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MALACOLOGIA ^^ 

International Journal of Malacology 



Vol. 48(1-2) 






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MALACOLOGIA 
http://malacologia.fmnh.org 

EDITOR-IN-CHIEF: 
GEORGE M. DAVIS 



Editorial Office: 

Malacologia 

P.O. Box 1222 

West Falmouth, MA 02574-1222 

georgedavls99@hotmail.com 

Copy Editor: 

EUGENE COAN 

California Academy of Sciences 

San Francisco, CA 

gene.coan@sierraclub.org 



Business & Subscription Office: 

Malacologia 

P.O. Box 385 

Haddonfield, NJ 08033-0309 

malacolog@jersey. net 

Associate Editor: 

JOHN B. BURCH 

University of Michigan 

Ann Arbor 
jbburch@umich.edu 



Managing Editor: 
CARYL HESTERMAN 

Haddonfield, NJ 
malacolog@jersey.net 



Graphics Editor: 

THOMAS WILKE 

Justus Liebig University 

Giessen, Germany 

tom.wilke@allzool.bio.uni-giessen.de 



Composition Editor: 

CLAUDIA WILKE 

Wettenberg, Germany 

claudiawilke@hotmail.com 



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



RÜDIGER BIELER 

Vice President 

Field Museum, Chicago 

JOHN BURCH 

University of Michigan, Ann Arbor 

MELBOURNE R. CARRIKER 
University of Delaware, Lewes 

GEORGE M. DAVIS 
Secretary and Treasurer 

CAROLE S. HICKMAN 
University of California, Berkeley 



ALAN KOHN 

President Elect 

University of Washington, Seattle 

JAMES NYBAKKEN 

President 

Moss Landing Marine Laboratory, California 

CLYDE F. E. ROPER 

Smithsonian Institution, Washington, D.C. 

SHI-KUEIWU 

University of Colorado Museum, Boulder 

DIARMAID Ó FOIGHIL 
University of Michigan, Ann Arbor 



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 © 2006 by the Institute of Malacology 
ISSN: 0076-2997 



2006 
EDITORIAL BOARD 



J.A.ALLEN 

Marine Biological Station 

Millport. United Kingdom 

E.E.BINDER 

Muséum d'Histoire Naturelle 

Geneve, Switzerland 

D. BLAIR 

James Cook University 

Townsville, Australia 

P. BOUCHET 

Muséum National d'Histoire Naturelle 

Paris, France 

P. CALOW 

University of Sheffield 
Sheffield, United Kingdom 

RA. D.CAMERON 
University of Sheffield 
Sheffield. United Kingdom 

J. G. CARTER 

University of North Carolina 

Chapel Hill, NC 

M. CHARRIER 
Université de Rennes 
Rennes, France 

R. H.COWIE 
University of Hawaii 
Honolulu, HI 

A. H.CLARKE, Jr. 
Portland. TX 



B. С CLARKE 
University of Nottingham 
Nottingham, United Kingdom 

R.T DILLON, Jr. 
College of Charleston 
Charleston, SC 

C.J. DUNCAN 
University of Liverpool 
Liverpool. United Kingdom 

G. DUSSART 

Canterbury Christ Church University College 

Kent, United Kingdom 

D. J.EERNISSE 

California State University Fullerton 

Fullerton, CA 



E. GITTENBERGER HA^V^^Sy 

Rijksmuseum van NatuurlijkfM^\lnfi^^5'\ i * 
Leiden, Netherlands 

f. GIUSTI 
Université di Siena 
Siena. Italy 

M. GLAUBRECHT 

Museum of Natural History Berlin 

Germany 

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

A. V. GROSSU 
Universitatea Bucaresti 
Romania 

J. HABE 

Tokai University 

Shimizu, Japan 

R. T HANLON 

Marine Biological Laboratory 

Woods Hole. MA 

G. HASZPRUNAR 

Zoologische Staatssammlung München 

München. Germany 

J. M. HEALY 
Queensland Museum 
South Brisbane, Australia 

D. M.HILLIS 
University of Texas 
Austin. TX 

K. E. HOAGLAND 
/. M. Systems Group 
Rockville. MA 

B. HUBENDICK 
Naturhistoriska Museet 
Göteborg, Sweden 

S.HUNT 

University of Central Lancashire 

Lancashire. United Kingdom 

R.JANSSEN 

Forschungsinstitut Senckenberg 
Frankfurt am Main. Germany 

M.S.JOHNSON 

University of Western Australia 

Crawley, Australia 



R. N.KILBURN 
Natal Museum 
Pietermaritzburg. South Africa 

J.KNUDSEN 
Zoologisk Museum 
Kobenhavn. Denmark 



Ql Z. Y. 

Academia Sinica 

Qingdao. People's Republic of China 

D. G. REID 

The Natural History Museum 
London. United Kingdom 



C. MEIER-BROOK 
Tübingen, Germany 

C. LYDEARD 
University of Alabama 
Tuscaloosa. AL 

H.K. MIENIS 

Hebrew University of Jerusalem 

Jerusalem. Israel 



S.G SEGERSTRALE 
Institute of Marine Research 
Helsinki. Finland 

L. R.L.SIMONE 

Museu de Zoología da Universidade 

de Sao Paulo. Brazil 

A. STANCYKOWSKA 
Siedice, Poland 



J. E. MORTON 
Auckland University 
Auckland. New Zealand 



F. STARMÜHLNER 

Zoologisches Institut der Universität Wien 

Wien, A и st h a 



J. J. MURRAY Jr. 
University of Virginia 
Charlottesville. VA 



J. STUARDO 

Universidad de Concepción 

Valparaiso. Chile 



R. NATARAJAN 
Mahne Biological Station 
Porto Novo. India 



C.THIRIOT 

University Pierre et Mane Cune 

Pahs, France 



D. OFOIGHIL 
University of Michigan 
Ann Arbor Ml 



S.TILLIER 

Muséum National d'Histoire Naturelle 

Pahs, France 



J. 0KLAND 
University of Oslo 
Oslo. Norway 



JAM. VAN DEN BIGGELAAR 
State University of Utrecht 
Utrecht, Nethehands 



T. OKUTANI 
University of Fisheries 
Tokyo. Japan 

W. L. PARAENSE 
Instituto Oswalde Cruz 
Rio de Janeiro. Brazil 



N. H. VERDONK 
Rijksuniversiteit 
Utrecht. Netherlands 

H.WÄGELE 

Ruhr-Universität Bochum 
Bochum. Germany 



J. J.PARODIZ 

Carnegie Museum of Natural History 

Pittsburgh. PA 



A WAREN 

Museum of Natural History 

Stockholm. Sweden 



R. PIPE 

The Marine Biological Association 

Plymouth. United Kingdom 



B.R. WILSON 

Conservation and Land Management 

Kallaroo. Western Australia 



J.P POINTIER 

Ecole Pratique des Hautes Etudes 

Perpignan Cedex. France 

W. F. PONDER 
Australian Museum 
Sydney Australia 



H.ZEISSLER 
Naturkundemuseum 
Leipzig. Germany 

A. ZILCH 

Forschungsinstitut Senckenberg 

Frankfurt am Main. Germany 



MALACOLOGIA, 2006, 48(1-2): 1-26 

ANATOMY OF SHINKAILEPAS MYOJINENSIS SASAKI, OKUTANI & FUJIKURA, 2003 

(GASTROPODA: NERITOPSINA) 

Takenori Sasaki\ Takashi Okutani^ & Katsunori Fujikura^ 

ABSTRACT 

The anatomy of Shinkailepas myojinensis Sasaki, Okutani & Fujikura, 2003, was exam- 
ined by gross dissection, scanning electron microscopy, and histological serial sections. 
The organization of the soft part conforms to general neritopsine pattern, especially in 
palliai complex, digestive system, reno-pericardial system, and nervous system. New char- 
acter states previously unknown in neritopsine gastropods were revealed mainly in female 
and male reproductive systems, sense organs, and glands in palliai cavity. Comparison of 
our observations with published descriptions of various gastropods confirmed that the 
species of Shinkailepas are assigned to the superfamily Neritoidea among Neritopsina. 
The inclusion of Siiinkailepas in the family Phenacolepadidae as in previous studies is 
also supported, although the number of their uniquely shared character is rather limited. 
Infrafamilial taxa of phenacolepadids so far anatomically studied are clearly divisible into 
deep-sea {Sliinkailepas and Olgasolaris) and shallow-water {Phenacolepas and 
Cinnalepeta) groups in characters of the shell, operculum, head-foot external morphology, 
mantle margin, digestive tract, and female reproductive organ. At species level, members 
of Shinkailepas are diagnosed by morphology of the eye stalks, epipodial fold, and penis, 
as well as shell, radular and opercular characters. 

Keywords: Shinkailepas, Phenacolepadidae, Neritopsina, comparative anatomy. 



INTRODUCTION 

In the recent systematics, Neritopsina in- 
cludes six to nine families, namely, Neritidae, 
Neritiliidae, Phenacolepadidae, Hydrocenidae, 
Neritopsidae, including Titiscaniidae, and three 
helicinoidean families (Helicinidae, Ceresidae, 
and Proserpinidae, or three subfamilies of 
Helicinidae) (Thompson, 1980; Ponder, 1998; 
Sasaki, 1998; Kano & Kase, 2000, 2002). They 
comprise a robust clade phylogenetically (Pon- 
der & Lindberg, 1997; Sasaki, 1998) and ex- 
hibit remarkable ecological diversification from 
deep-sea to terrestrial habitats (Kano et al., 
2002; Sasaki & Ishikawa, 2002). Among them, 
three genera, Shinkailepas, Olgasolaris, and 
Bathynerita. have been known only from deep- 
sea chemosynthesis-based biological commu- 
nities. The first two genera are currently 
assigned to the Phenacolepadidae (Beck, 
1992; Waren & Bouchet, 2001; Sasaki at al., 
2003), together with the shallow-water gen- 
era Phenacolepas and Cinnalepeta, and 
Bathynerita is regarded as a member of the 
Neritidae (Waren & Bouchet, 1993, 2001). 



They represent part of characteristic mollus- 
can elements endemic to vent/seep environ- 
ments. 

In the genus Shinkailepas Okutani, Salto & 
Hashimoto, 1 989, four species have been hith- 
erto described: (1) S. kaikatensis Okutani, 
Saito & Hashimoto, 1989, from Kaikata Sea- 
mount, off Ogasawara Islands, Japan, 470 m 
deep, (2) S. tufari Beck, 1992, from Manus 
Back-Arc Basin, 2,450-2,505 m deep, (3) S. 
br/and/ Waren & Bouchet, 2001, from Mid-At- 
lantic Ridge, Menez Gowen to Logatchev site, 
850-3,500 m deep, and (4) S. myojinensis 
Sasaki, Okutani & Fujikura, 2003, from Myojin 
Knoll, Ogasawara Ridge, Japan, 1 ,260-1 ,340 
m deep. In addition, unidentified species were 
also reported from Mariana Back-Arc Basin 
(Hasegawa et al., 1 997) and Okinawa Trough 
(Sasaki et al., 2003), suggesting the presence 
of more new species in the genus. 

Although some anatomical descriptions have 
been published (e.g., Fretter, 1984; Sasaki, 
1998; Kano & Kase, 2002), there is consider- 
able uncertainty in anatomical organization of 
phenacolepadids and other possibly related 



4he University Museum, The University of Tokyo, 7-3-1 , Hongo, Bunkyo-ku, Tokyo 11 3-0033, Japan; sasaki@urn.u-tokyo. ac.jp 
^Japan Agency for Marine-Earth Science and Technology, 2-15, Natsushima, Yokosuka City 237-0061, Japan 



SASAKI ETAL. 



neritoidean groups. Hence, further anatomical 
comparison is significant to understand rela- 
tionships among Shinkallepas. Olgasolaris, 
shallow-water neritoideans. and the remaining 
less known neritopsines. All of known species 
of Shinkallepas have been described based 
chiefly on the shell, radula. operculum, and 
external morphology of the animal (Okutani et 
al., 1989; Beck, 1992; Waren & Bouchet, 2001; 
Sasaki et al., 2003), and only limited anatomi- 
cal descriptions have been published for inter- 
nal organs of the genus. 

In this study, we attempted to provide de- 
tailed account of anatomical organization of 
Shinkallepas myojinensis by gross dissection, 
scanning electron microscopy, and histologi- 
cal serial sections. The results of observations 
are compared with existing knowledge of or- 
gan systems of other nertiopsines, and their 
significance is discussed in terms of compara- 
tive anatomy and systematics. 



Agency for Marine-Earth Science and Tech- 
nology (JAMSTEC). Details of sampling data 
are shown in the original description. Samples 
fixed in formalin were used for anatomical 
observations. After removed from the shell, soft 
parts of five specimens were dissected under 
a binocular microscope. Pieces of dissected 
soft parts were dried with a freeze-drier (Hitachi 
ES2030) and observed with SEM (Hitachi 
S2400). Whole animals of two females and 
three males were thin-sectioned at the thick- 
ness of 6 pm after embedding in paraffin. They 
were stained by a standard method of Mayer's 
Hematoxylin and Eosin staining. Whole series 
of sections (UMUT RM28647-28651 ) and 
SEM stub (UMUT RM28652) are deposited in 
the Department of Histological Geology and 
Paleontology, the University Museum, the 
University of Tokyo (UMUT). The terminology 
used in the descriptions chiefly followed that 
of Sasaki (1998) and Kano & Kase (2002). 



MATERIALSAND METHODS 

Materials examined in this study were se- 
lected from part of paratype series of S. myoji- 
nensis and those preserved at the Japan 



RESULTS 

Shell and Operculum 
See Sasaki et al. (2003). 




FIG. 1 . Dorsal view of animal, male, with mantle removed. 



ANATOMY OF SHINKAILEPAS 



External Anatomy 

The animal is dorsoventrally flattened and 
symmetrical in outline. The visceral mass is 
not spirally coiled (Fig. 1 ). The dorsal surface 
of the animal is covered with a thin mantle. 
The entire surface of the mantle carries dense 
filamentous mantle processes (mp: Fig. ЗА), 
which are multicellular and contain fiber-like 
structure internally (Fig. 4A). These processes 
are not branched and some of them penetrate 
through whole thickness of the shell at posi- 
tions corresponding to the shell pores (cf. 
Sasaki et al., 2003: fig. 120). The dorsal side 
of mantle margin also bears similar processes 
(Fig. 3B). 

The mantle margin (mm: Figs. 2, 40) is 
prominently thickened, divided into the outer 
and inner folds by the periostracal groove (pg: 
Figs. 3B, 40). Its inside contains muscle fi- 
bers and blood sinus. The portion of the mantle 
covering the palliai cavity is provided with ex- 
tensive blood sinus with numerous hemocytes 
inside (pis: Fig. 4F) and probably functions as 
a respiratory surface. 

Major part of the ventral side of the animal is 
occupied by a circular, flattened pedal sole (ps: 
Fig. 2). Its anterior margin is marked by the 
anterior pedal groove (apg: Figs. 2, 4B) with 
the opening of the anterior pedal gland (apd: 
Fig. 48). The ventral side of the head is ex- 
tended as the oral lappet (ol: Figs. 2, 30). The 
circumference of the mouth is thickened, papil- 
late, and wrinkled (Fig. 30). The lateral foot is 
smooth without any protrusive structure. The 
epipodial fold is extended between posterior 
dorsal rim of the foot and the mantle margin 
and provided with triangular tentacles (Figs. 
2, 3D). The epipodial tentacles lack 
micropapillae, sense organ and ciliated struc- 
ture. Their number ranges from 11 to 1 9, vary- 
ing among specimens examined. The 
operculum is firmly attached to the dorsal sur- 
face of the foot musculature below the visceral 
mass. 

The head consists of the snout, cephalic ten- 
tacles, cephalic lappets, and eye stalks (Fig. 
1). The snout is stout, short and not tapered. 
The cephalic tentacles are paired in equal form 
and size, symmetrically positioned on each 
side of the head, not covered with sensory 
papillae, and striated by internal longitudinal 
muscle. The cephalic lappets are symmetri- 
cal, equal in size in female, but the right one 
is greatly enlarged in the male as the penis 
(p: Fig. 1 ). The eye stalks are posterior to the 
cephalic tentacles, flattened, and morphologi- 



cally identical between both sexes. Only the 
posterior half of the right eye stalk is covered 
with a patch of tall glandular cells, which are 
here termed the "post-tentacular gland" (ptg: 
Figs. 1 , 4F). Black eye spots are visible exter- 
nally in anterior middle part of the eye stalks 
(Fig. 1). 

The shell muscle is divided into right and left 
portions (Ism, rsm: Fig. 1), leaving separated 
scars on the internal surface of the shell. Each 
muscle is not subdivided into bundles, nor 
penetrated by blood vessels. The head retrac- 
tor muscles are merged with shell muscles and 
do not produce independent attachment ar- 
eas. The mantle is devoid of particular retrac- 
tor muscle inserted on the shell, but instead 
attached to the shell by penetration of pallia! 
processes. 

Palliai Oavity 

The palliai cavity is deep and attains the level 
of the posterior end of both shell muscle at- 
tachments. It contains the ctenidium, 
osphradium (os: Fig. 4E), anus, the kidney 
opening (ko: Fig. 10O), and genital opening(s). 

The ctenidium is single, extended from the 
posterior left to the anterior right (Fig. 1). The 
ctenidial lamellae are bipectinate in alternating 




FIG. 2. Ventral view of animal. 



SASAKI ETAL. 







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FIG. 3. Scanning electron micrographs of external and internal organs. A. Mantle processes (mp) on 
surface of mantle covering visceral part. B. Dorsal view of mantle margin. С Ventral view of mouth 
(m) and oral lappet (ol). D. Ventral view of epipodial fold. E. Tooth of gastric shield (gst) inside of 
stomach. F. Left auricle (la) and ventircle (v) penetrated by intestine (i), removed from pericardium. 
A-F. UMUT RM28652. 



arrangement (Fig. 4D), ridged along midline (Fig. 
1), and not attached by afferent nor efferent 
ctenidial membranes. The skeletal rods and 
buhscles (sensory pockets) are absent (Fig. 4D). 
A hypobranchial gland is absent. Part of the 
mantle opposing the post-tentacular gland is 
covered with a tall glandular epithelium, which 
is termed the "anterior pallia! gland" (apl; Fig. 



4F). This gland is also extended to cover the 
distal tip of the palliai gonoduct. Dorsoventrally 
paired, two glands, the post-tentacular gland 
on ventral side and the anterior palliai gland 
on dorsal side, are developed in the same 
position and size in both sexes. The epithe- 
lium of equivalent part on the left side is not 
specialized as gland. 



ANATOMY OF SHINKAILEPAS 







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FIG. 4. Histology of external and palliai organs. A. Vertical section of mantle processes (mp) arising 
from mantle (mt) above ventricle (v). B. Longitudinal section of anterior tip of foot, showing anterior 
pedal gland (apd) and anterior pedal groove (apg). С Longitudinal section of posterior part of foot (f) 
and mantle margin (mm). Epipodial fold (ef) arises from part of foot. D. Longitudinal section of ctenidial 
lamellae. E. Cross section of osphradium (os) along left shell muscle (Ism). Arrowhead indicates 
longitudinal groove on osphradium. F. Cross section of two opposing glands in palliai cavity near 
right eye stalk. A, E-F. UMUT RM28648. B-D. UMUT RM28651. 



Digestive System 

The digestive system consists of the oral 
tube, sublingual pouch, buccal cavity, buccal 
mass, radula, esophagus, stomach, digestive 
glands, and intestine to anus (Fig. 5). 

The mouth opens ventrally (Fig. 1 ). The oral 
tube is considerably short in front of the buc- 
cal mass and followed by the sublingual pouch 



ventrally and by the buccal cavity dorsally. The 
anterior inner wall of the buccal cavity is par- 
ticularly thickened as the transverse buccal 
fold with a pair of distinctly cuticularized plates 
(cp: Fig. 1 0). The jaw with tooth-like elements 
is absent. 

The sublingual pouch is well developed be- 
low the buccal mass (sip: Fig. 7). Its epithe- 
lium is thin and smooth. Paired sublingual 



SASAKI ETAL. 




I mm ast 



FIG. 5. Configuration of digestive tract in dorsal view. 



glands project on each side of sublingual 
pouch and open into ventrolateral side of the 
oral tube (sig: Fig. 7). Their inner surfaces are 
roughened with irregular forms of glandular 



epithelium (Fig. 9B). The dorsal side of buc- 
cal cavity is thickened by a pair of well-devel- 
oped dorsal folds (df: Fig. 9D). Salivary glands 
are not differentiated around the buccal cav- 
ity. The radular diverticulum is deep below the 
esophageal valve. 

The buccal mass is elongated longitudinally 
and connected with body wall musculature with 
the lateral protractors (Ip), inner and outer pairs 
of the ventral protractors (ivp, ovp), median 
and dorsal levators (ml, dl), posterior depres- 
sors (pd), anterior levators (al), and anterior 
tensors (at) (Figs. 6, 7). The posterior part of 
the odontophore is separated on both sides 
of radular sac and closely tied by postdorsal 
buccal tensor (pdt: Fig. 6) on the dorsal sur- 
face. 

The buccal mass is internally supported by 
five odontophoral cartilages. The anterior 
cartilages are paired, elongated longitudinally, 
increasing in width backwards. The posterior 
cartilages are also paired, much smaller than 
the anterior pairs, firmly attached to the ante- 
rior pairs, and tapered posteriorly with pointed 
posterior ends. The median cartilage is un- 
paired, of almost the same length as the ante- 
rior cartilages, and lies between the anterior 
cartilages (Fig. 8). Its anterior one-fifth is de- 
marcated by dorsal constriction and is 
smoothly swollen ventrally (Fig. 8). Ventral 




FIG. 6. Drosal view of buccal mass, with anterior 
digestive tract intact. 



FIG. 7. Ventral view of buccal mass, with oral 
tube and sublingual pouch intact. 



ANATOMY OF SHINKAILEPAS 




0.5 mm 



A 



В 



FIG. 8. Odontophoral cartilages. A. Dorsal view. B. Ventral view. Outer 
approximator muscle (oap) is removed on left side. Anterior part of 
ventral approximator muscle (vap) is also removed (cf. Fig. 7). 



sides of the anterior cartilages are connected 
by the ventral approximator muscle (vap: Fig. 
7). The anterior and posterior cartilages are 
longitudinally united with the outer approxi- 
mator muscles (oap: Fig. 7). Sides of anterior 
cartilages are attached to the body wall by ten- 
sor muscles (tac: Figs. 6, 7), 

The radula is composed of an ensheathed 
part of the radular sac posterior to the buccal 
cavity, the subradular membrane spread over 
the buccal mass, and functional area exposed 
into the buccal cavity on the bending plane. 
The radular sac is extended backwards and 
not coiled in loops. Its posterior end is smooth 
and simple. The subradular membrane is at- 
tached by the median and lateral protractor 
muscles (mpr, Ipr: Fig. 7) anteriorly, and the 
retractor muscles (rsr: Fig. 7) laterally and 
posteriorly. The retractor muscles of radular 
sac (rrs: Fig. 7) originate from the posterior 
end of the posterior cartilages, insert on the 
radular sac ventrally. The radular teeth mor- 
phology was described by Sasaki et al. (2003). 

The esophagus begins from the postero- 
dorsal part of the buccal mass and comprises 
two parts, the anterior and posterior esophagi. 
The anterior esophagus is dorsoventrally de- 
pressed, almost consistent in width and curves 
towards the left over the buccal mass. Inside 
of the anterior esophagus is divided into the 
dorsal food channel (dfc) in the center and the 
lateral esophageal pouches (lep) on both sides 



by a pair of the dorsal folds (Figs. 9D, E). The 
inside of the lateral esophageal pouches is 
glandular, and therefore, it can also be called 
the anterior esophageal gland. Behind the 
buccal mass, a main part of the anterior 
esophagus slightly turns forward to form a very 
short loop. The posterior part of lateral 
oesphageal pouches are separated from dor- 
sal food channel, extending ventrally as the 
posterior esophageal gland, and surrounds the 
radular sac completely (peg: Fig. 9F). Their 
epithelia are heavily folded (Fig. 9F). The pos- 
terior esophagus is narrower than the ante- 
rior esophagus (Fig. 5), and defined by 
corrugated inner structure (Fig. 9G). It runs 
below the radular sac and other organs at the 
deepest level of the visceral cavity. 

The stomach is marked by the enlargement 
in diameter and folded into U shape. The in- 
side has large cuticularized area, short cres- 
cent-shaped gastric caecum (gc: Fig. 5), tooth 
of gastric shield (gst: Figs. 3E, 5), and paired 
typhlosoles on the ventral side. The digestive 
glands are well developed around the stom- 
ach and connected to the distal part of the 
stomach through four openings dorsally and 
two openings ventrally. The ducts of digestive 
glands are further divaricated complicatedly. 

The intestine is circular in cross section, 
nearly consistent in diameter along its entire 
length, turns in two sites, posterior to the buc- 
cal mass and below the pericardium (Fig. 5). 



SASAKI ETAL. 







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FIG. 9. Histology of digestive system. A. Longitudinal section of transverse buccal fold (tbf), cuticular- 
ized plate (cp), and radular teeth (rdt). B. Cross section of sublingual gland (sig). С Vertical section 
of anterior odontophoral cartilage. D. Cross section of left half of dorsal fold (df) and lateral esoph- 
ageal pouch (lep) over anterior part of buccal mass. E. Cross section of left half of anterior esopha- 
gus in more posterior part than in Fig. D. F. Cross section of posterior esophageal gland (peg) sur- 
rounding radular sac (rds). G. Cross section of posterior esophagus (pe). H. Cross section of stom- 
ach, showing openings to digestive glands (dgo) on dorsal and ventral (upper and lower in figure) 
sides. C, H. UMUT RM28647. B, D-F. UMUT RM28648. A, G. UMUT RM28651. 



ANATOMY OF SHINKAILEPAS 



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FIG. 10. Histology of circulatory and excretory systems and sense organs. A. Horizontal section of 
kidney (k) and pericardium (pc). B. Enlarged view of ventricle (v) penetrated by intestine (i). С Lon- 
gitudinal section of kidney (k), its opening (ко) and adjacent organs. D. Enlarged view of epithelium 
of kidney (k). E. Cross section of left eye (e). F. Cross section of statosysts (sta) containing statoconia 
(sc) below pleural commissure (pic). D. UMUT RM28647. E-F. UMUT Rl\/I28648. A-C. UMUT 
RM28651. 



Its epithelium is densely ciliated and composed 
of a layer of prismatic cells (Fig. 1 0B). Around 
the second turn, the intestine penetrates the 
ventricle and the pericardium. No anal (rectal) 
gland was found near the distal part of the in- 
testine. The anus opens on the right anterior 
side of the palliai cavity (Fig. 1 ). 



Circulatory System 

The heart is enclosed in the pericardium and 
consists of paired auricles of unequal size and 
a median ventricle (Figs. 1, 3F, 10A). The left 
auricle is much larger than the right and con- 
nected to blood vessels from the ctenidium and 



10 



SASAKI ETAL. 




FIG. 11 . Palliai oviduct of female, with intestine intact. A. Dorsal view. 
B. Ventral view. 



the mantle. The margin of the left auricle is rug- 
ose and constricted to be separated into a few 
chambers (Fig. 3F). The right auricle is vesti- 
gial and situated posterior to the ventricle (Fig. 
1). The ventricle is voluminous, penetrated by 
the intestine, stiffened by cardiac muscles in- 
side (Fig. 10B). An aorta from the ventricle is 
short and opens into haemocoel of the body. 
An aortic bulb is not formed. Large sinuses are 
developed among visceral organs and around 
the buccal mass. Considerable blood sinuses 
are also found near the lateral wall of the pedal 
musculature. Blood space adjacent to the kid- 
ney is connected anteriorly to the afferent 
ctenidial vessel. 

Excretory System 

The excretory organ consists of a single kid- 
ney and the pericardium. The kidney is on the 
posterior side of the ctenidium; its excretory 
opening is located below ctenidial base (ko: 
Fig. 10C). The inside of the kidney is partly 
partitioned into two branches on the anterior 
and right sides of the pericardium, respectively 
(Fig. 10A), but both are indistinguishable in 
histology. The epithelium of the kidney is not 
lamellated throughout the entire area and con- 
sists of single-layered papillate cells with basal 
nuclei (Fig. 10D). The renopericardial connec- 
tion is extended between the two branches of 
the kidney. 



Reproductive System 

The sexes are separate, and their reproduc- 
tive organs exhibit striking sexual dimorphism. 
Large part of the reproductive organ is occu- 
pied by the gonad and palliai gonoduct. In both 
sexes, the gonad develops dorsal to the diges- 
tive gland, the gonoduct is not connected to 
the kidney, and gonopericardial connection is 
not present. 

The female reproductive system consists of 
the ovary, a thin oviduct connecting the ovary 
and the palliai oviduct, a complex of palliai ovi- 
duct and associated glands, and the vaginal 
duct. The ovary and dorsal surface of the pal- 
liai oviduct are visible on the dorsal side of the 
animal through the mantle. The oviduct is thin 
and circular in cross section, arises from 
anteroventral part of the ovary, extends forward, 
and enters the palliai oviduct from its ventral 
side (Figs. 118, 12, 15C). 

The palliai oviduct is surrounded by two dif- 
ferently stained glands, a posteriorly situated 
albumen gland, and an anteriorly located cap- 
sule gland. The albumen gland covers poste- 
rior area of the palliai oviduct and is further 
subdivided into two portions, albumen gland I 
and II (agi, agil: Fig. 11). Both parts are not 
stained darkly, and the albumen gland I is obvi- 
ously more translucent than the albumen gland 
II (Fig. 15C). The palliai oviduct opens into the 
palliai cavity through its anterior tip (fo: Fig. 1 1 ). 



ANATOMY OF SHINKAILEPAS 



11 



cpg 




FIG. 12. Schematic drawing of ventral view of 
palliai oviduct, showing connection of various 
ducts and associated structures. 



The vaginal duct is ventral to the palliai ovi- 
duct, covered entirely with epithelium of pal- 
liai oviduct, elongated, and connected to the 
palliai oviduct posteroventrally. The terminal 
of the vaginal duct is expanded and opens as 
an oblique slit (vgo: Fig. IIB). Posterior to the 
vaginal opening, the receptaculum seminis is 
branched off from the vaginal duct. Sperma- 
tozoa are oriented in the receptaculum seminis 
towards its epithelium (Fig. 15A). The distal 
end of the receptaculum seminis is visible in 
ventral view (Fig. IIB). Near its connection to 
the palliai oviduct, the vaginal duct gives off 
another pouch-like structure. It is tentatively 
named "posterior sac" because of unknown 
function and uncertain homology with other 
reproductive structures. Its inside is charac- 
teristically roughened by many folds (ps: Figs. 
1 5B-E), and no spermatozoa was found there. 
A groove or ovipositor is not developed on the 
right side of the neck of the female. 

The male reproductive organ is composed 
of the testis (Fig. 15F), vas deference, semi- 
nal vesicle, and palliai male gonoduct (Fig. 1 3). 
The testis contains many cylindrical sectors 
in which growing sperms are arranged radi- 



mo 



mo 




FIG, 1 3. Pallia! gonoduct of male, with intestine intact. A. Dorsal view. 
B. Ventral view. 



12 



SASAKI ETAL. 



ally. A thin vas deference originates from the 
ventroanterior side of the testis and extends 
anteriorly. The distal part of the vas deference 
is complicatedly folded to form the seminal 
vesicle, which is filled with filamentous, com- 
pleted spermatozoa oriented in parallel (Figs. 
14C-D, 15G). The spermatophore was not 
observed in any section of vas deference. 

The palliai male gonoduct is greatly enlarged 
and surrounded by two different glands, the 
annex gland posteriorly and the prostate gland 
anteriorly (Figs. 13. 15H). In dorsal view, a part 
of the prostate pouch is visible on the outer 
surface (Fig. 13A). 

The penis arises from inner side of right 
cephalic tentacle, dorsoventrally depressed, 
lobate with a tapered tip (Fig. 1). Its dorsal 
surface is smooth, the right margin is grooved 
throughout, and the ventral side has a single 
ciliated papilla (Figs. 14A, B). A ciliated groove 
was not found between the gonoduct opening 
and the penis. 



Nervous System 

The nervous system consists mainly of the 
circumesophageal nerve ring, pedal cords, 
and visceral nerve. The circumesopahgeal 
nerve ring is formed by pairs of three major 
ganglia, namely, pleural, pedal, and cerebral 
ganglia. Their configuration is of hypoathroid 
type, namely pleural and pedal ganglia are 
more adjacent than cerebral ganglia (Fig. 16). 

The cerebral ganglia are located on the 
bases of the cephalic tentacles and send 
nerves to the cephalic tentacles and oral 
area. The paired ganglia are connected to 
each other by the cerebral commissure over 
the oral tube and the labial commissure be- 
low the sublingual pouch. Labial ganglia are 
not formed on the labial commissure. The 
buccal ganglia are obliquely extended at the 
base of the anterior esophagus and con- 
nected to the cerebral ganglia through thin 
connectives. 




FIG. 14. Scannning electron micrographs of male reproductive organs. A. Ventral view of penis re- 
moved from head. Arrowhead indicates groove along right margin. B. Enlarged view of ciliated pa- 
pilla on right ventral side. С Seminal vesicle in convoluted part of vas deference. D. Spermatozoa 
contained in seminal vesicle. Part of epithelium of seminal vesicle is removed to show inside. A-D. 
UMUT RM28652. 



ANATOMY OF SHINKAILEPAS 



13 








dps 






nc\ 200 nji 








ÄS().n.Li]..|3 






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ang .. 







FIG. 15. Histology of reproductive organs of female (A-E) and male (F-H). A. Cross section of semi- 
nal receptacle. B. Longitudinal section of posteroventral part of palliai oviduct. С Longitudinal sec- 
tion of palliai oviduct where oviduct (ovd) is connected to lumen of palliai oviduct. D. Longitudinal 
section of posterior sac (ps) of vaginal duct and surrounding glands. E. Enlarged view of longitudinal 
section of posterior sac (ps) of vaginal duct. F. Vertical section of testis. G. Vetical section of seminal 
vesicle (sv) containing completed spermatozoa. H. Horizontal section of glands of palliai gonoduct of 
male. A. UMUT RM28648. F-H. UMUT RM28650. B-E. UMUT RM28651. 



14 



SASAKI ETAL. 



le ce 




FIG. 16. Configuration of nervous system in dor- 
sal view. 



The pleural ganglia are situated below the 
posterior part of the buccal mass, and they 
are the largest among all ganglia and con- 
nected by a rather thin pleural commissure. 
Two distinct nerves are extended from the 
pleural ganglia to the body wall musculature. 

The pedal ganglia underlie pleural ganglia 
and are connected by the pedal commissure. 
The pleural and pedal ganglia are juxtaposed 
closely, forming almost fused complexes, but 
they receive separate cerebropleural 
connectives and cerebropedal connectives, 
respectively, from the cerebral ganglia. The 
pedal ganglia give off thin nerves anteriorly 
and thick pedal cords posteriorly. Pedal cross 
connection was not detected in the present 
sections. 

The visceral nerve is not a closed loop or 
streptoneurous and represented only by the 
subesophageal part, which arises from the 
right pleural ganglion toward base of ctenidium 
along the palliai cavity wall. The supraesopha- 
geal part is totally missing. 

Sense Organs 

Possible sensory organs include cephalic 
tentacles, oral lappets, papillae on the outer 
lip of the mouth, posterior epipodial fold, 
osphradium, statocysts, and eyes. The most 
of external structures are described above. 
Epipodial sense organs, and subradular or- 
gans are absent. 



The osphradium is elongated as a vermiform 
ridge along the left shell muscle, weakly de- 
veloped, and two-folded with a longitudinal 
central groove (os: Fig. 4E). Eyes are rudi- 
mentary without cornea and lens, simply rep- 
resented by pigmented cells, and sunken in 
connective tissue of eye stalks. Statocysts are 
attached to the dorsal side of pedal ganglia 
and below pleural commissure (pic: Fig. 10F) 
and contain several statoconia (sc: Fig. 10F). 



DISCUSSION 
Comparative Morphology 

The morphology of neritopsine gastropods, 
in which Shinkailepas is included, is highly di- 
verse in both shell and soft parts. Their similar- 
ity and dissimilarities have been employed for 
systematics at various taxonomic levels (e.g. 
Sasaki, 1998) and also used as characters for 
phylogenetic analysis (Ponder & Lindberg, 
1997: Sasaki, 1998). The results of observa- 
tions on Shinkailepas myojinensis in this study 
are compared with descriptions of relatively 
well-investigated genera (Tables 1, 2). 

Shell: The shell of Shinkailepas is character- 
ized by (1) a limpet form with the apex on the 
posterior center, (2) a multispiral globular 
protoconch with growth lines, (3) the septum 
extended between the visceral mass and foot, 
and (4) microscopic canal structures with 
crowded openings inside and sparse outside. 
Among these, (1) and (3) are also found in 
Phenacolepas and Septaria. (2) is shared by 
aquatic neritopsines in general (Sasaki, 1998). 
The shell of another vent-associated 
neritopsine limpet, Olgasolaris, differs from that 
of Shinkailepas in a more centrally situated 
apex, many regular and fine radial riblets, and 
thick periostracum overhanging the shell mar- 
gin. Phenacolepas and related shallow-water 
forms are most similar to Shinkailepas among 
Neritopsina in above characters, except (4). 

It is well known that numerous thin canals 
and corresponding pores are produced inde- 
pendently in the shells or valves of all 
polyplacophorans, part of bivalves and gastro- 
pods, and brachiopods (reviewed by Reindl & 
Haszprunar, 1996). In Gastropoda, canals and 
mantle processes (caeca) are found in the 
Fissurellidae (Reindl & Haszpruanr, 1994), 
Neritiliidae (Kano & Kase, 2002), Shinkailepas 
(Beck. 1992: Sasaki et al., 2003; this study), 
and Olgasolaris (Beck, 1992). Shell canals of 
Shinkailepas (and possibly also Olgasolaris) 



ANATOMY OF SHINKAILEPAS 



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SASAKI ETAL. 



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ANATOMY OF SHINKAILEPAS 



17 



are different from those of above-mentioned 
taxa in two points: (1) The diameter, cross- 
sectional form, and density of the canals are 
more prominently variable in Shinkailepas 
(Sasaki et al., 2003: fig. 120), and (2) some 
of canals penetrate the shell completely, but 
many of them do not. In the Fissurellidae, all 
canals penetrate the shell at least in an early 
ontogenetic stage with a rather constant di- 
ameter (e.g., Sasaki, 1998: fig. 40d). In the 
Neritiliidae, canals never reach the outer sur- 
face of the shell (Kano & Kase, 2002). Similar 
canals are not found in any section of the shell 
in other neritopsines, such as Nerita and 
Cinnalepeta at microstructural level (Sasaki, 
2001). 

Operculum: Most neritopsines are operculate, 
except the Oeresidae, Proserpinidae (Thomp- 



son, 1980) and shell-less Titiscania. The com- 
mon features of the opercula of Shinkailepas 
(and Olgasolaris) include (1 ) its position on the 
foot musculature below the visceral mass, (2) 
a trapezoidal, nail-like shape in outline, (3) two- 
layered structure, namely, calcified and cor- 
neous parts, (4) the nucleus located on the left 
side relative to animal's longitudinal axis, (5) 
the division into initially spiral and subsequently 
non-spiral parts, possibly of pre- and post- 
metamorphic stages, by different modes of 
growth line formation, and (6) the absence of 
an apophysis (Okutani et al., 1989: fig. 12; 
Beck, 1992: pi. 1, fig. 4, pi. 5, fig. 4; Sasaki et 
al., 2003: fig. 12D). Among neritopsines, the 
opercula of Shinkailepas and Olgasolahs are 
most similar to that of Septana in (1 ) and (2), 
but different in the remaining features. The 
demarcation of nucleus (possibly of pre-meta- 



TABLE 2. Comparison of character states among four phenacolepadid genera. 



Genus 


Shinkailepas 


Olgasolahs 


Phenacolepas 


Cinnalepeta 


Reference 


this study 


Beck (1992) 


Fretter(1984) 


Sasaki (1998) 


canal structures of 
shell 


partly penetrating 
shell completely 


penetrating shell 


absent 


absent 


operculum shape 


trapezoidal 


subtrapezoidal 


semicircular, 
vestigial 


absent 


apophysis of 
operculum 


absent 


absent 


? 


- 


circumpallial 
microtentacles 


absent 


absent 


present 


present 


epipodial flap 


present 


present 


absent 


absent 


cephalic lappet 


present 


present 


absent 


absent 


posterior 
esopahgeal 
glands 


tightly enclosing 
radular sac 


? 


lateral to radular 
sac? 


lateral to radular 
sac 


intestine 


2 loops 


2 loops 


3 loops? 


5 loops 


glandular and non- 


undifferentiated 


? 


differentiated 


differentiated 


glandular regions 
of kidney 










erythrocytes 


spherical? 


? 


biconcave 


biconcave 


vaginal opening 


transversely slit- 
like, anterior 


transversely slit- 
like, anterior 


small pore 


small pore, anterior 


receptaculum 
seminis 


posterior to vaginal 
opening 


? 


present 


connected to dorsal 
albumen gland 


posterior sac of 
vaginal duct 


present 


? 


absent 


absent 


female flap 


absent 


present? 


present as 
ovipositor? 


absent? 


seminal groove on 


lateral 


dorsal 


? 


? 


penis 










eyes 


vestigial, closed 


? 


well-developed, 
closed 


well-developed, 
closed 



18 



SASAKI ETAL. 



morphic part) is also known in {he Neritiliidae 
(Kano & Kase. 2000, 2003, in press; Kano et 
al., 2003), Bathynerita (Waren & Bouchet, 
1993), and Phenacolepas (Kimura & Kimura, 
1999: fig. 7C-D), and therefore it is not char- 
acteristic of Shinakailepas and Olgasoralls. 
The possession of apophysis in the opercu- 
lunn is rather common throughout neritopsines 
except the Helicinidae, Shinkailepas. and 
Olgasolaris (cf. Sasaki, 2001 ). The number of 
microstructual layers varies from two to four 
in neritopsine opercula (Suzuki et al., 1991; 
Sasaki, 2001). but its phylogenetic or adap- 
tive significance is uncertain. 

External Anatomy: The mantle margin is 
normally simple without projective or massive 
glandular structure in neritopsines, but it is 
modified in shallow-water phenacolepadids. 
The inner fold of the mantle margin is charac- 
teristically fringed with retractile microtentacles 
in Phenacolepas and Cinnalepeta (Fretter, 
1984; fig. 5; Sasaki, 1998; fig. 85b). In addi- 
tion, a thick layer of glandular tissue is devel- 
oped on ventral surface in Phenacolepas 
(Fretter, 1984; fig. 5). But, in contrast, such a 
specialization does not occur in Shinkailepas. 
The mantle margin morphology is, therefore, 
a distinctive character between shallow-wa- 
ter and deep-water phenacolepadids. 

Various forms of folds or tentacles are de- 
veloped in gastropod epipodium as in vetig- 
astropods, cocculinifom limpets, "vent-taxa," 
deep-sea phenacolepadids, and part of 
cerithioideans (Ponder & Lindberg, 1997). In 
Shinkailepas and Olgasolaris, the epipodial 
fold arises from the latero- to postero-dorsal 
part of the foot in Shinkailepas (Okutani et al., 
1989; Beck, 1992; Waren & Bouchet, 2001; 
fig. 33; this study), whereas comparable struc- 
ture is totally absent in other neritospines in- 
cluding shallow-water phenacolepadids 
(Tables 1 , 2). Because the degree of epipodial 
fold development is different from species to 
species, it is a useful taxonomic character at 
species level in Shinkailepas (see below). 

The cephalic lappets are small flaps projected 
on the inner side of the cephalic tentacles. They 
are found in part of the vetigastropods and 
neritopsines and may be all regarded as ho- 
mologue in terms of position and innervation. 
It is a common feature that the lappets are 
enlarged and used as a penis in male in 
neritopsines if present. The cephalic lappets 
are present in Shinkailepas (Fig. 1 ; Okutani et 
al., 1989; fig. 10), Olgasolaris, and Bathynerita 
(Waren & Bouchet, 2001), but absent in the 



Neritiliidae, shallow-water phenacolepadids, 
and the Neritidae excluding Bathynerita 
(Table 1). 

Palliai Organs: The palliai cavity of Shin- 
kailepas contains a set of palliai organs com- 
mon to the aquatic Neritopsina, namely the 
ctenidium, osphradium, anus, palliai gonod- 
uct with genital opening(s), and excretory pore, 
but a hypobranchial gland is absent. 

The ctenidium of S. myojinensis has typical 
neritopsine elements; (1) a single ctenidium 
is situated on the left side, (2) ctenidial lamel- 
lae are bipectinate in opposing arrangement 
on either side of ctenidial axis, (3) ctenidial 
lamellae are centrally ridged, (4) skeletal rods 
and (5) sensory pockets are absent. These 
features are not distinguishable from those of 
other neritopsines, except for a greatly re- 
duced ctenidium of the Neritiliidae (Kano & 
Kase, 2002) and its total absence of in terres- 
trial groups. A wart-like structure termed "ves- 
tigial gill" on the right side of the palliai cavity 
(Fretter, 1965; fig. 1c; Sasaki, 1998; fig. 77c) 
is lacking in S. myojinensis. The occurrence 
of the structure is restricted to members of the 
genus Nerita and not universal to neritopsines. 

The hypobranchial gland is present on the 
right palliai roof in some neritopsines, for ex- 
ample, the Neritidae (Fretter, 1965; Berry et 
al., 1973; Sasaki, 1998), Neritiliidae (Kano & 
Kase, 2002), and Helicinidae (Thompson, 
1980; Sasaki, 1998). However, it is absent in 
corresponding position in S. myojinensis, 
Phenacolepas (Fretter, 1984), and Cinnale- 
peta (Sasaki, 1998). 

In S. myojinensis, two glandular zones (the 
post-tentacular gland and anterior palliai gland) 
are observed on the right anterior corner of 
the palliai cavity. These glands may be analo- 
gous to the hypobranchial gland of other gas- 
tropods, but probably do not fulfill the function 
as a normal hypobranchial gland due to their 
restricted position at the anterior right. The true 
hypobranchial gland develops in deeper posi- 
tion in the palliai cavity in other neritoideans. 
The two glandular zones in S. myojinensis 
possibly have a function related to reproduc- 
tion, since the glands are developed near the 
gonoduct opening in both sexes. 

Digestive System: Its has been generally 
accepted that neritopsines lack true jaws 
(Fretter, 1965). However, paired cuticularized 
plates apparently develop on the inner wall of 
the oral tube in S. myojinensis (Fig. 9A). Iden- 
tical plates are also present generally in other 



ANATOMY OF SHINKAILEPAS 



19 



neritopsines. Sasaki (1998) described these 
plates as jaws, because they are located at a 
position corresponding to those of other gas- 
tropods. They may not be regarded as the jaws 
in that they lack scaly microelements, but such 
microelements are also lacking in the jaws of 
Cocculina and Patellogastropods (Sasaki, 
1998). Although the term "jaw" was not used 
in the present description, it is also possible 
that they represent a reduced state of the jaws. 
Homology can be established between jaws 
and cuticularized plates under positional cri- 
terion but is uncertain under structural crite- 
rion. More extensive comparison should be 
made among gastropod jaws for further dis- 
cussion. 

Buccal mass structure is remarkably useful 
character to define higher taxa in basal groups 
of gastropods (Sasaki, 1998). For example, 
patellogastropods, vetigastropods, and 
neritopsines each have their own composition 
of musculature and cartilages. The buccal 
musculature of Shinkailepas belongs to 
neritoidean type described for Nerita, Septana, 
and Cinnalepeta, and is partially different from 
that of Waldemaha (Sasaki, 1998: table 5). 

The configuration of odontophoral cartilages 
is invariable throughout the Neritopsina 
(Sasaki, 1998; Kano & Kase, 2002). Large 
anterior and small posterior cartilages are 
paired and connected by ventral approximator 
muscle, and the median cartilage is situated 
between the anterior cartilages. The structure 
of odontophoral cartilages of S. myojienensis 
conforms entirely to this pattern. 

The presence of a pair of sublingual glands 
beside the sublingual pouches is a distinctive 
character of neritoideans, including the 
Neritidae, Neritiliidae, and Phenacolepadidae 
(Sasaki, 1998: Kano & Kase, 2002). They are 
absent in the Helicinidae (Sasaki, 1998) or 
unknown in the rest of neritopsine members. 
The salivary glands are absent throughout the 
Neritopsina without exception. Probably their 
absence is compensated by the development 
of sublingual glands and anterior esophageal 
glands. 

The radular formula of Shinkailepas is the 
same as that of most members of neritoideans. 
The radula of Shinkailepas and that of 
Olgasolarls (Beck, 1992) are typified by the 
following six features: (1) the central tooth is 
present, (2) the first lateral teeth are obliquely 
elongate, (3) the second and third lateral teeth 
are small, (4) the fourth lateral teeth are longi- 
tudinally elongate without serration in their 
cusps, (5) lateromarginal plates are absent. 



and (6) the marginal teeth have small triangu- 
lar projection below their cusps (Sasaki et al., 
2003: figs. 12, 14). 

Major differences in radular characters exist 
mainly in the central and lateral teeth among 
Neritopsina. In contrast to most neritopsines, 
including Shinkailepas, the central tooth is 
absent in the Neritiliidae (Kano & Kase, 2002, 
2003, in press; Kano et al., 2003), Nerltopsis 
(Waren & Bouchet, 1993: fig. 3D), and 
TItiscanIa (Bergh, 1890; Taki, 1955). The lat- 
eral teeth morphology is considerably variable 
among neritopsines and it is difficult to gener- 
alize. For example, in the Neritidae, the first 
laterals are transversely elongate, and the 
fourth laterals are thickened in shield-like form. 
The Neritiliidae (Kano & Kase, 2002, 2003, in 
press; Kano et al., 2003) and most helicinoide- 
ans (e.g., Thompson, 1980; Sasaki, 1998; 
Richling, 2004) have obliquely elongate out- 
ermost lateral teeth with sharp serrations, and 
lateral teeth are more reduced in Nerltopsis, 
TItiscanIa, and the Hydrocenidae (Ponder, 
1 998: fig. 1 5.76). The presence of prominence 
below cusps in the marginal teeth (Kano & 
Kase, 2002: fig. 8) is known in the Neritiliidae, 
Nerltopsis, and TItiscanIa (Kano & Kase, 2000, 
2002), but not in others. 

The configuration of the radular sac has not 
hitherto been focused in the studies of the 
Neritopsina. Recently, Kano & Kase (2002) 
pointed out that in the Neritopsina the length 
of radular sac can be categorized into two 
groups. A long, looped radular sac is typical of 
the Neritiliidae, Nerltopsis and TItiscanIa; by 
contrast, a short straight type occurs in the 
Neritidae, Phenacolepadidae, and Helicinidae 
(Kano & Kase, 2002). Shinkailepas myojinen- 
sis (Fig. 5), S. kaikatensis (Okutani etal., 1989: 
fig. 15), and Olgasolans tollmanni {Beck, 1992: 
fig. 3B) have the latter type. 

The esophagus of the Neritopsina exhibits 
similar structure throughout the group (Sasaki, 
1 998): (1 ) It consists of the anterior and pos- 
terior esophagi only, lacking the differentiation 
of a mid-esophagus, (2) the anterior esopha- 
gus is sectioned into a centrally situated, dor- 
sal food channel and lateral esophageal 
pouches with the anterior esophageal glands 
inside, and (3) the posterior part of the lateral 
esophageal pouches are separated as the 
posterior esophageal glands. The esophagus 
of S. myojinensis matches this generalization 
well. 

A peculiar shaped esophagus was recently 
described in the Neritiliidae by Kano & Kase 
(2002). The anterior esophageal glands are 



20 



SASAKI ETAL. 



extremely elongated posteriorly as separated 
pouches and overlie the posterior esophageal 
glands. This double structure of esophageal 
glands is not known in other neritopsines in- 
cluding Shinkailepas. 

The posterior esophageal glands in S. 
myojinensis are complicatedly infolded and 
tightly enclose the radular sac with a narrow 
interstitial space (Fig. 9F). This morphology 
apparently differs from that of other 
neritopsines, including the Neritiliidae (Kano 
& Kase. 2002: fig. 5D). Hence, the structure 
of this part may be of sufficient systematic 
value, but cross-sectional morphology of the 
glands have not been described for compari- 
son in the rest of neritopsines. 

Another distinctive character of the 
Neritiliidae is the presence of the floor glands 
arising from anterior esophageal floor (Kano 
& Kase, 2002). The glands are paired blind 
sacs that open from the posterior side of the 
floor of the anterior esophagus and consist of 
two glandular cells ciliated differently and 
stained differentially with haematoxylin (Kano 
& Kase. 2002: fig. 3C). The identical glands 
were not found in sections of equivalent posi- 
tion in S. myojinensis. 

The stomach of the Neritopsina consists of 
a cuticularized area, gastric shield with a short 
reflected tooth, paired (major and minor) 
typholosoles on the ventral side, a ciliated in- 
testinal groove between the typhlosoles, and 
a short gastric caecum (Fretter, 1965; Sasaki, 
1998: Kano & Kase, 2002). Differences in the 
stomach structure among the Neritopsina is 
not very conspicuous. The Neritiliidae have 
only a single connection between stomach and 
digestive glands (Kano & Kase, 2002), 
whereas two or more openings of digestive 
glands occur near sorting area in the stomach 
of other neritopsines (e.g.. Bourne, 1909, 
1911; Fretter, 1965, 1984; Sasaki 1998). It is 
uncertain at present whether the number of 
the openings is dependent on body size or 
phylogentetically fixed. 

Excretory System: Neritopsine excretory 
system is composed of the auricle with 
podocytes as an ultrafiltration site, renoperi- 
cardial duct as a conduit of primary urine, and 
a single left kidney for osmoregulation and 
excretion (Estabrooks et al., 1 999). Within the 
Neritopsina, two types of kidneys are known 
to date: (1) In the Neritidae, Phenacolepa- 
didae, and Helicinidae, the kidney is composed 
of glandular region, non-glandular bladder, and 
short ureter (Little, 1972; Sasaki, 1998; Esta- 



brooks et al., 1999). (2) In the Neritiliidae, the 
wall of kidney is simple, not specialized into 
glandular and non-glandular areas (Kano & 
Kase, 2002). The kidney of S. myojinensis 
belongs to the latter type. Functional differ- 
ences of these two types are not clear, though 
development of infoldings in the glandular area 
of the former type is obviously related to func- 
tional advantage to increase its surface area. 

Circulatory System: In the heart of gastro- 
pods the ventricle is always single, but it is 
attached by paired or unpaired auricle(s), de- 
pending on taxa. In S. myojinensis, the peri- 
cardium contains two (right and left) auricles 
and a single median ventricle. The right au- 
ricle is obviously present in S. myojinensis but 
greatly reduced. A vestigial auricle is also 
present in Cinnalepeta (Sasaki, 1998) and 
Phenacolepas (Fretter, 1984). In other 
neritopsines, the right auricle is present in the 
Titiscanidae and Cerisidae of the Helicinidae 
but absent in the Hydrocenidae, Proserpi- 
ninae, and Helcinidae (Sasaki, 1998). The 
ventricle is penetrated by the rectum in the 
Neritopsina, except the Hydrocenidae and 
Helicinidae (Sasaki, 1998). 

Although details have not been studied 
hematologically, the presence of erythrocytes 
is a possible general feature of phenacole- 
padids. They are discoidal and biconcave in 
Phenacolepas (Fretter, 1984) and Cinnalepeta 
(Sasaki, 1988: fig, 85d). The animals of these 
two genera are red in fresh live condition, but 
immediatedly turned pale after the death by 
fixation. In Shinkailepas aff. kaikatensis from 
the Mariana Back-arc Basin, the red color is 
also very vivid only while living (Hasegawa et 
al., 1997). Hence, species of S/?/n/<a/7epas are 
presumed to have erythrocytes. The form of 
haemocytes in S. myojinensis, however, do not 
seem discoidal but spherical (Fig. 10B). 

Female Reproductive System: The female 
organs of neritopsines consist mainly of the 
ovary, oviduct, palliai oviduct with albumen and 
capsule glands, and vaginal duct with two sac- 
like appendages. 

It is well known that the number of repro- 
ductive opening(s) is different in female among 
neritopsine taxa. In S. myojinensis, palliai ovi- 
duct and vaginal duct have their own open- 
ings (diaulic). In contrast, female reproductive 
system is triaulic with an additional enigmatic 
duct in Septana (Sasaki, 1998) and monaulic 
in Titiscania (Marcus & Marcus, 1967) (neri- 
tiliids do not have a monaulic system as pre- 



ANATOMY OF SHINKAILEPAS 



21 



viously believed (Kano & Kase, 2002). A diaulic 
reproductive system is most common among 
Neritopsina. 

Separation of the vaginal duct from the pal- 
liai oviduct is a common feature in most 
neritopsines, and it is also true of S. myojinen- 
sis. Characteristically, the vaginal duct in S. 
myojinensis is associated v\/ith three struc- 
tures: (1 ) a transverse slit of the vaginal open- 
ing below the anterior part of pallia! oviduct, 
(2) the receptaculum seminis near the vaginal 
opening, and (3) the "posterior sac" below the 
posterior part of the palliai oviduct. 

A slit-like vaginal opening in the anterior po- 
sition is also described in Olgasolaris (Beck, 
1992). In the Neritiliidae, the vaginal opening 
is also a slit but located posteriorly (Kano & 
Kase, 2002). It is a small pore, not a long slit, 
in P/?enaco/epas (Fretter, 1984), Cinnalepeta, 
the Neritidae, and Helicinidae (Sasaki, 1998). 

An anteriorly situated receptaculum seminis 
near the vaginal opening in S. myojinensis is 
unique among the Neritopsina. In other 
groups, receptaculum seminis has been iden- 
tified in various positions (e.g., Sasaki, 1998), 
but it has not always been verified on histo- 
logical basis. The presence of oriented sper- 
matozoa in its epithelium (Fig. 1 5A) is the most 
reliable criterion for this identification. 

The "posterior sac" of the vaginal duct may 
also be peculiar to S. myojinensis. Its inner 
wall is heavily folded characteristically. Be- 
cause no sperm or egg was contained in sec- 
tioned specimens, its actual function in 
reproduction could not be detected. An equiva- 
lent structure is unknown in Olgasolaris (Beck, 
1992) and Phenacolepas (Fretter, 1984), or 
lacking in Cinnalepeta (Sasaki, 1998: fig. 84). 
The spermatophore sac in the Neritidae and 
Cinnalepeta (Sasaki, 1998: figs. 76, 84) can- 
not be homologized due to the differences in 
topological relationships with other reproduc- 
tive organs. 

It is uncertain whether S. myojinensis pro- 
duces spermatophores or not. The spermato- 
phores are generally known to occur in 
neritopsine gastropods (Robertson, 1989, re- 
viewed gastropod spermatophores). In the 
Neritidae, intact spermatophores are often 
contained in the spermatophore sac in female 
(Sasaki, 1998: figs. 76c-d), and the formation 
of the spermatophore sheath is also observ- 
able in the seminal vesicle of the male. In this 
case, the formation of spermatozoa is un- 
doubted. But in S. myojinensis, no spermato- 
phore was found in any section of female and 
male reproductive systems. 



The palliai oviduct of S. myojinensis is en- 
closed by two kinds of glands that correspond 
to the albumen and capsule glands, as is gen- 
erally found in gastropods that produce egg 
capsules. The albumen gland is further divis- 
ible into two parts in S. myojienesis. The simi- 
lar division is also known in the Neritiliidae, 
Neritidae, and shallow-water phenacolepadids 
(Sasaki, 1998; Kano & Kase, 2002), but not in 
non-neritopsine gastropods. 

Some neritopsines are known to have a min- 
eral-containing "crystal sac" and cover egg 
capsule with minerals from the sac. The ab- 
sence of the crystal sac was verified in S. 
moyojinensis in this study and in the Neritiliidae 
by Kano & Kase (2002). Meanwhile, the sac 
is distended with calcified grains in the 
Neritidae (Sasaki, 1998: fig. 77h). Marcus & 
Marcus (1967) reported the crystal sac in 
Titiscania, but it is questionable (Kano & Kase, 
2002). Probably the possession of the crystal 
sac is restricted to the family Neritidae. 

Internally fertilizing gastropods may develop 
a particular structure conveying eggs from the 
female opening to the foot through the neck 
region. The Neritiliidae have the neck furrow 
and female flap on the right side in the female 
(Kano & Kase, 2002: figs. 2B, 15B), and pre- 
sumably eggs are conveyed along a ciliated 
furrow in oviposition. The female flap in 
Neritiliidae is possibly homologous to the struc- 
ture identified as the ovipositor in Phena- 
colepas by Fretter (1 984) (Kano & Kase, 2002). 
In S. myojinensis, a corresponding structure 
was not found in the right pedal region. Be- 
cause actual behavior of egg deposition has 
never been observed in phenacolepadids, 
functional significance of right neck-foot mor- 
phology is unclear. 

Male Reproductive System: Male reproduc- 
tive organs of neritopsines generally comprise 
the testis, vas deference, seminal vesicle, 
palliai male gonoduct with prostate, and pe- 
nis. All of these organs represent a common 
element of the male reproductive system pos- 
sessed universally by internally fertilizing gas- 
tropods. 

The seminal vesicle is formed in convoluted 
part of vas deference in some Neritopsina, for 
example, Neritidae including Bathynerita 
(Waren & Bouchet, 1993), shallow-water 
phenacolepadids (Sasaki, 1998), and 
Shinkallepas. By contrast, in the Neritiliidae, 
the seminal vesicle is double and different 
from tightly convoluted type (Kano & Kase, 
2002). In the Helicinidae, it is simple, not en- 



22 



SASAKI ETAL. 



tangled (Thompson, 1980; Sasaki, 1998). 
Thus, the configuration of vas deference is a 
useful character defining some higher taxa in 
Neritopsina. 

It is common pattern in Neritopsina that the 
male palliai gonoduct is covered with the an- 
nex gland posteriorly and the prostate anteri- 
orly. The formation of the prostate pouch on 
the dorsal side in Shinkailepas is a distinctive 
character not known in other neritoideans. 

The position and structure of copulatory or- 
gan in male is greatly variable across various 
groups of gastropods. In Neritopsina, the pe- 
nis arises unexceptionally from inner side of 
the right cephalic tentacle, if present. The 
seminal groove in the penis extends along right 
lateral margin in S. myojienesis (Fig. 14A) but 
on dorsal surface in Olgasolaris tollmanni 
(Beck, 1992: pi. 5, fig. 6). 

The penis in male arises in a position equiva- 
lent to the right cephalic lappet of female. This 
may suggest that the penis has arisen as a 
modified cephalic lappet. The development of 
the penis is, however, independent of that of 
the cephalic lappets, because neritids and 
Cinnalepeta lacking cephalic lappets have the 
penis in a similar position (Sasaki, 1998). In 
the Neritiliidae, which entirely lack the cepha- 
lic lappets, the penis is rudimentary in Pisulina 
or absent in Neritilia (Kano & Kase, 2002). 

A ciliated papilla on the ventral side of the 
penis (Figs. 14A-B) is unique to S. myojinensis 
among Neritopsina. But, the presence or ab- 
sence of equivalent structure in other 
neritopsines is actually uncertain, because the 
penis has not been observed from its ventral 
side in the previous studies. Its function re- 
mains entirely unidentified. 

Nervous System: The nervous system of S. 
myojinensis consists of a hypoathroid circum- 
esophageal nerve ring, non-streptoneurous 
visceral nerve with characteristic configuration, 
a pair of thick pedal cords, and other thin pe- 
ripheral nerves. 

The circumesophageal nerve ring of S. 
myojinensis is rather concentrated for that of 
neritopsines, compared to other anatomically 
examined species (Fretter, 1984; Sasaki, 
1998; Kano & Kase, 2002). Especially, pleu- 
ral and pedal ganglia are closely situated and 
almost fused with each other. The positions of 
cerebral and buccal ganglia are similar to those 
of various rhipidoglossate gastropods. Pleu- 
ral ganglia have their own commissure, which 
is a general character peculiar to neritopsine 
gastropods. 



The presence of labial commissure below 
sublingual pouch is also a common feature 
throughout the Neritopsina. A similar commis- 
sure also exists in the Patellogastropoda in 
general, but it is different from that of neritop- 
sines in that the labial ganglia are developed 
on the commissure. The labial commissure 
without ganglia also occurs in the Ampullariidae 
(Berthold, 1991), which shows a comparable 
state in a different clade of gastropods. 

It is interesting that the visceral part of ner- 
vous system does not form a complete loop in 
some neritopsines. Such a condition is also 
known in Phenacolepas (Fretter, 1984), 
neritiliids (Kano & Kase, 2002), and S. 
myojinensis. In the Neritidae, it is complete, 
and the supraesophageal loop can be traced 
over the anterior esophagus from the right 
pleural ganglion (e.g., Sasaki, 1998; fig. 79d). 
The incomplete visceral loop is possibly sec- 
ondary reduction rather than primary condi- 
tion, because remaining gastropods and other 
molluscs generally have a complete visceral/ 
lateral nerve loop. 

Sense Organs: The eyes are often reduced 
or secondarily lost in various organisms living 
in such dark environments as caves and the 
deep sea. Shinkailepas myojinensis is distinct 
from other neritopsines in that the eyes are 
certainly present but markedly vestigial. They 
are represented only by pigmented cells, lack- 
ing lens, and deeply embedded below epithe- 
lium of the eye stalks. In contrast, the eyes of 
shallow-water and terrestrial neritopsines are 
filled with the lens and covered with the cor- 
nea. Another exception to this generalization 
is the Neritiliidae which have open eyes with- 
out lens and cornea (Kano & Kase, 2002). They 
are considered to have been modified due to 
adaptation to cryptic habitat. The eyes in 
Shinkailepas are probably non-functional and 
reduced in the dark deep-sea environment. 

There seems to be some differences in the 
structure of osphradium among Neritopsina. 
In aquatic neritopsines, the osphradium is lo- 
cated along the left shell muscle, and defined 
by central zone, paired lateral zones, and pig- 
ment bodies at ultrastructural level (Haszpru- 
nar, 1985). However, it is two-folded with a 
longitudinal central groove and devoid of 
clearly ciliated lateral zones in S. myojinensis. 
A similar longitudinal groove on the central 
zone is also present in Bathynehta (Waren & 
Bouchet, 1993), but obviously absent in other 
neritopsines, such as Nerita (Haszprunar, 
1985; Sasaki, 1998; fig. 77a). 



ANATOMY OF SHINKAILEPAS 



23 



The position of the statocysts is somewhat 
variable among neritopsines. They are located 
on the posterodorsal side of the pedal ganglia 
in S. myojinensis. and also the Neritidae 
(Sasaki, 1 998) and Helicinidae (Bourne, 1911). 
In the Neritiliidae, their position is shifted more 
anteriorly (Kano & Kase, 2002). 

Each statocyst contains either single statolith 
or many statoconia, depending on taxa in gas- 
tropods (Ponder & Lindberg, 1997). Even 
within Neritopsina, there are both of these two 
types. At least two examples are known: sta- 
tocysts in the Neritiliidae (Kano & Kase, 2002) 
and statoconia in S. myojienesis. Their condi- 
tion in other taxa have not been clearly de- 
scribed based on histological observations. 
The systematic and functional significance of 
statocyst contents is still unclear throughout 
neritopsines. 

Systematic Implications 

The allocation of Shinkailepas to Neritopsina 
was corroborated by sufficient numbers of 
anatomical characters. The features shared 
with other neritopsines in general (Sasaki, 
1 998: 220-221 ) are: (1 ) a multispiral globular 
protoconch with growth lines in aquatic mem- 
bers, (2) a single left ctenidium lacking skel- 
etal rods and bursicles, (3) a single 
osphradium along the left shell muscle, (4) 
three (anterior, posterior, and median) ele- 
ments of odontophoral cartilages, (5) the dor- 
sal levator muscles of odontophore, (6) the 
tensor muscles of anterior cartilages, (7) the 
absence of the salivary gland, (8) the esoph- 
ageal glands separated from the esophagus 
posteriorly, (9) a small crescent-shaped gas- 
tric caecum, (10) the labial commissure with- 
out labial ganglia, (11) one-side origin of 
visceral loop from right side, and (12) the pleu- 
ral commissure. Some more characters were 
previously regarded as general neritopsine 
features (Sasaki, 1998), but at least two, eye 
and kidney structure, were rejected as re- 
vealed in recent studies. As discussed above, 
closed eyes with vitreous body is not found in 
Neritiliidae and Shinkailepas. The kidney in 
neritopsines is not always clearly difl'erentiated 
into glandular and non-glandular sections. 

Within Neritopsina, anatomical comparison 
suggests that Shinkailepas is included in the 
superfamily Neritoidea, which currently in- 
cludes the Neritidae and Phenacolepadidae. 
The Neritoidea is mainly diagnosed by char- 
acters of digestive and reproductive organs, 
such as (1) a well-developed oral lappet, (2) 



the sublingual glands, (3) the median levator 
muscles of odontophore, (4) the radular for- 
mula П-4-1-4-П, (5) two auricles with right one 
smaller, (6) the capsule and albumen glands 
in female, (7) the annex gland in male gonod- 
uct, and (8) the penis from the inner side of 
right cephalic tentacle. This definition of the 
superfamily with above characters is revised 
from that of Sasaki (1998). 

Concerning the relationships with other 
neritopsine families, Shinkailepas shares no 
anatomical characters uniquely with three 
helicinoidean families (Bourne, 1911; 
Thomson, 1980; Sasaki, 1998) or Neritiliidae 
(Kano & Kase, 2002). The remaining families, 
the Hydrocenidae, and the Neritopsidae (in- 
cluding Titiscanidae: Kano et al., 2002), have 
not been described sufficiently for comparison. 
In molecular characters, Kano et al. (2002) 
revealed the relationships (Neritopsidae 
(Hydrocenidae (Helicinidae + Neritiliidae) 
(Neritidae + Phenacolepadidae))) based on 
28S rRNA sequences. Therefore, it is highly 
likely that neritoidean groups including 
Shinkailepas are phylogenetically distinct from 
non-neritoidean families within the Neritopsina. 

The Neritoidea is currently divided into the 
Neritidae and Phenacolepadidae, and Shin- 
kailepas is assigned to the latter (Beck, 1992; 
Waren & Beuchet, 2001). Phenacolepadids, 
including shallow-water and vent/seep-en- 
demic groups, share a rather limited number 
of unique characters compared to other 
neritopsine families. Their common characters 
are: (1) transversely elongated first lateral 
teeth of the radula, (2) longitudinally tall fourth 
lateral teeth, and possibly (3) erythrocytes. The 
absence of hypobranchial gland may be an- 
other general character of the family, but its 
state is not certain in Olgasolaris. There are 
some more similarities, but in fact, they are 
not specific to phenacolepadids. For example, 
the cephalic lappets are also described in 
neritid Bathynerita (Waren & Bouchot, 1993), 
a cephalic penis also occurs in the Neritidae, 
two shell muscles without division are found 
in Septana (Sasaki, 1998). 

Within phenacolepadids, two major groups, 
deep-sea and shallow-water ones, can be 
clearly diagnosed. Two deep-sea vent/seep- 
associated genera, Shinkailepas and Olga- 
solaris, have similar characters in common 
(Table 2): (1 ) shell canals and pores penetrated 
by the mantle processes, (2) a trapezoidal, 
non-spiral, calcified operculum, (3) the cepha- 
lic lappets, (4) the epipodial fold, (5) the ab- 
sence of circumpallial tentacles, (6) the intestine 



24 



SASAKI ETAL. 



with two loops only, and (7) an anteriorly posi- 
tioned, slit-like vaginal opening. Most of these 
similarities are distinctive of these two genera, 
suggesting their close phylogenetic relation. 

In contrast to two deep-sea genera, shallow- 
water phenacolepadids {Phenacolepas + 
Cinnalepeta) are united by (1 ) the absence of 
shell pores and mantle processes, (2) a weakly 
developed, spiral operculum with apophysis, if 
present, (3) the absence of cephalic lappets, 
(4) the absence of epipodial folds, (5) the circum- 
pallial tentacles. (6) complex loops of intestine, 
and (7) a small pore-like vaginal opening. 

At the species level, there are some anatomi- 
cal differences among four described species 
of Shinkallepas. The comparison with S. 
myojinensis revealed that in S. bhandi Waren 
& Bouchet. 2001, (1) the eye stalks ("eye- 
lobes") are very weakly developed, (2) the 
epipodial fold in neck region is prominent on 
the left and right sides, (3) the penis is more 
elongated, and (4) the posterior margin of the 
epipodial fold is not divided into triangular ten- 
tacles. Likewise, in S. kaikatensis Okutani, Saito 
& Hashimoto. 1989, (1) the eye stalks are not 
developed. (2) the penis is more acutely 
pointed, and (3) the number of tentacles on the 
epipodial folds ("pedal papilla") is smaller (11 
in S. kaikatensis, 11-19 in S. myojinensis). In 
S. tufari Beck, 1992, the number of tentacles 
on epipodial folds is largest (20-22) among 
known species, but other characters have not 
been described in detail. Thus, eye stalks, 
epipodial folds in neck and posterior pedal re- 
gions, and penis are useful species-level taxo- 
nomic characters in the external features of the 
soft part. For shell, radular, and opercular char- 
acters at species level, see Sasaki et al. (2003). 



ACKNOWLEDGEMENTS 

We are grateful to Prof. George M. Davis 
(George Washington University Medical Cen- 
ter). Dr. Eugene Coan (California Academy of 
Sciences), anonymous reviewers, and Prof, 
Kazushige Tanabe (University of Tokyo) for in- 
valuable suggestions for this study and com- 
ments on the manuscript. The material 
examined in this study was collected in dives 
directed by Prof. Toshiyuki Yamaguchi (Chiba 
University) and Dr. Shinji Tsuchida (JAMSTEC). 
Sampling operations were kindly supported by 
crew of Shinkai 2000 and R/V Natsusliima of 
JAMSTEC. This study was partly supported by 
the Grant-in-Aid from the Japan Society of the 
Promotion of Science (No. 15740309). 



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Revised ms. accepted 28 May 2004 



APPENDIX: Abbreviations used in descriptions 

a = anus 

аса = anterior cartilage of odontophore 

acv = afferent ctenidial vein 

ag = albumen gland 

ag I = albumen gland I 

ag 11= albumen gland II 

al = anterior levator muscle of odontophore 

any = annex gland 

apd = anterior pedal gland 

apg = anterior pedal groove 

apl = anterior palliai gland 

at = anterior tensor muscle of odontophore 

bcv = buccal cavity 

bg = buccal ganglion 

с = ctenidium 

cc = cerebral commissure 

cdc = cerebropedal connective 

eg = cerebral ganglion 

dp = cephalic lappet 



26 



SASAKI ETAL. 



cp = cuticularized plate ovd 

cpc = cerebropleural connective ovp 
cpg = capsule gland 

ct = cephalic tentacle p 

dbt = dorsal buccal tensor muscle pc 

df = dorsal fold of esophagus pea 

dfc = dorsal food channel of anterior esophagus pcd 

dg = digestive gland pcv 

dgo = opening of digestive gland to stomach pd 
dl = dorsal levator muscle of odontophore 

dps = duct of posterior sac pds 

e = eye pdt 

ecv = efferent ctenidial vein pe 

ef = epipodial fold pg 

es = eye stalk pic 

ev = esophageal valve pig 

f = foot pis 

fo = female opening of gonoduct pn 

gc = gastric caecum po 

gst = tooth of gastric shield pr 

i = intestine prp 

if = inner fold of mantle margin ps 

ivp = inner ventral protractor muscle of ptg 

odontophore ra 

к = kidney rds 

ко = kidney opening rdt 

la = left auricle rev 

lea = labial cartilage rrs 

lep = lateral pouch of anterior esophagus rs 

In - labial nerve rsm 

Ip = lateral protractor muscle of odontophore rsr 
Ipr = lateral protractor muscle of subradular 

membrane sbv 

Ism = left shell muscle sc 

m = mouth sig 

mca = median cartilage of odontophore sip 

ml = median levator muscle of odontophore sn 

mm = mantle margin srm 

mo = male opening of gonoduct st 

mp = mantle process sta 

mpg= male palliai gonoduct sv 

mpr = median protractor muscle of subradular t 

membrane tac 

mt - mantle tbf 

oap = outer approximator muscle of cartilages tn 

of = outer fold of mantle margin v 

ol = oral lappet vad 

OS = osphradium vao 

ot = oral tube vap 



= oviduct 

= outer ventral protractor muscle of 

odontophore 
= penis 
= pericardium 

= posterior cartilage of odontophore 
= pedal cord 
= palliai cavity 
= posterior depressor muscle of 

odontophore 
= pedal sole 

= postdorsal buccal tensor muscle 
= posterior esophagus 
= periostracal groove 
= pleural commissure 
= pleural ganglion 
= palliai sinus 
= palliai nerve 
= palliai oviduct 
= prostate 
= prostate pouch 
= posterior sac of vaginal duct 
= post-tentacular gland 
= right auricle 
= radular sac 
= radular teeth 

= retractor muscle of esophageal valve 
= retractor muscle of radular sac 
= seminal receptacle 
= right shell muscle 
= retractor muscle of subradular 

membrane 
= subesophageal part of visceral loop 
= statoconia 
= sublingual gland 
= sublingual pouch 
= snout 

= subradular membrane 
= stomach 
= statocyst 
= seminal vesicle 
= testis 

= tensor muscle of anterior cartilage 
= transverse buccal fold 
= tentacular nerve 
= ventricle 
= vaginal duct 
= vaginal opening 
= ventral approximator muscle of cartilages 



MALACOLOGIA, 2006, 48(1-2): 27-34 

EFFECTS OF DISSOLVED LEAD AND COPPER ON THE FRESHWATER 
PROSOBRANCH LANISTES CARINATUS 

AbdAllah Tharwat AbdAllah 

Department of Zoology, Faculty of Science, 
Al-Azhar University, Assiut, 71524, Egypt; abd_allaht@yahoo.com 

ABSTRACT 

Lead and copper bioconcentration and toxicity to the freshwater prosobranch Lanistes 
carinatus (Olivier, 1804) were examined after single and connbined exposure. Metal 
bioaccumulation in the digestive gland of adult individuals was investigated after 21 days 
exposure to 100 pM lead nitrate, 10 pM copper sulphate, and 0.002 X (1 X = 36 pM lead: 
1 pM copper: a ratio matching that recorded in the snail's aquatic habitat). Lead was 
bioaccumulated higher than copper in the group exposed to the metal solution mixture. 
Elevated lead or copper concentrations were demonstrated in combined solution group 
relative to single metal solution examined individuals. Both metals accumulated over 50 
fold in single solution examined groups and more than 800 times in combined solution 
tested individuals. Acute toxicity experiment showed lower 24 hour LC50 for snails ex- 
posed to metal mixtures rather than single solutions studied individuals. Chronic toxicity 
study demonstrated more histopathological damage in the digestive tubules of individuals 
21 days exposed to combined metal solution relative to dissolved lead or copper exam- 
ined snails. The results revealed synergistic toxic effect of both metals on L. carinatus. 
Further investigations are currently going on to examine the potential value ofthat snail as 
biomonitor for aquatic pollutants. 

Key words: Lanistes, lead, copper, bioconcentration, toxicity levels, histopathology. 



INTRODUCTION 

Several authors have addressed heavy 
metal bioconcentration in aquatic molluscs 
(Sholz, 1980; Simkiss et al., 1982; Phillips & 
Rainbow, 1993; AbdAllah et al., 2003). The 
uptake of metals in freshwater bodies is a func- 
tion of different variables as membrane per- 
meability and physiological status of the 
organisms, pH, water temperature, water hard- 
ness, and acid radical of the metal salt. The 
concentration of a substance within the accu- 
mulator organism is the difference between the 
amount taken in and the amount released 
(Ravera, 2001 ). A mechanism operated in the 
digestive gland to detoxify metal pollutants, 
binding them with metallothionein (a sulph- 
hydryl-rich protein with low molecular weight) 
or with some other agent and storing them in 
the lysosomes (George, 1982; Simkiss & Ma- 
son, 1983). 

Heavy metal pollution has become the cause 
of serious concern and has attracted the at- 
tention of governmental authorities. Lasheen 
(1987) has described heavy metal pollution in 



Egypt. Generally, the ratio of heavy metals in 
the freshwater bodies is a function of the an- 
thropogenic spill and natural input. However, 
most of the available information regarding 
their accumulation and toxicity were based on 
single metal solution experiments. Parott & 
Sprague (1993) showed that combination of 
low copper concentrations with high concen- 
trations of zinc resulted in antagonistic effect 
on fathead minnows. He reported that heavy 
metals might interact antagonistically or syn- 
ergistically depending on the type of metals 
and species affected. Harrahy & Clements 
(1997) observed that removal of zinc from a 
synthetic sediment contaminated with a mix- 
ture of lead, copper, zinc, and cadmium re- 
sulted in a pronounced decrease in growth and 
egg laying rate and an increase in the survival 
rate of the midge Chironomus tentans. Lead 
is considered of the most toxic heavy metals 
to human health, affecting nervous and excre- 
tory systems (Hutton, 1987). Also, it affects 
the haem synthesis mainly through inhibiting 
the conversion of a-aminolevulinic acid to por- 
phobilinogen (Berry et al., 1974). Copper is a 



27 



28 



ABDALLAH 



trace element needed in minute amounts by 
aquatic molluscs to synthesize haemocyanin 
(Ghiretti & Ghiretti-Magaldi, 1975: Simkiss & 
Mason, 1983). Increase of the copper content 
within the molluscan tissues resulted in toxic 
effects at the target organs (WHO, 1989: 
AbdAllah. 2000). 

The toxicity studies are needed to establish 
the water pollution standards necessary to 
protect the aquatic life. Also, this kind of study 
can supply information about the effect of sud- 
den discharge of pollutants on the aquatic or- 
ganisms. In addition, it supplies information 
about their sensitivity to contaminants, deter- 
mining thereby the maximum permissible con- 
centrations for aquatic life (Clubbet al., 1975). 

An extensive literature has appeared re- 
cently documenting the use of molluscs as 
successful sentinel organisms screening the 
aquatic environment for metal contaminants 
(Simkiss et al., 1982: Cessa, 1989: AbdAllah 
et al.. 1999, 2003). Lanistes carinatus is a 
widely distributed gastropod in Egyptian fresh- 
water ecosystems (Brown, 1 995). It has a large 
enough size to provide sufficient tissue for 
metal analysis. Investigations concerning its 
storing capability of various metal pollutants, 
their histopathological changes, and influences 
on different biological activities ofthat species 
are required to set up its efficiency as bio- 
monitor to freshwater contaminants. 

The present work aims to investigate the 
bioaccumulation and toxicity of lead and cop- 
per for the freshwater snail Lanistes carinatus 
when examined singly and to depict the na- 
ture of toxicity of both metals together, whether 
synergistic or antagonistic, when mixed in a 
ratio resembles that in the inhabitant area. Also, 
the histopathological change in the digestive 
gland as a result of prolonged exposure to sub- 
lethal levels of these metals is described. 



Preparation of Test Solutions 

Test solutions for lead nitrate and copper 
sulphate were prepared in terms of molar con- 
centrations as mentioned by AbdAllah et al. 
(2000). Additionally, a mixture of both metals 
was made in the ratio of 18 : 0.5 for lead and 
copper respectively, to match their observed 
proportion in the native habitat (18 pm lead 
and 0.5 pm copper). 

Uptake of Lead and Copper 

Groups of 30 adult and healthy snails each 
were exposed to 100 pM lead nitrate, 10 pM 
copper sulphate, and 0.002 X for 21 days in 
three-liter aquaria. Snails were fed fresh let- 
tuce every other day. A space of 50 ml/snail 
was allowed to prevent competition of snails 
and to minimize the effect of snails' secretions 
(Thomas & Benjamin, 1 974). The aquaria were 
continuously aerated using electric air pumps. 
The solutions were changed twice a week. 
Twenty-one days later, ten snails were col- 
lected from each group and were prepared for 
subsequent digestion. 

Sample Preparation for Heavy Metal Analysis 

The snails collected at the end of the previ- 
ous experiment were crushed in a petri plate. 
Shell pieces were removed and the soft tis- 
sues were dissected out to isolate the diges- 
tive gland using fine scissors. The dissected 
organ was rinsed in pure water and weighed 
to the nearest 0.005 mg using a Mettler bal- 
ance. Then, the excised organ was frozen at 
-70°C for 24 h and digested according to 
McDaniel (1991) and AbdAllah et al. (2003). 

Determination of Heavy Metals in the Digested 
Tissues 



MATERIALS AND METHODS 

Sampling 

The freshwater prosobranch Lanistes 
carinatus was collected from El-Mansoureya 
Canal, Abou-Rawash, Giza. Before any treat- 
ment, the snails were washed in running wa- 
ter to remove any debris and maintained in 
three-liter aquaria for one week to be adapted 
to the laboratory conditions and to release their 
internal metal contents. The aquaria were aer- 
ated with electric air pumps, and the snails were 
fed every other day with fresh romain lettuce. 



Lead and copper were determined in the 
digested tissue using the graphite furnace 
spectroscopy, employing a Perkin-Elmer 
spectrometer with a specific-hollow cathode 
lamp for each metal (McDaniel, 1991; Pip, 
1992: Kraaketal., 1993). The metal concen- 
tration was calculated in pg/mg wet weight. 

Statistical Analysis 

Two-way ANOVA followed by Student's t-test 
comparison of least square means were done 
to test the significance of metal accumulation 
in the different examined groups using Super- 



EFFECTS OF LEAD AND COPPER ON LANISTES CARINATUS 



29 



TABLE 1 . Two-way ANOVA examining the effect of lead and copper interac- 
tion on bioconcentration of metals in the digestive gland of L. cahnatus. 



Source 



df Sum of squares Mean squares F-value P-value 



Metal 


1 


3891381 


13890000 


16.253 


0.0003 


Treatment 


1 


38575511 


38580000 


45.134 


0.0001 


MetahTreatment 


1 


13583318 


13580000 


15.893 


0.0003 


Residual 


36 


30768878 


854691 







ANOVA software computer program, Abacus 
Concept, Inc., Berkeley, California. Possible 
correlation relationship between lead and cop- 
per levels in the digestive gland of snails ex- 
posed to snail mixture was examined. Also, 
regression analysis was conducted to deter- 
mine the relationship between metal concen- 
tration within the digestive gland and organ 
weight. 

Determination of Toxic Levels 

Preliminary experiments were conducted to 
set the appropriate concentrations of each 
metal and the metal mixture for the toxicity stud- 
ies. The lead nitrate concentrations tested were 
100, 250, 500 |jM, 1 mM, and 5 mM, while the 
examined copper sulphate concentrations 
were, 20, 30, 50, 100, 500, and 1,000 pM. The 
toxicity of lead and copper interaction was stud- 
ied employing a mixture of lead nitrate and cop- 
per sulphate (1 X = 1 8 pM : 0.5 pM respectively). 
The concentrations selected for the toxicity 
study were 0.001 X, 0.005 X, 0.01 X, 0.05 X, 
and 0.1 X. Groups of ten adult healthy snails 
were exposed to each examined concentra- 
tion for 24 h. The number of dead snails was 
counted. Failure to respond to needle touch 
was considered as sign of death. The experi- 
ment was repeated three times. LC25, L-Csq, 
LC75, and LC95 were determined according to 
Finney (1971). 

Histopathological Study of Long-Term Toxicity 

The effects of chronic exposure for a period 
of three weeks to sublethal levels of lead ni- 
trate (100 pM), copper sulphate (10 pM), and 
a mixture of these metals (0.002 X) were in- 
vestigated histologically in the digestive gland. 
Moreover, normal histological features of con- 
trol snails were described. 

Following the exposure period, the exposed 
and control individuals were dissected and the 



examined organ was isolated. Paraffin blocks 
of that organ were prepared according to 
Bancroft & Stevens (1996). Five-pm thin sec- 
tions were made using a rotary microtome, 
stained with Haematoxylin and Eosin, dehy- 
drated in an ascending series of ethyl alcohol, 
cleared in xylene, and mounted in Canada 
balsam. The permanent preparations of diges- 
tive gland of exposed and control individuals 
were photographed using a 35 mm camera 
attached to a Zeiss light microscope. 



RESULTS 

Bioconcentration of Lead and Copper in Lanistes 

Lead and copper concentrations were com- 
pared in the digestive gland of the freshwater 
prosobranch Lanistes carinatus using a two- 
way analysis of of variance (ANOVA) (Table 
1). Significant differences (P < 0.001) were 
found between metal concentrations in the 
digestive glands of snails that underwent 
single and mixed exposure and between lead 
and copper levels. Also, the interaction of metal 
type and treatment had a significant effect on 
metal concentration in the examined organ 
(P < 0.001). Comparison of least square of 
means (Table 2) showed significant difference 
between lead (P < 0.05) or copper (P < 0.01) 
concentrations of snails exposed to single and 
combined metals and also demonstrated sig- 
nificant difference (P < 0.01) between capa- 
bility of lead and copper bioconcentration in 
the digestive gland of snails exposed to metal 
mixture. Lead showed higher bioaccumulation 
factor than copper (Table 3) even in presence 
of mixed metals. In all cases, lead and copper 
are concentrated over 50 fold in the digestive 
gland of the snails singly exposed and more 
than 800 fold for snails that exposed to com- 
bined metals compared to the surrounding 
water. Significant negative correlation relation- 



30 



ABDALLAH 



TABLE 2. Student's t-test (t-values) comparing the least square of means of 
lead and copper concentrations in the freshwater prosobranch L. cahnatus 
after single and mixed exposure. (* P < 0.05, ** P < 0.01) 







Single exposure 


Mixed 


exposure 






Cu Pb 


Cu 


Pb 


Single exposure 


Cu 


-0.032 


2.132* 


ND 




Pb 


- 


ND 


7.569** 


Mixed exposure 


Cu 
Pb 




- 


-5.670** 



ship (r = -0.95) was demonstrated between lead 
and copper uptake in the digestive gland of 
snails after 21 days exposure to combined met- 
als. Regression analysis between metal con- 



centration and weight of the examined organ 
showed a significant relationship r = -0.893, 
P < 0.05 for lead (Fig. 1) and r = -0.877, 
P < 0.02 for copper (Fig. 2). 



100 




0.1 0,2 0.3 

weight (g) 



0.4 



FIG. 1 . Linear regression relationship between copper 
concentration (pg/g) and weight of the digestive gland 
of L. carinatus (r = -0.877, P < 0.02). 



Q. 




0.1 0.2 0.3 

weight (g) 



FIG. 2. Linear regression relationship between lead 
concentration (mg/g) and weight of the digestive gland 
of L. carinatus (r = -0.893, P < 0.05). 



EFFECTS OF LEAD AND COPPER ON LANISTES CARINATUS 



31 



TABLE 3. Bioaccumulation factor of lead and 
copper in the digestive gland of L. cahnatus af- 
ter single and mixed long-term exposure. 



Single exposure Mixed exposure 



Copper 
Lead 



55.811 
68.953 



854.401 
3198.493 



Acute Toxicity of Lead and Copper 

Toxicity levels: LC25, LC50, LC75, and LC95 
of lead, copper, and a mixture of them are 
demonstrated in Table 4. The data demon- 
strated that copper was more toxic than lead. 
The mixture of both metals was highly toxic 
relative to single metals. 



Histological Structure of the Digestive Gland 
of Lanistes carinatus 

The digestive gland of control snails (Fig. 3) 
consists of ovoid to cylindrical shaped diges- 
tive tubules. Each tubule is composed of co- 
lumnar basophil cells with darkly stained 
granules and digestive secretory cells that 
exhibit the absorptive phase where the cells 
are partially disintegrated. These cells are 
rested on a basement membrane or the in- 
tegument. 

Histopathological Effect of Chronic Exposure 
to Metal Treatment 

Chronic exposure to 1 00 pM lead nitrate (Fig. 
4) resulted in presence of necrotic digestive 
and basophil cells in a wide tubular cavity with 





'ФГ.^^^(, 






■^l^ 



/. 



^1 



*50 



«> 



FIG. 3. Light structure of the digestive gland of 
L. carinatus; digestive cell (dc), digestive tubule 
(dt). Scale bar = 50 pm. 



FIG. 5. Transverse section of the digestive gland 
of L. carinatus exposed to 10 pM copper sulfate 
for three weeks; cell necrosis (en), digestive tu- 
bule (dt). Scale bar = 50 pm. 







Шеш 



sm^x^ •-} 



dt 



tc 



\_4a^ 



FIG. 4. Transverse section through the digestive 
gland of L. carinatus 3 weeks exposed to 100 
pM lead nitrate; tubular cavity (tc), cell necrosis 
(en). Scale bar - 50 pm. 



FIG. 6. Light micrograph of the cross-sectioned 
digestive gland excised from L. carinatus three 
weeks exposed to 0.002 X; digestive tubule (dt), 
tubular cavity (tc), residual necrotic cells (nc). 
Scale bar = 50 pm. 



32 



ABDALLAH 



TABLE 4. Toxicity levels of 24 h single and combined exposure to lead, copper for the freshwater 
snail L. carinatus. 



LC25 



LC5 



LCze 



LCq 



Lead 

Copper 

Lead-Copper 



562.34 mM 
39.81 |jM 
0.0035 X 



1.995 mM 

141.25 pM 

0.018 X 



6.761 mM 

251.19pM 

0.079 X 



21.379 mM 

1.778 mM 

0.708 X 



slight tubular deterioration. Digestive tubules 
of snails exposed to 10 pM copper sulphate 
for three weeks were almost filled with batches 
of damaged digestive and basophil cells. De- 
terioration of the digestive tubules was obvi- 
ous (Fig. 5). Destructive effect as a result of 
three weeks exposure to a mixture of both 
metals (0.002 X) was clearly illustrated (Fig. 
6). with the digestive tubules appearing almost 
vacuolated and enclosing rudiments of the dis- 
integrated basophil and digestive cells. 



DISCUSSION 

The long-term toxicity data of metal pollut- 
ants can supply valuable information about the 
sensitivity of exposed organisms to such pol- 
lutants. In addition, the combined effect of 
metals is a subject worthy of study, as the 
metals naturally exist together in the aquatic 
environment in variable ratios, depending on 
various input sources (Clubbeta!.. 1975). The 
present investigation demonstrated a variable 
response of the examined prosobranch snail 
toward the short-term exposure to sublethal 
levels of lead nitrate, copper sulphate and a 
mixture of both metals (18 pg/l lead nitrate: 
0.5 pg/l copper sulphate). The results are in 
agreement with the observations of Mathur et 
al. (1981), who found a variable acute toxicity 
effect of zinc, copper, and mercury on the 
freshwater pulmonate Lymnaea luteola. Re- 
corded toxicity levels revealed that the com- 
bined effect of the two metals was more toxic 
than that of individual metals. This finding is 
in accordance with the observations of Harrahy 
& Clements (1997), who found that the re- 
moval of zinc from a synthetic sediment incor- 
porated with mixture of cadmium, copper, lead, 
and zinc resulted in increasing the survival rate 
of Chlronomus tentans. However, the results 
are in contrast with those of Parrott & Sprague 
(1993), who showed that the combination of 
low copper concentrations with high concen- 



trations of zinc resulted in antagonistic effect 
on fathead minnows. 

It is well documented that the digestive gland 
is the major site of metal storage in molluscs 
(Simkiss et al., 1982: Simkiss & Mason, 1983; 
AbdAllah, 1999; AbdAllah & Moustafa, 2002). 
A mechanism of metal detoxification was suc- 
cessfully operated in that organ to phagocy- 
toze heavy metals after being chelated with a 
proper agent, specifically metallothionein for 
copper and cadmium, and carbonate or 
lipofucsin for lead (George, 1982; Simkiss & 
Mason, 1983; Philips & Rainbow, 1993). How- 
ever, this mechanism has a maximal thresh- 
old, at which the toxic signs started to be 
manifested in that organ at higher dosages. 
In the present work, the uptake studies dem- 
onstrated higher capability of lead to store in 
the digestive gland tissues even in the pres- 
ence of low copper concentrations and a sig- 
nificant negative correlation between lead and 
copper concentrations, which indicates an in- 
versely relationship between their bioaccu- 
mulation in the gland tissues. This finding is in 
accordance with results of a previous study 
(AbdAllah & Moustafa, 2002). 

Histological studies are effective as a bio- 
marker tool indicating the pathological effect 
of a toxicant upon living organisms (Landis & 
Yu, 1995; AbdAllah, 2003). Necrosis, lesions, 
in addition to the appearance of disorganized, 
and vacuolated cell masses are the prominent 
features of the histopathological influence of 
a specific toxicant (Sullivan & Cheng, 1976; 
Sunila, 1984: AbdAllah, 2000). The chronic 
toxicity of copper and lead followed similar 
pattern, in which necrotic cells appeared fill- 
ing the tubular cavity and being detached from 
the tegument in Lanistes. The finding is in 
agreement with observations of Tolba et al. 
(1 999) for the effect of chronic exposure of the 
schistosome vectors Blomphalaha alexandhna 
and Bulinus truncatus to copper sulphate. 
Other studies defined the toxicity status as the 
increase in diameter of the digestive tubule 



EFFECTS OF LEAD AND COPPER ON LANISTES CARINATUS 



33 



that accompanied the reduction in cellular 
length (George, 1 990). The effect of long-term 
exposure to sublethal concentrations of mixed 
concentrations of lead nitrate and copper sul- 
phate on the digestive gland of Lanistes 
carinatus was more toxic compared to that 
shown for the single metals, with degenera- 
tion of tubular cells, expansion of the tubular 
cavity and detachment of tubular tegument 
observed. This supports the toxicity data of 
previous studies (Harrahy & Clements, 1997; 
AbdAllah et al. 2000), and indicates that the 
interaction of lead and copper is more toxic, 
compared to that of each metal singly. It is 
worth mentioning that the findings of this ex- 
perimental study might not be valid for field 
investigations, because in the water canal 
other organic and inorganic substances are 
present. The interaction of these compounds 
with lead and copper might be antagonistic, 
minimizing or abolishing their toxicities. Also, 
the concentrations used are Vio of the calcu- 
lated LC50 and are fairly higher than that re- 
corded in the freshwater body (18 pm lead and 
0.5 pm copper). The results are also consis- 
tent with that of Wong (1 987), who described 
this type of effect as more than an additive 
effect and recommended the use of metal mix- 
tures for both chronic and acute toxicity stud- 
ies rather than single metal solutions, because 
they supply more valuable and realistic infor- 
mation about the nature of heavy metal toxici- 
ties in the aquatic ecosystem. Further studies 
on L. carinatus are currently going on concern- 
ing its storage capability and sensitivity to other 
heavy metals and organic pollutants to inves- 
tigate its potential value as biological monitor 
capable of screening the Egyptian freshwater 
ecosystem for various contaminants. 

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Revised ms. accepted 3 May 2004 



MALACOLOGIA, 2006, 48(1-2): 35-42 

PHYLOGENETIC ANALYSIS OF THE PERI-HYDROTHERMAL VENT BIVALVE 
BATHYPECTEN VULCAN! BASED ON 18S rRNA 

Suzanne С Dufour\ Gerhard Steiner^ & Peter G. Beninger^ 

ABSTRACT 

The species Bathypecten vulcani (Schein-Fatton, 1985), found at the periphery of hy- 
drothermal vents at the East Pacific Rise, possesses primitive shell microstructures, which 
have led to its characterization as a living fossil. The shell-based classification of B. vulcani 
within the Pectinoidea has been difficult, the species bearing similarities to both the 
Pectinidae and the Propeamussiidae; as a result, interpretations of the anatomy and biol- 
ogy of the species in an evolutionary and taxonomic context have been hindered. Here, an 
188 rRNA-based molecular phylogeny is used to compare B. i/u/can/ with other pectinoids. 
The molecular trees group ß. vulcani with the propeamussiid Parvamussium undisonum, 
in a clade distinct from all pectinids. These results support the inclusion of B. vulcani within 
the propeamussiid clade, making it the most well-studied representative of this poorly 
known group. 

Keywords: Bathypecten vulcani, Propeamussiidae, phylogeny, 183, hydrothermal. 



INTRODUCTION 

Several faunal species believed to be en- 
demic to hydrothermal vents possess anatomi- 
cal characters described as primitive or archaic 
(Newman, 1985). Among these is the bivalve 
Bathypecten vulcani, which has been found 
at the periphery of hydrothermal vents at the 
East Pacific Rise, at 9°N and 1 3°N. In its origi- 
nal description, 8. vulcani was classified as a 
pectinid, having shell structural and ultrastruc- 
tural characters reminiscent of Paleozoic 
pectinoids (Schein-Fatton, 1985). Based on 
these shell characters, the species was 
deemed a living fossil. 

An examination of the gill of Bathypecten 
vulcani revealed a simple, homorhabdic orga- 
nization, which is primitive in comparison to 
the heterorhabdic gills of all other described 
pectinids (Le Pennée et al., 1988), including 
Hemipecten forbesianus: the specimens origi- 
nally described by Yonge (1981) as having 
homorhabdic gills were recently found to have 
heterorhabdic gills (Beninger, pers. obs). 
Structural similarities between the gills of B. 
vulcani and those of early developmental 
stages of pectinids suggested that an evolu- 
tionary transition from homorhabdic to hetero- 



rhabdic gills had occurred within the Pectinidae 
(Beninger et al., 1994). 

Schein-Fatton (1988) re-evaluated the phylo- 
genetic position oi Bathypecten vulcani, as well 
as that of its newly renamed congener, B. 
eucymatus (Dall, 1898), collected at abyssal 
depths from the Bay of Biscay. A reexamination 
of the shell microstructure of both Bathypecten 
species showed differences between the two 
species, with 8. vulcani having more archaic 
features, and characters that could not be rec- 
onciled with either the Pectinidae or the 
Propeamussiidae. According to Waller (1972, 
1984), the major character allowing distinction 
between both groups is the ctenolium: it is 
present, at least in early stages, in all pectinids, 
but absent in propeamussiids. In 8. vulcani, the 
ctenolium is lacking (Schein-Fatton, 1988). 

The genus Bathypecten was eventually 
placed within the Pectinidae, in the subfamily 
Propeamussiinae (Schein, 1 989, from the fam- 
ily-group name Propeamussiidae Abbott, 
1954), a sister-group to the subfamily Pectini- 
nae. Diagnostic characters for the subfamily 
Propeamussiinae are the same as those for 
the family Propeamussiidae, sensu Waller 
(1978). It is important to note that in the clas- 
sification of Schein (1989), as in other classi- 



'Marine Biology Research Division, Scripps Institution of Oceanography, 9500 Oilman Drive, La Jolla, CA 92093-0202, U.S.A.; 

present address: ISMER, Université du Québec à Rimouski, 310 Allée des Ursulines. Rimouski, Quebec G5L3A1 Canada; 

Suzanne. dufour@uqar.qc.ca 

^Institute of Zoology, University of Vienna, Althanstr.14, A-1090 Vienna, Austria 

^ISOMER, Faculté des Sciences, Université de Nantes, 44322 Nantes Cedex 3, France 



35 



36 



DUFOUR ETAL. 



fications (e.g., Waller, 1978, 1984), both 
groups are considered to be sister taxa. In this 
paper, we will refer to these taxa by their fam- 
ily nannes, Pectinidae and Propeamussiidae, 
according to common usage, and to avoid 
confusion; this does not imply familial rather 
than subfamilial status. 

More recently, it was found that the ultrastruc- 
ture of the spermatozoa oí Bathypecten vulcani 
differed significantly from that of pectinids, given 
that it could not be classified into either of the 
pectinid structural categories of Le Pennée et 
al. (2002). Unfortunately, due to the absence 
of information on spermatozoan ultrastructure 
in propeamussiids (Healy et al., 2000), it is un- 
known whether the spermatozoa of B. vulcani 
resemble those of propeamussiids. Similarly, a 
more detailed analysis of the anatomy, ciliation, 
and mucocyte types and distribution of the gill 
in 8. vulcani has shown that it is substantially 
different from that of adult pectinids; however, 
it shows a number of similarities with the lim- 
ited information available for propeamussiid 
gills (Beninger et al., 2003). 

In the end, the conclusions stemming from 
observations of Bathypecten vulcani anatomy 
(e.g., the gill of B. vulcani represents the an- 
cestral pectinid condition - Beninger et al., 
1994) could not be confidently interpreted in a 
taxonomic and evolutionary context, due to the 
uncertain phylogeny of this species based on 
shell characters alone. In order to better clas- 
sify B. vulcani W\{h\r\ the Pectinoidea, the 18S 
rRNA sequence is here obtained and compared 
with that of other pectinids and propeamussiids. 



MATERIALSAND METHODS 
Sample Collection 

Two specimens of Bathypecten vulcani were 
collected in May 2000 from the periphery of 
hydrothermal vents at 9''N along the East Pa- 
cific Rise, at the sites Tubeworm Pillar 
(949. 6'N, 104°17.38'W, depth; 2,540 m) and 
Marker 141 (9'49.8'N. 104°17.4'W, depth; 
2,530 m). At Tubeworm Pillar, the bivalves 
were within about 10 m from active smokers; 
at Marker 141, they were about 350 m from 
the closest smokers, which were at Tubeworm 
Pillar. Upon arrival at the surface, the bivalves 
were immediately fixed in absolute ethanol. 

Ethanol-preserved specimens of Parva- 
mussium undisonum (Dijkstraw, 1995) were 
obtained from the Muséum National d'Histoire 
Naturelle, Paris (Norfolk 1 expedition, station 
DW 1699, coll. M. Boisselier). 



DNA Extraction and Amplification 

The ethanol-preserved animals were 
washed in distilled water prior to DNA extrac- 
tion. Genomic DNA was extracted from the 
adductor muscle and gills with the "DNeasy 
Tissue Kit"® (Qiagen). The near-complete 18S 
rRNA gene was amplified using the primers 
1 8A1 (5- CCT ACC TGG TTG АТС CTG CCA 
G-3')and1800r(5'-ATGATCCTTCCGCAG 
GTT CAC С - 3). The PCR-reactions were 
made on a Robocycler 96 (Stratagene) in a 
30 pi reaction mix (1.5 mM MgCI,, each dNTP 
at 250pM, each primer at 0.5 pM, 0.6 units 
Biotaq Red polymerase [Bioline] and the sup- 
plied reaction buffer at 1 x concentration). The 
PCR cycle conditions were; initial denaturation 
step of 2 min at 94°C, 36 cycles of 30 sec de- 
naturation at 94°C, 45 sec annealing at 50°C, 
and 2 min primer extension at a 72°C, followed 
by a final primer extension step of 10 min at 
72°C. PCR products were purified with the 
Concert Rapid PCR Purification System (Life 
Technologies) and sequenced with a range of 
primers (Steiner & Dreyer, 2003) on an ABI 
3700 at VBC-Genomics Bioscience Research 
GmbH, Vienna. 

Choice of Taxa, Alignment and Phylogenetic 
Analysis 

The 18S rRNA sequences of Bathypecten 
vulcani and Parvamussium undisonum were 
aligned with those of all available species of 
Pectinidae, Spondylidae, Limidae (excluding 
the species of Limatula because of their di- 
vergent sequences), Anomiidae, and 
Plicatulidae (Table 1 ). According to Steiner & 
Hammer (2000), Giribet & Wheeler (2002), 
and Matsumoto (2003), the latter three fam- 
ily-groups comprise the closest relatives to the 
Pectinoidea. Additional outgroup taxa were 
selected from the Pinnidae and Arcoidea 
(Table 1). The computer-aided alignment of 
these 34 sequences produced by CLUSTAL 
X 1.8 (Thompson et al., 1997) using default 
parameters and subsequent manual correc- 
tions is available from the authors (GS). 

Unweighted heuristic parsimony (MP) 
searches were made with PAUP* 4.0b10 
(Swofford, 1998) on a PC with 50 random ad- 
dition sequences and TBR branch swapping. 
Bootstrap support (BP) was assessed by 1 ,000 
replicates, each with three random sequence 
additions. The program MODELTEST 3.06 
(Posada & Crandall, 1998) determined the 
GTR+I+Ä model as most suitable for maxi- 
mum-likelihood analyses (ML). The param- 



PHYLOGENY OF BATHYPECTEN VULCANI 



37 



eters estimated from the data were set for a 
ML search submitting the parsimony strict con- 
sensus tree to SPR branch swapping with re- 
arrangements limited to cross four branches 
in PAUP*. We tested the phylogenetic signal 
and the robustness of the ML tree with the 
quartet-puzzling program TREE-PUZZLE 5.0 
(Schmidt et al., 2002) under the same model 
as the ML analysis and parameters estimated 



by the program and with 100,000 puzzling 
steps. In addition, we analyzed phylogenetic 
relationships with Bayesian inference imple- 
mented in MRBAYES 3.0b4 (Huelsenbeck & 
Ronquist, 2001). We ran six chains through 
200,000 generations under the GTR+I+Ä 
model starting with random trees. The first 300 
trees were discarded as burn-in for the calcu- 
lation of posterior probabilities. 



TABLE 1 . Systematic list of species used in the phylogenetic analysis, with the GenBank accession 
number of the 18S rRNA sequences. 



Systematic position 



Species 



Accession Number 



Arcoidea 
Arcidae 



Noetiidae 
Glycymerididae 

Pinnoidea 
Pinnidae 

Anomioidea 
Anomiidae 



Plicatuloidea 
Plicatulidae 

Limoidea 
Limidae 



Pectinoidea 
Spondylidae 



Propeamussiidae 
Pectinidae 



Area noae (Linné, 1758) 
Acer plicata (Dillwyn, 1817) 
Barbatia virescens (Reeve, 1844) 
Stharca láctea (Linné, 1758) 
Glycymehs pedunculus (Linné, 1758) 
Glycymeris sp. 

Pinna muricata (Linné, 1758) 
Atrina pectinata (Linné, 1767) 

Anomia ephippium (Linné, 1758) 
Pododesmus caelata (Reeve, 1859) 
Pododesmus macrochisma (Deshayes, 1839) 

Plicatula plicata (Linné, 1767) 
Plicatula australis (Lamarck, 1819) 



Lima lima (Linné, 1758) 
L/mar/a /7/'ans (Gmelin, 1791) 
Ctenoides annulatus (Lamarck, 



1819) 



Spondylus crassisquamatus (Lamarck, 181 9) 
Spondylus hystrix (Röding, 1798) 
Spondylus sinensis (Schreibers, 1793) 
Bathypecten i/u/can/ (Schein-Fatton, 1985) 
Parvamussium undlsonum (Dijkstra, 1995) 
Pectén maximus (Linné, 1758) 
Placopecten magellanicus (Gmelin, 1791) 
Adamussium colbecki (E. A. Smith. 1902) 
Flexopecten glaber (Unné, 1758) 
Argopecten irradians (Lamarck, 1819) 
Argopecten gibbus (Linné, 1758) 
Chlamys islándica (Müller О. F., 1776) 
Chlamys hastata (Sowerby, 1843) 
Mimachlamys varia (Linné, 1758) 
Crassadoma gigantea (Gray, 1825) 
Exellichlamys spectabilis (Reeve, 1853) 
Pedum spondyloideum (Gmelin, 1791) 



X90960 

AJ389630 

X9197 

AF120531 

AJ389631 

X91978 

AJ389636 
X90961 

AJ389661 
AJ389650 



AJ389651 
AF229626 

AJ 389652 
AFI 20534 
AJ389653 

AJ389646 

AJ389647 

AF229629 

AY557608 

AY557607" 

L49053 

X53899 

AJ242534 

AJ389662 

L11265 

AF074389 

L11232 

L49049 

L49051 

L49050 

AJ389648 

AJ389649 



*The partial 28S sequence of Parvamussium undisonum is deposited under the accession number AY557609, 



38 



DUFOUR ETAL. 



RESULTS AND DISCUSSION 

188 Sequence and Molecular Phylogeny 

The alignment resulted in a data matrix with 
1 ,973 characters, of which 215 are parsimony- 
informative. The parsimony search yielded 1 1 2 
shortest trees of 51 7 steps (CI = 0.594, RC = 
0.478). The topology of the resulting strict con- 
sensus tree (Fig. 1) differs only slightly from 
the single maximum-likelihood tree (-InL = 
6386.38877) (Fig. 2). All analyses firmly sup- 
port the taxa Propeamussiidae {Parvamussi- 
um + Bathypecten). Pectinidae, and the 
Spondylidae. The monophyly of the Pectinoi- 
dea is always recovered, albeit with varying 
branch support. The Propeamussiidae and 
Pectinidae always appear as sister taxa with 
low support. This distinction is corroborated 
by the analysis of the mitochondrial gene, cy- 
tochrome-oxidase-l (Matsumoto, 2003), which 
supports pectinoid monophyly but yields a sis- 
ter group relationship of Propeamussiidae to 
the clade (Spondylidae + Pectinidae). The two 



propeamussid species have similar and highly 
divergent sequences and, accordingly, an ex- 
tremely long common branch. Although the 
limid species have similarly long branches, 
there is no indication of a long-branch attrac- 
tion effect. 

The molecular information is therefore con- 
sistent with the inclusion of Bathypecten 
vulcani in the propeamussiid group, rather than 
with the pectinid group. 

Soft Anatomical and Spermatozoan Characters 

Comparisons of new and published data 
concerning anatomical and spermatozoan 
characteristics of Bathypecten vulcani, 
pectinids, propeamussiids, and spondylids 
reveals that B. vulcani shares more affinities 
with the Propeamussiidae. Some of these 
anatomical characters may be apomorphies 
of propeamussiids, others are likely to be 
plesiomorphies, as discussed below. 

The gill structure of Bathypecten vulcani is 
much simpler than that of pectinids (Beninger 



96/92 



100/90 



1.00 



1.00 



100/90 



1.00 
100/95 



71/22 



0.97 



1.00 
100/98 



65/79 



0.99 



1.00 



100/98 



1.00 



100/97 



50/50 



0.94 



1.00 



100/99 



1.00 



35/- 



0.83 



50/- 



0.99 



76/56 



64/72 



0.67 



1,00 



89/96 



1.00 



ARCOIDEA 
PINNIDAE 
PLICATULIDAE 
ANOMIIDAE 

LIMIDAE 

Spondylus sinensis 
Spondylus crassisquamatus 
Spondylus hystrix 

Parvamussium undisonum 
Bathypecten vulcani 
Placopecten magellanicus 
Adamussium colbecki 
Exellichlamys spectabilis 
Crassadoma gigantea 
Chlamys islándica 
Pedum spondyloideum 
Mimachlamys vana 
Chlamys hastata 
Peden maximus 
Aequipecten opercularis 
Flexopecten glaber 
Argopecten gibbus 
Argopecten irradians 



FIG. 1. Strict consensus of 112 most parsimonious trees. Bootstrap and ML-puzzling supports are 
above, posterior probabilities below branches. 



PHYLOGENY OF BATHYPECTEN VULCANI 



39 



& Le Pennée, 1991), spondylids, and limids 
(Ridewood, 1903; Dakin, 1928): it is non-pli- 
cate, homorhabdic, has a non-reflected outer 
demibranch, and lacks latero-frontal cilia, in- 
ter-filamentarjunctions, and interlamellarjunc- 
tions (Beninger et al., 2003). The relatively 
poorly known propeamussiid gills have many 
similar features. The organization of Bathy- 
pecten vulcani gill filaments does not corre- 
spond to the inverted arrangement reported 
for Propeamussium lucidum, in which the fron- 
tal ciliary tracts were deemed to be located in 
the suprabranchial chamber (Morton & 
Thurston, 1989). However, this atypical orga- 
nization could easily have been misinterpreted, 
given that filaments without junctions are eas- 
ily disorganized and entangled during fixation 
(Morton & Thurston, 1989). 

Further examinations of propeamussiid gills 
would be needed to determine how the 8. 
vulcani gill organization compares to other 
members of this family. If other propeamussi- 
ids are found to share the simple gill structure 



of B. vulcani, then some character-states 
(homorhabdy, lack of plicae, and lack of 
filamentar and lamellar junctions) are likely to 
be plesiomorphic for the Propeamussiidae; 
similar character-states are found in anomiids, 
plicatulids, and arcids, with some variation in 
the extent of interfilamentar and interlamellar 
junctions (Ridewood, 1903; Yonge, 1973). In 
addition, the small labial palps and non-arbores- 
cent lips observed in B. vulcani (Beninger et 
al., 2003), and described in some prope- 
amussiids (Yonge, 1 981 ), may be plesimorphies 
for the Propeamussiidae (as compared to the 
condition in the Pectinidae and Spondylidae - 
Dakin, 1928; Yonge, 1973; Beninger & Le 
Pennée, 1991 ). The lack of laterofrontal cilia, 
as described in B. vulcani (Beninger et al., 
2003) and in Propeamussium lucidum (Morton 
& Thurston, 1989), is likely to be apomorphic 
for propeamussiids, as it has been described 
for no other pteriomorph to date. Also, the 
unique spermatozoan type described for ß. 
vulcani (Le Pennée et al., 2002) may be 



Arca noae 

Acar plicata 



— Barbatia virescens 
Striarca láctea 
Glycymeris sp 

Glycymeris pedunculata 



— Pinna muricata 
Atrina pectinata 

p Plicatula plicata 

Plicatula australis 
Pododesmus caelata 
Anomia ephippium 
Pododesmus macrochisma 



Limarla hians 



Ctenoides annulatus 



Spondylus sinensis 

Spondylus crassisquamatüs 
Spondylus hystrix 



^ 



0.01 



Placopecten magellanicus 
~ Crassadoma gigantea 
Chlamys islándica 
I Pedum spondyloideum 
" Mimactilamys varia 
Chlamys tiastata 
Adamussium colbecki 

Exellictilamys spectabilis 

Flexopecten glaber 
Pacten maximus 
Aequipecten opercularis 
Argopecten gibbus 
Argopecten irradians 



Parvamussium undisonum 
— Bathypecten vulcani 



FIG. 2. Maximum likelihood tree (-In L = 6386.38877) found under the GTR+I +Г model. Model param- 
eters estimated by MODELTEST: substitution rate matrix A-C = 2.2511 , A-G = 2.9955, A-T = 1 .6583, 
C-G = 1 .31 61 , C-T = 4.9707, G-T = 1 .00); nucleotide frequencies A = 0.2472, С = 0.221 3, G = 0.2724, 
Т = 0.2591; assumed proportion of invariable sites, pinvar = 0.5835; gamma distribution of rates at 
variable sites in four categories with shape parameter, alpha = 0.6141. 



40 



DUFOUR ETAL. 



apomorphic for propeamussiids, if a similar 
structure was found in this group. Further ana- 
tomical observations are required to confirm 
the evolutionary status of these characters. 

The presence of prismatic calcite on the left 
valve, such as found in Bathypecten vulcani, 
is only known from a group of Paleozoic fos- 
sils ancestral to the Propeamussiidae, the 
Pterinopectinidae and the Aviculopectinidae 
(Newell. 1938). Bathypecten vulcani may 
therefore have retained primitive characters, 
either by early phyletic divergence from other 
propeamussiids. or by paedomorphosis. 

Propeamussiid Anatomy and Habitat 

Several of the anatomical characters of 
Bathypecten vulcani and of other prope- 
amussiids are likely to be related to their deep- 
sea habitat. As described by Allen (1981), 
deep-sea bivalves are commonly small in size, 
and have reduced gills; this miniaturization is 
thought to be associated with the small 
amounts of available food at great depths. 
Most propeamussiids are found at depths 
greater than 1 50 m, and were probably deep- 
sea inhabitants in the Mesozoic and Cenozoic 
(Waller, 1972). 

One of the possible consequences of the 
deep-sea habitat of propeamussiids, and a 
possible outcome of their small body size, is 
the simplification of the gill. At the present state 
of knowledge, all propeamussiids have 
homorhabdic gills, with, at least in Bathypecten 
vulcani. few filaments. Due to the size restric- 
tion, it might not be possible for a gill with only 
approximately 50 filaments to become plicate, 
and by extension, heterorhabdic. Although the 
gills of developing postlarvae of pectinids are 
known to develop principal filaments at about 
4 mm body size, and plication at about 7 mm 
(Beninger et a!., 1994; Veniot et a!., 2003), 
these growing pectinids contain at least three 
times as many filaments of the same diameter 
per gill as B. vulcani (Veniot et al., 2003). Al- 
though a bivalve the size of a typical prope- 
amussiid can thus have a gill with enough 
filaments to become plicate and heterorhabdic, 
this may not be the most efficient organization 
for an adult bivalve, given the space limitation. 

To date. Bathypecten vulcani has only been 
found in the proximity of hydrothermal vents: 
however, no particular effort has been made 
to collect this species at other sites. Bathy- 
pecten vulcani may not be restricted to vent 
environments, given its feeding regime, which 
is largely dependent on particulate food ongi- 



nating from surface waters (Le Pennée et al., 
2003). Other Bathypecten species have been 
collected from bathyal and abyssal sediments 
in the Bay of Biscay and in the western Pa- 
cific (Schein, 1989), and do not appear to be 
found at vents. The discovery of B. vulcani in 
environments outside hydrothermal vent sites 
would confirm that its presence at vents is 
largely opportunistic; B. vulcani may simply be 
taking advantage of the relatively high amounts 
of particulate matter that are available at vents 
(Enright et al., 1981; Gage & Tyler, 1991). 



CONCLUSIONS 

The results of the present molecular phylo- 
genetic analysis are consistent with 
Bathypecten vulcani being a member of the 
family Propeamussiidae. This placement is in 
concordance with the classification of B. 
vulcani based the absence of a ctenolium, this 
being the major criterion used to distinguish 
pectinids from propeamussiids (Waller, 1984). 
Interpretations of the biology of B. vulcani 
should thus be recast in the light of its prope- 
amussiid status, rather than with reference to 
the pectinids (Beninger et al., 2003; Le Pennée 
et al., 1 988, 2002). Although far from complete, 
this body of work thus represents the most 
considerable amount of knowledge concern- 
ing any propeamussiid to date. 



ACKNOWLEDGEMENTS 

Horst Felbeck provided SCD with the oppor- 
tunity to participate on the hydrothermal vent 
cruise where the specimens were collected. 
The help of Hermann Dreyer (University of 
Vienna) in the molecular lab is gratefully ac- 
knowledged. 



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42 DUFOURETAL. 

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Revised ms. accepted 14 July 2004 



MALACOLOGIA, 2006, 48(1-2): 43-64 

THE GENUS STRICTISPIRA IN THE WESTERN ATLANTIC 
(GASTROPODA: CONOIDEA) 

Donn L. Tippett 

10281 Gainsborough Road Potomac, Maryland 20854-4038, U.S.A. 
donntipp2@ verizon.net 



ABSTRACT 

The genus Strictispira [formerly Turridae, now Strictispiridae] in the western Atlantic area is 
reviewed. Two new species, S. redferni anä S. coltrorum. are proposed. Crassispira quadri- 
fasciata (Reeve, 1843) is reassigned to Strictispira. Three additional species - S. drangai 
(Schwengel, 1951), S. paxillus (Reeve, 1845), and S. solida (С. В. Adams, 1850) - are 
discussed. Drillia acurugata Dall, 1890, regarded as a Recent species as well as fossil and 
as a Strictispira, is shown to be fossil only, with Recent specimens considered to be that 
species here regarded as S. redferni. Similarly, Drillia ebenina Dall, 1890, initially a fossil 
species and often considered to be Recent and a synonym of S. solida, is shown to be fossil 
only. Recent specimens identified as S. ebenina are regarded as S. solida. Characteristics 
of the genus and species were studied, and are here described and illustrated, including 
shell morphology, opercula, and anatomy - especially foregut anatomy and radular struc- 
ture. Comparisons are made with similar-appearing species, both within the genus and in 
other genera. The feeding mechanism of strictispirids is probably by ingestion aided by 
grasping of the prey by extruded radular teeth, followed by rasping and tearing of the prey by 
the teeth. The protoconch is paucispiral, indicating direct development. The genus has a 
western Atlantic distribution from the lower eastern Carolinian province to the Caribbean/ 
West Indian province, including both sides of Florida, the Florida Keys, Mexico and Central 
America, the Greater Antilles, Virgin Islands, Lesser Antilles, lower Caribbean, and the Bra- 
zilian province. 

Key words: Strictispira, taxonomy, shell morphology, radular structure, foregut anatomy. 



INTRODUCTION 

The genus Strictispira was established by 
McLean (1971a: 125) for crassispirine-like 
tropical eastern Pacific species bearing a dis- 
tinctive radular structure and teeth. The genus 
was placed in a new subfamily Strictispirinae 
of the family Turridae. The family Turridae has 
recently been reclassified by Taylor et al. 
(1993), with some of the subfamilies elevated 
to family level, the Strictispiridae among them. 
This classification is used here. McLean 
(1971a: 123-125)described the subfamily and 
the genus, described and illustrated the radu- 
lae of the eastern Pacific species (1 971 a: figs. 
86, 87), and pointed out the characteristics of 
the sinus structure of the group. 

Discussing Strictispirinae, McLean com- 
mented (1971a: 124) that he was "much in- 
debted to Virginia Maes for an exchange of 
ideas concerning the group, of which she has 
for some time been aware." The late Virginia 



Maes had specialized in the family Turridae 
for some years, and, although she published 
only sparsely, became one of the authorities 
on that large group. She meticulously curated 
the turrid collection at the ANSP. With regards 
Strictispira species, McLean commented 
(1971a: 125) that Dn///a еЬеп/па Dall, 1890, a 
western Atlantic species originally described 
as fossil, is also a member of the genus. He 
said (1971b: 730), with regard to the eastern 
Pacific Strictispira stillmani Shasky, 1971, 
"Strictispira ebenina is a related Caribbean 
species". There has been confusion as to 
whether Drillia ebenina and Fleurotoma solida 
С. В. Adams, 1850, are conspecific, I believe 
that Recent specimens identified as S. ebenina 
are in fact S, solida. Collections at both the 
USNM and the ANSP show mixing of the two 
identifications. At the ANSP, Recent material 
considered S. ebenina was maintained sepa- 
rate but following S. solida. Review of these 
shows that they are S. solida. It is probable 



43 



44 



TIPPETT 



that Maes had identified S. solida as stricti- 
spirid, because a specimen (ANSP 282214) 
from Belize with soft parts had been collected 
in 1961 by Robert Robinson of the ANSP. 
Maes's card files contain a card with photo- 
graphs of the shell and one showing the radula. 
which bears typical strictispirid teeth. Although 
now assigned to Sthctlspira, the specimen was 
without identification originally. It was located 
in the S. ebenina section, probably having ini- 
tially been considered that on Maes curating 
this material. 

In the course of her studies Maes had syn- 
onymized various species. These synonymies 
were seldom published, but have been listed 
in Malacolog, the online database of the west- 
ern Atlantic molluscan fauna created at the 
ANSP by Gary Rosenberg, and have there- 
fore circulated among malacologists. Pleuro- 
toma solida with Drillia ebenina as a synonym 
is an example. 

Further, Maes (1983) identified Pleurotoma 
paxillus (Reeve, 1845) as a Sthctispira and 
demonstrated other significant strictispirid ana- 
tomical features, including the lack of a poi- 
son gland and bulb. She also pointed out that 
the characteristic sinus structure ("turrid 
notch", on the shoulder slope in this case, not 
to be confused with the "stromboid notch" on 
the lower lip) of the group is not restricted to 
the strictispirids but also occurs in some west- 
ern Atlantic crassispirine species. She further 
commented about the difficulty differentiating 
the shells of S. paxillus and S. solida, plus such 
other similar-appearing species as Crassi- 
splrella fuscescens (Reeve, 1843) and 
Crassiclava apicata (Reeve, 1845). Kantor et 
al. (1997), in a cladistic study based on con- 
siderable foregut research of crassispirine 
species, suggested that the conventional sub- 
genera of Crassispira be raised to generic 
level. This is followed here, thus Crassiclava 
and Crassispirella are assigned generic level, 
Crassispira remaining at generic level but with- 
out subgenera. 

Taylor et al. (1993) reviewed the foregut 
anatomy of strictispirids, illustrating the radu- 
lar structure and teeth, noting absence of a 
poison apparatus, and showing that the buc- 
cal mass is positioned at the anterior end of 
the proboscis, the buccal tube being short, and 
they discussed the feeding mechanism. Kantor 
& Taylor (1994) reviewed S. pax/7/usin the light 
of a study of Maes's material, including analy- 
sis of serial sections, pointing out and illus- 
trating details of the foregut anatomy 
(compared here with the present findings). 



In the tropical eastern Pacific, Sthctispira con- 
tains two species, S. ericana (Hertlein & Strong, 
1951) and S. stillmani; the sister genus 
Cleospira contains only С ochsneri (Hertlein & 
Strong, 1949). The western Atlantic species, 
listed in Malacolog {fide Maes), are Sthctispira 
acurugata (Dall, 1890), S. dranga/ (Schwengel, 
1951), S. paxillus, and S. solida, with Dhllia 
ebenina as a synonym. The Recent material in 
the ANSP collection considered to be S. 
acurugata by Maes is here shown to be the new 
species S. redferni. Dhllia acurugata is restricted 
to fossil forms only, and is herein considered a 
probable member of the cochlespirine genus 
Pyrgospira. With the addition of two new taxa, 
Sthctispira redferni and Sthctispira coltrorum, 
reassignment of Crassispira quadhfasciata, 
which has been determined to be strictispirid, 
S. paxillus, S. solida, and possibly S. drangai. 
the number of Sthctispira species in the west- 
ern Atlantic is tentatively six. No members of 
Cleospira are yet known in this area. 

Fossil taxa considered to belong in the ge- 
nus on the basis of shell morphology are: S. 
acurugata (Dall, 1890), from the Upper 
Pliocene-Lower Pleistocene Caloosahatchee 
Formation of Florida; S. aurantia (Olsson, 
1 922) from the Late Miocene Gatun formation 
in Costa Rica; S. ebenina (Dall, 1890) from 
Upper Pliocene-Lower Pleistocene Caloosa- 
hatchee Formation and from the Middle 
Pliocene Pinecrest Beds of Florida; S. lomata 
(Woodring, 1928) and S. ponida (Woodring, 
1928), from the Upper Pliocene Bodwen For- 
mation of Jamaica; S. proebenina (Gardner, 
1937) from the Upper Middle Miocene Shoal 
River Formation of Florida - Maes ms notes; 
McLean (1971a; 125) included ponida and 
lomata on the basis of sinus structure - and 
Clavus (Crassispira) zizyphus Berry, 1940, 
from the Lower Pleistocene Hilltop Quary of 
San Pedro, California (pers. comm., McLean). 
As stated, S. acurugata and S. ebenina have 
been thought to be Recent as well as fossil, 
but are here considered fossil only and are 
further discussed below. Analysis of the other 
fossil taxa are outside the scope of this paper. 



MATERIALSAND METHODS 

Specimens of the genus Sthctispira, some 
with soft parts, from various geographic locali- 
ties, were examined as to shell morphology, 
and, where possible, as to anatomy, especially 
foregut and radular morphology. Photographs 
were made of representative shells. SEM 



THE GENUS STRICTISPIRA IN THE WESTERN ATLANTIC 



45 



preparations were made of protoconchs, oper- 
cula, and, in one instance, of a radular ribbon. 
Preserved specimens were dissected; dry 
specimens were treated with KOH and dis- 
sected as possible. Drawings of individual 
teeth were made from radular preparations, 
which had been slide mounted and stained. 
Serial sections were made in one instance. 
Type material and voucher specimens were 
deposited at the USNM, MORG, and other in- 
stitutions. Radular preparations were depos- 
ited at the USNM and the ANSP. 

Institutional abbreviations used: 

AMNH = American Museum of Natural History, 
New York, U.S.A. 

ANSP = Academy of Natural Sciences, Phila- 
delphia, U.S.A. 

DMNH = Delaware Museum of Natural History, 
Wilmington, Delaware, U.S.A. 

FMNH = Field Museum of Natural History, 
Chicago, Illinois, U.S.A. 

LACM = Los Angeles County Museum of 
Natural History, Los Angeles, Cali- 
fornia, U.S.A. 

MCZ = Museum of Comparative Zoology, 
Harvard University, Cambridge, Mas- 
sachusetts, U.S.A. 

MNHN = Muséum National d'Histoire Naturelle, 
Paris, France 

MORG = Museu Oceanógrafico do Rio Grande, 
Rio Grande, Brazil 

NHM = The Natural History Museum, London, 
England 

NM = Natal Museum, Pietermaritzburg, 
South Africa 

USGS = United States Geological Survey, 
Washington, D.C., U.S.A. 

USNM = National Museum of Natural History, 
Smithsonian Institution, Washington, 
D.C., U.S.A. 

Other abbreviations: 

spec. = specimen(s) 

colln. = collection 

Exp. = Expedition 

Stn. = Station 

SYSTEMATICS 

Strictispiridae McLean, 1971 

Genus Strictlspira McLean, 1971 

Type species: Crassispira ericana Hertlein & 

Strong, 1951 



Description 

Shells of small size (approximately 10-25 
mm), drilliiform, dark colored, sculptured with 
axial ribs and spiral cords or threads; concave 
sulcus with subsutural cord; laterally directed, 
U-shaped sinus with projecting parietal tu- 
bercle; protoconch smooth, of approximately 
two whorls; operculum ovoid, with terminal 
nucleus; animal with large radular ribbon bear- 
ing numerous rows of paired marginal teeth 
of pistol shape, with median flange; lacking 
poison apparatus. 

Strictlspira coltrorum, new species 

Figures 1-3, 19, 25, 36 

Description 

Shell small (to approximately 11 mm), 
drilliiform, elongate, turreted, moderately high 
spired; body whorl about half shell length; an- 
terior canal short, open, unnotched. Color 
medium brown overall to color form with vari- 
ably lighter spiral banding of shell periphery, 
lighter outer lip and parietal tubercle. 
Protoconch (Fig. 19) of two smooth whorls, 
tip protruding; teleoconch 6-6У2 whorls, mod- 
erately strong subsutural cord, occasionally 
lighter colored than rest of shell; shoulder sul- 
cus sharply concave; shoulder tabulate; axial 
ribs, with blunt posterior ends, extending an- 
teriorly, forming flat whorl profile to following 
whorl. Body whorl with flat peripheral region, 
ribs curving around moderately convex base, 
disappearing at moderately concave junction 
with canal. Suture rising slightly onto preced- 
ing whorl at end of body whorl. Ribs rounded, 
slightly less in width than interspaces, slightly 
opisthocline, 13-14 to varix on body whorl, 18- 
20 on penultimate whorl and spire whorls. Last 
rib enlarged to form modest varix % whorl or 
less back from thin, curved lip edge. Occa- 
sional specimens with varix formed of two 
joined ribs. Spiral cords rounded, evenly 
spaced, 4-5 on spire whorls, not crossing ribs 
or doing so only faintly until below periphery, 
6-7 forming slightly laterally elongate beads 
on crossing axials, beads becoming stronger 
anteriorly, 5-6 strong cords on canal. Moder- 
ately deep, U-shaped sinus on sulcus, apex 
at mid point, projecting parietal tubercle nar- 
rowing sinus entrance. Sinus tracks present 
on sulcus. Three distinct spiral threads always 
present on sulcus. Very shallow stromboid 
notch always present. 



46 



TIPPETT 




FIGS. 1-9. Shells of Strictispira spp. FIGS. 1-3: Strictispira coltrorum. holotype, MORG 43415, 
Escavalda Id., Guarapari, Espirito Santo, Brasil, 10.9 x 4.0 mm; FIGS. 4-6: Strictispira redferni, 
holotype. USNM 1010771, Abaco Id., Bahamas. 9.3 x 3.6 mm; FIG. 7: Strictispira redferni yanety, 
ANSP 221823. Vaca Key, Florida Keys, 14.1 x 4.6 mm; FIG. 8: Strictispira redferni y aueXy, ANSP 
355797, Exuma, Bahamas, 10.9 x 4.0 mm; FIG. 9; Drillia acurugata, holotype, USNM 97320, 
Caloosahatchee Riv., Upper Pliocene, Florida, 21.0 x 7.8 mm. Scale bar = 10 mm. 



THE GENUS STRICTISPIRA IN THE WESTERN ATLANTIC 



47 



Anatomy 

One specimen containing dried animal with 
operculum available. Animal with foot, head 
and mantle/siphon mottled black. Foot 
elongate. Head small, with two tentacles 
bearing eyes distally and laterally. Large 
siphon on left continuous with thin mantle. 
Mantle edge behind tentacle bases dorsally, 
bearing sinus indentation on right. Mantle 
semitransparent; gills and osphradium visible 
on left and penis on right, originating behind 
right tentacle, reflected backwards under 
mantle. Foregut anatomy difficult to discern but 
showing large rhynchodeum and moderate 
sized proboscis, both with circular internal 
folding due to retraction. Structure of buccal 
tube and cavity could not be determined. 
Massive odontophore dominating body cavity. 
No poison gland or bulb present. No salivary 
gland seen. Odontophore of paired cartilages, 
strong subradular membrane and paired, 
marginal radular teeth present. Partial radular 
ribbon with approximately 80 pairs of teeth. 
Teeth (Fig. 36) approximately 190 pm, solid, 
pistol-shaped, with pointed anterior end and 
median flange. Operculum (Fig. 25) ovate, 
elongate, with pointed anterior end and 
terminal nucleus. 

Type Material and Locality 

Holotype, MORG 43415, Escavalda Id., 
Guarapari, Espirito Santo, Brasil (20°42'S, 
40°25'W), dredged at 25-30 m, on bryo- 
zoans, Dec. 1993, A. Bodart!; paratypes, 
same data as holotype: 2 spec, USNM 
1011351; 1 spec, USNM 1011352; 1 spec 
each at AMNH, ANSP, DMNH, FMNH, 
LACM, MCZ, MNHN, MORG, NHM, NM (ma- 
terial ex author's colln.). 

Distribution 

Known only from the type locality. 

Discussion 

This is a very uniform group of shells, 
undoubtedly a population sample. One 
specimen has 4 fairly strong spiral lirae inside 
the outer lip extending back into the shell for 
about Va whorl. 

Strictispira coltrorum is nearest Strictlspira 
redferni, but is typically smaller (the holotype 
of S. redferni, selected because of its excellent 
condition, is smaller than the holotype of S. 



coltrorum). It is more elongate, has more and 
narrower axial ribs than S. redferni, has a 
stronger parietal tubercle of typical strictispirid 
form, different protoconch - two whorls, with 
protruding tip vs. 1У2 whorls with a partially 
immersed tip in S. redferni, and different color 
- medium brown vs. black brown for S. redferni. 
The radular structure and radular teeth are 
essentially the same in both species. 

Strictispira coltrorum is similar to Crassiclava 
apicata (Fig. 16) in shell morphology, differing 
by being smaller, having a strictispirid sinus, 
having more and closer ribs and more concave 
sulcus, different protoconch - two whorls with 
protruding laterally placed tip rather than 2- 
2У2 whorls with flat lateral tip, and of different 
color-medium brown vs. dark brown. 

Etymology 

The species is named after José and Marcus 
Coltro for their kind donation of specimens and 
their contributions to malacology. 

Strictispira c/ranga/ (Schwengel, 1951) 
Figures 10, 20, 26 

Crass/sp/radranga/ Schwengel, 1951: 116, pi. 
8,fig. 1. 

Crassispira (Crassispirella) drangai 
(Schwengel, 1951) - Abbott, 1974: 273, 
species 3056, list ("Very close to Clathrodrillia 
solida С. В. Adams."); Redfern, 2001: 125, 
species 519, pi. 56. 

Strictispira drangai (Schwengel, 1951) - 
Malacolog, 2004, list. 

Description 

Shell fusiform, turreted, moderately tall 
spired, spire angle 32°, length to approximately 
25 mm; body whorl somewhat truncate 
anteriorly, slight basal constriction. Protoconch 
(Fig. 20) of two smooth, brown whorls; 
teleoconch approximately 8 whorls. Whorl 
outline flattish below sulcus. Sulcus narrow, 
concave, bearing fine spiral striae and curved 
sinus traces, preceded by strong, sharply 
crested subsutural cord somewhat distant from 
suture. Sculpture of narrow axial ribs, 17-22 
on penultimate whorl, producing whorl 
shoulder, interspaces wider, disappearing at 
bottom of base. Four or five regularly spaced, 
widely separated spiral cords, crossing axials 
weakly on periphery, 4-6 more prominent, 
basal cords below periphery, producing 
beading on crossing axials, 5-6 cords down 



48 



TIPPETT 



canal. Fine secondary spiral threads between 
primaries overall. Sculpture forms pattern of 
rectangular spaces with enclosed spiral 
threads. Enlarged axial or two forming varix 
behind outer lip. Aperture parallel-sided, 
ending in short, open, slightly notched anterior 
canal bent slightly right. Small stromboid 
notch. Lip edge fluted. Columellar callus thin, 



emarginate. Sinus deep, U-shaped, with 
moderately projecting parietal tubercle, most 
specimens with vertical groove behind distal 
end of tubercle (see Discussion below). Color 
shiny dark brown when fresh, rib interspaces 
usually lighter colored, especially on body. 
Operculum (Fig. 26) ovoid, with pointed 
anterior end and terminal nucleus. 




FIGS. 10-18. Shells of Strictispira. Crassispira, Dhllia, Crassiclava. Crassispirella, Pyrgospira spp. 
FIG. 10: Strictispira drangai. holotype. ANSP 247104, Hastings, Barbados, 17.7 x 6.7 mm: FIG. 11: 
Strictispira solida. USNM 900424, Key West, 16.0 x 5.9 mm; FIG. 12: Crassispira sp, ANSP 368728, 
Bahamas, 16.0 x 6.4 mm; FIG. 13; Drillia ebenina. figured syntype, USNM 97318, Caloosahatchee 
Riv., Florida, Upper Pliocene, 16.5 x 7.0 mm; FIG. 14: Strictispira paxillus, specimen figured by Maes 
(1983: fig. 10), ANSP 342987. White Bay, Guana Id., British Virgin Ids., 10.0 x 4.4 mm; FIG. 15: 
Strictispira quadrifasciata, USNM 902242, Jamaica, 9.6 x 3.9 mm; FIG. 16: Crassiclava apicata, 
specimen illustrated by Maes (1983: fig. 15), ANSP 355011, White Bay Guana Id., British Virgin Ids., 
15.8x5.6 mm: FIG. 17: Crassispirella fuscescens, USNM 900978, off Stiltsville, Miami, Florida, 16.9 
X 6.9 mm; FIG. 18; Pyrgospira ostrearum, specimen illustrated by Tryon (1884, pi. 34, fig, 79), ANSP 
15470, Boca Ciega Bay, Florida, 13.4 x 4.9 mm. Scale bar = 10 mm. 



THE GENUS STRICTISPIRA IN THE WESTERN ATLANTIC 



49 



Type Material and Locality 

Holotype, ANSP 247104, Hastings, Barbados, 
T Dranga!, 1950, ex Schwengel colin. Shell 
length 17.7 mm, not 12.5 mm, as stated by 
Schwengel. Measurements: 17.7 x 6.7 x 9.3 
(body whorl length) x 5.8 (aperture length) mm. 

Distribution 

West Florida (site not given), off Miami, Ba- 
hamas, Greater Antilles, St. Thomas, Barbados. 

Material Examined 

ANSP: holotype, 247104, Barbados; 1 spec, 
355567, Grand Bahama Id., 26°38'N, 
78°25'W, J. Worsfold!, ex Worsfold colln.; 1 
spec, 298408, reef, NE of North Point, El- 
bow (Little Guana) Cay, Abaco, Bahama Ids., 
7 ft (2 m), under dead Acropora palmata, R. 
Robertson!, 4 Aug. 1953; 1 spec, 193696, 
off Miami, 27 fms (48 m), rocky, T L. Moise!, 
30 Apr. 1954; 1 spec, 62760, W. Florida, С 
W. Johnson!, 1890; 1 spec, 374474, Grand 
Bahama Id., 26°31'N, 78°46'30"W, J. 
Worsfold!, ex Worsfold colln. 

USNM: 1 spec, 64398, Jamaica; 1 spec, 
102967a, St. Thomas; 1 spec, 411904, 
Ensenada de Cochinos, Cuba, J. В. 
Henderson!; 1 spec, 411908, Cochinos Bay, 
Cuba, rocky shore, J. B. Henderson!; 1 
spec, 900980, Egmont Key, Florida, Gulf of 
Mexico, 45 ft (13.5 m), P. Williams!, 25 May 
1985; 1 spec, 1023063, shoreline NW of 
Thurstone Bay, Abaco, Bahamas, 
26°43'03"N, 77°19'85"W, live collected from 
underside of rock, 0.5 m, 1 July 1997, С 
Redfern!, ex Redfern colln. (last two lots ex 
author's colln.). 

Discussion 

Crassispira drangai was included as a mem- 
ber of the genus Strictispira by Maes on the 
basis of shell morphology, a reasonable loca- 
tion in view of its similarity to Strictispira solida, 
but questionable on the basis of the parietal 
tubercle, which is crassispirine. A preserved 
specimen (USNM 1023063, 15.0 x 6.2 mm), 
that figured by Redfern (2001 ), and the source 
of the protoconch and operculum figures here 
shown, was kindly made available by that au- 
thor. However, although some animal features 
could be discerned, a radula was not retrieved. 
Therefore, the current assignment is tentative, 
based on shell morphology, and definitive ge- 



neric assignment must await anatomical study. 

Shells of S. solida and S. drangai are very 
similar, differing chiefly on the basis of one 
character, the pattern formed by the periph- 
eral spiral cord structure. In S. solida (Fig. 11), 
there is a variable number of regularly spaced 
cords, crossing the ribs as well as between 
them, with no formation of rectangular spaces. 
In S. dranga/ (Fig. 10), the primary spirals are 
fewer, narrower, and more widely spaced, and 
rectangular spaces are produced between 
them and the axials. Three or four fine sec- 
ondary spiral threads are present between the 
primary spiral cords. This formation is absent 
in S. solida. Schwengel noted the fewer spi- 
rals on S. drangai, with finer secondary spiral 
threads in the interspaces. On the shell base, 
there are variably rectangular to square spaces 
formed in both species, this not being a differ- 
entiating feature. All other shell characters are 
variably present in both S. drangai and S. 
solida. Strictispira drangai is generally larger, 
M = 18.2 mm in length for S. drangai, 14.8 
mm for S. solida, and the body whorl/shell 
length ratio is smaller for S. drangai, 46% vs. 
61% for S. solida. Overlapping is present 
though for both measurements. When fresh, 
S. solida has a black shell; S. drangai is very 
dark brown. The lighter intercostal coloring is 
applicable to both species and is not a differ- 
entiating character. 

Crassispirella fuscescens (Reeve, 1843) 
(Fig. 17) is perhaps more likely confused with 
S. drangai, being quite similar to it. It differs in 
having a stubbier shell, with less basal con- 
striction, and a slightly larger body whorl (56% 
vs. 51%). The sulcus is less concave. There 
are more axial ribs with more prominent bead- 
ing on the basal segment. The peripheral 
sculpture pattern is less prominent in C. 
fuscescens because the spirals are closer to- 
gether, but is essentially the same as in S. 
drangai. The axial interspaces are always of 
lighter color in C. fuscescens, although faint 
in some specimens. It may be absent in 
drangai. Kaicher(1984: card 3906) figured the 
illustrated syntype of С fuscescens, a worn, 
faded shell, but the peripheral sculpture is 
evident in this photo, a hand lens being nec- 
essary to see the fine threads. De Jong & 
Coomans (1988: 109, species 582, pi. 43) re- 
port specimens of С fuscescens from 
Curaçao, reaching 24 mm. Their illustration is 
excellent. 

A lot in the ANSP (368728, 13 specimens, 
including 4 preserved, from the Bahamas) that 
had been considered to be S. solida, although 



50 



TIPPETT 



closer to S. drangai. turns out to be a 
Crassispira (Fig. 12); anatomical study of the 
preserved specimens revealed a radula of the 
duplex type similar to that of Crassispira (Kantor 
et al., 1997). The shell has a moderately ex- 
tended canal and a general form similar to 
Crassiclava apicata. There is a vertical groove 
behind the forefront of the parietal tubercle, as 
seen in Crassispira. therefore assignment to 
Crassispira is likely. The shells share the rect- 
angular peripheral sculptural pattern of S. 
drangai and С fuscescens. differentiation be- 
ing based on the shell form and extended ca- 
nal. This form is apparently undescribed. It is 
not further considered here, rather being in- 
cluded for differentiation from the present taxa. 

Etymology 

Named after Mr. Ted Dranga, the discoverer 
of the type specimen. 



Strictispira paxillus (Reeve, 1845) 
Figures 14, 21, 27, 37 

Pleurotoma paxillus Reeve, 1845: pi. 31, spe- 
cies 285, 

Drillia {Crassispira) paxillus (Reeve, 1845) - 
Tryon, 1884: 194, pi. 14, fig. 92 [repetition 
of Reeve's fig.]. 

Crassispira paxillus (Reeve, 1845) - de Jong 
& Coomans, 1988: 109, species 581, de- 
scription and figure. 

Strictispira paxillus (Reeve, 1845) - Maes, 
1983: 318, figs. 10, 21, 29, 43, 47; Redfern, 
2001: 127, species 526, pi. 57; Malacolog, 
2004, list. 

Clathrodrlllia solida (С. В. Adams, 1830 [sic]) 
- RÍOS, 1975: 130, pi., 39, fig. 583 [a 
misidentification, fide Maes, 1983: 318, "The 
Brasilian shell figured is S. paxillus']. 

Pleurotoma nigrescens Reeve, 1845, ex Gray 
MS: pi. 26, species 235. 




FIGS. 19-24. Protoconchs of Strictispira spp. FIG. 19: Strictispira coltrorum, USNM 1011352; FIG. 
20: Strictispira drangai, USNM 1023063; FIG. 21 : Strictipira paxillus, specimen, one of two, juvenile, 
shell 7.1 x 3.3 mm, Redfern colin.; FIG. 22: Strictispira quadriíasciata. USNM 902243; FIG. 23: 
Strictispira redferni. USNM 1010773; FIG. 24: Strictispira solida. USNM 900428. Scale bar = 0.5 mm. 



THE GENUS STRICTISPIRA IN THE WESTERN ATLANTIC 



51 



Pleurotoma jamaicensis Guppy, 1866: 290, pi. 

16, fig. 6. 
Drillia jamaicensis (Guppy) - Pilsbry, 1922: 

320, list and text [synonymized Drillia 

ebenina Dall, 1890]. 

Material Examined 

2 spec, one mature, one juvenile. Chub 
Rocks, Abaco, Bahamas, live collected on 
underside of rocks, 9 m, С Redfern!, 10 Oct., 
1982, Redfern colin. 

ANSP: 1 spec, 342987, White Bay, Guana Id., 
British Virgin Ids., 2-3 m, in drifted sand on 
rocks, V. O. Maes!, 15-28 Feb., 1975 (speci- 
men in Maes, 1983: fig.1); 1 spec, 15317, 
no locality data, ex R. Swift colln.; 3 spec, 
15487, "St. Thomas, W. I. (Krebs)" R. Swift; 1 
spec, 249182, Jack Bay, Anegada, Virgin 
Ids., 0-8 ft (0-2.4 m), sand, stones, coral, Stn. 
770,A. J.&J.C. Ostheimer!, 18 Mr., 1960; 1 
spec, 249316, 0.25 to 2 mi. SE of East Point, 
Anegada, Virgin Ids., 6-20 ft (1 .8-6 m), mostly 
sand, Stn. 774, A. J. & J. С Ostheimer!, 20 
Mr., 1960; 1 spec, 313121, Guantanamo Bay, 
Cuba, outer beaches, R. T & S. Abbott!, May 
1967; 1 spec, 331166, 0.5-1 mi. SSWofThe 
Bluff, Beef Id., British Virgin Ids., 12-14 fms 
(21.6-25.2 m), R. Robertson!, 11 Dec 1973; 
4 spec, 350580, Reef south of Bellamy Cay, 
Trellis Bay, Beef Id., British Virgin Ids., 1-5 
m, R. Robertson & V. O. Maes!, 16-21 Feb. 
1973; 1 spec, 350784, Pointe des Chateaux, 
Grande Terre Id., Guadeloupe, R. A. & V. O. 
Maes!, Feb. 1967; 1 spec, 355363, Enmedio 
Reef, Vera Cruz, Mexico, J. W. Tunell!, 17 June 
1973; 1 spec, 355364, Isla de Lobos Reef, 
Vera Cruz, Mexico, J. W. Tunell!, 9 June 1 973. 

Paleontológica! colin.: 6 spec, ANSP 3773, 
Jamaica, H. Vendryes!, ex Guppy colln. 

USNM: 2 spec, 161147, Mayaguez Harbor, 
Puerto Rico, U.S. Fish Comm.; 1 spec, 
502569, off Falmouth, Antigua, beach. Uni- 
versity of Illinois Exp., J. B. Henderson!, 
1918; 1 spec, 702318, Van Thiel, Curaçao, 
1 ft (3 m), underside of rocks at low tide, ex 
Mrs. D. Meyer colln., 20 Jan. 1981; 2 spec, 

90041 6, Curtain Bluff, Antigua and Barbuda, 
5-15 ft (1.5-4.5 m), Sept. 1981; 2 spec, 

90041 7, Curtain Bluff, Antigua and Barbuda, 
20 ft (6 m), S. Jazwinski!; 2 spec, 900418, 
Samana, Las Galeras, Dominican Republic, 
4-7 ft (1.2-2 m), G. Duffy!, Aug. 1994; 2 
spec, 900419, Cabo Rojo, Bahia Salinas, 
Puerto Rico, 18 ft (5.5 m), night collected, 
G. Duffy!, 18 May 1996 (last four lots ex 
author's colln.). 



Distribution 

Guantanamo, Cuba, east to Dominican Re- 
public Puerto Rico, Virgin Ids., Guadeloupe 
in Leeward Ids.; Mexico, Atlantic coast of Costa 
Rica (Robinson & Montoya, 1987: 391, list); 
Curaçao, Aruba, Bonaire area (de Jong & 
Coomans, 1988: 109); Brazil (Rios, 1975: 130, 
583, pi. 39, as Clathrodrillia solida, fide Maes, 
1983: 318); Colombia (Diaz & Puyana, 1994: 
222, 875, description and fig., as Crassispira 
{Strictispira) paxillus). 

Description 

Shell broadly biconic fusiform, spire angle 
39°, length to approximately 10 mm (reported 
to 1 5 mm by de Jong & Coomans, 1 988: 1 09); 
spire outline slightly concave; body whorl large, 
truncate anteriorly with little basal constriction; 
anterior canal absent. Protoconch (Fig. 21) of 
two smooth whorls, teleoconch approximately 
seven whorls. Whorls slightly rounded below 
sulcus on later whorls. Subsutural sulcus 
ftattish, subsutural cord projecting little, ftnely 
doubled. Sculpture of approximately 20 slightly 
opisthocline axial ribs on body whorl, forming 
a shoulder below sulcus, fading at base, and 
evenly spaced spiral threads between axials, 
becoming stronger and crossing axials with 
beading below shell periphery. Fine spirals and 
curved sinus traces on sulcus. Varix behind 
outer lip. No stromboid notch. Sinus U-shaped, 
deep, with protruding parietal tubercle some- 
what constricting sinus entrance. Color uni- 
formly shiny black to dark brown, with rib 
interspaces same color in fresh shells. 

Animal, according to Maes, with head and 
foot similar to crassispirines, covered with 
sooty blotches, with a muscular foregut, lack- 
ing a poison apparatus, and with characteris- 
tic radular teeth that protrude "from the buccal 
mass-like a pair of ice-tongs". Radular teeth 
(Fig. 37) pistol-shaped, slender for genus, with 
ftange slightly posterior from midpoint. Oper- 
culum (Fig. 27) semitransparent, reddish-or- 
ange, ovoid, with pointed anterior end and 
terminal nucleus. 

Discussion 

Described from an unknown locality, 
Strictispira paxillus was not identified as west- 
ern Atlantic until Maes's work, although Tryon 
had thought that it was in all likelihood a syn- 
onym of the western Atlantic Drillia {Crassi- 
spira) fuscescens (Reeve, 1843). Maes 



52 



TIPPETT 



examined Reeve's NHM paxillus material. On 
the type label, "West Indies" had been written 
in. She recognized it as the same as certain 
western Atlantic specimens, these therefore 
being paxillus. A note with S. paxillus ANSP 
15317 states, "agrees with type BM. V. O. M. 
7/3/68". Identified as "D. {Drillia) paxillus", Maes 
had penciled over this "Crasslspira". showing 
she was not thinking of Strictlspira at that time. 
Her later Guana Id. anatomical material clearly 
identified the species as strictispirid. It is worth 
noting how similar Reeve's excellent illustra- 
tion of the species is to S. paxillus specimens 
in the USNM and ANSP collections, including 
Maes' Guana Id. material. 

Maes (1983: 31 8f) described S. paxillus 
briefiy, figured it, including the shell, proto- 
conch, and a radular section, plus foregut 



anatomy, stomach and male reproductive sys- 
tem, reference to which is here made for de- 
tails. She considered Pleurotoma nigrescens 
Reeve, 1845, and P. jamaicensis Guppy, 1866, 
the latter from the Upper Pliocene of Jamaica, 
as synonyms, these both being high-spired 
forms. Pilsbry (1922) discussed Drillia 
jamaicensis from the Guppy collection at the 
ANSP, and these six specimens were exam- 
ined (Paleo, colin. 3773). They are clearly S. 
paxillus. The illustrated specimen from Maes 
(1983) is shown in Figure 14. Maes pointed 
out that there are a number of species of simi- 
lar general appearance, both within the 
strictispirids, as well as in other families, such 
as Crassispirella fuscescens and Crassiclava 
apicata. Thus, literature records are not reli- 
able unless voucher material is available. 




FIGS. 25-30. Opercula of Strictlspira spp. FIG. 25: Strictlspira coltrorum, USNM 
1011351; FIG. 26: Strictlspira drangai, USNM 1023063; FIG. 27: Strictlspira 
paxillus. as with Fig. 21; Fig. 28: Strictispira quadrlfasciata. USNM 902243; 
FIG. 29: Strictispira redferni, USNM 1010775; FIG. 30: Strictispira solida, USNM 
411922. Scale bar = 1.0 mm. 



THE GENUS STRICTISPIRA IN THE WESTERN ATLANTIC 



53 



Differentiation from other species, as she 
points out, is based on the broad shell, flat 
sulcus, and numerous ribs in paxillus. Addi- 
tionally, the slightly concave spire outline, ab- 
sence or near absence of an anterior canal, 
flat basal profile, and doubled subsutural cord 
are characteristic. The radula readily distin- 
guishes S. paxillus, and other strictispirids, 
from similar species in other genera, such as 
Crassispirella fuscescens (Fig. 17) and 
Crassiclava apicata (Fig. 16). 

Distinguishing shell features include larger 
size for both С fuscescens and C. apicata. 
Crassispirella fuscescens has a sculptural 
pattern on the shell periphery of rectangular 
spaces, as described above with S. drangai, 
stronger beading on the base, and lighter color 
between the axials. Crassiclava apicata has a 
narrower shell with a higher spire, longer an- 
terior canal with stronger basal constriction, 
and axial ribs curving onto preceding sulcus. 
For differentiation from other strictispirids, see 
following. 

Strictispira quadrifasciata (Reeve, 1845) 
Figures 15, 22, 28, 38 

Pleurotoma quadrifasciata Reeve, 1845, pi. 28, 
species 251. 

Drillia (Crassispira) quadrifasciata (Reeve, 
1845) - Tryon, 1884: 195, pi. 14, fig. 82 [re- 
peat of Reeve's fig.]. 

Crassispira quadrifasciata (Reeve, 1845) - 
Kaicher, 1984: card 3896; Leal, 1991: 189, 
pi. 24, fig. G.; Rosenberg, 1992: 105, illus- 
trated; Malacolog, 2004, list. 

Crassispira {Crassispirella) quadrifasciata 
(Reeve, 1845) - Humfrey, 1975: 183, pi. 22, 
fig. 12. 

Material Examined 

1 spec. Curtain Bluff, Antigua, 5-15 ft (1.5- 
4.5 m), Sept. 1981, sacrificed to obtain 
radula. 

USNM: 1 spec, 19046, no locality, U.S. Ex- 
ploring Exp.; 1 spec, 86869, Samana Beach, 
Santo Domingo, 16 fms (29 m), Blake Exp.; 
1 spec, 367064, no locality, ex T L. Casey 
colln.; 1 spec, 502561, Pelican Id., Barba- 
dos, shallow, on coral. Southern University 
of Illinois Exp., 1918; 20 spec, 598487, E 
side Buccoo Reef, Tobago, R. W. Foster!, Apr. 
1951 ; 2 spec, 6821 94, Buccoo Reef, Tobago, 
Smithsonian Bredin (SB!) Exp., Stn. 8, 5Apr. 
1959, 9:30AM-12:30 PM; 25 spec, 682219, 
Buccoo Reef, Tobago, SBI Exp. Stn. 15, 



middle portion of reef, off high ground, dry at 
low tide, 6 Apr. 1959, 7-9 AM; 10 spec, 
682294, Buccoo Reef, Tobago, SBI Exp. Stn. 
26, shallow, 9 Apr. 1959; 1 spec, 682318, 
Buccoo Reef, Tobago; 6 spec, 902240, Cur- 
tain Bluff, Antigua, 5-1 5 ft (1.5-4.5 m), SepL 
1981; 1 spec, 902241, offCat Id., Bahamas, 
3-6 ft (0.5-1 .8 m); 1 spec, 902242, between 
Montega Bay and Tryall, Jamaica, 20-40 ft 
(6-12 m), Dec. 1989; 2 spec, 902243, south 
coast of Dominican Republic, 1-3 m, G. 
Duffy!; 1 spec, 902244, Roatan Id., Hondu- 
ras, 10 ft (3 m), P. Williams!, 1985 (last five 
lots ex author's colln.). 
ANSP: 8 spec. , 1 95808, Buccoo Reef, Tobago, 
label reads "compared with type in BM, V. O. 
M., 4 July 1968"; 1 spec, 240097, off Morro 
de Pto. Moreno, Isla de Margarita, Venezu- 
ela, 4-50 ft (1.2-15 m), W. M. Hellman!, 4 
Feb. 1959, Stn. 21; 1 spec, 291178, 1 mi. N 
of Holetown, Barbados, 3-20 ft (1-6 m), reef 
and sand, R. & V. O. Maes!, Dec. 1963 (fig- 
ured in Encyclopedia of Seashells, G. 
Rosenberg, 1992: 105); 1 spec, 300152, 
Genipabú, Natal, Rio Grande do Norte, Bra- 
zil, dry to 3 ft (to 1 m), sand, rock outcrop, 
grass, G. & M. Kline!, 3 Dec. 1963, Stn. 582; 
2 spec, 313113, outer beaches, Guantanamo 
Bay, Cuba, R. T & S. Abbott!, May 1967; 10 
spec, 1 mi. N of Pointe des Chateaux, 
Guadeloupe, 3-10 ft (1-3 m), weed on coral 
rock, V. O. Maes!, live animal photographed; 
1 spec, 351090, Kralendijk, Bonaire, 
12°09'N, 68°18'W, 25 Feb. 1970. 

Distribution 

Bahamas, Greater Antilles, Lesser Antilles, 
Tobago, Venezuela, Brazil, Honduras. 

Description 

Shell small (to approximately 12 mm), elon- 
gate-biconic, turreted, gradually narrowing 
below periphery with little basal constriction 
to truncate, open, anterior canal. Body whorl 
half shell length. Prominent subsutural cord, 
narrow, concave shoulder sulcus. Protoconch 
(Fig. 22) shiny chestnut colored, low and squat, 
1У2 smooth whorls, followed by Va whorl with 
quickly enlarging axial riblets blending into 
adult sculpture. Teleoconch whorls 5/4-7. 
Numerous (approximately 20 on penultimate 
whorl, 15 to varix on body whorl), rounded, 
narrow, straight, slightly opisthocline axial ribs 
with wider interspaces, extending from bottom 
of sulcus to following suture on spire and, of 



54 



TIPPETT 



decreasing strength below periphery on body 
whorl, to junction with anterior canal. Regu- 
larly spaced spiral cords, 3-4, weak on spire, 
stronger on later whorls. On body whorl a fifth 
below periphery, followed by 2-3 more; on an- 
terior canal, 4-5 more or less "packed ", close- 
set, strong cords, appearing set-off from 
sculpture above. Beads formed on spirals 
crossing ribs. Fine secondary spiral threads 
overall. Aperture ovoid with U-shaped sinus 
at upper end and projecting parietal tubercle 
that may narrow entrance to sinus. Low tooth- 
like swelling may be present below sinus in- 
side outer lip. Varixof one or two enlarged ribs 
behind outer lip. Shallow stromboid notch in 
some specimens. Distinctive color pattern, 
white base, variable chestnut banding, typi- 
cally producing prominent white banding on 
sulcus, on fifth spiral cord region below pe- 
riphery, the anterior canal tip, and on the beads 
on ribs or entire rib white. Some material with 
no white on sulcus or on beads. Variable pat- 
tern down shell base, 

Radular teeth (Fig. 38) pistol-shaped 
marginals, approximately 180 pm, pointed 
anterior end, flange about 1/3 forward from 
spatulate posterior end. Thirty-five pairs of 
teeth on fragmented radula sections on slide. 
Operculum (Fig. 28) amber, roundly ovoid with 
moderately pointed anterior end and terminal 
nucleus. 

Discussion 

Shells rather uniform in appearance, differ- 
ing mainly in color patterning as noted, other- 
wise occasional specimens lack defined 
primary spirals on the shell periphery. It is not 
likely to be confused with any other species. 

Conventionally considered crassispirine, 
availability of a specimen with the soft parts 
permitted radular study showing that S. 
quadrifasciata is strictispirid, the teeth being 
characteristic of the family. Whereas the other 
members of the genus are all somewhat simi- 
lar in appearance, this species has pro- 
nounced color patterning, plus a protoconch 
and an operculum that differs significantly from 
the others - protoconch squat with axial riblets 
terminally, operculum broader with anterior 
end broad and rounded rather than pointed. 
Yet there is no difference in radular tooth struc- 
ture from other strictispirids. I considered pro- 
posing a new genus for this species, but a 
conservative position seems best, assigning 
it to Strictispira pending study of further mate- 
rial. 



Strictispira redferni, new species 

Figures 4-8, 23, 29, 31-35 

Strictispira sp. - Redfern, 2001: 127, species 

528, pi. 57. 
Strictispira acurugata (Dall, 1 890) - Malacolog, 

2004, list. 

Description 

(Based on type material, except shell length, 
which includes all material examined.) Shell 
small (to approximately 17.5 mm), drilliiform, 
turreted, body whorl about 60% shell length, 
anterior canal short, open, unnotched. Color 
light chestnut, fading to medium brown in 
beach specimens (the majority of the mate- 
rial), axial ribs slightly lighter at upper ends. 
Protoconch (Fig. 23) 1У2 smooth whorls with 
partially immersed tip, 6-6% teleoconch whorls 
with moderately strong subsutural cord, which 
is occasionally somewhat darker than rest of 
shell, followed by strongly concave shoulder 
sulcus, shoulder tabulate, axial ribs to follow- 
ing suture forming flat whorl profile. On body 
whorl, after flat peripheral region, ribs curving 
around moderately convex base and end at 
moderately concave junction with canal. Ribs 
blunt posteriorly, rounded, slightly opisthocline, 
of equal width to interspaces, 9-1 3 to varix on 
body whorl, 12-15 on penultimate whorl. Last 
rib or two enlarged forming moderate-sized 
varix % whorl back from thin, curved lip edge. 
Spiral cords rounded, evenly spaced, 5-7 on 
spire whorls, not crossing ribs until below pe- 
riphery, 5-9 across base, and 5-7 strong cords 
on canal. Slightly laterally elongate beads on 
spirals crossing basal axials, becoming stron- 
ger anteriorly. Moderately deep U-shaped si- 
nus on sulcus, apex at mid point, upper edge 
forming slightly projecting parietal tubercle on 
joining body whorl. Sinus tracks present on 
sulcus. Spiral threads 3-5 on sulcus, always 
present but varying from moderately strong to 
faint. Occasional fine spiral lirae extending 
somewhat back into shell below sinus inside 
outer lip. No stromboid notch, except slight 
curvature occasionally on mature specimens. 

Anatomy 

Animal whitish overall or with black mottled 
foot, head, and mantle/siphon complex. Foot 
elongate. Head small, bearing two tentacles 
with eyes distally and laterally. Mantle edge 
behind tentacle bases dorsally, bearing sinus 
indentation on right. Mantle thin, semitrans- 



THE GENUS STRICTISPIRA IN THE WESTERN ATLANTIC 



55 




rstm 



FIG. 31. Strictispira redferni. Semidiagrammatic sagital section of head and 
foregut, from serial section. Shell 10.1 x 3.8 mm, sacrificed: be = buccal cavity, 
cm = columellar muscle, con = circumoral nerve ring, m = mouth, od - 
odontophore, oe = oesophagus, orm - odontophoral/radular retractor muscle, 
p = proboscis, rcoel - rhynchocoel, rstm - rhynchostome, s = buccal septum, 
sg = salivary gland, sgdl = left salivary gland duct, sgdr = right salivary gland 
duct, tm = transverse muscle bundle. Scale bar = 1mm. 



parent, gills and osphradium visible on left and 
penis on right, originating behind and lateral 
to right cephalic tentacle, reflected backwards 
beneath mantle in male. Foregut anatomy (Fig. 
31 ) showing rhynchostome medial, just below 
tentacle bases. Rhynchodeum large, with walls 
compressed longitudinally from retraction, pro- 
ducing strong circular, folding interiorly. Heavy 
longitudinal musculature throughout length, 
continuous with columellar muscle ventrally 
and extending posteriorly in body cavity. Ra- 
dial and circular musculature interspersed in 
rhynchodeal walls, especially anteriorly, but no 
distinct rhynchostomal sphincter. High colum- 
nar rhynchodeal epithelium becoming flat 
cuboidal posteriorly. Moderately sized, mus- 
cular proboscis with strong folding due to re- 
traction and with circular fold around mouth 
opening. Mouth opening into short buccal tube, 
which enlarges rapidly forming buccal cavity 
demarcated posteriorly from opening to oe- 
sophagus by muscular septum. Epithelium of 
proboscis same as rhynchodeal. Massive 
odontophore and radular structure dominat- 
ing body cavity. Radula opening into proximal 
oesophagus, curving from ventrally and right. 
Strong radular membrane with doubled odon- 
tophoral cartilages curve posteriorly through 



body cavity. Radula of approximately 1 20 pairs 
of solid, pistol-shaped, pointed marginal teeth 
with median flange, measuring approximately 
200 pm (Figs. 34, 35). Radular and odonto- 
phoral muscle heavy, extending posteriorly, 
joining with rhynchodeal, proboscis, and col- 
umellar muscle, interspersed with prominent 
transverse muscle bundles. Coiled salivary 
gland composed of single layer of ciliated 
cuboidal cells ventral to anterior odontophore 
and oesophagus, splitting into two ducts, left 
curving around oesophagus and opening into 
oesophagus just posterior to buccal septum. 
Right duct termination not seen due to slide 
defect. Poison gland or bulb absent. Oesopha- 
gus circular initially, becoming flattened due to 
compression between bundles of circumoral 
nerve ring (not shown in figure), lined by single 
layer of ciliated cuboidal cells. Operculum (Fig. 
29) ovate, elongate, with flat columellar side, 
narrowed anteriorly and pointed, with terminal 
nucleus. 

Type Material & Locality 

Holotype, USNM 1010771, lee side of Guana 
Cay, Abaco Id., Bahamas (26°41'50"N, 
77°9'35"W), dredged live, 12 ft (3,6 m), 9 July 



56 



TIPPETT 




FIGS. 32-39, Radular ribbons and teeth of Strictispira spp. FIG. 32: 
Strictispira redfemi, USNM 1010773, slide preparation, light-transmitted, 
ribbon section; FIG, 33: Strictispira redfemi, USNM 1010775, SEM 
preparation, nbbon section; FIG. 34: Strictispira redferni, USNM 1010775, 
SEM preparation, radular teeth, ventral view; FIG. 35: Strictispira redferni, 
ANSP A9421 , Tavernier Key, Florida Keys; FIG. 36: Strictispira coltrorum, 
USNM 1011351; FIG. 37: Sír/cf/sp /ла pax/Z/us, drawing of tooth from Kantor 
& Taylor (1994: fig. 2C, using Maes's material), data as with Fig. 14; FIG. 
38: Strictispira quadhfasciata, Antigua, shell 7.9 x 4.1 mm, sacrificed; 
FIG. 39; Strictispira solida, USNM 411922. Scale bar = approximately 50 
|jm (Fig. 32), 100 |jm (Figs. 33-39). 



1994, С Redfern!; paratypes; 38 spec, 
USNM 1010772, sandbank, lee side Guana 
Cay, Abaco, Bahamas, 9 July 1992, С 
Redfern!, and 11 spec, USNM 1010773, 
spoil bank, Guana Cay, Abaco, Bahamas, 



14 Aug. 1989, С Redfern!; 1 spec, with data 
as per 1010772, at each of the following; 
AMNH.ANSP DMNH, FMNH, LACM, MCZ, 
MNHN, MORG, NHM, NM (material ex 
author's colln.). 



THE GENUS STRICTISPIRA IN THE WESTERN ATLANTIC 



57 



Additional Material Examined 

USNM: 2 spec, 53452, No Name Key, Florida 
Keys, in grass below 2 m, H. Hemphill!; 1 
spec, 27650, Lower Matecumbe Key, Florida, 
H. Hemphill!; 1 spec, 1021270 [ex 272674], 
Newfound Harbor Key, Florida Keys, P. 
Bartsch!; 12 spec, 1021140 [ex 411865], N 
shore Key West, Florida, beach, J. B. 
Henderson!; 3 spec, 1021137 [ex 411870], 
Upper Matecumbe Key, Florida, beach, J. B. 
Henderson!; 2 spec, 411953, Key West, 
Florida, 4.5 fms (8 m), J. B. Henderson!; 1 
spec, 412158, Tortugas, Florida, 16 fms (29 
m), J. B. Henderson!; 1 spec, 668097, off 
Dog Id., Florida, Gulf of Mexico, near Clear- 
water, 4-6 fms (7-11 m), in Astropecten 
articúlala stomach, Oct. 1962, G. Radwin!; 2 
spec, 601681, Jamaica. (USNM specimens 
were separated from large suite of Pyrgospira 
ostrearum specimens.) 

ANSP, as Strictispira acurugata: approximately 
400 spec, 221823, Boot Key Harbor, Vaca 
Key, Florida Keys, B. R. Bales!, Jan. -March 
1945, ex Schwengel colin, (originally identi- 
fied as Crassispira tampaensis): approxi- 
mately 75 spec, 313080 (ex 221702), 
Bonefish Key, Florida Keys, B. R. Bales!, ex 
Schwengel colin.; 1 spec, 314456, 0.5 mi. 
SE of Burnt Point, Crawl Key, Florida Keys, 
in sand pockets among weed and rock, 2-4 
ft (0.5-1.25 m), V. O. Maes!, 27 April 1968 
(originally identified as Crassispira sp.); 1 
spec, 31 3084 (ex 264988), Boca Ciega Bay, 
near St. Petersburg, Florida, ex J. D. Parker 
colln.; 3 spec, 368733, Hotel, W end Grand 
Bahama Id., Bahama Ids., 26°42'15"N, 
78°59'50"W, J. Worsfold!, ex Worfold colln.; 
1 spec, 368499, McLean Town, Grand 
Bahama Id., Bahama Ids., 26°38'45"N, 
77°57'30"W, 3 ft (1 m), J. Worsfold!, ex 
Worsfold colln.; 1 spec, 355797, Wardwick 
Wells Key, Exuma, Bahama Ids., 24°22'N, 
76°36'W, intertidal sand, D. Cosman!, ex 
Cosman colln.; 5 spec. White Sound, Elbow 
Cay, Great Abaco Id., Bahama Ids., 26°32'N, 
76°58'W,W.G. Lyons!, 1972, ex Lyons colln.; 
1 spec. 355798, Whale Cay, Abaco Id., 
Bahama Ids., 26°43'N, 77°14'W, D. Cosman!, 
Aug. 1979, ex Cosman colln.; 1 spec, 
329768, Bimini Lagoon, near Bailey Town, 
Bimini Ids., R. Robertson!, 1957-58; 6 spec, 
370553, North Hawksville Creek, Bahama 
Ids., 26°32'N, 78°45'W, 1-3 ft (0.3-1 m), J. 
Worsfold!, ex Worsfold colln.; 2 spec, 
374473, Grand Bahama Id., Bahama Ids., J. 
Worsfold!, ex Worsfold colln.; 4 spec, alco- 



hol presePk/ed, A9421 , between Tavernier Key 
and channel to Tavernier Creek, Florida Keys, 
25°2'N, 80°30'W, on Thalassia, 18 June 
1971, ex Florida Marine Research Lab. 
Drillia acurugata examined: USNM: holotype, 
97320, Caloosahatchee Riv., Florida; 1 spec, 
113153, Shell Creek, Florida; 1 spec, un- 
numbered, rock pit 3.5 mi. W of La Belle, 
Florida, N side of Caloosahatchee Riv. 
Author's colln: 1 spec, Caloosahatchee Riv. 

Distribution 

Lower west coast of Florida, Florida Keys to 
Tortugas, Bahamas, Bimini, Jamaica. 

Discussion 

Although a common, even abundant, spe- 
cies judging by its frequency at Abaco and its 
having been collected at other, rather widely 
separated sites, often in large numbers, 
Strictispira redferni has not been recognized 
as a separate species, generally being identi- 
fied as small specimens of Pyrgospira 
ostrearum (Stearns, 1872), or as Strictispira 
acurugata. Strictispira redfern/ differs from P. 
ostrearum firstly by the shell of P. ostrearum 
(Fig. 18) having no parietal tubercle (although 
old specimens may have an accumulation of 
gerontic callus at this site) or varix, secondly 
by P. ostrearum being more strongly beaded, 
the spirals crossing more numerous and nar- 
rower ribs, being larger, taller, narrower, and 
by a beaded subsutural cord. However, im- 
mature specimens of S. redferni lacking a varix 
and parietal tubercle can be difficult to differ- 
entiate, although the ribs are usually wider and 
lack beading in redferni. Pyrgospira tampa- 
ensls (Bartsch & Rehder, 1939: 136, pi. 17, 
figs. 5, 1 3), which I consider to be a form of P. 
ostrearum, differs from P. ostrearum mainly in 
fewer axials, and subdued beading. It inter- 
grades with P. ostrearum. 

Maes segregated 12 lots of shells and one 
lot of alcohol-preserved specimens in the 
ANSP under the name Strictispira acu- 
rugata (Dall, 1890), and this was subse- 
quently carried in Malacolog under that 
name. She apparently considered them living 
representatives of the Florida Pliocene fossil, 
and strictispirids on the basis of shell morphol- 
ogy. As her identifications have circulated, col- 
lectors have identified specimens as S. 
acurugata. Examination shows that these are 
not that species, but rather S. redferni, includ- 
ing a large form of that species. As seen in 



58 



TIPPETT 



Figure 9, true S. acurugata from the Upper 
Pliocene/Lower Plesitocene is larger, has a 
nearly flat, broad shoulder sulcus, spirals that 
are flat, wide bands separated by grooves 
rather than rounded cords, and there is no 
varix or parietal tubercle. There is a distinct 
stromboid notch, and the subsutural cord is 
weak, hugs the suture, and undulates with the 
previous ribs. The Recent "S. acurugata" 
specimens do not share these features but 
correspond with S. redferni. some being iden- 
tical to the type material and of the same size, 
others larger, reaching 13-14 mm in length. A 
few (Fig. 8) resemble S. acurugata superfi- 
cially. A large form (Fig. 7, see below) is nar- 
row and reaches 17.5 mm. However, there is 
complete intergrading of forms. It is noted that 
Maes considered them all to be the same spe- 
cies. 

It is worth noting that the generic position of 
the fossil species, assigned to Drillia by Dall, 
is in fact uncertain, appearing on the basis of 
the available material to more likely be of a 
group, such as the subfamily Cochlespirinae, 
which lacks a varix or an elaborated sinus at 
maturity. The genus Pyrgospira is a likely as- 
signment. 

Review of Recent ANSP material segregated 
as "S. acurugata" permits its being divided into 
two groups. The first consists of two lots with 
many specimens, 221823 and 313080. ANSP 
221823 is composed of shells of rather uni- 
form morphology (Fig. 7), mature specimens 
being larger (largest specimen 15.7 mm, yet 
an 11.5 mm specimen is still juvenile) than the 
type series of S. redferni. They are narrower, 
have a shallowly concave sulcus, less pro- 
nounced ribs with a tendency to intercalary 
axial ribs or enlarged growth markings on the 
body whorl. They are considered a variety of 
S. redferni. In this lot, and to a greater degree 
in ANSP 313080, there is intergrading with the 
type series. ANSP 313080 contains a number 
of these large forms, one of 17.5 mm, plus 
others of sizes to that of the type lot, all show- 
ing intergrading with the types of S. redferni. 

Maes separated a number of specimens in 
good condition from each of the two lots as 
representative. Random selection of a num- 
ber of specimens from these forms group 1. 
The second group consists of a number of lots 
showing a full range of intergrading between 
the first group and the type series, a number 
of the shells being identical to the type lot. The 
second group is combined with additional 
USNM material to form a transition grouping 
from the type lot to the large variety. These 



groups plus Pyrgospira acuruguta specimens 
were examined for possibly significant shell 
morphology differences, as show in Table 1. 

Although the statistics for the uncommon P. 
acurugata are of limited reliability due to the 
low N and the fact that two, perhaps the third 
also, of the four specimens are immature, thus 
skewing shell measurements, nevertheless the 
findings tend to substantiate the differentiation 
of S. redferni and Pyrgospira? acurugata. 
Pyrgospira acurugata is larger, with a lower 
body length/shell length ratio (the 21 mm ho- 
lotype is larger than the mean, 55%, but still 
smaller than S. redferni), more axials usually, 
fewer spirals. Qualitative rather than quanti- 
tative features are more important in differen- 
tiating these taxa, the differentiating features 
being noted above. (The number and charac- 
ter of axials is the same on the early whorls 
as on the mature whorls, and this is applicable 
to all taxa noted here.) 

With regards the species generally, S, 
redferni shows a weakly defined sinus struc- 
ture for the genus in that the parietal tubercle 
does not protrude markedly so as to narrow 
the sinus opening as seen in other species of 
the genus. However, occasional specimens of 
the large varietal group have more extended 
parietal tubercle roofs. 

Maes (1983) and Kantor & Taylor (1994), 
who restudied Maes's material, including se- 
rial sections, described and discussed the fo- 
regut anatomy of Strictispira paxillus. 
Strictispira redferni can be compared with their 
findings. The two species are basically the 
same, their major features agreeing - absence 
of poison apparatus, large odontophore with 
corollary large retractor muscles, same radu- 
lar tooth structure and salivary duct structure. 
Different is the presence of a buccal cavity 
area, followed by a septum, separating it from 
the oesophagus in S. redferni, as opposed to 
the large proboscis and essentially absent 
buccal tube and cavity in S. paxillus, in which 
the odontophore and radular ribbon occupy the 
entirety of the proboscis. In the serial-sec- 
tioned specimen of S. redferni, the radular 
structure curves from below the oesophagus, 
ending posterior to the buccal region behind 
the septum at the beginning of the oesopha- 
gus. However, in a dissected Abaco specimen, 
the radula was positioned at the proboscis 
mouth. It must be assumed that the arrange- 
ment in the specimen of S. redferni that was 
serial sectioned represents a further retracted 
state than that in the described specimen of 
S. paxillus, rather than an anatomical differ- 



THE GENUS STRICTISPIRA IN THE WESTERN ATLANTIC 



59 



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60 



TIPPETT 



ence. The alimentary musculature of these 
animals is obviously very powerful. The short- 
ened proboscis in S. redferni. in contrast to 
the larger organ noted in S. paxillus, suggests 
heightened retraction. 

Radular studies of S. redferni show findings 
very similar to S. paxillus. The large, paired 
odontophore, robust ribbon with strong mem- 
brane are equivalent. Teeth are the same, 
those of S. redferni (Figs. 34, 35), showing 
only minor differences from S. paxillus (Fig. 
37), as seen in Kantor & Taylor (1994), that of 
S. paxillus being more slender. 

Of interest is the seeming discrepancy be- 
tween SEM and light-transmitted images of the 
teeth. As seen in Figure 32, light transmitted 
slide preparations show the flange strongly, 
giving the impression that it wraps around the 
shaft, "collar-like". However, the SEM prepa- 
ration (Fig. 33) shows the flange simply pro- 
truding slightly from underneath the shaft 
(arrow). Thus, the flange is shown to attach 
on the lower/ventral side of the tooth. McLean 
recognized this, as indicated by his comment 
that the projecting collar-like structure was on 
the inside (ventral side) of the tooth (1971b: 
729). The attachment is sturdy, and extends 
from the tooth base to the flange. The de- 
pressed region on the underside of the tooth 
at the bend might be noted (also seen by 
Kantor & Taylor, 1994; fig. 2C). It appears to 
result from the pressure of the adjacent tooth's 
flange. 

Etymology 

The species is named for Mr. Colin Redfern, 
who collected the type material, and has been 
both generous and extremely helpful in assist- 
ing the author in this work. 

Stnctispira solida (С. В. Adams, 1850) 
Figures 11, 24, 30, 39 

Pleurotoma solida С. В. Adams, 1850: 61: 
Clench & Turner, 1 950: 342, pi. 29, fig. 8 [lec- 
totype designated]. 

Strictispira solida (С. В. Adams, 1 850) - Maes, 
1983: 320, text with Strictispira paxillus; 
Kaicher 1984: card 3917; Redfern, 2001: 
species 527, pi. 57; Malacolog, 2004, list. 

Crassispira {Crassispirella) fuscescens 
(Reeve, 1843) -Abbott, 1958: 94 [list and 
description plus text; synonyms: Pleurotoma 
solida С. В. Adams, 1850, Drillia ebenina 
Dall, 1890]. 



Not Dnilia ebenina Dall, 1890: 33, pi. 2, fig. 8; 
Abbott, 1974: 270, species 2997 [reprint of 
Dall's 1890 figure], as a synonym of "Drillia 
(Clathrodrillia) solida"; Malacolog, 2004, list, 
as synonym of S. solida. 

Not "Clathrodrillia solida" (С. В. Adams, 1830 
[sic]) - Ríos, 1975: 130, pi., 39, fig. 583 [a 
misidentification, fide Maes, 1983: 318, "The 
Brasilian shell figured is S. paxillus"]. 

Not Dnilia solida (С. В. Adams, 1 850) - Bändel, 
1984: 166, fig. 309, pi. 20, fig. 8. 

7Clathrodnllia solida С. В. Adams, 1830 [sic] 
-Ríos, 1985: 136, species 621, pi. 46 [Dall's 
figure of еЬеп/па]; Rios, 1994; 159, species 
712, pi. 53 [uncertain whether this is S, solida 
or not]. 

Description 

Shell broadly biconic, fusiform, spire angle 
37°, length approximately 19 mm, body large, 
somewhat truncate anteriorly, little basal con- 
striction. Protoconch (Fig. 24) two smooth 
whorls, teleoconch approximately eight whorls. 
Sulcus narrow, concave, bearing fine spiral 
striae and curved sinus traces, preceded by a 
strong, sharply crested subsutural cord some- 
what distant from suture. Whorl outline flatfish 
below sulcus. Body whorl riding up variably 
on preceding whorl terminally. Sculpture of 
approximately 18 narrow axial ribs extending 
slightly onto preceding sulcus, producing a 
shoulder of variable strength, with wider 
interspaces, disappearing on base, and 7-16 
regularly spaced spiral threads between axials, 
more prominent and wider spaced below shell 
periphery, producing some weak beading on 
crossing the axials. Enlarged axial or two form- 
ing a varix behind outer lip. Aperture parallel- 
sided, ending in short, open, slightly notched 
anterior canal bent slightly right. Lip broken 
back, usually healed, just following varix in 
about half of the specimens. Weak stromboid 
notch. Sinus deep, U-shaped, with parietal 
tubercle projecting as flat roof-like structure 
nearly closing opening. Color shiny black when 
fresh. 

Animal with conventional structures exter- 
nally - foot, head and siphon grayish-amber, 
mottled with sooty black. Tentacles with eyes 
placed laterally halfway to tips. Rhynchostome 
below and midway between tentacles. 
Rhynchocoel large, muscular walls folded 
transversely and irregularly, large proboscis 
folded on itself. Body cavity dominated by large 
radular ribbon. No poison apparatus. Section 



THE GENUS STRICTISPIRA IN THE WESTERN ATLANTIC 



61 



of ribbon has approximately 80 pairs of mar- 
ginal teeth. Teeth (Fig. 39) approximately 190 
|jm, pistol-shaped, with flange near tooth base, 
"pistol grip" short. Operculum (Fig. 30) ovoid, 
with pointed anterior end and terminal nucleus. 

Material Examined 

Lectotype, MCZ 186005, Jamaica. 

USNM: 2 spec, 27644, Lower Matecumbe 
Key, H. Hemphill!, identified as "ebeninä\ 
"type" penciled in on label (see Dall's com- 
ment concerning these specimens under 
Discussion below); 1 spec, 95943, Abrolhos 
Ids., off east Brazil; 4 spec, 102967, St. 
Thomas; 1 spec, 130465, Antilles, ex Lea 
colln.; 1 spec, 214978, St. Thomas, ex 
Carnegie Institute colln.; 1 spec, 366729, 
Jamaica?, Vendryes!, ex Orcutt colln.; 1 
spec, 383177, Jeremie, Haiti, Orcutt colln.; 
1 spec, 411903, Key West, 2 fms (3.5 m), J. 
B. Henderson! (identified as Drillia ebenina); 
1 spec, 411910, Tortugas, 16 fms (29 m), J. 
B. Henderson!, Eolis Stn. 33, 1911 (identi- 
fied as Drillia ebenina); 1 spec, 411911, 
Tortugas, 15 fms (27 m), J. B. Henderson!, 
Eolis Stn. 34, 1911; 1 spec, 411913a, off 
Miami, 10fms(18m), J. B. Henderson!, Eolis 
Stn. 70, 1913; 1 spec, 411914, Key West, 
J. B. Henderson!, Eolis Stn. 73 (identified as 
Drillia ebenina); 1 spec, 411915, off Gov- 
ernment Cut, Miami, Florida, 3 fms (5.5 m), 
J. B. Henderson!, Eolis Stn. 83, 1913; 1 
spec, 411918, Santa Lucia, Cuba, 2-4 fms 
(3.5-7 m). Barrera Exp., Stn. 200; 1 spec, 
411920, Cabanas Harbor, 25 fms (45 m). 
Barrera Exp., Stn. 202; 1 spec, 411921, 
Cabanas Harbor, Cuba, 3-12 fms (5.5-21.5 
m). Barrera Exp., Stn. 203: 5 spec, 411922, 
Santa Rosa, Cuba, 3-6 fms (5.5-11 m), 
Barrera Exp., Stn. 209; 10 spec, 411923, 
Esperanza, Cuba, 2-3 fms (3.5-5.5 m). 
Barrera Exp., Stn. 210; 1 spec, 411924, 
Cape San Antonio, Cuba, Barrera Exp., Stn. 
224; 1 spec, 843357, off west Florida 
(Naples), 26°03'ir'N, 82°27'27"W, 17 m. 
Continental Shelf Associates for MMS/BLM, 
scuba, 1 June 1983; 1 spec, 900421, Pea- 
nut Id., Lake Worth, Florida, 3 May 1969; 1 
spec, 900422, SW of Key West, 114 fms 
(205 m), R. Black!, 1975; 2 spec. Finger 
Channels, off Stiltsville, Miami, Florida, 2-3 
ft (0.5-1 m); 1 spec, 900424, W side of 
Fleming Id., Key West, Florida, 16 ft (4.8 m), 
22 Sept. 1995; 1 spec, 900425, E side 
Marquesas Keys, Florida Keys, 12 ft (3.5 m), 
scuba at night, 12 July 1991; 1 spec. 



900426, Tourmaline Reef, Mayaguez, Puerto 
Rico, 40 ft (12 m), 10 March 1993; 1 spec, 

900427, Isla Morro, Pelotas, Venezuela, 24 
ft (7 m); 1 spec, 900428, Cayo Levisa, 
Oriente, Cuba, 15 ft (4.5 m), scuba at night, 
7 Aug., 1995; 1 spec, 900429, Isla Coche, 
Venezuela, 50 ft (15 m), scuba at night, 16 
July 1993; 3 spec, 1004124, W side Fleming 
Id., Key West, Florida, 20 ft (6 m), scuba at 
night, 20 Dec, 1995; 3 spec, 1004125, 
Tambor Cay, Atlantic Panama, 40 ft (12 m), 
scuba at night, 1 1 Oct. 1 992 (last ten lots ex 
author's colln.) 

ANSP: 1 spec, 84478, St. Johns, Antigua, 
Silas L. Schumo!, 1903; 1 spec, 194117, 
off Garden Cove, Key Largo, Florida, 3 fms 
(5.4 m), T L. Moise!; 1 spec, 198968, NW 
of Water Pt., North Sound, Grand Cayman 
Id., A. J. Ostheimer 3^^!, Stn. D31; 1 spec, 
232571, off Palm Beach, Florida, J. S. 
Schwengel!, 24April 1940; 1 spec, 281650, 
SE end of McBride Cay, Belize, Stn. 1 06, R. 
Robertson!, 25 Aug. 1961; 1 spec, 282214, 
mouth of Monkey Riv., Belize, 12 ft (3.5 m), 
coarse quartz sand, 16°21'45"N, 
88°29'00"W, R. Robertson!, 21 Aug. 1961; 
4 spec, 284033, oft^ mouth of Mullins Riv., 
Belize, Stn. 62, R. Robertson!, 1-2 Aug. 
1961; 1 spec, 313036, outer beaches, 
Guantanamo Bay, Cuba, R. T. & S. Abbott!, 
May 1 947; 1 spec, 31 3083 (ex 221 702, split 
from lot of Crassispira cubana), Bonefish 
Key, Florida, J. S. Schwengel!; 1 spec, 
320964, St. Thomas, W. I., R. Swift!; 1 spec, 
337481, Key West, Florida, С L. Richard- 
son!; 1 spec, 368352, Tamarind, Grand 
Bahama Id., 26°30'45"N, 78°36'01"W, J. 
Worsfold!, ex Worsfold colln.; 2 spec, 
368588, Settlement Pt., W end. Grand 
Bahama Id., 1 ft (0.3 m), live, at night, J. 
Worsfold!; 13 spec, 368728, hotel, W end. 
Grand Bahama Id., 2-4 ft (0.5-1 .2 m), live, 
on sand and rocks, at night, J. Worsfold!; 4 
spec, 374475, Grand Bahama Id., J. 
Worsfold! 

Drillia ebenina examined: USNM: figured 
syntype , 97318, plus 10 further syntypes of 
same lot, one larger than figured specimen, 
Caloosahatchee Riv., Florida, Pliocene; 3 
spec, 23983, Caloosahatchee Riv., 
Pliocene; 5 (of 9) spec, 1 1 31 50, Shell Creek, 
Florida, Pliocene; ANSP: large batch, 18058, 
N. St. Petersburg, Florida, Pliocene, W. G 
Fargo!, 8 Oct. 1946, ex Fargo colln.; 1 spec, 
58371, no locality, 21 Mr, 1984. 

Author's colln.: 2 spec, Pinecrest beds, 
Sarasota, Florida, Middle Pliocene. 



62 



TIPPETT 



Distribution 

Palm Beach to Miami, Florida, to Florida 
Keys and Tortugas: off Naples, west Florida, 
Florida Bay (Tabb & Manning, 1961: 581. list, 
as Crassispira ebenina): Bahamas, Cuba, 
Grand Cayman, Jamaica. Puerto Rico, St. 
Thomas, Antigua: Belize, Colombia (Diaz & 
Puyana, 1994: 222, species 875, description 
and fig., as Crassispira (Strictispira) cf. solida, 
and Diaz, 1994: 40, list, as Strictispira solida), 
Venezuela, Brazil. 

Discussion 

Crassispira ebenina has been confused with 
S. solida for many years. It is probable that 
literature records of Recent specimens of the 
fossil C. ebenina are in all likelihood S. solida, 
and that position is adopted here. Drillia 
ebenina was described by Dal! from the Up- 
per Pliocene-Lower Pleistocene of Florida, and 
was considered by him as Recent also. He 
noted it found in shallow water in the Florida 
Keys by Hemphill, and gave it a distribution of 
Gulf of Mexico from Florida to Vera Cruz. Ex- 
cept for reporting one specimen from Puerto 
Rico (Dali & Simpson, 1901 : 387). Dall did not 
mention S. solida. Other authors (e.g., Mazyck, 
1913: 8: Abbott, 1954: 268: Tabb & Manning, 
1961: 581) continued this identification, con- 
sidering D. ebenina as Recent, listing it from 
S. Carolina, E. Florida, the West Indies. For 
whatever reason. S. solida was not consid- 
ered a valid or important species. It was listed 
only (Krebs, 1864: 12: Simpson, 1887: 54), or 
considered a synonym of Crassispirella 
fuscescens (Reeve, 1843) (Tryon, 1884: 193: 
Abbott, 1958: 94: Warmke & Abbott, 1962: 
134), Finally, Abbott (1974: 270) considered 
S. solida a valid species, nevertheless con- 
sidering С ebenina a synonym. Abbott's 1958 
misidentification of S. solida as С fuscescens 
is based upon ANSP 1 98968 from Grand Cay- 
man Island, Abbott included a slip with the shell 
stating, [it] "matches solida CBA OK". Maes 
indicated this was written approximately 1 957. 
The shell is S. solida, and was determined as 
that by Maes (5 Oct. 1977). It measures 14.6 
X 6.2 mm, and is a typical specimen. It ap- 
pears Abbott recognized the shell as S. solida, 
but through some error reported it as S. 
fuscescens in the publication. Comparison of 
S. solida and С ebenina (Figs. 11, 13) shows 
that they are not conspecific. Although similar 
in appearance, the sinus of C. ebenina is not 
strictispirid but crassispirine, probably a mem- 
ber of the genus Glossispira, at least on the 



basis of the sinus and parietal tubercle struc- 
ture (McLean, 1971a: 121: 1971b: 720) (see 
previous comment about conventional crassi- 
spirid subgenera). The shell of Glossispira 
ebenina is broader (although there are nar- 
rower forms, otherwise identical), there are 
more axial ribs, the spirals are more robust, 
and the subsutural cord is somewhat weaker, 
slightly rounded, and weakly beaded. 

For separation of S. solida from С fusces- 
cens, see features for С fuscescens noted 
above with S. drangai and S. paxillus. Addi- 
tionally, С fuscescens, has a non-crested 
subsutural cord. Separation from S. drangai 
is also discussed above. 

Strictispira solida may be differentiated from 
S. paxillus by its larger size, narrowly concave 
sulcus, fewer and more robust, orthocline axial 
ribs, and stronger, sharply crested subsutural 
cord a bit distant from the suture. A common 
species, Pleurotoma solida was assigned to the 
genus Strictispira by Maes (1983: 320) in the 
text with Pleurotoma paxillus, which she had 
discovered to be strictispirid by virtue of its radu- 
lar teeth, although the radula of S. solida was 
not mentioned. However, her identification card 
includes a photograph of a radular slide prepa- 
ration of a specimen of S. solida showing pairs 
of marginal teeth of strictispirid form (ANSP 
282214). The radular study shown here con- 
firms this assignment, the radular teeth being 
typical of the genus. The basal segment is 
slightly less flexed and a bit shorter than typi- 
cal of the genus. This is seen in Maes's slide 
figure also. This is not to the degree seen in 
the eastern Pacific sister genus Cleospira, as 
illustrated in McLean (1971a: fig. 88), wherein 
the flexing is still less and the flange less promi- 
nent. Bandel's figures of what is purported to 
be S. solida do not conform to the present find- 
ings, but rather show teeth very much like those 
of Cleospira. At this time, no representatives of 
Cleospira are known in the western Atlantic. 
Bändel does not figure or describe the shell(s) 
identified as S. solida, consequently in view of 
his radular findings, it is possible that there is a 
form of the genus Cleospira in this region. 



CONCLUSIONS 

The discovery of the existence of five or six 
species of Strictispira in the western Atlantic 
demonstrates that the genus is more common 
than realized, and more morphologically di- 
verse. It is likely that further members of the 
group will be recognized upon availability and 
study of the animals. 



THE GENUS STRICTISPIRA IN THE WESTERN ATLANTIC 



63 



The strictispirid radular structure findings 
further suggest, as stated by Taylor et al. 
(1993), that the feeding mechanism of the 
strictispirids involves rasping and tearing of 
prey by a protruding radula. In the present 
species, the radula can be protruded through 
the buccal cavity to the anterior proboscis, as 
with S. paxillus (Maes, 1983: 320; Kantor & 
Taylor, 1994: 343), thereby obtaining access 
to the prey. The radula could serve as a grasp- 
ing organ, assisting in propelling food to the 
buccal cavity and oesophagus by the teeth 
splaying out after crossing the bending plane 
and then coming together, like Maes's "ice- 
tongs" metaphor, in a grasp of the prey on re- 
traction. No food remnants were present in 
examined specimens. 

The protoconch structure indicates direct 
development, consequently there would prob- 
ably be no planktonic dispersion. This suggests 
that there is higher likelihood of different forms 
having developed from common ancestors. 

It is evident that correct systematic assign- 
ment of crassispirine-like taxa requires knowl- 
edge of the animal, especially radular 
information. Correct generic location of S. 
quadrifasciata, S. redfemi, and S. coltrorum 
would not have been suspected without the 
radula. There is no specific shell morphology 
that signifies the genus Strictispira, although 
the members do usually share a drilliiform shell 
with a strictspirid sinus structure. Until the 
radula is known, generic location can be as- 
signed only on a tentative basis. 

There is little available information concern- 
ing habitat, shallow, rocky areas with sand and 
occasionally vegetation being the reported 
features. Usually of shallow water, S. solida 
was dredged at 200 m. 



ACKNOWLEDGMENTS 

The author thanks the Department of Sys- 
tematic Biology, Smithsonian Institution for the 
opportunity of working with the mollusc col- 
lection and use of the museum's equipment. 
Similarly, the ANSP provided the opportunity 
to visit that institution and research parts of 
the paper, for which the author is very appre- 
ciative. Dr. Gary Rosenberg of the ANSP was 
most gracious and helpful in reviewing the 
paper and making highly valuable suggestions. 
Paul Callomon was especially helpful in work- 
ing on the ANSP collection. Dr. Thomas R. 
Waller and Warren Blow provided access to 
the NMNH Cenozoic mollusk collection and 
assisted in researching that material. Dr. Yuri 



Kantor kindly read the paper and made help- 
ful suggestions. Dr. Eugene Coan expertly 
edited the paper and made valuable recom- 
mendations. Colin Redfern supplied the ma- 
terial for Strictispira redferni and S. paxillus, 
José and Marcus Coltro that for Strictispira 
coltrorum. Yolanda Villacampa prepared the 
SEM illustrations, and Beth Fricano the serial 
section. Finally, Dr. Jerry Harasewych of the 
USNM was most kind with his assistance and 
support through the course of the work. The 
author is grateful to these individuals. 



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Revised ms. accepted 11 November 2004 



MALACOLOGIA, 2006, 48(1-2): 65-76 

GERM CELL DIFFERENTIATION AND SEXUAL MATURATION OF THE 

FEMALE NEPTÚNEA {BARBITONIA) ARTHRITICA CUMINGII (CROSSE, 1862) 

(GASTROPODA: BUCCINIDAE) 

Ее- Yung Chung\ Sung Yeon Kim^, Gab-Man Park^ & Jong Man Yoon"* 

ABSTRACT 

Oogenesis, the gonadosomatic index (GSI), reproductive cycle, and first sexual matura- 
tion of the female Neptúnea (Barbitonia) arthritica cumingii have been investigated by light 
and electron microscope observations. In the early vitellogenic oocyte, the Golgi complex 
and mitochondria were involved in the formation of glycogen, lipid droplets, and yolk gran- 
ules. In late vitellogenic oocytes, the rough endoplasmic reticulum and multivesicular bod- 
ies were involved in the formation of proteid yolk granules in the cytoplasm. In particular, 
compared with the results of other gastropods, it differs in that appearances of cortical 
granules at the cortical layer and microvilli on the vitelline envelope, which is associated 
with heterosynthetic vitellogenesis, were not observed in vitellogenic oocytes during oo- 
genesis. A mature yolk granule was composed of three components: main body (central 
core), superficial layer, and the limiting membrane. Monthly changes in the gonadosomatic 
index in females studied in 2002 and 2003 were closely associated with ovarian develop- 
mental phases. Spawning occurred between May and August in 2002 and 2003, and the 
main spawning occurred between June and July, when the seawater temperature rose to 
approximately 18-23°C. The female reproductive cycle can be classified into five succes- 
sive stages: early active stage (September to October), late active stage (November to 
February), ripe stage (February to June), partially spawned stage (May to August), and 
recovery stage (June to August). The rate of individuals reaching the first sexual maturity 
was 53.1% in femalesof 51.0 to 60.9 mm in shell height, and 100% in those over 61.0 mm. 

Key words: Neptúnea (Barbitonia) arthritica cumingii, oogenesis, germ cell differentia- 
tion, sexual maturation. 



INTRODUCTION 

Neptúnea arthritica cumingii {Crosse, 1862) 
is one of the most important edible gastropods 
in such East Asian countries as Korea, Japan, 
China, and Russia (Yoo, 1976; Kwon et al., 
1993). This species is especially found in silty 
sand of the subtidal zone of the west coast of 
Korea. Recently, as the standing stock of this 
species gradually decreased due to extensive 
reclamation projects and reckless over- 
harvesting, it has been designated as one of 
the important organisms in need of resource 
management. 

There have been some studies on Neptúnea 
species on aspects of reproduction, including 
the reproductive cycle (Takahashi et al., 1972; 
Takamaru & Fuji, 1981; Fujinaga, 1985; Kawai 
et al., 1994), and spawning (Miyawaki, 1953; 



Amio, 1963; Son, 2003), on aspects of ecol- 
ogy including distribution (Ito & Tachizawa, 
1981; Ito, 1982; Kwon et al., 1993), growth 
(Macintosh & Paul, 1977; Fujinaga, 1987; 
Suzuki et al., 1996) of N. arthritica, and feed- 
ing (Pearce & Thorson, 1967) of N. antigua. 
There has been one study on the spawning 
season of N. cumigii in the East China Sea 
(Amio, 1963). But, there are still gaps in our 
knowledge of its reproductive biology. So far, 
little information is available on ultrastructural 
study on germ cell differentiation and sexual 
maturation of N. arthritica cumingii in the Ko- 
rean waters and the Japan Sea (Chung & Kim, 
1996). However, there is some information on 
ultrastructural study of oogenesis in other gas- 
tropods (McCann-Coillier, 1977, 1979; Griffond 
& Gemot, 1979; Griffond, 1980; Hodgson & 
Eckelbarger, 2000; Pal & Hodgson, 2002). 



'School of Marine Life Science, Kunsan National University, Kunsan 573-701, Korea; eychung@kunsan.ac.kr 
^National Fisheries Development Institute, Busan 690-902, Korea 

^Department of Parasitology, Kwandong University College of Medicine, Gangnung 210-710, Korea 
■"Department of Aquatic Life Medicine, Kunsan National University, Kunsan 573-701, Korea 



65 



66 



CHUNG ETAL. 



Therefore, the results of ultrastructural stud- 
ies on germ cell differentiation of this species 
and other gastropods provides important infor- 
mation on the reproductive mechanisms. The 
reproductive cycles of the local populations in 
marine gastropods vary with such environmen- 
tal factors as water temperature and food avail- 
ability (Chung et al.. 2002). Understanding the 
reproductive cycle and the spawning period of 
N. arthritica cumingii will provide necessary 
information for natural spat collections or the 
recruitment period and age determination of 
this population. In addition, data on first sexual 
maturity and reproductive strategy of this popu- 
lation are very useful information for natural 
resource management. The main aim of the 
present study is to understand germ cell dif- 
ferentiation during oogenesis, the reproductive 
cycle and first sexual maturity of this species. 



MATERIALSAND METHODS 

Sampling 

Specimens of Neptúnea arthritica cumingii 
(Crosse, 1862) were collected monthly at the 
subtidal zone of Maldo, Kunsan, Korea, from 



January to December 2002 (Fig. 1). Snails 
ranging from 41 .0 to 106.8 mm in shell height 
were used for the present study. After the snails 
were transported alive to the laboratory, shell 
heights were immediately measured. 

Gonadosomatic Index (GSI) 

A total of 486 individuals were used for cal- 
culation of the GSI. Monthly changes in the 
mean gonadosomatic index (GSI) were cal- 
culated by the following equation (Chung et 
al., 2002) (Fig. 2): 



GSI = 



Thickness of the gonad x 100 
Diameter of posterior appendage in- 
cluding the gonad and digestive gland 



Germ Cell Differentiation by Electron Micro- 
scopic Observation 

For electron microscopical observations, 
excised pieces of the gonads were cut into 
small pieces and immediately fixed in 2.5% 
paraformaldehyde-glutaraldehyde in 0.1 M 
phosphate buffer (pH 7.4) for 2 h at 4°C. After 
initial fixation, the specimens were washed 
several times with the same buffer and then 



3600'N 



35°48"N 




Ci 



Gogunsangundo 



Sampling Area 



o^<Q 



<;::0 





O-^ oS^ 



126"12'E 126°24"E 

FIG. 1. Map showing the sampling area. 



126°36'E 



OOGENESIS AND SEXUAL MATURATION OF NEPTÚNEA 



67 




FIG. 2. Anatomy of Neptúnea arthritica cumingii, removed 
from its shell. Posterior appendage showing the ovary and 
digestive gland. X, Y and Z denote the sections for measure- 
ment of GSI. Three sections are spaced equally. Abbrevia- 
tions: DG, digestive gland; OV, ovary; ST, stomach; SC, stom- 
achal caecum. 



further fixed in 1% osmium tetroxide dissolved 
in 0.2 M phosphate buffer solution (pH 7.4) for 
1 h at 4°C. Specimens were then dehydrated 
in a series of increasing concentrations of etha- 
nol, cleared in propylene oxide, and embed- 
ded in Epon-Araldite mixture. Ultrathin sections 
of Epon-embedded specimens were cut with 
glass knives with a Sorvall MT-2 microtome and 
an LKB ultramicrotome at a thickness of about 
800-1,000 A. Tissue sections were mounted 
on collodion-coated copper grids, stained with 
uranyl acetate followed by lead citrate, and 
examined with a JEM 100 CX-2 (80 kV) elec- 
tron microscope. 

Gonadal Development by Histological Obser- 
vations 

For light microscopic examination of histo- 
logical preparations, a total of 456 individuals 
were used for histological analysis of the go- 
nads from January to December 2002. Gonad 
tissues were removed from shells and pre- 
served in Bouin's fixative for 24 h and then 
washed with running tap water for 24 h. Tis- 
sues were then dehydrated in alcohol and 
embedded in paraffin molds. Embedded tis- 
sues were sectioned at 5-7 pm thickness us- 
ing a rotary microtome. Sections were mounted 
on glass slides, stained with Hansen's hema- 



toxylin-0.5% eosin, Mallory's triple stain and 
PAS stain, and examined using a light micro- 
scope. 

First Sexual Maturity 

The first sexual maturation of a total of 187 
female individuals (31.4-90.5 mm in shell 
height) were investigated histologically in or- 
der to determine the shell heights of snails 
reaching maturation and participating in repro- 
duction from May (ripe stage) to late August 
(after spawning). 



RESULTS 

Position and Morphology of the Gonads 

Neptúnea arthritica cumingii is a dioecious 
species composed of well-defined female and 
male individuals. The ovary is located on the 
surface of the digestive gland in the spiral pos- 
terior region of the shell (Fig. 2). The ovary is 
composed of numerous oogenic follicles. As 
the ovary matured, it extended to the outer part 
of the digestive gland. As maturation pro- 
gresses, the sex of the snail can be distinguish- 
able easily by color: the ovary being pale 
yellow and testis yellowish-brown. At this time, 



68 



CHUNG ETAL. 




J FMAMJ JASONDJ FMAMJ JASOND 
2002 2003 

MONTH 



FIG. 3. Monthly changes in the gonadosomatic index of female 
Neptúnea arthritica cumingii, for two years from January 2002 
to December 2003. 



if it was slightly scratched with a razor, ripe 
eggs readily discharged from the ovary. But 
after spawning, the ovary degenerated, and it 
became difficult to distinguish their sexes by 
external color or dissection. 

Monthly Changes in the Gonadosomatic In- 
dex (GSI) 

Monthly GSI changes in females were 
showed in Figure 3. In 2002, the GSI slowly 
increased from September and reached the 
maximum (mean 3.11) in April when seawater 
temperature rapidly increased. The GSI rap- 
idly decreased after May, and the values 
reached the minimum in August, when spawn- 
ing was completely finished. Monthly changes 
in the GSI in 2003 showed similar patterns with 
those in 2002. 

Germ Cell Differentiation in the Ovary by Elec- 
tron Microscopic Observations 

Ultrastructural observations allow the germ 
cell developmental phases during oogenesis 
can be divided into 4 phases: (1) oogonial 
phase, (2) previtellogenic phase, (3) vitello- 
genic phase, and (4) mature phase. Charac- 
teristic features in each stage were as follows; 

Oogonial Phase: Oogenia in the oogonial 
phase, which propagated on the germinal epi- 
thelium (follicular wall), were oval and 15 pm 
in diameter. They commonly were single or 
formed a cluster on the germinal epithelium. 
Each oogonium had a large nucleus with chro- 
matin, several mitochondria, and the endo- 



plasmic reticulum, vacuoles in the cytoplasm 
(Fig. 4A). 

Previtellogenic Phase: Previtellogenic oo- 
cytes were 25-90 pm in diameter. With cyto- 
plasmic growth, several small mitochondria, 
a well-developed endoplasmic reticulum and 
several vacuoles were concentrated around 
the nucleus in the cytoplasm of the previtello- 
genic oocyte. The number of Golgi complexes, 
scattered from the perinuclear region to the 
cortical region of the oocyte, increased. At this 
time, many vacuoles formed by the Golgi com- 
plex appeared around the endoplasmic reticu- 
lum, several mitochondria, and large vesicles 
were present in the cytoplasm of the previtello- 
genic oocyte (Fig. 4B). 

Vitellogenic Phase: In the early vitellogenic 
oocyte, especially, well-developed endoplas- 
mic reticulum and vacuoles in the cytoplasm 
were concentrated around the nucleus hav- 
ing nucleoli. At this time, the follicle cell, which 
lied adjacent to the early vitellogenic oocyte, 
had an elongated nucleus. In particular, elec- 
tron-dense granules and several lipid droplets 
were accumulated in the cytoplasm of the fol- 
licle cell (Fig. 4C). With the initiation of yolk 
formation, lipid droplets were accumulated in 
the vacuoles formed by the Golgi complex in 
the perinuclear region. Lipid droplets diffused 
toward the cortical layer, and then glycogen 
particles appeared around the mitochondria 
at the cortical region of early vitellogenic oo- 
cytes (Fig. 4D). At this time, after electron- 
dense materials were accumulated in the Golgi 
complex (Golgi sac, Golgi vacuoles, and Golgi 
vesicles), lipid droplets were formed by secre- 



OOGENESIS AND SEXUAL MATURATION OF NEPTÚNEA 



69 




1. 


^•'' 


..>3 


» «ft < 


•I 





GS 



-, fr . ... • _ 



M 



LD 



•I ♦* 

i<> f/Ê Ö*^ 



ER 



m 



FIG. 4. Electron micrographs of the previtellogenic and early vitellogenic phases during oogenesis of 
Neptúnea arthhtica cumingii (A-F), A: Oogenia in the oogonial phase, with a large nucleus and 
several mitochondria in the cytoplasm; B: A previtellogenic oocyte, with a large nucleus with a few 
nucleolus and several mitochondria, the Golgi complex, and vacuoles in the cytoplasm; C: An early 
vitellogenic oocyte attached to a follicle cell, with a large nucleus containing chromatin and a number 
of vacuoles and well-developed endoplasmic reticulum in the cytoplasm; D; An early vitellogenic 
oocytes, with well-developed Golgi complex, glycogen particles and lipid droplets, E: An early 
vitellogenic oocyte, with lipid droplets formed by secretions in vacuoles and vesicles; F: An early 
vitellogenic oocyte, with a lipid droplet surrounded by the endoplasmic reticulum and the mitochon- 
dria. Abbreviations; CR, chromatin; ER, endoplasmic reticulum; G, Golgi complex; GS, Golgi sac; 
GVa, Golgi vacuole; GVe. Golgi vesicle; LD, lipid droplet; M, mitochondrion; N, nucleus; NO, Nucleo- 
lus; NU, nucleolus; 00, oocyte; OG, oogonium; ER, Endoplasmic reticulum; Va, vacuole; Ve, vesicle. 



70 



CHUNG ETAL. 



tion of electron-dense materials in the large 
vacuoles and small vesicles, which were 
formed by the Golgi vacuoles and Golgi 
vesicles (Fig. 4E). On the other hand, relatively 
large lipid droplet was surrounded by the en- 
doplasmic reticulum, the mitochondria and 
glycogen particles in the cytoplasm of the early 
vitellogenic oocyte (Fig. 4F). In the late vitello- 
genic oocyte, lots of yolk granules appeared 
between the rough endoplasmic reticulum and 
the mitochondria at the cortical layer in the 
cytoplasm (Fig. 5A). At this time, the 
multivesicular bodies, which were formed by 
the modified cristae of the mitochondria, ap- 



peared near the nuclear envelope of the 
nucleus in the late vitellogenic oocyte. Yolk 
precursors, such as glycogen particles, lipid 
droplets, yolk granules and multivesicular bod- 
ies, were accumulated in the cytoplasm (Fig. 
5B). Eventually, proteid yolk granules were 
formed by yolk granules and multivesicular 
bodies (Fig. 5C). 

Mature Phase: Mature oocytes were about 
180-250 X 300-450 pm in diameter. In the 
mature oocyte, various sizes of proteid yolk 
granules were intermingled with small lipid yolk 
granules, and it became a small mature yolk 
granule (Fig. 5C). Relatively small mature yolk 








FIG. 5. Electron micrographs of late vitellogenic and mature phases during oogenesis of Neptúnea 
arthritica cumingii (A-D). A: A late vitellogenic oocyte, with yolk granules between the rough 
endoplasmic reticulum and the the mitochondria; B: A late vitellogenic oocyte, with a number of 
multivesicular bodies formed by modified mitochondria; C; A late vitellogenic oocyte, with proteid yolk 
granules formed by yolk granules and multivesicular bodies; D; A mature oocyte, with a mature yolk 
granule being composed of the main body (central core), superficial layer and a limiting membrane of 
a yolk granule. Abbreviations: LD, lipid droplet; LM, limiting membrane; LYG, lipid yolk granule; M, 
mitochondrion; MBy, main body; MM. modified mitochondrion; MYG, mature yolk granule; MV, 
multivesicular body; N, nucleus; NE, nuclear envelope; NU, nucleolus; PYG, proteid yolk granule; 
rER, rough endoplasmic reticulum; SL, superficial layer. 



OOGENESIS AND SEXUAL MATURATION OF NEPTÚNEA 



71 




s Early active stage 
П Partially spawned 



A S О N D J F 
2003 

MONTH 

И Late active stage 
Ш Recovery stage 



N D 



I Ripe stage 



FIG. 6. Frequency of gonadal phases of Neptúnea arthhtica cumingii and the 
mean water temperatures, for two years, from January 2002 to December 2003. 



granules were continuously mixed with each 
other and became large mature yolk granules 
in the cytoplasm. A fully mature yolk granule 
is composed of three components: (1) main 
body, (2) superficial layer, and (3) a limiting 
membrane (Fig. 5D). 

Reproductive Cycle with the Gonad Develop- 
mental Stage 

Based on the morphological features and 
sizes of germ cells and the tissue cells around 
them, the reproductive cycle with gonadal 
phases can be classified into five stages in 
females. Especially, the reproductive cycle and 
monthly changes in water temperatures 
showed similar patterns in 2002 and 2003 (Fig. 
6). The criteria in defining of each stage are 
as follows: 

Early Active Stage: The gonadal volume was 
small, and the follicles occupied approximately 
25% of the gonad. The follicular walls were 
relatively thick. Oogonia and previtellogenic 
oocytes propagated along the oogenic follicu- 
lar walls and mesenchymal tissues of the ovary. 
The oogonia and previtellogenic oocytes are 



about 15-25 pm in size, respectively. At this 
time, early vitellogenic oocytes of 25-50 pm in 
diameter formed an egg-stalk attached to the 
walls (Fig. 7A). The individuals in the early 
active stage were found from September to 
October when seawater temperatures were 
gradually decreasing. 

Late Active Stage: This stage is character- 
ized by the presence of developing early 
vitellogenic oocytes. Follicular walls (germinal 
epithelium) were thin. A number of early 
vitellogenic oocytes of 100-140 pm in diam- 
eter were attached to the follicular walls 
through each egg-stalk. With the initiation of 
yolk formation, there were numerous yolk 
granules in the cytoplasm of late vitellogenic 
oocytes of 150-200 x 250-300 pm in diam- 
eter. Some fully mature oocytes were free in 
the lumen of the follicle (Fig. 7B, C). The indi- 
viduals in the late active stage appeared from 
November to February. 

Ripe Stage: In females, the majority of oo- 
cytes grew to 160-180 pm in diameter, occu- 
pied over 70% of the gonad, and follicular walls 
became very thin. Mature oocytes growing up 
to 180-250 X 300-450 pm in diameter became 



72 



CHUNG ETAL. 



tetragonal or polygonal in shape, and con- 
tained a number of mature yolk granules (Fig. 
7D). Mature or ripe ovaries were found in Feb- 
ruary through June, when seawater tempera- 
tures gradually increased. 



Partially Spawned Stage: Since about 50- 
70% of the oocytes in the follicles were dis- 
charged, the lumen of the follicles emptied. 
Spawned ovaries were characterized by the 
presence of a few undischarged vitellogenic 








FIG. 7. Photomicrographs of the gonadal phases of female Neptúnea arthritica cumingil. A: Transverse 
section of oogenic follicles in the early active stage; ВС; Section of follicles in the late active stage; D; 
Section of ripe oocytes in the ripe stage; E: Section of follicles in the partially spawned stage; F; 
Section of the follicles in the recovery stage. Scale bars - 50 pm. Abbreviations; LG, lipid granule; 
LM, lumen; MT, mesenchymal tissue; N, nucleus; NO, nucleolus; ОС, oocyte; OG, oogonium; V, 
vacuole; YG, yolk granule. 



OOGENESIS AND SEXUAL MATURATION OF NEPTÚNEA 



73 



TABLE 1. The shell height and first sexual maturity of ferлale Neptúnea 
(Barbitonia) arthhtica cumingii from May to August, 2002. 



Shell height 






Gonadal developmental stage 




(mm) 


EA 


LA 


Rl 


PS 


RE 


Total 


Mature (%) 


31.4-40.9 


34 










34 


0.0 


41.0-50.9 


26 


2 


2 






30 


13.3 


51.0-60.9 


15 


2 


10 


5 




32 


53.1 


61.0-70.9 




3 


21 


6 




30 


100.0 


71.0-80.9 




2 


22 


9 




33 


100.0 


81.0-90.5 






16 


12 




28 


100.0 


Total 












187 


100.0 



Abbreviations: EA, early active stage; LA, late active stage; Rl, ripe stage; PS, partially 
spawned stage; RE, recovery stage. 



oocytes, as well as prevltellogenic oocytes in 
the follicles (Fig. 7E). The individuals in this 
stage appeared from May to August, and the 
main spawning occurred between June and 
July when the seawater temperature rose to 
approximately 16-23°C. 

Recovery Stage: After spawning, the undis- 
charged vitellogenic oocytes in the lumen of 
the follicle undergo cytolysis, and each fol- 
licle was contracted, and then degeneration 
or resorption of undischarged vitellogenic or 
mature oocytes occurred. Thereafter, the re- 
arrangement of newly formed connective tis- 
sues, a few oogenia and prevltellogenic 
oocytes appeared on the newly formed folli- 
cular walls (Fig. 7F). The individuals in the re- 
covery stage appeared from June to August. 

First Sexual Maturity 

Before and after spawning, a total of 187 
female individuals (31.4-40.9 mm in shell 
height) were histologically examined to certify 
whether they reached maturity and partici- 
pated in reproduction. The rate of shells of dif- 
ferent sizes that reached first sexual maturity 
is summarized in Table 1. The breeding sea- 
son of this species was from May to August. 
In the case of some individuals with gonad 
developmental stage in the late active stage 
in May through August, it is supposed that they 
reach maturity, except for individuals in the 
early active stage during the breeding season. 
First sexual maturity was 0% in female snails 
of 31 .4-40.9 mm in shell height if they were at 
the early active stage during the breeding sea- 
son. 

The percentage of first sexual maturity of the 
female snail of 41 .0 to 50.9 mm in shell height 



was 13.3%. The percentages of first sexual 
maturity of the female individuals of 51.0 to 
60.9 cm in shell height were over 50%, all of 
which were at the late active, ripe or partially 
spawned stages. First sexual maturity was 
1 00% for snails over 61 .0 mm in height. 



DISCUSSION 

Germ Cell Development and Vitellogenesis 

As vitellogenesis commences, nuclei of the 
oocytes increased in size. Early vitellogenesis 
is characterized by proliferation of endoplas- 
mic reticulum and mitochondria, both of which 
are closely associated with lipid droplets. Ac- 
cording to our electron microscope observa- 
tions of early vitellogenic oocytes of N. 
arthritica cumingii, the Golgi apparatus is 
thought to be involved in a number of vacu- 
oles and small vesicles in the perinuclear re- 
gion in the cytoplasm, with carbohydrate 
(glycogen) particles filling the vacuoles. Lipid 
droplets and lipid yolk granules are then added 
to the vacuoles and vesicles formed by the 
Golgi complex (referred as autosynthetic by 
Taylor & Anderson, 1969), as in llyanassa 
obso/ete (Taylors Anderson, 1969), Biompha- 
laria glabrata (deJong-Brink et al., 1976), 
Mytilus edulis (Reverberi, 1971), Rapana 
venosa (Chung et al., 2002), Sipiíonaña 
capensis (Pal & Hodgson, 2002), Patella 
barbara, P. argenvillei, P. granulans, P. ocu- 
lus, P. miniata, and Halcion pectunculus 
(Hodgson & Eckelbarger, 2000). This study 
suggests that the Golgi complex and various 
sizes of vacuoles are involved in the forma- 
tion of lipid droplets in the early vitellogenic 



74 



CHUNG ETAL. 



oocytes. From our observations of oogenesis, 
it is assumed that the mitochondria and the 
endoplasmic reticulum near lipid droplets are 
involved in the formation of lipid droplets in 
the early vitellogenic oocyte. However, we did 
not find pinocytotic tubules, which are thought 
to be involved in yolk production as seen in 
the vitellogenic oocytes of Agriolimax reticu- 
lars (Hill & Bowen, 1976; Dohmen, 1983). In 
the late vitellogenic oocyte, we also did not 
observe microvilli on the vitelline envelope, 
which is thought to be involved in helping in 
absorption, transportation and secretion of egg 
envelopes (Norrevang, 1968), as seen in 
Mactra chinensis (Chung, 1997), M. veneri- 
formis (Chung & Ryou, 2000), and SIphonaria 
serriata (Pal & Hodgson, 2002). 

Formation of cortical granules is a prominent 
feature of late vitellogenic oocytes in most 
bivalves, such as Mactra chinensis (Chung, 
1997) and M. veneriformis (Chung & Ryou, 
2000). Regarding formation of cortical gran- 
ules during oogenesis, Hodgson & Eckel- 
barger (2000) described that Golgi complexes 
appeared predominately in the cortical region 
of the ooplasm and secrete electrone-dense, 
cortical granule-like organelles in the 
vitellogenic oocytes of Patella barbara, and 
they stated that Golgi complexes synthesize 
cortical granules. In the present study, how- 
ever, such structures were not observed in the 
vitellogenic oocytes, as in lllana obsoleta (Tay- 
lor & Anderson, 1969) and Rapana venosa 
(Chung et al.. 2002). Compared with Patella 
barbara, the lack of these structures is a promi- 
nent characteristic dunng oogenesis, repre- 
senting a significant difference in N. arthritica 
cumingii. In the present study, proteid yolk 
granules, which appeared near the rough en- 
doplasmic reticulum and modified mitochon- 
drial structure (multivesicular bodies), as seen 
in Hypselodoris tricolor and Godiva banyulen- 
sis (Medina et al., 1986), were observed at 
the cortical region of the cytoplasm. Accord- 



ingly, it is assumed that the endoplasmic reticu- 
lum and multivesicular bodies are involved in 
the formation of proteid yolk granules (Taylor 
& Anderson. 1969) as yolk precursor. In the 
present study, although the follicle cell, which 
lies adjacent vitellogenic oocyte, contains elec- 
tron-dense granules and lipid droplets, we 
could not observe clear evidence of secretion 
into the vitellogenic oocyte. Therefore, it is as- 
sumed that N. arthritica cumingii synthesize 
yolk autosynthetically, as in the majority of gas- 
tropods, exceptions being some gastropods 
{Planorbarius corneus, Lymnaea stagnalis, 
Hypselodoris tricolor. Godiva banyulensis. 
SIphonaria capensis. and S. serrata) that syn- 
thesize yolk autosynthetically and hetero- 
synthetically(Bottkeetal., 1982; Medina et al., 
1986; Pal & Hodgson, 2002). 

Gonadal Development and Maturation 

We observed that gametogenesis of N. 
arthritica cumingii begins at a temperature of 
about 3°C, with maximum gonadal maturation 
occurring in April 2002 and 2003, when water 
temperatures rose (Fig. 7) and phytoplank- 
ton was very abundant. Periods of high food 
abundance and gonad development were 
nearly coincident. In Korean coastal waters, 
growth and production of Meretrix lusorla and 
Ruditapes philippinarum are very high in the 
spring and early summer seasons (Kim et al., 
1977; Chung et al., 1994; Lee, 1995) due to 
the abundant phytoplankton that occurs with 
increasing water temperatures. Ruditapes 
philippinarum, Meretrix lusoria, and other 
clams are commonly used as food by N. 
arthritica cumingii. At this time, abundant food 
can be supplied to N. arthritica cumingii dur- 
ing the period of gonadal development and 
maturation. Therefore, it is suggested that 
gonadal development and maturation of N. 
arthritica cumingii is closely related to water 
temperature and food availability. 



TABLE 2. Comparisons of the spawning season of Buccinidae in each locality. 



Species 



Spawning season 



Locality 



Author 



Neptúnea arthritica cumingii May-August 

N. cumingii July-August 

N. arthritica May-June 

N. arthritica May-August 

N. constricta December 

Siphonalia assidariaeformis December 



Kunsan, Korea 

East China Sea, China 

Usu Bay, Hokkaido, Japan 

Saroma, Hokkaido, Japan 

East Sea, Korea 

East China Sea, China 



Present study 
Amio, 1963 
Fujinaga, 1985 
Kawaietal., 1994 
Son, 2003 
Habe, 1960 



OOGENESIS AND SEXUAL MATURATION OF NEPTÚNEA 



75 



Breeding Pattern 

As shown in Table 2, our histological obser- 
vations show that spawning of N. arthritica 
cumingli on the west coast of Korea occurs 
from late May to August in 2002 and 2003 
when sea water temperatures were high. The 
spawning season of N. cumingi collected by 
the trawl net in the East China Sea occurs 
between July and August (Amio, 1 963). Neptú- 
nea arthritica in Japan has been reported to 
spawn once a year between May and June in 
Usu Bay, Japan (Fujinaga, 1985). 

Therefore, it is assumed that the spawning 
period of N. arthritica cumingii on the west 
coast of Korea occurred somewhat earlier than 
that in the East China Sea. On the whole, N. 
arthritica cumingii in Korea is a summer 
breeder, based on the criteria outlined by 
Boolootian et al. (1962) for marine mollusks. 
In general, it is assumed that spawning of N. 
arthritica cumingii and N. arthritica in Korea 
and Japan occurs between May and August. 
However, spawning of N. constricta and 
Siphonalia assidariaeformis (Buccinidae) oc- 
curs during December, these species being 
winter breeders (Table 2). Therefore, the slight 
discrepancy in the spawning period between 
these studies might be related to geographic 
differences in water temperature and food 
availability (Chung et al., 2002). 

First Sexual Maturity with the Gonad Devel- 
opmental Stage 

From the result of histological observations, 
we found that although the specimens were 
collected during the breeding season, the go- 
nadal development of smaller individuals rang- 
ing from 31 .4 to 40.9 mm in shell height were 
in the early active stage as small number of 
oogenia and the previtellogenic oocytes were 
present in the follicle of the ovary. Judging from 
histological observations, it is supposed that 
the size of the oocyte could not have reached 
maturity until late August, when spawning 
ended. Snails of 51.0-60.9 mm high were in 
the late active, ripe and partially spawned 
stages, and more than 50% reached first 
sexual maturity. However, all snails in the late 
active, ripe, or partially spawned stages 
spawned if they were larger 61.0 mm. This 
means that larger individuals can reach matu- 
rity earlier than smaller individuals. In the as- 
pect of natural resources management, the 
present study suggests that because harvest- 
ing snails < 51.0 mm can potentially cause a 



drastic reduction in recruitment, a prohibitory 
measure should be taken for adequate re- 
source management. Henceforth, age deter- 
mination by size of the individuals should be 
investigated in detail for natural resources 
management of this species. 



ACKNOWLEDGEMENTS 

The authors are grateful to Dr. John B. Burch 
of the University of Michigan for helpful com- 
ments on the manuscript. This research was 
supported in part by the fund (2003) from the 
Coastal Research Center, Kunsan National 
University. 

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Revised ms. accepted 1 November 2004 



MALACOLOGIA, 2006, 48(1-2): 77-132 

REVISION OF THE GENUS ISLAMIA RADOMAN, 1973 (GASTROPODA, 

CAENOGASTROPODA, HYDROBIIDAE), ON THE IBERIAN PENINSULA AND 

DESCRIPTION OF TWO NEW GENERA AND THREE NEW SPECIES 

Beatriz Arconada & Maria-Angeles Ramos 

Museo Nacional de Ciencias Naturales (CSIC) 
José Gutiérrez Abascal 2, 28006 - Madrid. Spain: тспа31 3@mncn.csic.es 

ABSTRACT 

The presence of the genus Islamia Radoman, 1973, on the Iberian Peninsula is con- 
firmed based on the detailed study of a group of species, of which three were previously 
included in the genus Neohoratia Schutt, 1961. These species are most abundant in the 
south-southeastern Mediterranean region but also inhabit the northern Mediterranean ar- 
eas of the peninsula, with scattered populations in central and western Spain. Iberian 
Islamia currently includes /. globulus (Bofill, 1909), /. lagan (Altimira, 1960), and /. ateni 
(Boeters, 1969), plus two new species, /. pallida and /. henrici, the latter with two subspe- 
cies /. h. henrici and /. h. giennensis. Two new genera are also described, Milesiana and 
Josefus, each of which contains one species; M. schuelei (Boeters, 1981), which was 
previously assigned to Neohoratia, and most recently to Islamia, and a new species, Josefus 
aitanica, respectively. Histological study of the female genitalia confirmed the presence of 
two seminal receptacles and the absence of a bursa copulatrix in all species belonging to 
the three genera. In Islamia, the distal receptacle was once considered to be a reduced 
bursa copulatrix. We also confirm that there is no trace of glandular tissue on the penial 
lobe in any of the Islamia species for which histological evidence is available. 

Key words: Caenogastropoda, Hydrobiidae, Neohoratia, Islamia, Milesiana, Josefus, 
taxonomy, Spain, Iberian Peninsula. 



INTRODUCTION 

The European fauna of hydrobiids is par- 
ticularly rich in valvatiform species. However, 
their morphological study is challenging be- 
cause of their minute size. Many new genera 
and species have been described on the ba- 
sis of shell features, which are known to be 
highly convergent. Sometimes other anatomi- 
cal characters, which are frequently non-di- 
agnostic, are used in these descriptions. Data 
on character variability are absent or very 
rare. The result has been a much confused 
taxonomic picture that was recently reviewed 
and partially clarified by Bodon et al. (2001 ), 
who redescribed the type species of most of 
the European valvatiform genera based on 
new anatomical studies and data in the lit- 
erature. 

Preliminary studies on Iberian Peninsular 
valvatiform hydrobiids (Ramos et al., 1992, 
1 995; Arconada et al., 1 996) have shown con- 
siderable morphological diversity and high 



endemicity. Boeters (1988) recognized that 
species of two genera, Horatia Bourguignat, 
1887, and /\/eo/7orai/'a Schutt, 1961, inhabited 
this geographical area. An in-depth taxonomic 
review of the two genera is currently un- 
ravelling a very complex situation. Four new 
genera and several new species have been 
described in recent papers (Ramos et al., 
2000; Arconada & Ramos, 2001 , 2002). Some 
of the species in the new genera were previ- 
ously included in the above-mentioned gen- 
era. We continue these studies by revising the 
taxonomy of another group of species previ- 
ously assigned to Neohoratia by Boeters 
(1988). 

It has been difficult to distinguish the spe- 
cies of the genera Neohoratia Schutt 1961, 
and Islamia Radoman, 1973, given their mor- 
phological similarities (Bodon & Giovanelli, 
1994; Bodon et al., 1995; Manganelli et al., 
1998). The type species of Neohoratia is 
Valvata (?) subpiscinalis Kuscer 1932 (Figs. 
1-5, paratypes from the Biological Institute, 



77 



78 



ARCONADA & RAMOS 



Scientific Research Centre of Ljubljana. N 
1862, leg. Dr. J. Bole). This genus has under- 
gone several changes in its taxonomic status. 
It has been regarded as a subgenus of 
Hauffenia Pollonera, 1898, and of Horatia 
Bourguignat, 1887 (Schutt, 1961; Boeters. 
1974: Bodon & Giovanelli. 1994), and as a 
distinct genus (Bole & Velkovrh, 1 986; Boeters, 
1988; Bole. 1993). /\/eo/?oraf/a is characterised 
by having a rather short, flat, blunt or slightly 
pointed penis with 1-3 small, knob-like lateral 
lobes on its left side near the apex. The fe- 
male genitalia include a pin-like bursa copu- 
latrix and one proximal, small seminal 
receptacle (Bole, 1993; Bodon et al., 2001). 
Boeters (1988) and Boeters & Rolan (1988) 
overlooked these diagnostic characters while 
including several species from the Iberian 
Peninsula in this genus {Amnícola globulus 
Bofill, 1909; Microna ateni Boeters, 1969; 



Valvata coronado! Bourguignat, 1870; 
Hauffenia {Neohoratia) coronadol schuelei 
Boeters, 1981; \/a/uafa(rrop/d/>?a) fez/ Altimira, 
1960; Hauffenia {Neohoratia) gasulli Boeters 
1981; and Neohoratia azarum Boeters & 
Rolan, 1988). However, according to Boeters 
(1988), these Iberian species, apart from hav- 
ing a narrowing ('Einschnürung') of the outer 
side of the female oviduct glands (capsule + 
albumen glands), lacked a bursa copulatrix 
and had a renal oviduct with two seminal re- 
ceptacles. This combination of characters, in 
addition to a male genitalia with a penis usu- 
ally having one glandular lobe on its left side, 
has been described as typical of the genus 
Islamla (Bodon et al., 1995; Bodon et al., 
2001). Islamla is attributed to a wide geo- 
graphical distribution in the Mediterranean 
area [species are claimed to be from; Turkey 
(Schutt, 1964; Radoman, 1973b); the Balkanic 




FIGS. 1-5. Shell of Valvata subpisclnalis (IBCICL paratype n° 1862). 



REVISION OF THE GENUS ISLAMIA 



79 



Peninsula (Radoman, 1973a, b, 1978, 1983); 
Italy (Giusti & Pezzoli, 1981; Bodon et al., 
1 995, 1 996, 2001 ; Bodon & Cianfanelli, 2002); 
Israel (Schutt, 1991; Bodon et al., 1995); 
Greece (Radoman, 1973b, 1978); and France 
(cited as Hauffenia Pollonera, 1898) (Ber- 
nasconi, 1984)]. 

It was thus feasible that the species listed 
above from the Iberian Peninsula could be 
attributed to the genus Islamia (type species 
Hydrobia valvataeformis Möllendorff, 1873) or 
even to new genera. In fact, two of them, 
Hauffenia {Neohoratia) gasulli [N. (?) gasulli, 
sensu Boeters, 1988] and Valvata {Tropidina) 
fezi [N. (?) fezi, sensu Boeters, 1988] were 
recently allocated to two new genera, Tarra- 
conia Ramos & Arconada, 2000 (in Ramos et 
a!., 2000), and Spai/?ogyna Arconada & Ramos, 
2002, respectively. 

Here we describe three new species and 
redescribe the morphological characters (in- 
cluding previously unknown characters) of the 
above-mentioned species using a multi- 
disciplinary approach based on type speci- 
mens and a vast amount of recently collected 
material. Additionally, histological studies of 
these species provide evidence that the two 
sac-like structures on the renal oviduct are 
seminal receptacles and demonstrate the non- 
glandular nature of the penial lobe. 

We conclude that two of the "Neohoratia" 
species {sensu Boeters, 1988) from the Ibe- 
rian Peninsula {Amnícola globulus and 
Microna ateni) actually belong to the genus 
Islamia, as hypothesized by Bodon et al. 
(2001). Two other species, one of them with 
two subspecies, are described as new and 
placed into Islamia. Another species, Hauffenia 
{Neohoratia) coronadoi schuelei, reported as 
N. schuelei {\r\ Boeters, 1988) and as Islamia 
schuelei (in Bodon et al., 2001 ), is redeschbed 
and placed into a new genus Milesiana, and a 
third new species is described and placed in 
a new genus, Josefus. Neohoratia azarum has 
not been included here because still unpub- 
lished data (Arconada, 2000) clearly demon- 
strate that its anatomy is differs considerably 
from the genera and species described here. 

This paper increases the number of species 
and expands the distribution area of Islamia 
(Schutt, 1961; Radoman, 1973a, b; Giusti et 
al., 1981; Bernasconi, 1984; Bodon et al., 
1995) in Europe and reinforces the hypoth- 
esis that the Iberian Peninsula is one of the 
richest hydrobioid {sensu Davis, 1979) diver- 
sity areas in the Mediterranean Basin (Arco- 
nada & Ramos, 2003). 



MATERIAL AND METHODS 

Field collections, anatomical studies, histo- 
logical protocols, and morphometric measure- 
ments are described in Ramos et al. (2000) 
and Arconada & Ramos (2001). The number 
of specimens studied for histology and mor- 
phometry, localities and sampling dates for 
each species are indicated in the correspond- 
ing section in the text. The morphological de- 
scriptions are based on terminology from 
Hershler & Ponder (1998). Scanning Electron 
Microscope (SEM) photographs were made 
with a Philips XL20 following the methodol- 
ogy described in Ramos et al. (2000). Type 
material of Islamia globulus was photographed 
with a Environmental Scanning Electron Mi- 
croscope (ESEM) Philips Quanta 200 SEM at 
low vacuum mode, after being cleaned with 
ultrasound (Figs. 18, 20, 23, 25, 27, 30. 31, 
33. 34) or the periostracum removed by im- 
mersion in 5% sodium hypochlorite (Figs. 19, 
28). 

Paratypes of Islamia cianensis Bodon, 
Manganelli, Sparacio & Giusti, 1995 {n° 6732), 
and /. gaiteri Bodon, Manganelli, Sparacio & 
Giusti, 1995 (n° 6733), from the Museo 
Zoológico "La Specola" collection were used 
for comparisons. 

Localities are listed according to the code: 
stream or spring, municipality, province, UTM 
co-ordinates, sampling date, collector's initials, 
museum catalogue number and preservation 
conditions (see abbreviations below). Local- 
ity names and UTM co-ordinates were ob- 
tained from the official Army Geographical 
Service map (1;50.000 series). 

Statistical Analyses 

All statistics (mean value, standard devia- 
tion and coefficient of variation) were calcu- 
lated using STATVIEW for Macintosh, and 
standardized in order to avoid the effect of the 
measurement scale. 

A discriminant función analysis (DFA) was 
performed on nine shell measurements (no 
ratios) with STATISTICA v.6 for Windows in 
order to identify the morphological characters 
that best differentiated species when no or few 
anatomical data were available. There were 
no missing data. The effects of violating as- 
sumptions are minimized taking into account 
the robustness of the Ftest (Lindman, 1974). 
The significance of the overall discriminatory 
power of the analysis was tested using Wilk's 
Lambda. Canonical correlation was used to 



80 



ARCONADA & RAMOS 



measure interspecific variation. Classification 
functions were computed for each group 
(population) to determine, with the highest 
probability, which case belonged to which 
population. Cases were assigned to the group 
with the highest classification score. 

Abbreviations Used in the Text, Tables and 
Figures 

Shell and Operculum Characters: AH: aper- 
ture height; AL: aperture length: AW: aper- 



ture width: LBW. length of body whorl; NL: 
length of opercular nucleus: NW: width of 
opercular nucleus; NSW: number of spire 
whorls: OL: operculum length: OW: opercu- 
lum width: OLWL: length of the last whorl of 
the operculum: OLWW: width of the last whorl 
of the operculum: SL: shell length; SW: shell 
width; WAW: width of the antepenultimate 
whorl: WBW: width of the body whorl; WPW; 
width of the penultimate whorl; CV: coefficient 
of variation: SD: standard deviation. 
Anatomical Characters: Ag: albumen gland; Be: 




FIGS. 6-10. Histological sections of the anterior female genitalia of Milesians schuelei showing the 
position of the spermatozoids inside the seminal receptaculum. Note the heads of the spermatozoids 
attached to the ciliated epithelial cells of the seminal receptacles. FIGS. 6, 7: Proximal seminal 
receptaculum; FIGS. 8, 9: Distal seminal receptaculum; FIG. 10: Inner epithelium of the widened 
renal oviduct. Abbreviations: c: cilia; sp: spermatozoids. 



REVISION OF THE GENUS ISLAMIA 



81 



bursa copulatrix; Cg: capsule gland; DBG: 
duct of the bursa copulatrix; Os: osphradium; 
P: penis; PI: penial lobe; Po: palliai oviduct; 
Pp: pseudopenis; Pr: prostate; Ro: renal ovi- 
duct; SR1: distal seminal receptacle; SR2: 
proximal seminal receptacle; Ss: style sac; 
St: stomach; Vc: ventral channel of capsule 
gland; L: length; W: width. The concentration 
of the nervous system was determined by the 
"RPG" ratio (Davis et al., 1976): length of 
pleuro-supraesophageal connective divided 
by the sum of the lengths of right pleural gan- 
glion, pleuro-supraesophageal connective 
and supraesophageal ganglion. Following 
several studies, a synthesis of RPG ratios 
from diverse hydrobioid taxa indicates: dor- 
sal nerve ring concentrated (< 0.29); moder- 
ately concentrated (0.30-0.49); elongated 
(0.50-0.67); extremely elongated (> 0.68) 
(Davis et al., 1984, 1986, 1992). 

Collections: MNCN: Museo Nacional de Cien- 
cias Naturales, Madrid, Spain; MZB: Museu 
de Zoología, Barcelona, Spain; NNM: Na- 
tionaal Natuurhistorisch Museum, Leiden, 
Naturalis, The Netherlands; MHNG: Muséum 
d'Histoire Naturelle, Genève, Switzerland; 
SMF: Forschungsinstitut und Natur-Museum 
Senckenberg, Frankfurt, Germany; MZUF: 
Museo Zoológico "La Specola", Universita 
di Firenze, Italy; IBCICL: Slovenian Academy 
of Sciences and Arts, Ljubljana, Slovenia; 
NHMW: Naturhistorisches Museum, Wien, 
Austria. 

Collectors: R. A.: R. Araujo; B. A.: B. Arconada; 
J. A.: J. Astigarraga; A. В.: A. Bertrand; D. В.: 
D. Buckley; A. C.: A. Camacho; J. E.: J. 
Escobar; S. J.: S. Jiménez; N. M.: N. Martín; 
D. M.: D. Moreno; С. N.: С. Noreña; J. P: J. I. 
Pino; J. M. R.: J. M. Remón; J. R.: J. Roca; E. 
R.: E. Rolan; G. Т: G. Tapia. 



GENITAL HISTOLOGY 

Histological studies of 4 pm serial sections 
were conducted with special focus on female 
and male genital systems. For each species, 
the number and sex of specimens investigated 
are indicated in the corresponding texts. 

Considering the female genitalia of Islamia 
globulus, I. henrici henrici, Milesiana schuelei, 
and Josefus aitanica, histological evidence of 
"oriented sperm" in the two sac-like structures 
on the renal oviduct was obtained. The sper- 
matozoa are arranged with their heads an- 
chored to the cell surface among the cilia of 



the epithelial cells lining the lumen of the semi- 
nal receptacle (Figs. 6-9). This is the typical 
method for sperm storage in a molluscan semi- 
nal receptacle (Thompson & Bebbington, 
1 969; Giusti & Selmi, 1 985; Fretter & Graham, 
1994: 303-306) and is morphologically re- 
sponsible for the whitish-pearly réfringence 
characteristic of this structure. On the other 
hand, the bursa copulatrix (gametolytic gland) 
does not contain spermatozoa or contains few, 
non-oriented spermatozoa (its content is cen- 
trally located and never réfringent) (see also 
Ramos et al., 2000; Bodón et al., 2001 ). There- 
fore, morphological réfringence can be used 
to distinguish bursa copulatrix from seminal 
receptacles or even to infer the possible role 
of sperm storage deposit in widened parts of 
the renal oviduct (Davis et al., 1992; Ramos 
et al., 2000, and papers cited therein) when 
histological evidence is not available. The wid- 
ened portion of the renal oviduct has a thick, 
more developed inner epithelium in relation to 
the portion between proximal and distal semi- 
nal receptacles, giving rise to a stretched lu- 
men where the spermatozoids move (Fig. 10). 
Histological differences along the renal oviduct 
epithelium are similar to those described for 
Tarraconia gasulli (Ramos et al., 2000) and 
suggest that the widened part of the oviduct 
may act as an additional sperm storage. How- 
ever, we are not able to confirm this hypoth- 
esis, because we have not had evidence of 
oriented spermatozoa in any of the species 
studied. 

Careful analysis of serial sections of males 
belonging to /. globulus, I. pallida, M. schuelei, 
and J. aitanica reveals that the penis and pe- 
nial lobe are made up of a thick layer of exter- 
nal muscles beneath the outer epithelium 
(Figs. 11-15). The inner structure consists of 
numerous vascular spaces of reticulated 
connective tissue, denser along the periphery 
of the penis, with muscle fibres running be- 
tween them. There was no indication of any 
glandular tissue either on the penial lobe or 
on any other part of the penis. This structure 
is similar to that described for other molluscs 
(Fretter & Graham, 1994: 302). The undulat- 
ing penial duct can also be observed through- 
out the different sections of penis until it enters 
the nuchal area. Females of several species 
have a nuchal node or a pseudopenis located 
on the right side of the head, in a position simi- 
lar to that of the male penis. These females 
have fully functional genitalia with mature oo- 
cytes in the ovary (Fig. 16). 



82 



ARCONADA & RAMOS 



11/ 



12 






y T* 



f . .-. 



Ц 



Ч ^ 



sd 



/- 



/" 



sd 



f . 



'-> i'< 



13 






-i*jfc -- 



14 






* »» 



r. 



■ifi *^ 



i.\ 



/- . 




"Ч ■ ; 




0> 




^■^1^^ J. 






/ 



sd 



Ч 



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15 



X 



sd 



16 



FIGS, 11-16. Histological sections of the penis and its non-glandular lobe. FIGS. 11, 12: Islamia 
g/obu/tvs from Sopeira population; FIG. 13: /W//es/ana sc/7ue/e/ from Turrillas population; FIGS. 14, 15: 
ilosefus aitanica from Torremanzanas population (type locality); FIG. 16: Female gonad of /. henrici 
henrici from Guadalora River population (Hornachuelos), showing oocytes and yolk. Abbreviations: 
sd: sperm duct; i: rectum; Oo: oocytes; y: yolk. 



SYSTEMATIC DESCRIPTION 

Islamia Radoman, 1973 

Adriolitorea Radoman, 1973a: 234. 
Mienisiella Schutt, 1991: 134-136. 



Type Species 

Islamia valvataeformis (Möllendorff, 1873: 
59) = Horatia servaini Bourguignat, 1887 (by 
original designation). Horatia servaini \s a jun- 
ior synonym of Hydrobia valvataeformis Mol- 



REVISION OF THE GENUS ISLAMIA 



83 



lendorff according to Radoman, 1983, and ac- 
cepted by Bodon at al., 2001. 

Diagnosis 

Shell small or very small, ovoid or planispiral, 
rarely ovate-conic; operculum without peg; 
central tooth with one or two basal cusps on 
each side; penis with a well-developed non- 
glandular lobe on its left side; female genitalia 
with two seminal receptacles, proximal (SR2) 
larger and longer than distal (SRI); seminal 
receptacles located on opposite sides (or po- 
sitions) on unpigmented renal oviduct; they 
can arise either close to or rather distant from 
each other; proximal seminal receptacle (SR2) 
usually with evident duct and distal (SR1), 
usually without a duct evident; bursa copulathx 
absent. 

Islamia globulus (Bofill, 1909) 

Amnícola globulus ВоШ, 1909:205; 1915:57, 

58, pi. 6, fig. 6; 1917: 35. 
Amnícola anatína globulus (Bofill, 1 909) - Bofill 

& Haas, 1920: 50, 57, pi. Ill, figs. 19, 20. 
Amnícola similis (Draparnaud) - Haas, 1929: 

408, 409, fig. 163. 
Pseudamnicola similis globulus (Bofill, 1909) 

-Altimira, 1960: 10; 1963: 16. 



Neohoratia globulus globulus (Bofill, 1909) - 
Boeters, 1988: 214, figs. 137-144, 151-155, 
163-170, pi. 2, fig. 22; Bech, 1990: 61. 

Islamia globulus globulus (Bofill, 1 909) - Bodon 
et al., 2001: 179, figs. 195-200; Bodon & 
Cianfanelli, 2002: 20. 

Type Locality 

Font del Sot del Pinell, close to Forteilet del 
Montsech, Lérida, U.T.M.: GC16. 

Material Examined 

Type material: A lot containing 41 syntypes 
(dried) of Л. globulus collected by Artur Bofill 
at type locality were deposited in the MZB 
(Bofill, 1917), catalogue number: 80-1589. The 
specimen illustrated in Figs. 18, 23, 25, 27, 
30, 33, is here designated lectotype (ICZN, 
1999: Art. 74.7). The remaining syntypes are 
therefore paralectotypes. Lectotype (MZB 80- 
1 589a) and 29 paralectotypes from this lot are 
in the MZB collections and 9 in the MNCN 
collections with n° MNCN 15.05/46546. The 
second lot with around 1 ,000 syntypes (dried) 
is in the MZB collections (MZB 80-1628). 

Other populations examined: This species 
is widely distributed in the provinces of Lérida 
and Huesca (Fig. 17). Boeters (1988) also 




/. i>lohiilus 

2- Í. Idi^ari 

3- /. (itt'iii 

4- /. pcillidd 

5- /. Iienrici lienrici 

6- /. henrici ^ieimensis 

7- M. schuelei 
H- M. cf. schuelei 
9- J. (litan ica 



200 km 



FIG. 17. Map of localities of the genera Islamia, Milesiana and Josefus in the Iberian 
Peninsula. 



84 



ARCONADA & RAMOS 



cited it from Gerona, although we cannot con- 
firnn these data so far. One lot from Font La 
Figuereta (Lérida) population kept in the MZB 
(80-1629) was also examined and compared 
with that from the same locality kept in the 
MNCN (15.05/46540). Five specimens (etha- 
nol) from Laguarta population were donated 
to the MZB (n 2002-0537). 

Localities 

Spring in Amargosa, Aristot, Lérida, UTM: 
31TCG871948. 14 March 1999, B. A., MNCN 
15.05/46527 (ethanol and frozen material); 
Blanca spring, Vilanova de Meya, Lérida, UTM; 
31TCG371551, 25 Feb. 1986, J. R., MNCN 
15.05/46528 (ethanol, SEM preparation and 
frozen material); La Argentería spring, Baix 
Pallars, Lérida, UTM; 31TCG381842, 2 Oct. 
1986, J. R., MNCN 15.05/46529 (ethanol and 
SEM preparation); El Regué spring, Vilanova 
de Meya. Lérida, UTM; 31TCG304539, 27 Feb. 
1986, J. R., MNCN 15.05/46530 (ethanol); La 
Fayeda spring, Abella de la Conca, Lérida, 
UTM; 31TCG475668, 10 Oct. 1986, J. R., 
MNCN 15.05/46531 (ethanol); Fontanet spring, 
Abella de la Conca, Lérida, UTM; 31TCG4269, 
14 March, 1999, B.A.. MNCN 15.05/46593 
(ethanol and frozen material); Les Greixes 
spring, Sant Esteve de La Sarga, Lérida, UTM; 
31TCG126635, 8 May 1986. J. R., MNCN 
15.05/46532 (ethanol); Blanca spring, Gäbet 
de la Conca, Lérida. UTM; 31TCG301658, 13 
May 1986, J. R., MNCN 15.05/46533 (etha- 
nol); D'Arcallo spring, Baix Pallars, Lérida, 
UTM; 31TCG482818, 29 Sept. 1986, J. R., 
MNCN 15.05/46534 (ethanol); La Sarga 
spring. Gäbet de La Conca, Lérida, UTM; 
31TCG375567, 26 Feb. 1986, J. R., MNCN 
1 5.05/46535 (ethanol); Freda spring, Abella de 
la Conca, Lérida, UTM; 31TCG473677, 10 May 
1986, J. R., MNCN 15.05/46536 (ethanol), 14 
March 1999. B.A., MNCN 15.05/46616 (etha- 
nol and frozen material); Freda spring de Casa 
Pallas, Aren, Lérida; UTM; 31TCG065908, 28 
March 1987, J. R.. MNCN 15.05/46537 (etha- 
nol); Bordons spring, Aren, Huesca, UTM; 
31TCG085881, 31 March 1987, J. R., MNCN 
15.05/46538 (ethanol); Adraén, Cadi moun- 
tains, Lérida, UTM.; 31TCG767817, 15 Feb. 
1998, A. В., MNCN 15.05/46539 (ethanol and 
SEM preparation); 15 March 1999, B. A., 
MNCN 15.05/46541 (ethanol and frozen ma- 
tehal); La Figuereta spring, AIós de Balaguer, 
Lérida, UTM; 31TCG253439, 11 March 1986, 
J. R., MNCN 15.05/46540 (ethanol); Les Bulles 
spring, Isona, Lérida, UTM; 31TCG371667, 8 



May 1986, J. R., MNCN 15.05/46594; Laguar- 
ta. Huesca, UTM; 30TYM374998, 12 April 
1995; B. A., MNCN 15.05/46542 (ethanol and 
SEM preparation); 26 Oct. 1995, B.A. &E. R., 
MNCN 15.05/46543 (ethanol, SEM prepara- 
tion and frozen material); Grima spring, Gistain, 
Huesca. UTM; 31TBH799184, 13 April 1995, 
B. A., MNCN 15.05/46544 (ethanol); Sopeira 
spring, Huesca, UTM; 31TCG1487, 24 July 
1991 , R. A., D. M., J. M. R., MNCN 15.05/46545 
(ethanol and SEM preparation). 

Material Examined for Morphometry and 
Histology 

Shell and anatomical measurements (Tables 
1 , 3-7) correspond to populations from Lérida 
and Huesca; Operculum and radular measure- 
ments (Tables 2, 4) to Huesca (see table cap- 
tions). Male and females studied and 
measured were collected in the following 
months; Feb., March, April, May, July, and Oct. 
For histology, four females and three males 
were studied from a spring in Sopeira, Huesca 
(July 1991), and one female from Laguarta, 
Huesca (Oct. 1995). 

Diagnosis 

Shell ovate-conic, body whorl narrow; oper- 
culum ovate; central tooth of radula with a single 
basal cusp on each side; ctenidium well devel- 
oped; short pleuro-subesophageal connective; 
esophagus running straight underneath cere- 
bral commissure; bean-shaped prostate gland; 
big penis, usually black pigmented, with one 
large, unpigmented non-glandular lobe, com- 
monly protruding from the tip of penis; pyriform 
and pedunculated proximal seminal receptacle 
(SR2) and small, elongated, sessile distal semi- 
nal receptacle (SRI); receptacles emerge dis- 
tinctly separated from each other. 

Description 

(Figs. 18-29, 30-35, 42-49; Tables 1-7; 
Bodon et al., 2001; figs. 195-200) 

Shell: Shell ovate-conic, 4.1 whorls; sutures 
deep, aperture oval, slightly prosocline; 
peristome complete, slightly thickened at 
columelar margin, slightly reflected at lower 
and columelar margin; body whorl very nar- 
row, over V; of the total shell length; proto- 
conch consisting of 1.5 whorls; protoconch 
width and width of the nucleus are 380 pm 
and 140 pm, respectively (Figs. 30-35); 
protoconch pitted; umbilicus narrow, 130 pm 



REVISION OF THE GENUS ISLAMIA 



85 




FIGS. 18-29. Shells of Islamia globulus. FIGS. 18, 23, 25, 27: Lectotype (MZB 80-1 589a); FIG. 19: 
Paralectotype (MZB 80-1 589b); FIG. 20: Paralectotype (MNCN 1 5.05/46546); FIG. 28: Paralectotype 
(MZB 80-1 589c); FIGS. 21, 24,26, 29: Shells from Laguarta; FIG. 22: Shell from Sopeira population. 
Scale bar = 1 mm (FIGS. 18-26); 500 pm (FIGS. 27-29). 



86 



ARCONADA & RAMOS 



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^ s. 


o2. 


CD 2, 


^t s 


^° 


CM 2- 


•ф ^ 




ф > ^ 
20 


°°сз^ 


°-ю 


^co 


^^■^ 


^o 


^ CD 


P.- 


^in 


Po 


^ 00 


^co 


Q.~: 




о о 


•<- о 


о о 


О о 


о -^ 


о о 


о о 





,- 





ГО 


COS' 






о 


CD 


CD 


d 


d 


d 


d 


d 


d 


d 


d 


' ^^ — 




^-^ 


со 


óo" 


CM 


со 


Îl~ 


óo 


CD 


^ 


f^ 


f^ 





•^ С 




□ i 


^'^ 


см°° 


^"^ 


^-P 


in-P 


(b-p 


in-^ 


4^ 


сгГЧ 


CM ■^ 


inP 


■^^^ 




ел ^ ÍT 


о о 


т- (D 


о о 


о о 


о о 


о о 


о о 





о о 





Сч1 СО 


■^ пз 




+1 X т- 

го è с 


о ó 


CD 00 


CD 4 


d CD 


d 4 


d об 


d in 


d 00 


d in 


d со 


d in 


. 




+1 <=>. 


+|f^ 


+|P 


+1 ^ 


+1 P 


+1 P 


+1 P 


+1 P 


+1 P 


+|fN 


+1 ^ 


^~~. 




^ Z^ 


t^ I^ 


ooS 


^ о 


rsj S, 


со 2, 


cdS 


.r- 


CMS 


v 2. 


nZl 


о г- 




О 


°°а) 


О ^ 


^in 


p^ 


^00 


h- ^ 


'^■a> 


Pc3> 


PcD 


CM ^ 


^■^ 




о о 


'" •^ 


о о 


о о 


о о 


d i^ 


о о 








d ^ 


со о 


R5 






о 


CD 


CD 


d 


d 


d 


d 


d 


d 


d 


d 




,_. 


^ 


^ 


CM 


^ 


cÑT 


- • 


in 


со 


^ 


00 


in 




QÍ 


ài~ 


T-' ° 


0^^ 


dP 


,:.-p 


00 1^ 


C4Î^ 


d4 


ài^. 


ю '^ 


d^ 


о—; 




00^ — 


т- о 


CM T^ 


о о 


T- о 


T- CD 


T- CD 


T-; CD 


T- (D 








CM со 


о. . 
Il 

Э-Ф 




+1 ¿°° 

г^ _ го II 


о т^- 


Ö oô 


CD СО 


d 4 


d CD 


d CD 


d 00 


d 4 


d 4 


d 06 


d in 




+1 ^ 


+1 ^^. 


+lP 


+1 P 


+1 T 


+1 ^ 


+1 ^ 


+1 ^ 


+iP 


+\^. 


+1 ^ 




о ZZ- 


CD Z^ 


00 :i^ 


00 S 


о с. 


CD :c^ 


CJ5O 


ooS 


00° 


^ 


G. 




■^ о 


■^ 1^ 


^CD 


00 ^ 


Pin 


«^œ 


Poo 


P T- 


Pco 


^05 


й^ 


S^^ 








CD ^ 


о ,- 


о ,- 


о •,- 


d CM 


r- 


T- 


о см 


со о 


05 О) 




d 


CD 


CD 


d 


d 


d 


d 


d 


d 


d 


d 


1_ О) 




,^ 


^ 


r^ 


ir 


о 


4~ 


r^ 


oT 





h- 


S~ 





О fc 




QÍ 


ö^^ 


— CJ> 


CD^ 


cidP 


¿2^ 


0"°. 


nTP 


r--p 


r^-^^ 


ю^ 


00 P 


^ го 




00^ — 


СМ Y 


T- CD 


О -r^ 


о о 




T- CD 


CD 


P 9 





о о 


'S- 


Q.CÛ 




со Т! го II 
го ^ с 


о ó 


Ö N- 


Ö 4 


d in 


d N- 


d CD 


d со 


d ró 


d i^ 


d CD 


d in 


я о; 




+1^. 


+1 ^. 


+1 p 


+1 P 


+1 1^ 


+1 CN 


+1 P 


+1 P 


+1 f-. 


+1 ^. 


+1 ^ 


^■о 




oí^ 


O) Z^ 


CD Zl^ 


1^ ^ 


CD ::^ 


CD z::. 


o£ 


cdS 


CM 2. 


0° 


coS 


ф (Л 




1>^ 
"^ О 


^ см 


Po 


P^ 


Pcj> 


P T- 


Po 


t^ ^ 


CD ^ 


CD ^ 


"^.ю 


PcM 


-■о 




Ö 


о 


■^ О 
о 


о о 
d 


d 


d 


d ^ 
d 


d i^ 
d 


d •,- 

d 


о ■,- 
d 


^ 
d 


Ф 'í 




, . 


со 


óo 


in 


о 


Ó0 


Ó0 


in" 


Ó0 


Ó0 


in 


Ó0 




Q'i 


Cû^ 


díP 


ю p 


CD»^ 


cdP 


^-P 


c^-p 


c^-P 


^-P 


c^-p 


cWP 


(л го 

с:2 




W ^ — 


р "7 


о "7 


о т- 


о о 


о ■^ 


о о 


P T 








P V 







1Л 1! го II 


CD ГО 


CD uS 


О СП 


d in 


d ro 


d ó 


d CM 


d in 


d CD 


d CM 


d Ю 


.55 -Ф 




+1 ^ 


+1 ^ 


+1 '^ 


+1 P 


+1 ^ 


+1 P 


+1 P 


+1^ 


+1 P 


+1 P 


+1 f- 


Ф-' 




roi,c 


00 1^ 




■* ÎS 


(J^O 


00 ^ 


in Z^ 


C3^ 


cmS 


in I^ 


œS 


cm2- 


-Q го" 




1>^ 
"^ О 


^ю 


^S" 


Т '^ 


'^. ^ 


■^ in 


S^^ 


Й'^ 


^-^f 


s^^ 


^"^ 


!i ^ 


.2 Р 
с го 




•^ о 


■^ о 


-- о 


о о 


•^ о 


О о 


о о 
















Ь 


CD 


CD 


d 


d 


d 


d 


d 


d 


d 


d 


(В^'-' 




-^ — ^ 


CD 


CD 


óo 


óo 


о 


. S" 





cÑT 





CM 





^i3 




о| 


? ^ 


T-' ^ 


CD '^ 


4P 


Csl^ 


4' ^■ 


r^-p 


4^ 


4^ 


^-P 


C3ÍP 




со ^ — 




T— T— 




о о 




P T 














■>- ^ 


2^ 
Ф », 




"* _ го II 


CD <D 


CD CD 


CD in 


d о 


d ó 


d o6 


d C3 


d 4 


d CD 


d ^ 


d ó 




+1 Я 


+1 P 


+1 ^ 


+1 P 


+1 P 


+1 ^ 


+1 P 


+1 P 


+1 P 


+1 Ч 


+1 P 


^> 




roi,c 


ю es 


o) :s 


■^ C. 


со С- 


Ю I^ 




::^ 


o^S 


cdS 


CJ.O 


00 S 


3^1 




i> 


^ОО 


^00 


Pt- 


р^ 


-^00 


^■^ 


S=° 


!:::^c 


:^^" 


2° 


I: ^^ 


ч- (Л 




■^ О 


•^ о 


■^ О 


о о 


■^ о 


•^ о 











T- 


^ 


ош 




о 


CD 


CD 


d 


d 


d 


d 


d 


d 


d 


d 




,_^ 


CÑT 


CD 


CM 


S' 


CD 


о 


4^ 


CnT 


4^ 


Ó0 





z: ГО 
го , 




q| 


-Г-' «=5 


odP 


,C^ 


,;j«^ 


^-P 


CD^ 


CD P 


^'^. 


ih-f-. 


c^-" 


T-" P 




ÜO ^ - 


"Г "^ 


О ■^ 


О T^ 


о о 




P T 


P 9 








CD 


CM 4 


о ' 




+1 ¿°° 

го ГО II 

roi,^ 


о см 


CD ^i- 


CD r^ 


d CD 


d 4 


d CM 


d CNj 


d CD 


d 00 


d CD 


d CD 


о со 




+1 ^ 


+1 P 


+1 Ч 


+1 T 


+1 P 


+1 Ч 


+1 T 


+1 P 


+1 P 


+1 '^ 


+1 P 


ф го 




СЮ?^ 


h- C- 


r^ ZZ- 


^ ^ 


m i:^ 


cj) :s 


Z- 


t^ В 


.r- 


.^ 


00 s: 


5:1 
с^-ф 
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'^ о 


■^ m 


^CD 


Pin 


P^- 


^00 


^■ю 


PCD 


S^° 


s^° 


S^ 


^ю 




см о 


■^ О 


•^ о 


■^ о 


■^ о 


•^ о 


■^ 











'ï 




CD 


CD 


CD 


d 


d 


d 


d 


d 


d 


d 


d 


?5 


О) 


^_, 


So 


CM 


h- 


о 


CD 


óo 


CD 


CM 


Ó0 


CM 





ûi 


о ^ 


ài'P 


г--с^ 


in P 


r-'P 


i-'° 


CD p 


CD^ 


4P 


^-P 


r--p 


00 


+1 X т- 

^ С^ И 

го è с 




о T^ 


О ту 


P 9 


P "7 


о T- 





P 9 


CD 





t- <чГ 


" — TD 


Й 


Ö CD 


CD CD 


d s- 


d 00 


d CD 


d CN 


d ó 


d CD 


d 4 


d 4 


d CD 




+1 '^ 


+1 P 


+1 Ч 


+1 P 


+1 P 


+lp 


+1 T 


+1 P 


+1 P 


+1 '^ 


+1 p 


i2 ГО 


Ф 


^ Ç^ 


•^ C- 


in Z^ 


CD ^ 


'i ^ 


CM ZZ- 


CD i:^ 


Sí- 


cdS 


CJ5° 


CM s 


f, ^ 


С 


^ О 


^. ю 


'^. CD 


P in 


P in 


Pin 


^CD 


PcD 


Sí- 


^CD 


S° 


^in 


Ф о 
ф i5 


Ф 


■^ о 


T- о 


■<- о 


О о 


T- о 


•^ о 











•<- 


•^ 


D5 


CD 


Ö 


d 


d 


d 


d 


d 


d 


d 


d 


d 


^■> 


-с 


,^ 


óo 


CD 


CD 


cd' 


00 


00 


œ 


00 


S~ 


CM 





V) , 


-^ 


Q'i 


¿'2 


<3i^ 


^:CN 


cdP 


(b'f^ 


r-'f- 


CD ^ 


CD P 


4P 


^-P 


inP 


S^-n 


W ^ О 

+1 Í5 "^ 
го è с 




О -r;- 


о -r^ 


о о 


P T 


о CD 


CD 


P 9 








Гч| СО 


Е го 
— о 


С 


CD CD 


CD CD 


d CD 


d о 


d CM 


d 4 


d 4 


d CD 


d 4 


d ó 


d in 


ГО 


+1 '^ 


+1 P 


+1 P 


+1 '^ 


+1 ^ 


+1 P 


+iP 


+1 P 


+1 P 


+iP 


+1 f- 


00 


СО es 


c:) C, 


со Z^ 


t^ ^ 




1^ ZZ- 


CM il- 


00° 


'^ 


CJ50 


S 




^ о 


°^CD 


PcD 


"^■^л 


PcD 


P Ю 


T CD 


PcD 


^N. 


^ in 


Î4° 


T CÛ 


о 


■^ О 


■^ о 


•^ о 


О о 


■^ о 


■^ о 











T- 


^ 


с 


CD 


CD 


d 


d 


d 


d 


d 


d 


d 


d 


d 


_|ф 
со о. 
< о 


53 

-с 




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5 




X 

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5 


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CL 


5 

< 


z 



REVISION OF THE GENUS ISLAMIA 



87 



TABLE 2. Operculum measurements (in mm) of Islamia Iberian species. All populations from type 
locality except specimens of /. globulus (1 ) belonging to Laguarta population (Huesca). /. ateni (2), /. 
pallida (3), /. h. henhci (4) and /. h. giennensis (5). 





1 


2 


3 


4 


5 




Mean ± SD; 


Mean ±SD; 


Mean ± SD; 


Mean ±SD; 






CV(Max-Min) 


CV(Max-Min) 


CV(Max-Min) 


CV(Max-Min) 




OL 


0.88 ±0.04; 


0.58 ±0.02; 


0.45 ±0.03; 


0.52 ±0.01; 






0.05 (0.96-0.82) 


0.03(0.61-0.56) 


0.07 (0.47-0.42) 


0.02 (0.54-0.50) 


0.55 




(n = 9) 


(n = 4) 


(n = 2) 


(n = 5) 


(n = 1) 


OW 


0.41 ±0.01; 


0.41 ±0.02; 


0.38 ±0.01; 


0.43 ±0.02; 






0.06(0.73-0.61) 


0.06 (0.44-0.39) 


0.02 (0.39-0.38) 


0.06(0.48-0.41) 


0.45 




(n = 9) 


(n = 4) 


(n = 2) 


(n = 5) 


(n = 1) 


OLWL 


0.41 ±0.01; 


0.32 ±0.01; 




0.17 ±0.02; 






0.03 (0.43-0.40) 


0.03(0.32-0.31) 


0.16 


0.13(0.20-0.15) 


0.15 




(n = 4) 


(n = 2) 


(n = 1) 


(n = 5) 


(n = 1) 


OLWW 


0.28 ±0.04; 


0.20 ±0.04; 




0.13±0.01; 






0.03 (0.33-0.24) 


0.19(0.22-0.17) 


0.10 


0.08(0.14-0.11) 


0.13 




(n = 4) 


(n = 2) 


(n = 1) 


(n = 5) 


(n = 1) 


NL 


0.33 ± 0.06; 


0.17 ±0.02; 




0.27 ±0.02; 






0.20 (0.40-0.24) 


0.12(0.19-0.16) 


? 


0.10(0.29-0.23) 


0.27 




(n = 4) 


(n = 2) 




(n = 5) 


(n = 1) 


NW 


0.38 ±0.02; 


0.20 ±0.02; 




0.29 ±0.02; 






0.07 (0.42-0.36) 


0.12 (0.22-0.18) 


0.25 


0.08 (0.32-0.27) 


0.30 




(n = 4) 


(n = 2) 


(n = 1) 


(n = 5) 


(n = 1) 


OL/OW 


1.31 ±0.06; 


1.43 ±0.09; 


1.17±0.05; 


1.20 ±0.08; 






0.04(1.40-1.22) 


0.06(1.51-1.32) 


0.05(1.21-1.13) 


0.06(1.28-1.08) 


1.22 




(n = 4) 


(n = 4) 


(n = 2) 


(n = 5) 


(n = 1) 



in diameter (Figs. 27-29). In apical view, shell 
growth is quite regular and, consequently, the 
general shell shape is also regular. 

Operculum: Pale yellowish, ovate, submar- 
ginal nucleus (Figs. 36-38), with a muscle 
attachment area rounded or oval. 

Body: Head scarcely pigmented, with scat- 
tered pigment cells around the eye-spots 
(Fig. 46). External body pigmentation very 
dark, except last body whorl. 



Nervous System: With long pleuro-supra- 
esophageal and short pleuro-subesophageal 
connectives; RPG ratio is 0.43 (moderately 
concentrated). Esophagus running straight 
underneath cerebral commissure (Fig. 42). 

Ctenidium: With 12-13 well-developed 
lamellae (Fig. 43). Occupying nearly en- 
tire length of palliai cavity. Osphradium 
length two to three times longer than its 
width (Table 3). 



TABLE 3. Osphradium measurements (in mm) of several Islamia Iberian species. All populations 
from type localities except for /. globulus (1-2): 1 - Gäbet de la Conca (La Sarga spring), Lérida; 2 - 
Sopeira spring, Huesca. /. ateni (3), /. pallida (4), /. h. henhci (5) and /. h. giennensis (6). 



1 

Mean ± SD; 

CV(Max-Min) 

(n = 2) 



Mean ± SD; 

CV(Max-Min) 

(n-2) 



Mean ± SD; 

CV(Max-Min) 

(n = 13) 



Mean ± SD; 

CV(Max-Min) 

(n = 3) 



Mean ± SD; 

CV(Max-Min) 

(n = 4) 



Mean ±SD; 

CV(Max-Min) 

(n = 3) 



OsL 0.19 ±0.04; 0.33 ±0.01; 0.15 ±0.01; 0.12 ±0.01; 0.13 ±0.02; 0.17 ±0.04; 

0.23 (0.22-0.16) 0.03 (0.34-0.32) 0.07 (0.16-0.14) 0.05 (0.13-0.12) 0.12 (0.16-0.12) 0.22 (0.21-0.14) 

OsW 0.07 ±0.01; 0.08 ±0.01; 0.08 ±0.01; 0.06 ±0.01; 0.08 ±0.01; 0.08 ± 0.02; 
0.11 (0.07-0.06) 0.07(0.08-0.07) 0.11 (0.09-0.07) 0.20(0.07-0.05) 0.07(0.08-0.07) 0.24(0.11-0.07) 



88 



ARCONADA & RAMOS 



Stomach - Radula: Stomach length greater 
than width (Table 5): style sac protruding 
anteriorly into the intestinal loop (Fig. 44); 
rectum U-shaped, sometimes bending to- 
wards anterior portion of body (Fig. 45). 
Radula (Table 4) small (17%) relative to 
maximum shell dimension: central tooth 



(Figs. 39, 40) with a single basal cusp on 
each side; distance between cusps is ap- 
proximately 11 pm; central denticle long and 
wide, followed on each side by four small 
denticles in decreasing order of size; lateral 
teeth with 3-4 denticles on each side of a 
central one (Fig. 41). 




FIGS. 30-41. Protoconch, operculum and radula of Islamia globulus. FIGS. 30, 33: Lectotype (MZB 
80-1 589a); FIGS. 31-34: Paralectotype (lost specimen); FIGS. 32, 35, 36, 39-41: Shells, opercula 
and radula from Laguarta population; FIG. 38: Operculum from Sopeira population; FIGS. 30-35: 
Protoconch and microsculpture; FIGS. 36, 37: Inner side of the operculum; FIG. 38: Outer side of the 
operculum; FIG. 39: Transverse rows: FIG. 40: Central teeth; FIG. 41 : Lateral, outer and inner marginal 
teeth. Scale bar = 100 pm (FIGS. 30-32); 50 pm (FIGS. 33-35); 200 pm (FIGS. 36-38); 10 pm (FIG. 
39); 5 pm (FIGS. 40,41). 



REVISION OF THE GENUS ISLAMIA 



89 



TABLE 4. Radula formulae and measurements (in mm) of Islamia Iberian 
species. /. globulus (1 ) from Laguarta population. /. ateni (2) and /. h. henrici 
(3) populations from type localities. 



Central teeth 
Central teeth width 
Left lateral teeth 
Inner marginal teeth 
Outer marginal teeth 
Radula length 
Radula width 
Number of rows 



1 

4+C+4/1-1 

~ 9 |jm 

4+C+3 

~ 24 cusps 

~ 6 cusps 

~ 345 Mm 

~ 58 pm 

-50 



5+C+4(5)/1-1 

~ 7 pm 

6+C+3 

~ 24 cusps 

~ 10 cusps 

~ 364 pm 

~ 59 pm 

-62 



4+C+4/2-2 
~ 5.6 pm 
5+C+3 
~ 24 cusps 
~ 9 cusps 
~ 193 pm 
- 46 pm 

9 



Male Genitalia: With bean-shaped prostate 
gland (Table 6) leaning towards the poste- 
rior part of the rectal loop (Fig. 45); approxi- 
mately V3 of prostate gland extending Into 
palliai cavity; first lobes of testis spilling over 
onto posterior chamber of stomach and 
sometimes reaching anterior chamber; pe- 
nis large, usually darkly pigmented, with one 
large unpigmented glandular lobe located in 
medial position (Figs. 46, 47); penial duct in 
central position, at base, then running 
straight to penis tip. 

Female Genitalia: Renal oviduct makes a wide 
circle that overlies the albumen gland (Fig. 
48); almost ^/3 of the oviduct glands (albu- 
men + capsule glands) lie inside palliai cav- 
ity; oviduct glands (albumen + capsule 
glands) usually are not narrow, although 
some females have a discrete narrowing at 
their outer edge; albumen gland larger than 
capsule gland (Fig. 48); proximal seminal re- 



ceptacle (SR2) generally pyriform, peduncu- 
lated (Fig. 49); distal seminal receptacle 
(SRI) smaller, elongated, sessile; both a 
good distance from each other on opposite 
positions on renal oviduct; renal oviduct wid- 
ening posterior to SR2. 

Discussion 

Until now, no lectotype of Amnícola globulus 
has been designated. Since 1920 (Bofill & 
Haas, 1 920), the type material of this species 
has been referred to in the literature as "un- 
known". We traced the type material in the 
MZB collection and found it consists of two 
lots, one containing 41 specimens (MZB 80- 
1589) and the other over 1,000 specimens 
(MZB 80-1628). In the species description 
(Bofill, 1909) and in later papers, he mentions 
that "this species was extremely abundant". 
The first lot contains the original label and is 



TABLE 5. Digestive system measurements (in mm) of Islamia Iberian species. All populations from 
type localities, except for /. globulus (1) (Sopeira spring, Huesca). /. ateni (2), /. pallida (3), /. h. 
henrici (4) and /. h. giennensis (5). 





1 

Mean ± SD; 

CV(Max-Min) 

(n = 2) 


2 

Mean ± SD; 

CV(Max-Min) 

(n = 3) 


3 

Mean ±SD; 

CV (Max-Min) 

(n = 3) 


4 


5 

Mean ± SD; 

CV (Max-Min) 

(n = 3) 


SsL 


0.42 ±0.01; 
0.02(0.42-0.41) 


0.27 ±0.01; 
0.02 (0.28-0.27) 


0.19 ±0.02; 
0.11 (0.21-0.17) 


0.29 

(n = 1) 


0.27 ±0.01; 
0.05 (0.29-0.27) 


SsW 


0.33 ±0.04; 
0.11 (0.36-0.31) 


0.24 ±0.01; 
0.04 (0.25-0.23) 


0.18 ±0.02; 
0.12(0.20-0.16) 


0.28 

(n = 1) 


0.21 ±0.03; 
0.16(0.25-0.19) 


StL 


0.46 ±0.17; 
0.37 (0.58-0.34) 


0.40 ±0.07; 
0.18(0.45-0.32) 


0.29 ±0.04; 
0.15(0.34-0.26) 


0.26 
(n = 1) 


0.33 ±0.02; 
0.07 (0.36-0.32) 


stw 


0.47 ±0.04; 
0.09 (0.50-0.44) 


0.41 ±0.01; 
0.02 (0.42-0.40) 


0.28 ±0.04; 
0.14(0.31-0.23) 


0.19 
(n = 1) 


0.28 ±0.04; 
0.16(0.33-0.24) 



90 



ARCONADA & RAMOS 



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REVISION OF THE GENUS ISLAMIA 



91 





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Ro- 




SR2 



l-Vc 



FIGS. 42-49. Anatomy of Islamia globulus. FIG. 42: Partial nervous system; FIG. 43: Osphradium 
and ctenidium; FIG. 44: Stomach; FIG. 45: Prostate and rectum loop; FIGS. 46, 47: Head of a male 
and penis; FIG. 48: Anterior female genitalia; FIG. 49: Detail of the seminal receptacles; Abbreviations 
in text. Scale bar = 500 pm (FIGS. 42-48). 



92 



ARCONADA & RAMOS 



probably made up of a selection of the largest 
specimens: Bofill's descriptions and illustra- 
tions were likely based on this lot. After hav- 
ing determined that the specimens of both lots 
were conspecific, we realised it would be im- 
possible to identify the illustrated specimens 
(Bofill, 191 5: Bofill & Haas, 1 920). We selected 
a lectotype from this first lot. 

Islamia globulus is clearly distinguished from 
the other Islamia species by a combination of 
characters. Its ovate-conic shell easily distin- 
guishes it from both valvatiform (/. piristoma, I. 
trichoniana. etc.) and trochiform species (/. 
anatolica, I. bunarbasa). Other important char- 
acter states include a radula with only one basal 
cusp on each side and two separated seminal 
receptacles (SR2 large and pedunculated and 
SRI small, elongated and sessile). Differences 
and similarities with /. ateni and between /. 
globulus and /. lagari are discussed below. 



Islamia lagari (Altimira, 1960) 

Pseudamnicola /agar/ Altimira, 1960: 10, fig. 2. 
Neohoratia globulus lagari (Altimira, 1960) - 

Boeters, 1988: 216, figs. 145, 146, 156, 164, 

pi. 2, fig. 23: Bech, 1990: 61. 
Islamia globulus lagari (Altimira, 1960) - 

Bodon et al., 2001, 43: 179, figs. 201-206; 

Bodón & Cianfanelli, 2002: 20. 

Type Locality 

Sot de Can Parés, Gava, Barcelona, U.T.M.: 
31TDF120720(Fig. 17). 

Material Examined 

Type material: Lectotype (shell) of N. 
globulus lagari from the NNM (N 56466/1) 
(Figs. 50-54). Five dried specimens in the 




FIGS. 50-54. Shells of Islamia lagari (NNM 56466/1). Scale bar = 1 mm (FIGS. 50-53). 



REVISION OF THE GENUS ISLAMIA 



93 



NHMW (Vienna) (Coll. W. Klemm) (NHMW 
79000/K 45087) had a label with the same 
handwriting as that of lectotype. The text in 
both labels is the same "Pseudamnlcola lagan 
Alt. Can Parés. Gava. Barcelona. 11-59". In 
addition, the label in NHMW has number "7" 
also handwritten, thus suggesting that Altimira 
probably collected seven specimens in Nov. 
1959, one of which has not yet been located. 
Therefore, the specimens at the NHM should 
be paralectotypes after designation of the lec- 
totype by Boeters. 

Material Examined for Morphometry 

Shell measurements (Table 1) correspond 
to the lectotype and paralectotypes. 

Diagnosis 

Shell ovate-conic with large and inflated body 
whorl; operculum ovate; central tooth of radula 
with a single basal cusp on each side; 
ctenidium well developed; big penis, black pig- 
mented, with one unpigmented non-glandular 
lobe located in a subterminal position not pro- 
truding from penial tip; pin-like proximal semi- 
nal receptacle (SR2) with a long stalk and 
small, elongated, sessile distal seminal recep- 
tacle (SRI); receptacles emerge distinctly 
separated from one another. 

Description 
(Figs. 50-54; Table 1) 

Shell: Ovate-conic with 3.5 whorls; sutures 
deep, aperture oval to roundish, slightly 
prosocline, peristome complete, reflected at 
lower and columellar margin; body whorl 
large and inflated, over ^h of the total shell 
length; protoconch consisting of 1 .7 whorls; 
protoconch width and width of nucleus are 
370 pm and 130 (jm, respectively (Fig. 54); 
protoconch pitted; umbilicus narrow, about 
80 pm in diameter (Fig. 53), partially cov- 
ered by reflected columellar lip. In apical 
view, shell growth is rapid, especially body 
whorl, which has an inflated appearance. 
No specimens were available for anatomi- 
cal study. Anatomical data are shown in 
Bodon et al. (2001 : figs. 201-206). 

Discussion 

Islamia globulus and /. lagari have been con- 
sidered both good species and subspecies. 
The last treatment has prevailed since Boeters 



(1988) considered both to be subspecies of 
Neohoratia globulus. Based on morphological 
differences, we propose species status for both 
taxonomic entities. A detailed anatomical de- 
scription of /. globulus is given here. No etha- 
nol-preserved specimens of /. lagari were 
available for study. Therefore, only dried type 
material and illustrations from literature have 
been used to compare this species with /. 
globulus. We used the anatomical descriptions 
provided by Boeters (1988: figs. 156, 164) and 
Bodon et al. (2001: figs. 201-206). Morpho- 
logical differences between Islamia globulus 
and /. lagari (Boeters, 1988; Bodon et al., 
2001) are based on shell shape, penis size 
and size and shape of the glandular penial lobe 
and seminal receptacles. 

Shells of /. globulus are more compressed 
laterally and, consequently, are taller and nar- 
rower than those of /. lagari. The body whorl 
of /. globulus is proportionally smaller (shorter 
and narrower) than in /. /agar/ (Altimira, 1960). 
The latter species has an inflated body whorl 
and a relatively lower spire. The penis of /. 
globulus is larger and has a slightly flatter pe- 
nial lobe. The free part of penis towards the 
tip is also flatter, narrower and longer than in 
/. lagari. Islamia lagari has a smaller distal 
seminal recéptale (SR1) with a short stalk, 
which is not evident in /. globulus. Proximal 
seminal receptacle (SR2) of /. lagari is less 
developed than in /. globulus and has a longer 
and more slender stalk. 

The DFAalso confirmed differences between 
the two species. We analysed nine standard 
shell measurements from the four /. globulus 
populations and from the /. lagari type mate- 
rial. Of the 70 individuals classified, all of the 
/. lagari were correctly classified (100%) and 
perfectly discriminated from the rest by two 
highly significant functions (Wilk's lambda = 
0.039, F (36.211), p< 0.0001). The remaining 
/. globulus individuals were grouped into four 
overlapping clusters. For the first function, the 
characters that contributed most of the 83% 
explained variance were (in order): AL and AH. 
For the second function the order was: AW, 
LBW and WBW. Another DFA using all Islamia 
species studied herein yielded similar results. 
All of the /. lagari specimens were correctly 
classified and definitively discriminated from 
all the other species (see "Statistical Analysis 
of Islamia species" below and Fig. 138). 

Both taxa are allopatric, which does not help 
clarify their taxonomic status. However, both 
species are found in two different mountain 
chains that differ in geological origin and soil 



94 



ARCONADA & RAMOS 



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REVISION OF THE GENUS ISLAMIA 



95 



composition. Islamia globulus has a wide geo- 
graphical distribution in the provinces of Lérida 
and Huesca (cites in Gerona could not be con- 
firmed). This area is situated in the "Depresión 
del Ebro". It is of Oligocène origin and is com- 
posed of marls and sands on calcareous sub- 



strate. At a great distance away, more than 
150 km (Fig. 17), /. /agar/ is restricted to a small 
area in Sierra de Can Parés in the Garraf 
Massif (Barcelona), on Lower Triassic soils, 
where limestone, marls and sandstones pre- 
dominate. 




FIGS. 55-61 . Topotypes of Islamia ateni (MNCN 1 5.05/46547). FIGS. 55, 56: Frontal view; FIGS. 57, 
58: Lateral viev/; FIG. 59: Spire whorls; FIGS. 60, 61 : Protoconch microsculpture. Scale bar = 500 |jm 
(FIGS. 55-57, 59); 200 |jm (FIG. 58). 



96 



ARCONADA & RAMOS 



Islamia ateni (Boeters, 1969) 

MIcrona ateni Boeters, 1969: 70, figs. 6-8. 
Neohoratia ateni (Boeters, 1969) - Boeters, 

1988: 216, figs. 147, 148, 157, 158, 163, 288, 

pi. 2, fig. 24: Bech, 1990: 62. fig. 11. 
Islamia ateni (Boeters. 1969) - Bodon et al.. 

2001, 43: 178, figs. 189-194; Bodon & 

Cianfanelli, 2002: 20. 

Type Locality 

Balneario de San Vicente. Lérida, U.T.M.: 
CG89(Fig. 17). 

Type Specimens 

Holotype in NNM and paratypes NNM/37, 
SMF 194371/2 and ВОЕ 205 and 206. 

Material Examined 

The description of this species was made 
possible by studying topotypical material, 
kindly provided and deposited in MNCN by H. 
D. Boeters. There were 1 3 specimens in alco- 
hol [leg. Boeters coll. 514, 11/9/1972 (Figs. 55- 
61) MNCN 15.05/46547 (ethanol and SEM 
preparation)]. 

Morphometry 

All measurements correspond to specimens 
from the type locality. 

Diagnosis 

Shell ovate-conic; operculum ovate; central 
tooth of radula with a single basal cusp on each 
side; ctenidium well developed; esophagus 
running straight underneath cerebral commis- 
sure; small pear-shaped prostate gland; pe- 
nis long, unpigmented, with a large, flat, 
extended, unpigmented non-glandular lobe lo- 
cated near, but not protruding, from its tapered 
distal end; elongated, pedunculated proximal 
seminal receptacle (SR2) bending towards 
distal portion of renal oviduct and small, globu- 
lar, sessile distal seminal receptacle (SRI); 
seminal receptacles quite separated from one 
another. 

Description 
(Bodon et al., 2001: figs. 189-194) 

Shell: Ovate-conic, longer than wide, with 4 
whorls (Figs. 55-57, 59, Table 1); sutures 



deep: body whorl occupies more than ^/7 of 
total shell length: protoconch pitted (Figs. 60, 
61), consisting of 1.5 whorls; protoconch 
width and width of the nucleus are 280 pm 
and 120 pm, respectively; last whorl of 
teleoconch very narrow from apical perspec- 
tive (Fig. 59): aperture oval, orthocline or 
slightly prosocline; peristome thin at outer 
margin and slightly thickened at columellar 
margin, slightly reflected at lower and col- 
umellar margin; umbilicus very narrow; ex- 
ternal lip thin (Figs. 57, 58). 

Operculum: Yellowish, ovate (Figs. 62-64), 
with submarginal nucleus; muscle attach- 
ment area oval (Fig. 64). 

Body: Head dark pigmented from the middle 
of the tentacles to the eye lobes (Fig. 69); 
external body pigmentation very dark, except 
last body whorl. 

Nervous System: With long pleuro-supra- 
esophageal connective; no data on pleuro- 
subesophageal connectives were obtained 
due to the scarcity of specimens available 
for study; RPG ratio is 0.5 (elongated). Eso- 
phagus runs straight underneath the cere- 
bral commissure of the nervous system. 

Ctenidium - Osphradium: With approximately 
1 lamellae (Fig. 70), occupying Vb of length 
of palliai cavity. Osphradium oval and inter- 
mediate in size (Table 3). 

Stomach - Radula: Stomach almost as wide 
as it is long (Table 5, Fig. 71 ); style sac pro- 
truding anteriorly into intestinal loop; rectum 
U-shaped (Fig. 70). Radula medium-sized 
(21%) relative to maximum shell dimension 
(Table 4, Fig. 65); central tooth with a single 
basal cusp on each side (Fig. 66); distance 
between cusps approximately 6.7 pm; cen- 
tral denticle long, sharp, followed on each 
side by five small denticles in decreasing 
order of size; cutting edge markedly con- 
cave; lateral teeth with 5-6 denticles on each 
side of central tooth (Figs. 67, 68). 

Male Genitalia: Prostate gland small, pear- 
shaped (Table 6, Fig. 72); vas deferens en- 
tering posterior end of prostate, and palliai 
vas deferens exiting at its middle region, both 
are relatively close to each other; penis long, 
unpigmented (Fig. 73), with a large, flat ex- 
tended, non-glandular lobe near its tapered 
distal tip: undulating penial duct running 
along the right portion of penis and becom- 
ing straight before opening at penis tip. 

Female Genitalia: Distal seminal receptacle 
(SRI), globular and sessile, smaller than 
proximal (SR2), which is elongated and pe- 
dunculated, bending towards distal portion 



REVISION OF THE GENUS ISLAMIA 



97 



of renal oviduct (Fig. 75, Table 7); both semi- 
nal receptacles located at a distance from 
each other on opposite positions of renal 
oviduct; oviduct glands (albumen + capsule 
glands) with very weak or no narrowing, cap- 
sule gland smaller than albumen gland; re- 
nal oviduct forming a wide circle (Fig. 74) 
overlying albumen gland. 

Discussion 

Islamia ateni may be differentiated from the 
remaining European Islamia species by its 
peculiar ovate-conic or bythinelliform shell 



shape, a very small prostate gland relative to 
shell length, and by the rather large gap be- 
tween the two seminal receptacles. A single 
basal cusp on each side of the central tooth of 
the radula is a character state shared with /. 
valvataeformis, I. servaini, I. galten, I. pusllla 
and /. globulus. All other species described 
have two basal cusps. Its morphologically clos- 
est species is /. globulus. Main characters dif- 
ferentiating both species are related to shell 
size and shape (that of /. ateni are more slen- 
der than that of /. globulus), shape of the pe- 
nial lobe (more flattened and less extended in 
/. ateni, never protruding from penis tip), SR2 




FIGS. 62-68. Operculum and radula of Islamia ateni. FIGS. 62, 63: Outer side of the operculum; FIG. 
64: Inner side of the operculum; FIG. 65: Radula; FIG. 66: Central teeth; FIG. 67: Lateral, outer and 
inner marginal teeth; FIG. 68: Central and lateral teeth. Scale bar = 200 pm (FIGS. 62-64); 100 |jm 
(FIG. 65). 



98 



ARCONADA& RAMOS 



characteristically bending towards distal por- 
tion of renal oviduct, and the distance between 
seminal receptacles, which is longer in /. ateni. 

Islamia pallida Arconada & Ramos, n. sp. 

Type Specimens 

Holotype MNCN 15.05/46548 (SEM prepa- 
ration, Fig. 78) and paratypes (Figs. 82, 85, 
88, 90, 91) MNCN 15.05/46548, 5 April 1992, 
D. M. & N. M. (dried material, ethanol and SEM 
preparation). 

Type Locality 

Spring in Patones, Patones de Abajo, 
Madrid, UTM.; 30TVL603241. 



Specimens Examined 

The following specimens were also examined 
for comparative purposes: Lectotype, MHNG 
(Figs. 76, 80, 83, 86) and paralectotypes, 
MHNG (Figs. 77. 79, 81, 84, 87, 89) of Neo- 
horatia (?) coronado! (Bourguignat, 1 870) (origi- 
nally Valvata coronado!). 

Other localities: Province of Madrid (Fig. 17), 
e.g.. Spring in Patones, Patones de Abajo, 
Madrid (type locality), 29 June 1997, В. A. & 
D. В., MNCN 15.05/46550 (ethanol material); 
Jarama River, Patones, Madrid, UTM.: 
30TVL5824, 18 Jan. 1989, A. C; MNCN 15.05/ 
46549 (ethanol): 8 Aug. 1 989, A. С: La Parra 
channel, Patones, Madnd, UTM.: 30TVL603241, 
2 June 1996, B. A. & D. В., MNCN 15.05/46551 
(ethanol). 






r\y^^^ 


^°/ 


Ш 


w 


/LxSRl 




IvVc 



FIGS. 69-75. Anatomy of Islamia ateni. FIG. 69: Head pigmentation; FIG. 70: Rectum, osphradium 
and ctenidium; FIG. 71 : Stomach; FIG. 72: Prostate; FIG. 73: Penis; FIG. 74: Anterior female genitalia; 
FIG. 75: Detail of the seminal receptacles; Abbreviations in text. Scale bar = 500 pm (FIGS. 69-74). 



REVISION OF THE GENUS ISLAMIA 



99 




FIGS 76-91 Shells of Neohoratia (?) coronado! and Islamia pallida. FIGS. 76, 80, 83, 86: Lectotype 
of Neohoratia П) coronado! (MHNG); FIGS, 77, 79, 81, 84, 87, 89; Paralectotypes o^ Neohoratia {?) 
coronadoi (MHNG); FIG 78; Holotype of /. pallida (MNCN 15.05/46548); FIGS. 82, 85, 88, 90, 91; 
Paratypes of /. pallida. 



100 



ARCONADA & RAMOS 



Specimens Examined for Morphometry and 
Histology 

All measurements (Tables 1-3, 5-7) corre- 
spond to specimens from type locality (5/4/ 
1992). For histology, one male from type lo- 
cality (April, 1992) was studied. 

Etymology 

The name "pallida" refers to the fact that body 
is completely unpigmented. 



Diagnosis 

Shell depressed-trochiform or valvatiform; 
operculum circular; head and body unpig- 
mented; ctenidium well developed; short 
pleuro-subesophageal connective and small 
subesophageal ganglion; medium size pleuro- 
supraesophageal connective; esophagus run- 
ning straight underneath cerebral commissure; 
penis long, unpigmented, with a rounded and 
subterminal non-glandular lobe located near 
its distal end and protruding from penis tip; 





^^ 





FIGS. 92-99. Anatomy of Islamia pallida. FIG. 92: Partial nervous system; FIG. 93: Rectum, osphradium 
and ctenidium; FIG. 94: Stomach; FIGS. 95, 96: Head of a male and penis; FIG. 97: Anterior female 
genitalia; FIG. 98: Detail of the seminal receptacles; FIG. 99. Head of a female and pseudopenis. 
Abbreviations as in text. Scale bar = 500 pm (FIGS. 92-97, 99). 



REVISION OF THE GENUS ISLAMIA 



101 



penial duct undulates along entire length of 
central part of the penis; two elongated semi- 
nal receptacles located very close to each 
other on opposite sides of renal oviduct; fe- 
males with an unpigmented pseudopenis. 

Description 

Shell: Depressed-trochiform or valvatiform, 3.5 
whorls (Figs. 78, 85, Table 1); body whorl 
occupying more than V4 of total shell length; 
protoconch pitted (Fig. 91 ), consisting of 1 .5 
whorls (Fig. 90); protoconch width and width 
of the nucleus are 350 pm and 120 pm, re- 
spectively; aperture prosocline, rounded (Fig. 
78); peristome complete, thin (Fig. 82); um- 
bilicus narrow, 0.2 mm in diameter (Fig. 88); 
shells extremely fragile, some showing 
marked growth lines in teleoconch 
microsculpture. 

Operculum: Circular, yellowish, with central 
muscle attachment area on its inner surface 
(Table 2). 

Body: Head and body completely unpigmented 
(Figs. 95, 99). Eyes absent. 

Nervous System (Fig. 92): Medium sized 
supraesophageal and short pleural- 
subesophageal connective; subesophageal 
ganglion very small; RPG ratio is 0.42 (mod- 
erately concentrated). Esophagus runs 
straight underneath cerebral commissure. 

Ctenidium - Osphradium: Ctenidium with 9- 
10 long, narrow lamellae (Fig. 93); osphra- 
dium oval, length two times width (Table 3), 
located in opposite posterior part of 
ctenidium. 

Stomach - Radula: Stomach almost as long 
as it is wide. Style sac not protruding anteri- 
orly to intestinal loop (Fig. 94). Rectum mark- 
edly S-shaped, bending toward anterior 
portion of body (Fig. 93). Radula; unknown. 
No data on the radula were available due to 
its extreme fragility and the scarcity of avail- 
able specimens. 

Male Genitalia: Unpigmented penis almost as 
long as head (Table 6) with a rounded-trap- 
ezoidal, non-glandular, subterminal lobe 
(Figs. 95, 96) located parallel in ventral po- 
sition and near its blunt distal tip and pro- 
truding beyond tip of penis; penial duct 
strongly undulating along its length and near 
central part of penis. 

Female Genitalia: Minute with very small ovi- 
duct glands (albumen + capsule glands), 
without narrowing (Fig. 97), located approxi- 
mately V3 inside palliai cavity; renal oviduct 



making wide circle over albumen gland, 
which is larger than capsule gland; two elon- 
gated seminal receptacles equal in size (Fig. 
98, Table 7) very close to one another (al- 
most at the same level) on opposite sides of 
renal oviduct close to its loop, none of them 
with a stalk; females have an unpigmented 
pseudopenis (Fig. 99) measuring approxi- 
mately 0.20 mm, and occupies almost half 
length of head. 

Discussion 

The geographical distribution of this species 
corresponds to that of Neohoratia (?) 
coronadoi, described by Bourguignat (1870) 
as Valvata coronadoi "en los alrededores de 
Madrid, о, al menos, en algunos manantiales 
o arroyos de la provincia de Castilla La Nueva" 
[in Madrid's surroundings or, at least, in some 
springs or streams of the New Castillo Prov- 
ince]. There are no anatomical data available 
for Neohoratia (?) coronadoi, which has 
conchological characters that clearly differ 
from those of /. pallida. The shells of N. (?) 
coronadoi are large and planispiral, whereas 
those of /. pallida are small and trochiform. 
Boeters (1988) dubiously assigned the first 
species, V. coronadoi, to the genus Neohoratia 
[as N. (?) coronadoi], because of its similari- 
ties to Neohoratia schuelei {sensu Boeters, 
1 988). After several field samplings, we found 
no specimen of Valvata coronadoi, which is 
possibly now extinct. The presence of a 
pseudopenis in all females studied of /. pallida 
is a phenomenon that has also been reported 
and discussed in another Iberian valvatiform 
species {Spathogyna fez/Arconada & Ramos, 
2002). The development of male sexual char- 
acters in females has sometimes been related 
to parasitism (Rothschild, 1938), or even to 
imposex (Smith 1971; Fioroni et al., 1990). In 
the case of /. pallida, we did not find any sign 
of parasitism in any of the females studied. 

Juvenile specimens kept in an aquarium 
showed a monthly growth rate of 75% shell 
length and 87% width. They have a ciliated 
region in the propodium and at the tip of the 
tentacles (Figs. 100-103). 

Differences between /. pallida and other Ibe- 
rian Islamia species are based on a combina- 
tion of characters: the absence of head and 
body pigmentation, a very small subeso- 
phageal ganglion, two elongated seminal re- 
ceptacles, without stalk, very close to one 
another, located almost at the same level on 



102 



ARCONADA & RAMOS 



opposite sides on the renal oviduct close to 
its loop and a well-developed female 
pseudopenis. In relationship to other European 
Islamia species, most of the differences are 
related with the genitalia. In /. pallida the posi- 
tion of seminal receptacles is, in a way, simi- 
lar to that described for the type species, 
Islamia valvataeformis. However, in /. pallida 
both receptacles are smaller, not peduncu- 
lated, similar in size and shape, and are lo- 
cated close to the end of the renal oviduct loop 
(proximal position), whereas in /. valvatae- 
formis (Radoman, 1983: 124, fig. 69A, B; 
Bodon et al., 2001; 133) both seminal recep- 
tacles are "strongly developed " (the proximal 
one is larger, pyriform, and has an evident 
stalk), and emerge close to one another from 
the distal renal oviduct. In /. pallida, the penial 
lobe protrudes beyond the penis tip, similar to 
that described in species from the Balkan Pen- 
insula. Nevertheless, /. pallida has a blunt 
penis tip. In addition, the penial duct markedly 



undulates along its length and near the cen- 
tral part of penis. In the Balkan's species, the 
penial duct runs through the right part of the 
penis, undulating not so markedly from its base 
and becoming almost straight at the distal end. 
As in the Italian /. gaiteri and in the French /. 
minuta, I. globulina. I. consolationis and /. 
spirata. all /. pallida specimens studied lack 
eyes and have a completely unpigmented body. 
This may be related to living in an interstitial or 
underground water habitat (Bodon et al., 1995: 
47, 51, 52). 

Islamia benr/c/ Arconada & Ramos, n. sp. 

Type Specimens 

Holotype MNCN 15.05/46552 (Fig. 15B) 
(SEM preparation) and paratypes MNCN 
1 5.05/46552, 1 3 Oct. 1 992, E. R. (ethanol and 
SEM preparation - Figs. 106, 107, 109, 112, 
113, 116). 




FIGS. 100-103. Juveniles of Islamia pallida. FIGS. 100, 103: Complete body and operculum; FIG. 
101; Detail of the ciliated propodium; FIG. 102: Detail of the ciliated tentacles. 



REVISION OF THE GENUS ISLAMIA 



103 



Type Locality 



Diagnosis 



A tributary of the Guadalora River in Parque 
Natural de Hornachuelos, Córdoba, U.TM.: 
30STG9788. 

Etymology 

Dedicated to Enrique Arconada, whose given 
name has been Latinized as Henricus. 



Shell valvatiform or depressed-trochiform; 
central tooth with two basal cusps on each 
side; ctenidium scarcely developed or absent; 
esophagus curving posteriorly to cerebral com- 
missure; long pigmented penis with small non- 
glandular lobe located near its tip but not 
protruding from it; proximal seminal receptacle 
rounded, pedunculated or elongated, with 




FIGS. 104-116. Shells and penis of /s/am/a henrici. FIGS. 104, 108, 110, 111, 114, 115: Shells of /. 
henrici giennensishom La Iruela population; FIGS. 105-107, 109, 112, 113, 116: Shells and penis of 
/. henrici henrici from Hornachuelos population; FIG. 104: Holotype of /. henrici giennensis (MNCN 
15.05/46555); FIG. 105: Holotype of /. henrici henrici (MNCN 15.05/46552). Scale bar = 500 [^m 
(FIGS. 104-112). 



104 



ARCONADA & RAMOS 



swollen tip (SR2), bending towards distal por- 
tion of renal oviduct and distal seminal recep- 
tacle smaller, more or less globular and sessile 
(SRI). 

We consider that this species has two sub- 
species as follows: 

Islamia henrici henrici Arconada & Ramos, 
n. subsp. 

Populations Additional to Species Type Material 

This subspecies was found in the province 
of Córdoba (Fig. 17). A tributary of the 
Guadalora River, Parque Natural de Horna- 
chuelos, Córdoba (type locality). 16 April 1998, 
B. A., MNCN 15.05/46553 (ethanol and fro- 
zen material): La Almarja spring, Parque Natu- 
ral de Hornachuelos, Córdoba, U.T.M.: 
30SUG014869, 16 April 1998, B. A., MNCN 
15.05/46577 (ethanol. SEM preparation, and 
frozen material). 

Material Examined for Morphometry and His- 
tology 

All measurements of shell, operculum, 
osphradium, digestive, radular, female and 
male systems (Tables 1-7) correspond to 
specimens from the type locality (in Parque 
Natural de Hornachuelos). Male and females 
studied and measured were collected in Oct. 
One female from Guadalora River was stud- 
ied for histology. 

Diagnosis 

Long orangish pigmented penis with small 
non-glandular lobe located in distal position, 
but not protruding from penis tip: females hav- 
ing a nuchal node. 

Description 

Shell: Valvatiform ordepressed-trochiform, 3.5 
whorls (Table 1: Figs. 105, 106, 112); body 
whorl occupying approximately "/5 of total 
shell length: protoconch pitted consisting of 
more than 1.5 whorls (Fig. 113): protoconch 
width and width of nucleus are 290 and 120 
[jm, respectively; aperture rounded and 
orthocline or slightly prosocline, sometimes 
slightly oval descending (Figs. 105-107); 
peristome complete, thin, slightly reflected 
at columellar margin: external lip thin, inter- 
nal lip slightly reflected towards the umbili- 



cus: umbilicus medium-sized, 180 pm in di- 
ameter (Fig. 109). 

Operculum: Ovate with central nucleus (Figs. 
117-118): muscle attachment area rounded. 

Body: Head scarcely pigmented with scattered 
pigment cells at the base of tentacles around 
the eye-spots (Figs. 124, 129). 

Nervous System (Fig. 125): With a medium- 
sized pleuro-supraesophageal connective: 
RPG ratio is 0.3 (moderately concentrated). 
Esophagus curving posteriorly to cerebral 
commissure. 

Ctenidium - Osphradium: Ctenidium absent 
or very poorly developed, with 2-6 small 
lamellae (Fig. 126). Osphradium bean- 
shaped, length almost two times width (Table 

3). 

Stomach - Radula: Chambers almost equal 
in size. Style sac protruding anteriorly into 
intestinal loop (Table 5, Fig. 127). Rectum 
forming a marked S-loop and bends towards 
anterior portion of the body (Figs. 126, 128). 
Radula medium sized (23%) relative to maxi- 
mum shell dimension, with two basal cusps 
on each side of central tooth (Table 5, Figs. 
120-122); distance between its internal 
cusps is approximately 7 |jm; its central den- 
ticle long, sharp, followed on each side by 4 
long denticles in decreasing order of size; 
cutting edge of central tooth markedly con- 
cave; lateral teeth with 5-6 long, sharp den- 
ticles on each side of central denticle (Fig. 
123). 

Male Genitalia: With large bean-shaped pros- 
tate gland, narrow anteriorly (Fig. 128); less 
than 50% of prostate gland extending into 
palliai cavity; penis very long with small non- 
glandular lobe located in distal position (Figs. 
116, 129), showing a small réfringent area; 
penis orangish pigmented in live specimens; 
penial duct slightly undulating, close to cen- 
tral part of penis. 

Female Genitalia: With renal oviduct that 
makes a wide circle (Fig. 130); no narrow- 
ing of oviduct glands (albumen + capsule 
glands); capsule gland larger than albumen 
gland, occupying more than 50% of total 
palliai cavity length and narrowing at its dis- 
tal outer margin; proximal seminal receptacle 
(SR2) oval with a long stalk and slightly bent 
towards the distal part of renal oviduct (Fig. 
131, Table 7); distal seminal receptacle 
(SRI) smaller than proximal receptacle, 
globular, sessile; seminal receptacles lo- 
cated relatively far from one another on op- 
posite sides of renal oviduct. Some females 
have a dark nuchal node on the right side of 



REVISION OF THE GENUS ISLAMIA 



105 




FIGS. 117-123. Opercula and radula of Islamia henrici. FIGS. 117, 
118, 120-123: Opercula and radula of /. henrici tienhci from 
Hornachuelos population; FIG. 119: Operculum of /. Iienrici 
giennensis from La Iruela population; FIG. 117: Outer side of the 
operculum; FIGS. 118, 119: Inner side of the operculum; FIG. 120: 
Transverse rows; FIGS. 121, 122: Central teeth; FIG. 123: Lateral 
and inner marginal teeth. Scale bar = 200 pm (FIGS. 117-119). 



head (Fig. 124), which is approximately six 
times smaller than male penis, occupying 
20% of total head length. This nuchal node 



is usually simple, although it can some- 
times be bilobated, similar to the shape 
of the distal part of male penis. 



106 



ARCONADA& RAMOS 



Islamia henrici giennensis Arconada & 
Ramos, n. subsp. 

Type Specimens 

Holotype MNCN 15.05/46555 (SEM prepa- 
ration) (Fig. 104)andparatypesMNCN 15.05/ 
46555 (ethanol and SEM preparation, Figs. 
108, 110, 111, 114, 115, 119). 

Type Locality 

Spring facing the hotel "Sierra Cazoria ", La 
Iruela. Cazoria mountains. Jaén, UTM: 
30SWG005969. 

Etymology 

The subspecific epithet is a Latin adjective 
related to the province of Jaén (Latin Gienna). 

Other Specimens Examined 

This species was found in the province of 
Jaén (Fig. 17). La Toba spring. Jaén, U.T.M.; 
30SWH3826, 6 Oct. 1992, E. R., MNCN 15.05/ 

46558 (ethanol); 24 March 1998, B. A., MNCN 
15.05/46554 (ethanol); spring facing the hotel 
"Sierra Cazoria", La Iruela, Cazoria mountains, 
Jaén. UTM; 30SWG005969, E. R., MNCN 
15.05/46556 (ethanol); 30 April 1990, D. M. & 
N. M.. MNCN 15.05/46555 (ethanol); Madera 
River. La Fresnedilla. Segura mountains, Jaén, 
UTM.; 30SWH3644, 6 Oct. 1992, E. R., MNCN 
15.05/46557 (ethanol and SEM preparation); 
spring in Cazoria, Jaén, E. R., MNCN 15.05/ 

46559 (ethanol); La Nava de San Pedro, 
Cazoria, Jaén, UTM; 30SWG094948, 1 May 
1990, D. M. & N. M. 

Specimens Examined for Morphometry 

Shell, operculum, and anatomical measure- 
ments - osphradium, digestive, female and 
male systems (Tables 1 -3, 5-7) - correspond 
to type locality (La Iruela). Male and females 
studied and measured were collected in April. 

Diagnosis 

A slight varix near shell aperture in most of 
the specimens studied from all populations; 
long black pigmented penis with a small non- 
glandular lobe located in distal position but not 
protruding from penis tip, penis tip pointed; 
females have no nuchal node. 



Description 

Shell: Valvatiform or depressed-trochiform, 
with spire consisting of 2.75-3.5 whorls 
(Table 1; Figs. 104, 111); body whorl occu- 
pying approximately ■^L of total shell length; 
protoconch pitted consisting of more than 1 .5 
whorls (Figs. 114, 115); protoconch width and 
width of nucleus are 330 and 129 pm, re- 
spectively; aperture rounded, orthocline or 
slightly prosocline, sometimes slightly oval 
(Figs. 104, 108); peristome complete, thin, 
slightly reflected at columellar margin; most 
specimens have a slight varix near shell ap- 
erture (Fig. 108); external lip thin; internal lip 
reflected towards umbilicus; umbilicus me- 
dium-sized, 180 pm in diameter (Fig. 110). 

Operculum: Ovate yellowish with darker cen- 
tral nucleus (Fig. 119); muscle attachment 
area rounded. 

Body: Head scarcely pigmented with scattered 
pigment cells at base of tentacles around 
eye-spots. Mantle with dispersed pigmented 
areas. Pigmentation quite variable among 
specimens. 

Nervous System (Fig. 132); With a short 
pleuro-supraesophageal connective; RPG 
ratio is 0.14 (concentrated). Esophagus fre- 
quently making a curve posteriorly to cere- 
bral commissure. 

Ctenidium - Osphradiun: Ctenidium absent or 
very poorly developed, with 5-7 small lamel- 
lae (Fig. 133). Osphradium oval, length two 
times the width (Table 3). 

Stomach - Radula: Chambers almost equal 
in size, longer than they are wide. Style sac 
protruding anteriorly into intestinal loop 
(Table 5). Rectum forming a marked S-loop, 
bending toward anterior portion of body. 
Radula with two basal cusps on each side 
of central tooth; its central denticle long, 
sharp, followed on each side by 4 long den- 
ticles in decreasing order of size; cutting 
edge of the central tooth markedly concave; 
lateral teeth with 4-5 long, sharp denticles 
on each side of central denticle. 

Male Genitalia: With large, long prostate gland, 
narrowing towards anterior part (Fig. 134); 
less than 50% of prostate gland extending 
into palliai cavity; penis very long with a small 
non-glandular lobe located in a distal posi- 
tion (Fig. 135); penis black pigmented 
pointed at penis tip; penial duct slightly un- 
dulating, running close to central part. 

Female Genitalia: With renal oviduct making 
a wide circle (Fig. 136); oviduct glands (al- 



REVISION OF THE GENUS ISLAMIA 



107 



124 




125 





128 



127 





129 




130 




131 



.SR2 




SRI 



FIGS. 124-131. Anatomy of /. henrici henhci. FIG. 124: Head of a female and nuchal node; FIG. 125: 
Partial nervous system node; FIG. 126: Rectum, osphradium and ctenidium node; FIG. 127: Stomach; 
FIG. 128: Prostate and rectum; FIG. 129: Head of a male and penis; FIG. 130: Anterior female genitalia; 
FIG. 1 31 : Detail of the seminal receptacles; Abbreviations in text. Scale bar = 500 pm (FIGS. 1 24-1 30). 



bumen + capsule glands) sometimes show- 
ing a narrowing; capsule gland larger than 
albumen gland and showing a narrowing at 



its distal outer margin, occupying more than 
50% of total palliai cavity length; proximal 
seminal receptacle (SR2) elongated, with 



108 



ARCONADA & RAMOS 



132 





134 



135 




136 





137 



SRL 




SR2 



FIGS. 132-137. Anatomy of /. henhci giennensis. FIG. 132: Partial nervous system: FIG. 133: Rectum, 
osphradium and ctenidium if present: FIG. 134: Prostate and end of rectum: FIG. 135: Penis: FIG. 
136: Anterior female genitalia: FIG. 137: Detail of the seminal receptacles; Abbreviations in text. 
Scale bar = 500 pm. 



swollen tip and bending 90" towards distal 
part of renal oviduct (Fig. 137): distal semi- 
nal receptacle (SRI) much smaller than 
proximal receptacle (Table 7), elongated or 
pyriform without evident stalk; seminal re- 
ceptacles located not far from one another 
In opposite positions on renal oviduct. 

Discussion 

All /. h. henhci and /. h. giennensis popula- 
tions studied show identical anatomical char- 
acters. However, some anatomical differences 
permit us to distinguish two "groups": one that 
includes all populations from Córdoba Prov- 
ince, and the other comprising populations from 
Jaén. The Jaén (/, /?. giennensis) populations 
are characterised by a slight varix near the shell 
aperture in most of the specimens (no varix in 



/. /?. henhci), a short supraesophageal connec- 
tive, RPG ratio = 0.14 (medium-sized in /. h. 
henhci, RPG ratio = 0.30), an oval osphradium 
(bean-shaped in /. h. henhci), a prostate elon- 
gated pear-shaped (bean-shaped in /. h. 
henhci), a penial lobe without any réfringent 
area (a small réfringent area present in /. h. 
henrici), a black pigmented penis (penis 
orangish pigmented in /. h. henhci), long and 
slender proximal seminal receptacle (SR2) 
(elongated with swollen tip in /. h. henhci), and 
the absence of a nuchal node in females. A 
nuchal node is a constant character in all fe- 
male specimens from Córdoba (/. h. henhci). 
There is a notable geographic distance be- 
tween both "groups", which decreases the 
probabilities of gene flow. The anatomical dif- 
ferences together with the large geographic 
distances between the "groups", allow us to 



REVISION OF THE GENUS ISLAMIA 



109 




О /. globulus 
A /. lagari 
■ /. ateni 
D /. pallida 

I. h. henrici 
• /. h. giennensis 



FIG. 138. Plot of discriminant scores on the two canonical axes, obtained from DFA of shell 
measurements for all Iberian Islamia species and subspecies: /. globulus, I. ateni, I. lagari, I. pallida, 
I. henrici henrici and /. h. giennensis. Confidence interval for ellipses: 0.95. 



divide this species into two subspecies, /. 
henrici henrici (Córdoba populations) and /. 
henrici giennensis (Jaen populations). How- 
ever, more specimens need to be studied to 
better understand the taxonomical identity of 
both entities. Unfortunately, due to declining 
populations, sample sizes were very small. 

Islamia henrici can be distinguished from 
other European Islamia species by a group of 
characters: an under-developed or absent 
ctenidium (the same character is reported for 
the Italian /. gaiteri, Bodon et al., 1995: 51); a 
rather long, black or orangish pigmented pe- 
nis, with a small, pointed lobe, which does not 
extend the penis tip. This small penial lobe is 
similar to that described for other species, such 
as Islamia galten (Bodon et al., 1995: 51) and 
Islamia sp. form C, from the population of Monti 
della Calvana (Giusti et al., 1981 : 66). In this 
latter species, however, the lobe is larger, 
nearly reaching the tip of the penis. A very 
small or often indistinguishable area of réfrin- 
gent non-glandular tissue is found at the base 
of the penial lobe (Fig. 129). The shape of the 
esophagus posterior to the cerebral commis- 
sure is a character that has not previously been 
described in any Islamia species. It slightly 
curved in /. henrici, whereas markedly so in 
Josefus aitanica (see below). Other differences 



among Iberian Islamia are: the reduction or 
absence of lamellae in the ctenidium, a long 
stalk on the proximal seminal receptacle, an 
orangish pigmented penis, and a protuberance 
on the female heads (the same described for 
/. pallida) of several populations. 

Statistical Analysis of Islamia species 

Conchological differences between Islamia 
species were investigated by a discriminant 
function analysis using the nine standard shell 
measurements on Table 1 (all except NSW). 
For /. globulus, the Sopeira population was 
selected as it had the greatest number of well- 
classified specimens as well as the highest 
number of specimens measured (n = 30). Four 
highly significant discriminant functions were 
found (Wilk's lambda = 0.0018, F (45, 267) = 
18.27, p < 0.0001). The variables included in 
these functions were: SW, WBW, LBW, AL, 
AW, and WAW. For the first function that ac- 
counted for 84.5% of explained variance, the 
characters that contribute (highest weight) 
were (in order): SW, WBW and LBW. For the 
second function, the order was: AL, LBW, SW, 
WAW and AW. All discriminant functions were 
highly significant (p < 0.0001 ). Of the 73 indi- 
viduals classified, all of the /. ateni, I. globulus. 



110 



ARCONADA& RAMOS 



/. lagari. and /. h. henriciv^ere correctly classi- 
fied (100%); 62.5% of the /. pallida individuals 
and 85.71% of /. h. giennensis were also cor- 
rectly classified. On the scatterplot (Fig. 138), 
six clusters are observed. Three of them over- 
lap and correspond to the taxa that have the 
most depressed-trochiform or valvatiform 
shells and shorter and wider body-whorls (/. 
pallida. I. h. henrici, and /. h. giennensis). 

Milesiana Arconada & Ramos, n. gen. 

Type Species 

Hauffenia {Neohoratia) coronadoi schuelei 
Boeters, 1981: 56, figs. 3. 4. 

Etymology 

This subgenus is dedicated to the musician 
Miles Davis, for his great contribution to art 
and pleasure. 

Diagnosis 

This genus differs from all others by having 
a proximal receptacle (SR2) sessile and much 
smaller than distal (SRI), which has a long 
stalk: the seminal receptacles arise rather close 
to one another: a big non-glandular lobe is lo- 
cated in medial position of the penis; left pleu- 
ral and subesophageal ganglia are fused, the 
pleuro-subesophageal connective is absent in 
Milesiana. whereas it is present in all the other 
European genera for which information on this 
character is available (Radoman, 1 983), except 
in the genus Josefus described herein. Other 
features characterizing Milesiana are: shell 
small, ovoid or more usually planispiral; oper- 
culum without peg; central tooth with two basal 
cusps on each side; the two seminal receptacles 
are located on opposite sides on unpigmented 
renal oviduct; bursa copulatrix absent, 

Milesiana schuelei {Boeters. 1981) 

Hauffenia (Neohoratia) coronadoi schuelei 

Boeters, 1981: 56, figs. 3,4. 
Hauffenia schuelei (Boeters, 1981) - Ber- 

nasconi, 1985: 65. 
Neohoratia schuelei (Boeters, 1981) - Boe- 
ters, 1988: 217, figs. 135-136, 159, 171, 

288, pi. 2, fig. 26. 
Islamia schuelei {Boeters. 1981)-Bodon etal.. 

2001: 179; Bodon & Cianfanelli, 2002: 20. 
Horatia gatoa Boeters, 1980 - Only paratype 

in figure 6, which is here re-identified as M. 

schuelei. 



Type Locality 

"West of two springs between Galera and 
Orce, Granada" (Boeters, 1981). 

Type Specimens 

Holotype in SMF 253578/1, paratypes in 
SMF 253579/1 , NNM, Falkner, ВОЕ 222a and 
223, ex Falkner, 308 and 308b, ex Wirth, 548 
and 549, ex Bou. 

In the original description, Boeters (1981) 
mentioned the type locality but not that of the 
paratypes. The only available information is: 
i) that the material was collected by Ulrich 
Wirth/Bonn (1963), Gerard Falkner/Hörlkofen 
and München (1967) and Claude Bou/Moulis, 
AIbi (1972), and ii) that species distribution 
includes: Prov. Granada, Velez-Benaudalla, 
spring at the road from Motril to Granada 
(UTM: VF 57), two springs between Galera 
and Orce (UTM: WG 47). Prov. Teruel, close 
to Caminreal in ground waters from a tribu- 
tary of the Jiloca River (UTM: XL 42). Prov. 
Jaén, between Peal de Becerro and Úbeda, 
in ground waters of the Guadalquivir River. In 
his 1988 paper, Boeters confirmed type local- 
ity ("west of two springs between Galera and 
Orce, Prov. Granada", (WG 47) and completed 
information on paratypes as follows: SMF 
253579/1, RMNH, FALK (Galera/Orce), ВОЕ 
222a and 223a (Galera/Orce), 308a and 308b 
(Velez-Benaudalla), 548 (tributary of the Jiloca 
River) and 549 (tributary of the Fardés River). 

Specimens Examined 

Type Materia! Examined: Holotype in SMF 
253578/1 (Figs. 139-142, 154). 

Other Populations Examined. This species 
was found in the provinces of Cádiz, Almería, 
Granada and Málaga (Fig. 17). A population 
found far from its distribution range, in the 
Cáceres province, was provisionally assigned 
to this species as M. cf. schuelei. The species 
has not been found in Teruel Province. 

Localities: Algodonales, Cádiz, UTM.: 
30STF8584, 19 Oct. 1998, E. R., MNCN 15.05/ 
46495 (ethanol); El Nacimiento spring, 
Turnllas, Almería, UTM: 30SWF657975, 15 
Oct. 1990, D. M., MNCN 15.05/46496 (etha- 
nol, SEM preparation), 10 Oct. 1992, E. R., D. 
M., MNCN 1 5.05/46497 (ethanol); Los Minutos 
spnng, Turrillas, Almeria, UTM: 30SWF6598, 
10 Oct. 1992, D. M., N. M., MNCN 15.05/46591 
(SEM preparation); Andarax spring, river and 
channel, Laujar de Andarax, Almeria, UTM: 
30SWF0994, 11 Jan. 1992, D. M., N. M.; 11 



REVISION OF THE GENUS ISLAMIA 



111 



Oct. 1992, E. R., D. M., MNCN 15.05/46498 
(ethanol); Agua spring, Lucainena de Las 
Torres, Almería, UTM; 30SWF7199, 10 Oct. 
1992, E. R., D. M., MNCN 15.05/46499 (etha- 
nol); Vêlez Blanco, Almería, UTM: 
30SWG7972, E. R., MNCN 15.05/ 46592; 
Talama spring, Bayarcal, Almería, UTM: 
30SWF0098, 26 March 1994, D. M., N. M., 
MNCN 15.05/46500 (ethanol and SEM prepa- 
ration), 14 May 1994, D. M., N. M.. MNCN 
15.05/46501 (ethanol); El Marchai de Antón 
López, Almería, UTM: 30SWF3383, E. R.; 26 



March 1998, В. A., MNCN 15.05/46502 (etha- 
nol, SEM preparation and frozen material); 
Pool in Berchul, Félix, Almería, UTM: 
30SWF298813, E. R., MNCN 15.05/46503 
(ethanol and SEM preparation), 26 March 
1998, B. A., MNCN 1 5.05/46504 (ethanol and 
frozen material); spring near the pool in 
Berchul, Félix, Almería, UTM: 30SWF298813, 
26 March 1998, B. A., MNCN 15.05/46505 
(ethanol); spring in Conchar, Granada, UTM.: 
30SVF477912, 25 Sept. 1989, E. R., D. M., С 
A., MNCN 1 5.05/46506 (dried); Faldés spring, 




FIGS 139-153. Shells oí Milesiana schuelei. FIGS. 139-142: Holotype (SMF 253578/1); FIG. 143: 
Shell from Fuente del Mai Nombre, Fadul (Granada); FIGS. 144, 148, 150, 153: Shells from Gaucin 
(Málaga); FIGS. 145, 151: Shells from Fuente Talama, Bayarcal (Almería); FIGS. 146, 149, 152: 
Shells from Fuente Los Minutos, Turrillas (Almería); FIG. 147: Shell from Benaoján (Málaga). Scale 
bar = 500 pm. 



112 



ARCONADA & RAMOS 



Sierra Harana Granada, UTM.: 30SVG592308, 
23 April 1992, D. M., MNCN 1 5.05/46507 (etha- 
nol). 12 Oct. 1992, E. R., D. M., MNCN 15.05/ 
46508 (ethanol); 25 March 1998, B. A. MNCN 
15.05/46509 (ethanol): Los Caños spring, 
Graena, Granada, UTM.: 30SVG810285, 27 
Sept. 1989. E. R.. D. M., С A., MNCN 15.05/ 

46510 (dried, ethanol): Pilar del Mono spring, 
Durcal. Granada. UTM.: 30SVF493951, 25 
Sept. 1989, E. R., D. M., С A., MNCN 15.05/ 

46511 (dried, ethanol). 17 Oct. 1989, J. T, D. 
M., 27 March 1998, B. A.. MNCN 15.05/46512 
(ethanol): La Gitana spring. La Peza, Granada, 
UTM.: 30SVG703255, 25 March 1998, B. A., 
MNCN 15.05/46513 (ethanol): spring in Fadul, 
Granada, UTM.: 30SVF4497, 25 Sept. 1989, 
E. R.,D. M.,C.A., MNCN 15.05/46514 (etha- 
nol), 17 Oct. 1989, D. M.: 30 Sept. 1989, E. 
R., MNCN 15.05/46515 (ethanol. SEM prepa- 
ration): Mai Nombre spring. Fadul. Granada, 
UTM.: 30SUF445963. 27 March 1998. B. A.. 



MNCN 15.05/46516 (ethanol and frozen ma- 
terial): spring in Gaucin. Málaga, UTM.: 
30STF9244, 22 Nov. 1988: E. R,, MNCN 
15.05/46517 (ethanol, SEM preparation), 15 
Apnl 1998, B. A., MNCN 15.05/46518 (etha- 
nol and frozen material): Matiaña spring, El 
Chorro, Málaga, UTM.: 30SUF468824. E. R., 
MNCN 15.05/46519 (ethanol). 14 April 1998. 
B. A.. MNCN 15.05/46520 (ethanol and fro- 
zen material): Wet wall in El Chorro, Málaga, 
UTM.: 30SUF468824, E. R., MNCN 15.05/ 
46521 (ethanol), 14 April 1998, B. A., MNCN 
15.05/46522 (ethanol and frozen material): 
Cueva del Gato, Benaoján, Málaga, UTM.: 
30SVF003673, 24 April 1992, D.M., MNCN 
15.05/46523 (ethanol, SEM preparation): 15 
April 1998, B. A., MNCN 15.05/46524 (etha- 
nol and frozen material): Avellano River, La 
Cimada, Málaga. U.TM.: 30SUF0976, E. R., 
MNCN 15.05/46525 (ethanol and SEM prepa- 
ration). 



TABLE 8. Shell measurements (in mm) of Milesiana schuelei from the following populations: 1 - 
Turrillas (El Nacimiento). Almería: 2 - Turrillas (Los Minutos spring), Almeria: 3 - Fadul, Granada; 4 - 
El Chorro. Málaga: 5 - Benaoján, Málaga. 



1 

Mean ± SD: 

CV(Max-Min) 

(n = 15) 



Mean ± SD: 

CV (Max-Min) 

(n = 29) 



Mean ±SD: 

CV (Max-Min) 

(n = 10) 



Mean ±SD; 

CV (Max-Min) 

(n = 17) 



Mean ±SD: 

CV (Max-Min) 

(n = 27) 



SL 
SW 

SL/SW 

АН 

LBW 

WBW 

AL 

AW 

WPW 

WAW 

NSW 



0.75 ±0.05: 
0.07 (0.85-0.68 

1.21 ±0.08; 
0.07(1.35-1.10 

0.62 ±0.06; 
0.09 (0.73-0.51 

0.65 ±0.09; 
0.14 (0.82-0.55 

0.65 ±0.04; 
0.07 (0.75-0.57 

0.81 ±0.07: 
0.08 (0.92-0.70 

0.52 ±0.03; 
0.07 (0.60-0.46 

0.50 ±0,07; 
0.15(0.60-0.25 

0.33 ±0.03; 
0.10(0.40-0.28 

0.12 ±0.02; 
0.19(0.10-0.08 

3.22 ±0.19; 
0.06 (3.50-3.00 



0.80 ±0.06; 
0.07 (0.94-0.65 

1.27 ±0.08; 
0.06(1.42-1.01 



0.62 ± O 
0.08 (0.73- 

0.62 ± O 
0.07 (0.82- 

0.71 ±0 
0.07(0.81- 

0.82 ± O 
0.06(0.91 

0.54 ± O 
0.06(0.91 

0.53 ± O 
0.05 (0.60 

0.33 ± O 
0.13(0.40 

0.14±0 
0.18(0.18 

3.13±0 
0.04 (3.50 



05; 
0.53 

04; 
0.55 

05; 
■0.60 

05; 
■0.68 

03; 
•0.68 

02; 
•0.44 

04; 
•0.24 

02; 
■0.08 

14; 
-3.00 



0.68 ±0.04; 
0.07 (0.74-0.57 

1.09 ±0.07; 
0.06 (1.18-0.97 

0.62 ±0.03; 
0.06 (0.70-0.57 

0.52 ±0.02; 
0.04 (0.57-0.50 

0.43 ±0.04; 
0.10 (0.48-0.35 

0.69 ±0.04; 
0.06 (0.77-0.61 

0.49 ±0.03; 
0.07 (0.55-0.42 

0.48 ±0.02; 
0.04 (0.52-0.45 

0.27 ±0.03; 
0.12 (0.32-0.21 

0.12±0.01; 
0.15(0.14-0.10 

3.00 ±0.00; 
0.00 (3.00-3.00 



0.68 ±0.05; 
0.08(0.80-0.57 

1.13±0.08; 
0.07(1.27-1.04 

0.59 ±0.04; 
0.07 (0.68-0.52 

0.53 ±0.04; 
0.07 (0.60-0.47 

0.60 ±0.05; 
0.09(0.71-0.50 

0.72 ±0.05; 
0.08(0.85-0.62 

0.49 ±0.04; 
0.09(0.61-0.44 

0.49 ±0.04; 
0.09(0.61-0.44 

0.27 ±0.04; 
0.17(0.34-0.18 

0.09 ±0.01; 
0.15(0.12-0.07 

3.02 ±0.08; 
0.02 (3.25-3.00 



0.92 ± O 
0.11 (1.13- 

1.32 ± O 
0.07(1.54- 

0.70 ± O 
0.10(0.87- 

0.67 ± O 
0.06 (0.74- 

0.79 ± O 
0.13(0.98- 

0.88 ± O 
0.12(1.33- 

0.58 ± O 
0.08 (0.67- 

0.58 ± O 
0.07 (0.69- 

0.38 ± O 
0.10(0.47- 

0.15±0 
0.15(0.21- 

3.28 ± O 
0.06(3.50- 



.10; 
■0.75) 

.09; 
■1.17) 

.07; 
■0.58) 

.04; 
0.58) 

.10; 
•0.63) 

.11; 
•0.75) 

.05; 
•0.52) 

.04; 
•0.52) 

.04; 
•0.32) 

.02; 

■0.11) 

.21; 
•3.00) 



REVISION OF THE GENUS ISLAMIA 



113 



TABLE 9. Operculum measurements (in mm) of 
Milesiana schuelei from Gaucin population 
(Málaga). 



MeaniSD; 
CV(Max-Min) 



MeaniSD; 
CV(Max-Min) 



OL 



NL 



OW 



NW 



0.59 ±0.12; 

0.21 (0.78-0.46) 

(n-5) 

0.47 ±0.07; 

0.16(0.60-0,40) 

(n = 5) 

OLWL 0.21 ±0.10; OUOW 

0.47(0.36-0.11) 

(n-5) 

OLWW 0.15 ±0.06; 
0.42 (0.26-0.09) 
(n = 5) 



0.24 ± 0.04; 

0.16(0.27-0.17) 

(n = 5) 

0.31 ±0.01; 

0.05 (0.34-0.29) 

(n-5) 

1.23±0.10; 

0.08 (1.38-1.11) 

(n = 5) 



M. cf. schuelei: Robladillo de Gata, Cáceres, 
UTM: 29TQE0764, E. R., MNCN 15.05/46526 
(ethanol, SEM preparation). 

Material Examined for Morphometry and His- 
tology 

Shell measurements (Table 8) correspond to 
populations from Almería, Granada and 
Málaga. Operculum and radular measurements 
(Tables 9, 1 1 ) to Málaga and anatomical mea- 
surements (Tables 10, 12-14) to Almería, 
Granada, Málaga and Cáceres (more details 
in table captions). Male and females studied 
and measured were collected in the following 
months: March, May, Sept., Oct. and Nov. For 
histology, seven specimens preserved in etha- 
nol were studied: four females from Benaoján, 
Málaga (April 1992) and two males and one 
female from Turhllas, Almería (Oct. 1990). 



Diagnosis 

Shell small, planispiral or valvatiform; oper- 
culum circular; ctenidium well developed; pleu- 
ral-subesophageal connective absent; large 
pear-shaped prostate gland; penis slightly or 
completely unpigmented, with large, non-glan- 
dular penial lobe located in medial position; 
proximal seminal receptacle (SR2) small, 
sessile, rounded; distal seminal receptacle 
(SR1) always larger than SR2, pyriform, pe- 
dunculated; receptacles located very close to 
one another on opposite positions on renal 
oviduct. 

Description 

Shell: Planispiral or valvatiform (Figs. 139, 
143-147, Table 8), 3-3.5 whorls (Figs. 142, 
153); sutures deep; body whorl expanded 
near aperture; protoconch consisting of 1.5 
whorls; protoconch width and width of 
nucleus are 315 pm and 110-126 pm, re- 
spectively; protoconch pitted (Figs. 154- 
156); aperture prosocline, rounded (Figs. 
143-147); umbilicus wide, approximately 
240 |jm in diameter (Figs. 141, 150-152); 
outer peristome simple, thin, straight; inner 
peristome slightly reflected at columellar 
margin (Fig. 140, 148, 149). 

Operculum: Circular with large, central nucleus 
(Fig. 1 57); muscle attachment area rounded 
(Fig. 158). 

Body: Head (Fig. 170) with black pigmenta- 
tion extending from around the eyes to 
middle of tentacles. 

Nervous System: Pleuro-subesophageal con- 
nective absent, pleuro-supraesophageal con- 
nective middle-sized, RPG ratio 0.24 
(concentrated). Esophagus runs straight un- 
derneath cerebral commissure (Fig. 166). 



TABLE 10. Osphradium measurements (in mm) oí Milesiana schuelei Uom the following populations: 
1 - Turrillas (El Nacimiento), Almería; 2 - El Laujar de Andarax, Almería; 3 - La Cimada, Málaga; 4 - 
Fadul, Granada; 5 - El Marchai, Almería; 6 - Lucainena de Las Torres, Almería; 7 - Gaucin, Málaga. 



Mean ± SD; 


Mean ± SD; 


Mean ± SD; 


Mean ± SD; CV Mean ± SD; 






CV (Max-Min) 


CV (Max-Min) 


CV (Max-Min) 


(Max-Min) CV (Max-Min) 






(n = 8) 


(n = 3) 


(n = 3) 


(n = 2) (n = 4) 


(n = 1)(n = 


= 1 



OsL 0.17 ±0.02; 0.23 ± 0.03; 0.24 ±0.01; 0.16 ±0.02; 0.22 ± 0.03; 

0.13(0.19-0.13) 0.11(0.25-0.20) 0.05(0.26-0.23) 0.14(0.17-0.14) 0.14(0.27-0.20) 0.21 0.16 

OsW 0.08±0.01; 0.10±0.02; 0.11 ±0.04; 0.08 ± 0.02; 0.10±0.03; 

0.09(0.10-0.07) 0.16(0.11-0.09) 0.38(0.14-0.06) 0.28(0.09-0.06) 0.30(0.13-0.06) 0.08 0.09 



114 



ARCONADA& RAMOS 



Ctenidium - Osphradium: Ctenidium with 8- 
13 well-developed lamellae (Fig. 167). 
Osphradium oval, two to three times longer 
than it is wide (Table 10). 

Stomach - Radula: Anterior and posterior 
stomach chambers are of approximately 
same size. Style sac protruding slightly an- 
teriorly into intestinal loop (Fig. 168, Table 



12). Rectum strongly U-shaped (Fig. 167). 
Radula long (40%) relative to maximum shell 
dimension (Fig. 159); central tooth with two 
basal cusps on each side (Table 11, Figs. 
160-162), distance between internal cusps 
7-8 [jm approximately; central denticle long, 
tapered, followed on each side by four long, 
tapered denticles in decreasing order of size; 




^■:::^-- 






10 um 




FIGS. 154-165. Protoconch, operculum and radula of Milesians sctiuelei. FIG. 154: Holotype (SMF 
253578/1): FIGS. 155, 156, 158, 161, 162: Protoconchs, operculum and radula from Gaucin (Málaga); 
FIGS. 159, 160, 163: Radula from Marchai de Antón López (Almería); FIG. 158: Inner side of the 
operculum; FIGS. 159, 160: Transverse rows; FIGS. 161, 162: Central teeth; FIGS. 163, 164: Central, 
lateral and inner marginal teeth; FIG. 165: Inner and outer marginal teeth. 



REVISION OF THE GENUS ISLAMIA 



115 



TABLE 11 . Radula formulae and measurements 
(in mm) of Milesiana schuelei from Benaoján 
(Málaga) population. 



Radula characters 



Formulae and 
measurements (in mm) 



Central teeth 
Central teeth width 
Left lateral teeth 
Inner marginal teeth 
Outer marginal teeth 
Radula length 
Radula width 
Number of rows 



4-(3.5)+C+4(3)/2-2 

~ 8 jjm 

4-5+C+3 

~ 22 cusps 

~ 10 cusps 

-351 |jm 

-46 pm 

-85 



lateral teeth with 3-4 denticles on each side 
central one (Figs. 163, 164); denticles of in- 
ner marginal teeth larger than those of outer 
marginal teeth (Fig. 165). 

Male Genitalia: With pear-shaped prostate 
gland (Fig. 1 69) almost two times longer than 
it is wide (Table 1 3), partially covered by rec- 
tum in palliai cavity; penis (Figs. 170, 171) 
generally unpigmented or with a slight dark 
pigmentation at base, with a blunt distal tip 
and one unpigmented, big, non-glandular 
lobe located in medial position; penial duct 
slightly undulating at the base, then running 
straight close to outer edge. 

Female Genitalia: With renal oviduct making 
a narrow circle overlying the part between 
albumen and capsule glands (Fig. 172), ovi- 
duct glands (albumen + capsule glands) well 
developed, sometimes narrowing at outer 
edge between capsule and albumen glands; 



capsule gland larger than albumen gland; 
distal seminal receptacle (SRI ) much larger 
than proximal (SR2); SRI pyriform, pedun- 
culated, SR2 rounded, sessile (Figs. 172, 
173, Table 14), located rather close to one 
another; the renal oviduct widening distally 
with respect to SR2. 

Discussion 

Milesiana schuelei cannot be assigned to the 
genus Islamia because of differences in sev- 
eral diagnostic characters including some of 
the female genitalia and principally those re- 
lated to the seminal receptacles. The numer- 
ous females studied and collected throughout 
different months of the year from populations 
of Almería, Granada, Málaga and Cáceres 
provinces had a remarkably large and pedun- 
culated distal seminal receptacle (SRI), 
whereas the proximal one (SR2) was small 
and sessile. Moreover, illustrations in Boeters 
(1 988: 218) depict a pedunculated distal semi- 
nal receptacle and a rounded and sessile 
proximal receptacle apparently protruding from 
the widened part of the renal oviduct, in a po- 
sition corresponding to that of the proximal 
seminal receptacle. Both character states, a 
very large and pedunculated distal seminal 
receptacle (SRI) and a proximal one (SR2) 
small and sessile, are the opposite of those 
observed in Islamia (SRI is always smaller or 
equal in size than SR2, and in addition SRI is 
usually sessile while SR2 is always peduncu- 
lated). 

Bernasconi (1975, 1977, 1984, 1985) de- 
scribed a larger distal seminal receptacle for 



TABLE 12. Digestive system measurements (in mm) of Milesiana schuelei from the following 
populations: 1 - Turrillas (El Nacimiento), Almería; 2 - La Cimada, Málaga; 3 - Gaucín, Málaga.; 4 - El 
Laujar de Andarax, Almería.; 5 - Padul, Granada; 6 - El Marchai, Almería. 



1 

Mean ± SD; 

CV(Max-Min) 

(n=3) 



Mean ± SD; 

CV(Max-Min) 

(n=4) 



Mean ± SD; 

CV (Max-Min) 

(n=2) 



Mean ± SD; 

CV (Max-Min) 

(n=3) 



Mean ± SD; 

CV (Max-Min) 

(n=2) 



n = 1 



Ss 
L 

Ss 

W 

St 
L 

St 

W 



0.26 ±0.02; 
0.08 (0.28-0.23) 

0.19 ±0.02; 
0.14(0.22-0.17) 

0.35 ±0.02; 
0.07 (0.37-0.32) 

0.26 ±0.01; 
0.04 (0.27-0.25) 



0.30 ±0.02; 
0.07 (0.33-0.29) 

0.24 ± 0.04; 
0.15(0.27-0.19) 

0.41 ±0.03; 
0.07 (0.45-0.38) 

0.39 ±0.05; 
0.12(0.44-0.34) 



0.21 ±0.02; 
0.11 (0.22-0.19) 

0.17 ±0.03; 
0.18(0.19-0.15) 

0.26 ±0.02; 
0.09 (0.28-0.24) 

0.29 ± 0.04; 
0.13(0.32-0.27) 



0.26 ±0.01; 
0.05 (0.28-0.25) 

0.21 ±0.02; 
0.08(0.22-0.19) 

0.36 ±0.04; 
0.11 (0.39-0.32) 

0.33 ±0.01; 
0.04 (0.34-0.32) 



0.21 ±0.06; 
0.28(0.26-0.17) 

0.17±0.01; 
0.08(0.18-0.16) 

0.30 ±0.04; 
0.15(0.33-0.27) 

0.25 ±0.01; 
0.02 (0.26-0.25) 



0.26 



0.20 



0.32 



0.33 



116 



ARCONADA & RAMOS 



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REVISION OF THE GENUS ISLAMIA 



117 



French Islamia species, which later Bodon et 
al. (2001: 199) considered to be a misinter- 
pretation. 

Mileslana schuelei shows a wide range of 
inter-population variability in shell shape, body 
pigmentation, and narrowing between the ovi- 



duct glands. Even the size of SRI varies al- 
though it is always much larger than SR2. In 
addition to the size and shape of the seminal 
receptacles, other characters that distinguish 
M. schuelei Uovi) other Iberian Islamia species 
include: a flatter shell, larger umbilicus, a well- 




166 







,•ííS^^,, 




167 



168 




170 / 




169 




171 




172 



SRI 




FIGS. 166-173. Anatomy of Milesians schuelei. FIG. 166: Partial nervous system; FIG. 167: 
Osphradium and ctenidium; FIG. 168: Stomach; FIG. 169: Prostate; FIG. 170, 171: Head of a male 
and penis; FIG. 172: Anterior female genitalia; FIG. 173: Detailof the seminal receptacles; Abbreviations 
in text. Scale bar = 500 pm (FIGS. 166-172). 



118 



ARCONADA & RAMOS 



TABLE 14. Female genitalia measurements (in mm) of Milesiana schuelei from the following populations: 
1 - El Marchai, Almería: 2 - Turrillas (El Nacimiento), Almería: 3 - La Cimada, Malaga: 4 - Gaucin, 
Málaga: 5 - El Laujar de Andarax, Almería. 





1 


2 


3 


4 


5 




Mean ± SD: 


Mean±SD; 


Mean ± SD; 


Mean ±SD; 


Mean ±SD; 




CV(Max-Min) 


CV(Max-Min) 


CV(Max-Min) 


CV(Max-Min) 


CV(Max-Min) 


OpL 


0.72 ±0.15; 


0.56 ±0.13; 


0.87 ±0.02; 


0.76 ±0.14; 


0.63±0.11; 




0.21 (0.87-0.56) 


0.23(0.78-0.45) 


0.03 (0.88-0.85) 


0.19(0.89-0.61) 


0.18(0.80-0.56) 




(n = 3) 


(n = 6) 


(n = 2) 


(n = 3) 


(n=4) 


OpW 


0.29 ±0.04; 


0.26 ±0.04; 


0.30 ±0.02; 


0.32 ±0.08; 


0.27 ±0.02; 




0.15(0.33-0.24) 


0.15(0.31-0.21) 


0.07 (0.32-0.29) 


0.24 (0.38-0.23) 


0.08 (0.30-0.24) 




(n = 3) 


(n = 6) 


(n = 2) 


(n = 3) 


(n = 4) 


Ag. L 


0.28 ± 0.07; 


0.21 ±0.06; 




0.28 ±0.07; 


0.31 ±0.07; 




0.25(0.35-0.21) 


0.31 (0.28-0.16) 


0.34 (n = 1) 


0.25 (0.33-0.20) 


0.22 (0.38-0.25) 




(n = 3) 


(n = 3) 




(n = 3) 


(n = 3) 


Cg. L 


0.43 ±0.20: 


0.40 ±0.10; 




0.44 ±0.13; 


0.34 ±0.07; 




0.46 (0.66-0.27) 


0.26 (0.49-0.28) 


0.55 (n = 1) 


0.30(0.58-0.31) 


0.21 (0.41-0.28) 




(n = 3) 


(n = 3) 




(n = 3) 


(n = 3) 


SR1 L 


0.11 ±0.02; 


0.13 ±0.02; 


0.16±0.01; 


0.11 ±0.00; 


0.10 ±0.02; 




0.18(0.12-0.09) 


0.17(0.16-0.12) 


0.03(0.16-0.15) 


0.03(0.11-0.11) 


0.23(0.13-0.09) 




(n = 3) 


(n = 6) 


(n = 3) 


(n = 2) 


(n = 3) 


SR2L 


0.04 ±0.01; 


0.04 ±0.02; 


0.07 ±0.01; 


0.05 ±0.02; 


0.06 ±0.01; 




0.26 (0.04-0.03) 


0.46(0.06-0.01) 


0.20 (0.09-0.06) 


0.51 (0.07-0.03) 


0.22 (0.07-0.05) 




(n = 2) 


(n = 6) 


(n = 3) 


(n = 2) 


(n = 3) 



developed ctenidium with large lamellae, rec- 
tum U-shaped, central tooth with two basal 
cusps, and a penial lobe located in a medial 
position instead of close to the penial tip. The 
pleuro-subesophageal connective is absent in 
Milesiana. whereas it is present in all the other 
European genera for which information on this 
character is available (Radoman, 1983), ex- 
cept in the genus Josefus described herein. 

Due to the peculiar structure of the female 
genitalia, M. schuelei can only be compared 
with Pezzolia Bodon & Giusti, 1986, another 
European valvatiform genus from Liguria 
(Italy), which has a distal seminal receptacle 
equal to or larger than the proximal receptacle. 
Nevertheless, the distal seminal receptacle in 
Pezzolia has no evident duct. This genus may 
at times have a very reduced bursa copulatrix. 
It has neither eyes nor ctenidium, and has only 
one basal cusp on the central tooth of the 
radula. This genus and its type species, 
Pezzolia radapalladis Bodon & Giusti, 1986, 
were described using extremely variable di- 
agnostic genital characters (Bodon et al., 
2001; 147-149, 158, 166, 167). According to 
these authors, Pezzolia may have a simple 
penis (with no glandular lobe) or there may be 



one or two glandular lobes, located in a me- 
dial position or one in a medial position and 
the other near the base of the penis. Pezzolia 
female genitalia can lack a bursa copulatrix 
(or if present, it is very small), and proximal 
seminal receptacle that can be equal to or 
smaller than the distal seminal receptacle. This 
unusual and extreme anatomical variability 
suggests that in order to clarify their taxonomic 
status, the morphological characters of all 
known populations of the genus Pezzolia and 
particularly those of the species Pezzolia 
radapalladis, P. sp. 1 and P. sp. 2 need to be 
carefully reviewed and studied. 

The combination of two diagnostic charac- 
ters (a large and pedunculated distal seminal 
receptacle and a short and sessile proximal 
receptacle), which is consistent in all studied 
populations of this widely distributed species, 
together with the absence of bursa copulatrix, 
the absence of pleuro-supraesophageal con- 
nective and other distinguishing shell and ana- 
tomical features, differentiates M. schuelei 
from all other known European Hydrobiidae 
valvatiform species. Therefore, we consider it 
justified creating distinct supraspecific taxa for 
this species, which we have called Milesiana. 



REVISION OF THE GENUS ISLAMIA 



119 



Josefus Arconada & Ramos, n. gen. 

Type species 

Josefus aitanica, n. sp. 

Etymology 

In memorlam of our friend and colleague 
Jose Bedoya "Josefo", who, through his skills 
working with the SEM, helped us to discover 
the huge morphological diversity and complex- 
ity of this small fauna. 

Diagnosis 

Shell small valvatiform or depressed- 
trochiform; operculum without peg; central 
tooth with two basal cusps on each side; pe- 
nis with a non-glandular lobe located in distal 
position; female genitalia with two seminal re- 
ceptacles adjacent to one another, on the 
same side of unpigmented renal oviduct; bursa 
copulatrix absent. 

Josefus aitanica Arconada & Ramos, n. sp. 

Type Specimens 

Holotype MNCN 15.05/46560 (SEM prepa- 
ration) (Fig. 174), Paratypes MNCN 15.05/ 
46560, 3 May 1994, E. R. (ethanol and SEM 
preparation - Figs. 177, 181, 182, 184 - and 
ethanol). 

Type Locality 

Torremanzanas, Alicante, UTM.: 30SYH2476. 

Etymology 

The name aitanica refers to Sierra de Altana, 
a mountain chain in the distribution area of this 
species. 

Populations Studied 

This species was found in the provinces of 
Valencia and Alicante (Fig. 17). Lapica spring. 
Las Vihuelas, Valencia, UTM.: 30SXJ7155, 28 
May 1998, B. A. & J. A., MNCN 15.05/46561 
(dried and frozen material); La Granata, Taber- 
nes de La Valldigna, Valencia, UTM.: 
30SYJ358302, 21 March 1994, E. R., MNCN 
1 5.05/46562 (ethanol), 27 May 1 998, B. A. & J. 
A., MNCN 15.05/46563 (ethanol, SEM pre- 
paration and frozen material); Gamellons spring, 



Onteniente, Valencia, UTM.: 30SXH975942, 5 
Oct. 1994, E. R., MNCN 15.05/46564 (ethanol), 

29 May 1998, B. A., MNCN 15.05/46565 (etha- 
nol and frozen matehal); Gaspar spring, Beni- 
ganim, Valencia, UTM.: 30SYJ2113, 5 April 
1994, E. R., MNCN 15.05/46566 (ethanol), 29 
May 1998, B. A. & J. A., MNCN 15.05/46567 
(ethanol and frozen material). Pi spring, Beni- 
ganim, Valencia, UTM.: 30SYJ2113, 5 April 
1994, E. R., MNCN 15.05/46568 (ethanol); Ca- 
mello spring, Cuatretonda, Valencia, UTM.: 
30SYJ2514, 1 April 1994, G. T., MNCN 15.05/ 
46569 (ethanol and SEM preparation); La Mina 
source, Jarafuel, Valencia, UTM.: 
30SXJ645341, 28 May 1998, B. A. & J. A., 
MNCN.15.05/33290; Bella spring, Jarafuel, Va- 
lencia, Flores spring, Requena, Valencia, UTM: 
30SXJ615725, 29 March 1992, G. T, MNCN 
15.05/33263 (ethanol and SEM preparation), 27 
May 1998, B. A. & J. A., MNCN 15.05/33289 
(ethanol and frozen material); El Tollo spring, 
Requena, Valencia, UTM.: 30SXJ671513, 5 
May 1994, E.R., MNCN 15.05/46570 (ethanol); 
El Moro spring, L'Algar springs. Callosa d'en 
Sarria, Alicante, UTM.: 30SYH527831 , 8 Dec. 
1990, G. T, MNCN 15.05/46595 (ethanol); 30 
May 1998, B. A. & J. A., MNCN 15.05/46571 
(ethanol and frozen material); Reyinyosa 
spring, Bolulla, Alicante, UTM.: 30SYH5185, 

30 April 1994, E. R., MNCN 15.05/46572 (etha- 
nol), 30 May 1998, B. A. & J. A., MNCN 15.05/ 
46573 (ethanol); Moli Montes spring. Agres, 
Alicante, UTM.: 30SYH1595, 3 May 1994, E. 
R., MNCN 1 5.05/46574 (ethanol); Azut spring, 
Alfafar, Alicante, UTM.: 30SYH12394, 4 May 
1994, E. R., MNCN 15.05/46575 (ethanol), 29 
May 1998, B. A. & J. A., MNCN 15.05/46576 
(ethanol and frozen material). 

Specimens Examined for Morphometry and 
Histology 

Shell and anatomical measurements (Tables 
15, 17-19) correspond to populations from 
Alicante and Valencia. Operculum and radu- 
lar measurements (Tables 15, 16) correspond 
to the population from type locality (more de- 
tails in table captions). Male and females stud- 
ied and measured were collected in the 
following months: March, April, May and Oct. 
For histology, one male and two females from 
type locality (May 1995) were studied. 

Diagnosis 

Operculum ovate; ctenidium absent; central 
tooth with two basal cusps on each side; eso- 



120 



ARCONADA& RAMOS 



phagus making a loop to the left posterior to 
cerebral ganglion complex: pleuro-sub- 
esophageal connective absent: rhomboid- 
shaped prostate gland: long pigmented penis 
with large non-glandular lobe located in dis- 



tal position, never protruding from penis tip; 
two seminal receptacles small, sessile, 
rounded, equal in size, situated side by side 
on renal oviduct: all females with a nuchal 
node. 




FIGS. 174-184. Shells of Josefusa/ian/ca. FIGS. 174, 177, 181, 182, 184: Shells from Torremanzanas 
population (type locality): FIG. 174: Holotype (MNCN 15.05/46560): FIGS. 175, 176, 178^180, 183: 
Shells from Tabernes de la Valldigna population: FIGS. 181, 182: Varix separating protoconch and 
teleoconch. Scale bar = 500 pm (FIGS. 174-179). 



REVISION OF THE GENUS ISLAMIA 



121 



Description 

Shells: Valvatiform or depressed-trochlform 
(Table 1 5; Figs. 1 74, 1 75) with 3-3.5 whorls 
(Figs. 1 77, 1 78); about 1 .5 spire whorls (Figs. 
180, 181); highly developed body whorl 
(Figs. 177, 178); protoconch pitted (Figs. 
1 83, 1 84), with 1 .5 whorls; protoconch width 
300 pm and width of nucleus approximately 
1 05 pm; occasional varix observed at the end 
of protoconch seen in all populations (Figs. 
181,1 82); prosocline and rounded aperture; 
umbilicus of intermediate size, about 1 25 pm 
in diameter (Fig. 1 79); external lip (Figs. 1 76, 
1 77) sometimes becoming thinner at its outer 
margin. 



Operculum: Yellowish, oval, with rounded, big, 
central nucleus (Fig. 185); muscle attach- 
ment area rounded (Fig. 186). 

Body: Head with black-pigmented area from 
middle of tentacles to back of eye lobes (Figs. 
191, 197); external body pigmentation dark. 

Nervous System: Mid-sized pleuro-supra- 
esophageal connective; pleuro-subeso- 
phageal connective absent (Fig. 192); 
supaesophageal ganglion small; RPG ratio 
0.22 (concentrated). Esophagus making a 
marked loop posterior to left posterior to ce- 
rebral ganglia (Fig. 193). 

Ctenidium - Osphradium: Ctenidium absent 
(Fig. 1 94). Osphradium oval, two times longer 
than it is wide (Table 15). 



TABLE 15. Shell, operculum and osphradium measurements (in mm) of Josefus aitanica from the 
following populations: 1 - Callosa d'en Sarria, Alicante; 2 - Requena (Flores spring); 3 - type locality. 





Mean ± SD; 

CV(Max-Min) 

(n = 21) 


Mean ± SD; 

CV(Max-Min) 

(n = 10) 


SL 


0.96 ±0.06; 
0.06(1.07-0.83) 


1.35±0.17; 
0.13(1.53-0.97) 


SW 


1.08 ±0.08; 
0.08(1.24-0.83) 


1.33±0.14; 
0.11 (1.58-1.06) 


SL/SW 


0.89 ±0.10; 
0.11 (1.26-0.83) 


1.01 ±0.07; 
0.07(1.15-0.91) 


AH 


0.62 ±0.03; 
0.05 (0.68-0.57) 


0.84 ±0.09; 
0.11 (0.09-0.06) 


LBW 


0.85 ±0.05; 
0.06 (0.94-0.70) 


1.19±0.14; 
0.12(1.34-0.89) 


WBW 


0.75 ±0.05; 
0.07(0.91-0.64) 


1.07 ±0.12; 
0.11 (1.22-0.81) 


AL 


0.60 ±0.03; 
0.05 (0.64-0.53) 


0.79 ±0.08; 
0.10(0.90-0.62) 


AW 


0.53 ±0.03; 
0.06 (0.60-0.48) 


0.68 ±0.07; 
0.11 (0.78-0.54) 


WPW 


0.35 ±0.04; 
0.11 (0.41-0.24) 




WAW 
NSW 


0.14 ±0.02; 
0.17(0.21-0.08) 

3.15±0.18; 
0.06 (3.50-3.00) 


3.30 ±0.16; 
0.05(3.50-3.00) 



Mean ±SD; 
CV(Max-Min) 



OL 



OW 



OLWL 



OLWW 



NL 



NW 



OL/OW 



OsL 



Os W 



0.60 ±0.05; 

0.08 (0.64-0.57) 

(n = 2) 

0.47 ± 0.00; 

0.01 (0.47-0.46) 

(n = 2) 

0.21 ±0.04; 

0.21 (0.25-0.18) 

(n = 2) 

0.15±0.01; 

0.11 (0.16-0.13) 

(n = 2) 

0.27 ±0.00; 

0.00 (0.27-0.27) 

(n = 2) 

0.30 ±0.00; 

0.02 (0.30-0.29) 

(n = 2) 

1.29 ±0.12; 

0.09(1.38-1.20) 

(n = 2) 

0.19 ±0.08; 

0.39(0.30-0.10) 

(n = 5) 

0.08 ±0.03; 

0.36(0.12-0.05) 

(n = 5) 



122 



ARCONADA & RAMOS 




FIGS. 185-190. Operculum and radula of Josefusa/fan/ca. FIGS. 185, 186, 189, 190: Opercule and 
radula from Torremanzanas population (type locality); FIGS. 187, 188: Radula from Cuatretonda 
population; FIG. 185: Outer side of the operculum; FIG. 186: Inner side of the operculum; FIG. 187: 
Transverse rows; FIG. 188: Central and lateral teeth; FIG. 189: Central teeth; FIG. 190: Lateral, inner 
and outer marginal teeth. Scale bar = 200 pm (FIGS. 185, 186); 100 pm (FIG. 187). 



191 




REVISION OF THE GENUS ISLAMIA 

192 '^^^ 



123 






194 






195 



196 



197 





FIGS. 191-198. Anatomy o^ Josefus aitanica. FIG. 191: Head of a female and nuchal node; FIGS. 
192, 193: Partial nervous system; FIG. 194: Rectum and osphradium; FIG. 195: Stomach; FIG. 196: 
Prostate; FIG. 197: Head of a male and penis; FIG. 198: Anterior female genitalia; Abbreviations in 
text. Scale bar = 500 |jm. 



124 



ARCONADA & RAMOS 



TABLE 16. Radula formulae and measurements 
(in mm) of Josefus aitanica from type locality. 



Radula characters 



Formulae and 
measurements (in mm) 



Central teeth 
Central teeth width 
Left lateral teeth 
Inner marginal teeth 
Outer marginal teeth 
Radula length 
Radula width 
Number of rows 



5+C+5/2-2 

~ 6.3 |jm 

5+C+3 

~ 22 cusps 

- 24 cusps 

-400 |jm 

-43 Mm 

-85 



Stomach - Radula: Length and width equal, 
stomach chambers same size; style sac pro- 
truding anteriorly into the intestinal loop 
(Table 17, Fig. 195). Rectum U-shaped (Fig. 



TABLE 17. Digestive system measurements (in 
mm) of Josefus aitanica. Populations from: (a) 
Torremanzanas, Alicante (type locality); (b) Cal- 
losa d'en Sarria, Alicante; (с) Tabernes, Valen- 
cia; (d) Requena, Valencia. 



n = 1 


SsL 
SsW 
StL 
StW 


0.18(a) 
0.18(a) 
0.36(a) 
0.33(a) 


0.27(b); 0.26(c); 0.24(d) 
0.22(b); 0.14(c); 0.21(d) 
0.30(b); 0.28(c); 0.36(d) 
0.37(b); 0.28(c); 0.34(d) 



194). Radula (Table 16, Fig. 187) long (41%) 
relative to maximum shell dimension; cen- 
tral trapezoidal tooth with two basal cusps 
on each side that points towards the lateral 
margins (Figs. 188, 189); cutting edge mark- 
edly concave, five denticles in decresing or- 
der of size at each side of central denticle, 



TABLE 18. Male genitalia measurements (in mm) of Josefus aitanica from the following localities: 1 - 
Torremanzanas. Alicante (type locality); 2 - Beniganim, Valencia; 3 - Onteniente, Valencia; 4 - Tabernes, 
Valencia; 5 - Callosa den Sarria, Alicante; 6 - Requena (El Tollo), Valencia; 7 - Agres, Alicante. 





1 


2 


3 


4 


5 


6 


7 




Mean ± SD; 


Mean ± SD; 


Mean ± SD; 


Mean ± SD; 










CV(Max-Min) 


CV(Max-Min) 


CV(Max-Min) 


CV(Max-Min) 








PrL 


0.37 ±0.03; 

0.07 (0.39-0.36) 

(n = 2) 












0.44 
(n = 1) 


PrW 


0.19 ±0.03; 

0.16(0.21-0.17) 

(n = 2) 












0.22 

(n = 1) 


PL 


0.64 ± 0.24; 


0.73 ±0.12; 


0.93±0.11; 


0.70 ±0.04; 










0.37(0.94-0.34) 0.16(0.81-0.65) 
(n = 5) (n = 2) 


0.12(1.03-0.81) 0.05(0.72-0.66) 
(n = 3) (n = 3) 


0.39 
(n = 1) 


0.63 
(n = 1) 


1.01 
(n = 1) 


P W 


0.17 ±0.06; 


0.12 ±0.00; 


0.18 ±0.02; 


0.17 ±0.02; 










0.33(0.24-0.10) 
(n = 5) 


0.01 (0.12-0.12) 0.12(0.21-0.16) 0.13(0.19-0.15) 
(n = 2) (n = 3) (n = 2) 




0.15 
(n = 1) 


0.22 
(n = 1) 


Pl.L 


0.16 ±0.05; 


0.12 ±0.03; 


0.17 ±0.02; 


0.12 ±0.02; 


0.16 


0.14 


0.13 




0.29(0.23-0.10) 0.27(0.14-0.09) 0.09(0.19-0.16) 0.14(0.10-0.14) 
(n = 5) (n = 2) (n = 3) (n = 3) 


(n = 1) 


(n = 1) 


(n = 1) 


Pl.W 


0.11 ±0.04; 


0.08 ±0.00; 


0.14 ±0.02; 


0.08 ±0.02; 










0.38(0.18-0.07) 0.04(0.09-0.08) 0.12(0.15-0.12) 0.22(0.10-0.07) 
(n = 5) (n = 2) (n = 3) (n = 3) 


0.09 
(n = 1) 


0.10 
(n = 1) 


0.12 
(n = 1) 


Head 


0.57 ±0.13; 




0.74 ± 0.05; 


0.59 ±0.06; 








length 


0.23 (0.75-0.46) 
(n = 5) 


0.66 (n = 1) 


0.07 (0.77-0.70) 0.10 (0.66-0.54) 
(n = 2) (n-3) 


0.57 
(n = 1) 


0.46 
(n = 1) 


0.81 
(n = 1) 


PL/ 


0.87 ±0.52; 




1.3 5± 0.02; 


1.19±0.18; 








Head 
length 


0.14(1.57-0.14) 
(n = 5) 


0.98 (n = 1) 


0.01 (1.36-1.33) 0.15(1.35-1.00) 
(n = 2) (n = 3) 


0.69 
(n = 1) 


1.37 
(n = 1) 


1.25 
(n = 1) 



REVISION OF THE GENUS ISLAMIA 



125 



TABLE 1 9. Female genitalia measurements (in mm) of Josefus aitanica from the following populations: 
1 - Torremanzanas, Alicante (type locality); 2 - Callosa d'en Sarria, Alicante; 3 - Requena (Flores 
spring), Valencia; 4 - Tabernes, Valencia. 





1 

Mean ±SD; 

CV(Max-Min) 


2 

Mean ± SD; 

CV(Max-Min) 


3 

Mean ±SD; 

CV(Max-Min) 


4 
Mean ± SD; 
CV(Max-Min) 


OpL 


0.58 ±0.09; 

0.15(0.70-0.47) 

(n = 5) 


0.64 (n = 1) 


0.65 ±0.10; 

0.15(0.72-0.58) 

(n = 2) 


0.64 (n = 1) 


OpW 


0.20 ±0.03; 

0.15(0.16-0.15) 

(n = 5) 


0.26 (n = 1) 


0.27 ±0.04; 

0.14(0.30-0.24) 

(n = 2) 


0.28 (n = 1) 


Ag. L 


0.26 (n = 1) 


0.34 (n = 1) 


0.24 ± 0.02; 

0.09 (0.26-0.22) 

(n = 2) 


0.37 (n = 1) 


Cg. L 


0.36 (n = 1) 


0.30 (n = 1) 


0.42 ±0.12; 

0.29 (0.50-0.33) 

(n = 2) 


0.27 (n = 1) 


SRI L 


0.08 ±0.02; 

0.19(0.10-0.07) 

(n = 3) 


0.05 ±0.01; 

0.16(0.05-0.04) 

(n = 2) 


0.08 ±0.02; 

0.18(0.10-0.07) 

(n = 2) 


0.07 ±0.01; 
0.08 (0.07-0.06) 
(n = 2) 


SR2L 


0.05 (n = 1) 


0.05 ±0.01; 

0.16(0.05-0.04) 

(n = 2) 


0.06 ±0.01; 

0.13(0.06-0.05) 

(n = 2) 


0.06 (n = 1) 



lateral teeth with five denticles on each side 
a central one (Fig. 188); denticles of inner 
marginal teeth larger than those of outer 
marginal teeth (Fig. 190). 

Male Genitalia: Prostate gland (Fig. 196; Table 
18), almost rhomboidal, more slender ante- 
riorly and located quite posterior to rectum 
loop; posterior vas efferens entering near 
middle prostate region and anterior vas 
efferens exits close to this point; penis large, 
dark pigmented (Fig. 197), with a well-de- 
veloped, non-glandular, subterminal, unpig- 
mented lobe that is longer than penis tip; 
penial duct undulating along penis length at 
right edge. 

Female Genitalia: Two seminal receptacles, 
small, sessile, rounded, equal in size, aris- 
ing side by side on the renal oviduct facing 
the albumen gland (usual position where 
SR2 arises from proximal oviduct) (Fig. 1 98); 
renal oviduct not widening posteriorly to SR2 
and makes a tight circle over palliai oviduct; 
oviduct glands (albumen + capsule glands) 
do not usually narrow, although some fe- 
males narrow slightly at outer edge, between 
capsule and albumen gland; albumen gland 
smaller than capsule gland, and occupying 



approximately 40% of total length of palliai 
oviduct; ovary overlying posterior chamber 
of stomach. Unpigmented nuchal node (Fig. 
191) in an analogous position to that of pe- 
nis, occupying V4 of total head length, 0.14 
pm approximately. 

Discussion 

Josefus aitanica shows little interpopulation 
variability in the size of the oviduct glands, the 
presence/absence of narrowing between cap- 
sule and albumen glands, and the size and 
colour of the penis. All females studied and 
collected in different months throughout the 
year - March, April, May, Oct. - had a nuchal 
node, similar to that described in females of 
the genus Islamia. No cases of parasitism were 
detected. The esophagus forms a tight pleat 
below the left posterior portion of the pleuro- 
oesophagal ganglionic complex, whereas it is 
only slightly curved in /. henrici, the only 
Hydrobiidae species in which this character 
has been described. The new species can be 
distinguished from all the other Hydrobiidae 
by the shape and position of the seminal re- 
ceptacles, which are both sessile, equal in size 



126 



ARCONADA& RAMOS 



and emerge adjacent to each other on the 
same side of the renal oviduct. In the very few 
Islamia species where the two seminal recep- 
tacles have been observed close to one an- 
other (/. valvataeformis or /. pallida), they 
appear on opposite sides of the renal oviduct 
and, unlike in J. aitanica, are never equal in 
size and shape. The loop made by the renal 
oviduct is rather small and quite tight, and there 
is no widening of the oviduct before the loop. 



DISCUSSION 
Habitat Status and Conservation 

The species described here live in apparently 
non-polluted springs, rich in aquatic vegetation. 
Specimens can be found on vegetation, stones, 
wet walls and in mud. Milesiana schuelei has 
the widest geographical distribution range of 
the species studied. In the last decade, M. 
schuelei has been severely threatened in 
Almeria Province due to engineering projects 
aimed at optimising water resources in this ex- 
tremely arid area, thus depleting groundwater 
resources essential for hydrobiid survival. In 
contrast, Islamia globulus populations are well 
conserved, since water resources are sufficient 
in its distribution area. Islamia ateni is only 
known from its type locality (Balneario de San 
Vicente), a thermal spring that was seriously 
affected by the construction of a motorway. 
Since then, no specimens have been found, 
suggesting they are probably now extinct. 
Specimens of /. pallida. I. henrici henhci and /. 
h. giennensis are rare in the springs where they 
were discovered. Both species have a very 
narrow distribution and are highly threatened 
by human activities. The populations of the last 
two subspecies have been declining since they 
were first found. Channelization has dessicated 
many of the natural habitats of /. h. giennensis. 
The species has disappeared from some of the 
springs that previously held many of the bet- 
ter-conserved populations. 

The same is occurring with Josefus aitanica, 
although the majority of its populations are not 
yet threatened. Islamia lagari is restricted to a 
very small area (Sierra de Can Parés), al- 
though no live specimens have been collected 
for years. Following lUCN criteria we classify 
these species as follows: Extinct (EX) - Islamia 
ateni; Critically Endangered (CR) - Islamia 
pallida, I. lagari and both subspecies of /. 
henrici as: Lower Risk (LR) - Islamia globulus, 
Josefus aitanica and Conservation Dependent 
(cd) - Milesiana schuelei. 



Genital Morphology and Functionality 

Taxonomy at the rank of genus and family 
levels has been traditionally based on anatomi- 
cal characters, especially those of the male 
and female genitalia. Among these, penis 
structure and number and position of the sac- 
like structures associated with the renal ovi- 
duct have usually received more taxonomic 
weight as they are generally constant in spe- 
cies and species groups. 

The exact function of the sac-like structures 
on the renal oviduct of females of Islamia and 
Neohoratia has long been in question. It has 
been thought that these structures are either 
two seminal receptacles or a small bursa 
copulatrix and a seminal receptacle. In the 
past, authors described these structures in 
many species as a seminal receptacle and a 
pin-like or sessile bursa copulatrix (Bole, 1 970: 
Bernasconi, 1975). Histological observations 
and other direct morphological evidence have 
clarified many previous doubts regarding these 
structures. Pearly-whitish réfringence is un- 
doubtedly related to the way spermatozoa are 
organized in the seminal receptacles or in 
other sperm storage areas of the renal ovi- 
duct (Davis & Kang, 1990: Davis et al. 1990; 
Ramos et al., 2001). The bursa copulatrix is 
almost translucent and its contents are never 
réfringent. The location of the sac-like struc- 
tures in relation to the ovary and the palliai 
glands (albumen + capsule glands) is also 
useful for identification. When the bursa 
copulatrix is absent and there are two semi- 
nal receptacles, the proximal seminal recep- 
tacle (SR2) emerges from the oviduct close to 
the end of the loop, and the distal seminal re- 
ceptacle (SR1 ) originates at a point closer to 
where the oviduct enters the albumen gland, 
close to but more proximally located than the 
usual position of the bursa copulatrix (Bodon 
etal., 2001). 

The epithelium differs between the bursa and 
the seminal receptacles, as does the physi- 
ological function of these organs and the way 
spermatozoa are dispersed within them. In the 
receptacles, the spermatozoa face the cilia of 
the inner epithelial cells, while they have no 
directional pattern in the bursa (see Genital 
Histology above). Bodon et al. (2001) stated 
that Islamia ateni, I. globulus, and /. /agar/ have 
two seminal receptacles. Histological evidence 
and morphological observation of the female 
genitalia of Milesiana schuelei, Islamia 
globulus. I. h. henrici, and Josefus aitanica in- 
disputably confirm their assertion, and we ap- 
ply it to all the species studied herein. Given 



REVISION OF THE GENUS ISLAMIA 



127 



that the female genitalia of Neohoratia 
subpiscinalls (Kuscer, 1932) are currently de- 
scribed as having a poorly developed bursa 
copulatrix and a single seminal receptacle 
(Bole, 1993; Bodon et al., 2001 ), we redefine 
the taxonomic status of some Iberian taxa that 
were previously referred to and included in the 
genus Neohoratia (as N. globulus globulus, N. 
g. lagan, N. ateni) and ascribe them to Islamia, 
following previous papers (Bodon et al., 2001 ). 

Without providing real histological evidence 
(serial sections), some authors have inter- 
preted the réfringent area, or "banda 
traslucida", in the penial lobe of Islamia spe- 
cies to be a mass of glandular cells (Giusti et 
al., 1981: 51, Bodon et al., 2001: 133). This 
area can also be observed in the penis when 
mounted on microscope slides. This interpre- 
tation led Bodon et al. (2001 : 1 34) to conclude 
that Islamia had a "penis with one glandular 
(rarely non-glandular) lobe". This is the first 
study to investigate the penial lobe of Islamia 
species using histological serial sections. The 
males we observed show this refringency in 
the penial lobe (also seen in microscope 
slides), although it lacks glandular tissue. We 
conclude that morphological réfringence in 
penial structures cannot be attributed to a 
mass of glandular cells. 

Bodon et al. (2001) studied two males from 
the type locality of /. valvataeformis as well as 
/. globulus from two population of Huesca. He 
concluded that the réfringence observed in the 
penial lobe of both species was made up of a 
mass of glandular cells. We were unable to 
study specimens of the type species of the 
genus, but the serial sections of the /. globulus 
we examined clearly demonstrated that the 
réfringence observed in its penial lobe was of 
a non-glandular nature. In view of our findings, 
we suggest eliminating from the diagnosis of 
the genera any reference to the nature of the 
tissue observed in the réfringent area of the 
penial lobe if the tissue has not been studied 
using serial sections. Further histological stud- 
ies of this kind for the type species /. 
valvataeformis are particularly needed. 

Character Variability in the Genus Islamia 

Radoman (1973a) introduced the genus 
Islamia (type species: Horatia servaini Bour- 
guignat, 1887, a junior synonym of Hydrobia 
valvataeformis Möllendorf, 1873, according to 
Radoman, 1983, from Vrelo Bosne, near 
Sarajevo), with two subgenera, Islamia and 
Adriolitorea (type species: /. {Ad rio lito re a) 
zermanica Radoman 1973, from the Zrmanja 



River, in the middle freshwater section). Each 
subgenus contained two species from the 
Balkans: /. (Islamia) servaini (Bourguignat 
1887), /. (Islamia) bosniaca Radoman, 1973 
/. (Adriolitorea) zermanica Radoman, 1973 
and /. (Adriolitorea) latina Radoman, 1973. 
Radoman (1973a) stated that the four species 
are anatomically identical except for a slight 
difference in penis structure, which justified 
their separation into two groups Ç'Bien que 
I'anatomie de toutes ces espèces soit 
identique, il y a une légère difference dans la 
structure du pénis, ce qui les sépare en deux 
groupes"): The penis is slightly split at the top 
in Islamia, whereas the penial branches are 
longer and slightly more slender in Adriolitorea. 
Based on this difference the author suggested 
that there were two ancestors for these two 
groups of species, one from central Bosnia 
(Islamia s.s.) and the other from the coastal 
area (Adriolitorea). Later on, Radoman 
(1973b) included the following species in 
Islamia: a new species from Greece (/. graeca 
Radoman 1973), two new species from Tur- 
key (/. pseudorientalica Radoman 1973, and 
/. anatolica Radoman 1973), plus one previ- 
ously described species /. burnabasa (syn. 
Horatia burnabasa Schutt, 1964). The last 
three live in sympatry (type locality: Kirkgöz, 
Anatolia, Turkey). Although these descriptions 
were based on conchological characters, 
Radoman (1973b) concluded that all the spe- 
cies were anatomically identical to other spe- 
cies of the genus Islamia. In his 1983 paper, 
he assigns all eight above-mentioned species 
from Bosnia-Herzegovina, Croatia, Greece 
and Turkey plus /. trichoniana Radoman, 1978, 
from Greece to Islamia. The subgenus 
Adriolitorea was, therefore, regarded as a syn- 
onym of Islamia. According to Radoman 
(1973a, 1983) Islamia is characterised by: "(1) 
shell valvatoid, with a roundish-ovoid aperture 
and wide umbilicus, (2) central tooth of the 
radula with two basal cusps (one on each side, 
according to drawings of Radoman, 1973a), 
(3) a long pleuro-supraintestinal and a short 
pleuro-subintestinal connective, and (4) two 
seminal receptacles present (rsl and rs2), 
nearby at the same level, draining into the 
oviduct. A genital chamber absent." The penis 
is described as "very large, muscular, wide, 
split at the top, vas deferens draining at the 
point of the right branch. Near the penis point, 
on the ventral side, a muscular fold is present. 
Penis shape is to some extent variable in dif- 
ferent species of this genus" (Radoman, 1 983: 
124, figs. 69, 70). In fact, while the size and 
shape of the two penial branches differ among 



128 



ARCONADA& RAMOS 



these species, all possess a muscular pleat 
at the centre of the ventral side of the penis. 
Radoman did not nnention any glandular tis- 
sue inside the penis branches. Description of 
the female genital system was only provided 
for the type species (/. valvataeformis) 
(Radoman, 1973a, 1983), and according to 
Radoman's comments (1973a, b) female geni- 
talia do not seem to vary among species. In 
other words, only conchological and penial 
characters differ among Islamia species. 

The tenth species assigned to Islamia was 
Valvata pusilla Piersanti, 1952 (Giusti et al., 
1981). from Italy (type locality: Grotta delle 
Fontanelle, Napoli). In the description of this 
species, the authors introduced for the first 
time the concept that the translucid band ob- 
served on the penial lobe corresponded to a 
mass of glandular cells. They also described 
three other groups of populations as "Islamia 
sp. forma A", "forma B", and "forma C" from 
three different areas of Italy without giving 
them a taxonomical category. These four 
groups of populations, as well, were differen- 
tiated only by penial and conchological char- 
acters. 

According to Bodon et al (2001 ), Islamia in- 
cludes 19 species to date, in addition to those 
of Spain. In this paper, the authors considered 
Mienisiella Schutt, 1991, to be a junior syn- 
onym of Islamia, thus expanding the distribu- 
tion area of the genus to Lebanon - /. 
ga/V/ardof/ (Germain, 1911) - and to Israel - /. 
mienisi (Schutt, 1991), the type species of 
Mienisiella. Whereas the penial and 
conchological characters in these latter two 
species differ, they both have female genitalia 
that are similar to those previously described 
for Islamia species. 

Considering all these species, Bodon et al. 
(1995) distinguished a group comprised of 
"oriental" species from the Balkan Peninsula 
(Croatia, Bosnia, Greece) and Turkey and an 
"occidental " species' group located in France, 
Spain, and Italy. The oriental taxa shared two 
penial characters: a very well-developed glan- 
dular penial lobe and a non-glandular (mus- 
cular) pleat on the ventral side of the penis. 
These two characters are also found in Islamia 
pusilla (Piersanti, 1952), the unique species 
inhabiting south central Italy (Giusti et al., 
1981), and in /. cianensis Bodon et al., 1995, 
from Sicily, although the penial lobe is more 
reduced in the last species. The degree of 
development of the muscular pleat of the pe- 
nis and the distance between seminal recep- 
tacles in the female genitalia have sometimes 
been considered to be "minor anatomical fea- 



tures" (Bodon etal., 2001: 199) and at times, 
if constant, "sufficient to support the existence 
of two groups of species representing two dis- 
tinct branches in the radiation of Islamia" 
(Bodon et al., 2001: 201): The "oriental" spe- 
cies' group located in the Balkan Peninsula 
(including type species, /. valvataeformis), 
Turkey, Israel, and part of Italy (two species: /. 
pusilla and /. cianensis) have two seminal re- 
ceptacles that are very close to each other and 
a penis with a well-developed muscular pleat. 

The "occidental " species' group from France 
(/. minuta, I. consolationis, I. globulina, I. 
spirata) and Spain have two seminal recep- 
tacles that are generally substantially sepa- 
rated from each other and a penis with a less 
developed or completely absent muscular 
pleat. The Italian species, /. galten, is an ex- 
ception to this hypothesis, because it has two 
very closely adjacent seminal receptacles (as 
in most Islamia species), a penis with no mus- 
cular pleat, and a knob-like penial lobe that 
projects only slightly and without light micro- 
scope evidence of internal glandular tissue 
(Bodon et al., 1 995: 51 , figs. 20, 24-27). None 
of the Iberian species has a penis with mus- 
cular pleat. The degree of variation of this char- 
acter throughout the distribution area of 
Islamia suggests that an East-West sort of 
dine exists in the development of the muscu- 
lar pleat. It is prominent in oriental species, 
weakens westward and disappears completely 
in westernmost species. Variability observed 
in the female genitalia of Iberian species 
ranges from seminal receptacles that appear 
at the same point (/. pallida), are separated (/. 
globulus, I. lagan and /. henrici), or even at 
substantial distances from each other (/. ateni). 
The variability found in these two genital char- 
acters (distance between seminal receptacles 
and a penis with or without muscular pleat) 
among the supposedly "occidental" species' 
group suggests that neither of these features 
alone, nor a combination of these characters, 
are adequate enough to differentiate taxa at 
the genus or subgenus level. Therefore, it 
would be more appropriate to consider them 
as "species-specific anatomical features". 

In hydrobioid taxa, the structures associated 
with the renal oviduct in the female genitalia 
are relatively more important taxonomically 
than those of the male genitalia (Davis & 
Carney, 1 973). In a more recent study of Asian 
hydrobioids (Davis et al., 1992), involving 48 
informative anatomical characters, 33% were 
derived from the female reproductive system, 
23% from the male reproductive system, while 
only 1 9% were derived from the digestive sys- 



REVISION OF THE GENUS ISLAMIA 



129 



tern and 4% from the nervous system. 

Apart from the distance between seminal 
receptacles, other female genitalia characters 
of Iberian Islamia species also differ greatly, 
such as the size and shape of the two seminal 
receptacles. In general, the proximal seminal 
receptacle is larger than the distal receptacle 
(according to previously published diagnoses), 
but they can be almost equal in size in some, 
as they are in /. pallida. Another important char- 
acter that has yet to be considered is the in- 
sertion point of the seminal receptacles. Both 
receptacles emerge on opposite sides of the 
renal oviduct in all known Islamia species. This 
character may have been overlooked due to 
the minute size of the female genitalia and to 
the fact that the renal oviduct is contorted. 
However, it is worth noting that while the semi- 
nal receptacles of all the Islamia species in 
the literature seem to have been correctly 
drawn, they have been incorrectly simplified 
in taxonomic schemes (e.g., in Bodon et al., 
2001: figs. 180, 181). 

Another female genital characteristic, the 
presence of a narrowing at the outer margin 
of the palliai oviduct between the capsule and 
albumen gland, described by Boeters (1988) 
as diagnostic for the Iberian "Neohoratia" spe- 
cies, does not always hold true in all species. 
It is sometimes present in /. globulus, I. ateni, 
and /. h. gienensis and absent in /. pallida and 
/. h. henrici. The same situation was reported 
for Italian species: while /. cianensis and /. 
piristoma Bodon & Cianfanelli, 2002, show a 
slight narrowing in the transition area between 
the two oviduct glands, /. pusilla and /. galten 
lack this character (Giusti et al., 1981; Bodon 
et al., 1 995; Bodon & Cianfanelli, 2002). There- 
fore, even though this feature could be useful 
at the species level, it is obviously irrelevant 
at the supraspecific level. 

Other characters that are variable among 
Islamia species, although constant at the spe- 
cies level are: the number of basal cusps of 
the central tooth, the presence/absence of 
body or ocular pigmentation, and the pres- 
ence/absence of a nuchal node or a reduced 
non-functional penis-shaped structure on the 
head of females. Islamia h. henrici and /. 
pallida are the only known Islamia laxa that 
have this last character. Despite this unique- 
ness, and because the influence of environ- 
mental parameters on the development of this 
structure is still a matter of discussion, and 
because water parameters have not been 
measured in all localities, we prefer to adopt a 
conservative position and not consider this 
character to be diagnostic. If in fact this char- 



acter turns out to be diagnostic, a taxonomic 
re-arrangement of these species may be war- 
ranted. The absence/presence of ctenidium is 
also constant at the intraspecific level, except 
in the two /. /?enr/'c/ subspecies. The RPG ratio 
is also constant at the species level, except in 
the two /. henrici subspecies, but it is not quite 
useful at genus level, unless for the three gen- 
era here described. The nervous system is 
slightly elongated in /. ateni (although it has 
the smallest value in this category, 0.50), mod- 
erately concentrated in /. globulus (0.43), /. 
pallida (0.42) and /. h. henrici (0.30), and con- 
centrated in /. h. gienensis (0.14), M. schuelei 
(0.24) and J. aitanica (0.22). 

The shells of the Islamia species known to 
date (Radoman, 1973a, b, 1983; Giusti & 
Pezzoli, 1981; Schutt, 1991; Bodon et al., 
1995) vary little in shape. They are mostly 
valvatiform, although some French species 
have the spire raised to different degrees 
(Bodon et al., 2001). Islamia pallida and /. 
henrici also have planispiral or valvatiform 
shells, whereas shells of /. globulus, I. lagan 
and /. ateni are ovate-conic (bythinelliform). It 
is well known that shell features are not suffi- 
ciently diagnostic at the genus level if they are 
not supported by anatomical differences. 
Therefore, the variability here described 
should be included in the diagnosis of Islamia, 
which reinforces the need to review a number 
of species described from different sites in 
Europe and Turkey and assigned to Islamia 
on the basis of shell characters (Bodon et al., 
2001). This would probably lead to the con- 
clusion that Islamia is a taxonomic mess and 
probably polyphyletic, as unpublished molecu- 
lar genetic data suggests (Wilke, pers. comm.). 

An interesting character is the shape of the 
esophagus posterior to the pleuro-esophageal 
ganglionic complex of the nervous system, a 
character never mentioned nor figured to date 
for any Hydrobiidae species. The esophagus 
runs straight in all species studied in this pa- 
per except in /. henrici, in which it shows a 
weak curvature to the right side of body (Figs. 
17B, 18A), and in J. aitanica, in which it makes 
a marked loop to the left (Fig. 250). As the 
shape of the esophagus is constant in all stud- 
ied specimens of all the species, we rule out 
the possibility that curvatures are caused by 
manipulation or retraction of the animal dur- 
ing fixation. More research will reveal if this 
feature has potential taxonomic value or not. 

Islamia has been related genetically to other 
European genera: Alzonlella Giusti & Bodon, 
1984, Fissuria Boeters, 1981, and Avenionia 
Nicolas, 1882. These genera have been ten- 



130 



ARCONADA & RAMOS 



tatively assigned to the nominal subfamily 
Islamiinae Radoman, 1 973 (Wilke et al., 2001 ). 
Neverthess, important differences in morpho- 
logical character and character states clearly 
distinguish them from each other: Alzonlella 
has a conical or cylindrico-conical shell, a 
bursa copulatrix with a short to medium 
anterodorsal duct and two seminal recep- 
tacles, and a penis with one or more "glandu- 
lar" penial lobes located in its concave side 
(Giusti & Bodon, 1984: Bodon, 1988: Boeters, 
1999, 2000 ): Fissuria has a valvatiform shell, 
an oval bursa copulatrix of variable size, a 
short to long anterodorsal bursal duct, two 
equally-sized seminal receptacles, and a pe- 
nis with 3-4 lobes containing "mass of glan- 
dular tissue" (Bodon et al., 2001); Avenionia 
has a cylindro-conical, bythinelloid shell, a 
rudimentary gastric caecum, a penis with a 
very large subapical lobe, with three "glandu- 
lar" swellings on its apical border, a "glandu- 
lar" lobe located on the dorsal side of the penis 
close to the base of the subapical lobe, and 
female genitalia with a wide bursa copulatrix, 
a short and anteroventral bursal duct, and two 
seminal receptacles (Bodon et al., 2000). 
Islamia is also distinguished from the two new 
Iberian genera, Milesiana and Josefus, by a 
set of character and character states that have 
been previously discussed. 

Difficulties in defining synapomorphies be- 
tween the so-called "hydrobioids" (Davis, 
1979), together with the many conflicts that 
exist between morphological and molecular 
genetics (Wilke et al., 2001), call attention to 
the need for detailed anatomical studies de- 
signed to provide ways to accurately group 
species and to effectively distinguish closely 
related genera of this complex group. 



ACKNOWLEDGEMENTS 

We are gratefully indebted to Dr. F. Giusti, 
Dr. M. Haase and an anonymous reviewer for 
their helpful comments on the manuscript, 
which clearly helped to improve this version. 
We thank Dr. T. Wilke for his input on the phy- 
logeny of hydrobioids based on molecular se- 
quence data. We give heartfelt gratitude to Dr 
G. M. Davis who also made helpful sugges- 
tions along the process of revision and while 
editing the manuscript. We are also indebted 
to the following museum and curator teams: 
F. Uribe (MZB), S. Cianfanelli (MZUF, Italy), 
H. D. Boeters. E. Gittenberger (NNM, Nether- 
lands), R. Janssen (SMF, Germany), Y. Finet 
(MHNG, Switzerland), R. Slapnik (IBCICL, 



Slovenia), A. Eschner (NHMW, Austria). We 
also thank A. Camacho, R. Araujo, J. Asti- 
garraga, D. Buckley, J. Escobar, S. Jiménez, 
N. Martin, D. Moreno, С Noreña, J. I. Pino, J. 
M. Remón, J. Roca, E. Rolan and G. Tapia for 
providing us with field samples. J. Bedoya (t) 
from the MNCN prepared the SEM photomi- 
crographs. Drawings were re-done by I. Diaz 
Cortaberria. Dr. M. A. Alonso Zarazaga pro- 
vided advice on nomenclature. Anne Burton 
and James Watkins revised the English text. 
This work was funded by the "Fauna Ibérica" 
Project (DGES PB95-0235 and REN 2001- 
1956.C17.01/GLO). Beatriz Arconada was 
supported by a predoctoral fellowship from the 
Spanish Ministerio de Educación у Ciencia. 



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Revised ms. accepted 20 January 2005 



MALACOLOGIA, 2006, 48(1-2): 133-142 

THE MICHIGAN PHYSIDAE REVISITED: A POPULATION GENETIC SURVEY 
Robert T Dillon, Jr.' & Amy R. Wethington^ 

ABSTRACT 

We report an analysis of gene frequencies at 7 polymorphic allozyme-encoding loci in 1 6 
populations of physid snails collected from Michigan, surveyed as a step toward integrat- 
ing Te's (1978) influential classification of the Physidae with a more comprehensive sys- 
tem based on genetic interrelationships and breeding data. Analysis of a genetic distance 
matrix revealed three groups - two populations of Лр/еха hypnorum together, five popula- 
tions of Physa acuta together, and nine populations of P. gyrina, P. sayii, and P. parken 
combined. Allozyme divergence among the populations of this last cluster, referred to as 
the "gyrina group," was comparable to that seen among the five populations of the well- 
characterized P. acuta cluster, which breeding experiments have demonstrated biologi- 
cally conspecific. These results suggest that Michigan populations assigned to P. gyrina, 
P. sayii, and P. parl<eri may comprise a single biological species, the globose and often 
shouldered shell morphology of the latter resulting from local and perhaps phenotypically 
plastic responses to lacustrine environments. The 14 "taxonomic units" from Michigan that 
Те included in his analysis may represent as few as four biological species. A reduction in 
nominal higher levels of classification within the Physidae is called for. 

Key words: Gastropoda, Pulmonata, Ptiysella, allozyme polymorphism, protein electro- 
phoresis. 



INTRODUCTION 

The freshwater pulmonale family Physidae 
includes some of the more common and wide- 
spread gastropod species on earth (Burch, 
1989; Dillon, 2000; Dillon et al., 2002). In North 
America, the most influential classification of 
the family is currently that of George A. Те 
(1978, 1980). Te's analysis, based on 71 char- 
acters scored primarily from the shell and re- 
productive anatomy, suggested that the 85 
taxonomic units he recognized might be di- 
vided into four genera: Aplexa, Stenophysa, 
Physa and Ptiysella, the last genus with three 
subgenera {Petrophysa, Costatella, and 
Physella s.S.). This classification was adopted 
by Burch for his "North American Freshwater 
Snails" (Burch, 1989), and subsequently by 
Brown (1991), Turgeonetal. (1998), and many 
others. 

A wealth of data regarding genetic relation- 
ships among the North American physids has 
accumulated in the 25 years since Те proposed 
his classification. Reports have been published 
detailing gene frequencies at allozyme-encod- 
ing loci among a variety of nominal species 



(Buth & Sulloway, 1983; Liu, 1993; Dillon & 
Wethington, 1995; Jarne et al., 2000). More 
recently, data have become available on DNA 
sequence divergence (Remigio et al., 2001; 
Wethington & Guralnick, 2004; Wethington et 
al., in prep.) and microsatellite polmorphisms 
(Bousset et al., 2004). Controlled breeding 
studies have uncovered little reproductive iso- 
lation among physid populations long as- 
sumed to represent different species, 
prompting calls for a reappraisal of system- 
atic relationships within the family (Dillon et 
al., 2002, 2004; Dillon & Wethington, 2004; 
Dillon et al., in press 2). The classification sys- 
tem proposed by Wethington (2003; Wething- 
ton & Lydeard, in press) would return the 
number of genera to two - Physa and Aplexa. 
Ideally, a new classification of the Physidae 
would integrate Te's morphological observa- 
tions with more recent allozyme, DNA, and 
breeding data into a single unified system. 
Unfortunately, however, Te did not report col- 
lection localities or museum lot numbers for 
the 85 taxa upon which his 1978 classification 
was based, nor did he provide figures, keys, 
or any practical method by which the species 



department of Biology, College of Charleston, Charleston, South Carolina 29424, USA.; clillonr@cofc.edu 
^Science Department, Chowan College, 200 Jones Drive, Murfreesboro, North Carolina 27855, U.S.A. 



133 



134 



DILLON & WETHINGTON 



he recognized might be distinguished. Since 
any effort to modernize or update Te's system 
would ideally begin with a resampling of his 
taxa to gather correlative genetic information, 
progress in physid systematics has been 
slowed. 

Fortunately, Te (1975) did publish one pre- 
liminary paper, "Michigan Physidae. with sys- 
tematic notes on Physella and Physodon". 
Although limited to just the six species and 
eight subspecies he recognized in the state, 
Te provided figures, a dichotomous key (based 
on shell characters), anatomical notes, syn- 
onymy, range data, and a "partial phylogenetic 
tree" for this subset. The purpose of the 
present paper is to report the results of a sur- 
vey of genetic divergence at allozyme-encod- 
ing loci among a large sample of physid 
populations from Michigan, identified using the 
conchological key of Te (1975), as a step to- 
ward reconciling Te's 1978 classification with 
more recent classifications based on genetic 
data (Wethington, 2003; Wethington & 
Lydeard, in press). 

The physid fauna of Michigan includes three 
nominal species sharing the "type B" penial 
morphology, Physa gyrina, P. sayll, and P. 
parken, all assigned by Te to the subgenus 
"Physella . He noted some minor differences 
among these three species in the length ra- 
tios of the glandular and non-glandular por- 
tions of their penial sheaths, as well as the 
transparency of the non-glandular region and 
terminal swelling in the glandular. But Te 
(1975) wrote, "Physa gyrina, P. sayii and P. 
parken are all related in one species complex. 
As such, there are intermediate forms that may 
be difficult to place; this is especially a prob- 
lem between P. gyrina and P. sayii." 

Burch & Jung (1992) also found the Michi- 
gan species of the subgenus Physella difficult 
to distinguish. They wrote, "Our approach has 
been to note morphological groups that corre- 
spond to named entities (nominal species) that 
seem distinct enough to possibly be good spe- 
cies." Burch & Jung recognized four "named 
entities" of Physella (s.s.) inhabiting northern 
Michigan; globose, strongly shouldered P. 
parken, elliptical or elongate-ovate P. gyrina, 
ovate thin P. sayii, and ovate thick P. 
magnalacustris, which Te considered a sub- 
species of P. sayii. As the systematic relation- 
ships within this group have continued to prove 
especially problematic, populations of physids 
from the subgenus Physella were the objects 
of particular attention in the investigation re- 
ported here. 



METHODS 

Our field survey was designed to sample the 
physid species reported by Te (1975), identi- 
fied using the conchological key he provided, 
collected from their representative ranges 
across the state of Michigan. Ultimately, we 
sampled 16 populations, including two of 
Aplexa hypnorum, two of Physa sayii, three of 
Physa parkeri, four of Physa gyrina, and five 
of Physa acuta. The last-listed species was 
identified as "P. integra" by Te, a name that 
has subsequently been synonymized (Dillon 
et al., 2002). Sample sites are shown in Fig- 
ure 1 , with locality data and sample sizes listed 
in the Appendix. We were unable to collect the 
sixth species reported by Te, Physa jennessi, 
from any of the seven Michigan sites he listed. 

Whole-snail homogenates were centrifuged 
and analyzed via horizontal starch gel elec- 
trophoresis using methods and apparatus as 
described by Dillon (1 992). Multiple buffer sys- 
tems were employed where possible to screen 
for hidden variation (Coyne & Felton, 1978). 
The AP6 buffer system of Clayton & Tretiak 




FIG. 1 . Outline map of the state of Michigan, show- 
ing sample sites. A = Aplexa hypnorum, G = Physa 
gyrina, I = Physa acuta, P = Physa parkeri, S = 
Physa sayii. See Appendix for locality data. 



GENETICS OF MICHIGAN PHYSIDS 



135 



(1972) was used to resolve 6-phospho- 
gluconate dehydrogenase (6PGD), leucine 
aminopeptidase (LAP), glucose phosphate 
isomerase (GPI), and isocitrate dehydroge- 
nase (ISDH). We employed the TC6.8 buffer 
system of Mulvey & Vrijenhoek (1981) to re- 
solve GPI, ISDH, phosphoglucomutase 
(PGM2), and mannose phosphate isomerase 
(MPI). The TEB8 system (buffer III of Shaw & 
Prasad, 1970) was used to analyze LAP, 
6PGD, and the esterases (EST3). 

Our initial runs included control samples of 
the well-characterized P. acuta population in- 
habiting the main pond at Charles Towne 
Landing State Park, Charleston, South Caro- 
lina (population С or CTL in Dillon & Wething- 
ton, 1995; Dillon et al., 2002; Wethington & 
Dillon, 1991). Putative alleles were named 
according to the electrophoretic mobility of 
their allozyme products in millimeters, setting 
the mobility of the most common allele in popu- 
lation С to 100. Mendelian interpretation has 



been confirmed for EST3 and LAP by Dillon & 
Wethington (1994), and for GPI, PGM, and 
6PGD in planorbids by Mulvey & Vrijenhoek 
(1984) and Mulvey et al. (1988). 

Data analysis was performed using Biosys 
version 1.7 (Swofford & Selander, 1981). Be- 
cause large numbers of alleles were resolved 
at some loci, our sample sizes dictated that 
genotypes be pooled into three classes: ho- 
mozygotes for the most common allele, com- 
mon/rare heterozygotes, and rare homozygotes 
together with other heterozygotes before test- 
ing for Hardy-Weinberg equilibrium. Yates-cor- 
rected chi-square statistics were then employed 
for this purpose. We calculated matrices of Nei 
(1978) unbiased genetic identity and Cavalli- 
Sforza & Edwards (1967) chord distance. As 
distances of the latter type are Pythagorean in 
Euclidean space, they were used as the basis 
for an UPGMA cluster analysis (Wright, 1978) 
and a neighbor-joining tree (PAUP* 4.0b10; 
Swofford 1998). 




"^v G 





/ 



FIG. 2. Exemplar shells of the five physid species examined in this study. I - Physa acuta (population 
II), S - Physa say// (population Si), G - Physa gyrina (population Gl), к- Aplexa hypnorum (popula- 
tion A2), P - Physa parken (population PI ). See appendix for locality data. 



136 


P 


0.870 






[ 


DILLON &WETHINGTON 

Chord Distance 






13 


08 06 0.4 


0.2 

i 


ЩЩз 


15 

12 


Б.790 0.»13 

^.653 0.851 




" • 15 

• 12 


1" 


0.744, 0.718 


0.913 


0.809 


»11 


Gl 


0.090' 0.033 


0.000 


0.000 


0.033 




\ 


«Gl 


G4 
G3 


0.089 


0.033 


0.000 
0.000 


0.000 


0.033 


0.731 




V ^ — : 


• G4 


0.091 


0.034 


0.000 


0.034 


0.886 1 0.853 




^ \ / 


«03 


G2 
РЗ 


0.079 


0.029 


0.000 
0.019 


0.000 


0.02'j 


0848 0.752 0.872 




^\ ^ 


-»02 


0.092 


0.046 


0.017 


0.046 


'- ■ 1 :' iT 7R7 


851 887 




^^^ \ 


^^--^ P3 


P2 


0.087 


0.032 


o.ooo 


0.000 


0.032 


0.822 1 0.800 


0.917 


0.974 0.957 




0.984 \ 


III^-— ♦ P2 


SI 


0.087 


0.032 


0.000 


0.000 


0.032 


0.786 


0.792 


0.881 


0.939 977 


0.974 


-31 


S2 


0.081 


0.030 


0.000 


0.000 


0.030 


0.719 


0.742 


0.827 


0.874 0.973 


0.923 


^__._,-«S2 


PI 


0.082 


0.030 


0.000 


0.000 


0.030 


0.733 


0.729 


0.848 


0.838 0.956 


0.897 


,-,.,.-. 


991 


■~~— ~^P1 


A2 


0.006 


0.008 


0.007 


0.007 


0.008 


0.081 0.117 


0.074 


0.052 


0.048 


O.ObO 


O.Ubö 


U.Ü53 


0.Ü55 "^ 


\^A2 


AI 


0.000 


0000 


0000 


0.000 


0.000 


0.095 0.127 


0089 


0.066 


0.060 


0.064 


0.072 


0.067 


0069 1.000 


^^ AI 




14 


13 


15 


12 


11 


Gl G4 G3 


G2 


P3 


P2 S1 


S2 


PI A2 





FIG. 3. Neis (1978) unbiased genetic identities are shown below the diagonal, with nominally con- 
specific comparisons darkly shaded and other comparisons within the gyrina complex shaded lightly. 
Above the diagonal is the result of a UPGMA cluster analysis based on Cavalli-Sforza & Edwards 
(1967) chord distance. 



RESULTS 

We found Te's (1975) conchological key diffi- 
cult to apply to natural populations collected from 
the wild, failing entirely in smaller individuals. 
Although Aplexa and (generally) P. acuta could 
be distinguished unambiguously, shell morpho- 
logical variation within and among populations 
of Я gyrina, P. sayii, and P. parser/ often thwarted 
positive identification. Nor have any anatomical 
distinctions been subsequently described that 
might facilitate this process. We would have 
preferred to sample more populations of P. sayii 
in particular, but intergradation with both P. 
gyrina and P. parl<eri made identification of this 
taxon especially problematic. The shells cho- 
sen for illustration in Figure 2 are exemplars. 
Voucher specimens have been deposited in the 
University of Michigan Museum of Zoology. 

Allele frequencies at the seven enzyme-en- 
coding loci are given in Table 1 . Of the 1 6 x 7 = 
112 loci examined, a total of 54 were polymor- 
phic by the 95% criterion. Chi-square analysis 
revealed heterozygote deficits nominally signifi- 
cant at the 0.05 level in six of these cases - 
Est3 at population 14, Isdh in population 13, Est3 
in population G3, and three polymorphic loci in 
population 15: Est3, Lap, and Isdh. 




0.1 

Chord Distance 



FIG. 4. Neighbor-joining tree (PAUP*; Swofford 
1998) based on the matrix of Cavalli-Sforza & 
Edwards (1967) chord distance. 



GENETICS OF MICHIGAN PHYSIDS 



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GENETICS OF MICHIGAN PHYSIDS 



139 



Figure 3 shows the matrix of Nei's genetic 
identity among all pairs of populations and the 
results of an UPGMA cluster analysis based 
on Cavalli-Sforza and Edwards Chord distance. 
The cophenetic correlation (Sokal & Rohlf, 
1 962) for this analysis was very high, Гсд = 0.993 
(Sneath & Sokal, 1973; 304), indicating a good 
fit between the branch length and the original 
distance matrix. The neighbor-joining tree is 
shown in Figure 4. 



DISCUSSION 

Fits to Hardy-Weinberg expectation were 
good in almost all populations, with scattered 
nominally significant values of chi square prob- 
ably attributable to Type 1 statistical error. The 
exception was population 15, where significant 
heterozygote deficits were apparent at three 
of five polymorphic loci examined. Outcross- 
ing is strongly preferred in laboratory popula- 
tions of Physa acuta, self-fertilization resulting 
in a substantial fitness decrement (Wethington 
& Dillon, 1993, 1996, 1997). Evidence of in- 
breeding has nevertheless often been reported 
in natural populations of Physa (Dillon & 
Wethington, 1 995; Jarne et al., 2000) and other 
pulmonates (Jarne 1995). Some low level of 
self-fertilization may be an unavoidable con- 
sequence of the pulmonate reproductive sys- 
tem (Dillon et al., in press 1). At the 15 site, 
low population densities may have increased 
the frequency of self-fertilization beyond the 
background levels that were more difficult to 
detect in other populations at our sample sizes. 

Both the neighbor-joining tree and the 
UPGMA cluster analysis revealed three dis- 
tinct groups - the two populations of Aplexa 
together, the five populations of P. acuta to- 
gether, and the nine populations of P. gyrina, 
P. sayii, and P. parken combined (Figs. 3, 4). 
The five P. acuta populations, clustered at a 
chord distance of 0.37, showed a minimum 
genetic identity of 0.718. This is quite similar 
to the level of genetic divergence among the 
ten populations of P. acuta sampled from the 
Charleston area by Dillon & Wethington 
(1995). This level is also strikingly similar to 
that displayed within the nine populations of 
the gyrina/sayii/parkeh group, clustered at a 
chord distance of 0.43 with a minimum genetic 
identity of 0.715. The specific distinction be- 
tween P. gyrina, P. sayii, and P. parken, here- 
after referred to as the "gyrina group", is called 
into question. 



Physa gyrina ranges broadly across North 
America, throughout Canada and the United 
States as far south as Virginia and Kentucky. 
In Michigan, Те reported populations from a 
wide variety of shallow habitats - creeks, 
brooks, pools, ponds, and ditches. The ranges 
of Physa sayii and P. parken are more re- 
stricted to the Great Lakes region and to 
deeper waters. Те giving the habitat of the 
former as "lakes and rivers" and the habitat of 
the latter as "large lakes". 

Both Figures 3 and 4 depict the sayii/parkeri 
cluster as a subset within the larger gyrina 
group. This suggests to us that the generally 
larger, inflated, and globose shell that charac- 
terizes populations referred to these two 
nomena may be a regional (and possibly 
ecophenotypic) response to the colonization 
of lacustrine habitats by populations of the 
more typical P gyrina morphology. We hypoth- 
esize that individuals inhabiting larger lakes and 
rivers may tend to live longer, and hence grow 
larger of body, than individuals inhabiting ponds 
and creeks. It also possible that the rotund, glo- 
bose and often shouldered shell phenotype 
characterizing P. parken (and sometimes P. 
sayii) may be related to a deepwater habitat 
unaffected by current or wind. 

The tendency for physid snails to develop 
rotund shells as a phenotypically plastic re- 
sponse to the threat of fish prédation is well 
documented (DeWitt, 1998; DeWitt et al., 
1999, 2000; Langerhans & DeWitt, 2002). 
More recently, Britton & McMahon (2004) have 
reported that physids respond to increased 
water temperature by developing wider shell 
spire angle, a variable positively correlated 
with shell globosity. It seems clear that the 
minor differences in shell morphology upon 
which rest the distinctions among the several 
nominal species of the gyrina group need not 
reflect any heritable variance whatsoever. 

Breeding experiments would provide the 
ideal test to confirm that the three nominal 
species of the gyrina group inhabiting Michi- 
gan are in fact biologically conspecific. Dillon 
& Wethington (2004) reported the results of 
no-choice mating experiments between a line 
of P. parken from Douglas Lake and P. gyrina 
collected from its type locality near Council 
Bluffs, Iowa. Our control P parser/ hatched and 
reared under laboratory conditions did not 
develop the shoulder on their shell character- 
istic of wild-collected animals, remaining su- 
perficially indistinguishable from control P. 
gyrina. Control parken hybridized readily with 



140 



DILLON &WETHINGTON 



P. gyrina. producing viable F1 offspring. The 
growth, survival rate, and fecundity of P. 
parken were, however, significantly below 
those posted by control P. gyrina, in both the 
control pairs and in the outcross parken x 
gyrina experiment. We were ultimately unable 
to carry either control P. parkeri or parkeri x 
gyrina hybrids to the F2 generation under our 
culture conditions, leaving the question of re- 
productive isolation an open one. Our experi- 
ments nevertheless confirmed that the life 
history adaptations evolved by P. parkeri have 
a heritable basis, although some key aspects 
of shell morphology, upon which the taxonomy 
is based, may not. 

The overall form of the analyses shown in 
Figures 3 and 4 is consistent with the phylog- 
eny suggested by Wethington (2003) and 
Wethington & Lydeard (in press). Mitochon- 
drial COI and 16s sequence data, analyzed 
via parsimony, yielded a tree in which the gen- 
era Aplexa and Physa split first, followed by a 
split between the clade containing P. acuta and 
the clade containing the gyrina group. The 
analysis of Wethington & Lydeard also re- 
solved two clades within the gyrina group: a 
"typical" subset and a "globose" subset that 
included parkeri and say/7 (subspecies magna- 
lacusths.) The authors attributed this distinc- 
tion to geographical factors, however, not to 
reproductive isolation. 

Our allozyme data, taken together with the 
partial results of the Dillon & Wethington (2004) 
breeding experiments, suggest that the nomi- 
nal taxa P. parkeri and P. sayii may best be 
treated as junior synonyms of P gyrina. Final 
confirmation of this hypothesis will await care- 
ful analysis of reproductive interactions be- 
tween populations of these three nominal 
species in natural sympatry. Given the difficulty 
we and other workers have encountered dis- 
tinguishing members of the gyrina group in the 
field, however, it may materialize that no prac- 
tical site for such a study can be identified. 

The 85 taxonomic units upon which Te (1978, 
1980) based his classification included all 14 
of the taxa he recognized from Michigan: 
Aplexa Inypnorum (tryoni and hypnorum s.S.), 
Physa jennessi (subspecies skinneri), Physa 
gyrina {elliptica, hildrethiana, and gyrina s.S.), 
Physa sayii {magnalacustris, vinosa, and sayii 
s.S.), Physa parkeri {latchfordii and parkeri 
s.S.), and Physa integra (brevispira, walkeri, 
and integra s.S.). Including P. jennessi, the 
validity of which we have no reason to doubt, 
our allozyme data suggest that these 14 taxa 
may comprise just four biological species. It is 



clear that Tes analysis was based on a set of 
taxonomic units divided much more finely than 
biological species. This suggests to us that the 
revised classification of Wethington & Lydeard, 
returning the Physidae to a simpler two-genus 
system, has much to recommend it. 



ACKNOWLEDGEMENTS 

Field assistance was provided by Joseph 
Reznick, Zelda Wethington, Jennifer Walker, 
Jennifer Stephens, and Eileen Jokinen. J. B. 
Burch provided some insight regarding the 
identifications. This research was supported 
by a grant from the National Science Founda- 
tion, DEB-01 28964, and by a travel grant from 
the graduate student council at the University 
of Alabama. 



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DILLON, JR., in prep.. Allozyme. 16S, and 001 
sequence divergence among six populations of 
the cosmopolitan freshwater snail, Physa acuta. 

WRIGHT, S., 1978, Variability within and among 
natural populations. Vol. 4, Evolution and the 
genetics of populations. University of Chicago 
Press, Chicago, Illinois. 580 pp. 



Revised ms. accepted 1 September 2005 



APPENDIX 
Locality data and sample sizes 

A1 Aplexa hypnorum. Woodland pond at the 
Maple Bay access of Burt Lake, 
Cheboygan Co., Michigan. 45.4867°N, 
84.7088°W. N = 21. 

A2 Aplexa hypnorum. Houghton Lake at state 
campground, Roscommon Co., Michigan. 
44.3388^^N. 84.6648°W. N = 26. 

G1 Physa gyrina. Little Lake at state camp- 
ground, 1 km S of town of Little Lake, 



Marquette Co., Michigan. 46.2815°N, 

87.3337°W. N = 31. 
G2 Physa gyrina. Little Carp River at 

Hogsback Rd., 1 km N of Burt Lake, 

Cheyboygan Co., Michigan. 45.5520°N, 

84.6854°W, N = 28, 
G3 Physa gyrina. Turtle Lake at Miller Rd., 5 

km W of Bendon, Benzie Co., Michigan, 

44.6178°N, 85.9090°W. N = 24. 
G4 Physa gyrina. Twin Sun Lakes at Highgate 

Beach, Wixom, Oakland Co., Michigan. 

42.5466°N, 83.5085°W. N = 33. 

11 Physa acuta. Douglas Lake at the Uni- 
versity of Michigan Biological Station, 
Cheboygan Co., Michigan. 45.5634°N, 
84.6783°W. N = 32. 

12 Physa acuta. Higgins Lake near boat 
ramp at Sam О Set Blvd., Sharps Corners, 
Roscommon Co., Michigan. 44.4246°N, 
84.6942°W. N = 31. 

13 Physa acuta. Saginaw Bay at Quani- 
cassee Wildlife Area, Tuscola Co., Michi- 
gan. 43.5896°N, 83.6774°W. N = 57. 

14 Physa acuta. Pond near the junction of 
Mi 11 and Mi 37, Grand Rapids , Kent Co., 
Michigan. 42. 9168°N, 85. 577rW. N = 44. 

15 Physa acuta. Kent Lake at Kensington 
MetroPark, Oakland Co., Michigan. 
42.5336°N, 83.6462°W. N = 29. 

PI Physa parkeri. Douglas Lake at the Uni- 
versity of Michigan Biological Station, 
Cheboygan Co., Michigan. 45.5634°N, 
84.6783°W. N = 59. 

P2 Physa parkeri. Long Lake at Long Lake 
Rd., 10 km SE of Traverse City, Grand 
Traverse Co., Michigan. 44.7140°N, 
85.7316°W. N = 37. 

P3 Physa parkeri. Higgins Lake near boat 
ramp at Sam О Set Blvd., Sharps Corners, 
Roscommon Co., Michigan. 44.4246°N, 
84.6942°W. N = 47. 

51 Physa sayii. Lake Michigan at Wilderness 
State Park, Emmet Co., Michigan. 
45.7474°N, 84.9045°W. N = 49. 

52 Physa sayii. Crystal Lake 3 km N of Frank- 
fort, Benzie Co., Michigan. 44.6607°N, 
86.2320°W. N = 39. 



MALACOLOGIA, 2006, 48(1-2): 143-157 

EXTREME MITOCHONDRIAL SEQUENCE DIVERSITY IN THE INTERMEDIATE 

SCHISTOSOMIASIS HOST ONCOMELANIA HUPENSIS ROBERTSONI: 

ANOTHER CASE OF ANCESTRAL POLYMORPHISM? 

Thomas Wilke'*, George M. Davis^ Dongchuan Qiu^ & Robert С Spear* 

ABSTRACT 

Today, the human blood fluke. Schistosoma japonicum, is transmitted in China by two 
subspecies of the rissooidean snail taxon Oncomelania hupensis: O. h. hupensis and O. 
h. robertsoni. Whereas the eastern Chinese subspecies O. h. hupensis has been studied 
extensively using mitochondrial DNA sequences, very little data existsfor the western sub- 
species O. h. robertsoni. Preliminary phylogeographic studies indicate that the latter shows 
a very high degree of genetic diversity with Kimura 2 parameter distances in the cyto- 
chrome oxidase I (CGI) gene of up to 0.0932 (= 9.32%) among four sequences previously 
deposited in GenBank. Extreme degrees of intraspecific heterogeneity in gastropods have 
been reported before, and possible explanations include the presence of cryptic species 
complexes, isolation followed by secondary contact, heteroplasmy and duplications within 
the mitochondrial genome, the presence of "pseudogenes", and the retention of ancestral 
mitochondrial polymorphism. 

Given the great significance of understanding phylogeographic patterns in the interme- 
diate schistosomiasis host Oncomelania h. robertsoni for comprehending host/parasite 
relationships, DNA sequences of two mitochondrial genes (CGI and LSU rRNA) from 66 
O. hupensis robertsoni specimens are used to (1) assess the phylogenetic position, (2) 
study the degree of heterogeneity within and between "populations", (3) provide a prelimi- 
nary overview of the geographic distribution of major genetic groups and (4) study the 
phylogenetic concordance of the two gene fragments. 

Phylogenetic analyses, parametric bootstrapping and studies of sequence polymorphism 
show that: (1) all CGI sequences are fully protein-coding with no insertions or deletions, 
(2) both individual and combined analyses of the CGI and LSU rRNA genes show at least 
four distinct haplotype groups within O. h. robertsoni, (3) monophyly of the four clades 
cannot be confirmed, (4) there is high concordance in cluster patterns and arrangement of 
individual haplotypes of both gene fragments, (5) two of the genetic clades recovered 
appear to be localized, whereas the other two are widely distributed, and (6) sympatry of 
individuals belonging to different clades occurs. Moreover, based on preliminary AFLP 
analyses it could be shown that (7) there is no phylogenetic concordance between the 
mitochondrial and nuclear data presented here, and (8) the nuclear data from AFLP 
genotyping indicate a lack of clear population structure. 

Given the results of the present study, it is cautiously suggested that retention of ances- 
tral mitochondrial DNA polymorphism possibly in combination with some effects of sec- 
ondary contact (introgression) is the most probable explanation for the occurrence of deviant 
lineages in O. h. robertsoni. Gn the basis of nuclear, morphological, and ecological data, it 
is also suggested that there is no evidence of organismal subdivision in O. h. robertsoni. It 
is strongly recommended that future studies incorporate more data from nuclear loci in 
order to better understand phylogeography, population genetics, and host-parasite co- 
evolution in O. h. robertsoni. 

Keywords: schistosomiasis, Oncomelania, China, mitochondrial DNA, phylogeography, AFLP. 

'Justus Liebig University Giessen, Department of Animal Ecology and Systematics, Heinrich-Buff-Ring 26-32, D-35392 

Giessen, Germany; tom wilke@allzool,bio uni-giessen de 
■^Ttie George Washington University Medical Center, Department of Microbiology and Tropical Medicine, Wastiington, DC, 

U.S.A.; mtmgmd@gwumc.edu 

^Sichuan Institute of Parasitic Disease, Chiengdu, Sichuan, P. R. China 

"Center for Occupational and Environmental Health, School of Public Health, University of California, Berkeley, California, USA 
"Corresponding author 

143 



144 



WILKE ETAL. 



INTRODUCTION 

The human blood fluke. Schistosoma japoni- 
cum, responsible for one of the most serious 
disease problems in China, schistosomiasis, 
uses small dioecious rissooidean gastropods 
of the species Oncomelania hupensis as in- 
termediate hosts. Molecular and morphologi- 
cal analyses, together with breeding 
experiments and biogeographic studies of O. 
hupensis. indicate that there are three subspe- 
cies on the mainland of China (Davis, 1992; 
Davis et al., 1995, 1999). Oncomelania h. 
robertsoni is restricted to high elevations on 
the plateaus and mountains of Yunnan and 
Sichuan above the Three Gorges. Oncomela- 
nia h. hupensis is found throughout the Yangtze 
River drainage below the Three Gorges; it has 
spread to Guangxi Province probably via the 
Grand Canal from Hunan. Oncomelania h. 
tangi is restricted to Fujian Province along the 
coast. The latter subspecies has been eradi- 
cated except for two known populations, and 
the parasite presumably is extinct. 

Of the two wide-spread Chinese subspecies 
O. h. hupensis and O. h. robertsoni, the former 
has received considerable attention in genetic 
studies (allozymes and mitochondrial gene 
sequences) dealing with questions of popula- 
tion structure, phylogeography, infectivity and 
the nature of shell ribbing (e.g., Davis et al., 
1995; Wilke et al., 2000a; Shi et al., 2002). 

In contrast, very little is known about the ge- 
netics of the western Chinese subspecies O. 
h. robertsoni. In fact, whereas as of June 2005, 
140 nucleotide sequences are available for O. 
h. hupensisirom GenBank, only ten sequences 
(from a total of four specimens) exist for O. h. 
robertsoni. However, preliminary studies in a 
phylogeographic framework of other O. 
hupensis subspecies indicated a very high 
degree of genetic diversity within the few mito- 
chondrial sequences available for O. h. 
robertsoni. In fact, of the four sequences pub- 
lished for the mitochondrial cytochrome с oxi- 
dase subunit I (COI) gene (GenBank accession 
numbers AF213339, AF253075, AF253076, 
AF531547), two sequences (AF253075 and 
AF21 3339) differ by K2P (Kimura 2 parameter) 
distances of 0.0932. To give a comparison, the 
highest pairwise K2P distance among more 
than 100 CO! sequences for the eastern Chi- 
nese subspecies O. h. hupensis (which is re- 
garded as genetically highly diverse) is with 
0.0340 (GenBank accession numbers 
AF254484 and AF254509) only about 36% as 
high as in O. h. robertsoni. Moreover, in many 
phylogenetic studies of rissooidean gastro- 



pods, K2P distances in the COI gene compared 
to the amount found in O. h. robertsoni typi- 
cally reflect species, if not genus level relation- 
ships (e.g., Wilke et al., 2000b; Wilke, 2003). 
To complicate matters, in further studies involv- 
ing a single population of O. h. roberfson/ from 
the lower Anning River Valley in Sichuan (site 
A8, see below), we even found pairwise K2P 
divergences of up to 0.1027 within the site. 

Extreme degrees of intraspecific mitochon- 
drial heterogeneity in gastropods have been 
reported before, and potential explanations 
involve, among others, the presence of cryptic 
species complexes, isolation followed by sec- 
ondary contact, heteroplasmy and duplications 
within the mitochondrial genome, the presence 
of nuclear "pseudogenes", or the retention of 
ancestral mitochondrial polymorphism. 

In order to shed light on the problem of het- 
erogeneity within Oncomelania h. robertsoni, 
we here use mitochondrial DNA (mtDNA) se- 
quences from a larger data set of 66 specimens 
from 13 sites. In addition to the protein-coding 
COI gene, we study the mitochondrial gene for 
large subunit hbosomal RNA (LSU rRNA) to 
test for potential conflicts between these gene 
fragments that could help to reveal method- 
ological problems. 

The specific goals of this paper are; 
(1 ) to assess the phylogenetic position of On- 
comelania h. robertsoni within the frame- 
work of other O. hupensis ssp., 

(2) to study the degree of mitochondrial het- 
erogeneity within and between "popula- 
tions" of O. h. robertsoni. 

(3) to provide a preliminary overview of the 
geographic distribution of major mtDNA 
groups within O. h. robertsoni. and 

(4) to study the phylogenetic concordance of 
different mitochondrial gene fragments. 

We also use the results of preliminary AFLP 
(amplified fragment lengths polymorphism) 
genotyping of highly variable nuclear loci from 
a subset of 24 specimens to discuss the high 
degree of mtDNA diversity in the light of 
nuclear data (for a review of the performance 
of AFLP data in animal population genetics see 
Bensch & Akesson, 2005). 



MATERIALSAND METHODS 

Specimens Studied 

The current study includes 66 specimens of 
Oncomelania hupensis robertsoni Bartsch, 
1946, from 13 sites in Yunnan and Sichuan 
provinces, China (Table 1, Appendix). 



EXTREME SEQUENCE DIVERSITY IN ONCOMELANIA H. ROBERTSONI 



145 



TABLE 1: Locality information for Chinese specimens of Oncomelania hupensis robertsoni studied 
(M = Meishan Area, A = Anning River Valley, Y = Yunnan). 



Locality 
code 



Original 
locality # 



Latitude 
Longitude 



Locality 



No. specimens 
studied 



M 1 


D 98.16 


29.99163 °N 
103.41580 °E 


M2 


D 98.14 


30.13993 °N 
103.61167 °E 


M3 


MG 96.18 


30.0373 °N 
103.9002 °E 


M4 


- 


30.067 °N 
104.138 °E 


AI 


D 98.04 


27.93525 °N 
102.20540 °E 


A2 


D 98.03 


27.9318 °N 
102.1962 °E 


A3 


D 98.12 


27.87505 °N 
102.30867 °E 


A4 


D 98.09 


27.8000 °N 
102.204 °E 


A5 


D 98.07 


27.7995 °N 
102.3087 °E 


A6 


D 98.05 


27.7973 °N 
102.3157 °E 


A7 


D 98.11 


27.7468 °N 
102.1903 °E 


A8 


Xi Chang** 


26.9637 °N 
102.1328 °E 


Y1 


Dali** 


25.4510 °N 
100.2007 °E 



Sichuan, Danling County, Ernming 2 

Township, Xiaoqiao Village 

Sichuan, Dongpo County, Panao 10 

Township, Magau Group 2 Village 

Sichuan, Meishan County, Fusheng 5 

Township, Zhongfu Village 

Sichuan, Mianzhu 1* 

Sichuan, Xichang County, Xixiang 4 

Township, Gucheng Village 

Sichuan, Xichang County, Xxixiang 4 

Township, Gucheng Village 

Sichuan, Xichang County, Chaunxing 4 

Township, Minhe group 2 Village 

Sichuan, Xichang County, Jingjiu 4 

Township, Zhoutun Village 

Sichuan, Xichang County, Hainan 4 

Township, Gucheng group 2 Village 

Sichuan, Xichang County, Hainan 4 

Township, Gucheng group 5 Village 

Sichuan, Xichang County, Jingjiu 4 

Township, Jingjiu Village 

Sichuan, Miyi County, Panlian 12 

Township, Shuanggou Village 

Yunnan, Dali City, Da Jin Ping, Zi 8 

Ran Village 



* from GenBank (Attwood et al , 2003) 

** previously studied using allozyme electrophoresis by Davis et al. 



(1995) 



As primary outgroup taxon (which was used 
to root the mtDNA trees) served a yet unde- 
scribed representative of the genus Tricula 
{Tricula sp.; Davis et al., 1998) (GenBank 
AF213341, AF212895). Like Oncomelania, 
Tricula belongs to the family Pomatiopsidae. 
Additional outgroup taxa used in the current 
study are Oncomelania minima Bartsch, 1936 
(GenBank DQ212795, DQ212858), as well as 
four other subspecies of O. hupensis: O. h. 
hupensis {Gred\er, 1881) (GenBank AF254547, 
DQ212859), O. h. tang/ (Bartsch, 1936) (Gen- 
Bank DQ212796, DQ212860), O. h. formosana 
(Pilsbry & Mirase, 1905) (GenBank DQ112283, 
DQ212861), and O. h. Quadras/ (Moellendorff, 
1895) (GenBank DQ112287, DQ212862). 

DNA Isolation and Sequencing 

The method used for isolating DNA from snails 
was modified from that of Spolsky et al. (1996). 



Individual alcohol-preserved specimens were 
first soaked for 1 min in 1 ml ice-cold exchange 
buffer (0.02 M Tris base, 0.1 M EDTA, pH 8.0). 
Then, either the soft body of a whole specimens 
or part of the foot (depending on the size of the 
specimen) was cut in pieces and incubated 
overnight in a water bath at 58°C in 200 pi 
Turner lysis buffer (0.02 M Tris base, 0.1 M 
EDTA, 0.5% Sarkosyl, pH 8.0) and 3 pi of 20 
pg/pl Proteinase K. After digestion, 35 pi of 5 M 
NaCI and 35 pi of a 5% CTAB/0.5 M NaCI solu- 
tion were added. Extraction was carried out with 
270 pi chloroform. After centrifugation for 5 min 
at 9,000 rpm, the aqueous phase was trans- 
ferred into a new tube and 270 pi of СТАВ pre- 
cipitation buffer (1% СТАВ, 0.05 M Tris base, 
0.01 M EDTA) was added, mixed and placed 
at room temperature for 45 min. After pelleting 
the CTAB-DNAfor 10 min at 12,000 rpm, the 
supernatant was disposed and the pellet redis- 
solved in 100 pi of NaCI/TE (0.01 Tris base, 



146 



WILKE ETAL. 



0.001 M EDTA, 1 M NaCI. pH 8.0) and 1 |jl of 
10 mg/ml RNase. After incubation for 8 min at 
65°C, the DNA was precipitated over night at 
-20°C by adding 250 pi of ice-cold 96% etha- 
nol. After centhfugation for 15 min at 12,000 
rpm. the pellet was washed twice with 300 pi of 
ice-cold 70% ethanol, air-dried for 5-10 min and 
finally redissolved in approximately 50 pi HO. 
Quality and quantity of the isolated genomic 
DNA were checked on a 1% agarose gel. 

The primers used to amplify a fragment of 
the COI gene with a target length of 658 base 
pairs (excluding 51 bp phmer sequence) were 
LCO1490 and HC02198 as described by 
Folmer et al. (1994). The primers for amplifica- 
tion of a LSU rRNA fragment with a target length 
of 505-508 bp (excluding 42 bp pnmer se- 
quence) were 1 6Sar-L and 1 6Sbr-H of Palumbi 
et al. (1 991 ). Sequences (forward and reverse) 
were determined using the LI-COR (Lincoln, 
NE) DNA sequencer Long ReadIR 4200 and 
the Thermo Sequenase Fluorescent Labeled 
Primer Cycle Sequencing kit (Amersham 
Pharmacia Biotech, Piscataway, NJ). 

The COI sequences were aligned unambigu- 
ously by eye using BioEdit 5.0.9 (Hall, 1999). 
All sequences are fully protein-coding with no 
insertions or deletions. However, the first few 
base pairs (bp) behind the 3' end of each 
primer were difficult to read. We therefore uni- 
formly cut off the ftrst and last ten bp of each 
sequence, leaving a 638 bp-long completely 



overlapping fragment for the COI gene. Align- 
ment of LSU rRNA sequences was done using 
ClustalX (version 1.81; Thompson et al., 1997). 
No manual refinement was necessary as the 
alignment yielded only five gaps: three single- 
nucleotide gaps as well as one gap of up to 
two nucleotides and one gap of up to three 
nucleotides within a stretch of thymine bases. 
The total length of the aligned LSU rRNA is 510 
bp. All sequences are available from GenBank 
(for GenBank accession numbers and DNA 
voucher numbers see the Appendix). 

AFLP Genotyping 

Genomic DNA was digested with the fre- 
quent cutter restriction enzyme Mse\ (New 
England Biolab, NEB) and the rare cutter 
EcoRI (NEB). Adaptors (Table 2) were ligated 
to the genomic DNA using T4 ligase (NEB). 
Both digestion and ligation were carried out in 
a single reaction running for 12h at 37°C. 

The ligation product was used to perform a 
pre-selective PCR amplification with NEB Taq 
polymerase (for EcoRI and Mse\ primers see 
Table 2). The quality of the ligation/pre-ampli- 
fication was checked on a 1% agarose gel. 

Selective amplification was performed from 
1 :40 diluted pre-amp DNA as duplex PCR (one 
unlabeled Mse\ each with the two IRDye-la- 
beled EcoRI primers; Table 2). A total of 12 
primer combination was used for the PCR. 



TABLE 2: AFLP primers. 



Primer 



Sequence 



Adapters 

EcoRI 

Mse\ 
Pre-amplification primers 

E01 E-A (EcoRI) 

M02 M-C (Mse\) 



5'-CTC GTA GAG TGC GTA CO-CAT CTG ACG CAT GGT TAA-3' 
5'-GAC GAT GAG TCC TGA G-TA CTC AGG ACT CAT-3' 



5'-GAC TGC GTA CCA ATT CA-3' 
5-GAT GAG TCC TGA GTA AA-3' 



Selective amplification primers 



700 E-AAC 

800 E-AAG 

M-CGA 

M-CTT 

M-CTC 

M-CAT 

M-CTA 

M-CTG 



5-IRD700-GAC TGC GTA CCA ATT CAA C-3' 
5'-IRD800-GAC TGC GTA CCA ATT CAA G-3' 
5'-GAT GAG TCC TGA GTA ACG A-3' 
5'-GAT GAG TCC TGA GTA ACT T-3' 
5'-GAT GAG TCC TGA GTA ACT C-3' 
5'-GAT GAG TCC TGA GTA АСА T-3' 
5'-GAT GAG TCC TGA GTA ACT A-3' 
5'-GAT GAG TCC TGA GTA ACT G-3' 



EXTREME SEQUENCE DIVERSITY IN ONCOMELANIA H. ROBERTSONI 



147 



Labeled PCR products were separated on an 
8% acrylamide gel using the DNA sequencer 
LI-COR Long ReadIR 4200 and digitally cap- 
tured with the software package SAGA Genera- 
tion 2 (MX module version 3.2. 1 .) from LI-COR. 
We manually selected the most informative and 
consistent bands for analysis. These 102 poly- 
morphic loci were scored for 32 individuals of 
O. hupensis. Samples with > 10% ambiguities 
(more than ten ambiguous values for 102 loci 
scored) were removed from the data set and 
not included in any analyses, thus reducing the 
sample size from 32 to 24 individuals. 

Preliminary Statistical Analyses (mtDNA) 

Possible dissimilarities between the COI and 
LSU rRNA data sets are of primary interest for 
the current study. In order to test whether there 
were significant differences in incongruence- 
length between the CO! and LSU rRNA data 
sets, the HOMPART command in PAUP* v. 
4.0b10 (Swofford, 2002) was used to perform 
a partition-homogeneity test (Farris et al., 
1995). As the test did not reveal a significant 
conflict (P = 0.2580; 1 0,000 replicates), the two 
data sets were used in a combined analysis. 

Given the potential high degree of sequence 
diversity in Oncomelania hupensis robertsoni, 
as indicated by preliminary analyses, we used 
the test of Xia et al. (2003) implemented in 
the software package DAMBE 4.2.13 (Xia & 
Xie, 2001) to test for saturation prior to the 
phylogenetic analyses. The Xia et al. test did 
not reveal a significant degree of saturation 
(Iss = 0.301 , Iss.c = 0-801 , P = 0.0000). 

Nucleotide diversities and divergences (cor- 
rected according to the K2P-parameter-model) 
were calculated using MEGA 2.1 (Kumar et 
al., 2000) with standard errors estimated by 
1 ,000 bootstrap replications with pairwise de- 
letion of gaps and missing data. 

Phylogenetic Reconstruction (mtDNA) 

The performance of different phylogenetic 
methods is highly controversial, and as numer- 
ous factors such as degree of heterogeneity 
and sample size may affect the quality of phy- 
logenetic reconstruction (e.g., Huelsenbeck, 
1995; Wiens & Servedio, 1998; Kolaczkowski 
& Thornton, 2004), we here use both maxi- 
mum parsimony (MP) and Bayesian inference 
(Bl) based methods. 

Phylogenetic analyses based on the MP cri- 
terion were conducted in PAUP* 4.0b10 
(Swofford, 2002) using the heuristic search 
option with tree bisection reconnection branch- 



swapping, 1 00 replications of random stepwise 
additions, and MAXTREES set to 10,000. 
Node support was evaluated with 10,000 
bootstrapping replications. 

Phylogenetic reconstruction based on Bl was 
conducted using the software package 
MrBayes 3.0b4 (Huelsenbeck & Ronquist, 
2001 ). First, we compared several independent 
runs using the default random tree option to 
monitor the convergence of the -In likelihoods 
of the trees. The -log likelihoods started at 
around -8,1 00 and converged on a stable value 
of about -4,300 after approximately 60,000 gen- 
erations. We then did a final run using the Me- 
tropolis-coupled Markov chain Monte Carlo 
variant with four chains (one cold, three heated) 
and 1,000,000 sampled generations with the 
current tree saved at intervals of 10 genera- 
tions. A 50% majority rule tree was constructed 
from all sampled trees with the first 1 0,000 trees 
(100,000 generations) ignored as burn in. 

MP and Bl analyses were conducted with 
simple and optimal model of sequence evolu- 
tion (the latter based on the Akaike Informa- 
tion Criterion implemented in Modeltest 3.6; 
Posada & Crandall, 1998), respectively. 

Parametric Bootstrapping (mtDNA) 

A parametric bootstrapping approach was 
used to specifically test the monophyly of On- 
comelania h. robertsoni (for a review of the 
parametric bootstrap see Hillis et al., 1996). 
First we ran Modeltest to find the optimal 
model of sequence evolution for the aligned 
sequences of all O. h. robertsoni haplotypes. 
We then conducted maximum likelihood (ML) 
searches in PAUP* v. 4.0b10 under the con- 
straint that O. /?. robertsoni is NOT monophyl- 
etic (null hypothesis). The resulting tree was, 
together with the aligned sequences, imported 
into Seq-Gen 1.2.5. (Rambaut & Crassly, 
1997) to generate 100 random data sets 
based on the model suggested by Modeltest. 
We then analyzed in PAUP the differences in 
tree lengths between the constrained and un- 
constrained trees for each of the 100 replicates. 
The frequency of differences in tree lengths 
was plotted and compared to the tree length 
difference (constrained vs. unconstrained) of 
the original unpermutated data set. Finally, we 
estimated how likely it was that this difference 
could have been observed randomly. 

Intraspecific Genomic Polymorphism (AFLP) 

AFLP genotyping is used here in a first attempt 
to study the degree of nuclear polymorphism in 



148 



WILKE ETAL. 



О. h. robertsoni on the DNA fingerprint level. 
However, given the limited number of specimens 
used for AFLP genotyping, we restrict our analy- 
ses to estimating diversity indices and to com- 
puting a minimum spanning network (MSN) 
among genotypes in a preliminary assessment 
of genetic structure in our data set. 

A matrix of corrected average pairwise dif- 
ferences between Oncomelania h. hupensis 
and O. h. robertsoni as well as within O. h. 
robertsoni was calculated in Arlequin 2,0 
(Schneider et al., 2000), The matrix was also 
used to construct the MSN for the AFLP 
haplotypes via Arlequin 2.0. 



RESULTS 
MtDNA Sequence Polymorphism 

Among the 66 specimens of Oncomelania 
h. roberison/ studied, a total of 40 haplotypes 
was found for the combined COI/LSU rRNA 
fragments. The average nucleotide diversity 
(corrected to the K2P-model) among all indi- 
viduals of O. h. robertsoni is 0,046 ± 0.004 
with a pairwise maximum of 0.117 between 
individuals A8d (Anning River Valley) and M2e 
as well as M2g (both from Meishan Area). 

Within Oncomelania h. robertsoni. we de- 
tected four relatively distinct genetic groups 
(characterized by average genetic divergences 
of > 0.04 and numbered I, IIa, IIb, and Ile in Table 
3 and Fig. 1). The divergences among these 
groups range from 0.042 ± 0.006 (between 
groups IIa and lie) to 0.085 ± 0.010 (between 
groups I and lie). In comparison, the overall level 
of genetic divergence among representatives of 
other Oncomelania hupensis subspecies ranges 
from 0.0097 ± 0.0029 (between O. h. hupensis 
and O. h. formosana) to 0.1024 ± 0.0103 (be- 
tween O. h. formosana and O. /?, quadrasi). 



It should be noted that in phylogeographical 
studies, the evolutionary relationships above 
and below the species level are different in 
nature and their resolution requires a differ- 
ent set of methods (Posada & Crandall, 2001 ). 
Therefore, many workers use phylogeograph- 
ical tools (e,g., network, population structure 
and gene flow analyses) to infer within-spe- 
cies relationships. However, preliminary tests 
show that the diversity in our data set is too 
high for these analyses. Therefore, we have 
to restrict the following mtDNA analyses to 
standard phylogenetic tests (MP and Bl phy- 
logenetic reconstruction as well as paramet- 
ric bootstrapping), 

MtDNA Phylogenetic Analyses 

Given the great significance of data set con- 
gruence for addressing potential problems of 
heteroplasmy and NUMTs, we also performed 
and compared separate phylogenetic analy- 
ses with the individual COI and LSU rRNAdata 
sets, despite the fact that the partition-homo- 
geneity test did not reveal significant conflicts. 
Both MP and Bl analyses revealed four dis- 
tinct phylogenetic groups of O. hupensis 
robertsoni in the COI and LSU rRNA phylog- 
enies, A manual comparison of the two trees 
showed a high congruence between the clus- 
ter patterns in the COI and LSU rRNA trees, 
that is, in both gene trees, the same speci- 
mens clustered in the same groups (individual 
trees not shown here). However, there were 
differences in the trees relative to the mono- 
phyly of the four groups within O, hupensis 
robertsoni. Whereas MP and Bl analyses of 
the COI data set resulted in trees that showed 
the four major groups of O. h. robertsoni [o be 
monophyletic, in the LSU rRNA data set the 
four groups were either paraphyletic (Bl analy- 
sis: clade I clustered together with the other 



TABLE 3: Average K2P nucleotide divergences between four major ge- 
netic groups of Oncomelania h. robertsoni (below diagonal line) and 
average nucleotide diversities within major groups (diagonal line). For a 
geographic distribution of these groups, see Fig. 1. 



IIa 



Ile 



I 0.018 ±0.003 

IIa 0.083 ±0.009 

IIb 0.085 ±0.009 

Ile 0.085 ±0.010 



0.011 ±0.002 
0.043 ±0.006 
0.042 ±0.006 



0.005 ±0.001 

0.043 ±0.007 0.010 ±0.003 



EXTREME SEQUENCE DIVERSITY IN ONCOMELANIA H. ROBERTSONI 



149 




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150 



WILKE ETAL. 



four subspecies of O. hupensis) or unresolved 
(MP analysis). 

We then combined the two data sets and per- 
formed several phylogenetic analyses using MP 
and Bl. In all combined analyses, we could re- 
cover the four distinct clades of O. h. robertsoni. 
generally with good support values. However, 
both MP and Bl could not fully resolve the rela- 
tionships among these clades (similar to the MP 
analysis of the LSD rRNAdata set-see above): 
there is a trichotomy of (1 ) the clade comprising 
the four other subspecies of O. hupensis used 
in the present study, (2) clade I of O. h. robertsoni, 
and (3) a clade composed of sub-clades IIa, IIb, 
and Ile of О. h. robertsoni (Fig. 1 ). 

The most basal clade in O. ti. robertsoni 
(clade I) has a wide geographic distribution. 
Haplotypes belonging to this clade were found 
in all geographic areas sampled in the present 
study, that is. in Yunnan, in the southern Anning 
River Valley, and in eastern Meishan Area. In 
contrast, based on the limited data presented 
here, clade IIa appears to be a localized clade 
with haplotypes coming exclusively from locali- 
ties in the northern Anning River Valley. Clade 
lib has a wider distribution, ranging from the 



southern Anning River Valley to a single local- 
ity in Meishan Area. Finally, clade lie is a local- 
ized clade restricted to Meishan Area. 

Sympatric specimens belonging to different 
clades were found in two localities: at site A8 
(southern Anning River Valley): of the 12 speci- 
mens studied, nine belong to clade I and three 
to clade lib and at site M2 (central Meishan 
Area): from ten specimens studied, three be- 
long to clade lib and seven to clade lie (Fig. 1 ). 

Parametric Bootstrapping 

Given the inability to solve the problem of 
Oncomelania h. robertsoni monophyly using 
the phylogenetic methods above, a paramet- 
ric bootstrapping test was performed. 

The alternate hypothesis of non-monophyly 
cannot be rejected (P > 0.41 ) as the observed 
difference in tree lengths between the con- 
strained and unconstrained tree in the original 
data set is smaller than the observed difference 
in 59% of the simulated data sets (Fig. 2). In 
other words, a tree that has been forced to show 
O. h. robertsoni non-monophyletic is not sig- 
nificantly worse than an unconstrained tree. 



35- P>0.41 



30 



25 



20 



О 



15 



10 



observed 
■ difference 



lilllll.ll- ■ 

1 2 3 4 5 6 7 8 9 10 11 12 13 

Difference in tree lengths 

FIG. 2. Result of the parametric bootstrap analysis for the hypothesis of non-mono- 
phyly of Oncomelania fi. robertsoni. The black bars show the number of simulated 
data sets and the corresponding differences in tree lengths between the constrained 
(non-monophyly cnterion) and unconstrained trees. The dashed line shows the ob- 
served difference for the original data set. 41 of the 1 00 sampled data sets have tree 
length differences equal or smaller than in the original data set. Therefore, the null 
hypothesis of non-monophyly of O. ti. robertsoni cannot be rejected. 



EXTREME SEQUENCE DIVERSITY IN ONCOMELANIA H. ROBERTSONI 



151 



AFLP Analysis 



DISCUSSION 



All 24 specimens analyzed had unique AFLP 
fingerprints. Estimates of diversity indices (av- 
erage pairwise differences among all O. h. 
robertsoni genotypes divided by the total num- 
ber of loci scored as well as the average 
pairwise differences between O. h. hupensis 
and O. h. roberison/ divided by the total num- 
ber of loci) resulted in a within O. h. robertsoni 
diversity of 0. 1 1 3 ± 0.045 and in a divergence 
between the two respective subspecies of 
0.214 ±0.034. 

The MSN network (Fig. 3) shows most O. h. 
robertsoni genotypes clustering in a star-like 
pattern. The single O. h. iiupensis genotype 
scored is distinct from the O. /?. robertsoni 
genotypes. Within O. ti. robertsoni, some 
genotypes(e.g.,Y1f, M2g,A8g,M2b, andA2b) 
are relatively distinct as well. Given the star- 
like nature of the network, no clear population 
structure is recognizable and specimens from 
the same site do not cluster together in dis- 
tinct groups. Also, the four major mtDNA clades 
found in the present study (Fig. 1) are not re- 
flected in the AFLP data. 



Given the great significance of phylogeo- 
graphic patterns in the intermediate schisto- 
somiasis host Oncomelania h. robertsoni for 
understanding host/parasite relationships, 
there are several interesting findings in our 
study that potentially can help to shed some 
light on our observation of high rates of mtDNA 
sequence divergences within Oncomelania h. 
robertsoni: 

(1 ) all COI sequences are fully protein-coding 
with no insertions or deletions; 

(2) both individual and combined analyses of 
the mtDNA COI and LSU rRNA genes 
show four distinct haplotype groups within 
the subspecies of interest (note that given 
our still preliminary sampling design, it is 
well possible that more haplotype groups 
will be recovered in future studies); 

(3) neither the phylogenetic analyses nor the 
parametric bootstrapping test performed 
here are conclusive relative to the monophyly 
of the four O. h. robertsoni clades found; 

(4) both the partition-homogeneity test and vi- 
sual inspections of the individual CO! and 



Oncomelania 
h. hupensis 




Oncomelania h. robertsoni 



FIG. 3. Minimum spanning network for observed AFLP genotypes of Oncomelania h. robertsoni (large 
white circles) based on 102 polymorphic loci. For comparison, a specimen of O. h. hupensis (large 
black circle) was included. Small black circles indicate the scored differences between the haplotypes. 
For individual codes see the Appendix. 



152 



WILKE ETAL. 



LSU rRNA trees revealed high concor- 
dance in cluster patters and arrangement 
of individual mtDNA haplotypes; 

(5) two of the mtDNA clades recovered ap- 
pear to be localized, whereas two are 
widely distributed; 

(6) sympatry of individuals belonging to differ- 
ent mtDNA clades does occur: 

(7) there is no phylogenetic concordance be- 
tween the mitochondrial and preliminary 
nuclear data presented here: and 

(8) the nuclear data from AFLP genotyping in- 
dicate a lack of clear population structure 
in O. h. robertsonl. 

Based on these results, we will focus in our 
discussion on the high rates of intraspecific 
mtDNA variability and discuss some of the 
explanations found in the literature and their 
relevance for the O. h. robetisoni problem. 

Presence of a Cryptic Species Complex 

The presence of cryptic species radiations 
has been reported for many mollusc groups, 
though morphostasis seems to be particularly 
common in rissooidean gastropods (e.g., Pon- 
der et al., 1995: Hershleretal., 1999: Wilke& 
Pfenninger, 2002). In fact, several studies on 
snail hosts in SB Asia revealed cryptic radia- 
tions in the family Pomatiopsidae (e.g., Davis, 
1992: Attwood & Johnston, 2001). However, 
the taxon of concern in the present paper, 
Oncomelania hupensis, is one of the morpho- 
logically and ecologically best studied snail 
taxa in Southeast Asia. Particularly the three 
Chinese subspecies (O. h. hupensis, O. h. 
robertsoni. and O. h. tangí) were subject to 
extensive shell morphological and quantitative 
anatomical studies and comparative anatomi- 
cal analyses did not reveal significant differ- 
ences within these subspecies. In fact, a 
comparative anatomical study of O. h. robert- 
soni populations from Yunnan and Sichuan 
provinces showed that they are anatomically 
undistinguishable (George Davis, unpublished 
data). Moreover, an allozyme study (Davis et 
al., 1 995) of the same three populations of O. 
h. robertsoni hom Sichuan and Yunnan prov- 
inces showed low levels of heterogeneity 
within and between populations that are not 
indicative of a marked departure from the other 
subspecies. In fact, the allozyme heterogene- 
ity within 0. h. robertsoni was lower than in 
the eastern Chinese subspecies O. h. hupen- 
sis. This lack of population structure within O. 
h. robertsoni coula be confirmed in our AFLP 
study. Given these findings, the presence of a 



cryptic taxon complex within O. h. robertsoni 
can very likely be ruled out as a cause for the 
high degree of mtDNA diversity within this sub- 
species. 

Duplications within the Mitochondrial Genome 

Duplications of genes or gene fragments 
within the mitogenome involving protein-cod- 
ing genes are most often explained with the 
mechanism of tandem duplication of gene re- 
gions as a result of slipped strand mispairing, 
followed by the deletions of genes (Inoue et 
al., 2003, and references therein). Most dupli- 
cations involve short fragments where control 
regions and tRNA genes seem to be particu- 
larly prone to mispairing but there are also 
reported cases of duplication portions > 8 kbp 
(e.g., Moritz & Brown, 1 987: Inoue et al., 2003). 

If duplications of mtDNA genes were respon- 
sible for the observed mtDNA patterns in O. 
h. robertsoni, then this would involve a large 
portion of the mtDNA genome containing both 
CGI and LSU rRNA genes. While this does 
not seem to be impossible (see above), it is 
not very likely as this explanation requires a 
high number of assumptions. 

Presence of Nuclear Mitochondrial DNA 
(NUMT or "Pseudogenes") 

Nuclear copies of mitochondrial genes, so- 
called nuclear mitochondrial DNA (NUMT) or 
"pseudogenes" (e.g., Lopez et al., 1994; Ben- 
sasson et al., 2001) have been observed in 
many animal species and if unnoticed, can 
severely confound phylogenetic and popula- 
tion genetic studies (Zhang & Hewitt, 1996). 
According to Bensasson et al. (2001), symp- 
toms of NUMT contamination of mtDNA can 
include; (A) PCR ghost bands, (B) sequence 
ambiguities (e.g., if encountered in forward and 
reverse strands), (C) frame shift mutations, 
and (D) stop codons. None of these symptoms 
were observed in the sequence data gener- 
ated for the present study i.e., there were no 
ghost bands in the PCR products, there were 
no relevant alignment confticts in forward and 
reverse strands, there were no insertions or 
deletions in the alignment of the protein-cod- 
ing CGI gene, and the gene portion studied 
was free of stop codons. Moreover, as the in- 
dividual CGI and LSU rRNA phylogenies are 
concordant, both genes would have had to 
move simultaneously into the nuclear genome. 
Given all these facts, we can rule out the pres- 
ence of NUMTs in our data sets. 



EXTREME SEQUENCE DIVERSITY IN ONCOMELANIA H. ROBERTSONI 



153 



Heteroplasmy 

Heteroplasmy, that is, the presence of more 
than one type of mtDNA in males/female or 
even in the same organism, has been reported 
from several invertebrate species (e.g. Zouros 
et al., 1 992; Fujino et al., 1 995; van Herwerden 
et al., 2000; Steel et al., 2000). 

The mitochondrial genome is usually inher- 
ited maternally, but paternal 'leakage' and/or 
biparental inheritance patterns are common in 
some groups (e.g., Mytilus; Hoeh etal., 1991). 
In addition to biparental inheritance, animal 
mitochondrial heteroplasmy can also be 
caused by mutation of the genome within the 
individual or within the original oocyte (Steel et 
al., 2000). In most cases, heteroplasmy only 
involves variations in the number for repeats 
within the mitochondrial control region (e.g., 
Hoarau et al., 2002) but base substitutions in 
coding genes also have been found (e.g., van 
Непл /erden et al., 2000; Steel et al., 2000). 
While paternal or biparental inheritance can not 
be completely dismissed as cause for the mi- 
tochondrial diversity observed in O. h. robert- 
soni, it is not likely as, for example, all 28 
specimens studied from sites AI -7 belong to 
the same clade. Another possible scenario 
would be the existence of distinct populations 
of mitochondria due to non-concerted evolu- 
tion. Fujino et al. (1995) and van Herwerden 
et al. (2000) found heteroplasmy in the COI 
and ND1 genes, respectively, of several dige- 
netic trematode species. The workers sug- 
gested that structurally different forms of 
mitochondria are present in the tegumental and 
parenchymal cells of adults. Given the life style 
of the amphibious Oncomelania h. robertsoni, 
that is, the ability to closely shut the shell with 
its operculum to avoid dehydration during dry 
conditions and the fact that some freshwater 
snails (including snail hosts for schistosomia- 
sis) have been shown to be capable of switch- 
ing between aerobic and anaerobic respiration 
(Jurberg et al., 1997; van Hellemond et al., 
1995, 2003), structurally different types of mi- 
tochondria associated with different metabolic 
respiratory processes could, at least in theory, 
exist in Oncomelania h. robertsoni. However, 
as the full phylogenetic concordance of COI 
and LSU rDNA haplotypes (based on the com- 
parison of the individual CO! and LSU rDNA 
trees) does not support the existence of more 
than one type of mitochondrion in a single in- 
dividual, non-concerted evolution appears to 
be extremely unlikely as well. 



Temporal Isolation Followed by Secondary 
Contact 

An increasing number of studies shows that 
temporal isolation followed by secondary con- 
tact has deeply influenced the phylogeography 
of many Palearctic species (e.g. Taberlet et 
al., 1998; Hewitt, 2000). Particularly, pro- 
cesses resulting from fragmentation into gla- 
cial refuges followed by range expansions via 
postglacial colonization routes may lead to 
secondary contact zones among formerly dis- 
jointed lineages (e.g.. Pfenninger & Posada, 
2002). Pleistocene glaciations and climate 
changes certainly must have affected the riv- 
ers and streams of the area that is currently 
populated by O. h. robertsoni. However, we 
doubt that these phylogeographic processes 
alone are responsible for the extant mtDNA 
patterns seen today. The divergence between 
the major clades of O. h. robertsoni wúh K2P 
differences of up to 8.5% for the combined 
COI/LSR rDNA data set are indicative of much 
older divergence times than late Pleistocene 
or Holocene. Wilke (2003) suggested an av- 
erage COI local clock rate of 1.83 ± 0.21% 
uncorrected distance/my for Protostomia lin- 
eages that are not affected by saturation. 
Given an uncorrected average pairwise COI 
distance of 8.7% between clades I and II in 
our analysis (Fig. 1), the oldest split in O. /7. 
robertsoni is potentially some 4 my old (i.e., 
early Pliocene) and predates the split of all 
other O. hupensis subspecies. Secondary con- 
tact of formerly isolated population may there- 
fore not fully explain the patterns observed 
here, particularly as there is no compelling 
supporting evidence from our AFLP data or 
previous allozyme studies conducted by Davis 
etal. (1995). 

Retention of Ancestral mtDNA Polymorphism 

The conflict between our mtDNA und nuclear 
data sets combined with the potentially long 
age of the O. h. robertsoni clades, as dis- 
cussed above, may be indicative of a problem 
in some mtDNA analyses: retained ancestral 
polymorphism. 

A mtDNA phylogeny represents a gene tree 
that may not be congruent with the species tree 
(i.e., no reciprocal monophyly in the descen- 
dant taxa) because of the retention of ances- 
tral lineages due to stochastic processes (e.g.. 
Avise, 2000; Moore, 1995). This is particularly 
true for species with ancient divergences 



154 



WILKE ETAL. 



(Avise, 2000) and the problem cannot be 
solved using multiple mtDNA genes, as the 
animal mitochondrial genome is inherited as a 
single unit. Therefore, phylogenies derived 
from multiple mtDNA genes are not indepen- 
dent estimates of a species' phylogeny (Moore, 
1995; Page, 2000). 

Long-term substantial isolation among popu- 
lations of O. h. robertsoni could have disrupted 
gene flow and therefore allowed the retention 
of anciently separated matrilines. As pointed 
out by Avise (2000), the evolutionary continu- 
ance of isolated populations may buffer 
against the extinction of lineages within a spe- 
cies. However, this would not explain the oc- 
currence of different matrilines in sympatry as 
seen in sites M2 and A8 (Fig. 1 ). Perhaps there 
is secondary contact among these lines after 
all (i.e., introgression), either due to post-Pleis- 
tocene range expansions or human impact 
(like transport of snails or their eggs with rice 
plants). However, as our AFLP data (and pre- 
vious allozyme and morphological and eco- 
logical data) do not support the mtDNA 
matrilines, we suggest that there is no evi- 
dence of organismal subdivision in O. h. 
robertsoni (for a very similar case involving 
Drosphila simulans: Ballard et al., 2002). 

It is beyond the scope of this paper to dis- 
cuss the distinct selective forces acting on the 
mitochondrial and nuclear genomes. However, 
tests for deviation from a strictly neutral model 
of evolution in our mtDNA data sets based on 
Fu and Li's D*and F*(Fu & Li, 1993) as imple- 
mented in DnaSP 3.53 (J. Rozas & R, Rozas, 
1999) showed that the COI data set deviates 
significantly from expectations under neutral- 
ity both in Fu and Li's D* (1 .78, P < 0.02) and 
in Fu and Li's F* (1.80, P < 0.05). Neutrality 
was not rejected in the (smaller) LSU rDNA 
data set with values of 0.56 (P > 0,10) and 0.57 
(P > 0,10) for Fu and Li's D* and F*, respec- 
tively. At least the results for the COI data set 
suggest that selection and/or population level 
processes like expansion, contraction, or sub- 
division (Ballard & Whitlock, 2004) are acting 
upon the mtDNA in O. h. robertsoni. 

Interestingly, one of the extrinsic forces that 
has been shown to influence mtDNA evolution 
in natural populations are parasites (e.g., Turelli 
& Hoffmann, 1995; Ballard et al,, 2002). 
Whether, the parasite of O. h. robertsoni, Scfiis- 
tosoma sp., has a similar effect on the mtDNA 
evolution of its host would need to be tested in 
future studies. 

In the present paper, we offer DNA data from 
two mitochondrial gene fragments as well as 
preliminary data from AFLP genotyping as a 



first step to assess the problem of deviant lin- 
eages in O. h. robertsoni. We suggest that the 
presence of a cryptic species complex or the 
occurrence of NUMTs are unlikely to explain 
the phylogeographic patterns observed. 
Though, we cannot completely dismiss the 
occurrence of heteroplasmy or duplications 
within the mitochondrial genome, which have 
been observed in molluscs before, these ex- 
planations are unlikely as well. The most prob- 
able scenario is the retention of ancestral 
mtDNA polymorphism possibly in combination 
with some effects of secondary contact. Based 
on our preliminary AFLP data, we also sug- 
gest that there is no evidence of organismal 
subdivision in O. h. robertsoni. However, these 
hypotheses need to be tested thoroughly in 
future study. 

Nevertheless, we find it important to present 
our preliminary findings in order to draw at- 
tention to the problem observed. As interme- 
diate host for schistosomiasis in western 
China, Oncomelania h. robertsoni \s receiving 
growing attention in ecological and parasito- 
logical studies. It is strongly suggested that 
future studies incorporate more data from 
nuclear loci in order to better understand 
phylogeography, population genetics and host- 
parasite co-evolution in O. /?. robertsoni. 



ACKNOWLEDGEMENTS 

This work was supported in part by a United 
States NIH grant IP50-AI 3946 to the Shang- 
hai Tropical Medical Research Center, P. R. 
China (GMD, Co. P.I.) and in part by a grant of 
the German Science Foundation (Wl 1902/1- 
2) to TW. We thank Chunghong Yang of the 
Sichuan Institute of Parasitic Diseases for pro- 
viding detailed locality information. 

We are very grateful to Dr. Randy Hoeh (Kent 
State University, Kent, U.S.A.), Dr. David Blair 
(James Cook University, Townsville, U.S.A.), 
and Dr. Paul Brindley (Tulane University Health 
Sciences Center, New Orleans, U.S.A.) for their 
useful comments on a previous version of the 
paper. 



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Revised ms. accepted 30 September 2005 



EXTREME SEQUENCE DIVERSITY IN ONCOMELANIA H. ROBERTSONI 157 

APPENDIX 

Individual codes (M = Meishan Area, A = Anning River Valley, Y = Yunnan), DNA voucher num- 
bers and GenBank accession numbers for Chinese specimens of Oncomelania hupensis robertsoni 
studied. 



Individual 


DMA 


GenBank accession # 


Individual 


DNA 


GenBank accession # 


code 


voucher #** 


COI/LSU rRNA 


code 


voucher #** 


COI/LSU rRNA 


M1a 


0963 


DQ212797/- 


A4d 


0958 


DQ212828/DQ212883 


M1b 


0965 


DQ212798/- 


A5a 


0951 


DQ212829/DQ212884 


M2a 


0941 


DQ212799/DQ212863 


A5b 


0952 


DQ212830/DQ212885 


M2b 


0942 


DQ212800/DQ212864 


A5c 


0953 


DQ212831/DQ212886 


M2c 


0943 


DQ212801/DQ212865 


A5d 


0954 


DQ212832/- 


M2d 


1022 


DQ212802/- 


A6a 


0947 


DQ212833/DQ212887 


M2e 


1388 


DQ212803/DQ212866 


A6b 


0948 


DQ212834/- 


M2f 


1389 


DQ212804/DQ212867 


A6c 


0949 


DQ212835/- 


M2g 


1390 


DQ212805/DQ212868 


A6d 


0950 


DQ212836/DQ212888 


M2h 


1391 


DQ212806/DQ212869 


A7a 


0937 


DQ212837/DQ212889 


M2i 


1392 


DQ212807/DQ212870 


A7b 


0938 


DQ212838/- 


M2j 


1393 


DQ212808/DQ212871 


A7c 


0939 


DQ212839/DQ212890 


M3a 


MG14 


DQ212809/- 


A7d 


1021 


DQ212840/- 


M3b 


MG15 


DQ212810/- 


A8a 


0019 


DQ2 12841 /- 


M3c 


MG16 


DQ212811/- 


A8b 


0020 


DQ212842/DQ212891 


M3d 


MG30 


DQ212812/- 


A8c 


0021 


DQ212843/DQ212892 


M3e 


MG33 


DQ212813/- 


A8d 


0022 


DQ212844/DQ212893 


M4a 


-* 


AF531547/AF531545* 


A8e 


0023 


DQ212845/- 


A1a 


0932 


DQ212814/- 


A8f 


0026 


DQ212846/DQ212894 


A1b 


0934 


AF213339/AF212893 


A8g 


0028 


DQ21 284 7/DQ21 2895 


A1c 


0935 


DQ212815/- 


A8h 


0029 


DQ212848/DQ212896 


Aid 


1018 


DQ212816/- 


A8i 


0030 


DQ212849/- 


A2a 


0928 


DQ212817/DQ212872 


A8j 


0050 


DQ1 12252/- 


A2b 


0929 


DQ212818/DQ212873 


A8k 


0051 


DQ212850/DQ212897 


A2c 


0930 


DQ212819/DQ212874 


A8I 


0057 


DQ212851/DQ212898 


A2d 


0931 


DQ212820/DQ212875 


Via 


0045 


AF253074/DQ2 12899 


A3a 


0959 


DQ212821/DQ212876 


Ylb 


0046 


DQ212852/- 


A3b 


0960 


DQ212822/DQ212877 


Ylc 


0048 


AF253075/- 


A3c 


0961 


DQ212823/DQ212878 


Yid 


0055 


DQ212853/- 


A3d 


0962 


DQ212824/DQ212879 


Yie 


0066 


DQ212854/- 


A4a 


0955 


DQ212825/DQ212880 


Ylf 


1505 


DQ212855/DQ212900 


A4b 


0956 


DQ212826/DQ212881 


Y1g 


1506 


DQ212856/DQ212901 


A4c 


0957 


DQ212827/DQ212882 


Ylh 


1508 


DQ212857/DQ212902 



* from Attwood et al. (2003) 

** deposited at the DNA voucher collection of the Justus Liebig University, Giessen 



MALACOLOGIA, 2006, 48(1-2): 159-251 

A SYSTEMATIC REVISION OF THE SOUTHEAST ASIAN 
FRESHWATER GASTROPOD BROTIA (CERITHIOIDEA: PACHYCHILIDAE) 

Frank Köhler* & Matthias Glaubrecht 

Department of Malacology, Museum für Naturkunde, Humboldt University, 
Invalidenstr 43, D-1011 5 Berlin, Germany 

ABSTRACT 

We here present morphological and molecular genetic data on species of the Southeast 
Asian freshwater pachychilid Brotia, based on examination of material originating from 
various museum collections world wide, including type material, as well as material from 
field collections in Thailand and Indonesia. We show that a number of previous systematic 
assumptions about Brotia are in need of correction. Based on our analyses, we suggest a 
revised and more specific characterisation of this genus and outline the taxonomic and 
systematic implications of our findings. Accordingly, Brotia is restricted herein to vivipa- 
rous pachychilids possessing as morphological characteristics a subhaemocoelic brood 
pouch, a palliai oviduct with only a simple, deep, and papillated spermatophore bursa, as 
well as embryonic shells with a wrinkled apical whorl. This typical embryonic shell struc- 
ture results from a peculiar mode of ontogeny that includes a yolk sac protruding from the 
apical whorl during most stages of embryonic development, which are retained in the ma- 
ternal brood pouch. A molecular phylogeny based on two mitochondrial gene fragments 
(646 bp of COI and 826 bp of 16S) shows that Brotia as encompassed here forms a 
monophyletic group. The application of the revised concept results in a significantly re- 
duced number of species assigned to Brotia with implications also for a considerable re- 
duction of the distributional area covered by members of the genus. In total, 35 species 
are recognized; the systematic affinities of eight of them remain unclear, however. Data on 
the morphology, distribution and if known on the biology of these species is presented. 

Key words: taxonomy, systematics, Cehthioidea, Pachychilidae, Brotia, Melania, mor- 
phology, viviparity. 



INTRODUCTION 

It is a major challenge of modern biosystem- 
atic research to provide classifications that 
correctly reflect phylogenetic relationships 
among organisms. The complexity of work 
required to achieve this goal has inspired the 
allegory of an Herculean task, as so aptly for- 
mulated by Graf (2001) for his catalogue of 
North American Pleuroceridae. However, while 
in the Greek myth the job was finished by un- 
usual ways and means, in systematics accu- 
racy is needed. In addition to a well-founded 
hypothesis on the natural relationships be- 
tween taxa, a sound systematics also neces- 
sitates thorough revision of the taxonomy, 
which alone is a challenge as exemplified for 
various freshwater gastropods, such as 
Pleuroceridae (Graf, 2001), Pachychilidae 



(Köhler & Glaubrecht, 2002a), or Neotropic 
Ampullariidae (Cowie & Thiengo, 2003). In the 
current work, we attempt to combine both a 
taxonomic revision and a phylogenetic study, 
in order to improve our understanding of Asian 
freshwater snails of the genus Brotia H. 
Adams, 1866, which still are poorly known. 

Brotia is a member of the Pachychilidae, a 
family that was earlier incorporated within the 
so-called "melanians" or "Melaniidae", which 
represent a polyphyletic assemblage of fresh- 
water Cehthioidea (reviewed by Glaubrecht, 
1996, 1999; molecular phylogeny in Lydeard 
et al., 2002). Views on the correct familiar as- 
signment of Brotia have changed in recent 
decades due to our steadily improving knowl- 
edge. According to earlier systematic opinions, 
the genus was affiliated either with Thiaridae 
(e.g., Morrison, 1954; Brandt, 1968, 1974; 



'Corresponding author: frank.koehler@rz.hu-berlin.de 



159 



160 



KÖHLER & GLAUBRECHT 



Davis, 1971) or Pleurocehdae (e.g., Vaught. 
1989): this was reviewed by Kölnler & Glaub- 
recht (2002a). Molecular phylogenetic studies, 
however, show that Brotia is a pachychilid 
(Köhler et al., 2004), which in turn represent 
one of six cerithioidean freshwater clades next 
to, for example, the Thiaridae, Pleuroceridae, 
Melanopsidae. to name a few only here (for a 
molecular phylogeny of the Cerithioidea, see 
Lydeard etal., 2002). 

Freed from systematic misconceptions, 
Pachychilidae were recently highlighted as an 
¡deal focal group to address biological aspects 
of more general importance connected, for ex- 
ample, to processes of speciation and morpho- 
logical adaptation (Rintelen et al., 2004), the 
evolution of different modes of reproduction 
(Köhler et al., 2004), as well as biogeographi- 
cal problems (Glaubrecht, 2000; Glaubrecht & 
Rintelen, 2003). 

Taxonomy and systematics especially of the 
Asian Pachychilidae have remained confusing 
for a long time, as outlined by Köhler & Glaub- 
recht (2001, 2002a). Most Asian pachychilid 
species sooner or later were attributed to Brotia 
by one or the other author (e.g.. Martens, 1900; 
Thiele, 1928, 1929; Rensch, 1934; Abbott, 
1948; Solem, 1966; Brandt, 1968, 1974), which 
rendered Brotia a taxon frequently referred to 
in systematic literature but in turn most vaguely 
defined by means of its morphology, distribu- 
tion, and species composition. 

In an initial study, it was shown that Brotia as 
perceived up to then was an assemblage com- 
posed of four species groups characterized by 
the possession of different reproductive and 
embryonic shell morphologies (Köhler & Glaub- 
recht, 2001). In the meantime, it has been sub- 
stantiated that each of these groups indeed 
represents a distinct evolutionary lineage. The 
degree of morphological distinctiveness of these 
lineages has been considered large enough to 
justify the treatment as separate genera. Accord- 
ingly, in addition to Brotia, the following Asian 
pachychilid genera are currently recognized; 
Sulcospira Troschel, 1 858 (Köhler & Glaubrecht, 
2005), Pseudopotamis Martens, 1900 
(Glaubrecht & Rintelen, 2003), Tylomelania S. 
Sarasin & F. Sarasin, 1897 (Rintelen et al., 2004; 
Rintelen & Glaubrecht, 2005), Adamietta Brandt, 
1974, Paracrostoma Cossmann, 1900, and 
Jagora Köhler & Glaubrecht, 2003. Therefore, 
the status of various supraspecific pachychilid 
taxa was clarified in the last few years. Although 
we now know much better which species do 
not belong to Brotia, this has not necessarily 
improved our knowledge of Brotia itself. 



For this reason, the current work aims at a 
taxonomic and systematic revision of the ge- 
nus by comparative analysis of morphological 
and mitochondrial sequence data. This shall 
contribute to a stable and unequivocal tax- 
onomy and systematics of Brotia as a group 
frequently referred to in accounts on Asian 
freshwater gastropods and at the same time 
provide the fundament for future studies on 
the phylogeny, evolution, and biogeography 
of these promising model organisms. 



MATERIAL AND METHODS 

Nomenclatural Remarks 

The treatment of some species group names 
introduced by Troschel (1857) is subject to 
dispute. Bouchet & Rocroi (2005) argue that 
the non-hierarchical usage of the names 
"Thiarae" and "Pachychili" as well as 
"Bithyniae", "Lithoglyphi", "Hydrobiae", and 
"Ancyloti" by Troschel (1857) stands in con- \ 
trast with the procedure in the rest of his work, 
in which the ranks assigned to the formed 
names are indicated by formal endings, such 
as "-idea", "-ina", or "-acea". In case of the 
above-cited group names, Troschel (1857) 
explicitly refrained from such an assignment 
of family ranks for the somewhat ambiguous 
data he was faced with. For this reason, it was 
suggested by Bouchet & Rocroi (2005) to ig- 
nore these names. However, it has also been 
pointed out that some of these names, such 
as Bithyniidae, Thiaridae, or Hydrobiidae, are 
commonly published with Troschel as author. 
In contrast to the suggestion of Bouchet & 
Rocroi (2005) and unless it might otherwise 
be stipulated by an official decision of the 
ICZN, we prefer to further employ the names 
introduced by Troschel (1857), not only be- 
cause we regard them as available and valid 
irrespective of the circumstance that the au- 
thor refrained from the assignment of a spe- 
cific rank, but also in order to keep continuity 
in the use of zoological names. 

Material Examined 

This study is based on examination of mate- 
rial from various museum collections world 
wide (see under repositories). Most samples 
investigated comprise dry shells only; some 
others were fixed in 70-96% ethanol or in 
formalin. In many cases, preserved museum 
material was not suitable for a more detailed 



SYSTEMATIC REVISION OF BROTIA 



161 



examination of gross anatomy, for example, by 
histology, because the bodies were in a bad 
condition due to partial decay. It has further- 
more proven impossible to extract DNA from 
museum material. In orderte achieve a broader 
basis for our examinations, new collections 
were undertaken in Thailand and Indonesia. 
This voucher material is preserved in 75-96% 
ethanol, and is deposited with the Malacologi- 
cal Collection of the ZMB. To allow proper and 
quick fixation of the soft bodies, some shells 
were cracked prior to ethanol preservation. 
Consequently, the basis of available samples 
varies considerably between the different spe- 
cies in respect both to quality and quantity of 
available material. From some species hardly 
more than some dry shells were accessible, 
rendering it impossible to sufficiently assess 
the morphological and geographical range, 
whereas from others material was available 
suitable for various kinds of examinations. 



H— 




BW 



WA 



Morphological Examination 

Dimensions of adult and embryonic shells 
were measured with callipers to 0.1 mm using 
standard parameters (Figs. 1, 2). These pa- 
rameters were analysed using statistic software 
SPSS (vs 9.0). Anatomy was studied using a 
stereo microscope. Extracted radulae were 
cleaned as described by Holznagel (1 998) and 
mounted on stubs and coated with Gold-Pal- 
ladium for SEM examination with a Jeol FSM 
6300 scanning electron microscope. Embry- 
onic shells extracted from ethanol preserved 
specimens or from dried shells were cleaned 



FIG. 1 . Shell parameters used for morphometrical 
analyses. 

mechanically and by sonication and prepared 
for SEM as given for the radulae. Since in 
viviparous freshwater Cerithioidea a distinct 
transition from the larval or primary shell 
(= protoconch) to the adult or secondary shell 
(= teleoconch) is lacking for the loss of free 
larval stages, we apply the more general term 
"embryonic shell" for all shelled stages retained 
in the brood pouch. Embryonic shell param- 
eters were measured as shown in Figure 2. 
Soft tissues were treated with hexamethyl- 



H- 




FIG 2. Embryonic shell parameters used for morphometrical analyses. 



162 



KÖHLER & GLAUBRECHT 



disilazane prior to SEM as described by Na- 
tion (1983). Stomach morphology was exam- 
ined using the methodology and terminology 
described by Strong (2003). 

Molecular Genetics 

Two fragments of the mitochondrial genes of 
the Cytochrome Oxidase I ("COI". 646 bp) and 
the 1 6S rRNA ("1 6S", 826 bp) were sequenced. 
The data set contains 40 sequence pairs be- 
longing to 16 species of Brotia. five sequence 
pairs belonging to four species of Adamietta, 
and a sequence pair each of two species of 
Paracrostoma. Two additional sequence pairs 
belonging to Jagora were included as outgroup 
representatives. DNA was purified from about 
1-2 mm- of foot tissue from specimens pre- 
served in ethanol by СТАВ extraction (Winne- 
penninckx et al., 1993). PCR amplification of 
the fragments were performed in 25 \j\ volumes 
containing 1x Taq buffer, 1.5 mM MgCl2, 200 
pM each dNTP, 1 U Taq polymerase, approxi- 
mately 100 nM DNA and ddH20 up to volume 
on a Perkin Elmer GeneAmp 9600 or 2400 
thermocycler. After an initial denaturation step 
of 3 min at 95°C. cycling conditions were 35 
cycles of 1 min each at 95°C, 45-53°C, and 
72°C, with a final elongation step of 5 min. 
Primers used were LCO 1490 5'-GCTCAA 
CAAATCATAAAGATATT-3' and HC021 98 var. 
5-TAWACTTCTGGGTGKCCAAARAAAT-3' 
(Folmeretal., 1994, modification of HC021 98 
by A. B. Wilson) for COI, and 16SF 5'- 
CCGCACTTAGTGATAGCTAGTTTC-3' (Wil- 
son et a!.. 2004) and H3059-lnv 5'-CGGTYTG 
AACTCAGATCATGT-3' (Palumbi et al., 1991) 
for 16S. respectively. PCR products were pu- 
rified with QiaQuick PCR purification kits 
(Qiagen) following the standard QiaQuick PCR 
purification protocol. Both strands of the two 
genes were cycle sequenced with the original 
primers using ABI Prism BigDye^" terminator 
chemistry and visualized on an ABI Prism 377 
automated DNA sequencer. The resulting se- 
quence electropherograms of both strands 
were corrected manually for misreads and 
merged into one sequence file using BioEdit 
Version 5.0.1 (Hall, 1999). Sequences are ac- 
cessible via GenBank (accession numbers in 
Table 6). 

Sequence Analysis 

COI sequences were aligned manually and 
checked by translating the DNA sequences 
into amino acids in DAMBE 4.1 .19 (Xia & Xie, 



2001 ) using the genetic code for invertebrate 
mitochondrial DNA. 16S sequences were 
aligned using the online version of ClustalW 
provided by the hompeage of the Europaen 
Bioinformatics Institute (www.ebi.ac.uk/ 
clustalw/) (Thompson et al., 1994) using de- 
fault settings. A combined data set was con- 
structed by concatenating the sequences. 
Pair-wise genetic distances were calculated 
with PAUP* (Swofford, 1999). Phylogenetic 
trees were reconstructed using Neighbor Join- 
ing (NJ) (Saitou & Nei, 1987) and Maximum 
Parsimony (MP) as implemented in PAUP*. 
In addition, a Bayesian method of inference 
(Bl) was employed to estimate phylogenetic 
relationships (e.g., Huelsenbeck et al., 2002; 
Holder & Lewis, 2003) using MrBayes 3.0 
(Huelsenbeck & Ronquist, 2001). NJ analy- 
ses were conducted using the random initial 
seed option to break ties and under a general 
time reversible model of sequence evolution 
(GTR+1 +Г; Gu et al., 1995) to correct for mul- 
tiple substitutions. In the MP analyses, the 
heuristic search algorithm was employed with 
ten random additions of taxa and tree bisec- 
tion-reconstruction (TBR) branch swapping. 
Gaps were treated as fifth base. Other set- 
tings were left on default. Prior to Bl analyses, 
it was explored which substitution model fits 
best the sequence data set by running a hier- 
archical likelihood ration test implemented in 
MrModeltest (Nylander, 2002). For Bl analy- 
sis a Metropolis-coupled Markov chain Monte 
Carlo (4 chains, chain temperature = 0.2) was 
run for 750,000 generations. A 50% majority- 
rule consensus tree was constructed for the 
last 2,500 trees in order to assess the poste- 
rior clade probabilities for each node (bpp). 

Repositories and their Abbreviations 

AMS Australian Museum, Sydney, 

Australia 
ANSP Academy of Natural Sciences, 

Philadelphia, Pennsylvania, U.S.A. 
BMNH Natural History Museum, London, 

United Kingdom 
CAS California Academy of Sciences, 

San Francisco, California, U.S.A. 
IMC Indian Museum, Calcutta, India 
MCZ Museum of Comparative Zoology, 

Cambridge, Massachusetts, U.S.A. 
MHNG Muséum d'Histoire Naturelle, 

Genève, Switzerland 
MNHN Muséum National d'Histoire 

Naturelle, Paris, France 
MZB Zoological Museum, Bogor, Indonesia 



SYSTEMATIC REVISION OF BROTIA 



163 



NMB Naturhistorisches Museum, Basel, 
Switzerland 

RMNH Natural History Museum Naturalis, 
Leiden, The Netherlands 

SMF Senckenbergmuseum, Frankfurt/ 
Main, Germany 

ÜMB Überseemuseum, Bremen, Germany 

USNM National Museum of Natural 

History, Smithsonian Institution, 
Washington D.C., U.S.A. 

ZMA Zoologisch Museum, Amsterdam, 
The Netherlands 

ZMB Museum für Naturkunde, Humboldt- 
Universität Berlin [formerly Zoologi- 
sches Museum], Germany 

ZMH Zoologisches Museum und Institut, 
Universität Hamburg, Germany 

ZSI Zoological Survey of India, 

Calcutta, India 

ZSM Zoologische Staatssammlung, 
München, Germany 

ZMZ Zoologisches Museum, Zürich, 
Switzerland 

Abbreviations 

В breadth of shell 

BW height of the body whorl 

bp brood pouch 

bpp brood pouch pore 

с cerebral ganglion 

eg capsule gland 

cr crescent fold 

crt septate crescent thickening 

ct ctenidium 

DA diameter of apical whorl of embryonic 

shell 
dg digestive gland 
dgd digestive gland duct 
eg egg capsule 
ey eye 
ft foot 

gg genital groove 
gp gastric pad 
gs gastric shield 
H height of shell 
hd head 
int intestine 
kd kidney 

LA length of aperture 
If lateral fold 
II lateral lamina 
m median 
mc mantle cavity 
me mantle edge 
mf marginal fold 
ml medial lamina 



mr mantle roof 

N number of whorls 

oes oesophagus 

og oviductal groove 

op operculum 

ovd oviduct 

ovr ovary 

p pedal ganglion 

pi pleural ganglion 

rad radula 

s statocyst 

sa sorting area 

sb spermatophore bursa 

sbg sub-oesophageal ganglion 

sd standard deviation 

sg sperm gutter 

sn snout 

snn snout nerve 

spg supra-oesophageal ganglion 

ss style sac 

stomach 

major typhlosole 

minor typhlosole 

tentacle 

tentacular nerve 

testis 



St 

tn 

tnn 

ts 



WA width of aperture 

SYSTEMATIC ACCOUNT 

Pachychilidae Troschel, 1857 

Brotia H.Adams, 1866 

Brotia H. Adams, 1866. Type species, by 
monotypy: Melania pagodula Gould, 1847. 

Antimelania Fischer & Crosse, 1892. Type 
species, by subsequent designation in 
Pilsbry & Bequaert (1927): Melania variabilis 
Benson, 1836. 

Wanga Chen, 1943. Type species, by original 
designation: Melania henriettae Griffith & 
Pidgeon, 1834. 

Taxonomy and Systematics 

Brotia was originally established for the 
round and multispiral operculum of the type 
species, which however is a characteristic 
exhibited by a number of pachychilid taxa and 
not peculiar for Brotia (Köhler & Glaubrecht, 
2001, 2002a, 2003). In the 19»^ and 20'*^ cen- 
tury, a vast number of species were affiliated 
with Brotia by a number of authors without 
sufficient knowledge of their gross morphol- 
ogy (e.g., Brot, 1874-1879; Martens, 1897, 



164 



KÖHLER & GLAUBRECHT 



1900; Martens & Thiele, 1908; Abbott, 1948). 
This procedure has caused considerable sys- 
tematic confusion as to the taxonomy of Brotia 
and other described supraspecific taxa from 
Asia (see overview in Davis, 1971; 68, 69; 
Köhler & Glaubrecht, 2002a). A first, more 
comprehensive treatment of Brotia species 
based also on features of the soft body was 
presented by Brandt (1974). This author also 
suggested a subdivision of Brotia into three 
subgenera: (1 ) Brotia s. str., (2) Senci<enbergia 
Yen, 1939. and (3) Paracrostoma Cossmann, 
1900. This suggestion was refuted, however, 
by Köhler & Glaubrecht (2001), who argued 
that radular and opercular features alone are 
insufficient to differentiate supraspecific taxa 
among the Pachychilidae. Instead, it was 
shown that characters of the reproductive tract 
and embryonic shells are more informative at 
this level. Using these morphological struc- 
tures, a preliminary subdivision of Brotia into 
four species groups was suggested by Köhler 
& Glaubrecht (2001 ). Two of these groupings 
have since been established as genera inde- 
pendent of Brotia: Tylomelania endemic to 
Sulawesi (Rintelen et al., 2004; Rintelen & 
Glaubrecht, 2005) and Jagora endemic to the 
Philippines (Köhler & Glaubrecht, 2003). The 
status of the two remaining groupings, so- 
called "Brotia pagodula group" and "Brotia 
testudinaria group", have remained unresolved 
thus far. Only recently it has been suggested 
on basis of molecular genetic data that both 
species groups indeed form distinct monophyl- 
etic lineages (Köhler et al., 2004). According 
to this mitochondrial phylogeny, it was sug- 
gested to transfer all species of the "Brotia 
testudinaria group" designated by Köhler & 
Glaubrecht (2001) to Adamietta Brandt, 1974 
(Köhler et al., 2004; 2221). In regard to this 
suggestion, in the current study Brotia is re- 
stricted to the members of the "Brotia pagodula 
group" as delineated by Köhler & Glaubrecht 
(2001). Accordingly, morphological features 
characteristic for Brotia are (1) an irregularly 
wrinkled apical whorl of the embryonic shell 
and (2) a palliai oviduct possessing a simple, 
deep, and ciliated spermatophore bursa. 

Morphology and Differential Diagnosis 

Shell: Relatively large, often up to 4 or 5 cm. 
Moderately thick, broadly to elongate coni- 
cal, turreted spire, apex eroded or truncated. 
Sculpture variable comprising axial ribs, 
sometimes with nodules, and spiral ridges 
or lines. Body whorl comparatively large; 



aperture ovate, well rounded or angled be- 
low, pointed above. No features peculiar to 
Brotia. 

Embryonic Shell: Relatively large among vi- 
viparous pachychilids; average height 1 to 6 
mm, up to four whorls. Apical whorl asym- 
metrical, irregularly wrinkled; initial shell 
sharply delimited from subsequent whorls 
with more or less smooth sculpture (for pe- 
culiar ontogeny of Brotia causing wrinkles 
see below). 

Operculum (Fig. 3C); Either round, up to eight 
whorls, central nucleus or slightly oval for 
last whorl increasing in diameter with up to 
six whorls. 

External morphology and mantle cavity (Figs. 
ЗА, В): Animals light to dark brown, dark grey 
or black, often with light patches; broad, fur- 
rowed snout. Cephalic tentacles moderately 
long, each with tiny eye on side of base. 
Females with subhaemocoelic brood pouch; 
"egg transfer" or "genital groove" on right side 
of head connects palliai oviduct with brood 
pouch pore near base of right tentacle; 
present also in males. Mantle margin smooth; 
mantle cavity occupying approximately two 
thirds of first whorl. Osphradium delicate, 
slightly undulating, forming narrow ridge 
embedded in shallow trench, lying adjacent 
to anterior part of ctenidium. Ctenidium large, 
broad tapering posteriorly; beginning shortly 
behind mantle edge, extending posteriorly 
almost to end of cavity, on average twice as 
long as osphradium. Hypobranchial gland 
inconspicuous, adjacent to rectum. 

Radula: Taenioglossate, relatively large, ro- 
bust. Up to 30 mm long corresponding to half 
of shell height. Posteriorly embedded in con- 
nective tissue, coiled behind buccal mass in 
radular sac. Rachidian squarish, with one 
pronounced, more or less pointed central 
cusp flanked by up to three accessory den- 
ticles that taper in size; glabella well devel- 
oped. Anterior margin of rachidian concave 
or straight, lower rim concave by posteriorly 
extending glabella. Lateral teeth with 
rounded glabella; major cusp flanked by up 
to three smaller denticles on each side. In- 
ner marginal teeth with two, outer marginal 
with up to three denticles; hooked; simple 
flange or ledge at outer margin; more pro- 
nounced in outer marginal teeth. 

Nervous System (Fig. 3E): Cerebral commis- 
sure long, cerebro-pleural connectives short. 
Sub-oesophageal ganglion fused with left 
pleural ganglion. Pedal ganglia deeply em- 
bedded in propodium, connected to pleural 



SYSTEMATIC REVISION OF BROTIA 



165 




FIG. 3. Soft anatomy of Brotia. A: External anatomy of B. pagodula (Thailand, male); B: External 
anatomy of e. episcopalis (Sumatra, female); C: Opercula (from left to right: ß. pagodula, B. 
episcopalis, B. costula; D: Palliai oviduct of ß. pagodula; schematic reconstruction showing vari- 
ous cross-sections from anterior to posterior; E: Schematic reconstruction of nervous system of 
ß. pagodula. Scale bars = 10 mm. 



166 



KÖHLER & GLAUBRECHT 



and cerebral ganglia by relatively long 
connectives. Pedal ganglia closely joined, 
statocysts located basally. 
Alimentary System: Oesophagus longitudinally 
folded, transverse septae not present. Stom- 
ach typical pachychilid (Strong & Glaubrecht. 
1999). including presence of sorting area, 
single digestive gland duct, narrow glandu- 
lar pad, cuticular gastric shield, crescent 
ridge and groove (e.g.. Fig. 4, B. citrina). 
Major and minor typhlosole may be fused. 
Epithelium of style sac heavily ciliated with 
golden gloss. Crystalline style cylindrical or 
club-like. 

Reproductive System 

Gonochoristic with balanced sex ratio. 
Subhaemocoelic brood pouch occupying al- 
most entire visceral cavity, compartmentalized 
with lamellae of thin adventitious tissue em- 
bedding embryos (Figs. 5A-E for histological 
sections). Juveniles within pouch of same on- 
togenetic stage. Gonads comparatively large, 
comprising last two to three visceral whorls, 
adjacent to and dorsal of digestive gland. 
Ovary orange to light brown consisting of broad 
lobes (Fig. 6E). Testis light yellow consisting 
of highly branched thin tubes. Palliai gonod- 
uct open in both sexes. Palliai oviduct com- 
prising deep oviductal groove bounded by 
parallel laminae (Fig. 6A); ciliated sperm gut- 
ter forming along free edge of medial lamina, 
opening to papillated spermatophore bursa 
approximately at two thirds of oviductal length 
(Fig. 6B); large capsule gland comprises al- 




FIG. 4. Stomach anatomy of S. citrine (Thailand, 
Mae Sot; ZMB 200.212). Scale bar = 5 mm. 



most entire length of palliai oviduct; capacious, 
ciliated spermatophore bursa formed by me- 
dian lamina (Figs. 6C, D); Fig. 3D, schematic 
reconstruction of palliai oviduct). 

Habitat 

Most species inhabit small, clear mountain 
streams; some occur also in lakes. Often con- 
fined to specific habitats, such as upper course 
of rivers, and restricted to single rivers or river 
systems. Rarely more than two species co- 
occur with notable exception of endemic spe- 
cies flock in Kaek River, central Thailand 
(Glaubrecht & Köhler, 2004). 

Distribution 

Southeast Asia, from foot hills of Himalayas 
in northeast India and Bangladesh to 
Myanmar, Thailand, Malaysian Peninsula, 
Sumatra, Java, and Borneo. Reports from 
Java and Borneo are scarce, date back to 1 9"" 
century. Brotia as here defined does not oc- 
cur in most parts of Indochina, in Sulawesi, in 
the Philippines, or on the Smaller Sunda Is- 
lands. 

Fossil Record 

Fossil record in continental Southeast Asia 
extends back to middle Miocene. Gurung et 
al. (1997) report on Brotia species (e.g., B. 
palaeocostula and other undetermined spe- 
cies) from middle Miocene to Pliocene depos- 
its of Nepal (Churia group). Annandale (1919) 
mentions fossil Brotia from Miocene and Pleis- 
tocene sediments of Lower Burma, for ex- 
ample, "8. variabilis" from Miocene of Pegu, 
"в. baccata" from Lake Inié (Shan States) of 
presumably post-Pleistocene age. From lat- 
ter deposits, Bequaert (1943) noted three 
forms of Brotia and Sulcospira, respectively 
that persist to the Recent. His reference to 
Sulcospira is here attributed to Brotia, Sulco- 
spira being endemic to Java (Köhler & 
Glaubrecht, 2005). 

Affinities of Miocene and Pliocene fossils of 
Java reported by Martin (1914) and Oostingh 
(1 935) remain doubtful, not only since the dat- 
ing of these sediments was questioned 
(Oostingh, 1935; 2). Judging from figures in 
both publications, we consider the species in 
question, for example, "Brotia oppenoorthi', 
not congeneric with Recent Brotia. Instead, at 
least some species represent thiarids. 



SYSTEMATIC REVISION OF BROTIA 

bp 




FIG. 5. Brood pouch morphology. A-E: Histological sections of the head-foot of Brotia episcopalis 
(ZMH, Trang); A: Longitudinal section of head, showing the visceral cavity with radula, buccal mass, 
oesophagus, and anterior part of brood pouch situated just behind buccal mass; B: Cross-section at 
about mid head; brood pouch occupies most of visceral cavity, brood pouch pore visible; C: Cross- 
section some mm posterior to B; brood pouch filled with numerous egg capsules each embedded in 
thin membrane; D: Cross-section at posterior end of brood pouch; E: Detail of A; egg capsules in 
higher magnification; F: Macro-anatomical photograph of S. pseudosulcospira (ZMB 200.196); head 
of female with egg capsule sitting in genital groove just in front of brood pouch pore. 



168 

A 



KÖHLER & GLAUBRECHT 



mr 



ml 



В 



■ f ' 'Г- У' ;■-;.■■ '^-i 






#%,.:%■ 





юд 






dg' 






FIG. 6. Female reproductive anatomy. A-D: Histological sections of palliai oviduct of B. pagodula 
(ZMH: Myanmar); A: Cross-section at anterior end of palliai gonoduct; lateral lamina fused with mantle, 
simple medial lamina free: B: Cross-section at about one third of oviduct length; heavily ciliated 
sperm gutter formed by medial lamina; C; Cross-section at about half of oviduct length; spermato- 
phore bursa formed by medial lamina, capsule gland comprising base of oviductal groove; D: Cross- 
section at about tvuo thirds of oviduct length; ciliated spermatophore bursa; E; Cross-section of vis- 
ceral w/horl of S. episcopalis (ZMH, Trang); ovary filled with egg capsules, adjacent and posterior to 
digestive gland. Scale bars = 1 mm. 



Also fossil shells from Europe were attrib- 
uted to Brotia (Papp, 1953). The fossil taxon 
Tinnyea was treated as a subgenus of Brotia 
(Papp, 1953) and various species have been 
affiliated with this Southeast Asian taxon, such 



as '^rotla eschen' (Brongniart, 1822) and "S. 
vasarhelyif (Hantken, 1887) from Pannonian 
deposits near Budapest, Hungary - Upper 
Miocene (Lörenthey, 1902), Burgenland, Aus- 
tria - Upper Miocene (Fischer, 1994), and 



SYSTEMATIC REVISION OF BROTIA 



169 



Mainz Basin, Germany - Upper Oligocène to 
Lower Miocene (Kadolsky, 1995). Placement 
of these and other fossil species in Brotia does 
refer only to (rather superficial) shell similarity 
and ignores the uncertain freshwater origin of 
the deposits. Assignment of European fossils 
to Brotia is rejected here; whether some fos- 
sils might be included in the Pachychilidae 
awaits critical evaluation of the fossil material, 
which we have not examined yet. 



ACCOUNT OF RECENT SPECIES IN 
ALPHABETICAL ORDER 

Brotia ármate (Brandt, 1968) 
(Figs. 7, 8, 12A, B) 

Brotia (Paracrostoma) pseudosulcospira 
armata Brandt, 1968: 275, pi. 10, fig. 62 
("Maenam Kaek in Phitsanulok Prov. at 
Gaeng Song rapids, 45 km E Pitsanulok" = 
Thailand, Prov. Phitsanulok, Kaek River at 
Kaeng Song rapids, approximately 60 km E 
of Phitsanulok), holotype SMF 197380, 35 
paratypes ZMH; types seen. 

Paracrostoma pseudosulcospira armata - 
Brandt, 1974: 186, pi. 13, fig. 43; Köhler & 
Glaubrecht, 2002a: 144. 



Brotia armata - Glaubrecht & Köhler, 2004: 
283-287. 

Paracrostoma morrisoni Brandt, 1974: 188, 
189, pi. 14, fig. 47 ("Maenam Kaek at Sopa 
Falls, 71 km E of Pitsanulok" = Thailand, 
Prov. Phitsanulok, Kaek River at Sopha Falls, 
71 km E of Phitsanulok), holotype SMF 
215966, six paratypes SMF 215967, 12 
paratypes SMF 271191, 38 paratypes SMF 
193587, 11 paratypes BMNH 1976119, 14 
paratypes RMNH 55135/14; types seen; 
Köhler & Glaubrecht, 2002a: 141, 142. 

Paracrostoma paludiformis dubiosa Brandt, 
1974: 188, pi. 14, fig. 46 ("Kaek River, 80 km 
E of Pitsanulok" = Thailand, Prov. Phitsanulok, 
Kaek River, 80 km E of Phitsanulok), holo- 
type SMF 215964, six paratypes SMF 
21 5964, five paratypes RMNH 55284/5; types 
seen; Köhler & Glaubrecht, 2002a: 142. 

Taxonomy and Systematics 

Originally described as subspecies of ß. 
pseudosulcospira, it was transferred to Brotia 
and is treated as distinct species in the Kaek 
River species flock by Glaubrecht & Köhler 
(2004). According to these authors, P. morrisoni 
and P. paludiformis dubiosa are considered as 
synonyms. 




FIG. 7. Shell morphology of B. armata. A-D: Paratypes of P. pseudosulcospira armata SMF 193587; 
E-F: Paratypes of P. morrisoni SMF 215967. Scale bar = 10 mm. 



170 



KÖHLER & GLAUBRECHT 



Material Examined 



Description 



Thailand: Prov. Phitsanulok, Kaek River: 
Sakunothayan Falls, 33 km E of Phitsanulok 
(ZMB 200.265: ZMH); Kaeng Song rapids. 45 
km E of Phitsanulok (SMF 193587: ZMB 
200.193): resort. 53 km E of Phitsanulok (ZMB 
200.254): Poi Falls, 60 km E of Phitsanulok, 
16°50.75'N, 100°45.06'E (ZMB 200.268): 
Thung Salaeng Luang National Park, 90 km 
E of Phitsanulok, 16°52'N. 100°38"E (USNM 
794081: ZMB 200.252, 200.265). 

Differential Diganosis 

Shell relatively small, conical to oval, not 
more than three rather flattened whorls: one 
to three spiral cords supporting a spiral row of 
sometimes spiny nodules. 




Shell (Fig. 7): Relatively small, oval to coni- 
cal, up to three flattened to slightly convex 
whorls, tip eroded. One to three spiral cords, 
especially upper ones supporting spiral rows 
of spiny nodules: on body whorl often addi- 
tional cord visible. Some shells almost 
smooth. Aperture broadly ovate, large com- 
pared to shell, basal margin produced. Size: 
H = 26-38 mm, В = 18-24 mm. 

Embryonic Shell (Fig. 8): Smooth except for 
axial growth lines, sharp transition between 
apical area and penultimate whorl after about 
half of first whorl. Size of juveniles kept in 
brood pouch: 2.0-2.5 mm, 2.5 whorls. 

Operculum: Oval, up to four whorls that in- 
crease in diameter, sub-central nucleus. 

Radula (Figs. 12A, B): Length of ribbon: m = 
18.4 mm (sd = 4.4 mm; n = 15), up to 180 
rows of teeth. Central tooth with elongated 
main cusp and two or three much smaller 
accessory denticles on each side that taper 
in size; glabella narrow with straight lateral 
margins, rounded posterior rim that does not 
reach the basal rim of central tooth. Laterals 
with broad main cusp flanked by one to two 
accessory denticles on each side. Inner and 
outer marginals with large, broad outer cusp 
and spiny inner denticle. 

Stomach: Typical, as in B. binodosa (Fig. 13). 



2.4 














2.2 

2.0 












о 






1.8 




1.6 






















1.4 
























1 2 








о 


1.0 






N = 


1 
a ai 


73 
wata 




1 
В. bine 


'8 
:)dosa 





FIG. 8. Embryonic shell morphology of 
ß. armata. SEM images of embryonic 
shell removed from brood pouch 
(paratype BMNH 1976111); apical and 
front view. Scale bar = 1 mm. 



FIG. 9. Comparison of B. armata and B. binodosa 
by means of shell parameter H/B. Box plot dia- 
gram showing median, the 25%- and 75%-per- 
centile and largest non-extremes (less than 1.5 
times of box height). 



SYSTEMATIC REVISION OF BROTIA 



171 



TABLE 1 . Result of disriminant analysis of shel 
parameters of B. armata and 6. binodosa. 



Predicted group membership 



ß. armata 



B. binodosa 



B. armata 
B. binodosa 



134(97.8%) 
11 (14.1%) 



3 (2.2%) 
67 (85.9%) 



Distribution 

Thailand: Prov. Phitsanulok: Endemic to Kaek 
River; only in middle course between Sakuno- 
thayan Falls (33 km E Phitsanulok) and Thung 
Salaeng Luang NP (90 km E Phitsanulok). 

Remarks 

Brotia binodosa with similar sculpture is more 
turreted and slender possessing more whorls. 
Both species deviate mainly in proportion of 
shell height to width (H/B, Fig. 9), although not 
statistically significant (Table 1 ). Brotia pseudo- 
sulcospira lacks spines, is larger with a darker, 
thicker, smoother shell. 



in large rivers, Thailand; restricted to Sopha 
Falls, at the Kaek River near Phitsanulok by 
Brandt 1974: 175), holotype BMNH 
1903.2.28.2, paratype BMNH 1903.2.28.3 
(Figs. 8A, B); types seen. 

Brotia binodosa - Solem, 1966: 15, figs. 1a, 
b; Glaubrecht & Köhler, 2004: 287-289. 

Brotia (Brotia) binodosa binodosa - Brandt, 
1974: 174, 175, pi. 12, fig. 26. 

Brotia spinata - Köhler & Glaubrecht, 2002a: 
148 [partim]. 

Brotia (Brotia) binodosa spiralis Brandt, 1974: 
176, pi. 12, fig. 27 ("Thailand: Kaek River, 38.5 
km E Pitsanulok" = Thailand, Prov. Phitsanu- 
lok, Kaek River 38.5 km E of Phitsanulok), 
holotype SMF 220340; type seen. 

Brotia spinata spiralis - Köhler & Glaubrecht, 
2002a: 130. 

Taxonomy and Systematics 

Revised by Glaubrecht & Köhler (2004), who 
suggested 8. binodosa spiralis to represent a 
junior synonym. Member of the Kaek River 
species flock in Central Thailand. 

Material Examined 



Brotia binodosa (Blanford, 1903) 
(Figs. 10, 11, 12C, 13) 

Melania binodosa Blanford, 1903: 282, 283, 
pi. 8, fig. 2 ("Siam, in fluminibus majoribus" = 



Thailand: Prov. Phitsanulok: Chattrakan Fall, 
Kwae Noi River in the Chattrakan NP, N of 
Nakhon Thai (ZMB 200.202); Kaek River (SMF 
193577; RMNH 55288): Kaeng Song Falls 
(SMF 1 93874); resort, 53 km E of Phitsanulok 




FIG. 1 0. Shell morphology of B. binodosa. A: Holotype of M. binodosa BMNH 1 903.2.28.2; B: Paratype 
BMNH 1903.2.28.3; C: Thailand, Kaek River, Sopha Falls (ZSM 19983219). Scale bar = 10 mm. 



172 



KÖHLER & GLAUBRECHT 




FIG. 11. Embryonic shell morphology of В. binodosa. SEM images of 
embryonic shell removed from brood pouch (Thailand, Kaek River; ZSM 
19983219): apical and front view. Scale bar = 0.3 mm. 



9№И 




^И^^Ж wB Mr ^ 


m 


ШН^В ^^1^1шН^^^^^В1^^^Кик.л^ 


^•^7 





FIG. 12. Radular morphology of 8. armata, B. binodosa, and B. citrina. SEM images of radula seg- 
ments viewed from above. A; B. armata (Thailand, Kaek River; ZMB 200.252); B: S. armata (Thai- 
land, Kaek River; ZMB 200.254); C; B. binodosa (Thailand, Kaek River; ZMB 200.192); D; B. citrina 
(Thailand, Pa Charoen; ZMB 200.207). Scale bars = 100 pm. 



SYSTEMATIC REVISION OF BROTIA 

ss 



173 




FIG. 1 3. Stomach anatomy of B. binodosa (ZMB 200.269; Thailand, Kaek River) 



(ZMB 200.267); Poi Falls (ZMB 200.269; SMF 
205137); Sopha Falls (ZSM 19983214, 6, 8; 
RMNH 55288/6; SMF 193575, 220339; AMS 
146761); Thung Salaeng Luang NP (ZMB 
200.192; ZSM 19983217; SMF 193578; 
BMNH; AMS 146760); Tap Tami Falls (ZSM 
19983215; SMF 193576; ZHM). 

Differential Diganosis 

Shell elongately turreted, sculptured by two 
spiral rows of pointed nodules or tiny spines, 
each supported by a spiral cord. 

Description 

Shell (Fig. 1 0): Medium sized, spire elongately 
turreted with three to four whorls, eroded tip. 
Whorls convex with subsutural depression, 
separated by narrow, inconspicuous suture. 
Sculptured by more or less developed spiral 
ridges, most prominent at the base, and two 
spiral rows of pointed nodules or tiny spines, 
each supported by a spiral cord. Shell thin 
but solid; colour brown to red-brown, glossy 



surface. Basal whorl relatively large. Aperture 
oval, angled, produced below, inside white. 
Shell size: H = 25-35 mm. В = 14-18 mm. 

Embryonic Shell (Fig. 11); Conical, compris- 
ing up to 31/2 whorls. Sculpture smooth, faint 
growth lines. Spiral keel at about the centre 
of the whorl from third whorl on. In some 
specimens, this keel supports two spiral rows 
of smooth knobs. 

Operculum: Oval, with up to five whorls gradu- 
ally increasing in diameter; nearly central 
nucleus. 

Radula fFig. 12C): Length of ribbon: m = 20 
mm (sd = 1 ; n = 3), up to 1 90 rows of teeth. 
Very similar to B. armata, rachis tends to be 
more squarish in size. 

Stomach (Fig. 13): Typical, as in B. citrina 
(Fig. 4). 

Reproductive System 

Three dried shells contained between 131 
and 145 shelled juveniles varying in height 
between 1 and 3 mm, respectively (ZSM 
19983217). 



174 



KÖHLER & GLAUBRECHT 



Distribution 

Thailand: Prov. Phitsanulok: Only known 
from Каек River and adjacent Kwae Noi River. 

Remarks 

Very similar to B. spinata (Godwin-Austen, 
1872). B. binodosa is more slender, columella 
more curved (Blanford, 1903). Shell of B. 
armata is more conical possessing fewer 
whorls. Discriminant analysis of shell param- 
eters: Figure 9, Table 1. 

Brotia citrina (Brot, 1868) 
(Figs. 4, 12D, 14, 15) 

Melania citrina Brot, 1868: 11, 12, pi. 3, fig. 13 
("Siam" = Thailand), lectotype and three 
paralectotypes MHNG, coll. Brot (designated 
by Köhler and Glaubrecht, 2002a) (Figs. 
14A-C); types seen; Brot, 1875: 106, 107. 
pi. 13, fig. 5. 




Melania citnnoides Brot, 1886: 101, 102, pi. 5, 
fig. 4 ("Siam" = Thailand), lectotype and four 
paralectotypes MHNG, coll. Brot (designated 
by Köhler & Glaubrecht, 2002a) (Figs. 14D- 
H). 

Brotia citrina - Köhler & Glaubrecht, 2002a: 
131, fig. II (non Brandt, 1974). 

Taxonomy and Systematics 

Brandt (1974) based his diagnosis on mate- 
rial that we re-determined as 8. dautzen- 
bergiana. Material of B. citrina was apparently 
not available to him, except for the types. Con- 
sequently, Brandt's (1974) description of B. 
citrina and his conclusions in respect to its sys- 
tematics are refuted and attributed to B. 
dautzenbergiana, which is considered distinct 
(see below). Types of M. citrina and M. 
citnnoides do only differ in average shell height 
but not in respect to other morphological or 
morphometrical characteristics. This feature is 
not considered sufficient to indicate separate 



/ 



à 

f 



I 



\b. 



V 











'IWffjwïïïïj 



FIG. 14. Shell morphology of B. citrina. A: Lectotype of M. citrina MHNG, front and rear; B-C: 
Paralectotypes MHNG; D: Lectotype of M. citrinoides MHNG, front and rear; E-H: Paralectotypes 
MHNG; I: Thailand (ZMB 26.874); J: Thailand, Pa Charoen (ZMB 200.207). Scale bar = 10 mm. 



SYSTEMATIC REVISION OF BROTIA 



175 



status. For this reason, we agree with Brandt 
(1974) treating both taxa as synonyms. 

Material Examined 

Thailand; Prov. Kamphaeng Phet: Pa 
Charoen waterfall, S of Mae Sot, 16°30.5ГМ, 
98°44.89'E (ZMB 200.207); Nang Khruan wa- 
terfall near Mae Sot, 16°24.59'N, 98°39.27'E 
(ZMB 200.212). 

Differential Diganosis 

Highly turreted shell, thin but solid, smooth 
except for growth lines and fine, closely spaced 
spiral lirae; aperture wide, produced below; 
colour yellowish to olive-brown. Rachidian 
cusp relatively broad, upper rim well rounded. 




FIG. 15, Embryonic shell morphology 
of S. citrina. SEM images of embryonic 
shell removed from brood pouch (Thai- 
land, Pa Charoen; ZMB 200.207); api- 
cal and front view. Scale bar = 1 mm. 



Description 

Shell (Fig. 14): Elongately turreted, thin but 
solid, six to ten convex and regularly rounded 
whorls; narrow suture. Sculpture of regularly 
spaced spiral ridges becoming more promi- 
nent at the base, fine axial growth lines; 
some shells completely smooth. Colour yel- 
lowish to light olive brown, glossy surface. 
Aperture wide, oval, angled, produced be- 
low, pointed above, sharp to thin margin. 
Shell size; H = 21-63 mm, В = 9-22 mm. 

Embryonic Shell (Fig. 15): Smooth with faint 
growth and spiral lines; conspicuous 
subsutural depression; colour light greenish 
brown with broad chestnut brown spiral 
band. 

Operculum: Round, up to eight regular whorls, 
almost central nucleus. 

Radula (Fig. 12D): Central tooth relatively 
broad, basal margin well rounded. Central 
cusp flanked by three smaller denticles on 
each side. Inner marginals with two cusps, 
the outer one being broader. Outer marginals 
with mostly two, sometimes three cusps, 
outer one being broader. 

External Anatomy: Animal dark grey with light 
grey patches. Columellar muscle well devel- 
oped, relatively short and broad. 

Stomach (Fig. 4): Inner septate crescent pad 
of the sorting area weakly developed, outer 
one well developed, laminated part of sort- 
ing area with fine, densely arranged lami- 
nae; typhlosoles fused at 4/^ of style sac 
length. 

Reproductive System 

Females contained between 18 and 56 ju- 
veniles that varied in height between 2.2 and 
5.5 mm, up to 3.5 whorls (n = 3; ZMB 200.207). 
Large embryos lay anteriorly in the pouch. 

Distribution (Fig. 36) 

Thailand: Prov. Kamphaeng Phet: Two lo- 
calities in vicinity of Mae Sot as only known 
records. Not recorded by Brandt (1974) oth- 
erwise giving an excellent overview of the gas- 
tropod fauna of Thailand. 

Habitat 

Relatively cold, fast flowing, clear streams, 
well oxygenated, on limestone substratum. 
Buried in sand or mud, under rotten leaves or 
sunken wood presumably feeding on detritus. 



176 



KÖHLER & GLAUBRECHT 



TABLE 2. Result of disriminant analysis of shell 
parameters of B. citrina and B. dautzenbergiana. 



Predicted group membership 

B. dautzen- 
bergiana 



B. citrina 



В. citrina 

В. dautzen- 
bergiana 



19(95.0%) 
2 (4.7%) 



1 (5.0%) 
41 (95.3%) 



Remarks 

From S. dautzenbergiana to be distinguished 
by its more conical shell, uneroded spire, in 
average fewer whorls, and lack of dark brown 
spiral band; or by statistical analysis of shell 
parameters (Table 2, Fig. 16). 

Brotia costula (Rafinesque, 1833) 
(Figs. 17-19) 

Melania costula Rafinesque, 1833: 166 
("Ganges "); types not traced. 

Antimelania costula - Morrison, 1954: 15 
[partim]. 

Brotia costula - Benthem Jutting, 1956: 374- 
378, fig. 76 [partim]; 1959: 92-95 [partim]; 
Brandt, 1974: 175, pi. 13, figs. 37-39 
[partim]; Köhler & Glaubrecht, 2001: 295- 
299 [partim]; Köhler & Glaubrecht, 2002a: 
132 [partim]. 

Brotia costula episcopalis - Subba Rao & Dey, 
1986: 26 [partim]. 

Brotia {Antimelania) costula - Subba Rao, 
1989: 108, 109 [partim]. 

Melania carolinae Griffith & Pidgeon, 1834: 
598, pl. 13, fig. 3 ("India"), ex Gray ms, lecto- 
typeandparalectotypeBMNH 1874.10.12.11 
(designated by Köhler & Glaubrecht, 2002a) 
(Figs. 17B, С); types seen. 

Melania plicata \. Lea, 1835: 20, pl. 23, fig. 95 
{non M. plicata Menke, 1830) ("Bengal, 
Calcutta"). 

Melania variabilis Benson, 1836: 746, 747 {non 
M. variabilis Defrance, 1823) ("The river Gumti 
at Jonpur. and tolly's nullah near Calcutta" = 
Gomati River, Jaunpur, Uttar Pradesh, 2544'N, 
82ЧГЕ), lectotype BMNH 1872.12.2.2 (des- 
ignated by Köhler & Glaubrecht, 2002a) (Fig. 
17A); types seen; Souleyet, 1852: 545; Reeve, 
1860: species 204; Brot, 1870: 281 [partim]; 
Brot, 1 875: 85-87, pl. 1 0, figs. 1 , 1 a-d [partim]. 

Melania {Melanoides) variabilis- Nevill, 1885: 
251, 252 [partim]. 



14 
12 
10 



о 

Я 



N = 



20 

В. citrina 



31 



В. dautzenbergiana 



FIG. 16. Comparison of S. citrina and ß. dautzen- 
bergiana by means of number of whorls (N). Box 
plot diagram showing median, the 25%- and 
75%-percentile and largest non-extremes (less 
than 1.5 times of box height). 



Melanoides {Tiara) variabilis -Presión, 1915: 
23, 24, 

Acrostoma variabilis - Annanäa\e, 1920: 110; 
Annandale et al., 1921 : 560-562, pl. 6, figs. 
3-6; Prashad, 1921: 485-488 [partim]. 

Brotia variabilis- Rensch, 1934: 239 [partim]; 
Bequaert, 1943: 433, 434, pl. 33, figs. 11- 
16; Soiem, 1966: 15 [partim]. 

Brotia {Antimelania) variabilis - Adam & 
Leloup, 1938: 85, 86 [partim]. 

Melania \/ancosa Troschel, 1837: 174 ("Benga- 
lien, Ganges" = Bengal, Ganges), lectotype 
ZMB 2.226a (here designated for the stabili- 
sation of the name) (Fig. 17F) and 13 para- 
lectotypes ZMB 2.226b; types seen; Philippi, 
1844: 15, 16, pl. 3, fig. 2. 

Melanoides varicosa - H. Adams & A. Adams, 
1854: 297. 

Melania indica Souleyet, 1842: pl. 31 , figs. 12- 
15 ("India, Ganges"), five syntypes MNHN; 
types seen; Souleyet, 1852: 545. 

Melanoides indica - H. Adams & A. Adams, 
1853: pl. 31, figs. 5, 5a, b. 

Melania menkiana [sic !] I. Lea, 1842; 242 (re- 
placement name for M. plicata I. Lea, 1835, 
non M. plicata Menke, 1830; misspelled for 
intended "M. menkeana"); Brot, 1860: 280; 
Hanley& Theobald, 1874: 110. 

Melania menkeana Brot, 1875: 91, 92, pl. 11, 
fig. 1, la, b (replacement name for /W. шеп/</а- 
na Lea, 1842). 

Melania {Melanoides) variabilis menkeana - 
Nevill, 1885: 260. 



SYSTEMATIC REVISION OF BROTIA 

Й С 



177 




FIG 17 Shell morphology of B. costula. A: Lectotype of M. variabilis BMNH 1872.12.2.2; B: Lecto- 
type of /W carolinae BMNH 1874.10.12.11/A; C: Paralectotype BMNH 1874.10.12.11/B; D: Lectotype 
of /W spinosa BMNH 1907.10.28.79; E; Paralectotype BMNH 1907.10.28.80; F: Lectotype of M. 
varicosa (ZMB 2.226a); G: Syntype of M. hainesiana USNM 119741; H; Bangladesh, Chittagong 
(RMNH 76332). Scale bar = 10 mm. 



178 



KÖHLER & GLAUBRECHT 



Brotia menkeana -Yen, 1939: 59. pl. 5, fig. 13. 

Melania spinosa Hanley. 1854: pl. 1, fig. 7 
("River Jumna, Sylhet, British India" = River 
Jamuna, Sylhet, Prov. Chittagong, Bangla- 
desh, 24°53'N, 9Г52'Е) {non M. spinosa 
Gray, 1824), lectotype BMNH 1907.10.28.79 
and paralectotype BMNH 1907.10.28.80 
(Figs. 17D. E) (designated by Köhler & 
Glaubrecht, 2002a); types seen; Brot, 1875: 
92, 93, pl. 12, fig. 2. 




FIG. 18. Embryonic Shell morphology of ß. 
costula. SEM images of embryonic shell 
removed from dried material (ZMB 35.81 1 ); 
apical and front view. Scale bar = 1 mm. 



Melania variabilis var. spinosa - Hanley & 

Theobald, 1873: pl. 75, fig. 6. 
Melania halneslana I. Lea, 1856: 144 ("India"), 

ninesyntypesUSNM 119741 (Fig. 17G); types 

seen; I. Lea, 1864: 78, pl. 22, fig. 18; Brot, 

1875: 109, 110, pl. 14, fig. 4. 
Melania (Melanoldes) variabilis var. halneslana 

-Nevill, 1885: 255. 
Melania corrúgala Reeve, 1859: pl. 3, fig. 10 

("India, Java") {non M. corrúgala Lamarck, 

1822). 
Melania spinata - Brot, 1875: 89, 90, pl. 10, 

fig. 2a {non M. spinata Godwin-Austen, 1872). 
Melania episcopalls - Hanley & Theobald, 

1873: 31, 32, pl. 72, fig. 7, pl. 75, figs. 5, 7 

{non M. episcopalls H. Lea & I. Lea, 1850). 
Melania {Melanoldes) variabilis episcopalls - 

Nevill, 1885: 256 [partim]. 
Melania {Melanoldes) variabilis subvar. áspera 

Hanley & Theobald, 1874: pl. 109, fig. 6; 

Nevill, 1885: 252. 
Melania {Melanoldes) variabilis subvar. cincta 

Hanley & Theobald, 1874: pl. 109, fig. 5; 

Nevill, 1885: 252. 
Melania {Melanoldes) variabilis subvar. 

microstoma Nevill, 1885: 261 ("Sylhet"). 
Melania {Melanoldes) variabilis var. 

pseudospinosa Nevill, 1885: 258. 
Melania {Melanoldes) variabilis var. seml- 

laevlgata Nevill, 1885: 254 ("Cachar and 

Sylhet"). 
Melania {Melanoldes) variabilis subvar. 

subtuberculata Nevill, 1885: 252 ("Calcutta"). 
Melania {Melanoldes) variabilis subvar. 

subsplnosa Nevill, 1885: 252 ("Calcutta"). 

Taxonomy and Systematics 

This species was delineated in various ways 
by previous authors, the plethora of synonyms 
witnessing serious difficulties in species rec- 
ognition especially by 19'^ century authors. 
Presupposing that B. costula is highly variable, 
20'^ century authors frequently subsumed simi- 
lar taxa from across Southeast Asia under this 
name (e.g., Rensch, 1934; Benthem Jutting, 
1956; Brandt, 1974; Köhler & Glaubrecht, 
2001). Brandt (1974) hypothesised that B. 
costula forms a "rassenkreis" of three geo- 
graphical subspecies: (1) the nominate form 
ranging from NE India to Indochina, (2) S. с 
vancosa (Troschel, 1837), with suggested oc- 
currence on Sumatra, Java and Borneo, and 
(3) B. с peninsularis Brandt, 1974, restricted 
to the Malay Peninsula. This suggestion was 
refuted using comparative morphological and 



SYSTEMATIC REVISION OF BROTIA 



179 




FIG. 19. Radula morphology of B. costula. Radula segments viewed from above. A; India, Sikkim 
(ZMB 2.227); B: India, Manipur (BMNH). Scale bars = 100 pm. 



molecular genetic data (Köhler & Glaubrecht, 
2001). Köhler & Glaubrecht (2001) demon- 
strate that taxa from Borneo and Java, which 
were assumed to constitute the varicosa sub- 
species, among other features possess a dif- 
ferent embryonic shell morphology and, thus, 
are clearly distinct from 8. costula. In addi- 
tion, the molecular phylogeny shows that taxa 
from Sumatra, such as B. torquata, Malay 
Peninsula, such as B. episcopalis and B. 
peninsularis, and Myanmar, such as B. 
hercules, are also distinct (Figs. 78, 79). Brotia 
costula is encompassed here in a much more 
restricted way by means both of its distribu- 
tion and its morphology. Accordingly, under B. 
costula we subsume only those taxa described 
from northern India, especially from the 
Ganges plain and Bengal, that exhibit corre- 
sponding shells, opercula and radular patterns 
(if available). Forms possessing spiny axial 
ribs, such as M. menkiana, are tentatively con- 
sidered conspecific unless data on soft body 
morphology or molecular genetics may show 
otherwise. Here we follow Benson (1936: 747) 
who stated for M. variablis that "... several of 
these varieties [i.e., with or without spiny nod- 
ules] would, if viewed apart, be easily mistaken 
for distinct species, but they melt into each 
other so gradually, occasionally showing char- 
acters of more than one variety combined in 
the same shell, that no doubt remains of their 
blending in one species". 

Material Examined 

India (ZMB 200.044, 200.061 , 200.064; CAS 
6199); Ganges (ZMB 200.058, 200.062); 
Sikkim (ZMB 2.227, 200.078); Assam (ZMB 



200.042, 200.052; BMNH 1935.10.9.5-17, 
1888.12.4.1492-3), Guwahati (ZMZ 522377); 
Brahmaputra (ZMB 200.302-3); Durang 
(BMNH); Himalayas (BMNH 1841.7.23.9); 
Meghalaya; Jaintia-Khâsi hills (BMNH); 
Manipur (BMNH); Keladyne River (BMNH 
1899.12.4.1761-2); Kolkata (ZMB 20.738; 
200.063; BMNH; CAS 25326); Bengal (ZMB 
45.849; BMNH 1888.12.4.1480-2; ZMZ 
522371); Bengal, RiverTooIsi Ganga (BMNH); 
Bengal, River Atrai (BMNH); Settlepore (ZMZ 
522372); Madhya Pradesh; Jonapura (ZMZ 
522370); Bhutan: Duars, West Bhutan 
(BMNH); Bangladesh: Chittagong (BMNH; 
RMNH 71332; ZMB 35.811); Rajshahi: 
Basudebpur(BMNH); Malaudi (BMNH); River 
Jamuna (BMNH 1907.12.30.207); Sylhet 
(ZMB 200.071); Nepal: Prov. Narayani, 
Chitwan Distr., Bis Hajaar Lakes, 27°36.44'N, 
84°26.34'E (ZMB 112.783), Prov. Koshi, 
Sunsari Distr., Haripur, tributary of the Sapta 
Koshi, 26°33.28'N, 86°59.6'E (ZMB 112.660). 

Differential Diganosis 

Shell highly turreted, large, up to 12 whorls, 
sculptured by regularly spaced axial ribs 
throughout, only exceptionally these ribs may 
lack completely; in some specimens, ribs sup- 
port a spiral row of spiny nodules. 

Description 

Shell (Fig. 17): Medium sized to large, solid 
but not very thick, 6 to 12 whorls, pyramidal 
spire, frequently eroded tip; colour uniform 
light to olive-brown; whorls well rounded in 
diameter, separated by well-defined, thin 



180 



KÖHLER & GLAUBRECHT 



suture: sculpture of basal spiral ridges and 
regularly spaced axial ribs that occasionally 
support small, spiny nodules arranged in a 
spiral band at centre of whorl; some speci- 
mens smooth; aperture wide, well rounded 
at base, comprising about V5 of shell height. 
Size: H = 20-87 mm, В = 8-36 mm. 

Embryonic Shell (Fig. 18): Smooth except for 
fine growth lines. Maximum height 4 mm, 3.5 
whorls. Average proportions: H = 2.3 mm, В = 
1.1 mm, HA = 0.27 mm. BA = 0.48 mm, DA = 
0.63 mm (for n = 6). 

Operculum: Slightly oval, four to six whorls, 
central nucleus: almost fits aperture. 

External Morphology: Uniformly coloured, dark 
grey to black; grey foot sole with scattered 
light spots. 

Radula (Fig. 19): Ribbon length of up to 30 
mm, corresponding to about half of the shell 
height, about 180 rows of teeth. Rachidian 
with single main cusp, three smaller denticles 
on each side tapering in size; upper margin 
concave by inflated, rounded corners; lower 
rim rounded; glabella narrow, well rounded 
at its base, lateral margins slightly concave. 
Laterals with main cusp flanked by three 
smaller denticles. Inner and outer marginals 
with two to three denticles, somewhat 
pointed, of about same size and shape. 



Distribution (Fig. 20) 

Northeast India (Bihar, Uttar Pradesh, Madhya 
Pradesh, Manipur, Meghalaya, Mizoram, 
Sikkim, Assam, West-Bengal), Bangladesh, 
Bhutan, and Nepal. Namely, Ganges-Meghna- 
Brahmaputra River system with affluent rivers. 

Habitats 

Clear creeks with sandy bottoms, large riv- 
ers, and even ponds (Subba Rao, 1989). 

Remarks 

Reports from Sri Lanka (Annandale, 1920), 
Hainan and China (Yen, 1939), Sumatra and 
Java (Rensch, 1934; Benthem Jutting, 1956), 
Thailand, the Mekong, Borneo (Brandt, 1974), 
Melanesia (Abbott, 1948), and the Philippines 
(Bändel & Riedel, 1 998) refer to other species. 

Conchologically similar are B. episcopalis 
from the Malay Peninsula, B. sumatrensis 
from Sumatra, ß. /?ercu/ea from Myanmar, and 
B.jullieni Uom Cambodia; all were repeatedly 
synonymized with B. costula. Brotia 
episcopalis and B. sumatrensis tend to be 
smaller and more conical in shape. In ß. 
episcopalis, the upper whorls are smooth and 



80' 



90° 



.■ИЛЧ1'Л'и'ЛУ 



100° 
' 30° 




10° 



FIG. 20. Distribution of B. costula (closed circles) and B. hercúlea (open 
circles). 



SYSTEMATIC REVISION OF BROTIA 



181 




N = 34 

ß. costula 



B. hercúlea 



FIG. 21. Comparison of 8. costula and S. 
hercúlea by means of shell parameter H/LA. Box 
plot diagram showing median, the 25%- and 
75%-percentile and largest non-extremes (less 
than 1 .5 times of box height). 



axial ribs are more conspicuous and not as 
regularly spaced as in B. costula, in which 
closely spaced axial ribs are always present. 
В.уи///еп/ exhibits a larger, broader, and more 
conical shell, with a more pronounced spiral 
sculpture (e.g.. Figs. 21, 27, 41 for compari- 
son of shell parameters). 

Adamietta species formerly assigned to ß. 
costula, such as A. infracostata (Mousson, 
1849), differ in embryonic shell morphology 
(Köhler & Glaubrecht, 2001, for the "Brotia 
testudinarla-group"). 

Brotia dautzenbergiana (Morlet, 1884) 
(Figs. 22-24) 

Melania dautzenbergiana Morlet, 1884: 399, 
400, pi. 8, fig. la-c ("Les ruisseaux se jetant 
dans le Prec-Thenot, sur sa rive droite dans 
les environs de Kompong Tull" = streams 
discharging into the Prec-Thenot on its right 
bank near Kompong Tull, Cambodia), lecto- 
type and three paralectotypes MNHN (des- 
ignated by Köhler & Glaubrecht, 2002a) (Fig. 
22A); types seen; Fischer-Piette, 1950: 154. 




FIG. 22. Shell morphology of ß. dautzenbergiana. A: Lectotype of M. dautzenbergiana MNHN; B: 
Lectotype of M. dugasti MNHN; C-D: Paralectotypes of M. dugasti BMNH; E: Myanmar (ZMB 49.626); 
F-G: Thailand, Lampang (ZMB 200.229); H: Thailand, Thoern (ZMB 200.213). Scale = 10 mm. 



182 



KÖHLER & GLAUBRECHT 



Stenomelania dautzenbergiana - Habe, 1 964: 
55, pl. 1,fig. 19. 

Brotia dautzenbergiana - Köhler & Glaubrecht. 
2002a: 133, fig. 10. 

Melania dugasti Мог\е{, 1893: 153, 154, pl. 6, 
fig. 1 ("Laos, Nam-Si, affluent du Nam Moun" 
= Laos, River Nam Si, affluent of the Nam 
Moun), lectotype MNHN, four paralectotypes 
MNHN, three paralectotypes BMNH 
1893.12.8.117-119, three paralectotypes 
MHNG (designated by Köhler & Glaubrecht, 
2002a) (Figs. 22B-D): types seen: Fischer- 
Piette, 1950: 160. 

Brotia citrina - Brandt, 1974: 179, pl. 13, figs. 
33, 34 {non M. citrina Brot, 1868). 




FIG. 23. Embryonic shell morphology 
of В. dautzenbergiana. SEM images 
of embryonic shell removed from 
brood pouch (Thailand, Lampang; 
ZMB 200.229): apical and front view. 
Scale bar = 1 mm. 



Taxonomy and Systematics 

Melania dugasti is considered as a junior 
synonym of M. dautzenbergiana for the most 
similar shell. Brandt (1 974) assumed that both 
taxa are synonyms of 8. citrina. However, the 
species can be distinguished by several mor- 
phological features. Treatment as distinct spe- 
cies is corroborated by molecular phylogenetic 
data (Figs. 78, 79). 

Material Examined 

Myanmar: North Shan, affluent to the 
Salween, Meungyaw (ZMB 46.626, 200.293), 
Nampai nver, Lashio (ZMB 49.627); Mandalay 
(ZMB 200.264): Thailand: Prov. Chiang Mai, 
Lampang River in Lampang (ZMB 200.225- 
6), bridge 20 km from Lampang, highway to 
Uttaradit, 18°7.89'N, 99°97.33'E (ZMB 
200.229), bridge at highway 106 nearThoern, 
17°39.31'N, 98°7.9ГЕ (ZMB 200.213); Prov. 
Kamphaeng Phet, Huai Hin Fon near Мае Sot 
(ZMH); Prov. Nan, Thung Thing (RMNH 
71320); Huai Mae Lau (ZMH). 

Differential Diganosis 

Shell highly elongated, thin, slender; whorls 
well rounded; suture narrow, deeply incised. 
Sculpture smooth, only with growth lines and 
spiral lines, surface not glossy. Light brown, 
with dark brown patches or spiral band. 

Description 

Shell (Fig. 22): Medium sized, solid but not 
thick; elongately turreted, cylindrical, mostly 
truncated with five to ten remaining regular, 
convex whorls. Suture narrow, accompanied 
by subsutural depression. Upper whorls 
smooth except for growth lines, last whorls 
sculptured by numerous fine spiral lines 
forming regular pattern with crossing growth 
lines. Surface not glossy; colour of periderm 
yellowish to brownish green or olive, often 
with broad, dark brown spiral band, occa- 
sionally with dark axial flames at upper 
whorls. Shells often grey or black due to layer 
of mineral deposits. Aperture ovate with pro- 
tracted base. Size: H = 23-44 mm, В = 10- 
16 mm. 

Embryonic Shell (Fig. 23): Conical, smooth 
with faint growth lines; up to 2.5 mm high, 
2.0-2.5 whorls; average proportions: H = 1 .8 
mm, В = 1.1 mm, HA = 0.21, BA = 0.41, DA = 
0.66 (for n = 15). 



SYSTEMATIC REVISION OF BROTIA 



183 



Operculum: Slightly ovate, up to five fast in 
diameter increasing whorls, nucleus slightly 
eccentric. 

Radula (Fig. 24): Upper rim of rachidian slightly 
concave, lateral corners not excavated, 
lower rim rather straight, slightly convex; 
main cusp flanked by two or three smaller 
denticles on each side, glabella well rounded 
at the base, v-shaped, its lateral margins 
slightly concave. Lateral teeth with main 
cusp, flanked by two accessory cusps on 
each side tapering in size. Inner and outer 
marginal teeth with two cusps, outer cusp 
broad, rounded; inner cusp pointed, consid- 
erably smaller. Outer marginals with con- 
spicuous hooked outer flange. 

Stomach: Corresponds to B. citrina (Fig. 4). 

Reproductive System 

Females (n = 9) contained 11 to 275 juve- 
niles, height 1.0 to 2.5 mm. 

Distribution (Fig. 36) 

Myanmar, central, northern to eastern Thai- 
land, Laos, Cambodia, Vietnam. Widespread 
and fairly common in most parts of the 
Indochinese Peninsula. Few more precise lo- 
calities available, though. River systems of the 
Salween, and the Chao Praya, as well as some 
affluents of the Mekong, but not Mekong itself. 

Remarks 

Similar to B. citrina from which B. dautzen- 
bergiana is distinguished by its more elongated 
shell, eroded tip, dark brown spiral band. Both 



species can be discriminated by shell param- 
eters, although not statistically significant 
(Table 2). 

Brotia episcopalis (H. Lea & I. Lea, 1851) 
(Figs. 25, 26, 67A) 

Melania episcopalis H. Lea & I. Lea, 1 851 : 1 84 
("sluggish river, Malakka" = Melaka, Prov. 
Negeri Melaka; 2°12'N, 102°15'E), lectotype 
and paralectotype MCZ221841 (designated 
by Köhler & Glaubrecht, 2002a) (Figs. 25A, 
B); types seen; Hanley, 1854: pi. 3, fig. 27; 
Brot, 1875: 97,98, pi. 12, figs. 1, la. 

Melanoides episcopalis - H. Adams & A. 
Adams, 1854: 297. 

Melania (Melanoides) episcopalis - Chenu, 
1859: 288, fig. 1952. 

Melania (Melanoides) variabilis episcopalis - 
Nevill, 1885: 256 [partim]. 

Brotia costula episcopalis - Davis, 1 971 : 53- 
86. 

Brotia episcopalis - Köhler & Glaubrecht, 
2002a: 134, fig. 1P. 

Melania héros Brot, 1875.' 339, 400, pi. 34, 
fig. 8 (unknown locality), holotype MHNG 
(Fig. 25D); type seen. 

Sermyla perakensis Morgan, 1885: 421, pi. 8, 
figs. 14a-f ("Perak"), lectotype and para- 
lectotype MNHN (designated by Köhler & 
Glaubrecht, 2002a) (Fig. 25C); types seen. 

Brotia costula - Brandt, 1974: 175, pi. 13, fig. 
37-39 [partim]; Köhler & Glaubrecht, 2001: 
296-299, figs. ID, 10A-C, G, H [partim] (non 
M. costula Rafinesque, 1833). 

Brotia (Antimelania) costula - Subba Rao, 
1989: 108, 109 [partim] (non M. costula 
Rafinesque, 1833). 




FIG. 24. Radular morphology of 8. dautzenbergiana. SEM images of radula segments viewed from 
above. A: Thailand, Lampang (ZMB 200.229); В: Thailand, Thoern (ZMB 200.213). 



184 



KOHLER & GLAUBRECHT 




FIG. 25. Shell morphology of S. episcopalis. A: Lectotype of M. episcopalis MCZ 221841; B: 
Paralectotype MCZ 221841; C: Lectotype of Sermyla perakensis MNHN; D; Holotype of Melania 
héros MHNG. Scale bar = 10 mm. 



Brotia variabilis - Bequaert, 1943: 433, 434, 
pi. 33, figs. 11-16 [partim]; Solem, 1966: 15 
{non M. variablis Benson, 1836). 

Taxonomy and Systematics 

Frequently subsumed under В. costula by 
20'" century authors (Benthem Jutting, 1949, 
1956; Brandt. 1968, 1974; Köhler & Glaub- 
recht, 2001 ). However, molecular genetic data 
shows that B. episcopalis is distinct (Figs. 78, 
79). Melania héros and Sermyla perakensis 
are considered as synonyms. 

Material Examined 

Thailand; Prov. Trang; Trang (ZSM 
19983228). Prov. Nakhon Si Thammarat: 
Khiong Nga, Chawang (ZMH); Malaysia: Prov. 
Kedah; Baling, River to east coast (ZMA). 
Prov. Pahang, Taman Negara National Park 
(ZMA; ZMB 200.041, 200.047); Sungei 
Kenong (ZMB 200.139); Sungei Mantine, af- 
fluent to Sungei Serau (ANSP A8907). Prov. 
Selangor; Sungei Buaya - NW Rawang 
(ZMA); Sungei Kelang, 17 mi. S Kuala Lumpur 
(ZMA); rapidly flowing river, 16 mi. N of Kuala 
Lumpur (CAS 30197). Prov. Negeri Perak: 
Tong Temple near Ipoh (ZMA; ZMB 200.046); 
Perak River (ZMB 200.054). Prov. Negeri 
Melaka: Melaka (ZMB 52.656, 200.047, 
200.050, 200.306-7; MHNG). 



Differential Diganosis 

Shell large, solid; conic, up to 1 1 whorls, with 
strong axial ribs. Upper rim of the rachidian 
flanked by heavily excavated lateral corners. 

Description 

Shell (Fig. 25): Large, solid, pyramidal, fre- 
quently eroded, 6 to 11 convex, rounded 
whorls; strong axial ribs, and basal spiral 
ridges; colour light brown to olive-brown. 
Aperture wide, oval, well rounded below. 
Size: H = 34-57 mm, В = 15-24 mm. 

Embryonic Shell: No own data, but described 
and depicted by Davis (1971: 60, figs, 2h, i, 
11): up to 3.5, perhaps even 4.0 whorls, 2 
mm height, rather smooth. 

Operculum: Oval, multispiral, up to six whorls, 
sub-central nucleus. 

Radula (Fig. 67A): Up to 180 rows of teeth, 
length up to 20 mm, corresponding to about 
half of shell height. Upper margin of rachid- 
ian conspicuously concave, formed by two 
inflated, well rounded corners. Glabella 
slightly V-shaped, well rounded at its base, 
concave lateral margins. Main cusp flanked 
by mostly two smaller denticles on each side, 
sometimes only one. Laterals with short lat- 
eral extensions, pronounced inner flange, 
two main cusps flanked by two smaller den- 
ticles. Inner and outer marginal teeth with 



SYSTEMATIC REVISION OF BROTIA 



185 




FIG. 26. Stomach anatomy of 6. episcopalis 
(Thailand, Nakhon Si Thammarat; ZMH). Scale 
bar = 1 mm. 



two pointed cusps of about same size and 
shape. 
Stomach (Fig. 26): Typhlosoles fused at almost 
entire length of style sac; marginal fold nar- 
rowly angled posterior and underneath open- 
ing of intestine; flap-like posterior end of 



3.2 




8 
о 








,-i I) 








I 


2.8 










2.6 


















2 4 


ШЦ 


^»1 










■Ht 








2 2 


1 












2.0 
1 R 



















N=34 154 42 

ß. costula В. sumatrensis В. episcopalis 



FIG. 27. Comparison of B. costula, B. suma- 
trensis, and S. episcopalis by means of shell pa- 
rameter H/B. Box plot diagram showing me- 
dian, the 25%- and 75%-percentile and largest 
non-extremes (less than 1 .5 times of box height). 



major typhlosole flat, partially covering open- 
ing of style sac. 

Distribution (Fig. 68) 

Southern Thailand and Malaysia: Malay Pen- 
insula, S of Isthmus of Kra. Occurrence in 
Sumatra unclear. 



Habitat 

Streams and rivers, only rarely still waters 
(Davis, 1971; Kruatrachue et al., 1990). In 
great abundance in the Pahang River system 
in quiet, marginal waters together with ß. 
kelantanensis, which lives among rocks in rap- 
ids (Davis, 1982: 392, referring to B. costula 
and "a second spiny species"). 

Remarks 

Frequently confused with B. costula and B. 
sumatrensis. Brotia costula tends to have a 
more elongated shell with more closely spaced, 
regular ribs also on upper whorls. Brotia 
sumatrensis lacks marked transition from 
smooth upper whorls to strongly sculptured 
lower whorls and exhibits lesser pronounced 
axial ribs. Employing statistical analyses, ß. 
costula, B. episcopalis, and B. sumatrensis 
cannot be discriminated by their morphometry 
(Fig. 27). A detailed description of morphology, 
reproductive biology, growth rates, and rel- 
evance as intermediate host of the lung fluke 
Paragonimus westermanni is given by Davis 
(1971). Our observations fit well to the com- 
prehensive data reported in this paper. 

Brotia godwini (Brot, 1875) 
(Figs. 28, 31A) 

Melania (Melanoides) /?an/ey/ Godwin-Austen, 
1872: 514, 51 5, pi. 30, fig. 2 {non M. hanleyi 
Brot, 1860) ("Diyung River, North Cachar 
hills" = Diyung River, Jaintia-Khâsi hills N of 
Silchar, Meghalaya, India, 24°48'N, 92°46'E), 
lectotype BMNH 19991561/A and 
paralectotype BMNH 19991561/B (desig- 
nated by Köhler & Glaubrecht 2002a) (Figs. 
28A, B); types seen. 

Melania godwini Brot, 1875: 90, pi. 10, fig. 3 
(replacement name for M. hanleyi Godwin- 
Austen, 1872). 

Melania (Melanoides) variabilis var. 
binodulifera Nevill, 1885: 259 ("Khasi hills"). 

Brotia goc/w/n/ - Köhler & Glaubrecht, 2002a: 
136. 



186 



KÖHLER & GLAUBRECHT 



Taxonomy and Systematics 



Description 



Melania godwini Brot, 1875. was employed 
as replacement name for M. hanleyl Godwin- 
Austen, 1872, being preoccupied by M. hanleyl 
Brot, 1860. 

Material Examined 

India: Assam, Lamin (ZMB 94.722); Cachar 
(ZMB 20.737). 

Differential Diganosis 

Stepped whorls, deeply incised suture, two 
spiral ridges, lower one at about a third of 
whorls diameter, upper one at about two thirds, 
more pronounced. Upper ridge supports spi- 
ral row of spiny tubercles; some specimens 
with axial ribs; aperture very wide. 



Shell (Fig. 28): Small to medium sized, conical 
to turreted, four to five convex, stepped 
whorls, eroded; colour chestnut brown. Spi- 
ral row of spiny tubercles and spiral lines, 
most prominent at base of shell; last whorl 
large, inflated; aperture wide, ovate, produced 
below, comprising up to 1/3 of shell height. 

Radula (Fig. 31A): Ribbon with 120 rows of 
teeth. Central tooth squarish, anterior rim 
slightly concave, very large main cusp 
flanked by two smaller denticles on each 
side, glabella v-shaped, basely rounded; lat- 
eral teeth with main cusp and one acces- 
sory denticle on each side; inner and outer 
marginals with large, broad, spatula-shaped 
outer cusp and much smaller, pointed inner 
denticle; inner marginals broader than outer 
ones. 




FIG. 28. Shell morphology of a godwIni.A: Lectotype of /W. /?an/ey/ Godwin-Austen BMNH 19991561/ 
A; B: Paralectotype BMNH 1 9991 561/B; C: Assam, Cachar (ZMB 20.737); D-E: Assam (ZMB 97.422). 
Scale bar = 10 mm. 



SYSTEMATIC REVISION OF BROTIA 



187 



Embryonic shell morphology. Soft body 
anatomy Operculum: Unknown. 

Distribution 

India: Meghalaya, Assam, Manipur: tributar- 
ies of Brahmaputra (possibly also neighbouring 
regions of Myanmar and Bangladesh). Seem- 
ingly restricted to mountainous regions. 

Remarks 

Similar to spiny morphs of B. costula, but 
shell not as highly turreted, whorls much more 
stepped; radula differs in shape of glabella. 

Brotia henriettae (Griffith & Pidgeon, 1834) 
(Figs. 29, 30, 31B, C) 

Melania henriettae Griffith & Pidgeon, 1834: 
598, pi. 1 3, fig. 2 ("China"), ex Gray ms; lec- 
totype BMNH 19990495/Aand paralectotype 
BMNH 19990495/B (designated by Köhler 
& Glaubrecht, 2002a) (Figs. 29A, B); types 
seen; Reeve, 1859: 1. 

Semisulcospira henriettae - Yen, 1942: 204: 
pi. 15, fig. 66. 

Brotia henriettae - Köhler & Glaubrecht, 
2002a: 137, 138, fig. 2D. 

Melania baccata Gould, 1847: 219 
("Thoungyin River, branch of the Salween, 
Burma"), Lectotype MCZ 169052 and para- 
lectotype USNM 611239 (designated by 
Johnson, 1964) (Fig. 29E); types seen; Brot, 
1875: 81 , 82, pi. 9, fig. 6; Hanley & Theobald, 
1873: 32, pi. 75, figs. 1, 2, 4; Annandale, 
1918: 115, pi. 7, fig. 9; Johnson, 1964: 45. 

Melania (Melanoides) baccata - Nevill, 1885: 
262. 

Melania {Brotia) baccata - Martens, 1899: 35, 
36. 

Melanoides (Tiara) baccata - Preston, 1915: 
26. 

Melania baccata subsp. elongate Annandale, 
1918: 115, 116, pi. 7, figs. 3, 3a, 4-7 ("He- 
Ho Plain and Yawnghwe River" = He ho, N 
of Lake Inle, 20°44'N, 96°49'E), two syntypes 
ZSI 11155/2, according toAnnandale (1918); 
types not seen. 

Acrostoma elongatum - Annandale & Rao, 
1925: 117. 

Melania persculpta Ehrmann, 1922: 18-23, fig. 
8 ("Loikaw-Fluß, Süd-Schan-Staaten" = 
Loikaw River, Southern Shan States, 
Myanmar), lectotype SMF 221813, 20 para- 
lectotypes SMF 221814-5 (designated by 



Köhler & Glaubrecht, 2002a) (Fig. 29D); 

types seen. 
Acrostoma baccata - Rao, 1928: 442-445, 

figs. 17, 18. 
Brotia baccata - Bequaert, 1943: 431; 

Morrison, 1954: 384; Johnson, 1964: 45. 
Brotia (Brotia) baccata - Brandt, 1974: 178, 

pi. 13, fig. 32. 
Melania reticulata I. & H.C. Lea, 1851: 193 

("China"), holotype USNM 119663 (Fig. 

29C); type seen. 
Melanoides reticulata - H. Adams & A. Adams, 

1854: 297. 
Melania baccata var. pyramidalis Martens, 

1899: 36. 
Melania variabilis var. pyramidalis - Theobald, 

1865:274, fig. 7 
Melania variabilis var. g/abra Theobald, 1865: 

273. 
Melania variabilis war. wYíaía Theobald, 1865: 

273, fig. 4; Nevill, 1885:263. 
Melania variabilis yar. famfa Theobald, 1865: 

273, 274, fig. 5. 
Melania variabilis var. baccifera Theobald, 

1865:274, fig. 6. 
Melania (Melanoides) baccata subvar. recta 

Nevill, 1885: 262 ("Upper Salween"). 
Melania (Melanoides) subasperata Nevill, 

1885: 262 ("Shan States"). 
Melania (Melanoides) subasperata var. 

sublaevigata Nevill, 1885: 263 ("Shan 

States"). 

Taxonomy and Systematics 

Noticing the confusing variety of different 
shell forms that also lead 19'^ century authors 
to introduce a plethora of names, Annandale 
(1918) wondered whetherthese forms should 
be regarded as representing one highly vari- 
able species or a flock of morphologically simi- 
lar species. Indeed, the diversity of shell forms 
attributed to this species might be indicative for 
the existence of more then a single species. 
However, the question whether and, if so, how 
many different species are currently subsumed 
under the concept of ß. henriettae cannot be 
answered satisfactorily since only dry shell 
material is available from Myanmar. Unless 
more detailed morphological and molecular 
genetic data will show otherwise, we follow 
Brot (1875) considering these forms as con- 
specific. It remains unclear, however, why Brot 
(1875) referred to M. baccata but not to the 
older name M. henriettae. This treatment was 
followed by later authors, rendering M. baccata 



KÖHLER & GLAUBRECHT 

В с 




piWlwïïïïl 

1 

FIG. 29. Shell morphology of В. henriettae. A: Lectotype of M. henriettae BMNH 1999095/A; B: 
Paralectotype BMNH 1999095/B; C: Holotype of M. reticulata USNM 119663; D: Lectotype of M. 
persculpta SMF 221813; E: Lectotype of /W. baccata MCZ 169052; F: Thailand, Pai (ZMB 200.221); 
G: Myanmar, Lashio River (ZMB 49.612); H: Myanmar (ZMB 200.006); I: China, Hienshow (ZMB 
62.665); J; Myanmar, Myitnge (ZMB 49.613). Scale bar = 10 mm. 



SYSTEMATIC REVISION OF BROTIA 



189 



a name most commonly employed. None- 
theless, the name M. henhettae Griffith & 
Pidgeon, 1834, being available has priority 
over M. baccata. 

Brotia henhettae is type species of Wanga 
Chen, 1943, by original designation. This ge- 
nus is considered a junior synonym of Brotia. 

Material Examined 

China: Yunnan, Yaylayman (ZMB 27.511); 
Hienshow River (ZMB 52.665). Myanmar: 
Shan states (BMNH 1907.12.30.210; ZMB 
200006-7); North Shan States: Lashio River at 
Myitnge (ZMB 49.616), Lashio River (ZMB 
49.612, 200.076, BMNH 1899.6.21.72-5), 
Nampai River near Lashio (ZMB 49.61 1 , BMNH 




FIG. 30. Embryonic shell morphology 
of S. henhettae. SEM images of 
embryonic shell removed from dried 
shell (ZMB 49.613); apical and front 
view. Scale bar = 1 mm. 



1 899.6.21 .90-91 ), tributary of the Nampai near 
Lashio (ZMB 49.615), small stream at 
Meungyaw (ZMB 49.618), Myitnge (ZMB 
49.613, 200.004, 200.140), tributary of the 
Myitnge at Bagwyo nearThibaw (ZMB 49.614), 
small stream near Bangwyo (ZMB 200.002); 
ZMB 200.005); Chindwin, tributary of the 
Irawaddy at Matu (ZMB 49.620); affluent of the 
Salween near Lashio (ZMB 49.619, BMNH 
1899.6.21.76-79); tributary of the Salween 
(ZMB 200.000), Gotheik cave (ZMB 200.001 ), 
Thoungyin River (BMNH 1888.12.4.1767-8); 
Thailand: Prov. Chiang Mai, Pai River in Pai, 
19°21.57'N, 98°26.62'E (ZMB 200.221); Prov. 
Mae Hongsong, Som River at Ban Som (ZSM 
19983220); Prov. Kamphaeng Phet, Moei 
River, 30 km S of Mae Sot, boarder to 
Myanmar, 16°26.96'N, 98°39.27'E (ZMB 
200.210). 

Diagnostic Characteristics 

Pyramidal turreted, solid, flattened whorls, 
narrow suture; spiral lines support two or three 
spiral rows of closely spaced tubercles; in 
some specimens tubercles replaced by axial 
ribs; aperture well produced with sharp peris- 
tome; body whorl relatively large; operculum 
round with up to eight whorls, considerably 
smaller than aperture; embryonic shells with 
axial ribs from second whorl on. 

Description 

Shell (Fig. 29): Medium sized, solid; spire oval 
to cylindrical or highly turreted; six to eight 
flattened whorls, suture deeply incised; 
strong spiral cords support more or less dis- 
tinct nodules. Two to three nodules fre- 
quently arranged in vertical rows, sometimes 
forming axial ribs. Aperture rather narrow, 
peristome thin, sharp. Colour light to chest- 
nut brown. Size: H = 30-64 mm, В = 13-25 
mm. 

Embryonic Shell (Fig. 30): Conic to turreted, 
penultimate whorl with smooth sculpture, 
following whorls with strong axial ribs. Aver- 
age proportions: H = 3.0 mm, В = 1.9 mm, 
HA = 0.24 mm, BA = 0.40 mm, DA = 0.90 
mm (for n = 6) up to 3.5 whorls. 

Operculum: Round, up to eight regular whorls, 
almost central nucleus; much smaller than 
aperture. 

External Anatomy: Animal black with yellow- 
ish to light brown patches. 

Radula (Figs. 31 B, C): Up to 1 50 rows of teeth; 
radulae from different localities vary in breadth 



190 



KÖHLER & GLAUBRECHT 



and shape of main cusp. Generally, central 
tooth with concave upper rim, relatively broad 
central cusp flanked by two accessory den- 
ticles tapering in size, glabella with concave 
to angled lateral margins, basely well 
rounded. Lateral tooth with broad main den- 
ticle flanked by two inner and one or two outer 
accessory denticles. Inner and outer margin- 
als with two cusps, outer one broad, spatula- 
shaped, inner one small, pointed. Inner 
marginals broader. 
Stomach: Typical, as in ß. citrina (Fig. 4); 
typhlosoles unfused. 

Distribution (Fig. 36) 

China (southern China), particularly Yunnan; 
Myanmar (Northern and Southern Shan states); 
Thailand (northern and western Thailand); river 
system of the Irawaddy and Salween. 

Habitat 

Clear mountain rivers and streams with 
strong current, attached to stones and rocks. 



In the Maenam Moei (= Thoungyin River) co- 
occurring with B. pagodula and B. Iiercuiea. 

Fossil Record 

In Tertiary and Pleistocene cave deposits of 
Myanmar (Bequaert, 1943); sub-fossil shells 
reported byAnnandale (1918) from Myanmar. 

Remarks 

Similar sculpture in B. iravadica, frequently 
being smaller and more conical in shape, with 
fewer whorls. 

Brotia Iiercuiea (Gould, 1846) 
(Figs. 32-34) 

Melania hercúlea Gould, 1846; 100 ("Ta voy 
River, British Burma" = Tavoy, Myanmar, 
14°05'N, 98°12'E), lectotype MCZ 169436, 
two paralectotypes MCZ 87933, 17 para- 
lectotypes MCZ 169437, two paralectotypes 
USNM 611234 (designated by Johnson, 
1964) (Fig. 32A); types seen; Reeve, 1859: 




FIG. 31 . Radular morphology of B. godwini, B. henhettae, and B. jullieni. A: B. godwini (Assam; ZMB 
97.422); B: B. henhettae (Thailand, Pal; ZMB 200.221); C: B. henhettae (Thailand, Mae Sot; ZMB 
200.210); D: 8. yu/Z/en/ (Cambodia; ZMH). Scale bars = 0.1 mm. 



SYSTEMATIC REVISION OF BROTIA 



191 



pi. 2, fig. 4; Hanley & Theobald, 1873: 31 , pi. 

72, fig. 5; Johnson, 1964: 87, pi. 35, fig. 10. 
Melanoides hercúlea - H. Adams & A. Adams, 

1854: 297. 
Melania (Melanoides) /7erca/ea- Nevill, 1885: 

251. 



Melania balteata Reeve, 1860: pi. 20, species 
144 [non M. balteata Philippi, 1858) (no lo- 
cality given), lectotype ÜMB TK 304/1 and 
paralectotype ÜMB 308/1 (designated by 
Knipper, 1958, referring to M. reevei) (Figs. 
32B, C); types seen. 




FIG 32 Shell morphology of B. hercules. A: Lectotype of M. hercúlea MCZ 1 69436; 8: Lectotype of /If 
balteata Reeve ÜMB TK 304/1; C: Paralectotype ÜMB TK 308/1; D; Lectotype of M. gloriosa ANSP 
26363- E- Thailand, Raheng (MHNG); F: Thailand, Sai Yok (ZMB 200.235). Scale bar = 10 mm. 



192 



KÖHLER & GLAUBRECHT 



Melania reevei Brot. 1862: 46 (replacement 
name for /W. balteata Reeve): Brot, 1875: 95, 
96. pl. 11, figs. 4, 4a, pl. 13, fig. 6: Hanley & 
Theobald, 1876: 61, pl. 153, flg. 1. 

Melania (Melanoldes) reevei - Nevill, 1885: 
248. 

Melania (Melanoldes) reevei var. lanceolata 
Nevill. 1885: 248, 249 ("Mandalay: Hezada, 
Pegu; Thyet Myo"). 

Melania (Melanoldes) reevei var. imbrícala 
Hanley & Theobald. 1876: pl. 153. fig. 4 
(without locality): Nevill, 1885: 249. 

Melania (Melanoides) reevei var. sol id и seul a 
Nevill, 1885: 249, 250 ("Pegu, Noung-ben- 
Ziek"). 

Melania {Brotia ?) reevei- Martens, 1899: 36. 

Melania gloriosa Anihony, 1865: 207, pl. 18, 
fig. 3 ("Pegu" = Pegu. Myanmar), lectotype 
ANSP 26363, paralectotype MCZ 74106, 
three paralectotypes MCZ 74107. potential 
paralectotype MCZ 315666 (designated by 
Köhler & Glaubrecht, 2002a) (Fig. 32D); 
types seen: Brot, 1875: 94, 95, pl. 11, figs. 
3, 3a, b; Hanley & Theobald, 1873: 31, pl. 
72, figs. 1,2: Baker, 1964: 190. 

Melania (Melanoides) tourannensis var. 
gloriosa - Nevill. 1885: 250. 

Melania variabilis -Brol 1875: 85-87, pl. 10, 
fig. 1, 1a-d [partim]. 

Melania peguensis Hanley & Theobald, 1 873: 
31, pl. 72, fig. 6 [nomen nudum]. 

Melania (Melanoides) tourannensis var. 
peguensis- Nevill, 1885: 250. 

Melania (Melanoides) tourannensis var. com- 
pacta Nevill. 1885: 250, 251 ("Henzada, 
Pegu"). 

Melania (Melanoldes) tourannensis var. 
beddomeana Nevill, 1885: 251 ("near 
Moulmein"). 

Melania (Melanoides) variabilis subvar. 
subvaricosa Nevill, 1885: 252, 253 "Arakan, 
Pegu"). 

Melania (Melanoides) variabilis subvar. 
semllaevigata Nevill, 1885: 252. 

Brotia costula - Benthem Jutting, 1956: 374- 
378, fig. 76 [partim]; 1959: 92-95 [partim]; 
Brandt, 1974: 175, pl. 13, figs. 37-39 
[partim]; Köhler & Glaubrecht, 2001: 296- 
299, figs. 10D-F [partim]; Köhler & 
Glaubrecht, 2002a: 132 [partim]. 

Taxonomy and Systematics 

Treated as synonym of Brolla costula by, for 
example, Benthem Jutting (1956, 1959) and 
Brandt (1 974), this taxon is considered herein 
as a distinct species since B. costula and B. 



hercúlea occupy different positions in the phy- 
logenetic trees (Figs. 78, 79). Together with 
its distinct shell morphology, this is reason 
enough to not treat B. hercúlea conspecific 
with the former. 

A second taxon, Melania reevei Brot, has 
also frequently been considered a synonym 
of ß. costula by 20"' century authors. This 
name was employed as a replacement for M. 
balteata Reeve, being preoccupied by M. 
balteata Philippi. Most certainly identical with 
the latter are Melania gloriosa Anthony and 
M. peguensis Hanley & Theobald. The latter 
was introduced in error by Hanley & Theobald 
(1873), who intended to refer to Anthony's 
original figure but mixed up the legends of fig- 
ures 2 and 3 of pl. 18 of Anthony's work. So, 
they employed the name "M. peguensis", 
which however refered to a bivalve species of 
Monocondylaea, instead of "M. gloriosa", 
which would have been the correct reference 
for the species of Melania. 

Both M. reevei and M. gloriosa are tenta- 
tively subsumed under 6. hercúlea for their 
somewhat similar shell and since both origi- 
nate from the same area, Pegu. The type lots 
of M. hercúlea on one hand and the types of 
M. reevei and M. gloriosa, respectively, on the 
other hand mainly differ in the presence or 
absence of axial ribs. Examination of further 
series of dry shells from Pegu, though, reveals 
that the presence of ribs seems to be rather a 
variable feature, not sufficient to indicate the 
existence of two individual species. A more 
reliable decision on this aspect awaits the 
study of new alcohol preserved material, how- 
ever. 

Material Examined 

Myanmar (BMNH; ZMB 49.621, 200.059- 
60): Pegu (ZMB 41.199, 200.051, 200.060, 
200.065-6, 200.305; BMNH 1838.12.4.1757); 
Bassein District, Pegu (BMNH); Prome 
(MHNG); Mandalay (ZMB 47.125, 49.623; 
MHNG); Myadung (ZMB 27.512, 49.623); 
Yangon (ZMB 200.055-6, 200.067); 
Tenasserim (ZMB 200.304; BMNH); Chindwin 
near Matai (ZMB 49.624); Yu River, tributary 
of the Chindwin (ZMB 49.622, 49.625); Mu, 
tributary of the Irawaddy (ZMB 49.621 ); Thai- 
land: Prov. Mae Hong Song, Nam Mae Yuam 
near Mae Sariang (ZSM 19983228, 
19983247); Prov. Chiang Mai, Pai River ap- 
proximately 20 km E Pai, 19°17.83'N, 
98^^27. 93'E (ZMB 200.219), Pai River in Pai, 
19°21.57'N, 98°26.62'E (ZMB 200.220); Mae 



SYSTEMATIC REVISION OF BROTIA 



193 



Ping, 60 km N Chiang Mai (MNHN; AMS 
146766); bridge at the street from Samoeng 
to Chiang Mai, 18M4.23'N, 98°55.87'E (ZMB 
200.253); Prov. Kanchanaburi, Sai Yok Falls 
1 at Nam Ток, 14°14.16'N, 99°3.24'E (ZMB 
200.235-7); Prov. Kamphaeng Phet, Maenam 
Moei, about 30 km S Mae Sot, boarder to 
Myanmar, 16°26.96'N. 98°39.27'E (ZMB 
200.209); Prov. Так, Mae Dao River, Mae Sot 
(AMS 146762), Maenam Moei, 8 km N Mae 
Ramat(AMS 146765). 




FIG. 33. Embryonic shell morphology of 
ß. hercúlea. SEM images of embryonic 
shell removed from dried shell (ZMB 
49.623); apical and front view. Scale 
bar = 1 mm. 



Differential Diganosis 

Shell robust, highly turreted, up to 12 flat- 
tened whorls, the basal ones convex, more or 
less rounded in diameter; aperture wide with 
protracted base. Strong axial ribs, that may 
also lack completely; spiral lines. 

Description 

Shell (Fig. 32): Large to very large, shell solid 
to thick, spire pyramidal turreted, up to 12 
whorls, eroded tip; colour hazelnut to dark 
brown; spiral ridges most prominent at the 
base, in some specimens very conspicuous, 
in others almost completely absent; strong 
axial ribs may be present. Whorls flattened 
in diameter, with subsutural depression. 
Size: H= 28-98 mm, В = 10-34 mm. 

Embryonic Shell {F'\g. 33): Smooth, covered with 
axial wrinkles. Average proportions: H = 1.7 
mm, В = 1.0 mm, HA = 0.25 mm, BA = 0.40 
mm, DA = 0.61 mm (for n = 15), up to 3.5 
whorls. 

Operculum: Slightly oval, four to six whorls, 
central nucleus; almost fits aperture. 

External Morphology: Uniformly coloured, dark 
grey to black; grey foot sole with scattered 
light spots. 

Radula (Fig. 34): Ribbon length of up to 30 mm, 
corresponding to about half of the shell 
height, about 180 rows of teeth. Rachidian 
with single main cusp, three smaller denticles 
on each side tapering In size; upper margin 
concave by inflated, rounded corners; lower 
rim rounded; glabella narrow, well rounded 
at its base, lateral margins slightly concave. 
Laterals with main cusp flanked by three 
smaller denticles. Inner and outer marginals 
with two to three denticles, somewhat 
pointed, of about same size and shape. 

Stomach (Fig. 35): Typhlosoles fused at almost 
entire length of style sac; opening to style 
sac partly covered by fleshy, flap-like proxi- 
mal end of major typhlosole; proximal end of 
minor typhlosole thickened; crescent ridges 
below opening of digestive gland duct undu- 
lated; crescent pads adjacent to sorting area 
well developed, heavily undulated or ribbed. 

Distribution (Fig. 20) 

Myanmar and northwest Thailand: river sys- 
tems of the Irawaddy, Chindwin, and Salween 
(with Moei River), and Chao Praya (with Ping 
and Nan Rivers). 



194 



KÖHLER & GLAUBRECHT 




FIG. 34. Radula morphology of В. hercúlea. Radula segments viewed from above. A: Myanmar, Pegu 
(ZMB 41 .199); B: Thailand, Pai (ZMB 200.220); C; Thailand, Sai Yok Falls, Nam Ток (ZMB 200.237); 
D: Thailand, Pai (ZMB 200.219). Scale bars = 100 |jm. 




Habitat and Ecology 

Clear creeks and rivers on rock, mud, sand, 
roots, under and among piles of leaf litter in the 
water (Davis, 1 982, referring to B. costula), what 
can be confirmed from own observations in 
Thailand. May be infested by drilling sabellids 
(Nematoda). 

Remarks 

Largest species of the genus. B. costula dif- 
fers statistically significant in shell parameters 
H/B, H/LA, N (e.g.. Fig. 21). 

Brotia indragihca (Martens, 1900) 
(Fig. 37) 



FIG. 35. Stomach anatomy of a /lercu/ea (ZMB Melania indragirica Martens, 1900; 10, 11 
200.209; Thailand). Scale bar = 5 mm. ("Indragiri-Fluß, Sumatra" = Indragin River, 



90 



SYSTEMATIC REVISION OF BROTIA 
100° 



195 




FIG. 36. Distribution of S. citrina (open circles), B. dautzenbergiana (open squares) 
and B. henhettae (close circles). 



Sumatra (Indonesia), lectotype ZMB 51 .777a, 
three paralectotypes ZMB 51 .777b, five para- 
lectotypes NMB 1202q (designated by Köhler 
& Glaubrecht, 2002a) (Fig. 37); types seen; Bul- 
len, 1906: 14 (including an unnamed variety). 
Brotia indragirica - Köhler & Glaubrecht, 2002a: 
139, fig. 2L. 

Taxonomy and Systematics 

Only known from the types. For this reason, 
soft body, radula, and embryonic shells un- 
known. Shell clearly pachychilid being reason 
for affiliation with Brotia as the only pachychilid 
taxon known from Sumatra. 

Differential Diganosis 

Highly turreted, convex whorls flattened in 
diameter, keeled or angled; prominent, wavy 
spiral bands or ridges, along keel of the whorl 
spiral row of spiny nodules; aperture wide, well 
rounded. 



Description 

Shell (Fig. 37): Small, not thick but solid; spire 
turreted, eroded tip, four to five convex whorls. 




IWFFjwïïïïj 



FIG. 37. Shell morphology of 
B. indragirica. Lectotype of /W. 
indragirica ZMB 51 .777a. 



196 



KÖHLER & GLAUBRECHT 



Upper half of whorls flattened; conspicuous, 
wavy spiral ridges, weak axial ribs; spiral row 
of spiny nodules at centre of whorls where 
spiral ridge meets axial ribs. Aperture wide, 
ovate, produced below. Colour yellowish 
brown. Size; H = 23-36 mm, В = 10-15 mm. 
Embryonic Shell. Operculum. Radula. Son 
Body: Anatomy unknown. 

Distribution 

Sumatra (provinces of West-Sumatra and 
Riau); Indragiri River and its affluent Kwantan, 
discharging into South China Sea (approxi- 
mate centre of river at 0°33'S, 102°03'E). 

Brotia insólita (Brot, 1868) 
(Fig. 38) 

Melania insólita Brot, 1868; 11, pl. 3, fig. 4 
("Inde?"), lectotype and seven paralecto- 
types MHNG. Brot collection, "Siam" (desig- 
nated by Köhler & Glaubrecht, 2002a) (Fig. 
38); types seen; Brot, 1 875; 1 07, 1 08, pl. 1 3, 
fig. 7. 

Brotia (Brotia) insólita - Brandt, 1974; 176, 
177, pl. 13, figs. 29, 30. 

Brotia insólita - Köhler & Glaubrecht, 2002a; 
139, 140, fig. 2J. 

Taxonomy and Systematics 

Brot ( 1 868) stated that the species originated 
from India, which was later corrected to Thai- 
land (Brot, 1875). This corresponds with la- 
belling of the types. Because the type locality 
could not further be specified, the So Pa Falls, 




FIG. 38. Shell morphology of B. insolite. A: Lec- 
totype of M. insólita MHNG; B-D: Three para- 
lectotypes MHNG. Scale bar - 10 mm. 



Kaek River (Prov. Phitsanulok, central Thai- 
land) were subsequently designated as the 
type locality by Brandt (1974; 177). 

Material Examined 

Thailand (ZMB 31.172); Cambodia (ZMB 
26.870). Without locality (MHNG, Brot collec- 
tion; labelled "M. gloriosa"). 

Differential Diganosis 

Relatively small, conical, thin but solid; 
whorls well rounded; yellowish to greenish 
brown, dark brown spiral band may be present. 

Description 

Stiell (Fig. 38); Small, relatively thin but solid. 
Shell conical in shape, the body whorl is 
comparatively large and inflated, while sub- 
sequent whorls taper considerably in size. 
Spire with up to six whorls, eroded. Sculp- 
ture consisting of faint growth lines and deli- 
cate regularly spaced spiral ridges, in some 
specimens these ridges become stronger at 
the base, inconspicuous axial ribs may be 
present too. Surface glossy, colour yellow- 
ish brown a spiral band of darker coloration 
may be present at the mid of the whorls. 
Aperture wide, oval and well rounded and 
produced at the base. 

Embryonic Shell. Operculum, Radula, Soft 
Body: Anatomy unknown. 

Distribution 

Central Thailand to Cambodia, only vague. 

Remarks 

We were neither able to trace voucher ma- 
terial of Brandt from the Kaek River nor to find 
this species during our own field work. For this 
reason, we cannot confirm the occurrence in 
the Kaek River. Brandt (1974) described B. 
manningi, the shells of which are at best hard 
to distinguish from B. insólita. To complicate 
matters, B. insólita closely resembles some, 
but not all specimens of the type series of B. 
siamensis, among them the lectotype. The 
difficulties to reliably discriminate all these taxa 
will likely persist unless material suitable for 
studies on soft body morphology and molecu- 
lar genetic is available. For the time being, we 
follow the treatment of Brandt (1974). 



SYSTEMATIC REVISION OF BROTIA 



197 



Brotia siamensis tends to be more elongate, 
often exhibiting axial ribs at the upper whorls. 
Whorls of B. manningi are flattened in diam- 
eter. 

Brotia iravadica (Blanford, 1869) 
(Fig. 39) 

Melania iravadica Blanford, 1869: 445 ("Burma, 
Upper Irawaddy at Male and Bhamo"), three 
syntypes BMNH 1888.12.4.1808-10; types 
not seen; Hanley & Theobald, 1873: 30, pi. 
71, fig. 1. 

Melania irawadica [sic !] - Brot, 1 872: 34; Brot, 
1875: 111, 112, pi. 14, figs. 7, 7a. 

Melania [Melanoides) baccata var. iravadica 
-Nevill, 1885:262. 

Melania (Melanoides) iravadica - Nevill, 1885: 
33. 

Melania (Brotia) baccata var. iravadica - Mar- 
tens, 1899: 35, 36. 

Tiara (Melanoides) baccata var. irawadica 
[sic !]- Preston, 1915: 27. 

Acrostoma iravadica - Rao, 1928: 446, 447. 

Taxonomy and Systematics 

Mostly treated as subspecies or variety of 
morphologically relatively plastic B. henriettae. 
We suggest this taxon represents a distinct 
species because of its deviant shell. Excep- 
tionally known from the Irawaddy, but not from 
its tributaries where B. henriettae occurs. 
Whether both species occur in sympatry re- 
mains unclear. 




'PWPjwïïrj 










:fV 



FIG. 39. Shell morphology of 8, iravadica 
(Myanmar, Irawaddy; ZMB 49.617). 



Material Examined 

Myanmar: Irawaddy (BMNH 1899.21.6.76- 
79, ZMB 200.010), Irawaddy near Yenyang- 
young (ZMB 49.617, 200.005); Pegu (BMNH 
1871.9.23.49). Shan States (BMNH 
1888.12.4.1440). 

Differential Diganosis 

Relatively small, broadly conical, truncated, 
with two to four remaining whorls; two spiral 
bands of closely spaced nodules. 

Description 

Shell (Fig. 39): Relatively small, conical, trun- 
cated with two to four remaining whorls. Body 
whorl comparatively large compared to shell. 
Two spiral cords support rows of more or less 
developed nodules as well as some conspicu- 
ous spiral cords at base of shell. Aperture 
wide, produced below, columellar margin thin. 
Shell size: H = 18-33 mm, В = 9-19 mm. 

Operculum: Round, central nucleus, consid- 
erably smaller than aperture. 

Embryonic shell morphology, Radula, Soft 
body anatomy: Unknown. 

Remarks 

Can be distinguished from B. henriettae by 
its smaller and more conical shell and less 
pronounced sculpture. 

Brotia jullieni (Deshayes, 1874) 
(Figs. 34D, 40) 

Melania jullieni Deshayes, in Deshayes & 
Jullien, 1874: 115, pi. 7, figs. 7-9 ("Thio- 
Compih, Cambodge" = Thio Compih, Sâmbok 
at the Mekong, Cambodia, 12°34'N, 
106°01'E), lectotype and three paralectotypes 
MNHN (designated by Köhler & Glaubrecht, 
2002a) (Fig. 40A); types seen; Morlet, 1889: 
145. 

Melania julieni [sic] - Brot, 1875: 93, 94, pi. 
11, figs. 2, 2a. 

Taxonomy and Systematics 

Commonly treated as synonym of B. costula 
(e.g., Brandt, 1968, 1974; Davis, 1982), but 
herein considered distinct for its peculiar shell 
and radula. 



198 



KÖHLER & GLAUBRECHT 



Material Examined 

Laos: Muong-Bet sur le Song-Ma (MNHN); 
Mekong (MNHN). Cambodia: Mekong near 
Pakse (ZMH): Vietnam: Environs de Gang, 
Tonkin (MNHN): Song Ya near Yuong-Het, 
Tonkin (MNHN). 

Differential Diganosis 

Extraordinarily large and robust; aperture 
wide, basely produced; strong axial ribs, fine 
spiral lines. Radular teeth each with a very 
broad, rounded main denticle. 

Description 

Shell (Fig. 40); Large, broadly pyramidal, 
eroded tip; aperture wide, comprising about 



V4 of shell height; whorls rounded, suture 
thin: strong axial ribs and thin spiral ridges, 
at least at base of shell; colour yellowish to 
chestnut brown. Size: H = 55-65 mm, В = 
24-30 mm. 

Radula (Fig. 31 D): Lateral corners of rachid- 
ian conspicuously enlarged; very broad, 
spatula shaped main cusp flanked by two 
much smaller, pointed accessory denticles; 
glabella almost squarish, well rounded at its 
base, concave lateral edges. Lateral teeth 
with very broad main cusp flanked by two 
smaller denticles on each side, short lateral 
extensions. Inner and outer marginals with 
broadly rounded outer cusp and tiny pointed 
inner cusp. Inner marginals broader than 
outer ones. 

Embryonic shell morphology. Soft body 
anatomy: Unknown. 




"Wwr[wm 



Ч 



FIG. 40. Shell morphology of 8. jullieni. A: Lectotype of M. jullieni MHNH; B: 
Cambodia (ZMZ 522392); C: Laos, Pakse (ZMH). Scale = 10 mm. 



SYSTEMATIC REVISION OF BROTIA 



199 



4.0 














3.Ö 










3.6 








3.4 








3.2 














1 




3.0 








2.8 


























2.6 










^ 


2.4 
9 n 













34 
Б. с costula 



107 
S. с hercúlea 



6 
S. jullieni 



FIG. 41 . Comparison of S. costula and ß. jullieni 
by means of shell parameter H/LA. Box plot dia- 
gram showing median, the 25%- and 75%-per- 
centile and largest non-extremes (less than 1.5 
times of box height). 



Distribution 

Laos, Cambodia, Vietnam, perhaps also 
northeast Thailand; Mekong River system. 

Ecology 

Frequently infested by drilling sabellids 
{Caobangia spec, Nematoda). 

Remarks 

Only superficially similar with more elon- 
gated B. hercúlea (statistical analyses of shell 
parameters: Fig. 41). Main cusps of the radu- 
lar teeth and glabella of B. jullieni much 
broader, possessing only one accessory cusp 
instead of two in B. hercúlea. 

A report from the Ping River near Так, Thai- 
land, by Morlet (1891: "Riviere de Menam- 
Pinh, de Raheng à Xieng-Moi") refers to В. 
hercúlea. Brandt (1974) and Davis (1982), 
referring to B. costula, stated that this species 
is the only cerithioidean in the Mekong. 

Brotia kelantanensis (Preston, 1907) 
(Figs. 42, 43A) 

Melania kelantanensis Preston, 1907: 267, 
text-fig. ("Kelantan, Malay Peninsula"), types 
not seen. 



Taxonomy and Systematics 

Ignored by later authors, this species was 
reported only once by Davis (1982) mention- 
ing an unidentified, spiny species in the 
Pahang River system. Herein assigned to 
Brotia for it's characteristic morphology. 

Material Examined 

Malaysia: Pahang, Taman Negara National 
Park (ZMA). 

Differential Diganosis 

Shell comparatively small, broadly conical, 
no more than four whorls; prominent spiral cord 
at the centre of the whorls supporting spiral 
row of strong, pointed spines or nodules. 

Description 

Shell (Fig. 42): Medium sized, pyramidal, coni- 
cal, decollated, four remaining whorls; promi- 
nent spiral cord at centre of whorls supports 
spiral row of strong, pointed nodules, addi- 
tional, weak spiral ridge on upper sector. 
Colour chestnut brown. Aperture round, rela- 
tively small compared to body whorl, slightly 
produced below. Shell size: H = 31 mm, В = 
18 mm (n = 2). 

Operculum: Oval, four whorls, central nucleus. 

Radula (Fig. 43A): Ribbon 16 mm long, corre- 
sponding to about half of shell height, 100 
rows of teeth (n = 1). Rachidian with two 
conspicuously excavated upper corners, 
concave upper rim; main cusp flanked by two 
smaller, accessory denticles; glabella nar- 
row, well rounded below with concave lat- 
eral margins. Inner and outer marginals with 
two cusps, outer one broadly spatulate. 

Embryonic Shell: Unknown. 

Distribution 

Malaysia (Malay Peninsula): Federal State 
of Pahang; Pahang River system. 

Habitat 

On rocks in rapids (Davis, 1982: 392). 

Remarks 

Hardly to be mistaken for any other species. 
Occurs in sympatry with ß. episcopalis, which 
is more elongated and differs in average num- 
ber of whorls and sculpture. 



200 



KÖHLER & GLAUBRECHT 




'='" |WffjWTïïi| 
о 1 

FIG. 42. Shell morphology of S. kelantanensis (Malaysia, Pahang; ZMA). 



Brotia manningi Brandt, 1968 
(Figs. 43В, 44) 

Brotia (Brotia) manningi Brandt, 1 968: 272, pi. 
10, fig. 58 (Thailand: Huai Lan at Ban Dam 
Pon, Lorn Sak District, Phetchabun Prov- 
ince"), holotype SMF 197376, 22 paratypes 
MCZ 288652, 22 paratypes ZSM 1 9983239, 
20 paratypes RMNH 55289/20 (Fig. 44); 
types seen: Brandt, 1974: 179, 180, pi. 13, 
fig. 35. 

Brotia manningi- Köhler & Glaubrecht, 2002a: 
141, 

Taxonomy and Systematics 

In absence of additional information and 
material, we follow the statement of Brandt 
(1968, 1974). 



Differential Diganosis 

Shell elongate conic with flattened, slightly 
convex whorls: aperture produced: almost 
smooth, only with faint spiral lines and growth 
lines. 

Description 

Siiell (Fig. 44): Medium sized, spire conic with 
up to seven flattened whorls; suture narrow; 
smooth, faint growth lines; colour brown to 
olive, dark brown spiral band may be present. 
Aperture oval, well rounded to produced be- 
low. Size: H = 24-38 mm, В = 11-15 mm. 

Operculum: Oval, up to four fast in diameter 
increasing whorls, sub-central nucleus. 

Radula (Fig. 43B): Ribbon about 12 mm long 
with 80 rows of teeth (n = 1 ). Rachidian elon- 




FIG. 43. Radula morphology of B. I<elantanensis and B. manningi. A: B. kelantanensis (Malaysia, 
Pahang; ZMA); B: B. manningi, paratype ZSM 19983239). Scale bars = 100 |jm. 



SYSTEMATIC REVISION OF BROTIA 



201 




FIG. 44. Shell morphology of S. manningi.A-C: Paratypes ZSM 19983239. Scale = 10 mm. 



gate, anterior rim slightly concave, incon- 
spicuously excavated upper lateral corners; 
cutting edge with pronounced main denticle 
flanked by two, much smaller accessory 
denticles on each side; glabella narrow, 
rounded below, not reaching basal margin 
of rachidian. Laterals with very large main 
denticle. Inner and outer marginals relatively 
long, slender, broad outer cusp, smaller, 
spiny inner denticle. 
Embryonic Shell: Unknown. 

Distribution 



Luang NP, Prov. Phitsanulok), holotype SMF 
1 97378/1 , 1 paratypes SMF 205356/1 (Fig. 
45); types seen; Köhler & Glaubrecht, 2002a: 
141; Glaubrecht & Köhler, 2004: 289-291. 
Brotia (Brotia) microsculpta - Brandt, 1974: 
180, pi. 13, fig. 36. 

Taxonomy and Systematics 

Revised by Glaubrecht & Köhler (2004) based 
on morphological and molecular genetic data. 
Accordingly, B. microsculpta belongs to the 
Kaek River species flock in Central Thailand. 



Thailand: Central Thailand, Provinces of 
Nan, Loei, and Phetchabun (Brandt, 1974). 

Remarks 

Belongs to a group of taxa from central Thai- 
land with similar shells. To be distinguished 
from 8. insólita and ß. siamensis only by subtle 
morphological differences. Distinct status re- 
quires confirmation by examination of further 
material suitable for morphological and mo- 
lecular genetic studies. We were not able to 
trace material from the Kaek River, central 
Thailand, a locality reported by Brandt (1974). 

Brotia microsculpta Brandt, 1968 
(Figs. 45, 46A) 

Brotia microsculpta Brandt, 1968: 272, pi. 10, 
fig. 59 ("Thailand: Maenam Kaek, in Thung 
Salaeng Luang Botanical Garden, 80 km E 
of Pitsanulok" = Kaek River, Thung Salaeng 



Material Examined 

Thailand: Prov. Phitsanulok, Kaek River: 
Resort 53 km E Phitsanulok (ZMB 200.266); 
Poi Falls (ZMB 200.200); Sopha Falls, 71 km 
E of Phitsanulok (ZSM 19983240); Thung 
Salaeng Luang NP (ZMB 200.191). 

Differential Diganosis 

Shell small, conical to elongated, mostly 
three remaining, slightly rounded whorls; 
smooth sculpture. Aperture round, not pro- 
duced. Operculum round, not oval as other 
Kaek River species. Radula relatively short, 
closely spaced rows of teeth, marginal teeth 
prolonged. 

Description 

Shell (Fig. 45): Relatively small, conic to elon- 
gate conic, not thick but solid; truncated, 



202 



KÖHLER & GLAUBRECHT 




FIG. 45. Shell morphology of S. 
microsculpta. Holotype SMF 
197378/1. Scalen 10 mm. 



mostly three remaining, convex whorls; 
smooth, fine axial growth lines, faint spiral 
lines. Aperture almost round, relatively small 
compared to shell, basely rounded but not 
produced. Size: H = 1 0-25 mm. В = 8-1 5 mm. 

Operculum: Round to only slightly oval, 5-6 
regular whorls, central nucleus. 

Radula (Fig. 46A): Length of ribbon m = 11.8 
mm (sd = 1 .7 mm; n = 3), about 190 closely 
spaced rows of teeth. Radular teeth com- 
paratively small. Rachidian relatively broad, 
main cusp flanked by three accessory den- 
ticles on each side, glabella narrow, with 
straight lateral margin, cut basal rim, not 
reaching base of rachidian. Inner and outer 
marginals very long, narrow, curved, large, 
broad outer cusp, one to three tiny inner 
accessory denticles. 

Stomach: Typical, as in B. citrina (Fig. 4). 

Embryonic Shell: Morphology unknown. 

Habitat 

Buried into sandy substrata in quiet parts of 
the swift river. 

Distribution 

Thailand: Prov. Phitsanulok: Endemic to 
Kaek River and its northern tributary Huai 
Chieng Nam (Brandt, 1974). 

Remarks 

Recognizable by its smaller shell, round 
operculum, and typical radula. Brotia pseudo- 
sulcosplra is more conical, thicker, whorls 



more flattened. Only Kaek River species oc- 
curring on soft substrata. 

Brotia pagodula (Gould, 1847) 
(Figs. 46B, 47, 48) 

Melania pagodula Gould, 1847: 219 {non M. 
pagodulus Reeve, 1860) ("Thoungyin-River, 
tributary of the Salween River, Burma"), 
lectotype MCZ 169276 and paralectotype 
USNM 611238 (designated by Johnson, 
1964) (Fig. 47A); types seen; Brot, 1875: 
102, 103, pi. 13, fig. 2, Hanley & Theobald, 
1876: 61, pi. 153, fig. 3. 

/o pagodula - H. Adams & A. Adams, 1854: 
300; Reeve, 1859: pi. 3, fig. 10. 

Tiara (Acrostoma) pagodula - Preston, 1915: 
32. 

Brotia pagodula - Morrison, 1954: 382; 
Johnson, 1964: 121, pi. 44, fig. 2; Köhler & 
Glaubrecht, 2001: 292-295, ftgs. 1A, 9A-F; 
Köhler & Glaubrecht, 2002a: 142; Glaubrecht 
& Köhler, 2004: 283. 

Brotia (Brotia) pagodula - Brandt, 1974: 173, 
174, pl. 12, fig. 25. 

Taxonomy and Systematics 

Type species of Brotia. 

Material Examined 

Myanmar: (ZMB 26.708); Salween River, 
Tavoy (BMNH); Thailand: Prov. Kamphaeng 
Phet: Maenam Moei approximately 20 km E 
Mae Sot, 16°45.82'N, 98°45.14'E (ZMB 
200.205), Maenam Moei approximately 30 km 
S Mae Sot, boarder to Myanmar, 16°26.96'N, 
98°39.27'E (ZMB 200.208), Maenam Moei 
(USNM 776062), Maenam Moei, 8 km W of 
Mae Ramat (ZSM 19983241; ZMH; RMNH 
71319); soft bodies already removed from the 
shells, without location (ZMH). 

Differential Diganosis 

Conical shell sculptured by spiral row of con- 
spicuous spines; aperture wide, rhomboid, well 
produced below; radular teeth with very broad, 
enlarged main cusp; comparatively large ju- 
veniles in brood pouch. 

Description 

Shell (Fig. 47): Medium sized, spire broadly 
conical, decollated, up to five flattened whorls, 
narrow suture, spiral row of long, pointed 



SYSTEMATIC REVISION OF BROTIA 



203 




FIG. 46. Radular morphology of several Brotia species. A: B. microsculpta (Thailand, Kaek River- ZMB 
200.200); B: в. pagodula (Thailand, Moei River; ZMH); C: a paludiformis (Thailand, Kaek River SMF 
215963); D: B. peninsularis {Tháúand, Sural Thani; ZMB 200,242); E: B. praetermissa Paratype BMNH 
20010482/B; F: B. pseudosulcospira (Thailand, Kaek River; ZMH); G: B. solemiana (Thailand Ponq 
River; SMF 193585); H; B. subglohosa Paratype ZSM 19983219. Scale bars = 1 mm 



204 



KÖHLER & GLAUBRECHT 




FIG. 47. Shell morphology of ß. pagodula. A: Lectotype of M. pagodula (MCZ 169276; Thougyin); B: 
Thailand (ZMB 26.708); C: Thailand, Moei River (ZMB 200.205); D: Thailand, Moei River (ZMB 
200.208). Scale = 10 mm. 



spines: fine spiral lines at base of shell; light to 
chestnut brown colour, dark brown spiral band 
may be present. Aperture ovate with angular 
margin below, inside greyish white with brown 
bands. Size; H = 18-44 mm, В = 13-26 mm. 

Embryonic Shell (Fig. 48); Smooth; up to four 
rapidly increasing whorls, comparatively 
large compared to adult as well as to other 
species. 

Operculum: Round, 6 to 8 regularly increas- 
ing whorls; central nucleus; clearly smaller 
than aperture. 

Radula (Fig. 46B); 125 to 170 rows of teeth, 
length of up to 20 mm, corresponding to half 
of shell height. Rachidian with straight up- 
per rim, base convex by basally extending, 
broad glabella with more or less straight lat- 
eral margins and cut lower rim; very large 
main denticle flanked by two smaller den- 
ticles on each side. Laterals with large, 
broadly triangular main cusp flanked by two 
or three minute denticles on inner side and 
one or two at outer side. Inner and outer 
marginals broadly spatulate, with large main 
cusp and tiny inner denticle. 

Stomach: Stomach as in B. citrina (Fig. 4), 
except for typhlosoles fused at almost en- 
tire length of style sac. 



Reproductive System 

Females contain between 1 and 50 juveniles 
(n = 6) varying in height between 3.5 and 6 mm. 

Habitat 

Attached to rocks in sectors with swift current. 

Ecology 

Specimens collected during a field trip in 
2001 frequently infested with drilling sabellids 
{Caobangia spec, Nematoda). 

Distribution (Fig. 49) 

Myanmar, Thailand; Restricted to Salween 
and its tributary Thoungyin (= Maenam Moei), 
forming the border between Thailand and 
Myanmar. 

Remarks 

Can hardly be confused with any other spe- 
cies for its spiny shell. Spines of other species 
are considerably smaller (e.g., B. binodosa, 
B. costula, B. spinata). 



SYSTEMATIC REVISION OF BROTIA 



205 




FIG. 48. Embryonic shell morphology of B. pagodula. SEM images of 
embryonic shell removed from brood pouch (ZMH); apical and front 
view. Scale bar = 1 mm. 



90° 



100° 




FIG. 49. Distribution of в. peninsularis (close circles), ß. siamensis (open 
B. pagodula (close rectangle) and B. wykoffi (open rectangle). 



0° 

circle), 



206 



KÖHLER & GLAUBRECHT 



Brotia paludiformis (Solem, 1966) 
(Figs. 46C, 50) 

Paracrostoma paludiformis Solem, 1966: 17. 
pL 1 . figs. H-J, text-fig. 2 {non Semlsulcospira 
paludiformis Yen, 1939) ("Thailand, Provinz 
Phitsanulok: Kaek River at the Thung Salaeng 
Luang Falls"); types not seen. 

Paracrostoma paludiformis paludiformis - 
Brandt, 1974: 187, pl. 14, fig. 45. 

Paracrostoma paludiformis - Köhler & Glaub- 
recht, 2002a: 121-156. 

Brotia paludiformis - Glaubrecht & Köhler, 
2004: 291, 292. 

Taxonomy and Systematics 

For specimens from the Kaek River, the name 
"Paracrostoma paludiformis" was first em- 
ployed by Solem (1966) in reference to a pre- 
sumably pleurocerid species from Hainan 
described by Yen (1939). Although, Solem 
(1 966) erred in assuming that both taxa are con- 
specific, the name introduced by him is avail- 
able as the species epitheton has been used 
in context with a changed generic affiliation. 

This species belongs to the Kaek River spe- 
cies flock in Central Thailand and was revised 
and transferred to Brotia by Glaubrecht & 
Köhler (2004). 



Material Examined 

Thailand: Prov. Phitsanulok: Kaek River: 
Sopha Falls, 71 km E of Phitsanulok (ZMH; 
BMNH: SMF 215963). 

Differential Diganosis 

Shell conical, thick, very robust; two or three 
convexly rounded whorls; body whorl con- 
spicuously inflated; entirely smooth except for 
growth lines; aperture broadly oval. 

Description 

Shell (Fig. 50): Medium sized to large, broadly 
ovate, two or three well rounded, convex 
whorls; spire eroded; body whorl large, inflated; 
smooth sculpture consisting of faint growth 
lines, only rarely with spiral row of small, 
rounded nodules; colour chestnut brown; ap- 
erture wide, oval, well rounded below. Shell 
size: H = 24-30 mm, В = 18-22 mm. 

Operculum: Oval to slightly elongated, up to 
three whorls fast increasing in diameter, sub- 
central nucleus. 

Radula (Fig. 46C): Length of ribbon: m = 23.4 
mm (sd = 1.3 mm; n = 3), about 190 rows of 
teeth. Denticle morphology corresponding to 
B. armata. 




1 

FIG. 50. Shell morphology of B. paludiformis. A-B: Thailand, Kaek River, Sopha Falls (SMF 215963); 
C-D: Thailand, Kaek River, Sopha Falls (ZMH). Scale = 10 mm. 



SYSTEMATIC REVISION OF BROTIA 



207 



Embryonic shell morphology. Soft body 
anatomy: Unknown. 

Distribution 

Thailand: Prov. Phitsanulok: Endemic to Kaek 
River; exclusively known from Sopha waterfalls. 

Remarks 

Very distinct species in it's globular shape 
and inflated body whorl. Somewhat similar is 
B. pseudosulcospira, which differs most con- 
spicuously by its more flattened whorls. 

Brotia peninsularis (Brandt, 1974) 
(Figs. 46D, 51, 52) 

Brotia (Brotia) costula peninsularis Brandt, 
1974: 183, pi. 1, fig. 17 ("Thailand: Maenam 
Lampa, Province of Pattalung" = River Lampa, 
Prov. Phattalung), holotype SMF 220570, 17 
paratypes SMF 220571, six paratypes SMF 
220572, paratypes ZSM 19983232, paratypes 
ZMH; types seen. 

Taxonomy and Systematics 

Brandt (1974) mentioned a series of 50 
paratypes (Brandt collection 496). Thus, ad- 
ditional type material may exist that was not 
traced. This taxon has been described as a 
subspecies of в. costula. However, it is con- 
sidered here as distinct based on morphologi- 
cal and molecular genetic data. 



Material Examined 

Thailand: Prov. Surat Thani, Wiphawadi wa- 
terfalls, bridge at highway 401 to Nakhon Si 
Thammarat, 20 km off Surat Thani, 9°5.88'N, 
99°46.33'E (ZMB 200.041-2), Pum Pin near 
Takuha, km 63.5 (ZMH; ZSM 19983231); Prov. 
Phang Nga, Khiong Ipan, bridge at street 4035 
between Ao Luk and Phrasaeng (ZMB 200.043), 
Bok Ka Ra Ni falls near Phang Nga (ZMH; ZSM 
19983233); Prov. Krabi, street 4 at Ao Luk, 
8°91.44'N, 98°34.90'E (ZMB 200.046), creek 
between Krabi and Baling (ZSM 19982334), 
Klong Nga opposite Krabi (ZSM 19983230); 
Klong Sag, Ban Nai Sra (MCZ 288636; marked 
as paratypes); Yala, creek at new mine, NW Na 
Pupo (ZMH; ZSM 19983229). 

Differential Diganosis 

Shell rather small, thin but solid, conical; 
body whorl relatively large; regular spiral lines, 
rarely axial ribs. 

Description 

Shell (f\g. 51): Small, spire oval to conical tur- 
reted, moderately thick, up to eight flattened 
to rounded whorls, narrow suture; regular 
spiral ridges crossed by growth lines pre- 
dominant sculpture; rarely, small spiny nod- 
ules formed on spiral ridges; colour lightly 
brown to olive-brown. Aperture oval, well 
rounded below, pointed above. 




FIG. 51. Shell morphology of B. peninsularis. A-B: Paratypes ZMH; C: Paratype ZSM 19983232; D: 
Thailand, Surat Thani (ZMB 200.242). Scale = 10 mm. 



208 



KÖHLER & GLAUBRECHT 



Embryonic Shell (Fig. 52): Subsequent whorls 
smooth, sculptured only by growth lines. Av- 
erage proportions: H = 3.2 mm, В = 0.4 mm, 
HA = 0.18 mm, BA = 0.33 mm, DA = 0.65 mm 
(forn = 10). 

Operculum: Round to slightly oval, five to six 
whorls gradually increasing in diameter. 

Radula (Fig. 46D): Rachidian with slightly con- 
cave upper rim, glabella well developed, 
rounded below, concave lateral margins: 
main cusp flanked by three smaller denticles 
on each side. Lateral cusp formula 2-13. 
Inner and outer marginals with two cusps, 
the outer one being broader: inner marginal 
teeth generally broader than outer ones. 




Stomach: Typical (as in B. citrina: Fig. 4): ex- 
cept for both typhlosoles unfused at entire 
length of style sac. 

Reproductive System 

One female contained 23 juveniles (ZMB 
200.242). 

Habitat 

Rather small, swift streams on limestone; 
attached to rocks and boulders, sitting directly 
in the water current. 

Distribution (Fig. 49) 

Thailand, Malaysia: Malay Peninsula S of 
Isthmus of Kra (Thai provinces Chumphon, 
Surat Thani, Krabi, Phang Nga, and Nakhon 
Si Thammarat as well as province of Pahang, 
Malaysia: Brandt, 1974). 

Remarks 

Type specimens of B. siamensis are very 
similar but can be discriminated statistically 
significant by parameters N and H/B (Table 3, 
Fig. 53). 



2 6 1 




2.5. 








2.4. 
2 3 Í 






_. 


^H 


2.2- 




Í 




2.1- 




m 


2.0' 














1.9 J 


1 



32 
B. peninsularis 



18 
6. siamensis 



FIG. 52. Embryonic shell morphology 
of S. peninsularis. SEM images of em- 
bryonic shell removed from brood 
pouch (paratype ZMH): apical and 
front view. Scale bar = 0.3 mm. 



FIG. 53. Comparison of B. peninsularis and B. 
siamensis by means of shell parameter H/B. Box 
plot diagram showing median, the 25%- and 
75%-percentile and largest non-extremes (less 
than 1 .5 times of box height). 



SYSTEMATIC REVISION OF BROTIA 



209 



Brotia praetermissa Köhler & Glaubrecht, 2002 
(Figs. 46E, 54-56) 

Brotia praetermissa Köhler & Glaubrecht, 
2002b: 353-355 ("Borneo"), holotype BMNH 
20010482/A; three paratypes BMNH 
20010482/B (Fig. 54); types seen. 

Taxonomy and Systematics 

This species was described from material in 
the BMNH and is one of two Brotia species 
recorded from Borneo, even though the local- 
ity data is vague. 

Differential Diganosis 

Shell highly turreted, with stepped whorls, 
conspicuous spiral ridges, one or two spiral 
rows of spiny nodules; operculum round, rela- 
tively small; inner and outer marginal teeth with 
very broad, oval main tooth, only some outer 
marginals with accessory cusp at inner side. 

Description 

Shell (Fig. 54): Highly turreted, about eight 
stepped whorls, covered by thick calcareous 




W^f^ 



[WffjWFïïj 



FIG. 54. Shell morphology of S. praetermissa. 
Holotype BMNH 20010482/A. Scale bar = 10 mm. 



deposit; tip eroded; relatively deep suture; 
sculpture of six strong spiral ridges, most 
prominent at the base, one or two spiral rows 
of spiny nodules most prominent at second 
whorl; early whorls smooth or sculptured by 
inconspicuous axial ribs only. Colour hazel- 
nut brown to yellowish brown (probably 
leached due to conservation). Average shell 
dimensions: H = 58.2 mm, В = 22.3 mm. 

Embryonic Shell ('Fig. 55): Turreted, flattened 
whorls, smooth texture, faint spiral lines, 
regular growth lines; about 4 mm in height. 

Operculum: Round, up to 10 whorls, central 
nucleus; considerably smaller than aperture. 




FIG. 55. Embryonic shell mor- 
phology of B. praetermissa. SEM 
images of embryonic shell re- 
moved from brood pouch (para- 
type, BMNH 20010482/B); api- 
cal and front view. Scale bar = 
1 mm. 



210 



KÖHLER & GLAUBRECHT 



Radula (Fig. 46E): Ribbon about 20 mm long 
with 120 rows of teeth: rachidian with one 
main cusp flanked by two smaller denticles 
on each side that taper in size, glabella well 
developed with rounded basal margin; an- 
terior rim of rachidian slightly concave by 
slightly excavated lateral corners, basal rim 
rounded. Main cusp of laterals flanked by 
two accessory denticles on each side, gla- 
bella well developed comparatively long lat- 
eral extensions. Inner marginal tooth with 
one very broad, spatula-shaped cusp: some 
of outer marginals in addition possess ac- 
cessory cusp at inner side. Both, inner and 
outer marginals, curved or knee-shaped, 
outer ones with lateral flange at exterior side. 

Stomach (Fig. 56): Major and minor typhlosole 
unfused, gastric pad large, sorting area with 
two well developed crescent septate thick- 
enings. 

Reproductive System 

One female contained 18 shelled juveniles. 
Distribution 

Borneo (locality data vague). 

Remarks 

Somewhat similar is Jagora asperata from 
the Philippines, which can be distinguished by 
its different soft body, embryonic shell, and 
radular morphology (Köhler & Glaubrecht, 
2003). 



Brotia pseudoasperata Brandt, 1968 
(Figs. 57-59) 

Brotia (Brotia) pseudoasperata Brandt, 1968: 
270. 271 , pi. 10, fig. 57, text-fig, 39 ("Maenam 
San and its tributary Huai Kao Man", Prov. 
Loei. Thailand), holotype SMF 197375 ("Huai 
Kao Man, Phung Song, Loei"), 18 paratypes 
SMF 19381, 12 paratypes ZSM 19983244, 
nine paratypes ZSM 19983245, five para- 
types RMNH 5240/5, 14 paratypes BMNH 
1976072 (Fig. 57): types seen: Brandt, 1974: 
177, 178, pi. 13, fig. 31. 

Brotia pseudoasperata - Köhler & Glaubrecht, 
2002a: 144. 

Taxonomy and Systematics 

Brandt (1968) stated that shells from Annam 
(China) and Laos erroneously attributed to 
"Melania asperata" belong to his species. An- 
other lot of similar shells is known from Mt. Carin 
(Pegu, Myanmar: ZMB 47.129). However, it is 
still questionable whether all these references 
can really be attributed to this species. We 
rather suspect that B. pseudoasperata is re- 
stricted to the Heung River system. Species lim- 
its by means both of morphology and geo- 
graphical distribution remain dubious unless 
material suitable for morphological and molecu- 
lar genetic analyses will be available. 

Differential Diganosis 

Shell elongate turreted: closely spaced axial 
ribs that support one to three spiral rows of 





FIG. 56. Stomach morphology of 8. praetermissa, 
paratype BMNH 20G10482/B. 



FIG. 57. Shell morphology of 6. pseudoasperata. 
A-B: Paratypes ZSM 1 9983244. Scale bar = 10 mm. 



SYSTEMATIC REVISION OF BROTIA 



211 



spiny nodules; operculum round with up to 
eight whorls. 

Description 

Shell (Fig. 57): Medium sized, thin but solid, 
elongate turreted, tip eroded, up to seven 
convex whorls, narrow suture; thin, regularly 
spaced axial ribs that support one to three 
spiral rows of spiny nodules, the first approxi- 
mately at mid of whorls, the second, if 
present, at upper half of whorls; upper whorls 
may be smooth; spiral ridges at base. Colour 




FIG. 58. Embryonic shell morphology 
of B. pseudoasperata. SEM images of 
embryonic shell removed from dried 
shell (Thailand, Huai Kao Man, ZSM 
1 9983244); apical and front view. Scale 
bar = 1 mm. 



hazelnut brown, a dark brown spiral band 
may be present. Aperture broad, wide, well 
rounded, produced below. Size; H = 20-27 
mm, В = 8-12 mm. 

Embryonic Shell (Fig. 58); Ovate, smooth ex- 
cept for faint growth lines, comprising 2.0- 
2.5 whorls. Average proportions; H = 2 mm, 
В = 1 .6 mm, HA = 0.22 mm, BA = 0.33 mm, 
DA = 0.81 mm (for n = 3). 

Operculum: Round, up to eight gradually in- 
creasing whorls, central nucleus; clearly 
smaller than aperture. 

Radula (Fig. 59); Ribbon with 90 to 120 rows 
of teeth. Upper rim of rachidian concave by 
inflated lateral corners, lower rim almost 
straight; cutting edge with one main denticle 
flanked by two smaller ones; glabella nar- 
row, well rounded at base not exceeding 
lower rim of rachidian, v-shaped with con- 
cave lateral margins. Main cusp of laterals 
flanked by two accessory denticles on each 
side. Inner and outer marginal teeth with two 
cusps, outer one broad, rounded, inner one 
small, pointed. 

Reproductive System 

One female contained 19 shelled juveniles 
(ZSM 19983244). 

Distribution 

Thailand; With certainty known only from 
type locality (San River, affluent of Heung 
River, collecting area of the Mekong), and its 
tributary Huai Kao Man (Brandt, 1974). Re- 
ports from Laos, Vietnam, Myanmar should be 
treated with caution. 




FIG. 59. Radular morphology of B. pseudo- 
asperata, paratype ZMH. Scale bar = 100 |jm. 



212 



KÖHLER & GLAUBRECHT 



Brotia pseudosulcospira (Brandt, 1968) 
(Figs. 46 F. 60,61) 

Brotia (Paracrostoma) pseudosulcospira 
Brandt. 1968; 274, 275, pl.10, fig. 61, text- 
fig. 40 ("Maenam Kaek in Pitsanulok Prov., 
at Wang Nok Nang Aen. Wang Tong District, 
Thailand" = Thailand, Provinz Phitsanulok, 
Wang Tong District, Kaek River at Wang Nok 
Nang Aen), holotype SMF 197379; 23 
paratypes SMF 193586; five paratypes SMF 
194061; 11 paratypes BMNH 1976120; 12 
paratypes ZMH; 11 paratypes ZMH (alc); 
types seen. 

Paracrostoma pseudosulcospira pseudo- 
sulcospira -Bгanä{^974■. 185, pl. 13, fig. 42. 

Paracrostoma pseudosulcospira - Köhler & 
Glaubrecht, 2002a; 144. 

Brotia pseudosulcospira - Glaubrecht & 
Köhler, 2004; 292. 

Taxonomy and Systematics 

Brandt (1968) described a second subspe- 
cies, P. p. armata, which is considered distinct. 
A systematic revision based on morphologi- 
cal and molecular genetic data was presented 
by Glaubrecht & Köhler (2004). 



Material Examined 

Thailand; Prov. Phitsanulok, Kaek River; 
Sakunothayan Falls, 33 km E of Phitsanulok 
(ZMB 200.196, 200.299). 

Differential Diganosis 

Shell conical, up to three flattened whorls, 
rather smooth with growth lines, occasionally 
spiral cords at the base. Aperture widely ovate 
well rounded. 

Description 

Shell (Fig. 60); Medium sized, conical, robust, 
frequently with eroded spire, only two re- 
maining, flattened whorls; smooth sculpture 
except for growth lines, occasionally more 
or less developed, regularly spaced spiral 
cords, but not at base of shell. Aperture 
widely ovate well rounded, slightly produced 
below. Size; H = 26-40 mm, В = 18-24 mm. 

Embryonic Shell (Fig. 61); Smooth, with faint 
growth lines only; size of 2.0-2.5 mm, 2.5 
whorls. 

Operculum: Oval, up to four whorls fast in- 
creasing in diameter, sub-central nucleus. 




Illllllll 



FIG. 60. Shell morphology of B. pseudosulcospira. A: Paratype SMF 193586; B~C; Paratypes ZMH; 
D-E; Paratypes ZMH (ale); F; Paratype SMF 194061; G-H: Kaek River, Sakunothayan Falls (ZMB 
200.299). Scale bar = 10 mm. 



SYSTEMATIC REVISION OF BROTIA 



213 



Radula (Fig. 46F): Length of ribbon: m = 25 mm 
(sd = 2.5 mm; n = 3), up to 180 rows of teeth. 
Central tooth comparatively broad, glabella 
very narrow; otherwise similar to B. armata. 

Distribution 

Thailand: Prov. Phitsanulok: Endemic to 
Kaek River, restricted to its westernmost por- 
tion (Wang Nok Nang Aen, E of Wang Tong 
and Sakunothayan Falls close by). 

Remarks 

The shell of B. pseudosulcospira is very 
characteristic. Brotia paludiformis, also being 
smooth, exhibits convexly rounded whorls and 
an inflated body whorl. It latter lacks spiral lirae 




FIG. 61. Embryonic shell morphology of 
S. pseudosulcospira. SEM images of 
embryonic shell removed from dried shell 
(paratype ZMH); apical and front view. 
Scale bar = 1 mm. 



as observed at least in some specimens of B. 
pseudosulcospira. Brotia armata has spiny 
nodules. 

Brotia siamensis (Brot, 1886) 
(Fig. 62) 

Melania siamensis Brot, 1886: 90, 91, pi. 7, 
figs. 3-3b ("Raheng, Siam" = Так, Prov. Так, 
Thailand), lectotype and 18 paralectotypes 
MHNG, coll. Brot (designated by Köhler & 
Glaubrecht, 2002a) (Fig. 62); types seen. 

Brotia siamensis - Köhler & Glaubrecht, 
2002a: 147, fig. 3H. 

Taxonomy and Systematics 

Treated in various ways by previous authors, 
this taxon was considered conspecific with M. 
tiamonvillei Brot, 1887, by Bavay & Dautzen- 
berg (1910) for the similar shell. This assump- 
tion was also followed by Köhler & Glaubrecht 
(2002a). In fact, both taxon names are used 
interchangeably for material in various museum 
collections (own observations). However, in 
spite of their conchological similarity, the taxa 
are not conspecific as is revealed by a differ- 
ent embryonic shell morphology (unpubl. data). 
Melania hamonvillei possesses a protoconch 
typical for species of Adamietta and certainly 
is not member of Brotia. 

Melania siamensis was further been stated 
to be identical with M.jullieni by Morlet (1891) 
and B. costula by Brandt (1968, 1971). Also 
Köhler & Glaubrecht (2002a) noticed that 
some type specimens of M. siamensis are 
similar to 8. costula, whereas some others are 
not (Fig. 57). However, this superficial simi- 
larity is no reason to assume that both taxa 
are conspecific, since their distributional ar- 
eas are separated by a considerable geo- 
graphic distance. Re-examination of Brandt's 
voucher material reveals that the author was 
also not sure how to distinguish between B. 
siamensis and 8. peninsularis. The latter taxon 
was treated by him as a subspecies of 8. 
costula. Some lots of this species were labelled 
by him with 8. siamensis, however. Both taxa 
are indeed similar. 8. peninsularis as consid- 
ered here is restricted to the Malay Peninsula 
south of the Isthmus of Kra. The only confirmed 
record of 6. siamensis is the type locality. Так, 
about 700 km N of this isthmus. A reliable de- 
cision on the relationships of 8. siamensis and 
6. peninsularis awaits the examination of well- 
preserved material from the area of Так. For 



214 



KÖHLER & GLAUBRECHT 



TABLE 3. Result of disriminant analysis of shell 
parameters of B. peninsularis and B. siamensis. 



Predicted group membership 
B. peninsularis B. siamensis 



B. peninsularis 
B. siamensis 



29 (93.5%) 
(0%) 



2 (6.5%) 
18(100%) 



the time being, we consider both as distinct 
species, because they can be discriminated 
by statistical analyses of shell parameters with 
significance (Table 3). 

Differential Diganosis 

Shell variable, rather small, elongate tur- 
reted: apex frequently truncated: regularly 
spaced spiral ridges, sometimes axial ribs, 
mostly only on upper whorls; greenish to olive 
brown or dark brown to almost black, brown 
spiral band may be visible. 

Description 

Shell (Fig. 62): Medium sized, conical to elon- 
gate turreted, up to six convex whorls, apex 



frequently truncated: regularly spaced spiral 
ridges, most prominent at the base, axial ribs, 
mostly on upper whorls, may be lacking. 
Colour greenish to olive brown or dark brown 
to almost black: dark brown spiral band may 
be visible. Size: H = 2639 mm, В = 11-16 
mm. 
Embryonic Shell. Radula. Operculum, Soft 
Body: Unknown. 

Remarks 

Similar ß. peninsularis tends to have larger 
body whorl compared to the shell height, whorls 
more rounded in diameter. Brotiajullieni has a 
much larger shell, larger body whorl, wider 
aperture, protracted basal lip. Brotia costula is 
larger, not truncated, pyramidal turreted, more 
elongated in shape, different sculpture. "Mela- 
nia hamonvilleí' has distinct embryonic shell 
structure, resembling, for example, B. testu- 
dinaria (Köhler & Glaubrecht, 2001). 

Distribution (Fig. 49) 

Thailand: Type locality only known reference: 
Так (Prov. Так, north-central Thailand) at 
banks of Ping River. 








[WPPjWFm 



FIG. 62. Shell morphology of 8. siamensis. A: Lectotype MHNG (front and rear); B-P: Paralectotypes 
MHNG. Scale bar = 10 mm. 



SYSTEMATIC REVISION OF BROTIA 



215 



Brotia solemiana (Brandt, 1968) 
(Figs. 46G, 63) 

Brotia (Paracrostoma) solemiana Brandt, 
1968: 273, pi. 10, fig. 60 ("Maenam Pong at 
Ban Pa Nok Kao, Loei Prov." = Thailand, 
Prov. Loei, Pong River bei Ban Nok Kao), 
holotype SMF 197377, seven paratypes 
SMF 193583, six paratypes SMF 193585, 
two paratypes RMNH 55233/2 (Fig. 63); 
types seen. 

Paracrostoma solemiana - Brandt 1974: 186, 
pi. 1 3, fig. 44; Köhler & Glaubrecht, 2002a: 147. 

Brotia solemiana - Glaubrecht & Köhler, 2004: 
292, 293. 

Taxonomy and Systematics 

Brandt (1968, 1974) stated a slender shell, 
flattened whorls, an elongated aperture to be 
characteristic for this species. Furthermore, he 
assumed that it is endemic to the Pong River, 



between the provinces of Loei and Kon Kaen, 
central to western Thailand. Glaubrecht & 
Köhler (2004) attributed specimens also from 
the Kaek drainage to this species mainly due 
to a corresponding shell morphology. The de- 
scription of soft body features is mainly based 
on these specimens. 

Material Examined 

Thailand: Prov. Loei, Loei River: Tat Kok Falls 
at the road 2216 near Wang Saphung (ZMB 
200.174); Prov. Phitsanulok, upper course of 
the Kaek River at Sri Dit Falls (ZMB 200.203). 

Differential Diganosis 

Shell conical, two or three flattened whorls, 
smooth sculpture except for growth lines and 
occasionally fine spiral ridges, spiral lirae lack 
at base of shell; aperture widely ovate, acute 
or produced below. 




FIG. 63. Shell morphology of ß. solemiana. A-B: Paratypes 
SMF 193585; C: Prov. Loei, Tat Kok Falls (ZMB 200.174); D: 
Kaek River, Sri Dit falls (ZMB 200.203). Scale bar = 10 mm. 



216 



KÖHLER & GLAUBRECHT 



Description 

SA7e//(Fig. 63): Medium sized, conical, robust, 
with two or three flattened whorls, tip eroded; 
smooth sculpture except for growth lines, in 
some specimens inconspicuous spiral 
ridges, spiral lirae lack at base of shell; ap- 
erture widely ovate, acute or produced be- 
low. Colour yellowish to greenish brown. 
Size; H = 26-40 mm, В = 18-24 mm. 

Operculum: Oval, up to four whorls, sub-cen- 
tral nucleus. 

Radula (Fig. 46G); Length of the ribbon; m = 
1 6.0 mm (sd = 3.4 mm; n = 4), 1 50-1 60 rows 
of teeth. Rachidian relatively narrow, other- 
wise widely corresponding to B. armata. 

Stomach: Typical, as described for B. citrina 
(Fig. 4). 

Embryonic Sliell: Unknown. 

Distribution 

Thailand; Loei Prov.; Pong River, Prov. 
Phitsanulok; Kaek River at Sri Dit Falls in west- 
ern most headwater. 

Remarks 

Brotia pseudosulcospira with more flattened 
whorls, more conical shell; B. subglorlosa 
generally larger, more turreted; B. microsculpta 
with smaller body whorl, rounded aperture, 
circular operculum. Radula of ß. solemlana 
shorter as in other Kaek River species. 

Brotia subglorlosa (Brandt, 1968) 
(Figs. 46H, 64, 65) 

Brotia binodosa subglorlosa Brandt, 1968; 269, 
pi. 10, fig. 56, text-fig. 38 ("Thailand; Huai 
Chieng Nam, tributary of the Kaek River, about 
92 km E of Pitsanulok at the bridge of the 
Friendship Highway"), holotype SMF 19737, 
20 paratypes SMF 193572, paratype ZSM 
19983213, six paratypes ZSM 19983219, 11 
paratypes ZMH (Fig. 64); types seen. 

Brotia {Brotia) binodosa subglorlosa - Brandt, 
1974; 175. 176, pi. 13, fig. 28. 

Brotia splnata subglorlosa - Köhler & Glaub- 
recht 2002a; 129. 

Brotia subglorlosa - Glaubrecht & Köhler, 
2004; 293. 

Taxonomy and Systematics 

Described as a subspecies of 8. binodosa, it 
was stated that both taxa are connected by in- 



termediate morphs (Brandt, 1968). Such inter- 
mediates were not found by us among the 
voucher material examined; their existence is 
thus contended herein. According to Brandt 
(1968, 1974), ß. subglorlosa and ß. binodosa 
occur sympatrically in parts of the Kaek River, 
which conflicts a relation as geographical sub- 
species. For this reason, B. subglorlosa is con- 
sidered as distinct species, perhaps closely 
related to B. binodosa. According to Glaubrecht 
& Köhler (2004) this species likely is member 
of the Kaek River species flock. 

Differential Diganosis 

Shell elongate turreted, entirely smooth, 
aperture elongate produced and relatively 
narrow. 

Description 

Shell {F\g. 64); Medium sized, solid, elongate 
turreted; up to five convex, rounded whorls, 
truncated tip; smooth except for thin growth 
lines. Colour olive-brown, often covered with 
dark mineral deposits. Basal whorl relatively 
large. Aperture wide, elongate, produced 
below. Size; H = 25-45 mm, В = 16-24 mm. 

Embryonic Shell (Fig. 65); Conical, up to 3.5 
whorls; smooth sculpture with faint growth 
lines. 




FIG. 64. Shell morphology of B. 
subglorlosa. Paratype ZSM 19983213. 
Scale bar = 10 mm. 



SYSTEMATIC REVISION OF BROTIA 



217 



Operculum: Oval, up to five whorls gradually 
increasing in diameter, nearly central nu- 
cleus. 

Radula (Fig. 46H): Length of ribbon: 18 mm 
(n = 1), 220 rows of teeth. Central tooth com- 
paratively broad, glabella very narrow; other- 
wise corresponding to the radula of 8. armata. 

Reproductive System 

Two dried shells (ZSM 1 998321 9) contained 
130 and 156 shelled juveniles, respectively 
that varied in height between 0.5 and 1 .5 mm. 




FIG. 65. Embryonic shell morphology of B. 
subgloriosa. SEM images of embryonic shell 
removed from dried shell (paratype ZSM 
1 998321 3); apical and front view. Scale bars = 
0.1 mm (above), and 1 mm (below). 



Distribution 

Thailand: Endemic to Kaek River, between 
65 km (at Sopha Falls) and 92 km E of 
Phitsanulok, and tributary Huai Chieng Nam 
(Brandt, 1968:270). 

Remarks 

Superficially similar to other Thai species 
with smooth shells. Brotia microsculpta much 
smaller, with comparatively smaller, rounded 
aperture and operculum; B. pseudosulcospira 
more conical in shape with flattened whorls; 
B. solemiana more compact with compara- 
tively broader but shorter shell. 

Brotia sumatrensis (Brot, 1875) 
(Figs. 66, 67B-E) 

Melania (Melanoides) sumatrensis Brot, 1875: 
87, pi. 1 0, fig. 2b, pi. 1 3, figs, la, b ("Sumatra: 
Palembang"), three syntypes MHNG, Brot 
collection, one syntype MCZ 112689 (Figs. 
66A-C); types seen. 

Melania sumatrensis - Schepman, 1886: 13. 

Melania boeana Brot, 1881: 154, 155, pi. 6, 
fig. 1 ("Boea, Sumatra" = Bua, Sumatra), lec- 
totype and four paralectotypes MHNG, Brot 
collection (designated by Köhler & 
Glaubrecht, 2002a) (Figs. 66F-J); types 
seen. 

Melania (Brotia) episcopalis- Martens, 1900: 
10. 

Melania (Melanoides) palembangensis 
Strubell, 1897: 12 ("Südsumatra" = South 
Sumatra); types not seen. 

Brotia costula - Benthem Jutting, 1956: 374- 
378, fig. 76 [partim]; Benthem Jutting, 1959: 
92-95 [partim]; Brandt, 1974: 175, pi. 13, 
figs. 37-39 [partim]; Köhler & Glaubrecht, 
2001: 296-299, figs. ID, 10A-C, G, H 
[partim] (non M. costula Rafinesque, 1833). 

Brotia (Antimelania) costula - Subba Rao, 
1989: 108, 109 [partim] (non M. costula 
Rafinesque, 1833). 

Brotia variabilis -Rensch, 1934: 239 [partim]; 
Bequaert, 1943: 433, 434, pi. 33, figs. 11- 
16 [partim]; Solem, 1966: 15 (non M. 
variabilis Benson, 1836). 

Taxonomy and Systematics 

Brotia sumatrensis has been subsumed un- 
der B. costula by most previous authors (see 
also under that species), but molecular genetic 



218 



KÖHLER & GLAUBRECHT 



data Shows that the Sumatran species is dis- 
tinct. Problems of earlier authors to satisfacto- 
rily diagnose this species persist to the present 
due to lack of well-preserved soft body mate- 
rial. Shells examined from various museum col- 
lections are remarkably plastic, which may 
indicate the existence of yet undiscovered, 
morphologically similar species on Sumatra. 
This renders a correct characterisation and de- 
lineation of S. sumatrensis problematic and pro- 
visional. For the time being, we assign similar 
shells to B. sumatrensis, as representing the 



oldest available name. Future studies may re- 
veal a higher diversity of similar Brotia species 
on Sumatra. Brot (1875) struggled with the di- 
agnosis of B. sumatrensis and was unsure 
whether this species should instead be consid- 
ered a synonym of M. infracostata from Java. 
Schepman (1886: 13, 14, pl.1, figs. 3a, b, 4a, 
b) described and depicted a new var. mitescens 
for material with smooth shells, using a manu- 
script name of Martens. This variety is consid- 
ered a synonym of M. torquata for it's rather 
round, small operculum and fragile shell. Mela- 




FIG. 66. Shell morphology of B. sumatrensis. A-C: Syntypes of M. sumatrensis MHNG; D-E: Sumatra 
(MHNG, coll. Brot); F: Lectotype of /W. boeana MHNG, front and rear view; G-J; Paralectotypes 
MHNG; K; Sumatra, Lake Ranau (MZB); L; Sumatra, Jambi (MZB 9013); M; Sumatra, Lake Toba, 
Parapat(ZMB 200.119). 



SYSTEMATIC REVISION OF BROTIA 



219 



nia boeana Brot, 1881, is considered a syn- 
onym of B. sumatrensis, because we are not 
able to establish a significant distinction. The 
original series in the MHNG comprises in total 
seven specimens. Two of them originally are 
assigned to a var. b and, thus, not qualified as 
types (ICZN, Art. 72.4.1.). The type locality 
"Bua" is a common local village name that oc- 
curs several times in Sumatra. Consequently, 
the type locality of this taxon cannot be speci- 
fied more accurately. 

Material Examined 

Indonesia: Sumatra (ZMB 200.045; BMNH 
1890.2.21.1-4): Tandjungdjatti(ZMA);Suengei 
Ketil, Kampung (ZMA); Sungei Mentjirin near 
Kampung (ZMA); Kepahiang (ZMB 26.715 
200.039; MHNG); Bengkajang (ZMB 200.040) 
Sungei Kalau (ZMA); Sungei Minahol (ZMA) 
Tibitinggi (ZMB 26.717, ZMB 27.680) 
Demarguri (ZMB 35.819). Prov. Aceh (ZMB 
76671 ; ZMA; MZB 8786): Tributary of Alas River, 
Ketombe, SE Aceh (MZB 8624); LakeTakengon 
(ZMB 76.673, 200.136). Prov. Sumatera Utara: 
Trans-Sumatra highway, bridge 150 km N 
Bukittingi, r28.28'N, 99°19.41'E (ZMB 
200.116); Lake Toba, harbour of Parapat, 
2°49.17'N, 98°56.22'E (ZMB 200.119); Trans- 
Sumatra highway, r40.04'N, 99°10.05'E (ZMB 
200.120); Sungei Belawan (ZMB 51.776); 
Sungei Kopas, Kisaran, east coast (ZMA); 
Tandjung Langkat (ZMA); Laut Tawar, N 
Sibangun (ZMA; ZMB 87.409, 200.124); Bukit 
Lawang, at the Wisma Cottage (ZMB 200.125); 
Bukit Lawang (MZB 7058); Bohorok river (MZB; 
ZMA); Berastagi, Mt. Sinabung, Gunung Leuser 
NP (ZMB 200124); Medan (NMB); Sungei 
Rambai near Langkat (ZMA), Sungei Deli near 
Medan (ZMA). Prov. Sumatera Barat: small 
stream at Pajakumbuh, N Bukittingi, 0°27.3rS, 
1 00°36.2'E (ZMB 200. 1 22); Danau di Atas (ZMB 
200.069, 200.154; RMNH; ZMA); Sumpur 
(MZB); Ambulutu (MZB 4361); river in 
Pajakumbuh, N Bukittingi, 0°27.31'S, 
100°36.2'E (ZMB 200.122); Pajakumbuh 
(RMNH); Lake Manindjau (MZB 8632); Lake 
Singkarah (ZMA). Prov. Riau: Arau River (MZB 
9009); Kampar River, Pulau Jadang (MZB 
9010). Prov. Jambi (NMB): Lake Kerinci (RMNH; 
MZB 4901, 9022); Sungei Merangiu, Gunung 
Raya (MZB 9013). Prov. Sumatera Selatan, 
Pagaralam (ZMA), Palembang (RMNH; NMB); 
Lake Ranau (ZMB 76.288-9; MZB); Sungei 
Lepan, Langkat (ZMA); Simpang (ZMB 76.296); 
Sumani (ZMB 200.070); Sungei Musi, Muara 
Klingi (ZMB 76.295), Air Putih, Tjurup (ZMB 
76.298). Prov. Lampung (MZB 7028). 



Differential Diganosis 

Shell elongate turreted, thin but solid, slen- 
der, up to nine whorls; sculpture variable, from 
smooth to ribbed; no marked transition from 
smooth to ribbed whorls. 

Description 

Shell (Fig. 66): Relatively large, elongate tur- 
reted, slender in shape, up to nine whorls, 
rather thin. Sculpture variable; whorls either 
smooth or with axial ribs; no transition from 
smooth upper to ribbed lower whorls. One 
colour, chestnut brown. Shell size: H = 24- 
75 mm, В = 10-27 mm. 

Operculum: Oval, four to six whorls, central 
nucleus. 

Radula (Figs. 67B-E): Up to 200 rows of teeth; 
ribbon length up to 30 mm, corresponding 
to more than half of shell height. Upper mar- 
gin of rachidian concave by two inflated, well 
rounded corners; lower corners slightly 
angled; glabella slightly v-shaped, narrow, 
well rounded at base, its lateral margins con- 
cave. Cutting edge of rachidian with single 
main cusp and two or three smaller denticles 
on each side of it; some specimens with 
single flanking denticle. Laterals with short 
lateral extensions, pronounced inner flange, 
two main cusps flanked by two smaller den- 
ticles. Inner and outer marginals with two 
cusps, pointed, of about same size and 
shape. 

Stomach: Corresponds to B. episcopalis (Fig. 
26). 

Embryonic Shell: Unknown. 

Distribution (Fig. 68) 

Indonesia: Sumatra. 

Habitat 

From fast running, clear forest streams with 
sandy or stony bottom to muddy irrigation 
channels in rice fields; even in lakes with pol- 
luted waters (e.g., harbour of Parapat, Lake 
Toba). 

Remarks 

Brotia costula tends to be larger and more 
elongate, axial ribs are regular; ß. episcopalis 
differs mainly by a marked transition from 
smooth upper whorls to strongly sculptured 
lower whorls, with lesser pronounced axial 
ribs. 



220 



KÖHLER & GLAUBRECHT 




FIG. 67. Radular morphology of S. episcopalis and в. sumatrensis. А: В. episcopalis, Thailand, Nakhon 
Si Thammarat (ZMH); В: В. sumatrensis (Sumatra, Lampung; MZB 7028); C: South Sumatra (ZMB 
200.116); D: Sumara, Lake Toba (ZMB 200.119); E: Trans-Sumatra highway (ZMB 200.120). 




10° 



FIG. 68. Distribution of 8. sumatrensis (close circles) and B. episcopalis 
(open circles). 



SYSTEMATIC REVISION OF BROTIA 



221 



Brotia torquata (Busch, 1842) 
(Figs. 69, 70, 71A) 

Melania torquata Busch, 1842 - In: Philippi, 
1842:3, pi. 1,fig. 18 ("Java"), lectotype ÜMB 
TK 291/1 (designated by Knipper, 1958) (Fig. 
69A); type seen; Mousson, 1849: 70; Brot, 
1870: 281; Brot, 1875: 110, 111, pi. 14, fig. 
5, 5a [partim]. 

Melanoides torquata - H. Adams & A. Adams, 
1854:297. 

Brotia torquata - Köhler & Glaubrecht, 2002a: 
150. 

Melania zollingeri Brot, 1868: 42, pi. 2, fig. 4 
("Java"), holotype MHNG, coll. Brot (Fig. 69 
B); type seen; Brot, 1875: 111, pi. 14, fig. 6; 



Schepman, 1886: 14; Leschke, 1914: 252; 
Degner, 1928: 374; Benthem Jutting, 1959: 
93. 

Brotia zollingeri- Köhler & Glaubrecht, 2002a: 
152, fig. 30. 

Melania subplicata Schepman, 1886: 14, pi. 
1, fig. 6 ("Bedar Alam" = Sumatra, SW part 
of Riau, Bedar Alam, 0°45'S, 102°15'E), lec- 
totype ZMA and four paralectotypes RMNH 
71330 (designated by Köhler & Glaubrecht, 
2002a) (Figs. 69E, F); types seen; Martens, 
1897: 37, pi. 2, fig. 15, pi. 4, fig. 26; Bullen, 
1906: 15; Leschke, 1914: 218, 252; Degner, 
1928: 374; Benthem Jutting, 1959: 93. 

Melania sumatrensis var. mitescens Schep- 
man, 1886: 13, 14 ("Soepajang en nabij 




FIG. 69. Shell morphology of S. torquata. A: Lectotype of M. torquata ÜMB TK 291/1 ; B: Holotype of 
M. zollingeri MHNG; C: Lectotype of M. cun/icosta ZMA; D: Paralectotype of M. curvicosta ZMA; E: 
Lectotype of M. subplicata ZMA; F: Paralectotype of M. subplicata ZMA; G-H: West Sumatra, Fort 
Kok (RMNH 71331). Scale bar = 10 mm. 



222 



KÖHLER & GLAUBRECHT 



Alahan pandjang" = Supajang and Alahan 
pandjang, nearby); types not seen. 

Melania curvicosta Martens. 1897: 36. pi. 2, 
fig. 14, pL 4, fig. 27 ("See von Manindjau, 
Sumatra = Laker Manindjau, Sumatra), lec- 
totype, paralectotype ZMA, 12 paralecto- 
types ZMA (ale), three paralectotypes ZMB 
54.364 (designated by Köhler & Glaubrecht, 
2002a) (Figs. 69C, D): types seen; Bullen, 
1906: 15; Degner, 1928: 374. 

Melania curvicosta var. prestoniana Bullen. 
1 906: 1 5, pi. 2. fig. 8; types not seen; Degner, 
1928: 374. 

Brotia costula - Knipper, 1958 [partim]. 

Taxonomy and Systematics 

Busch (1842) stated type locality to be Java. 
However, the type is not accompanied by an 
original label. A newer label states 'Bengal", 
which is not believed to represent the type lo- 
cality. Likely due to this confusion, Brot (1875) 
stated that M. torquata is conspecific with M. 
terebra Benson, 1836, from Bengal. This is 
rejected herein, since the latter is a thiarid, 
neither sympatric with nor even similar to B. 
torquata. Studies of shell series show that sev- 
eral described taxa fall into a joint morphospace 
and that ribbed and smooth specimens occur 
syntopically. connected by intermediates. For 
this reason, these taxa are synonymized 
herein. Benthem Jutting (1956, 1959), Knipper 
(1958), and Brandt (1 974) treated M. torquata, 
M. zollingeri, M. subplicata, and /W. cun/icosta 
as synonyms of B. costula. However, 8. 
torquata can be distinguished from the latter 
by its different morphology: additional support 
is gained from molecular genetics. Rensch 
(1934: 233) affiliated "M. zollingeri" with 
"Tiaropsis". Re-examination of his voucher 
material in the ZMB shows that Rensch dealt 
with a thiarid, likely Melania subcancellata 
Boettger, 1890. Consequently, his systematic 
conclusions are obsolete. 

Material Examined 

Indonesia: Sumatra (ZMB 200.156). Prov. 
Sumatera Barat: Rao at the Trans-Sumatra 
highway, 0^26. 7N, 100°2.4'E (ZMB 200.121); 
Lake Manindjau (ZMB 54.360. 200.123, 
200.147-50, ZMB 200.053; ZMA); Lake 
Manindjau near Banjur (ZMB 200.131); Lake 
Manindjau at Manindjau, 0°19'S, 100^^22'E 
(ZMB 200.117); Fort Kok (RMNH 71331). 



Differential Diganosis 

Mostly small, delicate to thin, smooth or 
sculptured by convex, closely spaced axial 
ribs; operculum round; embryonic shells with 
more or less developed axial ribs from sec- 
ond whorl on; cutting edge of inner marginal 
teeth with two accessory cusps at inner side. 

Description 

Shell (Fig. 69): Small, thin, often even fragile, 
highly turreted, spire mostly eroded; three 
to five whorls; strong, closely spaced axial 
ribs, spiral striae at base, or entirely smooth. 




FIG. 70. Embryonic shell mor- 
phology of B. torquata. SEM im- 
ages of embryonic shell removed 
from brood pouch (Sumatra, Lake 
Manindjau; ZMB 200.117); apical 
and front view. Scale bar = 1 mm. 



SYSTEMATIC REVISION OF BROTIA 



223 




FIG. 71 . Radular morphology of B. torquata and B. verbecki. A: 8. torquata (Sumatra, Lake Manindjau; 
ZMB 200.117); B: 8. verbecki (Sumatra, Lake Singkarah; ZMB 200.118). Scale bars = 10 pm. 



When present, axial ribs curved across en- 
tire whorl from one suture to the other. Colour 
chestnut to dark brown. Aperture wide, 
basely rounded, pointed above. Size: H = 
25-48 mm, В = 6-23 mm. 

Embryonic Shell (Fig. 70): Conical turreted, 
three to four whorls; fine vertical growth lines, 
in some specimens distant axial ribs from the 
second whorl on. Average proportions: H = 
2.4 mm, В = 1.7 mm, HA = 0.22 mm, BA = 
0.34 mm, DA = 0.68 mm (for n = 9). 

Operculum: Round, with about six regularly 
increasing whorls, central nucleus, flat, 
clearly smaller than aperture. 

External Morphology: Animal small, up to four 
whorls; one colour dark grey to black; egg 
transfer groove beneath right tentacle incon- 
spicuous and short; mantle cavity short oc- 
cupying about 2/3 of first whorl; osphradium 
relatively short corresponding to V3 to 1/2 of 
length of ctenidium. 

Radula (Fig. 71 A): Ribbon with about 100 rows 
of teeth. Upper margin of rachidian concave 
by two inflated, rounded corners; lower cor- 
ners of basal plate rounded; glabella well 
rounded at base, its lateral margins concave; 
cutting edge of rachidian with single main 
cusp flanked by two accessory cups on each 
side that taper in size. Laterals with main 
cusp flanked by two inner and three outer 
denticles. Outer marginals with two pointed 
denticles, almost equal; inner marginals with 
pointed outer cusp and one or two inner ac- 
cessory denticles. 

Stomach: Typical, as in B. citrina (Fig. 4); ex- 
cept for unfused typhlosoles at entire length 
of style sac. 



Distribution 

Indonesia: Java, West-Sumatra. The only 
known reports from Java refer to types of M. 
torquata and M. zollingeri. Either these reports 
are in error or the species have become ex- 
tinct or extremely rare. All other material from 
West-Sumatra. 

Remarks 

Somewhat similar to B. verbecki when con- 
sidering similar shape and size of shell. Some 
specimens of ß. verbecki even exhibit marked 
axial sculpture otherwise typical for most 
specimens of ß. torquata. Shells of ß. torquata 
are thinner and generally lack more pro- 
nounced spiral elements except for basal lirae. 
The two species can significantly be discrimi- 
nated by shell morphometry (Table 4). The 
report of Leschke (1914) on M. subplicata from 
Bogor is incorrect; the voucher material in the 
ZMB is re-determined as Adamietta testudina- 
ria. 



TABLE 4. Result of disriminant analysis of shell 
parameters of 8. torquata and 6. verbecki. 



Predicted group membership 
8. torquata 8. verbecki 



B. torquata 
8. verbecki 



10(90.9%) 
1 (2.6%) 



1 (9.1%) 
37 (97.4%) 



224 



KÖHLER & GLAUBRECHT 



Brotia verbecki {Вю{. 1886) 
(Figs. 71 В, 72, 73) 

Melania verbecki Brot, 1886: 90, pi. 6, figs. 9- 
9b ("Lac de Singkarah, gouvernement de 
Padang, Sumatra occid." = Lake Singkarah, 
Padang distr.. West Sumatra), lectotype, 11 
paralectotypes MHNG, coll. Brot, paralecto- 
type MCZ 112682 (designated by Köhler & 
Glaubrecht, 2002a) (Figs. 72A-D); types 
seen: Martens, 1897: 38. 



Melania verbecki var. laevis Martens, 1897: 
38 (Lake Singkarah), 32 syntypes ZMB 
200.152. 

Brotia verbecki -КоЫег & Glaubrecht, 2002a: 
151, 152, fig. 3P. 

Melania papulosa Martens, 1897: 38, 39, pi. 
2, fig. 21 ("See Singkarah, Sumatra" = Lake 
Singkarah), lectotype ZMA, 18 paralecto- 
types ZMA, 1 5 paralectotypes ZMB 200.025 
(designated by Köhler & Glaubrecht, 2002a), 
(Figs. 72F-K); types seen. 







FIG. 72. Shell morphology B. verbecki. A: Lectotype of M. verbecki MHNG; B-D: 
Paralectotypes MHNG: E: Lectotype of /W. strictlcosta ZMB 200.102: F: Lectotype 
of M. papulosa ZMA: G-K: Paralectotypes ZMB 200.103: L-0: Sumatra, Lake 
Singkarah (ZMB 200.118). Scale bar 10 mm. 



SYSTEMATIC REVISION OF BROTIA 



225 



Melania stricticosta Martens, 1897: 39, 40, pi. 
2, figs. 22-26 ("See Singkarah, Sumatra" = 
Lake Singkarah, Sumatra), lectotype and two 
paralectotypes of M. stricticosta ZMB 
200.102, 16 paralectotypes ZMB 200.103, 
two potential paralectotypes ZMB 200.151 
(designated by Köhler & Glaubrecht, 2002a) 
(Fig. 72 E); types seen. 

Taxonomy and Systematics 

Brot described this species from Lake 
Singkarah, West-Sumatra (Indonesia) using a 
manuscript name of Boettger. From the same 
locality. Martens (1897) described not only a 
new van, laevis, but also two new species, M. 
papulosa and M. stricticosta, for subtle 
conchological differences. Examination of the 
type series shows that the named taxa only 
delineate conchological varieties of a single, 
albeit somewhat variable species. The differ- 
ent morphs occur frequently and syntopically 
in the lake with intermediate forms, and there 
is no evidence that they represent distinct spe- 
cies. 

Material Examined 

Indonesia, Sumatra: Lake Singkarah (ZMB 
76.290-4, 200.118, 200.126, 200.155, 
200.157-60). 

Differential Diganosis 

Shell small, thin but solid, conical to elon- 
gate turreted, pronounced sculpture of either 
strong spiral ridges, axial ribs, or combination 
of both; frequently with spiny nodules where 
spiral lines and axial ribs meet; operculum 
round; embryonic shells frequently with two 
spiral rows of nodules from second whorl on. 

Description 

Shell (Fig. 72): Small, relatively thin to deli- 
cate, broadly conic to elongate turreted, up 
to five flattened whorls; more or less promi- 
nent spiral lines or cords, especially at base, 
in some specimens dominating; mostly with 
strong axial ribs. In several specimens axial 
rows of three to four tubercles where spiral 
lines and axial ribs meet. Colour yellowish 
brown to olive brown. Aperture widely oval, 
well rounded to slightly produced below. 
Size: H = 12-36 mm, В = 6-16 mm. 



Embryonic Shell (Fig. 73): Conical turreted, 
three to four whorls; fine vertical growth lines, 
in many specimens, double spiral row of dis- 
tant, rounded nodules from second whorl on. 
Average proportions: H = 2.9 mm, В = 1.7 
mm, HA = 0.17 mm, BA = 0.32 mm, DA = 
0.66 mm (for n = 6). 

Operculum: Round, up to eight whorls, cen- 
tral nucleus, smaller than aperture. 

Radula (Fig. 71 B): Ribbon with about 1 00 rows 
of teeth. Rachidian with slightly concave 
upper rim by only slightly inflated lateral cor- 
ners. Cutting edge with main cusp flanked 





FIG. 73. Embryonic shell morphol- 
ogy of B. verbecki. SEM image of 
shell removed from brood pouch 
(Sumatra, Lake Singkarah; ZMB 
200.118). Scale bar = 1 mm. 



226 



KÖHLER & GLAUBRECHT 



by three smaller denticles on each side. Gla- 
bella rather straight at its basal end, with 
concave lateral margins. Lateral teeth with 
main cusp flanked by two to three smaller 
denticles. Inner and outer marginals with two 
pointed cusps, the outer one being broader. 
Stomach: Typical, as in B. citrina (Fig. 4); ex- 
cept for both typhlosoles unfused at entire 
length of style sac. 

Reproductive System 

Females contained between 19 and 75 
shelled juveniles (n = 4), forming cohorts with 
heights between 1.5 and 2.5 mm. 

Distribution 

Indonesia, West-Sumatra; Only known from 
Lake Singkarah. 

Brotia wy/io/f/ (Brandt, 1974) 
(Figs. 74-76) 

Brotia (Senckenbergia) wykoffi Brandt, 1974: 
184. pi. 13, fig. 41 ("Creek at Sai Yok, Kan- 
chanaburi Province"), holotype SMF 197268, 
four paratypes RMNH 55244/4; types seen. 

Brotia wykoffi - Köhler & Glaubrecht, 2002a: 
152. 




ЩЩ 



ÏÏÏÏW 



1 



FIG. 74. Shell morphology of 
8, wykoffi (Thailand, Nam 
Ток; ZMB 200.132). 



Taxonomy and Systematics 

Brandt (1974) affiliated this species to 
Senckenbergia Yen, 1939, and treated this 
taxon as a subgenus of Brotia. However, 
Senckenbergia is not considered a pachychilid 
(Köhler & Glaubrecht, 2002a). 

Material Examined 

Thailand: Prov. Kanchanaburi, Sai Yok Falls 
2 (Sai Yok NP), Nam Ток, 14°26.3'N, 98°51 .0'E 
(ZMB 200.131-2). 




FIG. 75. Embryonic shell morphology of 
B. wykoffi. SEM images of embryonic 
shell removed from brood pouch (Thai- 
land, Nam Ток; ZMB 200.1 32); apical and 
front view. Scale bar - 1 mm. 



SYSTEMATIC REVISION OF BROTIA 



227 



Differential Diganosis 

Shell smooth, rather small, thin but solid, 
conical turreted; whorls flattened, only slightly 
convex; colour olive brown with lightly green 
spiral bands; aperture inside olive green with 
yellowish bands. 

Description 

Shell (Fig. 74): Small, thin but solid; spire coni- 
cal turreted, up to eight flattened, only slightly 
convex whorls; smooth sculpture except for 
growth lines, weak spiral lirae at base. Colour 
olive brown with lightly green spiral bands. 
Aperture relatively narrow, angled below, 
pointed above, inside olive green with yel- 
lowish bands. H = 22-30 mm, В = 9-11 mm. 

Embryonic Shell {F\g. 75): Broadly ovate, three 
whorls; smooth sculpture; aperture wide. 
Average proportions: H = 2.0 mm, В = 1.4 
mm, HA = 0.15 mm, BA = 0.28 mm, DA = 
0.62 mm (forn = 10). 

Operculum: Round, up to eight regular whorls, 
central nucleus. 

Radula (Fig. 76): Ribbon with about 120 rows 
of teeth. Rachidian with slightly concave up- 
per rim by inflated lateral corners. Cutting 
edge with one main cusp flanked by two 
smaller denticles on each side. Glabella 
straight at its base, relatively long, with 
straight lateral margins. Laterals with main 
cusp flanked by two smaller denticles on each 
side, rather long lateral extensions. Inner and 
outer marginal teeth with two to three pointed 
cusps, the outer one being broader. 

Stomach: Typical, as in ß. citrina (Fig. 4); ex- 
cept for both typhlosoles unfused at entire 
length of style sac. 




FIG. 76. Radular morphology of в. wykoffi. SEM 
image of segment viewed from above (Thailand, 
Nam Ток; ZMB 200.132). Scale bar = 100 pm. 



Reproductive System 

A female contained 21 juveniles of more or 
less of same size (ZMB 200.232). 

Distribution 

Thailand: Prov. Kanchanaburi, known only 
from type locality (Sai Yok Falls, Nam Ток). 

Habitat 

Small, tangled, swift stream discharging into 
Kwae Noi River. 

Remarks 

Somewhat similar to ß. dautzenbergiana, 
which is much larger, juveniles more slender. 



INCERTAE SEDIS 

In the following, a number of taxa are listed 
that are members of the Pachychilidae, as can 
be judged from features of their shell, opercu- 
lum and/or radula. However, an unequivocal 
affiliation with Brotia is not possible for lack of 
crucial information on diagnostic features, 
such as embryonic shell morphology or repro- 
ductive anatomy. Because the species origi- 
nate from localities where other pachychilid 
genera may occur, such as, for example, 
Adamietta, we refrain from a formal treatment 
under Brotia, although it appears plausible for 
most of the following taxa that they are mem- 
bers of this genus. 

Brotia (?) angulifera (Brot, 1872) 
(Figs. 77A, B) 

Melania (Pachychilus) angulifera Brot, 1872: 
32, pi. 2, fig. 9 ("Java"), lectotype and para- 
lectotype MHNG, coll. Brot (designated by 
Köhler & Glaubrecht, 2002a) (Figs. 77A, B); 
types seen; Brot, 1875: 51, 52, pi. 6, fig. 5. 

Brotia angulifera - Köhler & Glaubrecht, 
2002a: 126, 127, fig. IB. 

Taxonomy and Systematics 

Benthem Jutting (1956) considered this spe- 
cies a synonym of "ß. testudinaria . However, 
the shells of both species are easy to distin- 
guish. B. angulifera is considered here a dis- 
tinct species. Details of soft body, radula, and 
embryonic shell remain unknown, which hin- 



228 



KÖHLER & GLAUBRECHT 



ders a systematic decision. Not identical with 
Melania {Plotia) scabra var. angullfera Mar- 
tens. 1897. 

Differential Diganosis 

Shell conical turreted. one colour dark green- 
ish to olive brown with convex, rounded to 
slightly shouldered whorls, sculptured by fine 
spiral lirae: spiral depression below suture. 

Description 

Shell (Figs. 77A, B): Medium sized, oval to 
conical turreted, solid, with six convex, well 
rounded to slightly shouldered whorls, nar- 
row suture; with fine spiral lirae and faint 
vertical growth lines. Colour greenish to ol- 
ive brown. Body whorl comparatively large. 
Aperture medium sized, oval, well rounded, 
slightly produced below. Columella thick. 
Size of lectotype: H = 33 mm, В = 14 mm. 

Embryonic shell morphology. Operculum. 
Radula. Soft body anatomy: Unknown. 

Distribution 

Indonesia: Java, the type locality as only 
known record. 

Brotia (?) assamensis (Nevill, 1885) 
(Figs. 77C, D) 

Melania (Acrostoma) assamensis Nevill, 1885: 
271 ("Delaima River, North Cachar"), four 
syntypes IMC, according to Nevill (1885): 
types not seen. 

Tiara (Acrostoma) assamensis - Preston, 
1915: 31. 

Paracrostoma assamensis - Köhler & Glaub- 
recht. 2002a: 128. 

Taxonomy and Systematics 

Specimens in the BMNH apparently were not 
available to Nevill (1885), who mentioned only 
four specimens in the IMC. Placed in 
Paracrostoma because of a close similarity to 
its type species, P. /7uege//(Philippi, 1843), by 
Köhler & Glaubrecht (2002a) and because 
Acrostoma Brot, 1870, is a synonym of 
Paracrostoma Cossmann, 1900 (Köhler & 
Glaubrecht, 2002a). However, Paracrostoma 
is endemic to southern India (federal states of 
Karnataka, Kerala, and Tamil Nadu) and most 
likely does not occur in Assam, from where 
otherwise some Brotia species are known 
(unpubl. data). For this circumstantial evidence, 



we suggest to treat this species as a member 
of Brotia. 

Material Examined 

India: Assam, Delaima River, North Cachar 
(= Delaima River, N of Silchar; BMNH 
19991534 (12 shells originating from the 
Godwin-Austen collection, same series as 
types; Figs. 77C, D). 

Differential Diganosis 

Shell elongate, conical with rounded whorls, 
surface smooth and glossy; one colour dark 
brown, sculptured by faint spiral lirae and 
growth lines only; aperture wide, angularly 
produced below; body whorl comparatively 
large compared to shell height. 

Description 

Shell (Figs. 77C, D): Medium sized, spire coni- 
cally turreted, eroded, with three to five 
slightly convex to flattened whorls, sculpture 
smooth except for faint spiral lines and 
growth lines. All one colour, chestnut brown. 
Aperture elongate oval with produced to 
slightly angled lower margin, columellar 
margin inconspicuous, peristome sharp. 

Embryonic Shell. Operculum. Radula. Soft 
Body: Anatomy unknown. 

Distribution 

India: Assam, Delaima River as the only 
known locality. 

Remarks 

Similar to Paracrostoma huegeli, but more 
slender in shape, coloration lacks spiral 
flames, body whorl not as inflated as in the 
former. P. huegeli lacks glossy surface. 

Brotia (?) beaumetzi (Brot, 1887) 
(Fig. 77H) 

Melania beaumetzi Brot, 1887: 34, 35 ("Baie 
du Touranne", in error, replaced by "environs 
de Than Moi" by Dautzenberg & Hamonville, 
1887 = Thanh Moi, about 200 km NE of 
Hanoi, Vietnam, 2r37'N, 106°32'E), holo- 
type MNHN (Fig. 77H); type seen; Dautzen- 
berg & Hamonville, 1 887: 219; Fischer-Piette, 
1950: 160, pi. 5, fig. 4. 

Brotia beaumetzi - Köhler & Glaubrecht, 
2002a: 129, fig. ID. 



SYSTEMATIC REVISION OF BROTIA 



229 




FIG. 77. Shell morphology of several species with uncertain classification. A; Lectotype of /W. angulifera 
MHNG- B- Paralectotype MHNG; C-D: Brotia (?) assamensis (Assam, North Cachar, Deíaima River; 
BMNH '19991534)- E; Holotype of /W. bomeens/s RM N H 71325; F: Lectotype of /W. cylindrus MHHG; 
G- Lectotype of M. subcylindrica MHNG; H: Holotype of M. beaumetzi MNHN; I: Lectotype of M. 
zonata von dem Busch, 1842 LIMB ТК 271/1 ; J-K: Paralectotypes LIMB TK 272/2; L-M: Syntypes of 
Melania canaliculata Reeve, 1869 (BMNH 20050105, ex coli. Cuming, = Melania sooloensis Reeve, 
1860); N: Brotia (?) solooensis (Philippines, Sulu Islands; ZMB 59.161). 



230 



KÖHLER & GLAUBRECHT 



Taxonomy and Systematics 

Brot (1887: 34) gave "Baie du Touranne" as 
the type locality. This was stated to be incor- 
rect and replaced by "Environs de Than-Moi 
(leg. M. de Merlaincourt)" (Dautzenberg & 
Hamonville, 1887), which corresponds to the 
label of the type material. The species was 
transferred to Brotia by Köhler & Glaubrecht 
(2002a) because of its characteristic shell. 

Differential Diganosis 

Shell small, robust, broadly conical with flat- 
tened whorls: thin, regularly spaced spiral lirae: 
distinct by its conical shape, tiny size, keeled 
basal whorl, fine and regular spiral sculpture. 

Description 

Shell (Fig. 77H): Small, conical turreted with 
five flattened whorls, inconspicuous suture; 
regular spiral lirae. Colour light brownish ol- 
ive. Aperture oval, angled, produced below, 
pointed above. Size of holotype: H = 20 mm, 
8 = 10 mm. 

Embryonic shell morphology. Operculum, 
Radula. Soft body anatomy: Unknown. 

Distribution 

Vietnam: Type locality only known record. 

Brotia (?) borneensis (Schepman, 1896) 
(Fig.77E) 

Melania borneensis Schepman, 1896: 137, 
138, pi. 2, fig. 4 ("Borneo"), holotype RMNH 
71325 (Fig. 77E): type seen. 

Brotia borneensis - Köhler & Glaubrecht, 
2002a: 130. 

Taxonomy and Systematics 

Transferred to Brotia by Köhler & Glaubrecht 
(2002a) because of its characteristic shell. 
Next to S. praetermissa, it would be the sec- 
ond Brotia species from Borneo. 

Differential Diganosis 

Shell relatively large, highly turreted with 
convex, well rounded whorls, sculptured by 
spiral lines most conspicuously below suture, 
and faint axial growth lines; aperture wide, well 
produced below. 



Description 

Shell (Fig. 77E): Large, spire elongate turreted, 
five remaining, regularly rounded, convex 
whorls; shell solid to thick; colour yellowish- 
olive; upper whorls sculptured by numerous 
spiral striae, more conspicuous on last whorl, 
growth lines inconspicuous. Aperture ovate, 
well rounded, produced below, pointed 
above; columellar margin thin, moderately 
curved; inferior of aperture bluish white. Size 
of holotype: H = 54.7 mm, В = 20.6 mm. 

Embryonic shell morphology, Operculum, 
Radula, Soft body anatomy: Unknown. 

Distribution 

Borneo: Type locality only known record. 

Brotia (?) cylindrus (Brot, 1886) 
(Figs. 77F, G) 

Melania cylindrus Brot, 1886: 92, 93, pi. 6, figs. 
7, 7a ("Siam" = Thailand), lectotype and two 
paralectotypes MHNG, paralectotype MCZ 
11268 (designated by Köhler & Glaubrecht, 
2002a) (Fig. 77F); types seen. 

Melania subcylindrica Brot, 1886: 102, 103, 
pi. 6, figs. 2, 2a ("Chine" = China), lectotype 
and two paralectotypes MHNG (designated 
by Köhler & Glaubrecht, 2002a) (Fig. 77G); 
types seen. 

Taxonomy and Systematics 

The two taxa described by Brot (1886) were 
considered identical for their similar shell and 
assigned to Brotia by Köhler & Glaubrecht 
(2002a). Because the types represent the only 
available material and most morphological 
properties are unknown, systematics is uncer- 
tain. 

Differential Diganosis 

Turreted shell, truncated spire, well-rounded 
whorls, sculptured by regularly spaced, fine 
spiral lines; aperture well rounded, relatively 
small; one colour dark brown to black. 

Description 

Shell (Figs. 77F, G): Highly turreted, frequently 
truncated after second or third whorl, whorls 
well rounded in diameter, sculptured by regu- 
larly spaced, fine spiral lines; aperture well 



SYSTEMATIC REVISION OF BROTIA 



231 



rounded below, relatively small compared to 
body whorl; one colour dark brown to black. 
Shell size: H = 27.5-42 mm, В = 13.5-19.7 
mm. 
Embryonic shell morphology, Operculum. 
Radula. Soft body anatomy: Unknown. 



Distribution 



Vague: 
records. 



'Siam" and "China" as only known 



Brotia (?) sooloensis (Reeve, 1859) 
(Figs. 77L, M) 

Melania canaliculata Reeve, 1859: pi. 6, spe- 
cies 31 {non M. canaliculata Say, 1821) 
("Sooloo Islands" = Sulu Islands, Philippines); 
two syntypes BMNH 20050105 (Sulu Islands, 
ex coll. Cuming) (Fig. 77L, M), types seen. 

Melania sooloensis Reeve, 1860: errata; Brot, 
1870: 281; Brot, 1875: 105, 106, pi. 14, fig. 3. 

Brotia sooloensis - Köhler & Glaubrecht, 
2002a: 147, 148. 

Taxonomy and Systematics 

The name M. sooloensis was employed by 
Reeve (1 860: errata) as replacement name for 
M. canaliculata Reeve, 1859, being preoccu- 
pied by M. canaliculata Say, 1821. The sys- 
tematic affinity is suspicious due to unknown 
properties of soft body, embryonic shell, 
radula, and operculum. Herein preliminarily 
affiliated with Brotia, but this treatment requires 
critical revision as the Zulu Archipelago, Phil- 
ippines, is not within the range of Brotia as 
defined here. 

Material Examined 

Philippines: Sulu Islands (MHNG, coll. Tay- 
lor; ZMB 59.161); Cagayan (MHNG; coll. 
Norris); Isabella (MHNG; leg. Semper), herein 
restricted to Isabela, Basilan (6°41'N, 
118°58'E). 

Differential Diganosis 

Shape of shell unmistakable; in particular 
elongate spire, stepped whorls, subsutural 
depression or shoulder. 

Description 

Shell {F\g. 77L-N): Elongate turreted, solid but 
not thick, up to six whorls, deep suture. 



mostly truncated tip; whorls well rounded at 
base, upper whorls convex but more flat- 
tened than basal ones; subsutural depres- 
sion, most prominent on last two or three 
whorls; smooth sculpture, basal spiral ridges, 
faint growth lines, faint spiral lines; surface 
glossy. Aperture oval, well rounded below. 
Shell size: H = 31-38 mm, В = 13-15 mm. 
Embryonic shell morphology, Radula, Soft 
body anatomy: Unknown. 

Distribution 

Reports on this species refer to Sulu Islands, 
Philippines; neither known from Mindanao nor 
Borneo. Two islands in the Sulu Sea are 
named Cagayan. The island Cagayan-Sulu 
(material in MNHG) in N of Borneo (Sarawak), 
more than 300 km W of Sulu archipelago 
(6°59'N, 118°28'E); Cagayan Island in central 
Sulu Sea is even more remote, between 
Palawan and Negros (9°35'N, 12Г28'Е), 
about 600 km NW of the Sulu archipelago. 
Occurrence on both islands seems dubious 
and requires confirmation. 

Remarks 

Somewhat similar are species of 
Pseudopotamis (Glaubrecht & Rintelen, 2003). 
Well preserved material of B. sooloensis is 
needed to clarify its systematic position. 

Brotia (7) spinata (Godwin-Austen, 1872) 

/We/ano/dessp/nate Godwin-Austen, 1872: 514, 
pi. 30, figs. 1,1a ("Kopili River, North Cachar 
hills, a tributary of the Brahmaputra" = Kopili 
River, Jaintia-Khâsi hills N of Silchar, federal 
state of Meghalaya, India); types not seen; 
Hanley & Theobald, 1 874: pi. 1 09, fig. 1 . 

Melania spinata - Brot, 1875: 89, 90, pi. 10, 
figs. 2, 2a. 

Melania (Melanoides) spinata - Nevill, 1885: 
261. 

Brotia spinata - Köhler & Glaubrecht, 2002a: 
148 [partim]. 

Taxonomy and Systematics 

Type material was not traced. Shell only 
known from original figure. Attributed to Brotia 
by Köhler & Glaubrecht (2002a) as being typi- 
cal for Brotia. Geographical distribution well 
within range of the genus. Köhler & Glaubrecht 
(2002a) assumed that B. binodosa is conspe- 
cific for the similar shell. However, in their re- 



232 



KÖHLER & GLAUBRECHT 



vision of the Каек River species flock. Glaub- 
recht & Köhler (2004) show that B. binodosa 
is endemic to central Thailand and, thus, not 
conspecific with 8. splnata. 

Differential Diganosis 

Highly turreted shell with two spiral rows of 
spiny nodules supported by more or less 
prominent spiral cords; body whorl large com- 
pared to shell; aperture wide, produced be- 
low. 

Distribution 

India, Meghalaya: Known from type locality only 

Remarks 

Similar to B. binodosa. which has a more 
slender shell. 

Brotia (7) zonata (Benson, 1836) 
(Figs. 771-K) 

Melania zonata Benson, 1836; 747 (no figure); 

types not seen. 
l\/lelania zonata Busch, 1842 - In; Philippi, 

1842; 3, pi. 1, fig. 12 ("Bengalia"), lectotype 

ÜMB TK 271/1 , two paralectotypes ÜMB TK 

272/2 (designated by Knipper, 1958) (Figs. 

771-K); types seen. 
Melanella zonata - H. Adams & A. Adams, 

1854; 296. 
Brotia zonata - Köhler & Glaubrecht, 2002a; 

152. 

Taxonomy and Systematics 

Benson described this species from a col- 
lection of freshwater shells originating from 
Bengal and Sylhet, but did not explicitly men- 
tion a type locality. Melania zonata Busch 
(1842) was stated to be junior synonym by 
objective homonymy (Reeve, 1859; Brot, 
1875; Knipper, 1958; Köhler & Glaubrecht, 
2002a). 

Differential Diganosis 

Shell rather small, broadly conical, truncated 
after third whorl, strong, sculpture smooth ex- 
cept for growth lines, glossy surface, two 
chestnut brown spiral bands, aperture widely 
oval and well produced below. 



Description 

Shell {F\gs. 771-K); Relatively small, broadly 
conical with three whorls, shell robust; sculp- 
ture smooth except for faint growth lines, 
body whorl comparatively large; colour 
greenish brown with chestnut brown spiral 
bands; aperture oval, wide inside whitish with 
brown bands. 

Embryonic shell morphology. Radula. Soft 
body anatomy: Unknown. 

Distribution 

India, Bangladesh; Bengal. 

Remarks 

Similar to B. pseudosulcospira and B. 
microsculpta in its smooth and conical shell; 
the spiral brown band being unique, though. 



MOLECULAR GENETICS 

Sequence Analysis 

Separate sequence alignments comprise 
646 bp (CGI) and 826 bp (16S), respectively. 
Plotting rates of transitions (s) and 
transversions (v) against sequence divergence 
for both genes separately indicates that se- 
quences are not saturated and, thus, accom- 
modate phylogenetic analyses. A partition 
homogeneity test as implemented in PAUP* 
showed that the two data partitions (CGI and 
16S) are not significantly incongruent at the 
99% level (P < 0.01). The analysis software 
MrModeltest (Nylander, 2002) revealed an in- 
variant + gamma distributed model of se- 
quence evolution (GTR+1 + Г; Gu et al., 1995) 
as the best fitting model for both sequence 
data sets. Accordingly, this model was cho- 
sen to calculate pair wise genetic distances 
shown in Table 5. The model was also imple- 
mented in distance based analyses (NJ and 
Bl). Pair wise genetic distances were calcu- 
lated separately for each of the partial genes. 
With one exception, in CGI infraspecific dis- 
tances usually do not exceed 16% (in 8. 
citrina) and mostly range between and 6%. 
The high sequence divergence in B. 
sumatrensis is very striking. Since a similar 
divergence is not observed in 16S, we assume 
that the one sequence of 6. sumatrensis high- 



SYSTEMATIC REVISION OF BROTIA 



233 



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234 KÖHLER & GLAUBRECHT 

TABLE 6. Sequence data analysed in this study with GenBank accessions and inventory numbers. 



Genus 


Species 


Inventory No. 


Origin 


CGI 


16S 


Adamietta A. 


hainanensis 


ZMB 200.301 


Hong Kong 


AY 330827 


AY 330778 


A. 


housei 


ZMB 200.165 


Thailand 


AY 330823 


AY 330774 


A. 


provisoria 


ZMB 200.053 


Borneo 


AY 242951 


AH 012869 


A. 


testudinaria 


ZMB 190.415 


Java 


AY 330825 


AY 330777 






ZMB 190.416 


Java 


AY 330826 


AY 330776 






ZMB 200.099 


Java 


AY 330824 


AY 330775 






ZMB 200.100 


Java 


AY 242950 


AY 242949 


Brotia B. 


armata 


ZMB 200.193 


Thailand 


AY 330853 


AY 330810 






ZMB 200.252 


Thailand 


AY 330854 


AY 330809 






ZMB 200.254 


Thailand 


AY 330834 


AY 330808 






ZMB 200.265 


Thailand 


AY 330855 


AY 330806 






ZMB 200.268 


Thailand 


AY 330837 


AY 330807 






ZMB 200.268a 


Thailand 


AY 330856 


AY 330811 


B. 


binodosa 


ZMB 200.192 


Thailand 


AY 330857 


AY 330815 






ZMB 200.202 


Thailand 


AY 330859 


AY 330819 






ZMB 200.267 


Thailand 


AY 330860 


AY 330818 






ZMB 200.269 


Thailand 


AY 330861 


AY 330820 






ZMB 200.328 


Thailand 


AY 330858 


AY 330816 


B. 


citrina 


ZMB 200.207 


Thailand 


AY 330829 


AY 330798 






ZMB 200.212 


Thailand 


AY 330830 


AY 330799 


B. 


costula 


ZMB 112.660 


Nepal 


DO 284985 


DO 284986 


B. 


dautzenbergiana 


ZMB 200.226 


Thailand 


AY 330831 


AY 330802 






ZMB 200.229 


Thailand 


AY 330832 


AY 330800 


B. 


lienriettae 


ZMB 200.210 


Thailand 


AY 330845 


AY 330793 






ZMB 200.221 


Thailand 


AY 330846 


AY 330794 


B. 


hercúlea 


ZMB 200.206 


Thailand 


AY 330841 


AY 330787 






ZMB 200.209 


Thailand 


AY 330842 


AY 330789 






ZMB 200.219 


Thailand 


AY 330843 


AY 330790 






ZMB 200.220 


Thailand 


AY 242972 


AY 242971 






ZMB 200.253 


Thailand 


AY 330844 


AY 330788 


B. 


microsculpta 


ZMB 200.191 


Thailand 


AY 330836 


AY 330805 






ZMB 200.200 


Thailand 


AY 330833 


AY 330804 






ZMB 200.266 


Thailand 


AY 330835 


AY 330803 


B. 


pagodula 


ZMB 200.205 


Thailand 


AY 330847 


AY 330795 






ZMB 200.208 


Thailand 


AY 172453 


AY 172443 


B. 


peninsularis 


ZMB 200.046 


Thailand 


AY 330850 


AY 330792 






ZMB 200.242 


Thailand 


AY 330841 


AY 330791 


B. 


pseudosulcospira 


ZMB 200.196 


Thailand 


AY 330862 


AY 330797 


B. 


solemiana 


ZMB 200.174 


Thailand 


AY 330849 


AY 330814 






ZMB 200.203 


Thailand 


AY 330848 


AY 330812 


B. 


sumatrensis 


ZMB 200.1 16 


Sumatra 


AY 330838 


AY 330784 






ZMB 200.1 19 


Sumatra 


AY 330840 


AY 330785 


B. 


torquata 


ZMB 200.117 


Sumatra 


AY 330864 


AY 330781 






ZMB 200.121 


Sumatra 


AY 330865 


AY 330782 


в 


verbecki 


ZMB 200.118 


Sumatra 


AY 330863 


AY 330779 


в 


wykoffi 


ZMB 200.232 


Thailand 


AY 330866 


AY 330796 


Paracrostoma P 


spec. 


ZMB 200.318 


South India 


AY 330821 


AY 330770 


P 


spec. 


ZMB 200.322 


South India 


AY 330822 


AY 330773 


Jagora J. 


asperata 


ZMB 200.311 


Philippines 


AY 172447 


AY 172439 


J. 


dactylus 


ZMB 200.109 


Philippines 


AY 172444 


AY 172438 



SYSTEMATIC REVISION OF BROTIA 



235 



100 

■100' 
100 



100 

100' 

100 



Jagora asperata ZMB 200. 11 1 
Jagora dactyl us ZMB 200.1 109 



83 



100 
■100 
100 



81 
76 

h 001 



7 

00, 

ooJ 

100^ — 

100 . 

-100—1 
100 ' — 



52 



99 
И 001 
100 



100 
-100- 
100 



100 
-100- 
100 



-94- 
100 



100 
-100- 



99 

-100- 

100 



94 
-100- 
100 



87 
■97- 
87 



100 



100 
100 



Adamietta house! ZMB 200.165 

CAdamietta hainanensis ZMB 200.301 
Adamietta cf schmidti ZMB 200.053 
Adamietta testudinaha ZMB 200.099 
Adamietta testudinaria ZMB 200.100 
Paracrostoma spec. ZMB 200.318 
Paracrostoma spec. ZMB 200.322 
Brotia armata ZMB 200.193 
Brotia armata ZMB 200.254 
Brotia armata ZMB 200.268 
Brotia armata ZMB 200.268a 
Brotia pseudosulcospira ZMB 200.196 
Brotia binodosa ZMB 200.267 
Brotia binodosa ZMB 200.269 
Brotia binodosa ZMB 200.328 

— Brotia microsculpta ZMB 200.200 

— Brotia microsculpta ZMB 200.266 
Brotia armata ZMB 200.265 
Brotia binodosa ZMB 200.202 
Brotia binodosa ZMB 200.192 
Brotia armata ZMB 200.252 
Brotia microsculpta ZMB 200.191 
Brotia solemiana ZMB 200.203 
Brotia solemiana ZMB 200.174 
Brotia sumatrensis ZMB 200. 1 1 6 
Brotia sumatrensis ZMB 200.1 19 

В 200.117 
Brotia torquata ZMB 200.121 
Brotia verbecki ZMB 200.1 18 
Brotia costula ZMB 1 12.660 
Brotia henriettae ZMB 200.210 
Brotia henriettae ZMB 200.221 
Brotia wykoffi ZMB 200.232 
Brotia peninsularis ZMB 200.046 
Brotia peninsularis ZMB 200.242 
Brotia citrina ZMB 200.212 
Sroi/a c/ir/na ZMB 200.207 
Brotia pagodula ZMB 200.205 
Sroi/a pagodula ZMB 200.208 
Sroi/a dautzenbergiana ZMB 200.213 
Sroi/a dautzenbergiana ZMB 200.229 
Srof/a dautzenbergiana ZMB 200.226 
Srof/a hercúlea ZMB 200.206 
Srof/a hercúlea ZMB 200.209 
Srof/a hercúlea ZMB 200.219 
Srof/a hercúlea ZMB 200.220 
Srof/a hercúlea ZMB 200.253 



Г-100— I 



100 1 — Srof/a torquata Z 
^qqIqqI Brotia torauata Z 

iooL_ 







FIG. 78. Confidence limits on the topology of the MP strict consensus cladogram of concatenated 
data set of 1 68 and COI expressed by branch support values mapped on respective branches (above: 
MP bootstrap values, middle: NJ bootstrap values, below: Bl posterior clade probabilities). 



236 



KÖHLER & GLAUBRECHT 



— Jagora asperata ZMB 200.1 1 1 
Jagora dactylus ZMB 200 1 109 



Adamietta hainanensis ZMB 200 301 



■Adamietta cf schmidti ZMB 200.053 



r Adamietta testudinaria ZMB 200.099 

•■ Adamietta testudinaria ZMB 200.100 
Adamietta housei ZMB 200 165 



Paracrostoma spec. ZMB 200.318 

Paracrostoma spec ZMB 200.322 

Brotia armata ZMB 200.193 
г r Brotia armata ZMB 200.254 
4 Brotia armata ZMB 200.268 
Brotia armata ZMB 200.268a 
•■ Brotia pseudosulcospira ZMB 200.196 
■ Brotia binodosa ZMB 200 267 
Brotia binodosa ZMB 200.269 
\ Brotia binodosa ZMB 200.328 
Brotia microsculpta ZMB 200 200 
Brotia microsculpta ZMB 200.266 
- Brotia binodosa ZMB 200.202 
Brotia armata ZMB 200.265 
I- Brotia binodosa ZMB 200.192 
— Brotia armata ZMB 200.252 
Brotia microsculpta ZMB 200.191 
Brotia solemiana ZMB 200.203 
I — Brotia solemiana ZMB 200.174 

Brotia sumatrensis ZMB 200.1 16 

Brotia sumatrensis ZMB 200. 11 9 
Brotia torquata ZMB 200.117 
Brotia torquata ZMB 200.121 
Brotia verbecki ZMB 200.1 18 
Brotia costula ZMB 1 12.660 

- Brotia henriettae ZMB 200.210 

- Brotia henriettae ZMB 200.221 
ßfof/a wykoffi ZMB 200.232 

Brotia peninsularis ZMB 200.046 
Brotia peninsularis ZMB 200.242 
Brotia citrina ZMB 200.207 

— Brof/ac/fr/na ZMB 200.212 

— Brotia pagodula ZMB 200.205 

Brotia pagodula ZMB 200.208 




f- 



Brotia dautzenbergiana ZMB 200.213 

Brotia dautzenbergiana ZMB 200.229 
Brotia dautzenbergiana ZMB 200.226 
r Brotia hercúlea ZMB 200.206 
1 Brotia hercúlea ZMB 200.209 

I Brotia hercúlea ZMB 200.219 
J Brotia hercúlea ZMB 200.220 

I Brotia hercúlea ZMB 200.253 



50 changes 



FIG. 79. Bayesian inference phylogram of concatenated data set of 16S and COL 



SYSTEMATIC REVISION OF BROTIA 



237 



lighted as genetically very distinct is deficient. 
It will thus not be considered in further discus- 
sion. 

Pair wise genetic distances between differ- 
ent Brotia species range between the maxima 
of 8 and 29% when considering species from 
outside the Kaek River, and between and 
8% when comparing endemic species in the 
Kaek River. 

In COI, pair wise genetic distances between 
species from different genera are even higher, 
with 28-40%, when comparing Jagora and 
Brotia, 20-47% when comparing Adamietta 
and Brotia, and 24-42% when comparing 
Paracrostoma and Brotia. 

Similar genetic distances are observed also 
in 16S with an infraspecific level of sequence 
divergence of up to 10%, interspecific dis- 
tances of up to 8% among Kaek River spe- 
cies and up to 25% between other Brotia 
species as well as divergence rates between 
30 and 56% when comparing species of dif- 
ferent genera with each other. 

Phylogenetic Analyses 

The concatenated sequence data set was 
analysed using MP, NJ, and Bl methodology. 

603 positions of the concatenated data set 
with a total length of 1,472 bp are constant, 
222 variable but parsimony uninformative, and 
647 variable and parsimony informative. MP 
analysis delivers 6 most parsimonious trees; 
the strict consensus tree is shown in Fig. 78 
(numbers mapped on the tree indicate branch 
support for the depicted topology by MP boot- 
strap values [above lines], NJ bootstrap val- 
ues [on lines], and Bayesian posterior clade 
probabilities [below lines], respectively). All 
trees were rooted with species of Jagora as 
outgroup since this genus is among the most 
basal groups among the Pachychilidae (Köhler 
et al., 2004). The topologies of two distance 
based trees, the NJ phylogram (not shown) 
and the Bl phylogram (Fig. 79), do widely cor- 
respond to the MP tree. However, in contrast 
to the MP tree, these reconstruction show both 
Adamietta and B. citrina as monophyletic 
groupings, the latter being sister to B. 
pagodula. 

All trees corroborate the monophyly of Brotia 
as delineated according to morphological char- 
acteristics in respect to the other pachychilid 
genera included into the analysis, that is, 
Jagora, Paracrostoma, and Adamietta. The 
monophyly of Adamietta is not unambiguously 



corroborated since it is not shown as a 
monophylum in the MP tree. However, this is 
not relevant in regard to the monophyly of 
Brotia. Within Brotia some well supported sub- 
groupings are shown, such as the Kaek River 
species flock, and a Sumatra clade. Only the 
species of the Kaek River species flock do 
not appear as monophyletic entities in either 
of the trees. These species also show low ge- 
netic distances, as has been mentioned 
above. 



DISCUSSION 
Evaluation of Morphological Characters 

(1) Adult Shell 

Traditionally, in classifications and taxon 
descriptions of gastropods the shell is empha- 
sized. It bears many characters that are most 
convenient for taxonomic purposes and that 
are accessible even from dry material and fos- 
sils (for examples. Smith, 1981; Ridgeway et 
al., 1998). Also in pachychilids, shell features 
are essential to distinguish species, and mor- 
phometry is often useful for species discrimi- 
nation. On the other hand, the shell may 
particularly be prone to environmental pres- 
sures such as wave action (Reid, 1986: 8 for 
Littorina) or prédation (Vermeij & Covich, 
1978) by birds (Reed & Janzen, 1999) or by 
crabs and crayfish (Reid, 1992; Warner, 
1996). Therefore, divergent shells may repre- 
sent just phenotypic variation. In addition to 
that, even relatively complex shell structures 
may have evolved in analogy as has been dis- 
cussed for the clausilial apparatus in the 
Clausiliidae (Moorsel et al., 2000). 

Shell features, such as shape, size, thick- 
ness, and sculpture, vary considerably among 
species of Brotia. This diversity provoked ear- 
lier authors to describe many new species 
based solely on the shell, and a few of them 
carried this to excess by introducing a vast 
number of taxonomic names for subtle 
conchological differences (e.g., Nevill, 1885). 
This procedure reflects the essentialist view 
of many systematists at this time (see Haffer, 
1997 for examples from ornithology; 
Glaubrecht, 2004, for malacology). 

Since the 1930's, when authors began to 
acknowledge the existence of infraspecific 
variation, it has frequently been assumed that 



238 



KÖHLER & GLAUBRECHT 



Brotia species are remarkably plastic not only 
in their phenotypic appearance. Similar taxa 
have in the following been considered conspe- 
cific, which has bloated the synonymies (e.g., 
Rensch, 1934; Benthem Jutting, 1956; Brandt, 
1974). However, in many cases it remained 
unclear (and unattended) to which extent shell 
parameters really varied within single species. 
Most recent data suggests that intraspecific 
variability of morphological characters includ- 
ing the shell frequently was overemphasized, 
which has lead to erroneous taxonomic con- 
clusions. This has been exemplified also for 
other pachychilids, such as Jagora by Köhler 
& Glaubrecht (2003). Consequently, one of the 
main results of the current study is the con- 
clusion that in Brotia, 20"^ century authors have 
frequently gone too far in synonymizing taxa 
for exhibiting a similar shell. Instead, a quite 
contrasting picture is revealed herein show- 
ing that Brotia species in general are much 
more restricted be means of their morphologi- 
cal variability as well as their distributional 
range than assumed before. 

Shell Shape: Most pachychilid species have 
highly turreted shells with about up to 12 
whorls. This feature is found in all major 
clades as a predominant character. Few 
species have conical or even globular shells, 
such as B. armata, B. paludiformis, or B. 
pagodula. These species live attached to 
stones and boulders in swiftly flowing 
streams while other species are found bur- 
ied in or crawling on substrata of all kinds. 

It has been shown by Urabe (1998) for 
Semisulcospira reiniana that individuals in- 
habiting riverine habitats have a more coni- 
cal shell than specimens from stagnant 
waters as a phenotypic response to environ- 
mental pressures. Although this observation 
refers to phenotypic responses only, a coni- 
cal shell can be considered as adaptation to 
strong water currents repeatedly obtained by 
Asian pachychilids. 

Size and Thickness: In general, shell size and 
thickness may be controlled by the availabil- 
ity of nutrients (Frömming, 1956), but also 
by the harshness of physical environmental 
factors (Vermeij, 1972), parasitism (Wright, 
1 966), or prédation (Zipser & Vermeij, 1 978; 
Reimchen, 1982; Reid, 1986). Nevertheless, 
there is substantial evidence that shell 
growth rate and adult size are also under 
genetic control (Vermeij, 1980). In Brotia, 
variability in shell size among conspecific 



specimens of same age is considered lower 
than formerly supposed. Only in few cases, 
shells may vary for about the twofold be- 
tween populations from different environ- 
ments: Specimens of B. torquata from Lake 
Manindjau are considerably smaller than 
those from adjacent rivers. In other cases, 
however, inhabitants of lakes are larger than 
riverine forms (e.g., 8. sumatrensis from 
Lake Toba). A possible explanation could 
include the limitation of certain nutrients due 
to interspecific competition in one case and 
the presence of predators, such as shell 
crushing crabs as discussed for Tylomelania 
in Sulawesi (Rintelen et al,, 2004) or simply 
the fact that large shells are prone to 
dislodgement in rivers but not in lakes in the 
other case. 

Sculpture: Freshwater gastropods in general 
are notorious for their plasticity in form and 
sculpture (e.g., Davis, 1971; Fretter & Gra- 
ham, 1984; Urabe, 2000). Similarly, among 
Brotia shell sculptures vary considerably and 
are used as a conspicuous feature to distin- 
guish among species. Shells may be com- 
pletely smooth or sculptured by strong axial 
ribs, spiral cords, spiny nodules, and/or 
spines. The degree of intraspecific variabil- 
ity, however, seems to differ greatly. In gen- 
eral, variation of the shell morphology, and 
thus also sculpture, has been considered to 
have a genetic basis and a strong sculpture 
shall be adaptive against predators or physi- 
cal environmental factors (e.g.. West & 
Cohen, 1996). It has been shown that sculp- 
tured shells are more tolerant of a crushing 
load than are smooth shells with the same 
shell mass (Urabe, 2000). Some studies 
have further demonstrated that shell mor- 
phology shows a great deal of phenotypic 
plasticity controlled by physical or biological 
factors (e.g.. De Wolf et al., 1997), such as 
the substratum (Urabe, 2000). While phe- 
notypic plasticity within single species has 
not been addressed in this study, it can be 
confirmed that shell form and sculpture are 
correlated to the substratum; species with 
smooth shells were always found on sandy 
or pebble substrata, whereas species with 
armed shells live on gravel, stony bottoms 
or sit on boulders (Glaubrecht & Köhler, 
2004, for Brotia species of the Kaek River). 
It is assumed that a sculpture not only pre- 
vents the animals from being preyed upon, 
which seems to be a rather imaginary threat 
when sitting directly in the water current, but 



SYSTEMATIC REVISION OF BROTIA 



239 



from the influence of physical forces. A well- 
developed sculpture, however, is unfavour- 
able when crawling in the sand as it would 
increase the friction with the substratum. 
Accordingly, different shell sculptures may 
have evolved as result of ecological and 
morphological diversification, in some cases 
induced by competitive interaction between 
the different species. 

Colour. In Brotia shell colour is uniform, from 
yellowish brown to olive brown, dark brown 
or almost black and overall not very helpful 
for species recognition. In some species, dark 
spiral bands may be present; axial flames that 
can be observed in other pachychilids, such 
as Pachychilus, Adamietta, and Paracro- 
stoma, are generally lacking. 

(2) Embryonic Shell 

Brotia shows a remarkable modification of 
the ontogeny that is also imprinted in the em- 
bryonic shell structure (Köhler & Glaubrecht, 
2001 ): In early ontogenetic stages, soft tissue 
protrudes from the apical whorl of the forming 
shell. This tissue is believed to have nutritive 
function for the encapsulated embryo. A sec- 
ondary shell layer closes at the apex not be- 
fore this tissue is entirely consumed. 
Protruding tissue and uncalcified apex was first 
noted by Morrison (1954) in embryonic shells 
of '^Brotia baccata" (= B. henhettae). The 
uncalcified apex was called by him an "open" 
or "soft apex" and stated to be a characteristic 
feature of ßrai/a. Subsequently, Solem (1966: 
16, ftg. 1) depicted several embryonic stages 
of B. binodosa with protruding soft tissue and 
open apex; this was followed by a report an 
"asymmetric" apical portion of the juvenile shell 
of 8. episcopalis (Davis, 1971: fig. 11). 

A storage structure similar to the tissue ob- 
served in Brotia was described for numerous 
other "prosobranchs", functioning as a sub- 
structure for the formation of the digestive gland 
(Fioroni & Schmekel, 1976: 129 ft.). The "yolk 
sac" of Brotia, which originates from the yolk 
supply of the egg capsule, is believed to be of 
same morphological and functional origin. 

Riedel (1 993) has hypothesized that delayed 
calciflcation of the apical whorl and a shrink- 
ing visceral mass in Melanoides tuberculata 
result in a wrinkled shell structure. This pat- 
tern is also observed in Brotia, in which the 
process of shell calcification is retarded and 
overlaps with the shrinking of the yolk sac by 
consumption of nutritive material. However, 



while delayed shell calcification is known from 
a number of gastropods (Eyster, 1986: 224- 
226), among them also some Thiaridae 
(Riedel, 1993; Glaubrecht, 1996), nutrition via 
a large, protruding yolk sac is unique among 
freshwater gastropods in the pachychilid gen- 
era Brotia and Jagora (description of the lat- 
ter: Köhler & Glaubrecht, 2003). However, the 
phylogenetic relationships between these two 
genera indicate that open apex and protrud- 
ing yolk sac have evolved independently 
(Köhler et al., 2004). This is suggested also 
by a different appearance of the apical por- 
tion of the embryonic shell in the two taxa. 
While in Brotia the apical whorl is wrinkled and 
appears irregular when viewed from above, in 
Jagora it is comprised by a lid-like structure 
that does not resemble a whorl at all (ftgured 
in Köhler & Glaubrecht, 2003). 

Embryonic shells of all other Asian Pachy- 
chilidae can easily be distinguished from Brotia 
by the lack of wrinkles. A comparative over- 
view of different embryonic shell morphologies 
in the Pachychilidae is provided by Köhler & 
Glaubrecht (2005). Consequently, in all other 
pachychilid taxa shell calcification is not re- 
tarded, but complete and continuous, a pro- 
truding yolk sac is not present. 

Operculum 

Next to the shell, the operculum is a feature 
that has long been used as diagnostic charac- 
ter for the classification of "melaniid" gastro- 
pods. For example, Troschel (1 857-58) based 
his classiftcation of the "Melaniidae" in part on 
opercular features. P. Sarasin & F. Sarasin 
(1898) distinguished between "Neomelanien" 
and "Palaeomelanien" on basis of a different 
operculum. Later, all palaeomelanian species 
were transferred to Brotia by Thiele (1928, 
1 929). While this decision has proven errone- 
ous, the two species groups delineated by P. 
Sarasin & F. Sarasin (1898) still are consid- 
ered to largely represent groups recognised 
by modern systematics: Pachychilidae and 
Thiaridae, respectively (Glaubrecht, 1999). 
Even taxa more closely related to the 
Pachychilidae, such as Faunas ater and the 
Melanopsidae, possess a paucispiral opercu- 
lum (Houbrick, 1991; Glaubrecht, 1996). Con- 
sequently, a multispiral operculum with a 
central or subcentral nucleus is considered as 
autapomorphy of the Pachychilidae. Within this 
family, however, operculum morphology is a 
conservative character, and only in some spe- 
cies it may be used for species determination. 



240 



KÖHLER & GLAUBRECHT 



Radula 

In general, the molluscan radula is consid- 
ered a conservative character with little varia- 
tion on the species level (Fretter & Graham, 
1994). Nevertheless, the importance of radu- 
lar characteristics, at least in higher level clas- 
sifications, has been acknowledged early on 
(Troschel, 1856-1863: Thiele, 1928, 1929- 
1935). At high levels of taxonomic hierarchy, 
several of the radular patterns first described 
by 19'" century morphologists still correspond 
largely or entirely with monophyletic clades 
recognised by modern cladistic analyses. Also 
at lower levels, recent cladistic analyses of 
morphology have frequently included radular 
characters (Glaubrecht, 1996; Reid, 1996: 
Ponder & Lindberg, 1997: Simone, 2001: 
Strong, 2003). Though, it became evident that 
radular characters, as any other morphological 
feature, may be prone to adaptation, parallel- 
ism and convergence and that intraspecific 
variability and plasticity may be considerable 
(Padilla, 1998: Reid & Мак, 1999: Reid, 2000). 
Therefore, before radular features can be used 
in phylogenetic studies, the extent of intraspe- 
cific variation must be carefully assessed, as is 
standard practise for shell characters. 

The pachychilid radula is of the generalised 
taenioglossate type. Each row consists of a 
central rachidian, flanked on each side by a 
lateral and an inner and outer marginal tooth. 
All these teeth bear a number of cusps. Com- 
parison of radulae of different pachychilid gen- 
era, such as Pachychilus (Troschel, 1858: pi. 
9: Fischer & Crosse, 1892: pi. 49, fig. 14: 
Simone, 2001: figs, 95, 96), Doryssa (Simone, 
2001: figs. 89-92), Potadoma (Glaubrecht, 
1996: pi. 5, figs. 7, 8), Jagora (Köhler & 
Glaubrecht, 2003), Sulcospira (Troschel, 
1858: pi. 9, fig. 6: Köhler & Glaubrecht, 2005), 
Pseudopotamis (Glaubrecht & Rintelen, 
2003), and Tylomelania (Rintelen & Glaub- 
recht, 2005), shows little variation of radula 
patterns within the family. Nonetheless, it has 
also been shown that denticle shape and size 
as well as radular length may vary consider- 
able even between closely related species if 
they occur in sympatry but feed on different 
substrata (Glaubrecht & Köhler, 2004: 
Rintelen et al., 2004: Rintelen & Glaubrecht, 
2005). 

The generalized radular pattern observed in 
most Brotia species comprises a central tooth 
with a well-developed glabella and a cutting 
edge comprising one main denticle flanked by 
up to three accessory cusps that taper in size, 



a lateral tooth exhibiting a glabella and a main 
denticle flanked frequently by two inner and 
two to three outer accessory cusps, as well as 
the inner and outer marginals, each with two 
cusps. These cusps may be rather of the 
same size or the outer cusp is enlarged. There 
are several other structures, for example, lat- 
eral extensions of the central and lateral tooth 
or a lateral flange of the marginal teeth that 
show a certain degree of variability among dif- 
ferent species. Furthermore, the shape of the 
glabella of the main denticle varies among 
species. In general, the range of variation 
within Brotia is rather small, though, and only 
rarely some radular features are species spe- 
cific. Most conspicuous modifications of the 
radula are connected to the substratum 
(Glaubrecht & Köhler, 2004: Rintelen et al., 
2004). In rock-dwelling species, cusps may be 
enlarged, blunt or broadly round (e.g., in B. 
pagodula), whereas species living on soft sub- 
strata may possess much smaller denticles as 
well as radular teeth (e.g., ß. microsculpta). 

Gross Anatomy 

The general appearance of the soft body and 
general organisation of the mantle cavity is 
rather constant among southeast Asian 
pachychilids and corresponds largely to the 
description given for Brotia. A feature typical 
for the Pachychilidae is the smooth mantle 
edge, which clearly differs from the papillated 
mantle edge found in Thiaridae. Among Pa- 
chychilidae, Jagora. Tylomelania. Melanatria, 
and Pachychilus differ from Brotia. Adamietta, 
and Paracrostoma in possessing a fleshy flap 
at the inner surface of the mantle roof. It has 
been suggested that this flap has a function 
for the formation of clutch masses during egg 
laying: therefore, it would have no function in 
viviparous species (Houbrick, 1 991 ). In Jagora, 
it still might be functional, perhaps to prevent 
egg capsules and juveniles from becoming dis- 
located from the mantle cavity in which they 
are retained. Another structure connected to 
reproduction is the genital groove at the right 
side of the head, which is found not only in 
pachychilids, but also Melanopsidae and 
Potamididae. While in egg laying species, this 
groove is involved in egg deposition, in Brotia 
it is needed to transfer eggs from the palliai 
oviduct to the brood pouch (Fig. 5F). 

Reproductive Organs: These are the most in- 
formative for pachychilid systematics (Köhler 
et al., 2004). Although all Asian Pachy- 



SYSTEMATIC REVISION OF BROTIA 



241 



chilidae are viviparous, brooding structures 
are not homologous among several groups. 
The subhaemocoelic brood pouch found in 
ßroi/a was first mentioned by Martens (1897: 
29). Later, Moore (1899: 161, 162; pi. 14, 
fig. 13; pi. 16, fig. 2), Morrison (1954: 383), 
Davis (1971: 69), and Köhler & Glaubrecht 
(2001) described this pouch in more detail. 
A homologous brood pouch is found in 
Adamietta (Brandt, 1974) and Paracrostoma 
(unpubl. data) and is considered as a 
synapomorphy of the Asia mainland clade 
among the Pachychilidae (Köhler et al., 
2004). 

No homologous incubatory structures are 
possessed by other Asian Pachychilidae or 
other freshwater cerithioideans. The Philip- 
pine pachychilid Jagora broods in the mantle 
cavity (Köhler & Glaubrecht, 2003), while 
pachychilid Pseudopotamis ana Tylomelania 
possess a uterine brood pouch (Glaubrecht 
& Rintelen, 2003; Rintelen & Glaubrecht, 
2005). Since oviparity is suggested to rep- 
resent a plesiomorphic character state in the 
Pachychilidae, brooding in turn must have 
evolved three times independently in this 
family (Köhler et al., 2004). In the Thiaridae 
and viviparous Planaxidae, a subhaemo- 
coelic brood pouch very similar to that of 
Brotia is found. While this brood pouch was 
discussed as representing a possible 
synapomorphy of a clade comprising 
Planaxidae and Thiaridae (e.g., Houbrick, 
1988; Glaubrecht, 1996; Simone, 2001), re- 
cent phylogenetic studies suggest that these 
are convergent (Lydeard et al., 2002; Köhler 
et al., 2004), The presence of a subhaemo- 
coelic brood pouch in Brotia was furthermore 
a reason for erroneously placing Brotia within 
the Thiaridae (e.g., Morrison, 1954; Benthem 
Jutting, 1956; Brandt, 1968, 1974). 

Other informative structures of the repro- 
ductive morphology include the palliai ovi- 
duct and the arrangement of the gonads. 
Among Asian Pachychilidae, Brotia pos- 
sesses the simplest palliai oviduct. 
Paracrostoma differs by a distinct 
organisation of the sperm gutter (unpubl. 
data), which is located more posteriorly. 
Adamietta possesses a seminal receptacle 
in addition to a spermatophore bursa, which 
is present also in Brotia (Köhler & 
Glaubrecht, 2001 , for the Brotia testudinaria 
group). Again, Jagora, Pseudopotamis, and 
Tylomelania possess oviduct morphologies 
that significantly deviate from Brotia 
(Glaubrecht & Rintelen, 2003; Köhler & 



Glaubrecht, 2003; Rintelen & Glaubrecht, 
2005). 

Stomach: Midgut morphology recently 
emerged as an yet untapped source of phy- 
logenetic information, at least when groups 
of higher taxonomic ranks are compared 
(e.g., Simone, 2001; Strong, 2003). Various 
features of the stomach, such as a laminated 
crescent sorting area with two adjacent cres- 
cent and septate thickenings, a lateral and 
marginal fold, a single digestive gland duct, 
and two crescent ridges posterior to the open- 
ing of the digestive gland duct are consid- 
ered synapomorphic among Pachychilidae 
(Strong & Glaubrecht, 1999). However, these 
features show little variation among the dif- 
ferent genera as can be judged from the fig- 
ures and descriptions for Potadoma (Binder, 
1959), Pachychilus (Simone, 2001), Jagora 
(Köhler & Glaubrecht, 2003), and Tylomela- 
nia (Rintelen & Glaubrecht, 2005), and we 
were not able to identify characters that can 
be considered as diagnostic for species of 
Brotia. 

Molecular Phylogeny of Brotia 

The number of species included into phylo- 
genetic analyses of molecular data is limited 
because of the restricted availability of mate- 
rial suitable for sequencing. For instance, it 
was not possible to extract high molecular DNA 
from preserved museum material. Nonethe- 
less, mitochondrial DNA from a total of 48 
samples of 1 6 Thai and Sumatran Brotia taxa, 
as well as 6 further pachychilid taxa from Asia 
mainland, were sequenced and analysed. 
Sequences of two Jagora species were in- 
cluded as outgroup representatives. 

The mitochondrial trees unambiguously cor- 
roborate the monophyly of Brotia as restricted 
herein by morphology with regard to other 
pachychilid genera included in the analyses 
(i.e., Jagora, Paracrostoma, Adamietta). In this 
respect, it is important to bear in mind that also 
the concepts of the latter two genera - 
Paracrostoma and Adamietta - are subject to 
changes in regard to previous treatments, for 
example, by Solem (1966) and Brandt (1968, 
1974). For instance, some species that were 
affiliated with Paracrostoma because of their 
concial shell were transferred to Brotia by 
Glaubrecht & Köhler (2004). Paracrostoma is 
now restricted to its type species, P. huegelii, 
and some yet undescribed species (Köhler, 
unpubl. data) endemic to southern India. 



242 



KÖHLER & GLAUBRECHT 



While on generic level the phylogeny strongly 
supports the classification based on the mor- 
phology, problems mainly occur as to the iden- 
tification of some species-level taxa, in 
particular among the Kaek River radiation 
(Figs. 78, 79). This radiation comprises at least 
seven species recognized by a divergent shell 
and radular morphology, such as 8. armata, 
B. binodosa. and B. microsculpta. However, 
sequence divergence among these taxa is 
very low, which is considered the main rea- 
son for the observed mismatch between the 
topology of the mitochondrial gene tree and 
the presumed species identity of these taxa 
as based on their morphology. Low genetic 
divergence indicates a relatively recent origin 
of the Kaek River radiation, and incomplete 
lineage sorting is the most likely explanation 
for the unresolved mitochondrial gene tree 
(Glaubrecht & Köhler, 2004). In order to get 
better resolved molecular reconstructions, it 
has been suggested to analyse different ge- 
netic markers and to use a different method- 
ology, that is. AFLP genotyping. 

Looking beyond the Kaek River species 
flock, all other Brotia species recognized by 
their morphology are also resolved as mono- 
phyletic entities in the mitochondrial gene 
trees. There is only one exception, B. citrina, 
the two sequences of which are shown as a 
paraphylum in the MP tree. In the distance- 
based trees, however, these sequences clus- 
ter together as a sister pair, which supports 
our treatment of the two populations as being 
conspecific. The mismatch in the MP tree 
therefore is no reason to doubt in the correct 
determination of B. citrina. 

Infraspecific sequence divergence among 
Brotia species calculated unter the GTR+1 +Г 
model of sequence evolution does not exceed 
a maximum of 16% in CGI and 29% in 16S, 
but mostly values are clearly smaller. Not con- 
sidered is the unusual high sequence diver- 
gence of one of the two sequences of B. 
sumatrensis, which is caused by numerous 
peculiar substitutions in this sequence. Since 
a similar divergence is not observed in 16S, 
technical failure in sequencing cannot be ruled 
out. 

Rates of sequence divergence reported here 
for Brotia, although difficult to compare since 
different models of gene evolution were ap- 
plied by different studies, does exceed the lim- 
its observed in other freshwater cerithioideans 
(e.g., Pleuroceridae; Lydeard et al. 1997; 
Holznagel & Lydeard, 2000), but is similar to 
infaspecific sequence divergences observed 



in other Pachychilidae (e.g., Köhler & Glaub- 
recht, 2003; Glaubrecht & Rintelen, 2003). 

Interestingly, in Brotia morphological dispar- 
ity and genetic differentiation obviously are not 
linked to each other. Instead, two extremes are 
observed with the morphologically diverse but 
genetically rather undifferentiated Kaek River 
species flock on one hand and with species 
such as 6. citrina and B. pagodula on the other, 
which show a low degree of morphological plas- 
ticity but a high degree of genetic differentia- 
tion. This phenomenon can probably be 
explained by strong competition and low 
prezygotic isolation (by means of geographical 
separation) between different sympatric taxa in 
the first case and absence of competition and 
relatively strong geographical separation be- 
tween different conspecific populations in the 
latter case. This significant variation of infraspe- 
cific sequence divergences among Brotia 
shows with which problems approaches are 
fraught that aim at delimiting species only by 
the use of genetic distances (for further discus- 
sion of the merits and limits of DNA taxonomy 
the reader is refered to the contributions of, e.g., 
Lipscomb et al., 2003; Seberg et al., 2003; Tautz 
étal., 2003; Blaxter, 2004). 

Systematic Implications 

(1) Family Placement 

The familiar placement of Brotia was sub- 
ject to controversy caused by a mélange of 
rival systematic opinions as well as taxonomic 
difficulties. In an attempt to clarify the confu- 
sion, we shortly revise phylogenetic and sys- 
tematic aspects on one hand and taxonomic 
issues on the other. 

In the first attempts to classify what we call 
today cerithioidean freshwater gastropods all 
species were placed in a single group called 
Melanien or melanians, later also Melaniidae 
(e.g., Lamarck, 1822; Brot, 1874). This huge 
assemblage was subsequently subdivided into 
different groupings according to diagnostic 
features of their shell, operculum, and radula 
(e.g.,Troschel, 1856-1863; Fischer & Crosse, 
1891 1892; Thiele, 1928, 1929-1935); but 
Melaniidae were still considered a large 
monophylum. Fischer & Crosse (1891-1892) 
as well as Thiele (1928, 1929-1935) recog- 
nized six different lineages within the 
Melaniidae, among them a group already char- 
acterized by Troschel (1857) as "Pachychili" 
that comprises, for example, Pacliyctiilus, 
Potadoma, Melanatria, and Sulcospira. This 



SYSTEMATIC REVISION OF BROTIA 



243 



group was ranked as a subfamily Pachy- 
chilinae of the Melaniidae according to the 
name introduced byTroschel. Morrison (1954), 
however, who strongly influenced most 20'" 
century authors, recognized only three lin- 
eages and placed representatives of the 
"Pachychili" within two different clades, that 
is, the Pleuroceridae {Pachychilus and 
Potadoma) and the Thiaridae (Sulcospira, 
Antimelania, and Brotia). Later authors fol- 
lowed Morrison and treated Neotropical taxa 
as member of the Pleuroceridae (e.g., Vaught, 
1989; Simone, 2001), but Asian taxa as 
Thiaridae (e.g., Solem, 1966; Davis, 1971; 
Brandt, 1968, 1974; Burch, 1980). This con- 
cept initially seemed to gain support even from 
a first cladistic analysis of morphological data 
presented by Houbrick (1988). In this analy- 
sis, which was to a large part based on mor- 
phological data presented by Morrison (1954), 
two major and independent freshwater lin- 
eages within the Cerithioidea were recognized, 
that is (1) Pleuroceridae + Melanopsidae and 
(2) Thiaridae. Brotia was affiliated with the lat- 
ter for possessing a subhaemocoelic brood 
pouch. First doubts in this view have been 
raised by another cladistic analysis of morpho- 
logical data (Glaubrecht, 1996), which re- 
vealed a new group besides Thiaridae and 
Melanopsidae (while Pleuroceridae were not 
included): the Pachychilidae. However, in this 
study only the oviparous taxa Pachychilus, 
Doryssa, Melanatha, and Potadoma \Nere sub- 
sumed under the Pachychilidae, whereas the 
viviparous Brotia still was considered a thiarid. 
A third cladistic analysis of morphological data 
(Simone, 2001 ) supports the existence of ex- 
actly this monophyletic freshwater group com- 
prising Pachychilus and Doryssa as being 
clearly distinct from the Thiaridae (with 
Melanoides and Aylacostoma). However, in 
this study the (wrong) name "Pleuroceridae" 
was employed for this lineage. 

Molecular genetic studies helped much to 
clarify aspects of cerithioidean phylogeny. The 
most comprehensive phylogeny based on mi- 
tochondrial sequence data was so far pre- 
sented by Lydeard et al. (2002). This study 
provided further evidence for the existence of 
at least three distinct freshwater lineages, (1 ) 
the Thiaridae, (2) the Melanopsidae + 
Pleuroceridae, and (3) an unnamed group 
comprising Pachychilus and Paracrostoma. 
This clear evidence unfortunately was ob- 
scured by application of a misleading tax- 
onomy: Although forming a distinct lineage, 



Pachychilus and Paracrostoma were 
uncritically treated as members of Pleuro- 
ceridae and Thiaridae, respectively. As a con- 
sequence, all freshwater cerithioidean 
lineages were seemingly rendered polyphyl- 
etic, while a more restricted application of 
names would have unmistakably shown that 
they are in fact all monophyletic. 

Direct comparison of the different phyloge- 
netic studies is complicated by their deviant 
taxon composition. However, a closer look 
reveals that there is strong evidence for the 
existence of a monophyletic freshwater lineage 
beside the (1 ) Thiaridae and (2) Pleuroceridae 
+ Melanopsidae, constituted by taxa such as 
Pachychilus, Doryssa, or Paracrostoma 
(Glaubrecht, 1996; Simone, 2001; Lydeard et 
al., 2002). All three studies failed to name this 
lineage properly, though. The names Thiaridae 
and Pleuroceridae although formerly used 
certainly are not available for this group, since 
they refer to the other two freshwater clades. 

As the oldest name for this "new" lineage 
the name "Pachychili" was introduced by 
Troschel (1857) and later used as 
Pachychilinae by Fischer & Crosse (1891). 
Thiele (1 921 ), who believed the name Pachy- 
chilinae to be invalid since he erroneously 
considered the generic name Pachychilus Lea, 
1850, for neotropical "melaniids" as being pre- 
occupied by Pachychila Eschscholtz, 1831, 
also recognized this taxon but suggested 
"Melanatriinae" as a replacement name. For 
a different reasoning against the validity of the 
name "Pachychilidae" with Troschel (1857) as 
author, see Bouchet & Rocroi (2005) as well 
as the introductory remarks in this article. 

In contrast to Thiele (1921, 1925, 1928) we 
consider the name Pachychilidae as available 
and valid. Consequently, Melanatriinae is a 
synonym of Pachychilidae (Köhler & Glaub- 
recht, 2002, 2002a). 

Eventually, Thiele (1925: 83) noticed that 
Brotia is member of this group besides, for 
example, Pachychilus and Melanatria, based 
on radular and opercular features. This is sup- 
ported by a molecular phylogeny showing the 
close affinity of the Asian taxa, such as Brotia, 
with the Neotropical taxa, such as Pachychilus. 
This provided strong evidence for the exist- 
ence of the clade named Pachychilidae 
(Köhler et al., 2004). As a consequence, the 
view of Morrison (1 954) and Houbrick (1 988), 
who strongly emphasized features of the soft 
body, in particular of the reproductive tract, on 
the systematic position of Brotia is refuted. 



244 



KÖHLER & GLAUBRECHT 



Morphological comparison of Brotia with other 
freshwater cerithioideans reveals that it shares 
as a synapomorphic character a widely corre- 
sponding operculum and radular morphology 
with oviparous pachychilids. such as Pachy- 
chilus. This also means that a subhaemocoelic 
brood pouch in Brotia has evolved in conver- 
gence to a similar structure found in the 
Thiahdae (Köhler & Glaubrecht. 2001 ; Köhler 
et al., 2004). 

(2) Phylogenese Relationships among Asian 
Pachychilidae 

Traditionally almost all Asian pachychilid 
species sooner or later were attributed to 
Brotia by one or the other author. This was 
done in absence of a phylogenetic reconstruc- 
tion, which would allow to identify autapo- 
morphic features and lead to an inflated 
concept of Brotia. which in its conventional 
understanding by Rensch (1934), Abbott 
(1948), Benthem Jutting (1956), Brandt (1968, 
1974), and Davis (1971) is rendered a poly- 
phyletic grouping. 

In a preliminary study, Köhler & Glaubrecht 
(2001 ) identified four different species groups 
among what was previously considered as 
constituting Brotia. which most conspicuously 
are characterized by peculiarities of their re- 
productive tract, their incubatory anatomy, and 
their embryonic shell. In concert with molecu- 
lar genetic analyses it has been shown that 
these groups represent independent and 
monophyletic evolutionary lineages. The con- 
spicuous morphological differences between 
and different evolutionary histories of these lin- 
eages justify the treatment as independent 
genera (Köhler & Glaubrecht, 2003; Glaub- 
recht & Rintelen. 2003: Köhler et al., 2004; 
Rintelen & Glaubrecht, 2005). According to this 
revised and more specific concept, Brotia is 
here restricted to pachychilid species possess- 
ing diagnostic characteristics, such as a 
wrinkled apical whorl of the embryonic shell 
and a simple palliai oviduct with a deep, cili- 
ated spermatophore bursa but without a semi- 
nal receptacle. Besides Brotia there are six 
further pachychilid genera mainly recognized 
on basis of a divergent reproductive and em- 
bryonic shell morphology. Some of them have 
already been systematically revised, such as 
(1 ) Jagora endemic to the Philippines (Köhler 
& Glaubrecht, 2003), (2) Tylomelania endemic 
to Sulawesi (Rintelen & Glaubrecht, 2005), (3) 
Pseudopotamis endemic to the Torres Strait 
Islands (Glaubrecht & Rintelen, 2003), and (4) 



Sulcospira endemic to Java (Köhler & 
Glaubrecht, 2005). Irrespective of the fact that 
a formal revision of the two remaining genera, 
(5) Adamietta and (6) Paracrostoma. still is 
pending, it is suggested on basis of a molecu- 
lar phylogeny of the Pachychilidae that they 
are also distinct (Köhler et al., 2004). This sug- 
gestion is corroborated by published and also 
unpublished morphological data (Köhler & 
Glaubrecht, 2001; Köhler, unpubl, data). 

Together with these latter two genera Brotia 
forms a monophyletic lineage, the Southeast 
Asia mainland clade, which is characterized 
by possession of a subhaemocoelic brood 
pouch as synapomorphic feature (see Köhler 
et al., 2004). 

Revised Concept of Brotia 

What remains of Brotia under the restricted 
concept, still is a diverse group comprising at 
least 27 species that ranges from northeast 
India through Bangladesh, Myanmar, Thailand, 
and the Malaysian Peninsula to Sumatra, 
Borneo, and perhaps even Java. Systematic 
affinities of eight additional species remain to 
be clarified. 

A subdivision into three subgenera as sug- 
gested by Brandt (1 974) is refuted by the cur- 
rent study. Brandt suggested ranking two taxa, 
Paracrostoma and Senckenbergia, as subgen- 
era of Brotia. This treatment is supported nei- 
ther by morphological nor by molecular genetic 
data. In fact, Paracrostoma represents a 
monophyletic group closely related to Brotia 
but definitely distinct, as is revealed by the 
mitochondrial phylogeny (Figs. 78, 79; Köhler 
et al.. 2004). All Thai species affiliated with 
Paracrostoma by Solem (1966) and Brandt 
(1968, 1974) are members of Brof/a since they 
are not closely related to Paracrostoma from 
southern India but cluster together within 
Brotia (Glaubrecht & Köhler, 2004). 

Type species of Senckenbergia is Melania 
pleuroceroides Bavay & Dautzenberg, 1910, 
a species from the Yangtze-Kiang. This spe- 
cies was stated to possess an operculum simi- 
lar to Semisulcospira, which is a pleurocerid 
(Yen, 1939; 55). Since the Yangtze-Kiang is 
far out of the range of Brotia, and since also 
the operculum of its type species is of a 
pleurocerid type, Senckenbergia cannot be 
considered as a member of Brotia. A species 
originally assigned to Senckenbergia by 
Brandt (1974) is herein treated as Brotia 
wykoffi in regard to its morphology and posi- 
tion in the molecular trees. 



SYSTEMATIC REVISION OF BROTIA 



245 



In comparison to concepts used by former 
revising authors, that is, mainly Brandt (1968, 
1974), the current study shows that assump- 
tions on the morphological variability and geo- 
graphical range of single species were 
exaggerated. For instance, Rensch (1934), 
Benthem Jutting (1956), and Brandt (1974) 
believed B. costula to be a highly variable spe- 
cies that occurs across entire Southeast Asia 
from India to the Philippines and even on some 
oceanic islands. It has been shown, however, 
that this species is much more restricted in its 
occurrence and also in respect to its morpho- 
logical properties. Still, there are a number of 
named forms that preliminary remain as syn- 
onyms of this as well as of other species, al- 
though their distinct shells might indicate that 
they in fact represent independent species. 
This holds true, for example, for B. reevei 
(treated as synonym of B. hercúlea) and B. 
elongata (treated as synonym of B. henriettae). 
However, any decision on the status of these 
and other named forms in absence of prop- 
erly preserved material would be rendered 
rather a matter of opinion. For this reason and 
in order to not further complicate the taxonomy 
of this group, we here follow the usual treat- 
ment of those taxa by former authors. In this 
respect, we are convinced that future studies 
will be able to recognize further, yet vaguely 
defined or unknown species within Brotia. 

Conclusions 

In summary, 27 species of Brotia are recog- 
nized in this work and eight additional species 
are presented with uncertain affinities. Using 
morphological and molecular data, the char- 
acteristics of Brotia are specified, and many 
species are newly delimited. Former system- 
atic concepts are discussed and corrected 
accordingly. The current study results in an 
altered and more restricted concept of Brotia 
in comparison to former suggestions. It fur- 
ther shows that the subdivision into several 
subgenera as suggested by Brandt (1974) is 
erroneous. The new systematic concept is 
relevant also from a biogeographical perspec- 
tive. While it has been assumed before that 
the range of Brotia covers almost entire South 
and Southeast Asia, it now becomes clear that 
its distribution is actually much more restricted. 
Thus, Brotia appears to be distributed mainly 
to the west of continental Southeast Asia rang- 
ing from northeast India (Assam, Sikkim, 
Meghalaya) and Bangladesh to central Thai- 
land and the Malaysian Peninsula in the east. 



It is in the latter area where Brotia reaches its 
highest diversity. In the south, Sumatra, Java, 
and Borneo, comprising parts of former 
Sundaland, are within its distributional area. 
Among these three areas, Sumatra supports 
the highest diversity of species, forming a 
monophyletic subgroup, while from Java and 
Borneo only few species are known. As a rule, 
reports from Java and Borneo are not con- 
firmed by collections after about 1920. If and 
how far the distribution of Brotia ranges to- 
wards the east of continental Asia (to Laos, 
Cambodia, southern China, and Vietnam) re- 
mains to be studied. 



ACKNOWLEDGEMENTS 

For technical assistance, we record our grati- 
tude to the colleagues at the Natural History 
Museum, Berlin, in particular to Gabriele 
Drescher, Robert Schreiber, Sabine Schutt, 
and Christine Zorn. Special thanks we owe to 
Yves Finet (Geneva), David Reid and David 
Brown (London) for various advices in tracing 
material and literature as well as for valuable 
comments. Ellen Strong shared her insights 
into gastropod morphology, and Thomas von 
Rintelen his knowledge in molecular genetics. 

Types and various other materials examined 
in this study were provided from various mu- 
seum collections worldwide. Therefore, we 
thank Adam Baldinger (Cambridge, Mass.), 
Philippe Bouchet (Paris), Yves Finet (Geneva), 
Edmund Gittenberger, Jeroen Goud, and Wim 
Maassen (Leiden), Ambros Hänggi (Basel), 
Bernhard Hausdorf (Hamburg), Robert 
Hershler (Washington), Roland Janssen 
(Frankfurt/Main), Edward Kools (San Fran- 
cisco), Elisabeth Kuster-Wendenburg 
(Bremen), Ristiyanti Marwoto (Bogor), Trudi 
Meier (Zurich), Robert Moolenbeek (Amster- 
dam), Gary Rosenberg (Philadelphia), 
Bernhard Ruthensteiner (Munich), Winston 
Ponder and Ian Loch (Sydney) as well as Fred 
Naggs, Joan Pickering, and Kathie Way (Lon- 
don) for making available materials from col- 
lections in their charge. Ulrich Bößneck kindly 
provided some material from his private col- 
lection, which facilitated the study of Brotia 
costula. 

A number of photographs were kindly pro- 
vided for publication by Vera Heinrich (Ber- 
lin), Pierre Lozouet (Paris), C. Ratton 
(Geneva), and the Natural History Museum 
(London). Financially this study was supported 
by a post-graduate scholarship and a travel 



246 



KÖHLER & GLAUBRECHT 



grant of the Konrad-Adenauer-Stiftung to FK 
as well as by a research grant of the Deutsche 
Forschungsgemeinschaft (DFG) to MG (GL 
297/4-1 and 4-2), which is thankfully acknowl- 
edged. Visits of the first author to the Natural 
History Museum. London and the Museum 
National d'Histoire Naturelle. Paris were fi- 
nanced by the Bioresource and the Parsyst 
Programme of the European Union, respec- 
tively. David Reid. Chris Jones, and Zeta Field 
(London) as well as Philippe Bouchet and 
Virginie Héros (Paris) offered kind support 
during these visits. 

Most valuable comments of three referees, 
George M. Davis, and Eugene V. Coan helped 
much to improve the quality of this article. Their 
effort to critically and carefully read the entire 
manuscript is most thankfully acknowledged. 



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Revised ms. accepted 10 October 2005 



MALACOLOGIA, 2006, 48(1-2): 253-264 

ECOGENETICS OF SHELL SCULPTURE IN ONCOMELANIA (GASTROPODA) 

IN CANALS OF HUBEI, CHINA, AND RELEVANCE FOR 

SCHISTOSOME TRANSMISSION 

George M. Davis\ Wei-Ping Wu^ & Xing-Jian Xu^ 

ABSTRACT 

Oncomelania hupensis in China is well known as the intermediate host of the human 
blood parasite Schistosoma japonicum. There are three subspecies on the mainland of 
China with discrete patterns of distribution: above the Three Gorges of the Yangtze River 
(O. h. robertsoni) in Yunnan and Sichuan provinces; below the Three Gorges along the 
Yangtze River drainage (O. h. hupensis) with an incursion into Guangxi Province; and 
Rujian Province along the coast (O. h. tangi). Of these taxa, only O. h. hupensis has ribbed 
shells. Until now, O. h. hupensis has been shown to be dimorphic, with ribbed-shelled 
aggregates of individuals on flood plains and smooth-shelled populations in habitats el- 
evated above the effects of floods or removed from the effects of severe annual floods by 
barriers. Molecular population genetics and anatomical studies have shown that there are 
no significant genetic differences between the two O. h. hupensis morphs; they belong to 
the same species (Davis et al., 1999b; Shi et al., 2002). Evidence to date has also shown 
that the ribbed-shelled aggregates of individuals are not true populations and are highly 
susceptible to infection with the parasite, whereas smooth-shelled populations have lesser 
potential to be infected, grading to total resistance. 

We recently found in two canals of Hubei, well buffered from the annual Yangtze River 
floods, isolated populations that are truly polymorphic, with three to five classes of shell 
sculpture. The two canals were significantly different in their polymorphisms in 2001 (single 
sample per canal) and in 2004 (multiple samples within canals). We know the history of 
the construction of these canals (14 and 21 years ago, respectively), and the only avail- 
able pathway of colonization of these canals (from the Yangtze River through the Guan Yin 
flood gate into the primary Hong Chou Canal). The colonizing snails were most probably 
derived from strongly ribbed snails of the adjacent flood plains. The changes from heavily 
ribbed to nearly smooth had to occur within the short span of 14 to 21 years. There were 
significant differences within canals in 2004 when multiple samples were taken. 

The purpose of this paper is to present base-line data on this first reported case of shell 
sculptural polymorphism within O. h. hupensis, with the hypothesis that in the absence of 
sever flooding selection, this taxon will rapidly change from heavily-ribbed shells to slightly- 
ribbed to the smooth-shelled condition. Further, these changes give insight into questions 
of population evolution and coevolution with Schistosoma japonicum, in which smooth- 
ness is associated with genetic stability (defined in Davis, 1999a) that leads to the reduced 
potential to transmit the parasite (under coevolutionary pressure) and, in some instances, 
the evolved refractiveness to transmission. 

Keywords: schistosomiasis, Schistosoma japonicum, China, polymorphism, Oncomela- 
nia, evolution, population structure, coevolution, Red Queen. 

INTRODUCTION fluke Schistosoma japonicum afflicting man 

and other mammals. There are three subspe- 

Oncomelania hupensis in China is well cies with discrete patterns of distribution on 

known as the intermediate host for the blood the mainland of China (reviewed in Davis, 

'Department of Microbiology and Tropical Medicine, George Washington University Medical Center, Washington, D.C. 20037, 

U.S.A.; mtmgmd@gwumc.edu 
^National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, 207 Ruijin Er Lu. 200025 

Shanghai, China 
^Hubei Institute of Schistosomiasis Control, Zhoudaoquan, Bei Lu 6, Wuchang, 430079 Wuhan, Hubei, China 

253 



254 



DAVIS ETAL. 



1992, 1994: Davis et al., 1995, 1999a). Ail 
three transmit Schistosoma japonicum strains. 
Oncomelania hupensis hupensis is found 
throughout the Yangtze River drainage system 
below the Three Gorges of the river. Onco- 
melania h. robertsoni is located in the high- 
lands and mountains of Yunnan and Sichuan 
provinces above the Three Gorges. Onco- 
melania h. fang/ lives in coastal areas of Fujian 
Province, isolated from the Yangtze River by a 
mountain range. 

Oncomelania with its two species, and O. 
hupensis with several subspecies distributed 
in Japan, Philippines, Celebes, Taiwan, are 
taxa that, with one exception, have smooth 
shells, as seen in the other genera of the fam- 
ily Pomatiopsidae (Davis, 1979. 1980, 1992: 
Davis et al., 1999a). The exception is found in 
populations of O. hupensis hupensis of the 
flood plains of the Yangtze River and its tribu- 
tanes. Such flood-challenged populations have 
prominent ribs. Additionally, flood-plain O. h. 
hupensis has longer, heavier shells than the 
other subspecies, especially O. h. robertsoni, 
with very small shells and no vahx (special 
thicl<ening of the outer shell lip). 

Until now, O. h. hupensis has been shown 
to be dimorphic, with ribbed-shelled aggre- 
gates of individuals on flood plains and 
smooth-shelled populations in habitats el- 
evated above the effects of flooding. Molecu- 
lar population genetics and anatomical studies 
show that there are no significant genetic dif- 
ferences between the smooth-shelled and 
ribbed-shelled populations; they belong to the 
same subspecies (Davis et al., 1995, 1999b, 
using allozymes: Shi et al., 2002, using mito- 
chondrial COI gene sequencing). Based on 
breeding genetics, ribbing in Oncomelania 
hupensis is controlled by a single locus (Men- 
delian inheritance of a single gene) where nbs 



TABLE 1 . X^ comparison of shell polymorphisms 
of shell ribbing on Oncomelania hupensis 
hupensis snails from two Hubei Canals in 2001 , 
N = number of shells from living snails. Data 
given as % of N. P > 0.0001 . See text for details. 



Ma Ling 
(N = 101) 



M 

SL 

S-/S 



13.9 
61.3 
24.8 



Gu Hu 
(N = 105) 


43.8 
40.0 
16.2 



are dominant, smooth recessive, and with mul- 
tiple alleles ofthat gene (Davis & Ruff, 1973). 
Lil<ewise, size is controlled by alleles at a 
single locus. 

All evidence indicates that ribbing is an 
evolved response to heavy annual flooding, 
that is, ribbing is maintained by natural selec- 
tion. The hypothesis is that increased size and 
ribbing of flood plain individuals confer a se- 
lective advantage by way of strengthening the 
shells and enabling flotation to survive flood- 
ing (reviewed in Davis et al., 1999a, b). 

We are currently studying the ecogenetics 
of Schistosoma transmission in two selected 
inner "tertiary" canals of Hubei, because they 
have an environment that is the most buffered 
from the ravages of the annual floods of the 
Yangtze River. Additionally, numbers of these 
canals, at the same or lower elevation as the 
Yangtze River, are relatively recently con- 
structed and thus provide an opportunity to 
study a number of unique factors impacting 
disease transmission. 

In October 2001 , while selecting study sites, 
we found populations of Oncomelania hupen- 
sis hupensis in two unconnected canals, 2.7 
km apart, that had shells that were polymor- 
phic for ribbing. These canals were chosen 
because they are part of a Schistosoma japo- 
nicum endemic area, with infected snails in 
these canals, and the canals are far removed 
from the influence of the Yangtze River. We 
scored shells from a single population from 
each canal for strength of ribbing and found 
that three to four classes of ribbing could be 
identified. Further, the populations had signifi- 
cantly different frequencies of the morphs 
(Table 1, P> 0.0001). 

The purpose of this paper is to present ini- 
tial base-line data derived from analysis of 
shell ribbing in populations along these two 
Hubei canals (both the 2001 and 2004 data), 
and to demonstrate that within populations 
there are polymorphisms that we hypothesize 
to be stages of loss of ribbing in the absence 
of flooding selection. Further, the polymor- 
phisms found give insight into questions of 
population evolution and coevolution involv- 
ing (1) the timing of reversion from ribbing to 
smoothness: (2) the coevolution of Oncomela- 
nia hupensis hupensis with Schistosoma 
japonicum, in which smoothness is associated 
with genetic stability (defined in Davis et al., 
1999a) leading to the reduced potential to 
transmit the parasite, and in some situations, 
the evolved refractiveness to transmission. 



ECOGENETICS OF SHELL SCULPTURE IN ONCOMELANIA 



255 



METHODS 

Canal Locations and Descriptions 

The canals are located in the administrative 
villages of Gu Hu and Ma Ling of Sha Shi Dis- 
trict, Jingzhou City, Hubei. The Gu Hu Canal 
(30°19.129'N,112°23.386'E at mid-canal) is 1.6 
km long. It is oriented E-W (95°-275° true). 
The Ma Ling Canal (30°19.578'N; 112°21 .270'E 
at mid-canal) is 1.48 km long, with a N-S ori- 
entation (187°-8° true). Both canals are abso- 
lutely straight. We divide the Ma Ling Canal 
into two zones, the dividing point being where 
the canal is interrupted by a wider canal, the 
Wu Yi Canal. The right angle Intersection is 
open in all four directions. Water from the Ma 
Ling-Wu Yi juncture flows through a pipe un- 
der the road, which parallels the Wu Yi Canal, 
to flow N 1 km to dead-end at the end of zone 
1 of the Ma Ling Canal. At high water, this 
northern zone acts as a drainage canal. If 
water levels get too high, water is pumped from 
the northern end to the immense Si Hu Canal 
on the other side of a high dyke. The Si Hu, 
built pre 1 960, is a major drainage canal flow- 



ing east. Zone 2 runs south of the Wu Yi Ca- 
nal and at low water it is separated from the 
Wu Yi Canal by a very low, man-made earthen 
dam that holds back water of zone 2 to form a 
duck pond. Further south, the standing water 
meanders between low banks with thick marsh 
grass providing an ideal marshy environment 
for snails. Zone 2 is about 280 m long. 

Both canals are about 4 to 5 m wide. The 
canals are separated by 2.7 km, with the Gu 
Hu Canal due E from the Ma Ling Canal. The 
unconnected canals are 2.4 km S of the vast 
Chang Lake and 1 2 km E of the Yangtze River. 
The canals in question are less than 22 years 
old. The Ma Ling Canal was built in 1984 and 
Gu Hu Canal in 1992. 

Sampling and Scoring 

Polymorphism data were an unintended and 
surprising byproduct of the primary purpose of 
our research on the long-term consequences 
of environmental change on the genetics and 
infectivity patterns of snails in recently con- 
structed and highly protected canal systems. 
Data were taken from the 2001 mass collec- 




FIG. 1 . Degrees of shell ribbing of Oncomelania hupensis hupensis. The schematic drawings (below) 
make clear the degree of ribbing strength and height that are very difficult to adequately portray in 
photographs or SEM pictures of shells (above). A: Strong ribs (S); B: Medium ribs (M); C: Slightly 
ribbed (SL); D: Trace of ribbing (S-); E: Smooth (SM). In D and E the "bumps" on the shell may be low 
swellings of a growth line or slightly elongated very low nodes. In smooth shells, the shell may be 
entirely smooth or, in few individuals, there may be one or two very low nodes or swellings indicating 
a highly degraded rib. 



256 



DAVIS ETAL. 



tion of snails taken to see If the sites were fit- 
ting for future study and from the experimental 
study initiated in 2004. 

For our primary purpose, snails were col- 
lected each collection period (twice a year) 
from a 2 m^ (a half frame, see Davis et al., 
2002) positioned at 20 randomly selected sites 
on either side of the canal, enabling estimates 
of snail density per m^ for each canal. Where 
there were ten or more snails per 4 m^^ (a 
frame) or > 2.5 snails per m^, (snails pooled 
from both sides of the canal) there were suffi- 
cient snails to score shells for one of five pos- 
sible shell sculpture conditions (Figs. 1A-E): 
smooth (SM), smooth with negligible trace of 
ribs (S-), slightly ribbed (SL), medium ribbed 
(M), and strongly ribbed (S). Strong ribs are 
the type of ribbing found on the Yangtze River 
flood plains (heavy shells, tall and thick ribs 
regularly positioned on each whorl). 

Of the 40 sites sampled, only five had ten or 
more snails per frame. Smooth shells vary 
from having a completely smooth shell sur- 
face to one which may have slight irregulari- 
ties as a low swelling (bump) or an irregular 
growth line Figure 1E. Between these ex- 
tremes (smooth or heavily ribbed) are three 
intermediate conditions (1) Negligible ribbing 
(Fig. 1D): the shell surface varies from com- 
pletely smooth to having one or two scattered 
irregular nodes, or low thin rib lines on the body 
and penultimate whorl indicating the position 
where a rib might develop. (2) Slightly ribbed 
(Fig. 1С): The shell surface has some irregu- 
larly placed low, thin ribs with some rib-nodes 
(undeveloped ribs). (3) Medium ribbed (Fig. 
1B): The penultimate and body whorls have 
regularly positioned fully developed low ribs 
on the entire whorls. These ribs are consider- 
ably lower than those found on heavily ribbed 
shells. 



Statistical Analysis 

Microsoft Excel was used for X^ analyses of 
morph frequencies. Given the small numbers 
in some cells, the Fisher Exact Test was used. 



RESULTS 

Results of Initial 2001 Exploratory Canal Ex- 
aminations 

On 25 October 2001, we collected snails 
from the first V3 of each canal closest to the 
main road. The snails were collected from a 
small area, about 90 m^ along one side of each 
canal for the purpose of seeing what they 
looked like and if they were infected. Exami- 
nation with a dissecting microscope at rela- 
tively low power showed the shells to have 
different patterns of ribbing. We could easily 
discern four types that we classified at that 
time as (1 ) smooth to trace of ribs (= types D 
and E, Fig. 1), (2) slightly ribbed, (3) medium 
ribbing. There were no strongly ribbed shells. 
The results of the scoring and the X^ analysis 
given in Table 1 show a highly significant dif- 
ference between the canals (P > 0.0001 ). The 
Fisher Exact Tests did not change the results. 
Ma Ling had significantly more slightly ribbed 
shells and significantly fewer medium ribbed 
shells than Gu Hu. This was the first time, to 
our knowledge, that such polymorphisms 
within populations of Oncomelania were found 
to exist. 

Results of the September 2004 Collection 

Scores for the four Ma Ling and one Gu Hu 
sites are given in Table 2 both for actual num- 
bers of snails scored and % of snails in each 



TABLE 2. Scoring snails for all sites for number and % snails with each morph category. S = strong 
ribs; M = medium ribbing; SL = slightly ribbed; 8- = trace of ribbing; SM = smooth. 



Site 1 
(N = 15) 



la Ling 



Site 17 
(N = 12) 



Site 19 
(N = 122) 



Site 20 
(N = 96) 



Gu Hu 

Site 16 
(N = 77) 



S 

M 

SL 

S- 

SM 




15(100%) 








1 (8.3%) 

10(83.3%) 

1 (8.3%) 





2(1.6%) 
31 (25.4%) 
74 (60.7%) 
14(11.5%) 

1 (0.8%) 





39 (40.6%) 

52 (54.2%) 

5 (5.2%) 







38 (49.4%) 

37(48.1%) 

2 (2.6%) 





ECOGENETICS OF SHELL SCULPTURE IN ONCOMELANIA 



257 



120 



100 



0) 




Q. 




>4 








Г 




Q. 


HO 






О 




E 




£ 








^ 


6U 


(Я 




0) 




^ 




(П 


4П 






о 





20 



Sites 




1 








HI 
D17 
■ 19 
3 20 




















E 


1 






1 




= 




Щ 


1 


1 


Tl , 1 ^^ 



M SL S- 

5 categories of morphs 



SM 



FIG. 2. The percent of morph types in the Ma Ling sites. S = strongly ribbed; M = medium 
ribbing; SL = slightly ribbed; S- = trace of ribbing; SM = smooth. 



of the five morph classes. The percentage of 
each morph type in the four Ma Ling sites is 
graphed (Fig. 2). Ma Ling site one, at the north- 
ern end of the canal (zone 1 , 1 .0 km from site 
1 7 close to the Wu Yi intersection), was unique 



in having only medium-ribbed shells, but the 
number of snails collected (15) was low. The 
remaining sites were separated by distances 
ranging from 25 m to 50 m between them. 
Snails from site 17 (zone 1 close to the Wu Yi 





160 




140 


w 

"(5 

с 

(Я 

о 


120 

100 

80 




z 


60 




40 




20 








2001 



2004 



P= 0.00013 



E 



1 I г 



"T I г 



M 



SL S-/SM S 

Classes of polymorphisms 



M 



SL S-/SM 



FIG. 3. Comparing Ma Ling snails from 2001 and 2004 (sites 17, 19, 20 combined data) for 
numbers of snails in each of four shell ribbing classes. See Fig. 2 for abbreviations. X^ = highly 
significant difference. 



258 



DAVIS ETAL. 



TABLE 3. Cross comparison of canals and localities for years 2001 and 2004 to determine level of 
significant differences among sites for polymorphic classes of shell sculpture. NS = not significant; 
BNS = barely not significant: HSD - highly significant difference: SD = significant difference: VSD = very 
significant difference. 





2001 


2001 


2004 


2004 


2004 


2004 


2004 




Ma Ling 


Gu Hu 


Gu H 


Ma Ling 1 


Ma Ling 17 


Ma Ling 19 


Ma Ling 20 


2001 
Ma Ling 


- 


HSD 


HSD 


HSD 


NS 


SD 


SD 


2001 
GuHu 




- 


SD 


HSD 


SD 


VSD 


SD 


2004 
GuHu 






- 


HSD 


SD 


VSD 


NS 


2004 
Ma Ling 1 








- 


HS 


HSD 


HSD 


2004 
Ma Ling 17 

2004 
Ma Ling 19 












NS 


NS 

BNS 
(0.060) 


2004 
















Ma Ling 20 

















canal intersection) were unique in having 83% 
slightly ribbed shells (but again low numbers, 
i.e., 12). Sites 19and20 were in zone 2. Across 
comparison of sites for significant differences 



(Table 3) yielded only five comparisons that 
were not significantly different (or barely not 
significantly different. Sites at the southern end 
of the Ma Ling Canal in 2004 (sites 17, 19, 20) 




2004 



P = 0.0121 



M 



SL S-/SM S M 

Classes of polymorphisms 




SL S-/SM 



FIG. 4. Comparing Gu Hu snails from 2001 and 2004 for shell polymorphisms. See Fig. 2 for 
abbreviations. X^ = significant difference but barely so. 



ECOGENETICS OF SHELL SCULPTURE IN ONCOMELANIA 



259 




S M SL S- SM 



S M SL S- SM 
Classes of polymorphisms 



S M SL S- SM 



FIG. 5. Comparing Ma Ling 2004 sites 17, 19, 20 for five classes of shell ribbing polymorphisms. The % 
of each class is shown. See Fig. 2 for abbreviations. Dashed arrows indicate trend for decreasing 
slightly ribbed moving up the canal and increasing medium ribbed snails. 



were not significantly different or bordering not 
significantly different. 

To enable cross comparisons of 2001 and 
2004 data, we combined data S- and SM from 
2004. We also combined data from Ma Ling 
17, 19, and 20, as 2004 populations were ei- 
ther not significantly different or barely signifi- 
cantly different. The Ma Ling snails from 2001 
were highly significantly different from those of 
2004 (Fig. 3). The 2004 population had fewer 
S- class and more M class snails. The Gu Hu 
snails from 2001 and 2004 were significantly 
different but barely so (Fig. 4). Most noticeable 
was the decrease in 2004 of S- class snails. 

In comparing Ma Ling southern canal popu- 
lations (17, 19, 20), one notices two distinct 
trends. Moving from north to south and across 
the larger perpendicular canal, there Is distinct 
decrease in percentage of snails of the slightly 
ribbed class (Fig. 5). There is a distinct increase 
in snails of the medium-ribbed class. 

Entirely smooth snails are thus far rare in 
these canals. 



DISCUSSION 

The major findings of this study are as fol- 
lows. (1 ) For the first time shell sculptural poly- 
morphism at a single site is reported in On- 



comelania. (2) The polymorphism in degree 
of ribbing from strongly ribbed to smooth- 
shelled individuals in a relatively new canal 
environment is not static. Changes occur over 
a short period of time, that is less than 22 
years, from strong ribbing to reduced strength 
of ribbing, including loss of ribbing. Further, 
morph frequency changes have occurred over 
the short span of 2-3 years. (3) The occur- 
rence of shell sculpture polymorphism in iso- 
lated populations provides the first demon- 
strable linkage between ribbed-shelled and 
smooth-shelled O. hupensis hupensis popu- 
lations, the rate with which the transition from 
the ribbed to smooth states can occur, and 
enables a clearer understanding of population 
genetic structure and the potential for popula- 
tions to transmit Schistosoma japonicum. 

Shell Sculptural Polymorphism and Environ- 
mental Selection 

Polymorphism here is apparently dependent 
on a relatively new man-made environment 
that is affecting the genetic structure of these 
populations. Stability and removal from the 
annual floods of the Yangtze are the keys to 
change. Individuals in these relatively isolated 
canal habitats are changing from the heavily 
ribbed morphotype seen along the banks of 



260 



DAVIS ETAL. 



the Yangtze River, where their recent ances- 
tors originated. Ribbing, as a derived flood- 
induced character, must be an energetically 
expensive character to maintain, as it is lost 
so quickly once the environmental enforce- 
ment is removed. 

These canals are low land environments. 
Flow of water into the canals from the Yangtze 
is controlled by flood gates. The origin of the 
canal snails in this large region must be from 
the Yangtze River flood plains, dispersing into 
canals as the canals were built. The flotation 
of living snails from the Yangtze through the 
river embankment portals has been well docu- 
mented (Xu & Fang, 1988; Xu et al., 1989, 
1993; Yang et al., 1992). The only plausible 
origin for these snails is from the Yangtze River 
through the Guan Yin flood Gate into the pri- 
mary Hong Chou Canal, hence to the north 
trending secondary Nan Bei Canal. The Nan 
Bei Canal is piped under (reverse siphoning) 
the Huge Shi Gong (that takes waste water 
away from Sha Shi City) to flow north to Malin 
Village. The Wu Yi Canal turns west off of the 
Nan Bei Canal to transect the Ma Ling Canal 
of our study about a km away. 

Now we see deep in the tertiary interior ca- 
nals that there has been a considerable 
change over the past 20 or so years since 
these particular canals were made. In 2004, 
only 0.47% of the snails had strong ribs, while 
41% had slightly irregularly ribbed shells, and 
5% were close to smooth. The significant dif- 
ferences between canals and between years 
within a given canal demonstrate a dynamic 
process, such as seen in Figure 5 showing 
discrete trends of increasing and decreasing 
morph frequencies between relatively closely 
positioned sites. We do not know the reason(s) 
for these short-term differences (or trends). In 
these canals, snails have low vagility and are 
subject to regular perturbation by man and 
animals. Differences could be due to founder 
effects or local selective pressures of micro- 
area effects. 

The compelling argument is that flooding 
selection drives the selection for alleles favor- 
ing development of stronger shells, larger size, 
and ribs. Prédation does not appear to drive 
these genotypes. The only known predators 
of Oncomelania hupensis snails are ducks and 
perhaps some predatory fish. But in the pres- 
ence of ducks, Oncomelania in the southern 
zone of the Ma Ling Canal do not have strong 
ribs; they mostly have slightly ribbed shells (> 
50%) and there are many (9%) nearly smooth 
(S-) shells. It is unlikely that fish are a factor 



as adult Oncomelania is amphibious, living in 
the ecotone between water and dry land, a 
habitat not accessible to fish. 

Ecology, Population Genetics of Oncomelania 
hupensis hupensis and the Transmission of 
S. japonicum 

While there is no direct genetic linkage be- 
tween shell sculpture and the potential to trans- 
mit Schistosoma japonicum, there are 
significant differences between strongly 
ribbed-shelled aggregates of O. hupensis 
hupensis and smooth-shelled populations with 
regard to both population genetic structure and 
the potential to transmit Schistosoma japo- 
nicum. Empirical data have shown ribbed- 
shelled aggregates of snails to be both 
genetically unstable (thus not true populations) 
and highly susceptible to infection with Schis- 
tosoma japonicum. Smooth-shelled popula- 
tions have this far been shown to be genetically 
stable and have low to no capacity to transmit 
S.yapoA?/CL/m (Davis et al., 1999a; Wilkeetal., 
2000; Shi et al., 2002). The infectivity capac- 
ity has been hypothesized to be driven through 
coevolution with S. japonicum, with infectivity 
differences the basis for invoking the Red 
Queen hypothesis of coevolution of Van Valen 
(1973) (Davis, 1980, 1992; 193). 

Origin of Ribbing, Genetic Instability and In- 
fectivity 

Historically, the evolutionary developments 
have been; (1 ) The plesiomorphic state (primi- 
tive or basic state) is being small and with 
smooth shell (Davis, 1979). (2) With evolving 
river systems and dispersal down the Yangtze 
River of the smooth-shelled morph into the 
new environment of the evolving Yangtze River 
and onset of annual monsoon-floods, there 
evolved the presence of ribs, a thicker shell, 
and increased size to cope with environmen- 
tal challenge. Of all Oncomelania taxa, only 
the Yangtze River drainage (and derived drain- 
ages) developed ribbing on the shells. (3) 
Below the Three Gorges of the Yangtze River 
and dispersing up into habitats not affected 
by flooding, as well as dispersing to Taiwan 
and Japan, the snails reverted to (or main- 
tained) a smooth shell. (4) Becoming smooth 
and living in isolation from immigration enables 
genetic stability, a requirement for the Red 
Queen to operate. In isolation, and normal in- 
breeding, smooth-shelled individuals evolve 
under selection pressure of the parasite from 



ECOGENETICS OF SHELL SCULPTURE IN ONCOMELANIA 



261 



being highly susceptible to the parasite to low- 
ered susceptibility at the population level to 
totally resistant to infection. 

Genetic Structure and the Transmission of S. 
japonicum 

Population genetic stability vs. instability was 
defined (Davis et al., 1999a) using MtDNA 
sequence data. Low haplotype diversity (1 or 
2 haplotypes per > 10 individuals collected 
from a small area (e.g., 100 m^) is a surrogate 
for Hardy-Weinberg equilibrium or normal 
panmixis within a population over a period of 
years, that is, stability. Instability is indicated 
by high haplotype diversity for the same con- 
ditions above, where 6-10 haplotypes are 
found in < 10 individuals. Instability is indica- 
tive of aggregates of individuals ("populations") 
of recent immigration; that is, these are not 
part of a normal interbreeding population. 
Snails are swept together from different loca- 
tions, carried by flood waters. Hardy Weinberg 
is not attained. The example was given (Davis 
et al., 1 999a) where five "populations" around 
the shores of Dong Ting Lake, all subjected to 
severe annual flooding, had heavy ribbing. 
These had 6-10 haplotypes for ten individu- 
als thus all were unstable. The sixth popula- 
tion came from an elevation between 1 00 and 
500 m. The shells were smooth and had two 
haplotypes per ten individuals, that is, genetic 
stability. All the ribbed snail populations were 
highly susceptible to schistosome infection. 
The smooth-shelled population was not and 
could not be infected (Li, Hunan Institute of 
Parasitic Diseases, personal communication). 

A study of O. hupensis hupensis populations 
along the Yangtze River from Hunan and Hubei 
provinces through to Zhejiang and Jiangsu 
provinces involved questions of population 
evolution, haplotype diversity and ecology 
(Wilke et al., 2000). The data indicated that rib- 
bing is associated with annual floods along the 
flood plains of the Yangtze River, where snails 
are swept into aggregates of snails with high 
haplotype diversity. In areas not affected by 
flooding, the snails were generally smooth and 
genetic diversity decreased significantly. The 
one Jiangsu population was smooth and living 
essentially at sea level in a water network (one 
haplotype in six individuals). One Zhejiang 
population (elevation of 100 m) had a trace of 
ribbing and low haplotype diversity. One Anhui 
population living in the lowlands but potentially 
removed from flooding had slightly ribbed shells 
and three haplotypes per ten individuals. 



A study re-visiting the Miao River (Shi et al., 
2002) involved haplotype analysis and eco- 
logical setting to examine both the question of 
smooth-shelled vs. ribbed shelled and the re- 
lationship between these morphs and infec- 
tivity. There was a clear trend for decreasing 
haplotype diversity upstream from the mouth 
of the river. Nucleotide-sequence diversity was 
> 0.01 5 at sites A and В close to the mouth of 
the river and where the snails had heavily 
ribbed shells; it was < 0.0085 at site G at the 
top of the river, where the snails were smooth 
(sites above the flood level with smooth- 
shelled populations were D-G). With regard 
to infectivity, the down-stream ribbed-shelled 
"populations" had higher infection rates and 
higher susceptibility to infection with S. 
japonicum than did upstream smooth-shelled 
populations. The higher infectivity of down- 
stream "populations" was attributed to the im- 
portation and mixture of snails (i.e., aggre- 
gates) of different genotypes of snails and 
schistosomes in flooded areas increasing the 
possibility of multiple infections by schisto- 
somes of different genotypes. In upstream 
populations, low infectivity is probably due to 
isolation and attaining equilibrium, with the 
parasite at low frequencies of infection. 

The Red Queen and Decreasing Infectivity 

The Red Queen pertains to co-evolution in 
which the impact of a parasite on the host (in 
this case the intermediate snail host) elicits a 
genetic response of the host to repel the para- 
site. This in turn generates a genetic response 
in the parasite to overcome the defense of the 
host. Through time, the interaction becomes 
highly specific and convoluted. For this to hap- 
pen, the snail population must be, in fact, a 
true population with little or no immigration, 
that is, in Hardy-Weinberg equilibrium (geneti- 
cally stable). There are three possible end re- 
sults of this "genetic war". (1) The parasite 
wins, and the snail population goes extinct 
(witnessed in the decline and local extinction 
of Hydrobia trúncala in New England, USA 
(Davis et al., 1988). (2) The snail wins, and 
the parasite becomes extinct in the snail popu- 
lation. (3) The snail infectivity rate decreases 
until some equilibrium is reached at a low level 
of infectivity. 

We have uncovered a number of situations 
in which the smooth-shelled snail population 
has gone to fixation for completely warding off 
the parasite. Davis & Ruff (1973) hybridized 
smooth totally refractive Oncomelania hupen- 



262 



DAVIS ETAL. 



s/s from Taiwan with highly susceptible ribbed- 
shelled О. hupensis from the mainland of 
China. The hybrids could be infected; there was 
indeed a genetic component to transmission. 
On Taiwan, there are Oncomelania hupensis 
populations that are totally refractive, others 
are susceptible to non-human Schistosoma 
japonicum. and one population is not naturally 
infected with any schistosome but can be in- 
fected with all allopatric strains of S. japonicum. 

We have found one Anhui smooth-shelled 
population that is totally refractive to infection. 
The refractive population above Dong Ting 
Lake was mentioned. The Miao River study 
demonstrated the highly infectious nature of 
the genetically unstable downstream ribbed- 
shelled snails in contrast to the much less sus- 
ceptible upstream smooth-shelled populations. 

There is a large literature on cross infectiv- 
ity studies, based on the pioneering work of 
DeWitt (1954) involving permutations and 
combinations of S. japonicum from different 
localities and countries and Oncomelania 
hupensis from the corresponding localities. A 
sampling of a few papers on cross suscepti- 
bility studies are Moose & Williams (1963), Chi 
etal. (1971), Heetal. (1991), Linetal. (1994), 
Hong et al. (1995), and Sheng et al. (1995). 
These studies show that there is considerable 
evidence for evolutionary divergence among 
allopatric populations with regard to the ge- 
netic potential to transmit an allopatric S. 
japonicum. The results range from complete 
incompatibility to partial compatibility involv- 
ing allopatric pairs. 

We continue to maintain the hypothesis that 
genetically unstable aggregates of ribbed- 
shelled snails are more highly susceptible than 
isolated populations of smooth-shelled snails 
because the mixing of snails, due to importa- 
tion by flooding (with an array of genotypes 
relative to schistosome success or failure at 
infecting these snails), facilitates high success 
in infecting snails. In such an environment, the 
schistosomes have a "menu" of genotypes to 
choose from with regard to their success in 
infecting the snail. Given the unstable nature 
of the mixture of the moment, one sees little 
opportunity for selective pressures to act on 
these genotypes relative to the process of 
speciation or emerging new disease. However, 
the mixture is a potent cocktail of genotypes 
that present a dangerous situation relative to 
importing the mixture, with all its genetic di- 
versity, to a new environment. In genetically 
stable populations that are isolated and where 



the Red Queen is in action, it is possible that 
selective pressures on allopatric stable popu- 
lations could drive speciation and emerging 
disease. 

Oncomelania hupensis robertsoni - A Differ- 
ent Evolutionary Trajectory 

Oncomelania hupensis robertsoni has a dif- 
ferent history and involvement with Schisto- 
soma japonicum than O. h. hupensis. 
Oncomelania hupensis robertsoni is highly di- 
vergent genetically from O. h. hupensis (Davis 
et al., 1998; Wilke et al., 2000, 2006). As de- 
scribed above, this taxon, living in the moun- 
tains of Sichuan and Yunnan provinces, closer 
to the area of origin of the genus than O. 
hupensis hupensis. is not affected by the great 
annual floods of the Yangtze River, and has 
small, smooth shells and no varix. The varix, 
the thickening of the outer lip seen in O. h. 
hupensis, is equal to the terminal rib seen in 
all O. ^upens/s /?üpens/s populations, smooth 
or ribbed. Of note is that thus far no popula- 
tion of O. h. robertsoni has been found to be 
refractive to infection with the Yunnan-Sichuan 
strain of S. japonicum. A different dynamic 
seems to be at work here. It is also noted that 
robertsoni lives on the banks of small streams, 
irrigation ditches, and the base of retaining 
walls of agricultural terraces. These are gen- 
erally in a high gradient environment, where 
heavy rain wash snails downward. Following 
such rains, the snails, which are negative geo- 
tropic and negatively rheotropic, move relent- 
lessly upward. The net result is hypothesized 
to be considerable genetic mixing within a 
drainage system, that is, resulting in geneti- 
cally unstable aggregates of snails with high 
susceptibility to S. japonicum. Preliminary data 
support the instability argument as of 1 3 popu- 
lations studied in Wilke et al. (2006), 40 of 66 
specimens had different haplotypes, and of 24 
specimens studied, each had a unique AFLP 
fingerprint. These data indicate a lack of popu- 
lation structure due to great heterogeneity, not 
due to uniform panmixia. 

A great deal of work must be done with O. h. 
robertsoni on population genetics, population 
structure, natural patterns of infection, and 
laboratory infectivity studies before one can 
say much more. A tentative hypothesis is that 
robertsoni maintains the plesiomorphic recep- 
tiveness to infection and those environmen- 
tal-population factors do not promote the Red 
Queen. 



ECOGENETICS OF SHELL SCULPTURE IN ONCOMELANIA 



263 



ACKNOWLEDGEMENTS 

This work was supported by a U.S.A. Na- 
tional Institutes of Health grant Al- 
2P50AI39461-06A1 (GMD, Co. P.I. -Project II). 
This is part of the N.I.H. - Tropical Medical 
Research Center granted to the National In- 
stitute of Parasitic Diseases, Chinese Center 
for Disease Control and Prevention, Shang- 
hai, China. 

We are deeply appreciative of the reviews 
and comments on this paper by Drs. David 
Blair, Gail Williams, Paul Brindley, Randy 
Hoeh, and Thomas Wilke, who provided val- 
ued comment. We, the authors take responsi- 
bility for the final result. 



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Revised ms. accepted 6 November 2005 



MALACOLOGIA, 2006, 48(1-2): 265-282 

TAXONOMIC DISTRIBUTION AND PHYLOGENETIC UTILITY OF GENDER- 
ASSOCIATED MITOCHONDRIAL GENOMES IN THE UNIONOIDA (BIVALVIA) 

Jennifer M. Walker'*, Jason P. Curole^ Dan E. Wade', Eric G. Chapman', 
Arthur E. Bogan^, G. Thomas Walters" & Walter R. Hoeh^ 

ABSTRACT 

Unionoid bivalves exhibit a great diversity in reproductive characteristics. However, the 
lacl< of a robust phylogeny severely restricts evolutionary interpretations regarding the 
genesis and consequences of reproductive character state diversity within the order. The 
apparent high fidelity of unionoidean doubly uniparental inheritance of mtDNA (DUI), where 
distinct female- (F) and male-(M) transmitted mtDNA genomes are present, may allow for 
multiple, independent mtDNA-based estimates of phylogeny and thus contribute to the 
generation of more robust estimates of unionoid evolutionary history. However, the current 
lack of knowledge regarding mtDNA transmission patterns in the Etherioidea severely ham- 
pers our ability to evaluate the potential of DUI for explicating unionoid phylogeny. This 
situation prompted us to address the following questions in this study: (1) Is DUI found in 
the Etherioidea? (2) What is the relative phylogenetic utility of F, M, and concatenated F + 
M cytochrome с oxidase subunit I (cox1) sequences for elucidating higher level unionoid 
evolutionary relationships? (3) What can trees derived from F and M sequence analyses 
tell us about the evolution of unionoid DUI and other reproductive characters? 

Forty-seven species representing all six families within the Unionoida were evaluated, 
using PCR-based methods, for the presence of DUI. Phylogenetic analyses were carried 
out on unionoid species for which complementary F and M cox1 DNA sequences were 
available as well as on a much more taxonomically inclusive F cox1 data set. We deter- 
mined that (1) the Etherioidea likely lacks DUI; (2) M and F + M cox7-based analyses 
provide better resolved estimates of unionoidean relationships than do F cox7-based analy- 
ses; and (3) the F and M non-concatenated cox1 inclusive phylogenetic analyses suggest 
the inference that (a) the presence of DUI, glochidial larvae, and endobranchous brooding 
are the ancestral unionoid character states, (b) both DUI and glochidial larvae were lost in 
the ancestral etherioidean lineage, (c) margaritiferids are closely related to unionids and 
exhibit a derived suite of morphological characteristics, and (d) a clarification of the evolu- 
tionary dynamics of unionoid DUI and other reproductive characteristics will require a ro- 
bust phylogeny for the order that is based on multiple data sets. 

Key words: DUI, cox1, mtDNA, Hyriidae, Margaritiferidae, Unionidae, Iridinidae, 
Mycetopodidae. 



INTRODUCTION 

Freshwater unionoid bivalves exhibit signifi- 
cant taxonomic diversity (-175 genera) and a 
broad geographic distribution that includes all 
continents, with the exception of Antarctica 
(Simpson, 1896, 1900, 1914; Haas, 1969; Sta- 
robogatov, 1970). Following Parodiz & Bonetto 
(1963), the bivalve order Unionoida is comprised 



of six families contained within two superfamilies 
of freshwater mussels (Superfamily Etherioidea: 
Etheriidae, Iridinidae and Mycetopodidae; Su- 
perfamily Unionoidea: Hyriidae, Margaritiferidae, 
and Unionidae). However, the concept of the 
Etheriidae as a monophyletic group containing 
all cemented unionoid bivalves has been re- 
jected (Bogan & Hoeh, 2000), thus its usage 
herein is applied only to the genus Etheria. 



^Evolutionary, Population, and Systematic Biology Group, Department of Biological Sciences, Kent State University Kent, 
Ohio 44242, USA. 

•^University of Southern California, Los Angeles, California 90089, U.S.A. 
^North Carolina Museum of Natural Sciences, Raleigh, North Carolina 27607, USA. 

■'Department of Evolution, Ecology and Organismal Biology, Ohio State University, Columbus, Ohio 43212, U.S.A. 
'Corresponding author: jwalker4@kent.edu 

265 



266 



WALKER ETAL. 



Unionoid bivalves also exhibit great diversity, 
among higher taxa, in their reproductive char- 
acteristics, such as the morphology of their 
parasitic larvae as well as the larval brooding 
location. There are two types of parasitic 
unionoid larvae: the bivalved glochidium 
(Hyriidae, Margeritiferidae, and Unionidae) 
and the univalved lasidium (Iridinidae and 
Mycetopodidae) (Wächtler et al., 2001 ). It has 
been suggested that the extreme morphologi- 
cal divergence between these two types of lar- 
vae indicates that the unionoidean and 
etherioidean bivalves represent independently 



derived freshwater lineages and thus the 
Unionoida is a polyphyletic assemblage (Paro- 
diz & Bonetto, 1963). However, the results of 
recent unionoid phylogenetic analyses reject 
the latter hypothesis (e.g., Hoeh et al., 2001; 
Roe & Hoeh, 2003). In addition to the extreme 
distinctions in larval morphology, unionoid 
higher taxa also exhibit differences in larval 
brooding location. Unionoids use three general 
brooding locations. Tetragenous brooders uti- 
lize all four ctenidia as marsupia (Margaritiferi- 
dae and some Unionidae), endobranchous 
brooders utilize only the inner two ctenidia 



В 



Hyriidae 
Margarititeridae 
.Unionidae 

.Iridinidae 

.Mycetopodidae 



. Iridinidae 
Mycetopodidae 
Hyriidae 

Linionidae 
Maruarititeridae 



Margarititeridae 
Unionidae 

iridinidae 
Mycetopodidae 

Hyriidae 



D 



Unionidae 



I Mycetopodidae 

— hidinidae 
_ I lyriidae 



Maruaritiferidae 



FIG. 1 . Simplified representations of unionoid familial relationships based on the A: Classifications of 
Simpson (1900) and Parodiz & Bonetto (1963); B: Phylogenetic analyses of morphological characters 
after Graf (2000) and Hoeh et al. (2001); С: Phylogenetic analysis of combined morphological and 
molecular characters after Hoeh et al. (2001); D: Phylogenetic analysis of morphological and 
molecular characters after Roe & Hoeh (2003). 



DUl IN THE UNIONOIDA 



267 



(Hyriidae, Iridinidae, and Mycetopodidae), and 
ectobranchous brooders utilize only the outer 
two ctenidia (some Unionidae). Increasing our 
understanding regarding the evolution of this 
diversity in reproductive structures has been a 
major, if largely unrealized, goal of recent 
unionoid phylogenetic studies (e.g., Graf & Ó 
Foighil, 2000; Hoeh et al., 1 998a, 2001 ). 

Numerous hypotheses of unionoid evolution- 
ary relationships, based on analyses of both 
morphological and DNA characters, have been 
published recently (Bogan & Hoeh, 2000; Graf 
& Ó Foighil, 2000; Hoeh et al., 1998a, 2001; 
Graf, 2000; Roe & Hoeh, 2003). Despite this 
flurry of recent studies, when comparing hy- 
potheses of unionoid familial relationships, it 
becomes apparent that there is little agree- 
ment (Fig. 1 ). Parodiz & Bonetto's (1 963) clas- 
sification mirrors that of Simpson (1896, 
1900), in describing Mycetopodidae + Iridini- 
dae (Simpson's Mutelidae) as fundamentally 
distinct from the Unionidae + Margaritiferidae 
+ Hyriidae (Simpson's Unionidae) (Fig. 1A). 
Recent phylogenetic analyses offer neither 
corroboration of this view nor significant 
among-analysis congruence. Analyses of 
morphological characters presented by Graf 
(2000) and Hoeh et al. (2001) both return the 
Margaritiferidae as the basal unionoid lineage, 
a paraphyletic Unionidae, and the Hyriidae + 
Mycetopodidae + Iridinidae as a derived lin- 
eage (Fig. IB). In contrast, both the molecular 
(i.e., cox1 DNA sequences) and the combined 
analysis of morphological and molecular data 
presented by Hoeh et al. (1998a, 2001) (Fig. 
1С) place the Hyriidae as the basal unionoid 
lineage, with the Unionidae returned as 
paraphyletic. Yet another combined analysis 
of morphological and molecular (i.e., cox1 
DNA sequences) characters (Roe & Hoeh, 
2003; Fig. ID), this time using binary coding of 
the morphological data and a posteriori char- 
acter weighting, returns the Margaritiferidae 
as basal and a monophyletic Unionidae as 
sister to a Hyriidae + Iridinidae + Mycetopodi- 
dae clade. As is readily apparent from the 
above comparisons, ambiguity still remains 
when attempting to explain the evolution of 
unionoid diversity. This difficulty results from 
the fact that the only degree of stability exhib- 
ited across all of the relationship hypotheses 
above is that the Mycetopodidae and Iridini- 
dae are always returned as closely related. 
Importantly, the topological positions of the 
Hyriidae and Margaritiferidae do not remain 
stable across analyses and thus, we are left 



with the lack of a well-resolved phylogeny for 
the Unionoida. This situation severely restricts 
evolutionary interpretations regarding the gen- 
esis and consequences of reproductive char- 
acter state diversity within the order. 

A largely underutilized set of phylogeneti- 
cally informative characters exists in the male- 
transmitted mtDNA genomes within the 
Unionoidea, as a consequence of the pres- 
ence of doubly uniparental inheritance of 
mtDNA (DUl) in that taxon. DUl has been ob- 
served in two orders of marine bivalves 
(Mytiloida: Skibinski et al., 1994; Zouros et al., 
1994; Hoeh et al., 1996; and Veneroida: 
Passamonti & Scali, 2001) and the freshwater 
bivalve superfamily Unionoidea (Hoeh et al., 
1996; Liu et al., 1996). In species with this 
type of mtDNA inheritance, there are distinct 
female-(F) and male-(M) transmitted ge- 
nomes. Typically, females are homoplasmic 
for the F genome, whereas males are hetero- 
plasmic, that is, they contain both the F and M 
genomes (Skibinski et al., 1994; Zouros et al., 
1994). In males, these two distinct mtDNA ge- 
nomes segregate by tissue type. The F ge- 
nome predominates in somatic tissues while 
the M genome is concentrated in spermatoge- 
nic tissues (Stewart et al., 1995; Garrido- 
Ramos étal., 1998). Therefore, taxa possess- 
ing DUl transmit two distinct mtDNA genomes. 
Females pass on their F genome to both male 
and female progeny while males transmit their 
M genome only to male progeny. While earlier 
mtDNA-based analyses of unionoid phylogeny 
largely made use of F genome sequences 
(e.g., Graf & O' Foighil, 2000; Hoeh et al., 
1998a, 2001), more recent phylogenetic 
analyses of both F and M genome DNA se- 
quences, from exemplar species representing 
the Hyriidae, Margaritiferidae, and Unionidae, 
have produced evolutionary trees with distinct 
F and M clades exhibiting very similar topolo- 
gies (Curóle & Kocher, 2002, 2005; Hoeh et 
al., 2002). The observed reciprocal monophyly 
of these topologically similar F and M clades, 
in conjunction with fossil evidence, suggests 
that DUl has been operating at a high level of 
fidelity in the Unionoidea for more than 100 
my (Curóle & Kocher, 2002, 2005; Hoeh et al., 
1996, 2002). 

The fidelity of the DUl system is sometimes 
compromised in mytiloids. Evidence support- 
ing this view comes from phylogenetic analy- 
ses (e.g., Hoeh et al., 1997) and breeding 
studies (Fisher & Skibinski 1990; Zouros et 
al., 1994; Rawson et al., 1996; Saavedra et 



268 



WALKER ETAL. 



al., 1997; Quesada et al., 1999: Ladoukakis 
et al.. 2002). For example, taxonomically more 
inclusive phylogenetic analyses of F and M 
genomes in mytiloids have failed to recover 
distinct F and M clades (e.g.. Hoeh et al.. 1996. 
1997). As one example, some Mytilus M ge- 
nomes appear more closely related to F ge- 
nomes than to other similarly transmitted 
genomes (Hoeh et al. 1997). Failure to inherit 
the M genome may result in recruitment and 
masculinization of the F genome to function 
as a newly derived "M" genome (Hoeh et al., 
1996. 1997). Initially after masculinization. the 
F and the newly derived "M" genome have 
identical DNA sequences. Subsequently, di- 
vergence between the F and "M" genome be- 
gins de novo (Hoeh et al., 1996, 1997). 
Additionally, mytiloid masculinization events 
have been observed in laboratory crosses 
(Zouros et al.. 1994; Saavedra et al.. 1997) 
as well as in natural populations (Fisher & 
Skibinski. 1990; Rawsonetal.. 1996; Quesada 
etal.. 1999; Ladoukakis et a!.. 2002). This pre- 
sents a problem when using F and M genomes 
in a complementary manner for mytiloid phy- 
logenetic analyses as non-orthologous com- 
parisons could result. To date, feminization, 
or recruitment of an M genome to function as 
the F, has not been observed or inferred for 
any taxa with DUI. Unlike the situation in 
mytiloids. masculinization events have not 
been documented for the Unionoidea (Hoeh 
et al., 1996, 2002; Curóle & Kocher, 2002, 
2005). This apparent high fidelity of unio- 
noidean DUI may allow for multiple, indepen- 
dent mtDNA-based estimates of phylogeny 
and genetic variation within the order (Hoeh 
eta!.. 2002; Krebs, 2004). 

The presence of DUI in the hyriid, margariti- 
ferid, and unionid specimens sampled to date 
prompted us to address the following ques- 
tions in this study; (1) Is DUI found in the 
Etheriidae, Iridinidae and Mycetopodidae? If 
so, DUI likely represents the ancestral mtDNA 
transmission pattern for unionoid bivalves. If 
not. DUI presence/absence data may be in- 
formative regarding unionoid familial relation- 
ships. (2) What is the relative phylogenetic 
utility of F. M, and concatenated F + M cyto- 
chrome с oxidase subunit I {cox1) sequences 
for elucidating higher level unionoid evolution- 
ary relationships? (3) What can trees derived 
from F and M sequence analyses tell us about 
the evolution of unionoid DUI and reproduc- 
tive characters? 



MATERIALS AND METHODS 

Taxa sampled in this study for the presence 
of a male genome included 47 species repre- 
senting all six families within the Unionoida 
(Table 1). Gender was determined by micro- 
scopical examination of gonadal tissues. To- 
tal genomic DNA was isolated from either 
somatic (mantle or foot) or testis tissue using 
the Qiagen DNeasy animal kit. An approxi- 
mately 710 bp fragment of cox1 was ampli- 
fied from both the F and M mtDNA genomes 
using modified versions of the universal cox1 
primers (Folmer et al., 1994): LC022me2 5'- 
GGTCAACAAAYCATAARGATATTGG-3'; 
HCO700dy2. 5'-TCAGGGTGACCAAAAAAYCA- 
3'. To efficiently screen for the presence of the 
M genome, largely gender-specific cox2 prim- 
ers were used to amplify the cox2-cox1 frag- 
ment used by Curóle & Kocher (2002). These 
primers were chosen due to the size differ- 
ence exhibited between the F and M cox2- 
cox1 fragments as described by Curóle & 
Kocher (2002). The "male-specific" cox2 
primer was UNI0C0II.2 (Curóle, 2004) and a 
"female-specific" primer (UNIOCOII.2b, 5'- 
CAGTGRTATTGRRVDTAYGA-3') was derived 
from the UNI0C0II.2 primerand otherunionid 
F sequences available from GenBank. Both 
"gender-specific" primers were paired with 
HCO700dy2 to amplify the cox2-cox1 frag- 
ment. These primers typically amplified ap- 
proximately 1.1 Kbp of cox2-cox1 from F 
genomes and approximately 1.7 Kbp from M 
genomes. PCR reactions consisted of IX 
Qiagen PCR buffer, 0.2 mM each dNTP О.бцМ 
each primer, and Qiagen Taq. Reactions us- 
ing the cox1 primer pair were cycled at 94°C 
for 60 s, 40°C for 60 s, and 72°C for 60 s for a 
total of 40 cycles and reactions using the male- 
specific cox2 primer were cycled at 94°C for 
60 s, 50°C for 60 s, and 72°C for 120 s for a 
total of 40 cycles. Reactions involving the fe- 
male-specific cox2 primer followed the same 
profile given above for the male specific primer 
but were annealed at 46°C. Sequencing tem- 
plate purification was carried out following 
Folmer et al. (1994). The cox1 fragment 
yielded 61 9 bp of sequence via cycle sequenc- 
ing with Perkin Elmer AmpliCycle Sequencing 
Kits using ddNTP-dNTP ratios optimized for 
automated sequencing. Sequences were ob- 
tained from both strands of the cox1 fragment 
and the dye-labeled cox1 sequencing primers 
were of the same sequence as the PCR prim- 



DUl IN THE UNIONOIDA 



269 



TABLE 1 . Taxa evaluated for the presence/absence of F and M cox2-cox1 amplicons; + = amplifica- 
tion successful, - = amplification failed, NA - amplification not attempted. 



Family 



Species 



cox2-cox1 
amplicon F 



cox2-cox1 
amplicon M 



Unionidae 



Actinonaias ligamentina 
Amblema plicata 
Anodonta californiensis 
Cyprogenia aberti 
Cyrtonaias tampicoensis 
Dromus dromas 
Ellipsana lineolata 
Elliptio d a it ata 
Epioblasma brevidens 
Fusconaia flava 
Glebula rotundata 
Hamiota subrotundata 
Lampsilis cardium 
Lampsilis hydiana 
Lampsilis powellii 
Lampsilis reeveiana 
Lampsilis siliquoidea 
Lampsilis straminea 
Lampsilis strecken 
Lampsilis teres 
Leptodea fragilis 
Leptodea leptodon 
Ligumia recta 
Medionidus conradicus 
Obovaria olivaría 
Popenaias popeii 
Potamilus a latus 
Potamilus capax 
Potamilus ohiensis 
Potamilus purpuratus 
Ptychobranchus fasciolare 
Toxolasma glans 
Truncilla truncata 
Venustaconcha ellipsiformis 
Villosa irís 
Villosa lienosa 
Villosa villosa 



Margaritiferidae 


Cumberíandia monodonta 
Dahurínaia sp. 
Margaritifera margaritifera 


Hyriidae 


Hyndella menziesi 


Iridinidae 


Chambardia rubens 
Mutela dubia 


Etheriidae 


Ethería elliptica 


Mycetopodidae 


Anodontites guanarensis 
Tamsiella tamsiana 


Neotrigoniidae 


Neotrigonia margaritacea 



+ 
+ 
+ 
+ 

+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 



+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 

+ 
+ 

+ 
+ 
+ 



NA 



270 



WALKER ETAL. 



ers. The 3' portion of the M cox2-cox1 frag- 
ment was sometimes sequenced, using the 
HCO700dy2 sequencing primer, to confirm M 
cox1 sequences generated by the cox1 primer 
pair. Sequences were visualized using Li-Cor 
4200L-2 and 4200S-2 DNA sequencers and 
initial base calls were made by e-Seq v 2.0. 
Contiguous sequences were assembled and 
verified using AlignlR v2.0 and final sequence 
alignments were completed manually with 
MacClade v4.0. GenBank accession numbers 
for the cox1 sequences generated and/or ana- 
lyzed herein are given in Table 2. All testis 
extraction-derived cox1 sequences were 
added to a matrix containing confirmed F and 
M cox1 sequences and phylogenetic analy- 
ses were used to test the putative M status of 
the newly generated sequences. Subsequent 
to the initial attempts to amplify the M cox2- 
cox1 fragment from their testis-derived total 
DNAs, multiple attempts were made to am- 
plify an M mitochondrial fragment from mem- 
bers of the Iridinidae and Mycetopodidae using 
two, intragenic universal primer pairs (i.e., cox1 
and 16S [LR-J-12887, LR-N-13398; Simon et 
al., 1994]). 

Three complementary F and M sequence 
data sets, populated by the unionoid species 
from which both F and M cox1 sequences were 
obtained, were analyzed using the maximum 
likelihood (ML) and maximum parsimony (MP) 
algorithms contained in PAUP* (v.4.0b10; 
Swofford, 2001). Bayesian inference (Bl) 
analyses were carried out with MrBayes 
v3.0b4 (Huelsenbeck & Ronquist, 2003). The 
complementary F and M genome cox1 se- 
quences were analyzed both individually and 
in an F + M concatenated manner. Recent lit- 
erature indicates that a total evidence ap- 
proach can produce the best tree topologies 
(e.g., Collin, 2003; Creer et al., 2003; Hassanin 
& Douzery, 2003; Schwarz et al., 2003). Thus, 
the F + M cox1 concatenated trees were used 
as the best estimates of the phylogenetic re- 
lationships among the unionoid sequences 
examined. Additional phylogenetic analyses 
were carried out, using the Bl and MP algo- 
rithms, on an inclusive non-concatenated cox1 
DNA sequence data set that contained a much 
broader taxonomic sampling of the available 
F cox1 sequences as well as all available M 
cox1 sequences. Modeltest (v. 3.6: Posada & 
Crandall, 1998) was used to determine which 
model best fit the F, M, and F + M sequence 
data. The GTR + G + I model was used in all 
Bl and ML analyses. Neotrigonia margaritacea 



(Trigonioida) cox1 sequences were used to 
root the trees derived from the complemen- 
tary data set analyses (e.g., Hoeh et al., 
1998a), while a much broader sampling of taxa 
was used to root the trees derived from analy- 
ses of the inclusive data set (e.g., Hoeh et al., 
2002). 

A total of 29 cox7 sequences were included 
in the complementary data set phylogenetic 
analyses while 105 sequences were present 
in the inclusive non-concatenated M and F 
data set. Each of the four Bl analyses con- 
sisted of 10 chains, 5 million generations, and 
a 2 million generation burn-in. PAUP* was 
used to select, from among all of the 1,000 
saved Bl trees from each of the complemen- 
tary data set analyses, the topologies with the 
highest log likelihood scores. Due to the satu- 
ration of third position transitions (e.g., Hoeh 
etal., 1998a), all MP analyses were conducted 
on transformed cox1 sequences such that third 
position transitions were excluded from analy- 
ses. Multiple random terminal taxa addition 
sequence runs, combined with global branch 
rearrangement options, were employed when 
generating topologies, from the complemen- 
tary data sets, via the ML and MP algorithms. 
These options increased the probability of find- 
ing the actual best topology under each of 
these two optimality criteria (e.g., Hendy et al., 
1988; Maddison, 1991). Standard non-para- 
metric bootstrap (Felsenstein, 1985) analyses 
were carried out to evaluate the level of sup- 
port for particular nodes obtained from the ML 
(1,000 bootstrap replicates) and MP (10,000 
bootstrap replicates; 100,000 fast-heuristic 
replicates for the inclusive data set) analyses. 
A parsimony-based ILD test (Farris, 1994), as 
implemented in PAUP*, was used to test for 
incongruence between the F and M cox1 se- 
quences. 



RESULTS AND DISCUSSION 

What is the Taxonomic Distribution of DUI 
within the Unionoida? 

Definitively M genome cox2-cox1 fragments 
were amplified from 40 species representing 
three (Hyriidae, Margaritiferidae, and 
Unionidae) of the six unionoid families (Table 
1). Sequences from cox1 confirmed that the 
long cox2-cox1 PCR fragments obtained from 
testis-based DNA extractions were from M 
genomes. However, testis-based DNA extrae- 



DUl IN THE UNIONOIDA 



271 



TABLE 2. Source taxa and GenBank accession numbers for the cox1 DNA sequences used in phylo- 
genetic analyses. 



Family 



Species 



GenBank Accession No. 
F M 



Unionidae 



Margaritiferidae 



Actinonias ligamentina 


AF231730 


AF406796 


Cyrtonaias tampicoensis 


AF231749 


AF406798 


Fusconaia flava 


AF231733 


AF406799 


Gonidea angulata 


DQ206792 


DQ206794 


Lampsilis teres 


AF406803 


AF406794 


Ligumia recta 


AF231748 


AF406795 


Potamilus purpuratus 


AF406804 


AF406797 


Pseudodon vondembusctiianus 


DQ206793 


DQ206795 


Pyganodon fragilis 


AF406805 


AF406800 


Pyganodon grandis 


AF231734 


AF406801 


Cumberlandia monodonta 1 


AY785393 


AY785397 


Cumberlandia monodonta 2 


AF1 56498 




Cumberlandia monodonta 3 


AF1 56497 




Cumberlandia monodonta 4 


AY579131 




Dahurinaia dahurica 1 


AY579123 




Dahurinaia da ti и rica 2 




AY785400 


Dahurinaia dahurica 3 




DQ241802 


Margaritifera auricularia 1 


AY579125 




Margahtifera auricularia 2 


AF303312 




Margaritifera auricularia 3 


AF303313 




Margaritifera auricularia 4 


AF303315 




Margaritifera falcata 1 


AY579126 




Margaritifera falcata 2 


AY579128 




Margaritifera falcata 3 


AY579127 




Margaritifera laevis 


AY579124 




Margaritifera margaritifera 1 


AF303319 




Margaritifera margaritifera 2 


AF303320 




Margaritifera margaritifera 3 


AF303336 




Margaritifera margaritifera 4 


AF303341 




Margaritifera margaritifera 5 


AY579129 




Margaritifera margaritifera 6 


AY579130 




Margaritifera margaritifera 7 


AF303331 




Margaritifera margaritifera 8 


AF303332 




Margaritifera margaritifera 9 


AF303338 




Margaritifera margaritifera 1 


AF303340 




Margaritifera margaritifera 1 1 


AF303335 




Margaritifera margaritifera 1 2 


AF303337 




Margaritifera margaritifera 1 3 


U56847 




Margaritifera margaritifera 14 


DQ060171 




Margaritifera margaritifera 1 5 


AF303339 




Margaritifera margaritifera 1 6 




AY785399 


Margaritifera margaritifera durrovensis 1 


AF303344 




Margaritifera margaritifera durrovensis 2 


AF303345 




Margaritifera margaritifera durrovensis 3 


AF303346 




Margaritifera margaritifera durrovensis 4 


AF303347 




Margaritifera margaritifera durrovensis 5 


AF303342 




Margaritifera margaritifera durrovensis 6 


AF303343 








(continues) 



272 




WALKER 1 1 /C 


\L. 


(continued) 










GenBank Accession No. 


Family 


Species 


F iVI 


Hyriidae 


Alathyria jacksoni 1 


AY386977 




Alathyha jacksoni 2 


AY386981 




Alathyria jacksoni 3 


AY386970 




Alathyha jacksoni 4 


AY386974 




Castalia stevensi 


AF231736 




Diplodon deceptus 


AF231736 




Hyndella australis 


AF305367 




Hyridella depressa 1 


AF1 56496 




Hyndella depressa 2 


AF305368 




Hyndella menziesi 1 


AF231747 




Hyndella menziesi 2 


AF406802 




Lortiella rugata 


AF231746 




Velesunio ambiguus 1 


AF305371 




Velesunio ambiguus 2 


AF305372 




Velesunio ambiguus 3 


AY211582 




Velesunio ambiguus 4 


AY211586 




Velesunio angasi 


AF231743 




Velesunio sp. 1 


AY387018 




Velesunio sp 


2 


AY386999 




Velesunio sp 


A 1 


AY211550 




Velesunio sp 


A2 


AY211554 




Velesunio sp 


В 1 


AY211558 




Velesunio sp 


B2 


AY211566 




Velesunio sp 


D 1 


AY211587 




Velesunio sp 


D2 


AY211598 


Iridinidae 


Chambardia rubens 1 


DQ241807 




Chambardia rubens 2 


DQ241808 




Chambardia rubens 3 


AY785389 




Mutela dubia 1 


DQ241805 




Mutela dubia 2 


AY785388 




Mutela dubia 3 


DQ241806 




Mutela rostrata 1 


AY785387 




Mutela rostrata 2 


DQ241804 


Mycetopodidae 


Acostaea rivolii 


AF231739 




Anodontites guaranensis 


AY785383 




Anodontites trigonus 


AF231738 




Monocondylaea minuana 


AF231745 




Tamsiella tamsiana 


AY785384 


Etheriidae 


Etheria elliptica 1 


DQ241803 




Etheha elliptica 2 


AF231739 


Outgroup taxa 


Albinaha turrita 


X71393 




Dentalium sp. 


U56843 




Drosophila yakuba 


X03240 




Katharina sp. 


U56845 




Lepetodrilus elevatus 


U56846 




Neotrigonia margaritacea 


U56850 




Solemya velum 


U56852 



DUl IN THE UNIONOIDA 



273 



tions from all five species representing the 
Etheriidae, Iridinidae, and Mycetopodidae 
failed to yield the expected long M cox2-cox1 
fragment. Regarding the total DNAs extracted 
from representatives of these three families, 
all PCR attempts resulted in amplification of 
an F genome fragment from both mantle and 
testis DNA extractions. Subsequent sequenc- 
ing and phylogenetic analyses of these frag- 
ments confirmed that identical sequences (all 
from F genomes) had been amplified from both 
mantle and testis extractions from the same 
individuals. Furthermore, the cox1 and 16S 
intragenic primer pairs failed to produce an M 
genome fragment. This corroborates the fail- 
ure of the intergenic cox2-cox1 primers to 
amplify an M fragment from the sampled 
etherioidean individuals. 

The Etheriidae, Iridinidae, and Mycetopodi- 
dae represent closely related taxa, and have 
typically been given distinct superfamilial sta- 
tus, Etherioidea (Parodiz & Bonetto, 1963; 
Hoeh et al., 1998b, 2001; Bogan & Hoeh, 
2000; Roe & Hoeh, 2003). Given their phylo- 
genetic propinquity, it is not surprising that rep- 
resentatives of these three families would 
produce similar, yet unexpected, results; fail- 
ure to yield M genome amplicons. There ap- 
pear to be three possible explanations for the 
failure of representatives of the Etheriidae, 
Iridinidae and Mycetopodidae to yield M frag- 
ments. (1 ) Recent masculinization events have 
occurred such that the newly recruited "M ge- 
nomes" (originally F genomes) are amplified. 
(2) The primers failed to anneal to the M se- 
quence due to the rapidly evolving nature of 
the M genomes (Rawson & Hilbish, 1995; 
Stewart et al., 1995; Curóle & Kocher, 2002; 
Hoeh et al., 2002; Krebs, 2004), (3) These taxa 
do not possess DUl. 

Mitochondrial DNA masculinization events in 
DUI-containing taxa were first postulated for, 
and later supported with data from, Mytilus by 
Hoeh et al. (1 996, 1 997). Subsequently, other 
investigators have corroborated the existence 
of the mtDNA masculinization process in 
mytiloid but not in unionoid bivalves (e.g., 
Zouros et al., 1994; Hoeh et al., 1996, 2002; 
Saavedra et al., 1997). However, if the mas- 
culinization hypothesis is to be invoked as the 
explanation for our observations, we would 
expect to observe distinct etherioidean M 
mtDNA sequences that are more closely re- 
lated to F sequences than to other M se- 
quences. Our repeated observations of 
identical mtDNA sequences from testis- and 



mantle-derived DNA extractions from each 
etherioidean individual examined do not meet 
these expectations. Immediately after a mas- 
culinization event, it is predicted that the F and 
new "M" (i.e., recently converted from the fe- 
male- to the male-transmission route) ge- 
nomes will be identical. However, under the 
masculinization hypothesis, it is extremely 
unlikely that we would have observed identi- 
cal cox1 sequences from separate mantle and 
testis DNA extractions from individuals repre- 
senting five species as this would require 
multiple independent, approximately simulta- 
neous, and relatively recent masculinization 
events. 

The M genomes in both unionoid and 
mytiloid bivalves have a significantly greater 
rate of substitution relative to that estimated 
for the corresponding F genomes (SkibinskI 
et al., 1994; Hoeh et al., 1996, 2002; Liu et 
al., 1996; Stewart et al., 1996; Quesada et al., 
1998; Krebs, 2004). This may be due to a 
higher mutation rate for the M genomes, 
smaller effective population size for the M ge- 
nomes, positive selection for the M genomes, 
relaxed selection for the M genomes, or a com- 
bination of these processes (Stewart et al., 
1 996; Passamonti et al., 2003). Nevertheless, 
an elevated rate of substitution has been sug- 
gested as the explanation for the occasional 
failure of universal mtDNA primer pairs to 
amplify M genomes (Rawson & Hilbish, 1995; 
Stewart et al., 1995; Curóle & Kocher, 2002; 
Hoeh et al., 2002; Krebs, 2004). Our use of 
three distinct, conserved primer pairs to at- 
tempt amplification of etherioidean M ge- 
nomes, with failure to do so in each instance, 
suggests that either (1) all of the sampled 
etherioid specimens have very divergent M 
mtDNA sequences for 16S, cox1, and cox2 or 
(2) these specimens lack DUl. We believe that 
multiple failed M-fragment amplification at- 
tempts, using both intra- and inter-genic con- 
served primer pairs, render the former 
hypothesis unlikely. 

We thus believe that it is likely that DUl is 
absent from the Etherioidea. This leads to the 
question ofwhetherthe absence of DU! in this 
superfamily indicates a loss in the ancestral 
etherioidean lineage or a gain of DUl in the 
ancestral unionoidean lineage (after Parodiz 
& Bonetto, 1963). Unfortunately, due to the 
lack of a robust unionoid phylogeny and infor- 
mation regarding the presence/absence of DUl 
in Neotrigonia, there remains no a priori way 
to rigorously evaluate which condition is apo- 



274 



WALKER ETAL. 



morphic and thus, the phylogenetically infor- 
mative character state. If DUI was derived 
within the Unionoida. then it may represent an 
apomorphy for the Unionoidea. whereas a loss 
of DUI in the common etherioidean ancestor 
would represent an apomorphy for the 
etherioids and the presence of DUI would rep- 
resent a plesiomorphy for the Unionoida. Ro- 
bust inferences regarding the evolutionary 
dynamics of unionoid DUI depend upon the 
existence of a robust phylogeny for the Unio- 
noida. As mentioned previously, phylogenetic 
analyses to date, utilizing partial F cox1 se- 
quences (Folmer fragment), have been unable 
to robustly resolve unionoid familial relation- 
ships. However, M cox1 sequences have dem- 
onstrated the ability to increase topological 
resolution when analyzed alone or in conjunc- 
tion with F cox1 sequences (Hoeh et al., 2002). 
Utilization of the relatively new model-based 
Bayesian phylogenetic methods appears to 
reveal additional phylogenetic signal contained 
within existing F cox1 sequences. 

What Can Analyses of Complementary F and 
M cox1 Sequences Tell us About Higher Level 
Unionoidean Relationships? 

Failure to amplify M genome fragments from 
any etherioid taxa obviously prevents us from 
conducting any taxonomically inclusive M ge- 
nome-based higher level phylogenetic analy- 
sis of unionoid relationships. Given this sehous 
limitation, what can analyses of complemen- 
tary F and M cox1 sequence data sets tell us 
about higher level unionoidean {sensu Parodiz 
& Bonetto, 1963) relationships? Phylogenetic 
analyses of F cox1 sequences recover 
Hyridella as the basal unionoidean lineage; 
however, the Unionidae are not recovered as 
monophyletic (Fig. 2). Specifically, Cumberlan- 
dia, a margaritiferid, is depicted as the sister 
taxon to Fusconaia and this placement ren- 
ders the Unionidae paraphyletic. Nodal sup- 
port levels for the interfamilial relationships are 
relatively low as seen in previously published 
F cox1 analyses (e.g., Hoeh et al., 2001 ). Ro- 
bust intergeneric nodal support values are only 
observed for the clade containing the follow- 
ing four lampsiline taxa: Actinonaias, 
Lampsilis, Ligumia, and Potamilus. 

Analyses of M cox1 sequences recovered a 
robustly supported, monophyletic Unionidae 
but the relationships among the hyriid, 
margaritiferid, and unionid taxa were not well 
resolved (Fig. 3). As in the topology obtained 
from the F cox1 analysis, Hyridella is repre- 



sented as a descendent of the primary 
unionoidean cladogenic event. In general, the 
nodal support values derived from analyses 
of M cox1 sequences are significantly im- 
proved over those of the F cox1 analysis pre- 
sented herein (Fig. 2). These results were 
foreshadowed by previous comparative analy- 
ses of F and M sequences (e.g., Hoeh et al., 
2002; Krebs, 2004). 

Concatenating F and M cox1 sequences was 
legitimized by the lack of significant incongru- 
ence between the F and M sequences (as in- 
dicated by the ILD test, p = 0.271). Phylo- 
genetic analyses of the concatenated F and M 
cox1 sequences (Fig. 4) produced a topology 
very similar to that produced by the M cox1 
analyses (Fig. 3). This result was anticipated 
due to the greater number of parsimony-infor- 
mative sites in the M cox7 sequences (Hoeh 
et al., 2002) and the lack of significant incon- 
gruence between the F and M sequences. 
However, a fundamental difference between 
the results of the M and F + M analyses is the 
latter's increased nodal support for margariti- 
ferids (represented herein by Cumberlandia) 
as the sister taxon to the Unionidae. This re- 
sult strongly supports the basal position of 
hyriids (represented by Hyridella) within the 
Unionoidea. This basal placement of hyriids is 
independently supported by Graf's (2002) 
analyses of 28S sequences, which also lacked 
representatives of the Etherioidea. 

The basal position of Hyridella in all of the 
analyses presented herein is in conflict with 
the placement of the Margaritiferidae as the 
basal unionoid lineage as depicted in the mor- 
phology-based trees of Graf (2000) and Hoeh 
et al. (2001), as well as in the total evidence- 
based tree presented in Roe & Hoeh (2003). 
However, our topology is consistent with the 
molecular and total evidence-based topologies 
of Hoeh et al. (1998b, 2001) as well as with 
the hypothesis that the relatively "simple" 
anatomy of margaritiferids is a derived rather 
than an ancestral condition as often postulated. 
For example, the loss of ctenidial water tubes 
in margaritiferids may be the end result of se- 
lection for the release of a significantly larger 
conglutínate mass than that of its ancestor. 

If the relative evolutionary relationships pos- 
tulated from the complementary F and M con- 
catenated cox7 data set analyses are main- 
tained in subsequent more taxonomically 
inclusive phylogenetic analyses of the Unio- 
noida, where might a monophyletic Etherioi- 
dea attach to our unionoidean tree? Two pre- 
viously presented hypotheses of unionoid 



DUl IN THE UNIONOIDA 



275 



BIPP 

MP 
(ML) 



57 



100 



69 



63 



92 
(84) 



Actinonaias 



Lampsilis 



Ligumia 



Potamilus 



Cyrtonaias 



Fusconaia 



Unionidae 



Cumberlandia Margaritiferidae 



77 



Gonidea 



Pseudodon 



Inversidens 



100 




P> ganodon tr 


100 
(100) 




- Pyganodongr. 


- Anodo 


ita 



Unionidae 



Hvridella 



Hyriidae 



Neotrisonia 



0.5 substitutions/site 



FIG. 2. Best tree found by Bayesian analyses of F cox1 sequences (619 bp). When > 50, Bayesian 
posterior probabilities (x100) presented above internodes, MP bootstrap values and ML bootstrap 
values (in parentheses) are presented below internodes. 



276 



Bl PP 
MP 
(ML) 



86 



1 00 



45 
(84) 



WALKER ETAL. 
90 




100 



76 



100 



99 
(98) 



G on i dea 



Aclinonaias 



Lampsilis 



Liizumia 



C\rtoriaias 



Potamilus 



Fusconaia 



100 



100 
(99) 



Pyganodon tV. 



Pvoanodon ai". 



Anodonla 



Imersidens 



Pseudodon 



LJnionidae 



Cumberlandia 



H\ridella 



Maryaritiferidae 



Hyriidae 



Neotriuonia 



0.1 substitutions/site 



FIG. 3. Best tree found by Bayesian analyses of M cox1 sequences (619 bp). When > 50, Bayesian 
posterior probabilities (x100) presented above internodes, MP bootstrap values and ML bootstrap 
values (in parentheses) are presented below internodes. 



DUl IN THE UNIONOIDA 



277 



BIPP 

MP 
(ML) 



90 



89 



(52 



52 



94 



90 



69 
(60) 



96 

(77) 



56 



81 

(72) 

100 Ч 

81 

(98) 



100 

84 
(88) 



100 



100 



98 
(98) 



98 
(98) 



Actinonaias 



Lampsilis 



Liautnia 



Potamilus 



Cyrtonaias 



Fusconaia 



100 



95 



97 
(92) 



100 

(100) 



Pyganodon fr. 



Pyganodon gr. 



Unionidae 



Anodonta 



Inversidens 



— Gonidea 



Pseudodon 



Cumberlandia 



Hvridclla 



Margaritiferidae 



Hyriidac 



Neotrigonia 



0.1 subslitutions/site 



FIG 4 Best tree found by Bayesian analyses of concatenated F + M cox1 sequences (1238 bp). When 
> 50 Bayesian posterior probabilities (x100) presented above internodes, MP bootstrap values and 
ML bootstrap values (in parentheses) are presented below internodes. 



278 



WALKER ETAL. 



higher level relationships are consistent with 
the relative relationships presented herein. One 
possibility is that the etherioidean lineage is 
the sister taxon to a monophyletic Unionoidea 
(Fig. 5A). This topology is consistent with the 
Parodiz & Bonetto (1963) unionoid classifica- 
tion. Deductions from this topology regarding 
DUI evolutionary dynamics are dependent on 
whether or not the outgroup. Neotrigonia, pos- 
sesses DUI. If Neotrigonia lacks DUI, this to- 
pology would be consistent with the hypoth- 
esis of an ancestral unionoidean gain of DUI. 
Alternatively, \1 Neotrigonia possesses DUI. this 



topology would be consistent with a loss of DUI 
in the ancestral etherioidean lineage. Under 
this topology, endobranchy is hypothesized as 
the ancestral unionoid brooding strategy but 
the two principal larval character states can- 
not be polarized. In addition, under this topol- 
ogy, the Parodiz & Bonetto (1963) "indepen- 
dent invasions of freshwater" hypothesis for 
unionoidean and etherioidean bivalves is not 
robustly rejected. 

An alternative unionoid topology, that would 
maintain the relative evolutionary relationships 
represented in the trees from the complemen- 



Unionidae 



Margaritiferidae 



Hyriidae 



Etherioidea 



loss of endobran 



В 




gain of DUI 



gain of endobranchy 



Unionidae Margaritiferidae 



loss of endobranchy 



Etherioidea 

loss of glochidium 
loss of DUI 



Hyriidae 



ШП of glochidium 

gain of endobranchy 

gam of DU I 



FIG. 5. Hypothesized character state transitions for reproductive characters exhibited 
by unionoid bivalves. A: Hypothesized familial relationships after Parodiz & Bonetto 
(1963): B: Hypothesized familial relationships after Hoeh et al. (2001). The two DUI 
character state optimizations displayed herein are based on the assumption that 
Neotrigonia lacks DUI. 



DUl IN THE UNIONOIDA 



279 



tary analyses, is that the Etherioidea is sister 
taxon to the margaritiferid + unionid clade (Fig. 
5B). This particular unionoid topology was sup- 
ported by previous analyses using F cox1 se- 
quences (Hoeh et al., 1998b, 2001). This 
topological placement would support the fol- 
lowing hypotheses: (1) the ancestral etherioi- 
dean lineage lost DUl, (2) the glochidium is 
the ancestral larval type for the Unionoida, and 
(3) endobranchy is the ancestral brooding con- 
dition for the Unionoida. Additionally, this par- 
ticular unionoid topology would reject the 
monophyly of the Unionoidea {sensu Parodiz 
& Bonetto, 1 963) as well as Parodiz & Bonetto's 
"independent invasions of freshwater" hypoth- 
esis for unionoidean and etherioidean bivalves. 

What Can Taxonomically Inclusive Analyses 
of Non-concatenated F and M cox1 Se- 
quences Tell us About Higher Level Unionoid 
Relationships? 

The topology of the F genome portion of the 
inclusive Bl analysis (Fig. 6) strongly supports 
the hypothesis that etherioids are the sister 
taxon to a margaritiferid + unionid clade (PP = 
93), thus rendering the Unionoidea para- 
phyletic. This topology is congruent with the 
hypothesis of unionoid relationships presented 
in Figure 1С (after Hoeh et al., 1998b, 2001). 
It also strongly supports the monophyly of 
margaritiferid, mycetopodid, and etherioid 
bivalves (PP = 100 for each clade) and the 
paraphyly of the Unionidae (PP = 90). In con- 
trast, monophyly of the Hyriidae is weakly sup- 
ported (PP = 63) and iridinid monophyly was 
not supported. The results from the M-genome 
portion of the inclusive Bl-based phylogenetic 
analysis of the cox1 DNA sequences (Fig. 6) 
strongly support the sister taxa status (PP = 
96) for the margaritiferid and unionid clades 
(PP = 1 00 for each family). Furthermore, both 
the F and M genome portions of this Bl analy- 
sis strongly support the evolutionary propin- 
quity of Gonidea and Pseudodon (PPs = 98 
and 100, respectively). The topology obtained 
from the concatenated F and M cox1 sequence 
analysis (Fig. 4) is in agreement with the former 
but not the latter hypothesis. In general, the 
MP bootstrap analysis of the inclusive non- 
concatenated cox1 data set produced much 
lower nodal support values than did the Bl 
analysis (Fig. 6). 

The Bl analysis presented in Figure 6 strongly 
supports the character state dynamics hypoth- 
esized in Figure 5B: (1 ) The presence of DUl, 
glochidial larvae, and endobranchous brood- 
ing characterized the ancestral unionoid lin- 



eage, (2) the loss of DUl and glochidial larvae 
(i.e., the gain of standard maternal inheritance 
and lasidial larvae) occurred in the ancestral 
etherioidean lineage, and (3) the loss of 
endobranchy occurred in the ancestor of the 
margaritiferid + unionid clade. The hypothesis 
that the relatively "simple" anatomy of margari- 
tiferids is a derived rather than an ancestral 
condition is also supported. Furthermore, the 
topology presented in Figure 6 strongly rejects 
the hypothesis that unionoidean and 
etherioidean bivalves represent independent 
invasions of freshwater habitat (i.e., unionoid 
bivalve polyphyly, as suggested by Parodiz & 
Bonetto, 1963). Reciprocal monophyly for the 
Etherioidea and Unionoidea, which would be 
consistent with the independent invasion hy- 
pothesis, is rejected by the topology presented 
in Figure 6. 

At least two aspects of the phylogenetic re- 
sults presented in Figure 6 should serve as a 
caution to any attempt to canonize these re- 
sults: (1) only the Bl analysis provided strong 
support for many of the higher level unionoid 
bivalve relationships discussed above and (2) 
the phylogeny for the Unionoida indicated in 
the F clade of Figure 6 is based on a relatively 
small number of nucleotides (a maximum of 
619) from a single genetic locus (F cox1). In 
order to more rigorously evaluate these cen- 
tral yet, in our opinion, currently open ques- 
tions regarding unionoid higher level relation- 
ships and character state evolutionary 
dynamics, we are currently investigating the 
efficacy of incorporating DNA sequences from 
additional F mitochondrial genes (e.g., F cox2). 
Including information from multiple female- 
transmitted unionoid mtDNA genes has the 
potential to increase the topological resolution 
of taxonomically inclusive analyses. In addi- 
tion, nuclear genes (e.g., 28S, EF1-alpha, 
act42A) as well as morphological characters 
are being investigated to assess their poten- 
tial to facilitate the construction of a robust 
phylogeny for the Unionoida. 



ACKNOWLEDGEMENTS 

For aid in specimen procurement, the authors 
would like to thank S. Ahlstedt, С Barnhart, В. 
Butler, D. Campbell, A. Christian, K. Cummings, 
R. Dimock, K. Garo, M. Gordon, J. Harris, P. 
Hartfield, W. Heard, B. Howells, D. Hubbs, J. 
Jones, K. KuehnI, B. Lang, J. Maunder, T 
Myers, B. Posey, B. Sietman, D. Smith, G. 
Soliman, R. Tankersly R. Trdan, M. Vidrine, and 
D. Zanatta. We also thank D. Senyo, С 



280 



WALKER ETAL. 



BIPP 
MP 




Margaritifera du 1 
Margantifera du 2 
Marganlifera du 3 
Margantifera du 4 
Margantifera du 5 
Margantifera du 6 
Margantifera rna 1 
Margantifera ma 2 
Margantifera ma 3 
Margantifera ma 4 
Margantifera ma 5 
Margantifera ma 6 
Margantifera ma 7 
Margantifera rria 8 
Margantifera ma 9 
Margantifera ma 10 
Margantifera ma 1 1 
Margantifera ma 12 
Margantifera ma 13 
Margantifera ma '4 
Margantifera ma 15 
Datiunnaia da 1 
Margantifera fa 1 
Margantifera fa 2 
Margantifera fa 3 
Margantifera la 
Margantifera au 1 
Margantifera au 2 
Margantifera au 3 
Margantifera au 4 
Cumberlandia mo 1 
(¿umberlandia mo 2 
Cumberlandia mo 3 
Cumberlandia mo 4 
Ligumia re 
Actinonaias li 
Lampsilis le 
Potamilus pu 
Cyrtonaias la 
Fusconaia fl 
Gonidea an 
Pseudodon vo 
Pyganodon fr 
Pyganodon gr 
Anodonlites gu 
Anodontites tr 
Tamsiella ta 
Monocondylaea mi 
Acostaea n 
Ctiambardia ru 1 
Chambardia nj 2 
Ctiambardia ru 3 
Ettiena el 1 
Eltiena el- 2 
Muleladu. 1 
Mulela du- 2 
Muleta du. 3 
Mulela ro 1 
Mutela ro. 2 
Alathyria ja. 1 
Alathyriaja 2 
Alattiyriaja 3 
Alathyna ¡a 4 
Veiesunio В 1 
Velesunio В 2 
Veiesunio an 
Lortiella ru 
Veiesunio А 1 
Veiesunio А 2 
Veiesunio am 1 
Veiesunio am 2 
Veiesunio sp 1 
Veiesunio am 3 
Veiesunio am 4 
Veiesunio sp 2 
Veiesunio D 1 
Veiesunio D 2 
Hyndella me 1 
Hyndellade 1 
Hyndella de 2 
Hyndella au 
Castalia st 
Diplodon de 
Neotngonia ma 
Lampsilis te 
Actinonaias li 
Ligumia re 
Cyrtonaias ta 
Polamilus pu 
Fusconaia fl 
Pyganodon fr 
Pyganodon gr 
Gonidea an 
Psuedodon vo 
Margantifera ma 16 
Dahurinaiada 2 
Dahurinaiada 3 
Cumberlandia mo 
Hyndella me 2 
Solemya ve 
Lepetodrilus el. 
Kaltianna sp 
Oentalium sp 
Albinaria tu 
Drosopliila ya. 



Margaritiferidae 



Unionidae 



Mycetopotjidae 

Indinidae 
Etheriidae 

Iridinidae 



Unionoidea 



Etherioidea 



Hyriidae 



Unionoidea 



Neotrigonildae Tngonioidea Tngonioida 



Unionidae 



Margantifendae 
I Hyriidae 



Unionoidea 



M 



Outgroups 



FIG. 6. Fifty percent majority rule consensus tree, with posterior probabilities (x100, above internodes), 
obtained from the inclusive Bayesian analysis of non-concatenated cox1 F and M DNA sequences. 
When > 50, MP bootstrap values (ЮО.ОООх, fast-heuristic search) are presented under the internodes. 



DUl IN THE UNIONOIDA 



281 



Paustian, and S. Cindea for laboratory assis- 
tance, W. H. Heard for stimulating discussions 
regarding unionoid bivalve evolutionary biology, 
and M. E. Gordon for comments on this manu- 
script. We believe that the suggestions provided 
by the editor, George M. Davis, and two anony- 
mous reviewers significantly improved this 
manuscript. This work was partially supported 
by an NSF grant DEB-0237175 to WRH. 



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Revised ms. accepted 19 October 2005 



RESEARCH NOTES 



MALACOLOGIA, 2006, 48(1-2): 285-294 

THE HISTORICAL MISIDENTIFICATION OF 

MARGARITIFERA AURICULARIA FOR M. MARGARITIFERA 

(BIVALVIA, UNIONOIDEA) EXPLAINED BY THEIR ICONOGRAPHY 

Arturo Valledor de Lozoya' & Rafael Araujo^ 

ABSTRACT 

Throughout its history, Margahtifera auriculaha has been confused with its relative M. 
margahtifera. This paper compiles the early iconography of M. auricularia and reproduces 
the illustrations of this species. Our objective is to not only recapture the many interesting 
images of M. auricularia, but also to examine the historical errors that led to the confusion 
between the two species. After selecting valid representations of M. auricularia and its true 
synonyms, we see that this confusion has existed since Spengler (1793) first described 
the species. Indeed, we show that the first published image of M. auricularia, by Draparnaud 
(1805), was erroneously labeled as an image of M. margahtifera. We also reproduce sev- 
eral previously undiscovered illustrations of juvenile specimens of M. auricularia, as well 
as some interesting figures of M. margaritifera that were published before its description 
by Linnaeus (1 758). One of these illustrations, Magnus (1 555), is probably the first known 
image of a freshwater mussel. 



FIRST DESCRIPTION OF M. AURICULARIA 

AND ITS EARLY MISIDENTIFICATION WITH 

M. MARGARITIFERA 

The giant freshwater mussel, Margaritifera 
auricularia, is one of two European species of 
Margaritifera. Before its present rarity, it lived 
in the large, muddy rivers of western Europe 
and North Africa (Araujo & Ramos, 2000), 
whereas its relative M. margaritifera inhabited 
the smaller, colder rivers of northern Europe 
and North America. The characteristics of the 
fluvial habitat of M. auricularia have made it 
difficult to gather specimens. Thus, not only 
was this species discovered later, but it is less 
well known than M. margahtifera, which has 
been exploited since Roman times for its ca- 
pacity to produce small pearls (Bonnemère, 
1901). 

Margaritifera margaritifera was first de- 
scribed by Linnaeus (1758) as Mya margah- 
tifera. Margahtifera auhculaha was originally 
named as Unio auhculahus by the Danish 
malacologist Lorentz Spengler (1793: 54-55), 
who erroneously cited the East Indies as its 
type locality. Although Spengler did not illus- 
trate U. auhculahus, his description of its large 
dorsal teeth and the hinge clearly differenti- 
ate it from M. margaritifera. Lamarck (1819) 



described Unio sinuata (Fig. 1), which today 
is considered to be a synonym of M. auhcu- 
laha. 

Despite Spengler's description, both Euro- 
pean species of the genus Margaritifera have 
been misidentified many times, and the first 
author to do so was, curiously enough, 
Spengler himself. In his original description, 
he cited a figure in by Martin Lister's Histohae 
conchyliorum (1686: fig. 149) as an illustra- 
tion of Unio auhculahus. However, Lister's fig- 
ure shows the inside of a large, very sinuate 
M. margahtifera valve with pronounced cardi- 
nal teeth, and which at first glance resembles 
a valve of M. auhculaha (Fig. 2). To confirm 
this, we tried unsuccessfully to find this speci- 
men. Lister used shells from several collec- 
tions to illustrate his book, mainly from his 
collection and that of William Courten. Accord- 
ing to Wilkins (1953), the Courten collection 
was acquired by Hans Sloane, and the Sloane 
collection later became the nucleus of the Brit- 
ish Museum collection, now in The Natural 
History Museum. Nevertheless, this M. mar- 
garitifera valve is not among the shells in the 
Sloane collection that were illustrated by Lister 
(Wilkins, 1953). It is possible that this valve 
was part of the Lister collection that was first 
owned by the Ashmolean Museum, and which 



'Dr Federico Rubio 4, 28039 Madrid, Spain 

^Museo Nacional de Ciencias Naturales, José Gutiérrez Abascal 2, 28006 Madrid, Spain; rafael@mncn.csic.es 



285 



286 



VALLEDOR DE LOZOYA & ARAUJO 




FIGS. 1-5. FIG. 1: One of the syntypes of Unio sinuata Lamarck (MHNG 1086/75). Inscriptions by 
Lamarck are found in the interior of the valves: FIG. 2: Lister (1686: sheet of "plates", each a separate 
woodcut) with several freshwater bivalves and one right valve of M. margaritifera in pi. 149 (bottom). 
By permission of the Museo Nacional de Ciencias Naturales, Madrid, Spain: FIG. 3: A fishery of M. 
margaritifera by Magnus (1555). By permission of the Biblioteca Nacional, Madrid, Spain: FIG. 4: The 
illustration of M. margaritifera (upper left corner) by Pontoppidan (1755). By permission of the Museo 
Nacional de Ciencias Naturales. Madrid, Spain: FIG. 5: Type specimen and original label of M. 
auriculaha from the Spengler collection. 



ICONOGRAPHIE OF M. AURICULARIA 



287 



was later moved to the Oxford University Mu- 
seum of Natural History. However, Dance 
(1986) reported that none of the shells attrib- 
uted to the Lister collection were there. 

Simpson (1900) attributed Lister's figure to 
M. margaritifera, and Haas (1909), one of the 
most important researchers on freshwater 
mussels, also discovered Spengler's error, 
realizing that the lateral teeth were absent. 
This also meant that M. margahtifera had been 
illustrated by Lister nearly a century prior to 
its description by Linnaeus. There were at least 
two other authors who illustrated M. margari- 
tifera before Lister. The first of these was prob- 
ably Olaus Magnus (1555), a Swedish 
geographer, archbishop of Upsala and author 
of Historiae de gentibus septentrionalibus. His 
illustration of a catch of M. margahtifera (Fig. 
3) was the first rough image of this species 
and perhaps the first ever of a freshwater 
mussel. Pontoppidan (1755), a bishop of 
Bergen, also illustrated M. margaritifera in his 
The natural history of Norway (Fig. 4). (This 
same figure was probably in the original 1753 
edition, but we have not had an opportunity to 
examine it.) Other pre-Linnean authors, includ- 
ing Rondelet (1555) and Boussuet (1558), il- 
lustrated specimens of such other freshwater 
mussels as Anodonta. 

Haas (1913) confirmed true identity of Unio 
aunculanus in his paper on the Unio species 
described by Spengler. In an attempt to pre- 
vent future misidentification, he illustrated 
Spengler's polished specimen in the Natural 
History Museum of Copenhagen (Fig. 5). 

Several years prior to this, two European 
authors contributed to the confusion with their 
interpretation of freshwater mussel fossils 
discovered in Britain. Jackson & Kennard 
(1909) mistakenly attributed M. aunculaha 
shells from Pleistocene sediments of the 
Thames River to Unio {Margaritana) 
margaritifer (Linnaeus) (= M. margaritifera). 
(Margaritana is an objective synonym of 
Margahtifera.) These authors noted the ex- 
traordinary size of the shells and concluded 
that "Unio margaritifer was living abundantly 
in the Thames". Haas (1910) and Jackson 
(1911) soon rectified this error when they con- 
firmed that the fossils were actually Unio 
sinuatus (Lamarck) (= M. auhculaha). 

Just like their European counterparts. North 
American malacologists have also been con- 
fused by these Margaritifera species. For in- 
stance, Simpson (1900) used the names 
Margaritana margaritifera (Linnaeus) and 



Margaritana crassa (Retzius, 1788) to refer to 
M. auhculaha. Several years later, Kennard 
etal. (1925) suggested that this confusion was 
caused "partly through misidentification and 
partly because the later observers relied on 
the figures of their predecessors more than 
on their texts but chiefly because successive 
writers borrowed the synonymy of their fore- 
runners without checking it". Despite this ob- 
servation, however, they also continued to 
make the same errors themselves. According 
to these authors, the Mya margaritifera from 
Schröter's Die Geschichte der Flüssconchylien 
(1779: pi. 4, fig. 1) represents M. auhculaha 
when, in fact, it is M. margaritifera. It is likely 
that they did not examine this figure, given that 
they considered their identification "unmistak- 
able because of the strong lateral teeth and 
the peculiarities of the anterior muscular 
scars". These characters are absent in the 
above mentioned engraving, which clearly il- 
lustrates a specimen of M. margaritifera. After 
reading the authors' commentaries on another 
figure, we are certain that either they did not 
carefully study or did not understand 
Schröter's book. Schroter's specimen of Mya 
testa crassa is not, as they claim, a medium- 
sized specimen of M. margahtifera, but rather 
a normal specimen of Unio crassus (Fig. 6). 

We see then that the confusion began with 
Spengler's erroneous interpretation of Lister's 
figure and was later complicated by the equally 
incorrect interpretation of Mya testa crassa 
(Schröter) by Kennard et al. (1925). Simpson 
(1900: 677, note 4) makes the same error by 
including Mya testa crassa (Schröter) as a 
synonym for the species Margaritana crassa 
(Retzius) in his records of M. auhculaha. The 
confusion was perhaps caused by usage of 
the Latin crassus (meaning "very thick"), by 
both Lister, in his caption below the figure of 
M. margahtifera (Musculus niger, omnium 
longe crassisimus, conchae longae species 
Gesn. Aldrov.), and by Spengler in his descrip- 
tion of Unio auricularius (Testa crassa, 
oblonga, etc.). 

More interesting information is revealed 
about Lister's figure in his Histohae animalium 
Angliae (1681), some years prior to Histohae 
conchyliorum (1686). Here, Lister illustrates 
the same M. margaritifera valve that appears 
in the later work, along with valves from two 
other molluscs - Unio pictorum and Anodonta 
sp. The description of the M. margaritifera 
valve is only slightly different from that which 
appeared in Histohae conchyliorum: "Black 



288 



VALLEDOR DE LOZOYA & ARAUJO 



U^,r. 'П^ ■ f--"^ 





«oitrTTE ráUr. j î МОгаКТТК .mure. 



^ Ф ^ Ф • Ф 




FIGS. 6-9. FIG. 6; Plate 2 of Schröter (1779). Муа testa crassa in fig. 2 (upper left corner) 
is actually Unio crassus. By permission of the British Library; FIG. 7: Blainville's (1827: pi. 
67, fig. 3) figure of M. auriculaha (middle). By permission of the Museo Nacional de Ciencias 
Naturales, Madrid, Spain; FIG. 8; Plate 10 of Draparnaud (1805). This is the first known 
illustration of M. auriculaha (middle and bottom left). By permission of the Museo Nacional 
de Ciencias Naturales, Madrid, Spain; FIG. 9: Plate 23 by Dupuy (1851) representing one 
adult specimen (top) and the first known figure of a M. auhcularia juvenile (middle) in figs. 
7a and 7c, respectively. Fig. 7b (left) depicts the hinge of the adult. By permission of the 
Museo Nacional de Ciencias Naturales, Madrid, Spain. 



ICONOGRAPHIE OF M. AURICULARIA 



289 



mussel, entire shell very thick and very strong, 
from long shelled species after Gesner and 
Aldrovandi" [Musculus niger, omnium 
crassissima et ponderosissima testa, conchae 
longae species Gesn. Aldrov.]. However, fur- 
ther information written below the figure plainly 
pertains to M. margahtifera. For instance, 
Lister says: "It is sometimes fished with net in 
the deep whirpools of the Tees River in York- 
shire, not so far from Dinsdale" [In profundis 
voraginibus Fluvii Tees agri Eboracensis, non 
longe a Dinsdale, rete aliquando expiscatur]. 
We know today that only M. margahtifera lives 
in Yorkshire Rivers. 



ICONOGRAPHY OF 
MARGARITIFERA AURICULARIA 

We have reviewed all the early books on 
shells and malacology listed by Caprotti (1 994) 
and Barbero (1999) (Table 1), as well as 
Simpson's (1900) list of synonyms for 
Margaritana margahtifera and M. crassa. Hav- 
ing confirmed that Mya testa crassa (Schröter) 
did not correspond to M, auhculaha, the next 
author on Simpson's list to illustrate the spe- 
cies was Blainville (1827: pi. 67, fig. 3). In a 
lithography showing naiads (Fig. 7), Blainville 
identified the giant freshwater pearl mussel as 
Unio sinuata (or moulette sinuée). Neverthe- 
less, Azpeitia (1933) discovered that another 
author, Draparnaud (1805), illustrated M. au- 
hculaha several years prior in his Histoire 



naturelle des mollusques terrestres et 
fluviátiles de la France (Fig. 8). This image 
went unnoticed because Draparnaud mis- 
identified both species of Margahtifera and 
labeled his image Unio margahtifer, Moulette 
margaritifera, or Moule du Rhin, although its 
real identity can be proven by the hinge teeth. 
Locard (1895) also reported this mistake in his 
Étude sur la collection conchyliologique de 
Draparnaud: "Draparnaud has made an error 
in respect of this species. His Unio margahtifer, 
cited by him as Mya margaritifera after Linné 
and Müller, really is the Unio sinuatus of 
Lamarck. We have specimens proceeding 
from the Loire River which are exactly similar 
to the one figured by him." 

The next authors on Simpson's list to illus- 
trate M. auhculaha were Dupuy (1851) (Fig. 
9), who drew the first known figure of a juve- 
nile M. auhculaha, Küster (1855) (Fig. 10), 
Rossmässler (1 855) (Fig. 1 1 ), Moquin-Tandon 
(1855) (Fig. 12), Drouet (1857) (Fig. 13), G. B. 
Sowerby II (1868) (Fig. 14), and Locard (1893) 
(Fig. 15). Simpson also makes reference to: 
Bruguière (1797: pi. 248) [as "Deshayes, 
1827"], Pfeiffer (1821), Rossmässler (1836, 
1838, 1856), and Hanley (1856), but with the 
exception of Rossmässler (1856), the figures 
of these authors do no depict M. auhculaha. 
Simpson (1900) wrote that the alleged M. au- 
hculaha specimens illustrated by Bruguière 
(1797) "look something like a heavy inflated 
Lampsilis alatus Say" [now Potamilus alatus 
(Say, 1817)]. In any event, the figured outline 



TABLE 1. Historical illustrations of M. auhculaha. 



Author 


Date 


Figure(s) 


Cited as 


Draparnaud 


1805 


pi. 10, fig. 19 


Unio margaritifera 


Blainville 


1827 


pi. 67, fig. 3 


Unio sinuata 


Dupuy 


1851 


pi. 23, fig. 7a-c 


Unio sinuatus 


Küster 


1855 


p!. 37, fig. 1 


Unio sinuatus 


Rossmässler 


1855 


pi. 70, fig. 853 


Unio sinuatus 


Moquin-Tandon 


1855 


pi. 48, fig. 1 


Unio sinuatus 


Drouet 


1857 


pi. 2 


Unio sinuatus 


Sowerby 


1868 


pi. 62, fig. 311 


Unio sinuatus 


Locard 


1893 


figs. 163, 164 


Unio margaritanopsis & U. sinuatus 


Haas 


1913 


fig. 1 


Unio a и пси la nus 


Haas 


1916 


fig. 1 


Margaritana auhculaha 


Kennard et al. 


1925 


pi. 21, figs. 1-3 


Margaritana auhculaha 


Haas 


1929 


figs. 181, 182 


Margaritifera auhculaha 


Germain 


1930 


pi. 26, fig. 609, 615 


Margaritana auricularia & /W. ? margaritanopsis 


Azpeitia 


1933 


pi. 12, figs. 65, 66; pi. 


13, fig. 67 Margaritana auhculaha 


Huckriede & Berdau 


1970 


pi. 1 


Margaritifera auhculaha 


Fechter & Falkner 


1990 


color photo, p. 255 


Pseudunio auhculahus 


Falkner 


1994 


photo, fig. 1 


Pseudunio auhculahus 



290 



VALLEDOR DE LOZOYA & ARAUJO 




Л7 



11 





Qbiíi tuiuitui .m 



13 




FIGS. 10-13. FIG. 10: Plate 37 of Küster (1848). Top, M. auricularia: FIG. 11: Plate 70 by 
Rossmässler (1835) showing the hinge and a left valve of /W. auricularia. By permission of the 
Österreichische Nationalbibliothek: FIG. 12: Plate 48 of Moquin-Tandon (1855). Fig. 1 (top) is 
M. auricularia. By permission of the Museo Nacional de Ciencias Naturales, Madrid, Spain; FIG. 
13: Figure of M. auricularia in plate 2 by Drouet (1857). By permission of the Natural History 
Museum Picture Library. 



ICONOGRAPHIE OF M. AURICULARIA 



291 




15 






Uoio margará tanopGis, Locab 

Tri« di^pnmi}. réguii^pcnieiu ova- 
birc, aucz allogg^, u» peu diclhc; 
r£fiû>n »Dtfñcure arr. uJie, h pi»i¿- 
rïcare dvux Го» pli'-«1oiguP, â point 
un pra pliiN ¿troitr; ro^lre oblu» el 
тЫпа: borJ f>iip<.^ncur Д kÍii« nr- 
(]u¿; boni inférieur allons^-i oblti- 
!^jwal!>inurus;dt:atMblngi3no. forlc 
laOKtl« courte 1 teil 1>Ш1 sosbrí. — L. &U 

Kar« ; I« 1л\ ï A^uillvn (Lot' et-Gii«aae}. 




II. 32; £- 17 laillifflëtfo. 



С. ~ СгФврс tío rf. >1)«иа'>»*. 
Trírs gfttoftc laillc, subrirniftmiK; mi b^i £|ni^ 

Unió sinUatUS, DC l.ilHtRCK. 




Tiv» grand, íuíjrétiifwme-obloai;, comprimí; bord supérieur très 
>r.juí-, ГшГ&гыл1г tri-* »iouw«; wmmeî iri» anivricnr; rè^n post*- 



18a 




FIGS. 14-18. FIG. 14: Plate 62 by G. В. Sowerby II (1868). Fig. 311 (middle) is M. auhcularia. By 
permission of the British Library; FIG. 15: Page 151 of Locard (1893) showing a juvenile (top) 
and an adult specimen of M. auhcularia (figs. 163 and 164, respectively). By permission of the 
Museo Nacional de Ciencias Naturales, Madrid, Spain; FIG. 16: The juvenile specimen of M. 
auhcularia figured by Haas (1916). By permission of the Museo Nacional de Ciencias Naturales, 
Madrid, Spain; FIG. 1 7: Plate 26 of Germain (1 930). Figs. 609 (top) and 61 5 (bottom right corner) 
depict an adult and a juvenile specimen of M. auhculaha. By permission of the Museo Nacional 
de Ciencias Naturales, Madrid, Spain; FIG. 18a: M. auhculaha in Azpeitia (1933: pi. 12); FIG. 
18b: Adult (middle) and juvenile (bottom) specimens of /W. auhculaha in Azpeitia (1933: pi. 13). 



292 



VALLEDOR DE LOZOYA & ARAUJO 



of the shell and the presence of two siphons 
are characters that are completely absent in 
nnargaritiferids. The image by Pfeiffer (1821) 
is, in fact, Potomida littoralis (Lamarck, 1801 ). 
and it is the same species that Rossmässler 
(1836: pi. 13, fig. 195) drew and labeled Unio 
sinuatus. Rossmässler's (1838: pi. 35, fig. 493) 
figure of Unio gargottae Philippi, 1836, actu- 
ally depicts M. margaritifera, and Ross- 
mässler's (1856: pi. 80, fig. 853) is the same 
M. auhcularia he illustrated in 1855. Lastly, the 
shell illustrated by Hanley (1856) identified as 
Unio crassissimus Hanley, 1 843, another syn- 
onym of M. auricularia, may or may not be M. 
auricularia, as it is one of 60 very small illus- 
trations of freshwater mussels on the same 
plate. It is interesting to note that Unio mar- 
garitanopsis Locara. 1893 (Fig. 15), is really a 
juvenile /W. auricularia. Haas (1913, 1916. 
1929) (Fig. 16). Kennard et al. (1925), Germain 
(1930) (Fig. 17), and Azpeitia (1933) (Fig. 18a, 
b) are the last historical authors to figure the 
species. Curiously, three of these four authors 
illustrated juvenile specimens. Haas (1916) 
and Azpeitia (1933) did so intentionally, but 
Germain assigns this juvenile as the type for 
a different species - Margaritana mar- 
garitanopsis (Locard), from the locality of 
Aiguillon, Lot et Garonne, the same locality of 
Locard's synonymous Unio margaritanopsis. 
Thefirst of the figures by Haas (1913) depicts 
the polished type specimen from the Spengler 
collection, whereas the second (Haas, 1929) 
was reproduced from the figure by Dupuy 
(1851). 

Some fossil valves were figured by 
Huckriede & Berdau (1970), but a new illus- 
tration of Recent M. auricularia did not appear 
until almost 60 years after the image by 
Azpeitia (1933). a color photo in Fechter & 
Falkner's (1990) guide to European land and 
freshwater molluscs. Several years later, 
Falkner (1994) photographed Spengler's type 
specimen of M. auricularia and designated it 
as the lectotype of the species Pseudunio 
auricularius. (Margaritifera auricularia is the 
type species of Pseudunio Haas, 1 91 0, a sub- 
genus sometimes used for it.) Since the re- 
discovery of M. auricularia in Spain, and after 
almost 60 years without records, many new 
illustrations have depicted this endangered 
species in all stages of its development (Araujo 
et al., 2002), illustrations that are very differ- 
ent from the earlier, yet charming lithographies 
and hand-colored engravings. 



ACKNOWLEDGEMENTS 

The authors are indebted to the following 
institutions for the reproductions contained in 
this work: Museo Nacional de Ciencias Natu- 
rales (Madrid, Spain), Biblioteca Nacional 
(Madrid, Spain), Österreichische National- 
bibliothek (Vienna, Austria), The British Library 
(London, U.K.) and The Natural History Mu- 
seum Picture Library (London, U.K.). Special 
thanks to Yves Finet (Muséum d'Histoire 
Naturelle, Genève, Switzerland) for the images 
of the Unio sinuatus syntypes of Lamarck, to 
Tom Schlotte (Zoologisk Museum, Copen- 
hagen, Denmark) for loaning us the lectotype 
of Unio auricularius, to K. Pisvin (Ashmolean 
Museum, Oxford, U.K.), and to Mr. J. B. Davies 
(Zoological Collection of the Oxford Univer- 
sity Museum of Natural History) for informa- 
tion on Lister's specimens. We also thank the 
photograph services of the MNCN, Javier 
Conde de Saro and Eugene Coan for their very 
valuable comments on the manuscript, and 
James Watkins for correcting the English ver- 
sion. 



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Revised ms. accepted 2 June 2004 



MALACOLOGIA, 2006, 48(1-2): 295-298 

A FIELD STUDY OF THE LIFE HISTORY OF THE ENDEMIC HAWAIIAN SNAIL 

SUCCINEA NEWCOMBIANA 

Susan G. Brown, Judy M. Spain & Marci Arizumi 

Social Sciences Division. University of Hawaii at Hilo, 
200 W. Kawlll St., Hilo, Hawaii 96720-4091, U.S.A.; susanb@hawail.edu 



Although the Hawaiian land snail fauna is 
noted worldwide for its diversity and ende- 
mism, little is known about the life histories 
and ecology of most of the endemic Hawaiian 
species. Hadfield et al. (1993) described the 
life histories of a few achatinelline tree snail 
species and, more recently, the life history of 
a succineid, Succlnea thaanumi, has been 
described in laboratory (Rundell & Cowie, 
2003) and field studies (Brown et al., 2003a, 
b). More information on the ecology and life 
histories of land snail radiations would allow 
comparisons with other molluscs, such as the 
freshwater molluscs described by Dillon 
(2000), and would increase our understand- 
ing of allopatric and sympatric speciation 
(Coyne & Orr, 2004). 

In this paper, we report on the life history of 
Succlnea newcombiana. In contrast to S. 
thaanumi, which is found on the eastern side 
of the island of Hawaii from Volcano Village to 
the Hilo Forest Reserve, S. newcombiana is 
found on the northern side of the island in the 
Kohala Forest Reserve. The two habitats dif- 
fer in that S. thaanumi is found in rainforests 
including Puu Makaala, where we previously 
studied S. thaanumi, and which receives a 
median annual rainfall of 4,000 mm (Giam- 
belluca & Sanderson, 1993), whereas S. 
newcombiana is found in a cloud mist envi- 
ronment where the median annual rainfall is 
only 2,000 mm (Giambelluca & Sanderson, 
1993). Although not as abundant or as wide- 
spread as S. thaanumi, S. newcombiana is 
relatively common compared to other Hawai- 
ian land snails. The current study was con- 
ducted in the Kohala Forest Reserve in a 10 
m^ plot at an elevation of 907 m (20°3.339'N, 
1 55°37.51 5'W). The understory consisted pri- 
marily of an alien torch ginger of the family 
Zingiberaceae; hapuu, a Hawaiian tree fern 
{Cibotlum sp.); and ieie {Freyclnetia sp.). The 
overstory consisted primarily of ohia lehua, 
Metrosideros polymorpha, and was less dense 
than the overstory of Puu Makaala. 



Data were collected from 25 January 2003 
to 18 January 2004 for a total of 42 observa- 
tions (1/03 = 1 ; 2/03 = 2; 3/03 = 2; 4/03 = 2; 5/ 
03 = 3; 6/03 = 6; 7/03 = 4; 8/03 = 5; 9/03 = 4; 
10/03 = 4; 11/03 = 5; 12/03 = 3; 1/04 = 1). 
More observations were made during the sum- 
mer months because we were following egg 
masses. All plants, up to 1.8 m high, in the 
study area were examined for the presence 
of snails and egg masses by at least two ob- 
servers during each observation. Maximum 
shell length of all snails was measured with a 
ruler in situ with a minimum amount of con- 
tact. Additionally, we recorded snail activity. In 
the past, we recorded activity based on 
whether a snail was extended out of its shell 
(Brown et al., 2003a). However, because S. 
newcombiana could not completely retract 
their bodies into their shells, we based activ- 
ity on whether or not the snail's eye stalks were 
retracted. We recorded the snail's placement 
on a plant: top or bottom of a leaf, petiole, 
flower or stem. Only a few snails were ob- 
served on the petioles, flowers or stems of the 
plants, so we did not include these data in the 
behavioral analyses. Number of egg masses 
found and the number of embryos in each 
mass were recorded. Temperature and humid- 
ity data were gathered during each observa- 
tion period with a RadioShack temperature/ 
humidity gauge. 

To examine the relationship between behav- 
ior and the microclimate variables across the 
42 observations, we computed simple corre- 
lations between the total number of observed 
snails, the number of snails found on the top 
of a leaf with their eye stalks in, the number of 
snails found on the top of a leaf with their eye 
stalks out, the number of snails found on the 
bottom of a leaf with their eye stalks in, the 
number of snails on the bottom of a leaf with 
their eye stalks out, the total number of snails 
with their eye stalks out regardless of their lo- 
cation on a plant, and the microclimate vari- 
ables of temperature and humidity. 



295 



296 



BROWN ETAL. 






July 2003 30- 




December 2003 




12 3 4 



7 8 9 10 



123456789 10 



123456789 10 



Juiliii» 

1 2 3 4 5 6 7 8 9 10 



\ August 2003 "^- \ January /UU4 

IIil.AJ kililliiL 



123456789 10 



1 2 3 4 5 b 7 8 9 10 



April 2003 30 



[A 




II 



123456789 10 





\ May 2003 
2 



? 8 9 10 




FIG. 1 . Monthly frequency distributions of snail size. The Y-axis is the average number of snails of a 
particular size observed across a month (Number of observations per month: 1/03 = 1 ; 2/03 = 2; 3/03 
= 2; 4/03 = 2; 5/03 = 3; 6/03 = 6; 7/03 = 4; 8/03 = 5; 9/03 = 4; 10/03 = 4; 11/03 = 5; 12/03 = 3; 1/04= 1). 
The X-axis is snail shell size in mm. In August 2003, there were 52 snails 1 mm in size rather than 30. 



LIFE HISTORY OF SUCCINEA NEWCOMBIANA 



297 



15- 



10- 




J FMAMJ J ASONDJ 



FIG. 2. Frequency distribution of the mean num- 
ber of egg masses observed across a month. 
The letters on the X-axis are the first letters of a 
month beginning with January. Bars represent 
the standard errors of the mean. 



The snails and their egg masses were found 
primarily on the alien ginger plants. Snail sizes 
varied across the year and showed two dis- 
tinct lineages (Fig. 1). In January 2003, there 
were three cohorts of snails: cohort 1 con- 
tained adult snails, cohort 2 contained half- 
grown snails, and cohort 3 contained newly 



emerged snails. As the months proceeded, the 
adult snails in cohort 1 disappeared. Snails in 
cohort 2 grew from January through July, laid 
eggs in June and July (Fig. 2) and disappeared 
in August. Snails in cohort 3 emerged from 
their eggs masses from January through 
March, grew from March through August, laid 
eggs in December (Fig. 2), and formed a co- 
hort of adult snails in January 2004 similar to 
cohort 1 observed in January 2003. From July 
through September, snails emerged from egg 
masses laid by cohort 2 and became cohort 

4. Cohort 4 snails grew from August to Janu- 
ary 2004 and formed a cohort of half-grown 
snails similar to cohort 2 observed in January 
2003. Finally, cohort 5 consisted of snails 
emerging from egg masses laid by cohort 3 in 
December and was similar to cohort 3 ob- 
served in January 2003 (Fig. 1). Therefore, 
we observed two lineages: lineage 1 was 
formed from cohorts 1 , 3 and 5; lineage 2 from 
cohorts 2 and 4. 

Egg masses were translucent like those of 

5. thaanumi (Brown et al., 2003b). The num- 
ber of new egg masses declined from August 
through November but increased dramatically 
in December (Fig. 2), followed by a second 
decrease in January 2004. Although many 
fewer egg masses were laid in December (n = 
28) than from May to August (n = 185), the 
December egg masses contained significantly 
more embryos (Fig. 3) than the May to August 
masses (Хг^, = 22.16; p < 0.0001). 



t -2 




May-August 
December 



1-3 4-6 7-9 >10 

Number of Embryos per Egg Mass 



FIG. 3. Bar graph of the number of embryos in a clutch of eggs during 
the two major laying seasons. 



298 



BROWN ETAL. 



We interpreted the above growth patterns for 
S. newcombiana as indicating an annual, 
semelparous life cycle for two snail lineages. 
Most snails probably lived about 12 months. 
These life cycles were similar to the life cycle 
of S. thaanumi (Brown et al., 2003a), but the 
data on S. thaanumi reflected a single snail 
lineage. The egg masses of the two lineages 
of snails also differed. Snails in cohort 2 laid 
more egg masses with fewer embryos, 
whereas snails in cohort 3 laid fewer eggs 
masses with more embryos per mass. At 
present, we do not know if the two lineages 
have different haplotypes. 

Mating was observed five times during the 
study: 2/1/03, 5/22/03, 6/16/03, 6/30/03, and 
7/7/03. As we found with S. thaanumi (Brown 
et al., 2003a), the smaller snail acted as the 
male (succineids are hermaphrodites), but, 
unlike S. thaanumi. we observed mating only 
between dyads (no triads as we previously 
observed with S. thaanumi). 

Snail behavior was related to the microcli- 
mates of the study area. Snails were more likely 
to be found on the bottom of a leaf with their 
eyes stalks in when temperature was higher 
(r = 0.66; p < 0.01 ; N = 42 for all correlations) 
and humidity was lower (r = -0.72; p < 0.01). 
Snails found on the tops of leaves were also 
more likely to have their eye stalks in when 
temperature was higher (r = 0.49; p < 0.01) 
and humidity was lower (r = -0.50; p < 0.01). 
Snails with their eye stalks out that were ac- 
tive on the plants, however, were found at all 
temperatures (r - -0.03) and humidities (r = 
0.07). This differed noticeably from our obser- 
vations of S. thaanumi. In high temperature and 
low humidity conditions, we seldom observed 
active S. thaanumi. but we often observed ac- 
tive S. newcombiana in direct sunlight and low 
humidity conditions. The total number of snails 
observed was also not related to temperature 
(r = 0.18) or humidity (r = -0.16). Again, this 
differed from our previous observations of S. 
thaanumi. for which we observed fewer snails 
as the temperature increased, suggesting that 
the snails moved to a different part of their 
habitat. Succinea newcombiana cannot retract 
into its shell, whereas S. thaanumi can do so. 
Because of its inability to retract into its shell, 
one might conclude that S. newcombiana is 
more susceptible to high temperature and low 



humidity than S. thaanumi, but this was not 
the case. These behavioral differences to high 
temperature and low humidity might be related 
to the different ecotypes the two species oc- 
cupy. The population S. newcombiana is found 
in a cloud mist forest with relatively less rain- 
fall but more mist, whereas populations of S. 
thaanumi are found in rainforest habitat with 
relatively more rainfall and less mist. Succinea 
newcombiana might have lost the ability to re- 
tract into its shell because of the presence of 
abundant moisture in the air. 



ACKNOWLEDGMENTS 

We thank Naio Vivas and Kensley Raigeluw 
for help in initially finding the snails and B. 
Kalani Spain for help in data collection. Addi- 
tional thanks go to Robert Cowie for identify- 
ing S. newcombiana for us. This research was 
funded, in part, by NSF grant 0223040. 



LITERATURE CITED 

BROWN, 8. G., B. K. SPAIN & K. CROWELL, 
2003a, A field study of the life history of an 
endemic Hawaiian succineid land snail. 
Malacologia. 45: 175-178. 

BROWN, 8. G, K. CROWELL & P. KEENE, 
2003b, Oviposition behavior and offspring 
emergence patterns in Succinea thaanumi, an 
endemic Hawaiian land snail. Ethology. 109: 
905-910. 

COYNE, J. A. & H. A. ORR, 2004, Speciation. 
Sinauer Associates, Sunderland, Massachu- 
setts, xiii + 545 pp. 

DILLON, R. T, 2000, The ecology of freshwater 
molluscs. Cambridge University Press, Cam- 
bridge, U.K. xii + 509 pp. 

GIAMBELLUCA, T & M. SANDERSON, 1993, 
The water balance and climatic classification. 
Pp. 56-72, in: M SANDERSON, ed.. Prevailing 
trade winds: climate and weather in Hawaii. 
University of Hawaii Press, Honolulu, Hawaii, 
ix + 126 pp. 

HADFIELD, M. G., S. E. MILLER & A. H. 
CARWILE, 1993, The decimation of endemic 
Hawaiian tree snails by alien predators. Ameri- 
can Zoologist, 33: 610-622. 

RUNDELL, R. J. & R. COWIE, 2003, Growth and 
reproduction in Hawaiian succineid land snails. 
Journal of Molluscan Studies, 69: 288-289. 



Revised ms. accepted 7 May 2005 



MALACOLOGIA, 2006, 48(1-2): 299-304 

NATICID BOREHOLES ON A TERTIARY CYLICHNID GASTROPOD 
FROM SOUTHERN PATAGONIA 

Javier H. Signorelli\ Guido Pastorino^ & Miguel Griffin^ 



INTRODUCTION 

The fossil communities are in clear disadvan- 
tage when it comes to study the interactions 
among the species included in them. However, 
prédation on shells gives us the possibility to 
examine at least some of these interactions. 
The diameter of the borehole, its placement, 
and the volume of the prey are parameters that 
can be easily recorded. Diverse combinations 
of these may allow inference about the size of 
the predator and the time spent on the perfo- 
ration (Kitchen etal., 1981). 

The morphology of two types of boreholes 
was described by Carriker& Yochelson (1968) 
that later Bromley (1981) described as 
ichnofossils. Taylor (1980) and later Bromley 
(1981) pointed out that externally wide and 
internally narrow prédation marks with parabo- 
loid walls are produced by naticid gastropods 
{Olchnus paraboloides), whereas those cylin- 
drical with non-beveled edges are assigned 
to predators belonging to the Muricidae 
(Carriker, 1981). 

Kelley (1988) developed a model in which 
prédation of the Miocene naticids is stereo- 
typed and predictable. The observed pattern 
of prédation was revealed when analyzing the 
selection of perforation location and the size 
of the predators by means of the perforation 
diameter. Kitchell et al. (1 981 ) showed that the 
main purpose of such patterns is an adaptive 
behavior to maximize energy efficiency and 
to select by prey size. 



GEOLOGICAL SETTING 

The material studied was collected in Neo- 
gene rocks exposed along the Atlantic coast 
of southern Patagonia. This is the first record 
of drilled gastropod shells from the Monte León 
Formation. Along the coast there are spectacu- 
lar almost continuous outcrops of Tertiary rocks 
from the mouth of the Rio Negro in northern 
Patagonia to the Straits of Magellan, and many 



authors have visited this area as they contain 
a very rich fauna of continental mammals and 
marine invertebrates. These beds have been 
subdivided based on their fossil content. These 
subdivisions - as well as the ages proposed 
for these rocks - have been a matter of great 
controversy, which has not yet been com- 
pletely resolved. 

The samples containing the studied mate- 
rial come from a locality along the southern 
margin of the Santa Cruz River first visited by 
Charles Darwin and by him called Mount En- 
trance (Fig. 1). They were collected in a very 
thin shelly bed of loose sediment lying within 
the Monte Entrada Member of the Monte León 
Formation (Fig. 2). The unit is richly fossilifer- 
ous, but the smaller specimens have gener- 
ally escaped attention. The extraordinary 
abundance of Kaltoa in this particular bed has 
been overlooked, as most existing collections 
have only a few specimens. The Monte León 
Formation was formally introduced by Bertels 
(1970, 1978), and she subdivided it into a lower 
member (Monte Entrada) and an upper one 
(Monte Observación). The age of these rocks 
has been amply discussed and is presently 
believed to be late Oligocène to more prob- 
ably earliest Miocene (Barreda & Palamar- 
czuk, 2000). 

Kaitoa patagónica (Ihering, 1897) (Fig. 3) is 
a small cylichnid first described from the 
Superpatagonian beds of Yegua Quemada, in 
the province of Santa Cruz and included in 
Bulla. However, the shell shape, ornamenta- 
tion and columellar features suggest its affini- 
ties lie with Kaitoa Marwick, 1 931 (type species 
Kaitoa haroldi Мапл /ick, 1931), a genus de- 
scribed originally from Altonian (late early Mi- 
ocene) rocks in New Zealand and which 
according to Beu & Maxwell (1990) occurs 
there from the Otaian (mid-early Miocene) to 
the Waipipian (early-late Pliocene). The oc- 
currence of taxa peculiar to Australasia or 
Antarctica in South America has been variously 
recorded and this is just another example of 
such a connection (Beu et al., 1997). 



'Museo Argentino de Ciencias Naturales, Av. Angel Gallardo 470, 3° piso lab 57, C1405DJR Buenos Aires, Argentina; 

jsignorelli@macn govar 
^Facultad de Ciencias Exactas y Naturales, Universidad Nacional de La Pampa, Av. Uruguay 151, L6300CLB Santa 

Rosa, La Pampa, Argentina 

299 



300 



SIGNORELLI ETAL. 




•^.-Vii 



69 W <=э^«'. ■ 66 W 



С 

g 
£ 



c 
о 
ф 

_| 

CD 

-4— ' 

С 

о 




20 т 



От 



FIG. 1 . Location map of the sampling area. 



FIG. 2. Schematic Section of 
Monte León Formation. 



MATERIALS AND METHODS 

A total of 873 specimens of Kaitoa pata- 
gónica were considered in this study (Figs. 3- 
9). Internal and external diameters of 242 bore- 
holes were measured using a stereoscopic 
microscope. The total length was measured 
in all 873 specimens, including those that were 
perforated. These three parameters were used 
to build a frequency table. SEMs pictures were 
done at MACN with a Philips XL30. All pic- 
tures were digitally processed. 

The studied material is housed in the Depar- 
tamento de Ciencas Naturales, Facultad de 
Ciencias Exactas y Naturales, Universidad 
Nacional de La Pampa, under the numbers 
GHUNLPam26500, 26501 , 26502 and 26503, 
for the illustrated specimens and GHUNL 
Pam26504 for the others. 



RESULTS 

All boreholes showed the same morphology. 
The perforations are conical, with the larger 
diameter on the external surface of the shell 
and the smaller diameter on the internal sur- 
face (Fig. 4). 

Of all the boreholes measured in Kaitoa 
patagónica, 90% are placed on an area of the 
last whorl near the inner lip, that is, on the 
central part of the apertural side of the shell. 
The rest were found on the dorsal side of the 
shell (Fig. 5). 

A very low percentage of incomplete bore- 
holes were observed in the population. This 
does not allow us to draw any conclusion about 
predator behavior. However, the drilling mecha- 
nism was recognized due to the presence of a 
slightly prominent central boss (Fig. 6). 



BOREHOLES ON PATAGONIAN TERTIARY GASTROPODS 



301 




FIGS. 3-9. Kaitoa patagónica (Ihering, 1897). FIG. 3: UNLPam 26500; FIGS. 4-5: Two drilled specimens, 
UNLPam 26501, 26502. Scale bar = 2 mm (FIGS. 3-5); FIG. 6: Detail of an incomplete borehole, 
UNLPam 26503. Scale bar = 200 pm; FIGS. 7-8; Two complete boreholes. Scale bar = 200 pm; FIG. 9: 
Detail of the square from Fig. 8. Scale bar - 50 pm. 



302 



SIGNORELLI ETAL. 



90 

80 

70 

О 60 
z 
ш 50 

ш 40 
а: 
"- 30 

20 

10 

О 





















А 






1 1 


, , , ,1, .,1,1,1,- ,.,.,. , ,. ,. 



поп predated 
predated 



2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.5 12.5 13.5 

SIZE INTERVAL 

FIG. 10. Size intervals vs. number of specimens (predated and non-predated) of Kaitoa patagónica. 



The size distribution curve of the population 
is normal in both predated and non-predated 
individuals. The most frequent size in the 
population is 6-6.5 mm of total length, whereas 
the most predated size is 5-5.5 mm of total 
length (Fig. 10). The distribution of borehole 
sizes is normal. The most frequent borehole 
size is 0.6-0.8 mm considering its internal di- 
ameter (Fig. 11). This borehole size curve is 
displaced to the left. 

Correlation between internal diameter and 
the size-range of the population was analyzed 



with the software Statistica v. 4.0. The result 
was not significant (R^ = 0.1257; p < 0.000), 
but there is a trend suggesting that the larger 
predators produced holes with a larger diam- 
eter (Fig. 12). 



DISCUSSION 

The conical shape of the perforations agrees 
with the morphology of boreholes referred to 
gastropods belonging to the Naticidae 



90 
80 
70 

>60 
О 
g 50 

§40 
DU 
^^ 30 

20 

10 

О 













тг1 




-| 


1 


ч.. 



I internal diameter 
external diameter 



0.4-0.6 0.6-0.8 0.8-1.0 1.0 1.2 1.2-1.4 1.4-1.6 1.6-1.8 
BOREHOLE DIAMETER 



FIG. 11 . Distribution of diameters of boreholes present in Kaitoa patagónica. 



BOREHOLES ON PATAGONIAN TERTIARY GASTROPODS 



303 



2.0 



0.5 



0.0 



y = 0.0757x + 0.4775 




♦ 5»' ♦ ♦ ♦ ♦ ♦ ♦ 

»♦ * * *- fr» 



5 6 7 

LENGTH OF PREY (mm) 



10 



FIG. 12. Total shell length of prey vs. internal diameter of the borehole. 



(Carriker & Yochelson, 1968; Taylor et al., 
1980, among others). Among the species of 
this family described from coeval rocks in the 
same area are Polinices santacruzensis 
Ihering, 1897, and Natica subtenuis Ihering, 
1897. These species were based on gener- 
ally poorly preserved large adult shells, which 
seem unlikely to have been responsible for the 
borings on Kaitoa. There are numerous small 
juvenile naticid shells in this unit, but until fur- 
ther data become available on the different 
stages of the species described on the basis 
of large specimens, we cannot ascertain to 
which of them they may belong. Therefore, the 
identity of the Ka/Yoa-borer must remain as yet 
uncertain. 

Borehole diameter provided an excellent tool 
to estimate the size of the predator. Such a 
size selection is a common behavior in naticids 
(Calvet i Cata, 1989). As reported here, the 
most abundant size in the population is not 
the most intensely attacked by the predator 
(Fig. 1 0). The reason for this discrepancy may 
lie in the fact that the predator could have been 
the very small, equally abundant naticid juve- 
nile that appears in the same beds as Kaitoa 
patagónica. These presumably could prey on 
Kaitoa patagónica up to a certain size, but 
were somehow prevented of attacking the 
larger specimens, whether because of mor- 
phological constraints or because of a faster 
growth of the opisthobranch compared to 
naticids. 

The non-predated population may constitute 
a size-refuge, such as those described for 



other groups of mollusks commonly attacked 
by naticids (Kabat, 1990; Pastorino & Ivanov, 
1996). 

A location selection for perforations 
(Hofmann & Martinell, 1986) is very well de- 
fined in most drilled shells of the Patagonian 
species. The area adjacent to the parietal cal- 
lus carries the largest percentage of the per- 
forations. This ellicits the question as to why it 
is so if the normal way of living is with the 
apertural side down. The possible answer to 
this may rest in the way in which the predator 
manipulated the prey. Additionally, this place 
is the easiest way to kill the prey because be- 
neath the adapertural part of the shell, the most 
exposed, is where the foot is retracted, 
whereas a perforation on the ventral side as- 
sures the predator better chances of reaching 
vital organs and therefore enhancing its pos- 
sibilities of killing the prey with minimum ef- 
fort. 



ACKNOWLEDGMENTS 

We are grateful to Paula Mikkelsen (AMNH) 
for sharing information about living cylichnids. 
This work was supported in part by Project PICT 
No. 02-01-10975 from the National Agency for 
Scientific and Technological Promotion, Argen- 
tina. We acknowledge funding by the Consejo 
Nacional de Investigaciones Científicas y 
Técnicas (CONICET) of Argentina, to which M. 
G. and G. P. belong as members of the Carrera 
del Investigador Científico y Tecnológico. 



304 



SIGNORELLI ETAL. 



LITERATURE CITED 

BARREDA, V. & S. PALAMARCZUK, 2000, 
Palinomorfos continentales y marinos de la 
Formación Monte León en su área tipo, provin- 
cia de Santa Cruz, Argentina. Ameghiniana, 37: 
3-12. 

BERTELS, A., 1 970. Sobre el "Piso Patagoniano" 
y la representación de la época del Oligocène 
en Patagonia austral. República Argentina. 
Revista de la Asociación Geológica Argentina, 
25: 495-450. 

BERTELS, A., 1978. Estratigrafía y foraminiferos 
(Protozoa) bentónicos de la Formación Monte 
León (Oligocène) en su área tipo, provincia de 
Santa Cruz, República Argentina. /Acias. 2° 
Congreso Argentino de Paleontología y Bio- 
etratigrafía y 1""' Congreso Latinoamericano de 
Paleontología (Buenos Aires. 1978). 2: 213-273. 

BEU, A. G.. M. GRIFFIN & P A. MAXWELL, 1 997, 
Opening of Drake Passage gateway and Late 
Miocene to Pleistocene cooling reflected in 
Southern Ocean molluscan dispersal: evidence 
from New Zealand and Argentina. Tectono- 
ptiysics. 281: 83-97. 

BEU, A. G & P A. MAXWELL. 1990, Cenozoic 
molluscs from New Zealand, New Zealand 
Geological Survey Palaeontological Bulletin, 
58: 1-432 

BROMLEY, R. G., 1981, Concepts in ichno- 
taxonomy illustrated by small round holes in 
shells. Acta Geológica Hispánica. 16: 55-64. 

CALVET I CATA. C. 1989, Posiciones preferidas 
en las perforaciones de Naticarius hebraeus 
(Martyn, 1769) (Naticidae: Gastropoda) realiza- 
das en bivalvos de el Maresme (Barcelona). 



Revista de Biología de la Universidad de 
Oviedo. 7: 91-97. 

CARRIKER, M. R., 1981, Shell penetration and 
feeding by naticacean and muricacean preda- 
tory gastropods: a synthesis, Malacologia. 20: 
403-422. 

CARRIKER, M. R. & E. L. YOCHELSON, 1968, 
Recent gastropod boreholes and Ordovician 
cylindrical borings. Professional Papers of the 
United States Geological Sutvey. 593-B: 23. 

HOFFMAN, A. & J. MARTINELL, 1984, Prey 
selection by naticid gastropods in the Pliocene 
of Emporda (Northeast Spain). Neues Jahrbucti 
für Geologie und Paläontologie, 1984: 393- 
399. 

KABAT, A. R., 1990, Predatory ecology of naticid 
gastropods with a review of shell boring pré- 
dation. Malacologia. 32: 155-193. 

KELLEY, P. H., 1988, Prédation by Miocene gas- 
tropods of the Chesapeake Group: stereotyped 
and predictable. Palaios, 3: 436-448. 

KITCHELL, JA., С. H. BOGGS, J. F. KITCHELL 
& J. A. RICE, 1981, Prey selection by naticid 
gastropods: experimental tests and application 
to the fossil record. Paleobiology, 7: 532-552. 

MARWICK, J., 1931 , The Tertiary Mollusca of the 
Gisborne District. Palaeontological Bulletin 
(New Zealand), 13: 1-177. 

PASTORINCG &V. IVANOV, 1996, Marcas de 
predación en bivalvos del Cuaternario marino 
de la costa de provincia de Buenos Aires, Ar- 
gentina. Iberus, 14: 93-101. 

TAYLOR, J. D., N. J. MORRIS & С N. TAYLOR, 
1980, Food specialization and the evolution of 
predatory prosobranch gastropods. Paleontol- 
ogy. 23: 375-409. 



Revised ms. accepted 23 May 2005 



MALACOLOGIA, 2006, 48(1-2): 305-308 

GEOTACTIC BEHAVIOUR OF DREISSENA POLYMORPHA (BIVALVIA) 

Jarostaw Kobak 



Nicolaus Copernicus University, Institute of General and Molecular Biology 

Department of Invertebrate Zoology 87 100 Torun, Gagarina 9, Poland; 

jkob@biol. uni. torun.pl 



ABSTRACT 

Zebra mussel movement was studied in the laboratory, on a glass slope inclined at 2, 3, 
4 or 8° to the bottom, in darkness and in the light (the latter on the steepest slope only). 
Small mussels (< 10 mm) climbed upward on the 4 or 8° slopes in darkness (negative 
geotaxis) but showed no preferences on the other slopes and in the light. Large mussels 
(> 12 mm) moved downwards on the 4 and 8° slopes (also in the light) and showed no 
preferences in the other treatments. 



INTRODUCTION 

Dreissena polymorpha (Pallas, 1771), the 
zebra mussel, is a gregarious bivalve that 
strongly influences freshwater ecosystems 
and hydrotechnical devices (Lewandowski, 
2001; O'Neill, 1997). Its distribution is mainly 
determined by dispersal and settlement of 
planktonic larvae (Lewandowski, 2001 ; Kobak, 
2004), but may also be affected by post-settle- 
ment movement of mussels: they move up- 
wards to avoid poor chemical conditions at the 
base of a colony (Burks et al., 2002) and pre- 
fer shaded or dark substrata (Kobak, 2001; 
Toomey et al., 2002). 

A cue that could be useful for a moving mus- 
sel is gravity. It provides information to an ani- 
mal about its orientation in space, indepen- 
dent of photoperiod and geographic location. 
In the field, zebra mussels may prefer either 
the lower (Walz, 1973; Lewandowski, 2001) 
or upper (Marsden & Lansky, 2000) substrate 
side. Such distribution could result from both 
geo- and phototaxis, as well as the effects of 
water flow or prédation. To test the influence 
of gravity upon mussels, I studied their move- 
ment on a series of slopes in the laboratory. 
In the light of my field research (Kobak, 2004), 
I hypothesized that small mussels would move 
upwards and illumination would reverse this 
behaviour because of negative phototaxis. I 
also expected that large mussels, less mobile 
than small ones, would prefer the easier, 
downward direction. 



MATERIALSAND METHODS 

Mussels were collected by a diver from a dam 
wall of the Wtodawek Dam Reservoir (the Vistu- 
la River, central Poland) and kept in a 500 I 
aquarium filled with aerated, settled tap wa- 
ter, at ca. 20°C. Only individuals that reat- 
tached themselves in this aquarium were 
tested. They were used only once, not sooner 
than two weeks and not later than three 
months after collecting. The tested individu- 
als were divided into small mussels (mean 
shell length ± SD: 7.3 ± 1 .29 mm, range: 3.3- 
9.9 mm) and large ones (15.3 ± 1.55 mm, 
range: 12.2-22.6 mm). 

The experiment was run in a glass tank (480 
X 230 mm, water level: 240 mm) with settled 
(24 h) tap water (19-22. 5°C). A400 x 230 mm 
glass plate was put into the tank, with one of 
its longer edges resting on the bottom and the 
other leaning against the wall (Fig. 1). The 
aerator was placed below the plate level to 
avoid mussel disturbance by air bubbles. The 
mussels (13 individuals per tank in a single 
trial) were put onto the central long axis of the 
plate, with their long axes parallel to the tank's 
longer edge. Each mussel was covered with 
a glass tunnel (width and height: 25 mm, 
length: 220 mm, outlets closed with 1 mm ny- 
lon mesh) to avoid the impact of conspecifics 
on their behaviour (Mort! & Rothhaupt, 2003). 

To study geotaxis in the dark, I tested mus- 
sels on slopes inclined at 2, 3, 4 or 8° to the 
bottom, in a tank covered with a cardboard 



305 



306 



KOBAK 



Glass tunnel separating 
mussels from one another 




Glass slope 



FIG. 1 . Experimental tank, a in various treatments 
is 2, 3, 4 or 8°. 



box. Illumination under this box was below the 
detection limits of the luxometer (Sonopan L- 
20A) (i.e., < 0.1 Ix). To examine the impact of 



light on geotaxis, I tested mussels on a con- 
stantly illuminated slope (16 W bulb 0.5 m 
above the surface, incident illuminance at the 
surface ca. 700 Ix). Light was used only in 
experiments involving the steepest slope (8°), 
the most likely to evoke geotaxis. 

To check whether mussels could passively 
slide down the slope, 20 empty shells of each 
size group, filled with aquarium silicon glue to 
imitate a live mussel's shape and weight, were 
put on the steepest slope (8°) in various posi- 
tions (lying on the ventral or side shell sur- 
face, with the front, back, or side pointing 
down). I observed no passive relocations of 
these shells. 

I carried out ten 48-hour trials (13 mussels in 
each) for each treatment and size group. They 
were run consecutively, in a random sequence. 
The slope direction relative to the laboratory 
room was changed in the successive trials. At 
the end of each trial, distances moved by the 
mussels (measured to their anterior ends) were 
determined to the nearest 1 cm (the scores 
were from -11 at the bottom to +11 at the top). 

Numbers of mussels moving in opposite di- 
rections were compared using t-tests for paired 
data with the sequential Bonferroni correction. 



Treatment 

Angle Light 






















t-test results 


Small mussels (< 10 mm) 


t 

(df = 9) 

028 

11 
341 
551 
1.38 

0.72 
2.28 
388 
4 10 
4 05 


P 


2° Dark 




1- 






H 




0.7825 


3° Dark 




1- 




1 ' 


i 




09162 


4° Dark 






^^^^^^^^^B"* 


0078* 


8° Dark 






H^^I^HH" 




00004** 


8° Light 




1- 






H 






0.2015 




Large mussels (> 12 mm) 




2° Dark 






h 




-• 




04923 


3° Dark 




H 




-^ 


ч 






00487 


4° Dark 






H 






00037* 


8° Dark 




H 






00027* 


8° Light 




H 








00029* 


N = 10 [ 


8 6 A 
Downward mc 


2 ( 
)vement 


) 


и 


4 6 i 
pward movement 


i 





Average number of mussels per trial 



FIG. 2. Direction of zebra mussel movement on different slopes. Error 
bars are standard errors of mean. Black bars indicate a significant pref- 
erence for one direction in a given treatment. The asterisks show statis- 
tical significance of the t-tests for paired data after applying the sequen- 
tial Bonferroni correction; *p < 0.05, **p < 0.01. 



GEOTAXIS OF DREISSENA POLYMORPHA 



307 



Differences among the treatments were tested 
using the two-way ANOVA (factors: mussel size 
and slope type) of individual distances moved 
by the mussels (with downward distances 
coded as negative values). The Bonferroni- 
adjusted pairwise t-tests were used as post- 
hoc comparisons. Mussels that neither moved 
nor attached themselves to the plate were re- 
garded as being in a poor physical condition 
and not analysed. 



RESULTS 

On the darkened 4 and 8° slopes, the small 
mussels tended to move upwards, while the 
large individuals preferred the downward direc- 
tion. In the light preferences of the small mus- 
sels disappeared, while the large ones retained 
their downward preference. I observed no di- 
rectional reactions on the other slopes (Fig. 2). 

The interaction between mussel size and 
slope was significant in the ANOVA of the dis- 
tances (F4 1014 = ^-97, p < 0.001). The dis- 
tances moved by the small mussels on the 
darkened 4 and 8° slopes differed significantly 
from those measured in the other treatments, 
as well as from the distances moved by the 
large individuals in the same conditions. In the 
case of the large mussels, only the distances 
moved on the 2 and 8° slopes differed signifi- 
cantly from each other (Fig. 3). 



DISCUSSION 

To my knowledge, negative geotaxis of meta- 
morphosed bivalves has not been reported so 
far. Uryu et al. (1996) observed positive geo- 
taxis of a mussel Limnoperna fortunei. Nega- 
tive geotaxis of small zebra mussels, found 
here, could account for the aggregations of 
recruits along the upper edge of vertical settle- 
ment plates deployed in the field (Kobak, 
2004). 

Negative geotaxis could be beneficial in a 
dense colony, where water quality is poor. 
Burks et al. (2002) found an upward move- 
ment of mussels apparently stimulated by 
chemical gradients within a colony (e.g., oxy- 
gen and nitrate). In the present study, mus- 
sels moved upwards, although they were kept 
at low density and separated from one another, 
so such a gradient did not appear. Thus, in 
certain conditions, upward movement may 
occur without any chemical stimuli. In the field, 
negative geotaxis could help mussels to find 
a suitable site at the top of a colony. However, 
zebra mussels are Photophobie (Kobak, 2001 ; 
Toomey et al., 2002), which is contradictory to 
negative geotaxis: climbing up means ap- 
proaching the light source. Photophobie 
behaviour may explain why the upward move- 
ment of small mussels disappeared under il- 
lumination in the present study. Uryu et al. 
(1996) observed a light-induced change in 



N= 114 100 110 89 90 



3 -2 

TD 

03 ~0 



103 119 99 112 88 



ÇT 



Slope 2 3 4' 



Dark 



I I Small mussels (< 10 mr 



Large mussels (> 12 mm) 



a ac ac с с 



Т 



I I I 
J I 



Light 



Dark 



Light 



FIG. 3. Average net distances moved by zebra mussels on different 
slopes. Downward distances were counted as negative values. Treat- 
ments labelled with the same letter did not differ significantly from one 
another (Bonferroni-adjusted t-tests). The values above the chart are 
the numbers of mussels analysed in each treatment. 



308 



KOBAK 



behaviour of L. fortunei, which chose no di- 
rection in darkness and moved downwards in 
light. For both species, illumination reduced 
upward movement. Previously, I have shown 
that light (ca. 700 Ix) was a stronger cue than 
gravity: small mussels avoided the upper, illu- 
minated part of the slope when only its lower 
half was darkened (Kobak, 2002). 

Large mussels move less frequently and 
over shorter distances than smaller mussels, 
probably due to their heavier bodies (Toomey 
et al., 2002). Thus, larger individuals may pre- 
fer the downward direction because it de- 
mands less effort. Older mussels often bear 
other individuals attached to their shells, which 
further limits their locomotion and makes it less 
likely to be crucial to their survival. 

Anumberofstudies(e.g., Kobak, 2001,2002; 
Burks et al., 2002: Toomey et al., 2002), in- 
cluding the present one, show that small ze- 
bra mussels can use multiple environmental 
cues to select an attachment site by crawling 
over substratum. Thus, active movement of 
settled individuals may be an important factor 
affecting mussel distribution in the field. 

ACKNOWLEDGEMENTS 

I am grateful to Mr Andrzej Denis, Szymon 
Denis and Józef Liczkowski for collecting the 
mussels for the experiments. I also thank the 
two anonymous reviewers for their valuable 
comments that helped improve this text. 



LITERATURE CITED 

BURKS, R. L., N. С TUCHMAN, С A. CALL & 
J. E. MARSDEN, 2002, Colonial aggregates: 
Effects of spatial position on zebra mussel re- 
sponses to vertical gradients in interstitial wa- 



ter quality. Journal of the North American 
Benthological Society. 21: 64-75. 

KOBAK. J., 2001, Light, gravity and conspecif- 
ics as cues to site selection and attachment 
behaviour of juvenile and adult Dreissena 
polymorpha Pallas, 1771. Journal of Mollus- 
can Studies. 67: 183-189. 

KOBAK, J., 2002, Impact of light conditions on 
geotaxis behaviour of juvenile Dreissena 
polymorpha. Folia Malacologica. 10: 77-82. 

KOBAK, J., 2004, Recruitment and small-scale 
distribution of Dreissena polymorpha (Bivalvia) 
on artificial materials. Archiv für Hydrobiologie, 
160: 25-44. 

LEWANDOWSKI, К., 2001, Development of 
populations oWreissena polymorpha (Pall.) in 
lakes. Folia Malacologica. 9: 171-213. 

MARSDEN, J. E. & D. M. LANSKY, 2000, Sub- 
strate selection by settling zebra mussels, 
Dreissena polymorpha. relative to material, 
texture, orientation, and sunlight. Canadian 
Journal of Zoology. 78: 787-793. 

MORTL, M. & K. O. ROTHHAUPT, 2003, In- 
traspecific and interspecific effects of adult 
Dreissena polymorpha on settling juveniles and 
associated macroinvertebrates. International 
Review of Hydrobiology, 88: 561-569. 

O'NEILL, С R., 1997, Economic impact of ze- 
bra mussels - results of the 1995 National 
Zebra Mussel Information Clearinghouse study. 
Great Lakes Research Review. 3: 35-42. 

TOOMEY, M. В., D. MCCABE & J. E. MARSDEN, 
2002, Factors affecting the movement of adult 
zebra mussels {Dreissena polymorpha). Jour- 
nal of the North American Benthological Soci- 
ety. 2^■. 468-475. 

URYU, Y., K. IWASAKI & M. HINOUE, 1996, 
Laboratory experiments on behaviour and 
movement of a freshwater mussel, Limnoperna 
fortunei (Dunker). Journal of Molluscan Stud- 
ies. 62: 327-341. 

WALZ, N., 1973, Studies on the biology of 
Dreissena polymorpha in Lake Constance. 
Archiv für Hydrobiologie, Supplement, 42: 452- 
482. 



Revised ms. accepted 14 July 2005 



LETTERS TO THE EDITOR 



MALACOLOGIA, 2006, 48(1-2): 311-319 

VALID UNTIL SYNONYMIZED, OR INVALID UNTIL PROVEN VALID? 
A RESPONSE TO DAVIS (2004) ON SPECIES CHECK-LISTS 

Philippe Bouchet 

Muséum National d'Histoire Naturelle, 55 rue Buffon, 75005 Paris, France; 

pbouchet@mntin.fr 



The recent publication of taxonomic author- 
ity lists for the non-marine mollusks of France 
(Falkner et al., 2002) and of north and north- 
west Europe (Falkner et al., 2001) has elic- 
ited critical comments from Davis (2004). I 
have been involved in both works, for the 
French list as instigator and editor, for the 
European list as a provider of supraspecific 
nomenclature (Bank et al. 2001 ). The purpose 
of the present response is to place both lists 
in perspective, to justify the editorial decisions 
taken, and to defend the rationale behind the 
scientific decisions made. 



On Checklists and Taxonomic Authority Lists 

Taxonomic authority lists or checklists are 
as old as the science of systematics. However, 
unlike many other groups of animals, large or 
small, mollusks do not have a comprehensive 
catalogue of species, or even of names. The 
last academic attempt to list the Recent mol- 
lusks of the world was Tryon and Pilsbry's 
Manual of Conchology, now on average 100 
years old. With an accumulated load of per- 
haps 500,000 names and a synonymy ratio 
that is matched probably only in butterflies, the 
compilation of a global mollusk checklist is not 
a small task. The result is that we do not even 
know whether the number of valid named 
Recent species of mollusks is on the order of 
50,000 or 100,000, an uncertainty that is per- 
sistent throughout Recent and fossil biota but 
is seen as "particularly problematic" for mol- 
lusks (Hammond, 1995). 

Information technology has suddenly made 
it much easier to compile and update species 
catalogues that reflect changes in knowledge 
and thus taxonomic instability, whereas, simul- 
taneously, a growing corpus of legal texts and 
other documents, such as Red Lists, demand 
authoritative lists of names that will change 
little over time. Recently published regional 
checklists emphasize one or the other of these 
two approaches. For instance, Turgeon et al. 's 



(1 998) list was compiled to provide an authori- 
tative reference for U.S. federal and state con- 
servation texts, and it emphasizes stability and 
established knowledge over scientific inquisi- 
tiveness and controversial opinions. 

In Europe, there has been a long tradition of 
national checklists of non-marine mollusks 
(see, e.g., Bruyne et al., 1994; Manganelli et 
al., 1995;Kerney, 1999), but no continent-wide 
list has been published since Westerlund's 
catalogues of the 1870-1 880s. In 1998, pro- 
posals were made to issue a taxonomic au- 
thority list of the land and freshwater 
metazoans of geographical Europe. With fund- 
ing from the European Commission, Fauna 
Europaea was formally initiated in 2000 for a 
period of four years, and Ruud Bank was cho- 
sen to be the "Group Coordinator" (in Fauna 
Europaea parlance) for the gastropods. At the 
onset, it was estimated that there would be on 
the order of 3,000 valid molluscan terminal 
taxa (species and subspecies; see below), and 
it was also recognized that, because of the 
chaos caused by the Nouvelle Ecole, the 
French fauna was the major stumbling block 
in compiling a list of valid taxa. I then decided 
to contract Gerhard Falkner and Theo Ripken, 
both with an extensive knowledge of the 
French fauna, to produce a taxonomic author- 
ity list for France. The result is the Falkner et 
al. publication appeared in March 2002, but 
the species list had already been made avail- 
able for the CLECOM [Check List of the Euro- 
pean co ntinental Molluscs] catalogue 
(covering the countries of northern and north- 
western Europe), involving Falkner and Ripken 
as co-authors and published the year before 
on the occasion of the World Congress of Ma- 
lacology in Vienna in August 2001 . In turn, the 
CLECOM catalogue became the core of the 
Fauna Europaea checklist, released electroni- 
cally in October 2004 (Bank, 2004). Although 
the three products are embedded within each 
other and have complementary scientific con- 
tents, they differ in format in addition to geo- 
graphical scope. The French checklist comes 



311 



312 



BOUCHET 



with 110 pages of endnotes that justify taxo- 
nomic or nomenclatural decisions, or report 
new faunistic records. The CLECOM and 
Fauna Europaea lists do not have associated 
notes, the former is available on paper and 
electronically, and the latter only in electronic 
form. 

I should like to emphasize that taxonomic 
checklists are only as good as the quality of 
the science that is behind them. Davis' (2004) 
criticisms focus in particular on the poor tax- 
onomy of hydrobioids in the French and 
CLECOM checklists. I do not disagree. But the 
culprits are the systematists themselves, past 
and present, who have been and are estab- 
lishing new taxa without proper qualifications 
and comparisons. The French checklist was 
about to go to press when Bernasconi (2000) 
published a work on the Bythinella of south- 
western France in a non-peer reviewed publi- 
cation, with the description of seven new 
species. As editor of the list, I agreed with 
Falkner that the new species should be given 
the benefit of the doubt and be listed as valid 
until synonymized. Falkneretal. (2001. 2002), 
CLECOM and Fauna Europaea thus list 37 
valid species of Bythinella from France, but 
the French checklist emphasizes (endnote 78, 
page 86): "Bernasconis results and our own 
will have to be tested by using molecular char- 
acters. In the meantime, segregating 
morphotypes at the rank of species will better 
meet the needs of mapping and conservation 
programs that motivated the compiling of the 
present list." I believe that highlighting the 
problems does a better service to science than 
suppressing them. I agree with Davis that one 
may, or even should, view the listing of 36 
species and subspecies of Bythlospeum in 
Germany with disbelief. Davis' comment "I do 
not know of any molecular or detailed anatomi- 
cal study that has looked at variability within 
and between populations to infer possible ge- 
netic breaks in taxa of Bythlospeum" is, in my 
view, not a criticism of list compilers but a criti- 
cism of those who add to the confusion by 
establishing still more new species based on 
inadequate character analysis and compari- 
sons. To make a comparison with North 
America, 1 would suggest that a future edition 
of Turgeon et al. 's Common and scientific 
names of aquatic Invertebrates from the United 
States and Canada should list Physella 
hemphllll D. W. Taylor, 2003, and Physella 
winnlpegensis Pip, 2004, as valid, unless 
these nominal species have been synony- 
mized; by doing so, Turgeon et al. would not 



be making a judgement on Taylor's (2003) or 
Pip's (2004) work. Checklists are simply re- 
flections of the state of the art, and one should 
not "shoot the messenger" if the news is not 
good. 



On Bourguignat and Revalidation of the 
Nouvelle Ecole's Nominal Taxa 

Few personalities in the world of malacol- 
ogy have elicited so much criticism, and even 
hatred, as Jules-René Bourguignat. It is fair 
to recognize that Bourguignat had a sharp dis- 
criminating eye for characters, that his knowl- 
edge of the literature was immense, and that 
he had a network of correspondants that chan- 
nelled large amounts of valuable material to 
him from all over the western Palaearctic 
(Kuiper, 1 969). However, Bourguignat also had 
very personal views on what deserved to be 
ranked as a species, and he developed an 
undefensible system whereby he would rank 
as "species" specimens that would be diag- 
nosable by three characters. This, in combi- 
nation with a self-infatuated personality and 
personal attacks on his competitors, invited the 
wrath of established and influential European 
malacologists. Both Crosse and Kobelt used 
the journals they edited, Journal de Con- 
chyliologie in Paris and Nachrichtsblatt der 
Deutschen Malakozoologlschen Gesellschaft 
in Frankfurt respectively, to build a sanitary 
cordon around Bourguignat and his followers, 
the self declared "Nouvelle Ecole". Bour- 
guignat retaliated with a gifted pen and ridi- 
culed his enemies in "Lettres Malacologlques" 
and other polemic writings (Bourguignat, 
1882). The two camps being at war with each 
other, Bourguignat's school would not listen 
to any, justified or unjustified, criticism and 
went on unchecked to establish thousands of 
new nominal species. 

By the end of the 19'" century, Locard rec- 
ognized no less than 1850 valid non-marine 
mollusk species in the French fauna (Locard, 
1893), among them no less than 506 species 
of unionids (versus eight now regarded as valid 
at the species level). The uncompromising 
attitude of the "Nouvelle Ecole" was mirrored 
in the other camp by the rejection en masse 
of Bourguignat's works and species. In the 
decades that followed, any species named by 
Bourguignat, Locard, Mabille, or Caziot, to 
name just a few, was a priori suspected to be 
synonymous of an earlier "classical" species, 
that is, a species recognized by British or Ger- 



A RESPONSE TO DAVIS (2004) ON SPECIES CHECK-LISTS 



313 



man authors. The question then asked was 
not "Is this a valid species or a synonym?", 
but "Which species is this a synonym of?" The 
first decades of the 20'" century were thus a 
period of massive synonymization; to ridicule 
the insignificance of the species established 
by Locard, Coutagne (1929: 16) even created 
the word "locardies", a parallel to the 
"jordanons" of botanical literature. Germain 
started his career by co-authoring two papers 
with Locard, but later became the principal 
instrument of the synonymization of the 
Nouvelle Ecole's nominal species. Germain's 
two volumes of the Faune de France 
(Germain, 1 931 ) represent the culmination of 
bringing the French non-marine fauna into 
harmony with its time. Because of the chaos 
caused by the Nouvelle Ecole's oversplitting, 
Germain's "normalization" was received with 
much relief and his new synonymizing was 
gladly and uncritically accepted by his contem- 
poraries and followers. It was not until the 
1970s that the French fauna received new, 
critical attention from malacologists from the 
Netherlands, Germany, Italy, and Spain. In 
particular, the Rijksmuseum in Leiden (today 
Naturalis) made southwestern Europe its area 
of excellence, resumed comprehensive field 
work, and started to critically re-examine the 
systematics of the land snails from France, 
Spain and Portugal based on solid population- 
based species concepts and using anatomi- 
cal, and, later, molecular data. This led to the 
resurrection of several nominal species from 
the graveyard of synonymy, e.g., Abida 
occidentalis (Fagot, 1888), a local endemic 
from the central Pyrenees, resurrected from 
the synonymy of A. pyrenaearia (Michaud, 
1831) (Gittenberger, 1973), and Cernuella 
aginnica (Locard, 1894), broadly distributed in 
southern France, resurrected from the syn- 
onymy of С virgata (da Costa, 1778) (Clerx & 
Gittenberger, 1977). When revalidating Trichia 
phorochaetia (Bourguignat, 1864), endemic to 
the Grande Chartreuse and Vercors regions 
of the French Alps, Winter (1 990) commented: 
"Notwithstanding the good description and fig- 
ures provided by Bourguignat (1864), the spe- 
cies was placed by both Hesse (1921) and 
Germain (1930) in the synonymy of Trichia 
villosa, no doubt because of Bourguignat's 
reputation." 

It became clear in the 1 980s and 1 990s that, 
among the many superfluous names produced 
by the Nouvelle Ecole, not everything was a 
synonym, and in fact Bourguignat and his fol- 
lowers had named some perfectly valid spe- 



cies, often local endemics from the Alps, the 
Pyrenees or the Mediterranean region. What 
Falkner and Ripken did when working up the 
French checklist was to critically re-examine 
as many nominal species as possible, based 
on the original collections, including types, of 
Bourguignat (in Muséum d'Histoire Naturelle 
de la Ville de Genève), of Locard (in Muséum 
National d'Histoire Naturelle, Paris), and of 
Caziot (in Muséum d'Histoire Naturelle de 
Nice), an approach that, surprisingly, no one 
had done systematically before. In the over- 
whelming majority of the nominal species they 
re-examined, they confirmed earlier accepted 
synonymies. However, this work also revealed 
a number of taxa that they suspected repre- 
sent valid species: rather than pushing these 
into synonymy against the available evidence, 
they decided to give the benefit of the doubt 
to these taxa. For instance, the French check- 
list thus revalidated Oxychilus colliourensis 
(Locard, 1894) and O. adjaciensis (Caziot, 
1904), based on historical as well as newly 
collected material. It also tentatively listed as 
valid, for example, Umax granosus (Béren- 
guier, 1900) and /W/7axoc/?raceüs(Bérenguier, 
1900) because of their distinctive anatomy, 
despite their not having been found in the last 
100 years (but also, admittedly, they have not 
been searched for at the type localities). As 
the editor of the French checklist, I agreed that 
"giving their chance" to Umax granosus and 
Milax ociiraceus as potentially valid species 
was more likely to lead to hypothesis testing 
and falsification, than continuing to obliterate 
them as doubtful synonyms (and then, as syn- 
onyms of what?). 

To conclude on Bourguignat and the Nouvelle 
Ecole chapter, I would like to make a com- 
parison with another (I am afraid, also French!) 
malacologist of the 1 9'" century who has been 
the subject of much controversy on the other 
side of the Atlantic, I mean Constantin 
Schmaltz Rafinesque of course. It has been 
said that Rafinesque was his own worst en- 
emy, and the same could be said of 
Bourguignat. Their published works were so 
controversial, their personalities were so un- 
conventional, that they became ostracized to 
the point of suppression. For a long time, ma- 
lacologists in the United States ignored 
Rafinesque's names, while European mala- 
cologists who dared declare any of Bour- 
guignat's species as valid were stigmatized. 
Admittedly, Rafinesque and Bourguignat 
wreaked havoc on the systematics and no- 
menclature of North American freshwater 



314 



BOUCHET 



mollusks, and western Palearctic land snails, 
respectively. But the rules of nomenclature 
demand that names and works are evaluated 
on a case-by-case basis. Rehabilitating Trichia 
phorochaetia as a valid species is not a reha- 
bilitation of its author, personality or method. 
After 130 years of blanket ostracism of 
Bourguignat, let's acknowledge that some 
valid species were indeed named by 
Bourguignat, even if such cases fuel incendi- 
ary judgements on zoological nomenclature: 
"In other sciences the work of incompetents is 
merely ignored; in taxonomy, because of pri- 
ority, it is preserved" (Michener, cited by Gould, 
1992). 



On Subspecies 

Continental European, especially Germanic 
authors, have a long tradition of systematics 
formally recognizing subspecies for discrete 
geographical variations within species, and the 
CLECOM and French checklists, as well as 
Fauna Europaea. clearly belong to this school. 
As regional checklists recently published in 
Europe vary in how they treat geographical 
variation, some background information is 
useful to place the Germanic tradition in per- 
spective. 

(1) For decades, many authors have used 
indiscriminately the concepts of "variety" and 
"subspecies", which is reflected to this day in 
the International Code of Zoological Nomen- 
clature that regulates how varietal names can 
be rescued when applied to subspecies. In the 
19'^ century and well into the 20" century, the 
concept of "variety" was used to designate any 
kind of variation (size, colour, sculpture), with 
or without a geographical component, and at 
any scale (local, regional or global). Taxonomic 
and nomenclatural practice shifted when evo- 
lutionary thought changed our understanding 
of variation. Taxonomists started to treat spe- 
cies as groups of populations, rather than col- 
lections of individuals, and they observed that 
morphological gaps could occasionally be 
overlaid with geographical gaps. This ap- 
proach was first conceptualized in the 1930s 
by Bernhard Rensch and Ernst Mayr, at the 
time both working in the Berlin Museum. 
Mayr's well-known influence on bird system- 
atics was a reflection of the immense impact 
of his teaching and writing on geographical 
variation. Less well known abroad is the role 
of Rensch, who had taken over in Berlin after 
Thiele, before becoming a professor at the 



University of Münster. Rensch had a consid- 
erable influence on German evolutionary sys- 
tematics after WWII and, because he was also 
a malacologist (see, among others, Rensch, 
1 937), his impact on German malacology can- 
not be overestimated. 

(2) It certainly is no accident that the study 
of geographical variation had a much higher 
resonance in the heart of alpine Europe than 
elsewhere in Europe. Most of northern Europe 
was covered by ice during the glacial periods 
of the Quaternary, and its current fauna and 
ftora could become established there only as 
the land became free of ice: for example, in 
the British Isles, nearly all the species that live 
today arrived there less than 10,000 years ago. 
This recent colonization has two conse- 
quences: first, there are no endemic species 
in the British fauna and flora; and, second, 
there is no discernible geographical variation 
among the British populations of even broadly 
distributed species. If British (or Scandinavian) 
authors do not recognize subspecies, this is 
not because Rensch was wrong, but rather 
because there are no subspecies within the 
British (or Scandinavian) fauna. 

By contrast, glaciations have created in al- 
pine Europe a mosaic of refuges. Whereas the 
major valleys were occupied by glaciers, there 
emerged archipelagoes of unglaciated terri- 
tories, for example, slopes facing south, nu- 
nataks, thermal areas, and populations 
isolated in these refuges had time to geneti- 
cally diverge between two interglacial cycles. 
Superimposed on the complex topography and 
climatology of the mountain areas of southern 
Europe, the glacial cycles had the effect of 
breaking down territorial and genetic continu- 
ities, generating the many highly localized 
species and subspecies that today character- 
ize alpine and Mediterranean Europe. The Alps 
thus naturally became the playground of ma- 
lacologists applying the concepts of Mayr and 
Rensch's evolutionary systematics. 

How we translate geographical variation into 
classification is not just an academic exercise 
in nomenclature, but is embedded in evolu- 
tionary biology and has consequences for 
management and conservation. Subspecies 
based on morphology (just as well as species) 
are hypotheses of genetic relationships be- 
tween specimens considered representative 
of natural populations. In this respect, the al- 
lopatric subspecies of evolutionary system- 
atists are the 'Least-Inclusive Taxonomic 
Units" (LITU) of phylogenetic systematists 
(Pleijel & Rouse 2000). It does not really mat- 



A RESPONSE TO DAVIS (2004) ON SPECIES CHECK-LISTS 



315 



ter, and it is in fact a matter of personal choice, 
whether one wants to call these terminal taxa 
species or subspecies, and whether one bases 
such decisions in a Phylogenetic Species Con- 
cept or a Biological Species Concept. For that 
matter, Kottelat's (1995) "Pragmatic Species 
Concept" is not lurking far behind. I believe 
that George Davis and I do not stand far apart 
on this issue, and I agree that taxonomic prin- 
ciples inspiring the checklist compilers could 
have been made more explicit. In fact, the 
whole issue broadly falls within the hotly de- 
bated subject of taxonomic ranks. Isaac et al. 
(2004) have recently discussed how changes 
In species concept, rather than new discover- 
ies, are leading to raising known subspecies 
to species level, with consequences on 
macroecology and conservation biology. What 
really matters is that these terminal taxa should 
be seen as biological/evolutionary/manage- 
ment units, rather than the esoteric fancy of a 
taxonomic splitter. 

Davis (2004) defends the view that "it is in- 
appropriate to name subspecies as a conve- 
nience and in the absence of well-founded 
data". None can disagree with him on this 
point, although we may disagree on what con- 
stitutes "well-founded data". However, I believe 



that Davis' criticisms are not aimed at the right 
target and do not do justice to the state-of- 
the-art of European non-marine molluscan 
taxonomy. The checklists compilers are not 
working in a nomenclatural terra nullius, and 
the names are in fact already out there in the 
250 years of accumulated literature on Euro- 
pean non-marine mollusks, however brilliant 
or pathetic, modern or outdated. In the check- 
lists being discussed, the list compilers did not 
name any new subspecies, but they recorded 
the use of subspecies names in the latest au- 
thoritative publications on the subject. There 
is already a considerable body of literature on 
the geographical variation and the distribution 
of subspecies of continental European land 
snails. In this respect, the French checklist only 
reflects the state-of-the art of that existing lit- 
erature. For instance, the subspecific tax- 
onomy of Chilostoma zonatum (Studer, 1820) 
is based on Forcart (1933), thatof/Ab/cíaseca/e 
(Draparnaud, 1801) is based on Gittenberger 
(1973), and that of Clausula rugosa (Drapar- 
naud, 1801) is based on Nordsieck (1990). 
One of several taxonomic areas where the 
French checklist innovates is unionid system- 
atics below the species level. In that family, 
taxonomic stability had been reached several 



Pseudanodonta complanata ligehca 



Pseudanodonta complanata elongata 




Pseudanodonta complanata dorsuosa 



Pseudanodonta complanata grateloupeana 



FIG. 1. Variation in European unionids has been historically disconcerting and difficult to analyze, 
because it includes a high within-population component and a more discrete geographical, between- 
population, component. By segregating discrete subspecies, the recently published French checklist 
hypothesizes that the morphologically recognizable forms from major drainages do have biological 
significance that must be taken into account into biodiversity inventories and management schemes. 
For instance, hypothetical populations reinforcements should avoid translocations of individuals be- 
tween populations of Pseudanodonta complanata from the Moselle (a tributary of the Rhine), Seine- 
Loire, Garonne, and Saône (a tributary of the Rhône) drainages. [Copied from Bouchet, in Falkner et 
al., 2002: 12]. 



316 



BOUCHET 



decades ago at the species level, with all of 
the hundreds of Nouvelle Ecole names end- 
ing up in synonymy. However, recent genetic 
work provides grounds for recognizing "sub- 
species" within these morphologically defined 
taxa. For instance, Badino et al. (1991) com- 
mented that a phenogram of genetic distances 
between populations of Unio elongatulus С 
Pfeiffer, 1 825 [now L/. mancus Lamarck. 1819], 
and Unio pictorum (Linnaeus, 1758) showed 
an "almost perfect arrangement of populations 
according to the hydrographie basins". Rec- 
ognition of "subspecies" within the unionids of 
France reflects the hypothesis that different 
hydrographie basins are inhabited by distinct 
genetic stocks, as evidenced by discrete mor- 
phological differences between basins (Fig. 1 ). 
As editor of the French checklist, I agreed with 
Falkners decision to highlight such discrete 
differences by using subspecific names, so 
that ecologists and geneticists could test and 
challenge them, rather than merging all re- 
gional variants into a broad, apparently uni- 
form pool. I had earlier (Bouchet et al., 1999) 
advocated subspecies to be an appropriate 
level for establishing lists of protected taxa 
under the European legal instruments. 



Regional Species Checklists: What They Are Not 

There are also points where I do agree with 
Davis, in particular classification, when he 
writes "Check lists should not be a vehicle for 
promoting the reconstruction of phylogenetic 
history or promoting a particular phylogenetic 
hypothesis" (Davis, 2004: 230). I agree that 
regional checklists are not the place to pro- 
duce elaborate, finely dissected classifica- 
tions. A classification using subfamilies, tribes 
and subgenera may be necessary when the 
purpose is to catalogue hundreds of species 
of Enidae from the Middle East and central 
Asia, but it certainly is not necessary when 
there are only six species of Enidae in the 
French fauna. This criticism of the inappropri- 
ateness of overly elaborate classifications in 
country or regional checklists probably is most 
founded in the case of hydrobioids which, in 
addition to the necessity of addressing their 
classification in a global context, are also un- 
dergoing a phase of profound re-evaluation 
(e.g., Wilke et al., 2001). The usefulness of 
national and regional checklists is because 
their compilers have an intimate knowledge 
of a usually highly fragmented local literature. 



both in space and time, dealing with taxonomic 
status and distribution of the terminal taxa. 
(Almost 64% of the 3,000 references in the 
French check-list are papers, pamphlets and 
books published in France.) However, the body 
of literature dealing with higher classification 
is of an entirely different nature, has no geo- 
graphical borders, and is fast changing. In the 
currently very active phase of réévaluation of 
the phylogeny of the mollusks, any classifica- 
tion is certain to become rapidly outdated 
(Bouchet & Rocroi, 2005). 

It should also be recognized that, in the case 
of the European fauna, regional species 
checklists are faced with special difficulties, 
because of the huge synonymy load, conflict- 
ing or parallel taxonomic schools, and centu- 
ries of accumulated literature, opinions, and 
mistakes. For fewer than 5,000 terminal taxa, 
there may be somewhere around 50,000 
nomenclaturally available names. Under 
these circumstances, these European re- 
gional species checklists cannot be and were 
not intended to be taxonomic revisions nor 
comprehensive nomenclatural compilations, 
in which every name is listed and/or every 
taxonomic opinion is supported by facts and 
references. As such, the checklists are not 
themselves standard, falsifiable research 
products, although they do synthesize such 
research results. 



Taxonomic "Authority" Lists: Authoritative to 
Whom? The Tyranny of Users 

The discussion that is now taking place in 
Malacologia about the French and the 
CLECOM checklists raises the issue of the 
acceptance of such checklists by the rest of 
the malacological community and users in 
general. These two categories of users may 
in fact at times have diverging interests, and 
this is where problems of acceptance may 
arise. Just like the International Code of Zoo- 
logical Nomenclature, taxonomic authority lists 
only work as long as there is a majority con- 
sensus to accept them. The International Com- 
mission on Zoological Nomenclature does not 
have a police to enforce violations of the Code, 
and the general adherence to the Code 
marginalizes those zoologists who might re- 
ject the Code. In other words, the community 
of systematic zoologists will only accept those 
rules that it is prepared to follow. In turn, the 
International Commission on Zoological No- 



A RESPONSE TO DAVIS (2004) ON SPECIES CHECK-LISTS 



317 



menclature is led to propose compromises 
between consistency, for example, the "prior- 
ity rule", and established usage, that may in- 
consistently apply the rules. 

Taxonomic "authority" lists face the same 
kind of dilemma. If they list only those taxa 
that are accepted by an overwhelming major- 
ity (95%?) of systematists, they will be seen 
as perpetuating the worst conservatism and 
will give the impression that everything is 
known and no further research in taxonomy is 
necessary. If they list nominal taxa that have 
not yet been properly scrutinized by peers, 
they run the risk of disseminating instability. 
However, the role of checklists is not primarily 
to give a false impression of stability where 
there is not. When passing judgement on the 
authoritativeness of a checklist, one should not 
underestimate the tyranny of non-specialist 
users whose demand for "stability" is legitimate 
when it concerns the nomenclature of reason- 
ably understood biological entities, but is not 
legitimate when it closes the door to progress 
in knowledge, or even to ambiguity (see, e.g., 
Dubois, 1998). Likewise, compilers of check- 
lists must sometimes find compromises be- 
tween scientific rectitude and the expectations 
of users. I want to illustrate this point by an 
example involving George Davis' own re- 
search. 

For nearly 100 years, British and Irish mala- 
cologists have argued over the taxonomic sta- 
tus of populations of Margaritifera living in the 
river Nore in Ireland. Whereas Margaritifera 
margaritifera is a soft-water species every- 
where it lives in Europe, there are Irish popu- 
lations in the Nore basin that live in hard water, 
that have subtle shell differences that led to 
their segregation as a different species, 
Margaritifera durrovensis. Until the advent of 
molecular techniques, it remained disputed 
and unresolved whether Margaritifera 
durrovensis was just a hardwater variant of 
M. margaritifera, or a distinct species. Subse- 
quently, an allozyme study (Chesney et al., 
1 993) concluded that M. durrovensis was just 
an ecophenotypic variant. As a scientist, I of 
course accept the results of the molecular 
study, but as a conservationist I may under- 
stand the desire by Irish naturalists to treat the 
Nore river Margaritifera as a "conservation 
unit". In fact, Chesney et al. themselves noted 
that "the classification of M. durrovensis as an 
ecophenotype of M. margaritifera does not 
detract from its need to be conserved". How- 
ever, how does one give special conservation 



consideration to a "Margaritifera margaritifera 
Nore basin conservation unit"? Legislators and 
regulators have answered that question by 
placing "Margaritifera durrovensis" on Annex 
2 to the European Habitats Directive (the ED 
equivalent to the US Endangered Species Act). 
And they have done so in 1995, that is, after 
the results of the Chesney et a!. (1 993) study, 
which were known to the proponent of the list- 
ing. I am not, through this example, advocat- 
ing that it was justified to place the "species" 
Margaritifera durrovensis or even a "subspe- 
cies" Margaritifera margaritifera durrovensis on 
a list of protected species. But the fact is that 
it was listed, despite the advice of scientists 
(including myself) consulted by their national 
regulatory authorities. How was this to be re- 
flected in CLECOM? One course of action was 
to promote scientific rectitude, ignore the name 
durrovensis altogether, and run the risk of be- 
ing viewed as "irrelevant" or "useless" by the 
agencies using taxonomic authority lists for 
management. Another course of action was 
to promote consistency between regulatory 
texts and taxonomic authority lists, list 
Margaritifera durrovensis as valid, and run the 
risk of being viewed as "incompetent" by pro- 
fessional systematists. After much debate 
among its authors, CLECOM chose a middle 
course, and listed Margaritifera margaritifera 
durrovensis as a valid subspecies. It would not 
take much for me to accept that this compro- 
mise is eminently disputable. Just as a classi- 
fication does not reflect all the relationships 
between taxa (the tree does), names are, af- 
ter all, no more than a tag that people - scien- 
tists or non-scientists - find convenient to use 
to designate a biological "entity" or "unit", and 
communicate about its attributes and proper- 
ties. Listing Margaritifera margaritifera 
durrovensis in taxonomic authority lists is a 
convenient way to access information on its 
conservation status as well as the associated 
literature, including Chesney et al. (1993). 

This example reminds us that taxonomists 
and compilers of taxonomic authority lists are 
not working in a sociological vacuum. There 
is pressure from non-scientists to have names 
to designate management entities, even when 
these do not correspond to sound biological 
units. Davis (2004) defends the idea of using 
"conservation units instead of dubious subspe- 
cies". I believe scientists may argue ad nau- 
seam in academic journals on whether 
"conservation units" are concepts that should 
be preferred over "dubious subspecies". I am 



318 



BOUCHET 



afraid this is an esoteric debate that has lost 
sight of the sociology of users who prefer to 
use named entities over "conservation units". 
As the Irish Margaritifera demonstrates, one 
powerful reason is that only named entities can 
have a legal status (that may actually promote 
research on the status of the taxon in ques- 
tion). I believe that when there is a testable 
hypothesis that such an entity may be treated 
as a biologically significant unit and there al- 
ready exists a name for it, then we should 
definitely use that name. When a name does 
not exist already, I agree that it may be dis- 
putable whether it is justified to establish one, 
and I would defend the view that it depends 
on the scientific as well socio-historical con- 
text. For three generations, malacologists 
working on the systematics of European land 
snails have extensively and consistently used 
trinominal nomenclature, whereas by contrast, 
the analysis of geographical variation of North 
American land snails, and/or the way this varia- 
tion is traditionally expressed through names, 
has not led to trinominal nomenclature. 

To conclude, I would like to proselitize on 
the recently produced checklists of French and 
European non-marine mollusks. They repre- 
sent a huge effort of data collation and knowl- 
edge consolidation, and they represent the 
state-of-the art of species-level systematics by 
systematists who have a personal opinion on 
the validity of nominal taxa, rather than per- 
petuate the state-of-the-art of 30 or 70 years 
ago. That the current state-of-the-art is chal- 
lenging so many entrenched usage traditions 
is a reflection of the health of non-marine mol- 
luscan systematics in Europe. That the cur- 
rent state-of-the-art is highlighting so many 
unresolved problems also reflects the need for 
more research. This is why I entitled my intro- 
ductory chapter to the French checklist: Land 
and freshwater mollusks of France: a new 
taxonomic authority list, a new start, new per- 
spectives [«Mollusques terrestres et 
aquatiques de France: un nouveau référentiel 
taxonomique, un nouveau départ, de nouvelles 
perspectives»]. To close with a dose of humil- 
ity, I will quote Isaac et al. (2004) in their recent 
essay on "taxonomic inflation": "Taxonomic 
uncertainty is ultimately due to the evolution- 
ary nature of species, and is unlikely to be 
solved completely by standardization. For the 
moment, at least, users must acknowledge the 
limitations of taxonomic lists and avoid unre- 
alistic expectations of species lists." 



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Revised ms. accepted 22 July 2004 



MALACOLOGIA, 2006, 48(1-2): 321-327 



CHECK-LISTS AND CLECOM: A RESPONSE TO DAVIS (2004) 

Ruud A. Bank\ Gerhard Falkner^, Edmund Gittenberger^ 
Theo E. J. Ripken^ & Ted von Proschwitz^ 



Recently, Davis (2004) published a paper with 
the title "Species check-lists: death or revival 
of the Nouvelle École?". From his title it is clear 
that he considers the work of CLECOM as the 
revival of the Nouvelle École (since a "death" 
of that school - whatever it means - would 
not be worth mentioning). This is a serious 
indictment, and therefore we have taken the 
liberty to explain some of our work in a more 
detailed manner. 

CLECOM 

The acronym CLECOM stands for "Check- 
List of the European Continental Mollusca". 
The initiative dates back to June 1986, and in 
August 1995 the CLECOM Working Group 
was officially endorsed by the General Assem- 
bly of Unitas Malacologica (UM) at its 12"^ In- 
ternational Malacologial Congress in Vigo, 
Spain. The CLECOM Working Group acts un- 
der the umbrella and on behalf of UM; the 
Friedrich-Held-Gesellschaft (München) is the 
organisation responsible for the CLECOM 
Working Group (Falkner et al., 2001 ). 

One of the primary aims of the CLECOM ini- 
tiative is to produce a distributional check-list 
of accepted scientific names for all the non- 
marine molluscan (sub)specles recognised in 
Europe. The first CLECOM list covers north- 
ern, western and central Europe (Falkner et 
al., 2001). The second CLECOM list gives a 
supraspecific classification of the European 
non-marine molluscs (Bank et al., 2001a). The 
third CLECOM list covers Macaronesia (Bank 
et al., 2002). The lists have been welcomed 
by many malacologists. In fact, recent works 
have adapted their nomenclature/classification 
to a large extent to the CLECOM lists (e.g.. 
Alba et al., 2004; Glöer, 2002; Martínez-Orti 
& Robles, 2003; Moorkens & Speight, 2001; 
Olsen, 2002; Steffek & Grego, 2002; Vilella et 
al., 2003). By doing so, a much more uniform 



nomenclature becomes available within the 
malacological community: regional scientific 
names, that are still hampering communica- 
tion approximately 250 years after Linnaeus, 
are more difficult to defend now. This will fa- 
vor integration, for example, among data- 
bases. One such database is a product of the 
Fauna Europaea project (www.faunaeur.org), 
and uses the CLECOM lists as its basis for 
the Gastropoda. 

Although there have been some cautionary 
comments regarding use of the CLECOM lists 
(e.g., Cameron, 2003), we have the impres- 
sion that a certain level of traditionalism is in- 
volved. For example, Cameron (2003) still 
uses the name Oxyloma pfeifferi despite the 
fact that the epithet pfeifferi Rossmässler, 
1834, has been replaced for taxonomic rea- 
sons by the older name elegans Risso, 1826, 
for nearly half a century (Forcart, 1956; see 
also ICZN Opinion 336, 1955). It is difficult to 
find a paper published by a non-British or non- 
Scandinavian malacologist after 1970 that 
uses the name pfeifferi. Davis (2004), how- 
ever, questioned the scientific basis of the 
CLECOM lists as a whole. Although his criti- 
cism is only very general, we would like to re- 
spond to his comments. 

Organismal and Molecular Taxonomists 
Should Combine Their Forces in Biodiversity 
Research 

People have marvelled and puzzled over 
morphological diversity for thousands of years. 
According to Wheeler (2004) we now have the 
opportunity to put centuries of scholarship on 
morphology into perspective and share it with 
the world. One tool for achieving this is the 
preparation of species check-lists, which 
should be considered snapshots of our knowl- 
edge of the world's biodiversity at a given 
moment. Such check-lists are often needed 



'Graan voor Visch 15318, 2132 EL Hoofddorp, The Netherlands; R.Bank@wxs nl 

^Bayerische Staatssammlung für Paläontologie und historische Geologie, Richard-Wagner-Straße 10/11, 80333 München, 
Germany 

^Nationaal Natuurhistorisch Museum "Naturalis", P.O Box 9517, 2300 RA Leiden. The Netherlands 
"Prinses Margrietlaan 11, 2635 JE Den Hoorn, The Netherlands 
^Naturhistohska Museet, Dept. of Invertebrate Zoology, Box 7283, 40235 Göteborg, Sweden 



321 



322 



BANK ETAL. 



to attain an overview of the data available in 
the ever increasing taxonomic and faunistic 
literature. In fact, check-lists and bibliographies 
often boost research, as they provide insight 
on the status of current knowledge. 

Most of the taxonomic and faunistic litera- 
ture is based on morphological studies. Pro- 
fessionals and amateurs make lists of species 
from particular locations by identifying species 
mostly with a field guide based on observable 
morphology. According to Davis (2004: 228), 
"creating taxa simply on the basis of shell char- 
acters (qualitative and quantitative) should al- 
ways be done with extreme caution". He even 
states that "the situation may not substantially 
change even when other characters, anatomi- 
cal, eco-ethological are added". Davis is ad- 
vocating molecular research as the Holy Grail. 
This approach to biodiversity/systematics is a 
subject of serious concern (Lee, 2004; 
Wheeler, 2004; Will & Rubinoff, 2004; Wiens, 
2004). We advocate that all disciplines of tax- 
onomy should combine their forces to deci- 
pher evolutionary processes rather than each 
stressing its own superiority. What we do not 
advocate is the training of biologists who can 
only identify organisms after grinding them up 
and feeding them into a sequencing machine. 

Sequences, although highly informative, are 
not a priori better than morphological, anatomi- 
cal or eco-ethological characters. An example 
is the recently published molecular phylogeny 
of the western palaearctic Helicidae sensu lato 
(Steinke et al., 2004). The maximum likelihood 
phylogenetic tree (based on a combined 
dataset of COI, 16S, 18S and ITS-1 se- 
quences) fits almost perfectly with the family/ 
subfamily classification provided by CLECOM, 
a classification based on anatomical data. 
Where more than one species per genus was 
sampled, these genera were monophyletic. An 
exception was the nominal genus Cernuella, 
as the three species that were sequenced - 
virgata, neglecta and cespitum - are scattered 
within the phylogenetic tree of the 
Hygromiidae. Had the authors consulted the 
CLECOM check-list, they would have learned 
that cespitum is not a Cernuella, but belongs 
to the genus Xerosecta, subgenus Xeromagna, 
and that neglecta and virgata belong to differ- 
ent subgenera {Xerocincta and Cernuella, re- 
spectively) of the genus Cernuella. 
Unfortunately, scientists working with mol- 
ecules often use old taxonomic frameworks in 
their publications. 



Naming Species and the Biological Species 
Concept 

Species concepts have been the subject of 
voluminous debate. We adhere to the biologi- 
cal species concept (BSC), although we ad- 
mit that most of the entities that are currently 
recognized in malacology as "good species" 
are in fact morphospecies. It is widely ac- 
cepted, however, that the vast majority of the 
currently accepted morphospecies correspond 
to biological species. In fact, we are not aware 
of any example in malacology of widely ac- 
cepted morphospecies recognized half a cen- 
tury ago as living in Europe now being lumped 
together as a result of more "sophisticated" 
studies involving, for example, population ge- 
netics or molecular data. In fact, such studies 
often revealed higher diversity than was ex- 
pected on morphological criteria, for example, 
because of the presence of cryptic species. 
The number of taxa in check-lists presented 
as "good species" should therefore be con- 
sidered conservative rather than exaggerated. 

Care should be taken with the doctrines 
known as the "Nouvelle École" of Bourguignat 
or "Starobogatovism