MAL: Uhl? + MALACOLOGIA International Journal of Malacology КА“. E . RR ce 2407 MATE En к NY en а Vol. 48(1-2) 2006 , MALACOLOGIA http://malacologia.fmnh.org EDITOR-IN-CHIEF: GEORGE M. DAVIS Editorial Office: Malacologia P.O. Box 1222 West Falmouth, MA 02574-1222 georgedavis99@hotmail.com Copy Editor: EUGENE COAN California Academy of Sciences San Francisco, CA gene.coan@sierraclub.org Managing Editor: CARYL HESTERMAN Haddonfield, NJ malacolog@jersey.net Graphics Editor: THOMAS WILKE Justus Liebig University Giessen, Germany Business & Subscription Office: Malacologia Р.О. Box 385 Haddonfield, NJ 08033-0309 malacolog@jersey.net Associate Editor: JOHN B. 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FRANCES ALLEN, Emerita Environmental Protection Agency Washington, D.C. KENNETH J. BOSS Museum of Comparative Zoology Cambridge, Massachusetts JACKIE L. VAN GOETHEM Treasurer, UNITAS MALACOLOGICA Koninklijk Belgisch Instituut voor Natuurwetenschappen Brussel, Belgium Emeritus Members 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 MCZ 2006 LIBRARY EDITORIAL BOARD FEB 21 2006 J.A. ALLEN E. GITTENBERGER ARVARD 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 R.A. 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 Б. Н. COWIE University of Hawaii Honolulu, HI А. H. CLARKE, Jr. Portland, TX В. 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 Rijksmuseum van Natuurtikg RARE RSI У Leiden, Netherlands Е GIUSTI Università di Siena Siena, Italy M. GLAUBRECHT Museum of Natural History Berlin Germany A. N. GOLIKOV Zoological Institute St. Petersburg, Russia А. V. GROSSU Universitatea Bucaresti Romania T. HABE Tokai University Shimizu, Japan К. Т. HANLON Marine Biological Laboratory Woods Hole, MA С. HASZPRUNAR Zoologische Staatssammlung München Múnchen, Germany J. M. HEALY Queensland Museum South Brisbane, Australia D. M. HILLIS University of Texas Austin, TX К.Е. HOAGLAND |. M. Systems Group Rockville, MA B. HUBENDICK Naturhistoriska Museet Goteborg, Sweden S. HUNT University of Central Lancashire Lancashire, United Kingdom R. JANSSEN Forschungsinstitut Senckenberg Frankfurt am Main, Germany М. $. JOHNSON University of Western Australia Crawley, Australia R. N. KILBURN Natal Museum Pietermaritzburg, South Africa J. KNUDSEN Zoologisk Museum Kobenhavn, Denmark C. MEIER-BROOK Túbingen, Germany C. LYDEARD University of Alabama Tuscaloosa, AL Н. К. MIENIS Hebrew University of Jerusalem Jerusalem, Israel J. E. MORTON Auckland University Auckland, New Zealand J. J. MURRAY, Jr. University of Virginia Charlottesville, VA R. NATARAJAN Marine Biological Station Porto Novo, India D. Ó FOIGHIL University of Michigan Ann Arbor, MI J. OKLAND University of Oslo Oslo, Norway T. OKUTANI University of Fisheries Tokyo, Japan W. L. PARAENSE Instituto Oswaldo Cruz Rio de Janeiro, Brazil J. J. PARODIZ Carnegie Museum of Natural History Pittsburgh, PA R. PIPE The Marine Biological Association Plymouth, United Kingdom J.P. POINTIER Ecole Pratique des Hautes Etudes Perpignan Cedex, France W.F. PONDER Australian Museum Sydney, Australia QUIZA Academia Sinica Qingdao, People's Republic of China D. G. REID The Natural History Museum London, United Kingdom $. С. SEGERSTRÄLE Institute of Marine Research Helsinki, Finland Е. К. Е. SIMONE Museu de Zoologia da Universidade de Säo Paulo, Brazil А. STANCYKOWSKA Siedlce, Poland F. STARMUHLNER Zoologisches Institut der Universität Wien Wien, Austria J. STUARDO Universidad de Concepción Valparaiso, Chile С. THIRIOT University Pierre et Marie Curie Paris, France S'MÉLIER Muséum National d'Histoire Naturelle Paris, France J. А. M. VAN DEN BIGGELAAR State University of Utrecht Utrecht, Netherlands М. Н. VERDONK Rijksuniversiteit Utrecht, Netherlands H. WAGELE Ruhr-Universitat Bochum Bochum, Germany A. WAREN Museum of Natural History Stockholm, Sweden B.R. WILSON Conservation and Land Management Kallaroo, Western 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? 8 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 pallial 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 pallial 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 Shinkailepas 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 (Shinkailepas 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 et al. 2003), together with the shallow-water gen- era Phenacolepas and Cinnalepeta, and Bathynerita is regarded as a member of the Neritidae (Warén & Bouchet, 1993, 2001). They represent part of characteristic mollus- can elements endemic to vent/seep environ- ments. In the genus Shinkailepas Okutani, Saito & Hashimoto, 1989, 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 т deep, (3) $. briandi Warén & 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., 1997) 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 'The University Museum, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; sasaki@um.u-tokyo.ac.jp 2Japan Agency for Marine-Earth Science and Technology, 2-15, Natsushima, Yokosuka City 237-0061, Japan 2 SASAKI ET AL. neritoidean groups. Hence, further anatomical comparison is significant to understand rela- tionships among Shinkailepas, Olgasolaris, shallow-water neritoideans, and the remaining less known neritopsines. All of known species of Shinkailepas have been described based chiefly on the shell, radula, operculum, and external morphology of the animal (Okutani et al., 1989; Beck, 1992; Warén 8 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 Shinkailepas 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. MATERIALS AND METHODS Materials examined in this study were se- lected from part of paratype series of $. myoji- nensis and those preserved at the Japan 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 um 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). RESULTS Shell and Operculum See Sasaki et al. (2003). FIG. 1. Dorsal view of animal, male, with mantle removed. ANATOMY OF SHINKAILEPAS 3 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. 12C). The dorsal side of mantle margin also bears similar processes (Fig. 3B). The mantle margin (mm: Figs. 2, 4C) is prominently thickened, divided into the outer and inner folds by the periostracal groove (pg: Figs. 3B, 4C). Its inside contains muscle fi- bers and blood sinus. The portion о the mantle covering the pallial cavity is provided with ex- tensive blood sinus with numerous hemocytes inside (pls: 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. 4B). The ventral side of the head is ex- tended as the oral lappet (ol: Figs. 2, 3C). The circumference of the mouth is thickened, papil- late, and wrinkled (Fig. 3C). 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 19, 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 pallial processes. Pallial Cavity The pallial 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. 10C), 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. 4 SASAKI ET AL. FIG. 3. Scanning electron micrographs of external and internal organs. А. Mantle processes (тр) on surface of mantle covering visceral part. B. Dorsal view of mantle margin. C. 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 buriscles (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 pallial gland” (apl: Fig. 4F). This gland is also extended to cover the distal tip of the pallial gonoduct. Dorsoventrally paired, two glands, the post-tentacular gland on ventral side and the anterior pallial 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 5 “Ро * PS rurale te te te 7 - > £ 7 5 ER A ея ее - ae 200 mu" , to = auf nu a ‘ 3 “a + + 2 . “Wath Py’ . = TES A + À * -. <» 0° # : e? a Ar Ve pa ¥ y ” Ge Ki = us FIG. 4. Histology of external and pallial 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). C. 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 pallial 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. 10). The jaw with tooth-like elements is absent. The sublingual pouch is well developed be- low the buccal mass (slp: Fig. 7). Its epithe- lium is thin and smooth. Paired sublingual 6 SASAKI ET AL. ot bev va _ lep He, NP) à EN Nele et PIRE IE P —d / K ne 8 — | a ae a er T a A A 1 rds я x Y LS ] \ Y > A DR y x St Е > cu ee iy а l mm ost —— o- 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 (slg: Fig. 7). Their inner surfaces are roughened with irregular forms of glandular 0.5 mm rds FIG. 6. Drosal view of buccal mass, with anterior digestive tract intact. 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. 7. Ventral view of buccal mass, with oral tube and sublingual pouch intact. ANATOMY OF SHINKAILEPAS 7 vap 0.5 mm A 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 ofthe 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). 8 SASAKI ET AL. a NM 5 ES ur : С 3 Ei Vo ` ¡SIA В: À A LEA CRE u > 2 |: 9 МР N > E RN AY IAN > TA Fee N IR Du р 7 We АО, = Е - q ‘ . Sine AE y > COS & E E A E ет / 1% EN, 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). C. 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 9 FRE De ER e-* - Pa À é o oe os oe! Г e ts ware 2 te > e = ve 5 GA A : For x = 3 70 e < æ) a ee TS ь KS 4 RE ga Ww te 2 e ‘ € с. e, > > o LA <“ oss AY г e , a? AIX LT FX Kay u 3 . ; As 5 os at, в . » + - LI a> - 3 + #4: ” 4 г + ee be se. e? re tes o. '. e. ALTA . AO AS AN CA AN ef I; (): = MOET aye en tls ar) z LU: . 3 = te PEL: и fa N 2 Я irn Тс A N no > eet: ‘à ip et? > ALES. A BEN ER * sale 20 > Е г Des he + À > We à sie Ze Y N rn JE > + RD NS, Com se A > A ANA A FE oa LS EIS y ES A = ae oh DIR EE NES AY tigi > Biens SIE 3 € Roe, x - SS QE x > SO © SSS SSS. 2 Sn * * ef, ” > Pa 6 Р Gn je р Jet: ~ £ E: FA Ÿ # sta Ge ne ra +? a =e A e yb 2 te ren, ¢ Po E 7100 um A ae en ese A Dr 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). C. Lon- gitudinal section of kidney (k), its opening (ko) 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 (plc). D. UMUT RM28647. E-F. UMUT RM28648. A-C. UMUT RM28651. Its epithelium is densely ciliated and composed of a layer of prismatic cells (Fig. 10B). 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 pallial 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 ET AL. 0.5 mm A FIG. 11. Pallial 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 pallial 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 pallial oviduct, a complex of pallial ovi- duct and associated glands, and the vaginal duct. The ovary and dorsal surface of the pal- lial 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 pallial oviduct from its ventral side (Figs. 11B, 12, 15C). The pallial 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 pallial oviduct and is further subdivided into two portions, albumen gland | and Il (agl, agll: Fig. 11). Both parts are not stained darkly, and the albumen gland | is obvi- ously more translucent than the albumen gland II (Fig. 15C). The pallial oviduct opens into the pallial cavity through its anterior tip (fo: Fig. 11). ANATOMY OF SHINKAILEPAS 11 FIG. 12. Schematic drawing of ventral view of pallial oviduct, showing connection of various ducts and associated structures. The vaginal duct is ventral to the pallial ovi- duct, covered entirely with epithelium of pal- lial oviduct, elongated, and connected to the pallial oviduct posteroventrally. The terminal of the vaginal duct is expanded and opens as an oblique slit (vgo: Fig. 11B). Posterior to the vaginal opening, the receptaculum seminis is branched off from the vaginal duct. Зрегта- 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. 11B). Near its connection to the pallial 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. 15B-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 pallial male gonoduct (Fig. 13). The testis contains many cylindrical sectors in which growing sperms are arranged radi- FIG. 13. Pallial gonoduct of male, with intestine intact. A. Dorsal view. B. Ventral view. 12 SASAKI ET AL. ally. А 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 pallial 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). Aciliated groove was not found between the gonoduct opening and the penis. ES -500 um x Nervous System The nervous system consists mainly ofthe 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. C. 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 eu) Kr ar. AR RENT rm % И PEN 7 ‘x fh AT „№; D 4 ir rhe а 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 pallial oviduct. C. Longitudinal sec- tion of pallial oviduct where oviduct (ovd) is connected to lumen of pallial oviduct. D. Longitudinal section of posterior sac (ps) of vaginal duct and surrounding glands. Е. 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 pallial gonoduct of male. А. UMUT RM28648. Е-Н. UMUT RM28650. В-Е. UMUT RM28651. 14 SASAKI ET AL. lc EG = In cg bg pos == = LENS -tn > Pia YAA one = a, Sa ede pn = A) | —— — ple ple” sta a sbv ped 1 mm 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 pallial 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 (plc: 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, O/gasolaris, 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) 15 АМАТОМУ ОЕ SHINKAILEPAS (sanunuo9) oes лепрел oes spuejb oes Jenpe, о} ¡e/9]e] oes Je]npe. о} ¡e/a]e] oes лепре! о} ¡e/a]e] Buisojoue Ajesoo| — лепрел Buisojoue Anubn ¡eaBeydosa 10119]sod snbeydosa ¿juasqe éjuesge ¿juasqe juasaud juasqe лоиазие ul рие|б 100 pajeBuoja jou pajeBuoja jou pajeBuoja jou Auouaysod pajeBuoja рэзебио]э jou spue|6 |еэбецаозэ 1o1ajue ous ous yous био] yous oes Jejnpes juasqe juasqe juasqe juasaud juasqe a]e¡d ¡euBjeuo/a]e] oMIJ-PISIUS Аэзлэлзиед 9MIJ-PIOIUS Л|э$лэлзие anbijqo anbılqo ц}ээ} uno} 4199} y ino, 4199} y no, 499} (p.tyy) 49}NO ешрпубио| 4498} no} епре. jo yaa) [219]e] juasa.d juasaud juasaJd juasqe juasaJd епре. jo Y}OO} ¡e.1Jueo juasqe juasad juasad juasad juasad puej6 |епбицап$ u ¿ À) é juaseld pue|6 se;noeju9}-}sod E & B 7 juasaud puej6 jeıjjed 1o1ajue juasaJd juasaud juasad juasaud juasqe риеб ¡jeiyouelqodÁy juasqe juasaid juasqe juasqe juasqe apis зчби uo 1116 ¡erb1 san juasqe pado¡anap-|¡am рэЧо|элэр-пэм 1ебцзэл рэас|элэр-пэм unIipiuajo juasqe juasqe juasqe juasqe juasaud jadde¡ энецаээ juasqe juasqe juasqe juasqe juasad dey jeıpodıda juasqe juasaud juasad juasad juasqe un nosado jo sisAydode JE|NOIIDIWAS леполониа$ ¡epiozade.] лепэлоние$ jepıozoden adeys шппэлэдо I19ys Ájaja¡dwoo juasge juasqe juasqe jo apısuı Ajuo Buiuedo eus бицедецаа Ápued 194$ jo salnjon1]s ¡eueo payloo paj109 Joduui| paj109 jaduu!| WO} ||US (8661) Meses (8661) Meses (8661) Meses (2002) asey $ ouey Ápnys $14} a9uaJajay виешер!елл EJUON euedes EIRUEN ‘EUINSIA sedayjleyulys snues Seplulol|aH Sepan эер эм эер! эм eepipeds|oseueud Анше- ‘елэцэб auisdoyuau pipedajooeuayd-uou pue зе4э/елищс и! saje]s Jaj9eJeyo jo чозиедшо) ‘| FIGVL SASAKI ET AL. 16 Jouajsod ‘a1od |jews Jouajue “ajod |jews 6 С ¿ ¡es1opoJa]sod 1е$10ро1э}504 |е$10ро1э}5$04 - ajduuis 7 juasqe juasald juasald рэзо|о 'padojanap- ¡jam рэ$о|э‘рэдо|элэр-|эм рэзо|о‘рэдо|элэр-|эм ¿aja¡duwoo a]a¡duwo9 ajajduwoo Е С € juasqe juasaJd juasa.d a|duuis pa¡Buejua pa¡Buejua juasqe juasqe juasqe juasqe juasqe juasqe juasqe juasaJd juasaid juasqe juasqe juasqe pue¡B ичэшпае pue¡5 uswngje ¿juasqe ¡esJOp о} pajoauuoo езлор о} pajoauuoo Jouajue ‘эло4 jjews yyjoye}s лоиаие 6 juasaud uado ‘jeibysan aJe|duooul 6 juasqe Jo Аледиэцирпа pajlogun ‘ajqnop juasaJd juasa.d juasqe juasqe oes aJoydojeuads jo ped 10118}$04 ‘a3xi-JIIS EIUOOOJEIS ¡es10poJa]sod panrooJ6 Ajjeuipnyibuo} juasa.d pasojo “¡e1B1san aJe|duooul 12.9} е] Jussald paBuejua juasqe juasqe juasqe juasaid Huiluedo ¡euiBea 0} J0119]sod JoLIEJUe *DMI|-J1]S ]U9}U09 3sA90]8]s elbueb jepad о} элце!эл UONISOd ISÁDO]E]S unipesydso jo auoz |едиээ unipelydso saña doo} эллаи |2199SIA siuad uo 8A0016 jeulwas siuad энецаээ 3|9IS3A |EUIWUSS dey sjewa} э!еша;} Ul мот} yOoU эеша; ul эе$ |е}$А1э yonp jeuißen jo Des 1013}$04 Slulluas WnN2e)da9a1 Buiuado |ешбел sBuiuado энпе!р энпе!р энпец} энпе!р энпе!р aAnonpoidal dyed} juasqe juasqe juasqe juasqe juasaud Ajqissod sajA90JyjAJa juasqe juasaJd juasaJd juasaJd juasaid ajoune зубы Aaupıy jo suoıßa4 1еприе|б разециалаир разециалаир разециалаир разециалашрип pajeyualayipun -uou pue Jejnpuejb spuej6 элцзэбир pue yoe рэлед paued рэлеа a|Buls рэлеа -WO}s usamjaq (s)Buiuado (8661) Meses (8661) Meses (8661) Meses (2002) asey $ ouey Ápnys $14} 99U919J9H виешар!ем PUAN euejdas PI/NUAN ‘еип$!с sedayjileyulys snuas) эерциэцен Sepan Sepan Sept uan aepipedajo9euayd AIILUE 4 (рапициоэ) 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. 12C), 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 ofthe 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 Ceresidae, 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: pl. 1, fig. 4, pl. 5, fig. 4; Sasaki et al., 2003: fig. 12D). Among neritopsines, the opercula of Shinkailepas and Olgasolaris are most similar to that of Septaria 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 Olgasolaris Phenacolepas Cinnalepeta Reference this study Beck (1992) Fretter (1984) Sasaki (1998) canal structures of partly penetrating penetrating shell absent absent shell shell completely operculum shape trapezoidal subtrapezoidal semicircular, absent vestigial apophysis of absent absent % - operculum circumpallial absent absent present present microtentacles epipodial flap present present absent absent cephalic lappet present present absent absent posterior tightly enclosing lateral to radular lateral to radular esopahgeal radular sac sac? sac glands 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- transversely slit- small pore small pore, anterior like, anterior like, anterior receptaculum posterior to vaginal present connected to dorsal seminis opening albumen gland posterior sac of present absent absent vaginal duct female flap absent present? present as absent? ovipositor? seminal groove on lateral dorsal % ? penis eyes vestigial, closed well-developed, well-developed, closed closed 18 SASAKI ET AL. morphic part) is also known in the Neritiliidae (Капо & Kase, 2000, 2003, in press; Капо et al., 2003), Bathynerita (Warén & Bouchet, 1993), and Phenacolepas (Kimura & Kimura, 1999: fig. 7C-D), and therefore it is not char- acteristic of Shinakailepas and Olgasoralis. The possession of apophysis in the opercu- lum 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 8 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; Warén & 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 о 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 (Warén & Bouchet, 2001), but absent in the Neritiliidae, shallow-water phenacolepadids, and the Neritidae excluding Bathynerita (Table 1). Pallial Organs: The pallial cavity of Shin- kailepas contains a set of pallial organs com- mon to the aquatic Neritopsina, namely the ctenidium, osphradium, anus, pallial gonod- uct with genital opening(s), and excretory pore, but a hypobranchial gland is absent. The ctenidium of $. 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 pallial 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 pallial roof in some neritopsines, for ex- ample, the Neritidae (Fretter, 1965; Berry et al., 1973; Sasaki, 1998), Neritiliidae (Kano 8 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 pallial gland) are observed on the right anterior corner of the pallial 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 pallial 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: lts 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, Septaria, and Cinnalepeta, and is partially different from that of Waldemaria (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 Olgasolaris (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), Neritopsis (Warén & 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 Neritopsis, Titiscania, and the Hydrocenidae (Ponder, 1998: fig. 15.76). The presence of prominence below cusps in the marginal teeth (Kano & Kase, 2002: fig. 8) is known in the Neritiliidae, Neritopsis, 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. Along, looped radular sac is typical of the Neritiliidae, Neritopsis 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 et al., 1989: fig. 15), and Olgasolaris tollmanni (Beck, 1992: fig. 3B) have the latter type. The esophagus of the Neritopsina exhibits similar structure throughout the group (Sasaki, 1998): (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 Бу Kano & Kase (2002). The anterior esophageal glands are 20 SASAKI ET AL. 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 8 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 8 Kase, 2002: fig. 3C). The identical glands were not found in sections of equivalent posi- tion in $. 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., 1999). 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 8 Kase, 2002). The kidney of $. 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 $. myojinensis, the peri- cardium contains two (right and left) auricles and a single median ventricle. The right au- ricle is obviously present т $. 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 Shinkailepas 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, pallial 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, pallial ovi- duct and vaginal duct have their own open- ings (diaulic). In contrast, female reproductive system is triaulic with an additional enigmatic duct in Septaria (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- lial oviduct is a common feature in most neritopsines, and it is also true of $. myojinen- sis. Characteristically, the vaginal duct in $. myojinensis is associated with three struc- tures: (1) a transverse slit ofthe vaginal open- ing below the anterior part of pallial oviduct, (2) the receptaculum seminis near the vaginal opening, and (3) the “posterior sac” below the posterior part of the pallial oviduct. A slit-like vaginal opening in the anterior po- sition is also described in O/gasolaris (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 Phenacolepas (Fretter, 1984), Cinnalepeta, the Neritidae, and Helicinidae (Sasaki, 1998). An anteriorly situated receptaculum seminis near the vaginal opening in $. 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. 15A) is the most reliable criterion for this identification. The “posterior sac” of the vaginal duct may also be peculiar to S. myojinensis. lts 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 $. myojinensis, no spermato- phore was found in any section of female and male reproductive systems. The pallial 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 8 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 8 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 (1984) (Kano & Kase, 2002). In $. 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, pallial 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 (Warén 8 Bouchet, 1993), shallow-water phenacolepadids (Sasaki, 1998), and Shinkailepas. By contrast, in the Neritiliidae, the seminal vesicle is double and different from tightly convoluted type (Kano 8 Kase, 2002). In the Helicinidae, it is simple, not en- 22 SASAKI ET AL. 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 pallial 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: pl. 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. 14А-В) is unique to $. 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. lts 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 8 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 $. 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. Shinkallepas 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 Bathynerita (Waren 8 Bouchet, 1993), but obviously absent in other neritopsines, such as Nerita (Haszprunar, 1985; Sasaki, 1998: fig. 77d). 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, 1998) and Helicinidae (Bourne, 1911). In the Neritiliidae, their position is shifted more anteriorly (Kano 8 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 8 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, 1998: 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 Neritilidae and Shinkailepas. The kidney in neritopsines is not always clearly differentiated 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 n-4-1-4-n, (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 8 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 28$ 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; Warén 8 Bouchet, 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 (Warén & Bouchet, 1993), a cephalic penis also occurs in the Neritidae, two shell muscles without division are found in Septaria (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 ET AL. 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 Shinkailepas. The comparison with $5. myojinensis revealed that т $. briandi Warén 8 Bouchet, 2001, (1) the eye stalks (“еуе- 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 $. 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 $. kaikatensis, 11-19 in $. 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 Natsushima 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 а =anus aca = anterior cartilage of odontophore acv = afferent ctenidial vein ag = albumen gland ag | = albumen gland | ag |= albumen gland II al = anterior levator muscle of odontophore ang = annex gland apd = anterior pedal gland apg = anterior pedal groove apl = anterior pallial gland at = anterior tensor muscle of odontophore bcv = buccal cavity bg = buccal ganglion с = ctenidium cc = cerebral commissure cdc = cerebropedal connective cg = cerebral ganglion ср = cephalic lappet 26 SASAKI ET AL. ср = cuticularized plate cpc = cerebropleural connective cpg = capsule gland ct = cephalic tentacle dbt = dorsal buccal tensor muscle df = dorsal fold of esophagus dfc = dorsal food channel of anterior esophagus dg = digestive gland dgo = opening of digestive gland to stomach di = dorsal levator muscle of odontophore dps = duct of posterior sac e =еуе ecv = efferent ctenidial vein ef = epipodial fold es =eye stalk ev = esophageal valve f = foot fo = female opening of gonoduct gc = gastric caecum gst = tooth of gastric shield i = intestine if = inner fold of mantle margin ivp = inner ventral protractor muscle of odontophore к =kidney Ко = kidney opening la = left auricle |са = labial cartilage lep = lateral pouch of anterior esophagus In = labial nerve Ip =lateral protractor muscle of odontophore |рг = lateral protractor muscle of subradular membrane Ism = left shell muscle т = mouth тса = median cartilage of odontophore ml = median levator muscle of odontophore mm = mantle margin mo = male opening of gonoduct mp = mantle process mpg = male pallial gonoduct mpr = median protractor muscle of subradular membrane mt = mantle оар = outer approximator muscle of cartilages of = outer fold of mantle margin ol = oral lappet os = osphradium ot = ога tube ovd = oviduct оур = ощег ventral protractor muscle of odontophore р =penis pc = pericardium pca = posterior cartilage of odontophore pcd = pedal cord рсу = pallial cavity pd = posterior depressor muscle of odontophore pds = pedal sole pdt = postdorsal buccal tensor muscle pe = posterior esophagus pg = periostracal groove plc = pleural commissure ра = pleural ganglion pls = pallial sinus pn = pallial nerve po = pallial oviduct pr = prostate = prostate pouch ps = posterior sac of vaginal duct ptg = post-tentacular gland ra = right auricle rds = radular sac rdt = radular teeth rev = retractor muscle of esophageal valve rrs = retractor muscle of radular sac rs = seminal receptacle rsm = right shell muscle rsr = retractor muscle of subradular membrane sbv = subesophageal part of visceral loop sc = statoconia sig = sublingual gland sip = sublingual pouch sn = snout srm = subradular membrane ке. — Le) | st = stomach sta = statocyst sv = seminal vesicle t = testis tac = tensor muscle of anterior cartilage tbf = transverse buccal fold tn = tentacular nerve у = ventricle vad = vaginal duct vao = vaginal opening vap = 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 combined exposure. Metal bioaccumulation in the digestive gland of adult individuals was investigated after 21 days exposure to 100 uM lead nitrate, 10 uM copper sulphate, and 0.002 X (1 X = 36 UM lead: 1 ЫМ 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 LC5g 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 of that 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 8 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 27. 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 28 ABDALLAH trace element needed in minute amounts by aquatic molluscs to synthesize haemocyanin (Ghiretti 4 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 (Clubb et 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; Cossa, 1989; AbdAllah et al., 1999, 2003). Lanistes carinatus is a widely distributed gastropod in Egyptian fresh- water ecosystems (Brown, 1995). 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 of that 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. 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. 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 um lead and 0.5 um copper). Uptake of Lead and Copper Groups of 30 adult and healthy snails each were exposed to 100 uM lead nitrate, 10 uM 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, 1974). 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 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; Kraak et al., 1993). The metal concen- tration was calculated in ug/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. carinatus. Source df бит of squares Mean squares F-value P-value Metal 1 3891381 13890000 16.253 0.0003 Treatment 1 38575511 38580000 45.134 0.0001 Metal* Treatment 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 uM, 1 mM, and 5 mM, while the examined copper sulphate concentrations were, 20, 30, 50, 100, 500, and 1,000 uM. The toxicity of lead and copper interaction was stud- ied employing a mixture о lead nitrate and cop- per sulphate (1 X= 18 uM: 0.5 uM 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, LCso, LC75, and LCgs 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 uM), copper sulphate (10 uM), 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-um 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. RESUS 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. carinatus after single and mixed exposure. (* P < 0.05, ** P < 0.01) Single exposure Cu Single exposure Cu = Pb Mixed exposure Cu Pb ship (г = -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- 100 90 80 70 60 50 40 30 20 10 0 Cu (ug/g) 0 0.1 Mixed exposure Pb Cu Pb -0.032 2.132" ND - ND 1.9697 - -5.670** centration and weight of the examined огдап showed a significant relationship r = -0.893, Р < 0.05 for lead (Fig. 1) and г = -0.877, P < 0.02 for copper (Fig. 2). 0.2 0.3 0.4 weight (9) FIG. 1. Linear regression relationship between copper concentration (ug/g) and weight of the digestive gland of L. carinatus (г = -0.877, < 0.02). 30 25 20 Pb (mg/g) a 0 0.1 0.2 0.3 0.4 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. carinatus af- ter single and mixed long-term exposure. Single exposure 55.911 68.953 Mixed exposure 854.401 3198.493 Copper Lead Acute Toxicity of Lead and Copper Toxicity levels: LC25, ЕСьо, LC75, and LCos 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 100 uM lead nitrate (Fig. 4) resulted in presence of necrotic digestive and basophil cells in a wide tubular cavity with FIG. 3. Light structure of the digestive gland of L. carinatus; digestive cell (dc), digestive tubule (dt). Scale bar = 50 pm. vs | a А”. eR eN Coat TES *; HET FIG. 4. Transverse section through the digestive gland of L. carinatus 3 weeks exposed to 100 uM lead nitrate; tubular cavity (tc), cell necrosis (cn). Scale bar = 50 um. FIG. 5. Transverse section of the digestive gland of L. carinatus exposed to 10 uM copper sulfate for three weeks; cell necrosis (cn), digestive tu- bule (dt). Scale bar = 50 um. 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 um. 32 ABDALLAH TABLE 4. Toxicity levels of 24 h single and combined exposure to lead, copper for the freshwater snail L. carinatus. LC25 LCso LC75 LCos Lead 562.34 uM 1.995 mM 6.761 mM 21.379 mM Copper 39.81 uM 141.25 uM 251.19 uM 1.778 mM Lead-Copper 0.0035 X 0.018 X 0.079 X 0.708 X slight tubular deterioration. Digestive tubules of snails exposed to 10 uM 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 (Clubb et al., 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 yug/l lead nitrate: 0.5 g/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 Chironomus 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). Amechanism 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. (1999) for the effect of chronic exposure of the schistosome vectors Biomphalaria alexandrina 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 ассотрате the reduction in cellular length (George, 1990). 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 1/49 of the calcu- lated LC5g and are fairly higher than that re- corded in the freshwater body (18 um lead and 0.5 um copper). 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WHO, 1989, Lead - Environmental aspects. En- vironmental Health Criteria, World Health Or- ganization. Geneva. 106 pp. WONG, P. T. S., 1987, Toxicity of cadmium to freshwater microorganisms, phytoplankton, and invertebrates. Pp. 117-138, in: J. O. NRIAGU & J. В SPRAGUE, eds., Cadmium in the aquatic environment. Wiley-Interscience, New York. xii +272 pp. Revised ms. accepted 3 May 2004 MALACOLOGIA, 2006, 48(1-2): 35-42 PHYLOGENETIC ANALYSIS OF THE PERI-HYDROTHERMAL VENT BIVALVE ВАТНУРЕСТЕМ VULCANI BASED ON 18$ rRNA Suzanne С. Dufour’, Gerhard Steiner? & Peter С. 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 В. 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 18S rRNA-based molecular phylogeny is used to compare B. vulcani with other pectinoids. The molecular trees group B. 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. Key words: Bathypecten vulcani, Propeamussiidae, phylogeny, 18$, 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 13°N. In its origi- nal description, B. 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 Pennec 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 of Bathypecten vulcani, as well as that of its newly renamed congener, B. eucymatus (Dall, 1898), collected at abyssal depths from the Bay of Biscay. Areexamination of the shell microstructure of both Bathypecten species showed differences between the two species, with B. 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 В. vulcani, the ctenolium is lacking (Schein-Fatton, 1988). The genus Bathypecten was eventually placed within the Pectinidae, in the subfamily Propeamussiinae (Schein, 1989, 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 Gilman Drive, La Jolla, CA 92093-0202, U.S.A.; present address: ISMER, Université du Québec a Rimouski, 310 Allée des Ursulines, Rimouski, Quebec G5L 3A1 Canada; suzanne.dufour@ugqar.qc.ca “Institute of Zoology, University of Vienna, Althanstr.14, A-1090 Vienna, Austria SISOMER, Faculté des Sciences, Université de Nantes, 44322 Nantes Cédex 3, France 36 DUFOUR ET AL. 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 names, Pectinidae and Propeamussiidae, according to common usage, and to avoid confusion; this does not imply familial rather than subfamilial status. More recently, itwas found that the ultrastruc- ture of the spermatozoa of Bathypecten vulcani differed significantly from that of pectinids, given that it could not be classified into either of the pectinid structural categories of Le Pennec 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 B. 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 within the Pectinoidea, the 18S rRNA sequence is here obtained and compared with that of other pectinids and propeamussiids. MATERIALS AND 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 (9°49.6’М, 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 т 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 18A1 (5'- CCT ACC ТСС TTGATC СТС CCA G - 3’) and 1800r (5'- ATG ATC CTT CCG CAG GTT CAC C - 3’). The PCR-reactions were made on a Robocycler 96 (Stratagene) in a 30 pl reaction mix (1.5 mM MgCl, each dNTP at 250uM, each primer at 0.5 uM, 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 18$ 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+A 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 3.064 (Huelsenbeck & Ronquist, 2001). We ran six chains through 200,000 generations under the GTR+I+A 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 18$ rRNA sequences. Systematic position Species Accession Number Arcoidea Arcidae Агса поае (Linné, 1758) X90960 Acar plicata (Dillwyn, 1817) AJ389630 Barbatia virescens (Reeve, 1844) X9197 Noetiidae Striarca lactea (Linné, 1758) AF120531 Glycymerididae Glycymeris pedunculus (Linné, 1758) AJ389631 Glycymeris sp. X91978 Pinnoidea Pinnidae Pinna muricata (Linné, 1758) AJ389636 Atrina pectinata (Linné, 1767) X90961 Anomioidea Anomiidae Anomia ephippium (Linné, 1758) AJ389661 Pododesmus caelata (Reeve, 1859) AJ389650 Pododesmus macrochisma (Deshayes, 1839) Plicatuloidea Plicatulidae Plicatula plicata (Linné, 1767) AJ389651 Plicatula australis (Lamarck, 1819) AF229626 Limoidea Limidae Lima lima (Linné, 1758) AJ389652 Limaria hians (Gmelin, 1791) AF 120534 Ctenoides annulatus (Lamarck, 1819) AJ389653 Pectinoidea Spondylidae Spondylus crassisquamatus (Lamarck, 1819) AJ389646 Spondylus hystrix (Róding, 1798) AJ389647 Spondylus sinensis (Schreibers, 1793) AF229629 Propeamussiidae Bathypecten vulcani (Schein-Fatton, 1985) AY557608 Parvamussium undisonum (Dijkstra, 1995) AY557607 Pectinidae Pecten maximus (Linné, 1758) L49053 Placopecten magellanicus (Gmelin, 1791) X53899 Adamussium colbecki (E. À. Smith, 1902) AJ242534 Flexopecten glaber (Linné, 1758) AJ389662 Argopecten irradians (Lamarck, 1819) L11265 Argopecten gibbus (Linné, 1758) AF074389 Chlamys islandica (Müller О. F., 1776) 1211232 Chlamys hastata (Sowerby, 1843) 149049 Mimachlamys varia (Linne, 1758) 149051 Crassadoma gigantea (Gray, 1825) 149050 Exellichlamys spectabilis (Reeve, 1853) AJ389648 Pedum spondyloideum (Gmelin, 1791) AJ389649 *The partial 28$ sequence of Parvamussium undisonum is deposited under the accession number AY557609. 38 DUFOUR ET AL. RESULTS AND DISCUSSION 18$ 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 112 shortest trees of 517 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-| (Matsumoto, 2003), which supports pectinoid monophyly but yields a sis- ter group relationship of Propeamussiidae to the clade (Spondylidae + Pectinidae). The two 96 / 92 1.00 100/90 100/90 1.00 1.00 100 / 95 1.00 71/22 100 / 98 0.97 1.00 65 / 79 0.99 50 / 50 0.94 100 / 99 1.00 35 / - 0.83 100 / 97 1.00 Bathypecten vulcani 76/56 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 ARCOIDEA PINNIDAE PLICATULIDAE ANOMIIDAE LIMIDAE Spondylus sinensis 100 / 98 Spondylus crassisquamatus 1.00 Spondylus hystrix Parvamussium undisonum Placopecten magellanicus Adamussium colbecki Exellichlamys spectabilis Crassadoma gigantea 20 / - Chlamys islandica 0.99 [64/72 Pedum spondyloideum 0.67 Mimachlamys varia Chlamys hastata Pecten maximus Aequipecten opercularis Flexopecten glaber 89 / 96 Argopecten gibbus 1.00 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 Pennec, 1991), spondylids, and limids (Ridewood, 1903; Dakin, 1928): it is non-pli- сае, homorhabdic, has а non-reflected ощег demibranch, and lacks latero-frontal cilia, in- ter-filamentar junctions, and interlamellar junc- 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 B. vulcani gill organization compares to other members of this family. If other propeamussi- ids are found to share the simple gill structure Arca noae Acar plicata Barbatia virescens Striarca lactea Glycymeris sp Glycymeris pedunculata Pinna muricata Atrina pectinata 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, 1981), may be plesimorphies for the Propeamussiidae (as compared to the condition in the Pectinidae and Spondylidae — Dakin, 1928; Yonge, 1973; Beninger & Le Pennec, 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 B. vulcani (Le Pennec et al., 2002) may be Plicatula plicata Plicatula australis Pododesmus caelata Anomia ephippium Pododesmus macrochisma Limaria hians Lima lima Ctenoides annulatus Spondylus sinensis Spondylus crassisquamatus Spondylus hystrix Parvamussium undisonum Bathypecten vulcani Placopecten magellanicus Crassadoma gigantea Chlamys islandica Pedum spondyloideum Mimachlamys varia Chlamys hastata Adamussium colbecki Exellichlamys spectabilis Flexopecten glaber Pecten maximus Aequipecten opercularis Argopecten gibbus 0.01 Argopecten irradians FIG. 2. Maximum likelihood tree (-In L = 6386.38877) found under the GTR+I+I model. Model param- eters estimated by MODELTEST: substitution rate matrix A-C = 2.2511, A-G = 2.9955, A-T = 1.6583, C-G = 1.3161, C-T = 4.9707, G-T = 1.00); nucleotide frequencies А = 0.2472, С = 0.2213, С = 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 ET AL: 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 150 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 al., 1994; Veniot et al., 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 origi- nating from surface waters (Le Pennec 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 Pennec et al., 1988, 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. LITERATURE CITED ALLEN, J. A., 1981, The ecology of deep-sea molluscs. Pp. 29-75, in: W. D. RUSSELL-HUNTER, ed., The Mollusca, Vol. 1, Ecology, Academic Press, Orlando, Florida. BENINGER.. Р.С. «Ss © DUFOUIR GP: DECOTTIGNIES & M. 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R., 1984, The ctenolium of scallop shells: functional morphology and evolution of a key family-level character in the Pectinacea (Mollusca: Bivalvia). Malacologia, 25: 203-219. YONGE, C. M., 1973, Functional morphology with particular reference to hinge and ligament in Spondylus and Plicatula and a discussion on relations within the superfamily Pectinacea (Mollusca: Bivalvia). Philosophical Transac- tions of the Royal Society of London, (B) 267: 173-208. УОМСЕ, С. M., 1981, On adaptive radiation in the Pectinacea with a description of Hemi- pecten forbesianus. Malacologia, 21: 23-34. 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, $. redferni and $. coltrorum, are proposed. Crassispira quadri- fasciata (Reeve, 1843) is reassigned to Strictispira. Three additional species — $. агапда! (Schwengel, 1951), S. paxillus (Reeve, 1845), and S. solida (C. B. Adams, 1850) — are discussed. Drillia асигида Пай, 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 $. redferni. Similarly, Drillia ebenina Dall, 1890, initially a fossil species and often considered to be Recent and a synonym of $. 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 ofthe 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 (1971a: 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 43 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 а the ANSP. With regards Strictispira species, McLean commented (1971a: 125) that Drillia ebenina 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 Pleurotoma solida C. B. Adams, 1850, are conspecific. | 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 $. solida. It is probable 44 TIRPETT 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 о the shell and one showing the radula, which bears typical strictispirid teeth. Although now assigned to Strictispira, 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 Strictispira 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- spirella 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 8 Taylor (1994) reviewed S. paxillus in 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, Strictispira con- tains two species, $. ericana (Hertlein & Strong, 1951) and S. stillmani; the sister genus Cleospira contains only C. ochsneri (Hertlein & Strong, 1949). The western Atlantic species, listed in Malacolog (fide Maes), are Strictispira acurugata (Dall, 1890), S. drangai (Schwengel, 1951), $. paxillus, and $. solida, with Drillia 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. Drillia 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, Strictispira redferni and Strictispira coltrorum, reassignment of Crassispira quadrifasciata, which has been determined to be strictispirid, $. paxillus, $. solida, and possibly $. drangai, the number of Strictispira 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, 1922) 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. MATERIALS AND METHODS Specimens of the genus Strictispira, 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. = Museum of Comparative Zoology, Harvard University, Cambridge, Mas- sachusetts, U.S.A. MNHN = Museum National d'Histoire Naturelle, Paris, France MORG = Museu Oceanografico do Rio Grande, Rio Grande, Brazil MCZ МНМ = 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, DES USA. Other abbreviations: spec. = specimen(s) colin. = collection Exp. = Expedition Stn. = Station SYSTEMATICS Strictispiridae McLean, 1971 Genus Strictispira 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. Strictispira 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 6-67 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 Y. 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. TIPPETT 9. Shells of Strictispira spp. FIGS. 1-3: Strictispira coltrorum, holotype, MORG 43415, valda Id., Guarapari, Espirito Santo, Brasil, 10.9 x 4.0 mm; FIGS. 4-6: Strictispira redferni, SNM 1010771, Abaco Id., Bahamas, 9.3 x 3.6 mm; FIG. 7: Strictispira redferni variety, Vaca Key, Florida Keys, 14.1 x 4.6 mm; FIG. 8: Strictispira redferni variety, ANSP ‚sahatchee 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 ld., Guarapari, Espirito Santo, Brasil (20°42’S, 40°25’W), dredged at 25-30 m, оп 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 % whorl. Strictispira coltrorum is nearest Strictispira 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. 172 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- 272 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 drangai (Schwengel, 1951) Figures 10, 20, 26 Crassispira drangai Schwengel, 1951: 116, pl. 8, figs 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, pl. 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 TIRPEMT inal. Fine secondary spiral threads between primaries overall. Sculpture forms pattern of rectangular spaces with enclosed spiral threads. Enlarged axial or two forming varix ehind outer lip. Aperture parallel-sided, anus 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, Drillia, 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 ld., British Virgin Ids., 10.0 x 4.4 тт; 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 ld., British Virgin Ids., 15.8 x 5.6 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, pl. 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 colln. 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 ld., 26°38'N, 7825'W, J. Worsfold!, ex Worsfold colln.; 1 spec., 298408, reef, NE of North Point, El- bow (Little Guana) Cay, Abaco, Bahama lds., 7 ft (2 m), under dead Acropora palmata, К. 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, C. W. Johnson!, 1890; 1 spec., 374474, Grand Bahama Id., 26°31’М, 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. B. 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"4303'N, 77%19'85"W, live collected from underside of rock, 0.5 m, 1 July 1997, C. Redfern!, ex Redfern colin. (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. drangai (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, $. solida has a black shell; $. 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 C. 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, pl. 43) re- port specimens of C. 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 $. drangai and C. fuscescens, differentiation be- ing based on the shell form and extended ca- nal. This form is apparently undescribed. lt 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: pl. 31, spe- cies 285. Drillia (Crassispira) paxillus (Reeve, 1845) — Tryon, 1884: 194, pl. 14, fig. 92 [repetition of Reeve's fig.]. Crassispira paxillus (Reeve, 1845) — de Jong 8 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, pl. 57; Malacolog, 2004, list. Clathrodrillia solida (C. B. Adams, 1830 [sic]) — Rios, 1975: 130, pl:, 39, На. эвэ [а misidentification, fide Maes, 1983: 318, “The Brasilian shell figured is $. paxillus”]. Pleurotoma nigrescens Reeve, 1845, ex Gray MS: pl. 26, species 235. FIGS. 19-24. Protoconchs of Strictispira spp. FIG. 19: Strictispira coltrorum, USNM 1011352; FIG. 20: Strictispira drangai, USNM 1023063; FIG. 21: Strictipira paxillus, specimen, one of two, juvenile, shell 7.1 x 3.3 mm, Redfern colln.; FIG. 22: Strictispira quadrifasciata, USNM 902243; FIG. 23: Strictispira redferni, USNM 1010773; FIG. 24: Strictispira solida, USNM 900428. Scale bar = 0.5 mm. THE GENUS STRICTISPIRA IN THE WESTERN ATLANTIC 51 Pleurotoma jamaicensis Guppy, 1866: 290, pl. 16, fig. 6. Drillia jamaicensis (Guppy) — Pilsbry, 1922: 320, list and text [synonymized Drillia ebenina Dall, 1890]. Material Examined 2 spec., one mature, one juvenile, Chub Rocks, Abaco, Bahamas, live collected on underside of rocks, 9 m, C. Redfern!, 10 Oct. 1982, Redfern colin. ANSP: 1 spec., 342987, White Bay, Guana ld., British Virgin 14$., 2-3 т, in drifted sand оп rocks, V. О. 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. |. (Krebs)” К. Swift; 1 spec., 249182, Jack Bay, Anegada, Virgin Ids., 0-8 ft (0-2.4 т), sand, stones, coral, Stn. 770, A. J. & J. С. 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. 8 J. С. Ostheimer!, 20 Mr., 1960; 1 spec., 313121, Guantanamo Bay, Cuba, outer beaches, R. T. 8 S. Abbott!, May 1967; 1 spec., 331166, 0.5-1 mi. SSW of The Bluff, Beef ld., 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, К. A. 8 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 1973. ‚ Paleontological colln.: 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. В. Henderson!, 1918; 1 spec., 702318, Van Thiel, Curaçao, 10 ft (3 m), underside of rocks at low tide, ex Mrs. D. Meyer colln., 20 Jan. 1981; 2 spec., 900416, Curtain Bluff, Antigua and Barbuda, 5-15 ft (1.5-4.5 т), Sept. 1981; 2 spec., 900417, Curtain Bluff, Antigua and Barbuda, 20 ft (6 т), $. 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); Curacao, Aruba, Bonaire area (de Jong & Coomans, 1988: 109); Brazil (Rios, 1975: 130, 583, pl. 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 15 mm by de Jong & Coomans, 1988: 109); 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 flattish, subsutural cord projecting little, finely 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 flange 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 amined 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 “Crassispira”, showing she was not thinking of Strictispira 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: 318f) described S. paxillus briefly, 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, colln. 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 Strictispira spp. FIG. 25: Strictispira coltrorum, USNM 1011351; FIG. 26: Strictispira drangai, USNM 1023063; FIG. 27: Strictispira paxillus, as with Fig. 21; Fig. 28: Strictispira quadrifasciata, 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 C. fuscescens and C. apicata. Crassispirella fuscescens has a sculptural pattern on the shell periphery of rectangular spaces, as described above with S. drangal, 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, pl. 28, species 251. Drillia (Crassispira) quadrifasciata (Reeve, 1845) — Tryon, 1884: 195, pl. 14, fig. 82 [re- peat of Reeve’s fig.]. Crassispira quadrifasciata (Reeve, 1845) — Kaicher, 1984: card 3896; Leal, 1991: 189, pl. 24, fig. G.; Rosenberg, 1992: 105, illus- trated; Malacolog, 2004, list. Crassispira (Crassispirella) quadrifasciata (Reeve, 1845) — Humfrey, 1975: 183, pl. 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 т), 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, К. W. Foster!, Apr. 1951; 2 spec., 682194, Buccoo Reef, Tobago, Smithsonian Bredin (SBI) Exp., Stn. 8, 5 Apr. 1959, 9:30 AM-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, 5-15 ft (1.5-4.5 m), Sept. 1981; 1 spec., 902241, off Cat 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., 195808, 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. 8 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’М, 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, 1% smooth whorls, followed by % whorl with quickly enlarging axial riblets blending into adult sculpture. Teleoconch whorls 5%-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. Varix of 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 um, 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. | 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 526 ph or. Strictispira acurugata (Dall, 1890) — 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) 1% 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, 9-13 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 95 WAAL IHG à DRE rcoel rstm FIG. 31. Strictispira redferni. Semidiagrammatic sagital section of head and foregut, from serial section. Shell 10.1 x 3.8 mm, sacrificed: bc = buccal cavity, cm = columellar muscle, con = circumoral nerve ring, m = mouth, od = odontophore, ое = vesophagus, огт = 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 120 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”М, 77°9'35’W), dredged live, 12 ft (3.6 m), 9 July 56 MEREDT 38 39 FIGS. 32-39. Radular ribbons and teeth of Strictispira spp. FIG. 32: Strictispira redferni, USNM 1010773, slide preparation, light-transmitted, ribbon section; FIG. 33: Strictispira redferni, USNM 1010775, SEM preparation, ribbon 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: Strictispira paxillus, drawing of tooth from Kantor & Taylor (1994: fig. 2C, using Maes's material), data as with Fig.14; FIG. 38: Strictispira quadrifasciata, Antigua, shell 7.9 x 4.1 mm, sacrificed; FIG. 39: Strictispira solida, USNM 411922. Scale bar = approximately 50 um (Fig. 32), 100 pm (Figs. 33-39). 1994, C. Redfern!; paratypes: 38 spec., 14 Aug. 1989, C. Redfern!; 1 spec., with data USNM 1010772, sandbank, lee side Guana as per 1010772, at each of the following: Cay, Abaco, Bahamas, 9 July 1992, C. AMNH, ANSP, DMNH, FMNH, LACM, MCZ, Redfern!, and 11 spec., USNM 1010773, MNHN, MORG, NHM, NM (material ex spoil bank, Guana Cay, Abaco, Bahamas, 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, Н. 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 т), J. В. 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 articulata 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, В. К. Bales!, Jan.-March 1945, ex Schwengel colln. (originally identi- fied as Crassispira tampaensis); approxi- mately 75 spec., 313080 (ex 221702), Bonefish Key, Florida Keys, B. R. Bales!, ex Schwengel colln.; 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., 313084 (ex 264988), Boca Ciega Bay, near St. Petersburg, Florida, ex J. D. Parker colln.; 3 spec., 368733, Hotel, W end Grand Bahama Id., Bahama 14$., 26°42'15”М, 7859'50"W, J. Worsfold!, ex Worfold colin.; 1 spec., 368499, McLean Town, Grand Bahama Id., Bahama 14$., 26°38’45”М, 77%57'30"W, 3 ft (1 m), J. Worsfold!, ex Worsfold colln.; 1 spec., 355797, Wardwick Wells Key, Exuma, Bahama 14$., 24°22’N, 76°36’W, intertidal sand, D. Cosman!, ex Cosman colln.; 5 spec., White Sound, Elbow Cay, Great Abaco Id., Bahama lds., 26°32’N, 76°58'W, W. С. Lyons!, 1972, ex Lyons colln.; 1 spec. 355798, Whale Cay, Abaco ld., Bahama lds., 26°43’М, 77°14’W, D. Созтап!, Aug. 1979, ex Cosman colln.; 1 spec., 329768, Bimini Lagoon, near Bailey Town, Bimini Ids., К. Robertson!, 1957-58; 6 spec., 370553, North Hawksville Creek, Bahama Ids., 26°32’N, 78°45’W, 1-3 ft (0.3-1 т), J. Worsfold!, ex Worsfold colln.; 2 spec., 374473, Grand Bahama ld., Bahama lds., J. Worsfold!, ex Worsfold colln.; 4 spec., alco- hol preserved, 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 redferni 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- ensis (Bartsch & Rehder, 1939: 136, pl. 17, figs. 5, 13), which | 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. А 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 Вай, 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 “$. 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- 59 THE GENUS STRICTISPIRA IN THE WESTERN ATLANTIC GS-6E BE 81-91 21-91 6-8 9209 82-29 (ON m2 Fl SL 94 = И 6'015'85 =И YSOFO9L=N ZOFEII=SN €0FS8=MN SIFZB=M 10+9`9=И ELFZLEN взебпипэе b=N b=N РЕМ РЕМ v=N РЕМ b=N t=N endsoblÁg 69-66 65—65 CEO 81-21 8-9 859 279€ Gvl-8'6 (jeuonıppe) vVLFO9=M LOFLE=W €LFO8BL=N GLFS8bHL=W ZOFZ9=M BOFOL=AW LOFEV=EW ELFZLL=IM IUJe/pel bl=N bl=N bl=N bl=N bl=N bes IN vl =N bl=N ends os 29-219 16-69 Те 91-61 6'/-6/'9 0'6-S'2 vt p 8 vl-yizL (Nauen) G'OF 09 = W GOFVSE=W 60FP8L=WN 80F/+L=W ZOFLLEW SOFL8=N У0+87=И IOFrEL=WN IUI8Jpel L=N ЛЕМ ZN MEN LEN JSN EIN LN 219542145 69-19 07-95 22-91 GL-&L G/ 9-09 9'/-8'6 pt 9€ AA (sadAy) 60F£9-M QOFGLE=W €LFEG8BL=N LOFGEL=W $0+89=и 90F99=WN УО+У=ИМ 60F90L=MW IUe/pel OL=N OL=N OL=N OL=N OP =SIN OL=N OL=N OL=N ends os (%) Apog/uBue] (%) HOUAA HOUM эешц зрочцмоээ1 (uu) Ápog (ww) yipim (WW) yBue7 upM/UBue7 Apog $1ел9$ -¡nuag sjeixy ‘эбиел рэллэзао $! OUl| J3MOT ‘иоцелэр рлерчцЕ}з = + ‘UBB = | ‘зцэциоэа$ jo лэдшти = М ‘$лэзелечцо jjays ee6ninoe ¿endsobi Ay pue /wajpas ела$111$ | 37191 60 TIPPER 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. Strictispira solida (C. B. Adams, 1850) Figures 11, 24, 30, 39 Pleurotoma solida C. B. Adams, 1850: 61; Clench & Turner, 1950: 342, pl. 29, fig. 8 [lec- totype designated]. Strictispira solida (С. В. Adams, 1850) — Maes, 1983: 320, text with Strictispira paxillus; Kaicher 1984: card 3917; Redfern, 2001: species 527, pl. 57; Malacolog, 2004, list. Crassispira (Crassispirella) fuscescens (Reeve, 1843) — Abbott, 1958: 94 [list and description plus text; synonyms: Pleurotoma solida C. B. Adams, 1850, Drillia ebenina Dall, 1890]. Not Drillia ebenina Dall, 1890: 33, pl. 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 $. solida. Not “Clathrodrillia solida” (С. В. Adams, 1830 [sic]) — Rios, 1975: 130, pl., 39, fig. 583 [a misidentification, fide Maes, 1983: 318, “The Brasilian shell figured is S. paxillus”]. Not Drillia solida (C. B. Adams, 1850) — Bandel, 1984: 166, fig. 309, pl. 20, fig. 8. ?Clathrodrillia solida C. В. Adams, 1830 [sic] — Rios, 1985: 136, species 621, pl. 46 [Dall's figure of ebenina]; Rios, 1994: 159, species 712, pl. 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 flattish 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 half way 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 um, 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 “ebenina”, “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 т), J. В. Henderson!, Eolis Stn. 34, 1911; 1 spec., 411913a, off Miami, 10 fms (18 m), J. В. 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 т), 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°03’11”М, 82°27'27"W, 17 т, 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 т), 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, 11 Oct. 1992 (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: 5. Schwengel!, 24 April 1940; 1 spec., 281650, SE end of McBride Cay, Belize, Stn. 106, 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, К. Robertson!, 21 Aug. 1961; 4 spec., 284033, off 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 1947; 1 spec., 313083 (ex 221702, split from lot of Crassispira cubana), Bonefish Key, Florida, J. $. Schwengel!; 1 spec., 320964, St. Thomas, W. |., К. Swift!; 1 spec., 337481, Key West, Florida, C. 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 ld., 2-4 ft (0.5-1.2 m), live, on sand and rocks, at night, J. Worsfold!; 4 spec., 374475, Grand Bahama ld., 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., 113150, 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 TIPPERT 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 Dall 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 (Dall & 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 8 Abbott, 1962: 134). Finally, Abbott (1974: 270) considered S. solida a valid species, nevertheless con- sidering C. ebenina a synonym. Abbott's 1958 misidentification of $. solida as С. fuscescens is based upon ANSP 198968 from Grand Cay- man Island. Abbott included a slip with the shell stating, [it] “matches solida CBA OK”. Maes indicated this was written approximately 1957. 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. lt 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 C. 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 C. fusces- cens, see features for C. fuscescens noted above with $. drangai and $. paxillus. Addi- tionally, C. fuscescens, has a non-crested subsutural cord. Separation from $. агапда! 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 $. 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. Bandel 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. lt 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. redferni, 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. LITERATURE CITED ABBOTT, R. T., 1954, American Seashells. D. Van Nostrand Co., Princeton, New Jersey. 541 pp., 40 pls. ABBOTT, R. T., 1958, The marine mollusks of Grand Cayman Island, British West Indies. Monograph of the Academy of Natural Sci- ences of Philadelphia, 11: 138 pp., 5 pls. ABBOTT, К. T., 1974, American seashells, 2% ed. Van Nostrand Reinhold Co., New York etc. 663 pp., 24 pls. ADAMS, C. 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The Veliger, 14(1): 67-72. SIMPSON, С. Т., 1887, Contributions to the Mol- lusca of Florida. Proceedings ofthe Davenport Academy of Natural Science, 5: 45-72. STEARNS, R. Е. C., 1872, Descriptions of new species of marine mollusks from the coast of Florida. Proceedings of the Boston Society of Natural History, 15: 21-24. ТАВВ, D. С. & К. В. MANNING, 1961, А check- list of the Йога and fauna of northern Florida Bay and adjacent brackish waters of the Florida mainland collected during the period July, 1957 through September, 1960. Bulletin of Marine Science of the Gulf and Caribbean, 11(4): 552- 649. TAYLOR, J. D., Y. 1. KANTOR & А. V. SYSOEV, 1993, Foregut anatomy, feeding mechanisms, relationships and classification of the Conoidea (= Toxoglossa) (Gastropoda). Bulletin of the Natural History Museum, London (Zoology), 59(2): 125-170. TRYON, С. W., 1884, Manual of conchology, Pts. 23, 24. Philadelphia. Pp. 151-413, 34 pls. WARMKE, С. L. & R. T. ABBOTT, 1962, Carib- bean seashells, 2° printing. Livingston Publish- ing, Narberth, Pennsylvania. 348 pp., 44 pls. WOODRING, W. P., 1928, Miocene mollusks from Bowden, Jamaica, Pt. Il. Gastropods and dis- cussion of results. Carnegie Institution of Wash- ington, Washington, D.C. vii + 564 pp., 40 pls. Revised ms. accepted 11 November 2004 MALACOLOGIA, 2006, 48(1-2): 65-76 GERM CELL DIFFERENTIATION AND SEXUAL MATURATION OF THE FEMALE NEPTUNEA (BARBITONIA) ARTHRITICA CUMINGII (CROSSE, 1862) (GASTROPODA: BUCCINIDAE) Ee-Yung Chung', Sung Yeon Kim’, Gab-Man Park* 8 Jong Man Yoon‘ ABSTRACT Oogenesis, the gonadosomatic index (GSI), reproductive cycle, and first sexual matura- tion of the female Neptunea (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 оо- 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 females of 51.0 to 60.9 mm in shell height, and 100% in those over 61.0 mm. Key words: Neptunea (Barbitonia) arthritica cumingii, oogenesis, germ cell differentia- tion, sexual maturation. INTRODUCTION Neptunea 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 Neptunea 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 8 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. antiqua. There has been one study on the spawning season of N. cumigii т 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 & Gomot, 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 66 CHUNG ET AL. 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. MATERIALS AND METHODS Sampling Specimens of Neptunea arthritica ситтди (Crosse, 1862) were collected monthly at the subtidal zone of Maldo, Kunsan, Korea, from 36 00'N Gogunsangundo Sampling Area Се ye 35°48'N À - 126 24'E 126 12'E FIG. 1. Map showing the sampling area. 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): Thickness of the gonad x 100 Diameter of posterior appendage in- cluding the gonad and digestive gland GSI = 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 126°36'E OOGENESIS AND SEXUAL MATURATION OF NEPTUNEA 67 FIG. 2. Anatomy of Neptunea arthritica cumingii, removed from its shell. Posterior appendage showing the ovary and digestive gland. X, У and 2 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 aseries 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 СХ-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 ит 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 Neptunea 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 ET AL. 3.5 1.5 0.5 GONADOSOMATIC INDEX JFMAMJJASONDJFMAMJJASOND 2002 2003 MONTH FIG. 3. Monthly changes in the gonadosomatic index of female Neptunea arthritica cumingii, for two years from January 2002 to December 2003. if it was slightly scratched with a razor, пре 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: Oogonia in the oogonial phase, which propagated on the germinal epi- thelium (follicular wall), were oval and 15 um 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 оо- cytes were 25-90 um 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 оо- 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 NEPTUNEA 69 FIG. 4. Electron micrographs of the previtellogenic and early vitellogenic phases during oogenesis of Neptunea arthritica cumingii (A-F). A: Oogonia in the oogonial phase, with a large nucleus and several mitochondria in the cytoplasm; В: А previtellogenic oocyte, with a large nucleus with a few nucleolus and several mitochondria, the Golgi complex, and vacuoles in the cytoplasm; С: 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; OC, oocyte; OG, oogonium; ER, Endoplasmic reticulum; Va, vacuole; Ve, vesicle. 70 CHUNG ET AL. 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- AE 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 um 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 | 7 La | FIG. 5. Electron micrographs of late vitellogenic and mature phases during oogenesis of Neptunea arthritica cumingii (A-D). A: A late vitellogenic oocyte, with yolk granules between the rough endoplasmic reticulum and the the mitochondria; В: A late vitellogenic oocyte, with a number of multivesicular bodies formed by modified mitochondria; С: Alate 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 NEPTUNEA TA < o nN о т W. TEMP. (°C) 5 80 ) 3 © o FREQUENCY (% a o . . . N Early active stage O Partially spawned Ш Late active stage E Recovery stage S PAS | ¡SS УЕ МАМ 9 Л А ЗАО 2003 MONTH B Ripe stage FIG. 6. Frequency of gonadal phases of Neptunea arthritica 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 um in size, respectively. At this time, early vitellogenic oocytes of 25-50 um 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 um 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 um 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 оо- cytes grew to 160-180 um 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 um in diameter became 12 CHUNG ET AL. 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 Neptunea arthritica cumingii. A: Transverse section of oogenic follicles in the early active stage; BC: 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 um. Abbreviations: LG, lipid granule; LM, lumen; MT, mesenchymal tissue; N, nucleus; NO, nucleolus; OC, oocyte; OG, oogonium; V, vacuole; YG, yolk granule. OOGENESIS AND SEXUAL MATURATION OF NEPTUNEA 73 TABLE 1. The shell height and first sexual maturity of female Neptunea (Barbitonia) arthritica cumingii from May to August, 2002. Shell height (mm) EA LA RI 31.4—40.9 34 41.0-50.9 26 2 2 51.0-60.9 15 2 10 61.0-70.9 3 21 71.0-80.9 2 22 81.0-90.5 16 Total Abbreviations: EA, early active stage; LA, spawned stage; RE, recovery stage. oocytes, as well as previtellogenic 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 oogonia and previtellogenic 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, itis 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 ifthey 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 Gonadal developmental stage PS RE Total Mature (%) 34 0.0 30 13.3 5 92 591 6 30 100.0 9 33 100.0 12 28 100.0 187 100.0 late active stage; RI, ripe stage; PS, partially 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 100% 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 //yanassa obsoleta (Taylor 8 Anderson, 1969), Biompha- laria glabrata (deJong-Brink et al., 1976), Mytilus edulis (Reverberi, 1971), Rapana venosa (Chung et al., 2002), Siphonaria capensis (Pal & Hodgson, 2002), Patella barbara, P. argenvillei, P. granularis, P. ocu- lus, P. miniata, and Helcion pectunculus (Hodgson 8 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 ET AL. 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- latus (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 8 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 8 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 lliana obsoleta (Tay- lor & Anderson, 1969) and Rapana venosa (Chung et al., 2002). Compared with Patella barbara, the lack of these structures is а promi- nent characteristic during 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 (Bottke et al., 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 lusoria 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. — Г&®›эги8кик„ко—д—————————————— —______—— Locality Author Species Spawning season Neptunea 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 Kawai et al., 1994 Son, 2003 Habe, 1960 OOGENESIS AND SEXUAL MATURATION OF NEPTUNEA 75 Breeding Pattern As shown in Table 2, our histological obser- vations show that spawning of N. arthritica cumingii 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, 1963). Neptu- 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 oogonia 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. 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YOO, J. S., 1976, Korean shells in color. \lgisa, Seoul. 196 pp. [in Korean]. Revised ms. accepted 1 November 2004 MALACOLOGIA, 2006, 48(1-2): 77-132 REVISION OF THE GENUS /SLAM/A RADOMAN, 1973 (GASTROPODA, CAENOGASTROPODA, HYDROBIIDAE), ON THE IBERIAN PENINSULA AND DESCRIPTION OF TWO NEW GENERA AND THREE NEW SPECIES Beatriz Arconada 8 Maria-Angeles Ramos Museo Nacional de Ciencias Naturales (CSIC) José Gutiérrez Abascal 2, 28006 - Madrid, Spain; mcna313@mncn.csic.es ABSTRACT The presence of the genus /s/amia 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 Schútt, 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), /. Jagari (Altimira, 1960), and /. ateni (Boeters, 1969), plus two new species, I. pallida and I. henrici, the latter with two subspe- cies I. В. henrici and I. В. giennensis. Two new genera are also described, Milesiana and Josefus, each of which contains one species: М. schuelei (Boeters, 1981), which was previously assigned to Neohoratia, and most recently to /slamia, 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 /slamia, 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 /s/amia 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, 1995; Arconada et al., 1996) have shown con- siderable morphological diversity and high УТ endemicity. Boeters (1988) recognized that species of two genera, Horatia Bourguignat, 1887, and Neohoratia Schütt, 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 /slamia Radoman, 1973, given their mor- phological similarities (Bodon 4 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, 78 ARCONADA & RAMOS Scientific Research Centre of Ljubljana, М 1862, leg. Dr. J. Bole). This genus has under- gone several changes in its taxonomic status. It has been regarded as a subgenus of auffenia Pollonera, 1898, and of Horatia ourguignat, 1887 (Schütt, 1961; Boeters, 974: Bodon & Giovanelli, 1994), and as а distinct genus (Bole & Velkovrh, 1986; Boeters, 1988; Bole, 1993). Neohoratia 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 & Rolán (1988) overlooked these diagnostic characters while including several species from the Iberian Peninsula in this genus (Amnicola globulus Bofill, 1909; Microna ateni Boeters, 1969; > (D TE Valvata coronadoi Bourguignat, 1870; Hauffenia (Neohoratia) coronadoi schuelei Boeters, 1981; Valvata (Tropidina) fezi Altimira, 1960; Hauffenia (Neohoratia) gasulli Boeters 1981; and Neohoratia azarum Boeters & Rolán, 1988). However, according to Boeters (1988), these Бепап species, apart from hav- ing a narrowing (‘Einschnurung’) 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 Islamia (Bodon et al., 1995; Bodon et al., 2001). /slamia 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 subpiscinalis (IBCICL paratype n° 1862). REVISION OF THE GENUS /SLAMIA 79 Peninsula (Radoman, 1973a, b, 1978, 1983); Italy (Giusti & Pezzoli, 1981; Bodon et al., 1995, 1996, 2001; Bodon & Cianfanelli, 2002); Israel (Schütt, 1991; Bodon et al., 1995); Greece (Radoman, 1973b, 1978); and France (cited as Hauffenia Pollonera, 1898) (Ber- nasconi, 1984)]. lt was thus feasible that the species listed above from the Iberian Peninsula could be attributed to the genus /slamia (type species Hydrobia valvataeformis Móllendorff, 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 al., 2000), and Spathogyna 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 (Amnicola 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 /slamia. Another species, Hauffenia (Neohoratia) coronadoi schuelei, reported as N. schuelei (in Boeters, 1988) and as /s/amia schuelei (in Bodon et al., 2001), is redescribed and placed into a new genus Milesiana, and а 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 /slamia (Schütt, 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 8 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 /slamia 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; Sa, 33, 34) or the periostracum removed Бу im- mersion in 5% sodium hypochlorite (Figs. 19, 28). Paratypes of Islamia cianensis Bodon, Manganelli, Sparacio 8 Giusti, 1995 (n° 6732), and /. gaiteri Bodon, Manganelli, Sparacio 8 Giusti, 1995 (n” 6733), from the Museo Zoologico “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 funcion 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 F test (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; Bc: FIGS. 6-10. Histological sections of the anterior female genitalia of Milesiana 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 /SLAMIA 81 bursa copulatrix; Cg: capsule gland; DBC: duct о the bursa copulatrix; Os: osphradium; P: penis; Pl: penial lobe; Po: pallial 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 ofthe 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 Zoologia, 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 Zoologico “La Specola”, Universita di Firenze, Italy; IBCICL: Slovenian Academy of Sciences and Arts, Ljubljana, Slovenia; NHMW: Naturhistorisches Museum, Wien, Austria. Collectors: К. А.: R. Araujo; B. A.: В. Arconada; J. A.: J. Astigarraga; А. B.: A. Bertrand; D. B.: D. Buckley; A. C.: A. Camacho; J. E.: J. Escobar; S. J.: S. Jiménez; N. M.: N. Martin; D. М.: D. Moreno; С. М.: С. Noreña; J. P.: J. 1. Pino; J. М. R.: J. М. Remon; J. R.: J. Roca; E. R.: Е. Rolan; С. Т.: ©. Tapia. GENITAL HISTOLOGY Histological studies of 4 um 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 /slamia globulus, 1. 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, 1969; Giusti & Selmi, 1985; Fretter & Graham, 1994: 303-306) and is morphologically re- sponsible for the whitish-pearly refringence 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 refringent) (see also Ramos et al., 2000; Bodon et al., 2001). There- fore, morphological refringence 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, |. 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 pa en A FIGS. 11-16. Histological sections of the penis and its non-glandular lobe. FIGS. 11, 12: Islamia globulus from Sopeira population; FIG. 13: Milesiana schuelei from Turrillas population; FIGS. 14, 15: Josefus 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 Type Species Islamia Radoman, 1973 Islamia valvataeformis (Móllendorff, 1873: 59) = Horatia servaini Bourguignat, 1887 (by Adriolitorea Radoman, 1973a: 234. original designation). Horatia servaini is a jun- Mienisiella Schutt, 1991: 134-136. ior synonym of Hydrobia valvataeformis Môl- REVISION OF THE GENUS /SLAMIA 83 lendorff according to Radoman, 1983, and ac- cepted by Bodon et al., 2001. Diagnosis Shell small or very small, ovoid or planispiral, rarely ovate-conic; operculum without ред; 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 (SR1); 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 copulatrix absent. Islamia globulus (Bofill, 1909) Amnicola globulus Bofill, 1909: 205; 1915: 57, 58 pl 6; пб. 6: 1917: 35. Amnicola апайпа globulus (Bofill, 1909) — Bofill 8 Haas, 1920: 50, 57, pl. Ш, figs. 19, 20. Amnicola 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, pl. 2, fig. 22; Bech, 1990: 61. Islamia globulus globulus (Bofill, 1909) — Bodon et al., 2001: 179, figs. 195-200; Bodon 8 Cianfanelli, 2002: 20. Type Locality Font del Sot del Pinell, close to Portellet del Montsech, Lérida, U.T.M.: GC16. Material Examined Type material: A lot containing 41 syntypes (dried) of A. 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- 1589a) 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. globulus I. lagari [. ateni I. pallida I. henrici henrici I. henrici giennensis M. schuelei M. cf. schuelei [= 2- ЗЕ 4- Se 6- TE $- 9- J. aitanica 200 km FIG. 17. Map of localities of the genera /slamia, Milesiana and Josefus in the Iberian Peninsula. 84 ARCONADA & RAMOS cited it from Gerona, although we cannot con- firm 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 ММСМ (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, В. 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, Gabet de la Conca, Lérida, UTM: 31TCG301658, 13 Мау 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, Gabet de La Conca, Lérida, UTM: 31TCG375567, 26 Feb. 1986, J. R., MNCN 15.05/46535 (ethanol); Freda spring, Abella de la Conca, Lérida, UTM: 31TCG473677, 10 May 1986, J. В., MNCN 15.05/46536 (ethanol), 14 March 1999, В. A., MNCN 15.05/46616 (etha- nol and frozen material); Freda spring de Casa Pallas, Arén, Lérida; UTM: 31TCG065908, 28 March 1987, J. R., MNCN 15.05/46537 (etha- nol); Bordons spring, Arén, Huesca, UTM: 31TCG085881, 31 March 1987, J. R., MNCN 15.05/46538 (ethanol); Adraén, Cadi moun- tains, Lérida, UTM.: 31TCG767817, 15 Feb. 1998, A. B., MNCN 15.05/46539 (ethanol and SEM preparation); 15 March 1999, B. A. ММСМ 15.05/46541 (ethanol and frozen ma- terial); La Figuereta spring, Alós de Balaguer, Lérida, UTM: 31TCG253439, 11 March 1986, J. В., 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, В.А. & E.R., MNCN 15.05/46543 (ethanol, SEM prepara- tion and frozen material); Grima spring, Gistaín, 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 (SR1); 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 °/, of the total shell length; proto- conch consisting of 1.5 whorls; protoconch width and width of the nucleus are 380 um and 140 рт, respectively (Figs. 30-35); protoconch pitted; umbilicus narrow, 130 um REVISION OF THE GENUS /SLAMIA 85 FIGS. 18-29. Shells of Islamia globulus. FIGS. 18, 23, 25, 27: Lectotype (MZB 80-1589a); FIG. 19: Paralectotype (MZB 80-1589b); FIG. 20: Paralectotype (ММСМ 15.05/46546); FIG. 28: Paralectotype (MZB 80-1589с); FIGS. 21, 24, 26, 29: Shells from Laguarta; FIG. 22: Shell from Sopeira population. Scale bar = 1 mm (FIGS. 18-26); 500 um (FIGS. 27-29). 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Operculum measurements (in mm) of /s/amia Iberian species. All populations from type locality except specimens of /. globulus (1) belonging to Laguarta population (Huesca). /. ateni (2), I. pallida (3), 1. В. henrici (4) and /. В. 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 (п = 9) (n = 4) (п = 2) (n = 5) (n= 1) OW 0.41 + 0.01; O47 0:02: 0332001: 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) 0.4 (n = 9) (n = 4) (n = 2) (n = 5) (n= 1) OLWL 0.41 + 0.01; 0.32 = 0.01: ОИ == 10102: 0.03 (0.43-0.40) 0.03 (0.32-0.31) 0.16 0.13 (0.20-0.15) OM (n = 4) (n = 2) (n = 1) (n = 5) (n= 1) OLWW 0.28 + 0.04; 0.20 + 0.04; OMS ECO 0.03 (0.33-0.24) 0.19 (0.22-0.17) 0.10 0.08 (0.14-0.11) 0.13 (n = 4) (п = 2) (n = 1) (n = 5) (n = 1) NL 0.33 + 0.06; OMe O102: 0.27 + 0.02; 0.20 (0.40-0.24) 0.12 (0.19-0.16) ? 0.10 (0.29-0.23) 0.2 (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 (п = 4) (n = 2) (n = 1) (n = 5) (n = 1) OL/OW 1312006: 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 (п = 4) (n = 4) (п = 2) (п=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 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 pallial cavity. Osphradium length two to three times longer than its dark, except last body whorl. width (Table 3). TABLE 3. Osphradium measurements (in mm) of several /s/amia Iberian species. All populations from type localities except for /. globulus (1-2): 1 - Gabet de la Conca (La Sarga spring), Lérida; 2 - Sopeira spring, Huesca. I. ateni (3), I. pallida (4), I. В. henrici (5) and I. В. giennensis (6). 1 2 3 4 5 6 Mean + SD: Mean + SD: Mean + SD: Mean + $0; Mean + SD; Mean + $0; CV (Мах-Мт) CV (Max-Min) CV (Max-Min) CV (Max-Min) CV (Max-Min) CV (Max-Min) (n= 2) (n = 2) (n = 13) (n = 3) (п = 4) (n = 3) ОЗ 0.19 + 0.04: 0.33 + 0.01; OMS AUDIO 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) Os W 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 um; 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 80-1589a); 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 орегсшит; FIG. 39: Transverse rows; FIG. 40: Central teeth; FIG. 41: Lateral, outer and inner marginal teeth. Scale bar = 100 рт (FIGS. 30-32); 50 um (FIGS. 33-35); 200 um (FIGS. 36-38); 10 pm (FIG. 39); 5 um (FIGS. 40, 41). REVISION OF THE GENUS /SLAMIA 89 TABLE 4. Radula formulae and measurements (in mm) of /s/amia Iberian species. /. globulus (1) from Laguarta population. /. ateni (2) and /. В. henrici (3) populations from type localities. 1 2 3 Central teeth 4+C+4/1-1 5+C+4(5)/1-1 4+C+4/2-2 Central teeth width — 9 um =i um ~5.6 um Left lateral teeth 4+C+3 6+C+3 5+:C+3 Inner marginal teeth — 24 cusps — 24 cusps — 24 cusps Outer marginal teeth ~ 6 cusps ~ 10 cusps ~ 9 cusps Radula length ~ 345 um ~ 364 um ~ 193 um Radula width OO umm ~ 59 um — 46 um Number of rows 90 102 % Male Genitalia: With bean-shaped prostate ceptacle (SR2) generally pyriform, peduncu- gland (Table 6) leaning towards the poste- rior part of the rectal loop (Fig. 45); approxi- mately ‘/; of prostate gland extending into pallial cavity; first lobes of testis spilling over onto posterior chamber of stomach and lated (Fig. 49); distal seminal receptacle (SR1) smaller, elongated, sessile; both a good distance from each other on opposite positions on renal oviduct; renal oviduct wid- ening posterior to SR2. 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 ?/; of the oviduct glands (albu- men + capsule glands) lie inside pallial 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- Discussion Until now, no lectotype of Amnicola globulus has been designated. Since 1920 (Bofill & Haas, 1920), 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 /s/amia Iberian species. All populations from type localities, except for /. globulus (1) (Sopeira spring, Huesca). /. ateni (2), I. pallida (3), I. В. henrici (4) and I. В. giennensis (5). 1 2 3 4 5 Mean + SD; Mean + SD; Mean + $0; Mean + SD; CV (Max-Min) CV (Max-Min) CV (Max-Min) CV (Max-Min) (n = 2) (n = 3) (n = 3) (n = 3) Se E 0.42 + 0.01; 027001: 0.19 + 0.02; 0.29 0127 == 0101: 0.02 (0.42-0.41) 0.02 (0.28-0.27) 0.11 (0.21-0.17) (n=1) 0.05 (0.29-0.27) $$ W 0.33 + 0.04; 0.24 + 0.01: 0.18 + 0.02; 0.28 0.210108: 0.11 (0.36-0.31) 0.04 (0.25-0.23) 0.12 (0.20-0.16) (n=1) 0.16 (0.25-0.19) Эви 0.46 + 0.17; 0.40 + 0.07; 0.29 + 0.04: 0.26 03 10102: 0.37 (0.58-0.34) 0.18 (0.45-0.32) 0.15 (0.34-0.26) (п=1) 0.07 (0.36-0.32) St W 0.47 + 0.04; ОТ = 0:01: 0.28 + 0.04; 0.19 0.28 + 0.04: 0.09 (0.50-0.44) 0.02 (0.42-0.40) 0.14 (0.31-0.23) (n = 1) 0.16 (0.33-0.24) ARCONADA & RAMOS 90 (Z =u) (<=) (P= u) (FS) (EURE ЕЕ ОЕ (| =U) (P= uy) =) (=u) у бие! 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PAIE NA Ie pre 43 wal 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 um (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, 1915; Bofill 8 Haas, 1920). We selected a lectotype from this first lot. Islamia globulus is clearly distinguished from the other /slamia species by a combination of characters. lts ovate-conic shell easily distin- guishes it from both valvatiform (I. piristoma, I. trichoniana, etc.) and trochiform species (I. anatolica, |. 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 SR1 small, elongated and sessile). Differences and similarities with /. ateni and between /. globulus and I. lagari are discussed below. Islamia lagari (Altimira, 1960) Pseudamnicola lagari Altimira, 1960: 10, fig. 2. Neohoratia globulus lagari (Altimira, 1960) — Boeters, 1988: 216, figs. 145, 146, 156, 164, pl. 2, fig. 23; Bech, 1990: 61. Islamia globulus lagari (Altimira, 1960) — Bodon et al., 2001, 43: 179, figs. 201-206; Bodon & Cianfanelli, 2002: 20. Type Locality Sot de Can Parés, Gavá, Barcelona, U.T.M.: 311DF120720(Fig: 17): Material Examined Type material: Lectotype (shell) of М. 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 /SLAMIA 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 “Pseudamnicola lagari 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 (SR1); 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 ?/, of the total shell length; protoconch consisting of 1.7 whorls; protoconch width and width of nucleus are 370 um and 130 um, respectively (Fig. 54); protoconch pitted; umbilicus narrow, about 80 um 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 I. 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 /slamia globulus and /. lagari (Boeters, 1988; Bodon et al., 2001) are based on shell shape, penis size and size and shape о 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 /. lagari (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 receptale (SR1) with a short stalk, which is not evident in /. globulus. Proximal seminal receptacle (SR2) of I. lagari is less developed than in /. globulus and has a longer and more slender stalk. The DFA also confirmed differences between the two species. We analysed nine standard shell measurements from the four /. globulus populations and from the I. lagari type mate- rial. Of the 70 individuals classified, all of the I. lagari were correctly classified (100%) and perfectly discriminated from the rest by two highly significant functions (Wilk’s lambda = 0.039, F (36.211), р < 0.0001). The remaining I. 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 /slamia species studied herein yielded similar results. All of the I. 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. 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(g-1) snjngojb “| :sarads uelaq| E/LEJS] ¡eJanas JO (ww ui) sjuawalnseau ецелиэб ajeway “2 3719V1 94 REVISION OF THE GENUS /SLAMIA 95 composition. /slamia 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 Oligocene origin and is com- posed of marls and sands on calcareous sub- Strate. At a great distance away, more than 150 km (Fig. 17), /. lagariis 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 15.05/46547). FIGS. 55, 56: Frontal view; FIGS. 57, 58: Lateral view; FIG. 59: Spire whorls; FIGS. 60, 61: Protoconch microsculpture. Scale bar = 500 um (FIGS. 55-57, 59); 200 um (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, pl. 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 BOE 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 13 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 (SR1); 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 ?/, of total shell length; protoconch pitted (Figs. 60, 61), consisting of 1.5 whorls; protoconch width and width of the nucleus are 280 um and 120 um, 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 10 lamellae (Fig. 70), occupying ‘/ of length of pallial 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 um; 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 pallial 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 (SR1), globular and sessile, smaller than proximal (SR2), which is elongated and pe- dunculated, bending towards distal portion REVISION OF THE GENUS /SLAMIA 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 /slamia 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, |. servaini, |. gaiteri, I. pusilla 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 I. ateni, never protruding from penis tip), SR2 FIGS. 62-68. Operculum and radula of /s/amia 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 pm (FIG; 65). 98 ARCONADA & RAMOS characteristically bending towards distal por- tion of renal oviduct, and the distance between seminal receptacles, which is longer in I. ateni. Islamia pallida Arconada & Ramos, n. sp. Type Specimens Holotype ММСМ 15.05/46548 (ЗЕМ prepa- ration, Fig. 78) and paratypes (Figs. 82, 85, 88, 90, 91) ММСМ 15.05/46548, 5 April 1992, D. М. & М. 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 (?) coronadoi (Bourguignat, 1870) (origi- nally Valvata coronadoi). Other localities: Province of Madrid (Fig. 17), e.g., Spring in Patones, Patones de Abajo, Madrid (type locality), 29 June 1997, B.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. 1989, A. C.; La Parra channel, Patones, Madrid, UTM.: 30TVL603241, 2 June 1996, В.А. & D. B., MNCN 15.05/46551 (ethanol). 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 um (FIGS. 69-74). REVISION OF THE GENUS /SLAMIA 99 FIGS. 76-91. Shells of Neohoratia (?) coronadoi and Islamia pallida. FIGS. 76, 80, 83, 86: Lectotype of Neohoratia (?) coronadoi (MHNG); FIGS. 77, 79, 81, 84, 87, 89: Paralectotypes of Neohoratia (?) coronadoi (MHNG); FIG. 78: Holotype of /. pallida (UNCN 15.05/46548); FIGS. 82, 85, 88, 90, 91: Paratypes of /. pallida. 100 ARCONADA & RAMOS Specimens Examined for Morphometry and Histology А! 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 um (FIGS. 92-97, 99). REVISION OF THE GENUS /SLAMIA 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 ?/, of total shell length; protoconch pitted (Fig. 91), consisting of 1.5 whorls (Fig. 90); protoconch width and width of the nucleus are 350 um 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 '/, inside pallial 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, 0, al menos, en algunos manantiales о arroyos de la provincia de Castilla La Nueva” [in Madrid’s surroundings or, at least, in some springs or streams of the New Castille 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 [аз N. (?) coronadol], because of its similari- ties to Neohoratia schuelei (sensu Boeters, 1988). 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 aphenomenon that has also been reported and discussed in another Iberian valvatiform species (Spathogyna fezi 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 /slamia 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, |. globulina, |. consolationis and I. spirata, all I. 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: AT, 51352); Islamia henrici Arconada & Ramos, n. sp. Type Specimens Holotype ММСМ 15.05/46552 (Fig. 15B) (SEM preparation) and paratypes MNCN 15.05/46552, 13 Oct. 1992, Е. R. (ethanol and SEM preparation — Figs. 106, 107, 109, 112, 1134116) 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 /SLAMIA 103 Type Locality A tributary of the Guadalora River in Parque Natural de Hornachuelos, Córdoba, U.T.M.: 30STG9788. Etymology Dedicated to Enrique Arconada, whose given name has been Latinized as Henricus. Diagnosis 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 /slamia henrici. FIGS. 104, 108, 110, 111, 114, 115: Shells of I. henrici giennensis from La lruela population; FIGS. 105-107, 109, 112, 113, 116: Shells and penis of I. henrici henrici from Hornachuelos population; FIG. 104: Holotype of /. henrici giennensis (MNCN 15.05/46555); FIG. 105: Holotype of /. henrici henrici (ММСМ 15.05/46552). Scale bar = 500 um (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 (SR1). We consider that this species has two sub- species as follows: Islamia henrici henrici Arconada 4 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, В. 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, В. А., 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 or depressed-trochiform, 3.5 whorls (Table 1; Figs. 105, 106, 112); body whorl occupying approximately */; 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 um, 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 um; 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 pallial cavity; penis very long with small non- glandular lobe located in distal position (Figs. 116, 129), showing a small refringent 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 pallial 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 (SR1) 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 /SLAMIA 105 FIGS. 117-123. Opercula and radula of Islamia henrici. FIGS. 117, 118, 120-123: Opercula and radula of /. henrici henrici from Hornachuelos population; FIG. 119: Operculum of /. henrici 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 um (FIGS. 117-119). head (Fig. 124), which is approximately six is usually simple, although it can some- times smaller than male penis, occupying times be bilobated, similar to the shape 20% of total head length. This nuchal node of the distal part of male penis. 106 ARCONADA & RAMOS Islamia henrici giennensis Arconada 8 Ramos, n. subsp. Type Specimens Holotype MNCN 15.05/46555 (SEM prepa- ration) (Fig. 104) and paratypes MNCN 15.05/ 46555 (ethanol and SEM preparation, Figs. 108,110, 1144 ¿114115 4119) Type Locality Spring facing the hotel “Sierra Cazorla”, La lruela, Cazorla 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, В.А., MNCN 15.05/46554 (ethanol); spring facing the hotel “Sierra Cazorla”, La lruela, Cazorla 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 Cazorla, Jaén, E. R., MNCN 15.05/ 46559 (ethanol); La Nava de San Pedro, Cazorla, 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- рута approximately “/; 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 um 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 pallial 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 /SLAMIA 107 124 128 131 129 E _SR2 FIGS. 124-131. Anatomy of /. henrici henrici. 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. 131: Detail of the seminal receptacles; Abbreviations in text. Scale bar = 500 um (FIGS. 124-130). 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 pallial cavity length; proximal seminal receptacle (SR2) elongated, with 108 ARCONADA & RAMOS FIGS. 132-137. Anatomy of I. henrici 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 um. swollen tip and bending 90° towards distal part of renal oviduct (Fig. 137); distal semi- nal receptacle (SR1) 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. henrici and 1. В. 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 (1. В. giennensis) populations are characterised by a slight varix near the shell aperture in most of the specimens (no varix in I. В. henrici), а short supraesophageal соппес- tive, RPG ratio = 0.14 (medium-sized in /. h. henrici, RPG ratio = 0.30), an oval osphradium (bean-shaped in /. h. henrici), a prostate elon- gated pear-shaped (bean-shaped in /. h. henrici), a penial lobe without any refringent area (a small refringent area present in /. h. henrici), a black pigmented penis (penis orangish pigmented in /. h. henrici), long and slender proximal seminal receptacle (SR2) (elongated with swollen tip in /. h. henrici), and the absence of a nuchal node in females. A nuchal node is a constant character in all fe- male specimens from Córdoba (I. h. henrici). 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 /SLAMIA 109 Root 2 Root 1 O I. globulus A |. lagari Ш / ateni D I. pallida I. В. henrici I. В. 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, |. pallida, Lhenrici henrici and I. В. 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 /s/amia 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 gaiteri (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 refrin- gent non-glandular tissue is found а 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 /slamia species. It slightly curved in /. henrici, whereas markedly so in Josefus aitanica (see below). Other differences among Iberian /slamia 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 I. pallida) of several populations. Statistical Analysis of /slamia species Conchological differences between /slamia 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 I. ateni, I. globulus, 110 ARCONADA & RAMOS I. lagari, and I. В. henrici were correctly classi- fied (100%); 62.5% of the /. pallida individuals and 85.71% of I. В. 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 (I. pallida, |. В. henrici, and I. В. 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 (SR1), 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, 1983), 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, pl. 2, fig. 26. Islamia schuelei (Boeters, 1981) — Водоп 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, BOE 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 Munchen (1967) and Claude Bou/Moulis, Albi (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 Ubeda, 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), BOE 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 Material Examined: Holotype in SMF 253578/1 (Figs. 139-142, 154). Other Populations Examined. This species was found in the provinces of Cadiz, Almeria, Granada and Malaga (Fig. 17). A population found far from its distribution range, in the Caceres province, was provisionally assigned to this species as M. cf. schuelei. The species has not been found in Teruel Province. Localities: Algodonales, Cadiz, UTM.: 30STF8584, 19 Oct. 1998, E. R., MNCN 15.05/ 46495 (ethanol); El Nacimiento spring, Turrillas, Almeria, UTM: 30SWF657975, 15 Oct. 1990, D. M., MNCN 15.05/46496 (etha- nol, SEM preparation), 10 Oct. 1992, Е. R., D. M., MNCN 15.05/46497 (ethanol); Los Minutos spring, Turrillas, Almeria, UTM: 30SWF6598, 10 Oct. 1992, D. M., М. М., MNCN 15.05/46591 (SEM preparation); Andarax spring, river and channel, Laujar de Andarax, Almería, UTM: 30SWF0994, 11 Jan. 1992, D. M., М. M.; 11 REVISION OF THE GENUS /SLAMIA 111 Oct. 1992, Е. 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); Vélez Blanco, Almería, UTM: 30SWG7972, E. R., MNCN 15.05/ 46592; Talama spring, Bayarcal, Almería, UTM: 30SWF0098, 26 March 1994, D. M., М. M. ММСМ 15.05/46500 (ethanol and SEM prepa- ration), 14 May 1994, D. M., М. M., MNCN 15.05/46501 (ethanol); El Marchal de Antón López, Almería, UTM: 30SWF3383, Е. R.; 26 March 1998, B. A., MNCN 15.05/46502 (etha- nol, SEM preparation and frozen material); Pool in Berchul, Felix, Almeria, UTM: 30SWF298813, E. R., MNCN 15.05/46503 (ethanol and SEM preparation), 26 March 1998, B. A., MNCN 15.05/46504 (ethanol and frozen material); spring near the pool in Berchul, Félix, Almeria, 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., C. A., MNCN 15.05/46506 (dried); Faldes spring, FIGS. 139-153. Shells of Milesiana schuelei. FIGS. 139-142: Holotype (SMF 253578/1); FIG. 143: Shell from Fuente del Mal Nombre, Padul (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 um. 112 Sierra Harana Granada, UTM.: 30SVG592308, 23 April 1992, D. М., ММСМ 15.05/46507 (etha- nol), 12 Oct. 1992, Е. К., D. М., ММСМ 15.05/ 46508 (ethanol); 25 March 1998, В. А. MNCN 15.05/46509 (ethanol); Los Caños spring, Graena, Granada, UTM.: 30SVG810285, 27 Sept. 1989, E.R., D. M., C.A., MNCN 15.05/ 46510 (dried, ethanol); Pilar del Mono spring, Durcal, Granada, UTM.: 30SVF493951, 25 Sept. 1989, Е. К. D. M., C. A., ММСМ 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 Padul, 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., ММСМ 15.05/46515 (ethanol, SEM ргера- ration); Mal Nombre spring, Padul, Granada, UTM.: 30SUF445963, 27 March 1998, B. A., ARCONADA & RAMOS ММСМ 15.05/46516 (ethanol and frozen ma- terial); spring т Gaucín, Malaga, UTM.: 30STF9244, 22 Nov. 1988; E. R., MNCN 15.05/46517 (ethanol, SEM preparation), 15 April 1998, В. А., ММСМ 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.: 30SUF 468824, E. R., MNCN 15.05/ 46521 (ethanol), 14 April 1998, В. 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, В. А., MNCN 15.05/46524 (etha- nol and frozen material); Avellano River, La Cimada, Malaga, U.T.M.: 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), Almería; 3 - Padul, Granada; 4 - El Chorro, Málaga; 5 - Benaoján, Málaga. 1 2 3 4 5 Mean + SD; Mean + SD; Mean + $0; Mean + SD; Mean + $0; CV (Max-Min) CV (Max-Min) CV (Max-Min) CV (Max-Min) CV (Max-Min) (n = 15) (n = 29) (n = 10) (п = 17) (п = 27) SL 075 40:05: 0.80 + 0.06; 0.68 + 0.04; 0.68 + 0.05; 0.92 + 0.10; 0.07 (0.85-0.68) 0.07 (0.94-0.65) 0.07 (0.74-0.57) 0.08 (0.80-0.57) 0.11 (1.13-0.75) SW 1.21 + 0.08; 1-27. = 0:08: 1.09 + 0.07: 1.130108: 1.32. = 0109: 0.07 (1.35-1.10) 0.06 (1.42-1.01) 0.06 (1.18-0.97) 0.07 (1.27-1.04) 0.07 (1.54-1.17) SL/SW 0.62 + 0.06; 0.62 + 0.05; 0.62 + 0.03; 0.59 + 0.04: 0.70= 0:07: 0.09 (0.73-0.51) 0.08 (0.73-0.53) 0.06 (0.70-0.57) 0.07 (0.68-0.52) 0.10 (0.87-0.58) AH 0.65 + 0.09; 0.62 + 0.04; 0.52 =.0.02: 0.53 += 0/04: 0.67 + 0.04; 0.14 (0.82-0.55) 0.07 (0.82-0.55) 0.04 (0.57-0.50) 0.07 (0.60-0.47) 0.06 (0.74-0.58) LBW 0.65 + 0.04; 0.71 + 0.05; 0.43 + 0.04; 0.60 + 0.05; 0.79 + 0.10; 0.07 (0.75-0.57) 0.07 (0.81-0.60) 0.10 (0.48-0.35) 0.09 (0.71-0.50) 0.13 (0.98-0.63) WBW 0.81 = 0:07: 0.82 + 0.05; 0.69 + 0.04; 0.72 + 0.05: OLSSON 0.08 (0.92-0.70) 0.06 (0.91-0.68) 0.06 (0.77-0.61) 0.08 (0.85-0.62) 0.12 (1.33-0.75) AL 0.52 + 0.03: 0:54 +0.03; 0.49 + 0.03; 0.49 + 0.04; 0.58 + 0/05: 0.07 (0.60-0.46) 0.06 (0.91-0.68) 0.07 (0.55-0.42) 0.09 (0.61-0.44) 0.08 (0.67-0.52) AW 0.50 +.0.07: 0.53'+ 0.02; 0.48 + 0.02: 0.49 + 0.04; 0.58 + 0.04; 0.15 (0.60-0.25) 0.05 (0.60-0.44) 0.04 (0.52-0.45) 0.09 (0.61-0.44) 0.07 (0.69-0.52) WPW 0:33+ 0.03: 0.33 + 0.04; 0.27 + 0.03; 0.27 + 0.04; 0.38 + 0.04; 0.10 (0.40-0.28) 0.13 (0.40-0.24) 0.12 (0.32-0.21) 0.17 (0.34-0.18) 0.10 (0.47-0.32) WAW 0.12 = 0.02; 0.14 + 0.02; OZ: 102015 0.09 + 0.01; 0.15 + 0.02; 0.19 (0.10-0.08) 0.18 (0.18-0.08) 0.15 (0.14-0.10) 0.15 (0.12-0.07) 0.15 (0.21-0.11) NSW 3.22 + 0.19: 3.13 + 0.14; 3.00 + 0.00; 3.02 + 0.08; 3.28 + 0.21; 0.06 (3.50-3.00) 0.04 (3.50-3.00) 0.00 (3.00-3.00) 0.02 (3.25-3.00) 0.06 (3.50-3.00) REVISION OF THE GENUS /SLAMIA 113 TABLE 9. Operculum measurements (in mm) of Milesiana schuelei from Gaucin population (Málaga). Diagnosis Shell small, planispiral or valvatiform; oper- culum circular; ctenidium well developed; pleu- Меап+$0: Mean+SD; ral-subesophageal connective absent; large CV (Max-Min) CV (Max-Min) pear-shaped prostate gland; penis slightly or completely unpigmented, with large, non-glan- OL 0.59+0.12; NL 0.24 + 0.04; dular penial lobe located in medial position: 0.21 (0730.96) 0.16 (0:27-0:17) proximal зетта! receptacle (SR2) small, (n= 5) (NES) sessile, rounded; distal seminal receptacle OW 0.47 + 0.07; NW 0.31 + 0.01; (SR1) always larger than SR2, pyriform, ре- 0.16 (0.60-0.40) 0.05 (0.34-0.29) dunculated; receptacles located very close to (n= 5) (n = 5) one another on opposite positions on renal OLWL OA OM NOLOWMA23EE 00; oviduct. 0.47 (0.36-0.11) 0.08 (1.38-1.11) (п= 5) (n = 5) Description OLWW 0.15 + 0.06; 0.42 (0.26-0.09) Shell: Planispiral or valvatiform (Figs. 139, (n = 5) 143-147, Table 8), 3-3.5 whorls (Figs. 142, M. cf. schuelel: Robladillo de Gata, Cáceres, UTM: 29TQE0764, Е. В., 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, 11) 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 Turrillas, Almería (Oct. 1990). 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 um, re- spectively; protoconch pitted (Figs. 154- 156); aperture prosocline, rounded (Figs. 143-147); umbilicus wide, approximately 240 um 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. 157); 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) of Milesiana schuelei from the following populations: 1 - Turrillas (El Nacimiento), Almeria; 2 - El Laujar de Andarax, Almería; 3 - La Cimada, Malaga; 4 - Padul, Granada; 5 - El Marchal, Almería; 6 - Lucainena de Las Torres, Almería; 7 - Gaucín, Málaga. 1 2 3 4 5 6 7 Mean + SD; Mean + SD; Mean + SD; Mean + $0; CV Mean + SD: CV (Max-Min) CV (Max-Min) CV (Max-Min) (Max-Min) CV (Max-Min) (n = 8) (n = 3) (n = 3) (п = 2) (п = 4) (п = 1) (n= 1) OsL 0.17+0.02: 0.23+0.03; 024+001; 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 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 Os W 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 um approximately; central denticle long, tapered, followed on each side by four long, tapered denticles in decreasing order of size; FIGS. 154-165. Protoconch, operculum and radula of Milesiana schuelei. FIG. 154: Holotype (SMF 253578/1); FIGS. 155, 156, 158, 161, 162: Protoconchs, operculum and radula from Gaucín (Malaga); FIGS. 159, 160, 163: Radula from Marchal de Anton Lopez (Almeria); 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 /SLAMIA (MS TABLE 11. Radula formulae and measurements capsule gland larger than albumen gland; (in mm) of Milesiana schuelei from Benaoján distal seminal receptacle (SR 1) much larger (Málaga) population. than proximal (SR2); SR1 pyriform, pedun- culated, SR2 rounded, sessile (Figs. 172, 173, Table 14), located rather close to one Formulae and another; the renal oviduct widening distally Radula characters measurements (in mm) with respect to SR2. Central teeth 4-(3.5)+C+4(3)/2-2 Central teeth width — 8 um Discussion Left lateral teeth 4-5+C+3 Inner marginal teeth — 22 cusps Milesiana schuelei cannot be assigned to the Outer marginal teeth — 10 cusps genus /slamia because of differences in sev- Radula length ~ 351 um eral diagnostic characters including some of Radula width ~ 46 um the female genitalia and principally those re- Number of rows 69 lated to the seminal receptacles. The numer- ous females studied and collected throughout different months of the year from populations lateral teeth with 3-4 denticles on each side of Almería, Granada, Málaga and Cáceres central one (Figs. 163, 164); denticles of in- provinces had a remarkably large and pedun- ner marginal teeth larger than those of outer culated distal seminal receptacle (SR1), marginal teeth (Fig. 165). whereas the proximal one (SR2) was small Male Genitalia: With pear-shaped prostate and sessile. Moreover, illustrations in Boeters gland (Fig. 169) almost two times longer than (1988: 218) depict a pedunculated distal semi- itis wide (Table 13), partially covered by rec- nal receptacle and a rounded and sessile tum in pallial cavity; penis (Figs. 170, 171) proximal receptacle apparently protruding from generally unpigmented or with a slight dark the widened part of the renal oviduct, in a po- pigmentation at base, with a blunt distal tip sition corresponding to that of the proximal and one unpigmented, big, non-glandular seminal receptacle. Both character states, a lobe located in medial position; penial duct very large and pedunculated distal seminal slightly undulating at the base, then running receptacle (SR1) and a proximal one (SR2) straight close to outer edge. small and sessile, are the opposite of those Female Genitalia: With renal oviduct making observed in /slamia (SR1 is always smaller or a narrow circle overlying the part between equal in size than SR2, and in addition SR1 is albumen and capsule glands (Fig. 172), ovi- usually sessile while SR2 is always peduncu- duct glands (albumen + capsule glands) well lated). developed, sometimes narrowing at outer Bernasconi (1975, 1977, 1984, 1985) de- edge between capsule and albumen glands; 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 Marchal, Almería. 1 2 3 4 5 6 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) (n=3) (n=4) (n=2) (n=3) (n=2) n=1 Ss 0:26 5/0/02; 0.30 + 0.02; 0:21 5002: 0.26 + 0.01; 0.21 + 006% 2026 L 0.08 (0.28-0.23) 0.07 (0.33-0.29) 0.11 (0.22-0.19) 0.05 (0.28-0.25) 0.28 (0.26-0.17) Ss 0.19 + 0.02; 0.24 + 0.04; 0.17 0103: 0:2] =0.02: 010500: 3020 W 0.14 (0.22-0.17) 0.15 (0.27-0.19) 0.18 (0.19-0.15) 0.08 (0.22-0.19) 0.08 (0.18-0.16) St 0.35 + 0.02; 0.41 + 0.03; 0.26 + 0.02; 0.36 + 0.04; 0.30 + 0.04; 0.32 L 0.07 (0.37-0.32) 0.07 (0.45-0.38) 0.09 (0.28-0.24) 0.11 (0.39-0.32) 0.15 (0.33-0.27) $1 0.26 + 0.01; 0.39 + 0.05; 0.29 + 0.04; 0.33 + 0.01; 0.25 + 0:01; 2033 W 0.04 (0.27-0.25) 0.12 (0.44-0.34) 0.13 (0.32-0.27) 0.04 (0.34-0.32) 0.02 (0.26-0.25) ARCONADA & RAMOS [de] = = (SM) (=) (=) (e =u) (£ =u) (EU) (=U) (p =u) Gel ZO'L LOL (620-€9'1) 07'0 (89`0-96`0) 210 (2190-911) 220 ($9`0-76`0) $20 (LS°0-0b'L) 68`0 yyBua] ‘GVOF LLL ЕО 180 ‘GG 0 + 160 ‘LLOFVZ0 ‘2€ 0 + +6 0 peeH/qd (MUSA (SU) (e =u) (e =u) (CE) (£ =u) (p =u) 6£ 0 420) 650 (/5`0-88`0) уго (0S0-86 0) 17’0 (25 0-/80)zcO ($9`0-с9`0) 00 (6£ 0-25 0) ¿1'0 yySua| :SLOFELO ‘470 + 990 ‘9L'0 F 02 0 :G0'0 + 990 ‘600 + 670 реэн (р) (€ =u) (£ =u) (£ = u) (p = u) LLO 9,0 (LL'O-ZL 0) 800 (EL'0-SL'0)60 0 (80'0-S1L'0) Z£ 0 (tL'0-LZ'0) 6L'0 :LO‘O F LLO ‘LOO ЕО :70`0 + LLO '£E00F 110 M ‘Id (SU) =) (=U) (£ =u) (¢=u) (p =u) £c 0 6LO (LL'O-0Z 0)2£ 0 (220-420) 100 (510-150) OF'0 (01'0-S1'0) 61'0 ‘G0'0 F tL 0 ‘00 0 + ¿ZO ‘60 0 + 25`0 ZOOFELO 1 Id MS = U) ZU) (f=) (=) ($=4) = (y =u) 910 50 20 10 (8l'o-zzo)zl'o (510-610) 820 (010-810) 820 (510-810) УГО (LL'0-91'0) 2LO ‘<0 0 + 05`0 ‘+0 0 + 9L'0 ‘y0'0 F GL'O ZOOFSLO ZOOFrL'O M d (peu) (L = (Pwr (36) GE) (g=Uu) (€ = u) (y =u) (y =u) aa) es'0 67'0 090 (£S'o-Lz L)+rO (19‘0-19`0) $20 (990-920) 210 ($5'0-19`0) 0€ 0 (6Z 0-SS'0) 67 0 ‘GEO FL 80 'ELVOFZGO :LLOFEIO УГО + 87`0 ‘€L'0 F SHO 1d (L=u) (=u) (=U) (z =u) (¢=) 910 8c 0 910 (6;'0-65`0) 670 (8L'0-22'0) 91'0 :YLOF6ZO ‘€0 0 + 0Z 0 M id (1 =u) (10) (L=u) (z =u) (= u) 320 870 6c 0 (Lv'0-S6'0) 95`0 (2ÿ'0-Et'0) Z0'0 :8£'0 F 89 0 ‘LOO €r0 74а (UIN-XEN) AD (UIN-XEN) AD (UIN-XEN) AD (UIN-XEN) AD (UIN-XEN) AD ‘as + чеэи ¿US + UB9SN ‘Gs + UE9W ‘as + Чеэи aks} + UB9/N OL 6 8 1 9 G y € С L ‘ереце.о ‘пред - OL 'ецэщу ‘S2110] SE] ap ечэшеэпл - 6 ‘емаци\у sein] ‘Huds SONNUIW $901 - 8 ‘S919929 ‘EEO эр оире!аоч - / ‘ерецело‘ецелен ‘$ 'apuels) ajuany - 9 ‘ePejen ‘ueloeusg - 6 !eßejeyy ‘ulones - y ‘ebejey ‘ерешио e7 - $ ‘емэщ\у ‘(озиэшиоем 13) ет | - Z ‘емэициу ‘jeyouey [3 - | :зэциеэо| Buimoyjo} ay] шо /9/anyos eueisajıyy jo (ww и!) syjuawanseau ецеиэб aJen ‘EL 3719VL REVISION OF THE GENUS /SLAMIA 117 French /slamia species, which later Bodon et al. (2001: 199) considered to be a misinter- pretation. Milesiana 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 SR1 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 from other Iberian /slamia species include: a flatter shell, larger umbilicus, a well- FIGS. 166-173. Anatomy of Milesiana 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: Detail ofthe seminal receptacles; Abbreviations in text. Scale bar = 500 um (FIGS. 166-172). 118 ARCONADA & RAMOS TABLE 14. Female genitalia measurements (in mm) of Milesiana schuelei from the following populations: 1 - El Marchal, Almería; 2 - Turrillas (El Nacimiento), Almería; 3 - La Cimada, Málaga; 4 - Gaucín, Málaga; 5 - El Laujar de Andarax, Almería. 1 2 3 4 5 Mean + SD: Mean + SD; Mean + SD; Mean + $0; Mean + SD; CV (Max-Min) CV (Max-Min) CV (Max-Min) CV (Max-Min) CV (Max-Min) Op L 0.72#.0:15; 0.56 = 0:13; 0.87 + 0.02; 0.76 = 0.14: 0.633.011: 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) Op W 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) (п = 2) (n = 3) (n = 4) Ag. L 0.28 + 0:07: 0.21 + 0.06; 028 0:07: 0.31 +0107; 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) SRE 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) (п = 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 Р 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 /SLAMIA 119 Josefus Arconada & Ramos, n. gen. Type species Josefus aitanica, n. Sp. Etymology In memoriam 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 айапгса refers to Sierra de Aitana, a mountain chain in the distribution area ofthis species. Populations Studied This species was found in the provinces of Valencia and Alicante (Fig. 17). Lapica spring, Las Viñuelas, Valencia, UTM.: 30SXJ7155, 28 May 1998, В. 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 15.05/46562 (ethanol), 27 May 1998, В.А. & J. A., MNCN 15.05/46563 (ethanol, SEM pre- paration and frozen material); Gamellons spring, Onteniente, Valencia, UTM.: 30SXH975942, 5 Oct. 1994, E.R., ММСМ 15.05/46564 (ethanol), 29 May 1998, B. A., MNCN 15.05/46565 (etha- nol and frozen material); Gaspar spring, Beni- ganim, Valencia, UTM.: 30SYJ2113, 5 April 1994, Е. R., ММСМ 15.05/46566 (ethanol), 29 Мау 1998, В.А. & J. A., ММСМ 15.05/46567 (ethanol and frozen material), Pi spring, Beni- ganim, Valencia, UTM.: 30SYJ2113, 5 April 1994, E. R., MNCN 15.05/46568 (ethanol); Ga- mello spring, Cuatretonda, Valencia, UTM.: 30SYJ2514, 1 April 1994, С. T., MNCN 15.05/ 46569 (ethanol and SEM preparation); La Mina source, Jarafuel, Valencia, UTM.: 30SXJ645341, 28 May 1998, В. A. 8 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 Мау 1998, В. А. & 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 Sarriá, Alicante, UTM.: 30SYH527831, 8 Dec. 1990, G. T., MNCN 15.05/46595 (ethanol); 30 May 1998, B.A. 8 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, В.А. & J. A., MNCN 15.05/ 46573 (ethanol); Molí Montes spring, Agres, Alicante, UTM.: 30SYH1595, 3 May 1994, E. R., MNCN 15.05/46574 (ethanol); Azut spring, Alfafar, Alicante, UTM.: 30SYH12394, 4 May 1994, Е. К., MNCN 15.05/46575 (ethanol), 29 May 1998, В.А. & 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 hagus making a loop to the left posterior to ral ganglion complex; pleuro-sub- >sophageal connective absent: rhomboid- shaped prostate gland; long pigmented penis ith large non-glandular lobe located in dis- rebı NO D 9 TD =) an (40) 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 Ag Shells of Josefus aitanica. 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 um (FIGS. 174-179). REVISION OF THE GENUS /SLAMIA 121 Description Shells: Valvatiform or depressed-trochiform (Table 15; Figs. 174, 175) with 3-3.5 whorls (Figs. 177, 178); about 1.5 spire whorls (Figs. 180, 181); highly developed body whorl (Figs. 177, 178); protoconch pitted (Figs. 183, 184), with 1.5 whorls; protoconch width 300 um and width of nucleus approximately 105 pm; occasional varix observed at the end of protoconch seen in all populations (Figs. 181, 182); prosocline and rounded aperture; umbilicus of intermediate size, about 125 um in diameter (Fig. 179); external lip (Figs. 176, 177) 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. 194). 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 Sarriá, Alicante; 2 - Requena (Flores spring); 3 - type locality. 1 2 3 Mean + SD; Mean + SD; Mean + SD; CV (Max-Min) CV (Max-Min) CV (Max-Min) (n = 21) (n = 10) SL 0.96 + 0.06; 1.35) 23 0817: OL 0.60 + 0.05; 0.06 (1.07-0.83) 0.13 (1.53-0.97) 0.08 (0.64-0.57) (n = 2) SW 1.08 + 0.08; ЗЕЕ OW 0.47 + 0.00; 0.08 (1.24-0.83) 0.11 (1.58-1.06) 0.01 (0.47-0.46) (ni=2) SL/SW 0.89 + 0.10; 1.01 0.0 OLWL 0.21 + 0.04; 0.11 (1.26-0.83) 0.07 (1.15-0.91) 0.21 (0.25-0.18) (n=2) AH 0.62 + 0.03; 0.84 + 0.09; OLWW OMS (0107) 0.05 (0.68-0.57) 0.11 (0.09-0.06) 0.11 (0.16-0.13) (1—2) LBW 018510105: 1.19 + 0.14; МЕ 0.27 + 0.00: 0.06 (0.94-0.70) 0.12 (1.34-0.89) 0.00 (0.27-0.27) (= 2) WBW 07332 0/05 ТОЙ = 0.12: NW 0.30 + 0.00: 0.07 (0.91-0.64) 0.11 (1.22-0.81) 0.02 (0.30-0.29) (0152) AL 0.60 + 0.03; 0.79 + 0.08; OL/OW 1#29 012; 0.05 (0.64-0.53) 0.10 (0.90-0.62) 0.09 (1.38-1.20) (eZ) AW 0.53=#:10.03; 0.68 + 0.07; О 021910108: 0.06 (0.60-0.48) 0.11 (0.78-0.54) 0.39 (0.30-0.10) (n =5) WPW 0.35 + 0.04; Os W 0.08 + 0.03; 0.11 (0.41-0.24) 0.36(0.12-0.05) (n = 5) WAW 0.14 + 0.02; 0.17 (0.21-0.08) NSW 315 OMS: 330 0516; 0.06 (3.50-3.00) 0.05 (3.50-3.00) 122 ARCONADA & RAMOS À FIGS. 185-190. Operculum and radula of Josefus aitanica. FIGS. 185, 186, 189, 190: Opercula 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 um (FIGS. 185, 186); 100 um (FIG. 187). REVISION OF THE GENUS /SLAMIA 123 1 191 => 195 196 FIGS. 191-198. Anatomy of 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 um. 124 ARCONADA & RAMOS TABLE 16. Radula formulae and measurements (in mm) of Josefus aitanica from type locality. Formulae and measurements (in mm) Radula characters Central teeth 5+C+5/2-2 Central teeth width =.6:3 Um Left lateral teeth 5+C+3 Inner marginal teeth — 22 cusps Outer marginal teeth — 24 cusps Radula length ~ 400 um Radula width ~ 43 um Number of rows — 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 Sarriá, Alicante; (c) Tabernes, Valen- cia; (d) Requena, Valencia. n=1 Ss L 0.18(a); 0.27(b); 0.26(c); 0.24(d) Ss W 0.18(а); 0.22(b); 0.14(c); 0.21(d) StL 0.36(a); 0.30(b); 0.28(c); 0.36(d) St W 0.33(a); 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 d’en Sarria, Alicante; 6 - Requena (El Tollo), Valencia; 7 - Agres, Alicante. 1 2 4 5 6 1 Mean + SD; Mean + $0; Mean + SD; Mean + SD; CV (Мах-Мт) CV (Max-Min) CV (Max-Min) CV (Max-Min) Pre 02005: 0.07 (0.39-0.36) 0.44 (n = 2) (=) Pr W 0.19 + 0.03; 0.16 (0.21-0.17) 0.22 (п= 2) (п-т) РЕ 0.64 + 0.24; 0.730. 12: 0.93 + 0.11; 0.70 + 0.04; 0.37 (0.94-0.34) 0.16 (0.81-0.65) 0.12 (1.03-0.81) 0.05 (0.72-0.66) 0.39 0.63 1.01 (n=5) (n=2) (n=3) (n=3) (n=1) (n=1) (n=1) PW 0.17 + 0.06; 0210100: 0.18 + 0.02; 0:17 = 0.02: 0.33 (0.24-0.10) 0.01 (0.12-0.12) 0.12 (0.21-0.16) 0.13 (0.19-0.15) 0.15 022 (n = 5) (m= 2) (n = 3) (0572) = Ш) PILE OMG 0:05: 012 20103; OME 0102: 04210102: 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=1) (n=1) (n=1) (n=5) (n = 2) (n = 3) (n = 3) PI.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) 0.09 0.10 0.12 (n = 5) (n = 2) (n= 3) (n = 3) (y=) at) Head 0.57 + 0.13; 0.74 + 0.05; 0.59 +0.06; length 0.23 (0.75-0.46) 0.66 (п=1) 0.07 (0.77-0.70) 0.10 (0.66-0.54) 0.57 046 0.81 (n = 5) (n = 2) (n = 3) nz an) РЕ) 0.87 + 0.52; 135-002 1.192.048; Head 0.14 (1.57-0.14) 0.98(n=1) 0.01 (1.36-1.33) 0.15 (1.35-1.00) 0.69 1.37 1.25 length (n=5) (n=2) (n = 3) ЕП м-р REVISION OF THE GENUS /SLAMIA 125 TABLE 19. Female genitalia measurements (in mm) of Josefus aitanica from the following populations: 1 - Torremanzanas, Alicante (type locality); 2 - Callosa d'en Sarriá, Alicante; 3 - Requena (Flores spring), Valencia; 4 - Tabernes, Valencia. 1 2 3 4 Mean + SD: Mean + SD: Mean + SD: Mean + SD: CV (Max-Min) CV (Max-Min) CV (Max-Min) CV (Max-Min) Op L 0.58 + 0.09; 0.65 + 0.10; 0.15 (0.70-0.47) 0.64 (п = 1) 0.15 (0.72-0.58) 0.64 (п = 1) (n= 5) (n = 2) Op W 0:20'= 0103: 027210104: 0.15 (0.16-0.15) 0.26 (n= 1) 0.14 (0.30-0.24) 0.28 (п = 1) (n=5) (n=2) Ag. L 0.24 + 0.02; 0.26 (п = 1) 0.34 (п = 1) 0.09 (0.26-0.22) 0.37 (n= 1) (132) Са. L 0420412 0.36 (n = 1) 0.30 (n = 1) 0.29 (0.50-0.33) 0.27 (n=1) (n=2) SR1L 0.08 + 0.02; 0.05 + 0.01; 0.08 + 0.02; 0.07 + 0.01; 0.19 (0.10-0.07) 0.16 (0.05-0.04) 0.18 (0.10-0.07) 0.08 (0.07-0.06) (n = 3) (n = 2) (n = 2) (n = 2) SR? 0.05 + 0.01; 0.06 + 0.01; 0.05 (п = 1) 0.16 (0.05-0.04) 0.13 (0.06-0.05) 0.06 (n = 1) (n = 2) (n = 2) 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. 198); renal oviduct not widening posteriorly to SR2 and makes a tight circle over pallial 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 pallial oviduct; ovary overlying posterior chamber of stomach. Unpigmented nuchal node (Fig. 191) in an analogous position to that of pe- nis, occupying '/, of total head length, 0.14 um 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 /slamia. 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 I. 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, /slamia globulus populations are well conserved, since water resources are sufficient in its distribution area. /slamia 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 I. pallida, I. henrici henrici and I. 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 I. h. giennensis. The species has disappeared from some ofthe 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. /slamia lagar is restricted to a very small area (Sierra de Can Parés), al- though no live specimens have been collected for years. Following IUCN criteria we classify these species as follows: Extinct (EX) — /slamia ateni; Critically Endangered (CR) — /slamia pallida, |. lagari and both subspecies of /. henrici as; Lower Risk (LR) — /slamia 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, 1970; Bernasconi, 1975). Histological observations and other direct morphological evidence have clarified many previous doubts regarding these structures. Pearly-whitish refringence 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 refringent. The location of the sac-like struc- tures in relation to the ovary and the pallial 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 et al., 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 /slamia ateni, I. globulus, and I. lagari have two seminal receptacles. Histological evidence and morphological observation of the female genitalia of Milesiana schuelei, Islamia globulus, |. 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 /SLAMIA 127 that the female genitalia of Neohoratia subpiscinalis (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, М. д. lagari, М. ateni) and ascribe them to /s/amia, following previous papers (Bodon et al., 2001). Without providing real histological evidence (serial sections), some authors have inter- preted the refringent area, or “banda traslucida”, in the penial lobe of /s/amia 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: 134) 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 /s/amia 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 refringence 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 refringence 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 refringence 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 refringent 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 /slamia Radoman (1973a) introduced the genus Islamia (type species: Horatia servaini Bour- guignat, 1887, a junior synonym of Hydrobia valvataeformis Môllendorf, 1873, according to Radoman, 1983, from Vrelo Bosne, near Sarajevo), with two subgenera, /slamia and Adriolitorea (type species: /. (Adriolitorea) zermanica Radoman 1973, from the Zrmanja River, in the middle freshwater section). Each subgenus contained two species from the Balkans: /. (/slamia) servaini (Bourguignat, 1887), I. (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 (“Bien que l'anatomie de toutes ces espéces 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 |. апаюйса Radoman 1973), plus one previ- ously described species /. burnabasa (syn. Horatia burnabasa Schútt, 1964). The last three live in sympatry (type locality: Kirkgóz, 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 /slamia. 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 /slamia. The subgenus Adriolitorea was, therefore, regarded as a syn- onym of Islamia. According to Radoman (1973a, 1983) /slamia 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 (rs1 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, 1983: 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 mention 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. п other words, only conchological and penial characters differ among /slamia species. The tenth species assigned to /s/amia 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 “/slamia sp. forma А”, “forma В”, and “forma С” 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), /s/amia in- cludes 19 species to date, in addition to those of Spain. In this paper, the authors considered Mienisiella Schütt, 1991, to be а junior syn- onym of /s/amia, thus expanding the distribu- tion area of the genus to Lebanon - /. gaillardoti (Germain, 1911) — and to Israel — /. mienisi (Schütt, 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 /slamia 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 /s/amia 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 et al., 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 /slamia” (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 I. 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 (l. minuta, I. consolationis, I. globulina, 1. 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, I. gaiteri, is an ex- ception to this hypothesis, because it has two very closely adjacent seminal receptacles (as in most /slamia species), а 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., 1995: 51, figs. 20, 24-27). None of the Бепап 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 cline 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 (I. globulus, |. lagari and I. 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, 1973). 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 19% were derived from the digestive sys- REVISION OF THE GENUS /SLAMIA 129 tem and 4% from the nervous system. Apart from the distance between seminal receptacles, other female genitalia characters of Iberian /s/amia species also differ greatly, such аз 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 т- sertion point of the seminal receptacles. Both receptacles emerge on opposite sides of the renal oviduct in all known /slamia 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 /s/amia 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 pallial 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 I. globulus, I. ateni, and /. h. gienensis and absent in I. pallida and |. В. henrici. The same situation was reported for Italian species: while /. cianensis and I. piristoma Bodon & Cianfanelli, 2002, show a slight narrowing in the transition area between the two oviduct glands, /. pusilla and I. gaiteri lack this character (Giusti et al., 1981; Bodon et al., 1995; 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. /slamia В. henrici and I. pallida are the only known Islamia taxa 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 /. henrici 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), 1. pallida (0.42) and /. В. henrici (0.30), and con- centrated in /. h. gienensis (0.14), M. schuelei (0.24) and J. aitanica (0.22). The shells of the /s/amia species known to date (Radoman, 1973a, b, 1983; Giusti & Pezzoli, 1981; Schütt, 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). /slamia pallida and I. henrici also have planispiral or valvatiform shells, whereas shells of /. globulus, I. lagari 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 /s/amia, which reinforces the need to review a number of species described from different sites in Europe and Turkey and assigned to /slamia on the basis of shell characters (Bodon et al., 2001). This would probably lead to the con- clusion that /s/amia 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. 25C). 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: A/zoniella 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, 1973 (Wilke et al., 2001). Neverthess, important differences in morpho- logical character and character states clearly distinguish them from each other: A/zoniella 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 (МММ, 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. 1. Pino, J. M. Remón, J. Roca, E. Rolán and G. Tapia for providing us with field samples. J. Bedoya (+) from the MNCN prepared the SEM photomi- crographs. Drawings were re-done by |. Díaz Cortaberría. 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). 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SZAROWSKA, 2001, Molecular systematics of Hydrobiidae (Mol- lusca: Gastropoda: Rissooidea): testing mono- phyly and phylogenetic relationships. Proceedings of the Academy of Natural Sci- ences of Philadelphia, 151: 1-21. 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 К. Wethington? ABSTRACT We report an analysis of gene frequencies at 7 polymorphic allozyme-encoding loci in 16 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 Aplexa hypnorum together, five popula- tions of Physa acuta together, and nine populations of P. gyrina, P. sayii, and P. parkeri 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, Р sayii, and P. parkeri 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 Te 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, Physella, allozyme polymorphism, protein electro- phoresis. INTRODUCTION The freshwater pulmonate 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. Te (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 Physella, 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), Turgeon et al. (1998), and many others. A wealth of data regarding genetic relation- ships among the North American physids has accumulated in the 25 years since Te 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, U.S.A.; dillonr@cofc.edu ¿Science Department, Chowan College, 200 Jones Drive, Murfreesboro, North Carolina 27855, U.S.A. 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 8 Lydeard, in press). The physid fauna of Michigan includes three nominal species sharing the “type B” penial morphology, Physa gyrina, P. sayii, and P. parkeri, 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. parkeri 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 Р 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 Р. parkeri, elliptical or elongate-ovate P. gyrina, ovate thin P. sayii, and ovate thick Р. 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 “Р 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 (1992). Multiple buffer sys- tems were employed where possible to screen for hidden variation (Coyne 8 Felton, 1978). The AP6 buffer system of Clayton & Tretiak О 100 km FIG. 1. Outline map of the state of Michigan, show- ing sample sites. A= Aplexa hypnorum, G = Physa gyrina, | = Physa acuta, P = Physa parkeri, S = Physa зауй. 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 Ш of Shaw & Prasad, 1970) was used to analyze LAP, 6PGD, and the esterases (EST3). Our initial runs included control samples of the well-characterized Р acuta population in- habiting the main pond at Charles Towne Landing State Park, Charleston, South Caro- lina (population C 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 C to 100. Mendelian interpretation has | ст been confirmed for EST3 and ГАР 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). FIG. 2. Exemplar shells of the five physid species examined in this study. | - Physa acuta (population 11), $ — Physa sayii (population $1), © — Physa gyrina (population G1), A— Aplexa hypnorum (popula- tion A2), P — Physa parkeri (population P1). See appendix for locality data. 136 DILLON & WETHINGTON Chord Distance о. 0.8 0.6 0.4 0.2 14 13 15 12 И 0.090 | 0.033| 0.000 ( 0.033 G1 0 089 | 0.033] o 000 | 0.000 | 0.033 G4 0.091} 0.034} 0000! 0.000! 0.034 G3 0.079 | 0.029 | 0.000 | 0.000 | 0.029 0.752 | 0.872 G2 0.092| 0.046| 0.019] 0.017| 0.046 | 0.715 0.767 | 0.851 | 0.887 P3 0.087| 0.032 | o. 000 “0.000 0.032 | 0.822| 0.800 | 0.917 | 0.974 | 0.957 P2 0.087| 0.032] 0.000! 0.000! 0.032 | 0.786 | 0.792 | 0.881 | 0.939 0.977 | 0.974 $1 0081| 0.030 | 0.000 | 0.000 | 0.030 | 0.719| 0.742 | 0.827 | 0.874 | 0.973 | 0.923 .984 S2 0.082| 0.030| 0.000| 0.000 0.030 | 0.733 | 0.729 | 0.848 | 0.838 .256| 0.897 | 0.991 | P1 0.006 0.008 | 0.007 0.007 | 0.008 0.081 | 0117 | 0.074 | 0.082 | 0.048 | 0.050 | 0.058 | 0.053 | 0.055 y A2 0.000} 0.000! 0000! 0.000! 0.000 | 0.095| 0.127 | 0.089 | 0.066 | 0.060 | 0.064 | 0.072 | 0.067 | 0.069 E A1 FIG. 3. Nei’s (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. > 01 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 P gyrina, Р $ауй, and Р parkeri 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 parkeri 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. Ofthe 16 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. FIG. 4. Neighbor-joining tree (PAUP*; Swofford 1998) based on the matrix of Cavalli-Sforza 8 Edwards (1967) chord distance. 137 GENETICS OF MICHIGAN PHYSIDS (sanunuo9) 0000 500 5500 0000 0100 0000 0000 0000 0000 0000 €680 1150 0000 1550 8/70 0080 98 1860 3560 8960 000! 0660 0000 0000 0000 0000 0000 0000 €820 0001 $790 0810 56/10 06 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 c6 5100 0500 0000 0000 0000 0000 0000 0000 0000 0000 Z0L0 0000 0000 0000 8750 SzZEO v6 0000 0000 0000 0000 0000 6100 0S20 9500 0000 0000 0000 0000 0000 0000 0000 0000 G6 0000 0000 0000 0000 0000 5760 05/0 7960 0001 000! 0000 0000 0000 0000 0000 0000 001 a949 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0001 0001 c8 0000 $5900 7/00 Cÿ00 0100 0000 0000 0000 0000 0000 1250 6620 0000 050 0000 0000 88 000 8560 9260 8560 0660 0000 0000 0000 0000 0000 6/90 L//0 000+ 0580 0000 0000 06 0000 0000 0000 0000 0000 0000 0000 8£00 0000 0000 0000 0000 0000 0000 0000 0000 сор 0000 0000 0000 0000 0000 8080 38550 8690 51/0 880 0000 0000 0000 0000 0000 0000 SOL 0000 0000 0000 0000 0000 C6LO 2290 920 8820 €180 0000 0000 0000 0000 0000 0000 001 dv] 0000 1700 0000 8100 0000 0000 0000 0000 0000 0000 0000 0000 0000 1590 0000 0000 v8 6cLO €c00 ЧЕГО 5510 $5750 0000 0000 0000 0000 0000 6520 0E90 0000 6/50 0000 0000 18 1180 6780 6580 41580 1190 0000 0000 0000 0000 0000 17/0 1120 2860 0000 0000 0000 68 0000 0000 0000 0000 0000 0000 0000 0000 ¿900 2600 0000 0000 0000 0000 0000 0000 06 0000 1800 0000 0000 9700 0000 0000 0000 0000 0000 0000 2510 8100 0000 0000 0000 L6 0000 0000 0000 0000 0000 920 0000 2200 €€60 6e80 0000 0000 0000 0000 0000 0000 v6 0000 0000 0000 0000 0000 5750 +980 €950 0000 S900 0000 0000 0000 0000 0000 0000 96 0000 0000 0000 0000 0000 6/50 9570 0 0000 0000 0000 0000 0000 0000 0000 0000 001 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0001 000'L vol 5153 ee AAA AAA AAA A A AAA Е Е ЕР we ее ee E eee cS LS €d са Ld S| vl el cl И тЭ $9 cd LD СУ LV Sally EEE ‘UeBIUOIN шоз sjleus pisAud jo suonejndod 9} ui 1901 эшАхиа oiydiowAjod uanas je Salouanbal} aus) | FIGVL DILLON & WETHINGTON 138 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0001 0001 96 0000 $550 95960 150 0000 0000 0000 0000 0000 0000 01990 0050 0000 0000 0000 0000 v6 ÿ900 9620 LILO 41790 0000 0000 0000 0000 0000 0000 ¿¿Z0 8/ÿ0 C860 0001 0000 0000 86 0000 0000 0000 0000 0000 000! 000! 000! 0001 0001 0000 0000 0000 0000 0000 0000 001 9560 1/90 8290 6ÿLO 0001 0000 0000 0000 0000 0000 9010 2200 8100 0000 0000 0000 cOL 1949 9100 0000 8050 2/00 0000 0000 0000 0000 0000 0000 21100 0000 0000 0000 0000 0000 c8 7860 cr60 9190 88/0 000! 0000 0000 0000 0000 0000 €860 000! 0001 0001 9020 0SCO 58 0000 8500 1/00 5510 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 06 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 76/0 05/0 c6 0000 0000 0000 0000 0000 1750 76/0 6850 0000 6090 0000 0000 0000 0000 0000 0000 v6 0000 0000 0000 0000 0000 8950 9210 2090 0001 1650 0000 0000 0000 0000 0000 0000 001 0000 0000 0000 0000 0000 1600 6200 6000 0000 0000 0000 0000 0000 0000 0000 0000 vor HSI 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0001 0001 86 0000 0000 0000 0000 0000 9020 6290 $5080 70/0 0000 0000 0000 0000 0000 0000 0000 001 0000 0000 0000 0000 0000 76/0 1/50 610 9620 000! 0000 0000 0000 0000 0000 0000 $01 0000 2200 $800 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 OIL 0001 8/60 ¿960 0001, 000! 0000 0000 0000 0000 0000 0001 0001 000! 9690 0000 0000 all 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 $7050 0000 0000 рр €CWOd 0000 0000 0000 0000 0000 0000 2200 8200 9100 2100 0000 0000 0000 0000 0000 0000 96 0000 0000 9600 0000 0000 0001 1250 2080 1860 2960 0000 0000 0000 0000 0000 0000 00! 0001, 000! +060 000! 000! 0000 9770 0170 0000 41100 0001, 0001, 000! 000! 0000 0000 vol 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 000! 0001 OLL IdW cS LS ed са ld SI vl el cl И тЭ $9 cd LS cv LV 91911V (panunuos) GENETICS OF MICHIGAN PHYSIDS 139 Figure 3 shows the matrix of Neïs 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, 1962) for this analysis was very high, гс = 0.993 (Sneath 8 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 8 Dillon, 1993, 1996, 1997). Evidence of in- breeding has nevertheless often been reported in natural populations of Physa (Dillon 8 Wethington, 1995; 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. parkeri 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/sayli/parkeri group, clustered at a chord distance of 0.43 with a minimum genetic identity of 0.715. The specific distinction be- tween Р gyrina, P. sayii, and P. parkeri, 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, Te reported populations from a wide variety of shallow habitats — creeks, brooks, pools, ponds, and ditches. The ranges of Physa sayii and P. parkeri are more re- stricted to the Great Lakes region and to deeper waters, Te 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 Р 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. parkeri (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 predation 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. parkeri from Douglas Lake and P. gyrina collected from its type locality near Council Bluffs, Iowa. Our control P parkeri 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 parkeri hybridized readily with 140 DILLON & WETHINGTON P. gyrina, producing viable F1 offspring. The growth, survival rate, and fecundity of P. parkeri were, however, significantly below those posted by control P. gyrina, in both the control pairs and in the outcross parkeri 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 8 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 8 Lydeard also re- solved two clades within the gyrina group: a “typical” subset and a “globose” subset that included parkeri and sayii (subspecies magna- lacustris.) 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 Р 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 hypnorum (tryoni and hypnorum S.s.), Physa jennessi (subspecies skinneri), Physa gyrina (elliptica, hildrethiana, and gyrina s.s.), Physa зауй (magnalacustris, vinosa, and sayii s.s.), Physa parkeri (latchfordii and parkeri s.s.), and Physa integra (brevispira, walkeri, and integra s.s.). Including Р 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 Te’s analysis was based on a set of taxonomic units divided much more finely than biological species. 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Dissertation, University of Alabama, Tuscaloosa, Alabama. 119 pp. WETHINGTON, А. К. & К. Т. DILLON, JR., 1991, Sperm storage and evidence for multiple in- 142 semination in a natural population of the fresh- water snail, Physa. American Malacological Bulletin, 9: 99-102. WETHINGTON, А. Б. & К. T. DILLON, JR., 1993, Reproductive development in the hermaphro- ditic freshwater snail, Physa, monitored with complementing albino lines. Proceedings of the Royal Society of London (B), 252: 109-114. WETHINGTON,A. Б. & К. T. DILLON, JR., 1996, Gender choice and gender conflict in a non- reciprocally mating simultaneous hermaphro- dite, the freshwater snail, Physa. Animal Behaviour, 51: 1107-1118. М/ЕТНИМСТОМ, А. К. & К. T. DILLON, JR., 1997, Selfing, outcrossing, and mixed mating in the freshwater snail Physa heterostropha: lifetime fitness and inbreeding depression. Invertebrate Biology, 116: 192-199. WETHINGTON, А. R. & К. GURALNICK, 2004, Are populations of physids from different hot springs distinctive lineages? American Mala- cological Bulletin, 19: 135-144. WETHINGTON, А. К. 8 С. LYDEARD, in press, A molecular phylogeny of Physidae (Gastropoda: Basommatophora) based on mitochondrial DNA sequences. Journal of Molluscan Studies. WETHINGTON, А. R., J. M. RHETT & R. T. DILLON, JR., in prep., Allozyme, 16$, and CO1 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 Ареха 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 $ of town of Little Lake, G2 G3 G4 Pal P2 P3 S1 S2 DILLON & WETHINGTON Marquette Co., Michigan. 46.2815°N, 87.3337°W. М = 31. Physa gyrina. Little Carp River at Hogsback Rd., 1 km М of Burt Lake, Cheyboygan Co., Michigan. 45.5520°N, 84.6854°W. М = 28. Physa gyrina. Turtle Lake at Miller Rd., 5 km W of Bendon, Benzie Co., Michigan. 44.6178°N, 85.9090°W. М = 24. Physa gyrina. Twin Sun Lakes at Highgate Beach, Wixom, Oakland Co., Michigan. 42.5466°N, 83.5085°W. N = 33. Physa acuta. Douglas Lake at the Uni- versity of Michigan Biological Station, Cheboygan Co., Michigan. 45.5634°N, 84.6783°W. N = 32. Physa acuta. Higgins Lake near boat ramp at Sam O Set Blvd., Sharps Corners, Roscommon Co., Michigan. 44.4246°N, 84.6942°W. N = 31. Physa acuta. Saginaw Bay at Quani- cassee Wildlife Area, Tuscola Co., Michi- gan. 43.5896°N, 83.6774°W. N = 57. Physa acuta. Pond near the junction of Mi 11 and Mi 37, Grand Rapids , Kent Co., Michigan. 42.9168°N, 85.5771°W. N = 44. Physa acuta. Kent Lake at Kensington MetroPark, Oakland Co., Michigan. 42.5336°N, 83.6462°W. N = 29. Physa parkeri. Douglas Lake at the Uni- versity of Michigan Biological Station, Cheboygan Co., Michigan. 45.5634°N, 84.6783°W. N = 59. 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. Physa parkeri. Higgins Lake near boat ramp at Sam O Set Blvd., Sharps Corners, Roscommon Co., Michigan. 44.4246°N, 84.6942°W. N = 47. Physa sayii. Lake Michigan at Wilderness State Park, Emmet Co., Michigan. 45.7474°N, 84.9045°W. N = 49. 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 Ош? & 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 | (COI) 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 (COI 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 COI sequences are fully protein-coding with no insertions or deletions, (2) both individual and combined analyses of the COI 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. On 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 ©. h. robertsoni. Key words: 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 “The George Washington University Medical Center, Department of Microbiology and Tropical Medicine, Washington, DC, U.S.A.; mtmgmd@gwumc.edu 3Sichuan Institute of Parasitic Disease, Chengdu, Sichuan, Р. К. China “Center for Occupational and Environmental Health, School of Public Health, University of California, Berkeley, California, U.S.A. *Corresponding author 143 144 WILKE ET AL. 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- nía 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. В. hupensis from 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 c oxi- dase subunit | (СО!) gene (GenBank accession numbers AF213339, AF253075, AF253076, AF531547), two sequences (AF253075 and AF213339) differ by K2P (Kimura 2 parameter) distances of 0.0932. To give a comparison, the highest pairwise K2P distance among more than 100 COI 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. robertsoni 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 СО! gene, we study the mitochondrial gene for large subunit ribosomal 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). MATERIALS AND 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, À = Anning River Valley, Y = Yunnan). Locality Original Latitude No. specimens code locality # Longitude Locality studied M 1 D 98.16 29.99163 °N Sichuan, Danling County, Ernming 2 103.41580 °Е Township, Xiaogiao Village M2 D 98.14 30.13993 °N Sichuan, Dongpo County, Panao 10 103161167 E Township, Magau Group 2 Village M3 MG 96.18 30.0373 °N Sichuan, Meishan County, Fusheng 5 103.9002 *E Township, Zhongfu Village M 4 - 30.067 °М Sichuan, Mianzhu A” 104.138 °E A1 D 98.04 27.93525 °N Sichuan, Xichang County, Xixiang 4 102.20540 *E Township, Gucheng Village A2 D 98.03 27.9318 °М Sichuan, Xichang County, Xxixiang 4 102.1962 "E Township, Gucheng Village A3 D 98.12 27.87505 °М Sichuan, Xichang County, Chaunxing 4 102.30867 *E Township, Minhe group 2 Village A4 D 98.09 27.8000 °М Sichuan, Xichang County, Jingjiu 4 102204 Е Township, Zhoutun Village A5 D 98.07 27.7995 °М Sichuan, Xichang County, Натап 4 102.3087 *E Township, Gucheng group 2 Village A 6 D 98.05 27.7973 °N Sichuan, Xichang County, Hainan 4 102307 Е Township, Gucheng group 5 Village A7 D 98.11 27.7468 °М Sichuan, Xichang County, Jingjiu 4 102.1903 °E Township, Jingjiu Village A8 Xi Chang** 26.9637 °N Sichuan, Miyi County, Panlian 12 102.18328 TE Township, Shuanggou Village Vail Dali** 25.4510 °N Yunnan, Dali City, Da Jin Ping, Zi 8 100.2007 °E 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: О. В. hupensis (Gredler, 1881) (GenBank AF254547, DQ212859), O. h. tangi (Bartsch, 1936) (Gen- Bank DQ212796, DQ212860), O. h. formosana (Pilsbry & Hirasé, 1905) (GenBank DQ112283, DQ212861), and O. h. quadrasi (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 10 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 ul Turner lysis buffer (0.02 M Tris base, 0.1 M EDTA, 0.5% Sarkosyl, pH 8.0) and 3 ul of 20 ug/ul Proteinase К. After digestion, 35 pl of 5M NaCl and 35 ul of a 5% CTAB/0.5 М NaCl solu- tion were added. Extraction was carried out with 270 ul chloroform. After centrifugation for 5 min at 9,000 rpm, the aqueous phase was trans- ferred into a new tube and 270 ul of CTAB pre- cipitation buffer (1% CTAB, 0.05 M Tris base, 0.01 M EDTA) was added, mixed and placed at room temperature for 45 min. After pelleting the CTAB-DNA for 10 min at 12,000 rpm, the supernatant was disposed and the pellet redis- solved in 100 ul of МаСИТЕ (0.01 Tris base, 146 WILKE ET AL. 0.001 M EDTA, 1 M NaCl, рн 8.0) and 1 ul 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 ul of ice-cold 96% etha- nol. After centrifugation for 15 min at 12,000 rpm, the pellet was washed twice with 300 ul of ice-cold 70% ethanol, air-dried for 5-10 min and finally redissolved in approximately 50 pl H,0. 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 primer sequence) were LCO1490 and HCO2198 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 primer se- quence) were 16Sar-L and 16Sbr-H of Palumbi etal. (1991). Sequences (forward and reverse) were determined using the LI-COR (Lincoln, МЕ) 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 first and last ten bp of each sequence, leaving a 638 bp-long completely TABLE 2: AFLP primers. 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 rRNAis 510 bp. А! 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 Msel (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 Msel 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 Msel each with the two IRDye-la- beled EcoRI primers; Table 2). A total of 12 primer combination was used for the PCR. Primer Sequence Adapters EcoRI 5'-CTC СТА САС TGC СТА СС-САТ СТС ACG CAT GGT TAA-3' Msel 5'-GAC GAT GAG TCC TGA G-TA CTC AGG АСТ CAT-3' Pre-amplification primers E01 E-A (EcoRI) 5'-GAC TGC GTA CCA ATT CA-3' M02 M-C (Msel) 5'-САТ GAG TCC TGA GTA AA-3’ Selective amplification primers 700 E-AAC 5'-IRD700-GAC TGC GTA CCA ATT CAA C-3’ 800 E-AAG 5'-IRD800-GAC ТСС GTA CCA ATT CAA G-3' M-CGA 5'-GAT GAG TCC TGA GTA AGG A-3' M-CTT 5'-САТ САС TCC TGA СТА ACT T-3' M-CTC 5'-САТ GAG TCC TGA GTA ACT C-3' M-CAT 5'-САТ GAG TCC TGA GTA ACA T-3' M-CTA 5'-GAT GAG TCC TGA GTA АСТ A-3' M-CTG 5'-GAT GAG TCC TGA СТА 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 COI 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; 10,000 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 8 Xie, 2001) to test for saturation prior to the phylogenetic analyses. The Xia et al. test did not reveal a significant degree of saturation (lss = 0.301, Igs.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 8 Servedio, 1998; Kolaczkowski 8 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, 100 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 о the —In likelihoods of the trees. The -log likelihoods started at around —8,100 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. A50% majority rule tree was constructed from all sampled trees with the first 10,000 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 8 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. 4.0610 under the con- straint that О. h. robertsoni is NOT monophyl- etic (null hypothesis). The resulting tree was, together with the aligned sequences, imported into Seq-Gen 1.2.5. (Rambaut & Grassly, 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 ET AL. О. В. 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 сот- puting a minimum spanning network (MSN) among genotypes in a preliminary assessment of genetic structure in our data set. А matrix of corrected average pairwise dif- ferences between Oncomelania h. hupensis and О. h. robertsoni аз well as within О. В. 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. robertsoni 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 |, Па, ПБ, and Ист Table 3 and Fig. 1). The divergences among these groups range from 0.042 + 0.006 (between groups lla and Ис) to 0.085 + 0.010 (between groups | and IIc). п 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. h. quadrasi). It should be noted that т 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 BI 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 rRNA data 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 СО! data set resulted in trees that showed the four major groups of O. h. robertsoni to be monophyletic, in the LSU rRNA data set the four groups were either paraphyletic (Bl analy- sis: clade | 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. | На ИБ Ис | 0.018 + 0.003 lla 0.083 + 0.009 0.011 + 0.002 Ib 0.085+0.009 0.043 + 0.006 0.005 + 0.001 Ic 0.085 +0.010 0.042+0.006 0.043 +0.007 0.010 + 0.003 149 EXTREME SEQUENCE DIVERSITY IN ONCOMELANIA H. ROBERTSONI ‘xipuaddy au} 988 sapos |еприл!ри! 104 ‘Jo19S0od e ээд eu} шо} рэлошэл sem “ds ena, ‘uewuiseds dnolbjno Алеша ayy “(easy ueysIen = pas ‘Аэнел Jenıy бишиу = эта ‘чеципл = чээ.6) рэроэ JOJO эле sapos |еприлири! BuIpuodsa1109 pue зеэле эцаебоэо ‘dew au} чо UMOUS SI SAPejo asay) jo иоцпащер эчаелбоэб eu ‘эп ‘аи ‘ell ‘| Paleqe| эле /UOS/8q01 “y ‘O UI sepejo Jolew 1n0} ay | ‘эрео yea Jo, рар!ло.4 эле зэци!аедола 1011104 ‘ренаае uonnjons ээцэпЬэ$ jo |эрош eu} о} BuIp1099e ais sad зиоцп}зап$ au} sJusseidel Jeg ajeos ay] ‘perdues saibojodo] jo snsuasuos эп.-АзоГеш 0.09 au} Bumous зэиэб YN YA NST PUE 109 Ay} yO зиэшбел рэшашоэ jo dq gp, ‘| чо paseq ‘dss sisuadny еиеэшозио 10} weiBojAud ие!зэлея "| 914 ec wan Pe IN ‘GE W ‘eg IN 550 SAN af er и [66`0 Ly tw ам ‘че IZ ZN ez м \ PZ,IN УБЕ ‘az IN TOOL 5 az uw (27 ABN Be y jay og vee v y ue 00 L À 58 м 4 dex L ¿PPM PP voor Г. oyw apes lazo j PLV V 2 Y eo lot DL Y PH v.EL val u | 00'E ЗЕ: 00-1 E ot Z OVA“PLA OL A ‘GL A FOOL 1.17 Thu 4-03 $ sisuadny ‘y ‘O0 | РиЕ50Ш/0} ‘y O, sespenb”y "O y ешииш вшеошюэио 00 E 150 WILKE ET AL. four subspecies of О. hupensis) or unresolved (MP analysis). We then combined the two data sets and per- formed several phylogenetic analyses using MP and BI. 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 BI could not fully resolve the rela- tionships among these clades (similar to the MP analysis of the LSU rRNA data set-see above): there is a trichotomy of (1) the clade comprising the four other subspecies of О. hupensis used in the present study, (2) clade | of O. h. robertsoni, and (3) a clade composed of sub-clades Па, IIb, and llc of О. h. robertsoni (Fig. 1). The most basal clade in O. h. robertsoni (clade |) 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 Па appears to be a localized clade with haplotypes coming exclusively from locali- ties in the northern Anning River Valley. Clade ИБ has a wider distribution, ranging from the 30: 25: | | 1 | 1 1 | 1 1 observed Wa difference 20: Count 157 10 southern Anning River Valley to a single local- ity in Meishan Area. Finally, clade Пс 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 | and three to clade ПБ and at site M2 (central Meishan Area): from ten specimens studied, three be- long to clade ПБ and seven to clade IIc (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. 0 1 ol | he | A | lS 2 3 4 5 6 TÍ 8 9 110) 1 | 12 13 Difference in tree lengths FIG. 2. Result of the parametric bootstrap analysis for the hypothesis of non-mono- phyly of Oncomelania h. robertsoni. The black bars show the number of simulated data sets and the corresponding differences in tree lengths between the constrained (non-monophyly criterion) and unconstrained trees. The dashed line shows the ob- served difference for the original data set. 41 of the 100 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. h. robertsoni cannot be rejected. EXTREME SEQUENCE DIVERSITY IN ONCOMELANIA H. ROBERTSONI 151 AFLP Analysis 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. robertsoni divided by the total num- ber of loci) resulted in a within O. h. robertsoni diversity of 0.113 + 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. hupensis genotype scored is distinct from the O. h. robertsoni genotypes. Within O. h. robertsoni, some genotypes (e.g., Y1f, M2g, A8g, M2b, and A2b) 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. Oncomelania h. hupensis DISCUSSION 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 COI and Oncomelania h. robertsoni © M2b 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 ET AL. 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 оссиг; (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. robertsoni. 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. robertsoni 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; Hershler et al., 1999; Wilke & Pfenninger, 2002). In fact, several studies on snail hosts in SE 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. tangi) 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., 1995) of the same three populations of O. р. robertsoni from 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 O. h. robertsoni was lower than in the eastern Chinese subspecies O. h. hupen- sis. This lack of population structure within O. h. robertsoni could 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, 1987; 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 СО! 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 ¡.e., there were no ghost bands in the PCR products, there were no relevant alignment conflicts in forward and reverse strands, there were no insertions or deletions in the alignment of the protein-cod- ing СО! gene, and the gene portion studied was free of stop codons. Moreover, as the in- dividual COI 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., 1992; Fujino et al., 1995; 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 et al., 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 Herwerden 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 A1—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 СО! 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 В. 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 СО! 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 with 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 СО! 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 | and II in our analysis (Fig. 1), the oldest split in O. h. robertsoni is potentially some 4 my old (1.е., 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 et al. (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 ET AL. (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 Lis D* and F* (Fu 8 Li, 1993) as imple- mented in DnaSP 3.53 (J. Rozas 8 К. Rozas, 1999) showed that the СО! data set deviates significantly from expectations under neutral- ity both in Fu and Lis D* (1.78, Р < 0.02) and in Fu and Lis 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 Lis 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 8 Hoffmann, 1995; Ballard et al., 2002). Whether, the parasite of O. h. robertsoni, Schis- 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 is 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. h. robertsoni. ACKNOWLEDGEMENTS This work was supported in part by a United States NIH grant IP50-Al 3946 to the Shang- hai Tropical Medical Research Center, P. R. 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WANG, 2003, An index of substitution saturation and its application. Molecular Phylogenetics and Evolution, 26: 1-7. ZHANG, D.-X. & С. М. HEWITT, 1996, Nuclear integrations: challenges for mitochondrial DNA markers. Trends in Ecology and Evolution, 11: 247-251. ZOUROS, E., A. O. BALL, С. SAAVEDRA & К. R. FREEMAN, 1994, An unusual type of mito- chondrial DNA inheritance in the blue mussel Mytilus. Proceedings of the National Academy of Sciences (USA), 91: 7463-7467. Revised ms. accepted 30 September 2005 EXTREME SEQUENCE DIVERSITY IN ONCOMELANIA H. ROBERTSONI 157 APPENDIX Individual codes (M = Meishan Area, À = Anning River Valley, Y = Yunnan), DNA voucher num- bers and GenBank accession numbers for Chinese specimens of Oncomelania hupensis robertsoni studied. Individual DNA GenBank accession # Individual DNA GenBank accession # code voucher #** COVLSU rRNA code voucher #** COI/LSU rRNA Mía 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/- Аба 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/- М2] 1393 00212808/00212871 А7с 0939 DQ212839/DQ212890 M3a MG14 DQ212809/- A7d 1021 DQ212840/- M3b МС15 DQ212810/- A8a 0019 DQ212841/- M3c MG16 DQ212811/- A8b 0020 DQ212842/DQ212891 M3d MG30 DQ212812/- A8c 0021 DQ212843/DQ212892 M3e MG33 DQ212813/- A8d 0022 DQ212844/DQ212893 М4а -* AF531547/AF531545* A8e 0023 DQ212845/- Ala 0932 DQ212814/- АВЕ 0026 DQ212846/DQ212894 A1b 0934 AF213339/AF212893 A8g 0028 DQ212847/DQ212895 Alc 0935 DQ212815/- Ash 0029 DQ212848/DQ212896 Aid 1018 DQ212816/- A8i 0030 DQ212849/- Ala 0928 DQ212817/DQ212872 A8j 0050 DQ112252/- A2b 0929 DQ212818/DQ212873 A8k 0051 DQ212850/DQ212897 A2c 0930 DQ212819/DQ212874 A8l 0057 DQ212851/DQ212898 A2d 0931 DQ212820/DQ212875 Yia 0045 AF253074/DQ212899 A3a 0959 DQ212821/DQ212876 Y1b 0046 DQ212852/- A3b 0960 DQ212822/DQ212877 Yic 0048 AF253075/- A3c 0961 DQ212823/DQ212878 Y1d 0055 DQ212853/- A3d 0962 DQ212824/DQ212879 Yie 0066 DQ212854/- A4a 0955 DQ212825/DQ212880 ait 1505 DQ212855/DQ212900 A4b 0956 DQ212826/DQ212881 Y1g 1506 DQ212856/DQ212901 А4с 0957 DQ212827/DQ212882 Yah 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 fúr Naturkunde, Humboldt University, Invalidenstr. 43, D-10115 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 pallial 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 СО! and 826 bp of 16$) 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, Cerithioidea, 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 “Corresponding author: frank.koehler@rz.hu-berlin.de 159 (Kohler & 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 Cerithioidea (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; 160 KOHLER 8 GLAUBRECHT Davis, 1971) or Pleuroceridae (e.g., Vaught, 1989); this was reviewed by Köhler 4 Glaub- recht (2002a). Molecular phylogenetic studies, however, show that Brotia is a pachychilid (Kôhler 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 et al., 2002). Freed from systematic misconceptions, Pachychilidae were recently highlighted as an ideal 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 (Kóhler 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 8 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 (Kóhler & 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, 1858 (Köhler & Glaubrecht, 2005), Pseudopotamis Martens, 1900 (Glaubrecht & Rintelen, 2003), Tylomelania S. Sarasin & Е. Sarasin, 1897 (Rintelen et al., 2004; Rintelen & Glaubrecht, 2005), Adamietta Brandt, 1974, Paracrostoma Cossmann, 1900, and Jagora Kohler & 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 & Косго! (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 8 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 8 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 order to 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. 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 (1998) 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- FIG. 2. Embryonic shell parameters used for morphometrical analyses. 162 KÓHLER 8 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 | (“COI”, 646 bp) and the 16S rRNA (“165”, 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 пт? of foot tissue from specimens рге- served in ethanol by CTAB extraction (Winne- penninckx et al., 1993). PCR amplification of the fragments were performed in 25 ul volumes containing 1x Taq buffer, 1.5 mM MgCl, 200 UM each dNTP, 1 U Tag 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 HCO2198 var. 5'-TAWACTTCTGGGTGKCCAAARAAAT-3" (Folmer et al., 1994, modification of HCO2198 by А. В. Wilson) for СОТ, and 16SF 5’- CCGCACTTAGTGATAGCTAGTTTC-3’ (Wil- son et al., 2004) and H3059-Inv 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 (В!) 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+I+T; 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 BI 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 BI 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 UMB Überseemuseum, Bremen, Germany USNM National Museum of Natural History, Smithsonian Institution, Washington D.C., U.S.A. ZMA Zoëlogisch Museum, Amsterdam, The Netherlands ZMB Museum für Naturkunde, Humboldt- Universitat Berlin [formerly Zoologi- sches Museum], Germany ZMH Zoologisches Museum und Institut, Universitát Hamburg, Germany ZSI Zoological Survey of India, Calcutta, India ZSM Zoologische Staatssammlung, Múnchen, Germany ZMZ Zoologisches Museum, Zúrich, Switzerland Abbreviations В breadth of shell BW height of the body whorl bp brood pouch bpp brood pouch pore с cerebral ganglion cg 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 ll 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 ор operculum ovd oviduct ovr ovary р pedal ganglion pl pleural ganglion rad radula $ 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 st stomach t, major typhlosole t, minor typhlosole tn tentacle tnn tentacular nerve ts testis 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 anumber 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 KOHLER 8 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) Вгова $. str., (2) Senckenbergia Yen, 1939, and (3) Paracrostoma Cossmann, 1900. This suggestion was refuted, however, by Köhler 4 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 Brotía into four species groups was suggested by Kohler 8 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 8 Glaubrecht, 2005) and Jagora endemic to the Philippines (Kohler & 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 (Kohler et al., 2004). According to this mitochondrial phylogeny, it was sug- gested to transfer all species of the “Brotia testudinaria group” designated by Köhler 8 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 Kóhler & Glaubrecht (2001). Accordingly, morphological features characteristic for Brotia are (1) an irregularly wrinkled apical whorl of the embryonic shell and (2) a pallial 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. 3A, B): 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 pallial 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 В. pagodula (Thailand, male); В: External anatomy of В. episcopalis (Sumatra, female); С: Opercula (from left to right: В. pagodula, В. episcopalis, В. costula; D: Раша! oviduct of В. pagodula; schematic reconstruction showing vari- ous cross-sections from anterior to posterior; Е: Schematic reconstruction of nervous system of B. pagodula. Scale bars = 10 mm. 166 KOHLER & 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. Pallial gonod- uct open in both sexes. Pallial 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 B. citrina (Thailand, Mae Sot; ZMB 200.212). Scale bar = 5 mm. most entire length of pallial oviduct; capacious, ciliated spermatophore bursa formed by me- dian lamina (Figs. 6C, D); Fig. 3D, schematic reconstruction of pallial 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 8 Kóhler, 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 19" 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, “B. variabilis” from Miocene of Pegu, “B. baccata” from Lake Inlé (Shan States) of presumably post-Pleistocene age. From lat- ter deposits, Bequaert (1943) noted three forms of Brotía and Sulcospira, respectively that persist to the Recent. His reference to Sulcospira is here attributed to Brotia, Sulco- spira being endemic to Java (Kóhler 8 Glaubrecht, 2005). Affinities of Miocene and Pliocene fossils of Java reported by Martin (1914) and Oostingh (1935) 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 167 atin NG NE еле FES 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 B. pseudosulcospira (ZMB 200.196); head of female with egg capsule sitting in genital groove just in front of brood pouch pore. 168 KOHLER & GLAUBRECHT A * a CL | TS МАЕ < 77 2 FIG. 6. Female reproductive anatomy. A-D: Histological sections of pallial oviduct of B. pagodula (ZMH; Myanmar); A: Cross-section at anterior end of pallial 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 two thirds of oviduct length; ciliated spermatophore bursa; E: Cross-section of vis- ceral whorl of B. 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 “Brotia escheri’ (Brongniart, 1822) and “В. vasarhelyi” (Hantken, 1887) from Pannonian deposits near Budapest, Hungary — Upper Miocene (Lórenthey, 1902), Burgenland, Aus- tria — Upper Miocene (Fischer, 1994), and SYSTEMATIC REVISION OF BROTIA 169 Mainz Basin, Germany — Upper Oligocene 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 armata (Brandt, 1968) (Figs. 7, 8, 12A, В) Brotia (Paracrostoma) pseudosulcospira armata Brandt, 1968: 275, pl. 10, fig. 62 (“Маепат Kaek т 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, pl. 13, fig. 43; Köhler 8 Glaubrecht, 2002a: 144. Brotia armata — Glaubrecht & Köhler, 2004: 283-287. Paracrostoma morrisoni Brandt, 1974: 188, 189, pl. 14, fig. 47 (“Маепат Kaek at Sopa Falls, 71 km E of Pitsanulok” = Thailand, Prov. Phitsanulok, Kaek River at Sopha Falls, 71 km Е 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, pl. 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 215964, five paratypes RMNH 55284/5; types seen; Kohler & Glaubrecht, 2002a: 142. Taxonomy and Systematics Originally described as subspecies of B. pseudosulcospira, it was transferred to Brotia and is treated as distinct species in the Kaek River species flock by Glaubrecht & Kóhler (2004). According to these authors, P morrisoni and Р paludiformis dubiosa are considered as synonyms. FIG. 7. Shell morphology of В. armata. A-D: Paratypes of P pseudosulcospira armata SMF 193587; E-F: Paratypes of P morrisoni SMF 215967. Scale bar = 10 mm. 170 KOHLER & GLAUBRECHT Material Examined 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. FIG. 8. Embryonic shell morphology of B. armata. SEM images of embryonic shell removed from brood pouch (paratype BMNH 1976111); apical and front view. Scale bar = 1 mm. Description 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, B = 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 тт; п = 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). N= 173 78 B. armata B. binodosa 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 AW TABLE 1. Result of disriminant analysis of shell parameters of B. armata and B. binodosa. Predicted group membership B. armata B. binodosa B. armata 134 (97.8%) 3 (2.2%) B. binodosa 11 (14.1%) 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. Brotia binodosa (Blanford, 1903) (Figs 1105 11, 12С. 13) Melania binodosa Blanford, 1903: 282, 283, pl. 8, fig. 2 (“Siam , in fluminibus majoribus” = 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 8 Kóhler, 2004: 287-289. Brotia (Brotia) binodosa binodosa — Brandt, 1974: 174, 175, pl. 12, fig. 26: Brotia spinata — Köhler & Glaubrecht, 2002a: 148 [partim]. Brotia (Brotia) binodosa spiralis Brandt, 1974: 176, pl. 12, fig. 27 (“Thailand: Kaek River, 38.5 km Е Pitsanulok” = Thailand, Prov. Phitsanu- lok, Kaek River 38.5 km E of Phitsanulok), holotype SMF 220340; type seen. Brotia spinata spiralis — Kóhler & Glaubrecht, 2002a: 130. Taxonomy and Systematics Revised by Glaubrecht 8 Köhler (2004), who suggested B. binodosa spiralis to represent a junior synonym. Member of the Kaek River species flock in Central Thailand. Material Examined 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 193874); resort, 53 km E of Phitsanulok FIG. 10. Shell morphology of B. binodosa. A: Holotype of M. binodosa BMNH 1903.2.28.2; B: Paratype BMNH 1903.2.28.3; C: Thailand, Kaek River, Sopha Falls (2SM 19983219). Scale bar = 10 mm. 172 KOHLER & GLAUBRECHT FIG. 11. Embryonic shell morphology of B. binodosa. SEM images of embryonic shell removed from brood pouch (Thailand, Kaek River; ZSM 19983219): apical and front view. Scale bar = 0.3 mm. FIG. 12. Radular morphology of B. armata, B. binodosa, and B. citrina. SEM images of radula seg- ments viewed from above. A: В. armata (Thailand, Kaek River; ZMB 200.252); В: В. 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 um. SYSTEMATIC REVISION OF BROTIA 173 FIG. 13. 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. 10): 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. B = 14-18 mm. Embryonic Shell (Fig. 11): Conical, compris- ing up to 31/5 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 (Fig. 12C): Length of ribbon: m = 20 mm (sd = 1;n = 3), up to 190 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 KOHLER 8 GLAUBRECHT Distribution Thailand: Prov. Phitsanulok: Only known from Kaek 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, pl. 3, fig. 13 (“Siam” = Thailand), lectotype and three paralectotypes МНМС, coll. Brot (designated by Köhler and Glaubrecht, 2002a) (Figs. 14А-С); types seen; Brot, 1875: 106, 107, pl13, пд: 5: Melania citrinoides Brot, 1886: 101, 102, pl. 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. 11 (non Brandt, 1974). Taxonomy and Systematics Brandt (1974) based his diagnosis on mate- rial that we re-determined as B. dautzen- bergiana. Material of B. citrina was apparently not available to him, except for the types. Con- sequently, Brandt's (1974) description of В. 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. citrinoides 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 FIG. 14. Shell morphology of B. citrina. A: Lectotype of M. citrina MHNG, front and rear; B-C: Paralectotypes MHNG; D: Lectotyp e of M. citrinoides MHNG, front and rear; E-H: Paralectotypes МНМС; |: Thailand (ZMB 26.874); J: Thailand, Pa Charoen (ZMB 200.207). Scale bar = 10 тт. SYSTEMATIC REVISION OF BROTIA 17.3 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°30.51'N, 98°44.89'Е (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 B. 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, B = 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 4/5 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 KOHLER 8 GLAUBRECHT TABLE 2. Result of disriminant analysis of shell parameters of B. citrina and B. dautzenbergiana. Predicted group membership = B. dautzen- B. citrina bergiana B. citrina 19 (95.0%) 1 (5.0%) В. daulzen- 2 (4.7%) 41 (95.3%) bergiana Remarks From B. 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, pl. 13, figs. 37-39 [partim]; Köhler & Glaubrecht, 2001: 295- 299 [partim]; Köhler 8 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- type and paralectotype BMNH 1874.10.12.11 (designated by Kohler & Glaubrecht, 2002a) (Figs. 17B, C); 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, 25°44'N, 82°41'E), lectotype BMNH 1872.12.2.2 (des- ignated by Kohler & Glaubrecht, 2002a) (Fig. 17A); types seen; Souleyet, 1852: 545; Reeve, 1860: species 204; Brot, 1870: 281 [partim]; Brot, 1875: 85-87, pl. 10, figs. 1, 1a—d [partim]. Melania (Melanoides) variabilis — Nevill, 1885: 251, 252 [partim]. 14 12 9 10 М = 20 31 В. citrina В. dautzenbergiana FIG. 16. Comparison of В. citrina and В. 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 — Preston, 1915: 23, 24. Acrostoma variabilis — Annandale, 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; Solem, 1966: 15 [partim]. Brotia (Antimelania) variabilis — Adam & Leloup, 1938: 85, 86 [partim]. Melania varicosa Troschel, 1837: 174 (“Вепда- lien, Ganges” = Bengal, Ganges), lectotype ZMB 2.226a (here designated for the stabili- sation of the name) (Fig. 17F) and 13 para- lectotypes ZMB 2.2265; 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 ММНМ; types seen; Souleyet, 1852: 545. Melanoides indica — H. Adams & A. Adams, 1853: pl. 31, figs25, 94:10: Melania menkiana [sic !] |. Lea, 1842: 242 (re- placement name for M. plicata |. 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, 1a, b (replacement name for M. menkia- na Lea, 1842). Melania (Melanoides) variabilis menkeana — Nevill, 1885: 260. SYSTEMATIC REVISION OF BROTIA 177 FIG. 17. Shell morphology of B. costula. A: Lectotype of M. variabilis BMNH 1872.12227B7Eecto- type of M. carolinae BMNH 1874.10.12.11/A; C: Paralectotype BMNH 1874.10.12.11/B; D: Lectotype of M. spinosa BMNH 1907.10.28.79; Е: Paralectotype ВММН 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 KOHLER 8 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, 91°52'E) (non M. spinosa Gray, 1824), lectotype BMNH 1907.10.28.79 and paralectotype BMNH 1907.10.28.80 (Figs. 17D, Е) (designated by Köhler 8 Glaubrecht, 2002a); types seen; Brot, 1875: 92-93pl212 fig: 2: FIG. 18. Embryonic shell morphology of B. costula. SEM images of embryonic shell removed from dried material (ZMB 35.811); apical and front view. Scale bar = 1 mm. Melania variabilis var. spinosa — Hanley & Theobald, 1873: pl. 75, fig. 6. Melania hainesiana |. Lea, 1856: 144 (“India”), nine syntypes USNM 119741 (Fig. 17G); types seen; |. Lea, 1864: 78, pl. 22, fig. 18; Brot, 1875: 109, 110, pl. 14, fig. 4. Melania (Melanoides) variabilis var. hainesiana — Nevill, 1885: 255. Melania corrugata Reeve, 1859: pl. 3, fig. 10 (India, Java”) (non M. corrugata Lamarck, 1822). Melania spinata — Brot, 1875: 89, 90, pl. 10, fig. 2a (non M. spinata Godwin-Austen, 1872). Melania episcopalis — Hanley & Theobald, 1873: 31,32. pl: 72, На. 7, pers. ЭЙ (поп М. episcopalis H. Lea 4 |. Lea, 1850). Melania (Melanoides) variabilis episcopalis — Nevill, 1885: 256 [partim]. Melania (Melanoides) variabilis subvar. aspera Hanley & Theobald, 1874: pl. 109, fig. 6; Nevill, 1885: 252. Melania (Melanoides) variabilis subvar. cincta Hanley 8 Theobald, 1874: pl. 109, fig. 5; Nevill, 1885: 252. Melania (Melanoides) variabilis subvar. microstoma Nevill, 1885: 261 (“Sylhet”). Melania (Melanoides) variabilis var. pseudospinosa Nevill, 1885: 258. Melania (Melanoides) variabilis var. semi- laevigata Nevill, 1885: 254 (“Cachar and Sylhet”). Melania (Melanoides) variabilis subvar. subtuberculata Nevill, 1885: 252 (“Calcutta”). Melania (Melanoides) variabilis subvar. subspinosa 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; Kohler & 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) B. c. varicosa (Troschel, 1837), with suggested oc- currence on Sumatra, Java and Borneo, and (3) B. c. 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); В: India, Manipur (BMNH). Scale bars = 100 um. 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 В. 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. herculea, 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 (ВММН 1841.7.23.9); Meghalaya: Jaintia-Khási 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, River Toolsi Ganga (ВММН); 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 (ВММН); Malaudi (BMNH); River Jamuna (ВММН 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'М, 86°59.6'Е (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 KÔHLER & 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 1/5 of shell height. Size: H = 20-87 mm, B = 8-36 mm. Embryonic Shell (Fig. 18): Smooth except for fine growth lines. Maximum height 4 mm, 3.5 whorls. Average proportions: Н = 2.3 тт, В = 1.1 mm, НА = 0.27 тт, ВА = 0.48 mm, ОА = 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. 80° 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 (Bandel & Riedel, 1998) refer to other species. Conchologically similar are В. episcopalis from the Malay Peninsula, B. sumatrensis from Sumatra, B. herculea from Myanmar, and B. jullieni from Cambodia; all were repeatedly synonymized with B. costula. Brotia episcopalis and B. sumatrensis tend to be smaller and more conical in shape. In B. episcopalis, the upper whorls are smooth and = 20° LOS FIG. 20. Distribution of B. costula (closed circles) and B. herculea (open circles). SYSTEMATIC REVISION OF BROTIA 181 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 М = 34 108 В. costula В. herculea FIG. 21. Comparison of В. costula and В. herculea 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. B. jullieni 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 В. costula, such as А. infracostata (Mousson, 1849), differ in embryonic shell morphology (Kôhler & Glaubrecht, 2001, for the “Brotia testudinaria-group”). Brotia dautzenbergiana (Morlet, 1884) (Figs. 22-24) Melania dautzenbergiana Morlet, 1884: 399, 400, pl. 8, fig. 1a—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 8 Glaubrecht, 2002a) (Fig. 22A); types seen; Fischer-Piette, 1950: 154. FIG. 22. Shell morphology of B. 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 KOHLER & GLAUBRECHT Stenomelania dautzenbergiana — Habe, 1964: 55, pl. 1, fig. 19. Brotia dautzenbergiana — Kohler & Glaubrecht, 2002a: 133, fig. 10. Melania dugasti Morlet, 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 Kohler & 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 B. 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 (1974) assumed that both taxa are synonyms of B. 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 river, 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 near Thoern, 17°39.31'N, 98°7.91'E (ZMB 200.213); Prov. Kamphaeng Phet, Huai Hin Fon near Mae 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, B = 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, B= 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 & |. Lea, 1851) (Figs. 25, 26, 67A) Melania episcopalis H. Lea & |. Lea, 1851: 184 (“sluggish river, Malakka” = Melaka, Prov. Negeri Melaka; 2°12'N, 102°15'E), lectotype and paralectotype MCZ 221841 (designated by Kohler & Glaubrecht, 2002a) (Figs. 25A, B); types seen; Hanley, 1854: pl. 3, fig. 27; Brot; 1875: 97, 98, pl. 12; figs 1; Та Melanoides episcopalis — H. Adams & А. Adams, 1854: 297. Melania (Melanoides) episcopalis — Chenu, 1859: 288, fig. 1952. Melania (Melanoides) variabilis episcopalis — Nevill, 1885: 256 [partim]. Brotia costula episcopalis — Davis, 1971: 53- 86. Brotia episcopalis — Köhler 8 Glaubrecht, 2002а: 134, fig. 1P. Melania heros Brot, 1875: 339, 400, pl. 34, fig. 8 (unknown locality), holotype MHNG (Fig. 25D); type seen. Sermyla perakensis Morgan, 1885: 421, pl. 8, figs. 14a-f (“Perak”), lectotype and para- lectotype MNHN (designated by Kohler & Glaubrecht, 2002a) (Fig. 25C); types seen. Brotia costula — Brandt, 1974: 175, pl. 13, fig. 37-39 [partim]; Köhler & Glaubrecht, 2001: 296-299, figs. 1D, 10А-С, С, H [рай] (non M. costula Rafinesque, 1833). Brotia (Antimelania) costula — Subba Rao, 1989: 108, 109 [partim] (non M. costula Rafinesque, 1833). FIG. 24. Radular morphology of B. dautzenbergiana. SEM images of radula segments viewed from above. A: Thailand, Lampang (ZMB 200.229); B: Thailand, Thoern (ZMB 200.213). 184 KOHLER & GLAUBRECHT FIG. 25. Shell morphology of В. episcopalis. А: Lectotype of M. episcopalis MCZ 221841; В: Paralectotype MCZ 221841; С: Lectotype of Sermyla perakensis ММНМ; D: Holotype of Melania heros МНМС. Scale bar = 10 mm. Brotia variabilis — Bequaert, 1943: 433, 434, pl. 33, figs. 11-16 [partim]; Solem, 1966: 15 (non M. variablis Benson, 1836). Taxonomy and Systematics Frequently subsumed under B. costula by 20" century authors (Benthem Jutting, 1949, 1956; Brandt, 1968, 1974; Kôhler & Glaub- recht, 2001). However, molecular genetic data shows that В. episcopalis is distinct (Figs. 78, 79). Melania heros and Sermyla perakensis are considered as synonyms. Material Examined Thailand: Prov. Trang: Trang (ZSM 19983228). Prov. Nakhon Si Thammarat: Khlong 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 11 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, B = 15-24 mm. Embryonic Shell: No own data, but described and depicted by Davis (1971: 60, figs. 2h, 1, 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 sa ST U > N и, FIG. 26. Stomach anatomy of В. 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 3.4 3.2 8 O 3.0 2.8 2.6 2.4 2.2 N= 34 154 42 B. costula B. sumatrensis B. episcopalis FIG. 27. Comparison of B. costula, B. suma- trensis, and B. 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 B. 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, B. 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) hanleyi Godwin-Austen, 1872: 514, 515, pl. 30, fig. 2 (non M. hanleyi Brot, 1860) (“Diyung River, North Cachar hills” = Diyung River, Jaintia-Khäsi hills N of Silchar, Meghalaya, India, 24°48'N, 92°46'E), lectotype BMNH 19991561/A and paralectotype BMNH 19991561/B (desig- nated by Kohler & Glaubrecht 2002a) (Figs. 28A, B); types seen. Melania godwini Brot, 1875: 90, pl. 10, fig. 3 (replacement name for M. hanleyi Godwin- Austen, 1872). Melania (Melanoides) variabilis var. binodulifera Nevill, 1885: 259 (“Khasi hills”). Brotia godwini — Köhler & Glaubrecht, 2002a: 136. 186 KÔHLER & GLAUBRECHT Taxonomy and Systematics Melania godwini Brot, 1875, was employed as replacement name for M. hanleyi Godwin- Austen, 1872, being preoccupied by M. hanleyi 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. Description 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 В. godwini. A: Lectotype of M. hanleyi Godwin-Austen BMNH 19991561/ A; B: Paralectotype BMNH 19991561/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, pl. 13, fig. 2 (“China”), ex Gray ms; lec- totype BMNH 19990495/A and paralectotype ВММН 19990495/B (designated by Köhler & Glaubrecht, 2002a) (Figs. 29A, B); types seen; Reeve, 1859: 1. Semisulcospira henriettae — Yen, 1942: 204: ple 15.19. 66: Brotia henriettae — Kohler & 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, pl. 9, fig. 6; Hanley & Theobald, 1873: 32, pl. 75, figs. 1, 2, 4; Annandale, 1918: 115, pl. 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. elongata Annandale, 1918: 115, 116, pl. 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 to Annandale (1918); types not seen. Acrostoma elongatum — Annandale & Rao, 192511 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 — Branat, 1974: 178, pl 13, fig.232. Melania reticulata |. & 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. glabra Theobald, 1865: 273. Melania variabilis var. vittata Theobald, 1865: 273, fig. 4; Nevill, 1885: 263. Melania variabilis var. turrita Theobald, 1865: 273, 274, fig. 5. Melania variabilis var. baccifera Theobald, 1865: 274, fig. 6. Melania (Melanoides) baccata subvar. recta Nevill, 1885: 262 (“Upper Замееп”). 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 whether these 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 B. 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 188 KOHLER & GLAUBRECHT FIG. 29. Shell morphology of В. henriettae. А: Lectotype of M. henriettae ВММН 1999095/А; В: Paralectotype BMNH 1999095/B; C: Holotype of M. reticulata USNM 119663; D: Lectotype of M. persculpta SMF 221813; E: Lectotype of M. baccata MCZ 169052; F: Thailand, Pai (ZMB 200.221); С: 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 тт. SYSTEMATIC REVISION OF BROTIA 189 a name most commonly employed. None- theless, the name М. henriettae Griffith & Pidgeon, 1834, being available has priority over M. baccata. Brotia henriettae 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 49.611, BMNH FIG. 30. Embryonic shell morphology of B. henriettae. SEM images of embryonic shell removed from dried shell (ZMB 49.613); apical and front view. Scale bar = 1 mm. 1899.6.21.90-91), 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 near Thibaw (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 $ 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, B = 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, B = 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. 31B, С): Up to 150 rows of teeth; radulae from different localities vary in breadth 190 KOHLER 8 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 B. 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. herculea. Fossil Record In Tertiary and Pleistocene cave deposits of Myanmar (Bequaert, 1943); sub-fossil shells reported by Annandale (1918) from Myanmar. Remarks Similar sculpture in B. iravadica, frequently being smaller and more conical in shape, with fewer whorls. Brotia herculea (Gould, 1846) (Figs. 32-34) Melania herculea Gould, 1846: 100 (“Tavoy 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. henriettae, and B. jullieni. A: B. godwini (Assam; ZMB 97.422): B: B. henriettae (Thailand, Pai; ZMB 200.221); C: B. henriettae (Thailand, Mae Sot; ZMB 200.210); D: B. jullieni (Cambodia; ZMH). Scale bars = 0.1 mm. SYSTEMATIC REVISION OF BROTIA 191 pl. 2, fig. 4; Hanley & Theobald, 1873: 31, pl. Melania balteata Reeve, 1860: pl. 20, species 72, fig. 5; Johnson, 1964: 87, pl. 35, fig. 10. 144 (non M. balteata Philippi, 1858) (no lo- Melanoides herculea — H. Adams & A. Adams, cality given), lectotype ОМВ TK 304/1 and 1854: 297. paralectotype ÜMB 308/1 (designated by Melania (Melanoides) herculea — Nevill, 1885: Knipper, 1958, referring to M. reevei) (Figs. 251: 32B, С); types seen. FIG. 32. Shell morphology of B. herculea. A: Lectotype of M. herculea MCZ 169436; B: Lectotype of M. balteata Reeve ОМВ TK 304/1: С: Paralectotype ОМВ TK 308/1; D: Lectotype of M. gloriosa ANSP 26363; E: Thailand, Raheng (MHNG); Е: Thailand, Sai Yok (ZMB 200.235). Scale bar = 10 mm. 192 KÔHLER & GLAUBRECHT Melania reevei Brot, 1862: 46 (replacement name for M. balteata Reeve); Brot, 1875: 95, 96, pl. 11, figs. 4, 4a, pl. 13, fig. 6; Hanley 8 Theobald, 1876: 61, pl. 153, fig. 1. Melania (Melanoides) reevei — Nevill, 1885: 248. Melania (Melanoides) reevei var. lanceolata Nevill, 1885: 248, 249 (“Mandalay; Hezada, Pegu; Thyet Myo”). Melania (Melanoides) reevei var. imbricata Hanley & Theobald, 1876: pl. 153, fig. 4 (without locality); Nevill, 1885: 249. Melania (Melanoides) reevei var. soliduscula Nevill, 1885: 249, 250 (“Pegu, Noung-ben- Ziek”). Melania (Brotia ?) reevei— Martens, 1899: 36. Melania gloriosa Anthony, 1865: 207, pl. 18, fig. 3 (“Реди” = 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 — Brot, 1875: 85-87, pl. 10, fig. 1, 1a-d [partim]. Melania peguensis Hanley 8 Theobald, 1873: 31, pl. 72, fig. 6 [nomen пиаит]. Melania (Melanoides) tourannensis var. peguensis — Nevill, 1885: 250. Melania (Melanoides) tourannensis var. com- pacta Nevill, 1885: 250, 251 (“Henzada, Pegu”). Melania (Melanoides) tourannensis var. beddomeana Nevill, 1885: 251 (“near Moulmein”). Melania (Melanoides) variabilis subvar. subvaricosa Nevill, 1885: 252, 253 “Arakan, Pegu”). Melania (Melanoides) variabilis subvar. semilaevigata 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 8 Glaubrecht, 2002a: 132 [partim]. Taxonomy and Systematics Treated as synonym of Brotia costula by, for example, Benthem Jutting (1956, 1959) and Brandt (1974), this taxon is considered herein as a distinct species since B. costula and B. herculea 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. herculea conspecific with the former. A second taxon, Melania reevei Brot, has also frequently been considered a synonym of B. 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 8 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 М. reevei and M. gloriosa are tenta- tively subsumed under B. herculea for their somewhat similar shell and since both origi- nate from the same area, Pegu. The type lots of M. herculea 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 Pal, 19°21.57'N, 98°26.62'E (ZMB 200.220); Mae SYSTEMATIC REVISION OF BROTIA 193 Ping, 60 km М Chiang Ма! (ММНМ; АМ$ 146766); bridge at the street from Samoeng to Chiang Mai, 18°44.23'N, 98°55.87'Е (ZMB 200.253); Prov. Kanchanaburi, Sai Yok Falls 1 at Nam Ток, 14°14.16'М, 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. Tak, Mae Dao River, Mae Sot (AMS 146762), Maenam Moei, 8 km N Mae Ramat (AMS 146765). FIG. 33. Embryonic shell morphology of B. herculea. 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, B = 10-34 mm. Embryonic Shell (Fig. 33): Smooth, covered with axial wrinkles. Average proportions: H = 1.7 mm, B = 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 lrawaddy, Chindwin, and Salween (with Moei River), and Chao Praya (with Ping and Nan Rivers). 194 KOHLER 8 GLAUBRECHT FIG. 34. Radula morphology of B. herculea. Radula segments viewed from above. A: Myanmar, Pegu (ZMB 41.199): B: Thailand, Pai (ZMB 200.220); C: Thailand, Sai Yok Falls, Nam Tok (ZMB 200.237); D: Thailand, Pai (ZMB 200.219). Scale bars = 100 um. FIG. 35. Stomach anatomy of B. herculea (ZMB 200.209; Thailand). Scale bar = 5 mm. Habitat and Ecology Clear creeks and rivers on rock, mud, sand, roots, under and among piles of leaf litter in the water (Davis, 1982, 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 indragirica (Martens, 1900) (Fig. 37) Melania indragirica Martens, 1900: 10, 11 (“Indragiri-Flu&, Sumatra” = Indragiri River, SYSTEMATIC REVISION OF BROTIA 195 FIG. 36. Distribution of B. citrina (open circles), B. dautzenbergiana (open squares) and B. henriettae (close circles). Sumatra (Indonesia), lectotype ZMB 51.777a, three paralectotypes ZMB 51.777b, five para- lectotypes NMB 1202q (designated by Kóhler & Glaubrecht, 2002a) (Fig. 37); types seen; Bul- len, 1906: 14 (including an unnamed variety). Brotia indragirica — Kóhler 8 Glaubrecht, 2002a: 139. figs ZI. 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, FIG. 37. Shell morphology of B. indragirica. Lectotype of M. indragirica ZMB 51.777a. 196 KOHLER & 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, B = 10-15 mm. Embryonic Shell, Operculum, Radula, Soft 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 insolita (Brot, 1868) (Fig. 38) Melania insolita Brot, 1868: 11, pl. 3, fig. 4 (“Inde?”), lectotype and seven paralecto- types МНМС, Brot collection, “Siam” (desig- nated by Kohler & Glaubrecht, 2002a) (Fig. 38); types seen; Brot, 1875: 107, 108, pl. 13, fig. 7. Brotia (Brotia) insolita — Brandt, 1974: 176, 177, pl. 13.105. 29; 30. Brotia insolita — Kóhler & Glaubrecht, 2002a: 139, 140, fig. 2J. Taxonomy and Systematics Brot (1868) 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, A D FIG. 38. Shell morphology of В. insolita. А: Lec- totype of М. insolita MHNG; B-D: Three para- lectotypes МНМС. Scale bar = 10 mm. Kaek River (Prov. Phitsanulok, central Thai- land) were subsequently designated as the type locality by Branat (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 Shell (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. insolita. To complicate matters, B. insolita 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, pl. И 1: Melania irawadica [sic !] — Brot, 1872: 34; Brot, 1875; 111, 112; pl. 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. FIG. 39. Shell morphology of B. 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, B = 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, pl. 7, figs. 7-9 (“Thio- Compih, Cambodge” = Thio Compih, Sámbok 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, pl. 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 KOHLER & GLAUBRECHT Material Examined Laos: Muong-Bet sur le Song-Ma (ММНМ); 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 1/4 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, B = 24-30 mm. Radula (Fig. 31D): 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. FIG. 40. Shell morphology of В. jullieni. A: Lectotype of M. jullieni MHNH; В: Cambodia (ZMZ 522392); C: Laos, Pakse (ZMH). Scale = 10 mm. SYSTEMATIC REVISION OF BROTIA 199 N= 34 107 6 B. c. costula B. c. herculea В. jullieni FIG. 41. Comparison of B. costula and B. 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. herculea (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. herculea. A report from the Ping River near Tak, Thai- land, by Morlet (1891: “Riviere de Menam- Pinh, de Raheng à Xieng-Moi”) refers to В. herculea. 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, B = 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 B. episcopalis, which is more elongated and differs in average num- ber of whorls and sculpture. 200 KOHLER & GLAUBRECHT FIG. 42. Shell morphology of B. kelantanensis (Malaysia, Pahang; ZMA). Brotia manningi Brandt, 1968 (Figs. 43B, 44) Brotia (Brotia) manningi Brandt, 1968: 272, pl. 10, fig. 58 (“Thailand: Huai Lan at Ban Dam Pon, Lom Sak District, Phetchabun Prov- ince”), holotype SMF 197376, 22 paratypes MCZ 288652, 22 paratypes ZSM 19983239, 20 paratypes RMNH 55289/20 (Fig. 44); types seen; Brandt, 1974: 179, 180, pl. 13, 19.25: 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 Shell (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, B = 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 (п = 1). Rachidian elon- FIG. 43. Radula morphology of B. kelantanensis and B. manningi. A: B. kelantanensis (Malaysia, Pahang; ZMA); B: B. manningi, paratype ZSM 19983239). Scale bars = 100 um. SYSTEMATIC REVISION OF BROTIA FIG. 44. Shell morphology of В. manningi. А-С 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 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 B. insolita and B. 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 Branat (1974). Brotia microsculpta Brandt, 1968 (Figs. 45, 46A) Brotia microsculpta Brandt, 1968: 272, pl. 10, fig. 59 (“Thailand: Маепат Kaek, in Thung Salaeng Luang Botanical Garden, 80 km E of Pitsanulok” = Kaek River, Thung Salaeng 201 sa a u _ a EN > aS \ = Be a : Paratypes ZSM 19983239. Scale = 10 mm. Luang NP, Prov. Phitsanulok), holotype SMF 197378/1, 10 paratypes SMF 205356/10 (Fig. 45); types seen; Kohler & Glaubrecht, 2002a: 141; Glaubrecht & Kohler, 2004: 289-291. Brotia (Brotia) microsculpta — Brandt, 1974: 180: pl 13, fig. 36. Taxonomy and Systematics Revised by Glaubrecht & Kôhler (2004) based on morphological and molecular genetic data. Accordingly, B. microsculpta belongs to the Kaek River species flock in Central Thailand. 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 KOHLER & GLAUBRECHT FIG. 45. Shell morphology of B. microsculpta. Holotype SMF 197378/1. Scale = 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 = 10-25 mm. В = 8-15 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- sulcospira 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, pl. 13, fig. 2, Hanley & Theobald, 1876: 61, pl. 153, fig: 3: lo pagodula — H. Adams & A. Adams, 1854: 300; Reeve, 1859: pl. 3, fig. 10. Tiara (Acrostoma) pagodula — Preston, 1915: 32: Brotia pagodula — Morrison, 1954: 382; Johnson, 1964: 121, pl. 44, fig. 2; Köhler & Glaubrecht, 2001: 292-295, figs. 1A, 9A-F; Köhler & Glaubrecht, 2002a: 142; Glaubrecht & Köhler, 2004: 283. Brotia (Brotia) pagodula — Brandt, 1974: 173, 174%pl12;fig:25; Taxonomy and Systematics Type species of Brotia. Material Examined Myanmar: (ZMB 26.708); Salween River, Tavoy (ВММН); Thailand: Ргоу. Kamphaeng Phet: Маепат Мое! approximately 20 km E Mae Sot, 16°45.82’М, 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), Маепат Мое! (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 ВРОТ/А FIG. 46. Radular morphology of several Brotia species. А: В. microsculpta (Thailand, Kaek River; ZMB 200.200); B: B. pagodula (Thailand, Moei River: ZMH); C: B. paludiformis (Thailand, Kaek River; SMF 215963); D: B. peninsularis (Thailand, Surat Thani: ZMB 200.242); E: B. praetermissa, Paratype BMNH 20010482/B; F: B. pseudosulcospira (Thailand, Kaek River: ZMH); G: B. solemiana (Thailand, Pong River; SMF 193585); H: B. subgloriosa Paratype ZSM 19983219. Scale bars = 0.1 mm. 204 KOHLER 8 GLAUBRECHT FIG. 47. Shell morphology of B. 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, B = 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 (Caobangía 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., В. 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. 902 20° 10 FIG. 49. Distribution of В. peninsularis (close circles), В. siamensis (open circle), B. pagodula (close rectangle) and B. wykoffi (open rectangle). 206 KOHLER & GLAUBRECHT Brotia paludiformis (Solem, 1966) (Figs. 46C, 50) Paracrostoma paludiformis Solem, 1966: 17, pl. 1, figs. H-J, text-fig. 2 (non Semisulcospira 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 — Kóhler 8 Glaub- recht, 2002a: 121-156. Brotia paludiformis — Glaubrecht 8 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 (1966) 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 8 Kohler (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, B = 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 ($4 = 1.3 mm; п = 3), about 190 rows of teeth. Denticle morphology corresponding to B. armata. 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, pl. 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 B. 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’М, 99°46.33’Е (ZMB 200.041-2), Pum Pin near Takuha, km 63.5 (ZMH; ZSM 19983231); Prov. Phang Nga, Khlong 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 (Fig. 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 KOHLER & GLAUBRECHT Embryonic Shell (Fig. 52): Subsequent whorls smooth, sculptured only by growth lines. Av- erage proportions: H = 3.2 mm, B=0.4 mm, НА = 0.18 mm, ВА = 0.33 mm, DA = 0.65 mm (for n = 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. FIG. 52. Embryonic shell morphology of B. peninsularis. SEM images of em- bryonic shell removed from brood pouch (paratype ZMH); apical and front view. Scale bar = 0.3 mm. 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, ЕЮ. 53). М = 32 18 В. peninsularis В. siamensis FIG. 53. Comparison of В. peninsularis and В. 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 Kohler & Glaubrecht, 2002 (Figs. 46E, 54-56) Brotia praetermissa Kóhler 8 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 FIG. 54. Shell morphology of B. praetermissa. Holotype BMNH 20010482/A. Scale bar = 10 mm. deposit; tip eroded; relatively deep suture; sculpture of six strong spiral ridges, most prominent а 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, B = 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 KOHLER 8 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 (Kóhler & Glaubrecht, 2003). Brotia pseudoasperata Brandt, 1968 (Figs. 57-59) Brotia (Brotia) pseudoasperata Brandt, 1968: 270, 271, pl. 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: TT FATE ASAS Brotia pseudoasperata — Kohler & 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 B. praetermissa, paratype BMNH 20010482/B. FIG. 57. Shell morphology of B. pseudoasperata. А-В: Paratypes ZSM 19983244. 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 19983244); 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, B = 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 pm. 212 KOHLER 8 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 — Brandt 1974: 185, pl. 13, fig. 42. Paracrostoma pseudosulcospira — Kóhler 8 Glaubrecht, 2002a: 144. Brotia pseudosulcospira — Glaubrecht & Kohler, 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 8 Kóhler (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, B = 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. FIG. 60. Shell morphology of B. pseudosulcospira. A: Paratype SMF 193586; B-C: Paratypes ZMH; D-E: Paratypes ZMH (alc.); 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: т = 25 mm (sd = 2.5 mm; п = 3), up to 180 rows of teeth. Central tooth comparatively broad, glabella very narrow; otherwise similar to В. 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 B. 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, pl. 7, figs. 3-3b (“Raheng, Siam” = Tak, Prov. Tak, Thailand), lectotype and 18 paralectotypes MHNG, coll. Brot (designated by Kohler & Glaubrecht, 2002a) (Fig. 62); types seen. Brotia siamensis — Kohler & Glaubrecht, 2002a: 147, fig. 3H. Taxonomy and Systematics Treated in various ways by previous authors, this taxon was considered conspecific with M. hamonvillei 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 Kohler & Glaubrecht (2002a) noticed that some type specimens of M. siamensis are similar to B. 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 B. peninsularis. The latter taxon was treated by him as a subspecies of B. costula. Some lots of this species were labelled by him with B. siamensis, however. Both taxa are indeed similar. B. peninsularis as consid- ered here is restricted to the Malay Peninsula south of the Isthmus of Kra. The only confirmed record of B. siamensis is the type locality, Tak, about 700 km N of this isthmus. A reliable de- cision on the relationships of B. siamensis and В. peninsularis awaits the examination of well- preserved material from the area of Tak. For 214 KOHLER & GLAUBRECHT TABLE 3. Result of disriminant analysis of shell parameters of B. peninsularis and B. siamensis. Predicted group membership В. peninsularis В. siamensis 29 (93.5%) 2 (6.5%) 0 (0%) 18 (100%) B. peninsularis B. siamensis 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 C 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, B = 11-16 mm. Embryonic Shell, Radula, Operculum, Soft Body: Unknown. Remarks Similar B. peninsularis tends to have larger body whorl compared to the shell height, whorls more rounded in diameter. Brotia jullieni 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 hamonwvillei has distinct embryonic shell structure, resembling, for example, B. testu- dinaria (Kôhler 8 Glaubrecht, 2001). Distribution (Fig. 49) Thailand: Type locality only known reference: Tak (Prov. Tak, north-central Thailand) at banks of Ping River. FIG. 62. Shell morphology of В. siamensis. А: Lectotype МНМС (front and геаг); В-Р: Paralectotypes МНМС. Scale bar = 10 mm. SYSTEMATIC REVISION OF BROTIA 215 Вгойа solemiana (Brandt, 1968) (Figs. 46G, 63) Brotia (Paracrostoma) solemiana Brandt, 1968: 273, pl.10, fig. 60 (“Маепат 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, pl. 13, fig. 44; Kóhler 8 Glaubrecht, 2002a: 147. Brotia solemiana — Glaubrecht 8 Kóhler, 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 8 Kôhler (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 В. solemiana. А-В: 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 KÔHLER & GLAUBRECHT Description Shell (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, B = 18-24 mm. Operculum: Oval, up to four whorls, sub-cen- tral nucleus. Radula (Fig. 46G): Length of the ribbon: m = 16.0 mm (sd = 3.4 mm; n = 4), 150-160 rows of teeth. Rachidian relatively narrow, other- wise widely corresponding to B. armata. Stomach: Typical, as described for B. citrina (Fig. 4). Embryonic Shell: 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. subgloriosa generally larger, more turreted; B. microsculpta with smaller body whorl, rounded aperture, circular operculum. Radula of B. solemiana shorter as in other Kaek River species. Brotia subgloriosa (Brandt, 1968) (Figs. 46H, 64, 65) Brotia binodosa subgloriosa Brandt, 1968: 269, pl. 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 subgloriosa — Branat, 1974: 175,176, pl. 13, fig. 28: Brotia spinata subgloriosa — Kóhler & Glaub- recht 2002a: 129. Brotia subgloriosa — Glaubrecht & Köhler, 2004: 293. Taxonomy and Systematics Described as a subspecies of В. 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 Branat (1968, 1974), B. subgloriosa and B. binodosa occur sympatrically in parts of the Kaek River, which conflicts a relation as geographical sub- species. Forthis reason, B. subgloriosa is con- sidered as distinct species, perhaps closely related to B. binodosa. According to Glaubrecht 8 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 (Fig. 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, B = 16-24 mm. Embryonic Shell (Fig. 65): Conical, up to 3.5 whorls; smooth sculpture with faint growth lines. FIG. 64. Shell subgloriosa. Paratype ZSM 19983213. Scale bar = 10 mm. morphology of B. 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 B. armata. Reproductive System Two dried shells (ZSM 19983219) 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 19983213); 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, pl. 10, fig. 2b, pl. 13, figs. 1a, b (“Sumatra: Palembang’), three syntypes MHNG, Brot collection, one syntype MCZ 112689 (Figs. 66А-С); types seen. Melania sumatrensis — Schepman, 1886: 13. Melania boeana Brot, 1881: 154, 155, pl. 6, fig. 1 (“Boea, Sumatra” = Bua, Sumatra), lec- totype and four paralectotypes МНМС, Brot collection (designated by Kôhler & 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, pl. 13, figs. 37-39 [partim]; Kóhler & Glaubrecht, 2001: 296-299, figs. 1D, 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, pl. 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 KOHLER 8 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 B. 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 M. boeana MHNG, front and rear view; G-J: Paralectotypes MHNG; К: Sumatra, Lake Ranau (MZB); |: 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 МНМС 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): Tandjung djatti (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); Lake Takengon (ZMB 76.673, 200.136). Prov. Sumatera Utara: Trans-Sumatra highway, bridge 150 km N Bukittingi, 1°28.28’М, 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, 1°40.04’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; ГМА); 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°27.31'S, 100°36.2’E (ZMB 200.122); 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’Е (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, B = 10-27 mm. Operculum: Oval, four to six whorls, central nucleus. Radula (Figs. 67В-Е): 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; B. episcopalis differs mainly by a marked transition from smooth upper whorls to strongly sculptured lower whorls, with lesser pronounced axial ribs. 220 KOHLER & GLAUBRECHT FIG. 67. Radular morphology of B. episcopalis and B. sumatrensis. À: B. episcopalis, Thailand, Nakhon Si Thammarat (ZMH); В: В. sumatrensis (Sumatra, Lampung; MZB 7028); С: South Sumatra (ZMB 200.116); D: Sumara, Lake Toba (ZMB 200.119); E: Trans-Sumatra highway (ZMB 200.120). 902 1002 1408 Е : ар LO? LOS FIG. 68. Distribution of B. 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, pl. 1, fig. 18 (“Java”), lectotype ÜMB ТК 291/1 (designated by Knipper, 1958) (Fig. 69A); type seen; Mousson, 1849: 70; Brot, 1870281 Brot 1879: 110, 111, pl 14; fig. 5, 5a [partim]. Melanoides torquata — H. Adams & A. Adams, 1854: 297. Brotia torquata — Köhler 8 Glaubrecht, 2002a: 150. Melania zollingeri Brot, 1868: 42, pl. 2, fig. 4 (“Java”), holotype MHNG, coll. Brot (Fig. 69 B); type seen; Brot, 1875: 111, pl. 14, fig. 6; Schepman, 1886: 14; Leschke, 1914: 252; Degner, 1928: 374; Benthem Jutting, 1959: 93. Brotia zollingeri— Köhler & Glaubrecht, 2002a: 152, tig: 30: Melania subplicata Schepman, 1886: 14, pl. 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 Kohler & Glaubrecht, 2002a) (Figs. 69E, F); types seen; Martens, 1897: 37, pl. 2, fig. 15, pl. 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 B. torquata. A: Lectotype of M. torquata UMB TK 291/1; B: Holotype of M. zollingeri MHNG; С: Lectotype of M. curvicosta ZMA; D: Paralectotype of M. curvicosta ZMA; E: Lectotype of M. subplicata ZMA; Е: Paralectotype of M. subplicata ZMA; G-H: West Sumatra, Fort Kok (RMNH 71331). Scale bar = 10 mm. 222 KOHLER & GLAUBRECHT Alahan pandjang” = Supajang and Alahan pandjang, nearby); types not seen. Melania curvicosta Martens, 1897: 36, pl. 2, fig. 14, pl. 4, fig. 27 (“See von Manindjau, Sumatra = Laker Manindjau, Sumatra), lec- totype, paralectotype ZMA, 12 paralecto- types ZMA (alc.), three paralectotypes ZMB 54.364 (designated by Kôhler & Glaubrecht, 2002a) (Figs. 69C, D); types seen; Bullen, 1906: 15; Degner, 1928: 374. Melania curvicosta var. prestoniana Bullen, 1906: 15, pl. 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 (1974) treated M. torquata, M. zollingeri, M. subplicata, and M. curvicosta as synonyms of B. costula. However, B. 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°26.7’М, 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 A М! EEE IL \ FIG. 71. Radular morphology of В. torquata and В. verbecki. А: В. torquata (Sumatra, Lake Manindjau; ZMB 200.117); B: B. verbecki (Sumatra, Lake Singkarah; ZMB 200.118). Scale bars = 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, B = 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, B = 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 1/3 to 1/2 of length of ctenidium. Radula (Fig. 71A): Ribbon with about 100 rows ofteeth. 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. 10 um. 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 B. verbecki even exhibit marked axial sculpture otherwise typical for most specimens of B. torquata. Shells of B. 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 ofLeschke (1914) on M. subplicata from Bogor is incorrect; the voucher material in the ZMB is re-determined as Adamietta testudina- ría. TABLE 4. Result of disriminant analysis of shell parameters of B. torquata and B. verbecki. Predicted group membership B. torquata B. verbecki B. torquata 10 (90.9%) 1 (9.1%) В. verbecki 1 (2.6%) 37 (97.4%) 224 KOHLER 8 GLAUBRECHT Brotia verbecki (Brot, 1886) [5195708 92.73) Melania verbecki Brot, 1886: 90, pl. 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 Kóhler & 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 — Köhler 8 Glaubrecht, 2002a: 151, 152. НО. oP. Melania papillosa Martens, 1897: 38, 39, pl. 2, fig. 21 (“See Singkarah, Sumatra” = Lake Singkarah), lectotype ZMA, 18 paralecto- types ZMA, 15 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 M. stricticosta ZMB 200.102; F: Lectotype of M. papillosa ZMA; G-K: Paralectotypes ZMB 200.103; L-O: Sumatra, Lake Singkarah (ZMB 200.118). Scale bar 10 mm. SYSTEMATIC REVISION OF BROTIA 225 Melania stricticosta Martens, 1897: 39, 40, pl. 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 Kohler & 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 var., laevis, but also two new species, M. papillosa 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 RÉ 2I0 48200 418-200 126; 2007155; 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, B = 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, B = 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. 71B): Ribbon with about 100 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 KOHLER & 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 мукой! (Brandt, 1974) (Figs. 74-76) Brotia (Senckenbergia) мукой Brandt, 1974: 184, pl. 13, fig. 41 (“Creek at Sai Yok, Kan- chanaburi Province”), holotype SMF 197268, four paratypes RMNH 55244/4; types seen. Brotia wykoffi — Kóhler & Glaubrecht, 2002a: 132: FIG. 74. Shell morphology of B. wykoffi (Thailand, Nam Tok; 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), Мат Tok, 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 Tok; ZMB 200.132); apical and front view. Scale bar = 1 mm. SYSTEMATIC REVISION OF BROTIA 22 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, B = 9-11 mm. Embryonic Shell (Fig. 75): Broadly ovate, three whorls; smooth sculpture; aperture wide. Average proportions: H = 2.0 mm, B = 1.4 mm, HA = 0.15 mm, BA = 0.28 mm, DA = 0.62 mm (for n = 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 B. citrina (Fig. 4); ex- cept for both typhlosoles unfused at entire length of style sac. FIG. 76. Radular morphology of B. wykoffi. SEM image of segment viewed from above (Thailand, Nam Tok; ZMB 200.132). Scale bar = 100 um. 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 Tok). Habitat Small, tangled, swift stream discharging into Kwae Noi River. Remarks Somewhat similar to B. 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, pl. 2, fig. 9 (“Java”), lectotype and para- lectotype MHNG, coll. Brot (designated by Kôhler 8 Glaubrecht, 2002a) (Figs. 77A, B); types seen; Brot, 1875: 51, 52, pl. 6, fig. 5. Brotia angulifera — Kóhler 8 Glaubrecht, 2002a: 126, 127, fig. 1B. Taxonomy and Systematics Benthem Jutting (1956) considered this spe- cies a synonym of “B. 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 KOHLER 8 GLAUBRECHT ders a systematic decision. Not identical with Melania (Plotia) scabra var. angulifera 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, B = 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, 191531: Paracrostoma assamensis — Köhler 8 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. huegeli (Philippi, 1843), by Kohler & Glaubrecht (2002a) and because Acrostoma Brot, 1870, is a synonym of Paracrostoma Cossmann, 1900 (Köhler 8 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, 21°37’N, 106°32’E), holo- type MNHN (Fig. 77H); type seen; Dautzen- berg & Hamonville, 1887: 219; Fischer-Piette, 1950: 160, pl. 5, fig. 4. Brotia beaumetzi — Kohler & Glaubrecht, 2002a: 129, fig. 1D. SYSTEMATIC REVISION OF BROTIA 229 FIG. 77. Shell morphology of several species with uncertain classification. A: Lectotype of M. angulifera MHNG; В: Paralectotype MHNG; C-D: Brotia (?) assamensis (Assam, North Cachar, Delaima River; BMNH 19991534); E: Holotype of M. borneensis RMNH 71325; F: Lectotype of M. cylindrus MHNG; С: Lectotype of M. subcylindrica MHNG; H: Holotype of M. beaumetzi ММНМ; |: Lectotype of M. zonata von dem Busch, 1842 ÚMB TK 271/1; J-K: Paralectotypes ÜMB TK 272/2; L-M: Syntypes of Melania canaliculata Reeve, 1869 (BMNH 20050105, ex coll. Cuming, = Melania sooloensis Reeve, 1860); N: Brotia (?) solooensis (Philippines, Sulu Islands; ZMB 59.161). 230 KOHLER 8 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. М. de Merlaincourt)” (Dautzenberg 8 Hamonville, 1887), which corresponds to the label of the type material. The species was transferred to Вгойа by Köhler 8 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: Н = 20 mm, В = 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, pl. 2, fig. 4 (“Borneo”), holotype RMNH 71325 (Fig. 77E); type seen. Brotia borneensis — Kohler & Glaubrecht, 2002a: 130. Taxonomy and Systematics Transferred to Brotia by Kohler & Glaubrecht (2002a) because of its characteristic shell. Next to B. 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, B = 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, ©) Melania cylindrus Brot, 1886: 92, 93, pl. 6, figs. 7, Та (“Siam” = Thailand), lectotype and two paralectotypes MHNG, paralectotype MCZ 11268 (designated by Kohler & Glaubrecht, 2002a) (Fig. 77F); types seen. Melania subcylindrica Brot, 1886: 102, 103, pl. 6, figs. 2, 2a (“Chine” = China), lectotype and two paralectotypes MHNG (designated by Kohler & 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 Kohler & 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: Н = 27.5-42 mm, В = 13.5-19.7 mm. Embryonic shell morphology, Operculum, Radula, Soft body anatomy: Unknown. Distribution Vague: “Siam” and “China” as only known records. Brotia (?) sooloensis (Reeve, 1859) (Figs. 77L, M) Melania canaliculata Reeve, 1859: pl. 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, pl. 14, fig.3. Brotia sooloensis — Kóhler & Glaubrecht, 2002a: 147, 148. Taxonomy and Systematics The name M. sooloensis was employed by Reeve (1860: 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 Brotía, 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 (МНМС; coll. Norris); Isabella (MHNG; leg. Semper), herein restricted to Isabela, Basilan (6°41'N, 118758 B)! Differential Diganosis Shape of shell unmistakable; in particular elongate spire, stepped whorls, subsutural depression or shoulder. Description Shell (Fig. 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, B = 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’М, 121°28’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 (?) spinata (Godwin-Austen, 1872) Melanoides spinata Godwin-Austen, 1872: 514, pl. 30, figs. 1, 1a (“Kopili River, North Cachar hills, a tributary of the Brahmaputra” = Kopili River, Jaintia-Khási hills N of Silchar, federal state of Meghalaya, India); types not seen; Hanley 8 Theobald, 1874: pl. 109, fig. 1. Melania spinata — Brot, 1875: 89, 90, pl. 10, figs. 2, 2a. Melania (Melanoides) spinata — Nevill, 1885: 261. Brotia spinata — Kóhler & Glaubrecht, 2002a: 148 [partim]. Taxonomy and Systematics Type material was not traced. Shell only known from original figure. Attributed to Brotia by Kôhler 8 Glaubrecht (2002a) as being typi- cal for Brotia. Geographical distribution well within range of the genus. Köhler 8 Glaubrecht (2002a) assumed that B. binodosa is conspe- cific for the similar shell. However, in their re- 232 KOHLER & GLAUBRECHT vision of the Kaek River species flock, Glaub- recht & Kóhler (2004) show that B. binodosa is endemic to central Thailand and, thus, not conspecific with B. spinata. 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 (?) zonata (Benson, 1836) (Figs. 771-K) Melania zonata Benson, 1836: 747 (no figure); types not seen. Melania zonata Busch, 1842 — In: Philippi, 1842: 3, pl. 1, fig. 12 (“Вепдайа”), lectotype UMB TK 271/1, two paralectotypes UMB TK 272/2 (designated by Knipper, 1958) (Figs. 771-К); types seen. Melanella zonata - H. Adams £ A. Adams, 1854: 296. Brotia zonata — Kohler & 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, 2002а). 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 (Figs. 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 (СО!) and 826 bp (16$), 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 (COI 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 (СТК+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 СО! infraspecific dis- tances usually do not exceed 16% (in B. citrina) and mostly range between 0 and 6%. The high sequence divergence in B. sumatrensis is very striking. Since a similar divergence is not observed in 16$, we assume that the one sequence of B. sumatrensis high- 233 SYSTEMATIC REVISION OF BROTIA 92 0-Sc 0 cc 0 ¿10 810 ST 0-E20 210 610 ЕО ve 0 02 0-8L'0 1210 210-010 | 591 K 100 «LG 1610 120-80 1170 120-720 20-120 820-120 120-60 220-020 20-60 220-120 220-170 109 HONG 90 0-G0'0 80 0 OL O0 6LO 600100 10-010 150060 150-060 9¢20-S20 150-810 LL'O LL'O-OL'0 | 591 90 0 *E6 0010 610-810 CcO-ICO 960-800 020-210 tve0-€20 0Z'0-61'0 (0740 6c 0-02 0 210 10-210 109 11994191 £0"0-0 [4 591 90'0-0 109 ejenb10] 60'0-80'0 #00 0-0 С 591 86 0-01 0 +67 0-0 109 sısuaewns 00 OL O0 10`0-0 С 591 810-910 «LE l-ZL 0 90'0-0 109 euela/os (Ame) LL'O Z0'0-10'0 GL'O 020-810 200-100 060-610 9,0 £cO-cc O0 21L0-SL0 €00-100 700-000 | 591 6LO-/LO ,6c1-8L0 ¿0'0-90'0 610-910 820-220 900-200 960-120 ccO-8L 0 ce 0 LLO-pL'O co 0 90 0-00 0 109 endsoo¡nsopnasd 61'0-81'0 020 eb 07210 Ss00'0-0 @ 591 210-90 69140 050-90 z0'0-0 109 syejnsujued Gc 0-€¢2 0 e2 07020 810-910 020-810 OL 0-0 С SOL $< 0-80 „8916,0 820-720 810-410 50-0 109 eınpobed ZL 00,0 LL'O-OL'O c0 0-0 GLO-€L0 6L'0-91'0 z0'0-0 E 591 020-210 „ЗЕ 1210 80 0-0 OZ'0-91'0 620-920 S0 0-0 109 взатозолоши vZ0-2Z'0 Léi0-0¢0 (905910 0500 050-810 80-920 s0'0-0 S 591 960-100 co EGO 960150 90-40 160100 760-050 60'0-0 109 eanalsy Eco EAN 0 60-30 90-50. 60-70’ Céi0=l60 “SHO-SlO0 60—10 z0'0-0 @ 591 ОсОО ЕЛЕ О 200-80’ Роб 70a ECONO" ET 0200 s0'0-0 109 BEYOUUBY 62 0-220 VGi0nECIOL 12028107 700-500 620-0610’ 5610-0910 SOLOS 6610020 200-0 € 591 80-90 89717610 020-810 SLO 70-150 cc0-020 Zt 0-910 cc 0 z0'0-0 109 eue/blaquez пер ÿL'O AO) OL'O 9,0 020 O) 610 910 921075210 0-90 1110-0710 10-00 | 591 810-910 ,9€'L-8L'0 vc 0 6LO-ZL'0 eco ÿcO-CcO 920-S20 920-520 Gz 0 AO OA 7002640 109 E/NJSO9 ÿc 0-02 0 760-020 SOS TOM SHOSZL0 910-00 170-70 60-0 050-600’ 650-610 OL 0-0 С sg, 920-810 .byc-ZLO 220-810 810-710 8LO-FL0 920-210 9550-80 “60-6170 020-210 910-0 109 gun AO NANA AN AAA O) c0 0-0 6} 0-10 910-90 €20-00 “210-70 z0 0-0 G 591 ÿL'O-EL'O 020-210 80'0-S0'0 0Z'0-91'0 860-750 90 0-0 G6 0-26 0) 2Li0,81i0 190020 97079110 GL'0-0 109 esopouiq cL O-LL'O GLiO-OL OM 3801022007 “0-70 76020 20i0-1010, 02072502 2059107500 081070601 0=7 010 £0°0-0 9 SOL 610-270 :,8£ 1-20 800-900 050-970 620-720 900-000 ryce0-6l0 650-810 660610 960—410 90 0-0 10 0-0 109 als sisuan] ejdjnos euelbiaq ejyenbio} -pwins eueiwajos зиепзииэа ejnpobed OJO eejnoley Sepauusy -veszmep BULI}IO esopoulq ejeuwe N ‘(UOISSNOSIP Bas ‘anjeA зпо!апр , 'SADUEBISIP эшоэдзеци! = pjog ul pajulid ¿sargads jad pasÁjeue saouanbas jo лэашпи = N) (SMOA Jamo]) SOL pue (SMO Jaddn) 109 JO} sardads 2701g иээмэа pue UIUJIM (I+1+H19) Sa9uejsip эцаиэ9 ‘6 FIGVL 234 KOHLER 8 GLAUBRECHT TABLE 6. Sequence data analysed in this study with GenBank accessions and inventory numbers. Genus Species Inventory No. Origin COI 16$ 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 DQ 284985 DQ 284986 В. dautzenbergiana ZMB 200.226 Thailand AY 330831 AY 330802 ZMB 200.229 Thailand AY 330832 AY 330800 B. henriettae ZMB 200.210 Thailand AY 330845 AY 330793 ZMB 200.221 Thailand AY 330846 AY 330794 B. herculea 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 200.116 Sumatra AY 330838 AY 330784 ZMB 200.119 Sumatra AY 330840 AY 330785 B. torquata ZMB 200.117 Sumatra AY 330864 AY 330781 ZMB 200.121 Sumatra AY 330865 AY 330782 B. verbecki ZMB 200.118 Sumatra AY 330863 AY 330779 B. 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 ine iE Jagora asperata ZMB 200.111 Jagora dactylus ZMB 200.1109 Adamietta housei ZMB 200.165 sof Adamietta hainanensis ZMB 200.301 = 71 Adamietta cf schmidti ZMB 200.053 00 de Adamietta testudinaria ZMB 200.099 100 Adamietta testudinaria ZMB 200.100 ‘oof Paracrostoma spec. ZMB 200.318 100 Paracrostoma spec. ZMB 200.322 Brotia armata ZMB 200.193 A Brotia armata ZMB 200.254 HOO} 99 | 70 j = | sof Brotia armata ZMB 200.268 100] - Brotia armata ZMB 200.268a Brotia pseudosulcospira ZMB 200.196 > Brotia binodosa ZMB 200.267 a7 Brotia binodosa ZMB 200.269 97 Brotia binodosa ZMB 200.328 a Brotia microsculpta ZMB 200.200 Brotia microsculpta ZMB 200.266 я Brotia armata ZMB 200.265 | Brotia binodosa ZMB 200.202 65 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 ‘oof — Brotia sumatrensis ZMB 200.116 100 Brotia sumatrensis ZMB 200.119 100 Brotia torquata ZMB 200.117 Brotia torquata ZMB 200.121 Brotia verbecki ZMB 200.118 Brotia costula ZMB 112.660 roof 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 Ш Brotia citrina Г МВ 200.207 > {se Brotia pagodula ZMB 200.205 À sel Brotia pagodula ZMB 200.208 Brotia dautzenbergiana ZMB 200.213 ‘ap Brotia dautzenbergiana ZMB 200.229 100 Brotia dautzenbergiana ZMB 200.226 100 Brotia herculea ZMB 200.206 00400 Brotia herculea ZMB 200.209 2 E Brotia herculea ZMB 200.219 Brotia herculea ZMB 200.220 Brotia herculea ZMB 200.253 FIG. 78. Confidence limits on the topology of the MP strict consensus cladogram of concatenated data set of 16S 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 KOHLER 8 GLAUBRECHT Jagora asperata ZMB 200.111 Jagora dactylus ZMB 200.1109 Adamietta hainanensis ZMB 200.301 Adamietta cf schmidti ZMB 200.053 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 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 binodosa ZMB 200.202 Brotia armata ZMB 200.265 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 200.116 Brotia sumatrensis ZMB 200.119 Brotia torquata ZMB 200.117 Brotia torquata ZMB 200.121 Brotia verbecki ZMB 200.118 Brotia costula ZMB 112.660 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 200.207 Brotia citrina ZMB 200.212 Brotia pagodula ZMB 200.205 Brotia pagodula ZMB 200.208 Brotia dautzenbergiana ZMB 200.213 Brotia dautzenbergiana ZMB 200.229 Brotia dautzenbergiana ZMB 200.226 Brotia herculea ZMB 200.206 Brotia herculea ZMB 200.209 Brotia herculea ZMB 200.219 Brotia herculea ZMB 200.220 Brotia herculea ZMB 200.253 50 changes FIG. 79. Bayesian inference phylogram of concatenated data set of 16$ and COI. SYSTEMATIC REVISION OF BROTIA 231 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 0 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 (Kóhler 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 1$, 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 ofthe 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. lt 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 predation (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 intraspecific variation, it has frequently been assumed that 238 KÔHLER & 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 Kóhler 8 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 (Frómming, 1956), but also by the harshness of physical environmental factors (Vermeij, 1972), parasitism (Wright, 1966), or predation (Zipser 8 Vermeij, 1978; 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., B. 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 (Kôhler & 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. henriettae). The uncalcified apex was called by him an “open” or “soft apex” and stated to be a characteristic feature of Brotia. Subsequently, Solem (1966: 16, fig. 1) depicted several embryonic stages of B. binodosa with protruding soft tissue and open арех; this was followed by a report an “asymmetric” apical portion of the juvenile shell of B. 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 ff.). 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 (1993) has hypothesized that delayed calcification 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: Kóhler 8 Glaubrecht, 2003). However, the phylogenetic relationships between these two genera indicate that open apex and protrud- ing yolk sac have evolved independently (Kôhler 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 (figured in Kohler & 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 Kôhler & 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 (1857-58) based his classification 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, 1929). 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 Faunus 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 о 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 KOHLER 8 GLAUBRECHT Radula In general, the molluscan radula is consid- ered a conservative character with little varia- tion on the species level (Fretter 8 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 8 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 8 Mak, 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: pl. 9; Fischer & Crosse, 1892: pl. 49, fig. 14; Simone, 2001: figs. 95, 96), Doryssa (Simone, 2001: figs. 89-92), Potadoma (Glaubrecht, 1996: pl. 5, figs. 7, 8), Jagora (Kóhler 8 Glaubrecht, 2003), Sulcospira (Troschel, 1858: pl. 9, fig. 6; Köhler 8 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., B. 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, 1991). 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 pallial oviduct to the brood pouch (Fig. SF). Reproductive Organs: These are the most in- formative for pachychilid systematics (Kôhler 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 Brotia was first mentioned by Martens (1897: 29). Later, Moore (1899: 161, 162; pl. 14, fig. 13; pl. 16, fig. 2), Morrison (1954: 383), Davis (1971: 69), and Kôhler & Glaubrecht (2001) described this pouch in more detail. А homologous brood pouch is found т Adamietta (Brandt, 1974) and Paracrostoma (unpubl. data) and is considered as а synapomorphy of the Asia mainland clade among the Pachychilidae (Kôhler 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 (Kóhler & Glaubrecht, 2003), while pachychilid Pseudopotamis and 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 (Kôhler 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; Kôhler 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 pallial ovi- duct and the arrangement of the gonads. Among Asian Pachychilidae, Brotia pos- sesses the simplest pallial 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 (Kóhler & Glaubrecht, 2001, for the Brotia testudinaria group). Again, Jagora, Pseudopotamis, and Tylomelania possess oviduct morphologies that significantly deviate from Brotia (Glaubrecht 4 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 4 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 (Kohler 8 Glaubrecht, 2003), and Tylomela- nia (Rintelen 8 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 16 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 Branat (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 (Kôhler, unpubl. data) endemic to southern India. 242 KÔHLER & 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 В. 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 & Kôhler, 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+I+I model of sequence evolution does not exceed a maximum of 16% in COI 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., Kóhler & 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 B. 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 et al., 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, Pachychilus, 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 by Troschel. 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, Melanatria, and Potadoma were 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 (1921), 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 (Kohler et al., 2004). As a consequence, the view of Morrison (1954) and Houbrick (1988), who strongly emphasized features of the soft body, in particular of the reproductive tract, on the systematic position of Brotia is refuted. 244 KÔHLER & 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 Thiaridae (Köhler 8 Glaubrecht, 2001; Köhler et al., 2004). (2) Phylogenetic 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, Kóhler & Glaubrecht (2001) identified four different species groups among what was previously considered as constituting Brotía, 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 (Kóhler & Glaubrecht, 2003; Glaub- recht & Rintelen, 2003; Köhler et al., 2004; Rintelen 8 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 pallial 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 (Kóhler 8 Glaubrecht, 2003), (2) Tylomelania endemic to Sulawesi (Rintelen 8 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 (Kôhler et al., 2004). This sug- gestion is corroborated by published and also unpublished morphological data (Kóhler 8 Glaubrecht, 2001; Kóhler, 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 Kóhler 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 (1974) 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; Kohler et al., 2004). All Thai species affiliated with Paracrostoma by Solem (1966) and Brandt (1968, 1974) are members of Brotia since they are not closely related to Paracrostoma from southern India but cluster together within Brotia (Glaubrecht & Kohler, 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. lt 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. herculea) 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 Schütt, 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 Hánggi (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 lan 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 KÔHLER & 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. LITERATURE CITED ABBOTT, R. 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Proceedings of the Malacological Society London, 24: 170- 289. ZIPSER, Е. & С. J. VERMEIJ, 1978, Crushing behaviour in tropical and temperate crabs. Journal of Experimental Marine Biology and Ecology, 31: 155-172. 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 Fujian 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. Key words: schistosomiasis, Schistosoma japonicum, China, polymorphism, Oncomela- nía, 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 ET AL. 1992, 1994; Davis et al., 1995, 1999a). All 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. tangi 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- taries. 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 varix (special thickening 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 CO1 gene sequencing). Based on breeding genetics, ribbing in Oncomelania hupensis is controlled by a single locus (Men- delian inheritance of a single gene) where ribs TABLE 1. X comparison of shell polymorphisms of shell ribbing on Oncomelania hupensis hupensis snails from two Hubei Canals in 2001. = number of shells from living snails. Data given as % of N. P> 0.0001. See text for details. Ma Ling Gu Hu (N = 101) (N = 105) M 13.9 43.8 SL 61-3 40.0 S-/S 24.8 16.2 are dominant, smooth recessive, and with mul- tiple alleles of that gene (Davis & Ruff, 1973). Likewise, size is controlled by alleles at a single locus. АП 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 Си Ни Canal (30°19.129’N, 112°23.386’Е 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 1960, 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 12 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 ET AL. 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 ofthe 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 рег 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. 1C): 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 Х? 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 1/3 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 Х? 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; S- = trace of ribbing; SM = smooth. Ma Ling Gu Hu Site 1 Site 17 Site 19 Site 20 Site 16 (N = 15) (N = 12) (N = 122) (N = 96) (N = 77) S 0 0 2 (16%) 0 0 М 15 (100%) 1 (8.3%) 31 (25.4%) 39 (40.6%) 38 (49.4%) SL 0 10 (83.3%) 74 (60.7% 52 (54.2%) 37 (48.1%) S- 0 1 (8.3%) 14 (11.5%) 5 (5.2%) 2 (2.6%) $М 0 0 1 (0.8%) 0 0 ECOGENETICS OF SHELL SCULPTURE IN ONCOMELANIA РТ 120 Sites 100 80 60 40 % of shells with morph type 20 МЕ _ 5 categories of morphs FIG. 2. The percent of morph types in the Ma Ling sites. $ = strongly ribbed; М = 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 17 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 2001 140 + 120 2004 О 0.00013 80,77 No. of snails 60 40 20 = A + = = S M SL S-/SM S M SL S-/SM Classes of polymorphisms 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 ET AL. 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 Ma Ling 2001 Gu Hu 2001 Ma Ling 2001 Gu Hu 2004 Gu Hu 2004 Ma Ling 1 2004 Ma Ling 17 2004 Ma Ling 19 2004 Ma Ling 20 - HSD canal intersection) were unique in having 83% slightly ribbed shells (but again low numbers, i.e., 12). Sites 19 and 20 were in zone 2. A cross comparison of sites for significant differences 50 2004 Ma Ling 1 HSD HSD 2004 2004 2004 Ma Ling 17 Ma Ling 19 Ma Ling 20 NS SD SD SD VSD SD SD VSD NS HS HSD HSD - NS NS : BNS (0.060) (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) 2001 2004 Р = 0.0121 No. of snails SL S-/SM S Classes of polymorphisms M S-/SM FIG. 4. Comparing Gu Hu snails from 2001 and 2004 for shell polymorphisms. See Fig. 2 for abbreviations. Х? = significant difference but barely so. % of snails ECOGENETICS OF SHELL SCULPTURE IN ONCOMELANIA 259 19 20 ne erg MA ; T T A | T T T # T S M SL S- SM 5 М SL S- SM Classes of polymorphisms 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 ET AL. 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. Predation 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 $. japonicum (Davis et al., 1999a; Wilke et al., 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., 1999a) 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 100 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 > 0.015 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 truncata 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 ET AL. sis 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 $. 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 8 Williams (1963), Chi et al. (1971), He et al. (1991), Lin et al. (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 1$ 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. hupensis hupensis 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 13 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- 2P50A139461-06A1 (GMD, Co.P.l.-Project II). This is part of the М.1.Н. - 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. LITERATURE CITED CHI, L. W., E. D. WAGNER & N. WOLD, 1971, Susceptibility of Oncomelania hybrid snails to various geographic strains of Schistosoma japonicum. 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Chinese Journal of Zoology, 27(4): 12-14. 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 Е. Wade", Eric С. Chapman, Arthur E. Bogan’, С. Thomas Watters* & Walter К. Hoeh' ABSTRACT Unionoid bivalves exhibit a great diversity in reproductive characteristics. However, the lack 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- (Е) 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) ls DUI found in the Etherioidea? (2) What is the relative phylogenetic utility of F, M, and concatenated F + M cytochrome с oxidase subunit | (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 М coxT DNA sequences were available as well as on a much more taxonomically inclusive F cox7 data set. We deter- mined that (1) the Etherioidea likely lacks DUI; (2) M and F + М cox1-based analyses provide better resolved estimates of unionoidean relationships than do F cox1-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, U.S.A. 2University of Southern California, Los Angeles, California 90089, U.S.A. ¿North Carolina Museum of Natural Sciences, Raleigh, North Carolina 27607, U.S.A. “Department of Evolution, Ecology and Organismal Biology, Ohio State University, Columbus, Ohio 43212, U.S.A. “Corresponding author: jwalker4@kent.edu 266 WALKER ET AL. 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 Нугидае Margaritiferidae Unionidae Iridinidae Mycetopodidae E Margaritiferidae Unionidae Iridinidae Mycetopodidae Hyriidae derived freshwater lineages and thus the Unionoida is a polyphyletic assemblage (Paro- diz 8 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 Iridinidae Mycetopodidae Hyriidae Unionidae Margaritiferidae D Unionidae Mycetopodidae Iridinidae Hyriidae Margaritiferidae FIG. 1. Simplified representations of unionoid familial relationships based on the A: Classifications of Simpson (1900) and Parodiz 8 Bonetto (1963); В: Phylogenetic analyses of morphological characters after Graf (2000) and Hoeh et al. (2001); C: 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). DUI 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 & О Foighil, 2000; Hoeh et al., 1998a, 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 8 O Foighil, 2000; Hoeh et al., 1998a, 2001; Graf, 2000; Roe 8 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 8 Bonetto's (1963) 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. 1B). In contrast, both the molecular (i.e., cox? DNA sequences) and the combined analysis of morphological and molecular data presented by Hoeh et al. (1998a, 2001) (Fig. 1C) place the Hyriidae as the basal unionoid lineage, with the Unionidae returned as paraphyletic. Yet another combined analysis of morphological and molecular (1.е., cox1 DNA sequences) characters (Roe & Hoeh, 2003; Fig. 1D), 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 (DUI) in that taxon. DUI 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 et al., 1998). Therefore, taxa possess- ing DUI 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 & О’ 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 (Curole & 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 DUI has been operating at a high level of fidelity in the Unionoidea for more than 100 my (Curole & Kocher, 2002, 2005; Hoeh et al. 1996, 2002). The fidelity of the DUI 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 ET AL. 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 “М” 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 “М” 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; Rawson et al., 1996; Quesada et al., 1999; Ladoukakis et al., 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; Curole 8 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 et al., 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 miDNA 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 c oxidase subunit | (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): LCO22me2 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 Curole & Kocher (2002). These primers were chosen due to the size differ- ence exhibited between the F and M cox2- cox1 fragments as described by Curole 8 Kocher (2002). The “male-specific” cox2 primer was UNIOCOII.2 (Curole, 2004) and а “female-specific” primer (UNIOCOII.2b, 5'- CAGTGRTATTGRRVDTAYGA-3') was derived from the UNIOCOII.2 primer and other unionid 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 1X Qiagen PCR buffer, 0.2 mM each dNTP, 0.5uM each primer, and Qiagen Тад. Reactions us- ing the cox1 primer pair were cycled at 94°C for 60 $, 40°C for 60 $, and 72°C for 60 $ 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 120sfor 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 619 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- DUI IN THE UNIONOIDA 269 TABLE 1. Таха evaluated for the presence/absence of F and M cox2-cox1 amplicons; + = amplifica- tion successful, - = amplification failed, NA = amplification not attempted. cox2-cox1 сох2-сох1 Family Species amplicon F amplicon M Unionidae Actinonalas ligamentina Amblema plicata Anodonta californiensis Cyprogenia aberti Cyrtonaias tampicoensis Dromus dromas Ellipsaria lineolata Elliptio dilitata Epioblasma brevidens Fusconaia flava Glebula rotundata Hamiota subrotundata Lampsilis cardium Lampsilis hydiana Lampsilis powellii Lampsilis reeveiana Lampsilis siliquoidea Lampsilis straminea Lampsilis streckeri Lampsilis teres Leptodea fragilis Leptodea leptodon Ligumia recta Medionidus conradicus Obovaria olivaria Popenaias popeli Potamilus alatus Potamilus capax Potamilus ohiensis Potamilus purpuratus Ptychobranchus fasciolare Toxolasma glans Truncilla truncata Venustaconcha ellipsiformis Villosa iris ++++ D CR ep Е ОР Е Е ЕЕ PRO Е ЕЕ и Villosa lienosa + Villosa villosa + Margaritiferidae Cumberlandia monodonta + Dahurinaia sp. - 5 Margaritifera margaritifera = + Hyriidae Hyridella menziesi + + Iridinidae Chambardia rubens + = Mutela dubia + = Etheriidae Etheria elliptica + = Mycetopodidae Anodontites guanarensis + = Tamsiella tamsiana + = Neotrigoniidae Neotrigonia margaritacea - NA oo 270 WALKER ET AL. ers. The 3' portion of the M cox2-cox1 frag- ment was sometimes sequenced, using the HCO700dy2 sequencing primer, to confirm М cox1 sequences generated by the cox7 primer pair. Sequences were visualized using Li-Cor 4200L-2 and 4200$-2 DNA sequencers and initial base calls were made by e-Seq v 2.0. Contiguous sequences were assembled and verified using AlignIR v2.0 and final sequence alignments were completed manually with MacClade v4.0. GenBank accession numbers for the cox1 sequences generated and/or ana- lyzed herein are given in Table 2. All testis extraction-derived cox1 sequences were added to a matrix containing confirmed F and M cox1 sequences and phylogenetic analy- ses were used to test the putative M status of the newly generated sequences. Subsequent to the initial attempts to amplify the M cox2- cox1 fragment from their testis-derived total DNAs, multiple attempts were made to am- plify an M mitochondrial fragment from mem- bers of the Iridinidae and Mycetopodidae using two, intragenic universal primer pairs (i.e., cox1 and 16$ [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 cox7 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 Е and М genome сох1 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 BI 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 cox? 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 + | 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 cox? 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 BI 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 BI 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 et al., 1998a), all MP analyses were conducted on transformed cox? 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 cox7 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 extrac- DUI IN THE UNIONOIDA 271 TABLE 2. Source taxa and GenBank accession numbers for the cox1 DNA sequences used in phylo- genetic analyses. Family Unionidae Margaritiferidae Species Actinonias ligamentina Cyrtonaias tampicoensis Fusconaia flava Gonidea angulata Lampsilis teres Ligumia recta Potamilus purpuratus Pseudodon vondembuschianus Pyganodon fragilis Pyganodon grandis Cumberlandia monodonta 1 Cumberlandia monodonta 2 Cumberlandia monodonta 3 Cumberlandia monodonta 4 Dahurinaia dahurica 1 Dahurinaia dahurica 2 Dahurinaia dahurica 3 Margaritifera auricularia 1 Margaritifera auricularia 2 Margaritifera auricularia 3 Margaritifera auricularia 4 Margaritifera falcata 1 Margaritifera falcata 2 Margaritifera falcata 3 Margaritifera laevis Margaritifera margaritifera 1 Margaritifera margaritifera 2 Margaritifera margaritifera 3 Margaritifera margaritifera 4 Margaritifera margaritifera 5 Margaritifera margaritifera 6 Margaritifera margaritifera 7 Margaritifera margaritifera 8 Margaritifera margaritifera 9 Margaritifera margaritifera 10 Margaritifera margaritifera 11 Margaritifera margaritifera 12 Margaritifera margaritifera 13 Margaritifera margaritifera 14 Margaritifera margaritifera 15 Margaritifera margaritifera 16 Margaritifera margaritifera durrovensis 1 Margaritifera margaritifera durrovensis 2 Margaritifera margaritifera durrovensis 3 Margaritifera margaritifera durrovensis 4 Margaritifera margaritifera durrovensis 5 Margaritifera margaritifera durrovensis 6 GenBank Accession No. Е AF231730 AF231749 AF231733 DQ206792 AF406803 AF231748 AF406804 DQ206793 AF406805 AF231734 AY785393 AF156498 AF156497 AY579131 AY579123 AY579125 AF303312 AF303313 AF303315 AY579126 AY579128 AY579127 AY579124 AF303319 AF303320 AF303336 AF303341 AY579129 AY579130 AF303331 AF303332 AF303338 AF303340 AF303335 AF303337 U56847 DQ060171 AF303339 AF303344 AF303345 AF303346 AF303347 AF303342 AF303343 M AF406796 AF406798 AF406799 DQ206794 AF406794 AF406795 AF406797 DQ206795 AF406800 AF406801 AY785397 AY785400 DQ241802 AY785399 (continues) 272 WALKER ET AL. (continued) ААА GenBank Accession No. Family Species F M Hyriidae Alathyria jacksoni 1 AY386977 Alathyria jacksoni 2 AY386981 Alathyria jacksoni 3 AY386970 Alathyria jacksoni 4 AY386974 Castalia stevensi AF231736 Diplodon deceptus AF231736 Hyridella australis AF305367 Hyridella depressa 1 AF 156496 Hyridella depressa 2 AF305368 Hyridella menziesi 1 AF231747 Hyridella 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. A 2 AY 211554 Velesunio sp. B 1 AY211558 Velesunio sp. B 2 AY211566 Velesunio sp. D 1 AY 211587 Velesunio sp. D 2 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 Etheria elliptica 2 AF231739 Outgroup taxa Albinaria turrita X71393 Dentalium sp. U56843 Drosophila yakuba X03240 Katharina sp. U56845 Lepetodrilus elevatus U56846 Neotrigonia margaritacea U56850 Solemya velum U56852 ee DUI 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 16$ 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 8 Bonetto, 1963; Hoeh et al., 1998b, 2001; Bogan & Hoeh, 2000; Roe & Hoeh, 2003). Given their phylo- genetic propinquity, itis 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; Curole 8 Kocher, 2002; Hoeh et al., 2002; Krebs, 2004). (3) These taxa do not possess DUI. Mitochondrial DNA masculinization events in DUI-containing taxa were first postulated for, and later supported with data from, Mytilus by Hoeh et al. (1996, 1997). 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 “М” (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 cox? 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., 1996; 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; Curole & 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 76S, cox1, and cox2 or (2) these specimens lack DUI. 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 DUI is absent from the Etherioidea. This leads to the question of whether the absence of DUI in this superfamily indicates a loss in the ancestral etherioidean lineage or a gain of DUI 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 DUI in Neotrigonia, there remains no a priori way to rigorously evaluate which condition is apo- 274 WALKER ET AL. 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 Е coxT se- quences (Folmer fragment), have been unable to robustly resolve unionoid familial relation- ships. However, M cox? sequences have dem- onstrated the ability to increase topological resolution when analyzed alone or in conjunc- tion with F cox? 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 cox7 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 serious 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 cox? 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 Е cox? 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 cox? 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 М cox? sequences are significantly im- proved over those of the F cox7 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 М cox? 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 conglutinate mass than that of its ancestor. If the relative evolutionary relationships pos- tulated from the complementary F and M con- catenated cox1 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 DUI IN THE UNIONOIDA Actinonaias BI PP iL slis МР. 57 ampsiis (ML) 100 Ligumia 92 (84) 78 Potamilus 60 Cyrtonaias Fusconaia 12 Cumberlandia Gonidea 69 77 Pseudodon Inversidens 63 Pyganodon fr. 100 100 (100) Pyganodon gr. Anodonta Hyridella Neotrigonia 0.5 substitutions/site 275 Unionidae Margaritiferidae Unionidae Hyriidae 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 BI PP MP (ML) 61 100 95 (84) 0.1 substitutions/site 86 WALKER ET AL. 90 70 Actinonaias (72) a 100 Lampsilis 75 (89) 60 Ligumia 100 Cyrtonaias 90 у (89) 100 Potamilus 96 (96) Fusconaia 100 Unionidae 76 . Pyganodon fr. 100 100 (99) c о 100 Pyganodon gr. 99 (98) Anodonta Inversidens Gonidea Pseudodon Cumberlandia Margaritiferidae Hyriidae Hyridella Neotrigonia FIG. 3. Best tree found by Bayesian analyses of M cox1 sequences (619 bp). When > 50, Bayesian posterior probabilities (х100) presented above internodes, MP bootstrap values and ML bootstrap values (in parentheses) are presented below internodes. DUI IN THE UNIONOIDA 211 81 (72) Actinonaias 100 < BI PP ee 7 MP Е re Lampsilis (ML) 100 84 (88) A Ligumia 100 Potamilus 98 (98) 100 Cyrtonalas 98 (98) Fusconaia 20 Unionidae 56 Pyganodon fr. 100 100 (100) El À 80 95 Pyganodon эт. 97 (52 (92) Anodonta 52 Inversidens 94 96 Gonidea (77) 90 69 Pseudodon (60) Cumberlandia Margaritiferidae Hyridella Hyriidae Neotrigonia — 0.1 substitutions/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 ET AL. 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, if Neotrigonia possesses DUI, this A Unionidae loss of endobranchy B Margaritiferidae Unionidae loss of endobranchy gain of DUI 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- Margaritiferidae Hyriidae Etherioidea gain of endobranchy Etherioidea loss of glochidium loss of DUI Hyriidae gain of glochidium gain of endobranchy gain of DUI FIG. 5. Hypothesized character state transitions for reproductive characters exhibited by unionoid bivalves. A: Hypothesized familial relationships after Parodiz 8 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. DUI 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 DUI, (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 8 Bonetto, 1963) as well as Parodiz 8 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 1C (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 = 100 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 DUI, glochidial larvae, and endobranchous brood- ing characterized the ancestral unionoid lin- eage, (2) the loss of DUI 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 BI 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 cox7). 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, C. Barnhart, B. 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. Kuehnl, B. Lang, J. Maunder, T. Myers, В. Posey, В. Sietman, D. Smith, С. Soliman, R. Tankersly, R. Trdan, M. Vidrine, and D. Zanatta. We also thank D. Senyo, C. 280 BI PP MP «km wor DB WALKER ET AL. 99 94 Q 99 00 = 35 100 Ann 88 100 65 08 79 100 100 00 Fa 100 88 4 100 0 53 00 % BA 61 96 00 ; 61 400 чм [ 81 78 Fr 92 10 100 А 97 ps LS an 100 D 65 97 100 ann 93 100 ann 100 dB Margaritifera du Margaritifera du Margaritifera du Margaritifera du Margaritifera du Margaritifera du Margaritifera ma Margaritifera ma Margaritifera ma Margaritifera ma Margaritifera ma Margaritifera ma Margaritifera ma Margaritifera ma Margaritifera ma Margaritifera ma Margaritifera ma Margaritifera ma. 12 Margaritifera ma. 13 Margaritifera ma. 14 Margaritifera ma. 15 Dahurinaia da. 1 Margaritifera fa. 1 Margaritifera fa. 2 Margaritifera fa. 3 Margaritifera la Margaritifera au. 1 Margaritifera au. 2 Margaritifera au. 3 Margaritifera au. 4 Cumberlandia mo. 1 Cumberlandia mo. 2 Cumberlandia mo. 3 Cumberlandia mo. 4 Ligumia re Actinonaias li Lampsilis te Potamilus pu Cyrtonaias ta Fusconaia fl Gonidea an Pseudodon vo Pyganodon fr Pyganodon gr F Anodontites qu Anodontites tr Tamsiella ta Monocondylaea mi Acostaea п Chambardia ru. 1 Chambardia ru. 2 Chambardia ru. 3 Etheria el. 1 Etheria el. 2 Mutela du. 1 Mutela du. 2 Mutela du. 3 Mutela ro. 1 Mutela ro. 2 Alathyria ja. 1 Alathyria ja. 2 Alathyria ja. 3 Alathyria ja. 4 Velesunio B 1 Velesunio B 2 Velesunio an Lortiella ru Velesunio À 1 Velesunio À 2 Velesunio am. 1 Velesunio am. 2 Velesunio sp. 1 Velesunio am. 3 Velesunio am. 4 Velesunio sp. 2 Velesunio D 1 Velesunio D 2 Hyridella me. 1 Hyridella de. 1 Hyridella de. 2 Hyridella au Castalia st Diplodon de Neotrigonia ma Lampsilis te Actinonaias li Ligumia re Cyrtonaias ta Potamilus pu Unionidae Fusconaia fl Pyganodon fr ae M Pyganodon gr Unionoidea Gonidea an Psuedodon vo Margaritifera ma. 16 Dahurinaia da. 2 Dahurinaia da. 3 Cumberlandia mo Hyridella me. 2 Solemya ve Lepetodrilus el Katharina sp Dentalium sp Albinaria tu Drosophila ya x — (D O0 -4 © о BA ma PA DA о |Margaritiferidae Unionoidea Unionidae Mycetopodidae [riánidae Etherioidea |Etheriidae Iridinidae Unionoidea Hyriidae Neotrigoniidae Trigonioidea Trigonioida Margaritiferidae I Hyriidae Outgroups FIG. 6. 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RICHTER, 2001, Larval types and early post- larval biology in Naiads (Unionoida). Pp. 93- 125, in: G BAUER & К. WACHTLER, eds., Ecology and evolutionary biology of the freshwater mussels, Unionoida. Ecological Studies, Vol. 145. Springer-Verlag, Berlin. ZOUROS, E., A. O. BALL, C. SAAVEDRA & K. R. FREEMAN, 1994, An unusual type of mito- chondrial DNA inheritance in the blue mussel Mytilus. Proceedings of the National Academy of Sciences USA, 91: 7463-7467. 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, Margaritifera auricularia has been confused with its relative M. margaritifera. 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. margaritifera. 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 (1758). One of these illustrations, Magnus (1555), 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. margaritifera, which has been exploited since Roman times for its ca- pacity to produce small pearls (Bonnemere, 1901). Margaritifera margaritifera was first de- scribed by Linnaeus (1758) as Mya margari- tifera. Margaritifera auricularia was originally named as Unio auricularius 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. auricularius, his description of its large dorsal teeth and the hinge clearly differenti- ate it from M. margaritifera. Lamarck (1819) ‘Dr. Federico Rubio 4, 28039 Madrid, Spain described Unio sinuata (Fig. 1), which today is considered to be a synonym of M. auricu- laria. 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 Historiae conchyliorum (1686: fig. 149) as an illustra- tion of Unio auricularius. However, Lister's fig- ure shows the inside of a large, very sinuate M. margaritifera valve with pronounced cardi- nal teeth, and which at first glance resembles a valve of M. auricularia (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 ¿Museo Nacional de Ciencias Naturales, José Gutiérrez Abascal 2, 28006 Madrid, Spain; rafael@mnen.csic.es 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 pl. 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. auricularia 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. margaritifera 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 ofthese 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. margaritifera (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 auricularius 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. auricularia shells from Pleistocene sediments of the Thames River to Unio (Margaritana) margaritifer (Linnaeus) (= M. margaritifera). (Margaritana is an objective synonym of Margaritifera.) 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. auricularia). 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. auricularia. 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 Schróter's Die Geschichte der Flüssconchylien (1779: pl. 4, fig. 1) represents M. auricularia 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 Schróter's book. Schroter's specimen of Mya testa crassa is not, as they claim, a medium- sized specimen of M. margaritifera, 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 (Schróter) by Kennard et al. (1925). Simpson (1900: 677, note 4) makes the same error by including Mya testa crassa (Schróter) as a synonym for the species Margaritana crassa (Retzius) in his records of M. auricularia. 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. margaritifera (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 Historiae animalium Angliae (1681), some years prior to Historiae 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 Historiae conchyliorum: “Black 288 VALLEDOR DE LOZOYA & ARAUJO A — en = 0 MOULETTE ridee. | $. MOULETTK sinuee Caatahic des Peintres | $. —— FIGS. 6-9. FIG. 6: Plate 2 of Schrôter (1779). Mya testa crassa in fig. 2 (upper left corner) is actually Unio crassus. By permission of the British Library; FIG. 7: Blainville’s (1827: pl. 67, fig. 3) figure of M. auricularia (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. auricularia (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. auricularia 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 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. margaritifera. 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” [/n profundis voraginibus Fluvii Tees agri Eboracensis, non longe a Dinsdale, rete aliquando expiscatur]. We know today that only M. margaritifera lives in Yorkshire Rivers. ICONOGRAPHY OF MARGARITIFERA AURICULARIA We have reviewed all the early books on shells and malacology listed by Caprotti (1994) and Barbero (1999) (Table 1), as well as Simpson's (1900) list of synonyms for Margaritana margaritifera and M. crassa. Hav- ing confirmed that Mya testa crassa (Schrôter) did not correspond to M. auricularia, the next author on Simpson’s list to illustrate the spe- cies was Blainville (1827: pl. 67, fig. 3). Ina 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- ricularia several years prior in his Histoire TABLE 1. Historical illustrations of M. auricularia. Author Date Figure(s) Draparnaud 1805 pl. 10, fig. 19 Blainville 1827 pl 671193 Dupuy 1851 pl. 23, fig. 7a—c Küster 1855 pl swage Rossmassler Moquin-Tandon 1855 pl. 70, fig. 853 1855 pl. 48, fig. 1 Drouet 1857 р: 2 Sowerby 1868 pl. 62, fig. 311 Locard 1893 figs. 163, 164 Haas 1913 fig. 1 Haas 1916 fig. 1 Kennard et al. 1925 pl. 21, figs. 1-3 Haas 1929 figs. 181, 182 Germain 1930 pl. 26, fig. 609, 615 Azpeitia 1933 Huckriede 8 Berdau 1970 pl. 1 Fechter 8 Falkner 1990 color photo, p. 255 Falkner 1994 photo, fig. 1 289 naturelle des mollusques terrestres et fluviatiles de la France (Fig. 8). This image went unnoticed because Draparnaud mis- identified both species of Margaritifera and labeled his image Unio margaritifer, 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 Etude sur la collection conchyliologique de Draparnaud: “Draparnaud has made an error in respect of this species. His Unio margaritifer, 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. auricularia were Dupuy (1851) (Fig. 9), who drew the first known figure of a juve- nile M. auricularia, Kúster (1855) (Fig. 10), Rossmássler (1855) (Fig. 11), Moquin-Tandon (1855) (Fig. 12), Drouet (1857) (Fig. 13), G. B. Sowerby И (1868) (Fig. 14), and Locard (1893) (Fig. 15). Simpson also makes reference to: Bruguière (1797: pl. 248) [as “Deshayes, 1827”], Pfeiffer (1821), Rossmássler (1836, 1838, 1856), and Hanley (1856), but with the exception of Rossmássler (1856), the figures of these authors do no depict M. auricularia. Simpson (1900) wrote that the alleged M. au- ricularia 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 Cited as Unio margaritifera Unio sinuata Unio sinuatus Unio sinuatus Unio sinuatus Unio sinuatus Unio sinuatus Unio sinuatus Unio margaritanopsis & U. sinuatus Unio auricularius Margaritana auricularia Margaritana auricularia Margaritifera auricularia Margantana aunculana 8 M.? margantanopsis pl. 12, figs. 65, 66; pl. 13, fig. 67 Margaritana auricularia Margaritifera auricularia Pseudunio auricularius Pseudunio auricularius 290 VALLEDOR DE LOZOYA & ARAUJO 11 Pg En FIGS. 10-13. FIG. 10: Plate 37 of Kúster (1848). Top, M. auricularia; FIG. 11: Plate 70 by Rossmássler (1835) showing the hinge and a left valve of M. auricularia. By permission of the Osterreichische 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 Galbo de Marguritana avec charnière d'Unio. Unio margaritanopsis, Loca» FIGS. 14-18. FIG. 14: Plate 62 by С. В. Sowerby II (1868). Fig. 311 (middle) is M. auricularia. By permission of the British Library; FIG. 15: Page 151 of Locard (1893) showing a juvenile (top) and an adult specimen of M. auricularia (figs. 163 and 164, respectively). By permission of the Museo Nacional de Ciencias Naturales, Madrid, Spain; FIG. 16: The juvenile specimen of M. auricularia figured by Haas (1916). By permission of the Museo Nacional de Ciencias Naturales, Madrid, Spain; FIG. 17: Plate 26 of Germain (1930). Figs. 609 (top) and 615 (bottom right corner) depict an adult and a juvenile specimen of M. auricularia. By permission of the Museo Nacional de Ciencias Naturales, Madrid, Spain; FIG. 18a: M. auricularia in Azpeitia (1933: pl. 12); FIG. 18b: Adult (middle) and juvenile (bottom) specimens of M. auricularia in Azpeitia (1933: pl. 13). 292 VALLEDOR DE LOZOYA & ARAUJO of the shell and the presence of two siphons are characters that are completely absent in margaritiferids. The image by Pfeiffer (1821) is, in fact, Potomida littoralis (Lamarck, 1801), and it is the same species that Rossmässler (1836: pl. 13, fig. 195) drew and labeled Unio sinuatus. Rossmássler's (1838: pl. 35, fig. 493) figure of Unio gargottae Philippi, 1836, actu- ally depicts M. margaritifera, and Ross- mássler's (1856: pl. 80, fig. 853) is the same M. аипсшапа he illustrated in 1855. Lastly, the shell illustrated by Hanley (1856) identified as Unio crassissimus Hanley, 1843, 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 Locard, 1893 (Fig. 15), is really a juvenile M. 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. The first 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, 1910, 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), Osterreichische 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 Schiotte (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. LITERATURE CITED ARAUJO, R. & M. A. RAMOS, 2000, A critical revision of the historical distribution of Margaritifera auricularia (Spengler, 1782) (Mol- lusca: Margaritiferidae) based on museum specimens. Journal of Conchology, 37: 49-59. ARAUJO, R., N. CAMARA & M. A. RAMOS, 2002, Glochidium metamorphosis in the endan- gered freshwater mussel Margaritifera auricu- laria (Spengler, 1793). A histological and scanning electron microscopy study. Journal of Morphology, 254: 259-265. AZPEITIA, F., 1933, Conchas bivalvas de agua dulce de España y Portugal. Memorias del Instituto Geologico y Minero de Espana, Vol. 2. Madrid. 763 pp., 36 pls. 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A., 1835-1854, /conogra- phie der Land- und Sússwasser-Mollusken, mit vorzúglicher Berúcksichtigung der Europá- ischen noch nicht abgebildeten Arten, Vols. 1- 3. Arnold, Dresden 4 Leipzig. Vol. 1: vi + 132 + [6] + 26 + [4] + 33 + [4] + 27 + [4] + 70 pp., 30 pls. [1835-1837]; Vol. 2: [4] + 44 + iv + 46 + iv + 15 + iv + 37 pp., folding table, pls. 31-37 [1838-1844]; Vol. 3: viii + 1-40 + viii + 47-78 + [2] + viii + 81-140 pp., pls. 61-90 [1854-1859]. SCHROTER, Ч. S., 1779, Die Geschichte der Flússconchylien. Gebauer, Halle. vi + 434 pp., 11 leaves of pls. SIMPSON, C. T., 1900, Synopsis of the Naiades or pearly fresh-water mussels. Proceedings of the United States National Museum, 22(1205): 501-1044. SOWERBY, С. B., Il, 1868 [1864-1868], Mono- graph of the genus Unio. In: ©. В. SOWERBY Il, ed., Conchologica Iconica,16: 96 pls., [8 pp.] [pl. 62 was published in 1868]. SPENGLER, L., 1793, Beskrivelse over et nyt Slaegt af de toskallede Konkylier, forhen af mig kaldet Chaena saa og over det Linneiske Slaegt Mya, hvilket noeiere bestemmes, og inddeles i tvende Slagter. Skrifter af Naturhistorie Selskabet, 3(1): 16-69, pl. 2. WILKINS, С. L., 1953, A catalogue and histori- cal account of the Sloane shell collection. Bul- letin of the British Museum (Natural History), Historical Series, 1: 147. 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 8 Marci Arizumi Social Sciences Division, University of Hawaii at Hilo, 200 W. Kawili St., Hilo, Hawaii 96720-4091, U.S.A.; susanb@hawaii.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, Succinea 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 Succinea 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, 155°37.515'W). The understory consisted pri- marily of an alien torch ginger of the family Zingiberaceae; hapuu, a Hawaiian tree fern (Cibotium sp.); and ieie (Freycinetia sp.). The overstory consisted primarily of ohia lehua, Metrosideros polymorpha, and was less dense than the overstory of Puu Makaala. 295 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. 296 BROWN ET AL. ecember 2003 4 e Fi ao wteels. alll. 8 4 € ) 5 3 4 6 7 8 ) 10 | | : | | 2 | 4 | 4 € 7 8 9 10 1 2 3 1 6 7 8 January 2004 3 9 10 | | Ill. h... a x April PERS \ \ № | Ali. lu. > À > > a ) 2 3 4 Е 7 8 ) 10 Mills ll Ih.. 4 ‘ - ; 4 ‘ 8 ) ( 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- УЕ МАМУЛАЗОМЬ J FIG. 2. Frequency distribution о the mean пит- 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 S. 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 (X?¿, = 22.16; р < 0.0001). 51 Proportion of Egg Masses E May August December 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 ET AL. 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 $. 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 $. 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 $. 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, S. 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, $. С., К. 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. xili + 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, М.. С., 5. E. MILLER: & А. В: CARWILE, 1993, The decimation of endemic Hawaiian tree snails by alien predators. Атеп- can Zoologist, 33: 610-622. RUNDELL, К. J. & К. 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, predation 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 (Kitchell et al., 1981). The morphology of two types of boreholes was described by Carriker 8 Yochelson (1968) that later Bromley (1981) described as ichnofossils. Taylor (1980) and later Bromley (1981) pointed out that externally wide and internally narrow predation marks with parabo- loid walls are produced by naticid gastropods (Oichnus 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 predation of the Miocene naticids is stereo- typed and predictable. The observed pattern of predation was revealed when analyzing the selection of perforation location and the size of the predators by means of the perforation diameter. Kitchell et al. (1981) 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 Río 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 Kaitoa 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 Oligocene to more prob- ably earliest Miocene (Barreda 8 Palamar- czuk, 2000). Kaitoa patagonica (lhering, 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, 1931 (type species Kaitoa haroldi Marwick, 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.gov.ar “Facultad de Ciencias Exactas y Naturales, Universidad Nacional de La Pampa, Av. Uruguay 151, L6300CLB Santa Rosa, La Pampa, Argentina 300 SIGNORELLI ET AL. > A A > F4 7 Rivadavia | Puerto Deseado FIG. 1. Location map of the sampling area. MATERIALS AND METHODS A total of 873 specimens of Кайоа pata- gonica 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. olfo San Jorge Ra mo u TE № © y Monte Leon Formation FIG. 2. Schematic Section of Monte León Formation. 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 patagonica, 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 FIGS. 3-9. Kaitoa patagonica (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 um; FIGS. 7-8: Two complete boreholes. Scale bar = 200 um; FIG. 9: Detail of the square from Fig. 8. Scale bar = 50 um. 302 SIGNORELLI ET AL. Ze FREQUENCY E non predated predated 25) 35 45 255, 65 15 585 SIZE INTERVAL 957 10:5 11:5 125 135 FIG. 10. Size intervals vs. number of specimens (predated and non-predated) of Kaitoa patagonica. 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 90 with the software Statistica v. 4.0. The result was not significant (К? = 0.1257; р < 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 80 +— 105 6071 FREQUENCY u Ш internal diameter 40 + | external diameter 30 | 20 10 0 мм AM || 0.40.6 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 patagonica. BOREHOLES ON PATAGONIAN TERTIARY GASTROPODS 303 2.0 + у = 0.0757x + 0.4775 e A р => + E 1.5 ; Le, — + X = LL = < (a) — < = X ul m = 0.0 LL T T = | 7 8 9 10 LENGTH OF PREY (mm) 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 lhering, 1897. These species were based on gener- ally poorly preserved large adult shells, which seem unlikely to have been responsible for the borings on Кайоа. 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 Кайоа-Богег 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 Catà, 1989). As reported here, the most abundant size in the population is not the most intensely attacked by the predator (Fig. 10). 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 Кайоа patagonica. These presumably could prey on Кайоа patagonica 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 4 Ivanov, 1996). A location selection for perforations (Hofmann 8 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 ET AL. LITERATURE CITED BARREDA, V. & $. 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., 1970, Sobre el “Piso Patagoniano” y la representacion de la epoca del Oligoceno en Patagonia austral, Republica Argentina. Revista de la Asociacion Geologica Argentina, 25: 495-450. BERTELS, A., 1978, Estratigrafía y foraminiferos (Protozoa) bentónicos de la Formación Monte León (Oligoceno) en su área tipo, provincia de Santa Cruz, República Argentina. Actas, 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 & Р.А. MAXWELL, 1997, Opening of Drake Passage gateway and Late Miocene to Pleistocene cooling reflected in Southern Ocean molluscan dispersal: evidence from New Zealand and Argentina. Tectono- physics, 281: 83-97. BEU, А. С. 8 Р.А. 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 | 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, М. Б. 8 E. L. YOCHELSON, 1968, Recent gastropod boreholes and Ordovician cylindrical borings. Professional Papers of the United States Geological Survey, 593-B: 23. HOFFMAN, A. 8 J. MARTINELL, 1984, Prey selection by naticid gastropods in the Pliocene of Emporda (Northeast Spain). Neues Jahrbuch fúr Geologie und Paláontologie, 1984: 393- 399. КАВАТ, A.R., 1990, Predatory ecology of naticid gastropods with a review of shell boring pre- dation. Malacologia, 32: 155-193. KELLEY, P. H., 1988, Predation by Miocene gas- tropods of the Chesapeake Group: stereotyped and predictable. Palaios, 3: 436—448. KITCHELL, J.A:, €. H. BOGGS, J..F. KITCHEEE & 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. PASTORINO, G. 8 V. IVANOV, 1996, Marcas de predación en bivalvos del Cuaternario marino de la costa de provincia de Buenos Aires, Ar- gentina. /berus, 14: 93-101. TAYLOR, J. D., М. J. MORRIS & С. М. 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, хебга 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 predation. To test the influence of gravity upon mussels, | studied their move- ment on a series of slopes in the laboratory. In the light of my field research (Kobak, 2004), | hypothesized that small mussels would move upwards and illumination would reverse this behaviour because of negative phototaxis. | also expected that large mussels, less mobile than small ones, would prefer the easier, downward direction. 305 MATERIALS AND METHODS Mussels were collected by a diver from a dam wall of the Wtoctawek Dam Reservoir (the Vistu- la River, central Poland) and kept in a 500 | 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 п) 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 (Мб & Rothhaupt, 2003). To study geotaxis in the dark, | tested mus- sels on slopes inclined at 2, 3, 4 or 8” to the bottom, in a tank covered with a cardboard 306 KOBAK Aerator's position: - equal distances to both side walls - near the floor near the back <=, of a mussel FIG. 1. Experimental tank. & in various treatments 15/2. 3,4 or 8°: box. lllumination under this box was below the detection limits of the luxometer (Sonopan L- 20А) (i.e., < 0.1 Ix). To examine the impact of light on geotaxis, | tested mussels on a con- stantly illuminated slope (16 W bulb 0.5 m above the surface, incident illuminance at the surface ca. 700 |x). 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). | observed no passive relocations of these shells. | 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. t-test results en Small mussels (< 10 mm) pale 9) 2 Dark 0.28 0.7825 3 Dark Г. 0.11 0.9162 4% Dark 3.41 0.0078* 8° Dark 5.51 0.0004** 8° Light 1.38 0.2015 Large mussels (> 12 mm) 2° Dark 0.72 0.4923 3 Dark 2.28 0.0487 4 Dark | 3.88 0.0037* 8 Dark | 4.10 0.0027* 8° Light 405 0.0029* 8 6 4 2 0 Downward movement — 2 4 6 8 Upward movement 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: *р < 0.05, **р < 0.01. СЕОТАХ!$ 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. | 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 = 7.97, 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). N= 114 100 110 89 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 photophobic (Kobak, 2001; Toomey et al., 2002), which is contradictory to negative geotaxis: climbing up means ap- proaching the light source. Photophobic 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 103 119 99 112 88 2 Small mussels (< 10 mm) Fe Large mussels (> 12 mm) Net distance moved by mussels (cm) (o) Slope 2 3 2 8 Dark 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, | have shown that light (са. 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. A number of studies (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 | am grateful to Mr Andrzej Denis, Szymon Denis and Józef Liczkowski for collecting the mussels for the experiments. | also thank the two anonymous reviewers for their valuable comments that helped improve this text. LITERATURE CITED BURKS, К. L., М. С. TUCHMAN, С. A. CALL 8 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 fur Hydrobiologie, 160: 25-44. LEWANDOWSKI, K., 2001, Development of populations of Dreissena 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. /nternational 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, М. B., 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, 21: 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 10 THE EDINOKR 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@mnhn.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). | 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 311 (1998) 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 1870-1880s. 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. | 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 COntinental 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 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. | 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. | 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, | agreed with Falkner that the new species should be given the benefit of the doubt and be listed as valid until synonymized. Falkner et al. (2001, 2002), CLECOM and Fauna Europaea thus list 37 valid species of Bythinella from France, but the French checklist emphasizes (endnote 78, page 86): “Bernasconi's 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.” | believe that highlighting the problems does a better service to science than suppressing them. | agree with Davis that one may, or even should, view the listing of 36 species and subspecies of Bythiospeum in Germany with disbelief. Davis’ comment "| 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 Bythiospeum” 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, | 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 hemphilli D. W. Taylor, 2003, and Physella winnipegensis 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, 1969). 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 Malakozoologischen 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 Malacologiques” 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- А RESPONSE TO DAVIS (2004) ON SPECIES CHECK-LISTS 313 man authors. The question then asked was not “Is this a valid species ог a зупопут?”, 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, 1931) 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. lt 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 C. virgata (da Costa, 1778) (Clerx 8 Gittenberger, 1977). When revalidating Trichia phorochaetia (Bourguignat, 1864), endemic to the Grande Chartreuse and Vercors regions of the French Alps, Winter (1990) соттещеа: “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 1980s and 1990s 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, Limax granosus (Béren- guier, 1900) and Milax ochraceus (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, | agreed that “giving their chance” to Limax granosus and Milax ochraceus 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, | would like to make a com- parison with another (| am afraid, also French!) malacologist of the 19" century who has been the subject of much controversy on the other side of the Atlantic, | 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 Múnster. Rensch had a consid- erable influence on German evolutionary sys- tematics after WWII and, because he was also a malacologist (see, among others, Rensch, 1937), 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 flora 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- А RESPONSE TO DAVIS (2004) ON SPECIES CHECK-LISTS 315 ter, and К 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. | believe that George Davis and | do not stand far apart on this issue, and | 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, | believe Pseudanodonta complanata ligerica Pseudanodonta complanata grateloupeana 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), that of Abida secale (Draparnaud, 1801) is based on Gittenberger (1973), and that of Clausilia 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 elongata Pseudanodonta complanata dorsuosa 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 C. Pfeiffer, 1825 [now U. mancus Lamarck, 1819], and Unio pictorum (Linnaeus, 1758) showed an “almost perfect arrangement of populations according to the hydrographic basins”. Rec- ognition of “subspecies” within the unionids of France reflects the hypothesis that different hydrographic basins are inhabited by distinct genetic stocks, as evidenced by discrete mor- phological differences between basins (Fig. 1). As editor of the French checklist, | agreed with Falkner’s 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. | 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 | 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). | agree that regional checklists are not the place to pro- duce elaborate, finely dissected classifica- tions. Aclassification 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 reevaluation 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 /nternational 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- А 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. | 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., 1993) concluded that M. durrovensis was just an ecophenotypic variant. As a scientist, | of course accept the results of the molecular study, but as a conservationist | 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 EU equivalent to the US Endangered Species Act). And they have done so in 1995, that is, after the results of the Chesney et al. (1993) study, which were known to the proponent of the list- ing. | 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”. | believe scientists may argue ad nau- seam in academic journals on whether “conservation units” are concepts that should be preferred over “dubious subspecies”. | 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). | 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, | agree that it may be dis- putable whether it is justified to establish one, and | 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, | 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 | 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, | 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.” LITERATURE CITED BADINO, G., G. CELEBRANO 4 K.-O. NAGEL, 1991, Unio elongatulus and Unio pictorum (Bivalvia: Unionidae): molecular genetics and relationships of Italian and central European populations. Bollettino, Museo Regionale di Scienze Naturali, Torino, 9(2): 261-274. BANK, R. A., P. BOUCHET, G. FALKNER, E. GITTENBERGER, В. HAUSDORF, T. VON PROSCHWITZ 8 T. E. J. RIPKEN, 2001, Supraspecific classification of European non- marine Mollusca (CLECOM Sections I+ll). Heldia, 4: 77-128. BANK, R.A. [Group Coordinator], 2004, Fauna Europaea: Gastropoda. www.faunaeur.org BERNASCONI, R., 2000, Revision du genre Bythinella (Moquin-Tandon, 1855) (Gastropoda Prosobranchia Hydrobiidae: Amnicolinae Bythinellini) de la France du centre-ouest, du Midi et des Pyrénées. Documents Malaco- logiques, hors série 1: 1-126. BOUCHET, P., С. FALKNER & M. SEDDON, 1999, Lists of protected land and freshwater molluscs in the Bern Convention and European Habitats Directive: are they relevant to conser- vation? Biological Conservation, 90: 21-31. BOUCHET, P. & J. P. ROCROI, 2005, Classifi- cation and nomenclator of gastropod families. Malacologia, 47(1-2): 1-397. BOURGUIGNAT, J. R., 1882, Lettres malacologiques a MM. Brusina d’Agram et Kobelt de Francfort. Tremblay, Paris. 55 pp., 1 pl. BRUYNE, К. H. DE, К. A. BANK, J. P. Н. M. ADEMA & F. A. PERK, 1994, Nederlandse Naamlijst van de weekdieren (Mollusca) van Nederland en Belgié. Backhuys, Oegstgeest. 149 pp. CHESNEY, Н. С. С.,Р. С. OLIVER & С. M. DAVIS, 1993, Margaritifera durrovensis Phillips, 1928: taxonomic status, ecology and conservation. Journal of Conchology, 34: 267-299. CLERX, J. P. M. & E. GITTENBERGER, 1977, Einiges über Cernuella (Pulmonata, Helicidae). Zoologische Mededelingen, 52(4): 27-56. COUTAGNE, G., 1929, La faune malacologique de la Tarentaise. Annales de la Société Linnéenne de Lyon, new ser., 74(2): 7-79. DAVIS, С. M., 2004, Species check-lists: death or revival of the Nouvelle Ecole? Malacologia, 46(1): 227-231. DUBOIS, A., 1998, Lists of European species of amphibians and reptiles: will we soon be reach- ing “stability”? Amphibia-Reptilia, 19: 1-28. FALKNER, G., В. А. BANK 8 Т. VON PROSCH- WITZ, 2001, Check-list ofthe non-marine mol- luscan species-group taxa of the states of northern, Atlantic and central Europe (CLECOM 1). Heldia, 4: 1-76. [Also available electronically www.gnm.se/gnm/clecom/ clecom.asp] FALKNER, G., T. RIPKEN 8 M. FALKNER, 2002, Mollusques continentaux de France. Liste de A RESPONSE TO DAVIS (2004) ON SPECIES CHECK-LISTS 319 référence annotée et bibliographie. Patrimoines Naturels, 52: 1-350. FORCART, L., 1933, Revision des Rassen- kreises Helicigona (Chilostoma) zonata Studer. Verhandlungen der Naturforschenden Gesellschaft in Basel, 44(2): 53-107, 7 pls. GERMAIN, L., 1931, Mollusques terrestres et fluviatiles. Faune de France, 21: 1-477, pls. 1-13; 22: i-xiv, 479-897, pls. 14-26. Lechevalier, Paris. GITTENBERGER, E., 1973, Beitrage zur Kennt- nis der Pupillacea, III. Chondrininae. Zoo- logische Verhandelingen, 127: 1-267. GOULD, S. J., 1992, Bully for Brontosaurus. Norton and Co., New York & London. 540 pp. HAMMOND, P. M. [lead author], 1995, The cur- rent magnitude of biodiversity. Pp. 113-138, in: V.H. HEYWOOD &R. T. WATSON, eds., Global Biodiversity Assessment. Cambridge Univer- sity Press. ISAAC, М. J. B., 4. MALLET & С. М. MACE, 2004, Taxonomic inflation: its influence on macro- ecology and conservation. Trends in Ecology and Evolution, 19(9): 464-469. KERNEY, M., 1999, Atlas of the land and fresh- water molluscs of Britain and Ireland. Harley Books, Great Horkesley, Colchester. 264 pp. KOTTELAT, M., 1995, Systematic studies and biodiversity: the need for a pragmatic approach. Journal of Natural History, 29: 565-569. KUIPER, J. G J., 1969, Schetsen uit de mala- cologische geschiedenis van Frankrijk. Correspondentieblad von de Nederlandse Malacologische Vereniging, 133: 1424-1474. LOCARD, A., 1893, Coquilles des eaux douces et saumátres de France. Rey, Lyon. 327 pp. MANGANELLI, G., M. BODON, L. FAVILLI & F. GIUSTI, 1995, Gastropoda Pulmonata. In: A. MINELLI ETAL., eds., Checklist delle specie della fauna italiana, 16: 1-60. Calderini, Bologna. NORDSIECK, H., 1990, Revision der Gattung Clausilia Draparnaud, besonders der Arten in SW-Europa (Das Clausilia rugosa-problem) (Gastropoda: Stylommatophora: Clausiliidae). Archiv für Molluskenkunde, 118(4-6): 133-179. РР, Е., 2004, А new species of Physella (Gas- tropoda: Physidae) endemic to Lake Winnipeg, Canada. Visaya, 2: 42-48. РЕЕШЕЕ, Е. & С. W. ROUSE, 2000, Least-inclu- sive taxonomic unit: a new taxonomic concept for biology. Proceedings of the Royal Society of London, ser. B, Biological Sciences, 267: 627-630. RENSCH, B., 1937, Untersuchungen über Ras- senbildung und Erblichkeit von Rassen- merkmalen bei sizilianischen Landschnecken. Zeitschrift für Induktive Abstammungs- und Vererbungslehre, 72: 564-588. TAYLOR, D. W., 2003, Introduction to Physidae (Gastropoda: Hygrophila); biogeography, clas- sification, morphology. Revista de Biologia Tropical, 51, Suppl. 1: 289 pp. TURGEON, D. D., J. Е. QUINN, А. Е. BOGAN, Е. V. СОАМ, Е. С. HOCHBERG, W. С. LYONS, Р. М. MIKKELSEN, К. J. NEVES, С. КОРЕК, С. ROSENBERG, В. ROTH, А. SCHELTEMA, Е. С. THOMPSON, М. VECCHIONE & J. D. WILLIAMS, 1998, Common and scientific names of aquatic invertebrates from the United States and Canada, ed. 2. American Fisheries Society Special Publication 26. 526 pp. WILKE, T., G. M. DAVIS, A. FALNIOWSKI, F. GIUSTI, M. BODON & M. SZAROWSKA, 2001, Molecular systematics of Hydrobiidae (Mol- lusca: Gastropoda: Rissooidea): testing mono- phyly and phylogenetic relationships. Proceedings of the Academy of Natural Sci- ences of Philadelphia, 151: 1-21. WINTER, А. J. DE, 1990, Little known land snails from the French Alps (Pulmonata). Basteria, 54(4-6): 227-237. Revised ms. accepted 22 July 2004 DN UD A о . 7 a Pp u u = Dt as u в NAF, pro =» © Le u . ol o o os AAA) LUE e р ee в nz D. им в = Е д а =) LR. = = ~~ =e = a as TO Го И Zn DI ZA A ВО 5 u — ce = = “=e? a Y u MALACOLOGIA, 2006, 48(1-2): 321-327 CHECK-LISTS AND CLECOM: A RESPONSE TO DAVIS (2004) Ruud A. Вапк', 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 Ecole?”. From his title it is clear that he considers the work of CLECOM as the revival of the Nouvelle Ecole (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)species 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; Glóer, 2002; Martinez-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 Rossmassler, 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 °Naturhistoriska Museet, Dept. of Invertebrate Zoology, Вох 7283, 40235 Göteborg, Sweden 322 BANK ET AL. 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 Ecole” of Bourguignat or “Starobogatovismus”. Practitioners of both doctrines should realize that they do not con- tribute to a better understanding of biodiversity (Dance, 1970; Reischütz, 1994). However, as one can see from our CLECOM list, we have evaluated the numerous taxa recognized by the “Nouvelle Ecole” of Bourguignat or the Starobogatov doctrine and found most taxa not to be taxonomically sound. Thus, only a few names have entered our lists. We would like to stress once more, that we have critically investigated, case by case, the evidence on which a morphospecies has been based be- fore we decided to accept it in our check-list. So we did exactly what Davis describes as the necessary methodological procedure: sort through publications, look at voucher speci- mens and field records (in order to assess the biological validity of formally described taxa), consider contradictory information, make de- cisions, and ensure compliance with the In- ternational Code of Zoological Nomenclature. We agree with Davis that there is some sub- jectivity in this procedure. This subjectivity is likely to be minimized through the collabora- tion of independent specialists: as clearly stated in the introduction, the CLECOM check- list is not the product of a single person. We hope that Davis does not expect us to be in- CHECK-LISTS AND CLECOM 323 fallible. Of course, errors, wrong decisions, and omissions happened; and sometimes — to spare time — we were not critical enough and relied on secondary literature of which we judged the authors to be thorough and reliable. To correct such errors and to include new find- ings we formally institutionalised the instrument of Updates for the CLECOM-lists, of which the first appeared т 2001 (Bank et al., 2001b) and the second is now ready for printing. The Use of Subspecies К is well-known that many species vary in space. They may consist of (groups of) recog- nizable populations inhabiting different geo- graphical areas. Such populations may have been initially described as separate species. They are combined as polytypic species, ob- jectively whenever hybrid zones connect the diagnosable population groups, or subjectively on the — weak indeed — basis of similarity and analogy to comparable cases. In land snails the subspecies concept has resulted in a de- crease in the number of so-called species. Given the importance of the polytypic species concept, it is remarkable that it is widely ig- nored in faunistic studies (but often not in taxo- nomical work!). Davis (2004: 229) argues that a check-list that is based on faunistic litera- ture should ignore subspecies, as in faunistic literature the subspecies is widely neglected. However, our check-list is based on taxonomic literature. We see no good reason to ignore the subspecies level of biodiversity in faunis- tic studies. Subspecies in the BSC are termi- nal taxa in the phylogenetic species concept (see the response of Bouchet, 2006). Too Many (Sub)Species? Some authors feel uncomfortable about the large number of species and subspecies men- tioned in the CLECOM list or in the Fauna Europaea database. An example is the large list of Clausiliidae from Greece. However, molecular data on some of the most speciose genera are in agreement with the large radia- tions that were unraveled by morphological studies (Schilthuizen, 1994; Moorsel, 2001; Uit de Weerd, 2004). The diversification of the helicid genus Arianta, based on morphologi- cal features and postulated over a decade ago (Gittenberger, 1991), has now been confirmed by molecular data (Gittenberger et al., 2004). Species complexes within the Arionidae, rec- ognized on the basis of morphological and anatomical characters, are becoming con- firmed by molecular data as well (Pinceel et al., 2004). We thus feel that in the vast major- ity of cases the CLECOM list reflects the biodiversity of continental molluscs as present in Europe. Of course, gaps in our knowledge will certainly result in incomplete or wrong data in our list. The list can only be as good as the science on which it is based. We still have a long way to go to achieve a complete assess- ment of the actual snail diversity of Europe. Currently, some 25 new (sub)species are de- scribed on a yearly basis from Europe: on av- erage one new taxon every two weeks. The Case of Bythiospeum Bythiospeum is admittedly a major problem in the faunal lists (and Red Lists) of Germany in general and the federal states of Baden- Württemberg and Bayern in particular, which hampers the evaluation of the fauna with re- spect to its degree of endemism and the fixa- tion of conservation priorities. So far, 58 names have been introduced to designate the differ- ent forms of Bythiospeum in Germany. The validity of these nominal taxa has been a mat- ter of intensive discussion, but so far no con- sensus has been reached (also not among the CLECOM authors!). Obviously however, the three polytypic species complexes of Bolling (1966) are not a reflection of the true amount of differentiation in this genus in Germany. Nevertheless, Bolling was followed in the suc- cessive editions of the freshwater mollusc guide of Glöer et al. (1978-1992) and Glóer 8 Meier-Brook (1994—2003). Perpetuation of that practice was considered by us as unaccept- able. We also did not follow the example of Boeters (1998), who simply provided a list of all available names. Although this exhaustive listing has the advantage of being objective, it is purely technical and by definition excludes any modification or incorporation of new bio- logical results with the exception of the addi- tion of new names. Such a list is surely useful but is not compatible with our aim of depicting “real world” biological diversity. We will explain how we established the list of Bythiospeum taxa as presented in our CLECOM list. First, we accepted the hypoth- esis of Geyer (1908) that in several species parallel converging dwarf forms occur, which may be so different from their stem form that they give the impression of being distinct spe- 324 BANK ET AL. cies (in some cases syntopic occurrences of distinct species may really be hidden under this phenomenon). Second, we took into ac- count the fact that several species can be clearly distinguished by their anatomy. In this context, it is of interest to note that the three species that are differentiated by Boeters (1984) were regarded by Bolling (1966) as belonging to one “species” (acicula sensu Bolling) and that two of them (suevicum and exiguum) represented his subspecies clessini. This shows that even a provisional maintainance of Bolling's system was impos- sible. In the Bavarian alpine foreland, В. acicula and B. heldii (occurring in the same hydrological system at Obernach on the Walchensee) are anatomically separable (Boeters, 2002). The other nominal species occurring in the Bavarian alpine foreland, namely rougemonti, carychiodes, aciculoides and algoviensis, were considered provision- ally by us as synonyms of acicula or heldii; В. alzense was considered a separate species. For Baden-Württemberg, we accepted in gen- eral the classification of Geyer (1908), being the result of extensive and carefully planned fieldwork by Geyer, producing a hitherto un- surpassed wealth of material. The classifica- tion system was elaborated by Geyer in a self-critical way following the modern species concept (i.e., anticipating the biological spe- cies concept). He based his revision on thor- ough conchological examination but also stressed the need for anatomical studies. From his papers, it is clear that Geyer recognised variability and ecophenotypical variation, and that he had well-founded ideas on geographi- cal barriers and the genetical coherence of populations. Geyer was also aware of the pit- falls of Bourguignatism, which he rejected: “Ich ... hútete mich aber vor der Art der neufranzó- sischen Schule, welche den Lebenszusam- menhang mißachtet” (1908: 595). In our opinion, the work of Geyer is the best that has ever been done on Bythiospeum and we see no reasons to abandon his system, which is scientific because it allows falsification and amendment. We have revalidated Bythiospeum pellucidum, the type-species of the genus, which was shown by Boeters (1984) to be a good species, different from B. quenstedti to which it had been attributed by Geyer as a mere ecophenotype (Geyer, who named the species with inclusion of its alleged derivate quenstedti, was apparently unaware of the nomenclatural consequences, as were all his followers). Other deviations from Geyer are: (1) we neglected all subspecies that could not be attributed to geographical regions or were said to occur side by side in the same cave or spring; (2) we treated /amperti, taxisi, and senefelderi as full species, following Ehrmann (1933); (3) for Mainfranken, we followed the concept of two species (clessini and puerkhaueri) with several subspecies and pro- visionally treated moenanum, elongatum and по! as synonyms, based on Noll & Hásslein (1952). In summary, we rejected the Bythiospeum species constructs of Bolling and adopted the more traditional (non-Bourguignatian) classi- fication of Geyer (1904-1908) with modifica- tions based on arguments of later authors. Taking into account the relevant literature on this topic, we listed for Germany 26 species, 6 of them polytypic (a total of 36 names). We have thus “neglected” (synonymised) 22 names. This will certainly be the subject of further changes after additional research. But it remains to be demonstrated — by DNA se- quencing for example — whether this is indeed a case of “splitting”. In this context, it is of in- terest to note that Haase (1995), after a de- tailed morphometric study of the then eight recognized Bythiospeum taxa from Austria, accepted them all (albeit provisionally) as separate species. Value of Molecular Data: Systematics of Hydrobiidae as an Example We acknowledge the great value of molecu- lar data for systematic work and, as a conse- quence, also for check-lists, and have used these data whenever possible. We are sur- prised to learn from Davis that we have ig- nored the molecular work from his group. He writes (2004: 230) “... the molecular work ту group has done in the past ten years on sev- eral European taxa in the superfamily Rissooidea largely has been ignored by CLECOM. Instead, the rissooidean systemat- ics of that list is still mainly based on tradi- tional (mostly shell-based) data. It is not that the molecular data have been ignored, be- cause they often contradict the findings of members of the CLECOM team. Rather, it is important that CLECOM incorporates new find- ings based on genetic data much more quickly, even if these findings are inconvenient’. First, we would like to stress that our classification is not mostly shell-based but mainly based on CHECK-LISTS AND CLECOM 325 anatomical characters, namely on the genital organs from a large number of genera (as pro- vided by Radoman, 1983). Second, we are well aware of the excellent work of Davis and co-workers on Rissooidea, and have inte- grated their results in our check-list. So we are eager to learn, where our list deviates from his work published before the beginning of 2001, the year that our list was published. Unfortunately, no example(s) is(are) given. An interesting paper by Wilke et al. (December 2001), with Davis as one of the authors, de- scribed the molecular systematics of the Hydrobiidae. This paper is the most recent work of Davis dealing with the classification of a part of the European taxa generally as- signed to the Hydrobiidae. Interestingly, the presented classification essentially reflects the classification used by CLECOM. We noted the following differences: the Lithoglyphidae, Cochliopidae and Amnicolidae are dealt with by CLECOM as subfamilies within the Hydrobiidae, rather than families. As already stated by Wilke et al. (2001: 11), “There is no universal definition for “family”.” In this con- text, it is of interest to note, that Davis et al. (1982, 1985) ranked these families, as CLECOM did, before 2001 as subfamilies. Thus, we “lumped” rather than “split” the suprageneric hierarchical units: certainly no revival of the “Nouvelle Ecole”! The subdivision of Hydrobiidae into several clades by Wilke et al. (2001) (that is, the maxi- mum likelihood tree based on combined COI and 18S sequences - their fig. 3), also essen- tially reflects our classification. Adrioinsulana, Pseudamnicola, Adriohydrobia, Hydrobia, Peringia and Ventrosia [correct name: Ecrobia] cluster together, separate from Graziana, Belgrandia, Horatia, Sadleriana, Orientalina, Hauffenia, Fissuria, Alzoniella, Avenionia and Islamia. These two clades are designated Hydrobiinae and Belgrandiinae, respectively, in our classification. The only two surprises in their figure 3 are (1) Mercuria, which we clas- sified within the Hydrobiinae, not Belgrandiinae, and (2) the classification of Bythiospeum within the Moitessieriidae. With respect to Bythiospeum, there are massive anatomical data suggesting that Bythiospeum and Moitessieria are not closely related (Boeters, 1972; Boeters 8 Gittenberger, 1990; Boeters, 2003). Unfortunately, the sequences are based on an unidentified “Bythiospeum spec.” collected in southern France, which does not allow verification of the diagnostic characters given by Boeters (2003). The fam- ily status of the Moitessieriidae itself has been defended by some authors for many years and denied by others: here DNA demonstrates its value. All this shows the importance of a good (morphological) taxonomic framework to start with. Incorrect identifications result in wrong conclusions. With respect to Mercuria, we noticed that in the maximum likelihood tree based on the mitochondrial COI only (their fig. 2B), Mercuria seems to be more closely re- lated to the Hydrobiinae than to the Belgrandiinae, whereas in the maximum like- linood tree based on the nuclear 18$ (their fig. 2A) the reverse is seen. This is a good example of the well-know fact that gene trees and phylogenetic trees may differ significantly from one another. What is needed is an inte- grated approach. It is this approach that was used while preparing the CLECOM check-list. LITERATURE CITED ALBA, D. М., А. TARRUELLA, J. CORBELLA, М, VILELLA, ©. GUILEEN, 2. 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Revised ms. accepted 22 August 2005 D MALACOLOGIA, 2006, 48(1-2): 329-342 INDEX Taxa in bold are new; pages in italic indicate figures of taxa. aberti, Cyprogenia 269 Abida occidentalis 313 pyrenaearia 313 secale 315 Acar plicata 37, 39 acicula, Bythiospeum 324 aciculoides, Bythiospeum 324 Acostaea rivolii 272, 280 Acrostoma 228 baccata 187 elongatum 187 iravadica 197 variabilis 176 Actinonaias 274-277 ligamentina 269, 271, 280 acurugata, Drillia 43-44, 46, 57 Pyrgospira 58-59 Strictispira 44, 54, 57-58 acuta, Physa 133-136, 134-135, 139 Adamietta 160, 162, 164, 181, 213, 227, 237, 239-241, 244 hainanensis 234-236 housei 234-236 infracostata 181 provisoria 234 schmidti 235-236 testudinaria 223, 234-236 Adamussium colbecki 37-39 adjaciensis, Oxychilus 313 Adriohydrobia 325 Adrioinsulana 325 Adriolitorea 82, 127 Aequipecten opercularis 38-39 aginnica, Cernuella 313 Agriolimax reticulatus 74 aitanica, Josefus 77, 81, 82-83, 109, 119, 120, 121, 122-123, 124-126, 129 Alathyria jacksoni 272, 280 alatus, Lampsilis 289 Potamilus 269, 289 Albinaria turrita 272, 280 alexandrina, Biomphalaria 32 algoviensis, Bythiospeum 324 alzense, Bythiospeum 324 Alzoniella 129-130, 325 ambiguus, Velesunio 272, 280 Amblema plicata 269 Amnicola anatina globulus 83 329 globulus 79, 83, 89 similis 83 Amnicolidae 325 Ampullariidae 22, 159 anatolica, Islamia 92, 127 angasi, Velesunio 272, 280 angulata, Gonidea 271, 280 angulifera, Brotia 227 Melania 229 Melania (Pachychilus) 227 Melania (Plotia) scabra var. 228 annulatus, Ctenoides 37, 39 Anodonta 275-277, 287 californiensis 269 Anodontites guanarensis 269, 272, 280 trigonus 272, 280 Anomia ephippium 37, 39 Anomiidae 36-38 Anomioidea 37 Antimelania 163, 243 costula 176 antiqua, Neptunea 65 apicata, Crassiclava 44, 47, 48, 52-53 Aplexa 133, 136, 139140 hypnorum 133-134, 134-135, 140 hypnorum hypnorum 140 hypnorum tryoni 140 Arca noae 37, 39 Arcidae 37 Arcoidea 36-38 argenvillei, Patella 73 Argopecten gibbus 37-39 irradians 37-39 Arianta 323 Arionidae 323 armata, Brotia 169-171, 169-170, 172, 173- 174, 206, 213, 216-217, 233-236, 238, 242 Brotia (Paracrostoma) pseudosulcospira 169 Paracrostoma pseudosulcospira 169, 169, 212 arthritica, Neptunea 65, 74-75 aspera, Melania (Melanoides) variabilis subvar. 178 asperata, Jagora 210, 234-236 Melania 210 assamensis, Brotia 228, 229, Melania (Acrostoma) 228 Paracrostoma 228 Tiara (Acrostoma) 228 assidariaeformis, Siphonalia 74-75 ateni, Islamia 77, 83, 86-87, 89-90, 92, 94, 95, 96-98, 97-98, 109, 109, 126, 128-129 330 INDEX Microna 78-79, 96 Neohoratia 96, 127 ater, Faunus 239 Atrina pectinata 37, 39 aurantia, Strictispira 44 auricularia, Margaritana 289 Margaritifera 271, 280, 285, 286, 287, 288, 289, 290-291, 292 auricularius, Pseudunio 289, 292 Unio 285, 287, 289, 292 australis, Hyridella 272, 280 Plicatula 37, 39 Avenionia 129-130, 325 Aviculopectinidae 40 Aylacostoma 243 azarum, Neohoratia 78-79 baccata, Acrostoma 187 Brotia 166, 187, 239 Brotia (Brotia) 187 Melania 187, 188, 189 Melania (Brotia) 187 Melania (Melanoides) 187 Melanoides (Tiara) 187 baccifera, Melania variabilis var. 187 balteata, Melania 191-192, 191 banyulensis, Godiva 74 barbara, Patella agenvillei 73-74 Barbatia virescens 37, 39 Bathynerita 1, 18, 21-23 Bathypecten 35, 38, 40 eucymatus 35 vulcani 35-40 beaumetzi, Brotia 228 Melania 228, 229 beddomeana, Melania (Melanoides) tourannen- sis var. 192 Belgrandia 325 Belgrandiinae 325 binodosa, Brotia 170-171, 170-173, 174, 204, 216, 231-236, 239, 242 Brotia (Brotia) binodosa 171 Melania 171, 171 binodulifera, Melania (Melanoides) variabilis var. 185 Biomphalaria alexandrina 32 glabrata 73 Bithyniae 160 Bithyniidae 160 boeana, Melania 217-219, 218 borneensis, Brotia 230 Melania 229, 230 bosniaca, Islamia (Islamia) 127 brevidens, Epioblasma 269 brevispira, Physa integra 140 briandi, Shinkailepas 1, 24 Brotia 159-160, 162-164, 165, 166, 168-169, 189, 195, 199, 202, 203, 206, 209, 213, 218, 226-228, 230-231, 233, 237-245 angulifera 227 (Antimelania) costula 176, 183, 217 (Antimelania) variabilis 176 armata 169-171, 169-170, 172, 173-174, 206, 213, 216-217, 233-236, 238, 242 assamensis 228, 229 baccata 166, 187, 239 beaumetzi 228 binodosa 170-171, 170-173, 174, 204, 216, 231-236, 239, 242 binodosa spiralis 171 binodosa subgloriosa 216 borneensis 230 (Brotia) baccata 187 (Brotia) binodosa binodosa 171 (Brotia) binodosa spiralis 171 (Brotia) binodosa subgloriosa 216 (Brotia) costula peninsularis 207 (Brotia) insolita 196 (Brotia) manningi 200 (Brotia) microsculpta 201 (Brotia) pagodula 202 (Brotia) pseudoasperata 210 citrina 166, 166, 172, 173-174, 174-176, 176, 182-183, 190, 195, 202, 204, 208, 216, 223, 226-227, 232-237, 242 costula 165, 176, 177-181, 178-181, 183- 185, 185, 187, 192, 194, 197, 199, 199, 204, 207, 213-214, 217, 219, 222, 233- 236; 245 costula episcopalis 176, 183 costula peninsularis 178 costula varicosa 178 cylindrus 230 dautzenbergiana 174, 176, 176, 181-183, 181-183, 195, 227, 233-236 elongata 245 episcopalis 165, 167-168, 179-180, 183- 185, 184-185, 199, 219, 220, 239 escheri 168 godwini 185, 186, 190 henriettae 187, 188-189, 189, 190, 195, 197, 233-236, 239, 245 herculea 179-180, 181, 190, 191, 192, 193- 194, 199, 233-236, 245 indragirica 194-195, 195 insolita 196, 196, 201 iravadica 190, 197, 197 jullieni 180-181, 190, 197, 198-199, 199, 214 kelantanensis 185, 199, 200 manningi 196-197, 200, 200-201 menkeana 178 INDEX 391 microsculpta 201, 202-203, 216-217, 232- 236, 240, 242 oppenoorthi 166 pagodula 164, 165, 168, 190, 202, 203-205, 233-238, 240, 242 palaeocostula 166 paludiformis 203, 206, 206, 213, 238 (Paracrostoma) pseudosulcospira 212 (Paracrostoma) pseudosulcospira armata 169 (Paracrostoma) solemiana 215 peninsularis 179, 203, 205, 207-208, 207- 208, 213-214, 233-236 praetermissa 203, 209, 209-210, 230 pseudoasperata 210, 210-211 pseudosulcospira 167, 169, 171, 202, 203, 207, 212-213, 212-213, 216-217, 232- 236 reevel 245 (Senckenbergia) wykoffi 226 siamensis 196-197, 201, 205, 208, 208, 213-214, 214 solemiana 203, 215-217, 215, 233-236 sooloensis 229, 231 spinata 171, 174, 204, 231-232 spinata spiralis 171 spinata subgloriosa 216 subgloriosa 203, 216, 216-217, sumatrensis 180, 185, 185, 217-219, 218, 220, 232-236, 238, 242 testudinaria 164, 181, 214, 227, 241 torquata 179, 221-223, 221-223, 233-236, 238 variabilis 166, 176, 184, 217 vasarhelyii 168 verbecki 223-224, 223-225, 233-236 wykoffi 205, 226, 226-227, 233-236, 244 zollingeri 221 zonata 232 lamperti 324 moenanum 324 по! 324 pellucidum 324 puerkhaueri 324 quenstedti 324 rougemonti 324 senefelderi 324 suevicum 324 taxisi 324 caelata, Pododesmus 37, 39 californiensis, Anodonta 269 canaliculata, Melania 229, 231 Caobangia 204 capax, Potamilus 269 capensis, Siphonaria 73-74 cardium, Lampsilis 269 carinatus, Lanistes 27-33, 31 carolinae, Melania 176, 177 carychiodes, Bythiospeum 324 Castalia stevensi 272, 280 Ceresidae 1, 17, 20 Cernuella 322 aginnica 313 (Cernuella) virgata 322 cespitum 322 neglecta 322 virgata 313, 322 (Xerocincta) neglecta 322 cespitum, Cernuella 322 Chambardia rubens 269, 272, 280 Chilostoma zonatum 315 chinensis, Mactra 74 Chlamys hastata 37-39 islandica 37-39 cianensis, Islamia 79, 128-129 cincta, Melania (Melanoides) variabilis subvar. 178 Buccinidae 65, 75 Bulinus truncatus 32 Bulla 299 bunarbasa, Islamia 92 burnabasa, Horatia 127 Islamia 127 Bythinella 312 Bythiospeum 312, 323, 325 acicula 324 aciculoides 324 algoviensis 324 alzense 324 carychiodes 324 clessini 324 elongatum 324 exiguum 324 heldii 324 Cinnalepeta 1, 17-22, 24 citrina, Brotia 166, 166, 172, 173-174, 174- 176, 176, 182-183, 190, 195, 202, 204, 208, 216, 223, 226-227, 232-237, 242 Melania 174, 174, 182 citrinoides, Melania 174, 174 Clathrodrillia solida 47, 50-51, 60 Clausilia rugosa 315 Clausiliidae 237, 323 Clavus (Crassispira) zizyphus 44 Cleospira 44, 62 ochsneri 44 clessini, Bythiospeum 324 Cocculina 19 Cochliopidae 325 colbecki, Adamussium 37-39 colliourensis, Oxychilus 313 332 INDEX coltrorum, Strictispira 43-45, 46, 47, 50, 52, 56, 63 compacta, Melania (Melanoides) tourannensis var. 192 complanata, Pseudanodonta 315 Conoidea 43 conradicus, Medionidus 269 consolationis, Islamia 102, 128 constricta, Neptunea 74-75 corneus, Planorbarius 74 coronadoi, Neohoratia 98, 99, 101 Valvata 78, 98, 101 corrugata, Melania 178 Costatella 133 costula, Antimelania 176 Brotia 165, 176, 177-181, 178-181, 183- 185, 185, 187, 192, 194, 197, 199, 199, 204, 207, 213-214, 217, 219, 222, 233- 236, 245 Brotia (Antimelania) 176, 183, 217 Melania 176, 183, 217 crassa, Margaritana 287, 289 Mya testa 287, 288, 289 Testa 287 Crassadoma gigantea 37-39 Crassiclava 44, 48, 50 apicata 44, 47, 48, 52-53 crassisimus, Musculus omnium longe 287 crassissimus, Unio 292 Crassispira 44, 48, 50, 52, 57 (Crassispirella) drangai 47 (Crassispirella) fuscescens 60 (Crassispirella) quadrifasciata 53 cubana 61 drangai 47, 49 ebenina 62 ericana 45 fuscescens 62 paxillus 50 quadrifasciata 43-44, 53 (Strictispira) paxillus 51 (Strictispira) cf. solida 62 tampaensis 57 Crassispirella 44, 48 fuscescens 44, 48, 49-50, 52-53, 62 crassisquamatus, Spondylus 37-39 crassus, Unio 287, 288, Ctenoides annulatus 37, 39 cubana, Crassispira 61 Cumberlandia 274-277 monodonta 269, 271, 280 cumingii, Neptunea 65, 74-75 Neptunea arthritica 65-67, 67-72, 73-75 Neptunea (Barbitonia) arthritica 65 curvicosta, Melania 221, 222 cylindrus, Brotia 230 Melania 229, 230 Cyprogenia aberti 269 Cyrtonaias 275-277 tampicoensis 269, 271, 280 dactylus, Jagora 234-236 dahurica, Dahurinaia 271, 280 Dahurinaia 269 dahurica 271, 280 dautzenbergiana, Brotia 174, 176, 176, 181-183, 181-183, 195, 227, 233-236 Melania 181-182, 181 Stenomelania 182 deceptus, Diplodon 272, 280 Dentalium 272, 280 depressa, Hyridella 272, 280 dilitata, Elliptio 269 Diplodon deceptus 272, 280 Doryssa 240, 243 drangai, Crassispira 47, 49 Crassispira (Crassispirella) 47 Strictispira 43-44, 47, 48, 49-50, 50, 52, 53, 62 Dreissena polymorpha 305 Drillia 48, 58 acurugata 43-44, 46, 57 (Clathrodrillia) solida 60 (Crassispira) fuscescens 51 (Crassispira) paxillus 50 (Crassispira) quadrifasciata 53 (Drillia) paxillus 52 ebenina 43-44, 48, 51, 60-62 jamaicensis 51-52 solida 60 Dromus dromas 269 dubia, Mutela 269, 272, 280 dubiosa, Paracrostoma paludiformis 169 dugasti, Melania 181, 182 durrovensis, Margaritifera 317 Margaritifera margaritifera 271, 280, 317 ebenina, Crassispira 62 Drillia 43-44, 48, 51, 60-62 Glossispira 62 Strictispira 43-44 Ecrobia 325 edulis, Mytilus 73 elegans, Oxyloma 321 elevatus, Lepetodrilus 272, 280 Ellipsaria lineolata 269 ellipsiformis, Venustaconcha 269 elliptica, Etheria 269, 272, 280 Physa gyrina 140 Elliptio dilitata 269 elongata, Brotia 245 Melania baccata subsp. 187 elongatulus, Unio 316 elongatum, Acrostoma 187 Bythiospeum 324 INDEX 333 Enidae 316 ephippium, Anomia 37, 39 Epioblasma brevidens 269 episcopalis, Brotia 165, 167-168, 179-180, 183-185, 184-185, 199, 219, 220, 239 Brotia costula 176, 183 Melania 178, 183, 184 Melania (Brotia) 217 Melania (Melanoides) 183 Melania (Melanoides) variabilis 178, 183 Melanoides 183 ericana, Crassispira 45 Strictispira 44 escheri, Brotia 168 Etheria 265 elliptica 269, 272, 280 Etheriidae 265, 268-269, 272-273, 280 Etherioidea 265, 273-274, 278-280 eucymatus, Bathypecten 35 Exellichlamys spectabilis 37-39 exiguum, Bythiospeum 324 falcata, Margaritifera 271, 280 fasciolare, Ptychobranchus 269 Faunus ater 239 fezi, Spathogyna 101 Valvata (Tropidina) 78-79 Fissurellidae 14, 17 Fissuria 129-130, 325 flava, Fusconaia 269, 271, 280 Flexopecten glaber 37-39 forbesianus, Hemipecten 35 formosana, Oncomelania hupensis 145, 148 fortunei, Limnoperna 307-308 fragilis, Leptodea 269 Pyganodon 271, 275-277, 280 fuscescens, Crassispira 62 Crassispira (Crassispirella) 60 Crassispirella 44, 48, 49-50, 52-53, 62 Drillia (Crassispira) 51 Strictispira 62 Fusconaia 274-277 flava 269, 271, 280 gaillardoti, Islamia 128 gaiteri, Islamia 79, 97, 102, 109, 128-129 gargottae, Unio 292 gasulli, Hauffenia (Neohoratia) 78-79 Neohoratia 79 Tarraconia 81 gatoa, Horatia 110 gibbus, Argopecten 37-39 giennensis, Islamia henrici 77, 83, 86-87, 89- 90, 94, 103, 105, 106, 108-110, 108-109, 126, 129 gigantea, Crassadoma 37-39 glaber, Flexopecten 37-39 glabra, Melania variabilis var. 187 glabrata, Biomphalaria 73 glans, Toxolasma 269 Glebula rotundata 269 globulina, Islamia 102, 128 globulus, Amnicola 79, 83, 89 Amnicola anatina 83 Islamia 77, 79, 81, 82-83, 83, 85, 86-87, 88, 89-90, 91, 92-95, 97, 109, 109, 126- 129 Islamia globulus 83 Neohoratia 93 Neohoratia globulus 83, 127 Pseudamnicola similis 83 gloriosa, Melania 191, 192, 196 Melania (Melanoides) var. gloriosa 192 Glossispira 62 ebenina 62 Glycymeris 37, 39 pedunculata 39 pedunculus 37, 39 Glycymerididae 37 Godiva banyulensis 74 godwini, Brotia 185, 186, 190 Melania 185-186 Gonidea 275-277, 279 angulata 271, 280 graeca, Islamia 127 grandis, Pyganodon 271, 275-277, 280 granosus, Limax 313 granularis, Patella 73 Graziana 325 guanarensis, Anodontites 269, 272, 280 gyrina, Physa 133-134, 134-136, 136, 139-140 Physa gyrina 140 hainanensis, Adamietta 234-236 hainesiana, Melania 177, 178 Melania (Melanoides) variabilis var. 177 Hamiota subrotundata 269 hamonvillei, Melania 213-214 hanleyi, Melania 185-186, 186 Melania (Melanoides) 185 haroldi, Kaitoa 299 hastata, Chlamys 37-39 Hauffenia 78-79, 325 (Neohoratia) coronadoi schuelei 78-79, 110 (Neohoratia) gasulli 78-79 schuelei 110 Helcion pectunculus 73 heldii, Bythiospeum 324 Helicidae 322 Helicinidae 1, 15, 18-21, 23 Hemipecten forbesianus 35 hemphilli, Physella 312 334 INDEX henrici, Islamia 77, 102, 103, 105, 109, 125- 126, 128-129 Islamia henrici 77, 81, 82-83, 86-87, 89- 90, 94, 103, 104, 105, 107, 108-110, 109, 126, 129 henriettae, Brotia 187, 188-189, 189, 190, 195, 197, 233-236, 239, 245 Melania 163, 187, 188, 189 Semisulcospira 187 herculea, Brotia 179-180, 181, 190, 191, 192, 193-194, 199, 233-236, 245 Melania 190, 191, 192 Melania (Melanoides) 191 Melanoides 191 heros, Melania 183-184, 184 hians, Limaria 37, 39 hildrethiana, Physa gyrina 140 Horatia 77-78, 325 burnabasa 127 gatoa 110 servaini 82, 127 housei, Adamietta 234-236 huegeli, Paracrostoma 228, 241 hupensis, Oncomelania 143-145, 147-148, 149, 150, 152-153, 253-254, 260-262 Oncomelania hupensis 143-145, 148, 151- 152, 151, 253-254, 255, 259-262 hydiana, Lampsilis 269 Hydrobia 325 truncata 261 valvataeformis 79, 82, 127 Hydrobiae 160 Hydrobiidae 77, 118, 125, 160, 325 Hydrobiinae 325 Hydrocenidae 1, 19-20, 23 Hygromiidae 322 hypnorum, Aplexa 133-134, 134-135, 140 Hypselodoris tricolor 74 Hyridella 274-277 australis 272, 280 depressa 272, 280 menziesi 269, 272, 280 Hyriidae 265-267, 269-270, 275-280 hystrix, Spondylus 37-39 lliana obsoleta 74 llyanassa obsoleta 73 imbricata, Melania (Melanoides) reevei var. 192 indica, Melania 176 Melanoides 176 indragirica, Brotia 194-195, 195 Melania 194, 195 infracostata, Adamietta 181 Melania 218 insolita Brotia 196, 196, 201 Brotia (Brotia) 196 Melania 196, 196 integra, Physa 134, 140 Physa integra 140 Inversidens 275-277 lo pagodula 202 iravadica, Acrostoma 197 Brotia 190, 197, 197 Melania 197 Melania (Brotia) baccata var. 197 Melania (Melanoides) 197 Melania (Melanoides) baccata var. 197 irawadica, Melania 197 Tiara (Melanoides) baccata var. 197 Iridinidae 265-270, 272-273, 280 iris, Villosa 269 irradians, Argopecten 37-39 Islamia 77-79, 82, 83, 86-87, 89-90, 93, 97, 101-102, 109, 109, 115, 117, 125-130, 325 (Adriolitorea) latina 127 (Adriolitorea) zermanica 127 anatolica 92, 127 ateni 77, 83, 86-87, 89-90, 92, 94, 95, 96- 98, 97-98, 109, 109, 126, 128-129 bunarbasa 92 burnabasa 127 cianensis 79, 128-129 consolationis 102, 128 gaillardoti 128 дайеп 79, 97, 102, 109, 128-129 globulina 102, 128 globulus 77, 79, 81, 82-83, 83, 85, 86-87, 88, 89-90, 91, 92-95, 97, 109, 109, 126- 129 globulus globulus 83 globulus lagari 92 graeca 127 henrici 77, 102, 103, 105, 109, 125-126, 128-129 henrici giennensis 77, 83, 86-87, 89-90, 94, 103, 105, 106, 108-110, 108-109, 126, 129 henrici henrici 77, 81, 82-83, 86-87, 89- 90, 94, 103, 104, 105, 107, 108-110, 109, 126, 129 (Islamia) bosniaca 127 (Islamia) servaini 127 lagari 77, 83, 86, 92-93, 92, 95, 109, 110, 126, 128-129 mienisi 128 minuta 102, 128 pallida 77, 81, 83, 86-87, 89-90, 94, 98, 99-100, 100-102, 102, 109-110, 109, 126, 128-129 piristoma 92, 129 INDEX 335 pseudorientalica 127 pusilla 97, 128-129 schuelei 79, 110 servaini 97 spirata 102, 128 trichoniana 92, 127 valvataeformis 82, 97, 102, 126-128 islandica, Chlamys 37-39 jacksoni, Alathyria 272, 280 Jagora 160, 162, 164, 237-241, 244 asperata 210, 234-236 dactylus 234-236 jamaicensis, Drillia 51-52 Pleurotoma 51-52 japonicum, Schistosoma 143-144, 253-254, 259-262 jennessi, Physa 134, 140 Josefus 77, 79, 83, 110, 118-119, 130 aitanica 77,81, 82-83, 109, 119, 120, 121, 122-123, 124-126, 129 julieni, Melania 197 jullieni, Brotia 180-181, 190, 197, 198-199, 199, 214 Melania 197, 198, 213 kaikatensis, Shinkailepas 1, 19-20, 24 Кайоа 299, 303 haroldi 299 patagonica 299300, 301-302, 303 Katharina 272, 280 kelantanensis, Brotia 185, 199, 200 Melania 199 lactea, Striarca 37, 39 laevis, Margaritifera 271, 280 Melania verbecki var. 224-225 lagari, Islamia 77, 83, 86, 92-93, 92, 95, 109, 110, 126, 128-129 Islamia globulus 92 Neohoratia globulus 92, 127 Pseudamnicola 92-93 lamperti, Bythiospeum 324 Lampsilis 274-277 alatus 289 cardium 269 hydiana 269 powellii 269 reeveiana 269 siliquoidea 269 straminea 269 streckeri 269 teres 269, 271, 280 lanceolata, Melania (Melanoides) reevei var. 192 Lanistes 27, 29, 32 carinatus 27-33, 31 latina, Islamia (Adriolitorea) 127 latchfordii, Physa parkeri 140 Lepetodrilus elevatus 272, 280 Leptodea fragilis 269 leptodon 269 leptodon, Leptodea 269 lienosa, Villosa 269 ligamentina, Actinonaias 269, 271, 280 Ligumia 274277 recta 269, 271, 280 Lima lima 37, 39 lima, Lima 37, 39 Limaria hians 37, 39 Limatula 36 Limax granosus 313 Limidae 36-38 Limnoperna fortunei 307-308 Limoidea 37 lineolata, Ellipsaria 269 Lithoglyphi 160 Lithoglyphidae 325 littoralis, Potomida 292 Littorina 237 lomata, Strictispira 44 longae, Musculus conchae 287, 289 Lortiella rugata 272, 280 lucidum, Propeamussium 39 lusoria, Meretrix 74 luteola, Lymnaea 32 Lymnaea luteola 32 stagnalis 74 macrochisma, Pododesmus 37, 39 Mactra chinensis 74 veneriformis 74 magellanicus, Placopecten 37-39 magnalacustris, Physa 134, 140 Physa sayii 140 mancus, Unio 316 manningi, Brotia 196-197, 200, 200-201 Brotia (Brotia) 200 margaritacea, Neotrigonia 269-270, 272, 280 Margaritana 287 auricularia 289 crassa 287, 289 margaritanopsis 289, 292 margaritifera 287, 289 margaritanopsis, Margaritana 289, 292 Unio 289, 292 margaritifer, Unio 287, 289 Unio (Margaritana) 287 Margaritifera 285, 287, 289, 317-318 auricularia 271, 280, 285, 286, 287, 288, 289, 290-291, 292 durrovensis 317 falcata 271, 280 laevis 271, 280 336 margaritifera 269, 271, 280, 285, 286, 287, 289, 292, 317 margaritifera durrovensis 271, 280, 317 margaritifera, Margaritana 287, 289 Margaritifera 269, 271, 280, 285, 286, 287, 289, 292, 317 Mya 285, 287, 289 Unio 289 Margaritiferidae 265-267, 269-271,274-278, 280 maximus, Pecten 37-39 Medionidus conradicus 269 Melanatria 240, 242-243 Melanatriinae 243 Melanella zonata 232 Melania 159 (Acrostoma) assamensis 228 angulifera 229 asperata 210 baccata 187, 188, 189 baccata subsp. elongata 187 baccata var. pyramidalis 187 balteata 191192, 191 beaumetzi 228, 229 binodosa 171, 171 boeana 217-219, 218 borneensis 229, 230 (Brotia) baccata 187 (Brotia) baccata var. iravadica 197 (Brotia) episcopalis 217 (Brotia) reevei 192 canaliculata 229, 231 carolinae 176, 177 citrina 174, 174, 182 citrinoides 174, 174 corrugata 178 costula 176, 183, 217 curvicosta 221, 222 curvicosta var. prestoniana 222 cylindrus 229, 230 dautzenbergiana 181-182, 181 dugasti 181, 182 episcopalis 178, 183, 184 gloriosa 191, 192, 196 godwini 185186 hainesiana 177, 178 hamonvillei 213214 hanleyi 185186, 186 henriettae 163, 187, 188, 189 herculea 190, 191, 192 heros 183-184, 184 indica 176 indragirica 194, 195 infracostata 218 iravadica 197 гамма са 197 insolita 196, 196 julieni 197 jullieni 197, 198, 213 kelantanensis 199 (Melanoides) baccata 187 (Melanoides) baccata subvar. recta 187 (Melanoides) baccata var. iravadica 197 (Melanoides) episcopalis 183 (Melanoides) hanleyi 185 (Melanoides) herculea 191 (Melanoides) iravadica 197 (Melanoides) palembangensis 217 (Melanoides) reevei 192 (Melanoides) reevei var. imbricata 192 (Melanoides) reevei var. lanceolata 192 (Melanoides) reevei var. soliduscula 192 (Melanoides) spinata 231 (Melanoides) subasperata 187 (Melanoides) subasperata var. sublaevigata 187 (Melanoides) sumatrensis 217 (Melanoides) tourannensis var. beddo- meana 192 (Melanoides) tourannensis var. compacta 192 (Melanoides) tourannensis var. gloriosa 192 (Melanoides) tourannensis var. peguensis 192 (Melanoides) variabilis 176 (Melanoides) variabilis episcopalis 178, 183 (Melanoides) variabilis menkeana 176 (Melanoides) variabilis subvar. aspera 178 (Melanoides) variabilis subvar. cincta 178 (Melanoides) variabilis subvar. microstoma 178 (Melanoides) variabilis subvar. pseudo- spinosa 178 (Melanoides) variabilis subvar. semi- laevigata 192 (Melanoides) variabilis subvar. subspinosa 178 (Melanoides) variabilis subvar. subtuber- culata 178 (Melanoides) variabilis subvar. subvaricosa 192 (Melanoides) variabilis var. binodulifera 185 (Melanoides) variabilis var. hainesiana 177 (Melanoides) variabilis var. semilaevigata 178 menkeana 176 menkiana 176, 179 (Pachychilus) angulifera 227 pagodula 163, 202, 204 pagodulus 202 papillosa 224-225, 224 peguensis 192 INDEX 337 persculpta 187, 188 pleuroceroides 244 plicata 176 (Plotia) scabra var. angulifera 228 reevei 191-192 reticulata 187, 188 siamensis 213 sooloensis 229, 231 spinata 178, 231 spinosa 177, 178 stricticosta 224, 225 subcancellata 222 subcylindrica 229, 230 subplicata 221-223, 221 sumatrensis 217-218, 218 sumatrensis var. mitescens 218, 221 terebra 222 torquata 218, 221-223, 221 variabilis 163, 176, 177, 179, 184, 192, 217 variabilis var. baccifera 187 variabilis var. glabra 187 variabilis var. pyramidalis 187 variabilis var. spinosa 178 variabilis var. turrita 187 variabilis var. vittata 187 varicosa 176, 177 verbecki 224, 224 verbecki маг. laevis 224-225 zollingeri 221-223, 221 zonata 229, 232 Melaniidae 159, 239, 242-243 Melanoides 243 episcopalis 183 herculea 191 indica 176 reticulata 187 spinata 231 (Tiara) baccata 187 (Tiara) variabilis 176 torquata 221 tuberculata 239 varicosa 176 Melanopsidae 160, 239-240, 243 menkeana, Brotia 178 Melania 176 Melania (Melanoides) variabilis 176 menkiana, Melania 176, 179 menziesi, Hyridella 269, 272, 280 Mercuria 325 Meretrix lusoria 74 Microna ateni 78-79, 96 microsculpta, Brotia 201, 202-203, 216-217, 232-236, 240, 242 Brotia (Brotia) 201 microstoma, Melania (Melanoides) variabilis subvar. 178 mienisi, Islamia 128 Mienisiella 82, 128 Milax ochraceus 313 Milesiana 77, 79, 83, 110, 118, 130 schuelei 77, 80, 81, 82-83, 110, 111, 112- 113, 114, 115-118, 117, 126, 129 cf. schuelei 83, 110, 113 Mimachlamys varia 37-39 miniata, Patella 73 minima, Oncomelania 145 minuana, Monocondylaea 272, 280 minuta, Islamia 102, 128 mitescens, Melania sumatrensis var. 218, 221 moenanum, Bythiospeum 324 Moitessieria 325 Moitessieriidae 325 Monocondylaea minuana 272, 280 monodonta, Cumberlandia 269, 271, 280 morrisoni, Paracrostoma 169, 169 muricata, Pinna 37, 39 Muricidae 299 Musculus niger 287, 289 conchae longae 287, 289 omnium longe crassisimus 287 Mutela dubia 269, 272, 280 rostrata 272, 280 Mutelidae 267 Mya margaritifera 285, 287, 289 testa crassa 287, 288, 289 Mycetopodidae 265-270, 272-273, 280 myojinensis, Shinkailepas 1-2, 14, 18-24 Mytilus 153, 268, 273 edulis 73 Natica subtenuis 303 Naticidae 302 neglecta, Cernuella 322 Cernuella (Xerocincta) 322 Neohoratia 77-79, 101, 126-127, 129 ateni 96, 127 azarum 78—79 coronadoi 98, 99, 101 gasulli 79 globulus 93 globulus globulus 83, 127 globulus lagari 92, 127 schuelei 79, 101, 110 subpiscinalis 127 Neotrigonia 273, 275-278 margaritacea 269-270, 272, 280 Neotrigoniidae 269, 280 Neptunea 65 antiqua 65 arthritica 65, 74-75 arthritica cumingii 65-67, 67-72, 73-75 (Barbitonia) arthritica cumingii 65 338 INDEX constricta 74-75 cumingii 65, 74-75 Nerita 15-19 Neritidae 1, 15-16, 18-23 Neritilia 15-16, 22 Neritiliidae 1, 14-23 Neritoidea 1, 23 Neritopsidae 1, 23 Neritopsis 19 newcombiana, Succinea 295, 298 niger, Musculus 287, 289 nigrescens, Pleurotoma 50, 52 noae, Arca 37, 39 Noetiidae 37 nolli, Bythiospeum 324 oblonga, Testa 287 Obovaria olivaria 269 obsoleta, lliana 74 llyanassa 73 occidentalis, Abida 313 ochraceus, Milax 313 ochsneri, Cleospira 44 oculus, Patella 73 ohiensis, Potamilus 269 Oichnus paraboloides 299 Olgasolaris 12, 14, 17-19, 21, 23 tollmanni 19, 22 olivaria, Obovaria 269 Oncomelania 143, 145, 253-254, 256, 259- 260 hupensis 143-145, 147-148, 149, 150, 152-153, 253-254, 260-262 hupensis hupensis 143-145, 148, 151 152, 151, 253-254, 255, 259-262 hupensis formosana 145, 148 hupensis quadrasi 145, 148 hupensis robertsoni 143-145, 147-148, 149-151, 150-154, 157, 253-254, 262 hupensis tangi 144-145, 152, 253-254 minima 145 opercularis, Aequipecten 38-39 oppenoorthi, Brotia 166 Orientalina 325 ostrearum, Pyrgospira 48, 57 Oxychilus adjaciensis 313 colliourensis 313 Oxyloma elegans 321 pfeifferi 321 Pachychila 243 Pachychilidae 159-160, 163-164, 169, 227, 237, 239-244 Pachychili 160, 242-243 Pachychilinae 243 Pachychilus 239-244 pagodula, Brotia 164, 165, 168, 190, 202, 203- 205, 233-238, 240, 242 Brotia (Brotia) 202 lo 202 Melania 163, 202, 204 Tiara (Acrostoma) 202 pagodulus, Melania 202 palaeocostula, Brotia 166 palembangensis, Melania (Melanoides) 217 pallida, Islamia 77,81, 83, 86-87, 89-90, 94, 98, 99-100, 100-102, 102, 109-110, 109, 126, 128-129 paludiformis, Brotia 203, 206, 206, 213, 238 Paracrostoma 206 Paracrostoma paludiformis 206 Semisulcospira 206 papillosa, Melania 224-225, 224 paraboloides, Oichnus 299 Paracrostoma 160, 162, 164, 228, 234-237, 239-241, 243-244 assamensis 228 huegeli 228, 241 morrisoni 169, 169 paludiformis 206 paludiformis dubiosa 169 paludiformis paludiformis 206 pseudosulcospira 212 pseudosulcospira armata 169, 169, 212 pseudosulcospira pseudosulcospira 212 solemiana 215 Paragonimus westermanni 185 parkeri, Physa 133-134, 134-135, 136, 139- 140 Physa parkeri 140 Parvamussium 38 undisonum 35-39 patagonica, Kaitoa 299-300, 301-302, 303 Patella argenvillei 73 barbara 73-74 granularis 73 miniata 73 oculus 73 paxillus, Crassispira 50 Crassispira (Strictispira) 51 Drillia (Crassispira) 50 Drillia (Drillia) 52 Pleurotoma 44, 50, 62 Strictispira 43-44, 48, 50, 50-53, 52, 56, 58, 60, 62-63 Pecten maximus 37-39 pectinata, Atrina 37, 39 Pectinidae 35-39 Pectininae 35 Pectinoidea 35-38 pectunculus, Helcion 73 Pedum spondyloideum 37-39 INDEX 339 pedunculata, Glycymeris 39 pedunculus, Glycymeris 37 peguensis, Melania 192 Melania (Melanoides) tourannensis var. 192 pellucidum, Bythiospeum 324 peninsularis, Brotia 179, 203, 205, 207-208, 207-208, 213-214, 233-236 Brotia (Brotia) costula 207 Brotia costula 178 perakensis, Sermyla 183-184, 184 Peringia 325 persculpta, Melania 187, 188 Petrophysa 133 Pezzolia 118 radapalladis 118 pfeifferi, Oxyloma 321 Phenacolepadidae 1, 15-16, 19-20, 23 Phenacolepas 1, 14, 17-18, 20-22, 24 phorochaetia, Trichia 313-314 Physa 133, 139-140 acuta 133-136, 134-135, 139 gyrina 133-134, 134-136, 136, 139-140 gyrina elliptica 140 gyrina gyrina 140 gyrina hildrethiana 140 integra 134, 140 integra brevispira 140 integra integra 140 integra walkeri 140 jennessi 134, 140 jennessi skinneri 140 magnalacustris 134, 140 parkeri 133-134, 134-135, 136, 139-140 рагкеп latchfordii 140 parkeri parkeri 140 sayii 133-134, 134-135, 136, 139-140 sayii magnalacustris 140 Sayli sayii 140 sayii vinosa 140 Physella 133-134 hemphilli 312 winnipegensis 312 Physidae 133-134 Physodon 134 pictorum, Unio 287, 316 Pinna muricata 37, 39 Pinnidae 36-38 Pinnoidea 37 piristoma, Islamia 92, 129 Pisulina 15-16, 22 Placopecten magellanicus 37-39 Planaxidae 241 Planorbarius corneus 74 Pleuroceridae 159-160, 242-243 pleuroceroides, Melania 244 Pleurotoma jamaicensis 51-52 nigrescens 50, 52 paxillus 44, 50, 62 quadrifasciata 53 solida 43-44, 60, 62 plicata, Acar 37, 39 Amblema 269 Melania 176 Plicatula 37, 39 Plicatula plicata 37, 39 australis 37, 39 Plicatulidae 36-38 Plicatuloidea 37 Pododesmus caelata 37, 39 macrochisma 37, 39 Polinices santacruzensis 303 polymorpha, Dreissena 305 Pomatiopsidae 145, 254 ponida, Strictispira 44 popeii, Popenaias 269 Popenaias popeii 269 Potadoma 240-243 Potamididae 240 Potamilus 274-277 alatus 269, 289 capax 269 ohiensis 269 purpuratus 269, 271, 280 Potomida littoralis 292 powellii, Lampsilis 269 praetermissa, Brotia 203, 209, 209-210, 230 prestoniana, Melania curvicosta var. 222 proebenina, Strictispira 44 Propeamussiidae 35—40 Propeamussiinae 35 Propeamussium lucidum 39 Proserpinidae 1, 17 provisoria, Adamietta 234 Pseudamnicola 325 lagari 92-93 similis globulus 83 Pseudanodonta complanata 315 pseudoasperata, Brotia 210, 210-211 Brotia (Brotia) 210 Pseudodon 275-277, 279 vondembuschianus 271, 280 Pseudopotamis 160, 231, 240-241, 244 pseudorientalica, Islamia 127 pseudospinosa, Melania (Melanoides) vari- abilis subvar. 178 pseudosulcospira, Brotia 167, 169, 171, 202, 203, 207, 212-213, 212-213, 216-217, 232-236 Brotia (Paracrostoma) 212 Paracrostoma 212 Paracrostoma pseudosulcospira 212 Pseudunio 292 auricularius 289, 292 Pterinopectinidae 40 340 INDEX Ptychobranchus, fasciolare, 269 puerkhaueri, Bythiospeum 324 purpuratus, Potamilus 269, 271, 280 pusilla, Islamia 97, 128-129 Valvata 128 Pyganodon fragilis 271, 275-277, 280 grandis 271, 275-277, 280 pyramidalis, Melania baccata var. 187 Melania variabilis var. 187 pyrenaearia, Abida 313 Pyrgospira 44, 48, 58 acurugata 58-59 ostrearum 48, 57 quadrasi, Oncomelania hupensis 145, 148 quadrifasciata, Crassispira 43-44, 53 Crassispira (Crassispirella) 53 Drillia (Crassispira) 53 Pleurotoma 53 Strictispira 48, 50, 52, 53-54, 56, 63 quenstedti, Bythiospeum 324 radapalladis, Pezzolia 118 Rapana venosa 73-74 recta, Ligumia 269, 271, 280 Melania (Melanoides) baccata subvar. 187 redferni, Strictispira 43-44, 46, 47, 50, 52, 54, 55-56, 57-60, 63 reevei, Brotia 245 Melania 191-192 Melania (Brotia) 192 Melania (Melanoides) 192 reeveiana , Lampsilis 269 reiniana, Semisulcospira 238 reticulata, Melania 187, 188 Melanoides 187 reticulatus, Agriolimax 74 Rissooidea 324-325 rivolii, Acostaea 272, 280 robertsoni, Oncomelania hupensis 143-145, 147-148, 149-151, 150-154, 157, 253-254, 262 rostrata, Mutela 272, 280 rotundata, Glebula 269 rougemonti, Bythiospeum 324 rubens, Chambardia 269, 272, 280 rugata, Lortiella 272, 280 rugosa, Clausilia 315 Sadleriana 325 santacruzensis, Polínices 303 sayii, Physa 133-134, 134-135, 136, 139-140 Physa sayii 140 Schistosoma 154 japonicum 143-144, 253-254, 259-262 schmidti, Adamietta 235-236 schuelei, Hauffenia 110 Hauffenia (Neohoratia) coronadoi 78-79, 110 Islamia 79, 110 Milesiana 77, 80, 81, 82-83, 110, 111, 112- 113, 114, 115=118,.117,126, 129 Milesiana cf. 83, 110, 113 Neohoratia 79, 101, 110 secale, Abida 315 semilaevigata, Melania (Melanoides) variabilis subvar. 192 Melania (Melanoides) variabilis var. 178 Semisulcospira 244 henriettae 187 paludiformis 206 reiniana 238 Senckenbergia 164, 226, 244 senefelderi, Bythiospeum 324 Septaria 14-17, 19-20, 23 Sermyla perakensis 183-184, 184 serriata, Siphonaria 74 servaini, Horatia 82, 127 Islamia 97 Islamia (Islamia) 127 Shinkailepas 1-2, 14-24 briandi 1, 24 kaikatensis 1, 19-20, 24 myojinensis 1-2, 14, 18-24 tufari 1, 24 siamensis, Brotia 196-197, 201, 205, 208, 208, 213-214, 214 Melania 213 siliquoidea, Lampsilis 269 similis, Amnicola 83 sinensis, Spondylus 37-39 sinuata, Unio 285, 286, 289 sinuatus, Unio 287, 289, 292 Siphonalia assidariaeformis 74-75 Siphonaria capensis 73-74 serriata 74 skinneri, Physa jennessi 140 solemiana, Brotia 203, 215-217, 215, 233-236 Brotia (Paracrostoma) 215 Paracrostoma 215 Solemya velum 272, 280 solida, Clathrodrillia 47, 50-51, 60 Crassispira (Strictispira) cf. 62 Drillia 60 Drillia (Clathrodrillia) 60 Pleurotoma 43-44, 60, 62 Strictispira 43-44, 48, 49, 50, 52, 56, 60, 62-63 soliduscula, Melania (Melanoides) reevei var. 192 sooloensis, Brotia 229, 231 Melania 229, 231 INDEX 341 Spathogyna 79 fezi 101 spectabilis, Exellichlamys 37-39 spinata, Brotia 171, 174, 204, 231-232 Melania 178, 231 Melania (Melanoides) 231 Melanoides 231 spinosa, Melania 177, 178 Melania variabilis var. 178 spiralis, Brotia binodosa 171 Brotia (Brotia) binodosa 171 Brotia spinata 171 spirata, Islamia 102, 128 Spondylidae 36-39 spondyloideum, Pedum 37-39 Spondylus crassisquamatus 37-39 hystrix 37-39 sinensis 37-39 stagnalis, Lymnaea 74 Stenomelania dautzenbergiana 182 Stenophysa 133 stevensi, Castalia 272, 280 stillmani, Strictispira 43-44 straminea, Lampsilis 269 streckeri, Lampsilis 269 Striarca lactea 37, 39 stricticosta, Melania 224, 225 Strictispira 43-45, 46, 48, 49, 50, 52, 52, 54, 56, 62-63 acurugata 44, 54, 57-58 aurantia 44 coltrorum 43-45, 46, 47, 50, 52, 56, 63 drangai 43-44, 47, 48, 49-50, 50, 52, 53, 62 ebenina 43-44 ericana 44 fuscescens 62 lomata 44 paxillus 43-44, 48, 50, 50-53, 52, 56, 58, 60, 62-63 ponida 44 proebenina 44 quadrifasciata 48, 50, 52, 53-54, 56, 63 redferni 43-44, 46, 47, 50, 52, 54, 55-56, 57-60, 63 solida 43-44, 48, 49, 50, 52, 56, 60, 62-63 stillmani 43—44 Strictispiridae 43, 45 Strictispirinae 43 subasperata, Melania (Melanoides) 187 subcancellata, Melania 222 subcylindrica, Melania 229, 230 subgloriosa, Brotia 203, 216, 216-217 Brotia binodosa 216 Brotia (Brotia) binodosa 216 Brotia spinata 216 sublaevigata, Melania (Melanoides) sub- asperata var. 187 subpiscinalis, Neohoratia 127 Valvata 77, 78 subplicata, Melania 221-223, 221 subrotundata, Hamiota 269 subspinosa, Melania (Melanoides) variabilis subvar. 178 subtenuis, Natica 303 subtuberculata, Melania (Melanoides) vari- abilis subvar. 178 subvaricosa, Melania (Melanoides) variabilis subvar. 192 Succinea newcombiana 295, 298 thaanumi 295, 297-298 suevicum, Bythiospeum 324 Sulcospira 160, 166, 240, 242-244 sumatrensis, Brotia 180, 185, 185, 217-219, 218, 220, 232-236, 238, 242 Melania 217-218, 218 Melania (Melanoides) 217 tampaensis, Crassispira 57 tampicoensis, Cyrtonaias 269, 271, 280 tamsiana, Tamsiella 269, 272, 280 Tamsiella tamsiana 269, 272, 280 tangi, Oncomelania hupensis 144-145, 152, 253-254 Tarraconia 79 gasulli 81 taxisi, Bythiospeum 324 terebra, Melania 222 teres, Lampsilis 269, 271, 280 Testa crassa 287 oblonga 287 testudinaria, Adamietta 223, 234-236 Brotia 164, 181, 214, 227, 241 thaanumi, Succinea 295, 297-298 Thiarae 160 Thiaridae 159-160, 239-241, 243-244 Tiara (Acrostoma) assamensis 228 (Acrostoma) pagodula 202 (Melanoides) baccata var. irawadica 197 Tiaropsis 222 Tinnyea 168 Titiscania 17, 19-21 Titiscanidae 20, 23 Titiscaniidae 1 tollmanni, Olgasolaris 19, 22 torquata, Вгойа 179, 221-223, 221-223, 233- 236, 238 Melania 218, 221-223, 221 Melanoides 221 Toxolasma glans 269 Trichia phorochaetia 313-314 villosa 313 342 INDEX trichoniana, Islamia 92, 127 tricolor, Hypselodoris 74 Tricula 145, 149 Trigonioidea 280 trigonus, Anodontites 272, 280 truncata, Hydrobia 261 Truncilla 269 truncatus, Bulinus 32 Truncilla truncata 269 tryoni, Aplexa hypnorum 140 tuberculata, Melanoides 239 tufari, Shinkailepas 1, 24 Turridae 43 turrita, Albinaria 272, 280 Melania variabilis var. 187 Tylomelania 160, 164, 238, 240-241, 244 undisonum, Parvamussium 35-39 Unio 287 auricularius 285, 287, 289, 292 crassissimus 292 crassus 287, 288 elongatulus 316 gargottae 292 mancus 316 (Margaritana) margaritifer 287 margaritanopsis 289, 292 margaritifer 287, 289 margaritifera 289 pictorum 287, 316 sinuata 285, 286, 289 sinuatus 287, 289, 292 Unionidae 265-271, 274-280 Unionidea 274, 278-280, 285 Valvata coronadoi 78, 98, 101 pusilla 128 subpiscinalis 77, 78 (Tropidina) fezi 78-79 valvataeformis, Hydrobia 79, 82, 127 Islamia 82, 97, 102, 126-128 varia, Mimachlamys 37-39 variabilis, Acrostoma 176 Brotia 166, 176, 184, 217 Brotia (Antimelania) 176 Melania 163, 176, 177, 179, 184, 192, 217 Melania (Melanoides) 176 Melanoides (Tiara) 176 varicosa, Brotia costula 178 Melania 176, 177 Melanoides 176 vasarhelyii, Brotia 168 Velesunio 272, 280 ambiguus 272, 280 angasi 272, 280 velum, Solemya 272, 280 veneriformis,Mactra 74 venosa, Rapana 73-74 Ventrosia 325 Venustaconcha ellipsiformis 269 verbecki, Brotia 223-224, 223-225, 233-236 Melania 224, 224 villosa, Trichia 313 Villosa 269 Villosa iris 269 lienosa 269 villosa 269 vinosa, Physa sayii 140 virescens, Barbatia 37, 39 virgata, Cernuella 313, 322 Cernuella (Cernuella) 322 vittata, Melania variabilis var. 187 vondembuschianus, Pseudodon 271, 280 vulcani, Bathypecten 35-40 Waldemaria 15-16, 19 walkeri, Physa integra 140 Wanga 163, 189 westermanni, Paragonimus 185 winnipegensis, Physella 312 wykoffi, Brotia 205, 226, 226-227, 233-236, 244 Brotia (Senckenbergia) 226 Xerocincta 322 Xeromagna 322 Xerosecta 322 zermanica, Islamia (Adriolitorea) 127 zizyphus, Clavus (Crassispira) 44 zollingeri, Brotia 221 Melania 221-223, 221 zonata, Brotia 232 Melanella 232 Melania 229, 232 zonatum, Chilostoma 315 MALACOLOGIA International Journal of Malacology Vol. 48(1-2) 2006 Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Publication dates , No. 17 Dec. 13 May 17 Dec. 22 Sep. 31 Dec. 18 Oct. 20 Aug. 8 Feb. 30 Aug. 29 Aug. 22 Mar. 23 Aug. 30 Dec. 20 July 1996 1998 1998 1999 1999 2000 2001 2002 2002 2003 2004 2004 2004 2005 VOL. 48, NO. 1-2 MALACOLOGIA 2006 CONTENTS ABDALLAH THARWAT ABDALLAH Effects of Dissolved Lead and Copper on the Freshwater Prosobranch Paste CMA ARR O CRE Nr TN 27, BEATRIZ ARCONADA & MARIA-ANGELES RAMOS Revision of the Genus /slamia Radoman, 1973 (Gastropoda, Caeno- gastropoda, Hydrobiidae) on the Iberian Peninsula and Description of Two NewaGenerasand Three New Species 0........... u... Tf RUUD A. BANK, GERHARD FALKNER, EDMUND GITTENBERGER, THEO E. J. RIPKEN & TED VON PROSCHWITZ Check-Lists and CLECOM: A Response to Davis (2004) ............. 321 PHILIPPE BOUCHET Valid Until Synonymized, or Invalid Until Proven Valid? A Response to Davis:(2004)'on Species Check=Lists . coords o 311 SUSAN G. BROWN, JUDY M. SPAIN & MARCI ARIZUMI A Field Study of the Life History of the Endemic Hawaiian Snail Succinea NEWCOMDIANG ATT RE TE E AE AI AA et AAA 295 EE-YUNG CHUNG, SUNG YEON KIM, GAB-MAN PARK & JONG MAN YOON Germ Cell Differentiation and Sexual Maturation of the Female Neptunea (Barbitonia) arthritica cumingii (Crosse, 1862) (Gastropoda: Buccinidae) . . . 65 GEORGE M. DAVIS, WEI-PING WU, XING-JIAN XU Ecogenetics of Shell Sculpture in Oncomelania (Gastropoda) in Canals of Hubei, China, Relevance for Schistosome Transmission ............. 253 ROBERT T. DILLON, JR. & AMY R. WETHINGTON The Michigan Physidae Revisited: a Population Genetic Survey ....... 133 SUZANNE С. DUFOUR, GERHARD STEINER & PETER С. BENINGER Phylogenetic Analysis of the Peri-hydrothermal Vent Bivalve Bathypecten уса вазе 8 RNA 2.2.0000 2er о 35 JAROSLAW КОВАК Geotactic Behaviour of Dreissena polymorpha (Bivalvia) ............. 305 FRANK KÔHLER & MATTHIAS GLAUBRECHT A Systematic Revision of the Southeast Asian Freshwater Gastropod Brotia (Ceniblordea:.Rachychllldae). a NO CR 159 TAKENORI SASAKI, TAKASHI OKUTANI & KATSUNORI FUJIKURA Anatomy of Shinkailepas myojinensis Sasaki, Okutani & Fujikura, 2003 (GaStopoda”NernitopSina) к... о а 1 JAVIER Н. SIGNORELLI, GUIDO PASTORINO & MIGUEL GRIFFIN Naticid Boreholes on a Tertiary Cylichnid Gastropod from Southern Patagonia. har Auen enr MO A perl cal EA 299 DONN Е. TIPPETT The Genus Strictispira in the Western Atlantic (Gastropoda: Conoidea) . . . . 43 ARTURO VALLEDOR DE LOZOYA & RAFAEL ARAUJO The Historical Misidentification of Margaritifera auricularia for M. margaritifera (Bivalvia, Unionoidea) Explained by their Iconography .... 285 JENNIFER M. WALKER, JASON P. CUROLE, DAN E. WADE, ERIC G. CHAPMAN, ARTHUR E. BOGAN, G. THOMAS WATTERS & WALTER R. HOEH Taxonomic Distribution and Phylogenetic Utility of Gender-Associated Mi- tochondrial Genomes in the Unionoida (Bivalvia) ................... 265 THOMAS WILKE, GEORGE M. DAVIS, DONGCHUAN QIU & ROBERT C. 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VOL. 48, NO. 1-2 MALACOLOGIA 2006 CONTENTS TAKENORI SASAKI, TAKASHI OKUTANI & KATSUNORI FUJIKURA Anatomy of Shinkailepas myojinensis Sasaki, Okutani 8 Fujikura, 2003 (Gastopada: MertaBSina) -- = 2: 2. + ¿ie ee 1 ABDALLAH THARWAT ABDALLAH Effects of Dissolved Lead and Copper on the Freshwater Prosobranch сое = ) 5.205. BEY ne er ee REP 27T SUZANNE C. DUFOUR, GERHARD STEINER & PETER G. BENINGER Phylogenetic Analysis of the Peri-hydrothermal Vent Bivalve Bathypecten мени Basen on TES FRNA... u. ее оо 35 DONN L. TIPPETT The Genus Strictispira in the Western Atlantic (Gastropoda: Conoidea) .... 43 EE-YUNG CHUNG, SUNG YEON KIM, GAB-MAN PARK & JONG MAN YOON Germ Cell Differentiation and Sexual Maturation of the Female Neptunea (Barbitonia) arthritica cumingii (Crosse, 1862) (Gastropoda: Buccinidae) . . . 65 BEATRIZ ARCONADA & MARIA-ANGELES RAMOS Revision of the Genus /slamia Radoman, 1973 (Gastropoda, Caeno- gastropoda, Hydrobiidae) on the Iberian Peninsula and Description of Two New Genera and Three New Species ............................ Tr ROBERT T. DILLON, JR. 8 AMY R. WETHINGTON The Michigan Physidae Revisited: a Population Genetic Survey ....... 133 THOMAS WILKE, GEORGE M. DAVIS, DONGCHUAN QIU & ROBERT C. SPEAR Extreme Mitochondrial Sequence Diversity in the Intermediate Schistoso- miasis Host Oncomelania hupensis robertsoni: Another Case of Ancestral тс os aura. rennes ce ee Ae ee 143 FRANK KÔHLER & MATTHIAS GLAUBRECHT А Systematic Revision of the Southeast Asian Freshwater Gastropod Brotia (Centhicidea: Pachychilidae) :: = 2.2. u. ао. 159 GEORGE M. DAVIS, WEI-PING WU, XING-JIAN XU Ecogenetics of Shell Sculpture in Oncomelania (Gastropoda) in Canals of Hubei, China, Relevance for Schistosome Transmission ............. 253 JENNIFER M. WALKER, JASON P. CUROLE, DAN E. WADE, ERIC G. CHAPMAN, ARTHUR E. BOGAN, G. THOMAS WATTERS & WALTER R. HOEH Taxonomic Distribution and Phylogenetic Utility of Gender-Associated Mi- tochondrial Genomes in the Unionoida (Bivalvia) ................... 265 RESEARCH NOTES ARTURO VALLEDOR DE LOZOYA & RAFAEL ARAUJO The Historical Misidentification of Margaritifera auricularia for M. margaritifera (Bivalvia, Unionoidea) Explained by their Iconography .... 285 SUSAN G. BROWN, JUDY M. SPAIN & MARCI ARIZUMI A Field Study of the Life History of the Endemic Hawaiian Snail Succinea COWCO : <0 05 aaa oe ооо hod eed hae о 295 JAVIER H. SIGNORELLI, GUIDO PASTORINO & MIGUEL GRIFFIN Naticid Boreholes on a Tertiary Cylichnid Gastropod from Southern РОН EE PE ae es one Sead eee 299 JAROSLAW KOBAK Geotactic Behaviour of Dreissena polymorpha (Bivalvia) ............. 305 LETTERS TO THE EDITOR PHILIPPE BOUCHET Valid Until Synonymized, or Invalid Until Proven Valid? A Response to Davis (2004) on Species Check-Lists ...cococononsrcicarorsa a 311 RUUD A. BANK, GERHARD FALKNER, EDMUND GITTENBERGER, THEO E. J. RIPKEN 8 TED VON PROSCHWITZ Check-Lists and CLECOM: A Response to Davis (2004) ............. 321