 
dia Meek, 1871, may be a senior synonym of 
Panacea; if true, and if Panacea and Parilimya 
were also synonymized, Procardia would be 
the earliest name. 

Until a comprehensive comparative review 
is made of all the Recent species, together 



with consideration of the genera based on 
fossil taxa, I hesitate to propose further syn- 
onymies at the generic level. 

Genus Panacea Dall, 1905 

Panacea africana (Locard, 1898: 165-167, 
474, pi. 7, figs. 42-45, ex Fischer ms) [Pho- 
ladomya], from 2,083-2,324 m off the west 
coast of Morocco, is closest to P. chilensis and 
differs in having a more rounded, less pro- 
duced anterior end and 10-12 major radial 
ribs with smaller ribs between them (Fig. 3). 

Panacea arrala (Verrill & Smith, in Verrill, 
1881: 301-302) [Pholadomya], type species 
of Panacea, from 126-238 m off Martha's 
Vineyard, Massachusetts, has much finer 
sculpture. See also Verrill (1882: 567, 587, pi. 
58, fig. 37; 1884: 278, 292, pi. 30, figs. 4-6), 
Dall (1889: 64, pi. 45, figs. 4-6, pi. 65, figs. 
133-134), Morton (1982: 162, pi. 1), and 
Johnson (1989:22) (Fig. 4). 

Panacea locardi (Dall, 1903: 1532) [Pho- 
ladomya], based on " Pholadomya arata Verrill 
& Smith, in Verrill, 1881," of Locard (1898: 
167-168, 475, pi. 8, figs. 1-5), from 1,130 m 
off west Africa, like P. arrala, is covered with 
finer sculpture (Fig. 5). This was a new 
species based on Locard's description and 
figures and not the renaming of a homonym, 
as indicated by Boss et al. (1968: 190). 

Panacea sumatranaTh\e\e & Jaeckel, 1931 
(pp. 244-245, pi. 5, fig. 123), from 175 m off 
Siberut Island, Indonesia, differs in being 
more trigonal, and in having 19 radial ribs, 
which extend onto the anterior slope (Fig. 6). 

Panacea tasmanica (Hedley & May, 1914: 
132-133, 3 text-figs.) [Pholadomya], type 
species of Notomya Cotton, 1 931 (non M'Coy, 
1847), from 91 m off Port Arthur, Tasmania, 
Australia, is trigonal and also differs in having 
19 radial ribs, which extend onto the posterior 
slope (Fig. 7). 

Genus Parilimya Melvill & Standen, 1899 

Parilimya Melvill & Standen, 1899. Type 
species (monotypy): Pholadomya (Parilimya) 
haddoniMe\v\\\ & Standen, 1899. Recent, Tor- 
res Straits. 

Nipponopanacca Habe, 1977: 303. Type 
species (original designation): Pholadomya 
(Panacea) sakuraii Habe, 1958a. Recent, 
Japan. 

Species that remain here are ovate to 
ovate-elongate, rather than having the 
cunéate shape of Panacea, and they do not 




FIG. 1,2. 1 , Panacea chilensis, new species; right valve, holotype, LACM 2876; length, 21 .0 mm. 2, camera- 
lucida sketch showing positions of adductor muscle scars, palliai line and sinus, and taenioid muscle scar 
(near center). 

FIG. 3. Panacea africana (Locard, 1898), right valve, from Locard (1898: pi. 7, fig. 42); original specimen 
length, 13.7 mm. 

FIG. 4. Panacea anata (Verrill & Smith, in Verrill, 1881), right valve, from Verrill (1882: pi. 58, fig. 37); origi- 
nal specimen length, 36 mm. 

FIG. 5. Panacea locardi (Dall, 1903), right valve, from Locard (1898: pi. 8, fig. 1); original specimen length, 
26.7 mm. 

FIG. 6. Panacea sumatrana Thiele & Jaeckel, 1931, left valve, from Thiele & Jaeckel (1931: pi. 5, fig. 123); 
original specimen length, 32.5 mm. 

FIG. 7. Panacea tasmanica (Hedley & May, 1914), right valve, from Hedley & May (1914: 133, text-fig.); orig- 
inal specimen length, 34 mm. 



168 



COAN 



have its sinuous anterior margin. Several 
have more numerous fine radial ribs. 

Pahlimya fragilis (Grieg, 1920: 8, pi. 1, figs. 
5-7) [Pholadomya], from 1,100 m off Nova 
Scotia is thin, ovate-trigonal, with 18 radial 
ribs concentrated on the central slope. See 
also Soot-Ryen (1966: 12-15, pi. 1, fig. 6, 
text-figs. 6, 8-10) and Morton (1982). 

Pahlimya haddoni (Melvill & Standen, 1899: 
203-206, pi. 11, fig. 22) [Pholadomya (Pahl- 
imya)], from 10 m off Warrior Island, Torres 
Straits, is ovate-subquadrate, smooth at both 
ends, with 20 fine radial threads. See also 
Morton (1982: 168-170, pi. 4 and figs. 4, 5). 

Pahlimya levicaudata (Matsukuma, 1989: 
211-213, 214-215, 218-219, pi. 1, figs. 7- 
12, 220-221, pi. 2, figs. 7, 8) [Pholadomya 
(Nipponopanacca)], from 50-60 m off Cape 
Noshap, Wakkanai, Hokkaido, is ovate-elon- 
gate, with a long posterior end and is covered 
by fine radial threads. 

Pahlimya loveni (Jeffreys, 1882: 934, pi. 70, 
fig. 7) [Pholadomya], from stations off Portu- 
gal and Spain (type locality not yet restricted) 
in 523-1,314 m, is ovate and covered by 32 
fine radial ribs. See also Waren (1980: 51) 
and Morton (1982: 166, fig. 3). 

Pahlimya maoria (Dell, 1963: 206-207, pi. 
1, figs. 1, 2) [Pholadomya], from 622 m be- 
tween Alderman and Red Mercury islands, 
North Island, New Zealand, is ovate-elongate, 
with 13 radial ribs concentrated on the central 
slope and with radial threads throughout. See 
also Powell (1979: 431, pi. 78, fig. 16) and 
Morton (1982: 166-167, pi. 3 and fig. 2). 

Pahlimya pacifica (Dali, 1907: 172-173) 
[Pholadomya], from 80 m off Hakodate and 
196 m off Nagasaki, Japan, is oval, heavy, 
smooth on both ends, and has 11 strong radial 
ribs. See also Dall (1909a: 115-117; 1909b: 
142-143; 1925: 24, pi. 29, figs. 8, 9), Habe 
(1958a: 275, pi. 13, fig. 7), Boss et al. (1968: 
235), Morton (1 982: 1 71 -1 72, pi. 5 and fig. 6), 
and Matsukuma (1989: 209-210, 214, 220- 
221, pi. 2, figs. 4-6). 

Pahlimya sinica (Xu, 1992: 211-213, text 
fig.) [Pholadomya (Nipponopanacca)], from an 
unknown depth in the Okinawa Trough, East 
China Sea, is ovate-elongate, with low corn- 
marginal lamellae and pustules radially ar- 
ranged only on the beaks. See also Xu (1999: 
111, fig. 71 ), but with the journal and pagination 
of the original description misquoted. 

Pahlimya sukuraii (Habe, 1958a: 179-180, 
176, fig. 4) [Pholadomya (Panacea)], from 
200 m off Honshu, Japan, is ovate, slightly 
truncate anteriorly, and has fine radial 



threads. See also Habe (1977: 303-304, pi. 
64, fig. 3) and Matsukuma (1989: 210-211, 
214, 218-219, text-fig. 1, pi. 1 , figs. 1-6). 

Pahlimya sp. from an unknown depth off 
Taiwan is oval, with commarginal undulations 
and radial rows of pustules (Matsukuma, 
1989: 213-214, 215, 220-221, pi. 2, figs. 
1 -3, as ''Pholadomya (Nipponopanacca) '. 

Family Pholadomyidae Gray, 1847: 194 

Genus Pholadomya G. B. Sowerby I, 1823 

Pholadomya G. B. Sowerby I, 1823: 2 pp., 
1 pi. Type species (subsequent designation of 
Gray, 1847: 194): Pholadomya candida G. В. 
Sowerby I, 1823: 2 pp., 1 pi. Recent, Carib- 
bean. See also Runnegar (1972, 1979), Mor- 
ton (1980), Gibson-Smith & Gibson-Smith 
(1980), and Diaz & Borrero (1995). Morton 
(1980) contains a detailed anatomical ac- 
count. 



ACKNOWLEDGMENTS 

I appreciate the assistance of Lindsey 
Groves of the Natural History Museum of Los 
Angeles County in making the specimen 
available to me. Alan R. Kabat provided 
copies of needed literature. Bruce Runnegar 
provided some information. One anonymous 
reviewer provided several helpful sugges- 
tions. Sharon Williams made up the plate. 



LITERATURE CITED 

BOSS, K. J., J. ROSEWATER & F. A. RUHOFF, 
1968, The zoological taxa of William Healey Dall. 
Bulletin of the United States National Museum. 
287: 427 pp. 

COTTON, В. O, 1931, Pelecypoda of the Flinder- 
sian region, southern Australia. No. 2. Records of 
the South Australian Museum, 4(3): 333-354. 

COX, L. R., with additions by N. D. NEWELL, 1969. 
Family Pholadomyidae Gray, 1847. Pp. 827- 
838, in: L. R. cox et al., eds., Treatise on Inverte- 
brate Paleontology. Part N (Mollusca 6)(2): 
491 -952. Lawrence, Kansas, Geological Society 
of American & University of Kansas 

DALL, W. H., 1889, A preliminary catalogue of the 
shell-bearing marine mollusks and brachiopods 
of the south-eastern coast of the United States, 
with illustrations of many of the new species. Bul- 
letin of the United States National Museum, 37: 
221 pp., 74 pis. 

DALL. W. H., 1903. Contributions to the Tertiary 
fauna of Florida, with especial reference to the 



NEW SPECIES OF PANACCA 



169 



silex beds of Tampa and the Pliocene beds of the 
Caloosahatchie River, including in many cases a 
complete revision of the generic groups treated of 
and their American Tertiary species. Part VI. Con- 
cluding the work. Transactions of the Wagner 
Free Institute of Science of Philadelphia, 3(6): xiv 
+ 1219-1654 pp., pls.48-60. 

DALL, W. H., 1905, Note on the genus Aporema 
Dall. The Nautilus, 18(12): 143. 

DALL, W. H., 1907, Descriptions of new species of 
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Revised ms accepted 1 April 2000. 



MALACOLOGIA, 2000, 42(1): 171-201 

A REVISION OF THE GENUS BELGRANDIA, WITH THE DESCRIPTION OF A NEW 
SPECIES FROM FRANCE (CAENOGASTROPODA: HYDROBIIDAE) 

Martin Haase 

Institut für Natur-, Landschafts- und Umweltschutz, Universität Basel, St. Johanns- Vorstadt 10, 
CH-4056 Basel, Switzerland; martin, haase @unibas.ch 

ABSTRACT 

A new species of the hydrobiid genus Belgrandia Bourguignat, 1869, B. gfrast, nov. sp., from 
the nature reserve Petite Camargue Alsacienne in France is described. In order to define the new 
species, it was necessary to investigate the syntype series or topotypes of the known nominal 
species. This revision is based on morphometric analyses of the shells and demonstrates that 
the number of diagnosable species is much higher than 20th century revisions have suggested. 
On the other hand, several nominal species attributed to Belgrandia turned out to belong to 
Bythinella Moquin-Tandon, 1856. I recognize 16 species and one subspecies — this figure does 
not include Italian taxa, which are the subject of a revision by Italian colleagues — as belonging 
to Belgrandia. The range of this genus covers the Iberian Peninsula, France, Italy, Croatia, and 
Greece. The highest diversity is found in southern France, with nine species. 

Morphologically, the new species is characterized by the combination of a pointed apex, con- 
vex whorls, a single varix on the body whorl, which is mostly close to the aperture but may lie up 
to half a whorl behind it, and a ratio of shell height to shell width of 1.78 (mean). Anatomically 
diagnostic features are the wide visceral oviduct and the penis, with a wide, rounded muscular 
lobe on the left side in the distal half. Occasionally, there is a second, smaller lobe on the right 
side of the penis. The generic allocation is justified through the shell varix and the distal female 
genitalia with two receptacula seminis and a bursa copulatrix originating from the renal oviduct, 
which describes a loop of 270°. All specimens investigated anatomically had peritrich ciliates of 
the genus Trichodina Ehrenberg, 1830, in their mantle cavities. 

The new species is only known from a single locality. It lives in constantly cool water 
(11.3-12.3°C), which has high concentrations of calcium and magnesium. However, the con- 
centrations of nitrate, chloride and potassium are also considerable. The sources of this conta- 
mination are probably the nearby settlements and agriculture. 

Key words: anatomy, Belgrandia, Hydrobiidae, morphology, systematics. 



INTRODUCTION 

The Petite Camargue Alsacienne is a rem- 
nant of the former wetlands on the left bank of 
the river Rhine about 5 km north of Basel in 
the southeast of Alsace, France (Fig. 1). 
These wetlands have been largely destroyed 
through the construction of two channels, the 
Canal de Huningue and the Grand Canal 
d'Alsace, and of embankments during the 
19th and early 20th centuries. These mea- 
sures cut off the wetlands from the Rhine. 
Regular flooding was prevented, and the 
groundwater level dropped. But the Petite 
Camargue Alsacienne partly kept its charac- 
ter as wetland owing to a number of springs 
emerging in the southwest and water diverted 
from the Canal de Huningue (Schenker, 
1992). The Petite Camargue Alsacienne is 
characterized by a high diversity of habitats 



and hence plants and animals and therefore 
has been declared nature reserve in 1982. 
Since then, measures for the revitalisation 
and management have been taken, and an 
inventory of flora and fauna is being made 
(Schenker, 1992; Dürrer, 1998). 

During pilot studies for the inventory of mol- 
lusks an hitherto undescribed hydrobiid 
species belonging to the genus Belgrandia 
Bourguignat, 1869, was found. This genus 
has been established for species with conical 
shells bearing one or more varices close to 
the aperture (Bourguignat, 1869). Later, few 
anatomical observations also of populations 
attributed to the type species, B. gibba 
(Draparnaud, 1805), allowed the diagnosis to 
be extended (Giusti & Pezzoli, 1980). 
However, comprehensive anatomical studies 
of representatives of this genus are still 
scarce, so that it is still not well defined. And 



171 



172 



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1 km 



SWITZERLAND 



FIG. 1. Situation of the Petite Camargue Alsacienne (stippled area). Arrow indicates type locality of 
Belgrandia gfrast, nov. sp. 



REVISION OF BELGRANDIA 



173 



the varix is not an apomorphy restricted to 
Belgrandia. It is also found in representatives 
of the genus Bythinella Moquin-Tandon, 1856 
(Boeters, 1998; see Discussion). 

The Recent distribution of Belgrandia 
ranges from the Iberian Peninsula (Boeters, 
1 988) over southern and central France (Ger- 
main, 1931) to Italy (Giusti & Pezzoli, 1980), 
Croatia (Schutt, 1961), and Greece (Bodon et 
al., 1999). Fossil shells attributed to Bel- 
grandia were found near Paris (Bourguignat, 
1869), in Great Britain (Sandberger, 1880), 
eastern Germany (Sandberger, 1870-1875), 
and Latium, Italy (Settepassi & Verdel, 1965). 

In order to delimitate the new species, it has 
been necessary to reinvestigate type material 
of nominal species at one or another time 
attributed to Belgrandia (Appendix 1). The 
resulting revision is based only on shell mor- 
phology. Therefore, because of the lack of 
anatomical data, no definite statements can 
be made about the true generic allocation of 
species retained in Belgrandia and about 
potential synonymies. On the other hand, it 
has been possible to exclude and reallocate a 
number of nominal species. In some cases, 
the original material could not be traced, or 
the respective collections were not accessible 
because of shortage of personnel. In these 
cases, topotypes were investigated or, where 
not available, I had to rely on the original 
descriptions. The species known from Italy 
are not explicitly treated in the present paper, 
because they are subject of a revision pre- 
pared by Cianfanelli, Bodon, Manganelli & 
Giusti (pers. comm.). However, I have also 
investigated the syntypes or topotypes of 
most of these species for the delimitation of 
the new species. For this purpose, published, 
mostly qualitative characters proved to be suf- 
ficient, so that new observations on these 
Italian taxa need not be incorporated in the 
present account. The same holds for the com- 
parison with Litthabitella chilodia iónica 
Schutt, 1980, recently tentatively attributed to 
Belgrandia (Bodon et al., 1999), which was 
not available for investigation. 

The detailed description of the new species 
is based on character sets and state defini- 
tions provided by Hershler & Ponder (1998), 
who attempt to standardize the terminology 
and interpretation of characters used in taxo- 
nomic descriptions of hydrobioid gastropods 
{sensu Davis, 1979) as a basis for a phyloge- 
netic analysis of this poorly understood group. 
In addition, data of ten physicochemical para- 
meters measured in the type locality of the 



new species by Berger (1993) are provided in 
order to characterize its habitat in terms of 
abiotic factors. 



MATERIAL & METHODS 

More than 100 specimens of the new 
species were collected on July 6th, 1998, in 
the origin of the Chenal des Sources (spring 
channel) in the Petite Camargue Alsacienne 
(Fig. 1). The animals were fixed either in 70% 
ethanol or Bouin's solution. Some of the 
snails were relaxed with menthol prior to fixa- 
tion. Shells were measured using a dissecting 
microscope equipped with an ocular microm- 
eter. The number of whorls was estimated to 
the nearest eighth of a whorl. Morphometric 
analyses of nine shell parameters were per- 
formed using the computer programs SYN- 
TAX 5.02 (Podani, 1993) and StatView 5.0 
(SAS Institute Inc., 1998). Principal compo- 
nent analyses (PCA) were performed starting 
from correlation matrices. Dendrograms 
(UPGMA, unweighted pairgroup method 
using arithmetic averaging; MST, minimum 
spanning tree) are based on Euclidean dis- 
tances computed from z-standardized means 
or individual values where only a single spec- 
imen was available. The anatomy was inves- 
tigated by dissection and reconstruction of 
histological serial sections using the computer 
program SURFdriver versions 2.5.5 and 3.0 
(Moody & Lozanoff, 1997). The hardparts, 
which were cleaned with sodium hypochlorite, 
as well as external features of the critical- 
point-dried soft body were sputtered with plat- 
inum and examined with a Jeol JSM 6300F 
scanning electron microscope (SEM). The 
physicochemical data of the origin of the 
spring channel, the type locality of the new 
species, presented following the systematic 
description are taken from Berger (1 993), who 
measured ten parameters from March to 
December 1990 in weekly intervals. 

Abbreviations of museum collections: 

ВОЕ, private collection of H. D. Boeters, 
Munich; GNM, Göteborgs Naturhistoriska Mu- 
seum (Gothenburg); MHNG, Museum d'His- 
toire Naturelle (Bourguignat collection), 
Genève (Geneva); MNHN, Museum National 
d'Histoire Naturelle, Paris; NHMW, Natur- 
historisches Museum Wien (Vienna); NMB, 
Naturhistorisches Museum Basel; SMF, Na- 
turmuseum und Forschungsanstalt Sencken- 



174 



HAASE 



berg, Frankfurt; ZSM, Zoologische Staats- 
sammlung München (Munich). 

RESULTS 
The Species of Belgrandia 

As mentioned in the Introduction, several 
old collections are currently not accessible. 
This holds also for Paladilhe's collection, 
which is housed in the Medical Faculty of the 
University of Montpellier. However, Paladilhe 
extensively exchanged material with Bourgui- 
gnat, so that many samples of Paladilhe's 
original material, often syntypes, can be found 
in Geneva, where Bourguignat's collection 
has been deposited. 

In the following short descriptions, a section 
Material Examined is inserted in cases where 
the original material was not available or if a 
specification is necessary because only part 
of the type material was investigated. The 
Descriptions are based on the material exam- 
ined unless otherwise stated. Additional infor- 
mation from the literature may be given in the 
Remarks. Lectotypes are designated in order 
to unambiguously define the respective 
species. The morphometric data are summa- 
rized in Table 1 . I do not refer to this table 
each time. Only those parameters character- 
izing a species are mentioned. The power of 
the statistical tests is quite low in most cases, 
for which only a small number of individuals 
was available. Therefore, the Bonferroni pro- 
cedure correcting a in multiple parallel tests 
has to be considered conservative. The 
Remarks discuss only species-specific, taxo- 
nomic issues. Other issues are put into a 
wider context in the Discussion. 

Belgrandia 
Bourguignat, 1869 

Belgrandia Bourguignat, 1869: 13-15 

Thermhydrobia Paulucci, 1878 -Clessin, 
1882: 137 f. 

Type Species: Cyclostoma gibbum Drap- 
arnaud, 1805, by subsequent designation 
(Kobelt, 1878). 

Diagnosis: Shell conical, with pointed apex; 
one or several varices on the body whorl, 
occasionally there is no varix or it is replaced 
by folds; aperture oval; ctenidium with about 
ten filaments; radula with a single pair of basal 



cusps on the central tooth; penis with one 
muscular lobe on the left side, occasionally a 
second lobe on the right side; female genital 
system with a sac-shaped bursa copulatrix 
and two seminal receptacles, the proximal 
one arising from the proximal-most point of 
the loop of the renal oviduct, the distal one 
shortly before the origin of the bursal duct; 
renal oviduct unpigmented, describing a sim- 
ple, wide loop of 270°. 

Remarks: This diagnosis is largely based 
on the findings of Giusti & Pezzoli (1980). As 
already mentioned before, only few (nominal) 
species have been investigated anatomically, 
so that this diagnosis has to be considered 
preliminary and will probably be extended 
when more data become available. At present 
the following characters allow to differentiate 
species: shell shape; number, position, and 
formation of varices; shape of penis; number, 
position, and formation of penial lobes. 



Belgrandia gibba 
(Drapamaud, 1805) 

Cyclostoma gibbum Draparnaud, 1805: 38 

Belgrandia varica (Paget, 1854)-Germain, 
1913:301 

Belgrandia moitessieri (Bourguignat, 
1866)-Germain, 1913: 301 

Belgrandia gibberula Paladilhe, 1869 
Germain, 1913: 301 

Belgrandia cazioti Locard, 1 892 - Germain, 
1913:301 

Hydrobia paladilhi Moitessier, 1869 
Germain, 1931: 632 

Belgrandia bourguignati de Saint-Simon, 
1870 -Germain, 1931: 632 

Type Material: lectotype NHMW 100519, 
here designated in order to define the 
species; paralectotypes NHMW 100520 (3) 

Type Locality: France, not further specified 

Description: Shell (Fig. 2A) high (mean 2.13 
mm), moderately slender, with up to 4.375 
whorls; deep sutures separate the markedly 
convex whorls; up to five very prominent 
varices on body whorl up to 1/2 a whorl behind 
outer lip; aperture small relative to shell; 
umbilicus very narrow. 

Remarks: The precise origin of Drapar- 
naud's specimens is not known. Draparnaud 
lived in Montpellier, and therefore it is usually 
assumed that the species he described were 
collected in southern France. Giusti & Pezzoli 
(1980) investigated individuals from two 



REVISION OF BELGRANDIA 



175 



French populations they attributed to B. gibba 
and described the penis as being conical and 
pointed, with a short, pointed lobe on the left 
side close to the tip. 

The synonymization of six nominal species 
with B. gibba by Germain (1913, 1931) is 
problematical, because he did not give argu- 
ments. The only feature all these species 
have in common is their occurrence in south- 
ern France. However, the morphometric 
analysis (Figs. 4, 5; Appendix 2) indicates that 
probably none of them is B. gibba, and most 
of them may be good species. This issue is 
further discussed for each nominal species 
under its respective description. 

Moguin-Tandon (1856) has introduced the 
three varieties uniplicata, marginata and 
aplexa, which differ only in the number and 
position of the varices. Taxonomically, this dif- 
ferentiation is certainly not warranted, since 
all three varieties are from a single locality, the 
source of the river Lez near Montpellier. To 
which species these varieties really belong 
could not be determined, because the where- 
abouts of Moquin-Tandon's syntypes are not 
known. Therefore, I tentatively consider all 
three varieties as synonyms of B. gibba. 

Belgrandia marginata 
(Michaud, 1831) 

Paludina marginata Michaud, 1831 : 98 

Belgrandia boscae Salvana (1877)-Boe- 
ters, 1988:223 

Type Material: not traced 

Type Locality: Draguignan, river Var, France 

Material Examined: Foux de Draguignan, 
France, ex ВОЕ 291a (10) 

Description: Shell (Fig. 2B) very slender, on 
average 1 .83 mm high and thus rather small, 
with slightly more than four markedly convex 
whorls; spire high relative to aperture height; 
aperture almost as high as wide with a 
straight outer lip (frontal view); one prominent 
varix close to lip; umbilicus a very narrow slit. 

Remarks: Belgrandia boscae should not be 
regarded as synonym for reasons outlined 
below. 

Belgrandia conoidea 
(Reynies, 1844) 

Paludina conoidea Reynies, 1844: 4 f. 
Type Material: not traced 
Type Locality: Ardus sur I'Aveyron, near 
Montauban, France 



Material Examined: Le Lez à Montpellier 
MHNG (2) 

Description: Shell (Fig. 2C) broadly conical, 
high (larger specimen 2.14 mm), with about 
four convex whorls; aperture high relative to 
shell height; body whorl without varices; 
umbilicus a more or less narrow slit. 

Remarks: This species has been largely 
neglected in 20th century revisions of the 
French malacofauna. Recently, it has been 
attributed to Belgrandia (Falkner et al., in 
prep.). However, since this species lacks 
varices, this allocation must remain doubtful 
until confirmation through anatomical investi- 
gations. It is of course possible that B. 
conoidea has lost the varices and hence this 
state is apomorphic. Reynies' syntypes could 
not be traced. The material examined from 
Bourguignat's collection is probably a sample 
mentioned and attributed to this species, 
albeit under the genus Hydrobia Hartmann, 
1821, by Paladilhe (1870). Thus, it is ques- 
tionable that this sample really belongs to B. 
conoidea. 



Belgrandia varica 
(Paget, 1854) 

Hydrobia varica Paget 1 854: 454 f. 

Belgrandia gibba (Draparnaud, 1805)- 
Germain, 1913: 301 

Type Material: not traced 

Type Locality: river Var near Nizza below 
the mill of Davigo and a ditch at the 
Grenouillères 

Description: Shell ovate-conical, up to 2 
mm high and 1.25 mm wide with up to 4 
whorls, which are separated by deep sutures; 
last whorl ventricose, not detached, a single 
varix close to outer lip; aperture obligue, with 
an obtuse angle. 

Remarks: This description is based on 
Paget's (1854) original. His collection is 
apparently lost. Although Paget quite clearly 
distinguished his species from B. gibba, and 
although the type localities of these species 
are distant from each other, Germain (1913) 
considered B. varica as junior synonym of the 
latter. Considering the type locality of B. var- 
ica, one might rather assume affinities with B. 
marginata. Both hypotheses, however, would 
need verification through the investigation of 
topotypical material, which requires that the 
type locality is still intact. 



176 



HAASE 



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REVISION OF BELGRANDIA 177 



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178 



HAASE 




FIG. 2. Shells. А, В. gibba, lectotype (left) and 2 paralectotypes; B, B. marginata. topotypes; С, В. conoidea 
D, В. moitessieri, lectotype (left) and 2 paralectotypes; E, B. gibberula, lectotype (left) and 2 paralectotypes- 
F, B. gibberula var.; G, B. gfrast, nov. sp., 3 paratypes. Scale bar = 2 mm. 



Belgrandia moitessieri 
(Bourguignat, 1866) 

Hydrobia moitessieri Bourguignat, 1866' 
191 

Belgrandia gibba (Drapamaud, 1805) 
Germain, 1913: 301 



Type Material: lectotype MHNG, here des- 
ignated in order to define the species; para- 
lectotypes MHNG (11) 

Type Locality: source of the river Martinet 
near Montpellier, France 

Description: Shell (Fig. 2D) broadly conical, 
with a height of on average 1.68 mm very 



REVISION OF BELGRANDIA 



179 



small, up to 3.75 rapidly growing, convex 
whorls; body whorl inflated; almost round 
aperture wide relative to shell width; 0-3 
varices not more than 1/4 whorl behind the 
outer lip; umbilicus a narrow slit. 

Remarks: This species can readily be dis- 
tinguished from B. gibba in all parameters 
measured but aperture width and the ratio 
shell width/aperture width (Mann Whitney Li- 
test, P < 0.05; however, after Bonferroni-cor- 
rection the three other ratios are also no 
longer significant), and is very remote from all 
other species in the morphospace displayed 
by the PCA in Fig. 5 (for details about the 
PCA, see Appendix 2). Consequently, B. 
moitessieri should be treated as a valid 
species until anatomical data might prove the 
contrary. 



Belgrandia lusitanica 
(Paladilhe, 1867) 

Paludina gibba (Michaud, 1831)-Morelet, 
1845:91 

Hydrobia lusitanica Paladilhe, 1867: 60 f 

Belgrandia occidentalis Clessin, 1878 
Clessin, 1882: 136 

Hydrobia gibba (Draparnaud, 1805)- 
Nobre, 1885: 58 

Type Material: lectotype [MHNG], here des- 
ignated in order to define the species; para- 
lectotypes [MHNG (5)] 

Type Locality: Fonte das Lagrimas near 
Coimbra, Portugal 

Description: Shell (Fig. 3E) slender conical, 
small, with a height of on average 1.69 mm; 
slightly more than four not very convex 
whorls; aperture adapically with a more or 
less distinct angle; one distinct vahx closely 
behind outer lip; umbilicus a very narrow slit; 
some individuals carry egg capsules mainly 
on sutures. 

Remarks: Belgrandia lusitanica cannot be 
confused with B. gibba, because it is much 
smaller and has constantly a single varix 
behind the outer lip, contrary to B. gibba, 
where up to five varices which are, in addition, 
much more prominent, can be found. Clessin 
(1878a) overlooked Paladilhe's description 
when he introduced B. occidentalis and sub- 
sequently put his own name in synonymy 
(Clessin, 1882). Very similar and geographi- 
cally closest is B. heussi heussi Boettger, 
1963 (Fig. 5). The differentiation of these 
species is discussed in the Remarks to the 
latter. 



Belgrandia bigorriensis 
Paladilhe, 1869 

Belgrandia bigorriensis Paladilhe, 1869: 
125 f 

Belgrandia vitrea (Draparnaud, 1801)- 
Germain, 1913: 303 

Type Material: lectotype MHNG, here des- 
ignated in order to define the species; para- 
lectotype MHNG (1) 

Type Locality: a ferruginous spring near 
Bigorre, France 

Description: Conical shell (Fig. ЗА) rela- 
tively large, with a height of 2.44 mm; the lec- 
totype has 4.625 convex whorls; aperture nar- 
row relative to shell width; one not very 
prominent varix close to outer lip; umbilicus 
an open slit. 

Remarks: Only the lectotype could be 
investigated, because the paralectotype once 
had apparently been glued onto cardboard 
and still has remnants of the glue. According 
to Paladilhe's (1969) original description, 
there can be a second varix. The most similar 
species is B. coutagnei Locard, 1892. 
However, this species is smaller, more slen- 
der and has a much wider aperture relative to 
shell size. Belgrandia bigorriensis is clearly a 
distinct species. 

Belgrandia vitrea (Cyclostoma vitreum) has 
been identified as belonging to Moitessieria 
Bourguignat, 863 (Boeters, 1969, 1972), 
which is conchologically characterized by a 
very slender, turriform shell with fine spiral 
sculpture. Therefore, B. bigorriensis cannot 
be a junior synonym of Draparnaud's species. 



Belgrandia gibberula 
Paladilhe, 1869 

Belgrandia gibberula Paladilhe, 1869: 126- 
128 

Hydrobia paladilhi Dubrueil, 1869 -Pal- 
adilhe, 1870:228 

Belgrandia gibba (Draparnaud, 1805)- 
Germain, 1913: 301 

Type Material: lectotype MHNG, here des- 
ignated in order to define the species; para- 
lectotypes MHNG (>50) 

Type Locality: Source de Verdun near Saint 
Guilhem-le-Désert, France 

Description: Shell (Fig. 2E) on average 1 .89 
mm high, ovate conical; mean number of 
whorls (3.54), which are convex and slightly 
shouldered, low; aperture in both directions 
measured wide relative to shell size; 0-2 



180 



HAASE 




FIG. 3. Shells. А, В. bigorriensis, lectotype; В, B. coutagnei, lectotype (left) and 2 paralectotypes; С, B. cazi- 
of/ Westerlund, lectotype; D, B. cazioti Locard non Westerlund, syntypes; E, ß. lusitanica, lectotype (left) and 
2 paralectotypes; F, B. boscae, topotypes; G, В. heussi heussi, paratypes; H, B. heussi alcoaensis, paratype; 
I, B. torifera, topotypes. Scale bar = 2 mm. 



rather flat varices on the last 0.25 whorls; 
umbilicus a very narrow slit or completely 
covered. 

Remarks: Belgrandia gibberula is morpho- 
metrically unique (Figs. 4, 5) and can hardly 
be confused with any other species. That 



holds also for a comparison with B. gibba, 
which is larger, more slender, has a smaller 
aperture, non-shouldered whorls, and up to 
five very prominent varices. Therefore, a syn- 
onymization of both species is unwarranted. 
Paladilhe (1870) considered Hydrobia pal- 



REVISION OF BELGRANDIA 

Distance 



181 



W 
Y 

T 
I 

Е 

U 
L 
M 

Z 
К 

в 

G 
А 

V 
H 
D 



FIG. 4. UPGMA dendrogram based on z-standardized Euclidean distances computed from the nine shell 
parameters of Table 1 . For abbreviations of populations, see Table 1 . 



182 



HAASE 



PC2 

2 



СИ 



-2 




F— D 



W 



3 -2 



з PC1 



РСЗ 

1 



2 




3 



2 



PC1 



FIG. 5. Minimum spanning tree based on z-standardized Euclidean distances computed from the nine shell 
parameters of Table 1 superimposed on ordinations following a principal component analysis of these param- 
eters. For abbreviations of populations, see Table 1; PC, principal component. 



REVISION OF BELGRANDIA 



183 



adilhi Dubrueil, 1869 [Germain (1931: 632) 
erroneously attributed the authorship of this 
taxon to Moitessier, 1869], as juveniles of B. 
gibberula. Dubrueil's syntypes from Frouzet 
near St. Martin de Londres, at least a sub- 
sample, have been found in Bourguignat's 
collection (MHNG). This sample consists of 
five juvenile shells. However, it is impossible 
to decide whether they represent a good 
species or belong to an older taxon. 

Two more samples from Bourguignat's col- 
lection, one labelled as B. gibberula Pala- 
dilhe, Département Hérault, the other one B. 
gibberula Paladilhe Var. from St. Jean de Fos, 
Hérault (Fig. 2F), were investigated, primarily 
in order to get an idea of the variability of B. 
gibberula for comparisons with the new 
species described below, which is, on a first 
glance, similar to B. gibberula. This compari- 
son is discussed in detail in the Remarks to 
the new species. In both samples, egg cap- 
sules attached to shells were found. 



Belgrandia paladilhi 
(Dubrueil, 1869) 

See Remarks to B. gibberula. 

Belgrandia occidentalis 
Clessin, 1878 

See Remarks to B. lusitanica. 

Belgrandia germanica 
Clessin, 1878 

Belgrandia margínala (Michaud, 1831 ) 
Sandberger, 1870-1875: 915 f. 

Belgrandia germanica Clessin, 1878b: 101 

Type Material: not traced 

Type Locality: upper Pleistocene tuffs of 
Weimar, Gräfentonna and Mühlhausen, Ger- 
many 

Description: Shell cylindro-conical, up to 
2.25 mm high and 1 .25 mm wide; apex blunt, 
up to six convex whorls separated by deep 
sutures; body whorl almost as high as spire; 
aperture almost round; a single varix behind 
outer lip; umbilicus almost completely cov- 
ered. 

Remarks: Sandberger's original material is 
probably lost. Therefore, the above descrip- 
tion is a reproduction of the original. 
Sandberger (1870-1875) identified his fossil 
species from eastern Germany as B. mar- 



gínala. Hence, Clessin (1878b) introduced the 
new name B. germanica and later differenti- 
ated it from B. margínala through its shorter 
and more conical spire (Clessin, 1882). 



Belgrandia boscae 
(Salvana, 1887) 

Hydrobia boscae Salvana, 1887: 141 

Belgrandia cí. margínala (Michaud, 1831 ) 
Boeters, 1988: 223 

Moitessieria locardi Coutagne, 1 883 - Bofill 
Poch, 1924: 99 

Type Material: not traced 

Type Locality: springs at Gandia, Valencia, 
Spain 

Material Examined: Gandia, Acequia El 
Bañador, Spain, ex ВОЕ 1455 (5) 

Description: Shell (Fig. 3F) small (1 .83 mm 
high), moderately slender; up to four convex 
whorls separated by deep sutures; aperture 
ovate, peristome slightly reflected; a single, 
not very prominent varix closely behind outer 
lip; umbilicus a moderately narrow slit. 

Remarks: According to Boeters (1988), the 
varix may be very weak or even absent. 
Although B. boscae is very similar to B. mar- 
gínala as regards shell size and shape, there 
are some qualitative states that distinguish 
both taxa, such as the reflected peristome 
and the less prominent varix in B. boscae, or 
the straight outer lip and the narrower umbili- 
cus in B. margínala, so that these nominal 
species are probably not conspecific. In addi- 
tion, it is very unlikely that a crenobiontic 
species has a range from southeastern 
France across the Pyrenees to western 
Spain. Therefore, I suggest that B. boscae is 
a distinct species. 



Belgrandia cazioti 
Westerlund, 1890 

Paludinella {Belgrandia) cazioti Wester- 
lund, 1890: 154 

Type Material: lectotype GNM 4417, here 
designated in order to define the species; 
paralectotype GNM 4417a (1) 

Type Locality: Les Angles, France 

Description: Shell (Fig. 3C) very broadly 
conical, lectotype 2.02 mm high and 1 .35 mm 
wide; 3.875 very convex and rapidly growing 
whorls; aperture ovate; lectotype has four 
very prominent varices on the last 0.25 
whorls; umbilicus an open slit. 



184 



HAASE 



Remarks: The paralectotype is damaged so 
that measurements could not be taken and 
the total number of varices not be determined. 
Through its gross appearance this species 
cannot be mistaken with any other congeneric 
species. Belgrandia cazioti Loca rd, 1892, is a 
different species and thus a junior homonym 
of B. cazioti Weste rl und, 1892 (see below). 

Belgrandia cazioti 
Locard, 1892, non Westerlund, 1890 

Belgrandia cazioti Locard, 1892: 3 

Belgrandia gibba (Draparnaud, 1805)- 
Germain, 1913: 301 

Type Material: syntypes MNHN 

Type Locality: quarter Champfleuri near 
Avignon, France 

Description: Shell (Fig. 3D) large, conical, 
with a mean height of 2.19 mm; on average 
4.2 little convex whorls; aperture wide relative 
to shell width; one prominent varix near but 
not immediately behind aperture; umbilicus a 
slit. 

Remarks: There may be two varices 
according to the original description (Locard, 
1892). 

Belgrandia cazioti Locard, 1892, is clearly 
not identical with B. cazioti Westerlund, 1890, 
and, hence, is a junior homonym of the latter. 
Figures 4 and 5 indicate that it might well be a 
separate species, with B. coutagnei Locard, 
1 892, as the most similar congeneric. It differs 
significantly from B. coutagnei in number of 
whorls and the shape indicators shell 
height/shell width and shell height/aperture 
height (Mann-Whitney U-test, P < 0.05). 
However, after Bonferroni correction, these 
comparisons are no longer significant. 
According to Locard (1892), both species are 
sympatric. It is thus not unlikely that Locard 
separated the slender and stouter shells of a 
single population and ascribed them to differ- 
ent taxa. Because of this circumstantial evi- 
dence, I suggest to consider B. cazioti Locard 
as synonym of B. coutagnei. 

Belgrandia coutagnei 
Locard, 1892 

Belgrandia coutagnei Locard, 1892: 3 

Belgrandia vitrea (Draparnaud, 1801 ) 
Germain, 1913: 303 

Belgrandia cazioti Locard, 1892 -present 
paper 

Type Material: Lectotype MNHN, here des- 



ignated in order to define the species; para- 
lectotypes MNHN (3) 

Type Locality: quarter Champfleuri near 
Avignon, France 

Description: Shell (Fig. 3B) large, with an 
average height of 2.33 mm, slender conical 
with up to 5 (mean 4.59) convex whorls; aper- 
ture ovate, small compared to shell height; 
one varix immediately behind outer lip; umbili- 
cus a narrow, but distinct slit. 

Remarks: Locard (1892, 1893) described 
1 -2 varices in this species. However, all four 
syntypes have only one. The most similar 
species is B. bigorriensis (Figs. 4, 5). The dif- 
ferentiation of both species is already given in 
the Remarks to B. bigorriensis. 

As to the potential synonymy with B. vitrea, 
see Remarks to B. bigorriensis Paladilhe, 
1869. 

Belgrandia torifera 
Schutt, 1961 

Belgrandia torifera Schutt, 1961 : 143 f. 

Type Material: holotype SMF 164357; 
paratypes SMF 164358, private collection of 
H. Schutt 

Type Locality: spring Stenjevac near 
Vrgorac, Croatia 

Material Examined: topotypes NHMW 
16716(1), NHMW 48796 (5) 

Description: Shell (Fig. 3I) small (mean 
1.64 mm), broadly conical, with on average 
3.75 rapidly and regularly growing, convex 
whorls; aperture practically as wide as high, 
large relative to shell size; one prominent 
varix immediately behind outer lip and occa- 
sionally a second, weaker one up to 0.75 
whorls behind the outer lip; umbilicus almost 
completely covered. 

Remarks: Both samples were pooled for 
the analysis but one shell from the larger lot 
was excluded because it was very different in 
size and shape, which may be due to para- 
sitism. In size, B. torifera is similar to B. 
moitessieri, but it differs from the latter in its 
regular growth, the larger aperture and the 
formation and position of the varices. In gen- 
eral, B. torifera is isolated in the morphospace 
of Figure 5. 

This species is the eastern-most represen- 
tative of the genus and up to now the only one 
known from the Balkans. Its allocation to 
Belgrandia is of course, as holds for the 
majority of the species discussed in this 
paper, tentative and needs confirmation 
through anatomical data. 



REVISION OF BELGRANDIA 



185 



Beigrandia heussi heussi 
Boettger, 1963 

Belgrandia heussi heussi Boettger. 1963: 
40-42 

Type Material: holotype SMF 167898; 
paratypes SMF 167898 (18) 

Type Locality: Rio Liz, Portugal 

Material Examined: two paratypes ex SMF 
167898 

Description: Shell (Fig. 3G) ovate-conical, 
larger specimen 1.86 mm high, 4.25 hardly 
convex whorls; aperture with straight outer lip 
and a distinct angle between parietal and 
palatal walls; the larger specimen had no 
varix, the smaller one a very inconspicuous 
varix close to outer lip; umbilicus a very nar- 
row slit. 

Remarks: The generic allocation has been 
confirmed by Boeters (1988). Both seminal 
receptacles are about equally long, the bursal 
duct is short, and the bursa copulatrix is a sim- 
ple, slender sac. The penis has a distinct lobe 
pointing anteriorly in the middle of the left 
side; the penial tip tapers gradually and has a 
black spot in its right half. Conchologically, B. 
h. heussi is similar to its Portuguese "fellow- 
species" B. lusitanica. These species differ 
primarily in the convexity of the whorls and in 
that B. lusitanica has always a distinct varix. 
Belgrandia h. heussi \s wider and has a higher 
aperture than B. lusitanica (see also Figs. 4, 
5), but these differences are not significant 
after Bonferroni correction (Mann Whitney Li- 
test). Anatomical differences may be the short 
proximal receptacle and the smaller penial 
lobe in B. lusitanica. However, Boeters (1988) 
has apparently investigated only one speci- 
men of each sex in each species. More data 
would be desirable. 

Belgrandia heussi alcoaensis 
Boettger. 1963 

Belgrandia heussi alcoaensis Boettger, 
1963:42 

Type Material: holotype SMF 176900: 
paratypes SMF 176901 (14) 

Type Locality: Rio Alcoa, Portugal 

Material Examined: one paratype ex SMF 
176901 

Description: The single specimen investi- 
gated (Fig. 3H) is 1.9 mm high and has four 
basally keeled whorls, giving the shell the 
shape of an Asian temple; the outer lip is 
straight; no varix; umbilicus an open slit. 

Remarks: The keel can be variably distinct 



according to the original description (Boettger. 
1963). Otherwise, neither Boettger nor 
Boeters (1988) found further differences to 
the nominate subspecies. However, probably 
due to the keel B. heussi alcoaensis appears 
to be much wider, which is also reflected in the 
PCA (Fig. 5; Appendix 2). Unfortunately, only 
very few specimens of each subspecies were 
at my disposal, so that no statistical confirma- 
tion has been possible. However, B. h. 
alcoaensis is unambiguously diagnosable 
because of its keel. Its status as subspecies 
might even be questioned. Because I cannot 
add substantial new data, I refrain from 
changing its rank. 

Belgrandia iónica 
(Schutt, 1980) 

Remarks: This species from the Greek 
island Corfu, originally described as Littha- 
bitella chilodia iónica Schutt. 1980, has only 
recently and tentatively been attributed to 
Belgrandia by Bodon et al. (1999). Schütt's 
holotype turned out to be most likely a 
Belgrandia, while the paratypes from the 
Island of Levkas belong to Litthabitella 
Boeters. 1970. The description of B. iónica is 
very vague. Therefore, I refrain from a further 
treatment and discussion and refer to Schutt 
(1980) and Bodon et al. (1999) for more 
details. 



SPECIES ONCE ERRONEOUSLY 
ALLOCATED TO BELGRANDIA 

Practically all European nominal species 
with one or more varices behind the outer lip 
have been attributed to Belgrandia at one 
time or another. However, Clessin (1882) real- 
ized that the formation of varices may not 
be restricted to Belgrandia and moved B. 
guranensis Paladilhe, 1870. B. simoniana 
Paladilhe, 1870 1 . and B. subovata Paladilhe, 
1876, to Bythinella. Later authors (e.g., 

1 ln fact, this name has been introduced by Moquin- 
Tandon (1856: 518) as Bythinia marginata var. 
simoniana: Paladilhe (1870) elevated its rank, and 
since then most subsequent authors ascribed the 
authorship to Paladilhe. Nevertheless, Moquin- 
Tandon's original combination frequently appears in 
the synonymy (e.g.. Paladilhe. 1870: Clessin. 1882; 
Locard. 1882. 1893). Locard (1893) changed the 
name to Belgrandia saint-simoniana Paladilhe, 
1870: Germain (1913, 1931) adopted Locards 
name in the slightly altered version B. saint-simoni. 
but cited Moquin-Tandon as author. 



186 



HAASE 



Locard, 1893; Germain, 1913, 1931) did not 
follow Clessin. However, my own investiga- 
tion (Bernasconi, in prep.) of the syntypes of 
B. guranensis and B. simoniana (both MHNG) 
confirmed Clessin's view as to these taxa. 
(His other reallocations cannot be followed.). 
The syntypes of B. subovata could not be 
traced, but the original description emphasiz- 
ing a very blunt apex suggests that this 
species is a Bythinella. 

Further species once assumed to belong to 
Belgrandia and later placed into other genera 
are Cyclostoma vitreum Draparnaud, 1801, 
and B. cylindracea Paladilhe, 1869. Cy- 
clostoma vitreum has been identified as 
Moitessieha Bourguignat, 1863 (Boeters, 
1969, 1972). Boeters (1998) has also recog- 
nized that B. cylindracea is a Bythinella and 
declared both Belgrandia sequanica Pal- 
adilhe, 1870, and B. tricassina Locard, 1893, 
synonyms. Bythinella lanceolata Locard, 
1893, and B. riparia Locard, 1893, are treated 
as forms of Bythinella cylindracea, a category 
excluded from the provisions of the Interna- 
tional Code of Zoological Nomenclature (Arti- 
cle 15.2, ICZN, 1999). 

Bithinia diaphana Dupuy, 1849, was con- 
sidered to be a synonym of Belgrandia vitrea 
(Draparnaud, 1805) by Germain (1931). Dra- 
parnaud's species has turned out to be a 
Moitessieha (see above), and Paludina di- 
aphana Michaud, 1831, which is the original 
name -Dupuy is only the author of the 
combination Bithinia diaphana— belongs to 
Bythiospeum Bourguignat, 1882 (Bernasconi, 
1985). 

The seven fossil species from alluvia of the 
river Seine described by Bourguignat in 1869, 
Belgrandia joinvillensis, B. desnoyersi, B. 
lartetiana, B. archaea, B. deshayesiana, B. 
edwardsiana, and B. dumesniliana, all share 
a blunt apex (Bourguignat, 1869). The syn- 
types were not traceable, but Bourguignat 
(1869) used the same expression for the 
description of the apex of those seven 
species as Paladilhe (1869) for B. cylindracea 
(Paladilhe, 1869) and for B. sequanica 
Paladilhe, 1870 (Paladilhe, 1870), and Locard 
(1893) for B. lanceolata, B. riparia, and B. tri- 
cassina, that is, obtusus (Latin) or obtus 
(French), which mean obtuse, blunt, truncate. 
The syntypes of these five nominal species 
still exist (MHNG). They all have a blunt apex 
as it is typical for the genus Bythinella and 
have already been identified as such as 
stated above. Therefore, I consider also 
Bourguignat's seven fossil species, for which 



he in fact has introduced the name 
Belgrandia, to belong to Bythinella. 

Belgrandia bourguignat! de Saint-Simon, 
1870, from Bourrassol may be a similar case. 
Also this species is rather a Bythinella judging 
form the original description (de Saint-Simon, 
1870). Syntypes are not known. Germain 
(1931) treated it as a variety of Belgrandia 
cylindracea, which turned out to be a 
Bythinella (see above). This may be addi- 
tional circumstantial evidence. 

Again similar is the case of Belgrandia nana 
Sandberger, 1880, a fossil species from Great 
Britain, the syntypes of which are probably 
also lost. Sandberger (1880-1881) described 
the apex as blunt and identified B. sequanica 
as closest relative. Sandberger's figure (tab. 
XII, fig. 5, 5b) shows a shell with pointed apex. 
However, this discrepancy between descrip- 
tion and illustration exists also in the original 
publications introducing the above listed 
French species. Hence, B. nana is probably 
also a Bythinella. 

Another fossil British species was attributed 
to Belgrandia by Sandberger (1880-1881), 
Paludina minuta Strickland. This nominal 
species is listed as synonym of B. margínala 
by Wood (1850-1856) and found in a list in 
Murchison (1839). However, I have been 
unable to trace an original description by 
Strickland, and it is therefore likely that P. mi- 
nuta is a nomen nudum. In addition, this 
species might also belong to Bythinella judg- 
ing from the description given by Wood 
(1850-1856). 

Bourguignat (1877) and later authors (e.g., 
Wagner, 1914) have also attributed a number 
of species primarily from the Balkan area with 
pupiform shell but lacking a varix to 
Belgrandia. Today, these species belong to 
Belgrandiella Wagner, 1928, and related or 
similar genera (Wagner, 1928; Radoman, 
1983; Haase, 1994). They are not further dis- 
cussed here nor included in Appendix 1. 

Belgrandia g f rast, nov. sp. 

Diagnosis: B. gfrast, nov. sp., differs from all 
other congeners with a single varix on the 
body whorl by its unique shell shape. It is 
rather broadly conical, with a pointed apex 
and convex whorls. The proportion of shell 
height to shell width is 1.78 ± 0.08 (mean ± 
standard deviation). The peristome may be 
detached and the varix up to half a whorl 
behind the outer lip. The new species is fur- 
ther characterized by a wide lobe on the left 



REVISION OF BELGRANDIA 



187 



side in the distal half of the penis. An occa- 
sional second lobe opposite to the first one is 
smaller. Also unique is the wide visceral 
oviduct. 

Holotype: NMB 11473 (Fig. 6) 

Paratypes: MNHN (15 specimens), NHMW 
90924 (15 specimens), NMB 11474 (> 50 
specimens), ZSM 19990017-1999001724 
(series of eight histological sections) 

Type Locality: Origin of the Chenal des 
Sources (47°36.9'N, 7°32.3'E; 242 m above 
sea level) in the Petite Camargue, Alsace, 
France. 

Etymology: Gfrast, used as noun in apposi- 
tion, is Viennese dialect and describes- 
depending on the context -a charmingly or 
really impudent person. The new species is 
dedicated to my friend Kathrin Wunderle, who 
has been giving me a wonderful time since we 
met in Switzerland. 

Shell: The shell is conical with up to 4.375 
convex whorls, of which the protoconch com- 
prises somewhat more than one whorl (Figs. 
2G, 7, 8). Measurements are given in Table 1 . 
Males and females cannot be distinguished 
by their shells. The aperture is oval and can 
be slightly detached from the penultimate 
whorl, leaving a narrow, but open umbilicus. 
The outer lip is orthocline, with an adapical 
sinus (Fig. 7B). There is a prominent varix 
mostly immediately, sometimes up to half a 
whorl behind the thin outer lip (Fig. 7B). The 
protoconch is pitted (Fig. 8B), while the teleo- 
conch shows no structure except growth 
lines. The shell has no colour and the perio- 
stracum is light brown. 

Operculum: The operculum is elongate- 
ellipsoidal, paucispiral, corneous, dark yellow, 
and its nucleus is submarginal and orange 
(Fig. 9A) 

Radula: The radula (Fig. 9B) is six to seven 
times longer than wide. The ribbon is beneath 
the buccal mass, and the radular sac forms a 
simple U-shaped loop behind the buccal 
mass. The dorsal part of this loop contains, 
like the radula bolsters, black pigment. The 
central tooth is trapezoidal. Its basal tongue is 
as long as the lateral margins and broadly V- 
shaped with curved edges. The prominent 
pair of basal cusps arises from the lateral 
margins. The face of the lateral teeth is taller 
than wide and has a prominent basal projec- 
tion. The lateral wing of the lateral teeth is 
much longer than the cutting edge. The cusps 
on the inner marginal teeth are slightly larger 







FIG. 6. Holotype (female) of Belgrandia gfrast, nov. 
sp. Shell height = 2.34 mm. 



than on the outer marginal teeth. The cutting 
edge of the inner marginal teeth is longer than 
25% of the total tooth length. The denticula- 
tion of the radula is described by the formula 
R:6-7 1 6-7/1 1, L: 5 1 5, M1: 24-28, M2: 
28-32. 

Non-Genital Anatomy: The epidermis is 
uniformly black, except for the mantle collar, 
the lips and the foot sole. The cephalic tenta- 
cles are black, with a white, ciliated line in the 
median (Fig. 10A) and a white tip. Black gran- 
ules are found in the connective tissue of the 
head and foot. The snout is approximately as 
long as wide, slightly tapering and distally 
weakly lobate. There is no palliai tentacle, but 
a strongly ciliated sensory field on the right 
side of the mantle edge (Fig. 10B, C). 

The ctenidium extends through about 75% 
of the mantle cavity and consists of six to 
eleven filaments, which are as broad as high. 
The osphradium is ellipsoid, more than two 
times longer than wide and lies somewhat 
behind the middle of the ctenidium. The hypo- 
branchial gland is massively developed, and 
its distal end almost reaches the tip of the pal- 
liai oviduct. 

Both supra- and suboesophageal connec- 
tives are long, and the oesophageal ganglia 
are not fused with the pleural ganglia. 
Cerebral, pleural and pedal ganglia contain 
black pigment granules. 



HAASE 




FIG. 7. Shell of Belgrandia gfrast, nov. sp. A, paratype; B. apical view of aperture of same specimen show- 
ing sinus and varix. Scale bars = 250 urn in A, 100 urn in B. 




FIG. 8. Protoconch of Belgrandia gfrast. nov. sp. A, whole protoconch; B, surface structure. Scale bars = 100 
urn in A, 10 urn in B. 



In the proximal whorls, the digestive gland 
is a simple tube. Distally it has a series of 
lobes, the length of which increases with their 
distance from the apex. These lobes lie paral- 
lel to the columella and have their blind ends 
adapical (Fig. 11). The digestive gland has 
only a single opening into the stomach. The 
stomach has no coecum, and its anterior and 
posterior chambers are about equally long. 
The intestine coils around the style sac and 
runs straight along the palliai oviduct (Fig. 11) 



or the prostate, respectively. The anus lies 
behind the mantle edge. 

Female Reproductive System: The ovary is 
a simple sac (Fig. 11) occupying more than 
66% of the visceral coil behind the stomach 
and slightly overlaps the posterior chamber of 
the stomach. The visceral oviduct is excep- 
tionally wide (Figs. 11-12). The renal oviduct 
is not pigmented and forms a simple loop of 
270° lying against the bursa copulatrix. There 



REVISION OF BELGRANDIA 




FIG. 9. Operculum and Radula of Belgrandia gfrast, nov. sp. A, inner side of operculum; В, left half of the 
radular ribbon, 9 transverse rows. Scale bars = 100 u.m in A, 10 цт in B. 



are two elongate seminal receptacles with 
short ducts. The proximal one arises from the 
proximal-most part of the straight section of 
the renal oviduct and bends around the ven- 
troposterior edge of the bursa copulatrix. The 
distal receptacle is much smaller. It branches 
off at about the origin of the bursa copulatrix 
and lies against the latter. The bursa copula- 
trix is an elongate sac distally lying against the 
albumen gland. The posterior, blind end 
extends the albumen gland. The bursal duct 
lies on the longitudinal axis of the bursa and is 
considerably shorter than the latter. Both, 
capsule and albumen glands are bipartite. 
The posterior capsule gland and the anterior 
albumen gland are much shorter than the 
anterior capsule gland and the posterior albu- 
men gland, respectively. The ventral channel 
forms a small vestibulum. The gonopore is a 
short slit shortly behind the distal end of the 
ventral channel (Fig. 12). 

Egg Capsules: Egg capsules are lenticular 
and have an ellipsoid circumference. They 
are laid singly on the shells of their con- 
specifics, preferably, but not exclusively, 
along the sutures (Fig. 13). Whether eggs are 
deposited also on other firm substrates is not 
known. 

Male Reproductive System: The testis has 
simple, vertical lobes. It begins almost at the 
tip of the visceral coil and distally covers the 
posterior chamber of the stomach. The poste- 
rior vas deferens coils immediately after it 
emerges from the middle region of the testis, 
forming a seminal vesicle. Before entering the 
prostate at its posterior end, the vas deferens 
makes an S-shaped loop. The prostate is 
bean-shaped. Its ventral surface is closed; 
there is no connection with the mantle cavity. 



The palliai vas deferens exits the prostate at 
its distal end. It is almost straight and has only 
weak musculature (Fig. 14). The resting penis 
is U-shaped. In extended condition it has par- 
allel sides. It tapers only distal to the wide, 
muscular lobe on the left side in the distal half 
of the penis. Opposite to this left lobe there 
may be also a second, smaller lobe on the 
right side (Fig. 15). There is a black spot in the 
distal half of the penis. The pigment granula 
are in the connective tissue. 

Additional Observations: Of 50 sexed snails 
23 (46%) were males and 27 (54%) females. 
In the type locality, the density of adult snails 
was 6,400/m 2 in October 1999. About 120 m 
downstream, the population density was 
17,807 adults/m 2 (Mosimann, pers. comm.). 
The mantle cavity of each animal investigated 
anatomically contained peritrich ciliates of the 
genus Trichodina Ehrenberg, 1830, with 
diameters of about 60 urn 

Remarks: Shell morphology and anatomy 
of B. gfrast correspond well with the diagnosis 
of Belgrandia, so that the generic allocation of 
the new species is unambiguous. In order to 
confirm its status as a separate, hitherto 
undescribed species B. gfrast had to be com- 
pared to the known (nominal) species. 
According to multivariate analyses [PCA 
(Appendix 2), UPGMA] based on the nine 
morphometric parameters (Table 1), the most 
similar species were (Figs. 4, 5): B. conoidea, 
В. gibberula, В. gibba, and B. heussi alcoaen- 
sis. From these species, B. heussi alcoaensis 
was excluded from further statistical analy- 
ses, because only a single shell was avail- 
able. Besides, it is clearly differentiated by its 
keel. In case of B. gibberula, it must be spec- 
ified that the syntypes are rather remote from 



190 



HAASE 




FIG. 10. Ciliation of Belgrandia gfrast, nov. sp. A, 
line of cilia on the right cephalic tentacle; В, C, cil- 
iary sensory field on the right side of the mantle col- 
lar (between arrow heads). In С the arrow indicates 
a tuft of cilia, not a tentacle. Scale bars = 50 urn. 



the other two samples ascribed to this 
species. A Kruskal-Wallis Test over all six 
samples was significant for all parameters (df 
= 5,P< 0.0036, Bonferroni corrected) but the 
ratio aperture height/aperture width. Omitting 
the syntypes of B. gibberula, significant differ- 
ences were found only in the size parameters 
and the number of whorls (df = 4, P < 0.027, 
Bonferroni corrected). Of these remaining 
four samples compared to B. gfrast, only B. 
gibba is a name-bearing syntype series. As 



already mentioned, both samples ascribed to 
B. gibberula are very different from the syn- 
types of B. gibberula. The sample from St. 
Jean de Fos may represent a separate 
species, whereas B. gibberula from Hérault, 
which has up to three varices within the last 
0.25 whorls, has affinities to B. gibba. 
Belgrandia gfrast is distinguished from both in 
that it has always one varix, which varies in 
position from closely behind to up to 0.5 
whorls behind the outer lip. The syntypes of B. 
gibba have, in addition, a significantly smaller 
aperture, which is also smaller in relation to 
shell height (Mann Whitney U-Tests, P< 0.05, 
Bonferroni corrected) compared to B. gfrast. 
Anatomically, B. gfrast can readily be distin- 
guished from B. gibba by its triangular penial 
lobe on the left side and the occasional pres- 
ence of a small, right lobe. In B. gibba, there 
is only the left penial lobe, and this is smaller, 
pointed and directed anteriorly (Giusti & 
Pezzoli, 1980). 

For a delimitation of B. gfrast from B. varica, 
I can only refer to its original description 
(Paget, 1854). Distinguishing features of B. 
varica are the much stouter shell, the peris- 
tome being never detached, and a single varix 
that is always closely behind the outer lip. 

Also among the Italian nominal species, 
currently comprehensively revised by Cian- 
fanelli, Bodon, Manganelli, & Giusti (pers. 
comm.) and why the following comparisons 
are kept to a minimum, none can be confused 
with the new species from the Petite 
Camargue Alsacienne. Generally, the single, 
variably positioned varix of B. gfrast is unique 
so far and allows its unambiguous identifica- 
tion even when compared to the less well- 
known, nominal species, that is, species lack- 
ing anatomical data, from Italy (Appendix 1). 
Currently, anatomical descriptions exist for 
only three nominal Italian species, B. minus- 
cola (Paulucci, 1881), B. caprai Giusti, 1970, 
which is synonymized with B. thermalis 
(Linnaeus, 1767) by Bodon et al. (1995), and 
В. mariatheresiae Giusti & Pezzoli, 1972 
(Giusti, 1970; Giusti & Pezzoli, 1972, 1980). 
Comparing only the genital anatomy, B. 
caprai appears to be very similar to B. gfrast. 
However, in B. caprai the cutting edge of the 
central radular tooth has fewer denticles, and 
the shell is much stouter and has an ample 
and unregularely shaped aperture (Giusti, 
1970). Also, B. mariatheresiae has a rather 
conical, stout shell and, in addition, folds 
rather than varices, and its penis is much 
more slender with a single, round lobe on the 



REVISION OF BELGRANDIA 



191 




FIG. 11. Digestive and reproductive systems of a female of Belgrandia gfrast, nov. sp., foregut omitted, aa, 
anterior albumen gland; ac, anterior capsule gland; an, anus; be, bursa copulatrix; dg, digestive gland; od, 
oviduct; ov, ovary; pa, posterior albumen gland; pc, posterior capsule gland; pr, proximal seminal receptacle; 
re, rectum; st, stomach; ve, vestibulum. Scale bar = 200 ¡am. 



left side (Giusti & Pezzoli, 1972). Belgrandia 
minuscola, finally, lacks varices and penial 
lobes (Giusti & Pezzoli, 1980). 

Abiotic Characterization of the Type Local- 
ity: The type locality of B. gfrast is the first of 
about 20 springs emerging on the edge of a 
low gravel terrace in the southwest of the 



Petite Camargue Alsacienne. These springs 
discharge into the 1.3 km long Chenal des 
Sources, which flows along the edge of the 
terrace towards north with 1 -3 cm/s (Berger, 
1993), primarily on gley. The springs are fed 
by water of brooks oozing away in the 
Sundgau west and southwest of the Petite 
Camargue Alsacienne (Schenker, 1992). 




FIG. 12. Distal female genitalia of Belgrandia gfrast, nov. sp. aa, anterior albumen gland; ac, anterior cap- 
sule gland; be, bursa copulatrix; bd, bursal duct; dr distal seminal receptacle; go, genital opening; od, oviduct; 
pa, posterior albumen gland; pc, posterior capsule gland; pr, proximal seminal receptacle; vc, ventral chan- 
nel; ve, vestibulum. Scale bar = 100 цт. 




FIG. 13. Egg capsules of Belgrandia gfrast, nov. sp. A, on a subadult shell, arrow indicates egg capsule 
shown in B; B, single egg capsule. Scale bars = 200 urn in A, 50 цт in B. 



The temperature was rather constant over 
the whole period of observation oscillating 
between 11.3°C and 12.3°C. The electric 
conductivity had its maximum in March with 
810 uS/cm and steadily decreased until 
December 1990 down to 730 uS/cm. The pH 
varied between 6.80 and 7.19, with the lower 



values falling into the warmer period of the 
year. Ammonium was practically not de- 
tectable, while the concentration of nitrate 
was considerable with a minimum of 14.34 
mg/l and a maximum of 37.48 mg/l. The con- 
centration of phosphate was rather low during 
the year, with values between undetectable 



REVISION OF BELGRANDIA 



193 




FIG. 14. Male genital system except penis of Belgrandia gfrast, nov. sp. pt, prostate; te, testis; vd, vas def- 
erens: vs, vesicula seminalis. Scale bar = 200 (im. 



and 0.06 mg/l. Only in late fall higher values 
up to 0.13 mg/l were measured. Chloride had 
its highest concentrations during the early 
months of the year, with a maximum of 44.1 
mg/l, which decreased towards fall below 39 
mg/l. The concentration of potassium oscil- 



lated between 1.1 mg/l and 5.1 mg/l over the 
period of observation with no detectable 
trend. The concentration of calcium was high- 
est in winter and early spring, with a peak of 
121.5 mg/l and decreased during the warmer 
period until fall to 48.4 mg/l. In November and 



194 



HAASE 




FIG. 15. Penis of Belgrandia gfrast, nov. sp. A, with- 
drawn; B, extended, only with left lobe; C, 
extended, arrow indicates small right lobe. Scale 
bars = 100 |.im. 



December, the concentration increased 
again. Magnesium, finally, showed an oppo- 
site trend with higher values during summer 
reaching up to 33.15 mg/l and lower concen- 
trations in winter with a minimum of 8.65 mg/l. 

DISCUSSION 

The latest extensive revision of the French 
malacofauna recognizes only five extant 



species as belonging to the genus Belgrandia 
(Germain, 1931), namely B. gibba, B. saint- 
simoni (= B. simoniana, see above), B. mar- 
gínala, В. vitrea and В. cylindracea. The latter 
two were later shown to be a Moitessieria and 
a Bythinella, respectively (Boeters, 1969, 
1972, 1998), and Belgrandia saint-simoni has 
also to be transferred to Bythinella as outlined 
above. The present account, on the other 
hand, recognizes eight French species, all of 
them extant. Three species occur on the 
Iberian Peninsula, and one each is from 
Croatia and Greece. Belgrandia germanica 
from eastern Germany is one of only three 
fossil species -there are two more fossil 
species from Italy described in Settepassi & 
Verdel (1965) (see Appendix 1)-being a true 
Belgrandia as far as can be judged from the 
original description (Sandberger, 1870-1875; 
Clessin, 1878b, 1882; the whereabouts of its 
syntypes are unknown, see above). 

The discrepancy between Germain's 
(1931) and my conclusions is, of course, a 
consequence of our different approaches to 
analyze similarity and to interpret differences 
taxonomically. Compared with other groups of 
gastropods, the differences between con- 
generic hydrobiid species may appear rather 
negligible. However, considering that due to 
the miniaturization of hydrobiids the number 
of taxonomically useful characters is reduced 
(Hershler & Ponder, 1998), which means that 
only few characters can vary in the course of 
evolution, "slight" differences, be they quanti- 
tative or qualitative, gain comparatively higher 
taxonomic significance. 

The morphological investigations have 
shown that there is considerable variance 
among French Belgrandia. This basic account 
should stimulate field studies, followed by 
anatomical studies and phylogenetic analy- 
ses, in order to learn more about the evolution 
and relationships of these species and, fur- 
thermore and probably even more important 
in our days, to evaluate the actual situation of 
this diverse fauna of spring snails, whose 
existence is only too often threatened through 
habitat destruction due to human activities 
(e.g., Ponder, 1994; Haase, 1996, Haase et 
al. in press; Hershler, 1996; Bouchet, 1997). 

Belgrandia gfrast is the northern-most re- 
cent representative of the genus. The center of 
diversification of Belgrandia is southwest Eu- 
rope (Giusti & Pezzoli, 1980; Germain, 1931; 
Boeters, 1988). The new species is thus fairly 
isolated from its recent congeners. However, 
during the Pleistocene, Belgrandia had appar- 



REVISION OF BELGRANDIA 



195 



ently a far wider distribution, reaching as far as 
eastern Germany (Sandberger, 1870-1875, 
1 880; Clessin, 1 878b, 1 882). Also the Dalma- 
tian В. torifera appears to be remote from re- 
lated species. But the Dalmatian spring snail 
fauna has more biogeographical affinities with 
western areas as indicated by the distribution 
of, for example, Pyrgula annulata (Linnaeus, 
1767), which is sympatric with B. torifera 
(Schutt, 1961), and the genera Islamia Rado- 
man, 1973, or Orientalina Randoman, 1978 
(Schutt, 1 961 ; Bodón et al. 1 992; Bodón et al. 
1 994). The same holds for the Ionian Islands, 
where B. iónica occurs (Bodon et al., 1999). 
The distribution of the three Iberian species in 
the far west and the far east of the peninsula, 
respectively, suggests that there are more 
species to be discovered bridging this gap. 

The phenetic analyses do not of course 
indicate systematic relationships. On the con- 
trary, the high similarity of geographically 
remote species suggests convergent shell 
evolution rather than common ancestry, con- 
sidering that the most common mode of spe- 
ciation in crenobiontic hydrobiids is probably 
parapatric. 

Whether B. gfrast and other Belgrandia 
species (B. lusitanica and В cf. gibberula) at- 
tach their egg capsules exclusively on shells of 
conspecifics or also on other hard substrata is 
not known. This behaviour, however, may 
probably be considered a further autapomor- 
phy of Belgrandia paralleled by only few other, 
remotely related crenobiontic genera (e.g., 
Haase & Bouchet, 1998). Attaching eggs on 
conspecifics ensures that the eggs remain in 
their appropriate habitat in case the origin of a 
brook changes down- or upstream over a sea- 
son depending on rainfall, provided the snails 
can follow these changes. 

As probably the majority of crenobiontic 
hydrobiid species, B. gfrast lives in constantly 
cool and slowly running water (cf. Haase et 
al., in press). The water of the type locality is 
rich with calcium and magnesium, which 
reach similar concentrations as in natural car- 
bonate brooks (Otto & Braukmann, 1983). 
However, the pH is much lower than in natural 
carbonate brooks, and the high values for 
conductivity and the high concentrations of 
chloride, nitrate and potassium indicate that 
the Chenal des Sources is fairly contami- 
nated. The sources of this contamination are 
probably the nearby settlements and agricul- 
ture (Berger, 1993). A more comprehensive 
comparison of the autecology of B. gfrast with 
other species is hardly possible because of 



the lack of comparable data (Haase et al., in 
press). However, we begin to learn that creno- 
biontic hydrobiids do not necessarily indicate 
good water quality (Patzner, 1994). 

Several epibiontic, peritrich ciliates of the 
genus Trichodina are known to be parasites, 
especially on fish (Görtz, 1996). But Tricho- 
dina is also found on a variety of invertebrates, 
among them several molluscs (e.g., van Ban- 
ning, 1979; Raut, 1980; Basson & van As, 
1992). Koval & Chernogorenko (1978) report 
a Trichodina sp. parasitizing on another hy- 
drobiid gastropod, Lithoglyphus naticoides (C. 
Pfeiffer, 1828). Whether the ciliates encoun- 
tered in the mantle cavity of B. gfrast are harm- 
ful to the snails or only commensalistic cannot 
be told from the sections. 



ACKNOWLEDGEMENTS 

I am grateful to P. Knibiely (Petite Camargue 
Alsacienne) for providing the collection permit. 
I thank H. D. Boeters (Munich), P. Bouchet 
(Paris), S. Cianfanelli (Florence), A. Eschner 
(Vienna), Y. Finet (Geneva), M. Glaubrecht 
(Berlin), R. Janssen (Frankfurt), and T von 
Proschwitz, who loaned material from their 
collections for my comparisons. I thank О 
Mosimann (Basel) for sharing her population 
density data with me prior to publication. I am 
indebted to G. and M. Falkner (Hörlkofen) and 
T. E. J. Ripken (Paris) for helpful comments 
and providing their unpublished data. The fol- 
lowing people are acknowledged for providing 
literature: P. Bouchet (Paris), H. Dürrer 
(Basel), A. Eschner, Y. Finet, M. Geisthardt 
(Wiesbaden), V. Heros (Paris), and H. Satt- 
mann (Vienna). Figure 1 has been largely pre- 
pared by M. Wurtz (Basel). H. Frefel (Basel) 
did part of the dark room work. I thank D. 
Matthys (Basel) for his technical assistance at 
the SEM. And finally, the referees are ac- 
knowledged for their helpful comments. 

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Revised ms. accepted 2 May 2000 



APPENDIX 1 



TABLE 2. Nominal species attributed, at one stage or another, to Belgrandia. Species now attributed to 
Belgrandiella or related genera are excluded. 



Nominal species, subspecies, variety Type locality 



Allocation to Belgrandia and 
present allocation 



Turbo thermalis Linnaeus, 1767 



Pisa. Italy 



Cyclostoma vitreum Draparnaud, 


France 


1801 




Cyclostoma gibbum Draparnaud, 


France 


1805 




Paludina margínala Michaud, 1831 


Foux de Draguignan, 




Var, France 


Paludina minuta Strickland, in 


Cropthorn. Great 


Murchison, 1839 


Britain 


Paludina conoidea Reynies, 1844 


Ardus sur l'Aveyron, 




près Montauban. 




France 


Hydrobia vahea Paget. 1854 


Nice, Alpes Maritimes 




France 


Bythinia gibba var. uniplicata 


source of the river 


Moquin-Tandon, 1856 


Lez near 




Montpellier 


Bythinia gibba var. marginata 


source of the river 


Moquin-Tandon, 1856 


Lez near 




Montpellier 


Bythinia gibba var. aplexa 


source of the river 


Moquin-Tandon, 1856 


Lez near 




Montpellier 



Belgrandia thermalis (Linnaeus. 1758)— 

Clessin (1882), Paulucci (1882): 

Cianfanelli et al., in prep. 
Moitessieria (Spiralix) vitrea (Draparnaud. 

1801)-Boeters (1969, 1972) 
Belgrandia gibba (Draparnaud, 1805)— 

(Bourguignat, 1869) 
Belgrandia marginata (Michaud, 1831)— 

Paladilhe(1870) 
Belgrandia minuta (Strickland)— 

Sandberger (1880-1881)- probably a 

nomen nudum, present paper 
Belgrandia conoidea (Reynies. 1844)— 

Falkner, Ripken & Falkner, in prep. 

Belgrandia vahea (Paget, 1854)— 
Paladilhe(1869) 

Belgrandia gibba var. uniplicata 
(Moquin-Tandon, 1856)— Clessin 
(1882); synonym of Belgrandia gibba 
(Draparnaud, 1805)— present paper 

Belgrandia gibba var. marginata 
(Moquin-Tandon, 1856)— Clessin 
(1882); synonym of Belgrandia gibba 
(Draparnaud, 1805)— present paper 

Belgrandia gibba var. aplexa 
(Moquin-Tandon. 1856)— Clessin 
(1882); synonym of Belgrandia gibba 
(Draparnaud, 1805)— present paper 



REVISION OF BELGRANDIA 



199 



TABLE 2. Continued. 



Nominal species, subspecies, variety Type locality 



Allocation to Belgrandia and 
present allocation 



Bythinia marginata var. simoniana 
Moquin-Tandon, 1856 



Hydrobia moitessieri Bourguignat, 
1866 

Bythinia saviana Issel, 1866 



Hydrobia lusitanica Paladilhe, 1867 

Belgrandia joinvillensis Bourguignat. 
1869 

Belgrandia desnoyersi Bourguignat. 
1869 

Belgrandia lartetiana Bourguignat. 
1869 

Belgrandia archaea Bourguignat, 
1869 

Belgrandia deshayesiana 
Bourguignat. 1869 

Belgrandia edwardsiana 
Bourguignat, 1869 

Belgrandia dumenisliana 
Bourguignat. 1869 

Hydrobia paladilhi Dubrueil, 1869 
1869 

Belgrandia cylindracea Paladilhe, 

1869 
Belgrandia bigorriensis Paladilhe. 

1869 
Belgrandia gibberula Paladilhe. 1869 

Belgrandia guranensis Paladilhe. 

1870 
Belgrandia sequanica Paladilhe, 1870 

Belgrandia bourguignat! de 

Saint-Simon. 1870 
Belgrandia subovata Paladilhe. 1876 



near Cierp. France 



source of the river 

Martinet near 

Montpellier, France 
San Giuliano near 

Pisa, Italy 



Fonte das Lagrimas. 

Coimbra, Portugal 
Joinville-le-Pont, 

Val-de-Marne. 

France 
Canonville, 

Val-de-Marne, 

France 
Joinville-le-Pont, 

Val-de-Marne, 

France 
Joinville-le-Pont, 

Val-de-Marne, 

France 
Joinville-le-Pont, 

Val-de-Marne. 

France 
Canonville. 

Val-de-Marne, 

France 
Canonville, 

Val-de-Marne, 

France 
Frouzet near St. 

Martin de 

Londres, France 
environs of Amanees. 

France 
Bigorre, France 

St. Guilhem le 
Désert, France 

Guran near Cierp. 
France 

river Seine near 
Verrières 

Bourassol, France 

Clausel, France 



Belgrandia occidentalis Clessin. 1878 Coimbra. Portugal 

Belgrandia germanica Clessin, 1878 Weimar, 

Gräfentonna and 

Mühlhausen. 

Germany 



Belgrandia simoniana (Moquin-Tandon, 
1856)- Paladilhe, 1870; Bythinella 
simoniana (Moquin-Tandon, 1856)— 
(Clessin. 1882) 

Belgrandia moitessieri (Bourguignat. 
1 866)- Bourguignat (1 869) 

synonym of Thermhydrobia thermalis 
(Linnaeus. 1767)-Paulucci, 1878; 
Belgrandia saviana (Issel, 1866)— 
Clessin (1878c); synonym of B. ther- 
malis (Linnaeus, 1767)- Clessin (1882); 
see also Cianfanelli, Bodon, Manganelli 
& Giusti in prep. 

Belgrandia lusitanica (Paladilhe. 1867)— 
Paladilhe (1869) 

Bythinella joinvillensis (Bourguignat. 
1869)— present paper 

Bythinella desnoyersi (Bourguignat, 
1869)— present paper 

Bythinella lartetiana (Bourguignat, 1869)— 
present paper 

Bythinella archaea (Bourguignat, 1869)— 
present paper 

Bythinella deshayesiana (Bourguignat. 
1869)— present paper 

Bythinella edwardsiana (Bourguignat. 
1869)— present paper 

Bythinella dumenisliana (Bourguignat. 
1869)— present paper 

synonym of Belgrandia gibberula 
Paladilhe, 1869-Paladilhe (1870) 

Bythinella cylindracea (Paladilhe. 1869)— 

Boeters (1998) 
Belgrandia bigorriensis Paladilhe, 1869 

Belgrandia gibberula Paladilhe. 1869 

Bythinella guranensis (Paladilhe. 1870)— 

Clessin (1882) 
synonym of Bythinella cylindracea 

(Paladilhe, 1869)- Boeters (1998) 
Bythinella bourguignat! (de Saint-Simon, 

1870)— present paper 
Bythinella subovata (Paladilhe, 1876)- 

Clessin (1882) 
synonym of Belgrandia lusitanica 

(Paladilhe, 1867)-Clessin (1882) 
Belgrandia germanica Clessin, 1878 



200 

TABLE 2. Continued. 



HAASE 



Nominal species, subspecies, variety Type locality 



Allocation to Belgrandia and 
present allocation 



Belgrandia bonelliana de Stefani, 

1879 
Belgrandia thermalis var. 

controversa Del Prête, 1879 

Thermhydrobia thermalis var. 

minuta Paulucci, 1880 
Belgrandia nana Sandberger, 1880 

Hydrobia minuscola Paulucci, 1881 

Hydrobia minuscola var. curta 

Paulucci. 1881 
Belgrandia targioniana Clessin, 1882 
Belgrandia delpretiana Clessin, 1882 



Hydrobia boscae Salvana 1 887 



Belgrandia cazioti ' Westerlund, 1890 
Belgrandia cazioti Locard, 1892 



Belgrandia coutagnei Locard. 1 892 



Belgrandia lanceolata Locard, 1893 



Belgrandia riparia Locard, 1893 
Belgrandia tricassina Locard, 1893 

Belgrandia heussi heussi 

С Boettger, 1963 
Belgrandia heussi alcoaensis 

С Boettger, 1963 
Belgrandia latina Settepassi, in 

Settepassi and Verdel, 1965 
Belgrandia zilchi Settepassi, in 

Settepassi and Verdel, 1965 
Belgrandia caprai Giusti, 1970 



Belgrandia mariatheresiae Giusti 

& Pezzoli, 1972 
Litthabitella chilodia iónica Schutt, 

1980 



Sarteano, Italy 
Bozzano, Italy 



near Sesto, Italy 

Mundesley, 
Great Britain 

S. Ágata Mátese, 
Italy 

Caramanico, Italy 

Firenze, Italy 
Viareggio, Italy 



springs near 
Gandia, Spain 

Les Angles, France 
quarter Champfleuri 

near Avignon, 

France 

quarter Champfleuri 
near Avignon. 
France 

river Seine near 
Verrières 



river Seine near 

Verrières 
river Seine near 

Verrières 
Rio Liz, Portugal 

Rio Alcoa, Portugal 

Valle del Liri, Latium, 

Italy 
Ponte di Castelluccio, 

Latium, Italy 
Rosia, Toscana, Italy 



Fonte di S. Cassiano 
near Fabriano, Italy 

Messongi river, Corfu, 
Greece 



Cianfanelli et al., in prep. 

synonym of Belgrandia thermalis 
(Linnaeus, 1767)-Bodon et al. (1995); 
Cianfanelli et al., in prep. 

Cianfanelli et al., in prep. 

Bythinella nana (Sandberger, 

1880)— present paper 
Belgrandia minuscola— Giusti & Pezzoli 

(1980); Cianfanelli et al., in prep. 
Cianfanelli et al., in prep. 

Cianfanelli et al., in prep, 
synonym of Belgrandia thermalis 

(Linnaeus, 1767)-Bodon et al. (1995); 

see also Cianfanelli et al., in prep. 
synonym of Belgrandia cf. marginata— 

Boeters (1988): B. boscae— present 

paper 
Belgrandia cazioti Westerlund (1890) 
primary junior homonym of Belgrandia 

caz/of/ Westerlund, 1890, and probably 

synonym of B. coutagnei— present 

paper 
Belgrandia coutagnei Locard (1892) 



synonym of Belgrandia cylindracea 
Paladilhe, 1869 -Germain (1913); 
synonym (forma) of Bythinella cylin- 
dracea (Paladilhe, 1 869)- Boeters 
(1998) 

synonym (forma) of Bythinella cylindracea 
(Paladilhe, 1 869)- Boeters (1998) 

synonym (forma) of Bythinella cylindracea 
(Paladilhe, 1869)- Boeters (1998) 

Belgrandia heussi heussi Boettger, 1963 

Belgrandia heussi alcoaensis Boettger, 

1963 
Cianfanelli et al., in prep. 

see Cianfanelli et al., in prep. 

synonym of Belgrandia thermalis 
(Linnaeus, 1767)-Bodon et al. (1995); 
Cianfanelli et al., in prep. 

Cianfaneli et al., in prep. 

Belgrandia iónica (Schutt, 1980)— Bodón 
étal. (1999) 



REVISION OF BELGRANDIA 



201 



APPENDIX 2. 
PRINCIPAL COMPONENT ANALYSIS 

The principal component analysis based on 
the correlation matrix of all nine shell parame- 
ters yielded eight principal components with 
positive Eigenvalues (Table 3). Principal com- 
ponents 1 -3 represent 93.66% of the total 
variance. Therefore, the three dimensional 



ordination of the samples in Figure 5 repre- 
sents fairly well the originally nine dimen- 
sional morphospace. PC1 is primarily com- 
posed of the size parameters. PC2 mainly 
represents the ratios sh/ah, sh/sw, which indi- 
cate shell shape, and the number of whorls. 
Two shape parameters, sw/aw and ah/aw, 
and aperture width have the highest weights 
on PC3. 



TABLE 3. Principal component analysis based on a matrix of correlations of the shell parameters of Table 
1. cv, cumulative variance in %; E, Eigenvalue; PC, principal component; v, variance in %; w, weights of 
parameters in Eigenvectors. 





PC1 


PC2 


PC3 


PC4 


PC5 


PC6 


PC7 


PC8 


E 


4.34200 


2.95000 


1.13700 


0.40390 


0.15770 


0.00781 


0.00085 


0.00029 


V 


48.25 


32.78 


12.64 


4.49 


1.75 


0.09 


0.00 


0.00 


cv 


48.25 


81.02 


93.66 


98.15 


99.90 


99.99 


100 


100 




w 


w 


w 


w 


w 


w 


w 


w 


sh 


0.44555 


0.16659 


-0.18683 


-0.06913 


-0.24356 


-0.81054 


-0.11577 


0.06170 


sw 


0.44319 


-0.20835 


-0.06515 


0.17195 


-0.09382 


0.28777 


-0.56142 


-0.07638 


ah 


0.44581 


-0.16978 


-0.15358 


-0.24710 


0.00128 


0.20837 


0.13284 


-0.66924 


aw 


0.38565 


-0.24662 


-0.38925 


-0.06271 


0.02411 


0.25191 


0.42380 


0.62385 


W 


0.25847 


0.43338 


-0.13050 


0.29662 


0.80005 


-0.00604 


0.00638 


-0.01161 


sh/sw 


-0.00279 


0.55966 


-0.15731 


-0.31557 


-0.20449 


0.33419 


-0.47157 


0.24402 


ah/aw 


0.28457 


0.16978 


0.59988 


-0.61089 


0.14339 


0.03350 


0.18808 


0.09094 


sh/ah 


0.06477 


0.55588 


-0.09126 


0.27162 


-0.44021 


0.18664 


0.46435 


-0.23348 


sw/aw 


0.32496 


-0.01802 


0.61301 


0.51588 


-0.18720 


0.07591 


-0.02715 


0.17556 



MALACOLOGIA, 2000, 42(1-2): 203-206 



LETTERS TO THE EDITOR 



A QUICK, SIMPLE, AND INEXPENSIVE MOLLUSC DNA EXTRACTION 
PROTOCOL FOR PCR-BASED TECHNIQUES 

Coline H. M. van Moorsel 1 , W. Johan van Nes 1 & Hendrik-Jan Megens 1 ' 2 



Frequently, molecular systematicists and 
population geneticists must extract DNA from 
many specimens — a time consuming and la- 
borious task. This time investment is particu- 
larly high with land snails and other molluscs 
that come loaded with enormous quantities of 
unwanted, slimy mucopolysaccharides. More- 
over, current extraction techniques, designed 
to remove the mucus, use reagents that are 
hazardous to the environment as well as to the 
researcher (Douris et al., 1998; Kyle & Bould- 
ing, 1998; Lydeard et al., 1998; Winnepen- 
ninckxetal., 1993). 

Here we present a new DNA extraction pro- 
tocol for molluscs that both saves time and 
money and avoids such harmful reagents as 
chloroform or phenol. With some alterations, 
this protocol borrows procedures from Walbot 
& Warren (1 988), designed for plant DNA, and 
Flook et al. (1 992), designed for insect DNA. It 
was further optimized for use on clausiliid land 
snails (Gastropoda; Pulmonata, Clausiliidae), 
and extracts from these were found to be use- 
ful in several PCR-based techniques. While in 
some cases these extracts are found to expire 
sooner than those from traditional, phenol- 
based methods, the time required to complete 
the extraction is considerably shorter, and, fur- 
thermore, the reagents are inexpensive and 
easily available in most molecular labs. 

Extraction Procedure 

Complete clausiliid snails, with their shells 
attached, are suitable for homogenizing. One 
advantage of using the entire animal is that 
parasites, hidden in the upper reaches of the 
shell, are detectable using parasite-specific 
primers. If, on the other hand, the shell is not 
included, it is advisable to add a small amount 



of autoclaved sand to assist in tissue homog- 
enization. 

DNA Extraction: 400 ul of homogenizing 
buffer (0.1 M NaCI; 0.2 M Sucrose; 0.1 M Tris- 
HCI, pH 8.0; 0.05 M EDTA, pH 9.1 ) was added 
to a 1 .5 ml Eppendorf tube containing a single 
snail. The sample was homogenized with a 
micropestle and centrifuged at 13,000 rpm for 
5 min. The supernatant was removed by de- 
canting the tube, thus getting rid of excess 
mucus while retaining DNA in the pellet. Then 
320 ul of SDS buffer (0.02 M Tris; 0.01 M 
EDTA; 1% SDS) was added, and the sample 
was vortexed for about four sec. If the tissue 
failed to redistribute in the solution, a sterile 
toothpick was used to stir it up. The tube was 
then incubated in a 65°C water bath for one h. 
Subsequently, 30 ul of 8M chilled potassium 
acetate was added, the tube vortexed for 
about one sec, and then placed on ice for one 
h. The tube was centrifuged at 13,000 rpm for 
5 min and the supernatant was poured in a 
new tube containing 700 ul of chilled absolute 
ethanol. The tube was stored at -20°C for at 
least 30 min to allow the DNA to precipitate. 
After that, the tube was centrifuged for 1 min 
at 13,000 rpm in a chilled (4°C) machine. The 
supernatant was removed, and the pellet was 
dried in a speedvac. The pellet was resus- 
pended in 30 ul ddH 2 with 1 ul of RNAse 
(200 ng/ul) and incubated in a water bath at 
37°C for 1 h. The extraction was stored at 
-80°C at a concentration of 10 MM Tris-HCI. 
We found that tissues without excess 
amounts of mucopolysaccharides could be 
homogenized simultaneously with the ho- 
mogenizing buffer and the SDS buffer. The 
homogenate from the combined buffer is then 
incubated at 65°C for 60 min, and the stan- 
dard protocol continues thereon. 



Institute of Evolutionary and Ecological Sciences. University of Leiden, PO Box 9516, 2300 RA Leiden, The Netherlands; 
coline@rulsfb.leidenuniv.nl 
2 National Museum of Natural History, Naturalis, PO Box 9517, 2300 RA Leiden, The Netherlands 

203 



204 



VAN MOORSELETAL. 




4 5 6 7 8 9 10 11 12 13 14 15 16 



FIG. 1 . 5 ц1 of each DNA extraction was loaded on a 1% agarose gel, electroforesed for half an hour at 70 V 
and stained with ethidium bromide. From left to right Albinaria spratti: SJ 1 to 16. 



Testing for the Presence of DNA: From each 
extract, 5 ul were loaded on a 1% agarose gel 
stained with ethidium bromide, and then elec- 
trophoresed for 30 min at 70 volts. Viewed 
under ultraviolet light, the gel revealed clear, 
heavy bands of intact DNA, smears of de- 
graded DNA, and some small RNA fragments 
(Fig. 1). To block the damaging activity of en- 
donucleases that remain in the extract, the 
final product can be brought to a concentra- 
tion of 0.1 mM EDTA. However, this preserva- 
tive might also inhibit polymerase activity in 
PCR and is therefore not advised if the DNA 
is to be used immediately. To further prevent 
degradation of the DNA, the extract can be 
heated to 95°C for 15 min, and then gradually 
cooled to room temperature. Since heating 
will also cleave the DNA, this treatment 
should be used if only smaller DNA fragments 
(< 1000 bp) are needed (Wade, pers. comm.). 

Testing the DNA in PCR: The extracted DNA 
was used to amplify multi- and single-copy 
nuclear and mitochondrial DNA. The following 
primers were used to amplify the complete 
first Internal Transcribed Spacer (ITS1) of the 
ribosomal DNA: 18d, 5'-cacaccgcccgtcgctac- 
tacc-3' (Hillis & Dixon, 1991), and 5.80K, 5'- 
attgactcgccgaccc-3' (designed by the au- 
thors). To amplify the complete nuclear single 
copy Calmodulin intron, the primers cam-1, 
5'-acatgatcaatgaagtggatgc-3' and cam-2, 5'- 
catcatggtgaggaattctgg-3' (both designed by 
M. Schilthuizen) were used. For the mito- 
chondrial DNA, we amplified a part of the 16S 
ribosomal DNA corresponding to positions 
12898-13382 of the Albinaria coerulea mito- 
chondrial genome (Hatzoglou et al., 1995). In 
this case, we used the primers 16Sar, 5'-cgc- 
ctgtttatcaaaaacat-3', and 16Sbr, 5'-ccggtct- 
gaactcagatcacgt-3' (Simon et al., 1994). 



The results of the PCR were viewed under 
U V light after running on a 1 % agarose gel and 
stained with ethidium bromide (Fig. 2). In each 
case, the length of the PCR fragment matched 
the expected size according to known se- 
quences within the genus Albinaria. This 
showed that the DNA extracted with this pro- 
cedure can be used for amplification of both 
nuclear, multi- and single-copy, and mitochon- 
drial DNA fragments. Extraction and PCR 
amplification using this method has been re- 
peated by M. Schilthuizen (Agricultural Uni- 
versity of Wageningen) with Malaysian land 
snails of the genera Gyliotrachela (Gastro- 
poda: Pulmonata, Vertiginidae), Diplomma- 
tina, and Opisthostoma (Gastropoda: Proso- 
branchia, Diplommatinidae). Both the PCR 
and sequencing of the ITS-1 region and the 
Calmodulin intron were successful (Schil- 
thuizen, personal communication). In addition, 
this method has been repeated by M. Haase 
(University of Basel), who added 10 u.l Pro- 
teinase К (10 mg/ml) to the homogenizing 
buffer. Haase extracted DNA from Arianta ar- 
bustorum (Gastropoda: Pulmonata, Helici- 
dae) and from Bradybaena frucicum (Gas- 
tropoda: Pulmonata, Bradybaenidae). These 
isolations performed well in both PCR of COI 
and RAPD analysis (Haase, personal commu- 
nication), and once again the results under- 
score the applicability of gastropod DNA, 
obtained by the described protocol, in PCR- 
based techniques. 

Often we found that the isolated DNA was 
slightly viscose, the pellet would not always 
dissolve well, and the DNA was sometimes 
even a little degraded. But not surprisingly, 
these shortcomings did not cause any prob- 
lems with PCR. However, we caution that for 
methods that require intact high molecular 
weight DNA, such as in restriction enzyme 



MOLLUSC DNA EXTRACTION PROTOCOL FOR PCR-BASED TECHNIQUES 205 




1 



8 



10 11 



FIG. 2. PCR-products of three primer pairs for three different Albinaria species. Lane 2 to 4: Calmodulin in- 
tron; lane 5 to 7: 16S region; lane 8 to 10: ITS1 . Each PCR was performed on A. coerulea, A. spratti and A. 
corrúgala respectively. To check the length of the PCR-products a ladder is represented in the first lane. The 
500 bp band is stressed in intensity: above 500 bp a band at every 100 bp, below 500 bp a band at every 50 
bp. The last lane contains a standardmarker (л DNA digested with EcoRI and Hindi II). 



mapping, this protocol is not advisable. The 
presence of DNA fragments of different 
lengths, and the inability of complete diges- 
tion, may cause such experiments to fail. 

This protocol is relatively simple, ommitting 
chloroform/phenol steps, and quick: the ex- 
traction takes about 4 h, of which 3.5 h of wait- 
ing periods of 30 min or more. Furthermore, it 
is extremely inexpensive: the costs of the eas- 
ily available reagents of the buffer and the ac- 
etate are approximately just US$ 0.50 per 1 00 
extractions. And the DNA has proven to be 
usefull in several PCR-based techniques for 
both mitochondrial and nuclear gene amplifi- 
cations. 

Acknowledgements 

We would like to thank M. Momose for valu- 
able suggestions concerning the protocol and 
W. H. Piel for revising the manuscript. 



LITERATURE CITED 

DOURIS V., S. GIOKAS, R. LECANIDOU, M. MY- 
LONAS & G. С RODAKIS, 1998, Phylogenetic 



analysis of mitochondrial DNA and morphological 
characters suggest a need for taxonomic re-eval- 
uation within the Alopiinae (Gastropoda: Clausili- 
idae). Journal of Molluscan Studies, 64: 81 -92. 
FLOOK, P. K., M. D. WILSON & R. J. POST, 1992, 
The use of repetitive DNA probes in the analysis 
of natural populations of insects and parasites. 

Pp. 484-486, in R. S. BERRY, T. J. CRAWFORD & 

G. M. MEWiTT, eds., Genes in ecology, Blackwell 
Scientific Publications, Oxford. 

HATZOGLOU, E., G. С RODAKIS & R. 
LECANIDOU, 1995, Complete sequence and 
gene organization of the mitochondrial genome 
of the land snail Albinaria coerulea. Genetics, 
140: 1353-1366. 

HILLIS, D. M. & M. T DIXON, 1991, Ribosomal 
DNA: Molecular evolution and phylogenetic infer- 
ence. The Quarterly Review of Biology, 66: 
411-453. 

KYLE, С J., & E. G. BOULDING, 1998, Molecular 
genetic evidence for parallel evolution in a marine 
gastropod, Littorina subrotundata. Proceedings 
of the Royal Society of London, (B) 265: 
303-308. 

LYDEARD, C, J. H. YODER, W. E. HOLZNAGEL, 
F G. THOMPSON & P. HARTFIELD, 1998, Phy- 
logenetic utility of the 5'-half of mitochondrial 1 6S 
rDNA gene sequences for inferring relationships 
of Elimia (Cerithioidea: Pleuroceridae). Malacolo- 
gia, 39: 183-193. 



206 



VAN MOORSELETAL. 



SIMON, C, F. FRATI, A. BECKENBACH, В. 
CRESPI, H. LIU & P. FLOCK, 1994, Evolution, 
weighting and phylogenetic utility of mitochondr- 
ial gene sequences and a compilation of con- 
served polymerase chain reaction primers. An- 
nals of the Entomological Society of America, 87: 
651-701. 

WALBOT, V. & С WARREN, 1988, Regulation of 
the Mu element copy number in maize lines with 
an active or inactive Mutator transposable ele- 
ment system. Molecular & General Genetics, 
211:27-34. 

WINNEPENNINCKX, В., T. BACKELJAU & R. DE 
WÄCHTER, 1993, Extraction of high molecular 
weight DNA from molluscs. Trends in Genetics, 9: 
409. 



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, P.O. Box 1222, West 
Falmouth, MA 02574-1222, U.S.A. 



Revised ms. accepted 18 August 1999 



MALACOLOGIA, 2000, 42(1-2): 207-215 



INDEX 



Taxa in bold are new; pages in italic indicate 
figures of taxa. 

abyssorum, Neolepton 1 28 
Achatina fúlica 107 
affinis, Psammobia 21 
africana, Panacea 166, 167 
Ag hol i max reticu latus 1 09 
Albinaria 204-205 

coerulea 204-205 

corrugata 205 

spratti 204-205 
albopalliatus, Madeirovithna 55, 61 
Amphichaena kindermanni2\ 

Gari 9 
annularis, Phenacolimax AA 
annulata, Pyrgula 1 95 
antárctica, Graneledone 65, 74 
antipodum, Neolepton 1 27 
aplexa, Belgrandia 175, 198 

Bythinia 198 
Aporema 1 65 
arabica, Vitrina 41 , 60 
Arabivitrina 39-42, 44, 55-56, 60 

darnaudiW 

Janssen i АО, 58, 60 
arate, Pholadomya 165-166 
arbustorum, Arianta 204 
archaea, Belgrandia 186, 199 

Bythinella 199 
Arcopagia 1 4 
Arianta arbustorum 204 
Arion ater 107 
arrate, Panacea 166, 767 
Asaphis 2 
aspersa, Helix 1 07 
ater, /4r/'on 1 07 
atlántica, Plutonia 45-46, 55, 62 

Viquesnelia 41 , 44 

balthica, Macoma 21 

Belgrandia 86, 93, 171-195, 198-201 

aplexa Mb, 198 

archaea 186, 199 

bigorhensiS 176, 179, 180-182, 184, 199 

bonelliana 200 

boscae 175-176, 180-182, 183, 200 

bourguignati 1 74, 1 86, 1 99 

capra/ 1 90, 200 

caz/oí/ 174, 176, 180-182, 183-184, 200 

conoidea 175-176, 778, 181-182, 189, 198 



controversa 200 

coutagnei MS, 179, 180-182, 184, 200 

cylindracea 186, 194, 199-200 

delpretiana 200 

deshayesiana 1 86, 1 99 

desnoyersi 1 86, 1 99 

dumenisliana 1 86, 1 99 

edwardsiana 186, 199 

germanica 183, 194, 199 

gfrastM^, 172, 176, 778, 181-182, 186- 

195, 787-754 

g/bba 171, 174-180, 778, 181-182, 184, 

189-190, 194, 198 

gibberulaMA, 176-177, 778, 179-180, 787- 

782, 183, 189-190, 195, 199 

guranensis 185-186, 199 

fteuss/ alcoaensis 1 77, 180-182, 1 85, 1 89, 

200 

heussiheussiM7, 179, 180-182, 185, 200 

/on/ca 185, 195, 200 

/o/nw/tens/s 186, 199 

lanceolata 200 

lartetiana 186, 199 

/af/'na 200 

lusitanica 177, 179, 180-182, 183, 185, 195, 

199 

marginataMS, 177, 778, 181-182, 183, 

186, 194, 198,200 

mariatheresiae 1 90, 200 

m i ñuscóla 190-191, 200 

minuta 1 98 

moitessieri MA, 177-179, 778, 181-182, 

184, 199 

пала 1 86, 200 

occidental M9, 183, 199 

paladilhi 1 83 

фаг/'а 186, 200 

saint-si топ i 1 85, 1 94 

saint-simoniana 1 85 

saviana 1 99 

sequanica 1 86, 1 99 

simoniana 185-186, 194, 199 

subovata 185-186, 199 

targioniana 200 

thermalis 1 90, 1 98-200 

toriferaM7, 180-182, 184, 195 

tricassina 186, 200 

uniplicata 175, 198 

var/ca 174-175, 190, 198 

vitrea M9, 184, 186, 194 

z/Vc/7/200 



207 



208 



INDEX 



Belgrandiella 1 86 

Benthoctopus 67 

Bernardinidae 123 

bigorriensis, Belgrandia 176, 179, 180-182, 

184, 199 
bimaculatus, Heterodonax2\ 
biradiata, Psammobia 21 
Bithinia diaphana 1 86 
blainvillei, Solecurtus 22 
blainvillii, Solecurtus 22 
blauneri, Vitrina 39 
bonelliana, Belgrandia 200 
boreopacifica, Graneledone 66, 68, 73 
boscae, Belgrandia 175-176, 180-182, 183, 

200 

Hydrobia 183, 200 
bourguignati, Belgrandia 174, 186, 199 

Bythinella 1 99 
bowdichi, Psammobia 22 
Bradybaena frucicum 204 
Bradybaenidae 204 
Buhminidae 57 
burmeisteri, Psammobia 23 
Bythinella 171, 173, 185-186, 194 

archaea 1 99 

bourguignati 1 99 

cylindracea 186, 199-200 

deshayesiana 1 99 

desnoyersi 1 99 

dumenisliana 1 99 

edwardsiana 1 99 

guranensis 1 99 

joinvillensis 1 99 

lanceolata 1 86 

lartetiana 1 99 

nana 200 

simoniana 1 99 

subovata 1 99 
Bythinia aplexa 1 98 

g/'bba 198 

marginata\ 85, 198-199 

saviana 1 99 

simoniana 1 85, 1 99 

uniplicata 1 98 
Bythiospeum 1 86 

Calidivitrina 41 

California, Sanguinolaria 21 

californica, Gari 4-7, 5, 10-12, 20 

(Gobraeus) Gari 1,3,17 

Psammobia 3, 5, 21 
Calyptogena 68 
canadensis, Insul ¡vitrina 61 



Wrina 39 
Canarivitrina 39-58, 53 
(Canarivitrina) Plutonia 39, 41 , 56 

cancellatum, Neolepton 1 28 

candida, Pholadomya 1 68 

capra/, Belgrandia 190, 200 

Cardium 1 09 

carniolica, Vitrina 60 

casia, Psammobia 21 

cayennensis, Psammobia 21 

caz/'of/, Belgrandia 174, 176, 180-182, 183- 

184,200 

(Belgrandia) Paludinella 1 83 
cerina, Psammobia 21 
challengeri, Graneledone 66, 72 
Chi lens is, Panacea 165-166, 767 
chilodia, iónica Litthabitella 173, 185, 200 
christinae, Guerrina 39, 61 
c/'rce, (Dysmea) Gari 1 9 

Gar/1, 78, 19,21 

Psammobia 1 9 
claibornense, (Graum) Psammobia 22 
Clausilüdae 57, 203 
coerulea, Albinaria 204-205 

Pate//a 131-132 
cognata, ТетпосопсЬа2Л 
columbiana, Psammobia 22 
conoidea, Belgrandia 1 75- 1 76 , 7 7S, 181-182, 

189, 198 

Paludina 175, 198 

Wfn'na 41 
constrieta, Macoma 21 
controversa, Belgrandia 200 
corrúgala, Albinaria 205 
coutagnei, Belgrandia 176, 179, 180-182, 

184,200 
cowlitzensis, Psammobia 22 
Cran/a 131-132 
crassa, Psammobia 1,16 
Crassostrea gigas 1 1 3 

virginica 1 1 3 
Crassulobia 2 
curta, Hydrobia 200 
cutícula, Guerrina 61 

(Guerrina) Plutonia 45 

Helix 39, 41 
Cyamioidea 123, 128 
Cyclostoma gibbum 1 74, 1 98 

vitreumMQ, 186, 198 
cylindracea, Belgrandia 1 86, 1 94, 1 99-200 

Bythinella 186, 199-200 
cylindrica, Psammobia 22 



INDEX 



209 



dalli, Gari 22 
darnaudi, Arabivitrina 61 
darwini, Psammobia 22 
declivis, Psammobia 21 

Sofen 21 
decora, Psammobia 21 
delpretiana, Belgrandia 200 
Dentalium 1 44 
depressa, ТеШпаЗ 
deshayesiana, Belgrandia 186, 199 

Bythinella 1 99 
desnoyersi, Belgrandia 186, 199 

ßyi/7/ne//a199 
dianae, Plutonia 47-49, 51 
diaphana, Bithinia 1 86 

Paludina 1 86 
diegoensis, Psammobia 22 
Diplommatina 204 
Diplommatinidae 204 
dombei, Leporimetis2\ 
Donax (Amphichaena) kindermanni2\ 
Dreissena polymorpha 113-114, 1 1 9-1 20 
dumenisliana, Belgrandia 1 86, 1 99 

Bythinella 1 99 
Dysmea 1-2,17 

Gar/ 17, 19 

eborea, Psammobia 22 
eceroensis, Insulivitrina 39 
edentula, Gari 7, 9 

Siliquaria 1 , 7, S, 9 
ec№, /Wytf/us 113, 160 

Os/rea 113 
edwardsiana, Belgrandia 1 86, 1 99 

Bythinella 1 99 
emmersoni, Insulivitrina 39 
endentulus, Gari 7 
eoundulata, Gari 22 
Eucobresia 41 , 46, 62 

falcifer, Plutonia 52 

falcifera, Plutonia 47-49, 52, 54 

fasciatus, Liguus 31 

fasciolata, Vitrina 39 

Ferrisia 93 

fez/, Neohoratia 96 

//'/osa, Psammobia 22 

/rag/7/s, Parilimya 1 65, 1 68 

frucicum, Bradybaena 204 

fecate, Gari 2-4, 7, 8, 9-10, 20, 22 

(Gobraeus) Gari 1 , 7-9 

7e///'na 7, S, 9 
/u//'ca, Achatina 1 07 



fuscata, Gari 7 

Gallandia 39-42, 44, 55-56, 61 

/ecfe/v 55 

olympica 41 , 44, 55 
Gammatricula 99 
дал/, ТеШпаЗ 
Gari \ -23,20 

(Amphichaena) 9 

californica 4-7 ', 5, 10-12, 20 

c/rce1, 70, 19,21 

da///' 22 

(Dysmea) 17, 19 

(Dysmea) circe 1 9 

(Dysmea) helenae 1 , 10, 17 

(Dysmea) occidens 1 9 

edentula 7, 9 

endentulus 7 

eoundulata 22 

fucata 2-4, 7, 8, 9-10, -20, 22 

fuscata 7 

(Gari) 19 

(Gobraeus) 1,3, 9-11, 16 

(Gobraeus) californica 1,3, 17 

(Gobraeus) fucata 1 , 7-9 

(Gobraeus) helenae 1 7 

(Gobraeus) lata 1,9-11 

(Gobraeus) listrota hadratera 22 

(Gobraeus) listrota listrota 22 

(Gobraeus) maxima 1,11 

(Gobraeus) panamensis 1,13 

(Gobraeus) solida 1,14 

(Gobraeus) vaginatus 21 

(Gobraeus) wagneri 1 3 

helenae 4, 17, 75, 19,20 

/?om/7 umpquaensis 22 

jacksonensis 22 

kazusensis 1 , 7 

kazusensis atsumiensis 1 

lata 2, 4, 10, 11-13,20 

linharesl, 17, 70, 19 

maxima 2, 4,7, 10-13, 72,20 

occidens 17, 19 

panamensis 4, 12-14, 74, 20, 22 

(Psammobia) texta 22 

(Psammacola) 11,21 

(Psam mocóla) 9, 1 1 , 17, 21 

regularis^, 3,7, 10, 17 

so//da 2, 4, 75, 20, 22 

vulgaris 3 

wagneri 74, 14 
gasulli, Neohoratia 75, 79, 96 

(Neohoratia) Häuften ia 75, 79, 95 



210 



INDEX 



Tarraconia 76, 79, 80-81, 82, 83, 84-87, 85, 

88-90, 92, 94-98, 96-97 
germanica, Belgrandia 183, 194, 199 
gfrast, Belgrandia 171, 172, 176, 778, 181- 

182, 186-195, 187-194 
gibba, Belgrandia 171, 174-180, 778, 787- 

782, 184, 189-190, 194, 198 

Bythinia 1 98 

Hydrobia 1 79 

Paludina 1 79 
gibberula, Belgrandia 174, 176-177, 778, 179- 

180, 181-182, 183, 189-190, 195, 199 
gibbum, Cyclostoma 1 74, 1 98 
gibbus, Tagelus21 
gigas, Crassostrea 1 1 3 
glacialis, Vitrina 46 
Glossoidea 128 
Gobraeus 1-3, 17 
(Gobraeus) Gari 1 , 3, 9-1 1 , 1 6 

Psammobia 3, 7, 9, 11 
gomerensis, Insulivitrina 39 
Graneledone 74 
Graneledone 63-68, 65, 72-74 

antárctica 65, 74 

boreopacifica 66, 68, 73 

challengeri 66, 72 

macrotyla 65, 74 

pacifica 64, 67, 72 

verrucosa 66, 67 
guranensis, Belgrandia 185-186, 199 

Bythinella 1 99 
guassu, Psammobia 23 
guassus, Psammobia 23 
gubematoria, Psammobia 22 

da///' Psammobia 22 
Guerrina 39-42, 44-46, 55-56, 61 

christinae 39, 61 

cutícula 61 
(Guerrina), Plutonia 56 
Gyliotrachela 204 

haddoni, Parilimya 1 68 

(Parilimya) Pholadomya 1 66 
Halodraka 123 
hanetianus, Solenocurtus 22 
harrisi, Psammobia 22 
Hauffenia 75, 96, 98-99 

(Neohoratia) gasulli 75, 79, 95 

te///'n/75 
helenae, (Dysmea) Gari 1 , 10, 17 

Gar/ 4, 17, 76, 19,20 

(Gobraeus) Gari 1 7 
Helicidae 204 



Helicolimax lamarckii 39, 41 

ma/or 41 
He//x aspersa 1 07 

cutícula 39, 41 

I uco ru m : 07, 110 

sem/7/max41 
Heterodonax 1 

bimaculatus 21 

paci ficus 21 
Heteroglypta 2 
heussi, alcoaensis Belgrandia 177, 180-182, 

185, 189,200 

heussi Belgrandia U7, 179, 180-182, 185, 

200 
Híspanla Tarraconensis 79 
hodgei, Isocardia 1 65 
Horaf/'a 75, 96, 98-99 
/70/77/7, Tellina 22 

umpquaensis Gari 22 
Hydrobia 175 

ooscae 183, 200 

curta 200 

gr /'ЬЬа 1 79 

lusitanica 1 79, 1 99 

minuscola 200 

moitessieri 1 78, 199 

pa/ad///7/174, 179-180, 183, 199 

va/7'ca 175, 198 
Hydrobiidae 75, 97, 99, 171 
Hydrobiinae 99 

Insulivitrina 39-42, 44-46, 55-56, 61-62 

canariensis 61 

eceroensis 39 

emmersoni 39 

gomerensis 39 

machadoi 39 

mascaensis 39, 54 

nogalesi 39 

oromii 39 

tamaranensis 39 

tu bereu lata 39 
(Insulivitrina), Plutonia 56 
/bn/'ca, Belgrandia 185, 195, 200 
Islamia 1 95 
Isocardia hodgei 165 

jacksonensls, Gari 22 
jansseni, Arabivithna 40, 58, 60 
joinvillensis, Belgrandia 1 86, 1 99 
ßyi/7/rte//a199 

kazusensis, Gari 1 , 7 



INDEX 



211 



atsumiensis Gari 1 

atsumiensis Psammobia 3, 6 

Psammobia 3, 7 

Psammocola 5 
Kelliella 128 
Kelliellidae 123, 128 
Kermadysmea 2 
kindermanni, Amphichaena 21 

Donax (Amphichaena) 21 

Psammobia 21 
kotulae, Semilimax 55, 60 

lamarckii, Hei icol i max 39, 41 

(Insulivitrina) Plutonia 45 
lanceolata, Belgrandia 200 

Bythinella 1 86 
/ap///üs, Mvce//a 103, 105-106, 107, 70S, 109- 

110 
lartetiana, Belgrandia 186, 199 

Bythinella 199 
Lasaea 1 09 
/ate, Gari 2, 4, 10, 11-13,20 

(Gobraeus) Gari 1,9-11 

Psammobia 9-10, 70 
latebasis, Vitrina 39 
lateralis, Mulinia 1 1 3 
/af/'na, Belgrandia 200 
latisiliqua, Lysiloma 33 
/ecter/, Trochovitrina 44 

Gallandia 55 
Lepohmetis dombei2~\ 
Lepton sulcatulum 1 23 
levicaudata, Pahlimya 1 68 
L/fiWUS 31 -32, 32,34,36 

fasciatus 31 
lilacina, Psammobia 1 , 3 
Umacoidea 39, 41 
//nea, Psammobia 22 
linhares, Gari:, 17, 75, 19 
listrota, listrota (Gobraeus) Gari 22 

hadratera (Gobraeus) Gari 22 
Lithoglyphus naticoides 1 95 
Litthabitella 1 85 

chilodia iónica 173, 185, 200 
locardi, Moitessieria 1 83 

Panacea 166, 767 
/oven/, Pahlimya 1 68 
lucorum, He//'x107, 110 
lusitanica, Belgrandia 177, 179, 180-182, 183, 

185, 195, 199 

Hydrobia 179, 199 
lusoria, Psammobia 21 
Lymnaea 86, 93 



Lysiloma 31, 33-36 
latisiliqua 33 

machadoi, Insulivitrina 39 
Macoma balthica 21 

constrieta 21 

tente 21 
macrotyla, Graneledone 65, 74 
Macfra 161 

venehiormis\ 49, 151-155, 757-750,159- 

162, 757 
Mactridae 149 
maculata, Semperula 1 07 
Madeirovitrina 39-42, 44, 55-56, 61 

albopalliatus 55, 61 

mareida 40 

п/7 /da 40 
(Madeirovitrina), Plutonia 56 
magellanicus, Placopecten 1 1 3 
тау'ол, Hei icol i max 41 

Phenacolimax 40, 61 
maoria, Pahlimya 1 68 
mareida, Madeirovitrina 40 
marginata, Belgrandia 175, 177, 775, 757- 

752, 183, 186, 194, 198,200 

BythiniaWb, 198-199 

Paludina 175, 198 
mahatheresiae, Belgrandia 190, 200 
martini, Psammobia 21 
mascaensis, Insulivitrina 39, 52 

Plutonia 47-49, 50, 52, 54 
maxima, Gari 2, 4, 7, 10-13, 72, 20 

(Gobraeus) Gari 1,11 

Psammobia 11-12, 72 
Megadesmidae 165 
Melanopsis 86, 93 
minuscola, Belgrandia 190-191, 200 

Hydrobia 200 
minuta, Belgrandia 1 98 

Paludina 186, 198 

Thermhydrobia 200 
mississippiensis, Psammobia 22 
moitessieh, Belgrandia 174, 177-179, 7 75, 

181-182, 184, 199 

Hydrobia 178, 199 
Moitessieria 98, 179, 186, 194 

/oearef/ 183 

(Spiralix) vitrea 1 98 
Mulinia lateralis 1 1 3 
MytilusWS, 131-132, 135, 143-145 

edu//s 113, 160 

nana, Belgrandia 1 86, 200 



212 



INDEX 



Bythinella 200 
naticoides, Lithoglyphus 1 95 
Neohoratia 75-76, 95-96, 98 

fez/ 96 

gasulli 75, 79, 96 
Neolepton 123-129 

abyssorum 1 28 

antipodum 1 27 

cancellatum 1 28 

profundorum 1 23, 725- 729, 1 27- 1 28 

sulcatulum 1 23 
Neoleptonidae 123 
Nipponopanacca 1 66 
ri/f/da, Madeirovitrina 40 

l//'7r/r?a 41 
nogalesi, Insulivitrina 39 
Notolepton 123 
Notomya 165-166 
A/uce//a 103-110 

/ap/'/tos 103, 105-106, 107, 708, 109-110 
Nuttallia 1 , 21 

nuttallii2: 
nuttallii, Nuttallia 21 

obscura, Psammobia 22 
occidentalis, Belgrandia 179, 183, 199 
occidens, (Dysmea) Gari 1 9 

Gar/ 17, 19 

So/en 17 
Octopodidae 63-64 
olequahensis, Psammobia 22 
Oligoli max 39, 41 , 44, 56 
olympica, Gallandia 41 , 44, 55 
Opisthostoma 204 
Orientalina 1 95 
oromii, Insulivitrina 39 
Osfrea edi///'s 1 1 3 
ova//'s, Spisula 1 1 3 
ozarkana, Psammobia 22 

Pachydrobiini 99 

pacifica, Graneledone 64, 67, 72 

Parilimya 1 68 

Psammobia 21 
pacificus, Heterodonax2\ 
paladilhi, Belgrandia 1 83 

Hydrobia 174, 179-180, 183, 199 
Paludina (Belgrandia) cazioti 1 83 

conoidea 175, 198 

£/PPa179 

marginata 175, 198 

minuta '\ 86, 198 
Panacea 165-168 



africana 166, 767 

arraía 166, 767 

chilensis 165-166, 767 

tocard/ 166, 767 

sumatrana 166, 767 

tasmanica 166, 767 
panamensis, Gari 4, 12-14, 73, 20, 22 

(Gobraeus) Gari 1 , 13 
papyria, Psammobia 22 
Parilimya 166, 168 

f ragil is '\ 65, 168 

haddoni 1 68 

levicaudata 1 68 

toi/en/ 1 68 

maoria 1 68 

pacifica 1 68 

s/n/'ca 1 68 

sukuraii 1 68 
Paralimyidae 165 
Parilimyidae 165-166 
parry/, WYr/'na 39 
patagónica, Psammobia 23 
Patella ^3^, 137, 143-146 

соеш/еа 131-132 

vulgata -\07, 109 
paulucciae, Vitrina 41 , 44 
peruanus, Tagelus 1 6 
Phenacolimacinae 41-42, 44 
Phenacolimax 39-42, 44, 55-56, 62 

annularis 44 

major 40, 61 
Pholadomya 166-168 

arafe 165-166 

candida 1 68 

(Nipponopanacca) 1 68 

(Panacea) 168 

(Panacea) sakuraii 1 66 

(Parilimya) 168 

(Parilimya) haddoni 1 66 

tasmanica 1 65 
Pholadomyidae 165, 168 
Pholadomyoidea 165 
Pr/ysa 93 
Pisidium 86, 93 
Placopecten magellanicus 1 1 3 
p/ana, Scrobicularia 1 09 
Platynereis 131 
Plutonia 39-42, 44, 46, 50, 55-56, 58, 62 

atlántica 45-46, 55, 62 

(Canarivitrina) 39, 41 , 56 

(Canarivitrina) taburientensis 41 , 56 

dianae 47-49, 51 

fe/c/fer 52 



INDEX 



213 



falcifera 47-49, 52, 54 

(Guerrina) 56 

(Guerrina) cutícula 45 

(Insulivitrina) 56 

(Insulivitrina) lamarcki 45 

(Madeirovitrina) 56 

mascaensis 47-49, 50, 52, 54 

(Plutonia) 56 

ripkeni 47-49, 50-52, 58 

taburientensis 46, 47-49, 50-52, 58 
(Plutonia), Plutonia 56 
Plutoniainae 44 
Plutoniinae 39-58 
Plutonium 44 
Plutoniuminae 44 

polymorpha, Dreissena 113-114, 1 19-120 
Pomatiopsidae 75, 99 
Potamopyrgus 93 
Procardia 165-166 
profundorum, Neolepton 123, 125-129, 127- 

128 
(Psammacola) rubrolineata 3 
Psammobella 2 
Psammobia 2-3, 14, 21 

aff/n/'s 21 

(Amphichaena) regularis 9 

biradiata 21 

bowdichi 22 

burmeisteri 23 

cali fornica 3, 21 

casia 21 

cayennensis 21 

cer /ла 21 

c/'rce 1 9 

columbiana 22 

cowlitzensis 22 

crassa 1,16 

cylindrica 22 

darwini 22 

declivis 21 

decora 21 

diegoensis 22 

eborea 22 

fi/osa 22 

(Gobraeus) 3, 7, 9, 1 1 

(Gobraeus) vaginatus 1 1 

(Gobraeus) vaginoides 1 1 

(Graum) claibomense 22 

guassu 23 

guassus 23 

gubernatoria dalli 22 

Yarn's/ 22 

kazusensis 3, 7 



kazusensis atsumiensis 3, 6 

kindermanni2\ 

lata 9-10, 70 

lilacina 1 , 3 

//nea 22 

lusoria 21 

martini 21 

maxima 11-12, 72 

mississippiensis 22 

obscura 22 

olequahensis 22 

ozarkana 22 

pacifica 21 

papyria 22 

patagónica 23 

(Psammobia) 7, 21 

purpureomaculata 21 

régularisai, 10, 10 

rubroradiata 1 , 3 

sm/f/7/22 

solida 1, 75, 16 

squamosa 19, 21 

sqvamosa 19, 21 

tehuelcha 22 

vaginoides 21 
Psammobüdae 1-3 
(Psammacola) 9 

Gar/ 11,21 
(Psammocola) 9 

Gar/ 9, 11, 17,21 

kazusensis 5 
Psammodonax 2 
Psammotaena 2 
Pseudamnicola 86, 93, 99 
punicea, Trochydrobia 98 
purpureomaculata, Psammobia 21 
pyrenaicus, Sem///max 61 
Pyrgula annulata 1 95 
Pyrgulidae 75, 99 

regularis, Gari\, 3, 7, 10, 17 

(Amphichaena) Psammobia 9 

Psammobia 1, 70, 10 
reticulata, Vitrina 39 
reticulatus, Agriolimax 1 09 
r/par/a, Belgrandia 186, 200 
ripkeni, Plutonia 47-49, 50-52, 58 
fí/ssoa 109 
го/ал/, Tarraconia 75, 76, 82, 84, 86-87, 97- 

94,91-97,96-97 
rubrolineata, (Psammacola) 3 
rubroradiata, Psammobia 1 , 3 

Sanguinolaria 3 



214 



INDEX 



saint-simoniana, Belgrandia 1 85 
saint-simoni, Belgrandia 185, 194 
sakuraii, (Panacea) Pholadomya 1 66 
Sanguinolaria california 21 

rubroradiata 3 
saviana, Belgrandia 1 99 

Bythinia 1 99 
Scalpomactra 23 
Scrobicularia plana 1 09 
Semilimacella 44 
Semilimacinae 41-42 
Semilimax 39-45, 43, 54-56, 60-61 

/cofu/ae 55, 60 

pyrenaicus 61 

semilimax 55, 60-61 
semilimax, Helix 41 

Semilimax 55, 60-61 
Semperula macúlala 1 07 
sequanica, Belgrandia 1 86, 1 99 
Siliquaria 1 6 
simoniana, Belgrandia 185-186, 194, 199 

Bythinella 199 

Bythinia 185, 199 
s/n/ca, Parilimya 1 68 
sm/ï/j/, Psammobia 22 
Solecurtus blainvillei 22 

blainvillii 22 

solidus 1 6 
So /ел declivis 21 

occidens 1 7 

solidus 15 

(Solenocurtus) solidus 1 4 

vespertinus 3 
Solenocurtus hanetianus 22 
Soleotellina 2 
solida, Gari 2, 4, 15,20,22 

(Gobraeus) Gari 1,14 

Psammobia 1 , У 5, 1 6 
solidus, Solecurtus 1 6 

(Solenocurtus) Solen 1 4 

So/en 15 
Spisula ovalis 1 1 3 
spratf/, Albinaria 204-205 
squamosa, Psammobia 19, 21 
sqvamosa, Psammobia 19, 21 
subovata, Belgrandia 185-186, 199 

Bythinella 199 
sukuraii, Parilimya 1 68 
sulcatulum, Lepton 123 

Neolepton 123 
sumatrana, Panacea 166, 767 

taburientensis, Plutonia 46, 47-49, 50-52, 58 



(Canarivitrina) Plutonia 41 , 56 
Tagelus gibbus 21 

peruanus 1 6 
tamaranensis, Insulivitrina 39 
targioniana, Belgrandia 200 
Tarraconensis, Híspanla 79 
Tarraconia 75, 79, 97-99 

gasu/// 76, 79, SO-SÍ, 82, S3, 84-87, 35, 33- 

50, 92, 94-98, 93-97 

rolani 75, 76, 82, 84, 86-87, 91-97, 97-94, 

93-97 
tasmanica, Panacea 166, 737 

Pholadomya 1 65 
tehuelcha, Psammobia 22 
Ге///ла 21 -22 

depressa 3 

fucate 7, 3, 9 

gar/ 3 

nom// 22 

truncata 3 
te///n/, Hauffenia 75 
Tellinidae 21 
Tellinoidea 1 

Temnoconcha cognata 21 
tenía, Psammobia 21 
texte, (Psammobia) Gari 22 
Theodoxus 86, 93 
thermalis, Belgrandia 190, 198-200 

Thermhydrobia 1 99-200 

Turbo 1 98 
Thermhydrobia 1 74 

m/nute 200 

thermalis 1 99-200 
tentera, Belgrandia 177 ', 180-182, 184, 195 
tricassina, Belgrandia 1 86, 200 
Trichodina 171, 189, 195 
Triculinae 99 
Trochovitrina 41-42, 44 

/ecter/ 44 
Trochydrobia 98 

punicea 98 
tuberculata, Insulivitrina 39 
Turbo thermalis 1 98 
truncata, ТеШпаЗ 

uniplicata, Belgrandia 1 75, 1 98 
Bythinia 1 98 

vaginatus, (Gobraeus) Psammobia 1 1 

(Gobraeus) Gari2\ 
vaginoides, (Gobraeus) Psammobia 1 1 

Psammobia 21 
var/ca, Belgrandia 174-175, 190, 198 



INDEX 



215 



Hydrobia 175, 198 
Veneridae 128 
veneriformis, Mactra 149, 151-155, 157-159, 

159-162, 161 
verrucosa, Graneledone 66, 67 
Vertiginidae 204 
vespertinus, Solen 3 
Viquesnelia atlántica 41 , 44 
virginica, Crassostrea 1 1 3 
vitrea, Belgrandia 179, 184, 186, 194 

(Spiraux) Moitessieria 1 98 
vitreum, Cyclostoma 179, 186, 198 
Vitrina 41 , 45 

arabica 41 , 60 

blauneri 39 

bonelli 44 

canariensis 39 

camiolica 60 

conoidea 41 



fasciolata 39 

glacialis 46 

latebasis 39 

n/ï/cte 41 

par/y/ 39 

paulucciae 41 , 44 

reticulata 39 
Vitrinidae 39, 41-44, 56 
Vitrininae 41-42 
Vithnobrachium 41 
Vitriplutoniinae 44 
vulgaris, Gari2> 
vulgata, Patella 107, 109 

wagneri, Gari 14, 14 
(Gobraeus) Gari 1 3 

z/Vc/7/, Belgrandia 200 



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VOL. 42, NO. 1-2 MALACOLOGIA 2000 

CONTENTS 

EUGENE V. COAN 

The Eastern Pacific Recent Species of the Bivalve Genus Gari (Tellinoidea: 
Psammobiidae), with Notes on Western Atlantic and Fossil Taxa 1 

ROBERT E. BENNETTS, STEVEN A. SPARKS & DEBORAH JANSEN 

Factors Influencing Movement Probabilities of Florida Tree Snails Liguus 
fasciatus (Müller) in Big Cypress National Preserve Following Hurricane 
Andrew 31 

MARÍA R. ALONSO, MANUEL J. VALIDO, KLAUS GROH & MIGUEL IBÁÑEZ 

Plutonia (Canarivitrina), New Subgenus, from the Canary Islands, and the 
Phylogenetic Relationships of the Subfamily Plutoniinae (Gastropoda: 
Limacoidea: Vitrinidae) 39 

JANET R.VOIGHT 

The Distribution of Octopuses of Graneledone (Cephalopoda: Octopodidae) 

in Reference to Deep-Sea Features 63 

M. A. RAMOS, B. ARCONADA, E. ROLAN & D. MORENO 

A New Genus and a New Species of Hydrobiid Snail (Mollusca: Gastropoda: 
Hydrobiidae) from Eastern Spain 75 

VASILIS K. DIMITRIADIS & ELIZABETH B. ANDREWS 

Ultrastructural and Cytochemical Study of the Digestive Gland Cells of the 
Marine Prosobranch Mollusc Nucella lapillus (L.) in Relation to Function ... 103 

KRISTEN M. LEWIS, JEFFREY L. FEDER, THOMAS G. HORVATH & 

GARY A. LAMBERTI 

Heterozygosity and Fitness: No Strong Association in Great Lakes 
Populations of Zebra Mussel, Dreissena polymorpha (Pallas) 113 

J.A.ALLEN 

A New Deep-Sea Species of the Genus Neolepton (Bivalvia; Cyamioidea; 
Neoleptonidae) from the Argentine Basin 123 

MARGHERITA RAINERI 

Early Neurogenesis Pattern in Patella coerulea (Patellogastropoda) and its 
Possible Phylogenetic Implications 131 

EE-YUNG CHUNG & DONG-KI RYOU 

Gametogenesis and Sexual Maturation of the Surf Clam Mactra veneriformis 

on the West Coast of Korea 1 49 

EUGENE V COAN 

A New Species of Panacea from Chile (Bivalvia: Pholadomyoidea: 
Paralimyidae) 165 

MARTIN HAASE 

A Revision of the Genus Belgrandia, with the Description of a New Species 

from France (Caenogastropoda: Hydrobiidae) 171 

COLINE H. M. VAN MOORSEL, W. JOHAN VAN NES & HENDRIK-JAN MEGENS 

Letters to the Editor 203