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MALACOLOGIA 




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International Journal of Malacology 




Vol. 46^1) 





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MALACOLOGIA 
http:\\nnalacologia.f mnh.org 



EDITOR-IN-CHIEF: 
GEORGE M. DAVIS 



Editorial Office 

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



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Field Museum, Chicago 

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University of Delaware, Lewes 

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ALAN KOHN 

Vice President 

University of Washington, Seattle 

JAMES NYBAKKEN 

President Elect 

Moss Landing Marine Laboratory, California 

CLYDE F E. ROPER 

Smithsonian Institution, Washington, D.C. 

SHI-KUEIWU 

University of Colorado Museum, Boulder 



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Secretary, UNITAS MALACOLOGICA 
The Natural History Museum 
London, United Kingdom 



JACKIE L. VAN GOETHEM 
Treasurer, UNITAS MALACOLOGICA 
Koninklijk Belgisch Instituut 
voor Natuurwetenschappen 
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Environmental Protection Agency 
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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 © 2004 by the Institute of Malacology 
ISSN: 0076-2997 



2004 
EDITORIAL BOARD 



J. A. ALLEN 

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

E.E.BINDER 

Museum d'Histoire Naturelle 

Geneve, Switzerland 

P. BOUCHET 

Muséum National d'Histoire Naturelle 

Paris, France 

bouchet@cimrs l.mnhn. fr 

P. CALOW 

University of Stieffield 
United Kingdom 

R.CAMERON 

Stieffieid 

United Kingdom 

R. Cameron @ Sheffield, ac. uk 

J. G. CARTER 

University of Nortti Carolina 

Chapel Hill, U.S.A. 

MARYVONNE CHARRIER 
Université de Rennes 
France 
Maryvonne.Charrier@univ-rennes1.fr 

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

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

B. 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. 

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Rijksmuseum van Natuurlijke Historie 
Leiden, Netherlands 
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R GIUSTI 

Universita di Siena, Italy 

giustif@unisi.it 



A. N. GOLIKOV 
Zoological Institute 
St Petersburg, Russia 

A.V.GROSSU 
Universitatea Bucuresti 
Romania 

T. HABE 

Tokai University 

Shimizu, Japan 

R. HANLON 

Marine Biological Laboratory 

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



G. HASZPRUNAR 

Zoologische Staatssammlung Muenchen 

Muenchen, Germany 

haszi@zi.biologie.uni-muenchen.de 

J. M. HEALY 

University of Queensland 

Australia 

jhealy@zoology.uq.edu.au 

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

K. E. HOAGLAND 

Council for Undergraduate Research 

Washington, DC, U.S.A. 

Elaine@curorg 

B. HUBENDICK 
Naturhistohska Museet 
Göteborg, Sweden 

S. HUNT 
Lancashire 
United Kingdom 

R.JANSSEN 

Forschungsinstitut Senckenberg, 
Frankfurt am Main, Germany 

M.S.JOHNSON 
University of Western Australia 
Nedlands, WA, Australia 
msj@cyllene. uwa. edu.au 

R. N.KILBURN 
Natal Museum 
Pietermaritzburg, South Africa 

M. A. KLAPPENBACH 

Museo Nacional de Historia Natural 

Montevideo, Uruguay 

J. KNUDSEN 

Zoologisk Institut Museum 

Kobenhavn, Denmark 



MCZ , 
LIBRARY 

HARVARD 
UNIVERSITY 



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

С MEIER-BROOK 
Tropenmedizinisches Institut 
Tubingen, Germany 

H. K. 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.0KLAND 
University of Oslo 
Norway 

T. OKUTANI 
University of Fisheries 
Tokyo, Japan 

W. L. PARAENSE 

Instituto Oswalde Cruz, Rio de Janeiro 

Brazil 

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

R. PIPE 

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

J. R POINTIER 

Ecole Pratique des Hautes Etudes 

Perpignan Cedex, France 

pointier@gala.univ-perp.fr 

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 

A. STAÑCZYKOWSKA 
Siedlce, Poland 

F. STARMÜHLNER 

Zoologisches Institut der Universität 

Wien, Austria 

Y I. STAROBOGATOV 
Zoological Institute 
St. Petersburg, Russia 

J. STUARDO 
Universidad de Chile 
Valparaiso 

C.THIRIOT 

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

S.TILLIER 

Museum National d'Histoire Naturelle 

Paris, France 

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

N. H.VERDONK 
Rijksuniversiteit 
Utrecht, Netherlands 

H. WÄG ELE 

Ruhr-Universität Bochum 

Germany 

Heike. Waegele@ruhr-uni-bochum.de 

ANDERS WAREN 

Swedish Museum of Natural History 

Stockholm, Sweden 

B.R.WILSON 

Dept. Conservation and Land Management 

Kallaroo, Western Australia 

H.ZEISSLER 
Leipzig, Germany 

A. ZILCH 

Forschungsinstitut Senckenberg 

Frankfurt am Main, Germany 



MALACOLOGIA, 2004, 46(1): 1-17 

ANATOMY OF A SMALL CLAM, ALVEINUS OJIANUS (BIVALVIA: KELLIELLIDAE), 
WITH A DISCUSSION ON THE TAXONOMIC STATUS OF THE FAMILY 

George A. Evseev, Natalya K. Kolotukhina & Olga Ya. Semenikhina 

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



ABSTRACT 

The anatomy of the small bivalve Alveinus ojianus (Yokoyama, 1927) of 1 .8-2.0 mm shell 
length from shallow bays of the northwestern Pacific was studied. The morphological fea- 
tures, in which A. ojianus differs from other bivalve species occurring in the shelf waters 
include: a reduced gill consisting of the inner demibranchs, the posterior filaments of which 
are replaced by a membrane lining the cloaca; one siphon, the walls of which consist of the 
cuticular epithelium lining the mantle cavity; the inner mantle fold of the adult is thickened 
and, in addition to the mantle gland, contains extracellular granules; the labial palps have 
no sorting ridges, and the gill flexure is without a food groove; the stomach has a thickened 
cuticular lining and a helicoid structure, which are homologues of the gastric shield of other 
bivalves; and there are no sorting areas, digestive pouch, caecum or dorsal hood in the 
stomach. Comparison of its internal morphological features with those of other bivalve taxa 
demonstrates the close similarity of the Kelliellidae to the deep-water Verticordiidae but not 
Veneroida, to which Kelliellidae are usually referred. However, comparison of Kelliellidae 
and Verticordiidae based on shell morphology shows that differences between them makes 
their relationship improbable. One of the reasons for these contradictions appears to be the 
paedomorphic development of Л. ojianus and a mixed nature of the morphological features 
used for the taxonomy. 

Keywords: anatomy, mantle, extracellular granules, gill, stomach, paedomorphic features. 



INTRODUCTION 

The mollusks with a shell length no more 
than 3-5 mm hold a special position in the 
taxonomic systems of the Bivalvia. In their 
morphological features, they can be divided 
into two groups. One group is the miniature 
adult bivalves - many species of Propeamus- 
siidae, Carditinae, Circinae and others - in 
which features are homologous to those of 
larger taxa, and which are usually included in 
a subfamily with those larger taxa (Habe, 
1977; Dijkstra & Kastoro, 1997; Oliver & 
Zuschin, 2001). Small bivalves of the second 
group belong to families or superfamilies - 
Crenellidae, Montacutidae, Lasaeidae, Philo- 
bryidae, Condylocardiidae and others - in 
which the macroforms are usually absent 
(Habe, 1977; Morton, 1978; Morton & Scott, 
1989; Middelfart, 2000). Their shell morpho- 
logical features are usually characterized by 
hypoplasia, and among their internal organs 
there are structures that are unknown in 
macroforms (Oldfield, 1961; Morton, 1981; 
Mikkelsen & Bieler, 1989, 1992; Allen, 2000). 
As a result of this, in most cases the taxo- 
nomic features of the adult small forms are 



difficult to compare with those of the larger 
taxa, on which one of the most prevalent sys- 
tems of Heterodonta is based (Cox, 1969; 
Keen, 1969). The taxonomic status and phylo- 
genetic relationships some of them are still 
being discussed (Ockelmann, 1964; Cosel & 
Salas, 2001). 

Alveinus ojianus (Yokoyama, 1927) belongs 
to the otherwise deep-water family Kelliellidae, 
a member of the second group of the small 
bivalves. In addition to A. ojianus and another 
species of this genus recently found in the 
Red and Arabian seas (Oliver & Zuschin, 
2001), in the family there are 12-14 species of 
Kelliella mainly inhabiting the waters of the 
eastern and southern Pacific (Knudsen, 1970; 
Bernard, 1989). Allen (2001) has provided a 
detailed description of this genus, including 
internal morphology, as well as the taxonomic 
history of the Kelliellidae and its relationships 
with other deep-water taxa. In contrast to 
deep-water Kelliella, species of the genus 
Alveinus occur both in shallow bays and open 
parts of the coastal zone (Miyadi & Habe, 
1957; Oliver & Zuschin, 2001). 

Some data on biology oí A. ojianus are to be 
found in general studies on the ecology and 



EVSEEVETAL. 



taxonomy of bivalves (Habe, 1950, 1973, 
1977; Scarlato, 1981; Evseev, 2000). Accord- 
ing to these works, A. ojianus is widely distrib- 
uted in Peter the Great Bay and eastern part 
of the Sea of Japan, as well as off the Pacific 
coast of Japan and the South Kuril Islands. It 
is common in sandy mud at a depth of 2-22 
m, where its density may exceed 1,000 speci- 
mens per m^. The bivalves attach to the sand 
grains with 1-2 byssus threads. The shell 
length of Л. ojianus rarely exceeds 2 mm. The 
mollusks are easily identified by their triangu- 
lar, lustrous brownish shell. 

In this study, we examined the anatomy of /A. 
ojianus, no data on which were found in the 
literature. There is also no information on the 
phylogenetic relationships of the Kelliellidae 
with its sister families. In this connection, we 
also attempted to estimate the taxonomic and 
phylogenetic significance of the internal fea- 
tures of A. ojianus for their use as additional 
taxonomic characters for the family Kellielli- 
dae, for which the taxonomy is almost com- 
pletely based on shell morphology. 



MATERIALS AND METHODS 

The adult and juvenile specimens of Л. ojianus 
from the Amursky Bay and Vostok Bay, as well 
as the open part of Peter the Great Bay, the Sea 
of Japan, were used in this study. The bivalves 
were collected with a dredge from the research 
vessel "Lugovoye" in September 1999 and 
sampled by SCUBA-diving to 12-14 m depth in 
July-September 2000-2001 and in March 
2002. Most of the specimens investigated 
measured from 350-1,000 цт in length. 

The anatomy was studied by means of serial 
histological sections. The mollusks were pre- 
liminarily fixed in 96% ethanol. The shell valves 
were removed with a fine needle. The speci- 
mens were embedded in paraffin using routine 
methods, but the holding time in alcohol, chlo- 
roform and paraffin was greatly reduced. The 
sections were made from 7-10 цт with a slid- 
ing microtome, mounted on glass slides and 
stained with Eriich and Boomer's haemo- 
toxylin. A light microscope was used to exam- 
ine the sections. 



1 
3a 



ABBREVIATIONS IN THE FIGURES 

first cardinal tooth 

third anterior cardinal tooth 



3b third posterior cardinal tooth 

aa anterior adductor 

abf ascending branchial filaments 

ag apical gland of the foot 

a/ line of attachment of the inner demibranch 

to the visceral mass 

an anus 

ar anterior retractor 

ba branchial axis 

bg byssal gland 

с cloaca 

eg terminal cutícula of the gill 

els cutucular lining of the stomach 

cpg cerebral-pleural ganglion 

cso crystalline style opening 

ct loose connective tissue 

dd digestive diverticula 

ea exhalant aperture 

ec excretory cells 

eg extracellular granules of the mantle lobe 

f foot 

g mantle gland 

gg extracellular granules of the apical gland 

g/7 gastric helicoid structure 

gr gastric ridges as "traffic circles" of the 

crystalline style 

/a inhalant aperture 

ici intracapillaceous inclusions 

ice inner ciliated epithelium 

icg intracellular granules 

id inner demibranch 

ifc interfilamentary connective 

ig intestinal groove 

ip inhalant sensory papillae 

ipe inner pavement epithelium 

iue inner unciliated epithelium 

/i kidney 

/c branchial lateral cilia 

If longitudinal muscle fibres 

Ifc branchial latero-frontal cilia 

Ig marginal groove of the lunule 

// pit of the internal ligament 

Iml left mantle lobe 

le external ligament 

Ip labial palps 

mg mid-gut 

mm mucous masses 
mmf middle mantle fold 

n nimpha 

о oesophagus 

obc opening of the byssogenous canal 

omf outer mantle fold 

ose outer stratified epithelium 

ov ovary 

p pericardium 

pa posterior adductor 



ANATOMY OF ALVEINUS OJIANUS 



pd post-apertural dilatation of the mid-gut 

pg pedal ganglion 

pj posterior mantle junction 

pp posterior sensory papillae 

pr posterior retractor 

ps passage from the stomach to the post- 
apertural dilatation of mid-gut 

pss passage from the style sac to the post- 
apertural dilatation of mid-gut 

pt1 posterior lateral tooth 

pvj postero-ventral mantle junction 

r rectum 

re rotary cilia of the crystalline style sac 

rml right mantle lobe 

rsw right stomach wall 

s stomach 

sc statocyst capsule 

si exhalant siphon 

skr branchial skeletal rods 

sla slit-like aperture of the crystalline style sac 

ss crystalline style sac 

st statocyst 

sti statolith 

t testis 

th tooth of the gastric helicoid structure 

ty typhlosole 

ucf branchial unciliated filaments 

vj ventral mantle fusion 

vsw ventral stomach wall 



RESULTS 



Cuticular tissue forms very thin, semi-trans- 
parent walls of the siphon. Like the base of the 
inhalant aperture, the base of the siphon is 
thickened and its posterior edge bears three 
papillae. The total number of sensory papillae 
on both sides of the inhalant and exhalant 
apertures amounts to 17. In juveniles, there 
may be fewer papillae; for example, in a speci- 
men of approximately 500 |am shell length, 
two pairs of papillae were found at the lateral 
edges of the inhalant aperture, two pairs of 
common papillae and one pair of short papil- 
lae were located between the inhalant and 
exhalant apertures. As in adults, three papillae 
were present dorsal to the siphon. 




pvj ip 



A general view, details of the right shell, and 
the internal topography of A. ojianus are 
shown in Figures 1-4. 

Mantle 

The mantle edge has three folds. The thin 
outer fold is located along the posterior, ven- 
tral and anterior shell margin. The thin middle 
fold fuses near the anterior adductor and ven- 
trally (Fig. 1, vj) and forms a broad pedal- 
byssal gape occupying most of the ventral 
mantle edge. The inhalant aperture of the 
mantle cavity is separated from the exhalant 
aperture by the postero-ventral fusion of the 
fold (Fig. 1, pvj). The thickened edges of the 
inhalant aperture bear laterally three marginal 
sensory papillae each. There are one to three 
short guard cilia at the papillae tips. 

Each side of the thickened, fused part of the 
middle mantle fold has four similar lateral pa- 
pillae between the inhalant and exhalant aper- 
tures. The exhalant aperture terminates in a 
conic siphon with a smooth, tapering opening. 



li le 




FIGS. 1, 2. Alveinus ojianus. FIG. 1. Lateral and 
ventral views of living specimen. Bar = 500 цт. 
FIG. 2. Internal view of right shell valve showing 
hinge teeth. Bar = 300 цт. 



omf 




®^ mmf rml 



FIG. 3. Topography of internal organs as seen from left side with 
left shell valve and left mantle lobe removed. Bar = 200 цт. 



pr к pa an eg 




Щ~оЫ 



ip ^S^^^g^gg 

FIG. 4. Internal morphology and configuration as seen from left side (sagittal section). Bar = 100 цт. 



ANATOMY OF ALVEINUS OJIANUS 



The inner mantle fold of the adult is usually 
thickened (20-40 цт), whereas the thickness 
of the other proximal mantle part does not 
exceed 6-8 цт. In width, the thickened fold 
may vary from relatively narrow ridge to the 
crescent belt, which starts ventral to the ante- 
rior adductor, continues on either side of the 
pedal-byssal gape and ends close to the inhal- 
ant aperture. 

Sections made near the posterior edge of 
the pedal-byssal gape (Fig. 5) and through the 
central part of the inner thickened fold (Fig. 6) 
show that the thickening and the proximal di- 
latation of this fold are formed by connective 
and glandular tissue, as well as by yellowish 
extracellular granules. 

The surface of the inner wall of the mantle 
represents a smooth transparent epithelium 
consisting of polygonal pavement cells with 



mm 



1^ eg 




mm 



ose 



FIGS. 5, 6. Extracellular granules of mantle. Bar = 
50 цт. FIG. 5. Transverse section through 
posterior part of layer of granules. Fig 6. Diagonal 
section through central part of their layer. 



marked nuclei and nucleoli. The wrinkled outer 
layer of the fold consists of a stratified semi- 
transparent epithelium with large cells and 
clearly distinguishable nuclei. Within the thick- 
ened fold, there is a layer of deeply stained, 
vesicular floccular glandular tissue, under 
which the oval or oblong-angular grayish or 
yellowish granules are located. 

The granules usually form one, sometimes 
two layers. They are enveloped in the homoge- 
neous milk-white masses, which fuse into a 
single substance resembling the mucous se- 
cretion of the labial palps. Where granules form 
two layers, their size ranges from 20-30 цт in 
the upper layer and from 1 0-1 2 to 1 5-20 цт in 
the lower layer. If granules are in one layer, 
their length amounts to 35-40 цт, and they are 
large and roundly elongate. The granules are 
joined to the outer or inner epithelium by their 
short sides. At the same time, contacting sides 
of the granules are often free of the milk-white 
masses. The glandular tissue sometimes sepa- 
rates the granule layers. 

The granules occur inside the mantle lobes 
in both males and females. In adults, the total 
number of granules is 60-80. On left and right 
lobes of the same individual, the arrangement 
may be asymmetrical and varying in shape, 
size and quantity of the granules. These ap- 
pear to be different stages in their formation. 
In juveniles of 350-400 цт shell length, gran- 
ules were not found. Adults 500-700 цт long 
collected in October-November have from 
20-25 to 40-55 granules in the anterior part of 
the mantle cavity, where they were located in 
a single layer. No granules were found in 
bivalves sampled at the end of March. 

Muscular System 

The anterior adductor is elongate and nar- 
rowed towards the retractor. The posterior 
adductor is larger and more rounded in shape. 
As in Turtonia minuta (Fabricius, 1780) and 
Lasaea rubra (Montagu, 1803) (Oldfield, 
1955), both adductors consist of smooth, 
bundled muscle fibres (Figs. 3, 4). In other 
bivalves, muscle fibres of this type usually 
form the outer portion of adductor, which is 
responsible for maintaining valve closure 
(Yonge, 1936). An inner adductor component 
of "quick" cross-striated muscle fibres is lack- 
ing in A. ojianus. 

A system of circular, longitudinal and diago- 
nal muscle fibres is located within the foot. 
The longitudinal and diagonal fibres continue 



EVSEEV ET AL. 



dorsally and form a pair of anterior and a pair 
of posterior pedal retractors, wliicli do not dif- 
fer from adductors in their color and structure. 
The other muscles are represented by smaller 
bundles and fine fibres scattered within the 
mantle and the visceral mass. 

Gills 

The subquadrate gills cover practically all 
the visceral mass. They consist of inner left 
and right demibranchs, each with long de- 
scending and short ascending lamellae. The 
descending lamellae extend from the bran- 
chial axes. The latter run almost parallel to the 
external ligament between the subumbonal 
enlargement of the visceral mass and the pos- 
terior adductor (Fig. 3, ba). The ascending 
lamella terminates dorsally in arcuate chiti- 
nous bridges attached to the visceral mass 
along a line between the adductors. The de- 
scending and ascending lamellae join ven- 
trally in a flexure with one or two interlamellar 
connectives. Thin, rounded, chitinous inter- 




FIGS. 7, 8. Branchial filaments. Bar = 30 цт. 
FIG. 7. Descending filaments. FIG. 8. Ascending 
filaments. 



filamentar connectives that join skeletal rods of 
adjacent filaments are rare and irregular. 

The homorhabdic gill filaments are oval in a 
transverse section, with two skeletal rods join- 
ing abfrontally (Figs. 7, 8, skr). The filament 
walls consist of a one-layer ciliary epithelium 
with indistinct cell borders. The blood vessels 
are without muscle septa. Rare blood cells 
and deeply stained organic inclusions, which 
appear to be bacterial in a nature, occur within 
some vessels. The interfilamentar space is 
filled with long cilia, of which the latero-frontal 
cilia are the most pronounced. Laterally, each 
filament bears symmetrical thick cilia (?) of 
unclear function, the orientation of which is 
opposite that of the remaining cilia. The former 
are similar to the "anomalous" latero-frontal cilia 
delimiting the water fluxes (Atkins, 1938) but 
differ from them in direction and location. The 
diameter of the filament is about 30^0 |am; the 
filament number varies from 17 to 22 in an 
adult demibranch. 

Anteriorly, the demibranch is located be- 
tween the labial palps. However, the ventral 
marginal food groove is indistinct. The height 
of the ascending lamella decreases posteri- 
orly, and it is lacking behind the visceral mass. 
In its place, the descending lamella of the left 
demibranch joins the descending lamella of 
the right demibranch to form the reno-anal 
cavity (cloaca or suprabranchial cavity) 
(Pelseneer, 1906). Anteriorly, the lateral walls 
of the cloaca are formed by the distal limbs of 
the kidney. The posterior region of the lateral 
walls and the posterior wall of the cavity are 
lined internally with an elastic cuticular tissue 
(Figs. 3, 4, eg). The anus opens into the cloaca 
through the posterior wall; the cloaca commu- 
nicates ventrally with the siphon through the 
exhalant aperture. The postero-ventral wall of 
the cloaca joins the inner fold of the mantle 
between the inhalant and exhalant apertures. 

Foot 

The elongate foot is comparatively large. Its 
middle part is cylindrical; the apical part is 
pointed and ciliated. At its base, the foot usu- 
ally expands abruptly (Fig. 4). The postero- 
ventral part of the foot forms a well-marked 
heel. In vivo, the foot may greatly expand, 
becoming about twice or three times as long 
as the shell. In fixed specimens, the foot is 
usually directed towards the labial palps. The 
outer wrinkled surface of the foot is covered 
with a stratified epithelium. 



ANATOMY OF ALVEINUS OJIANUS 



There are two glands in the foot. The byssal 
gland (Fig. 4, bg) lies in the posterior part of 
the foot and consists of large, deeply stained 
cells fornning the secreting lamellae. The latter 
are separated by narrow passages, which are 
filled with a homogeneous, poorly staining 
secretion. This system of the lamellae and 
passages converges ventrally to a broad 
byssogenous canal that opens anterior to the 
heel. The byssogenous canal is lined with one 
layer of similarly staining cells. The byssus 
threads are very thin (5-7 цт) and semi-trans- 
parent. Their proximal part is club-shaped; the 
distal tip bears a small terminal disc, which is 
attached to large grains of sand or gravel. 
There are usually one or two byssus threads. 

The second apical gland (Fig. 4, ag) lies in 
the central and distal, or only in the distal parts 
of the foot. It is separated from the byssal 
gland by a layer of loose connective tissue 
and by a system of the blood lacunae, within 
which connective tissue islets and rare radial 
muscle fibres are scattered. The gland cells 
also form secreting lamellae, but these are 
narrower, more compact and less intensely 
stained in some places than the byssal gland 
cells. In the intercellular space of the apical 
gland, isometric or elongate homogenous, 
faintly stained granules sometimes occur. 
These are similar to the mantle cavity gran- 
ules in shape and dimensions. Unlike the 
byssal gland cells, the cells of the apical gland 
are small and rare in some individuals. In oth- 
ers, the secreting cells may be absent, and 
only the axial part of the foot consists of rela- 
tively well-stained glandular tissue surrounded 
by numerous lacunae. 

The byssal groove is not marked on the ven- 
tral side of the foot, and no openings are 
found in the distal and middle parts of the foot. 
The canal of the apical gland appears to open 



either in the byssogenous canal or in the short 
byssal slit. 

Statocysts 

These are symmetrical sense organs repre- 
sented by spherical capsules approximately 
30 |дт diameter. A spherical yellow or brown- 
ish statolith measuring up to 16-17 цт in di- 
ameter lies within each statocyst (Fig. 9). Cilia 
were not observed on the inner capsule walls. 
The statolith structure appears to be radial. A 
dark dot is sometimes seen in the center of 
statolith. 

The capsules are located in the anterior part 
of the foot lateral to the pedal ganglion. They 
belong to the type B2 occurring in the 
Anomalodesmata (Morton, 1985). 

Labial Palps 

These are parallel anterior and posterior 
lamellae attached dorsal to the visceral mass 
(Fig. 4, /p). The lamellae are ventrally elon- 
gated. Near their base is a broad funnel- 
shaped mouth that opens into a long 
oesophagus. The smooth inner surface of the 
palps and walls of the oesophagus are cov- 
ered by long, dense cilia, which are inclined 
proximally. Sorting ridges on the palps are 
lacking. The ciliary area is usually covered 
with dense mucus. Under the ciliated epithe- 
lium there are scattered, intensely staining 
mucous gland cells, as well as loose connec- 
tive tissue with numerous lacunae. The outer 
surface of the palps is unciliated. 

In fixed adults, the length of the anterior 
palps, including bases, is 250-300 цт, 
whereas the length of the free ends does not 
usually exceed 100-150 цт. However, in liv- 
ing specimens the anterior palps are capable 
of expanding posteriorly up to 400-500 цт. 




FIG. 9. Pedal ganglion and statocysts. Bar = 30 цт. 



Alimentary Canal 

A long oesophagus enters the stomach 
antero-ventrally (Fig. 4, o). The oesophageal 
walls consist of one layer of columnar ciliated 
epithelial cells, the long, dense cilia of which 
are directed towards the stomach. Mucous 
gland cells are scattered among the epithelial 
cells. Deep folds, three to four in number, may 
be seen in a transverse section of the oe- 
sophagus (Fig. 10). 

The stomach is hemispherical (Fig. 4, s), 
consisting of anterior and posterior sections. 



EVSEEVETAL. 




ice 



FIG. 10. Oesophagus (transverse 
section). Bar = 30 цт. 

The lateral walls and antero-ventral section 
are thin and lined with the columnar digestive 
(?) epithelial cells containing numerous inclu- 
sions of different shape and density. The cell 
borders on the inside of stomach cavity are 
usually indistinct. Ciliated cells were not found. 



The dorsal and right-dorsal walls of the ante- 
rior section are composed of a thickened cu- 
ticular tissue (Fig. 4, els) that is devoid of the 
inner covering of the digestive cells. 

In the center of the anterior section of the 
stomach there is a cup-shaped organ 100 цт 
in diameter joined to the right-dorsal wall of 
the stomach (Fig. 11, gh). It is lined by the 
same thick cuticular tissue as the wall. In a 
transverse section, the organ is shaped like a 
convoluted or helicoid lamina. The initial bifur- 
cated part appears to function as the erosive 
tooth of the gastric shield. The distal height of 
the wall of the helicoid structure is 30-40 цт. 
The antero-ventral wall of the helicoid struc- 
ture is absent, and ventrally there are the 
curved ridges (Fig. 1 1 , gr) running towards the 
intestinal groove. 

The contents of the anterior section of the 
stomach consist of a mucous secretion and 
algae, among which the diatoms Thalassiosira, 
Pyxidicula, Odantella predominate. The dia- 
toms are represented both by the whole 




# ig 



FIG. 11. Antero-dorsal wall of stomach and food contents (left view). Bar = 50 цт. 



ANATOMY OF ALVEINUS OJIANUS 



rsw 




FIG. 12. Crystalline style sac. Transverse section through dorsal part. Bar = 50 цпп. 



VSW.' 




rsw 



FIG. 13. Crystalline style sac. Transverse section through middle 
part and ventral wall of stomach. Bar = 50 цт. 



10 



EVSEEV ET AL. 



ig PS 




FIG. 14. Crystalline style sac. Transverse section through 
ventral wall and mid-gut opening. Bar = 50 цт. 



thecas (25-30 цт length) and large fragments 
forming assemblages near the oesophagial 
opening. The small fragments forming two to 
three diffuse spots usually occur anterior to the 
helicoid structure, where one of the ducts of the 
digestive diverticula appears to be located. 




FIG. 15. Mid-gut (transverse section). Bar = 20 цт. 



The relatively large crystalline style sac 
forms the posterior section of the stomach. 
The single layer of epithelial cells forming the 
sac walls has large nuclei and numerous in- 
clusions (Figs. 12-14). The inner surface of 
the sac is densely lined with deeply staining 
cilia, which rotate the short thickened style 
clockwise. A thin membrane coat continuous 
with the similar coat of the stomach wall cov- 
ers the outer surface of the sac. 

The sac communicates with the stomach by 
means of a longitudinal slit-like aperture (Fig. 
12, sla). The dorsal part of the aperture, where 
the style projects from the sac, is broad, 
whereas the middle part, to which the intesti- 
nal groove leads from the stomach, is nar- 
rower (Fig. 13, sla). In the middle part of the 
sac, the slit-like aperture is formed from the 
edges of the anterior sac wall, which are 
turned inward. Ventrally, the slit-like aperture 
extends to the bottom of the sac. In a trans- 
verse section through this area (Fig. 14), the 



ANATOMY OF ALVEINUS OJIANUS 



11 



left inward turned side of the aperture looks 
like the typhlosole, which is curved like a small 
tongue. It runs in a spiral along the posterior 
edge of the opening and the right wall of the 
sac into the post-apertural dilatation of the 
mid-gut. In other words, there are two pas- 
sages that lead into the post-apertural dilata- 
tion of the gut. One of them (Fig. 14, ps) 
communicates gut with stomach via the intes- 
tinal groove. The other (Fig. 14, pss) leads 
from the sac cavity into the gut through the 
slit-like aperture. 

The mid-gut runs from the bottom of the 
style sac towards the oesophagus (Fig. 4). At 
the anterior part of the visceral mass, it imme- 
diately loops and runs back to the base of the 
foot. Then the gut ascends dorsally along the 
posterior wall of the visceral mass to the peri- 
cardial cavity. The hindgut rounds the adduc- 
tor not only on its dorsal side, as in all 
bivalves, but also to the ventral side. The rec- 
tum turns anteriorly and dorsally. 

There are no differences in transverse sec- 
tions of the gut immediately posterior to the 
stomach (Fig. 15) and across the ascending 
part (Figs. 12-14). The gut wall consists of a 
single layer of large cylindrical cells with dis- 
tinct nuclei and transparent or stained cyto- 
plasm. In diagonal and longitudinal sections, 
the cell aggregations look like twisted, in- 
tensely staining fibrous bands with the dense 
cytoplasmic inclusions. The outer wall of the 
gut is covered by a thin membrane. A similar 
membrane appears to line the inner gut wall. 
No cilia and no typhlosole were found in the 
lumen of the intestine. 

The food wastes in the beginning of the mid- 
gut represent a thickened grayish mass con- 
sisting of fragments of algal thecas. Contents 
of the ascending section are brownish in color. 
Gaps outlining the borders of the pellets ap- 
pear in the contents. There are isolated oval 
dark or brownish pellets in the hindgut. 

The digestive diverticula consist of two 
lobes, which unite under the umbo and sur- 
round the stomach laterally. The lobes taper 
ventrally and extend almost to the base of the 
foot. In some individuals, large triangular- 
rounded cells of the diverticular gland together 
form oval rosettes. In sections transverse to 
the stomach wall, a branching net of canals 
located among the cell groups and converging 
towards the stomach wall can be observed. 
There are vacuoles, faintly staining nuclei and 
small dark cytoplasmic granules within the 
cells. In other individuals (Fig. 11), the thin 
gland has short broad canals and large cells. 



the borders of which are indistinct. This ap- 
pears to be caused by autolysis. In this case, 
the gland cells contain numerous transparent 
brownish granules, among which the deeply 
stained granules form aggregations. Digestive 
ducts and tubules typical of most bivalves 
were not found. 

Pericardium and Kidneys 

The pericardial cavity lies anterior and dor- 
sal to the posterior adductor (Figs. 3, 4). 
Within the cavity, there is a transparent ven- 
tricle, through which the rectum passes, and 
two thin-walled postero-ventral auricles. The 
kidney consists of two elongated distal limbs 
and lies posterior to the pericardium. Within 
the limbs, faintly staining floccular tissue and 
large excretory cells with the brownish or dark 
granules occur (Fig. 16). Each distal limb ex- 
tends laterally to the terminal cutícula of the 
gill forming the cloaca and anteriorly to the 
posterior wall of the visceral mass. In the ven- 




FIG. 16. Kidney. Transverse section through 
distal part of left limb and terminal cuticula of gill. 
Bar = 50 |.im. 



12 



EVSEEVETAL. 



tral part of the cloaca, the kidney limbs only 
join the terminal cutícula of the gill, and the 
renal openings are located near the base of 
the exhalant siphon. 

Reproductive System 

The species is dioecious; it becomes mature 
at a shell length of approximately 0.8-1 .0 mm. 
The ovary lies in the visceral mass and sur- 
rounds the digestive diverticula and the stom- 
ach laterally and posteriorly. The anterior part 
of the ovary is located in the subumbonal en- 
largement of the visceral mass. Ventrally, the 
ovary borders the base of the foot. The testis 
is similarly located in the visceral mass. 

The walls of the follicle of the ovary or testis 
are very thin. In the ovary, the oval and oval- 
angular oocytes were predominant among the 
detached oocytes. The large growing oocytes 
of 30-40 |am, sometimes 45 цт, diameter with 
well-marked nuclei and nucleoli were attached 
to the follicle wall. No ripe oocytes were found 
in sections. Unlike the ovary, the testis was 
filled by both spermatocytes I and II and by 
nutritional cells. Ripe spermatozoa with the 
large heads occurred in center of the follicle. 
The largest spermatocytes were 6-7 цт in 
diameter. Their large nuclei were lightly 
stained, with small nucleoli and granules of 
chromatin. Gonial and nutritional ceils were 
attached to the wall. 



DISCUSSION 

Alveinus ojianus is a member of the family 
Kellieilidae, which appears to consist of 3-4 
genera and 23-25 species, including fossil 
taxa (Habe, 1953, 1977; Keen, 1969; Bernard, 
1989; Hayami & Kase, 1993; Allen, 2001; 
Oliver & Zuschin, 2001). The taxonomic status 
and phylogenetic relationships of this family 
still remain insufficiently well-founded and this 
could be the result of poor data on the internal 
comparative and functional morphology both 
of this species and the family as a whole. As 
a result, the taxonomic significance of some 
internal features of the Kellieilidae that could 
also be useful phylogenetic characters is not 
yet determined. 

The mantle and apical glands as well as the 
extracellular mantle granules of >A. ojianus are 
examples of such features of unclear taxo- 
nomic significance. A mantle gland that is 
structurally and topologically similar to that of 



A. ojianus is known not only in such sister 
families as the Vesicomyidae (Morton, 1986; 
Allen, 2001), but also in the more distant 
Hiatellidae, Crassatellidae, Carditidae, Thyasi- 
ridae and Verticordiidae (Pelseneer, 1906; 
Allen, 1968; Yonge, 1969, 1971; Allen & 
Turner, 1974). In these taxa, mucus secreted 
by the gland is used for pseudofaeces forma- 
tion, for attaching sand grains to a shell and, 
probably in the Verticordiidae, for encapsula- 
tion of motile prey caught in the mantle cavity. 

There are also species (for example, 
Turtonia minuta), in which only the female 
mantle gland takes part in the formation of 
brooding capsules (Oldfield, 1955, 1963). This 
species also has an apical gland. None of the 
above taxa, including T. minuta, contain extra- 
cellular granules, such as are found in the 
thickened inner mantle fold of A. ojianus and 
possibly in other species of Kellieilidae 
(Clausen, 1958). 

These granules begin to form at the end of 
September or the beginning of October when 
the shell length of the juvenile A. ojianus ex- 
ceeds 350-400 цт. In sections through the 
mantle of adults, the granules are located both 
on and under the mantle fold and on the epi- 
thelium. In some sections of the gland, there 
are "empty places", which are similar to the 
granules in shape, size and location. Brood- 
ing, in which these granules might be used for 
nutrition of the young, as for instance in the 
eggs of some gastropods (Thorson, 1936), is 
absent in A. ojianus. In this species, there is 
no hypobranchial gland that can be used for 
nutrition of the brooded young (Owen, 1961; 
Morton, 1977, 1982). The pelagic larvae of /A. 
ojianus occur in Peter the Great Bay in August 
and settle at a shell length about 230-240 цт. 
Formation of the granules at the beginning of 
the thermal minimum and their absence in 
spring indicate that the granules can be used 
as a food resource not for the young, but for 
the adult mollusk itself during the winter game- 
togenesis. Their presence is also independent 
of sex. 

The stomach is another organ that is impor- 
tant in the taxonomy of A. ojianus. Its morphol- 
ogy noticeably differs from those in related 
families Arcticidae, Glossidae, Trapezidae and 
Veneridae (Purchon, 1960; Reid, 1965). There 
is no cuticular lining or helicoid structure in the 
stomachs of members of these families, but 
they possess digestive pouch, dorsal hood, 
caecum, gastric shield, and sorting areas con- 
sisting of ridges and grooves. The stomach of 



ANATOMY OF ALVEINUS OJIANUS 



13 



Vesicomyidae has not been studied. But in a 
transverse section, the digestive tubules of 
large vesicomyids, for example Calyptogena 
(Morton, 1986), are similar to the tubules of 
most bivalves. The mid-gut, hindgut and rec- 
tum of Calyptogena differ from the gut of A. 
ojianus in having a folded, ciliated epithelium 
on the inner wall and in the presence of a 
typhlosole in the rectum or by absence of the 
anterior loop of the gut as, for instance, in 
Isorropodon (Cosel & Salas, 2001). The slit- 
like aperture of the style sac opening is not in 
the mid-gut, as in many bivalves, but in the 
posterior section of the stomach. It is a re- 
markable distinguishing feature of the alimen- 
tary canal of A. ojianus and, possibly, other 
members of Kelliellidae. Taking into account 
the general configuration and composition of 
functionally important sections of the stomach, 
which have been used as taxonomic charac- 
ters of suborders or orders in the Bivalvia 
(Purchon, 1978, 1987; Starobogatov, 1992), 
as well as other internal features - siphons, 
papillae, mantle apertures, labial palps, termi- 
nal cutícula of the gill, foot and its glands 
(Table 1) - the inclusion of Kelliellidae in the 
same subdivision of the Veneroida as the 
above families may be considered as insuffi- 
ciently founded. 

On the other hand, some morphological fea- 
tures of A. ojianus that seem to be taxonomi- 
cally important in comparison to veneroid 
families, occur in more distant phylogenetic 
lines. For instance, the cuticular lining of the 
stomach wall of Л. ojianus and, possibly, other 
species of Kelliellidae is most similar either to 
that of more primitive Nucinellidae, Sole- 
myiidae and Nuculidae, or of the specialized 
Verticordiidae, Poromyidae and Cuspidariidae 
(Starobogatov, 1992). A more detailed com- 
parison shows that, like A. ojianus, the stom- 
ach of primitive taxa has no caecum, no 
digestive pouch, and no large ciliary fields, but 
has sorting ridges, large pouch-like digestive 
tubules, and small embayments in the right- 
dorsal wall resembling the dorsal hood 
(Purchon, 1956; Allen & Sanders, 1969). In 
addition to the cuticular lining occupying a 
small part of the stomach wall, a common gas- 
tric shield joined with the underlying columnar 
cells by means of pseudociliary cuticular 
connectives may also occur in stomach of the 
primitive mollusks (Halten & Owen, 1968). 
However, the cup-shaped helicoid structure is 
lacking, and the general configuration of the 
stomach of the primitive bivalve differs mark- 



edly from that of A. ojianus and other taxa, 
such that the former could be separated as a 
special subclass based on their morphology 
and digestion features alone (Purchon, 1987). 

As in A. ojianus, the stomachs of specialized 
taxa (e.g., Verticordiidae) have no dorsal 
hood, caecum, sorting areas and digestive 
tubules similar to those of other bivalves (Allen 
& Turner, 1974). The cuticular (scleroprotein) 
lining may bear the irregular, almost parallel 
ridges covering the most part of the anterior 
section of the stomach or fan-like divergent 
curved ridges resembling those located at the 
bottom of the A. ojianus helicoid structure. The 
cuticular lining of the verticordiid stomach also 
has spiral structures (Allen & Turner, 1974: fig. 
63), which are comparable with the helicoid 
structure of A. ojianus in shape and location. 
In addition to the dorsal opening, the style sac 
seems to have an anterior slit-like aperture 
that opens into the stomach, as in A. ojianus 
(Allen & Turner, 1974: figs. 19, 77). Other simi- 
larities to A. ojianus include a mid-gut consist- 
ing of columnar cells lacking cilia, although in 
some verticordiids a typhlosole may occur in- 
side the gut. Also, the labial palps of the 
Verticordiidae are usually funnel-shaped and 
without the sorting ridges, but possess wing- 
shaped processes, buccal cavities or special 
glands used for capture and partial digestion 
of motile prey. 

In addition to the above features, as well as 
valves and additional tentacles in the mantle 
apertures that are features concerned with 
nutritional specialization at specific or generic 
levels (Allen & Turner, 1974), the Verticor- 
diidae have important characters at the famil- 
ial level that are absent in A. ojianus. These 
are a thickened muscular wall surrounding the 
stomach and oesophagus, dilatation of the 
hindgut - an analogue of the "masticatory 
stomach" of Cephalaspidea (Ivanov, 1 985) - a 
radial mantle gland consisting of separate is- 
lets, and a shell, which morphologically and 
structurally differs from the Kelliellidae, such 
that inclusion of these families within the same 
order is not possible. 

Therefore, although there is no relationship 
between the Kelliellidae and the Verticordii- 
dae, a similar evolutionary route can cause a 
high degree of likeness in the internal mor- 
phological features of these taxa (Table 1). 
One of the features of this evolutionary path is 
their small body size as, for instance, in many 
species of Kelliellidae, Verticordiidae, and 
Vesicomyidae, in which the average shell 



14 



EVSEEVETAL. 



TABLE 1. The internal morphological features of the Kelliellidae, Veneroida and Verticordiidae and 
their significance in the taxonomy and evolutionary development of Kelliellidae. (+) - feature present; 
(±) - feature may be absent; (-) - feature absent; (*) - feature has significance; (ф) - feature has no 
significance; (?) - significance of feature not determined. 







Taxa 




Significance 












Paedo- 


Morphological Features 


Kelliellidae Veneroida Verticordiidae Taxonomy 


тофЬо515 


Mantle Organs 


Mantle gland 


+ 


± 


± 


* 


Ф 


Extracellular granules 


+ 


- 


- 


* 


* 


Only exhalant siphon 


+ 


- 


+ 


Ф 


* 


Apical siphonal papillae 


- 


+ 


- 


Ф 


Ф 


Basal siphonal papillae 


+ 


+ 


+ 


* 


* 


Ciliated mantle lobes 


- 


+ 


- 


Ф 


? 


Mantle Cavity Organs 


Inner and outer branchial lamellae differ 












in size 


- 


± 


± 


* 


* 


Only inner branchial lamellae 


+ 


- 


± 


* 


* 


Ventral marginal food groove 


+ 


+ 


± 


* 


? 


Free posterior gill end 


- 


+ 


- 


? 


? 


Homorhabdic branchial filaments 


+ 


- 


+ 


7^ 


* 


Adductor consisting of one portion 


± 


- 


+ 


* 


* 


Visceral Mass Organs 


Byssal gland 


+ 


- 


+ 


Ф 


? 


Apical gland 


+ 


- 


- 


* 


Ф 


Ventral foot groove 


- 


- 


± 


* 


Ф 


Ciliated foot 


+ 


+ 


- 


? 


Ф 


Statocysts of B2-type 


+ 


? 


+ 


Ф 


? 






Alimentary Canal Orga 


ns 




Funnel-shaped labial palps 


+ 


- 


± 


Ф 


* 


Sorting ridges of labial palps 


± 


+ 


± 


* 


* 


Labial muscle rim 


- 


+ 


- 


Ф 


* 


Cuticular lining of stomach 


+ 


- 


+ 


* 


? 


Gastric helicoid structure 


+ 


- 


? 


* 


? 


Digestive pouch 


- 


+ 


- 


Ф 


* 


Dorsal hood 


- 


+ 


± 


Ф 


* 


Caecum of stomach 


- 


+ 


- 


Ф 


* 


Sorting areas of stomach 


- 


+ 


- 


Ф 


* 


Typhlosoles of stomach 


± 


+ 


± 


* 


? 


Digestive tubules 


± 


+ 


+ 


? 


* 


Crystalline style sac combined with gut 


- 


± 


? 


Ф 


Ф 


Crystalline style sac combined with 












stomach 


+ 


- 


? 


* 


* 


Typhlosole of gut 


± 


+ 


± 


9 


* 


Dilatation of gut 


- 


- 


+ 


Ф 


Ф 



ANATOMY OF ALVEINUS OJIANUS 



15 



length does not exceed 4-5 mm (Knudsen, 
1970; Allen & Turner, 1974; Bernard, 1989; 
Waren, 1989; HayamI & Kase, 1993; Allen, 
2001 ; Cosel & Salas, 2001 ). Mollusks of such 
a size usually have either only an inner 
demibranch or an inner and underdeveloped 
outer demibranch. The number of the gill bran- 
chial filaments rarely exceeds 22-23. At the 
same time, the branchial axis is located dor- 
sally, and the posterior filaments are replaced 
by a membrane or differ from other filaments 
in their shape and ciliary system (Pelseneer, 
1906). The adductors of these bivalves some- 
times consist only of the outer portion of 
smooth muscle fibres, which in ontogenesis 
were previously the internal portion of cross- 
striated fibres (Oldfield, 1955). In these mol- 
lusks, the pedal gape of the mantle cavity may 
be not separated from the inhalant aperture 
(Clausen, 1958). In cases in which the pedal 
gape is separate, the inhalant siphon is usu- 
ally absent, and the exhalant siphon repre- 
sents a simple tube without the apical papillae, 
as in juvenile Veneridae (Ansell, 1962). 

The alimentary canal is also characterized 
by such "juvenilization" (Table), that is, paedo- 
morphosis (De Beer, 1958). The paedomor- 
phic features include the funnel-shaped labial 
palps, the total or partial absence of the sort- 
ing ridges on the palps, the morphologically 
indistinct mouth without the labial muscle rim, 
the epithelium of the oesophagus covered 
with long cilia, which occurs in macroforms in 
the veliger or pediveliger stages, the absence 
of the digestive pouch, caecae and sorting 
areas as, for example, in Erycinidae and other 
bivalve families (Oldfield, 1955, 1963; Chanley 
& Andrews, 1971;Alataloetal., 1984). These 
features as well as the underdeveloped gill 
and adductors, the primitive inhalant aperture 
without protection of the mantle cavity by 
valves; the unciliated epithelium of the mantle 
cavity and intestinal tract and cloaca, and the 
accelerated reproductive development, which 
in A. ojianus matures at a shell length of about 
1 mm, suggest incomplete somatic develop- 
ment of not only the internal organ, but also 
the outer skeletal organ, the shell. 

Thus, the Verticordiidae and the Kelliellidae 
are of different phylogenetic lines, but with a 
similar pattern of evolutionary development - 
paedomorphosis, in which the post-juvenile 
stages prove to be "cut" (De Beer, 1958; 
Gould, 1977) in taxa of both families. There- 
fore, based on internal morphological charac- 
ters, an attempt to determine the place of 



Kelliellidae in the taxonomic system rested on 
the conchological features of adults, meets 
with failure. This is caused by insufficiently 
studied internal organs of bivalves as a whole. 
In conchological features, the family Kellielli- 
dae appears to be among the most primitive 
Heterodonta, but it is impossible to determine 
its place more distinctly because of poorly 
studied juvenile stages of Heterodonta with 
morphological characters similar to those of 
adult kelliellids. 



ACNOWLEDGEMENTS 

We greatly appreciate Prof. John A. Allen 
(University Marine Biological Station, Millport) 
for critical reading the manuscript and very 
useful comments. We also thank him for send- 
ing reprints of papers. We are very grateful to 
Dr. Paula M. Mikkelsen (American Museum of 
Natural History, New York), Dr. Eugene V. 
Coan (California Academy of Sciences, San- 
Francisco) and anonymous reviewer for their 
helpful comments, beneficial criticism and cor- 
rections. We thank Dr. Konstantin A. Lutaenko 
(Institute of Marine Biology, Vladivostok), who 
provided us with benthic samples from the 
research vessel "Lugovoye". We are grateful 
to Dr. Vladimir V. Malakhov (Moscow State 
University, Moscow) for his consultation and 
support of our work. We thank Dr. Alexander I. 
Kafanov (Institute of Marine Biology, Vladi- 
vostok), Dr. P. Graham Oliver (National Mu- 
seum of Wales, Cardiff), and Dr. Rudo von 
Cosel (Museum National D'Histoire Naturelle, 
Paris) who provided us with reprints of papers. 

This study was funded by grant 02-04- 
49470 from the Russian Foundation for Basic 
Research. 



LITERATURE CITED 

ALATALO, P., С J. BERG & С N. D'ASARO, 
1984, Reproduction and development in the 
Lucinid clam Codakia orbicularis (Linne, 1758). 
Bulletin of Marine Science, 34: 424-434. 

ALLEN, J. A., 1968, The functional morphology 
of Crassinella mactracea (Linsley) (Bivalvia: 
Astartacea). Proceedings of the Malacological 
Society of London, 38: 27-40. 

ALLEN, J. A., 2000, An unususal suctorial 
montacutid bivalve from the deep Atlantic. Jour- 
nal of the Marine Biological Association of the 
United Kingdom, 80: 827-834. 

ALLEN, J. A., 2001, The family Kelliellidae 
(Bivalvia: Heterodonta) from the deep Atlantic 



16 



EVSEEV ET AL. 



and its relationship witli the family 
Vesicomyidae. Zoological Journal of the Lin- 
nean Society, London, 131(2): 199-226. 

ALLEN, J. A. & H. L. SANDERS, 1969, Nucinella 
serrei Lamy (Bivalvia: Protobranchia), a 
monomyarian solemyid and possible living 
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Revised ms. accepted 4 July 2003 



MALACOLOGIA, 2004, 46(1): 19-35 

MORPHOLOGICAL AND MOLECULAR ANALYSIS OF THE STATUS AND 

RELATIONSHIPS OF OXYCHILUS PAULUCCIAE (DE STEFANI, 1883) 

(GASTROPODA: PULMONATA: ZONITIDAE) 

Giuseppe Manganelli\ Simone Cianfanelli^, Nicola Salomonen & Polco Giusti^ 

ABSTRACT 

Morphological data shows that O. paulucciae (De Stefani, 1883) belongs to Oxychilus (s. 
Str.), sensu Giusti & Manganelli (1999), and is distinguished from sympatric, similarly 
shelled species, such as O. draparnaudi {Beck, 1837) and O. meridionalis (Paulucci, 1881), 
by its larger shell (diameter: 13.9-17.4 mm), smaller umbilicus (about 1/8 of shell diameter), 
narrow mid-penial region, internal ornamentation of proximal penis consisting of longitudinal 
pleats, and a less developed vaginal gland, often forming an incomplete ring around 
proximal vagina. DNA sequence data, analysing the ITS-1 region in two specimens of 
O. paulucciae and representatives of several other species occurring in Tuscany, O. 
draparnaudi, O. ша/оп (Paulucci, 1886), O. meridionalis, O. pilula (Paulucci, 1886) and 
O. uzielli (Issel, 1873), indicates that O. paulucciae represents a well-differentiated 
evolutionary lineage and suggests it has close relationships with O. meridionalis and 
O. uziellii. Finally, analysis of morphological characters and DNA sequencing data 
demonstrates that Oxychilus lanzai Forcart, 1967, is a junior synonym of O. paulucciae. 

Key words: Zonitidae, Oxychilus paulucciae, Oxychilus lanzai, systematics. 



INTRODUCTION 

During the 1960s, the leading Swiss 
malacologist L. Forcart received many 
specimens of Tuscan Oxychilus, most 
collected by Prof. B. Lanza (Museo Zoológico 
de "La Specola", Université di Firenze) in NW 
Tuscany (provinces of Massa Carrara, Lucca 
and Florence). Based on these specimens, 
Forcart (1967) produced a first revision of 
many taxa of the species group described 
from Tuscany in the nineteenth century (Table 
1), and described a new species: O. (Ortizius) 
lanzai Forcart, 1967. 

In the late 1960s, one of us (FG), together 
with M. Mazzini, was involved in the study of 
the malacofauna of the Apuan Alps (NW 
Tuscany) as part of a project promoted by the 
Società Italiana di Biogeografia (Giusti & 
Mazzini, 1971). The study of the Oxychilus 
material collected on this occasion led to 
revision ofthat studied by L. Forcart (1967, 
1968). It became evident that Forcart, using 
diaphanized preparations of the whole distal 
genitalia mounted on glass slides, sometimes 



misinterpreted the internal structure of the 
penis, which was then considered very 
important for the diagnosis of subgenera: 
parallel rows of papillae for Oxychilus s. str.; 
parallel uninterrupted pleats for Ortizius 
Forcart, 1957. This happened for specimens 
from Tana di Magnano, Garfagnana, Province 
of Lucca, which he assigned to O. {Oxychilus) 
paulucciae (De Stefani, 1883), but when re- 
examined turned out to be a species of 
Ortizius, anatomically identical to that from the 
Grotta della Risvolta, Apuan Alps, which he 
assigned to O. lanzai. 

Giusti & Mazzini (1971) declined to express 
formal synonymy between O. lanzai and O. 
paulucciae, because they realized that since 
Forcart did not have topotypical specimens of 
the classic Tuscan taxa for anatomical inves- 
tigation, he based his study on spirit material 
with shells similar to those of the types, thus 
misinterpreting some classic species (shell 
shape is very rarely diagnostic in species of 
Oxychilus). They merely stated that the entire 
group of nominal species described for NW 
Tuscany required revision before the problem 



■'Dipartimento di Scienze Ambientali, Université di Siena, Via Mattioii 4, 53100 Siena, Italy; manganelli@unisi.it 
^Museo Zoológico de "La Specola", Sezione del Museo di Storia Naturale dell'Università di Firenze, Via Romana 17, 
1-50125 Firenze, Italy 
^ipartimento di Biología Evolutiva, Université di Siena, Via Mattioii 4, 53100 Siena, Italy 



19 



20 



MANGANELLI ETAL. 



TABLE 1. Nominal taxa of the species group introduced for Tuscan Oxychilus (excluding those 
established for species living in the Tuscan Archipelago) (for syntypes kept in the Museo di Zoología 
"La Specola", the collection number is followed by the number of specimens). 



Nominal taxon 



Status 



Zonites Uziellii \sse\, 1872: 60-61. 
Type material: lectotype (MZUF 689) and one paralectotype 

(MZUF 1 1 521 ) in Paulucci collection. 
Type locality: "Fra i detriti del Gombo, presse Pisa", but see 

Manganelli & Giusti (2000). 
Hyalina scotophila De Stefani, 1879: 38-39. 
Type material: 4 syntypes; one in Paulucci collection (MZUF 

738) and three in Museo di Storia Naturale dell'Accademia dei 

Fisiocritici in Siena. 
Type locality: "Siena, in un profondo condotto sotterraneo" 
Hyalinia meridionalis Paulucci, 1881: 78-79, pi. 1, fig. 6. 
Type material: lectotype (MZUF 13187) and 30 paralectotypes 

in Paulucci collection (781/12, 828/7, 829/1, 830/3, 832/2, 

13188/5). MZUF 781, 829, 830, 832, 833 belong to other 

species (Manganelli & Giusti, 2001). 
Type locality: "Fabbriche presso i Bagni di Lucca". 
Hyalinia Isseliana Paulucci, 1882: 165-168, pi. 9, fig. 13. 
Type material: lectotype (MZUF 687) and 4 paralectotypes 

(MZUF 688/3, 13346/1) in Paulucci collection (Manganelli & 

Giusti, 2001). 
Type locality: "Fabbriche presso i Bagni di Lucca (Lucca; 

Toscana)". 
Hyalinia Guidoni De Stefani, 1883: 35, 1888: fig. 3. 
Type material: unknown. 
Type locality: "Forno Volasco, 480 [m]". 
Hyalinia Paulucciae De Stefani, 1883: 35-36, 1888: fig. 1. 
Type material: no syntype being known, a neotype was 

designated (Fig. 1). The neotype (a spirit specimen) is in the 

Museo Zoológico de "La Specola", Sezione del Museo di 

Storia Naturale dell'Università di Firenze (Italy) (MZUF 17597) 
Type locality: "Alp. E. Vagli 850" (p. 36) and "Strada nazionale 

presso il Ponte di Ceserana" (caption of un-numbered plate). 

"Alp. E" is for "Pendici orientali délie AIpi Apuane dall'alveo de! 

fiume Serchio e deU'Aulella fino alla crina" (p. 17). Following 

the designation of the neotype, the type locality becomes 

"Vagli di sopra. Valle Arnetola, 930 m asi (Vagli di sotto, 

Lucca), 32TPP0084". 
Hyalina scotophila yar. notha Paulucci, 1886: 12-13, pi. 1, fig. 2. 
Type material: 27 syntypes (MZUF 788/4, 789/9, 790/5, 791/7, 

792/1, 13347/1) in Paulucci collection. The shell MZUF 792 is 

that illustred by Paulucci (1886). 
Type locality: "alia Fonte dell'Appetito presso Porto Santo 

Stefano, presso la vetta del Telégrafo, sopra al Convento de' 

Passionisti, in vicinanza delle scogliere di Calagrande ... 

AH'isola del Giglio in una località denominata «Franco»". 



Oxychilus uziellii (Issel, 1872) 
(Manganelli & Giusti, 1985, 
1993,2001) 



probably junior synonym of 
Oxychilus drapa maud i (Beck, 
1837) (Manganelli & Giusti, 
2001) 

Oxychilus meridionalis 
(Paulucci, 1881) (Manganelli 
& Giusti, 2001) 



junior synonym of Oxychilus 
meridionalis {Paü\ucc\, 1881 
(Manganelli & Giusti, 2001) 



nominal taxon in need of 
revision 

Oxychilus paulucciae (De 
Stefani, 1883) (this paper) 



junior synonym of Oxychilus 
draparnaudi (Beck, 1837) 
(Giusti, 1968; Manganelli et 
al., 1995) 



(Continues) 



REVISION OF OXYCHILUS PAULUCCIAE 



21 



(Continued) 



Nominal taxon 



Status 



Hyalinia nitidula var. amiatae Westerlund, 1886: 57. 

Type material: 33 syntypes in Paulucci collection (MZUF 
807/12, 19290/10, 19291/11). 

Type locality: "Italien, M. Amiata" [in località La Scarpa]. 
/-/ya//n/a sy/wco/a Westerlund, 1886: 59. 

Type material: 8 syntypes in Paulucci collection (MZUF 805). 

Type locality: "Italien, Bosco di San Vittore in Toskana". 
[= Pozza délie Monache, Bosco di San Vettere]. 
Hyalinia blauneri var. c/oacarum Westerlund, 1886: 61. 

Type material: 8 syntypes in Paulucci collection (MZUF 804) 

Type locality: "Italien b. Volterra" [= Fogna délia Chiesa di 
Camporbiano]. 
IHyalina scotophila var. dilátala Westerlund, 1886: 61. 

Type material: 8 syntypes in Paulucci collection (MZUF 19289). 

Type locality: "Ital., San Martine b. Palma" [= San Martine alla 
Palma] 
Oxychilus {Ortizius) lanzai Forcañ, 1967: 114-115, fig. 1, pi. 1, fig. 1. 

Type material.- The holotype (spirit specimen, MZUF 462), 
seven paratypes (spirit specimens, MZUF 454/2, 463/5 from 
"Grotta del Buggine" and six paratypes (four spirit specimens, 
MZUF 683; two shells, MZUF 691) from "Grotta della Risvolta" 
are in the Museo Zoológico de "La Specola", Sezione del 
Museo di Storia Naturale dell'Università di Firenze (Italy). Four 
other paratypes from "Grotta del Buggine" (two spirit 
specimens, NMB 6562; two spirit specimens, MFP) are in the 
Naturhistorisches Museum Basel (Switzerland) and in the 
Museo "Felice Poey", La Habana (Cuba) respectively. 

Type locality.' "Toskana, Prov. Lucca, Apuaner Alpen, Grotta del 
Buggine 315 m (N. 166 T.) bei Cardoso Stazzemese". 
Oxychilus (Ortizius) tongiorgii G\us\\, 1969: 367-369, figs. 1-2, 

5A, pi. 1,figs. 1, 2. 

Type material: holotype and 9 paratypes in Giusti collection. 

Type locality: "Grotta del Ladri (n. 262 T. Pi) Monti Pisani nei 
pressi di Asciano". 
Oxychilus {Ortizius) forcartianus Giusti, 1969a: 369-371 , figs 3, 4, 

5B, pi. 1,figs. 3, 4. 

Type material: holotype and 3 paratypes in Giusti collection. 

Type locality: "Grotta del Fiorentini presse Pomarance (Grosseto)". 



junior synonym of Oxychilus 
draparnaudi {Beck, 1837) 
(Manganellietal., 1995) 

nominal taxon in need of 
revision 



nominal taxon in need of 
revision 



nominal taxon in need of 
revision 



junior synonym of Oxychilus 
paulucciae (De Stefani, 1883) 
(this paper) 



junior synonym of Oxychilus 
meridionalis ( Pa u I u cci , 1881) 
(Manganelli& Giusti, 2001) 



junior synonym of Oxychilus 
meridionalis ( Pa u I u cci , 1881' 
(Manganelli & Giusti, 2001) 



of the relationships between O. paulucciae 
and O. /anza/ could be tackled. 

The oldest established Tuscan Oxychilus 
species - Zonites uziellii Issel, 1872; Hyalinia 
meridionalis Paulucci, 1881; and Hyalinia 
isseliana Paulucci, 1881 - have now been re- 
vised (Manganelli & Giusti, 1985, 1993, 2000, 
2001), and it is therefore possible to revise O. 
paulucciae, to clarify its relationships with 
other Tuscan Oxychilus and to resolve the 
problem of its synonymy with O. lanzai. 



The first problem with Hyalinia paulucciae is 
that its type-material has not been traced. The 
malacological collection of De Stefani, which 
remained in Pisa when he moved to Siena 
and then Florence, was irreparably damaged 
during the Second World War. In order to de- 
fine this nominal taxon objectively, we have 
selected a neotype, because the "qualifying 
conditions", required for the designation of a 
neotype, exist (ICZN, 1999: Art. 75.3). The 
neotype, the shell and genitalia of which are 



22 



MANGANELLI ETAL. 



shown in Figure 1 (shell) and Figures 3-4 
(genitalia), is deposited in the Museo 
Zoológico de "La Specola", Sezione del Museo 
di Storia Naturale dell'Università di Firenze, 
Italy (catalogue no. 17597). It is a spirit speci- 
men collected near Vagli, one of the two locali- 
ties where De Stefani reported his species. Its 
shell matches the original description perfectly. 



MATERIAL AND METHODS 
Morphological Analysis 

Whole shells were photographed under the 
light microscope (Wild M5A). All dimensions - 
NW number of whorls (Ehrmann, 1933: fig. 
12), SD shell diameter, SH shell height and 
UD umbilicus diameter - were measured us- 
ing a micrometer. 

Live specimens were drowned in water, then 
fixed and preserved in 75% ethanol buffered 
with sodium carbonate. The bodies were iso- 
lated after crushing the shells and dissected 
under the light microscope (Wild M5A) using 
thin-pointed watchmaker's tweezers. Anatomi- 
cal details were drawn using a Wild camera 
lucida. Some parts of the genital organs - duct 
of bursa copulatrix, distal vagina, epiphallus, 
flagellum, proximal portion of penis, "bottle- 
neck", distal penis and penial sheath - were 
measured by micrometer. 

Radulae were extracted manually from buc- 
cal bulbs, washed in 75% ethanol, mounted 
on copper stubs with electronconductive glue, 
sputter-coated with gold and photographed 
using a Philips 505 SEM. 

All specimens listed in material examined 
belong to anatomically determined popula- 
tions. The material examined is listed as fol- 
lows: locality, municipality and province names 
in parenthesis, UTM reference, collector(s), 
date, number of specimens in parenthesis (sp 
spirit preserved specimen/s, sh shell/s) and 
bibliographical reference, in parenthesis, if 
they are voucher specimens. Locality names 
and UTM references are according to the of- 
ficial 1:25,000 scale map of Italy (series M 
891). 

Key to museum and collection acronyms: 
FGC, collection F. Giusti, Dipartimento di 
Scienze Ambientali, University of Siena, Italy; 
NMB, Naturhistorisches Museum Basel, Swit- 
zerland; MFP, Museo "Felice Poey", La 
Habana, Cuba; MZUF, Museo Zoológico "La 



Specola", Sezione del Museo di Storia Natu- 
rale dell'Università di Firenze, Italy; SCC, S. 
Cianfanelli collection, Firenze, Italy. 

Key to acronyms in figures: B, "bottle-neck"; 
ВС, bursa copulatrix; BS, "bottle-neck" sheath; 
BW, body wall; DBC, duct of bursa copulatrix; 
DP, distal portion of penis; E, epiphallus; EO, 
epiphallus opening; F, flagellum; FO, free ovi- 
duct; PCS, prostatic portion of ovispermiduct; 
PP, proximal portion of penis; PR, penial re- 
tractor; PS, penial sheath; UOS, uterine por- 
tion of ovispermiduct; V, vagina; VD, vas 
deferens; VG, vaginal gland. 

Molecular Analysis 

DNA Extraction, PCR and Sequencing 

Two specimens of O. paulucciae and others 
of several species occurring in Tuscany - O. 
draparnaudi {Beck, 1837), O. тауог/ (Paulucci, 
1886), O. meridionalis (Paulucci, 1881), O. 
pilula (Paulucci, 1886), and O. uzielli (Issel, 
1 873) - were used for molecular analysis. One 
specimen of O. paulucciae (O. paulucciae 1) 
was collected in the type locality of this species 
and the other (O. paulucciae 2) in a cave where 
part of the type material of O. lanzai was col- 
lected. Collection sites and codes of the 
samples used for molecular analysis are indi- 
cated in Table 2. Total DNA was extracted from 
fresh foot muscle using standard phenol/chlo- 
roform and ethanol precipitation methods as 
described in Salomone et al. (2002). We ampli- 
fied the ITS-1 region by PCR using the primer 
pair CS249 (5'TCGTAACAAGGTTTCCG3') 
and DT421 (5'GCTGCGTTCTTCATCG3') 
(Schlötterer et al., 1994). PCR amplification 
was performed in a reaction volume of 50 pi 
following a profile consisting of 25 cycles with 
temperatures of 95°C for 20", 55°C for 30" and 
72°C for 30", plus a final extension step at 72°C 
for 5'. The products obtained using these con- 
ditions were very clean single bands, showing 
no evidence of double or ambiguous bands. 
After elimination of excess nucleotides and 
primers by gel separation and purification with 
Nucleospin Extract (Genenco) columns, both 
strands of the final products were sequenced 
using the two amplification primers. Sequenc- 
ing reactions were performed at the core facil- 
ity of MWG-BIOTECH, Ebersberg, Germany. 
All sequences were checked manually for se- 
quencing errors and submitted to GenBank 
(Accession Nos. AY373635-AY373645). 



REVISION OF OXYCHILUS PAULUCCIAE 
TABLE 2. Material examined for DNA sequencing. 



23 



Taxon 



Locality 



Oxychilus draparnaudi 1 
Oxychilus draparnaudi 2 
Oxychilus majori 

Oxychilus meridionalis 1 
Oxychilus meridionalis 2 
Oxychilus meridionalis 3 
Oxychilus paulucciae 1 
Oxychilus paulucciae 2 
Oxychilus pilula 
Oxychilus uziellii 

Retinella olivetorum 



Castalio di Brollo (Gaiole in Chianti, Siena), 32TPP9909, G. 

Manganelli & L. Manganelli leg. 01.10.2000 
Giglio Island: Giglio Castello (Isola del Giglio, Grosseto), 32TPM5692, 

V. Vignoli leg. 30.06.2000 
Monte Argentarlo: Grotta di Punta degli Stretti 250 T/GR (Monte 

Argentarlo, Grosseto), 32TPN7800, S. Cianfanelli & G. Manganelli 

leg. 07.10.2001 
Capanno (Castelnuovo Berardenga, Siena), 32TPP9308, G. 

Manganelli & L. Manganelli leg. 28.05.2000 
Passe délia Calla (Santa Sofia, Forlî), 32TQP2060, S. Cianfanelli & G. 

Manganelli leg. 16.09.2001 
Fabbriche di Bagni di Lucca (Bagni di Lucca, Lucca), 32TPP3175, S. 

Cianfanelli & E. Lori leg. 15.04.2003 
Apuane Alps: Vagli di sopra. Valle Arnetola (Vagli di sotto, Lucca), 

32TPP0084, S. Cianfanelli & M. Calcagno leg. 24.06.2000 
Apuane Alps, Grotta délia Risvolta 158T/LU (Stazzema, Lucca), 

32TPP0372, S. Cianfanelli & M. Calcagno leg. 14.10.2001 
Capraia Island: Il Laghetto (Capraia Isola, Livorno), 32TNN6665, F. 

Barbagli & S. Lotti leg. 22.06.2001 
Fosso délie Filicaie, San Giusto in Saldo (Gaiole in Chianti - Radda in 

Chianti, Siena), 32TPP9115, G. Manganelli & L. Manganelli leg. 

28.05.2000 
Fosso délie Filicaie, San Giusto in Saldo (Gaiole in Chianti - Radda in 

Chianti, Siena), 32TPP9115, G. Manganelli & L. Manganelli leg. 

28.05.2000 



Phylogenetic analysis 

Sequences were aligned using Clustal W 
(Thompson et al., 1994) and slightly modified 
by eye. Boundaries of the ITS-1 region were 
estimated by comparison with those deter- 
mined for /A/b/nar/a caerulea (Deshayes, 1835) 
(GenbankAcc. No. AFI 3601 2). Phylogenetic 
analyses were performed with PAUP* (version 
4.0b10; Swofford, 2001) with Retinella 
olivetorum (Gmelin, 1791) as outgroup, using 
maximum parsimony (MP) and maximum like- 
lihood (ML). MP reconstruction was performed 
by an exhaustive search of the most parsimo- 
nious tree(s) with equal weighting of all charac- 
ters. Gaps were treated as missing data. For 
ML analysis, the appropriate substitution model 
of DNA evolution that best fitted the data set 
was determined by the likelihood ratio test and 
the Akaike information criterion (AlC; Akaike, 
1974) with Modeltest 3.04 (Posada & Crandall, 
1998). A ML heuristic search (Step-Add ran- 
dom; TBR branch swapping) was then run un- 
der the likelihood setting estimated by 



Modeltest. Support for individual nodes was 
evaluated by bootstrap analysis (heuristic 
search) with 1000 replications. 



RESULTS 

Redescription of O. paulucciae (De Stefani, 1883) 

Identification 

A medium-sized species of Oxychilus (s. 
Str.), sensu Giusti & Manganelli (1999), a "sub- 
genus" of Oxychilus characterized by: penis 
with flagellum; penial retractor inserted at 
apex of flagellum; internal ornamentation of 
proximal penis consisting of pleats or pleats 
and rows of papillae without apical thorns; 
epiphallus usually longer than proximal penis, 
its internal wall with slender longitudinal 
pleats; mucous gland mainly vaginal; long 
mesocone of central tooth). Oxychilus 
paulucciae is identified with respect to similar- 
shelled sympatric species (O. draparnaudi 



24 



MANGANELLI ETAL. 



and О. meridionalis) by a larger shell (shell 
diameter: 13.9-17.4 тгл) with small umbilicus 
(about 1/8 of shell diameter), narrow mid-pe- 
nial region, internal ornamentation of proximal 
penis consisting of longitudinal pleats, and 
vaginal gland often forming incomplete ring 
around proximal vagina. 



Description 

Body pale gray in colour; neck and upper 
part of sides with variably wide areas with pits 
(with phylacites); foot slender, of aulacopod 
type, with sole longitudinally tripartite (central 
part whitish, lateral parts pale gray); eyes 




FIGS. 1 , 2. Two shells of Oxychilus paulucciae (De Stefani, 1883) from Vagli di sopra, Valla Arnetola, 
930 m asi (Vagli di sotte, Lucca), 32TPP0084, S. Cianfanelli & M. Calcagno leg. 8.10.2000 (MZUF 
17597, neotype) (FIG. 1) and Grotta della Risvolta, 220 m asi, no. 158T/LU (Stazzema, Lucca), 
32TPP0372, M. Bodon & S. Cianfanelli leg. 9.11.1997 (FIG. 2). 



REVISION OF OXYCHILUS PAULUCCIAE 



25 




FIGS. 3-5. Distal genitalia (FIG. 3) and internal ornamentation of flagellum and proximal penis (FIGS. 
4, 5) in specimens of Oxychilus paulucciae (De Stefani, 1883) from Vagli di sopra, Valle Arnetola, 930 
m asi (Vagli di sotto, Lucca), 32TPP0084, S. Cianfanelli & M. Calcagno leg. 8.10.2000 (MZUF 17597, 
neotype) (FIGS. 3-4) and S. Cianfanelli & M. Calcagno leg. 24.6.2000 (FIG. 5). 



26 



MANGANELLI ETAL. 



present, normal in size; kidney sigmurethrous; 
jaw oxygnathous. 

Slieil (Figs. 1, 2; Forcart, 1967: pi. 1, fig.1, 
figs. 1, 1a-1c [as O. lanzai], figs. 4, 4a-4c) 
dextral, medium in size, discoidal, de- 
pressed, thin and fragile, subtransparent, 
glossy when fresh, whitish-yellow or pale 
greenish, sometimes opalescent below; sur- 
face smooth, with variably evident growth 
lines and microsculpture consisting of very 
fine wavy spiral lines; spire usually tectiform, 
5 1/12-5 1/2 whorls, gradually increasing in 
size, last whorl dilated near aperture, its last 
quarter descending slightly or not at all, 
rarely slightly angled at periphery; sutures 
shallow; umbilicus small, about 1.4-2.6 mm 
wide (usually 1/8-1/9, rarely 1/7 and in only 
one case 1/5 of maximum shell diameter), 
sometimes eccentric; aperture oval, oblique; 
peristome interrupted, simple, not thickened 
or reflected, its superior vertex starting at, or 
slightly above, periphery of last whorl. Di- 
mensions (30 shells measured). Number of 
whorls: 5 1/4 ± 1/6 (5-5 5/6); shell diameter: 
15.5 ± 1.0 mm (13.9-17.4); height: 6.1 ± 0.6 
mm (5.3-7.2); umbilicus diameter: 1.9 ± 0.2 
mm (1.4-2.6). 

Genitalia (Figs. 3-12; Forcart, 1967: fig. 1 
[as O. lanzai], fig. 3). General scheme of geni- 
talia as in Oxychilus (s. str.), sensu Giusti & 
Manganelli (1999). Only distal genitalia are 
described here (a total of 21 adult specimens 
were dissected for study of genital structure 
during the various phases of the research). 
Female genitalia include free-oviduct, bursa 
copulatrix and its duct, and vagina. Distal free 
oviduct and most proximal vagina enveloped 
by muff of spongy glandular tissue forming 
vaginal gland; vaginal gland relatively unde- 
veloped, sometimes enough to form continu- 
ous ring around wall of most proximal part of 
vagina, distal part of free oviduct and of duct 
of bursa copulatrix, sometimes reduced to 
cover only one side (that facing free-oviduct) 
of proximal vagina and of distal duct of bursa 
copulatrix (large portion of wall on opposite 
side is uncovered); in both cases, vaginal 
gland often envelopes one side (that facing 
free oviduct) of distal canal of bursa copulatrix; 
duct of bursa copulatrix long (7.5 mm; n: 2), 
initially moderately flared, narrowing before 
entering oval or pyriform bursa copulatrix; dis- 
tal vagina (that without glandular muff) vari- 
ably long (2.7-4.9 mm; n: 2) and wide. 



reducing in calibre slightly or not at all near 
genital atrium. 

Male distal genitalia include vas deferens, 
epiphallus and penial complex (flagellum and 
penis). Epiphallus variably long (7.3-10.3 
mm; n: 2) and slender, internal walls bearing 
series of very slender longitudinal pleats. Fla- 
gellum usually very long (3.7-4.7 mm; n: 2), 
with penial retractor muscle ending at apex 
(sometimes thin muscular branch extends on 
one side to end at about half flagellum 
length). Penis variably long (9.9-14.3 mm; n: 
2) with clear distinction into proximal and dis- 
tal parts due to "bottle-neck" (terminal, slen- 
der part of proximal penis: minimum caliber 
recorded 0.5-0.62 mm: n: 4), enveloped by 
thin, distinct, translucent sheath. Proximal 
penis rather short (5.4-5.9 mm; n: 2). Distal 
penis variable in length (4.5-8.4 mm; n: 2), 
enveloped by variably long (2.5-4.1 mm; n: 
2) penial sheath, proximally very thin, tra- 
versed on one side by vas deferens, then 
slightly thicker for rest of length. Internal sur- 
face of flagellum and proximal penis sur- 
rounding opening of epiphallus into penis 
with many small radially disposed pleats, 
sometimes fragmented into rows of variably 
large papillae; lateral surface, and that oppo- 
site opening of epiphallus into penis, having 
slender longitudinal pleats with jagged sides, 
frequently fragmented into rows of variably 
large papillae. A variable number (9-12) of 
these pleats continues on rest of proximal 
penis, converging, fusing and reducing in 
number before continuing, with a more or 
less marked interruption at "bottle-neck", in- 
side distal penis, where they are usually 
wider with jagged sides. Very short, thin- 
walled duct connects distal penis (level with 
where penial sheath originates) to genital 
atrium in which vagina also ends. 

Radula consisting of many rows of about 
31-35 teeth, according to formula: 11-13 M/1 
+ 0-1 LM/2 + 3-4 L/3 + C/3 + 3-4 L/3 + 0-1 
LM/2 + 11-13 M/1 (6 specimens examined). 
Central teeth with well-developed basal plate, 
apical portion of which V-like, with pointed 
vertices; body of tooth wide, providing base for 
long, slender, pointed mesocone flanked by 
two very short ectocones. On both sides of 
each central tooth, three-four lateral tricuspid 
teeth, sometimes one latero-marginal bicuspid 
tooth and series of monocuspid marginal teeth 
in decreasing order of size. 



REVISION OF OXYCHILUS PAULUCCIAE 



27 




FIGS. 6, 7. Distal genitalia (FIG. 6) and internal ornamentation of flagellum and proximal penis (FIG. 7) 
in a specimen of Oxychilus paulucciae (De Stefani, 1883) from Grotta del Buggine Stazzemese, 315 
m asi, no. 166 T/LU (Stazzema, Lucca), 32TPP0573, B. Lanza & P. Lanza leg. 1960 (NMB 6562-a, 
paratype). 



28 



MANGANELLI ETAL. 




9-10 



2 mm 



FIGS. 8-10. Distal genitalia (FIG. 8), internal ornamentation of flagellum and proximal penis (FIG. 9) 
and mid-penis region (FIG. 10) in a specimen of Oxychilus paulucciae (De Stefani, 1883) from Tana 
del Pollone di Magnano, 565 m asi, no. 1017 T/LU (Villa Collemandina, Lucca), 32TPP1592, B. Lanza 
leg. 30.4.72 (MZUF 15856). 



REVISION OF OXYCHILUS PAULUCCIAE 



29 




11 



4 mm 



12 



2 mm 



FIGS. 11, 12. Distal genitalia (FIG. 11) and mid-penis region (FIG. 12) in a specimen of Oxychilus 
paulucciae (De Stefani, 1883) from Grotta della Risvolta, 220 m asi, no. 158 T/LU (Stazzema, Lucca), 
32TPP0372, M. Bodon & S. Cianfanelli leg. 9.11.1997. 



Material Examined 

NP98 Buca della Freddana, 650 m asi, no. 
230 T/MS (Massa, Massa Carrara), 
32TNP9783, G. Comotti leg. 12.7.85 
(1 sp, 2 sh, FGC). 



PP07 Grotta del Buggine Stazzemese, 315 m 
asi, no. 166 T/LU (Stazzema, Lucca), 
32TPP0573, no collector and date (2 
sp, FGC), B. Lanza leg. 18.10.59 (2 sp 
[paratypes of Oxycliilus lanzai], MZUF 
454); B. Lanza & R Lanza leg. 1960 (1 sp 



30 



MANGANELLI ETAL. 



[holotype of Oxychilus lanzai], MZUF 
462; 5 sp [paratypes], MZUF 463; 2 sp 
[para types of Oxychilus lanzai], NMB 
6562-a). Grotta della Risvolta, 220 m 
asi, no. 158 T/LU (Stazzema, Lucca), 
32TPP0372, B. Lanza leg. 23.7.61 (4 sp 
[paratypes of Oxychilus lanzai], MZUF 
683; 2 sh [paratypes of Oxychilus 
lanzai], MZUF 691); B. Lanza leg. 
15.3.64 (3 sp, FGC); B. Lanza leg. 
21.12.69 (1 sp, FGC); M. Bodon & S. 
Cianfanelll leg. 9.1 1 .97 (5 sp, 3 sh, SCC 
8097/1742); S. Cianfanelll leg. 14.10.01 
(1 sp, 29sh, SCC 11647/2819). 

PP08 Vagli di sopra, Valle Arnetola, 930 m asi 
(Vagli dl sotto, Lucca), 32TPP0084, S. 
Clanfanelli & M. Calcagno leg. 24.06. 
2000 (2 sp, 2 sh, FGC; 22 sh, SCC 
9328/2291 ; 7 sh, SCC 9330/2290; 1 sh, 
SCC 9485/2400), S. Cianfanelll & M. 
Calcagno leg. 8.10.2000 (1 sp, MZUF 
17597 [neotype of O. paulucciae]; 7 
sh SCC 9484/2399). 

PP17 Buca delle Fate di San Rocco, 635 m 
asi, no. 362 T/LU (Pescaglia, Lucca), 
32TPP1170, P. Magrini leg. 22.8.79 (1 
sp, 3 sh, FGC). 

PP19 Grotta della Faglia, Pania di Corfino 
(Villa Collemandina, Lucca), F. Utili leg. 
24. 1 1 .63 (2 sp, FGC). Tana del Gracchi 
di Sasso Rosso, 755 m asi, no. 289 T/ 
LU (Villa Collemandina, Lucca), 
32TPP1293, F. Utili leg. 22.11 .63 (1 sp, 
FGC). Tana del Pollone di Magnano, 
565 m asi, no. 1017 T/LU (Villa 
Collemandina, Lucca), 32TPP1592, B. 
Lanza leg. 30.4.72 (8 sp, MZUF 15856; 
1 sp, MZUF 15858). Tana di Magnano, 
635 m asi, no. 162 T/LU (Villa 
Collemandina, Lucca), 32TPP1592, B. 
Lanza & B. Malkin leg. 30.11.59 (1 sp 
det. O. paulucciae by Forcart, 1967, 
1968, NMB 6561 -a; 1 sp det O. 
paulucciae by Forcart, 1968, MZUF 
445), F. Utili leg. 24.11.63 (5 sp, 1 sh, 
FGC), B. Lanza leg. 15.3.64 (2 sp, 2 
sh, FGC), B. Lanza leg. 24.10.65 (1 sp, 
FGC), F. Utili leg. 5.12.65 (2 sp, 1 sh, 
FGC); P. Brignoli & A. Vigna Taglianti 
leg. 3.11.67 (3 sp, 3 sh, FGC); B. 
Lanza leg. 17.3.68 (11 sp, 20 sh, FGC). 

PP28 Grotta dell'Iseretta, 650 m asi, no. 823 
T/LU (Bagni di Lucca, Lucca), 
32TPP2882, P. Magrini leg. 6.74 (2 
sp, FGC). 



Etymology 

De Stefani (1883) named this species after 
the famous Italian malacologist. Marquise 
Marianna Paulucci (1835-1919) and Forcart 
(1967) after Prof. Benedetto Lanza, former di- 
rector of the Museo Zoológico de "La Specola" 
(Florence), who collected the specimens used 
for the description. 

Habitat 

All the specimens of O. paulucciae were 
collected inside caves. The area inhabited by 
the species is karstic. The species is presum- 
ably adapted to subterranean life and may be 
defined as troglobie. 

Geographical Distribution 

Species with reduced distribution, limited to 
northwestern Tuscany (Fig. 13). 

Status and Conservation 

Not globally threatened. Despite its limited 
distribution, O. paulucciae does not seem to 
be under any particular threat at present. 

Molecular Data 

Sequence Data Analysis 

The ITS-1 region ranged from 544 (O. 
uziellii) to 593 bp (O. meridionalis 1 and O. 
meridionalis 2) in length. After deletion of am- 
biguously aligned positions, the data-set in- 
cluded a total of 660 nucleotide positions, 61 
of which were phylogenetically informative 
under the parsimony criterion. Uncorrected 
percentage sequence divergence (p-distance) 
between the two O. paulucciae specimens (1, 
Vagli di sopra; 2, Grotta della Risvolta) was 
1.1%. Oxychilus meridionalis 1 (Capanno) and 
O. meridionalis 2 (Passo della Calla) are 
separated by a sequence divergence 0.3%, 
whereas a divergence of 2.3% distinguished 
them from O. meridionalis 3 (Bagni di Lucca). 
Sequence difference between O. draparnaudi 
1 (Castello di Brollo) and O. draparnaudi 2 
(Giglio Castello) was 1.3%. Genetic distances 
between morphologically defined species 
ranged from 2.5% (O. meridionalis 2 and O. 
paulucciae 2) to 7.9% (O. draparnaudi 2 and 
O. majori). 



REVISION OF OXYCHILUS PAULUCCIAE 

N PQTU V W XYBC 



31 



M 



\~^~^- 1 -- 


■^ 


1 _L14V— - 




Ьш^^^ 




Й^^Д^С-- 




IL 


V 


J^^ZlZHÏl 


r 




m 


Ir^rl 


-h4V 




^î^* 


ïsrr- 
Гаг 


5 




^ — \ r^ 




i°] 


.M 


1 




C2 




(I 


/ 


—^^^Ц^ 


И -Il 



M 



L MN PQTU VW XYB 

FIG. 13. The distribution of Oxychilus paulucciae (De Stefani, 1883) on UTM nnap of central-northern Italy. 



Phylogenetic Analysis 

Maxinnum parsimony analysis supported a 
single best tree shown in Figure 14 (tree 
length: 297, CI: 0.926, Rl: 0.788). The likeli- 
hood ratio test and AlC fronn Modeltest sup- 
ported the HKY+G model (Hasegawa et al., 
1985, including among-site rate heterogene- 
ity) as the best fit substitution model for the 
data. Parameters estimated for this model 
were: Ti : Tv ratio = 0.789, gamma shape pa- 
rameter = 1.02 and base frequencies A = 
0.1994, T = 0.2592, G = 0.26248, С = 0.2790. 
A ML analysis incorporating these parameters 
generated a tree with a likelihood score (-InL) 
of 2193.43 (Fig. 15). 

Parsimony and likelihood analyses pro- 
duced essentially the same topologies. The 
clade grouping, in MP topology, the two O. 
draparnaudi specimens with O. pilula repre- 
sents the only difference between the two 
reconstructions. In both topologies, all con- 
specific specimens grouped together in clades 
strongly supported by bootstrap values. The 
two reconstructions also suggest close phylo- 
genetic relationships between O. majori, О. 
paulucciae, О. uziellii and O. meridionalis. 



DISCUSSION 

Hyalinia paulucciae and Oxychilus lanzai 

Re-examination of the specimens from Grotta 
della Risvolta (Figs. 2, 11, 12) and Grotta del 
Buggine (Figs. 6, 7), which Forcart (1967) as- 
signed to O. {Ortizius) lanzai, and those from 
Tana di Magnano, which Forcart (1967) as- 
signed to O. {Oxychilus) paulucciae, confirmed 
that they belong to a single species. These 
specimens have the same characters as those 
collected near Vagli, the type locality of O. 
paulucciae. Similar conclusions were provided 
by molecular data. The two specimens (one 
from Grotta della Risvolta and one from Vagli) 
analysed were genetically very similar, forming 
a well differentiated evolutionary lineage with 
respect to all the other Oxychilus examined. The 
level of genetic divergence observed was corre- 
lated with geographic sampling and fall within 
the range observed for the other conspecific 
Oxychilus. The observed congruence between 
morphological and molecular data definitively 
demonstrates that O. lanzai is a junior synonym 
of O. paulucciae, as hypothesized by Giusti & 
Mazzini (1971) and Riedel (1980, 1997, 1998). 



32 



MANGANELLI ETAL. 

I— О. paulucciae 1 



80 



84 



94 



_ О. paulucciae 2 



97 



99 



86 



г О. meñdionalis 1 



• О. meñdionalis 2 



О. meñdionalis 3 



О. uziellii 



О. majori 



О. pilula 



I — О. drapamaudi 1 



97 



О. drapamaudi 2 



Retinella olivetorum 



10 changes 



FIG. 14. Most parsimonious tree calculated from ITS-1 sequence data. 
Bootstrap values are indicated at nodes (1,000 replications). 



Morphological Analysis 

Oxychilus paulucciae belongs to Oxychilus 
(s. Str.), sensu Giusti & Manganelli (1999), 
being characterized by: penis with flagellum; 
penial retractor inserted at apex of flagellum; 
internal ornamentation of penis consisting of 
pleats or rows of papillae without apical 
thorns; epiphallus long, usually longer than 
proximal penis; internal wall of epiphallus 
with slender longitudinal pleats; mucous 
gland mainly vaginal; long mesocone of cen- 
tral tooth. 

Among Oxychilus (s. str.) species it holds an 
intermediate position, sharing a narrow mid- 
penial portion ("bottle-neck") enveloped by a 



thin sheath with O. diductus (Westerlund, 
1886), O. drapamaudi, O. majori, О. mortilleti 
(Pfeiffer, 1859), О. oglasicola Giusti, 1968, 
and O. oppressus (Shuttleworth, 1878), but 
unlike them it has internal ornamentation of 
proximal penis consisting of longitudinal pleats 
very similar to that of O. meñdionalis (О. 
diductus: Manganelli et al., 2002: figs. 7-11; 
О. drapamaudi: Giusti & Manganelli, 1997: 
figs. 15-30; Manganelli & Giusti, 1998: figs. 
19-22; O. majori: figs. 4-8; O. meridionalis: 
Manganelli & Giusti, 2001: figs. 9-31; O. 
mortilleti: Manganelli & Giusti, 1998: figs. 5- 
17; O. oglasicola: Manganelli etal., 1999: figs 
12-14; O. oppressus: Riedel, 1967: figs. 1, 2; 
personal unpublished data). 



REVISION OF OXYCHILUS PAULUCCIAE 

- G. paulucciae 1 



33 



71 



82 



94 



- 0. paulucciae 2 



95 



96 



85 



0. meridionalis 1 
O. mendionalis 2 
- O. meridionalis 3 
O. uziellii 



O. majori 



О. pilula 



T— o. draparnaudi 1 



70 



- O. draparnaudi 2 



Retinella olivetorum 



0.05 substitutions/site 



FIG. 15. Maximum likelihood tree calculated from ITS-1 sequence data. Bootstrap 
values are indicated at nodes (1,000 replications). 



Consequently, internal ornannentation of the 
proximal penis consisting of longitudinal 
pleats readily distinguishes O. paulucciae 
from O. diductus, O. draparnaudi, O. majori, 
О. mortilleti, О. oglasicola and О. oppressus, 
and the narrow mid-penial region ("bottle- 
neck") distinguishes it from O. meridionalis. 
Besides the structure of the mid-penial region, 
O. paulucciae can be distinguished from O. 
meridionalis by its pale grey body, larger shell 
(shell diameter: 15.5 ± 1.0 mm) with small 
umbilicus (about 1/8 of shell maximum diam- 
eter), and vaginal gland that often forms in- 
complete ring around proximal vagina (body 
slate blue in colour; smaller shell: shell diam- 
eter: 13.1 ± 2.0 mm, with larger umbilicus. 



about 1/6-1/7 of shell diameter; vaginal 
gland always forming complete ring around 
proximal vagina; for detailed description of O. 
meridionalis: Manganelli & Giusti, 2001). 

Oxychilus paulucciae also shares the inter- 
nal ornamentation of the proximal penis, con- 
sisting of longitudinal pleats, with the species 
traditionally assigned to Ortizius Forcart, 1957 
(type species: Hyalina {Polita) helvetica Blum, 
1 881 ). Only one of the 28 species assigned by 
Riedel (1 980, 1 998) to this subgenus (Giusti & 
Manganelli, 2002: table 1) occurs within the 
area inhabited by O. paulucciae: O. clarus 
(Held, 1838). It is impossible to confuse the 
two species: O. clarus has a very small whit- 
ish shell (Kerney et al., 1983: pi. 10). 



34 



MANGANELLI ETAL 



Phylogenetic Relationships 

Our pliylogenetic reconstructions indicate 
that the species analysed in this study do in- 
deed represent well-differentiated evolutionary 
lineages. In particular, the three species for 
which multiple specimens were available al- 
ways formed monophyletic clades supported 
by bootstrap analysis, suggesting good overall 
resolution of the data set. Molecular data also 
indicated that O. paulucciae is genetically close 
to O. meridionalis, the two taxa separated by a 
sequence divergence ranging from 2.5 to 3.7%. 
This result is congruent with morphological evi- 
dence, if it is admitted that the internal structure 
of proximal penis, not the narrow mid-penis 
region, supports taxonomic relationships within 
the genus. Another interesting finding of this 
study was the close relationship between O. 
paulucciae, O. meridionalis, O. uziellii, and O. 
majori, which supports the existence of a 
Tuscan radiation of the genus. The last two 
taxa, О. uziellii, and O. majori, are character- 
ized by some highly derived features in the 
penial complex. This makes it difficult to unam- 
biguously infer their systematic affinities based 
on morphological evidence. Morphologically 
derived taxa represent a challenge for system- 
atics that can only be addressed by a molecu- 
lar approach. Finally, molecular data indicated 
that O. pilula and O. draparnaudi are distantly 
related taxa with respect to the above species. 



CONCLUSION 

Both morphological and ITS-1 sequence 
data indicates that O. paulucciae is close, but 
distinct from O. meridionalis, a widespread 
Tuscan species. The molecular results also 
showed that O. uziellii and O. majori, two 
other morphologically highly derived Tuscan 
species, are closely related to O. meridionalis 
and O. paulucciae and distinct from the most 
common O. draparnaudi. 

Combined morphological and molecular 
analysis of wider taxonomic sample, espe- 
cially type species of the many subgenera of 
Oxychilus, will further clarify the taxonomy of 
the genus and the relationships within 
oxychiline zonitids. 



ACKNOWLEDGMENTS 

We thank Antonella Daviddi and Leonardo 
Gamberucci for technical assistance, Helen 
Ampt for revising the English, Marco Bodon 



(Genoa, Italy), Micaela Calcagno (Florence, 
Italy), Gianni Comotti (Nembro, Bergamo, Italy), 
Elisabetta Lori (Pistoia, Italy), Paolo Magrini 
(Florence, Italy), Augusto Vigna Taglianti 
(Rome, Italy), and Franco Utili (Florence, Italy) 
for field collection and Benedetto Lanza (Flo- 
rence, Italy), Ambros Hänggi and Jolanda 
Ineichen-Riedi (Basel, Switzerland) for informa- 
tion about or loan of material from the 
Naturhistorisches Museum Basel (Switzerland). 
This research was funded by grants from 
"Bioitaly Tuscany", "EEC Regulation 2081/93- 
Objective 5/b" projects and by Università di 
Siena (PAR 2001, project "I molluschi non 
marini della fauna italiana: filogenesi, 
sistemática, faunistica, zoogeografía, conser- 
vazione) and Museo Zoológico de "La 
Specola", Sezione del Museo di Storia Naturale 
deirUniversità di Firenze (Italy). 



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Revised ms. accepted 4 July 2003 



MALACOLOGIA, 2004, 46(1): 37-55 

THE BIOLOGY AND FUNCTIONAL MORPHOLOGY OF 

FOEGIA NOVAEZELANDIAE (BIVALVIA: ANOMALODESMATA: 

CLAVAGELLOIDEA) FROM WESTERN AUSTRALIA 

Brian Morton 

Western Australian Museum, Francis Street, Pertii, Western Australia 6000, Australia; 

prof_bsmorton@hotmail. com 

ABSTRACT 

As more representatives of the adventitious, tube-building anomalodesnnatan 
Clavagelloidea are examined, a pattern of extraordinary adaptive radiation is being re- 
vealed. Despite its name, Foegia novaezelandiae is known only from the Holocene and 
Recent of Western Australia and is thus possibly very modern. A few tubes are held in the 
collections of the Western Australian Museum, Perth, and a single living individual has been 
collected from a hypoxic beach at Dampier, Western Australia. Like other clavagelloids, 
using a muscular pedal disc, F. novaezelandiae pumps interstitial water into its mantle cavity 
via the pedal gape, and hence the pedal slit and tubules of its anterior "watering pot" com- 
ponent of the adventitious tube. 

Foegia novaezelandiae is similar to Brechites vaginiferus in being amyarian, except for 
minute anterior pedal retractor muscles in the latter. As with B. vaginiferus also, palliai re- 
tractor muscles effect siphonal and pedal disc retraction. The adventitious tube of F. 
novaezelandiae is more complex in that the shell valves are recessed and largely hidden 
externally by additional bulbous concretions of tube material secreted from anterior and 
posterior palliai crests. Also like B. vaginiferus, F novaezelandiae pumps interstitial water 
into the mantle cavity, probably collecting interstitial bacteria and dissolved organic mate- 
rial as nutritional supplements. Unlike B. vaginiferus, however, F. novaezelandiae has an 
agglomeration of organic material and bacteria adhering to its highly convoluted 
periostracum anteriorly, particularly that of the pedal disc and thus inside the adventitious 
tube. Such bacteria may help detoxify the hydrogen sulphide contained in the interstitial 
water of the hypoxic sediment that F. novaezelandiae inhabits. However, F novaezelandiae 
has a full complement of mantle cavity and intestinal organs for the processing of food fil- 
tered from the seawater above. 

Key words: Foegia novaezelandiae, Clavagelloidea, adventitious tube formation, 
anatomy, tube function, watering-pot shell. 



INTRODUCTION 

The adventitious tubes of the diverse repre- 
sentatives of the Clavagelloidea d'Orbigny, 
1843, constitute some of the weirdest and rar- 
est bivalve structures. The most recent cla- 
distic analysis of the Anomalodesmata by 
Harper et al. (2000) did not identify sister 
groups but noted that Clavagella and its allies 
first appeared in the Cretaceous, whereas 
Brechites and its allies are known from the 
Oligocène onwards. Savazzi (2000) also noted 
that representatives of the Clavagelloidea 
seem to fall into two groups comprising those 
that (i) have their left valve united into the fab- 
ric of an adventitious tube in the case of 



endobenthic (Stirpulina) and epibenthic 
{Dianadema) genera, or a crypt in the case of 
nestling and boring species {Clavagella, 
Bryopa), with the right valve free inside it, and 
(ii) those in which both valves are incorporated 
into the structure of an adventitious tube, again 
in the case of endobenthic (Brechites, Foegia) 
and epibenthic (Humphreyia) genera. 

The anatomies of species of Clavagella, 
Bryopa and Dianadema have been described 
by Soliman (1971), Savazzi (1999, 2000) and 
Morton (1984a, 2003), respectively, and those 
of representatives of Brechites and 
Humphreyia by Morton (1984b, 2002a, b). Two 
of the above genera, that is, Dianadema and 
Humphreyia, are known only from Australia, 



37 



38 



MORTON 



and Smith (1 971 , 1 976, 1 998) and Lamprell & 
Healy (1998) catalogue the species recorded 
from that continent. These authors consider 
that in Australia the genus Brechites comprises 
three subgenera, that is, Brechites, s.S., plus 
Penicillus and Foegia, the second subgenus 
being represented by ß. (P.) philippinensis 
(Chenu, 1843) and the third by B. (F.) 
novaezelandiae (Bruguière, 1789) and B. (F.) 
veitchi Smith, 1971. Brechites (F.) novae- 
zelandiae is the type species of Foegia but, 
as noted by Smith (1971), other than for a 
description of its adventitious tube, virtually 
nothing else is known about it and there are 
no extant specimens with tissues available 
for study. 

During January 2000, a research trip was 
made to Western Australia and a single living 
individual of Foegia novaezelandiae was col- 
lected. On this and subsequent visits, the small 
collection of tubes of this species in the West- 
ern Australian Museum was examined. Obser- 
vations on the living animal and the collection 
of examined tubes are herein reported upon 
to provide an insight into the biology and 
anatomy of one of the strangest species, of 
one of the strangest superfamilies 
(Clavagelloidea) within the Bivalvia (Morton, 
1981a, 1985a). 



MATERIALS AND METHODS 

The specimen of Foegia novaezelandiae 
was collected from intertidal mud on the beach 
adjacent to the leased property of Dampier Salt 
Co. Ltd., Karratha, Western Australia. It was 
buried anterior end down, that is, the watering 
pot, with the posterior tube projecting just 
above the mud surface. 

As described for Brechites vaginiferus 
(Chenu, 1843) by Morton (2002a), the ante- 
rior end only of the adventitious tube of Foegia 
novaezelandiae was placed within a transpar- 
ent tub with a lid that had a central hole in it to 
hold the tube in place and containing a sus- 
pension of Ehrlich's haematoxylin in seawa- 
ter. The whole animal and tub was then placed 
in a much larger, also transparent, container 
of filtered seawater and left overnight. The liv- 
ing animal was subsequently dissected and 
the ciliary currents of the organs of the mantle 
cavity studied by application, again, of a sea- 
water suspension of Ehrlich's haematoxylin. 
The specimen was fixed in 5% formalin even- 
tually and, following routine histological pro- 



cedures, sectioned transversely at 6 pm and 
every tenth section retained. Alternate slides 
were stained in either Ehrlich's haematoxylin 
and eosin or Masson's trichrome. 

The nine specimens of Foegia novae- 
zelandiae contained in the collections of the 
Western Australian Museum, Perth, were ex- 
amined and the dimensions of all intact tubes 
measured to the nearest 1 mm. These were: 
greatest width, total length and length to the 
first growth (or possibly repair) increment. 



ABBREVIATIONS USED IN FIGURES 



AC 


Anterior concretion 


AN 


Anus 


APC 


Anterior palliai crest 


APCC 


Anterior palliai crest cavity 


AU 


Auricle 


CA 


Ctenidial axis 


CF 


Cuticular fusion 


CM 


Circular muscle 


CP 


Ctenidial plica 


C-P-V-CONN Cerebro-pleural visceral connective 


DD 


Digestive diverticula 


DK 


Distal limb of the kidney 


ES 


Exhalant siphon 


F 


Foot 


FPA 


Fourth palliai aperture 


H 


Heart 


HA 


Haemocoel 


HG 


Hypobranchial gland 


IBC 


Infra-branchial chamber 


ID 


Inner demibranch 


IE 


Inner epithelium 


ILP 


Inner labial palp 


IP 


Inner layer of periostracum 


IS 


Inhalant siphon 


К 


Kidney 


КС 


Kidney concretion 


KT 


Kidney tubule 


LM 


Longitudinal muscle 


LV 


Left shell valve 


MG 


Mid gut 


N 


Nerve 





Oesophagus 


OA 


Organic agglomeration 


OD 


Outer demibranch 


OE 


Outer epithelium 


OLP 


Outer labial palp 


OP 


Outer layer of periostracum 


OS 


Osphradium 


OV 


Ovary 


P 


Periostracum 


PC 


Posterior concretion 



BIOLOGY OF FOEGIA 



39 



PD 


Pedal disc 


PE 


Pericardium 


PEG 


Periostracal groove 


PG 


Pedal gape 


PK 


Proximal limb of the kidney 


PL 


Palliai line 


PPG 


Posterior pallia! crest 


PPCC 


Posterior palliai crest cavity 


PRM 


Palliai retractor muscle 


R 


Rectum 


RA 


Renal aperture 


RV 


Right shell valve 


S 


Siphons 


SA 


Saddle 


SBC 


Supra-branchial chamber 


SC 


Sensory cell 


SN 


Siphonal nerve 


SVI 


Shell valve impression 


TE 


Testes 


TMP 


Transverse muscle fibres 


V 


Ventricle 


VM 


Visceral mass 


VMG 


Ventral marginal food groove 



TAXONOMIC CONSIDERATIONS 

Smith (1971) discussed the taxonomy of 
Brechites {Foegia) novaezelandiae (Bruguière, 
1789). He regarded Aspergillum agglutinans 
Lamarck, 1818 (p. 430), and A. novae- 
hollandiae Chenu, 1843 (p. 3, pi. 4, fig. 8), to 
be synonyms. Penicillus novae Zelandiae 
Bruguière, 1789 (p. 129-130), was based on 
an ambiguous illustration in Favanne de 
Montcervelle & Favanne de Montcervelle 
(1780: 642, plate 79, fig. E), and misattributed 
to New Zealand. A neotype may be needed to 
stabilize the concept, because the original 
material has not come to light. No type mate- 
rial of A. agglutinans has been found. Two 
syntypes of Aspergillum novaehollandiae 
Chenu, 1843, are held in the collections of the 
Natural History Museum, London (1968668), 
and these are figured here (Fig. 1). 

Gray (1858a: 313) differentiated Foegia 
Gray, 1847 (p. 188), from other genera in his 
Aspergillidae Gray, 1858, a junior synonym of 
the Clavagellidae Orbigny, 1844, in several 
important respects: "Umbo more or less cov- 
ered with a swollen prominence in front; the 
whole of the valves except the umbo or 
nucleus enclosed in the tube; fringe indistinct, 
formed like the hole in the disk, of short thick 
separate tubes". The above description is gen- 
erally correct, and because of other anatomi- 



cal differences, I agree with Gray (1858a) that 
the genus Foegia is valid. It is possible that 
Foegia might date from Gray (1 842: 77), where 
there is a definition but no named species. In 
any event, the type species of Foegia is Peni- 
cillus novae Zelandiae Bruguière, 1789, by 
monotypy in Gray (1847). The species under 
consideration is, therefore, Foegia novae- 
zelandiae (Bruguière, 1789). 








%^ 



'"N>, 



^^лЧ 




20 mm 



FIG. 1. Foegia novaezelandiae. The two syn- 
types of Aspergillum novaehollandiae (NHM 
London 1968668). 



40 



MORTON 



DISTRIBUTION 

In the collection of the Department of Earth 
and Planetary Sciences of the Western Aus- 
tralian Museum are a number of local Ho- 
locene subfossils of Foegia novaezelandiae: 
LKwinana (south of Perth). Dredged from 
Cockburn Sound, 3 specimens (WAM 
69.1070a, b, c). 
2.Fremantle. Dredged from a fishing anchor- 
age, 1 specimen (WAM 70.2034). 
In the collection of Recent Mollusca in the 
Western Australian Museum are 16 specimens 
of Foegia novaezelandiae collected from ei- 
ther Cockburn Sound, South Fremantle, 
Woodman's Point or Leighton Beach, all loca- 
tions again just south of Perth. One was 
dredged from 1-2 fathoms (2-4 m), and all 
were dead when collected. Only nine tubes 
are intact. 

Smith (1971) records that Foegia novae- 
zelandiae occurs along "The central and south 
west coast of Western Australia and two speci- 
mens from the north coast of Queensland" (p. 
152). Cotton (1961) does not record the spe- 
cies from South Australia, nor do Wells & Bryce 
(2000) from Western Australia, presumably 
because of Its rarity. Smith (1976) illustrates 
(p. 201 , map 3) the range of F. novaezelandiae. 
Lamprell & Healy (1998) agree with this distri- 
bution pattern and report that the species oc- 
curs from depths of 3-22 m In sand. The record 
herein, from Dampier, though intertidal, is 
within the distribution range described, and 
therefore F. novaezelandiae is a Southern 
Hemisphere, warm temperate-tropical species. 



BIOLOGY 

The single specimen from the Dampier Salt 
Co. Ltd. lease at Karratha, Western Australia, 
was collected from the intertidal of an un- 
named muddy beach, the landward drainage 
onto which has been restricted by construc- 
tion of a bund to create solar salt pond "0". 
The seaward remnant of the original creek 
which drained onto the beach, lies opposite 
and is divided into two outlets by West Inter- 
course Island. Mangroves fringe the beach: 
an Avicennia forest to the seaward is followed 
landward, in succession, by Rhizophora scrub, 
Avicennia scrub, and (locally) Ceriops- 
Avicennia heath grading into a salt flat. The 
main water influence here is the tides because 
the hypersaline (salinities > 40%o), drainage 
from the land, as reported upon by Morton 



(2002a) for this part of Western Australia, has 
been halted by construction of the bund and 
causeway for pond "0". This has thus in turn 
adversely impacted not only beach dynamics 
but also interstitial water character. Whether 




FIG. 2. Foegia novaezelandiae. A. The adventitious 
tube; B. the siphonal tube as seen from the 
posterior aspect; 0. a closer view of the tube 
showing the calcareous tube and periostracum 
beneath the adhering detritus and D, the watering 
pot as seen from the anterior aspect. Note the 
dorso-ventrally aligned pedal slit (for abbreviations 
see pp. 38-39). 



BIOLOGY OF FOEGIA 



41 



natural or perturbed, the substratum of sandy- 
mud in this Foegia novaezelandiae habitat is 
hypoxic, and the specimen was oriented ver- 
tically in it with the posterior end of the tube 
projecting above the sediment surface by 
some 10 mm. Semeniuk & Wurm (1987) de- 
scribe in broad terms the characteristics of the 
shore seaward of pond "0" and provide basic 
maps (figs. 21, 22) of the area. 



ANATOMY 

Adventitious Tube 

The nine tubes of Foegia novaezelandiae in 
the collections of the Western Australian Mu- 
seum range in total length from 69-98 mm and 
in maximum width from 13-16 mm. The living 
individual from Dampier was 1 30 mm long and 
1 5 mm wide. Some tubes in the collection have 
either a single growth increment or a repair at 
a length ranging from 80-94 mm. The Dampier 
individual has two (Fig. 2A). The relationships 
between tube width and total tube length and 
length to the first growth increment or repair 
are illustrated in Figure 3. Where there is no 
growth increment, the two measurements are 



the same. Although the correlations are poor, 
the lines of best fit are similar. Four individu- 
als, each with one growth or repair mark on 
the tube, lie on the right side of the plot sug- 
gesting that any such increment occurs at a 
length of between ~ 85-100 mm. The above 
implies that the adventitious tube is secreted 
but once when the contained animal becomes 
an adult, but that it can be subsequently ex- 
tended or repaired posteriorly, as in Brectiites 
vaginiferus (Morton, 2002a). 

The tube of the living Foegia novaezelandiae 
is illustrated in Figure 2. The main shaft of the 
tube (Fig. 2A) is covered in sand grains and 
other hard detritus, except posteriorly and 
anteriorly at the watering pot disc. Posteriorly, 
there are two growth (or repair) increments, 
both secreted internal to the preceding one. 
These are covered sparsely in detritus and 
raised above the sediment surface. Viewed 
from the posterior aspect (Fig. 2B), the tube 
aperture is 8-shaped in cross-section match- 
ing the configuration of the siphons, which 
project up into it. In places, the shell debris is 
worn away from the tube beneath exposing 
the calcareous tube with a thin adhering film 
of periostracum (Fig. 20). Seen from the an- 
terior end (Fig. 2D), the watering pot disc has 





16-1 








• O" 


's 


15- 


У = -0.0156x+ 15.315 
^ R^ = 0.0355 


o^ 


E 












14- 


Ф- 


:^z;=^=^ 


=■ -^^^u^-^JlZr "<^ 


Ф 










3 


13- 
12 ■ 


y = 0.0039x+ 13.79 
R^ = 0.0029 




#• 


♦ ♦ 






O- 


Tube width versus total tube length 












Tube width versus to first growth increment/repair 
Tubes with one growth increment/repair 




11 • 






•ir 











50 



55 



60 



65 



70 75 80 

Tube length (mm) 



85 



90 



95 



100 



FIG. 3. Foegia novaezelandiae. The relationship between adventitious tube width and (i), total length and 
(ii), length to the first growth (or repair) increment. 



42 



MORTON 

PC 



■ ■<■■■■•: •-■ >iV.'.*í.-A-W>, 


















/, 






* 


'"^^^ 


r,v 




•V4 




■'»: 


.;*Ч* ••■'-■ ■ 


^í: 


* 


.¿■^> 









2.5 mm 



AC 



FIG. 4. Foegia novaezelandiae. AV\e\N of the dorsal surface of the adventitious tube 
showing the true shell valves and enclosing anterior and posterior bulbous 
projections (for abbreviations see pp. 38-39). 



a dorso-ventrally aligned pedal slit and an ar- 
ray of open tubules which, as shown by Gray 
(1858a), do not have a distinct "fringe" sepa- 
rating it from the tube's shaft, as is the case in 
Brechites vaginiferus and where it is identi- 
fied as a distinct "line" (Morton, 2002a: fig. 1). 

Tube Function 

When the watering pot of the living individual 
of Foegia novaezelandiae was placed in a 
suspension of Ehrlich's haematoxylin in sea- 
water, the animal clarified it within 12 hours. 
Thus, as with Brecliites vaginiferus (Morton, 
2002a), F. novaezelandiae pumps interstitial 
water into the mantle cavity through the pedal 
slit and tubules that constitute the watering pot. 

Shell 

As noted by Gray (1 858a), the shell of Foegia 
novaezelandiae is covered by two, anterior 



and posterior, bulbous secretions and Is gen- 
erally hidden within the fabric of the adventi- 
tious tube. However in one specimen in the 
Western Australian Museum collection (from 
Cockburn Sound, (i) of (iv) specimens col- 
lected In 1965; broken base only; S 14232), 
the shell valves are partly visible. This speci- 
men was cleaned carefully with dilute nitric 
acid, to remove sand grains and other debris 
and is illustrated in Figure 4. The two shell 
valves have parted and are ~ 3 mm long. They 
are equivalve and inequilateral, that is, anteri- 
orly foreshortened and posteriorly elongate, 
and thus of the same general form as in all 
clavagelloids hitherto described, for example, 
Brechites vaginiferus, Humphreyia strangei 
and Dianadema multangularis (Morton, 2002a, 
b, 2003). The umbones are slightly pointed, 
and there Is a trace of a radial sculpture of 
periostracal spinules, similar to those de- 
scribed for Lyonsia hyalina by Prezant (1 979a) 
and for the clavagelloids listed above. Around 



BIOLOGY OF FOEGIA 

PPCC 



43 



SVI 




APCC 



2 mm 



FIG. 5. Foegia novaezelandiae. An internal view of the adventitious tube showing 
the positions of the true shell valves and palliai line lying below the saddle (for 
abbreviations see pp. 38-39). 



the two shell valves and uniting them, Is a 
"saddle" of secondarily secreted shell which 
has fine concentric growth lines also seen in 
other clavagelloids (see above). Shell and 
saddle are sunk into the general fabric of the 
adventitious tube. A thick, bulbous concretion 
covers the antero-dorsal region of the right 
valve, and a second, similarly bulbous con- 
cretion is present posteriorly. 

Internally, the shell, saddle and adventi- 
tious tube of the Dampier specimen (Fig. 5) 
are united and covered by a smooth calcar- 
eous concretion. The positions of the valves 
appear as depressions surrounded by raised 
borders of secondarily and internally se- 
creted calcium carbonate. Pockets where 
anterior and posterior palliai crests are in- 
serted above the valves to create the bul- 
bous secretions covering them are also 
evident. Two crescentric pallial-line scars 
encircle the antero-lateral sides of the shell 
valve impressions. The Pilbarra region of 
Western Australia is mineral rich and the in- 
ternal surface of the anterior watering pot 
was stained brown with iron oxide. 



Internal Anatomy 

The living animal of Foegia novaezelandiae 
was removed from its tube and is illustrated in 
Figure 6A-C. The siphons have contracted. 
The entire body is enclosed in periostracum 
secreted by the general mantle epithelium. 
Covering the mantle immediately beneath the 
true shell and, therefore, approximately en- 
compassing the pericardium, the periostracum 
is a transparent skin (this is illustrated as a 
light stippling in Figure 60). Elsewhere, cov- 
ering siphons, pedal disc and the general 
mantle surface, the light brown periostracum 
is thick and wrinkled. From the dorsal view 
(Fig. 6A), the pericardium contains a heart, 
which comprises a central ventricle, pen- 
etrated by the rectum, and lateral auricles. 
Posteriorly, there are paired kidneys, over 
which the rectum passes. Anteriorly, the vis- 
ceral mass contains the digestive diverticula 
and the paired ovaries. From what is the 
crescentric remnant of a palliai line, palliai re- 
tractor muscles pass into the mantle in ante- 
rior, ventral and posterior directions to effect 



44 



MORTON 

PRM R 



V 




К f^ ^f ^ PPC APC 

PRM \ \ \ I / / /PL 




FPA 

FIG. 6. Foegia novaeze/and/ae. А generalized picture of the anatomy, as seen from A, dorsal; 
B, ventral and C, right lateral aspects. Note that in С the periostracum surrounding the 
pericardium is illustrated with a light stippling as in Figure 2C: elsewhere the periostracum 
is brown, thick and wrinkled (for abbreviations see pp. 38-39). 



BIOLOGY OF FOEGIA 45 

К R H PPC APC 




FIG. 7. Foegia novaezelandiae. An interval view of the organs and ciliary currents of the mantle cavity as 
seen from the right side (for abbreviations see pp. 38-39). 



contraction of the body within its adventitious 
tube. There are no other muscles. Also seen 
dorsally, above the visceral mass, are ante- 
rior and posterior palliai crests. 

From the ventral view (Fig. 6B), the 
periostracum-covered pedal disc lies antero- 
ventrally, and in its centre is a dorso-ventrally 
aligned pedal gape. Where the siphons meet 
the remainder of the mantle, there is a mid- 
ventral fourth palliai aperture. The animal, as 
seen from the right side (Fig. 60), shows the 
heart within the pericardium and the rectum 
passing over the kidneys, the palliai retractor 
muscles and the anterior pedal disc and gape. 
Also seen are the fourth palliai aperture and 
the anterior and posterior palliai crests. 

Organs and Ciliary Currents of the Mantle 
Cavity 

The extended body of Foegia novaezelandiae 
is shown in Figure 7 after being opened on the 
right side. The most obvious feature is the long 
paired ctenidia, each of which consists of a com- 
plete inner demibranch and the dorsally directed 
descending lamella only of the outer. The 
ctenidia extend into the apex of the siphons and 
thus separate supra- from infra-branchial cham- 
bers. The ciliary currents of the ctenidia are of 
Type E (Atkins, 1937a) and pass collected par- 
ticles anteriorly towards the mouth in the 
ctenidial axis and in the ventral marginal food 
groove of the inner demibranch via small labial 
palps. 

The visceral mass is small with a little foot 
antero-ventrally. No statocysts have been iden- 
tified, although they occur in most anomalo- 



desmatans (Morton, 1985b), but were similarly 
not seen in Dianadema multangularis (Morton, 
2003). Their absence in this specimen may 
be because only every 10'^ transverse sec- 
tion was kept but this would mean any missed 
statocysts would be very small, that is, < 60 
|jm in length. Within the visceral mass, dorsal 
ovaries are separate from ventral testes. 

The ciliary currents of the visceral mass are 
directed towards its postero-ventral edge where 
unwanted particles fall onto the mantle mid- 
ventrally. As in Breciiites vaginiferus (Morton, 
2002a), the ciliary currents on the internal sur- 
face of the pedal disc radiate outwards and 
downwards from the pedal gape. The ciliary 
currents on the internal surface of the mantle 
are downward, complementing those of the vis- 
ceral mass but, mid-ventrally, strong ciliary cur- 
rents transfer unwanted material posteriorly, 
where it is ejected from the inhalant siphon as 
pseudofaeces There are also posteriorly di- 
rected ciliary currents in the supra-branchial 
chamber and which presumably help to trans- 
fer faeces to the exhalant aperture because the 
anus is located deep inside the siphons on the 
posterior surface of the paired kidneys. 

Musculature 

Foegia novaezelandiae has no adductor and 
pedal retractor muscles. The palliai line is 
short, ~ 3 mm, on each side of the body and 
from it arise palliai retractor muscles that ex- 
tend anteriorly, ventrally and posteriorly. The 
attachment of the palliai retractor muscles to 
the adventitious tube, at the palliai line, is 
shown in transverse section in Figure 8. 



46 



MORTON 

DD О 



C-P-V-CONN 
OLP 




FIG. 8. Foegia novaezelandiae. A transverse section through the visceral nnass and 
mantle (for abbreviations see pp. 38-39). 







FIG. 9. Foegia novaezelandiae. A transverse section through the outer mantle 
epithelium of the pedal disc showing the periostracum and agglomeration of adhering 
organic material and bacterial cells (for abbreviations see pp. 38-39). 



BIOLOGY OF FOEGIA 



47 




FIG. 10. Foegia novaezelandiae. A SEM micrograph of the outer surface of 
the pedal disc, that Is, Inside the adventitious tube, showing attached 
Inorganic and organic detritus and rod-shaped bacteria. 



Mantle 

The mantle margin of Foegia novaezelandiae 
is shown in transverse section in Figure 8. 
Mantle fusion is of Type С (Yonge, 1982), that 
Is, Inner, middle and Inner surfaces of the outer 
mantle folds, so that virtually everywhere the 
outer surface of the general mantle Is enclosed 
In thick perlostracum. The palliai retractor 
muscles extend into the mantle (Fig. 7) and 
posteriorly form longitudinal fibres that retract 
the siphons. Laterally, the mantle has a capa- 
cious haemocoel and circular muscles from 
both the left and right assist In palliai contrac- 
tion. 

The mantle of the pedal disc Is shown In 
transverse section In Figure 9. The outer epi- 
thelium Is thrown into many folds and at the 
apex of each pleat there Is a swollen cell ~ 8 
|jm in diameter which Is Innervated by tiny 
subepithelial nerves. The epithelium also se- 
cretes the perlostracum, which comprises two 
layers. The Inner is thick, up to 50 pm and 
stains blue in Masson's trichrome. It is prob- 
ably mucoid. The outer layer Is thin (2 pm), 
stains red In Masson's trichrome and is thrown 
into complex fibrous folds and strands. Around 
the pedal disc but diminishing towards the si- 
phons, the outer surface of the perlostracum 
is covered in an agglomeration of organic 
material. Within this are slightly curved, rod- 
shaped bacteria, ~ 1.5-2 pm In length, and 
which do not stain In either Masson's trichrome 
or Ehrllch's haematoxylln, but shine a bright 
yellow-green. This agglomeration of organic 



material and bacteria attached to the pedal 
disc, being Inside the adventitious tube, is in 
darkness. It Is not present In the similarly 
endobenthic Brechites vaginiferus (Morton, 
1984a: fig 16a). The agglomeration of inor- 
ganic and organic detritus with the bacteria 
attached to the pedal disc, as seen under the 
SEM, Is illustrated In Figure 10. 

Siphons 

As Is typical of all clavagellolds studied hith- 
erto (Morton, 1984a, b, 2002a, b, 2003), and 
for other anomalodesmatans (Prezant, 1 979b; 
Morton, 1981b), radial mantle glands at the 
apices of the siphons of F. novaezelandiae 
produce a secretion which attaches sand 
grains and other detritus to the thick 
perlostracum of their outer surfaces to cam- 
ouflage them. The siphons are shown In trans- 
verse section in Figure IIA. Internally, there 
are 1 6 palliai nerves that, in other clavagellolds, 
for example, Brechites vaginiferus (Morton, 
2002a) relate to the number of sensory papil- 
lae, which surround the siphonal orifices. 

The siphonal wall is Illustrated In greater 
detail In Figure IIB. Externally, are outer and 
Inner layers of the perlostracum. Internal to 
the outer epithelium Is a haemocoel and Inter- 
nal to this are successive layers of longitudi- 
nal, circular, longitudinal and circular muscles. 
Criss-crossing the longitudinal muscle blocks 
are transverse and oblique fibres that must 
create the tonus which extends and contracts 
the siphons. In cooperation with the other 



48 



MORTON 




НА OE 



P<^/ V^rf^'^v vJ'u. 



Ч • • 




CM 



FIG. 11. Foegia novaezelandiae. Transverse sections through A, the siphons showing the thick perio- 
stracum and pallia! nerves and B, the siphonal wall in greater detail (for abbreviations see pp. 38-39). 



muscles and blood-filled haemocoels of the 
mantle. In terms of its muscular complexity, 
the siphons of Foegia novaezelandiae are very 
similar to those of Brecfiites vaginiferus 
(Morton, 1984a: fig. 14) and Humpiireyia 
strangei (Morton, 2002b: fig. 12). 

Ctenidia 

The long, homorhabdic ctenidia (Fig. 7) are 
also illustrated diagrammatically in transverse 
section in Figure 12. Approximately five pli- 
cae make up the descending lamella of the 
outer demibranch and about eight both lamel- 
lae of the inner. There is a ventral marginal 
food groove in the latter. Each plica comprises 
a maximum of 20 filaments anteriorly, but only 
two as the ctenidia decline in size posteriorly 
(Fig. 7). 

As in other clavagelloids, for example, 
Brechites vaginiferus (Morton, 1984a, 2002a), 
the epithelium ventral to the kidneys and which 
forms the dorsal surface of the supra-branchial 
chamber of the outer demibranch is modified 
into a hypobranchial gland. The descending 
lamella of the outer demibranch attaches to 
the visceral mass by a cuticular junction, as 
does the ascending lamella of the inner 
(Atkins, 1937b). This was first described for 
an anomalodesmatan, that is, Laternula 
truncata, by Morton (1973) and is considered 
characteristic of all representatives. 



Medially, adjacent to the cuticular junction is 
an osphradium that has not hitherto been de- 
scribed for any anomalodesmatan, although 
it has been reported in other bivalves, for ex- 
ample, Corbicula fluminea (Kraemer, 1981). 
Left and right osphradia (Fig. 12) extend from 
the labial palps to the posterior end of the vis- 
ceral mass. In transverse section (Fig. 13), 
each osphradium lies between the cuticular 
junction of the outer demibranch with the vis- 
ceral mass and the hypobranchial gland. It 
comprises a central core of cells between 
which nerve fibres pass towards the periph- 
ery. The outer epithelium is thin (4 pm) but 
periodically along its margin there are swollen 
sensory cells ~ 8 pm tall and towards which 
the nerves are oriented. 

Pericardium and Kidneys 

The pericardium and kidneys are illustrated 
in Figure 6A and С and in transverse section 
in Figure 12. The rectum is enclosed by the 
ventricle of the heart (in turn surrounded by 
the pericardium) but lies dorsal to the paired 
kidneys. Each kidney comprises a capacious 
distal limb and a bag-like proximal limb that 
opens into the supra-branchial chamber of the 
inner demibranch at ciliated renal apertures 
(Fig. 12). There are no pericardial proprio- 
receptors such as occur in Humpiireyia 
strangei and Dianadema multangularis 



BIOLOGY OF FOEGIA 

PE R V 



49 




2.5 mm 



FIG. 12. Foegia novaeze /алсУ/ае. A transverse section through the paired kidneys 
showing the renal apertures, the ctenidia and the position of the paired 
hypobranchial glands and osphradia within the supra-branchial chamber of the 
outer demibranch (for abbreviations see pp. 38-39). 



SBC 




FIG. 1 3. Foegia novaezelandiae. A transverse section through the hypobranchial 
gland and osphradium in the supra-branchial chamber of the outer demibranch 
(for abbreviations see pp. 38-39). 



50 



MORTON 




КС 



20 цт 



FIG. 14. Foegia novaezelandiae. А transverse section 
through two distal limb tubules of the kidney showing the 
contained concretions (for abbreviations see pp. 38-39). 



(Morton, 2002b, 2003) probably because there 
are no remnants of the posterior pedal retrac- 
tor muscles as in Brechites vaginiferus, which 
similarly does not have such sense organs 
(Morton, 2002a). 

Distal kidney tubules are illustrated in trans- 
verse section in Figure 14. The cells are some 
10 pm tall, largely vacuolated, and contain 
approximately spherical concretions, between 
6-8 pm in diameter and which stain blue in 
Masson's trichrome but with a lighter staining 
core. Such concretions also occur in the lu- 
mina of the distal limb tubules. 



DISCUSSION 

The first, detailed description of a tube-dwell- 
ing clavagelloid {Aspergillum dich oto m urn) 
was by Lacaze-Duthiers (1883). Three-quar- 
ters of a century later, Purchon (1956, 1960) 
described Brechites penis and, later, Smith 
(1971, 1976, 1998) produced simple illustra- 
tions of Australian species, but not Foegia 
novaezelandiae. Subsequently, Morton 
(1984a, 2002a, b) described Brechites 
vaginiferus and the cemented Humphreyia 
strangei. Clavagelloids that unite only the left 
valve into the fabric of a crypt {Clavagella and 
Bryopa) have been described by Owen (1835), 
Soliman (1971) and Morton (1984b). The 
strange, cemented species, Dianadema 
multangularis, with tubules that form a crown 
over the dorsal part of the shell and adventi- 
tious tube, was described by Morton (2003) 
and suggested to be similar functionally to the 
North American, Late Cretaceous Ascaulo- 
cardium armatum (Pojeta & Sohl, 1987). 



Savazzi (1982, 1999) described adaptations 
of clavagelloids to a tube-dwelling mode of life, 
and Carter (1978) described how the tubes of 
gastrochaenids are formed. The gastro- 
chaenids Cucurbitula and Eufistulana (Morton, 
1982, 1983) are convergently very similar to 
Dianadema and Brechites, respectively, in 
forming adventitious tubes. However, the shell 
valves of gastrochaenids do not unite with the 
tubes. Also, there is no anterior pedal slit nor 
are there tubules giving access to interstitial 
waters. Morton (1984a, 2002a) speculated on 
the process of tube formation in Brechites 
vaginiferus as, earlier, had Gray (1858b) and 
Smith (1978). These authors agree that the 
adventitious tube is secreted but once and that 
posterior extension is possible either as the 
animal grows or has to extend itself either to 
keep pace with an accreting habitat or to ef- 
fect repair. Because the whole body internal 
to the tube is covered in thick periostracum, 
Morton (1984a, 2002a) believed erroneously 
that the tube of ß. vaginiferus was created by 
a secretion produced from glands in the apex 
of the siphons pouring down the outside of the 
periostracum-covered adult, between it and 
the burrow, to form a structure that matched 
the configuration and surface structure of the 
burrow wall. Subsequently, Morton (2002b, 
2003) showed that the tubes of Humphreyia 
strangei and Dianadema multangularis could 
not be secreted in this way, since both are 
cemented epibenthically with no burrow tem- 
plate. Formation probably results from the 
mantle epithelium secreting sequentially either 
periostracum or adventitious tube, in a man- 
ner similar to that described by Savazzi (2000) 
for the ligament of Bryopa. 



BIOLOGY OF FOEGIA 



51 



A 







Burrow wall 
Mantle 



в 






^::r< j 



.*"•'•♦•' 






V . + » • .• .* i, ' . »- f 



Burrow wall 



Mantle 







Burrow wall 



Adventitious tube 



>;•: î- -'.Vy^^lslrl/ :^' '"' ^'-^'^*'^.^Í^¿^'a/^ 'S Mantle 







Burrow wall 



Adventitious tube 



Mantle 



ш^шшЁШ^^^тшЕ 



?щш&^ 

ФМ 







Burrow wall 



Adventitious tube 



Mantle 



FIG. 15. Foegia novaezelandiae. Generalized illustrations of longitudinal sections through 
the shell, saddle and adventitous tube showing the postulated method of construction (for 
abbreviations see pp. 38-39). 



52 



MORTON 



In Foegia novaezelandiae the process of 
tube formation is more complicated than that 
of other clavagelloids and is illustrated in Fig- 
ure 15A-E. Initially, the tube is secreted the 
same way as in Brechites vaginiferus (Morton, 
1 984a, 2002a), in that the juvenile shell is cov- 
ered by periostracum: (Fig. 1 5A, ano\N 1 ). The 
animal expands hydrodynamically enlarging its 
burrow to full adult size, and a second layer of 
periostracum is then secreted by the mantle 
and covers the whole body. The anterior and 
posterior palliai crests secrete this too over the 
tiny shell valves (Fig. 15B, arrow 2). Secre- 
tion of periostracum 2 having halted, the ad- 
ventitious tube is then produced by the mantle 
(Fig. 1 50). Extra secretions of the tube by the 
palliai crests produce the bulbous protuber- 
ances above the true shell valves largely hid- 
ing them. Internally too, further secretions by 
the dorsal mantle unite shell valves, saddle 
and tube, creating the situation whereby the 
former are effectively incorporated into the total 
structure of the adventitious tube (Fig. 15D). 
Finally (Fig. 15E, arrow 3), a further layer of 
periostracum is produced by the mantle so that 
the whole animal, within its tube, is now cov- 
ered in periostracum which is thin and trans- 
parent dorsally (small arrow 3), and thick and 
wrinkled all over the rest of the mantle (large 
arrow 3). 

The secretion of the adventitious tube of 
Foegia novaezelandiae is thus highly complex 
involving the mantle in a sequence of secre- 
tions of different properties to produce: (i) shell 
and saddle (covered by periostracum), (ii) a 
second layer of periostracum, (iii) the main 
component of the adventitious tube and, finally, 
(iv) a third layer of periostracum. This results 
in the peculiar situation wherein the animal is 
encased within periostracum, within a tube, 
within periostracum and within a burrow. 

The hydrodynamic forces within the mantle 
and siphons of Foegia novaezelandiae which 
pump the animal up to its full size before pro- 
duction of the adventitous tube and subse- 
quently extend the siphons following 
contraction, must, as postulated for Brechites 
vaginiferus (Morton, 2002a), in the absence 
of any adductor muscles, be created by con- 
tractions of the pedal disc. In F. novae- 
zelandiae, the pedal disc must also create 
the hydrodynamic forces in the haemocoels 
of the mantle and siphons, acting agonisti- 
cally with the circular, longitudinal and trans- 
verse muscles within the latter, to effect 
siphonal extension. The paired supra-bran- 



chial osphradia of F. novaezelandiae are of 
interest in this respect. Bivalve osphradia are 
usually simple structures and generally be- 
lieved to monitor water flow through the 
ctenidia (Kraemer, 1981). However, in the 
case of F. novaezelandiae, perhaps they 
monitor the complex hydrodynamic forces in 
the mantle cavity and assist in their 
synchronisation. 

Foegia novaezelandiae is also of interest 
in another respect. The thick anterior cover- 
ing of periostracum, especially around the 
pedal disc, is thrown into complex folds not 
seen in other tube-dwelling clavagelloids, for 
example, Brechites vaginiferus (Morton, 
1 984a: fig. 1 2). It also possesses an external 
covering of an agglomeration of organic ma- 
terial and anucleate "cells". Sand grains and 
other inorganic detritus covering the siphonal 
apices exposed to light are not present in the 
pedal disc agglomeration in the dark. The 
"cells" are bacteria: might they be sulphide 
oxidizing? Foegia novaezelandiae is unusual 
in that it occupies hypoxic mud. Is it possible 
that it has within the base of its tube and into 
which interstitial water is pumped, a collec- 
tion of symbiotic bacteria that help to detoxify 
the sulphide in the incoming water? Might 
such bacteria also provide it with a supple- 
mentary source of nutrition in the form of re- 
duced carbon and amino acids fixed and 
produced by them, respectively? This may 
reduce dependence on short-term inputs of 
organic matter from the tropical, nutrient de- 
ficient waters above (Rochford, 1980). This 
study cannot answer these questions until 
more intact specimens are available for study. 

Reid (1990) surveyed the occurrence of 
chemoautotrophic sulphide oxidizing bacteria 
in the Bivalvia and showed that they occur 
within the ctenidial filaments in specialized 
bacteriocytes and are characteristic of hydro- 
thermal vent species, for example, Calyptogena 
and Bathy modiolus, shallow water represen- 
tatives of the Lucinoidea (Taylor & Glover, 
2000) and Solemyoidea, many of which inhabit 
sulphur-rich sediments (Dando et al., 1986). 
Foegia novaezelandiae does not have intra- 
cellular, ctenidial bacteria, but the record of 
free-living bacteria with characteristics of sul- 
phide-oxidizing ones on the pedal disc 
periostracum is of interest and deserves fur- 
ther study. 

It is now known that the adventitious tubes 
of clavagelloids fulfil a number of functions. 
These are: 



BIOLOGY OF FOEGIA 



53 



(i) Creating the rigid external skeleton 
against which the pedal disc can pump 
interstitial water into and out of the mantle 
cavity to generate the hydrodynamic pres- 
sures necessary in the palliai haemocoels 
to extend the siphons following retraction, 
(il) The same pumping action may supply the 
animal with interstitial bacteria and dis- 
solved organic material and mineral salts, 
which probably act as sources of nutrients 
accessory to the material collected by 
suspension feeding from the tropical, nu- 
trient poor overlying water (Rochford, 
1980). 
(iii) Aeration of the interstitial water may be 
achieved by pumping mantle cavity water 
obtained from the sea above via the si- 
phons into the burrow heading, 
(iv) Possible detoxification of interstitial wa- 
ter, by burrow aeration, 
(v) Possible detoxification of hydrogen sul- 
phide in the incoming interstitial water by 
(loosely symbiotic?) chemoautotrophic 
bacteria and the supply of reduced car- 
bon and amino acids to the host. 
Our understanding of the adaptive radiation 
of the Clavagelloidea increases with each new 
species studied. It stems from initial, but sepa- 
rate, adaptations in the Cretaceous 
(Clavagellidae: Clavagella and Dianadema) 
and Oligocène (Penicillidae: Brechites and 
Foegia) to life within a tube but how such ad- 
aptations arose and from what ancestor(s) are 
unknown (Harper et al., 2000). 



ACKNOWLEDGEMENTS 

I am grateful to F. E. Wells, D. S. Jones and 
M. Hewitt of the Western Australian Museum, 
Perth, Western Australia, for organizing field 
research at Dampier, Western Australia, in 
2000, to S. Slack-Smith and G. W. Kendrick, 
also of the Western Australian Museum, for 
assistance in accessing the collections of Re- 
cent and fossil Mollusca, respectively. E. V. 
Coan of Palo Alto, California, and J. D. Taylor, 
Natural History Museum, London, are thanked 
for assistance with the complex taxonomy of 
Foegia novaezelandiae, and the latter is fur- 
ther thanked for providing the SEM photograph 
(Fig. 10) of the pedal disc. 

The Director and staff of the Western Aus- 
tralian Museum are thanked for the provision 
of facilities and many kinds of help and hospi- 
tality, respectively. 



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Revised ms. accepted 18 August 2003 



MALACOLOGIA, 2004, 46(1): 57-71 

NEW SPECIES OF THE GENUS KELLIA (BIVALVIA: KELLIIDAE) 

FROM THE COMMANDER ISLANDS, 

WITH NOTES ON KELLIA COMANDORICA SCARLATO, 1981 

Gennady M. Kamenev 

The Institute of Marine Biology, Russian Academy of Science, Vladivostok 690041, Russia; 
kamenev@mail333. com, inmarbio@mail.primorye. ru 

ABSTRACT 

A new species, Kellia kussakini, is described from the Commander Islands. This species 
has a small (to 4.8 mm), translucent, pear-shaped, very inflated, almost globular shell 
(shell length, height, and width almost equal), with a slightly polished, yellowish-gray 
periostracum and posteriorly placed beaks. It was found in the subtidal zone (depth 5-20 m) 
of Bering and Medny islands, on a rocky platform, with population density to 1,190 speci- 
mens/m^. Scarlato (1981) described Kellia comandorica Scarlato, 1981, from the Com- 
mander Islands after study of a small amount of material (10 specimens). Later, Coan et 
al. (2000) synonymized К. comandorica with K. suborbicularis (Montagu, 1803). A study of 
extensive material (146 specimens) has shown that K. comandorica is a separate species 
having characters that distinguish it from other species of Kellia. An expanded description 
of K. comandorica is given. 

Key words: Kellia, Kelliidae, Bivalvia, Commander Islands, morphology, distribution. 



INTRODUCTION 



The bivalve mollusk fauna of the Com- 
mander Islands shelf has been poorly stud- 
ied. The most complete species list of bivalve 
mollusks of the Commander Islands was pub- 
lished after examination of the extensive ma- 
terial collected by two joint expeditions of 
IMB-PRIFO (the sealer "Krylatka", 1972; RV 
"Rakitnoye", 1973) to these islands, as well 
as an analysis of previous investigations 
(Kamenev, 1995). However, there still were a 
number species requiring additional investiga- 
tion and more accurate identification. Subse- 
quently, a few papers devoted to the study of 
these species were published (Kamenev, 
1996, 2002; Kamenev & Nadtochy, 2000). 
Further examination of bivalve mollusks col- 
lected in the shelf zone of the Commander 
Islands revealed one new species of the ge- 
nus Kellia which was erroneously identified as 
Kellia suborbicularis (Montagu, 1803) 
(Kamenev, 1 995). In addition, another species 
of this genus, Kellia comandorica Scarlato, 
1981, described by Scarlato (1981) based on 
a small amount of material, is abundant in the 
intertidal and subtidal zones of the Com- 
mander Islands. Scarlato (1981) described К 



comandorica in detail and provided a compara- 
tive diagnosis with distinguishing characters 
of this species, and photos of the holotype. 
Coan et al. (2000) considered this species as 
a synonym of K. suborbicularis. A study of a 
large quantity of K. comandorica, which is a 
common mollusk in the Commander Islands, 
has clearly shown that it is a well-identiftable, 
separate species of Kellia. The goal of this 
paper is to describe the new species and ex- 
pand the description of K. comandorica, with 
new data on its morphology, ecology, and geo- 
graphical distribution. 



MATERIAL AND METHODS 

In this study I used the material collected by 
the joint expeditions of IMB-PRIFO in the 
subtidal zone of the Kuril Islands (the sealer 
"Krylatka", September-October 1969) and 
Commander Islands (the sealer "Krylatka", 
July 1972; RV "Rakitnoe", August-October 
1973) and the expedition of 1MB in the inter- 
tidal zone of the Commander Islands (June- 
August 1 972). The material of the new species 
and of K. comandorica from the subtidal zone 
of the Commander Islands was fixed and 
stored in 70% ethanol in 1MB. Material of K. 



57 



58 



KAMENEV 



comandoríca from the Kuril Islands and the 
intertidal zone of the Commander Islands was 
fixed in 70% ethanol and stored dry in 1MB. 

For comparison purposes, collections of K. 
suborbicularis - 88 specimens from the North 
Atlantic (CAS, NHM, NMW) and more 300 
specimens from the northeastern Pacific 
(CAS, UW); of Kellia japónica Pilsbry, 1895 
- 2 specimens from Japan (NSMT Mo 73530) 
and 16 specimens from the Pacific seas of 
Russia (MIMB); of Kellia porculus Pilsbry, 
1904 - 1 specimen from Japan (NSMT Mo 
73531); and of Kellia subrotundata (Dunker, 
1882) - 1 specimen from (NSMT Mo 73532) 
were used. All material of these species was 
stored dry. 

Shell Measurements 

Figure 1 shows the shell morphology mea- 
surements. Shell length (L), anterior end length 
(A), height (H), width (W) (not shown) were 
measured for each valve. The ratios of these 
parameters to shell length (A/L, H/L, W/L, re- 
spectively) were determined. Shell measure- 
ments were made using a caliper and an ocular 
micrometer with an accuracy of 0.1 mm. 
The following material was measured: 

(1) 85 specimens, 1 right and 5 left valves of 
К comandorica from Urup Island, Kuril Is- 
lands, (MIMB, 15 specimens, 1 right, 5 left 
valves) and the Commander Islands 
(MIMB, 1MB, 70 specimens). 

(2) 97 specimens of the new species from the 
Commander Islands (1MB). 

(3) 44 specimens of K. suborbicularis from the 
North Atlantic: Weymouth, Dorset, Sea area 
16, United Kingdom (NMW 1953.183, 24 
specimens); Tenby, Pembrokeshire, Sea 
area 21, United Kingdom (NMW 1953.183, 
9 specimens); Guernsey, Channel Is., Sea 
area 17, United Kingdom (NMW 1953.183, 
3 specimens); Plymouth, United Kingdom 
(NHM 20030382, 2 specimens); Isle of 
Herm, Guernsey, United Kingdom (NHM 
20030383, 2 specimens); England (CAS 

165845, 2 specimens); England (CAS 

165846, 2 specimens). 

(4) 21 specimens of K. suborbicularis from the 
northeastern Pacific: Monterey Bay, Cali- 
fornia (CAS 161254, 8 specimens); Oreas 
Island, San Juan Islands, San Juan County, 
Puget Sound, Washington (CAS 161256, 
7 specimens); Alaska (CAS 161255, 6 
specimens). 



Statistics 

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

The calculated indices (A/L; H/L; W/L) are 
less susceptible to change compared with 
other measured parameters. Therefore, the 
statistical analysis was performed using only 
these characteristics. All data was tested with 
a Kolmogorov test for their fit to a normal dis- 
tribution. The distribution of some indices was 
different from the norm. Therefore all analy- 
ses were performed on log^^ transformations 
of the original variables. All indices for pairs of 
different valves of Kellia species were com- 
pared using the Student (T) parametric test and 
one-way analysis of variance (ANOVA). 

Throughout this study, statistical significance 
was defined as P < 0.05. 

Abbreviations 

The following abbreviations are used in the 
paper: CAS - California Academy of Sciences, 
San Francisco; 1MB - Institute of Marine Biol- 
ogy, Russian Academy of Sciences, Vladivostok; 
MIMB - Museum of the Institute of Marine Biol- 
ogy, Vladivostok; NHM - The Natural History 
Museum, London; NMW - National Museums 
& Galleries of Wales, Cardiff; NSMT - National 




FIG. 1 . Placement of shell measurements: L - shell 
length; H - height; A - anterior end length. 



KELLIA IN THE COMMANDER ISLANDS 



59 



Science Museum, Tokyo; PRIFO - Pacific Re- 
search Institute of Fisliehes and Oceanograpliy, 
Vladivostok; UW - University of Washington, 
Seattle; ZIN- Zoological Institute, Russian Acad- 
emy of Sciences, St.-Petersburg. 



Type Material and Locality 

Holotype (ZIN 9372), Commander Islands, 
Coll. E. F. Gurjanova, 1930 (Scarlato, 1981). 

Material Examined 



SYSTEMATICS 

Family Lasaeidae Gray, 1842 
Genus Kellia Turton, 1822 

Type species: Mya suborbicularis Montagu, 1803 

Diagnosis 

Shell small (< 30 mm), thin, ovate to globu- 
lar, inflated, inequilateral, equivalve. Surface 
with growth lines. Periostracum thin, adher- 
ent, colorless, gray, green to yellow. Beaks 
prosogyrate, almost central. Hinge plate nar- 
row. Right valve with one cardinal tooth and 
posterior lateral tooth; left valve with two car- 
dinal teeth and posterior lateral tooth. Liga- 
ment internal, partly lodged in a lanceolate 
resilifer, situated between cardinal and lateral 
teeth. Palliai line without palliai sinus. 

Kellia comandorica Scarlato, 1981 
(Figs. 2-20, Table 1) 

Kellia comandorica Scarlato, 1981: 321, pis. 

284 (holotype), 285. 
Kellia suborbicularis (Montagu, 1803), Coan 

et al., 2000: 323 (partim). 



16 lots (MIMB 2989, 2990, 2992, 2994, 
2996-3000, 3047-3050, 3052, 3053, 3055) 
from the tidal zone of Urup Island, Kuril Is- 
lands (15 specimens, 1 right, 5 left valves); 63 
lots (MIMB 2993, 3001, 3002, 3052, 3054, 
1MB) from the intertidal and tidal zones of the 
Commander Islands (131 specimens). Total of 
146 specimens, 1 right, and 5 left valves. 

Description (expanded from that of Scariato,1981) 

Exterior. Shell small (to 16.8 mm), ovate-an- 
gular, high (H/L = 0.735-0.976), equivalve, in- 
flated (W/L of valve 0.198-0.397), inequilateral, 
thin, solid. Surface with conspicuous, often 
rather rough growth lines. Periostracum thin, 
adherent, non-polished, colorless or gray, ex- 
tending into inner surface. Beaks small, mod- 
erately projecting above dorsal margin, slightly 
anterior to midline (sometimes central) (A/L = 
0.314-0.5), rounded, prosogyrate. Anterior and 
posterior ends rounded. Anterodorsal margin 
slightly convex, gently descending ventrally, 
smoothly transitioning to slightly curved ante- 
rior margin. Ventral margin slightly curved. 
Posterodorsal margin slightly convex, rather 
steeply descending to rounded posterior mar- 
gin. 



TABLE 1. Kellia comandorica Scarlato, 1981. Summary statistics of the shell measurements (mm) 
and indices: L - shell length; A - anterior end length; H - height; W - width. Numerator indicates the 
summary statistics for the right valve, denominator - for the left valve. 



Statistics 


L 


A 


H 


W 


A/L 


H/L 


W/L 


Mean 


9.31 


4.26 


7.91 


2.47 


0.458 


0.846 


0.264 




9.21 


4.21 


7.82 


2.47 


0.458 


0.846 


0.265 


SD 


0.33 


0.15 


0.29 


0.10 


0.003 


0.005 


0.003 




0.32 


0.15 


0.28 


0.10 


0.003 


0.005 


0.003 


SE 


3.04 


1.41 


2.72 


0.92 


0.027 


0.049 


0.031 




2.99 


1.39 


2.69 


0.93 


0.027 


0.048 


0.030 


Min 


3.4 


1.6 


2.5 


0.9 


0.314 


0.735 


0.198 




3.4 


1.6 


2.5 


0.9 


0.314 


0.735 


0.198 


Max 


16.8 


7.8 


15.2 


5.1 


0.500 


0.976 


0.389 




16.8 


7.8 


15.2 


5.2 


0.500 


0,976 


0.397 


n 


86 


86 


86 


86 


86 


86 


86 




90 


90 


90 


90 


90 


90 


90 



60 



KAMENEV 



Interior. Right valve with one cardinal tooth 
and posterior lateral tooth; left valve with two 
cardinal teeth and posterior lateral tooth. In 
right valve, cardinal tooth large, elongate, flat- 
tened, with a flat top, anteroventrally directed, 
situated at edge of inner part of anterodorsal 
shell margin; posterior lateral tooth large, long, 
extending along posterodorsal shell margin. 



In left valve, anterior cardinal tooth large, elon- 
gate, flattened, often triangular, anteroventrally 
directed, situated at edge of inner part of 
anterodorsal shell margin; posterior cardinal 
tooth small, rounded, isolated, fingerlike, with 
rounded top, situated exactly under beak; pos- 
terior lateral tooth large, long, extending along 
posterodorsal shell margin. Internal ligament 




FIGS. 2-10. Kellia comandorica Scarlato, 1981. 2-4: MIMB (3002), Gladky Cape, Medny Island, 
Commander Islands, intertidal zone, shell length 16.4 mm. 5: MIMB (2994), Lidina Cape, Urup Is- 
land, Kuril Islands, 20 m, shell length 16.0 mm. 6: Poludennaya Bight, Medny Island, Commander 
Islands, 20 m, shell length 11.8 mm. 7; Poludennaya Bight, Medny Island, Commander Islands, 20 
m, shell length 12.2 mm. 8: Peschany Cape, Medny Island, Commander Islands, shell length 13.1 
mm. 9: MIMB (2998), Van-der-Linda Cape, Urup Island, Kuril Islands, 10 m, shell length 14.7 mm. 10: 
Polovina Cape, Bering Island, Commander Islands, 5 m, dorsal view of both valves of a young speci- 
men. Bar = 1 mm. 



КЕША IN THE COMMANDER ISLANDS 



61 




FIGS. 11-20. Kellia comandorica Scarlato, 1981. 11-14: Peschany Cape, Medny Island, Commander 
Islands, 15 m. 11, 12: Right and left valves of an adult specimen. 13, 14: Hinge of right and left valves. 
15-20. Phedoskina Cape, Bering Island, Commander Islands, 5 m. 15, 16: Right and left valves of a 
young specimen. 17, 18: Hinge of right and left valves. 19, 20: Ventral view of hinge of right and left 
valves showing resilifer. Bar = 500 цт. 



62 



KAMENEV 



well-developed, large, situated between car- 
dinal and lateral teeth, posteriorly directed, 
partly lodged in lanceolate resilifer extending 
obliquely posterior to beaks. Anterior adduc- 
tor muscle scar large, rounded; posterior 
muscle scar large, ovate-angular, longer and 
wider than anterior scar. Palliai line without 
palliai sinus. Shell interior with conspicuous 
radial rows of fossae extending to palliai line. 



ally the beaks are anteriorly placed but some- 
times they occupy the central position. The 
sizes and shape of cardinal and lateral teeth in 
both valves vary little. All investigated speci- 
mens, independent of the age, habitat, and 
geographic area, had conspicuous radial rows 
of fossae on the inner shell wall. 

Distribution and Habitat (Fig. 21) 



Variability 

Shell shape and proportions, as well as width 
of the valves vary markedly (Table 1 , Figs. 5- 
7). The shell shape varies from ovate-elongate 
with relatively small shell height to rounded with 
height almost equal to shell length. The shell is 
most often slightly angular but sometimes it is 
regularly ovate without angles. The specimens 
frequently have a deformed shell because of 
living in small holes and crevices of boulders 
and rocky platforms, preventing normal growth. 
The position of the beaks is also variable. Usu- 



Kellia comandorica occurs near the Com- 
mander Islands and Urup Island (Kuril Islands). 
Near Bering Island and Medny Island (Com- 
mander Islands), K. comandorica is a com- 
mon species of the bottom fauna. It was 
recorded from the intertidal zone to 20 m 
depth, on boulders and rocky platforms, at a 
bottom temperature from 4.0 to 10.2°C, with 
population density to 170 specimens/m^. Near 
Urup Island (Kuril Islands) this species was 
found at depth from 5 to 20 m, on boulders 
and rocky platforms, with population density 
to 40 specimens/m^. 







140° 150° 


^^jr 


- 60° 


RUSSIA / 

/ / 

у Sea of Okhotsk 


/If 

/ Bering Sea 




Où 


Commander Islands 

Г 


- 50° 


(] \ ' ^ 

J In 

/ ¡Л ^ .J 
J Ч " yf Ump Is. 

y ^JAPANlJ " 


J 

50° - 

Pacific Ocean 


Л 


1 L _. 


160° 



FIG. 21. Distribution of Kellia comandorica, Scarlato 1981. 



KELLIA IN THE COMMANDER ISLANDS 



63 




FIGS. 22-39. Shells of Kellia species. 22-27. Kellia suborbicularis (Montagu, 1803) from the north- 
eastern Pacific. 22-25: CAS (161254), Monterey Bay, California, 18-22 m, shell length 26.1 mm. 
26-27: CAS (1 61 255), Alaska, left valve, shell length 1 3.8 mm. 28-33. Kellia suborbicularis (Montagu, 
1803) from the North Atlantic. 28-29: NMW (1953.183), Tenby, Pembrokeshire, Sea area 21 , United 
Kingdom, right valve, shell length 11.7 mm. 30-31: NHM (20030382), Plymouth, United Kingdom, 
right valve, shell length 9.0 mm. 32-33: CAS (165845), England, right valve, shell length 8.9 mm. 
34-35: Kellia japónica Pilsbry, 1895, NSMT (Mo 73530), Nagashima, Mie Prefecture, Japan, right 
valve, shell length 11.5 mm. 36-37: Kellia subrotundata (Dunker, 1882), NSMT (Mo 73532), Nosappu 
Cape, Hokkaido, Japan, right valve, shell length 11.4 mm. 38-39: Kellia porculus Pilsbry, 1904, NSMT 
(Mo 73531), Ushimado, Okayamo Prefecture, Seto Inland Sea, Japan, right valve, shell length 8.3 mm. 



64 



KAMENEV 



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KELLIA IN THE COMMANDER ISLANDS 



65 



Comparisons 



Remarks 



Kellia comandorica is easily distinguished 
from other species of this genus by its shell 
with radial rows of fossae on the inner wall 
(Table 2). Moreover, K. comandorica differs 
from Kellia kussakini by its larger, lower, and 
less inflated ovate-angular shell with anteri- 
orly placed beaks. 

In shell shape and proportion K. coman- 
dorica is similar to K. suborbicularis from the 
North Atlantic. However, with the exception of 
the above-mentioned distinguishing character, 
in contrast to K. suborbicularis from the North 
Atlantic, this species has a less inflated shell 
with the non-polished periostracum and less 
anteriorly placed beaks (Table 1, 3). Mean 
values and variances of the indices charac- 
terizing the position of beaks (A/L) and the 
relative width (W/L) were significantly differ- 
ent in these species (Table 4, Figs. 28-33). 

Unlike K. comandorica, the shell of K. 
suborbicularis from the northeastern Pacific is 
non-angular, rounded-ovate or ovate-elongate 
with faint growth lines and the polished yellow 
or grayish-yellow periostracum, interiorly 
smooth, sometimes with the faint radial striae 
especially noticeable along the ventral shell 
margin (Tables 1, 2, 4, 5, Figs. 22-27). 

In contrast to K. japónica, К. porculus and K. 
subrotundata, K. comandorica has a less inflated 
shell with a non-polished gray periostracum, 
conspicuous growth lines, and less anteriorly 
placed beaks (Table 2, Figs. 34-39). 



In the northwestern Pacific, the Russian and 
Japanese malacologists recognize another 
species K. japónica (Scarlato, 1981; Kafanov, 
1 991 ; Okutani, 2000), which Coan et al. (2000) 
also considered a synonym of K. suborbi- 
cularis. However, the very wide geographic 
distribution of K. suborbicularis may suggest 
that it is a composite that includes several 
species (Scarlato, 1981 ). The placement of K. 
japónica in synonymy with K. suborbicularis 
further extends the range of K. suborbicularis. 

The study of different-age individuals (from 
0.3 mm to 1 1 .7 mm) of K. suborbicularis from 
the North Atlantic showed that the shell shape 
varies from ovate-elongate to globular (Figs. 
28-33). However, the proportion of speci- 
mens with the ovate-elongate shell was small. 
On the whole, in comparison with K. sub- 
orbicularis from the northeastern Pacific, in- 
dividuals of this species from the North 
Atlantic have a relatively small (< 12 mm), 
markedly more rounded, higher, and more in- 
flated shell with less anteriorly placed beaks 
and a slightly polished yellowish-gray 
periostracum (Tables 3, 5). Mean values and 
variances of the indices characterizing the 
position of beaks (A/L), the relative height (H/ 
L) and width (W/L) in K. suborbicularis from 
the North Atlantic were significantly different 
from the mean values and variances of the 
same indices of this species from the north- 
eastern Pacific (Table 4). 



TABLE 3. Kellia suborbicularis (Montagu, 1803) from the North Atlantic (NMW 1953.183; NHM 
20030382, 20030383; CAS 165845, 165846). Summary statistics of the shell measurements (mm) 
and indices: L - shell length; A - anterior end length; H - height; W - width. Numerator indicates the 
summary statistics for the right valve, denominator - for the left valve. 



Statistics 


L 


A 


H 


W 


A/L 


H/L 


W/L 


Mean 


6.91 


3.02 


5.92 


2.12 


0.443 


0.857 


0.303 




6.92 


3.02 


5.92 


2.13 


0.442 


0.857 


0.304 


SD 


0.32 


0.12 


0.27 


0.11 


0.005 


0,006 


0.005 




0.32 


0.12 


0.27 


0.12 


0.005 


0.006 


0.005 


SE 


2.11 


0.81 


1.80 


0.75 


0.033 


0.037 


0.035 




2.11 


0.81 


1.80 


0.77 


0.034 


0.037 


0.036 


Min 


2.7 


1.2 


2.1 


0.6 


0.346 


0.776 


0.211 




2.7 


1.2 


2.1 


0.6 


0.337 


0.776 


0.211 


Max 


11.7 


4.6 


9.4 


3.8 


0.500 


0.944 


0.367 




11.7 


4.6 


9.4 


3.8 


0.500 


0.944 


0.367 


n 


44 


44 


44 


44 


44 


44 


44 




44 


44 


44 


44 


44 


44 


44 



66 



KAMENEV 



TABLE 4. Results of comparison by pairs of mean values (Student (T) test) and variances (ANOVA) 
of indices of the right and left valves of Kellia comandorica, K. kussakini, and K. suborbicularis: L - 
shell length; A - anterior end length; H - height; W - width; P - probability that index values in K. 
comandorica, K. kussakini, and K. suborbicularis are drawn from the same population; n - number of 
valves of compared species, respectively; * - significant difference. 



Indices 



A/L* 
H/L 
W/L* 

A/L* 
H/L* 
W/L 

A/L* 
H/L* 
W/L* 

A/L* 
H/L* 
W/L* 

A/L* 
H/L* 
W/L* 

A/L* 
H/L* 
W/L* 



Right valves 



Left valves 



K. comandorica and K. suborbicularis (North Atlantic) 

0.006 7.66 0.006 86/44 -2.73 0.004 8.75 0.004 

0.080 1.77 0.187 86/44 1.44 0.008 1.76 0.187 

< 0.001 42.69 < 0.001 86/44 6.23 < 0.001 43.38 < 0.001 

K. comandorica and K. suborbicularis (Northeastern Pacific) 

< 0.001 48.43 < 0.001 86/21 8.43 < 0.001 51.50 < 0.001 

< 0.001 10.62 0.002 86/21 3.87 < 0.001 11.24 0.001 
0.205 0.47 0.492 86/21 0.87 0.196 0.49 0.486 

K. kussakini and K. comandorica 



-2.60 
1.45 
6.24 



8.23 
3.78 
0.83 



13.46 
11.02 
15.99 



-12.39 
-8.88 
-5.79 

18.60 
11.88 
13.55 

K. 

4.34 
4.82 
6.09 



< 0.001 178.21 <0.001 86/97 13.59 < 0.001 179.79 < 

< 0.001 125.47 <0.001 86/97 11.25 < 0.001 128.38 < 

< 0.001 252.99 <0.001 86/97 16.04 < 0.001 253.16 < 

K. kussakini and K. suborbicularis (North Atlantic) 

< 0.001 160.41 < 0.001 97/44 -12.26 < 0.001 159.50 < 

< 0.001 76.69 < 0.001 97/44 -8,85 < 0.001 79.36 < 

< 0.001 35.06 < 0.001 97/44 -5.65 < 0.001 33.44 < 

K. kussakini and K. suborbicularis (Northeastern Pacific) 

< 0.001 205.68 < 0.001 97/21 18.60 < 0.001 205.68 < 

< 0.001 147.89 < 0.001 97/21 11.89 < 0.001 148.39 < 

< 0.001 111.36 < 0.001 97/21 13.80 < 0.001 109.01 < 
suborbicularis (North Atlantic) and K. suborbicularis (Northeastern 

< 0.001 13.74 < 0.001 44/21 4.24 < 0.001 12.95 < 

< 0.001 23.61 < 0.001 44/21 4.84 < 0.001 23.76 < 

< 0.001 27.30 < 0.001 44/21 6.16 < 0.001 27.03 < 



0.001 
0.001 
0.001 



0.001 
0.001 
0.001 



0.001 
0.001 
0.001 

Pacific) 

0.001 
0.001 
0.001 



90/44 
90/44 
90/44 

90/21 
90/21 
90/21 

86/97 
86/97 
86/97 

97/44 
97/44 
97/44 



97/21 
97/21 
97/21 

44/21 
44/21 
44/21 



Most specimens of K. suborbicularis from the 
northeastern Pacific had a ovate-elongate, 
markedly less inflated shell, reaching more 
than 26 mm in length, with more anteriorly 
placed beaks and yellow, strongly polished 
periostracum (Figs. 22-27, Table 5). The 
specimens with an analogous shell shape 
(more 20 mm in length) were also recorded 
off the Kuril Islands (materials from the expe- 
dition with the R/V "Akademik Oparin", 1 July 
- 4 August 2003). All these morphological char- 
acteristics are observed in Kellia laperousii 
(Deshayes, 1839) (Oldroyd, 1925; Scartato, 
1981), which Coan et al. (2000) also consid- 
ered to be a synonym of K. suborbicularis. 
They stated that no consistent difference from 
the North Atlantic K. suborbicularis can be 
found when similar-sized specimens are com- 



pared. However, a detailed study of this ques- 
tion is needed. Taking into account the results 
of comparison of specimens of K. suborbicula- 
ris from the North Atlantic and the northeast- 
ern Pacific, I think that most likely a separate 
species K. laperousii occurs in the North Pa- 
cific. 

The species of the genus Kellia - K. 
japónica, К. porculus and K. subrotundata - 
living off the coast of Japan (Okutani, 2000), 
are most similar to K. suborbicularis in shape 
and shell proportions (Tables 2, 3, Figs. 28- 
39). In addition, the species from Japan are 
also very similar to each other. Some differ- 
ences between these species exist in the de- 
gree of inflation of the shell, the position and 
form of the beaks, and the color of the 
periostracum (Table 2). However, the differ- 



KELLIA IN THE COMMANDER ISLANDS 



67 



TABLE 5. Kellia suborbicularis (Montagu, 1803) from the northeastern Pacific (CAS 1612564, 161255, 161256). 
Sumnnary statistics of the shell measurements (mm) and indices: L - shell length; A- anterior end length; H - 
height; W - width. Numerator indicates the summary statistics for the right valve, denominator - for the left valve. 



Statistics 


L 


A 


H 


W 


A/L 


H/L 


W/L 


Mean 


15.56 


6.40 


12.55 


4.06 


0.414 


0.809 


0.259 




15.56 


6.40 


12.55 


4.08 


0.414 


0.809 


0.260 


SD 


0.97 


0.37 


0.75 


0.30 


0.004 


0.008 


0.005 




0.97 


0,37 


0.75 


0.30 


0.004 


0.008 


0,005 


SE 


4.47 
4.47 


1.70 
1.70 


3.44 
3.44 


1.36 
1.37 


0.020 
0.020 


0.038 
0.038 


0.023 
0.021 


Min 


9.4 


4.2 


7.3 


2.0 


0.380 


0.743 


0.213 




9.4 


4.2 


7.3 


2.0 


0.380 


0.743 


0.213 


Max 


26.1 


10.4 


19.4 


8.2 


0.463 


0.873 


0.314 




26.1 


10.4 


19.4 


8.2 


0.463 


0.873 


0.314 


n 


21 


21 


21 


21 


21 


21 


21 




21 


21 


21 


21 


21 


21 


21 



ence in shell proportions can be attributed to 
the individual variability of one species. Be- 
cause only a very limited material on Kellia 
species from Japan was at my disposal, no 
final conclusion can be made. It is not improb- 
able that after a detailed study of more exten- 
sive material, these species prove to be a 
synonym of one species different from K. 
suborbicularis or K. laperousii. 

Kellia kussakini Kamenev, new species 

Figs. 40-53, Table 6 

Type Material and Locality 

Holotype (MIMB 7770), Phedoskina Cape, 
Bering Island, Commander Islands, Pacific 
Ocean, 5 m, rocky platform, bottom water tem- 
perature of 1 0.0°C, Coll. V. I. Lukin, 23-IX-1 973 
(RV "Rakitnoye"); paratypes (96) (MIMB 7771 ) 
from holotype locality. 

Other Material Examined 

38 specimens with slightly damaged shells 
from type locality; 1 specimen from Najushka 
Bight, Bering Island, Commander Islands, 
Pacific Ocean, 20 m, rocky platform, bottom 
water temperature of 9.8°C, Coll. G. T Beloko- 
nev, 24-IX-1973 (RV "Rakitnoye"); 1 specimen 
from Nerpichy Cape, Bering Island, Com- 
mander Islands, Pacific Ocean, 10 m, rocky 
platform, bottom water temperature of 7.4°C, 
Coll. V. I. Lukin, 21-VII-1972 (the sealer 
"Krylatka"); 2 specimens from Peschany Cape, 



Medny Island, Commander Island, Bering Sea, 
10 m, boulders, bottom water temperature 
of 5. OX, Coll. V. P. Kashenko, 10-VII-1972 
(the sealer "Krylatka"); 5 specimens from 
Poludennaya Bight, Medny Island, Com- 
mander Island, Pacific Ocean, 15 m, rocky 
platform, bottom water temperature of 4.2°C, 
Coll. V. I. Lukin, 17-VII-1972 (the sealer 
"Krylatka"); 1 right valve from Vodopadsky 
Cape, Medny Island, Commander Islands, 
Pacific Ocean, 20 m, rocky platform, bottom 
water temperature of 7.6°C, Coll. G. T. 
Belokonev, 11-IX-1973 (RV "Rakitnoye"). To- 
tal of 47 specimens and 1 right valve. 

Description 

Exterior. Shell very small (to 4.8 mm), pear- 
shaped, with slightly narrowing anterior end, 
almost globular, very high (H/L = 0.833-1 .036, 
shell height almost equal to or sometimes 
greater than length), equivalve, very inflated 
(W/L of valve = 0.278-0.429, shell width al- 
most equal to length), inequilateral, thin, frag- 
ile, translucent. Surface with faint growth lines. 
Periostracum thin, adherent, slightly polished, 
yellowish-gray, extending into inner surface. 
Beaks small, high, strongly projecting above 
dorsal margin, slightly posterior, sometimes 
central or slightly anterior (A/L = 0.371-0.579), 
rounded, prosogyrate. Anterior end slightly 
narrowed, rounded, lower than posterior shell 
end. Posterior end rounded. Anterodorsal 
margin slightly convex, rather steeply de- 
scending ventrally, smoothly transitioning to 



68 



KAMENEV 



strongly curved anterior margin. Ventral mar- 
gin strongly curved. Posterodorsal margin 
slightly convex, steeply descending ventrally, 
smoothly transitioning to curved posterior 
margin. 

Interior. Right valve with one cardinal tooth 
and posterior lateral tooth; left valve with two 
cardinal teeth and posterior lateral tooth. In 
right valve, cardinal tooth large, elongate, flat- 
tened, with rounded or flat top, anteroventrally 



directed, situated at edge of inner part of 
anterodorsal shell margin; posterior lateral 
tooth large, long, extending along postero- 
dorsal shell margin. In left valve, anterior car- 
dinal tooth larger, than posterior, elongate, 
flattened, with rounded top, anteroventrally 
directed, situated at edge of inner part of 
anterodorsal shell margin; posterior cardinal 
tooth smaller, rounded, isolated, fingerlike, 
with rounded top, situated exactly under 
beak; posterior lateral tooth large, long, ex- 




FIGS. 40-48. Kellia kussakini Kamenev, new species. 40, 41: Holotype (MIMB 7770), Phedoskina 
Cape, Bering Island, Commander Islands, Pacific Ocean, 5 m. 42-48: Paratypes (MIMB 7771) from 
holotype locality. 42: Dorsal view of both valves. 43, 44: Right and left valves of an adult specimen. 45, 
46: Right and left valves of a young specimen. 47, 48: Right and left valves without ligament. Bar = 1 mm. 



КЕША IN THE COMMANDER ISLANDS 



69 



tending along posterodorsal shell margin. 
Internal ligament well developed, large, situ- 
ated between cardinal and lateral teeth, pos- 
teriorly directed, partly lodged in lanceolate 
resilifer extending obliquely posterior to 
beaks. Anterior adductor muscle scar large, 
rounded; posterior muscle scar large, ovate- 
angular, longer and wider than anterior scar. 
Palliai line without palliai sinus. Shell interior 
smooth, without radial rows of fossae or 
striae. 

Variability 

Shell shape and proportions change little with 
age. In young specimens (< 2.5 mm), the shell is 
less high, less inflated, and beaks are more pos- 
teriorly placed. In adults, shell height and width, 
the position of beaks vary slightly (Table 6, Figs. 
43-46). As a whole, shell remains pear-shaped, 
with more narrow anterior end, and almost globu- 
lar because of almost absolute equality of the 
length, height, and width. The beaks are mostly 



posteriorly placed (the beaks of 8 out of 97 mea- 
sured specimens are anteriorly placed). The sizes 
and shape of cardinal and lateral teeth in both 
valves vary little. A few specimens had three car- 
dinal teeth in the left valve (Fig. 53). 

Distribution and Habitat (Fig. 54) 

This species was recorded near Bering and 
Medny islands. Commander Islands, at a 
depth from 5 to 20 m, on boulders and rocky 
platforms, at a bottom water temperature from 
4.2°C to 10.0°C, with population density to 
1,190 specimens/m^ (type locality). 

Comparisons 

This species is easily distinguished from 
other species of this genus by its small, al- 
most globular, very high, and inflated shell with 
posteriorly placed high beaks (Table 2). Mean 
values and variances of the indices charac- 
terizing the position of beaks (A/L), the rela- 




FIGS. 49-53. Kellia kussakini Kamenev, new species, paratypes (MIMB 7771) from holotype locality. 
49, 50: Hinge of right and left valves. 51, 52: Ventral view of hinge of right and left valves showing 
resilifer. 53: Hinge of left valves with three cardinal teeth. Bar = ЗООцт. 



70 



KAMENEV 



'^^ 



Bering Sea '^ 



Commander Islands 



166 ° 



167' 



RUSSIA 



55' 





Bering Sea 




Pacific Ocean 



166 



167 



55°. 




168' 



FIG. 54. Distribution of Kellia kussakini 



type locality). 



TABLE 6. Kellia kussakini Kamenev, new species. Sinei! measurements (mm), indices of holotype 
(MIMB 7770), and summary statistics of holotype and paratypes (MIMB 7771) characters: L - shell 
length; A - anterior end length; H - height; W - width. Numerator indicates shell measurements, 
indices, and summary statistics for the right valve, denominator - for the left valve. 



Statistics 


L 


A 


H 


W 


A/L 


H/L 


W/L 










Holotype 










4.0 


2.1 


3.8 


1.4 


0.525 


0.950 


0.350 




4.0 


2.1 


3.8 


1.4 


0.525 


0.950 


0.350 








Holotype and paratypes 






Mean 


2.95 


1.52 


2.71 


1.01 


0.517 


0.917 


0.340 




2.95 


1.52 


2.71 


1.01 


0.517 


0.917 


0.340 


D 


0.06 


0.03 


0.06 


0.03 


0.003 


0.004 


0.003 




0.06 


0.03 


0.06 


0.03 


0.003 


0.004 


0.003 


SE 


0.60 


0.29 


0.59 


0.27 


0.027 


0.037 


0.034 




0.60 


0.29 


0.59 


0.27 


0.027 


0.037 


0.034 


Min 


1.4 


0.8 


1.2 


0.4 


0.444 


0.833 


0.278 




1.4 


0.8 


1.2 


0.4 


0.444 


0.833 


0.278 


Max 


4.8 


2.6 


4.6 


1.8 


0.579 


1.036 


0.429 




4.8 


2.6 


4.6 


1.8 


0.579 


1.036 


0.429 


n 


97 


97 


97 


97 


97 


97 


97 




97 


97 


97 


97 


97 


97 


97 



KELLIA IN THE COMMANDER ISLANDS 



71 



tive height (H/L) and width (W/L) in K. kussakini 
were significantly different from the mean val- 
ues and variances of the same indices of K. 
comandorica and K. suborbicularis (Table 4). 
Besides, unlike K. comandorica, it has a 
smooth interior shell wall without the radial 
rows of fossae. 

In shell shape, K. kussakini is close to K. 
porculus (Figs. 38, 39), living off the coast of 
Japan, from which it is distinguished in having a 
more high, very inflated, fragile and translucent 
shell with posteriorly placed beaks and a slightly 
polished, yellowish-gray periostracum (Table 2). 

Etymology 

The specific name honors Oleg G. Kussakin, 
Academician of the Russian Academy of Sci- 
ences, a famous Russian researcher of the 
marine fauna of the intertidal zone of Russian 
Pacific seas and world isopod fauna, who de- 
voted all his life to the study of the northwest- 
ern Pacific fauna. 



ACKNOWLEDGMENTS 

I am very grateful to Dr. V. A. Nadtochy 
(PRIFO, Vladivostok) and Mrs. N. V. Kameneva 
(MIMB, Vladivostok) for great help during work 
on this manuscript; to Professor T W. Pietsch 
and Dr. K. Stiles (UW, Seattle) for arrangement 
of my visit to the UW and work with the bivalve 
mollusk collection, for all-round, very kind, and 
friendly help during my life and work in Seattle; 
to Professor A. J. Kohn and Dr. G. Jensen (UW, 
Seattle) for great help during work with the bi- 
valve mollusks collection at the UW; to Mr. Gary 
Cook (Berkeley) for all-round, very kind and 
friendly help during my life and work in San Fran- 
cisco; to Dr. P. D. Roopnahne and Miss E. Kools 
(Department of Invertebrate Zoology, CAS, San 
Francisco) for arrangement of my work with the 
bivalve mollusks collection at the CAS and great 
help during this work; to Dr. H. Saito (NSMT, 
Tokyo) for sending the specimens of K. japónica, 
К. porculus, and K. subrotundata; to Dr. Gra- 
ham Oliver, Ms. Harriet Wood (NMW, CardifO, 
Dr. David G. Reid, and Mrs. Amelia MacLellan 
(NHM, London) for sending the specimens of 
K. suborbicularis from the North Atlantic; to Dr. 
E. V. Coan (Department of Invertebrate Zool- 
ogy, CAS, San Francisco) for consultations, help 
in communication with other specialists, and 
comments on the manuscript; to Professor G. 
J. Vermeij (University of California at Davis, 
Davis) for help in communication with other spe- 



cialists; to Mr. D. V. Fomin (1MB, Vladivostok) 
for help in work with the scanning microscope; 
to Ms. T N. Kaznova (1MB, Vladivostok) for help 
with translating of the manuscript into English; 
to Dr. George M. Davis for help in the publica- 
tion of the manuscript; to Dr. J. A. Allen for com- 
ments on the manuscript. 

This research was supported by Grant 01- 
04-48010 from the Russian Foundation for 
Basic Research. 



LITERATURE CITED 

BOROVIKOV, V. P. & I. R BOROVIKOV, 1997, 
"STATISTICA". Statistical analysis and 
processing of data using "WINDOWS". Filin 
Press, Moscow, 608 pp. [in Russian]. 

COAN, E. v., R H. SCOTT & F R. BERNARD, 
2000, Bivalve seashells of western North 
America. Marine bivalve mollusks from Arctic 
Alaska to Baja California. Santa Barbara 
Museum of Natural History, 764 pp. 

KAFANOV, A. I., 1991, Shelf and continental 
slope bivalve molluscs of the northern Pacific 
Ocean: a check-list. Far Eastern Branch of the 
Academy of Sciences of the USSR, 
Vladivostok, 200 pp. [in Russian, with English 
summary]. 

KAMENEV, G. M., 1995, Species composition 
and distribution of bivalve mollusks on the 
Commander Islands shelf. Malacological 
Review, 28: 1-23. 

KAMENEV, G. M., 1996, Additional data on 
morphology and geographic distribution of 
Adontorhina cyclia Berry, 1947 (Bivalvia: 
Thyasiridae), newly reported from the 
northwestern Pacific. The Veliger, 39(3): 213- 
219. 

KAMENEV, G. M., 2002, Genus Parvithracia 
(Bivalvia: Thraciidae) with descriptions of a new 
subgenus and two new species from the 
northwestern Pacific. Malacologia, 44(1): 107- 
134. 

KAMENEV, G. M. & V. A. NADTOCHY, 2000, 
Mendicula ferruginosa (Forbes, 1844) 
(Bivalvia, Thyasiridae) from the Far Eastern 
Seas of Russia. Ruthenica, 10(2): 147-152. [in 
Russian, with English abstract]. 

OKUTANI, T, 2000, Marine mollusks in Japan, 
lokal University, Tokyo, Japan, xlvii + 1175 pp., 
incl. 542 pis. 

OLDROYD, I. S., 1925, The marine shells of 
the west coast of North America, Vol. 1 
[Bivalvia]. Stanford University, Publications, 
University Series, Geological Sciences, 1(1): 
247 pp., 57 pis. 

SCARLATO, O. A., 1981, Bivalve mollusks of 
temperate waters of the northwestern Pacific. 
"Nauka" Publ. House, Leningrad, 480 pp. [in 
Russian]. 



Revised ms. accepted 28 August 2003 



MALACOLOGIA, 2004, 46(1): 73-78 

REVISION OF THE REPRODUCTIVE MORPHOLOGY OF THREE 

LEPTAXIS SPECIES (GASTROPODA, PULMONATA, HYGROMIIDAE) 

AND ITS IMPLICATION ON DART EVOLUTION 

Joris M. Koene^ & Igor V. Muratov^ 

ABSTRACT 

Many species of land snails have one or more sharp, calcareous "love darts" that are 
used to stab the partner during mating. These darts are produced and stored in special- 
ized organs called stylophores. Because their number and position varies among species, 
stylophores are often used for identification and classification, especially in the family 
Hygromiidae. Having several stylophores, and thus several darts, is presumably the an- 
cestral state from which species with one stylophore evolved. Species with small acces- 
sory sacs or rudimentary stylophores located above the functional stylophore are therefore 
thought to represent intermediate forms between species with double and single stylophores. 
We investigated the stylophores, darts, and associated reproductive organs of three spe- 
cies of the hygromiid genus Leptaxis - L. erubescens, L nivosa and L. undata. In all the 
specimens of the investigated species, a small sac located just above the stylophore was 
found to be present. We conclude that this previously overlooked organ represents a rudi- 
ment of a stylophore, leading us to conclude that Leptaxis should be considered as an 
intermediate form in the evolution towards a single stylophore in the Hygromiidae. 

Keywords: love dart, dart sac, stylophore, snail, stylommatophora, Helicoidea, rudiment. 



INTRODUCTION 

In the reproductive system of many her- 
maphroditic land snail species, one or more 
sharp, calcareous structures are present. 
When these are produced in a specialized or- 
gan, the stylophore (also referred to as dart 
sac), they are called darts. In many species, 
these "love darts" are stabbed through the 
partner's skin during mating (Adamo & Chase, 
1990; Reyes Tur et al., 2000). In Helix aspersa 
(Müller, 1774) -often called Cornu aspersum, 
Cryptomphalus aspersus, or Cantareus 
aspersus - this "dart shooting" results in the 
transfer of an allohormone that inhibits sperm 
digestion and thereby increases sperm storage 
and fertilization success (Koene & Ter Maat 2001 ; 
Koene & Chase, 1 998a; Rogers & Chase, 2001 , 
2002; Landolfa et al., 2001; Landolfa, 2002). 

Recently, a comparative study demonstrated 
that the evolution of darts may be driven by 
sexual conflict (Koene & Schulenburg, submit- 
ted), thus explaining the diversity in number 
and shape of darts. For example, Trichia has 
two conical darts without blades (Schileyko, 



1 978a); Leptaxis and Hygromia each have one 
dart with two (differently arranged) blades (re- 
spectively: Spence, 1 91 1 ; Giusti & Manganelli, 
1 987); IHelix has a dart with four blades (Masse 
et al., 2002); and Monactioides has one dart 
with seven blades (Koene & Schulenburg, 
submitted). Some species, such as Cepaea 
nemoralis and С hortensis, which are other- 
wise remarkably similar, can most easily be 
distinguished by the shape of their darts 
(Kerney et al., 1983). Despite the large diver- 
sity in shapes, darts are rarely used for taxo- 
nomic purposes. Conversely, stylophores are 
traditionally used for identiflcation and classi- 
fication of land snails within the superfamily 
Helicoidea (Nordsieck, 1987; Schileyko, 
1 989). Species with one stylophore are thought 
to have evolved from ancestral species bear- 
ing several stylophores (Schileyko, 1989). 
When more than one stylophore is present, 
different arrangements are possible. Several 
stylophores can be arranged around the vagi- 
nal duct (e.g., Humboldtiana: Thompson & 
Brewer, 2000). Two pairs of stylophores can 
be present on opposing sites of the vaginal 



Taculty of Earth and Life Sciences, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands; 
joris.l<oene@falw.vu.nl 
^Department of Malacology, The Academy of Natural Sciences, Philadelphia, Pennsylvania, USA 



73 



74 



KOENE & MURATOV 



duct (e.g., Tñchia: Schileyko, 1978a). Alterna- 
tively, only one pair of stylophores can be 
present {Hygromia: Giusti & Manganelli, 1987). 
In these latter two cases, within each 
stylophore pair, the stylophore that is furthest 
away from the genital opening normally does 
not contain a dart, but see Taniushkin et al. 
(1999) for a possible case of atavistic devel- 
opment of darts in the upper stylophores of 
Xeropicta krynickii (Krynicki, 1833). 

There are also species with morphologies 
that clearly represent intermediate stages be- 
tween the above-mentioned forms. In such 
cases, a stylophore has become reduced in 
size and no longer produces a dart; such a 
rudimentary organ is then often referred to as 
an accessory sac (Nordsieck, 1993), internal 
dart sac (Giusti & Manganelli, 1987), or upper 
stylophore (Schileyko, 1989). Because these 
terms all describe the same organ, probably 



at different stages of reduction, we have cho- 
sen to use the term upper stylophore through- 
out the rest of this paper. Obviously, these 
intermediates provide important information 
about the course of evolution of the stylo- 
phore(s). Because rudimentary organs can be 
greatly reduced in size, they have sometimes 
been overlooked in previous studies. This is 
the case for the genus Leptaxis, which is why 
we redescribe the morphology of the 
stylophores, darts and associated reproduc- 
tive organs of three species of this genus. 



MATERIAL AND METHODS 

Species of the genus Leptaxis inhabit 
Macaronesia, which includes 32 islands 
grouped in five major archipelagos: Azores, 
Canaries, Cape Verde, Madeira, and Selvagens 




FIG. 1. Shells of the investigated Leptaxis species. 



LEPTAXIS DART EVOLUTION 



75 



(Mitchell-Thome, 1976). We have focused on 
the reproductive morphology of species en- 
demic to the Madeiran archipelago. Among 
these, Leptaxis erubescens (Lowe, 1831) is 
the only species of this genus that occurs on 
all the different island groups - Madeira, Porto 
Santo, and The Desertas - of this volcanic 
archipelago (Cook, 1996). The other Leptaxis 
species are confined to one of the island 
groups (Cook, 1996). Of these species we in- 
vestigated, L. undata (Lowe, 1831) from Ma- 
deira and L nivosa (Sowerby, 1 824) from Porto 
Santo. Figure 1 shows the shells of the inves- 
tigated species. 



Dry and alcohol preserved specimens of L. 
erubescens (N = 2) were obtained from the 
malacological collection of the Academy of 
Natural Sciences of Philadelphia (ANSP 
128459 A9427H). Several specimens (frozen 
at -80°C) of this species (N = 4), as well as of 
L. undata (N = 4) and L. nivosa (N = 5), were 
generously made available to us by P. Van Riel 
(Royal Belgian Institute of Natural Sciences, 
Brussels). 

The specimens of each species, which were 
all adult, were dissected to remove the repro- 
ductive tract. Subsequently, the reproductive 
organs were drawn using a camera lucida. To 



Leptaxis erubescens 



L. nivosa 





/:Z7 



L. undata 



0.2 cm 





ZIZZ 



0.2 cm 



FIG. 2. Comparison of the position and relative size of the upper and lower stylophores of Leptaxis 
erubescens, L. undata , and L. nivosa. The reproductive system of L. erubescens is shown to depict 
the other reproductive structures that are mentioned in the text. Abbreviations: A, appendage; AG, 
albumen gland; ВС, bursa copulatrix; ВТ, bursa tract; DG, digitiform gland; FL, flagellum; FPSC, 
fertilization pouch-spermathecal complex; G, genital pore; HD, hermaphroditic duct; LS, lower 
stylophore; P, penis; PRM, penis retractor muscle, SO, spermoviduct; US, upper stylophore; V, vaginal 
duct; VD, vas deferens. 



76 



KOENE & MURATOV 



avoid damage of the darts, the stylophores 
were carefully cut out of the reproductive tracts 
and placed overnight in 1N NaOH, which dis- 
solved all the tissue and mucus but left the 
dart intact. For cross-sections darts were care- 
fully broken in two. The intact and broken darts 
were consecutively prepared for electron mi- 
croscopy by placing them on small aluminium 
plates with an electrically conducting adhesive 
(Leit-Tab, Piano). Subsequently, they were 
coated with gold using a Metalloplan (Leitz). 
The darts were then placed under a scanning 
electron microscope (S-530 SEM, Hitachi) and 
photos were taken. 



RESULTS 

In all the mature specimens of each species, 
one large stylophore, containing one dart, was 
present. This stylophore was positioned in such 



a way that it curved slightly around the vaginal 
duct. Besides this stylophore, we also found a 
small sac situated between the larger stylophore 
and the vaginal duct in all species. The position 
of this organ suggests that we are dealing with 
the rudiment of an upper stylophore. Addition- 
ally, a flattened, non-hollow appendage at the 
base of the vagina is present. Figure 2 gives an 
overview of the morphology of the investigated 
Leptaxis species showing the positions and rela- 
tive sizes of the stylophores, the small sac (i.e., 
upper stylophore) and the appendage. The two 
mucus glands of each of the species are situ- 
ated above the stylophore around the vaginal 
duct. Each of these digitiform glands has sev- 
eral branches that join at the base. These glands, 
as well as the rest of the reproductive system, 
are only depicted for L. erubescens. 

The darts of all three species have a round 
base and a broad corona by which they are at- 
tached to a tubercle in the stylophore (Fig. 3). 





L. 


erubescens 






500 |ji 




^^тЁЕИЕЕЙЕЕЕЕЕШт 


Ы 






TI 


i 




L. undata 




L. 


nivosa 


Щ 


^W 


fm sà 


^ 


ш^ 




^ 


250 Mm 250 |jm 




г •"■* «^3 



FIG. 3. Electron microscopic pictures of the darts of Leptaxis 
erubescens, L undata, and L nivosa. 



LEPTAXIS DART EVOLUTION 



77 



Approximately halfway towards the tip of the 
dart the curved shaft broadens and flattens out, 
thus forming two large blades (Fig. 3). The dart 
is curved and lightly contorted, which is illus- 
trated by the electron microscopic picture of the 
side view of a dart of L. erubescens (Fig. 3), 
also reflecting the shape of the stylophore. 



DISCUSSION 

It has long been thought, based on morpho- 
logical data, that the genus Leptaxis fully con- 
forms to the European Hygromiidae with one 
stylophore (Mandahl-Barth, 1943; Backhuys, 
1975; Schileyko, 1989). Interestingly, Pilsbry 
(1894: 292-293) stated after having investi- 
gated several Leptaxis species: "I had ex- 
pected to find in Leptaxis some archaic 
characters preserved; for its geographic posi- 
tion and the shell-peculiarities argue for the 
group an ancient origin; but the evidence 
shows that however remote in the past the type 
was derived from the continental fauna, the 
main anatomical features of modern European 
Helices were then well established". Our find- 
ing of the small organ just above the stylophore 
in the investigated species of Leptaxis sug- 
gests that Pilsbry was correct in expecting 
some ancient characters. 

The position of the previously overlooked 
organ is consistent with the position of the 
upper stylophore in the genus Trichia 
(Schileyko, 1978a) and the internal or acces- 
sory dart sac in the genus Hygromia (Giusti & 
Manganelli, 1987; Nordsieck, 1987). There- 
fore, we conclude that the investigated spe- 
cies of the Leptaxis genus possess a rudiment 
of an upper stylophore. This rudiment has 
probably been overlooked for so long because 
the small organ is well hidden in connective 
tissue between the vaginal duct and the much 
larger functional stylophore that contains the 
contorted dart. Nevertheless, the presence of 
the upper stylophore in Leptaxis has impor- 
tant implications for the phylogenetic position 
of this genus within the Hygromiidae. Much of 
the molluscan phylogeny is heavily based on 
traits of the reproductive morphology and, es- 
pecially within the Hygromiidae, the presence 
and number of (reduced) stylophores play an 
important role in the classification within the 
family (Nordsieck, 1987, 1993; Schileyko, 
1989). 

Several observations can be made with re- 
spect to the reproductive morphology of the 



family Hygromiidae. There are clear morpho- 
logical differences between the phylogeneti- 
cally older subfamily Trichiinae and the 
younger subfamily Hygromiinae. All Trichiinae 
have two pairs of stylophores, that is, two up- 
per and two lower stylophores (e.g., Trictiia: 
Schileyko, 1978a). Most of the differences in 
the stylophore morphology between genera of 
Trichiinae are relatively small, while important 
morphological changes are found within the 
Hygromiinae. In this subfamily, one pair of 
stylophores has been lost, consequently many 
species have one upper and one lower 
stylophore (e.g., ¡Hygromia: Giusti & Manganelli, 
1987). Additionally, a further reduction of the 
upper stylophore and an enlargement of the 
lower stylophore occurred (e.g., Leptaxis: this 
paper; Lindliolmomneme: Schileyko, 1978b), 
culminating with total loss of the upper 
stylophore (e.g., Monachoides: Schileyko, 
1978b, 1989). 

Simultaneously with this evolution towards 
a single stylophore, the dart seems to be- 
come more elaborate. Perpendicular blades 
on the dart occur in several genera of 
Hygromiinae, resulting in different dart 
shapes, and increasing the dart's surface 
area. Presumably, this allows the dart to 
transfer larger amounts of the product from 
the mucus glands (Fedoseeva, 1994; Adamo 
& Chase, 1996; Koene & Schulenburg, sub- 
mitted). However, it is still unclear whether 
the hygromiid dart is used in a similar way 
as the helicid dart (Koene & Chase, 1998a, 
b; Rogers & Chase, 2001; Landolfa et al., 
2001) to transfer an allohormone (Koene & 
Ter Maat, 2001, 2002). Hence, behavioural 
data are required to determine how the 
Leptaxis dart is used. Observations of the 
mating behaviour of Leptaxis may also shed 
light on the function of the appendage at the 
base of the genital system (see also 
Mandahl-Barth, 1943). 

To conclude, we found the rudiment of an 
upper stylophore in three species of Leptaxis, 
which has previously gone unnoticed 
(Mandahl-Barth, 1943; Backhuys, 1975, 
Schileyko, 1989). The presence of this small 
organ is of importance because it indicates that 
Leptaxis links Hygromiinae with two (upper 
and lower) stylophores (e.g., Lindiiolm- 
omneme) and Hygromiinae with single 
stylophores (e.g., Monachoides). Therefore, 
our findings lead us to conclude that this ge- 
nus is an intermediate form in the evolution 
towards a single stylophore. 



78 



KOENE & MURATOV 



ACKNOWLEDGEMENTS 

We are grateful to P. Van Riel for generously 
providing some of the specimens. We thank H. 
Reise, P. Van Riel, and H. Schulenburg for valu- 
able comments and discussion, and J. Lange 
and С Levesque for their technical assistance. 
JMK was supported by a Jessup-McHenry 
Award of the Academy of Natural Sciences of 
Philadelphia and a Casimir-Ziegler Stipend of 
the Royal Netherlands Academy of Arts and 
Sciences. 



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Florida Museum of Natural History, 43: 49-77. 



Revised ms. accepted 15 December 2003 



MALACOLOGIA, 2004, 46(1): 79-125 

PRELIMINARY PHYLOGENETIC STUDY OF BRADYBAENIDAE (GASTROPODA: 
STYLOMMATOPHORA: HELICOIDEA) 

Min Wu 

Hebei University, IHezuolu 1, Baoding 071002, Ciiina; 
minwu@mail.libu.edu.cn 

ABSTRACT 

Morphological variation in the terminal genitalia of genera of Bradybaeninae is compared 
and discussed. This is the first attempt to study the anatomy of the endemic Chinese 
bradybaenids Catiiaica {Pliocattiaica), Pseudiberus (Platypetasus), and l\/letodontia.Apre- 
liminary phylogenetic analysis of bradybaenids was performed based on the character 
matrix from the present study. The focus was primarily on the terminal genitalia. He/zx 
(Helicidae) and Camaena (Camaenidae) were used as outgroups. The results suggest that 
several previous taxonomic arrangements for the subdivision of this family, based on the 
analyses using shell features and/or superficial anatomy of genital system, are unsuitable. 
The cladistic analysis suggests that the use of the subfamily Helicostylinae, sensu lato, 
might not be suitable for use as the sister group of the known Bradybaeninae. 

Two new endemic genera from western China are described based on the comparison 
of the terminal genitalia: Aegistohadra n. gen. and Eueuhadra n. gen. They are monophyl- 
etic and are readily distinguished from other bradybaenids by a synapomorphy, the pres- 
ence of penial caecum. Nanina delavayana Heude, 1885, is designated as the type species 
of Aegistohadra. The type species of Eueuhadra is a new species, E. gonggashanensis. 

Key words: Stylommatophora, Helicoidea, Bradybaenidae, China, terminal genitalia, 
phylogeny, phylogenetic analysis, new taxa. 



INTRODUCTION 

The Bradybaenidae (= Bradybaenidae + 
Helicostylidae, sensu Schileyko 1991) are a 
large group of terrestrial snails widely distrib- 
uted in eastern Asia, with one species in Eu- 
rope. Historically, more than 150 authors 
(Richardson, 1983; Wu, unpublished cata- 
logue) have published on Chinese 
bradybaenids. However, most work on the 
classification of higher taxa of China was 
based on shell, not anatomical characters 
(Pilsbry, 1888-1894; Möllendorff, 1899; 
Dautzenberg, 1914-1915; Bavay & 
Dautzenberg, 1900, 1915; Blume, 1925; Ping 
& Yen, 1932; Yen, 1939; Zilch, 1940; almost 
all previous work). Therefore, knowledge on 
the bradybaenid systematics has remained 
unsatisfactory. 

The monograph by Wiegmann (1900), in 
which species from 12 genera and subgenera 
are described, was the first study dealing spe- 
cifically with the anatomy of bradybaenid geni- 
talia. More recently, some malacologists have 
made comparative studies of the genital mor- 



phology, mainly based on their native 
bradybaenid taxa (Schileyko, 1978; Azuma, 
1982; literature of Japanese workers, cited by 
Nordsieck, 2002; Lee & Kwon, 1993, 1994; 
Wu, 2001; Wu & Quo, 2003). Many authors 
have focused on the general structures, such 
as the size of dart sac, the presence/absence 
of a flagellum, and the number of mucous 
glands. Schileyko (1978) gave a much more 
precise, detailed description of the terminal 
genitalia of Russian bradybaenids that in- 
cludes the above traditionally described char- 
acter and internal dissections of the penis and 
dart apparatus. More recently, Nordsieck 
(1 987) stated that the bradybaenid groups are 
characterized by apomorphies of the genital 
organs. 

However, similar work covering most en- 
demic Chinese bradybaenid taxa, which is 
essential for understanding the general 
anatomy of bradybaenids and construction of 
a sound taxonomic framework, has been lack- 
ing. 

The present work compares the structure of 
the terminal genitalia of some genera of the 



79 



80 



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Bradybaenidae based on dissection of their 
type species or non-type congeners. Two new 
bradybaenid genera are proposed based on 
anatomical and sliell characters. A preliminary 
phylogenetic analysis is performed based on 
the data obtained from these dissection re- 
sults. This phylogeny is compared to the three 
bradybaenid subdivision plans comprehen- 
sively reviewed recently by Nordsieck (2002), 
widely used thus far in China (e.g., Yen, 1 939; 
Zilch, 1960), Russia (e.g., Schileyko, 1978), 
and Japan (e.g., Kuroda & Habe, 1949; Minato, 
1988). 



MATERIALS AND METHODS 

This study is based on specimens from the 
collections of the Zoological Museum, Insti- 
tute of Zoology, Chinese Academy of Sciences 
(IZCAS), and from those belonging to 
Forschungsinstitut und Naturmuseum 
Senckenberg (SMF). Many genera are repre- 
sented by the non-type congeners rather than 
by the type species, because of the paucity of 
alcohol-preserved specimens in museums and 
the absence of specimens from type localities. 

All examined specimens (except specimens 
of IZCAS00067, which were first fixed in for- 
malin before being placed in 70% ethanol) are 
preserved in 70% ethanol. For preparing the 
dissections, a tiny hole was carefully drilled 
into the shell apex to assist removal of the soft 
parts of the snail using water pressure. All the 
illustrations were drawn using a stereo micro- 
scope and camera lucida. Shell and genital 
measurements were taken with 0.01 mm and 
0.1mm accuracy respectively for the new taxa 
described. Whorl number was counted as de- 
scribed by Kerney & Cameron (1979) and was 
taken with 1/8 whorl accuracy. Both color and 
length of soft parts in the descriptions refer to 
those observed and measured after alcohol 
preservation. Type specimens of the new spe- 
cies are deposited in IZCAS, Beijing. 

Taxa studied are listed in Appendix I along 
with locality data and museum accession num- 
bers. Descriptions of new taxa are given in 
Appendix II. 

Abbreviations 

The abbreviations used in the text and in the 
illustrations are explained as: ADC - channel 
connecting accessory sac and dart sac; AG - 
albumen gland; App - vaginal empty appen- 



dicula; AS - accessory sac (= inner stylophore 
in Giusti et al., 1992); ASC - accessory sac 
chamber; At - atrium; ВС - bursa copulatrix; 
BCD - bursa copulatrix duct; C23 - chamber 
produced by V2 and V3 in dart sac; DS - dart 
sac (= outer stylophore in Giusti et a!., 1992); 
Dt - love dart; DtC - chamber containing the 
dart = dart sac chamber; DVM - membranous 
sac surrounding dart sac and/or distal region 
of vagina near atrium (= basal genital sheath 
in Cuezzo, 1998). When preparing the genita- 
lia for observation, the structures were care- 
fully preserved for future examination. 

Ep - epiphallus, the region between the pe- 
nis and the insertion of the vas deferens. The 
delimitation is esily recognized when the 
ephiphallic papilla (= verge in Cuezzo, 1998: 
102) is present. When the epiphallic papilla is 
lacking, the continuous ridge structure can 
help to distinguish the epiphallic region from 
that of penis (Cuezzo, 1998). It is notable that 
the concept used by Cuezzo (1998) differs 
from that used by Giusti et a!. (1992), who 
defined the epiphallus as "from end of vas 
deferens to point of attachment of penial re- 
tractor". The term epiphallus of Cuezzo (1 998) 
is used here, because the point of attachment 
of penial retractor varies among different 
bradybaenid groups, and in most cases it is 
not level with the ephiphallic papilla. 

EpP - epiphallic papilla (= "penial" verge in 
Schileyko, 1 991 ); MAC - mucous gland-acces- 
sory sac channel; MG - mucous gland (= dart 
gland in Nordsieck, 2002); OD - oviduct; Ov - 
ovotestis; P - penis; according to the epiphallus 
concept used by Cuezzo (1998), the term pe- 
nis used in this study refers to the region be- 
tween the epiphallic papilla and the atrium, or 
when the epiphallic papilla is absent, it refers 
to the region close to the atrium and internally 
possesses the similar and continuous pilaster/ 
ridge structure. 

PLs - polylayered structure in dart sac and/ 
or accessory sac, produced by wavy and 
spongy connective tissue. PLs is not separate, 
but connected tightly with neighboring tissue, 
and if present, is visible when the dart appa- 
ratus is dissected sagittally. This structure oc- 
curs as occupying most part of dart sac (e.g., 
in Fig. 14C) or a small and limited region (e.g., 
in Fig. 1 1 B) in the dart apparatus; PR - penial 
retractor muscle; PS - penial sheath; PP - 
penial pilaster(s)/ridge(s); SPC - simple pe- 
nial caecum; T - talon; UV - free oviduct; Va - 
vagina; VD - vas deferens; VI - a valvule op- 
posite the opening of mucous glands, in sag- 



PHYLOGENY OF BRADYBAENIDAE 



81 



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ittal plane of dart sac (as in Fig. 6D); V2 - a 
valvule opposite V1 and closest to atrium, to- 
gether with V1 forming a muscular tube con- 
taining dart(s) (as in Fig. 6D); V3 - a valvule 
between V2 and V4, in sagittal plane of dart 



sac (as in Fig. 6D); V4 - most inner/proximal 
valvule in DC, together with V1 forming a 
chamber containing love dart(s) (as in Fig. 6D). 
Terms V1-V4 are employed, for convenience 
only, to show the sagittal plane of dart sac. 




FIG. 1. Fruticicola fruticum (O. F. Müller, 1774), IZCAS01009-2. A, general view of genitalia; B, dart 
sac and part of vagina, sagittal section, with cross-section of accessory sac. Structured DVM indicated 
by a thick solid arrow; C, penis, opened, with cross-section, showing a fold formed by the penial 
pilasters. A& В showing the elongated vagina section between dart sac and atrium. Bars equal 1 mm. 



PHYLOGENY OF BRADYBAENIDAE 



83 



Each view of the three dimensional portrayal 
represents only one part of the boundary of 
the chamber near it. 

Cladistic Analysis 

Cladistic analyses were performed using the 
computer program Hennig86 Version 1.5 
(Farris, 1988) and program Winclada Version 
1.00.08 (Nixon, 2002). The analysis of the 
character distribution on the cladograms was 
carried out using the program Winclada. 

All the 28 characters used, observed from 
terminal genitalia except Character 27 from 
mantle, are based on a selection made after 
my study of the representatives for terminal 
groups. Of the characaters (0-27), seven bi- 
nary characters and the remaining multistate 
characters were coded as non-additive. To 
avoid artificial judgement, character polarity is 
obtained as one of the results of the analysis 
rather than as an apriori assumption (Nixon & 



Carpenter, 1993). Therefore, all characters 
involved are treated as undirected and unor- 
dered. No missing character state occurred in 
the examined terminals (Table 1). Consider- 
ing that fused coding involves a loss of phylo- 
genetic information (Lee & Bryant, 1999), the 
inapplicable characters (e.g., coding of char- 
acter 1 was separated from that fused with 
character 0) were separately coded when the 
character-variable is inapplicable in some taxa. 

Selection of the Ingroup and Outgroup Taxa 

Besides including two newly proposed gen- 
era, the ingroup bradybaenid taxa considered 
were those included in the subfamily 
Bradybaeninae by Richardson (1983), except 
Armandiella Ancey, Tricheulota Pilsbry, 
Plecteulota Möllendorff, Neseulota Ehrmann, 
Archaeoxesta Kobelt, Coccoglypta Pilsbry, 
Coneulota Pfeffer, Dolicheulota Pilsbry, and 
Ponsadenia Schileyko, because alcohol-pre- 



DVM 





в t 

MG Va 



^UV 



BCD 




FIG. 2. Bradybaena similaris {Rang, 1831), IZCAS01072-1. A, lateral view of dart apparatus and peniai 
complex; B, basal view of dart apparatus; C, penis, opened; D, dart sac, sagittal section. DVM 
indicated by thick solid arrows in A & D. Bar equals 1 mm. 



wu 



served material was unavailable. Semi- 
buliminus Möllendorff was excluded because 
it was recently grouped into Metodontia (Wu, 
in review). Halolimnohelix Germain, Haplohelix 
Pilsbry, Urguessella Preston, and Vicarihelix 
Pilsbry listed in Richardson's bradybaenid 
catalog (1 983; also in Thiele, 1 931 ) were ex- 
cluded because they are, on the basis of 
anatomy, non-bradybaenid helicoids 
(Nordsieck, 1986, 1987; Schileyko, 1991). 

Results of previous cladistic analyses for 
Helicoidea were used as the departure point 
for outgroup selection. According to the cla- 
dogram based on a molecular database 



(Wade et al., 2001 : fig. 3c), the Camaenidae- 
Helicidae-Polygridae group forms the sister 
group of Bradybaenidae. In another anatomy- 
based cladistic analysis of Xanthonychidae 
(= Helminthoglypidae; Cuezzo, 1998), the 
sister relationship of the Bradybaenidae and 
Xanthonychidae-Helicidae groups are sup- 
ported by four synapomorphies. Therefore, 
in this study, the Helicidae and Camaenidae 
were chosen as outgroups. 

All the 23 genera and subgenera were 
treated as separated terminal taxa. The type 
species was available for only 11 ingroup and 
one outgroup genera. These are: Acusta, 




MG V4 



FIG. 3. Karaftohelix weyrichii (Schrenck, 1867), IZCAS01 080-2. A, B, lateral views of dart sac, DVM 
indicated by thick solid arrows; C, dart sac, sagittal section, with cross-section showing DVM, DVM 
indicated by thick solid arrows, neck-structure indicated by thick hollow arrows; D, penis, opened, with 
cross-sections, fold formed by the penial pilasters indicated by thick solid arrows. Bar equals 1 mm. 



PHYLOGENY OF BRADYBAENIDAE 



85 



Bradybaena, Cathaica, Fruticicola, Mastigeu- 
lota, Pseudaspasita, Nesiohelix, Aegistohadra 
n. gen., Eueuhadra n. gen., Trichobradybaena, 
Pfeifferia, and Helix. Otherwise, only those spe- 
cies commonly accepted in a group were used 
as the representatives for their generic group. 



RESULTS 
Character Descriptions 

Character 0: Presence of the membranate sac 
surrounding the dart sac and/or the distal 
region of the vagina near to the atrium 
(DVM). 

(0) absent; (1 ) present (Figs. 1 B, 2A, 2D, 9A, 
indicated by thick solid arrows). 
Remarks: The dart sac is inserted on the va- 
gina. In very few cases, the dart sac is ba- 
sally wrapped by a layer of membrane, which 
sometimes appears to be sac-like (Fig. 3A- 
C, indicated by thick solid arrows) near the 
atrium, completely or partially. 

Character 1: The DVM inernally simple or 
structured: 

(0) not applicable because DVM absent; (1) 
DVM present, internally simple (Figs. 2A, 2D, 
9A, indicated by thick solid arrows); (2) DVM 
present, internally structured, with numerous 
cells (Figs. 1B, 3C, indicated by thick solid 
arrows). 

Character 2: The proximal dart sac and/or the 
distal region of the vagina are/is wholy en- 
circled by the DVM or not: 
(0) not applicable because DVM absent; (1) 
DVM present, proximal dart sac partially 
encircled by DVM (Figs. 1 B, 3B, C, indicated 
by thick solid arrows); (2) DVM present, 
proximal dart sac wholly wrapped by DVM 
(Fig. 2A, indicated by thick solid arrows). 

Character 3: Presence of the penial sheath: 
(0) absent; (1) present. 
Remarks: In Nesiohelix swinhoei, Aegisto- 
hadra n. gen., Eueuhadra n. gen., Pfeifferia 
micans, and Calocochlea coccomelos, the 
penial sheath is lacking (Figs. 5D, 6A, C, 7A, 
B, 8A, B, 9A, D). In the other genera, the 
penial sheath is always present (e.g.. Azuma, 
1982). In the outgroup Helix pomatia, the 
penial sheath is present and developed, wrap- 
ping the whole penis and the basal part of 
penial retractor (Fig. 10D, E, indicated by thick 
solid arrows). In bradybaenid genera, the 
penial sheath, if present, cannot be morpho- 
logically distinguished from that of Helix. 



Character 4: Differentiation status of the pe- 
nial pilasters: 

(0) penial pilasters not differentiated; (1) pe- 
nial pilasters differentiated near epiphallus; 
(2) penial pilasters differentiated near atrium. 
Remarks: Differentiated penial pilasters are 
those thickened, deep, and/or morphologi- 
cally distinguishable from the neighboring 
zig-zag ones of moderate thickness. In most 
species examined, the penial pilasters are 
somewhat thickened near the epiphallus, 
becoming thinner near the atrium (e.g.. Figs. 
2C, 11C, 12C, 13C, 14E, 15D). It is charac- 
teristic that the pilasters on the penial inner 
wall differentiate towards the atrium or to- 




MG 



FIG. 4. Metodontia yantaiensis (Crosse & 
Debeaux, 1863), IZCAS00131-1. A, general view 
of genitalia, with cross-sections of penial sheath 
and penis; B, dart sac, sagittal section. Bar 
equals 1 mm. 



86 



WU 



wards the epiphallus. In Fruticicola fruticum 
and Karaftohelix weyrichii, the penial pilas- 
ters differentiate near the epiphallus and 
form an asymmetrically projecting fold (Figs. 
1С, 3D), which is similar to the asymmetri- 
cal epiphallic papilla of Nesiohelix swinhoei 



(Fig. 5E), and assumed to serve as the 
epiphallic papilla. In Stilpnodiscus moellen- 
doiffi (Fig. 16B), S. yeni, S. entochilus, and 
Laeocathaica {Laeocathaica) subsimilis (Fig. 
17B), the penial pilasters become thickened 
and differentiated near the atrium. Especially 




FIG. 5. Nesiohelix swinhoei (L. Pfeiffer, 1865), IZCAS00055-2. A, dart sac, sagittal section; B, sagittal 
section of accessory sac; C, cross section of mucous glands insertion on accessory sac, mucous tube 
entrance indicated by a thick solid arrow; D, penis and epiphallus, sagittal section, with cross-section 
of epiphallus; E, sagittal section of penis-epiphallus region, diagrammatic, with cross-section; F, 
epiphallus and flagellum, opened, with cross-sections; G, cross-section of dart sac, showing two 
pieces of dart; H, a piece of dart, with cross-sections. Bars equal 1 mm. 



PHYLOGENY OF BRADYBAENIDAE 



87 



in Stilpnodiscus, such differentiated pilasters 
are high and valvule-shaped. 
Character 5: Presence of the epiphallic papilla: 

(0) epiphallic papilla absent (e.g., Fig. 11C); 

(1) present (e.g., Fig. 12C). 



Character 6: Symmetry of the epiphallic pa- 
pilla (EpP): 

(0) not applicable because epiphallic papilla 
absent; (1) epiphallic papilla present and 
symmetric (e.g.. Figs. 12C, 13F); (2) 




FIG. 6. Aegistohadra delavayana n. gen. & comb., IZCAS00132-3. A, general view of terminal genitalia, 
the sac of vagina opposite to dart sac indicated by a thick solid arrow (left), penial caecum indicated 
by a thick solid arrow (right); B, basal view of dart sac, diagrammatic; 0, penial complex, opened, with 
cross-sections, penial caecum indicated by a thick solid arrow; D, dart sac, sagittal section, the sac 
of vagina opposite to dart sac indicated by a thick solid arrow. Bar equals 1 mm. 



wu 




FIG. 7. Eueuhadra gonggashanensis, n. gen. & sp., IZCAS00067-13, Paratype. A, general view of 
terminal genitalia; В, penial complex, penis and partially epiphallus opened, with cross-sections' С 
epiphallus and flagellum, opened, with cross-sections; D, dart sac, sagittal section; E, section of penis- 
epiphallus region, diagrammatic. Bar equals 1 mm 



PHYLOGENY OF BRADYBAENIDAE 



89 



epiphallic papilla present and asymmetric 
(Figs. 5E, 8D, 9D). 

Remarks: In Nesiohelix swinhoei, Aegisto- 
hadra n. gen., Eueuhadra n. gen., Pfeifferia 
micans, Calocochlea coccomelos, Cathaica 
(Pliocathaica) gansuica, Aegista (Aegista) 
accrescens, Aegista (Plectotropis) gerlachi, 



Laeocathaica (Laeocathaica) subsimilis, 
Acusta ravida, Trishopiita dacostae, and 
Eutiadra herklotsi (Figs. 5D, 6C, 7B, 8D, 9D, 
12C, 13F, 14E, 178, 18D, 19D, 20C), a more 
or less protruding epiphallic papilla is present. 
In the remaining bradybaenid genera the 
epiphallic papilla is depressed or missing. 




FIG. 8. Calococlilea coccomelos (Sowerby, 1840), SMF 323619. A, general view of genitalia; B, dart 
sac, sagittal section; C, mucous glands, sagittal section; D, above: Penis-epiphallus region, opened; 
below: cross-section of penis-epiphallus transition, diagrammatic; valve-shaped epiphallic papilla 
indicated by thick solid arrows. Bars equal 1 mm. 



90 



wu 



Character 7: The epiphallic papilla valve- 
shaped or papilla-shaped: 
(0) not applicable because epiphallic papilla 
absent; (1) epiphallic papilla present, valve- 
shaped (Figs. 8D & 9D, indicated by thick 
solid arrows); (2) epiphallic papilla present, 
papilla-shaped (e.g.. Figs. 5D, 10D). 

Character 8: Presence of the penial caecum: 
(0) absent; (1) present (e.g., Figs. 6A, 6C, 
indicated by thick solid arrows). 
Remarks: This structure can be easily dis- 
tinguished from the following simple penial 



caecum (SPC) by the PC pilasters, which 
are differentiated from those of the caecum. 
In the simple penial caecum (SPC), which 
characterizes the genera Trichobradybaena 
and Mastigeulota, the penial pilasters form- 
ing the inner wall of caecum are just the ex- 
tended parts from its outer/entering pilasters. 

Character 9: The simple penial caecum: 
(0) absent; (1 ) present (Figs. 1 1 B, 23A, indi- 
cated by thick solid arrows). 

Character 10: Presence of the flagellum: 
(0) absent; (1) present. 




FIG. 9. Pfeifferia micans Pfeiffer, 1845, SMF 323620. A, general view of genitalia; B, dart sac, sagittal 
section except mucous glands; C, mucous glands, sagittal section; D, penis-epiphallus region, 
opened, valve-shaped epiphallic papilla indicated by a thick solid arrow. Bars equal 1 mm. 



PHYLOGENY OF BRADYBAENIDAE 

Ep 



91 




MG 



FIG. ^0. Helix pomatia {Lmné, 1758), IZCAS00188-1. A, general view of terminal genitalia, bar equals 
2 mm; B, dart sac, sagittal section with mucous glands removed; C, section of partial dart sac, with 
cross-section; D, penial complex, opened, with cross-sections, middle two thick solid arrows indicating 
penis-epiphallus chambers; E, section of penis-epiphallus region, diagrammatic, penial sheath 
indicated by a thick solid arrow; F, distal penis near atrium opening, opened; G, basal view of dart sac, 
diagrammatic. 



92 



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Remarks: The flagellum and the vas defer- 
ens insertion structure are almost the same 
in the species examined. Flagellum, if 
present, with inner ridges simple or somewhat 
complexly arranged. Insertion of vas defer- 
ens on flagellum inwardly forms a more or 
less distinct C-shaped (in cross-section) fold 
towards the tip of flagellum (Figs. 5F, 7C, 10D, 
13D, 14F, 19E, 20A). The only exception is 
Aegistohadra delavayana, n. comb., in which 
a depressed pilaster instead of the distinct 
C-shaped fold is present (Fig. 6C). These 
structures are the same in bradybaenid gen- 
era and in Helix pomatia. Therefore, if present, 
the flagellum of the various groups examined 
might be considered homologous. 
Character 11: Presence of the polylayered 
structure (PLs) in accessory sac: 
(0) PLs absent (e.g., Figs. IB, 5A, 8B); (1) 
PLs present (e.g.. Figs. 2D, IOC, IIB, 13C, 
14C, 22D); (2) not applicable because dart 
sac absent. 

Remarks: In Metodontia yantaiensis, the ac- 
cessory sac has some wavy and spongy 



connective tissue (polylayered structure, 
PLs) (Fig. 4B). In Pseudaspasita binodata, 
such structure seems to be weakly devel- 
oped (Fig. 21 C). This kind of structure can 
be easily distinguished from the folds/pilas- 
ters on the inner wall of the accessory sac 
(Fig. 16E) by the compactness and paral- 
lelism in the arrangement of its filaments/ 
layers. In Bradybaena similaris and 
Cathaica (Cathaica) fasciola, the structure 
is much developed and situated between 
insertion of mucous glands and vagina (Figs. 
2D, 22D). In Aegista (Aegista) it is highly 
developed and uppermost, and it wraps the 
dart chamber (DtC) (Figs. 13C, 14C). In 
Aegista (Plectotropis), PLs occupies the 
whole accessory sac that is externally vis- 
ible and the region between dart sac and 
the vagina. Interestingly, in Helix pomatia, 
the polylayered structure is also present, at 
the pit formed by both dart the sac and each 
of the accessory sacs/trunk of basal mucous 
stalks (Fig. IOC). The observed PLs of the 
taxa studied are provisionally assumed to 



V2 V3 V4 PLs 



DtC 




SPG 



FIG. 11. Mastigeulota kiangsinensis (E. Martens, 1875), IZCAS00003-1. A, lateral view of dart 
apparatus; B, dart sac, sagittal section; C, penis, opened, simple penial caecum (SPG) indicated by 
a thick solid arrow. Bars equal 1 mm. 



PHYLOGENY OF BRADYBAENIDAE 



93 



be homologous in origin, because they oc- 
cur only in the specific region in dart appa- 
ratus, and assumed to be related to dart 
shooting or pumping the mucus out during 
copulation. 
Character 12: Distribution of polylayered struc- 
ture (PLs) in accessory sac: 
(0) not applicable because PLs absent; (1) 
distributed between mucous glands insertion 



and vagina (region I) (e.g.. Fig. 22D); (2) PLs 
present, distributed between mucous glands 
insertion and dart caecum (DtC; region II) 
(e.g., Figs. 4B, 10C, 11B); (3) PLs present 
at region I & II (e.g., Fig. 14C). 
Character 13: The common entrance of mu- 
cous glands: 

(0) mucous glands without common entrance 
(e.g., Figs. 19B, 20D); (1) with common en- 




FIG. 12. Cathaica {Pliocathaica) gansuica (Möllendorff, 1899), IZCAS00210-1. A, basal view of dart sac 
and penial complex; 8, lateral view of dart sac and penial complex; C, penis, opened; D, dart sac, sagittal 
section, neck-structure region of dart sac indicated by four thick hollow arrows. Bar equals 1 mm. 



94 



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trance; (2) not applicable because dart sac 
absent. 

Remarks: There are two ways by which the 
mucous glands are inserted on the acces- 
sory sac, which can only be observed when 
the accessory sac is cut open sagittally. Usu- 
ally, the mucous glands open into the ac- 
cessory sac through a common duct (Figs. 
1B, 2D, 4B, 6D, 7D, 11B, 12D, 13C, 14C, 
15C, 16C, 17C, 18B, 21C, 22D, 23E, 24B). 
Another situation was found in Karaftohelix 
weyrichii, Trishoplita dacostae, Euhadra 



herklotsi, and Nesiohelix swinhoei {F\gs. 3C, 
5C, 19B, 20D), with two to numerous sepa- 
rate tubes rather than a common tube open- 
ing into the accessory sac. 
Character 14: The distinguishability of the ac- 
cessory sac from outside of the dart sac: 
(0) indistinct from outside of the dart sac; (1 ) 
distinct from outside of the dart sac; (2) not 
applicable because dart sac absent. 
Remarks: The accessory sac cannot always 
be distinguished externally by an apparent 
external boundary from the dart sac (e.g.. Fig. 




FIG. 13. Aegista (Aegista) accrescens (Heude, 1882), IZCAS00027-4. A, B, lateral views of dart sac; 
C, dart sac, sagittal section; D, flagellum, opened, with cross-sections; E, penial complex, penis 
opened, with cross-sections; F, section of penis-epiphallus region, diagrammatic. Bar equals 1 mm. 



PHYLOGENY OF BRADYBAENIDAE 



95 




FIG. 14. Aegista (Plectotropis) gerlachi (E. Martens, 1881), IZCAS00044-2. A, basal view of dart sac; 
B, lateral view of dart sac, outer tissue partially removed to show the polylayered structure inside dart 
sac; C, dart sac, sagittal section; D, polylayered structure in accessory sac, magnified; E, penis and 
epiphallus, opened; F, penial complex, with cross-sections. Bar equals 1 mm. 



96 



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2В). Various genera show different patterns 
of tine accessory sac, which has little relation- 
ship with its size from external view. The ac- 
cessory sac is situated usually on the bottom 
of dart sac, except in Acusta, where it is situ- 
ated near the top of dart sac (Fig. 18B). The 
structurally simplest accessory sac is an empty 
sac, only with a few depressed folds (= pilas- 
ters) on its inner wall (e.g., Figs. 1 B, 7D & 1 6C). 

Character 15: The accessory sac is bipartite 
(e.g.. Figs. 14A, 22C) or undivided (e.g., 
Figs. 5A, 7A): 

(0) accessory sac divided into two parts; (1) 
accessory sac undivided; (2) not applicable 
because dart sac absent. 

Character 16: Presence of V1-V4 in the dart 
apparatus (= V2 is present): 
(0) VI -V4 indistinct (= V2 is indistinct/ab- 
sent) (e.g.. Figs. 8B, 9B); (1) V1-V4 distinct 
(= V2 is distinct) (e.g.. Figs. 7D, 23E); (2) 
not applicable because dart sac absent. 
Remarks: Inside the dart sac, several 
valvules (VI -V4) form a tube that contains 



one love dart (or two in Nesiohelix) serving 
as mating-related organ (e.g., Figs. 7D, 23E). 
According to this study, the position and the 
number of the valvules are intraspecifically 
stable but vary among the genera studied. 
The term valvule is used here for the first 
time in land snail anatomy. It is a small valve- 
like structure that describes the nature of the 
chamber boundry (dart sac, accessory sac 
chanmber), visible in sagittal section. How- 
ever, the position of VI-V4 can easily be 
determind even when V2 is absent, because: 
(1 ) VI and V4 always form the opening of a 
muscular tube containing the love dart(s); (2) 
the space between V4 and V3 is usually the 
opening of the accessory sac (the only ex- 
ception is in Acusta, in which the accessory 
sac is situated on the top of dart sac); (3) 
the space between V3 and V2 is C23, which 
varies from presence as a pronounced 
chamber to totally absence. Such absence 
means V2 is lacking morphologically. For this 
reason, the complexity of the development 




FIG. 15. Pseudobuliminus {Pseudobuliminus) piligerus (Möllendorff, 1899), IZCAS00085-21. A, В 
lateral views of terminal genitalia; C, dart sac, sagittal section; D, penial complex, penis opened, with 
cross-sections. Bar equals 1 mm. 



PHYLOGENY OF BRADYBAENIDAE 
DS 



97 




FIG. 16. Stilpnodiscus. A, B, С Stilpnodiscus moellendorffi \Nu, 2001, IZCAS00081-4, Paratype. A, 
lateral view of terminal genitalia; B, penis, opened, with cross-sections; C, dart sac, sagittal section' 
D, E Stilpnodiscus entochilus Möllendorff, 1899, IZCAS00076-2. D, basal view of dart sac" E dart sac' 
sagittal section, in detail, with cross-sections of penis. Bar equals 1 mm. 



98 



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of the dart sac inner structure is considered, 
described, and employed for the first time 
as an important and necesssary character 
for the dart sac in the Bradybaenidae. 
Character 17: Presence of a papilla within 
accessory sac formed by the mucous glands 
insertion: 

(0) without papilla; (1) with a papilla; (2) not 
applicable because dart sac absent. 
Remarks: A papilla with a tiny pore or sev- 
eral tiny pores for the entrance of mucous 
from mucous glands into the accessory sac 
is sometimes present. If the mucous gland 
ducts merge into one common tube, the pa- 
pilla also has one pore, as in Acusta ravida 
(Fig. 18B, G). When the mucous glands en- 
ter the accessory sac separately, two papil- 
lae are present, as in Trishoplita dacostae 
(Fig. 19B, indicated by two lower thick solid 
arrows) or a somewhat complex structure 



with numerous pores as in Nesiohelix 
swinhoei and Euhadra herklotsi (Figs. 5C, 
20D). In most genera, such a structure is 
absent (other Figs.). 

Character 18: Presence of the structure de- 
rived from mucous glands entering papilla 
leading to DtC: 

(0) not applicable because mucous glands 
entrance papilla absent; (1) mucous glands 
entrance papilla present, its derived part 
does not lead to DtC; (2) mucous glands 
entrance papilla present, its derived part 
leads to DtC (Figs. 19B, 20D, respectively 
indicated by a upper thick solid arrow). 

Character 1 9: Number of branches of mucous 
glands: 

(0) numerous mucous branches; (1) one 
spherical mucous gland (Figs. 8, 9); (2) two 
branches of mucous glands; (3) not appli- 
cable because dart sac absent. 





FIG. 17. Laeocathaica (Laeocathaica) filippina (Heude, 1882), IZCAS00006. A, В IZCAS00006-5. A, 
lateral view of terminal genitalia; B, penial complex, penis opened; C, IZCAS00006-6, dart sac, sagittal 
section. Bar equals 1 mm. 



PHYLOGENY OF BRADYBAENIDAE 



99 



Character 20: The length of vaginal region 
between dart sac and atrium: 
(0) region short; (1) region pronouncedly 
elongated (e.g., Figs. 9A, 18B); (3) not ap- 
plicable because dart sac absent. 

Character 21: Proximal part of dart sac elon- 
gated, forming a neck-structure. 
(0) neck-structure absent; (1 ) neck-structure 
present (Figs. ЗА, 12D); (2) not applicable 
because dart sac absent. 

Character 22: Presence of penis-epiphallus 
chamber(s): 
(0) absent; (1) a simple chamber present 



(Fig. 78, E); (2) more chambers present (Fig. 
10D, E). 

Remarks: The penis-epiphallus chamber oc- 
curs in the wall of penis-epiphallus junction. 
Dissection shows that there are three cases 
of differentiation. (1) It is solid (i.e., without 
any chamber within) between the epiphallic 
papilla and its wall. (2) There is only a simple 
chamber between the epiphallic papilla and 
its wall. (3) As seen in Helix pomatia 
(Helicidae) (Fig. 10D, E), more than one 
chamber is developed in this area, and some 
of them extend into the penial wall. All 




FIG. 18. Acusta ravida (Benson, 1842), IZCAS00944-2. A, general view of genitalia; B, dart sac and 
part of vagina, sagittal section, with cross-section of vagina; C, region near penial sheath; D, proximal 
region of penis, opened, showing epiphallic papilla; E, section of penis-epiphallus region, 
diagrammatic; F, distal region of penis, opened; G, section of accessory sac, papilla of entrance for 
mucous tubes indicated by a thick solid arrow, diagrammatic. Bar equals 1 mm. 



100 




FIG 19 Trishoplita dacostae Gude, 1900. IZCAS00174-2. A, lateral view of dart apparatus; B, dart 
sac sagittal section, upper thick solid arrow indicating the structure derived from the mucous glands 
entering papilla leading to dart chamber, two lower thick solid arrows indicating the mucous glands 
entering papilla; C, the mucous glands entering papillae and the derived structure; D, pernal complex, 
with penis opened; E, epiphallus and flagellum, opened, with cross-sections. Bar equals 1 mm. 



PHYLOGENY OF BRADYBAENIDAE 



101 




PR 



-MG 



FIG. 20. Euhadra herklotsi {E. Martens, 1861), IZCAS01076-1. A, epiphallus and flagellum, with cross- 
sections; B, section of penis-epipiiaJlus region, diagrammatic; C, penis-epiphalius region, penis 
opened; D, dart sac, sagittal section, upper thick solid arrow indicating the structure derived from the 
mucous glands entering papilla leading to dart chamber, lower thick solid arrow indicating the structure 
of mucous glands entering papilla. Bar equals 1 mm. 



102 



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FIG. 21. Pseudaspasita binodata (Möllendorff, 
1886), IZCAS01 075-2. A, general view of 
terminal genitalia; B, basal view of dart sac, 
diagrammatic; C, dart sac, sagittal section. Bar 
equals 1 mm. 





Dt DtC PLs V4 V1 ^2 





V3 C23 



FIG. 22. Cathaica {Cathaica) fasciola (Draparnaud, 1801), IZCAS01074-6. A, lateral view of dart sac; 
B, lateral view of dart sac, with cross-sections of penial sheath and penis; C, basal view of terminal 
genitalia; D, dart sac, sagittal section; E, polylayered structure in accessory sac. Bar equals 1 mm. 



I 



PHYLOGENY OF BRADYBAENIDAE 



103 



bradybaenid genera examined, except 
Eueuhadra, n. gen., fall into the first case. 
Character 23: Number of darts per dart sac: 
(0) dart sac containing 1 dart; (1) dart sac 
containing 2 darts (Fig. 5G, H); (2) not ap- 
plicable because dart sac absent. 
Remarks: In Nesiohelix swinhoei (type spe- 
cies of the genus Nesiohelix; Richardson, 
1983, the type species mistakenly given as 
Nesiohelix caspari; see original introduction 
of genus by Kuroda & Emura, 1943), the 
dart sac contains two darts, each of which 
is wrapped by a muscular tube. These two 
muscular tubes are attached closely but dis- 
tinctly divided. In this study, the two darts 
are the same length rather than "one larger, 
the other smaller" (Kuroda & Emura, 1943: 
text-fig. 1), semi-circled in cross-sections, 
and blunt apically. In some other congeneric 
species of Nesiohelix, such as N. 
samarangae (Kuroda & Miyanaga, 1942) 
and N. moreletiana (Heude, 1882), the dart 



sac contains two darts (Habe, 1 945, not fig- 
ured), which is confirmed in this study and 
is an important synapomorphy characteriz- 
ing Nesiohelix. 

Character 24: Internal pilaster of accessory 
sac differentiated or not: 
(0) not differentiated; (1) differentiated (Fig. 
IB); (2) not applicable because dart sac 
absent. 

Character 25: Position where the accessory 
sac is inserted on dart sac: 
(0) accessory sac inserted on the bottom of 
dart sac; (1 ) accessory sac on the upper side 
of dart sac (Fig. 18B); (2) not applicable be- 
cause dart sac absent. 
Remarks: The accessory sac is usually situ- 
ated on the bottom of dart sac, except in 
Acusta, where it is situated at/near the top 
of dart sac (Fig. 18B, compared to its nor- 
mal position, e.g., that shown in Fig. 9A). 
The abnormal position of accessory sac can 
be observed in all anatomically known 




FIG. 23. Trichobradybaena submissa (Deshayes, 1873), IZCAS00010-3. A, general view of genitalia, 
simple penial caecum (SPG) indicated by a thick solid arrow; B, C, lateral views of dart sac; D, penis, 
partially opened; E, dart sac, sagittal section. Bars equal 1 mm (after Wu & Guo, 2003). 



104 



wu 



Acusta species and is preliminarily consid- 
ered as the inverse of accessory sac in po- 
sition (Wu, unpublished paper on Acusta). 
Character 26: Presence of sacs inserted on 
vagina oppsite to dart sac: 
(0) absent; (1 ) present (Fig. 6A, D, indicated 



by thick solid arrows); (2) not applicable be- 
cause dart sac absent. 
Character 27: Relation of the mantle to the shell. 

(0) shell is not partially enclosed by mantle; 

(1) the mantle partially enclosing shell (ob- 
served in Pfeifferia micans, SMF323620). 




FIG. 24. Pseudiberus (Platypetasus) chentingensis Yen, 1935, IZCAS00163. A, lateral view of terminal 
genitalia, with cross-sections of epiphallus and vas deferens; B, dart sac, sagittal section; C, penial 
complex, with penis opened. Bar equals 1 mm. 



PHYLOGENY OF BRADYBAENIDAE 



105 




FIG. 25. Camaena platyodon (L. Pfeiffer, 1846), IZCAS00833. Male section of terminal 
gentalia, left, showing saggital section of penial wall; right, showing cross-sections 
of penis, epiphallus and flagellum. 



Cladistic Analysis 

The data matrix (Table 1 ) was submitted to 
HENN1G86 and Winclada. All the observed 
apomorphies were Included among the char- 



acters because they are useful for the char- 
acterization of certain terminals, although they 
are not informative for the construction of the 
phylogenies. The removal of apomorphies in 
the analysis will decrease the steps in con- 



106 



wu 



structing a cladogram, but will not Influence 
its reliability. Two types of analyses were per- 
formed. In the first type of analysis, all char- 
acters were weighted equally. Data sets were 
calculated with an exact algorithm (implicit 
enumeration). Another type of analysis used 
the successive weighting function provided by 
the Hennig86 program, which is considered 
by Carpenter (1988) to be the best method for 
weighting characters and choosing among 
equally parsimonious cladograms. The first 
analysis produced 11 equally parsimonious 
trees (EPTs) with length of 96, consistency 
index (CI) 0.54, and retention index (Rl) 0.67. 
Extended branch swapping was then applied 
to the initial tree using the branch-breaking 



(bb*) command, producing 3,502 bb trees. A 
strict consensus tree (SCT) (Fig. 26) was then 
summarized from these 3,502 trees with 
Winclada in order to find the most unambigu- 
ous monophylies. 

In the second analysis, after two iterations 
of successive approximations weighting and 
branch-breaking, 87 trees were retained, each 
with length 274, CI = 0.73, Rl = 0.87. The cla- 
dograms obtained by the first and the second 
type of analyses were then introduced to 
Winclada for rerooting and mapping the dis- 
tribution of characters. Based on the trees re- 
sulting from the second type of analysis, the 
rooted SCT was produced using Winclada 
(Fig. 27). 



Camaena 
Helix 



Acusta 



Karañohelix 
Pliocathaica 
Laeocathaica 
Aegistohadra n. gen. 
Eueuhadra n. gen. 
Nesiohelix 



Trishoplita 
Euhadra 



Aegista 

Plectotopis 

Pseudaspasita 

Mastigeulota 

Metodontía 

Cathaica 

Platypetasus 

Stílpnodiscus spa 

Stílpnodiscus spb 

Pseudobuliminus 

Bradybaena 

Trichobradybaena 

Fruticicola 



Calocochlea 
Pfeifferia 



FIG. 26. Rooted SCT resulted from 3,502 EPTs (L = 96, CI = 54, Rl 
characters. 



67) based on equally weighted 



PHYLOGENY OF BRADYBAENIDAE 



107 



In the 3,502 EPTs constructed based on the 
equally weighted characters, 51 trees were 
found to be exactly equal respectively, in to- 
pology, to those obtained by philosophy of 
successive approximations weighting. In other 



words, 51 out of 87 trees based on weighted 
characters had the exact topology with the cla- 
dograms from the first analysis. When the 
rooted SCT (Fig. 27) was summarized from 
the 87 trees from the second analysis, only 



3 1113141516171819202123242626 
r—O« >•»••■ ••■•■>• 
022222233222222 
1112 

o<^ Helix 

1 2 

4 1017182025 



■Camaena 




Nesiohelix 
Trishoplita 
Eu h adra 



Aegistohadra п. gen. 
Eueuhadra n. gen. 



I — Aegista 



Plectoiropis 
Pseudaspasita 



Laeocathaica 

12 4 1316 
12 110 

Pliocathaica 

111215 
1 1 



Karaftohelix 



Cathaica 



Stilpnodiscus spa 

9 1112 

-ooo — Mastige ulo ta 
— Platypetasus — 



Stilpnodiscus spb 

111216 

-ooo — Metodontia 

1 2 

— Pseudobuliminus 

111216 
1 1 



Bradybaena 



Trictiobradybaena 

1 2 4 1624 



■ Fruticicola 



3 5 

"— CX> 



5 6 7 19 I ^-' 

O««» 27 

12 11 Li_ 



Cabcochlea 
Pfeifferia 



R J N 

■ B— B— B- 

■B— E— E- 
A— A— A- 

B— E— E- 



B- 



A— A— A- 
A— A— A- 
A- 

B B- 

B— B— B- 

B- 

B- 
B- 
B- 
B- 
B- 
B- 
B- 
B- 



B- 



В 

В— В- 



В- 



В- 



FIG. 27. Left: Rooted SCT resulted from 87 EPTs (L = 274, CI = 73, Rl = 87) based on weighted 
characters, showing the distribution of character states. Solid circle - nonhomoplasious change; 
empty circle - homoplasious change. Right: Showing suprageneric classification by different authors: 
A - Aegistinae/Aegistini, В - Bradybaeninae/Bradybaenini, E - Euhadrinae/Euhadrini; H - 
Helicostylidae/Helicostylinae; R - Russian authors, J - Japanese authors, N - Nordsieck (Nordsieck, 
2002). 



108 



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one node collapsed, so this rooted SCT was 
thought to be informative and proper for being 
used both to interpret the present bradybaenid 
phylogeny and to indicate the reliability of the 
monophyletic groups. 

As shown in the rooted SCT obtained by us- 
ing the second type of analysis (Fig. 27), the 
ingroup was well defined by three nonhomo- 
plasious synapomorphies characters 15(1), 
16(1), and 22(0) (Fig. 27). Eight clearly distin- 
guished monophylies supported by nonhomo- 
plasious synapomorphy/synapomorphies are 
as follows: 

(a) The clade {Trishoplita, Euhadra), was sup- 
ported by character state 1 8(2). The mono- 
phyly of this clade was also confirmed by 
the SCT 

(b) The clade composed of Aegistohadra and 
Eueuhadra, supported by character state 
8(1); 

(c) The monophyly {Aegista, Plectotropis, 
Pseudaspasita), supported by character 
state 12(3). The monophyly of this clade 
was also confirmed by the SCT; 

(d) The clade composed of Karaftohelix and 
Pliocathaica, supported by synapomorphic 
character state 21(1); 

(e) The clade composed of Cathaica, 
Stilpnodiscus, Mastigeulota, Platypetasus, 
Metodontia, Pseudobuliminus, Brady- 
baena, Trichobradybaena, Fruticicola, 
Calocochlea, and Pfeifferia, supported by 
synapomorphic character states 5(0), 6(0) 
and 7(0); 

(f) The clade embedded in (e), Mastigeulota, 
Platypetasus, Stilpnodiscus spb, 
Metodontia, Pseudobuliminus, Brady- 
baena, Trichobradybaena, Fruticicola, 
Calocochlea, and Pfeifferia, supported by 
synapomorphic character state 14(0). The 
monophyly of this clade was also confirmed 
by the SCT 

(g) The clade composed by Bradybaena, 
Trichobradybaena, Fruticicola, Calococh- 
lea, and Pfeifferia, supported by 
synapomorphic character states 1(1) and 
2(2). The monophyly of this clade was also 
confirmed by the SCT; 

(h) The clade Calocochlea and Pfeifferia, sup- 
ported by synapomorphic character states 
6(2), 7(1 ), and 19(1). The monophyly of this 
clade was also confirmed by the SCT. 
Based on the characters extracted from ter- 
minal genitalia, only part of the examined 
bradybaenid genera could be characterized by 
their autapomorphies. The terminal taxa 



Trishoplita, Euhadra, Aegista, Pliocathaica, 
Calocochlea, Pseudobuliminus, and Platy- 
petasus had the opposite situation, that is, 
considering the anatomy of terminal genitalia, 
they were not characterized by derived char- 
acters. As indicated in Figure 27, they seemed 
to be defined by the "loss of character state(s)" 
rather than autapomorphies that could be di- 
rectly observed. 



DISCUSSION 

The proposed phylogeny of bradybaenid 
genera has almost no similarity with the previ- 
ous systems reviewed by Nordsieck (2002). 
Previously, knowledge of bradybaenid system- 
atics came from the shell and very few genital 
features, and resulted from methodologically 
subjective analyses. The present cladograms, 
with too many branches, are not strongly sup- 
ported, indicating that this is a preliminary re- 
sult, providing a testable hypothesis of 
realtionships among bradybaenid genera. The 
hypothesis reflected by the cladogram in Fig- 
ure 27 is preferred, because it represents the 
best testable systematic hypothesis explain- 
ing the present data set. 

While the hypothesis presented is limited and 
requires the addition of data from many un- 
studied taxa, focus on some monophyletic 
branches with relatively strong support shows 
convincing results. The monophyletic clade of 
Calocochlea and Pfeifferia, representatives of 
the Helicostylinae Ihering, 1909, is well nested 
in the ingroup, suggesting that Helicostylinae 
are a bradybaenid group rather than a sepa- 
rate family (Helicostylidae sensu Schileyko, 
1991). In the definition of the family 
Helicostylidae sensu Schileyko (Schileyko, 
1991: 221), "the flagellum is variously devel- 
oped but is always present" is a dubious char- 
acter, because the present work shows that 
both Pfeifferia micans (type species of 
Pfeifferia) and Calocochlea coccomelos have 
no flagellum (Figs. 8, 9). 

The present phylogeny, by artificially exclud- 
ing helicostyline groups, is more or less com- 
patible with the tripartite plan of bradybaenid 
genera, that is, tripartitite classification of (1) 
modified Aegistini (= subfamily Aegistinae 
sensu Kuroda & Habe, 1949, listed as a tribe 
by Nordsieck, 2002; partial Aegistinae sensu 
Schileyko, 1991), (2) Euhadhni (= Euhadhnae 
Minato, 1988, listed as a tribe by Nordsieck, 
2002: including both Nesiohelix anä Euhadra), 



PHYLOGENY OF BRADYBAENIDAE 



109 



and (3) Bradybaenini (= Bradybaeninae sensu 
Kuroda & Habe, 1949, listed as a tribe by 
Nordsieck, 2002) (Fig. 27). The Aegistini was 
distinguished from Bradybaenini by the pres- 
ence of the flagellum. The present phyloge- 
netic hypothesis suggests the flagellum has 
been at least convergently lost in Brady- 
baenidae twice (Fig. 27). Accordingly, this 
character should not be employed as the 
proper character defining Aegistinae as used 
in the original designation (Kuroda & Habe, 
1949). The present hypothesis shows the reli- 
ability for the monophyly of {Thshoplita, 
Euhadra), which are distributed in both 
Aegistini (including genus Tishoplita: Kuroda 
&Habe, 1949;Minato, 1988; Schileyko, 1991; 
Nordsieck, 2002) and Euhadrini. Therefore, 
Aegistini should be condisered a paraphyletic 
group as indicated by the evidence that 
Trishoplita is embedded in the clade of 
Euhadrini. 

As clearly indicated by the all ETPs (3,502 
ETPs, not figured) from the first analysis and 
cladogramed based on the weighted charac- 
ters (in all 87 ETPs, not figured), Acusta oc- 
curred most basally in the cladograms. Also 
indicated by the rooted SCTs (Figs. 26, 27), 
Acusta, which was placed in the Bradybaenini 
(= Bradybaeninae sensu Russian and Japa- 
nese authors), was confirmed as the sister 
group to all the remaining bradybaenids 
examinated. Thus, the Bradybaeninae is a 
paraphyletic group, and Acusta should not be 
placed in Bradybaenini {sensu Nordsieck, 
2002). In summary, the result obtained dem- 
onstrates the Bradybaenini {sensu Nordsieck, 
2002) is not monophyletic. 

Cathaica was divided into several subgen- 
era byAndreae (1911) based on shell charac- 
ters. This study examined two of them, 
Cathaica (s. str.) and Cathaica {Pliocathaica). 
The results here show that Cathaica (s. str.) 
has a much closer relationship to the termi- 
nals in Clade (e), than to Pliocathaica, which 
is closest to Karaftohelix (Fig. 27). Accordingly, 
subgroups of Cathaica may be polyphyletic. 

Some characters used by other authors are 
thought to be unreliable after careful dissec- 
tions and thus are omitted from the present 
study. The widening of the basal bursa 
copulatrix duct, which was used by Schileyko 
(1991) as a diagnostic character of Brady- 
baenidae sensu Schileyko, is not included in 
the present data set, because this part varies 
in thickness according to physiological state, 
for example in Aegista accresens (Heude, 



1882), as observed by the author. Some char- 
acters once used to describe the genitalia are 
ambiguous and thus should be avoid being 
used. For example, the development status 
of the accessory sac, which is an autapo- 
morphy in the diagnostic definition for Helico- 
stylidae {sensu Schileyko, 1991) as "an 
accessory sac is weakly developed or lack- 
ing", seems not to be so definite and conse- 
quently is less informative or misleading. In 
the Bradybaeninae {sensu Nordsieck, 2002), 
the accessory sac shows a variety of devel- 
opment states, such as size range, differen- 
tiation of the internal pilasters, and occurrence 
of the mucous glands entering papilla. There- 
fore, the accessory sac comprises a series of 
characters instead of a character with several 
character states. In Helicostylinae (= Helico- 
stylidae sensu Schileyko, 1991), the genera 
Calocochlea and Pfeifferia (Figs. 8B, 9B) have 
an accessory sac with similar structure as 
those seen in the bradybaenine, for example, 
in Trichobradybaena (Fig. 23E) and in 
Pliocathaica (Fig. 12D). This suggests some 
characters, such as seen in the non-homo- 
plasious characters (both synapomorphies 
and autapomorphies) in this study, should be 
given special attention as to whether they are 
shared by or transformed into certain states 
in any other bradybaenid genus not covered 
in this work. Careful consideration of this prob- 
lem will enhance the reliability of the phylog- 
eny obtained. 

Cuezzo (1 998) points out that there are three 
different problems seen in the published lit- 
erature of the Xanthonychidae (= Helmintho- 
glyptidae). I see the same problems in the 
current the study of the Bradybaenidae. Virtu- 
ally in all the published literature, the system- 
atics of Bradybaenidae is established on 
"arbitrary narrative character transformations". 
Any effort to make a predictive classification 
of the Bradybaenidae (or any other group), as 
Nordsieck (2002) suggests, should be iDased 
on testable hypotheses, and after as many 
species as possible are examined. The present 
work does not aim to provide a définitive clas- 
siftcation of the Bradybaenidae, as many gen- 
era and and many other important characters, 
for example, anatomical (besides terminal 
genitalia), molecular, and chromosomal, are 
not included in the data set. However, it does 
suggest that the phylogeny of the Brady- 
baenidae is complex and considerable further 
work on the systematics for this group is 
needed. 



110 



wu 



ACKNOWLEDGEMENTS 

The author is in debt to iVIr. Hartmut Nord- 
sieck and Dr. Ronald Janssen (Natural His- 
tory Museum of Senckenberg, Germany) for 
helpful opinions on this the manuscript and for 
lending important specimens. Prof. Foico 
Giusti (Dipartimento di Scienze Ambientali, 
Italy) and Prof. Anatoly A. Schileyko (A. N. 
Severtzov Institute of Problems of Evolution, 
Russia) reviewed the manuscript and provided 
valued and helpful comments. Dr. Larisa A. 
Prozorova (Russian Academy of Sciences) 
provided some critical specimens. Dr. 
Bernhard Hausdorf (Universität Hamburg, 
Germany) shared the author his experience 
in the construction of phylogenies. Prof. 
Edmund Gittenberger (National Museum of 
Natural History, Netherlands) has provided lit- 
erature. Prof. Andrzej Wiktor (Wroclaw Univer- 
sity, Poland) offered good suggestions and 
shared his experience in dissection. Prof. 
George M. Davis (George Washington Univer- 
sity Medical Center, the United States) showed 
care to the author at all times, as well as his 
great patience in dealing with this manuscript 
as an editor. The study was financially sup- 
ported by the grants from the Natural Science 
Foundation of China (NSFC, No. 30100017) 
and the grant from Hebei University. 



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Revised ms. accepted 11 January 2004 



APPENDIX I: Taxa studied 

Acusta ravida (Benson, 1842), type species of 
the genus: IZCAS00944, Jiangning County, 
Jiangsu Province, coll. unknown. Four adult 
specimens (two dissected) and two young 
specimens. 

Aegista {Aegista) accrescens (Heude, 1882): 
IZCAS00027, Xiushan County (28.4°N, 
108. 9°E), Sichuan Province, 1986-VII-21, 
coll. De-Niu Chen & Jia-Xiang Gao. Six adult 
specimens (three dissected). 

Aegista {Plectotropis) gerlachi (E. Martens, 
1881): IZCAS00044, Guangdong Province, 
other collection data unknown. Nine adult 
(two dissected) and three young specimens. 

Bradybaena similahs (Rang, 1831) (not A. 
Férussac 1821; see Nordsieck, 2002), type 
species of the genus: IZCAS01072, Fuzhou, 
Fujian Province, 1975-X-16, coll. unknown. 
Forty adult (two dissected) and 17 young 
specimens. 

Cathaica (Cathaica) fasciola (Draparnaud, 
1801), type species of the genus: 
IZCAS01074, Pi County, Xuzhou, Jiangsu 
Province, 2000-V-2, coll. Qi-Lian Gin. Nu- 
merous specimens (three dissected). 

Cathaica {Pliocathaica) gansuica (Möllendorff, 
1899): IZCAS00210, Dachuanxiang, Zhouqu 
County, Gansu Province, 1200 m alt., 1998- 
V-9, coll. De-niu Chen & Guo-Qing Zhang. 
166 specimens (two dissected). 

Fruticicola fruticum (O. F. Müller, 1774), type 
species of the genus: IZCAS01009, lime- 
stone quarry near Klodzko, Wapniarka Mt., 
Lower Silesia, Poland, 1999-VI-26, coll. Min 
Wu & Andrzej Wiktor. Seven adult speci- 
mens (two dissected) and one young speci- 
men. 

Karaftohelix weyhchii (Schrenck, 1867): 
IZCAS01080, near Yushno-Sakhalinsk City, 
Sakhalin Island, Russia, 2001-VII-29, coll. 
Larisa A. Prozorova. One adult (dissected) 
and four young specimens. 

Mastigeulota kiangsinensis (E. Martens, 
1875), type species of the genus: 
IZCAS00003, Huangnipo, Badong County 
(31.0°N, 110.3°E), Hubei Province, coll. De- 
Niu Chen, 1984-VI-29. Six adult (two dis- 
sected) and one young specimen. 

Metodontia yantaiensis (Crosse & Debeaux, 
1863): IZCAS00131, Quyang County, Hubei 
Province, coll. Min Wu. Fifteen adult speci- 
mens (two dissected) and 19 young speci- 
mens. 



112 



WU 



Pseudaspasita binodata (Möllendorff, 1886), 
type species of the genus: IZCAS01075, 
Beiquan Park, Beipei, Chongqin, 1964-V-12. 
Twenty-seven adults (two dissected) and 12 
young specimens. 

Pseudiberus (Platypetasus) chentingensis 
Yen, 1935: IZCAS00163, Jiaozuo, Henan 
Province, 1999-VII-22, coll. Guang-Wen 
Chen. Six adults (two dissected) and 17 
young specimens. 

Stilpnodiscus moellendorffi Wu, 2001: 
IZCAS00081, type specimens, Shanggou, 
Shawanxiang, Dangchang County, Gansu 
Province, 1998-VI-6, coll. De-Niu Chen & 
Guo-Qing Zhang. 

Stilpnodiscus entochilus Möllendorff, 1899: 
IZCAS00076, Guoyuanxiang, Nanping 
County (now Jiuzhaigou County) (33.2°N, 
104. 2°E), Sichuan Province, 1000 m alt., 
coll. De-Niu Chen & Guo-Qing Zhang, 1998- 
V-18. 25 adult (four dissected) and 17 young 
specimens. 

Laeocathaica {Laeocathaica) subsimilis 
(Deshayes, 1873): IZCAS00006, Xingjian- 
xiang, Nanchong (30.8°N, 106.ГЕ), Sichuan 
Province, coll. unknown, 1964-V-20. Eleven 
adult specimens (four dissected). 

Pseudobuliminus (Pseudobuliminus) piligerus 
(Möllendorff, 1899): IZCAS00085, Anchang- 
hexiang. Wen County (33.0°N, 104. 6°E), 
Gansu Province, 1200 alt., coll. De-Niu 
Chen & Guo-Qing Zhang, 1998-V-19. 287 
specimens (three dissected). 

Trishoplita dacostae Gude, 1900: 
IZCAS00174, Kobayashi Miyazaki, Japan, 
coll. unknown, 1998-X. Six adult (three dis- 
sected) and six young specimens. 

Euhadra herklotsi (E. Martens, 1861): 
IZCAS01076, Ishigakijima, 1931-VII, coll. 
Shikanu (?). Two adult specimens (one dis- 
sected). 

Nesiohelix swinhoei (L. Pfeiffer, 1865), type 
species of the genus (in Richardson, 1983, 
the type species mistakenly given as 
Nesiohelix caspari; Kuroda & Emura, 1943): 
IZCAS00055, Yilan County (24.7°N, 
121.7°E), Taiwan Province, coll. unknown, 
1896-X. Two adult specimens dissected. 

Aegistohadra delavayana (Heude 1885) n. 
gen. and comb.: IZCAS00132, Zhibenshan 
Mt., Baoshan (26.3°N, 104.4°E), Yunnan 
Province, coll. De-Niu Chen, 1981-VI-23. 
Four adult specimens (two dissected) and 
three young specimens. Paratypes, IZCAS- 
type-2902-1 and IZCAS-type-2902-2, Fa 
Kouan Tchen, coll. Unknown. 



Aegistohadra seraphinica (Heude, 1889): 
paratypes, IZCAS-type-3071-1 and IZCAS- 
type-3071-2, Si-lin, Guangxi, coll. Unknown. 

Eueuhadra gonggashanensis, n. gen. & sp., 
type species of the genus: IZCAS00067, 
west slope of Gonggashan Mt., Kangding 
County (30.0°N, 101. 9°E), Sichuan Prov- 
ince; coll. De-Niu Chen & Jia-Xiang Gao, 
1982-IX-9. Fifteen adult (four dissected) and 
seven young specimens; IZCAS01061, bor- 
der of Jiuzaigou County and Songpan 
County (33°02'14.4"N, 103°42'32.1"E), 
Sichuan Province, coll. Min Wu. One adult 
specimen dissected. 

Trichobradybaena submissa (Deshayes, 
1873), type species of the genus: 
IZCAS00010, Hanzhong, Shaanxi Province, 
1992-IV-15, coll. De-Niu Chen. Numerous 
specimens (three dissected). 

Helix pomatia (Linné, 1758) (Helicidae), type 
species of the genus: IZCAS00188, lime- 
stone quarry near Klodzko, Wapniarka Mt., 
Lower Silesia, Poland, 1999-VI-26, coll. Min 
Wu & Andrzej Wiktor. One adult specimen 
dissected. 

Calocochlea coccomelos (Sowerby, 1840): 
SMF323619, Philippines: Sibuyan, ex 
Moellendorff. One specimen dissected. 

Pfeifferia micans Pfeiffer, 1845, type species 
of the genus: SMF323620, Philippinen: 
Cagayan, Pamplona, O. v. Moellendorff. 
One adult specimen dissected. 

Camaena platyodon (L. Pfeiffer, 1846) 
(Camaenidae): IZCAS00833, Hainan, other 
collection data lacking. Eleven adult (three 
dissected) and one young specimens. 



APPENDIX II: New taxa 

Aegistohadra, n. gen. 

Type species: Nanina delavayana Heude, 

1885: 102, pi. xxvi, fig. 8. 

Aegistohadra delavayana (Heude, 1885), 

n. gen. & comb. 

(Figs. 6, 28-31; Table 2) 

Material 

Four adults (IZCAS001 32-1 -4) of which two 
are full grown but broken and three young 
shells were examined, Zhibenshan Mt., 
Baoshan (26.3°N, 104. 4°E) ("Yunlong 
County" in original label is a printing error), 
Yunnan Province; coll. De-Niu Chen, 1981- 
VI-23. 



PHYLOGENY OF BRADYBAENIDAE 



113 



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Two paratype specimens of Nanina 
delavayana H., IZCAS-type-2902-1 and IZCAS- 
type-2902-2 (Fig. 32), Fa Kouan Tchen, coll. 
unknown. Two paratype specinnens of Helix 
seraphinica Heude, 1889 (Fig. 33). 

Etymology 

The genus name is derived from the names 
of two bradybaenid genera Aegista and 
Euhadra. 

Diagnosis 

Female part of genitalia with sac-shaped 
structure on vagina opposite to dart sac. 

Short Description 

Shell strongly depressed, sinistral, thick and 
solid. Umbilicus broad. Protoconch with radial 
wrinkles. Penial sheath absent; penis with a 
penial caecum near penial retractor; epiphallus 
with a flagellum; penis-epiphallus chamber 
absent; accessory sac undivided; in dart ap- 
paratus polylayered structure absent; VI -V4 
in the dart apparatus present; two sacs in- 
serted on vagina opposite to dart sac. 



Full Description 

Shell sinistral, thin but solid. Apex distinct. 
Whorls convex. Suture strongly impressed. 
Umbilicus narrow to moderately wide. Col- 
umella oblique; columellar lip dilated, slightly 
covering umbilicus. Adult shell and young shell 
with smooth surface, spiral furrows irregularly 
and sparsely present, ribs absent; growth lines 
not accompanied by irregular thickenings, 
background microscopic ripples absent. 
Protoconch with radial wrinkles. Immature 
shells unkeeled and unangulated. Body whorl 
large, unkeeled, weakly descending in front, 
with convex bottom. Aperture rounded, ob- 
lique. Lip toothless, equally expanded, thin 
within. Peristome reflexed equally. Parietal 
callus indistinct. Shell dull, opaque; yellowish 
brown with two brown bands, one above and 
one beneath periphery, the lower sometimes 
not as distinct as the upper. Bottom of body 
whorl yellowish brown (Figs. 29, 30). 

Animal uniformly gray. Jaw arcuate with 7- 
8 ribs dentating the concave margin, ribs con- 
tiguous, wide. In a paratype (IZCAS00132-2), 
radula with 169 rows of teeth, each with one 




FIG. 28. Distribution map. Square: Aegistohadra delavayana n. gen. & comb.; dots: 
Eueuhadra gonggashanensis n. gen. & sp. 



PHYLOGENY OF BRADYBAENIDAE 



115 



central tooth and 53 lateral teeth at each side; 
central tooth and lateral teeth L^-L^g unicus- 
pid; L20-L53 each with an endocone and an 
ectocone (Fig. 31 E). 

Genitalia: Penial sheath absent (Figs. 6A, 
31 A), except for some basal connective tis- 
sue present near atrium covering penis. Pe- 
nis short, slender, with a finger-shaped penial 
caecum near penial retractor (Figs. 6A, 6C, 
31 A, 31 B). Retractor simple, thin or thick, short 




or in moderate length. Epiphallus thick, short, 
with more or less protruding symmetrical 
epiphallic papilla (Figs. 6C, 31C). Penis- 
epiphallus chamber absent. Flagellum roundly 
blunt at end, with fairly smooth surface, thick, 
short (Figs. 6A, 6C, 31 A, 31 B); innerly folds 
not forming a C-shaped open tube towards fla- 
gellum or epiphallus (Figs. 6C). Pore of penial 
papilla located near the pore leading to penial 
caecum, mainly built by two pilasters derived 
from four thicker ones longitudinally arranged 
along penial inner wall (Figs. 6C, 31 C). Dart 
sac developed, with an accessory sac below. 
Accessory sac large in size, slightly elongated 
(Figs. 6A, 6D, 31A). Dart sac containing one 
dart. Dart about 7.0 mm in length, almost 
straight, slightly expanding basally; cross sec- 
tion of dart throughout rounded or ovate at 
lower part, upper 1/4 with 2 opposite sharp 
ridges. (Fig. 31 F). Inside dart sac, ADC shar- 
ing same entrance with DtC; VI -V4 present, 
V2 merged into a pilaster towards vagina; VI , 
V3 and V4 forming DtC; C23 present, but 
opened to vagina (Fig. 6D). Two sacs on va- 
gina opposite dart apparatus, one with two 
highly ridged pilasters, and another just be- 
neath the first one and with connective tissue 
inside, of unknown function (Figs. 6A, В & D); 
DVM absent. Mucous glands with two lobules, 
each as long as dart sac, stalks distinct, sepa- 
rated from dart sac and tied tightly to the trunk 
of vagina, inserting near base of dart sac (Fig. 
31A). Lobules simply branched, distally sac- 
shaped. Bursa copulatrix slightly elongated, 




FIG. 29. Aegistohadra delavayana n. gen. & 
comb., shell near mature, IZCAS00132-1. A, 
apical view; B, basal view; C, apertural view. Bar 
equals 5 mm. 



FIG. 30. Aegistohadra delavayana (Heude, 
1885), n. gen. & comb., shell, IZCAS00132-3. A 
broken but adult shell, showing aperture 
structure. Bar equals 5 mm. 



116 



wu 




FIG. 31. Aegistohadra delavayana (Heude, 1885), n. gen. & comb., IZCAS00132-2. A, general view 
of genitalia; B, penial complex; C, penis and epiphallus, opened; D, penial caecum (PC), opened; E, 
teeth of radula, bar equals 25 pm; F, dart, with cross-sections; G, a leaf of ovotestis. A-D, F, G, bars 
equal 1 mm. 



PHYLOGENY OF BRADYBAENIDAE 



117 



well differentiated from its duct (Fig. 31A). 
Bursa copulatrix duct moderately long, insert- 
ing low on vagina (Fig. 31A). Ovotestis palm- 
shaped, with single stalk (Fig. 31 G). Holotype: 
dart sac 10.0 mm in length, 2.5 mm in width, 
ratio of width to length 0.3; mucous duct length 
9.3 mm; vagina length 13.8 mm; bursa 



copulatrix duct 18.0 mm long, basal width 1.3 
mm; transverse diameter (maj.) of bursa 
copulatrix 1.8 mm, sagittal diameter (maj.) of 
bursa copulatrix 3.5 mm; vas deferens length 
16.3 mm; penis length 12.0 mm; flagellum 
length 5.8 mm; epiphallus 3.8 mm in length; 
penial retractor 3.8 mm long (Fig. 31 G). 




FIG. 32. Aegistohadra delavayana (Heude, 1885), n. gen. & comb., shell, paratypes, A-C, IZCAS-type- 
2902-1; D-F, IZCAS-type-2902-2. Bar equals 10 mm. 



118 



WU 



Range 
Southwestern China (Fig. 28). 

Remarks 

This species can be distinguished from all 
known bradybaenid species in that the female 
part of the genitalia has a sac-shaped struc- 
ture on the vagina opposite to the dart sac. It 
also differs from all bradybaenids, except 



Eueuhadra gonggashanensis, n. gen. & sp., 
in having a pronounced penial caecum. 

Based on shell features, although distinctly 
larger (diam. maj.: 55 mm; min.: 48 mm; alt.: 
30 mm), it is possible that Helix seraphinica 
Heude, 1889, from Silin (as "Xilin" in today's 
spelling, Guangxi Province) should be placed 
in Aegistohadra because of their similar shell 
shape (Fig. 33), as suggested by H. Nordsieck 




FIG. 33. Helix seraphinica Heude, 1889, shell, paratypes, A-C, IZCAS-type-3071-1; D-F, IZCAS-type- 
3071-2. Bars equal 5 mm. 



PHYLOGENY OF BRADYBAENIDAE 



119 



(pers. comm.). However, Helix seraphinica we 
cannot be certain until its anatomy is known, 
considering the great morphological diversity 
shown in helicoid shells. 

Eueuhadra n. gen. 
Type species: Eueuhadra 
gonggashanensis, n. sp. 

Eueuhadra gonggashanensis, n. sp. 

(Figs. 7, 28, 34-39; Table 3) 

Material 

Holotype (IZCAS00067-1), West slope of 
Gonggashan Mt., Kangding County (30.0°N, 
101.9°E), Sichuan Province; coll. De-Niu Chen 
& Jia-Xiang Gao, 1982-IX-9. Paratypes 14 
(IZCAS00067-2-15), the same data as holo- 
type; seven young specimens (IZCAS00067- 
16-22) were also examined; paratype 1 
(IZCAS01061), border of Jiuzaigou County 
and Songpan County (33°02'14.4"N, 
103°42'32.1"E), Sichuan Province; 3311 m a. 
s. I.; coll. Min Wu, 2001 -X-4. 

Etymology 

The genus name comes from "eu-" (real) 
and the bradybaenid genus Euhadra. The 
species is named after the holotype locality: 
Gonggashan Mountains. 

Diagnosis 

A simple penis-epiphallus chamber present; 
dart sac with multiple mucous branches. 

Short Description 

Shell depressed, dextral, thin but solid. Um- 
bilicus very narrow and more or less covered 
by columellar margin of the peristome. 
Protoconch shell granulöse. 

Penial sheath absent; penis distally with an 
outstanding tube-shaped penial caecum; 
epiphallus with a flagellum; a simple penis- 
epiphallus chamber present; dart sac with a 
distinct and relatively large accessory sac on 
the end on which a bundle of mucous glands 
is inserted per one common duct; accessory 
sac undivided; in dart apparatus, polylayered 
structure absent, VI -V4 present. 

Range 
W China. 

Full Description 

Shell dextral, depressed, thin but solid. Apex 
distinct. Whorls convex. Suture impressed. 



Umbilicus very narrow and more or less cov- 
ered by columellar margin of peristome. Col- 
umella very oblique. Spiral furrows absent, 
without ribs, growth lines not accompanied by 
irregular thickenings, microscopic ripples ab- 
sent. Protoconch finely granulöse, granulation 
regularly arranged. Teleoconch finely and un- 
evenly granulöse on upper spire. Immature 
shells bluntly angulated. Whorls increasing 
rapidly; body whorl fairly large, unkeeled. 




FIG. 34. Eueuhadra gonggashanensis, n. gen. & 
sp., shell, IZCAS00067-1, Holotype, A, apical 
view; B, basal view; C, apertural view. Bar equals 
5 mm. 



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PHYLOGENY OF BRADYBAENIDAE 



121 



slightly descended in front, with convex base. 
Aperture rather broadly lunate, more or less 
oblique. Lip toothless, uniformly thickened 
within, forming a ring-like thickening. Peris- 
tome thin, uniformly reflexed. Parietal callus 
distinct. Periostracum uniformly in greenish 
brown, bandless. Bottom of body whorl with 
same or lighter colour (Figs. 34, 35 & 39; 
Table 3). 

Animal with numerous brown spots on the 
anterior half. Jaw arcuate with 10-12 ribs 




FIG. 35. Eueuhadra gonggashanensis, n. gen. & 
sp., shell, IZCAS00067-5, Paratype, a shell with 
periostracum on. A, apical view; B, basal view; 
C, apertural view. Bar equals 5 mm. 



dentating the concave margin, ribs wide and 
almost contiguous. Radula of holotype with 
133 rows of teeth, each with one central tooth 
and 44 lateral teeth on both sides; central 
tooth with 1 tiny cusp at each side; lateral 
teeth L^-L^^ each with an ectocone; L^g-Lj^ 
each with a tiny endocone and an ectocone; 
main cones and ectocones of Lj^-L^^ bicuspid 
respectively, two cusps of ectocone roundly 
blunt (Fig. 36D). 

Genitalia: Penial sheath absent (Figs. 7A, 
36A, 37A). Penis of moderate length, swollen, 
with a tube-like penial caecum (PC) near pe- 
nial retractor. Penial retractor short. Epiphallus 
thick, short. Epiphallic papilla depressed, sym- 
metrical. Penis internally with three thick pe- 
nial pilasters and two thinner ones among 
them (Figs. 36B, 370). Near to the pore lead- 
ing to penial caecum, a papilla, built partially 
by above-mentioned pilasters present (Fig. 
7B). Flagellum thick, short, smooth, abruptly 
tapering and forming a vermiform appendix 
(Fig. 36A). Penis-epiphallus chamber present, 
small, simple (Fig. 7E). Vas deferens inserted 
on flagellum, with inner folds forming a 0- 
shaped open tube towards flagellum (Fig. 70). 
Dart sac containing one dart. Dart approxi- 
mately 2 mm, medially rounded, apically trap- 
ezoid in cross section (Fig. 37E, only seen 
from the spirit material of IZOAS01061; in all 
dissected specimens of IZOAS00067, the 
darts are completely eroded, because of hav- 
ing been first fixed in formalin before being 
preserved in alcohol). Mucous glands longer 
than dart sac, inserted at the end of accessory 
sac; with 11-13 (in two specimens of 
IZOAS00067) mucous lobules radially ar- 
ranged, stalk of lobule indistinct; each lobule 
simply branched and consisting of slightly ex- 
panded vesicles, not expanded distally (Fig. 
36A). Accessory sac developed, innerly 
simple except for some narrow pilasters; a 
bundle of mucous glands inserting on the end 
of AS, from a common entering tube, its inner 
entrance without papilla (Fig. 7D); ADO share 
the same entrance with DtO; \/1-V4 present; 
V1, V2 and V4 forming DtO; 023 present and 
tiny, with entrance leading to ADO (Figure 7D). 
DVM absent. Polylayered structure (PLs) ab- 
sent. Bursa copulatrix ovate, not well differen- 
tiated from bursa copulatrix duct (Fig. 36A). 
Bursa copulatrix duct of moderate length, 
wide, inserting high on vagina (Figs. 36A, 37A). 
Ovotestis palm-shaped, distinctly branched 
and two stalks closely arranged and having a 
common duct; in holotype, ovotestis embed- 



122 



WU 



PC 



PR 



MG 




FIG. 36. Eueuhadra gonggashanensis, n. gen. & sp., IZCAS00067-1, Holotype, A, general view of 
genitalia, bar equals 2 mm; B, penis, opened; C, penial caecum, opened; D, teeth of radula, bar equals 
25 |jm; E, a leaf of ovotestis, bar equals 0.5 mm; F, ovotestis matrix, magnified. B, C, bars equal 1 mm. 



PHYLOGENY OF BRADYBAENIDAE 

Fl Ер PR 



123 




FIG. 37. Eueuhadra gonggashanensis, n. gen. & sp., genitalia, IZCAS01061 , paratype, A, general view 
of genitalia; B, a branch of mucous glands; C, penis, penial caecum (PC), and flagellum, opened; D, 
ovotestis; E, dart with cross sections. Bars equal 1 mm. 



124 



WU 



ded in matrix composed of disordered fibers 
(Fig. 36F; in the otlner examined specimens 
the matrix normal); stalks fairly long (Figs. 
36E, 37D). Holotype: dart sac 3.3 mm in 
length, 1 .8 mm in width, ratio of width to length 
0.5; mucous duct length 7.3 mm; vagina 
length 4.6 mm; free oviduct 12.2 mm; bursa 
copulatrix duct length 10.3 mm, bursa 
copulatrix duct basal width 1 .5 mm; transverse 
diameter (maj.) of bursa copulatrix 1.5 mm, 
sagittal diameter (maj.) 2.2 mm; vas deferens 
length 13.7 mm; penis length 7.0 mm; 
epiphallus 6.4 mm; flagellum length 3.0 mm; 
PR length 2.2 mm. 

Range 

Western Sichuan, the species was known 
only from two localities where type and holo- 
type material were collected (Fig. 28). 

Remarks 

This species differs from all known 
bradybaenids by having a simple penis- 



epiphallus chamber. The sisterhood of this 
species and Aegistohadra delavayana 
(Heude, 1885), n. gen. & comb., is suggested 
by their common derived character the penial 
caecum. 

Ecology 

This species (IZCAS01061) inhabits high 
mountains (Fig. 38B), in very low density. The 
environment is extremely wet, inside a dark fir 
forest, where the stones and fallen trunks are 
covered by a thick layer of lichen and moss 
which sometimes reaches the thickness of 
approximately 50 cm (Fig. 38C, D). The speci- 
men (IZCAS01061), the only collection after 
careful search of about 500 m^ in the forest 
was found inactive under moss. The popula- 
tion in Jiuzaigou seems to be isolated from 
another known population in the Gonggashan 
Mountains. Based on 30-days field work cov- 
ering the area from Dujiangyan to Jiuzaigou 
along Minjiang River, it has been confirmed 
that these two populations are fairly sepa- 




FIG. 38. Habitat of Eueuhadra gonggashanensis, n. gen. & sp. A, Paratype, IZCAS01 061 , in its habitat; 
B-D, natural environment conditions of locality for Paratype IZCAS01061. 



PHYLOGENY OF BRADYBAENIDAE 



125 



rated. In the same area, no helicoid snails 
were found and only the non-helicoid snail 
Deroceras (Deroceras) altaicum (Simroth, 
1 886) (Wiktor et al., 2000), which is widely dis- 
tributed in the whole vally of Minjiang River 
and its neighboring mountains. It is also inter- 
esting that no conchologically similar species 
was recorded in this region before (e.g., 
Pilsbry, 1934). 




FIG. 39. Eueuhadra gonggashanensis, n. gen. & 
sp., shell, IZCAS01061, paratype. A, apical view; 
B, basal view; C, apertural view. Bar equals 5 mm. 



MALACOLOGIA, 2004, 46(1): 127-156 

TOWARD COMPREHENSIVENESS: INCREASED MOLECULAR SAMPLING 
WITHIN CYPRAEIDAE AND ITS PHYLOGENETIC IMPLICATIONS 

Christopher P. Meyer 

Florida Museum of Natural History, University of Florida, 
Gainesville, Florida 32611 USA; cmeyer@flmnh.ufl.edu 



ABSTRACT 

This paper introduces 73 additional taxa to the existing mitochondrial molecular data- 
base of 202 taxa for the Cypraeidae and addresses the systematic implications of their 
inclusion. Five outgroup members from the Ovulidae are also added. Sequence data are 
included from all previously missing extant named genera (Propustularia, Barycypraea 
and Schilderia), completing the overall "generic-level" framework for living cowries. Newly 
added taxa include 47 recognized species, 25 subspecies, and six undescribed taxa. Phy- 
logenetic results generally are consistent with previous arrangements, with few minor ad- 
justments. The most significant findings are that: (1) currently recognized Nesiocypraea is 
broken into two disparate clades, a deeply rooting Nesiocypraea sensu stricto group and 
the more derived Austrasiatica (Lorenz, 1989). (2) Two newly included Barycypraea taxa 
are sister to Zoila, reaffirming the validity of the subfamilial clade Bernayinae. (3) The 
inclusion of a significant number of added Erroneini taxa (N = 24) creates a phylogenetic 
challenge because of poor support and recovered relationships inconsistent at first glance 
with traditionally recognized affinities. In order to maintain nomenclatural consistency. 
Errónea is maintained at a generic level, whereas Adusta is dropped to subgeneric status 
within Errónea. Greater than 90% of currently recognized species are included, and 93% 
of these are supported by molecular criteria. Moreover, more than 70% of the tested, 
recognized subspecies are distinct. The phylogeny provides one of the most comprehen- 
sive, species-level frameworks to date for testing diversification theories in the marine 
tropics. 

Key words: Cypraeidae, molecular systematics, taxon sampling, Cypraea. 



INTRODUCTION 

Cowries (Gastropoda: Cypraeidae) are taxo- 
nomically one of the best known of all mollus- 
can groups, and have been used frequently to 
examine speciation and biogeographic pat- 
terns in the marine tropics (Schilder, 1965, 
1969; Foin, 1976; Kay, 1984, 1990; Meyer, 
2003). A wealth of taxonomic (Schilder & 
Schilder, 1938, 1971; Schilder, 1939; Lorenz & 
Hubert, 1993; Groves, 1994; Lorenz, 2002), 
anatomical (Troschel, 1863; Vayssière, 1923, 
1927; Riese, 1931; Risbec, 1937; Schilder, 
1936; Kay, 1957, 1960, 1963, 1985, 1996; 
Bradner & Kay, 1996; Lorenz, 2000), biogeo- 
graphic (Schilder, 1965, 1969; Foin, 1976; 
Burgess, 1985; Liltved, 1989; Lorenz & 
Hubert, 1993; Lorenz, 2002) and fossil data 
(Schilder & Schilder, 1971; Kay, 1990, 1996; 
Groves, 1994) is available for the group; how- 
ever, what has been lacking is a well-resolved, 
comprehensive species-level phylogeny. 



These phylogenetic hypotheses of relationship 
establish sister pairs at the appropriate taxo- 
nomic level and provide the framework to test 
diversification theories. Meyer (2003) introduced 
molecular data for 234 taxa in Cypraeidae and 
generated phylogenetic hypotheses for most 
major clades as well as sister-group relation- 
ships for most species. Systematics for 
Cypraeidae were reviewed in light of the results 
and diversification patterns within the tropics 
were addressed. The study presented herein 
significantly increases the comprehensiveness 
of taxon sampling in the group by introducing 73 
Cypraeidae and five Ovulidae taxa to the exist- 
ing molecular dataset and discusses their sys- 
tematic implications. In addition to broader 
taxonomic sampling, this paper presents the re- 
sults of broader geographic sampling. The ap- 
pendix lists 147 localities added across the 
various taxa. Five outgroup taxa from six locali- 
ties are included, and 67 recognized cypraeid 
species or subspecies are added from 75 locali- 



127 



128 



MEYER 



ties. The remaining 66 localities were added to 
supposedly known taxa, but revealed six previ- 
ously unrecognized taxa, some of which may 
correspond to names currently in synonymy 
upon review of type localities. 



Molecular Methods 

Most methods follow protocols detailed in 
Meyer (2003) for all aspects of preservation, 
extraction, amplification, and sequencing. Tis- 
sue samples were acquired from a variety of 



MATERIALSAND METHODS 



Recognition Criteria: ESU versus OTU 

The ultimate goal of this project is to con- 
struct a comprehensive phylogeny of cypraeid 
gastropods at the appropriate level for diversi- 
fication studies. As such, the operational taxo- 
nomic unit (OTU) chosen for phylogenetic 
analyses generally represents an evolution- 
arily significant unit (ESU) that must fulfill 
some minimal criteria established through 
genetic scrutiny. First, mtDNA haplotypes of 
sampled individuals must represent a mono- 
phyletic clade; yet this alone is not sufficient, 
because any phylogeny has a plethora of 
monophyletic groups, because a clade re- 
quires only two individuals. Thus, auxiliary cri- 
teria are required to delineate significant units. 
Within cowries, these additional criteria are (1) 
geographic distinction or allopatry, (2) signifi- 
cant genetic distance from the sister group 
such that pairwise distance comparisons yield 
a bimodal distribution, and/or (3) taxonomic 
recognition by previous workers. An OTU is 
included in analyses only if at least two of 
these three criteria are met. Most OTUs fulfill 
all three criteria and are considered evolution- 
arily significant units (ESUs) {sensu Moritz, 
1994). These criteria are erected in order to 
delineate independent evolutionary trajecto- 
ries, but do not guarantee that the units are 
reproductively isolated. In a few instances, two 
of the three criteria (genetic separation and 
taxonomic recognition) are not supported by 
the third (exclusive geographic signatures). 
While the genetic differences (monophyly) 
between populations indicate some indepen- 
dent period of evolutionary history between 
geographic regions, it appears that, on occa- 
sion, haplotypes from outlying regions can mix 
back into the sister gene pool. The few cases 
where all three criteria are not fulfilled always 
occur on the periphery of regions (e.g., 
Marquesas, Hawaii) and show asymmetrical, 
"downstream", dispersal events (Fig. 1). As 
circumscribed, all ESUs discussed indicate 
independent evolutionary histories, but alter- 
native criteria, such as either nuclear markers 
or breeding experiments, are needed to verify 
reproductive isolation. 



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gaskoini 976 
astaryl2136 ^ 

cumingii TIK 2138 "yf 
astaryl 1472 
astaryl 2134 

astaryl 2135 
astaryl 2137 ^ 

cumingii TIK 1970 '^f 
astaryi 1105 
astaryi 1471 
astaryi 2133 

GARCIAI2413 "^ 

cumingii TIK 2140 
— cumingii RR 1966 
■ cumingii RR 2144 

L cumingii RR 2143 
cumingii RR 1968 
cumingii TIK 1969 
cumingii TIK 2139 
cumingii RR 2141 
' cumingii RR 2142 
cumingii RR 1967 
' cumingii HUA 700 



FIG. 1. ESU vs. OTU criteria. Phylogram show- 
ing the relationships among members of the 
Pacific Cribrarula subclade, with bootstrap val- 
ues for major groups. Four distinct clades are 
evident, and the names presented on the right: 
Cribrarula catholicorum, C. gaskoini, С astaryi, 
and C. cumingii. Note that single individuals of 
two newly included taxa, С. taitae and С gardai 
(white stars), nest within two of the major clades 
and show little variation (a single mutation). 
These two new taxa are introduced as OTUs, 
because of their distinct morphology and geog- 
raphy (American Samoa and Easter Island, re- 
spectively), but are currently not considered 
ESUs by molecular criteria. All individuals from 
the Marquesas are С astaryi; however, two in- 
dividuals of С cumingii possess haplotypes be- 
longing to the C. astaryi clade as well (dark stars). 
While the two haplotype clusters are distinct, the 
pattern indicates uni-directional exchange of lar- 
vae downstream from the Marquesas (C. astaryi). 
Molecular criteria recognize these two clades as 
ESUs with historically limited exchange. (TIK = 
Tikehau, RR = Rangiroa, HUA = Huahine, all С 
astaryi from Marquesas, all C. gaskoini from 
Hawaii, and all C. catholicorum from Solomon 
Islands) 



INCREASED MOLECULAR SAMPLING IN CYPRAEIDAE 



129 



sources and locations (listed in the 
acknowledgements and appendix). Most 
sannples were preserved in 95% ethanol. DNA 
extraction was perfornned using DNAzol 
(Chomczynski et al., 1 997) using one-half vol- 
umes and following the manufacturer's proto- 
col (Molecular Research Center, Inc.) with the 
exception that the digestion step was in- 
creased by an additional 24 or 48 h. PCR was 
performed as described in Meyer (2003). COI 
primers were as follow (from Folmer et al., 
1994): LCO-1490 (5-3') GOT CAA CAA АТС 
ATAAAG ATATTG G, and HCO-2198 (5'-3') 
TAA ACT TCA GGG TGA CCA AAA АТС A. 
For problematic taxa, these primers were de- 
generated as follows: dgLCO-1490 (5'-3') 
GGT CAA CAA АТС ATAAAG AYA TYG G, and 
dgHCO-21 98 (5'-3') TAA ACT TCA GGG TGA 
CCA AAR AAY CA. Two internal primers were 
designed for small amplifications of degraded 
DNA: InCypLCO (5'-3') CGT YTA AAT AAT 
ATAAGYTTYTG, and InCypHCO (5'-3')CGT 
ATA TTA ATA ATT GTT GTA AT. Palumbi's 
(1996) 16Sar and 16Sbr primers were used 
for16S:16Sar(5'-3')CGCCTGTTTATCAAA 
AAC AT and 1 6Sbr (5'-3') CCG GTC TGA ACT 
CAG АТС ACG T Two internal primers were 
designed for small amplifications of degraded 
DNA: I n 1 6Sar (5'-3') GGG СТА GTA TGA ATG 
GTT TGA, and ln16Sbr (5'-3') ATG CTG TTA 
TCC CTATGG TAA CT. The polymerase chain 
reaction was carried out in 50 pi volumes, us- 
ing 1 pi of template. Each reaction included 5 
pi 10X PCR buffer, 5 pi dNTPs (lOmM stock), 
2 pi of each primer (lOpM stock), 3 pi MgClj 
solution (25 mM stock), 0.2 pi Taq (5 Units/pl 
stock) and 31.8 pi ddH20. Reactions were run 
for 35-40 cycles with the following parameters: 
an initial one min denaturation at 95°C; then 
cycled at 95°C for 40 sec (denaturation), 40°C 
to 44°C (COI) or 50°C to 54°C (1 6S) for 40 sec 
(annealing), and 72°C for 60 sec (extension). 
Successfully amplified products were cleaned 
for cycle sequencing using Wizard'^' PCR Preps 
(Promega). Sequencing also followed Meyer 
(2003) with all new sequences generated using 
ABI chemistry and sequencers. Sequences were 
generated from the resulting electrophenograms 
using Sequencher (Gene Codes). 

All primer sequences, aligned COI and 16S 
sequences and Nexus files are available at 
the archived data web pages of the Florida 
Museum of Natural History Malacology Depart- 
ment (http://www.flmnh.ufl.edu/malacology/ 
archdata/Meyer2004), and new sequences are 
deposited in Genbank under accession num- 
bers AY534351 through AY534503. 



Phylogenetic Analyses 

The 297 operational taxonomic units (OTUs) 
presented in this paper were selected from an 
extensive database comprised of over 2,000 
sequenced individuals. In general, taxa are 
included if they exhibit distinctive geographic 
and/or genetic signatures. In most instances, 
new OTUs are recognized in the literature as 
either species (N = 47) or subspecies (N = 
25). This paper introduces six previously un- 
recognized taxa. 

The increasing size of this dataset presents 
computational and heuristic challenges for 
phylogenetic analyses. Two weighted trans- 
version bias parsimony searches (3:1 and 5:1 ) 
were performed on the complete dataset us- 
ing PAUP* (Swofford, 1998). At first, 250 ran- 
dom-addition replicate searches were 
performed, but with a tree limit of ten imposed 
to minimize search time on suboptimal islands. 
After 250 replicates, the most parsimonious 
topologies were used as starting trees for ex- 
haustive searches without tree limits. This 
strategy was employed for both weighted 
analyses, and the most parsimonious topolo- 
gies were pooled and evaluated using likeli- 
hood criteria. ModelTest v. 3.06 (Posada & 
Crandall, 1998) was used to select the most 
appropriate model for likelihood parameters. 
The most likely weighted parsimonious trees 
were then compared using consensus meth- 
ods. 

A two-tiered, compartmentalized strategy 
was adopted that followed Meyer (2003) for 
levels of topological support. The strict con- 
sensus topology derived from the most likely 
overall analyses was divided into four 
subequal components called basal, midi, 
mid2, and derived. Because the basal, mid1 
and mid2 cohorts are necessarily paraphyletic 
groups that include the common ancestor and 
some, but not all, of its descendants, repre- 
sentative derived clades were included in the 
paraphyletic analyses. In this way multiple 
derived member clades overlapped between 
more basal and derived analyses, and the 
overall topology could be "scaffolded" together 
by linking clades shared in both basal and 
derived compartments. 

Within each of the four subanalyses, parsi- 
mony searches were performed using a 5:1 
transversion bias. Both bootstrap (Felsenstein, 
1985) analyses (1,000 replicates) and decay 
(Bremer, 1994) analyses (TreeRot v2; 
Sorenson, 1999) were performed to establish 
levels of support. Results from Bayesian meth- 



130 



MEYER 



ods (Mr. Bayes v3.04b) are not reported in this 
paper, but were generated for the four sub- 
groups and compared to the combined parsi- 
mony/likelihood methods utilized in PAUP*. 
Overwhelmingly, they were consistent with the 
results presented here, but on few occasions 
differed in hypotheses of relationship. The 
scaffolded parsimony global topologies were 
compared to the scaffolded Bayesian topol- 
ogy using likelihood criteria in PAUP*. The 
combined topology derived from the compart- 
mentalized Bayesian subsets was less likely 
than the overall topologies found using the 
combined parsimony/likelihood criteria. It ap- 
pears that Bayesian results depended on taxon 
sampling and outgroup inclusion. While this 
finding may be of interest to the general sys- 
tematic community, it is not a point specifically 
addressed in this paper. 



RESULTS 

The final culled dataset contained 297 OTUs 
and 1 , 1 07 characters, 493 base pairs from 1 6S 
and 614 bases from COI. For 16S, alignment 
followed those presented in Meyer (2003) 
based on secondary structure. Weighted par- 
simony searches resulted in 512 equally most 
parsimonious trees (MPTs) for 3:1 Ti:Tv and 480 
trees for 5:1 searches. Derived portions of the 
comprehensive topology were consistent. Thus, 
all named clades (subfamilies, tribes and gen- 
era) presented in Figure 2 are found in all to- 
pologies, except one mentioned below. 
However, the topologies recovered from alter- 
nate weightings differed in five deeper regions, 
all of which are poorly supported regardless of 
methodology. First, 5:1 topologies placed the 
clade consisting of Propustularia/Nesiocypraea/ 
Ipsa basal as sister to all other cowries. In 3:1 
topologies this clade moves up one node and 
is sister to Erosariinae. Second, the pustulose 
clade consisting of Nucleolaria/Cryptocypraea/ 
Staphylaea is monophyletic in 5:1 trees, while 
in 3:1 topologies these genera are a basal 
paraphyletic grade leading to the clade includ- 
ing Monetaria/ Perisserosa/Erosaria. Third, in 
5:1 topologies Perisserosa is sister to Erosaria, 
whereas in 3:1 trees, Perisserosa is sister to 
IVIonetaria. Fourth, the arrangement of major 
groups along the backbone from Umbiliini to 
Cypraeovulinae conflicts. Results from 5:1 
searches are shown in Figure 2, whereas in 
3:1 topologies, Notocypraea and Cypraeovula 
(Cypraeovulinae) are a basal sister grade lead- 
ing to more derived member groups. Finally, 
the basal arrangement within Erroneini is dif- 



ferent. In 3:1 topologies Purpuradusta is more 
basal, while in 5:1 trees. Errónea is more basal. 

When alternative topologies were evaluated 
using ModelTest, the GTR+I+G model was se- 
lected as the best-fit model. When both the 3:1 
MPTs and 5:1 MPTs were evaluated using the 
selected likelihood criteria [Iset base = 
(0.315128 0.136452 0.111915), Nst = 6, 
Rmat = (0.99559 41.36057 1.0461 1.68935 
22.78834), rates = gamma, shape = 0.562423, 
Pinvar = 0.48426], the 5:1 subset was signifi- 
cantly more likely (ANOVA: p < 0.001 , average 
-In likelihood = 49513.8). Therefore, results 
from the 5:1 searches are presented herein. 

The overall relationships among major sub- 
groups recovered in the 5:1 MPTs are more 
consistent with both morphological and fossil 
evidence in addition to being more likely based 
on molecular data. In particular, a monophyl- 
etic pustulose clade is more parsimonious for 
conchological and anatomical features, be- 
cause it is more likely that a bumpy shell was 
derived a single time, rather than being derived 
either twice independently, or derived once then 
lost. Also, the basal, paraphyletic status of 
Notocypraea and Cypraeovula within the 3:1 
topologies is inconsistent with the fossil record 
for both groups relative to more derived mem- 
bers of the 3:1 MPTs (i.e., Umbilia, Barycypraea, 
and Zoila), which appear earlier in the record 
and root more deeply in the 5:1 topologies. Also, 
the sister-group relationship of the two genera 
is more consistent with paleobiogeography (the 
breakup of Gondwanaland) and recognized 
affinities based on both conchological and de- 
velopmental criteria. The other major discrep- 
ancies between the 3:1 and 5:1 MPTs (most 
basal cowries, Perisserosa affinities, and posi- 
tion of Purpuradusta) are more ambiguous 
based on alternate criteria (morphological or 
paleontological). 

Suprageneric Relationships (Fig. 2) 

Overall, suprageneric results were consistent 
with previous systematic findings (Meyer, 
2003), with two exceptions. First, Ipsa falls out- 
side Erosariinae and is no longer sister to 
Erosariini, but instead is allied with newly in- 
cluded Propustularia and Nesiocypraea sensu 
stricto. New sequence data from Nesiocypraea 
teramachii neocaledonica did not result in an 
affinity with other recognized '^ Nesiocypraea" 
species (Л/, hirasei, N. sakurai and N. langfordi). 
Instead, Nesiocypraea teramachii roots more 
deeply in the phylogeny as a distant sister to 
Ipsa childreni, within a clade that includes both 
Ipsa and Propustularia. Thus, the inclusion of 



INCREASED MOLECULAR SAMPLING IN CYPRAEIDAE 



131 



Cypraeidae 




Ovulidae 

PROPUSTULARIA 

NESIOCYPRAEA 

Ipsa 



Cryptocypraea 

Nucleolaria 

Staphylaea 

Monetaria 

Perisserosa 

Erosaria 



Umbilia 



Muracypraea 
Cypraea 



Macrocypraea 
Leporicypraea 
Mauritia 



BARYCYPRAEA 

Zoila 



Talparia 
Luria 



Trena 

Annepona 

Chelycypraea 

Austrocypraea 

Arestoides 

Lyncina^ 



Pustulaha 
Neobernaya 
Pseudozonaria 
SCHILDERIA 
Zonaria 
r- Notocypraea 
^ Cypraeovula 



Palmadusta 

Bistolida 

Ovatipsa 

Talostolida 

Chbrarula 



Erronelnae 



Austrasiatica^ 

Pamulacypraea 

Errónea'^ 

Purpuradusta 

Contradusta 

Notad usta 

Eclogavena 

Melicerona 

Blasicrura 



Erosariini 

Umbiliini 
Cypraeini 

Mauritiini 

Luriini 
Austrocypraeini 



BASAL 



MIDI 



Bistolidini 



Erroneini 



MID 2 



DERIVED 



FIG. 2. Strict suprageneric consensus topology of 480 most parsimonious trees derived from a 
5:1 Ti:Tv weighted search strategy of all 297 OTUs. Subfamilies are indicated with arrows and 
tribes are listed to the right. The four compartments for further subanalyses are bracketed to the 
right. The four newly added genera are capitalized and bolded. ^Lyncina includes the subclades 
Callistocypraea, Miolyncina and Lyncina as reported in Meyer (2003). ^Austrasiatica replaces 
the prior use of Nesiocypraea for the same clade. ^Errónea now includes Adusta, formerly rec- 
ognized as the sister taxon. 



132 



MEYER 



two new ancient lineages {Propustularia and 
Nesiocypraea) affects the relative position of 
Ipsa. Moreover, the finding that Nesiocypraea 
teramachii is not related to other previously rec- 
ognized Nesiocypraea, compels me to recog- 
nize the clade Austrasiatica proposed by Lorenz 
(1 989) at the generic level for the group includ- 
ing Austrasiatica hirasei, A. sal<urai, and A. 
langfordi. There are some conchological and 
anatomical features that support this separa- 
tion. The left posterior terminal ridge in 
Nesiocypraea is more produced and separate 
from the body of the shell, whereas in 
Aur^rasiatica, the ridge is continuous with the 
booy. Lorenz (pers. comm.) also states that (1 ) 
Nesiocypraea lacks a distinct embryonic band- 
ing, having instead only a darker middorsal 
zone, (2) Nesiocypraea have a proportionally 
larger spire, and (3) the darker pattern of the 
shell is absent in juvenile Austrasiatica, only 
gained after the deflection of the labral margin; 
whereas, the darker pattern can be part of ju- 
venile Nesiocypraea shells. Additionally, the 
rachidian tooth of Nesiocypraea lacks the 
prominent paired basal denticles present in the 
three Austrasiatica taxa, and the tooth shape 
is less elongated and squared, whereas the 
rachidian in Austrasiatica narrows toward the 
cusps (Bradner & Kay, 1996). The fact that 
Austrasiatica was erected to differentiate the 
three species (albeit incorrectly aligned with 
Sctiilderia) is also an indication that the two lin- 
eages possess independent histories. The deep 
position of Propustularia within the cowrie phy- 
togeny is not surprising because it is one of the 
oldest of extant taxa, extending back to the 
Lower Eocene (Kay, 1996). 

The second suprageneric difference concerns 
the relative position of Zoila in the overall phy- 
logeny and is caused by the inclusion of se- 
quence data for two taxa from the ancient 
lineage Barycypraea. These new data indicate 
that Barycypraea teulerei and Barycypraea 
fultoni are sister taxa, and they are sister to 
Zo/7a. This Barycypraea/Zoila clade is recog- 
nized as the extant members of the subfamily 
Bernayinae, a group that includes many extinct 
fossil members and extends back into the Me- 
sozoic (Kay, 1996). These new data change 
the relative position of Zo/7a to Cypraeinae 
(Meyer, 2003); however, the topology in this 
region of the phylogeny is poorly supported. 

The final suprageneric addition to the molecu- 
lar database is the inclusion of sequence data 
from Schilderia achatidea, the single, living rep- 
resentative from an older, more diverse genus 
of European affinities. Previously, the 
paraphyletic arrangement of the genera 



Pseudozonaria and Zonaria was a surprising 
result (Meyer, 2003). These new data for 
Schilderia place the genus as sister to Zonaria 
to the exclusion of Pseudozonaria (and 
Neobernaya), and phylogenetic results main- 
tain their independent, paraphyletic status. 
These finding are more consistent with geo- 
graphic affinities than recognized taxonomic 
affinities (Pseudozonaria is often considered a 
subgenus of Zonaria), as both Neobernaya and 
Pseudozonaria are currently restricted to the 
eastern Pacific whereas Schilderia and Zonaria 
are restricted to the western Atlantic. 

Basal Compartment (Fig. 3) 

Five Ovulidae taxa are added in these analy- 
ses: Pseudocypraea exquisita, Volva volva, 
Primovula concinna, Dentiovula takeoi, and 
Prosimnia semperi. Within Ovulidae, only a few 
major clades are well supported and may be 
the results of poor taxon sampling. First, the 
clade Eocypraeinae appears well supported 
and includes Pedicularia, Jenneria and 
Pseudocypraea. Eocypraeinae is sister to a 
strongly supported clade (Ovulinae) that in- 
cludes the remaining Ovulidae. Within the 
Ovulinae, two subgroups are well supported 
and represent the major clades Volvini and 
Ovulini. Of the added Ovulidae, Volva falls into 
Volvini, but Prosimnia unexpectedly falls into 
Ovulini as do Primovula and Dentiovula. These 
results are generally consistent with Gate's 
(1974) arrangement of higher-level relation- 
ships within the Ovulidae. Cyphoma gibbosum 
falls basal to these two sisters in the strict con- 
sensus topology; however, its position is poorly 
supported, and it is expected to move within 
the Volvini with the inclusion of more taxa. 
Monophyly of Ovulidae is not addressed herein 
and would require the inclusion of more distant 
representatives from Lamellaridae, Triviidae 
and Eratoidae. 

The Cypraeidae basal group includes the 
genera Propustularia, Nesiocypraea, Ipsa, 
Cryptocypraea, Nucleolaria, Staphylaea, 
Monetaria, Perisserosa, and Erosaria. 
Propustularia, Nesiocypraea, and Ipsa form a 
clade that roots deeply within the phylogeny and 
is sister to all other cowries. Each of the three 
genera is represented by only a single taxon, 
and only Nesiocypraea contains additional rec- 
ognized species missing from the dataset 
{Nesiocypraea midwayensis, N. lisetae and N. 
aenigma). While sharing a most recent com- 
mon ancestor, the three genera are highly di- 
vergent from each other, representing 
significant periods of independent history. Two 



INCREASED MOLECULAR SAMPLING IN CYPRAEIDAE 



133 



Pedicularia pacifica 

Jenneria pustulata 
Pseudocypraea adamsonii 
Pseudocypraea exquisita 
Cyphoma gibbosum 

Neosimnia aequalis 

1^ - " Volva volva 

1 I Phenacovolva weaveri 



Phenacovolva tokioi 
Ovula ovum 

Serratovolva dondani 




Calpurnus verrucosus 

Crenavolva rosewateri 
^^^^— Primovula concinna 
Procalpurnus lacteus 
Crenavolva tokuoi 
volva cf rosewateri 
Dentiovuta takeoi 
Prionovolva brevis 

Adamantia flonda 
Prosimnia semperi 



chJIdreni 
djilwynj 
nucleus 
granúlala' 
limacina interstincta 
limacina limacina 
staphylaea laevigata 
staphylaea staphylaea 
semiplota 
annulus 
obvelata* 
mo neta 
caputophJdii 
caputserpentis 
^aputdré 
guttata 

margjnalis 
citrina 

helvola helvola 
helvola cf. callista 
helvola hawaiiensis 
tardus 
irrorata 
albuginosa 
poraria 
beckii 

macandrewi 
igierti 
kingae 
thomasi 
cernica 
spurca 
acicularis 
nebrites 
erosa (10) 
(PO) 
labrolineata 
boivinii 
ocellata 
gangranosa 
miliaris 
ebúrnea* 

lamarckii cf. redimita 
lamarckii lamarckii 
^^ armeniaca 
^^ hesitata 
1 pcapricornica 
^^ cf. petilirostrls 

J — tigris (10) 

Li ^ tigris (PO) 

~ pantherina 

talpa 
exusta 

pulchra 
cinerea 
lurida 
tessellata 
Isabella 
isabellamexicana 



FIG. 3. Basal Compartment cladogram and phylogram. Bootstrap values are presented above 
branches in the cladogram and rescaled decay values below. Bolded taxa are new additions to the 
data set. Their identity number shown in parentheses follows the listing in the Appendix. Generic or 
suprageneric groupings are indicated to the right of the cladogram. OTUs with an asterisk (*) are not 
ESUs based on molecular criteria. Phylogram to the right is based on likelihood distances using a 
GTR+I+G model of sequence evolution. Note that the scaling for branch lengths changes between 
Ovulidae and Cypraeidae. 



134 



lEYER 



are known exclusively from the Indo-Pacific 
{Nesiocypraea and Ipsa) and one 
(Propustularia) from the western Atlantic, but 
has a fossil record from North America, the 
Caribbean, and Europe (Kay. 1996). The splits 
among these ancient groups are among the 
earliest of all extant species and may have oc- 
curred in the Mesozoic. While reasonably sup- 
ported as a clade. this basal group is not 
strongly supported as the most basal sister, and 
in other analyses (3:1) moves up to become 
sister of the remaining basal taxa (Erosariinae). 
The final six genera from the basal compart- 
ment form the strongly supported clade 
Erosariinae and is the sister group to all remain- 
ing extant species. Membership and relation- 
ships within the Erosariinae are consistent with 
previous findings (Meyer. 2003). Five taxa from 
Erosaria are added: Erosaria marginalis. E. 
citrina. E. Iielvola cf. callista. E. macandrewi. 
and E. englerti. Ten independent lineages are 
strongly supported (bootstraps > 90/decays > 
6) within Erosaria. but interrelationships among 
them are not (< 50/< 4). Erosaria marginalis 
and E. citrina, both from the western Indian 
Ocean, are strongly supported as sister taxa. 
This clade is poorly supported as sister to the 
E. helvola complex. Within Erosaria helvola. 
three ESUs are identifiable: E. helvola 
hawaiiensis from Hawaii. E helvola cf. callista 
from the Marquesas, and E. helvola helvola 
from the remainder of the IndoPacific. The 
newly included ESU. E helvola cf. callista, may 
need a new name, because the type locality of 
E. helvola callista is Tahiti (Shaw, 1909). not 
the Marquesas. These five taxa are sister to 
the remaining Erosaria: however, the basal po- 
sition is poorly supported. Erosaria turdus is a 
monotypic. deeply divergent lineage. Newly 
added Erosaria irrorata. a species restricted to 
the oceanic islands of the Pacific, is poorly sup- 
ported as sister to a strongly supported clade 
(97/12) including E albuginosa and E poraria. 
These three taxa are sister to a well-supported 
lineage (92/6) of eight taxa that I tentatively rec- 
ognize as Paulonaria at the subgeneric level. 
New sequence data from Erosaria macandrewi. 
a Red Sea taxon. closely ally that species with 
E. beckii. These two species are sister to the 
remaining Paulonana taxa. The final additional 
taxon within Paulonana is Erosaria englerti. a 
species endemic to Easter Island and Sala y 
Gomez. Erosaria englerti shares a more recent 
common ancestor with the remaining five 
Paulonaria taxa. All other relationships within 
Erosaria are the same as those presented in 
Meyer (2003) and are indicated in Figure 3. 
Newly added haplotypes from E. lamarckii 



lamarckii populations of the western Indian 
Ocean exhibit a recent divergence from the 
previously recorded E lamarckii cf. redimita of 
the Andaman Sea. One final finding from addi- 
tional Erosaria sequence data is that haplotypes 
from Erosaria miliaris and E ebúrnea individu- 
als interfinger. indicating that either the diver- 
gence between these two taxa is very recent 
and lineage sorting has not occurred, or that 
these two taxa represent a dine across the 
western Pacific from a colored dorsum in the 
west to white shells in the east. 

Midi Compartment (Fig. 4) 

The second paraphyletic compartment con- 
tains mostly large-shelled taxa from the follow- 
ing tribes: Umbiliini, Cypraeini. Mauritiini. Luriini. 
Austrocypraeini. and the genus Pustularia. All 
six clades are well supported (> 70/> 5) except 
for Austrocypraeini. As in Meyer (2003). inter- 
relationships among these major suprageneric 
clades are resolved in the consensus, but pooriy 
supported. Austrocypraeini and Luriini are sis- 
ters and recognized as the subfamily Luriinae. 
Barycypraea and Zoila are sisters and recog- 
nized as the subfamily Bernayinae. Cypraeini 
and Mauritiini are sisters and recognized as the 
subfamily Cypraeinae. In the current topology. 
Pustularia and all remaining cowries share a 
more recent common ancestor, This large clade 
is sister to Luriinae, which in turn is sister to 
Bernayinae, and this inclusive clade is sister to 
Cypraeinae, As in Meyer (2003), Umbiliini is sis- 
terto all remaining midi, mid2 and derived taxa. 

Within the midi compartment, 13 taxa are 
added to the sequence database. The first addi- 
tion falls within the genus Umbilia and is tenta- 
tively recognized as Umbilia cf. petilirostris. A 
single divergent sequence was generated from 
tissue samples collected from the deep waters 
in the Capricorn Channel off Queensland. Aus- 
tralia. Seven sequenced individuals were com- 
pletely identical, while an eighth sample from a 
subadult shell was significantly divergent. This 
single sample may represent the newly de- 
scribed Umbilia petilirostris Darragh. 2002: how- 
ever, authors disagree on its taxonomic status 
(Wilson & Clarkson. in press). Until more com- 
prehensive sampling is done in the region. I 
present the divergent sequence as a different 
ESU. which does not preclude it from being 
lumped within U. capricornica at a later date 
with more exhaustive sampling. The relation- 
ships within Umbilia remain as in previous 
analyses (Meyer. 2003). 

The second taxon added to midi is Lepon- 
cypraea mappa aliv^alensis from Natal. South 



INCREASED MOLECULAR SAMPLING IN CYPRAEIDAE 



135 



ran и lata' 

limacina interstincta 

limacina limacina 

staph laevigata 

staph staphylaea 

semiplota 



mappa mappa 
^^^— mappa aliwalensis 

- mappa rosea 
geographica 




mauiensis 

bistrinotata keeljngensis 
bistrinotata bistrinotata 
bistrinotata subiaevis 



0.05 substitutions/site 



FIG. 4. Mid 1 Compartment cladogram and phylogram. All other information as in Fig. 3. 



136 



lEYER 



Africa, and falls as sister to Leporicypraea 
mappa rosea. Lorenz (2002) has recently re- 
vised the taxonomy of the mappa group in light 
of molecular findings. Importantly, the names I 
associated previously with ESUs have 
changed, and those changes are reflected in 
the Appendix and also discussed herein. The 
taxon I previously recognized as Leporicypraea 
mappa viridis from SE Polynesia is now recog- 
nized as Leporicypraea admirabais. The taxon 
I previously recognized as Leporicypraea 
mappa panerythra from the non-continental 
portions of the western Pacific is now recog- 
nized as Leporicypraea mappa viridis. The other 
taxon names remain the same. Sequences of 
L. mappa "rewa" from Pacific localities (Fiji, 
Vanuatu, Palau, and South China Sea) 
interfinger with haplotypes of L. mappa 
geograpliica individuals from Indian Ocean lo- 
calities (NW Australia, Phuket, Seychelles, and 
Zanzibar). Therefore, I recognize only a single 
taxon, L. mappa geographica, for this clade. 
Because of its conchological distinctiveness 
and sympatry with conspecifics, Lorenz (2002) 
elevated L. mappa geographica to specific sta- 
tus with Indian and Pacific subspecies. Based 
on the genetic difference between mappa-com- 
plex conspecifics and geographic overlap, spe- 
cific status is certainly acceptable. However, the 
remaining L. mappa subspecies are para- 
phyletic. The phylogeny Lorenz (2002: 27) pre- 
sents is correct and reflects this arrangement. 
Certainly, other recognized cowrie species are 
derived from paraphyletic parent species (e.g., 
Eclogavena coxeni and others; see Meyer, 
2003: table 4, and cases herein), and L. 
geographica would have to be added to this 
list. These results suggest a third species sis- 
ter to L. geographica should be recognized that 
would include both L. mappa viridis and L. 
mappa admirabais. L mappa geographica in- 
dividuals have been found sympatrically with 
both L. mappa mappa and L mappa viridis in- 
dividuals in the Pacific Ocean. However, as yet, 
L. mappa mappa and L. mappa viridis haplo- 
types have not been found together. 

One new undescribed taxon is added to 
Mauritia. Haplotypes of M. arabica individuals 
from American Samoa cluster independently 
from haplotypes of M. arabica individuals from 
other Pacific localities. Shells from Samoan in- 
dividuals tend to be smaller, more heavily mar- 
gined and more circular than individuals from 
other Pacific localities. Results from increased 
sampling in both M. depressa depressa (N = 
10) and M. depressa dispersa (N = 10) main- 
tain their independent, reciprocally monophyl- 
etic status, albeit recently diverged. As in 



previous findings, the interrelationships among 
major lineages in Mauritia are poorly supported. 
Consensus methods and poor support result 
in two polytomies (Fig. 4). Further genetic data 
will be needed to address this region of the phy- 
logeny as all extant taxa have been sampled. 

New sequence data from Barycypraea 
teulerei and B. fultoni place them as sister taxa 
and align them with the genus Zoila to form the 
group Bernayinae. Sequence data presented 
for Barycypraea fultoni are of B. fultoni amorimi 
from Mozambique. The Australian Zoila 
marginata complex is split into two ESUs as 
increased sampling indicates fixed molecular 
differences between populations separated by 
the Southwest Cape region between capes 
Naturaliste and Leeuwin. Further sampling di- 
rectly within this region may uncover interme- 
diate haplotypes that would link the two ESUs 
and suggest a dine instead of two independent 
lineages. Such a finding is the case in the Zoila 
friendii complex. However, as none have been 
discovered yet, I present the data as two tenta- 
tive ESUs: Zoila marginata marginata to the 
south and Z marginata ketyana to the west. 
Other described Z. marginata taxa (Lorenz, 
2001; 2002) within each ESU interfinger, and 
do not fulfill molecular criteria for recognition. 
Sequence data from Zoila mariellae are the fi- 
nal addition to the Bernayinae clade. While the 
exact provenance of the animal sequenced is 
unknown, it is likely from the northwestern shelf 
of Australia. Molecular results place Z. mariellae 
as a distinct sister to Z. decipiens, also from 
the northwestern shelf, as expected. 

Following along the phylogeny, the clade 
Luriinae comes next. Talparia and Luria are 
strongly supported as the clade Luriini. A small 
fragment from 16S was amplified from a de- 
graded Talparia exusta specimen, and as ex- 
pected, the taxon is sister to the more 
widespread Talparia talpa. Surprisingly, se- 
quence divergence between the two species 
appears to be relative small, indicating a more 
recent divergence than expected. Better-pre- 
served material from T. exusta is needed be- 
fore these relative results can be confidently 
assessed. The inclusion of four new taxa to the 
Austrocypraeini {Arestoides argus contrastriata, 
Lyncina broderipii, L. ventriculus from the In- 
dian Ocean, and L. kuroharai) does not help in 
resolving interrelationships among member 
taxa. Arestoides argus is broken into a Pacific 
clade, A. argus argus, and a western Indian 
Ocean clade, A. argus contrastriata, based on 
additional sequence data from the Indian 
Ocean. Lyncina broderipii appears as sister to 
L. nivosa within the Callistocypraea clade, as 



INCREASED MOLECULAR SAMPLING IN CYPRAEIDAE 



137 



predicted in Meyer (2003). A single sampled 
individual of L. ventriculus from Christmas Is- 
land in the Indian Ocean falls significantly out- 
side the haplotype cluster of individuals (N = 6) 
from various regions of the Pacific basin. 
Lyncina ventriculus is an oceanic taxon, and 
because of the geographic gap between sites 
across continental Southeast Asia, I choose to 
present the Christmas Island form as new, 
undescribed, distinct ESU. Further sampling of 
individuals from Christmas Island may change 
this interpretation, but they are currently lack- 
ing. A single sample of Lyncina i<uroliarai was 
sequenced and the results place it closely re- 
lated to L. sulcidentata, an endemic Hawaiian 
taxon. The shallow split between these two taxa 
indicates a relatively recent common ancestor. 
Faunal ties have been documented in other 
cowrie species between Hawaii and Japan, 
most notably in Luria isabella, and the close 
affinities between L. kurofiarai and L. 
sulcidentata represent another example of this 
biogeographic link. 

The final two ESUs added within the midi 
compartment are members of the genus 
Pustularia, and more specifically are recognized 
subspecies of Pustularia bistrinotata. A single 
P. bistrinotata keelingensis individual was se- 
quenced, is distinct, and appears as sister to 
the remaining P. bistrinotata complex. Further- 
more, P. bistrinotata sublaevis individuals (N = 
5) from southeast Polynesia (Tuamotu and 
Societies) cluster together, forming a third ESU 
within P. bistrinotata. 

Mld2 Compartment (Fig. 5) 

The third phylogenetic compartment, mid2, 
contains members from the genera 
Neobernaya, Pseudozonaria, Schilderia, 
Zonaria, the subfamily Cypraeovulinae, and the 
tribe Bistolidini of the subfamily Erroneinae. 
Interrelationships among member clades are 
consistent with previous findings (Meyer, 2003). 
Neobernaya and Pseudozonaria are sisters, 
and that clade is sister to the remaining cow- 
ries. The inclusion of sequence data from the 
genus Schilderia (S. achatidea), place the 
group as sister to Zonaria, and together this 
clade shares a more recent ancestor with the 
remaining taxa. The subfamily Cypraeovulinae 
includes both the South African Cypraeovula 
and South Australian Notocypraea and is sis- 
ter to the western IndoPacific Erroneinae, which 
is composed of two tribes: Bistolidini and 
Erroneini. 

Within the mid2 compartment, 25 taxa are 
added to the existing sequence database; at 



least one ESU is added within each genus ex- 
cept the monotypic Neobernaya. Pseudo- 
zonaria nigropunctata, a Galapagos endemic, 
falls into the eastern Pacific clade as a diver- 
gent sister to P. arabicula, although not strongly 
supported. The position of Schilderia achatidea 
has been mentioned previously as sister to 
Zonaria, now found exclusively in the eastern 
Atlantic. Two taxa are added from Zonaria. 
Zonaria picta from the Cape Verde Islands falls 
near the base of Zonaria, and its relationship 
with other Zonarid taxa is ambiguous, resulting 
in a polytomy at the base of the group. Alterna- 
tive phylogenetic reconstructions at the base 
of the group show small internodes, indicative 
of a short radiative burst, with little divergence 
since. New sequence data from Pseudozonaria 
angelicae are extremely similar to haplotypes 
from P. pyrum (both P. pyrum angolensis and 
P. pyrum senegalensis). I include P. angelicae 
as a taxon in the phylogeny, but prefer to con- 
sider it at most a subspecies until further se- 
quence data are available within the P. pyrum 
complex, as I have reservations concerning di- 
vergences along the mostly continuous West 
African/Mediterranean coastline. 

Sequence data from six additional taxa are 
included within Cypraeovulinae, two from 
Notocypraea and four from Cypraeovula. In 
Notocypraea, I tentatively recognize two ESUs 
within Notocypraea angustata, with a phyloge- 
netic break somewhere between Port Lincoln 
and Port Macdonnel, South Australia. Two di- 
vergent haplotype clusters exist without inter- 
mediate states. Again, further data may change 
this interpretation, but at present I chose to rep- 
resent these as different ESUs indicating dis- 
tinct evolutionary trajectories. Sequence data 
from a single specimen of Notocypraea 
hartsmithi, a rare species from southeastern 
Australia, indicate that the species is sister to 
all remaining Notocypraea taxa. Within 
Cypraeovula, four taxa are added, but their in- 
clusion does not change previous interpreta- 
tions that the group is composed of 
predominately four divergent lineages with mi- 
nor differences within each. New sequence data 
from both Cypraeovula fuscorubra and С 
fuscodentata closely align these taxa with С 
capensis. New sequence data from С mikeharti 
and С algoensis closely align those taxa with 
C. edentula and C. alfredensis. Noting the shal- 
low divergences among recognized species in 
Figure 5, I am doubtful that many of the de- 
scribed subspecies within Cypraeovula (sum- 
marized in Lorenz, 2002) will fulfill my 
molecular criteria for ESU status. As some 
species are differentiated currently by only a 



138 



1EYER 






12 
97 


38^— 






10 


гЦц — 




76 


1 


^^*fc 


7 




9 


9 1 




¡00 


'» Щ- 




— 

5 






95 1 


Li 

3 






ej 6 \— 




globulus globulus 
globulus brevJrostris 
cicércula 
margarita 



bistrinotata keelrngensis 

bistrinotata bistrinotata 
bistrinotata subl^evig 



annettae 
robertsi 

nigropunctata (29) 



hartsmjthj (33) 

piperita 
pulicana 
comptom 

angustata (P. Lincoln) (34) 

anguslata 



nelli 
casta nea 

lUtSUl 

fuscorubra (35) 

capensis 

fuscodentata (36) 
coronata 
edentula 
alfredensis 
mikeharti' (37) 
alaoensis (38) 



lutea 

saulae 

lentiginosa 

ziczac misella 
contammata distans 
contaminata contaminata 
asellus bitaeniata 
asellus vespacea 
asellus asellus 
diluculum 
arluffelí 

clandestina candida 
cland. clandestina (39) 
clandestina passenna 



stolida stolida 
stolida clavicula 
stolida diagues (40) 

stolida rubiginosa 



i (41) 



erythi 

lallii 
hirundo 

(WP) 
ursetius (Andaman) (42) 

ЭГ1 kieneri 

depnesten A 
kien depriesteri В 



coloba 

amiges (43) 

chinensis chinensis 



subteres (44) 
Hior (Л5\ 



gaski 
taitae' (46) 
catholicorum 
astaryi 
garciaj* (47) 
cumingii 

pellisserpentís (48) 
cribraria comma 
esontropia francescoi (49) 
esontropia esontropia 
fallax 
gaspardi 

fibrana cf australiensis 
cribraria cf. abaliena (50) 
exmouthensis exmouthensis 
exmouth. magnífica (51) 
cribraria rottnestensis 
cribraria melwardi (52) 
cnbrana cribraria 
ibraria abrolhensis (53) 



langfordi cavatoensis 



serru litera 

mtnoridens 

oryzaeformis 

hammondae 
icrodon microdon 
licrodon chrysalis 

gracilis gracilis 

gracilis notata 

fimbriata fimbriata 

fimbriata cf unifasciata 




Neobernaya 

Pseudozonaria 

Schilderia 

Zonaha 



Notocypraea 



Cypraeovula 



globulus globulus 

globulus brevirostria 
cicércula 

rgarita 



I spac 



bistrinotata keelingensis 

bistrinotata bistrinotata 
bistrinotata sublaevis 
spadtcea 
annettae 
robertsi 
arabicula 

nigropunctata 
hatidea 



Ovatipsa 
Talostolida 



Austrasiatica 
Palmulacypraea 



Purpuradusta 




0.05 substitutions/site 



microdon chrysalis 

gracilis gracilis 
gracilis notata 

fimbriata fimbriata 
fimbriata cf unifasciata 
fímbriata marquesana 
fimbriata waikikiensis 



FIG. 5. Mid 2 Compartment cladogram and phylogram. All other information as in Fig. 3. 



INCREASED MOLECULAR SAMPLING IN CYPRAEIDAE 



139 



single mutation (e.g., Cypraeovula mikeharti/ 
C. algoensis or C. castanea/C. iutsui), there 
simply is not enough room for differences to 
have accumulated between taxa. This is not 
to say that described entities are not indepen- 
dent. Indeed, because Cypraeovula taxa are 
direct developers with limited dispersal and 
gene flow, regional differences are expected 
on small geographic scales, much like the 
South Australian endemic clades Umbilia, 
Zoila, and Notocypraea. However, based on the 
genetic similarity among sampled member 
Cypraeovula, much of this variation has to be 
very recently derived. This pattern is borne out 
in the South Australian direct developers that 
have been more extensively sampled. 

The tribe Bistolidini within Erroneinae is com- 
posed of members from five genera: 
Palmadusta, Bistolida, Ovatipsa, Talostolida 
and Cribrarula. As in Meyer (2003), the basal 
root of Bistolidini is poorly resolved. Overall 
analyses place either Palmadusta as sister to 
the other four genera or Palmadusta and 
Bistolida as a clade, sister to the remaining 
three. Compartmentalized analyses place 
Palmadusta at the base, although poorly sup- 
ported. The addition of 15 ESUs did not help in 
resolving this issue. Only one taxon is added 
to the Palmadusta clade, but it alters the sub- 
specific designations previously ascribed 
(Meyer, 2003). New haplotypes from Andaman 
Sea P. clandestina individuals form a distinct 
monophyletic clade. This new ESU is sister to 
the western Indian Ocean P. clandestina 
passerina, and the two of them are sister to the 
Pacific P. clandestina clade and the Japanese 
endemic P. artuffeli. Based on a review of P. 
clandestine subspecies and type localities, the 
Pacific clade that I had formerly (Meyer, 2003) 
recognized as P. clandestina clandestina should 
be P. clandestina candida, and the new P. 
clandestina clade from the Andaman Sea now 
bears the name P clandestina clandestina. I 
also reviewed the subspecies and type locali- 
ties for the three P. asellus ESUs previously 
unnamed (Meyer, 2003). Based on increased 
sampling and conchological comparisons, I ten- 
tatively ascribe the following subspecific des- 
ignations for the three clades: P asellus asellus 
for the western Indian Ocean clade, P asellus 
vespacea for the Seychelles to western Pacific 
clade, and P. asellus bitaeniata for the 
Melanesian and Pacific clade (Fig. 5, Appen- 
dix). Unfortunately, the addition of P clandestina 
clandestina does not help in resolving the basal 
nodes of Palmadusta. As shown in Figure 5, 
the base of Palmadusta is poorly resolved and 
sister group assignments are ambiguous. A few 



lineages remain strongly supported (P asellus, 
P. clandestlna/diluculum, P. ziczac and P. 
contaminata), but confident hypotheses of other 
interrelationships require further data. 

Three taxa are added to Bistolida: B. stolida 
diagues, B. owenii and an undescribed, dis- 
tinct eastern Indian Ocean clade of B. ursellus. 
Individuals of B. stolida diagues from the 
Seychelles fall as sister to B. stolida 
rubiginosa. Bistolida owenii, a western Indian 
Ocean taxon, is sister to the Red Sea endemic 
B. erythraeensis. A new Bistolida ursellus se- 
quence from the Andaman Sea is poorly sup- 
ported as sister to the remaining B. ursellus 
taxon from the Pacific basin. Its placement is 
equally parsimonious as either sister to B. 
ursellus (Pacific) or forming a B. ursellus grade 
leading to the B. kieneri lineage. The topology 
of the two B. ursellus taxa as sisters is more 
likely and consistent with morphology. 

One taxon is added to Ovatipsa and two taxa 
to Talostolida. Within Ovatipsa, the subspe- 
cies O. chinensis amiges from the Pacific ba- 
sin and Western Australia is distinct from O. 
chinensis chinensis from the Philippines west- 
ward through the Indian Ocean to the east 
coast of Africa. Various other O. chinensis 
subspecies have been described within the 
Indian Ocean (e.g., Lorenz & Hubert, 1993), 
and preliminary data indicate that these Indian 
Ocean subspecies may represent very recent 
divergences within what I am currently recog- 
nizing as O. chinensis chinensis. However, 
until more individuals are sampled, I maintain 
them all under the taxon Ovatipsa chinensis 
chinensis. Within Talostolida, two taxa are 
added that appear as sisters to each other: T 
subteres from southeastern Polynesia and T 
/ai/or from Hawaii. These two taxa are sister 
to Talostolida pellucens. All four taxa currently 
included within Talostolida are deeply divergent 
independent ESUs. A single haplotype of 
Talostolida teres "alveolus" (sensu Lorenz, 
2002) is completely identical to haplotypes of 
T teres teres individuals from both the Society 
Islands and the Tuamotu. Moreover, T teres 
individuals from SE Polynesia have been de- 
scribed by Lorenz (2002) as a distinct subspe- 
cies T teres "janae"; however sampled 
individuals of T teres from SE Polynesia 
interfinger with individuals sampled from the 
Western Pacific (Papua New Guinea and 
Guam). Therefore, the data do not support T 
teres "janae" as a valid taxon, based on my 
criteria. All Marquesan individuals sequenced 
possess T pellucens haplotypes, whereas all 
T. teres-like individuals from the remainder of 
SE Polynesia possess T teres haplotypes. 



140 



lEYER 



The Cribrarula clade includes eight additional 
taxa, making it the nnost diverse genus within 
Bistolidini. Two taxa, Cribrarula taitae from 
American Samoa and С gardai from Easter 
Island, are added to the deeply divergent Pa- 
cific subclade. Both taxa are recently divergent 
members from their respective sister taxon. 
Cribrarula taitae appears as a closely related 
sister to C. cathollcorum, and C. gardai is 
closely related to C. cumingii. Only a single in- 
dividual from each of the two taxa was included 
in these analyses, and the results would be 
better addressed with multiple samples. Two 
members are added to the Western Indian 
Ocean subclade: Cribrarula pellisserpentis and 
С esontropia francescoi, both from Madagas- 
car. Cribrarula esontropia francescoi is a closely 
related sister to С esontropia esontropia, which 
includes C. esontropia cribellum (Meyer, 2003). 
Cribrarula pellisserpentis is a deeply divergent 
member within the western Indian Ocean 
subclade and is sister to the other three ESUs. 
Four taxa are added to the remaining Cribrarula 
member clade. A single individual of C. cribraria 
from Masirah, Oman, appears significantly di- 
vergent from population samples of the previ- 
ously unnamed С cribraria ESU from the 
Andaman Sea. Conchologically, this individual 
approximates the western Indian Ocean taxon 
C. cribraria abaliena and is tentatively recog- 
nized as such. A single individual of С cribraria 
australiensis from Western Australia falls within 
the Andaman С cribraria cluster; therefore, I 
tentatively adopt the name C. cribraria cf. 
"australiensis" for a taxon that extends from the 
Andaman Sea southward to Western Austra- 
lia. More exhaustive sampling is required to 
confirm these geographic patterns. A single in- 
dividual of C. exmouthensis magnifica from 
Broome is significantly different from samples 
of С exmouthensis exmouthensis from the 
Exmouth Gulf region, therefore validating the 
status of that taxon. Additional samples of С 
cribraria rottnestensis (N = 3) further validate 
the taxon's uniqueness. Eight individuals of С 
melwardi from northeastern Australia all share 
a common ancestor and are reciprocally mono- 
phyletic with respect to the remaining C. 
cribraria individuals. Moreover, a single C. 
cribraria cribraria individual from the same reef 
(Lament Reef in the Bunker Group) clusters as 
expected with other Pacific C. cribraria cribraria 
individuals. The final taxon included is C. 
cribraria abrolhensis (N = 3), and haplotypes 
are shallowly divergent but reciprocally mono- 
phyletic with respect to samples of C. cribraria 
cribraria (N = 30) from predominately western 
Pacific localities (Appendix). More thorough 



analyses and discussion of this fascinating, 
species-rich group is in preparation (Meyer et 
al., in prep.). 

Derived Compartment (Fig. 6) 

The final compartment analyzed is the derived 
monophyletic clade recognized as the tribe 
Erroneini. This clade includes the following nine 
genera: Austrasiatica, Palmulacypraea, 
Errónea, Purpuradusta, Contradusta, Nota- 
dusta, Eclogavena, Melicerona and Blasicrura. 
Many (25) taxa are added within the tribe, and 
phylogenetic analyses result in some surpris- 
ing affinities. For the most part, major genera 
are well supported, but their interrelationships 
are not. Three taxa currently ascribed to 
Austrasiatica were included in previous analy- 
ses (Meyer, 2003); however, they were consid- 
ered as representatives of the genus 
Nesiocypraea. As discussed earlier, the find- 
ing that Nesiocypraea teramachii is distantly 
related raises the subgenus Austrasiatica to 
generic status for the clade that includes 
Austrasiatica langfordi, A. hirasei and A. 
sakurai. As in Meyer (2003), Austrasiatica is 
sister to all other Erroneini taxa, followed by 
Pamulacypraea as sister to the remainder. As 
predicted in Meyer (2003), the newly added 
Pamulacypraea musumea falls as sister to P. 
katsuae. Even with the addition of 24 taxa (a 
67% increase), the topology among the rest of 
the major Erroneini lineages is ambiguous. Six 
added "Errónea" species form a basal grade 
leading to the Adusta/ Errónea split previously 
recognized in Meyer (2003). I take a conserva- 
tive approach and redefine Errónea to include 
all these taxa and subsume Adusta to a well- 
supported subclade within the group, as the 
new data demonstrate that Adusta and Errónea 
(including the more recent additions) are not 
equivalent (sisters). U Adusta were to be main- 
tained at equivalent generic status. Errónea 
would represent a paraphyletic group. 
Purpuradusta, Eclogavena, Melicerona and 
Blasicrura are all well-supported monophyletic 
lineages. As in Meyer (2003), Notadusta is well 
supported only if restricted to members of the 
Notadusta punctata complex. However, be- 
cause Notadusta martini is often considered a 
member of Notadusta, I include it within 
Notadusta here, although poorly supported. In 
a similarly conservative manner, I include two 
of the added taxa within Contradusta, although 
again poorly supported. Support for relation- 
ships among these seven genera is poor and 
is likely because of the short internode length 
between divergent lineages. 



INCREASED MOLECULAR SAMPLING IN CYPRAEIDAE 



141 



100 I 

I a 1 73 1 







100 I 

■^l""|ioor— 




goodallii 
hirundo 
stolida stolida 
stolida clavicola 
stolida diagues 
stolida rubiginosa 
owenii vasta 
erythraeensjs 
ursellus 

ursellus (Andaman) 
kienen kieneri 
kien depriesteri A 
kien depriesteri В 



langfordj cavatoensis 

hirasei 

sakuraii 



katsuae 
musumea (54) 



xanthodon (55) 
pallida (56) 
vredenburgi (57) 
rabaulensis (58) 
fernandoi (59) 
onyx 
adusta 

subviridis subviridis 
onyx melanesiae 
subviridis dorsalis 
pyriformis (60) 
cylindrica lenella(61) 
cylindrica cylindrica 
ovum ovum (62) 
ovum palauensis 
caurica elongata (63) 
caurica draceana 
caurica spp. 1 (64) 
caurica quinquefasciata 
caurica spp. 2 (65) 
caunca derosa 
erronés 

ovum chrysostoma 
caurica samoensis (66) 
caurica caurica 



serrulifera 
minoridens 
oryzaeformis (67) 
hammondae 
microdon microdon 
microdon chrysalis (68) 
gracilis gracilis 
gracilis notata 
fimbriate fimbnata 
fimbriata cf unifasciata 
fímbriata marquesana (69) 
fimbriata waikikiensis (70) 



walken 
bregenana 
barclayi (71) 
pulchella (72) 



martini 

hungerfordi (73) 
punctata berinii В 
punctata berinii A 
punctata (Andaman) 
punctata trizonata (74) 
punctata punctata 



dayritiana 

quadnmaculata thielei 
coxeni 

quad- quadrimaculata 



listen melvilli (75) 

llsteri 

felina (76) 



interrupta 
pallidula pallidula 
pallidula rhinoceros 
pallidula cf. vivía (77) 
summersi (78) 



Bistolida 



Austrasiatica 
Palmulacypraea 



Tj-sI 

^ si 






I' ■- lar 

MZZ 



stolida stolida 
stolida clavicola 
stolida diagues 

stolida rubiginosa 

— owenii vasta 

^^ erythraeensis 

— goodallii 
hirundo 

ursellus 

ursellus (Andaman) 

kieneri kieneri 
kien depnesteri A 
kien depnesteri В 

langfordi cavatoensis 
hirasei 



sakuraii 



katsuae 



■ musumea 



Purpuradusta 



Contradusta 



Notad usta 



Eclogavena 



Melicerona 



xanthodon 
pallida 
vredenburgi 

baulensis 
fernandoi 



\_r-^. 

I ^— subvi 
^ f onyx I 



г cyli 

If 



I 



onyx 
adusta 
subviridis subviridis 
onyx melanesiae 
subviridis dorsalis 
pyriformis 
cylindrica lenella 
cylindnca cylindrica 
ovum ovum 
ovum palauensis 
caurica elongata 
caunca draceana 
caurica spp. 1 
caurica quinquefasciata 
caurica spp. 2 
caurica derosa 
erronés 
ovum chrysostoma 



caunca samoensis 

caurica caurica 



^ 



• minoridens 
^—oryzaeformis 



£ 



micr. microdon 
-micr. chrysalis 



gracilis gracilis 
gracilis notata 

fimbriata fimbnata 
fimbriata cf- unifasciata 
fimbriata marquesana 
fímbriata waikikiensis 



f: 



walkeri 

- bregeriana 
barclayi 

— pulchella 
martini 



-O 



I — PUI 
I *- pur 

— '' 

1— L' 



R. 



hungerfordi 
punctata berinii A 
punctata berinii В 
punctata (Andaman) 
punctata trizonata 
punctata punctata 
dayritiana 

quadrimaculata thielei 
coxeni 
quadrimaculata quadrimaculata 
^^ listen melvilli 
~~|r listen listeri 

L felina 
I —■ ■ interrupta 
I pallidula pallidula 
I ^ pallidula rhinoceros 
* I pallidula cf. vivia 
^ summersi 



0,05 substitutions/site 



FIG. 6. Derived Compartment cladogram and phylogram. All other information as in Fig. 3. 



142 



lEYER 



Twelve additional taxa are added to Errónea. 
Six of the additions are traditionally recognized 
as distinct species, four have been recognized 
as subspecies, and two are newly discovered, 
but may have names associated with them that 
have been placed into synonymy. Of the new 
species, three form a relatively well-supported 
clade: Errónea rabaulensis shares a more re- 
cent common ancestor with E. fernandoi (80/1 ), 
and those two are sister to E. vredenburgi (84/ 
3). The three additional Errónea species all nest 
deeply within the clade, and their relationships 
are not well supported. Errónea pallida appears 
as sister to the clade of the previously described 
three species and Adusta. Errónea pyriformis 
is relatively well supported (81/6) as the sister 
to the clade previously recognized as Errónea 
(Meyer, 2003). Finally, Errónea xanthodon falls 
at the base of Errónea and is sister to all other 
Errónea taxa. Within the crown Errónea 
subclade, six taxa are added that are all tradi- 
tionally recognized at the subspecific level. In- 
dividuals of Errónea cylindrica lenella (N = 8, 
all from New Caledonia) form a monophyletic 
group strongly supported (91/6) as sister to the 
clade including the remaining E. cylindrica in- 
dividuals plus two subspecies of E. ovum. 
These results imply that E. cylindrica at the 
specific level is a paraphyletic taxon. Newly 
added individuals of Errónea ovum ovum from 
both Singapore and the Philippines (N = 15) 
form a monophyletic group sister to E. ovum 
palauensis (N = 7). The four remaining, newly 
added taxa are all members of the Errónea 
caurica complex. First, individuals (N = 7) of 
the newly described E. caurica samoensis ap- 
pear as a distinct lineage sister to individuals 
(N = 15) from the remainder of the Pacific and 
Western Australia (E. caurica caurica). Four 
geographically structured haplotype clades are 
found exclusively in the Western Indian Ocean. 
Errónea caurica dracaena is currently restricted 
to the Seychelles based on sampling. Newly 
added individuals from East Africa and Mada- 
gascar form a haplotype clade that I recognize 
as Errónea caurica elongata. Individuals of E. 
caurica quinquefasciata from the Red Sea, East 
Africa and Oman form the third monophyletic 
group. Finally, newly sequenced individuals 
from Masirah (N = 7) form a private haplotype 
clade (E caurica ssp. #1) sister to E caurica 
quinquefasciata. The final, newly added taxon 
(E. caurica ssp. #2) within the E. caurica com- 
plex is a clade (N = 18) that includes individu- 
als primarily from India, but with a few 
individuals from Masirah, Oman. This haplo- 
type clade is sister to the clade recognized pre- 
viously as E. caurica cf. derosa from the 



Andaman Sea (Meyer, 2003). The Errónea 
caurica complex and the associated E. 
cylindrica, E. ovum and E. erronés species will 
be more thoroughly addressed in another pa- 
per (Meyer, in prep.) as the group exhibits re- 
markable geographic structuring, polyphyly of 
recognized species (E. ovum), and evidence 
of introgression based on nuclear markers. 

Purpuradusta is well supported and contains 
four newly added taxa that fall in expected re- 
lationships. The southeastern Polynesian en- 
demic species Purpuradusta oryzaeformis is 
distinct and sister to P. minoridens that ranges 
throughout the remainder of the western 
IndoPacific. A single specimen of P. microdon 
from East Africa falls outside the haplotype 
clade of other sampled individuals from the 
Pacific basin (N = 5). This East African popula- 
tion is recognized as Purpuradusta microdon 
chrysalis. Two peripheral populations of 
Purpuradusta fimbriata in the Pacific Basin are 
introduced. First, Hawaiian populations of P. 
fimbriata are distinct (N = 7) and were previ- 
ously recognized as P. fimbriata waikikiensis; 
thus this name is resurrected as a valid entity. 
Second, individuals from the Marquesas are 
also distinct genetically, consistent with the 
subspecies designation of Lorenz (2002), P. 
fimbriata marquesana (N = 14). Both of these 
Pacific P. fimbriata subclades share a more 
recent history with the widespread Pacific sub- 
species P. fimbriata unifasciata, as expected. 

Two newly added species, "Errónea" barclayi 
and "Errónea" pulchella, come out as sister 
species in phylogenetic analyses. Moreover, 
these two taxa appear as sister to Contradusta 
in the most likely topology. Because of these 
results, and the poorly supported nature of their 
relationships, I tentatively place the two taxa in 
the genus Contradusta, with the caveat that 
they may be removed with future data. These 
results are somewhat surprising, particularly 
because "Contradusta" pulchella is thought to 
be closely related to Errónea pyriformis be- 
cause of the darkly stained columellar denti- 
tion and overall conchological similarities. The 
sister relationship between Contradusta 
pulchella and C. barclayi is more acceptable 
as their divergence is deep, and the phyloge- 
netic affiliations of С barclayi were more diffi- 
cult to predict based on morphological criteria. 
Another surprising result is the sister relation- 
ship between Notadusta martini and "Errónea" 
hungerfordi. Given these phylogenetic results, 
I tentatively place "Errónea" hungerfordi \N\\.h'\n 
Notadusta, but with little confidence, although 
it is reasonably supported (73/4), and suspect 
that it may be removed with more samples and 



INCREASED MOLECULAR SAMPLING IN CYPRAEIDAE 



143 



sequence data. Within the remaining Notadusta 
complex, individuals of N. punctata trizonata 
(N = 9) form a monophyletic group sister to the 
Pacific N. punctata punctata clade. Finally, in 
regards to Notadusta, "Notadusta" rabaulensis 
was mentioned previously as a member of 
Errónea and "Notadusta" musumea as 
Palmulacypraea, further reducing the member- 
ship of Notadusta (Meyer, 2003). 

The final four additions to the dataset fall into 
Melicerona and Blasicrura. First, two taxa are 
added to Melicerona. Samples of Melicerona 
listen melvilli (N = 5) from Queensland, Austra- 
lia, form a monophyletic group sister to the re- 
maining Melicerona taxa. (Two rostrate and 
melanistic individuals interfinger among the 
other three haplotypes indicating that the tera- 
tology is likely driven by phenotypic responses 
to environmental conditions rather than having 
a genetic basis.) Samples of Melicerona felina 
from both Oman and East Africa form a mono- 
phyletic group, and because the haplotypes 
from the two regions interfinger, there is no 
evidence for a distinction between the subspe- 
cies M. felina felina and M. felina fabula. Within 
Blasicrura, two taxa are added, based on the 
sequencing results. First, samples of S/as/crura 
summersi, a Fijian and Tonga endemic, appear 
as a recently divergent sister to the also newly 
included B. pallidula cf. vivia from American 
Samoa. This clade is sister to the Melanesian 
subspecies Blasicrura pallidula rhinoceros, as 
expected based on geography. This resulting 
topology indicates that the Blasicrura pallidula 
complex is paraphyletic. 



DISCUSSION 

The ultimate goal of this project is to construct 
a comprehensive phylogeny of cypraeid gas- 
tropods at the appropriate level for diversifica- 
tion studies. From a molecular perspective, all 
ESUs presented are effectively equal units of 
diversity, whether they are currently recognized 
as species, subspecies or some other level. 
There are some noted exceptions as OTUs 
were used on occasion that represented un- 
sorted or clinal variation within an ESU (e.g., 
Erosaria miliaris! ebúrnea). However, on a gen- 
eral scale, each taxon shown in the phylogenies 
(Figs. 3-6) represents an independent evolu- 
tionary trajectory. 

Because so much taxonomic information is 
available for cowries, it is informative to see 
how molecular criteria compare with recog- 
nized taxonomic entities. The most recent com- 
pilation of the cowries is that of Lorenz (2002), 



and I will use his checklist (pp. 250-291) as a 
benchmark for comparisons. Lorenz recog- 
nizes 232 species, of which I have sequenced 
210 (> 90%), and they are presented herein. 
The missing species are as follows: 
Nesiocypraea aenigma, N. lisetae, N. midway- 
ensis, Austrasiatica alexhuberti, Erosaria 
ostergaardi, Zoila perlae, Lyncina camelopar- 
dis, L. joycae, Pustularia chiapponii, Cypra- 
eovula col I ig ata, С. cruickshanki, С. immelmani, 
Palmad usta androyensis, P. johnsonorum, 
Austrasiatica deforgesi, Palmulacypraea 
boucheti, P. omii, Eclogavena luchuana, 
Errónea (?) angioyorum, and E. nymphae. Se- 
quences from samples of both Purpuradusta 
barbieri and "Talostolida" rashleighana have 
been obtained, but were too late for inclusion in 
these analyses. All missing species are rare, 
with small ranges located generally at the pe- 
riphery of their putative sister species based on 
conchological and anatomical characters. Of 
the 210 sequenced species, phylogenetic com- 
parisons and molecular criteria support all but 
15 (93%) as ESUs. The 15 recognized species 
not supported by my criteria are discussed be- 
low. For Nucleolaria granulata. Monetaria 
obvelata, Erosaria ebúrnea, Zoila orientalis, Z. 
thersites, Luria controversa, L. gilvella, 
Notocypraea occidentalis, and Palmadusta 
humphreysii, multiple individuals were se- 
quenced and the haplotypes interfingered 
within their closest relative. For the next six 
species that I do not support, only a single in- 
dividual was sequenced, thus they may indeed 
represent a very young independent trajectory. 
However, when compared to the genetic diver- 
sity within their closest relative, the genetic dif- 
ference is unremarkable, and in some 
instances, only a single mutation different from 
putative conspecifics: Zonaria angelicae, Z. 
petitiana, Cypraeovula mikeharti, Bistolida 
brevidentata, Cribrarula gardai, and C. taitae. 
While genetic data are overall broadly con- 
sistent with taxa recognized at the specific level, 
the results are even more remarkable when 
compared among taxa recognized at subspe- 
cific levels. Lorenz recognizes 260 taxa at the 
subspecific level. Of those 260 subspecies, I 
have sequenced at least two individuals from 
160 in order to assess their validity. Molecular 
criteria support 113 (> 70%) of these taxa as 
legitimate ESUs. Moreover, sequence results 
indicate an additional 20 distinct ESUs not 
recognized as subspecies by Lorenz (but 
sometimes mentioned as important varieties 
or forms). A full listing of sampled taxa and 
their current ESU status as indicated by the 
prior criteria can be found at the Cowrie Ge- 



144 



MEYER 



netic Database Project Website (http:// 
www.flmnh.ufl.edu/cowries). The website in- 
cludes other information, such as localities 
sampled, numbers of individuals for each taxon, 
and photographs of the specimens sequenced. 
Overwhelming molecular support for tradition- 
ally recognized taxa, both at specific and sub- 
specific levels, is extremely encouraging. First, 
from a taxonomic standpoint, these molecular 
results corroborate the excellent work done by 
centuries of malacological researchers, at both 
professional and amateur levels. Similar mo- 
lecular surveys of other diverse groups will pro- 
vide valuable comparisons in order to assess 
taxonomic congruence (e.g., Jackson & 
Cheetham, 1990) and address concordant di- 
versification patterns. Second, from a molecu- 
lar perspective, sequence data provide a 
suitable, objective, relative metric for circum- 
scribing appropriate evolutionary units. Assum- 
ing rate constancy in the molecules (COI only, 
in prep.), molecular divergences can constrain 
the tempo of diversification and assess the dis- 
tinctiveness of purported taxa. A growing body 
of molecular data across the diversity of life 
undoubtedly will provide insight to some of our 
most fundamental evolutionary questions. 



ACKNOWLEDGEMENTS 



Mtumwa Mwadini, Peter Ng, Steve Norby, 
Shuichi Ohashi, Yoshihiro Omi, Ina Park, Marcel 
Pin, Cory Pittman, Xavier Pochon, Matt Rich- 
mond, Raphael Ritson-Williams, Gonçalo Rosa, 
Gary Rosenberg, Teina Rongo, Fred Schroeder, 
Mike Severns, Pauline Severns, Hung-Long Shi, 
Brian Simison, Michael Small, John Starmer, 
Steve Tettlebach, David Touitou, Martin Wallace, 
Chia-Hsiang Wang, Dave Watts, Barry Wilson, 
Woody Woodman, Shu-Ho Wu. The following 
institutions are acknowledged: Florida Museum 
of Natural History; University of California Mu- 
seum of Paleontology; Academy of Natural Sci- 
ences of Philadelphia; Bernice P. Bishop 
Museum, Honolulu, Hawaii; California Academy 
of Sciences; Institute of Marine Sciences, Zan- 
zibar; University of Dar es Salaam; Jackson- 
ville Shell Club; Musée National d'Histoire 
Naturelle, Paris, France; National Museum of 
Natural History Naturalis, Leiden, The Nether- 
lands; Santa Barbara Museum of Natural His- 
tory; National Museum of Natural History; and 
Suganthi Devadason Marine Research Institute. 
I also would like to thank Felix Lorenz, Jr., for 
his thoughtful comments, as well as the reviews 
of four anonymous reviewers. Final decisions 
and opinions are wholly mine. 

This research has been financially supported 
by the following NSF grants: DEB-9807316, 
DEB 0196049, and OCE-0221382. 



An ever-growing number of individual and 
institutions have contributed and supported this 
ongoing research. Without their assistance, the 
work would not be possible. The following per- 
sons are recognized: Nonoy Alonzo, Vicente 
Azurin, Paul Barber, Don Barclay, Marty Beals, 
Victor Bonito, Philippe Bouchet, Michel Beutet, 
Roy Caldwell, Carlos Carvalho, Hank Chaney, 
John Chester, Peter Clarkson, Lori Bell Colin, 
Pat Colin, Allen Collins, Harry Conley, Vince 
Crayssac, Carolyn Cruz, Donald Dan, Martyn 
Day, Bruno de Bruin, Helen deJode, John Earle, 
Andrew Edinger, Mark Erdmann, Melissa Frey, 
Michel Garcia, Bill Gibbs, Serge Gofas, Terry 
Gosliner, Jeroen Goud, Robert Gourguet, 
Fabien Goûtai, Paulo Granja, Kibata Mussa Haji, 
Jerry Harasewych, Itaru Hayami, Brian Hayes, 
Claus Hedegaard, Ed Heiman, Bert Hoeksema, 
John Hoover, John Jackson, Maurice Jay, Scott 
Johnson, Paul Kanner, Yasunori Kano, Tomoki 
Kase, Norbert Kayombo, Shigemitsu Kinjo, Lisa 
Kirkendale, Kitona Kombo Kitona, Utih Kukun, 
Senthil Kumar, Jean Paul Lefort, Bill Liltved, 
Hung-Chang Liu, Charlotte Lloyd, Felix Lorenz, 
Jr., Felix Lorenz, Sr., Larry Madrigal, Marlene 
Martinez, Gerald McCormack, Mohammed 
Mohammed, Hugh Morrison, Gowele Mtoka, 



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Revised ms. accepted 19 January 2004 



146 



MEYER 




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MALACOLOGIA, 2004, 46(1): 157-168 

NEW SPECIES OF THE GENUS ABRINA (BIVALVIA: SEMELIDAE) 
FROM THE COMMANDER AND KURIL ISLANDS 

Gennady M. Kamenev 

The Institute of Marine Biology, Russian Academy of Science, Vladivostok 690041, Russia; 
kamenev@mail333. com, inmarbio@mail.primorye.ru 



ABSTRACT 

A new species, Abrina scarlatoi, is described from tiie Commander and Kuril islands. 
This species has a small (to 11.2 mm), ovate-trigonal, high, almost equilateral shell with a 
non-polished, gray or light brown periostracum and conspicuous growth lines. The external 
ligament is attached to a short, wide nymph. The internal ligament is lodged in an ovate- 
elongate resilifer, which extends obliquely posterior to the beaks. Abrina scarlatoi was 
found in shelf zones of the Commander Islands (depth 3-100 m) and Kunl Islands (intertidal 
zone to 120 m), on rocky platforms and boulders, covered by a thick layer of lime red 
algae, brown algae, and sponges, with a population density up to 30 specimens/m^. The 
taxonomic status oí Abrina magna Scarlato, 1965, and A. hainanensis Scarlato, 1965, is 
also discussed. 

Key words: Abrina, Semelidae, Bivalvia, Commander and Kuril islands. 



INTRODUCTION 



Previously, four species of the genus Abrina 
-A. cuneipyga Scarlato, 1981; A. sachalinica 
Scarlato, 1981; A. shiashkotanika Scarlato, 
1981; and A. tatarica Scarlato, 1981 - have 
been listed in Russian fauna (Scarlato, 1981). 
However, examination has shown that they are 
species of Macoma Leach, 1819 (Tellinidae) 
(Kamenev & Nadtochy, 1999). 

Study of the bivalve fauna of the Commander 
Islands shelf revealed an unknown species 
tentatively assigned to the genus Abrina Habe, 
1952 (Kamenev, 1995; Bujanovsky, 1997). 
Detailed examination of the material from the 
Commander Islands and additional specimens 
from the Kurils has led me to regard it as a 
new species of Abrina. 



MATERIAL AND METHODS 

In this study, I have used the material col- 
lected by the 1MB intertidal expedition to the 
Kuril Islands (June-July, 1 967), joint IMB-PRIFO 
expeditions to the Commander Islands (8-28 
July 1972, sealer "Krylatka"; 30 August-6 Octo- 
ber 1973, RA/ "Rakytnoe") and the Kuril Islands 
(July-November 1987, RA/ "Tikhookeansky"), 



and joint 1MB - PIBOC expedition to Sakhalin 
Island and the Kuril Islands (1 July-4 August 
2003, R/V "Akademik Oparin"). 

For comparison purposes, collections of the 
following taxa were used: Abrina lunella 
(Gould, 1861) (NSMT); A. kinoshitai (Kuroda 
& Habe, 1958) (NSMT NSMI); A. declivis 
(Sowerby, 1868) (SBMNH); A. magna 
Scarlato, 1965, and A. hainanensis Scarlato, 
1965 (bothZIN), and different species of other 
genera of the Semelidae (UW, CAS, USNM). 
Abrina declivis was stored in 70% ethanol. All 
other materials were stored dry. 

Shell Measurements 

Figure 1 shows the position of the shell mor- 
phology measurements. Shell length (L), 
height (H), width of each valve (W) not shown, 
anterior end length (A), maximal distance from 
posterior shell margin to top of palliai sinus 
(LI), and minimal distance from top palliai si- 
nus (L2) to anterior adductor muscle scar (L2) 
were measured for each valve. The ratios of 
these parameters to shell length (H/L, W/L, /V 
L, L1/L, L2/L, respectively) were determined. 
Shell measurements were made using an ocu- 
lar micrometer with an accuracy of 0.1 mm. I 
made measurements of 34 specimens of the 
new species. 



157 



158 



KAMENEV 



t< A h 



H 



'~^'^i~c^ 




LI 



FIG. 1. Placement of shell measurements: L - 
shell length; H - height; A - anterior end length; 
L1 - maximal distance from posterior shell 
margin to top of palliai sinus; L2 - minimal 
distance from top of palliai sinus to anterior 
adductor muscle scar. 



Diagnosis 

Shell small (< 20 mm), thin to medium in 
thickness, moderately inflated, subtrigonal, 
ovate-trigonal or ovate, white, equivalve or 
with right valve sometimes more inflated, 
equilateral to longer anteriorly. Posterior end 
attenuate, with radial ridge along postero- 
dorsal margin, sometimes flexed to right. 
Periostracum thin, adherent or dehiscent, 
silky to dull, colorless, tan, gray, light brown. 
Surface with faint or conspicuous growth 
lines. Beaks orthogyrate, central or posterior. 
Hinge weak, two cardinal teeth in each valve; 
lateral teeth absent. Ligament opisthodetic, 
parivincular, both external and internal; ex- 
ternal seated on a nymph not projecting 
above dorsal margin; internal lodged in ob- 
lique resilifer posterior to cardinal teeth. Pal- 
liai sinus long, sometimes slightly different 
length and form in each valve, partly confluent 
with palliai line. 

Abrina scarlatoi Kamenev, new species 
Figs. 2-19, Table 1 



Abbreviations 

The following abbreviations are used in the 
paper: CAS - California Academy of Sciences, 
San Francisco; 1MB - Institute of Marine Biol- 
ogy, Russian Academy of Sciences, 
Vladivostok; MIMB - Museum of the Institute 
of Marine Biology, Vladivostok; NHMI - Natu- 
ral History Museum and Institute, Chiba; 
NSMT - National Science Museum, Tokyo; 
PIBOC - Pacific Institute of Bioorganic Chem- 
istry, Russian Academy of Sciences, 
Vladivostok; PRIFO - Pacific Research Insti- 
tute of Fisheries and Oceanography, 
Vladivostok; SBMNH - Santa Barbara Mu- 
seum of Natural History, Santa Barbara; 
USNM - United States National Museum of 
Natural History, Smithsonian Institute, Wash- 
ington, D.C.; UW - University of Washington, 
Seattle; ZIN - Zoological Institute, Russian 
Academy of Sciences, St. Petersburg. 



SYSTEMATICS 

Family Semelidae Stoliczka, 1870 

Genus Abrina Habe, 1952 

Type species: Abra kanamarui Kuroda, 1951; 

= Macoma lunella Gould, 1861 



Type Material and Locality 

Holotype (MIMB 9529), Polovina Bight, 
Bering Island, Commander Islands, Bering 
Sea, 3 m, rocky platform, bottom water tem- 
perature of 8.0°C, Coll. V. N. Romanov, 26- 
VII-1972 (sealer "Krylatka"); paratypes (30): 
paratypes (2) (MIMB 9530) from the holotype 
locality; paratypes (5) (MIMB 9531), Tonky 
Cape, Bering Island, Commander Islands, 
Bering Sea, 10 m, rocky platform, bottom wa- 
ter temperature of 9.1°C, Coll. S. D. Vavilin, 
1 3-IX-1 973 (RA/ "Rakitnoye"); paratype (MIMB 
9532), Kamni Bobrovye - Kitolovnaya Bed, 
Medny Island, Commander Islands, Bering 
Sea (54°58.0'N, 1 67°21 .5'E), 1 00 m, rocky plat- 
form, Coll. V. I. Lukin, 18-IX-1973 (R/V 
"Rakitnoye"); paratypes (2) (MIMB 9533), 
Cherny Cape, Medny Island, Commander Is- 
lands, Bering Sea, 15 m, rocky platform, bot- 
tom water temperature of 9.4°C, Coll. V. I. 
Lukin, 17-IX-1973 (R/V "Rakitnoye"); 
paratypes (2) (MIMB 9534), Palata Cape, 
Medny Island, Commander Islands, Pacific 
Ocean, 20 m, rocky platform, bottom water 
temperature of 5.0°C, Coll. V. I. Lukin, 16-VII- 
1972 (sealer "Krylatka"); paratype (MIMB 
9535), Sivuchy Kamen, Medny Island, Bering 
Sea, 10 m, boulders, bottom water tempera- 



NEW SPECIES OF THE BIVALVE GENUS ABRINA 



159 



ture of 8.6°C, Coll. V. I. Lukin, 2-X-1973 (R/V 
"Rakytnoe"); paratypes (3) (MIMB 9536), Tonky 
Cape, Bering Island, Commander Islands, 
Bering Sea, 20 m, rocky platform, bottom wa- 



ter temperature of 9.0°C, Coll. G. T. Belokonev, 
13-IX-1973 (RA/ "Rakytnoye"); paratypes (2) 
(MIMB 9537), Vodopadskogo Cape, Medny Is- 
land, Commander Islands, Pacific Ocean 




FIGS. 2-13. Abrina scarlatoi Kamenev, new species. FIGS. 2-5: Holotype (MIMB 9529), Polovina 
Bight, Bering Island, Commander Islands, Bering Sea, 3 m, shell length 9.8 mm. FIGS. 6, 7: Paratype 
(MIMB 9538), Phedoskina Cape, Bering Island, Commander Islands, Pacific Ocean, 5 m, right and 
left valves of a young specimen. FIGS. 8, 9: Paratype (MIMB 9531), Tonky Cape, Bering Island, 
Commander Islands, Bering Sea, 10 m, right and left valves with ligament. FIG. 10: Paratype (MIMB 
9530), from holotype locality, right valve without ligament. FIG. 11: Paratype (MIMB 9534), Palata 
Cape, Medny Island, Commander Islands, Pacific Ocean, 20 m, left valve without ligament. FIG. 12: 
Paratype (MIMB 9533), Cherny Cape, 15 m, Medny Island, Commander Islands, Bering Sea, right 
valve without ligament. FIG. 13: MIMB 9549, Nadezda Strait (Rashua Island - Matua Island), Kuril 
Islands, 48°00'N, 153°15'E, 50 m. Bar = 1 mm. 



160 



KAMENEV 



(54°38.6'N, 167°43.5'E), 40 m, rocky platform, 
Coll. V. I. Lukin, 3-X-1973 (R/V "Rakytnoe"); 
paratypes (3) (MIMB 9538), Phedoskina Cape, 
Bering Island, Commander Islands, Pacific 
Ocean, 5-15 m, rocky platform, bottom water 
temperature of 9.8-1 0.0°C, Coll. V. I. Lukin, 23- 
IX-1973 (R/V "Rakytnoe"); paratypes (3) (MIMB 
9539), Peregrebnogo Cape, Bering Island, 
Commander Islands, Bering Sea, 15-20 m, 
rocky platform, bottom water temperature of 
10.0°C, Coll. B.I. Sirenko, 5-IX-1973 (R/V 
"Rakytnoe"); paratype (MIMB 9540), Bujan 
Bight, Bering Island, Commander Islands, 
Bering Sea, 5 m, rocky platform, bottom water 
temperature of 7.6°C, Coll. V. I. Lukin, 28-VII- 
1 972 (sealer "Krylatka"); paratype (MIMB 9541 ), 
Poloviny Bay, Bering Island, Commander Is- 
lands, Bering Sea, 10 m, rocky platform, bot- 
tom water temperature of 9. 8°C, Coll. V. I. Lukin, 
27-IX-1973 (R/V "Rakytnoe"); paratypes (4) 
(MIMB 9542) Ushishir Islands, Kuril Islands 
(42°30.2'N, 152°51.0'E), 87-120 m, boulders 
covered by Spongia, Coll. G. M. Kamenev, 19- 
VII-2003 (R/V "Akademik Oparin"). 

Other Material Examined 

One slightly damaged specimen (MIMB 
9543), Korabelnaya Bight, Medny Island, Com- 
mander Islands, Bering Sea, 5 m, rocky plat- 
form, bottom water temperature of 6.8°C, Coll. 
V.l. Lukin, 14-VII-1 972 (sealer "Krylatka"); one 
slightly damaged specimen (MIMB 9544), 
Kamny Bobrovye, Medny Island, Commander 
Island, Bering Sea, 5 m, bottom water tem- 
perature of 5.6°C, Coll. V. I. Lukin, 1 3-VII-1 972 
(sealer "Krylatka"); one left valve (MIMB 9545), 
Palata Cape, Medny Island, Commander Is- 
lands, Pacific Ocean, 15 m, rocky platform, 
bottom water temperature of 5.2°C, Coll. V. I. 
Lukin, 16-VII-1972 (sealer "Krylatka"); one left 
valve (MIMB 9546), Phedoskina Cape, Bering 
Island, Commander Islands, 20 m, rocky plat- 
form, bottom water temperature of 9.9°C, Coll. 
V. I. Lukin, 23-IX-1973 (R/V "Rakitnoye"); one 
slightly damaged specimen (MIMB 9547), 
Utesnaya Bight, Second Kuril Strait, 
Paramushir Island, Kuril Islands, intertidal 
zone, boulders with brown algae of the gen- 
era Fucus and Alaria, Coll. M. B. Ivanova, 7- 
VII-1967; one specimen (MIMB 9548), 
Burevestnik Village, Iturup Island, Kuril Islands, 
Sea of Okhotsk, intertidal zone, boulders with 
brown algae of the genus Alaria, Coll. O. G. 
Kusakin, 24-VII-1967; one specimen (MIMB 
9549), Nadezda Strait (Rashua Island - Matua 



Island), Kuril Islands (48°00'N, 153°15'E), 50 
m, rocky platform. Coll. V. I. Lukin, 19-VIII-1987 
(R/V "Tikhookeansky"). Total of 5 specimens 
and 2 left valves. 

Description 

Exterior. Shell small (to 11.2 mm), ovate- 
trigonal, high (H/L = 0.740-0.827), equivalve, 
moderately inflated (W/L = 0.181-0.235), al- 
most equilateral (slightly longer anteriorly, 
sometimes equilateral or longer posteriorly), 
thin, solid, white under periostracum. Surface 
with conspicuous growth lines. Periostracum 
non-polished, gray, sometimes light brown, 
dehiscent, easily peeled off near beaks, ex- 
tending into inner surface, thrown into small 
wrinkles, more conspicuous at shell margins. 
Beaks orthogyrate, small, slightly rounded, 
moderately projecting above dorsal margin, 
slightly posterior or anterior to midline, some- 
times central (A/L = 0.474-0.583). Anterior end 
rounded. Posterior end narrow, obliquely 
subtruncate, with faint radial ridge from beaks 
to ventral limit of posterior end. Anterodorsal 
margin slightly convex, gently descending ven- 
trally, smoothly transiting to rounded anterior 
end. Ventral margin slightly curved. 
Posterodorsal margin short, straight, gently 
descending ventrally, forming noticeable angle 
at transition to posterior margin. Posterior 
margin straight, rather steeply descending 
ventrally, forming rounded angle at transition 
to ventral margin. External ligament short (1/2 
posterodorsal margin length), attached to 
short, wide nymph not projecting above dor- 
sal margin. 

Interior. Hinge plate wide, sometimes pro- 
jecting into shell cavity in area of cardinal teeth. 
Hinge weak, with two cardinal teeth in each 
valve. In left valve, anterior tooth wide, long, 
reaching edge of hinge plate; posterior tooth 
very narrow, lamellate, shorter, not reaching 
edge of hinge plate, almost parallel to anterior 
tooth. In right valve, anterior and posterior 
teeth almost same length and width (anterior 
tooth slightly shorter and wider). Internal liga- 
ment well developed, reaching edge of hinge 
plate, lodged in ovate-trigonal or ovate-elon- 
gate resilifer, which extends obliquely poste- 
rior to beaks. Anterior adductor muscle scar 
large, ovate, vertically extended; posterior 
adductor scar large, rounded, shorter and 
wider than anterior scar. Palliai sinus distinct, 
moderate, reaching past midline (L1/L = 
0.603-0.698), broad, rounded anteriorly, of 



NEW SPECIES OF THE BIVALVE GENUS ABRINA 



161 




FIGS. 14-19. The hinge of the different age specimens oí Abrina scarlatoi Kamenev, new species. 
FIGS. 14-16. Hinge of right valve. FIG. 14: Paratype (MIMB 9538), Phedosklna Cape, Bering Island, 
Commander Islands, Pacific Ocean, 10 m, shell length 6.2 mm. FIG. 15: Paratype (MIMB 9536), Tonky 
Cape, Medny Island, Commander Islands, Bering Sea, 20 m, shell length 6.8 mm. FIG. 16: Paratype 
(MIMB 9533), Cherny Cape, Medny Island, Commander Islands, Bering Sea, shell length 7.6 mm. 
FIGS. 17-19. Hinge of left valve. FIG. 17: Paratype (MIMB 9530), from holotype locality, shell length 
6.2 mm. FIG. 18: Paratype (MIMB 9534), Palata Cape, Medny Island, Commander Islands, Pacific 
Ocean, 20 m, shell length 7.5 mm. FIG. 19: Paratype (MIMB 9531), Tonky Cape, Bering Island, 
Commander Islands, Bering Sea, 10 m, shell length 8.5 mm. Bar = 500 цт. 



same shape and size in both valves (L1/L and 
L2/L of left valve 0.655 and 0.184; L1/L and 
L2/L of right valve 0.654 and 0.187), substan- 
tially detached, confluent with palliai line for 
more than 1/2 of its length. Shell interior often 
with faint radial striae. 

Variability 

Shell shape and proportions change with 
age. In young specimens (< 4 mm), in con- 
trast to adults, the shell is more elongate and 
angular; the posterodorsal margin at the tran- 
sition to the posterior margin forms a distinct 
angle; the posterior margin more steeply de- 
scends ventrally, forming a pointed acute 
angle at the transition to ventral margin; the 
ventral margin is almost straight; the beaks 



are placed more posteriorly (A/L = 0.54- 
0.583). The periostracum of young speci- 
mens has very fine, short, discontinuous 
radial lines in the area of the beaks. In young 
and adult specimens, the relative length, 
shape, and degree of confluence of the pal- 
liai sinus with the palliai line vary slightly. 
Sometimes, the length and shape of palliai 
sinus of right and left valves are slightly dif- 
ferent (Table 1). 

Distribution and Habitat (Fig. 20) 

Commander Islands: Bering Island and 
Medny Island; Kuril Islands: Paramushir Is- 
land; Nadezda Strait (Rashua Island - Matua 
Island) (48°00'N, 153°15'E); Ushishir Islands 
(42°30.2'N, 152°51.0'E); Iturup Island. 



162 



KAMENEV 



Near the Commander Islands, this species was 
found at depths from 3 m (Polovina Bight, Bering 
Island) to 100 m (Kamni Bobrovye - Kitolovnaya 
Bed, Medny Island, 54°58'N, 167°2Г5Е) on a 
rocky platform and boulders covered by a thick 
layer of lime red algae, with a population den- 
sity up to 30 specimens/m^; near the Kuril Is- 
lands - from the intertidal zone (Paramushir 
Island, Iturup Island) to 120 m (Ushishir Islands) 
on boulders covered by brown algae of the gen- 
era Fucus and Alaria or sponges. 

Comparisons 

In contrast to other species of Abrina, A. 
scarlatoi has the shell with rough, conspicu- 
ous growth lines, gray, a non-polished, dehis- 
cent periostracum, wide hinge plate, and a 
short, wide nymph (Table 2). Moreover, A. 
scarlatoi differs from A. lunella (Figs. 21-28) 
in its smaller, higher shell with less posteriorly 
placed beaks and in having the hinge with non- 
bifid cardinal teeth and a very narrow, lamel- 



late posterior cardinal tooth in the left valve 
(Gould, 1861; Kuroda, 1951; Habe, 1952, 1977 
1981; Kuroda et al., 1971; Ito et al., 1986 
Kamenev & Nadtochy, 1999; Okutani, 2000) 
from A. kinoshitai, in a smaller, higher, more 
inflated, ovate-trigonal shell without a flexure 
of the posterior end, with less posteriorly placed 
beaks, a shorter palliai sinus of the same shape 
and size in both valves, and in having the hinge 
with non-bifid cardinal teeth and a very narrow, 
lamellate posterior cardinal tooth in the left valve 
(Ito, 1967, 1989; Kuroda et al., 1971; Habe, 
1977; Tsuchida & Kurozumi, 1995; Kamenev & 
Nadtochy, 1999); from A. declivis, in the more 
elongate shell with a much less attenuate pos- 
terior end and in having the hinge with non- 
bifid cardinal teeth and a very narrow, lamellate 
posterior cardinal tooth in the left valve (Scott, 
1994); from A. sibogai (Prashad, 1932), A. 
/пал /s (Prashad, 1932), and Л. weber/ (Prashad, 
1932), in the shell with less posteriorly placed 
beaks and lacking lunule and a escutcheon 
(Prashard, 1932). 



60° 



RUSSIA 




Bering Sea 



Commander 
Islands 



Paramushir Is. 



^^^ c.^ 



Nadezda Strait 



ÜAPAN 



^ ^0 I Usliishir Is 
»*^ Iturup Is. 



Pacific Ocean 



FIG. 20. Distribution of Abrina scarlatoi. 



NEW SPECIES OF THE BIVALVE GENUS ABRINA 163 

TABLE 1. Abrina scarlatoi Kamenev, new species. Shell measurements (mm), indices and summary 
statistics of all characteristics: L - shell length; H - height; W - width; A - anterior end length; LI - 
maximal distance from the posterior shell margin to the top of palliai sinus; L2 - minimal distance from 
the top of palliai sinus to the anterior adductor muscle scar. Numerator indicates shell measurements 
and indices for the left valve, denominator - for the right valve. 



Depository L H W A L1 L2 H/L W/L A/L L1/L L2/L 

Holotype MIMB 9529 9^ M 2^ 47 6^ Zl 0.827 0.224 0.480 0.653 0.214 

9.8 8.1 2.2 4.7 6.4 2.1 0.827 0.224 0.480 0.653 0.214 

Paratype MIMB 9530 L6 6^ 16 3^ 5^ 1_4 0.789 0.211 0.513 0.658 0.184 

7.6 6.0 1.6 3.9 5.0 1.4 0.789 0.211 0.513 0.658 0.184 

Paratype MIMB 9530 ^2 47 12 M 4J. LI 0.758 0.194 0.548 0.661 0.194 

6.2 4.7 1.2 3.4 4.1 1.2 0.758 0.194 0.548 0.661 0.194 
Paratype MIMB 9531 M 6^ L9 13 57 Ц 0.776 0.224 0.506 0.671 0.200 

8.5 6.6 1.9 4.3 5.7 1.7 0.776 0.224 0.506 0.671 0.200 
Paratype MIMB 9531 8^ a4 18 40 5^ 16 0.800 0.225 0.500 0.663 0.200 

8.0 6.4 1.8 4.0 5.3 1.6 0.800 0.225 0.500 0.663 0.200 

Paratype MIMB 9531 L6 6^ 17 3^ M 13 0.789 0.224 0.513 0.671 0.171 

7.6 6.0 1.7 3.9 5.1 1.3 0.789 0.224 0.513 0.671 0.171 
Paratype MIMB 9531 6^ 5^ 16 M IZ 10 0.794 0.235 05 0.691 0.147 

6.8 5.4 1.6 3.4 4.5 1.2 0.794 0.235 0.5 0.662 0.176 

Paratype MIMB 9531 5^ 13 Ц 3^ M 0_9 0.768 0.196 0.536 0.643 0.161 

5.6 4.3 1.1 3.0 3.6 0.9 0.768 0.196 0.536 0.643 0.161 
Paratype MIMB 9532 77 57 14 3^ 48 16 0.740 0.182 0.506 0.623 0.208 

7.7 5.7 1.4 3.9 5.0 1.5 0.740 0.182 0.506 0.649 0.195 
Paratype MIMB 9533 L6 6J 16 3^ 5J_ 12 0.803 0.211 0.474 0.671 0.158 

7.6 6.1 1.6 3.6 5.0 1.2 0.803 0.211 0.474 0.658 0.158 

Paratype MIMB 9533 06 M 14 M M 12 0.758 0.212 0.485 0.667 0.182 

6.6 5.0 1.4 3.2 4.3 1.3 0.758 0.212 0.485 0.652 0.197 
Paratype MIMB 9534 L5 09 15 09 5^ 15 0.787 0.200 0.520 0.693 0.200 

7.5 5.9 1.5 3.9 5.0 1.7 0.787 0.200 0.520 0.667 0.227 

Paratype MIMB 9534 04 40 Ц 27 05 10 0.741 0.204 0.500 0.648 0.185 

5.4 4.0 1.1 2.7 3.5 1.0 0.741 0.204 0.500 0.648 0.185 
Paratype MIMB 9535 L3 05 16 37 IZ U 0.753 0.219 0.507 0.644 0.178 

7.3 5.5 1.6 3.5 4.5 1.4 0.753 0.219 0.479 0.616 0.192 
Paratype MIMB 9536 7\2 06 14 06 19 15 0.778 0.194 0.500 0.681 0.208 

7.2 5.6 1.4 3.6 4.8 1.5 0.778 0.194 0.500 0.667 0.208 

Paratype MIMB 9536 08 SJ. 14 06 13 16 0.750 0.206 0.529 0.632 0.235 

6.8 5.1 1.4 3.6 4.4 1.4 0.750 0.206 0.529 0.647 0.206 
Paratype MIMB 9536 45 05 09 Z4 M M 0.778 0.200 0.533 0.667 0.200 

4.5 3.5 0.9 2.4 3.0 0.9 0.778 0.200 0.533 0.667 0.200 
Paratype MIMB 9537 L2 04 14 05 16 1^ 0.750 0.194 0.486 0.639 0.208 

7.2 5.4 1.4 3.5 4.6 1.5 0.750 0.194 0.486 0.639 0.208 

Paratype MIMB 9537 09 45 12 OO 37 12 0763 0.203 0.508 0.627 0.203 

5.9 4.5 1.2 3.0 3.7 1.2 0.763 0.203 0.508 0.627 0.203 
Paratype MIMB 9538 37 2^ 07 ZO Z3 07 0.757 0.189 0.541 0.627 0.189 

3.7 2.8 0.7 2.0 2.3 0.7 0.757 0.189 0.541 0.622 0.189 
Paratype MIMB 9538 05 49 14 3^ 4^ 1^ 0.754 0.215 0.508 0.646 0.185 

6.5 4.9 1.4 3.3 4.2 1.2 0.754 0.215 0.508 0.646 0.185 

Paratype MIMB 9538 02 48 13 SJ. M 13 0.774 0.210 0.500 0.613 0.210 

6.2 4.8 1.3 3.1 3.9 1.3 0.774 0.210 0.500 0.629 0.210 
Paratype MIMB 9539 57 44 12 OO M M 0.772 0.211 0.526 0.632 0.140 

5.7 4.4 1.2 3.0 3.8 0.8 0.772 0.211 0.526 0.667 0.140 

Paratype MIMB 9539 37 Z8 07 ZO 2J: M 0757 0.189 0.541 0.649 0.162 

3.7 2.8 0.7 2.0 2.4 0.6 0.757 0.189 0.541 0.649 0.162 

Paratype MIMB 9539 Z4 19 05 14 16 04 0.792 0.208 0.583 0.667 0.167 

2.4 1.9 0.5 1.4 1.6 0.4 0.792 0.208 0.583 0.667 0.167 
Paratype MIMB9540 03 43 Ц Z8 37 08 0.811 0.208 0.528 0.698 0.151 

5.3 4.3 1.1 2.8 3.6 0.9 0.811 0.208 0.528 0.679 0.170 

(continues) 



164 

(continued) 



KAMENEV 



Depository L H W A L1 L2 H/L W/L A/L L1/L L2/L 

Paratype MIMB 9541 3^ M 0^ 2^ 2^ 0^ 0.795 0.205 0.564 0.641 0.154 

3.9 3.1 0.8 2.2 2.5 0.6 0.795 0.205 0.564 0.641 0.154 

Paratype MIMB 9542 1_L2 9^ Z4 5^ L2 17 0.782 0.214 0.491 0.643 0.152 

11.2 9.0 2.4 5.5 7.2 1.8 0.782 0.214 0.491 0.643 0.160 

Paratype MIMB 9542 105 M 2^ 5^ LO 2^ 0.804 0.219 0.505 0.667 0.210 

10.5 8.5 2.3 5.3 7.0 2.2 0.804 0.219 0.505 0.667 0.210 

Paratype MIMB 9542 Ц^ 9J. 2^ 5J. M LZ 0.813 0.214 0.455 0.696 0.152 

11.2 9.1 2.4 5.1 7.8 1.8 0.813 0.214 0.455 0.696 0.161 

Paratype MIMB 9542 4J. 3^ 10 Z2 27 OS 0.791 0.244 0.537 0.659 0.195 

4.1 3.3 1.0 2.2 2.7 0.8 0.791 0.244 0.537 0.659 0.195 

MIMB 9547 aO 18 13 3^ 10 11 0.800 0.217 0.550 0.667 0.183 

6.0 4.8 1.3 3.3 4.0 1.1 0.800 0.217 0.550 0.667 0.183 

MIMB 9548 L8 6J. 16 10 IZ 13 0.782 0.205 0.513 0.603 0.167 

7.8 6.1 1.6 4.0 4.7 1.3 0.782 0.205 0.513 0.603 0.167 

MIMB 9549 9^ 7J. 17 17 6^ 19 0.755 0.181 0.500 0.670 0.202 

9.4 7.1 1.7 4.7 6.3 2.0 0.755 0.181 0.500 0.670 0.213 

Statistics L H W A LI L2 H/L W/L A/L L1/L L2/L 

Mean 6^ 5^ 142 3A6 4A7 L2§. 0.778 0.208 0.514 0.655 0.184 

6.81 5.31 1.42 3.45 4.46 1.28 0.778 0.208 0.514 0.654 0.187 

SD 2J0 172 M8 QM 143 0ЛЗ 0.023 0.014 0.026 0.024 0.024 

2.10 1.72 0.48 0.96 1.42 0.44 0.021 0.014 0.027 0.019 0.021 

SB 036 030 O08 016 024 O07 0.004 0.002 0.005 0.003 0.004 

0.36 0.30 0.08 0.16 0.24 0.07 0.004 0.002 0.005 0.003 0.004 

Min Z4 19 05 14 16 0Л 0.740 0.181 0.455 0.603 0.140 

2.4 1.9 0.5 1.4 1.6 0.4 O740 0.181 0.455 0.603 0.140 

Max 1L2 9J. 2^ 5^ L8 2^ 0.827 0.244 0.583 0.698 0.235 

11.2 9.1 2.4 5.5 7.8 2.2 0827 0.244 0.583 0.696 0.227 
N 3434343434343434343434 

34 34 34 34 34 34 34 34 34 34 34 



Etymology 

The specific name honors Orest A. Scarlato, 
Academician of the Russian Academy of Sci- 
ences, a famous Russian researcher of the 
marine bivalve fauna of Russia. 

Remarks 

The genus Abrina also includes species A. 
magna Scarlato, 1965, and A. hainanensis 
Scarlato, 1965, described by Scarlato (1965) 
from Hainan Island, South China Sea, China. 
All the material of Л. magna (4 specimens and 
103 shells) was collected from assemblages of 
empty shells on sandy beaches of Hainan Is- 
land and northern Vietnam. The material of A. 
hainanensis (the holotype and 10 additional 
specimens) is much smaller, but with the ex- 
ception of the holotype, was also sampled from 
assemblages of empty shells on sandy shores 
Hainan Island and the Gulf of Thailand 
(Bangkok). The holotype was collected in the 



intertidal zone of Hainan Island in the estuary 
of river, on silty sand among the mangroves. 

Having studied all materials relating to these 
species in the ZIN collection, I think that 
Scarlato (1965) erroneously assigned these 
species to Abrina. The hinge plate in these spe- 
cies is very wide, projects into the shell cavity 
in the area of resilifer. The hinge is weak, with 
two cardinal teeth in the right valve and two 
cardinal teeth in the left valve of A. hainanensis, 
and one cardinal tooth in the left valve of A. 
magna (Figs. 29-34). External and internal liga- 
ments are very large. The external ligament is 
deeply sunken, almost internal, separated from 
the resilium by a slight ridge. The resilium is 
lodged in a large, trigonal resilifer behind the 
cardinal teeth. Lateral teeth are absent. 

Thus, the hinge of both species is identical to 
the hinge of the genus Psammotreta (Tellinidae) 
(Keen, 1969), except that the left valve of A. 
magna bears one cardinal tooth instead of two. 
To all appearances, the posterior cardinal tooth 
on the left valve of A. magna is partly or com- 



NEW SPECIES OF THE BIVALVE GENUS ABRINA 



165 




FIGS. 21-34. Shells oí Abrina species. FIGS. 21-28. Abrina lunella (Gould, 1861), NSMT (Mo 73503), 
Shiroko, Suzuka-shi, Mie Prefecture, Japan. FIGS. 21-24: Shell length 12.8 mm. FIGS. 25, 26: Shell 
length 10.2 mm. FIGS. 27, 28: Hinge of left and right valves. Bar = 1 mm. FIGS. 29-34. Abrina magna 
Scarlato, 1965. FIGS. 29, 30: ZIN (17), Tonkin Bay, North Vietnam, South China Sea, right valve, 
length 63.0 mm. FIGS. 31-34. ZIN (20), North Vietnam, South China Sea. FIGS. 31, 32: Right valve, 
length 49.6 mm. FIGS. 33, 34: Left valve, length 45.5 mm. 



166 



KAMENEV 



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NEW SPECIES OF THE BIVALVE GENUS ABRINA 



167 



pletely reduced with age. Unlike Л. hainanensis, 
all valves of A. magna were very large, 42 to 
73 mm long (valves of A. hainanensis are 9.5 
to 22.3 mm in length). Moreover, since all ma- 
terial on this species was collected in assem- 
blages of empty shells, almost on all left and 
right valves the cardinal teeth were partly or 
completely destroyed, and the ligament and 
periostracum were lacking. Therefore, it is not 
inconceivable that the thin and weak posterior 
cardinal tooth was broken in all left valves. 

Examination of the descriptions and figures 
of members of the genus Psammotreta (Keen, 
1969, 1971; Habe, 1977; Lamprell & White- 
head, 1992; Okutani, 2000) shows that A. ma- 
gna is most likely a synonym of Psammotreta 
(Tellinimactra) edentula (Spengler, 1798), in- 
habiting the intertidal and upper subtidal zones 
of Japan, South China, North Vietnam and Aus- 
tralia. Abrina magna is identical to P. (T.) 
edentula in hinge structure and morphology of 
the external and internal ligaments. Moreover, 
it has similar shape, proportions and size of the 
shell, a very deep palliai sinus in both valves, 
and scars of the anterior and posterior adduc- 
tors differing in shape and size (Figs. 29-34). It 
is possible that the material of A. hainanensis 
comprises young specimens of P. (T.) edentula. 
However, it is not unlikely that A. hainanensis 
is a separate species of the same subgenus. A 
more thorough study of specimens of different 
species of Psammotreta is needed to make a 
correct identification of A. magna and A. 
hainanensis. 



DISCUSSION 

Scarlato (1981) described new species of 
Abrina on the basis of a study of young speci- 
mens of Macoma (Kamenev & Nadtochy, 
1999). The main morphological characteristic 
on the basis of which these species were pre- 
viously included in Abrina, was the presence 
of an internal ligament in an oblique resilifer 
posterior to the cardinal teeth. The genera 
Abrina and Macoma are similar in most mor- 
phological characteristics. The main distin- 
guishing characteristic of Abrina is the 
presence of a well-developed internal ligament 
in the resilifer, a narrow groove posterior to 
the cardinal teeth. In Macoma, an internal liga- 
ment is absent. However, studies of the com- 
mon northwestern Pacific Macoma species - 
M. /oven/ (Jensen, 1905), M. calcárea (Gmelin, 
1791), M. balthica (Linne, 1758), M. crassula 
(Deshayes, 1855), M. lama Bartsch, 1921, M. 
incongrua (Martens, 1865) - show the pres- 



ence of an internal ligament in young speci- 
mens (Kamenev & Nadtochy, 1999). Thus, a 
well-developed internal ligament lodged in 
oblique resilifer in representatives of the ge- 
nus Macoma is a juvenile characteristic that 
is preserved in Abrina during its entire life. 

Morphological similarity of the genera Abrina 
and Macoma, and the presence of an internal 
ligament in young specimens of species of 
Macoma, at first leads one to suggest that the 
present species is a juvenile of species of 
Macoma. In Macoma, a well-developed 
resilium is found only in individuals up to 5-6 
mm in shell length, whereas in specimens with 
a shell length more than 10 mm, it is lacking 
(Kamenev & Nadtochy, 1999). A study of A. 
scarlatoi of different ages showed that both 
young and adult specimens of this species 
have a well-developed resilium, lodged in the 
oblique resilifer posterior to the cardinal teeth. 
The shape of the resilifer changes with age, 
but its position and relative size remain un- 
changed. Furthermore, /A. scar/ato/ differs from 
most species of Macoma (Scarlato, 1981; 
Coan et al., 2000) in the lack of a flexure to 
the right of the posterior shell margin and by 
having palliai sinuses of similar shape and size 
in both valves. Therefore, I think that the spe- 
cies described herein belongs to the genus 
Abrina, not to Macoma. 



ACKNOWLEDGMENTS 

I am very grateful to Mrs. N. V. Kameneva 
(MIMB, Vladivostok) for great help during work 
on this manuscript; to Professor T W. Pietsch 
and Dr. K. Stiles (UW, Seattle) for arrangement 
of my visit to the UW and work with the bivalve 
mollusks collection, for all-round, very kind, and 
friendly help during my stay and work in Se- 
attle; to Professor A. J. Kohn and Dr. G. Jensen 
(UW, Seattle) for great help during work with 
the bivalve mollusks collection at the UW; to 
Dr. P. D. Roopnarine and Miss. E. Kools (De- 
partment of Invertebrate Zoology, CAS, San 
Francisco) for arrangement of my work with the 
bivalve mollusks collection at the CAS and great 
help during this work; to Mr. Gary Cook (Ber- 
keley) for all-round, very kind and friendly help 
during my stay and work in San Francisco; to 
Drs. B. I. Sirenko and A. V. Martynov and all 
collaborators of Marine Research Laboratory 
(ZIN, St. Petersburg) for sending of the speci- 
mens of Abrina species and help during work 
with collection of bivalve mollusks of ZIN; to 
Dr. H. Saito (NSMT, Tokyo) for sending of the 
specimens of /A. lunella and A. kinoshitai; to Drs. 



168 



KAMENEV 



T. Kurozumi and E. Tsuchida (NHMI, Chiba) for 
sending of tlie specimens of A. kinoshitai and 
reprints of necessary papers; to Dr. K. Amano 
(Joetsu University of Education, Joetsu) for 
sending of the reprints of scientific papers nec- 
essary for work; Ms. R. N. Germon (USNM, 
Washington) for sending the specimens of dif- 
ferent species from the Semelidae; to Dr. E. V. 
Coan (Department of Invertebrate Zoology, 
CAS, San Francisco) and Paul Valentich Scott 
(Department of Invertebrate Zoology, SBMNH, 
Santa Barbara) for consultations and sending 
of the copies of scientific papers necessary for 
work; to Mr. D. V. Fomin (1MB, Vladivostok) for 
help in work with the scanning microscope; to 
Ms. T. N. Kaznova (1MB, Vladivostok) for help 
with translating of the manuscript into English; 
to Professor George M. Davis for help in the 
publication of the manuscript; and to two anony- 
mous reviewers for comments on the manu- 
script. 

This research was supported by Grant 01- 
04-48010 from the Russian Foundation for 
Basic Research. 



LITERATURE CITED 

BUJANOVSKY, A. I., 1997, On the fauna and 
ecology of the bivalves of the shallow water 
shelf zone of the Commander Islands. Pp. 
242-253, in: A. V. RZHAVSKY, ed., Benthic flora 
and fauna of the shelf zone of the Commander 
Islands. Vladivostok: Dalnauka Press. 270 pp. 
[in Russian, with English abstract]. 

COAN, E. v., P H. SCOTT & F. R. BERNARD, 
2000, Bivalve seashells of western North 
America. Marine bivalve mollusks from Arctic 
Alaska to Baja California. Santa Barbara Mu- 
seum of Natural History, viii + 764 pp. 

GOULD, A. A., 1861, Descriptions of shells col- 
lected by the North Pacific Exploring Expedi- 
tion. Proceeding Boston Society Natural 
History, 8: 14-40. 

HABE, T., 1952, Genera of Japanese shells. N 
3. Pelecypoda, 187-280. [in Japanese]. 

HABE, T., 1977, Systematics of Mollusca in Ja- 
pan. Bivalvia and Scaphopoda. Tokyo. 372 pp. 
[in Japanese]. 

HABE, T., 1 981 , Bivalvia. A catalogue of molluscs 
of Wakayma Prefecture, the Province of Kii. I. 
Bivalvia, Scaphopoda and Cephalopoda. Pub- 
lications of the Seto Mahne Biological Labora- 
tory, Special Publication Series, 7(1): 25-224. 

ITO, K., 1 967, A catalogue of the marine mollus- 
can shell-fish collected on the coast of and off 
Tajima, Hyogo Prefecture. Bulletin of the Ja- 
pan Sea Regional Fisheries Research Labo- 
ratory, 18: 39-91 [in Japanese, with English 
abstract]. 

ITO, K., 1989, Distribution of molluscan shells in 
the coastal areas of Chuetsu, Kaetsu and Sado 
Island, Niigata Prefecture, Japan. Bulletin of 



the Japan Sea Regional Fisheries Research 
Laboratory, 39: 37-133 [in Japanese, with En- 
glish abstract]. 

ITO, K., Y. MATANO, Y YAMADA& S. IGARASHI, 
1986, Shell species cught S/S Rokko-Maru off 
the coast Ishikawa Prefecture. Bulletin of the 
Ishikawa Prefectural Fisheries Experimental 
Station, 4: 1-179 [in Japanese, with English 
abstract]. 

KAMENEV, G. M., 1995, Species composition 
and distribution of bivalve mollusks on the 
Commander Islands shelf. Malacological Re- 
view, 28: 1-23. 

KAMENEV G. M. & V. A. NADTOCHY, 1999, 
Species of Macoma (Bivalvia: Tellinidae) from 
the Pacific coast of Russia, previously de- 
scribed as Abrina (Bivalvia: Semelidae). 
Malacologia, 41(1): 209-230. 

KEEN, A. M., 1969, Superfamily Tellinacea. Pp. 
613-643, in: R. с MOORE, ed.. Treatise on In- 
vertebrate Paleontology: Mollusca 6, Part N 
(Bivalvia). Lawrence, Kansas: University of 
Kansas Press, xxxviii + 952 pp. 

KEEN, A. M., 1971, Sea shells of tropical west 
America; marine mollusks from Baja Califor- 
nia to Peru. Stanford, California: Stanford Uni- 
versity Press, xvi + 1064 pp. 

KURODA, T, 1951, Descriptions of a new ge- 
nus of a marine gastropod, Kanamurua, gen. 
п., and a new species of a bivalve. Abra 
kanamarui, sp. п., dedicated to Mr. T. 
Kanamaru on his 60'^ birthday. Venus, 16: 68- 
72 [in Japanese]. 

KURODA, T, T HABE & К. OYAMA, 1971, Sea 
shells of Sagami Bay. Tokyo: Maruzeh. xiv + 
741 [in Japanese] + 489 pp. [in English]. 

LAMPRELL, K. & T. WHITEHEAD, 1992, 
Bivalves of Australia. Vol. 1. Bathurst: Crawford 
House Press. 182 pp. 

OKUTANI, T, 2000, Marine mollusks in Japan. 
Tokyo: Tokai University, xlvii + 1175 pp. 

PRASHAD, В., 1932, The Lamellibranchia of the 
Siboga Expedition. Systematic part II: 
Pelecypoda (exclusive of the Pectinidae), in: 
M. WEBER, ed., Siboga-Expeditie 34(53c). 
Leiden. 353 pp. 

SCARLATO, O.A., 1965, Bivalve mollusks of the 
superfamily Tellinacea of the Chinese seas. 
Studia Marina Sinica, 8: 27-114 [in Chinese, 
with Russian summary]. 

SCARLATO, O. A., 1981, Bivalve mollusks of 
temperate waters of the northwestern Pacific. 
Leningrad: NAUKA Press. 480 pp. [in Russian]. 

SCOTT, P H., 1994, Bivalve molluscs from the 
southeastern waters of Hong Kong. Pp. 55-1 00, 
in: B. MORTON, ed.. The malacofauna of Hong 
Kong and southern China III: Proceedings of 
the Third International Workshop on the Malac- 
ofauna of Hong Kong and Southern China, Hong 
Kong, 13April-1 May 1992. Hong Kong: Hong 
Kong University Press, xxii + 504 pp. 

TSUCHIDA, E. & T KUROZUMI, 1995, Fauna 
of marine mollusks of sea around Otsuchi Bay, 
Iwate Prefecture (5) BIVALVIA-2. Otsuchi Ma- 
rine Research Center Report, 20: 13-42 [in 
Japanese]. 

Revised ms. accepted 8 February 2004 



MALACOLOGIA, 2004, 46(1): 169-183 

SHELL STRUCTURES OF SELECTED GASTROPODS 
FROM HYDROTHERMAL VENTS AND SEEPS 

Steffen Kiel 

Freie Universität Berlin, Institut für Geologische Wissenschaften, Fachrichtung Paläontologie, 
Malteserstrasse 74-100, 12249 Berlin, Germany; steffen.kiel@gmx.de 

ABSTRACT 



Shell structures of 24 gastropod species from hydrothermal vents and seeps are elec- 
tron microscopically investigated, and the ecological and phylogenetic implications of their 
shell structures are discussed. The presence of prismatic complex crossed lamellar, and 
regularly foliated structure in the Neolepetopsidae provides further evidence for their posi- 
tion as sister group of the Acmaeidae. The Lepetodriloidea are considered to be derived 
from, or to have a common ancestor with the Fissurellidae based on their complex crossed 
lamellar structure and on the presence of shell pores. The earlier hypothesis that Pelto- 
spiridae derived from Neomphalidae by reduction of complex crossed lamellar structure 
cannot be supported; both groups show the same array of shell structures. It is shown that 
shell pores are a frequent feature in Neomphalidae and Peltospiridae. Dissolution of the 
inner shell walls is documented for Bathynerita naticoides. The trend that small and thin- 
shelled gastropod groups tend to reduce their shell structure to intersected crossed platy, 
can also be observed in the vent/seep gastropods. Generally, their shell structures appear 
to reflect those of the phylogenetic group to which they belong, rather than being influ- 
enced by the peculiarities of the extreme environment they inhabit. 

Keywords: Gastropoda, shell structure, deep-sea, hydrothermal vent, cold seep, 
phylogeny. 



INTRODUCTION 

Chemosynthetic ecosystems in the deep-sea 
harbor highly endemic faunas (Tunnicliffe et 
al., 1996). The gastropods that live there are 
no exception to this: 95-98% of the species 
and 70% of the genera are endemic to vents 
and seeps, and five families are found exclu- 
sively here (Waren & Bouchet, 2001). Origin 
and phylogenetic relationships of many of the 
endemic taxa are still debated. 

Shell structures have only been described 
for three out of the about 1 25 gastropods spe- 
cies known from chemosynthetic ecosystems: 
Neomphalus fretterae (Batten, 1984), Melano- 
drymia aurantiaca (Hickman, 1984), and 
Lepetodrilus elevatus (Hunt, 1 993). The scope 
of the present study is to provide an overview 
over the shell structures of the gastropod fami- 
lies present at chemosynthetic ecosystems, 
and to discuss their ecological and phyloge- 
netic implications. Additionally, these data can 
help to clarify the identity of fossil vent and 
seep gastropods. 



MATERIALSAND METHODS 

The majority of the material used here is 
from the study of Waren & Bouchet (2001 ), 
and was provided by the Muséum National d' 
Histoire Naturelle in Paris (MNHN). Three ad- 
ditional species were provided by the Natural 
History Museum of Los Angeles County 
(LACM). 

All investigated specimens had the size of 
adult specimens as reported in the literature. 
The shell structure of protoconchs and onto- 
genetic changes in shell structures were not 
the subject of this study. Shell mineralogy was 
not studied, and is only inferred from the 
known mineralogy in related groups or is 
noted in cases when the structures have an 
unequivocal mineralogy. To observe the shell 
structures, pieces of shell were broken off the 
apertural region to obtain fresh fracture zones. 
The material was then mounted on stubs, 
coated with gold, and observed with several 
scanning electron microscopes in Paris and 
Hamburg. 



169 



170 



KIEL 



The different types of shell structures were 
determined following the scheme of Carter & 
Clark (1985) and Hedegaard (1990, 1997). All 
figures in this study are oriented in a way that 
the outer side of the shell is up. The shell 
structures present in each species are listed 
from the outer side of the shell towards the 
inside. The taxonomic framework is that of 
Waren & Bouchet (2001). 

Abbrevations in Figures 

ccl complex crossed lamellar 

hom homogenous 

ica intersected crossed acicular 

icp intersected crossed platy 

пае nacre 

pec prismatic complex crossed lamellar 

per organic periostracum 

rfo regularly foliated 

rsp regular spherulitic prismatic 

sc! simple crossed lamellar 

spr simple prismatic 




FIGS. 1, 2. Neolepetopsis of. gordensis. FIG. 1: 
Upper side of shell with the outer complex 
crossed lamellar layer and the prismatic complex 
crossed lamellar layer below (bar = 1 GO |jm). FIG. 
2: Detail of the prismatic complex crossed 
lamellar layer (bar = 10 |jm). 



RESULTS 

Subclass Patellogastropoda 
Family Neolepetopsidae 

The prismatic complex crossed lamellar and 
regularly foliated structures are always com- 
posed entirely of calcite (Hedegaard, 1990). 

Neolepetopsis cf. gordensis McLean, 1990 

- complex crossed lamellar (Fig. 1) 

- prismatic complex crossed lamellar (Figs. 
1,2) 

Mid-America Trench, Jalisco Block, 
18°22'N-104°23'W; seep in 3,000-3,300 m 
(MNHN). 

Eulepetopsis vitrea McLean, 1990 

- prismatic complex crossed lamellar (Figs. 
3,4) 

- regularly foliated (Fig. 5) 

- simple prismatic 

East Pacific Rise, NE of I'llle de Paques, site 
Rehu, 17°24'S-113°12'W; vent in 2,578 m 
(MNHN). 

The prismatic complex crossed lamellar layer 
in this species has a very similar appearance 
as the "outer calcific crossed lamellar" layer 
of Patella crenata described by Bändel & 
Geldmacher (1996). These authors com- 
pared their terminology only to those of 
B0ggild (1930) and MacClintock (1967), but 
not to those of the more recent works of 
Lindberg (1986, 1988) and Hedegaard 
(1990). However, due to their similarity the 
"outer calcific crossed lamellar" layer of Pa- 
tella crenata is here considered the same 
structure as prismatic complex crossed 
lamellar. 

Paralepetopsis ferrugivora Waren & Bouchet, 
2001 

- prismatic complex crossed lamellar (Figs. 
6,7) 

Mid-Atlantic Ridge, Lucky Strike; vent in 
about 1,650 m (MNHN). This specimen 
lacked any further details on its label, the 
depth is derived from the description of the 
Lucky Strike vent field (Van Dover et al., 1996). 
Hedegaard (1990) noted that the prisms of 
the prismatic complex crossed lamellar struc- 
ture are always convex towards the outer 
side of the shell. This is also observed in the 
three neolepetopsids investigated here. 
Hedegaard (1990) also pointed out that 
these prisms show ribbed surfaces, which 



SHELL STRUCTURES OF VENTAND SEEP GASTROPODS 



171 



he interpreted as the edges of the second 
order lamellae. This is also observed here, 
and these ribs have quite different appear- 
ances: in Neolepetopsis cf. gordensis they 
are fine tubercles (Fig. 2), in Paralepetopsis 
ferrugivora they are coarse and irregular 
(Fig. 7), and in Eulepetopsis vitrea they form 
a distinct grid-like pattern with tuberculate 
intersections (Fig. 4). 




Subclass Cocculiniformia 
Family Pyropeltidae 

Pyropelta musaica McLean & Haszprunar, 1 987 

- simple prismatic (Figs. 8, 9) 

- simple crossed lamellar (Figs. 8, 9) 

- simple prismatic (Fig. 8) 

California, Santa Catalina Basin, between 
San Clemente and Santa Santa Catalina, 
33°12'N, 118°30'W; whale bone from 1,240 
m; (LACM 146909). 

The shell consists of at least five alternating 
layers of simple crossed lamellar and simple 
prismatic structure, with the simple crossed 
lamellar layer becoming progressively 
thicker towards the outer side of the shell. 
The microcrystals of the simple crossed 
lamellar layers are not very densely packed. 

Subclass Vetigastropoda 
Family uncertain 

Sahlingia xandaros Waren & Beuchet, 2001 

- simple prismatic (Fig. 10) 

- intersected crossed acicular or platy (Fig. 10) 

- homogenous (Fig. 10) 




FIGS. 3-5. Eulepetopsis vitrea. FIG. 3: Outer 
prismatic complex crossed lamellar layer (bar = 
10 |jm). FIG. 4: Close-up on the grid-like, ribbed 
surface of the prisms (bar = 1 |jm). FIG. 5: 
Regularly foliated layer (bar =10 |jm). 



FIGS. 6, 7. Paralepetopsis ferrugivora. FIG. 6: 
Overview showing that the entire shell is composed 
of prismatic complex crossed lamellar structure 
(bar = 10 pm). FIG. 7: Close-up on the prisms, 
showing their ribbed surface (bar = 10 pm). 



172 



KIEL 







FIGS. 8, 9. Pyropelta musaica. FIG. 8: Cross- 
section showing five layers with simple crossed 
lamellar structure, with four thin layers with simple 
prismatic structure between them, marked by thin 
white bars (bar = 1 [jm). FIG. 9: Close-up on three 
simple crossed lamellar layers (bar = 10 |jm). 



Alaska, Aleutian Trench, Kodiak Seep, 
56°55.65'N, 149°32.90'W (LACM 1999-45); 
seep in 4,430 m. 

The crossed layer has a granular appearance 
making it difficult to distinguish between in- 
tersected crossed acicular or platy structure. 



FIGS. 11, 12. Protolira valvatoides. FIG. 11: 
Organic periostracum, simple prismatic, and 
intersected crossed platy structure (bar = 1 pm). 
FIG. 12: Outer side of shell with organic 
periostracum and the intersected crossed platy 
layer (bar =10 pm). 




FIG. 10. Sahlingia xandaros, showing the thin 
outer layer of simple prismatic structure, the 
remaining shell is composed of intersected 
crossed acicular or platy structure (bar = 1 pm). 



FIG. 13. Bruceiella athlia, showing the intersected 
crossed platy and simple prismatic layers, and 
the intersected crossed acicular layer below (bar 
= 10 pm). 



SHELL STRUCTURES OF VENTAND SEEP GASTROPODS 



173 



Family Skeneidae 

Protolira valvatoides Waren & Bouchet, 1993 

- simple prismatic (Fig. 11) 

- intersected crossed platy (Figs. 11, 12) 

- simple prismatic (Fig. 11) 
Mid-Atlantic Ridge, Lucky Strike, site Pago- 
das, 54°18.32'N-32°1 6.51 W; vent in 1,685 m 
(MNHN). 

Many of the microcrystals have a granular 
appearance, and show cavities between 
each other. 

Bruceiella athlia Waren & Bouchet, 1 993 

- intersected crossed platy (Fig. 13) 

- simple prismatic (Fig. 13) 

- intersected crossed acicular (Fig. 13) 
Aleutian Trench, site Shumagin, 54°18.06'N- 
157°12.irW; seep in 2,524 m (MNHN). 
Many of the microcrystals have a granular 
appearance, and show cavities between 
each other. 

Family Sutilizonidae 

Sutilizona theca McLean, 1989 

- simple prismatic (Fig. 14) 

- intersected crossed platy (Fig. 14) 

East Pacific Rise, ir46'N, 103°47'W; vent 
in 2,715 m (Paratype LACM 2355). 

Family Lepetodrilidae 

Hunt (1 992) used powder diffraction to show 
that the shell of Lepetodrilus elevatus is com- 
posed entirely of aragonite. It is therefore as- 
sumed that the shells of the lepetodrilids 
investigated here are also composed of ara- 
gonite. 




Lepetodrilus pustulosas McLean, 1988 

- simple prismatic (Fig. 15) 

- complex crossed lamellar (Figs. 15, 16) 
East Pacific Rise, sites Parigo, Genesis, 
Elsa, 12°48.52'N-103°56.48'W; vent in 
4,808 m (MNHN). 

There are occasionally fine pores perpen- 
dicular to the shell's surface, with an aver- 
age diameter of 1 pm (Fig. 16). 

Pseudorimula midatlantica McLean, 1992 

- homogenous 

- complex crossed lamellar 
Mid-Atlantic Ridge, Snake Pit, site Elan, 
23°23'N-44°56'W; vent in 3,520 m (MNHN). 
There are occasionally fine pores perpen- 
dicular to the shell's surface, with an aver- 
age diameter of 1-2 pm. 

Family Trochidae 

Bathymargarites symplector Waren & Bouchet, 
1989 




FIG. 14. Sutilizona theca, showing the thin outer 
layer of simple prismatic structure, the remaining 
shell is composed of intersected crossed platy 
structure (bar = 3 pm). 



FIGS. 15, 16. Lepetodrilus pustulosas. FIG. 15: 
Cross section showing the outer simple prismatic 
layer, and the inner complex crossed lamellar 
layer (bar = 100 pm). FIG. 16: Close-up on the 
crossed lamellar layer, arrows indicate the fine, 
vertical pores (bar = 10 pm). 



174 



KIEL 





FIGS. 17, 18. Bathymargarites symplector. FIG. 
17: Overview showing the regular spherulitic 
prismatic upper layer, and the nacreous inner 
layer (bar = 50 [jm). FIG. 18: Close-up on the 
outer side of the shell showing the thin 
homogenous layer and the upper part of the 
regular simple prismatic layer (bar = 10 pm). 



- homogenous (Fig. 18) 

- regular spherulitic prismatic (Figs. 17, 18) 

- columnar nacre (Fig. 17) 

East Pacific Rise 13°N; the label in the box 
indicates "same as Waren & Bouchet, 1993: 
11-13, figs. 10A-E, 11A-B"; it is thus likely 
to be from a vent in 2,61 6-2,635 m (MNHN). 




FIG. 19. Retiskenea diploura, the homogenous, 
simple prismatic, and intersected crossed platy 
structures merge into each other (bar = 10 pm). 



FIG. 20. Melanodrymia aurantiaca, view on the 
simple crossed lamellar structure (bar = 10 цт). 



Subclass uncertain 
Family Neomphalidae 

Retiskenea diploura Waren & Bouchet, 2001 

- homogenous (Fig. 19) 

- simple prismatic (Fig. 19) 

- intersected crossed platy (Fig. 19) 

- simple prismatic 

Aleutian Trench, site Shumargin, 
54°18.17'N-157°11.82'W; seep in 4,808 m 
(Paratype, MNHN). 

The microcrystals of the crossed platy layer 
are not very densely packed and have a 
granular appearance. 

Melanodrymia aurantiaca Hickman, 1984 

- simple prismatic (Fig. 20) 

- simple crossed lamellar (Fig. 20) 

- simple prismatic (Fig. 20) 

East Pacific Rise, sites Parigo, Pogosud, 
Genesis, 12°48.52'N-103°56.48'W; vent in 
2,630 m (MNHN). 

Hickman (1984) reported a thick layer with 
complex prismatic structure in this species. 
Hedegaard (1990) pointed out that Mac 
Clintock's (1967) "complex prismatic" struc- 
ture is identical with the "simple crossed 
lamellar" structure of Carter & Clark (1985). 
Thus, the superficial differences between 



SHELL STRUCTURES OF VENTAND SEEP GASTROPODS 



175 




FIG. 21. Cyathermia naticoides, upper part of a 
cross-section with simple prismatic and complex 
crossed lamellar structure, arrow indicates a shell 
pore (bar =10 |jm). 



Hickman's (1 984) and my descriptions of the 
shell structures of Melanodrymia aurantiaca 
is likely to be only a difference in terminology. 

Cyathermia naticoides \Naren & Bouchet, 1989 

- simple prismatic (Fig. 21) 

- complex cross lamellar (Fig. 21) 





FIG. 22. Pachydermia laevis, homogenous and 
complex crossed lamellar structure, arrow 
indicates a shell pore (bar = 100 pm). 



FIG. 23. Peltospira smaragdina has an outer layer 
with simple prismatic structure, and an inner layer 
with complex crossed lamellar structure, arrows 
indicate the broad shell pores (bar = 10 pm). 



East Pacific Rise, sites Julie, Genesis, 
Parigo, 12°48.96'N-103°46.62'W; vent in 
2,630 m (MNHN). 

Pachydermia laevis Waren & Bouchet, 1989 

- homogenous (Fig. 22) 

- complex crossed lamellar (Fig. 22) 

East Pacific Rise, site Genesis, 12°48.56'N- 
103°46.58'W; vent in 2,630 m (MNHN). 
There are occasionally fine pores perpen- 
dicular to the shell's surface with an aver- 
age diameter of 3 pm (Fig. 22). 

Planorbidella planispira (Waren & Bouchet, 
1989) 

- homogenous 

- complex crossed lamellar 

- simple prismatic 

East Pacific Rise, site Elsa, 12°48.09'N- 
103°46.34'W; vent in 2,630 m (MNHN). 

Family Peltospihdae 

Peltospira smaragdinaVslarén & Bouchet, 2001 

- simple prismatic (Fig. 23) 

- complex cross lamellar (Fig. 23) 
Mid-Atlantic Ridge, Lucky Strike, site Sintra, 
37°17.50'N-32°16.47'W; vent in 1,622 m 
(MNHN). 

There are occasionally fine pores perpen- 
dicular to the shell's surface, with an aver- 
age diameter of 4 pm (Fig. 23). 

Ctenopelta porifera Waren & Bouchet, 1993 

- homogenous, with traces of unidentified 
shell structures (Figs. 24, 25) 

- simple prismatic (Fig. 25) 

East Pacific Rise, sites Totem, Genesis, 
Elsa, 12°48.71'N-103°56.53'W; vent in 
2,630 m (MNHN). 



176 



KIEL 



The shell is perforated by fine pores with an 
average diameter of 4 pm (Figs. 24, 25); 
these pores have not been observed in the 
internal septum. 

Lirapex costellata Waren & Bouchet, 2001 

- simple prismatic 

- complex crossed lamellar 

- homogenous 

- simple prismatic 

Mid-Atlantic Ridge, Lucky Strike, site Tour 
Eiffel, 37°17.32N-32°16.5rW; vent in 1,685 
m (MNHN). 

Hedegaard (1990) presented shell structure 
data for three species he considered 
peltospirids. Among these, Hyalogyrina glabra 
has subsequently been assigned to the 
heterobranch family Hyalogyrinidae (Waren & 
Bouchet, 2001). The other two - Xyloskenea 
costulifera and Bathyxylophila excelsa - were 
placed in the Skeneidae (Marshall, 1988), and 



no subsequent work has been done on them. 
However, the Skeneidae are a heterogenous 
group of small-shelled trochoids, and the as- 
signment of Xyloskenea costulifera and 
Bathyxylophila excelsa to either the Skeneidae 
or the Peltospiridae is still uncertain (Marshall, 
pers. comm., 2003). 

Subclass Neritimorpha 
Family Neritidae 

Bathynerita naticoidea Clarke, 1989 

- homogenous (Fig. 26) 

- simple cross lamellar (Fig. 26) 

- simple prismatic 

Louisiana Slope, Bush Hill Seep, 
27°46.91'N-9r30.34'W; seep in 540-580 m 
(MNHN). 




FIGS. 24, 25. Ctenopelta porifera. FIG. 24: Outer 
layer with homogenous, and remnants of an 
unidentified structure, arrow indicates a shell pore 
(bar = 10 |jm). FIG. 25: Lower side of shell with 
homogenous?, and simple prismatic structure, 
arrow indicates a shell pore (bar = 10 pm). 



FIGS. 26, 27. Bathynerita naticoidea. FIG. 26: 
Mainly complex crossed lamellar structure, and 
thin, homogenous outer layer (bar = 100 pm). 
FIG. 27: View on the shell's interior showing that 
the inner walls are dissolved (bar = 100 pm). 



SHELL STRUCTURES OF VENTAND SEEP GASTROPODS 



177 



Hedegaard (1990) found the outer, homog- 
enous layers of the five Neritidae investigated 
by him to be composed of calcite. It is thus 
likely that the thin homogenous outer layer of 
Bathynerita naticoidea is also composed of 
calcite. The inner shell walls of Bathynerita 
naticoidea are dissolved (Fig. 27). Dissolution 
of the inner shell walls is characteristic for the 
Neritidae, but has apparently never been docu- 
mented for Bathynerita naticoidea. 

Family Phenacolepidae 

Shinkaiiepas briandi \Naren & Bouchet, 2001 

- homogenous (dense) (Fig. 29) 

- homogenous (granular) (Fig. 29) 

- complex crossed lamellar (Figs. 28, 29) 

- intersected crossed platy (Fig. 28) 

- simple prismatic 

Mid-Atlantic Ridge, Lucky Strike, site Sintra, 
37°17.50'N-32°16.47'W; vent in 1,622 m 
(MNHN). 



The homogenous outer layer that builds the 
ridges on the shell surface is similar to the 
outer layer described for Phenacoiepas 
pulchellus by Hedegaard (1990). Hedegaard 
(1990) assumed that this layer has calcific 
shell mineralogy, which is also likely in the 
species investigated here. 




FIGS. 28, 29. Shinl<ailepas briandi. FIG. 28: Inner 
side of the shell, showing the transition from 
complex crossed lamellar to intersected crossed 
platy structure (bar = 10 |jm). FIG. 29: Outer side 
of the shell showing the homogenous outer layer; 
the homogenous layer is very dense in the rib, and 
more granular away from the rib (bar = 100 цт). 



FIGS. 30-32. Provanna variabilis. FIG. 30: 
Overview showing the inner and outer, simple 
prismatic layers, and a central layer with complex 
crossed lamellar structure (bar = 10 pm). FIG. 
31: Close-up on the upper side of the shell, 
showing the slightly detached organic 
periostracum, and the transition from the outer 
simple prismatic to the complex crossed lamellar 
layer (bar = 10 pm). FIG. 32: Inner simple 
prismatic layer is absent in this part of the shell 
(bar = 50 pm). 



178 



KIEL 




■'Щ\- 



FIG. 33. Alviniconcha hessleri, the organic 
periostracum is about two anda half times thicker 
than the shell (bar = 100 |jm). 



Subclass Caenogastropoda 
Family Provannidae 

Provanna variabilis Waren & Beuchet, 1986 

- simple prismatic (Figs. 30-32) 

- complex crossed lamellar (Figs. 30-32) 

- simple prismatic (Fig. 30) 



Juan de Fuca Ridge, 47°57'N-129°04'W; 
vents in 2,212 m (MNHN). 
In the innermost portion of the complex 
crossed lamellar layer the microcrystais are 
sometimes only loosely packed, although they 
are densely packed in the remaining part of 
the layer (Fig. 30). The inner simple prismatic 
layer may be present or absent at different 
parts of the shell (compare Figs. 30 and 32). 

Alviniconciia hessleri Okutani & Ohta, 1988 

- simple prismatic 

- complex crossed lamellar (Fig. 33) 

- simple prismatic 

Mariana Back Arc Basin, site Alice Springs, 
18°12.59'N-144°42.43'E; vent in 3,630- 
3,655 m (MNHN). 

Subclass Heterobranchia 
Family Xylodisculidae 

Xylodiscula análoga Waren & Bouchot, 2001 

- intersected crossed platy (Fig. 34) 

- simple prismatic (Fig. 34) 
Mid-Atlantic Ridge, Lucky Strike, site Tour 




FIG. 34. Xylodiscula analogs has mainly intersected crossed platy structure, and a thin layer with 
simple prismatic structure at the inner side of the shell. The "smeared" area in the center of the 
picture and the loose packing of microcrystais probably indicate a high content of organic material 
(bar = 10 |jm). 



SHELL STRUCTURES OF VENTAND SEEP GASTROPODS 



179 



Eiffel, 37°17.32'N-32°16.51'W; vent in 

1,685m (MNHN). 

The microcrystals are not very densely 

packed. 

Family Hyalogyhnidae 

Hyalogyrina umbellifera Waren & Bouchet, 2001 

- simple prismatic (Figs. 35, 36) 

- intersected crossed platy (Figs. 35, 36) 
Aleutian Trench, siteShumagin, 54°18.17'N- 
157°11.82'W; seep in 4,808 m (Paratype, 
MNHN). 

This composition of shell structure is similar 
to that described for Hyalogyrina glabra 
(Hedegaard, 1990). The microcrystals are 
not very densely packed. 







FIGS. 35, 36. Hyalogyrina umbellifera. FIG. 35: 
Thin outer layer with simple prismatic structure, 
and intersected crossed platy structure below 
(bar = 1 |jm). FIG. 36: Close-up on the transition 
from simple prismatic to intersected crossed platy 
structure, note the loose packing of the 
microcrystals (bar = 10 pm). 



DISCUSSION 

Among the purposes of this study was to 
investigate whether the peculiarities of the 
vent/seep environment influence the shell 
structures of the gastropods groups living 
there. The microcrystals that build the shell 
structures are not very densely packed in sev- 
eral species (e.g., in Retisl<enea diploura, 
Hyalogyrina umbellifera, Xylodiscula análoga, 
and to a lesser extend also in Pyropelta 
musaica, Protolira valvatoides, and Bruceiella 
athlia). Such loose packing is rarely observed 
in gastropods from shallow-marine environ- 
ments, even in very thin-shelled species 
(Bändel, pers. comm. 2003; pers. observa- 
tions). This loose packing is most probably the 
result of a high organic content in the shell. 
Loose packing occurs most frequently in small, 
thin shells with intersected crossed platy struc- 
ture. This makes it at present impossible to 
distinguish whether it is related to the extreme 
vent/seep habitat, or to shell thickness and 
structure, or both. 

An obvious correlation, although not related 
to the vent/seep habitat, is that between the 
presence of intersected crossed platy struc- 
ture and shell thickness. This structure occurs 
more frequently in small, thin-shelled species 
than in larger, thicker-shelled ones. Hede- 
gaard (1990) noted that among the Archaeo- 
gastropoda, intersected crossed platy 
structure dominates in species from small- 
shelled groups. In case of the vent/seep gas- 
tropods investigated here, this tendency can 
not only be observed among archaeogastro- 
pods, but also among the Heterobranchia, in 
the families Hyalogyhnidae and Xylodisculidae. 

There are no apparent correlations between 
shell structures and depth or habitat. 

Neolepetopsidae 

Anatomical and molecular data indicate a 
sister group relationship of Neolepetopsidae 
and Acmaeidae (Lindberg, 1998; Harasewych 
& McArthur, 2000). The three neolepetopsids 
investigated here have prismatic complex 
crossed lamellar shell structure, which Hede- 
gaard (1990) considered as apomorphy of the 
Acmaeidae. Hedegaard (1990) also pointed 
out that regularly foliated structure is present 
only in few acmaeids, and considered the re- 
duction of this structure as an apomorphy of 
the Acmaeidae sensu stricto. Regularly foil- 



180 



KIEL 



ated structure is present in Eulepetopsis vitrea, 
but absent in Neolepetopsis cf. gordensis and 
Paralepetopsis ferrugivora. The position of the 
Neolepetopsidae as sister group of the 
Acmaeidae within the Acmaeoidea can thus 
be supported. However, it should be noted that 
the patellid Patella crenata also has prismatic 
complex crossed lamellar structure (Bändel & 
Geldmacher, 1996), raising some doubt 
whether this shell structure can actually be 
considered as an apomorphy of the Acmaeidae. 

Pyropeltidae 

Pyropelta musaica has simple crossed 
lamellar structure like the three cocculinids 
investigated by Hedegaard (1 990), but a multi- 
layered occurrence of this structure separated 
by thin layers of a different structure as in 
Pyropelta musaica was not described. Neither 
does a fossil cocculinid from the Cretaceous 
show such a pattern (my data). A total of five 
investigated Cocculiniformia are far too few 
to propose this alternation of shell structures 
as an apomorphy of the Pyropeltidae. How- 
ever, when future research confirms that this 
pattern does not occur in other cocculiniforms, 
it could be used for phylogenetic purposes, 
and also to identify members of the Pyropel- 
tidae in the fossil record. 

Sahlingia 

Sahlingia xandaros has only simple prismatic 
and intersected crossed platy structures, which 
are not very conclusive for phylogenetic pur- 
poses. 

Skeneidae 

The two skeneids have the same shell struc- 
tures as the three skeneids investigated by 
Hedegaard (1990). 

Lepetodrilidae and Sutilizonidae 

The Lepetodrilidae are considered here to 
be derived from, or to have a common ances- 
tor with the Fissurellidae. The two decisive 
factors are their complex crossed lamellar 
structure, and their fine shell pores. Among 
the slit-bearing Vetigastropoda, the Pleuro- 
tomariidae, Haliotidae, and Seguenziidae have 
a nacreous shells (Boggild, 1930; Erben & 
Krampiz, 1972; Bändel, 1979; Hedegaard, 
1990; Harasewych, 2002) and are thus less 



likely to be related. This also pertains to the 
Palaeozoic slit-bearing Porcellidae, for which 
nacre is inferred from the presence of nacre 
in its Mesozoic sister group, the Cirridae (Kiel 
& Fryda, 2004). The two remaining slit-bear- 
ing groups, Fissurellidae and Scissurellidae, 
have crossed lamellar structure (Batten, 1975; 
Bändel, 1998), have shell morphologies simi- 
lar to those of the lepetodrilids, and are ana- 
tomically similar (Waren, pers. comm., 2003). 
Among these, only the fissurellids show shell 
pores as found in the lepetodrilids. Shell pores 
evolved independently in several groups of 
mollusks (Reindl & Haszprunar, 1996), can be 
present or absent in genera of the same fam- 
ily (e.g., Peltospiridae or Neomphalidae as 
shown herein), and may even be present or 
absent in species of the same genus - for 
example, Shinkailepas briandi without pores 
(herein) and Shinkailepas myojinensis with 
pores (Sasaki et al., 2003). However, shell 
pores have never been reported in scissurellids 
but frequently in fissurellids. Thus, the coinci- 
dence of similar shell shape, shell structure, 
and the presence of shell pores in both groups 
allows me to propose a close phylogenetic 
relationship between Fissurellidae and 
Lepetodrilidae. 

The sutilizonid Sutilizona theca has inter- 
sected crossed platy structure, which is nei- 
ther very conclusive for phylogenetic analysis, 
nor does it contradict previously suggested 
relationships (Haszprunar, 1988; Ponder & 
Lindberg, 1997; Waren & Beuchet, 2001). 

Trochidae 

Bathymargarites symplector has columnar 
nacre which is a common shell structure 
among the Trochidae (Wise, 1970; Erben, 
1974; Hedegaard, 1990; Hickman & McLean, 
1990). It is, however, the only gastropod with 
nacreous shell investigated here. Among other 
trochids from vents and seeps, nacre was re- 
ported from Cataegis meroglypta (McLean & 
Quinn, 1987; Waren & Bouchet, 1993), and 
from species of Falsimargarita (Waren & 
Bouchet, 2001). 

Neomphalidae and Peltospiridae 

Hedegaard (1990) proposed that the Pelto- 
spiridae is derived from the Neomphalidae by 
reduction of crossed lamellar structure. How- 
ever, this was based on the incorrect higher 
taxonomic placement of his species - none of 



SHELL STRUCTURES OF VENTAND SEEP GASTROPODS 



181 



them appears to belong to the Peltospihdae. 
Two of the three peltospihds investigated here 
show complex crossed lamellar structure, 
whereas two of the six Neomphalidae with 
known shell structure lack complex crossed 
lamellar structure. These new observations 
negate derivation of the Peltospihdae from the 
Neomphalidae, but do provide additional evi- 
dence that both families are related, as indi- 
cated by anatomical and molecular studies 
(Israelsson, 1998; McArthur & Tunnicliffe, 
1998; Waren & Beuchet, 2001). 

Shell pores in the Neomphalidae were first 
reported from Neomphalus fretterae, which has 
two types of pores, averaging 0.1 pm and 1.0 
pm in diameter (Batten, 1984). Four out of the 
nine species of the Neomphalidae and 
Peltospiridae investigated here (including 
Neomphalus fretterae) have pores in their 
shells. In Peltospira smaragdina, Pachydermia 
laevis, and Ctenopelta porifera, the pores have 
an average diameter of 1.0-4.0 pm. The latter 
species has additional macropores 30.0-70.0 
pm in diameter (Waren & Beuchet, 1993). The 
function of such shell pores is still controver- 
sial (Reindl & Haszprunar, 1996). Batten (1984) 
found the highest concentration of pores in 
Neomphalus fretterae around muscle insertion 
fields, and therefore interpreted them as muscle 
insertions. In the case of Ctenopelta porifera, 
Waren & Beuchet (1993, 2001) suggested the 
macropores to be related to chemosymbiosis. 

Although the shell structure of the proto- 
conch is not the scope of this study. Batten's 
(1984) interpretation of the multi-layered 
protoconch of Neomphalus fretterae deserves 
comment. Batten (1984) speculated that the 
three shell layers of the protoconch "may indi- 
cate that the veliger larval stage may have an 
extended planktonic mode." Calcification of the 
protoconch in archaeogastropods, however, 
takes place at the beginning of their benthic 
life, after the velum has been discarded 
(Bändel, 1982), and thus after the free-swim- 
ming larval stage. The additional inner layers 
have thus been built by the benthic juvenile or 
adult, possibly to strengthen the apical por- 
tion of the sub-limpet shell. 

Neritidae and Phenacolepidae 

Both investigated species, Bathynerita 
naticoidea and Shinkailepas briandi, have 
crossed lamellar structures like their shallow- 
marine relatives. Likewise, both species have 
a homogenous outer layer with presumably 



calcific mineralogy. In this respect, both spe- 
cies differ from the neritilliid Pisulina, in which 
the thin outer layer has simple prismatic struc- 
ture (Kano & Kase, 2000). In contrast to the 
classification of Waren & Beuchet (2001), 
Bathynerita has recently been considered to 
be more closely related to the Phenacolepidae 
than to the Neritidae (Hodgson et al., 1998; 
Капо et al., 2002). Unfortunately, shell struc- 
tures are too uniform among the two groups 
to provide further evidence to this hypothesis. 
The inner shell walls of Bathynerita naticoidea 
are dissolved, a feature that is characteristic 
for all known neritoids, but had not yet been 
demonstrated for Bathynerita. 

Provannidae 

The observed shells structures in the two 
provannids are similar to those of other 
caenogastropods (Bändel, 1990). 

Hyalogyhnidae and Xylodisculidae 

Both investigated species have homogenous 
and intersected crossed platy structure, whereas 
all other known heterobranchs have crossed 
lamellar structure (Bändel, 1990). This devia- 
tion might result from their small and thin shells, 
rather than being of phylogenetic importance. 

In sum, the shell structures of the vent and 
seep gastropods appear to reflect those of the 
phylogenetic group to which they belong, 
rather than being influenced by the peculiari- 
ties of the extreme environment they inhabit. 



ACKNOWLEDGMENTS 

I would like to thank P. Bouchet, Paris, who 
made my visit to the MNHN in Paris possible; 
V. Heros and R. von Cosel, Paris, for their help 
with the collection in Paris; J. McLean and L 
Groves, Los Angeles, for making shell material 
from the NHM available to me; С Chancogne, 
and G. Mascaren, Paris, for their help with the 
SEM; A. Waren, Stockholm, for discussion and 
for sharing unpublished data; С T. S. Little, 
Leeds, for linguistic improvements; K. Bändel, 
Hamburg, for discussion of shell structures 
and shell formation in gastropods, and two 
anonymous reviewers for their critical reading 
of the manuscript. This study was financially 
supported by the COLPARSYST- program at 
the MNHN Paris. 



182 



KIEL 



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Revised ms. accepted 2 April 2004 



MALACOLOGIA, 2004, 46(1): 185-202 

CREPIDULA CACHIMILLA (MOLLUSCA: GASTROPODA), 
A NEW SPECIES FROM PATAGONIA, ARGENTINA 

Maximiliano Cledón^ 2* l^j^ Ricardo L. Simone^ & Pablo E. Penchaszadeh^ 



ABSTRACT 

A new species, Crepidula cachimilla, is described based on a population from 15 m 
deptfi in San Antonio Oeste, Argentina. Shell length ranged from 5.4 to 28.5 mm for males 
and from 9.6 to 52.2 mm for females. The minimum shell length recorded for a brooding 
female was 23.5 mm, and the maximum shell length was 49.3 mm. A detailed anatomical 
description is given, showing as main characters of the species a relative thick columellar 
muscle, a greater closure of the palliai cavity aperture by a fusion of the mantle border, a 
very small osphradium, with about 16 broad filaments, endostyle divided by a middle lon- 
gitudinal furrow, very large salivary glands, duplication of both gastric ducts to the diges- 
tive gland, male seminal vesicle very long and with irregular walls, pallia! oviduct with a 
broad vaginal duct and a tall papilla originating both from palliai floor and roof. Brood egg 
masses of mature females contained from 15 to 65 egg capsules. The triangular-shaped 
egg capsules measured between 2.2 and 3.4 mm in length and between 2.3 and 3.8 mm 
in width. Each egg capsule contained between 129 and 563 eggs. The number of eggs per 
capsule and the egg diameter did not correlate with female shell length. Uncleaved eggs 
measured between 180 and 200 pm in diameter. They all developed synchronously within 
the egg capsules. Prehatching veliger shells measured between 260 and 300 pm in length. 
After hatching at the veliger stage, protoconch length during metamorphosis ranged be- 
tween 700 and 800 pm. These parameters neither coincide with those reported by Hoagland 
(1977) for the similar Californian Crepidula onyx, nor with the reproductive characters 
reported by Miloslavich & Penchaszadeh (2001) for Crepidula aplysioides, which suppos- 
edly occurs in the region. 

Key words: Crepidula cachimilla, new species, Calyptraeoidea, anatomy, reproduction, 
southwestern Atlantic, Patagonia, hermaphroditism. 



INTRODUCTION 

According to Dall (1909: 234), Crepidula 
onyx (G. B. Sowerby I, 1824) occurs along the 
Pacific coast from North America to Chile. 
Based on shell and radular morphology, 
Parodiz (1939) reported this species on the 
Atlantic coast of Argentina, from San Matías 
Gulf to Punta Norte, and Aguirre & Fahnati 
(2000) recorded fossils of this species from 
the Quaternary period in northeastern Argen- 
tina. Hoagland (1977) suggested that the At- 
lantic material studied by Parodiz (1939) 
should be attributed to C. aplysioides. 
Crepidula aplysioides has been defined both 
anatomically (Simone, 2002) and by reproduc- 
tive patterns (Miloslavich & Penchaszadeh, 



2001 ). Based on the differences with the stud- 
ied sample, we conclude that our material from 
San Antonio Oeste, Argentina, belongs to an 
undescribed species. In this paper, we de- 
scribe this new species, which is restricted to 
an area of Patagonia, southwestern Atlantic. 
The study on the calyptraeids has grown 
considerately in the last few years with the 
addition of knowledge on the anatomy (e.g., 
Simone, 2002), molecular biology (e.g., Collin, 
2000), and reproductive strategies (e.g., 
Miloslavich & Penchaszadeh, 2001 ). From the 
eastern coast of Americas, knowledge of the 
informally defined "Crepidula plana complex" 
is of particular importance (Collin, 2000; 
Simone, submitted; Simone et al., 2000), of 
which this paper is a part. 



'Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina 
^Alfred Wegener Institut für Polar und Meeresforschung, Bremerhaven, Germany 

^Museu de Zoología da Universidade de Säo Paulo, Caixa Postal 42594, 04299-970 Säe Paulo, SR Brazil 
*To whom correspondence should be addressed; mcledon@bg.fcen.uba.ar 



185 



186 



CLEDON ETAL 



MATERIALS AND METHODS 

Three samples were collected in March, May, 
and August 2001 at 15 m depth at Playa 
Orengo, San Antonio Oeste (40°53'S, 64°36'W), 
Argentina, by SCUBA diving. The animals 
were attached to the bivalves Atrina seminuda 
(d'Orbigny, 1 846) and Aulacomya atra (Molina, 
1 782) and to stones. Approximately 370 speci- 
mens were collected. 

Live individuals were carried to the labora- 
tory, carefully detached from their substratum, 
measured to the nearest 0.1 mm precision with 
a digital vernier calliper, and some specimens 
dissected for anatomical description in vivo. 

Shell parameters were measured following 
Hoagland's (1 977) definitions. "D" refers to the 
length of the shell arc, whereas convexity is 
the relation between shell arc and shell length; 
"SL" refers to shell length. 

The sexual characteristics of the population 
were determined by the presence or absence 
of a penis. 

A total number of 47 egg masses was found 
and fixed in 5% seawater-formalin. Four ran- 
domly chosen egg capsules per egg mass 
were detached, and their length and width 
were measured under a stereomicroscope. 
Eggs and embryos contained within these egg 
capsules were counted and measured, and the 
presence or absence of cannibalism or nurse 
eggs was analyzed with a Kruskal-Wallis test. 

Settlement size was estimated by measur- 
ing the protoconch length under SEM. 

Simple linear regression type 2 following 
natural logarithmic (In) transformations was 
carried out to identify the parameters of taxo- 
nomic value. 

Radular characteristics of six individuals of 
different sizes were also studied with SEM. 

The anatomical study was performed using 
standard methodology, with non-narcotized 
specimens fixed in 70% ETOH. Dissections 
were performed under a stereomicroscope, with 
the specimens immersed in fixative. All draw- 
ings were done with the aid of a camera lucida. 

Abbreviations of anatomical structures are as 
follows: aa, anterior aorta; ab, auricle region 
beyond ventricle connection; ac, anterior ex- 
tremity of gill on mantle border; ad, adrectal 
sinus; af, afferent gill vessel; ag, albumen 
gland; an, anus; ap, aperture of visceral vas 
deferens into palliai cavity; au, auricle; bg, buc- 
cal ganglion; ce, cerebro-pleural ganglia; eg, 
capsule gland; cm, columellar muscle; cv, 
ctenidial vein; dd, duct to digestive gland; dg, 
digestive gland; di, septum separating 



haemocoel from visceral mass; dm, dorsal shell 
muscle; dp, posterior duct to digestive gland; 
en, endostyle; es, esophagus; ey, eye; fd, foot 
dorsal surface; ff, female folds of genital pa- 
pilla; fg, food groove; fl, female papilla; fp, fe- 
male pore; gd, gonopericardial duct; gf, gastric 
fold; gi, gill; gp, pedal ganglion; gs, gastric 
shield; ig, probable ingesting gland; in, intes- 
tine; iu, "U"-shaped loop of intestine on palliai 
roof; ki, kidney; II, left lateral expansion (flap) 
of neck; Im, lateral shell muscle; m1-m14, 
odontophore muscles; mb, mantle border; ml, 
mantle region restricting palliai cavity; mo, 
mouth; ne, nephrostome; ng, nephhdial gland; 
nr, nerve ring; od, odontophore; os, osphra- 
dium; ov, palliai oviduct; oy, ovary; pb, probos- 
cis; PC pericardium; pd, penis sperm groove; 
pe, penis; pp, penis papilla; pr, propodium; py, 
palliai cavity; rg, repugnatohal gland; rl, right 
lateral expansion (flap) of neck; rn, radular 
nucleus; rs, radular sac; rt, rectum; sa, sali- 
vary gland duct; sd, palliai sperm groove; se, 
subesophageal ganglion; sg, salivary gland; si, 
siphon-like fold; sr, seminal receptacles; ss, 
style sac; st, stomach; su, supraesophageal 
glangion; sv, seminal vesicle; sy, statocyst; te, 
cephalic tentacle; tg, integument; tm, net of 
transversal muscles of haemocoel; ts, testis, 
ve, ventricle; vg, vaginal duct; vm, visceral 
mass; vo, visceral oviduct. 

Abbreviations of institutions: AMNH, Ameri- 
can Museum of Natural History, New York, 
New York, USA; FMNH, Field Museum of 
Natural History, Chicago, Illinois, USA; MACN, 
Museo Argentino de Ciencias Naturales "В. 
Rivadavia", Buenos Aires, Argentina; MZSP, 
Museu de Zoología da Universidade de Sao 
Paulo, Sao Paulo, Brazil. 



RESULTS 

Crepidula cachimilla, new species 
(Figs. 1-44) 

Crepidula onyx Sowerby: Parodiz, 1939: 701, 

pi. 1, fig. 1; Scarabino, 1977: 185, pi. 3, fig. 

5 {non G. В. Sowerby I, 1824). 
Crepidula aplysioides Reeve: Hoagland, 1977: 

369 (Argentinean material only) [non Reeve, 

1859). 

Type Material 

Holotype: AMNH 306947. Paratypes: AMNH 
306957 to 306961 , 14 paratypes (5 dry speci- 
mens); AMNH 306948 to 306956, 9 paratypes 



CREPI DULA CACHI MILLA N. SP. 



187 



(4 females, 5 males preserved In ethanol); 
MZSP 41427 (15 paratypes); FMNH (10 para- 
types). 

Type Locality 

Rio Negro, San Antonio Oeste, Playa 
Orengo, Argentina (40°53'S, 64°36'W), 15 m 
depth, on shells of Atrina seminuda and 
Aulacomya atra and on stones (Figs. 1-3). 

Etymology 

The name of the species alludes to the 
mapuche word meaning great friend and is dedi- 
cated to our colleagues at the Invertebrates I 
Laboratory of the Facultad de Ciencias Exactas 
y Naturales, Universidad de Buenos Aires. 




FIGS. 1-3. Crepidula cachimilla on different 
substrata. FIG. ^■. Aulacomya atra. FIG. 2: Atrina 
seminuda. FIG. 3: Rock. Scale bar = 3 cm. f: 
female; m: male. 



Diagnosis 

Shell outer surface smooth, lacking 
periostracum; apex projecting posteriorly, 
slightly away from posterior shell edge. Col- 
umellar muscle somewhat thick. Palliai cavity 
aperture restricted at right by a closure of mantle 
edge. Osphradium small, approximatgely 1/8 
of mantle aperture length, with about 16-17 
broad, closely spaced filaments. Endostyle 
divided by a middle longitudinal furrow. Hypo- 
branchial gland greatly reduced. Transversal 
fold of kidney at level of nephrostome. Sali- 
vary glands very large, slightly larger than 
haemocoel. Both gastric ducts to digestive 
gland duplicated. Male seminal vesicle very 
large, coiled, wall markedly irregular. Female 
seminal receptacles reunited in a same region, 
mostly 4-5; vaginal duct long, broad; genital 
papilla tall, with a pair of separate longitudinal 
folds, ending subterminally. 

Description 

Shell (Figs. 1-15): To 50 mm in length and 
38 mm in width; walls with 0.46-0.60 mm thick; 
slightly to strongly convex (convexity = 1 .095- 
1.350) (Table 1, including other measure- 
ments). Growth lines covering entire shell and 
septum. Color opaque-brown, internally bright- 
chocolate brown. In males, always opaque- 
brown externally and bright-brown internally. 
Few individuals (about 1%) with a white shell. 
Periostracum totally deciduous. Male speci- 
mens with very thin, brittle, flattened shells 
(Figs. 4-15). Protoconch smooth, with 1.4 
whorls; transition to teleoconch not clearly 
defined. Aperture elliptical or subcircular. Apex 
solid, generally prominent, turned to right in 
females, almost central in males, slightly 
above margin, never reaching margin in males, 
extending beyond it in females. Flattened sep- 
tum never convex, ridge central, margin with 
a clear central notch, covering less than half 
of ventral surface. Septal edge translucent, 
sinuous, slight turned towards right. Muscle 
scars inconspicuous. 

Head-Foot (Figs. 16-18): Head differenti- 
ated, on long, dorsoventrally flattened neck, 
about half length of foot. Proboscis short, cy- 
lindrical. Tentacles long, stubby, apex some- 
what bifid. Eyes dark, small, located on 
obsolete ommatophores in basal region of lat- 
eral margin of tentacles. Neck with pair of lat- 
eral, flattened lappets (nuchal lobes); left 



1i 



CLEDON ETAL. 



expansion narrower than right; right expansion 
bringing low food groove along its dorsal limit 
with head (Fig. 17: fg). Foot very ample, oc- 
cupying about 3/4 of shell concavity, dorsoven- 
trally greatly flattened, thin; clear longitudinal 
inner sinus running in median line; shell sep- 
tum as dorsal foot limit. Mantle fusing with 
dorsal surface of foot, protruding beyond its 
borders. Furrow of pedal glands transverse, 
in anterior margin of foot; anterior margin of 
foot covered dorsally by posterior region of 
neck ventral surface. Columellar muscle 
somewhat reduced, small, but somewhat 
thickened, contouring whole anterior border of 
shell septum, slightly taller at right (Figs. 17, 
40: cm). Inner haemocoel cavity narrow, run- 



ning approximately in center of neck region. 
Inner space almost filled by great quantity of 
transverse, very slender muscular fibers; these 
fibers connecting ventral surface of dorsal 
haemocoel wall with dorsal surface of its ven- 
tral wall, contouring salivary glands and 
esophagus (Fig. 18: tm). No vestiges of oper- 
culum except in very young specimens, being 
circular, paucispiral, thin, semi-transparent, 
flexible. 

Mantle Organs (Figs. 16, 19-22): Mantle 
border thick, slightly hollow due to broad collar 
sinuses (Fig. 21). Mantle border surrounding 
entire shell ventral margin, free in anterior third, 
attaching to foot borders in posterior 2/3, situ- 




FIGS. 4-15. Crepidula cachimilla. FIGS. 4 6: Female holotype, AMNH 306947. FIGS. 7-9: Female 
paratype 1, AMNH 306949. FIGS. 10-12: Female paratype 2, AMNH 306950. FIGS. 13-15: Male 
paratype 5, AMNH 306955. Scale bar for FIGS. 4-12 = 4 cm. Scale bar for FIGS. 13-15 = 2 cm. 



CREPI DU LA CACHIMILLA N. SP. 



189 



atad slightly away from foot edge, connecting 
to it by a thin, semi-transparent portion. Mantle 
border without appendages, but entirely edged 
by series of minute repugnatorial glands, im- 
mersed in central region of mantle edge (Fig. 
21 : rg). Mantle border with special arrangement 
of folds in middle region of palliai cavity aper- 
ture, a somewhat narrow fold located from gill 
anterior end running towards left, decreasing 
and disappearing abruptly at level of 
osphradium, its broader region with a broad 
central furrow, its posterior edge expanding 
weakly beyond mantle border covering ven- 
trally anterior region of gill, its anterior edge 
slightly projecting, but not extending beyond 
mantle edge (Figs. 19, 20, 22). 

Dorsal shell muscle well developed (Fig. 16: 
dm), origin small, in about middle-right region 
of shell, just anterior to septum, its fibers run- 
ning anteriorly, spraying like fan, inserting in 
adjacent anterior region of dorsal surface of 
palliai cavity. Lateral shell muscle (Figs. 16, 
1 9, 20: Im) small, fan-like, located close to right 
side of mantle border, in region where palliai 
cavity penetrates shell septum chamber, with 
a differentiated muscular branch running to- 



wards mantle border, thickness restricting pal- 
liai aperture (Fig. 20). Palliai cavity aperture 
occupying about 2/3 of right-anterior half of 
shell border (compared to a clock in dorsal 
view, with head at 12 o'clock, palliai aperture 
from 11 to 2 o'clock) (Fig. 19); right region of 
palliai cavity aperture restricted by a broad clo- 
sure of mantle border, forming a transverse 
septum (Fig. 20). Palliai cavity deep, broad, 
triangular, arched, dorsoventrally flattened. 
Anterior extremity of palliai cavity a little larger 
than its aperture because of closure in left and 
right extremities produced by fusion of mantle 
and foot (Figs. 19, 20: ml). Palliai cavity nar- 
rowing gradually towards posterior, penetrat- 
ing at left of visceral mass; cavity length about 
2/3 length of animal (Figs. 16, 19). 

Osphradium small, monopectinate, located 
between anterior half of gill and mantle bor- 
der, at some distance from gill anterior end, 
located about in left region of palliai aperture 
somewhat perpendicular to longitudinal axis 
of body (Figs. 1 9, 20). Osphradium length little 
more than 1/8 of palliai aperture length, inform 
of a small fold, attached to mantle, separated 
from gill structures. Osphradium leaflets cy- 



TABLE 1. Measurements in mm of the holotype and paratypes. 



Specimen 


Total 
length (L) 


Height 


Width 


D 


Septum 
length 


Septum free 
shell length 


Convexity 
(D/L) 


Holotype AMNH 
306947 (female) 


52.5 


8.5 


36.4 


59.3 


24.9 


18.35 


1.13 


Paratype 1 AMNH 
306948 (female) 


30.9 


8.4 


21.8 


35.4 


12.2 


16.1 


1.14 


Paratype 2 AMNH 
306949 (female) 


42.8 


9.2 


29.5 


47.5 


28.8 


22.3 


1.11 


Paratype 3 AMNH 
306950 (female) 


38.7 


7.8 


27.4 


41.3 


11.3 


19.1 


1.07 


Paratype 4 AMNH 
306951 (female) 


31.9 


12.6 


19.8 


42.1 


13.3 


15.8 


1.34 


Paratype 5 AMNH 
306952 (male) 


20.1 


6.4 


14.7 


23.8 


8.8 


9.3 


1.18 


Paratype 6 AMNH 
306953 (male) 


23.3 


5.7 


17.7 


25.7 


10.9 


10.3 


1.10 


Paratype 7 AMNH 
306954 (male) 


15.3 


3.7 


11.6 


17.2 


5.8 


8.3 


1.12 


Paratype 8 AMNH 
306955 (male) 


12.3 


3.9 


10.2 


14.2 


3.9 


6.4 


1.15 


Paratype 9 AMNH 
306956 (male) 


26.6 


8.2 


20.2 


30.9 


12.1 


13.3 


1.16 



190 



CLEDON ETAL. 



lindrical, close from each other, somewhat 
thick, low, about 1 6-1 7 in number (Fig. 22: os) 
in females. Osphradium ganglion narrow. 

Gill very large, its base narrow, edging ante- 
rior and left margin of palliai cavity almost the 
entirety of its length; anterior gill extremity in 



right-anterior region of palliai cavity aperture, 
near its right limit, on thick mantle border; gill 
posterior extremity in posterior end of palliai 
cavity (Fig. 20). Gill filaments triangular at their 
base and with very long, almost straight, nar- 
row, stiff rod turned to right (Fig. 21: gi); rods 




FIGS. 16-21. Crepidula cachimilla female anatomy. FIG. 16: Whole dorsal view, specimen extracted 
from shell. FIG. 17: Same, head-foot, dorsal view, visceral mass and palliai structures removed. FIG. 
18: Head and haemocoel, ventral view, foot and neck "sole" removed. FIG. 19: Palliai cavity and 
visceral mass extracted, ventral view. FIG. 20: Same, left palliai connection sectioned, ventral portion 
of visceral mass deflected, most gill filaments artificially sectioned. FIG. 21 : palliai cavity roof, transversal 
section in region tangent to rectum. Scales = 2 mm. 



CREPI DU LA CACHIMILLA N. SP. 



191 



extending about three times longer than their 
triangular, membranous base; rods beginning 
in ctenidial vein region, in left margin of cavity 
roof, and touching food groove of head-foot, 
in right margin of cavity floor; rod apex 
rounded, preceded by thicker region. Gill fila- 
ments connected to each other by cilia, mainly 
in their thicker apical region, holding them in a 
somewhat firm position. Gill filaments longer 
in central gill region, shorting gradually in both 
extremities; gill anterior extremity with short 
filaments, abruptly turning forwards, located 
on mantle border (Fig. 22). Ctenidial vein nar- 
row, with uniform width along its entire length. 
Endostyle well developed (Figs. 20, 21, 22: 
en), yellowish, in form of broad, flat glandular 
ridge located in middle level of ventral surface 
of ctenidial vein along its entire length. Endo- 
style divided longitudinally by a shallow middle 
furrow. Hypobranchial gland extremely thin, 
practically absent. About 1/3 of visceral mass 
encroaching on palliai cavity roof (Fig. 20), 
occupying about 1/3 of this cavity in poste- 
rior-right region; pericardium and kidney lo- 
cated posteriorly; a long intestinal loop, anus 
and palliai oviduct located anteriorly. 

Visceral Mass (Figs. 16, 19, 20): Form of a 
dorsoventrally flattened cone, housed in shell 
chamber produced by septum, which sepa- 
rates visceral mass from dorsal surface of foot. 
Left and anterior region of visceral mass oc- 
cupied by palliai cavity (Figs. 16, 19, 20). Re- 
maining regions of visceral mass with stomach 
as central structure, immediately surrounded 
by greenish-beige digestive gland (except in 
some ventral and dorsal areas). Gonad sur- 
rounding digestive gland, more concentrated 
anteriorly and at left. Visceral mass encroach- 
ing on right-posterior region of palliai cavity 
roof, possessing another ventral flap as pal- 
liai cavity floor (Fig. 20: vm). Anterior extrem- 
ity of visceral mass ventral flap ending at 
anterior border of shell septum, covering col- 
umellar muscle (Fig. 17). 

Circulatory and Excretory Systems (Figs. 20, 
25): Pericardium somewhat triangular, broad, 
oblique to longitudinal axis of animal (Fig. 16: 
pc). Pericardium left region very narrow, in 
form of a vein connecting gill with auricle, be- 
ginning at posterior extremity of gill in poste- 
rior-left end of palliai cavity, running to surround 
area where visceral mass encroaches into 
palliai cavity, gradually increasing towards 
anterior and right (Fig. 25). Remaining peri- 



cardium limits: (1) anterior and ventral - pal- 
liai cavity; (2) posterior - visceral mass (go- 
nad generally); (3) dorsal - mantle; (4) right - 
kidney. Auricle thin walled, long, narrow, run- 
ning all along pericardium length, attached to 
its anterior and dorsal inner surfaces (Fig. 25), 
connecting with ventricle approximately in its 
middle portion; auricle having a broad portion 
beyond ventricle as blind sac (Fig. 25: ab), 
bearing orifice to nephridial gland. Ventricle 
elliptical, very muscular; its connection with 
auricle located about in middle region of its 
anterior surface, on opposite side bearing ori- 
gin of aortas. Anterior aorta broad, running 
towards opposite side from posterior aorta. 
Anterior aorta running towards right, surround- 
ing posterior inner pericardium surface, then 
penetrating head haemocoel. 

Kidney occupying about half of area of vis- 
ceral mass within palliai cavity (Fig. 20). Kid- 
ney limits: (1) dorsal - mantle; (2) ventral - 
palliai cavity; (3) posterior-right- visceral mass 
(gonad generally); (4) posterior-left - pericar- 
dium; (5) anterior - an intestinal loop; (6) lat- 
eral-right - intestine and oviduct (when 
present). Kidney central region hollow, with 
single anterior lobe (Fig. 25). Kidney lobe 
slightly uniform, covering dorsal surface, in- 
testinal region passing through kidney cham- 
ber, and about 1/4 of inner space of kidney 
adjacent to intestine. Renal lobe having longi- 
tudinal, branching, narrow folds; a larger fold 
located at left of nephrostome having a series 
of anterior branches situated somewhat uni- 
formly; another transversal, somewhat tall fold 
located at level of nephrostome. Nephridial 
gland in renal limit with pericardium, very small, 
having a series of triangular, transversal, nar- 
row folds connected with dorsal renal lobe (Fig. 
25: ng). Nephrostome a very small slit located 
in central region of ventral wall (Figs. 20, 25: 
ne), in anterior region of hollow portion of kid- 
ney; no inner glandular folds close to it. 
Adrectal sinus very broad, edging externally 
intestine loop exposed in palliai cavity, con- 
nected to main kidney chamber but separated 
by a thin septum (Figs. 20, 25: ad). 

Digestive System (Figs. 16, 26-28): Probos- 
cis short, broad (Figs. 16-18: pb). Pair of nar- 
row ventral proboscis retractor muscles very 
thin, immersed in proboscis wall. Mouth lon- 
gitudinal, in center of anterior proboscis sur- 
face. Buccal mass very large, occupying most 
of proboscis inner space and short portion of 
haemocoel posterior to it. Jaw plates in dorsal 



192 



CLEDON ETAL. 



wall of buccal mass thin, almost vestigial, 
broader laterally, short longitudinally. Pair of 
broad, low dorsal folds beginning well poste- 
rior to jaws; dorsal chamber between these 
folds shallow. Odontophore large, occupying 
most of buccal mass. 

Odontophore muscles similar to other spe- 
cies of Crepidula (Simone, 2002) (Figs. 26, 
27: m1); several very narrow jugal muscles 



connecting buccal mass with adjacent wall of 
snout, more concentrated anteriorly around 
mouth: m1b pair of dorsal protractor muscles 
narrow, thin, superficial, originating in 
anterodorsal region of mouth, close to median 
line, inserting in posterodorsal-lateral region 
of odontophore; m1 v similar to m1 b but located 
in ventral surface; m2 pair of retractor muscles 
of buccal mass (retractor of pharynx) broad. 




iu ad 



FIGS. 22-28. Crepidula cachimilla anatomy. FIG. 22: Anterior portion of palliai cavity, close to mantle 
border, mantle border slightly deflected for showing anterior gill region. FIG. 23: Central nervous 
system (nerve ring), ventral view. FIG. 24: Same, dorsal view. FIG. 25: Middle and posterior-right 
region of palliai roof, ventral view, ventral wall of pericardium and kidney partially removed. FIG. 26: 
Buccal mass, ventral view. FIG. 27: Same, dorsal view, salivary gland (sg) fully shown. FIG. 28: 
Middle and distal digestive tubes shown as in situ, ventral view, some adjacent structures also 
represented. Scales = 1 mm. 



CREPIDULA CACHIMILLA N. SP. 



193 



originating in lateral-ventral region of 
haemocoel just posterior to snout (Fig. 18), 
running towards anterior, inserting in lateral- 
posterodorsal region of odontophore 
cartilages; m2a pair of dorsal tensor muscles 
of radula, continuation of m2 after insertion in 
cartilages, running towards anterior, inserting 
in subradular cartilage in middle region of its 
dorsal inner surface; mt dorsal transversal 
muscle, or approximator muscle of cartilages, 
connecting dorsally both posterodorsal-lateral 
surfaces of cartilages, lying between superfi- 
cial membrane that covers odontophore and 
tissue on middle region of radula; m4 pair of 
median dorsal tensor muscle of radula very 
large, thick, originating in ventral-middle-pos- 
terior region of odontophore cartilages, run- 
ning towards medial, contouring medial-ventral 
surface of cartilages, running on their dorsal 
surface, inserting in subradular cartilage dor- 
sal-posterior-medial extremities; m5 pair of 
median radular tensor muscle thick, originat- 
ing in median-posterodorsal region of 
odontophore cartilages, near side of m2 in- 
sertion and m2a origin, covering perpendicu- 
larly m4 middle region, running medially, 
inserting along both sides of radular sac (each 
m5 branch covering a side of radular sac, 
medially and dorsally); m6 horizontal muscle 
very thin, uniting anterior half of odontophore 
cartilages, inserting on their dorsal margin; m7 
pair of ventral tensor muscle of radula thin, 
narrow, originating inside radular sac ventral 
surface close to each other, running anteriorly, 
separating gradually from each other, insert- 
ing in radula ventral border; m8 pair of strong 
muscles originating in posterodorsal-lateral 
regions of odontophore cartilages near inser- 
tion of m2, running attached to dorsal margin 
of odontophore cartilages, inserting in their 
anterodorsal region close to horizontal muscle 
(m6); m9 pair of dorsal-medial tensor muscle 
of radula broad, thin, originating along dorsal- 
median surface of radular sac (in its region 
internal to odontophore), crossing to dorsal 
surface, inserting in dorsal-ventral border of 
subradular cartilage; mj jaws and peribuccal 
muscles somewhat thick, surrounding lateral 
and dorsal wall of buccal mass, originating 
around mouth, inserting in middle level of lat- 
eral and dorsal wall of odontophore; m11 pair 
of ventral tensor muscles of radula weakly 
present; m14 pair broad, thin, originating in 
posterodorsal region of odontophore, close to 
m2 and m5 origins, running towards ventral 
and anterior, inserting in snout inner ventral 



surface in about middle level of odontophore; 
tissue covering middle region of radula within 
odontophore, on its dorsal surface. 

Radula short, little more than odontophore 
length (Figs. 29, 30); rachidian tooth narrow, 
strongly curved inwards, central cusp large, 
sharp, secondary cusps 2-4 similar-sized pairs 
(formula 2-1-2/0-0 to 4-1-4/0-0), weak pair of 
lateral reinforcements on its borders; lateral 
tooth broad (about three times broader than 
rachidian), curved inward, with about 7-10 
short, triangular cusps, along edge on mar- 
ginal side and 1-3 very weak cusps on edge 
on rachidian side, cusps decreasing laterally, 
disappearing about in middle region of tooth, 
with thick, arched border (formula from 1-1-7/ 
0-0 to 3-1-10/0-0); inner marginal tooth long, 
curved, tall, tip sharply pointed (cusp formula 
0-1-5/0-0 to 2-1-7/0-0); outer marginal tooth 
narrower than inner marginal tooth, thin, and 
with two small cusps along its inner margin 
only (cup formula 0-1-2/0-0 to 0-1-3/0-0). 

Pair of buccal ganglia large, close to each 
other near median line (Fig. 26: bg), located 
between buccal mass and adjacent esopha- 




FIGS. 29, 30. Radula of Crepidula cachimilla. 
FIG. 29: General view of the radula. Scale bar = 
100 |jm. FIG. 30: Detail of the central and lateral 
tooth. Scale bar = 64 |jm. 



194 



CLEDON ETAL. 



gus. Salivary glands not passing through nerve 
ring, longer than haemocoel, fitting inside it, 
bent (Figs. 18, 27: sg); distal end rounded, of 
about 1/3 of haemocoel width, running towards 
anterior possessing approximately same width 
along its length, narrowing close to buccal 
mass. Ducts of salivary glands broad, sinuous 
(Fig. 27: sa), running in dorsal surface of buc- 
cal mass, penetrating adjacent buccal mass 
wall a short distance, apertures small, in ante- 
rior region of dorsal folds of buccal mass. 

Esophagus (Figs. 18, 28: es) narrow, long; 
anterior esophagus inner surface with pair of 



broad folds, running straight posteriorly, be- 
coming gradually slender. Stomach (Fig. 28) 
somewhat conical, large, occupying about half 
of visceral mass size; esophagus inserting in 
left side of its posterior-left region, close to shell 
apex. Anterior duct to digestive gland located 
in region of stomach ventral surface preced- 
ing style sac, separated into two similar-sized, 
well-spaced ducts, each running in opposite 
directions, highly dichotomic. Posterior duct to 
digestive gland also duplicated (distance be- 
tween this pair greater than that of anterior 
ducts), each one running in opposite direc- 



31 te 




PP Pd 



FIGS. 31-35. Crepidula cachimilla anatomy. FIG. 31: Head-foot, male, dorsal view, palliai structures 
and visceral mass removed. FIG. 32: Visceral mass and adjacent part of palliai cavity, male, ventral 
view. FIG. 33: Penis and adjacent structures, dorsal view, penis deflected. FIG. 34: Visceral vas 
deferens extracted, seminal vesicle (sv) uncoiled. FIG. 35: Palliai oviduct, ventral view as in situ, 
most integument and palliai cover removed (except close to papilla). Scale bars = 1 mm. 



CREPI DU LA CACHI M ILLA N. SP. 



195 



tions, both very narrow, located in ventral re- 
gion of stomach almost at Its posterior end, 
one of them turned posteriorly. 

Stomach gradually narrowing towards an- 
terior and left, arriving close to left-posterior 
extremity of palliai cavity. Gastric shield oc- 
cupying about 1/3 of stomach inner surface, 
located in its right side (Fig. 28: gs). Pair of 
longitudinal folds separating intestine from 
style sac running at left (Fig. 28: gf), in re- 
gion anterior to anterior ducts to digestive 
glands, abruptly separating one another per- 
pendicularly, in a T-fashion, surrounding en- 
tire stomach circumference in this region, 
forming a low, narrow fold separating style 
sac from main gastric chamber. A weak con- 
striction marking region between style sac 
and main gastric chamber, clearer at right. 
Digestive gland pale brown in color, surround- 
ing stomach except some areas on dorsal and 
ventral surfaces (Figs. 16, 20, 24). Intestine 
narrow, sinuous (Fig. 28: in), running on an- 
terior border of visceral mass from left to right, 
initially in its ventral region, slightly near me- 
dian line cross to its dorsal region and run- 
ning up to right-anterior extremity of visceral 
mass (Fig. 28); running towards left in this 
region, becoming broader and exposed in 
palliai cavity, surrounding right and anterior 
border of kidney, abruptly running towards 
right in a U-shape, parallel to preceding loop 
(Figs. 16, 21, 28, 25: iu). Anus small, si- 
phoned, located In right region of palliai cav- 
ity close to mantle border (Figs. 21, 28, 25). 
Final intestine loops filled with several small, 
elliptical fecal pellets. 

Male Genital System (Figs. 31-34): Mature 
males up to 28 mm In shell length. Testis white, 
located mostly In anterior region of visceral 
mass (Fig. 32: ts). Sperm duct differentlable 
in region of testis just at right of esophagus 
penetration Into visceral mass. Seminal vesicle 
Intensely coiled, locally accumulated In ante- 
rior-right region of visceral mass (Fig. 32: sv); 
If uncoiled, presenting about same length as 
visceral mass; wall glandular, greatly irregu- 
lar, varying from broad to very narrow along 
its length (Fig. 34). Seminal vesicle abruptly 
narrowing near palliai cavity, having a very nar- 
row aperture located in right-posterior end of 
this cavity (Figs. 32, 34: ap). Palliai sperm 
groove starting immediately below this aper- 
ture, running as a relatively deep, narrow fur- 
row with elevated edges. Palliai sperm groove 
running along right neck lobe close to its edge 



(Fig. 31: sd), slightly dorsal; abruptly curving 
towards left close to penis base, connecting 
to its posterior base region (Fig. 33). Penis lo- 
cated behind right cephalic tentacle, curved 
in same direction, of about 3-4 times its size 
(Fig. 31). Distal papilla long, about 1/4 of length 
of remaining penis region, about 1 /5 of Its width 
(Fig. 33). Penis groove deep, central, running 
along ventral surface up to penis papilla tip 
(Fig. 33). 

Female Genital System: Ovary cream yellow, 
surrounding digestive gland, more concen- 
trated In anterior region of visceral mass (Fig. 
19: oy); when mature, oocytes distinguishable 
by their transparency. Visceral oviduct formed 
by gradual decrease from right-anterior end of 
ovary. Gonopehcardial duct narrow, relatively 
short, originating in right-ventral extremity of 
pericardium, running ventral to visceral glands 
in area in which visceral mass encroaches to- 
ward palliai roof, inserting in posterior extrem- 
ity of palliai oviduct, joined with insertion of 
visceral oviduct (Fig. 35: gd). Palliai oviduct 
narrow, located in right-anterior end of palliai 
cavity (Figs. 16, 20: ov). Seminal receptacles 
(sr) located in right side of last portion of vis- 
ceral oviduct, four to five in number, with three 
always significantly larger (Fig. 35: sr); each a 
small sac; duct very narrow, long; their inser- 
tion preceding albumen gland, on right surface. 
Albumen gland long, narrow, whitish, its walls 
thick, glandular; located in anterior-right ex- 
tremity of visceral mass, about half size of cap- 
sule gland (Fig. 35: ag). Separating albumen 
from capsule glands a narrow differentlable, 
paler colored tissue, most probably an ingest- 
ing gland (Fig. 35: ig). Capsule gland a con- 
tinuation of albumen gland, but situated 
perpendicular and slightly dorsal to it, broad, 
spherical (Fig. 35: eg); walls thick glandular, 
pale brown; inner duct narrow, U-shaped, 
length about 1/8 ofpallial cavity aperture. Vagi- 
nal duct (vg) relatively broad, equal In size to 
albumen gland. Genital pore preceded by tall, 
long papilla close to mantle border, at right and 
slightly removed from anus (Fig. 20: fl). Geni- 
tal papilla with broader base and somewhat 
conical form; pair of well-spaced low folds run- 
ning along its posterior-left side; both start 
gradually in papilla base and terminate at some 
distance from pore (Fig. 35: ff); posterior fold 
originating on surface of palliai cavity floor; 
anterior fold originating from palliai roof. Geni- 
tal pore a transverse apical slit, perpendicular 
to papilla folds (Fig. 35: fp). 



196 



CLEDON ETAL 



Reproduction 

Animals categorized into four sexual phases: 
(1) undifferentiated juveniles, (2) males, (3) 
transitional individuals, and (4) females. These 
are easily recognizable under a microscope by 
observation of the external development of the 
reproductive organs. Juveniles are without vis- 
ible sexual organs. Males have a well-devel- 



oped penis. Transitional individuals have a 
penis in retraction phase and a developing 
genital papilla. Females lack a penis and have 
an easily distinguishable papilla. 

Undifferentiated juveniles were between 3.7 
and 5.1 mm SL (mean: 4.3 SD: 0.4 N: 11). 
Males were 5.4-28.5 mm SL (mean: 14.1 SD: 
0.8 N: 103), always attached to larger individu- 
als. Females were 9.6-52.2 mm SL (mean: 



March 



15 



>. 12 
u 

с 
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3 

с- 9 

<D 



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*^ 

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10 16 22 28 34 

Shell length (mm) 



40 



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52 



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er 9 



го 
ф 
a: 



3 - 



November 



i\ 



It 




III 



10 16 22 28 34 

Shell length (mm) 



40 



46 



52 



FIG. 36. Crepidula cachimilla sex proportion in March (N: 270) and November (N: 
286). White: males; black: females. 



CREPI DU LA CACHI MILLA N. SP. 



197 



37.3 SD: 0.9 N: 252), forming stacks of 2-5 
individuals. Tine smallest brooding female was 
23.5 mm SL; the largest was 49.5 mm SL. 

Peak of female development from August to 
April (observed in 47 brooding females). No- 
table period of reproductive rest between 
March and November. No juveniles were en- 
countered in the field during winter, being re- 
flected in the diminution in the proportion of 
males in the population and their larger shell 
length in comparison with the summer (Fig. 36). 

Egg masses with 15-65 capsules (Fig. 37) 
(mean: 35, SD: 14, N: 47). Egg capsules (Fig. 
38) 2.2-3.3 mm in length (mean: 2.8, SD: 0.5, 
N: 148) and 2.3-3.4 mm in width (mean: 2.6, 
SD: 0.4, N: 148). Each egg capsule containing 
129-441 uncleaved eggs (Fig. 39) (mean: 226, 
SD: 57, N: 148) in a whitish viscous liquid. All 
eggs developing into veliger larvae (Fig. 40) 
and hatching. It was not possible to measure 
hatching time. No nurse eggs or cannibalism 
was observed. No differences in number of 
embryos between initial and late brood stages 



in females of same size found. 

Neither In-transformed mean capsule size (r^ = 
0.01) nor In-transformed capsule number per 
mass (r^ = 0.13) correlated with shell length of 
brooding females. There was no positive corre- 
lation between In-transformed egg number per 
capsule and In-transformed capsule size (r^ = 
0.33) or In-transformed female size (r^ = 0.02). 

Uncleaved egg diameter 180-200 |jm (mean: 
191.7; SD: 7.2, N: 20). Protoconch length of 
juvenile shells (Figs. 41, 42) 700-800 |jm 
(mean: 760 SD: 65 N: 11). 

Habitat 

Between 10 and 20 m depth, attached to 
hard substrata. 

Distribution 

Known only from northeastern Patagonia 
including the records from Golfo San Matías 
to Punta Norte of Parodiz (1939). 




FIGS. 37-42. Crepidula cachimilla egg mass and protoconch. FIG. 37: Egg mass. Scale bar = 7 mm. 
FIG. 38: First and late stage egg capsule. Scale bar = 3.5 mm. FIG. 39: Eggs in first division stages. 
Scale bar = 500 |jm. FIG. 40: Prehatching stage. Scale bar = 350 |jm. FIG. 41: Protoconch on an adult 
shell. Scale bar = 800|jm. FIG. 42: Detail with SEM of protoconch of an adult shell. Scale bar = 1,100 pm. 



198 



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200 



CLEDON ETAL. 



DISCUSSION 

The shell of Crepidula cachimilla is similar 
to species occurring in the western Atlantic 
belonging to the "Crepidula plana complex" 
(Collin, 2000; Simone, submitted). Its most 
distinctive characters are the projecting apex, 
located somewhat away from the posterior 
shell base, and the absence of periostracum. 

The characters of Crepidula cachimilla pre- 
sented in the Diagnosis, mostly morphologi- 
cal, as well as those summarized in the Table 
2, are the main basis differentiating this spe- 
cies. That set of characters easily separates 
the new species from the remaining South 
American taxa. From the Atlantic species with 
known anatomy, С cachimilla has a thicker 
columellar muscle, a condition found only in 
other species in early stages of the develop- 
ment, after which the columellar muscle be- 
comes reduced. As stated by Simone (2002), 
based on comparison of the ontogeny and 
phylogeny, the lateral and dorsal shell muscles 
are also derived from the columellar muscle, 
and are both thick in С cachimilla; however, 
the respective scars in the shell are incon- 
spicuous. The restriction of the palliai cavity 
aperture is one of the synapomorphies of the 
family Calyptraeidae (Simone, 2002); how- 
ever, in С cachimilla this state is still more 
developed, as it is greatly restricted on the right 
side by a broad fusion of the mantle border. 
The hypobranchial gland is normally reduced 
in Crepidula, being a thin glandular layer sur- 
rounding the visceral structures encroached 
into palliai cavity roof (Simone et al., 2000; 
Simone, 2002); however, С. cachimilla has 
practically no developed hypobranchial gland, 
the region where it would occur being thin and 
transparent. The contrary happens with the 
salivary glands, which are normally small; in 
С cachimilla, these glands are longer than the 
haemocoel, being folded inside this cavity. This 
state is comparable with that of Bostrycapulus 
aculeatus (Gmelin, 1791) (also known as 
Crepidula aculeata); however, in that species, 
these glands are still larger (Simone, 2002). 
Other notable feature of С cachimilla is the 
duplication of both ducts to digestive gland in 
the stomach. 

Despite the conchological peculiarities of C. 
cachimilla, shell characters alone do not clearly 
distinguish it from C. onyx, which it resembles 
in shape, color, and size. This similarity led 
Parodiz (1939) to assume that the studied 
species was C. onyx. Such misidentifications 



are common in this family, with C. argentina 
(Simone et al., 2000) having been confused 
with C. protea in Argentina. 

There are subtle differences in shell shape 
between С cachimilla and C. onyx. Crepidula 
cachimilla tends to have a more pointed apex, 
and the shell also seems to be less convex, 
but these features can be strongly affected by 
the substratum. 

Anatomical differences between С 
cachimilla and C. onyx are not yet known, 
because there has not been a detailed ana- 
tomical study of the latter. However, the radu- 
lar morphology of С onyx (Hoagland, 1977) 
is markedly different from that of the studied 
species, in which the central tooth has 2-4 
cusps (formula 2-1-2/0-0 to 4-1-4/0-0); the lat- 
eral tooth 7-10 cusps, (formula 7-1-0/0-0 to 
10-1-0/0-0); and the inner marginal tooth has 
1 -3 cusps (1-1 -0/0-0 to 3-1 -0/0-0). In addition, 
the uncleaved eggs of the Argentinean mate- 
rial are larger than those of the Californian С 
onyx population described by Hoagland 
(1986). The main difference between the spe- 
cies is the occurrence of six "malformed", or 
nurse eggs per sac (Hoagland, 1986) and the 
fact that "frequently fully half the entire num- 
ber of embryos disintegrate within the capsules 
and are used as food by the survivors" (Сое, 
1942). Although we are unable to assess the 
frequency of this phenomenon in California, 
such malformed eggs or disintegrating em- 
bryos were absent in the studied Argentinean 
material. 

Aguirre & Farinati (2000) reported the pres- 
ence of C. onyx among other Crepidula spe- 
cies from Quaternary sediments in Argentina. 
Because of the shell of the species described 
here is very similar to that of C. onyx, it is rea- 
sonable to assume that these fossil records 
belong to the species described here. The 
occurrence of these fossils proves that this is 
not an exotic species recently introduced to 
the area. Additional differences between the 
reproductive parameters reported by 
Hoagland (1986) and Сое (1942) for С onyx 
and C. cachimilla are: the larger egg diameters 
and the complete lack of nurse eggs or canni- 
balism in C. cachimilla, and the different radu- 
lar morphology. On this basis, the material 
studied by Parodiz (1939) should be assigned 
to the new species described here instead of 
being assigned to C. aplysioides, as proposed 
by Hoagland (1977). 

Crepidula cachimilla also differs from other 
species in many reproductive strategy char- 



CREPIDULA CACHIMILLA N. SP. 



201 



acteristics. Crepidula aplysioides Reeve, 1859, 
is a small (up to 2.0 cm SL; brooding female 
between 9.4 and 18.2 mm SL) tropical and 
subtropical species with egg capsules contain- 
ing fewer eggs than C. cachimilla (Hoagland, 
1 977). Further reproductive characteristics are 
given by Miloslavich & Penchaszadeh (2001). 
The number of eggs per capsule separates C. 
cachimilla from C. coquimbensis Brown & 
Olivares, 1996; С. dilatata Lamarck, 1822; and 
C. protea Orbigny, 1841 (Table 2). In С 
cachimilla (Table 2), the egg diameter clearly 
differs from that of C. argentina (Cledón & 
Penchaszadeh, 2001), С. philippiana 
(Gallardo, 1977, 1996), С. fecunda (Gallardo, 
1979) and С dilatata (Gallardo, 1977; 
Chaparro & Paschke, 1990) (Table 2). In С. 
cachimilla (Table 2), eggs per capsule are 
more numerous, and both males and females 
are larger than those of C. protea (Hoagland, 
1983). The larval shell at hatching and the 
protoconch of juveniles are larger in C. 
cachimilla (Table 2) than in C. argentina 
(Cledón & Penchaszadeh, 2001) (Table 2). 

According to our observations on C. 
cachimilla, broods containing a large number 
of capsules (more than 40) always belong to 
females larger than 31 mm SL. Because of 
the number of eggs per capsule does not de- 
pend on the female size, we used this param- 
eter as species representative. 

A more extensive comparison of the mor- 
phology of C. cachimilla with other species of 
the "Crepidula plana complex" is being pub- 
lished elsewhere (Simone, submitted), with a 
phylogenetic analysis of all known species 
occurring from Florida to Patagonia. Crepidula 
cachimilla is separated from the remaining 
species by such plesiomorphies as the thick- 
ness of the shell muscles (columellar, lateral 
and dorsal muscles), which are very thin in 
the other species; the nephridial gland having 
clearly transverse septa, whereas in the re- 
maining species this gland has irregular lon- 
gitudinal folds; the larger size of the salivary 
glands, which are normally reduced; and re- 
tention of the ventral tensor muscle of the 
radula (mil), mostly lost in other species. 



ACKNOWLEDGEMENTS 

This study was financially supported by 
SECyT-Argentina Pict 98-01 -04321 (P. E. Pen- 
chaszadeh p. i.), UBACyT-X917 (P. E. Pen- 
chaszadeh p. i.), Comisión de Investigación 



Científica de la Provincia de Buenos Aires 
(CIC), SECyT-Argentina Pict 01-7222 (A. Rumi 
p. i.), Deutscher Akademischer Austausch- 
dienst (DAAD) and Fundación Antorchas (M. 
Scelzo p. i.). 

We thank Guido Pastorino and Fabrizio 
Scarabino for their useful comments to an ear- 
lier MS version. We are especially grateful to 
Paula Mikkelsen and Sonja Guetz for improv- 
ing the present version. The morphological 
study was also part supported by the Fundaçâo 
de Amparo à Pesquisa do Estado de Sao Paulo, 
process # 00/11074-5 and 00/11357-7. 



LITERATURE CITED 

AGUIRRE, M. L. & E. A. FARINATI, 2000, Molus- 
cos del cuaternario marino de la Argentina. 
Boletín de la Academia Nacional de Ciencias, 
Córdoba, Rep. Argentina, 64: 235-333. 

BROWN, D. I. & С. А. OLIVARES, 1996, А new 
species of Crepidula (Mollusca: Meso- 
gastropoda: Calyptraeidae) from Chile: addi- 
tional characters for the identification of eastern 
Pacific planar Crepidula group. Journal of Natu- 
ral History, 30: 1443-1458. 

CHAPARRO, О. R. & К. A. PASCHKE, 1990, 
Nurse egg feeding and energy balance in em- 
bryos of Crepidula dilatata (Gastropoda: 
Calyptraeidae) during intracapsular develop- 
ment. Marine Ecology Progress Series, 65: 
183-191. 

CLEDÓN, M. & R E. PENCHASZADEH, 2001, 
Reproduction and brooding of Crepidula 
argentina Simone, Pastorino & Penchaszadeh, 
2000 (Gastropoda: Calyptraeidae). 777e Nau- 
tilus, 115: 15-21. 

СОЕ, W. R., 1942, The reproductive organs of 
the prosobranch mollusk Crepidula onyx and 
their transformation during the change from 
male to female phase. Journal of Morphology 
70: 501-512. 

COLLIN, R., 2000, Phylogeny of the Crepidula 
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GALLARDO, С, 1979, Especies gemelas del 
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GALLARDO, С, 1996, Reproduction in Cre- 
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127-141. 



Revised ms. accepted 16 February 2004 



RESEARCH NOTES 



MALACOLOGIA, 2004, 46(1): 205-209 

CHROMOSOMES OF THE CHINESE MUSSEL 

ANODONTA WOODIANA (LEA 1834) (BIVALVIA, UNIONIDAE) 

FROM THE HEATED KONIN LAKES SYSTEM IN POLAND 

Pawel Woznicki 

Department of Evolutionary Genetics, University in Olsztyn, Oczapowsl<iego 5, 
10-718 Olsztyn, Poland; pwozn@uwm.edu.pl 

ABSTRACT 

The chromosome complement of freshwater mussel Anodonte woodiana was investigated 
using Giemsa, Ag-NOR and chromomycin A3 staining. The diploid chromosome number of 
this species is 2n = 38, and the arm number (FN) = 76. Nucleolar organizer region (NOR) 
was found on one chromosome pair, and it was connected to GC-rich chromatin, as 
visualized by CMA3 staining. 

Key words: Anodonta woodiana, chromosomes, freshwater bivalve, karyotype, NOR. 



INTRODUCTION 

The freshwater bivalve mollusk Anodonta 
woodiana is native to eastern Asia. In recent 
years, it has been discovered in Europe (Kiss 
& Pekli, 1988; Beran, 1997) and on several 
Indonesian islands (Watters, 1997). It has also 
been collected in the wild in the Dominican 
Republic and Costa Rica (Watters, 1997). 

In the heated Konin Lakes of Poland, 
Anodonta woodiana appeared in the mid- 
1 980s following the introduction of silver carp, 
Hypophthalmichthys molitrix (Val.), from Hun- 
gary (Afanasjev et al. 2001; Kraszewski & 
Zdanowski, 2001). Anodonta woodiana was 
observed as a dominant species in some parts 
of this system of lakes (Protasov et al., 1 994). 

Within Unionidae, the chromosome number 
is known for 26 species, and most of them 
have 38 (reviewed in Nakamura, 1985; 
Barsiene, 1994; Thiriot-Quiévreux, 2002). Five 
species of Anodonta have been studied cyto- 
genetically, but only diploid chromosome num- 
ber (2n = 38) and fundamental arm number 
(FN = 76) have been established (Nakamura, 
1985; Barsiene, 1994). 

The present report describes the karyotype 
and location of nucleolar organizer regions 
(NORs) o^ Anodonta woodiana from Poland. 



MATERIALS AND METHODS 

Nineteen specimens oí Anodonta woodiana 
from the Konin Lakes in central Poland were 
studied for chromosome complement. 

A 0.4% solution of cobalt chloride was injected 
in vivo (0.05-0.1 ml per specimen, depending 
on shell length, which ranged from 1 to 1 7 cm). 
Cobalt chloride blocks two major steps of cel- 
lular respiration. As the result of tissue hypoxia 
it stimulates cell proliferation (Webb, 1 962, cited 
by Cucchi & Baruffaldi, 1989). 

After 60 h, 0.1% colchicine solution was in- 
jected to the mussel's foot in vivo for 6 h. From 
0.5 to 1 .0 ml of colchicine solution were used 
(depending on the mussel's size). Gills were 
dissected, homogenized in distilled water, and 
hypotonized for 60 min in distilled water. Cell 
suspensions were fixed by 3:1 methanol/ace- 
tic acid and centrifuged three times at 1 ,000 
rpm. Each slide preparation was made using 
air-drying technique (Thiriot-Quiévreux & 
Ayraud, 1982). 

For conventional karyotypes, chromosome 
preparations were stained with 5% Giemsa in 
distilled water for 20 min. CMA3 staining was 
done according to Sola et al. (1992) and Ag- 
NOR staining as described by Howell & Black 
(1980). 



205 



206 



WOZNICKI 



TABLE 1. Relative lengths (RL) and centromeric indices (CI) of Anodonte woodiana 
chromosomes. 



Chromosome 












pair no. 


RL 


SD 


CI 


SD 


Classification 


1 


3.75 


±0.06 


46.50 


±4.39 


m 


2 


3.39 


±0.08 


49.09 


±0.50 


m 


3 


2.98 


±0.06 


43.34 


±2.83 


m 


4 


2.60 


±0.03 


43.93 


±3.20 


m 


5 


2.53 


±0.07 


43.06 


±2.21 


m 


6 


2.41 


±0.04 


45.65 


±0.99 


m 


7 


2.42 


±0.01 


45.19 


±5.49 


m 


8 


2.31 


±0.13 


45.38 


±1.19 


m 


9 


2.21 


±0.07 


43.39 


±2.46 


m 


10 


2.15 


±0.09 


43.76 


±1.96 


m 


11 


3.20 


±0.02 


35.78 


±2.34 


sm 


12 


2.96 


±0.14 


37.26 


±1.45 


m-sm 


13 


2.82 


±0.03 


33.47 


±1.07 


sm 


14 


2.60 


±0.08 


36.02 


±2.51 


sm 


15 


2.51 


±0.09 


29.65 


±4.91 


sm 


16 


2.50 


±0.17 


37.31 


±1.19 


m-sm 


17 


2.22 


±0.11 


38.11 


±0.68 


m-sm 


18 


2.16 


±0.01 


38.27 


±2.27 


m-sm 


19 


2.28 


±0.04 


31.96 


±2.01 


sm 




«11 »1 



m 



lili i« 



6 7 8 

msm|| II II 

11 12 13 



m-sm 



m ж 1 ц 

16 17 18 



ti шл 

9 10 

m §i 

14 15 
19 



FIG. 1 . Karyotype of Chinese mussel {Anodonta woodiana). m - metacentric 
chromosomes, m-sm - meta-submetacentric and sm - submetacentric chro- 
mosomes. NOR-bearing chromosome pair is framed. Scale bar equals 5 pm. 



ANODONTA WOODIANA CHROMOSOMES 



207 



Chromosome spreads were analyzed under 
a Nikon Optiphot 2 fluorescent microscope 
equipped with UV filters for identification of 
fluorescent signals and photographed by 
Coolpix 995 camera. 

Ten metaphase plates were karyotyped. Mor- 
phometric measurements of chromosomes 
were made using the freeware computer appli- 
cation MicroMeasure version 3.3 available on 
the Internetat: http://www.colostate.edu/Depts/ 
Biology/MicroMeasure. The relative length 
(RL) (100x chromosome length/total haploid 
length) and the centromeric index (CI) (100x 
length of the short arm/total chromosome 
length) were calculated. Chromosomes were 
classified according to Levan et al. (1964). In 
case of six animals sequential staining CMA3/ 
Ag-NOR was done, and at least three 
metaphase plates from each specimen were 
analysed. About 50 interphase nuclei were 
observed from the same six individuals after 
silver staining. 



RESULTS 

From 19 individuals oi Anodonta woodiana, 
211 Giemsa-stained metaphase plates were 
analysed, showing that the diploid chromo- 
some number was 2n = 38 (Fig. 1). Relative 
length ranged from 3.74 to 2. 1 5 (Table 1 ), and 
the karyotype consisted of 10 pairs of meta- 
centric, five pairs of meta-submetacentric, and 
four pairs of submetacentric chromosomes 
(FN = 76) (Fig. 1, Table 1). 

Staining with fluorochrome CMA3 revealed 
bright positive bands at terminal position on 
the short arm of one chromosome pair of 
Anodonta woodiana (Fig. 2). The same results 
were obtained using silver staining (Ag-NOR). 
Sequential СМАз /Ag-NOR staining procedure 
of the same metaphases showed that the 
CMA3 and silver positive signals appeared at 
the same chromosome site of metacentric 
chromosome pair no. 6 (Fig. 3). The number 
of silver-stained interphase nucleoli in A. 
woodiana cells never exceeded two nucleoli 
per cell (Fig. 4). 




DISCUSSION 

The karyotype of the Chinese mussel has 
been described for the first time in the present 
paper. The chromosome number of Anodonta 
woodiana, 2n = 38 (Fig. 1), is coincident with 



FIGS. 2-4. Anodonta woodiana. FIG. 2: 
Metaphase chromosomes of after CMAg-stain- 
ing. Arrows indicate NOR chromosomes. Scale 
bar equals 5 |jm. FIG. 3: Metaphase chromo- 
somes of after Ag-staining. Arrows indicate NOR 
chromosomes. FIG. 4: Silver stained interphase 
nuclei with two active nucleoli. 



208 



WOZNICKI 



that reported for other Anodonta spp. - A. 
anatina, A. grandis, A. piscinalis, A. cygnea, 
A. subcircularis (Nakamura,1985; Barsiene, 
1994). Such a diploid chromosome number is 
the most frequent one among the bivalve spe- 
cies previously studied. About 47% of species 
within the class Bivalvia, particularly from 
Palaeoheterodonta and Heterodonta possess 
38 chromosomes (Thiriot-Quiévreux, 1994). 

Fundamental chromosome arm number (FN) 
reported for four Unionidae species equaled 
76 (Nakamura, 1985). The same value of FN 
was observed in Anodonta woodiana, because 
only bi-armed chromosomes (meta- and sub- 
metacentrics) were found (Fig. 1; Table 1). 

In eukaryotes, the 18S, 5.8S, and 28S ribo- 
somal RNA genes (called major rDNA) are 
present in high copy number and are clustered 
as tandem repeats at one or more chromo- 
somal sites, termed nucleolar organizer re- 
gions (NORs) (Long & David, 1980). These 
clusters can be visualized indirectly by stain- 
ing complex of residual acidic protein associ- 
ated with the fibril center of the nucleolus 
(Ag-NORs) (Jordan, 1987) or using 
chromomycin A3 (CMA3) staining, which binds 
to GC rich chromatin (Amemiya & Gold, 1986). 
These methods do not detect the regions con- 
taining 5S (minor) rDNA, another multicopy ri- 
bosomal gene not involved in the formation of 
the nucleolus (Little & Braaten, 1989). 

The single NOR locus in Chinese mussel (Fig. 
3) represents one of the NOR patterns observed 
in bivalves. The number of NOR-bearing chro- 
mosome pairs in these mollusks varies from 
one in Mya arenaría (Thiriot-Quiévreux et al., 
1998), Donaxímncu/üs (Martinez et al., 2002) 
and Brachidontes pharaonis (Vitturi et al., 
2000) to three in Mytilus californianus 
(Martinez-Lage et al., 1997; Gonzalez-Tizon 
et al., 2000) and M. trossulus (Martinez-Lage 
et al., 1997). The chromosomal location of 
NORs in most species was terminal, as was 
found in A. woodiana (Figs. 2, 3). It has been 
suggested that a single pair of chromosomal 
NORs located terminally may represent a 
plesiomorphic character (Amemiya & Gold, 
1990; Thiriot-Quiévreux, 1994). 

GC-rich CMA3 positive heterochromatin con- 
nected to NORs is typical offish and amphib- 
ians (Amemiya & Gold, 1986), although it has 
also been observed in bivalve mollusks 
(Martinez-Expositoetal., 1997; Martinez-Lage 
et al., 1 994). Staining with fluorochrome CMA3 
has revealed the existence of GC bands on 
one chromosome pair, at the same location 



as Ag-NOR in Anodonta woodiana (Figs. 2, 
3). Other bivalve species show CMA3 positive 
bands on two or more chromosome pairs. In 
mytilids, some CMA3 bands were present at 
the NOR sites but also СМАз -negative NORs 
were present and CMA3 bands not connected 
to NORs were found (Martinez-Lage et al., 
1995; Vitturi et al., 2000). The interstitial loca- 
tions of CMA3 bands were observed on Donax 
trunculus (Martinez et al., 2002) and Dreissena 
polymorpha chromosomes (Woznicki & Boron, 
2003). Apart from the single Ag-NOR site 
CMA3 positive signals were found in zebra 
mussel on almost all chromosomes except 
pairs 1, 5 and 16 (Woznicki & Boron 2003). 
The association of NORs with СМАз -bright 
bands shows that the use of combined, se- 
quential CMAß/Ag-NOR staining proved to be 
practicable and reliable for the detection of ri- 
bosomal regions of the bivalve mollusks chro- 
mosomes. 

Present findings provide an initial step in the 
cytogenetic characterization of invasive 
aquatic species, Anodonta woodiana and the 
first case of NORs description in the species 
from genus Anodonta. 



ACKNOWLEDGEMENTS 

The study was supported by project No. 
0804.0205 financed by the University of 
Warmia and Mazury in Olsztyn, Poland. 



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Revised ms. accepted 23 January 2004 



MALACOLOGIA, 2004, 46(1): 211-216 

LOCOMOTION IN HELIX ASPERSA 

Norbert Buyssens 

Morphology Unit, Laboratory of Pharmacology, 
University of Antwerp - UIA, Wilrijk, Belgium; liliane.vandeneynde@ua.ac.be 

ABSTRACT 

The pedal waves in Helix asperse move faster than the foot of the animal. On the other 
hand, a histological study of the foot could not identify an organized muscular structure 
expected to be capable of wave construction. Instead, a non-organized tissue with rela- 
tively few muscle cells and many collagen fibers mixed with vessels and empty cavities 
was found. 

We could demonstrate that the movement of the waves was uncoupled from the move- 
ment of the foot and that the forward displacement of the snail is due to rhythmic fluid 
accumulation under pressure. This pressure generates a force in backward direction on 
the substratum, which in turn is used as push off by the animal to move in a forward 
direction. The snail does not crawl, the waves move independently from the foot sole, and 
the animal glides smoothly without changing the length or the shape of the foot. 

The problem how waves are constructed starting from the available muscular material is 
not solved. We advance a cautious hypothesis that it happens by a cyclic reversible re- 
cruitment of cells at the moving front and a corresponding dropping off at the rear. 

Key words: Helix, locomotion, haemolymph. 



INTRODUCTION 

During a study of the changes in the shape 
of smooth muscle cells when undergoing con- 
traction, our attention was drawn to the foot of 
the snail as a possible suitable model. Com- 
mon garden snails were caught, fixed and pro- 
cessed for histological examination. This 
showed that the foot did not contain one or 
more large muscles orientated in one direc- 
tion, but many discrete muscle cells and fi- 
bers distributed in an irregular three- 
dimensional pattern. Moreover, collagen fibers 
and empty clefts or holes made up the larger 
part of the space. When watching the charac- 
teristic waves on the foot sole, the question 
arose how an apparently unorganized group 
of individual muscle cells and fibers could 
manage to assemble ordered unidirectional 
waves. Closer examination showed that the 
waves moved faster than the foot, which led 
to the conclusion that the movement of the 
waves was uncoupled from the advancing 
movement of the foot. A transmission by a fluid 
interphase, in this case the haemolymph, was 
considered as a likely possibility and became 
the aim of this study. Because we do no not 
know exactly how these waves are structured. 



the use of the term is purely descriptive. What 
we see are narrow, dark, transverse stripes 
separated by wider light segments, which we 
will call "junctions" for convenience. 



MATERIALS AND METHODS 

Snails (n = 150) of the species Helix aspersa 
were collected in spring and early summer. 
They were housed in plastic boxes and fed lettuce. 
Their average weight was 5.2 g (3.9-7.2 g). After 
dissection, tissues of 20 animals were fixed in 
a 4% aqueous solution of formalin. Bouin's, 
methacarn, and isopropanol fixatives were 
used when appropriate. Sections of paraffin- 
embedded material were stained with Sirius red 
haematoxylin. Additional stains were Masson's 
trichrome, PAS, PAS with amylase digestion, 
haematoxylin and eosin, alcian blue at pH 4.2, 
mucicarmin. Von Kossa's for calcium, 
Kernechtrot and Fontana's silverstaining for 
melanin. A contracted foot of 21 mm length and 
an expanded one of 32 mm length were cut In 
uninterrupted serial sections. 

Speeds of the animals were recorded while glid- 
ing on реф1ех plates over a distance of 50 mm, 
and for each animal five consecutive displace- 



211 



212 



BUYSSENS 



ments were timed. Videotapes were made 
from animals in different positions, on different 
substrata and under different angles of illumi- 
nation. Standard and scanning radiographs of 
resting and moving animals were taken. Injec- 
tions of Indian ink droplets in the foot of anaes- 
thetized animals were studied by videotape 
and after killing by histology in order to estab- 
lish the exact location in the foot. 

Repeated attempts to record electrical ac- 
tivity were unsuccessful. 



RESULTS 



Histology 



The foot contains from the tip to the tail a 
moderate number of slender discrete smooth 
muscle cells or fibers embedded in a loose 
network of many collagen fibers. Fibers lie in 
longitudinal, circular, and vertical directions 
without any preferential pattern. They make 
many short contacts with each other (Fig. 1). 
Many vacuolated interstitial cells and many 
empty spaces occupy the interstitium. Thin- 
walled muscular vessels (arteries) and spaces 
lined by flattened endothelial type cells, prob- 
ably representing veins, can be identified. 
Many empty spaces not lined by cells are dis- 
tributed throughout and are particularly con- 
centrated at the margins of the foot sole. The 
existence of a ramified communicating system 
of channels can be demonstrated convincingly 
by the injection of Evans blue in the tail of a 
fixed foot. The dye spreads diffusely in the core 
of the foot and then moves to the margins 
where it is easily recognized because of their 
thinness. If sufficient pressure is applied, the 



dye fills the head and induces the eversión of 
the antennae. 

Few nerve fibers can be detected by routine 
stains. We used a Mab against acetylated tubu- 
lin (Sigma T 6793, clone 6-11 B-1) and DAB as 
chromogen to demonstrate a nervous network 
making contact with the smooth muscles. We 
only found thin ramifications extending to the 
base of the epithelial cells of the sole that had 
all the characteristics of sensitive nerve endings. 
In addition, the sole shows transverse lines regu- 
larly spaced at 1 .0-1 .5 mm intervals in a con- 
tracted foot of 25 mm length. Histology reveals 
that they are condensations of collagen fibers 
closely apposed to the sole epithelium. We did 
not find mention of these structures in the litera- 
ture. Findings on mucus cells are not reported 
because they are not relevant to this study. 

Displacement of the Waves 

The snail Helix aspersa moves according to 
a monotaxic anterograde "wave" pattern. The 
waves move faster than the animal. The junc- 
tion length is ± 4 mm, the thickness of the wave 
is 1 .0-1 .5 mm. The wave is composed of two 
layers, a cranial one which is lighter and a 
darker caudal one. Generally 10 to 12 waves 
can be counted at one point in time. Table 1 
shows figures that allow calculation of the ra- 
tio of the speed of the waves to the speed of 
the animal. The waves move 2.3 times faster 
than the animal. Prior & Gelperin (1 974) found 
in Umax maximus a ratio of 2.2. Jones & 
Trueman (1970) detected in Patella vulgata a 
ratio of 3.5. Bonse (1935) reports ratios be- 
tween 0.93 and 1.48 in Helix pomatia. Unfor- 
tunately, these authors did not elaborate on 
this phenomenon in later studies. 



TABLE 1. Speed of animals versus speed of waves. 



Speed of animals (n = 90 individuals) 



Distance covered in 30 sec 
Speed per second 



S = 50 mm 
1,6 mm 



Speed of waves (n = 60 individuals) 



Length of junction 
Number of waves in 30 sec 
Distance covered in 30 sec 
Speed per second 



S = 4 mm 

S = 27.5 

4 mm x27.5 = 110 mm 

3.7 mm 



Ratio speed of waves/speed of animals 2.3 



HELIX ASPERSA LOCOMOTION 



213 



A 



лгчг'^ячиаь-'» "Ж "^ « '%^ 




FIG. 1. Section of foot. A. Irregular distribution of muscle fibers in red and collagen fibers in green. 
Masson's trichrome stain. Scale bar = 200 |jnn. B. Collagen fibers are red and are closely apposed to 
the muscle fibers which show many contacts. Sirius Red Hematoxylin. Scale bar = 50 |jm. 



Because the progression of waves is not 
matched by a corresponding progression of the 
foot, the waves cannot act as "toes" on which 
the snail could lean to move ahead. Hence the 
problem of how the waves command foot move- 



ment must be addressed. 

Independent from the waves, other irregular 
undulations are seen at the very margins of the 
foot over a width less than 1 mm. They move in 
a caudal direction at a pace of 1 mm every 1-3 




FIG. 2. Scanning radiograph showing the tight apposition of the foot on the substratum. A. Frontal 
view. B. Lateral view. 



214 



BUYSSENS 



TABLE 2. Time to pull different weights over a 
distance of 50 mm in three snails (A-C) of com- 
parable body weight (body weight is given in 
parentheses). 







Time in sec 




Weight 


A (5.9 g) 


В (5.4 g) 


С (5.3 g) 


9g 

15g 
21 g 


29 
40 
90 


35 
41 
65 


27 
42 
70 



sec and reach the tail. They were mentioned 
by Bonse (1 935), and their function is unknown. 

Before a wave appears, the tip of the foot is 
dilated by fluid accumulation. In this dilated 
portion, the first wave is formed. Successive 
waves develop in caudal direction, but at the 
very moment they are formed they start to 
move in cranial direction. It is clear that this is 
not comparable to a peristaltic wave system 
originating in the tail and rushing to the tip. 
When the animals stops, the waves also stop, 
the most caudally situated first. There is no 
jamming at the tail. Accidental amputation of 
the tail does not stop emergence of waves. 

When the moving animal is grasped by the 
observer and turned upside down waves con- 
tinue for a few minutes. Visual observation 
confirmed by videotaping show that the waves 
do not cause a retraction of the foot surface 
but that the junctions bulge as they are filled 
with fluid. It is clear that the junctions by their 
expansion push on the substratum and that 
they are the propulsive elements. Bonse 
(1935) and Lissmann (1945) have demon- 
strated that the pressure of the foot on the 
substratum decreased when the wave passed 
and resumed to normal when the junction 
moved over the recorder plate. 

Waves can persist in feet severed from the 
body. They can last for 15 min and thereafter 
continue as irregular slow movements for two 
hours. These contractions come to an end in 
the tip of the foot where they first originated: 
primum movens, ultimum moriens! The mar- 
ginal undulations were very resistant and 
could be observed up to six hours after sec- 
tioning. In the slug Umax maximus. Prior & 
Gelperin (1 974) detected waves after decapi- 
tation and demonstrated that the presence 
of the central nervous system was necessary 
to initiate them. However, once started, the 
waves could form independently. The impor- 
tant conclusion of these findings is that waves 



can develop and function without a pulsating 
heart. 

In our material, the snail does not lift its foot 
in a detectable amplitude during forward move- 
ment. We could substantiate this in three ways. 
First, by visual observation of the moving ani- 
mals with an amputated tail. Second, by al- 
lowing the snail to glide on black paper and 
studying the mucus trail. Over distances from 
50 cm to 1 m this is a straight ribbon without 
any irregularities either in the centre or at the 
margins. Third, by scanning radiography of 
resting and moving animals, which demon- 
strates that the foot really sticks over its whole 
length to the substratum (Fig. 2). 

The snail glides on its own slime. The bulg- 
ing junction exerts a backward pressure par- 
allel to the substratum. This can convincingly 
be demonstrated by videotaping: when the 
snail rests on a thin plastic strip and is held by 
an observer, the marker lines on the strip move 
backwards. With the same method, it is pos- 
sible to measure the energy that the snail uses 
for forward displacement. When we attach to 
the strip a string with a weight that we let hang 
over the border of a table, we can measure 
the time in which the animal can push back- 
wards the plastic strip with its attached weight. 
Table 2 shows the figures for three snails of 
comparable weight. It appears that animal A, 
for instance, can pull a weight of 9 g over a 
distance of 50 mm in 29 sec, which is the same 
speed as a free moving animal. When the 
weight increases, the speed decreases ac- 
cordingly. Calculating the energy in our first 
example, we arrive at 0.0015 milliwatt. 

The displacement of haemolymph in the foot 
as demonstrated by the bulging of the junc- 
tions is not visible when the foot rests on the 
substratum, because it is a pressure mecha- 
nism and not a crawling mechanism. The pres- 
sure generates a force that is parallel to the 
substratum and directed backwards. These 
are the conditions for developing shear stress. 
However, due to the fact that the mass of the 
substratum is too big to be displaced, the dis- 
placement or shear strain, occurs in the op- 
posite direction. This may be an explanation 
why a backward moving force induces a for- 
ward displacement. 

Finally, the behavior of injected Indian ink 
should be reported. Histological examination 
showed that the droplets were generally 
present in pre-existing clefts in the core of the 
foot. Sometimes they accumulated in the nu- 
merous spaces close to the epithelium. A lin- 
ear deposit of a few mm in longitudinal 



HELIX ASPERSA LOCOMOTION 



215 



direction is particularly suited for examination. 
When the arriving wave hits the posterior end 
of the ink deposit, it is slightly pushed forward 
and during the passage of the wave slightly 
stretched. Once the wave has passed, the de- 
posit resumes its original shape and position. 
The image looks like the wobbling of a leaf on 
the waves in a pond. During these small 
changes in the shape and position of the ink 
deposits special attention was paid to the 
shape of the foot and to the continuity of the 
forward movement. Analysis of the videotapes 
could not disclose any change, leading to the 
conclusion that the behavior of the ink mate- 
rial is an internal event and is not coupled to 
the foot sole. 



DISCUSSION 

Our study demonstrates that in Helix aspersa 
the pedal waves move faster than the foot. The 
shape of the foot and the continuity of the for- 
ward movement do not show any temporal or 
topographic relationship with the wave move- 
ment. The displacement and deformation of 
Indian ink droplets is not coupled to similar 
changes in the foot sole. These findings allow 
the conclusion that the waves act indirectly by 
the intermediary of the haemolymph. This con- 
clusion is also corroborated by the observa- 
tion of the filling with fluid of the segments 
("junctions") between the waves, resulting in 
the pressure on the substratum, leading in turn 
to the forward movement. In addition, the tem- 
poral persistence of waves after severing the 
foot from the body indicates that a pulsating 
heart is not necessary to maintain waves. 

The question of how waves are built up from 
non-organized muscles is not solved. Authors 
who describe muscle bundles in different fixed 
directions are fortunate and use these to ex- 
plain the locomotion of the foot in snails and 
slugs. Jones (1975) published an exhaustive 
report on locomotion in Pulmonata. However, 
he did not address the problems of our present 
study. To the best of our knowledge, he is the 
first author who succeeded in catching mov- 
ing waves. He described in Agrolimax 
reticulatus the fixation of waves by immersing 
the moving animals in liquid nitrogen (Jones, 
1973). In cryostat sections, he reported a com- 
pression of oblique muscles and an almost com- 
plete occlusion of the haemocoel. He presented 
a micrograph showing muscle fibers that are 
reduced to strings, lying in many directions and 
occupying a very small fraction of the tissue 
area. For a morphologist, it is difficult to corre- 



late this tissular arrangement with waves. In 
Helix pomatia, he described the foot as highly 
muscular. We did not study Helix pomatia, but 
we can confirm that in Helix aspersa and in 
several other snails this is not the case. 

In his review on locomotion of molluscs, 
Trueman (1983) described for Helix a model 
of locomotion based on a crawling mechanism. 
Crawling is a biphasic activity. A transient sta- 
tionary point or zone serves as an anchor for 
the contracting or elongating free moving and 
uplifting segment of the animal. When waves 
are present, it is assumed that the wave is the 
anchor and that the animal advances because 
of the successive anchoring of the waves. The 
author refers to the work of Denny (1 980), who 
studied the physicochemical properties of the 
mucus oí Agrolimax columbianus. This author 
proposed a dual reaction of mucus changing 
from a solid phase in the stationary state to a 
liquid phase in the moving state, acting like a 
material ratchet. These properties of the mu- 
cus may facilitate the crawling mechanism 
proposed by Trueman. 

Several other students of snail locomotion 
also propose the crawling mechanism for for- 
ward displacement: Trappman (1916), Miller 
(1974), Gainey (1976), and Moffett (1979). 

In a study on the histology of the foot and 
the locomotion of Gastropoda, Elves (1961) 
mentioned that in Discus rotundatus the mus- 
culature of the foot is not well developed, and 
muscle fibers are of small size and few in num- 
ber. He devoted a short description to Helix 
aspersa and described in the foot a reticulum 
of connective tissue fibers and large muscles 
which run both longitudinally and dorsoven- 
trally. However, in the accompanying diagram, 
he depicted the muscular component as a few 
scattered small bundles occupying a small 
fraction of the total transverse section area. In 
his discussion, he mentioned that the locomo- 
tion may be influenced by an interplay between 
haemocoel turgor and muscular waves. 

An interesting type of forward displacement 
in terrestrial gastropods was discussed by 
Pearce (1989). It was termed loping" (derived 
from galloping) and differs from the gliding pro- 
gression. In the loping motion, the gastropod 
lifts its head from the substratum and thrusts it 
forward, then replaces it on the substratum, 
forming a low arch in the sole behind the head 
through which the rest of the body flows to the 
new stationary point of contact. The mucus trail 
left consists of more or less elongated dots, in 
contrast to the continuous mucus trail during 
gliding progression. Hence, loping is a perfect 
example of crawling and a strong argument 



216 



BUYSSENS 



for the existence of a different "ordinary" (ter- 
minology of Pearce) gliding mechanism. Inter- 
estingly, the waves of ordinary gliding are 
present with loping, but there is no interference. 

We can only speculate about what happens 
in Helix aspersa. The signal for wave forma- 
tion starts at the tip of the foot, resulting in the 
successive appearance of waves in the cau- 
dal direction. At the very moment the waves 
are induced, they start moving in cranial di- 
rection back to their initial inductive signal. The 
waves are not at all sinusoidal peristaltic con- 
tractions or pressure waves like in blood ves- 
sels. They resemble slice-like condensations 
of tissue (muscle, collagen, and interstitial 
cells), which move in an upright position along 
a horizontal plane parallel to the foot sole. A 
possible explanation how to visualize the for- 
mation of such a structure could be that the 
slices of condensed tissue while moving ac- 
quire cells at the advancing front and release 
them again at the trailing front. This phenom- 
enon of recruitment is known in the forward 
movement of cells in culture. Bretcher & 
Aguado-Velasco (1988) describe how 
lamellipodia, which are formed by cells when 
they start moving, recruit plasma membrane 
material at the expense of the trailing end of 
the cell. In fact, these cells do not advance by 
moving but by growing. The concept of recruit- 
ment can perhaps explain why waves are 
bilayered, the frontal directed half being the 
recruitment front and the rear half being the 
propulsion machine. 

Concerning the muscular structure of the foot 
an interesting micrograph is published by Ber- 
nard (1968). It shows in the foot of the large 
marine snail Polinices lewisi muscle and col- 
lagen fibers in a three-dimensional pattern 
reminiscent of the foot of Helix aspersa. Waves 
were not mentioned, and the foot aspirates and 
expels ambient water. 

Analyses of movie films of waves in Helix 
pomatia have been reported by Bonse (1935) 
and Lissman (1945) and in Patella vulgata by 
Jones & Trueman (1970). However, these ex- 
cellent studies do not address the basic ques- 
tion how we can understand the formation of 
waves or why they move faster than the ani- 
mals. 



ACKNOWLEDGMENTS 

The author wishes to thank Rita Van den 
Bossche for the histology work and Liliane Van 
den Eynde for logistical and secretarial work. 



Prof. A. Herman offered graciously his labora- 
tory facilities. Prof. A. De Schepper and his staff 
provided generously top quality radiographs. 



LITERATURE CITED 

BERNARD, F. R., 1968, The aquiferous system 
of Polinices lewisi. Journal of the Fisheries 
Research Board of Canada, 25(3): 541 -546. 

BONSE, H., 1935, Ein Beitrag zum Problem der 
Schneckenbewegung. Zoologische Jahr- 
bücher, Abteilung Allgemeine Zoologie und 
Physiologie der Tiere, 54: 349-384. 

BRETCHER, M. S. & С AGUADO-VELASCO, 
1998, Membrane traffic during cell locomotion. 
Current Opinion in Cell Biology, 10: 537-541. 

DENNY, M.L., 1980, The role of gastropod pedal 
mucus in locomotion. Nature, 285: 160-161. 

ELVES, M. W., 1961, The histology ofthe foot of 
Discus rotundatus and the locomotion of gas- 
tropod Mollusca. Proceedings of the Malaco- 
logical Society London, 34: 346-355. 

GAINEY, L. P., Jr., 1976, Locomotion in the Gas- 
tropoda: functional morphology of the foot in 
Neretina reclivata and Thais rustica. 
Malacologia, 15(2): 411^31. 

JONES, H. D., 1973, The mechanism of loco- 
motion of Agrolimax reticulatus (Mollusca: 
Gastropoda). Journal of Zoology, London, 171: 
489^98. 

JONES, H. D., 1975, Locomotion. Pp. 1-32, in: 
V. FRETTER & J. PEAKE, eds., Pulmonates, I, 
Functional anatomy and physiology. London: 
Academic Press. 

JONES, H. D. & E. R. TRUEMAN, 1970, Loco- 
motion ofthe limpet, Patella vulgata L. Journal 
of Experimental Biology, 52: 201-216. 

LISSMANN, H. W., 1945, The mechanism of lo- 
comotion in gastropod molluscs. 1. Kinemat- 
ics. Journal of Experimental Biology, 21: 
58-69. 

MILLER, S. L., 1974, Adaptive design of loco- 
motion and foot form in prosobranch gastro- 
pods. Journal of Experimental Marine Biology 
and Ecology, 14: 99-156. 

MOFFETT, S., 1979, Locomotion in the primi- 
tive pulmonate snail Melampus bidentatus: foot 
structure and function. Biological Bulletin, 57: 
306-319. 

PEARCE, T A., 1989, Loping locomotion in ter- 
restrial gastropods. Walkerana, 3(10): 229- 
237. 

PRIOR, D. J. &A. GELPERIN, 1974, Behavioral 
and physiological studies on locomotion in the 
giant garden slug Umax maximus. Malacologi- 
cal Review, 7: 50-51. 

TRAPPMAN, W., 1916, Die Muskulatur von He- 
lix pomatia L. Zeitschrift für Wissenschaftliche 
Zoologie, 115: 490-585. 

TRUEMAN, E. R., 1983, Locomotion in molluscs. 

Pp. 155-198, in: A. S. M. SALEUDDIN & К. M. 

WILBUR, eds., The Mollusca, Volume 4, Physi- 
ology. London: Academic Press. 

Revised ms. accepted 4 May 2004 



MALACOLOGIA, 2004, 46(1): 217-224 

COLORATION IN HELICINIDAE (MOLLUSCA: GASTROPODA: NERITOPSINA) 

Ira Richling 

Zoologisches Institut, Christian-Albrechts-Universität zu Kiel 
Olshausenstraße 40, 24098 Kiel, Germany; ira@richling.de 

ABSTRACT 

The coloration of Costa Rican Helicinidae has been studied, with special attention paid 
to the arboreal species. It is shown that either shell color or mantle pigmentation contribute 
to the coloration visible in the living animals. Ecological and systematic implications are 
given. This paper is supplementary to Richling (2004). 

Keywords: Helicinidae, Costa Rica, Central America, classification, coloration. 



INTRODUCTION 

During a recently published revision of the 
systematics and species differentiation in 
Costa Rican Helicinidae (Richling, 2004), the 
role of coloration was studied. As the plates 
were printed in black and white, I provide here 
the same plates in color and give additional 
notes on species in relationship to color pat- 
terns. Further details on the species, especially 
those in Figure 6, material and methods and 
literature are given in Richling (2004). 



RESULTS AND DISCUSSION 

Coloration in land snails is mainly determined 
by the need for camouflage. Thus, it depends 
strongly on the habitat of the respective spe- 
cies. The Costa Rican species inhabit tropical 
rain forests and can be split into two groups: 
arboreal species and ground dwellers crawl- 
ing in leaf litter. The arboreal species show a 
variable, bright coloration, as can be found in 
other arboreal land snails, for example, spe- 
cies of Liguus (Orthalicidae), Amphidromus 
(Camaenidae), and Cepaea (Helicidae), with 
yellow and red prevailing. They are seldomly 
greenish in adaptation to leaves. 

All Costa Rican species of the genus Helicina 
exhibit this pattern (Figs. 3, 4, 5A-D), with a 
greenish color developed in certain specimens 
of Helicina funcki L. Pfeiffer, 1849 (Fig. ЗА), 
and Helicina escondida Richling, 2004 (Fig. 
4H). The ground dwellers, Lucidella lirata (L. 
Pfeiffer, 1847), Aleadla hojarasca (Richling, 
2001 ), and Aleadla boeckeleri (Richling, 2001 ), 
are uniformly brownish, and in addition exhibit 



a rough surface, that is, pehostracal hairs or 
ridges on the shell (Figs. 20-0, 5F-H). 
Pyrgodomus microdinus (Morelet, 1 851 ) is the 
only exception in its strong association to sur- 
faces of calcareous rocks. In living individu- 
als, the bright yellow empty shell (Fig. 2R) 
becomes greenish-grayish because of the 
underlying dark pigmentation of the mantle. 
Furthermore, P. microdinus glues particles of 
detritus on its shell, thus perfectly resembling 
the rock surface (Fig. 5E). The same applies 
to the Jamaican Eutrochatella pulchella (Gray, 
1825), in which the active camouflage is func- 
tionally replaced by the white-yellowish mot- 
tling of the shell (Figs. 6M, P). 

When comparing the coloration of living ani- 
mals with empty shells, it becomes obvious 
that in the Costa Rican arboreal species, two 
different ways are utilized to produce the vari- 
able and bright coloration. On one hand, the 
species have a variable shell color combined 
with rather thick shells and a uniform mantle 
pigmentation, for example, Helicina funcki, 
which can even be nearly reddish; H. pitalensis 
Wagner, 1910; H. beatrix Angas, 1879; H. 
talamancensis (Richling, 2001); and H. 
punctisulcata cuericiensis Richling, 2004 (Figs. 
1A-E, L-M, 2A-E). On the other hand, the 
shells are thin and more or less transparent 
with exception of the outer lip (Figs. 1 F-K, 2F- 
N), but the mantle is variously mottled and 
causes the visible coloration, for example, 
Helicina tenuis L. Pfeiffer, 1849; H. gemma 
Preston, 1903; H. monteverdensis Richling, 
2004; H. escondida Richling, 2004; and H. 
chiquitica (Richling, 2001) (Figs. 3D-E, 4C- 
H, 5A-D). As an artifact in empty shells, the 
transparency becomes less, for example, com- 



217 



218 



RICHLING 



pare Figure 21 and Figure 4F. The latter way 
seems to liave evolved in connection with the 
very limited availability of calcium carbonate 
in Costa Rica. 

It seems that the optimal coloration of small- 
sized arboreal Helicinidae, about 3-4 mm, is 
dark. Evidence is given by the very small spe- 
cies H. chiquitica, in which most individuals 
are dark (Fig. 5C), populations of Helicina 
monteverdensis of a reduced average body 
size (Fig. 4G), and juveniles of Helicina funcki 
(Fig. 3B). 

Due to the high adaptability of the colora- 
tion, its value for systematics is limited, al- 
though the present study shows that in 
arboreal species the way to achieve the final 
coloration is typical for each species. For thin- 
shelled species, the varying and patterned 
mantle pigmentation is characteristic. When 
looking at a number of individuals, this pat- 
tern shows at certain specificity for different 
species, but single specimens might show 
exceptions. In some cases, it is even typical 
at population level, for example, in Helicina 



tenuis from Cabo Blanco on the Pacific plain 
(Fig. 3D) and La Selva on the Caribbean plain 
(Fig. 3E), or in Helicina monteverdensis in 
populations about 5 km from each other (Figs. 
4F, G). 

The coloration of head and foot seldom 
shows species specificity: the upper side is 
usually dark, especially towards the head and 
tentacles, whereas the lower side is light. 
Among the Costa Rican species, Helicina 
talamancensis represents the only exception. 
In all specimens studied, the whole body is 
whitish except for the sharply separated black 
tentacles (Fig. 48). 



LITERATURE CITED 

RICHLING, I., 2004, Classification of the Heli- 
cinidae: review of morphological characteris- 
tics based on a revision of the Costa Rican 
species and application to the arrangement of 
the Central American mainland taxa (Mollusca: 
Gastropoda: Neritopsina). Malacologie, 45(2): 
195-440. 



COLORATION IN HELICINIDAE 



219 




FIG. 1. Shell coloration of Costa Rican species. A-C. Helicina funcki. A. Rio Barbilla. В. Manzanillo. 
С. Santa Elena. D-E. H. pitalensis. D. Bajo Bonito. E. Península de Osa. F-l. H. tenuis. F-H. Cabo 
Blanco. I. La Selva. J-K. H. echandiensis Richling, 2004, campamento Echandi. L-M. H. punctisulcata 
cuericiensis, Estación Cuerici; scale bars = 4 mm (A-E), 3 mm (F-M). 



220 



RICHLING 




FIG. 2. Shell coloration of Costa Rican species. A. Helicina beatrix beatrix, Guayacán. B-C. H. b. 
confusa (Wagner, 1908). B. Uatsl. С Shiroles. D. H. b. riopejensis Richling, 2004, Rio Peje. E. H. 
talamancensis, Bajo Bonito. F-H. H. gemma. F. Cacao. G. Las Pavas. H. Siquirres. I-J. H. 
monteverdensis, Monteverde. K-M. H. escondida, Rio Barbilla. N. H. chiquitica, Río Barbilla. O. 
Alcadia hojarasca, Mirador Gerardo. P A. boeckelerí, Pitilla. Q. Lucidella lirata, Cahuita. R. Pyrgodomus 
microdinus, Fila de Cal; scale bars = 3 mm (A-M), 2 mm (N-P), 1.2 mm (Q-R). 



COLORATION IN HELICINIDAE 



221 











FIG. 3. Living animals of Costa Rican species. A. Helicina funcki, Cahuita. B. H. funcki, juvenile, 
Uatsi. С H. pitalensis, Bajo Bonito. D. H. tenuis, Cabo Blanco. E. H. tenuis, La Selva. F. H. beatríx 
confusa, Uatsi. G. H. beatrix confusa, Shiroles (photograph: Vollrath Wiese). H. H. beatríx riopejensis, 
Río Peje. 



222 



RICHLING 




FIG. 4. Living animals of Costa Rican species. A. Helicina beatrix beatrix, Guayacán. В. H. 
talamancensis, Bajo Bonito. С. H. gemma, Cacao. D. H. gemma, Las Pavas. E. H. gemma, Siquirres. 
F. H. monteverdensis, Monteverde. G. H. monteverdensis. Mirador Gerardo. H. H. escondida, Shiroles. 



COLORATION IN HELICINIDAE 



223 




FIG. 5. Living animals of Costa Rican species. A. Helicina escondida, Shiroles. B. H. escondida, Río 
Barbilla. С. H. chiquitica, Río Barbilla. D. H. cliiquitica, Río Pacuarito. E. Pyrgodomus microdinus, 
Fila de Cal (photograph: Vollrath Wiese). F. Alcadia liojarasca, Mirador Gerardo. G. A. boec¡<eleñ, 
Pitilla. H. Lucidella lirata, Cahuita. 



224 



RICHLING 




FIG. 6. Shell coloration. A. Helicina neritella Lamarck, 1799, Jamaica. B. H. platychila (Mühlfeldt, 
1816), Dominica. С H. orbiculata (Say, 1818), Florida. D. H. turbinata Wiegmann, 1831, Mexico. E. 
H. amoena L. Pfeiffer, 1849, Guatemala. F. H. dysoni L. Pfeiffer, 1849, Trinidad & Tobago. G. H. sericea 
Drouet, 1859, Suriname. H. Angulata brasiliensis (Gray, 1825), Brazil. I. Alcadia major {Gray, 1824), 
Jamaica. J. A. hollandi {C. B.Adams, 1849), Jamaica. K. A. jamaicensis (Sowerby, 1841), Jamaica. 
L. A. rotunda (Orbigny, 1841), Cuba. M. Eutrochatella pulchella, Jamaica. N. Lucidella aureola 
(Férussac, 1822), Jamaica. O. Schasicheila alata (L. Pfeiffer, 1848), Mexico. P. Eutrochatella 
pulchella, Jamaica; scale bar = 5 mm (A-0). 



LETTER FROM THE EDITOR 



MALACOLOGIA, 2004, 46(1): 227-231 

SPECIES CHECK-LISTS: DEATH OR REVIVAL OF THE NOUVELLE ÉCOLE? 

George M. Davis 

Department of Microbiology and Tropical Medicine 

George Washington University Medical Center 

Ross Hall 731, 2300 Eye Street NW, Washington DC 20037, U.S.A.; 

georgedavis99@hotmail.com 



With more than 100,000 described species, 
mollusks belong to the second largest phylum 
after the Arthropoda. Mollusks have attracted 
a large number of shell collectors, amateur 
malacologists, field biologists, conservation- 
ists, as well as evolutionary biologists, taxono- 
mists and systematists. 

As a result, there are huge amounts of ma- 
lacological publications available for most re- 
gions of our planet, and our knowledge of the 
group is increasing day by day. However, de- 
spite the extensive work that has been done, 
nomenclature and taxonomy of many groups 
are still in a confused state, and the systemat- 
ics of numerous taxa is embroiled in contro- 
versy. Moreover, the problems increase with 
the recent advent of new anatomical and mo- 
lecular methods, where the new types of data 
are often in conflict with the traditional, that is, 
shell-based taxonomy. 

This confusion affects most taxonomic lev- 
els from subspecies to higher taxa and makes 
it difficult for many non-biologists and even 
professional biologists to apply or understand 
the correct name for a taxon. The conse- 
quences are not-trivial, as an incorrect deter- 
mination or classification can be the deciding 
factor in many diverse scientific and non-sci- 
entific activities. 

To maintain order in the sometimes chaotic 
system of publications and taxa, species 
check-lists are often generated for individual 
biogeographic regional and/or systematic 
groups. For the uninitiated, it is difficult to imag- 
ine how much work is involved to generate a 
widely acceptable check-list. Often interna- 
tional groups of scientists have to sort through 
hundreds or even thousands of primary publi- 
cations, look at many voucher specimens and 
work through quantities of field records. They 
have to carefully consider frequently contra- 
dicting information, make educated decisions 
about the "correct" nomenclature and tax- 
onomy, and ensure compliance with the Inter- 
national Code of Zoological Nomenclature. 



And finally, these check-lists have to be up- 
dated on a regular basis in order to keep pace 
with the malacological research. 

Given the nature of species check-lists there 
are, however, some critical points I would like 
to discuss. This could possibly help to further 
improve the quality of those check-lists and/ 
or to point out some possible pitfalls the users 
should be made aware of. Although the fol- 
lowing points might apply to many species- 
check lists, I will focus my attention on the two 
most recent European lists, the "Mollusques 
continentaux de France: liste de référence 
annotée et bibliographie" by Falkner et al. 
(2002), and the "Check-list of the non-marine 
Molluscan species-group taxa of the states of 
Northern, Atlantic and Central Europe" 
(CLECOM I) by Falkner et al. (2001). 

(1) Naming Species 

Identifying a species, a "good species", has 
often been a very difficult task, due, in part, to 
the large variety of species concepts and in 
part to difficulties in the objective selection and 
interpretation of the characters used, often 
resulting in a personal view of what a species 
is. The famous malacologist W. Kobelt (1 881 ) 
once wrote [translated from German]: "/ obey 
a simple, practical rule, no matter how unsci- 
entific it may be. I call a good species what I 
can diagnose without long and careful com- 
parisons and measurements ...". More than 
half a century ago, matters changed due to 
the introduction of the biological species con- 
cept (BSC) (Mayr, 1940) and of other, pro- 
gressively more refined concepts (review by 
Hull, 1997). Many traditional taxonomists, nev- 
ertheless, continue working as in the past. 
They use an often arbitrary chosen level of 
morphological difference, frequently calcu- 
lated "at a glance" to decide what a new "good 
species" is. However, these good species are 
actually "morphospecies" (see Giusti & Man- 
ganelli, 1992), and it is usually tacitly taken 



227 



228 



DAVIS 



for granted that they correspond to biological 
species. 

Obviously, this practice is extremely subjec- 
tive. Extremism gave rise to "the lumpers", who 
require robust differences to segregate taxa, 
and in doing so, tend to accept very polymor- 
phic species. Others, "the splitters", base their 
taxonomic decisions on low levels of differen- 
tiation. More recently, subjective decisions 
were brought into clear view when molecular 
research revealed that both splitters and 
lumpers are frequently wrong, because the 
levels of morphological difference are often 
independent of genetic divergence (organisms 
with a low level of morphological difference 
may have high levels of genetic divergence, 
and vice versa). 

A quick glance at many papers shows that 
the authors continue to use superficial shell 
morphological/typological procedures. These 
are sometimes based on simple differences 
in shell height or diameter, or on the relation- 
ship height/diameter, differences that repeat- 
edly have been shown to be unsuitable for 
species differentiation. A first conclusion isthat 
creating taxa simply on the basis of shell char- 
acters (qualitative and quantitative) should al- 
ways be done with extreme caution (many 
cases of convergence in shell shape in not 
related groups are known, convergent evolu- 
tion is rampant; Davis, 1979), unless there are 
very compelling reasons (fossils, rare deep 
sea taxa, taxa of family-level groups notori- 
ous for being over-named and for having high 
intraspecific variation, etc.). The situation may 
not substantially change even when other 
characters, anatomical, eco-ethological, are 
added (anatomical characters are not always 
highly revealing; peculiar ecology, parasitol- 
ogy or ethology may be the source of certain 
shell differences). 

The situation is rendered more problematic 
by the so-called "fanatisme du nobis" (Dance, 
1970), that is, the introduction of new names 
to give "eternity" to one's own name. The more 
new names one introduces, the greater is the 
possibility one of them survives as that of a 
"good species" or "good subspecies". 

The International Code on Zoological No- 
menclature (ICZN) was created to manage, in 
a legalistic manner, the naming of species. But 
it has been abused and indirectly supports 
proliferation of names that cannot be sup- 
pressed and therefore must be considered 
valid and listed in check-lists until a future re- 
vision is made. The last edition of the ICZN 



(1999) called for adequate and rigorous spe- 
cies definitions (see also Hawskworth & Bisby, 
1988: 12-13), but since then, the literature 
shows that this call remains unheaded in many 
instances. 

Recent creation of new species based on 
dubious characters is the survival/revival of the 
school called "Nouvelle École", founded by the 
French malacologist Jules-René Bourguignat 
in the second half of the 19'^ century, accord- 
ing to which a species should be determined 
on arbitrarily chosen characters, which nearly 
always meant shell characters. If an individual 
was found to differ from all others by three 
characters or more, it should be considered 
to belong to a species new to science (Dance, 
1 986). This school has been unanimously con- 
demned, but never completely abandoned. As 
a matter of fact, many taxonomists officially 
criticize the Nouvelle École, but actually fol- 
low a very similar if not identical practice. Con- 
tinuation of this practice is fueled by the fact 
that at least some of the species described by 
the followers of the Nouvelle École subse- 
quently turn out to be "good species" (see 
above). Moreover, recently the method of the 
Nouvelle École is spreading again due to the 
strategy of the "valeur patrimoniale" of local 
faunas to support conservation programs 
(Falkner et al., 2001: 6; 2002: 19; Beuchet, 
2002: 8-12). According to such strategy, "It is 
extremely difficult to convince engineers or 
politicians of the value and need for protec- 
tion of a special, even unique, unnamed form, 
but from the moment that a name can be pro- 
vided there exists a recognizable unit which 
can be referred to" (Falkner et al., 2001: 6). 
Though these intentions are honorable, taxa 
of the species group should never be de- 
scribed for "political" reasons! Apart from the 
fact that that strategy opens doors to irrespon- 
sible students and amateurs, it risks to reduce 
taxonomy to a mere artifice possibly with grim 
consequences: "if the philosophy of the 
Nouvelle École had become widely popular its 
effect on systematic conchology, as may be 
imagined, could have been catastrophic" 
(Dance, 1986). 

While I reject the subjective and unethical 
practice just described, I am concerned about 
how one manages the many names given to 
taxa that have uncertain value. Each taxon 
correctly (legally) described has value unless 
the contrary is demonstrated. At the same time, 
it is a considerable disservice to science and 
society to inflate check-lists with numerous 



SPECIES CHECK-LISTS 



229 



dubious nominal species/subspecies! I am 
convinced that these taxa must be evaluated 
critically and that, when it is eventually decided 
they must be listed as potentially endangered 
or as a part of a special ecosystem, their un- 
certain taxonomic status should be made clear. 

(2) The Use of Subspecies 

Subspecies are even more difficult to define 
objectively than species as was well known from 
the late 19^^ century (Kobelt, 1881, continued 
the sentence given above as follows: "... That 
which I can distinguish only by precise mea- 
surements I call a variety"). Alas, some mala- 
cologists still name subspecies as it was done 
by Kobelt for varieties (minor variations), just to 
distinguish a little characterized local form, and 
as a tool to satisfy their "fanatisme du nobis". 

Difficulties in using subspecies come, first 
of all, from the fact that not all species con- 
cepts recognize the subspecies status. One 
species concept that explicitly does is the BSC. 
However, although often claimed by malacolo- 
gists, the BSC, in its true meaning, is rarely 
applied in malacology, because it places the 
taxonomy of natural species within the scheme 
of population genetics. Within the framework 
of the BSC, the concept of subspecies gradu- 
ally evolved from a simple "unit of conve- 
nience" (Blackwelder, 1967; Dobzhansky etal., 
1977; Mayr, 1963, 1982) to became applicable 
to populations that are kept isolated usually 
by geographical barriers and that exhibit rec- 
ognizable phylogenetic partitioning due to the 
time-dependent accumulation of genetic dif- 
ferences (O'Brien & Mayr, 1991). Therefore, 
subspecies should presently never be used 
without an immense amount of rigorous data 
that clearly demonstrate that the concept can 
be applied legitimately, that is, allopatric popu- 
lations that have diverged sufficiently geneti- 
cally (based on real genetic data) where they 
would be elevated to full species were it not 
for the complete sameness of the mate rec- 
ognition system and the full capacity to pro- 
duce a Fl and F2 generation if given the 
opportunity (Davis, 1994). 

It is obvious that most check-lists that use 
subspecies are not based on such rigorous 
work. To give an example from CLECOM, for 
Germany there are 36 (!) species and subspe- 
cies listed for the minute rissooidean genus 
Bythiospeum. I do not know of any molecular 
or detailed anatomical study that has looked 
at variability within and between populations 



to infer possible genetic breaks in taxa of 
Bythiospeum that, in turn, could be used to 
deduce reproductive isolation. Therefore, I 
would consider the splitting of Bythiospeum a 
simple matter of pure subjectivity. In fact, pre- 
liminary genetic data produced by one of my 
collaborators indicate that the number of 
Bythiospeum species in Europe is much lower 
than hitherto believed and that the genus ap- 
pears to be paraphyletic, demonstrating the 
diagnostic inadequacy of the morphological 
methods used up-to-day. 

Unfortunately, CLECOM gives contradicting 
information as to legitimacy of the subspecies 
listed. Whereas on page 3 of the introduction 
to CLECOM I it is said that "... the CLECOM 
check-list will contain the nomenclaturally cor- 
rect names of the species and subspecies that 
are considered to be valid", further down on 
the same page it is written "... Inclusion of 
named subspecies in the CLECOM database 
results inevitably in both well-founded and 
even spectacular forms being mentioned along 
with some 'weak' subspecies." Moreover, it is 
said that "In the exciting faunistic literature of 
northern, western and central Europe subspe- 
cies are widely, if not generally, neglected." If 
so, a check-list that is based on this literature 
should ignore subspecies as well. 

One reason for including subspecies in 
CLECOM is related to conservation purposes: 
"Our only tool to make this diversity apparent 
to conservation authorities, researchers in 
applied sciences and others who require 
recognising its existence is the application of 
trinominal nomenclature." Many conservation- 
ists are well aware of the need to protect in- 
traspecific diversity but adopt different 
strategies. In recent years, several approaches 
have been deployed using "conservation units" 
instead of dubious subspecies (e.g., Crandall 
et al., 2000; Fraser & Bernatchez, 2001). 

But surely it is inappropriate to name sub- 
species as a convenience and in the absence 
of well-founded data, no matter how honor- 
able the intentions are. 

(3) Data Sources 

In the introduction to the (sub)generic list of 
CLECOM l+ll by Bank et al. (in: Falkner et al., 
2001 ) it is said that "The list presented in this 
paper is based on the study of hundreds of 
publications ...; only a selection of them could 
be cited in the reference list." I can only imag- 
ine how many publications the CLECOM com- 



230 



DAVIS 



mittee must have had studied. But it certainly 
would be useful to list all those publications 
as they are the primary data source and as a 
check-list can only be as good as the publica- 
tions it is built upon. 

Another data source that is often used in 
check-lists is unpublished information. Philippe 
Bouchet specifically acknowledged this valu- 
able source in his introduction to the French 
list of continental molluscs. However, unpub- 
lished data are not subject to the scrutiny of 
the scientific community and its quality and 
reliability may vary. Therefore, if unpublished 
data are used in check-lists, they should be 
clearly marked as such. 

Another data source of great value for check- 
lists is molecular data. The advance of robust 
population genetics, phylogeographic and 
phylogenetic studies has lead to numerous re- 
assignments of species, genera and families. 
Yet, these data are (still) largely neglected in 
check-lists. To give an example, the molecu- 
lar work my group has done in the past ten 
years on several European taxa of the super- 
family Rissooidea largely has been ignored by 
CLECOM. Instead, the rissooidean systemat- 
ics of that list is still mainly based on tradi- 
tional (mostly shell-based) data. It is not that 
the molecular genetic data have been ignored 
because they often contradict the findings of 
members of the CLECOM team. Rather, it is 
important that CLECOM incorporate new find- 
ings based on genetic data much more quickly, 
even if these findings are inconvenient. One 
of the declared goals of the CLECOM list is to 
promote a stable nomenclature of European 
non-marine molluscs. But this can only be 
achieved by an objective assessment of all 
data available. 

With regard to promotion of a stable nomen- 
clature (one of the two primary goals of the 
ICZN), it is important that check-lists are not 
used to introduce changes in the names of 
various taxa, species in particular. Apparent 
novelties in this field must be the subject of 
careful evaluation by the scientific community 
before they are proposed as unquestionable 
to a vast public of non-specialists. Stability of 
nomenclature is not necessarily achieved by 
an uncritical application of the law of priority, 
but through the conservation of the names in 
use, no matter if they are not the oldest names 
available, as clearly seen in many findings of 
the ICZN over the past few decades. The 
adoption of new systematic hierarchical order- 
ing in check lists (as done by CLECOM) that 



were neither adopted before nor previously 
checked by the scientific community is another 
critical point we should raise. As such new 
systems are unknown in the scientific litera- 
ture or in private or public collections, they in- 
evitably are a source of confusion, particularly 
for non-specialists. Moreover, as experience 
shows, they are inevitably subject to rapid 
change when they are subjected to rigorous 
scientific evaluation. 

Check lists should be a catalogue of all the 
species of a certain group for a given geo- 
graphical area, not a vehicle for promoting the 
reconstruction of phylogenetic history or pro- 
moting a particular phylogenetic hypothesis. 

(4) Need for Further Research 

The introduction to CLECOM begins with the 
headline "The need for a uniform catalogue to 
promote biodiversity studies". And indeed, 
comparing the various activities ongoing at 
national levels and formulating coherent syn- 
theses that can be used by both scientists and 
policy-makers is a declared goal of the 
CLECOM committee. 

In order to indicate taxa that need further 
research, the CLECOM list uses question 
marks for those taxa. However, it appears as 
if those question marks are heavily under- 
utilized in CLECOM. To use the Bythiospeum 
example above, none of the 36 dubious spe- 
cies and subspecies listed for Germany car- 
ries a question mark. In fact, none of the 
numerous German taxa of the superfamily 
Rissooidea (a highly controversial group with 
many cryptic radiations) is, according to the 
CLECOM list, in need of further studies. But 
numerous recent publication using molecular 
markers have shown that many of these 
groups are in urgent need of revision. 

We do not know the reason the above omis- 
sions, but it seems that CLECOM is trying to 
suggest that almost everything is known about 
the systematics and taxonomy of the European 
non-marine mollusks. This certainly will not 
promote further research and it will not help to 
protect biodiversity either. In fact, it only dis- 
courages biologists from asking meaningful 
questions and therefore contradicts the de- 
clared goals of the same CLECOM. 

In conclusion, the above points highlight 
some of the problems I see with recently pub- 
lished check-lists. Success and acceptance of 
those lists depend not only on the quality and 



SPECIES CHECK-LISTS 



231 



quantity of the databases, but also on the abil- 
ity to consolidate conflicting information and 
ensure a critical assessment of their own work. 
In order to promote nomenclatural stability, it 
is also necessary to clearly state the subspe- 
cies and species concepts upon which the list 
is based and the operational criteria used to 
implement those concepts. 



LITERATURE CITED 

BLACKWELDER, R.E., 1967, Taxonomy. A text 
and reference book. John Wiley & Sons, New 
York, London, Sydney. 

BOUCHET, P., 2002, Mollusques terrestres et 
aquatiques de France: un nouveau référentiel 
taxonomique, un nouveau départ, de nouvelles 
perspectives. Pp. 5-20, in: G. FALKNER, T. E. J. 
RIPKEN & M. FALKNER, Mollusquos Continentaux 
de France. Liste de référence annotée et 
Bibliograpliie. Publications scientifiques du 
Muséum National d'Histoire Naturelle, Paris. 

CRANDALL, K. A., O. R. P BININDA-EMONDS, 
G. M. MAGE & R. K. WAYNE, 2000, Consider- 
ing evolutionary processes in conservation bi- 
ology. Trends in Ecology and Evolution, 15: 
290-295. 

DANCE, S. P., 1970, "Le fanatisme du nobis": a 
study of J.-R. Bourguignat and the "Nouvelle 
École". Journal of Conchology, 27: 65-86. 

DANCE, S. P, 1986, A history of shell collecting. 
E. J. Brill & W. Backhuis, Leiden. 

DAVIS, G. M., 1979, The origin and evolution of 
the gastropod family Pomatiopsidae, with em- 
phasis on the Mekong River Triculinae. Mono- 
graph of the Academy of Natural Sciences of 
Philadelphia, 20: i-viii, 1-120. 

DAVIS, G. M., 1994, Molecular genetics and 
taxonomic discrimination. Nautilus, 8, Suppl. 
2: 3-23. 

DOBZHANSKY, T, F. J. AYALA, G. L. STEBBINS 
& J. W. VALENTINE, 1977, Evolution. W.H. 
Freeman and Company, San Francisco. 

FALKNER, G., A. BANK & T v. PROSCHWITZ, 
2001, Clecom-Project. Check-list of the non- 



marine molluscan species-group taxa of the 
States of Northern, Atlantic and Central Europe 
(CLECOM I). Heldia, 4: 1-76. 

FALKNER, G., T E. J. RIPKEN & M. FALKNER, 
2002, Mollusques continentaux de France. 
Liste de référence annotée et bibliographie. 
Publications scientifiques du Muséum National 
d'Histoire Naturelle, Paris. 

FRASER, D. J. & L. BERNATCHEZ, 2001 , Adap- 
tive evolutionary conservation: towards a uni- 
fied concept for defining conservation units. 
Molecular Ecology, 10: 2741-2752. 

GIUSTI, F. & G. MANGANELLI, 1992, The prob- 
lem of the species in malacology after clear 
evidence of the limits of morphological system- 

atics. Pp. 153-172, in: E, GITTENBERGER & J. 

GOULD, eds.. Proceedings of the Ninth Inter- 
national Malacological Congress, Edinburgh, 
1992, Unitas Malacologica, Leiden. 

HAWKSWORTH, D. L. & F. A. BISBY 1 988, Sys- 
tematics the keystone of biology. Pp. 3-30, in: 
D.L. HAWKSWORTH, ed.. Prospects in System- 
atics. The Systematic Association, special vol- 
ume no. 36, Clarendon Press, Oxford. 

HULL, D. L., 1997, The ideal species concept - 
and why we can't get it. Pp. 357-380, in: м. f. 

CLARIDGE, H. A. DAWAH & M. R. WILSON, eds., 

Species: the units of biodiversity. Chapman & 
Hall, London. 

KOBELT, W., 1881, Exkursionen in Süditalien. 
Die Sicilianischen Iberus. Jahrbücher der Deut- 
schen Malakologischen Gesellschaft, 8: 50-67. 

MAYR, е., 1940, Speciation phenomena in birds. 
American Naturalist, 74: 249-278. 

MAYR, E., 1963, Animal species and evolution. 
The Belknap Press of Harvard University 
Press, Cambridge (Massachusetts, USA), Lon- 
don (England). 

MAYR, E., 1982, The growth of biological 
thought: diversity, evolution and inheritance. 
The Belknap Press of Harvard University 
Press, Cambridge (Massachusetts, USA), Lon- 
don (England). 

O'BRIEN, S. J. & E. MAYR, 1991, Bureaucratic 
mischief: recognizing endangered species and 
subspecies. Science, 251: 1187-1188. 

Revised ms. accepted 3 June 2004 



MALACOLOGIA 



International Journal of Malacology 





Vol. 46(1) 



2004 



Publication dates 

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 18 Oct. 2000 

Vol.43, No. 1-2 20 Aug. 2001 

Vol. 44, No. 1 8 Feb. 2002 

Vol. 44, No. 2 30 Aug. 2002 

Vol. 45, No. 1 29 Aug. 2003 

Vol. 45, No. 2 22 Mar. 2004 



VOL. 46, NO. 1 MALACOLOGIA 2004 

CONTENTS 

NORBERT BUYSSENS 

Locomotion in Helix aspersa 211 

MAXIMILIANO CLEDÓN, LUIZ RICARDO L SIMONE, 

& PABLO E. PENCHASZADEH 

Crepidula cachimilla (Mollusca: Gastropoda), A New Species from 
Patagonia, Argentina 1 85 

GEORGE M. DAVIS 

Species Check-Lists: Death or Revivlal of the Nouvelle École? 227 

GEORGE A. EVSEEV NATALYA K. KOLOTUKHINA, & OLGAYA. SEMENIKHINA 
Anatomy of a Small Clam, Alveinus ojianus (Bivalvia: Kelliellidae), with a 
Discussion on the Taxonomic Status of the Family 1 

GENNADY M. KAMENEV 

New Species of the Genus Kellia (Bivalvia: Kellidae) from the Commander 
Islands, with Notes on Kellia comandorica Scarlato, 1981 57 

GENNADY M. KAMENEV 

New Species of the Genus Abrina (Bivalvia: Semelidae) from the 
Commander and Kuril Islands 157 

STEFFEN KIEL 

Shell Structures of Selected Gastropods from Hydrothermal Vents and 
Seeps 169 

JORIS M. KOENE & IGOR V MURATOV 

Revision of the Reproductive Morphology of Three Leptaxis Species 
(Gastropoda, Pulmonata, Hygromiidae) and Its Implication on Dart Evolution 73 

GIUSEPPE MANGANELLI, SIMONE CIANFANELLI, NICOLA SALOMONE, 

& FOLCO GIUSTI 

Morphological and Molecular Analysis of the Status and Relationships 
of Oxychilus paulucciae (De Stefani, 1883) (Gastropoda: Pulmonata: 
Zonitidae) 19 

CHRISTOPHER P MEYER 

Toward Comprehensiveness: Increased Molecular Sampling within 
Cypraeidae and Its Phylogenetic Implications 127 

BRIAN MORTON 

The Biology and Functional Morphology of Foegia novaezelandiae (Bivalvia: 
Anomalodesmata: Clavagelloidea) from Western Australia 37 

IRARICHLING 

Coloration in Helicinidae (Mollusca: Gastropoda: Neritopsina) 217 

PAWEL WOZNICKI 

Chromosomes of the Chinese Mussel Anodonta woodiana (Lea 1834) 
(Bivalvia, Unionidae) from the Heated Konin Lakes System in Poland .... 205 

MINWU 

Preliminary Phylogenetic Study of Bradybaenidae (Gastropoda: 
Stylommatophora: Helicoidea) 79 



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VOL. 46, NO. 1 MALACOLOGIA 2004 

CONTENTS 

GEORGE A. EVSEEV, NATALYA K. KOLOTUKHINA, & OLGA YA. SEMENIKHINA 
Anatomy of a Small Clam, Alveinus ojianus (Bivalvia: Kelliellidae), with a 
Discussion on the Taxonomic Status of the Family 1 

GIUSEPPE MANGANELLI, SIMONE CIANFANELLI, NICOLA SALOMONE, 

& FOLCO GIUSTI 

Morphological and Molecular Analysis of the Status and Relationships 
of Oxychilus paulucciae (De Stefani, 1883) (Gastropoda: Pulmonata: 
Zonitidae) 19 

BRIAN MORTON 

The Biology and Functional Morphology of Foegia novaezelandiae (Bivalvia: 
Anomalodesmata: Clavagelloidea) from Western Australia 37 

GENNADY M. KAMENEV 

New Species of the Genus Kellia (Bivalvia: Kellidae) from the Commander 
Islands, with Notes on Kellia comandorica Scarlato, 1981 57 

JORIS M. KOENE & IGOR V. MURATOV 

Revision of the Reproductive Morphology of Three Leptaxis Species 
(Gastropoda, Pulmonata, Hygromiidae) and Its Implication on Dart Evolution 73 

MINWU 

Preliminary Phylogenetic Study of Bradybaenidae (Gastropoda: 
Stylommatophora: Helicoidea) 79 

CHRISTOPHER R MEYER 

Toward Comprehensiveness: Increased Molecular Sampling within 
Cypraeidae and Its Phylogenetic Implications 127 

GENNADY M. KAMENEV 

New Species of the Genus Abrina (Bivalvia: Semelidae) from the 
Commander and Kuril Islands 157 

STEFFEN KIEL 

Shell Structures of Selected Gastropods from Hydrothermal Vents and 
Seeps 169 

MAXIMILIANO CLEDÓN, LUIZ RICARDO L SIMONE, 

& PABLO E. PENCHASZADEH 

Crepidula cachimilla (Mollusca: Gastropoda), A New Species from 

Patagonia, Argentina 185 

RESEARCH NOTES 

PAWEL WOZNICKI 

Chromosomes of the Chinese Mussel Anodonta woodiana (Lea 1834) 
(Bivalvia, Unionidae) from the Heated Konin Lakes System in Poland .... 205 

NORBERT BUYSSENS 

Locomotion in Helix aspersa 211 

IRA RICHLING 

Coloration in Helicinidae (Mollusca: Gastropoda: Nehtopsina) 217 

LETTER FROM THE EDITOR 

GEORGE M. DAVIS 

Species Check-Lists: Death or Revival of the Nouvelle École? 227 



/ 1 i\ L. 




'f^ ■-■X 



MALACOLOGIA 

m 



ï 



International Journal of Malacology 




Bivalve Studies in the Florida Keys 

Proœedings of the International Marine Bivalve Wori^shop 
Long Key, Florida, July 2002 I 

Edited by Rüctoer Bieler and Paula M. Mikkelsen f 

Vol. 46(2) 




MALACOLOGIA 
http:\\malacologia.fmnh.org 



EDITOR-IN-CHIEF: 
GEORGE M. DAVIS 



Editorial Office 

Malacologie 

P.O. Box 1222 

West Falmouth, MA 02574- 1222 

Copy Editor: 

EUGENE COAN 

California Academy of Sciences 

San Francisco, CA 



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Malacologia 

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Haddonfield, NJ 08033-0309 

Associate Editor: 

JOHN B. BURCH 

University of Michigan 

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- Graphics Editor: 

THOMAS WILKE 

Justus Liebig University 

Giessen, Germany 



Assistant Business Managers: 

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Delaware Museum of Natural History 

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



RÜDIGER BIELER 
Field Museum, Chicago 

JOHN BURCH 

MELBOURNE R. CARRIKER 
University of Delaware, Lewes 

GEORGE M. DAVIS 
Secretary and Treasurer 

CAROLE S. HICKMAN 

President 

University of California, Berkeley 



ALAN KOHN 

Vice President 

University of Washington, Seattle 

JAMES NYBAKKEN 

President Elect 

Moss Landing Marine Laboratory, California 

CLYDE F. E. ROPER 

Smithsonian Institution, Washington, D.C. 

SHI-KUEIWU 

University of Colorado Museum, Boulder 



Participating Members 



PETER MORDAN 

Secretary, UNITAS MALACOLOGICA 
The Natural History Museum 
London, United Kingdom 



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 © 2004 by the Institute of Malacology 
ISSN:0076-2997 



2004 
EDITORIAL BOARD 



MCZ 
LIBRARY 



Ulh 



J.A.ALLEN 

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

E. E. BINDER 

Museum d'Histoire Naturelle 

Geneve, Switzerland 

P. BOUCHET 

Muséum National d'Histoire Naturelle 
Paris, France 
bouchet@cimrs 1. mnhn. fr 

P. CALOW 

University of Sheffield 
United Kingdom 

R. CAMERON 

Stieffield 

United Kingdom 

R. Cameron@sheffield. ac. uk 

J. G. CARTER 

University of North Carolina 

Chapel Hill, U.S.A. 

M. CHARRIER 

Universite de Rennes 

France 

maryvonne. charrier@univ-rennes 1 . fr 

R. H. CO WIE 
University of Hawaii 
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. 

С J. DUNCAN 

University of Liverpool 
United Kingdom 

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

E. GITTENBERGER 
Rijksmuseum van Natuurlijke Historie 
Leiden, Netherlands 
sbu2eg@rulsfb.leidenuniv.de 

F. GIUSTI 

Universita di Siena, Italy 
giustif@unisi.it 



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

A. V. GROSSU 
Universitatea Bucuresti 
Romania 

T. HABE 
Tokai University 
Shimizu, Japan 

R. HANLON 

Mahne Biological Laboratory 
Woods Hole, Mass., U.S.A. 



G. HASZPRUNAR 

Zoologische Staatssammlung München 

München, Germany 

haszi@zi. biologie, uni-muenchen. de 

J. M. HEALY 
University of Queensland 
Australia 
jhealy@zoology. uq. edu. au 

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

К. E. HOAGLAND 
West Falmouth, U.S.A. 

В. HUBENDICK 

Naturhistoriska Museet 
Göteborg, Sweden 

S. HUNT 
Lancashire 
United Kingdom 

R. JANSSEN 

Forschungsinstitut Senckenberg, 
Frankfurt am Main, Germany 

M. S. JOHNSON 
University of Westem Australia 
Nedlands, WA, Australia 
msj@cyllene. uwa. edu.au 

R. N. KILBURN 

Natal Museum 
Pietermaritzburg, South Africa 

M. A. KLAPPENBACH 

Museo Nacional de Historia Natural 

Montevideo, Uruguay 

J. KNUDSEN 

Zoologisk Institut Museum 

K0benhavn, Denmark 



im 1 2005 

HARVARD 
UNIVERSITY 



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

С MEIER-BROOK 
Tropenmedizinisches Institut 
Tübingen, 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 

D. ÓFOIGHIL 
University of t\/lichigan 
Ann Arbor, U.S.A. 

J.0KLAND 

University of Oslo 
Norway 

T. OKUTANI 
University of Fisheries 
Tokyo, Japan 

W. L PARAENSE 

Instituto Oswalde Cruz, Rio de Janeiro 

Brazil 

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

R. PIPE 

Plymouth Marine Laboratory 

Devon, United Kingdom 

RKPI@wpo.nerc.ac.uk 

J. P POINTIER 

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

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. SEGERSTRALE 

Institute of Marine Research 
Helsinki, Finland 

A. STAÑCZYKOWSKA 
Siedlce, Poland 

F. STARMÜHLNER 

Zoologisches Institut der Universität 
Wien, Austria 

Y I. STAROBOGATOV 
Zoological Institute 
St. Petersburg, Russia 

J. STUARDO 

Universidad de Chile 
Valparaiso 

C. THIRIOT 

University Pet M. Си rie 
Villefranche-sur -Меп France 
thiriot@obs-vlfrfr 

S. TILLIER 

Museum National d'Histoire Naturelle 

Paris, France 

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

N. H. VERDONK 

Rijksuniversiteit 
Utrecht, Netherlands 

H. WAGELE 

Ruhr-Universität Bochum 

Germany 

Heike. Waegele@ruhr-unl-bochum. de 

A. 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. Genvany 



BIVALVE STUDIES IN THE FLORIDA KEYS 

Proceedings of the International Marine Bivalve Workshop 
Long Key, Florida, July 2002 



GUEST EDITORS 



Rüdiger Bieler 



Department of Zoology (Invertebrates) 
Field Museum of Natural History, Chicago 



Paula M. Mikkelsen 

Division of Invertebrate Zoology 
American Museum of Natural History, New York 




MALACOLOGIA, 2004, 46(2): 241-248 



INTERNATIONAL MARINE BIVALVE WORKSHOP 2002: 
INTRODUCTION AND SUMMARY 

Paula M. Mikkelsen^ & Rüdiger Bieler^ 



In July 2002, a two-week workshop on ma- 
rine bivalves, with an emphasis on systemat- 
ics, anatomy, and natural history, was 
organized to further knowledge of living ma- 
rine bivalves and to train graduate-level stu- 
dents In this understudied field of modern 
malacology. With support from and in the spirit 
of the National Science Foundation's Partner- 
ships in Enhancing Expertise in Taxonomy 
(РЕЕТ) program, students worked one-on-one 
in teams with expert scientists on selected bi- 
valve species or groups of species. This vol- 
ume, for the most part comprising papers 
co-authored by the scientist-student research 
teams, represents the scientific results of 
projects initiated at the workshop. 



The Florida Keys at the southernmost tip of 
peninsular Florida, a region emphasized by the 
organizers' joint research program since 1994, 
formed a biologically diverse and logistically 
convenient site for a workshop of this type. As 
defined by this research venture, the Florida 
Keys includes the entire island chain and sur- 
rounding waters, from Broad Creek at the 
northern end of Key Largo (including Card and 
Barnes sounds, but not Biscayne Bay) through 
and including the Dry Tortugas, plus the ap- 
proximate southeastern half of Florida Bay (ex- 
cluding the more brackish areas in the outfall 
of the Florida Everglades), and offshore ar- 
eas to the reef line and beyond (with collec- 
tion and literature records to a maximum depth 




Atlantic 
Ocean 



Ulf of Mexico 



Florida Keys 





Big Pine Key 



655 
626-629, M^"' 
637 ж,**' 633 
k^^iH^ 634 



Key Vaca 



647, 657, 659 




620, 653 
660 1 



631 

♦ 



645 



622, 622A 4Í* ♦ 

фг' 4^56 640 

...,-'-'-""'623, \ 

636, Long Key 
652 



л,л 63Ô, ^ 

643, __oA »,>•' 

644 °3^2'^ 642, 646, 

Í* 646A, 6468 
♦ 
639 



♦ 
654 



♦ 
621 



♦ 
641, 648 



625 
624 ♦ 

♦ ♦ ♦ 

651 650 



FIG. 1. Field stations (black diamonds) sampled during the International Marine Bivalve Workshop, 
July 2002. Re-sampled or neighboring stations are here combined; see text for individual station 
data. Station FK-619 (Lake Surprise), about 48 km northeast, is not shown. West-to-east extent of 
the displayed area, ranging from Big Pine Key to Upper Matecumbe, is approximately 85 km. 



division of Invertebrate Zoology, American Museum of Natural History, Central Park West at 79th Street, New York, 

New York 10024-5192, U.S.A.; mlkkel@amnh.org 
^Department of Zoology, Division of Invertebrates, Field Museum of Natural History, 1400 S. Lake Shore Drive, Chicago, Illinois 

60605-2496, U.S.A.; bieler@fieldmuseum.org 



241 



242 



MIKKELSEN & BIELER 



of 300 m). Included within these limits is the 
Florida Keys National Marine Sanctuary, the 
second largest marine sanctuary in the United 
States, as well as a variety of other jurisdic- 
tions protecting vahous terrestrial and marine 
sites. The workshop was held at the Keys Ma- 
rine Laboratory on Long Key, a venue centrally 
located in the Keys allowing ready access to a 
large portion of the archipelago including off- 
shore and bayside habitats (Fig. 1). Easily ac- 
cessible habitats included intertidal rocks, sand 
and seagrass flats, rock ledges, seawalls, 
mangroves, mud channels, patch reefs, back 
reefs, artificial reefs (shipwrecks), and the only 
living near-shore coral reefs in the continental 
United States. 

Tourism is the leading industry of Florida and 
the Florida Keys has long been a favorite des- 
tination for ocean-oriented vacationing, diving, 
sport fishing, and shell collecting. In spite of a 
century of avid shell collecting and molluscan 
research in the Keys, Levy et al. (1996) noted, 
"except for a few ecological inventories that 
include mollusks, there is a lack of compre- 
hensive, ecosystem-wide species inventories 
in the Florida Keys." Coral reef conservation 
efforts stress corals, sponges, algae, spiny 
lobster and fish, and, except for a few mem- 
bers of the "charismatic macrofauna" [e.g., the 
queen conch, Sírombusg/gas Linnaeus, 1758, 
and Flamingo Tongue, Cyphoma gibbosum 
(Linnaeus, 1758)], regularly ignore mollusks. 
This deficiency has been acutely sensed since 
the establishment of the Florida Keys National 
Marine Sanctuary (FKNMS) in 1990, created 
to protect and restore fragile marine habitats 
from the environmental impact of human use. 
The FKNMS Draft Management Plan listed 
only 630 marine species (Lyons & Quinn, 
1995), only slightly fewer than a considerably 
earlier yet little-known, privately published list 
of 710 species by Lermond (1936). The mol- 
luscan species list compiled by the organiz- 
ers' research program has more than doubled 
to nearly 1,700 species through original field- 
work, museum collection surveys, and exten- 
sive literature research; approximately 400 of 
these species are bivalves (Mikkelsen & Bieler, 
2000; Bieler & Mikkelsen, 2004). 

Twelve international specialists in marine 
bivalve systematics participated by mentoring 
a student during the workshop. They included 
scientists with a wide range of specialties, 
spanning functional morphology, phylo- 
genetics, molecular biology, and faunal diver- 
sity research. A list of readily obtained Florida 



Keys bivalves was provided early in the plan- 
ning process, allowing each scientist to select 
and prepare materials to study a taxon in the 
field. The 1 2 student participants were selected 
from 49 applicants in response to notices dis- 
tributed at meetings and on internet listservers. 
Research teams were formed by pairing an 
alphabetical list of scientists with a reverse- 
alphabetical list of students. Together with the 
six members of the organizing team, this was 
a highly international group of 30 individuals 
representing 17 nations and five continents of 
origin or residence (Fig. 2). The participants 
were: 

Mr. Kyle Bennett, Rutgers University, New 
Brunswick, New Jersey, U.S.A. 

Dr. Rüdiger Bieler, Field Museum of Natural 
History, Chicago, Illinois, U.S.A. [organizer] 

Mr. Gregorio Bigatti, Universidad de Buenos 
Aires, Argentina. 

Mr. Matthew Campbell, Indiana University, 
Bloomington, Indiana, U.S.A. 

Mr. Anton Chichvarkhin, Rossiiskoi Akademii 
Nauk, Vladivostok, Russia. 

Ms. Louise Crowley, City University of New 
York and American Museum of Natural His- 
tory, New York, New York, U.S.A. [organiz- 
ing team]. 

Ms. Crete Dinesen, University of Aarhus, Den- 
mark. 

Dr. Osmar Domaneschi, Universidade de Sao 
Paulo, Brazil. 

Ms. Joanne Dougherty, Villanova University, 
Villanova, Pennsylvania, U.S.A. 

Dr. Emily Glover, The Natural History Museum, 
London, United Kingdom. 

Ms. Johanna Järnegren, Norges Teknisk- 
Naturvitenskapelige Universitet, Trondheim, 
Norway. 

Ms. Isabella Kappner, University of Illinois at 
Chicago and Field Museum of Natural History, 
Chicago, Illinois, U.S.A. [organizing team]. 

Ms. Lisa Kirkendale, Florida Museum of Natu- 
ral History, Gainesville, Florida, U.S.A. 

Ms. Martina Knapp, Universität Wien, Austria. 

Dr. José H. Leal, The Bailey-Matthews Shell 
Museum, Sanibel, Florida, U.S.A. 

Ms. Amy Maxmen, Harvard University, Cam- 
bridge, Massachusetts, U.S.A. 

Dr. Paula M. Mikkelsen, American Museum of 
Natural History, New York, New York, U.S.A. 
[organizer]. 

Dr. Russell Minton, Field Museum of Natural 
History, Chicago, Illinois, U.S.A. [organizing 
team]. 



IMBW 2002 - INTRODUCTION AND SUMMARY 



243 



Ms. Jurl A. Miyamae, Swarthmore College, 
Pennsylvania, and American Museum of 
Natural History, NSF-REU Summer intern 
Program, New York, New York, U.S.A. [or- 
ganizing team]. 

Prof. Brian Morton, Swire Institute of Marine 
Science, University of Hong Kong, China. 

Dr. Diarmaid Ó Foighil, Museum of Zoology, 
University of Michigan, Ann Arbor, Michigan, 
U.S.A. 

Dr. P. Graham Oliver, National Museum of 
Wales, Cardiff, United Kingdom. 

Ms. Melita Peharda, Institut za Oceanografiju 
i Ribarstvo, Split, Croatia. 

Dr. Jay Schneider, George Washington Uni- 
versity, Washington, DC, U.S.A. 

Ms. Elizabeth K. Shea, Bryn Mawr College, 
Bryn Mawr, Pennsylvania, U.S.A. 

Dr. Luiz Ricardo L. Simone, Museu de Zoología, 
Universidade de Sao Paulo, Brazil. 

Dr. Gerhard Steiner, Universität Wien, Austria. 

Prof. John Taylor, The Natural History Mu- 
seum, London, United Kingdom. 



Mr. Paul Valentich Scott, Santa Barbara Mu- 
seum of Natural History, Santa Barbara, 
California, U.S.A. 

Dr. Richard Willan, Northern Territory Museum 
of Arts & Sciences, Darwin, Australia. 

The workshop occupied nearly the entire 
Keys Marine Laboratory facility, including dor- 
mitories, wet laboratory for sorting, dry labo- 
ratory for microscope work and photography, 
classroom for presentations and discussion, 
and small boats for snorkeling trips. Various 
vehicles facilitated land travel for collecting and 
other group events; some scuba trips utilized 
commercial dive boats. Group and team ac- 
tivities included collecting by snorkeling, 
scuba, shovel-and-sieving (Fig. 3), and crack- 
ing dead coral rocks, followed by appropriate 
laboratory study (Fig. 4) and sharing their find- 
ings through discussions and presentations. 
Each research team included at least one cer- 
tified scuba diver and this allowed exploration 
of additional habitats, including an offshore 




FIG. 2. Participants of the IMBW gather for a group photograph at Pigeon Key (photograph by L. Simone). 



244 



MIKKELSEN & BIELER 




FIG. 3. Shovel-and-sieving typified collecting efforts for shallow-water bivalves in the Florida Keys. 




FIG. 4. Following a day's collecting efforts, the laboratory filled with a variety of study activities. 



IMBW 2002 - INTRODUCTION AND SUMMARY 



245 



wreck that had become a habitat for several 
deeper-water bivalve species. In deference to 
the sanctuary location, no dredging was con- 
ducted, and no protected species were col- 
lected; workshop activities were intentionally 
designed around relatively common, shallow- 
water species. Lectures were presented on 
most evenings, either by guest speakers or 
the participants. Each scientist spoke of his/ 
her research or laboratory, and toward the end 
of the workshop, each student summarized the 
results of their team investigations. 

The IMBW documented 121 species of 
bivalves from 48 field stations (Fig. 1), includ- 
ing several previously unrecognized taxa and 
others of poorly known distribution and habi- 
tat. Voucher specimens are deposited in the 
mollusk collections at AMNH, FMNH, and the 
home institutions of some participants (includ- 
ing Florida Museum of Natural History, 
Gainesville; Bailey-Matthews Shell Museum, 
Sanibel Island, Florida; Museum of Zoology, 
University of Michigan, Ann Arbor; Santa Bar- 
bara Museum of Natural History, California; The 
Natural History Museum, London, United King- 
dom; Museu de Zoología, Universidade de Sao 
Paulo, Brazil; and Northern Territory Museum 
of Arts & Sciences, Darwin, Australia). 



WORKSHOP STATIONS 

IMBW-FK-619, 14-VII-02, Lake Surprise, Key 
Largo, MM 107.5, NE end of U.S. Rte. 1 
causeway across lake, 25°10.9'N, 
80°23.0'W, off mangroves at side of road, 
by hand on shallow subtidal rocks. 

IMBW-FK-620, 16-& 18-VII-02, Old Dan Bank, 
bayside of Long Key, 24°50.45'N, 
80°49.63'W, Thalassia seagrass bed with 
Halimeda calcareous algae, Pontes finger- 
coral, sponges, hydroids, patches of sand/ 
Halimeda shell hash, by hand, 0.3-0.6 m, 
R/V FLORIDAYS. 

IMBW-FK-621, 17-VII-02, "Long Key Artificial 
Reefs", oceanside of Long Key, 24°44.78'N, 
80°50.00'W, sand plain with Thalassia/ 
Syringodeum seagrass patches, scuba, by 
hand, 7 m, RA/ FLORIDAYS. 

IMBW-FK-622, 20-VII-02, directly off Keys 
Marine Laboratory, bayside of Long Key, 
24°49.5'N, 80°48.9'W, seagrass bed with 
coral rubble, snorkeling, sieving, by hand, 
0-1.5 m. 

IMBW-FK-622A, 22-VII-02, off Keys Marine 
Laboratory, bayside of Long Key, 24°49.5'N, 



80°48.9'W, about 30 m from shore, thin sand 
over rock, with Halodule, Thalassia, Syringo- 
dium, Halimeda, shovel/sieving, 0.5-1 m. 

IMBW-FK-623, 20-VII-02, Long Key State 
Park, oceanside, 24°48.67'N, 80°49.68'W, 
seagrass bed (predominantly Thalassia) on 
muddy sand, snorkeling, by hand, 0-0.75 m. 

IMBW-FK-624, 20-VII-02, Horseshoe Reef, off 
Fat Deer Key, 24°39.91'N, 80°59.56'W, 
patch reef with sandy bottom, scuba, 7.3 m, 
MA/ SHUTTERBUG II. 

IMBW-FK-625, 20-VII-02, Coffins Patch Sanc- 
tuary Preservation Area, off Crawl Key, 
24°40.92' N, 80°58.26' W, patch reef with 
sand patches, gorgonian, pillar coral, scuba, 
6.4 m, MA/ SHUTTERBUG II. 

IMBW-FK-626, 21-VII-02, "The Horseshoe" 
site, bayside of West Summerland Key 
(Spanish Harbor Keys), MM 35, 24°39.3'N, 
81°18.2'W, "hole" at center of quarry, rock 
wall and soft sediment, snorkeling and 
scuba, to ca. 6.1 m. 

IMBW-FK-627, 21-VII-02, "The Horseshoe" 
site, bayside of West Summerland Key 
(Spanish Harbor Keys), MM 35, 24°39.3'N, 
81°18.2'W, mangrove area, soft sediment 
and detritus, hand dredge, < 1 m. 

IMBW-FK-628, 21-VII-02, "The Horseshoe" 
site, bayside of West Summerland Key 
(Spanish Harbor Keys), MM 35, 24°39.3'N, 
81°18.2'W, Thalassia seagrass, shovel/ 
sieve, ca. 1 m. 

IMBW-FK-629, 21- & 26-VII-02, "The Horse- 
shoe" site, bayside of West Summerland Key 
(Spanish Harbor Keys), MM 35, 24°39.3'N, 
81°18.2'W, among rocks along arms of 
quarry, by hand, snorkeling, to ca. 1 m. 

IMBW-FK-630, 22-VII-02, roadside quarry N 
of Keys Marine Laboratory, Long Key, 
24°49.78'N, 80°48.51'W, rock wall and 
scuzzy algae, hot (36°C) water layer, snor- 
keling, by hand, > 1 m to horizontal ledge on 
wall [total depth of quarry not assessed], S = 
31 ppt. 

IMBW-FK-631, 22-VII-02, Burnt Point, 
bayside, N point of Long Point Park, 
24°45.56' N, 80°59.14' W, rocky bottom, soft 
coral/sponges, patches of seagrass, snor- 
keling, 0.6-1.2 m [2.4 m in channel along 
shore]. 

IMBW-FK-632, 22-VII-02, Bahia Honda State 
Park, oceanside, just E of old bridge, 
24°39.25'N, 81°16.83'W, seagrass beds with 
sand blowholes, snorkeling at low tide, 0.6 m. 

IMBW-FK-633, 22-VII-02, Missouri Key, 
24°40.5'N, 81°14.3'W, coral rubble and 



246 



MIKKELSEN & BIELER 



seagrass beds, snorkeling and by hand, in- 
tertidal zone to 1 m. 

IMBW-FK-634, 22-VII-02, Bahia Honda State 
Park, oceanside, 24°39.69'N, 81°16.irW, at 
small roadway bridge over channel W before 
Sandspur Cannpground, sandy bottom at low 
tide, sparse seagrass, by hand, 0-0.1 m. 

IMBW-FK-635, 22-VII-02, Veteran's Beach, 
oceanside, Little Duck Key, 24°40.87'N, 
81°13.82'W, Thalassia/Halodule seagrass 
on silty sand, shovel/sieve, low intertidal 
zone to shallow subtidal. 

IMBW-FK-636, 22-VII-02, Long Key State 
Park, oceanside, 24°48.67'N, 80°49.68'W, 
sandy beach, seagrass, by hand, snorkel- 
ing, intertidal zone. 

IMBW-FK-637, 22-VII-02, "The Horseshoe" 
site, bayside of West Summerland Key 
(Spanish Harbor Keys), MM 35, 24°39.3'N, 
81°18.2'W, among rocks/rubble along arms 
of quarry, by hand, intertidal zone. 

IMBW-FK-638, 23-VII-02, Anne's Beach, 
oceanside, Craig Key, MM 72, 24°50.95'N, 
80°44.40'W, Thalassia/Halodule seagrass, 
shovel/sieve, by hand, 0.5-1 m. 

IMBW-FK-638A, 26-VII-02, Anne's Beach, 
oceanside, Craig Key, MM 72, 24°50.95'N, 
80°44.40'W, Thalassia/Halodule seagrass, 
shovel/sieve, by hand, 0.5-1 m. 

IMBW-FK-639, 23-VII-02, Coral Gardens in- 
shore patch reef, oceanside off Lower 
Matecumbe Key, 24°50.23'N, 80°43.77'W, 
snorkeling, 3.6-4.6 m, Keys Marine Labora- 
tory boat. 

IMBW-FK-640, 23-VII-02, oceanside off Craig 
Key, 24°49.81'N, 80°45.73'W, nearshore 
patch reef, hardbottom, snorkeling, 0.1-1.2 
m, Keys Marine Laboratory boat. 

IMBW-FK-641 , 23-VII-02, Tennessee Reef, off 
Long Key, 24°44.75'N, 80°46.95'W, hard 
bottom with coral, scuba, 7 m, R/V 
FLORIDAYS. 

IMBW-FK-642, 23-VII-02, "The Billboard" site, 
oceanside, Lower Matecumbe Key, MM 
74.5, 24°51.4'N, 80°43.7'W, thin sand cover 
on rock platform, small coral, Thalassia/ 
Halodule seagrass, Sargassum, wading, 
snorkeling, shovel/sieving, 0.5-1 m. 

IMBW-FK-643, 23-VII-02, Fiesta Key cause- 
way, oceanside, 24°50.4ГМ, 80°46.95'W, at 
turnoff W of Channel #5 bridge, rocky shore, 
sand, Thalassia seagrass, snorkeling, by 
hand, 0-3 m. 

IMBW-FK-644, 23-VII-02, Fiesta Key cause- 
way, bayside, 24°50.41'N, 80°46.95'W, at 
turnoff W of Channel #5 bridge, rocky shore, 
concrete pilings, snorkeling, by hand, 0-3 m. 



IMBW-FK-645, 24-VII-02, Grassy Key, 
oceanside, 24°46.60'N, 80°55.44'W, at turn- 
off before Tom's Harbor Channel, attached 
to underside of large, algal-encrusted boul- 
ders/rocks along shore, by hand, snorkeling, 
1-3 m. 

IMBW-FK-646, 25-VII-02, "The Billboard" site, 
oceanside. Lower Matecumbe Key, MM 
74.5, 24°51 .4'N, 80°43.7'W, rubble and sand 
with seagrass, wading, shovel/sieving, 0.5- 
0.75 m. 

IMBW-FK-646A, 24-VII-02, "The Billboard" site, 
oceanside. Lower Matecumbe Key, MM 74.5, 
24°51 .4'N, 80°43.7'W, thin sand on rock plat- 
form, with Thalassia, Halodule, Halimeda, 
Penicillus, shovel/sieving, 0.5-1 m. 

IMBW-FK-646B, 27-VII-02, "The Billboard" site, 
oceanside. Lower Matecumbe Key, MM 74.5, 
24°51 .4'N, 80°43.7'W, thin sand on rock plat- 
form, with Thalassia, Halodule, Halimeda, 
Penicillus, shovel/sieving, 0.5-1 m. 

IMBW-FK-647, 25-VII-02, W side of Pigeon 
Key, 24°42.2'N, 81°09.3'W, Thalassia/ 
Halodule/Syringodeum seagrass on sand/ 
rubble, concrete bridge piers, by hand, snor- 
keling, shovel/sieving, 0.5-1 m. 

IMBW-FK-648, 26-VII-02, Tennessee Reef 
Light, off Long Key, 24°44.75'N, 80°46.95'W, 
patch reef, sand, rubble, scuba, 4-7 m, RA/ 
FLORIDAYS. 

IMBW-FK-649, 27-VII-02, Sprigger Bank, 
bayside, just W of Everglades National Park 
border, 24°54.75'N, 80°56.24'W, Thalassia/ 
Syringodeum seagrass, snorkeling, shovel/ 
sieving, 0.1-0.9 m. Keys Marine Laboratory 
boat. 

IMBW-FK-650, 27-VÍI-02, wreck of "Thunder- 
bolt", approx. 6 nmi S of Marathon, 24°39.68'N, 
80°57.82'W, steel wreck with fouling 
bivalves, alcyonarians and hydroids, orange/ 
red sponge overcoating most specimens, 
scuba, 34.1 m, MA/ SHUTTERBUG II. 

IMBW-FK-651, 27-VII-02, "Samantha's patch 
reef", approx. 5 nmi S of Marathon, 
24°39.49'N, 81°00.32'W, coral rock inter- 
spersed with sandy channels, scuba, 7.6 m, 
MA/ SHUTTERBUG II. 

IMBW-FK-652, 27-VII-02, Long Key State 
Park, oceanside, 24°48.67'N, 80°49.68'W, 
seagrass bed (predominantly Thalassia) on 
muddy sand, wading, shovel/sieving, less 
than 1 m. 

IMBW-FK-653, 27-VII-02, Old Dan Bank, 
bayside of Long Key, 24°50.45'N, 
80°49.63'W, Thalassia seagrass bed with 
Halimeda calcareous algae, Pontes finger- 
coral, sponges, hydroids, patches of sand/ 



IMBW 2002 - INTRODUCTION AND SUMMARY 



247 



Halimeda shell hash, snorkeling, 0.3-1.5 m, 
R/V LAST MANGO. 

IMBW-FK-654, 28-VII-02, East Turtle Shoal, 
oceanside off Grassy Key, 24°43.49'N, 
80°56.00'W, at Marker "45" in Hawk Chan- 
nel, silty patch reef, scuba, 7.5 m, R/V 
FLORIDAYS. 

IMBW-FK-655, 28-VII-02, Veteran's Beach, 
oceanside. Little Duck Key, 24°40.87'N, 
81°13.82'W, Thalassia/Halodule seagrass 
on silty sand, shovel/sieve, low intertidal 
zone to shallow subtidal. 

IMBW-FK-656, 28-VII-02, mangrove channel 
near Goshen House, South Layton Drive, 
Layton, Long Key, 24°49.40'N, 80°48.77'W, 
red mangrove roots, snorkeling, 0.6-1.2 m. 

IMBW-FK-657, 28-VII-02, Pigeon Key, 
24°42.2'N, 81°09.3'W, seagrass, sand, 
rubble, by hand, wading, 0-0.5 m. 

IMBW-FK-658, 26-VII-02, E end of Big Pine 
Key, Spanish Harbor Channel, 24°38.89'N, 
81°19.80'W, pier/pilings, algae-covered 
rocks, snorkeling, hammer/chisel, 0-2 m. 

IMBW-FK-659, 28-VII-02, Pigeon Key, 
24°42.2'N, 81°09.3'W, seagrass, scuba, 0.6- 
1.2 m. 

IMBW-FK-660, 28-VII-02, Old Dan Bank, 
bayside of Long Key, 24°50.08'N, 
80°49.63'W, Thalassia seagrass bed with 
Halimeda calcareous algae, Pontes finger- 
coral, sponges, hydroids, patches of sand/ 
Halimeda shell hash, snorkeling, 0.3-1.5 m, 
RA/ LAST MANGO. 

The contributions to this proceedings vol- 
ume reflect the specialized interests of the par- 
ticipants. The projects were initiated at the 
12-day workshop but required substantial fol- 
low-up between scientist and student, often 
communicating and even visiting across con- 
tinents or oceans. The majority of the studies 
focused on detailed investigations of the com- 
parative and functional anatomy/morphology 
of exemplar species in the families Arcidae, 
Donacidae, Psammobiidae, Pteriidae, and 
Veneridae. Others took a somewhat broader 
taxonomic approach and developed regional 
systematic studies based on morphology and/ 
or molecules. These resulted in reviews of 
Florida Keys oysters (Gryphaeidae and 
Ostreidae), boring bivalves (Gastrochaenidae, 
Mytilidae, and Petricolidae), and western At- 
lantic Chamidae. Again other teams, using 
different experimental setups and analytical 
approaches, studied predator-prey interac- 
tions between naticid gastropods and venerid 



bivalves, or made fine- and ultrastructural in- 
vestigations into aspects of periostracal mor- 
phology, and oocyte and sperm development 
in the family Lucinidae. Two additional papers 
on the entire bivalve fauna of the region, com- 
piled from the organizers' long-term research 
program, provide a broader look at the diver- 
sity of the Florida Keys bivalve fauna. 



ACKNOWLEDGMENTS 

Major funding for this workshop was pro- 
vided by the National Science Foundation 
РЕЕТ program (DEB-9978119), as part of a 
grant on marine bivalves to RB and PMM. 
Additional support was provided by the Ber- 
tha LeBus Charitable Trust, Comer Science 
& Education Foundation, Field Museum's 
Women's Board, as well as other institutional 
funds from AMNH and FMNH. José Leal gen- 
erously provided supplemental transportation 
through use of the Bailey-Matthews Shell 
Museum vehicle. Collections were supported 
by permits from the Florida Keys National 
Marine Sanctuary (educational event permit 
FKNMS-2002-079), the State of Florida (in- 
dividual saltwater fishing licenses to all par- 
ticipants), Florida Department of 
Environmental Protection (FDEP 5-02-43; for 
Long Key and Bahia Honda State Parks), and 
Pigeon Key Foundation. The organizers 
thank members of the organizing team as 
listed above, as well as Julia Sigwart 
(AMNH), for their assistance with numerous 
aspects of planning and running the work- 
shop. Special thanks are extended to three 
internationally recognized bivalve specialists, 
Drs. John Allen (University Marine Biologi- 
cal Station Millport, Scotland), Kenneth J. 
Boss (Museum of Comparative Zoology, 
Harvard University), and Eugene V. Coan 
(Palo Alto, California), who assisted in re- 
viewing the student applicants' qualifications 
and choosing the awardees. Thanks are also 
due to the numerous colleagues who cri- 
tiqued and improved the manuscripts through 
rigorous peer review, and to George M. Davis 
and Eugene V. Coan who coordinated re- 
views of the editors' own papers. Workshop 
activities were facilitated and enhanced by 
Billy Causey (Superintendent, Florida Keys 
National Marine Sanctuary), Dr. Erich Mueller 
(Mote Marine Laboratory, Center for Tropi- 
cal Research) and the staff of Keys Marine 
Laboratory. 



248 



MIKKELSEN & BIELER 



LITERATURE CITED 

BIELER, R. & P. M. MIKKELSEN, 2004, Marine 
bivalves of the Florida Keys; a qualitative fau- 
nal analysis based on original collections, mu- 
seum holdings and literature data. In: R. bieler 
& p. M. MIKKELSEN, eds.. Bivalve Studies in the 
Florida Keys, Proceedings of the International 
Marine Bivalve Workshop, Long Key, Florida, 
July 2002. Malacologia, 46(2): 503-544. 

LERMOND, N. W., 1936, Check list of Florida 
marine shells. Privately published, Gulfport, 
Florida. 56 pp. 

LEVY, J. M., M. CHIAPPONE & K. M. SULLIVAN, 
1996, Invertebrate infauna and epifauna of the 
Florida Keys and Florida Bay. Site character- 
ization for the Florida Keys National Marine 
Sanctuary and environs, vol. 5: 1-166, The Na- 
ture Conservancy, Florida and Caribbean Ma- 



rine Conservation Science Center, University of 
Miami & The Preserver, Zenda, Wisconsin. 

LYONS, W. G. & J. F. QUINN, Jr., 1995, Appen- 
dix J. Marine and terrestrial species and algae: 
Phylum Mollusca. J-10 - J-26, in: Florida Keys 
National Marine Sanctuary Draft Management 
Plan/Environmental Impact Statement, Vol. III. 
United States Government Printing Office, 
Washington, D.C. 

MIKKELSEN, R M. & R. BIELER, 2000, Marine 
bivalves of the Florida Keys: discovered 
biodiversity. Pp. 367-387, in: The evolution- 
ary biology of the Bivalvia [Proceedings of Bi- 
ology & Evolution of the Bivalvia, an 
international symposium organized by the 
Malacological Society of London, 14-17 Sep- 
tember 1999, Cambridge, UK], E. M. harper, 
J. D. TAYLOR & J. A. CRAME, eds. Geological So- 
ciety, London, Special Publication 177. 



MALACOLOGIA, 2004, 46(2): 249-275 

SHELL MORPHOMETRY OF WESTERN ATLANTIC AND INDO-WEST PACIFIC 
ASAPHIS; FUNCTIONAL MORPHOLOGY AND ECOLOGICAL ASPECTS OF 
A. DEFLORATA FROM FLORIDA KEYS, U.S.A. (BIVALVIA: PSAMMOBIIDAE) 

Osmar Domaneschi^ & Elizabeth K. Shea^ 

ABSTRACT 

The genus Asaphis has long been considered monotypic, with A. deflorata having a 
worldwide, tropical distribution. Recent research has provided evidence for a tropical, 
western Atlantic species, A. deflorata, and a tropical Indo-West Pacific species, A. violascens. 
Ecological and other aspects of the biology of these species have been studied exten- 
sively but morpho-functional features have been known for the Indo-West Pacific species 
only, so that separation of them has been based on shell sculpture alone. This paper 
examines the shell morphometry of western Atlantic and Indo-West Pacific specimens of 
Asaphis, and the functional morphology and ecological aspects of a population of the 
genus present in the Florida Keys, USA. It is our aim to improve l<nowledge about the 
biology of the western Atlantic Asaphis and identify new characters that may support either 
their monotypy or the two valid species hypothesis. In addition to confirming that shell 
sculpture may be a good character in distinguishing both forms, our ecological and mor- 
pho-functional data also concur in validating A. deflorata as a distinct species. Growth 
rates, maturity levels and predominantly upper shore intertidal position of the Keys popu- 
lation are consistent with a previous study of the Bahamas population of Л. deflorata. In 
both studied areas, A. deflorata constitutes the sole bivalve present in the upper shore; 
conversely, most specimens of the Indo-West Pacific A. violascens occupy an intermedi- 
ate to subtidal position, and share the intertidal region horizontally and vertically with other 
species of bivalves. The functional anatomy of A. deflorata is very similar to that of A. 
violascens; however, the hind gut provides a useful parameter for separation of both spe- 
cies, as it progressively widens, coils and spirals in the Atlantic form, whereas it has an 
extraordinary dilation in its proximal end only, in the Indo-West Pacific A. violascens. 

Key words: functional morphology, shell morphometry, ecology, Asaphis, Psammobiidae, 
Bivalvia. 



INTRODUCTION 



Bivalves of the genus /\sap/7/s Modeer, 1793, 
are commonly found intertidally in gravelly sand, 
cobble-covered sediments (Depledge, 1985; 
Britten, 1985; Berg & Alatalo, 1985) or around 
mangrove roots (Coomans, 1969; Stanley, 
1970; Berg & Alatalo, 1985). Populations at- 
tain sufficiently high densities to support sus- 
tained collection for human consumption 
(Fisher, 1978; Berg & Alatalo, 1985; Wlllan, 
1993), and their viability as an aquaculture re- 
source has been investigated (Berg & Alatalo, 
1 981 , 1 985). The natural variability in shell color 
gives the Atlantic Asaphis its common name, 



gaudy asaphis, and a place in shellcraft indus- 
try (Abbott, 1974; Berg & Alatalo, 1985). 

Prashad (1932) considered the Indo-Pacific 
specimens of Asaphis as belonging to A. 
dichotonria (Anton, 1838), and distinct from 
those living in the Western Atlantic assigned 
to A. deflorata (Linné, 1758). Abbott (1950) 
considered both Indo-Pacific and western At- 
lantic forms to be conspecific, and proposed 
that the genus is monotypic, with A. deflorata 
having a worldwide, tropical distribution. This 
hypothesis was codified in Abbott (1974), but 
more recent research (Willan, 1993) provides 
evidence for a tropical western Atlantic spe- 
cies, A. deflorata, and a tropical Indo-West 
Pacific species, Л. wo/ascens (Forsskâl, 1775). 



departamento de Zoología, Instituto de Biociências, Universidade de Sao Paulo, PO Box 11461, CEP 05422-970, Sao 
Paulo (SP), Brazil; domanesc@ib.usp.br 
^Department of Biology, Bryn Mawr College, Bryn Mawr, Pennsylvania, U.S.A.; eshea@brynmawr.edu 



249 



250 



DOMANESCHI & SHEA 



The Indo-Pacific Asaphis was generally 
known as A. dichotoma until the early 1970s 
(Willan, 1993), when Cernohorsky (1972) al- 
tered Its name, without explanation, to A. 
violascens. Willan (1993) has vindicated such 
an alteration. In this paper, Willan observed 
that the separation between A. deflorata and 
A. violascens was based on shell sculpture 
alone. Actually, there have been no compre- 
hensive studies to date on the functional mor- 
phology of the Atlantic Asaphis that allow 
comparison with that performed by Purchon 
(1960) on the stomach, and by Narchi (1980) 
on the functional anatomy of the Indo-Pacific 
species. Specimens of >4sap/7/'sfrom the Indo- 
Pacific Ocean (Singapore?), were sent by 
Purchon to R. Tucker Abbott, Pennsylvania, 
USA, who identified them as A. deflorata 
(Purchon, 1960). 

Ecological and biological data on Asaphis 
spp. have been provided by Stanley (1970), 
Narchi (1980), Britton (1985), Depledge 
(1985), Berg &Alatalo (1985), Soemodihardjo 
& Matsukuma (1989), Willan (1993), and 
Kurihara et al. (2000, 2001). 

This paper examines the shell morphometry 
of the two Asaphis spp. sensu Willan (1993), 
and the functional morphology and ecological 
aspects of Asaphis deflorata from the Florida 
Keys, USA. It is also the aim of this study to 
identify characters that may support Abbott's 
(1950) or Willan's (1993) proposal. 



MATERIALS AND METHODS 

Survey Site 

Asaphis deflorata was collected during the 
International Marine Bivalve Workshop 
(IMBW) held in the Florida Keys, USA, 19-30 
July 2002 from Station IMBW-FK-629, "The 
Horseshoe" site, bayside of West Summerland 
Key (Spanish Harbor Keys), MM 35, Monroe 
County, Florida Keys 24°39.3'N, 8ri8.2'W. 
Mikkelsen & Bieler (2004) provide a listing of 
all stations and a map of the studied area. 
Collections were made in accordance with 
permit requirements of the State of Florida, 
under a Research/Collecting Permit issued by 
the U. S. Department of Commerce, National 
Oceanic and Atmospheric Administration, Na- 
tional Ocean Service to Drs. Paula Mikkelsen 
and Rüdiger Bieler (Permit FKNMS 2002-079). 
Individual Florida Saltwater Fishing Licenses 



(FSFL) numbers were "M-N1I2Y01 8675" (OD) 
and "M-N1A79018604" (EKS). 

Field Survey 

Sampling was performed at low tide when 
the clams' intertidal habitat was fully exposed; 
waters receded ~ 4 m from the high tide mark 
during the sampling period. Specimens were 
collected along a 4 m transect, from four 0.25 
m^ quadrats. The first quadrat was placed at 
the low water mark, and three additional 
samples were taken at 0.5 m intervals moving 
toward the high tide mark. The quadrat area 
was excavated to 10-12 cm, the maximum 
depth allowed by the local rocky gravel sub- 
stratum along the transect. All sediments were 
sieved through two sieves, with mesh open- 
ings of 10.0 mm and 0.4 mm, respectively. 

Behavior 

Behavior of /\. deflorata was observed in the 
Keys Marine Laboratory on Long Key, and tak- 
ing photographs in the field and snorkeling dur- 
ing high tide. Results were compared with those 
obtained by Stanley (1970) and Berg & Alatalo 
(1985) observing specimens both in laboratory 
and in field. Laboratory observations included 
analyses of the burrowing period (Stanley, 1970) 
of seven individuals each of 1.0, 1.1, 1.3, 1.8, 
2.5, 4.5, and 5.7 cm shell length, lying either on 
the right or left shell valve on a coarse sand 
substratum free from natural obstacles as 
pebbles, shells and rubbles. The ability of the 
species to surmount sediment deposits through 
maximum extension of the siphons was evalu- 
ated by firmly trapping ten specimens (shell 
length range: 2.5-5.7 cm) posterior end up 
among pebbles on the bottom of small aquaria. 
This procedure simulates the condition in which 
several specimens were found in nature: 
wedged and trapped both in crevices and 
among pebbles within the sediment. A new 1-2 
cm-thick layer of coarse sand was added every 
time the siphons tip reached sediment surface, 
simulating catastrophic burial by sand as it is 
known to happen in nature (Stanley, 1970; Berg 
& Alatalo, 1985). This procedure was repeated 
till the specimens failed to reach the water col- 
umn. Extrusion of both siphons from the sub- 
stratum and the ability of the inhalant to take 
either suspended or deposited material in were 
observed in ~ 30 specimens kept buried for eight 
days in clean, coarse sand in aquaria. 



MORPHOLOGY AND SHELL MORPHOMETRY OF ASAPHIS 



251 



Morphometries 

To evaluate if growth data are taxonomically 
Important In distinguishing the Atlantic A. 
deflorata from Indo-West Pacific A. 
violascens, shell length, height and width of 
complete shells from ten different localities 
in the Indo-West Pacific, and deposited at the 
Delaware Museum of Natural History - 
DMNH, USA (n = 23; shell length range (sir): 
28.5-74 mm), and of our material (n = 32; 
sir: 20.6-59.2 mm) were measured to 0. 1 mm 
with dial calipers, and recorded in an Excel 
spreadsheet. Such growth data were mod- 
eled using a Model II regression analysis In 
Systat V. 5.2.1 for Macintosh. To accomplish 
this, the loss function was changed from the 
ordinary least squares regression equation 
to: LOSS = (Y - (BO + B1*X))'^2/ABS (B1), 
where Y and X are the dependent and Inde- 
pendent variables, and BO and B1 are the two 
parameters to be estimated. 

To evaluate if radial ribs are taxonomically 
Important, ribs were counted in the best-pre- 
served shell valves toward the umbo and at 
the valve margin of the Atlantic Asaphis (n = 
10; shell length range: 35-59.2 mm) (our ma- 
terial) and the Indo-West Pacific Asaphis (n = 
10; sir: 41-56.5 mm) (the Delaware Museum 
of Natural History - DMNH, USA collection). 
The degree of branching was recorded as a 
ratio of margin/umbo ribs, where a larger num- 
ber Indicates more branching. 

Principal components and cluster analyses 
(McCune & Mefford, 1999) were used to as- 
sess the degree of similarity or difference be- 
tween the Atlantic and the I ndo-Paclfic Л sap/7/s 
spp. Six shell characters were Included In the 
analysis: shell length, height, width, number 
of ribs at margin, number of ribs at umbo, and 
the ratio of margln/umbo ribs. T-tests were 
used to assess significance In character dif- 
ferences. 

Museum Collections 

Asaphis violascens. Delaware Museum of 
Natural History, USA: Solomons Island, lots 
185776 (1 v., 2 spec.) and 129978 (1 v., 1 
spec); Fiji Islands, lots 185057 (1 spec), 
211943 (2 v.), 205760 (2 spec.) and 166675 
(2 spec); Japan, lots 185777 (2 spec), 1 09642 
(1 spec); Western Australia, lot 175710 (3 
spec); Guam Island, lot 174675 (1 spec); 



Philippines, lots 152564 (1 v.), 122836 (1 v.), 
122499 (1 V.) and 194390 (1 spec); Ambon 
Island, lot 168461 (4 spec); Palau, lot 211968 
(1 v., 1 spec); Northern Australia, lot 181892 
(1 spec); Malaysia, lot 201332 (1 spec); 
Singapore, lot 21 545 (2 v.); Moorea Island, lot 
152461 (1 v.). Instituto de Biociências, 
Universidade de Sao Paulo (IBUSP), Brazil: 
four whole specimens and fourteen single shell 
valves from Hong Kong, China, not numbered, 
on which Narchi (1980) based part of his work 
on A. violascens; material qualitatively ana- 
lyzed for shell features. 

The following museum molluscan database 
were examined In mid-January 2003 to assess 
the historical and recent distribution of A. 
deflorata: the Academy of Natural Sciences 
of Philadelphia (ANSP), National Museum of 
Natural History (USNM), Florida Museum of 
Natural History (FLMNH), the University of 
Miami Rosentiel School of Marine and Atmo- 
spheric Sciences (RSMAS), and the Florida 
Marine Research Institute (FMRI). 

Specimen identifications in the museum col- 
lections were assumed to be correct and were 
not verified by the authors; however, speci- 
men identifications of the RSMAS collection 
were verified by P. M. Mikkelsen, to whom we 
are grateful, those of the DMNH collection by 
E. K. Shea, and of the Instituto de Biociências, 
Universidade de Sao Paulo by О. Domaneschl. 

Anatomy 

Live specimens (shell length range: 6.9-59.2 
mm) were dissected and examined for the 
presence of developed gonads and eggs or 
sperm using a compound microscope. Speci- 
mens were recorded as immature when no 
gonad tissue could be located; mature males 
and females were identified when sperm or 
eggs were present. 

Studies of the anatomical features and draw- 
ings were made based on living and relaxed 
and preserved specimens. Magnesium sulfate 
and refrigeration were used as relaxing agents. 
Ciliary currents of feeding and cleansing were 
observed in live specimens using both colloi- 
dal graphite and carmine powder suspensions, 
carborundum grade F3 and graded sand par- 
ticles. Complete serial histological sections (4 
to 8 pm thick) were taken from a specimen 
1 .5 cm in shell length fixed In Bouin acetic and 
stained with Ehrlich's haematoxylin and eosin. 



252 



DOMANESCHI & SHEA 



RESULTS 



Ecology 



At the "The Horseshoe" site, A. deflorate is 
restricted to the intertidal zone, in patches of 
gravelly coarse sand covered with pebbles and 
rubble on a coral ground. The beach slope of 
the sampled area is slightly larger than 10° 
and waters were calm and receded 
approximately 4 m from the high tide mark 
during the studied period. 

Live, sparse unburied specimens were found 
lying by the high tide mark; buried specimens 
occurred till a maximum depth of 10-12 cm, 
the latter determined by the rocky, impenetrable 
substratum underneath. Crevices and restricted 
spaces among pebbles within the substratum 
were usually occupied by individuals. Depths 
of 5 to 1 5 cm (Berg & Alatalo, 1 985) and deeper 
(Stanley, 1970) have been registered for the 
species. Our experiments showed the species 
can extend their siphons as long as 1 .5 times 
the shell length. This allows us to predict that 
the largest specimens (~ 8 cm in shell length - 
Stanley, 1970; Berg & Alatalo, 1985) burrow as 
deep as ~ 20 cm. In pockets of sand crowded 



with Asaphis, larger and smaller specimens 
were intermingled indifferently, occupying the 
substratum without horizontal or vertical 
segregation according to size. Their normal life 
position was with the posterior end up and the 
longitudinal axis at an angle of between 10° to 
30° from the vertical. This angle increased to 
90° in some specimens buried shallowly both 
in crowded or shallow pockets of sand. This 
supports Stanley's (1970) statements that life 
position and burial depth of /A. deflorate in nature 
are in part controlled by boundary effects. 

Along the transect, each station (quadrat) 
sampled had a different sediment composition, 
and a correspondingly different population 
density and structure (Fig.1). Station 1 was 
composed of rubbles and pebbles and no living 
specimen was present (n = 0). Station 2 was 
composed of pebble-covered, gravelly silt; a 
few (n = 27) living specimens were present, 
intermingled among a larger number (not 
recorded) of buried, recently dead shells 
retained in their life position. Station 3 was 
composed of gravelly coarse sand with silt; in 
spite of being dominated by one extremely large 
piece of coral rubble, it yielded the largest 
number of specimens (n = 238). Station 4 was 




^ <5' 



AX.f.*y^\.^ 



1^ Ф 



é' 



^ ^^ tí? <f> <D? 

Size (mm) 

П station 1 П Station 2 «Station 3 ■ Station 4 



FIG. 1. Asaphis deflorata. Size distribution of live specimens collected at 
Stations (St.) 1-4 in July 2002, from the "The Horseshoe" site population, 
Florida Keys, USA. Total number of specimens by station: St. 1 (n = 0); St. 
2 (n = 27); St. 3 (n = 238); St. 4 (n = 137). 



MORPHOLOGY AND SHELL MORPHOMETRY OF ASAPHIS 



253 



composed of gravelly coarse sand and had a 
smaller yield (n = 134). Dead shells were 
scarce In stations 3 and 4. Over 93% of the 
399 specimens collected came from stations 
3 and 4, the nearest to the high tide mark and 
regularly exposed during ebb tides. 

Morphometries 

Shell length ranged between 6.9-59.2 mm 
(n = 399). Almost 70% of the specimens 
collected were 30-50 mm in shell length; 
approximately 25% of the population was 
between 5-30 mm, and only 2.5% was > 50 
mm. Based on the measurements of living and 



dead collected specimens, the height and 
width of A. deflorata grow gradually without 
obvious interruption or change in the growth 
trajectory over the size range of 6.9-59.2 mm 
(Fig. 2A). Overall, shell length of /\. deflorata 
is 1.54 times the height, and 2.26 times the 
width. 

Parameters of the growth equations that 
describe A. violascens (DMNH collection) fall 
within the 95% confidence intervals for A. 
deflorata, reflecting the overall similarity in 
ontogenetic trajectories (Fig. 2B); overall shell 
ratios are also similar, with the shell length of 
A. violascens 1.44 times the height and 2.22 
times the width. 



50 



^40 
^ 30 

0) 

E 

i 20^ 

(Л 

ш 

(U 

s 10 



15 



í = 0.640x + 1.039 




25 



35 45 

Length (mm) 



55 



65 



о height (mm) ■ width (mm) 



60 



___^^ 


50 


E 




E, 


40 






с 




(1) 




F 


30 


(i> 








3 
CO 


20 


ra 




(U 




S 


10 



В 




20 30 40 50 60 

Length (mm) 

о height (mm) • width (mm) 



70 



80 



FIG. 2. Model II regression analysis of height and width vs. length 
for: A, Asaphis deflorata from "The Horseshoe" site population, 
Florida Keys, and B, A. violascens at Delaware Museum of Natural 
History, U.S.A. Although the regression equations are different for 
each variable, the slope and intercept are within the 95% confidence 
intervals of each other, and thus the growth trajectories are 
essentially the same. 



254 



DOMANESCHI & SHEA 



TABLE 1. Comparison of observed rib counts (our data - D & S) made on well-pre- 
served shells oí Asaphis deflorata (n = 10; sir: 35-59.2 mm) from the "The Horseshoe" 
site population, Florida Keys, USA and of Л. violascens (n = 10; sir: 41-56.5 mm) in 
the collection at the Delaware Museum of Natural History, USA, and rib counts pre- 
dicted for both species by Willan (1993 - W); n, total number; SD, standard deviation; 
sir, shell length range; x, average. 



Shell character 



Asaphis deflorata 
range (x±SD) 



Asaphis violascens 
range (x ± SD) 



Rib number (1Л0 

Rib number - at umbo (D & S) 

Rib number - at margin (D & S) 

Rib branching (W) 

Rib branching index (D & S) 



60-90 

48-80 (56.3 ± 10.9) 

80-102 (92.8 ±7.9) 

less frequent 

1.35-2.58 (1.70 ±0.38) 



40-60 
23-80 (32.3 ±17.0) 
52-100 (68.4 ±12.9) 

more frequent 
1.25-2.91 (2.31 ±0.5) 



Rib counts both at the umbo and at margin of 
A. deflorata from the "The Horseshoe" site and 
of /A. violascens (DMNH collection) have a wider 
range than predicted for both species by Willan 
(1 993), and the ranges overlap (Table 1 ). In spite 
of this, principal components analysis (PCA) 
(Fig. 3) shows that two groups are consistently 
found: one that contains /A. deflorata specimens 
with a few A. violascens specimens, and one 



that is solely composed of A. violascens. It is 
also evident that A. violascens generally has 
fewer, more branching, ribs than A. deflorata, 
and these differences are significant at P = 0.05 
(t-test: ribs near umbo P = 0.002; ribs at margin 
P = 0.000; branching ratio P = 0.018). Cluster 
analysis (Fig. 4) shows that all A. deflorata 
specimens are clustered in a group that shares 
< 25% of the information with the majority of A. 



3- 



2- 



о 

■Û 1, 

с 
■? 



-1- 



CM 
(0 

'x 

(0 

< 
Ü 

Û. .2 















А. deflorata 






Fij 


4 W. Aus. 






♦ А. violascens 






Palau4 ^'^'''♦Fiji 
Sol.ls>^ N.Aus. ^ 

Malaysian ''' ♦ Japan 
















Japan Ф 


FL 

О 


FL 

О 




О "=•- 

FL О 
FL О О о 
Guam 

Japan 

♦ 

О FL 




Philip. 40 FL 
SoLls. ♦ 




■ 




1 


■ 




1 





2 

PCA axis 1 (size) 



FIG. 3. First two axes of the principal components analysis account for > 85% of the overall variance. 
Size (length, width and height) increases from left to right along axis 1. Umbo and margin rib counts 
decrease from bottom to top along axis 2, whereas the branching ratio increases from bottom to top. 



MORPHOLOGY AND SHELL MORPHOMETRY OF ASAPHIS 



255 



FL 
FL 



100 



75 



Information Remaining (%) 
50 



25 



FL о 

FL о 

FL О 

FL О 

FL О 

Guam •- 

FL o- 

Japan Фх 

FL o^ 

FL o- 

Sol. is. •- 

Philip. •- 

Japan •- 

W.Aus. •- 

Fiji •- 

Palau • 
Sol. Is. 
N. Aus. 



^ 



О А. deflorata 
ФА. violascens 



Fiji •-! , 

Fiji •-■ L 

Fiji •-! Г 

Japan •— ' 



Jap 
Malaysia«- 



FIG. 4. Cluster diagram (Jaccard distance measure, group average linkage) showing the overall 
similarities between each specimen in length, height, width, number of ribs at umbo, number of ribs 
at margin, and branching ratio. Branch points at indicate no relationship between variables; branch 
points at 100% mean the specimens were virtually identical overall. PL, Florida, USA; Sol. Is., Solomons 
Island; Philip., Philippines; W. Aus., Western Australia; N. Aus., Northern Australia. 



violascens; however, two anomalous specimens 
of A. violascens cluster within the A. deflorata 
branch. Thus, there is no relationship between 
the two major groups, even though PGA analysis 
shows that several A. violascens group within 
that of /A. deflorata. Regardless of these outliers, 
t-tests show that the number of ribs at the umbo 
and at margin, and the degree of branching 
distinguish the species and that size 
measurements do not. Other useful shell 
characters in distinguishing A. deflorata from A. 
violascens are: the presence of a discernible, 
rounded posterior radial ridge and posterior 
slope in the Atlantic Asaphis (our material), not 
discernible (Willan, 1993; IBUSP collection) in 
the Indo-West Pacific specimens; a smooth 
inner surface in the Atlantic /\sap/?/s, contrasting 
with the well-marked, ridged inner surface in 
specimens from Hong Kong (IBUSP collection). 

Sexual Maturity 

In the sampled population, all specimens < 
24 mm in shell length were immature, and all 
specimens > 32 mm had recognizable eggs 
or sperm in the gonad tissue. The gonads of 
most specimens > 24 mm in shell length were 



identifiable as male or female; just two 
specimens of 26 mm and 34 mm in shell length 
were not. 

Distribution 

Asaphis deflorata has historically been 
collected along the Atlantic and Gulf coasts of 
Florida, as far north as Saint Augustine Beach 
(FLMNH 16923), and as far south as the Dry 
Tortugas (FLMNH 16919) as well as in the 
Bahamas. Rios (1994) registered the species 
for Atol das Rocas, off northeast Brazil. Most 
of these records have collection dates between 
1878 and 1966. None of the collections 
assessed had specimens of A. deflorata 
collected after 1975, although extant 
populations exist at other Caribbean sites 
including the Bahamas (FLMNH 247323), 
Guba (ANSP 1 92408), and Trinidad & Tobago 
(FLMNH 226465). 

Functional Anatomy 

Shell: The shell of >A. deflorata from the "The 
Horseshoe" site population (Fig. 5), matches 
the general shell characterization described 



256 



DOMANESCHI & SHEA 



by Abbott (1974) and Rios (1994) for the 
species in Atlantic waters of the Caribbean 
region, Bermuda and off northeast Brazil. 

Shell oval-elongate, equivalve, moderately 
inflated; umbos subcentral anterior. Maximum 
shell height at the umbonal-ventral axis; 
maximum width at the level of umbos. 
Posterior margin truncate; anterior margin 
broadly rounded; ventral margin straight to 
slightly convex; shell ends gaping slightly. 
Shell with rounded posterior ridges and 
discernible posterior slope marked by stronger 



radial ribs; posterior ridges more conspicuous 
at the dorsal half of the shell, running 
diagonally to and fading as it meets the acute- 
rounded confluence of both posterior and 
ventral shell margins. Outer surface sculptured 
with numerous fine, radial ribs ranging in 
number from 80-102 (n = 10; shell length 
range: 35-59.2 mm) at shell margin (Table 1 ). 
Few ribs with a weak tendency to fork; radial 
ribs stronger, wider apart and scaly to slightly 
nodulose posteriorly. Fine commarginal ridges 
smoothly crossing the radial elements and 




FIG. 5. Asaphis deflorata. External view of the left shell valves of specimens from the "The Horseshoe" 
site population, Florida Keys, USA, showing radial ornamentation and little variation in shell outline. 
Scale bar = 5 cm. 



MORPHOLOGY AND SHELL MORPHOMETRY OF ASAPHIS 



257 



expanding into scale-like or nodulose 
processes on the posterior radial ribs. Exterior 
dull; yellowish to creamish white predominate 
in the population. Periostracum thin, dehiscent. 
Interior often glossy, brightly colored; radial 
ornamentation on the outer surface not 
interfering on the inner surface, which is 
smooth throughout. A deep violet blotch often 
present, spreading over from the nymph 
through the posterior margin; a similar, smaller, 
often fading blotch can be present at the level 
of the anterior adductor muscle scar. Anterior 
adductor muscle scar elliptical, elongate 
dorso-ventrally (Fig. 6); posterior one elliptical 
to rounded; anterior and posterior retractors 
pedal muscle scars fused dorsally to the 
corresponding adductor scars. Palliai line 
recessed deeply from within the smooth shell 
margin. Cruciform muscle scars faint to 
invisible. Palliai sinus broad, extending almost 
to the level with the posterior cardinal tooth; 



upper limb straight to slightly curved; anterior 
margin rounded; lower limb detached from 
palliai line, descending obliquely to coalesce 
with the latter far from the rear end of the palliai 
sinus. Extension of the fusion lower limb-pallial 
line corresponding to one third of the palliai 
sinus depth; rear end of this extension reaching 
level with the anterior half of the posterior 
adductor scar. Striking impression of the fan- 
shaped siphonal retractor muscle present within 
the palliai sinus. Hinge plate with two cardinal 
teeth in each valve (Fig. 7); nymph broad; 
ligament elongate, thick, tough. Right anterior 
cardinal tooth conspicuous, emerging from the 
hinge plate as a knob-like or thick, plate-like 
projection; right posterior cardinal stronger, 
elongate, deeply bifid and separated from the 
anterior tooth by a deep triangular socket. Left 
anterior cardinal tooth strong, elongated and 
deeply bifid; left posterior cardinal emerging as 
a knob-like or thick, plate-like projection. 



aas 




FIG. 6. Asaphis deflorata. Internal view of the right shell valve. Abbreviations: aas, anterior 
adductor muscle scar; act, anterior cardinal tooth; ars, anterior pedal retractor muscle 
scar; cms, cruciform muscle scars; I, ligament; n, nymph; pas, posterior adductor muscle 
scar; pet, posterior cardinal tooth; pi, palliai line; prs, posterior pedal retractor muscle scar; 
ps, palliai sinus; sri, siphonal retractor muscle impression; u, umbo. Scale bar = 1 cm. 



258 



DOMANESCHI & SHEA 



Mantle: The mantle lobes are thin and trans- 
lucent with the usual three folds along their free 
ventral edges. Asaphis deflorata lacks the ad- 
ditional folds that isolate the rejection channel 
for pseudofaeces (waste canal of Kellogg, 
1915), which is present in some Tellinidae and 
Semelidae (Yonge, 1949), and in species of 
Mesodesmatidae and Venehdae (Narchi, 1 981 , 
2002). Graham (1934a) and Domaneschi 
(1992) have given a detailed picture and de- 
scription of the mantle edges in species of Gari. 
The mantle edges in A. deflorata are histologi- 
cally similar to those described and illustrated 
in detail by Domaneschi (1992) for Gari solida 
(Gray, 1828). 

The outer fold is the least developed; the 
inner fold is moderately higher and the only to 
be involved in siphon formation. The middle is 
a huge, sensory fold, which bears a single row 
of cylindrical, cup-shaped tentacles and the 
best supplied with palliai muscles. When the 
foot and siphons are protracted, the middle 
folds are extended well beyond the limits of 
the shell valves, exhibiting an outer surface 
lined by a thin, translucent periostracum. The 
periostracal groove lies outside, adjacent and 
parallel to row of the tentacles. Similar organi- 
zation has been noted in the other Tellinoidea 
in the semelid Ervilia castanea (Montagu, 



1803) by Morton (1990) and in other 
Psammobiidae- in Gari solida by Domaneschi 
(1992) and in IHeterodonax bimaculatus 
(Linné, 1 758) by Narchi & Domaneschi (1 993). 
Each bundle of fibers that comprise the pal- 
liai retractor musculature splits into two sets 
immediately after they originate at the palliai 
line. Both sets supply mainly the middle fold 
and respective tentacles, and meet again ad- 
jacent to the periostracal groove, as in Gari 
tellinella (Lamarck, 1818) (Graham, 1934b) 
and G solida (Domaneschi, 1992). A few fi- 
bers that supply the outer mantle fold arise from 
the set running adjacent to the outer mantle 
epithelium. Muscle fibers that retract the inner 
mantle fold arise from the inner set underlying 
the inner mantle epithelium. In addition to these 
palliai retractors, there are bundles of fibers 
running transversely across the mantle edge 
and folds, and bundles of longitudinal fibers 
restricted to the inner and middle folds, with 
the bulk of them along the inner face of middle 
fold. The palliai nerve cord lies embedded in 
the connective tissue contained by the two sets 
of palliai retractors. Mucous-gland cells form a 
well-defined, glandular region along the pedal 
opening, just dorsal to the base of the inner 
mantle fold. Mucus-secreting cells are more 
abundant on both ends of the pedal opening. 




FIG. 7. Asaphis deflorata. Hinge plate morphology of the right (bottom) and left 
(top) shell valves. Abbreviations: act, anterior cardinal tooth; I, ligament; n, nymph; 
pet, posterior cardinal tooth. Scale bar = 2 mm. 



olp ^ 



MORPHOLOGY AND SHELL MORPHOMETRY OF ASAPHIS 
™ P^ Od 



259 



dh 



dd 



aam / ^ iP >\ ^ /^' 




ex 



e.^-.'ie» 







W'^ ,><:■ 



in 



SO 



FIG. 8. Asaphis deflorate. The animal viewed from the left side after removal of the left shell valve and 
mantle lobe. The siphons and foot are shown somewhat contracted. Arrows show the direction of the 
ciliary currents. Abbreviations: aam, anterior adductor muscle; cm, cruciform muscle; dd, digestive 
diverticula; dh, dorsal hood; ex, exhalant siphon; f, foot; id, inner demibranch; ilp, inner labial palp; in, 
inhalant siphon; Ig, lateral oral groove; mi, mantle isthmus; od, outer demibranch; olp, outer labial 
palp; pam, posterior adductor muscle; pm, palliai retractor muscles; pr, pericardial region; prm, posterior 
pedal retractor muscle; so, sense organ. Scale bar = 1 cm. 



The cruciform muscle with its specialized 
sensory organs occurs postero-ventrally, be- 
tween the base of the inhalant siphon and the 
pedal gape (Fig. 8). Both structures are diag- 
nostic for the Tellinoidea (Ihering, 1900; 
Frenkiel, 1979; Morton, 1984, 1990; Morton & 
Scott, 1990). The sensory organs, which lie at 
the posterior arms of the cross open directly 
to the siphonal space at the summit of minute 
papillae as described by Graham (1934a) and 
Domaneschi (1992) for Gari, and Domaneschi 
(1995) for Semele. The cruciform muscle and 
its sensory organs have been described by 
Mouëza & Frenkiel (1974, 1976, 1977), 
Frenkiel & Mouëza (1977, 1984), Frenkiel 
(1979), Morton (1990). 

Cilia are present all over the inner epithe- 
lium of the mantle lobes. They are more con- 
centrated along a well-defined, wide ciliated 
gutter that lies parallel to the origin of the pal- 
liai retractor muscles. Cleansing ciliary cur- 
rents (Fig. 9) sweep particles forward and 
downward from the dorsal area adjacent to the 
posterior adductor muscle to a vigorous C- 
shaped rejection tract. This rejection tract ex- 
actly follows the line of origin of the siphonal 



retractor muscle. Weak cleansing currents 
within the limits of the C-shaped tract, and on 
the dorsal area adjacent to the anterior ad- 
ductor muscle convey particles downward and 
backward to the base of the inhalant siphon. 
Minute, isolated particles coming into contact 
with inner mantle fold are carried slowly up- 
ward to join those coming downward on the 
mantle surface. 

Despite the presence of the ciliated gutter, 
A. deflorata lacks a well-defined, vigorous, 
rejection current running backward parallel to 
the mantle edge. Such a vigorous current is 
present in A. violascens, Heterodonax 
bimaculatus, and Gari solida (Psammobiidae) 
(Narchi, 1980; Narchi & Domaneschi, 1993; 
Domaneschi, 1992, respectively). 

Siphons: The siphons, type Aof Yonge (1957, 
1982), are wide, separate throughout their 
extent (Fig. 8) and up to 1.5 times the shell 
length. The inhalant is slightly longer, as in 
other Psammobiidae (Yonge, 1949; 
Domaneschi, 1992; Narchi & Domaneschi, 
1993). In a week's time in the laboratory, only 
the inhalant siphon was extruded clear of the 
sediment surface, but not as far as the 3-5 cm 



260 



DOMANESCHI & SHEA 




FIG. 9. Asaphis deflorata. Ciliary cleansing 
currents on the inner surface of the right mantle 
lobe. Scale bar = 1 cm. 



as reported by Berg & Alatalo (1985). When 
extruded and undisturbed, the inhalant was 
held passively for many hours, with the ring of 
tentacles either straight or curled inward; re- 
tracted, its aperture either flushed with or was 
kept below the sediment surface. This latter 
behavior is the rule for the exhalant tip. The 
inhalant was never seen bending down onto 
or along the bottom sediment sucking in de- 
posited material as in typical deposit-feeding 
tellinoideans described by Yonge (1 949). Many 
specimens buried within the sediment in 
aquaria had the tip of the inhalant siphon com- 
pletely covered by a 1 cm-thick layer of coarse 
sand, but maintained an active current flow- 
ing through the interstices of the sand grains, 
as confirmed by pouring carmine powder 
suspension into the water. Such an ability al- 
lows the species to survive buried within either 
rock-, or cobble-covered gravelly sediment 
without extruding the siphons into the water 
column. Berg & Alatalo (1 985) stated that they 
have never observed the tip of the inhalant 
exposed into the water column in the field. 

The inhalant aperture is fringed with twelve 
simple, finger-like tentacles, six longer alter- 
nating regularly with six shorter. From the dis- 
tal end of each longer tentacles arises a double 
row of minute, cylindrical, cup-shaped papil- 
lae which extend down the length of the si- 
phon. The exhalant aperture is fringed with 
eight longer tentacles interspersed with eight 
shorter tentacles. Single rows of minute papil- 
lae extend throughout the length of the exhal- 
ant siphon, each row associated with longer 
tentacles. 

Brilliant, golden-yellow pigmentation is 
present in the inner wall of both siphons. Pig- 
ments may simply create a speckled pattern. 



or be grouped into rounded-elongate, regularly 
arranged spots, or a mixture of both. The larger 
concentration of pigments in the tips of the si- 
phons gives them a vivid, golden-yellow color, 
which fades away toward the bases. Narchi 
(1980) and Domaneschi (1992) also noted a 
yellow color for the siphons of A. violascens 
and Gari solida, respectively. The siphons of 
A. deflorata are sensitive to touch. Although 
light sensitive, they exhibit a similar response 
either to high or low luminosity coming from 
an electronic flash, or a microscope illumina- 
tor, respectively. 

Labial Palps: The labial palps (Fig. 8) are 
approximately one half the shell length. When 
completely expanded, their free distal tips do 
reach and even surround the posterior border 
of the visceral mass. 

The ventral half of the inner surfaces of the 
palps are obliquely folded and separated from 
the dorsal, smooth half by a longitudinal fleshy 
cord that overhangs slightly the dorsal extremi- 
ties of the folds. The inner demibranchs of the 
ctenidia project deeply between the palps, but 
the ventral tips of their anteriormost filaments 
are not inserted into a distal oral groove. Thus, 
the labial palps-ctenidial junction is of Category 
III (Stasek, 1963). 

Exceedingly large palps, provided with dif- 
ferent ciliary tracts indicate that A. deflorata 
processes large amounts of particles in the 
mantle cavity. The palps play an important 
selective function, even though the wide 
ctenidia exert a previous selection of the bulk 
of material entering the mantle cavity as in 
other suspension feeding tellinoideans. The 
sorting mechanisms of the palps are shown in 
Figures 8 and 10. 

Particles coming into contact with the smooth 
outer face of the palps (Fig. 10A) are carried 
dorsalward (current "a") and then passed to 
the internal, smooth, dorsal half of the organs. 
Here, transverse, ventrally directed currents 
transfer them onto the folded area (Fig. 10B) 
to be selected. 

Transversely directed currents (b), operat- 
ing obliquely oralward and markedly 
ventralward across the crests of the folds, to 
either accept or reject particles, depending 
upon the size or total volume of particles. 

On the aboral face of the folds a current (c) 
carries isolated particles and small agglom- 
erations of particles dorsally. As they move 
dorsally, they are influenced by transverse 
currents "b" and removed anteriorly. 

Only minute, isolated particles are caught by 
cilia on the adorai faces of the folds and are 



MORPHOLOGY AND SHELL MORPHOMETRY OF ASAPHIS 



261 



carried dorsalward. Traveling on this current 
"d", particles reach the dorsal extremity of the 
folds, where a conspicuous oralward current 
(e) transports them onto the lateral oral groove, 
between the palps. 

Particles escaping from the action of the pre- 
vious currents "b", "c", "d", and reaching the 
floor of the grooves between adjacent folds, 
are driven ventrally (current "f ) onto a rejec- 
tion current (g) present along the narrow, 
smooth ventral edges of the palps. Excess 
material on current "b" also converges onto 
this current "g". 

Currents "c" and "d" function as resorting 
devices; in combination with current "b", they 
keep the food material away from rejection 
tracts, and allow the agglomerations to be 
disintegrated, resorted, and useful material in- 
gested prior to being discarded as pseudo- 
faeces. 

Muscular contractions of the palps increase 
their sorting efficiency in processing different 
amounts of particles drawn into the mantle 
cavity through the inhalant current. By bend- 
ing laterally, the inner palps touch the visceral 
mass epithelium where particles are being 
carried ventrally. Once trapped by currents "a", 
such particles are passed to the folded sur- 



face of the organ and sorted. The outer labial 
palps do the same, touching the mantle epi- 
thelium. Muscular activity is also responsible 
either for bringing folds closer or forcing them 
apart, and in keeping them erect or bending 
them over. Such devices permit total exposure 
of the ciliary tracts, favoring resorting and in- 
gestion of scarce profitable food, as well as 
their concealment, which favors rejection when 
the inhalant flow of water contains excess 
material. 

Foot and Visceral Mass: The foot is a huge, 
axe-shaped muscular organ that expands far 
beyond the anteroventral margin of the shell. 
At the posterior end of its narrow, ventral edge 
is a distinguishable slit-like opening, related to 
a shallow depression (duct), both remnant of 
the byssal complex, as identified by Pelseneer 
(1911), Graham (1934b), and Domaneschi 

(1992) for Gari spp. No trace of byssus gland 
was detected within the foot of A. deflorata. 
Pelseneer (1911) and Narchi & Domaneschi 

(1993) did not find even a byssal groove aper- 
ture in Asaphis violascens and Heterodonax 
bimaculatus, respectively. Narchi (1980) made 
no reference to byssal complex in A. 
violascens. Every time it was provided a soft 
substratum, the foot in all laboratory specimens 




adorai 



aboral 



FIG. 10. Asaphis deflorata. A, sketch of the left inner labial palp showing the 
ciliary currents (a), (e) and (g). B, diagrammatic representation of three folds 
and respective ciliary currents (b), (c), (d) and (f). For lettering (a) through (g), 
see text on labial palps. 



262 



DOMANESCHI & SHEA 



of A. deflorata (shell length range: 6.9 to 57 
mm) was used exclusively for burrowing, this 
being slower in larger individuals. Burrowing 
period (Stanley, 1970) lasted from 2 to 5 hours 
among individuals < 2.5 cm in shell length (n = 
5), and 23 hours and 35 hours in individuals 
4.5 and 5.7 cm in shell length, respectively. 

The upper, visceral portion of the foot is cov- 
ered with a low, smooth epithelium. Ciliary 
currents were detected in all dissected speci- 
mens, even though only sparse patches of cilia 
could be seen in histological sections. Intense 
cleansing ciliary currents on this visceral por- 
tion of the foot sweep and concentrate par- 
ticles downward over a narrow longitudinal 
area, juxtaposed with the outer dorsal mar- 
gins of the inner labial palps and the free edges 
of the inner demibranchs. Only a weak 
dorsalward current was observed on a nar- 
row area juxtaposed with the very proximal 
portion of the inner labial palps. Particles trav- 
eling on this current are caught by cilia on the 
outer, smooth face of the palps and passed to 
the opposite face of the organs (Fig. 10A). 

The most ventral, predominantly muscular 
portion of the foot is obliquely ridged and lined 
with a densely ciliated epithelium (cilia 5.7 pm 
long). Despite being intensively ciliated, no 
ciliary currents could be detected on this por- 
tion of the foot; Domaneschi (1982) stated they 
are absent in Gari solida. Only Pohio (1972: 
fig.1) has depicted dorsalward ciliary currents 
on the most ventral portion of the foot of a 
psammobiid. 

The portion of the foot juxtaposed mainly with 
the labial palps has a 28 pm-depth, longitudi- 
nally striated epithelium. Lack of cilia on this 
portion was confirmed through histological 
sections and by observing live specimens. 

Ctenidia: The ctenidia are large and occupy 
most of the mantle cavity when completely 
expanded (Fig. 8). They are eulamellibranch, 
plicate and heterorhabidic. The morphology of 
both demibranchs is much the same, the outer 
being more shallowly plicate. 

The ascending lamellae of both inner 
demibranchs are attached to the epithelium 
of the visceral region of the foot by ciliary junc- 
tions; behind the foot they connect to each side 
of a thin, wide triangular membrane the base 
of which surrounds and attaches to the foot 
by ciliary junctions. The ascending lamellae 
of both outer demibranchs connect to the vis- 
ceral mass epithelium by tissue fusion. 

Histological sections prepared from a speci- 
men 1.5 cm in shell length revealed that the 
deepest plicae (400 pm depth) of the inner 



demibranch are formed by 35 ordinary fila- 
ments. Each filament bears 4.3 pm-long, fron- 
tal cilia, these bordered latero-frontally by 17.2 
pm-long, latero-frontal cilia. The lateral cilia 
responsible for the inhalant current of water 
are roughly 11.5 pm long. Terminal cilia (23 
pm long) form a fringe along the lateral walls 
of the marginal food groove. This is 34.2 pm 
depth on average and evenly carpeted with 
5.7 pm-long cilia. 

The frontal surface of a principal filament 
varies in form throughout its extent; it is ridged 
near the free ventral margin of both 
demibranchs, and changes to a broad, shal- 
low gutter toward the ctenidial axis. 

The more stretched a particular region of the 
principal filament is, its frontal gutter is more 
flattened and broad, the sides of the filament 
frequently sloping away nearly at the same 
plane as that of the groove. In strongly con- 
tracted plicae, the frontal gutter of the princi- 
pal filament changes to a narrow, deep central 
groove. This is flanked by the sides of the fila- 
ment, which lie at the same plane of that of 
the frontal surface, as described by Ridewood 
(1903) for Gari vespertina (Gmelin, 1791). 

The morphology and respective ciliary 
mechanisms of the ctenidium (Fig. 11) are of 
type С (la) (Atkins, 1937), characteristic of a 
variety of eulamellibranchs, including the 
Tellinoidea, as in the Psammobiidae analyzed 
by Atkins (1937) and Narchi & Domaneschi 
(1 993). Acceptance oralward currents are thus 
restricted both to the marginal food groove of 
the inner demibranch and to the ctenidial axis. 
Frontal ciliary currents on both ordinary and 
principal filaments of the outer demibranch are 
exclusively ventralward on the ascending 
lamella, and round the bend at the free edge, 
and dorsalward on the descending lamella. 
Frontal currents are exclusively ventralward 
on both lamellae of the inner demibranch. 

There is a tendency for large particles or 
masses of particles that reach the free edge 
of the outer demibranch to be passed straight 
off onto the inner demibranch. However, an 
incipient oralward current was also registered 
along the free ventral margin of the outer 
demibranch. Particles on this current are car- 
ried for short distances anteriorly, then devi- 
ate ventralward under the influence of the 
frontal currents on the inner demibranch. 

Another incipient oralward current does ex- 
ist along and outside the marginal food groove 
of the inner demibranch. Material traveling on 
this current usually falls off on the rejections 
currents of the mantle epithelium. 



MORPHOLOGY AND SHELL MORPHOMETRY OF ASAPHIS 



263 




FIG. 11. Asaphis deflorata. Diagrammatic trans- 
verse section showing the form of the ctenidium 
and directions of the frontal ciliary currents (ar- 
rows). Solid circles, oralward currents; hollow 
circles, incipient oralward currents. 



Adductor Muscles: The anterior adductor 
muscle (aam) is elliptical and dorsoventrally 
elongate; the posterior adductor (pam) is 
subelliptical to rounded and thicker than the 
anterior (Fig. 12). 

Pedal Musculature: The extrinsic pedal mus- 
culature (Fig. 12) consists of bilateral pairs of 
almost equally developed anterior and poste- 
rior pedal retractors, one pair of anterior pedal 
protractor, and one pair of vestigial pedal el- 
evator muscles. 

On each side of the roof of the visceral mass, 
a thin layer of muscle fibers (el) converge dor- 
sally and insert deep on the umbonal cavity, 
where they leave a single, well-impressed 
scar, or more than a single scar. Such a mus- 
cular layer is functionally not significant com- 
pared to the other extrinsic pedal muscles; it 



corresponds by its insertion on the shell valve 
to the functional elevator pedal muscle in many 
other bivalves. In A. deflorata, the elevator pedal 
muscles are atrophied as in other Tellinoidea, 
for example, Garispp. (Pelseneer, 1911; Bloomer, 
1911; Domaneschi, 1992), Telllna foliácea (Linné, 
1 758) (Pelseneer, 1911), Semele spp. (Domaneschi, 
1982, 1995). Narchi (1980) and Narchi & 
Domaneschi (1993) did not find pedal elevators 
muscles in A. violascens and Heterodonax 
bimaculatus, respectively. 

The anterior and posterior pairs of pedal re- 
tractors are equally developed, each attached 
under the hinge plate internal and contiguous 
to the adductor, where their edges and respec- 
tive scars coalesce. The right and the left 
posterior pedal retractors (prm) pass antero- 
ventrally, converging and meeting in the sag- 
ittal plane, almost completely enveloped in the 
kidneys. Where these muscles meet, their 
most internal bundle of fibers intersect; the 
bundles coming from the right pass deeply into 
the left side of the foot and vice-versa. The 
most external bundles of each posterior re- 
tractor pass directly into the foot. As a whole, 
the posterior retractors form the innermost 
muscular layers within the foot. 

The anterior pedal retractors (arm) also con- 
verge on the sagittal plane, where a few inter- 
nal bundles of fibers intersect each other and 
pass deep into the opposite side of the foot. 
The majority of bundles from each muscle pass 
directly into the foot, on its corresponding side. 
Within the foot the anterior retractors form an 
outer layer in relation to the posterior pedal 
retractors. 

The outermost muscular layer of the foot is 
composed of fibers coming from a pair of an- 
terior pedal protractor muscles (ppm). Differ- 
ently from other psammobiids, on each side 
of A. deflorata the anterior protractor is com- 
posed of two separate sets (branches) of 
bundles: one ventral, slender set attaches to 
the shell juxtaposed to and slightly inserted in 
the postero-dorsal surface of the anterior ad- 
ductor; the other set, dorsally placed, gathers 
the bulk of the fibers. Fibers of the dorsal set 
insert on the shell valve embracing the ven- 
tral half of the origin of the anterior retractor. 

From its origin on the shell valve, the slen- 
der, ventral set of the protractor muscle passes 
horizontally into the foot, where the bulk of its 
fibers go backward, while the remainder twist 
abruptly, most downward and a few upward, 
spreading like a fan. Since the bulk of its fi- 
bers lies almost to the level with the base of 
the labial palps, this ventral branch of the pro- 



264 



DOMANESCHI & SHEA 



tractor may easily be гл15]иадес1 as related to 
the palps. However, careful dissections re- 
vealed the protractor fibers penetrating exclu- 
sively into the visceral region of the foot. 

The dorsal set of the protractor has the bulk 
of its fibers extending almost horizontally and 
running backward parallel to the longitudinal 
musculature of the ctenidial axis. Such a juxta- 
position makes the identification of both 
muscles almost impossible. The dorsal set has 
a large number of its fibers spreading fanwise 
downward, where they mask those coming from 
the ventral set. The remaining fibers of the dor- 
sal set spread fanwise dorsally and intermingle 
with those coming from the vestigial, "elevator" 
pedal muscle. Narchi (1980) stated that pro- 
tractor muscles are lacking in A. violascens. 

Apart from the extrinsic pedal muscles, the 
visceral mass and the most ventral parts of 
the foot contain a large number of isolated, 
transverse bundle of fibers (intrinsic muscles 
of Bloomer (1911)). The bundles crossing the 



visceral mass are particularly numerous, thick 
and long in A. deflorata. Such transverse 
bundles play an important role in moving blood, 
and moving materials within the organs. 

Ctenidial Retractor and Longitudinal, 
Ctenidial Axis Muscles: The ctenidial retrac- 
tors (cr) are a pair of thin, but conspicuous 
muscles, with an origin slightly posterior to the 
insertion of each vestigial "elevator" pedal 
muscle on the umbonal cavity. The ctenidial 
retractor fibers pass downward, meet and in- 
termingle with those muscular fibers running 
longitudinally throughout the ctenidial axis 
(cam). The combined action of both, ctenidial 
retractor and longitudinal, ctenidial axis 
muscles shortens the ctenidia as a whole and 
lifts especially the inner demibranchs. Bloomer 
(1907), Villarroel & Stuardo (1977), and 
Domaneschi (1982, 1995) described and de- 
pict a similar muscle in Tagelus divisus 
(Spengler, 1794), T. dombeii {Lamarck, 1818) 
and Sámele spp., respectively. 



el 



cam 



aam 








FIG. 12. Asaphis deflorata. Musculature, as seen from the left side. Abbreviations: aam, anterior 
adductor muscle; arm, anterior pedal retractor muscle; cam, longitudinal, ctenidial axis muscle; cr, 
ctenidial retractor muscle; el, "elevator muscle"; pam, posterior adductor muscle; ppm, ventral and 
dorsal sets of the pedal protactor muscles; prm, ventral and dorsal sets of the posterior pedal retractor 
muscle. Scale bar = 1 cm. 



MORPHOLOGY AND SHELL MORPHOMETRY OF ASAPHIS 



265 



Alimentary Canal: The stomach and style sac 
in A. deflorata follow the general psammobiid 
pattern described by Purchon (1960), Narchi 
(1980), Domaneschi (1992), and Narchi & 
Domaneschi (1993), whereas the intestine 
parallels only that of A. violascens, in which 
the hind gut dilates to store faeces (Purchon, 
1960; Narchi, 1980). 

The general configuration of the alimentary 
canal of >A. deflorata is shown in Figures 13 
and 14. The main difference distinguishing it 
from A. violascens lies in the hind gut, as 
shown in Table 2. The measurements provided 
below were taken from a transversely sec- 
tioned, 1 .5 cm-long specimen; dimensions and 
total numbers of faecal pellets were taken from 
a 5.2 cm-long specimen. 

Essentially the intestine comprises two coiled 
sections. The first section (mg) lies posterior 
and adjacent to the distal half of the style sac 
and is separated from this by gonad follicles, 
digestive diverticula, and a number of cross 
strands of muscle fibers. Its external diameter 



is almost uniform and very reduced (500 |jm 
in average), with a 36 pm-thick wall, the 25 
pm-tall columnar epithelium richly provided 
with 16 pm-long cilia. A feeble layer of circu- 
lar, muscular fibers is present around the mid 
gut walls. The cilia cause rotation and reloca- 
tion of material coming from the stomach and 
wrap it in a viscous mass. Muscular fibers may 
be responsible for peristalsis that contributes 
to compacting, molding and relocating dis- 
carded material. This way faecal pellets are 
completely formed within the mid gut. Faecal 
pellets are similar in appearance to the con- 
tents within the appendix, described below in 
the stomach section; however, they gather a 
larger number of inorganic particles that include 
whitish, hard corpuscles and small quantity of 
non-identified organic debris and microorgan- 
isms. Most sponge spicules and diatom frag- 
ments were not affected when the contents of 
the appendix of the stomach, and faecal pel- 
lets were submitted to weak acid solution (HCl); 
however, the whitish hard fragments dissolved 




FIG. 13. Asaphis deflorata. Configuration of the alimentary canal and part of the excretory and 
circulatory systems, as seen from the left side. Abbreviations: ab, aortic bulb; ax, appendix; dh, 
dorsal hood; f, foot; hg, hind gut; k, kidney; Ic, left caecum; Ip, left pouch; mg, mid gut; mo, mouth; o, 
esophagus; pc, pericardial cavity; r, rectum; ss, style-sac; st, stomach; v, ventricle. Scale bar = 1 cm. 



266 



DOMANESCHI & SHEA 



completely, revealing their calcareous compo- 
sition. 

Midway to the stomach (st), the mid gut por- 
tion ends and the intestine dilates to form its 
second coiled section, the hind gut (hg), dor- 
sally placed. This second section is more ex- 
tensive, more expanded and more intricately 
coiled throughout its extension (Fig. 13); its 
walls comprise a 5.5 pm-thick epithelium sur- 
rounded by an either equal or thinner layer of 
circular, muscular fibers, which allow widen- 
ing/narrowing of the hind gut diameter. Cilia 
could not be detected in histological sections 
of the hind gut. The section walls are so thin 
and so closely applied together that the ex- 
amination and tracking of the hind gut course 
was extremely difficult. Polarized light allowed 
confirmation of the presence of muscular fi- 
bers surrounding the alimentary canal. 

The hind gut accumulates small, very regu- 
lar, rod-shaped faecal pellets. From its proxi- 
mal end (Fig. 13) the hind gut extends dorsal- 
and backward to the floor of the pericardial 
cavity (pc). From here it passes downward to 



the right side of the animal (Fig. 14), spirals 
both in a tight and clockwise way, increasing 
to its maximum width of 6.6 mm, that is, about 
14 times wider than the mid gut (Fig. 13). This 
enormous swelling extends ventrally, where it 
reduces in diameter and turns abruptly both 
forward and dorsalward (Fig. 14) passing to 
the posterior right side of the appendix (ax) of 
the stomach. Here it returns to a narrow ex- 
ternal diameter (~ 250 pm, empty condition; 
lining epithelium deeply folded), penetrates the 
pericardium (pc) and terminates in the anal 
papillae on the posterodorsal face of the pos- 
terior adductor muscle. The epithelial cells lin- 
ing this very rear portion of the intestine are 
8-10 pm in height and densely ciliated (cilia 
8.3 pm long). Similar to the esophagus, this 
portion has a deep folded epithelium, sur- 
rounded by a thick fibrous-like layer, which 
includes circular muscular fibers; the muscu- 
lar layer is thicker around the esophagus. 

Approximately 650 faecal pellets, with a most 
frequent length of 1 .5 mm (range; 0.5-2.5 mm) 
were recovered from a 5.2 cm-long specimen. 




^v>. -A:^'' 




mo 



Ч 

\ \ 



Ic 



■ss 



hg mg 






FIG. 14. Asaphis deflorata. Configuration of the alimentary canal and part of the excretory and 
circulatory systems, as seen from the right side. Abbreviations: re, right caecum. For other lettering, 
see Fig. 13. Scale bar = 1 cm. 



MORPHOLOGY AND SHELL MORPHOMETRY OF ASAPHIS 



267 



bp salO mt gs mg cs e 




\ i 



ó ь 



//\ с 



FIG. 15. Asaphis deflorate. A. Interior of the stomach after being opened by a longitudinal incision in 
the dorsal wall. B, С Internal anatomies of the right and left caeca, respectively. С anatomical differ- 
ences found in two different specimens. Abbreviations: ax, appendix; bp, blind pocket; cs, crystalline 
style; dh, dorsal hood; e, semi-circular elevation on floor of the stomach; el, long forwardly projecting 
elevation; fp, fleshy pads within the appendix; gs, gastric shield; ig, intestinal groove; Ic, left caecum; 
Ip, left pouch; mg, mid gut opening; mt, minor typhlosole; o, esophagus; r, broad fold passing from the 
anterior floor of the stomach to the interior of the dorsal hood; r1 , ridge passing from the posteroventral 
blind pocket to the interior of the dorsal hood; re, right caecum; rm, rim to the esophageal orifice; rt, 
rejection tract; s, swelling on the left anterior wall of the stomach; sa, sa3, sa6, sa7, sa8, sa10, sail, 
sorting areas; ty, major typhlosole. 



268 



DOMANESCHI & SHEA 



Arranged end to end, all these pellets would 
perform a beaded, 98 cm-long thread (650 x 
1.5 mm). 

Stomach: The morphology and functioning 
of the stomach of /\. deflorata (Fig. 15) are so 
similar to those described in details by Purchon 
(1960)andNarchi (1980) for >A. wo/ascens that 
a complete description of the stomach of the 
former species is here assumed to be unes- 
sential. Table 2 shows the main differences 
between the organs in both species. Apartfrom 
those differences, the following aspects of the 
morphology and functioning of the stomach of 
A. deflorata deserve mention: 

- The semi-circular elevation (e) [= shortest 
branch of the major typhlosole (ty)] borders 
a shallow, supporting gutter for the rotating 
crystalline style (cs); the gastric shield (gs) 
protects this gutter against abrasive materi- 
als adhering to the style. 

- Entering the right caecum (re), the longest 
branch of the major typhlosole sends flares 
into the openings of five ducts coming from 



the digestive diverticula; entering the left 
caecum (Ic), it sends flares into the mouth 
of the largest of such ducts only. 

- The left caecum does not receive a constant 
number of ducts coming from the digestive 
diverticula in different specimens (Fig. 1 5C). 

- Freshly incised stomachs show that the fold 
"r" prolongs backward the gutter formed by 
the floor of the esophagus. The free edges 
of this fold touch both the crystalline style 
and the semi-circular branch of the major 
typhlosole, posteriorly, and a low, broad 
swelling (s) on the anterior left wall of the 
stomach. The fold "r" isolates ventrally the 
entrances of both right and left caeca, the 
entrance of the left pouch and the transverse 
section of the intestinal groove, as observed 
by Purchon (1960) in A. violascens. So stra- 
tegically positioned, the fold "r" favors mate- 
rial entering the esophagus to be caught by 
the rotating style and mixed with the disinte- 
grating, gelatinous tip of the latter. Thence, 
material is passed into the dorsal hood (dh), 



TABLE 2. Analysis of morphological variation in the alimentary canal of Asaptiis deflorata [our data] 
and A. violascens [Purchon's (1960 P) and Narchi's (1980 N) data]; r, fold; sa, sorting area on the 
inner wall of the stomach. 



A. deflorata 



A. violascens 



Hind gut widens progressively as it coils and 

spirals in a tight way throughout its 
extension; turns to a narrow diameter 
only within the heart 



(P) (Л/) extraordinarily ballooned in its 
proximal end, where mid gut enters 
dorsally and a narrow, non-coiled hind gut 
leaves ventrally 



Stomach 



"r" present; passes deep into the dorsal 

hood 
"r1" present; lying posterior to and 

throughout the extension of the long 

sorting area "sa3" 

"sa7" present; extending from below the 

esophagus opening deep into the left 
pouch 

"saß" present; at the stomach roof and anterior 

wall of the dorsal hood 

"sail" present; a single sorting area 



(P) (Л/) present; does not enter the dorsal 

hood 

(P) described and depicted a similar fold 

shorter than "sa3"; didn't name it 

(Л/) just depicted it throughout the 

extension of "sa3" 

(P) (Л/) present; just below the esophagus 

opening 

(P) (A/) neither described nor depicted it 

(P) (A/) present; two sorting areas 



Appendix outward ciliary currents present on its 

fleshy pads 
Left pouch sorting areas present (sa6, sa7) 

Blind pouch present; a wide cone-shaped depression 
on the stomach floor 



(P) (A/) no outward ciliary currents are 

present 

Sorting areas present [sa6 (P) (Л/)]; two 

others unnamed (P) 

(P) (Л/) did not refer to it 



MORPHOLOGY AND SHELL MORPHOMETRY OF ASAPHIS 



269 



following storage of excess material in the 
appendix (ax). Another important role of the 
fold "r" is to prevent material entering from 
the esophagus to be caught earlier by cilia 
on the intestinal groove. 

- The full packed appendix equals the volume 
of the main cavity of the stomach. A thick, 
gelatinous mass constitutes the bulk of ma- 
terial often present within this pocket. Lots 
of amorphous greenish-brown debris and 
minute, white, mineral corpuscles, many 
sponge microspicules and broken 
megascleres, some algae debris, diatoms, 
a few foraminiferans and unidentified micro- 
organisms and eggs were the most frequent 
material entangled in a such viscous mass. 
Outward ciliary currents are present on the 
two fleshy pads (fp) that protects the appen- 
dix entrance and neck respectively. Such 
currents are not vigorous enough to deal with 
(remove) the bulk of material and the vis- 
cous mass stored in the appendix. Contrac- 
tions of the appendix walls, as well as of the 
closest intrinsic, transverse musculature of 
the visceral portion of the foot probably play 
an important role in such emptying process. 
Purchon (1960) stated that the contents of 
the appendix of A. violascens are possibly 
discharged into the stomach by muscular 
contraction of its walls. 

- The sorting areas salO and sail are ill-de- 
fined and the least conspicuous within the 
stomach. The same is true for sa7, in its 
portion below the esophagus only. Only care- 
ful analyses of several live and preserved 
specimens allowed confirmation of their 
presence in the stomach of A. deflorata. 
Organs of the Pericardium: The heart lies at 

the level of the shell ligament (Figs. 13, 14). 
This organ comprises a ventricle (v) pen- 
etrated by the rear end of the hind gut, and a 
pair of auricles. The posterior aorta dilates just 
after its emergence from the ventricle to form 
the aortic bulb (ab), the latter as long as the 
ventricle (0.1 of the shell length). From the 
pericardium arise a pair of reno-pericardial 
apertures which drain primary urine into a pair 
of kidneys (k) located between the posterior 
retractor pedal muscles and the floor of the 
pericardium. The kidneys open into the supra- 
branchial chamber at the summit of minute 
papillae, between the ctenidial axis and the 
line of attachment of the ascending lamella of 
the inner demibranch to the visceral mass. 
Close and ventral to the renal apertures are 
the slit-like gonopores. 



DISCUSSION 

In considering the Atlantic and the Pacific 
specimens oiAsapliis to be conspecific, Abbott 
(1950) pointed out that, when or if differences 
can be demonstrated between them, it would 
be wise perhaps to retain the name A. deflorata 
Linné, 1758, for the western Atlantic speci- 
mens and apply Л. wo/ascens (Forsskâl, 1775) 
to the Pacific ones. Willan (1993) considered 
that the Atlantic and Pacific Asapfiis share a 
common ancestor from which divergence oc- 
curred relatively recently. Lacking anatomical 
studies on the Atlantic specimens has re- 
stricted separation between them exclusively 
on the basis of shell sculpture. 

The analysis of >Asap/7/'sfrom the "The Horse- 
shoe" site population. West Summerland Key, 
Florida, USA, has shown that, in addition to 
shell sculpture, as predicted by Prashad 
(1932) and Willan (1993), the western Atlan- 
tic Asaphis also has ecological and morpho- 
functional characters that distinguish it from 
its Indo-West Pacific, close relative. 

Such a population at the "The Horseshoe" 
site is restrict to the intertidal region, where it 
lives buried at moderate depths (0-12 cm) and 
densely aggregated in gravelly sand, cobble 
covered environments in the high intertidal 
zone. The species constitutes the sole bivalve 
present in the upper shore; no specimen was 
found subtidally. These characteristics are 
consistent with previous reports of this Atlan- 
tic species (Stanley, 1970; Berg & Alatalo, 
1985). In contrast, populations of /A. violascens 
in Hong Kong and Indonesia share the inter- 
tidal region both horizontally (Narchi, 1980; 
Britton, 1985; Depledge, 1985) and vertically 
(Soemodihardjo & Matsukuma, 1989J with 
other bivalve species, and may be found in 
sandy environments. Most specimens of A. 
violascens occupy an intermediate intertidal, 
or even subtidal position (Narchi, 1980; 
Soemodihardjo & Matsukuma, 1989; Willan, 
1993) and are found more deeply buried than 
specimens of other bivalve species, at an av- 
erage depth of 20 cm (Narchi, 1980; 
Soemodihardjo & Matsukuma, 1989). 

Zonation patterns in intertidal bivalves have 
been attributed to differences in physiological 
tolerances to desiccation, salinity and heat 
stress (Britton, 1985; Depledge, 1985, and ci- 
tations there). Because A. deflorata and A. 
violascens have different horizontal distribu- 
tions, they likely have different physiological 
tolerances. The responses of /\. violascens to 



270 



DOMANESCHI & SHEA 



temperature, salinity and desiccation have 
been assessed (Briton, 1985; Depledge, 
1985), but similar experiments have not been 
conducted on A. deflorata. 

When found in gravelly sand or cobble cov- 
ered substrata, both A. deflorata and A. vio- 
lascens are densely aggregated (Stanley, 1 970; 
Britton, 1985; Berg & Alatalo, 1985; Kurihara 
et al., 2000, 2001 ; our data). Asaphis violascens 
collected from sandy beaches, or beaches ei- 
ther with reduced cobble coverage or increas- 
ing deposition of sand were drastically lower in 
density (Soemodihardjo & Matsukuma, 1989; 
Kurihara et al., 2001). Willan (1993) observed 
that A. violascens is strictly intertidal and in- 
habits the lower shore where it prefers muddy 
sand substrata with incorporated gravel or coral 
rubble; uniform muddy or sandy substrata ap- 
pear inimical to habitation. Stanley (1970) re- 
ported a casual observation of Asaphis deflorata 
in Bermudan sand flats; Berg & Alatalo (1985) 
could not confirm such behavior for the spe- 
cies population living in the Bahamas beaches. 
These studies suggest Asaphis deflorata is 
more strongly adapted to a coarse gravelly, 
cobble-covered sediment than \sA. violascens. 

The presence of live, unbuhed specimens of 
A. deflorata lying by the high tide mark sug- 
gests that the "The Horseshoe" site population 
faced recent, natural disturbance of the sedi- 
ment and/or that specimens are able to move 
out spontaneously. Berg & Alatalo (1985) have 
never found live individuals lying on the sub- 
stratum surface in the field and considered this 
indicative of little natural disturbance in the sedi- 
ment. 

In the laboratory, a few specimens moved in 
and up within the coarse-sand substratum, this 
being free from natural obstacles such as 
pebbles, shells and rocks. Berg & Alatalo 
(1985) observed that the species can move in 
and out in disturbed substratum, such as in 
and around the tag-recapture plots in the field, 
but have difficulty penetrating the natural, un- 
disturbed, coarse gravel nearby. 

The large number of dead shells of A. 
deflorata retained in their life position in sta- 
tion 2, and their scarce presence in stations 3 
and 4 (present work) may indicate that mor- 
tality in station 2 occurred either from senes- 
cence, or possibly from recent, catastrophic 
modification of the substratum along the low 
tide water, leading to suffocation or starvation 
of the trapped animals. Future field investiga- 
tion on the life cycle of A. deflorata inhabiting 
Florida Keys are necessary to distinguish be- 
tween these alternatives. 



Catastrophic, long-term burial by sand is 
thought to kill entire local populations of A. 
deflorata (Stanley, 1970). Berg & Alatalo 
(1985) identified size-independent mortality 
caused by movements of sand over the habi- 
tat of A. deflorata in the Bahamas beaches. 
Long-term burial by a thick layer of sand 
makes it difficult for clams wedged among 
pebbles to move to the surface (Berg & 
Alatalo, 1985). 

Although restricted to two days of field work, 
during a 12-day workshop period, our quanti- 
tative data on the "The Horseshoe" site popu- 
lation of Asaphis provided reliable results, as 
they fit well with previous ecological and bio- 
logical data of Berg & Alatalo (1985) on A. 
deflorata population in the Bahamas. Com- 
pared with those data obtained from January 
1981 through January 1983 by Berg & Alatalo 
(1985) on the Bahamas population, our data 
reveal that both have very similar life history 
characteristics, including growth rates and 
spawning times. The population structure in 
July 2002 of the "The Horseshoe" site is bi- 
modal, as it is in the July population of the 
Bahamas (Berg & Alatalo, 1985), although the 
most frequently encountered size is smaller 
in the former. Size at maturity is similar for both 
populations, with maturation of the gonads 
occurring at approximately 24-25 mm in shell 
length, which corresponds to an age of 2-3 
years (Berg & Alatalo, 1985). Gonad condi- 
tions at the "The Horseshoe" site in July were 
consistent with the July data from Berg and 
Alatalo (1985) and indicate that spawning was 
imminent. 

Statistical analyses show that shell rib num- 
ber and tendency to fork are good characters 
in distinguishing A. deflorata and A. violascens, 
supporting previous qualitative assessment 
(Prashad, 1932; Willan, 1993) of these shell 
characters. However, we found both species 
have considerably larger rib ranges than pre- 
dicted, and that these ranges can overlap. 
Furthermore, the five outlier A. violascens 
specimens that nest within the A. deflorata 
specimens suggest that rib counts and indi- 
ces may be better regarded as emergent prop- 
erties of a population, not definitive characters 
of the individual. Further examination of the 
effects of environment and geography on rib 
number are warranted. 

Distinguishing characters other than shell 
sculpture are: the presence of a discernible, 
rounded posterior radial ridge and posterior 
slope in the Atlantic >Asap/7/s (our material), not 
discernible (Willan, 1993; IBUSP collection) in 



MORPHOLOGY AND SHELL MORPHOMETRY OF ASAPHIS 



271 



the Indo-West Pacific specimens, and a 
snnooth inner surface in the Atlantic Asaphis, 
contrasting with the well-marked, ridged inner 
surface in specimens from Hong Kong (IBUSP 
collection). The effects of the environment and 
geography on these characters also deserve 
further examination. 

Asaphis deflorata is a native species that has 
been periodically collected in southern Florida 
and the Florida Keys for over 100 years. 
Asaphis deflorata should be commonly en- 
countered in the Florida Keys because suit- 
able environmental condition occur, including 
gravelly sand, cobble rich intertidal habitat. The 
two-week planktonic larval period (Berg & 
Alatalo, 1985) should provide ample time for 
dispersal. Dispersal routes are dependent on 
local current regimes. Overall, current patterns 
in the Florida Keys move from Florida Bay, 
through the Keys and then join a southwest- 
erly countercurrent flow (http:// 
oceanexplorer.noaa.gov, accessed 14 Janu- 
ary 2003). These patterns suggest that north- 
ern populations could serve as a source of 
larvae to populate more southerly areas. 

Prior to the mid 1 970s, A. deflorata was com- 
monly collected from Key West to Key 
Biscayne (Miami), and possibly included the 
Dry Tortugas (FLMNH 16919, collected in 
1937). Many pre-1975 collection records sug- 
gest Key Biscayne had a robust population of 
adult A. deflorata specimens. Our search did 
not find any post-1975 records attributed to 
Key Biscayne. Although the lack of museum 
records does not prove Asaphis is not present 
in the field, the combination of no records and 
a single population found after extensive sam- 
pling of the molluscan fauna in the Florida Keys 
(P. Mikkelsen, pers. commun.) suggests the 
distribution of A. deflorata has diminished 
greatly. Although our data are preliminary, they 
point to a drastic decline in the distribution of 
Asaphis populations in the Florida Keys. Why 
the persistent "The Horseshoe" population 
does not act as a source population requires 
further investigation. The two-week larval 
stage of Asaphis should provide ample time 
for distribution to more southerly locations. 
However, if the spawning season does not 
coincide with sufficiently strong and appropri- 
ately oriented currents, the larvae may never 
be moved outside the confines of the sheltered 
"The Horseshoe" site. Because appropriate 
substrata and quiet waters are found through- 
out the Keys, this physical barrier is the most 
likely explanation for the lack of additional 
southerly populations. Future studies on the 



distribution of Asaphis deflorata should incor- 
porate a systematic examination of the sum- 
mertime current regime around the "The 
Horseshoe" site. 

The functional morphology of the Atlantic 
Asaphis is very similar to that of the Indo-West 
Pacific species as described by Narchi (1 980). 
The correct identification of both species 
based upon the tissues requires the analysis 
of the alimentary canal. Great similarities sup- 
port Willan's (1993) opinion that both species 
share a common ancestor from which morpho- 
logical and ecological divergence has occurred 
relatively recently. The most striking anatomi- 
cal difference lies in the hind gut configura- 
tion: progressively widening, and intricately 
coiled and spiraled throughout its extension 
in the Atlantic Asaphis (our data); extraordi- 
nary ballooning of its proximal end only, where 
the mid gut enters dorsally and a narrow, non- 
coiled hind gut leaves ventrally, in the Indo- 
West Pacific specimens (Purchon, 1960; 
Narchi, 1980). 

The long, wide, coiled hind gut of >A. deflorata 
and the dilation of that of A. violascens are 
both devices for retention of large amount of 
faecal pellets. It is difficult to understand how 
the compacted, randomly positioned faecal 
pellets are relocated from a wide compartment 
toward a narrow one, which is even more re- 
stricted in its passage through the ventricle. 
Peristalsis of the hind gut walls and the trans- 
verse musculature within the visceral portion 
of the foot may be the mechanism to achieve 
relocation of such compacted, rod-shaped fae- 
cal pellets. Expansion/contraction of the ven- 
tricle, peristalsis, and ciliary action of the very 
rear sector of the intestine force faecal pellets 
oriented end-to-end or in small groups to be 
expelled. 

Most specimens of /A. deflorata examined for 
alimentary canal morphology had the hind gut 
packed with faeces, these always fully formed 
in their origin within the midgut It is quite prob- 
able that faeces storage in Asaphis is not cor- 
related with the necessity to consolidate faecal 
pellets as proposed by Yonge (1949) for de- 
posit-feeding tellinoideans. 

Comparing the gross morphology of Abra 
profundorum (Smith, 1885) (Tellinoidea) and 
that of north Atlantic species of the genus, Allen 
& Sanders (1966) found a clear correlation 
between gut length and volume, size of de- 
posit-feeding bivalves and respective palps 
and ctenidia, and depth they inhabit A reduc- 
tion in thickness of the gut walls, an increase 
of the lumen diameter allowing faecal pellets 



272 



DOMANESCHI & SHEA 



to be randomly positioned, and tine gut tal<ing 
up an increasingly proportion of the body were 
correlated with depth by these authors. Allen 
& Sanders (1966) stated that among the spe- 
cies of Abra they compared, "the limit of evolu- 
tion of the hind gut is reached in A. profundorum 
where many of the adjoining walls of the coiled 
gut are lost and much of the posterior half of 
the body above the muscles of the foot be- 
comes a sac containing faeces". 

Asaphis deflorata inhabits shallow waters 
and preferentially the upper shore; neverthe- 
less, this species shares most of the morpho- 
logical features of the gut referred to by Allen 
& Sanders (1966) for tellinoideans inhabiting 
deep waters, except for a loss of walls where 
the coils and spiral are tightly applied to each 
other. 

Asaphis deflorata has a long intestine, com- 
pared with the suspension-feeding 
psammobiid Heterodonax bimaculatus from 
intertidal, coarse sand (Narchi & Domaneschi, 
1993), and only moderately long compared to 
Gari solida from subtidal coarse sand sub- 
strata (Domaneschi, 1992). The exceedingly 
large palps, the presence of simple, digitiform 
tentacles around the inhalant aperture, but 
especially the enormous amount of faecal 
pellets retained within the hind gut suggest that 
the species deals with and ingests large 
amounts of material entering via the inhalant 
siphon. Asaphis deflorata shares most of the 
morpho-functional features considered by 
Pohio (1982) to be characteristic of typical 
suspension feeding tellinoideans: large gills, 
no waste canal, outer demibranch not up- 
turned, marginal food groove present, animal 
lying in a vertical position within the sediment 
and the inhalant siphon does not take depos- 
ited material actively. Once extruded into the 
water column this siphon in A. deflorata is kept 
passively, much the same as in the suspen- 
sion-feeders tellinoideans studied by Yonge 
(1949), PohIo (1972), Domaneschi (1992, 
1995). However, its non-straining, curled in- 
ward tentacles may allow entrance of large 
amount of suspended material brought by the 
rising and falling tides, as well as of dense 
material lifted from the bottom. Living in the 
constraint of a cobble-covered sediment, the 
inhalant aperture more often is flush with, or 
slightly below the sediment surface. It contrib- 
utes to the intake of dense, mineral particles, 
benthonic microorganisms and organic and 
inorganic debris deposited either outside 
around the aperture of, or lining the passage 
of the inhalant siphon through the substratum. 



Such behavior supports Berg & Alatalo's 
(1985) statement that >A. deflorata is a suspen- 
sion and facultative deposit feeder. Phy- 
toplankton and C3 plants detritus constitute 
most of its diet (Berg & Alatalo, 1985). 

Like/\. wo/ascens (Narchi, 1980), Gari solida 
(Domaneschi, 1992), and other Tellinoidea 
(PohIo, 1982; Domaneschi, 1995), Л. deflorata 
shares a mosaic of morphological features with 
suspension and specialized deposit-feeding 
tellinoideans. Such a condition was considered 
to represent (PohIo, 1982) an intermediate 
step in the evolution of the Tellinoidea, the 
primitive forms represented by early suspen- 
sion-feeding species, and the most derived, 
represented by highly specialized, deposit- 
feeding species. 

When present in the beach, Asaphis 
deflorata has been detected to be the only bi- 
valve occupying the upper shore (Stanley, 
1970; Berg & Alatalo, 1985; our data). The 
possession of a huge appendix in the stom- 
ach, and of a capacious hind gut allows stor- 
age of excess, ingested food and retention of 
an exceedingly large amount of faecal pellets, 
respectively. Both features are, probably, ad- 
aptations to upper shore life and non-selec- 
tive feeding habit, as during high tides the 
species can make the most of the period of 
submersion, both to get rid of lots of faecal 
pellets and take into the mantle cavity a large 
amount of suspended and re-suspended ma- 
terial, processes and ingests it. /\sapÄ7/s feeds 
only during the period of submergence by high 
tides and fasts the remainder of the time (Berg 
& Alatalo, 1985). The huge appendix provides 
room for the bulk of material entering the stom- 
ach and mixed with enzymes liberated by the 
dissolving head of the style. It also prevents 
blockage of the main cavity of the stomach, 
allowing its normal functioning as already pro- 
posed by Yonge (1949) for the appendix of 
other tellinoideans. The capacious hind gut 
allows retention of a corresponding great vol- 
ume of material coming from the stomach and 
being molded into faecal pellets within the mid 
gut. Faeces have to be eliminated during sub- 
mergence periods only, when the exhalant 
current of water takes them far away from the 
animal; conversely, during low tides the ani- 
mal is at risk of faeces sedimentation within 
its own mantle cavity. Purchon (1960) consid- 
ered the capacious stomach and intestine of 
A. violascens a device for retention of food 
and faecal material during low tides, as well 
as for survival when the habitat is covered with 
sand. Capacious stomach provided with an 



MORPHOLOGY AND SHELL MORPHOMETRY OF ASAPHIS 



273 



appendix structurally and functionally similar 
to the appendix of the tellinoidean bivalves is 
also shared by the Pholadidae, Xylophaginidae 
and Teredinidae (Pholadoidea) (Purchon, 
1941, 1955), the appendix being exceedingly 
long and broad to store mainly wood particles 
in many teredinids (Lopes et a!., 2000, and 
citations there). Purchon (1941, 1955) con- 
cluded from that similarities that these struc- 
ture are homologous and provide reliable 
evidence of a relationship between these 
groups. Lopes et al. (2000) discussed the prob- 
able implications of the specialization of the 
appendix, as well as of the digestive system 
as a whole in the evolution of the xylophagous 
habit of two species of Teredinidae. 

The presence of a thin-walled, huge hind gut 
suggests that the compacted mass of faecal 
pellets of >A. deflorata cannot be promptly and 
completely eliminated. As retention is not re- 
lated to the necessity to consolidate faecal 
pellets, it may be that another purpose is 
served by such an intriguing hind gut than to 
store, then eliminate faeces during convenient 
periods: for example, to allow enough time for 
the breakdown and consumption of material 
with a food value present in the faecal pellets; 
the enzymes required for this process could 
be the same as those present in the stomach 
and passed onto the intestine along with dis- 
carded material. Similar storage of faeces oc- 
curs in some teredinid bivalves (Lopes et al., 
2000, and citations there). Long residence of 
faeces within the anal canal of Neoteredo 
reynei (Bartsch, 1920) was considered by 
Lopes et al. (2000) as a probable device al- 
lowing both enzymatic degradation of mate- 
rial of a food value and absorption to be 
continued; the presence of epithelial cells 
richly supplied with microvili, and the highly 
vascularized anal canal walls of the species 
giving support to this latter hypothesis. 

Allen & Sanders (1966) detected the pres- 
ence of large numbers of amoebocytes in the 
gut and surrounding the faecal pellets in Abra 
profundorum, "which would possibly indicate 
that the pellets provide a surface, upon which 
bacteria would thrive, attack and convert to a 
digestible form the carbon compounds indi- 
gestible to the mollusc." According to Allen & 
Sanders (1966), "Newell (1965) has shown 
that faecal pellets of both Hydrobia (gastro- 
pod) and Macoma (tellinoidean bivalve) are 
excellent substrates for bacteria", and "that the 
nitrogenous compounds built up by the growth 
of the bacteria can be digested by the mol- 
lusc". 



Neither amoebocytes nor bacteria could be 
identified within the hind gut of A. deflorata 
through the methodology adopted in histologi- 
cal preparations. This cannot be taken as a 
definitive result, nor the hypotheses of the 
presence of amoebocytes and of a possible 
relationship of the species with symbiotic bac- 
teria can be discarded. Morphological and 
functional similarities between the gut of this 
psammobiid and of A. profundorum address 
those hypotheses. Appropriate methodologies 
were not employed to test the occurrence of 
such living elements in the gut of /\. deflorata, 
as this was not the aim of the present work. 
The significance of pellet storage and the hy- 
pothesis that digestion and absorption of nu- 
trients continue within the hind gut of A. 
deflorata via endogenous enzymes and/or 
symbiosis remain to be elucidated; it cannot 
be fully explained until further experimental 
examination with live specimens has been 
carried out. 



ACKNOWLEDGMENTS 

We are greatly indebted to Dr. Paula 
Mikkelsen and Dr. Rüdiger Bieler for inviting us 
to participate in the International Marine Bivalve 
Workshop; to the members of the IMBW and 
Keys Marine Laboratory staff for the support 
and hospitality they provided us. The authors 
gratefully acknowledge the U. S. National Sci- 
ence Foundation - РЕЕТ Program (DEB- 
9978119) that provided major funding for this 
workshop, as part of a grant to the co-organiz- 
ers R. B. & P.M., as well as the Bertha LeBus 
Charitable Trust, the Comer Science & Educa- 
tion Foundation, the Field Museum of Natural 
History, and the American Museum of Natural 
History for additional support. We thank Enio 
Matos, IBUSP, for technical assistance in the 
histological preparations, and Flávio D. Passes, 
André F. Sartori, José E. R. Marian, Sonia M. 
Montini, advisees of the author (OD), for their 
support with photographs, in mounting plates 
and gathering literature. Dr. Shahroukh Mistry 
(Bryn Mawr College) provided essential advice 
on the use and interpretation of principal com- 
ponents and cluster analysis. Dr. Kevin Roe and 
Leslie Skibinski (DMNH) generously provided 
access to specimens, and Mark Leiby (FMRI) 
and Dr. Nancy Voss (RSMAS) both provided 
collections information. Special thanks to Dr. 
Rüdiger Bieler, Eugene V. Coan, and the two 
anonymous reviewers for their valuable com- 
ments on the manuscript. 



274 



DOMANESCHI & SHEA 



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Revised ms. accepted 31 October 2003 



MALACOLOGIA, 2004, 46(2): 277-294 

EXTRAORDINARY FLEXIBLE SHELL SCULPTURE: THE STRUCTURE 

AND FORMATION OF CALCIFIED PERIOSTRACAL LAMELLAE IN 

LUCINA PENSYLVANICA (BIVALVIA: LUCINIDAE) 

John D. Taylor^*, Emily Glover\ Melita Peharda^, Gregorio Bigatti^ & Alex BalP 

ABSTRACT 

The lucinid bivalve Lucina pensylvanica possesses an unusual flexible commarginal 
shell sculpture formed from calcified periostracal lamellae. The lamellae comprise thick, 
recurved, periostracal extensions with distal calcified scales. The periostracum is also 
densely embedded with calcareous granules around 2.0-2.5 |jm in diameter and a thin (10 
pm) layer of prismatic aragonite covers the ventral face of each lamella. Other species of 
Lucina in the western Atlantic possess calcified scales but with different morphologies and 
the continuous commarginal ridges of the eastern Atlantic Lucina adansoni and other Afri- 
can species are similarly constructed and homologous. The periostracal lamellae are a 
probable apomorphy of the genus Lucina and morphology of the calcified structures pro- 
vides a set of systematic characters of importance in the discrimination of species. 

Key words: Lucina pensylvanica, periostracum, calcification, shell growth, systematics. 



INTRODUCTION 



Lucina pensylvanica (Linnaeus, 1758) is one 
of ten species of chemosymbiotic lucinid 
bivalves inhabiting intertidal and shallow 
subtidal habitats in the middle Florida Keys. 
Remarkably, the shell sculpture consists of 
closely spaced commarginal lamellae, faced 
with triangular, calcareous scales that are 
slightly flexible in live animals. The scales and 
lamellae become brittle after death and in 
beach-collected shells the surface is white, 
relatively smooth with low, thin, commarginal 
ridges, sometimes with traces of periostracum. 
Our initial observations suggested that both 
lamellae and scales were a form of periostracal 
or extra-periostracal calcification, distinct from 
the normal shell. Because of the rarity of 
periostracal calcification in bivalves in general 
and the probable apomorphy of this character 
for Lucina spp., we decided to investigate the 
structure and formation of the lamellae in more 
detail and, if possible, determine the periodic- 
ity of their secretion. Additionally, we wanted 
to compare the form of the periostracal lamel- 
lae between Lucina species, both to establish 



the homology of these as well as investigate 
their possible use as systematic characters. 
Detailed understanding of lamellar formation 
may also suggest hypotheses about their pos- 
sible function. 

Periostracal and extraperiostracal calcifica- 
tion is an unusual feature of bivalves but has 
been described in different forms from a vari- 
ety of families. Usually in Lucinidae the 
periostracum is relatively thin (Harper, 1997), 
although exceptionally the genus Rasta has a 
dense, shaggy periostracum extended into 
numerous long pipes (Taylor & Glover, 1997). 
Prominent, sculpture-forming calcified 
periostracum appears restricted to the genus 
Lucina, of which L. pensylvanica is the type 
species (ICZN, 1977). The morphology of the 
calcified scales has been used by Gibson- 
Smith & Gibson-Smith (1982) as a character 
to divide "Lucina pensylvanica" of the west- 
ern Atlantic into four separate species. 
Amongst other bivalve families, Veneridae, 
such as Lioconcha and Callocardia possess 
encrustations formed of fine aragonitic needles 
projecting through the periostracum (Ohno, 
1996; Morton 2000); others such as 
Granicorium and Samarangia secrete extra- 



department of Zoology, The Natural History Museum, London SW7 5BD, United Kingdom 
^Institute of Oceanograpliy and Fisheries, PO Box 500, 21000 Split, Croatia 

^epartado Biología, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pab II, 
C1428EHA Buenos Aires, Argentina 
'Corresponding author; j.taylor@nhm.ac.uk 



277 



278 



TAYLOR ET AL. 



periostracal calcareous cements to form a 
crust of sediment on the shell (Taylor et al., 
1999; Braithwaite et al., 2000). Many 
Anomalodesmata, such as Laternula and 
Lyonsia, possess spines formed within the 
periostracum as do some Gastrochaenidae, 
such as Spengleria rostrata (Spengler) (Carter 
& Aller, 1975). Amongst the Mytilidae, 
intraperiostracal aragonltic granules and pro- 
jecting spikes have been described in 
Trichomya and Brachidontes (Carter & Aller, 
1975; Bottjer & Carter, 1980; Carter et al., 
1990), while intra- and extraperiostracal cal- 
cified structures are a feature of various spe- 
cies of Lithophaginae (Carter et al., 1990). 

Little is known of biology of Lucina 
pensylvanica. Stanley (1970) demonstrated 
using x-rays that animals burrowed with the 
anterior part of the shell lying uppermost in 
the sediment, an unusual life orientation for 
Lucinidae. The general anatomy was de- 
scribed by Allen (1958) and Gros et al. (1996) 
made a detailed description of the gill ultra- 
structure and chemosymbiotic bacteria. Addi- 
tionally, Taylor & Glover (2000) illustrated the 
large bipectinate mantle gills that lie alongside 
the pallia! blood vessel. 

Lucina pensylvanica and its close allies are 
often referred to in the literature under the 
generic name Linga. However, the name 
should correctly be Lucina as Lucina 
pensylvanica was designated the type species 
of the genus in 1977 (ICZN, 1977). 



MATERIALS AND METHODS 

Lucina pensylvanica was live collected from 
a number of oceanside intertidal and shallow 
water sites in the Florida Keys during the In- 
ternational Marine Bivalve Workshop (IMBW) 
in 2002 (Mikkelsen & Bieler, 2004, fig. 1 - 
map). Live animals were abundant only at Sta- 
tion IMBW-FK-642, mile marker 74.5 
(24°51.4'N, 80°43.7'W) on Lower Matecumbe 
Key. Here they occurred in low intertidal to 
shallow, subtidal pockets of medium to coarse 
sand, located on a wide, coral-rock platform. 
The area was vegetated with patches of 
Thalassia and Halodule, as well as growths of 
Penicillus and Halimeda. Despite similar col- 
lecting effort, Lucina pensylvanica was much 
less common at other sites, such as Anne's 
Beach, Upper Matecumbe Key (Station IMBW- 
FK-638) from Thalassia-coyered sand and 
Pigeon Key (Station IMBW-FK-657) in a tidal 
stream with Thalassia and Syringodium. No 



live animals were found at any bayside sta- 
tions. Animals were collected by extensive dig- 
ging and hand sieving. Voucher specimens 
held in BMNH, London. 

Live animals were fixed in 75% ethanol, 5% 
seawater formalin or Bouin's fluid. Tissue 
samples were also fixed in 2.5% solution of 
glutaraldehyde in phosphate buffer. Sections 
of mantle were stained with Mallory's triple. 
For optical microscopy of the shell, geological 
thin sections were made from fresh specimens 
embedded in resin. Pieces of the same em- 
bedded shell were also examined by scanning 
electron microscopy (SEM) after cutting, pol- 
ishing and etching in EDTA. 

Shell sections were also examined by con- 
focal microscopy using a Leica SP NT in re- 
flected light mode. Simultaneous images were 
collected at several different wavelengths, and 
a reference image was obtained with the trans- 
mitted light detector. We also carried out an 
initial test for autofluorescence using a wave- 
length (lambda) scan. The section was 
scanned at a single focal plane with each la- 
ser in turn. The detector was programmed to 
step through 25 pre-determined 10 nm-wide 
detection windows at wavelengths from 495- 
750 nm that produced an intensity profile for 
each emission wavelength. This optimised la- 
ser detector position and line. The best results 
were obtained with the 488 nm Argon laser 
and this was used for all subsequent imaging. 
No autofluorescence was detected from within 
the shell matrix, so the first detector window 
was set at 486-507 nm. This wavelength gave 
a direct reflection image of the sample and was 
false coloured in green. Strong 
autofluorescence from the periostracum was 
detected at around 550 nm, so the second 
detector window was set at 537-568 nm and 
the images coloured red. A stack of 30 images 
was collected at -0.4 pm intervals. Each frame 
was scanned three times and run through a 
frame-averaging filter to reduce background 
noise. For single images, the z-axis (depth) 
data from the entire stack was combined and 
the brightest pixel from each point computed 
and displayed (maximum projection image). 

Growth Periodicity 

Twenty valves from live collected animals 
were used to study growth periodicity. We 
embedded these in MET20 resin (Struers Ltd), 
sectioned them transversely from the umbo 
to the ventral edge. They were then ground, 
polished and etched for 20 min in 0.01 M HCl 



SHELL SCULPTURE IN LUCINA PENSYLVANICA 



279 



and acetate peel replicas prepared following 
Richardson (2001). Distances between suc- 
cessive periostracal lamellae were measured 
to the nearest 0.05 mm on 11 shells. Distinc- 
tive major lines in the outer and middle shell 
layers and in the umbonal region (Fig. 22) were 
correlated with the formation of closely spaced 
or uncalcified periostracal lamellae. Three 
separate observers used these major growth 
marks in both umbo and valve to estimate the 
age in years of the animal (Richardson, 1993). 
The major growth increments were treated as 
annual lines by comparison with a similar study 
of Codakia orbicularis from the Bahamas (Berg 
&Alatalo, 1984). 



RESULTS 

Shell Microstructure 

The shell consists of three aragonitic layers. 
The outermost layer is composed of a pris- 
matic layer of irregular acicular crystals, their 
long axes inclined towards the shell margin 
(irregular spherulitic structure of Carter & 
Clark, 1 985). This is followed by a middle layer 
of finely lamellate, crossed-lamellar structure 
and, within the palliai myostracum comprising 
irregular prisms, there is an inner layer formed 
of complex crossed-lamellar structure, inter- 
calated with thin prismatic sheets. This se- 




FIG. 1 . Lucina pensylvanica exterior of right valve 
showing commarginal periostracal lamellae with 
projecting calcareous scales. Shell height = 22.8 
mm. Station IMBW-FK-642, Mile Marker 74.5, 
24°51 .4'N, 80°43.7'W, on Lower Matecumbe Key. 



quence of shell layers resembles most other 
Lucinidae (Taylor et al., 1973). 

Calcified Periostracal Lamellae 

Periostracal lamellae (hereafter referred to 
as lamellae) consist of an extended 
periostracum sheet faced with prominent cal- 
cified scales (Fig. 1). The lamellae recurve 
dorsally and are regularly spaced at intervals 
of 400-1500 pm, extending about 1,000 pm 
from the shell surface. Interspaces between 
the lamellae are relatively smooth (Figs. 2, 3) 
and in live collected specimens are packed 
with sediment grains (Figs. 8, 9). The discrete, 
closely-spaced calcareous scales (Fig. 8) are 
around 600-1 ,000 pm in height and seemingly 
embedded into the periostracum. In shape, the 
scales are triangular to lanceolate, broad at 
the base (varying between 500-950 pm) and 
taper distally. When newly formed, they are 
usually pointed at the tips (Fig. 4) but become 
truncated with wear. Scale shape varies 
around the shell; those on the posterior dor- 
sal area are usually broader, more closely 
spaced and less recurved. Over most of the 
shell surface, lamellae recurve dorsally but 
when first formed they extend straight out from 
the shell margins, with the scales embedded 
in the sheet of periostracum (Fig. 4). Subse- 
quently, lamellae become progressively re- 
curved away from the commissure (Fig. 4), and 
the periostracum erodes away from the scales 
(Figs. 5, 6). 

On juvenile shells, the scales are differently 
shaped (Fig. 7) being lower and quadrate with 
narrower spaces between, so that they form 
an almost continuous ridge. The quadrate 
scales change to a triangular shape at a shell 
height of around 4.5-5.0 mm. 

Sections 

Optical, scanning and confocal microscopy 
shows that each lamella is composed of a 
periostracal extension in which the calcare- 
ous scales occupy the distal ventral face (Figs. 
8-11). Each lamella projects from a thin ridge 
in the true shell (Figs. 8-10). Within a lamella 
the periostracum is about 55 pm thick and 
continuous with that of the outer shell surface. 
Between successive lamellae the 
periostracum gradually increases in thickness 
from around 1-2 pm at the termination of one 
extension to about 50 pm at the base of the 
succeeding extension (Fig. 12). Higher mag- 
nification of the calcareous scales reveals a 



280 



TAYLOR ET AL. 




FIGS. 2-4. Lucina pensylvanica. FIG. 2: Surface view of successive commarginal lamellae with scales. 
Scale bar = 500 pm; FIG. 3: Pehostracal lamellae on posterior of shell with pointed scales with 
smooth pehostracal surface between lamellae. Scale bar = 500 |jm; FIG. 4: Site of formation of 
pehostracal lamellae at valve margins showing lamellae lying parallel with shell margin but becoming 
recurved dorsally away from the edge. Scale bar = 500 pm. 



SHELL SCULPTURE IN LUCINA PENSYLVANICA 



281 




FIGS. 5, 6. Lucina pensylvanica. FIG. 5: Ventral 
view of forming lamella at shell margin showing 
row of scales embedded in periostracum 
stretched between them, but in the preceding 
row this has disappeared. Scale bar = 250 pm; 
FIG. 6: View of posterior shell margin with 
pointed scales joined by a membrane of 
periostracum. Scale bar = 250 pm. 




FIG. 7. Lucina pensylvanica, juvenile shell (shell 
height 3.5 mm) with lamellae formed of closely 
spaced, quadrate scales. Scale bar = 200 pm. 



FIGS. 8, 9. Lucina pensylvanica. FIG. 8: 
Transverse section of shell showing two lamellae. 
Note ridges in shell and sediment trapped behind 
lamellae. Scale bar = 250 pm; FIG. 9: Transverse 
section of a single lamella. Scale bar = 250 pm. 



thin (1.5-2.0 pm) initial pehostracal sheet fol- 
lowed by a layer of aragonitic spherulitic mi- 
crostructure (Fig. 14). Each scale is about 220 
pm thick tapering distally. Within the spheru- 
litic layer of the scale, Interpenetrant bundles 
of long, thin crystals radiate from nucleation 
sites on the inner pehostracal surface. Fine 
growth lines indicate that the scales are se- 
creted incrementally. Another calcified layer 
(10-15 pm thick), of short, prismatic arago- 
nite crystals embedded in periostracum, forms 
the ventral face of each completed lamella 
(Figs. 11, 13, 18). 

Sections of the basal pehostracal part of the 
lamella show that it is densely embedded with 
tiny calcareous granules about 2-2.5 pm in 
diameter consisting of aggregations of crys- 
talline aragonite (Figs. 13, 16, 19). Granules 
are absent in the outermost of part of the 
pehostracum but at about 1 pm from the edge 
of the lamella increase in abundance (Fig. 12). 



282 



TAYLOR ET AL. 



pi 




SC 


tp -^ 




*.^ PS 






FIGS. 10, ^^. Lucina pensylvanica.F\G. 10: Confocal image of transverse section through a periostracal 
lamella. Periostracum red; calcified structures green. Scale bar = 100 |jm. Abbreviations: pf, calcified 
prismatic front of lamella; pi, periostracum of lamella; ps, periostracum above shell; r, ridge in outer 
shell layer; s, shell; sc, scale; FIG. 11: Confocal image of the proximal region of a periostracal lamella, 
showing detail of the periostracum and the calcified front of the lamella. Scale bar = 50 |jm. 
Abbreviations: as for Fig. 10; gz, granule zone; oz, outer granule-free periostracal zone. 




FIG. 12. Lucina pensylvanica, SEM image of transverse section through base of a lamella showing 
shell ridge and thinned periostracum that thickens towards the succeeding lamella. Scale bar = 100 
pm. Abbreviations: osl, outer shell layer; p, periostracum; pf, prismatic front of lamella; pi, periostracum 
of lamella; tp, thin periostracum. 



SHELL SCULPTURE IN LUCINA PENSYLVANICA 



283 



These granules are also present in the nor- 
mal periostracum secreted above the outer 
shell layer and gradually increase in frequency 
between successive lamellae. 

Sections of the junction between the calcar- 
eous scales and the periostracal lamella show 
that lines representing growth increments in- 
terdigitate from periostracum into the calcified 
scales and also that the granules increase in 



density and fuse at the transitional boundary 
(Figs. 13, 15). The calcified scales are thus 
secreted contemporaneously with the 
periostracal layers of the lamella and not laid 
down subsequent to it. Images clearly show a 
covering of periostracum eroding from the 
scale surfaces. We conclude from these ob- 
servations that both the granules and scales 
are forms of periostracal calcification. 




FIGS. 13-15. Lucina pensylvanica. FIG. 13: SEM image of a transverse section through junction 
between calcareous scale and proximal part of the lamella showing interdigitation of calcareous 
layer with periostracum and granules. Scale bar = 50 pm; FIG. 14: Section through a calcareous 
scale showing spherulitic crystal growth arising from thin periostracum layer below. Scale bar = 70 
цт; FIG. 15: Section through junction of calcareous scale and periostracum showing continuity of 
growth increments from the calcified portion into the periostracum. Scale bar = 50 pm. Abbreviations: 
gz, granule zone of periostracum; p, periostracum; sp, spherulitic crystal growth. 



284 



TAYLOR ET AL. 




FIGS. 16-19. Lucina pensylvanica. FIG. 16: Surface of a forming pehostracal lamella at shell margin 
showing aragonitic granules embedded in surface. Scale bar = 50 pm; FIG.17: Higher magnification 
image of granules showing crystalline form. Scale bar= 15 |jm; FIG.18: Section of periostracal lamella 
showing discrete aragonitic granules in periostracum and the fringe of prismatic aragonite crystals 
along the front of the lamella. Scale bar = 20 |jnn; FIG. 19: Detail of discrete granules embedded in 
periostracum. Scale bar = 2 |jm. 



Mantle Edge 

The mantle edge of L. pensylvanica is thick 
and divided into several folds (Fig. 20). The 
large outer fold (of) is thrown into deep corru- 



gations indicating the potential for consider- 
able extension. Epithelial cells at the margin 
are tall, with nuclei located towards the mid- 
point, but decrease in height dorsally to the 
short, cuboidal cells of the general outer 



SHELL SCULPTURE IN LUCINA PENSYLVANICA 



285 



ome 




FIG. 20. Lucina pensylvanica. Transverse section of anterior mantle edge. Mallory's triple stain. Scale 
bar = 250 |jm. Abbreviations: c, cuticle; fme, corrugated mantle epithelium of outer fold; gz, glandular 
zone; if, inner mantle fold; Im, longitudinal palliai muscles; mf(1) & mf(2), lobes of middle mantle fold; 
of, outer mantle fold; ome, outer mantle epithelium; p, periostracum; pg, periostracal groove; rm, 
radial palliai muscles. 



mantle surface. The outer fold is separated 
from the middle fold by a deep periostracal 
groove, with the forming periostracum lying 
against the outer surface of the middle fold. 
The middle fold is divided into two distinct 
lobes with the outermost of these (mf 1 ) form- 
ing a short, slender lobe whilst the other (mf 
2) is broad and longer. The inner fold (if) is a 
small, low ridge. Cells of the middle lobes are 
shorter than those of the outer fold and pos- 
sess basal nuclei. The epithelium of the middle 
folds is overlain by a thin cuticle (ct) that ex- 
tends almost to the inner fold. The mantle sur- 
face within the inner fold is ciliated. 

Two well-defined bundles of radial muscles 
extend into the outer and middle folds respec- 
tively and a thick bundle of longitudinal palliai 
muscles (Im) is located near the inner fold 
(seen in transverse section in Fig. 20). The 
inner part of the mantle within the inner fold is 



highly glandular with subepithelial gland cells 
opening to the inner mantle surface. Two types 
of gland cell are present; one type, staining 
blue, is located superficially while the other 
dark green type lie more deeply. 

Periodicity of Lamellae 

The lamellae appear regularly spaced but 
measurements taken from acetate peels of 
shell sections show that the increments are 
variable in width and furthermore change with 
age. Figure 21 demonstrates that for eight live- 
collected shells widths between successive 
lamellae increase steadily from around 200- 
450 pm to a maximum (up to 1,800 pm) at 
around 25-30 mm shell height. Thereafter, 
interlamellar spacing becomes much narrower 
but more variable. Observations of the outer 
surfaces of larger, dead-collected shells show 



286 



TAYLOR ET AL. 



Specimen 3 




1.8 • 
I 1.6- 

E 1-4 

tu 

- 1.2 



Specimen 5 




cumulative distance 



cumulative distance 





cumulative distance 



cumulative distance 



1.8 

1.6 ■ 




Specimen 12 




cumulative distance 



cumulative distance 





cumulative distance 



cumulative distance 



FIG. 21 . Lucina pensylvanica, interval between successive lamellae plotted against cumulative length 
around shell circumference for eight individual Lucina pensylvanica. Measurements made from acetate 
peels of transverse sections. 



SHELL SCULPTURE IN LUCINA PENSYLVANICA 



287 




V 



ШШМШ1:'::^г^а-:. 




FIG. 22. Lucina pensylvanica, acetate peel of transverse section of shell 
showing major growth line extending through outer and middle shell layers. 
Scale bar = 500 pm. 



that this change in the interlamellar interval is 
visible on all individuals at shell heights of 
around 22-27 mm. In older individuals the in- 
terval between major growth halts is narrower 
with fewer lamellae (Fig. 21: specimen 12). 



Frequently, major growth halts are marked by 
the secretion of a sequence of several un- 
calcified periostracal extensions (Figs. 22, 23). 
Our interpretation of this growth pattern is that 
shell accretes rapidly and uninterrupted to a 




FIG. 23. Lucina pensylvanica, semidiagrammatic summary drawing 
(based on camera lucida image) of transverse section through shell 
showing successive lamellae and two growth halts where only uncalcified 
periostracal sheets were secreted. Scale bar =1.0 mm. Abbreviations: 
cf, calcified front of lamella; msl, middle shell layer; osl, outer shell layer; 
ps, periostracum above shell; pi, periostracum of lamella; r, ridge in 
outer shell layer; sc, scales; upl, uncalcified periostracal lamellae. 



288 



TAYLOR ET AL. 




FIG. 24. Lucina pensylvanica, semidiagrammatic 
transverse section through a single periostracal 
lannella. Scale bar = 500 |jm. Abbreviations: gz, 
granule zone; osl, outer shell layer; pf, thin 
prismatic ventral fringe to lamella; tp, thin 
periostracum. 



size of around 25 mm. Thereafter, growth rates 
decline and become more variable. Study of 
gonads from our small sample indicates that 
sexual maturity occurs in these bivalves at 
shell heights of around 20-25 mm (Bigatti et 
al., 2004). The major change in shell growth 
pattern may thus coincide with time of first 
spawning. 

A study of growth in Codakia orbicularis 
(Linnaeus, 1758) from the Bahamas showed 
that prominent growth rings in the shell were 
annual (Berg & Alatalo, 1984). Following this, 
the major growth halt lines seen in shell sec- 
tions (Figs. 22, 23) in our sample could be ten- 
tatively interpreted as annual marks and used 
to estimate the ages of the animals. Table 1 
indicates that 20 sectioned shells show be- 
tween 0-4 major lines and the interpretation 
is that the animals vary between one and four 
years old. Proper age estimation should be 
done using marked and calibrated shells but 
this was impossible in the time available for 
the study. 



Sequence of Secretory Events 

The structure of the commarginal lamellae is 
summarized diagrammatically in Figures 23- 
24. Each commarginal lamella represents an 
extension of the mantle beyond the normal shell 
profile. Although the lamellae in L. pensylvanica 
are recurved dorsally, observations at the site 
of secretion show that the lamellae initially 
project more or less straight from the valve 
margin and curve dorsally later (Fig. 4). Thus, 
the mantle is not extended and reflected dor- 
sally as it would be if secreting commarginal 
lamellae formed from normal shell layers as 
seen in other bivalves such as the venerid 
Placamen calophyllum (Philippi, 1836) (Checa, 
2002). 

Initially, the mantle secretes a thin, 
periostracal sheet, followed by calcification of 
the distal portion with spherulitic aragonite crys- 
tals. Calcification of the distal edge of the lamella 
is localised, presumably to groups of cells, so 
that individual scales are formed. At the same 
time the proximal part of the lamella is laid down 
as periostracum, embedded with crystalline 
granules. Finally, the mantle withdraws from the 
extended position, leaving a thin layer of pris- 
matic crystals along the ventral face of the 
lamella. The withdrawal of the mantle is marked 
by a low, commarginal ridge in the shell profile 
(Figs. 12, 24). Following termination of a 
lamella, the periostracum is very thin but gradu- 
ally thickens and becomes densely embedded 
with granules prior to the next lamellar exten- 
sion (Fig. 24). Periodically, there are major 
growth breaks where only extended uncalcified 
periostracal sheets are formed (Fig. 23). 

Comparison with Lucina adansoni and Other 
Species 

An interesting comparison may be made with 
another species, Lucina adansoni (Orbigny, 
1839) from West Africa. This has a thick, 
subspherical shell, sculptured, with closely 
spaced, broad commarginal lamellae about 300 
pm in width (Figs. 25, 26). These are often 
eroded, detached or absent in dead-collected 
shells or museum specimens. Each lamella is 
divided into sections (up to 500 pm long) by 
narrow sutures aligned between successive 
lamellae. Interspaces between lamellae are 
often packed with sediment. Thin sections show 
that the lamellae are similarly constructed to 
those of Lucina pensylvanica (Figs. 27, 28) but 
instead of discrete scales, the calcified units 
are fused laterally to form a continuous ridge 



SHELL SCULPTURE IN LUCINA PENSYLVANICA 



289 



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that is triangular in cross section (Fig. 27). The 
lamellae are tilted towards the ventral shell 
margin rather than recurved dorsally as in L. 
pensylvanica. Each lamella is composed of a 
thick periostracal extension that terminates dis- 



tally in the calcified unit. This is more heavily 
calcified than the scales of L. pensylvanica but 
similarly constructed of spherulitic crystal 
growth. The periostracal extensions are shorter 
than L. pensylvanica but similarly embedded 




27 



FIGS. 25-27. Lucina adansoni. FIG. 25: Right valve (Leiden RMNH 12179). Cape Verde Islands, SB 
of Boa Vista 15°59'N, 22°44'W, depth 36 m. Shell height = 32.5 mm; FIG. 26: Detail of commarginal 
lamellae. Arrows mark suture lines between sections along lamellae. Note sediment grains packed 
into interspaces between lamellae. Scale bar =1.0 mm; FIG. 27: Transverse section of a commarginal 
lamella. Scale bar = 500 ijm. Abbreviations: pi, periostracal lamella; ps, periostracum above shell; s, 
shell; sc, calcareous scale. 



SHELL SCULPTURE IN LUCINA PENSYLVANICA 



291 



with calcareous granules about 2 pm in diam- 
eter (Fig. 28). Also, the periostracum gradu- 
ally increases in thickness between successive 
lamellae and then thins dramatically at their 
termination (Fig. 28). Beneath each lamella the 
outer shell layer forms a steep-faced lip (Fig. 
28) about 200 pm high. In worn shells this is 
the only shell sculpture remaining after the 
lamellae have become detached. 

Calcified periostracal commarginal lamellae 
similar to those of L. adansoni have been ob- 
served (BMNH collections) in the southern 
African species Lucina carnosa Dunker, 1858, 
and L. roscoeorum (Kilburn, 1974). The lamel- 
lae in the latter species are described (Kilburn, 
1974: 340-341, figs, 4, 5) as being "...apically 
imbricate, rendering their crests somewhat 
tabulate (i.e. in cross section each would re- 
semble an inverted "L")..." and "... the crests 
of the lamellae are regularly but superficially 
incised transversely...". 



DISCUSSION AND CONCLUSIONS 

We have demonstrated that the structurally 
complex commarginal shell sculpture of Lucina 
pensylvanica is a form of periostracal calcifi- 
cation, a rather unusual feature amongst 
bivalves. The calcareous granules within the 
periostracum were briefly mentioned by Bottjer 
& Carter (1 980), but no details were given. We 
are not aware of any similar structures in any 
other lucinid. Most Lucinidae lack prominent 
commarginal shell sculpture but two species 
of Lamellolucina, namely L. dentifera (Jonas, 
1846) from the Red Sea and L. gemma 
(Reeve, 1850) from the Philippines possess 
thin, elevated lamellae with spinose edges 
(Taylor & Glover, 2002: fig. 6) reminiscent of 
the lamellae in L. pensylvanica. However, the 
lamellae and spines of Lamellolucina are en- 
tirely calcareous and comprise extensions of 
the outer shell layer rather than periostracal 




FIG. 28. Lucina adansoni, confocal image of transverse section of a 
commarginal lamella. Periostracum red, calcareous components green. Scale 
bar = 1 00 |jm. Abbreviations: gz, granule zone of periostracum; pi, periostracal 
lamella; ps, periostracum above shell; r, ridge in outer shell at base of lamella; 
s, shell; sc, calcareous scale; tp, thin periostracum. 



292 



TAYLOR ET AL. 



structures. Similarly, Lucinisca species from 
the western Atlantic and eastern Pacific pos- 
sess spinose commarginal lamellae, but again 
these are formed from the outer shell layer 
rather than periostracum. 

A diversity of instances of periostracal calci- 
fication has been described from a wide range 
of different bivalve families (Carter & Aller, 
1975; Bottjer & Carter, 1980; Carter et al., 1990; 
Ohno, 1996; Morton, 2000), but none is com- 
parable with L. pensylvanica. Analogous cal- 
careous granules embedded in periostracum 
have been illustrated for the mytilids 
Brachidontes granulatus (Bottjer & Carter, 
1980: fig. 3) and Trichomya hirsuta (Carter & 
Aller, 1975: fig. 1c). Little attention has been 
paid to this calcification either functionally or 
as a set of systematic characters and in many 
cases it is routinely cleaned off specimens. 

Function of the Lamellae 

Although we have no experimental evidence, 
we suggest by analogy with sculpture on other 
bivalves that there might be at least three pos- 
sible functions of the commarginal lamellae. 
These include acting as a sculptural aid to 
burrowing, maintaining stability in the sediment 
and as a possible deterrent to predators. Un- 
usually amongst bivalves, the commarginal 
lamellae of L. pensylvanica are flexible in life 
and this property may have added but un- 
known functional significance. 

As demonstrated by Stanley (1970), some 
lucinids, including Lucina pensylvanica, bur- 
row into the sediment vertically with the hinge 
axis parallel to the sediment surface and rock 
from side to side to gain purchase into the 
sand. Unusually for lucinids, L. pensylvanica 
rotates posteriorly after penetrating the sedi- 
ment to lie with the anterior part of the shell 
uppermost. The recurved, flexible lamellae and 
scales might aid this process but we have no 
experimental evidence similar to that available 
for the divaricate-ribbed Divaricella 
quadrisulcata (Orbigny, 1846) (Stanley, 1970). 
However, the external lamellae of L. 
pensylvanica are easily removed to enable a 
comparison of burrowing performance to be 
made with and without the structures. 

In shallow burrowing bivalves, the ridges and 
spines on the shell surface have been shown 
to reduce the effects of scour and may pre- 
vent dislodgement from the sediment (Bottjer 
& Carter, 1980; Stanley, 1981). We have no 
experimental observations but in Lucina 
pensylvanica and L. adansoni the lamellae are 



extremely effective in trapping sediment close 
to the shell surface (Figs. 8, 26) and in most 
live-collected specimens the interlamellar 
spaces are full of sediment. Compared to other 
lucinids of similar size from the Florida Keys, 
Lucina pensylvanica is the most shallowly 
burrowed, living in medium to coarse, mobile 
sands rather than the thicker T/7a/ass/a-bound 
sediments favoured by Codakia orbicularis and 
Anodontia alba. 

A further possible function of the lamellae 
might be to deter prédation. Strong commar- 
ginal lamellae on the venerid Placamen 
calophylum have been shown to deter shell 
drilling predatory gastropods (Ansell & Morton, 
1 985). Any test of this suggestion would need 
experimental analysis. 

The function of the discrete aragonitic gran- 
ules embedded in the periostracum and 
periostracal extensions of L. pensylvanica and 
L. adansoni is unclear, but they may provide 
additional stiffness to the largely proteinaceous 
part of the lamellae that supports the more 
heavily calcified distal scales or ridge. Further- 
more, the thin calcified layer along the ventral 
face of the lamellae may also provide stiffness 
but, additionally, the differential mechanical 
properties on either face of the lamella may 
cause the lamellae to curve dorsally. 

Systematic Implications of Commarginal 
Lamellae in Lucina 

Although Lucina pensylvanica is thought to 
be widely distributed around the Western At- 
lantic and Caribbean area, from North Caro- 
lina to Brazil (Britton, 1970; Abbott, 1974; 
Bretsky, 1976), it is much more likely that a 
complex of several species exists. J. Gibson 
Smith & W. Gibson Smith (1982) used the 
morphology of the calcareous scales to divide 
the "L. pensylvanica" of the western Atlantic, 
naming three new species on the basis of dif- 
ferences in the form of the scales. These they 
distinguished from L. pensylvanica, assuming 
its type locality to be Florida. All the species 
are similar in general shell morphology but 
dift'er in the form of the calcified periostracal 
lamellae. We have examined the types of the 
Gibson-Smith species and also the syntypes 
of Lucina pensylvanica (Linnaeus, 1758), but 
unfortunately the latter material is heavily worn 
without any trace of lamellae. 

Firstly, Lucina belizana J Gibson-Smith & 
W Gibson-Smith, 1982 (Holotype: BMNH 
1 9801 03) from Belize is characterised by fine, 
close lamellae with delicately pointed, lightly 



SHELL SCULPTURE IN LUCINA PENSYLVANICA 



293 



calcified spines. Secondly, Lucina roquesana 
J Gibson-Smith & W Gibson-Smith, 1982 (Ho- 
lotype and paratype: BMNH 1980105/1-2) 
from Venezuela has calcified periostracal 
lamellae, but these bear broad closely spaced, 
blunt-ended scales that are arranged in a ra- 
dial rows in successive lamellae. Lucina 
podagrina caymanana J Gibson-Smith & W 
Gibson-Smith, 1982 (Holotype: BMNH 
1980104/1) from the Cayman Islands is simi- 
lar to L. roquesana, but the periostracum is 
pale brown and the shell less globose {Lucina 
podagrina podagrina Dall, 1903, is a Pliocene 
fossil species.). J. Gibson Smith & W. Gibson 
Smith (1 982) have undoubtedly highlighted the 
existence of a species complex within the 
former "Lucina pensylvanica", but in our opin- 
ion the taxonomy is even more complicated. 
For example, another species from the west- 
ern Atlantic, Lucina aurantia Deshayes, 1830, 
which is usually synonymised with L. 
pensylvanica (Abbott, 1974; Britton, 1971; 
Bretsky, 1976), has many distinctive shell char- 
acters including size and shape, dentition and 
colour. Some unworn shells have remnants of 
fine, pointed scales. We are confident that this 
is yet another unregarded species. Another 
likely distinct species from the Bahamas has 
been confused with L. pensylvanica but it can 
readily distinguished by extremely fine pointed 
scales (specimens from Blue Hole Cay, off 
Andres Is., collected by P. Mikkelsen and G. 
Hendler). Athorough systematic revision of the 
"Lucina pensylvanica" complex in the western 
Atlantic using live-collected animals with mor- 
phological and molecular analysis is needed. 

On the other side of the Atlantic, Lucina 
adansoni, L. carnosa, and L. rosceorum seem 
to form another possibly related clade, linked 
by the possession of calcified periostracal 
lamellae that form continuous ridges. As we 
have demonstrated, these ridges differ in mor- 
phology but are similarly constructed and thus 
homologous with the lamellae of the western 
Atlantic "L. pensylvanica" group. The relation- 
ships of the two clades need clarification. 

It should be emphasized that in museum 
specimens the periostracal calcified structures 
so diagnostic of these Lucina species are usu- 
ally damaged or in the case of beach collected 
shells, completely worn way. In dried shells, 
the periostracal lamellae become brittle and 
are easily damaged without special curatorial 
care. We recommend wet preservation as the 
most satisfactory method of preserving these 
structures. 



ACKNOWLEDGEMENTS 

We are indebted to Paula Mikkelsen and 
Rüdiger Bieler for organising and inviting us 
to participate in the International Marine Bio- 
logical Workshop. We thank Dave Cooper and 
Tony Wighton for making the thin sections of 
mantle and shells; A. Pallaoro and M. Kraljevic 
helped with analysis of shell sections, and A. 
Zuljevic produced Figure 22. We are grateful 
to E. Gittenberger and J. Goud (Nationaal 
Natuurhistorisch Museum, Leiden) for the loan 
of Lucina adansoni specimens and for permis- 
sion to section a shell. P. Mikkelsen kindly sent 
additional specimens from the Bahamas. Fi- 
nally, thanks are due to our colleagues at the 
workshop for good humoured company and 
advice on sampling locations. Specimens were 
collected under Permit FKNMS-2002.079. The 
International Marine Bivalve Workshop, held 
in the Florida Keys, 19-30 July 2002, was 
funded by U.S. National Science Foundation 
award DEB-9978119 (to co-organizers R. 
Bieler and P. M. Mikkelsen), as part of the 
Partnerships in Enhancing Expertise in Tax- 
onomy (РЕЕТ) Program. Additional support 
was provided by the Bertha LeBus Charitable 
Trust, the Comer Science & Education Foun- 
dation, the Field Museum of Natural History, 
and the American Museum of Natural History. 



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ALLEN, J. A., 1958, On the basic form and ad- 
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ANSELL, A. D. & B. MORTON, 1985, Aspects of 
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BERG, С J. & R ALATALO, 1984, Potential of 
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BIGATTI, G., M. PEHARDA & J. D. TAYLOR, 
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BOTTJER, D. J. & J. G. CARTER, 1980, Func- 
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BRAITHWAITE, С. J. R., J. D. TAYLOR & E. A. 
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Linnaeus, 1758, as type species of Lucina 
Bruguière, 1797 (Mollusca, Bivalvia). Bulletin 
of Zoological Nomenclature, 34: 150-154. 

KILBURN, R. N., 1974, Taxonomic notes on 
South African marine Mollusca (4): Bivalvia, 
with descriptions of new species of Lucinidae. 
Annals of the Natal Museum, 22: 335-348. 

MIKKELSEN, P M. & R. BIELER, 2004, Interna- 
tional Marine Bivalve Workshop 2002: Intro- 
duction and Summary. In: r. bieler & p. м. 



MIKKELSEN, eds.. Bivalve studies in the Florida 
Keys, Proceedings of the International Marine 
Bivalve Workshop, Long Key, Florida, July 
2002. Malacologia, 46(2): 241-248. 

MORTON, В., 2000, The anatomy of Callocardia 
hungerfordi (Bivalvia: Veneridae) and the ori- 
gin of its shell camouflage. Journal of Mollus- 
can Studies, 66: 21-31. 

OHNO, T, 1996, Intra-periostracal calcified 
needles of the bivalve family Veneridae. Bulle- 
tin de l'Institut Océanographique, Monaco, No. 
Spécial 14, 4: 305-314. 

RICHARDSON,C.A., 1993, Bivalve shells: chro- 
nometers of environmental change. Pp. 419- 
435, in: B. MORTON, ed., 777e marine biology of 
the South China Sea. Hong Kong University 
Press, Hong Kong. 

RICHARDSON, С A., 2001, Molluscs as ar- 
chives of environmental change. Oceanogra- 
phy and Marine Biology: an Annual Review, 
39: 103-164. 

STANLEY, S. M., 1970, Relation of shell form to 
life habits of the Bivalvia (Mollusca). Geologi- 
cal Society of America Memoir, 125: 1-296. 

STANLEY S. M., 1981, Infaunal survival: alter- 
native functions of shell ornamentation in the 
Bivalvia (Mollusca). Paleobiology, 7: 384-393. 

TAYLOR, J. D. & E. A. GLOVER, 1 997, A chemo- 
symbiotic lucinid bivalve (Bivalvia: Lucinoidea) 
with periostracal pipes: functional morphology 
and description of a new genus and species. 
Pp. 335-361, in: F. E. WELLS, ed., The marine 
flora and fauna of the Houtman Abrolhos, West- 
ern Australia, Western Australian Museum, 
Perth. 

TAYLOR, J. D. & E. A. GLOVER, 2000, Func- 
tional anatomy, chemosymbiosis and evolution 
of the Lucinida, in: E. M. harper, j. d. Taylor & 
J. A. crame, eds.. The evolutionary biology of 
the Bivalvia. Geological Society of London 
Special Publication, 177: 207-225. 

TAYLOR, J. D. & E. A. GLOVER, 2002, Lamello- 
lucina: a new genus of lucinid bivalve with four 
new species from the Indo-West Pacific. Jour- 
nal of Conchology, 37: 317-336. 

TAYLOR, J. D., E. A. GLOVER & С J. R. 
BRAITHWAITE, 1999, Bivalves with 'concrete 
overcoats', Granicorium anä Samarangia. Acta 
Zoológica, 80: 285-300. 

TAYLOR, J. D., W. J. KENNEDY & A. HALL, 
1973, The shell structure and mineralogy of the 
Bivalvia. II. Lucinacea- Clavagellacea, Conclu- 
sions. Bulletin of the British Museum (Natural 
History) Zoology Series, 22: 225-294. 



Revised ms. accepted 31 October 2003 



MALACOLOGIA, 2004, 46(2): 295-307 

PREDATOR-PREY INTERACTIONS BETWEEN CHIONE ELEVATA 

(BIVALVIA: CHIONINAE) AND NATICARIUS CANRENA 

(GASTROPODA: NATICIDAE) IN THE FLORIDA KEYS, U.S.A. 

Brian Morton^ & Martina Knappe 

ABSTRACT 

Field samples of Chione elevata (Veneridae) were collected from two sites (10 x 25 x 25 
cm quadrats) on the Atlantic coast of the Florida Keys at Long Key State Park, Long Key, 
and Anne's Beach, Lower Matecumbe Key. Shells were divided into living, empty (non- 
drilled) and drilled categories and measured along their greatest lengths. The shells of 
other bivalve species were also so separated. Generally, the С elevata samples from the 
four sites were similar to each other, except with regard to the numbers of drill holes, that 
is, with a significantly higher level of prédation at Long Key. 

The shells of nine species of bivalves, but especially Chione elevata, were drilled in an 
approximately stereotypical manner, that is, equally on both valves, usually postero-dor- 
sally. Most drillings were successful, and the predator is believed to be Naticarius canrena 
- this being the only naticid shown experimentally to be capable of drilling C. elevata in the 
postero-dorsal location. 

Chione elevata possesses an array of potential anti-predator shell defences, notably 
elevated shell lamellae, similar to those of Placamen calophyllum in the Indo-West Pacific. 
Placamen calophyllum is immune from naticid attack except by species of Polinices, which 
drill it at the valve margin. Naticarius canrena attacks С elevata by drilling between the 
shell lamella, at the interspaces, postero-dorsally. In this the thickest region of the bivalve 
shell, the few incomplete drill holes suggest that N. canrena is highly successful. There 
were very few marginal drill holes. Mid-ventrally, C. elevata possesses large, paired palliai 
glands, and it is possible that this is an anti-predator device inhibiting marginal drilling. 
Naticarius canrena, therefore, has to drill this commonest of all intertidal bivalves postero- 
dorsally but does so by attacking its prey selectively, that is, smaller individuals attacking 
smaller bivalves so that drill hole size is always narrower than interlamellar distance. 

Key words: Predator-prey interactions, Naticarius, Chione, Florida Keys, drill hole posi- 
tion, shell defenses. 



INTRODUCTION 

That many naticid gastropods drill holes in 
bivalve and some gastropod shells to access 
the tissues Inside is well documented (Taylor, 
1998) and has led to the concept of an "arms 
race" between predator and prey as the former 
strives to overcome the defences of the latter 
and the bivalve seeks to improve its ability to 
defend itself against the gastropod (Vermeij, 
1978, 1980). Species of Austroginella 
(Marginellidae) drill holes in their prey (Pon- 
der & Taylor, 1992), and it has been shown 
recently that juvenile Nassarius festivus 
(Nassariidae) can also drill the shells of con- 



specifics (Morton & Chan, 1997). There are, 
however, two major groups of intertidal drill- 
ing predatory gastropods: representatives of 
the Muricidae, generally, on rocky shores and 
species of Naticidae on soft ones. On Malay- 
sian shores, Anadara granosa is drilled by both 
Natica maculosa (Naticidae) and Thais 
carinifera (Muricidae) (Broom, 1981 ). Muricids 
drill vertically straight holes in shells of their 
prey, naticids countersunk ones, and both at- 
tack in a stereotypical way to gain access. 
Thus, Arua (1989) was able to show for an 
Eocene molluscan community from Nigeria 
that the most common prey of naticids was 
the epifaunal, strongly ornamented bivalve 



'The Swire Institute of Marine Science, The University of Hong Kong, Hong Kong, China; present address: Department of 
Aquatic Zoology, The Western Australian Museum, Perth, Western Australia; prof_bsmorton@hotmail.com 
^Institute of Zoology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria 



295 



296 



MORTON & KNAPP 



Tivelina newtoni, whereas the principal prey 
of muricids was the epifaunal, but also strongly 
ornamented gastropod Bonellitia amekiensis. 
Today, on a southwestern Australian rocky 
shore, the muricid Lepsiella flindersi attacks 
the mussel Xenostrobus pulex in a stereotypi- 
cal manner on the posterodorsal side of left 
and right valves equally (Morton, 1999). Simi- 
larly, in a southwestern Australian marsh, 
Lepsiella vinosa attacks another mussel, X. 
inconstans, also in a stereotypical manner 
posteriorly (Morton, 2004). In the Azores, Thais 
haemastoma drills the intertidal mussel 
Trichomusculus semigranatus only at the pos- 
terior margin (Morton, 1995). On Hong Kong 
shores. Morula musiva attacks an array of 
shallow-water bivalves by drilling at specific 
locations on each of their shells (Harper & 
Morton, 1997). 

Similar observations have been made upon 
naticids. In Malaysia, Berry (1982) showed that 
Natica maculosa drills the trochid gastropod 
Umbonium vestiarium. In Europe, Ansell 
(1982a, b) demonstrated that the bivalves 
Venus striatula and Tellina tenuis are drilled 
by Polinices alderi, whereas on the coast of 
Massachusetts, Edwards & Huebner, (1977) 
showed that Mya arenaria is drilled by P. 
duplicatus. 

Virtually all species of bivalves are vulner- 
able to either muricid or naticid attack and 
there has been much research conducted on 
the predator/prey relationship between them. 
This is because both are readily identifiable in 
the fossil record (Kitchell et al., 1981), drilled 
shells are obvious in modern field-collected 
bivalve assemblages, and the stereotypical 
feeding behaviour of the predator makes both 
it and its prey eminently suitable experimental 
animals. For example, shell height/length pa- 
rameters can be correlated readily with tissue 
weights. Thus, Taylor (1970), examined the 
feeding habits of predatory boring gastropods 
in a Tertiary mollusc assemblage, and Negus 
(1 975) was able to analyse the drill holes made 
by Natica catena in a collection of Donax 
vittatus shells. Morton (1990a) showed that in 
the Azores, the commonest shallow subtidal 
bivalve, Ervilia castanea, was drilled by 
Polinices alderi in a stereotypical, posterior, 
position. Ansell & Morton (1987) demonstrated 
that various species of Indo-West Pacific 
naticids attacked different species of bivalve 
prey in a different sequence according to in- 
tra-specific size, inter- and intra-specific shell 
thicknesses and at different locations on the 



shells. In an elegant series of laboratory stud- 
ies, Ansell (1982a, b, c) examined the ener- 
getic relationship between P. alderi (and P. 
catena) and its bivalve prey. 

With respect to the Florida Keys, Roopnahne 
& Beussink (1999) suggested that during a 
Pliocene-Pleistocene extinction, Chione erosa 
Dall, 1903, was replaced by С cancellata 
Linnaeus, 1767. When this occurred, there 
was an increase in the size of Chione selected 
by naticid predators, and С cancellata re- 
sponded to this by significantly increasing rela- 
tive shell thickness. Mikkelsen & Bieler (2000) 
identified three species of Chione from the 
Florida Keys, with C. cancellata being the most 
widespread. The paper on western Atlantic 
Chione by Roopnahne & Vermeij (2000), how- 
ever, suggests that С cancellata is a Caribbean 
species, whereas the Floridian species is C. 
elevata (Say, 1822). There is, thus, a large lit- 
erature on what was considered, in Florida and 
north to North Carolina, to be C. cancellata, but 
which actually refers to С elevata. 

Species of Chione (Chioninae) are charac- 
terized by elevated concentric lamellae on the 
shell valves. These are produced periodically 
and may represent a defense against drilling 
predators. For example, Atlantic species of 
Chione (Roopnarine & Vermeij, 2000) are very 
similar to Indo-West Pacific species o^ Bassina 
and Placamen (Matsukuma & Yoosukh, 1988), 
and it has been shown for P. calophyllum 
(Philippi, 1836) that its shell lamellae protect 
it from side-drilling naticid predators (Ansell & 
Morton, 1985; Morton, 1985). Polinices 
tumidus could, however, attack it by edge drill- 
ing. Moreover, when the lamellae of P. 
calophyllum were removed, side-drilling preda- 
tors could attack it with impunity. This may not 
be the case in species of Atlantic Chione, how- 
ever, as Roopnarine & Beussink (1999, fig. 1) 
illustrate a shell of Pliocene С erosa with a 
drill hole located between adjacent lamellae. 

During a ten-day period of study in the 
Florida Keys, one of the most commonly en- 
countered species of bivalves was Chione 
elevata. In field samples, moreover, many C. 
elevata shells were found to have been drilled 
by a naticid predator. It was decided, there- 
fore, to undertake a study of this bivalve, its 
drill holes and try to identify the predator that 
made them. The overall question that we at- 
tempted to answer, however, was: like 
Placamen calophyllum, do the shell lamellae 
of С elevata protect it from naticid prédation 
(Morton, 1985)? If not, why not? 



CHIONE ELEVATA-NATICARIUS CANRENA INTERACTIONS 



297 



MATERIALS AND METHODS 

For a short period from 20-29 July 2002, a 
research visit was paid to the Florida Keys. It 
was determined to research the anatomy of 
Chione elevata, undertake an analysis of drill 
holes in the shells of field-collected samples 
of bivalves and attempt to determine which 
predator made them. 

Anatomy 

Living individuals of Chione elevata were 
dissected. A single specimen was also fixed 
in 5% formalin, decalcified and, following rou- 
tine histological procedures, sectioned trans- 
versely at 6 pm, and alternate slides stained 
in Ehrlich's haematoxylin and eosin and 
Masson's trichrome. 

Field Studies 

On the southeastern coast of the keys, that 
is, the Atlantic side, two shores were chosen 
for study after reconnaissance of sites on both 
sides and when very few Chione elevata were 
collected from the Gulf of Mexico side. The 
two chosen shores were Long Key State Park 
on Long Key and Anne's Beach on Lower 
Matecumbe Key, that is, station numbers 
IMBW-FK-623 and IMBW-FK-638, respec- 
tively (Mikkelsen & Bieler, 2004: fig.1 ). At each 
of two sampling locations at these two sites, 
ten 25 X 25 cm quadrats were laid haphaz- 
ardly on the sandy shore covering the full 
range of tidal heights. A total of, thus, 40 quad- 
rats was examined by excavating their con- 
tents to a depth of ~ 10 cm and sieving them 
through a one millimeter mesh sieve. All living 
and empty bivalve shells (plus any naticid 
shells) were sorted from the samples. These 
were analyzed in the following manner. 

Shells and living individuals of Chione 
elevata were measured along their greatest 
lengths; in the case of empty valves, only right 
ones were measured. Empty valves were ex- 
amined for drill holes. Where these were en- 
countered, the following records were made: 
(i) which valve; (ii) the location of each drill 
hole was plotted on a master illustration of the 
valves; (iii) the outer diameter of the drill hole; 
and (iv) the distance between adjacent lamel- 
lae at the drilling location. 

All other bivalve shells with drill holes were 
identified to species and the locations of them 
on the left and right valves similarly plotted on 



master illustrations. Although several other 
species of naticids are known to inhabit the 
shallow waters of the Florida Keys (Lyons & 
Quinn, 1995; Bieler & Mikkelsen, pers. 
comm.), the only shells ever collected were 
those of Naticarius canrena (Linnaeus, 1758). 
Similarly, but one living individual of this spe- 
cies was collected from any habitat during the 
ten-day period of study. Voucher specimens 
of Chione elevata (108 preserved individuals) 
and N. canrena (16 shells) have been depos- 
ited in the collections of the Natural History 
Museum, London (Reg. No's: 20030605 and 
20030609, respectively). 

Statistical Analyses 

The dataset comprising the numbers of liv- 
ing, empty and drilled shell valves of Chione 
elevata among the four locations was tested 
for normality and homogeneity of variances 
using the Shapiro-Wilk test and Levene sta- 
tistic, respectively, both at the 0.05 level of sig- 
nificance before ANOVA. One-way ANOVAs 
were performed on the dataset to test the null 
hypothesis that there were no significant dif- 
ferences in these variables among locations. 
Where differences were detected, Student's 
Newman-Keuls (SNK) tests were carried out 
to identify where the differences lay. For the 
drilled shells, the relationships between shell 
length and (i) lamella distance and (ii) drill hole 
diameter were evaluated by regressions. 



RESULTS 



Anatomy 



Shell: The shell of Chione elevata is illus- 
trated from various perspectives in Figure 1 . 
In general terms, the shell is approximately 
equivalve and equilateral, and thus 
isomyarian, but slightly pointed posteriorly. 
There is a fine radial sculpture, and each valve 
is strongly commarginally lamellate (Fig. 1A). 
In anterior view (Fig. IB), there is a small an- 
terior lunule (as defined by Carter, 1967) each 
valve here interlocking by means of marginal 
denticles. In dorsal view (Fig. 1С), the poste- 
rior escutcheon (also as defined by Carter, 
1967) is much longer than the lunule and in 
the case of the former but not the latter, the 
dorsal edge of the left valve overlaps that of 
the right. This is also illustrated in posterior 
view (Fig. ID). Finally, the shell valve margins 




MORTON & KNAPP 



В 







10 mm 



FIG. 1. Chione elevata. The shell as seen from: A, the right side; B, anteriorly; C, dorsally; D, poste- 
riorly and E, ventrally. 



are interlocked ventrally by the expanding ra- 
dial ribs (Fig. 1E). The shell valves of С 
elevata are thus very difficult to separate. 

Siphons and Mantle Cavity: A living indi- 
vidual of Chione elevata is illustrated in Fig- 



ure 2 fronn the right side. The shell is, as de- 
scribed, radially striate and commarginally 
lamellate. Rays of light purple pattern the gen- 
erally white-cream outer surface. Anteriorly, 
there is a large digging foot. Posteriorly, there 
is a pair of separated siphons. The exhalant 



CHIONE ELEVATA-NATICARIUS CANRENA INTERACTIONS 



299 




FIG. 2. Chione elevata. An individual seen from the right side 
with siphons and foot extended. 



siphon is conical and a ring of short, thin ten- 
tacles sub-apically surrounds its aperture. The 
inhalant siphon is much larger in diameter, but 
shorter and is fringed apically by a circlet of 
long siphonal tentacles and papillae. Mid-ven- 
trally, the mantle possesses a line of papillae 



and palliai fusions, where they occur, are of 
the inner folds only, that is, type A (Yonge, 
1982). 

Jones (1979) provided additional details on 
the gross anatomy of Chione elevata (as C. 
cancellata) and other chionine species. 



300 



MORTON & KNAPP 




PRM 



MMF IMF(2) 



FIG. 3. Chione elevata. A transverse section through the left mantle 
lobe at the margin. H, haemocoel; IMF[1], inner component of the inner 
mantle fold; IMF[2], outer component of the inner mantle fold; MMF, 
middle mantle fold; OMF, outer mantle fold; P, periostracum; PL, pallia! 
line; PRM, palliai retractor muscle; PN, palliai nerve; RT, rejection tract; 
SC, secretory cell; TF, transverse fibres; VC, vacuolated cell. 



CHIONE ELEVATA-NATICARIUS CANRENA INTERACTIONS 



301 



Mantle Margin: In transverse section, the 
general surface of the mantle comprises epi- 
thelia, cross-connected by transverse fibers 
(Fig. 3, TF) and enclosing a capacious hae- 
mocoel (H). Jones (1979, fig. 21) provided a 
simple illustration of a transverse section 
through the mantle margin of Chione elevata 
(as С cancellata). Here, that of C. elevata is 
shown to be very large and complex. It com- 
prises the usual three folds (Yonge, 1 982). The 
outer fold (OMF) is large, and its inner sur- 
face secretes a very thin periostracum (P) 
against the template of the outer surface of 
the middle mantle fold (MMF). The template 
is a long thin sheet of tissue arising from the 
major element of the middle fold. The inner 
fold is divided into two components, a smaller 
inner (iMF[1]) and a larger outer (IMF[2]) that 
also gives rise to the mantle papillae which 
fringe the mantle margin especially ventrally. 

The greatest component of the mantle mar- 
gin of Chione elevata is a large gland com- 
prising elongate vacuolated cells (VC) that lie 
beneath the inner epithelium and stain a light 
red in Masson's trichrome: apically, such cells 
are actively secretory (SC). The gland does 
not therefore appear to be secreting mucus to 
bind up pseudofaeces: it is too large for such 
a purpose and the habitat of coarse coral sand 
would not necessitate such rejectory capabili- 
ties, but otherwise its function is unknown. The 
illustrations of the sectioned ventral mantle 



margins of C. elevata (as C. cancellata) and 
С undatella (Sowerby, 1835) by Jones (1979, 
figs. 21, 24) do not identify whether such a 
gland is present although they appear swol- 
len as described herein. 

Field Studies 

Overall, 2.3 (± 2.4) living, 13.7 (± 9.6) empty 
shells and 4.5 (± 4.40) drilled right valves of 
Chione elevata were encountered per quad- 
rat (Table 1 ). The numbers of living, empty and 
drilled valves differed among locations, how- 
ever (Table 2A, p < 0.05). 

Results of a Student-Newman-Keuls test 
(Table 3) showed that the two Anne's Beach 
samples were similar to Long Key State Park 
A, but also that the two Long Key State Park 
locations were similar to Anne's Beach A in 
terms of living Chione elevata. Similar results 
were obtained for the empty shells, that is, the 
two Anne's Beach sites were similar, as were 
the two Long Key State Park locations but 
these were also similar to Anne's Beach B. 
Only with regard to the drilled valves was a 
clear intersite difference obtained, that is, the 
two Long Key State Park locations were simi- 
lar (6.0 and 8.8 drilled valves-quadrat''), as 
were the Anne's Beach ones (1.4 and 1.7 
drilled valves-quadrat"^ ) (Table 1 ), but also that 
the two pairs of locations were different from 
each other (Table 3). That is, there appears to 



TABLE 1. Results of the statistical analysis of the Chione elevata shell dataset (living, empty 
and drilled) obtained from the four stations in the Florida Keys (10 x 25 cm x 25 cm quadrats). 



Shell Type 


Location 


Mean 


Standard Deviation 


Living 


Long Key State Park Site A 


2,7 


1.70 




Long Key State Park Site В 


3.4 


2.76 




Anne's Beach Site A 


2.5 


2,99 




Anne's Beach Site В 


0.6 


0,70 




Average 


2.3 


2.39 


Empty 


Long Key State Park Site A 


8,9 


8.21 




Long Key State Park Site В 


9,9 


8,14 




Anne's Beach Site A 


21.4 


8,62 




Anne's Beach Site В 


14.7 


9.04 




Average 


13.7 


9,59 


Drilled 


Long Key State Park Site A 


1.4 


1,07 




Long Key State Park Site В 


1.7 


1.77 




Anne's Beach Site A 


6,0 


2.98 




Anne's Beach Site В 


8,8 


5.31 




Average 


4.5 


4,40 



302 



MORTON & KNAPP 



TABLE 2. Results of a one-way ANOVA on the Chione elevata shell dataset (living, empty and drilled) 
obtained from the four stations in the Florida Keys. 



Shell Type 



Comparison 



df 



Mean Square 



F Significance (p = 0.05) 



Living 



Empty 



Drilled 



Between groups 


3 


14.33 


Within groups 


36 


4.98 


Total 


39 




Between groups 


3 


325.89 


Within groups 


36 


72.45 


Total 


39 




Between groups 


3 


127.29 


Within groups 


36 


10.34 


Total 


39 





4.50 



12.32 



0.049 



0.009 



0.000 



be a higher level of drilling prédation, possibly 
by Naticarius canrena, at the Long Key State 
Park than at the Anne's Beach locations. 

Location of Drill Holes: In addition to Ciiione 
elevata, the field-collected bivalve shells with 
naticid drill holes comprised nine species. 
Outlines of the shells of these species are il- 
lustrated in Figure 4. Also illustrated are the 
positions of the drill holes on both the left (o) 
and right (•) valves. 

The two lucinids, Ctena orbiculata (Montagu, 
1808) and Lucinisca nassula (Conrad, 1846), 
were both drilled close to the ventral shell 
margin. All seven other bivalves, that is, the 
glycymehd Tucetona pectinata {GmeWn, 1791), 
the carditid Pleuromeris tridentata (Say, 1832), 
the cardiid Laevicardium mortoni (Conrad, 
1830), the venerid Pitar simpsoni {DaW, 1895), 
and three tellinids - Tellina mera Say, 1834, 
T. iris Say, 1822, and T. similis J. Sowerby, 
1806, were all side drilled in most cases close 
to the umbones and, again mostly, posterior 
to them. 

Figure 5 illustrates diagrammatically the 
empty right (A) and left (B) valves of Ciiione 
elevata with the positions of the total numbers 
of drill holes on them identified. It is obvious 
that there is, first, an approximately equal dis- 
tribution of drill holes (and attempts) between 
the two valves, that is, right 99, left 98. Sec- 
ond, most of the drill holes were, again, ap- 
proximately equally, in terms of the two valves, 
distributed around the postero-dorsal region 
of the shell with a few scattered over the rest 
of the surface. A third important point is that 
there are very few failed drill holes, that is, 
two in the right and four in the left valves. 
Fourth, only a very few of the drill holes were 
over the shell lamellae, that is, two on the right 



and four on the left valves and, in the latter case, 
these were also at the marginal lamellae. 

Stiell Measurements: Figure 6A shows the 
relationship between shell length and the dis- 
tance between adjacent lamellae demarcat- 
ing a drill hole site on the shell of Chione 
elevata. The relationship is linear, suggesting 
that the predator is, for a prey of a particular 
size, choosing a site appropriate for drilling. 
That is, smaller predators (making smaller drill 
holes) chose positions on the C. elevata shells 
where there are smaller interlamellar distances 
in these younger, smaller bivalve prey. Con- 
versely, larger predators (making larger drill 
holes) chose positions on shells where there 
are larger interlamellar distances, that is, older, 
larger bivalve prey. This conclusion is substan- 
tiated in Figure 68, where it is further shown 
that drill hole diameter is correlated positively 
with shell length in C. elevata. That is, smaller 
predators, making smaller drill holes, attack 
smaller individuals of C. elevata. 

In summary, therefore, it would appear from 
this analysis of naticid-drilled shells that 



TABLE 3. Student-Newman-Keuls test grouping 
of the four Florida Keys locations (1 = Long Key 
State Park Site A, 2 = Long Key State Park Site 
B, 3 = Anne's Beach Site A, 4 = Anne's Beach 
Site B) into subsets in terms of the mean num- 
ber of living, empty and drilled shells of Chione 
elevata in an ascending order. 



Shell Type 



Subsets 



Living 
Empty 
Drilled 



4=3=1<3=1=2 
1=2=4<4=3 
1 =2<3 = 4 



CHIONE ELEVATA-NATICARIUS CANRENA INTERACTIONS 



303 



Tellina 
mera 





Tucetona 
pectinata 



Pitar 
simpsoni 





Pleuromeris 
tridentata 



Laevicardium 
mortoni 




Tellina 

iris 




Tellina 
similis 





Ctena 
orbiculata 



о Left valve 
• Right valve 




Liicinisca 
nassula 



FIG. 4. The positions of drill holes on the left and right valves of empty shells of nine other species of 
bivalves collected with the Chione elevata field samples. Also shown is a ventral view of Naticarius 
canrena and the drill hole one individual of this predator made in the shell of an aquarium-held 
Chione elevata. 



Chione elevata (and probably other resident 
bivalves) is attacked in a stereotypical man- 
ner at the posterodorsal margin, and at inter- 
lamellar spaces. There were virtually no 
examples of either marginal or lamellar drill- 



ing; that is, the predator avoids the structur- 
ally very similar margins and lamellar edges, 
unlike the situation of Placamen calophyllum 
in the Indo-West Pacific (Ansell & Morton, 
1985). 



304 



MORTON & KNAPP 





FIG. 5. Chione elevata. The composite pattern of drill holes in the field-collected left and right valves 
of ernpty shells (o non-lamella borehole, • attempted borehole, ♦ lamella borehole). 



J.3 -. 


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2 
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f 1-4- 

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у = 0.0406х + 0.2849 

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15 20 

Shell length (mm) 



30 



FIG. 6. Chione elevata. The relationships between shell length and A, the distance between two 
lamellae at the position of an interlamellar drill hole and B, the outer diameter of the drill hole. 



CHIONE ELEVATA-NATICARIUS CANRENA INTERACTIONS 



305 



Laboratory Studies 

Although 16 empty shells of Naticarius 
canrena were collected from the 40 quadrats, 
only one living individual was obtained. This 
was placed in an aquarium with small individu- 
als of Chione elevata and one drill hole was 
made, on the right valve postero-dorsally (Fig. 
4, C. elevata shell), that is, in the position typi- 
cal of the locations of the field collected 
bivalves of this species (Fig. 5). No other 
naticid species (not even shells) was ever col- 
lected alive. It thus seems possible, at least, 
that N. canrena made the drill holes on all the 
field-collected bivalves, but especially C. 
elevata, collected during the course of this 
study. 



DISCUSSION 

Chione elevata has a shell that, superficially, 
would appear to offer much protection. Pro- 
tective characteristics include a tightly fitting 
margin, with ventrally interlocking ribs, similar 
interlocking marginal denticles at the antero- 
dorsal lunule, a marginally overlapping es- 
cutcheon and internally large, strong cardinal 
teeth (Jones, 1979). Closed С elevata are 
extremely difficult to open, even by the authors! 
Each adductor muscle also has a large slow 
component for sustained adduction, and the 
palliai line is deeply inset within the shell mar- 
gin. With the commarginal lamellae on the 
outsideof a solid, thick shell, C. e/e\/aia would, 
superficially, appear to be impregnable. Such 
characters are also possessed by the Indo- 
West Pacific Placamen calophyllum and are 
very effective in protecting it from naticid 
predators (Ansell & Morton, 1985, 1987). 

Lamellar protection is not afforded to Chione 
elevata, however, and the only predator iden- 
tified as being capable of attacking and drill- 
ing this species at the study sites on the Florida 
Keys is Naticarius canrena and it does this at 
the interlamellar spaces. This is the opposite 
of the situation described for the Indo-West 
Pacific Placamen calophyllum, which is virtu- 
ally immune from attack by side-drilling 
naticids because of the shell lamellae but is 
vulnerable to the edge-drilling Polinices 
tumidus, as described by Ansell & Morton 
(1985). It is strange therefore that the lamel- 
lae of the Atlantic C. elevata do not confer any 
protection from N. canrena and further strange 
that there were very few identified attempts to 



drill the bivalve at the valve margins, espe- 
cially ventrally. The ventral mantle margin has 
a huge gland discharging onto the inner but 
widely open surfaces of the mantle: does the 
secretion from this constitute a further defen- 
sive adaptation? In the absence of any knowl- 
edge about the composition of the secretion 
from this gland, it is impossible to hypothesize 
further, but it does not stain for simple mucus. 

As noted above, it is thought possible that 
the major predator of Chione elevata on the 
Atlantic side of the Florida Keys is Naticarius 
canrena. In this study, one such aquarium-held 
predator did attack the bivalve successfully in 
the position identified for the great majority of 
the field-collected empty and drilled valves, 
that is, postero-dorsally. It is not known for 
certain, however, if this species made the drill 
holes at the marginal lamellae. However, since 
very few drillings represented failed attempts, 
it is clear that if N. canrena is the predator, it 
has apparently successfully overcome the 
seemingly impenetrable defenses of C. elevata 
by attacking it at the posterodorsal interlamel- 
lar spaces. Younger predators also attack 
younger prey as with juvenile Polinices 
duplicatus feeding on Gemma gemma, that is, 
drill hole diameter is related directly to preda- 
tor size (Wiltse, 1980). This has also been 
demonstrated for Polinices lewisii feeding on 
the littleneck clam, Protothaca staminea by 
Peitso et al. (1994). A thick shell characteristi- 
cally protects bivalves from drilling predators, 
for example, Corbula crassa in Hong Kong 
(Morton, 1990b), although Borzone (1988) 
showed that a species of Polinices, as dem- 
onstrated here for N. canrena, selectively 
drilled its prey, Venus antiqua, in the thickest 
region of the shell, that is, umbonally. Although 
no measurements were taken of C. elevata 
shell thickness at the drill hole sites, the 
posterodorsal region is the thickest, and N. 
canrena has therefore clearly overcome the 
bivalve's defenses by selective drilling accord- 
ing to relative prey size and shell location char- 
acteristics. 

The most interesting question derived from 
this study, however, is: how is it that Indo-Pa- 
cific species of Bassina and Placamen have 
evolved strong shell lamellae that protect them 
from side-drilling species of Natica but have 
been overcome by edge-boring species of 
Polinices (Morton, 1985; Ansell & Morton, 
1 985, 1 987), whereas the same shell defences 
of Chione offer no protection from side-drill- 
ing Naticarius in the western Atlantic? 



306 



MORTON & KNAPP 



ACKNOWLEDGEMENTS 

The International Marine Bivalve Workshop, 
held in the Florida Keys from 19-30 July 2002, 
was funded by the U.S. National Science Foun- 
dation award DEB-9978119 (to organizers R. 
Bieler and P. M. Mikkelsen), as part of the Part- 
nerships in Enhancing Expertise in Taxonomy 
[РЕЕТ] program. The Bertha LeBus Charitable 
Trust, the Comer Science and Education Foun- 
dation, the Field Museum of Natural History, 
and the American Museum of Natural History 
provided additional support. We are grateful to 
Paula Mikkelsen (American Museum of Natu- 
ral History, New York) and RiJdiger Bieler (Field 
Museum, Chicago) and their colleagues and 
students for organizing the workshop and much 
practical help and kindness during it. MK also 
thanks the organizers for providing a travel grant 
to Florida. BM thanks Dr. Katherine Lam (The 
Swire Institute of Marine Science, The Univer- 
sity of Hong Kong) for statistical advice and 
help. 



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ANSELL, A. D., 1982b, Experimental studies of 
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ARUA, I., 1989, Gastropod predators and their 
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BROOM, M. J., 1981, Size-selection, consump- 
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CARTER, R. M., 1967, On the nature and defini- 
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EDWARDS, D. С & J. D. HUEBNER, 1977, 
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HARPER, E. M. & B. MORTON, 1997, Muricid 
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JONES, С С, 1979, Anatomy of Chione 
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KITCHELL, J. A., С H. BOOGS, J. F. KITCHELL 
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LYONS, W. G. & J. R QUINN, JR., 1995, Appen- 
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MATSUKUMA, A. & W. YOOSUKH, 1988, Living 
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MIKKELSEN, P M. & R. BIELER, 2000, Mahne 
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Revised ms. accepted 4 February 2004 



MALACOLOGIA, 2004, 46(2): 309-326 

OYSTERS OF THE CONCH REPUBLIC (FLORIDA KEYS)" 

A MOLECULAR PHYLOGENETIC STUDY OF PARAHYOTISSA MCGINTYI 

TESKEYOSTREA WEBERI AND OSTREOLA EQUESTRIS 

Lisa Kirkendale\ Taehwan Lee^, Patrick Baker^ & Diarmaid Ó FoighiP 

ABSTRACT 

We investigated the evolutionary relationships of three species of Florida Keys oysters, 
Parahyotissa mcgintyi, Teskeyostrea weberi, and Ostreola equestris, using nuclear and 
mitochondrial (mt) phylogenetic trees. Both 28S (nuclear) and 16S (mt) ribosomal gene 
trees consistently recovered a paraphyletic Parahyotissa in which Я mcgintyi, the type 
species, was robustly sister to a tip clade containing P numisma and Hyotissa hyotis. This 
topology implies that there is no phylogenetic basis for Parahyotissa Harry, 1985, and we 
therefore recommend that all hyotissinid taxa be returned to the genus Hyoi/ssa Stenzel, 
1971. Phylogenetic placement of T. и/еЬел/ within brooding oyster mt 16S gene trees con- 
clusively demonstrated that it is a distinct ostreinid lineage, lacking any obvious candidate 
sister species, and falsified the hypothesis that it is a free-living ecomorph of the sponge 
commensal Cryptostrea permollis. Population-level mt COI sequence analysis of Ameri- 
can Ostreola equesths and New Zealand Ostrea aupouria revealed that these two globally 
disjunct ostreinids, though remarkably close relatives, are reciprocally monophyletic sister 
taxa. Unlike a large fraction of the Floridian nearshore marine biota, O. equesths shows no 
evidence of a vicahant phylogenetic break distinguishing Gulf of Mexico and Atlantic popu- 
lations. Our results imply that its present day Gulf/Atlantic distribution has been achieved 
by range extension from source Atlantic populations followed by a demographic growth 
pulse in the new Florida Keys/Gulf of Mexico habitats. Ostreola equesths individuals dis- 
play an impressive range of shell morphs and coloration, some externally resembling T. 
weben, and we present a plate of genotyped individuals that document this diversity. 

Keywords: Ostreidae, Gryphaeidae, systematics, biogeography, Florida, molecular phylogeny. 



INTRODUCTION 

The Florida Keys archipelago extends 362 
km SW from the tip of peninsular Florida, sepa- 
rating Florida Bay from the Straits of Florida. 
This subtropical island chain represents the 
exposed surface layer of a much larger car- 
bonate platform and has a rich bivalve fauna, 
estimated at approximately 325 species 
(Mikkelsen & Bieler, 2000). The strategic goal 
of the International Marine Bivalve Workshop, 
held at the Keys Marine Laboratory (Long Key) 
from 19-30 July 2002, was to expand our 
knowledge oftargeted segments of this fauna. 
We elected to study the local oyster taxa, or 
at least that fraction accessible by wading, 



snorkeling and SCUBA diving during our lim- 
ited sampling window. 

Although oysters are among the most stud- 
ied marine invertebrate taxa, their taxonomy 
and systematics is still fraught with uncer- 
tainty due to their xenomorphic post-larval 
growth patterns (Ranson, 1951; Quayle, 
1988; Yamaguchi, 1994), relative dearth of 
tractable anatomical characters, and exten- 
sive anthropogenic global transfer (Dinamani, 
1971; Edwards, 1976; Buroker et al., 1979; 
Chew, 1990; Carlton & Mann, 1996). Harry's 
(1985) ambitious taxonomic revision, based 
largely on morphology, represents the most 
recent comprehensive reclassification of liv- 
ing oysters. Subsequently, a number of de- 



^epartment of Malacology, Florida Museum of Natural History, University of Florida, Gainesville, Florida 32611, U.S.A. 
Museum of Zoology and Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor Michiaan 
48109, U.S.A. ' 

^Department of Fisheries and Aquatic Sciences, University of Florida, 7922 NW 71st St, Gainesville, Florida 32653 USA 
Corresponding author: diarmaid@umich.edu 



309 



310 



KIRKENDALE ETAL. 



tailed paleontológica! studies (Malchus, 1990; 
Malchus & Aberhan, 1998; Dhondt et al., 
1999), together with a steady trickle of mo- 
lecular phylogenetic analyses (Reeb & Avise, 
1990; Littlewood 1994; Banks et al., 1993, 
1 994; Anderson &Adlard, 1994; Hare & Avise, 
1998; Boudry et al., 1998; Ó Foighil et al., 
1998; Jozefowicz & Ó Foighil, 1998; Ó Foighil 
& Taylor, 2000; Campbell, 2000; Steiner & 
Hammer, 2000; Lam & Morton, 2001; Giribet 
& Wheeler, 2002; Lapegue et al., 2002), have 
significantly refined our understanding of many 
aspects of ostreoidean evolution and system- 
atics. 

The ostreoidean fauna of the Florida Keys 
is atypical in that the ecologically dominant 
cupped oysters of the adjacent Caribbean and 
Atlantic seaboards are almost completely ab- 
sent. Although isolated records occur in the 
Keys (Mikkelsen & Bieler, 2000), we did not 
encounter specimens of either the temperate 
Crassostrea virginica (Gmelin, 1791), or the 
tropical С rhizophorae (Guilding, 1828) 
(Ostreidae, Crassostreinae). Crassostrea 
virginica populations are critically dependent 
on estuarine conditions, absent from the Keys, 
where salinity variation acts to reduce biotic 
competition and parasitism (Galtsoff, 1964; 
Ford & Tripp, 1996; Shumway, 1996). 

Our sampling efforts yielded three distinct 
oyster groupings. By far the most common 
were small flat oysters (Ostreidae, Ostreinae), 
displaying an impressively diverse and over- 
lapping range of shell morphology and colora- 
tion. Based on shell phenotype, many of these 
were readily identifiable as either Ostreola 
equesths (Say, 1834) or Tesl<yostrea weberi 
(Olsson, 1951); however, quite a few individu- 
als were difficult to place with confidence. 
During dives, we encountered specimens of 
the gorgonian-associated Dendostrea irons 
(Linné, 1758) (Ostreidae, Lophinae) and the 
equally distinctive Paraliyotissa mcgintyi 
Harry, 1985 (Gryphaeidae, Pycnodonteinae). 
We focused our efforts on the gryphaeid and 
flat oysters as they require the most system- 
atic attention. In particular, we addressed the 
following four questions. 

Systematic Placement of Parahyotissa 
mcgintyi Harry, 1985 

Harry (1985) reorganized the gryphaeid 
(pycnodonteinid) tribe Hyotissini into the mo- 
notypic Indo-Pacific genus IHyotissa and a new 
genus Parahyotissa (containing three subgen- 



era and four species) which includes the tropi- 
cal Atlantic type species P. (Paraliyotissa) 
mcgintyi, and the Indo-West-Pacific P. 
(Numismoida) numisma (Lamarck, 1819). He 
distinguished among the two hyotisssinid gen- 
era mainly by the relative degree of opening 
of the left promyal passage: open but reduced 
in IHyotissa, closed in Paraliyotissa. We aimed 
to test the phylogenetic robustness of this ge- 
neric reorganization by constructing nuclear 
and mitochondrial ribosomal gene trees incor- 
porating these three taxa together with a 
neopycnodontinid gryphaeid, Neopycnodonte 
coclilear (Poll, 1795), that is sister to the 
Hyotissini (Ó Foighil & Taylor, 2000). 

Phylogenetic Status of Tesl<yostrea weberi 

Olsson (1951) considered Ostrea weberHo 
be the most distinctive regional species of 
oyster, and designated Key West as its type 
locatity. Harry (1985) supported its taxonomic 
distinctiveness, placing it in a monotypic new 
genus, Tesl<eyostrea. Alternatively, Abbott 
(1974) regarded T. weberi as a free-living 
ecophenotype, and junior synonym, of the 
sponge commensal Cryptostrea permollis (G. 
B. Sowerby II, 1871), and this taxonomic in- 
terpretation has been largely followed in the 
subsequent literature (Carriker & Gaffney, 
1996). Cryptostrea permollis is recorded from 
the northeastern Gulf of Mexico and off North 
Carolina (Harry, 1 985), and we did not encoun- 
ter it in the Florida Keys. There are multiple 
records of С permollis in the Florida Keys 
(Mikkelsen & Bieler, 2000); however, these 
refer to free-living, T. weberi (R. Bieler, pers. 
comm.). Jozefowicz & Ó Foighil (1998) incor- 
porated, for comparative purposes, Keys 
specimens they identified as T. weberi in their 
molecular study of Southern Hemisphere flat 
oysters. However, they were unaware that the 
range of shell ecomorphs produced by another 
Keys ostreinid, Ostreola equestris, overlaps 
with that of T. weberi. Subsequent unpublished 
work by one of the authors (P. Baker) showed 
conclusively that the "T. weberi" specimens 
sequenced by Jozefowicz & Ó Foighil (1998) 
were actually O. equestris. The phylogenetic 
placement of T. weberi therefore still remains 
to be established. We revisited this issue by 
generating mitochondrial genotypes - large 
ribosomal subunit (16S) - from authentic T. 
weberi and incorporating them, together with 
С permollis and O. equestris genotypes, into 
a phylogenetic analysis of brooding oysters. 



FLORIDA KEYS OYSTERS 



311 



Biogeographic Relationships of Ostreola eques- 
tris and Ostrea aupouria (Dinamani & Beu, 1 981 ) 

Jozefowicz & Ó Foighil (1998) uncovered a 
nunnber of unexpectedly close phylogenetic re- 
lationships among geographically disjunct 
ostreinid taxa. Their Keys Ostreola equestris 
samples (misidentified as Teskeyostrea weberi, 
see above) differed from specimens of the New 
Zealand O. aupouria by as little as a single trans- 
version in their mt 16S large subunit ribosomal 
gene fragments. We aimed to revisit this sur- 
prising biogeographic pairing by utilizing Cyto- 
chrome Oxidase I (COI), a faster-evolving mt 
gene fragment more useful in resolving oyster 
tip taxa (Ó Foighil et al., 1998), and by incorpo- 
rating samples of O. equestris spanning the well- 
defined Gulf/Atlantic marine biogeographic 
break in southeastern Florida (Avise, 1992, 
2000; Cunningham & Collins, 1994). In the ab- 
sence of post-separation gene flow, the process 
of lineage sorting is expected to sequentially 
lead newly formed daughter populations from 
initial polyphyly, to paraphyly, and ultimately to 
reciprocal monophyly (Avise, 2000). We were 
interested in establishing whether these disjunct 
New Zealand/American populations were recip- 
rocally monophyletic, or if one was a recent 
founder of the other. Another objective was to 
determine how the aupourial equestris genetic 
disjunction scaled relative to the anticipated 
Gulf/Atlantic break in O. equestris. Two hypo- 
thetical topologies, each containing an O. 
equestris Gulf/Atlantic disjunction, are presented 
as exemplars in Figure 1. There are of course 
many other topological possibilities. 

Shell Phenotype Variation in Ostreola equestris 

Ostreola equestris is commonly known as the 
"crested" oyster and, as its informal name im- 
plies, it is described as having a shell with raised 
crenulated margins (Abbott, 1974). We encoun- 
tered this morph in intertidal Keys habitat; how- 
ever, subtidal individuals, genotyped in this study 
for mt markers, were usually cemented to the 
substratum along their entire left valves, yield- 
ing a very thin, contour-hugging, morph that ex- 
hibited a wide variety of coloration and sculptural 
texture, some of which closely approximated the 
Teskeyostrea weberi phenotype (Olsson, 1951; 
Harry, 1985). Employing genotyped individuals 
only, we aimed to give a photographic summary 
of the impressive range of shell phenotypes dis- 
played by our samples of this species. 






-<^NZ 



FIG. 1. Two exemplary unrooted mitochondrial 
tree topologies predicted by distinct hypotheses 
of historical relationships among geographically 
disjunct sister populations of New Zealand 
{Ostrea aupouria) and American {Ostreola 
equestris) ostreinids. Both hypotheses assume 
a priori that O. equestris has undergone dado- 
genesis into distinct Atlantic (At) and Gulf (G) 
lineages, a well-documented pattern among 
coastal Floridian marine taxa (Avise, 1992, 
2000; Cunningham & Collins, 1994). There are 
of course many other hypothetical topologies 
that could be entertained, a, O. aupouria (NZ) 
represents a recent founder population of Gulf 
O. equestris (G) and genotypes of the former 
are predicted to nest within a Gulf tip clade; b, 
O. aupouria (NZ) has experienced a distinct 
evolutionary history that predates the origin of 
the Gulf/Atlantic disjunction in O. equestris and 
all three groupings are predicted to be recipro- 
cally monophyletic with the stem branch lead- 
ing to O. aupouria (NZ) being the most 
pronounced. 



MATERIALS AND METHODS 

A summary of sampling locations and of 
voucher specimen information is outlined in 
Table 1, and specific sampling details for Flo- 
ridian taxa are given in the following para- 
graphs. For specimens collected in the Florida 
Keys, all collections were made via snorkel- 
ing in depths from 1-5 m, except collections 
from IMBW-FK-650 where SCUBA was used 
to sample specimens from roughly 30 m. 
These specimens were preserved in 95% de- 
natured alcohol and then transferred to 95% 
non-denatured alcohol upon return to the De- 
partment of Malacology at the Florida Museum 
of Natural History. Specimens collected else- 
where were sampled from shore and pre- 
served in > 70% ethanol. 



312 



KIRKENDALE ETAL. 



TABLE 1 . Species identification and sampling locality data, together with voucher specimen information. 
UMMZ and FLMNH numbers respectively refer to the voucher specimen catalog numbers of the 
Mollusk Division, University of Michigan Museum of Zoology, and the Department of Malacology, 
Florida Museum of Natural History. See Mikkelsen & Bieler (2004) for specific details concerning the 
International Marine Bivalve Workshop (IMBW-FL) sampling stations. 



Taxa 



Location 



# of individuals 
sequenced 



Catalog # 



Family Gryphaeidae 
Subfamily Pycnodonteinae 
Parahyotissa mcgintyi 
Parahyotissa numisma 
Hyotissa hyotis 
Neopycnodonte cochlear 

Family Ostreidae 
Subfamily Ostreinae 

Teskeyostrea weberi 

Ostreola equesths 

Ostreola equestris 

Ostreola equestris 

Ostreola equestris 

Ostreola equestris 

Ostrea aupouria 

Cryptostrea permollis 
Subfamily Crassostreinae 

Crassostrea virginica 

Crassostrea virginica 



IMBW-FK-650 
Guam 
Guam 
Maui, Hawaii 



IMBW-FK-645 
IMBW-FK-629 
IMBW-FK-644 
IMBW-FK-649 
Skidaway River, Georgia 
Cedar Key, Florida 
Hauraki Gulf, New Zealand 
Panacea, Florida 

Skidaway River, Georgia 
Panacea, Florida 



1 


UMMZ 300092 


1 . 


UMMZ 265996 


1 


UMMZ 265995 


1 


UMMZ 265997 


4 


FLMNH 298644 


g 


FLMNH 298643 


1 


FLMNH 298645 


1 


FLMNH 298640 


11 


UMMZ 300093 


10 


UMMZ 300094 


12 


UMMZ 255404 


2 


UMMZ 255410 


3 


UMMZ 300095 


2 


UMMZ 300096 



Parahyotissa mcgintyi 

Numerous specimens of the gryphaeid 
Parahyotissa mcgintyi were sampled (by L. 
Kirkendale and G. Steiner) from the superstruc- 
ture epibenthos of the sunken vessel Thunder- 
bolt (IMBW-FK-650; Table 1) - apparently this 
species' first record from the Florida Keys 
(Mikkelsen & Bieler, 2000). Parahyotissa 
mcgintyi is easily distinguished from other re- 
gional oysters by its frequently plicated shell 
margins, absence of clasper spines, typically 
pycnodonteinid vesicular shell structure (Fig. 
2), and presence (in live adult specimens) of a 
bright orange pigment in ovarian tissue (Harry, 
1 985). In order to test Harry's (1 985) taxonomic 
rearrangement of the Hyotissini, we sequenced 
a 941 nt (post-alignment length) fragment of 
nuclear 28S rDNA, added it to Ó Foighil & 
Taylor's (2000) homologous 288 ostreoidean 
matrix, and analyzed the resulting dataset uti- 
lizing pterioid outgroups (Giribet & Distel, 
2003). A complementary gryphaeid mt IBS 
rDNA data set was constructed and then phy- 
logenetically analyzed using Neopycnodonte 
cochlear, a sister taxon to the Hyotissini (Ó 
Foighil & Taylor, 2000), as an outgroup. 



Teskeyostrea weberi 

Specimens of Teskeyostrea weberi were re- 
covered (by L. Kirkendale) from one of our 
sampling sites: the ocean-side shore of Grassy 
Key (IMBW-FK-645; Table 1), where it was 
locally abundant attached to the underside of 
large boulders at depths of 1-3 m. Positive 
identification of this species was made not only 
on the basis of its shell characters - flat, thin 
apricot-colored shell ornamented with fine ra- 
dial ribbing and thin lamellose extensions 
(Olsson, 1951; Harry, 1985) - but also on its 
lack of an anal appendage, a prominent ana- 
tomical feature of Ostreola equestris (Harry, 
1985). To place Teskeyostrea weberi phylo- 
genetically, we generated mt IBS sequences 
for four individuals, yielding two haplotypes, 
which were incorporated into Jozefowicz & Ó 
Foighil's (1998) brooding oyster IBS matrix. 
This matrix was further supplemented by IBS 
sequences (two haplotypes) generated from 
11 Florida Keys Ostreola equestris specimens 
sampled from three locations in the Florida 
Keys (Table 1 ). These latter specimens collec- 
tively displayed a wide variety of shell morphs, 
including T. weberi look-alikes, but exhibited 



FLORIDA KEYS OYSTERS 



313 




FIG. 2. Views of gross shell morphologies of adult and juvenile Parahyotissa mcgintyi specimens 
sampled from IMB\/V-FK-650. a, internal view of the left valve of an adult preserved in 95% ethanol 
after shucking (Note the prominent plication of the ventral valve margin); b, external view of the right 
valve of specimen depicted in 2a (Note the heavy fouling which obscures the valve outline); c, detail 
of anterio-dorsal inner edge of left valve of adult (see boxed area in 2a) showing the distinctive 
vesicular substructure characteristic of pycnodonteinid gryphaeids (Harry, 1985); d, external view of 
intact juvenile (note straight hinge line, flattened D-shaped profile and the vesicular substructure 
evident in abraded surface areas). 



distinct anal appendages (mainly digitform, 
some more cardiform in outline). Finally, we 
added to the single available 16S haplotype 
of the sponge commensal Cryptostrea 
permollis by sequencing two additional speci- 
mens (Table 1). 



Ostreola equestris 

In order to more fully resolve the phyloge- 
netic relationships of these geographically dis- 
junct, polytomous (at least for 16S, Fig. 4), 
New Zealand/American tip taxa, a mt COI 



314 



KIRKENDALE ETAL. 



gene fragment (626 nt) data set was gener- 
ated for a total of 44 individual oysters. Twelve 
New Zealand Ostrea aupouria - reliably dis- 
tinguished by their possession of an anal ap- 
pendage (Dinamani & Beu, 1981) from the 
co-occurring Ostrea chilensis (Philippi, 1844) 
- were sequenced, yielding 6 haplotypes, as 
were 32 Ostreola equestris specimens which 
collectively contained 15 haplotypes. 

We were interested in establishing if Ostreola 
equestris exhibits a regional Gulf/Atlantic ge- 
netic break in southeastern Florida in common 
with many other co-occurring nearshore ma- 
rine taxa (Avise, 1992, 2000; Cunningham & 
Collins, 1994) and, if so, how it might scale 
relative to the equestrisi aupouria disjunction. 
In addition to Florida Keys specimens (N = 11, 
six haplotypes), our 32 O. equestris individu- 
als sequenced for CGI also included speci- 
mens from the northeastern Gulf of Mexico 
(Cedar Key, N = 11, seven haplotypes) and 
from the Atlantic coast of Georgia (Skidaway 
River estuary, N = 10, six haplotypes). To pro- 
vide a phylogeographic yardstick, we also 
generated homologous CO! sequences (598 
nt) for a token number of replicate Gulf (Pana- 
cea, Florida Panhandle, N = 2, one haplotype) 
and Atlantic (Skidaway River, N = 3,2 haplo- 
types) specimens of the cupped oyster 
Crassostrea virginica. This ecologically domi- 
nant regional oyster species displays a well- 
characterized Gulf/Atlantic mt disjunction 
centered on southeastern Florida (Reeb & 
Avise, 1990). 

Molecular Methods 

Specimens utilized in this study were pro- 
cessed for molecular characterization either 
at the University of Florida (by L. Kirkendale) 
or the University of Michigan (by T. Lee). As 
a result, there were some minor methodologi- 
cal distinctions associated with DNA template 
preparation and PCR amplification as re- 
ferred to below. All novel DNA sequences 
were generated at the University of 
Michigan's DNA Sequencing Core and have 
been deposited in GenBank (Accession #s 
AY376596-AY376635). 

Genomic extractions and amplifications of 
flat oyster samples collected during the Florida 
Keys Bivalve Workshop were conducted by 
L. Kirkendale at the Florida Museum of Natu- 
ral History Molecular Phylogenetics Lab at the 
University of Florida (UF). Total genomic DNA 
was obtained from ethanol-preserved mantle 



tissue using modifications of standard proto- 
cols. Roughly 20-30 mg of tissue was finely 
cut, ground with a mortar and pestle and 
placed in 750 pL of DNAzol with 5-20 pL of 5- 
20 mg/ml proteinase К (Molecular Research 
Center, Inc.). Tissue was gently shaken over- 
night on an orbital shaker and following three 
rounds of ethanol extraction and centrifuga- 
tion, the pellet was eluted in 100 mL ddH20 
(for further details of DNAzol extraction pro- 
cedure, refer to Chomczynski et al. 1997). 
Universal primers were used to amplify 1 6S and 
CGI gene regions sequenced from the above- 
mentioned samples and were as follows: 1 6Sar 
5'-CGCCTGTTTATCAAAAACAT-3' and 16Sbr 
5'-GCCGGTCTGAACTCAGATCACGT-3' 
(Kessing et al. 1989) and LCO1490 5'- 
GGTCAACAAATCATAAAGATATTGG-3' and 
HC02198 5'-TAAACTTCAGGGTGACCA 
AAAAATCA-3' (Folmer et al., 1994). Reactions 
included IpL of genomic DNA template and 
31 .8 pL ddH20, 5 pL of 1 0X TAQ PCR buffer 
(Perkin Elmer), 5 pL of dNTPS (10 mM stock), 
2 pL of each primer (10 pM stock), 3 pL of 
MgCI2 solution (25 mM stock, Perkin Elmer) 
and 0.2 pL TAQ enzyme (Perkin Elmer). Re- 
actions for 1 6S were initially denatured at 96°C 
for 150 sec, followed by 37 cycles of 94°C for 
40 sec, 52°C for 35 sec, and 72°C for 60 sec. 
Reactions for COI were handled similarly ex- 
cept that the initial denaturation step was at 
95°C for 120 sec and that 40 cycles of ampli- 
fication were employed with a 40°C annealing 
temperature. All amplifications were run with 
positive and negative (no template) controls. 
PCR products were visualized by electro- 
phoresis on 1% TBE agarose gels, stained 
with ethidium bromide solution and photo- 
documented. Successful PCR products were 
cleaned for cycle sequencing using Wizard 
PCR Preps (Promega), following described 
protocols. Verification of the cleaned PCR 
product occurred in the same manner as for 
initial PCR products. 

Ostreola equestris samples from Cedar Key 
were extracted at UF, as above, but amplified at 
the Museum of Zoology, University of Michigan 
(UMMZ), by T Lee, along with Skidaway River 
O. equestris samples, using specifically de- 
signed CO! primers: 5'-GATATTGGACGGTTTT 
ATAT-3' and 5'-CCAAAAATCAAAACAATGCT- 
3' (Lee, unpublished). DNA template prepara- 
tion methods utilized at the UMMZ are detailed 
in Lee & Ó Foighil (2003). Other target gene 
fragments amplified at the UMMZ were mt 16S 
from Cryptostrea permollis and from the four 



FLORIDA KEYS OYSTERS 



315 



gryphaeid study species (Table 1) using 
Kessing et al. (1989) primers, 28S nuclear ri- 
bosomal domains 1-3 from Parahyotissa 
mcgintyi using Ó Foighil & Taylor's (2000) 
primer set, and mt COI from Ostrea aupouria, 
and Crassostrea virginica Gulf (Panacea) and 
Atlantic (Skidaway River) samples using 
Folmer et al. (1994) primers. A touchdown 
(Palumbi, 1996) protocol was used for all 
UMMZ PCR reactions [after 4 min denaturation 
at 94°C, the initial annealing temperature of 
65°C was decreased by 2°C/cycle (40 sec 
denaturing at 94°C, 40 sec annealing and 1.5 
min extension at 72°C) until the final anneal- 
ing temperature (45°C for COI, 50°C for 16S 
and 52°C for 28S) was reached and subse- 
quently maintained for an additional 30 cycles]. 

Phylogenetic Methods 

Initial alignments were constructed using 
Clustal X (Thompson et al., 1997) using de- 
fault parameters and then adjusted by eye to 
minimize mismatches in the ribosomal gene 
datasets. Phylogenetic analyses were con- 
ducted on each of six molecular datasets - 
(1) gryphaeid 28S, (2) gryphaeid 16S, (3) 
Ostreid/Lophinid 16S, (4) Ostrea aupouria/ 
Ostreola equestris COI, (5) О. equestris COI, 
and (6) Crassostrea virginica COI - under the 
maximum parsimony (MP) optimality criterion 
using PAUPM.OblO (Swofford 2002). While 
unrooted anayses were performed on COI 
datasets, the pterioid taxa, Neopycnodonte 
cochlear, and lophinid taxa were designated 
as outgroup for gryphaeid 28S, gryphaeidi 6S 
and ostreid 16S datasets respectively. MP 
analyses were performed using heuristic 
search option with 100 random stepwise ad- 
ditions and tree bisection-reconnection (TBR) 
branch-swapping. Gaps were treated as a 
missing state, character states were treated 
as unordered and equal weights were as- 
sumed. Branch support was estimated by 
bootstrapping (Felsenstein, 1985) (500 repli- 
cates, heuristic searches, 10 random additions 
each) and decay indices (Bremer, 1994), gen- 
erated in TreeRot (Sorenson, 1996). 

We wished to construct unrooted gene net- 
works for three CO! datasets [Ostreola 
equestris and O. aupouria; O. equestris alone, 
Crassostrea virginica alone) and took a Maxi- 
mum likelihood (ML) approach because two 
of the three (O. equestris and O. aupouria; O. 
equestris alone) produced multiple equally 
most parsimonious trees. A MP tree was first 



used to estimate the log-likelihood scores us- 
ing PAUP*. The best-fit ML model for each 
partition was then determined by hierarchical 
likelihood ratio tests (hLRTs) using Modeltest 
3.06 (Posada & Crandall, 1998). ML analyses 
were conducted using a heuristic search op- 
tion in which the parameter values under the 
best-fit model were fixed and a MP tree was 
used as a starting point for TBR branch swap- 
ping. The K81uf model [K81 model (Kimura, 
1981) with unequal base frequencies] + Г 
[gamma-distributed heterogeneity of the sub- 
stitution rate across sites (Yang, 1994)] was 
chosen as the best-fit model for the combined 
Ostreola equestris and O. aupouria dataset. For 
the O. equestris and C. virginica COI datasets, 
the respective best-fit models chosen were 
K81uf and HKY (Hasegawa et al., 1985). 



RESULTS 

Systematic Placement of Parahyotissa mcgintyi 

Figure 3 shows the most parsimonious gene 
tree obtained when a P. mcgintyi 28S geno- 
type was added to, and analyzed with, Ó 
Foighil & Taylor's (2000) ostreoidean 28S 
dataset. We obtained a paraphyletic 
Parahyotissa and a robust terminal sister re- 
lationship for the two Pacific Hyotissini: P. 
numisma and Hyotissa hyotis. A congruent 
topology was recovered when the 16S se- 
quences for the four gryphaeid taxa at our dis- 
posal (Table 1) were subjected to a maximum 
parsimony analysis (Fig. 3). The earlier study 
(Ó Foighil & Taylor, 2000) should be consulted 
for a detailed discussion of the ostreid clade 
topology. 

Phylogenetic Status of Teskyostrea weberi 

Figure 4 shows the strict consensus topol- 
ogy of the 54 most parsimonious trees obtained 
when the brooding oyster 16S matrix was ana- 
lyzed using the lophine taxa as outgroups. Major 
elements of the topology are congruent with that 
obtained, and discussed at length, in an earlier 
study (Jozefowicz & Ó Foighil, 1998) and will 
not be reiterated here. The salient features of 
the topology concern the relative placement of 
the three Floridian flat oyster taxa (labeled in 
bold text). All three occur in distinct, well-sup- 
ported terminal clades: Teskeyostrea weberi on 
its own, Ostreola equestris in a terminal 
polytomy with the New Zealand Ostrea 



316 



KIRKENDALE ETAL. 



hogiiomoii alatlis 



Pvictada iinbricata 



100 



100 



60 



10 changes 



100 



31 



28S 



99 



12 



100 



11 



Gryphaeidae 

Neopyctiodonte cochlear 
Paraît} 'О tissa mac git ityi 
Hyotissa hyotis 
^ Paraliyotissa fawiisnia 



16S 



100 



15 



100 



Crassostrea rJtizophorae 
С virginica 

Striostrea viargaritacea 



66 



64 

4 



100 



100 



14 



99 



78 



Г Saccostrea comviercialis 
Saccostrea cuciiUata 
~ Crassostrea ariakeiisis 
С gigas 

Ostrea cliilensis 

9 
O. atigasi 

' O. edulis 

O. cofidiap/iila 

O. piwldiaiia 

O. deriselcanellosa 

~ O. algoeiisis 

Dendostrea frons 



Ostreidae 



100 



10 



61 



100 



14 



D. folhmt 

A]ectryo}iella plicattila 
Lopiia cristagalli 



FIG. 3. The single most parsimonious tree (809 steps, CI = 0.668, Rl = 0.779) obtained by heuristic 
unweighted searches of 28S genotypes for 22 oyster taxa, including 4 gryphaeid species, with the two 
pterioids, Pinctada and Isognomon, designated as outgroups. See also the juxtaposed single most 
parsimonious tree (173 steps, CI = 0.948, Rl = 0.710) obtained by heuristic unweighted searches of 
gryphaeid mt 16S genotypes, in which Neopycnodonte cochlear \Nas the designated outgroup. Numbers 
above the branches represent bootstrap values (> 50) and numbers below indicate decay index values. 



a и pou ría, Cryptostrea permollis in a terminal 
polytomy with the Argentine Ostrea puelchana. 
A prominent basal ostreinid (+ Dendostrea 



frons) polytomy captures the branch support- 
ing the T. weben tip clade (Fig. 4), thereby ob- 
scuring its sister relationships. 



FLORIDA KEYS OYSTERS 



317 



61 



95 



75 



66 



99 



63 



85 



100 



79 



100 



10 



100 



58 



100 



10 



100 



■ Ostrea cmpouria 1 

■ O. ctupouria 2 

■ O. ctupouria 3 

■ Ostreola equestris 1 (N=10) 

■ Ostreola equestris 2 

■ Cryptostrea permollis 1 (N=2) 

• C. permollis 2 

■ C. permollis 3 

■ Ostrea puelcluma 

■ O. defuelamellosa 

■ Ostreola concliaphila 

■ Teskeyostrea weberi 1 (N=3) 

■ T. weberi 2 

■ Ostrea angasi 1 

■ O. angasi 2 

■ O. angasi 3 

■ O. edulis 1 

• O. edulis 2 

■ O. edulis 3 

• O. chilensis 

■ Ostrea algoensis 1 

• O. algoensis 2 
Defidostreafrons 1 

■D. from 2 
D. frons 3 
D. folium 1 
D. folium 2 

Alectryonella plicatula 
Lopha cristagalli 



FIG. 4. Strict consensus of 54 equally most parsimonious trees (174 steps, CI = 0.6379, Rl = 0.8437) 
resulting from heuristic unweighted searches of 29 brooding oyster 16S genotypes. The lophine taxa 
D. folium, D. frons, A. plicatula and L. cristagalli were designated as outgroups. Florida Keys ostreinid 
taxa are in boldface. Bootstrap values (> 50) and decay indices are shown above and below the 
branches, respectively. 



318 



KIRKENDALE ETAL. 



Biogeographic Relationships of Ostreola 
equestris and Ostrea aupouria 

A maximum-lil<elihood analysis of the com- 
bined American Ostreola equestris and New 
Zealand Ostrea aupouria COI dataset is shown 
as an unrooted network in Figure 5. New 
Zealand and American samples were recipro- 
cally, and robustly, monophyletic. Note however, 
that the minimum cumulative branch lengths 
separating members of the two clades was less 
than that of the maximum branch lengths sepa- 
rating within-clade O. equestris haplotypes. 

Figure 6 concerns only American taxa and 
shows the unrooted maximum-likelihood Gulf/ 
Atlantic COI networks for both Ostreola 
equestris and Crassostrea virginica. The 
Crassostrea virginica Gulf/ Atlantic phylogenetic 



split, estimated by Reeb & Avise (1990) from 
whole mt genome RFLP assays at approxi- 
mately 2.5% divergence, was also recovered 
from our token sample of Gulf/Atlantic CO I 
gene fragment sequences (1.8%; 11 substitu- 
tions over 598 nt). In sharp contrast, no such 
disjunction was evident in Ostreola equestris. 
Two haplotypes were found in all three regional 
populations (Table 2, Fig. 6), including by far 
the most common mt COI genotype (AFG1; N 
= 13). This latter mt genotype was numerically 
predominant in both Gulf (Cedar Key, 6/1 1 ) and 
Florida Keys (5/11) samples of Ostreola 
equestris, but not among our Atlantic (Skidaway 
River sample; 2/10) specimens. If we consider 
the former two samples in isolation, the numeri- 
cally predominant haplotype was centrally 
placed and connected to all but one (F4) of the 




0.001 substitutions/site 



FIG. 5. Maximum likelihood network (-In = 1 073.4044) of Ostrea aupouria (New Zealand) and Ostreola 
equestris (American) CGI haplotypes. Numbers on the branches are MP bootstrap values. 



FLORIDA KEYS OYSTERS 



319 



Skidaway River estuary 





^AtFG2 



Ostreola equestris COI Network 

At# Atlantic haplotypes (Skidaway River) 
F4 F О Florida Keys haplotypes 

G Gulf haplotypes (Cedar Key) 

At4 



0.001 substitution/site 



Ч 



Florida Keys 



Gulf/ Atlantic Crassostrea virginica COI disjunction 



Gulfhaplotype 
(Panacea) 



Atlantic haplotype 
(Skidaway River) 



FIG. 6. Regional map showing our collection sites for Gulf/Atlantic Ostreola equestris and Crassostrea 
virginica samples and also the superimposed maximum likelihood networks of the resulting O. equestris 
(-In = 985.5091) and С virginica (-In = 878.0842) CO! haplotypes. 



TABLE 2. Relative distribution of the 16 COI genotypes recovered from the three regional Gulf/ 
Atlantic Ostreola equestris sampling locations. The prefixes At, F, G and AtFG, respectively indicate 
haplotypes found solely in the Atlantic (Skidaway River) site, solely in the Florida Keys sites, solely in 
the Gulf (Cedar Key) site, and finally, those recovered from all three sites. See Figure 6 for map 
showing sampling site locations and the inferred topological relationships among the COI haplotypes. 



^ 


CM 






























(D 


О 






























LL 


LL 


•^ 


CN 


oo 


'ф 


.J_ 


CM 


CO 


^i 


T- 


CM 


CO 


'í 


un 


CD 


< 


< 


< 


< 


< 


< 


LL 


LL 


LL 


LL 


o 


О 


о 


О 


<D 


О 



Skidaway River 
Florida Keys 
Cedar Key 



2 14 111 ---------- 

6 1 - - - - 1 1 1 1 ----- - 

5 1 -------- 1 1 1 1 1 1 



320 



KIRKENDALE ETAL. 



other 10 COI genotypes recovered from the 
Gulf (Cedar Key) and Florida Keys populations 
by single substitutions (Fig. 6). Our Atlantic 
(Skidaway River) sample exhibited a different 
topological pattern characterized by a relatively 
extensive network in which the constituent 
haplotypes showed more pronounced collec- 
tive phylogenetic definition (Fig. 6). 

Shell Phenotype Variation in Ostreola equestris 

An impressive diversity of O. equestris shell 
phenotypes was recovered from the Florida 
Keys, and indeed also from single sampling 
sites, such as the Summerland Key Horse- 
shoe. Intertidal Horseshoe specimens exhib- 
ited a shell morphology that is typically 
associated with this species: gray oval shells 
with raised crenulated margins (Abbott, 1974). 
Figure 7a shows a cluster of specimens show- 
ing this morphology, sampled in this particu- 
lar case from the Skidaway River study 
population. Subtidal Florida Keys specimens 
were generally flatter in appearance, in some 
cases markedly so, and frequently incorpo- 
rated a diversity of pigmentation colors and 
patterns, some of which are presented in Fig- 
ure 7 (b-f). Exemplars spanning the range of 
O. equestris shell phenotypes found in the 
Horseshoe site, and other locations in the 
Keys, were genotyped using mt (1 6S and COI) 
markers and no evidence for genetic differ- 
entiation was evident among them. A minor- 
ity of O. equestris individuals displayed shell 
phenotypes that resembled Teskeyostrea 
weberi in external appearance: very thin shells 
with golden brown pigmentation sculptured 
with fine radial ribbing and lamellose exten- 
sions (Fig. 7). 



DISCUSSION 

Systematic Placement of Parahyotissa mcgintyi 

Our nuclear and mt ribosomal gene trees 
consistently recovered a paraphyletic 
Paraiiyotissa in which P. mcgintyi, the type 
species, was robustly sister to a tip clade con- 
taining P. numisma and IHyotissa hyotis. This 
topology implies that the character state used 
by Harry (1985) to distinguish Parafiyotissa 
(closed left promyal passage) is plesiomorphic 
in extant Hyotissini, rather than a 
synapomorphy diagnosing a Parahyotissa 
clade, and that the condition in the monotypic 



genus i-iyotissa (open but reduced left promyal 
passage) is autapomorphic. Based on avail- 
able information, there seems to be no phylo- 
genetic basis for Harry's Parafiyotissa. Future 
research incorporating P. {Paraiiyotissa) 
imbricata (Lamarck, 1819) and P. (Plioliyotissa) 
quercinus (G. B. Sowerby II, 1871), may un- 
cover more than one natural (i.e., monophyl- 
etic) group within the Hyotissini that can be 
defined by morphological synapomorphies and 
warrant generic status. Until then, we recom- 
mend that all hyotissinid taxa be returned to 
the genus IHyotissa Stenzel, 1971. 

Phylogenetic Status of Teskeyostrea weberi 

Our 16S strict consensus tree topology (Fig. 
3) conclusively demonstrates that this species 
is not a free-living ecomorph of the sponge 
commensal Cryptostrea permollis, as thought 
by Abbott (1974), but is instead a distinct 
ostreinid lineage lacking (at present) any ob- 
vious candidate sister species. Olsson (1 951 ) 
had proposed the eastern Pacific "Ostrea 
iridescens", synonymized with Striostrea 
prismática (Gray, 1825) by Harry (1985), as a 
putative sister species to T. weberi, based on 
the similarity of the former's juvenile shell phe- 
notype to that of the adult T. weberi. However, 
S. prismatica's taxonomic placement in the 
cupped oyster subfamily Crassostreinae 
(Harry, 1985), which is supported by prelimi- 
nary molecular data (Lee & Ó Foighil, unpub- 
lished), rules this out. A more comprehensive 
sampling of brooding oyster global diversity, 
including data from genes other than 16S, is 
required to better resolve T. weberi's phylo- 
genetic position within the Ostreinae/Lophinae. 

Although Teskyostrea weberi and Ostreola 
equestris represent very distinct lineages (Fig. 
3), they co-occur in the Florida Keys, and a 
fraction of latter species resemble T. weberi 
in their external appearance (Fig. 7). Fortu- 
nately, these O. equestris M/eber/-lookalikes 
can be distinguished upon dissection by their 
distinct anal appendage (Harry, 1985), and 
their relatively larger adductor muscle. Based 
on our preliminary observations, there may 
also be ecological and larval settlement dif- 
ferences among these two ostreinid taxa in 
the Florida Keys. All of the I weberi speci- 
mens we encountered were attached to the 
underside of rocks (Harry, 1985: fig. 25) in an 
oceanside location, whereas O. equestris were 
commonly sampled from the exposed hard 
surfaces in bayside locations. 



FLORIDA KEYS OYSTERS 



321 




FIG. 7. Shell phenotypes. a-f, displayed by genotyped Ostreola equestris sampled from the Skidaway 
River, Georgia (a, cluster of individuals), and from 2 sites in the Florida Keys (b-e, IMBW-FK-629 
from rock surfaces and f, IMBW-FK-649 epifaunal on Pinna); g, a specimen of Ostrea aupouria, New 
Zealand sister species of Ostreola equestns (UMMZ 255404); h, a specimen of the sponge commen- 
sal Cryptostrea permollis from Panacea, Florida Gulf Coast (UMMZ 255410); i and j, individuals of 
Teskeyostrea weberi sampled from IMBW-FK-645. 



322 



KIRKENDALE ETAL. 



Biogeographic Relationships of Ostreola 
equestris and Ostrea aupouria 

The COI gene tree topology (Fig. 5) demon- 
strates that our respective study populations 
of New Zealand Ostrea aupouria and Gulf/At- 
lantic Ostreola equestris are reciprocally 
monophyletic. This result is sufficient, at least 
for now, for retention of their respective spe- 
cific status. Coan et al. (2000) rejected the 
separation of Ostreola from Ostrea based on 
morphological characters and the phylogenetic 
validity of Harry's (1985) Ostreola is question- 
able given that two of his three constituent 
species (O. equestris and O. conchaphila) are 
not sister taxa in our gene trees (Fig. 3). How- 
ever, a definitive generic designation for 
equestris and aupouria requires data from the 
Mediterranean/African-Atlantic type species 
Ostreola stentina (Payraudeau, 1826). 

Two lines of evidence indicate that the 
Ostreola equestris/0. aupouria disjunction re- 
sults from evolutionarily recent dispersal rather 
than ancient vicariance. Maximum within- 
population CO! genetic divergence for the 
Skidaway River sample exceeds the minimum 
New Zealand/American divergences obtained 
(Fig. 5). This result implies that the age of the 
O. equestris/O. aupouria disjunction may be 
less than the haplotypic lineage sorting time 
window for the Atlantic population of the O. 
equestris. Although we do not have a fossil- 
calibrated lineage-specific clock for any oys- 
ter, the well-studied Gulf/Atlantic Crassostrea 
virginica divergence has been dated, using 
"conventional calibrations" to approximately 
1.2 myr (Reeb & Avise, 1990). Parsimony 
analysis of our token samples of Gulf/Atlantic 
C. virginica COI sequences found that they 
differed by 1 1 steps (1 .83% of the 598 nt frag- 
ment). The minimum number of substitutions 
separating the New Zealand and American 
COI clades in parsimony analyses is six steps 
(0.95% of the 626 nt fragment). Although the 
resulting age estimate of 0.625 myr for the O. 
equestris/O. aupouria disjunction is undoubt- 
edly crude, it is over two orders of magnitude 
less than the vicariant separation of New 
Zealand from Gondwanaland (Weissel & 
Hayes, 1977). 

The Ostreola equestris/O. aupouria geo- 
graphic disjunction is but one of three such 
cases involving tip taxa in the brooding oyster 
16S gene tree (Fig. 3); the other two involve 
Ostrea edulis/0. angasi and Crypytostrea 
permollis/ Ostrea puelchana and are discussed 



in Jozefowicz & Ó Foighil (1998). Although an- 
thropogenic transoceanic oyster introductions 
have occurred on numerous occasions 
(Dinamani, 1971; Edwards, 1976; Buroker et 
al., 1979; Chew, 1990; Carlton & Mann, 1996; 
Boudry et al., 1998; Ó Foighil et al., 1998), we 
can, with some confidence, rule out such his- 
toric transfers among the New Zealand/Ameri- 
can study populations (Fig. 4). This conclusion 
is based on their lack of shared COI haplotypes 
and on their reciprocal monophyly (Fig. 5), a 
phylogenetic relationship that is characteristic 
of populations that have not experienced evo- 
lutionary recent gene flow (Avise, 2000). It is 
possible, however, that such an event may have 
occurred involving yet-to-be-sampled, geneti- 
cally differentiated portions of either species' 
ranges -according to Harry (1985), O. equestris 
occurs from North Carolina to Argentina. 

Genetic Structuring of Gulf/Atlantic Ostreola 
equestris and Crassostrea virginica 

Genetic characterization of near-shore ma- 
rine taxa found on either flank of the Floridian 
peninsula have revealed cryptic phylogenetic 
disjunctions among diverse Gulf-Atlantic Caro- 
linian faunal elements (Saunders et al., 1986; 
Bert, 1986; Avise et al., 1987; Bert & Harrison, 
1988; Dillon & Manzi, 1989; Brown & 
Wolfingbarger, 1989; Cunningham et al., 1991; 
Sarver et al., 1992; Cunningham & Collins, 
1994; Felder & Staton, 1994; Bert & Arnold, 
1995; Duggins et al., 1995; Ó Foighil et al., 
1 996; Schizas et al. , 1 999; Avise, 2000; Collin, 
2001, 2002), with by far the most intensively 
studied exemplar being the American oyster 
Crassostrea virginica (Reeb & Avise, 1990; 
Karl & Avise, 1992; McDonald et al., 1996; 
Hare & Avise, 1996, 1998; Hare et al., 1996). 
Ostreola equestris occurs in micro-sympatry 
with С virginica throughout regional estuar- 
ies, although prior research has shown that 
O. equestris tends to be abundant only at high 
salinity portions of estuaries (Hoese, 1960). 
Surprisingly, our O. equestris mt COI data (Fig. 
5, Table 2) show that this oyster species dif- 
fers from C. virginica, and from a large frac- 
tion of the regional marine biota, in lacking a 
Gulf/Atlantic mt genetic disjunction. Absence 
of genetic structuring among Gulf and Atlantic 
populations is not unique to O. equestris (Gold 
& Richardson, 1998; Avise, 2000); however, 
our results indicate that these two co-occur- 
ring oyster species have experienced signifi- 
cantly different regional histories. 



FLORIDA KEYS OYSTERS 



323 



Another discrepancy among the two oyster 
mt datasets concerns the relative topological 
definition of Gulf and Atlantic populations. 
Beckenbach (1994) performed a cladistic 
analysis of Reeb & Avise's (1990) extensive 
(N = 232) C. virginica mt RFLP dataset and 
found that both Gulf and Atlantic populations 
were dominated by one or two common 
haplotypes. These occupied central positions 
in their respective clades and were separated 
by single steps from a large number of termi- 
nally positioned rare haplotypes. Our Gulf (Ce- 
dar Key) and Florida Keys samples of Ostreola 
equestris showed (either separately or jointly) 
essentially a similar topology; however, the 
Atlantic (Skidaway River) sample did not (Fig. 
5). In the absence of significant homoplasy, 
the relative lengths of individual branches 
within a molecular phylogenetic tree topology 
are rough proxies for evolutionary time. In this 
context, it is interesting to note the markedly 
longer collective branch lengths interconnect- 
ing Ostreola equesir/s Atlantic haplotypes rela- 
tive to the truncated area of the COI topology 
occupied by Gulf and Florida Keys haplotypes 
(Fig. 5). This topological distinction is consis- 
tent with an older evolutionary history for this 
species in the Atlantic section of its present- 
day regional range. The compact star-like hap- 
lotypic topology produced by Gulf (Cedar Key) 
and Florida Keys CO! genotypes (Fig. 5) is 
characteristic of a population founded more 
recently by one ancestral type, presumably 
represented by the numerically predominant, 
topologically central, well-connected 
(Castelloe & Templeton, 1994) haplotype 
AFG1, found in all three study populations. 
Such a topology is also indicative of popula- 
tions that have experienced a phase of rapid 
demographic growth, a process associated 
with lowered stochastic elimination of novel/ 
rare lineages (Avise et al., 1984; Slatkin & 
Hudson, 1991; Moritz, 1996). 

Our mt COI data for the three study popula- 
tions of Ostreola equestris paint a regional his- 
tory that differs in important respects from that 
of Crassostrea virginica and also from a large 
fraction of the local marine biota. The domi- 
nant regional theme is the presence of a Gulf- 
Atlantic phylogeographic break characterized 
by considerable geographic concordance in 
genetic structuring across diverse faunistic 
elements (Avise, 2000). This implies a coher- 
ent spatial patterning of vicariance and sec- 
ondary contact events. In contrast, O. 
equestris shows no evidence of a vicariant 



imprint and our results imply that its present 
day Gulf/Atlantic distribution has been 
achieved by range extension from source At- 
lantic populations followed by a demographic 
growth pulse in the new Florida Keys/Gulf of 
Mexico habitats. 

Shell Phenotype Variation in Ostreola 
equestris 

Though forearmed with an awareness of the 
fabled xenomorphism of oysters, we were sur- 
prised at the extent to which O. equestris, the 
most commonly encountered ostreid in the 
Florida Keys, exhibited a multitude of shell 
phenotypes - a repertoire far from exhausted 
by our limited presentation in Figure 6. This 
facility is also a characteristic of Ostrea 
aupouria, its New Zealand sister taxon 
(Dinamani & Beu, 1981). Although genetic 
characterization is a reliable method for dis- 
tinguishing co-occurring oyster species with 
overlapping shell morphs, the presence of a 
distinct anal appendage in O. equestris (Harry, 
1985; but not all are digitiform) and in O. 
aupouria (Dinamani & Beu, 1981) is also 
particularily useful in this regard. It is unclear 
to what degree the phenotypic variation we 
observed in O. equestris reflects populational 
allelic diversity and/or local micro-environmen- 
tal parameters, or what contribution this plas- 
ticity makes to the local ecological success of 
this small species - the numerically dominant 
Florida Keys oyster. 



ACKNOWLEDGEMENTS 

Our thanks to Paula Mikkelsen and Rüdiger 
Bieler for organizing the International Marine 
Bivalve Workshop and to Russ Minton, Louise 
Crowley and Isabella Kappner for their unstint- 
ing field assistance. We are grateful to our fel- 
low workshop attendees who facilitated our 
efforts in numerous ways and to two anony- 
mous reviewers whose detailed comments sig- 
nificantly improved the manuscript. Liath 
Appleton provided expert help with the photo- 
graphic plates. Supplementary specimens were 
kindly provided by Gustav Paulay (Pacific 
gryphaeids), Andrew Jeffs {Ostrea aupouria) 
and Randy Walker (Skidaway River Ostreola 
equestris and Crassostrea virginica). Supported 
by NSF awards DEB-99781 1 9 to R. Bieler & P 
M. Mikkelsen and by OCE-0099084 to D. Ó 
Foighil. Additional support was provided by the 



324 



KIRKENDALE ETAL. 



Bertha LeBus Charitable Trust, the Comer Sci- 
ence & Education Foundation, the Field Mu- 
seum of Natural History, the American Museum 
of Natural History and the Mollusk Division of 
the University of Michigan Museum of Zoology. 



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Revised ms. accepted 31 October 2003 



MALACOLOGIA, 2004, 46(2): 327-338 

HOW RELIABLE IS MORPHOLOGY BASED SPECIES TAXONOMY 

IN THE BIVALVIA? A CASE STUDY ON ARCOPSIS ADAMSI 

(BIVALVIA: ARCOIDEA) FROM THE FLORIDA KEYS 

P. Graham Oliver^ & Johanna Järnegren^ 

ABSTRACT 

A morphological study of Л rcops/s adamsi was made on three populations from contrast- 
ing biotopes, supralittoral, sublittoral (1 m depth) and offshore (8-10 m depth). Shell 
morphometries gave statistically significant differences and these were supported by sub- 
jective observations on shell sculpture, periostracum. and haemoglobin content. The ob- 
servations form the core of a discussion on the reliability of morphological characters when 
defining species taxonomy. It must still be recognised that bivalve growth can strongly 
affect shell morphology and that sculptural and anatomical differences can be the result of 
environmental differences. The influence of geographical separation or isolation of popu- 
lations probably unduly influences taxonomic decision making. Such decision making re- 
quires consideration of ecological factors and should now be more widely supported by 
molecular studies at the population level. 

Key words: Arcopsis, morphology, species taxonomy, growth effects. 



INTRODUCTION 

This paper is a result of a taxonomic training 
workshop organised under the U.S. National 
Science Foundation FEET Initiative and re- 
flects on one issue of bivalve taxonomy, namely 
the reliability of morphological characters at the 
species level. 

Current bivalve species taxonomy remains 
primarily based on morphological characters and 
species identification relies mostly on shell char- 
acters. Introducing students to bivalve taxonomy 
is often problematic because many characters, 
both shell and anatomical, are subtle parts of a 
continuum and not discreet. Compounding this 
are the widespread phenomena of 
ecophenotypic and geographic clinal variation. 

Arcopsis adamsi (Dall, 1886) is a relatively 
common species inhabiting both intertidal and 
sublittoral biotopes (Abbott, 1974) and was 
chosen because it or similar taxa had already 
appeared in morphometric analyses (Marko & 
Jackson, 2001 ; Oliver & Cosel, 1 992). In these 
instances, the comparisons were made across 
isolation barriers or along large geographical 
ranges. Analysing morphological variation at a 
much smaller geographical scale has not been 
done within the Arcoidea. Arcopsis adamsi is a 
common component of intertidal, sublittoral and 



offshore mixed rock and sand biotopes to re- 
corded depths of 10 m. It is generally found 
attached by a weak byssus to the undersides 
of rocks resting on sand. It is widespread in the 
subtropical and tropical western Atlantic and 
Caribbean Sea ranging from Brazil to North 
Carolina. In the tropical eastern Atlantic it has 
a sister species, Arcopsis afra (Gmelin, 1791) 
(Figs. 8, 9), that occurs along with the closely 
related Striarca láctea (Linnaeus, 1758) (Figs. 
14, 15). In the western Atlantic, in contrast, the 
genus Striarca is not represented in the Re- 
cent fauna. A similar situation occurs in the 
Pacific Panamic region where Arcopsis solida 
(Sowerby, 1833) is the sole representative of 
the Striaciinae. Arcopsis species may therefore 
fill a much broader niche in the Americas and 
may have radiated into cryptic species. 

This study examines the biotope range of 
Arcopsis adamsi in the Florida Keys and com- 
pares the morphology of populations from dif- 
ferent biotopes. The primary aim of this paper is 
to draw attention to issues that students will en- 
counter when undertaking species-level tax- 
onomy studies and is intentionally discursive in 
nature. Many questions about Arcopsis tax- 
onomy are raised by this study and both authors 
are aware that the solutions are not provided 
although methodologies are proposed. 



^Department of Biodiversity & Systematic Biology, National Museum of Wales, Cardiff, United Kingdom; 
graham.oliver@nmgw.ac.uk 
^Trondjem Biological Station, Trondjem University of Science & Technology, Norway 



327 



328 



OLIVER & JARNEGREN 



MATERIALS AND METHODS 
Materials 

The materials used in the morphometric 
analysis are housed in the National Museum 
of Wales, Cardiff, under the specific numbers 
cited below. All additional materials are also 
housed in the National Museum of Wales un- 
der NMW.Z.2003.075. 

Arcopsis adams/ was found in four biotopes. 

(1) Supralittoral crevices 

Collection site: IMBW-FK-629, 21 & 26-VII-02, 
"The Horseshoe" bayside of West Summerland 
Key, MM35, Monroe County, Florida Keys, 
24°39.3'N, 8ri8.2'W; NMW.Z.2003.075.1. 

At the "The Horseshoe" site, the supralittoral 
fringe along the south side of the quarry con- 
sists in places of highly eroded friable limestones 
with many cavities and crevices that remained 
damp throughout the tidal cycle. These cavities 
were populated by many Arcopsis a few 
Brachidontes and the occasional Area imbricata 
and Isognomon. The presence of the "terres- 
trial" snails Laemodonta cubensis (Pfeiffer, 1 854) 
and Truncatella pulchella Pfeiffer, 1839, is highly 
indicative of the supralittoral environment. 

(2) Shallow sublittoral rubble on sand and 
lower littoral 

Collection site: IMBW-FK-629, 21 &26-VII-02, 
"The Horseshoe" bayside of West Summerland 
Key, MM35, Monroe County, Florida Keys, 
24°39.3'N, 8ri8.2'W; NMW.Z.2003.075.2. 

Other sites: IMBW-FK-622, IMBW-FK-657 

On the north side of the Horseshoe quarry 
the very low vertical face drops onto a sloping 
face covered in muddy sand and blocks of 
quarry rubble. Arcopsis was found frequently 
attached to the undersides of the rubble blocks 
in 0.5 to 1.5 m water depth. This area was 
never exposed at low tide and can be consid- 
ered sublittoral. The upper surfaces of the 
blocks were covered, predominantly by large 
Ctiama, Area, and oysters. The lower surfaces 
were mostly bare with an assemblage of small 
gastropods, Rissoidae, Cerithidae, Muricidae. 

Arcopsis can also be found attached to the 
undersides of rocks in the lower littoral often 
with Acar domingensis. 

(3) Offshore coralline sands and rubble 
Collection sites: IMBW-FK-651, 27-VII-02, 



"Samantha's patch reef 5 nmi S of Marathon, 
Monroe County, Florida, 24°39.49'N, 
81 °00.32'W, 7.6 m; NMW.Z.2003.075.3. IMBW- 
FK-641, 23-VII-02, Tennessee Reef Light, off 
Long Key, Monroe County, Florida, 24°44.75'N 
80°46.95'E, 6.1 m; NMW.Z.2003.075.4. IMBW- 
FK-624, 20-VII-02, Horseshoe Reef, off Fat 
Deer Key, Monroe County, Florida, 24°39.91'N, 
80°59.56'E, 7.3 m; NMW.Z.2003.075.5 

These sites are all of patch reefs with sandy 
bottoms and rubble blocks. Arcopsis are found 
attached to the undersides of the blocks lying 
on sand. 

(4) Sea grasses 

Arcopsis were also collected from a number 
of sea grass biotopes but not in sufficient num- 
bers for analyses. They were found attached 
to the base of shoots and exposed rhizomes. 

Methods 

Shell morphometric analyses were based on 
samples of at least 30 individuals, represent- 
ing the total size ranges, taken from the 
supralittoral, sublittoral, and offshore sites. In 
the hand, specimens from each biotope had a 
different appearance generally in the appar- 
ent inflation and elongation of the valves. Pa- 
rameters used focussed on these outward 
subjective views and included: shell length, 
shell height, and shell tumidity. The ligament 
appeared to be smaller in the offshore popu- 
lation, and the parameters inter-umbonal dis- 
tance, ligament length, and number of ligament 
bands were measured. During the process, it 
was suspected that the inflation of the right 
and left valves was not equal and consequently 
left and right valve tumidity were included. This 
analysis was not applied to the small number 
offshore specimens available, as the separa- 
tion of the valves would have destroyed the 
soft tissues, which were needed for anatomi- 
cal study. Paired parameter comparisons 
were made rather than a multivariate ap- 
proach in orderte reveal specific differences. 
Arcoid bivalves are known to exhibit allomet- 
ric growth of the ligament (Thomas, 1975, 
1976) and it was decided that other shell pa- 
rameters should be examined in relation to 
ontogeny. All data were analysed using 
Statview™. 

Shell sculpture and structure were examined 
using a scanning electron microscope. 

Anatomical comparisons were made from 
both living and preserved specimens. 



ARCOPSIS MORPHOLOGY AND TAXONOMY 



329 



RESULTS 

Shell Morphometries 

Maximum size: The maximum size of the 
shells differed between samples with the larg- 
est shells occurring in the shallow sublittoral 
and reaching 13.5 mm in length. In the 
supralittoral the maximum size recorded was 
1 0.5 mm and for offshore it was 9.5 mm. Sample 
sizes here for the supralittoral and sublittoral 
populations exceeded 100 individuals but for 
the offshore population only 30 were collected. 



Comparisons ofSliell Parameter Ratios: The 
most apparent difference in the hand was the 
shorter, more inflated form of the supralittoral 
shells, and to test this the length: height (Fig. 
1 ) and length: tumidity (Fig. 2) ratios were com- 
pared. An ANOVA test (Fisher's PSLD) of the 
two ratios showed that the supralittoral popu- 
lation did have proportionately shorter and 
more tumid shells as compared to the other 
populations (significant at p = 0.0005 to < 
0.0001) but that the difference between the 
sublittoral and offshore populations was not 
significant. 



1 



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FIGS. 1-5. Box plots comparing shell morphometric ratios for populations of Arcopsis adamsi from 
the Florida Keys. FIG. 1: Shell length to shell height; FIG. 2: Shell length to shell tumidity; FIG. 3: Shell 
length to inter-umbonal distance; FIG. 4: Shell length to ligament length; FIG. 5: Left valve to right 
valve tumidity. 



330 



OLIVER & JARNEGREN 













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FIGS. 6, 7. Comparisons of growth curves in three populations of /Arcops/s adams/ from 
the Florida Keys. FIG. 6: Plot of ligament length against shell length; FIG. 7: Plot of shell 
tumidity against shell length. Diamond, supralittoral; dot, offshore; +, sublittoral. 



ARCOPSIS MORPHOLOGY AND TAXONOMY 



331 



Comparisons of the inter-umbonal distance 
(length: inter-umbonal distance) gave the same 
pattern of results (Fig. 3) with significance at p 
< 0.0001 . Comparison of the relative size of the 
ligament (length: ligament length) gave signifi- 
cant differences between all three populations 
(Fig. 4) except that this was less between the 
supra and sublittoral populations, p only 0.0106. 

Comparing the tumidity of left and right 
valves did reveal that Arcopsis adamsi is 
slightly inequivalve (Fig. 5) and that the 
supralittoral population is significantly more 
inequivalve than the sublittoral population, p 
= 0.02. 

Comparisons of Growth Curves: The regres- 
sion plot of ligament length to shell length fits 
an exponential curve (Fig. 6) and confirms al- 
lometric growth of the ligament. Similar plots 
of tumidity (Fig. 7), interumbonal distance and 
number of ligament bands all reveal a similar 
pattern. 

In all cases, the point at which the curve most 
rapidly climbs is at a smaller shell size in the 
supralittoral population. In the offshore popu- 
lations the curves are least steep. 

Shell Sculpture and Structures 

Sculpture: On outward appearance the 
shells from the three biotopes have different 
aspects. The offshore shells (Figs. 10, 20) 
appear delicate, are clean with no apparent 
periostracum, white in colour with a sculpture 
of interlacing radial and concentric tracery. The 
density of the sculpture appears less in the 
offshore population, with the posterior area 
lacking the distinct semi-erect nodules seen 
in the sub and supralittoral populations. The 
sublittoral shells (Figs. 12, 18) are dirty with a 
pilose periostracum that retains fine sediment. 
The sculpture is partly obscured but when re- 
vealed appears primarily radial but is cancel- 
late. The supralittoral shells (Figs. 13, 16) are 
grubby, dirty white with the periostracum ei- 
ther lacking or persistent around the margins 
only. The sculpture appears primarily radial but 
is cancellate. 

A detailed examination using the scanning 
electron microscope reveals that all three 
populations have the same sculptural pattern 
(compare Figs. 16, 18, 20) but that the appar- 
ent differences are related to the expression 
of the concentric element. The sculpture con- 
sists of radial rows of elongate teardrop 
shaped pustules, the expanded portion coin- 



ciding with the intersection of the concentric 
element (compare Figs. 17, 19, 21). The con- 
centric sculpture consists of elevated bands 
attached to the radial elements but not to the 
interspaces, so when entire there is a lattice 
effect. The observed differences consequently 
appear to be related to the degree and rapid- 
ity of erosion of the non-attached interspace 
concentric cords. 

Periostracum: The periostracum consists of 
flimsy concentric lamellae with fine hairs. In 
the offshore population, the periostracum is 
not apparent in the hand but, when magnified, 
appears as a thin concentrically striated cov- 
ering expanded into thin lamellae along the 
lower edges of the concentric shell sculpture 
(Fig. 26). The lamellae bear thin, slightly thick- 
ened strap-like hairs at regular intervals. This 
contrasts with the obvious brown pilose cov- 
ering on the sublittoral and supralittoral shells 
(Fig. 25) where both the adherent portion and 
the lamellae are more strongly developed. 

Sliell Pores: As with other arcoid bivalves, 
Arcopsis valves possess numerous pores 
(caeca) (Reindl & Haszprunar, 1996). These 
tubules traverse the valves and can be ob- 
served on the inner and outer surfaces. On 
the external surface the pores are visible be- 
tween the raised sculpture and under low 
magnification were most obvious in the 
supralittoral population. Scanning electron 
microscopy shows that the pores in all three 
populations are similar in size but that in the 
supralittoral shells the area around each pore 
is more heavily eroded and thus gives the 
appearance of being larger. 

Pore density was also examined and was 
observed to vary over the shell with a radial 
pattern present and a decrease in density to- 
wards the ventral margins. Comparisons be- 
tween populations were made by examining 
a strip on the internal surfaces directly below 
the ligament, and then at the same point on 
each strip (compare Figs. 27, 28, 29). A visual 
comparison suggests that the pore density is 
greater in the supralittoral population. 

Larval Shell: The larval shells in all three 
populations are of the same form and same 
size (compare Figs. 22-24). There is a 
Prodissoconch I that is 105-106 pm in width 
and is smooth. There is a Prodissoconch II 
that is 174-178 pm in width and has concen- 
tric sculpture of widely spaced raised lines. 



332 



OLIVER & JÄRNEGREN 




FIGS. 8-15. Scanning electron micrographs of Arcopsis and Striarca shells. FIGS. 8, 9: Arcopsis 
afra, Angola; FIGS. 10, 11: >Arcops/s adamsi, offshore population, IMBW-FK-651; FIG. 12: A. adamsi, 
sublittoral population, IMBW-FK-629; FIG. 13: A. adamsi, supralittoral population, IMBW-FK-629; 
FIGS. 14, 15: Striarca láctea, Banyuls, Mediterranean Sea. 



ARCOPSIS MORPHOLOGY AND TAXONOMY 



333 



i»0- 



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4i 




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FIGS. 16-24. Arcopsis adamsi, all scanning electron micrographs. FIGS. 16, 17: Cleaned supralittoral 
shell and detail of sculpture; FIGS. 18, 19: Cleaned subiittoral shell and detail; FIGS. 20, 21: Cleaned 
offshore shell and detail; FIGS. 22-24: Larval shells, supralittoral, sublittoral and offshore. 



334 



OLIVER & JÄRNEGREN 



Anatomy 

Gross Anatomy: The gross anatomy of 
Arcopsis adamsi is in all respects very similar to 
that of the Indo-Pacific, epibyssate Striarca 
symmetrica (Reeve, 1844) (Oliver, 1985) and the 
eastern Atlantic Striarca láctea (Oliver, pers.obs). 

Offshore Population (Fig. 30) - The adductor 
muscles are approximately of equal size, both 
with quick and catch portions. The posterior 
pedal (byssus) retractor is prominent but not 
large, some 5 x the size of the anterior pedal 
retractor. 

The mantle is thick, and the mantle edges 
are free, with the main inhalant and exhalant 
regions at the posterior; the frilled anterior 
mantle edge suggests that there is an ante- 
rior inhalant current. 



The foot has a developed toe and a smaller 
heel; the byssal groove runs along the ante- 
rior and median regions. 

The gills are paired, with both demibranchs 
well developed, the inner being larger than the 
outer. The labial palps are moderately large, 
with 12 to 15 well-developed palp ridges. 

The anus is attached to the underside of the 
posterior adductor muscle and is accompanied 
by a pair of abdominal sense organs each in 
the form of a simple dome. 

Sublittoral (Fig. 32) and Supralittoral (Fig. 31 ) 
Populations - As above, with the only appar- 
ent difference seen in the supralittoral popu- 
lation, where the inner demibranch is smaller 
and almost the same as the outer. It is not 
possible to discount differential contraction 
through the fixation process. 







200|jm 



FIGS. 25-29. Arcopsis adamsi, SEM of periostracum. FIG. 25: Supralittoral shell; FIG. 26: Offshore 
shell; FIGS. 27-29: Arcopsis adamsi, SEM of shell pores at the median area below the beaks; FIG. 
27: Supralittoral; FIG. 28: Sublittoral; FIG. 29: Offshore. 



ARCOPSIS MORPHOLOGY AND TAXONOMY 

LP 

PPR/BR 



335 




FIGS. 30-35. Anatomy of Arcopsisadamsi. FIG. 30: Gross anatomy (left mantle removed), Samantha's 
Patch Reef (offshore population). A, anus; AA, anterior adductor muscle; APR, anterior pedal retrac- 
tor muscle; ASO, abdominal sense organ; CT, ctenidium; F, foot; ID. inner demibranch of ctedium; LP, 
labial palps; ME, mantle edge; ОС, ocelli; OD, outer demibranch of ctenidium; PA, posterior adductor 
muscle; PPR/BR, posterior pedal/byssus retractor muscle; FIGS. 31 , 32: Gross anatomy of supralittoral 
(FIG. 31) and sublittoral (FIG. 32) specimens; FIGS. 33-35: Left mantle after shell removal showing 
areas of haemoglobin staining (stippled areas); FIG. 33: Offshore; FIG. 34: Sublittoral; FIG. 35: 
Supralittoral. 



336 



OLIVER & JÄRNEGREN 



Haemoglobin: The Arcoidea are one of the 
few bivalve superfamllies in which haemoglo- 
bin cells are a characteristic component of the 
haemocoelomic fluid. Arcopsis ac/ams/ tissues, 
especially those of the mantle, are tinged pink 
to blood red indicating the presence of hae- 
moglobin. The variation in colour intensity in- 
dicates that the haemoglobin concentrations 
differ between populations, but given the con- 
straints of the workshop we were unable to 
measure the actual concentrations. We ob- 
served that the living tissues of the offshore 
population are scarcely tinged pink and in fixed 
material a rust coloured band is present only 
along the mantle edge (Fig. 33). This contrasts 
completely with the supralittoral population, in 
which the living tissues, primarily the mantle, 
are dark blood red and in the fixed state the 
pigmentation covers most of the mantle (Fig. 
35). The sublittoral population is intermediate 
in appearance but very distinctly with haemo- 
globin and thus more like the supralittoral 
population (Fig. 34). 



DISCUSSION 

The question for the taxonomist is of the 
degree of significance of these observations, 
could they reflect different species or are they 
result of ecophenotypic variation related to the 
different biotopes inhabited by the Arcopsis 
populations? 

Marko & Jackson (2001) using shell 
morphometries concluded that the Pacific 
taxon Arcopsis solida was different from the 
Caribbean A. adamsi, but that this difference 
was primarily one of size rather than shape. 
Nevertheless, they nowhere suggested that 
the two were conspecific. Given that the two 
taxa are now isolated by the Panamanian isth- 
mus, their conclusion to maintain them as 
separate species was probably influenced by 
the geographical separation of the taxa as 
much as the morphological differences. Marko 
(2002) later showed that at the molecular level 
his populations of /\. solida and A. ac/ams/ were 
distinct. The anatomy of A. solida and A. 
adamsi has been described by Heath (1941), 
but no direct comparisons were made, and his 
observations were inconclusive in relation to 
differences between these species. 

Oliver & Cose! (1992), studying Striarca 
láctea populations along the West African 
coast, also used morphometries to justify the 
erection of subspecies, giving outline and 
sculpture most credence. They did discuss the 



problems of erecting new taxa on such evi- 
dence but again were influenced by the geo- 
graphical separation of the populations. 

The morphometric data from these studies 
are comparable with those presented here but 
if they were used to create new taxa such de- 
cisions would be met with severe scepticism. 
The proximity of the populations and the data 
available would suggest to many that 
ecophenotypic variation was being observed. 
However, it is now widely accepted that, for 
example in the Littorinidae, that a number of 
species can live in adjacent microhabitats in 
close proximity to each other (Reid, 1 986). This 
is accepted because the differences cited are 
from disjunct characters, such as the struc- 
tures of the genitalia and radula, rather than 
being based on statistical analyses of gradi- 
ents such as shell shape. Unfortunately, 
bivalves display few characters of this kind and 
one of the major problems in bivalve species 
taxonomy is the gradation of many characters. 
Greater character definition of these gradients 
would strengthen bivalve taxonomy, but in 
using statistical methods at what levels of sig- 
nificance do we attach a species level or popu- 
lation level distinction? 

In 1992 Oliver & Cosel did discuss the af- 
finities of the West African taxon A. afra and 
A. adamsi. They indicated sculptural differ- 
ences, but given the variation now seen in A. 
adamsi such differences are not so conclu- 
sive. Arcopsis afra appears more umbonate 
with a stronger sculpture, but in most respects 
difficult to separate from A. adamsi. Once 
again, the geographic isolation of the two taxa 
is giving the weight to their separation. 

It is therefore essential to examine in detail 
the possible causes of the morphological dif- 
ferences observed in populations that only 
show character gradients. 

Thomas (1975, 1976; Thomas et al., 2000) 
showed that the allomethc growth of the arcoid 
ligament was essential to maintain a function- 
ing hinge. This allometry is expressed either 
in the progressive invasion of the ligament 
ventrally into the hinge plate or by further and 
further separation of the beaks. In Arcopsis 
the allometry is displayed in the separation of 
the beaks, and this allometry occurs at smaller 
shell length in the supralittoral population and 
is least marked in the offshore population. The 
growth function of the ligament is correlated 
not only to the relative inter-umbonal distance, 
ligament width and number of ligament bands, 
but probably also to the relative differences in 
shell tumidity and the degree of the inequiva- 



ARCOPSIS MORPHOLOGY AND TAXONOMY 



337 



Ive condition. Consequently, the morpliometric 
data presented are all a function of growth and 
may be related to environmental parameters. 

Of the three biotopes sampled, the supra- 
littoral provides the most extreme conditions 
for a suspension feeding organism as feeding 
time is restricted to a short period at high tide 
times. Long periods of exposure will also cause 
potential stress from desiccation and restricted 
respiration. It is reasonable to suggest that 
restrictions in feeding time will reduce the 
growth rate in the supralittoral population and 
will result in the decrease in maximum size 
observed. If the allometry is age-related rather 
than size-related, then different growth rates 
will produce shells of different shapes where 
the parameters are linked to umbonal separa- 
tion. The increased relative tumidity and in- 
equivalve condition seen in the supralittoral 
population is likely to be growth-related and 
therefore ecophenotypic. However, the off- 
shore population has a similar maximum size 
but shows little allometry and is smaller than 
the sublittoral population. If we were to use 
ecophenotypic variation as an explanation of 
morphological difference, then it would be sup- 
portive if we could link the differences to envi- 
ronmental parameters. The taxonomist, 
therefore, should also be aware of the ecol- 
ogy and habitats of the taxa under study. Here 
we can only speculate at the ecological differ- 
ences between the habitats occupied by 
Arcopsis, but changes in growth rate and maxi- 
mum size are likely to be controlled by food 
availability. 

The function of the shell pores has been ten- 
tatively linked to respiration and haemoglobin 
is known to be a more efficient oxygen carrier 
than haemocyanin. The density of shell pores 
and the haemoglobin concentration are great- 
est in the supralittoral population and may be 
physiological responses to the greater time 
spent out of the water. When considering the 
sublittoral and offshore populations, it is more 
difficult to apply the same reasoning as both 
populations are permanently submerged. The 
sublittoral population lives under rocks embed- 
ded in muddy sand and may suffer oxygen 
depletion in the very warm surface waters 
along the margins of the Keys. At the offshore 
sites, the rocks sit on clean sand and water 
flow is probably much greater over the ani- 
mals. These differences may account for the 
different densities of shell pores and haemo- 
globin seen in the two submerged populations. 

Consequently, the characters that define the 
supralittoral population can all be related to 



environmental effects on growth rate and 
physiology, but a similar argument cannot ex- 
plain all the differences between the offshore 
population and the two shallow populations. 
Additionally, it is difficult to explain the differ- 
ence in periostracum simply through abrasion 
as that on the offshore population is not 
abraded only very thin. The sculptural density 
is also not readily explained by differential 
growth rates. 

Without disjunct characters, the taxonomic 
process has been led more and more towards 
ecology and physiology and increasing at- 
tempts to discover the functionality of the 
characters under review. Most of the discus- 
sion above is subjective and would require 
substantial experimentation to confirm 
whether or not morphology and physiology 
were responding to environment. Taxono- 
mists traditionally have not or were not able 
to support their decisions concerning species 
discrimination and intraspecific variation. 
Morphometric analyses do give statistical 
support to observations but do not resolve 
the issue. Environment does affect shell form 
and growth rate, and these may alter sculp- 
tural density reinforcing the need for the tax- 
onomist to be aware of ecology. At least in 
the arcoids, the ligament growth is related to 
the expression of many other shell charac- 
ters so that what may appear to be an array 
of characters is in reality a single one. 

Although morphological characters are the 
traditional tools of the bivalve taxonomist they 
must be used carefully, because many are 
gradients and many are influenced by envi- 
ronment. Without additional data from ecology, 
including physiology and reproductive biology, 
the interpretation of characters is difficult. The 
application of molecular techniques needs to 
become routine especially when ecophenotyic 
or geographic variation is suspected. It must 
be recognised that most bivalve species tax- 
onomy remains at the morphospecies concept 
and that a good species remains the product 
of a good taxonomist! A molecular study of the 
populations discussed here is in progress but 
the wider application of this technique now 
requires access to correctly preserved mate- 
rial, which precludes most of the collections 
in the worlds museums. Morphological based 
taxonomy will remain widespread as will the 
need to identify species based on readily ob- 
servable characters. Although molecular tech- 
niques are necessary, the routine use of 
molecular characters for identification is prob- 
ably a long way off. 



338 



OLIVER & JÄRNEGREN 



CONCLUSIONS 

Morphological characters in bivalves need 
careful assessment before conferring spe- 
cies level significance to differences in them. 

Many characters form gradients and need sta- 
tistical analyses to substantiate observations. 

Statistical differences in gradient characters do 
not necessarily indicate species difference. 

Many shell characters are inter-dependent and 
can all be altered by simple changes in growth. 

Where possible, ecology should be an inte- 
gral part of taxonomic studies. 

Molecular techniques need to be applied to 
complex problems to give better resolution. 



ACKNOWLEDGEMENTS 

The authors wish to thank the organisers of 
the International Marine Bivalve Workshop, Dr. 
Rüdiger Bieler and Dr. Paula M. Mikkelsen for 
the invitations to attend and to them and their 
colleagues for their considerable efforts to 
accommodate our research. The Workshop, 
held in the Florida Keys, 1 9-30 July 2002, was 
funded by U.S. National Science Foundation 
award DEB-9978119 to co-organisers R. Bieler 
and P. M. Mikkelsen, as part of the Partner- 
ships in Enhancing Expertise in Taxonomy 
[РЕЕТ] Program. Additional support was pro- 
vided by the Bertha LeBus Charitable Trust, 
the Comer Science & Education Foundation, 
the Field Museum of Natural History, and the 
American Museum of Natural History. 

Thanks are also due to the National Museum 
of Wales for additional support and facilities 
to complete this study. 



OLIVER, P. G., 1 985, A comparative study of two 
species of Striarciinae from Hong Kong with 
comments on specific and generic systemat- 

ics. Pp. 283-310, in: B. MORTON & D. DUDGEON, 

eds.. Proceedings of the Second International 
Workshop on the Malacofauna of Hong Kong 
and Southern China, 1983. Hong Kong Uni- 
versity Press, Hong Kong. 

OLIVER, P G. & R. VON COSEL, 1992, Tax- 
onomy of tropical West African bivalves. V. 
Noetiidae. Bulletin du Muséum National 
d'Histoire Naturelle, Paris, (4)14[A](3-4); 655- 
691. 

REÍD, D. G, 1986, The littorinid molluscs of 
mangrove forests in the Indo-Pacific Region. 
British Museum (Natural History) Publication 
978, London. 227 pp. 

REINDL, S. & G HASZPRUNAR, 1996, Fine 
structure of caeca and mantle of arcoid and 
limopsoid bivalves (Mollusca, Ptehomorpha). 
The Veliger, 39: 10-116. 

THOMAS, R. D. K., 1975, Functional morphol- 
ogy, ecology and evolutionary conservatism in 
the Glycymerididae. Palaeontology, 18: 217- 
254. 

THOMAS, R. D. K., 1976, Constraints of liga- 
ment growth, form and function on evolution in 
the Arcoida (Mollusca: Bivalvia). Paleobiology, 
2(1): 64-83. 

THOMAS, R. D. K., A. MADZVAMUSE, P K. MAINI 
& A. J. WATHEN, 2000, Growth patterns of 
noetiid ligaments: implications of developmen- 
tal models for the origin of an evolutionary nov- 
elty among arcoid bivalves. Pp. 279-289, in: E. 

M. HARPER, J. D. TAYLOR & J. A. CRAME, eds.. The 

evolutionary biology of the Bivalvia. Geological 
Society of London, Special Publication 177. 



Revised ms. accepted 18 March 2004 



LITERATURE CITED 



ABBOTT, R.T, 1974, American Seashells. Van 
Nostrand Reinhold, New York. 663 pp. 

HEATH, H., 1941, The anatomy of the pelecy- 
pod family Arcidae. Transactions of the Ameri- 
can Philosophical Society, (n.s.) 31(5): 
287-319, 22 pis. 

MARKO, P В., 2002, Fossil calibration of mo- 
lecular clocks and on the divergence times of 
geminate species pairs separated by the Isth- 
mus of Panama. Molecular Biology and Evo- 
lution, 19(11): 2005-2021. 

MARKO, P B. & J. B. С JACKSON, 2001, Pat- 
terns of morphological diversity among and 
within arcid bivalve species pairs separated by 
the Isthmus of Panama. Journal of Paleontol- 
ogy, 75(3): 590-606. 



MALACOLOGIA, 2004, 46(2): 339-354 



ROCK AND CORAL BORING BIVALVIA (MOLLUSCA) 
OF THE MIDDLE FLORIDA KEYS, U.S.A. 

Paul Valentich-Scotf & Crete Elisabeth Dinesen^ 



ABSTRACT 

Eight species from three bivalve families were collected and/or observed in the Middle 
Florida Keys. Diagnoses based on shell characters are given for Botula fusca, Lithophaga 
antillarum, L. aristata, and L. bisulcata in the Mytilidae, and Gastrochaena hians in the 
Gastrochaenidae. Shell and anatomical comparisons are made for three members of the 
Petricolidae, Petricola lapicida, Choristodon robustum, and Ctioristodon sp. A, which is 
not attributable to a described Recent Choristodon species. 

These bivalves bore into limestone and dead coral, and in one case into living coral. 
Observations substantiated previous findings of primary chemical boring processes in Botula 
and Petricola. 

Key words: Botula, Lithophaga, Petricola, Choristodon, Gastrochaena, endolithic, boring 
bivalves, Florida Keys. 



INTRODUCTION 

As an expansion of the general bivalve 
biodiversity study initiated by Mikkelsen & 
Bieler (2000), we here describe the rock and 
coral boring bivalve fauna of the Middle Florida 
Keys. The goal of this publication is to provide 
a guide to the identification of the rock and 
coral boring bivalves in the Middle Keys re- 
gion. Where possible, we have made obser- 
vations and comparisons of the living animal, 
the anatomy, and the habitat of each species. 

Middle Keys boring bivalves are represented 
in the families Mytilidae, Petricolidae, and 
Gastrochaenidae. Turner & Boss (1962) de- 
scribed the lithophagan mytilids throughout the 
western Atlantic, including the Florida Keys. 
Coan's (1997) treatment of the eastern Pa- 
cific Ocean Petricolidae discussed species that 
are also found in the Caribbean/Atlantic re- 
gion. The taxonomy and biology of the 
Gastrochaenidae are well documented in 
Carter (1978). Carter also provided a list of 
coral boring bivalves from Soldier Key, Dade 
County, Florida, which is only 100 km north of 
the site of this study (West Summerland Key). 
Including members in the three aforemen- 
tioned families, Kleemann (1980, 1990a) dis- 
cussed the methods of chemical boring of 



these bivalves in the Caribbean, eastern Pa- 
cific Ocean and the Great Barrier Reef. Morton 
(1990) presented a global overview of coral- 
boring bivalves, including those in the west- 
ern Atlantic Ocean. 



MATERIALS AND METHODS 

Limestone and coral habitats were examined 
for boring bivalves, intertidally and subtidally 
to 3 m in the Middle Florida Keys in July 2002 
(Mikkelsen & Bieler, 2004, provide a station 
listing and map). Individuals were observed 
and/or collected primarily from the Florida Bay 
side of West Summerland Key (24°39'N). The 
limestone at this site is thought to be Key Largo 
Limestone, which in some cases is overlain 
by the Miami Oolite fades (M. Campbell, pers. 
comm., March 2003). Boring bivalves were 
collected from limestone and dead coral sub- 
stratum with a rock hammer and chisel. 
Bivalves occurring in living coral were exam- 
ined, but not collected. Field observations of 
the living animal and their burrows were made. 
In addition, bivalve borers were observed at 
Bahia Honda State Park (24°39'N), Fat Deer 
Key (24°40'N), Crawl Key (24°41'N), Grassy 
Key (24°44'N), Long Key (24°45'N), Planta- 



^epartment of Invertebrate Zoology, Santa Barbara Museum of Natural History, 2559 Puesta del Sol Road, Santa Barbara, 

California 93105 U.S.A.; pvscott@sbnature2.org 
^Institute of Biological Sciences, Department of Marine Ecology University of Aarhus, Finlandsgade 14, DK-8200 Aarhus N, 

Denmark; grete.dinesen@biology.au.dk 



339 



340 



VALENTICH-SCOTT & DINESEN 



tion Key (24°50'N), and Lower Matecumbe Key 
(24°50'N). 

Live animals were removed from their bur- 
rows, and relaxed in 7% MgCl2. Observations 
of the shell, ligament, siphons, mantle, and foot 
were made while the living animal was in a 
relaxed state. The right shell valve was care- 
fully removed with a scalpel inserted between 
the mantle margin and the shell. For selected 
species, the morphology of the labial palps, 
ctenidia, and siphons were recorded. 

Relaxed specimens were placed in 4% for- 
malin solution, and transferred to 70% ethyl 
alcohol. Ctenidial and labial palp filament 
counts were compared between living and pre- 
served specimens. 

Voucher specimens for all species collected 
in this study have been deposited at the Santa 
Barbara Museum of Natural History (SBMNH). 

Each species description includes a short 
diagnosis, followed by an expanded descrip- 
tion of the shell morphology and, where ob- 
served, the anatomy. Measurements and 
localities of specimens examined are given, 
along with habitats where the species were 
observed and collected. Additional literature 
citations are provided for each species, and 
when necessary additional remarks on the 
taxonomy or biology of the species are given. 

The following abbreviations are used in the 
text: AMNH, American Museum of Natural His- 
tory, New York, New York, USA; BMSM, Bailey- 
Mathews Shell Museum, Sanibel, Florida, 
USA; FMNH, Field Museum of Natural History, 
Chicago, Illinois, USA; SBMNH, Santa Barbara 
Museum of Natural History, Santa Barbara, 
California, USA. Station numbers listed in the 
following text refer to International Bivalve 
Workshop - Florida Keys (IMBW-FK) stations, 
as maintained by AMNH and FMNH 
(Mikkelsen & Bieler, 2004). 



SYSTEMATIC ACCOUNT 

Mytilidae Rafinesque, 1815 

Botula fusca (Gmelin, 1791) 

Figures 1-4 

Diagnosis 

Shell highly inflated; exterior dark brown; 
periostracum silky; beaks terminal, inturned, 
projecting beyond anterior margin; sculpture 
of commarginal striae only; without calcare- 
ous incrustations on exterior of shell; length 
of shell to 40 mm. 



Description 

Exterior - Lateral View: Shell subquadrate-elon- 
gate, slightly bent in the middle, slightly flar- 
ing posteriorly; beaks terminal, prosogyrate, 
inturned, pronounced, inflated; region ventral 
of umbones straight; posterior end rounded; 
broadly inflated from umbones to posterior 
margin, with rounded shoulders radiating from 
umbones to anteroventral and posterior re- 
gions, middle region slightly depressed; ven- 
tral margin incurved; byssus visible; sculpture 
of commarginal striae; periostracum chestnut 
brown, lighter brown in small specimens, 
strongly adherent; milky white mucus rem- 
nants attached to shell. 

Dorsal View: Ligament sunken, long, dark 
brown portion of ligament split for much of 
length; shell highly inflated. 

Ventral View: Umbones and prodissoconch 
visible from ventral side; ventral margin 
smooth; commarginal striae more pro- 
nounced near posterior margin. 

Interior. Shell pearly white internally; periostra- 
cum covering hinge under beaks; long thin, 
sharp posterior lateral tooth; edentulous un- 
der umbones; ligament deeply sunken, at- 
tached to a rolled nymph on the anterior 
portion, and a shelf-like nymph posteriorly. 

Anatomy 

Dorsal View: Foot orange anteriorly, white 
posteriorly, depressed in an anterior poste- 
rior direction, with small heel; distal portion 
of foot triangular, black; byssus projecting 
from base of foot; mantle unfused for most 
of ventral length; posterior rim of mantle is 
dark brown, remainder of mantle milky white. 

Lateral View (with left valve and mantle re- 
moved): Anterior adductor muscle large for 
size of shell; posterior adductor circular, larger 
than anterior; inner fold of mantle margin very 
muscular, middle fold thin; labial palps short. 

Measurements 

Length 29 mm, height 13 mm, width 15 mm; 
length 17 mm, height 8 mm, width 9 mm; both 
specimens from West Summerland Key, 
IMBW-FK-629, 24°39.3'N, 8ri8.2'W, col- 
lected by R Valentich-Scott and G. Elisabeth 
Dinesen (SBMNH 350547, 350548). Additional 
observations were made at Crawl Key and 
Bahia Honda State Park. Four additional lots 
of dry specimens from the Florida Keys were 
examined (SBMNH). 



ROCK AND CORAL BORING BIVALVIA 



341 



Habitat 

In a mucus nest, boring in soft limestone. 
Carter (1978) reported in dead coral [Diploria]. 

Remarks 

The limestone burrows of several specimens 
were found with dorsal keels, or with anterior 



notches in the limestone under the umbones 
(Fig. 4). Mechanical boring would not allow 
these keels or notches to be formed in the 
borehole. These findings correspond with Wil- 
son & Tait (1984), who suggested that Botula 
fusca only uses chemical means for boring. 

There has been much nomenclatural debate 
as to the correct name for the species in the 
western Atlantic Ocean. Wilson & Tait (1984) 




FIGS. 1-4. Botula fusca. FIGS. 1, 2: External left valve, internal right valve. West Summerland Key, 
Monroe County, Florida; 24°39.3'N, 8ri8.2'W; subtidal; Station 629; length 28.8 mm; SBMNH 350546; 
FIG. 3: Dorsal view. Grassy Key, Monroe County, Florida; 24°45'46"N, 80°57'11"W; length 39.6 mm; 
SBMNH 53503; FIG. 4: In limestone substratum; arrows denote invagination below umbones and 
corresponding notch in limestone; locality data the same as figures 1-2; length 26.1 mm; SBMNH 
350547. 



342 



VALENTICH-SCOTT & DINESEN 



used Botula fusca (Gmelin, 1791) as a single 
global species distributed in the Indian, Atlantic, 
and Pacific Oceans, and placed B. cinnamonea 
(Gmelin, 1 791 ) in synonymy. Nielsen (1 986) con- 
trasted this viewpoint, seeing B. cinnamonea as 
valid, with a broad Northern Hemisphere distri- 
bution. In addition, Nielsen designated a lecto- 
type for 8. cinnamonea, and restricting the type 
locality of this species to the Nicobar Islands. 



Additional morphological, anatomical and genetic 
studies are needed to solve this global issue. 

Literature 

Abbott (1974: 436), Keen (1971: 74), 
Mikkelsen & Bieler (2000), Nielsen (1976), 
Redfern (2001: 201), Soot-Ryen (1955: 86), 
Wilson & Tait (1984). 




FIGS. 5-8. Lithopliaga antillarum. Missouri Key Monroe County, Florida; 24°40.6'N, 81 °14.3'W; 
length 77.9 mm; SBMNH 350549. FIG. 5: Dorsal view; FIG. 6: External left valve; FIG. 7: 
Internal right valve; FIG. 8: Ventral view. 



ROCK AND CORAL BORING BIVALVIA 



343 



Lithophaga antillarum (Orbigny, 1853) 
Figures 5-8 

Diagnosis 

Shell elongate, cylindrical; beaks subtermi- 
nal, but not extending past anterior end; 
periostracum light to medium brown, dehiscent; 
sculpture of fine vertical lines over most of shell, 
and heavy commarginal undulations postero- 
dorsally; without calcareous incrustations on 
exterior of shell; length of shell to 120 mm. 

Description 

Exterior- Lateral View: Shell cylindrical, some- 
what compressed laterally, sharply rounded 
anteriorly, broadly rounded posteriorly, 
slightly flaring in the middle portion; beaks 
subterminal, small; sculpture of fine vertical 
lines over entire surface except narrow re- 
gion from beaks to posterior end, and irregu- 
lar commarginal striae, commarginal 
undulations posterodorsally; large portions 
of shell eroded, especially anteriorly; 
periostracum dehiscent, medium brown; cal- 
careous incrustations not present on shell, 
no encrusting extensions. 

Dorsal View. Beaks small, not inflated or pro- 
truding; dorsal margin not gaping; ligament 
not visible from dorsal surface; with long nar- 
row escutcheon; lunule not well demarcated; 
shell widest near midline, tapering posteriorly. 

Ventral View: Shell tightly closing, except for 
very narrow, short pedal gape, and very 
slight posterior gape; ventral margin slightly 
beveled inward. 

Interior: Interior pearly white, translucent; 
edentulous; ligament dark brown, deeply 
sunken, extending from umbones nearly to 
the shell midline. 

Anatomy 

Not examined. 

Measurements 

Length 85 mm, height 25 mm, width 21 mm; 
specimen collected by José Leal (26 July 
2002) at West Summerland Key, IMBW-FK- 
629, 24°39.3'N, 8Г18.2'\Л/ at 3 m depth, in 
soft limestone; deposited as a voucher speci- 
men at the Zoological Museum, University of 
Copenhagen, Denmark. Also obsen/ed at Fat 
Deer Key. Eight additional lots examined from 



Missouri Key (24°40'N) (SBMNH 350549), 
Vaca Key (24°46'N), and Barbados (all 
SBMNH), and Lower Matecumbe Key and 
Townsend Island (BMSM). 

Habitat 

Boring into soft limestone. Carter (1978) re- 
ported in dead coral (Diploria), and Scott 
(1988a) observed in dead coral and rock. 

Literature 

Turner & Boss (1962), Kleemann (1983, 
1984, 1990a, b, 1996), Mikkelsen & Bieler 
(2000), Morton (1990), Redfern (2001: 201), 
Warmke & Abbott (1971: 164). 

Lithophaga aristata (Dillwyn, 1817) 
Figures 9-11 

Diagnosis 

Shell inflated, cylindrical; beaks subterminal; 
with heavy calcareous incrustations over most 
of shell; elongated incrustations posteriorly, 
forming overlapping scissors-like "forceps"; 
length of shell to 33 mm. 

Description 

Exterior- Lateral View: Shell elongate ovate 
to cylindrical, sharply rounded anteriorly, ta- 
pering posteriorly; beaks subterminal, usu- 
ally eroded; sculpture of fine commarginal 
striae; periostracum dark brown; heavy cal- 
careous incrustations over entire surface, 
eroded in some spots; incrustations extend- 
ing past the posterior end, forming overlap- 
ping, scissors-like projections. 

Dorsal View: Beaks usually eroded, not ex- 
tending past the anterior margin; ligament 
black, sunken anteriorly, becoming visible 
near shell midline. 

Ventral View: Shell tightly closing, without vis- 
ible pedal gape; ventral margin nearly 
straight; posterior scissors-like incrustations 
easily viewed from this orientation. 

Interior. Shell very thin, fragile, translucent, 
slightly pearly white, slightly flaring dorsally; 
edentulous; posterior end of shell tapering, 
with calcareous extensions. 

Anatomy not examined. Morton (1993) dis- 
cussed various aspects of the anatomy, in- 
cluding a discussion on the formation of the 
scissors-like "forceps". 



344 



VALENTICH-SCOTT & DINESEN 




FIGS. 9-11. Lithophaga aristata. West Summerland Key, Monroe County, Florida; 24°39.3'N, 
8ri8.2'W; subtidal; Station 629; length 9.9 mm; SBMNH 350550. FIG. 9: Dorsal view; FIG. 10: 
Ventral view; FIG. 11: External left valve. 



Measurements 

Length 9.9 mm, height 4.1 mm; specimen 
collected by Diarmaid O'Foighil (27 July 2002) 
at West Summerland Key, IMBW-FK-629, 
24°39.3'N, 8ri8.2'W (SBMNH 350550). Two 
additional Florida lots were examined, along 
with 50 lots from the eastern Pacific Ocean 
(all SBMNH). 

Habitat 

Boring into limestone and coral. Coan et al. 
(2000) reported boring into shell in the east- 
ern Pacific Ocean. 

Literature 

Coan et al. (2000: 181), Keen (1971: 70), 
Kleemann (1983, 1990a, b, 1996), Mikkelsen 
& Bieler (2000), Morton (1 993), Redfern (2001 : 
202), Turner & Boss (1962), Yonge (1955). 

Lithophaga bisulcata (Orbigny, 1853) 
Figures 12-15 

Diagnosis 

Shell cylindrical, with flare along dorsal mar- 
gin, tapering posteriorly; with feathery calcar- 



eous incrustations along posterodorsal slope; 
incrustations extending evenly past posterior 
end of shell; length of shell to 45 mm. 

Description 

Exterior - Lateral View: Shell cylindrical, ta- 
pering posteriorly, anteriorly rounded; dor- 
sal and ventral margin parallel for the anterior 
half of the shell, flaring posterodorsally and 
then tapering posteriorly; beaks broad, 
slightly projecting, near anterior end, not ter- 
minal; sculpture of fine commarginal striae, 
with broad keel running from just posterior 
of beaks to posterior end; periostracum 
chestnut dark brown; surface anterior of keel 
with fine granular concretions except in the 
umbonal region and ventral margin, poste- 
rior of keel with heavy concretions, concre- 
tions becoming heavier posteriorly, feathery 
concretions posteriorly, posterior concretion 
extension short with fine granules. 

Dorsal View: Ligament deeply sunken in deep 
long escutcheon; anterior end triangular; 
gaping posteriorly; concretions along entire 
dorsal surface. 

Ventral View: Ventral margin slightly incurved 
to slightly bowed, smooth; shell narrowly 
gaping posteriorly; posteroventral calcare- 
ous incrustations with zipper-like pattern. 



ROCK AND CORAL BORING BIVALVIA 



345 




FIGS. 12-15. Lithophaga bisulcata. Missouri Key, Monroe County, Florida; 24°40.6'N, 
8Г14.3' W; length 45.0 mm; SBMNH 350551. FIG. 12: Dorsal view; FIG. 13: External left 
valve; FIG. 14: Internal right valve; FIG. 15: Ventral view. 



Interior. Shell dark brown, translucent, with 
slight sheen; edentulous; ligament deeply 
sunken, extending from beaks to the end of 
dorsal flare (well posterior of midline); beaks 
near anterior end, but not subterminal; an- 
terior end broadly rounded, dorsal flaring, 
posterior tapering; calcareous incrustations 



straight off posterior end, not forming for- 
ceps. 

Anatomy 

Not examined. Scott (1988a) detailed much 
of the anatomy of this species. 



346 



VALENTICH-SCOTT & DINESEN 



Measurements 

Length 21 mm (4 mm are the forceps concre- 
tions); height 6.5 mm; width 6 mm; specimen 
collected by Diarmaid O'Foighil (27 July 2002) 
at West Summerland Key, IMBW-FK-629, 
24°39.3'N, 8Г1 8.2'W. Six additional Florida lots 
examined (SBMNH), including specimens from 
Missouri Key (SBMNH 350551). 

Habitat 

Boring into limestone. Scott (1 988a) reported 
from living and dead coral, and rock. 

Literature 

Kleeman (1983, 1990a, b, 1996), Mikkelsen 
& Bieler (2000), Morton (1 990), Redfern (2001 : 
202), Scott (1985, 1988a, b), Turner & Boss 
(1962), Warmke & Abbott (1971; 164). 



Petricolidae Orbigny, 1840 

Choristodon robustum (G. B. Sowerby I, 1834) 

Figures 16-19, Table 1 

Diagnosis 

Shell ovate-elongate to trigonal, moderately 
inflated; inequilateral, posterior end much 
longer; anterior broadly rounded, posterior end 
tapering; sculpture of strong, irregular radial 
ribs, most prominent on the central portion of 
the shell; anterior and posterior ends gaping; 
siphons fused for nearly half length; length of 
shell to 43 mm. 

Description 

Exterior- Lateral View. Shell ovate to trigonal; 
moderately inflated; inequilateral, posterior 
end much longer; anterior end rounded, pos- 
terior end attenuate; umbones prosogyrate; 




FIGS. 16-19. Ciioristodon robustum. West Summeriand Key, Monroe County, Florida; 24°39.3'N, 
8ri8.2'W; subtidal; Station 629. FIGS. 16, 17: Length 20.2 mm; SBMNH 350554; FIG. 16: External 
left valve; FIG. 17: Internal right valve; FIG. 18: Length 19.8 mm; dorsal view; SBMMH 350553; FIG. 
19: Siphons of living animal, demarcation showing region of siphonal fusion; SBMNH 350552. 



ROCK AND CORAL BORING BIVALVIA 



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348 



VALENTICH-SCOTT & DINESEN 



beaks broad, projecting; sculpture of irregu- 
lar radial ribs, weak anteriorly, strong posteri- 
orly; posterior ribs prominent, broad, rounded, 
groove between ribs shallow, broad; anterior 
ribs very weak, barely visible at anterior end; 
mid portion ribs gradually increasing in size 
and height, sharp, thin; commarginal striae 
closely spaced, making surface weakly can- 
cellate, slightly lamellate posteriorly. 

Dorsal View: Shell moderately inflated, slightly 
compressed posteriorly; shell inequivalve, 
right side larger; shell gaping anterior and 
posterior of beaks; ligament short, external, 
sunken, on nymph; lunule small, deep; 
prodissoconch large. 

Ventral view: Widely gaping except for midline; 
terminal end of radial ribs intermesh at mid- 
line; right valve convex posteriorly; left valve 
concave posteriorly; posterior end twisted to 
the left; inequivalve, left valve smaller. 

Interior. Hinge plate short; three cardinal teeth 
in left valve - two anterior teeth short, stout, 
posterior tooth larger, plate-like, pointing pos- 
teriorly; right valve with two cardinal teeth - 
anterior tooth short, wide, stout, posterior 
tooth very small, thin, plate-like; ligament in 
two parts, outer section beginning just below 
beaks, light brown, inner section attached to 
nymph, black. 

Palliai sinus broad, shallow, not extending to 
beaks (about 1/3 distance between adduc- 
tors); right valve palliai sinus slightly broader 
than that of left valve; palliai line continuous 
in sinus region, patchy along ventral margin; 
anterior adductor muscle scar long, moder- 
ate in width, pointed dorsally and ventrally; 
posterior adductor muscle scar nearly circu- 
lar; left valve with two small pedal retractor 
scars posterodorsally; inside shell surface 
chalky; inner margin weakly and irregularly 
crenulate behind umbonal middline, inner 
margin non-crenulate anteriorly. 

Anatomy (Table 1 ) 

External View: Siphons translucent pale yellow/ 
orange on outer section, milky white near 
mantle, with small white granules in tissue, 
and brown streaks and blotches; exhalant si- 
phon much narrower than inhalant; inhalant 
siphon with short, simple papillae along rim; 
exhalant with simple papillae along rim; 
middle mantle fold light orange distally, rim 
plicate. 

Internal View: Outer and inner mantle fold very 
thick milky white, middle fold plicate, light or- 
ange with sporadic white granules towards 
the siphons, middle mantle fold near siphons 



dark brown; mantle fused from siphons to line 
below umbones; pedal gape relatively short 
extending from below beaks to anterior mar- 
gin; labial palps small, with 14 plicae; siphons 
fused for approximately half of their length; 
ctenidia creamy white; ctenidial plicae paral- 
lel to dorsal margin; outer demibranch 2/3 
length of inner demibranch; plicae much 
larger and wider than P. lapicida, approxi- 
mately 20 plicae on outer demibranch, about 
20 on inner demibranch; foot white, laterally 
compressed, long sole, toe small, sharp, 
pointed; with small pointed heel. 

Measurements 

Length 19 mm, height 14 mm, width 10 mm; 
specimen collected by P. Valentich-Scott and 
G. Elisabeth Dinesen at West Summerland Key, 
IMBW-FK-629, 24°39.3'N, 8ri8.2'W (SBMNH 
350552). 

Habitat 

Shallow, unlined burrows in limestone rocks. 
Carter (1978) reported (as Rupellaria typica) in 
dead coral (Diploria). 

Literature 

Coan (1 997), Keen (1 971 : 1 99), Lamy (1 923), 
Redfern (2001: 240), Warmke & Abbott (1971: 
199, as Rupellaria typica). 

Choristodon sp. A 
Figures 20-23, Table 1 

Diagnosis 

Shell ovate, inflated; subequilateral; anterior 
end with flange, extending well beyond inner 
shell margin; sculpture of flne radial ribs on 
anterior portion, stronger radial ribs posteriorly; 
anterior end slighfly gaping, posterior end tightly 
closed; siphons only fused basally; length of 
shell to 23 mm. 

Description 

Exterior- Lateral View: Shell ovate, highly in- 
flated, anterior end broad, posterior end 
slightly tapered; subequilateral, posterior end 
slightly longer; posterodorsal margin straight; 
anteroventral margin flared laterally; beaks 
broad, inflated, prosogyrate; lunule deep; 
sculpture of pronounced radial flat-topped 
ribs, interspaces deep, wide, overlain by flne 
commarginal striae. 



ROCK AND CORAL BORING BIVALVIA 



349 




FIGS. 20-23. Choñstodon sp. A. West Summerland Key, Monroe County, Florida; 24°39.3'N, 
8ri8.2'W; subtidal; Station 629; length 22.8 mm; SBMNH 350556. FIG. 20: External right valve; FIG. 
21: Internal left valve; FIG. 22: Dorsal view; FIG. 23: Siphons of the living animal, arrow showing 
fusion only at base of siphon (SBMNH 350555). 



Dorsal View: Ligament deeply sunken, short; 
shell gaping anteriorly, but closed posteriorly; 
equivalve. 

Ventral View. Anterior end slightly gaping, pos- 
terior end tightly closed. 

Interior. Right valve with two cardinal teeth, with 
large stout anterior tooth, fairly large plate- 
like posterior tooth; left valve with 3 cardinal 
teeth, anterior tooth small, stout, middle tooth 
large stout, posterior tooth thin plate-like. 

Anatomy (Table 1 ) 

Siphons small, short; space between siphons 
dark brown, dorsal of exhalent siphon dark 
brown, remaining area around siphons white; rim 
of both siphons with simple, short papillae with 
dark brown spots; inner papillae white, flower- 
like; siphons only fused for a short distance be- 



yond mantle; posterior portion of mantle very dark 
brown; outer fold thick, smooth; middle mantle 
fold thinner than outer, slightly plicate, pigmented 
towards siphons; inner mantle fold thick, milky 
white, smooth; pedal gape short anteriorly; inner 
mantle fold unfused along anterior margin, but 
fused for remainder of ventral margin; labial palps 
small, short, with 16 plicae; ctenidia pale pink to 
creamy white; outer demibranch with 12 plicae, 
inner demibranch with 16 plicae; foot thick, broad, 
without heel, with broad, short anterior end. 

Measurements 

Length 25 mm, height 20 mm, width 16 mm; 
specimen collected by R Valentich-Scott and 
G. Elisabeth Dinesen at West Summerland 
Key, IMBW-FK-629, 24°39.3'N, 8ri8.2'W 
(SBMNH 350555). 



350 



VALENTICH-SCOTT & DINESEN 



Habitat 

Boring into limestone, adjacent to 
Choristodon robustum. We found many shells 
of this species to be heavily bored by sponges 
and polychaetes. 

Remarks: Coan (1997) placed Choristodon 
typica Jonas, 1844, in synonymy with C. 
robustum, based on the figure provided by 
Jonas (Coan, 1997: fig. 43). The species we 
describe above is distinct, conchologically and 
anatomically, from C. robustum (e.g., shell out- 
line and sculpture, siphonal fusion, siphonal 
tentacles). As yet, we have not found a de- 
scribed species to correspond with our mate- 
rial. However, our specimens are very similar 
to the species illustrated by Narchi (1974), 
which he identified as С typica. The Florida 
Keys species is not the same at Redfern's 
(2001: 240) Petricoia sp. from the Bahamas, 



nor P. stellae (Narchi, 1975) from Brazil 
(Narchi, 1975). 

Table 1 compares anatomical characters of the 
two species of Choristodon found in the Middle 
Florida Keys, along with Petricoia lapicida. 

Literature 

Narchi (1974). 

Petricoia lapicida (Gmelin, 1791) 
Figures 24-27 

Diagnosis 

Shell subquadrate; inequilateral, posterior 
end much longer; sculpture of fine divaricate 
ribs over entire surface, and partial radial ribs 
near the posterior margin; siphons not fused; 
length of shell to 30 mm. 




FIGS. 24-27. Petricoia lapicida. West Summerland Key, Monroe County, Florida; 24°39.3'N, 8ri8.2'W; 
subtidal; Station 629; length 27.3 mm; SBMNH 350343. FIG. 24: External left valve; FIG. 25: Internal 
right valve; FIG. 26: Detail of external of right valve showing divaricate markings; FIG. 27: Preserved 
animal in limestone burrow, arrows denote burrow tightly fitting around animal (anteriorly), and the 
constricted posterior portion of the burrow; SBMNH 350559. 



ROCK AND CORAL BORING BIVALVIA 



351 



Description 

Exterior - Lateral View: Shell subquadrate, 
strongly prosogyrate, beaks broad, inflated; 
inequilateral, posterior end nnuch longer, 
beaks almost at anterior end; anterior end 
rounded; posterior end truncate; postero- 
dorsal margin nearly straight; sculpture very 
fine, divaricate ribs over most of surface (Fig. 
26); posterodorsal region with few pro- 
nounced, sharp radial ribs, terminating be- 
fore margin, sometimes wavy near ventral 
margin (eroded in some), interspaces be- 
tween radial ribs wide, flat. 

Dorsal View: Inflated anteriorly, more com- 
pressed posteriorly; equivalve; ligament 
deeply sunken, short; lunule deeply exca- 
vated posteriorly beneath ligament. 

Ventral View: Without ventral gape. 

Interior: Hinge plate short, triangular; peri- 
ostracum in lunular region; ligament deeply 
sunken, seated on a elongate infolded 
nymph, in two sections, both dark brown; left 
valve with two teeth, anterior tooth large, 
rectangular, posterior tooth small, thin, plate- 
like; right valve with two teeth, anterior small 
peg-like, posterior larger but plate-like; palliai 
sinus very broad, shallow, not reaching beaks; 
ventral palliai line slightly patchy, continuous 
in sinus area; anterior adductor muscle scar 
long, narrow (slightly broader than C. 
robustum); posterior adductor circular. 

Anatomy (Table 1 ) 

Mantle fused from beak to anteroventral 
margin, small fusion just anterior of inhalant 
siphon; mantle open over entire ventral re- 
gion from inhalant siphon to anterior margin; 
mantle without papillae; most of mantle milky 
white, except near siphons where it is dark 
brown in color; mantle swollen antero- 
ventrally, possibly a palliai gland; outer mantle 
fold very thin; middle fold muscular, tapering 
on margin, wavy, inner fold thin; mantle filled 
with white granules; labial palps white, me- 
dium length, pointed ventrally, with 32 plicae; 
smooth dorsally and anterior portion of palp; 
ctenidia dark orange; plicae parallel to dor- 
sal surface; outer demibranch extending to 
middle of inner demibranch; plicae number 
on demibranchs - inner 52, outer 40; siphons 
transparent dark gray, with embedded white 
granules; exhalant siphon circular in outline 
with tentacles of several types, some with 
simple bifurcations, others heavily branched; 
inhalant siphon elongate-ovate, gray with white 



spots; inhalant siphonal tentacles of two types, 
large compared to exhalant, simple, pointed 
(not bifurcate), with small lobes on side; also 
very short tentacle nobs projecting; inhalant 
siphon three times as large in diameter as 
exhalant; siphons barely extending beyond 
shell margin; foot compressed laterally, peach 
color, very flexible, pointed at tip. 

Measurements 

Length 27 mm, height 20 mm, width 13 mm, 
specimen collected by P. Valentich Scott and 
G. Elisabeth Dinesen at West Summerland 
Key, IMBW-FK-629, 24°39.3'N, 81°18.2'W 
(SBMNH 350558). 

Remarks 

Field observations of the burrow of Petricola 
lapicida have shown it lives in a constricted, 
flat burrow (Fig. 27). This strongly suggests 
the species burrows through chemical means 
only, and agrees with the findings of Morton 
& Scott (1988) and Morton (1990). Compari- 
sons between the functional morphology of 
P. lapicida and P. pholadiformis were pre- 
sented by Purchon (1955). 

Habitat 

Shallow burrow in limestone. Carter (1978) 
reported this species in dead coral {Diploria). 

Literature 

Abbott (1974), Bromley (1978), Kleemann 
(1 990a), Lamy (1 923), Morton (1 990), Morton 
& Scott (1988), Redfern (2001: 240), 
Robertson 1963, Warmke & Abbott (1971: 
191). 

Gastrochaenidae Gray, 1840 

Gastrochaena hians (Gmelin, 1791) 

Figures 28-30 

Diagnosis 

Shell ovate, white; incurved and widely gap- 
ing ventrally; widely gaping posteriorly; beaks 
terminal. 

Description 

Exterior - Lateral View: Shell inflated, ovate 
elongate; posterior end rounded, flaring; an- 
terior end narrow pointed; beaks terminal. 



352 



VALENTICH-SCOTT & DINESEN 



pointed, prosogyrate; prodissococh large, 
smooth; widely gaping anteroventrally, invagi- 
nate; shell color translucent white; sculpture 
of commarginal striae, stronger antero- 
ventrally, without radial elements; shell thicker 
along ventral gape. 

Dorsal View: Highly inflated, more compressed 
posteriorly; right valve overlapping the left; left 
valve slightly concave posteriorly; ligament 
external, protruding, long, one third of shell 
length; valves slightly gaping posterior to liga- 
ment. 

Ventral View: Periostracum thin, milky white, 
translucent, dehiscent; outer mantle fold thick, 
projecting beyond valve margin, wide gape, 
not fused for half shell length; middle mantle 
edge fused except for small pedal gape near 
shell midline; posterior end tightly closed, right 
valve overlapping left; posteriorly periostracum 
projecting beyond shell margin. 

Interior. Not examined. 

Anatomy 

See Carter (1978) for discussion of Gastro- 
chaena anatomy and shell features, along with 
diagnostic characters of related species. 



Measurements 

Length 1 1 mm, maximum height 6 mm, width 
4.5 mm, ligament length 4.5 mm, gape length 
8 mm, gape width 4 mm; specimen collected 
by Lisa Kirkendale on 27 July 2002, at Fiesta 
Key, IMBW-FK-644, 24°50.4'N, 80°47.0'W 
(SBMNH 350345). Three additional specimens 
were collected by the authors from West 
Summerland Key, IMBW-FK-629. 

Habitat 

In calcareous lined burrows in living and dead 
coral, and limestone. Carter (1978) reported in 
dead coral {Dlploha). 

Remarks 

Coan, et al. (2000: 494, left specimen) illus- 
trated a Florida specimen of Gastrochaena as 
G. ovata, but this specimen is actually G. hians. 

Literature 

Carter (1978), Morton (1983, 1990), Redfern 
(2001: 242). 




FIGS. 28-30. Gastrochaena hIans. Fiesta Key Causeway, Monroe County, Flohda Keys; 24°50.4'N, 
80°47.0'W; subtidal; station 644; length 11.8 mm; SBMNH 350345. FIG. 28: Dorsal view; FIG. 29: 
Ventral view; FIG. 30: Lateral view of left side. 



ROCK AND CORAL BORING BIVALVIA 



353 



DISCUSSION 



ACKNOWLEDGEMENTS 



The rock and coral boring bivalves of the 
Middle Florida Keys are diverse and numerous. 
With a modest sampling effort, eight species 
representing three families were observed and 
collected. While quantitative studies were not 
undertaken, several limestone rocks had more 
than 50 individuals/m^. However, there was a 
distinct patchiness to the distribution of these 
borers, even with seemingly identical substrata 
in adjacent areas. Often, large limestone boul- 
ders were completely void of bivalve borers, 
where adjacent rocks were riddled with 
pethcolids, mytilids, and gastrochaenids. 

Careful examination of the living bivalves and 
boreholes has confirmed the boring mecha- 
nisms of two species. In agreement with Wil- 
son & Tate (1984) and Kleemann (1990a), our 
observations indicate that Botula fusca is a 
chemical borer (Fig. 3). Similarly we have found 
strong indications of chemical boring in 
Petricola lapicida (Fig. 27), concurring with 
Morton & Scott (1 988). Lithophagans were rela- 
tively rare in our sampling areas, and we were 
unable to make definitive conclusions on habi- 
tat or boring mechanisms of these species. 

Far outside the scope of this paper are con- 
clusions about the localized or global distribu- 
tions of many boring bivalve species. Among the 
different lineages of boring bivalves, several are 
thought to be represented by a single genus with 
one or only a few species, and distributed world- 
wide (Morton, 1990). Morton further discussed 
the evolutionary events and implications, which 
could explain both the presence of true cosmo- 
politanism of some species and restricted re- 
gional distribution of other species. 

Nomenclatural inconsistency by researchers 
may account for confusion between cosmopoli- 
tan distributions and localized endemism within 
the boring bivalve lineages. This is easily un- 
derstood, as the majority of boring bivalve spe- 
cies names (and most marine bivalves) were 
originally designated exclusively based on shell 
characters. The shell morphology of boring 
bivalves has shown intraregional variation as 
large as interregional variation (Coan, 1997). 
This could be due to worldwide conspecifity, 
as has been suggested for Botula fusca by 
Wilson & Tait (1984), with shell plasticity as a 
consequence of individual morphometric adap- 
tation to their boring habitat. The use of shell 
features to discriminate between the species 
within different lineages of boring bivalves still 
needs confirmation from other methods (e.g., 
gross anatomy and histology, molecular se- 
quencing and analyses). 



We deeply appreciate all of the efforts in or- 
ganizing the international workshop on the 
Bivalvia by Rüdiger Bieler (FMNH), Paula 
Mikkelsen (AMNH), Russ Minton (FMNH), 
Isabella Kappner (FMNH), and the staff at the 
Keys Marine Laboratory. Important specimens 
for this project were collected by Liz Kirkendale 
(University of Florida), and José Leal (BMSM), 
who provided specimens and photographs. 
Eugene Coan gave a very helpful review of the 
first draft of this manuscript. Henry Chaney and 
Patricia Sadeghian, both of SBMNH, gave use- 
ful comments on a later draft of this paper. 
Thanks go to Claus Nielsen, Zoological Mu- 
seum of Copenhagen, for valuable discussion 
and help with Gmelin and Chemnitz type ma- 
terial and designations. 

The International Marine Bivalve Workshop, 
held in the Florida Keys, 19-30 July 2002, was 
funded by U.S. National Science Foundation 
award DEB-9978119 (to co-organizers R. Bieler 
and P. M. Mikkelsen), as part of the Partner- 
ships in Enhancing Expertise in Taxonomy 
[РЕЕТ] Program. Additional support was pro- 
vided by the Bertha LeBus Charitable Trust, the 
Comer Science & Education Foundation, the 
Field Museum of Natural History, and the 
American Museum of Natural History. 

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Revised ms. accepted 31 October 2003 



MALACOLOGIA, 2004, 46(2): 355-379 

COMPARATIVE MORPHOLOGICAL STUDY OF FOUR SPECIES OF BARBATIA 
OCCURRING ON THE SOUTHERN FLORIDA COAST (ARCOIDEA, ARCIDAE) 

Luiz Ricardo L. Simone^ & Anton Chichvarkhin^ 

ABSTRACT 

A detailed study on the morphology of the arcid genus Barbatia s.l. is performed, based 
on the common species occurring in Florida, complemented by samples from Brazil. The 
species are: B. cancellaria, B. candida, В. dominguensis, and B. teñera. The primary goal 
of this project is to collect comparative morphological data (especially on the internal anatomy) 
suitable for use in phylogenetic analysis. A complete descriptive and systematic treatment 
of these species is presented. A small phylogenetic analysis, based on nine characters (19 
states) and seven taxa, demonstrates a closer relationship of B. cancellaria with 6. can- 
dida, and of ß. dominguensis with B. teñera. Barbatia s.l. is found to be monophyletic. 

Keywords: specific differentiation, phylogeny, distribution, systematics. 



INTRODUCTION 

It is often thought that bivalve anatomy is 
very conservative and therefore of limited use 
for resolving systematic problems. To test this 
hypothesis, a study including four sympatric 
species that most malacologists consider to 
belong to a single arcid genus was undertaken. 
The genus is Barbatia Gray, 1842 (type spe- 
cies Area barbate Linné, 1 758, by subsequent 
designation of Gray, 1857, from the Mediter- 
ranean). The four species examined are com- 
monly collected in intertidal and subtidal areas 
of rocky environments on the Florida coast, 
as well as in the tropical western Atlantic. Ad- 
ditional information about arcid biology and 
systematics was provided by Lamy (1907), 
Reinhart (1935), Heath (1941), Sullivan 
(1961), Coelho & Campos (1975), and Boyd 
(1998). 

The taxonomy of the various members of 
Arcidae is mostly based on conchology, as the 
anatomical knowledge is sparse and present 
in few papers (e.g.. Heath, 1941). The four 
Barbatia species studied here are commonly 
allocated in different subgenera, with B. 
cancellaria and ß. candida mostly referred to 
the subgenus Barbatia s.s. (alternatively, B. 
candida has been placed in Cucullaearca 
Conrad, 1865; type species: Byssoarca lima 
Conrad, 1848, by the subsequent designation 
of Stoliczka, 1871; Upper Cretaceous, east- 
ern United States), ß. dominguensis to the 



subgenus Лсаг Gray, 1865 (type species: >Arca 
gradata Broderip & G В. Sowerby I, 1829, by 
the subsequent designation of Stoliczka, 1 871 ; 
eastern Pacific), and B. teñera to the subge- 
nus Fugleria Reinhart, 1937 (type species by 
original designation: F. pseudoillota Reinhart, 
1937; Pliocene of Florida). Our paper provides 
detailed comparative descriptions of the mor- 
phology of some arcid representatives, taxo- 
nomic treatments, and a preliminary application 
of these data in a small phylogenetic study. 

The objective of this paper is to perform a 
detailed anatomical study, developing the 
analysis of the data under a comparative sce- 
nario, showing that the study of the anatomy 
is valuable in comparative biology and impor- 
tant source of data. 



MATERIALS AND METHODS 

The specimens were collected during snor- 
keling and scuba dives in several localities of 
the Florida Keys (Mikkelsen & Bieler, 2004: fig. 
1 ), and were maintained alive for several days. 
Some of the specimens were narcotized using 
magnesium hydroxide in the field laboratory 
and subsequently dissected. Other specimens 
were fixed in 96% ETON, and after some days 
transferred to 70% ETOH for dissection at 
MZSP or FMNH. The dissections were per- 
formed using standard techniques, with speci- 
mens immersed in sea water or fixative. Images 



'Museu de Zoología da Universidade de Sao Paulo, Cx. Postal 42594, 04299-970 Sao Paulo, SP Brazil; lrsJmone@usp.br 
^Russian Academy of Sciences, Vladivostok, Russia 



355 



356 



SIMONE & CHICHVARKHIN 



were obtained using a standard digital camera 
directly or though a microscope; drawings were 
made with the aid of a camera lucida. For shell 
measurements, length indicates the anteropos- 
terior distance; lateral indicates the maximum 
inflation of the articulated valves; height indi- 
cates the dorsoventral distance originating from 
highest region of the umbo. 

The phylogenetic analysis was performed 
using the computer program Hennig86 (Farris, 
1 988) by means of the interface Tree Gardener 
(Ramos, 1998). Two arcid species were also 
included to test monophyly of the genus 
Barb at ¡a: 

(1) Area zebra (Swainson, 1833). U.S.A.; 
Florida; Florida Keys, Monroe County, 
24°39.3'N, 81 °1 8.2'W, "The Horseshoe" site, 
bayside of West Summerland Key (Spanish 
Harbor Keys), 4 specimens, MZSP 36100 
(FK-626, Simone coll. 26/vii/02). 

(2) Anadara notabilis {Rödlng, 1798). BRAZIL; 
Bahia; Salvador, Ribeira beach, MZSP 
28481, 15 specimens (Simone coll., 24-27/ 
ii/1997). 

A non-arcid filibranch was used as an 
outgroup: Isognomon bicolor (С. В. Adams, 
1845), Isognomonidae (Martins, 2000). This 
species is operationally used as the outgroup 
(rooting), the other two arcids {A. zebra, A. 
notabilis) are operationally analyzed as part 
of the ingroup. This procedure is undertaken 
for testing the monophyly of the genus 
Barbatia, as represented here. The anatomi- 
cal study of these three non-Barbatia species 
was performed in detail similar to that of the 
Barbatia species described herein. 

The following abbreviations are used in the 
figure captions: aa, anterior adductor muscle; 
an, anus; ao, abdominal organ; ap, anterior 
pedal protractor muscle; ar, anterior retractor 
muscle of foot; au, auricle; bf, byssal furrow of 
foot; by, byssus; ce, cerebral ganglion; cp, 
central gastric pad; cv, ctenidial vein; dd, ducts 
to digestive diverticula; dh, dorsal hood; er, 
esophageal rim; es, esophagus; ey, eye of 
mantle edge; ft, foot; ga, gill edge attached by 
cilia; gi, gill; gm, gill longitudinal muscle; gp, 
gill projection; gs, gastric shield; hi, hinge; ia, 
intestinal and style sac apertures; id, inner 
demibranch; if, inner fold of mantle edge; ih, 
inner hemipalp; in, intestine; ki, kidney; mb, 
mantle border; mf, middle fold of mantle edge; 
ml, mantle lobe; mo, mouth; ne, nephrostome; 
nv, nerve; od, outer demibranch; of, outer fold 
of mantle edge; oh, outer hemipalp; pa, pos- 
terior adductor muscle; pc, pericardium; pe, 
periostracum; pg, pedal ganglion; pm, palliai 



muscle; pp, palps; pr, posterior retractor 
muscle of foot; pt, pedal tentacle; rt, rectum; 
sa, gastric sorting area; sh, shell; ss, style sac 
and proximal portion of intestine; st, stomach; 
tm, connective between cerebral-pleural gan- 
glia with visceral ganglion; ty, pair of typhlo- 
soles separating intestine and style sac; um, 
umbo; ve, ventricle; vg, visceral ganglia; vm, 
visceral mass. 

Abbreviations of institutions: AMNH, Ameri- 
can Museum of Natural History, New York; 
FMNH, Field Museum of Natural History, Chi- 
cago; MNRJ, Museu Nacional da Universidade 
Federal do Rio de Janeiro; MZSP, Museu de 
Zoología da Universidade de Sao Paulo. 



SYSTEMATICS 

Barbatia cancellaria (Lamarck, 1819) 
(Figs. 1-7, 33-36,43-51) 

For additional synonymy, see Lamy (1907: 55). 
?Barbatia barbata: Heath, 1941: 294, pi. 5, 

figs. 2, 7; pi. 15, fig. 12 {non Linné, 1758). 
Barbatia (Barbatia) cancellaria: Warmke & 

Abbott, 1 962: 1 58, pi. 30, fig. j; Ríos, 1 970: 1 51 ; 

Andrews, 1971: 150, fig.; Abbott, 1974: 421- 

422, fig. 4966; Rios, 1975: 192, pi. 61 , fig. 939; 

Humfrey, 1975: 210, pi. 23, fig. 11; Rios 1985: 

208, pi. 75, fig. 1061; 1994: 230, pi. 80, fig. 

1136; Redfern, 2001: 203, pi. 83, fig. 831. 
Barbatia cancellaria: Diaz & Puyana, 1994: 46, 

fig. 24. 

Description 

Shell (Figs. 1 -7). Medium to large size, to 1 00 
mm. Color pale reddish brown. Main sculp- 
ture composed of narrow radial threads, 
separated from each other by furrows of 
approximately same width as threads (Fig. 
3); commarginal sculpture weak, composed 
mainly of undulations. Periostracum some- 
what thick, with many scales along radial 
threads; scales longer close to shell border. 
Most specimens with a pattern (most clear 
in posterior region) of one row of long scales 
followed by five rows of short scales (Figs. 
1-5). Periostracum extending beyond shell 
edges (Fig. 5). Umbos flat, located between 
anterior and middle thirds of hinge. Inner 
surface glossy, brownish violet on borders, 
becoming paler towards umbo. Hinge vari- 
able, but generally with about 30 teeth lo- 
cated just anterior to umbo and posteriorly 
(Figs. 5-7); three anteriormost teeth broader, 



FLORIDIAN BARBATIA MORPHOLOGY 



357 



weakly arched, tilted towards anterior; next 
three teeth with similar shape to those de- 
scribed, but narrower; following 8-10 teeth 
abruptly different, shorter, narrower, situated 
perpendicularly to outer edge of hinge, 
gradually becoming thicker, slightly longer 
and more tilted towards posterior; last 5-6 
teeth broader, almost horizontal. 

Soft Part Color (Figs. 33-36): Mantle border 
with a mixture of dark and pale brown spots, 



being darker towards edge (Fig. 33); some 
pale cream spots randomly distributed 
mostly along middle region of border. Pos- 
terior region of mantle border more pig- 
mented than more anterior region, including 
inner surface to base of gills; this posterior- 
inner surface pigmented uniform beige, hav- 
ing irregularly sized, randomly distributed 
white spots (Fig. 33). This pattern also cov- 
ering posterior and dorsal surface of poste- 
rior adductor muscle and rectum; anal papilla 




FIGS. 1-7. Barbatia cancellaria shell. FIG. 1: Left view, MZSP 36105; FIG. 2: Same, dorsal view; FIG. 
3: Same, detail of sculpture in posterior region; FIG. 4: Outer view of both valves, MZSP 32336; FIG. 
5: Same, inner view; FIG. 6: Right valve; MZSP 36105, detail of hinge; FIG. 7: Same, MZSP 36211. 
Scale bars = 5 mm. 



358 



SIMONE & CHICHVARKHIN 



abruptly preceded by a white region. Foot 
and ventral region of visceral mass pig- 
mented by a mosaic of brownish purple, coa- 
lescent spo