J. A. ALLEN Marine Biological Station Millport, United Kingdom jallen @udcf.gla.ac.uk E. E. BINDER Museum d’Histoire Naturelle Geneve, Switzerland P. BOUCHET Museum National d'Histoire Naturelle Paris, France bouchet @cimrs1.mnhn.fr P. CALOW University of Sheffield United Kingdom R. CAMERON Sheffield United Kingdom R.Cameron @sheffield.ac.uk J. G. CARTER University of North Carolina Chapel Hill, U.S.A. MARYVONNE CHARRIER Universite de Rennes France Maryvonne.Charrier@univ-rennes1.fr В. H. COWIE University of Hawaii Honolulu, HI., U.S.A. A. H. CLARKE, Jr. Portland, Texas, U.S.A. B. C. CLARKE University of Nottingham United Kingdom R. DILLON College of Charleston SC, U.S.A. C. J. DUNCAN University of Liverpool United Kingdom D. J. EERNISSE California State University Fullerton, U.S.A. E. GITTENBERGER Rijksmuseum van Natuurlijke Historie Leiden, Netherlands sbu2eg @rulsfb.leidenuniv.de F. GIUSTI Universita di Siena, Italy giustif @ unisi.it 2001 EDITORIAL BOARD A. N. GOLIKOV Zoological Institute St. Petersburg, Russia S. J. GOULD Harvard University Cambridge, Mass., U.S.A. A. V. GROSSU Universitatea Bucuresti Romania T. HABE Tokai University Shimizu, Japan R. HANLON Marine Biological Laboratory Woods Hole, Mass., U.S.A. G. HASZPRUNAR Zoologische Staatssammlung Muenchen Muenchen, Germany haszi @zi.biologie. uni-muenchen.de J. M. HEALY University of Queensland Australia jhealy O zoology.uq.edu.au D. M. HILLIS University of Texas Austin, U.S.A. K. E. HOAGLAND MCZ LIBRARY FEB 14 2002 Council for Undergraduate Research Washington, DC, U.S.A. Elaine @cur.org B. HUBENDICK Naturhistoriska Museet Goteborg, Sweden S. HUNT Lancashire United Kingdom R. JANSSEN Forschungsinstitut Senckenberg, Frankfurt am Main, Germany M. S. JOHNSON University of Western Australia Nedlands, WA, Australia msj @cyllene.uwa.edu.au R. N. KILBURN Natal Museum Pietermaritzburg, South Africa M. A. KLAPPENBACH HARVARD UNIVERSITY Museo Nacional de Historia Natural Montevideo, Uruguay J. KNUDSEN Zoologisk Institut Museum Kobenhavn, Denmark C. LYDEARD University of Alabama Tuscaloosa, U.S.A. clydeard@biology.as.ua.edu C. MEIER-BROOK Tropenmedizinisches Institut Tubingen, Germany Н. К. MIENIS Hebrew University of Jerusalem Israel J. E. MORTON The University Auckland, New Zealand J. J. MURRAY, Jr. University of Virginia Charlottesville, U.S.A. R. NATARAJAN Marine Biological Station Porto Novo, India DIARMAID O’FOIGHIL University of Michigan Ann Arbor, U.S.A. J. OKLAND University of Oslo Norway T. OKUTANI University of Fisheries Tokyo, Japan W. L. PARAENSE Instituto Oswaldo Cruz, Rio de Janeiro Brazil J. J. PARODIZ Carnegie Museum Pittsburgh, U.S.A. R. PIPE Plymouth Marine Laboratory Devon, United Kingdom RKPI@wpo.nerc.ac.uk J. P. POINTIER Ecole Pratique des Hautes Etudes Perpignan Cedex, France pointier @ gala.univ-perp.fr М.Е. PONDER Australian Museum Sydney QIFZ. У. Academia Sinica Qingdao, People’s Republic of China D. G. REID The Natural History Museum London, United Kingdom $. G. SEGERSTRALE Institute of Marine Research Helsinki, Finland A. STANCZYKOWSKA Siedice, Poland F. STARMUHLNER Zoologisches Institut der Universitat Wien, Austria Y. |. STAROBOGATOV Zoological Institute St. Petersburg, Russia J. STUARDO Universidad de Chile Valparaiso C. THIRIOT University P. et M. Curie Villefranche-sur-Mer, France thiriot @ obs-vifr.fr S. TILLIER Museum National d'Histoire Naturelle Paris, France J.A.M. VAN DEN BIGGELAAR University of Utrecht The Netherlands N. Н. VERDONK Rijksuniversiteit Utrecht, Netherlands H. WÄGELE Ruhr-Universität Bochum Germany Heike. Waegele @ruhr-uni-bochum.de ANDERS WAREN Swedish Museum of Natural History Stockholm, Sweden B. R. WILSON Dept. Conservation and Land Management Kallaroo, Western Australia H. ZEISSLER Leipzig, Germany A. ZILCH Forschungsinstitut Senckenberg Frankfurt am Main, Germany MALACOLOGIA, 2002, 44(1): 1-15 SMALL-SCALE MUSSEL SETTLEMENT PATTERNS WITHIN MORPHOLOGICALLY DISTINCT SUBSTRATA AT NINETY MILE BEACH, NORTHERN NEW ZEALAND Andrea С. Alfaro! & Andrew G. Jeffs? ABSTRACT Microscale settlement patterns of juveniles of the mussel Perna canaliculus were investigated within drift material at Ninety Mile Beach, northern New Zealand. Size- and site-specific selec- tivity on various morphologically distinct algal and hydroid species were identified within drift ma- terial and corroborated in laboratory experiments with similar artificial substrata. Mussel spat densities were greater within fine-branching natural (-28-57%) and artificial materials (-13-20%) compared to medium- and coarse-branching natural (-7-8%) and artificial (-2-3%) materials. Size-frequency distributions of mussel spat within natural and artificial ma- terials suggested a relationship of increasing mussel size with decreased branching of substrata. Field and laboratory investigations indicated higher settlement of 1.5-2.0 mm mussel size classes in coarse-branching substrata, whereas fine-branching substrata had greater settlement of mussels within the <0.5 mm size class. Mussel settlement comparisons within node and inter- node areas of all substrata in the field and in the laboratory indicated a strong preference of set- tlement in node areas over inter-node areas. The microscale settlement patterns observed in this study are argued to be indicative of a life strategy to maximize juvenile mussel survival within the dynamic environment of drift material in oceanic currents, before the potential arrival and re-set- tlement to rocky coastal areas. The present study is the first to elucidate settlement patterns of Perna canaliculus on drift material that washes up on Ninety Mile Beach, where >70 tonnes/year of this material is collected and supplied to the New Zealand aquaculture industry to seed mus- sel farms. Key words: mussels, small-scale settlement patterns, micro-habitat selection, size-frequency distribution, drift algae. INTRODUCTION The complexity and diversity of planktonic larval settlement patterns has received a great deal of attention within various temporal and spatial scales (Butman & Grassle, 1992; Bourget & Harvey, 1998). Physical and bio- logical factors have been investigated as po- tential contributors to passive and active set- tlement outcomes (Butman, 1987; Butman & Grassle, 1992; Grassle et al., 1992). For ma- rine invertebrates, such as mussels, larval settlement on filamentous substrata repre- sents an important intermediate step to the eventual recruitment to the adult population (Bayne, 1964; Davies, 1974; Highsmith, 1985; King et al., 1990; Harvey et al., 1993; Hunt & Scheibling, 1996). However, the dy- namics of these interactions are not well un- derstood for most broadcast spawners (Dame, 1996). Bayne (1964) first demonstrated that larvae of the mussel Mytilus edulis tended to settle on filamentous algae (primary settlement) and later moved to established adult mussel beds as secondary settlers (1-2 mm) through a bysso-pelagic phase. Bysso-pelagic drifting has since been shown to be a common means of dispersal among pelagic larvae and juveniles (Lane et al., 1985; Martel, 1993; Martel et al., 1994; Buchanan & Babcock, 1997). Buchanan & Babcock (1997) also found a size-specific settlement pattern for the New Zealand green-lipped mussel Perna canaliculus on various intertidal algae, hy- droids, and adult mussel beds at Piha Beach, North Island, New Zealand. In their study, Buchanan & Babcock (1997) observed that mussels <0.5 mm (primary settlers) were more abundant in attached filamentous algae and a hydroid species, whereas secondary settlers (>0.5 mm) were numerous in coarser- "Corresponding author. School of Environmental and Marine Sciences, The University of Auckland, Auckland, New Zealand; a.alfaro@auckland.ac.nz “National Institute of Water 8 Atmospheric Research Ltd., P. O. Box 109695, Newmarket, Auckland, New Zealand; a.jeffs O niwa.cri.nz 2 ALFARO & JEFFS branching algae and the adjacent intertidal mussel bed. Elsewhere in northern New Zealand, at Ninety Mile Beach (NMB), early juveniles or spat of Perna canaliculus also are found associated with floating algae and hy- droids. Large quantities (>70 t/y) of spat, at- tached to this drift material, are used to seed the extensive mussel farms in New Zealand, after collection from the surf zone (Jeffs et al., 2000). Nowhere else in New Zealand but along the 90 km stretch of NMB, are such vast amounts of wild spat concentrated in one place on detached material that can be con- veniently and economically harvested to sup- ply the mussel industry (Jeffs et al., 2000). Distinctive patches of filamentous substrata that arrive at the beach (wash-up events) may vary in substratum abundance and density, and spat size-frequency distribution (Hick- man, 1982). A single wash-up at NMB may contain up to 70 tonnes of substratum and spat material (C. Hensley, personal communi- cation). These filamentous substrata may de- rive from intertidal to deep subtidal regions in particular areas along NMB, and mussel set- tlement onto these substrata may take place before or after detachment of the alga or hy- droid. Regardless of the timing of settlement, algal and hydroid materials appear to be es- sential to the transport of mussel spat, as they may not only provide a means for long- distance dispersal, but also an alternative habitat to maintain or increase physiological processes (Highsmith, 1985; Schneider & Mann, 1991a). If a size-specific mussel settle- ment pattern occurs on the filamentous sub- strata found within a wash-up event at NMB, it is likely that substratum selection is the pre- dominant force behind mussel settlement pat- terns, rather than random physical encoun- ters along this particularly high energy portion of the New Zealand coastline. The persis- tence of size-specific settlement patterns of mussels on drift material, while exposed to the energetic hydrodynamic regimes often en- countered in the ocean, suggests a complex life history strategy. The aim of the present work is to elucidate microscale (cm) settle- ment patterns on the substrata found in wash- up events that occur along the 90 km length of NMB. Thus, characterization of substratum types, and size- and site- specific settlement patterns upon various drift substrata, may provide a first step to understanding spat-sub- stratum interactions at various spatial and temporal scales. METHODS AND MATERIALS Study Site and Terminology Samples of Perna canaliculus spat and as- sociated substrata were collected from the surf zone at various locations along Ninety Mile Beach (NMB), Northland, New Zealand (Fig. 1). Each sample was collected from dis- tinct wash-up events on 17 October 1998, 22 December 1998, and 16 May 1999. Wash-up samples were collected by net from the surf along the beach. Once collected, all samples were immediately frozen for later laboratory analyses at the University of Auckland, Auck- land, New Zealand. Recruitment is an operational word that does reflect a unique and specific biological event (Hunt & Scheibling, 1997). There is no standard size nor age at which mussels are defined to be settling or recruiting. Bayne (1964) suggests that primary settlers are mussels <0.5 mm in shell length, and sec- ondary settlers are mussels between 0.5-2.0 mm in shell length. Because mussels can, and often do re-settle even at shell lengths of up to 25 mm, it is difficult to suggest a cut-off line between settlement and recruitment. The mussels that were found attached to algae and hydroids in our experiments are likely to have used the drifting substrata as an inter- mediate settlement step before recruiting to the adult mussel beds in suitable rocky sub- strata. We use the term settlement in this study, because the majority of the mussels we encountered in the drift samples were <5 mm in shell length. Natural Substrata As afirst step to investigate small-scale set- tlement patterns of mussel spat on various drifting natural substrata, two 100-g wet weight subsamples were taken from each of three wash-up samples. The original wash-up samples were between 1-5 t (wet weight) of very homogeneous substratum and mussel wash-up material. All distinctive algal and hy- droid substrata within each subsample were separated and grouped into four morphologi- cally distinctive categories, with algal cate- gories based on the level or degree of branch- ing. The categories were: coarse-branching algae (Osmundaria colensoi, Carpophyllum angustifolium, and Rhodymenia dichotama); medium-branching algae (Melanthalia ab- SMALL-SCALE MUSSEL SETTLEMENT 3 Cape Reinga NEW ZEALAND Great Exhibition Bay Cape Karikari „Kaitaia Ahipara Tonatona Beach 173° 00 E FIG. 1. Location of study site at Ninety Mile Beach, Northland, New Zealand. Rocky intertidal areas are found at Tonatona Beach, The Bluff, and Scott Point. Spatfall collection sites on 17 October 1998, 22 December 1998, and 16 May 1999 are marked (x) along the beach. scissa, Laurencia thyrsifera, Pterocladia lu- cida, P. capillacea, Gigartina marginifera, G. alveata, and Pachymenia lusoria); fine- branching algae (Champia laingii, Plocamium costatum, Haliptilon roseum, and Corallina of- ficinalis); and hydroids (Amphisbetia bispi- nosa, Dictyocladium moniliferum, Craterit- heca insignis, Aglaophenia acanthocarpa, and Lytocarpia incisa). The distribution of these algae and hydroids varies from below the low water mark of spring tides to deep subtidal, with the exception of Gigartina sp. and Pachymenia lusoria, which also can be found in the low intertidal zone (Morton & Miller, 1968; Adams, 1994). All algal species found in the samples were red algae, except 4 ALFARO & JEFFS Carpophyllum angustifolium, which is a brown alga (Adams, 1994) and constituted <5% of each sample. The remaining material (shells, wood fragments, and other debris) did not contain attached mussels and was discarded. Mussels within each algal and hydroid sub- stratum type were removed by vigorous agita- tion in water, and the removal of remaining mussels was achieved with forceps (Buchanan & Babcock, 1997). Mussels within five different size-classes (<0.49, 0.5-0.99, 1.0-1.49, 1.5-1.99, >2.0 mm in length) were separated with a set of sieves of appropriate sizes. Previous research had verified this technique as an effective means of reliably sorting mussel material (A. Alfaro, unpub- lished data). The mussels were then dried to constant weight in an oven at 80°C. The num- ber of mussels within each size class was ini- tially determined by counting under the micro- scope. The number of dried mussels of each size class per unit volume was calculated, and volume subsequently was used to esti- mate numbers of mussels. Several test sam- ples were processed initially to ensure the ac- curacy of the methodology. The surface area of flattened algal and hydroid samples was determined from video-computer images using NIH Image 1.61 software for the Macin- tosh (Harvey et al., 1993). All algae and hy- droids were then dried in an oven at 80°C and weighed. Percent data sets of natural sub- stratum abundance and mussel densities (A) were arcsine transformed to normalize the data. An additional 20 samples of ~25 g wet weight were taken from each wash-up sample to determine the number of mussels attached to nodes and internodes of each of the four types of substrata (Fig. 2a). Anode area was defined as a 1 cm? area including at least one axil, and an inter-node area as a 1 cm? area without an axil (Fig. 2b). Node and internode areas were randomly chosen from a large set of suitable areas. All mussels within node and internode areas on each substratum sample were removed with forceps and counted. The exact area occupied by each substratum within the 1 cm? region was determined from image analyses using NIH Image 1.61 soft- ware. The total number of mussels per unit area of node and internode was calculated for each sample of algal and hydroid substrata. Artificial Substrata In order to further examine the settlement dynamics of mussel spat onto morphologically distinct substrata, settling patterns onto artifi- cial substrata of different shapes were moni- tored in the laboratory. Five to six 10-cm-long plastic aquarium plants of six different types were placed in separate seawater tanks of 1.5 liters. The artificial substrata were standard aquatic plant replicas and, for consistency, also were classified as coarse-branching < Internode (B) FIG. 2. (A) Diagram depicting node and internode areas on a filamentous substratum. (B) Close-up of node and inter-node area (1 cm’). SMALL-SCALE MUSSEL SETTLEMENT 5 (Myriophylium verticillatum, Ceratophyllum demersum, and Cabomba aquatica), medium- branching (Elodea densa, Elodea sp., and Ro- tala indica), and fine-branching (Vallisneria americana, V. spiralis, and Ceratopteris thalic- troides) substrata to facilitate comparisons with the natural substratum experiments. Al- though the aquatic plants were not identical to the algae found in the wash-up samples, the surface area and degree of branching were very similar to the natural substrata, and the degrees of branching were distinctive among the three experimental groups. Mussel spat were collected from intertidal habitats at NMB, detached from their original algal substrata, and placed in a water tank with seawater on the same day. The density of mussels of dif- ferent sizes was adjusted to be similar among size classes. From this mixture, subsamples of about 500 mussels, representing all sizes within 0.5 to 3.0 mm in length, were separated using a standard plankton splitter. The mussel subsamples were then placed in each of the 50 tanks containing artificial substrata. Each tank was aerated to ensure constant resus- pension of unattached mussels. The water temperature was maintained similar to the col- lection site at 15°C, and no food was added to the tanks during these short-term experi- ments. The light regime was set to mimic out- door conditions. After two days, mussel abun- dance, size-frequency distribution, site of 90 80 70 60 50 - 40 30 Available Substrate Area (%) 20 Coarse-branching algae algae Medium-branching mussel re-settlement, and substratum area were determined with the procedures outlined for natural substrata. Percent mussel spat densities were arcsine transformed to normal- ize the data. RESULTS Natural Substrata Substratum characterization in wash-up samples revealed overall differences in the total abundance of the four different settle- ment substrata, possibly due to seasonal changes in algal and hydroid productivity. The total amount of algal and hydroid material (+ SE) from the original 100 g subsample for the three wash-up samples was 96 + 2.3% in Oc- tober 1998, 31 + 5.2% in December 1998, and 64 + 3.5% in May 1999. However, the proportional algal and hydroid substratum comparisons among replicates and wash-up samples indicated that coarse-branching algae were more abundant than any other substratum for all three wash-up events (Fig. 3). The mean percent area (+ SE) of coarse- branching algae was 69.1 + 2.7%, followed by medium-branching algae (14.1 + 1.4%), hydroids (12.1 + 1.4%), and fine-branching algae (4.6 + 1.2%). Atwo-way ANOVA of per- cent substratum among wash-up events (arc- sine transformed data) showed no statistical 17-Oct-98 0 22-Dec-98 16-May-99 Fine-branching algae Hydroids Substratum Type FIG. 3. Percent area of four natural substratum types among three spatfall events. Non-significant Tukey tests for substratum differences are shown (*). 6 ALFARO & JEFFS significance among wash-up events (ANOVA; F5 42) = 0.35, р > 0.05), but a significant dif- ference among substratum types (ANOVA; F 5 42) = 217.33, р < 0.05). The interaction be- tween wash-up and substratum type was not statistically significant (ANOVA; Fi, 12) = 1.91, р > 0.05). Tukey’s pairwise comparisons of means failed to find significant differences be- tween medium-branching algae and hydroids (Tukey test; p = 0.799). The total number of mussels differed greatly among samples of different wash-up events. The first sample, collected on 17 Oc- tober 1998, yielded mussel numbers ranging from 12 to 3,866 mussels/cm? of substratum, and a mean (+ SE) of 658.7 + 230.7 mus- sels/cm? of substratum. The second wash-up sample of 22 December 1998 contained very few mussels, ranging from 0 to 12 mussels/cm* of substratum and a mean (+ SE) of 2.6 + 0.7 mussels/cm? of substratum. Finally, the third sample of 16 May 1999 hada range of 9 to 1,632 mussels/cm“ of substra- tum and a mean (+ SE) of 255.2 + 123.5 mussels/cm? of substratum. While the ab- solute number of mussels among the three wash-up samples was very different, the per- cent of mussels within each substratum type was similar for all wash-up samples (Fig. 4). The mean percent of mussels between repli- 70 - Ш 19-Oct-98 60 0 22-Dec-98 = 16-May-99 E о 50 = & > 40 == 2) о 30 Q = * ® ® 20 5 = o Coarse-branching Algae Algae MA Er Medium-branching cates and among wash-up events indicates that hydroid substrata consistently accumu- lated the greatest number of mussels com- pared to all other substrata (Fig. 4). The mean percent of mussels/cm? (+ SE) among sub- strata was 57 + 4, 28 + 2,8 + 2% and 7 + 2% mussels/cm? for hydroids, fine-branching, medium-branching, and coarse-branching algae, respectively. Results from a two-way ANOVA on the arcsine transformed data, with wash-up event and substratum type as fac- tors, indicated that there was no statistically significant ВЕ among wash-up events tion (ANOVA; Fe = 4.41, р > 0:05), al- though there was a significant difference among substratum types (ANOVA; E, ¿> = 419.97, p < 0.05). Tukey’s test comparisons failed to find significant differences between coarse- and medium-branching substrata (Tukey test; p = 0.714). Results from the size-frequency distribution of mussels within each substratum type re- vealed a relationship between increasing mussel size and decreased degree of branch- ing of substratum. Coarse-branching algae had a greater percent of large mussels (1.5-1.99 mm in length), whereas fine- branching algae and hydroids had the great- est percent of small mussels (<0.5 mm in Fine-branching Algae Hydroids Substratum Type FIG. 4. Mussel densities within four natural substrate types and three events samples. Non-significant Tukey tests between substrates are shown (*). SMALL-SCALE MUSSEL SETTLEMENT 100 Coarse-branching Algae Medium-branching Algae 60 > o № o o an o (>) Fine-branching Algae Mussel Density (% / cm?) 3 <0.49 0.5-0.99 Hydroids <0.49 0.5-0.99 1.0-1.49 1.0-1.49 № 17-Oct-98 О22-Оес-98 2 16-May-99 Ш 17-Oct-98 D22-Dec-98 216-Мау-99 1.5-1.99 >2.0 @ 17-Oct-98 О22-Оес-98 16-May-99 1.5-1.99 >2.0 17-Oct-98 O22-Dec-98 E116-May-99 1.5-1.99 >2.0 Mussel Size Class (mm) FIG. 5. Size-frequency distribution of mussels within five size classes and three spatfall events for four nat- ural substratum types. Pairwise comparisons (Tukey test) were performed for substrata that showed non-sig- nificant interactions (coarse-branching algae, medium-branching algae, and hydroids) in an overall ANOVA test and are shown (*). length) (Fig. 5, Table 1). Two-way ANOVA's, with mussel size class and wash-up event as treatment, were performed for each substra- tum (Table 1). Results from mussel density comparisons between node and inter-node areas within each of four natural substrata indicate that mussels were more abundant in node areas in all substratum types (Fig. 6). Furthermore, the relationship between increasing mussel abundance and decreased degree of substra- tum branching was generally apparent in ALFARO & JEFFS TABLE 1. Mean (+ SE) percent mussel density within each of five size classes for four natural substratum types. Two-way ANOVAs are shown for each substratum type. Data were arcsine transformed for statistical analyses and back transformed to obtain the means. Mussel Size ANOVA Class Mean + SE Substratum Type (mm) %/cm? Source df Е pa=0.05 Coarse-branching <0.49 5.62 + 3.50 Sample 2 0.20 0.819 ns Algae 0.5-0.99 6.74+ 3.10 Size 4 17.63 0.000 1.0-1.49 23.31 + 6.30 Sample x Size 8 2.33 0:075 15 1.5-1.99 52.63 3:34 Error 15 >2.0 2.75 = 3197 Medium-branching <0.49 15.15 + 4.94 Sample 2 0.10 0.906 ns Algae 0.5-0.99 14.77 + 3.64 Size 4 10.44 0.000 1.0-1.49 27.35 + 2.37 Sample x Size 8 3.52 0.017 1.5-1.99 31.85 + 4.23 Error 15 >2.0 2.57 = 4:35 Fine-branching <0.49 50.48 + 1.26 Sample 2 1.08 0.365 ns Algae 0.5-0.99 30.57 + 1.46 Size 4 167.10 0.000 1.0-1.49 15.57 + 1.10 Sample x Size 8 2.23 0.086 ns 1.5-1.99 1576 #228 Error 15 >2.0 0.17 = 1.76 Hydroids <0.49 42.81 + 0.96 Sample 2 0.11 0.894 ns 0.5-0.99 29.27 + 1.15 Size 4 173.34 0.000 1.0-1.49 17.60 + 0.71 Sample x Size 8 1.82 0.150 ns 1.5-1.99 9:50) 2.22 ¡EMO! 15 >2.0 0.05 + 0.48 1200 e = £ Ш Nodes 2 1000 Internodes 9 © = Z 800 > 5 £ + 600 г 17 = 400 © Q © =. 200 5 = 0 Coarse-branching Algae FIG. 6. Medium-branching Algae Fine-branching Algae Hydroids Substratum Type these samples as well (Fig. 6). However, no difference between coarse- and medium- branching algae was observed (Fig. 6). The mean number of mussels within node areas were 4.7 + 0.9, 52.4 + 9.5, 557.1 + 33.9, and 1,055.0 + 41.5 individuals/cm?, and within in- Mussel densities within node and internode areas within four natural substratum types (n = 20). ternode areas were (0.4 + 0.3, 23.7 + 6.0, 61.8 + 13.9, and 581.2 + 145.6 individuals/ cm?) for coarse-, medium-, and fine-branch- ing, and hydroid substrata, respectively. Al- though size classes were not differentiated in this part of the study, the fact that the pattern SMALL-SCALE MUSSEL SETTLEMENT 9 is similar for all substratum types (which con- tained different predominate mussel size classes) suggests that mussels of all size classes were more abundant within node areas. In samples with low total mussel abun- dances, clumps of mussels within node areas were often clearly visible. As the total density of the sample increased, the clumps of mus- sels extended from internodes along the branches, and in some cases, the entire algal or hydroid substratum was covered with mus- sels. A two-way ANOVA, with site of attach- ment (node versus internode) and substratum type, showed statistical Signlucangs for site of attachment (ANOVA; Fi, 475, = 248.74, р < 0.05) and substratum type (ANOVA; F 3 475) = 562.03, p < 0.05). A statistically significant In- teraction (ANOVA; Fu 72) = 72.54, p< 0.05) reflected the inability. to differentiate settle- ment patterns between coarse- and medium- branching algae. Artificial Substrata While the absolute number of mussels that settled on the artificial substrata was much lower than the numbers recorded from the natural material found in wash-up samples, the general trend among morphologically dis- tinct groups was consistent with the natural substrata (Fig. 7). Mussel settlement within 100 - 90 80 70 - 60 - 50 40 30 Mussel Density (% / cm?) 20 Coarse-branching substrate Medium-branching substrate coarse-branching artificial substrata ranged from O to 2 mussels/cm? of substratum and had a mean of 0.21 + 0.05 mussels/cm? of substratum. Medium-branching material had a settlement range from 0 to 6 mussels/cm* and a mean of 0.38 + 0.09 mussels/cm? of substratum. Finally, fine-branching material had a range of 0 to 25 mussels/cm? of sub- stratum and had a mean settlement of 3.23 + 0.45 mussels/cm? of substratum. Three one- way ANOVAs, with artificial plant type as treat- ment, were run on the arcsine transformed data to test for settlement differences among plastic aquarium plants within each of the three experimental branching groups. The re- sults of these tests showed no significant dif- ferences among any of the groups (ANOVA; F5 = 0.02, р > 0.05, ANOVA; F (2 15) = 0.07, p > 0.05, and ANOVA; Гель) = 0. 17, P= 0105 respectively for coarse-, medium-, and fine- branching substrata) (Fig. 7). However, a one- way ANOVA comparing mussel settlement on artificial substrata with different branching de- grees found significant settlement differences among substrata (ANOVA; F4 51, = 80.17, р < 0.05) (Fig. 7). A one-way ANOVA comparing all coarse- and medium-branching substrata, regardless of plant species, resulted in non- significant substratum effects (ANOVA; Р 34) — 1106, р> 0:05); Frequency distribution results from the arti- Fine-branching substrate Substratum Type FIG. 7. Mussel densities within three experimental group containing artificial substrata. A significant ANOVA test between coarse- and medium-branching algal substrata only are shown (*) (n = 6). See text for further explanations. 10 ALFARO & JEFFS Coarse-branching Substrata <0.49 0.5-0.99 1.0-1.49 1.5-1.99 >2.0 Medium-branching Substrata Mussel Density (% / cm’) RR <0.49 0.5-0.99 1.0-1.49 1.5-1.99 >2.0 Fine-branching Substrata Mussel Size Class (mm) FIG. 8. Size-frequency distribution of mussels within five size classes and three experimental groups of arti- ficial substrata. Pairwise comparisons (Tukey test) were performed for substrates that showed non-signifi- cant interactions (coarse- and medium-branching substrata) in an overall ANOVA test and are shown (*). ficial substratum experiments also indicated size class, whereas coarse- and medium- greater settlement on fine-branching material branching substrata had a similarly high per- than coarse- and medium-branching material centage of > 1.5 mm mussels, and very little (Fig. 8). Generally, fine-branching substrata to no settlement of < 1.5 sized mussels (Fig. had a higher percentage of < 0.99 mm mussel 8). Results from statistical analyses, including SMALL-SCALE MUSSEL SETTLEMENT ial two-way ANOVAs for each experimental group (degree of branching and mussel size as treatment), are shown in Table 2. Settlement comparisons between nodes and internodes areas within artificial substrata also resulted in greater mussel densities in node areas than in internode areas (Fig. 9). Again, no settlement differences between coarse- and medium-branching substrata were observed, but fine-branching substrata did contain greater settlement than the other two substrata (Fig. 9). Mussels often were ob- served extruding their foot to test surrounding substrata. In some cases, mussels were ob- served moving from the tip of an artificial plant to the nearest node. This migratory behavior sometimes resulted in clumping of mussels. A two-way ANOVA, with site of attachment (node versus internode) and substratum type, showed significant differences between node and internode attachment sites (ANOVA; Fo 402) = 13.40, p < 0.05) among artificial sub- TABLE 2. Mean (+ SE) percent mussel density within each of five size classes for three artificial substratum types. Two-way ANOVAs are shown for each substratum type. Data were arcsine transformed for statistical analyses and back transformed to obtain the means. Source df E Mussel Size Class Mean + SE Substratum Type (mm) %/cm? Coarse-branching <0.49 0.00 + 0.00 Substrata 0.5-0.99 0.00 + 0.00 1.0-1.49 0.00 + 0.00 1.5-1.99 0.29 + 0.06 >2.0 3.66 + 0.09 Medium-branching <0.49 0.01 + 0.01 Substrata 0.5-0.99 0.01 + 0.01 1.0-1.49 0.05 + 0.03 1.5-1.99 0.46 + 0.07 >2.0 5231013 Fine-branching <0.49 47.26 + 0.30 Substrata 0.5-0.99 18.79 + 0.54 1.0-1.49 2165 0:15 1.5-1.99 2.15 028 >2.0 1.44 + 0.09 Ш Nodes O Internodes Mussel Density ( # individuals / cm?) Coarse-branching substratum Medium-branching substratum ANOVA pa=0.05 Substrate 2 0.00 0.998 ns Size 4 20.61 0.000 Sample x Size 8 0.12 0.998 ns Error 75 Substrate 2 0.24 0.786 ns Size 4 15.79 0.000 Sample x Size 8 O83) 01953" ins Error 75 Substrate 2 3.14 0.049 Size 4 41.75 0.000 Sample x Size 8 5.34 0.000 Error 715 Fine-branching substratum Mussel Size Class (mm) FIG. 9. Mussel densities within node and inter-node areas within three artificial substratum types (n = 6). 12 ALFARO & JEFFS stratum types (ANOVA; F, 192 = 41.60, р < 0.05), and interaction (ANOVA: E (2,102) — 11.00, p < 0.05). DISCUSSION One of the great interests in the field of in- vertebrate ecology is the question of how ini- tial settlement patterns affect the distribution of adult broadcast spawners, such as mus- sels. While oceanic-current dispersal of lar- vae and juveniles is a passive means of trans- port, some degree of habitat choice is exercised by individuals that encounter and attach to drift material. This initial settlement process on various morphologically distinct drift materials may be of significance to the survival and successful resettlement of juve- niles to the adult population. Thus, elucidation of microscale settlement patterns of mussel spat on natural drift substrata may enhance our understanding of the process of spat transport and arrival to coastal areas. Substratum characterization within three wash-up events at NMB indicated that coarse-branching algal substrata consistently comprised the majority of settlement sub- strata found associated with wash-up events. Medium-branching algae, fine-branching algae, and hydroids were less abundant. Drift algal material may originate from rocky inter- tidal and subtidal areas that may have been detached from their natural substrata by storms or strong ocean currents. These mate- rials may become aggregated by local oceanographic conditions. However, the ma- jority of the material (mostly red algae) is sub- tidal in origin (Osmundaria colensoi, Car- pophyllum angustifolium, and Rhodymenia dichotoma; Adams, 1994; Steneck & Dethier, 1994), indicating the importance of subtidal sources. The large amount of subtidal mater- ialin NMB wash-up samples also may be a re- sult of the scarcity of rocky intertidal areas that could provide intertidal algal sources along the beach (Fig. 1). Furthermore, none of the intertidal areas at or near NMB contain the same species of algae found washed-up at NMB in enough abundance to indicate a likely source (A. Alfaro, personal observation). The often fresh and intact nature of the mate- rial that arrives at the beach suggests that the subtidal material comes from rocks situated just offshore (<35 m water depth) of three rocky outcrops along the beach (Tauroa Point, The Bluff, and Scott Point). Indeed, algal beds have been observed off Tauroa Point (S. Hooker, unpublished), and extensive rocky substrata, likely to harbor algal material, have been noted by local fishers off Scott Point. Furthermore, spawning peaks of intertidal and subtidal mussels (< 35 m water depth), as well as algal seasonal cycles, may strongly influ- ence the abundance, dispersal and coloniza- tion potential of spat within temporal and spa- tial scales (Alfaro et al., in review). However, more samples collected throughout the year are necessary to clarify these patterns. Quantification of mussel spat among algal and hydroid substrata indicated that a greater number of spat settle on hydroids, followed by fine-branching algae; whereas fewer spat set- tle on medium- and coarse-branching algae. Laboratory settlement experiments with artifi- cial substrata also resulted in greater settle- ment on fine-branching material over coarse- and medium-branching substrata. Buchanan & Babcock (1997) found a similar size- frequency distribution of mussel spat on inter- tidal algae at Piha, North Island, New Zealand. In their study, the authors found that primary settlement of mussels (< 0.4999 mm in length) was mostly on the hydroid Amphis- Бена bispinosa, and fine- and medium- branching algae, whereas larger mussels in the dispersal (0.5-5.4999 mm in length) and stable (> 5.0 mm in length) stages of their life history tended to resettle onto coarser- branching algae and the adult mussel bed. By contrast, the present study included consider- ation of a wider range of free-drifting algal- branching types (coarser red and brown algae) that are likely of mostly subtidal origin. Nonetheless, the size-frequency distribution of mussel spat on natural (in the field at NMB) and artificial (in the lab) substrata strongly corroborates observations at Piha by Bu- chanan & Babcock (1997). The field experi- ments at NMB indicated that the percent of mussel spat among the four algal and hydroid substrata was similar for all three wash-up samples and may indicate that settlement pat- terns are not affected by absolute variations in substratum abundance. Furthermore, mussel spat will settle predominantly on less abun- dant natural material even when existing spat densities may be quite high (~3,800 individu- als/cm? on hydroid material). This strong se- lectivity has been observed elsewhere (But- man, 1987; Schneider & Mann, 1991 a, b; Butman & Grassle, 1992; Grassle et al., 1992; Harvey et al., 1993; Buchanan & Babcock, 1997). Schneider & Mann (1991a) found that SMALL-SCALE MUSSEL SETTLEMENT 13 there was a strong selectivity of epifaunal in- vertebrates to macroalgae with varying de- grees of branching. These authors concluded that the relationship between invertebrate species and algal morphology benefited the associated invertebrates in terms of food source and living space provisions. It is un- clear as to which different ecological proper- ties may be provided by different substrata (such as coarse-branching algae versus hy- droids) to the associated mussel spat, but it is likely that the relationship is based primarily on attaining a structurally secure place to in- habit. Indeed, results from the artificial sub- stratum experiments suggested that mussels will preferentially settle onto filamentous sub- strata on the basis of their physical shape. While these experiments with artificial sub- strata do not rule out the possibility that chem- ical cues exuded by natural algae affect mus- sel settlement patterns, they do support the idea that substratum morphology alone strongly influences settlement of different- sized mussels. The observed movement of mussels from the tips of artificial aquarium plants to node areas suggest that attraction cues among mussels may exist, as well as substratum selectivity. The size-frequency distribution of mussel spat on the various natural substrata within NMB wash-up samples (Fig. 5), and artificial substrata (Fig. 8), shows a general inverse re- lationship between spat size and degree of substratum branching. Thus, larger mussels (1.5-2.0 mm) appear to settle predominantly on coarse-branching substrata, whereas smaller mussels (< 0.5 mm) preferentially set- tle on fine-branching material (Figs. 5, 8). Buchanan & Babcock (1997) suggested that such size-specific settlement on morphologi- cally distinct substrata in the intertidal zone may be largely a result of recolonization of natural substrata. Our results suggest that the selection process also may take place in sub- tidal habitats and in the water column, possi- bly within accumulated patches of drift algae and hydroids. Furthermore, this size-specific selection persists even after the mussels have been transported large distances by dy- namic oceanographic conditions evident at NMB. The possibility exists that the observed relationship between spat size and substra- tum type is a result of differential growth rates that allow the less abundant spat on coarse- and medium-branching algae to attain a larger size due to lower crowding effect. How- ever, the fact that all substratum types and mussel spat size classes were consistently represented in all three ММВ wash-up sam- ples suggests that differential growth rates of spat within patches of drift material is unlikely. Furthermore, artificial substratum experi- ments resulted in similar trends after a period of only two days, which was not long enough to note size differences due to growth. A size-specific settlement pattern may rep- resent a choice for scaled physical stability, where, for example, larger mussels may re- quire larger morphologically stable substrata, such as the coarse- and medium-branching algae. However, smaller-scale selectivity also may contribute to stability requirements of mussel spat. Comparisons of settlement den- sities between node and inter-node areas within four different natural substrata (Fig. 6), and three artificial plant substrata (Fig. 9), in- dicate that node settlement is preferred by mussels settling in all types of substrata. Micro-scale selectivity has been demon- strated in laboratory flume experiments (Har- vey et al, 1993; Harvey & Bourget, 1995; Bourget & Harvey, 1998). Bourget & Harvey (1998) found that recruits of nine marine in- vertebrates, including six bivalve species, were more abundant in nodes as compared to internodes, and that the rate of recruitment in- dicated that passive deposition alone was not sufficient to obtain such an outcome. The au- thors ruled out the influence of differential ero- sion and mortality on higher settlement pat- terns for nodes over linear areas. Evidence has been accumulating that points to behav- ioral responses to small-scale (<1 cm) sub- stratum irregularities, such as pits, grooves, and depressions, as determinants in mi- croscale settlement preferences (Bourget et al., 1994; Nellis & Bourget, 1996; Bourget & Harvey, 1998). While chemical cues between spat and substratum, and among individual mussels, cannot be excluded as factors in the site-settlement preference experiments re- ported here, it is likely that morphology is the driving force to the strong habitat selectivity observed in this study. The large volume of wash-up material (up to 70,000 t/y; C. Hensley, personal communi- cation) that can be collected only along NMB suggests that settlement on drift material may be an important part of the life history strategy for Perna canaliculus. Indeed, floating algal clumps have been found to provide alterna- tive habitats and transport of invertebrates (Schneider & Mann, 1991a; Highsmith, 1985; Bologna & Heck, 1999). Micro-scale selectiv- 14 ALFARO & JEFFS ity of mussel spat of different sizes onto mor- phologically distinctive substratum types may reflect a delicate level of organization within the transitional environment of drift material. Transport by drift material may be crucial to the invasion and retention of mussel spat onto new intertidal and subtidal sites (Highsmith, 1985). Pulses of new mussel settlements have been observed at Scott Point, NMB after large accumulations of drift material on the rocky shore (C. Hensley, personal communi- cation). It is possible that entire rocky shore mussel communities may depend on the peri- odic arrival of spat from drift material. In the case of NMB, where rocky habitats constitute a very small portion of the coastal area, it ap- pears that most wash-up events may arrive on the sandy beach where mussels have little chance of survival, unless collected by spat collectors and transported to mussel farms. Additional research on substratum availability and the dynamics of oceanographic transport of drift material at NMB is critical to further un- derstanding of mussel dispersal, colonization, and maintenance of adult populations at vari- ous temporal and spatial scales. AKNOWLEDGMENTS We thank C. and R. Hensley, K. Campbell, and D. Tasker for their invaluable assistance with sample collections in the field. We are grateful for the technical support of A. Turner, |. MacDonald, V. Ward, and D. Todd. Algal identifications were done by W. Nelson, and S. O’Shea conducted the hydroid identifica- tions. The Geology Department at the Univer- sity of Auckland provided lab space and equipment for the size-frequency analyses. We thank K. Campbell and two anonymous reviewers for their comments on the manu- script. LITERATURE CITED ADAMS, M., 1994, Seaweeds of New Zealand. Christchurch, New Zealand: Canterbury Univer- sity Press. 360 pp. ALFARO, A. C. A. G. JEFFS & S. H. HOOKER, in review, Reproductive behavior of the green- lipped mussel, Perna canaliculus, in northern New Zealand. BAYNE, B. 1964, Primary and secondary settle- ment in Mytilus edulis L. Journal of Animal Ecol- ogy, 33: 513-523. BOLOGNA, P. & K. HECK, 1999, Macrofaunal as- sociations with seagrass epiphytes relative im- portance of trophic and structural characteristics. Journal of Experimental Marine Biology and Ecol- ogy, 242: 21-39. BOURGET, E., J. DeGUISE & G. DAIGLE, 1994, Scale of substratum heterogeneity, structural complexity, and the early establishment of a ma- rine epibenthic community. Journal of Experi- mental Marine Biology and Ecology, 181: 31-51. BOURGET, E. & M. HARVEY, 1998, Spatial analy- sis of recruitment of marine invertebrates on ar- borescent substrata. Biofouling, 12: 45-55. BUCHANAN, S. & R. BABCOCK, 1997, Primary and secondary settlement by the greenshell mus- sel Perna canaliculus. Journal of Shellfish Re- search, 16: 71-76. BUTMAN, C. 1987, Larval settlement of soft-sedi- ment invertebrates: the spatial scales of patterns explained by active habitat selection and the emerging role of hydrodynamical processes. Oceanography and Marine Annual Reviews, 25: 113-165. BUTMAN, C. & J. GRASSLE, 1992, Active habitat selection by Capotella sp. | larvae. |. Two-choice experiments in still water and flume flows. Jour- nal of Marine Research, 50: 669-715. DAME, R. F. 1996, Ecology of marine bivalves: an ecosystem approach. New York: CRC Press. 254 pp. DAVIES, G. 1974, Amethod for monitoring the spat- fall of mussels (Mytilus edulis L.). Journal du Conseil International pour l'Exploration de la Mer, 36: 27-34. GRASSLE, J., С. BUTMAN & $. MILLS, 1992, Ac- tive habitat selection by Capotella sp. | larvae. Il. Multiple-choice experiments in still water and flume flows. Journal of Marine Research, 50: 717-743. HARVEY, M. & E. BOURGET, 1995, Experimental evidence of passive accumulation of marine bi- valve larvae on filamentous epibenthic struc- tures. Limnology and Oceanography, 40: 94- 104. HARVEY, M., E. BOURGET & G. MIRON, 1993, Settlement of Iceland scallop spat (Chlamys is- landica) in response to hydroids and filamentous red algae: field observations and laboratory ex- periments. Marine Ecology Progress Series, 99: 283-292. HICKMAN, B. 1982, Watch kept on mussel seed. Catch, 9: 15. HIGHSMITH, R. 1985, Floating and algal rafting as potential dispersal mechanisms in brooding in- vertebrates. Marine Ecology Progress Series, 25: 169-179. HUNT, H. & R. SCHEIBLING, 1996, Physical and biological factors influencing mussel (Mytilus trossulus, M. edulis) settlement on a wave-ex- posed rocky shore. Marine Ecology Progress Se- ries, 142: 135-145. HUNT, H. & R. SCHEIBLING, 1997, Role of early post-settlement mortality in recruitment of benthic SMALL-SCALE MUSSEL SETTLEMENT 15 marine invertebrates. Marine Ecology Progress Series, 155: 269-301. JEFFS, A., R. HOLLAND, S. HOOKER & B. HAY- DEN, 2000, Overview and bibliography of re- search on the greenshell mussel, Perna canalicu- lus, from New Zealand waters. Journal of Shellfish Research, 18: 1-14. KING, P., D. MCGRATH & W. BRITTON, 1990, The use of artificial substrates in monitoring mussel (Mytilus edulis L.) settlement on an exposed rocky shore in the west of Ireland. Journal of the Marine Biological Association of the United King- dom, 70: 371-380. LANE, D., A. BEAUMONT & J. HUNTER, 1985, Byssus drifting and the drifting threads of the young post larval mussel Mytilus edulis. Marine Biology, 84: 301-308. MARTEL, A. 1993, Dispersal and recruitment of zebra mussel (Dreissena polymorpha) in a nearshore area in west-central Lake Erie: the sig- nificance of postmetamorphic drifting. Canadian Journal of Fisheries and Aquatic Sciences, 50: 3-12. MARTEL, A., A. MATHIEU, S. FINDLAY, S. NEP- SZY & J. LEACH, 1994, Daily settlement rates of the zebra mussel, Dreissena polymorpha, on an artificial substrate correlate with veliger abun- dance. Canadian Journal of Fisheries and Aquatic Sciences, 51: 856-861. MORTON, J. & M. MILLER, 1968, The New Zealand sea shore. Auckland: Collins. 638 pp. NELLIS, P. & E. BOURGET, 1996, Influence of physical and chemical factors on settlement and recruitment of the hydroid Tubularia larynx. Ma- rine Ecology Progress Series, 140: 123-139. SCHNEIDER, F. & K. MANN, 1991a, Species spe- cific relationship of invertebrates to vegetation in a seagrass bed. |. Correlational studies. Journal of Experimental Marine Biology and Ecology, 145: 101-117. SCHNEIDER, F. & K. MANN, 1991b, Species spe- cific relationship of invertebrates to vegetation in a seagrass bed. Il. Experiments on the impor- tance of macrophyte shape, epiphyte cover and predation. Journal of Experimental Marine Biol- ogy and Ecology, 145: 119-139. STENECK, R. & M. DETHIER, 1994, A functional group approach to the structure of algal-domi- nated communities. Oikos, 69: 476-498. Revised ms. accepted 29 March 2001 MALACOLOGIA, 2002, 44(1): 17-22 DETECTION OF PHENOLOXIDASE (PO) IN HEMOCYTES OF THE CLAM VENUS ANTIQUA Luis A. Mercado', Sergio H. Marshall & Gloria M. Arenas? Laboratorio de Genetica e Inmunologia Molecular, Instituto de Biologia, Facultad de Ciencias Bäsicas y Matemäticas, Universidad Catolica de Valparaiso, Chile ABSTRACT The prophenoloxidase (proPO) system, particularly studied in Arthropoda, is one of the im- portant mechanisms of non-self recognition in the immunological response of some inverte- brates. Under natural conditions, the activation of the system involves the stimulation of serine proteases by the presence of LPS and ß-1,3 glycans on the surface of microorganisms. It results in the triggering of proPO, which in turn generates peptides responsible of opsonizing and Iytic actions. We report here the preliminary analysis of this type of response in hemocytes of the bi- valve mollusc Venus antiqua, both in intact cells and in crude lysates. Our strategy was first to monitor PO activity in whole hemocytes. Second, to confirm its presence in hemocyte Iysate su- pernatant (HLS) by Dot and Western blot analysis using polyclonal antibodies against tyrosinase, the commercially available form of PO. Third, to measure its enzymatic activity in HLS by com- paring the oxidation of the substrate L-DOPA in the presence and absence of known activators or inhibitors. In this report, we demonstrate PO activity in granular cell subpopulations. In HLS, we quantitate the specific activity of the enzyme by spectromethomertric methods. Finally, im- munological characterization of HLS suggests the existence of two PO-like polypeptides, one of 45 kDa and a second one slightly higher. Key words: mollusc immunity, clam hemocyte, PO activity, immunodetection. INTRODUCTION The proPO system, best studied in arthro- pods, corresponds to a complex of he- molymph enzymes and associated protein factors that activate the phenoloxidase (PO) cascade generating small peptides, which act as mediators of the host immune response (Rattcliffe, 1991; Kwon et al., 1997; Perazzolo & Barracco, 1997). The system has been best characterized in crustaceans and other se- lected invertebrates (Johansson & Söderhäll, 1989; Iwanaga et al., 1992; Hoffmann & Reichhart, 1997). The proPO system has also been described in such molluscs as Bio- phalaria glabrata (De Aragao & Bacila, 1976), Mytilus edulis (Coles & Pipe, 1994; Ren- wrantz et al., 1996), and Perna viridis (Asokan et al., 1997). Notwithstanding, the marine bi- valve Perna perna did not show PO activity (Barracco et al., 1999). The proPO system is primarily activated by polysaccharides from the microorganisms surface, of which B-1,3-glucans from fungal cell wall and LPS-peptidoglycan complexes are considered to be the most classical acti- vators (Unestam & Söderhäll,) 1977; Char- alambidis et al., 1996). In arthropods, the proPO has been reported as a 76 kDa mole- cule (Aspan & Söderhäll, 1991), which is turned into an active form (PO) by ppA, a ser- ine protease of 36 kDa (Aspan et al., 1990). Other endogenous proteinases activate proPO in a way that could very well represent an invertebrate counterpart of the vertebrate complement system (Hernandez-Lopez et al. 1996). Once PO is formed, a number of meta- bolic pathways are activated oxidazing L- DOPA to melanin as the final common end- product (Nappi et al., 1995; Marmaras et al., 1996). A great variety of molecules have been described during these activated intermediate steps. Among them, aglutinines (lwanaga et al., 1998; Gollas-Galvan et al., 1999), adhe- sive proteins (Söderhäll et al., 1990), and quinones (Ratcliffe, 1991). "Present Address: Departamento de Bioquimica y Biologia Molecular, Facultad de Veterinaria, Universidad Santiago de Compostela, Lugo, Spain. Address correspondence to: Instituto de Biologia, Universidad Catölica de Valparaiso, Avenida Brasil 2950, Casilla 4059, Valparaiso. Chile; garenas @ucv.cl 18 MERCADO ET AL. Considering that the proPO system is well established in arthopods and insects and that there is some controversy in molluscs, we de- cided to verify if this relevant innate immune response could be assayed in another marine bivalve. In this report using the clam Venus antiqua as model system, we demonstrate its activity. Moreover, by introducing immunolog- ical detection as an innovative tool, we also demonstrate its presence and potential evolu- tionary conservation. MATERIALS AND METHODS Recovery of Hemolymph Twelve adult specimens of Venus antiqua were collected from natural grounds in the coast of the V Region in Chile. Hemolymph was drained from the posterior adductor mus- cle of each individual (2.0 ml) using sterile plastic seringes (Friebel & Renwrantz, 1995). Resulting hemolymph was pooled in a polypropylene tube and kept at 4°C. Recovery of Hemocytes The hemolymph was centrifuged in a clini- cal centrifuge at 300 x g for 5 min at 4°C. The plasma was stored at 4°C until processed and the cellular pellet carefully resuspended in ca- codylate buffer (10 mM sodium cacodylate buffer (NCB): 0.45 M NaCl, 20 mM CaCl,, 10 mM MgCl, pH 7.4) and intact hemocytes were washed twice and resuspended in the same buffer. Hemocyte Lysate Supernatant (HLS) Hemocytes in 800 ul of NCB buffer were fully homogenized in a glass-plungered ho- mogenator. The homogenate was centrifuged at 35.000 x g (Sorvall RC-5B centrifuge; HB-4 rotor) for 20 min at 4°C and the pellet dis- carded. The supernatant constitutes HLS, the source for PO evaluation. Cytochemistry Intact hemolymph diluted 2:1 in NCB buffer (pH 7.4) buffer were fixed with 5% formalde- hyde on a polylysine-treated (0.1%) slide. After 15 min, slides were exposed to L-DOPA (0.1% in NCB 150 mM NaCl) for 12 h at room temperature in the dark (Renwrantz et al., 1996). In control samples, L-DOPA was om- mitted. PO Activity in HLS Triplicates of 50 ul of HLS were incubated with inducers, either 50 ul of trypsin (1 mg/ml) or SDS (1 mg/ml), for 5 min at 20°C. Then, 50 ul of L-DOPA (3 mg/ml), with or without 10 mM CaCl,, a PO cofactor, were added and further incubated for 30 min. In paralel, samples con- taining either inducer were supplemented with 50 ul of 0.2 mM phenilthiourea (PTU) as a PO inhibitor to rule out endogenous peroxidase activity as well as to determine the real PO specific activity. Reactions were ended by di- lutions with deionized water to a final volume of 1.0 ml. PO activity was determined spec- trophotometrically by measuring oxidation of the L-DOPA into a DOPA-chrome detected at 490 nm. The specific activity of the enzyme (S.A.) is expressed as the ratio of the ab- sorbance value (Abs) per time per amount of protein. S.A. = Abs x min ! x mg prot! (Smith & Söderhäll, 1991; Perazzolo & Barracco, 1997). Final S.A. was expressed as average values + SD, according to the t-test for inde- pendent samples (p < 0.05) (Statistica 5.0). Immunological Tests Polyclonal monospecific anti-tyrosinase (commercial mushrom PO, Sigma) were elicited as previously described (Mercado & Arenas, 1997) and used in all immunological detections. Protein concentration was deter- mined by the method of Bradford (1976) using Bovine Serum Albumin (BSA) as a standard. Dot Blot Analysis 5 ug of HLS and plasma; 1 ug of tyrosinase (Sigma) and horseradish peroxidase (HRP) (Sigma) were spotted onto nitrocellulose membranes and incubated for 2 h at room temperature with anti-tyrosinase antibody (1:3,000) in TBST buffer (0.05% Tween 20 in TBS, pH 7.4), in the presence of 3% BSA and 0.2% sodium azide. Second antibody was rabbit anti mouse IgG-alkaline phosphatase (PA) (1:30,000). After 1 h incubation the mem- brane was developed with. BCN-NBT Fast (Sigma) (Dyson, 1991). DETECTION OF PHENOLOXIDASE IN HEMOCYTES OF THE CLAM 19 Western Blot Analysis 20 ug of HLS resolved into a 12% SDS- PAGE (Laemmli, 1970) was electrotransfered to an immobilon-P membrane (PVDF, Milli- pore) (Towbin et al., 1979) and processed as described above. Non-specific sites were blocked with 3.0% BSA in TBST. RESULTS The existence of the proPO activating sys- tem is demonstrated in hemocytes of the clam Venus antiqua. 58-60% of intact granular he- mocytes showed a positive PO reaction by the conversion of the initial substrate to L- DOPA into a dark end product (Fig. 1A), as opposed to the 100% absence in the negative controls (Fig. 1B). In HLS fractions, pigment formation was also indicative of PO activity, although a slight positive reaction is detected in plasma exposed to L-DOPA. Nontheless, there is no dark precipitate in the presence of the enzyme inhibitor PTU (Fig. 2). In enzymatic activity assays, proPO in HLS was activated by both inducers trypsin and SDS, with a slighter higher value for the latter in the presence of calcium (Fig. 3). Dot blot analysis demonstrate the speci- ficity of our antityrosinase antibody (Fig. 4). The polyclonal antibody showed no cross-re- activity with either HRP or the negative con- trol, while an intense reaction is seen in the HLS sample in contrast to a feeble one in plasma. Finally, Western blot shows in HLS, two im- munoreactive bands: a very intensive one with a mobility of approximately 45 kDa, anda less reactive slightly higher molecular weight band, which correlates with the stained profile of the corresponding gel (Figs. 5: lanes 3 and 2, respectively). A e? | 2 3 4 FIG. 2. Qualitative assay of PO activity in V. antigua hemolymph. (1) activated HLS, (2) activated HLS in the presence of 0.2 mM PTU, (3) non activated plasma, (4) 10 mM Na* cacodilate buffer. DISCUSSION The presence of the PO system among dif- ferent groups of invertebrates is under dis- cussion. In arthropods, it has been described exclusively in the hemocytes of some cray- fish, such as Penaeus californiensis and P. paulensis (Hernändez-Löpez et al., 1996; Perazzolo & Barracco, 1997), or solely in the plasma of insects Bombyx moriand Manduca sexta (Ashida et al., 1983; Saul et al., 1987). In bivalve molluscs, Renwrantz et al. (1996) described PO activity in hemocytes of Mytilus edulis, whereas in Perna viridis Asokan et al. (1997) found it in both plasma and hemo- cytes. Our study in clams supports the putative existence of PO activity in marine inverte- brates. We demonstrate that a high percent- age of bulk granular hemocytes display the activity (Fig. 1). Moreover, statistically signifi- cant spectrophotometric assays measuring the oxidation of L-DOPA to DOPA-chrome seem to be good indicators of PO activity in HLS from Venus antiqua (Fig. 3). The basal activity detected in plasma (Fig. 2), might be interpreted as a reflection of the physiological stage of the specimens analyzed. Finally, our novel immunological approach suggest the existence of two size-specific polypeptides FIG. 1. A, Hemocytes subpopulations from Venus antiqua with a positive PO oxidizing reaction. Monolayer incubated with L-DOPA as substrate, 1.000 x. B, Hemocytes incubated in the absence of substrate. 20 MERCADO ET AL. Control Trypsin — Ca” * SDS — Ca” * Trypsin ** SDS ** Trypsin - 0.2 mM PTU SDS - 0.2 mM PTU PO activity in HLS. Abs 490 nm (min! x mg prot! 0.064 + 0.016 0.133 + 0.033 0.277 + 0.104 0.018 + 0.012 0.037 + 0.009 0.006 + 0.002 0.007 + 0.001 The enzymatic activity values are expressed as average + SD. * The substrate L-DOPA was added in the presence of 10 mM Call, ** The substrate L DOPA was Ca” free added. FIG. 3. Differential effect of Trypsin, SDS, Ca?* and PTU over PO activity in HLS. Spectrophotometric quan- titation of PO specific activity. MEAN + | 2 3 À FIG. 4. PO immunodetection in Dot blot. (1) acti- vated HLS and (2) non activated plasma, (3) com- mercial tyrosinase, (4) HRP. Each dot contains 1 ug of protein. kDa LS 66—}| 2 4 PA 15 a + <— 45 ++ FIG. 5. SDS/PAGE characterization of HLS and Western blot analysis. (1) HLS, 50 ug (10% gel) Coomassie blue stained, (2) HLS, 20 ug (12% stained gel). (3) Western blot of a parallel gel shown in lane 2. Reactive bands indicated by slim arrows. Relative molecular weights in kDa indicated on the left of each lane. (50 and 45 kDa), although different from those of crayfish molecule (Aspan & Söderhäll, 1991), are highly conserved and presumably responsible for the detected PO activity in Venus antiqua. In Mytilus edulis, the native enzyme characterized in non denaturant gels also resulted in two proteins with relative mol- ecular weights of 381 + 13.7 and 316 + 11.1 kDa, respectively (Renwrantz et al., 1996). This might very well mean that the two Venus antiqua polypeptides could represent sub- units of a larger enzyme. In spite of the fact that the PO values re- ported for HLS of Venus antiqua are low com- pared to other species, we think they are real. PO activity fully responds to serine proteases, such as trypsin (Fig. 3), in agreement with other reports (Nellaiappan & Sugumaran, 1996). Additionally, the same figure summa- rizes the expected activating effect of SDS (Moore & Flurkey, 1990). Also, for both cases, the inducing effect of Са?* as a co-factor, as well as the fully inhibitory action of PTU (Sug- umaran et al., 1988). Further studies tending to optimize the measurement of PO activity in Venus antiqua or in other mollusc model sys- tem might contribute to asses the observa- tions reported here. The specific recognition of two polypeptides in hemocytes of Venus antiqua using antibod- ies elicited agaist a commercial enzyme of mushrom origin clearly suggests the exis- tence of common epitopes of evolutionary conserved enzymes, situation expected for in- nate immunological responses. SUMMARY AND CONCLUSIONS The PO activating system was studied in hemocytes of the bivalve mollusc Venus anti- qua. Qualitative assays were used to demon- strate the formation of dark precipitates in in- tact hemocytes and quantitative assays of enzyme activity measured by oxidation of L- DETECTION OF PHENOLOXIDASE IN HEMOCYTES OF THE CLAM 21 DOPA to DOPA-chrome formation. Immuno- logical detection was incorporated in order to characterize the putative polypeptides in- volved in the activation process in mollusc. We conclude that the PO system is present mostly in hemocytes of Venus antiqua. The putative polypeptides recognized by antibod- ies elicited to a mushrom enzyme, suggest a high degree of evolutionary conservation. Al- though they do not match the expected molecular weights described for crayfish, they might correspond to subunits of the native en- zyme described in Mytillus edulis. In sum- mary, our results tend to indicate a structured innate immune response active in a species of marine bivalve. ACKNOWLEDGMENTS The present work was financed by the Di- recciön de Investigaciön, Vice Rectoria de In- vestigaciön y Estudios Avanzados, Universi- dad Catolica de Valparaiso, Chile. LITERATURE CITED ASHIDA, M., Y. ISHIZAKI & H. IWAHANA, 1983, Activation of prophenoloxidase by bacterial cell walls or B-1,3-glucans in plasma of the silkworn, Bombyx mori. Biochemical and Biophysical Re- search Communications, 113: 562-568. ASOKAN, R., M. ARUMUGAM & P. 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RIVERA, 1988, Differential mechanism of oxidation of N-acetyldopamine and N-acetylnorepinephrine by cuticular phe- noloxidase from Sarcophaga bullata. Archives of Insect Biochemistry and Physiology, 8: 229-241. TOWBIN, H., Т. STAEHELIN & J. GORDON, 1979. Electrophoretic transfer of proteins from poly- acrylamide gels to nitrocellulose sheets: proce- dure and some applications. Proceedings of the National Academy of Sciences USA, 76: 4350- 4354. UNESTAM, T. & K. SÖDERHÄLL, 1977, Soluble fragments from fungal cell walls elicit defence re- actions in crayfish. Nature, 267: 45-46. Revised ms accepted 25 March 2001 MALACOLOGIA, 2002, 44(1): 23-31 A SURVEY OF GENETIC VARIATION AT ALLOZYME LOCI AMONG GONIOBASIS POPULATIONS INHABITING ATLANTIC DRAINAGES OF THE CAROLINAS Robert T. Dillon, Jr. & Andrew J. Reed Department of Biology, College of Charleston, Charleston, South Carolina 29424, U.S.A.; dillonr@cofc.edu ABSTRACT We estimated gene frequencies at eight polymorphic enzyme loci in 12 populations of Go- niobasis from Atlantic drainages of North Carolina, South Carolina, and Georgia. Our sample in- cluded four populations of G. proxima from the piedmont, five populations of G. catenaria cate- naria, one population of G. catenaria postellii, and two populations of G. catenaria dislocata from the coastal plain. The fit to Hardy-Weinberg expectation was good within all populations of both species (F,, = 0.042 proxima, 0.035 catenaria), while levels of interpopulation divergence were high (F,, = 0.461 proxima, 0.564 catenaria). In spite of slightly overlapping geographic distribu- tions, and instances of striking similarity in shell morphology between the two species, G. prox- ima and G. catenaria were genetically distinct (average values of Nei’s D near 1.0), with no evi- dence of hybridization. Although their ranges are fragmented into numerous isolated and genetically distinct populations, both species remain broadly recognizable across states, drainages, and physiographic regions. The relationship between G. catenaria postelliiand seven other nominal species and subspecies of Goniobasis sometime recognized from Atlantic drainages of Georgia is called into question. Key words: electrophoresis, isozymes,freshwater, Carolina, Georgia, gastropods, pleuroceri- dae, Elimia. INTRODUCTION Freshwater snails of the family Pleuroceri- dae are abundant, widespread, and important elements of the grazing and shredding com- munity in lotic ecosystems throughout the southeastern United States (Power et al., 1988; McCormick & Stevenson, 1991; Hill et al., 1995; Huryn et al., 1995; reviews by Fem- inella & Hawkins, 1995; Dillon, 2000). Their di- verse and often isolated populations, easily sampled year around, have made pleuro- cerids attractive models for the study of diver- gence and speciation (Chambers 1980, 1982; Dillon 1989, 1991; Bianchi et al., 1994; Dillon & Lydeard, 1998; Lydeard et al., 1997; Holz- nagel & Lydeard, 2000). Yet the pleurocerid fauna of much of the southeastern United States remains obscure and poorly docu- mented, and their systematic relationships unclear. Goodrich (1942) listed three pleurocerid genera, 15 species, and 26 subspecies in- habiting Atlantic drainages from New England to Texas. Four of these taxa will be treated in the present work: Goniobasis proxima (Say, 1825) from the “highlands of North and South 23 Carolina’; G. catenaria dislocata (Ravenel, 1834) inhabiting “headstreams” in Virginia, east-central North Carolina, and South Car- olina; G. catenaria catenaria (Say, 1822) from “springs of eastern South Carolina”; and G. catenaria postellii (Lea, 1858) from the “Al- tamaha, Ogeechee, and Canoochee Rivers” of Georgia. Goniobasis catenaria and G. proxima were two of the first four species of this diverse North American genus to reach formal description. Although altering the generic name to “Elimia,” Burch (1982) and Burch & Totten- ham (1980) generally adopted Goodrich’s un- derstanding of the pleurocerid fauna of North America as outlined above. But the system- atic relationships among the Georgia repre- sentatives of this group have also been re- viewed by Clench & Turner (1956), Krieger (1977), Chambers (1990), and Mihalchik (1998). Specific nomina applied to popula- tions from Georgia Atlantic drainages have in- cluded Goniobasis bentoniensis (Lea, 1862), G. boykiniana viennaensis (Lea, 1862), G. mutabilis (Lea, 1862), G. mutabilis timida (Goodrich, 1942), G. timida (Goodrich, fide Mihalchik, 1998), G. postellii (Lea, 1858), and 24 DILLON & REED G. suturalis (Haldeman, 1840). All these names would be junior synonyms of G. cate- naria, to the extent that G. catenaria extends into Georgia, as suggested by Goodrich. Between 1986 and 1995, Dillon & Keferl (2000) surveyed 629 aquatic sites distributed evenly throughout South Carolina, document- ing approximately 30 pleurocerid populations at 44 sites. They found ten populations of G. proxima in small streams in or near the Ap- palachian foothills, confirming Goodrich’s (1942) report. Ten populations of G. catenaria catenaria were found at 26 sites in streams and rivers of varying size through the South Carolina midlands, expanding the known range of that species substantially from that suggested by Goodrich. The disjunct distribu- tion of these populations suggested to Dillon & Keferl that this species may have been highly impacted by agricultural siltation and impoundment. Dillon & Keferl also reported six G. catenaria dislocata populations at eight sites in small streams of the coastal plain. Goniobasis catenaria populations were oc- casionally found well into the South Carolina piedmont, closely neighboring populations of G. proxima in upstate tributaries of the Broad/Santee and Catawba/Santee. And the similarity in shell morphology between G. proxima and G. catenaria dislocata of the coastal plain was striking. The shells of both species were identically shaped and marked by a single spiral carina. Goniobasis catenaria dislocata shells differed from G. proxima only by the presence of faint axial costae near the apex, disappearing after the first 2-3 whorls. Populations of both species inhabit small, rapidly flowing streams, G. catenaria dislo- cata generally being found in those rather un- usual regions of the coastal plain (often bor- dering large rivers) with slope and marl exposure. But whether the gross shell similar- ity of G. proxima and G. catenaria dislocata might reflect a genuine genetic relationship, or might be the result of convergence, Dillon & Keferl could not determine. The genetics of North Carolina and Virginia populations of G. proxima have been the sub- ject of intensive study for 20 years (Dillon & Davis, 1980; Dillon, 1984a; Stiven & Kreiser, 1994). Intrapopulation variation seems to be unusually low, and interpopulation divergence high, as might be expected from populations of such a poorly mobile animal isolated in small streams of the piedmont and mountains (Dillon, 1984b). Despite the occurrence of fixed differences at multiple enzyme loci, how- ever, transplants and artificial introductions have revealed no evidence of reproductive isolation among G. proxima populations (Dil- lon, 1986, 1988a). The purposes of the present work are threefold. First we compare the levels of intra- and interpopulation genetic variance ob- served in G. catenaria to the better-known G. proxima. Second, we evaluate the genetic distinctiveness of G. proxima and G. cate- naria, paying particular attention to the possi- bility of hybridization. Third, we examine evi- dence that populations of G. catenaria dislocata might be extralimital G. proxima, rather than G. catenaria with shell morphol- ogy convergent on G. proxima. METHODS We sampled five populations of G. cate- папа catenaria, two populations of G. cate- папа dislocata from eastern South Carolina, and one G. catenaria postellii from Georgia. The four populations of G. proxima we sam- pled included Yad from northwestern North Carolina, which we have previously compared to other populations of Goniobasis (as Yad in Dillon & Davis, 1980; Yad7 in Dillon, 1984b, 1988b) and which served as a control for the present investigation. Our Georgia site was identical to “Station X” of Krieger & Burbanck (1976) and YELD of Krieger (1977), inhabited by a population identified as “G. suturalis” in the former publication and “G. boykiniana vi- ennaensis’ in the latter. Population Cirk of G. catenaria and population Bull of G. proxima were sampled from tributaries of the Broad River separated by only about 15 km through water. The sites at which we collected all 12 populations may be located in Figure 1. Lo- cality data for all sites are provided in the Ap- pendix. Voucher specimens have been de- posited in the Academy of Natural Sciences of Philadelphia. At each site we collected at least 30 indi- vidual snails, which were cracked and stored in Tris-phosphate 7.4 tissue buffer at -70°C upon return. Allozyme variation was resolved from whole-animal homogenates by horizon- tal starch gel electrophoresis, using equip- ment and techniques as previously described (Dillon, 1985, 1992; Dillon & Lydeard, 1998). We initially screened ten enzyme systems and six buffers by requiring that polymor- phism interpretable as the product of codomi- nant Mendelian alleles be expressed in a GONIOBASIS POPULATION GENETICS 25 Pee Dee Santee Altamaha FIG. 1. The study area, showing primary drainages and sample sites. Circles indicate G. catenaria, and squares G. proxima. comparison of G. proxima population Yad, G. catenaria postelliipopulation Yel, and G. cate- naria dislocata population Srp. Ultimately, we compared all populations on the basis of eight loci, most resolved on two buffer systems, as follows. The AP6 buffer of Clayton & Tretiak (1972) was used to resolve mannose-phosphate isomerase (MPI, EC 5.3.1.8), 6-phosphogluconate dehydroge- nase (6PGD, EC 1.1.1.44) and isocitrate de- hydrogenase (IDHF and IDHS, EC 1.1.1.42 — cathodal and anodal loci, respectively). A ТЕВ8 buffer (Shaw & Prasad 1970) was used for esterases (EST1, ЕС 3.1.1.2 —the strong, slow locus only), xanthine dehydrogenase (XDH, EC 1.2.1.37), MPI, and IDHF. The Poulik (1957) buffer was used for octopine de- hydrogenase (ODH, EC 1.5.1.11), glucose- phosphate isomerase (GPI, EC 5.3.1.9) and EST1. Chambers (1980) demonstrated that inher- itance of allozyme phenotype is Mendelian at the 6PGD locus in G. floridensis. Dillon (1986) verified Mendelian inheritance at GPI, ODH, and EST1 by mother-offspring analysis in G. proxima. The designations of putative alleles used for population Yad were retained from the system of Dillon (19846) for GPI, MPI, ODH, EST1 and XDH. For those loci which had not previously been examined for popula- tion Yad (6PGD, ISDHF, ISDHS), the most common Yad allele was named “100.” Then 26 DILLON & REED alleles in all other populations were named according to the mobility of their allozymes in millimeters relative to the Yad standard. Data were initially analyzed using BIOSYS- 1 (Release 1.7; Swofford & Selander, 1981). Genotype frequencies at all polymorphic loci were tested for conformance to Hardy-Wein- berg expectation within populations using chi- square statistics, with Yates correction in 2 x 2 cases, pooling rare classes as required. Mean F-statistics (Wright, 1978) were calcu- lated across loci for the four populations of G. proxima and the eight populations of G. cate- naria separately. Values of Nei’s (1978) unbi- ased genetic similarity and distance were cal- culated pairwise over all populations, as well as the chord distance of Cavalli-Sforza & Ed- wards (1967). The matrix of chord distances, which are Pythagorean in Euclidean space (Wright, 1978), was input to STATISTICA (Re- lease 5.0, Statsoft, Inc.) and clustered using the method of unweighted pair-group averag- ing (UPGMA). RESULTS Example shells are shown in Figure 2. Typ- ical G. catenaria bore shells with both spiral costae (or cords) and axial costae (or ribs) in- tersecting to form nodules. The shells of G. proxima had no axial costae and only a single spiral costa (or carina) becoming obsolete with age. Typical G. catenaria shells also tended to be wider per unit length than G. proxima. The shells of the Ye/ population, nominally G. catenaria postellii, did not differ noticeably from those of typical G. catenaria catenaria. But as noted by Dillon & Keferl (2000), the shells of G. catenaria dislocata were narrower and lacked both spiral and axial costae on later whorls, rendering them more similar to G. proxima than typical G. catenaria (Dillon & Keferl 2000). For allozyme analysis, sample sizes aver- aged across loci ranged from N = 29 at West to N = 43 at Yad (Appendix). Sample sizes at all 12 x 8 loci examined were greater than 30, except N = 29 for Bull (XDH), N = 26 for Bull (EST1), N = 14 for West (IDH), N = 20 for Morg, (IDH), and N = 26 for Morg (GPI, XDH). Allele frequencies are given in Table 1. Over all observations, a total of 26 loci were polymorphic by the 95% criterion, a value somewhat higher than has been observed in comparable surveys of pleurocerid population genetics previously published (Chambers, 1980; Dillon & Davis, 1980; Dillon, 1984b; Dil- lon & Lydeard, 1998). Fits to Hardy-Weinberg expectation were excellent within popula- tions. Chi-square tests uncovered no signifi- cant differences between observed genotypic frequencies and those expected from Hardy- Weinberg equilibrium in any case. The values ofF. for both G. proxima and G. catenaria av- eraged over eight loci were negligible (Table 2). As might be expected from their highly iso- lated population structure, divergence among the four G. proxima populations was high (F., = 0.461; Table 2). Table 1 shows fixed (or nearly fixed) differences at a minimum of one locus between all pairs of G. proxima, except Morg —Bull. Divergence among the eight G. catenaria was not as striking upon first exam- ination, the trio of populations from the middle FIG. 2. Example shells from the four taxa studied. From left, Goniobasis catenaria catenaria (Cirk), G. cate- naria dislocata (Srp), G. proxima (Bull), and G. catenaria postellii (Yel). The standard length of the G. cate- naria catenaria shell is 1.75 cm, the remaining shells are figured at the same scale. GONIOBASIS POPULATION GENETICS 27 TABLE 1. Allele frequencies at 8 enzyme loci т 12 populations of Goniobasis from southern Atlantic drainages of the United States. G. catenaria Locus allele Yad West Morg Bull Сик Uwh Lnch Cola Sant ES 100; 0.167 0.191 0.513 0.219 0.188 103 0.802 0.794 0.474 0.885 1.000 0.734 0.813 1.000 1.000 0.679 0.789 0.524 а. proxima Srp MeC Yel 106 0.031 0.015 0.013 0.115 0.047 0.321 0.211 0.012 107 0.463 GPI 98 0.014 0.976 100 1.000 1.000 1.000 1.000 0.986 0.024 0.162 1.000 102 1.000 1.000 1.000 1.000 105 0.784 110 0.054 IDHF 93 0.953 97 0.125 99 0.016 0.319 100 1.000 1.000 0.750 1.000 103 0.050 105 0.075 0.047 0.984 1.000 1.000 0.681 1.000 1.000 1.000 IDHS 100 1.000 1.000 1.000 1.000 104 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 MPI 95 0.833 0.200 0.750 100 0.167 1.000 0.800 0.250 1.000 1.000 1.000 1.000 1.000 0.989 1.000 1.000 103 0.011 ODH 106 0.738 1.000 0.145 109F 0.125 0.855 1.000 111 0.118 0.431 113F 0.138 114 1.000 0.969 1.000 1.000 1.000 1.000 0.882 0.569 118 0.031 6PGD 100 1.000 0.882 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.522 105 0.118 0.422 110 0.056 XDH 97 1.000 0.731 0.603 0.015 0.875 0.390 98 1.000 0.269 0.397 1.000 1.000 1.000 0.985 0.125 0.610 1.000 0.568 100 0.432 TABLE 2. Values of Wright’s (1978) F-statistics averaged over eight loci for four populations of G. proxima and eight populations of G. catenaria. the eight G. catenaria populations over the three-state region was 0.564, similar to that observed for G. proxima. Nei’s statistics among all populations are SE Szealenana presented in Table 3, and the result of the EX 0.042 0.035 UPGMA cluster analysis based on interpopu- Fr 0.484 0.580 lation chord distances is shown in Figure 3. == 0.461 0.564 Each G. catenaria dislocata population was of the study area (Uwh-—Lnch-—Cola) being genetically indistinguishable. But the south- ern populations were more divergent. The Yel population of G. catenaria postellii had un- usual alleles in high frequency at three loci (EST1, 6PGD, XDH), and both the Srp popu- lation of G. catenaria dislocata and the McC population of G. catenaria catenaria were strikingly distinct at the GPI locus. Population Cirk was distinguished by a unique IDHF al- lele. Consequently, the mean value of F,, for more similar to its neighboring G. catenaria catenaria population (| = 0.86, 0.89) than the pair of dislocata were to each other (| = 0.81). This tends to support the hypothesis that the two populations of G. catenaria dislocata have lost shell sculpture independently. Reinforcing the impression left from the F- statistics, Figure 3 suggests that the levels of interpopulation divergence within G. proxima and G. catenaria are comparably high. The four G. proxima populations are nevertheless quite distinct from the eight G. catenaria pop- ulations, sharing no alleles at three loci (GPI, 28 DILLON & REED TABLE 3. Nei’s (1978) statistics among pairs of Goniobasis populations. Unbiased genetic iden- tity is shown below the diagonal, and unbiased genetic distance above. Yel McC Srp Sant Cola Lnch Uwh Сик Bull Morg West Yad Yel == 0.22. 70.29.”.0.21 ¿0:11 0.11” ¿0.107 027 - 1.481.215 124132 McC 0.81 = 015 0:24 0.10 0.10 0.11 0.24 1.08 :0:96 11:06, 0:92 Srp 0.75 0.86 = 0.21 0.17 0.17 0.17 .0.33 1.08 0.91 0.95. 1:10 Sant 0.81 0.78 0.81 ак 0.12 0.13 0.13 0.21, 0.96 0.80 0.73 1.23 Cola 0.90 0.91 0.84 0.89 zu 0.00 0.01 0.12 1.08 0.98 1.05 0.93 Lnch 0.90 0.91 0.84 0.88 1.00 = 0.00 0.13 1.13 096 1.08 0.94 Uwh 0.90 0.90 0.84 0.88 0.99 1.00 = 0.13 1.14 0.95 “1:09 095 Ск 0.76 0.79 0.72 0.81 0.89 0.88 0.88 == 1.07 1:00 1:05 0792 Bull 0.23 0.34 0.34 0.38 0.34 0.32 0.32 0.34 = 0:09" 0:28 70:16 Morg 0.30 0.38 0.40 045 0.38 0.38 0.39 0.37 0.91 = 0:15 70/26 West 0.29 0.35 0.39 0.48 035 0.34 034 0.35 0.76 0.86 Le 0.28 Yad 0.26 0.40 0.33 0.29 0.40 040 0.39 040 0.85 0.77 0.76 0:7 0.6 0.5 0.4 0.3 Cavalli-Sforza & Edwards Chord Distance G. proxima 0.2 G. catenaria catenaria | G. catenaria dislocata | С. catenaria postellii 0.1 0.0 FIG. 3. UPGMA Cluster analysis of the 12 study populations, based on a matrix of genetic distances calcu- lated using the chord method of Cavalli-Sforza & Edwards (1967). ODH, IDHS) and almost completely distinct at IDHF. Table 1 shows no evidence of hy- bridization between the species, in the Broad/Santee drainage between Cirk and Bull or elsewhere. DISCUSSION Although inhabiting a more moderate cli- mate and a less mountainous environment, the population genetic structure of G. cate- naria does indeed resemble that of the better- studied G. proxima. The similarity of the G. catenaria populations inhabiting the Pee Dee drainage (Uwh and Lnch) and the (apparently isolated) Santee populations (particularly Cola) was unexpectedly high. But the overall values of Wright’s F-statistics and most val- ues of Nei’s genetic distances among G. cate- naria populations were comparable to values obtained from G. proxima. Values of genetic similarity and distance weakly suggest that our two dislocata popula- tions may have independently lost shell sculp- turing, Sant deriving from Cola and Srp from McC. But as indicated in Figure 3 and Table 3, the two putative source G. catenaria catenaria populations, Cola and McC, are more similar to each other than either is to its G. catenaria dislocata neighbor. Sant and Srp do share a unique allele (GP198) not found in any other population, and are also more similar to each other at the XDH locus than either is to G. catenaria catenaria. Additional data will be re- quired before the origin of G. catenaria dislo- cata can be proposed with any confidence. It is clear, in any case, that G. catenaria dis- locata populations are not extralimital G. prox- ima, nor do they bear more genetic similarity GONIOBASIS POPULATION GENETICS 29 to G. proxima than to other G. catenaria of more typical shell morphology. We found no evidence of hybridization between the two species, in the upper Santee tributaries where they are closely neighboring or elsewhere. Goniobasis proxima and G. catenaria are quite distinct. As might be predicted from its geographic situation on the periphery of the study area, population Ye/ was the most genetically dis- tinctive of the populations here identified as G. catenaria (Fig. 3). But the level of diver- gence displayed clearly does not warrent the removal of this population to another specific name. Population West is more different from other G. proxima than population Yel is from other G. catenaria. Goodrich’s (1942) sugges- tion that Goniobasis populations from this re- gion of Georgia be referred to G. catenaria as a subspecies postellii, rather than distin- guished as a separate species, would seem to have considerable merit. Based on a comparison of his freshly col- lected shells to shells in the University of Michigan collections, Krieger (1977) identified Georgia populations of Goniobasis from the Atlantic drainages as G. postellii in the Oconee River, G. suturalis (= G. mutabilis) in the Yellow River above Porterdale, and G. boykiniana viennaensis in the Apalachee River and in the Yellow/Ocmulgee River below Porterdale. Mihalcik (1998) identified a population of Goniobasis from Snapping Shoals on the South River, a tributary of the Ocmulgee 20 km west of Porterdale, as G. mutabilis. She identified a population from an Ocmulgee tributary downstream about 350 km from Porterdale as G. timida, and sug- gested that a third population from the Atlantic tributaries of Georgia, Rocky Creek of the Oconee drainage, might be an undescribed “Species C.” Mihalcik referred Goniobasis populations from the Savannah River to G. bentoniensis. 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PRASAD, 1970, Starch gel elec- trophoresis of enzymes—a compilation of recipes. Biochemical Genetics, 4: 297-320. STIVEN, А. E., & В. В. KREISER, 1994, Ecological and genetic differentiation among populations of the gastropod Goniobasis proxima (Say) in streams separated by a reservoir in the piedmont of North Carolina. Journal of the Elisha Mitchell Science Society, 110: 53-67. SWOFFORD, D. L. & R. B. SELANDER, 1981, BIOSYS-1: a FORTRAN program for the com- prehensive analysis of electrophoretic data in population genetics and systematics. Journal of Heredity, 72: 281-283. WRIGHT, S., 1978, Variability within and among natural populations. Vol. 4, Evolution and the ge- netics of populations. Chicago: University of Chicago Press, Chicago, Illinois 580 pp. Revised ms. accepted 25 March 2001 APPENDIX Identification and locality data for study popu- lations. The sample sizes given are averaged over the 8 enzyme loci reported in Table 1. Bull —G. proxima (N = 31). Bullock's Creek at SC 889 bridge (Boheler Road), 10.9 km NNW of York, York Co., South Carolina. 81°19’W, 35°05'N. Same as site 20p of Dillon & Keferl (2000). Cirk —G. catenaria саепапа (N = 32). Clarks Fork Creek at SC 55 bridge, 16.1 km NW of York, York Co., South Carolina. 81°24'W, 35°05’N. Same as site 17c of Dillon & Keferl (2000). GONIOBASIS POPULATION GENETICS 31 Cola—G. catenaria catenaria (N = 34). Big Cedar Creek at SC 59 bridge (Wildflower Road), 21.3 km N of Forest Acres, Rich- land Co., South Carolina. 81°06’W, 34°11'N. Same as site 23c of Dillon & Ke- ferl (2000). Lnch—G. catenaria catenaria (N = 32). Lynches River at SC 265 bridge, 3.8 km SW of Jefferson, Chesterfield/Lancaster Cos., South Carolina. 80°26’W, 34°38'N. Same as site 40c of Dillon & Keferl (2000). McC—G. catenaria catenaria (N = 38). Stevens Creek at SC 23 bridge, 24.6 km WSW of Edgefield, Edgefield/McCormick Cos., South Carolina. 82°11'W, 33°44'N. Same as site 4c of Dillon & Keferl (2000). Morg — G. proxima (N = 33). Small tributary of Clear Creek at bridge 50 m N of U.S. 64, 5 km S of Morganton, Burke Co., North Carolina. 81°45'W, 35°40'N. Sant —G. catenaria dislocata (N = 36). Head of Chapel Branch, Santee, Orangeburg Co., South Carolina 80°29'W, 33°30'N. Same as site 28d of Dillon & Keferl (2000). Srp—G. catenaria dislocata (N = 39). Lower Three-Runs Ck at SC 70 bridge, 9 km $ of Snelling, Barnwell Co., South Carolina. 81°27'W, 33°11'М. Same as site 6d of Dillon & Keferl (2000). Uwh-G. catenaria catenaria (N = 32). Uwharrie River at NC 109 bridge, 1 km NW of Uwharrie, Montgomery Co., North Carolina. 80°01’W, 35°26'N. West -G. proxima (N = 29). Small tributary of the Chauga River at SC 196 bridge, 1km W of Mountain Rest, Oconee Co., South Carolina. 83°10'W, 34°52'N. Yad—G. proxima (N = 43). Naked Creek at NC 1154 bridge, 5.2 km N of Ferguson, Wilkes Co., МС. 81°22’W, 36°09’N. Same as Yadk of Dillon & Davis (1980), Yadl of Dillon (1984). Yel—G. catenaria postellii (N = 40). Yellow River below dam at Porterdale, Newton Co., Georgia. 83°53'W, 33°34'N. MALACOLOGIA, 2002, 44(1): 33-46 SPATIAL DISTRIBUTION, DENSITY AND LIFE HISTORY IN FOUR ALBINARIA SPECIES (GASTROPODA, PULMONATA, CLAUSILIIDAE) Sinos Giokas' & Moysis Mylonas? ABSTRACT In this study, we analysed field sequential data on density, mortality, and spatial distribution of four Albinaria species. We also recorded and compared a series of life-history traits, including the onset and end of aestivation, copulation, oviposition, and hatching. Our aim was to obtain more information on the ecology of Albinaria, to reveal possible life-history patterns, and to un- derstand the association of life-history characteristics with both extrinsic (density independent) and intrinsic (density dependent) factors and microevolutionary processes. The onset of aesti- vation period occurs always in April and is independent of the end of rainfall, whereas awaken- ing and copulation is actually synchronous and occurs after the first autumn rains. The duration ofthe oviposition and hatching period is short, but oviposition can be prolonged under favourable climatic conditions. Density is high, but its fluctuations were not related to climatic or time factors. Any density dependent pattern resulting from competition was not detected. Higher mortality did not coincide with aestivation, and usually relatively high mortality results after hatching. Juveniles usually exhibit higher mortality than adults. Spatial distribution of these rock-dwelling snails is al- ways highly contagious. Clustering behaviour was probably influenced by the substratum, mo- saic or uniform, and the occurrence of crevices. Predictions about the population dynamics of these iteroparous, long-lived species were not possible because of sampling inadequacies and/or because fluctuations of population structure, density, mortality and spatial distribution were random. Key words: Albinaria, Greece, life history, density, mortality, spatial distribution. INTRODUCTION Population dynamics is affected, either di- rectly or indirectly, by such density-indepen- dent factors as climate, and/or such density- dependent factors as predation, disease and competition. Life-history studies are essential for understanding the role of these factors and the action of natural selection (Stearns, 1992). However, in land snails the influence of these factors is often abstruse or inadequately un- derstood. Though regulated life cycles are not likely to be distinguishable from random den- sity fluctuations (May, 1975; Cook, 1990), sev- eral studies indicate the effect of environmen- tal conditions on abundance (Baur & Raboud, 1988; Heller & Dolev, 1994; Lazaridou-Dimitri- adou & Sgardelis, 1997), or the influence of intraspecific crowding on density, via its direct or indirect affect on individual fecundity, sur- vival, locomotion, adult size, and growth rate (Yom-Tov, 1972; Cameron & Carter, 1979; Dan & Bailey, 1982; Tilling, 1985; B. Baur, 1988; Staikou & Lazaridou-Dimitriadou, 1989; A. Baur & B. Baur, 1992). Resource or inter- ference competition is another controversial subject in land snails (Pearce, 1997). Albinaria is a pulmonate genus distributed around the northeastern coasts of the Mediter- ranean, exhibiting a high degree of morpho- logical and molecular differentiation. More than 90, in some cases dubious (Douris et al., 1995, 1998a, b), nominal species have been described (Nordsieck, 1999). Ecological stud- ies of Albinaria populations can be very infor- mative about evolutionary processes in this re- gion, which has a complex palaeogeographic and climatic history (Paepe, 1986; Anasta- sakis & Dermitzakis, 1990; Westeway, 1994), because the high species number does not appear to be associated with proportionate ecological differentiation (Gittenberger, 1991). However, although the biogeography and sys- tematics of Albinaria have been well consid- ered (for a review, see Giokas, 2000), and A/- binaria species constitute about 15% of the total Greek land-snail species, studies of their life history are lacking. Similarly, very few data ‘Corresponding author — Zoological Museum, Department of Biology, University of Athens, GR-15784, Athens, Greece; sinosg O biol.uoa.gr Natural History Museum, Department of Biology, University of Crete, GR-71409, lrakleion, Greece; director @nhmc.uch.gr 34 GIOKAS & MYLONAS are available (Warburg, 1972; A. Baur, 1990; B. Baur & A. Baur, 1990; 1991, 1992; Heller & Dolev, 1994) on the biology and life history of the Clausiliidae. Albinaria snails live on limestone substrata (Schilthuizen, 1994; Giokas, 1996). They feed on the microflora, that is, lichens and bryophytes. They are active only during the wet season, which in southern Greece is from late October through the end of April. Eggs are laid in clutches of about 5-7 eggs, gener- ally shortly after the beginning of the wet sea- son. During the intervening dry periods, juve- niles and adults aestivate on the rock surfaces, in rock crevices, and occasionally on shrubs, or under stones. Especially during aestivation, aggregates are often formed, sometimes including many tens of individuals. Albinaria live about seven years. The devel- opment from a juvenile (juveniles do not have a lip or well-formed internal lamellae) to a fully grown snail takes two and occasionally three wet seasons. Shell development precedes maturation, which occurs during the last dry season after enlargement of genitalia size. Copulation then takes place during the first weeks of autumn rains. Population densities can sometimes be very high, in spite of mor- tality caused by desiccation, especially during aestivation, or of occasionally heavy preda- tion by larvae of the beetle family Drilidae (Schilthuizen et al., 1994), or by typical preda- tors of snails, such as rodents and birds. This is the first comparative field study of both qualitative and quantitative life history characteristics for Albinaria. We analysed se- quential density, mortality, and spatial distri- bution data of four well-defined Albinaria species. We also recorded and compared a series of life-history traits, including the onset and end of aestivation, copulation, oviposi- tion, hatching, and aggregation. Our aim was to obtain more information on the ecology of Albinaria, to reveal possible life-history pat- terns and to understand the association of life-history characteristics with both extrinsic (density independent) and intrinsic (density dependent) factors and microevolutionary processes. MATERIALS AND METHODS Species Studied Four different Albinaria species, with each one population per species, were studied (Fig. 1). These were A. coerulea (Deshayes, 1835)— а{ Vravrona, Attiki, on the Acropolis hill beside the archaelogical site, A. turrita (Pfeif- fer, 1850) — Kea island, N. W. Kyklades, around the Monastery of Panagia Kastriani, A. discolor (Pfeiffer, 1846) — Aigina Island, Ar- gosaronikos Gulf, 5 km east from Agios Nek- tarios Monastery, and A. voithii (Rossmassler, 1836) — at Parori, Lakonia, near Sparti, at the entrance of the gorge. Habitat and Bioclimatic Characteristics of the Studied Sites Table 1 shows the main bioclimatic (Mavrommatis, 1978) and habitat characteris- tics of the studied sites. A/binaria coerulea, A. turrita and A. discolor face the same biocli- matic conditions, which generally can be characterised as arid. However, A. voithii lives in a more humid habitat and the dominant vegetation type at Parori is maquis and not phrygana as in the other areas. Aigina, though arid, has a mixed vegetation (phrygana and maquis). Other climatic data with unambigu- ous seasonality (temperature, precipitation, humidity) are not presented because they were uncorrelated with the examined life his- tory parameters (see Results). All the above- studied populations are very localized and re- stricted to the sampled areas, which can be as small as that of Parori. Though the sub- Strata at all sites is calcareous, sites are dif- ferentiated by the presence and density of crevices and calcareous plates, and the con- tinuous or mosaic allocation of rock bulks. Sampling Procedure We adopted the quadrat random sampling procedure (Krebs, 1989; Chalmers & Parker, 1986; Williams, 1987). Within each site, sam- ples were taken every month for A. coerulea (April 1992-April 1994) and on a seasonal basis for A. voithii (April 1992-August 1994), A. turrita (August 1992-April 1994), and A. discolor (July 1992-April 1994). The size of the sampling quadrats (50 cm x 50 cm) was determined after preliminary sampling work (Krebs, 1989). A 20% confidence limit was chosen (Elliot, 1971; Hayek & Buzas, 1997) to set the number of sampling quadrats for each studied species. The number of quadrats on each sampling occasion was 15 in Vravrona, and 20 in Parori, Kea and Aigina. During aes- tivation we sampled once. All samplings were LIFE HISTORY IN ALBINARIA 35 a > TURKEY FIG. 1. The distribution of the genus Albinaria (shaded area) and the studied sites. done on non-rainy days, at approximately 9.00 a.m. In each quadrat, all the adult and ju- venile specimens, both alive and dead, were recorded; we counted only the dead speci- mens still attached to the substratum and not the empty shells fallen to the ground. Dead specimens were removed after each sam- pling. One month before the start and the end of the aestivation period and one month after that period we were visiting the sampling sites more often (every ten days at Vravrona) in order to record more precisely alterations of associated life-history traits (aestivation onset and ending, copulatory activity, oviposition, hatching). Statistical Analysis Besides descriptive statistics, we used ANOVA and Tukey HSD tests in order to analyse density fluctuations for the alive and dead specimens (adults and juveniles). We used log-transformed values. We examined ratio (adults/juveniles mortality) differences with contingency table analysis (G-test, Fisher exact test) (Zar, 1984). Additionally, we used time-series analysis (Wilkinson, 1989), in order to investigate possible autocorrela- tion and cross-correlation patterns for the density series of alive and dead specimens (adults and juveniles). Spatial distribution was estimated using: (a) Green’s index of dispersion Gl = (s?/m) — 1/(n — 1), (m = mean number of individuals, s* = variance, and n = total number of individuals in sample), and (b) Taylor’s Power Law (s? = am?) (a and bare population parameters) (El- liot, 1971; Ludwig & Reynolds, 1988; Hayek & Buzas, 1997). Parameter b, in Taylor’s Power Law, can vary from zero to infinity. When b > 1 the distribution is contagious, when b= 1 the distribution is random and when b < 1 the dis- tribution is uniform. Finally, we used MANOVA and multiple re- gression analysis in order to examine possi- ble effects of climatic (precipitation, humidity, temperature) and time (month, season, year) factors to density, mortality and spatial distri- bution. GIOKAS & MYLONAS 36 EE O 2 /<ÉÁ<Á Gazelle) ajesadula) piuny-qns (Oz) WIEM pue-ILU9S (GO M) WIEM рие-циэ$ WO) WIEM PUE-IUIOS Joojj-qns 100} эцец0!8 91yeu1]901g (001 > > GZ) ueeuel -1эйрэш-озэш esuajul (OS| > > 921) veaueN -лэурэш-ошаэц} esuajul (OSL > > вер ueeurei -1эурэш-ощиэц} esuajul (OS| > x > вер) ueeurei —лэурэаш-ошаэц} esuajul adj эцешиоо!я sınbew sinbew y eueBluyd eueBÁlyd eueBAiud эал} цоцеэбэдл заэлэло ма} чим 3901 зубиап snoeiesjeo сш 06 HYJIOA “y ‘иолед S991A919 цим SH901 Sayed J0J09SIP “Y зпоэлез[еэ P3119YEIS zu OOS ‘eulbiy S991A919 MJ чим $3904 sayje¡d зпоэлееэ zui 009 PIN] “y “eay S991A819 чим $3204 seed P9/N1809 “y snoa/e9¡e9 SNONUNUOD zw 0001 “RUOJABIA unye1asaqns pole pajdwes ogg ‘(1884 ay) JO YJUOW 3зэр!оэ ay} jo эле -Jodua] шпили ueaw :“ | ‘skep pue AjpeoiBojoig Jo Joquunu :X) SAYS parpnys ay) jo зоцзиаоелецо эцещиоса pue JEHQEH “| FIAVL LIFE HISTORY IN ALBINARIA 37 RESULTS Aestivation, Awakening, Copulation, Oviposi- tion, Hatching, Activity Aestivation always began in April. However, at the more arid sites of Vravrona and Kea aestivation began earlier (first week of April) than at Aigina and Parori (last week of April). Specifically, at Vravrona, on average for the three sampling years, in the middle of April 77.4% of adults had formed epiphragm, while at the end of April 100% of them were fully aestivating. The onset of the aestivation pe- riod was always independent of the end of the rainfall period. It is notable that aestivation, both in the arid springs of 1992 and on the much more humid and rainy of years 1993 and 1994, started approximately at the same time (April) for all species. Awakening and copulation occurred only after the first autumn rain (in 1992 in the mid- dle of October and in 1993 in early Novem- ber). However, destruction of the epiphragm started in early October. More precisely at Vravrona, whereas in mid-September we did not observe any specimens with fragmenting epiphragm, in the beginning of October, on average for the two years, 86% of adults had an epiphragm with some holes, and at the end of October all specimens had no trace of epiphragm. Copulation is actually immediate and synchronous, and it is the main activity of Albinaria specimens after awakening. Copu- lation period lasts for about a week on aver- age. Oviposition starts about 20 days after cop- ulation and stops after ten days, but at the more humid site of Parori continued almost until the end of the wet period. Eggs were usu- ally laid in crevices or under stones in clutches of about five to eight eggs. Hatching occurred approximately two to three weeks after oviposition. During the wet period, on the sampling days, only a portion of the population was ac- tive, feeding on the microflora. However, we did not estimate that portion. Density Table 2 shows the mean densities (speci- mens per 0.25 m?) of the live specimens of the four studied species, and the significant differ- ences (between the sequential samples of each species) are indicated, Figure 2 shows density fluctuations during sampling. Autocor- TABLE 2. Mean density of alive specimens (speci- mens/0.25 m?) of the studied Albinaria species. Asterisks indicate significant (at o. = 0.05) density differences between sequential samples of each population. total adults juveniles A»coerulear 14.132417 9.1=24” 5:0) 0:7* A. turrita 84-15 44-07 4:0 ls A. discolor OS (6:9 1.2 MIE 105 A. voithii 9.4 = 1.57 5:0 = 0:9* 4.4 =0:8* relation analysis showed that these were гап- dom series. It is notable that mean densities can be quite high. Higher densities were usu- ally observed during the aestivation period. However, in neither species, the differences between sequential samples that were sug- gested by the Tukey HSD test could be attrib- uted (MANOVA, Multiple regression) to cli- matic (temperature, precipitation, humidity) and time (year, season, month) sources of variation, and consequently are not pre- sented. Significant mean density differences (adults plus juveniles) were not found among the four studied populations (ANOVA, p = 0.05). Like- wise, the mean densities of the adult speci- mens did not differ significantly (ANOVA, p = 0.09), but mean densities of juveniles differed significantly (ANOVA, p = 0.003), and the Tukey HSD test indicated significant differ- ences between juveniles at Aigina with those at Vravrona and Parori. Mortality Mean densities of dead specimens (speci- mens per 0.25 m?) of the four studied species, and significant differences (between the se- quential samples of each species) are shown in Table 3; Figure 3 shows density fluctua- tions during sampling. Adults and juveniles usually exhibit high mortality after hatching period; however, differences between se- quential samples were not caused by climatic and time sources of variation (MANOVA, Mul- tiple regression). Significant differences between the mean densities of dead individuals (adults plus ju- veniles) were not found among the four stud- ied populations (ANOVA, p = 0.05). Mean densities of adult dead specimens differed (ANOVA, p = 0.015), and the Tukey HSD test showed differences between adults at Aigina and those at Kea and Parori. Densities of 38 GIOKAS & MYLONAS A соеплеа 45 specimens/0.25m* specimens/0 25m? 8! specimens/0 25m с 6} | | | 4) | > 2} | | 0 = ==) Jul Dec Mar Jun Dec Mar 1992 nes months FIG. 2. Density fluctuations of alive specimens for the studied Albinaria species (in bars black stands for adults and white for juveniles). LIFE HISTORY IN ALBINARIA A coerulea specimens/0 25m? specimens/0 25m° specimens/0 25m? specimens/0 25m° FIG. 3. Density fluctuations of dead specimens for the studied Albinaria species (In bars black stands for adults and white for juveniles). 39 40 GIOKAS & MYLONAS dead juveniles (ANOVA: p = 0.016) also dif- fered significantly, and the Tukey H.S.D. test indicated significant differences between Kea and Parori. Mean mortality ratio (dead specimens/ (dead + living specimens)) can vary signifi- cantly (p < 0.0001) between sequential sam- ples (Table 3, Fig. 4). Juvenile mortality (the ratio of dead juveniles to dead + living juve- niles) was significantly greater (p < 0.05) than adult mortality (dead adults/dead + living adults), across time, for A. coerulea and A. voithii and greater, however not significantly for A. discolor. In A. turrita, juvenile and adult mortality did not differ significantly, even though juvenile mortality was lower than that of adults. Spatial Distribution Spatial distribution was always contagious (Table 4), according to the parameters a and b of Taylor's Power Law and Green’s Index, except in the case of living adults of A. turrita. Values of b were (except for A. turrita) high (above 2). Higher values of b (for total and adults) were estimated for À. discolor and A. coerulea. For juveniles, A. coerulea had the lower b value. Adults were more aggregated than juveniles, except for A. turrita. For dead specimens, values of b were more or less of the same order. However, comparison of slopes bis not always justified, because bcan be affected by scale changes (Hayek & Buzas, 1997). DISCUSSION The start of the aestivation period, regard- less of site, year or species, was not corre- TABLE 3. Mean density of dead specimens (speci- mens/0.25 m?) of the studied Albinaria species. Asterisks indicate significant (at с = 0.05) density differences between sequential samples of each species. In parentheses mean mortality ratio (dead specimens/dead + living specimens). total adults juveniles A. coerulea 1.4 = 0.2 07+01 0.6 = 0.1* (9%) (7.1%) (10.7%) A. turrita 0.5=0.1 "103: =:0.1/. O12 01 (5.6%) (6.4%) (4.8%) A. discolor 1.38 015" 1.3 =0:4% 05 +101 (17.3%) (15.8%) (22.7%) A. voithii 1.2: 0:3* 70:4 0/2" 0.3071” (11.3%) (7.4%) (15.4%) lated directly to certain climatic conditions (rain, temperature and humidity). Even though it is not possible to separate geo- graphical, species and population effects, probably this implies that aestivation onset in Albinaria is relatively independent from these fluctuating environmental factors, and the possible cardinal action of an unknown intrin- sic physiological mechanism possibly associ- ated with day-length perception. However, the end of the aestivation period and the onset of copulation require rainfall (yet, not before the end of September). Heller & Dolev (1994) re- ported similar observations for the clausiliid Cristataria genezarethana. These life-history characteristics have been observed for sev- eral other (32) Albinaria populations belong- ing to 13 species sampled from 1990 to 1995 throughout Greece (Giokas, 1996). Addition- ally, laboratory experiments with 25 Albinaria populations belonging to 12 species (among them the studied species of A. coerulea, A. turrita, A. discolor and A. voithil) proved that awakening is actually synchronous (on aver- age 90% of snails awake within five hours) and that copulation is the main activity of Albi- naria specimens for the first 36 hours after awakening (Giokas, 1996). According to Heller & Dolev (1994) and Lazaridou-Dimitri- adou & Sgardelis (1997), the rapid awakening response of aestivating snails to autumn rains is advantageous in seasonally dry habitats, which prevail in Mediterranean climate condi- tions. In that way, Snails avoid adverse effects during unfavourable conditions. Several Albi- naria species, for example, those belonging to the grisea group, which aestivate under stones or deep in crevices and have a thin, transparent shell, awake later (in late Novem- ber) (Giokas, 1996), and Giokas et al. (2000), support that awakening is partly associated with aestivation behaviour and morphological features, for example, thin shell. Oviposition and hatching stopped after the first month of the wet period at the more arid sites of Vravrona, Kea and Aigina. However, at the more humid and inland Parori site, oviposition and hatching continued through- out the active period, though mating was re- stricted to its beginning. Likewise, according to Lazaridou-Dimitriadou & Sgardelis (1997), coupling and oviposition are more or less con- temporaneous within populations of land snails living in coastal habitats, where the suitable period is relatively short, whereas breeding lasts longer for the inland species. Perhaps egg laying period can be prolonged 41 LIFE HISTORY IN ALBINARIA A coerulea Анерош % months A. tumta months A. discolor months A. voithii Jul months FIG. 4. Fluctuations of mortality ratio (dead specimens/(dead + living specimens)) for the studied populations of Albinaria (black bars stand for total, hatched bars for adults, and white bars for juveniles). GIOKAS & MYLONAS 42 at an a ne ee ne a AER E ee Se es «810 + 4781 =9 „УЕ’0 = 6651 =4 ДР = ‚Ho = ELS'E=4 20.8 ‚sro + y29'1 =9 8v6'0=8 ¿120 = 6691 = q 1860 =e + + + a „090 «VEO i= + + 880 =Ee r003=4 ZO b= AA G/80=E ¿coc = 4 c80=E «130 «980 PAS) „950 + + TS + rss, =q 86`0 =в Lec =4q 270 be soss=q \r2’0=Ee v80's = 4 r80=e «610 +0€'0 EE 0 (saliueanf) реэа (synpe) реэа (2101) реэа + q + (sajlusan) элиу (seluean) реа (sinpe)peog ___ (ею) рееа (senueandanıy — (smpejomy (PRO ONY .£t 0 + €/0 © = 9 £0/'0=8 «900 + pco'E = ро =е 890 + 15/'0 =9 86 7 =k «900 + 98d = 4 8670 = e (synpe) any 960 7 210 =а 6990=E yon Y «¿90 + v1493=4q 6/&0=2 4JOJOISIP “Y sv 0 + ZI =4 8321 =е PUN] “y x00 0' = ZZE°¢ =4 soro=e eajnla09 “y (1R10}) SAI ‘(S0'0 > d ayesıpu! , ‘78 +) me] лемод злое! 10 q pue e злэешелед uoneindod эцу о} Buipiooe selseds eueurgyy paıpms ay, Jo чоцпаще!р тееа$ "y FIAWL LIFE HISTORY IN ALBINARIA 43 under favourable conditions for the survival of the juveniles. Giokas (1996) reports that A/bi- naria specimens collected during aestivation laid eggs during captivity in the laboratory without copulation (among them A. coerulea and A. voithii), suggesting that sperm or fer- tilised eggs can be retained as in other land snails (Duncan, 1975; Tompa, 1984). Mean values of density (Table 2) are high and comparable with those reported for other clausillids. For example, Vestia elata has 400 specimens/m?, with a maturation period of five years (Piechocki, 1982), and Balea per- versa has 70 specimens/m”, with a matura- tion period of two years (B. Baur & A. Baur, 1992). The Israeli Alopiinae land snail Crista- taria genezarethana (Heller & Dolev, 1994) has a mean density of 150-200 individu- als/m?. Heller & Dolev (1994) attribute those high densities to the presence of crevices, the absence of predators and competitive spe- cies, and to the snails’ small size. Additionally, they consider that low copulation frequencies, as a response to lack of food, and growth in- hibition (11 years to mature), as a response to high population density through limited activ- ity (prevention of simultaneous demand for food, lichens with extremely low growth rate, in quantities that the rock surface may not be able to supply) or competitive action of adult specimens, may regulate population size. However, studies of resource and interfer- ence competition in land snails have resulted to argumentative outcomes (Pearce, 1997). These particular density-dependent mecha- nisms suggested by Heller & Dolev (1994) were not proved to act in Albinaria, in which (Schilthuizen, 1994; Giokas, 1996): copula- tion is synchronised, maturation comes after 1.5-2 years, Albinaria specimens are of the same size (on average 18 mm height) as Cristataria genezarethana, have the same diet preferences and similar activity pattern, and food (lichens and bryophytes) was al- ways abundant (however only a portion of specimens was active simultaneously). Addi- tionally, in neither case, at any time lag, higher mortality as a consequence of high density was observed. Therefore, in Albinaria intra- population competition for food and space or interference competition that would influence survival and abundance is still ambiguous (but of course needs further field and labora- tory examination). The abundance of the four studied Albinaria species fluctuated almost randomly through- out the sampling period, exhibiting no de- tectable trend, despite the stable oscillations of the environmental parameters. Lazaridou- Dimitriadou & Sgardelis (1997) have reported a similar pattern for Bradybaena fruticum and Eobania vermiculata. Though Lazaridou-Dim- itriadou & Sgardelis (1997) also demonstrated several examples of predictable phenological oscillation patterns, they claimed that the lat- ter are more evident for semelparous and short-lived species. Albinaria species are iteroparous and relative long-lived, as are Bradybaena fruticum and Eobania vermicu- lata, and population fluctuations could not be attributed directly to extrinsic environmental conditions. However, the role of intrinsic fac- tors remained unclear, in part because regu- lated life cycles are often difficult to distinguish from random density fluctuations (May, 1975; Cook, 1990), or because our sampling tech- niques failed to estimate with accuracy popu- lation dynamics. Density estimates of several Albinaria populations (Giokas, 1996) con- firmed the established notion (Lazaridou-Dim- itriadou & Sgardelis 1997) that population densities of different snail species may differ considerably, and that even populations of the same species do not always exhibit a peak density at the same time of the year. Mean estimated mortality percentage was generally low (Table 3) yet higher than the es- timation of 5% for Cristataria genezarethana (Heller & Dolev, 1994) and the estimation of 2.5% for marked aestivating specimens of A. discolor (Giokas et al., 2000). However, higher mortality did not coincide with aestiva- tion period (Fig. 4). Besides sampling defi- ciencies, we can suggest that these rock- dwelling snails do not suffer considerably during the adverse hot and dry period, taking into account the tendency of Albinaria speci- mens to form clusters near deep and narrow crevices, the thickness and the white colour of the shell, and the protective function of the clausilium. However, high mortality, especially of adult specimens, was usually reported after hatching (Fig. 4). Probably this indicates that a major mortality cause is weakening after parental investment. Lazaridou-Dimitriadou (1995) came to a similar conclusion for Heli- cella pappi. Juveniles also exhibit high mor- tality after hatching, probably because of low intolerance. Albinaria turrita and A. discolor juveniles did not exhibit significantly higher mortality than adults, even though A. discolor juveniles show higher mortality than adults. Heller and Dolev (1994) state the same for Cristataria a GIOKAS & MYLONAS genezarethana, whereas A. Baur (1990) re- ports higher mortality for the juveniles of Balea perversa. That was unexpected, be- cause Albinaria juveniles lack some internal shell morphological features (lamellae and door like clausilium) that may prevent desic- cation (Gittenberger & Schilthuizen, 1996), even though their significance is disputed (Arad et al., 1995; Giokas, et al., 2000). Per- haps aggregation and crevice-dwelling can be in some cases important for juvenile sur- vival, even when juvenile mortality is higher than adult mortality (A. coerulea and A. voithii). The contagious spatial distribution of the four studied Albinaria species conformed to the general attitude of the xero-thermophilic species (Lazaridou-Dimitriadou, 1981). This behaviour seems to be important for rock- dwelling snails (Arad et al., 1995), and espe- cially for Clausiliidae (Heller & Dolev, 1994; Arad et al., 1995), and Albinaria (Warburg, 1972; Kemperman & Gittenberger, 1988; Schilthuizen & Lombaerts, 1994), as in this way loss of water and predation is inhibited. However, the fluctuations and differences of the contagious spatial distribution are not eas- ily explainable given the problems that con- cern the sampling procedure and the indices used. Nevertheless, higher contagious distri- bution could possibly be attributed to the frag- mentation of the biotopes and the presence of crevices. Parameter b of Taylor's Power Law had higher values on Aigina and Vravrona. Rocks on the site of Aigina are scattered, and on Vravrona crevices are very abundant. On the contrary, on Kea and Parori the suitable and favourable rock habitats are united, and there is a relative lack of crevices. Unfortunately, the relatively instant picture, on which we had to rely, as in most short-term life-history studies, does not allow us to fore- cast, because on investigating the effect of in- trinsic and extrinsic factors we have to con- sider several aspects that prevail or change in time and space. Consequently, some popula- tion characteristics, such as density, mortality, and spatial distribution, cannot be predicted per se, and we must be skeptical about fore- casting. ACKNOWLEDGMENTS We are grateful to M. Lazaridou-Dimitri- adou, from the Aristotle University of Thessa- loniki, for her helpful comments, to the editor and two anonymous referees for their con- structive suggestions, and to Stephen Roberts for grammatical suggestions. LITERATURE CITED ANASTASAKIS, G. C. & M. DERMITZAKIS, 1990, Post-Middle-Miocene paleogeographic evolution of the Central Aegean Sea and detailed. Quater- nary reconstruction of the region. 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MALACOLOGIA, 2002, 44(1): 47-105 THE EASTERN PACIFIC RECENT SPECIES OF THE CORBULIDAE (BIVALVIA) Eugene V. Coan Department of Invertebrate Zoology & Geology,' California Academy of Sciences, Golden Gate Park, San Francisco, California 94118-4599, U.S.A.; gene.coan @sierraclub.org ABSTRACT There are 18 Recent species of the Corbulidae in the eastern Pacific, of which one has been introduced from the northwestern Pacific. Division of Corbula into additional genera is premature without new characters and a formal cladistic analysis. Seven subgenera are utilized, with six species remaining in Corbula, s. |. Three new species are described: C. (Caryocorbula) otra, C. (Varocorbula) grovesi, and Corbula ($. |.) colimensis. One neotype and 14 lectotypes are desig- nated. The distributions and habitats of the species are documented, along with their fossil oc- currences and the relationships to other Recent and fossil species. Key words: Corbulidae, Corbula, eastern Pacific, western Atlantic. INTRODUCTION Previous Treatments Deshayes (1845-1848: 212-237, pls. 20, 21) described and illustrated the anatomy of Corbula gibba (Olivi, 1792) (as “C. stri- ata” Fleming, 1828, ex Walker ms, one of its synonyms) and Lentidium mediterraneum (Costa, 1829). White (1942) described but did not figure the pericardial area of C. gibba, and Yonge (1946) discussed the anatomy of C. gibba (without reference to Deshayes’ earlier treatment). Morton (1986) discussed the biol- ogy and functional morphology of C. crassa Reeve, 1843, at the same time questioning whether the Corbulidae are properly placed in the Myoidea. However, the only molecular phylogenies thus far published that have in- cluded a corbulid closely ally Corbula to Mya (Adamkewicz et al., 1997; Steiner & Hammer, 2000). Important early papers describing a number of species of Corbula at the same time are those of G. B. Sowerby | (1833), Hinds (1843), and C. B. Adams (1852a, b). Reeve (1843-1844) published the only comprehen- sive monograph on Corbula. Tryon (1869) listed the then-known species. Lamy (1926) discussed the species of Lamarck, and then gave synonymies for many of the known species (Lamy, 1941). Dall (1898, 1900) discussed the genera of the family in connection with his review of the fossil species of the eastern United States. Vokes (1945) and Keen (1969b) also covered the genera of the family. Zhuang & Cai (1983) treated the species of China, and Habe (1977: 280-284) those of Japan. Anderson (1994, 1996) discussed the species of the Neogene of the Dominican Re- public, and showed that the eastern Pacific corbulids now average larger than those of the western Atlantic (Anderson, 2001). McLean (1942) discussed sculptural differ- ences between very inequivalve and less in- equivalve species of Corbula. Bacesco et al. (1957) and Gomoiu (1965) treated popula- tions of Lentidium mediterraneum (Costa, 1829), and Hrs-Brenko (1981) those of Cor- bula gibba. Lewy & Samtleben (1979), de Cauwer (1985), Morton (1986), Anderson (1992), Harper (1994), and Kardon (1998) discussed predation of corbulids and the role of the thick layer of conchiolin in combatting it. A preliminary outline of the results of the present study is given in Coan & Skoglund (2001). Format In the following treatment, each valid taxon is followed by a synonymy, information on type specimens and type localities, notes on distribution and habitat, and an additional dis- cussion. Mailing address: 891 San Jude Avenue, Palo Alto, California, 94306-2640, U.S.A.; also Research Associate, Santa Barbara Museum of Natural History and Los Angeles County Museum of Natural History. 48 COAN The synonymies include all major accounts about the species, but not most minor men- tions in the literature. The entries are arranged in chronological order under each species name, with changes in generic allo- cation from the previous entry, if any, and other notes given in brackets. The distributional information is based on Recent specimens | have examined, except as noted. Fossil occurrences are taken from the literature, except as noted. References are provided in the Literature Cited for all works and taxa mentioned. Abbreviations The following abbreviations are used in the text: AMNH, American Museum of Natural History, New York, New York, USA; ANSP, Academy of Natural Sciences of Philadelphia, Philadelphia, Pennsylvania, USA; BMNH, British Museum (Natural History) collection, The Natural History Museum, London, Eng- land; BMSM, Bailey-Matthews Shell Museum, Sanibel, Florida, USA; CAS, California Acad- emy of Sciences, San Francisco, California, USA; ICZN, International Commission on Zo- ological Nomenclature; LACM, Natural His- tory Museum of Los Angeles County, Califor- nia, USA; MCZ, Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts, USA; SBMNH, Santa Bar- bara Museum of Natural History, Santa Bar- bara, California, USA; UCMP, University of California Museum of Paleontology, Berkeley, California, USA; UMML, University of Miami Marine Laboratory, Rosenstiel School of Ma- rine and Atmospheric Sciences, Miami, Florida, USA; and USNM, United States Na- tional Museum collection, National Museum of Natural History, Smithsonian Institution, Washington, DC, USA. The eastern Pacific Corbulidae in the pri- vate collections of Carol C. Skoglund, Phoenix, Arizona, USA; and Kirstie L. Kaiser, Puerto Vallarta, Jalisco, Mexico, were also ex- amined. Morphological Characters In spite of the morphological plasticity of corbulids, there are a number of characters that are useful in distinguishing among the species. Some of these characters are sum- marized in Table 1. Overall shape is a useful criterion, with some species oval, some trigonal, and some other shapes. All corbulids are inequivalve, with the right valve larger than the left, which fits into it, overlapping most conspicuously posteroventrally. Most eastern Pacific species are only slightly inequivalve, and only three very inequivalve. Some species may become thick-shelled as adults, whereas others are never greatly thickened. The anterior end is rounded in all species, but more broadly in some and more sharply in others. The shape of the posterior end is more characteristic of each. In some, it may be extended by a short, shelly “spout” (e.g., Figs. 2, 3), but this may be present in only some specimens; other species never have a spout. A useful charac- ter is the nature of the division between the central and posterior slopes. In some, it is de- lineated by a carina, a ridge, a change in angle, and/or a change in sculpture; in others, is it scarcely set off. Similarly, a narrow, elon- gate escutcheon of different degrees of prominence may be present; it is generally widest and more evident in the right valve. Sculpture, while variable in some species, can nonetheless be of diagnostic value. Com- marginal sculpture predominates, but radial ribs are present in some taxa. Color provides a useful character for several species. In some taxa, the hinge plate is broad, in others narrow. In general, the conspicuous tooth in the right valve is too variable in shape to be a useful diagnostic tool. The resilifer in the right valve may be visible on the hinge plate, or recessed beneath it. The chon- drophore in the left valve may be very project- ing or only slightly projecting, and it may be conspicuously divided into sections in some. At its posterior margin, a small tooth may be present, which varies in prominence among species. (It articulates on the posterior side of a small tooth in the resilifer of the right valve.) The pallial line, and its sinus, if present, have informative characters, as shown in the draw- ings (Figs. 40-57). A small sinus is evident in some taxa, not in others; in some, a small posterior extension is visible at the pos- teroventral corner of the pallial line (e.g., Fig. 40). SYSTEMATIC ACCOUNT Family Corbulidae Lamarck, 1818 Lamarck, 1818: 493, as “corbulidees” (ac- cepted under ICZN Code, 1999: Art. 11.7.2) 49 EASTERN PACIFIC CORBULIDAE L2G‘? :ре!рп]$ SIO] lEJOL squ anbijgo ‘ге|эше] pal ‘MO| ‘eul} — A] :SUOIjE] JEL 902 Ajuo sejiueanf ul эбри рэрипол -19Â о} энум -npun yeulBsewWwoo— AH jeuoß1-syeno Kian esomads ‘9 pero yeulBseuwoo 662 2:8 молеи чиэрмэ эбри рэрипол ‘зпомел бицеприп ‘эзелэрош ajelpenbqns-ajeno Ánubis ввоицивш'Э 181 c'OL }иэрмэ эбри рэрипол quid 10 энум геиблешиоэ eu, ajeno ÁnyBis O) cg geL эбри Aq рэццер эбри dıeys энум гемблешииоэ Buons syeipenbans-syeno ÁpuybBis вл I 8 O'pL peuyap Амеэм эбри pepunoi sıym Jjeulßlewwoo э]елэрош ajeno ARUBIS sisuauwjo9 9 QWOS Ul энум yeulBuew 82 8'02 эбри Aq peulep эбри dıeys 'sauoz 10J09 = - WOO Ul} о} э]елерош ajeBuoja-ajeno — Aus вдерелд ‘7 ешблешиоэ au — A] нешблецщииоэ Aneay 85 Je] juapina JOU эбри рэрипол OJUM — AH ;sieipel— syeaq эуело Kian взадо 9 SJeIpe, jure, ‘YJOOLUS — A] L Oa juasald uoneledas ou anym чешблешиоэ au — AH эуело-[ецоби} Алэл IS@AOIB ‘7 05 S bz 189 Aq pauyap эбие dıeys энум еиблешиоэ эиц ayeupenbqns-ayebuoja = Апцчбиз sinua] 'I Mu с 161 juapina jou ajBue ‘AH ul jou auuym yjoows ajeno Арэроэр = sisuaunwe ‘7 8 0'S€ jsou и! juapina эбри мо] эиум еиблешиоэ aul jeuoß1 oy ajeno ÁpnyBis PBSOOLIJUBA 'I эбри 152 0er dieus Aq pouyap эбри dıeys alum jeulßlewwoo эуелэроц syeipenbans-syeno Ayßils gJeuneaig ‘7 suolyejnpun 9 G22 эбри Aq peuyap эбри dıeys энум еиблешишоэ peoiq ajebuoja-ajeno Anubis — ер/елэшза 9 эбри 89 2:01 dueys Aq peuyap эбри Buoys auym jeulbsewwoo эуелэрош ajeipenbqns-ajeno ÁpnuBis ejjsa1od ‘Dd 89 262 эбри Aq peuep эбри мо] эиум гемблешиоэ э]елэрош ajebuoja-ajeno AnuBls вепло 9 Aeıpau 101 L'9c рэицер Амеэм эбри рэрипол ajdind Геиблешишоэ э]елэрош ajebuoja-ajeno Anubis eno ‘7 6/8 ysl эбри Aq peulep эбри sıym гешблешиоэ э]елэрош эуебио|э-э]ело Ajlybijs eınseu 9 19 8'0€ эбри mo] Аа pauljep эбри eye1epouu ajdind ¡euibreuwuwoo э]елэрош ¡euoBuy-ajeno Anubis eunsayi4ue ‘9 paıpnıs sjo| ww uosyamos3 sado¡s Jola3sod 10[09 aın]dinas adeys ssauaNnjen ‘08d '3 "ON ‘azis 'xeyy я |211U99 UBEMJEq [eulajx3 -ınbauj uoneledes BINQIOD эщоед Wiaysez Jo Aouenbe14 pue ‘ezis ‘suajoeieyD Bunenueleyiq Aey “| 3719vL 50 COAN Two Recent subfamilies are current recog- nized, the Corbulinae and the Lentidiinae Vokes, 1945 (pp. 6, 23-24), the latter con- taining only the living genus Lentidium, which is briefly discussed below. Subfamily Corbulinae Lamarck, 1818 Genus Corbula Bruguiere, 1797 Corbula Bruguiere, 1797: pl. 230, genus with- out named species (ICZN Code, 1999: Art. 12.2.7); description by Lamarck (1799: 89); original list by Lamarck (1801: 237) (ICZN Code, 1999: Art. 67.2.2). Type species (subsequent designation of Schmidt, 1818: 77, 177): Corbula sulcata Lamarck, 1801: 137 (species based on Bruguiere, 1797: pl. 230, fig. 1a-c). Re- cent; west Africa. to be confused with Corbula Röding, 1798: 184-185, the type species of which (subsequent designation of Winck- worth, 1930: 15) is Corbula anomala Röding, 1798, a junior synonym of Venus deflorata Linnaeus, 1758: 687. Corbula Röding is thus a synonym of Asaphis Modeer, 1793 (pp. 176, 182), a member of the Psammobiidae (Keen, 1969a: 633; Willan, 1993: 5-6) Aloides Megerle von Mühlfeld, 1811: 67 Type species (monotypy): A. guineensis Megerle von Mühlfeld, 1811: 67; = Cor- bula sulcata Lamarck, 1801, making Aloides an objective synonym of Corbula. The genus Corbula Bruguiere was long a source of nomenclatural confusion, and a number of authors, lacking key pieces of liter- ature as well as the modern nomenclatural rules, attempted to sort out the taxonomic tan- gle (Vokes, 1945; J. Q. Burch, 1960). The for- mula given above represents the consensus under the current rules. Corbula sulcata, which occurs from Mauri- tania to Angola, is large, heavy, and sube- quivalve. It has heavy commarginal sculpture in the right valve and finer commarginal sculp- ture in the left valve. Its posterior end has two radial ridges, one between the central and posterior slopes and another defining a broad escutcheon. The hinge is very heavy. None of the Recent species in the New World is closely similar to the type species of Corbula. There are a bewildering array of specific and generic taxa in this subfamily, with char- acter sets that do not covary. Many ofthe gen- era were established based mostly on single characters, such as Anisocorbula Iredale, No — 1930 (p. 404), for an elongate species with a sharp posterior keel, Solidicorbula Habe, 1949 (p. 2), for a thick-shelled species, and Minicorbula Habe, 1977 (p. 282), for a small- sized species. Subsequent authors have at- tempted to shoehorn every species into vari- ous subgenera, no matter how uncomfortable the fit. Efforts to make a meaningful arrange- ment of genera and subgenera will be fraught with inconsistencies until additional charac- ters become available and are rationally eval- uated. In light of this, elevating subgenera to genera, as many authors have done (includ- ing by Coan et al., 2000: 478-480), is prema- ture. Here, a conservative effort has been made to group similar species under some of the named subgenera and leaving several species in Corbula, s. 1, but even these groupings should not be given much weight until a full-scale revision of the family is un- dertaken. Grant & Gale (1931) placed Corbula luteola and C. porcella in Corbula (Lentidium), and the first assignment was followed by some other authors. The type species by monotypy of Lentidium Cristofori 4 Jan, 1832 (De- scrizione, p. 9; Disposito, p. [ii]; Conchylia ter- restria, p. 8; Mantissa, p. 4), is Tellina mediter- ranea Costa, 1829 (pp. 14, 26-27, 131, pl. 1, fig. 6), which is small, thin, shiny, translucent, and shaped like a Tellina. It is sufficiently dif- ferent from other corbulids that it has been placed in a separate subfamily, the Lentidi- inae Vokes, 1945 (p. 23). No eastern Pacific species is similar. Lamy (1941) placed Corbula fragilis and C. luteola in Corbula (Corbulomya). Corbulomya Nyst, 1845 (p. 59), has as its type species (subsequent designation of Herrmannsen, 1847: 308) Corbula complanata J. Sowerby, 1822 (p. 86, pl. 362, figs. 7, 8), from the upper Pliocene of England. This genus is now re- garded as a synonym of Lentidium (Vokes, 1945: 23, 26; Glibert & van de Poel, 1966: 58; Keen, 1969b: 696). The eastern Pacific Corbulidae include the following two species (or species complexes) of formidable morphological diversity, which cannot be fully resolved without new lines of evidence not available in the current study: (1) Corbula (Caryocorbula) nasuta is by far the most common species in collections, and it occurs in enormous populations on soft- bottoms in shallow-water. Individuals of this species can attain a fairly large size, but most populations contain small individuals. Al- EASTERN PACIFIC CORBULIDAE 51 though there is a wide range in morphologies, there is as yet no reliable evidence that more than one species is present, and specimens of intermediate morphologies can be found. (2) Corbula biradiata, while not having as much morphological plasticity as C. nasuta, varies considerably with respect to the posi- tion of its beaks, the strength of its commar- ginal sculpture, and its color. Corbulids are not only abundant, but there are many collection lots in alcohol or with dried animals. Thus, the group would be a prime candidate for biochemical genetic stud- ies, not only to test for possible additional di- versity at the species level but also to under- stand phylogenetic relationships in the family as a whole. Subgenus (Caryocorbula) Gardner, 1926 Caryocorbula Gardner, 1926: 46 Type species (original designation): Corbula alabamiensis Lea, 1833 (reference below, under C. nasuta Discussion). Eocene, eastern United States. Serracorbula Olsson, 1961: 433 Type species (original designation): S. tu- maca Olsson, 1961, = Corbula nasuta С. В. Sowerby I, 1833 (references below) Small to moderate in size, subequivalve, subequilateral, with a strong to weak ridge be- tween central and posterior slopes. Sculpture of strong to weak commarginal ribs, similar on both valves. Specimens of some species with “marginal” ribs along the ventral and dorsal margins; these are further discussed below. | include five eastern Pacific species in this subgenus. Other authors have placed addi- tional species here as well. Lamy (1941) placed Corbula nasuta in Cor- bula (Cuneocorbula). Cuneocorbula Coss- mann, 1886 (p. 49 [reprint: 37]), has as its type species (subsequent designation by Dall, 1898: 836) Corbula biangulata Deshayes, 1857 (p. 231, pl. 13, figs. 19-23).? This species, from the Paleocene of France, is thin, elongate, and birostrate. “Glibert & van de Poel (1966: 55) renamed this species Cuneocorbula pelseneeri on the grounds that it was a junior homonym of “Corbula biangulata Sowerby, 1833,” but there is no such senior homonym. They must have misread either Corbula bicarinata G. B. Sowerby I, 1833, or C. biradiata G. B. Sowerby I, 1833. Corbula (Caryocorbula) amethystina (Olsson, 1961) Figures 1, 39 Caryocorbula (Caryocorbula) Olsson, 1961 Olsson, 1961: 431, 548, pl. 75, fig. 1-1c; Keen, 1971: 262, 264, fig. 674 [Corbula (Caryocorbula)] amethystina Type Materials & Localities ANSP 218902, holotype, pair; length, 27.6 mm; height, 18.1 mm; width, 13.8 mm (Fig. 1). “Tortutilla” [? = Isla Tortolita, Panamä Pro- vince], Panama (8.8°N); H. B. Johnson, 1958. In the plate explanation, Olsson gives the lo- cality as [Isla] “Taboguilla,” but both the text and label with the specimen say “Tortutilla”. UMML 30.11391, paratype, right valve; Puerto Mensabe, Los Santos Province, Panama. UMML 30.11368, paratype, left valve; San Carlos, Panamä Province, Panama. UMML 30.11389, paratypes, right valve, left valve; San Carlos, Panamä Province, Panama. UMML 30.11390, paratypes, 3 right valves, 2 left valves; El Lagartillo, Panamä Province, Panama. UMML 30.11388, paratype, right valve; Isla Gibraleön, Archipielago de las Per- las, Panamä. UMML 30.11326, paratype, left valve; Punta Cocos, Isla del Rey, Archipielago de las Perlas, Panama. UMML 30.11390, paratype, left valve; Puerto Callo, Manabi Province, Ecuador. UMML 30.11388, para- type, right valve; Santa Elena, Guayas Pro- vince, Ecuador. Description Ovate-trigonal, thick; right valve slightly larger than left; posterior end longer (beaks 37-43% from anterior end); anterior end rounded; posterior end pointed, slightly ex- tended by a spout in some specimens; poste- rior slope set off from central slope by a mod- erately sharp ridge that extends to the ventral margin. Escutcheon defined by a low ridge. Beaks with fine commarginal sculpture. Central slope with moderate commarginal ribs and much finer commarginal striae. Exterior color light to dark purple; interior color purple around margins, brown in some. Hinge plate broad; right valve with a large tooth; left valve with a broad, slightly projecting chondrophore and a small tooth. Posterior end of pallial line with small posterior extension (Fig. 39). 52 COAN FIG. 1. Corbula amythestina, holotype; ANSP 218902; length, 27.6 mm. FIG. 2. C. nasuta. Lectotype of C. nasuta; BMNH 1966565/1; length, 17.6 mm. EASTERN PACIFIC CORBULIDAE 53 Length to 30.8 mm (SBMNH 345487; Playas [de Villamil], Guayas Province, Ecuador). Distribution Mazatlan, Sinaloa (23.2°N) (UCMP E.8424; CAS 121790, 121793; LACM 152689, 152690, 63-11.78; SBMNH 126540), and La Paz, Baja California Sur (24.2°N) (CAS 121786; SBMNH 345503 [“Punta Coyote,” probably one of the two in this vicinity]), Mex- ico, to Playas [de Villamil], Guayas Province, Ecuador (2.6°S) (SBMNH 345487). It has been found living from the intertidal zone to 82 m (mean, 17.2 m; n = 27), on sand bottoms. | have seen 67 eastern Pacific lots, including the types. A single lot labeled as having come „from the western Atlantic at Islas Los Roques (as “Las Rochas 1$.”), Venezuela (BMSM 15008), seems improbable (R. Cipriani, e- mail, 30 Nov. 2000) and probably represents a labeling error. Discussion Corbula amythestina merits comparison with C. dominicensis Gabb, 1873b (p. 247), from the Miocene of the Dominican Republic (concerning the latter: Anderson, 1996: 14-15, pl. 1, figs. 9, 12, pl. 2, figs. 1, 2, 4, 5). Corbula (Caryocorbula) nasuta G. B. Sowerby I, 1833 Figures 2-7, 40 Corbula nasuta G. B. Sowerby |, 1833 G. B. Sowerby |, 1833: 35; Reeve, 1843: pl. 1, fig. 1; d’Orbigny, 1845: 571; Car- penter, 1857a: 183, 228, 300; 1864b: 537 [1872 reprint: 23]; Tryon, 1869: 65; Lamy, 1941: 233 [Corbula (Cuneocorbula)]; Hertlein & Strong, 1950: 240, 252, pl. 2, fig. 9 [Aloidis (Caryocorbula)]; Hertlein & Strong, 1955: 205-206; Soot-Ryen, 1957: 11; Keen, 1958: 209, fig. 527 [Cor- bula (Caryocorbula)]; Olsson, 1961: 429-430, 548, pl. 75, fig. 3-3e [Cary- ocorbula (Caryocorbula)]; Keen, 1971: 265-266, fig. 677 [Corbula (Caryocor- bula)]; Gemmell et al., 1987: 59, figs. 72, 73 [as Corbula cf. nasuta] Corbula nuciformis G. B. Sowerby |, 1833 G. B. Sowerby I, 1833: 35; Reeve, 1843: pl. 2, fig. 9; Carpenter, 1857a: 183, 300; 1864b: 537, 668 [1872 reprint: 23, 154]; Tryon, 1869: 65; Lamy, 1941: 234 [Cor- bula (Cuneocorbula)]; Olsson, 1961: 430-431, 548, 549, pl. 75, figs. 7, 8; pl. 76, fig. 7 [Caryocorbula (Caryocorbula)]; Keen, 1971: 265-266, fig. 678 [Corbula (Caryocorbula)] First revision herein [NON Corbula nuciformis, auctt., which = C. obesa] Hertlein & Strong, 1950: 241, pl. 3, fig. 1 [Aloidis (Caryocorbula)]; Keen, 1958: 209, fig. 528 [Corbula (Caryocorbula)] [not to be confused with Corbulolumna nuci- formis Vokes, 1945: 9-11, pl. 2, figs. 5-8, from the Cretaceous of Lebanon.] Corbula fragilis Hinds, 1843 Hinds, 1843: 56; Reeve, 1844: pl. 3, fig. 13; Hinds, 1845: 68, pl. 20, fig. 11; Car- penter, 1857a: 207, 300; Tryon, 1869: 64; Lamy, 1941: 240 [Corbula (Corbu- lomya)]; Hertlein 8 Strong, 1950: 243- 244 [Aloidis (Tenuicorbula)]; Keen, 1958: 210, 211, fig. 537; Olsson, 1961: 430 [as a synonym of Caryocorbula nasuta]; Keen, 1966: 268, pl. 4, fig. 3 Corbula alba Philippi, 1846 Philippi, 1846: 19; Carpenter, 1857a: 224, 244; 1857b: 534, 547 [as asynonym of Corbula bicarinata]; Tryon, 1869: 63 Corbula pustulosa Carpenter, 1857 Carpenter, 1857a: 244, 300 [nomen nudum]; 1857b: 22-23; 1864a: 368 [1872 reprint: 204]; 1864b: 553 [1872 reprint: 39]; Tryon, 1869: 65; Lamy, 1941: 143; Hertlein & Strong, 1950: 240 [as a synonym of Aloidis (Caryocorbula) na- suta]; Palmer, 1951: 13; Brann, 1962: 29, pl. 4, fig. 32; Keen, 1968: 402, pl. 56, fig. 25 [as a synonym of Corbula nasuta] Serracorbula tumaca Olsson, 1961 Olsson, 1961: 433, 549, pl. 76, fig. 4-4d; Keen, 1971: 268-269, fig. 690 [as Cor- bula (Serracorbula)] Type Materials & Localities Corbula nasuta-BMNH 1966565/1, lecto- type here designated, open pair, the speci- men closest to the original length measure- ment; length, 17.6 mm; height, 11.5 mm; width, 12.0 mm (Fig. 2). BMNH 1966565/2-4, paralectotypes: closed pair, length, 18.4 mm; closed pair labeled “4”, length, 16.0 mm; open pair, length 15.3 mm, perhaps the specimen figured by Reeve (1843).”Xipixipi” [Jipijapa; Puerto de Cayo], Manabi Province, Ecuador 54 COAN FIGS. 3, 4. Corbula nasuta. FIG. 3. Lectotype of C. nuciformis; BMNH 1966566/1; length, 13.4 mm. FIG. 4a. Lectotype of C. fragilis; BMNH 1966234/1; length, 7.3 mm. FIG. 4b. Paralectotype of C. fragilis; BMNH 1966234/2; length, 6.4 mm. (1.3°S); 10 fms. [18 m], sandy mud; Hugh Cuming. G. B. Sowerby | (1833) also reported some small specimens, tentatively assigned to this species, from the Golfo de Nicoya, Puntarenas Province, Costa Rica. Corbula nuciformis-BMNH 1966566/1, lectotype here designated, open pair, la- beled “6”, probably the specimen figured by Reeve (1843); length, 13.4 mm; height, 8.4 mm; width, 9.4 mm (Fig. 3). ANSP 50875, possible paralectotypes, 2 small, closed pairs. “Real Llejos” [Rio Realejo; Corinto], Chinan- dega Province, Nicaragua (12.5°N), 6 fms. [11 m], sandy mud; Hugh Cuming. G. B. Sowerby EASTERN PACIFIC CORBULIDAE 55 | (1833) also cited fossil material from near Guayaquil, Guayas Province, Ecuador. Corbula fragilis- 1966234/1, lectotype here designated, right valve; length, 7.3 mm; height, 4.3 mm; width, 1.9 mm (Fig. 4a). BMNH 1966234/2, paralectotype, left valve; length, 6.4 mm (Fig. 4b). A larger specimen cited by Hinds (1843) is not in the BMNH. Ve- ragua[s] Province, Panama (approximately 7.7°N), 18 fms. [33 m]; Edward Belcher. Corbula alba—Presumably lost. The origi- nal specimen measured 13.0 mm in length, 8.2 mm in height, and 7.6 mm in width. Mazatlan, Sinaloa, Mexico (32.2°N); Kinder- man. Corbula pustulosa-BMNH 1857.6.4.77, lectotype (Keen, 1968: 402), closed pair mounted on a glass slide; length, 4.2 mm; height, 3.2 mm; width, approximately 2.0 mm (difficult to measure because of being glued to the slide) (Fig. 5). USNM 715647, paralecto- type, left valve (glued to a glass slide); length, 3.4 mm. Mazatlän, Sinaloa, México (32.2°N); Frederick Reigen. Serracorbula tumaca—ANSP 218948, lec- totype here designated, right valve; length, 12.4 mm; height, 8.4 mm; width, 5.4 mm (Fig. 6a). Of the material that had been in the ANSP lot labeled “holotype,” this valve comes clos- est to the holotype measurements in Olsson’s text, but it does not match any of his illustra- tions labeled “holotype.” Olsson’s fig. 4, 4b, and 4c corresponds to a paralectotype pair measuring 12.0 mm in length (ANSP 405291) (Fig. 6b). His fig. 4d is a paralectotype left valve measuring 11.8 mm in length (also in ANSP 405291). The left valve shown in his fig. 4a was not present in the lot. Tumaco, Narino Province, Colombia (1.8°N). The ANSP lot also contained a paralectotype right valve from San Miguel, Isla del Rey, Archipielago de las Perlas, Panamä (now ANSP 405292). No type material was located in the Olsson col- lection in the UMML. Description Ovate-elongate, heavy; right valve slightly larger than left; longer posteriorly to slightly longer anteriorly (beaks 34-51% from ante- rior end). Posterior end somewhat pointed ventrally, sometimes extended by a spout. Posterior slope set off by a well-defined ridge, often with a slight radial sulcus just anterior to it. Ventral margin sometimes growing medially to form an inflated, flattened ventral surface. Margins, particularly the ventral margin, sometimes with “marginal”? ribs, resulting in a serrate edge. Escutcheon defined by a ridge, more evident in right valve. Beaks relatively smooth, with fine, pustules. Sculpture on central and posterior slopes of closely spaced moderate commarginal ribs and fine, radially arrayed pustules. Periostracum tan. External color white; in- ternal color white, but yellow or tan in some specimens. Hinge plate heavy; right valve with a large tooth; left valve with a broad chon- drophore, projecting in some, less in others; tooth small (Fig. 40). Length to 18.4 mm (a paralectotype of C. nasuta). Distribution Isla Natividad, Baja California Sur (27.9°N) (SBMNH 345488); Bahia Magdalena, Baja California Sur (24.5°N) (CAS 121538), into and throughout the Golfo de California to its head at Puerto Peñasco, Sonora (31.4°N) (UCMP E8431; CAS 115324, 121917, 121919, 122072; SBMNH 21350, 116514, 119426; USNM 212789, 707996; Skoglund Collection; and many other lots), Mexico, to Callao, Lima Province, Peru (12.1°S) (LACM 35-152.1, 35-153.1, 35-177.2); Isla del Coco, Costa Rica (SBMNH 345489, 345490, 345491; LACM 38-180.2); Isla Santa Cruz, Islas Galapagos, Ecuador (LACM 38-193.13); from the intertidal zone to 152 m (mean, 28.7 m; n = 328), on mud or sand. (A single valve with an indicated depth of 384 m—LACM 35- 177.2—is probably the result of drift from shallower water.) | have seen 875 eastern Pa- cific lots, including the types. Two lots labeled as having come from “Monterey, California” (USNM 21480, 58346), probably represent la- beling errors, and one of them may be the source of the Monterey record of C. fragilis by Dall (1921: 53) and Oldroyd (1925: 203). This species has also been recorded in beds of Pleistocene age at Puerto Penasco, Sonora (Hertlein & Emerson, 1956: 165), and at Bahia Magdalena, Baja California Sur (Jor- dan & Hertlein, 1936: 111, as “C. porcella” and as “C. fragilis’; CAS Loc. 754), México, in beds of Pliocene age of the Loreto Basin, ЗТпезе ribs appear along the outer ventral surface near the shell margins (Fig. 6a, b). Olsson (1961) referred only to “marginal serrations”. They are not radial ribs, because they do not radiate from the beaks. | settled on the term “marginal ribs” because they run laterally along the shell margin. 56 COAN FIGS. 5-7. Corbula nasuta. FIG. 5. Lectotype of C. pustulosa; BMNH 1857.6.4.77; length, 4.2 mm. FIG. 6a. Lectotype of Serracorbula tumaca; ANSP 218948; length, 12.4 mm. FIG. 6b. Paralectotype of S. tumaca; ANSP 405291; length, 12.0 mm. FIG. 7. SBMNH 345490; Bahia Chatham, Isla del Coco, Costa Rica; 46-69 m; largest specimen length, 5.0 mm. EASTERN PACIFIC CORBULIDAE 57 Baja California Sur, Mexico (Piazza & Robba, 1998: 238, 248, 260, as “C. nuciformis”), and in the middle to late Pliocene Canoa Forma- tion at Punta Blanca, Manabi Province, Ecuador (Pilsbry & Olsson, 1941: 11, 75). G. B. Sowerby | (1833) cited fossil material of unknown age from Guayaquil, Guayas Province, Ecuador. Discussion Corbula nasuta is the most variable of the eastern Pacific taxa. When small (< 10 mm), specimens are thin shelled, with conspicuous fine pustulose radial sculpture. This is the morphology named C. fragilis. As specimens grow, the ventral margin may soon become flattened, yielding the short, inflated morphol- ogy that was named C. nuciformis and C. pus- tulosa. The size at which material thickens and forms a flattened base varies greatly among populations, and this can occur in specimens as small as 5 mm. Corbula nasuta typically becomes rostrate posteriorly, and a short spout may be added in some speci- mens. In some large specimens, marginal ribs may be developed at the margins, especially the ventral margin. This is the form that was named Serracorbula tumaca. The type lots of both C. nasuta and C. nuciformis also contain specimens with such ribs. | believe that Corbula alba belongs here as a synonym in that Philippi’s description speaks of the posterior slope being acute and subrostrate, and because the two European Mio-Pliocene species with which he com- pared C. alba-C. revoluta (Brocchi, 1814: 516, 685, pl. 12, fig. 6 —orginally described as Tellina) and the closely related C. carinata Du- jardin, 1837 (p. 257)—are much more similar to C. nasuta than to C. bicarinata, with which it was previously synonymized. Some material from the northwestern coast of South America is shorter, more rounded, without much of a posterior sulcus setting off the posterior end, and it has finer sculpture than material from further north. A similar mor- phology is seen in material from Isla del Coco, Costa Rica (lots cited in the Distribution), which is uniformly small, rounded, and smooth (Fig. 7). This might be regarded as a separable species or subspecies, but given the variability of this species (or species com- plex) as a whole, it is premature to bestow ad- ditional names. Corbula nasuta is most similar to the later- named western Atlantic C. swiftiana C. B. Adams, 1852c (pp. 236-237), which has been reported from Massachusetts to Ar- gentina. Given the variability of these two taxa, | doubt that the two can be told apart on a morphological basis, and perhaps they should be synonymized until more evidence is available. Synonyms in the western Atlantic may include: C. kjoeriana C. B. Adams, 1852c (p. 237), C. barrattiana C. B. Adams, 1852c (pp. 237-238), C. chittyana C. B. Adams, 1852c (p. 238), C. fulva C. B. Adams, 1852c (pp. 240-241), С. caribaea d’Orbigny, 1853“ (p. 284 [Spanish ed., p. 323], pl. 27, figs. 5-8), C. lavalleana d’Orbigny, 1853 (p. 284 [Span- ish ed., p. 323], pl. 27, figs. 9-12), and C. uruguayensis Marshall, 1928 (p. 5, pl. 4, figs. 7-9). Records of C. nasuta from the western Atlantic, such as those of Dall (1889b: 70, pl. 2, fig. 6-6c, as С. nasuta “Say”) and Haas (1953: 204), were presumably based on C. swiftiana. Records of C. nasuta from Australia (Angas, 1867: 913, 1878: 869; Hedley, 1918: M31) were based on specimens of C. coxi Pilsbry, 1897 (pp. 363-364, pl. 9, figs. 1-3). Dautzenberg (1912: 99) reported Corbula nasuta Sowerby from several localities in west Africa. This African species was also de- scribed as С. lyrata Е. A. Smith, 1872 (р. 729, pl. 75, fig. 2), a junior homonym of С. Iyrata J. de C. Sowerby, 1840 (expl. to pl. 21), and Smith’s species was renamed C. dautzenberg Lamy, 1941 (pp. 235-236), the name cur- rently in use. Corbula nasuta Sowerby, 1833 (17 May), is not to be confused with Corbula nasuta Con- rad, 1833 (3 Sept.) (p. 38; unpublished pl. 19, fig. 4 [not “pl. 20, fig. 2,” as stated in text]), from the Middle Eocene Claiborne Formation of Alabama, for which the name Corbula al- abamiensis Lea, 1833 (Dec.) (p. 45, pl. 1, fig. 12), is now used (Palmer & Brann, 1965: 74). Corbula nasuta Conrad was later reported by Conrad (1857: 161, pl. 19, fig. 4) from the Ter- “Dall (1889b: 18), followed by Aguayo (1943: 38), maintained that the entire Atlas to d’Orbigny’s monograph on the mollusks of Cuba appeared in 1842, with the French text on the bivalves appear- ing between 1847 and 1853. Aguayo also thought that the entire Spanish text might have appeared in 1845; Keen (1971: 1006) dated the Spanish text as 1846. However, there is no evidence that any ofthe bivalves were published, in text or plates, and in ei- ther edition, before 1853, when citations to the species begin to appear in other works. 58 COAN tiary of western Texas, and this Texas mater- ial was later named C. conradi by Dall (1898: 842). Small specimens of this species commonly occur in shallow water with C. marmorata. These species may most easily be distin- guished as follows: nasuta marmorata Posterior end tapered, subquadrate, pointed pointed pos- posteriorly teroventrally Exterior color white mottled, with purplish blotches near beaks Interior color white magenta, espe- cially around margins The complex of nasuta-like taxa has been vastly overnamed in the Pliocene and Miocene of the western Atlantic region. A par- tial list of taxa that belong in this complex in- cludes: C. (Cuneocorbula) sarda Dall, 1898 (pp. 847-848; 1900: pl. 36, fig. 14); C. (C.) whitfieldi Dall, 1898 (p. 849; 1900: pl. 36, fig. 18); C. (Caryocorbula) whitfieldi stika Gard- ner, 1928 (pp. 232-233, pl. 35, figs. 8, 9); and C. (C.) whitfieldi boyntoni Gardner, 1928 (p. 233, pl. 35, figs. 10-13), from the Miocene of Florida; C. (Cuneocorbula) sericea Dall, 1898 (pp. 848-849; 1900: pl. 36, fig. 8) [synonyms: C. (C.) cercadica Maury, 1917: 396-397 [= 232-233], pl. 65 [= 39], figs. 16, 17; C. (C.) caimitica Maury, 1917: 395 [= 233], pl. 65 [= 39], figs. 18, 19] (concerning: Anderson, 1996: 15-17, pl. 2, figs. 7-21); C. (C.) hele- nae Maury, 1912 (p. 62, pl. 9, fig. 25) [syn- onyms: C. (C.) smithiana Maury, 1912: 63, pl. 9, figs. 29, 30; C. (C.) caribaea pergrata Maury, 1925: 255 [= 103], pl. 20, fig. 8; C. (C.) daphnis Maury, 1925: 256 [= 104], pl. 20, figs. 10-11] (concerning: Jung, 1969: 407-409, pl. 38, figs. 12, 13, pl. 39, figs. 1-9), from the Miocene and Pliocene of the Caribbean; Caryocorbula (Caryocorbula) prenasuta Ols- son, 1964 (p. 70, pl. 9, fig. 10), from the Miocene of Ecuador; C. oropendula dolicha Woodring, 1982 (p. 712, pl. 119, figs. 4-6), from the Miocene of Panama; Corbula (Cu- neocorbula) swiftiana harrisii Dall, 1898 (p. °Corbula conradi Dall is not the renaming of a homonym, as assumed by Boss et al. (1968: 88), but rather a new species based on Conrad’s mate- rial (USNM 9899). 855), from the Miocene of Texas; and C. inae- qualis Say, 1824 (p. 153, pl. 13, fig. 2), from the Pliocene of Maryland and Virginia [syn- onym: C. inaequalis mansfieldi Richards, 1947: 32, pl. 11, figs. 27, 31] (concerning: Campbell, 1993: 48, pl. 21, fig. 191). Corbula (Caryocorbula) otra Coan, new species Figures 8, 41 Corbula ovulata, auctt., in part, non G. B. Sowerby |, 1833 G. B. Sowerby |, 1833: 35-36 [material cited from Mazatlan, México, and Corinto, Nicaragua]; Hanley, 1843: 47, 4 (pl. expl.), pl. 10, fig. 52; 1856: 344; Reeve, 1843 [material cited from Mazatlan, México, and Corinto, Nicaragua]; C. B. Adams, 1852a: 522-523 [1852b: 298-299] [in part; the large pair cited from Isla Taboga, Panama]; Carpenter, 1857b: 23 [speci- men cited from Mazatlan]; Lamy, 1941: 127-128; Hertlein & Strong, 1950: 241, 252, pl. 2, fig. 11 [Aloidis (Caryocorbula)]; Hertlein & Strong, 1955: 206; Keen, 1958: 209, fig. 530 [Corbula (Caryocorbula)]; Olsson, 1961: 428-429, 548, pl. 75, fig. 2c [Caryocorbula (Caryocorbula)]; Keen, 1971: 265, 266, fig. 680 [lower right fig. only] [Corbula (Caryocorbula)] Type Material & Locality SBMNH 345493, holotype, pair; length, 22.8 mm; height, 13.6 mm; width, 12.0 mm, including portion of dried soft parts (Figs. 8, 41). SBMNH 345494, paratypes; open pair, length, 23.3 mm; closed pair, length, 21.0 mm; right valve, length, 21.5 mm. Manzanillo, Colima, México (19.1°N, 104.3°W); 30-45 m; Carl & Laura Shy; ex Skoglund Collection. Description Ovate-elongate; heavy; right valve slightly larger than left; longer posteriorly (beaks at 40-43% from anterior end). Anterior end rounded; posterior end tapered, slightly up- turned posteriorly, often extended by a short spout. Posterior slope set off by a rounded ridge, becoming less evident ventrally. Es- cutcheon weakly defined by a low ridge, most evident in right valve. Beaks smooth; most of surface covered by well-spaced, rounded moderate commarginal EASTERN PACIFIC CORBULIDAE 59 FIG. 8. Corbula otra, holotype; SBMNH 345493; length, 22.8 mm. FIG. 9. C. ovulata, lectotype; BMNH 1967946/1; length, 26.7 mm. 60 COAN ribs and very fine commarginal striae. Poste- rior slope with much less conspicuous ribs. Ventral and dorsal margins with marginal ribs in some specimens, such as in the holotype. Periostracum light tan. Exterior color white on anterior and posterior ends and ventrally, purple to pink medially; beaks white. Interior white to purple. Hinge plate heavy; right valve with large tooth; left valve with chondrophore not very extended and a small tooth. Posterior end of pallial line with short posterior extension (Fig. 41). Length to 26.1 mm (Bahia Chamela, Jalisco, México; Skoglund Collection). Distribution Isla Carmen, Baja California Sur (26.0°N) (CAS 121884), and Guaymas, Sonora (27.9°N) (UCMP E.8425, CAS 121883), Mex- ico, to La Libertad, Guayas Province, Ecuador (2.2°S) (SBMNH 109827). A lot in the USNM 21479 labeled as “Monterey, California,” is un- doubtedly a labeling error. A few valves num- bered with a locality in the Islas Galapagos, Ecuador, now isolated as CAS 121890, are thought to represent numbering errors on a few shells that had been a large lot from Cor- into, Nicaragua. Recorded depths of live col- lected material are from the intertidal zone to 55 m (mean, 17.9 т; п = 31), on mud or sand. | have seen 107 lots. Etymology The species name is the Spanish word for “other.” Discussion This new species is closest to C. ovulata, and their distributions overlap from Costa Rica to Ecuador. Corbula ovulata is more elongate, even as a juvenile, more produced posteriorly, and it is always white in color. The radial rib between the central and posterior slope is more pronounced, and the commar- ginal sculpture is denser and more evenly dis- tributed. Corbula (Carycorbula) ovulata G. B. Sowerby I, 1833 Figures 9, 42 Corbula ovulata, G. B. Sowerby |, 1833 G. B. Sowerby |, 1833: 35-36; Hanley, 1843: 47 [in part; not the fig.]; Reeve, 1843: pl. 1, fig. 7 [in part]; d’Orbigny, 1845: 571-572; C. B. Adams, 1852a: 522-523 [1852b: 298-299] [in part]; Carpenter, 1857a: 183, 228, 244, 280, 300; 1857b: 23 [in part]; 1864a: 368 [1872 reprint: 204]; 1864b: 537, 668 [1872 reprint: 23, 154]; Tryon, 1869: 65; Lamy, 1941: 127-128 [in part]; Hertlein & Strong, 1950: 241 [Aloidis (Caryocorbula)] [in part; not the fig.]; Hertlein & Strong, 1955: 206; Keen, 1958: 209 [Corbula (Cary- ocorbula)] [in part; not the fig.]; Olsson, 1961: 428-429, 548; pl. 75, fig. 2-2b [Caryocorbula (Caryocorbula)] [in part; not fig. 2c]; Keen, 1971: 265-266, fig. 680 [Corbula (Caryocorbula)] [in part; not lower right fig. ] Type Material & Locality BMNH 1967946/1, lectotype here desig- nated, open pair, the largest specimen, that figured by Reeve (1843); length, 26.7 mm; height, 15.1 mm; width, 12.1 mm (Fig. 9). BMNH 1967946/2-3, paralectotypes, two other pairs: closed pair, length, 24.3 mm; open pair, length, 24.1 mm. “Caraccas” [Bahía de Caraques], Manabi Province, Ecuador (0.6°S). Acollective depth of 7-17 fms. [13-31 m] was given for ail the original material. Five localities were originally mentioned. The two northernmost—Mazatlan, Sinaloa, Mexico, and “Real Llejos” [ = Rio Realejos; Corinto], Chinendega Province, Nicaragua — specifically refer to the species [“beautiful pink color’] here described as Corbula otra and are not represented by material in the BMNH col- lection, nor is material present from the Golfo de Montijo, Veraguas Province, Panama, or from the other cited Ecuadorian locality — ”Xipixapi” [Jipijapa; Puerto de Cayo], Manabí Province. Without examining type material, Hertlein & Strong (1950: 241) “designated” the latter as the type locality, but this is now superceded by the locality of the lectotype designated here (ICZN Code, 1999: Art. 76.2): Description Ovate-elongate, heavy; right valve slightly larger than left; equilateral to longer posteri- orly (40-50% from anterior end); anterior end rounded; posterior end produced, extended by a spout in large specimens. Posterior slope set off from central slope by a very low ridge EASTERN PACIFIC CORBULIDAE 61 that disappears ventrally. Escutcheon well de- fined by a ridge. Beaks smooth; most of surface with closely spaced, moderate commarginal ribs and very fine commarginal striae. Ventral margin with marginal ribs in some material. Escutcheon smooth. Periostracum tan. Exterior white; interior with brown patches and brown marginal color in large specimens. Hinge plate heavy; right valve with a large tooth; left valve with a broad, not very projecting chondrophore and a small tooth. Posterior end of pallial line with posterior extension (Fig. 42). Length to 29.2 mm (Skoglund Collection; Playas, Guayas Province, Ecuador). Distribution Bahia Juanilla, Guanacaste Province, Costa Rica (10.9°N) (LACM 72-13.30), to Cabo Blanco, Piura Province, Peru (4.3°S) (CAS 121777), from the intertidal zone to 55 m (mean, 8.2 т; п = 13), on sand bottoms. | have seen 58 lots, including the types. Reported on the Pleistocene third terrace on the Peninsula de Santa Elena (Hoffstetter, 1948: 81) and, with question, as a subfossil at Laguna de Salinas (Hoffstetter, 1952: 44), both in Guayas Province, Ecuador. Also re- ported in the early Pliocene Jama Formation at Puerto Jama, Manabi Province, Ecuador (Pilsbry & Olsson, 1941: 11, 75), in the Pliocene at Rio la Vaca and Rio Blanca, Burica Peninsula, Puntarenas Province, Costa Rica (Olsson, 1942: 171, 172), and in the Miocene Tumbes Formation at Zorritos, Tumbes Province, Peru (Olsson, 1932: 140). Discussion See under C. otra. Corbula (Caryocorbula) porcella Dall, 1916 Figures 10, 43 Corbula porcella Dall, 1916 Dall, 1916a: 41 [nomen nudum); Dall, 1916b: 415-416; Oldroyd, 1925: 204; Grant & Gale, 1931: 422 [Corbula (Len- tidium)]; Lamy, 1941: 242 [as С. “porcel- lio”); Hertlein & Strong, 1950: 242, 252, pl. 2, figs. 13, 15 [Aloidis (Caryocorbula)]; Keen, 1958: 210-211, fig. 531 [Corbula (Caryocorbula)]; Olsson, 1961: 430, 549, pl. 76, fig. 8 [Caryocorbula (Caryocor- bula)]; Keen, 1971: 265-266, fig. 681 [Corbula (Caryocorbula)]; Coan et al., 2000: 478, pl. 102 [Caryocorbula] Corbula fragilis Hinds, auctt., non Hinds, 1843 Dickerson, 1922: 563; Grant & Gale, 1931: 422 [Corbula (Lentidium)] Type Material & Locality USNM 97039, lectotype here designated, open pair; length, 7.9 mm; height, 5.2 mm; width, 4.9 mm (Fig. 10). USNM 880652, para- lectotypes, 1 closed pair, 6 right valves, 8 left valves, and assorted fragments. USFC Sin. 2838, off the east side of Isla Cedros, Baja California, México (28.2°N, 115.2°W), 44 fms. [99 m], green mud; May 5, 1888. Description Shell ovate-subquadrate, of moderate thickness for size; right valve slightly more in- flated than left; beaks closer to anterior end (32-34% from anterior end). Anterior end rounded. Posterior end almost vertically trun- cate. Posterior slope separated from central slope by a fairly strong radial ridge. Es- cutcheon sharply defined by a ridge. Beaks smooth. Central slope with moder- ate, irregular commarginal ribs, lamellar on posterior slope; posterior end also with radial rows of pustules in many specimens. Color white exteriorly and interiorly. Hinge plate broad; right valve with a narrow tooth; left valve a narrow, non-projecting chon- drophore and a small tooth (Fig. 43). Length to 10.2 mm (MCZ 260684; S of Laguna San Ignacio, Baja California Sur). Distribution & Habitat Esteros Bay, San Luis Obispo County, Cal- ifornia (35.4°N) (USNM 207636), to Punta Magdalena, Baja California Sur, Mexico (24.6°N) (LACM 71-13.5), in 27 to 210 m (mean, 80.3 m; n = 55), on mud and sand. | have seen 68 lots, including the types. The record of C. porcella from Bahia Bal- lena, Puntarenas Province, Costa Rica, by Hertlein & Strong (1950: 242) was based on specimens of C. nasuta (CAS 121941). Also recorded in the late Pleistocene Miller- ton Formation at Tomales Bay, Marin County, California (Dickerson, 1922: 563, as “C. frag- ilis — CAS Loc. 561; Valentine, 1961: 390, as “C. fragilis’; R. G. Johnson, 1962: 115-120; J. 62 COAN FIG. 10. Corbula porcella, lectotype; USNM 97039; length, 7.9 mm. FIG. 11. C. esmeralda. FIG. 11a. Lecto- type; ANSP 218903; length, 20.6 mm. FIG. 11b. Paralectotype; ANSP 403198; length, 21.9 mm. EASTERN PACIFIC CORBULIDAE 63 E. Johnson, 1987: 116, as both C. porcella and “C. fragilis’). The record of this species in the Pleistocene at Bahia Magdalena, Baja California Sur, México (Loc. 754; Jordan, 1936: 111), was based instead on specimens of C. nasuta. Discussion Corbula procella may be distinguished from C. luteola, which is sympatric but in shallower water, in that the latter is more elongate and longer anteriorly, is frequently colored, has finer sculpture, is less inflated, has a less pro- nounced rib separating the central slope from the posterior slope, and has a less sharply de- fined escutcheon. Hertlein & Strong (1950), followed by Soot- Вуеп (1957:11), Keen (1958, 1971), and Ols- son (1961), suggested a relationship of C. porcella to the Panamic C. obesa Hinds, 1843. However, C. obesa is very distinct, at- taining a larger size, being very inflated, hav- ing a proportionately more inflated right valve, having radial ribs on its umbones, in lacking an escutcheon, in being colored interiorly, and in having a heavier periostracum. Specimens of the highly variable C. nasuta account for Panamic records of C. porcella. They differ as follows: C. nasuta more pointed poster- oventral corner, often set off by a shallow radial sulcus just anterior to it radial ridge fairly sharp all the way to ventral margin tinted yellow to brown escutcheon indistinct sometime with a poste- rior spout C. porcella even, truncate poste- rior end radial ridge becoming indistinct near ventral margin white eschtcheon defined by a ridge never with a posterior spout Subgenus (Hexacorbula) Olsson, 1932 Hexacorbula Olsson, 1932: 140 Type species (original designation): Corbula hexacyma Brown & Pilsbry, 1913, = C. gatunensis Toula, 1909 (references in Oddly, Soot-Ryen (1957) made С. obesa Hinds, 1843, a junior synonym of C. porcella Dall, 1916b, in reporting the latter from Panama. His specimen was probably one of the variable C. nasuta. Discussion below). Middle Miocene, Panama. Medium-sized, heavy, ovate to ovate-elon- gate, subequivalve, subequilateral; sculpture of strong commarginal undulations. Woodring (1982: 713) and Anderson (1996: 12) suggested that Hexacorbula is very simi- lar in sculpture to Bothrocorbula Gabb, 1873a (p. 274, pl. 10, fig. 3, 3a); its type species, by monotypy, is Corbula viminea Guppy, 1866a (pp. 293, 295, pl. 18, fig. 11), from the middle Miocene of Jamaica and the Dominican Re- public. Bothrocorbula differs from Hexa- corbula in having a lunular pit. Corbula (Hexacorbula) esmeralda (Olsson, 1961) Figures 11, 44 Caryocorbula (Hexacorbula) esmeralda Ols- son, 1961 Olsson, 1961: 432-433, 549, pl. 76, fig. 3-3c; Keen, 1971: 266-267, fig. 683 [Corbula (Hexacorbula)] Type Material & Locality ANSP 218903, lectotype here desig- nated, right valve, the specimen shown in Olsson’s fig. 3a, c; length, 20.6 mm; height, 12.4 mm; width, 5.0 mm (Fig. 11a). ANSP 403198, paralectotypes —left valve, shown in Olsson’s fig. 3b, length, 21.9 mm; left valve, shown in Olsson’s fig. 3; length, 20.6 mm (similar to holotype in size and shape, but they are not a pair) (Fig. 11b); right valve, not figured in Olsson (1961), length, 20.3 mm. UMML 30.11326, paralectotypes, 3 right valves, 9 left valves. Esmeraldas, Esmeraldas Province, Ecuador (1.0°N); 20 ft. [5 m]; Axel A. Olsson, 1958. Olsson (1961) illustrated an unmatched pair of valves as the “holotype”, necessitating a lectotype selection. Description Shell ovate-elongate, thin in small speci- mens to very thick in large specimens; sube- quivalve; longer posteriorly (beaks at 33-39% from anterior end). Central slope with a broad radial sulcus. Posterior slope set off from cen- tral slope by a relatively sharp ridge. Es- cutcheon set off by a ridge. Beaks relatively smooth, with fine commar- ginal ribs and still finer radial rows of pustules. 64 COAN Most of surface with broad commarginal un- dulations and finer commarginal ribs. Poste- rior slope with only fine commarginal sculp- ture. Color white exteriorly and interiorly. Right valve with a large tooth; left valve with a broad chondrophore, which is not very projecting, and a small tooth (Fig. 44). Length to 22.5 mm (2.5 km S of river at Esmeraldas; Skoglund Collection). Distribution Esmeraldas, Esmeraldas Province (1.0°N) (type locality), to Chone, Bahia de Caraquez, Manabi Province (0.6°S) (USNM 709895), Ecuador, in 5-43 m (mean, 23.8; n = 4); no bottom types have been recorded. | have seen only 6 lots, including the types. Discussion Corbula esmeralda differs from the middle Miocene C. (H.) gatunensis Toula, 1909 (p. 733, pl. 27, fig. 12), in being more elongate and in having its posterior end less set off by a sulcus. In addition to C. hexacyma Brown 8 Pilsbry, 1913 (p. 518, pl. 26, fig. 4), Woodring (1982: 714) listed C. (Carycorbula) buenavis- tana F. Hodson, in F. Hodson & H. Hodson, 1931 (p. 24, pl. 8, fig. 6, pl. 12, figs. 8-13), from the Miocene of Venezeula as a synonym of H. gatunensis. Corbula (H.) cruziana Olsson, 1932 (p. 141, pl. 3, fig. 5, pl. 4, fig. 9), from the early Mio- cene of Perú and Panamá is apparently the oldest member of this subgenus. Subgenus (Juliacorbula) Olsson & Harbison, 1953 Juliacorbula Olsson & Harbison, 1953: 148- 149 Type species (original desigation): Corbula cubaniana d'Orbigny, 1853; = C. knoxi- anaC. B. Adams, 1852c; = C. aequivalvis Philippi, 1836 (references in Discussion below). Recent, western Atlantic. Shell small to medium in size, heavy, sube- quivalve; beaks just posterior to midline; pos- terior end truncate; with a strong ridge be- tween central and posterior slopes and another outlining the escutcheon. Sculpture of strong commarginal ribs in both valves. After saying that their new genus contained “several” species, Olsson & Harbison (1953) referred only three to it: the type species, C. scutata Gardner, 1944 (reference below), and the eastern Pacific C. biradiata; the latter was perhaps an error for C. bicarinata, which is very similar if not identical to the type species. Corbula (Juliacorbula) bicarinata G. B. Sowerby |, 1833 Figures 12, 13, 45 Corbula bicarinata G. B. Sowerby |, 1833 С. В. Sowerby |, 1833: 35; Hanley, 1843: 46, 6, pl. 12, fig. 31; 1856: 344; Reeve, 1844: pl. 3, fig. 23; d’Orbigny, 1845: 571; C. B. Adams, 1852a: 521 [1852b: 297]; Carpenter, 1857a: 183, 224, 228, 244, 280, 281, 300; 1857b: 21-22, 547: 1864a: 368 [1872 reprint: 204]; 1864b: 537 [1872 reprint: 23]; Tryon, 1869: 63; Lamy, 1941: 128-129; Hertlein & Strong, 1950: 238 [Aloidis (Caryocorbula)]; Keen, 1958: 208-209, fig. 523 [Corbula (Caryocorbula)]; Olsson, 1961: 436, 548, pl. 75, fig. 6-6b [Juliacorbula]; Keen, 1971: 266-268, fig. 684 [Corbula (Julia- corbula)]; Gemmell et al., 1987: 60 Type Material & Locality BMNH 1966567/1, lectotype here desig- nated, closed pair, the specimen figured by Reeve (1844); length, 10.6 mm; height, 8.5 mm; width, 6.6 mm (Fig. 12). BMNH 1966567/2-3, paralectotypes, 2 open pairs, lengths, 10.9, 10.5 mm. BMNH 1907.12.30.102, paralectotype, open pair, length 9.6 mm. Four localities in “Columbiae Occidentalis” [West Colombia] were given by С. В. Sowerby |—Panama; “Real Llejos” [= Rio Realejos; Corinto], Chinendega Province, Nicaragua; and “Caraccas” [Bahia Carä- quez], Manabi Province, Ecuador; and Santa Elena, Guayas Province, Ecuador. Neither of the two lots in the BMNH collection has a spe- cific locality, so the type locality is here clar- ified (ICZN Code, Recommendation 76a) as being Playa Kobbe, Panama Province, Panama (8.9°N), where the species is com- mon (Skoglund Collection). A collective depth for the original material was given as 7-17 fms. [13-31 m], in sandy mud; Hugh Cuming. Description Ovate-subquadrate, moderately heavy; right valve slightly larger than left; posterior EASTERN PACIFIC CORBULIDAE 65 FIGS. 12, 13. Corbula bicarinata. FIG. 12. Lectotype; BMNH 1966567/1; length, 10.6 mm. FIG. 13. CAS 120689; San Felipe, Baja California, Mexico; length, 11.2 mm. end longer (beaks at 38-40% from anterior end). Anterior end rounded; posterior end abruptly truncate. Posterior slope set off from central slope by a sharp ridge that becomes somewhat more rounded ventrally. Es- cutcheon set off by a similarly sharp ridge. Beaks relatively smooth. Most of surface with moderate, rounded commarginal ribs, which continue onto posterior slope, and very fine radial striae. Escutcheon with fine com- marginal ribs. Periostracum light tan. White exteriorly; in- terior white to yellowish. Hinge plate narrow; right valve with a large tooth; left valve with a Narrow chondrophore and a very inconspicu- ous tooth (Figs. 13, 45). Length to 13.0 mm (Bahia Cholla, Puerto Penasco, Sonora, Mex- ico; Skoglund Collection). Distribution Head of the Golfo de California at Puerto Penasco, Sonora, México (31.4°N) (Skoglund Collection; UCMP B.6008, E.8416, E.8431; LACM 60-11.33, 62-22.28, 63-56.38; SBMNH 113654), south to Zorritos, Tumbes Province, Peru (3.7°S) (UMML 30.11424); Isla Santa Maria, Islas Galapagos, Ecuador (LACM 32- 2.4); from the intertidal zone on undersides of rocks to 110 m, in rubble (mean, 12.4 m; п = 61). | have seen 257 lots, including the types. This species has been reported in the Pleis- 66 COAN tocene at Bahia Santa Inez and Isla Carmen (Hertlein, 1957: 63), and in the Pliocene on Isla Carmen (Emerson & Hertlein, 1964: 341), Baja California Sur, Mexico. Discussion Carpenter (1857b: 547) and some other au- thors have synonymized C. alba Philippi, 1846, with this species. However, Philippi’s description, together with shapes of the Euro- pean fossil species with which he compared it, indicate that C. alba should instead be re- garded as a synonym of C. nasuta (see Dis- cussion under the latter). Olsson (1961) suggested that C. ira Dall, 1908, described from Panama, might be a synonym. Although superficially similar in shape, C. ira is longer and narrower posteri- orly, there is no rib defining the escutcheon, and the commarginal ribs are fewer and much more prominent. This species is similar to the western At- lantic type species of the subgenus, C. ae- quivalvis Philippi, 1836 (pp. 227-228, pl. 7, fig. 4), which has been reported from Florida to Panamä. Synonyms of C. aequivalvis in- clude C. knoxiana C. B. Adams, 1852c (pp. 238-239), and C. cubaniana d’Orbigny, 1853 (p. 283 [Spanish ed., p. 322], pl. 26, figs. 51-54). Corbula aequivalvis seems to have finer commarginal sculpture than C. bicari- nata (based on comparison with CAS 130494; Salinas Papaya, near Ensenada, southwest coast, Puerto Rico). Corbula aequivalvis was discussed by Jung (1969: 410-411, pl. 39, figs. 11-15) and by Anderson (1996: 17-18, pl. 2, figs. 22-26), who compared it with closely related fossil taxa, including C. knoxi- ana fossilis Pilsbry, 1922 (pp. 427, 435, pl. 46, fig. 14), from the Miocene of the Dominican Republic; C. aequivalvis stainforthi Rutsch, 1942 (p. 124-125, pl. 3, figs. 8, 9), from the Miocene of Trinidad; and C. scutata Gardner, 1944 (pp. 140-141, pl. 23, figs. 16, 30-32), from the Plio-Pleistocene of Florida and North Carolina. Subgenus (Panamicorbula) Pilsbry, 1932 Panamicorbula Pilsbry, 1932: 105 Type species (original designation): Pota- momya inflata C. B. Adams, 1852a; = Corbula ventricosa A. Adams & Reeve, 1850 (references below); tropical eastern Pacific, in mangrove swamps. Shell large, thin in most material to thick in largest specimens; subequivalve; beaks just anterior to midline; left valve with submarginal marginal ridges; with fine commarginal sculp- ture; chondrophore broad, conspicuously di- vided. C. B. Adams (1852a, b) described his three synonymous taxa in the genus Potamomya J. de C. Sowerby, 1835 (p. 241). Its type species, by subsequent designation of Keen (1969b: 698), is Mya plana J. Sowerby, 1814 (pp. 173-174, pl. 76, fig. 2), from the Eocene and Oligocene of Europe. Potamomya is now considered to be a synonym of Erodona Bosc, 1801, ex Daudin ms (vol. 2:329-330, pl. 6, fig. 2) (Keen, 1969b: 698). Erodona, placed in its own family, the Erodonidae, within the My- oidea, is still living in brackish waters on the east coasts of Central and South America. It has a projecting chondrophore in the left valve similar to that of Mya. Tryon (1869) placed Potamomya aequalis in Corbula (Azara). Azara d’Orbigny, 1842 (p. 161, pl. 7), is an ob- jective synonym of Erodona (Vokes, 1945: 26; Keen, 1969b: 698). Corbula (Panamicorbula) ventricosa A. Adams & Reeve, 1850 Figures 14-19, 46 Corbula ventricosa A. Adams & Reeve, 1850 A. Adams & Reeve, 1850: 83, pl. 13, fig. 12; Carpenter, 1857a: 284, 300; Tryon, 1869: 66 NOT C. ventricosa A. Adams & Reeve, auctt. [= C. colimensis Coan, n. sp.] Hertlein & Strong, 1950: 242-243, 251, pl. 2, figs. 3, 4; Keen, 1958: 210, 211, fig. 532; Olsson, 1961: 428, 549, pl. 76, fig. 9; Keen, 1971: 266-267, fig. 682 Potamomya aequalis C. B. Adams, 1852 C. B. Adams, 1852a: 519-520, 547-548 [1852b: 295-296, 323-324]; Carpenter, 1857a: 280, 300; 1864a: 363 [1872 reprint: 204]; Tryon, 1869: 67 [Corbula (Azara)]; Turner, 1956: 28, 128, pl. 19, figs. 5, 6 [Potamomya]; Keen, 1958: 210- 211, fig. 533 [Corbula (Panamicorbula)] Potamomya inflata C. B. Adams, 1852 C. B. Adams, 1852a: 520, 548 [1852b: 296, 324]; Carpenter, 1857a: 280, 300; 1864a: 363 [1872 reprint: 204] [as a ju- nior synonym of Р aequalis]; Tryon, 1869: 67 [as a junior synonym of C. ae- qualis]; Pilsbry, 1932: 105 [Corbula (Panamicorbula)]; Vokes, 1945: 9, 11- 12, pl. 2, figs. 1-4; Turner, 1956: 56, 126, pl. 17, figs. 12, 13 [Potamomya]; Keen, EASTERN PACIFIC CORBULIDAE 67 1958: 210-211, fig. 535 [Corbula (Pana- micorbula);]; Olsson, 1961: 434-435, 549, pl. 76, fig. 1-1c [Panamicorbula]; Keen, 1971: 268-269, fig. 689 [Corbula (Panamicorbula)] Potamomya trigonalis C. B. Adams, 1852 C. B. Adams, 1852a: 520, 548 [1852b: FIGS. 14, 15. Corbula ventricosa. FIG. 14. Lectotype of С. ventricosa; BMNH 1967980/1: length, 22.0 mm. FIG. 15. Holotype of Potamomya aequalis; MCZ 186325; length, 19.4 mm. 296, 324]; Carpenter, 1857a: 280, 300; 1864b: 363 [1872 reprint: 204] [as proba- ble synonym of P. aequalis]; Tryon, 1869: 67 [as probable synonym of Corbula ae- qualis]; Turner, 1956: 93, 128 [Pota- тотуа; as *triagonalis”], pl. 18, figs. 3, 4; Hoffstetter, 1952: 44, fig. 10 [Pana- 68 COAN FIGS. 16, 17. Corbula ventricosa. FIG. 16. Lectotype of Potamomya inflata; MCZ 186315; length, 17.2 mm. FIG. 17. Lectotype of P trigonalis; MCZ 186314; length, 23.8 mm. micorbula]; Keen, 1958: 210-211, fig. 214358]; Olsson, 1961: 435 [as a syn- 536 [Corbula (Panamicorbula)] onym of P inflata] Corbula macdonaldi Dall, 1912 Panamicorbula cylindrica Morrison, 1946 Dall, 1912: 3; Dall, 1925: 15, pl. 17, figs. Morrison, 1946: 47, pl. 1, figs. 15, 17; 1, 3 [catalogue number misquoted as Keen, 1958: 210-211, fig. 534 [Corbula EASTERN PACIFIC CORBULIDAE 69 FIGS. 18, 19. Corbula ventricosa. FIG. 18. Lectotype of Corbula macdonaldi; USNM 214353; length, 20.7 mm. FIG. 19. Holotype of Panamicorbula cylindrica; USNM 542186; length, 13.3 mm. FIG. 20. Corbula amurensis; CAS 089104; Martinez, Contra Costa County, California; length, 16.4 mm. 70 COAN (Panamicorbula)]; Olsson, 1961: 435, 549, pl. 76, fig. 2, 2a [Panamicorbula]; Keen, 1971: 267-268, fig. 688 [Corbula (Panamicorbula)] ? Corbula ustulata Reeve, auctt., non Reeve, 1844 Menke, 1847: 191; Carpenter, 1857a: 236; 1857b: 539 [non Reeve, 1844 — reference in Discussion of next species] Type Materials & Localities Corbula ventricosa-BMNH 1967980/1, lectotype here designated, pair, the larger of two specimens, probably that figured by A. Adams & Reeve (1850); length, 22.0 mm; height, 16.8 mm; width, 13.6 mm (Fig. 14). BMNH 1967980/2, paralectotype, pair; length, 16.0 mm. “China Sea,” but type local- ity here clarified as the mangrove swamp at Paitilla, near Panama City, Panama Province, Panama (9.0°N), where this species is known to occur (ANSP 155409). Potamomya aequalis-MCZ 186325, holo- type, pair; length, 19.4 mm; height, 16.5 mm; width, 10.2 mm (Fig. 15). Mangrove thicket, 2.5 miles [6.5 km] E of Panama City, Panama Province, Panama (9.0°N); soft mud; C. B. Adams;, 27 Nov. 1850-2 Jan. 1851. Potamomya inflata-MCZ 186315, lecto- type (Turner, 1956: 126), pair; length, 17.2 mm; height, 13.8 mm; width, 12.4 mm (Fig. 16). MCZ 186316, paralectotypes, 2 pairs Same locality as P. aequalis. Potamomya trigonalis—MCZ 186314, lecto- type (Turner, 1956: 128), pair; length, 23.8 mm; height, 19.8 mm; width, 13.6 mm (Fig. 17). There are no paralectotypes in the MCZ. Same locality as P aequalis. Corbula macdonaldi-USNM 214353, lec- totype here designated, the specimen fig- ured by Dall (1925), left valve; length, 20.5 mm; height, 16.5 mm; width, 5.7 mm (Fig. 18). USNM 517479, paralectotype, right valve; length, 22.7 mm; and pair; length, 18.6 mm. “Loc. 5848; Pleistocene muck beds at Colon,” but label says “Miraflores Locks,” and Olsson (1961: 435) says “near Panama City,” Panama Province, Panama (9.0°N). Panamicorbula cylindrica- USNM 542186, holotype, pair; length, 13.3 mm; height, 9.3 mm; width, 6.3 mm (Fig. 19). USNM 542187, paratypes: pair, length, 12.5 mm; right valve, length, 20.6 mm; broken right valve, length, approximately 21.2 mm; left valve, length, 19.3 mm. Rio Marina mangrove swamp, Isla San José, Archipielago de las Perlas, Panamä (8.3°N); J. P. E. Morrison, 19 February 1944. Description Shell variable in shape, from ovate to trigo- nal, from thin- to thick-shelled; right valve slightly larger than left. Posterior end slightly longer (beaks 46% from anterior end). Ante- rior end rounded; posterior end obliquely sub- truncate. Posterior slope set off from central slope by a low ridge, more pronounced in some specimens than in others. Escutcheon present in most specimens, set off by a slight ridge and a change in sculpture. Anterior and posterior slopes of juvenile portion of unworn specimens with fine, regu- lar commarginal ribs. Central and posterior slopes of larger specimens with fine, irregular commarginal sculpture and fine radial striae. Periostracum brown to greenish, generally eroded away. White exteriorly and interiorly. Right valve with a large, triangular tooth and elongate submarginal ridges resembling lat- eral teeth situated well away from cardinal tooth; left valve with a large, projecting, di- vided chondrophore, having a moderate tooth on its posterior end; anteroventral hinge mar- gin swollen into a small tooth medially. Pallial sinus absent (Fig. 46). Length to 35.0 mm (USNM 612203; Indian kitchen midden at Val- divia, Guayas Province, Ecuador). Distribution Medano Blanco, N of Topolobampo, Sinaloa, Mexico (25.7°N) (Skoglund Collec- tion), to Puerto Pizarro, Tumbes Province, Perú (3.5°S) (SBMNH 127876; UMML 30.11329, 11338). All living material has been collected from intertidal mudflats, generally in mangrove swamps. | have seen 78 lots, in- cluding the types of the various synonyms. Parker’s (1964: 162) west Mexican offshore records of C. ventricosa were based on spec- imens of C. ira (MCZ 260660, 260668), C. na- suta (MCZ 253667), and C. porcella (MCZ 260013, 260679, 260684). This species was recorded as a subfossil at Laguna de Salinas, Guayas Province, Ecua- dor (Hoffstetter, 1952: 44, fig. 10, as “Pana- micorbula trigonalis”). Anderson (1996: 18-19, pl. 3, figs. 1-10) described C. (P) canae from the upper Mio- cene Cercado Formation of the Dominican Republic. It is more rostrate and acuminate than the Recent species. She also discussed EASTERN PACIFIC CORBULIDAE 71 one other possible new species from the same formation. Discussion Carpenter (1857a: 284, 300) first sug- gested that Corbula ventricosa came from the eastern Pacific. Hertlein & Strong (1950) also discussed the likelihood that the type material of C. ventricosa actually came from the Panamic Province, rather than the “China Sea,” as originally indicated.” This lot did in- deed come from the eastern Pacific, but it is not the species Herilein & Strong thought. Given the fact that C. B. Adams described three synonymous taxa from a single station, subsequent workers might have exercised more caution in proposing additional names without a better understanding of the variabil- ity of this species of Corbula (Panamicorbula). Although material of this species remains un- common in most collections, because few workers have collected in mangrove swamps, abundant specimens from single stations at the Instituto Nacional de Biodiversidad (INBio) in Costa Rica clearly demonstrate that only a single taxon is present. The largest specimens tend to become trigonal. Speci- mens in which the ventral margin turns medi- ally at a smaller size become inflated and more cylindrical. Subgenus (Potamocorbula) Habe, 1955 Potamocorbula Habe, 1955: 272 Type species (original designation): Corbula amurensis Schrenck, 1861. Recent, northwestern Pacific. Small sized, thin, subequivalve, smooth. Left valve with a more prominent radial ridge than the right valve. Left valve with a project- ing chondrophore. Corbula (Potamocorbula) amurensis Schrenck, 1861 Figures 20, 47 Corbula amurensis Schrenck, 1861 Schrenck, 1861: columns 412-413; Schrenck, 1867: 584-586, pl. 25, figs. "Although the Samarang did not stop in the eastern Pacific, the earlier Sulphur expedition, on which Ed- ward Belcher also served, did so and was probably the source of the eastern Pacific material mixed into the Samarang collection. This confusion is dis- cussed by Carpenter (1857a: 224; 1864b: 534) and Hertlein & Strong (1950: 241). 5-8; Lamy, 1941: 247-248 [as Corbula (Erodona)]; Oyama, 1980: 116, pl. 55, figs. 6, 8, 10, 13 [as Potamocorbula]; Scarlato, 1981: 392-393, pl. figs. 415-417, text fig. 14; Zhuang & Cai, 1983: 65, fig. 12; Coan et al., 2000: 479-480, pl. 102. Corbula amplexa A. Adams, 1862 A. Adams, 1862: 223-224 Corbula frequens Yokoyama, 1922 Yokoyama, 1922: 123, pl. 6, figs. 16, 17 Corbula pustulosa Yokoyama, 1922 [non Car- penter, 1857] Yokoyama, 1922: 123-124, pl. 6, fig. 18 [non Carpenter, 1857b: 22-23] Corbula sematensis Yokoyama, 1922 Yokoyama, 1922: 124-125, pl. 6, fig. 19 [not fig. 20, which is a Poromya (Oyama, 1980: 120)] Corbula vladivostokensis Bartsch, 1929 Bartsch, 1929: 133, pl. 2, figs. 1-7 Corbula amurensis takatuayamaensis Ando, 1965 Ando, 1965: 209, fig. 28 Type Materials & Localities Under study in a separate project by an- other worker. Description Ovate, thin; right valve decidedly larger than left valve; beaks anterior to midline (ap- proximately 41% from anterior end); anterior end sharply rounded; posterior end sharply rounded. Posterior end not set off from central slope in right valve, but set off by an angle in left valve. Escutcheon not evident. Beaks smooth; rest of surface with low, ir- regular commarginal ribs. Periostracum tan. Shell white exteriorly and interiorly (Fig. 20). Hinge plate narrow; right valve with a nar- row tooth, attached to shell wall below hinge- line; left valve with a long, projecting chon- drophore that is conspicuously divided, and with a very small tooth on its posterior end; anteroventral hinge margin swollen into a low tooth medially. Pallial line with a small sinus (Fig. 47). Length to 19.7 mm (CAS 121534; Carquinez Strait, San Francisco Bay, Contra Costa County, California). Distribution This introduced species is thus far found only in San Francisco Bay, California, its ecol- 72 COAN ogy there discussed by Carlton et al. (1990), Nichols et al. (1990), and Duda (1994). It oc- curs from the intertidal zone to 8 m, on mud, sand or clay. Discussion Another worker in a separate project is at- tempting to understand the taxonomy of the Asian species attributed to this subgenus. Corbula laevis Hinds, 1843 (p. 59), and C. us- tulata Reeve, 1844 (pl. 4, fig. 25), are earlier names for members of this species complex. Resolution of this group will require access not only to the type material of the nominai species but also suites of specimens from several Asian localities to fully understand variability and distributions. Material of the San Francisco Bay import shows significant variability in shape and thickness. For exam- ple, CAS 121534 contains specimens that are ovate, subtrigonal, and ovate-elongate. А comparison between С. (Potamocorbula) amurensis and С. (Panamicorbula) ventri- cosa is instructive because they both inhabit brackish waters: C. ventricosa subequivalve subquadrate C. amurensis inequivalve ovate, ovate-elongate, to subtrigonal smooth, with fine posterior radials in right valve length to 20 mm stronger commarginal sculpture length to 35 mm has a small pallial sinus division between central and posterior slopes pallial sinus not evident division between central and posterior slopes evident only in left in both valves valve no lateral ridges on lateral ridges in right hinge valve tooth in right valve seated deeply under hinge plate chondrophore very projecting tooth less deeply seated less projecting Subgenus (Tenuicorbula) Olsson, 1932 Tenuicorbula Olsson, 1932: 141 Type species (original designation): Corbula tenuis G. B. Sowerby |, 1833 (reference below). Recent, eastern Pacific. Thin, subequivalve, longer posteriorly. With a strong keel separating posterior and central slopes and another keel defining a lunule. Posterior end truncate. Sculpture of fine, raised commarginal ribs, strongest on poste- rior slope. Corbula (Tenuicorbula) tenuis G. B. Sowerby I, 1833 Figs. 21, 22, 48 Corbula tenuis G. B. Sowerby |, 1833 G. B. Sowerby I, 1833: 36; Hanley, 1843: 47; Reeve, 1843: pl. 2, fig. 13; C. B. Adams, 1852a: 523-524 [1852b: 299- 300]; Carpenter, 1857a: 183, 228, 244, 280, 300; 1864a: 363 [1872 reprint: 204]; 1864b: 537 [1872 reprint: 23]; Tryon, 1869: 66; Lamy, 1941: 143-144; Vokes, 1945: 9, 14-15, pl. 2, figs. 10, 11; Keen, 1958: 211, fig. 538 [Corbula (Tenui- corbula)]; Olsson, 1961: 433-434, 550, pl. 77, fig. 3, За [Tenuicorbula]; Keen, 1971: 268-270, fig. 691 [Corbula (Tenui- corbula)] Corbula glypta Li, 1930 Li, 1930: 264, pl. 5, fig. 38, 38a; Pilsbry, 1931: 431 [as a synonym of C. tenuis] Type Materials & Localities Corbula tenuis- BMNH 1966563, holotype, pair; length, 22.8 mm; height, 12.1 mm; width, 10.2 mm (Fig. 21). Bay of [Golfo de] Montijo, Veraguas Province, Panama (7.7°N); 12 fms. [22 m]; Hugh Cuming. Corbula glypta- AMNH 268094 [formerly Columbia University 22098], holotype, pair; length, 23.9 mm; height, 13.4 mm; width, 10.6 mm (Fig. 22). Mouth of Rio Grande near La Boca, Panamä Province, Panamä (8.9°N); 10-40 ft. [3-12 m]; D. F. MacDonald, 1907. As “Miocene,” but actually Recent (Pilsbry, 1931: 428, 431). Description Shell elongate-subquadrate, thin, sube- quivalve; longer posteriorly (beaks at 40% from anterior end). Posterior end slightly ta- pered ventrally, truncate, sharply turned dor- sally. Posterior slope set off from central slope by a carina. Escutcheon well defined by a sharp angle. Beaks, central and posterior slopes with fine, dense commarginal ribs, strongest on posterior slope. Escutcheon much smoother. Periostracum tan; white exteriorly and inte- riorly. Right valve with a long, narrow tooth. Left valve with a projecting chondrophore; tooth EASTERN PACIFIC CORBULIDAE 73 FIGS. 21, 22. Corbula tenuis. FIG. 21. Holotype of C. tenuis; BMNH 1966563; length, 22.8 mm. FIG. 22. Holotype of C. glypta; AMNH 268094; length, 23.9 mm. not evident (Fig. 48). Length to 24.5 mm (Ve- nado Beach, Panama Province, Panama; Skoglund Collection). Distribution Southeastern coast of Isla Tiburon, Sonora, Mexico (28.9°N) (MCZ 319592), to Zorritos, Tumbes Province, Peru (3.7°S) (UMML 3011808 411858 11359) e11S95a" 11S96: 11397, 11401), from the intertidal zone to 73 m (mean 12.9 m; n = 8), on mud or sand. | have seen 30 lots, including the types. Although Olsson (1932) mentioned a spec- imen from an old collection at Cornell Univer- sity labeled as having come from Mazatlan, 74 COAN Sinaloa, México, no specimens from north of Panama have been collected in recent years other than the single specimen from the cen- tral Golfo de California in the MCZ cited here as the northern record (MCZ 319592).® An- other specimen in the MCZ (319593) from an old collection is labeled has having come from “Margarita Bay, California,” but there is no such place in California or Baja California, and this specimen may instead have come from Panama. The record by DuShane (1962: 44) of С. tenuis from Puertecitos, Baja Cali- fornia, México, cannot be verified, because the specimen has not been located in the AMNH (J. Cordero, e-mail, 17 January 2001), the present location of the bulk of the DuShane collection. This species has also been recorded on the Pleistocene third terrace, Peninsula de Santa Elena, Guayas Province, Ecuador (Hoffstet- ter, 1948: 81). Discussion Corbula tenuis lupina Olsson (1932: 143, pl. 14, figs. 7, 10), described from the Mio- cene Tumbes Formation at Quebrada Tucillal, Zorritos, Tumbes Province, Peru, was said to differ in being heavier, more narrowly elon- gate, and more coarsely sculptured. Jung (1965: 477, 620, pl. 62, figs. 8, 9) subse- quently reported this subspecies from upper middie Miocene beds on the Paraguana Peninsula of Venezuela, and he described the similar Tenuicorbula melajoensis from the late Miocene of Trinidad (Jung, 1969: 413-414, pl. 40, figs. 7-9); it was said to differ in having heavier sculpture. These fossil taxa merit comparison with C. acutirostra Spieker, 1922 (pp. 176-177, pl. 10, figs. 18, 19), described from the late Miocene Zorritos Formation of Peru. Subgenus (Varicorbula) Grant & Gale, 1931 Varicorbula Grant & Gale, 1931: 420, foot- note 1 Тре voucher collection in the MCZ from Parker's (1964) study of the benthic fauna of the west Mexi- can coast was incomplete, and the labels with the remaining specimens do not always correspond to the publication. For example, the single specimen that now constitutes the northern record of this species was not cited in the report, and the only other extant Parker voucher lot labeled C. tenuis (his station 1) contained a mixture of C. nasuta (MCZ 253549) and C. biradiata (MCZ 320412). Type species (original designation): Tellina gibba Olivi, 1792: 101. Recent, Mediter- ranean. Shell of medium size, very inequivalve, with right valve larger, higher, more inflated and more rostrate. Right valve with pronounced commarginal sculpture; left valve with sub- dued commarginal sculpture and often with sparse radial ribs. Corbula (Varicorbula) grovesi Coan, new species Figures 23, 49 Type Materials & Locality LACM 2891, holotype, pair; length, 11.0 mm; height, 10.1 mm; width, 6.7 mm (Figs. 23, 50). LACM 2892, paratype, pair; length, 11.0 mm; height, 9.0 mm; width, 6.0 mm. Sal Si Puedes Basin, S end of Isla San Lorenzo, Baja California Sur, Mexico (28.7°N, 113.0°W), in 732 m (LACM 67-135). Description Trigonal-ovate; right valve much larger than left; left valve fitting inside right valve, leaving a wide margin; subequilateral (beaks at 48% from anterior end). Anterior end rounded; pos- terior end narrowed, subtruncate, without a radial rib between the central and posterior slopes. Escutcheon present, but not defined by a rib. Beaks smooth. Right valve with dense, closely set commarginal ribs. Left valve with 5-6 faint radial ribs; otherwise without sculp- ture. Periostracum very thin. White externally; in- ternally white to yellowish. Hinge plate broad; right valve with a prominent tooth; left valve with a large, vertically projecting chon- drophore and a prominent, elongate tooth (Fig. 49). Length to 11.0 mm (holotype and paratype). Distribution Thus far known from only the type lot con- taining two pairs. Etymology This species is named for Lindsey T. Groves of the Natural History Museum of Los EASTERN PACIFIC CORBULIDAE 75 FIG. 23. Corbula grovesi, holotype; LACM 2891; length, 11.0 mm. Angeles County, who has helped with this and many other projects. Discussion A similar western Atlantic Recent species is C. operculata Philippi, 1848 (p. 13), which oc- curs from North Carolina to Brazil. Synonyms of С. operculata include С. krebsiana С. В. Adams, 1852c (p. 234), C. disparilis d’Or- bigny, 1853 (p. 283 [Spanish ed., p. 322], pl. 27, figs. 1-4), and C. philippii E. A. Smith, 1885 (p. 33, pl. 7, fig. 4-4b [not “pl. 8,” as stated in text]). Acommensal foraminiferan of C. operculata was discussed by Bock & Moore (1968). Corbula caloosae Dall, 1898 (p. 853), 1900 (pl. 36, fig. 16), from the Plio- Pleistocene Caloossahatchee Formation of Florida, merits close comparison with C. oper- culata. The two Recent species differ as fol- lows (based on a comparison with BMSM 15009; W of Cape Romano, Collier County, Florida; and BMSM 15011; Boca Grande, Lee County, Florida): grovesi operculata rounded posteriorly truncate posteriorly fine sculpture in left heavier sculpture in left valve valve 76 COAN periostracum inconspic- periostracum shiny, light uous, transparent tan right valve smooth, with right valve with moderate fine radial rays commarginal sculpture and still finer radial rays beaks less prominent beaks prominent Of Recent eastern Pacific species, C. grovesiis closest to C. obesa, differing in hav- ing a thin, transparent periostracum, being more inequivalve, in lacking radial ribs on the beaks, in having a smoother left valve with faint radial ribs, and in having a proportion- ately broader hinge plate in the left valve with larger teeth. Corbula grovesi differs from the European type species of the subgenus Varicorbula— С. gibba- in being more triangular, less truncate posteriorly, and white in color. There are two Pliocene species of Corbula (Varicorbula) in western North America. Cor- bula gibbiformis Grant & Gale, 1931 (pp. 420-421, 920, pl. 19, figs. 4-6), was de- scribed from the early Pliocene upper Etchegoin Formation (near lower boundary of San Joaquin Formation) at Southern Califor- nia Gas Company Well 1-4 (Sec. 4, T. 28 S., R. 23 E.; approximately 35.5°N, 119.5°W), Kern County, California, at a depth of 3,951- 3,952 feet. Examination of the holotype of C. gibbiformis (SDMNH 172, right valve; length, about 13 mm; height, about 13 mm) demon- strates that it is very different from C. grovesi, with heavy sculpture, bulbous, projecting beaks, and more prominent radial sculpture in the left valve. This Pliocene species is also re- ported from the late Pliocene San Joaquin For- mation, Kettleman Hills, Kings County (Grant & Gale, 1931; Woodring, 1938: 55-56, pl. 6, figs. 8, 9; Woodring et al., 1941: opposite p. 78), the Niguel Formation, Orange County (Vedder, 1960: 326), and the San Diego For- mation, San Diego County (Hertlein & Grant, 1972: 323-324, pl. 57, figs. 3, 4), California, and northwestern Baja California (Rowland, 1972:29, as С. “gibbiformis (Sowerby, 1833)”), and from the middle Pliocene Pico [and/or Saugus] Formations, Ventura County (Watts, in Grant & Gale, 1931; Woodring et al., in Win- terer & Durham, 1962: 304-305), the Santa “Clara” [Clarita] Valley, “Ventura” [Los Ange- les] County (Grant & Gale, 1931), and the East Coyote field, USGS Loc. 13873, Los Angeles Basin, Los Angeles County (Woodring, 1938; USNM 496103), California. Corbula (Varicorbula) granti Olsson, 1942 (p. 197 [=45], 238 [=86], pl. 15 [=2], figs. 8, 9), was described from the Pliocene Charco Azul Formation, Quebrada Penitas, Burica Penin- sula, Costa Rica. The type material (PRI 5505, 5506) is now missing (W. Allmon, e- mail, 11 August 2000). It has a more rostrate posterior end and heavier sculpture in the right valve than C. grovesi, and it has no radial sculpture on the left valve. The Pliocene species merit further compar- ison with several other species from New World Pliocene, Miocene, and Oligocene strata, including Corbula chowanensis Bailey, 1977 (pp. 129-130), from the Pliocene of North Carolina, and C. caloosae Dall, 1898 (p. 853; 1900: pl. 36, fig. 16), from the Pliocene of Florida. Corbula bradleyi Nelson, 1870 (p. 200), from the Miocene Tumbes Formation at Zorritos, Tumbes Province, Peru, illustrated and discussed by Spieker (1922: 171-172, 196, pl. 10, figs. 13, 14), was subsequently tentatively identified from the Pliocene Charco Azul Formation at Quebrada Melissa, Burica Peninsula, Panama (Olsson, 1942: 197- 198). Other taxa include: Corbula chipolana Gardner, 1928 (p. 229, pl. 34, figs. 13-17); C. chipolana carolina Richards, 1977 (p. 33, pl. 11, figs. 34, 35); C. waltonensis Gardner, 1928 (p. 229, pl. 34, figs. 18-22), and C. wal- tonensis rubisiniana Mansfield, 1932 (pp. 156-157, pl. 34, figs. 2-4), from the Miocene of Florida; C. sanctidominici Maury, 1925 (pp. 98-99, pl. 19, fig. 2), from the upper Miocene of the Dominican Republic; C. vieta Guppy, 1866b (pp. 580-581, 590, pl. 26, fig. 8), and C. islatrinitalis Maury, 1925 (p. 101, pl. 19, figs. 8-10), from the Miocene of Trinidad; C. heterogena Dall, 1898, ex Guppy ms (p. 850; 1900: pl. 36, fig. 15), from the Miocene of Panama; C. carrizalana F. Hodson, in F. Hod- son & H. Hodson, 1931 (p. 23-24, pl. 10, figs. 4, 6-9), from the Miocene of Venezuela; C. prenucia Speiker, 1922 (p. 172, pl. 10, fig. 12), from the Miocene of Peru, and C. zuliana F. Hodson, in F. Hodson & H. Hodson, 1931 (pp. 22-23, pl. 10, figs. 1-3, 5), from the upper Oligocene of Venezeula. (For a discussion of some of these species: Woodring, 1982: 715-717; Anderson, 1996: 20-22). It it likely that there are fewer species than there are names. Corbula ( Varicorbula) obesa Hinds, 1843 Figures 24, 25, 50 Corbula obesa Hinds, 1843 Hinds, 1843: 57 [1844 reprint: 230]; Reeve, 1844: pl. 5, fig. 38 [in part; text but EASTERN PACIFIC CORBULIDAE 77 FIGS. 24, 25. Corbula obesa. FIG. 24. Neotype of С. obesa; SBMNH 345495; length, 12.7 mm. FIG. 25. Fig- ure of C. obesa from Hinds (1845). not fig., which = C. nasuta]; Hinds, 1845: 68, pl. 20, fig. 12; C. B. Adams, 1852a: 522 [1852b: 298]; Carpenter, 1857a: 207, 280, 300; 1864a: 368 [1872 reprint: 204]; 1864b: 537, 668 [1872 reprint: 23, 154]; Tryon, 1869: 65; Oldroyd, 1925: 202 [in part]; Lamy, 1941: 133; Keen, 1958: 209, fig. 529 [Corbula (Caryocorbula)]; Keen, 1966: 268; Keen, 1971: 265-266, fig. 679 Corbula nuciformis G. B. Sowerby |, auctt., non G. B. Sowerby I, 1833 Hertlein & Strong, 1950: 241, 251, pl. 2, fig. 1; Keen, 1958: 209, fig. 528 Type Materials & Localities Corbula obesa- SBMNH 345495, neotype here designated, pair; length, 12.7 mm; height, 11.0 mm; width, 9.0 mm, with dried soft parts (Figs. 24, 50). Mazatlän, Sinaloa, Mexico (23.2°N, 106.4°W); 91-128 m; Don- ald R. Shasky, October 3, 1961; ex Skoglund Collection. (Other material from this station in the Skoglund and museum collections has no type status.) Two localities were originally pro- vided for this species—San Blas, Nayarit, México (21.5°N), and Veragua[s] Province, Panama (approximately 7.7°N); the collective 78 COAN depth distribution originally given was 22-33 fms. [38-60 m], on mud; Edward Belcher. No material from either station has been located in the BMNH collection. Workers have guessed about the identity of this species. Reeve (1844) illustrated a spec- imen of C. nasuta as C. obesa. Hertlein & Strong (1950), followed by Keen (1958), fig- ured the present species as C. nuciformis, which is here regarded as a synonym of C. nasuta. Keen (1958, 1971) speculated on a possible relationship between C. porcella and C. obesa, but they are not very similar (see Discussion under C. porcella). À neotype is therefore necessary to stabilize nomencla- ture. Hinds’ description and subsequent figure (Fig. 25) are in accord with the neotype here designated, which is about twice as large as the originally measured specimen. Mazatlan is approximately 225 km north of San Blas, but this species occurs still further north. Description Shell ovate, very inflated, rotund; right valve much more inflated than left; equilateral (beaks at about 50% from anterior end). Pos- terior end narrow, subtruncate, only slightly extended by a spout. Posterior slope set off from central slope by a broadly rounded ridge. Escutcheon not evident. Beaks of both valves with commarginal and radial ribs. Right valve with heavy commar- ginal sculpture; left valve with much finer com- marginal sculpture. Periostracum heavy, light brown. Exterior surface white; interior surface white, suffused light brown; adductor muscle scars and pallial line stained brown in some. Right valve with a large tooth; left valve with a narrow, non-projecting chondrophore and a projecting ridge on its posterior end (Fig. 50). Length to 12.7 mm (neotype). Distribution Isla Espiritu Santo, Baja California Sur (25.5°N) (CAS 121638; LACM 60-6.27; SBMNH 996; Skoglund Collection), and Mazatlan, Sinaloa (23.2°N) (SBMNH 129866, 21358, 21362), México, to near Isla Coiba, Veraguas Province, Panama (7.4°N) (Kaiser Collection); from 14-205 m depth (mean, 100.9 m; n = 33), on mud bottoms. There is a lot in the CAS labeled as having been col- lected on Isla Cedros, Baja California (28.2°N) (CAS 121638), presumably in beach drift. This locality requires further verification. | have seen 38 lots. The record of C. obesa from Santa Catalina Island, California (Dall, 1921: 53), was based on specimens of C. nasuta (USNM 199001), and also these were probably from Isla Santa Catalina in the southern Golfo de California, a labeling error that has resulted in other mis- taken Californian records of Panamic taxa. Discussion Corbula granti Olsson, 1942, from the Pliocene of Costa Rica, may be a synonym of C. obesa (reference in Discussion of C. grovesi). Olsson’s material was admittedly worn, and the radial sculpture on the beaks may not have been visible. Short of rediscov- ery of the now-missing type material, topo- typic specimes would be required to make a convincing case. This species has some resemblance to C. patagonica d’Orbigny, 1845 (p. 570; 1847: pl. 82, figs. 18-22), which occurs from Brazil to Argentina. Corbula patagonica attains a larger size, has a narrower, more produced posterior end, and has less bulbous beaks that lack the prominent radial sculpture pres- ent on those of C. obesa. Corbula patago- nica also has sparse radial ribs on its right valve. Corbula, $.1. | am hesitant to place any of the following species in named subgenera. Much more work must be done to demonstrate true lin- eages within this family. Corbula biradiata G. B. Sowerby |, 1833 Fig. 26-31, 51 Corbula biradiata G. B. Sowerby |, 1833 G. B. Sowerby I, 1833: 35; Hanley, 1843: 47, 4, pl. 10, fig. 51; 1856: 344; Reeve, 1843: pl. 1, fig. 3; d’Orbigny, 1845: 571; C. B. Adams, 1852a: 521-522 [1852b: 297-298]; Carpenter, 1857a: 183, 244, 280, 300; 1857b: 22; 1864a: 368, 369 [1872 reprint: 204, 205]; 1864b: 534, 537, 553, 637 [1872 reprint: 20, 23, 39, 123]; Tryon, 1869: 63; Lamy, 1941: 134; Hertlein & Strong, 1950: 238 [Aloidis (Caryocorbula)]; Keen, 1958: 208-209, EASTERN PACIFIC CORBULIDAE 79 FIGS. 26-28. Corbula biradiata. FIG. 26. Lectotype of C. biradiata: BMNH 1966564/1; length, 16.2 mm. FIG. 27. Holotype of C. rubra; MCZ 186313; length, 12.5 mm. FIG. 28. Holotype of C. ecuabula; ANSP 14486; length, 16.3 mm. 80 COAN FIGS. 29-31. Corbula biradiata. FIG. 29. Holotype of Juliacorbula elenensis; ANSP 218913; length, 17.0 mm. FIG. 30. Paratypes of J. elenensis; UMML 30.11384; lengths, 11.5 mm, 9.4 mm. FIG. 31. LACM 34- 318.2; Isla La Plata, Manabi Province, Ecuador; 13-18 m; length, 12.3 mm. fig. 524 [poor redrawing of Reeve's fig- ure] [Corbula (Caryocorbula)]; Olsson, 1961: 437, 548, pl. 75, figs. 6-6b [Juli- acorbula]; Keen, 1971: 267-268, fig. 685 [Corbula (Juliacorbula)] Corbula rubra C. B. Adams, 1852 C. B. Adams, 1852a: 523, 548 [1852b: 299, 324]; Carpenter, 1857a: 280, 300; 1864a: 363 [1872 reprint: 204] [as a syn- onym of C. biradiata]; 1864b: 553 [1872 reprint: 39]; Turner, 1956: 82-83, 126, pl. 17, figs. 8, 9 Corbula polychroma Gould & Carpenter, 1857 Gould 8 Carpenter, 1857: 198-199; Car- penter, 1857a: 226, 228, 300; 1864a: 31 [1872 reprint: 205] [as a synonym of C. biradiata]; Carpenter, 1864b: 534, 553 [1872 reprint: 20, 39]; Palmer, 1958: 117; 1963: 318; R. I. Johnson, 1964: 129 Corbula ecuabula Pilsbry & Olsson, 1941 EASTERN PACIFIC CORBULIDAE 81 Pilsbry 8 Olsson, 1941: 75, 78, pl. 12, figs. 3-5; Olsson, 1961: 437 [Juliacorbula] Juliacorbula elenensis Olsson, 1961 Olsson, 1961: 438, 550, pl. 77, fig. 5; Keen, 1971: 267, 268, fig. 686 [Corbula (Juliacorbula)] Type Materials 4 Localities Corbula biradiata-BMNH 1966564/1, lec- totype here designated, closed pair, num- bered “5” on shell, possibly the originally mea- sured specimen and that figured by Reeve (1843); length, 16.2 mm; height, 11.1 mm; width, 8.7 mm (Fig. 26). BMNH 1966564/2, paralectotype, open pair, length 15.2 mm. BMNH 1966564/3, paralectotype, closed pair; length, 14.7 mm; BMNH 1966564/4, open pair; length, 14.1 mm. [Golfo de] Chiriquí, Ve- raguas Province, Panamá (8.0°N); Hugh Cuming. BMNH 1907.12.30.118, paralecto- type, open pair, that figured by Hanley (1843); length, 9.5 mm. “Bay of Caraccas” [Bahia de Caräquez], Manabi Province, Ecuador; Hugh Cuming. A collective habitat of 3-6 fms. [5-11 m], on mud and sand, was given for this species. Corbula rubra-MCZ 186313, holotype, pair; length, 12.5 mm; height, 4.6 mm; width, 3.6 mm (Fig. 27). Panamä, presumably near Panamä City, Panamä Province (about 9.0°N), Panama; C. B. Adams, 27 Nov. 1850- 2 Jan. 1851. Corbula polychroma-Not located. The originally cited Gould collection specimens, stated to have come from Santa Barbara, Cal- ifornia, might be expected to be either in the MCZ or the USNM, but they have not been lo- cated. Carpenter (1864a:31) later said that these specimens probably instead came from either Acapulco, México, or Panamä. Speci- mens were also originally cited from the Cum- ing Collection obtained from the Golfo de Cal- ifornia, and these might be expected in the BMNH, but they have not been located. The originally stated measurements were: length, 13.4 mm; height, 6.8 mm; width, 9.4 mm (height and width were probably reversed). Corbula ecuabula- ANSP 14486, holotype, right valve; length, 16.3 mm; height, 11.5 mm; width, 4.2 mm (Fig. 28). ANSP 78998, paratype, left valve; length, 11.2 mm. Punta Blanca, Manabi Province, Ecuador (1.1°S); Canoa Formation, middle to late Pliocene. UMML 30.11455, paratypes, 1 right valve, 2 left valves; Puerto Callo, Manabi Province, Ecuador (1.3°S); Recent. Juliacorbula elenensis-ANSP 218913, holotype, right valve; length, 17.0 mm; height, 12.0 mm; width, 4.4 mm (Fig. 29). UMML 30.11341, paratypes, 5 right valves, 1 left valve; [Salinas], Santa Elena Peninsula, Guayas Province, Ecuador (2.2°S). UMML 30.11384, paratypes, 5 right valves, 1 left valve (Fig. 30); Puerto Callo, Manabi Province, Ecuador (1.3°S). UMML 30.11456, paratype, 1 right valve; Zorritos, Tumbes Province, Peru (3.7°S) Description Ovate-elongate, thin to moderately heavy; right valve slightly larger than left; posterior end usually longer (beaks at 41-48% from anterior end), but anterior end slightly longer in some specimens (beaks at 54-55% from anterior end). Anterior end rounded; posterior end narrowed, obliquely subtruncate, ext- ended by a small spout in some specimens; bluntly subtruncate in some specimens. Pos- terior slope set off from central slope by a fairly sharp ridge that continues to ventral margin. Escutcheon well defined by a ridge. Beaks, central and posterior slopes with moderate commarginal ribs and very fine ra- dial striae. Some material with only fine com- marginal striae. Escutcheon smooth. Exterior light tan to orange, with light radial color bands about a third of the way to ends in small specimens; ends purple. Interior red- dish-brown to purple, particularly around mar- gins, but some material nearly colorless. Hinge plate broad; right valve with a large tooth; left valve with a narrow chondrophore and a conspicuous tooth (Fig. 51). Length to 20.8 mm (Bucaro, Los Santos Province, Panama; UMML 30.11442). Distribution El Solita, Laguna Ojo de Liebre [Scam- mons], Baja California (27.8°N) (SBMNH 31519); Bahia San Luis Gonzaga, Baja Cali- fornia (29.8°N) (LACM 40-36.5), and Isla San Jorge, Sonora (31.0°N) (Skoglund Collec- tion), México, to Punta Pena Mala, Piura Province, Peru (4.2°S) (UMML 30.11449); Isla Santa Cruz (LACM 38-193.14) and Isla San Cristobal (LACM 34-43.22), Islas Galapagos, Ecuador; from the intertidal zone to 57 m (mean, 7.3 m; n = 86), on mud or sand. | have seen 248 lots, including the types of the vari- ous synonyms. 82 COAN This species is reported in beds of Pleis- tocene age at Puerto Libertad, Sonora, Mex- ico (Stump, 1975: 182, 186, 193, 195), and from the Pleistocene Armuelles Formation, Burica Peninsula, Chiriqui Province, Panamä (Olsson, 1942: 162). It is also recorded from the middle to late Pliocene Canoa Formation at Punta Blanca, Manabi Province, Ecuador (Pilsbry & Olsson, 1941: 11, 75, as both C. bi- radiata and C. ecuabula; UMML 30.11380). Rutten (1931: 661) reported but did not fig- ure this species (as “cf.”) from the Quaternary of Surinam, based on an unpublished thesis, a record that requires further verification. Discussion | have come to the conclusion that C. ecuabula is inseparable from C. biradiata. The key feature of Corbula ecuabula, which Pilsbry & Olsson (1941) and Olsson (1961) also reported from the Recent fauna, was that it is significantly longer anteriorly. Olsson de- scribed the beaks of C. ecuabula as being in the “posterior third”, but even in his type ma- terial, the beaks are only 4-5% behind the midline. Occasional specimens may be found throughout the distribution of C. biradiata that have a slightly longer anterior end. | then concluded that Juliacorbula elenen- sis is also a synonym. This species was based on beach-drift material that is light in color, rounded, and with even commarginal sculpture. | have found similar material from the Golfo de California (LACM 78-30.12, from Bahia San Carlos, Sonora, and LACM 79- 111.14, from Bahia de los Angeles, Baja Cali- fornia), and a specimen from Esmeraldas, Ecuador (UMML 30.11321), came to light with subdued sculpture in the left valve and “ele- nensis”-like sculpture in the right valve. An off- shore specimen from Isla La Plata, Manabi Province, Ecuador (LACM 34-318.8), is thin, white, and almost equilateral (Fig. 31). In the fossil fauna, this Corbula biradiata merits comparison with C. (Caryocorbula) urumacoensis F. Hodson, in F. Hodson & H. Hodson, 1931 (pp. 25-26, pl. 12, figs. 1-7), and C. (C.) democraciana F. Hodson, in F. Hodson & H. Hodson, 1931 (pp. 26-27, pl. 11, figs. 1-6), both described from the middle Miocene of Venezuela, and perhaps to C. (C.) retusa Gardner, 1944 (p. 140, pl. 23, figs. 33, 34) [synonym: C. (C.) conradi Gardner, 1944: 139-140, pl. 23, figs. 27, 28, non Dall, 1898), from the Pliocene of Virginia and North Car- olina (concerning C. retusa: Campbell, 1993: 47-48, pl. 21, fig. 190). Corbula colimensis Coan, new species Figures 32, 52 Corbula ventricosa A. Adams & Reeve, auctt., non À. Adams & Reeve, 1850 Hertlein & Strong, 1950: 242-243, pl. 2, figs. 3, 4 [Aloidis (Caryocorbula)]; Keen, 1958: 210-211, fig. 532 [Corbula (Cary- ocorbula)}; Olsson, 1961: 428, 549, pl. 76, fig. 9 [Caryocorbula (Caryocorbula)]; Keen, 1971: 266-267, fig. 682 [Corbula (Caryocorbula)] Type Material & Locality SBMNH 345496, holotype, pair; length, 13.7 mm; height, 9.5 mm; width, 7.7 mm (Figs. 32, 52). SBMNH 345497, paratypes, 3 closed pairs; lengths, 14.0 mm, 13.4 mm, 3.1 mm. Las Ventanas, Manzanillo, Colima, Méx- ico (19.0°N, 104.3°W), 42 m; Carl & Laury Shy, 1969-1970; ex Skoglund Collection Description Shell ovate, thin to moderate in thickess; right valve slightly larger than left; posterior end longer (beaks at 32% from anterior end). Posterior end subtruncate, extended by a short spout in some specimens; posterior slope set off from central slope by a rounded ridge. Escutcheon faint, set off by a slight ridge in right valve. Beaks with fine commarginal ribs; most of surface with moderate commarginal ribs. Pe- riostracum light brown. Shell white exteriorly and interiorly. Hinge plate of moderate thick- ness; right valve with a large tooth; left valve with a chondrophore of medium width, which is not very projecting, and a moderate tooth. Pallial sinus with a somewhat sharp bend posteriorly (Fig. 53). Length to 14.0 mm (a paratype). Distribution Los Corchos, Nayarit (21.7°N) (MCZ 260451), to Bahia Tangola Tangola, Oaxaca (15.8°N) (LACM 34-240.5), Mexico, in 29-112 m (mean, 55.9 m; n = 7), on mud bottoms. This species is thus far known from 8 lots. The lot cited by Hertlein & Strong (1950) from off EASTERN PACIFIC CORBULIDAE 83 FIG. 32. Corbula colimensis, holotype; SBMNH 345496; length. 13.7 mm. FIG. 33. C. ira, lectotype; USNM 122944; length, 11.4 mm. 84 COAN Bahia Tangola Tangola has not been located inthe CAS or AMNH. Etymology This species is named for the Mexican state of Colima. Discussion Corbula colimensis is most similar in size, shape, and color to C. obesa, but the new species is equivalve, thinner, and less in- flated, and it has no radial sculpture. Corbula ira Dall, 1908 Figures 33, 53 Corbula (Cuneocorbula) ira Dall, 1908 Dall, 1908: 423; Lamy, 1941: 233-234; Keen, 1958: 210 [as a synonym of С. ven- tricosa]; Olsson, 1961: 436, 549, pl. 76, fig. 5 [Juliacorbula]; Keen, 1971: 267- 268, fig. 687 [Corbula (Juliacorbula)] Type Material & Locality USNM 122944, lectotype here desig- nated, right valve; length, 11.4 mm; height, 8.6 mm; width, 3.0 mm (Fig. 33). USNM 880651, paralectotypes, 1 left valve, length, 11.8 mm (close in size to lectotype, but they are not a pair); right valve, length, 11.0 mm. Albatross Stn. 3355; Dall (1908), followed by other authors, reported this as “Gulf of Panama,” but the log and map (Townsend, 1901: 412) place this station at 7.2°N, 80.9°W, just off Punta Mariato, Peninsula de Azuero, Veraguas Province, Panama; 182 fms. [333 m], mud; Feb. 23, 1891. Description Ovate-subquadrate, moderately heavy; right valve slightly larger than left; beaks closer to anterior end (beaks at 30-34% from anterior end). Ventral surface sometimes flat- tened. Anterior end sharply rounded. Poste- rior end tapered, obliquely truncate. Posterior slope set off from central slope by a sharp ridge that runs almost to ventral margin. Es- cutcheon defined by a ridge. Beaks relatively smooth. Central slope with strong, rounded, shingle-like commarginal ribs and very fine radial ribs. Posterior slope with finer commarginal ribs. Periostracum tan. Exterior white; interior white, sometimes suffused with purple or brown. Hinge plate broad; right valve with a large tooth; left valve with a very small chon- drophore and a small tooth (Fig. 53). Length to 13.6 mm (USNM 810921; Cabo Lobos, Sonora, México). Distribution & Habitat Cabo Lobos, Sonora (29.9°N) (USNM 212797; MCZ 260668), and Bahia San Luis Gonzaga, Baja California (29.8°N) (LACM 37- 199.10, 40-36.6), México, to Callao, Lima Province, Peru (12.1°S) (LACM 35-153.2, 35- 177.3); Isla del Coco, Costa Rica (SBMNH 345530), from 15 to 388 m (mean, 98.4 m; n = 60), on sand and mud. | have seen 85 lots, including the types. Discussion This species can be distinguished from C. marmorata of similar size in being more quadrate and elongate, more truncate poste- riorly, with a stronger radial rib, and commar- ginal ribs that are higher and narrower. It may also become ventrally flattened, as in C. na- suta, which is never true of C. marmorata. Corbula luteola Carpenter, 1864 Figures 34, 35, 54 Corbula luteola Carpenter, 1864 Carpenter, 1864b: 611, 637 [1872 reprint: 97, 123]; 1865: 207; Tryon, 1869: 65; Arnold, 1903: 181, pl. 17, fig. 11 [a poor figure]; Oldroyd, 1925: 203; Grant & Gale, 1931: 421-422, 920, pl. 19, figs. 2, 7 [Corbula (Lentidium)]; Lamy, 1941: 240-241 [Corbula (Corbuloyma)|; Hertlein & Strong, 1950: 239 [Aloidis (Caryocorbula)]; Keen, 1958: 209 [in part; not fig. 525, which = C. marmorata] [Corbula (Caryocorbula)]; Palmer, 1958: 117-118, 340, pl. 15, fig. 13-18 [Corbula (Lentidium)]; Keen, 1971: 264 [in part; not fig. 675, which = C. marmorata] [Cor- bula (Caryocorbula)]; Hertlein & Grant, 1972: 324-325, pl. 55, figs. 1, 2, 5, 6, 15 [Corbula (Lentidium)]; Coan et al., 2000: 479, pl. 102 [Juliacorbula] Corbula luteola rosea Williamson, 1905, non T. Brown, 1843 (or earlier), non Reeve, 1844 Williamson, 1905: 120; Coan, 1989: 298 EASTERN PACIFIC CORBULIDAE 85 FIGS. 34, 35. Corbula luteola. FIG. 34. Lectotype of С. luteola; USNM 14897; length, 10.2 mm. FIG. 35. Holo- type of C. luteola rosea; LACM 1421; length, 7.0 mm. [as a synonym of С. luteola] [non Corbula rosea T. Brown, 1843 (or earlier): 105, pl. 42, fig. 6; non Corbula rosea Reeve, 1844: pl. 5, fig. 26] Type Materials & Localities Corbula luteola-USNM 14897, lectotype here designated, the largest pair, closest to Carpenter’s (1865) measurement; length, 10.2 mm; height, 6.8 mm; width, 4.5 mm (Fig. 34). USNM 880650, paralectotypes: pair, length, 8.4 mm (the specimen figured by Palmer, 1958); right valve, length, 8.3 mm; left valve, length 5.9 mm; pair, length, 5.7 mm. San Pedro, Los Angeles County, California (33.7°N); James G. Cooper. (Evidently, at some point in the past, USNM 15668 had been combined into this lot, because this number is written on the back of the label.) USNM 73457, paralectotypes, 1 right valve, 1 left valve; San Diego, San Diego County, Cal- ifornia (32.7°N); James G. Cooper. Lots in other collections from Cooper material are not types, because they were not studied by Car- penter. Corbula luteola rosea Williamson —LACM 1421, holotype, pair; length, 7.0 mm; height 4.6 mm; width, 2.3 mm (Fig. 35). Terminal Is- land, San Pedro, California (33.7°N); on an 86 COAN anemone in a rock pool on the old breakwa- ter; Martha Burton Williamson. The original description specified a single valve, but what is in the type lot is a matched pair of valves. Description Shell ovate, solid; right valve slightly larger than left; posterior end longer (approximately 42-48% from anterior end). Posterior end vertically truncate. Posterior slope set off from central slope by a rounded ridge that be- comes obsolete ventrally. Escutcheon evident but not sharply defined. Beaks, central and posterior slopes with fine commarginal sculpture. Exterior white or pink, sometimes with pur- ple patches on either side of beaks. Interior white or tan. Hinge plate relatively narrow; right valve with a triangular tooth; left valve with relatively broad chondrophore and a small tooth (Fig. 54). Length to 10V.2 mm (holotype; CAS 121848; Point Loma, San Diego County, California). Distribution Monterey, Monterey County, California, U.S.A. (36.7°N) (CAS 121792), possibly a re- sult of larval settlement in an El Nino year; Topanga Creek, Los Angeles County, Cali- fornia (33.7°N) (LACM 68-192.21), south to Bahia Magdalena, Baja Californ Sur (CAS 121791; USNM 217823; Skoglund Collec- tion); from the intertidal zone to 80 m (mean, 16.1 т; п = 47), in rubble. | have seen 157 Re- cent lots, including the types. Records of this species from the Golfo de California were based on misidentified C. marmorata, or, in the case of Parker (1964: 161), C. nasuta (MCZ 253667, 259948, 260263) and C. ira (MCZ 260703). This species has been widely reported as a fossil in California and Baja California. In the late Pleistocene, there are records from ter- races on Santa Rosa Island, Channel Islands, Santa Barbara County (A. G. Smith, in Orr, 1960: 1117, A. G. Smith, 1968: 22); Playa del Rey (Willett, 1937: 391), the Baldwin Hills (B. L. Burch, 1947: 9), terraces in the Palos Verdes Hills (Chace, 1966: 169; Marincovich, 1976: 20), San Pedro (DeLong, 1941: oppo- site p. 244; Valentine, 1961: 375, 376, 377), Huntington Beach (Valentine, 1959: 54), Los Angeles County; Newport Bay, Orange County (Kanakoff & Emerson, 1959: 22); San Diego, San Diego County (Dall, 1878a: 11, 1878b: 27; Emerson & Chace, 1959: 338; Valentine, 1961: 357; Valentine & Meade, 1961: 10; Kern et al., 1971: 333), California; northwestern Baja California (Valentine, 1957: 297; Valentine & Rowland, 1969: 518); Bahia San Quintin (Dall, in Orcutt, 1921: 24; Jordan, 1926: 245; Manger, 1934: 293), Baja California, Laguna San Ignacio (Hertlein, 1934: 64), Bahia San Bartolomé [Turtle Bay] (Emerson, 1980: 72; Emerson et al., 1981: 111), and Bahia Magdalena (Jordan, 1936: 111), Baja California Sur. Records of this species from the Pleistocene of the southern Golfo de California (Durham, 1950) were based on specimens of C. marmorata (see under the latter). It has been recorded in strata of early Pleis- tocene age in Santa Monica (Woodring, in Hoots, 1931: 121; Valentine, 1956: 196; Rodda, 1957: 2484) and San Pedro (DeLong, 1941: opposite p. 244), Los Angeles County, California. Cooper, in Watts (1897: 79) and Woodring, in Hoots (1931: 116) reported this species from late Pliocene strata in Los Angeles County, and Hertlein & Grant (1972: see syn- onymy) reported it from the late Pliocene San Diego Formation, San Diego County, Cali- fornia. There is a record in the early Pliocene Towsley Formation of Los Angeles County, California (Woodring, in Winterer & Durham, 1962: 304-305). There are also records from the late Miocene Santa Margarita Formation (Gale, in Preston, 1931: 15) of central California, the late Miocene Castaic Formation of southern California (Stanton, 1966: 23), from the early to middle Miocene Temblor Formation (Ade- goke, 1969: 153) of central California, and from the Miocene Isidro Formation near Bahia Magdalena, Baja California Sur (Beal, 1948: 66). Discussion This species is most similar to C. mar- morata, which accounts for Panamic records of C. luteola. The two differ as follows: C. luteola C. marmorata oval pointed posteroventrally larger (to 10.2 mm) smaller (to 8.2 mm) beaks closer to midline longer posteriorly solid color mottled color without a medial sulcus with a medial sulcus radial ridge becomes radial ridge strong to obscure ventrally ventral margin chondrophore larger chondrophore smaller EASTERN PACIFIC CORBULIDAE 87 hinge plate narrower at wider hinge plate same size finer, sharper sculpture heavier, more undulating sculpture beaks sculptured beaks less sculptured For comparison with the sympatric C. por- cella, see under the latter. Of the western Atlantic taxa, С. luteola is most similar to C. contracta Say, 1822 (p. 312), differing from it in being more inflated, having a much more prominent ridge between the central and posterior slopes, and having more lamellar sculpture, which extends onto the beaks. Corbula marmorata Hinds, 1843 Figures 36, 37, 55 Corbula marmorata Hinds, 1843 Hinds, 1843: 58 [1844 reprint: 231]; Reeve, 1844: pl. 5, fig. 39; Hinds, 1845: 69, pl. 20, fig. 13; Carpenter, 1857a: 207, 300; Tryon, 1869: 65; Lamy, 1941: 133- 134; Hertlein & Strong, 1950: 239-240, 252, pl. 2, fig. 17 [Aloidis (Caryocorbula)]; Keen, 1958: 209, fig. 526 [Corbula (Cary- ocorbula)]; Olsson, 1961: 431-432, 548, pl. 75, fig. 5 [Caryocorbula (Caryocor- bula)]; Keen, 1966: 268 [Corbula]; Keen, 1971: 264-265, fig. 676 [Corbula (Cary- ocorbula)]; Gemmell et al., 1987: 58-59 [Corbula] Corbula luteola Carpenter, auctt., non Car- penter, 1864 Durham, 1950: 94, 170, pl. 25, figs. 15, 16; Keen, 1958: 209, fig. 525 [Panamic records and the fig.]; Keen, 1971: 265, fig. 675 [Panamic records and the fig.] Type Material & Locality Not located in the BMNH collection (Keen, 1966). The description and Hinds’ subsequent figure, however, are sufficiently clear and un- ambiguous that a neotype is not required. A copy of the figure from Hinds (1845) is given here (Fig. 36). The originally measured spec- imen was 4.2 mm in length, 2.8 mm in height, and 2.1 mm in width. Veraguafs] Province, Panama (approximately 7.7°N); 26 fms. [48 m], mud; Edward Belcher. Description Shell ovate-subquadrate, moderately heavy for size; right valve slightly larger than left; beaks in anterior third of shell (beaks 28-36% from anterior end). Anterior end rounded. Shallow medial radial sulcus pres- ent. Posterior end truncate, pointed pos- teroventrally. Posterior slope set off from central slope by a radial ridge that runs to ven- tral margin. Escutcheon evident, narrow, strongest in right valve. Beaks relatively smooth; central slope with moderate, undulating commarginal ribs. Sculpture more lamellar on posterior slope. Exterior white, brown, or red, mottled with ma- genta or white, and with a magenta patch an- terior to beaks. Interior magenta, particularly around margins. Right valve with a triangular tooth; ligament under valve margin. Left valve with a narrow chondrophore; tooth small (Figs. 37, 55). Length to 8.2 mm (Bahia Cholla, Sonora, México; Skoglund Collec- tion). Distribution Bahia Magdalena, Baja California Sur (24.6°N) (CAS 121641; LACM 67-70.26), and throughout the Golfo de California to its head at Puerto Penasco, Sonora (31.4°N) (UCMP E.8419; CAS 121646; Skoglund Collection), México, south to Callao, Lima Province, Perú (12.195) (LACM 35-153.3). This species also occurs in the western Atlantic, where speci- mens are sometimes labeled as C. blandiana C. B. Adams, 1852, which is a synonym of C. dietziana C. B. Adams, 1852 (see Discussion under C. speciosa), as C. barrattiana C. B. Adams, 1852, a synonym of C. swiftiana C. B. Adams. 1852 (see Discussion under C. na- suta), or C. contracta Say, 1822 (p. 312), a distinct, uncolored, more inflated, more heav- ily sculptured species that occurs as far north as New England. Corbula marmorata occurs from the intertidal zone to 137 m (mean, 19.9 m; n = 115), in rubble and on the undersides of rocks. | have seen 299 eastern Pacific lots. The record of this species from the Galapa- gos Islands (Kaiser, 1997: 25) was based on a specimen of C. bicarinata (LACM 34-43). Also present in the Pleistocene at Bahía Santa Inez, Baja California Sur, México (Durham, 1950: 94, as “С. luteola”). Discussion Corbula marmorata sometimes occurs at the same stations as C. speciosa. Small spec- imens of the latter are similar to C. marmorata in having a mottled color pattern and a purple patch in front of the beaks. In specimens of 88 COAN FIGS. 36, 37. Corbula marmorata. FIG. 36. Figure of C. marmorata from Hinds (1845). FIG. 37. SBMNH 345499; Bahia Cholla, Sonora, Mexico; length, 6.8 mm. FIG. 38. C. speciosa, lectotype; BMNH 1967945/1; length, 19.7 mm. EASTERN PACIFIC CORBULIDAE 89 similar size, C. speciosa is more elongate; longer, broader, and more truncate posteri- orly; has a stronger rib separating the central from the posterior slope; has a more pro- nounced medial radial sulcus; and the pos- terodorsal margin is more elevated and flange-like. Species that are possibly ancestral to C. marmorata include: C. engonata burnsii Dall, 1898 (p. 847), C. (Caryocorbula) franci Gard- ner, 1928 (pp. 231-232, pl. 35, figs. 1-4), C. (C.) wakullensis Gardner, 1928 (p. 232, pl. 35, figs. 5, 6), and C. barrattiana leonensis Mans- field, 1932 (p. 160, pl. 33, figs. 1, 3), from the Miocene of Florida, and C. cuneata Say, 1824 (pp. 152-153, pl. 13, fig. 3), from the Pliocene of Maryland and Virginia. Corbula speciosa G. B. Sowerby |, 1833 Figures 38, 56 Corbula radiata G. В. Sowerby I, 1833, non Deshayes, 1824 G. B. Sowerby I, 1833: 36; Hanley, 1843: 47 [non Deshayes, 1824: 58-59, pl. 9, figs. dientes] Corbula speciosa Reeve, 1843 Reeve, 1843 (Aug.): pl. 1, fig. 6; Hinds, 1843 (Nov.): 57 [1844 reprint: 230]; Hinds, 1845: 68-69, pl. 20, figs. 7, 8; Carpenter, 1857a: 207, 300; Tryon, 1869: 66; Lamy, 1941: 133; Hertlein & Strong, 1950: 237 [Aloidis (Aloidis)]; Keen, 1958: 208-209, fig. 522 [Corbula (Corbula)); Olsson, 1961: 438-439, 550, pl. 77, fig. 7-7c [Varicorbula]; Keen, 1971: 269- 270, fig. 692 [Corbula (Varicorbula)] Type Materials & Localities Corbula radiata—Not located in the BMNH. The original measurements were: length, 8.9 mm; height, 6.3 mm; width, 4.3 mm. Acapulco, Guerrero, México (16.9°N); Hugh Cuming. Corbula speciosa-BMNH 1967945/1, lec- totype here designated, the largest speci- men and closest to Reeve’s figure; length, 19.7 mm; height, 15.4 mm; width, 11.6 mm (Fig. 38). BMNH 1967945/2, paralectotype, left valve; length, 18.4 mm. BMNH 1967945/3, paralectotype, left valve; length, 15.3 mm. Golfo de Nicoya, Costa Rica (approximately 9.9°N); Edward Belcher. BMNH 1879.2.26.91, Not in Brocchi (1814), as Reeve (1843) claimed. Deshayes’ species is a Cardiomya. pair; length, 17.8 тт. Panama, 6 fms. [11 m], mud; Edward Belcher. The latter seems to be the specimen figured in Hinds (1845), but ma- terial from Panama was not mentioned by Reeve (1843), who made the name available three months before Hinds. Description Shell subtrigonal as an adult, heavy; right valve much larger than left, very inflated. Pos- terior end truncate. Posterior slope set off from central slope by a rounded ridge. Es- cutcheon apparent in some specimens only. Juvenile shell set off by a remarkable change in growth direction, shape, sculpture, and color. Subquadrate juvenile shell flat- tened, with commarginal undulations, a me- dial radial sulcus, fine radial ribs, and a mot- tled color pattern. Right valve of adult with commarginal un- dulations; left valve with finer, lamellar, some- what oblique sculpture. Exterior color of red- dish-brown radial ribs. Periostracum light to dark brown. Interior white to yellowish, with red ribs visible ventrally. Right valve with a large tooth; left valve with a small, non-pro- jecting chondrophore, with the ligament con- fined to a small area medial to a large poste- rior cardinal (Fig. 56). Length to 20.6 mm (Bahia Tenacatita, Jalisco, Mexico; LACM 34- 146.14). Distribution San Felipe, Baja California (31.0°N) (LACM 40-279.3), and Isla Tiburon, Sonora (LACM 36-80.45) (28.7°N), Mexico, to Punta Utria, Choco Province, Colombia (6.0°N) (LACM 35-63.18, 35-170.7); Isla Socorro, Islas Revil- lagigedo, Mexico (LACM 34-5.17; Kaiser col- lection); from the intertidal zone to 125 m (mean, 40.2 m; n = 98), on sand or gravel, or in rubble. | have seen 137 lots, including the types. This species has been reported in late Miocene deposits of the Imperial Formation in Riverside County, California (Powell, 1988: 16). Discussion This species is very similar and perhaps identical to the later-named western Atlantic C. dietziana C. B. Adams, 1852c (pp. 235- 236), which occurs from North Carolina to Brazil. Synonyms of C. dietziana include C. 90 COAN blandiana C. B. Adams, 1852c (pp. 234-235), and C. cymella Dall, 1881 (p. 115) (based on comparison with BMSM 15012; Boca Grande, Lee County, Florida). Small specimens of Corbula speciosa can be distinguished from C. ira, with which it sometimes occurs, by its more quadrate out- line, longer posterior end, mottled color, less prominent sculpture, and medial radial sulcus. Some authors have placed this species in the subgenus Varicorbula because it is in- equivalve, but I think that this allocation is pre- mature. Corbula speciosa differs from other species of Varicorbula in having a highly dif- ferentiated juvenile shell. Indeed, in several respects small specimens of C. speciosa are closest to C. marmorata (for a comparison, see Discussion under C. marmorata). ADDITIONAL NOTES & RECORDS Corbula altirostris Li, 1930 (pp. 263-264, pl. 5, fig. 37), supposedly from an offshore outcrop of the Miocene Gatun Formation in the Bay of Panamä, was based on a Recent specimen of the mactrid Mulinia pallida (Broderip & Sowerby, 1829: 360, as Mactra) (Pilsbry, 1931: 431). Carpenter (1857a: 300; 1860: 2) listed a Corbula boivinei, without authorship, from Central America. This is a nomen nudum. The record of “Corbula cf. collazica Maury,” 1920 (pp. 44-45, pl. 6, figs. 10, 11), suppos- edly from an offshore outcrop of the Gatun Formation in the Bay of Panama (Li, 1930: 263, pl. 5, fig. 36, 36a), was actually based on a Recent specimen of Corbula ovulata, ac- cording to Pilsbry (1931: 431). Corbula gibbosa Broderip & Sowerby, 1829 (p. 361), from the Arctic coast of Alaska, is a probable synonym of Mya truncata Linnaeus, 1758 (p. 670) (Coan et al., 2000: 471). Corbula kelseyi Dall, 1916b (p. 416; Dall, 1916a: 41, nomen nudum), was based on a worn specimen of Cumingia californica Con- rad, 1837: 234 (Coan & Scott, 1991; Coan et al., 2000: 437, 478). The record of “Corbula cf. swiftiana C. B. Adams,” 1852c (pp. 236-237), supposedly from an offshore outcrop of the Gatun Forma- tion in the Bay of Panama (Li, 1930: 264, pl. 5, fig. 39), was based on a Recent specimen of the mactrid Миша pallida (Broderip & Sowerby, 1829) (Pilsbry, 1931: 431). Corbula tenuis Moody, 1916 (pp. 59, 62, pl. 2, fig. 4a, 4b), поп ©. В. Sowerby |, 1833, = С. binominata Hanna, 1924 (p. 163), from the Pliocene Fernando Formation in downtown Los Angeles, California [holotype: UCMP 11087; Loc. 3030], is not a corbulid. Although broken and partially sediment- and glue-filled, the holotype seems to be a poromyid, closest and probably identical to the Recent Der- matomya mactrioides (Dall, 1889a: 448, as Poromya (Dermatomya)); Coan et al. (2000: 572) treated this Dermatomya. Corbula bi- nominata was also later recorded from the Pliocene of the Los Angeles Basin by Soper & Grant (1932: 1060). NOTE ADDED IN PROOF A paper has recently appeared that sheds new light on the western Atlantic species of Corbula (Varicorbula) (Mikkelsen & Bieler, 2001). Using Varicorbula as a full genus, they adopt the traditional early dates of d’Orbigny’s species of Corbula, with the result that V. dis- paralis (d’Orbigny, 1842) is regarded as the valid name for the species closest to Corbula (Varicorbula) grovesinamed here. Asynonym that | had not noted, Corbula limatula Conrad, 1846 (p. 25, pl. 1, fig. 2), would be the valid name if the dates of d’Orbigny are as late as | now think. They regard Corbula operculata Philippi, 1848, the name | used for this species, as a nomen dubium, because of the absence of type material. Clearly, reaching a definitive conclusion about the dates of the all- important d’Orbigny work should be a high pri- ority for workers on the western Atlantic fauna. CONRAD, T. A., 1846, Descriptions of new species of fossil and Recent shells and corals. Proceed- ings of the Academy of Natural Sciences of Philadelphia, 3(1): 19-27, pl. 1 MIKKELSEN, P. M. & R. BIELER, 2001, Varicorbula (Bivalvia: Corbulidae) of the western Atlantic: tax- onomy, anatomy, life habits, and distribution. The Veliger, 44(3): 271-293 ACKNOWLEDGMENTS | appreciated the help of the following cura- tors, other personnel and their institutions, who made specimens, literature, and informa- tion available: Warren D. Allmon, Paleonto- logical Research Institution, Ithaca, New York, USA; Laurie C. Anderson, Louisiana State University, Baton Rouge, Louisiana, USA; Adam J. Baldinger & Kenneth J. Boss, Mu- seum of Comparative Zoology, Harvard Uni- versity, Cambridge, Massachusetts, USA; Warren Blow, Tyjuana Nickens, and Thomas Waller, National Museum of Natural History, EASTERN PACIFIC CORBULIDAE Ji 42 FIGS. 39-42. Diagramatic interior views of eastern Pacific species of Corbula, showing hinge, pallial sinus, and adductor scars of right and left valves. Stippling in left valve indicates socket for tooth of right valve. Cross-hatching indicates areas of ligament attachment on chondrophore of left valve and visible areas of at- tachment on hinge margin of right valve. FIG. 39. C. amethystina; SBMNH 345487: Playas de Villimil, Guayas, Ecuador; trawled; length, 30.8 mm. FIG. 40. C. nasuta; SBMNH 345492, between Santa Cruz and Platinitos, Nayarit, Mexico; 6-18 m; length, 15.9 mm. FIG. 41. C. otra, new species; SBMNH 345493, holo- type; Manazanillo, Colima, Mexico; 30-45 m; length, 22.8 mm. FIG. 42. C. ovulata; SBMNH 345498; Playas de Villamil, Guayas, Ecuador; trawled; length, 29.2 mm. 92 COAN FIGS. 43-46. Diagramatic interior views of eastern Pacific species of Corbula, showing hinge, pallial sinus, and adductor scars of right and left valves. FIG. 43. C. porcella; CAS 142446; E end of Isla Cedros, Baja Cal- ifornia, Mexico; 82 m; unpaired valves: right valve, 7.4 mm; left valve, 7.2 mm. FIG. 44. C. esmeralda; ANSP 218903, 403198, lectotype and paralectotype; Esmeraldas, Esmeraldas, Ecuador; lengths, 20.6 mm. FIG. 45. C. bicarinata; CAS 120689; San Felipe, Baja California, Mexico; length, 11.2 mm. FIG. 46. C. ventricosa; CAS 120699; Panama; length, 15.3 mm. EASTERN PACIFIC CORBULIDAE 93 SS < © SS FIGS. 47-50. Diagramatic interior views of eastern Pacific species of Corbula, showing hinge, pallial sinus, and adductor scars of right and left valves. FIG. 47. C. amurensis; CAS 089104: Martinez, Contra Costa County, California; length, 16.4 mm. FIG. 48. C. tenuis; CAS 120702; Isla Taboga, Panama, Panamá; length, 19.1 mm. FIG. 49. C. grovesi; LACM 2891, holotype; S end of Isla San Lorenzo, Baja California Sur, Mex- ico; 732 m; length, 11.0 mm. FIG. 50. C. obesa; SBMNH 345495, neotype; Mazatlan, Sinaloa, México; length, 12.7 mm. 50 94 COAN se eos 54 FIGS. 51-54. Diagramatic interior views of eastern Pacific species of Corbula, showing hinge, pallial sinus, and adductor scars of right and left valves. FIG. 51. C. biradiata, SBMNH 345500; Playa Kobbe, Panamá, Panamá; length, 16.8 mm. FIG. 52. C. colimensis, new species; SBMNH 345496, holotype; Las Ventanas, Manzanillo, México; length, 13.7 mm. FIG. 53. C. ira; SBMNH 345501; Bahía Carazal, Colima, México; 75 m; length, 11.4 mm. FIG. 54. C. luteola; CAS 120696; Arch Rock, Corona del Mar, Orange County, Califor- nia; length, 10.4 mm. EASTERN PACIFIC CORBULIDAE 95 TI an II FIGS. 55, 56. Diagramatic interior views of eastern Pacific species of Corbula, showing hinge, pallial sinus, and adductor scars of right and left valves. FIG. 55. C. marmorata; CAS 120688; Mazatlan, Sinaloa, México; length, 5.8 mm. FIG. 56. C. speciosa; CAS 120695; Bahia Santa Inez, Baja California Sur, Mexico; 55-64 m; length, 17.5 mm Washington, DC, USA; Yolanda Camacho, In- stituto Nacional de Biodiversidad, Santo Domingo, Heredia, Costa Rica; James Cordeiro and Paula Mikkelsen, American Mu- seum of Natural History, New York, New York, USA; Thomas A. Deméré and N. S. Rugh, San Diego Natural History Museum, San Diego, California, USA; Charlene Fricker, Mark Kitson, and Gary Rosenberg, of the Academy of Natural Sciences, Philadelphia, Pennsylvania, USA; Lindsey T. Groves and James H. McLean, Natural History Museum of Los Angeles County, Los Angeles, Califor- nia, USA; Elizabeth Kools, Department of In- vertebrate Zoology, California Academy of Sciences, Golden Gate Park, San Francisco, California, USA; Rafael La Perna, Universita degle Studi di Bari, Bari, Italy; José Leal, Bai- ley-Matthews Shell Museum, Sanibel, Florida, USA; David R. Lindberg and Karen Wetmore, Museum of Paleontology, Univer- sity of California, Berkeley, California, USA; Joan Pickering and Kathie Way, The Natural History Museum, London, England, UK; Patri- cia Sadeghian and Paul Valentich Scott, Santa Barbara Museum of Natural History, Santa Barbara, California, USA; Nancy Voss, University of Miami, Miami, Florida, USA. Carol C. Skoglund and Kirstie L. Kaiser gen- erously made available material or informa- tion from their collections. David Campbell, Roberto Cipriani, Juan Diaz, Carole M. Hertz, Alan R. Kabat, and Konstantin Lutaenko pro- vided some information, and Richard Petit supplied copies of scarce literature. Laurie C. Anderson, Lindsey T. Groves, Carol C. Skoglund, and Paul Valentich Scott made many helpful comments on the manuscript. Sharon Williams helped to prepare the plates. LITERATURE CITED ADAMKEWICZ, S. L., M. G. HARASEWYCH, J. BLAKE, D. SAUDEK & С. J. BULT, 1997, Amolec- ular phylogeny of the bivalve mollusks. Molecular Biology and Evolution, 14(6): 619-629. ADAMS, A, 1862, On some new species of acephalous Mollusca from the Sea of Japan. An- nals and Magazine of Natural History, (3)9(51): 223-230. 96 COAN ADAMS, A. & L. A. REEVE, 1848-1850, Mollusca. x + 87 pp., 24 pls., in: A. ADAMS, ed., The zoology of the voyage of H. M. S. Samarang, under the command of Captain Sir Edward Belcher, . . . during the years 1843-1846. London (Reeve, Benham & Reeve) [pp. 1-24, pls. 1-9, Novem- ber 1848; pp. 25-44, pls. 10-13, May 1850; pp. 45-87, i-x, pls. 14-24, August 1850]. 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Vers, coquilles, mollusques, et polypiers. Paris (Agasse), viii + 180 + 16 pp., 488 pls. [pls. 190-286, 1797, by Bruguiere]. BURCH, B. L., 1947, Comparison of the molluscs of three Pleistocene beds with the Recent fauna of Los Angeles County, California. Minutes of the Conchological Club of Southern California, 73: 1-18 [charts reterred to on p. 18 never pub- lished]. BURCH, J. Q., 1960, Notes on the taxonomy of pelecypod genus Corbula. The Veliger, 1(2): 33-34. CAMPBELL, L. D., 1993, Pliocene molluscs from the Yorktown and Chowan River formations in Virginia. Virginia Division of Mineral Resources Publication, 127: vii + 259 pp., 43 pls. CARETONS Ji i, JE К THOMPSON; (LE. E. SCHEMEL & F. H. NICHOLS, 1990, Remarkable invasion of San Francisco Bay (California, USA) by the Asian clam Potamocorbula amurensis. |. Introduction and dispersal. Marine Ecology — Progress Series, 66: 81-94. CARPENTER, P. 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Proceedings of the Zo- ological Society of London, for 1863(3): 339-369 [reprinted in Carpenter, 1872: 173-205]. CARPENTER, P. P., 1864b, Supplementary report on the present state of our knowledge with regard to the Mollusca of the west coast of North Amer- ica. Report of the British Association for the Ad- vancement of Science, 33[for 1863]: 517-686 [reprinted: Carpenter, 1872: 1-172]. CARPENTER, P. P., 1865 [1864-1866], Descrip- tions of new marine shells from the coast of Cali- fornia. Parts I-Ill. Proceedings of the California Academy of Sciences, 3: |: 155-159 (July 1864); I: 175-176 (December 1864); 177 (January 1865); Ill: 207-208 (post-4 September 1865); 209-224 (February 1866). CARPENTER, P. P., 1872. The mollusks of western North America. Embracing the second report made to the British Association on this subject, with other papers; reprinted by permission, with a general index. Smithsonian Miscellaneous Col- lections, 10(252): xii + 325 + 13-121. CHACE, E. P., 1966, Pleistocene Mollusca from the second terrace at San Pedro, California. Trans- actions of the San Diego Society of Natural His- tory, 14(13): 169-172 COAN, E. V., 1989, The malacological papers and taxa of Martha Burton Woodhead Williamson, 1843-1922, and the Isaac Lea Chapter of the Agassiz Association. The Veliger, 32(3): 296-301. COAN, Е. V. & Р. Н. SCOTT, 1991, Corbula kelseyi unmasked: a Cumingia (Bivalvia). The Veliger, 34(4): 366. COAN, E. V. & C. SKOGLUND, 2001, Family Cor- bulidae. Pp. 82-85, in C. SKOGLUND, Panamic Province molluscan literature. Additions and changes from 1971 to 2000. |. Bivalvia. The Fes- tivus, Suppl.: 119 pp. COAN, Е. V., Р. VALENTICH SCOTT & E R: BERNARD, 2000, Bivalve seashells of western North America. Marine bivalve mollusks from Arc- tic Alaska to Baja California. Santa Barbara Mu- seum of Natural History, Monographs, 2: viii + 764 pp. CONRAD, T. A., 1833 [1832-1837]. Fossil shells of the Tertiary formations of North America, illus- trated by figures drawn on stone, from nature. Philadelphia (Dobson), viii + 9-46, 29-56 pp., 18 pls., 1 map [1(3): 29-38, September 1833] [reprinted by Harris, 1893, which was reprinted in 1963]. CONRAD, T. A., 1857, Descriptions of Cretaceous and Tertiary fossils, in W. H. EMORY, Report of the United States and Mexican Boundary Survey [U. $. 34" Congress, 1° Session, Senate Executive Document 108 and House Executive Document 135], vol. 1(2): 141-174, pls. 1- 21. COSSMANN, A. Е. M., 1886, Catalogue illustré des coquilles fossiles de l’Eocene des environs de Paris. Premier fascicule. Annales de la Societe Malacologique de Belgique, 21[(4)1]: 15-186, pls. 1-8 [reprint: 174 pp., 8 pls.]. COSTA, O. G., 1829, Catalogo sistematico e ra- gionato de’ Testacei delle due Sicilie. Napoli (Min- erva), 8 + cxxxii pp., 2 pls. CRISTOFORI, G. & G. JAN, 1832, Catalogus in IV. sectiones divisus rerum naturalium in museo ex- stantium Josephi de Cristophori et Georgii Jan . complectens abumbrationem oryctognosiae et geognosiae atque prodrumum faunae et florae Italiae superioris, Sect. II— Conchliologia. Parma (Carmignani). This is a complex, multi-part work, of which only relevant sections are cited here. The parts are inconsistently numbered or unnum- bered. Descrizione dei generi degli animali per 98 COAN servire d’introduzione al prodormo delle fauna del'ltalia superiore compreso nei cataloghi del Museo di Storia Naturale. . . . vi + 10 pp. (March); Pars 1a. Conspectus methodicus mol- luscorum. Fasc. lus. Testacea terrestria et fluvi- atilia. Dispositio methodica generum. . . . —[ii] pp. Conchylia terrestria et fluviatilia. . . . —8 pp. Mantissa in secondam partem catalogi testaceo- rum existantium in collectione quam possident de-Cristofori et Jan. . . . —4 pp. (April) DALL, W. 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United States Geological Sur- dom, 26(3): 258-276 vey, Professional Paper, 195: 170 pp., 57 pls. [as ZHUANG, Q. & Y. CAl, 1983, Studies on the Cor- “1940”] bulidae (Bivalvia) off Chinese coasts. Transac- YOKOYAMA, M., 1922, Fossils from the Upper tions of the Chinese Society of Malacology, 1: Musashino of Kazusa and Shimosa. Journal of 57-68 the College of Science, Tokyo [Imperial] Univer- sity, 44(1): 200 + viii, 17 pls. Revised ms. accepted 10 June 2001 MALACOLOGIA, 2002, 44(1): 107-134 GENUS PARVITHRACIA (BIVALVIA: THRACIIDAE) WITH DESCRIPTIONS OF ANEW SUBGENUS AND TWO NEW SPECIES FROM THE NORTHWESTERN PACIFIC Gennady M. Kamenev Institute of Marine Biology, Russian Academy of Sciences, Vladivostok 690041, Russia; inmarbio @ mail.primorye.ru ABSTRACT Anew subgenus, Pseudoasthenothaerus, of the genus Parvithracia Finlay, 1927, and two new species, Parvithracia (Pseudoasthenothaerus) lukini and P. (Pseudoasthenothaerus) sirenkoi, are described from the Pacific seas of Russia. Previously representatives of the genus Parvithra- cia, With the type species Parvithracia suteri Finlay, 1927, were found only off New Zealand. Study of thraciid genera showed that Japanese species assigned to the genus Asthenothaerus Carpenter, 1864, should be placed in the genus Parvithracia. For Asthenothaerus sematana (Yokoyama, 1922) and A. isaotakii Okutani, 1964, new combinations are suggested: Parvithra- cia (Pseudoasthenothaerus) sematana (Yokoyama, 1922) and Р (Pseudoasthenothaerus) isao- takii (Okutani, 1964). The main morphological characteristics of the new subgenus are the pres- ence of conspicuous lateral teeth in both valves and a subumbonal plate for the attachment of the lithodesma that is supported by pillars. Expanded descriptions of P. suteri, Р (Pseudoas- thenothaerus) sematana, and P. (Pseudoasthenothaerus) isaotakii are given. Key words: Parvithracia, Thraciidae, Bivalvia, northwestern Pacific, morphology, anatomy. INTRODUCTION The bivalve family Thraciidae Stoliczka, 1870, in the Pacific seas of Russia has long been represented by a sole genus Thracia Blainville, 1824 (Scarlato, 1981; Kafanov, 1991). Recently, members of the genus Lam- peia MacGinitie, 1959, previously recorded from America (MacGinitie, 1959; Coan, 1990), were found in this region (Kamenev & Nad- tochy, 1998). Examination of thraciids col- lected by expeditions in the shelf and bathyal zones of the Pacific seas of Russia revealed two new species. Based on the shell mor- phology and anatomy, these species were as- signed to a new subgenus of the genus Parvithracia Finlay, 1927, previousiy known only from the South Pacific (Finlay, 1927a, b; Powell, 1937, 1955, 1979). Study of the rep- resentatives of other genera of the Thraciidae showed that Japanese species previously as- signed to the genus Asthenothaerus Carpen- ter, 1864, should also be placed in this sub- genus. Diagnoses and descriptions of the genus Parvithracia and description of the type species given in the literature are brief, some- times inaccurate, and often illustrated only by schematic figures of Parvithracia suteri Finlay, 107 1927 (Suter, 1913; Finlay, 1927a, b; Keen, 1969; Powell, 1955, 1979). Thorough study of shell morphology of P. suteri and of north- western Pacific species has enabled an ex- panded and modified diagnosis of the genus and an improved description of P. suteri to be made. This paper provides new data on its shell morphology, and describes a new sub- genus of Parvithracia and four species, two of which are new. For Asthenothaerus sematana (Yokoyama, 1922) and A. isaotakii Okutani, 1964, new combinations are suggested. MATERIALS AND METHODS In this study, | have used the material col- lected by numerous expeditions to the shelf and bathyal zones of Pacific seas of Russia from 1931 to 1995. Mollusks were fixed in 70% ethanol or 4% formaldehyde. The mate- rial was stored in 70% ethanol and in the dry form in ZIN, IMB, and PRIFO. For comparison purposes, collections of the following taxa were used: Asthenothaerus diegensis (Dall, 1915) (USNM); A. hemphillii Dall, 1866 (USNM); А. isaotakii Okutani, 1964 (NSMT); A. sematana (Yokoyama, 1922) (NSMT, CAS); Eximiothracia concinna (Gould, 108 KAMENEV 1861) (NSMT); Lampeia adamsi (MacGinitie, 1959) (UAM; MIMB); L. triangula Kamenev & Nadtochy, 1998 (MIMB); L. posteroresecta Kamenev, 1998, in Kamenev & Nadtochy, 1998 (MIMB); Thracia curta Conrad, 1837 (USNM); T. myopsis Moller, 1842 (MIMB, ZIN); T. kakumana (Yokoyama, 1927) (MIMB, ZIN); T. seminuda Scarlato, 1981 (ZIN); T. septentrionalis Jeffreys, 1872 (USNM; ZIN); Thracidora japonica Habe, 1952 (NSMT); Trigonothracia pusilla (Gould, 1861) (NSMT); Parvithracia suteri Finlay, 1927 (MNZ and ma- terial of R. Asher (CIN)). Asthenothaerus isao- takiiand Lampeia species from Pacific seas of Russia were fixed and stored in 70% ethanol. All other material was stored dry. The gross anatomy of the new species has been described from whole mount speci- mens. Shell Measurements Figure 1 shows the position of the shell morphology measurements. Shell length (L), height (H), width of each valve (W) (not shown), anterior end length (A), maximal dis- tance from the posterior end to the top of pal- lial sinus (S), and lithodesma length (L1) were measured for each valve. The ratios of these parameters to shell length (H/L, W/L, A/L, S/L, L1/L, respectively) were determined. The L FIG. 1. Placement of shell measurements: L — shell length; H —height; А — anterior end length; L1 —lith- odesma length; S—maximal distance from poste- rior shell margin to the top of pallial sinus. number of pillars (N) supporting the subum- bonal plate for lithodesma in each valve was also recorded. Shell measurements were made using a caliper and an ocular microme- ter with an accuracy of 0.1 mm. Statistics Statistical analysis of the material used a package of statistical programs STATISTICA (Borovikov & Borovikov, 1997) and Data Analysis Module of MS Excel 97. The calculated indices (H/L; W/L; A/L; S/L and L1/L) are less susceptible to change as compared with other measured parameters. Therefore the statistical analysis was per- formed using only these characteristics. All data was tested with a Kolmogorov test for their fit to a normal distribution. The distribu- tion of some indices was different from the norm. Therefore all analyses were performed on log,, transformations of the original vari- ables. All indices for pairs of different valves of new species were compared using the Stu- dent (t) parametric test and one-way analysis of variance (ANOVA). A discriminant analysis was used to test the validity of the hypothesis of division of all studied specimens into two groups. Throughout this study, statistical signifi- cance was defined as P < 0.05. Morphological Terminology Subumbonal plate is a plate on the inner shell wall to which the internal ligament and lithodesma are attached. It is located ventral to the umbo and is partially or completely at- tached to the shell wall. Various specialists designate it either as a chondrophore (Ya- mamoto & Habe, 1959, for the genus Trigo- nothracia Yamamoto & Habe, 1959; Keen, 1969, for the genus Lampeia; Xu, 1980, for the genus Trigonothracia), buttressed struc- ture (Coan, 1990; Kamenev & Nadtochy, 1998, for the genus Lampeia), or resilifer (Coan et al., 2000, for the genus Lampeia)). Resilifer is a depression or projection of the hinge plate not attached to the inner shell wall that serves for the attachment of the internal ligament. Abbreviations The following abbreviations are used in the paper: CAS--California Academy of Sci- THE BIVALVE GENUS PARVITHRACIA 109 ences, San Francisco; CIN —Cawthron Insti- tute, Nelson; IMB — Institute of Marine Biol- ogy, Russian Academy of Sciences, Vladi- vostok; MIMB—Museum of the Institute of Marine Biology, Vladivostok; MNZ — Museum of New Zealand, Wellington; NIGNS — Ма- tional Institute of Geological and Nuclear Sci- ences, Lower Hutt; NSMT — National Science Museum, Tokyo; PRIFO—Pacific Research Institute of Fisheries and Oceanography, Vladivostok; UAM — University of Alaska Mu- seum, Fairbanks; USNM--United States National Museum of Natural History, Smith- sonian Institute, Washington, D.C.; ZIN— Zoological Institute, Russian Academy of Sci- ences, St.-Petersburg. SYSTEMATICS Order Pholadomyoida Newell, 1965 Superfamily Thracioidea Stoliczka, 1870 Family Thraciidae Stoliczka, 1870 Genus Parvithracia Finlay, 1927 Type species: Montacuta triquetra Suter, 1913: 915, non Verrill & Bush, 1898: 782, 783, pl. 91, fig. 3; = Parvithracia suteri Finlay, 1927b: 529 Diagnosis Shell small (< 11 mm), very thin and fragile to medium in thickness, ovate-elongate to subtrigonal, inequivalve; right valve larger and more inflated. Beaks central or posterior. Pos- terior end truncate, with a faint radial ridge. Periostracum thin, adherent, colorless or tan. Surface with conspicuous growth lines and very fine granules. Escutcheon present; lunule present in some. Ligament internal, supported by a strong lithodesma attached to elongate-trigonal subumbonal plate that is sometimes supported by a series of pillars. Hinge plate weak, with large, short anterior and more or less conspicuous, elongate pos- terior lateral teeth in right valve, and long, lamellate, anterior lateral tooth in left valve. Pallial line with deep pallial sinus of same shape and size in both valves, sometimes reaching midline. Pallial sinus not confluent with pallial line. Remarks Previously this genus included two species: Р suteri and Parvithracia cuneata Powell, 1937 (Powell, 1937, 1955, 1979). However, studies of Bruce A. Marshall (MNZ) have shown that P. cuneata belongs to a different genus (Bruce A. Marshall, personal communi- cation). Subgenus Parvithracia, s.s. Diagnosis Shell small (< 5 mm), thin, fragile, subtrigo- nal, inequivalve; right valve more inflated. Beaks slightly posterior. Posterior end trun- cate, with very faint radial ridge. Periostracum thin, adherent, colorless. Surface with faint growth lines and very fine granules. Es- cutcheon and lunule present. Ligament inter- nal, supported by a strong lithodesma, at- tached to elongate-trigonal subumbonal plate. Hinge plate weak, with large, high ante- rior and more or less conspicuous, elongate, posterior lateral teeth in right valve and long, lamellate, anterior lateral tooth in left valve; in- ternal part of anterodorsal and posterodorsal margins with long grooves. Pallial line with deep pallial sinus of same shape and size in both valves, reaching midline. Pallial sinus not confluent with pallial line. Parvithracia (Parvithracia) suteri Finlay, 1927 Figs. 2-10, Table 1 Montacuta triquetra Suter, 1913: 915, pl. 53, fig. 7a, non Verrill & Bush, 1898: 782, 783, pl. 91, fig. 3; Finlay, 1927a: 461. Parvithracia suteri Finlay, 1927b: 529; Pow- ell, 1955: 45; Powell, 1979: 434, fig. 117. Type Material and Locality Lectotype (NIGNS TM 399) and 4 paralec- totypes (NIGNS TM 405-409) (Boreham, 1959; Bruce A. Marshall, personal communi- cation). Port Pegasus, Stewart Island, New Zealand, 18 fathoms (approximately 33 m) (Suter, 1913; Powell, 1955, 1979). Material Examined 1 lot (MNZ ex M.44790) from North Arm, Port Pegasus, Stewart Island, New Zealand (47° 11'S, 167° 41'E), 37-44 m, mud, 22 Feb- ruary 1972 (R/V “Acheron”) (3 spec.); 1 lot (CIN) from Tasman Bay, Nelson, New Zealand (40°35'S, 173°54’E), 60 m, mud/sand, Coll. Rod Asher, January 1997 (1 damaged spec.). 110 KAMENEV FIGS. 2-10. Parvithracia (Parvithracia) suteri Finlay, 1927 (MNZ ex M.44790), North Arm, Port Pegasus, Stewart Island, New Zealand (47°11'S, 167°41’E), 37-44 m. 2, 3: Left and right valves (bar = 1 mm). 4: Gran- ules on the shell surface (bar = 100 um). 5: Hinge of the right valve (bar = 300 um). 6: Hinge of the left valve (bar = 300 um). 7: Dorsal view of teeth on the right valve (bar = 100 um). 8: Close-up of a left valve showing the subumbonal plate and the lateral tooth on the anterodorsal shell margin, ventral view (bar = 300 um). 9: Close-up of a right valve showing inner part of antero- and posterodorsal shell margins with a shallow groove (bar = 1 mm). 10: Ventral view of lithodesma (bar = 100 um). Description (right valve more inflated), inflated (W/L of right valve 0.281-0.303; W/L of left valve Expanded and modified from Suter (1913) — 0.235-0.250), slightly inequilateral, thin, Exterior: Shell small (< 5.0 mm), subtrigonal, solid. Surface with faint growth lines and very high (H/L = 0.824-0.892), slightly inequivalve dense fine granules. Periostracum dull, thin, THE BIVALVE GENUS PARVITHRACIA 111 adherent, colorless. Beaks small, high, pro- jecting considerably above dorsal margin, slightly posterior to midline (A/L = 0.531- 0.588), somewhat sharp, orthogyrate. Ante- rior end narrow, rounded. Posterior end de- cidedly truncate, with a faint radial ridge ex- tending beaks to junction of posterior end with ventral margin. Anterodorsal margin straight or slightly convex, very steeply descending ventrally, smoothly transitioning to rounded anterior end. Ventral margin slightly curved (more curved in right valve). Posterodorsal margin straight or slightly convex, very steeply descending ventrally, forming a dis- tinct angle at transition to posterior end. Pos- terior end slightly curved, anteriorly directed, forming a rounded angle at transition to ven- tral margin. Lunule present only in left valve, narrow, well expressed along entire an- terodorsal margin, demarcated by a ridge. Es- cutcheon narrow, more expressed in left valve, demarcated by ridges extending along posterodorsal margin from beaks to posterior end. Interior: Right valve with anterior and pos- terior lateral teeth and long grooves on inner part of anterodorsal and posterodorsal mar- gins; left valve with anterior lateral tooth. In right valve, anterior lateral tooth very large, high, strongly projecting above inner part of anterodorsal margin, ventrally directed; pos- terior lateral tooth small, elongate, somewhat projecting or not projecting above inner part of posterodorsal margin, extending along pos- terodorsal margin; inner part of antero- and posterodorsal margins thickened, with a long, shallow groove more expressed in inner part of posterodorsal margin, running parallel to anterior and posterior dorsal slopes for almost their entire length. In left valve, anterior lateral tooth long, lamellate, noticeably projecting above inner part of anterodorsal margin; inner part of anterodorsal margin slightly concave between beak and tooth; inner part of pos- terodorsal margin straight. Subumbonal plate short, slightly elevated above inner shell sur- face, attached to shell wall, free along its anteroventral margin. Pillars absent. Litho- desma large (L1/L = 0.121-0.125), ovate- trapezoidal. Anterior adductor muscle scar large, elongate, kidney-shaped. Posterior ad- ductor scar small, rounded. Pallial sinus dis- tinct, of the same shape and size in both valves, deep, broad, rounded anteriorly, reaching midline (S/L = 0.455-0.5), anterior limit short of faint vertical line from beaks. Shell interior with faint radial striae. Variability Slight variations occur in shell shape, shell height, valve width, and position of the beaks (Table 1). There is also variation in the shape and size of the teeth, lithodesma shape, and pallial sinus length. Distribution and Habitat Parvithracia (P.) suteri occurs near New Zealand (Three Kings Islands; Cape Brett; Hen and Chickens Islands; Mayor Island; Tas- man Bay; Stewart Island, Port Pegasus; Bounty Islands) (Suter, 1913; Powell, 1955, 1979), from 33 m (Stewart Island, Port Pega- sus) to 260 m (Three Kings Islands), on mud and sandy mud. Pseudoasthenothaerus Kamenev, new subgenus Type species: Pseudoasthenothaerus lukini Kamenev, new species Description Shell small (< 11 mm), ovate-elongate to ovate-trigonal, inequivalve; right valve larger, more inflated. Beaks central or posterior. Pos- terior end truncate, with faint radial ridge. Pe- riostracum thin, adherent, colorless or tan. Surface with conspicuous growth lines and very fine granules. Lunule absent. Es- cutcheon narrow, well expressed. Ligament internal, supported by a strong lithodesma, at- tached to elongate-trigonal subumbonal plate that extends obliquely posterior from beaks, free along its anteroventral margin, where it is supported by a series of pillars separated by shallow or deep pits. Hinge plate weak, with large, short anterior and more or less con- spicuous, elongate posterior lateral teeth in right valve and long, lamellate anterior lateral tooth in left valve. Pallial line with deep pallial sinus, of same shape and size in both valves, not reaching midline. Mantle lobes fused, with small pedal and three pallial apertures. Siphons long, separate. Ctenidia consisting of two demibranchs; demibranchs subequal to inner demibranch much larger than outer. Labial palps broadly triangular. Stomach very large, to right of visceral mass; much of stom- ach to right of visceral mass not surrounded by digestive diverticula. Style sac joined to mid gut. Intestine passing through heart. Si- 112 KAMENEV TABLE 1. Parvithracia (Parvithracia) suteri Finlay, 1927. Shell measurements (mm), indices and summary statistics of all characteristics: L— shell length; H —height; W — width; A— anterior end length; $ — maximal distance from the posterior shell margin to the top of pallial sinus; L1 —lithodesma length. Numerator indi- cates shell measurements and indices for the right valve, denominator — for the left valve. ММ — not mea- sured. Statistics L H W A S 11 W/L AL SE EVE NM NM NM NM Depository MNZ ex M. 44790 0.303 0.576 0.485 NM MNZ ex M. 44790 0.303 0.576 0.485 0.121 MNZ ex M. 44790 0.281 0.531 0.500 0.125 MNZ ex M. 44790 NM NM NM NM NM NM NM 3:4: 2.8 10:8: 2 00017 0.824 3.3 2.8 1.0 1.9 1.6 ММ 0.848 NM NM NM NM м NM 3:3 2977 1:07 19 16 04. 0:879 3:3, 2.9 20.87 2192 1.5 0.879 Je 28 12 16 09 04 0875 ЗУ Е 5151018 0.844 Меап 3.27 2.83 0.97 1.83 1.60 0.4 0.867 3.30 2.80 0.80 1.87 1.57 0.849 $0 0.06 0.06 0.06 0.12 0 0 0.017 0.10 0.10 0 0:15; (0:12 0.028 ЗЕ 0.03 0.03 0.03 0.07 0 0 0.010 0.06 0.06 0 0.09 0.07 0.016 Min 3212.8 OA 1167 0:4 0848 3:20 02.7 (08 117 51.5 0.824 Мах 3322.95 1.07 1.9 1:6 0:4 0:879 3:4 2:95 10:8: 12:0 117 0.879 n 3 3 3 3 3 20:3 3 3 3 3 3 3 multaneous hermaphrodite. Testes occupying ventral position in visceral mass above foot. Paired ovaries occupying posterior position in visceral mass. Remarks The major morphological characters of the genera recognized within the Thraciidae by various specialists are presented in Table 2. The new subgenus has a combination of mor- phological characteristics not seen in any ex- isting thraciid genus. Parvithracia (Pseudoas- thenothaerus) differs from all other genera in having anterior and posterior lateral teeth in the right valve and an anterior lateral tooth in the left valve, and a subumbonal plate sup- ported by pillars. On the basis of external and internal shell morphology, as well as hinge structure, the new subgenus is most similar to Parvithracia. This subgenus differs from Parvithracia, s.s., in having pillars supporting the subumbonal plate, in lacking a groove on the inner part of the dorsal shell margin, and in having a lunule. In my opinion, these differences are less substantial compared to the differences among genera within this family (Table 2). Therefore, Pseudoasthenothaerus is as- signed as a subgenus of Parvithracia, with which it is most similar in shell morphology. On the basis of internal shell morphology, hinge structure, and relative size of the lithodesma, this subgenus is also similar to Lampeia. Only Lampeia and Parvithracia (Pseudoasthenothaerus) have a subumbonal plate supported by pillars. However, unlike Parvithracia (Pseudoasthenothaerus), Lam- peia has only an anterior tooth in the right valve. Moreover, Parvithracia (Pseudoas- thenothaerus) differs from Lampeia in lacking a thick brown periostracum, lunule, and exter- nal ligament, and in having granules on the shell surface. In shell form, proportions and morphology, Parvithracia (Pseudoasthenothaerus) resem- bles Asthenothaerus. For this reason, Japa- nese malacologists assigned representa- tives of Parvithracia (Pseudoasthenothaerus) found off the coast of Japan to Astheno- thaerus. However, Asthenothaerus has no lat- eral teeth, the subumbonal plate is tightly at- tached to the shell wall, and the lithodesma is less massive, more angular, with long, sharp ends. Perhaps, Asthenothaerus huanghaien- sis Xu, 1989, described from northern Huang- 113 THE BIVALVE GENUS PARVITHRACIA 6661 ‘0861 ‘NX :6961 ‘эаен Y озошешед 6961 ‘u98 ‘0061 ‘EN 3 9]e1 :8981 'sebuy 2161 ‘эаен 0003 “ye ja UBOD :0661 ‘иеоЭ +1961 UelIY 6961 ‘чээУ ‘7681 “aye :5881 “yuus 6/61 ‘!Эмоз ‘6961 'usay 'q ‘2/61 ‘Ае|и!- ‘66| “ems 8661 ‘Ачоозрем Y AU -9UIEY 0661 “UBO) ‘6S6L ‘HIUIDIEN 0002 ‘18 19 UBOD :0661 ем ¡Jays элоае pajenaja ‘Hig UMOUS JON juasqy }uesqy juesqy seid Aq payoddns ‘big IBM 194$ элоае pajenaja Big siellid Aq peuoddns ‘Big иблеш Jays [ES10PO19} -sod jo ved лэци! элоае би! -JoaloJd jou 'pspanıp Апедиэл ‘SOAJEA YJOQ UI YJOO} 10112]S0d элгел Yo] и! snjjed JO} uolsseidap ‘anıen 1yBu и! эбри JO UJ00} JejjaWıe| ломаие pue snjje9 juauluold 10119I504 ayejd эбич UO Ja11Is91 чим “ajejuap3 ayeıd эби шо лэизэл Bunoel -01d y]im saunauwos “ajejuapy ayeıd эби Wo 191111894 Ajjaus бицоэГола Big чим “ayejuapy элгел Ya] и! 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JUBSA1q Ju9SsQqy padeys-A|ua] па ло panno ‘Big Jeploz -ade1]-ajeno “Big pedeys-Ajp9} па ло peano “Big jeuieju| euiaju| ¡eusaju] ¡eusajul pue ¡eula)x3 ¡euJajul pue ¡eusa]x3 еше! ¡eulajul ¡euJajul pue ¡eula]x3 ¡euJajul ysıumolg JO SS9110]09 ‘uly} ¿sejnisnd pue saul] ц}молб snonoidsuo9 SS2|10[09 ‘UIU] ‘IPS [eIpel jure, pue saul] YMOAB jure AeJB 10 sse|10109 “uly] ‘sajnues6 pue ‘eels jeipeı “saul| ymo1B SnonoidsuoD umo1q ‘Aes6 “Ax]1s о} |пр ‘yolu} Jo uly} ¿sajnisnd ‘seul UMOJ6 jure} ло Snonoidsuog ysmym Аир “uyy ‘sajnues6 pue sql эщиээцоэ snonoidsuoy) umo.1q-3yB1] 10 quid 'moJ¡9Á “ssajJo¡o9 “uly] “sajnueb pue seul] ymolB snonoidsuog SS2|10/09 ‘и!ц} ‘Sein -uei6 pue saul] YMOAB jure y umolg ‘HOIU} “saul| yimo1B snonoidsuog se¡nisnd pue saul] YIMOJB 6961 ‘эаен © 00 WEWEA вовлцоиоби| 0061 Ави х 9]e/ SISdoI2e1yJ vc6l ‘эгерэл| 2/0DIEIU y vest ‘элите! е/22/41 7681 ‘eJe| вшзиошбелца (5плэв yjoueyseopneSd) BIOBIYJINIC 2261 ‘Aejul4 (вовцимеа) BIOBIYJINIE Y 6961 ‘эшиое\| eradwe7 ‘UE09 “1961 ‘ually juasqy 4ayıllseı бицоэГолА yym ‘ayeyuapy juasqy pue ¡eula]x3 o1juasguoy snonoidsuo) ZG8L ‘Чэеэл вех] eusapoy.l padeys ue] JO ssaj1o¡o9 “uly] ¿sejn] r261 ‘эерэ.| 0661 'ueo) juasqy 10; 1918 чим ajejd эбиц 'ajejuap3 -1eq ‘ба 10 [PUIS ¡eula]x3 -snd pue saul] ymoJ6 jure y PIOBIYJOIWIXF AIS ue] ‘и! 0002 “18 эуе!а эби wo} ¡euJajul ‘sainsnd pue sajnues6 yum 6r8L ‘релиоЭ 19 UBO) '066| ‘UeOD juasqy Jajisaj Bunoeloid um ‘eyejuepz ралапэ ‘PUS pue [ец/э}х3 зиоцеприп anbijgo Алеэн виороцелЭ эуеа eBuiy pue ¡Jem ¡Jays ssajo u3am]9q Hays ло ¡Jem ¡Jays uo ешаи! -|09 “Uly] ‘sajnysnd эиц pue 0661 ‘UeOD juasqy запэ jeiuoqwngns чим ‘ayejuepy padeys-1eq ‘big pue ¡eulayx3 Sql ощиеэиоэ зпопэа$и09 9881 ‘ed eiysng 0002 “¡e Нем 1194$ 0} padeys Aysnı “uly] ‘seynues6 "7981, Uajuadieo 19 UBO) “0661 'ueo)y payoeye Авибц Big 3]e]uap3 -Ájuayng “Big ела}! aul} pue saul] YIMOAB jure smaeyjouayjsy saJuaJajoy ajejd jeluoqunqns эбин ешзэрочцил зиэшебт unoesouad ‘eindinos JajoeseyyD\snues ‘эерноели Alle) eu} Jo елэиэб биоше зле}оелецо jeoıbojoydiow шеш jo иозивашо) ‘z 3719v1 114 KAMENEV hai Sea (China) (Xu, 1989), should also be placed in Parvithracia (Pseudoastheno- thaerus). The subgenus name is derived from the similarity of external shell morphology of species of this subgenus to members of the genus Asthenothaerus. Parvithracia (Pseudoasthenothaerus) lukini Kamenev, new species Figs. 11-34, Table 3 Type Material and Locality Holotype (MIMB 4047), Evreinova Strait (Onekotan Island — Makanrushi Island), Kuril Islands, 100 m, silty sand, Coll. V. I. Lukin and S. |. Grebelny, 26-VIII-1987 (R/V “Tikhookean- sky”); paratypes (15): paratype (MIMB 4048) from the type locality; paratype (MIMB 4049), Fourth Kuril Strait (Onekotan Island —Рага- mushir Island), Кий Islands (49°38'5N, 155°22'3E), 500 m, sandy silt, Coll. V. I. Lukin and S. |. Grebelny, 27-X-1987 (R/V “Tikhooke- ansky”); paratype (ZIN 1), southern Sea of Okhotsk (51°35’6N, 151°46E), 418 m, silty sand, bottom water temperature of +1.53°C, salinity 33.62%, Coll. Р. V. Ushakov, 10-VII- 1932 (R/V “Gagara”); paratype (ММВ 4050), west coast of Kamchatka, Sea of Okhotsk (52°20'N, 155°18’E), 142 m, sand + silt, Coll. V. A. Nadtochy, 10-V-1989 (R/V “Mys Dalny”); paratypes (4) (MIMB 4051), Black Cape, Medny Island, Commander Islands, Bering Sea (54°41.2'N, 167°59.7’E), 150-200 m, silt, Coll. V. I. Lukin, 16-IX-1973 (R/V “Rakyt- noye”); paratype (ZIN 2), Aniva Cape, Sakhalin Island, Sea of Okhotsk (45°58'N, 143°46.3E), 207 m, sandy silt, bottom water temperature of —0.4°C, Coll. Z. I. Kobjakova, 24-1Х-1947 (R/V “Toporok”); paratype (ZIN 3), Svobodnogo Cape, Sakhalin Island, Sea of Okhotsk (46°47.5'N, 143°52.3’E), 187 m, sandy silt, bottom water temperature of —0.4°C, Coll. Z. |. Kobjakova, 4-IX-1947 (R/V “Toporok”); paratype (ММВ 4052), Morzovaja Rock, Kunashir Island, Kuril Islands, Sea of Okhotsk (44°33’8N, 146°28’7E), 101 m, silty sand + gravel, Coll. V. I. Lukin and V. P. Pavluchkov, 23-VII-1987 (R/V “Tikhookean- sky”); paratypes (2) (MIMB 4053), Kunashir Is- land, Kuril Islands, Pacific Ocean (44°08'N, 146°54’4E), 189 m, silty sand + gravel, Coll. М. |. Lukin and V. P. Pavluchkov, 19-VII-1987 (R/V “Tikhookeansky”); paratypes (2) (ZIN 4), Peter the Great Bay, Sea of Japan (42°32.2’N, 131°13.5’E), 65 m, Coll. Tarasov, 2-IX-1932. Other Material Examined One left valve (ZIN 5) from Svobodnogo Cape, Sakhalin Island, Sea of Okhotsk (46°47.5'N, 143°52.3’E), 187 т, sandy silt, bottom water temperature of —0.4°C, Coll. Z. |. Kobjakova, 4-IX-1947 (R/V “Toporok”); one left valve (MIMB 4054) from east coast of Sakhalin Island, Sea of Okhotsk, 280 m, silty sand, Coll. V. N. Koblikov, 5-VIII-1977 (R/V “8- 452”); one left valve (ZIN 6) from Tatar Strait, Sakhalin Island, Sea of Japan (49°00.4’N, 141°44.2'E), 99 m, silty sand + gravel + peb- ble, bottom water temperature of +2.3°C, Coll. Skalkin, 28-VIII-1949 (R/V “Toporok”); one right valve (ZIN 7) from Pesherny Cape, Sea of Japan, 230-238 m, silt + sand, Coll. Ohromkin, 17-VII-1931 (R/V “Rossinante”); one specimen (ZIN 8) from Peter the Great Bay, Sea of Japan (42°32.2’N, 131°13.5’E), 65 m, Coll. Tarasov, 2-IX-1932; one left valve (MIMB 4055) from Bolshoy Peles Island, Peter the Great Bay, Sea of Japan (42°31'8N, 131°23'5E), 72 m, large-particle sand, Coll. V. V. Gulbin, 19-IX-1995 (R/V “Akademik Oparin”); one specimen and left valve (MIMB 4056) from Black Cape, Medny Island, Com- mander Islands, Bering Sea (54°41.2’N, 167°59.7'E), 150-200 m, silt, Coll. V. 1. Lukin, 16-1Х-1973 (R/V “Rakytnoye”); one right valve (MIMB 4057) from Yodny Cape, Iturup Island, Kuril Islands, Pacific Ocean (44°35'5N, 147°27'5Е), 200 m, sandy silt, Coll. V. 1. Lukin and S. |. Grebelny (R/V “Tikhookeansky”). Total of 2 specimens, 5 left, and 2 right valves. Description Exterior: Shell small (< 11.0 mm), ovate-an- gular, high (H/L = 0.785-0.936), slightly in- equivalve (right valve slightly higher, more in- flated), moderately inflated (W/L of right valve 0.185-0.273; W/L of left valve 0.185-0.267), inequilateral, thin, solid. Surface with conspic- uous growth lines and fine, very dense gran- ules. Periostracum dull, thin, adherent, color- less, yellowish, pinkish or light brown, with dark brown or black patches or crusts near beaks and on dorsal shell margin, extending into inner surface. Beaks small, moderately projecting above dorsal margin, posterior to midline (A/L = 0.585-0.765), slightly rounded, opisthogyrate. Anterior end rounded. Poste- rior end narrow, truncate, with faint radial ridge from beaks to ventral limit of posterior end. Anterodorsal margin straight or slightly convex, gently descending ventrally, smoothly THE BIVALVE GENUS PARVITHRACIA its FIGS. 11-24. Parvithracia (Pseudoasthenothaerus) lukini Kamenev, new species. 11-15: Holotype (MIMB 4047), Evreinova Strait (Onekotan Island — Makanrushi Island), Kuril Islands, 100 m, shell length 9.9 mm. 16: Lithodesma of holotype, ventral view (bar = 1 mm). 17-22: Paratypes (MIMB 4051), Black Cape, Medny Is- land, Commander Islands, Bering Sea (54°41.2’N, 167°59.7'E), 150-200 m. 17, 18: Left and right valves of a young specimen (bar = 1 mm). 19: Dorsal view of both valves of a young specimen (bar = 1 mm). 20, 21: Ventral view of right and left valves showing subumbonal plate and teeth (bar = 1 mm). 22: Ventral view of lithodesma (bar = 1 mm). 23: Paratype (MIMB 4050), west coast of Kamchatka, Sea of Okhotsk (52°20'N, 155°18’E), 142 m, ventral view of lithodesma (bar = 1 mm). 24: Paratype (MIMB 4053), Kunashir Island, Pa- cific Ocean (44°08’N, 146°54’4E), 189 m, close-up of right valve showing attachment of lithodesma (ventral view) to subumbonal plate and the tooth (bar = 1 mm). 116 KAMENEV FIGS. 25-32. Parvithracia (Pseudoasthenothaerus) lukini Kamenev, new species. FIGS. 25-28, 30. Paratypes (MIMB 4051), Black Cape, Medny Island, Commander Islands, Bering Sea (54°41.2’N, 167°59.7'E), 150-200 m. 25, 26: Dorsal view of teeth on right and left valves (bar = 1 mm). 27, 28: Hinge of right and left valves (bar = 1 mm). 29: Paratype (MIMB 4050), west coast of Kamchatka, Sea of Okhotsk (52°20'N, 155°18'E), 142 m, subumbonal plate with pillars (ventral view), right valve (bar = 1 mm). 30: Sub- umbonal plate with pillars (ventral view), left valve (bar = 1 mm). 31, 32: Right valve with of specimen with reversed hinge (ventral view), Black Cape, Medny Island, Commander Islands, Bering Sea (54°41.2’N, 167°59.7'E), 150-200 m (bar = 1 mm). THE BIVALVE GENUS PARVITHRACIA 117 FIG. 33. Parvithracia (Pseudoasthenothaerus) lukini. Organs of mantle cavity as seen from right side with right shell valve and mantle removed (shell length, 6.4 mm). AA, anterior adductor muscle; APR, anterior pedal retractor muscle; DD, digestive diverticula; E, exhalant siphon; EM, edge of mantle; F, foot; HG, hind gut; I, inhalant siphon; ID, inner demibranch; К, kidney; LP, labial palp; MC, mineralized concretions; OD, outer demibranch; PA, posterior adductor muscle; PPR, posterior pedal retractor muscle; SM, siphonal mus- culature. transitioning to rounded anterior end. Ventral margin slightly curved (more curved in right valve), sometimes straight in left valve. Pos- terodorsal margin slightly concave, rather steeply descending to form a smooth angle at posterior end. Posterior end slightly curved, almost vertical, only slightly turned anteriorly to form a rounded angle at ventral shell mar- gin. Escutcheon narrow, well defined, bor- dered by ridges along entire posterodorsal margin. Interior: Right valve with anterior and pos- terior lateral teeth; left valve with anterior lat- eral tooth. In right valve, anterior lateral tooth large, triangular, strongly projecting above inner part of anterodorsal margin, anteroven- trally directed; posterior lateral tooth small, elongate, only slightly projecting or not pro- jecting above inner part of posterodorsal mar- gin. In left valve, anterior lateral tooth long, lamellate, slightly projecting above inner part of anterodorsal margin; inner part of an- terodorsal margin straight between beak and tooth; inner part of posterodorsal margin straight. Subumbonal plate short, elevated above inner shell surface. Supporting pillars high, of varying thickness, generally of the same thickness throughout their individual lengths or becoming thinner ventrally; width and height of pillars, and width of pits between pillars decreasing posteriorly from anterodor- sal margin. Number of pillars from 2 to 7, usually equal in both valves. Lithodesma large (L1/L = 0.098-0.225), butterfly-shaped, sometimes trapezoidal, often asymmetrical. Anterior adductor muscle scar large, elon- gate, kidney-shaped. Posterior adductor scar small, rounded. Pallial sinus distinct, of the same shape and size in both valves, deep, rounded anteriorly, sometimes reaching mid- line (S/L = 0.395-0.54), anterior limit short of faint vertical line from beaks. Shell interior with faint radial striae. Anatomy: Gross morphology differs little 118 KAMENEV MG FIG. 34. Parvithracia (Pseudoasthenothaerus) lukini. Internal morphology as seen from right side with right shell valve, mantle, and ctenidium removed (shell length, 6.4 mm). A, anus; AA, anterior adductor muscle; APR, anterior pedal retractor muscle; DD, digestive diverticula; F, foot; H, heart; HG, hind gut; K, kidney; LP, labial palp; MC, mineralized concretions; MG, mid-gut; O, ovary; OE, oesophagus; PA, posterior adductor muscle; PPR, posterior pedal retractor muscle; S, stomach; SS, style sac; T, testis; VG, visceral ganglia. from previous descriptions of Thracia (Kiener, 1834; Deshayes, 1846). Mantle very thin, ex- cept at ventral margin, which is slightly thick- ened, completely fused except at extensive siphonal and pedal gapes, and small, round aperture ventral to inhalant siphon. Siphons long; inhalant siphon longer, larger. Anterior adductor muscle large, elongate dorsoven- trally, slightly curved parallel to anterior end. Posterior adductor muscle smaller, almost round. Foot small, laterally compressed, pro- jecting anteroventrally, its shape in preserved specimens variable depending on degree of contraction. Anterior pedal retractor muscles ascending almost vertically from base of foot, passing dorsally above labial palps, attaching dorsal to anterior adductor muscle. Posterior pair of narrow pedal retractor muscles pass- ing on either side of anus as single muscle sheets from base of foot, attaching dorsal to posterior adductor muscle. Ctenidia thin, con- sisting of two demibranchs; inner demibranch much larger than outer. Paired labial palps long, elongate-triangular, of equal length. Mouth small, situated close to anterior adduc- tor muscle, at basal junction of inner and outer palps. Oesophagus long, straight, extending parallel to anterodorsal side of body, opening into anterodorsal part of stomach. Stomach very large, oval, lying along dorsoventral axis, on right side of visceral mass; stomach wall very close to dorsal and right sides of visceral mass; much of stomach on right side not sur- rounded by digestive diverticula (stomach contents may be visible through the translu- cent walls), with a combined style sac and mid gut. Combined style sac and mid gut leaving stomach at posteroventral border, passing ventrally into visceral mass. Mid-gut separate from style sac, forming large number of short THE BIVALVE GENUS PARVITHRACIA 119 loops extending anteriorly ventral to stomach almost as far as mouth, turning back along dorsal margin of-foot to style sac. Hind gut thickened, turning dorsally between paired transparent ovaries, passing through heart and posteriorly along posterodorsal margin of body above kidney, partially circling posterior adductor muscle, terminating near exhalant siphon as an anal papilla free of attachments. Digestive diverticula surrounding oesophagus and greater part of stomach. Kidneys large, posterodorsal between posterior adductor muscle and ovaries, containing large number of mineralized concretions. Cerebral ganglia consisting of two small commissural bodies between mouth and anterior adductor muscle. Pedal ganglia large, Iying at interface of foot and viscera. Visceral ganglia large, conspicu- ous, lying ventral to kidney, in contact with in- testine near anus. This species is a simulta- neous hermaphrodite. Testes occupying a ventral position in visceral mass dorsal to foot. Paired ovaries occupying a posterior position in visceral mass, appearing as transparent sacs with large eggs (up to 150-200 um). Variability Shell shape and proportions vary markedly (Table 3). The shell shape varies from ovate- angular to rounded. Shell height and width, the position of the beaks, the degree of con- cavity of posterodorsal margin, and the rela- tive length of pallial sinus vary. The shape, length, and width of lithodesma is also markedly variable, but it is most often butter- fly-shaped. In young specimens, it is some- times trapezoidal. The number of pillars varies and, as a rule, increases with shell size; some of them bifurcate ventrally or are fused. The sizes and shapes of the lateral teeth in both valves, and the inclination of an- terior lateral tooth in right valve vary little. One specimen has the hinge reversed (Figs. 31, 32); its left valve has two lateral teeth, the shape and size of which are identical to the teeth of right valve of other specimens exam- ined. The right valve possesses an anterior lamellate tooth analogous to the tooth of left valve of normal specimens. Distribution and Habitat (Fig. 35) In the Bering Sea, Р (Pseudoastheno- thaerus) lukini occurs near Medny Island, Commander Islands; in the Sea of Okhotsk — near the west coast of Kamchatka, near Sakhalin Island, and in the southern part of the sea (51°35’6N, 151°46E); near the Kuril Islands —in Evreinova and Fourth Kuril Straits, near Iturup and the Kunashir Islands; in the Sea of Japan—in Tatar Strait (near Sakhalin Island), near Pesherny Cape, and in Peter the Great Bay. In the Bering Sea, this species was ob- tained at depth 150-200 m on silt; in the Sea of Okhotsk — from 142 m (west coast of Kam- chatka) to 418 m (southern Sea of Okhotsk) on silty sand and sandy silt at a bottom tem- perature from —0.4°C (Svobodnogo and Aniva Capes, Sakhalin Island, depth 187 and 207 m, respectively) to 1.53°C (southern Sea of Okhotsk, depth 418 m); near the Kuril Is- lands —from 100 m (Evreinova Strait) to 500 m (Fourth Kuril Strait) on silty sand and sandy silt sometimes with some admixture of gravel; in the Sea of Japan—from 65 m (Peter the Great Bay) to 238 m (Pesherny Cape) on large-particle sand, silty sand and silt some- times with admixture of gravel and pebbles at a bottom temperature 2.3°C (Tatar Strait, Sakhalin Island, depth 99 m). Comparisons Parvithracia (Pseudoasthenothaerus) lukini is easily distinguished from Parvithracia (Pseudoasthenothaerus) sematana and P (Pseudoasthenothaerus) isaotakii in having a thicker, heavier shell (Table 4). Moreover, in contrast to P. (Pseudoasthenothaerus) se- matana, this species has a higher, less elon- gate shell, with a highly elevated subumbonal plate and distinct, high pillars. Unlike Р (Pseudoasthenothaerus) isaotakii, it has a more oval shell, with a considerably less ex- panded posterior end, and it has opisthogy- rous beaks. This species is most similar to Parvithracia (Pseudoasthenothaerus) sirenkoi; but differs in having a more elongate, oval, lower, and less inflated shell. The shell of Parvithracia (Pseudoasthenothaerus) lukini is more ex- panded anteriorly, the beaks are more poste- rior, the lateral teeth in both valves are smaller, and there is a deeper pallial sinus. Etymology The specific name honors Dr. Vladimir 1. Lukin, a famous Russian researcher of the marine fauna of the Kuril Islands, who has col- 120 KAMENEV TABLE 3. Parvithracia (Pseudoasthenothaerus) lukini Kamenev, new species. Shell measurements (mm), indices and summary statistics of characters: L— shell length; H— height; W — width; А — anterior end length; $ — maximal distance from the posterior shell margin to the top of pallial sinus; L1 —lithodesma length; N — number of pillars under subumbonal plate. Numerator indicates shell measurements and indices for the right valve, denominator — for the left valve. NM — not measured. Statistics L H W A SA A W/L A/L S/L L1/L Depository Holotype 99 80 25 67 46 1.7 6 0808 0.253 0.677 0.465 0.172 MIMB 98 78 22 65 45 6 0.796 0.224 0.663 0.459 0.173 4047 Paratype 97 85 26 70 43 20 4 0.876 0.268 0.722 0.443 0.206 MIMB 97 82 24 70 4.0 4 0.845 0.247 0.722 0.412 0.206 4049 Paratype 81 6.5 1.5 5.7 35 14 Z 0802 0.85 0,704 0432 "0173 ММВ 8.1, 6.4 1:5 5:7 3.4 7 0.790 0.185 0.704 0.420 0.173 4050 Paratype 7.9 6.3 18 54 34 13 5 0.797 0.228 0.84 0.430 0.165 MIMB 79 62 18 55 32 5 0.785 0.228 0.696 0.405 0.165 4053 Paratype 67 58 18 41 28 NM 4 0.866 0.269 0.612 0.418 NM ZIN 2 67157 17 212.8 4 0.851 0.254 0.612 0.418 NM Paratype 6.5 5.6 13 42 30 10 5 0.862 0.200 0.646 0.462 0.154 ZIN 1 6.5 5.6 1.3 44 3.1 4 0.862 0.200 0.677 0.477 0.154 Paratype 6.4 55 17 40 28 12 3 0.859 0.266 0.625 0.438 0.188 ZIN 3 6.4 55 1.7 40 2.8 3 0.859 0.266 0.625 0.438 0.188 Paratype 6.4 53 15 42 32 12 5 0.828 0.234 0.656 0.500 0.188 ZIN 4 6.3 52 15 43 3.4 5 0.825 0.238 0.683 0.540 0.190 Paratype 6.3 56 15 4.1 28 1.1 4 0.889 0.238 0.651 0.444 0.175 MIMB 6.35.5 15 41 2:8 5 0.873 0.238 0.651 0.444 0.175 4051 Paratype 6.3 54 14 40 29 10 4 0857 0.222 0635 0.460 0.159 MIMB 6.3 53 15 40 29 4 0.841 0.238 0.635 0.460 0.159 4048 Paratype 58 53 13 37 27 09 4 0914 0.224 0.638 0.466 0.155 MIMB 5:88 15.3 ЗАЗ 2. 4 0.914 0.224 0.638 0.466 0.155 4051 Paratype 58 48 12 43 27 10 4 0828 0.207 0.741 0.466 0.172 MIMB 5.8 4.7 1.2 43 27 4 0.810 0.207 0.741 0.466 0.172 4053 Paratype 5.3 48 11 31 24 ММ 2 0.906 0.208 0.585 0.453 NM MIMB 5.34.7 1. 3.1 2.4 3 0.887 0.189 0.585 0.453 NM 4051 Paratype 47 44 10 28 22 08 2 0936 0.213 0.596 0.468 0.170 MIMB 4.7 4A 1.1 29 22 3 0.936 0.234 0.617 0.468 0.170 4051 Paratype 41 34 08 28 1.7 04 3 0.829 0.195 0.683 0.415 0.098 MIMB 410093408228 silt 7 0.829 0.195 0.683 0.415 0.098 4052 Paratype 32, 2.9 07 2.0 16 0.5 2 0906 10:219/ 0.625 0.500’ 10.156 ZIN 4 3:27 02.8: 2077052107 ALG 2 0.875 0.219 0.625 0.500 0.156 11.0 96 3.0 7.0 45 22 7 0.873 0.273 0.636 0.409 0.200 ММВ NM NM NM NM NM NM NM NM NM NM NM 4057 NM NM NM NM NM NM NM NM NM NM NM NM ZIN5 9.0 7.8 24 54 3.8 6 0.867 0.267 0.600 0.422 NM NM NM NM NM NM 2.0 NM NM NM NM NM NM MIMB 8:9. 77.8 2.0 26.41 4.0 6 0.876 0.225 0.685 0.449 0.225 4054 86 7.1 21 62 34 15 7 0826 0.244 70.721 (0/3895 0174 ZIN 8 8:5 7.0" (2/0). 6:5 8.7: 7 0.824 0.235 0.765 0.435 0.176 THE BIVALVE GENUS PARVITHRACIA 121 TABLE 3. (Continued) Le A М А Е М H/L W/L AL S/L L1/L Depository NM NM NM NM NM NM NM NM NM NM NM NM ZIN 6 MOTOS 1.9: 5:7 3.6 5 0.872 0.244 0.731 0.462 NM NM NM NM NM NM NM NM NM NM NM NM NM MIMB PARC TRI 43 3:2 5 0.859 0.239 0.606 0.451 NM 4056 6.0 50 13 4.0 29 ММ 4 0.833 0.217 0.667 0.483 NM ZIN 7 NM NM NM NM NM NM NM NM NM NM NM Mean 6.77 5.77 1.57 4.49 3.02 1.25 6.87 5.82 1.58 4.59 3.07 0.851 0.228 0.664 0.450 0.171 SD 2.00 1.65 0.64 1.46 0.82 0.51 0.040 0.027 0.043 0.029 0.024 1.78 1.43 0.48 1.35 0.74 0.039 0.023 0.051 0.032 0.027 SE 0.46 0.38 0.15 0.34 0.19 0.12 — 0.009 0.006 0.010 0.007 0.006 0.39 0.31 0.10 0.30 0.16 0.008 0.005 0.011 0.007 0.007 Min 32 72:8) 0:4 20 1.6 0:40’ 2 0797 (08185 0585 0.395 0.098 3:22 52:0) 0 20 16 2 0.785 0.185 0.585 0.405 0.098 Мах ЕО OIG. 308 5720), 4.6; 2.20 их 01936 0:273 074417 0.500) 0.206 OBE 8:2 24 70 45 } 0936’ 0:267 0.765 10.5407 0'225 п 19. 19. 10.180 18 1 10 8 19 19 19 16 PA) PA ah il РЯ 2 21 21 21 16 140° Russia Bering Sea Sea of Okhotsk y 2 Commander Islands Sakhalin Is. Makanrushi Is o Paramushir Is. A че Onekotan Is. 9 0 0 0 Tatar Stfait o I Iturup Is. JA Japan e Kunashir ls. / Peter the Great Bay Pacific Ocean 40° Sea of Japan ie LE | FIG. 35. Distribution of Parvithracia (Pseudoasthenothaerus) lukini (№ type locality). KAMENEV 122 RR == 0 2 — parno (6t'0) auipiuu Sulyoea, ‘Апезлор moJJeu yous juasqe рэрэлр Айед -иэл ‘бицоэо.а A/Buons moJJeu Алэл juasqe (g'o) eue ‘eye1ABoyyo ‘Bunoaloıd Alayeıapow Анеэшеэл pepuedxe papuno, Aldıeys (120) payeyuı Ajayesepow 67 (98`0) ayenogns JusJedsue4} jou ‘elle, pequosep jou (Z€°0) euljpiw би! -U9891 jou ‘Ajjesiop реола yous Алэл Jussqe pspanıp Апедиэлолэие ‘Bunoaloıd ABuous MOMEU Jussqe (590) auıpı о} souaysod ‘эуелАбоц$!Ао ‘Bunoaloıd А|эуелэрош Анеэошел pepuedxe jou papuno, Ajmoueu (vz'0) perequi Alayesspow L'9 (2/`0) ayebuo¡a-ajero }уэледзиед ‘elfe pedeys-Ajpeyng (6e'0) aunpiu би! -U9891 jou ‘Ajjesiop moJJeu yBly Jussqe pajoaJip Апед -иэл ‘бицоэГола ABuous эрим juasqe (€9°0) euypiuu 0} iouaysod ‘эуелАбо -yisido ‘бицоэ[ола ^!буо$ Анеэцел papuedxa papuno, Aldıeys (92°0) разеци! 6'8 (06:0) ечобц}-ауело juaJedsue,) jou “pijos редецз-Ашезпа (St'0) aupiu Buiyoeei seuljeuos ‘Alesıop peoiq yBiy Jussqe pspanıp Апедиэлолэие ‘бицоэола Авчби$ MOJBU juasqe (p9'0) eupiuu 0} Jo1a]sod ‘ayesABo -yisido ‘Bunoaloıd ABuous Alesıuan pepuedxe jou рэрипол (£z'0) payeyu! Alayeıapow Ott (58:0) Jeinbue-syeno }иэледзиед jou “pijos ерю7э4е-э}ело (870) эиирии бицоеэ/ saw} -эш0$ ‘А|е$лор peoiq juasqe juasaid pajoaJip Alle -u9A ‘Bunoaloid AlBuoNs MOLEU BAILA YO] Ul JUBSId (9s'0) aunprus о} 1011 -a1sod Ацчби$ ‘эуелАбо -yuo ‘бицоэГо. А ABuous Анеэцэл pepuedxe jou pepunoi Ajdieus (/2`0) payeyul 09 (980) ¡euoBuqns jualedsue,] JOU “pijos edeus eusapoyy7] (1/5) snuis reed syeıd ¡euoquinqns jo Sie]|Id ulBuew IIays jesiop jo ped jeulajul UO SAODOIH SALA 1yBu Jo 4400} JOLISJUY uoayamosy ajnun7 (1/v) syeag ulBsew [Jays 10118}S04 иблеш ||э4$ лоиаи\у (1/M) зэмел ww ‘цбча| ‘хеш |эц$ (7/H) edeys печ$ 194$ PON AS AP SS AP ee ee eee oo INyBJOBSI (snıseyJouayIseopnasg) BIDBIYJIAICY (2012120000) (snieeyjoueyseopnesd) BREIYJINIBZ JOyualls (snuaeyjoueyjseopnesd) ERBIYJINIEZ Jun] (snuaeyjouayjseopnesd) BIDBIYJIAIEg иадп$ (вэвелцилаед) вовцилеа sıepeleyg a A A it A AA A ee “snuis jeijjed jo do} eu} о} шблеш [Jays 1018} -sod au} шод энер jewıxew— $ ‘щбие!рие лонаие — y “yjpim эмел— M зубец — H ‘щбие! |ецз — 7 ‘вювлциллеч JO sergads jo s1eJoe1eyo Bueyususyig ‘+ 37191 THE BIVALVE GENUS PARVITHRACIA 123 lected many of the specimens examined in this paper. Parvithracia (Pseudoasthenothaerus) sirenkoi Kamenev, new species Figs. 36-54, Table 5 Type Material and Locality Holotype (MIMB 4058), Iturup Island, Ku- ril Islands, Pacific Ocean (44°52'N, 149° 27.7'E), 910-920 m, silty sand, Coll. B. I. Sirenko, 25-\1-1984 (R/V “Odissey”); paratypes (10): paratypes (2) (MIMB 4059) from holotype locality; paratypes (2) (ZIN 1), Кигир Island, Kuril Islands, Pacific Ocean (44°46.8’N, 149°06.7'E), 880 m, silty sand, Coll. B. |. Sirenko, 26-VII-1984 (R/V “Odis- sey”); paratype (ZIN 2), Iturup Island, Kuril Is- lands, Pacific Ocean (44°02.6'N, 148°11’E), 600 m, silty sand + gravel + pebbles, Coll. B. |. Sirenko, 22-IX-1984 (R/V “Odissey”); paratypes (4) (MIMB 4060), Kuril Islands, Coll. V. I. Lukin, 1987 (R/V “Tikhookeansky”); paratype (MIMB 4061), Yury Island, Kuril Is- lands, Pacific Ocean (43°10’N, 146°17’E), 490 m, silty sand + gravel + pebbles, bottom water temperature of +2.33°C, Coll. V. I. Lukin FIGS. 36-48. Parvithracia (Pseudoasthenothaerus) sirenkoi Kamenev, new species. 36-40: Holotype (ММВ 4058), Iturup Island, Kuril Islands, Pacific Ocean (44°52’N, 149°27.7'), 910-920 m, shell length 8.3 mm. 41-45: Paratype (MIMB 4060), Kuril Islands, shell length 8.0 mm. 46-48: Paratypes (MIMB 4059) from type locality. 46, 47: Right and left valves of a young specimen (bar = 1 mm). 48: Dorsal view of both valves of a young specimen (bar = 1 mm). 124 KAMENEV FIGS. 49-54. Parvithracia (Pseudoasthenothaerus) sirenkoi Kamenev, new species. 49, 50: Paratype (MIMB 4060), Kuril Islands, hinge of the left and right valves (bar = 1 mm). 51, 52: Paratypes (ZIN 2), Iturup Island, Kuril Islands, Pacific Ocean (44°02.6'N, 148°11’E), 600 m. 51: Subumbonal plate with pillars (ventral view), left valve (bar = 1 mm). 52: Close-up of right valve showing attachment of lithodesma (ventral view) to subumbonal plate and teeth (bar = 1 mm). 53: Paratype (MIMB 4060), Kuril Islands, ventral view of lith- odesma (bar = 1 mm). 54: Paratype (ZIN 1), Iturup Island, Kuril Islands, Pacific Ocean (44°46.8'N, 149°06.7'), 880 m, dorsal view of teeth on right valve (bar = 1 тт). and V. P. Pavluchkov, “Tikhookeansky”). 8-VII-1987 (R/V Other Material Examined Two right and one left valves (MIMB 4062) from type locality; one specimen (ZIN 3) from lturup Island, Kuril Islands, Pacific Ocean (44°59'N, 148°47'E), 450 m, silty sand + peb- bles, Coll. B. I. Sirenko, 24-IX-1984 (R/V “Odissey”); one specimen (ZIN 4) from south- western Sea of Okhotsk (45°06’N, 143°43.5'E), 188 m, silt, bottom water tem- perature of +0.5°C, Coll. Yu. I. Galkin, 25-VIII- 1948 (R/V “Toporok”); one left valve (ММВ 4063) from Morzovaja Rock, Kunashir Island, Kuril Islands, Sea of Okhotsk (44°33’8N, 146°28'7E), 101 m, silty sand + pebbles, Coll. V. |. Lukin and V. P. Pavluchkov, 23-VII-1987 (R/V “Tikhookeansky”). Total of 2 specimens, 2 left, and 2 right valves. Description Exterior: Shell small (<8.9 mm), ovate-trig- onal, high (H/L = 0.84-0.963), slightly in- equivalve (right valve slightly higher, more inflated), inflated (W/L of right valve 0.211- THE BIVALVE GENUS PARVITHRACIA 125 0.313; W/L of left valve 0.237-0.293), inequi- lateral, thin, solid. Surface with conspicuous growth lines and fine, very dense granules. Periostracum dull, thin, adherent, colorless, pinkish or light brown, with dark brown or black patches or crusts near beaks and dorsal shell margin, extending into inner surface. Beaks small, high, projecting considerably above dorsal margin, posterior to midline (A/L = 0.568-0.72), slightly rounded, opisthogy- rate. Anterior end sharply rounded. Posterior end higher than anterior, decidedly truncate, with a faint radial ridge extending from beaks to ventral limit of posterior end. Anterodorsal margin straight or slightly convex, very steeply descending, smoothly curving to rounded anterior end. Ventral margin slightly curved (more curved in right valve). Pos- terodorsal margin straight or slightly concave, descending and forming a distinct angle with posterior end. Posterior end slightly curved, descending ventrally or slightly anteriorly, forming a rounded angle with ventral shell margin. Escutcheon wide, well defined, de- marcated by ridges extending along pos- terodorsal shell margin from beaks to poste- rior end. Interior: Right valve with anterior and pos- terior lateral teeth; left valve with anterior lat- eral tooth. In right valve, anterior lateral tooth large, high, triangular, projecting considerably above inner part of anterodorsal margin, ven- trally (Sometimes anteroventrally) directed; right posterior lateral tooth small, elongate, slightly projecting above inner part of pos- terodorsal margin, extending along pos- terodorsal margin. In left valve, anterior lateral tooth long, lamellate, strongly projecting above inner part of anterodorsal margin; inner part of anterodorsal margin slightly concave between beak and tooth; inner part of pos- terodorsal margin slightly convex. Subum- bonal plate short, elevated above inner shell surface. Pillars supporting plate high, varying in thickness between each other, generally of same thickness throughout their individual lengths or becoming thinner ventrally; width and height of pillars, and width of pits between pillars decreasing posteriorly from anterodor- sal margin. Number of pillars from 2 to 6, usually equal in number in both valves. Lith- odesma large (L1/L = 0.067-0.225), butterfly- shaped, sometimes ovate-trapezoidal, often asymmetrical. Anterior adductor muscle scar large, elongate, kidney-shaped. Posterior ad- ductor scar small, rounded. Pallial sinus dis- tinct, of the same shape and size in both valves, deep, narrow and rounded anteriorly, not reaching midline (S/L = 0.2-0.447), ante- rior limit short of faint vertical line from beaks. Shell interior with dense, tiny pockmarks and faint radial striae. Anatomy: Internal morphology similar to that of Р (Pseudoasthenothaerus) lukini. Mantle very thin, except at ventral margin, which is slightly thickened, completely fused except for the small pedal and three pallial apertures. Siphons long; inhalant siphon longer, larger. Anterior adductor muscle large, elongate dorsoventrally, slightly curved paral- lel to anterior shell margin. Posterior adductor muscle smaller, almost round. Foot small, lat- erally compressed, projecting anteroventrally. Anterior pedal retractor muscles almost verti- cally from base of foot, passing above labial palps to attach dorsal to anterior adductor muscle. Posterior pair of narrow pedal retrac- tor muscles passing on either side of anus as a single muscle sheets from base of foot to at- tach dorsal to posterior adductor muscle. Ctenidia thin, consisting of two subequal demibranchs. Paired labial palps long, elon- gate-triangular, of equal length. Mouth small, close to anterior adductor muscle at basal junction of inner and outer palps. Oesopha- gus long, straight, extending parallel to an- terodorsal side of body, opening to anterodor- sal part of stomach. Stomach very large, oval, lying along dorsoventral axis, in right side of visceral mass; stomach wall located close to dorsal and right sides of visceral mass, much of stomach on right side not surrounded by di- gestive diverticula (stomach contents some- times visible through the translucent walls). Combined style sac and mid-gut leaving stomach at posteroventral border and passing ventrally into visceral mass. Hind gut thick, passing dorsally between paired, transparent ovaries, through heart, posteriorly along pos- terodorsal margin of body above kidney, passing over posterior adductor muscle, ter- minating near exhalant siphon. Anal papilla free. Digestive diverticula surrounding oe- sophagus and stomach. Kidney large, oc- cupying a posterodorsal position between posterior adductor muscle and ovaries. Vis- ceral ganglia large, conspicuous, lying below kidney, touching intestine near anus. This species is a simultaneous hermaphrodite. Testes occupying a ventral position in visceral mass dorsal to foot. Paired ovaries occupying a posterior position in visceral mass, appear- ing as transparent sacs with large eggs (up to 150-200 um). 126 KAMENEV Variability Shell shape and proportions change little with age. In young specimens (<6 mm), the shell is less inflated (Table 5). In adults, shell height, width, the position of beaks, and rela- tive length of the pallial sinus vary slightly. The sizes and shape of the lateral teeth in both valves, and the inclination of the anterior tooth in the right valve are fairly variable. The shape, length, and width of lithodesma can vary considerably, but it is generally butterfly- shaped. The number of pillars supporting the subumbonial plate varies and correlates with shell size. Distribution and Habitat (Fig. 55) South Kuril Islands: Yury Island (43°10’N, 146°17'E), Kunashir Island (44°33’8N, 146° 28'7E), Iturup Island (44°59'N, 148°47'E; 44°52'N, 149°27.7'E; 44°46.8'N, 149°06.7'E; 44°02.6'N, 148°11’E); southwestern Sea of Okhotsk (45°06'N, 143°43.5’E). This species was recorded off the South Kuril Islands at depth from 101 m (Kunashir Island) to 910-920 m (Iturup Island) on silty sand, sometimes with some pebbles and gravel; in the southwestern Sea of Okhotsk, it was present at a depth of 188 m, on silt, at a bottom water temperature of +0.5°C. Comparisons This species is easily distinguished from other species of this subgenus by its high, ovate-triangular shell with a narrow anterior end (Table 4). Moreover, P. (Pseudoastheno- thaerus) sirenkoi differs from P. (Pseudoas- thenothaerus) sematana and P. (Pseudoas- thenothaerus) isaotakii by its thicker, more solid shell with a larger lateral tooth. It is clos- est to Р (Pseudoasthenothaerus) lukini, dif- fering from it in having a higher, more inflated shell, higher and less posteriorly placed beaks, more steeply sloping anterodorsal and posterodorsal shell margins, a smaller apical angle, larger lateral teeth in both valves, the inner part of the posterodorsal margin convex in left valve, and a shallower pallial sinus. Mean values and variances of the indices characterizing the relative height (H/L) and width (W/L) of the shell, as well as the position of beaks (A/L), and the relative length of the pallial sinus (S/L) were significantly different in these species (Table 6). Discriminant analysis of all lots containing P. (Pseudoasthenothaerus) lukini and P. (Pseudoasthenothaerus) sirenkoi showed that these species differ significantly in a com- plex of parameters (Wilks Lambda = 0.3331, F-value = 15.3134, df = 3, 23, Squared Maha- lanobis Distance = 8.1063, p < 0.0001; Means of Cannonical variables: P. (Pseudoastheno- thaerus) lukini = —1.21764; Р (Pseudoas- thenothaerus) sirenkoi = 1.52205). Off 30 specimens analysed, 28 were accurately classified (93.33%), with two specimens mis- takenly classified as P. (Pseudoastheno- thaerus) lukini. The most significant indices for dividing all specimens into two species were S/L for right valves, and W/L, and A/L for left valves (Table 7). Etymology The specific name honors Dr. Boris |. Sirenko, Zoological Institute - St. Petersburg, who has collected almost all ofthe specimens of this species examined for this paper. Parvithracia (Pseudoasthenothaerus) sematana (Yokoyama, 1922) Figs. 56-60, 65, Table 8 Thracia sematana Yokoyama, 1922: 173, pl. 14, figs. 17, 18. Parvithracia sematana (Yokoyama, 1922), Oyama, 1973: 120, pl. 57, figs. 13, 14. Asthenothaerus sematensis (Yokoyama, 1922), Ito, 1989: 66, pl. 28, fig. 3. Asthenothaerus sematanus (Yokoyama, 1922), Tsuchida & Kurozumi, 1996: 15, 16. Asthenothaerus sematana (Yokoyama, 1922), Habe, 1977: 312; Higo et al., 1999: 524; Okutani, 2000: 1039, but not pl. 517, fig. 6, which is Trigonothracia pusilla Trigonothracia pusilla (Gould, 1861), Oku- tani, 2000: pl. 517, fig. 8 Type Material and Locality Lectotype (CM 21507, left valve) and para- lectotype (CM 21508, right valve) (Oyama, 1973). Shito, Semata, Ichihara City, Chiba Prefecture, central Honshu, Japan (Pleis- tocene) (Yokoyama, 1922; Higo et al., 1999). Material Examined 1 lot (NSMT Mo 48820) from Jyogashima, Miura Peninsula, Japan, 73-85 m (3 right and THE BIVALVE GENUS PARVITHRACIA 127 TABLE 5. Parvithracia (Pseudoasthenothaerus) sirenkoi Kamenev, new species. Shell measurements (mm), indices and summary statistics of all characteristics: L — shell length; H — height; W — width; А — anterior end length; S — maximal distance from the posterior shell margin to the top of pallial sinus; L1 —lithodesma length; N— number of pillars under the subumbonal plate. Numerator indicates shell measurements and indices for the right valve, denominator — for the left valve. NM — not measured. Statistics L МА М НЕ W/L AL S/L Li/L Depository Holotype SONO 2:5 Desc. 71.37 А 0876 0301 05663 0:398 0:155 ММВ 83172124 53 33:3 4 0.863 0.289 0.639 0.398 0.155 4058 Paratype 80 74 24 53 33 15 5 095 0.300 0.663 0.413 0.188 MIMB 8:07 73 2352 33 5 0.913 0.288 0.650 0.413 0.188 4060 Paratype Ви 2:5 5.0 32 18 5 0963 0.313’ (01625 0.400 0.225 ММВ 8097745723503 2 NM 0.925 0.288 0.625 0.400 0.225 4060 Paratype ПИ Э.В 29:5 2577 10948: 107299 00/6231 0377040195 MIMB ИИ ke 12:27 74:8) 72-6 4 0.935 0.286 0.623 0.338 0.195 4060 Paratype 74 67 20 46 31 13 4 0905 0270 0.622 0.419 0.176 MIMB 74 6.6 2.0 46 2.8 4 0.892 0.270 0.622 0.378 0.176 4060 Paratype 62 5.3 16 39 25 1.0 4 0855 0.258 0.629 0.403 0.161 ZIN 2 629587 1:5 39 2.5 4 0.855 0.242 0.629 0.403 0.161 Paratype 5.4 48 13 33 23 NM 3 0.889 0.241 0.611 0.426 NM ZIN 1 5.4 48 13 33 2.2 3 0.889 0.241 0.611 0.407 NM Paratype 54 47 13 34 23 08 2 0.870 0.241 0.630 0.426 0.148 MIMB 54-46113 23:47 22:3 2 0.852 0.241 0.630 0.426 0.148 4059 Paratype 0.860 0.240 0.640 0.400 0.160 MIMB 0.840 0.240 0.640 0.400 0.160 4059 Paratype 4.5 40 11 28 09 03 3 0.889 0.244 0.622 0.200 0.067 ZIN 1 5:0) 4.312 32 20 0:8 ND IN 4.5 40 1.1 28 0.9 3 0.889 0.244 0.622 0.200 0.067 Paratype 3.8 36 0.8 22 1.7 06 2 0.947 0.211 0.579 0.447 0.158 ММВ 3.8 36 0.9 22 17 2 0.947 0.237 0.579 0.447 0.158 4061 NM NM NM NM NM NM NM NM NM NM NM NM MIMB 89 80 24 52 3.8 6 0.899 0.270 0.584 0.427 NM 4063 NM NM NM NM NM NM NM NM NM NM NM NM MIMB SD ON NS ONE Er 2 0.878 0.293 0.720 0.378 NM 4062 6.6 6.0 18 44 25 ММ 4 0.909 0.273 0.667 0.379 NM MIMB NM NM NM NM NM NM NM NM NM NM NM 4062 58151 14 39 21 0913 0879’ 024100672062 04155 ZIN 3 5.8 51 14 39 2.1 3 0.879 0.241 0.672 0.362 0.155 31811315 08 2:47 1.5 ММ’ 2 101921 0241477 0163270895 NM MIMB NM NM NM NM NM NM NM NM NM NM NM 4062 Mean 6.27 5.68 1.69 3.95 2.47 1.13 — 0.904 0.262 0.630 0.392 0.168 6.71 5.99 1.79 4.22 2.62 0.890 0.263 0.628 0.387 0.168 SD 1.59 1.53 0.61 1.04 0.75 048 — 0.034 0.033 0.030 0.058 0.041 1.63 1.51 0.56 1.07 0.78 0.031 0.022 0.037 0.059 0.041 SE 0.41 0.39 0.16 0.27 0.19 0.14 — 0.009 0.009 0.008 0.015 0.012 0.42 0.39 0.14 0.28 0.20 0.008 0.006 0.010 0.015 0.012 Min 3:0) 535" 20:87 22 70:9 0°30) 277:0:85577.0211,.0:56877.0:200770!067 3.8 36 09 22 09 2 0.840 0.237 0.568 0.200 0.067 Max 8.3 7.7 25 55 35 180 5 0.963 0.313 0.672 0.447 0.225 8.97 8:0 24 59 38 6 0.947 0.293 0.720 0.447 0.225 п 1 Чо Ша Ша 1 1 Ч 18 15 15 15 12 —^ al _ a > a an a —^ a => al => a E a a a A a a № 128 KAMENEV Russia Bering Sea Sea of Okhotsk N N Commander Islands Sakhalin Is. re = mm Is. 9 | Japan CSN Kunashir Is. ; Pacific Ocean 40 | Sea of Japan bes ie 7. | ae eS) FIG. 55. Distribution of Parvithracia (Pseudoasthenothaerus) sirenkoi (№ type locality). TABLE 6. Results of comparison by pairs of mean values (Student (t) test) and variances (ANOVA) of indices of right and left valves of Parvithracia (Pseudoasthenothaerus) lukini and P. (Pseudoasthenothaerus) sirenkoi: L— shell length; H —height; W — width; A— anterior end length; S — maximal distance from posterior margin to the top of pallial sinus; L1 —lithodesma length. P.— probability that index values in Р (Pseudoasthenothaerus) lukini and P. (Pseudoasthenothaerus) sirenkoi are drawn from the same population; n— number of valves of P. (Pseudoasthenothaerus) lukini and P. (Pseudoasthenothaerus) sirenkoi, respectively. Indices Right valves Left valves ANOVA ANOVA t P F (1, 26) P n t P Е (25) P n H/L -3.59 0.001 9.93 0.004 19,15 -3.09 0.004 9.13 0.006 21, 14 W/L -3.16 0.003 11.36 0.002 19,15 -2.09 0.044 18.48 <0.001 21, 15 АЛ- 2.16 0.038 6.74 0.015 19, 15 2.27 0.030 11.36 0.002 21, 15 S/L 3.82 <0.001 10.93 0.003 19, 15 2.81 0.008 12.86 0.001 21, 15 L1/L 0.13 0.901 0.02 0.900 16,12 -0.76 0.454 <0.01 0.989 15, 12 THE BIVALVE GENUS PARVITHRACIA 129 TABLE 7. Results of discriminant analysis for the most significant characteristics for dividing all spec- imens into species of Parvithracia (Pseudoas- thenothaerus) lukini (17 spec.) and Р (Pseudo- asthenothaerus) sirenkoi (13 spec.): L—shell length; W—width; A —anterior end length; S— maximal distance from the posterior shell margin to the top of pallial sinus. Significant Standard- charac- ized Wilks Valves teristics coefficient Lambda P Right S/L —0.7335 0.4930 0.015 Left W/L 0.5985 0.4331 0.003 Left A/L -0.5970 0.4228 0.021 1 left valves); 1 lot (CAS 63354) from gami Bay, Honshu, Japan (2 right and 1 left valves). Total of 5 right and 2 left valves. Description Expanded from Yokoyama (1922)—Exte- rior: Shell small (<6.6 mm), elongate, ovate- elongate, moderately high (H/L = 0.656- 0.776), slightly inequivalve (right valve slightly higher), moderately inflated (W/L = 0.212- 0.283), inequilateral, thin, fragile, translucent, white. Surface with faint growth lines and very fine granules. Periostracum dull, thin, adher- ent, colorless. Beaks small, moderately pro- jecting above dorsal margin, posterior to mid- line (AL = 0.618-0.655), slightly rounded, opisthogyrate. Anterior end narrowly rounded. Posterior end truncate, with a faint radial ridge extending from beaks to where posterior end meets ventral margin. Anterodorsal margin straight or slightly convex, smoothly descend- ing and smoothly transitioning to rounded an- terior end. Ventral margin slightly curved, sometimes straight in left valve. Posterodor- sal margin slightly concave, smoothly de- scending, forming a smooth angle with poste- rior end. Posterior end slightly curved, almost vertical, only slightly turned anteriorly to form a rounded angle where it meets ventral mar- gin. Escutcheon narrow, well expressed, de- marcated by ridges along posterodorsal mar- gin from beaks to posterior end. Interior: Right valve with anterior and pos- terior lateral teeth; left valve with anterior lat- eral tooth. In right valve, anterior lateral tooth FIGS. 56-64. Shells of Parvithracia species from Japan. 56-60. Parvithracia (Pseudoasthenothaerus) se- matana (Yokoyama, 1922) (NSMT Mo 48820), Jyogashima, Miura Peninsula, Japan, 73-85 m. 56, 57: Left valve, length 6.2 mm. 58, 59: Right valve, length 5.8 mm. 60: Subumbonal plate with pillars (ventral view), left valve (bar = 1 mm). 61-64. Parvithracia (Pseudoasthenothaerus) isaotakii (Okutani, 1964) (NSMT-Mo 71463), Tosa Bay, Japan, Pacific Ocean (33°05,6'N, 133°41,4’E), 800 m, shell length 7.6 mm. 130 KAMENEV large, triangular, projecting considerably above inner part of anterodorsal margin, an- teroventrally directed; posterior lateral tooth small, elongate, slightly projecting above inner part of posterodorsal margin, extending along posterodorsal margin. In left valve, an- terior lateral tooth long, lamellate, slightly pro- jecting above inner part of anterodorsal mar- gin; inner part of anterodorsal shell margin slightly concave between beak and tooth; inner part of posterodorsal margin slightly convex. Subumbonal plate short, slightly ele- vated above inner shell surface, almost com- pletely attached to shell wall, free along its an- teroventral margin. Pillars very short, sometimes lying on shell wall, but not sup- porting subumbonal plate. Number of pillars from 2 to 4. Anterior adductor muscle scar large, elongate, kidney-shaped. Posterior ad- ductor scar small, rounded. Pallial sinus dis- tinct, of the same shape and size in both valves, deep, rounded anteriorly, not reaching to midline (S/L = 0.358-0.385), anterior limit short of faint vertical line from beaks. Shell in- terior with faint radial striae, noticeable along ventral shell margin. Variability Shell shape and proportions vary little (Table 8). Pillars supporting the subumbonal plate are sometimes difficult to see and count. Distribution and Habitat Parvithracia (Pseudoasthenothaerus) se- matana occurs off Japan: in the Pacific Ocean — in Otsushi Bay (Honshu) (Tsuchida & Kurozumi, 1996), near Boso Peninsula and TABLE 8. Parvithracia (Pseudoasthenothaerus) sematana (Yokoyama, 1922). Shell measurements (mm), indices and summary statistics of all characteristics: L— shell length; H — height; W — width; A — anterior end length; $ — maximal distance from the posterior shell margin to the top of pallial sinus; N — number of pillars under the subumbonal plate. Numerator indicates shell measurements and indices for the right valve, denominator — for the left valve. NM — not measured. Statistics L H W A S N H/L W/L AL S/L Depository Lectotype NM NM NM NM NM NM NM NM NM NM CM 21507 6.6 48 1.7 NM NM NM 0.27 0.258 NM NM Paralectotype 61 40 13 NM NM NM 0.656 0.213 NM NM CM 21508 NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NSMT Mo 48820 6.2 42 15 40 2.3 ММ 0.77 0.242 0.639 0.371 58 45 16 38 2.1 NM 0.76 0.276 0.655 0.362 NSMT Mo 48820 NM NM NM NM NM NM NM NM NM NM 5.5 39 14 34 2.1 NM 0.709 0.255 0.618 0.382 NSMT Mo 48820 NM NM NM NM NM NM NM NM NM NM 53 39 14 34 19 NM 0.736 0.283 0.642 0.358 NSMT Mo 48820 NM NM NM NM NM NM NM NM NM NM 52 40 11 34 20 3 0.769 0.212 0.654 0.385 CAS 63354 NM NM NM NM NM NM NM NM NM NM 52 40 11 34 20 3 O769 0212 0654 0385 CAS 63354 NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM CAS 63354 52 39 11 34 19 3 0750 0:212 0.654 0.365 Mean 5.52 4.05 1.33 348 2.02 — 0.736 0.242 0.645 0.374 6.00 4.30 1.43 3.70 2.10 0.718 0.237 0.650 0.368 SD 0.37 0.23 0.21 0.18 0.08 — 0.047 0.034 0.016 0.013 0.72 046 0.31 0.42 0.28 0.037 0.023 0.006 0.004 SE 0.15 0.92 0.08 0.08 0.04 — 0.019 0.014 0.007 0.006 0.42 0.26 0.18 0.30 0.20 0.022 0.013 0.005 0.003 Min 52 39 11 34 19 3 0.656 0212 0,618 0.358 5272397 11 84777109537 0677 0,2127 0:645), 0365 Max 6.1 45 16 38 21 3 0.776 0.283 0.655 0.385 6.6 48 17 40 23 3 0.750 0.258 0.654 0.371 n 6 6 6 5 5 5 6 6 5 5 3 3 3 2 2 23 3 2 2 THE BIVALVE GENUS PARVITHRACIA 131 southwards (Honshu) (Higo et al., 1999); in the Sea of Japan—near Oga Peninsula, off Honshu (Ito, 1989; Higo et al., 1999); in the Yellow Sea —off Kyushu (Higo et al., 1999). This species was recorded at a depth from 20 m to 300 m, on the fine sand (Tsuchida & Kurozumi, 1996; Habe, 1977; Higo et al., 1999). Comparisons This species is easily distinguished from other species of the subgenus in having a small, elongate, very thin translucent shell with the subumbonal plate almost completely attached to the shell wall and indistinct, short pillars (Table 4). Parvithracia (Pseudoasthenothaerus) isaotakii (Okutani, 1964) Figs. 61-64, Table 9 Asthenothaerus isaotakii Okutani, 1964: 84, 85, text fig. 6; Habe, 1977: 312; Higo et al. 1999: 524; Okutani, 2000: 1039, pl. 517, fig. 6 Type Material and Locality Holotype (shell length 7.9 mm), Sagami Bay, Japan (35°05.35N, 139°18.65E), 550 m, FIG. 65. The hinge and subumbonal plate of Parvithracia species from Japan by camera lucida. A, B. Parvithracia (Pseudoasthenothaerus) sematana (Yokoyama, 1922) (NSMT Mo 48820). A: Hinge of right valve. B: Hinge of left valve. C-F. Parvithracia (Pseudoasthenothaerus) isaotakii (Okutani, 1964) (NSMT-Mo 71463). C: Hinge of right valve. D: Hinge of the left valve (inner part of anterodorsal shell margin broken). E: Close-up of right valve showing attachment of lithodesma (ventral view) to subumbonal plate. F: Subumbonal plate with internal ligament and pillars (ventral view), left valve. Bar = 1 mm. 132 KAMENEV TABLE 9. Parvithracia (Pseudoasthenothaerus) isaotakii (Okutani, 1964). Shell measurements (mm) and indices: L— shell length; H — height; W — width; А — anterior end length; $ — maximal distance from the pos- terior shell margin to the top of pallial sinus; L1 — lithodesma length; N — number of pillars under the subum- bonal plate. Numerator indicates shell measurements and indices for the right valve, denominator — for the left valve, ?—ventral margin of the left valve is partly broken. NM — not measured. pa H W A S L1 N 7.6 6.5 1.6; 03:82, ЗУ 41-0 7-5 6.1? ММ 4.0 3.7 Coll. T. Okutani, 27-IX-1960 (R/V “Soyo- Maru”) (Okutani, 1964). Material Examined 1 lot (NSMT-Mo 71463) from Tosa Bay, Japan (33°05.6'N, 133°41.4'E), 800 m, 12- IX-1997 (R/V “Kotaka-Maru”) (1 spec.). Description Expanded from Okutani (1964)— Exterior: Shell small (<7.9 mm), subovate, high (H/L = 0.855), slightly inequivalve (right valve slightly longer and higher), moderately inflated (W/L = 0.211), thin, fragile, ashy white under perio- stracum. Surface rough, with conspicuous growth lines and very fine granules. Perio- stracum dull, thin, adherent, sometimes peel- ing near beaks, dark grayish, sometimes with dark brown or black patches or crusts near beaks and dorsal shell margin. Beaks small, moderately projecting above dorsal margin, central or slightly posterior to midline (A/L = 0.5-0.533), slightly rounded, orthogyrate. An- terior end sharply rounded. Posterior end ex- panded, vertical, higher than anterior, decid- edly truncate, with a faint radial ridge extending from beaks to junction of posterior end with ventral margin. Anterodorsal margin Straight, steeply descending, smoothly transi- tioning to rounded anterior end. Ventral mar- gin slightly curved. Posterodorsal margin straight, smoothly descending, forming a smooth, obtuse angle with posterior end. Pos- terior end straight or slightly curved, almost vertical, only slightly turned anteriorly to form a rounded angle where it meets ventral mar- gin. Escutcheon very narrow, well developed, demarcated by ridges extending along pos- terodorsal margin from beaks to posterior end. Interior: Right valve with anterior and pos- terior lateral teeth; left valve with anterior lat- eral tooth. In right valve, anterior lateral tooth W/L AL S/L Li/L Depository 6 0.855 0.211 0.500 0.487 0.132 NSMT 6 0.813? NM 0.533 0.493 0.133 Mo 71463 large, triangular, projecting considerably above inner part of anterodorsal margin, ven- trally directed; posterior lateral tooth small, elongate, slightly projecting above inner part of posterodorsal margin, extending along pos- terodorsal margin. In left valve, anterior lateral tooth long, lamellate, considerably projecting above inner part of anterodorsal margin; inner part of anterodorsal margin straight between beak and tooth; inner part of posterodorsal margin slightly convex. Subumbonal plate short, elevated above inner surface. Pillars supporting plate short, wide, partly fused, considerably tapering ventrally. Number of pil- lars in both valves 6. Lithodesma large (L1/L = 0.133), curved. Anterior adductor muscle scar large, elongate, kidney-shaped. Posterior ad- ductor scar small, rounded. Pallial sinus dis- tinct, of the same shape and size in both valves, deep, narrow and rounded anteriorly, reaching midline (S/L = 0.487-0.493). Shell interior polished, with faint radial striae. Distribution and Habitat Parvithracia (Pseudoasthenothaerus) isao- takii occurs on the Pacific coast of Japan: Tosa Bay (33° 05.6'N, 133°41.4’E), 800 m; Sagami Bay (35°05.35’N, 139°18.65’E; 35°09.9'N, 139°30.4'E), 550-700 m (Oku- tani, 1964); Sea of Enshu-Nada (34°25.7'N, 137°58.5'E), 620 m (Okutani, 1964). Comparisons In contrast to other species ofthe subgenus, P. (Pseudoasthenothaerus) isaotakii has a broader posterior end, and the beak is almost central and orthogyrate (Table 4). This species also differs from P. (Pseudoasthenothaerus) lukiniand P. (Pseudoasthenothaerus) sirenkoi in having a thin, fragile shell, and a curved lith- odesma that is not butterfly-shaped. Parvithra- cia (Pseudoasthenothaerus) isaotakii is dis- tinguished from P. (Pseudoasthenothaerus) THE BIVALVE GENUS PARVITHRACIA 133 sematana, which also has a thin and fragile shell, in having a highly elevated subumbonal plate, with distinct, high pillars. ACKNOWLEDGMENTS | am very grateful to Dr. V. A. Nadtochy (PRIFO, Vladivostok) and Mrs. N. V. Kameneva (IMB, Vladivostok) for great help during work on this manuscript; to Drs. V. B. Durkina, L. N. Usheva, M. A. Vaschenko, and |. G. Syasina (IMB, Vladivostok) for consulta- tions and help during study of anatomy of the new species; to Dr. H. Saito (NSMT, Tokyo), Ms. R. N. Germon (USNM, Washington), Dr. N. R. Foster (UAM, Fairbanks), Mr. R. Asher (CIN, Cawtron) and Dr. K. A. Lutaenko (IMB, Vladivostok) for providing at my disposal specimens of different species of Thraciidae genera; to Drs. B. |. Sirenko, A. V. Martynov and all collaborators of the Marine Research Laboratory (ZIN, St.-Petersburg) for help dur- ing work with collection of bivalve molluscs at the ZIN; to Dr. B. A. Marshall (MNZ, Welling- ton) for consultations and sending material of the genus Parvithracia and reprints of papers; to Dr. E. V. Coan (Department of Invertebrate Zoology, CAS, San Francisco) for consulta- tions, comments on the manuscript, and sending reprints of papers; to Drs. T. Kurozumi and E. Tsuchida (NHMI, Chiba), Dr. K. Amano (Joetsu University of Education, Joetsu) for sending reprints of papers; to Mr. E. V. Jakush (PRIFO, Vladivostok) and D. V. Fomin (IMB, Vladivostok) for help in work with the scanning microscope; to Mr. A. A. Omelyanenko (IMB, Vladivostok) for making photographs; to Ms. T. N. Kaznova (IMB, Vladivostok) for translating the manuscript into English; Dr. George M. Davis for help in the publication of the manuscript; two anony- mous reviewers for comments on the manu- script. This research was partly supported by Grants 98-04-48279, 01-04-48010, and 00- 15-97890 from the Russian Foundation for Basic Research. LITERATURE CITED ALLEN, J. A., 1961, The British species of Thracia (Eulamellibranchia). Journal of the Marine Bio- logical Association of the United Kingdom, 41: 723-735. 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Lamellibranchia (2). Bulletin of the Marine Biological Station of Asamushi, 9: 85-122. YOKOYAMA, M., 1922, Fossils from Upper Musa- shino of Karuza and Shimosa. Journal, College of Science, Imperial University of Tokyo, 44(1): 1- 200 Revised ms accepted 8 July 2001 MALACOLOGIA, 2002, 44(1): 135-143 LOCALIZATION OF NADPH-DIAPHORASE-POSITIVE ELEMENTS IN THE INTESTINE OF THE MUSSEL CRENOMYTILUS GRAYANUS (MOLLUSCA: BIVALVIA) Anatoly A. Varaksin'*, Eugenia A. Pimenova?, Galina $. Varaksina' & Lydia T. Frolova' ABSTRACT By NADPH-diaphorase histochemistry (Hope & Vincent, 1989), localization of nicotinamide adenine dinucleotide phosphate diaphorase-positive (NADPH-d-positive) elements was studied in the direct and recurrent loops of the midgut and hindgut of the mussel Crenomytilus grayanus. Intraepithelial NADPH-d-positive cells were found in the intestinal groove and major typhlosole of the direct loop and in the recurrent loop of the midgut, as well as in the hindgut. The cells are fusiform and lie separately or in minor groups. Their apical processes are directed to the gut lumen, and the basal processes contact with the basiepithelial plexus. The latter, in turn, sends separate NADPH-d-positive fibers to make contacts with subepithelial plexus. Both plexuses are well developed in the intestinal groove and major typhlosole of the direct loop and in the recur- rent loop of the midgut, as well as in the hindgut. In the minor typhlosole and crystalline style sac, both plexuses are less developed. In the ciliary groove of the hindgut, the basiepithelial plexus is absent. Possible role of NADPH-d-positive elements in regulation of the digestion in bivalve mollusks is discussed. Key words: histochemistry, NADPH-diaphorase, basiepithelial plexus, subepithelial plexus, in- testine, Crenomytilus grayanus (Dunker), Mollusca. INTRODUCTION Nitric oxide (NO) acts as a transcellular messenger in the mammalian nervous sys- tem (Garthwaite & Boulton, 1995; Rand & Li, 1995). NO, together with citrulline, is pro- duced by NO-synthase from L-arginine. For the reaction to proceed normally, O,, Cas and nicotinamide adenine dinucleotide phos- phate (NADPH) are required (Bredt 8 Snyder, 1990). In the course of the histochemical re- action, exogenous nitro blue tetrazolium is re- duced by NO-synthase to diformazan precipi- tate, which thereby marks cells containing the enzyme. At present, of all NADPH-dependent oxidoreductases, NADPH-diaphorase is only considered to retain activity after para- formaldehyde fixation (Matsumoto et al., 1993). Histochemical activity of NADPH-di- aphorase is thus believed to be a topographi- cal marker of NO-synthase-containing struc- tures in tissues of both vertebrates (Hope et al., 1991; Dawson et al., 1991) and inverte- brates (Moroz & Gillette, 1995, 1996; Moroz et al., 1996). To date, NO-ergic element localization and functional role in the mammalian alimentary tract have been intensively studied (Schleiffer 8 Raul, 1997; Huber et al., 1998). Among lower vertebrates, NO-synthase-containing neurons were found in the alimentary tract of the toad Bufo marinus (Li et al., 1992, 1993; Murphy et al., 1993); an agama lizard, Agama sp. (Knight & Burnstock, 1999); the rainbow trout, Salmo gairdneri (Li & Furness, 1993); the Atlantic cod, Gadus morhua; and the spiny dogfish, Squalus acanthias (Olsson & Karila, 1995). At the same time, NO-ergic element lo- calization and functional role in the inverte- brate digestive system are poorly studied. These elements are revealed in the cardiac stomach of the starfishes Marthasterias glacialis (Martinez et al., 1994) and Asterias rubens (Elphick & Melarange, 1998), and in the oesophagus of the opisthobranch Pleuro- branchaea californica (Moroz & Gillette, 1996). To date, no data have been present on NO-ergic elements in the alimentary tract of bivalve mollusks. The present work focused on NADPH-d- positive element localization in the direct and recurrent loops of the midgut, and in the hindgut of the mussel Crenomytilus grayanus (Dunker). "Institute of Marine Biology, Russian Academy of Sciences, Vladivostok 690041, Russia; inmarbio@mail.primorie.ru “Far East State University, Vladivostok 690600, Russia “corresponding author 135 136 VARAKSIN ET AL. MATERIALS & METHODS Samples were obtained from five adult mussels with shells 10.5-11.0 cm high and 4.5-5.0 cm wide. Mollusks were collected in Amurskii Bay, Sea of Japan, in November 1998 and kept in aerated aquaria for 2-3 days. During that time, the mollusks were not fed. NADPH-d-positive elements in the intestine were revealed by aNADPH-diaphorase histo- chemistry method (Hope & Vincent, 1989). Fragments of the direct and recurrent midgut loops and of the hindgut, shown in Figure 1, were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 2 h at 4°C. They were then rinsed in 3-4 changes of 15% sucrose prepared in 0.05 M Tris-HCl buffer (pH 8.0) for 24 h at the same tempera- ture. Both transverse and longitudinal, 15 um thick, cryostat sections were prepared and placed on to slides. NADPH-Diaphorase Histochemistry Sections were incubated in a medium con- taining 0.5 mM B-NADPH, 0.5 mM nitro blue tetrazolium, and 0.3% Triton X-100 in 0.15 M Tris-HCl buffer (pH 8.0) for one h at 37°C. Then, they were rapidly washed twice in distilled water, dehydrated in an ethanol se- ries of increasing concentrations, and mounted in Dammar Resin. Control sections h hg were incubated in the following media: (1) without B-NADPH, (2) without nitro blue tetra- zolium. Drugs Used Nitro blue tetrazolium, B- NADPH were sup- plied by Sigma Chemical Co, USA. Statistical Analysis Sections were observed and photographed using Olympus BH2-RFCA microscope (BHS model). NADPH-d-positive cells were drawn by means of a Carl Zeiss drawing tube ata total magnification of x400. Cell height and width were measured using a plotting scale. For each of the intestine parts studied, a total number of NADPH-d-positive cells was counted in ten transverse sections, and a mean and standard deviation per section was calculated. Statistical significance was tested by paired t-tests at the 0.05 confidence level. RESULTS Direct Loop of the Midgut The direct loop of the midgut consists of: the intestinal groove, the major and minor ty- rl sates KR 1 7m NA pa gl FIG. 1. Anatomy of the digestive system of Crenomytilus grayanus. Abbreviations: aa, anterior adductor; dg, digestive gland; di, direct loop of the midgut; gi, gills; h, heart; hg, hindgut; ml, mouth lobes; oe, oesophagus; pa, posterior adductor; rl, recurrent loop of the midgut; s, stomach. Scale bar, 10 mm. NADPH-DIAPHORASE-POSITIVE ELEMENTS OF THE MUSSEL 137 FIG. 2. Schematic localization of NADPH-d-positive elements in the intestine of Crenomytilus grayanus. A. Direct loop of the midgut; B. Recurrent loop of the midgut; C. Hindgut. Abbreviations: bep, basiepithelial NADPH-d-positive plexus; cg, ciliary groove; cs, crystalline style; css, crystalline style sac; ct, connective tis- sues with separate muscular cells; e, epithelium; gl, gut lumen; ic, intraepithelial NADPH-d-positive cells; ig, intestinal groove; sep, subepithelial NADPH-d-positive plexus; mat, major typhlosole; mit, minor typhlosole; rt, the region of the typhlosole, adjacent to the crystalline style sac. Scale bar, 1 mm. phlosoles, and the crystalline style sac (Fig. 2A). Among these structures, a considerable difference in intensity and pattern of NADPH- d-positive elements staining was observed. In the intestine groove wall, fairly developed basi- and subepithelial NADPH-d-positive plexuses were observed (Fig. 3A). The basiepithelial plexus was dense and inten- sively stained, with the maximum thickness of 13.7 + 4.6 um. In the intestine groove epithe- lium, there occur NADPH-d-positive cells av- eraging 2.6 + 1.9 per section (Table 1). The 138 VARAKSIN ЕТ AL. FIG. 3. NADPH-d-positive element localization in the direct loop of Crenomytilus grayanus midgut. A. In- testinal groove. B. Major typhlosole. C. Crystalline style sac. D. Intraepithelial NADPH-d-positive cell in the major typhlosole. Abbreviations: f, NADPH-d-positive fibers in the connective tissue of the intestinal groove; for other abbreviations, see caption to Fig. 2. Scale bar, A, C, D, 100 um; B, 150 um. cells are located mainly in the basal zone of the epithelium; they are fusiform, 10.9 + 2.0 um high and 3.6 + 0.9 um wide. The subep- ithelial plexus represents a dense, thick (30.1 + 8.2 um), interlacement of NADPH-d-posi- tive fibers (Fig. 3A). Both plexuses are con- nected to each other with separate thin fiber bundles. Occasional large, varicose fibers branch off from the subepithelial plexus to submerge to the adjacent connective tissue (Fig. 3A). In the major typhlosole, the basiepithelial plexus 6.7 + 2.8 um thick has the appearance of a rather sparse, slightly branched interlace- NADPH-DIAPHORASE-POSITIVE ELEMENTS OF THE MUSSEL 139 TABLE 1. The average proportion of NADPH-d-positive cells to the total number of epithelial cells per section in the Crenomytilus grayanus intestine. Total NADPH-d- NADPH-d- epithelial positive cell positive cell Intestine region cell number number proportion (%) Direct loop of the midgut Intestine groove 509 + 152 2.6 + 1.9 0.51 Major typhlosole 1349 + 457 17.3 + 10.4 1.28 Minor typhlosole 1109 + 295 cm = Crystalline style sac 585 + 154 = 7 Recurrent loop of the midgut 1810 + 452 15.1 + 4.8 0.83 Hindgut 1474 + 244 13 3E 461 0.92 Values represent the mean + the standard error of the mean. ment of fibers bearing varicosities (Fig. 3B). It is more prominent in the region of the ty- phlosole, adjacent to the crystalline style sac. In the major typhlosole epithelium, NADPH-d- positive cells also occur averaging 17.3 + 10.4 per section (Table 1). The cells are fusiform and lie diffusely, close to the gut lumen (Fig. 3B). They are 14.9 + 2.9 um high and 5.7 + 1.7 um wide (Fig. 3D). In the minor typhlosole, the basiepithelial NADPH-d-posi- tive plexus is formed by thin fibers, most of which are longitudinally oriented. In the region of the minor typhlosole, adjacent to the crys- talline style sac, at the epithelium base, only rare thin fibers occurred, no NADPH-d-posi- tive cells being observed. In both typhlosoles, the subepithelial plexus represents an inter- lacement of weakly branched NADPH-d-pos- itive fibers bearing varicosities. The subep- ithelial layers of the major and minor typhlosoles are, respectively, 31.9 + 10.7um and 12.0 + 7.2 um thick. In the adjacent con- nective tissue, occasional NADPH-d-positive fibers occur. Inthe area of the crystalline style sac, the basiepithelial plexus represents a thin (4.2 + 1.4 um) interlacement of fibers bearing vari- cosities (Fig. 3C). In the subepithelial plexus, which is 5.2 + 1.3 um thick, the longitudinal orientation of NADPH-d-positive fibers pre- vails. In the adjacent connective tissue, occa- sional fibers beaded with varicosities occur. Recurrent Loop of the Midgut In the recurrent loop of the midgut, NADPH- d-positive elements were found in the basal zone of the epithelium and in the underlying connective tissue (Fig. 2B). The basiepithelial FIG. 4. NADPH-d-positive element localization in the recurrent loop of Crenomytilus grayanus midgut. Abbreviations are the same as in Fig. 2. Scale bar, 70 um. NADPH-d-positive plexus represents a dense, intensively stained nerve fiber inter- lacement 23.7 + 4.7 um thick (Fig. 4). In the epithelium of the loop occur NADPH-d-posi- tive cells averaging 15.1 + 4.8 per section (Table 1). These lie separately or in groups of 140 VARAKSIN ET AL. 2-3 cells (Fig. 4). The cells are fusiform, 17.3 + 2.8 um high, and 7.9 + 1.3 um wide. The subepithelial plexus is 43.2 + 5.9 um thick. It is formed by densely interlacing NADPH-d- positive fibers (Fig. 4). Occasional fibers bear- ing varicosities leave the plexus to enter the adjacent connective tissue. Hindgut In the hindgut, NADPH-d-positive elements were found in both layers (Fig. 2C). The basiepithelial plexus represents a dense, in- tensively stained NADPH-d-positive fibers in- terlacement 14.3 + 3.7 um thick. (Fig. 5A-D). The only exception is the ciliary groove re- gion, which lacks a basiepithelial plexus (Fig. 5D). In the basal zone of the epithelium, sin- gle fusiform NADPH-d-positive cells are re- vealed (Fig. 5B, C) averaging 13.3 + 6.1 per section (Table 1). These are 17.9 + 4.6 um high and 5.7 + 0.9 um wide. The cells pos- sess apical processes directed to the gut lumen, their basal processes contacting the basiepithelial plexus (Fig. 5B, C). The subepithelial plexus is formed by an in- terlacement of NADPH-d-positive _ fibers, which are mainly longitudinally oriented (Fig. 5A, D). Its thickness reaches 27.2 + 11.2 um and 106.3 + 11.7 um at the concave and con- vex sides of the gut, respectively. At the con- vex side of the gut, the plexus is denser than at the concave side. Neither control test revealed NADPH-d- positive elements in any of the C. grayanus gut parts studied. DISCUSSION We revealed NADPH-d-positive cells lo- cated in the epithelium of the direct and re- current loops of the midgut, as well as in the hindgut of the mussel C. grayanus. In many invertebrate groups, specialized cells were found in the epithelial lining of the alimentary tract that are not directly involved in the process of digestion and seemingly serve reg- ulatory functions (Punin, 2000). In bivalve mollusks, the system regulating gut function- ing includes both nerve and endocrine cells. By using methylene blue staining, nerve cells were revealed in the Anodonta cellensis midgut epithelium (Gilev, 1952). Electron mi- croscopy studies of Arctica islandica and Mytilus edulis gut epithelium revealed intraep- ithelial cells with a fairly developed rough en- doplasmic reticulum, the Golgi apparatus of dictyosomic type, numerous granules, and first-order processes. The latter formed pro- nounced basiepithelial plexus. Based on their morphology, these cells were considered nerve cells (Punin, 1981, 1989). At the same time, endocrine cells in the gut epithelium of bivalve mollusks are characterized by larger cytoplasmic granules and the lack of processes (Punin, 2000). From the above facts, we believe that intraepithelial NADPH- d-positive cells in the C. grayanus gut are of nerve type. We revealed a dense, intensively stained plexus of NADPH-d-positive fibers in the basal zone о the mussel intestinal epithelium. A plexus of similar or even greater density and complexity was found in the subepithelial zone of the direct and recurrent midgut loops and of the hindgut. The bivalve alimentary tract is known to possess complicated systems comprised of the basi- and subepithelial nerve plexuses (Giusti, 1970; Punin, 1981, 1989). Based on electron microscopy data, both zones were shown to be to a certain degree autonomous (Punin & Konstantinova, 1988; Punin, 1989). The subepithelial plexus is assumed to be a peripheral component of the molluscan ner- vous system (Punin & Konstantinova, 1988). The basiepithelial plexus was earlier shown to consist of the processes of intraepithelial nerve cells (Punin, 1989). Nerve elements of the subepithelial plexus are also presumed to contribute to basiepithelial plexus formation. By light microscope studies of M. edulis (Punin & Konstantinova, 1988) and of A. is- landica (Punin, 1981), processes were found that penetrated into the neighbouring connec- tive tissue. Terminals of nerve processes can penetrate into the basal zone of the intestine epithelium from the surrounding connective tissue, as in the case in the Tapes waltingii hindgut (Dougan & McLean, 1970) and in the M. galloprovincialis midgut (Giusti, 1970). We repeatedly found processes connecting the basi- and subepithelial plexuses. This sug- gests that both plexuses were structurally and functionally integrated, and the putatively NO- ergic system of the C. grayanus intestine pos- sess a complex multilevel structure. It is worth noting that the basiepithelial plexus is relatively well developed in all parts of the gut. However, it contains few intraep- ithelial cells of putative NO-ergic nature. Nu- merous NO-ergic fibers were revealed in the neuropils of the C. grayanus ganglia, whereas NADPH-DIAPHORASE-POSITIVE ELEMENTS OF THE MUSSEL 141 FIG. 5. NADPH-d-positive element localization in the hindgut of Crenomytilus grayanus. A. Convex side of the gut. Scale bar, 150 um. В, С. Intraepithelial NADPH-d-positive cells. Scale bar, 50um. D. Concave side ofthe gut. Scale bar, 250 um. The solid arrow shows the apical process; the dashed arrow, the basal process; for other abbreviations, see caption to Fig. 2. the number of specifically stained perikarya was relatively small there (Annikova et al., 2000). A similar pattern of NO-positive stain- ing was observed in other gastropod and bi- valve mollusks and in echinoderms (Moroz & Gillette, 1996; Dyuizen et al., 1999). It is pro- posed that C. grayanus NO-ergic neurons are not only capable of NO synthesis in the cell body but also possess a system of local nitric oxide synthesis within the processes (An- nikova et al., 2000). The presence of numer- ous, putatively NO-ergic fibers bearing vari- cosities in the C. grayanus intestine supports this proposal. Thus, in the wall of the C. grayanus intes- tine, there exists an integrated system com- prised of intraepithelial, putatively NO-ergic nerve cells and of basi- and subepithelial nerve plexuses, too, of putative NO-ergic na- ture. 142 VARAKSIN ET AL. To date, the role of NO in regulation of in- vertebrate digestion is poorly studied. It is shown that in the oesophagus of Lymnaea stagnalis, nitric oxide is capable of functioning as a neuromodulator (Elphick et al., 1994; Moroz et al., 1993). NO is capable of modu- lating the frequency of epitheliocyte cilia beat- ing, for instance, in the ciliary epithelium ofthe mammalian respiratory system (Jain et al., 1993). In C. grayanus, NO-synthase activity is found to be high in the basiepithelial plexus of the intestine, the fibers of which establish di- rect contacts with ciliated epitheliocytes. We suppose that the NO role in controlling ep- itheliocyte locomotory activity consists in modulating the transport of water with food masses kept by epitheliocyte ciliary streaming and the transport of fecal masses through the intestine. However, in the epithelium of the crystalline style sac, where a highly intensive сша beating is maintained, basiepithelial plexus is unexpectedly ordinary developed. Obviously, NO does not play a principal role in regulating ciliary activity of epitheliocytes in the C. grayanus intestine. In the intestine of bivalve mollusks, epithe- liocytes secrete mucus, digestive enzymes, and protein-like substances of the crystalline style matrix (Owen, 1966; Reid, 1966; Giusti, 1970). The secretory activity is especially prominent in epitheliocytes of the region of the major typhlosole, adjacent to the crystalline style sac (Frolova, 1989). 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Zhurnal Evolyucionnoi Biokhimii i Fiziologii, 36: 586-599 [in Russian]. PUNIN, M. YU. & M. S. KONSTANTINOVA, 1988, Endocrine-like cells in the gut epithelium of the bi- valve mollusk Mytilus edulis. Tsitologiya, 30: 795-801 [in Russian, with English summary]. RAND, M. J. & С. G. LI, 1995, Nitric oxide as a neu- rotransmitter in peripheral nerves: Nature of transmitter and mechanism of transmission. An- nual Review of Physiology, 57: 659-682. REID, R. G. B., 1966, Digestive tract enzymes in the bivalves Lima hians Gmelin and Mya arenaria L. Comparative Biochemistry and Physiology, 17: 417-433. SCHLEIFFER, R. & F. RAUL, 1997, Nitric oxide and the digestive system in mammals and non-mam- malian vertebrates. Comparative Biochemistry and Physiology, 118A: 965-974. Revised ms. accepted 30 May 2001 MALACOLOGIA, 2002, 44(1): 145-151 ORIGIN OF THE EXCRETORY CELLS IN THE DIGESTIVE GLAND OF THE LAND SNAIL HELIX LUCORUM Vasilis K. Dimitriadis & Vasiliki Konstantinidou Department of Genetics, Development and Molecular Biology, School of Biology, Faculty of Sciences, Arisrotle University Of Thessaloniki, Thessaloniki 54006, Greece; vdimitr@bio.auth.gr ABSTRACT The examination of the digestive gland cells of the land snail Helix lucorum with light and elec- tron microscopes after starvation and subsequent periods of feeding supports the hypothesis that the so-called excretory cells are digestive cells in a final step of their cell cycle, rather than a distinct cell type. Results supporting this hypothesis are: (1) the fact that the so-called diges- tive cells containing heterolysosomes and residual bodies or the so-called excretory cells con- taining excretory vacuoles were not found in significant numbers in the digestive gland epithe- lium at the same time; (2) the fact that the decrease in the number and size of the heterolysosomes implicated in the endocytotic uptake and digestion of nutrients was combined with an increase in the number and size of the excretory vacuoles responsible for the excretory processes, and vice versa; and (3) the fact that starvation caused the appearance of a large number of excretory vacuoles and the reduction of the heterolysosomes in the digestive gland epithelium to a minimum. INTRODUCTION The digestive gland (or midgut gland, or he- patopancreas or liver) is the largest organ in the body of terrestrial gastropods. It consists of two lobes communicating with the stomach via large ducts, which branch to form smaller ducts, ductules, and complex branched blind tubules. It is an organ dealing with elaboration of enzymes, absorption of nutrients, endocy- tosis of food substances, food storage, and excretion (Runham, 1975). In spite of the numerous studies on the morphology and physiology of the digestive gland cells, there are conflicting reports about the cell types that constitute the digestive gland epithelium of terrestrial gastropods. Even at the ultrastructural level, undoubted in- terpretation of the results is still difficult be- cause of the changes in the fine structure as- sociated with phases of activity. Most of the findings are consistent with the hypothesis that the digestive gland epithelium is com- posed of three cell types: digestive cells, cal- cium cells, and excretory cells (Fig. 1), sur- rounded by connective tissues, muscular layers, and haemocoelic spaces (Sumner, 1965; Dimitriadis & Hondros, 1992). The pres- ence of a fourth type, the thin cells, is docu- mented in many instances, but these cells 145 are regarded as undifferentiated precursors of the other cell types (Sumner, 1965; Walker, 1970). Digestive cells are the most frequent cell type found in the digestive gland of snails and slugs. They are usually columnar in shape, varied in size, and usually display microvilli. Digestive cells are characterized by numer- ous granules usually located in the apical por- tion of the cells consisting of secondary lyso- somes, the heterolysosomes, as well as by vacuoles with dense cores consisting residual bodies, named green and yellow granules, re- spectively, after light microscopic observa- tions in Helix and Agriolimax (Sumner, 1965; Walker, 1970, 1972), or apical granules and cisternae with electron-dense cores after electron microscopic observations in Helix (Dimitriadis & Hondros, 1992; Dimitriadis & Liosi, 1992). The second cell type of the di- gestive gland, the calcium cells, are charac- terized by their pyramidal shape and the pres- ence of calcium granules (Sumner, 1965; Dimitriadis & Hondros, 1992). The third cell type, the excretory cells are larger in size than the digestive cells and are characterized by one or more large excretory vacuoles con- taining cores or amorphous mass of high electron density (Sumner, 1965; Dimitriadis & Hondros, 1992). 146 DIMITRIADIS & KONSTANTINIDOU FIG. 1. Drawing of the three cell type regarded by many authors that form the digestive gland epithe- lium of terrestrial gastropods, based on the original in Dimitriadis & Hondros (1992). Abbreviations: CC, calcium cell; DC, digestive cell; EC, excretory cell; Ev, excretory vacuole; HI, heterolysosome; N, nu- cleus; Rb, residual body. In spite of the view that the excretory cells are a separate celltype, there are also results supporting the view that these cells are either digestive cells differentiated towards excre- tory cells or cells of the final developmental stage of the digestive cells. The present study was designed to answer the questions related to the structure and function of the excretory cells. A previous study (Dimitriadis & Hondros, 1992) showed that the digestive gland cells of Helix lucorum, even after 40 days of starva- tion or 37 days hibernation, exhibited in- creased number of large excretory vacuoles, as well as heterolysosomes compared to con- trols, and this increase was attributed to the accumulation of residual indigestible material inside the tubule cells reflecting decreased di- gestive and excretory function due to starva- tion and hibernation. Similar results were also reported in starved Helix aspersa Muller (Sumner, 1965; Porcel et al., 1996), in which prolonged starvation caused a significant de- crease in the percentage of the digestive cells and an increase of the excretory and narrow cells. In addition, the carbohydrate cytochem- istry of the content of these lysosomal struc- tures did not change after the same periods of starvation and hibernation compared to con- trols (Dimitriadis & Liosi, 1992). Reports from a variety of molluscs show the existence of different phases of cell activity, each with a distinct appearance, the digestive and fragmentation state being the most fre- quent (Morton, 1974, 1975, 1979). In the lat- ter reports, the length of the cell cycle is ap- proximately 24 hours. Because preliminary observations in the digestive gland of H. luco- rum showed a substantially longer cell cycle and less extensive cell alteration during this cycle compared to other molluscs, another purpose of the present study was to present indications related to the digestive rhythm of the digestive gland cells of H. lucorum. MATERIALS & METHODS The snails used in the experiments were collected from a natural habitat of Helix luco- rum (Gastropoda, Pulmonata, Helicidae) in the Logos region of Edessa, northern Greece. The shell diameter of the snails varied from 20 to 40 mm, and the body weight was between 14 and 20 q. The animals were kept in the laboratory under stable and controlled conditions of pho- toperiod (L:D 13:11 h), temperature (22 + 2°C), and relative humidity (90 + 5%). They were fed lettuce, which is the most assimi- lated food for H. lucorum (Staikou & Lazari- dou-Dimitriadou, 1989). The controls were kept in the conditions described above, while others were starved for 14 days, being in identical conditions of photoperiod, tempera- ture, and humidity as the controls. During the starvation period, the animals developed a velum of mucus. After starvation, the animals were fed again with lettuce, and food was kept in the boxes during the whole experiment, while each of the animals was observed at subsequent periods. The snails started to move at about 1.5 h after feeding. Animals were examined after 0, 10, 22, 34, 45, 56, 65, 75 h from the beginning of food ingestion. For each experimental stage, the animals began feeding at the same time and were actively feeding for the whole experimental period. Five actively feeding animals from each ex- perimental stage were examined under the light and electron microscope. For light and electron microscopic observa- tions, samples were fixed in 3% glutaralde- hyde, postfixed in 2% osmium tetroxide, de- hydrated, and embedded in Spurr’s resin. Ultrathin sections were post-stained with uranyl acetate and lead citrate and examined under a JEOL 100B electron microscope op- erating at 80 KV. For light microscopic obser- vations, thick sections were stained with 1% toluidine blue. DIGESTIVE GLAND OF HELIX LUCORUM 147 RESULTS In the following paragraphs, the light and electron microscopic views of the digestive gland epithelium of snails Helix lucorum, which were starved for 14 days and then fed for 10, 22, 34, 45, 56, 65 and 75 h are de- scribed. At the beginning of food ingestion (animals after 14 days of starvation), as well as 10h from the beginning of food ingestion, exami- nation of transverse sections of digestive tubules under the light (Fig. 2A) or the elec- tron microscope (Fig. 2B) show a large num- ber of excretory vacuoles in the form of large vacuoles with dense cores, similar to that de- scribed in the excretory cells of Helix lucorum. These large vacuoles, the accumulation of which in the digestive gland epithelium is at- tributed to starvation, reach usually 2-8 um, while in certain cases reach 32 um. However, cells containing heterolysosomes in the form of apical granules, which is a main character- istic of the digestive cells, are very few. The apical plasma membranes show limited pinocytotic phenomena at 0 h, expressed by the formation of a moderate number of coated vesicles. However, at 10 h these phenomena become intensive and the formation of coated pinocytotic vesicles, and a network of micro- tubules and vesicles in the apical cytoplasm is apparent (not shown). The calcium cells lo- cated in the base of the epithelium (Fig. 2A) exhibit the usual morphology described for this cell type. The apical region of the cells show a large number of excretory vacuoles, as well as in- tensive pinocytotic phenomena 22 h from the beginning of food ingestion. Also, a small number of heterolysosomes is present in the tubule cells in the form of apical granules of moderate size (0.5-1, 5 um) filled with mater- ial with low electron density. After 34 h from the beginning of food ingestion, these het- erolysosomes are larger in size and numbers, reaching 1-2 um and are located near the apical border of the cells (Fig. 2C). The excre- tory vacuoles are now located adjacent to the apical region of the cells, being probably in a phase just before their excretion, while such vacuoles are present in the tubule lumen in certain cases (not shown). The ultrastructural observations show that micropinocytosis con- tinues at this stage. At the 45 h stage, a large number of het- erolysosomes, about 2 um in size, as well as an increased number of excretory vacuoles are obvious in the apical and middle portion of digestive gland cells under the light micro- scope. The heterolysosomes 56 h after the beginning of food ingestion are increased very much in number, and their size usually reaches 4 um (Fig. 2D). With the electron mi- croscope, the presence of recently formed large vacuoles with dense cores is apparent, while some of them are observed to fuse with each other (Fig. 3A). The calcium cells do not show any obvious alteration in the number and location, comparing to the previous stages. The digestive tubules cells 65 h and 75 h from the beginning of food ingestion are char- acterized by an increased number of excre- tory vacuoles, the size of which being more evident at 75 h (Fig. 3B). The apical het- erolysosomes decrease in number, being al- most absent at 75 h (Fig. 3B). No any visible change in the number and location of the cal- cium cells was noted. Considering the digestive gland cells of the control animals, that is the snails fed with let- tuce and kept in identical photoperiod, tem- perature, and humidity, their functional mor- phology resemble that already described for these snails examined under normal condi- tions. DISCUSSION There is a disagreement on the origin and the function of excretory cells in terrestrial gastropods. Fretter (1952), Billett & McGee- Russell (1955), Walker (1970), and Morton (1979) regarded these cells as differentiated digestive cells, whereas Thiele (1953) and Sumner (1965) suggested that they are de- generate calcium cells. In other studies, Abolins-Krogis (1961) found mucopolysac- charides, proteins, lipids, and small quantities of RNA inside the cisternae of excretory cells in Helix aspersa and suggested that these materials are implicated in shell repair. The results of the present study support the hypothesis that in the digestive gland, the so- called excretory cells are, simply, digestive cells in a final step of their cell cycle rather than a distinct cell type. The results showed that the so-called digestive cells containing heterolysosomes and residual bodies or the so-called excretory cells containing excretory vacuoles did not follow independent cell cy- cles, because they were not found in signifi- cant numbers at the same time. The increase 148 DIMITRIADIS & KONSTANTINIDOU FIG. 2A, B. Beginning of food ingestion. Transverse sections of digestive tubules. Under the light (arrows) or the electron microscope (asterisks), the digestive gland cells of the snail Helix lucorum show absence of het- erolysosomes, while abundance of large vacuoles with dense cores is noted. A number of calcium cells are located at the base of the epithelium. C. 34 h from the beginning of food ingestion. Large numbers of het- erolysosomes (apical granules) are present at the apical portion of the digestive gland cell (asterisks). D. 45 h from the beginning of food ingestion. Transverse sections of a digestive tubule. Under the light microscope, the cells contain large number of heterolysosomes (small arrows), as well as an increased number of ex- cretory vacuoles (large arrows). Abbreviations: CC, calcium cell; Lu, lumen; Mv, microvilli. Scale bars —A = 15 um, B= 1.5 um, C = 1 um, D = 16 um. DIGESTIVE GLAND OF HELIX LUCORUM 149 FIG. 3A. 56 h from the beginning of food ingestion. Under the electron microscope, the digestive gland cells show abundance of excretory vacuoles in the form of vacuoles containing dense cores (asteriks). The arrows show possible sites of fusion between adjacent vacuoles. B. 75 h from the beginning of food ingestion. Trans- verse section of a digestive tubule. The abundance of large vacuoles with dense cores (arrows) and the ab- sence of heterolysosomes are prominent in the digestive gland cells at this stage. Scale bars: À = 1 um, B = 25 um in the number and size of the heterolyso- somes and residual bodies implicated in the endocytotic uptake and digestion of nutrients from the gland lumen and their digestion was combined with a decrease in the number and size of the excretory vacuoles responsible for the excretory processes in the epithelium, and vice versa. The cell changes observed in the digestive gland epithelium should be re- garded as different phases of cell activity of the same cell type, the digestive cells. Another result that supports the above hy- pothesis was the fact that in the digestive gland cells starvation caused the appearance of a great number of excretory vacuoles and the reduction of the heterolysosomes to a minimum (Fig. 2A). It is also supported by re- sults in starved Helix lucorum (Dimitriadis & Hondros, 1992) and Helix aspersa (Porcel et al., 1996), in which prolonged starvation caused a decrease in the percentage of the digestive cells compared to the increased per- centage of the excretory cells. According to the above approach, the phe- nomena related to the endocytotic and excre- tory processes reported in the present study could exhibit the following sequence: at the beginning of food ingestion (end of 14 days starvation period), the digestive gland epithe- lium consists only of cells with excretory vac- uoles formed during the previous cell cycle and were accumulated in the cells during star- vation (Fig. 4C). In the following periods, the excretory vacuoles are relocated adjacent to the apical border of the cells and finally are extruded from the cells, which could be conclude by the presence of such vacuoles in the tubule lumen. The digestive activities progress and initially increase in number and size the heterolysosomes, that is, the cell elements implicated in the function of endocy- tosis and Iysosomic digestion of the endocy- tosed material (Fig. 4A) and later (approxi- mately at the 45 h), the residual bodies formed by further digestion and condensation of the content of the heterolysosomes (Fig. 4B). Digestion proceeds, and there is a de- crease in the number and size of both het- 150 DIMITRIADIS & KONSTANTINIDOU FIG. 4. Drawing of the various stages that the digestive cells possibly undergo during the endocytotic and excretory processes in the digestive gland epithelium. A. As the digestive activity progresses, in the diges- tive cells initially increase in number and size the heterolysosomes (Hl), the secondary Iysosomes implicated in the function of endocytosis and lysosomic digestion of the endocytosed material. B. Intralysosomal diges- tion in the heterolysosomes and condensation of their content leads to the formation of large number of resid- ual bodies (Rb) or heterolysosomes condensed to residual bodies C. Fusion of residual bodies (arrow) leads to the formation of large excretory vacuoles and to a decrease in the number and size of both heterolyso- somes and residual bodies. D. Cytoplasmic vessels containing excretory vacuoles, amongst other or- ganelles, are cut off from the apical region of digestive cells and are extruded to the lumen or the whole cell is degenerated and is extruded to the lumen. erolysosomes and residual bodies and an in- crease in number and size of the excretory vacuoles, which possibly arise after fusion of smaller residual bodies (Figs. 3A, 4C). The excretory vacuoles are finally extruded into the tubule lumen as described above. The proposed sequence is supported by the results of Walker (1970) in Agriolimax reticulatus, in which the excretory cells were also regarded as the final step in the develop- ment of the digestive cells. The excretory cells in Agriolimax contained the digestive rem- nants of ingested material, which is absorbed by the former cells, degraded by Iysosomal action and subsequently extruded to the lumen of the digestive gland. Reports show that in the land pulmonate Deroceras reticulatum (Walker, 1972; Triebs- korn & Florschutz, 1993), the food transit through the digestive tract takes 9-12 h, while in the freshwater pulmonates Lymnaea stag- nalis (L.) (Veldhuijzen, 1974) and Biophalaria grabrata (Say) (Florschutz & Becker, 1999) the digestive gland is emptied of food every 60-110 min, discharging undigested parti- cles. In the absence of information, the latter authors postulated the existence of a rhythmic activity of the basommatophoran digestive gland cells. On the other hand, reports froma variety of different molluscs, from the phy- tophagous pulmonate Amphibola cerenata (Morton, 1974) to the zoophagous pulmonate Deroceras caruanae (Morton, 1979) and the microphage bivalve Geloina proxima Prime, 1864 (Morton, 1975) showed the existence of different phases of cell activity, each with a distinct appearance, the digestive and frag- mentation state being the most frequent. In these reports, the length of the cell cycle is es- timated to be approximately 24 h. Although a different methodology would be required for studying cell turnover, the results of the pres- ent study display indications that in Helix lu- corum this cycle seems to be longer, reaching approximately 55 h. This could be concluded by the fact that at 0-10 h, as well as at 65-75 h from the beginning of food ingestion, identi- cal morphological features were observed in the digestive gland cells. In addition, the mor- phological changes in the cell that accompany the cell cycle were not as extensive as in other gastropods and were related to the presence or absence of certain lysosomic structures, rather than extensive changes, for example in the thickness of the epithelium. By using light and electron microscope ob- servations the present study provides infor- mation about the cell types and the physiol- ogy of the digestive gland cells of H. lucorum. However, more studies, using for example histochemistry in cryosections and immuno- cytochemistry are needed for the better un- DIGESTIVE GLAND OF HELIX LUCORUM 151 derstanding of the fine structure and function of these cell types. LITERATURE CITED ABOLINS-KROGIS, A., 1961, The histochemistry of the hepatopancreas of Helix pomatia (L.) in rela- tion to the regeneration of the shell. Arkiv fur Zo- ologie, 13: 159-201. BILLETT, F. & S. M. MCGEE-RUSSELL, 1955, The histochemical localization of B-glucuronidase in the digestive gland of the Roman snail Helix po- matia (L.) Quarterly Journal of Microscopical Sci- ences, 96: 35-48. DIMITRIADIS, V. K. & D. HONDROS, 1992, Effect of starvation and hibernation on the fine structural morphology of digestive gland cells of the snail Helix lucorum. Malacologia, 24: 63-73. DIMITRIADIS, V. K. & M. LIOSI, 1992, Ultrastruc- tural localization of periodate-reactive complex carbohydrates and acid-alkaline phosphatases in digestive gland cells of fed and hibernated Helix lucorum (Mollusca: Helicidae). Journal of Mollus- can Studies, 58: 233-243. FRETTER, V., 1952, Experiments with ®P and '3'| on species of Arion, Helix and Agriolimax. Quar- terly Journal of Microscopical Science, 93: 133-146. FLORSCHUTZ, A. & W. BECKER, 1999, Gastroin- testinal transit and digestive rhythm in Bio- phalaria glabrata (Say.) Journal of Molluscan Studies, 65: 163-170. MORTON, B. S., 1974, The seasonal variation in the feeding and digestive cycle of Amphibola cer- enata (Martin 1784) (Gastropoda: Pulmonata). Forma et Function, 8: 175-190. MORTON, B. S., 1975, The diurnal rhythm and the feeding responses of the South East Asian man- grove bivalve, Geloina proxima Prime 1864. (Bi- valvea: Corbiculacea). Forma et Functio, 54: 482-500. MORTON, B. S., 1979, The diurnal rhythm and the cycle of feeding and digestion in the slug Dero- ceras caruanae. Journal of Zoology, 187: 135- 152. PORCEL, D., J. D., BUENO & A. ALMENDROS, 1996, Alterations in the digestive gland and shell of the snail Helix aspersa Müller (Gastropoda, Pulmonata) after prolonged starvation. Compara- tive Biochemistry & Physiology, A [Physiology], 115: 11-17. RUNHAM, N. W. 1975, Alimentary canal. Pp. 53-105, in: FRETTER, V. and PEAKE, J., eds., Pul- monates. Vol. 1. Functional anatomy and physi- ology. London, Academic Press. STAIKOU, A. & M. LAZARIDOU-DIMITRIADOU, 1989, Feeding experiments on and energy flux in a natural population of the edible snail Helix luco- rum L. (Gastropoda: Pulmonata: Stylommato- phora) in Greece. Malacologia, 31: 217-227. SUMNER, A. T., 1965, The cytology ad cytochem- istry of the digestive gland cells of Helix. Quar- terly Journal of Microscopical Science, 106: 173- 192. THIELE, G., 1953, Vergleichende unterschuchun- gen uber den Feinbau und die Funktion der mit- terdarmdruse einheimischer Gastopoden. Zeit- schrift für Zellforschung und Microscopische Anatomie, 38: 87-93. 3 TRIEBSKORN, R. & A. FLORSCHUTZ, 1993, Transport of uncontaminated and molluscicide- containing food in the digestive tract of the slug Deroceras reticulatum (Müller). Journal of Mol- luscan Studies, 59: 35-42. VELDHUIJZEN, J. P., 1974, Sorting and retention time of particles in the digestive gland of Lym- naea stagnalis (L). Netherlands Journal of Zool- ogy, 24: 10-21. WALKER, G., 1970, The cytology, cytochemistry and ultrastructure of the cell types found in the di- gestive gland of the slug Agriolimax reticulatus (Muller.). Protoplasma, 71: 91-109. WALKER, G., 1972, The digestive system of the slug Agriolimax reticulatus (Müller.): Experiments on phagocytosis and nutrient absorption. Pro- ceedings of the Malacological Society London, 40: 33-43. Revised ms. accepted 25 April 2001 MALACOLOGIA, 2002, 44(1): 153-163 INTERPOPULATION VARIATION IN LIFE-HISTORY TRAITS OF POMACEA CANALICULATA (GASTROPODA: AMPULLARIIDAE) IN SOUTHWESTERN BUENOS AIRES PROVINCE, ARGENTINA Pablo R. Martin & Alejandra L. Estebenet Universidad Nacional del Sur, Departamento de Biologia, Bioquimica y Farmacia, San Juan 670, 8000 Bahia Blanca, Argentina; pmartin@criba.edu.ar ABSTRACT The Argentinean apple snail Pomacea canaliculata is a freshwater gastropod with a high inter- population variation in shell shape, size and thickness. Previous experimental studies have shown that many life-history traits are highly dependent on rearing conditions. Three natural populations located in one same drainage basin and climatic regime showed marked differences in birth, ma- turity and maximum sizes. Most of this variation disappeared when newborns from each popula- tion were reared under homogeneous conditions in the laboratory, indicating its ecophenotypic ori- gin. However, a significant variation in reproductive, growth and survival patterns attributable to genetic differences among the source populations was still discernible among laboratory cohorts. The three sites studied represent a marked gradient in stability and food availability. Females from the most unstable and poorer site showed a faster prematurity growth and a higher ovipo- sition rate than those from the most stable and productive site. This higher oviposition rate was associated with bigger clutches and a higher mortality rate. The different patterns of survival and somatic and reproductive allocation in the three populations, being heritable and adaptively cor- related to different environmental conditions, could be considered as parts of different life-history strategies. Since the 1980s, P. canaliculata has become a serious pest of paddy fields in most Southeast Asian countries. Nearby and recently isolated populations of Р canaliculata from the same basin showed genetically different life-history strategies, so that different populations of this pest could require different control programs. Key words: apple snail, life-history traits, ecophenotypic variation, genetic variation INTRODUCTION Pomacea canaliculata (Lamarck, 1822) isa freshwater prosobranch gastropod with a high polymorphism in shell shape, size and thick- ness (Cazzaniga, 1987). Its natural distribu- tion is basically tropical and subtropical, in- cluding the Amazonas and La Plata basins (Ihering, 1919), though in the last two decades it has invaded most Southeastern Asia coun- tries, becoming a serious rice pest (Wada, 1997). During an extensive survey in the southern limit of the native area of this species, the southernmost apple snail in the world (Martin et al., 2001), we observed a great in- terpopulation variation in maximum size, shell thickness and egg size, even at a small spatial scale and within the same drainage system. Experimental trials have shown that growth rates, longevity and age at maturity are highly dependent on temperature (Estebenet & Caz- zaniga, 1992), crowding (Cazzaniga & Es- tebenet, 1988; Tanaka et al., 1999), and food 153 (Lacanilao, 1990; Estebenet, 1995; Albrecht et al., 1999; Lach et al., 2000). This evidence and the strong intersite environmental varia- tion (Martin et al., 2001) seems to support the hypothesis of an ecophenotypic origin of the interpopulation variation. Cazzaniga (1987) reported that shells from lentic, soft-bottom water bodies are generally bigger and lighter than those from hard-bottom running waters. However, a genetic basis of this interpopula- tion variation cannot be ruled out. The aims of this study were to analyze the variation in birth, maturity and maximum sizes among populations of P. canaliculata from three environmentally different sites belonging to the same drainage basin, and to determine if they were ecophenotypic or genetic in origin. STUDY AREA The study area lay between 36°40’S and 37°35’S and 61°15’W and 63°10’W in south- 154 MARTIN & ESTEBENET western Buenos Aires Province, Argentina. The climate of the region is temperate, with a mean annual temperature and amplitude of 13.8°C and 15.1°C, respectively; mean an- nual rainfall is 733 mm, mainly concentrated in fall and spring. The rainfall is highly vari- able, with marked fluctuations between dry and wet years (Scian & Donnari, 1997). The three sites selected belong to the En- cadenadas del Oeste Basin (Fig. 1), a closed drainage system composed of a chain of six shallow lakes of increasing conductivity to- wards the west, from Alsina Lake (freshwater) to Epecuen Lake (hypersaline) and de la Sal Lake (temporary salt lake). Streams and creeks of pluvial hydrological regime, with scant and very variable water discharge, run northwards into the lakes from the Ventania Mountains. Alsina Lake is a permanent, eutrophic lake, with a surface area of 105 km”, maximum and mean depths of 6.4 m and 2.8 m respectively Alsina Lake ween EI Quilcé del Monte Lake ix x, El Ниазсаг + Cochicö Lake = del Venado Lake Epecuén Lake yo 1 Cochicö ; Rivulet a de la Bal Lake Curalmalal Stréam ` ` Ventani mr. y RT 2 и E ~~ Mountains г 1 — ? and a shoreline length of 62 km. The bottom is composed of fine sand and mud. The dom- inant macrophytes during the present study were the emergent Typha spp. and Schoeno- plectus californicus and the submerged Pota- mogeton sp. and Myriophyllum elatinoides, sometimes heavily colonized by cyanophytes. Curamalal Stream is a permanent stream approximately 91 km long that discharges in- termittently into Alsina Lake. The bottom is composed of mud with minor areas of lime- stone bed. The macrophytes were the emer- gent Cyperus spp. and S. californicus, the submerged Chara sp. and Potamogeton sp. and the floating-leaved Ludwigia sp. Cochico Rivulet is a 20-km-long creek with intermittent flow along most of its course. It discharges in the salty Cochicó Lake (con- ductivity 11.3 mS.cm '), and during high floods the Curamalal Stream overflows into it. The dominant substratum is mud with some areas of limestone bedrock and cobble. 4 .r Stre Las Tunas Stream kilometers FIG. 1. Map ofthe Encadenadas del Oeste basin showing the location (®) ofthe three study sites (from north to south Alsina Lake, Cochicö Rivulet and Curamalal Stream) (Inset: map of Argentina showing the study zone). LIFE-HISTORY VARIATION IN POMACEA 155 Emergent macrophytes were absent and only occasionally was the presence of scarce Lud- wigia sp., Potamogeton sp. and Enteromor- pha sp. detected. MATERIALS & METHODS The three sites were visited in February, July and October 1998 and February 1999. Two or three people searched for apple snails during up to four hours among the submerged vegetation, under stones, or buried in the sub- stratum; all snails larger than 20 mm were col- lected and their shell length from the apex to the basal extreme of the aperture was mea- sured. In October 1998 and February 1999 copulating snails (i.e., couples in which male penis sheath was inserted into female pallial cavity) were collected and sized. The pink, aerial and conspicuous egg masses, de- posited on riparian or emergent plants, were obtained in March and October 1998 and maintained until hatching in the laboratory. Temperature, conductivity (mS.cm !) and pH were determined in situ with a multimeter Horiba U-10. A subsurface water sample was collected at each site for further analyses. The concentrations of major ions and other limno- logical variables were measured as detailed in Martin et al. (2001). Progeny from at least five clutches from each site (March 1998) that hatched in a pe- riod of 72 hours were pooled and 100 new- born were randomly selected to generate three experimental cohorts. They were reared in sets of ten snails under homogeneous con- ditions (aereated tap water saturated with СО.Са, temperature of 25 + 3°C, permanent illumination and lettuce ad libitum). During the first two months the sets of ten snails were kept in 3 | aquaria and then trans- ferred to 15 | aquaria to avoid crowding (Caz- zaniga & Estebenet, 1988). When the first egg mass was observed in each 15 | aquarium, snails were sexed on the basis of shell shape (Cazzaniga, 1990; Estebenet, 1998) and color and appearance of the gonad (Andrews, 1964), visible through the thin shell. One fe- male and one male were randomly selected, and reared again in 3 | aquaria. The eight re- maining snails of each aquarium were sacri- ficed and stored for later morphometric analy- sis, which will be published elsewhere. Water was changed weekly and survivor- ship, shell length and number of clutches and eggs were recorded. Dead snails were re- placed by mature snails of the same sex with a shell length larger than 30 mm, obtained in the wild and maintained in the lab under the same rearing conditions, to keep rearing con- ditions constant for the survivors, especially mating possibilities and density. The oviposi- tion and spawning rates were calculated as the ratio of life-time production of eggs or egg masses to the length of the spawning period (from first to last spawn). Clutch size was es- timated as the ratio of the life-time production of eggs to the life-time production of egg masses. Field egg masses obtained in March and October 1998 and one egg mass from each couple from each cohort were incubated in the lab. After hatching, groups of 30-50 new- born were dried (80°C, 48 h), weighed to the nearest 0.1 mg, and reweighed after ignition (600°C, 4 h) to obtain total and ash dry weights. Shell lengths of ten hatchlings from field spawns and fifteen from laboratory spawns were measured under a stereoscopic microscope to the nearest 0.01 mm. To test for differences among sites in repro- ductive variables one way Anovas were per- formed. Two way Anovas were used for length and age at death, since sexual dimorphism in these traits has been recorded (Estebenet & Cazzaniga, 1998). For newborn and imma- ture snails nested Anovas were performed on shell length with egg masses or aquaria as the nested factor. RESULTS The values of the physico-chemical vari- ables measured in Alsina and Curamalal sites fall within the range of the water bodies of the Southern Buenos Aires province classified as “habitable for snails” by Martin et al., (2001). The Cochico, contrarily, showed marked fluctuations in conductivity (Fig. 2) and Na* concentration, reaching values out of the mentioned range. The variation in water tem- perature among the three sites was less than 3°C during most part of the year, except in summer, when temperatures 7°C higher were measured in Cochico. Maximum sizes differed markedly among the three populations. The mean shell length of the 15 larger snails on each sampling date is showed in Figure 3. Snails from Cochico were generally smaller than those from Alsina and Curamalal. Despite the similarity in maxi- 156 MARTIN & ESTEBENET & Alsina о {+ Curamalal = 41| e Cochicó _ Е o —® > > 2- 3 $ A 3 РЕН = о 17 o FIG. 2. Seasonal variation in conductivity in the studied sites of Alsina Lake, Cochicó Rivulet and Curamalal Stream. 60 = _ A $$ AS, A — à _ Е 50; 5 ti : Е 1 т = = | 2 | № Е: | = 40 + = À E ” 0 ¢ Feen ] - & Alsina 2 4 {+ Curamalal | | e Cochicó 30 ————— > > > — J FMAM J J AS ON D JS FM 1998 date 1999 FIG. 3. Seasonal variation in mean shell length of the fifteen larger snails in the studied sites of Alsina Lake, Cochicö Rivulet and Curamalal Stream (bars are 95% confidence intervals). mum sizes in spring, the size of the copulating snails was smaller in the Cochico population than in the Curamalal population in October 1998, and also in February 1999 (Fig. 4; t,, = 3.07, р < 0.01 and t,., = 6.03, р < 0.001, re- spectively). Couples were not observed in Alsina on those dates. Despite the highly sig- nificant variation in newborn shell length among clutches of the same site (Fy. 270 = 19.52, Р < 0.0001 and F, jog = 35.33, P < 0.0001, respectively), the intersite variation was highly significant both in March and Oc- tober 1998, Cochicö hatchlings being the smallest in both dates (Table 1). The new- borns from Cochicö were also lighter than those from Alsina and Curamalal, though dif- ferences were significant only in March 1998 50 + Ger = =] : {OCuramalal] | 0 Cochic 40 | 30 + shell length (mm) October 1998 February 1999 FIG. 4. Mean shell length of copulating snails in the Cochicö Rivulet and Curamalal Stream sites in Oc- tober 1998 and February 1999 (bars are 95% con- fidence intervals). (Table 1). Egg and clutch sizes were also evi- dently smaller in Cochico (pers. obs.). Growth, survivorship and oviposition pat- terns for the three laboratory cohorts are shown in Figure 5. The initial size differences disappeared before the age of two months (Table 2), suggesting a higher growth rate for the Cochico cohort. Growth curves were very similar from two months of age onwards, and female shell length at first spawn was not sig- nificantly different (Table 2; one female from Cochicé and Alsina each died accidentally and one from Curamalal that never spawned was excluded from analysis). Length at death was significantly different between sexes (males smaller than females) but not among cohorts (Table 3). Mortality was null during the pre-reproduc- tive phase in the three cohorts but after the beginning of spawning the mortality rate in- creased steadily. It was highest in the Cochico cohort, with a maximum longevity of 318 days. The Alsina cohort showed the lowest mortality, with some snails surviving up to 650 days. Differences in age at death (Table 3) were significant only between Cochico (253.53 days) and Alsina cohorts (343.58 days) (critical value for the SNK test = 71.33 days, p < 0.05). The survivorship pattern of the Curamalal cohort was intermediate, with a mean survival time of 300.37 days and a max- imum longevity of 627 days. Although the age at first spawning and the duration of the spawning period in the Alsina cohort were higher than in the Cochico and Curamalal ones, they were not significantly different (Table 2), probably due to the high variation in reproductive behavior in that co- LIFE-HISTORY VARIATION IN POMACEA TABLE 1. One-way Anova for length and dry weights of newborn from field egg masses (&: Nested Anova with four or five spawns (nested factor) per site and ten newborn per spawn). Underlined means are significantly different to the other two (SNK tests, p < 0.05). Means Variable M.S. 9.1. = P Cochico Curamalal Alsina March 1998 total weight (mg) 0.966 2,1 20.572 0.000 1.053 1.808 1.555 ash weight (mg) 0.411 2,16 9.459 0.002 0.329 0.824 0.648 shell length (mm)& 2.414 2,12 24.738 0.000 2.199 2.628 2.498 October 1998 total weight (mg) 0.181 2,9 2.564 0.131 0.920 1.236 1.356 ash weight (mg) 0.060 2,9 20705404182 0.524 0.735 0.768 shell length (mm)& 0.931 2,9 4.392 0.047 2.156 2.388 2.447 157 hort (Fig. 6). The life-time production of eggs and clutches, and the spawning rate were not significantly different among cohorts, al- though the oviposition rate was significantly lower in Alsina females. On the other hand, the clutch size from the Cochicö cohort was the highest (Table 2). The total and ash dry weights of newborns were not significantly different among the lab- oratory cohorts (Table 2). The variation in newborn shell length among clutches of the same cohort was highly significant (F,¿ 25, = 34.27, P < 0.0001) and greater than the inter- cohort variation (Table 2). Only for the Cochicö site there was a significant variation between newborn from egg masses laid in the field (March 1998) and their own progeny in the laboratory, both in length (nested Anova with five spawns —nested factor — per source and ten newborn per spawn: F, з = 9.62, P < 0.01) and weight (t,, = 3.97, р < 0.001 for total dry weight and t,, = 8.25, р < 0.0001 for ash dry weight). DISCUSSION Pomacea canaliculata shows great life-his- tory interpopulation variation, related mainly to different thermal regimes (Estebenet & Cazzaniga, 1992). Though the three natural populations studied here are located in the same drainage basin and under the same cli- matic regime, they showed marked differ- ences in some life-history traits. The variation in maximum sizes among the sites indicates different survivorship or growth rates. On the other hand, at the beginning of the reproduc- tive season (early spring, Hylton-Scott, 1958) the size of copulating snails differed between the two stream populations, though the size distributions were very similar, suggesting dif- ferences in size or age at maturity. Interpopu- lation variation in the size of eggs, clutches and newborn was also observed. Most of the interpopulation variation in birth, maturity and maximum sizes disappeared when the snails were reared under homoge- neous conditions in the laboratory, indicating an ecophenotypic origin of the variation. How- ever, notwithstanding the elimination of envi- ronmental differences, some variation in re- productive, growth and survival patterns was still discernible and attributable to genetic dif- ferences among populations. Although the maternal and environmental effects on the newborn used to initiate the cohorts cannot be discarded, their influence seems to be irrele- vant. The effect of site conditions during em- bryo development was probably null, since very fresh clutches (easily recognizable by their moisture and bright pink color) were used. On the other hand, the differences in newborn size, partially attributable to mater- nal size or condition (see below) soon disap- peared under the highly favorable rearing conditions in the laboratory. The three sites represent a marked gradi- ent in stability and food availability. The Cochicö is an intermittent creek, with very marked seasonal fluctuations in water level, the lower section being the only one that never dries completely; however, high con- ductivity peaks caused by the rise in Cochicö Lake water levels affect this section. These peaks exceed the maximum values of con- ductivity recorded for sites inhabited by Р canaliculata in this area (2.89 mS.cm !, Martin et al., 2001). Minimum discharge in Curamalal Stream is higher than in the Cochicö Rivulet, and the flow is continuous even during dry summers. Though the area of Alsina Lake is quite variable, water level fluc- tuations are comparatively slow and related 158 MARTIN & ESTEBENET 100 ersrarireraie ea ana - ee 600 fe eS DÍ DABAAAAN = Alsina SER ¡egg production 8 — = shell length Г 500 \ E survirvorship da = + 400 60 - Ñ — ТУ ee ЕН ee at Lalla Г 300 40 II ttt à + : AAA RAA AN 200 7 IR a 20 © ee | Il _n | 100 * | | Il | | ol o Oooo 1 | | HL ВД ола. In. 0 | 0 17 52 87 122 262 297 332 367 402 100 qatar AAA Ahh LA à — 600 Curamalal cg er \ oo 17 52 87 12 157 192 227 262 297 332 367 402 shell length (mm) or survivorship (%) egg production (eggs.female ‘.wk") 100 гео —— —— —- 600 — DA | NES | A an costed ds | | + | \ + 400 | A 4 | À +444 2722257 CH | 300 40 Y \ и ie + 200 $ \ | Ltstytytsh 20 =“ \ г 100 Аа НЙ \ 0 | ey u e RETA) 0 17 52 87 157 192 22 262 297 332 . 367 1 1402 age (days) FIG. 5. Growth, survivorship and oviposition patterns for the three laboratory cohorts from Alsina Lake, Cochicö Rivulet and Curamalal Stream (bars are 95% confidence intervals for shell length). LIFE-HISTORY VARIATION IN POMACEA 159 TABLE 2. One-way Anova for growth and reproductive variables from laboratory cohorts. Underlined means are significantly different to the other two (SNK tests, p < 0.05). (#: Nested Anova with ten aquaria (nested factor) per cohort and ten snails per aquarium; &: nested Anova with six females (nested factor) per cohort and fifteen newborn per female; +: data from some females were lost). Variable M.S. d.f. Length at 17 d (mm) + 27.883 2,27 Length at 65 d (mm) + 16367 2,27 Length at first spawn (mm) 32.490 2,24 Age at first spawn (days) 2806.48 2,24 Spawning period (days) Clutch size (eggs.clutch ') Oviposition rate (е99$.мК`') Spawning rate (clutches.wk~ ') 0.0191 2,24 Life-time egg production 126115.15 2,24 Life-time clutch production 195.44 2,24 Newborn shell length (mm) & 0.960 2,15 Newborn total weight (mg) 0.179 2, 18+ Newborn ash weight (mg) 0.018 2, 18+ 11322.11 2,24 25361.35 2, 24 49543.56 2, 24 TABLE 3. Two-way Anova for growth and survival of males and females from labo- ratory cohorts. Variable Source Length at death (mm) Site Sex Site x Sex Error Age at death (days) Site Sex Site x Sex Error — á—]<]á] á]á|l ———— ЕЕ Alsina oo ——ч > — —. —— —— 4 A, | Curamalal ranked females Cochicó m— — —— —) — —— a ——_—_— — -— т T T = T T T T T T 0 50 100 150 200 250 300 350 400 450 500 550 600 age (days) FIG. 6. Spawning period duration of females from the three laboratory cohorts from Alsina Lake, Cochicö Rivulet and Curamalal Stream ranked by age at first spawn. Means F Pp Cochicó Curamalal Alsina 13.354 0.000 5.07 6.00 Sal 2.052 0.148 23.70 23.99 23.19 1.598 0.223 47.27 47.82 50.80 0.752 0.482 136.78 164.55 169.55 2.655 0.091 90.00 92.78 152.78 6.696 0.005 264.75 204.29 158.94 4.576 0.021 375.10 346.36 234.66 0.748 0.484 1.47 1543 1.48 0.023 0.977 4554.22 4407.22 4641.45 1.261 0.302 19.00 22.45 28.21 3.501 0.056 2.48 2.46 2.65 2.333 07126 1137 1.41 1.67 0.618 0.550 0.78 0.80 0.88 M.S. 9.1. Е Р 52.87 2 2.761 0.073 222.80 1 11.633 0.001 17.56 2 0.917 0.406 19.15 51 38394.78 2 4.495 0.016 10938.61 1 1.281 0.263 1224.78 2 0.143 0.867 8541.13 51 mainly to multiannual cycles of rainfall (IATASA, 1994; Gonzälez Uriarte & Orioli, 1998). On the other hand, the scarcity of aquatic macrophytes and riparian vegetation suggest that trophic constraints are stronger in the Cochicö Rivulet. In this region, water bodies inhabited by P. canaliculata generally are quite shallow, quiet and turbid, with low Na*/(K* + Mg**) ratios as compared to the un- inhabited sites (Martin et al., 2001). The Cochicö Rivulet is a less suitable habitat for these snails than the other two sites. The lower maximum and maturity sizes in Cochicö Rivulet were probably due to lower growth rates, although the detrimental influ- ence of recurrent disturbance on longevity cannot be ruled out. The low food availability, and not the temperature, was probably the 160 MARTIN & ESTEBENET growth limiting factor, because laboratory snails from this source showed the highest growth rates during most of the pre-reproduc- tive period, and summer water temperature was higher at this site. Minimum female re- productive sizes of approximately 25 mm have been recorded for P. canaliculata grow- ing under very different densities, food and temperature conditions (Martin, 1986; Es- tebenet & Cazzaniga, 1992; Tanaka et al., 1999), age at first oviposition being hence highly dependent on growth rate. In this study, the first spawn of each cohort was observed at ages between 80 and 99 days, a very early age as compared to other cohorts at 25°C (Estebenet & Cazzaniga, 1992, 1998), but at 39 mm shell length, indicating that maturity depends not only on size, but that a minimum age is also required. The slow growth at the Cochicö site and the lack of genetic interpop- ulational variation in age and size at maturity suggest that differences in size of field copu- lating snails are not due to an earlier matura- tion in Cochicö. In this population, the mini- mum age is probably attained before the minimum size, whereas in the Curamalal the minimum size is reached before the minimum age. Hatching in P. canaliculata occurs by me- chanical fracture of the calcareous egg shell, after consuming all the perivitelline egg re- serve, at which stage the length of the new- born equals the diameter of the egg (Es- tebenet & Cazzaniga, 1993; Heras et al., 1998). Estebenet & Cazzaniga (1993) re- ported that the egg size variation among clutches from the same pond was greater than interpond variation. In the present study, on the contrary, the intersite variation was greater than intrasite variation. The size of newborn was the lowest in Cochicö Rivulet, probably limited by the small size of females, because newborns from laboratory reared, big-sized females from this source were big- ger and similar in size to those from the other two sources. Female size in the permanent ponds studied by Estebenet & Cazzaniga was greater than in Cochico and probably was not a constraint for egg size. Under homogeneous conditions, snails from the three sites showed variation in re- productive, growth and survival patterns. Fe- males from Cochicó showed a genetically fixed tendency to lay a higher number of eggs per week and packed in bigger clutches than those from Alsina. This higher oviposition rate implies a higher reproductive allocation asso- ciated with a higher mortality rate, probably owing to a competence between somatic maintenance and reproduction only (Jokela, 1997), because growth was almost null during most of the reproductive period. The different patterns of survival, somatic and reproductive allocation in the three popu- lations could be considered as parts of differ- ent life-history strategies, because they showed a genetic component and are corre- lated in an adaptive way to different environ- mental conditions (Calow, 1978, 1983). Under the thermal regime of the study zone, P canaliculata is iteroparous, with two or more reproductive periods limited to the warmer months (Estebenet & Cazzaniga, 1992, 1993). Fast prematurity growth and a higher reproductive output, despite the detrimental effect of the latter on survivorship and future egg production, seems to be adaptive in the Cochicö Rivulet. At this site, where frequent and unpredictable disturbance events cause mass mortality episodes, probably only a few mature snails survive to reproduce in a sec- ond season. In contrast, in the Alsina Lake, where the frequency and magnitude of the disturbances are lower, a restrained repro- ductive allocation can led to more reproduc- tive seasons, thus maximizing the lifetime egg production. The adaptive value of early maturation and high reproductive effort depends on a lower survival of parents relative to their offspring (Calow, 1983). Adults of Pomacea spp. from ephemeral but more predictable water bodies aestivate (Burky, 1974; Kushlan, 1975; Don- nay & Beissinger, 1993), but this behavior has not been described for P. canaliculata. Al- though water loss and susceptibility are prob- ably higher in small snails (Turner, 1996), their chances of fitting in humid microhabitats are also higher. Pomacea canaliculata mobility and feeding are highly reduced when water depth is lower than shell diameter (Wada, 1997), so post-drought survival is probably negatively related to size. Conductivity peaks and drought episodes shorter than the lapse between oviposition and hatching (about 17 days in this region) will affect parent survival without harmful effects on their aerial eggs. In- deed, despite their normal nocturnal spawn- ing behavior (Schnorbach, 1995; Albrecht et al., 1996), P. canaliculata females exposed to air for several hours frequently spawn, even during light hours (Cazzaniga, 1981; pers. obs.), probably increasing the chances of leaving progeny. Scouring by floods into the LIFE-HISTORY VARIATION IN POMACEA 161 salty Cochicö Lake is probably a major source of catastrophic mortality, specially for big snails, less able to withstand fast flow and to find in-stream flow refuges of adequate size (Calow, 1983; Dussart, 1987; Johnson & Brown, 1997). On the other hand, clutches can endure submersion for several days with- out deleterious effects on the embryos (pers. obs.). Apart from local adaptation, genetic drift is another probable cause of interpopulation ge- netic variation. Encadenadas del Oeste basin was once part of an allocthonous river running through the present Vallimanca River into the Salado River (Fig. 1). The chain of lakes was originated in its paleo-valley when discharge diminished after the end of the last glacial age and climate became increasingly arid in the re- gion (Frenguelli, 1945; Malagnino, 1989). Fos- sil records of P. canaliculata are common in the Salado basin (Camacho, 1966; Dangavs & Blasi, 1992), and the species was probably widespread in it when subtropical conditions advanced to southern Buenos Aires Province (8-6 Kyr BP; Iriondo, 1994). Since then, local populations probably became increasingly isolated because of increasing habitat discon- tinuity. The P. canaliculata populations in the studied sites do not seem to have originated from independent arrivals from a genetically heterogeneous source, rendering founder ef- fects unimportant. The maintenance of genetic differentiation among the studied populations is noteworthy, taking into account that their isolation was not complete in the recent past. The three study sites secularly connected each other in most peaks of the multiyear rainfall cycle, espe- cially when Alsina and Cochicö lakes merge and both present freshwater conditions (IATASA, 1994; Gonzalez Uriarte & Orioli, 1998). However, at present the spread of P canaliculata in this geographic region seems to be very slow, even within the same stream (Martin et al., 2001) and gene flow is probably strongly unidirectional, mostly due to down- stream drift. Since 1992, a roadway embank- ment with sluice gates divided Alsina and Cochico lakes, probably increasing their iso- lation. On the other hand, population size in the Cochicö Rivulet was probably very small, due to the small size of the habitable section and low food availability. These factors proba- bly account for the persistence of the genetic differentiation provoked by the strong selec- tive pressures, even in the absence of total habitat discontinuity. Asian pest managers have showed deep concern about the specific identity of apple snails that invaded paddy fields (Wada, 1997; Cowie, in press). However, close and recently isolated populations of P. canaliculata from the same basin showed genetically different life-history strategies, so even populations from the same species could require different control measures. The situation is compli- cated by the fact that most Asian countries suffered repeated introductions of apple snails (Litsinger & Estano, 1993; Wada, 1997; Cowie, in press), probably from distant geo- graphic regions, among which genetic in- traspecific variation is expected to be even greater. Moreover, since the early 1980s the control and culture practices in paddy fields have been exerting new and strong selective pressures that are probably already shaping the major features of Pomacea life history. ACKNOWLEDGMENTS We wish to express our gratitude to Natalia Pizani and Eduardo Albrecht for their assis- tance in field work. This study was funded with grants by CONICET (“Consejo Nacional de Investigaciones Cientificas y Técnicas”) and “Agencia Nacional de Promociön Cientifica y Técnica”, Argentina. ALE is a researcher in CONICET. LITERATURE CITED ALBRECHT, E. A., N. B. CARRENO & A. 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ESTANO, 1993, Manage- ment of the golden apple snail Pomacea canali- culata (Lamarck) in rice. Crop Protection, 12: 363-370. MALAGNINO, E. C., 1989, Evoluciön del sistema fluvial de la Provincia de Buenos Aires desde el Pleistoceno hasta la actualidad. Actas de las Se- gundas Jornadas Geológicas Bonaerenses: 201-211, Bahía Blanca. MARTIN, P. R., A. L. ESTEBENET & N. J. CAZ- ZANIGA, 2001, Factors affecting the distribution of Pomacea canaliculata (Gastropoda: Ampullari- LIFE-HISTORY VARIATION IN POMACEA 163 idae) along its southernmost natural limit. Mala- cologia, 43: 13-23. MARTIN, S. M., 1986, Ciclo reproductivo de Am- pullaria canaliculata (Gastropoda; Ampullariidae) en el ärea rioplatense. Neotröpica, 32: 171-181. SCHNORBACH, H. J., 1995, The golden apple snail (Pomacea canaliculata Lamarck) an increasingly important pest in rice, and methods of control with Bayluscid . Pflanzenschutz-Nachrichten Bayer, 48: 313-346. SCIAN, B. & M. DONNARI, 1997, Aplicaciön del indice Z de Palmer para la comparaciön de se- quias en las regiones trigueras II, IV y V sur de Argentina. Revista de la Facultad de Agronomia, Universidad de Buenos Aires, 17: 41-46. TANAKA, K., T. WATANABE, H. HIGUCHI, K. MIYAMOTO, Y. YUSA, T. KIYONAGA, H. KI- YOTA, Y. SUZUKI & T. WADA, 1999, Density de- pendent growth and reproduction of the apple snail, Pomacea canaliculata: a density manipula- tion experiment in a paddy field. Research in Population Ecology, 41: 253-262. TURNER, R. L., 1996, Use of stems of emergent plants for oviposition by the Florida applesnail, Pomacea paludosa, and implications for marsh management. Florida Scientist, 59: 34-49. WADA, T., 1997, Introduction of the apple snail Po- macea canaliculata and its impact on rice agri- culture. Proceedings of the International Work- shop on Biological Invasions of Ecosystems by Pests and Beneficial Organisms, pp. 170-180, Tsukuba. Revised ms. accepted 9 August 2001 MALACOLOGIA, 2002, 44(1): 165-168 CHROMOSOMES OF PISIDIUM COREANUM (BIVALVIA: VENEROIDA: CORBICULOIDEA: PISIDIIDAE), AKOREAN FRESHWATER CLAM Gab-Man Park,' Tai-Soon Yong,’ Kyung-il Im,* & John В. Burch? ABSTRACT Polyploidy is reported as occurring in Pisidiidae, being found in Pisidium coreanum from Korea. The chromosome number is n = 95 and 2n = 190. Because of the large number of its chro- mosomes, P. coreanum is obviously a polyploid species. The basic chromosome number for the superfamily Corbiculoidea is X = 18 and X = 19. The polyploid condition found in P. coreanum probably originally resulted from a single event. Once the polyploid condition was reached, the stability of this number has been maintained. Key words: Pisidium coreanum, chromosome, Pisidiidae, polyploid, Korea. INTRODUCTION The bivalve superfamily Corbiculoidea has two families of special interest, Corbiculidae and Pisidiidae, both with wide geographical and ecological distributions (Clarke, 1973; Burch, 1975; Kuiper, 1987; Kwon & Park, 1993). The Corbiculoidea are represented in Korea by six species of the Corbiculidae and two species of the Pisidiidae (Kwon et al., 1993). The Corbiculidae are Asian in origin, but in relatively recent times they have been spread to many other parts of the world through human commerce. Particularly in Asia, corbiculid clams are an important food source, not only for wildlife, including fish, but also for humans. Sphaeriid clams, on the other hand, are not economically important, other than providing an important link in various aquatic food chains. But from a biological standpoint, they are significant because of their peculiar lifestyles. They live in many dif- ferent habitats and environments, and some species seem to be nearly cosmopolitan (Burch, 1975). All the species presumably are hermaphroditic, with the same individuals pro- ducing both sperm and ova, although direct observations have been made for only a few species. Fertilization is internal, and the young are brooded in special gill chambers of the par- ent, in some species until the young are rela- tively large (Park & Kwon, 1993). However, ex- actly how fertilization and development take place, that is, whether by cross- or self-fertil- ization, or by some type of parthenogenesis, is not Known, nor is it Known how polyploidy came about (i.e., are the species autopoly- ploids or allopolyploids, and in the later case segmental or genomic?). In the marine bivalve genus Lasaea, which shares many similarities with the corbiculoidean clams (O Foighil & Smith, 1995), polyploidy is also a common oc- currence, and here, at least in the polyploid clones, parthenogenetic development, trig- gered by autosperm (without syngamy) is the rule (O Foighil & Thiriot-Quiévreux, 1991). Our preliminary observations on gametogenesis in sphaeriid clams suggest that it is likely that some, perhaps many, species of corbicu- loidean clams have similar reproductive strategies. Many species are known to be capable of propagation by self-fertilization when the more normal biparental mating behavior is prevented. In such groups, a large amount of polyploidy might be expected, but chromo- some surveys have shown that this is not the case. Natural polyploidy has also been ob- served in Argopecten purpuratus (Alvarez & Lozada, 1992), Sphaerium striatinum (Lee, 1999), in Lasaea spp. (Thiriot-Quiévreux et al., 1988, 1989; O Foighil & Thiriot-Quiévreux, 1999) and in Corbicula spp. (Park et al., 2000). The first Corbiculoidea clams in which polyploidy was detected was Corbicula (Cor- biculina) leana (Okamoto & Arimoto, 1986). The chromosome numbers have been deter- mined for several other species belonging to the order Corbiculoidea (Table 1). In these, the chromosome number varies from n = 12 to ‘Department of Parasitology, Yonsei University College of Medicine, Seoul 120-752, Korea; tsyong212@yumc.yonsei.ac.kr Museum of Zoology and Department of Biology, College of Literature, Science and the Arts, and School of Natural Re- sources, University of Michigan, Ann Arbor, Michigan 48109, USA 166 TABLE 1. Chromosome numbers in Corbiculoidea. PARKETAL. Species Chromosome number References Corbicula fluminea 54 (3n) Park et al., 2000 C. papyracea 54 (3n) Park et al., 2000 C. leana 54 (3n) Okamoto & Arimoto, 1986 C. colorata 38 (2n) Park et al., 2000 C. japonica 38 (2n) Okamoto & Arimoto, 1986 C. sandai 36 (2n) Okamoto & Arimoto, 1986 “C. leana” 24 Nadamitsu & Kanai, 1978 Musculium securis approx. 247 Burch et al., 1998 Sphaerium corneum 36 (2n) Keyl, 1956 S. occidentale approx. 209 Burch et al., 1998 S. striatinum approx. 68-98 Woods, 1931 S. striatinum approx. 152 Lee, 1999 Pisidium casertanum approx. 150, 180 Barsiene et al., 1996 P. casertanum approx. 190 Burch et al., 1998 P. coreanum 190 (2n) Present study n = 76. The chromosome number of C. flu- minea, C. papyracea and C. leana suggests that these species may be members of a 3n species that has been established by poly- ploid evolution. The chromosome numbers of Sphaeriidae have been reported for five species (Table 1). The chromosome numbers obtained in the Sphaeriidae are all very large (over 150 mitotic chromosomes), except for the European S. corneum (2n = 36). This sug- gests that the large numbers of chromosomes in this family represent polyploids. This study presents the chromosome analysis of P. coreanum based on mitotic metaphase chromosomes. MATERIALS AND METHODS Twenty-one specimens of Р coreanum were collected from a spring pond located in Bongmung-ri, Chunchon-city, Kangwon-do, Korea, from June to September 2000, and ex- amined shortly after collection. Chromosome preparations were made from gonadal tissues by the standard air-dry- ing method. The live specimens were set aside for one week in a petri-dish containing 10 ml of distilled water with 0.3 ml of 0.05% colchicine solution. The treated tissues were dissected and minced with needles in a hypo- tonic 0.01% NaCl solution. Separated cells were collected by centrifugation at 930 g for 10 min. These cells were fixed in freshly mixed modified Carnoy’s fixative (three parts methyl alcohol and one part glacial acetic acid). The supernatant was replaced by fresh fixative. The centrifugation (930 g, 10 min) was repeated two more times. A drop of the cell suspension was then pipetted by a micro- hematocrit capillary tube and dropped onto a clean slide glass pre-cooled to 4°C. The cells on the slide were air-dried and then stained for 10 min with 4% Giemsa (Gurr R66) solu- tion made up in 0.1 M phosphate buffer, pH 7.0. The prepared slides were observed under an Olympus (BX50F-3) microscope with a 100X (n.a. 1.25) oil immersion objective and a 10X ocular. Voucher specimens of the shells used in this investigation have been placed in the Department of Parasitology, Yonsei University College of Medicine, Korea. RESULTS The mean shell size determined in this study consisted of a measured shell length of 4.2 mm, shell height of 4.1 mm, and shell breath of 2.0 mm. Twelve individuals were ex- amined for chromosomes. Eighteen cells clearly had 190 chromosomes. Chromosome numbers of n = 95 and 2n = 190 were counted (Fig. 1). Ninety-five bivalents were observed during late prophase (diakinesis) (Fig. 1A). The chromosome types of this species con- sisted of metacentric, submetacentric and te- locentric chromosomes as shown (Fig. 1B). However, this chromosome figure was not sufficient for analyzing karyotypes, not only because of the large numbers but also the small size. The longest dimension of the largest metaphase bivalent was only 3.6 um. DISCUSSION Polyploidy is the multiplication of the normal chromosome number of an organism. Be- CHROMOSOME OF PISIDIUM COREANUM 167 A pp" tree As и % x ? | eo А ña * „+ Y AY a d ’ №, ARCHE > < Y e Y @ } Re de > >» “у tue tas & L En JS wh o/s ps un a FIG. 1. Chromosomes of Pisidium coreanum. A, diakinesis (n = 95). B, metaphase chromosomes (2n = 190) Scale bars indicate 5 um. cause of the inability of a newly derived poly- ploid to breed with its sibling diploid it is usu- ally chromosomally sterile. Accordingly, poly- ploidy is a method, and the only clearly established one, for instantaneous speciation (Wagner et al., 1993). The occurrence of triploid chromosomes in three species of the genus Corbicula has been reported (Okamoto & Arimoto, 1986; Park et al., 2000) (Table 1). From the numbers found in the three species of Corbicula, each of these species is poly- ploid. The family Pisidiidae contains three sub- families, the Pisidiinae, Sphaeriinae and Eu- perinae. In the Sphaeiinae, four species has been studied, Sphaerium corneum (Keyl, 1956), S. occidentale (Burch et al., 1998), S. striatinum (Woods, 1931; Lee, 1999) and Musculium securis (Burch et al., 1998), and the mitotic chromosome numbers of these species have been reported ranging from 36 to approximately 152. In the Pisidiinae, the chro- mosome numbers of P. casertanumis 2n = ap- proximately 247 as counted by Burch et al. (1998). This study, the first report of polyploidy in Pisidiidae clams in Korea, determined that the chromosome number of P. coreanumis 2n = 190. Polyploidy is probably an important method of evolution in the cosmopolitan and common genus Pisidium, and may account for the morphological patterns that have made taxonomy so difficult. Most molluscan groups are generally conservative with regard to chro- mosomal change (Patterson, 1969). However, the chromosome numbers are very variable among Mytilidae and Pectinidae (Nakamura, 1985). Also, the chromosome number within two families of the order Veneroidea, 18 to 95 pairs of chromosomes, is not constant. The origin of polyploidy in the Pisidiidae is still speculative. In mollusks, Burch & Huber (1966) suggested that polyploidy came about in the African-Near Eastern Bulinus by hy- bridization, followed by a doubling of the chro- mosome number, that is, allopolyploidy was involved. Cytological evidence presented by Goldman et al. (1983) tends to support this conclusion. Certainly the ecological conditions where most of the ploidy states in Bulinus occur — the Ethiopian highlands, located near the equator — provide the opportune physical characteristics in which polyploid events might be expected to occur (Patterson & Burch, 1988). Future studies of the chromosomes of other species of the Pisidiidae family are needed to document the variability of the chromosome number within this group. LITERATURE CITED ALVAREZ, S. & L. E. LOZADA, 1992, Spontaneous triploidy in Chilean scallop Argopecten purpura- tus (Lamark 1819) (Bivalvia, Pectinoidae). /nves- tigacion Pesqueras, 37: 119-126. BARSIENE, J., G. TAPIA & D. BARSYTE, 1996, Chromosomes of mollusks inhabiting some 168 PARKETAL. mountain springs of eastern Spain. Journal of Molluscan Study, 62: 539-543. BURCH, J. B., 1975, Freshwater sphaeriacean clams (Mollusca: Pelecypoda) of North America. Malacological Publications, Hamburg, Michigan. 96 pp. BURCH, J. B. & J. M. HUBER, 1966, Polyploidy in mollusks. Malacologia, 5: 41-43. BURCH, J. B., С. M. PARK & Е. У. CHUNG, 1998, Michigan’s polyploid clams [abstract]. Michigan Academician, 30: 351-352. CLARKE, A. H., 1973, The freshwater mollusca of the Canadian Interior Basin. Malacologia, 13: 1-509. GOLDMAN, M. A., Р. Т. LOVERDE & С. L. CHIRS- MAN, 1983, Hybrid origin of polyploidy in fresh- water snails of the genus Bulinus (Mollusca: Planorbidae). Evolution, 37: 592-600. KEYL, H. G., 1956, Boobachtungen über die ©- meiose der Muschel Sphaerium corneum. Chro- mosoma, 8: 12-17. KUIPER, J. ©. J., 1987, Systematic rank, synonymy and geographical distribution of Pisidium ob- tusale, P. rotundatum and P. ventricosum. Walk- erana, 2: 145-158. KWON, O. K. & J. C. PARK, 1993, Studies of the development and the spawning season of Pisid- ¡um (Neopisidium) coreanum (Bivalvia: Sphaeri- idae). The Korean Journal of Malacology, 9: 33-38. KWON, O. K., G. M. PARK & J. S. LEE, 1993, Coloured shells of Korea. Academy Publishing Company, Seoul. 221-224 pp. LEE, T., 1999, Polyploidy and meiosis in the fresh- water clam Sphaerium striatinum (Lamark) and chromosome numbers in the Sphaeriidae (Bi- valvia, Veneroida). Cytologia Tokyo, 64: 247- 252. NADAMITSU, S. & T. KANAI, 1978, On the chro- mosomes of the three species in two families in freshwater Bivalvia. Bulletin Hiroshima Women’s University, 10: 1-5. NAKAMURA, H. K., 1985, A review of molluscan cy- togenetic information based on the CISMOCH- Computerized Index System for Molluscan Chro- mosomes. Bivalvia, Polyplacophora and _ Cephalopoda. Venus, 44: 193-225. O FOIGHIL, D. & M. J. SMITH, 1995, Evolution of asexuality in the cosmopolitan marine clam _ Lasaea. Evolution, 49: 140-150. O FOIGHIL, D. & C. THIRIOT-QUIEVREUX, 1991, Ploidy and pronuclear interaction in northeastern Pacific Lasaea clones (Mollusca: Bivalvia). Bio- ‚ logical Bulletin, 181: 222-231. _ O FOIGHIL, D. & C. THIRIOT-QUIEVREUX, 1999, Sympatric Australian Lasaea species (Mollusca, Bivlavia) differ in their ploidy levels, reproductive modes and developmental modes. Zoology Jour- nal and Linnaeous Society, 127: 477-494. OKAMOTO, A. & B. ARIMOTO, 1986, Chromo- somes of Corbicula japonica, C. sandai and C. (Corbiculina) leana (Bivalvia: Corbiculidae). Venus, 45: 194-202. PARK, J. C. & O. K. KWON, 1993, Studies on the development and the spawning season of Pisid- ¡um (Neopisidium) coreanum (Bivalvia: Spheri- idae). The Korean Journal of Malacology, 9: 33-38. PARK, G. M., T. S. YONG., K. IM & E. Y. CHUNG, 2000, Karyotypes of three species of Corbicula (Bivalvia: Veneroida) in Korea. Journal of Shell- fish Research, 19: 979-982. PATTERSON, C. M., 1969, Chromosomes of mol- luscs. Proceedings of the Symposium on Mol- lusca, Marine Biological Association India, 2: 635-689. PATTERSON, C. M. & B. BURCH, 1988, Chromo- somes of pulmonate mollusks. Pp 171-217, in: v. FRETTER & J. PEAKE, eds., Pulmonates, Vol. 2A, Systematics, evolution and ecology. Academic Press, London, New York and San Francisco. 540 pp. | THIRIOT-QUIEVREUX C., A. M. INSUA POMBO & P. ALBERT, 1989, Polyploïdie chez un bivalve in- cubant, Lasaea rubra (Montagu). Comptes Ren- dus de Séances d’Academie de Science, Paris, 308: 115-120. THIRIOT-QUIEVREUX C., J. SOYER, F. de BOVEE & P. ALBERT, 1988, Unusual chromo- some complement in the brooding bivalve Lasaea consanguinea. Genetica, 76: 143-151. WAGNER, РВ. Р. М. P. MAGUIRE & В. L. STALLINGS, 1993, Variation in chromatin organi- zation and amount. Pp. 269-272, in: Chromo- somes: a synthesis. Wiley-Liss. New York. WOODS, F. H. 1931, History of the germ cells in Sphaerium striatnum (Lam.). Journal of Morphol- ogy and Physiology, 51: 545-595. Revised ms. accepted 15 August 2001 MALACOLOGIA International Journal of Malacology Vol. 44(1) 2002 Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Publication dates 34, No. 35, No. 35, No. 36, No. 37, No. 37, No. 38, No. 39, No. 40, No. 41, No. 41, No. 42, No. 43, No. 4 4 2 4 4 2 1 1 1 1 2 1 1 -2 -2 -2 -2 9 Sep. 14 Jul. 2 Dec. 8 Jan. 13 Nov. 8 Mar. 17 Dec. 13 May 17 Dec. 22 Sep. 31 Dec. 18 Oct. 20 Aug. 1992 1993 1993 1995 1995 1996 1996 1998 1998 1999 1999 2000 2001 VOL. 44, NO. 1 MALACOLOGIA 2002 CONTENTS ANDREA C. ALFARO & ANDREW G. JEFFS Small-scale Mussel Settlement Patterns Within Morphologically Distinct Substrata at Ninety Mile Beach, Northern New Zealand................ 1 EUGENE V. COAN The Eastern Pacific Recent Species of the Corbulidae (Bivalvia) ......... 47 ROBERT T. DILLON, JR., & ANDREW J. REED A Survey of Genetic Variation at Allozyme Loci among Goniobasis Populations Inhabiting Atlantic Drainages of the Carolinas .............. 23 VASILIS K. DIMITRIADIS & VASILIKI KONSTANTINIDOU Origin of the Excretory Cells in the Digestive Gland of the Land Snail ESTU Nike tanec Nene М а 145 SINOS GIOKAS & MOYSIS MYLONAS Spatial Distribution, Density and Life History in Four Albinaria Species (Gastropoda. Pulmonata, CGlausilidae) 2... 2... ...... 33 GENNADY M. KAMENEV Genus Parvithracia (Bivalvia: Thraciidae) with Descriptions of a New Subgenus and Two New Species from the Northwestern Pacific ......... 107 PABLO R. MARTIN & ALEJANDRA L. ESTEBENET Interpopulation Variation in Life-history Traits of Pomacea canaliculata (Gastropoda: Ampullariidae) in Southwestern Buenos Aires Province, ATOME ce RE N See ne ee me: 153 LUIS A. MERCADO, SERGIO H. MARSHALL, & GLORIA M. ARENAS Detection of Phenoloxidase (PO) in Hemocytes of the Clam Venus antiqua ТИ GAB-MAN PARK, TAI-SOON YONG, KYUNG-IL IM, & JOHN В. BURCH Chromosomes of Pisidium coreanum (Bivalvia: Veneroida: Corbiculoidea: Pisidiidae) a /KoreanFreshwatenClamit ar meer a IR: 165 ANATOLY A. VARAKSIN, EUGENIA А. PIMENOVA, GALINA $. VARAKSINA, & LYDIA T. FROLOVA Localization of Nadph-diaphorase-positive Elements in the Intestine of the Mussel Crenomytilus grayanus (Mollusca: Bivalvia) ................... 135 WHY NOT SUBSCRIBE TO MALACOLOGIA ORDER FORM Your name and address Send U.S. $56.00 for a personal subscription (per volume) or U.S. $70.00 for an institutional subscription. VISA and MASTERCARD are accepted for an addi- tional $2.00 fee. Checks must be drawn on an American bank and made payable to MALACOLOGIA. Address: Malacologia P.O. Box 385 Haddonfield, NJ 08033-0309 U.S.A. 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JEFFS Small-scale Mussel Settlement Patterns Within Morphologically Distinct Substrata at Ninety Mile Beach, Northern New Zealand................ LUIS A. MERCADO, SERGIO H. MARSHALL, & GLORIA M. ARENAS Detection of Phenoloxidase (PO) in Hemocytes of the Clam Venus antiqua ROBERT T. DILLON, JR., & ANDREW J. REED A Survey of Genetic Variation at Allozyme Loci among Goniobasis Populations Inhabiting Atlantic Drainages of the Carolinas .............. SINOS GIOKAS & MOYSIS MYLONAS Spatial Distribution, Density and Life History in Four Albinaria Species (Gastropoda, Pulmonata, Clausiliidae) .. 2... cic. 60 ее, oe Hee EUGENE V. COAN The Eastern Pacific Recent Species of the Corbulidae (Bivalvia) ......... GENNADY M. KAMENEV Genus Parvithracia (Bivalvia: Thraciidae) with Descriptions of a New Subgenus and Two New Species from the Northwestern Pacific ......... ANATOLY A. VARAKSIN, EUGENIA A. PIMENOVA, GALINA S. VARAKSINA, & LYDIA Т. FROLOVA Localization of Nadph-diaphorase-positive Elements in the Intestine of the Mussel Crenomytilus grayanus (Mollusca: Bivalvia) ................... VASILIS K. DIMITRIADIS & VASILIKI KONSTANTINIDOU Origin of the Excretory Cells in the Digestive Gland of the Land Snail IN A ene de AAA AO PABLO В. MARTÍN 8 ALEJANDRA L. ESTEBENET Interpopulation Variation in Life-history Traits of Pomacea canaliculata (Gastropoda: Ampullariidae) in Southwestern Buenos Aires Province, ее ee GAB-MAN PARK, TAI-SOON YONG, KYUNG-IL IM, & JOHN B. BURCH Chromosomes of Pisidium coreanum (Bivalvia: Veneroida: Corbiculoidea: Pisidiidae), a Korean Freshwater Clam ....::......:...:.:.,442 02% 2002 17 107 135 145 153 165 J. A. ALLEN Marine Biological Station Millport, United Kingdom jallen @ udcf.gla.ac.uk Е. Е. BINDER Museum d’Histoire Naturelle Geneve, Switzerland P. BOUCHET Museum National d’Histoire Naturelle Paris, France bouchet @cimrs1.mnhn.fr P. CALOW University of Sheffield United Kingdom R. CAMERON Sheffield United Kingdom R.Cameron @ sheffield.ac.uk J. G. CARTER University of North Carolina Chapel Hill, U.S.A. MARYVONNE CHARRIER Universite de Rennes France Maryvonne. Charrier @univ-rennes1.fr R. H. COWIE University of Hawaii Honolulu, HI., U.S.A. A. H. CLARKE, Jr. Portland, Texas, U.S.A. B. C. CLARKE University of Nottingham United Kingdom R. DILLON College of Charleston SC, U.S.A. C. J. DUNCAN University of Liverpool United Kingdom D. J. EERNISSE California State University Fullerton, U.S.A. E. GITTENBERGER Rijksmuseum van Natuurlijke Historie Leiden, Netherlands sbu2eg @rulsfb.leidenuniv.de F. GIUSTI Universita di Siena, Italy giustif @ unisi.it 2002 EDITORIAL BOARD А. N. GOLIKOV Zoological Institute St. Petersburg, Russia S. J. GOULD Harvard University Cambridge, Mass., U.S.A. A. V. GROSSU Universitatea Bucuresti Romania T. HABE Tokai University Shimizu, Japan R. HANLON Marine Biological Laboratory Woods Hole, Mass., U.S.A. G. HASZPRUNAR Zoologische Staatssammlung Muenchen Muenchen, Germany haszi @zi. biologie. uni-muenchen.de J. M. HEALY University of Queensland Australia jhealy O zoology.uq.edu.au D. M. HILLIS University of Texas Austin, U.S.A. K. E. HOAGLAND Council for Undergraduate Research Washington, DC, U.S.A. Elaine Ocur. org B. HUBENDICK Naturhistoriska Museet Goteborg, Sweden S. HUNT Lancashire United Kingdom R. JANSSEN Forschungsinstitut Senckenberg, Frankfurt am Main, Germany М. $. JOHNSON University of Western Australia Nedlands, WA, Australia msj@cyllene.uwa.edu.au R. N. KILBURN Natal Museum Pietermaritzburg, South Africa M. A. KLAPPENBACH Museo Nacional de Historia Natural Montevideo, Uruguay MCZ IBRARY HATVARD ıxiVERSITY J. KNUDSEN Zoologisk Institut Museum Kobenhavn, Denmark C. LYDEARD University of Alabama Tuscaloosa, U.S.A. clydeard @ biology.as.ua.edu C. MEIER-BROOK Tropenmedizinisches Institut Tubingen, Germany H. K. MIENIS Hebrew University of Jerusalem Israel J. Е. MORTON The University Auckland, New Zealand J. J. MURRAY, Jr. University of Virginia Charlottesville, U.S.A. R. NATARAJAN Marine Biological Station Porto Novo, India DIARMAID O’FOIGHIL University of Michigan Ann Arbor, U.S.A. J. OKLAND University of Oslo Norway T. OKUTANI University of Fisheries Tokyo, Japan W. L. PARAENSE Instituto Oswaldo Cruz, Rio de Janeiro Brazil J. J. PARODIZ Carnegie Museum Pittsburgh, U.S.A. R. PIPE Plymouth Marine Laboratory Devon, United Kingdom RKPI@wpo.nerc.ac.uk J. P. POINTIER Ecole Pratique des Hautes Etudes Perpignan Cedex, France pointier @ gala.univ-perp.fr М.Е. PONDER Australian Museum Sydney QUEZY Academia Sinica Qingdao, People's Republic of China D. G. REID The Natural History Museum London, United Kingdom S. G. SEGERSTRÄLE Institute of Marine Research Helsinki, Finland A. STANCZYKOWSKA Siedice, Poland F. STARMÜHLNER Zoologisches Institut der Universitat Wien, Austria У. |. STAROBOGATOV Zoological Institute St. Petersburg, Russia J. STUARDO Universidad de Chile Valparaiso C. THIRIOT University P. et M. Curie Villefranche-sur-Mer, France thiriot @ obs-vifr.fr S. TILLIER Museum National d'Histoire Naturelle Paris, France J.A.M. VAN DEN BIGGELAAR University of Utrecht The Netherlands N. H. VERDONK Rijksuniversiteit Utrecht, Netherlands H. WAGELE Ruhr-Universitat Bochum Germany Heike. Waegele O ruhr-uni-bochum.de ANDERS WAREN Swedish Museum of Natural History Stockholm, Sweden B. R. WILSON Dept. Conservation and Land Management Kallaroo, Western Australia H. ZEISSLER Leipzig, Germany A. ZILCH Forschungsinstitut Senckenberg Frankfurt am Main, Germany MALACOLOGIA, 2002, 44(2): 175-222 THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC (OCTOPODIDAE, CEPHALOPODA) Bent Muus Zoological Museum, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark; bbisballe @ zmuc.ku.dk ABSTRACT Profound confusion among deep-water octopods of the genera Bathypolypus and Benthocto- pus stems from misidentifications of the Atlantic Bathypolypus arcticus (Prosch, 1849), which is here shown to consist of at least two allopatric species—the nominal form is a truly arctic species; the other, Octopus bairdii Verrill, 1873, is a boreal cold-water form, which is reinstated as a Bathypolypus. A third species, Bathypolypus pugniger, n. sp., of uncertain systematic posi- tion, occurs in a narrow belt along the southern limit of B. arcticus. Synonyms are discussed, and evidence is advanced that all records of Benthoctopus pisca- torum (Verrill, 1879) are erroneous and that the type specimen is identical with В. bairdii. Using an amended generic diagnosis, a critical survey of all Bathypolypus species is given. Six species, all Atlantic, are recognized. Distribution and habitats are given, and a key and tables provided to facilitate identifications. Key words: Bathypolypus, Benthoctopus, North Atlantic, Bathypolypodinae (Cephalopoda). INTRODUCTION The octopod genera Bathypolypus and Benthoctopus comprise deep-water species that have caused much taxonomic confusion since Victor Prosch described the first species, Octopus arcticus, from southwest Greenland waters in 1849. The two genera were established in a rather casual way by Grimpe (1921), the respective type species being Bathypolypus arcticus (Prosch, 1849) and Benthoctopus piscatorum (Verrill, 1879). Robson (1924b, 1927) defined the two genera and then treated all known species in his monograph on octopods (1932). From Robson's work on B. arcticus, supple- mented by findings of Hoyle (1886), Pfeffer (1908), Joubin (1920), Grimpe (1933), Kon- dakov (1936), Adam (1939), Bruun (1945), Jaeckel (1958), Kumpf (1958), Macalaster (1976), Perez-Gandaras & Guerra (1978), Nesis (1987), and Voss (1988b), B. arcticus would appear to be a highly variable species with a huge geographic range in the North At- lantic. lt seems in fact to be the most abun- dant bottom-living octopod on the upper half of the continental slopes from Florida to West Greenland, along both sides of the ridge be- tween Greenland and Scotland and from the Bay of Biscay to the Barents Sea, Svalbard, and even the Kara Sea. 175 In a report on the cephalopods from the Danish Godthaab Expedition 1928 to Davis Strait and Baffin Bay (Muus, 1962), | dis- cussed specimens of B. arcticus from near the type locality. This species was easily iden- tified by means of the type material т ZMUC. However, | also described a new, rather abun- dant species, Bathypolypus proschi, which in spite of a superficial similarity diverged from B. arcticus, especially in eye size, ligula, radula, and spermatophores. When years later | inspected the Norwegian collections, | was baffled to find B. arcticus only in the arctic material and B. proschi, along the Norwegian West coast, labelled as В. arcticus. lt dawned upon me that there are still serious identification problems, not only with B. arcticus but, as | would learn, also with other species of the genera Bathypolypus and Benthoctopus, and that a revision was badly needed. Robson (1932) expressed the need for much larger collections to settle the questions left by his revision. Fortunately, growing trawl and dredge activity along the continental slopes has procured substantial additional material from most parts of the North Atlantic. Thus, | have had over 600 specimens at my disposal and ample opportunity to inspect rel- evant type material and most of the speci- mens treated by previous authors to settle 176 MUUS what seemed to be a case of confusion among sibling species. MATERIAL The main revision concerns North Atlantic material deposited in various museums as species placed in one or the other ofthe gen- era Bathypolypus and Benthoctopus, supple- mented with recently collected material from Faroese and Icelandic waters (the BIOFAR and BIOICE projects respectively) and from the deep-sea prawn fisheries off southwest- ern Greenland. The material is listed in Ap- pendix 1. METHODS Measurements, counts and calculation of indices were performed according to the stan- dard for descriptive characters of octopods given by Roper & Voss (1983). The soft body and variable state of preservation, however, make many measurements of octopods sub- jective, and repeated measurements of the same specimens often gave results deviating 5-10%. If different persons perform the mea- surements, the deviation may be even greater, which | noticed when | remeasured specimens treated by other authors. Meristic characters, such as number of suckers and lamellae copulatoriae of the hectocotylus, were often more reliable. Also the eye lens was useful, being a solid structure, and sup- plementing the dorsal mantle length as a standard of size. The lens was extracted with tweezers through a slit cut horizontally in the lower part of the eyeball. Remnants of the darkish primary cornea suspending the lens at “equator” was removed, and the diameter was measured gently with a calipers, or under a microscope. Due to the slightly subjective manner in which many measurements of octopods have to be conducted, all graphs and figures com- paring species are based on my own figures to minimize bias. Only in Figure 9, a few meristic data from Macalaster (1976) are in- cluded. For convenience, the standard measure- ments used in this work are presented below. In addition to conventional indices, | have added some new indices that might be useful in taxonomic work on other octopod genera. Note that as a slight deviation from stan- dards as given by Roper & Voss (1983), ligula and calamus length are measured from the center of last sucker, not from its rim. It is ac- curate and eases the use of calipers. Mantle length is always dorsal ML (midpoint between eyes to mantle apex). Measurements Indices AL: Arm length ALI: % of ML CaL: Calamus length Call: % of LigL ED: Diameter of eye ball EDI: % of ML HcL: Hectocotylized arm length HcLl: % of ML HW: Head width HWI: % of ML LD: Lens diameter LDEDI: LD % of ED (Muus) LigL: Ligula length LigLl: % of HcL ML: Mantle length MLTLI: % of TL MW: Mantle width MWI: % of ML OAI: HcL % of 3. left AL SD: Sucker diameter (largest) SDI: % of ML SDLDI: SD % of LD (Muus) SDEDI: SD % of ED (Muus) SpLI: % of ML SpRl: % of SpL SpWI: % of SpL SpL: Spermatophore length SpRL: Sperm reservoir length SpW: Spermatophore width TE: Total length WD: Web depth (web sectors: A,B,C,D,E) WDI: % of longest armpair WDMI: WD % of ML Counts LamC: Laminae copulatoriae LamCl: LamC % of SHcC (Muus) SHcC: Suckers on hectocotylized arm Acronyms of museum collections used: BMNH _ British Museum of Natural History, London, England IMNH Icelandic Museum of Natural History, Reykjavik, Iceland IRSNB Institut Royal des Sciences Naturelles de Belgique, Brussels, Belgium MNHN Museum National D'Histoire Naturelle, Paris, France MNHT Museum of Natural History, Torshavn, Faroe Islands, Denmark TMDZ Tromso Museum, Department of Zoology, Norway USNM National Museum of Natural History, Washington, DC, USA ZIASP Zoological Institute, Academy of Sciences, St. Petersburg, Russia ZMUB Zoological Museum University of Bergen, Norway ZMUC Zoological Museum University of Copenhagen, Denmark ZMUO Zoological Museum University of Oslo, Norway REDESCRIPTION OF BATHYPOLYPUS ARCTICUS (PROSCH, 1849) The earliest recognition of octopodids in Greenland was by Fabricius (1780: 353). His Sepia octopodia is, however, at most a nomen dubium, because the meagre description fits any of the now known species, and no type material exists. Sepia groenlandica Dewhurst (1834: 263) is a nomen nudum, because no description was given, and Octopus granula- THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC tus Möller (1843: 77) is both a nomen nudum and a junior homonym of О. granulatus Lamarck, 1798. The first direct reference to B. arcticus (under the name “Octopus granulatus”) was in Reinhardt & Prosch’s (1846) paper on the anatomy of Cirroteuthis mülleri, in which some specimens were used for anatomical comparison, and it was stated for the first time that both species are devoid of an ink sac. The authors mentioned that they had many specimens of the Greenland species at their disposal “different in sex, age and develop- ment” [my translation]. In 1849, Prosch described Octopus arcticus based on some of the specimens mentioned in 1846, which were sent from west Green- land in the early 1840s. He did not designate a type specimen, and the lectotype, and two paralectotypes described below were identi- fied as prosch’s original material and marked “types” about 1930 by the curator R. Spärck, among the specimens that were retained in the ZMUC. Material Examined ZMUC CEP-13: Lectotype of Bathypolypus arcticus (Prosch, 1849), called “holotype” in Kristensen & Knudsen (1983): Male, ML: 42 mm. Label: Greenland, K. M. Jörgensen, Au- gust 26, 1841; CEP-14, paralectotype: Fe- male, ML: about 50 mm. Label: Greenland, K. M. Jörgensen, July 27, 1840; CEP-15, paralectotype: The dissected parts of a male used by Prosch (1849) for his drawings (his figs. 1-3). Depository: ZMUC. Further 120 specimens from arctic and sub- arctic North Atlantic as listed in Appendix 1. Remarks on the Type Series The lectotype has been dissected and some measurements are not reliable. The funnel organ is lost, but was formerly present 177 and clearly VV-shaped. Measurements and indices are given in Table 1. Paralectotype CEP-14: Female, ML about 50 mm. Has been dissected, and most mea- surements are unreliable. SD: 2-6 mm, ED: 12 mm. Crop diverticulum present containing polychete bristles and crustacean remains. Gonads filled with eggs. Paralectotype CEP-15: The dissected parts of a male used by Prosch (1849) for his figs. 1-3. Hectocotylus abnormal: only left side of ligula developed, 14 laminae. Remnants of typical B. arcticus spermatophores (Fig. 2d). Material seen by Steenstrup (1856a) and probably by Prosch (1849): Male, ML: 32 mm, ED: 9.5 mm, LD: 3.8 mm, SD: 2.7 mm. Ligula 9 mm with 9 laminae. Funnel organ lost. Too fragile and flabby for measurements. Label: Holböl and Möller, Julianehab (southwestern Greenland, probably 1840). Three females in a jar. They have all been dissected but agree in all recognizable char- acters with the lectotypes (sucker size, eye- balls, crop). | extracted the beak and radula from one of the specimens. (Fig. 5e) Label: Octopus arcticus, Greenland (probably 1840). Synonymy Octopus arcticus Prosch, 1849: 55, pl. 2, figs. 1-3 Octopus grönlandicus Dewhurst: Steenstrup 1856a: 17, pl. 2, fig. 2; 1856b: 234, pl. 11, 19. 2. 1857297. pl 3-16: 2 Octopus piscatorum Мет: Hoyle, 1886: 91 Polypus piscatorum: Russell, 1922: 7, pl. 2, fig. 2 Polypus faeroensis Russell, 1909: 446; 1922: 5, pl. 1, fig. 1, pl. 2, figs. 4-6 Bathypolypus arcticus: Robson, 1927: 251, figs. 1a, 2A; 1932 [in part]: 286, pl. 6, figs. 1, 2, text-figs. 53-60; Kondakov, 1936: 61, figs. 1, 2; Adam, 1939 [in part]: 9, figs. 2-4; Bruun, 1945 [in part]: 6; Muus, 1959: 224, figs. 109A, C, 115; 1962: 10, figs. 1, 2, 4c TABLE 1. Measurements (mm) of the B. arcticus Lectotype. We 165 ML: 42 MLTLI: 25 HW: 27 HWI: 64 MW: (38) MWI: (90) ED: 9 EDI: 21 LD: 5 ODED: 50 SD: 2) ЗЕ Y LigL: 19 наи: 20 LamC: 14 SHcC: 42 AL: | Il Ш IV r 119 112 93 114 | 1207113 112 110 WD: A B C D E 33.5 332.305 32 29 38 35 35 178 MUUS Benthoctopus piscatorum: Robson, 1927: 254, figs. 2B, 3; 1932: 224, figs. 31, 34, 35 Benthoctopus sasakii Robson, 1927: 257, fig. 8. Bathypolypus faeroensis: Toll, 1985: 598, figs. 12 Diagnosis Body egg-shaped, papillated with minute warts often in a stellate pattern on small light spots; head narrower than body; each eye with a verrucose supraocular cirrus; funnel organ double, a clear-cut VV; hectocotylus with about 40 suckers and a deeply exca- vated ligula with 10-16 laminae. Radula usu- ally with irregularly multicuspid, seldom ho- modont rachidians. Esophagus with crop diverticulum. Total length rarely over 200 mm. Description Skin and Colors: In freshly caught speci- mens, the skin is violet to purple strewn with lighter yellowish subcircular spots with minute warts, often surrounding a central slightly big- ger wart in concentric rings, as observed by Prosch (1849). Ventral side paler, with few or no warts. Over each eye is an erectile stout and verrucose cirrus often with adjacent smaller protuberances. The cirrus may be about 10 mm long but often more or less re- tracted in preserved specimens. Color and sculpture patterns of the skin vary with state of preservation. In some specimens, the skin is smooth with no evident sculpture. This is often the case with preserved juvenile speci- mens (ML: < 30 mm.) Bodily Proportions: The mantle is ovoid in outline (MWI: 65-90) slightly constricted be- hind the eyes (Fig. 1). ML rarely over 60-70 mm., TL rarely over 200 mm. The head is nar- rower than the mantle. Due to allometric growth, HWI decreases from 60-85 at ML 10, to 40-70 at ML 60. The eyeballs are not very prominent. They decrease in relative size with age, EDI being 28-40 at ML 10, 20-33 at ML 60 (Fig. 4). The lens measures about 35% of the ED (LDEDI: 30-40). The mantle aperture is 40-50% of the cir- cumference of the neck. The funnel is free of the mantle for about 50%. The funnel organ is VV-shaped. Typically its limbs appear as narrow swollen “ropes” that are easily detached from the funnel wall. They may vary in form (Fig. 18). The gills are reduced and have 6-7(8) gill filaments on each demibranch. The brachial complex is stout, with the arm order I. Il. Ш. IV. The ML constitutes 25-35% of the TL, which leaves 65-75% to the brachial complex. Arm length Index: | 231, II 218, Ill 202, IV 189 (mean of 12 specimens). The web extends along the arms almost to the tips. The web sectors A, B, C and D are subequal in most specimens (WDI: 33-34 at an average), E usually slightly shallower (WDI: mean 30). In a beautifully preserved fe- male with fully extended umbrella, the WDI was: A: 38, B: 46/46, C: 46/46, D: 46/43, E: 41 (ML: 24 mm, M/K “Asterias”, Svalbard, ZMUO). The suckers are biserial, rather small and well spaced, SDI: 6.5-7.3-8.4 depending on degree of expansion. They are of the same sizes on all arms, and there is no sexual di- morphism. They number 80-90 on the dorsal arms, 60-70 on the ventral. The hectocotylized third right arm is some- what shorter than the corresponding left arm (OAl: 75-86-100) and carries about 40 suck- ers (Fig. 8). The number is individually con- stant throughout life (Fig. 23). The ligula was not described by Prosch (1849) probably be- cause he had only one male with an abnormal hectocotylus at his disposal when he finally described B. arcticus (paralectotype CEP- 15). But Steenstrup (1856a, b, 1857) pictured a male with hectocotylized arm and, based on five specimens, he stated that the arm carries 41-43 suckers and ligula 13-17 transverse laminae. The ligula is aspoon-shaped pointed organ with inrolled curved sides (Fig. 1). The width is about 50-70% of the length. It has a central ridge and 11-16 (17) deep, well- separated laminae (variation shown in Fig. 9). The number of laminae is individually con- stant from the onset of maturity (Fig. 22). LigLl: 9-23, the observed variation also de- pending on state of sexual maturity (Fig. 3). Calamus is short and pointed, CaLl: approx. 20. Spermatophoral groove well developed, the strong membrane curling the arm inwards in preserved specimens. Already at ML 16 mm the ligula may be discernible as a 1.5 mm-long undifferentiated tip of the third right arm. Female Organs: The ovary is large with big globular and heavily pigmented oviducal glands (Fig. 2c). Proximally, the united THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 179 FIG. 1. Bathypolypus arcticus (Prosch, 1849). a: hectocotylus, ML: 40 mm; b: upper and lower beak, Q ML 46 mm, “Godthaab” Stn. 87, ZMUC; с: ©’ ML 54 mm, South Greenland, Just & Vibe Stn. 45, ZMUC. oviducts are partly covered by the outer mem- brane of the ovary; distally they are short, very stout and leaving the oviducal glands forming a right angle. In ripe females, the gonad oc- cupies 30-40% of the mantle cavity and is stuffed with 60-80 yellow or brownish eggs. Ripe eggs measure 16-18 mm in length. They are smooth, with fine longitudinal lines. The pointed end of each egg has a short stalk, and the stalks of all eggs are attached to the same small area of the ovary wall. Male Organs: The penis has a well-devel- oped diverticulum (Fig. 2b). Its size and shape very much depend on presence or absence of spermatophores, and it was not measured. Needham’s sac was often distended by 3-6 spermatophores, as in Figure 2b. The large brownish spermatophores are very character- istic (Fig. 2d; Prosch 1849: fig. 2): the sperm reservoir occupies only about one third of the total length, and the oral end is a long, stout horn. The casing is very opaque, and details of the reservoir and middle piece could not be obtained. Зри: 105-130, SpWI: approx. 18, SpRI: 26-32 (data from six males). Beaks and Radula: The beaks do not have distinctive features. The radula seems to be very variable. In most cases, the central teeth 180 MUUS FiG. 2. Bathypolypus arcticus, a: digestive tract; ant.sal.gl.: anterior salivary glands; cae.: spiral caecum; dig.gl.: digestive or hepatic gland; post.sal.gl.: posterior salivary glands; stom.: stomach. b: male reproduc- tive organs; acc.gl.: accessory gland; needh.: Needham’s sac containing ripe spermatophores; pen.div.: penis diverticulum; sem.ves.: seminal vesicles; test.: testis; vas.def.: vas deferens. c: female reproductive or- gans; ov.gl.: oviducal gland; d: spermatophore. a,b and а нот $ ML 43 mm, Ymer Island, East Greenland, 1932, ZMUC; c: 2 ML 42 mm, BIOFAR Stn. 274, ZMUC. THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 181 B.arcticus n=49 10 20 30 40 50 ML mm FIG. 3. Ligula length versus mantle length in B. arcticus. Open circles: juveniles. Maturity is reached at about ML 30 mm. Inserted lectotype of В. arcticus (encircled dot) and the : Уре” of В. faeroensis in ZMUC (dot in square). Juv.: y=-1.90 + 0.225x, г? = 0.81; ad.: у = -3.9 + 0.340x, г? = 0.39. о B.bairdii n=142 B.arcticus n=92 10 20 30 40 50 60 ML mm FIG. 4. Eye diameter index versus mantle length in B. bairdii and B. arcticus. No sex „difference was found. Allometric growth is discernible, but correlation is weak; bairdii: у = 52.68 — 0.2204x, r = 0.249; arcticus: у = 36.7 — 0.199x, r? = 0.321. are clearly multicuspid (Fig. 5), with a seri- regular rough knots. The ectocones may also ation of 2-5 symmetrical (Fig. 5c) or asym- be reduced to a certain lateral ruggedness of metrical teeth (Fig. 5b, d-f). In some speci- the rachis teeth even in presumed unworn mens, the ectocones are rather delicate, parts of the radula. This may lead to quasi ho- uniform thorns (Fig. 5b); in others they form ir- modont rachidians, but completely homodont FIG. 5. Radula and rachidians of В. arcticus; a and b: Y ML 54 тт; с: Y ML 37 mm; d: Y ML 44 mm; e: Y ML approx. 50 mm; fand 9: ФФ ML 33, 52 тт. Caught at various positions off northern Iceland, except e: southwestern Greenland, probably 1840. Scale: 100 um. rachidians are sometimes found. A striking ex- ample is demonstrated by two otherwise typi- cal arcticus females (Fig. 5f, g) taken at BIOICE Stn. 2751. One has multicuspid, the other one homodont rachidians. Digestive Tract: The esophagus has a well- developed crop diverticulum (Fig. 2a) on which the posterior salivary glands are loosely attached when in situ. The spiral caecum is reduced to at most one turn. The liver is large THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 183 and bulbous, about the same size as the testis. There is no ink sac. Comparison with Prosch’s Description The material examined fits well with the types and Prosch's description, which is sup- plemented by Steenstrup’s (1856a, b, 1857) remarks and illustrations of the species. One serious discrepancy, however, is the state- ment by Prosch that arcticus has no crop. This error of Prosch is hard to explain unless one of the specimens dissected during the study of the anatomy of Cirroteuthis (Reinhardt & Prosch, 1846) was B. bairdii, which is very common in West Greenland. | have however no evidence for this. An important point that the present investi- gation confirms is that the rachidians are usu- ally heterodont (Muus, 1962: 11, fig. 1A), though much more variable than | previously thought. Junior Synonyms and Supposed Synonyms of B. arcticus Three species which have contributed con- siderably to the prevailing taxonomic confu- sion are discussed below: Bathypolypus faeroensis (Russell, 1909), which was re- moved from the junior synonomy of B. arcticus by Toll (1985), and the two species that had been placed in Benthoctopus- sasakii (Rob- son, 1927) and piscatorum (Verrill, 1879). Bathypolypus faeroensis (Russell, 1909) Russell (1909) described Polypus faeroen- sis based on two males and a female trawled in the Faeroe Channel by the Fishery Cruiser “Goldseeker” Stn. 19a, 60°40'N, 4°50’W at 1030 m. August 24, 1908. In 1922, Russell re- peated his earlier description based on the same three specimens, adding illustrations of radula, ligula, a papillary area of the skin, and a good photograph of the whole animal. Russell stated that faeroensis is closely al- lied to the squat form that he knew as *bairdii = arcticus”, from which it differs in certain well- defined ways: it has a better marked neck, a narrow head, and a shorter ligula, with more numerous laminae. The skin has a character- istic papillation with tiny warts arranged in a stellate pattern on light, circular spots. Bathypolypus faeroensis has been consid- ered a synonym of B. arcticus (Prosch, 1849) by Robson (1932, with hesitation), Jaeckel (1958), Kumpf (1958), Roper et al. (1984), and Nesis (1987). Toll (1985) redescribed B. faeroensis and reinstated it as a valid species based on a female specimen taken by FFS Walther Herwig in Denmark Strait, 67°21, 5'N, 23°30’W, 480-485 m, September 9, 1973. Because Toll was unable to trace the types of B. faeroensis, this specimen was designated neotype. In ZMUC | found a misplaced jar labelled “Polypus faeroensis, Type, 61°27'N, 1°47'W, 1240 m, July 25, 1909. Russell det. Received from A. C. Stephen, the Fishery Board of Scot- land, March 25, 1924.” The specimen, a male with ML 34 mm, was caught a year after the specimens used for the description by Russell 1909 (and later 1922). | am inclined to believe it was received as a donation to ZMUC, but it is unknown whether Russell or the ZMUC cu- rator (R. Spärck) is responsible for the “type” label. | found in BMNH a female specimen taken in the same haul as the above-men- tioned male. Because it had been misplaced, the ZMUC-type is not recorded in the ZMUC type list (Kristensen & Knudsen, 1983). The “type” in ZMUC might have been a bet- ter choice than the neotype designated by Toll, because it is a male, caught at the type locality and identified by the describer. Table 2 shows measurements of the specimen in ZMUC. The ZMUC “type” has a VV-funnel organ. Hectocotylized arm with 39 suckers. The buc- cal mass has been removed, so the radula and beaks are missing. The esophagus has a TABLE 2. Measurements (mm) of the B. faeroensis “type” in ZMUC. mE 105 ML: 34 HW: 20 HWI: 59 MW: 27 MWI: 79 ED: 12 EDI: 35 LD: 372. SDEDIZ 18:3 SD: 2 SDI: 6.5 LigL: 7 LigLl: 11815 LamC: 11 SHcC: 42 AL: | Il Ш IV r 67 64 520056 | 70 62 DY 2 25 21 184 crop diverticulum. Needham's sac with four unripe spermatophores. SpL: 22 mm, SpRi 31, of the characteristic arcticus type (SpRI approx. 30; Fig. 2d). In all measurable characters, the ZMUC specimen agrees with the general descrip- tions by Russell (1909, 1922) and Toll (1985). Neither Russell nor Toll examined type ma- terial of B. arcticus. Both authors assumed that the “bairdii’-form is the true В. arcticus. Ironically, however, the species described by Russell and the faeroensis specimen in ZMUC is the genuine Bathypolypus arcticus, and all the data on B. faeroensis fit measure- ments and indices as indicated in the present redescription of that species. Toll's careful re- description of faeroensis might as well be a redescription of a female arcticus. The ho- modont rachidians pictured by Toll (his fig. 1b) are unusual but not unknown in arcticus. Ho- modont rachidians were found in one of two females (Fig. 5f, g) caught in the same haul at 68°00'N, 20°40'W (BIOICE Stn. 2751). In all aspects, Toll's specimen is indistinguishable from B. arcticus. Benthoctopus sasakii Robson, 1927 Material examined: Types in BMNH: 89.1.24.33-34. A male and a female labelled: “Benthoctopus sasakii Robson, HMS ‘Triton’ Stn. 9, Faroe Channel, 60'5'N, 6'21'W, 608 fms. August 23, 1882”. Male: ML about 32 mm, distorted and dis- sected. Hectocotylized arm 59 mm with 44 suckers, LigL: 7.8 mm. Ligula with 12-15 in- distinct lamellae. SD: 2.3 mm, ED: 10 mm, SpL: 34 mm, SpRL: 9 mm. Funnel organ VV with the outer limbs somewhat shorter than the median limbs. Skin smooth. Female: TL: 100 mm, ML: 27, MW: 24, HW: 20, SD: 2.3 mm, ED: 10 mm. Funnel organ fragmented. Robson (1927: 262, fig. 8) depicted the radula, which shows a more or less irregular seriation of multicuspid rachidians. Both spec- MUUS imens are typical of juvenile B. arcticus, as ev- idenced by the hectocotylus, the funnel organ and the indices: SDEDI: 23 and SpRl: 26 (Table 9). They were furthermore trawled in arctic water masses at the same depth and lo- cality as B. faeroensis (= B. arcticus). Robson became doubtful about his sasakii and later (1932) treated it as a junior synonym of Ben- thoctopus piscatorum (Verrill, 1879), which prompted me to reexamination of the type of piscatorum in USNM. Benthoctopus piscatorum (Verrill, 1879) Material examined: Holotype USNM 574641. From western part of Le Have Bank off Nova Scotia, October 1879, 120 fms. (219 m). Female, ML: 39. Measurements are give in Table 3. The holotype was a unique speci- men (Roper & Sweeney, 1978). Description: The skin is smooth, with no evi- dent sculpture. A much contracted cirrus de- tectable over the right eye, no trace over the left eye. The funnel organ partly missing but of bairdii type (Fig. 18). Esophagus without crop diverticulum, only with a slight swelling. Number of suckers on each arm 65-75. Radula and jaws not available. Verrill's description (1879: 470) is not very exhaustive, as noted by Robson (1932). In Verrill's opinion, piscatorum was easily distin- guished from Octopus bairdii “by a more elon- gated body, longer arms, shorter web, lack of supraocular cirrus and by its smoothness.” | believe that the type specimen of Octopus piscatorum is a somewhat aberrant specimen of Bathypolypus bairdii (Verrill, 1873), as is the case with his О. lentus and О. obesus (see below). None of the bodily proportions are distinctive. The only index that falls out- side the natural variation of bairdii is SDEDI: 14 (Table 9), but this is compensated for by SDLDI: 28, which is a more reliable index, being based on the firm eye lense as standard for eye size (Fig. 16). TABLE 3. Measurements of holotype of Benthoctopus piscatorum. MESAS ML: 39 HW: 28 HWI: 71 MW: 33 MWI: 85 ED: 15 EDI: 38 ÉD: 75 ЗОШ 28 $0: 2.1 = SDI: 5.4 AL: | Il ll IV r 93 75 82+ 78 | 98 102 77+ 75+ WD: д Ме NID QUE 25 16 27 24 12? 32 32 25 THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 185 | believe that the smoothness of the body is due to contraction of all warts or flaccidity at the time of death, as seen in varying degrees in many preserved specimens of both bairdii and arcticus. Traces of a retracted cirrus over the right eye suggest that the smoothness over the left eye hides a potential cirrus. The identity with bairdii is further substanti- ated by the lack of a crop diverticulum. This contrasts with Robson’s statement that pisca- torum has “a tolerably well-developed crop” (1932: 225) and his figure 31. However, Rob- son based this statement on dissections of B. sasakii (= arcticus). In 1981, | discussed the Bathypolypus-Ben- thoctopus problems with Gilbert Voss, who also examined the type specimen of piscato- rum. He concurred with my opinion that Verrill (1879) described a somewhat atypical speci- men of Bathypolypus and consequently the genus Benthoctopus Grimpe, 1921, would be indistinguishable. (Voss & Pearcy 1990). East Atlantic “piscatorum” The true identity of piscatorum raises seri- ous doubts as to the identity of the many specimens attributed to piscatorum from the East Atlantic by Hoyle (1886), Appellof (1893), Pfeffer (1908), Massy (1909), Russell (1922), Robson (1932), Grieg (1933), and Grimpe (1933). The first to record piscatorum from the Faroe Channel was Hoyle (1886). | examined his specimens in BMNH, and they are identi- cal with the specimens later described as Benthoctopus sasakii by Robson (1927) and once again as piscatorum by Robson (1932). As argued above, the type specimens of sasakii are conspecific with arcticus. Appellöf (1893), inspired by Hoyle (1886), identified three very juvenile specimens from 66°41'N, 6°59’E and 78°2’N, 9°25’E as pis- catorum. He does not give particulars, except that the body is smooth and that mantle and head of the male measure 14 mm. One of the specimens was caught with a juvenile male “Octopus lentus” (junior synonym of bairdii), and later identified by Grieg (1933) as pisca- torum. Because piscatorum specimens from the Svalbard-Barents Sea area are all caught in cold water (-0.9°-+0.8°C), | strongly sus- pect them to be smooth and partly juvenile specimens of arcticus sensu stricto. | dismiss the records as unreliable, although | have not seen the specimens. Pfeffer (1908) merely repeated the Ameri- can records of Verrill and the East Atlantic records of Hoyle (1886) and Appellöf (1893). He repeated Verrill’s pictures of piscatorum and for unknown reasons synonymized Ben- thoctopus ergasticus with piscatorum. As shown below, В. ergasticus is a distinct species. In conclusion, Pfeffer’s records of piscato- rum are unreliable. Massy (1907) described Polypus normani, which she later, having consulted Pfeffer, de- cided was a piscatorum (Massy, 1909). It is a male, TL: 206 mm, trawled off Ireland at 51°15'N, 11°47’W, 707-710 fms., September 1907. | have not examined the type, but Massy’s measurements and drawings (1909: pl. Il, figs. 3, 4) allow the conclusion that it is not conspecific with either the type of Verrill's pis- catorum nor bairdii or arcticus: the hectocotyl- ized arm has 64 suckers, far beyond the num- ber found in bairdii (Fig. 8). OAl is 64.6, which is off the range of variation in bairdii and arcti- cus, in which OAI is about 80-90. SDI is 9.5, which means that the suckers are larger than in bairdii and arcticus. The smoothness of the skin and the bodily proportions given by Massy are not distinctive, but the shortness of the hectocotylized arm and its relatively high number of suckers reminiscent of Benth- octopus ergasticus (P. Fischer & H. Fischer, 1892). Two males and a female of this species were caught in the same haul as normani. Still, B. normani may be a valid species. Massy was advised by Pfeffer who, as men- tioned above, considered ergasticus synony- mous with piscatorum. The ligula and cala- mus of normani is clearly juvenile (Massy 1909: figs. 3, 4). Her figure was erroneously used by Nesis (1987: 318, fig. 84H) to illus- trate the calamus and ligula of piscatorum. Russell (1922) recorded three male and four female Polypus piscatorum taken in the Faeroe Channel in the same haul as his Р faeroensis (“Goldseeker” Stn. 19a). All seven specimens are juvenile. His measurements do not allow the species to be even tentatively identified. But if his measure “Posterior end to eye” is accepted as expression of the ML, the SDI of the largest female (ML: 32 mm) and the largest male (ML: 27 mm) is 9.4 and 7.4 re- spectively, beyond the sucker size of bairdii, but compatible with arcticus. The large female faeroensis (= arcticus) taken in the same haul (Russell, 1922: 6) has SDI: 8.3. The ligula of the largest male is depicted and shows a 6.5- mm-long typical juvenile arcticus ligula with 186 “half a dozen indistinct transverse ridges” (Russell, 1922: fig. 7). Like many of the earlier writers, Russell has attached undue weight to the smoothness of the skin of his “piscatorum” specimens. | con- sider it indubitable that they are juvenile arcti- cus, as all the other specimens | have exam- ined from the arctic water masses in the depth of the Faeroe Channel (Fig. 19). Robson (1932) did not examine the type of piscatorum. He based his revision on three specimens, two of which are the specimens originally described as piscatorum by Hoyle (1886), later as sasakii by Robson (1927). As argued above, sasakii is a junior synonym of arcticus. The only other specimen seen by Robson is a female (Robson’s C30) surprisingly caught in the same haul as the “type” of faeroensis found in ZMUC: Faroe Channel 61°27'N, 1°47'W, 681 fms., July 25, 1909. | have ex- amined the specimen at BMNH: the body is ovoid, the skin smooth with no trace of warts. The funnel organ is not well preserved but shows VV, the inner limbs are however weakly joined anteriorly. Measurements are given in Table 4. Robson (1932: 225, fig. 35) shows the radula of this specimen. The rachidians are heterodont, showing irregular lateral cusps and a seriation of about four teeth. The esoph- agus has a well-developed crop diverticulum. The multicuspid rachis teeth, the presence of a crop diverticulum, as well as the mea- surements and indices in Table 4 show that Robson’s piscatorum is really the smooth- skinned specimen of B. arcticus. In conclusion, Benthoctopus piscatorum sensu Verrill, 1879, is a junior synonym of Ba- thypolypus bairdii, whereas В. piscatorum sensu Hoyle (1886), Russell (1922), and Rob- son (1932) is smooth-skinned and often juve- nile Bathypolypus arcticus. A large female octopod caught 1967 at a depth of 60 fathoms in Placenta Bay, New- foundiand, and identified as Benthoctopus piscatorum by Aldrich & Lu (1968: table 1, MUUS figs. 1, 2) is undoubtedly misidentified and should be reexamined. With a TL of 362 mm, ML 89 mm and HWI of only 35, the specimen deviates considerably from both B. bairdii and B. arcticus. The authors were aware of the in- consistency with Robson’s description of pis- catorum but put it down to its poor state of preservation. Nixon (1991), studied the eggs of this specimen (as “piscatorum” eggs). Revision of B. arcticus by Robson (1932) Robson (1932) was very uncertain about the large number of forms ascribed to the arcticus-bairdii complex. Though with hesita- tion, he concluded that faeroensis with the ovoid body and narrow head at the one ex- treme and the bairdii form with the saccular body and large ligula at the other were one and the same species, B. arcticus. Robson presented two excellent photos of the two forms (1932: pl. VI, figs. 1, 2), one of which is a “type”-specimen of B. arcticus from Greenland on loan from ZMUC. | have tried in vain to identify the specimen in the Copen- hagen collections, but the photo is easily rec- ognizable as B. arcticus, sensu stricto. The other photo depicts a typical B. bairdii, the square-bodied boreal form, which is rein- stated as a distinct species below. Because the two species were confounded, Robson's text is of limited use. Besides the photo, he provided (1932: 290-291) three fig- ures of a B. arcticus, sensu stricto, from the Barents Sea: outline of mantle (fig. 54), an oc- ular cirrus (fig. 55), and a funnel organ (fig. 56). The radulae (fig. 58) are from B. bairdii specimens. Robson (1932) enforced the prevailing tax- onomic confusion by his erroneous identifica- tion of smooth skinned and juvenile B. arcti- cus as Benthoctopus piscatorum. Records of B. arcticus by Adam (1939) Adam (1939: 6, table, figs. 2-4) records six juvenile specimens of B. arcticus from TABLE 4. Measurements of Robson’s Benthoctopus piscatorum. a 185 ML 43 HW: 34 HWI: 79 MW: 48 MWI: 112 ED: 14 EDI: 33 SD: 38: :5Dl: 7.6 SDEDI 24 AL: | Il Ш IV r 133 130 131 130 | 12911136 % 134 ANSE = 30 33 38 38 30 35 36 39 THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 187 the Iceland-Faroe area (about 66°25’М, 12°30'W). | reinspected the material on loan from IRSNB and found, that four of the speci- mens (Adam's specimens b and с) were iden- tical with arcticus as here redescribed. They all have the typical VV funnel organ and the diagnostic index SDLDI is: 53, 53, 53 and 59, respectively, which distinguishes arcticus from B. bairdii and B. pugniger, n. sp. (with SDLDI < 40). Two of the specimens are erro- neously identified as females (specimens b: ML 14 and 16 mm). They are juvenile male arcticus with undifferentiated ligulae of 1.5 and 1.9 mm. Adam’s drawing of the radula of a female arcticus (p. 12, fig. 4, specimen c) shows a homodont rachidian reminiscent of the radula from “В. faeroensis” pictured by Toll (1985: 600, fig. 1) and my Figure 5g. It under- lines the fact that B. arcticus has a very vari- able radula. The remaining two specimens of the mate- rial treated by Adam (1939: specimens a, figs. 2, 3), | consider to be B. pugniger, n. sp., and are discussed below. REINSTATEMENT AND REDESCRIPTION OF BATHYPOLYPUS BAIRDII (VERRILL, 1873) Material examined: about 500 specimens from boreal parts of the North Atlantic as listed in Appendix |. Types examined: Octopus bairdii Verrill, 1873 (syntype, USNM 574638) Octopus piscatorum Verrill, 1879 (holotype, USNM 574641) Octopus lentus Verrill, 1880 (holotype, USNM 34223) Octopus obesus Verrill, 1880 (holotype, USNM 382469). Synonymy Octopus bairdii Verrill, 1873a: 5; 1873b: 394, figs. 76, 77; 1881: 107, pl. 2, fig. 4, pl. 4, fig. 1; 1882: 368, pl. 33, fig. 1, 1a, pl. 34, figs. 5, 6, pl. 36, fig. 10, pl. 38, fig. 8, pl. 49, fig. 4, 4a, pl. 51, fig. 1, 1a; Sars, 1878: 339, pl. 17, fig. 8, pl. 33, figs. 1-10; Kumpf, 1958 (passim) Octopus piscatorum Verrill, 1879: 470; 1882: 377, pl. 36, figs. 1, 2 Octopus obesus Verrill, 1880: 137; 1882: 379, pl. 36, figs. 3, 4; Robson, 1932: 299; Kumpf, 1958 (passim). Octopus lentus Verrill, 1880: 138; 1881: 108, ple 4; fig: 2; 1882: 351, 375, pl: 35, figs: 15 ры, Нд. 12; Robson; 1932:=297; Kumpf, 1958 (passim) Octopus arcticus: Norman, 1890: 466; Joubin 1920: 32, pl. 7, figs. 4, 5 Polypus arcticus: Pfeffer, 1908: 16, fig. 6 Polypus lentus: Pfeffer, 1908: 17, figs. 7, 8 Bathypolypus arcticus: Robson, 1932 [in part]: 286, figs. 53-60, pl. 6, figs. 1, 2; Thiele, 1935: 992, fig. 890; Boone, 1938: 360, pl. 152; Bruun, 1945 [in part]: 6; Kumpf, 1958: 1-135; Jaeckel, 1958: 563: Cairns, 1976: 261; Macalaster, 1976; Perez-Gandaras & Guerra, 1978: 201, figs. 6-8; O’Dor & Macalaster, 1983: 401, fig. 24.1, 24.2; Roper et al., 1984: 222; Nesis, 1987: 315, fig. 83B-E; Guerra, 1992: 251, fig. 89 Bathypolypus proschi Muus, 1962: 13, figs. 2-4 Diagnosis Square-bodied, with papillated skin; arms short; head broad, with large eyeballs, each with a supraocular cirrus; funnel organ dou- ble, pad-like; hectocotylized arm with about 40 suckers; ligula very large deeply exca- vated with 8-12 prominent laminae. Radula with homodont central teeth. Esophagus with- out crop diverticulum. Total length rarely over 200 mm. Description Skin and Colors: In newly caught specimens, the skin is violet to purple, often without any conspicuous spots or patterns, but sometimes speckled with small greyish spots. In pre- served specimens, the skin may be smooth, but more often the dorsal surface is papil- lated, especially in the antero-dorsal region. Single warts or aggregations occur, the latter sometimes in a stellate pattern, a number of small warts encircling a larger one, similar to B. arcticus. Over each eye is an erectile pointed cirrus with adjacent smaller protuber- ances. Erected it measures 5-10 mm. Bodily Proportions: Square-bodied, with broad head and huge eyeballs. HWI de- creases from 80-100 at ML 10 to 60-90 at ML 60 mm. TL rarely over 200 mm. The eyeballs are very prominent. They de- crease in relative size from EDI 40-60 at ML 10, to 30-45 at ML 60 (Fig. 4). The lens mea- sures on an average 41% of the ED (LDEDI: 35-46). The mantle aperture is 36-42% of the cir- 188 MUUS FIG. 6. Bathypolypus bairdii (Verrill, 1873). a: habitus (after Vecchione et al., 1989); b: upper and lower beak; с: hectocotylus of с’ ML 42 mm, Greenl. Fish. Invest. Stn. 5047, ZMUC. cumference of the mantle. The funnel is free of the mantle for about 50%. The funnel organ consists typically of two pad-like structures with a very variable ap- pearance (Fig. 18). They are never strictly VV- shaped, but may have an anterior indentation like a heart or be broken up into bars, sug- gesting an evolutionary past as VV-shaped organs. They are rather loosely connected with the funnel wall and easily lost. The gills are reduced and have 6-8 gill fila- ments on each demibranch. The arm order is I.Il.IIl.IV. The ML consti- tutes 28-38% of the TL, which leaves 62- 73% to the brachial complex. ALI: | 201, Il 184, Ill 174, IV 168 (mean of 21 specimens). The web extends along the arms. The web sectors B, C and D are subequal. WDI: A 32, B 35, C 36, D 35, E 27. WDMI: A: 54, B: 59, C: 60, D: 59, E: 46 (mean of 21 specimens). The suckers are biserial, small and well spaced. SDI: 2.9-3.9-4.8 depending on de- gree of expansion. They are similar in size on all arms, and there is no sexual dimorphism. The hectocotylized third right arm is shorter than the left (OAI: mean, 88). Spermatophoral groove well developed. The number of suck- ers on the hectocotylized arm shows geo- graphical variation: the east American popula- tion (S of 45°N) has 26-40 suckers, whereas specimens from western Greenland and the eastern Atlantic have 35-49 (Fig. 8). The number is individually constant throughout life (Fig. 23). The ligula is a large spoon-shaped organ, which in ripe animals (ML > 30 mm) is often THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 189 FIG. 7. В. bairdii. a: digestive tract; b: reproductive organs; с: spermatophore of с’ ML 41 mm, Dana Stn. 11648, МОС; d: female reproductive organs, ML 41 mm, Greenl. Fish. Invest. Stn. 5112; e: hectocotylus of juvenile с’ ML 23 mm, Dana Stn. 2346, ZMUC. rather square, with almost parallel sides dis- tally ending with rounded flaps and a small pointed tip (Fig. 6). The width is 50-75% of the length. It has a central ridge and a number of deep and well-separated laminae. The num- ber is individually constant from the onset of maturity. It has a total range of 7-13, but shows a geographical variation that is very ap- parent in the western Atlantic, the Newfound- land waters being a transitional area between the American population and the populations off Labrador and western Greenland (Fig. 9). Also, the size of the ligula in ripe animals shows geographical variation: at a ML > 30 mm, LigLI in the eastern American population is 24-44, whereas in western Greenland and the rest of the North Atlantic, it is 18-38. The American population apparently reaches ma- turity ata ML of 25-35 mm, whereas in the rest of the Atlantic maturity is generally reached at ML 30-40 (Fig. 10). Calamus is short and pointed (CaLl approx. 190 MUUS per cent B.arcticus 30 n=40 10- | РЕ B.bairdii 301 EAt n=40 10 B.b. 30 W.Greenl n=61 10 30 10 27 30 33 36 39 42 45 48 Suckers FIG. 8. The American population of B. bairdii (< 45°N) deviates in mean number of suckers on the hecto- cotylus from the western Greenland population (P = 0.95); mean: 32.6, SE 2.6 versus 41.4, SE 3.4. The east- ern Atlantic population differs only slightly from Greenland (mean: 42.7, SE 3.1). Inserted: arcticus, mean: 39.7, SE 3.1. 20). Already at ML 11 mm the ligula may be identified as a 1.2-mm-long undifferentiated tip of the third right arm. Female Organs: The ovary is large, with con- spicuous globular blackish oviducal glands (Fig. 7d). Ripe females with 60-85 yellowish eggs measuring 15-18 mm in length. The eggs are smooth with fine longitudinal lines. Male Organs: The penis with well-developed diverticulum (Fig. 7b). In Needham's sac was found up to six spermatophores. The sausage- shaped sperm reservoir of the spermatophore occupies about half of the total length, and the oral end is a slim, rather stiff and curved tube (Fig. 7c; Verrill, 1882: pl. 36, fig. 10). The sper- matophore is glossy and opaque; the interior details are difficult to make out. SpLl: 60-90- 115, SpWI about 19, SpRI 50-60. Jaws and Radula: The beaks do not present distinctive features. The radula is homodont, the rachidians having smooth concave sides THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 191 per cent Г] Lab.-Greenl. => | n=92 Newf. 29 n=65 оз: О | Am<45°N 40 n=121 65587107512 46 758 628 7077274 N <= B.bairdii B.pugniger n.sp n=17 | | B.arcticus n=36 10 12 14 16LamC FIG. 9. Number of laminae copulatoriae. B. bairdii shows geographic variation with a highly significant dif- ference (P: > 0.99, z-test) in mean number between the American (< 45°N) and the western Greenland pop- ulation (mean: 10.12, SE 0.109 versus 8.38, SE 0.3). The Newfoundland area forms a transitional zone (mean: 9.56, SE 0.2). East Atlantic (mean: 9.89, SE 0.2) versus western Greenland shows a similar signifi- cant difference. Variation in B. pugniger, n. sp., and B. arcticus inserted for comparison. and never showing any sign of ectocones (Fig. 11; Verrill, 1882: pl. 49, fig. 4). Digestive Tract: The esophagus has no crop diverticulum, only a slight swelling where the posterior salivary glands are fastened when in situ (Fig. 7a). The spiral caecum is reduced to less than one turn. The liver is large, about the same size as the testis. Junior Synonyms of B. bairdii. Robson (1932) strongly suspected that Oc- topus lentus Verrill, 1880, and ©. obesus Ver- rill, 1880, were conspecific with the typical bairdii form, because he was not able to find critical differences. Not having seen the type specimens, however, he hesitated to place them in synonomy. In a master’s thesis, Kumpf (1958) tried to sort out the Bathypolypus arcticus-bairdii- lentus-obesus complex. He had a large amount of material, 178 specimens, caught off the eastern American coast, including the types of Verrill’s Octopus bairdii, O. lentus and O. obesus. In addition, he used measure- ments of seven “В. arcticus” specimens ex- amined by Gilbert Voss in the BMNH. Kumpf applied the standard of measure- ments of Robson (1927, 1932) and amplified by Pickford (1945, 1949). He thoroughly com- pared his material with the types of bairdii, lentus and obesus and concluded that these species are conspecific and that in spite of all variation, only one Bathypolypus species is represented in his material. | can confirm this part of his conclusion. The bulk of Kumpf’s material stems from between 192 MUUS B.bairdii East America (45°N n=54 B.bairdii W- Greenland - Europe n=98 10 20 30 40 50 60 70 ML mm FIG. 10. Ligula length versus mantle length in B. bairdii. Open circles: juveniles. Lower figure: pooled data for western Greenland and NE Atlantic (juv.: у = —3.34 + 0.333x, г? = 0.58; ad.: y =-3.20 + 0.505x, г? = 0.66). Upper figure: Eastern America < 45°N (juv.: у = -0.49 + 0.248x, 1” = 0.39; ad.: у = 4.90 + 0.452x, г? = 0.58). In eastern America, maturity is often reached already at ML 25 mm and ligula grows larger than in the North Atlantic. Dot in circle: syntype of bairdii (USNM 574638). Dot in square: holotype of B. obesus (Verrill, 1882) (USNM 382469). THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 193 FIG. 11. Radula and central teeth of В. bairdii. с’ ML 55 mm., Davis Strait 65°12’N, 56°21’W. Scale: 100 um. 30°N and 45°N, and | examined and remea- sured most of it, including the types of bairdii, obesus and lentus, during a visit to USNM in 1975. The data are included in the present re- vision. Kumpfs main conclusion, however, that Verrill's species are junior synonyms of arcti- cus, is erroneous. The genuine arcticus is a more northerly species, not found off the east coast of USA (Fig. 20). Paradoxically, Kumpf never saw the species he purports to revise. To understand B. arcticus, he had to rely on the literature, especially on Robson’s (1932) revision, with all its ambiguities and errors. Kumpf focused on the squat “bairdii-type” as being the typical arcticus, thus missing the point that Verrill's bairdiiis a separate species. Kumpf (1958) was incorrect in stating that Verrill did not know of Prosch's arcticus. In a footnote describing bairdii, Verrill (1873a: 5) wrote that his species differed from O. groen- landicus Dewhurst, which has a smaller hec- tocotylus “with more numerous folds, and the basal part bears 41-43 suckers”. This is a ci- tation from Steenstrup's then well-known paper (1856a, or the translations of it: 1856b, 1857), in which O. groenlandicus is syn- onymized with arcticus and in which the species and its hectocotylized arm are figured (see also Verrill, 1880: 138, footnote). The knowledge of West Atlantic Bathypoly- pus was much extended and elaborated upon by Macalaster (1976), who studied no less than 750 Bathypolypus specimens collected in Canadian waters from Georges Bank to the Labrador Sea. Macalaster was warned by me that “arcticus” covered more than one species, but through morphometric analyses 194 MUUS she showed convincingly that only one species occurs in Canadian waters and that it is identical with Kumpf's “В. arcticus”. Leaning on КитрР$ revision, she excusably put the wrong species in the title of her otherwise valuable contribution, instead of B. bairdii. This mistake was repeated by O’Dor & Macalaster (1983) and by Wood et al. (1998), which are in fact accounts of life cycle and breeding ecology, respectively, of B. bairdii, not of B. arcticus, as indicated in the titles. Macalaster’s primary interest was not tax- onomy but the opportunity to study the life cycle, growth, and reproduction of Bathypoly- pus, including observations of live animals kept in aquaria. Of principal interest to the present revision, however, is Macalaster’s comprehensive measurements, which sup- plement Kumpf's (1958) and my own Ameri- can data and which show that B. bairdii is commonly distributed along the upper part of the slopes from Florida to the Labrador Sea and that the genuine B. arcticus seems to be absent. | included some of her data as a sup- plement to my own to demonstrate morpho- logical differences between the southern and northern populations of В. bairdii. Octopus piscatorum Verrill, 1879 A full account of this nominal species and description of the holotype is given above under the synonymy of B. arcticus, which has been the species most often mistaken for pis- catorum. It is here shown beyond reasonable doubt that O. piscatorum is a junior synonym of O. bairdii Verrill, 1873. It is also shown that Robson's concept of piscatorum was based upon misidentified arcticus specimens from the Faroe Channel and that consequently characters of B. arcticus, for example, pres- ence of crop diverticulum, multicuspid radula, have crept into his diagnosis of piscatorum. This of course has repercussions on the con- cept of the genus Benthoctopus, of which pis- catorum is the type species. Bathypolypus proschi Muus, 1962 Holotype: Male TL 115 mm, “Dana” Stn. 10018, 65°02’5”М, 56°00’W, Davis Strait, 730-740 m, trawl, July 19, 1956. Depository: ZMUC. The unanimous claim among earlier au- thors (exception: Sars, 1878) that bairdii is synonymous with arcticus led me to describe proschi as a new species, because it was ob- viously distinct from the type material of B. arcticus (Muus 1962: 18, table III). The large amount of material now available and examination of Verrill’s types of bairdii made it evident that proschi is identical with the here-reinstated bairdii and is a junior syn- onym of that species. It should however be pointed out that there are (subspecific?) mor- phological differences between the American and West Greenland populations (see below). Bathypolypus Species Recorded from Northwestern Spain A paper by Perez-Gandaras & Guerra (1978) recorded for the first time Bathypoly- pus from 120-439 m off the Galician coast. Of 22 specimens, 14 were identified as B spon- Salis, three as B arcticus, another three (type A) with hesitation as B. proschi, while two de- fective specimens could not be assigned to any Known species. The present revision shows that it is highly improbable that arcticus, a true arctic species (Fig. 20), would be found off Spain. The bo- real bairdii, formerly confused with arcticus, is a more likely candidate and could be ex- pected to have its southern East Atlantic limit somewhere in the Biscayan neighbourhood. The data for “arcticus” given by Perez-Gan- daras & Guerra (Table 5) seem to support this expectation. The specimens concerned are two males, ML: 27-39 mm, and a female, ML: 37 mm. With few probably insignificant deviations, the index values lie within the range of varia- tion of bairdii (Table 9) and exclude the other here recognized Bathypolypus species. Such important meristic characters as number of laminae copulatoriae (8-10) and suckers on the hectocotylized arm (31-35) confirm affin- ity to bairdii. One male specimen figured by Guerra (1992: 252, fig. 89) shows a typical bairdii form, apart from the ligula, which has a pointed tip different from the broad-tipped norm for bairdii known from eastern America and the Scotland-Greenland Ridge (Fig. 6). Some other characters described by Perez- Gandaras & Guerra (1992) are ambiguous. The rachidian (their fig. 7) is described as multicuspid on account of two very small cusps at the base. This is unusual in the northern populations of bairdii, which have smooth bases (Fig. 11). Further the sper- THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 195 TABLE 5. Measurements (mm), indices and counts of three dubious “arcticus” from the Galican coast extracted from data of Perez-Gandaras & Guerra (1978). Sex male male female mE 78 102 113 ML: 27 39 42 MLTLI: 35 33 ЗИ НМ: 85 62 62 EDI: 44 36 31 SDI: 5.2 4.4 Sr SDEDI: 12 12 18 ALI: | 178 179 150 Il 174 179 152 Ш 174 179 136 IV 167 167 131 HeLl: 130 115 = OAI: 74 64 = наи: 20 18 = LamC 9-10 8-9 = SHcC: 31 35 = Зри: — 144 = SpRI: = 31 = matophore (their fig. 8) has a reservoir occu- pying only 31% of the spermatophore length, which among the Bathypolypus species is a value known only from arcticus (Table 9). Apart from the longer oral end, the sper- matophore has proportions similar to what is found in bairdii, for example, the characteris- tic light swelling seen in the cement gland por- tion. In arcticus, the oral end of the sper- matophore is a thick, opaque tube with no swelling where the cement gland is located (Fig. 2d). Unfortunately, funnel organ and digestive tract were not described. Until further material can be studied, | think the three specimens should be assigned to B. bairdii, but with reservation. “Type A”, described by Perez-Gandaras & Guerra (1978: figs. 9-11) includes one female and two males, ML: 34-40 mm, that could not be assigned to a known species with any cer- tainty. The authors suggest B. proschi, but that species is now shown to be synonymous with bairdii. The specimens possess a pecu- liar supraocular cirrus, which is smooth, cylin- drical and raised at the anterior edge of a hemispherical wart (their fig. 9). The ligula (their fig. 10) is a simple spoon-shaped organ with midrib and 9-12 low laminae and a small pointed calamus, LigLl: 16-18. Compared with Багой, it looks juvenile. Proportions of body, spermatophore and most indices (their table 4) are compatible with bairdii. The rachidians have two small denticles at their bases like the “arcticus” specimens described above, and one radula is abnormal, having nine rows of teeth (their fig. 11). One male (Guerra, 1992: 254, fig. 90) has much smaller eyeballs (EDI: approx. 27, measured on the drawing) than indicated by Perez-Gandaras & Guerra (1978: table 4), in which EDI is given as 35-41, compatible with bairdii. | think “type A” has to retain its dubious po- sition until a larger series of specimens be- comes available. Bathypolypus pugniger, n. sp. Material examined: 31 specimens from the Iceland-Faroe area as listed in Appendix 1. Holotype: Male, ML 32 mm, Icelandic Fish- ery Investigations, haul B5-78-44, 64°58'N, 27°44'W, 860-870 m, March 14, 1978. Mea- surements: Table 5, specimen 470. Deposi- tory: IMNH 19990971. Paratypes: 9 males, 10 females as listed and measured in Tables 6 and 7. Synonymy Bathypolypus arcticus: Adam, part]: 9, figs. 2, 3 1939 [in Diagnosis Ovoid or square-bodied with papillated skin; arms very short, arm order III:IV:II:1 or of subequal length; head broad, with large eyes, each with a supraocular erectile cirrus; hecto- cotylized arm with about 35 suckers; ligula globular fleshy and deeply excavated, with 4-6 laminae; funnel organ double and pad- like. Radula with broad homodont central teeth; esophagus without crop diverticulum. Total length rarely over 150 mm. Description Skin and Colors: Freshly preserved, the skin is violet to purple dorsally, ventrally lighter and yellowish or brownish. The antero-dorsal re- gion more or less equally strewn with numer- ous small warts. Over each eye is a 3-7 mm- long single verrucose cirrus, which may be bifurcated, the anterior branch being shortest, or there are two closely set cirri. 196 MUUS Es = | ’ 1.4, any = “à FIG. 12. Holotype of Bathypolypus pugniger, п. sp., © ML 32 mm. Depository: IMNH 19990971. Bodily Proportions: Ovoid (Fig. 12) or more square-bodied, like bairdii (Fig. 13), with large eyeballs. MWI: males 67-92-106, females 73- 96-118. HWI: males 73-85-100, females 48- 79-96. TL rarely 150 mm, ML rarely over 50 mm. MLTLI: 33-37.5-42. EDI for both sexes 30-44-50, highest for young specimens. The lens measures on an average 37% ofthe eye diameter. The rough diagnostic index SDEDI, useful to discriminate arcticus and bairdii, shows pugniger to lie between the two species (Table 9). The corresponding relation between SD and LD is supposed to be more accurate, but still shows some overlap with young specimens of bairdii (Fig. 16). The mantle aperture is about 40% of the mantle circumference, and the funnel is free for about 50%. The funnel organ is a couple of pad-like structures sometimes heart-shaped but vari- able as in В. bairdii (Fig. 18). The gills are reduced and have 6-7 gill fila- ments on each demibranch. The arm length order is IIl:IV:Il:| or sube- qual (Tables 6, 7). This is reverse from arm order of В. arcticus and В. Батай. The ML constitutes 33-42% of the TL making В. pug- niger the most short-armed of the three species. ALI: I 145, Il 150, Ill 155, IV 154 (means in Tables 6, 7). The web extends along the arms. WDMI: A 53, B56, C 59, D 60, E 51 (mean of 18 spec- imens). In well-preserved specimens, web sectors C and D tend to be subequal. The suckers are biserial, small, and well spaced. Each arm with 65-75 suckers. They are of the same order of size on all arms, and there is no sexual dimorphism. SDI: 4.7-5. 8-7.6. The hectocotylized third right arm is usually slightly shorter than the left opposite arm. OAI: 83-91-106. It has 31-35-45 suckers. Spermatophoral groove well developed. The ligula is fleshy, short, and broad, with firmly inrolled borders, giving it a globular ap- pearance as a clenched fist (Figs. 12, 13c). LigLI: 22-27-34 in 11 specimens > 25 mm ML. The ligula has a tiny pointed tip between the broadly rounded anterior flaps (Fig. 13a). The width is 80-100% of the length. The spoon has a central ridge and 4-6 deep, well-sepa- rated laminae (Figs. 9, 13a). Calamus is stout, CLI: 26-38-51. Already at a ML of 6 mm, the nectocotylized arm may be identified by the THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 197 FIG. 13. Bathypolypus pugniger, п. sp. a: distal hectocotylus of young © ML 18 mm. The “clenched” ligula was forced open to show the laminae; b: upper and lower beak of с’ ML: 33 mm; с: paratype ZMUC CEP- 17, ML 54 mm. Dana Stn. 16437, southwestern Greenland. 0.8-mm-long, as-yet undifferentiated ligula (Table 6: no. 77). Female Organs: The ovary is large with blackish globular oviducal glands (Fig. 14a, b). Proximally the oviducts are joined, distally they are short, stout, leaving the oviducal glands forming right angles. Supposedly ripe eggs measure about 10 mm. They are smooth, with fine longitudinal lines. Male Organs: The penis with well-developed diverticulum (Fig. 14f). The spermatophores are of bairdii type, with sausage-shaped sperm reservoir occupying about 55% of the total length. SpLI: 60-100. Beaks and Radula: The beaks do not present distinctive features. Only four radulae have been investigated. The rachidians deviate considerably from both arcticus and bairdii (Fig. 15), whereas the lateral teeth show no distinct features. Basically, the rachidians are homodont, but they may be very broad with highly set or drooping shoulders (Fig. 15b, e, respectively) or just stocky and pointed ho- modont, with almost straight sides (Fig. 15c, f), but different from the slim, slightly concave homodont teeth of bairdii. Digestive Tract The esophagus has no crop diverticulum, but swells to double diameter from the point 198 MUUS TABLE 6. Measurements (mm) of male Bathypolypus pugniger, n. sp. (Note that in Tables 6 and 7 arm length is given as average of paired arms, except for males, where AL Ill represent the left arm only. Web depth for sectors B, C and D are likewise averages.) No 77 76 75 659 653 463 648 470 453 122 mi 11 30 38 51 67 76 88 90 91 146 ML: 6.2 11 15 18 25 30 33 32 36 54 HW: 5.9 10 12 15 25 22 27 31 29 45 MW: 5.5 9.3 12 19 28 20 34 32 33 45 ED: 2.0 4.5 6.2 10 13 11 13 16 16 22 ED: 1.0 2.0 = 37 5.0 4.2 55 5.8 6.0 7.4 $0: 0.4 0.7 0.8 1,2 1.9 1:5 1.8 2.0 2.0 2.5 AL: | 8.5 15 17 26 36 39 40 55 50 85 Il 8.5 15 19 26 8% 41 48 55 55 87 Ш 8.5 14 19 29 37 42 45 56 53 89 IV 9.0 = 19 28 40 43 44 55 55 87 HcL: 9.0 18 16 24 34 37 38 52 50 83 LigL: 0.8 2.4 3.1 3.5 10 8.3 13 15 13 18 LamC: 0 4 4 5 6 5 5 6 5 6 SHcC 35 Sil 32 34 31 42 34 40 40 45 WD: A: 3.8 — = 9.5 14 14 14 20 17 33 B: 3.8 = = 11 14 15 16 19 19 35 C: 3.9 — = 10 15 15 16 20 20 38 D: 3.4 = — 10 16 15 16 20 23 40 = 3.0 — = 7.8 14 14 10 15 18 29 TABLE 7. Measures of females attributed to B. pugniger, n. sp. No 74 649 71 455 461 466 465 456 457 464 q 38 46 55 83 c 90 100 97 99 103 c115 ML: 15 18 28 27, c 32 33 36 39 40 c 44 HW: 12 16 19 26 = 30 30 33 30 21 MW: 11 17 21 31 a 39 34 35 35 34 ED: 6.6 11 10 13 15 14 14 16 16 13 LD: — 4.0 3.6 GS 5.3 = = 5.8 5.8 5.0 SD: 0.8 1.1 1.1 21 2.0 1.9 1.9 2.0 2.2 2.1 AL: | 18 26 29 47 53 60 58 50 58 64 Il 19 25 32 52 56 60 66 50 55 69 Ш 22 28 33 55 58 68 62 50 59 64 iv 19 26 32 55 59 60 61 52 56 65 WD: A: 7 10 12 19 117 19 17 21 14 21 B: 6.5 11 13 16 20 18 18 23 21 21 C: 1.5 10 18 19 22 24 22 26 20 22 D: 8.5 8.5 12 19 23 23 22 22 23 24 Е: 8 8 11 20 19 19 19 Uz 16 20 where the leaf-like second salivary glands are fastened and down to the stomach (Fig. 14c). The two ducts from the posterior salivary glands are separate for more than half of the distance to the buccal mass (Fig. 14c-e). In arcticus and bairdii, the united excretory canal is relatively longer (Figs. 2a, 7a). When in situ, the very large stomach rests in a deep groove of the liver, which is almost bilobed. The spiral caecum is scarcely coiled. There is no ink sac. Etymology pugniger, derived from the Latin pugnus, a fist, alludes to the characteristic boxing glove appearance of ligula. THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 199 FIG. 14. Bathypolypus pugniger, п. sp. a: reproductive organs of Y ML 40 mm; b: egg from same specimen, Stn. B5-77-46, IMNH; c-e: digestive tract; f: reproductive organs of с’ ML 38 mm. A spermatophore is at- tached to the penis. MNHT, haul 25, July 19, 1979, now ZMUC. FIG. 15. Radula and rachidians of В. pugniger, п. sp. a and b: с’ ML 31 mm., BIOFAR Stn. 269; с: с’ ML 36 mm, Iceland Fish. Invest., haul B5-77-46; d and e: с’ ML 33 mm, BIOFAR Stn. 269; f: Y ML 33 mm, Iceland Fish. Invest., haul B5-77-48. Scale: 100 um. Synonyms of B. pugniger, n. sp. A revision of specimens identified by Adam (1939) as B. arcticus showed two specimens to be identical to B. pugniger, n. sp. They are juvenile, a male (ML: 22 mm) and a female (ML: 18 mm) caught in 1938 off the east coast of Iceland (66°23'N, 12°53’W) in 200-250 т. Adam (1939: 10, specimens a, figs. 2, 3) fig- ures the reproduction organs of the male and the globose ligula with five distinct laminae typical of B. pugniger. THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 201 TABLE 8. Measurements (mm) of a male B. pugniger, n. sp., identified as B. arcticus by Adam (1939). Adam’s 60-year-old measurements in parenthesis. We 59 (-) Indices ML: 22 (25) MLTLI: 37 HW: И (19) HWI: ИТ MW: 20 (18.5) MWI: 91 ED: 90 (-) EDI: 40 SD: 1 SES) SDI: 5.9 LigL: 62 (6:5) LigLl: 22 Table 8 shows measurements and indices of the male specimen. Comparison with Adam’s measurements, which were done while the material was fresh, show consider- able preservational shrinkage. Recognition of B. pugniger as a New Species The first specimens seen were in fact iden- tified as slightly abnormal B. bairdii. The glo- bose ligula of the males at first believed to be an accidental deformity or a regeneration phe- nomenon turned out to be a regularly occur- ring stable structure with 4-6 laminae, thus deviating significantly from North and East At- lantic B. bairdii (Fig. 9). This character, in com- bination with a reverse arm order as compared with bairdii and arcticus, deviating rachidians (Fig. 15), and sucker and eye sizes (Fig. 16) justified establishment of a new species. As is usual in octopods, the hectocotylized males offer the best distinguishing characters. The females were only recognized after re- newed inspection of all material from the Arm Length right left ) | 32 (33) 34 (36) ) || 30 (33) 35 (36) Ш 28 (32) 33 (34) IV = (-) = (28+) Faroe-Iceland area that | had previously iden- tified as B. bairdii. The search for females of pugniger was based on the assumption that, like in other Bathypolypus species, there would be no sex- ual dimorphism. This means that bodily pro- portions, arm order and size of eyes and suckers should match pugniger males of sim- ilar sizes. In this way, 12 females could be at- tributed to B. pugniger. A circumstantial evi- dence for the plausibility of the result is the sex ratio of 39% females, close to the sex ratio found in bairdii (38% females; n = 235). Morphologically pugniger is very close to bairdii. As yet few characters distinguishing these species have been found. Among juve- niles and females mistaken identity between pugniger and bairdii is possible, because the state of preservation can blur the differences in arm order and size of suckers and eyes. The rough diagnostic index SDEDI useful to discriminate between arcticus and baïirdii, shows pugniger to lie between the two species (Table 9). The corresponding relation between SD and LD should be more accu- TABLE 9. Range of distinctive indices and counts of the Bathypolypus species based on material examined. The sparse data for valdiviae are extracted from text and drawings of Chun & Thiele (1915), Robson (1924b), and Sanchez & Moli (1984). Data for sponsalis from the Mediterranean population. arcticus bairdii ergasticus pugniger sponsalis valdiviae Call 15-25 15-25 30-40 26-38-51 33-42 28-38 EDI 20-35 30-55 31-41-47 30-44-50 30-41-47 40 LamC 11-16 7-12 7 (п=4) 4-5-6 4-6-7 4-5 LigLI 9-23 18-44 6-9-12 22-27-32 10-12-15 15-18 MLTLI 24-29-35 27-33-38 13-17-23 33-38-42 16-22-26 30 OAI 83-89-100 71-88-104 53-60-70 83-91-106 47-63-73 75 SDEDI 18-26-34 6-9-13 9-11-13 11-13-16 4-6.5-8.5 13 SDI 6-7-8.4 3-4-5 3.5-4-5.5 4.7-5.1-6 2.1-3-3.7 5 SDLDI 50-70-1100 19-25-36 22-25 (n = 2) 32-35-40 15-19-22 = SHcC 32-49 36-49 73-77-83 31-35-45 52-57-61 45 SpLI 105-130 60-90-115 110-150 90 (n = 3) 50-70 100 SpRI 26-32 50-60 50-60 50-60 50-60 50 Eye cirri present present absent present absent present Crop divert. present absent present absent absent ? 202 MUUS rate, but still shows some overlap with bairdii, especially with younger specimens (Fig. 16) In bodily proportions and hectocotylus, B. pugniger has a striking resemblance to the South African Bathypolypus valdiviae (Chun & Thiele, 1915), known from the Agulhas Bank and off the Namibian coast. With one exception from West Greenland (Fig. 13c, Table 6: no. 122), all specimens of pugniger are caught on the Faroe-lceland plateau in warm Atlantic water. The zoogeo- graphical and ecological perspectives are dis- cussed below. HISTORICAL REVIEW AND PRESENT STATUS OF BATHYPOLYPUS AND BENTHOCTOPUS. In 1921, Grimpe erected two new genera in the subfamily Octopodinae to accommodate some characteristic octopods without ink sac: Octopus arcticus Prosch, 1849, was desig- nated type species of Bathypolypus, and O. piscatorum Verrill, 1879, type species of Ben- thoctopus. Grimpe just stated that the two species “erheblich verschieden” [differ con- siderably], but he did not otherwise define the genera. To accommodate two new species (Bath- ypolypus grimpei and Benthoctopus berryi), Robson (1924a, b) defined the genera this way: “Bathypolypus Grimpe: Deepwater poly- pods with broad and long hectocotylus and unicuspidate rhachidian teeth. The skin is usually covered with warts and may be gelati- nous. There is no ink sac. Type: B. arcticus.” “Benthoctopus Grimpe: Abyssal polypods with small hectocotylus, multicuspid rhachid- ian teeth and smooth skin. There is no ink sac. Type: B. piscatorum.” Without discussing details, Grimpe (1925: 100, footnote) declared himself in complete agreement with Robson’s generic definitions. With small modifications these definitions have persisted (for example, Thiele, 1935; Mangold & Portmann, 1989). Robson (1927) amended the generic definitions slightly and later (1928) erected a new subfamily, Bathy- polypodinae, comprising the two genera and simply defined as: “Octopods mainly abyssal in habitat and devoid of an ink sac”. In his monograph, however, Robson (1929, 1932) moved Benthoctopus with 14 nominal species back to the subfamily Octopodinae, while Bathypolypus with six nominal species was retained in Bathypolypodinae. With the characters of the subfamily (Robson, 1932: 286), Bathypolypus was now defined as: “Abyssal octopodids devoid of an ink sac and in which the crop is usually reduced and sometimes absent. Eggs and vaginae are large and spermatophores large and few in number. The mantle aperture is very narrow and the general habit squat and short armed.” Robson removed Benthoctopus to Octo- podinae because he felt that some of the species resembled ordinary forms of Octopus and that “were it not for the lack of the ink-sac one would place them in that genus” (Robson, 1932: 51). On the other hand, Robson real- ized that some species have traits of both genera. The latter problem has caused some taxo- nomic confusion. After a redescription of Bathypolypus sponsalis, it prompted Wirz (1955: 146) to declare that the strict distinc- tion between the genera Benthoctopus and Bathypolypus made by Robson was unjusti- fied considering the small number of differ- ences. Thiele (1935) reunited the two genera in Bathypolypodinae. As presented by Robson, the two genera represent an array of species that in varying degrees demonstrate traits believed to reflect adaptation to benthic life in deep water-ab- sence of ink sac; reduction of gills, radula, and crop; increasing size of eggs and sper- matophores; elaboration of ligula; shortening of the arm complex; and funnel organ tending to be double. In general, the least specialized species, that is, those most similar to Octopus, seem to be accommodated in Benthoctopus, the most divergent in Bathypolypus. The present revision shows that most of the existing confusion derives from misidentifica- tions that have led to errors in the concept of the two type species and subsequent misun- derstandings in the generic definitions of Ba- thypolypus and Benthoctopus. The choice by Grimpe (1921) of Octopus piscatorum as type for Benthoctopus was es- pecially unlucky, because an examination of the type shows it to be identical with the here- reinstated Bathypolypus bairdii (Verrill, 1873). Aware of this fact and in connection with the description of five new species of Benth- octopus from the Pacific, Voss & Pearcy (1990) plead for the preservation of the name Benthoctopus, which now includes 20 species worldwide (Sweeney & Roper, 1998). Voss (1988a, b) restricted the subfamily Ba- THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 203 B.arcticus n=33 B.arcticus B.pugniger n.sp. B.bairdii 1 2 3 4 5 6 7 8 9 10 11 FIG. 16. Sucker diameter versus lens diameter in arcticus, pugniger, n. sp., and bairdii. Reduced major axis regressions show that the relation differs among arcticus and bairdii and among arcticus and pugniger at the P = 0.01 level of significance. Among pugniger and bairdii at = > 0.1 < 0.05. Regression slopes with 95% confidence intervals and coefficient of determination: 0.8372 (0.7177-0.9766; 0.9766) for arcticus; 0.4112 (0.3541-0.4774; 0.9025) for pugniger, and 0.2643 (0.2294-9.3045; 0.7921) for bairdii. The regression in- tercepts are 0.1614, 0.0641, and 0.0592 respectively and not considered different in pairwise comparison among species. Inserted in upper figure: data for the type of B. arcticus (crossed square), the “type” of B. faeroensis found in ZMUC (star) and the holotype of Benthoctopus piscatorum (cross in circle). 204 MUUS thypolypodinae to benthic octopods with bise- rial suckers and devoid of ink sac and pro- posed the following definition of Benthoctopus Grimpe, 1921: “Deepwater octopods of normal Octopus- like appearance with short to long arms, suck- ers biserial; hectocotylus Octopus-like, ligula slightly to moderately excavated with indis- tinct midrib, smooth or bearing low, often in- distinct rugae, never laminate; crop present, usually with diverticulum; ink sac absent: radula with strongly, seldom weakly, multicus- pid rachidian; body entirely smooth, papillae or ocular cirri absent. Type species Octopus piscatorum Verrill, 1879, by original designa- tion.” If the genus Benthoctopus is to be pre- served, it would need a different type species to be designated by the International Com- mission on Zoological Nomenclature. A sensi- ble choice would be Benthoctopus januarii (Hoyle, 1885). This species is revised and thoroughly redescribed by Toll (1981) based on a series of recently caught male and fe- male specimens. It conforms to the diagnosis of Benthoctopus suggested by Voss and fur- thermore has the advantage of being a wide- spread species in the Gulf of Mexico and off Brazil. The final decision and the plea to the Inter- national Commission on Zoological Nomen- clature, however, should be made by a reviser of the Benthoctopus species. The genus Bathypolypus should under any circumstances be preserved. Robson, being the first revisor of the two genera, acknowl- edged Octopus arcticus as type of a group of species characterized by large lamellated ligulae, as opposed to another group of species characterized by small Octopus-like ligulae, the present Benthoctopus group. To uphold the distinction between the two groups and to match the definition of Benthoctopus by Voss & Pearcy (1990). | propose the fol- lowing modification of Robson’s definition of Bathypolypus in an attempt to make an oper- ational distinction between the two genera: Bathypolypus Grimpe, 1921. Type species: Octopus arcticus Prosch, 1849, by original designation. Deepwater octopods of normal Octopus- like appearance, with stout body and gener- ally with short arms; suckers biserial; hecto- cotylus with deeply excavated ligula bearing a number of well-defined laminae; crop, if pres- ent, seldom with diverticulum; ink sac absent; radula with homodont or weakly and irregu- larly multicuspid rachidians; skin usually with papillae; supraocular cirri often present. GENERAL SURVEY OF THE BATHYPOLYPUS SPECIES The elaborated hectocotylus, with a promi- nent, deeply excavated, laminated ligula, is the most distinctive character in the amended generic definition of Bathypolypus. In addi- tion, there are reductions in digestive tract, gills, radula, relative arm length, and the en- largement of eyes exhibited by the species in varying degrees. A tendency to develop some of these traits is found in Benthoctopus. The difference between the hectocotyli of the Ba- thypolypus and the Benthoctopus species provides sufficient substance to justify pre- serving the two genera, if only as a prelimi- nary measure. For the time being, our igno- rance regarding evolutionary sequence and weighting of derived characters in Octopodi- dae precludes serious phylogenetic consider- ations. Bathypolypus comprises six species in the most recent list of accepted cephalopod taxa (Sweeney & Roper, 1998): arcticus (Prosch, 1849); faeroensis (Russell, 1909); proschi Muus, 1962; salebrosus (Sasaki, 1920); sponsalis (P. Fischer & H. Fischer, 1892); and valdiviae (Chun & Thiele, 1915). The species proschi and faeroensis are here shown to be synonyms of the reinstated bairdii and arcticus respectively, whereas pugniger is recognized as new. It remains, however, to reconsider the remaining three of the hitherto recognized species in the light of the amended generic definitions of Bathypoly- pus and Benthoctopus. Also, the position of Benthoctopus ergasticus (P. Fisher & H. Fischer, 1892) is reconsidered. Benthoctopus salebrosus (Sasaki, 1920), new comb. Polypus salebrosus Sasaki, 1920: 182; 1929: 99, text-fig. 54, pl. 6, figs. 5, 6. Bathypolypus salebrosus: Robson, 1929: 41; 1932: 302; Akimushkin, 1965: 134, fig. 34; Nesis, 1987: 315, fig. 83A. Type and paratype: two females, USNM 332969, TL: 153 mm, ML: 45 mm; USNM 332970, TL: 77 mm, ML: 23 mm (not ex- amined). THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 205 A characteristic, though not well-known species from the Okhotsk Sea and off the Japanese Pacific coast in 212-1070 m. Based on the female types, Robson (1929, 1932) assigned salebrosus to Bathypolypus with hesitation, mostly on account of its short arms, rather deep web (33%), and because it is sculptured with closely set, well-defined, roundish warts. He found that the rachidians were weakly multicuspid, with at most one denticle on each side, and he established the absence of an ink sac. The hectocotylus was later described by Akimushkin (1965: fig. 34) and Nesis (1987: fig. 83A). The ligula is narrow, conical and pointed, LigLi about 14. The central groove is shallow and transversely striated with numer- ous indistinct rugae. Calamus is short and pointed. Benthoctopus salebrosus has no supraocu- lar cirri. The funnel organ is figured by Sasaki (1929: text-fig. 54) as a single W, the outer legs being the shortest. Gills not much re- duced, each demibranch with 9-10 filaments, the mantle aperture correspondingly moder- ate (B-C in Robson’s terminology). The slim, striated ligula unknown to Robson is of a type seen in some Benthoctopus species but very different from the highly spe- cialized ligulae of Bathypolypus arcticus and B. bairdii. | conclude that salebrosus is best placed in the genus Benthoctopus due to the simple non-laminated ligula and the single funnel organ, irrespective of its warty skin, which is unusual among other species of that genus. No other character contradicts this opinion. Bathypolypus sponsalis (P. Fischer & H. Fischer, 1892). Octopus sponsalis P. Fischer & H. Fischer, 1892: 297, fig. A; Fischer & Joubin 1907: 322, pl. 22, figs. 5-11 Bathypolypus sponsalis: Robson, 1927: 252; 1932: 300, figs. 61-63; Wirz, 1954: 139, figs. 1-5; 1955: 129, figs. 1-12; Adam, 1960: 504, fig. 2; Mangold-Wirz, 1963: 49; Perez-Gandaras & Guerra, 1978: 195, figs. 2-5; Nesis, 1987: 315, fig. 831; Guerra, 1992: 249, figs. 88, 91. Material examined: Syntypes of Octopus sponsalis P. Fischer & H. Fischer, 1892, MNHN 571097: 4 males ML: 27-41 mm, Exp. du “Talisman”, 332-1250 m, NW- Africa; in ZMUC, 8 males, ML: 33-49 mm and 3 females ML: 27-38 mm, Catalan Sea, western Mediterranean, June- September 1954 and June-September 1955, 200-500 m; in BMNH: one female, ML: 36 mm, Exp. du “Talisman”, July 12, 1885. This species was originally caught off west- ern Sahara (22°N, 19°46’E) at the same lo- cality as В. ergasticus. It has since been re- ported as a common mesobenthic species in the western Mediterranean (Wirz, 1954, 1955), the Aegean Sea (D’Onghia et al., 1993), and off Portugal and Galicia (Perez- Gandaras & Guerra, 1978). Good pictures of the whole animal and the hectocotylus are presented by Fischer & Joubin (1907: pl. 22), radula and internal or- gans by Robson (1932: figs. 61-63), and Wirz (1954: figs. 3-5; 1955: figs. 5-12). Robson (1927, 1932) placed sponsalis in Bathypolypus because of its apparent affini- ties to the arcticus-group: a squat, relatively short-armed body with huge eyes, small suck- ers, double funnel organ, a reduced radula with homodont rhachidians, lack of crop, and a ligula that is deeply excavated, with 6-7 laminae. The morphometrics and major features of the life cycle of the Mediterranean population of sponsalis are well known (Wirz, 1955; Mangold-Wirz, 1963) based on about 600 specimens caught at all times of the year. Table 9 gives the main distinctive indices based on my own measurements of speci- mens from the Catalan Sea. There are, however, some discrepancies between these data and descriptions of spec- imens from the type locality (Cape Verde Is- lands). Examination of the syntypes (MNHN 571097) shows that the Mediterranean popu- lation deviates in several important traits: Two spermatophores extracted from one of the syntypes confirmed Robson’s observation (1932) that the sperm reservoir is long, SpRI: 65-70 (versus 50-60 in the Mediterranean). The short oral end has a distinct swelling in the middle, making it spindle-shaped, unlike any other spermatophores of the genus (Fig. 17). The apical end is swollen compared with the smaller and more cylindrical reservoir in Mediterranean specimens. Calamus is very large and fleshy (Fischer & Joubin, 1907: pl. 22, fig. 6). In four syntypes, | found CaLl: 57-71 (Medit.: 33-42). The dif- ference was noted by Wirz (1955). The ligula is larger in the type series, LigLI: 14-22 (n = 5) (Medit.: 10-15), and the num- 206 MUUS 1cm FIG. 17. Spermatophores of B. sponsalis. Left: syn- type, ML 41 mm., Cape Verde. Right: specimen from the Catalan Sea, western Mediterranean, ML 44 mm. ber of suckers on the hectocotylus is low, SHcC: 44-50 (n = 4) (Medit.: 57-61). The type specimens have warts or pustules over the eyes and often on the antero-dorsal region. Adam (1960: fig. 2) found supraocular cirri, as well as a mixture of multifid and sim- ple warts sprinkled dorsally on two specimens from the Cape Verde Plateau. In contrast, the Mediterranean and Galician specimens have smooth skin without traces of warts. | conclude that the morphological devia- tions between populations may be rooted in subspecific variation or even represent unrec- ognized sibling species. Not least, the mor- phology of hectocotylus and spermatophores speak for the latter possibility. The Mediter- ranean population is well documented, but more specimens from the Cape Verde Pla- teau are needed. Regardless, the specimens hitherto identified as sponsalis clearly belong to the genus Bathypolypus. Bathypolypus valdiviae (Chun & Thiele, 1915) Polypus valdiviae Chun & Thiele, 1915: 485, text-figs 52, 53, pl. 80, figs. 1-5 Bathypolypus grimpei Robson, 1924a: 208; 1924b: 663, text-figs. 37-41, pl. 2, fig. 10 Bathypolypus valdiviae: Massy, 1927: 165; Robson, 1932: 303, figs. 64-68; San- chez & Moli, 1984: 19, fig. 18; Nesis, 1987: 315, fig. 83F-H Type: Z. M. Humboldt Univ. Berlin. Male, ML: approx. 30 mm (not examined). This species is known from the South African Agulhas Bank and off the Namibian coast. Bathypolypus valdiviae has a remarkable resemblance to B. pugniger, n. sp, being a short-armed, big-eyed, warty species usually with supraocular cirri and having the same kind of characteristic hectocotylus. Ligula is globular and deeply excavated with 4-5 well developed laminae. LigLl: 15-18. The hecto- cotylized arm bears about 45 suckers. HcLl: approx. 75. The arms are of subequal length (Chun & Thiele, 1915: pl. 1, fig. 4; Robson, 1924b: fig. 37, as “B. grimpei”). Also in other characters valdiviae shows affinity to pugniger and bairdii. The funnel organ is a pair of widely separated V-shaped pads (Robson, 1932: text-fig. 66); the rhachid- ians are homodont, but more pointed than the broad teeth in pugniger; the spermatophore is a true copy of bairdii and pugniger sper- matophores (Robson 1924b: text-figs. 39, 41). | conclude that placement of valdiviae in Bathypolypus is justified. Bathypolypus ergasticus (P. Fischer & H. Fischer, 1892), new comb. Octopus ergasticus P. Fischer & H. Fischer, 1892: 298, fig. B; Fischer & Joubin, 1907: 325, fig. 2A, pl. 22, figs. 1-4 Octopus profundicola Massy, 1907: 277. Polypus ergasticus: Massy, 1909: 7, pl. 1, figs. 1-3, pl. 2, fig. 1; 1913: 1; Robson, 1924b: 668 Benthoctopus ergasticus: Grimpe, 1921: 300; Robson, 1927: 255, figs. 4-6, 9; 1932: 244, figs. 44, 45; Nesis, 1987: 320, fig. 84E, F. Material examined: Syntypes of Octopus er- gasticus P. Fischer & H. Fischer 1892, MNHN 5811: 3 juv. males ML: approx. 17-35 mm, Exp. du “Talisman” 1883, 830 m, NW-Africa; in BMNH: syntypes of Polypus profundicola Massy, 1907: 3 males, ML: 50-80 mm; 3 females, ML: 57-61 mm. “Helga” stations 363, 365, 369, 400, 477 and 489, approx. 51°25'N, 11-12°W, 385-720 fath. One male er- THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 207 gasticus, ML: 45 mm, NW-Africa (gift from Prof. Joubin). This characteristic species originally caught off western Sahara (22°24'N, 19°46’E, 860 m) is well described by Fischer & Joubin (1907: pl. 22) and by Massy (1907, as O. pro- fundicola; 1909: pls. 1, 2) based on speci- mens caught off western Ireland. Robson has described reproductive organs, radula (1927: figs. 4-6, 9), and the digestive tract (1932: figs. 44, 45). The main indices of ergasticus are given in Table 9 based on my own measurements. Ad- ditional data in Massy (1909). Robson found it difficult to accommodate ergasticus in either Bathypolypus or Benth- octopus. He decided on the latter genus, how- ever, mainly because he, in contrast to Massy, found that the rachidians are multicuspid and because the esophagus has a crop diverticu- lum. As mentioned earlier, Robson confused the type species of the genus, Benthoctopus piscatorum, with specimens of Bathypolypus arcticus from the Faroe Channel. Multicuspid rhachidians and a crop diverticulum are found in the latter species and are thus not unique to Benthoctopus. Robson (1932: 247) admits that the hecto- cotylus of ergasticus is unlike those of other Benthoctopus species. The hectocotylized arm is short (OAI: 60) with a well-developed, deep, spoon-shaped ligula with about 7 strong laminae (LigLI: 9). Calamus prominent (CaLl: 30-40). The hectocotylus has a strong likeness to that of sponsalis. Chun & Thiele (1915: 485) consider it obvious that ergasti- cus, sponsalis, and valdiviae are related due to the form of the hectocotylus. Another character showing affinities to the Bathypolypus species is the enormous sper- matophore. One specimen with a total length of 111 mm, had a sperm reservoir of 51 mm in length and 8 mm in width. Apart from the size, the proportions are very much like the sper- matophores of bairdii and pugniger (Robson, 1927: 255, fig. 4). The funnel organ is double, and in the spec- imens from Ireland consists of two very char- acteristic subquadratic or pentagonal pads (Fig. 18), a type unknown in Benthoctopus, but not far from the structures in bairdii and pugniger. In the specimen from West Africa, the funnel organs each had a slight indenta- tion anteriorly, making them more heart- shaped. In the light of the amended generic diag- noses given, ergasticus is best accommo- dated in genus Bathypolypus. The genus Bathypolypus, as here recog- nized, thus includes only the following six At- lantic species: arcticus (Prosch, 1849), bairdii (Verrill, 1873), pugniger, n. sp., sponsalis (P. Fischer & H. Fischer, 1892), valdiviae (Chun & Thiele, 1915), and ergasticus (P. Fischer & H. Fischer, 1892). The species salebrosus (Sasaki, 1920) is moved to Benthoctopus on account of the simple, non-laminated hecto- cotylus and single funnel organ. DISTRIBUTION OF BATHYPOLYPUS Bathypolypus arcticus, s. str., are confined to Norwegian Sea Deep Water (NSW), whereas the squat, broad-headed species (B. bairdii, B. pugniger, n. sp.) are found in the warmer Atlantic water south of, or on top of, the Greenland-Scotland Ridge. This is brought out in Figure 19, which is based on the updated hydrographic and topo- graphic rewiew papers by Hansen (1985) and Westerberg (1990) in connection with the BIOFAR benthic projects. It is seen that sub- zero NSW fills the trough of the Faroe-Shet- land Canal 500-700 m under the north-going warm Atlantic Current. Almost barred in the south by the Wyville Thomson Ridge, the main flow of NSW is forced to make a bend round the southern end of the Faroe Plateau and flows through the Faroe Bank Canal to reach, and eventually slide down, the south- ern slopes of the Iceland-Faroe Ridge mixed with Atlantic water. Northern species — В. arcticus, В. bairdli, B. pugniger, n. Sp. Bathypolypus arcticus Horizontal Distribution: Figure 19 clearly demonstrates the stenothermal arctic affinity of arcticus. Specimens caught outside NSW are invariably in places intermittently exposed to overflow of cold water, which on the Faroe- Iceland Ridge often is a mixture of NSW and Arctic Intermediate Water (Al) generated in the Iceland and Greenland seas. The arctic affinity of arcticus is confirmed by its wider distribution in the cold parts of the Barents Sea, off eastern Greenland, and in the cold threshold fiords of southwestern 208 MUUS B.bairdii CO 0000 00 Uv JO CI OD Vo 000 B.pugniger 000000 OV MY q pp B.arcticus У VVVVYV Уиуму м B.ergasticus VO VOUJDOOD OQ B.sponsalis VAYAVIVAVAVAVIVA VEUVE, FIG. 18. Variability of funnel organ in five North Atlantic Bathypolypus species. The left pair in each row is considered typical of the species. Greenland, not influenced by the warm Irminger Current (Fig. 20). According to Kon- dakov (1936), arcticus occurs in the Kara Sea (78°01'N, 105°27'W, 175 m, —0.64°C). | have not had the opportunity to verify this identifi- cation, but Kondakov’s drawing looks con- vincing. Bruun (1945: 8) in his treatise of Icelandic cephalopods, though following Robson’s sensu lato concept of arcticus, is the only au- thor who noticed that the narrow-headed specimens occur off northern Iceland, whereas the “bairdii form” is found off the south and southeast coast of Iceland, that is south of the North Atlantic Ridge. The material of adult arcticus are too few to disclose possible geographic variation in meristic characters. THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 209 5 4 3 2 == D ZEN 7 Q / Foula 60 4 (SHETLANDS) р / AH 72 | \ + am Bee E > N FIG. 19. Distribution in Faroese waters of B. arcticus (squares), B. bairdii (dots) and B. pugniger, n. sp. (stars). Arctic deep water with negative temperatures darkly screened. In northwest occasional overflow of arctic water is indicated. Bathymetrical Distribution: Based on the present material, the depth range of arcticus is 37-565-1210 m. Only in the northernmost localities is the species caught in less than 100 m. In the south, optimal low temperatures are found at greater depth, usually over 400 m. Juveniles and sexually mature animals are evenly scattered at all depths. Bathypolypus bairdii Horizontal Distribution: This species seems to prefer Atlantic water masses with tempera- tures in the range of 2-8°C (Figs. 19, 21). Due to the warm Norwegian Current, bairdiiranges from the northern North Sea and Skagerrak along the Norwegian Coast to the southern parts of the Barents Sea. It is common on the southern slopes of the Iceland-Greenland Ridge, but is probably barred from southern Greenland by the eastern Greenland Polar Current. The southeastern Atlantic limit for bairdii seems to be northwestern Iberian wa- ters (Perez-Gandaras & Guerra 1978). In western Greenland, bairdii is found in water tempered by the warm Irminger Cur- rent, and it is common on the prawn trawling grounds and fishing banks south of the ridge FIG. 20. Distribution of Bathypolypus arcticus (Prosch, 1849). Open circles: positions not precisely known. separating Davis Strait and Baffin Bay. There are fewer records along the Labrador coast, but the Newfoundland area seems to offer the species excellent conditions. The Labrador Current, with admixture of warmer Atlantic water, fills the deeper parts (> 100-200 m) of the Laurentian Channel with water that rather constantly holds 2-5°C (Brunel et al., 1998). Macalaster (1976, as “B. arcticus’) records an average temperature of 4.3°C (SD: 1.7°C) for stations where the species was caught. The species was reported as far south as Fowey Rocks, Miami (Boone, 1938). Cairns (1976) showed bairdii to be the most abun- dant cephalopod in the Straits of Florida. Geographic variation: Bathypolypus bairdii is distributed in a many thousand km long, nar- row band largely following the upper 180- 1000 m of the continental slopes of the North Atlantic (Fig. 21). As it probably is a rather sta- tionary animal and having non-pelagic young, a certain clinal geographic variation could be expected. In bodily proportions, there does not seem to be variation. But characters of the hectocotylus show statistically significant geographic variation. Populations south of 45°N have larger ligu- lae (Fig. 10), with more laminae (Fig. 9) but fewer suckers on the hectocotylized arms (Fig. 8) than populations from Labrador and western Greenland. As regards the number of laminae, the Newfoundland area acts as a transitionary zone (Fig. 9). The clinal variation may be due to genetic differencies, or it may be a response to different ecological or hydro- graphic regimes. Small but perceptible geographic variation exists between the population from Labrador and western Greenland and their eastern At- lantic fellows. The Cape Farewell area at the southern tip of Greenland seems to form a distributional gap (Fig. 21). The occurrence of B. arcticus in this area (Fig. 20) suggests that the eastern Greenland Polar Current, which sweeps the slopes causes the absence of bairdii. In the eastern Atlantic, the ligula has more laminae (Fig. 9) and the hectocotylus slightly fewer suckers than found in Labrador and western Greenland. The relative size of THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 211 FIG. 21. Distribution of Bathypolypus bairdii (Verrill, 1873). Dots: specimens examined by the author, open circles: specimens treated by Kumpf (1958) or Macalaster (1976), but not examined by the author. ligula (LigLl) is the same for both populations (Fig. 10). The geographic variation is established for the males only, and it is most distinct in the western Atlantic population south of New- foundland. At the present state of knowledge | do not think a subspecific division is advis- able. The two major dividing areas, New- foundland and Cape Farewell, southern Greenland, have led to three slightly different populations. Bathymetric Distribution: Kumpf (1958) states the depth range of his large western Atlantic material of bairdii to be 20-350-1545 m. In the Strait of Florida, Cairns (1976) found the depth range to be 190-365-674 m. The southernmost record (29%45'N, 30°09’W) was also the deepest. Similar depth ranges were found off Greenland (maximum 1100 m) and in the East Atlantic (maximum 910 m). Juveniles and sexually mature animals are found at all depths, and there are no evidence of vertical migrations. Habitat and Biology: The vast majority of specimens are caught in trawling grounds on muddy or sand-mixed bottoms, which seem to be its natural habitat. The catches suggest a rather even dispersion. Underwater photos from the western Greenland prawn grounds showed bairdiiin its natural surroundings. The animal was seen resting unprotected in an ev- idently self-made shallow depression with the arms neatly curled along its sides. The stom- ach contents of simultaneously caught speci- mens showed a variety of polychete bristles and crustacean remains, including Pandalus. Based on aquaria observations, O’Dor & Macalaster (1983) suggest that bairdii prac- tises a sit-and-wait feeding strategy, and they list food items demonstrating its omnivorous nature. Underwater photos confirm the supposed feeding strategy. In the prawn trawling grounds, bairdii is surrounded by a rich food supply of roaming crustaceans, polychetes, and molluscs. Mortality must be very low. O’Dor & Macal- aster (1983) show that a three-year lifespan, including one reproduction period, is probable, but that a longer life cannot be ruled out. They estimate that due to the few eggs (at most 100) and even fewer hatchlings, about 4% of the lat- ter have to survive to replace the parents. To deposit and guard the eggs, the females 212 MUUS need a firm substratum. This may explain why | found a skewed sex ratio on the level trawl- ing grounds poor in shelters (38% females, n = 235). Hiding egg-guarding females are less apt to be caught in a trawl. Brooding females may be found in rocks (Macalaster, 1976) or in cans and containers discharged from ships (my observation) and even in plastic bags (Bergstrom, fide Macal- aster, 1976). Wood et al. (1998) observed mating behav- iour and brooding of B. bairdii (called “B. arcti- cus’) from Fundy Bay, Canada. In aquaria, brooding of eggs lasted over 400 days (at 6-10°C) or roughly one-third of the stipulated lifetime of bairdii. Hatchlings had a mantle length of 6 mm. Bathypolypus pugniger, n. sp. Horizontal Distribution: While arcticus and bairdii present a neat picture as distinct al- lopatric species, B. pugniger, n. sp., brings new problems through its enigmatic system- atic position and peculiar distribution. The sin- gle find from western Greenland (Table 6: no. 122) and the specimens from the Faroe-Ice- land Ridge are all caught in waters with posi- tive temperatures but in localities episodically exposed to overflow of arctic water from the realm of arcticus. | find this distribution sug- gestive and propose three different specula- tive explanations: (1) В. pugniger is a stunted form of В. bairdii. Adult bairdii living on the Faroe-Iceland Ridge may endure periods of overflow with arctic water. Brooding females probably stay on. Is it possible that negative temperatures during embryonic development result in stunted development? If this is the case, why are there not transitional stages between bairdii and pugniger? (2) В. pugniger is a hybrid between bairdii and arcticus. The similarity in lifestyle, habitat, and mor- phology of the two species suggests that mat- ing behaviour may also be similar. Is it possi- ble that male arcticus in places with episodic overflow of arctic water extend their territory and seduce (rape?) female bairdii left behind and perhaps less resistant, chilled as they are? This and the previous suggestion could explain the peculiar distribution of pugniger in a narrow zone bordering the arcticus habitat. (3) B. pugniger is a valid species. It has been overlooked and confused with bairdii. The Bathypolypus species of the East Atlantic are not yet well Known and more ma- terial will show a wider distribution of pug- niger. The question can best be solved by molec- ular approaches, such as protein elec- trophoresis or DNA-sequencing using fresh material not previously preserved in formalin. Bathymetric Distribution: The depth range of 20 stations where B. pugniger was caught is 200-610-1000 m. The Southern Species-B. sponsalis, B. ergasticus, B. valdiviae Bathypolypus sponsalis is an East Atlantic species, which replaces bairdii from the Gali- cian coast to the Cape Verde Islands. It is widespread in the Mediterranean. It seems to prefer the same habitats as bairdii and has a similar bathymetric distribution: 170-1250 m (P. Fischer & H. Fischer, 1892; Wirz, 1955; Perez-Gandaras & Guerra, 1978). Some evi- dence of upslope ontogenetic migration was found in the eastern Mediterranean by Vil- lanueva (1992). Bathypolypus ergasticus occurs off south- western Ireland and the Cape Verde Islands but is not known from the Mediterranean. In its northern distribution, it overlaps bairdii and further south sponsalis. However, ergasticus prefers deeper water than its congeners: 704-1350 m off Ireland (Massy, 1909), 932- 1139 m off Cape Verde Islands (P. Fischer & H. Fischer, 1892). The three species are para- patric. Bathypolypus valdiviae is the only repre- sentative of the genus known from the south- ern hemisphere. Off the Namibian coast and on the Agulhas Bank, the species does not seem to be rare on soft bottoms. Bathymetric range: 500-900 m (Chun & Thiele, 1915; Massy, 1927; Roeleveld, 1974; Sanchez & Moli, 1984). REMARKS ON THE IDENTIFICATION OF SPECIES The application of bivariate ratios (indices) in multivariate statistical analysis is inadvis- able (Atchley et al., 1976), but ratios are use- ful in taxonomic work, such as in keys. They are also used here for comparison with earlier THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 213 published data. Ratios based on variables with isometric or approximately isometric growth (e.g., SDI, LDI, ОА!) are acceptable tools for identification of species and of course so are indices based on size-indepen- dent meristic characters. Allometric growth, which may impair the usefulness of indices, is not prominent in the Bathypolypus species within the relevant size range of adolescent and adult animals (ML 20-60 mm). It is mod- est even where it can best be demonstrated (EDI, Fig. 8, HWI), and generally intraspecific inherent variability and artifacts far exceed and mask variation due to allometric growth in these phenetically similar species. For pur- poses of species identification, the allometric variation was less than overall variation and was not calculated. Regrettably, interspecific overlap is often unavoidable (Table 9). The parameters are normally or approximately normally distributed, and the application of several indices tends to obliterate marginal values and leads to clearer discrimination of species, supplementing other, more tangible distinctive characters (funnel organ, crop, radula, sculpture, meristic characters). An important point that the present revision brought out is that the number of suckers on the hectocotylus is individually constant, even from the juvenile stage, and that the number of laminae is constant from the onset of matu- rity, irrespective of later increase in size (Figs. 22, 23) Counts of suckers and laminae are good size-independent meristic characters to use in concert with other characters, even if cases of overlap exist (Fig. 9, Table 9). The relative constancy of SHcC was also demon- strated by Toll (1988), who used differences in Laminae о B.arcticus n=35 e B.bairdii n=48 10 20 30 SHcC as additional argument for taxonomic separation of Atlantic and Pacific Scaeurgus. In the radula, only the rachidians are dis- tinctive in the Bathypolypus species. They may be very uniform (bairdii, sponsalis), in others very variable (arcticus, pugniger, n. sp.). A striking example of similar variability is seen in Graneledone pacifica, in which the rachidians range from heterodont B8 seriation to simple homodont or even degenerate con- dition (Voss & Pearcy, 1990: 87, fig. 19). KEY TO THE NORTH ATLANTIC BATHYPOLYPUS SPECIES The majority of specimens may be identi- fied by outer distinctive features, supple- mented by inspection of the funnel organ (Fig. 18). Additional help may be gained from Table 9, which shows ranges of essential indices and counts. For juveniles, females, aberrant males, and less well-preserved specimens, however, inspection of radula, digestive tract, and spermatophores may be necessary. 1a. Mantle length usually more than 25% of total length. Arms with fewer than 100 suckers. Length of hectocotylus at least 70% of opposite (3, left) arm and with fewer than 50 suckers. Skin often warty and with a pointed cirrus overeach eye .2 1b. Mantle length usually less than 25% of total length. Arms with over 140 suckers. Length of hectocotylus at most 70% of op- posite arm and with over 50 suckers. Skin smooth, supraocular cirri absent ...... 4 2a. Body egg-shaped, with a constriction be- hind the head. Diameter of eyes less than 33% of ML. Largest sucker diameter 40 50 60 ML FIG. 22. In Bathypolypus species the number of laminae on ligula seems to be individually constant from the onset of maturity. Regress. slopes: < +/- 0.023, 1”: < 0.015 (data for bairdii from western Greenland). 214 MUUS Suckers hect. arm B.arcticus 10 20 30 40 50 ML mm FIG. 23. In Bathypolypus species, the number of suckers on the hectocotylized arm seems to be constant throughout life. Regress. slopes: < +/— 0.03, 6.5-8.5% of ML and 18-33% of eye di- ameter. Ligula with 11-16 laminae; funnel organ a clear-cut VV (Fig. 18); esophagus with prominent crop diverticulum; radula usually with heterodont rachidians (Fig. 5); seminal reservoir occupying only 30% of the spermatophore length .. . .arcticus 2b. Body squat; diameter of eye-balls 30- 45% of ML; largest sucker diameter less than 6.5% of ML and at most 6-16% of eye diameter. Funnel organ pad-like, never a clear-cut VV. Ligula with less than 13 laminae. Esophagus without crop di- verticulum; radula homodont; seminal reservoir occupying about 50% of sper- matophore length 3a. Arm length order usually: 1.2.3.4. Ligula large, oblong, with 7-12 laminae; cala- mus 15-25% of ligula length; radula with long, slim homodont rachidians (Fig. 11) AM НИЕ bairdii 3b. Arm length order usually 3.4.2.1 or sube- qual. Ligula globular, fleshy, deeply hol- lowed, with 4-6 laminae; calamus 26-51% of ligula length; radula variable but with broad homodont rachidians (Fig. | =) Pa eee a ere eC ren pugniger, n. sp. 4a. Hectocotylus with 70-85 suckers; other arms usually with over 200 suckers. Fun- nel organ almost square pads (Fig. 18). Esophagus with crop diverticulum. Sper- matophore very large, longer than ML .er- gasticus г = 0.02. 4b. Hectocotylus with 50-65 suckers; other arms with 140-200 suckers. Funnel organ VV (Fig. 18); esophagus without crop di- verticulum. Spermatophore shorter than MIE ee sponsalis ACKNOWLEDGEMENTS | am indebted to the staff of the BIOFAR and BIOICE projects for access to the valu- able recent collections from the Greenland- Scotland ridge and the Faroe-Shetland Canal. Dr. C. C. Lu kindly supplied me with speci- mens of B. bairdii from Canadian waters and Dr. Katharina Mangold blessed me (and ZMUC) with a collection of B. sponsalis from the Catalan Sea. With gratitude | have re- ceived advice and/or valuable information from Dr. G. Perez-Gandaras, Dr. K. Nesis, Dr. C.F.E. Roper, and, not least, from the late Dr. G. Voss, with whom | had fruitful discussions. Finally, | wish to thank the curators of mol- luscs at the zoological museums in Bergen, Berlin, London, Oslo, and Washington for pleasant stays and help at various phases of my work. In ZMUC Stine Elle, Tom Schiotte and Gert Brovad offered me invaluable tech- nical assistance. My wife Kirsten Muus and two unknown but meticulous referees have THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 215 improved the first version of the script in nu- merous ways. LITERATURE CITED ADAM, W., 1939, Sur quelques cephalopodes de la mer d'Islande. 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ML: 8.—Udsire Hole, 58°58'N, 03°37’E, May 15, 1912. © МЕ; 35.—71°50М№ 282107Е; Мау 1, 1975, 180 fms., Ф ML: approx. 50. MNHT: Haul 46, 61°25’N, 05°13’W, July 7, 1979, 481 т, © ML: 23.—BIOFAR project: Stn. 117, 62°00.7'N, 09°4.63’W, July 25, 1987, 481 m, © ML: 16.—Stn. 124, 62°16.94'N, 09°38.93'W, July 26, 1987, 600 m, © МЕ 35-5. 158. 61387 05-38 W, Мау 7, 1988, 322 m, с’ МЕ: 36.—Stn. 419, 62°25'N, 10°38.17'W, June, 1, 1989, 702 m, 2 oo ML: 40, 42.-Stn. 420, 62°32'81N 10°27.57'W, June 1, 1989, 597 m, © ML: 33.—Stn. 482, 61°01.94’N, 05°13.94'W, July 22, 1989, 509 m, Ф ML: 24. IMNH: B5-77-?, 65°42'N, 27°53’W, February 24, 1977, 750-820 m, 2 99 ML: 23, 33.— B5-77-48, 65°38’N, 29°27’W, March 23, 1977, 450 m, © ML: 41, Q ML: 33.—B5- 77-49, 65°37'N, 29°32’W, March 23, 218 MUUS 1977, 420-430 m, С’ ML: 41, ФФ ML: 21, 50.—B5-77-50, 63°38'N, 29°27'W, March 23, 1977, 450-460 m, 2 So ML: 35, 45, 2 ФФ ML: 24, 36.—B5-77-73, 63°38'N, 26°14'W, March 27, 1977, 575 m, Ф ML: 35.—B5-78-07, 63°48'N, 27°00'W, March 10, 1978, 1110-1095 т, O” ML: 63.—B5-78-22, 64°56’N, 27°59'W, March 14, 1978, 970-1030 m, Q ML: 47.—DALB-1-80-13, 66°39'N, 28°43’W, October 21, 1980, 415 m, © ML: approx. 64.—B3-81-34, 65°22’М, 32°37'W, February 25, 1981, 910 m, © ML: 75.—BIOICE project: Stn. 2299, 63°00'10N, 22°39'61W, September 10, 1992, 775 m, juv. ML: 28.—Stn. 2346, 63°23'N 12°88'W, May 6, 1993, 501 т, oO ML: 39, Y ML: 18. ZIASP: Nr. 25, near Spitsbergen, 215 m, © ML: 35. ZMUB: Norwegian North-Atl. Exp. Stn. 290, 72°27'N, 20°51’E, July 7, 1878, 349 m, © ML: 26, Q ML: 18.— Outer part of Laksefjord, Fin- mark (approx. 71°N, 27°E), August 27, 1900, 280 m, © ML: 24, Q ML: 39.—R.V. “Michael Sars”: Stn. 56, 70°09’N, 31°00’E, May 21, 1901, 200 m, juv. © ML: 23.—Stn. 108, 70°32'N, 18°17’E, June 18, 1909, 300 m, juv. с’ ML: 11.—Stn. 5, 70°07'N, 30°53’E, June 4, 1914, © ML: approx. 44.—Stn. 28, 70°16'N, 32°20’E, June 24, 1914, © ML: approx. 30.-MS “Armauer Hansen” Stn. 2, outer part of Sognefjord (N of Bergen), March 10, 1917, juv. с’ ML: 15.-Salhus (N of Bergen), July 17, 1916, 400-500 m, © ML: 55.—42837, Salhusfjord (N of Ber- gen), July 12, 1934, 2 F'T' ML: 40, 42.— Mangerfjord (N of Bergen), March 18, 1931, 300-400 m, 2 So ML: 36, 38.— 53336, Kvinnheradsfjord, 59°59'50’N, 05°54'E, December 7, 1956, 687-672 m, 3 00 ML: 34-42, 3 ФФ ML: 22-36.— Hardangerfjord, NE of Varaldsoy, Stn. F 103, November 8, 1957, 690 m, ©’ ML: 40.— 50281, Sognefjord, 61°03'N, 06°25'E, May 3, 1966, 1238-1228 m, © ML: 56, 3 ФФ ML: 28-47.-R.V. “G.O. Sars”, S of Bear Island (Björnöya), August 4, 1974, 500 m, © ML: 74. ZMUC: Trondhjemsfjord, Norway, March 4, 1891, Storm leg., Y ML: 26.- Trondhjemsfjord, September 19, 1934, 180-220 m, © ML: 16.— Skager-rak, NNW of Skagen, July 28, 1897, 210 Tms., 200 ML: 29;,49.— Skagerrak, July 28, 1897, C. G. Joh. Pe- tersen leg., 275 fms., 2 juv. ML: 7, 7.—Sk- agerrak, about 14 n.m. NW of Hirtshals, June 21, 1911, 313 т, © ML: 41.— Trond- hjemsfjord, Norway, N of Tatra, Septem- ber 19, 1934, 180-220 m, Stephensen leg, © МЕ: 15.—“Thor: Sin. 167, 63°05'N, 20%07'W, July 14, 1903, 557 m, Ф м. ML: 9.—Stn. 223, 64°30'N, 12°25'W, March 17, 1904, 535 m, © ML: 60.— Stn. 99, 61°35'N, 9°35’W, May 22, 1904, 900 т, © ML: 38.—Stn. 274, Sk- agerrak, NW of Hirtshals, October 9, 1904, 660 m, © juv. ML: 23.—Stn. 1074, Skagerrak, 4.5 n.m. S of Okso light- house, May 28, 1907, 480 m, © ML: 43, Ф ML: 45.—Stn. 1570, Skagerrak, 53 n.m. N of Hanstholm, June 23, 1911, 525-550 m, © ML: 58, juv. 11.—“Dana”: Stn. 6001, 63°33’N, 11°25’W, July 24, 1938, 322 m, с’ ML: 33.—Stn. 6004, 63°06'N, 10°40’W, July 24, 1938, 437 т, Oo ML: 43.-Stn. 11643, 57°44'N, 07°40'W, April 17, 1961, 440 т, 2 00 ML: 50, 55.-Stn. 13320, 58°10’М, 04°22'E, March 12, 1965, 270 m, с’ ML: 42.—Stn. 15194, 57°35'N, 08°08’E, Sep- tember 19, 1969, 210 m, 2 SO ML: 27, 29.—Stn. ?, 61°03’N, 05°04'W, April 20, 1988, 800-600 m, © ML: 38. ZMUO: Oslofjord, off Filtvet lighthouse, April 26, 1910, 100 fms., juv. ML: 13.—Oslofjord, Filtvet, Мау 3, 1966, 150-280 m, Ф ML: 61.— 32291, Oslofjord, Torbjornskaer light- house, с’ ML: 39.—Oslofjord, Drobak, © ML: 43, Y ML: 31.- Oslofjord, Vestmedet, August 14, 1937 Greenland ZMUC: “Ingolf”: Stn. 28, 65°14’N, 55°42’W, July 1, 1895, 420 fms., © ML: 37.—Stn. 32, 66°35'N, 56°38’W, July 11, 1895, 2 99 ML: 18, 62 + 4 juv.—Stn. 35, 65°16’N, THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 219 55°05’W, July 18, 1895, 682 m, 2 juv. ML: 8, 9.—“Tjalfe”: Stn. 337, 64°05’N, 55°20'W, Мау 8, 1909, 1100 m, Ф ML: 54.—Stn. 428, 63°54'N, 53°15’W, June 8, 1909, 988 m, © ML: 21.—“Rink”: Stn. 45, Bredefjord, SW Greenl. (approx. 60°49'М, 46°45’W), July 18, 1912, 430-450 m, С’ ML: 52.—“Dana”: Stn. 2346, 66°37'N, 56°37'W, June 22, 1925, 450 m, 6 So ML: 16-68, 2 ФФ ML: 32, 44, 3 juv.— Stn. 2361, 68°08'N, 57°30'W, June 26, 1925, 398 m, 3 O'O' ML: 46-55, COMMERS4= Sins 10018. 65°02’N, 56°00’W, July 19, 1957, 730-740 т, © ML: 45 (type of B. proschi Muus, 1962).— Stn. 13662, 64°24'N, 52°57'W, July 27, 1966, 450-510 m, с’ МЕ: 32.—Greenl. Fish. Invest: Stn. 4539, 64°19’N, 52°55’W, June 4, 1971, 470-570 m, 29 do ML: 26-50, 10 ФФ ML: 25-54.— Stn. 5043, 63°57'N, 52°21’W, June 18, 1975, 300 m, © ML: 33 + juv.— Stn. 5047, 64°21'N, 52%58'W, June 24, 1975, 550-580 m, 8 So ML: 15-49, 8 ФФ ML: 24-42.-Stn. 5101, 66°34'N, 54°15’W, August 10, 1975, 335-340 m, © ML: 20.— St. 5112, 64°23’N, 52°58’W, August 21, 1975, 450-510 m, 5 Jo ML: 32-41, Oe Mizar A = Stns 75206, 63°58:N; 52°21'W, June 3, 1976, 300-310 m, © ML: 38.—Stn. 5209, 63°58’N, 52°21'W, June 8, 1976, 300-308 m, © ML: 34, Y ML: 46.—Stn. 5215, 64°21'N, 52°59'W, June 10, 1976, 510-520 m, 4 oo ML: 29-45, 5 ФФ ML: 37-41.—Stn. 5384, 64°21'N, 52°59'W, April 22, 1977, 485- 510 m, © ML: 44.—“Elias Kleist”: Stn. 791010/3, 67°57'N, 57°10'W, October 10, 1979, 310-100 m, Ф ML: 47. American East Coast BMNH: Off Marthas Vineyard, Massachusetts, May 21, 1898, 200-388 fms., © ML: 35 (la- belled Octopus bairdii). USNM: 34223, Le Have Bank, Nova Scotia, 120 fms., holotype of Octopus lentus Verrill, 1880.— 382469, fishing banks off Massachusetts, 36 miles E of NE Light, Sable Island, from halibut stomach, holotype of Octopus obesus Verrill, 1880, © ML: 44.- 39943, off New Jersey, 39°49'30N, 71°10’W, Au- gust 3, 1884, 420 fms., Y ML: 51.—52979, off New Jersey, 39°50'N, 71°43’W, Sep- tember 18, 1885, 137 fms., © ML: ap- prox. 41.-574638, Bay of Fundy, paratype of Octopus bairdii Verrill, 1873, O ML: 24. 575271, 43° 38'М, 69°13’W, August 2, 1912, 60 fms., Ф МЕ20.—575274, Gulf of Maine, August 16) 1878, Ys'ims, 31010) MER2IF26 — 575275, off Саре Cod, August-Septem- ber 1878, 70-94 fms., 6 G'O ML: 12-26, 4 ФФ ML: 10-18.—575276, off Salem, 41°58'30N, 69°44’W, September 18, 1379: 4 Gio МЕ О-о МЕ 39 = 575277, off Salem, August 1877, 49 fms., 2.©:@.ME: 27, 3951) 9 МЕ: 10575278, 42°31'N, 70°20’W, September 2, 1878, 98 fms., 3 specimens.—575279, off Martha’s Vineyard, 39°46’N, 71°05’W, February 10, 1880, 487 fms., Ф ML: 39, juv. ML: 9.—575280, 40°02'N, 70°23'6W, September 4, 1880, 192 fms., © ML: 14, Q ML: 35.—575281, 37°24'N, 74°17'W, November 16, 1880, 300 fms., 2 Jo’ ML: 26, 23, 3 ФФ МЕ 17-27.—575282, 39°49'N, 70°54'W, September 13, 1880, 225-252. -imss #2: 10/61 МЕ: -20,-38:— 575285, 37°07'50N, 74°34'20W, Novem- ber 18, 1884, 167 fms., © ML: 37.— 575288, off Martha’s Vineyard, 39°53’30N, 71°13’30W, August 9, 1881, 319 fms., 2 So ML: 50, one damaged, Y ML: 50.—575289, 40°04’N, 69°29’ 30W, September 28, 1884, 58 fms., © ML: 39.—575293, 39°52'20N, 70°58'W, October 2, 1880, 372 fms., © ML: 28.— 575294, 39°53’N, 70°58’30W, October 2, 1880, 365 fms., 3 oc ML: 38-46.— 575306, off Gay Head, Martha’s Vine- yard, 39°57'N, 70°31’30W, August 23, 1881, 225-396 fms., © ML: 40,3 Y Q ML: 21-49.-575971, 39°57'N, 70°58’W, Au- gust 25, 1879, 175-200 fms., O' ML: 44.— RV “Oregon”: Stn. 6800, 29°48'N, 80°09'05W, July 20, 1967, 183 fms., 2 qo.—Gosnold cruise: Stn. 105, 39° 51'N, 70°56’W, August 10, 1972, 875- 880 m, ©’ ML: 51.—Stn. 120, 39°50'N, 70°32'W, August 16, 1972, 750-775 m, CO ML: 65.-RV “Chain”: Stn. 243, 39°30'N, 72°20’W, February 28, 1973, 474-529 m, © ML: 21, Q ML: 27.—Stn. 244, 39°28'N, 72°18'W, February 28, 1973, 260-342 m, Ф ML: 44.—Stn. 254, MUUS 39°51'N, 70°47'W, March 2, 1973, 768-947 m, © ML: 49, Q ML: 56.— Stn.?, 39°31'N, 78°18'W, February 26, 1973, 810-900 m, 2 ML: 41.—Stn.?, 39°31’N, 75°23'W, February 28, 1973, 420-590 m, Q ML: 26.—RV “Knorr”: Stn. 300, 39°40’N 72°27'W, November 13, 1973, 110-182 m, С’ ML: 26.-Stn. 301, 39°32’N, 72°24'W, November 13, 1973, 475-520 т, 40°C ML: 20-33, Y ML: 28.—GI-75- 08: Stn. 10, 37°53’N, 74°40'08W, Sep- tember 9, 1975, 290-340 т, 2 сс’ 99% (distorted).—Stn. 18, 37°04'07N, 74° 34'03W, September 10, 1975, 200-215 m, 3 do, 19.—St. 19, 36°58’04N 74°37'05W, September 11, 1975, 190- 250 т, 2 00,3 9 9 .—St. 22, 36°58’06N, 74°33'08W, September 11, 1975, 880- 920 т, 3 oo ML: 24-42.-Stn. 99, 36°36'05М 74°39'W, September 20, 1975, 850-1000 m, Y ML: 50. ZMUC: “Albatros IV”, cruise 76-02: Stn. 221, 40°07'N, 69°04'W, March 28, 1976, 465 m, Q ML: 30.—Stn. 237, 41°44'N, 69°46’W, March 30, 1976, 78 m, © ML: 27.—Stn. 288, 41°53’N, 67°52'W, April 6, 1976, 52 m, с’ ML: 19.-Stn. 300, 42°05'N, 68°15’W, April 7, 1976, 189 m, Ф ML: 25.—Stn. 305, 42°18'N, 6912'W, April 8, 1976, 230 m, Ф ML: 38.— Stn. 309, 42°26'М, 70°06'W, April 8, 1976, 84m, © МЕ: 42.- Stn. 321, 42°45'N, 70°00'W, April 14, 1976, 163 m, Ф ML: 41.—Stn. 322, 42°46'N, 69°35'W, April 14, 1976, 163 m, © ML: 25, Y ML: 43.—Stn. 330, 42°59'N, 68°39'W, April 17, 1976, 187 m, 2 99 ML: 20, 22.-Stn. 332, 42°45'N, 67°47'W, April 17, 1976, 200 m, 2 So ML: 38, 42.- Stn. 333, 42°34’N, 67°58'W, April 17, 1976, 206 m, 2 ML: 20.—Stn. 339, 42°17'N, 66°49’W, April 18, 1976, 270 m, С’ ML: 26.—Stn. 341, 42°19’N, 66°12'W, April 18, 1976, 262 m, 1 spm.— Stn. 405, 43°26'N, 67°34’W, April 29, 1976, 218 m, © ML: 37, © ML: 37.—Stn. 406, 43°06'N, 67°48'W, April 29, 1976, 200 m, 2 ML: 29.—Stn. 409, 43°39’N, 68°17'W, April 29, 1976, 190 m, 3 SO ML: 18-29.—Stn. 426, 43°29'N, 69° 01’W, Мау 6, 1976, 139 m, 2 ML: 50.— Notre Dame Bay, Newfoundland: October 1975, 3 JO ML: 67-74.—USSR “Belo- gorsk”, cruise 74-11: Stn. 142, 41°16'N, 68°41'W, October 12, 1974, 80 m, Y ML: 32. Bathypolypus arcticus (Prosch) East Atlantic BMNH: 60°03'N, 05°51'W, August 17, 1880, 540 fms. (labelled В. faeroensis) S ML: 35, © ML: 30.-HMS “Triton”, Stn. 9, 60°05’М, 06°21'W, August 23, 1882, 608 fms. (syntypes of Benthoctopus sasakii Rob- son), © ML: approx. 32, Y ML: 27.—Stn. 40, 57°34'N, 00°01'W, November 27, 1904, Tow net 100 m, © ML: 30.— 61°27'N, 01°47'W, July 25, 1909, 1240 m, © ML: 43 (labelled Benthoctopus pis- catorum) + Ф ML: 20.—61°42’N, 02°00'W, July 25, 1909, 1236 m, Y ML: 31 (labelled B. faeroensis).—Stn. 77, 75°16'N, 24°46’E, June 1956, 85 m, Е. Holt, 2 ФФ ML: 28, 29.—Cruise IV, Stn. 37, 60°25’N, 04°31'W-60°29'N, 04° 22'W, W of Foula, 1973, 940-910 m, 2 ФФ ML: 20, 28. IRSNB: (vide Adam, 1939) 66°20’N, 12°28'W, June 21, 1938, 180 —220 m, 2 SO juv. ML: 16, 10, Y ML: 13.—66°20'N, 12°28'W, June 22, 1938, 180-220 m, Ф ML: са. 18. MNHT: Haul 96, 62°59'N, 09°54'W, July 23, 1979, 481 m, Y ML: 46.—BIOFAR project: Stn. 15, 62°37'68N, 04%40'37W, July 17, 1987, 683 m, juv. МЕ 12.—Stn. 95, 60%41'51N, 05°18’63W, July 23, 1987, 803 m, Y ML: 21.—Stn. 274, 63°00'79N, 07°49'22W, May 16, 1988, 698 m, Ф ML: 42.—Stn. 294, 60°26'N, 07°28’W, July 17, 1988, 1096 m, © ML: 30.—Stn. 502, 60°30'26’N, 08°04'W, July 25, 1989, 890 m, o МЫ 30.—Stn:~ 589, 60°40'N; 10°00’W, April 9, 1990, 250 m, Ф ML: 15.—Stn. 726, 60°39’N, 06°54'W, Sep- tember 29, 1990, 400 m, Ф ML: 27. IMNH: 06-80-50, 67°11’N, 18°30'W, April 20, 1980, 430 m, С’ ML: approx. 41.-B16-80, 66°50'N, 20°26'W, October 29, 1980, 415 m, Y ML: approx. 37.—B3-81-34, THE BATHYPOLYPUS-BENTHOCTOPUS PROBLEM OF THE NORTH ATLANTIC 221 62°59’N, 20°23'W, March 7, 1981, 910 m, Q ML: 95.—B4-81-137, 66°37'N, 12°58’W, April 1, 1981, 320 m, Y ML: 31.-BIOICE project: Stn. 2080, 67°22'79N, 17°20'77W, July 4, 1992, 897 m, © ML: 46.-Stn. 2085, 67°15’67N, 17°26’41W, July 4, 1992, 754 m, Ф ML: 61.—Stn. 2088, 67°02'35N, 13°25’05W, July 4, 1992, 903 m, с’ ML: 40.—Stn. 2134, 66°44'46N, 18°54'93W, July 8, 1992, 504 m, © МЕ: 17.—Stn. 2135, 66°44'37N, 18°57'32W, July 8, 1992, 418 m, с’ juv. ML: 26.—Stn. 2320, 64°02'N, 09°44'W, May 2, 1993, 758 m, © ML: 49.—Stn. 2322, 63°55'N, 10°04’W, Мау 3, 1993, 627 m, Ф ML: 45.—Stn. 2326, 63°44'N, 10°09'W, May 3, 1993, 563 m, Q ML: 33.—Stn. 2328, 63°20'N, 10°57'W, May 3, 1993, 430 m, Y ML: 54. TMDZ: M/K “Asterias”: Isfjord, Svalbard, July 18, 1955, 242-228 m, Ф ML: 26.—Isfjord, Svalbard, August 14, 1958, 230-215 m, GO juv. ML: 23, Y ML: 23. ZIASP: 92, Barents Sea, 140-150 m, Kondakov det., O” juv. ML: approx. 12.-119, between Spitsbergen and Frantz Josef Land, 210 m, Kondakov det., с’ ML: 31.— 120, Bar- ents Sea, 201 m, © ML: 19.—Nr. 607, Novaya Zemlya, 37 m, 2 ML: 39. ZMUB: ESE of Vardo (about 70°N, 32°E), August 10, 1902, © ML: approx. 49.—“Solveig |”, Stn. 52, Kongsfjord, Svalbard, June 22, 1938, 290-333 m, Ф МЕ: 42.- 42838, Stn. 64, Svalbard, 78°25'N, 12°06’E, July 7, 1939, © ML: 44.- 36145, “Sotra”, Stn. 250, 78°04'N, 14°10'E, September 911930: © МЕ: 38 Ток» эм. 10, 77°44'N, 11°45’E, June 30, 1925, 185-228 m, © juv. ML: 13.-78°03’N, 14°10’E, August 30, 1931, 147 m, 2 ML: 31.-75°00’N 37°20'E, September 1, 1968, 100-150 m, Ф ML: 57.- 18678, “Michael Sars”, Stn. 37, 62°43’N, 01°26’E, June 29, 1902, 775 m, © ML: 36. ZMUC: 61°27'N, 01°27'W, July 25, 1909, 1240 m, la- belled Polypus faeroensis Russell Type. (leg. A. C. Stephen). Greenland ZMUC: Lectotype of Octopus arcticus Prosch, 1849: С’ ML: 42, SW Greenland, August 26, 1841, К. M. Jorgensen leg.— Paralecto- type ©, partly dissected, SW Greenland, July 27, 1840, К. M. Jorgensen leg.— Dis- sected male organs pictured by Prosch (1849: figs. 1-3)—3 ФФ in bad shape and partly dissected labelled Greenland and evidently from the 1840s.—Hol- steinsborg, SW Greenland, 1892, Trau- stedt leg., Y ML: 64.—“Ingolf”: Stn. 124, 67°40’N, 15°40’W, July 28, 1896, 932 m, 2 do ML: 44, 44.- Near Nanortalik, SW Greenland, April 15, 1906, © ML: 31.— Lichtenau fjord, SW Greenland, October 14, 1910, from stomach of Greenland halibut, Ф ML: 45.—Lichtenau fjord, June 6, 1914, 220 fms., Ф ML: 41.—Lichtenau fjord, July 3, 1947, Poul Hansen leg., Ф ML: 51.—“Godthab”: Stn. 81, 75°35’N, 65°41’W, August 1, 1928, 490 m, с’ ML: 54, Q МЕ 23:=Sin. 87, 77°05 № 71°13’W, August 4, 1928, 790 m, 14 00° ML: 32-58, 2 ФФ ML: 32-46, 1 juv. ML; : 22.—Stn. 90, 77°17'N, 69°59’W, August 5, 1928, 930 m, o ML: 46, Y ML: 38.— Stn. 116, 76°08’N, 80°53’W, August 17, 1928, 80 m, © juv. ML: 13.—“Godthab’s summer cruise, Stn. 341, off Kap Hacker, Jameson Land, E Greenland, August 27, 1933, juv. ML: 15.—Ella Island, Kong Oscar Fjord, approx. 73°N, 25°W, Octo- ber 10, 1931, 67-68 m, Y ML: 35.— Ymer Island, Frantz Joseph Fjord, E Greenland (approx. 73°20'N), August 8, 1932, © ML: 43.—Frantz Joseph Fjord, off Eng- dalen, August 7, 1931, 45-36 m, © ML: 52.—Lindenow Fjord, 60°30'N, 43°25'W, July 17, 1935, 100-150 m, Bertelsen leg., O” ML: 36.—Amerdlok Fjord, near Holsteinsborg (Sisimiut), July 26, 1938, approx. 500 m, ® ML: 54.—Skovfjord, SW Greenland, June 16, 1948, P. Hansen leg., с’ ML: 58.— Young Sound, off Daneborg, approx. 74%15'N, 20°W, July 1947, Ф ML: approx. 58.—Bylot 222 MUUS Sound, 76°31'N, 69°09’W, August 20, 1968, 240 m, Just & Vibe Stn. 45, ©’ ML: 54.-Kap Farvel Exp.: Stn. 56, 60°13'N, 44°13’W, July 31, 1970, 400-420 m, © ML: 35.—Stn. 128, 60°01’М, 43°59'W, August 21, 1970, 530 m, Y ML: 51.-W Greenland, 66°21'N, 54°56’W, August 2, 1975, 280-290 m, Max Andersen leg., © ML: 12.-Greenland Fish. Invest.: Stn. 3978, 63°53'N, 51°27'W, May 9, 1968, 230-250 m, Ф ML: 35.—Stn. 4360, May 21, 1970, 240-250 m, © ML: 43, © ML: 34.—Stn. 5101, 66°34'N, 54°15’W, Au- gust 10, 1975, 300 m, 2 So ML: 33 + juv. ZMUO: Hoel’s Greenland Exp.: Stn. 1101, Kong Oscar Fjord, E Greenland (approx. 72°20'N), August 12, 1930, 55-100 m, © juv. ML: 17.-Stn. 1116, Kong Oscar Fjord, August 15, 1930, 250 m, © ML: 30.—Stn. 1118, Kong Oscar Fjord, August 16, 1930, 120 m, 1 м. ML: 26.—Stn. 1119, Kong Oscar Fjord, Vegasund, Au- gust 17, 1930, 190-250 m, 4 So ML: 37-44. American East Coast No material. Bathypolypus pugniger n. sp. IRSNB: 66°23'М 12°53’W, June 14-15, 1938, 200-250 т, o ML: 22, Q ML: 18 (vide Adam 1939). MNHT: Haul 25, 63°16’N, 09°30’W, July 6, 1979, 500 т, 2 °C ML: 27, 28.-BIOFAR project: Stn. 47, 61°02’31”N, 05°54'W, July 19, 1987, 280 m, Ф ML: 19.—Stn. 80, 60° 38'89N, 08°27'93W, July 22, 1987, 678 m, Ф МЕ: 18.—Stn. 269, 62°49'84N, 08° 15'55W, May 15, 1988, 510 m, © ML: 33.—Stn. 503, 60°38'02N, 08°33’54W, July 25, 1989, 513 m, © ML: 18.— Sin. 734, 60%10'06N, 07°57'03W, May 8, 1990, 634 m, © ML: 31.—Stn. 738, 62°19’N, 10°13’W, October 1, 1990, 749 mo ML: 25.-Stn: 74076229IN7102 02'W, October 1, 1990, 597 m, o ML: 18. IMNH: B5-77-36, 65°40'N, 28°20'W, March 21, 1977, 1000-970 m, Ф ML: 36.—B5-77- 40, 65°36'N 29°17’W, March 22, 1977, 870-910 m, 9 ML: 33.—B5-77-42, 65°34'N, 29°29’W, March 22, 1977, 750-760 m, © ML: approx. 44.—B5-77- 46, 65°29'N, 29°33’W, March 23, 1977, 960 m, 2 SO ML: 36, 30, 3 ФФ ML: 27-40.—B5-77-50, 65°38'N, 29°27’W, March 23, 1977, 450-460 m, Ф ML: ap- prox. 32, O' ML: 30.—В5-78-44, 64°58'N, 27°44'W, March 14, 1978, 860-870 m, holotype 19990971. © ML: 32. ZMUC: “Dana” Stn. 5840, 62°44'N, 06°06'W, May 14, 1938, 330 m, © ML: 19.—Stn. 6001, 63°33’N, 11°25’W, July 24, 1938, 322 m, 5 do (3 juv.) ML: 6-13, 2 99 (1 juv.) МЕ: 15, 23.-Stn. 16437, 64°14’N, 57°26'W, July 24, 1974, 760 m, © ML: 54.—Stn. B5-77-36, 65°40'N, 28°20'W, March 21, 1977, 1000-970 m, Q ML: 36.— Haul 25, 63°16'N, 09*30'W, July 6, 1979, 500 m, 2 0 ML: 27, 38. BIOFAR project: Stn. 503, 60°38'N, 08°33’54W, July 25, 1989, 513 m, с’ ML: 18.—Stn. 738, 62°19'N, 10°13’W, October 1, 1990, 749 т, © ML: 25.—Stn. 734, 60°10’06N 07°57'03W, Мау 8, 1990, 634 m, © ML: 31, MALACOLOGIA, 2002, 44(2): 223-239 ULTRASTRUCTURE OF THE SUPPORTING CELLS AND SECRETORY CELLS OF THE ALIMENTARY CANAL OF THE SLUGS, LEHMANNIA MARGINATA AND BOETTGERILLA PALLENS (PULMONATA: STYLOMMATOPHORA: LIMACOIDEA)' Ana Maria Leal-Zanchet Laboratorio de Histologia, Centro de Ciéncias da Saude, Universidade do Vale do Rio dos Sinos, Av. UNISINOS, 950, 93022-000 Sao Leopoldo- RS, Brasil; ipp @ bios. unisinos.br ABSTRACT The supporting cells and the secretory cells of the alimentary canal of two slugs of the super- family Limacoidea, Lehmannia marginata and Boettgerilla pallens, were studied by electronmi- croscopy. From the esophagus to the third intestinal region, and in the rectum, ciliated and/or nonciliated columnar cells occur. In the fourth intestinal region and in the intestinal caecum, the latter present only in the intestine of L. marginata, there are nonciliated squamous cells. The ul- trastructure of the columnar cells of the crop, stomach and first and second intestinal regions in- dicate that these cells are involved in food uptake, digestion and storage of reserve materials. Ultrastructural features of ciliated cells of the typhlosoles and the leader folds of the alimentary canal indicate the involvement of these cells in the production of currents, thus aiding food and faeces transportation. The ultrastructure of the squamous cells of the fourth intestinal regions and caecum point to one of the roles of these organs in the absorption of water and ions from the faecal pellets. Eight secretory cell types in the alimentary canal of L. marginata and six secretory cell types in B. pallens are distinguished by their ultrastructural features, such as diameter and contents of secretion granules, development and contents of rough endoplasmatic reticulum, as well as diameter of their cisternae, and development of the agranular endoplasmatic reticulum. Key words: epithelium, secretion, digestive system, glands cells, intestine, caecum, Limaci- dae, Boettgerillidae. RESUMO As células de revestimento e as células secretoras do tubo digestivo de duas lesmas da su- perfamilia Limacoidea, Lehmannia marginata e Boettgerilla pallens, sao analisadas ultraestru- turalmente. Células cilindricas ciliadas e/ou nao ciliadas sao observadas desde o esöfago até a terceira regiao intestinal bem como no reto. Células pavimentosas nao ciliadas ocorrem na quarta regiao intestinal e no ceco intestinal, este ultimo presente apenas em L. marginata. As caracteristicas ultraestruturais das células cilindricas do papo, do est6mago e da primeira e se- gunda regiôes intestinais indicam que essas células auxiliam na absorcäo, na digestao e no ar- mazenamento de material de reserva. Caracteres ultraestruturais das células ciliadas presentes nas tiflossoles e dobras condutoras do tubo digestivo indicam que essas células atuam na pro- ducäo de correntes, auxiliando no transporte do bolo alimentar e fecal. A ultraestrutura das célu- las pavimentosas da quarta regiao intestinal e do ceco sugerem que uma das funcöes desses Orgaos é a absorcao de agua e ions do bolo fecal. Oito tipos de células secretoras ocorrem no tubo digestivo de L. marginata e seis tipos de células secretoras, em B. pallens, sendo diferen- ciadas por caracteristicas ultraestruturais, tais como diametro e conteudo dos granulos secre- tores, forma, diametro e conteudo das cisternas do reticulo endoplasmatico granular, bem como forma e abundancia relativa das cisternas do reticulo endoplasmatico agranular, dentre outras caracteristicas. "Part of a thesis submitted to the Lehrstuhl Spezielle Zoologie of the Eberhard-Karls Univertitat Tubingen, Germany, in par- tial fulfilment of the requirements for the degree of Doctor of Natural Sciences and supported by a fellowship of the Brazil- ian Research Council (CNPq). 223 224 LEAL-ZANCHET INTRODUCTION Knowledge of the role of the alimentary canal of pulmonates in digestion and absorp- tion is still incipient, this related to the paucity of available information concerning the ultra- structure its organs. Babula & Wielinska (1988) described the crop and intestine of De- roceras reticulatum; Angulo & Moya (1989) studied the intestine of Arion ater; Boer & Kits (1990) examined the whole alimentary canal of Lymnaea stagnalis; and Franchini & Otta- viani (1992) studied intestinal cell types of Planorbarius corneus. In addition, Triebskorn (1989), Triebskorn & Künast (1990), and Triebskorn & Köhler (1992) analysed ultra- structural changes in different cell types of the alimentary canal of D. reticulatum and/or Arion lusitanicus as a background for obser- vations on the effects of molluscicides. Dero- ceras reticulatum and L. stagnalis, among other species of pulmonates, were also the object of anatomical and histological investi- gations of the alimentary canal (reviewed by Runham, 1975, and Luchtel et al., 1997). The present work completes a series of anatomical and histological (Leal-Zanchet, 1998), histochemical (Leal-Zanchet, 1999) and ultrastructural studies of the alimentary canal of limacoid slugs. Leal-Zanchet (1998) analysed comparatively the anatomy and his- tology ofthe alimentary canal of six species of the superfamily Limacoidea (Deroceras laeve, D. rodnae, D. reticulatum, Lehmannia marginata, Malacolimax tenellus, Boettgerilla pallens) and the milacid slug Tandonia bu- dapestensis, and described the following re- gions in this canal: esophagus, crop, stom- ach, first, second, third and fourth intestinal regions and the rectum, besides an intestinal caecum for D. rodnae, D. reticulatum and L. marginata (Leal-Zanchet, 1998). Inthe limacoid slugs cited above, as well as in 7. budapestensis, the epithelium of the ali- mentary canal is composed of supporting cells and secretory cells, the former being cil- iated and/or nonciliated columnar cells from the oesophagus to the third intestinal region, and in the rectum. The supporting cells of the fourth intestinal region and intestinal caecum are nonciliated squamous to cuboidal (Leal- Zanchet, 1998). Eight secretory cell types could be distin- guished in the alimentary canal of L. mar- ginata (Leal-Zanchet, 1998). Two types of mucous cells occur in the proximal and me- dian segments of the alimentary tract, type | (esophagus, crop, stomach and second in- testinal region) and type Il (stomach and first intestinal region). Two types of secretory cells, namely intestinal secreting cells oftypes | and Il, occur in the second and third intesti- nal regions, respectively. Besides four other types of secretory cells, mucous cells of types Ш, IV and V, and cystic cells, occur in the dis- tal regions of the tract (third and fourth intesti- nal regions, intestinal caecum and rectum). In B. pallens, both mucous cells of type V and the cystic cells are absent. Most of the limacoid slugs are herbivorous (Frömming, 1954), but B. pallens seems to be zoophagous (Leal-Zanchet, 1995). Because of the apparently diverse feeding habits, as well as the differing complexity of the alimen- tary canal (length of the alimentary canal, presence/ absence of an intestinal caecum and number of secretory cell types), Lehman- nia marginata (Limacidae) and Boettgerilla pallens (Boettgerillidae) were selected for the present investigation. MATERIAL AND METHODS The animals were collected near the town of Tübingen, Baden-Württemberg, Germany, Key to Lettering on Figures aer: agranular endoplasmic reticulum ar: apical region bb: basal bodies bf: basal feet ci: cilia cr: cilia rootlets cl connective tissue fe fold 9: Golgi bodies gl: glycogen interdigitations li: lipids lu: lumen m: mitochondria mc: muscle cells mi: microvilli n: nucleus ne: nerve ending nu: nucleolus г ribosomes rb: rootlike basal extensions rer: rough endoplasmic reticulum sg: secretory granules sm: secretion mass sv: synaptical vesicles V: vesicles Va: vacuoles vi: vacuoles of type | vil: vacuoles of type Il za: zonula adhaerens 75: zonula septata ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 225 and kept in a cool room at 15°C. They were maintained humid with decanted water in the laboratory for approximately two weeks in petri dishes with a certain amount of natural soil and litter from the collecting site. Lehman- nia marginata was raised on lettuce, carrot and cabbage. Boettgerilla pallens consumed eggs of limacoid slugs and also accepted carrot. Before preparation, the slugs were starved for three days. For previous micro- anatomical, histological and histochemical studies, eight adult specimens of each L. mar- ginata and B. pallens were analysed. For present ultrastructural studies, two adult specimens of each species were examined. They were anaesthetised in 5% menthol for one hour and killed in the fixative solution (2% paraformaldehyde in 0.05 M phosphate buffer and 2% glutaraldehyde, pH 7.2) (Plattnert, 1975), in which they were dissected. The whole alimentary tract was transferred to a fresh amount of fixative solution, and small pieces of the various parts of the alimentary canal were separated, labelled and main- tained for one hour in a new quantity of fixa- tive solution. The material was washed in Sörensen’s phosphate buffer (Ruthmann, 1966), post-fixed in 2% osmium tetroxide in 0.05 M phosphate buffer, washed in 0.025 M phosphate buffer, dehydrated in a graded se- ries of ethanol, treated with propylene oxide and embedded in Epon 812 (Serva). Ultrathin sections (60-90 nm), obtained with a LKB-UI- trotome | ultramicrotome, were stained with Reynolds’ (1963) lead citrate and uranyl ac- etate. The sections were examined in a Siemens Elmiskop 102 transmission electron microscope. RESULTS Columnar Cells Both ciliated (Figs. 1, 12) and nonciliated (Fig. 13) columnar cells of the alimentary tract of L. marginata and B. pallens show microvilli. The apical zone of the cytoplasm (Fig. 18) is devoid of cell organelles, with the exception of vesicles and cisternae of the agranular endo- plasmic reticulum. In the optical microscope, in sections stained by routine histological methods, this zone appears as a clear area (Leal-Zanchet, 1998). In the ciliated columnar cells, the cilia are anchored to the cytoplasm by basal bodies from which cross-striated rootlets extend into the cytoplasm (Table 2). Below the apical zone, there are numerous mitochondria (Figs. 17, 18), as well as small vesicles and multi-vesicular bodies. The mito- chondria present an oval to oblong form. In the rest of the cytoplasm, mitochondria are only sparsely scattered. The supranuclear cy- toplasm of the columnar cells contains nu- merous large and slightly electron-dense fat droplets, the mean diameter of which is 2.2 + 0.4 um in the crop of L. marginata and 3.2 + 0.4 um in the crop of B. pallens, being more plentiful in the crop and stomach of both species (Figs. 14, 15). Glycogenrosettes were often observed in the cytoplasm of the colum- nar cells of B. pallens (Fig. 17). There are also membrane-limited vacuoles in the supranu- clear cytoplasm, which can be categorized into two types. The vacuoles of type | (Figs. 12, 14, 21) show a electron-dense peripheral zone and a lesser electron-dense central core. The mean diameter of the vacuoles is 1.3 + 0.3 and 1.3 + 0.1 umin the crop and 0.7 + 0.2 and 0.9 + 0.2 um in the intestine of L. marginata and B. pallens, respectively. In L. marginata, the peripheral part has mainly het- erogeneous, granular contents (Fig. 14). The relative number of vacuoles of type | de- creases in the second intestinal region of both species. The vacuoles of type Il (1.2 + 0.3 um in diameter) were observed only in the colum- nar cells of the crop, stomach and first and second intestinal regions of B. pallens. They contain granular, slightly electron-dense ma- terial (Fig. 21). In the basal cytoplasm, some mitochondria, Golgi bodies, rough endoplas- mic reticulum, and fat droplets are present. In the optical microscope, in sections stained by routine histological methods, type | vacuoles show cromophobe contents and type II vac- uoles appear acidophillic (Leal-Zanchet, 1998). Neighbouring cells of the alimentary canal are interconnected apically by the zonula ad- haerens and zonula septata (Fig. 18). The zonula adhaerens is located close to the tran- sition of the apical to the lateral membranes of the cells, followed by the zonula septata, which is partly transversed by interdigitations (Fig. 18). In the middle and basal zones of the epithelium, the cells present large intercellular spaces, wherein occur connections uniting the epithelial cells. In the first, second and third intestinal re- gions the height of the columnar cells gradu- ally decreases (Leal-Zanchet, 1998), the mi- crovilli and the cilia rootlets becoming smaller as well (Tables 1, 2). 226 LEAL-ZANCHET HE 3 UU UU, ci rer So = = poe 7 ГИЯ C= o rer FIGS. 1-3. Schematic drawings of a ciliated columnar cell of the first intestinal region (Fig. 1), a ciliated columnar cell of the rectum (Fig. 2) and a squamous cell of the fourth intestinal region (Fig. 3). In the ciliated columnar cells of the ty- phlosoles and of the leader groove of the stomach, the cilia are very numerous and densely arranged (Figs. 15-17). Between two neighbouring cilia only three microvilli are present. The basal bodies of the cilia are in- terconnected by well-developed basal feet, and the rootlets are thick and very long (Table 2). In the apical cytoplasm of these cells, the mitochondria are more numerous than in the ciliated columnar cells of other regions, being situated close to the cilia rootlets (Fig. 17). In the leader folds of the rectum of B. pal- lens, as well as in the ciliated columnar cells of the rectum of L. marginata, the microvilli and the cilia rootlets are long (Tables 1, 2). The apical cytoplasm consists of vesicles, channels of the agranular endoplasmic reticu- lum (Fig. 19) and ribosomes. In the leader folds, the mitochondria zone occupies most of the supranuclear cytoplasm, and the mito- chondria are arranged parallel to the cilia rootlets. In the rectum of Lehmannia mar- ginata, where specialized leader folds are ab- sent, the mitochondria of the ciliated cells do not show such an arrangement, being less abundant and scattered in the supranuclear cytoplasm (Fig. 2). Numerous poliribosomes and some channels of the agranular endo- plasmic reticulum are found between the mi- tochondria. Poliribosomes and mitochondria in a scattered distribution occur in the basal cytoplasm. Some vesicles and Golgi bodies are found in the zone of the nucleus. ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA PPT FIGS. 4-7. Schematic drawings of mucous cells of types | (Fig. 4) and Il (Fig. 5) and intestinal secreting cells of types | (Fig. 6) and II (Fig. 7). Squamous to Cuboidal Cells the supranuclear zone is narrow. Mitochon- dria are sparsely scattered in the cytoplasm. In the squamous, nonciliated cells of the The apical and middle cell zones show chan- fourth intestinal region and the intestinal cae- nels of the agranular endoplasmic reticulum cum, the microvilli are short (Fig. 3, Table 1). as well as vesicles. Golgi bodies are found in An apical mitochondria zone is absent, and the middle zone of the cells. In infranuclear 228 LEAL-ZANCHET 10 hee JR / FIGS. 8-11. Schematic drawings of the cell body of mucous cells of type III (Fig. 8), type IV (Fig. 9), type V (Fig. 10) and the cystic cell (Fig. 11). TABLE 1. Length of microvilli in the alimentary canal of Lehmannia mar- ginata and Boettgerilla pallens (in um).-: absent;--: not measured. L. marginata Crop 1.6+0.2 Stomach (typhlosoles and canal) HO O Stomach (other areas) SEO First intestinal region 0.7 + 0.1 Second intestinal region O17 0:1 Third intestinal region 0.4 + 0.1 Fourth intestinal region 0.523031 Intestinal caecum 0.5 + 0.06 Rectum 0.7 = 0:07 В. pallens 37-05 SEO 107208 1.028102 0.9 + 0.2 0.6 + 0.08 0.4 + 0.1 ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 229 TABLE 2. Length of cilia rootlets in the alimentary canal of Lehmannia marginata and Boettgerilla pallens (in шт).-: absent;--: not measured. Oesophagus Crop Stomach (typhlosoles and canal) Stomach (other areas) First intestinal region Second intestinal region Third intestinal region Rectum cytoplasm, there are small vacuoles of type | (0.5 # 0.1 um in diameter) and ribosomes. The granular endoplasmic reticulum is poorly developed. The basal membrane shows nu- merous folds (107 + 27 nm wide), which form rootlike basal extensions (Fig. 22), containing mitochondria, ribosomes and vesicles with poor electron-dense contents (83 + 16.7 nm in diameter). These epithelial basal exten- sions come close to folds of the muscle cells. The muscle folds are wider (0.25 + 0.06 um) than the epithelial basal extensions and show numerous vesicles (83 + 16.7 nm) with elec- tron-dense contents. The connective tissue between the folds present collagen fibres, which are arranged parallel to the epithelial folds. Secretory Cells The mucous cells of type |, the mucous cells of type Il, the intestinal secreting cells of type I, and the intestinal secreting cells of type Il are intraepithelial. The mucous cells of type Ш, the mucous cells of type IV, the mucous cells of type V, and the cystic cells show a subepithelially located cell body and a cell neck that extends to the surface of the epithe- lium. Cell extensions emerge from the basal part of the intraepithelial secretory cells, which enter into the connective tissue. Nerve endings, which contain electron-dense as well as electron-lucent synaptical vesicles, come close to these cell extensions (Fig. 23). By the subepithelial secretory cells, nerve endings make contact with the cell body or with the subepithelial part of the cell neck (Fig. 30). In the resting phase, the secretory cells show microvilli. In the process of secretion, the api- cal cytoplasm forms a camber and the mi- crovilli are reabsorbed (Figs. 25, 28). L. marginata B. pallens 0.9 + 0.2 = 8.7 = 1:3 6.0 + 1.0 2.9 + 0.4 2.0+0.5 1.6 = 0:3 1.7 = 0.4 0.7 = 0:1 5 0:3 0.4 + 0.1 -- 0/9 = 0.3 0:6 = 0.1 Mucous Cells of Type | The mucous cells of type | (Fig. 4) have a widened basal part that contains the nucleus and most of the cell organelles (Golgi bodies, granular endoplasmic reticulum and mito- chondria) (Fig. 24), as well as some secretion granules. The middle and apical cell zones are performed with secretion granules. Vesi- cles containing electron-dense material occur between the granules and at the margins of the cell. Multi-vesicular bodies and numerous ribosomes are found throughout the cyto- plasm. Golgi zones are well developed. In L. marginata, the Golgi lamellae show a width of 40 + 10 nm. Vesicles and recently formed se- cretion granules are often associated with the Golgi bodies. In B. pallens, the Golgi bodies usually consist of minimally widened lamellae (40 + 10 nm) with electron-dense contents. The rough endoplasmic reticulum (RER) shows distended (0.4 + 0.17 um), branched cisternae that contain tubuli (24 + 4 nm). Usu- ally mitochondria occur close to the cisternae (Fig. 24). The secretion granules of L. mar- ginata (2.1 + 0.3 um in diameter) are slightly electron-dense, with dense, tube-like scat- tered structures, containing at the margins a concentration of strong electron-dense mate- rial (Fig. 25). In B. pallens, the secretion gran- ules (1.7 + 0.2 um in diameter) are electron- lucent contents with speckles of granular, moderately electron-dense material. The granules usually form large secretion masses that are membrane-limited (Fig. 26). Mucous Cells of Type Il The mucous cells of type II (Fig. 5) present an elongated basal part, which contains very numerous RER cisternae as well as numer- 230 LEAL-ZANCHET FIG. 12. Ciliated columnar cell of the oesophagus of Lehmannia marginata. Note the numerous vacuoles of type | in supranuclear cytoplasm. Scale bar, 3 um. FIG. 13. Columnar cell of the crop of Lehmannia mar- ginata showing long microvilli. Scale bar, 1 um. FIG. 14. Supranuclear zone of the cytoplasm of a columnar cell of the crop of Boettgerilla pallens. Scale bar, 1 um. FIG. 15. Apical zone of the cytoplasm of a ciliated columnar cell of the typhlosole of the stomach of Lehmannia marginata. The long cilia rootlets are visible. Scale bar, 2 um. FIG. 16. Apical zone of the cytoplasm of a ciliated columnar cell of the typhlosole of the stomach of Lehmannia marginata. Note the basal bodies interconnected by the well-developed basal feet. Scale bar, 0.3 um. FIG. 17. Apical zone of the cytoplasm of a ciliated columnar cell of the typhlosole of the stomach of Boettgerilla pallens. Scale bar, 0.7 um. FIG. 18. Apical zone of the cytoplasm of a ciliated colum- nar cell out of the typhlosoles of the stomach of Lehmannia marginata. Scale bar, 1.3 um. ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 231 FIG. 19. Apical cytoplasm of a cuboidal cell of the leader fold of the rectum of Boettgerilla pallens. The arrow shows channels of the agranular endoplasmic reticulum. Scale bar, 0.3 um. FIG. 20. Nonciliated cuboidal cell of the fourth intestinal region of Lehmannia marginata. Scale bar, 3 um. FIG. 21. Supranuclear cytoplasm of a columnar cell and part of a mucous cell of type Il of the first intestinal region of Boettgerilla pallens. Scale bar, 1 um. FIG. 22. Rootlike basal extensions of the basal part of a squamous cell of the intestinal caecum of Lehmannia marginata and corresponding folds of the muscle cells. Scale bar, 1 um. FIG. 23. Basal cyto- plasm of a mucous cell of type Il of the stomach of Lehmannia marginata. The arrow shows tubuli of the rough endoplasmic reticulum. Nerve endings come close to the cell. Scale bar, 0.6 um. 232 LEAL-ZANCHET FIG. 24. Basal zone of the cytoplasm of a mucous cell of type | of the oesophagus of Lehmannia marginata. Scale bar, 1 ит. FIG. 25. Apocrine secretion of a mucous cell of type | of the oesophagus of Lehmannia mar- ginata. Scale bar, 1.3 um. FIG. 26. Supranuclear cytoplasm of a mucous cell of type | of the crop of Boettge- rilla pallens. Scale bar, 2 um. FIG. 27. Supranuclear cytoplasm of a mucous cell of type II of the typhlosole of the stomach of Lehmannia marginata. Scale bar, 1 um. FIG. 28. Apical part of a mucous cell of type II of the stomach of Boettgerilla pallens. The apical cytoplasm forms a camber and the microvilli are reabsorbed (arrow). Scale bar, 0.5 um. ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 233 ous Golgi bodies and some mitochondria. The RER cisternae are dilated (0.4 + 0.1 um) and also contain tubuli (28 + 4 nm in diameter). In the basal cytoplasm occur large vacuoles (1.5 + 0.1 um in diameter) containing finely gran- ular, electron-dense material (Fig. 27). Occa- sionally some secretion granules are present in the infranuclear cytoplasm. In the supranu- clear cytoplasm, however, the granules are very numerous. In L. marginata, the granules are electron-lucent with clumps of osmiophillic material (Fig. 27). In B. pallens, the granules are electron-lucent with flocculent contents (Fig. 28). In both species, the granules are very small (0.9 + 0.2 and 0.8 + 0.2 um in L. marginata and B. pallens, respectively). Vesi- cles and channels of the agranular endoplas- mic reticulum as well as ribosomes are pres- FIG. 29. Cell body of a mucous cell of type III of the fourth intestinal region of Boettgerilla pallens. Scale bar, 2.2 ит. FIG. 30. Subepithelial part of a cell neck of a mucous cell of type III of the fourth intestinal region of Lehmannia marginata. Nerve endings make contact with the cell neck. Scale bar, 0.7 um. FIG. 31. Cell body of a mucous cell of type IV of the rectum of Lehmannia marginata. Scale bar, 2 um. FIG. 32. Detail of the cy- toplasm of the cell body of a mucous cell of type IV of the rectum of Boettgerilla pallens. Scale bar, 0.2 ит. FIG. 33. Detail of the cytoplasm of the cell body of a mucous cell of type V of the third intestinal region of Lehmannia marginata. Scale bar, 2 um. FIG. 34. Detail of the cytoplasm of the cell body of a cystic cell of the third intestinal region of Lehmannia marginata. Scale bar, 2.2 um. 234 LEAL-ZANCHET ent between the granules. Mitochondria occur in the marginal cytoplasm. Mucous Cells of Type Ill The cell body of the mucous cells of type Ill (Figs. 8, 29) is occupied by secretion granules (1.2 + 0.1 um in diameter in L. marginata and 1.8 + 0.4 um in diameter in B. pallens). These granules often fuse to form larger granules. In L. marginata as well as in B. pallens, their contents show different electron densities, slightly to moderately electron-dense (Fig. 29). The granules are so closely located that seldom are other organelles present. The RER cisternae occur in the marginal cyto- plasm and close to the nucleus. The cisternae are distended (0.5 + 0.2 in L. marginata or 0.25 + 0.1 um in diameter in B. pallens) and contain tubuli (Fig. 30). Golgi bodies and mi- tochondria are present at the cell margins. Very numerous RER cisternae and some mi- tochondria and channels of the agranular en- doplasmic reticulum occur in the proximal part of the cell neck (Fig. 30). In the apical part of the cell, the secretory granules form a large, electron-lucent secretory mass. Mucous Cells of Type IV The secretion granules are smaller (Figs. 9, 31) than the granules of the mucous cells of type III (0.6 + 0.1 um in diameter in L. таг- ginata and 1.3 + 0.3 um in diameter in B. pal- lens). In both species, the secretion granules show homogenous, electron-dense contents (Fig. 31). Usually the granules do not form se- cretion masses. Numerous RER cisternae, which contain finely granular, electron-dense material, occur in the cytoplasm between the granules (Fig. 32). The cisternae are usually thin (51 + 14 пт L. marginata and 115 + 54 nm in B. pallens), but in L. marginata some cisternae reach 0.17 + 0.1 um in diameter. Golgi bodies are present at the cell margins and close to the nucleus. The apical part of the cell neck shows electron-dense secretion granules that fuse distal to discharging in the lumen. Vesicles and polyribosomes occur in the apical cytoplasm. Mucous Cells of Type V This cell type does not occur in the alimen- tary tract of B. pallens. A secretion mass, formed by the coalescence of the secretion granules, often occupies most of the cell, so that the organelles (polyribosomes, mitochon- dria and RER cisternae) and the nucleus are found in the basal part of the cell body (Fig. 10). The secretion mass is electron-lucent and flocculent (Fig. 33). Channels and vesi- cles of the agranular endoplasmic reticulum are numerous in the peripheral cytoplasm. The RER cisternae are thin (75 + 20 nm in di- ameter) and contain electron-lucent material. Golgi complexes are well developed (Fig. 33). Occasionally, recently formed secretion gran- ules are associated with the Golgi bodies. Cystic Cells This cell type is absent in the alimentary tract of B. pallens. The cell body of the cystic cell is occupied by a granular, electron-dense secretion mass surrounded by a membrane (Figs. 11, 34). Small vacuoles showing a sim- ilar content as well as vacuoles containing tubular structures are observed in basal and marginal cytoplasm (Figs. 34, 35). Granules (0.5 + 0.4 um in diameter) with a homoge- neous, stronger electron-dense material also occur in the cytoplasm (Fig. 35). The RER is often highly developed and shows thin cister- nae (113 + 82 nm). Polyribosomes and chan- nels of the agranular endoplasmic reticulum are abundant (Fig. 35). Some channels are connected to the outer cell membrane. Intestinal Secreting Cells of Type | The cytoplasm is occupied by the highly de- veloped RER and abundant free ribosomes (Figs. 6, 38). Some RER cisternae are thin (33 + 5 nm) and show a platelike structure. Most of the RER cisternae are, however, di- lated (0.22 + 0.02 um in diameter) and occur scattered throughout the cytoplasm. They contain a finely granular, slightly electron- dense material. Golgi bodies occur in the in- franuclear cytoplasm as well as around the nucleus (Fig. 36). Their lamellae (0.1 + 0.05 nm) have an electron-dense material. The cy- toplasm also presents channels of the agran- ular endoplasmic reticulum, small vesicles and vacuoles with heterogeneous electron- dense contents (Fig. 38). The round secretion granules (2.3 + 0.2 um in diameter in L. mar- ginata and 1.5 + 0.15 um in diameter in B. pallens) are usually present in the supranu- clear cytoplasm. Their content is homoge- ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 235 FIG. 35. Detail of the cell body of a cystic cell of the third intestinal region of Lehmannia marginata. The arrow shows a channel of the agranular endoplasmic reticulum. Arrowheads indicate tubular structures in a vac- uole. Scale bar, 0.3 um. FIG. 36. Detail of the cytoplasm of an intestinal secreting cell of type | of the second intestinal region of Boettgerilla pallens. Scale bar, 0.7 um. FIG. 37. Apical cytoplasm of an intestinal secret- ing cell of type | of the second intestinal region of Lehmannia marginata. Scale bar, 0.6 um. FIG. 38. Supranu- clear cytoplasm of an intestinal secreting cell of type | of the second intestinal region of Boettgerilla pallens. Scale bar, 1.0 um. FIG. 39. Supranuclear cytoplasm of an intestinal secreting cell of type Il of the third in- testinal region of Boettgerilla pallens. Scale bar, 1.0 um. 236 LEAL-ZANCHET neous electron-dense (Figs. 36, 37). Numer- ous mitochondria occur in the apical cyto- plasm. Intestinal Secreting Cells of Type Il The intestinal secreting cells of type II (Fig. 7) also possess an extensive RER and abun- dant polyribosomes (Fig. 39). The RER cister- nae are thinner (119 + 37 nm) compared to those of the intestinal secreting cells of type I. They contain finely granular, electron-dense material. Golgi bodies are found around the nucleus. They show dilated lamellae (67 + 29 nm in diameter) with electron-transparent ma- terial (Fig. 39). Vesicles that contain electron- dense material are often associated with the Golgi bodies. The secretion granules that show a distinctive limiting membrane are larger than those of the intestinal secreting cells of type | (3.8 + 1.0 um in diameter in L. marginata and 2.5 + 0.5 um in diameter in B. pallens) and may form secretion masses. They contain a flocculent, electron-transpar- ent material as well as membrane-like struc- tures (Fig. 39). Mitochondria and polyribo- somes are often present in the apical cytoplasm. Channels of the agranular endo- plasmic reticulum and vesicles may also be present. DISCUSSION According to Boer & Kits (1990) the colum- nar cells of the alimentary tract of Lymnaea Stagnalis are involved in the absorption, di- gestion and storage of reserve material. Mi- crovilli were demonstrated on the surface of the columnar and squamous cells of the ali- mentary canal of L. marginata and B. pallens. However, the absorption of food particles should occur mainly through the columnar cells of the crop, the stomach, and the first and second intestinal regions, where the mi- crovilli are long, and numerous mitochondria are present in the apical cell zones (Pacheco & Scorza, 1971). This would be consistent with the presence of alkaline phosphatase in the epithelial cells of the crop and intestine of Deroceras reticulatum (Babula & Skowron- ska-Wendland, 1988) and with the findings of Walker (1972), who showed in D. reticulatum an uptake of glucose, glycine and palmitic acid by the epithelial cells of the crop and in- testine. A type of vacuole with electron-dense con- tents (vacuoles of type 1) occurs in the cyto- plasm of the columnar cells of the crop, stom- ach and first and second intestinal regions of B. pallens and L. marginata. Bowen (1970) and Babula & Wielinska (1988) also observed vacuoles with similar contents by ultrastruc- tural studies of epithelial cells of the crop and intestine of Arion ater and D. reticulatum, re- spectively. According to Bowen (1970) and Angulo et al. (1986), such vacuoles are liso- somes, presenting a positive reaction to acid phosphatase. The unavailability of histochem- ical studies of enzymes in the slugs B. pallens and L. marginata make it impossible to point out the functions of type | and type II vacuoles. The storage of reserve material, such as glycogen and lipids, was observed in the columnar cells of B. pallens and L. marginata. Evidence of the storage of lipids was ob- served mainly in the crop, stomach and first intestinal region. The storage of glycogen was demonstrated by ultrastructural studies, as well as histochemically (Leal-Zanchet, 1999). Some columnar cells of the esophagus, crop, stomach, and intestine of D. reticulatum were designated by Triebskorn (1989) as storage cells, because of their high lipid and glycogen content. Ciliated cells occur along the entire alimen- tary tract of B. pallens and L. marginata, ex- cept for the crop of both species, the fourth in- testinal region of B. pallens and the intestinal caecum of L. marginata (Leal-Zanchet, 1998). In some regions of the stomach and rectum, such as the typhlosoles and the transversal fold present in the stomach of limacoid slugs and the leader folds of the rectum of B. pal- lens, we observed that the ciliated cells show distinctive features, namely the cilia are longer, very numerous and densely arranged (Leal-Zanchet, 1998). Furthermore, they pre- sent very long cilia rootlets, being intercon- nected by well-developed basal feet on the basal bodies. According to Zylstra (1972), such a contact between the basal bodies would be involved in the coordination of ciliary movements, and this coordination would be crucial in regions involved in the production of currents, such as the leader system of the stomach. As a matter of fact, Walker (1972) studied the ciliary tracts of the stomach of D. reticulatum and found three main ciliary path- ways: a sorting mechanism in the folds sur- rounding the openings of the digestive glands; extrusion pathways for material that becomes bound up into faecal pellets; and a circulation pathway particularly for fine material. After ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 237 Walker (1972), the cilia on the typhlosoles and accessory fold (= transversal fold) beat mainly towards the gastric channels and intestinal groove, and cilia in the basis of the gastric channels (channels between each tiphlosole and the transversal fold) and anterior intesti- nal groove (channel between the tiphlosoles) transport material posteriorly, whereas cilia of the more posterior region of the intestinal groove transport material anteriorly. Where these two opposite directional movements meet, faecal pellets are formed (Walker, 1972). Physiological studies are not available for the rectal leader folds of limacoid slugs, but their similar ultrastructural morphology may indicate that such folds could be better suited to aid faeces transportation than are the usual folds (Leal-Zanchet, 1998). The squamous cells of the fourth intestinal region, the intestinal caecum, and the rectum show microvilli on the apical surface, apart from numerous folds of the basal membrane, which form rootlike basal prolongations be- sides well-developed interconnections be- tween neighbouring cells. These features are characteristic of water- and ion-transporting epithelia and would be consistent with the findings of Deypup-Olsen & Martin (1987), who verified that the distal part of the intestine of Ariolimax columbianus plays a significant role in osmoregulation. As suggested by Boer & Kits (1990) for Lymnaea stagnalis, this would indicate that water and ions are ab- sorbed from the faecal pellets. In limacoid slugs that have an intestinal caecum, espe- cially in L. marginata in which this caecum is long, this region could provide an additional site for water and ions absorption. The occurrence of five mucous cell types was detected in the alimentary canal of L. marginata, as well as in such other limacoid slugs as Malacolimax tenellus, Deroceras laeve, D. reticulatum, and D. rodnae (Leal- Zanchet, 1998). By optical microscopical analysis, these cell types were distinguished by their distribution along the alimentary tract, their position related to the epithelium (intra- or subepithelial location), the mean diameter of their secretory granules, the morphology of the basal part or cell body of the cells, and their appearance after histochemical reac- tions for protein and mucopolyssaccharides (Leal-Zanchet, 1998, 1999). Thus, type | and II mucous cells were distinguished on the basis of the mean diameter of the secretory granules and the morphology of the basal part of the cells, characteristics observed by opti- cal microscopy and confirmed here. Besides, type II secretory cells show a restricted distri- bution in the stomach and first intestinal re- gion, whereas type | are present in the esoph- agus, crop and second intestinal region. Both types secret acid mucopolyssaccharides (Leal-Zanchet, 1999), but their morphological distinctive features and distribution in the ali- mentary canal indicate that a more accurate histochemical analysis could confirm them as different types. Types Ш, IV and V mucous cells, restricted to the distal regions of the ali- mentary tract (third and fourth intestinal re- gions, intestinal caecum and rectum) were distinguished by optical microscopical studies due to the different mean diameter of their granules and their reaction to trichromical stains. Previous histochemical studies showed that the three types are involved in the secretion of neutral mucopolysaccha- rides, but type Ill is also involved in the secre- tion of acid mucopolysaccharides. Besides, in B. pallens, where type V mucous cells and cystic cells are absent, type IV mucous cells could be histochemically distinguished be- cause they show positive reaction for protein and neutral mucopolysaccharides (Leal- Zanchet, 1999). The present investigation shows that the granules of types III and IV, in both species, have a different electron-den- sity and that their rough endoplasmatic reticu- lum has a different morphology and distribu- tion in the cytoplasm. Type V mucous cell is distinguishable by optical studies through the elongate morphology of the secretory gran- ules. The present ultrastructural studies show that they form a eletron-lucent secretion mass occupying most of the cell. The mucous cells of type |, Il and Ill show tubuli in the cisternae of the rough endoplas- mic reticulum. The presence of tubuli in the rough endoplasmic reticulum has been de- scribed by Wondrak (1967), Moya & Rallo (1975), Kessel & Beams (1984) and Angulo & Moya (1989). According to Kessel & Beams (1984), tubular structures within the cisternae of the endoplasmatic reticulum appear to be a feature of more than one cell type. Further- more, according to these authors, their preva- lence within secretory cells suggests that these tubular structures could be secretory products of the cells. The secretion of the cystic cells and of the intestinal secreting cells of type | and Il gave positive results for protein (Leal-Zanchet, 1999). This would be consistent with the pres- ence of a highly developed rough endoplas- 238 LEAL-ZANCHET mic reticulum, observed in the present inves- tigation. The secreting cells of type |, occur- ring in the second intestinal region, where the columnar cells show ultrastructural features that indicate involvement in the absorption of food particles, imply that the secretion of this cell type is probably of an enzymatic nature and may play a role in digestion (Leal- Zanchet, 1998). However, additional histo- chemical studies, including enzyme histo- chemistry, are needed to clarify the role of these secretory cells. Considering that data obtained in labora- tory experiments assume that B. pallens is carnivorous, whereas Lehmannia marginata is herbivorous, this living on a specialised diet of lichens (Wiktor, 1973), it could be expected to find more distinguishing morphological fea- tures in the alimentary canal of the two species. However, B. pallens shows a mere shortening of the intestinal regions (Leal- Zanchet, 1998). ACKNOWLEDGEMENTS Dr. Wolfgang Rähle supervised the doctoral thesis. Prof. Dr. Wolfgang Maier is thanked for accommodation in his department. Prof. Dr. Christian F. Bardele gave his permission to use the Laboratory of Electronmicroscopy. Ms. Siegrid Schultheiss is thanked for the EM technical assistance. Mr. Fernando Carbayo is thanked for the preparation of the drawings. Mr. Edward Benya and Mr. Ramon A. Clark corrected the English version of the manu- script. Thanks are also due to the anonymous referees for the valuable suggestions. LITERATURE CITED ANGULO, E. & J. MOYA, 1989, Ultrastructural as- pects of the intestinal epithelium of Arion ater (L.) (Gastropoda: Pulmonata). Неа, 1(5/6): 161-164, pls. 22-24. ANGULO, E., J. MOYA & I. LA VEGA, 1986, Histo- chemical observations on the intestinal epi- thelium of the slug Arion ater (Linnaeus, 1758) (Mollusca, Pulmonata, Stylommatophora). Cua- dernos de Investigation Biologica, 9: 59-65. BABULA, A. & L. WIELINSKA, 1988, Ultrastructural studies of the digestive system of the slug Dero- ceras reticulatus (Müller) (Pulmonata). Bulletin de la Societe des Amis des Sciences et des Let- tres de Poznan, (D)26: 73-78. BABULA, A. & D. SKOWRONSKA-WENDLAND, 1988, Histological and histochemical studies of the digestive system of the slug Deroceras retic- ulatus (Müller) (Pulmonata). Bulletin de la So- ciete des Amis des Sciences et des Lettres de Poznan, (D)26: 65-71. BOER,H.H. & К. $. KITS, 1990, Histochemical and ultrastructural study of the alimentary system of the freshwater snail Lymnaea stagnalis. Journal of Morphology, 205: 97-111. BOWEN, I. D., 1970, The fine structural localisation of acid phosphatase in the slug, Arion ater (L.). Protoplasma, 70: 247-260. DEYRUP-OLSEN, |. & W. MARTIN, 1987, Os- molyte prosessing in the gut and an important role of the rectum in the land slug, Ariolimax columbianus (Pulmonata; Arionidae). Journal of Experimental Zoology, 243: 33-38. FRANCHINI, A. & E. OTTAVIANI, 1992, Intestinal cell types in the freshwater snail Planorbarius corneus: histochemical, immunocytochemical and ultrastructural observations. Tissue and Cell, 24(3): 387-396. FROMMING, E., 1954, Biologie der mitteleuropäi- schen Landgastropoden. Berlin: Duncker & Hum- blot. 404 pp. KESSEL, R.G. & H.W. BEAMS, 1984, Intracisternal tubules and intramitochondrial filaments in cells of a snail, Limnaea. Tissue and Cell, 16(3): 405-410. LEAL-ZANCHET, A. M. 1995. Vergleichende Anato- mie, Histologie, Ultrastruktur des Verdauungssys- tems limacoider und milacoider Nacktschnecken (Pulmonata: Stylommatophora). Unpublished D. Nat. Sci. Thesis, Tübingen University. 134 pp. LEAL-ZANCHET, A. M., 1998, Comparative studies on the anatomy and histology of the alimentary canal of the Limacoidea and Milacidae (Pul- monata: Stylommatophora). Malacologia, 39 (1-2): 39-57. LEAL-ZANCHET, А. M., 1999, Histochemical study of the gland cells of the alimentary canal of Lima- coidea (Gastropoda, Pulmonata). Brazilian Jour- nal of Morphological Sciences 16(1): 113-118. LUCHTEL, D., A. W. MARTIN, I. DEYRUP-OLSEN & H. H. BOER, 1997, Gastropoda: Pulmonata. Pp. 545-591, in F. W. HARRISON & A. J. KOHN, eds., Microscopic anatomy of invertebrates. Vol. 6B: Mollusca Il, New York: John Wiley & Sons. 828 pp. MOYA, J. & A. M. RALLO, 1975, Intracisternal poly- cylinders: a cytoplasmatic structure in cells of the terrestrial slug, Arion empiricorum Ferussac (Pul- monata, Stylommatophora). Cell and Tissue Re- search, 159: 423-433. PACHECO, J. & J. V. SCORZA, 1971, Estudio al microscopio electronico del epitelio anterior de Pomacea urceus (Mollusca, Gastropoda). Acta Biologica Venezuelana, 7, 4: 399-420. PLATTNERT, N., 1975, Die chemische Fixierung biologischer Objekte für die Eletronenmikro- skopie. Pp. 1-47, in G. SCHIMMEL & W. VOGELL, eds., Methodensammlung der Elektronenmikro- skopie. Stuttgart: Wissenschaftliche Verlagsge- sellschaft mbH. 307 pp. ULTRASTRUCTURE OF ALIMENTARY CANAL OF LIMACOIDEA 239 REYNOLDS, E. S., 1963, The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Journal of Cell Biology, 17: 208. ВУМНАМ, N. W. 1975, Alimentary canal. Pp. 54- 104, in V. FRETTER & J. PEAKE, eds., Pulmonates. V. 1: Functional anatomy and physiology. Lon- don: Academic Press. 417 pp. RUTHMAN, A., 1966, Methoden der Zellforschung. Franckh'sche Verlagshandlung, Stuttgart: W. Keller & Co. 301 pp. TRIEBSKORN, R., 1989, Ultrastructural changes in the digestive system of Deroceras reticulatum (Müller) induced by a carbamate molluscicide and by metaldehyde. Malacologia, 31(1): 141- 156. TRIEBSKORN, R. & C. KÜNAST, 1990, Ultrastruc- tural changes in the digestive system of Dero- ceras reticulatum (Mollusca Gastropoda) in- duced by lethal and sublethal concentrations of the carbamate molluscicide Cloethocarb. Mala- cologia, 32: 89-106. A TRIEBSKORN, R. 8 H.-R. KOHLER, 1992, Plastic- ity of the endoplasmic reticulum in three cell types of slugs poisoned by molluscicides. Proto- plasma, 169: 120-129. WALKER, G., 1972, The digestive system of the slug, Agriolimax reticulatus (Múller). Proceedings of the Malacological Society of London, 40: 33-43. WIKTOR, A. 1973, Die Nacktschnecken Polens. Ar- ionidae, Milacidae, Limacidae (Gastropoda, Sty- lommatophora). Kraköv: Monografie Fauny Pol- ski, 182 + 97 pp. WONDRAK, G., 1967, Die exoepithelialen Schleim- drúsenzellen von Arion empiricorum (Fér.). Zeitschrift für Zellforschung und Mikroskopische Anatomie, 76: 287-294. ZYLSTRA, U., 1972, Histochemistry and ultrastruc- ture ofthe epidermis and the subepidermal gland cells of the freshwater snails Lymnaea stagnalis and Biomphalaria pfeifferi. Zeitschrift für Zell- forschung und Mikroskopische Anatomie, 130: 93-134. Revised ms. accepted 6 September 2001 MALACOLOGIA, 2002, 44(2): 241-257 SEXUAL MATURATION, SPAWNING, AND DEPOSITION OF THE EGG CAPSULES OF THE FEMALE PURPLE SHELL, RAPANA VENOSA (GASTROPODA: MURICIDAE) Ee-Yung Chung’, Sung-Yeon Kim?, Kwan Ha Park! & Gab-Man Park? ABSTRACT Germ cell development, sexual maturation, and fecundity of female Rapana venosa (Valenci- ennes) have been examined by light and electron microscope observations. The Golgi appara- tus, vacuoles and mitochondria were involved in the formation of glycogen particles, lipid droplets and yolk granules in early vitellogenic oocytes. The modified mitochondria and endoplasmic reticulum in late vitellogenic oocytes were involved in the formation of proteid yolk granules near the cortical layer. A mature yolk granule was composed of three components: main body (central core), superficial layer, and limiting membrane. Monthly changes in the gonadosomatic index were closely associated with ovarian developmental phases. Spawning occurred between May and early August, when the seawater temperature rose to 18-26°C. The female reproductive cycle of this species was divided into five successive stages: early active stage (September to February), late active stage (October to April), ripe stage (March to July), partially spawned stage (May to August), and recovery stage (June to September). The rate of individuals reaching the first sexual maturity was 51.6% in females of 7.1-8.0 cm in shell height, and 100% in those > 10.1 cm. The total number of egg capsules per individual and the mean number of eggs in an egg capsule were 184 to 410 and 976, respectively. Fecundity ranged approximately from 179,000 to 400,000 eggs/individual with two to four broods (spawning frequencies) during the spawning season. The duration of development in egg capsules was 15-17 days at about 20°C. Rapana venosa is a species with embryos that hatch as veliger larvae. The sex ratio of female: male was not significantly different from 1:1. Key word: Rapana venosa, gametogenesis, reproductive cycle, first sexual maturity, fecundity, egg capsule, sex ratio. INTRODUCTION The purple shell, Rapana venosa (Gas- tropoda: Muricidae), one of the most impor- tant edible gastropods in Asia (Yoo, 1976; Kwon et al., 1993), is abundant along the coasts of Korea, China and Japan, especially, in silty sand of the intertidal and subtidal zones. However, the standing stock of this species has gradually been decreasing due to extensive loss of habitats from reclamation projects and reckless over-harvesting. Thus, R. venosa has been identified as a target or- ganism that should be carefully managed. Prior studies have considered various as- pects of the biology of R. venosa, including classification (Kuroda & Habe, 1952; Habe, 1969; Tsi et al., 1983; Wu, 1988; Choe, 1986, 1992; Higo et al., 1999), morphology (Lee & Kim, 1988), biochemical aspects (Yoon, 1986; Yoo et al., 1991; Hwang et al., 1991), distribu- tion (Gomoiu, 1972; Zolotarev, 1996), habitat and age estimation (Chukhchin, 1984; Hard- ing & Mann, 1999), reproductive cycle (Chung et al., 1993; Chung & Kim, 1997), and spawn- ing and egg capsule (Hirase, 1928; Habe, 1960; Amio, 1963; Smagowicz, 1989; Harding & Mann, 1999). However, there is still uncer- tainty in many aspects of reproductive biology of the purple shell. For example, little informa- tion is available on ultrastructure of germ cell differentiation during oogenesis, the repro- ductive cycle, first sexual maturity and fecun- dity. Understanding the reproductive cycle and the spawning period of R. venosa will pro- vide necessary informations needed for the determination of age and recruitment period of natural populations. Additional information on first sexual maturity, fecundity associated with egg capsules, sex ratio and reproductive strategy are needed to facilitate effective management of this important natural re- "Faculty of Marine Life Science, Kunsan National University, Kunsan 573-701, Korea; eychung@kunsan.ac.kr National Fisheries Research and Development Institute, Pusan 619-902, Korea “Department of Parasitology, Kwandong University College of Medicine, Kanghung 210-710, Korea 241 242 CHUNG ET AL. source. Therefore, the main aim of the pres- ent study was to describe vitellogenesis dur- ing oogenesis, the reproductive cycle and spawning period, first sexual maturity, fecun- dity, and sex ratio of R. venosa inhabiting the west coast of Korea. MATERIALS AND METHODS Sampling Specimens of the purple shell, R. venosa (Valenciennes 1846), were collected monthly by subtidal dredging near Biung-do, Chol- labuk-do Korea, for one year from June 1994 to July 1995 (Fig. 1). Purple shells ranging from 3.1 cm to 16.9 cm in shell height were used for the present study. After the purple shells were transported alive to the laboratory, shell heights and total body weights were im- mediately measured. Gonadosomatic Index Monthly changes in the mean gonadoso- matic index (GSI) (Fig. 2) were calculated by the following equation (Chung et al., 1993): Br en KOREA D 9 >> м МИ >. SS er ö Gogunsangun-Do °V > 126° 15' 126° 30' FIG. 1. Map showing the sampling area. Biung-Do > Sampling area GSI = Thickness of gonad x 100 Diameter of posterior appendage including gonad and digestive gland Light and Electron Microscope Observations For light microscopic examination of histo- logical preparations, gonad tissues were re- moved from shells and preserved in Bouin’s fixative for 24 h, and then washed with run- ning tap water for 24 h. Tissues were then de- hydrated in alcohol and embedded in paraffin molds. Embedded tissues were sectioned at 5 — 7 um thickness using a rotary microtome. Sections were mounted on glass slides and stained with Hansen’s hematoxylin-0.5% eosin, Mallory’s triple stain and PAS stain, and examined using a light microscope. For electron microscopical observations, excised pieces of the gonads were cut into small pieces and immediately prefixed in 2.5% paraformaldehyde-glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for2hat4°C. After prefixation, the tissues were washed several times with the same buffer and then postfixed in 1% osmium tetroxide dissolved in Chungcheongnam-Do 36°00' „. Simje-Gun ; Buan-Gun 35° 45° 126° 45" REPRODUCTION IN RAPANA 243 FIG. 2. Anatomy of the female purple shell, Rapana venosa, removed from its shell. A, reproductive organ of female; B, posterior appendage showing the gonad and liver. X, Y and Z denote the sections for measurement of GSI, Three sections are spaced equally. Abbreviations: A, anus; DG, digestive gland; F, foot; MA, mantle; MO, mouth; OP, opercu- lum; OV, ovary; PA, posterior appendage; S, stom- ach; SC, stomachal caecum. 0.2 M phosphate buffer solution (pH 7.4) for 1 В at 4°C. Tissues were then dehydrated in a series of increasing concentrations of ethanol, cleared in propylene oxide and embedded 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 ~ 1000 A. Tissue sections were mounted on collodion-coated copper grids, doubly stained with uranyl acetate followed by lead citrate, and examined with a JEM 100 CX-2 (80 kv) electron microscope. First Sexual Maturity A total of 214 female purple shells (3.1-17.0 cm in shell height) were used for the histological study of first sexual maturity. The percentage of sexual mature was deter- mined for shell heights that reached maturity during the breeding season (from May to late August). Egg Capsule and Fecundity To investigate the number of egg capsules per individual and the number of eggs in an egg capsule in the laboratory, a total of 25 adult females of 11.2-14.2 cm in shell height were used for observation of spawning be- havior in an FRP aquarium (80 cm x 60 cm x 60 cm) bedded with sand and small gravels. An established filtration and aeration appara- tus was employed. Purple shells used for this experiment were those collected in May to June 1994. Length and width of egg capsules of the adult purple shells were measured and their egg developmental processes were checked under a light microscope. The purple shells were reared at the spe- cific gravity of 1.021 and water temperatures of 18.4-23.8°C. Seawater in the rearing FRP aquarium was changed every 3 days. Suffi- cient amounts of bivalves (Ruditapes philip- pinarum, Meretrix lusoria, and Mactra veneri- formis) were supplied as food for R. venosa during the rearing period. Sex Ratio Sex ratios in a total of 445 sexually mature individuals (>7.1 cm in shell height) were in- vestigated from June 1994 to May 1995. Sex of individuals was determined by visual ob- servation of the presence/absence of the penis. Female individuals do not have any genital organ (penis) near the tentacles. To confirm a clear sexuality for each individual, both sexes were confirmed by light micro- scopic examination of histological prepara- tions of the gonads. A Chi square test on goodness-of-fit was used to test the hypothe- sis of equal representation of each sex. RESULTS Morphology and Internal Structures of Re- productive Organs The purple shell Rapana venosa is dioe- cious, and the ovary is located on the surface of the digestive gland in the spiral posterior re- gion of the shell. The ovary is composed of 244 numerous oogenic follicles. With gonad matu- ration, the external color of the ovary be- comes pale yellow. Female individuals do not have the genital organ (penis) near the tenta- cles (Fig. 2) and can therefore be usually dif- ferentiated from males. Changes in the Gonadosomatic Index (GSI) and Water Temperature Monthly GSI changes were measured in fe- male samples collected between June 1994 and May 1995 (Fig. 3). The GSI values slowly increased from October through February, when seawater temperatures gradually de- creased. However, the values rapidly in- creased from March, when seawater began to increase again and then reached the maxi- mum value (mean 17.0) in April, when seawa- ter temperature rapidly increased. Thereafter, the GSI showed relatively lower values in May to August, when relatively higher water tem- peratures were maintained and spawning oc- curred. And the value temporarily reached the minimum level in September, when most of gonads were completely degenerated or re- sorbed after spawning. CHUNG ETAL. Electron Microscope Observations on Germ Cell Development during Oogenesis Ultrastructural observations allow the germ cell developmental phases during oogenesis to be divided into four phases: (1) oogonial phase, (2) previtellogenic phase, (3) vitel- logenic phase, and (4) mature phase. Char- acteristic features in each phase during ooge- nesis were as follows: Oogonial Phase: Oogonia in the oogonial phase, which propagated on the follicular wall, were oval in shape and 15 um in diame- ter. They were single or formed a cluster in the oogenic follicle. Each oogonium had a large nucleus with chromatin, several mitochondria, and the endoplasmic reticulum in the cyto- plasm (Fig. 4A). At this stage, the granular cells and mesenchymal cells were present around the oogonia (Fig. 4B). Previtellogenic Phase: With cytoplasmic growth, several small mitochondria, a well-de- veloped endoplasmic reticulum and several vacuoles were concentrated around the nu- cleus in the cytoplasm of the previtellogenic oocyte (Fig. 4C, D). The number of Golgi ap- 20 30 AN © x 1 5 —1 E — O Lao dz À 20 > = tf < < Ge = 10-4 O re Ф > O LL] о 4 HE > i [= Os ar oO E LL] O-5- E Е == @ --@-- Water temperature e —f- Gonadosomatic index SAS LOAN" DA Me AN 1994 (895 MONTH FIG. 3. Monthly changes in the gonadosomatic index in Rapana venosa and the mean water temperatures. Vertical bars represent standard error. REPRODUCTION IN RAPANA 245 ЕЕ ER + “el FIG. 4. Electron micrographs of oogenesis of Rapana venosa (A-F). A, An oogonium, with a large nucleus and several mitochondria in the cytoplasm, scale bar = 2 um; B, the mesenchymal tissue and the granular cells near the oogonia, scale bar = 2 um; C, a previtellogenic oocyte, with a large nucleus containing chro- matin, and several mitochondria, a number of vacuoles and well-developed endoplasmic reticula in the cy- toplasm, scale bar = 2 um; D, E, the previtellogenic oocytes, with the Golgi apparatus and glycogen particles in the vacuoles, scale bars = 4 um; Е, an early vitellogenic oocyte, with a number of the vacuoles and lipid droplets and yolk granules near the nucleus, scale bar = 2 um. (Figure continues.) 246 CHUNG ET AL. FIG. 4. (Continued) G, a late vitellogenic oocyte, with yolk granules and proteid yolk granules, scale bar = 2 um; H, a late vitellogenic oocyte, with a number of proteid yolk granules and immature yolk granules; |, a ma- ture oocyte, with mature yolk granules near the nucleus, scale bar = 2 um; J, 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, scale bar = 2 um. Abbreviations: CR, chromatin; ER, endoplasmic reticulum; G, Golgi appa- ratus; GC, granular cell; GR, granule; IYG, immature yolk granule; LD, lipid droplet; LM, limiting membrane of mature yolk granule; M, mitochondrion; MBy, main body; MC, mesenchymal cell; MYG, mature yolk gran- ule; N, nucleus; OC, oocyte; OG, oogonium; PYG, proteid yolk granule; SL, superficial layer; V, vacuole; YG, yolk granule. paratus, scattered from the perinuclear region to the cortical region of the oocyte in the pre- vitellogenic phase, increased (Fig. 4D). At this time many vacuoles formed by the Golgi ap- paratus appeared near the endoplasmic retic- ulum and some mitochondria (Fig. 4E). Vitellogenic Phase: In the early vitellogenic oocyte, especially with the initiation of yolk formation, glycogen particles, lipid droplets and a few yolk granules were found in the vac- uoles formed by the Golgi apparatus in the perinuclear region. Yolk granules diffused to- ward the cortical layer, and yolk granules ap- peared around the well-developed cortical re- gion of early vitellogenic oocytes (Fig. 4F). In the late vitellogenic oocyte, accumulation of yolk granules occurred in the cortical layer. Particularly, yolk granules, proteid yolk gran- ules, and glycogen particles were apparent in the cytoplasm (Fig. 4G). In addition, a number of modified mitochondria and endoplasmic reticulum were involved in the formation of proteid yolk granules. Glycogen particles, yolk granules and proteid yolk granules were ac- cumulated in the cytoplasm. Eventually, their yolk granules containing several different components were intermingled and became REPRODUCTION IN RAPANA 247 immature yolk granules in the oocyte in the vitellogenic phase (Fig. 4H). Mature Phase: In the oocyte in the mature phase, small immature yolk granules were continuousiy mixed and became larger ma- ture yolk granules in the cytoplasm (Fig. 41). A mature yolk granule was composed of three components: (1) main body (central core), (2) superficial layer; and (3) a limiting membrane of a mature yolk granule (Fig. 4J). Occurrence of Germ Cells during Oogenesis and the Reproductive Cycle Based on electron microscopic and histo- logical observations of the germ cells and 60 FREQUENCY (%) > о N о 1994 Early active M Ripe E Recovery other surrounding cells, the gonadal phases were classified into five successive stages (Fig. 5), which show an annual cycle. Occur- rence of germ cells of various phases at each stage and their characteristics are as follows: Early Active Stage: The gonadal volume was small, and the follicles occupied approxi- mately 25% of the gonad. The follicular walls (germinal epithelium) were relatively thick. A few oogonia of 15 um in diameter were pre- sent along the follicular walls ofthe ovary, and the previtellogenic oocytes appeared. Previ- tellogenic oocytes of 60-70 um in diameter formed an egg-stalk attached to the follicular wall (Fig. 6A). The individuals in the early ac- tive stage were found from September to ES] Late active Partially spawned FIG. 5. Frequency of gonadal phases of Rapana venosa from June 1994 to May 1995. 248 CHUNG ET AL. FIG. 6. Photomicrographs of the histological gonadal phases of the female purple shell, Rapana venosa. À, Transverse section of oogenic follicles in the early active stage, scale bar = 60 um; B, C, section of follicles in the late active stage, scale bars = 60 ит; D, section of ripe oocytes in the пре stage, scale bar = 60 um; E, section of follicles in the partially spawned stage, scale bar = 60 um; F, section of the follicles in the re- covery stage, scale bar = 60 um. February when seawater temperatures were gradually decreasing. Late Active Stage: This stage is character- ized by the presence of developing early vitel- logenic oocytes. Follicular walls (germinal ep- ithelium) of oogenic follicles were thin. A number of early vitellogenic oocytes of 120-140 um in diameter were attached to the follicular walls through each egg-stalk. With the initiation of yolk formation, there were nu- merous yolk granules in the cytoplasm of late vitellogenic oocytes of 150-190 um. Some mature oocytes were free in the lumen of the follicle (Fig. 6B, C). The individuals in the late active stage appeared from October to April. Ripe Stage: Follicles occupied over 70% of the gonad, and follicular walls became very thin. Mature oocytes growing up to 230-250 REPRODUCTION IN RAPANA um diameter became polygonal in shape, and contained a number of mature yolk granules (Fig. 6D). Mature or ripe ovaries were found in March through July, when seawater tempera- tures were gradually increasing. Partially Spawned Stage: The lumen of the oogenic follicle became considerably empty because approximately 50-60% of ripe oocytes in the lumen were discharged. Spawned ovaries were characterized by the presence of a few undischarged vitellogenic oocytes as well as previtellogenic oocytes in the follicle (Fig. 6E). This stage appeared in the individuals collected from May to early Au- gust, when seawater temperatures were 18-26°C. Recovery Stage: After spawning, each folli- cle shrank, and then degeneration or resorp- tion of undischarged vitellogenic or mature oocytes occurred. Thereafter, the connective tissues and a few oogonia and oocytes ap- peared on the newly formed follicular walls (Fig. 6F). The individuals in the recovery stage were found from June to September. First Sexual Maturity During the breeding season, a total of 214 individuals (3.1-17.0 cm in shell height) were histologically examined to check whether they reached maturity and participated in repro- 249 duction. The rate of shells of different size that reached the first sexual maturity is summa- rized in Table 1. The breeding season of R. venosa was found to be from May to August. Inthe case of some individuals with gonad de- velopmental stage in the late active stage in May through July, it is supposed that they can reach maturity except for individuals in the early active stage during the breeding sea- son. First sexual maturity was 0% in female purple shells of 3.1 to 5.0 cm high if they were at the early active stage during the breeding season. The percentages of first sexual matu- rity of female snails of 5.1-6.0 cm and 6.1-7.0 cm in shell height were 14.3% and 27.3%, respectively: most of the individuals were still in the early active stage. Percentage of first maturity in 7.1 to 8.0 cm in shell height were over 50%, all of which were at the late active, ripe or partially spawned stages. Sex- ual maturity was 100% for shells over 10.1 cm in height. Copulatory Behavior and Spawning In the experimental aquaria in the labora- tory, copulatory interaction between females and males could be observed from early April. During this time, it was confirmed from histo- logical examinations that most of the ovaries were filled with a number of late vitellogenic oocytes or mature oocytes in the follicle rep- TABLE 1. The shell height and first sexual maturity of female purple shell, Rapana venosa from early August 1994 to May 1995. Number of individuals by gonadal stage* Shell height (cm) EA LA RI 3.1-4.0 6 4.1 ~ 5.0 8 5.1 ~ 6.0 12 2 6.1 ~ 7.0 8 2 1 7.1 ~ 8.0 15 2 11 8.1 - 9.0 5 2 9 9.1 - 10.0 3 16 10.1 ~ 11.0 11 11.1 - 12.0 9 12.1 - 13.0 5 13.1 ~ 14.0 5 14.1 ~ 15.0 5 15.1 ~ 16.0 5 16.1 ~ 17.0 2 Total PS RE Total Mature (%) 6 0.0 8 0.0 14 14.3 11 2703 iS 31 51.6 4 20 75.0 9 28 89.3 9 4 24 100.0 9 2 20 100.0 4 1 10 100.0 5 2 12 100.0 6 2 13 100.0 6 3 14 100.0 1 3 100.0 214 *Gonadal stage: EA, early active stage; LA, late active stage; RI, ripe stage; PS, partially spawned stage; RE, recovery stage. 250 CHUNG ET AL. resenting the late active or ripe stages. In the peak spawning period (June to July), how- ever, spawning in female individuals occurred 15-30 days later after copulation. This obser- vation indicates that copulation occurs one to two months earlier than the spawning in fe- male individuals. Spawning and Number of Spawning Frequencies (Broods) in Aquarium Egg capsules and spawning intervals of Я. venosa were counted by visual observation (Table 2, Fig. 7). Five out of twenty individuals successfully spawned in the experimental aquarium kept in the laboratory from May to August 1994: a total of 301 egg capsules were spawned by No. 1 adult female at inter- vals of 1-4 days (three broods); 410 capsules by No. 2 at 1-2 days (four broods); 306 cap- sules by No. 3 at 1-2 days (three broods); 184 capsules by No. 4 at 2 days (two broods); and No. 5 spawned a total number of 386 egg cap- sules at intervals of 1-3 days (four broods). The average time required for a spawning of this species was 5.5 h (1-6 h). According to the observation in the laboratory, most spawning occurred from night to early morn- ing. FIG. 7. External feature of deposition of egg cap- sules on the shell of Rapana venosa. Fecundity, Egg Size, Hatching Shell Length and Mode of Development The total number of egg capsules per indi- vidual and the mean number of eggs in an egg capsule of R. venosa were 184 to 410 and 976, respectively (Table 2). Thus, fecundity TABLE 2. Spawning frequency and the number of egg capsules of Rapana venosa observed from May to July 1995. Shell Ind. height Spawning Spawning No. (cm) date hours 1 13.5 Мау 23 1994 18:34-24:06 Мау 24 1994 02:36-08:18 Мау 28 1994 01:16-07:08 2 14.6 Мау 24 1994 23:16-05:16 Мау 25 1994 16:31-21:52 Мау 27 1994 16:26-22:10 Мау 28 1994 04:34-10:08 3 13.6 June 15 1994 22:24-03:50 June 16 1994 01:04-06:36 June 18 1994 03:14-08:05 4 14-2 June 20 1994 23:34-04:30 June 22 1994 05:48-18:18 5 14.4 July 24 1994 22:10-03:09 July 25 1994 19:18-23:50 July 28 1994 16:06-20:43 July 29 1994 05:04-09:43 Water No. of Spawning Interval temp. egg frequency (day) (°C) capsules (No. of broods) 18.4 113 18.6 96 20.5 92 (301) 3 1-4 19.4 118 20.1 107 20.0 94 20.4 91 (410) 4 1-2 21.6 114 21.8 98 22.3 94 (306) 3 1-2 22.5 96 22.8 88 (184) 2 2 22.9 112 23.0 95 23.6 92 23.8 87 REPRODUCTION IN RAPANA was estimated to be 179,000 to 400,000 eggs per individual. The egg size and hatching shell length of R. venosa in Korea were 230-250 um and 0.41 x 0.30 mm, respec- tively (Table 3). The duration of development from fertilized eggs to veliger larvae in egg capsules just before hatching of R. venosa, were 15 to 17 days at about 20°C under labo- ratory conditions (Fig. 8). The mode of devel- 251 opment of this species is a planktotrophic veliger larval pattern because the embryos hatch as planktotrophic veliger larvae. Sex Ratio Of total 445 purple shells, 228 were fe- males and 217 males (Table 4). There was no significant difference in the prevalence of TABLE 3. Comparison of the sizes of eggs and egg capsules, and larval shells of some species in the family Muricidae. Shell length Number of Egg Duration of of larvae at Shell length at Egg size eggs in an capsule development haching hatching (mm) (mm) egg capsule size (mm) (days) (mm) Source Hatch as veligers: Rapana venosa 0.26 790 - 1300 30x25 13 0.41 x 0.29 Amio, 1963 R. venosa 0.24 772 - 1190 30x26 15 - 17 0.41 x 0.30 Present study R. bulbosa 0.28 - 0.32 0.42 Thorson, 1940 Bedevina birileffi 0.19 60 ~ 90 3.0 x 0.85 14 0.30 ~ 0.32 Amio, 1963 Thais clavigera 0.19 160-220 4.5x1.2 0.30 - 0.32 Amio, 1963 T. bronni 0.20 160-360 1.0x1.5 0.32 - 0.34 Amio, 1963 T. tissoti 0.19 75 11 0.32 Middelfart, 1996 T. floridana 0.11 0.13 D’Asaro, 1966 T. carinifera 0.20 - 0.22 0.32 - 0.34 Thorson, 1940 Purpura patula 0.24 0.4 Lewis, 1960 FIG. 8. Morphology of veliger larvae before hatching of Rapana venosa. Scale bar = 0.4 mm. 252 CHUNG ET AL. TABLE 4. Monthly variations in sex ratio of Rapana venosa. No. of No. of Month Females Males June 1994 24 20 July 1994 20 26 August 1994 23 19 September 1994 22 1174 October 1994 18 24 November 1994 18 15 December 1994 14 18 January 1995 14 19 February 1995 18 13 March 1995 21 18 April 1995 19 15 May 1995 17 13 Total 228 217 Total Sex Ratio x (Chi Numbers (F/(F + M)) squared)* 44 0.55 0.37 46 0.43 0.78 42 0.55 0.38 39 0.56 0.64 42 0.43 0.86 33 0.55 0.27 32 0.44 0.50 33 0.42 0.76 31 0.58 0.81 39 0.54 0.23 34 0.56 0.47 30 0.57 0.53 445 0.51 0.27 “The critical value for x goodness-of-fit test of equal numbers of females and males (1 df) at 95% sig- nificance is 3.84. each sex (not different from a 1:1 sex ratio, X = 0.27, p > 0.05), and monthly comparisons showed no statistical differences. DISCUSSION Germ Cell Development and Vitellogenesis Our electron microscope observations of early vitellogenic oocytes of Rapana venosa indicate that the Golgi apparatus is involved in the formation of a number of glycogen particles filled vacuoles and small vesicles in the perinuclear region of the cytoplasm. Lipid droplets and lipid granules are then added to the vacuoles and vesicles formed by the Golgi apparatus (referred as autosynthetic), as in llyanassa obsoleta (Taylor 8 Anderson, 1969), Biomphalaria glabrata (de Jong-Brink et al., 1976), Mytilus edulis (Reverberi, 1971), and Mactra veneriformis (Chung & Ryou, 2000). Therefore, our observation suggests that the Golgi apparatus and vacuoles present in the perinuclear region are involved in the forma- tion of lipid droplets and lipid granules in the early vitellogenic oocytes. Some mitochondria in late vitellogenic oocytes of R. venosa ap- pear to undergo a process of transformation during late vitellogenesis, and some evidence affirms that the yolk precursors are derived from modified mitochondrial structures found in the cytoplasm. The endoplasmic reticulum and modified mitochondria are present near the proteid yolk granules. However, we could not find pinocytotic tubules, such as those in- volved in yolk production in the vitellogenic oocytes of Agriolimax reticulatus (Hill 8 Bowen, 1976; Dohmen, 1983), or multivesicu- lar bodies as seen in the opisthobranchs Hypselodoris tricolor and Godiva banyulensis (Medina et al., 1986), and another snail, Physa acuta (Terakado, 1974). Accordingly, it is as- sumed that the endoplasmic reticulum and modified mitochondria are involved in the for- mation of proteid yolk granules (Taylor & An- derson, 1969), with R. venosa only synthesiz- ing yolk autosynthetically, as seems to occur in the majority of gastropods. Exceptions include gastropod species (Planorbarius corneus, Lymnaea stagnalis, Hypselodoris tricolor, and Gordiva banyulensis) that synthesize yolkbya combination of autosynthetic and heterosyn- thetic processes (Bottke et al., 1982; Medina et al., 1986). Occurrence and Resorption of Germ Cells during Oogenesis and the Reproductive Cycle As in most other marine mollusks (Chung et al., 1993; Chung & Ryou, 2000), gonad de- velopmental phases of R. venosa show an annual periodicity (Chung et al., 1993; Chung & Kim, 1997), with cyclic change of germ cell development during the reproductive cycle. According to occurrence of germ cells with the gonad developmental stage, the oogonia and previtellogenic oocytes appeared in the early active stage, and the early and late vitel- logenic oocytes occurred in the late active stage. Numerous fully mature oocytes in the ripe stage were released in the partially spawned stage. After spawning, small num- bers of undischarged vitellogenic and mature REPRODUCTION IN RAPANA 253 oocytes were degenerated and resorbed. Newly formed oogonia and previtellogenic oocytes occurred in the recovery stage. Regarding reproductive energy allocated to the production of gametes, in particular, the continuous production and resorption of germ cells may be regard as an adaptation to envi- ronmental temperature and food availability (Morvan & Ansell, 1988; Paulet, 1990). If the reproductive energy allocated to the produc- tion of gametes is too large, nutritive reserves may not be enough to allow all eggs to reach the critical size for spawning and fertilization. In this case, the products of gamete atresia may be resorbed and the energy reallocated to still-developing oocytes or used for other metabolic purpose by marine moliusks (Dor- ange & LePennec, 1989; Motavkine & Varak- sine, 1989). Therefore, it is supposed that R. venosa should have a reproductive mecha- nism to resorb and utilize the high nutritive substances rather than releasing hopeless gametes. Gonadal Development and Maturation It is well known that development and mat- uration of gametes of prosobranchs are gen- erally influenced by several factors, such as temperature, food availability, illumination (day length), and hormones (Boolootian et al. 1962; Fretter, 1984). We observed that gametogenesis of Я. venosa initiates at a temperature of about 3.0°C, with maximum gonadal maturation oc- curring in June, when temperatures were high and phytoplankton were very abundant. Ac- cording to Segal (1956) and Sutherland (1970), in temperate prosobranchs, gonadal maturation and spawning occur in rapid suc- cession during the summer. The importance of food is obvious in the limpet Acmaea spp.: gametogenic activity of upshore populations with less available food is more restricted than those of the same species lower on the shore. As suggested by Sastry (1963) from a study with bay scallops, water temperature seems to be the most important parameter for main- tenance of gonadal development. In Korean coastal waters, growth and production of bi- valves that are predated upon by R. venosa is relatively high from spring to early summer seasons (Kim et al., 1977; Chung et al., 1994; Lee, 1995) due to the abundance in phyto- plankton. Thus, abundant food supply (e.g., bivalves) is available to R. venosa during the period of gonadal development and matura- tion. Therefore, it is suggested that gonadal development and maturation of Korean R. venosa is closely related to temperature change and food availability. Fretter (1984) observed that in temperate zones, the sea- sonal temperature fluctuation associated with changing illumination is a controlling factor in gametogenesis. In consequence, gonadal de- velopment and maturation of this species may be retarded under low illumination, due to the decrease in food (bivalves) availability caused by diminished primary production of phyto- plankton. First Sexual Maturity with the Gonad Developmental Stage All specimens of 3.1-5.0 cm high were in the early active stage although collected dur- ing breeding seasons, and our gonad histol- ogy indicates that none of them could fully de- velop: only small numbers of oogonia and previtellogenic oocytes were present in the oogenic follicle. The size of the oocyte indi- cates that they could not have reached matu- rity until late August, when spawning was ended. Accordingly, the percentage of first sexual maturity of female snails ranging from 3.1-5.0 cm in shell height is 0%. However, in- dividuals 7.1-8.0 cm in shell height belonged to one of the early active, late active, ripe and partially spawned stages during the breeding season. Sixteen individuals in the late active, ripe, and partially spawned stage underwent gonadal development, whereas 15 individuals in the early active stage did not. It was ob- served that among snails of 7.1-8.0 cm high and in the late active stage more than 50% reached the first sexual maturity. However all snails, in late active, ripe, or partially spawned stages, reached it ifthey were larger 10.1 cm. This means that larger individuals can reach maturity earlier than smaller individuals. The results suggests that because catching purple shells < 7.1 cm can potentially cause a dras- tic reduction in recruitment, a prohibitory mea- sure should be taken for adequate natural re- sources management. Breeding Pattern Our histological observations on the purple shell show that spawning of R. venosa on the west coast of Korea occurs from late May to August. Japanese R. venosa have been re- ported to spawn once a year from June to Au- gust in the Ariake Sea, Japan (Amio, 1963). 254 CHUNG ET AL. Our results agree well with those described by Amio (1963). The slight discrepancy in the spawning period between these two studies might be related to geographic differences in water temperature (Chung et al., 1993). As the spawning of R. venosa in Korea occurs from late May to August, this species may be classified as asummer breeder (Boolootian et al., 1962). Morphology of Egg Capsule The surface of the exit part ofthe egg cap- sule of R. venosa is flattened and has a chiti- nous capsular form. The egg capsule in the genus Rapana have a curved sickle shape, with a long cylindrical stem, and an albume- nous substance being contained in the egg capsules. These findings coincide with Amio’s observation from R. venosa in Japan (Amio, 1963). Rawlings (1999) described that the morphology of egg capsules varies within the marine neogastropods, showing differences in shape, size, surface texture within families (D’Asaro, 1970, 1988, 1991, 1993, 1997; Bandel, 1973). Some species in a genus (Per- ron & Corpuz, 1982; Palmer et al., 1990; Mid- delfart, 1996) and sometimes even individuals of the same species population (Rawlings, 1990, 1994, 1995, 1999) show distinct char- acteristics. These morphological differences in neogastropods may be associated with en- vironmental factors such as physical stresses or geographical latitude. Number of Eggs in an Egg Capsule In the present study, R. venosa in Korea spawned 184 — 410 egg capsules each, and the mean number of eggs from an egg cap- sule was 976. Thus, the fecundity (the total number of eggs) from each individual ranged from 179,000 to 400,000 eggs. Amio (1963) described that the number of eggs in each egg capsule of R. venosa in Japan ranged from 790 to 1,300. These results indicate a close similarity in reproductive output be- tween the Korean and the Japanese purple shells. Egg Size and Hatching Shell Length The egg sizes of R. venosa collected in Korea (230-250 um) is slightly smaller than those of R. venosa (260 um) in Japan, and shell lengths of veliger larvae at hatching of the Korean and Japanese purple shells were 0.41 x 0.30 mm and 0.41 x 0.29 mm, respec- tively. The sizes of eggs and shell length of veliger larvae at hatching of R. venosa in Korea were similar to R. venosa in Japan, but the egg sizes and hatching shell lengths of veliger larvae of R. venosa were larger than those of Thais clavigera, T. bronni, and Be- devina birileffi (Table 3). Spight (1976) and Middelfart (1996) described that egg size in the egg capsule and shell length of veliger lar- vae at hatching are very similar for genera within Muricidae (Table 3). The factors affect- ing egg size and hatching shell length of R. venosa have not been studied yet. Duration of Development in the Egg Capsule We observed that the duration of develop- ment from fertilized eggs to hatching veligers ranged from 15-17 days at about 20°C in the laboratory. However, Amio (1963) described that of R. venosa in Japan took 12 days to de- velop from trochophore larvae to veliger larvae (i.e., about 13 days after fertilization). In gen- eral, our results were similar to those of Amio’s (1963) in the duration of development in egg capsules (Table 3). It seems that the duration of development in egg capsules, from deposi- tion of an egg mass to hatched larvae, varies with both internal and external factors, as pro- posed by Amio (1963). Middelfart (1996) re- ported that duration of development of Thais tissotiwas 11 days. Accordingly, it is assumed that duration of development of this species is shorter than R. venosa (Table 3). No Evidence of Nurse Eggs as Larval Food According to our microscopic observations, approximately 80% of eggs in a capsule were normally fertilized, and these embryos hatched as veliger larvae. The remaining 20% failed to develop, but they did not serve as nurse eggs for the normally developing lar- vae. Spight (1976) has classified marine gas- tropods into two groups based on hatching modes: (1) species with embryos that hatch as planktotrophic veliger larvae; and (2) species with embryos that hatch as crawling young snails (juveniles). Spight (1976) also differentiated developing embryos into those that consume nurse eggs and those do not consume nurse eggs within egg capsules in the process of embryonic development. REPRODUCTION IN RAPANA 255 Of Muricidae species, Siratus senegalensis (Knudsen, 1950), Chicoreus torrefactus (Cer- nohorsky, 1965), and Thais emarginata (Le Boeuf, 1971; Middelfart, 1994) have been re- ported to hatch as young snails. According to Spight (1976), among Muricidae, 16 species hatch as snails consuming nurse cells, and 17 species hatch as snails without nurse cells. Twenty-two species hatch as veligers without nurse cells, but four hatch as veliger with nurse cells. These species consume 5.9-91.4 nurse eggs per embryo during development (Spight, 1976), and the size of newly hatched young snails is usually large (> 1.15 mm). In Pisania maculosa, a species with embryos that do not consume nurse eggs, 90% of the eggs were not fertilized, 8% became nurse eggs, and only 2% developed normally (Staiger, 1950). If the encapsulated embryos should share a common nutrient of albumen, developmentally retarded eggs are likely to be fed by surviving embryos (Staiger, 1951). It has been suggested that in case of hatching size of veliger is < 0.5 mm, R. venosa do not use nurse eggs, whereas young snails > 1.0 mm may use nurse eggs for development. Shell size of the veliger larvae of R. venosa was relatively small (< 0.41 x 0.30 mm), indi- cating that the purple shell hatches as veligers without utilization of nurse eggs dur- ing development. In view of our present findings and those observed in other prosobranch gastropods (Spight, 1976), an important postulate can be made: species that hatch as large (> 1.0 mm) veligers or juvenile snails consume nurse eggs during development in the egg capsules, whereas those with smaller veligers (< 0.5 mm) do not. ACKNOWLEDGEMENTS The authors are grateful to Dr. John B. Burch of the University of Michigan and to the referees for helpful comments on the manu- script. The authors are grateful to the Fish- eries Science Institute of Kunsan National University for equipment and fund in the pro- gram year of 2001. 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Bulletin of Korean Fisheries Society, 19: 446-452 [in Korean]. ZOLOTAREV, V., 1996, The Black Sea ecosystem change related to the introduction of new mollusk species. Marine Ecology, 17: 227-236. Revised ms. received 10 September 2001 MALACOLOGIA, 2002, 44(2): 259-272 IMPACT OF SOIL CHEMISTRY ON THE DISTRIBUTION OF ONCOMELANIA HUPENSIS (GASTROPODA: POMATIOPSIDAE) IN CHINA Edmund У. W. Seto', Weiping Wu?, Dongchuan Qiu’, Hongyun Ми“, Xueguang Gu’, Honggen Chen“, Robert С. Spear” & George М. Davis? ABSTRACT Oncomelania hupensis subspecies serve as intermediate hosts for the human Schistosoma parasite in China. In this study we present a multivariate analysis and comparison soil chemistry data associated with the presence or absence of two subspecies, O. hupensis robertsoni and O. hupensis hupensis, that are associated with schistosomiasis disease transmission upstream and downstream, respectively, of the soon to be completed Three Gorges Dam on the Yangtze River. Soil and water samples were collected and analyzed from the Anning River Valley, an 800 km? area in the mountainous region of Sichuan Province and from the Poyang Lake floodplain, a 4,647 km? area in Jiangxi Province. A multivariate classification of soil data was able to discrim- inate between snail habitat and non-habitat in the Anning River Valley with 85.5% accuracy, and revealed that the lack of critical soil conditions potentially excluded O. hupensis robertsoni from some sites. The soil classification was also able to correctly identify 72.4% of marginal habitat sites that were misclassified by field ecologists. We found that O. hupensis hupensis was less dependent upon soil conditions for sites on the Poyang Lake floodplain, and hypothesize that changes in water level associated with seasonal flooding may play a larger role than soil in dis- criminating between snail habitat and non-habitat in this environment. Key words: classification and regression trees, China, Oncomelania hupensis, schistosomia- sis, Schistosoma japonica, soil chemistry, snail ecology, Poyang Lake, Anning River, Sichuan, Jiangxi. INTRODUCTION Despite the successful eradication of schis- tosomiasis in some regions of China, it still re- mains a serious infectious disease that is en- demic in 118 counties, infecting nearly one million Chinese (Chen & Zheng, 1999). It is estimated that over 40 million are at risk of de- veloping schistosomiasis in China. Many of those at risk live along the Yangtze River, which stretches across China, from the moun- tains in the west to the coast in the east. Along this span exist a multitude of terrains and en- vironments, some of which are ideal habitats for subspecies of Oncomelania hupensis, which serve as the intermediate snail hosts for the Schistosoma japonica parasite that causes schistosomiasis. Current research attempts to better charac- terize the different ecologies related to schis- tosomiasis transmission (Davis et al., 1999; Zhou et al., 2001). Such work is of critical im- portance, as the construction of the new Three Gorges Dam on the Yangtze will poten- tially result in the emergence of schistosomia- sis in entirely new areas (Davis et al., 1999; Hotez et al., 1997). The Three Gorges Dam will be the world’s largest dam. The reservoir created by the dam may impact global cli- mate, cause dramatic change in the flow pat- terns of the Yangtze River that will increase snail habitat, and increase the probability of snail migration so dramatically that the geo- graphic distribution of snail habitats may change. A better understanding of which envi- ronments can and cannot support snails, and how these environments are distributed spa- tially now and in the future can potentially lead to effective control strategies. This is particu- larly true in China, where historically, snail control has been an integral part of success- ful disease eradication (Chen & Zheng, 1999; Sleigh et al., 1998). The need for a better ecological under- School of Public Health, University of California, Berkeley, California 94720, U.S.A.; seto @uclink.berkeley.edu. "Institute of Parasitic Diseases, Chinese Academy of Preventive Medicine, 207 Rui Jin Er Liu, Shanghai 200025, P.R.C. “Sichuan Institute of Parasitic Diseases, Chengdu 610041 Sichuan, P.R.C. “Jiangxi Provincial Institute of Parasitic Diseases, Nanchang, P.R.C. School of Public Health, University of California, Berkeley, California 94720, U.S.A. Department of Microbiology and Tropical Medicine, The George Washington University Medical Center, Washington, D.C. 20037, U.S.A. 260 SETOETAL. standing of O. hupensis is most evident in cur- rent research in the use of remote sensing technologies to identify snail habitats (Spear et al., 1999; Zhou et al., 2001). Such studies, motivated in large part by the construction of the Three Gorges Dam, have been carried out both upstream above the dam and down- stream below the dam, in current schistoso- miasis endemic areas. In such studies it is im- portant to realize that different subspecies of O. hupensis exist in different regions, and that these subspecies have evolved to adapt to their particular ecological niches and human- parasite interactions. Oncomelania hupensis robertsoni exists upstream of the Three Gorges Dam site, in the mountains of Sichuan Province where the snails live just above the waterline in the moist soil and shaded by the vegetation of irrigation ditch networks feeding agricultural fields. Oncomelania hupensis hu- pensis exists downstream of the dam, in the lower Yangtze basin, where the snails live in the marshes and grasslands of Poyang Lake, a low elevation plain, completely enclosed by an impressive dyke system, and affected by strong seasonal flooding. The Anning River Valley in southern Si- chuan Province characterizes the mountain- ous environment where O. hupensis robert- soni live (Figs. 1, 2). The valley, at an elevation of 1,500 m, is approximately 48 km in length, 24 km at its widest point, represent- Three Gorges Area ing approximately 400 km? of potential snail habitat. Here, land is predominantly irrigated agriculture, with two growing seasons and rice, wheat, corn, tobacco, and various export vegetables the major crops grown. Domestic animals play a minor role in the agriculture of the valley and in schistosomiasis transmis- sion. Snails do not live within fields, but rather, along the edges of vegetated irrigation ditches. Recent prevalence surveys con- ducted in the summer of 2000 by collabora- tors from the Xichang County Anti-Schistoso- miasis Station have shown that the variance in human schistosomiasis infection is quite large in the valley, ranging from below 10% in some areas to above 70% in other areas. On- comelania hupensis robertsoni that live in this environment have a smooth shell, which has no varix on the outer lip and is relatively short (Davis et al., 1995). The subspecies is re- stricted to Sichuan and Yunnan provinces above the Three Gorges. Conversely, the Poyang Lake environment where O. hupensis hupensis live, as de- scribed by Davis et al. (1999), is vastly differ- ent. Poyang Lake of Jiangxi Province is the largest lake in China (Figs. 1, 3). During the flooding season it regularly fills with water and is held back from towns and villages by a se- ries of dikes. The time that flooding begins depends on the onset of the annual monsoon. Typically the flooding is May through October. Poyang Lake Dongting Lake EN Endemic area Provincial boundary FIG. 1. Map of schistosomiasis-endemic areas along the Yangtze River. The Anning River Valley, Poyang Lake and Three Gorges Dam Area are shown. SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 261 FIG. 2. Landsat TM satellite image of the Anning River Valley. During this period only fisherman can venture out on the lake when it resembles a vast sea. The flooding covers all of the marshland used by cattle for grazing. After the rainy season, the lake empties, losing as much as 90% of its water. All snails are found on the flood plains and on the numerous flat marshy islands in- side the dikes. All transmission occurs within the lake basin, where the cattle graze on the marshlands; there are no snails outside the dikes. Cattle play a much larger role in trans- mission in this environment than in the moun- tains of Sichuan Province. The flooding drowns adult snails, which cannot withstand continuous submersion. During drought, the adults move down into the soil and aestivate. Oncomelania hupensis hupensis has pri- marily ribbed shells in habitats regularly flooded and smooth shells in habitats that are not flooded (Davis et al., 1995). All popula- tions have a varix. The length of shell is, on average, greater than that of O. hupensis robertsoni. The shell allometry is the same as that of O. hupensis robertsoni. The life ex- pectancy for snails in this environment is about one year (Zhang et al., 1996). On- comelania hupensis hupensis is restricted to the Yangtze River drainage below the Three Gorges; it has spread to Guangxi Province, presumably by way of the grand canal from Hunan. We have studied the characteristic soil con- ditions for both environments. We apply mul- tivariate statistics to determine which soil con- ditions are capable of discriminating between areas where O. hupensis exist and where they do not. For the Sichuan environment, we also look at soil conditions for what we call “marginal habitat’, which are areas that to the field ecologist look suitable as snail habitat, but where snails were not found. METHODS Soil samples from sites in the Anning River Valley were collected as part of a remote sensing validation study in the summer of 1998 (Spear et al., 1999). Each site was de- fined by randomly generated geographic co- ordinates. A global positioning system (GPS) receiver was used to navigate to each site. Each site consisted of a 30 x 30 m area, where a Snail survey was conducted and soil was collected. A total of 111 sites were visited (35 snail, 47 non-snail, and 29 marginal sites). Snail status was determined in two manners. A snail survey was performed in the sur- rounding 30 x 30 m area. The snail survey is a systematic search through the ditches for the presence of snails (Gu, 1990). The pres- ence of a single snail in the survey constitutes a positive snail site. In addition to the snail survey, a judgment was made by a field ecol- ogist as to whether the habitat seemed suit- able for snails. There exists sites where no snails were found, but where field ecologists felt the snails could exist. For each site visited, approximately 1,000 cm? of soil was collected by taking multiple samples along ditches within the 30 x 30 m area. Soil samples for each site were com- bined, dried arid well-mixed to constitute a sin- gle soil sample for each site. These samples were later analyzed using Lamotte Company Electronic Soil Lab, Model DCL-12, Code 1985-01. Measurements were made for pH, conductivity, nitrate nitrogen, nitrite nitrogen, ammonia nitrogen, phosphorus, potassium, 262 SETO ET AL. FIG. 3. Landsat TM satellite image of Poyang Lake at low water, showing TMRC study sites. sulfur, copper, iron, manganese, zinc, calcium, magnesium, and chlorides. Soil moisture was measured using a common garden soil mois- ture meter. Soil texture was analyzed using Lamotte Company Texture Kit, Code 1067, which provided the fractions of sand, silt, and clay present in the soil. Water samples were also collected at each site from the nearest irrigation ditch within the 30 x 30 m area. Water samples were ana- lyzed in the field using Lamotte Company GREEN Water Monitoring Kit Code 5848. Measurements were made for pH, phospho- rus, turbidity, temperature, dissolved oxygen, and nitrate. The Tropical Medical Research Center based in Shanghai conducted snail surveys at 98 sites in the Poyang Lake environment in 1999 and 2000. Sites were chosen based on a new randomized snail survey protocol. Ac- cording to this protocol, buffalo grazing ranges were mapped, and 10,000 m? “squares” were located throughout the grazing ranges to pro- vide adequate coverage of the different areas of the ranges. Each square is divided into numbered grids of 10 m x 10 m, of which 20 are selected at random each collecting sea- son. A frame of 4 m? is placed in the center of each randomly selected grid-square and all snails are collected. AGPS record is made of each grid-square. The percent area with no snails is calculated from the cells with no snails. For the area calculated to have snails, the mean and standard deviation of snails per m? and the frequency of infections are recorded. Usable ecological data and soil con- ditions were determined for 87 ofthe 98 sites. Snails were found at 50 of the 87 sites. Eco- logical and soil data measured include ambi- ent air and soil temperature, humidity, light, saturation, pH, nitrate nitrogen, phosphorus, potassium, zinc, ammonia nitrogen, calcium, chloride, copper, ferric iron, magnesium, man- ganese, nitrite nitrogen, sulfate, and soil tex- ture. Soil conditions were measured using the same Lamotte equipment and methods as that used in Sichuan Province for the Anning River Valley. Univariate statistics were calculated for each ecological property measured, allowing for comparison between the Anning River Val- ley and Poyang Lake environments. To test for significant univariate differences in the dis- tribution of ecological properties between the two environments, Kolmogorov-Smirnov dis- tance statistics were calculated (Conover, SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 263 1980). The Kolmogorov-Smirnov statistic is a non-parametric test of the equality of two dis- tributions, and was calculated using the Stata statistical analysis software (Stata Corpora- tion, 2000). Multivariate statistics were used to consider possible interactions between ecological properties in discriminating between snail habitat and non-habitat sites. The Classifica- tion and Regression Trees (CART) method (Breiman, 1984) was used to create a classi- fication of habitat versus non-habitat for both the Anning River Valley and Poyang Lake en- vironments. CART produces optimal binary decision trees that describe how different fac- tors interact to produce particular classifica- tion outcomes. Trees for the Anning River Val- ley and Poyang Lake were compared. For data from the Anning River Valley, information exists on snail habitat, non-habitat, and mar- ginal habitat. Only habitat and non-habitat sites were used in the CART analysis so that the decision tree could subsequently be used to predict the outcome for marginal habitat. CART analyses were performed using the CART for Windows program from Salford Systems (Steinberg & Colla, 1997). RESULTS Univariate statistics for the ecological vari- ables related to snail habitats and non-habi- tats for the Anning River Valley and Poyang Lake environments are presented in Table 1. In general, there is greater overall variation in soil conditions for the Anning River Valley than in those for Poyang Lake. Snail habitat within the Anning River Valley is more variable than that in Poyang Lake, as the standard de- viations for all but four soil properties (nitrate nitrogen, potassium, chloride, and nitrite nitro- gen) are larger for snail habitat in the Anning River Valley. Similarly, non-habitats within the Anning River Valley are more variable than those in Poyang Lake, as the standard devia- tions for all but four soil properties (potassium, ammonia nitrogen, chloride, and nitrite nitro- gen) are larger for non-habitats in the Anning River Valley. In both regions though, snail habitat may be complex, as generally higher variances exist in soil conditions associated with snail habitat than with non-habitat. Table 1 shows that there is overlap in the minimum and maximum ranges of soil proper- ties for snail habitat and non-habitat, suggest- ing that there is no clear univariate method for discriminating between habitat and non-habi- tat. However, the presence of overlap in the ranges does not necessarily indicate that the distributions of soil property for snail habitat and non-habitat are the same. Kolmogorov- Smirnov distance statistics are better suited to determining the difference between two distri- butions. The distance statistic assesses the difference in cumulative distributions between snail habitat and non-habitat sites for a partic- ular soil property. As an example, the cumula- tive distributions for sulfate measurements at snail habitat and non-habitat sites in both the Anning River Valley and at Poyang Lake are shown in Figure 4. It is clear from this figure that there is a significant difference in sulfate levels for snail habitat versus non-habitat in the Anning River Valley. This difference in sul- fate is not apparent for Poyang Lake. Corre- spondingly, the Kolmogorov-Smirnov statis- tics presented in Table 2 reveal a relatively large, statistically significant (P < 0.05) D-sta- tistic for sulfate in the Anning River Valley ver- sus a small, statistically insignificant D-statis- tic for sulfate in Poyang Lake. Overally, for the Anning River Valley, three soil properties have statistically significant (P < 0.05) distances (sulfate, ammonia nitrogen, and chloride), whereas twice as many distances are statisti- cally significant for the Poyang Lake environ- ment (zinc, potassium, nitrite nitrogen, silt, sand, and calcium). This suggests that a dis- crimination of snail habitat from non-habitat will depend upon a different set of soil proper- ties in the Anning River Valley versus Poyang Lake. A graphical illustration of the ranges of the ecological variables for snail habitat sites in the Anning River Valley and Poyang Lake is presented in Figure 5. Comparing the ranges for snail habitat for the two subspecies, it is apparent that for most soil properties there is overlap. However, there are distinct differ- ences in the range of pH and calcium. More- over, although there is some overlap, the lev- els of silt, phosphorus, ferric iron, magnesium, nitrite nitrogen and sulfate seem different for the two subspecies. Again, rather than only looking at ranges, Kolmogorov-Smirnov sta- tistics were used to compare the distributions of snail habitat for the two areas (Table 3). All but one of the distance statistics (chloride) were statistically significant, reinforcing the fact that the two snail subspecies live in very different environments. To understand how multivariate interactions work to discriminate between snail habitat and 0'29€ SES orl 9°08 8'vcl [981 g'ec 0'082 S'0€S Он! 00001 0'00€ 0'09 5'5 019 eve 0'001 SETOETAL. 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ER Uh = / À FT = 8 3 4 Poyang Lake 50 ES а Kolmogorov-Smirnov D = 0.2484 | Р = 0.170 .020408 — | | | 2.9 525 Sulfate (ppm) FIG. 4. Cumulative distributions and Kolmogorov-Smirnov statistics for snail habitat versus non-habitat for (A) Anning River Valley and (B) Poyang Lake. 266 SETOETAL. TABLE 2. Kolmogorov-Smirnov distance between snail habitat and non-habitat for each soil property. A comparison of Kolmogorov-Smirnov distances for the Anning River Valley and Poyang Lake suggest that the discrimination of snail habitat and non-habitat for the two environments depend upon different soil properties. Statistically significant results (P < 0.05) are shown in bold type. Anning River Valley Poyang Lake Soil Moisture 0.2558 Sand (%) 0.1318 Silt (%) 0.1792 Clay (%) 0.1045 pH 0.2208 Nitrate Nitrogen (Ibs/acre) 0.1695 Phosphorus (Ibs/acre) 0.2000 Potassium (lbs/acre) 0.1604 Zinc (ppm) 0.1091 Ammonia Nitrogen (lbs/acre) 0.3623 Chloride (lbs/acre) 0.3266 Copper (ppm) 0.1257 Ferric Iron (ppm) 0.1435 Magnesium (lbs/acre) 0.2916 Manganese (ppm) 0.1864 Nitrite Nitrogen (lbs/acre) 0.1381 Sulfate (ppm) 0.4013 Calcium (lbs/acre) 0.2942 P value D P value 0.138 0.831 0.3455 0.019 0.483 0.3455 0.018 0.962 0.1480 0.742 0.242 0.2674 0.119 0.554 0.2343 0.225 0.356 0.1534 0.714 0.626 0.4005 0.005 0.945 0.4370 0.001 0.008 0.2020 0.379 0.024 0.0647 1.000 0.874 0.2175 0.298 0.747 0.2470 0.174 0.057 0.2456 0.178 0.436 0.2428 0.192 0.777 0.3695 0.009 0.003 0.2484 0.170 0.052 0.3082 0.048 non-habitat for each subspecies of snail, CART analyses were performed. The CART tree forthe Anning River Valley is shown in Fig- ure 6. The binary decision tree is read from the top down. All 83 data observations are repre- sented at the topmost node of the tree, where 42% of the sites are snail habitat. CART fol- lows a brute force algorithm, where every pos- sible binary split criterion for every soil prop- erty is considered. The criterion that produces the best outcome in terms of splitting snail habitat from non-habitat is then chosen to split the top node into two child nodes. For the An- ning River Valley, magnesium is chosen to be the best first split. If the level of magnesium at a particular site is less than or equal to 1671.8, that site is passed to the left child. Otherwise, the site is passed to the right child. After this split, 73 of the original 83 sites are passed to the left child node, of which 34.2% are snail habitat sites. The remaining ten sites are passed to the right child node, where 100% of the sites are snail habitat. Since all ten sites in the right child node are snail habitat, there is no need for further splits. However, for the left child node with 73 sites, CART determined that a criterion based on sulfur can further dis- criminate between snail habitat and non-habi- tat sites. This criterion leads to the creation of two additional child nodes. This procedure continues recursively until child nodes can no longer be reliably split by a soil criterion. In the Anning River Valley tree magnesium, sulfate, phosphorus, and silt are used. Actually, not displayed in the tree, but still important are contributions from chloride and potassium, the values of which were used when values of the other four soil properties were missing. The six-parameter tree produces a remark- ably high 85.5% cross-validated accuracy level. To independently test the classification, marginal habitat sites — those sites from the Anning River Valley where field ecologists felt snails could exist, however, where snails were not found — were subsequently passed down the CART tree. 72.4% of these sites were cor- rectly classified as non-habitat. Anode of par- ticular interest is terminal node T1, where a large number of non-habitat sites fall. This node represents low magnesium, sulfate, phosphorus, and silt conditions. For the Poyang Lake environment, a CART classification of snail habitat versus non-habi- tat revealed that a single soil property, zinc was able to correctly classify 63% (cross-vali- dated accuracy) of the sites (Fig. 7). The ma- jority of the snail sites (39 of 50) fall into ter- minal node T2, where zinc levels are between 2.7 and 6.2 ppm. Recall from the univariate analyses that zinc was also identified through SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 267 Sand ARV 3 63 PL | | a em Bene ee ee Silt ARV 24.7 72 ПИ м Clay ARV 4 67 PL и а рН ARV 6 8.5 PL 41M : Nitrate ARV 5 76 PL ОЕ ek A o оо P ARV 0.2 693 PL 0: e K ARV 128.8 647.4 Amm. Nit. ARV 1174 Cl ARV 20 200 411 Cu ARV 0 8 PL RSR CARR SERA ne = Fe ARV 0 786 PL 0. 4B 28.2 Mg ARV 185.8 9907.2 РЕ 14.4— 248 Мп ARV 0 1002 PL MA A Nitrite ARV 0.1 3.7 PL A A RS eo) | Sulfate ARV 7.2 245.5 ее >> + Ca ARV 2270.4 14035.2 PL BD 288 FIG. 5. Illustration of the approximate ranges of soil properties associated with snail habitat in the Anning River Valley (ARV) and Poyang Lake (PL) environments. Kolmogorov-Smirnov statistics to have a large statistically significant distance between snail habitat and non-habitat distributions. Aseparate CART classification (Fig. 8) was run for the Poyang Lake sites using the same six soil parameters as those found to be im- portant in discriminating between snail and non-snail sites in the Anning River Valley. The result of this analysis was a considerably more complex eight-terminal node tree. How- ever, the accuracy for this complex tree was only slightly better (65.6%) than that of the simple three-terminal node zinc tree (63%). The interactions between the soil parameters in Poyang Lake seem to be different from those seen in Sichuan. However, the one fac- tor that remains important for snails in both re- gions is the sufficient availability of silt. Soil moisture measurements were col- lected for the Anning River Valley. Kol- mogorov-Smirnov statistics indicate that the difference in the distribution of soil moisture 268 SETOIETAL: TABLE 3. Kolmogorov-Smirnov distance between snail habitat in the Anning River Valley and snail habitat in Poyang Lake for each soil property. All soil properties with the exception of chloride are statis- tically significant to P < 0.05, which suggests that the Anning River Valley and Poyang Lake environ- ments are very different. D P value Sand (%) 1.0000 0.000 Silt (%) 1.0000 0.000 Clay (%) 1.0000 0.000 pH 1.0000 0.000 Nitrate Nitrogen (Ibs/acre) 0.4327 0.001 Phosphorus (Ibs/acre) 0.5306 0.000 Potassium (Ibs/acre) 0.3333 0.016 Zinc (ppm) 0.4163 0.001 Ammonia Nitrogen (lbs/acre) 0.5102 0.000 Chloride (lbs/acre) 0.2286 0.187 Copper (ppm) 0.4748 0.000 Ferric Iron (ppm) 0.6857 0.000 Magnesium (Ibs/acre) 1.0000 0.000 Manganese (ppm) 0.7959 0.000 Nitrite Nitrogen (lbs/acre) 0.9714 0.000 Sulfate (ppm) 0.5878 0.000 Calcium (lbs/acre) 1.0000 0.000 between snail habitat and non-habitat sites is not significant (Table 2). However, range of soil moisture for snail habitat sites is much narrower than for non-habitat sites, with snails favoring high moisture environments (Table 1). Corresponding data were not available for Poyang Lake. Both univariate and multivariate ap- proaches were used to analyze water sam- ples from the Anning River Valley. Univariate statistics for the water samples are presented in Table 1. There is considerable overlap in the ranges of measured water characteristics. However, Kolmogorov-Smirnov statistics indi- cate that the distribution of water turbidity for snail habitat sites is significantly different (P < 0.05) from non-habitat sites (Table 4). Multi- variate CART analysis produced a decision tree based on water turbidity and temperature values. However, there was significant mis- classification with this tree, suggesting that the variance in water conditions does not sig- nificantly affect the existence of snails. Corre- sponding water data for Poyang Lake were not collected. DISCUSSION Based on the Kolmogorov-Smirnov statis- tics of Table 3, the Anning River Valley is very different from Poyang Lake, which is not sur- prising given the differences in general ecolo- gies for these two regions. Perhaps the most important differences between the two envi- ronments are pH and calcium levels, for which no overlap exists in the ranges for these soil factors. Despite these differences, we cannot conclude that O. hupensis robertsoni would only be able to survive in the mountains of Sichuan, and O. hupensis hupensis in Poyang Lake. It can be argued that even though high pH and high calcium soils do not exist in Poyang Lake, O. hupensis hupensis may still find such environments suitable, and might be able to survive or adapt to the Anning River Valley environment. The CART classification of snail habitat ver- sus non-habitat relies on six soil properties, suggesting that the lack of critical levels of magnesium, sulfate, phosphorus, and silt may prevent the establishment of snails. However, one must be careful in interpreting such re- sults, as it is difficult to say with certainty that there is biological significance to these partic- ular soil properties. This is especially true in light of the number of different variables con- sidered here, many of which are probably cor- related. One should also be cautioned that as a non-parametric discriminant analysis, CART can only create decision rules based upon data given. As a consequence, variables that do not show up in the tree may not nec- essarily be unimportant to snail survival. Such variables may indeed be vitally important at extremely high or low levels. However, CART does not take these extremes into considera- tion, as such data do not exist within our datasets. Nevertheless, the Anning River Valley clas- sification tree is remarkably accurate, particu- larly in light of its performance in classifying marginal habitat sites. Marginal habitat sites should be considered as very difficult sites to classify correctly since field ecologists essen- tially misclassified them as snail habitat. The ability of CART to correctly classify 72.4% of these sites as truly non-habitat suggests that soil conditions are in fact very important, and that there is merit to the notion that the lack of particular soil conditions can make what would look like great snail habitat, marginal at best. Such a result is relevant to the changing ecologies associated with the Three Gorges Dam, where areas suspected of being snail habitat may be eliminated as habitat for the O. hupensis robertsoni subspecies based on the lack of critical soil conditions. Moreover, exist- SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 269 Percentage of snail sites Number of sites within node Mg <= 1671.8 À PEA Mg > 1671.8 Е Е $ <= 21 SEAS Number of sites within terminal node re) Lea en P> 168.9 Р <= 54.15 Pe S455 Km г KE 100 x 14 SILT / Ne >62.5 4 7 05 P= 92205 ey SS ma ва eo 2 ва Mg <= => | Mg > 681.1 ni 13 ni 0 6 FIG. 6. CART classification of snail habitat versus non-habitat for the Anning River Valley. Units for silt is per- cent; magnesium and phosphorus are Ibs/acre; and sulfate is ppm. ing regional soil maps may be studied in the future for potential correlation with important soil factors and snail occurrence, which may explain historical patterns of disease-endemic areas. The comparisons of the variation in soil properties between the Anning River Valley and Poyang Lake environments suggest that the Anning River Valley is amore complex en- vironment than Poyang Lake, both in general, as well as with respect to snail habitat. Kol- mogorov-Smirnov statistics suggest that from a univariate standpoint, it is easier to discrim- inate between snail habitat and non-habitat in the Poyang Lake environment. This is also re- flected in the multivariate analyses, in which a more complicated multivariate eight-terminal node tree was needed to discriminate be- tween snail habitat and non-habitat in the An- ning River Valley, whereas a more compli- cated multivariate eight-terminal node tree performed just slightly better than the univari- ate three-terminal node classification tree. One might expect Sichuan mountain environ- 270 Number of sites within node Terminal Node ID Percentage of snail sites Number of sites within terminal node Zinc <= 2.7 SETO ET AL. EX Zinc => 23) FIG. 7. CART classification of snail habitat versus non-habitat for Poyang Lake. Zinc split criteria are shown in units of ppm. TABLE 4. Kolmogorov-Smirnov distance between snail habitat and non habitat for each water prop- erty in the Anning River Valley. Only the distance for turbidity is statistically significant to P < 0.05, which suggests that turbidity may play a role in discrimi- nating between snail habitat and non-habitat sites in the Anning River Valley. D P value pH 0.1382 0.826 Dissolved Oxygen 0.0794 1.000 Nitrate 0.1563 0.717 Phosphorus 0.2401 0.221 Temperature 0.2451 0.203 Turbidity 0.3240 0.039 ments to be more complex because of highly variable geology and earth processes associ- ated with mountainous terrain. The Poyang Lake environment is clearly composed of de- positional sediments from flooding, and thus might be expected to be more uniform, given that these sediments have been accumulating from the same sources and mixed over sev- eral centuries. The Anning River Valley is a much more stable environment for snails than Poyang Lake, due to the relative lack of seasonal flooding. This stability may lead to the estab- lishment of different niche habitats for snails in the valley. Recent trips to the field reinforce this hypothesis. Different snail densities were found at different elevations, where different land-use and related farming practices soil conditions existed at different elevations. In the lowlands, more paddy fields and long lin- ear irrigation ditches were found, whereas in the terraced highlands more complex field and ditch structure and considerably higher snail density were found. These land-use dif- ferences within the valley may correspond to the complexity seen in the snail habitat for this area. As a large flood basin, we hypothesize that there is less spatial variation in soil conditions for Poyang Lake than for the Anning River Val- ley due in part to the deposition, accumula- tion, mixing, and dispersal of soil and nutri- ents that occurs with flooding over time. It is in fact the flooding itself that is most likely the largest determinant of snail habitat versus non-habitat. Areas that are either too wet or too dry for too much of the year will exclude snails. Moreover, strong evidence is provided by Davis et al. (in press) that the severity of annual floods can affect whether snails will be found in an area one year but not another. Soil conditions only play a small role in discrimina- tion of snail habitat versus non-habitat for this area. This is supported by the fact that both the simple univariate CART tree based on zinc levels, as well as the complex multivari- ate CART tree were only able to achieve 65.6% cross-validated accuracy, in compari- son to the 85.5% accuracy of the tree for the Anning River Valley. There are implications of these findings for SOIL CHEMISTRY AND ONCOMELANIA DISTRIBUTION 271 Percentage of snail sites Number of sites within node ESE Number of sites within terminal node Mg<=62.4 K <= 85.6 SS ae 96 K > 85.6 PEN oe pie ee Serial Mg>62.4 S<=21.7 S 217 Bee aes Silt <= 6.8 ne FIG. 8. CART classification of snail habitat versus non-habitat for Poyang Lake using same six parameters used in the Anning River Valley classification. Units for silt is percent; magnesium, potassium, and phos- phorus are Ibs/acre; and sulfate is ppm. snail control and future research. For O. hu- pensis robertsoni in the mountains, the strong relationships between land-use, soil condi- tions, and snail habitat suggest that the sur- veillance of disease transmission may be very much related to the monitoring of changing land-use and farming practice. If the level or risk associated with particular types of land- use is better understood, then land-use change, such as those associated with envi- ronmental modification and different agricul- tural practices, may be effective control strategies for O. hupensis robertsoni. Remote sensing studies associated with this sub- species and environment should focus on characterizing differences in land-use and crop types, identifying which land cover types are most associated with high snail density, and determining the spatial relationship be- tween these particular land cover types and sites of high human exposure. For O. hupen- sis hupensis in Poyang Lake, the lack of a strong relationship between soil conditions and snail habitat suggest that relatively simple 272 SETO ET AL. remote sensing studies focused on identifying grasslands and their relationship to variances in water levels and to human exposure sites would be most appropriate. Studies of Poyang Lake must take into account chang- ing water levels associated with the seasonal flooding in order to understand the geo- graphic distribution of snail habitats over time. ACKNOWLEDGEMENTS This study was supported by NASA-Ames Joint Research Interchange (NCC2-5102). NIEHS Mutagenesis Center at the University of California, Berkeley (5 P30 ES 01896-20 ZESI), NIH-NIAID (1 ROI-Al43961-01A1), NSF of China (49825511), the University of California Pacific Rim Research Program, and М.1.Н. grant 1 P50 Al3946, the Tropical Medical Research Center, Shanghai, China (1 P50 AI3946). We acknowledge Ms Zhang Jing of the Jiangxi Institute of Parasitic Dis- eases for doing the soil chemistry, and the su- port of that institute for the intensive GIS field work. We thank the Xichang County Anti- Schistosomiasis Station for their field support. LITERATURE CITED BREIMAN, L., 1984, Classification and regression trees. Belmont, California Wadsworth Interna- tional Group. CHEN, M. & F. ZHENG. 1999, Schistosomiasis control in China. Parasitology International, 48: 11-19. CONOVER, W. J., 1980, Statistics of the Kol- mogorov-Smirnov type. Pp. 344-385, in: Practi- cal nonparametric statistics, New York Wilay. DAVIS, G. M., T. WILKE, Y. ZHANG, X. J. XU, C. P. QIU, C. SPOLSKY, D. C. QIU, Y. S. LI. M. Y. XIA & Z. FENG. 1999, Snail-Schistosoma, Parago- nimus interactions in China: population ecology, genetic diversity, coevolution and emerging dis- eases. Malacologia, 41: 355-377. DAVIS, G. M., W. WU, H. CHEN, H. LIU, J. GUO, D. LIN, S. LIV, G. WILLIAMS, A. SLEIGH, F. Z. & D. P. MCMANUS, 2002, A baseline study of the importance of bovines for human Schistosoma japonicum infections around Poyang Lake, China: villages studied and snail sampling strat- egy. American Journal of Tropical Medicine and Hygiene; in press. DAVIS, С. M., 2. Yl, ©. Y. HUA & С. SPOLSKY, 1995, Population genetics and systematic status of Oncomelania hupensis (Gastropoda, Poma- tiopsidae) throughout china. Malacologia, 37: 133-156. GU, X. G., 1990, Epidemiological investigation of schistosomiasis in Minhe and Hexing villages, Xi- chang, Sichuan Province. Sichuan Institute of An- tiparasite Diseases. HOTEZ, P. J., F. ZHENG, X. LONG-QI, C. MING- GANG, X. SHU-HUA, L. SHUXIAN, D. BLAIR, D. P. MCMANUS & G. M. DAVIS, 1997, Emerg- ing and reemerging helminthiases and the public health of China. Emerging Infectious Diseases, 3: 303-310. SLEIGH. A., X. M. LI, $. JACKSON & К. Е. HUANG, 1998, Eradication of schistosomiasis in Guangxi, China. Part 1: Setting, strategies, operations, and outcomes, 1953-92. Bulletin of the World Health Organization, 76: 361-372. SPEAR, R., P. GONG, Е. SETO, Y. ZHOU,.BEXU; D. MASZLE, S. LIANG, G. DAVIS & X. GU, 1999, Remote sensing and GIS for schistosomiasis control in mountainous areas in Sichuan, China. Geographic Information Sciences, 4: 14-22. STATA CORPORATION, 2000, /ntercooled Stata 7.0 for Windows 98/95/NT. Stata Corporation. College Station, Texas, USA. STEINBERG, D. & P. COLLA, 1997, CART for Win- dows 3.6,3. Salford Systems. San Diego, Califor- nia, USA. ZHANG, $. J., Z. D. LIU & L. S. HU, 1996, Studies on snail ecology in the marshlands of Poyang Lake region. Journal of Nanchang University (Natural Science), 20 Supplement Dec: 25-34. ZHOU. Х. М., 1. В. MALONE, Т. К. KRISTENSEN & N. В. BERGQUIST, 2001, Application of део- graphic information systems and remote sensing to schistosomiasis control in China. Acta Tropica, 79: 97-106. Revised ms. accepted 1 December 2001 MALACOLOGIA, 2002, 44(2): 273-288 ECOLOGY OF POTENTIAL HOSTS OF SCHISTOSOMIASIS IN URBAN ENVIRONMENTS OF CHACO, ARGENTINA A. Rumi', J. A. Bechara’, M. I. Hamann? & M. Ostrowski de Nufez* ABSTRACT Some of the Biomphalaria species living in Chaco, such as B. straminea and B. tenagophila, are natural transmitters of schistosomiasis in Brazil, while those of the genus Drepanotrema are not intermediate hosts ofthe disease. The aim ofthe present work was to analyze the importance of a selected set of environmental variables in explaining patterns of distributions and relative abundance of planorbid gastropod assemblages. The study sites were located in urban areas of Resistencia City, Chaco Province, and the environmental variables measured were substratum (macrophytes), water quality (pH, O,, nutrients, among others), as well as other gastropods (An- cylidae, Hydrobiidae and Ampullaridae). Seasonal samplings were carried out in four distinct en- vironments. Thirty-one quantitative samples of gastropods and environmental variables were ob- tained. In canonical correspondence analysis (CCA), seven environmental variables were retained after a stepwise forward selection, from a total of 26, including [N-NH,*], O,%, and the macrophytes Eichhornia crassipes, Pistia stratiotes, Panicum elephantipes, Hydrocotyle ranun- culoides and Canna glauca. They explained 62% of the variation in planorbid association. Canna glauca was the most significant variable, being positively correlated with all of the species of Drepanotrema. Axis | separates B. tenagophila from B. straminea, along a gradient related to in- creasing O,% and P. elephantipes abundance, as well as decreasing [N-NH,"] and P. stratiotes. Axis Il separates D. lucidum, D. anatinum and D. cimex from the other planorbid species along a gradient associated with decreasing abundances of H. ranunculoides and C. glauca. Some common aquatic macrophytes, and to a lesser extent, dissolved oxygen and ammonium in water, may be useful indicators of favorable environmental conditions for potential intermediate hosts of schistosomiasis in Chaco Region. Key words: Gastropoda, Planorbidae, vector-ecology, schistosomiasis, intermediate-hosts, Chaco Region, Parana River. INTRODUCTION The southern expansion of mansonic schis- tosomiasis in the Neotropical region (Pa- raense & Corréa, 1987) necessitates devel- oping preventive strategies in those areas with major risk of disease penetration. In Ar- gentina, these zones are related to the Guyano-Brazilian subregion, mainly occupied by the Del Plata Basin, that includes geo- graphic areas such as Chaco, Mesopotamia and Pampas (Bonetto, 1994). The most prob- able colonization path is along the sub-basin of the Parana River, since the most recent in- festation foci of Schistosoma mansoni Sam- bon, 1907 (Trematoda: Digenea), discovered in southern Brazil, were found in localities within that sub-basin in proximity to Argentina. One of these foci was located at San Fran- cisco do Sul, Santa Catarina State, near the headwaters of the Iguazu River (Bernardini & Machado, 1981), with Biomphalaria tenagophila (d’Orbigny, 1835) as intermediate host. The other was located in the Piquiri River, a tributary of the Parana River (Pa- raense, 1986), with B. glabrata (Say, 1818) the intermediate host. Up to now, the latter host has not been detected in Argentina. Bio- mphalaria tenagophila was described with type locality in “Canton de las Ensenadas”, Corrientes Province, Argentina (d’Orbigny, 1835), and Biomphalaria straminea from Ca- racas, Venezuela (Dunker, 1848), was re- corded here about 30 years ago. These two "CONICET, Facultad de Ciencias Naturales y Museo, División Zoologia Invertebrados, Universidad Nacional de La Plata, Paseo del Bosque s/n, 1900, La Plata, Buenos Aires, Argentina; alerumi@museo.fenym.unlp.edu.ar 2CONICET, Instituto de Ictiología del Nordeste, Facultad de Ciencias Veterinarias, Universidad Nacional del Nordeste. S. Cabral 2139, 3400, Corrientes Argentina. “CONICET, Centro de Ecología Aplicada del Litoral, Ruta 5, km 2,5, Laguna Brava, 3400 Corrientes, Argentina. “CONICET, Facultad de Ciencias Exactas y Naturales, Departamento Biología, Universidad Nacional de Buenos Aires, Pa- bellón 2, Ciudad Universitaria, 1428, Capital Federal, Buenos Aires, Argentina. 274 RUMI ET AL. latter species are very common in freshwater habitats of northeastern Argentina. A detailed knowledge of the biology of pos- sible hosts of schistosomiasis, as well as the host-parasite relationships established in both natural and urban environments, is nec- essary for any control strategy (Rumi et al. 1997). Several research studies have been conducted in the Parana and Uruguay river basins, including species identification, ecol- ogy, demography, population dynamics, and spatial distribution of potential intermediate hosts of S. mansoni (Bonetto et al., 1982, 1990; Olazarri, 1978, 1984; Rumi, 1991, 1993; Rumi & Hamann, 1990, 1992; Rumi & Tassara, 1985; Tassara & Bechara, 1983). These potential hosts are always species of the genus Biomphalaria Preston, 1910 (Mol- lusca: Gastropoda: Planorbidae), though the studies also treated non-transmitter planor- bids of the genus Drepanotrema Preston, 1910. Other studies consider the host-para- site relationships with autochthonous trema- todes in natural and urban environments (Os- trowski de Nunez et al., 1989, 1991; Rumi & Hamann, 1990, 1992). Urban environments, like those analyzed in the present paper, are particularly interesting because of the high probability of schistoso- miasis transmission to humans, in case this parasitism was found in Argentina. In the humid portion of the Chaco Plains, the Paranä River and its tributaries frequently generate an extensive plain of meanders with hundreds of lakes. Many human settlements are well established at those places, including small towns and medium-sized cities. Therefore, the identification of environmental variables that would allow a rapid evaluation of the po- tential of a habitat to support planorbids might be a useful strategy to prevent proliferation of the disease. As a general rule, the environmental alter- ations produced by human activities modify the adaptive capacities of host species, as well as their abilities to transmit the disease. Consequently, it is necessary to study these hosts in natural and urban environments in order to contrast their dynamics in different conditions. According to ter Braak & Prentice (1988), most species occupy a limited area of the available habitat, tending to be more abundant in their optimal conditions. Conse- quently, assemblage composition shifts along gradients of environmental variables, and species replacements tend to follow the pat- tern of spatial and temporal variation of those variables. Gradients are not necessarily phys- ically continuous in space or time, but it is a useful abstraction to explain organism distri- butions (Austin, 1985). The concept of spatial partition of the life zone also implies the sep- aration of species along “resource gradients” (Tilman, 1982), “regulator gradients” or “com- plex gradients” (Huston, 1994). This scheme resulted in two classical models: the environ- mental control, in which environmental vari- ables regulate the presence and abundance of organisms (Whittaker, 1956), and biotic control, in which the relationships among or- ganisms, such as competition and predation (Connell, 1983), are considered as the main factors that structure communities. In an al- ternative model, Quinn & Dunham (1983) pro- posed that they are not mutually exclusive, but both contribute. This paper aims to identify and analyze, in different planorbid assemblages found in urban environments in the city of Resistencia, Chaco Province, Argentina. The most impor- tant biotic and abiotic variables that explain their distribution and patterns of relative abun- dance. MATERIAL AND METHODS Study Area The sampling area was located between Resistencia and Barranqueras (59°15'S, 27°44'W), Department of San Fernando, Chaco Province (Fig. 1). According to Drago (1990), the environments involved in the pres- ent study are within in the Parana River Basin, which encompasses the second most impor- tant watercourse in South America after the Amazon. These environments are on the right margin of the alluvial plain of the Middle Parana River. A large plain of meanders, with very low slope (10 to 12 cm km”?), runs across this sector of the plain. Soils are brown silty- clay, with abundant calcareous material in some places, and elevated alkalinity. An im- portant feature of this alluvial system is the great density of subcircular, shallow water- bodies (< 5 m depth), organized in a drainage arrangement that shows different degrees of connection between lotic and lentic waterbod- ies. Commonly, they are oxbow lakes and other meander-generated lakes and wet- lands, locally named “madrejones”. Most of them are rich in floating, submerged or settled POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 275 о = 2 5 < FIG. 1. Geographic location of the study area and sampled biotopes. Scale bar = 1km. aquatic vegetation, surrounded by hydrophilic gallery forests. In the plain of meanders, lakes are found in different states of succession, be- ginning with the active meandering river chan- nel, followed by recently generated lakes, ending in the almost complete coverage of the pond basin with palustrine vegetation and sediments. Usually these ponds are shallow (up to 2 m deep). The climate of eastern Chaco is subtropi- cal, subhumid mesothermal, with a mean an- nual temperature of 23°C, and monthly aver- ages between 17°C in July and 28°C in January. The mean annual rainfall ranges be- tween 950 and 1,100 mm, distributed almost uniformly around the year, with peaks in spring (September-October) and fall (March- April). 276 RUMI ET AL. Sampling Scheme Selection of variables, identified a priori as presumably important for colonization and de- velopment of planorbid populations, was based on three criteria: (1) Relationships with the substratum — mainly composed of aquatic vegetation, and described at the scale of their rela- tive abundances. The pulmonate snail uses macrophytes as sites for reproduc- tion, feeding, and shelter. Consequently, a decrease in abundance of macrophytes generally results in a decrease of gastro- pod populations (Thomas & Daldorph, 1994). (2) Relationships with water quality vari- ables — including indicators of the trophic state of the selected habitats (nutrient concentrations), as well as other vari- ables related to the snail abundance along regulator gradients (pH, oxygen, and calcium concentrations). (3) Relationships with other members of the mollusk assemblages — also referred to a scale of relative abundance, represent- ing potential competitors or making up a part of multispecific associations. To include the four climatic seasons, sam- plings were carried out quarterly, between February 2, 1988, and October 23, 1989. This area was first widely surveyed to inventory habitats. Four sampling sites were selected based on their planorbid richness: Negro River, at the Municipal Beach of Resistencia City; Paikin, a oxbow-lake related to a suburb of the same name, used as garbage deposit by people living in a group of huts without a sewer system; Rural Society, a pond within the “Rural Society” of Chaco Province, be- longing to the Araza River corridor, which was partially channeled underground; and Golf Club, a pond near the Resistencia “Golf Club”, being probably an old oxbow-lake of the Negro River. The Negro and the Araza rivers cross the city of Resistencia NE-SW toward the city of Barranqueras, draining into the Parana River (Fig. 1). The present human population inhabiting both cities is around 300,000 people. Gastropod sampling was carried out with a mean collection time of 41 min (standard devi- ation, 18 min) within a range of about 6.5 h (from 9:30 to 16:00); snail abundance was de- termined as capture per unit effort (CPUE) in terms of specimens hour '. Collections were made with simple meshed, standard samplers (Thiengo, 1995), locally named “copos” (1 mm mesh size, and 13.5 cm frame diameter). Thirty-one samples were obtained from the lit- toral zone of the habitats (0.5-1.0 m deep). Collected material was carried alive to the laboratory and prepared for identification, dis- section of soft parts, counting and measuring. The planorbid specimens were relaxed in a Nembutal solution 0.01% for approximately 24 h, according to the method described by Paraense (1986). Afterwards, they were sac- rificed by immersion in water at 65-70°C for 45-55 sec, and submerged in fresh water equilibrated with air temperature. Finally, the prepared material was fixed in a Raillet-Henry solution (Paraense, 1976), to facilitate its identification by dissection of the soft parts under a stereoscopic microscope. At each sampling site, physical and chemi- cal variables were monitored immediately be- fore the capture of gastropods. Temperature, pH and conductivity were measured using thermistors, electrode pH-meters, and battery conductivity-meters, respectively. In addition, water samples were taken with a peristaltic pump and transported refrigerated to the lab- oratory, where the following variables were measured: dissolved oxygen concentration (Winkler method), oxygen percentage satura- tion, nutrients (nitrites + nitrates, total soluble nitrogen, ammonium and total soluble reac- tive phosphorus), calcium, hardness, and al- kalinity. Chemical analyses where performed according to Standard Methods (1980) and Golterman et al. (1978). Regarding vegetation, the criterion of rela- tive dominance was employed, based on vi- sual inspection of the sampling area. Plants were coded between 0 and 4 for the data ma- trix, according to the following scale: 0 = ab- sent (0%); 1 = rare (1-25%); 2 = frequent (25%-50%); 3 = co-dominance (50-99%); and, 4 = complete dominance (100%). The surveyed macrophytes belonged to free-float- ing covers: Eichhornia crassipes (Mart.) Solms. (Pontederiaceae), Pistia stratiotes L. (Araceae), Spirodela intermedia W. Koch. (Lemnaceae), Salvinia herzogii de la Sota, and S. rotundifolia Willd. (Salvinaceae); set- tled with floating leaves: Victoria cruziana d’Orbigny (Ninfaceae); settled: Enhydra ana- gallis Gardn. (Compositae), Hydrocotyle ra- nunculoides L. (Umbeliferae), and Ludwigia peploides (H.B.K.) Hara (Onagraceae); emer- gent: Panicum elephantipes Nees (Grami- neae), Canna glauca L. (Cannaceae); and POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 277 unidentified cespitose hydrophilic Gramineae (Table 3). Statistical Analyses Data on gastropod abundance and limno- logical variables were transformed to decimal logarithm (log(x+1)) to linearize relationships among the variables. The multivariate tech- nique chosen to explore the relationships be- tween environmental variables (independent variables) and snails taxocenosis (dependent variables) was canonical correspondence analysis (CCA) (ter Braak, 1986; ter Braak & Prentice, 1988). The program CANOCO 4 (ter Braak & Smilauer, 1998) was employed to perform the analyses, including adjustment of model parameters, goodness of fit tests, and plotting the results. This canonical ordination technique is a combination of ordination in re- duced space and multiple regression. It im- plies the representation of samples and species constrained by environmental vari- ables in the reduced space of orthogonal axes, which are in turn linear combinations of species relative abundance and environmen- tal gradients. This method assume both uni- modal (Gaussian) or linear relationships be- tween species and environmental variables, for long or short environmental gradients, re- spectively. It shows the major trends of varia- tion of multidimensional data within a reduced space of linearly independent canonical axes, and it is a useful tool to understand commu- nity composition in terms of species distribu- tion along different environmental gradients. Therefore, given the unimodal proprieties of CCA, linear relationships between a given en- vironmental variable and species are not re- quired, such as in most canonical multivariate techniques. The unimodal properties of the model allow the estimation of optimum and tolerances of the species, both indicators of niche attrib- utes. Optimum can be approximated by the CANOCO 4 output named “spaces-by-envi- ronmental table”, which are weighted aver- ages of species with respect to standardized environmental variables. Results are repre- sented graphically in a reduced space of or- thogonal axes, displaying the distribution of species, samples, and/or environmental gra- dients in the same bidimentional space (bi- plots and triplots). Since triplots with species scorers and environmental scorers may be in error because they contain the main patterns only, this table is useful to verify that infer- ences drawn from the plots hold true in the ac- tual data on weighted averages (ter Braak & Smilauer, 1998). Among the 26 environmental variables sur- veyed, it was necessary to preselect those of higher contribution to the total variability in snail relative abundance in order to reduce the possibility of an artificial increase in the explained variance by environmental factors, as well as multicolinearity. Stepwise forward selection procedure was employed, using Monte Carlo permutation tests, as imple- mented in CANOCO 4. A critical rejection level of P < 0.10 was applied. The amount of variance explained by the whole model and the first ordination axes was calculated em- ploying Monte Carlo permutation tests, with a critical rejection level of P < 0.05. RESULTS Planorbid Species The following planorbid species were found (Table 1): Biomphalaria tenagophila (d’Or- bigny, 1835), B. straminea (Dunker, 1848), Drepanotrema anatinum (d’Orbigny, 1835), D. lucidum (Pfeiffer, 1839), D. kermatoides (d’Or- bigny, 1835), and D. cimex (Moricand, 1937). In three samples, no planorbids were found; these were omitted from the subsequent sta- tistical analysis. Biomphalaria straminea occurred more fre- quently than B. tenagophila (48% and 35%, respectively), but the latter showed much higher abundances, especially in 1989. Distri- butional patterns in space were also different, with B. straminea being more common in Negro River and Golf Club Pond, whereas B. tenagophila was more abundant in Rural So- ciety and Paikin ponds. Both species are in- termediate hosts of schistosomiasis in Brazil. Species of Drepanotrema were much less frequent than Biomphalaria (10-16%), but abundances were highly variable in time and space, with values similar in order of magni- tude to those of B. straminea, and never reaching the high levels of B. tenagophila. Drepanotrema cimex and D. anatinum were found almost exclusively in Paikin Pond, a biotope where the other species of Drepa- notrema also showed the highest frequency. In the remaining environments, this genus was rarely found, with D. anatinun in Rural Society Pond (one presence), D. lucidum in Negro River (two presences), and D. kerma- toides in Golf Club Pond (two presences). RUMI ET AL. 278 0'0 00 0'0 vo 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 $ 0 0 0 0 €'9¢ Lye 6 bc O'SE 0 cl 0 0 09 0 0 0 0 OL 0 0 vi 901 0 vl OL 0 90! cel 9c 0 0 8 vi 0 89 L6 0'0 0'0 O'EL 0'0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 € 0 0 0 c9 0 00 GOL 0'0 0'0 0 St 0 0 0 0 0 0 0 0 0 0 0 69 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 хаило ‘а зарюеииах ‘а unpion| ‘а шпицеие 'Q £ OL 6891 ueaN 0 6/1 68/10/13 0 cco 68/70/50 0 191 68/20/17 0 ges 88/11/91 09 0 88/80/56 ct 9 88/90/90 0 cl 88/20/63 L'6L ÿ SSL UESW 0 ecc 68/01/82 0 0S9 68/20/42 0 vol 68/70/50 0 0 68/20/17 0 0 88/80/Ec 0 0 88/90/90 vel LZ 88/20/20 08r 00 уеэи 8 0 68/20/43 Le 0 68/20/13 Lp 0 88/80/56 OL 0 88/90/90 vs! 0 88/20/63 5'79 BCL ueoN 98 0 68/0 L/EZ 961 0 68/20/43 orl 518 68/70/50 0 0 68/20/13 9€ 0 88/11/91 8 6 88/80/Ec с 0 88/90/90 Le 0 88/20/62 eaumwens ‘g enydobeua)] '9 sayeg puod ‘90S [eu1ny Puod шмеа J8AIH OIBIN puog ANID 409 sedojoig ‘soyep бинашез je ul A9 er9ua]sisay WO. ‘spuod qniO j09 pue AJ8100S jeiny ‘axe| mogxo uned ‘JAH олбэм ay, и! pajdwes (,_1nou sjenpiaipui) sarads piqioue]d jo aouepunqy *| 378V1 POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 279 Environmental Variables For all sampling periods and sites, physical and chemical variables are shown in Table 2. Water temperature varied between 8°C in June 1988 and 32°C in February 1988. Aver- ages values were similar among sampling sites, being only 1-2°C higher in the Rural Society Pond and Golf Club Pond, respec- tively. These differences may be partly be- cause sampling hours were not the same for all biotopes. The pH ranged from 6.3 to 8.9 (October 1989 and February 1988, respec- tively), being alkaline in most sampling dates in Rural Society Pond, and closer to neutral point in the others waterbodies. Specific con- ductivity showed marked spatial and temporal variations within a range of 90 and 1,600 uS cm | (August 1988 and November 1988, re- spectively). The highest conductivities were measured in Golf Club Pond and the lowest in Negro River. Oxygen concentration [O,] and oxygen saturation percentage (%O,) were low or undetectable during the warmest peri- ods, increasing during winter and spring, with figures as high as 8.1 mg I" and 90% satura- tion. Calcium concentration [Ca*] was also highly variable (Table 2), ranging from 9.5 to 119.4 mg Г' in Paikin Pond (July 1989 and February 1989, respectively). According to Dussart’s (1976) classification of mollusk habitat regarding calcium concentration, the highest mollusk abundance generally corre- sponds to the hardest waters ([Ca*] > 40 mg Г'), while the highest diversity is usually ob- served in mid-range waters ([Ca*] between 5-40 mg Г’). Sampled environments of Chaco generally had waters of medium hard- ness, although hard waters were observed on some occasions (Table 2). Calcium concen- tration was positively correlated with hard- ness (r = 0.677, n = 31, P < 0.01), a variable that ranged between 36 and 329 mg Г" (June 1989 and February 1989, respectively). The range of alkalinity due to [HCO °] was 45-357 mg Г' (October 1989 and August 1988, re- spectively). Rural Society Pond generally showed the highest levels of alkalinity, while Negro River had the lowest. Concerning nutrient concentrations (Table 2), nitrates and nitrites [N-NO, + N-NO,], were usually below 0.4 mg Г", reaching exception- ally 9.6 mg Г' on November 1988 in Paikin Pond. Ammonium concentration [N-NH,?] showed maximum levels in Paikin Pond dur- ing 1989, and minimum levels in Rural Soci- ety Pond most of the time. This nutrient also tended to increase in all waterbodies during the second sampling year. Total soluble nitro- gen (N-SOL) followed the same pattern of ammonium. Finally, phosphate concentration [P-PO,] was generally below 1.0 mg Г", reaching exceptionally high levels in one sam- pling date in Paikin Pond and Rural Society Pond. In Negro River, phosphates had the lowest concentrations of all waterbodies, never being over 0.3 mg Г". Relative abundances of aquatic plants are shown in Table 3. Among the most impor- tant emergent macrophytes, Panicum ele- phantipes was frequent and dominant in Golf Club Pond and Negro River, but absent in the other waterbodies. On the other hand, Canna glauca was always dominant in Paikin Pond, and completely absent from the other biotopes. Among other settled macrophytes, Enhydra anagallis was frequent and important in Paikin and Rural Society ponds, while Hy- drocotile ranunculoides was present only on some dates in Rural Society Pond and Paikin Pond. Of free-floating plants (Table 3), Pistia stratiotes was dominant in Rural Society Pond and during 1989 in Golf Club Pond. Eichhor- nia crassipes was completely absent from Negro River, and present in variable abun- dance and frequency in the remainder of the waterbodies. Other gastropods that were present with planorbids (Table 3), were Heleobias spp. (Hy- drobiidae), Stenophysa marmorata (Guilding, 1828) (Physidae), Gundlachia moricandi (d’Orbigny, 1835) (Ancylidae), and species of the family Ampullariidae, mainly Pomacea canaliculata (Lamark, 1801) and P. scalaris (d’Orbigny, 1835). All the gastropods found with planorbids were eliminated in the step- wise forward selection of independent vari- ables. Pomacea canaliculata was the most frequent gastropod considering all samples. This species has also a wide distribution in del Plata Basin, especially in the Argentinean area. Canonical Correspondence Analysis Seven of the 26 environmental variables used in the canonical correspondence analy- sis (CCA, Table 4) were retained in the step- wise forward selection procedure. 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(LH) GE? иеэи G02 68/2/23 Ge 68/v/S ve 68/28/12 66° 88/11/91 Le 88/8/Ec el 88/9/9 Le 88/2/62 PUOd 90S JeINd L'ec ues 83 68/0 L/EZ da 68/Z/LC сс 68/7/58 ve 68/c/1cC ve 88/8/€3 LL 88/9/9 87 88/2/63 puod ulIed E03 UBaIN La 68/L/LC es 68/c/1c 174 88/8/Ec el 88/9/9 ce 88/2/62 J93MY олбэм 8r2 UBaN LE 68/0 L/EZ 9! 68/2/43 Le 68/t/S 8c 68/c/LcC 05 88/11/91 8c 88/8/€3 8 88/9/9 6c 88/2/62 PUOd ANID F0) (9.) due} sayeq sedojoig JoyeM 280 ajqelieAe jou eJep = , ‘ajqejoajap jou = p/u ‘sejep Huljdwes ||е и! A9 eroua]sisay шод spuod ато Jos) pue AJei90S ¡emy “axe mogxo UIHIEd ‘эл! олбэм ay, Ul painseaw sajqenea ¡eonuayo pue ¡eoisÁyd jo sonen ‘с FIGVL 281 POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 68/10/15 68/70/SO 68/20/15 88/L1/91 88/80/87 88/90/90 88/20/65 PUOd “905 JeINd 68/0 L/ES 68/10/13 68/70/50 68/<0/1+< 88/80/56 88/90/90 88/<0/<0 puod UMIBd 68/10/13 68/c0/Lc 88/80/Ec 88/90/90 88/20/63 JOAIH oben 68/01/56 68/10/15 68/v0/S0 68/20/L 3 88/11/91 88/80/EcC 88/90/90 88/20/62 PUOd ANID HOH Zr UCI Ue 1 15а тэ 5ееа sedojoig COIN =) (=>) <>) >) O0 0 Oro: est NOOO QOS Or Oli O° OT 1916010 001010100010 01OMmMONOONOIOOMOIOCIOIOIS SION 0 00010 0 0010070 NON OOOO OOOO EDRISIOISLSISESELSTSITSISHSISTSIZSISISITSTSISTESISTSISIES SIOILSTSTSISISTSHSLSIOLSISHIEIEIIISESZONNESISTSISZSISES ооо сбое OO) ооо NUN NO 'Oi@ MSM Socie OI ооо ооо ооо HR ON ON зо ооо ооо (>) (= (=>) >) >) >) (S) St SP И OS (>) esse (Sy) (©) (>>> (СЫ (eve) ФИ) fe) ее хр se) SS О (gp) (ge) TN] AS (¡SMS SMS MSM ©оеооо сост SOOO (= Же ее ИИ S (> (>) (>) (elie See >>>) ee) (se ey) («> SH I SD DITSIO SH "OSOS (а) <>) (<>) (>) > ел) (<>) <> (©) (<> (<>) (<>) <>) <>) (=>) (<>) SHC Se OOO O11 © © о ‘9 Do AE sysnjjouw Jeyemysel se]Auydoisen ‘suejeos y À вепоцеиево eaoewog “puesuow enyoejpune) ‘велоиивш esAydouaS “ds seigoajaH. :SASNIION ‘eone/6 eBUuURD pue зарю/пэипие/ эМюэолрАН ‘eueiznio ви ‘sedyueydeja шпэшед ‘sapiojded eıßımpn7 ‘eeeuueis ‘sıjebeue вр/Ациз 'eıpaw -19]ul вэролас “sajones ensig “sedisseJo enuJoyyor3 :se\Aydosoey ‘seyep бинашез ¡je и! ‘AID eIousjsısay шод spuod ап! пою pue AJe190S ¡emy “exe моахо un eg AAI олбэм By} ul рэлпзеэш ‘sysn|jow piqoue|d-uou pue sayAydoleu и! рэллэ$4о ээчеишор элцеэн ‘€ 7719VL 282 RUMI ET AL. TABLE 4. Summary of major results of the canonical correspondence analysis relating planorbid species to the selected environmental variables. Canonical Axes Eigenvalues 0.469 0.401 0.354 Cumulative percentage of variance of species data 23.4 43.4 61.0 Cumulative percentage variance of species-environment relation 37.6 69.8 98.2 Species-environment correlation 0.840 0.837 0.838 Correlation of the environmental variables with the axes P. elephantipes 0.543 —0.346 —0.190 С. glauca 0.095 0.684 0.413 H. ranunculoides —0.139 0.461 —0.036 E. crassipes —0.221 —0.006 —0.068 P. stratiotes 0.385 —0.274 —0.587 [N-NH47] 0.408 0.133 0.310 %O, —0.291 0.268 —0.237 Total of unconstrained eigenvalues 2.006 Total of canonical eigenvalues 1,247 (62% of explained variance) avoiding multicolinearity and over-explanation (ter Braak and Smilauer, 1998). Ammonium [N-NH,*] and percentage saturation of D.O. (%0,) were the water quality variables re- tained. The remaining environmental vari- ables were all macrophytes, including P. stra- tiotes, E. crassipes, H. ranunculoides, P. elephantipes, and C. glauca. The selected en- vironmental variables explain 62% of the total variation in planorbid species relative abun- dance. Moreover, the first three canonical axes account for 98% of the variance ex- plained by these variables. The species-envi- ronment relationship was highly significant, according to the Monte Carlo permutational test (F = 4.46; P < 0.001; 999 permutations), as well as the first canonical axis (F = 5.80; P < 0.01; 999 permutations). In three samples from Negro River and one from Rural Society Pond, planorbids were not found, being ex- cluded by the CCA program. The graphic representation of the species positions along the environmental gradients in the reduced space of the first three canonical axes, yielded the following results (Figs. 2, 3): (1) Axis | separates B. straminea and D. lu- cidum from B. tenagophila and D. kerma- toides, along an increasing gradient of [N-NH,*] and P. stratiotes, as well as a decreasing gradient of %O, and P. ele- phantipes. The remaining species of Drepanotrema occupy intermediate posi- tions along the first axis gradient. (2) Axis Il separates D. lucidum, D. anatinum and D. cimex from the species of Biom- phalaria and D. kermatoides, mainly through a decreasing gradient of H. ra- nunculoides and C. glauca, as well as an increasing gradient of P. elephantipes. (3) Finally, along axis Ш, species of Drepa- notrema appear more clearly distributed, with D. kermatoides on the top, D. anat- inum and D. cimex in the middle, finishing with D. lucidum at the opposite end, close to B. straminea. Biomphalaria tenago- phila is placed on the opposite negative side of the planorbid distribution along the third axis. This axis is mainly related to the independent variables C. glauca and [N-NH,'] (positively) and Р. stratiotes (negatively). In addition, employing the environment-by- species table provided by the program CANOCO 4 (Table 5), the position of the opti- mum of planorbid species along gradients of environmental variables can be better ana- lyzed, independently of their correlations with canonical axes. With this alternative analytical approach, the following results were obtained: (1) Canna glauca was the most important in- dependent variable in the stepwise for- ward selection. All species of Drepan- otrema appear on the positive side ofthe gradient, with D. anatinum and D. cimex placed atthe edge. Biomphalaria species were on the negative side, with B. straminea at the outermost end of the gradient. (2) Pistia stratiotes, the second most im- POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 283 an > O | Dre ana Can gla Dre cim Dre luc x = | a < z = = ES ARS E ic cra Е Вю ten Y x O 2 N-NH4@ 9 © | 2 Biostr # Dre ker* Pis str @ Pan ele | a) a a SES CANONICAL AXIS | +2.0 FIG. 2. Spatial representation of the samples, species and environmental variables in the space defined by the two first canonical axes. Circles: Paikín pond. Diamonds: Rural Society Pond. Squares: Negro River. Tri- angles: Golf Club Pond. Empty symbols: 1988. Filled symbols: 1989. Arrows: environmental variables. Stars: planorbid species. Bio str: Biomphalaria straminea; Bio ten: B. tenagophila; Dre ana: Drepanotrema ana- tinum; Dre cim: D. cimex; D. ker: D. kermatoides; Dre luc: D. lucidum; Can gla: Canna glauca; Eic cra: Eich- hornia crassipes; Hyd ran: Hydrocolyle ranunculoides; Pan ele: Panicum elephantipes and Pis str: Pistia stratiotes. portant independent variable, had B. tenagophila at the positive end of the dis- tribution, whereas all species of Dre- panotrema were on the negative side. Biomphalaria straminea was placed close to the center of the distribution. (3) Along the gradient of [N-NH,?], both species of Biomphalaria were clearly segregated, with B. tenagophila having its optimum at higher concentrations, to- gether with D. kermatoides. Moreover, B. straminea was placed on the opposite end, close to D. lucidum, while D. cimex occupied the center of the distribution. (4) With respect to %O,, planorbid species responded inversely as [М-МН, "|. (5) Dense stands of P. elephantipes sup- ported the highest abundances of B. straminea, together with D. lucidum. (6) Hydrocotile ranunculoides had a ten- dency to house populations of D. ana- tinum, D. lucidum and D. cimex prefer- ably, while Biomphalaria species and D. kermatoides were at the negative side of the gradient. (7) Finally, along the E. crassipes gradient, B. straminea and B. anatinum were on the positive side, D. cimex, close to the center, and the remainder on the nega- tive side. With respect to sample distribution in the re- duced space of the first three axes, four groups were observed, each one character- ized by a kind of vegetation and a particular planorbid assemblage (Figs. 2, 3): (1) Samples in Paikín in 1988, characterized mainly by C. glauca and H. ranuncu- 284 vw, A + | A le WY x Dre ana < Pan ele a < О ET EEE 3 = | xDre luc = Eic era 5 9,02 | S = | -1.5 CANONICAL AXIS | RUMI ET AL. Dre ker - Can gla @ > N-NH4 Dre cim Bio ten $80 Pis str $ $ 720 FIG. 3. Spatial representation of the samples, species and environmental variables in the space defined by canonical axes | апа Ш. Circles: Paikin oxbow lake. Diamonds: Rural Society Pond. Squares: Negro River. Triangles: Golf Club Pond. Empty symbols: 1988. Filled symbols: 1989. Arrows: environmental variables. Stars: planorbid species. Bio str: Biomphalaria straminea; Bio ten: B. tenagophila; Dre ana: Drepanotrema anatinum; Dre cim: D. cimex; D. ker: D. kermatoides; Dre luc: D. lucidum; Can gla: Canna glauca; Eic cra: Eichhornia crassipes; Pan ele: Panicum elephantipes and Pis str: Pistia stratiotes. loides. The most common species were D. anatinum, D. lucidum and D. cimex. The genus Biomphalaria was rare. Samples from Rural Society Pond and from Paikin Pond in 1989, characterized by the presence of Р stratiotes and the highest concentrations of ammonium, with B. tenagophila and D. kermatoides as the most common planorbid species. Samples from Rural Society Pond in 1988, with low-density stands of E. cras- sipes, and the presence of B. straminea and B. tenagophila also in low densities. Samples from Negro River and Golf Club Pond, characterized by the presence of P. elephantipes, and the highest concen- trations of %O,. Biomphalaria straminea was the most typical species, while the other planorbids were negatively related to this kind of vegetation. Within this group, two separated clusters of samples can be observed, one of them more POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 285 TABLE 5. Environment-by-species table showing weighted means and standard deviations of environmen- tal factors, weighted averages of planorbid species with respect to the seven standardized variables (“optima”), and back-transformed data to original format as in Tables 2 and 3 (in parentheses). Note that for limnological variables, original data were transformed to decimal logarithm. Stand B. B. D. D. D. D. Mean dev. tenagophila straminea anatinum lucidum kermatoides cimex C. glauca 1.460 1.881 —0.288 —0.584 1.163 0.536 0.426 0.839 (0.9) (0.4) (3.6) (2.5) (2.3) (3.0) H. ranunculoides 0.393 0.930 —0.196 —0.158 0.772 0.622 —0.423 0.3826 (0.2) (0.2) (ED (1.0) (0.0) (0.7) P. elephantipes 0.884 1.648 —0.434 0.688 —0.536 0.393 0.009 —0.5361 (0.2) (2.0) (0.0) (1.5) (0.9) (0.0) E. crassipes 1055 11.558 —0.185 0.274 0.203 —0.112 —0.301 —0.0004 (0.8) (1:5) (1.4) (0.9) (0.6) (1.1) Р stratiotes №325 81.775 0.726 —0.068 —0.646 -0.745 —0.745 —0.2038 (2.6) (1.2) (0.2) (0.0) (0.0) (1.0) [N-NH4*] 0.238 0.276 0.211 —0.328 —0.136 —-0.709 0.884 0.0696 (0.977) (0.403) (0.586) (0.102) (2.034) (0.807) %0, 1.534 0.493 0.193 0.158 0.435 0.499 0.789 0.2062 (26.5) (39.9) (55.0) (59.2) (13.0) (42.2) clumped, belonging to 1988, and the other more dispersed, from 1989. These results indicate that there is not a clearly defined seasonal variation in the struc- ture of planorbid assemblages. The most im- portant patterns were spatial or inter-annual, as is shown in the triplot of axes | and Il (Fig. 2). There is a defined tendency of most sam- ples from all waterbodies to move in a top-left to bottom-right direction from 1988 to 1989, following a gradient of increasing ammonium concentration and decreasing oxygen satura- tion. DISCUSSION The empirical model developed in this paper explains a relatively high percentage of ob- served variance compared with other commu- nity studies (Borcard et al., 1992), and is in ad- dition highly significant. As a general pattern, the multivariate model shows that Biom- phalaria straminea tends to be relatively more important in well-oxygenated environments with reophilic vegetation, such as Panicum elephantipes. Junk (1973) also found that this planorbid species was abundant in the roots of “floating meadows” (Paspalum repens Berg, and Echinochloa polystachya (H.B.K.)) in várzea lakes ofthe Amazon River. Conversely, Biomphalaria tenagophila, the other potential transmitter of schistosomiasis, is more com- mon in less oxygenated waters and higher am- monium concentrations, conditions that, to- gether with pH over 8 and temperatures higher than 25°C, may be extremely toxic for aquatic animals due to the high proportion of ammonia (МН.) (Boyd, 1990). These particular limno- logical features are generally associated with continuous floating covers of Pistia stratiotes, with roots that would function as substratum and refuge for B. tenagophila. Drepanotrema kermatoides follows a similar pattern to that of B. tenagophila, while the association of B. straminea with species of Drepanotrema was unclear, except for D. lucidum. This latter species was also found by Junk (1973) occu- pying the same habitat as B. straminea. In this sense, the dominance of Canna glauca and Hydrocotyle ranunculoides also indicates the dominance of Drepanotrema species, as well as lower densities of Biomphalaria species, particularly B. straminea. Among the environmental factors taken into account in the CCA, the regulatory effect of water temperature on population dynamics was already known, as was the importance of calcium concentration related to pH, hardness and macrophytes, on the distribution and abundance of mollusks (Dussart, 1976; Pigott & Dussart, 1995; Thomas, 1982; Rumi & Hamann, 1992). However, only ammonium concentration and percentage of oxygen sat- uration were retained as significant limnologi- cal variables in the process of forward selec- 286 RUMI ET AL. tion, and they were negatively correlated with each other. Biomphalaria tenagophila and D. kermatoides were distributed along this lim- nological gradient, mainly at the highest am- monium concentrations, with optima at 0.98 and 2.00 mg Г', respectively, as well as the lowest oxygen saturation, with optima at 26.6% and 13.0%, respectively. These results suggest that the variance explained by the ex- cluded variables was either unimportant to be selected in the stepwise procedure, or that this variation was better explained by oxygen and ammonium, or even aquatic plants, which were retained first by the statistical method employed, due to their larger explained vari- ance. Therefore, these results do not neces- sarily mean that variables not retained were not relevant for planorbids. The lack of clear seasonal trends in planor- bid communities may be explained because the environments in which this study was Car- ried out are of permanent waters, unlike oth- ers of tropical areas, with well-defined sea- sonality in rainfall. These observations agree with Rumi & Hamann (1992), who compared, in another permanent pond of eastern Chaco in Corrientes Province, rainfall pattern with the relative abundance of Biomphalaria occi- dentalis Paraense, 1981, finding also a very low correlation between those variables. However, in this region of Chaco, inter-annual variability in water availability can be very im- portant, particularly the surplus after evapora- tion, soil infiltration and scouring (Bruniard, 1997). Since environmental variables as well as planorbid population dynamics are related to climatic changes, large variations in planor- bid composition and abundance in samples of the same biotopes from one year to the next may be due to different rainy and drought pe- riods. For example, different amounts of pre- cipitation can affect total biotope area, pro- ducing an increase in planorbid density during dry periods due to retraction in pond area, or an opposite decrease during wet periods due to dilution effects. Furthermore, although ponds are physically isolated most of the time, extensive superficial interconnections among wetlands always take place during rainy episodes or river floods. Thus, migration events in snail populations would be expected at those times. These two processes could provoke rapid changes in abundance, and could partially explain the marked seasonal modifications in snail capture observed in the present paper. Until not long ago, the urban ponds were employed for rubbish deposits, which affected the water quality and planorbid population size, especially during dry climatic events. In addition, populations of B. straminea were much more abundant during the first year, while B. tenagophila showed an increase in population densities during the second year. The non-explained variability in planorbid relative abundance could be attributed to a rather extensive list of possibilities, including the colonization patterns in space and time, sampling biases, and environmental variables not included (i.e., fish and invertebrate preda- tors). First, given the limited capacity of planorbids to disperse and the time required for settled populations to grow, it is possible that even with suitable environmental condi- tions, populations could not reach maximum or stable densities within the sampling period. Second, the observed variation range of envi- ronmental variables and of planorbid assem- blages could be related to the specific envi- ronments selected for sampling, as well as the sampling technique employed. Thus, in- cluding more biotopes and making a greater sampling effort could have resulted in a larger explained variance or in a somewhat different distributional pattern. Finally, the role of predators as controls of planorbid populations cannot be ignored, given that eastern Chaco environments usually hold an abundant and diverse fish fauna (Menni et. al., 1992), with large proportions of invertebrate-eating, in- cluding mollusc-eating, species, such as small to medium-sized silurids, characins, ci- chlids and gymnotids. Therefore, given similar environmental conditions, the presence or ab- sence of predatory fishes could result in dif- ferent planorbid densities and species com- position. From a sanitary standpoint, it is remarkable that the species B. tenagophila and B. straminea, living in urban environments in the Chaco, are natural transmitters of schistoso- miasis in Brazil. Because the presence and abundance of these two planorbid species is explained chiefly by some species of aquatic macrophytes, these variables may be em- ployed as indicators for a rapid assessment of potential sites of proliferation of B. tenago- phila and B. straminea. However, these re- sults must be supplemented with new sam- plings, as well as experimental field and laboratory tests, to confirm the observed trends. POTENTIAL HOSTS OF SCHISTOSOMIASIS IN CHACO 287 ACKNOWLEDGEMENTS This study was made possible by a grant supplied by the Consejo Nacional de Investi- gaciones Cientificas y Tecnicas (CONICET) (AGENCIA PICTO1-03453), in Argentina. Fieldwork and water analyses were facilitated by the logistic support of the Centro de Ecologia Aplicada del Litoral (CECOAL). Lic. Cecilia Longoni de Meabe supervised water quality analyses. We also thank Mr. Luis Benetti by his valuable collaboration in field- work. LITERATURE CITED AUSTIN, M. 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Revised ms. accepted 22 December 2001 MALACOLOGIA, 2002, 44(2): 289-305 EGG MASSES OF CHROMODORID NUDIBRANCHS (MOLLUSCA: GASTROPODA: OPISTHOBRANCHIA) Nerida G. Wilson Centre for Marine Studies, University of Queensland, St Lucia 4072, Queensland, Australia; nwilson O marine.uq.edu.au ABSTRACT The egg mass characteristics of 20 species of chromodorid nudibranchs are presented, rep- resenting the genera Chromodoris, Digidentis, Diversidoris, Glossodoris, Hypselodoris, Noumea and Pectenodoris. The egg mass details for 14 of the included species appear previously un- recorded. These results combined with observations from the literature (comprising a total of 67 species from 15 genera) indicate that most genera in the Chromodorididae show only one type of egg mass. The exception to this is the genus Chromodoris, which includes all three egg mass types outlined in this study. Based on anatomical evidence, a group of flat-spawning Chro- modoris species has been suggested to belong to a monophyletic clade, which indicates that egg mass structure can reflect phylogenetic signal. Genera considered to be more derived among the Chromodorididae are more likely to lay an egg mass that is outward sloping or crenulated. Cadlinella shows spawning traits (flat egg mass containing extra-capsular yolk) that indicate it may be ancestral to the Chromodoris lineage. Developmental data is presented for 11 species of Chromodoris, seven of which utilized extra-capsular yolk in their egg masses. The presence of extra-capsular yolk appears to correlate with upright egg masses within this genus. However, the occurrence of extra-capsular yolk appears to be restricted to Indo-Pacific and Red Sea Chro- modoris species, while upright spawns from Caribbean species lack this feature. Key words: nudibranch, egg mass, chromodorid, reproduction, extra-capsular yolk INTRODUCTION All doridinean nudibranch egg masses, in- cluding those of chromodorids, take the form of a spiral ribbon that is attached to the sub- stratum and consists of embryos embedded in a gelatinous matrix. The shape of the egg mass has been considered to a certain extent to characterize particular groupings (Eliot, 1910; Ostergaard, 1960; Hurst, 1967; Bandel, 1976; Fernandez-Ovies, 1981; Soliman, 1987), and some authors have equated the taxonomic value of the structure of egg cap- sules (and associated coverings) in shelled gastropods with shell, radular and opercular characters (Andrews, 1935). In a recent cladistic analysis, Mikkelsen (1996) confirmed that the opisthobranch order Anaspidea had a particular egg mass type (string) common to all investigated members of the group. An extensive literature exists on the anatomy and systematics of chromodorids. However, comparatively little is known about chromodorid reproduction aside from the mor- phology of the reproductive system itself (Rudman, 1984). In particular, very little is 289 known about the structure of the chromodorid egg mass in species occurring in Australian waters, apart from a few brief descriptions, il- lustrations or unpublished theses (Kenny, 1970; Thompson, 1972; Rose, 1981, 1985; Avern, 1986; Marshall & Willan, 1999). Often descriptions of chromodorid egg masses are brief and include such ambiguous expres- sions as “tangled coils” or “loosely coiled” (MacGinitie & MacGinitie, 1949; Boucher, 1983; Johnson & Boucher, 1983). This gives no indication as to whether these terms per- tain to the distance between the coils, the reg- ularity of coiling, or to the actual attachment of individual whorls of the spiral mass to the sub- stratum. Numerous papers from Japan have significantly improved our understanding of opisthobranch egg masses (Baba & Hama- tani, 1961; Hamatani, 1960a, b, 1961, 1962). However, only one account described chro- modorid nudibranch egg masses (Baba et al., 1956). Other studies may inadequately iden- tify the parent species or give conflicting data, thus rendering the resulting egg mass infor- mation less useful (Bandel, 1976; Barash & Zanziper, 1980; Rose, 1985). However, many 290 WILSON photographic nudibranch identification guides have helped highlight substantial diversity in nudibranch egg mass structure (Gosliner, 1987; Coleman, 1989; Behrens, 1991; Wells & Bryce, 1993; Debelius, 1998). More re- cently, websites have also contributed signifi- cantly to our knowledge of egg masses (for example, www.seaslugforum.net). Opisthobranch egg masses were classified according to a scheme introduced by Hurst (1967). This scheme aimed to describe egg masses in a format that would facilitate com- parison between taxa. As it was based on opisthobranch taxa from the cold temperate northwest coast of the USA, chromodorids were not included, although nearly all egg mass types laid by chromodorids were repre- sented in the scheme by other dorids. Hurst’s scheme consists of four categories, which show some taxonomic correlations, for exam- ple, Type C (jelly bag attached by string) com- mon amongst cephalaspideans and Type D (sac-like structure) typical of very small aeolid nudibranchs. Doridinean spawns were only present in one category that Hurst defined as Type A: “The egg mass is in the form of a ribbon at- tached along the length of one edge, capsules occurring throughout most of it. This is com- mon amongst dorids, which whilst laying may grip the mass between foot and mantle edge tending to flatten it, as mentioned by Fretter & Graham (1964). This is probably not the sole cause of the flattened shape” (Hurst, 1967: 256). Hurst (1967) noted differences in relative lengths of the free edge compared to the at- tached edge of the masses, but did not incor- porate this variation in her classification. Con- sequently, any descriptions of doridinean spawn masses that employ Hurst’s categories define the ribbon as simply being upright. This does not provide enough information to make any comparisons at or below the familial level. Bandel (1976) briefly discussed Hurst’s scheme and proposed 12 of his own group- ings. Many of these groupings were based on a single opisthobranch species, and all dorid- inean egg masses remained in one group, pro- viding no improvement over Hurst's original scheme. Some subcategories were added to Hurst's classification by Fernandez-Ovies (1981), who recognised that the scheme did not adequately describe variation within each type’. While these subcategories were a sig- nificant improvement, they still did not account for all the variation caused by differences in the length of the free edge of upright egg masses and did not account for flat egg masses. In fact, Fernandez-Ovies incorrectly listed Chro- modoris orientalis (as Glossodoris pallescens) laying an upright egg mass, whereas it is actu- ally laid flat (Baba et al., 1956). MacGinitie & MacGinitie (1949) report that some dorids lay flat egg masses, but it is not until much later that this shape is formally recognised in a clas- sification (Soliman, 1987). An unusual feature of egg masses that often goes unnoted is the existence of yolk reserves external to the capsule, but still con- tained within the egg mass. Boucher (1983) termed this “extra-capsular yolk” in a paper that reported the phenomenon in the saco- glossan genera Elysia Risso (Elysiidae) and Bosellia Trinchese (Polybranchiidae) and the chromodorid genera Chromodoris Alder & Hancock and Cadlinella Thiele (Chromodori- didae). Risbec (1928) erroneously interpreted extra-capsular yolk inthe egg masses of Cad- linella ornatissima Risbec as crustacean ova. A few brief descriptions or illustrations have highlighted the presence of extra-capsular de- posits in the egg masses of opisthobranchs (Gohar & Aboul-Ela, 1957; Marcus & Burch, 1965; Gohar & Soliman, 1967a; Kay & Young, 1969), but Thompson (1972) appears to be the first author to formally recognize these bodies as being composed of “yolky material”. The first and only paper to call attention to the widespread occurrence of extra-capsular yolk resulted from a taxonomic survey of opistho- branchs in the Marshall Islands (Boucher, 1983). Since that study, no other literature has specifically addressed the subject of extra- capsular yolk in nudibranch egg masses. The actual deposition of extra-capsular yolk varies between major taxa, and may take the form of granules, caps or blobs in chromodorid nudi- branchs (Boucher, 1983) or yolk strings in sacoglossans (Clark et al., 1979; Clark & Jensen, 1981; Boucher, 1983). Goddard (1991) renamed the phenomenon “extrazy- gotic” and “extra-embryonic” instead of “extra- capsular” in order to include his observation of extra yolky polar bodies inside aeolid nudi- branch capsules. It is unlikely that all of these extra yolk sources are homologous. Although most nudibranchs spawn readily in captivity, perhaps in response to capture and handling stress (Hadfield & Switzer-Dun- lap, 1984), there are few detailed descriptions of egg masses. The aims of the present study are to increase awareness of structural varia- tion in chromodorid nudibranch egg masses, CHROMODORID EGG MASSES 291 and to highlight the presence of extra-capsu- lar yolk in certain taxa. Knowledge of egg mass types and extra-capsular yolk may pro- vide new data to help evaluate existing phylo- genies. MATERIALS AND METHODS Egg Mass Collection, Maintenance and Measurement A total of 20 species were collected for this study from the east coast of Australia during 1998-2001. The austral summer is repre- sented by the months December, January, February; autumn by March, April, May; win- ter by June, July, August, and spring by Sep- tember, October, and November. The majority of specimens were collected using SCUBA, although some material was collected in the intertidal zone. Animals were kept in holding tanks with gentle aeration and flowing seawa- ter. There is some evidence to suggest that egg masses produced after a prolonged pe- riod of stress can be atypical of the species, that is, when organ systems begin to deterio- rate (J. Havenhand, pers. comm.). To mini- mize the likelihood of this effect, only egg masses that were produced in the first week after capture were used in the study. A portion of each egg mass was excised with a scalpel and preserved in either glutaraldehyde (3% prepared in 0.1M sodium phosphate buffer containing 10% w/v sucrose) or neutral- buffered formalin (10%). The remainder of each egg mass was maintained in a holding tank at a water temperature equivalent to that of the collection locality. Portions of the egg mass were observed under a compound mi- croscope at least every second day, and more frequently closer to hatching. The develop- mental period was deemed to cease on the first day that hatching was observed. Un- cleaved ova were measured either from black and white photographs taken using an Olym- pus BH-2 Nomarski contrast compound mi- croscope fitted with an Olympus OM-2N cam- era attachment and Kodak T-max 100 ASA film, or from heat images generated from a National F10 CCD Video attachment to the above microscope, printed on a Sony video graphic printer UP-811. To increase the sam- ple size for comparisons at generic levels, de- scriptions and photographs from the literature were included. However, any data that con- tained ambiguous terminology or was difficult to interpret was excluded. Definition of Egg Mass Types The egg masses examined in this study were classified into three types (Fig. 1) based on whether or not they are attached flat on the substratum or whether they are attached along one edge. Type A egg masses are at- tached to the substratum by the broad side of the ribbon, and are therefore flat. Type B egg masses had a free edge that was either shorter than the attached edge, causing the ribbon to slope toward the centre of the spiral or was equal in length to the attached edge, standing upright. Type C egg masses had a free edge that was either slightly longer than the attached edge, causing the ribbon to slope away from the centre of the spiral or much longer than the attached edge, causing undulations or waves along the ribbon in addition to an outward slope. In some egg masses, the free edge was so long that the ribbon showed tight crenula- tions. Extra-Capsular Yolk Categories Boucher (1983) recognised two categories of extra-capsular yolk in nudibranchs and these are applied here with some modifica- tions. Boucher (1983) defined Type 1 as “cap- like bodies associated with individual cap- sules”. In this study, caps falling into this category were further subdivided into (A) caps that were distributed equally and (B) caps that were distributed unequally (Fig. 2). Type 2, as defined by Boucher (1983), was “discrete yolk bodies strewn throughout the egg mass”, and were not observed during this study, although are known to occur within the genera Cadlinella and Chromodoris (Risbec, 1928; Gohar & Aboul-Ela, 1957; Boucher, 1983). Developmental Type Definitions Where possible, larvae were examined by light microscope (Olympus BH-2 Nomarski contrast compound microscope) and catego- rized into developmental types according to the definitions proposed by Thompson (1967) and Bonar (1978). A well-developed velum and larval retractor muscle, small ova and short embryonic period identified plank- totrophic larvae, whereas lecithotrophic lar- vae typically exhibited a less developed but recognisable velum and larval retractor mus- cle, prominent propodium, and large ova with 292 WILSON FIG. 1. Stylized drawing of the three types of chromodorid egg masses. Type A is laid flat on the substratum, Type B is laid upright and may also slope inwards, and Type C is laid upright, slopes outwards and may be crenulated. FIG. 2. Extra-capsular yolk. A, equally distributed. B, unequally distributed. Scale bar = 100 um. a longer embryonic period (Thompson, 1967). RESULTS Intracapsular development was determined to be either metamorphic (undergoing capslar The developmental characteristics of metamorphosis) or ametamorphic (not under- twenty chromodorid species are described going a typical veliger stage) (Bonar, 1978). below and summarized in Table 1. 293 CHROMODORID EGG MASSES = = — | ou (g =u) LL + 905 yuid g eJe9u!11] SHOPOU8}28d corte (2) vL-21 o1uopyue|d | ou (ZL =Uu) e + 58 wea19 g eqlou вашпом 12 (ks aluoyyue]d = ou = SUM 7) eJAydez sliopojesdAH 22-02 (г) 11-6 oiydonou}19e| | ou (01 =U) + + 9$ эбиело 9 ‘ds suopojasdAH 92-32 ‘ее (Mr (2) 01-6 oiuopqueid | ou (IL =U)G + POL эиум 9 2119540 SUOPOJSSAÄH = = = L ou = MOJ|9Á 9 1Y20]INq SUOPOJSSAÄH 22-21 (1) 99 olydiouejJeue | ou (OL =U) 6L + 00€ weald а edsan иоро$$0/|Е) Gz (1) 8 olydo1jou}I98| L ou = eJIUM q взо/проиоциелте SUOPISISNIT >. = = | ou (1=u) ZL = L6p эбиело а sninqie jo зциар!б!а =. = = ez Vi = eBue1o q ELIOJOUN SUOPOWOIYD фе (1) 8 oluoyyue]|d L ou (LL = U) z + 08 weed У в]еби]$ SUOPOWOIYD 22-02 (1) LE oluoyyue]d Pre al (Ol =U) 8 + LOL WEeaWm-eBueo Я 10901 SUOPOWOIYI се (L) 6 aiydornoyque|d | ou = WEI У 1490] SUOPOWOIYI 22-02 (1) vk oluoyxue]d Z10 | gl (OL =U)E + PO] Wealo-aBuelo а зпрлваоз| зиороишо1ц) 22-02 (1) SL oiuoyyue|d с al (Z=U)Zg +601 шеэл-эбиею g Jaluny SUOPOWOIYI 82-82 Le (1) 9 (1) 9 oluoyyue|d | ou weald v 119yny SUOPOWOIYI ele (1) OL aluoyyue]d L al (g=U)/+28 —швало-эбиело а вошешовб SUOPOWOIYUI 22-12 22 (8) 01-8 (09° otuopquerd | ou (GL =u) © + €6 WeaJo У вищедез!е SUOPOWOIYZ 92-52 (1) 9 Sıydonopyuejd | Vi (8 =u) + 821 Weald g auydep иорошо/цЭ ez (1) 9 oJUoOpuejd | vi -- MOJ|9Á g „роомбииоэ SUOPOWOIYZ (9%) (sassew adh} ensdeo adh} жол (un) azis eno 1nN0[09 adh} salods ainjelodue] 66a :ou) jetuswdojenag jad Je¡nsdeo ssew sÁep ul роиэа ело -211X3 663 91U0Á1qUIY ‘ON a AA __—_—_—_—_— зцоцеларпи риорошолцо эшоз jo sonsiajoeJeyo pejuauwdojanag ‘| FIGVL 294 WILSON Chromodorididae Chromodoris Alder & Hancock Chromodoris collingwoodi Rudman, 1987 A single egg mass was observed in March from an animal collected at North Stradbroke Island, Queensland. The egg mass was up- right, consisting of two whorls slightly sloping inward to centre of spiral. Ova were yellow, and associated with extracapsular yolk of Type 1A. Each capsule contained a single embryo. The embryonic period was six days at 23°C. Larvae were planktonic, although the exact developmental type was not determined. Chromodoris daphne (Angas, 1864) A single egg mass was observed in No- vember from an animal collected at Welling- ton Point, Moreton Bay, Queensland. The egg mass was upright, with the broad side of rib- bon constricted slightly toward centre of spi- ral, giving it a slightly “hour-glass” shape. Two whorls were present, containing cream ova arranged linearly within the spawn mass. Un- cleaved ova were 123 + 5 um in diameter (n = 8). Extra-capsular yolk of Type 1A was present, and each capsule contained a single embryo. The embryonic period lasted six days at 25-26°C, and the veligers that were re- leased were planktotrophic. Chromodoris elisabethina Bergh, 1877 Four egg masses observed from three indi- viduals. The adult nudibranchs were all col- lected from the Gneering Shoals, Mooloolaba, Queensland, and the egg masses laid in Jan- uary and May. All egg masses were laid flat, and while all were spiral in shape, they often ended askew. The egg masses ranged from three to five whorls. Ova were cream and there was no extra-capsular yolk observed. The rate of deposition of the egg mass for one individual was measured at 1.58 mm/min. Un- cleaved ova were 93 + 5 um in diameter (n = 15). Each capsule contained a single embryo. The embryonic period lasted 5-6 days at ap- proximately 27°C (n = 1) and 8-10 days at 20.5-22°C (n = 3). Larvae were planktonic, al- though the exact developmental type was not determined. Chromodoris geometrica Risbec, 1928 Two incomplete egg masses were ob- served in May from two individuals collected at the Gneering Shoals, Mooloolaba, Queens- land. Egg masses were upright and consisted of only a partial whorl (animals tried to lay their spawn on the air-water interface, which could not sustain the weight of more than a partial egg mass). Ova were cream and extra-cap- sular yolk Type 1B was present in the egg mass. The ova were 82 + 7 um (n = 8) in di- ameter. Each capsule contained a single em- bryo. The embryonic period of one of the pieces lasted 10 days at 20.5-22°C. Larvae were planktonic, although the exact develop- mental type was not determined. Chromodoris kuiteri Rudman, 1982 Three egg masses were observed from in- dividuals from Cook Island, New South Wales (n = 1), and Heron Island, Great Barrier Reef, Queensland (n = 2). These were laid in De- cember and March respectively. Egg masses were laid flat, and consisted of 2-5 whorls. The regularity of the coiling varied greatly be- tween egg masses laid by different individu- als. The ova were pale orange in colour, arranged linearly, and no extra-capsular yolk was observed. Each capsule contained a sin- gle embryo. The embryonic period lasted six days at 27°C, and also six days at a variable temperature range of 23-28°C. Larvae were planktonic, although the exact developmental type was not determined. Chromodoris kuniei Pruvot-Fol, 1930 A single egg mass was observed in August from an individual collected from Heron Is- land, Great Barrier Reef, Queensland. The egg mass was upright, consisting of two whorls. The ova were orange and were asso- ciated with extra-capsular yolk Type 1B. The uncleaved ova were 109 + 2 um (n = 7) in di- ameter. Each capsule contained two em- bryos. The embryonic period was 15 days at 20-22°C. Larvae were planktonic, although the exact developmental type remains unde- termined. Chromodoris leopardus Rudman, 1987 A single egg mass was observed in Sep- tember from an individual collected in the Gneering Shoals, Mooloolaba, Queensland. The egg mass was upright, consisting of two whorls. The ova were orange and were asso- ciated with extra-capsular yolk Type 1B. The uncleaved ova were 104 + 3 um in diameter CHROMODORID EGG MASSES 295 (n = 10). Each capsule contained one or two embryos. The embryonic period lasted 14 days at 20-22°C. Larvae were planktonic, al- though the exact developmental type was not determined. Chromodoris lochi Rudman, 1982 A single egg mass was observed in April from a specimen collected at Ribbon Reef Number 10, Great Barrier Reef, Queensland. The egg mass was laid flat and consisted of three whorls. The ova were cream in colour, and no extra-capsular yolk was present. Each capsule contained a single embryo. The em- bryonic period lasted nine days at 25°C, and larvae showed a planktotrophic developmen- tal pattern. Chromodoris roboi Gosliner & Behrens, 1998 Two egg masses have been observed from two individuals collected from Heron Island, and another egg mass was observed from the Whitsunday Islands, Great Barrier Reef, Queensland. The egg masses from Heron Is- land were laid in March and September, and were upright and consisted of two whorls. The ova were orange and were associated with extra-capsular yolk of Type 1B. The ova were 101 + 8 um in diameter (n = 10). Many cap- sules contained multiple embryos; typically they contained two, but up to four were ob- served. The embryonic period of one egg mass lasted 11 days at 20-22°C. Larvae were planktonic, although the exact developmental type remains undetermined. The egg mass observed in the Whitsundays was laid in Au- gust and consisted of one whorl. This egg mass was upright but laid in an irregular spi- ral, so that the whorl consisted of short, straight sections with a distinct kink that joined to another short, straight section. Thus, the egg mass appeared to be crenulated, but both the free edge and attached edge were equal in length. Chromodoris strigata Rudman, 1982 A single egg mass was observed in October from an animal collected on Heron Island, Great Barrier Reef, Queensland. The egg mass was flat, and consisted of five whorls. The ova were pale orange and no extra-cap- sular yolk was observed. The ova measured 80 + 2 um in diameter (n = 11). Each capsule contained a single embryo. The embryonic period lasted eight days at 24°C. Larvae were planktonic, although the exact developmental type remains undetermined. Chromodoris tinctoria (Rüppell & Leuckart, 1828) A single egg mass was observed in March from North Stradbroke Island, Queensland. The egg mass was upright and consisted of three whorls. The orange ova were arranged linearly within the egg mass and were associ- ated with extra-capsular yolk of Type 1A. Each capsule contained two to three em- bryos. The embryos died after 11 days at 22-24°C, and no further details of develop- ment could be ascertained. Digidentis Rudman Digidentis cf. arbutus (Burn, 1961) A single egg mass was observed in Febru- ary from Pt Puer, Tasmania. The animal was disturbed while laying, so the egg mass was incomplete. However, the spawn was upright and quite firm. The ova were orange and no extra-capsular yolk was observed. The ova measured 491 + 12 um (n = 7) in diameter and each capsule contained a single embryo. The developmental type could not be deter- mined, although the large size of the ova po- tentially indicates direct development. Diversidoris Rudman Diversidoris aurantionodulosa Rudman, 1987 Four egg masses were observed in April from Pt. Cartwright, Mooloolaba, Queensland. The egg masses were upright and consisted of one to two whorls each. Two egg masses laid in the laboratory sloped inwards very slightly while those laid in the field appeared typically upright. The ova were cream-white in colour, no extra-capsular yolk was observed and each capsule contained a single embryo. The em- bryonic period lasted 8 days at 25°C, with the larvae showing a lecithotrophic developmen- tal pattern. Glossodoris Ehrenberg Glossodoris vespa Rudman, 1990 A single egg mass was observed in May from an individual collected in the Gneering Shoals, Mooloolaba, Queensland. The egg mass was upright, very firm and consisted of 296 WILSON two whorls. The cream ova measured 300 + 19 um (n = 10) and no extra-capsular yolk was observed. The embryonic period lasted for 56 days at 17-22°C, and the development pat- tern was ametamorphic direct. Hypselodoris Stimpson Hypselodoris bullocki (Collingwood, 1881) Two egg masses were observed from two individuals during November on Orpheus Is- land, Great Barrier Reef, Queensland. They were upright but with the free edge ofthe egg mass sloping away from the centre of the spi- ral. Ova were yellow and no extra-capsular yolk was observed. Each capsule contained a single embryo, but the developmental type re- mains unknown. Hypselodoris obscura (Stimpson, 1855) Four egg masses were observed in total from three individuals. Three egg masses were observed from a pair of nudibranchs col- lected in April from Amity Point, North Strad- broke Island, Queensland, and one egg mass was observed in November from an individual collected from Wellington Point, Moreton Bay, Queensland. The egg masses consisted of 2-5 whorls. Allegg masses were upright but ranged from being slightly outward sloping to having a crenulated free edge. The ova were white, arranged linearly in the egg mass and no extra-capsular yolk was observed. The ova from one egg mass measured 104 + 5 um (n = 11). Each capsule contained a single em- bryo. The embryonic period lasted 9-10 days at 22°C (п = 2) and 4-5 days at 25-26°C (п = 1). Veligers were planktonic, but the exact de- velopment type remains undetermined. Hypselodoris sp. Chromodoris geometrica Coleman, 1981: 32. Misidentified Four egg masses were observed in total from three individuals, all from the Gneering Shoals, Mooloolaba, Queensland. Three egg masses were laid in September and one in January. The egg masses ranged from 1-3 whorls and were upright with the free edge crenulated. Ova were dark orange and no extra-capsular yolk was observed. Ova were 146 + 4um in diameter (n = 10). Each capsule contained a single embryo. The embryonic pe- riod took 9-11 days at 20-22°C (n = 2), and the resulting veligers were lecithotrophic. Hypselodoris zephyra Gosliner & Johnson, 1999 Two egg masses were observed from a single animal in December from Cook Island, New South Wales. The egg masses ranged from 2-3 whorls and were upright with the free edge crenulated. Ova were white and the embryonic period took five days at 27°C. Veligers were planktonic, although exact de- velopmental type was not determined. Noumea Risbec Noumea norba Marcus & Marcus, 1970 Four egg masses were observed from two individuals in May, from the Gneering Shoals, Mooloolaba, Queensland. The egg masses ranged from 2-3 whorls and ranged from up- right to having the free edge sloping toward the centre of the spiral. Ova were cream, arranged linearly and measured 83 + 3 um in diameter (n = 12). Each capsule contained a single embryo. The embryonic period was 12-14 days at 20.5-22°C (n = 2). Veligers were planktonic, although the exact develop- mental type was not determined. Pectenodoris Rudman Pectenodoris trilineata (Adams & Reeve, 1850) One egg mass was observed in August on Heron Island, Great Barrier Reef, Queens- land. The egg mass was firm, and consisted of one whorl. The ova were pale pink in colour and measured 205 + 11 um (n = 8). Each capsule contained a single embryo. Develop- mental details were not recorded. DISCUSSION Although only a small fraction of the spawn- ing details of chromodorid species is known, there is some evidence to suggest that egg mass morphology is consistent within genera and even groups of genera (Table 2). The ob- vious exception to this is the presence of mul- tiple egg mass types within Chromodoris, the TABLE 2. Egg mass types of chromodorid species CHROMODORID EGG MASSES 297 Species Cadlina luteomarginata Cadlina modesta Cadlina pellucida Cadlinella ornatissima Cadlinella sp. Tyrinna nobilis Chromodoris aspersa Chromodoris africana Chromodoris annulata Chromodoris aureopurpurea Chromodoris binza Chromodoris coi Chromodoris collingwoodi Chromodoris clenchi Chromodoris daphne Chromodoris elisabethina Chromodoris geometrica Chromodoris geometrica Chromodoris kuniei Chromodoris kuiteri Chromodoris leopardus Chromodoris lineolata Chromodoris lochi Chromodoris magnifica Chromodoris orientalis Chromodoris perola Chromodoris roboi Chromodoris strigata Chromodoris tinctoria Chromodoris willani Chromodoris woodwardae Glossodoris cincta Glossodoris pallida Glossodoris plumbea Glossodoris sibogae Glossodoris sp. Glossodoris sp. Glossodoris vespa Noumea decussata Noumea haliclona Noumea norba Noumea simplex Verconia verconis Pectenodoris trilineata Digidentis cf arbutus Diversidoris aurantionodulosa Ceratosoma amoena Ceratosoma brevicaudatum Ceratosoma magnifica Mexichromis cf multituberculata Thorunna australis Thorunna daniellae Thorunna florens Thorunna montrouzieri Hypselodoris bullocki Hypselodoris emma A B Egg Mass Type C Source Dehnel & Kong, 1979 Behrens, 1991 Fernandez-Ovies, 1981 Boucher, 1983 Debelius, 1998 Muniain et al., 1996 Gohar & Soliman, 1967b, as C. inornata Pease Gohar & Aboul-Ela, 1957, as C. quadricolor (Ruppell & Leuckart) Gohar & Aboul-Ela, 1957 Baba et al., 1956, as Glossodoris Ortea et al., 1994 Taylor, 2001 present study Ortea et al., 1994 present study Johnson & Boucher, 1983; present study Johnson & Boucher, 1983; Chuk, 2001; present study Rose, 1981; Fraser, 2001a present study; Adams, 2001; Warren, 2001 present study present study Kenny, 1970 present study Klussman-Kolb & Wagele, 2001 Baba et al., 1956, as Glossodoris pallescens Bergh Bandel, 1976 present study present study present study Gill, 2001 Rudman, 1998a Gohar & Soliman, 1967c, as C. obseleta (Rüppell & Leuckart) Soliman, 1987 Gohar & Aboul-Ela, 1959, as G. atromarginata Cuvier Baba et al., 1956 Fraser, 2001b Ostergaard, 1960 present study Johnson, 2001a Avern, 1986; present study present study Johnson, 2001b Debelius, 1998 present study present study present study Coleman, 2001 Smith et al., 1989 Jamieson, 1999, as Miamira Miller, 2001a S. Johnson, pers. comm. Miller, 2001b Coleman, 2001 Rudman, 1998b present study Marshall & Willan, 1999 (continues) 298 TABLE 2. (Continued) Egg Mass Type Species A B C WILSON Source Hypselodoris festiva . Hypselodoris kanga . Hypselodoris maculosa . Hypselodoris obscura ° Hypselodoris sp. . Hypselodoris whitei . Hypselodoris zebra . Hypselodoris zephyra Risbecia ghardagana Risbecia pulchella Risbecia tryoni largest genus in the Chromodorididae. Cur- rently, it is estimated that Chromodoris con- tains approximately 200 species (Gosliner 8 Draheim, 1996), whereas most other chro- modorid genera are considerably less spe- ciose and some are monotypic (eg., Diversi- doris, Verconia). Although the total percentage of Chromodoris species sampled within the present study is very low, all three types of egg mass structure were detected (Table 2). The nine Chromodoris species that are known to exhibit flat egg masses (Type A) occur in two colour groups. Rudman (1977, 1982, 1983) described these groups in order to facilitate identification of similarly coloured species. The first of these groups, the Chro- modoris quadricolor colour group, contains all but two of these flat-spawning species. Based on the distribution of mantle glands and on re- productive characters, it has been suggested that this colour group may represent a dis- crete clade within the genus Chromodoris (Gosliner & Behrens, 1998). This provides fur- ther evidence that egg masses can potentially reflect phylogenetic influence. The remaining species that lay a flat egg mass, Chromodoris aspersa (Gould) and C. orientalis Rudman, both belong to the C. aspersa colour group (Rudman, 1983). These species have long been confused, although external colouration can be used to reliably separate them (Rud- man, 1983). The notal spots in C. orientalis are black, whereas in C. aspersa they are deep purple. The precise nature of the rela- tionship between the two colour groups re- mains to be investigated, but it is interesting to note that most recorded Type A spawners in Chromodoris share a band of orange around the mantle. They also typically possess translucent orange gills and/or rhinophores, and all but C. aspersa share the presence of Baba et al., 1956, as Glossodoris Rudman, 1999 Johnson, 2000 present study present study Johnson & Boucher, 1983, as H. mouaci (Risbec) Geiger, 1999 present study Gohar & Aboul-Ela, 1957 Gohar & Aboul-Ela, 1957 Johnson & Boucher, 1983, as Chromodoris black pigment (present as stripes or back- ground colour in the С. quadricolor colour group and as spots in C. orientalis). Upright egg masses (Type B) were present in at least 13 of the 24 species of Chromodoris species listed in Table 2. There was some dif- ficulty in classifying the egg masses of Chro- modoris coi, C. kuniei and C. roboi. These species all lay ribbons that in most cases are upright, but are often attached in short kinks that cause them to appear outward sloping. These egg masses may also have grooves on the broad side of the ribbon running parallel to attachment, although the significance of this is unclear. The two reports of aclearly crenulated egg mass (Type C) occurring in Chromodoris warrant further attention. Chromodoris mag- nifica falls into the C. quadricolor colour group of Rudman and would thus be predicted to lay a flat egg mass similar to all other known mem- bers of the group. However, Klussmann-Kolb & Wagele (2001) report C. magnifica laying an upright and crenulated egg mass, although further observations are desirable to confirm the report. Similarly, conflicting reports occur regarding the egg mass of Chromodoris geo- metrica. Boucher (1983) recorded C. geomet- rica in the Marshall Islands with an upright, or- ange egg mass containing extra-capsular yolk. This study confirmed that report for spec- imens from subtropical eastern Australia, and an upright orange ribbon was also reported from Papua New Guinea (Chuk, 2001). How- ever, Rose (1985) recorded some spawning details of C. geometrica from temperate east- ern Australia and reported an absence of extra-capsular yolk. It is only in his unpub- lished thesis (1981) that he describes the egg mass as being fluted. Although it is quite pos- sible that the single specimen that Rose col- lected was misidentified, Fraser (2001a) also CHROMODORID EGG MASSES shows an egg mass of C. geometrica that is clearly crenulated. This latter observation from South Africa also differs from all previous ac- counts in that the colour of the egg mass is white. It is likely that this egg mass lacked extra-capsular yolk as well, as the resulting or- ange hue is usually visibletothe naked eye. As both Rose (1981) and Fraser (2001a) made their observations at similar latitudes in the Pa- cific and Indian oceans respectively (approxi- mately the southermost limits for C. geomet- rica), it is possible that the production of extra-capsular yolk reserves is related to tem- perature. An upright egg mass structure (Type B) was found in all species of Glossodoris, Noumea, Verconia, Pectenodoris, Digidentis and Diver- Ceratosoma FN = Pectenodoris _ Glossodoris 0) Verconia Ardeodoris ? x Chromodoris ~ Noumea 299 sidoris represented in Table 2. According to the first phylogeny proposed for the Chro- modorididae (Rudman, 1984), all these gen- era are typically considered in the “mid re- gion” of evolution within the family, neither basal nor highly derived (Fig. 3). Gosliner & Johnson's (1999) cladistic analysis found no resolution between the lineage containing Chromodoris, Ceratosoma and Glossodoris and the one containing Noumea, Pecten- odoris, Verconia, Thorunna and Digidentis (Fig. 4). However, both phylogenies agree that the crown group within one lineage con- sists of Risbecia + Hypselodoris. This crown group (with the exception of Hypselodoris zebra) all show egg masses that are either outwardly sloping or crenulated. This indi- Hypselodoris Risbecio uses) 2 Ourvilledoris Mexichromis = x x \ \ x Thorunna DE Digidentis x { ; | EZ : N x ‘ Cadlina Codlinella C= N re) )) LAS: FIG. 3. Egg mass shape mapped onto hypothesized phylogeny of the Chromodorididae (from Rudman, 1984). 300 St a Pao’ | < У < > > os RS Se x so Sl WILSON Nm) AP a ¿Y x © Se ESOS ES LEN N RE ER Resigns oe № Qe con с © У ce «3 ce FIG. 4. Egg mass shape mapped onto hypothesized phylogeny of the Chromodorididae (from Gosliner & Johnson, 1999). Broken lines indicate that the spawn type illustrated above is also present in the genus. cates that the most highly derived forms are more likely to lay egg masses that have the free edge of the ribbon lengthened, resulting in outward sloping or crenulated egg masses. As the structure of an egg mass is said to re- flect the degree of anatomical complexity of the reproductive system (Fretter & Ko Bun, 1984), it is likely that more highly derived gen- era would lay more complex egg masses. The single Known exception in Hypselodoris (H. zebra) shows an upright egg mass. This is the only egg mass known for a member of the At- lantic/Eastern Pacific clade of the genus. Hypselodoris is known to consist of two clades, the above-mentioned clade and an Indo-Pacific clade (Gosliner & Johnson, 1999), which is represented by all the other Hypselodoris egg masses observed in this study. The genus Thorunna is considered rela- tively derived in both phylogenies of the Chro- modorididae, and the egg mass data from Table 1 appear to sustain this. Thorunna has been proposed as a sister group to both Digi- dentis (Rudman, 1984) and Durvilledoris (Gosliner & Johnson, 1999). Observations on the egg mass of Digidentis offers no firm evi- dence in support of this relationship, and the egg mass structure of Durvilledoris species remains unknown. Ceratosoma is closely allied to Chro- modoris and Glossodoris, according to the phylogeny of Gosliner & Johnson (1999). This contrasts with Rudman (1984), who places it in the “hypselodorid” subgroup (also contain- ing Digidentis, Thorunna, Durvilledoris, Mexi- chromis, Risbecia and Hypselodoris), consid- ering Ceratosoma to have a Chromodoris ancestor but having diverged early in hypselo- dorid evolution. Here, the egg masses of three Ceratosoma species are reported to be outwardly sloping or crenulated, indicating a derived condition. Extra-capsular yolk reserves have so far been recorded in only two chromodorid gen- era, Cadlinella and Chromodoris, with five new records in the present study (Table 3). While extra-capsular yolk is found in the flat egg masses of Cadlinella, the flat egg masses of Chromodoris never contain these reserves. It is only in the upright egg masses of Indo-Pa- cific Chromodoris species that extra-capsular yolk is present. Chromodoris binza and Chro- modoris clenchi, both found in the Caribbean, lay upright egg masses but do not incorporate extra-capsular yolk reserves into the egg CHROMODORID EGG MASSES 301 TABLE 3. Chromodorids that produce extra-capsular yolk. Species Cadlinella ornatissima Chromodoris albopunctatus Chromodoris albopustulosa 1A Chromodoris annulata Chromodoris collingwoodi 1A Chromodoris daphne 1A Chromodoris decora 1A Chromodoris E-6 1A Chromodoris fidelis 1A Chromodoris galactos 1A Chromodoris geometrica 1B Chromodoris kuniei 1B Chromodoris leopardus 1B Chromodoris marginata 1A Chromodoris preciosa Chromodoris горо! 1B Chromodoris rubrocornuta Chromodoris E-328 Chromodoris E-48 Chromodoris thompsoni 1A Chromodoris tinctoria 1A Chromodoris vibrata mass (Ortea et al., 1994). It will be of great in- terest to determine whether extra-capsular yolk is restricted solely to Indo-Pacific and Red Sea Chromodoris. While Cadlinella sp. from the Red Sea does lay flat egg masses, they have not yet been examined to deter- mine if they also contain extra-capsular yolk like Cadlinella ornatissima. While Cadlina, Tyrinna and Cadlinella are all currently considered basal within the Chro- modorididae, there appears to be no indica- tion that these three genera are themselves closely related (Rudman, 1984). It is therefore no surprise that these genera exhibit different egg mass types. While varying hypotheses regarding the basal chromodorids have been proposed or supported (Rudman, 1984; Muni- ain et al., 1996; Gosliner & Johnson, 1999), the most recent discussion concludes only that the phylogeny of these basal groups re- mains unclear (Schródl & Millen, 2001). Given that Cadlinella shares a flat egg mass and extra-capsular yolk with varying Chromodoris species, it is likely that Cadlinella gave rise to the Chromodoris lineage. There is some concern that egg mass structure may reflect environmental rather than phylogenetic influences (Wagele & Willan, 2000), and is therefore not suitable to be used as a Character in phylogenetic analy- ses. Observations on spawning in the field and laboratory have shown some differences, Yolk type 2, small & pale 2, large & orange 2, large & orange not determined not determined not determined not determined not determined Original source Risbec, 1928 Boucher, 1983 Kay & Young, 1969 Gohar & Aboul-Ela, 1957 present study present study Kay & Young, 1969 Boucher, 1983 Marcus & Burch, 1965 Boucher, 1983, as E-57 Boucher, 1983 present study present study Boucher, 1983 S. Johnson, pers. comm. present study S. Johnson, pers. comm. S. Johnson, pers. comm. S. Johnson, pers. comm. Thompson, 1972, as C. loringi Gohar & Soliman, 1967 S. Johnson, pers. comm. which have been incorporated into the egg mass classification in this study. Specimens of Noumea norba and Diversidoris aurantio- nodulosa laid upright ribbons in the field, while the same specimens in the laboratory laid egg masses that sloped inward. Specimens of Hypselodoris obscura lay egg masses that range from outward sloping to crenulated. Some egg masses, particularly those that are thin and flaccid, can appear slightly fluted when laid on irregular or uneven substrata. However, it is possible to differentiate be- tween these and egg masses that are truly outward sloping or crenulated by comparing the length of both the free and attached edges. The regularity of the coiling, that is, the space between the whorls of one egg mass, differed greatly within a species, suggesting this may be affected by environmental condi- tions or perhaps the reproductive history of the parent. It is not yet possible to make any correlation between egg mass type and the habitat of the parent nudibranch, since there is little information regarding movement within the latter. Many species of nudibranch are found in both intertidal and subtidal environ- ments without showing any obvious change in egg mass structure. However, controlled ex- periments varying such factors as tempera- ture, salinity and water flow are desirable to test this idea. There are apparently conflicting reports 302 WILSON where the colour of aegg mass has been re- ported to differ between localities, even when extra-capsular yolk is absent. Johnson & Boucher (1983) reported that Hypselodoris maculosa lays a pale pink egg mass, whereas Marshall & Willan (1999) recorded it as white. While it is possible that a change in prey items may trigger a corresponding change in ova colour, differences may also reflect subjective interpretation of colour. It is also important to know whether the animal laying the egg mass is identified correctly. Hypselodoris maculosa individuals are known to be variable in colour, and there is the possibility that a complex of species is currently identified as a single species. Egg masses may have the potential to help separate these complexes but need to be used in conjunction with morphological data from the parent specimens. Another fac- tor that can influence the colour of an egg mass is the amount of time that has elapsed since it was laid. Gradual colour changes occur as the developing embryos use up the available yolk. However, while small changes in colour may be attributed to such factors, real disagreement in colour may reflect differ- ences in the identity of the parent. Risbecia tryoni has been reported to lay a rose-pink egg mass with a crenulated free edge (John- son & Boucher, 1983), whereas Marshall & Willan (1999) reported the mass as orange but do not describe its structure. Marshall & Willan (1999) also incorrectly cited Johnson & Boucher as attributing extra-capsular yolk to this species. While the general form of the egg mass is usually characteristic of a species or genus, Rudman & Avern (1989) found both upright and crenulated egg mass types in the rela- tively small genus Rostanga (approximately 13 species). This was also the case for Acan- thodoris (Hurst, 1967), in which both upright and crenulated egg masses were recorded. This indicates that some caution may be nec- essary when interpreting phylogenetic signal from egg mass structure, as it may be useful at different taxonomic levels in different groups. The absence of a fossil record means there is currently no reliable method of dating genera. It may only be in more recent genera that egg mass structure remains conservative throughout. It is possible that the trend to- wards crenulation of egg masses in the more derived Chromodorididae is also seen within a single “older” genus that has had more time to evolve. Soliman (1987) recognized the potential taxonomic value of egg mass type among gastropods, but he recommended that when interpreting phylogenetic relationships, pri- mary consideration should be given to ana- tomical, palaeontological or ecological evi- dence. Because no fossil record exists for the Nudibranchia, and accurate ecological infor- mation is still scarce for most groups, alterna- tive characters may be found in reproductive data. Egg mass structure may help confirm or challenge phylogenetic hypotheses based solely on anatomical data, but much work is still required to understand the underlying causes of observed variation. ACKNOWLEDGEMENTS Support for this project was obtained from a University of Queensland Research Grant, a Mollusc Research Grant from the Malacologi- cal Society of Australasia, and the Undersea Explorer, Port Douglas. | would like to thank many people for assistance in collecting ani- mals, namely Dan Jackson, Suzie Green, David Harris, Shane Litherland and Shireen Fahey. Bill Rudman assisted with identifica- tions, and Scott Johnson provided valuable discussion and observations. Maria del Car- men Gömez-Cabrera and Rosa Garcia Novoa are thanked for their assistance in translating the work of Fernandez-Ovies. This manu- script was improved by comments from John Healy, Bill Rudman and two anonymous re- viewers. | would like to acknowledge the Great Barrier Reef Marine Park Authority (G98/110), Queensland Parks and Wildlife Service (QSE99/489) and the Department of Primary Industries, Water and Environment, Hobart (P99/00-121) for allowing respective collection permits. 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Western Australian Museum, Perth, 184 pp. Revised ms. accepted 13 February 2001 MALACOLOGIA, 2002, 44(2): 307-324 ALLOZYMIC TAXONOMY WITHIN THE GENUS MELANOPSIS (GASTROPODA: CERITHIACEA) IN ISRAEL: A CASE IN WHICH SLIGHT DIFFERENCES ARE CONGRUENT Andrzej Falniowski', Joseph Heller’, Magdalena Szarowska', & Krystyna Mazan-Mamczarz' ABSTRACT Molecular differences between the species of Melanopsis distinguished by Heller were stud- ied by means of cellulose acetate gel allozyme electrophoresis on 26 Melanopsis populations from Israel: 6 M. buccinoidea Olivier, 1801; 8 M. saulcyi Bourguignat, 1853; 1 M. meiostoma Heller & Sivan, 2001; and 11 M. costata Olivier, 1804, represented by two subspecies: M. costata costata Olivier, 1804, and M. costata jordanica Roth, 1839. Fourteen loci (9 polymorphic) were scorable: Aat, Alp, Est-1, Est-2, Gpi, Hbdh, Idh-1, Idh-2, Iddh, Mdh, Mdhp, Mpi, Pgdh, Pgm. Mean sample size was 28.983; mean number of alleles per locus: 1.45 (1.1-1.7); polymorphic loci per population: 36.5% (14.3-57.1); mean observed heterozygosity: 0.085 (0.041-0.121); mean ex- pected heterozygosity: 0.112 (0.045-0.170). Nei genetic distance (0.000-0.232, mean 0.0761, no significant association with geographic distance) and Cavalli-Sforza and Edwards arc genetic distance (0.054-0.441, mean 0.240, significant association with geographic distance) were com- puted for pairs of populations. Interpopulation differences were analyzed phenetically (corre- spondence analysis, UPGMA), and phylogenetically (neighbor-joining and Fitch-Margoliash ad- ditive trees). Despite the slight differentiation of the taxa, the results confirmed the distinctness of M. costata costata, M. costata jordanica and M. saulcyi although the speciation processes may not yet be completed. The molecular variability of М. buccinoidea was wider than that of all the other studied taxa together, and overlapped the variation ranges of each. It may be ancestral species to the others, which may have speciated as peripheral isolates. The position of M. meios- toma as a distinct species was not confirmed. Key words: freshwater snail, species, allozymes, electrophoresis, phenetics, phylogenetics. INTRODUCTION The freshwater snail genus Melanopsis (Ferussac, 1807) (Prosobranchia: Melanopsi- dae) is one of the most abundant mollusks in the lakes, rivers and springs of the Middle East, and is widely distributed throughout the circum-Mediterranean (Germain, 1921-22; Bilgin, 1973; Tchernov, 1973, 1975; Schütt & Bilgin, 1974; Banarescu, 1990-95). Its anat- omy was described by Bilgin (1973) and Al- taba (1991), sperm structure by Afzelius et al. (1989), radula variation by Glaubrecht (1993), and allozyme variation in the western Mediterranean by Altaba (1991), who found considerable genetic differentiation over short distances. There are numerous studies on mollusk allozymes, devoted to molecular tax- onomy (Woodruff et al., 1988; Borsa & Ben- zie, 1993; Ponder et al., 1993; Davis, 1994; Haase, 1994; Falniowski et al., 1996), includ- ing in Melanopsis (Altaba, 1991; Altman & Ritte, 1996). In Melanopsis, the shell is extremely vari- able, with smooth and costate, elongate and stout, banded and all-black forms found in a relatively small area. Heller et al. (1999) in- vestigated the systematics, distribution and extent of presumed hybridization of Melanop- sis in the central and northern Jordan Valley. Basing their study on the shell morphometry of 700 snails collected at 35 sites, they recog- nized three species: snails with smooth shells (M. buccinoidea Olivier, 1801); with straight- ribbed shells that almost always extend the entire length of the whorl (M. costata Olivier, 1804); and with tubercle-ribbed shells that do not extend the entire length (M. saulcyi Bour- guignat, 1853). Melanopsis buccinoidea is common in a wide variety of aquatic habitats, from small trickles to springs and streams; M. costata occurs in the upper Jordan River, "Department of Malacology, Institute of Zoology, Jagiellonian University, ul. Ingardena 6, 30-060 Kraköw, Poland; faln @ zuk.iz.uj.edu.pl Department of Evolution, Systematics and Ecology, Hebrew University, Jerusalem, Israel 308 FALNIOWSKI ET AL. Lake Kinneret and lower Jordan River; M. saulcyi in streams that border swamps. Heller et al. (1999) also reported the occur- rence of presumed hybrids characterized by shells with weak ribs, thus defined based on shell characters alone. According to those au- thors, hybrids were found in narrow zones of contact, both between M. buccinoidea and M. costata, and between M. buccinoidea and M. saulcyi. Outside these zones, the species re- tain their distinct conchological identities. The narrowness of the hybrid zones suggested to Heller et al. (1999) that selection was main- taining the parapatric distribution of the species involved, with the narrow hybrid zones acting as partial barriers to gene flow. Between M. buccinoidea and M. costata, the hybrid zones were at habitat transition, from streams to lake. Within M. costata, Heller et al. (1999) dis- tinguished three subspecies. Two of them are dealt with in the present paper: M. costata costata Olivier, 1804 (shell elongate; the upper Jordan River, on mud and submerged vegetation) and M. costata jordanica Roth, 1839 (shell usually stout; Lake Kinneret, on stones and boulders). The stout shell of M. costata jordanica seems correlated with a stormy habitat. Recently a new species, M. meiostoma, was described from Israel (Heller & Sivan, 2000) characterized by a small, slender shell with relatively small mouth and weak callus. The aim of the present study is to test whether the morphological differences be- tween the species and subspecies distin- guished by Heller et al. (1999) are accompa- nied by the molecular differences. The morphologies are rather finely differentiated: in fact, the differences are marked in charac- ters that are prone to environmental factors (e.g., Gostan, 1966; Palmer, 1985; Falniow- ski, 1988). Thus, our null hypothesis is that the entire studied group is a single, concho- logically variable species, with shell variation determined environmentally, not genotypi- cally. The latter is not a rare phenomenon within the Gastropoda. It is essential in any discussion on species distinctness to specify the species concept applied. In this study, we follow the cohesion species concept, as de- fined by Templeton (1989), considering not only genetic but also demographic and eco- logical factors. In particular, speciation is re- garded as the development of cohesion mechanisms, not isolation mechanisms. The cohesion concept assumes, as cohesion mechanisms, not only genetic exchangeabil- ity, but also demographic exchangeability. The latter means that all the populations of a species must share the same fundamental niche, as defined by Hutchinson (1965). Thus, to demonstrate that the fundamental niches of two taxa are not the same means to prove their species distinctness. The cohesion con- cept covers the full range of biological reali- ties, from completely closed systems of asex- ually reproducing taxa to open systems of syngameons, with species defined practically based solely on demographic exchangeabil- ity. It must be stressed that speciation is rather a process, not an event (Templeton, 1989). Hence, wherever the process is still going on and not completed, there obviously will be the “bad” species undergoing speciation as op- posed to the “good”, well-defined ones. The cohesion concept is, therefore, what applies best to such cases. MATERIAL AND METHODS Locality Description The distribution of the localities is shown in Figure 1. Melanopsis buccinoidea (1) Enot Huga (IG —New Israel Grid—200- 213). Within a recreation site, a small spring that irrigates by way of a recently constructed channel into a small pool consisting of boulders and cobble; snails collected from the channel, at a depth of 2-3 cm. (2) En Hanatziv (IG 197-208). A spring within a large natural pool; snails col- lected from dead, floating vegetation and firm boulders. (3) En Raqgat, spring (IG 199-245). Emerg- ing 15 m from the shore of Lake Kinneret, this spring trickles down into the lake among boulders and sparse, swampy vegetation; snails collected along the trickle, at a depth of 2-3 cm. (4) Dan (IG 212-295). A lowland stream that forms the principal tributary of the Jordan River; the physico-chemical aquatic envi- ronment extremely stable: annual as well as diurnal water temperatures virtually constant, at 15.5°C (+ 0.5%), chemical parameters (NH,, PO,, NO,) vary little, dissolved oxygen concentrations uni- formly high at 95% of saturation (Heller et al., 1997); snails collected from several ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 309 ha Y E [> 4 £ a 2 Е © Lake Kinneret FIG. 1. Distribution of studied localities. Black arrow in the small map at the bottom indicates the area presented in the bigger map. Population numbers: see text. Symbols of taxa: see Fig. 2. small brooks near the spring, from boul- ders at a depth of 5-10 cm. (6) Banias (IG 214-294). A cool spring form- ing a major source of the Jordan River; snails collected from the artificial pool surrounding the spring, at a depth of down to 30 cm. (7) Nahal Tavor (IG 201-223). A narrow, shallow stream covered by dense vege- tation; snails collected from within the stream, on the muddy substratum, at a depth of 2-3 cm. Melanopsis meiostoma (8) Kefar Haruv (IG 211-240). A small, spring that pours into a small cement pool; brackishwater (a high, 670 mg/l, content of chloride anions); snails col- lected from the walls of the pool, at a depth of 30 cm. Melanopsis costata jordanica Lake Kinneret (21 km x 12 km, 170 km). In its natural condition, the water level of Lake Kinneret used to fluctuate annually at an av- erage of 0.8-1.0 m. In 1964, the lake was dammed, its level increased by 1 m, and was left at peak levels for much longer periods than in its natural condition. Further, in its nat- ural condition, the lake’s salinity was almost 400 mg/l (chlorinity). Recent diverting of saline springs from the lake has decreased its salinity to about 200 mg/l. Daily storms fre- quent the lake, the shores of which consist of gravel, cobble, stones, mud, sand, and silt. There were six localities at Lake Kinneret: (9) Ginossar, Kinneret (IG 209-250). A nat- ural shore of boulders and stones; snails collected along the shore at a depth of 5-10 cm. (10) 208, Kinneret, (IG 209-253). A natural shore of boulders and stones; snails col- lected at a depth of 5-10 cm. (11) En Raggat, Kinneret (IG 199-245). A nat- ural shore of boulders and stones; snails collected along the shore at a depth of 5-10 cm. (12) En Sheva, Kinneret (IG 201-252). A nat- ural shore of boulders and stones, snails collected along the shore at a depth of 3-10 cm. (13) Ha’on, Kinneret (IG 208-237). A shore consisting of muddy sand, with a few submerged dead reeds; snails collected from the reeds, at water level. (14) Bet Gavriel, Kinneret (IG 205-234). A re- cently constructed retaining wall con- 310 FALNIOWSKI ET AL. sisted of large basalt boulders; snails col- lected at a depth of 5-10 cm. Melanopsis costata costata (15) Gesher Benot Ya'aqov, upper Jordan River (IG 209-269). Muddy substratum, willows along the banks; snails collected from vegetation, a few cm above the water line. (16) Gesher Lehavot, upper Jordan River (IG 209-283). Muddy substratum, willows along the bank; snails collected from veg- etation a few cm above the water line. (17) Eastern Canal of the upper Jordan River, (IG 208-278). Muddy substratum, wil- lows along the banks; snails collected from the muddy substratum. (18) Bet Hillel (IG 207-289). A tributary of the Jordan River, mainly with mud and cob- ble along the banks; snails collected from the cobble. (19) Sede Nehemia (IG 208-288). A tributary of the Jordan River; within a recreation site; snails collected from the mud and from submerged stems, branches and tree trunks. Melanopsis saulcyi (5) Sede Eliyahu (IG 198-204). A small arti- ficial pool, with a muddy substratum; snails collected from dead vegetation floating in the water. (20) The same locality as 2. (21) Sheluhot (IG 196-209). A cement chan- nel in which water from several springs flows with a rather strong current; snails collected from the walls of the channel, at a depth of 30 cm. (22) Hamat Gader, 15 m from spring (IG 213-232). A small artificial pool into which the Hamat Gader spring flows; snails collected from boulders and stones along the shores of the pool. (23) The same locality as 7. (24) The same locality as 1. (25) Hamat Gader, spring (IG 213-232). A pool in which a spring gushes; snails col- lected off logs and various dead branches, floating in the water. (26) En Malkoah (IG 198-198), a small stag- nant water body; on substratum and plants. Electrophoretic Techniques The snails were transported alive to Poland and then frozen and stored in a deep freezer, at —80°С. Unsexed individuals for electro- phoresis were dissected on ice, and their he- pato-pancreas tissue was taken for homoge- nization. The tissue was homogenized on ice, in a glass homogenizer in 50-100 ul of ho- mogenizing solution (100 ml distilled water, 10 mg NADP, 10 mg NAD, 100 ul B-mercap- toethanol), depending on snail size. The ho- mogenates were centrifuged at 11,000 rpm for 10 min, then used immediately for cellu- lose acetate electrophoresis following the pro- tocol of Richardson et al. (1986). The follow- ing running buffers were used, depending on enzyme system: citrate-phosphate pH 6.4 (buffer A), phosphate pH 7.0 (buffer В), or tris- maleate pH 7.8 (buffer C). The position of ori- gin was cathode, the duration of run 1.5 h at 220 V. Staining protocols followed Richardson et al. (1986). The cellogels were from MALTA, Italy, all other chemicals from SIGMA, USA. Enzyme names and EC numbers are after Murphy et al. (1996). Numerical Techniques Genetic variability parameters and genetic distances were computed with BIOSYS-1 (Swofford & Selander, 1981). One of the two distances we had chosen was Nei unbiased distance. This is the most commonly used ge- netic distance, and so it is the best for com- parisons with the literature. The other one was Cavalli-Sforza and Edwards arc distance; this is not affected by intrapopulation polymor- phism and is the most appropriate when a group of rather closely related populations is concerned. GENEPOP (Raymond & Rousset 1995) was used to compute exact tests for HWE (Guo & Thompson 1992; Rousset & Raymond 1995), as well as f (equaling F, of Wright) for each population and for each locus. Following the notation and computa- tional procedure introduced by Weir & Cock- erham (1984) and Weir (1990), F-statistics was computed over all populations with Weir’s FSTAT (Weir, 1990; Goudet, 1995), the confidence intervals estimated with jackknife and bootstrapping techniques (Weir, 1990). The same procedure was applied to each of the taxa represented by more than one popu- lation (i.e., except М. meiostoma). Mantel tests, correspondence analysis, nonlinear multidimensional scaling, minimum-spanning tree, clustering, and cophenetic distances were computed with NTSYSpc v.2.0 (Rohlf, 1998). Mantel tests were used for testing the association between the genetic and geo- ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 311 graphic distances between the populations, as well as for evaluation of the phenograms and testing the association between the origi- nal and cophenetic distances. Additive neigh- bor-joining trees (Saitou & Nei, 1987) and Fitch-Margoliash trees (Edwards & Cavalli- Sforza, 1964; Fitch & Margoliash, 1967; Swof- ford & Olsen, 1990; Swofford et al., 1996) were computed with NEIGHBOR and FITCH, respectively, of the PHYLIP 3.6 package (Felsenstein, 1990). From the same package, the program SEQBOOT was applied to gen- erate 200 bootstrap pseudosamples. For each ofthem, Cavalli-Sforza and Edwards arc distances were computed with GENDIST (the source code of Felsenstein modified for arc instead of chord version of that distance), and the resulted file with 200 pseudosamples of distance matrices processed with FITCH with the global search option. Finally, the extended majority rule consensus tree was computed with CONSENSE of PHYLIP 3.6 package. RESULTS Twelve enzyme systems coded by 14 loci gave well resolved and always interpretable results: Aat (aspartate aminotransferase, EC 2.6.1.1., buffer B), Alp (alkaline phosphatase, EC 3.1.3.1, buffer C), Est-1 (esterase, locus 1, nonspecific, buffer A), Est-2 (esterase, locus 2, non-specific, buffer A), Gpi (glucose-6- phosphate isomerase, EC 5.3.1.9, buffers A, C), Hbdh (3-hydroxybutyrate dehydrogenase, EC 1.1.1.30, buffer C), Idh-1 (isocitrate dehy- drogenase, locus 1, EC 1.1.1.42, buffer C), Idh-2 (isocitrate dehydrogenase, locus 2, EC 1.1.1.42, buffer C), Iddh (L-iditol dehydroge- nase, EC 1.1.1.14, buffer C), Mdh (malate dehydrogenase, EC 1.1.1.37, buffer C), Mdhp [malate dehydrogenase (NADP*), EC 1.1.1.40, buffer C], Mpi (mannose-6-phos- phate isomerase, EC 5.3.1.8, buffers B, C), Pgdh (phosphogluconate dehydrogenase, EC 1.1.1.44, buffers B, C), and Pgm (phos- phoglucomutase, EC 5.4.2.2, buffer C). Nine of the loci (64.3%) were polymorphic: Alp, Est- 1, Est-2, Gpi, Hbdh, Idh-1, Idh-2, Pgdh, and Pgm. Allele frequencies in those loci are pre- sented in Table 1. Mean sample size was 28.983 (Table 2). Mean number of alleles per locus averaged 1.45, varying from 1.10 in population 8 to 1.7 in population 12. Mean values for the taxa were 1.43 for Melanopsis buccinoidea, 1.10 for M. meiostoma, 1.50 for M. costata jorda- nica and M. costata costata, and 1.43 for M. saulcyi. On average there was 36.5% of the polymorphic loci per population, varying from 14.3% in population 8 to 57.1% in population 11 (Table 2). Mean values for the taxa were: 39.3% for M. buccinoidea, 14.3% for M. meiostoma, 40.5% for M. costata jordanica, 32.9% for M. costata costata, and 36.6% for M. saulcyi. Mean observed heterozygosity (Table 2) varied from 0.041 in population 13 to 0.121 in population 10, mean for all the popu- lations: 0.085. Mean expected heterozygosity (Table 2) ranged from 0.045 in population 8 to 0.170 in population 22, mean for all the popu- lations: 0.112. Departures from Hardy-Wein- berg equilibrium (HWE), F-statistics, linkage disequilibrium, estimates of gene flow, and detailed analysis of the genetic structure of the studied populations are presented in Fal- niowski et al. (in press). The multi-locus (by population) tests per- formed with GENEPOP showed statistically significant heterozygote deficits in most popu- lations. The multi-population (by locus) tests indicated a statistically significant deficiency of heterozygotes at all loci, except Idh-1 and Pgdh. Four populations (8, 17, 18, 25) were at HWE or had all loci monomorphic. Heterozy- gote excess was observed at Est-2 in popula- tion 10, at Gpi in 21, and at Pgdh in 1. All the other departures from HWE were heterozy- gote deficits. The ratio of the number of popu- lations at disequilibrium to the number of pop- ulations with a given locus polymorphic varied from 9.1% at Idh-1 to 59.1% at Est-1. Statisti- cally significant values of f were found in 13 populations for Est-1, in 9 for Gpi, in 8 for Hbdh, in 7 for Est-2, in 3 for Alp and Pgm, in 2 for Idh-2 and Pgdh, and in 1 for Idh-1 (Fal- niowski et al., in press). The results of FSTAT for all populations indicated HWE for Est-2, Idh-1, and Pgdh: for all those loci f values were not statistically significant and F approx- imated 6. The highest values of f were found for Idh-2, Alp and Est-1. For all the polymor- phic loci, both Е and 6 differed significantly from 0. The highest 6 was found for Pgm (0.770), the lowest for Idh-2, Alp and Gpi. The values and confidence intervals for particular loci did not overlap. The highest values of F were found for Pgm, Est-1 and Idh-2, the low- est for Pgdh. 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Genetic variability at 14 loci in all populations (standard errors in parentheses). Mean heterozygosity Mean Mean no. Percentage sample size of alleles per of loci HdyWbg Population per locus locus polymorphic* Direct count expected”. Melanopsis buccinoidea 1. Enot Huga 31-910 1) 1.5102) 42.9 0.116 (+0.053) 0.133 (0.053) 2. En Hanatziv 8.0(+0.0) 1.45 (= 0:2) 28.6 0.089 (+0.062) 0.140 (+0.063) 3. En Rakkat spring 41.0 (+2.4) 1.4 (+0.1) 42.9 0.092 (+0.032) 0.131 (+0.049) 4. Dan 30:9(=0:9)= 1.501) 50.0 0.090 (+0.050) 0.090 (+0.039) 6. Banias 29.4 (+0.3) 1.4 (+0.2) SET 0.068 (+0.033) 0.117 (+0.048) 7. Nahal Tavor AAA 16) - 1.4204) 357 0.053 (+0.023) 0.102 (+0.046) M. meiostoma 8. Kefar Haruv 19/0119) 1.1 (E05) 14.3 0.042 (+0.029) 0.045 (+0.032) M. costata jordanica 9. Ginnossar 361111) 1.4(Е0:2) 35.7. 0.074 (+0.030) 0.106 (+0.043) 10. 208 SEO A) 1.202) 35% 0.121 (+0.069) 0.122 (+0.055) 11. En Rakkat Kinneret 38:41 (0.9) 6 (6-04) Hal 0.116 (+0.041) 0.165 (+0.055) 12. En Sheva Sigi E01) Wat. (02) 50.0 0.104 (+0.038) 0.126 (+0.042) 13. Ha’on 3116 (=0.2) 14 (02) 28.6 0.041 (+0.019) 0.101 (+0.049) 14. Bet Gavriel SIMEONE 1.502) 357 0.085 (+0.036) 0.101 (0.042) M. costata costata 15. Gesher Benot Ya'agov 32.0{+0.0) 1.6 (+0.3) 28.6 0.080 (0.044) 0.093 (+0.044) 16. Gesher Lehavot SOON = 21:57(=02) 3577 0.055 (+0.034) 0.081 (+0.033) 17. Eastern Canal 30:0 (2.5) 1.4.0.2) 28.6 0.104 (+0.049) 0.099 (+0.048) 18. Bet Hillel S2AOMEOO) 717502) 35%. 0.112 (+0.055) 0.102 (+0.050) 19. Sede Nehemia SMED 21.512072) 35.7 0.106 (+0.055) 0.114 (+0.055) M. saulcyi 5. Sede Eliahu San E NI (EE OA) 35.7 0.059 (=0.031) 0.117 (+0.048) 20. En Hanatziv 3177.(=02) 1-6, (0:2) 42.9 0.091 (=0.040) 0.122 (+0.050) 21. Sheluhot 32.0 (+0.0) 1.4 (+0.2) 35.7 0.063 (=0.040) 0.078 (+0.038) 22. Hamat Gader 15 mf.s. 21.0 (+1.3) 1.4 (+0.1) 42.9 0.106 (+0.042) 0.170 (+0.060) 23. Мапа! Tavor РИ 5 (EE 02) 42.9 0.109 (+0.049) 0.155 (+0.059) 24. En Huga 31:7 (0:2) 5 (02) 42.9 0.054 (+0.022) 0.117 (+0.047) 25. Hamat Gader spring 10:07=0:6) 7 1:24( 01) 21.4 0.113 (+0.074) 0.077 (+0.044) 26. En Malkoah 41.3 (=0.3) 1.4 (+0.2) 28.6 0.067 (+0.033) 0.111 (+0.050) *A locus is considered polymorphic if more than one allele was detected; ** unbiased estimate (see Nei, 1978) tance (Table 3). The mean value of Cavalli- Sforza and Edwards arc distance equaled 0.240, and the Mantel test showed a signifi- cant association between the matrices of this distance and the geographic distance (r = 0.52133, t = 8.0717, p = 0.0002). The lowest four values were found between populations 17 and 18 (0.054), 15 and 16 (0.072), 15 and 18 (0.081), 18 and 19 (0.081), and 15 and 19 (0.091). The highest values (exceeding 0.4) were between population 2 and the following populations: 18 (0.441), 19 (0.438), 6 (0.435), 17 (0.433), 14 (0.429), 4, 16, 15 and 13. The mean value of Nei distance equaled 0.076, and the Mantel test showed no significant as- sociation between Nei distance and geo- graphic distance (r = 0.47674, t = 7.6041, p = 0.3778). The lowest values were found be- tween populations 24 and 25 (0.000); 15 and 16, 15 and 18, 17 and 18 (0.001); and 18 and 19 (0.002). The highest values, exceeding 0.2, were all found for population 2 and the fol- lowing: 14 (0.232), 18 (0.229), 16 (0.228), 19 (0.224), 17 (0.219), 4, 15, 12, 13 and 6. The means and ranges of Cavalli-Sforza and Edwards arc distance and Nei unbiased distance, within and between the studied taxa, are listed in Table 4. In general, the val- ues are low, and the intraspecific ranges are not necessarily narrower than the interspecific ones. The mean values of the distances be- tween the two subspecies of M. costata are similar to the mean intraspecific values for the other species. The interpopulation allozymic differentiation was analyzed both phenetically and phyloge- netically. The phenetic analysis included cor- respondence analysis of the allele frequen- cies and clustering based on the genetic distances. The correspondence analysis re- FALNIOWSKI ET AL. 314 „u [ОКО v6L'0 E00 sun... LOLO 550`0 0000 su... 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Thus, we decided to present the grouping in only the first two dimensions that explained together 51.81% of the total variability. The eigenval- ues were 0.1583 and 0.0520, and the per- centages of variability explained were 39.00 and 12.81, respectively. The plot of the col- umn factors in the first two-dimension space (Fig. 2) shows distinct groups representing M. saulcyi (populations 5 and 20-26) and M. costata (populations 9-19). Within the latter, M. costata jordanica (populations 9-14) are not mixed with M. costata costata (popula- tions 15-19), although they all form a single, compact group. Melanopsis meiostoma (pop- ulation 8) lies close to M. saulcyi. On the other hand, the populations of M. buccinoidea (1-4 and 6-7) are scattered across all the plot, mixed with both M. saulcyi and M. costata. Nearly the same picture was observed in all the ten dimensions analyzed. The UPGMA clustering was computed on 0 50 025 000 the genetic distances (Fig. 3). For each UPGMA tree, the cophenetic distances were calculated, and Mantel tests performed for association between the matrices of original versus phenetic distances, the so-called cophenetic correlation. For Cavalli-Sforza and Edwards arc distance r = 0.85446; for Nei unbiased distance r = 0.84413. As the as- sumptions of the Mantel test were violated (the matrices were not independent), only the subjective interpretation of the r-values was allowed (Rohlf, 1998). The values above 0.8 and below 0.9 were considered a good fit. Both the two phenograms (Fig. 3) show the same picture, resembling also the one ob- tained with correspondence analysis. Mela- nopsis costata costata always forms a cluster, and M. costata jordanica forms another clus- ter; the two clusters are placed together. Also M. saulcyiis grouped in one cluster, which in- cludes M. meiostoma. On the other hand, the populations that represent M. buccinoidea are scattered in the M. saulcyi as well as M. costata jordanica clusters. Similar results, al- though less clear, and with the stress values belonging to the Ча!” range, gave nonlinear multidimensional scaling, with the minimum- 0.25 050 075 1.00 FIG. 2. Correspondence analysis, populations projected on 1st and 2nd dimension. Population numbers: see text and Fig. 1. A— Melanopsis buccinoidea Olivier, 1801, B— M. meiostoma Heller & Sivan, 2001, C—M. costata jordanica Roth, 1839, D—M. costata costata Olivier, 1804, E and F — M. saulcyi Bourguignat, 1853. ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 317 0.00 0.03 0.06 0.09 0.12 FIG. 3. UPGMA clustering trees, based on Cavalli-Sforza and Edwards arc genetic distance (above) and Nei unbiased genetic distance (below). Symbols of taxa: see Fig. 2. spanning tree superimposed on the plot, so we do not present it here. Phylogenetic analysis included distance- based additive trees, computed with two tech- niques: Neighbor-joining and Fitch-Margo- liash (Figs. 4, 5). Surprisingly, the tree topologies, which means not the branch lengths but the branching patterns, showed by neighbor-joining are similar to those re- vealed by clustering: the populations repre- senting M. saulcyi form one cluster, whereas the other includes all the populations of M. costata. In the latter, one subspecies is not mixed with the other. Melanopsis meiostoma falls within the cluster of M. saulcyi, and M. buccinoidea is scattered in both clusters. The Fitch-Margoliash additive tree technique based on Cavalli-Sforza and Edwards arc dis- tance resulted in a tree (Fig. 5) with an aver- age percent standard deviation of 9.5672 (6,339 trees analyzed). For unbiased Nei dis- tance (Fig. 5), the value was much higher: 23.2768 (7,083 trees analyzed). Thus, the most reliable phylogeny reconstruction is the one based on Cavalli-Sforza and Edwards arc distance. The phylogenetic trees are drawn in the form of a phylogram (Maddison & Maddi- son, 1992), with the branch lengths made pro- portional to the amount of change, thus en- abling one to observe not only the tree 318 FALNIOWSKI ET AL. FIG. 4. Neighbor-joining additive trees, presented in the form of phylograms, based on Cavalli-Sforza and Edwards arc genetic distance (above) and Nei unbiased genetic distance (below). Symbols of taxa: see Big. 2. topology, but also the amount of anagenetic evolution. Again, there is a distinct group of closely related populations that represents M. costata jordanica and joins with the somewhat less close M. costata costata group. Another big cluster includes all the populations of M. saulcyi, but also the single population that represents M. meiostoma. Also here M. buc- cinoidea populations are scattered in both clusters, some of them terminating long branches. It must be stressed that allthe trees are unrooted. The Fitch-Margoliash tree based on Ca- valli-Sforza and Edwards arc distance was evaluated using the bootstrap of the original frequencies, followed by calculating new dis- tances, and Fitch-Margoliash technique ap- plied for each one of the new 200 distance sets. The extended majority-rule consensus tree was similar in topology to the original one; it also showed the M. costata costata, M. costata jordanica, and M. saulcyi population groups as distinct from each, and M. bucci- noidea populations scattered all over the tree. However, the support of the branches was low, about 30-40%. ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 319 a 4 2 A 2 А 7 + 2324 zch + 20 140 + + 154 150 1>@ 17@ ERS о 135 O AT a A EN 14 ы | sch _ 23 - EN 21 À „+ Г 110 er ae ates =0 FIG. 5. Fitch-Margoliash additive trees, presented in the form of phylograms, based on Cavalli-Sforza and Edwards arc genetic distance (above) and Nei unbiased genetic distance (below). Symbols of taxa: see 810. 2: DISCUSSION The proportion of polymorphic loci in fresh- water gastropods may approach 62% (Woodruff et al., 1988); in the freshwater Vi- viparus, it ranged from 0 to 33.3% (Falniowski et al., 1996); in Bythinella, 11.1 to 55.6% (Fal- niowski et al., 1998); from 3.1 to 53.1% in Dalhousia; and from 0 to 16% in Fluvidona (Ponder et al., 1996). Hence, the values found in Melanopsis, although relatively high for prosobranchs, do not exceed the range of freshwater gastropods. The value of mean ex- pected heterozygosity for all the mollusks studied so far is 0.148 (Lazaridou-Dimitriadou et al., 1994). In fresh- or brackishwater gas- tropods, the values are usually lower: 0.002- 0.136 or 0.021-0.049 in Hydrobia (Haase, 1993; Davis et al., 1988, respectively) and 0.04-0.19 on average for various species of Oncomelania (Woodruff et al., 1988). On the other hand, in Bythinella mean expected het- erozygosity ranged from 0.018 to 0.229 (Fal- niowski et al., 1998). Thus, the mean ex- pected gene diversity in Melanopsis was among the highest noted in prosobranchs. Despite the criticism concerning Nei ge- netic distance (e.g., Swofford & Olsen, 1990; 320 FALNIOWSKI ET AL. Davis, 1994; Falniowski et al., 1996; Swofford et al., 1996), it is the most commonly used statistic, thus using it is convenient because one can easily make comparisons with the rich literature. In this study, contrary to Cavalli- Sforza and Edwards arc distance, unbiased Nei distance was not correlated with geo- graphic distance. This can be explained by the fact that Nei distance is intended to reflect the number of codon substitutions, thus muta- tions, as the main source of variation, and it is heavily affected by intrapopulation genotypic polymorphism. Thus, it is certainly not appro- priate where there is a low level of genetic drift, and not necessarily realistic as a quan- tificator of the time and/or amount of evolu- tionary change after coalescence time. As concerns mutations, the highest number should be detectable among species, but the studied taxa are not geographically distinct: their distribution does not show any geo- graphic pattern. On the other hand, the high level of both genetic drift (shown by the re- sults of Ohta’s D-statistics and estimates of gene flow calculated from 0: Falniowski et al. in press) and polymorphism in the studied populations has undoubtedly biased the val- ues of Nei distance. The values of intraspecific Nei distances found in Viviparus contectus (Falniowski et al., 1996) ranged from 0.0014 to 0.0397, mean 0.0166. In his survey of over 7,000 compar- isons of conspecific populations of plants and animals, Thorpe (1983) revealed that only 2% of the intraspecific Nei distance estimates ex- ceeded 0.10. The highest intraspecific value for sexually reproducing gastropod (0.63) was reported in Cepaea nemoralis (Johnson et al., 1984). Usually the values are lower, for exam- ple, 0.001-0.131 in Helix (Lazaridou-Dimitri- adou etal., 1994), 0.001 -0.051 in Trochus and Tectus (Borsa & Benzie, 1993), 0.000-0.007 in Haliotis (Brown, 1993), 0.000-0.013 in Hy- drobia (Davis et al., 1988), 0.051-0.191 in On- comelania (Davis et al., 1994). Thus, the val- ues found within the presumed species of Melanopsis are typical intraspecific distances of a rather weakly to quite considerably genet- ically differentiated gastropod species. The Nei distances between the presumed species were also low. They even did not ex- ceed in all cases the intraspecific values. In M. buccinoidea, indeed, the intraspecific dis- tances were often higher than almost any in- terspecific distance. The average values were about 0.08 between M. buccinoidea and all the other species, about 0.05 between M. meiostoma and M. saulcyi, about 0.1 between M. meiostoma and M. costata, and about 0.12 between M. costata and M. saulcyi. The val- ues of interspecific distances in the European Viviparus (Falniowski et al., 1996) were be- tween 0.2306 and 0.9888, mean 0.6871. Thorpe (1983) reviewed 900 comparisons of interspecific Nei distance for congeners in plants and animals and reported a mean dis- tance of about 0.40 (from 0.03 to more than 1); Davis (1994) listed a number of distances for mollusks. For gastropods, it may be as high as 5.383 in Haliotis (Brown, 1993), or 1.726 in Trochus (Borsa & Benzie, 1993), but also may not exceed 0.199 for Cristilabrum (Woodruff & Solem, 1990), or 0.077 for Stag- nicola (Rudolph & Burch, 1989). The above figures indicate that there is no general rule as to how high the value of Nei distance must be to prove species distinct- ness. However, the values in Melanopsis are rather of conspecific level. The range of in- traspecific values, exceeding the ones be- tween the taxa, does not confirm the species distinctness of the taxa, either. We can con- sider them either conspecific, thus not reject- ing our null hypothesis, or consider them dis- tinct species but the speciation of which caused very little change in allozyme pattern. A similar case was observed in Cerion (Gould & Woodruff, 1986). Secondly, morphology may not reflect speciation (Larson, 1989). A third possibility that by chance the loci we studied are not representative and do not re- flect reproductive isolation cannot be ad- dressed with our data. Thus, we restrict the discussion to the question: are they finely al- lozymatically differentiated yet distinct spe- cies (or at least distinct biological, units of not necessarily a species level) or else, are the studied populations conspecific and all their shell variation is environmentally induced, as postulated by the null hypothesis? As it can be seen in Figure 2, the concho- logical differences between the studied taxa, although rather well marked, are all ex- pressed in such characters as the shell di- mensions, proportions, and sculpture. There is a vast literature describing high, ecologi- cally determined variation of shell characters. For instance, many prosobranchs that live in stormy habitats, where the water movement is strong, have ribbed shells (e.g., Wigham, 1975; Verduin, 1976; Palmer, 1985; Fal- niowski, 1988). There were some fine mor- ALLOZYMIC TAXONOMY WITHIN MELANOPSIS 321 phometric differences in the radulae of the studied taxa (Mazan et al., 2001). On the other hand, one can hardly expect well- marked differences in radular characters be- tween so closely related taxa. Finally, the anatomy of Melanopsis is simplified, the snails lacking copulatory organs. We did not find anatomical differences between the stud- ied taxa. Hence, it is the molecular characters that are decisive. As stated above, the cohesion concept of species assumes that the fundamental niches of two species cannot be identical. From field observations of one of us (J. H.), it seems that the fundamental niches of the studied taxa are not identical, although the differences are rather fine. Thus, the demographic exchange- ability of the taxa may be rejected. On the other hand, the reasoning may be just re- versed: slight differences in ecology may create morphology differences between the organisms that are basically the same. How- ever, in such a case, the morphological and ecological differences would not be accompa- nied by molecular differences. Although there are a few well-documented exceptions (Sza- rowska et al., 1998), the neutral theory holds, and the vast majority of allozymes seems se- lectively neutral; thus, one cannot expect eco- logical differentiation in allozyme pattern. The results of either phenetic or phyloge- netic analyses present a surprisingly congru- ent pattern of distinct but close clusters repre- senting M. costata costata and M. costata jordanica, as opposed to another cluster formed by all the populations of М. saulcyi. Melanopsis meiostoma invariably adjoins the cluster of M. saulcyi. On the other hand, M. buccinoidea is scattered in both big clusters. Correspondence analysis shows the same picture; it also shows that the variability range of M. buccinoidea is wider than the variability of all the other taxa together. On the other hand, the support indices provided by boot- strap were low. This may have been due to the slight differentiation of the studied popula- tions, and thus the high sensitivity of the in- ferred trees to any modification of the data. It must also be stressed that the number of characters, that is the number of alleles found, is far too low for the bootstrap to give reliable estimates (e.g., Swofford et al., 1996). It is unusual that clustering and phyloge- netic techniques of additive trees give such congruent results. As a rule, when the dis- tances are short and differentiation in general is weak, each technique applied will give such different results that it does not make sense to compute a consensus tree, because too much information will then be lost. In Melanopsis, however, though the differences are small, the different techniques all yield exactly the same results. Thus, at least M. costata costata, M. costata jordanica, and M. saulcyi, each mor- phologically distinct, appear also allozymati- cally as distinct biological units, though very close to each other. The level of molecular dif- ferences between them suggests that they speciated not long ago, or even that the speci- ation process is still not completed. The latter possibility, as stated above, is a good expla- nation of those species being not “good”. Whatever the taxonomic decision, it is evident that in the studied case, the allozymic differ- entiation accompanies differentiation in shell form, thus our null hypothesis of a single, con- chologically variable species must be re- jected. The two putative subspecies of M. costata are indeed allozymatically, most close to each other. As concerns M. meiostoma, its species distinctness cannot be confirmed by the present data: more specimens and more loci must be studied to confirm this. In all trees, М. meiostoma falls within M. saulcyi. The most problematic case is M. bucci- noidea. Its smooth shell is very characteristic, but specimens are found in which the shell has weak costae, suggesting hybridization with other, ribbed species. Again, it can be in- terpreted as a proof of the conspecifity of all the Melanopsis populations studied. How- ever, conspecifity would hardly result in such clearly and univocally distinguishable allozy- matic groups of populations as M. saulcyi and M. costata. A better explanation of the ob- served pattern of distribution of the popula- tions of M. buccinoidea in trees and plots is that M. buccinoidea is a distinct species, with perhaps some level of hybridization with other species, and some admixture of hybrids in the studied populations (some level of genetic in- trogression, which nevertheless cannot be proved without genetic markers). However, we emphasize again that M. buccinoidea is notwithstanding far more variable than all the other taxa together. Its wide variability, includ- ing all the alleles found in the studied taxa, indicates rather that it is not a phenotypic, thus polyphyletic group consisting of smooth, unribbed forms of both M. costata and M. saulcyi. Perhaps the most consistent hypothesis 322 FALNIOWSKI ET AL. with our data is that M. buccinoidea, with its high genetic variability, is the ancestral species from which the other species in this study eventually speciated: M. costata (with two subspecies diverging later), and M. saulcyi. Genetic variation in each of the pre- sumed daughter species is much narrower than in M. buccinoidea, which suggests a founder effect (Barton, 1989) and the al- lopatric speciation of peripheral isolates. On the other hand, the founder effect must not have been too severe, as the high levels of polymorphism are still maintained in the daughter species. Later, M. buccinoidea be- came sympatric with its daughter species. Speciation in Melanopsis, as in Hydrobia (Davis, 1993), is perhaps a morphostatic one, with little differentiation in morphology, as well as in allozymes and ecological niches. Such speciation seems not uncommon in gas- tropods (Falniowski, 1987, 1992; Giusti & Manganelli, 1992). On the other hand, speci- ation might have been sympatric, with selec- tion restricting the range of genotypic, thus also phenotypic variation, which resulted in the restricted ranges of environmental vari- ability experienced by the taxa. As concerns the potential hybridization, our study has not been designed adequately to answer the question. However, even if the hybridization occurs, it has not resulted in merging the ge- netic pools of the studied taxa. Thus, its pos- sible occurrence does not reject the species distinctness of M. buccinoidea. ACKNOWLEDGMENTS The molecular work was supported by a grant from the Polish Committee of the Scien- tific Research BW/IZ/UJ/98 to A. Falniowski, and a travel grant from the Hebrew University of Israel to J. Heller. LITERATURE CITED AFZELIUS, В. А., В. DALLAI & G. CALLANI, 1989, Spermiogenesis and spermatozoa in Melanopsis (Mesogastropoda, Mollusca). Journal of Submi- croscopic Cytology and Pathology, 21: 187-200. ALTABA, С. 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YUAN, 1988, Genetic variation in Oncomelania hupensis: Schistosoma japonicum transmitting snails in China and the Philippines are distinct species. Malacologia, 29: 347-361. Revised ms. accepted 5 March 2002 MALACOLOGIA, 2002, 44(2): 325-331 MORPHOLOGICAL AND CYTOCHEMICAL CHARACTERISTICS OF HEMOCYTES OF MERETRIX LUSORIA (Bivalvia: Veneridae) Sung-Woo Park, Kang-Soo Lee & Ee-Yung Chung School of Marine Life Science, College of Ocean Science & Technology, Kunsan National University, Kunsan 573-701, Korea; eychung@kunsan.ac.kr ABSTRACT To help understand the cellular defense mechanism of Meretrix lusoria hemocytes withdrawn from the posterior adductor muscle were classified on the basis of their morphology, cytochem- istry and phagocytic characteristics under a light microscope. Two cell types, granulocytes and hyalinocytes, were found in the hemoctyes. Granulocytes, 13.1 + 2.6 um - 16.2 + 3.3 um in size, contain some large or small eosinophlic granules in their cytoplasm. Granuloctyes were subclassified into small eosinophilic granulocytes and large eosinophilic granulocytes based on the size of the granules. Granulocytes were positive to PAS, acid phosphatase and phenoloxidase but negative to Sudan black B. Granulocytes showed pos- itive phagocytic activity to zymosan particles. Hyalinocytes, that is, agranulocytes that did not contain granules in the basophilic small cyto- plasm, were 11.3 + 2.8 um in diameter and had an oval or irregular nucleus at the center of the cell. The ratio of nucleus to the cytoplasm was very high. Hyalinocytes were weakly positive to PAS but negative to Sudan black B and acid phosphatase. Phenoloxidase acivity was not detected in hyalinocytes. Hyalinocytes phagocytized zymosan particles but the activity was very low compared with that of granulocytes. Key words: Meretrix lusoria, hemocytes, morphology, cytochemical characteristics. INTRODUCTION Meretrix lusoria (Röding, 1798) (Bivalvia: Veneridae) is one of the commonly cultured bivalve species in the western coastal areas of Korea. It grows well in shallow water, which may be brackish or full salinity seawater. Juvenile clams taken from natural beds had been transferred onto culturing beds restricted with net fences to protect their escape and to allow them to grow to commer- cial size. Because mass mortality occurred from July to August in 1973 and continued during the following 2 or 3 years, their com- mercial production ceased because natural juvenile clams were not available. Yoo et al. (1975) reported some possible causes of the mortality, such as bacterial infections by Vibrio angillarum, Pseudomoans ichyodermidis and Acromobacter dermidis, inflow of agricultural pesticides to the beds, and environmental deterioration due to high water temperature and high salinity, but the exact cause still remains unclear. In recent years, it has not been difficult to obtain clams larger than 5 cm in shell size, which indicates a gradual increase of the natural population in the nat- 325 ural beds. Fishermen have transplanted juve- nile clams onto new culturing beds where it has not yet been cultured, but have not gotten good survival there. Bivalve hemocytes play various important roles in defense mechanisms, such as wound healing and shell repair, nutrient digestion and transportation, and excretion (Cheng, 1981). Two types of bivalve hemocytes, granulocytes and hyanilocytes, have been documented in several species of bivalve on the basis of mor- phological, cytochemical, and functional crite- ria. Most studies on bivalve hemocytes have been carried out using oysters or mussels (Foley & Cheng, 1972; Bachere et al., 1991; Carballal et al., 1997b; Lopez et al., 1977; Nakayama et al., 1997), but there are a few studies on the hemocytes and their functions in Meretrix lusoria. On the basis of staining and morphological characteristics, the hemo- cytes of M. lusoria were classified into four type of cells by Wen et al. (1994) and three types of cells by Shih et al. (1996), but there were no studies on the cytochemistry or phagocytosis of Meretrix lusoria hemocytes. The phagocytic ability of bivalve hemocytes is one of the effective cellular defense mecha- 326 PARKETAL. nisms of killing bacterial invaders, and granu- locytes have much higher phagocytic ability than hyalinocytes, which posses few or a few granules containing bacteriolytic enzymes (Foley & Cheng, 1975). Phenoloxidase was detected in many bivalves hemocytes (Coles & Pipe, 1994; Asokan et al., 1997; Carballal et al,, 1997a), but its role in defense mecha- nisms has not been revealed. Paillard et al. (1994) suggested that this enzyme system could take part in the defense mechanisms in Venerupis philippinarum against Vibrio P1. It is important to understand the roles of Meretrix hemocytes in defense, and in evalu- ating the clam’s adaptation to new beds as well as avoiding damages by epizootics or from environmental conditions. This study provides data on the morphological and cyto- chemical characteristics of Meretrix lusoria hemocytes as seen under a light microscope. MATERIALS AND METHODS Specimens of Meretrix lusoria, 43.8 mm in mean shell length and 36.1 mm in mean shell width, were collected from a shellfish farm in Simpo Bay, Chonbuk, Korea. The clams were transferred to the laboratory in an ice bag and washed several times with filtered sea water (FSW) before using them for hemolymph col- lection. After making a small hole in the shell margin near the posterior adductor muscle with an iron drill, hemolymph was directly withdrawn from the posterior adductor muscle with a 2.5-ml plastic syringe containing either cold Modified Alsever’s Solution (MAS; Bachere et al., 1991) as anti-aggregant, or Baker’s formol calcium or MAS containing neutral formalin at 10% (MAS10) as fixative. Morphology Hemolymph collected 1: 3 in MAS were spread in thin films on glass slides, cover- slipped, and immediately observed for the morphology of live hemocytes under a phase contrast microscope. Hemolymph collected in MAS10 was transferred into a siliconized glass tube and then fixed at room temperature for 10 min. The hemolymph was centrifuged at 250xg for 10 min, the supernatant decanted, and the pellet was resuspended in MAS. The cell suspension was smeared on glass slides by centrifuging in a cell-collecting apparatus (Hanil, Korea) at 80xg for 4 min at room temperature. After staining the smears with May-Grünwald Giemsa, the cell size of hemocytes was measured under a light microscope. Cytochemistry Lipids were demonstrated using Sudan black B stain, as described by Barracco et al. (1999). Hemocytes smears were air-dried and fixed with 50% ethanol for 3 min and then immersed in Sudan black B solution in 70% ethanol for 30 min. After dipping several times in 50% ethanol, the smears were counter- stained with Giemsa solution. Polysaccharides were detected by the peri- odic acid-Schiff reaction (PAS). The air-dried hemocyte smears were fixed with fixative solution (6 acetone: 3 formalin: 1 acetic acid) for 10 min and rinsed with running tap water for 5 min. The fixed smears were treated with 1% periodic acid for 10 min, rinsed with dis- tilled water for 15 min, and immersed in a commercial Schiffs reagent (Sigma) for 30 min in a refrigerator. After rinsing for 5 min in each of three consecutive sulfurous solution, the smears were kept in running tap water for 30 min, counterstained with Mayer hema- toxylin for 10 min, and mounted after washing in distilled water for 10 min. Control smears were treated twice with saliva (4 ml of saliva in 1 ml of 0.1 M phosphate buffered solution, pH 7.0) for 15 min at 37°C in a wet chamber to confirm if the positive materials were glyco- gen before being treated with periodic acid. Acid phosphatase was revealed using Gomori’s technique described by Barracco et al. (1999) or a commercial kit for leucocytes acid phosphatase (Sigma). Hemolymph was collected 1:3 in Baker’s formol calcium con- taining 2% NaCl, allowed to fix for 20 min at room temperature and washed with distilled water. Hemocyte smears were prepared by the methods described above and air-dried. The smears were incubated for 2 hr in the Gomoris medium which had preheated over night at 37°C, washed with distilled water, stained with 1% ammonium sulfide for 2 min, and finally counterstained with methylene blue (Carballal et al., 1997a). Controls were incubated in a medium without the substrate. Phenoloxidase was demonstrated by the method of Coles & Pipe (1994) with a minor modification. Hemolymph was withdrawn from the posterior adductor muscles of five clams, diluted 1:1 in 10% Baker’s formol cal- cium containing 2% NaCl, and allowed to fix for 10 min at room temperature. After washing CHARACTERISTICS OF HEMOCYTES OF MERETRIX LUSORIA 327 three times in 0.01 M phosphate buffered solution, the hemocytes were smeared on glass slides by the methods as described above. The smears were air-dried and incu- bated in a staining jar with 0.01 M phosphate buffered solution containing 2 mg/ml of L-3, 4 dihydroxyphenyl-alanine (L-dopa) for 2 hr at 30°C. After being washed with distilled water, the smears were stained with Giemsa solution for 10 min at room temperature to differentiate cell types. Control slides were incubated in phosphate buffered solution without the sub- strate. Phagocytosis Zymosan particles (Sigma) were sus- pended in filtered sea water at a concentra- tion of 1 ug/ml, heated for 30 min at 100°C, washed and resuspended in MAS. One drop of the hemocytes pooled from 3-4 individuals was placed on glass slides and allowed to adhere on the surface of the glass slides in a wet chamber for 20 min at 25°C. The glass slides were then washed with MAS to remove unattached hemocytes and incubated with zymosan suspension for 1 hr at 25°C. After incubation uningested zymosan particles were removed by several washings of the glass slides with MAS. The air-dried glass slides were stained with May-Grünwald Giemsa and observed for phagocytic ability under a light microscope. RESULTS Morphology of Hemocytes Two types of hemocytes are easily identi- fied in the hemolymph of M. lusoria on the basis of the presence or absence of cytoplas- mic granules under a phase contrast micro- scope. Granulocytes possessing some large or small granules in the cytoplasm were oval or round large cells and were darkly seen due to the presence of the granules (Fig. 1a). As the large granules in granulocytes were round in shape and were observed brightly, their presence were easily revealed under a phase contrast microscope. The small granules in granulocytes were evenly distributed in the cytoplasm. The granulocytes with the large granules were larger in cell size than those with the small granules. Hyalinocytes with none or a few granules in the cytoplasm were round in shape, smaller than granulocytes in size, and most abundant in number. The cen- ter of hyalinocytes, containing the nucleus was seen more darkly than the cytoplasm (Fig. 1b). The morphological and cytochemical char- acteristics, and phagocytic ability of Meretrix hemocytes, are shown in Table 1 and Figure 2. Meretrix lusoria hemocytes were divided into two types of cells based on the staining affinity and the presence of the granules in the cytoplasm after staining with May-Grünwald Giemsa (Fig. 1). Granulocytes were round, ovoid or irregular cells (13-16 um) and the nucleus was commonly eccentric. They con- tained numerous large or small eosinophilic granules in the basophilic cytoplasm. Granulocytes could be divided into large or small eosinophilic granulocytes based on the granule size. The large eosinophilic granulo- cytes (Lgs) were larger than the small eosinophilic granulocytes (Sgs) in size (Table 1). As the granules in Lgs were compactly packed in the cytoplasm, it was difficult to see the nucleus. The Sgs had basophilic affinity in the peripheral cytoplasm and their granules were more plentiful in the peripheral part of the cells than around the nucleus. Hyalinocytes possessing none or a few granules in the cytoplasm were round cells (11.3 + 2.8 um) and the nucleus was eccen- tric. But, only a few hyalinocyes had cytoplas- mic granules. The ratio of nucleus:cytoplasm of hyalinocyes was higher than that of granu- locytes, especially much higher in the cells with a central nucleus than in the cells with eccentric nucleus. The cytoplasm of hyalino- cytes was more basophilic than that of the granulocytes. A few large hyalinocyes had large cytoplasmic vacuoles. Cytochemistry and Phagocytosis of Clam Hemocytes Both granulocytes and hyalinocytes were negative in Sudan black B. Only granulocytes showed PAS-positive reaction in their cyto- plasm. The PAS-positive materials were evenly distributed in the cytoplasm but some strongly PAS-positive granules were also observed in the peripheral cytoplasm. After digestion with saliva prior to reacting with Schiffs reagent, the PAS-positive reaction was not altered, suggesting the positive mate- rials in the cytoplasm were different from glycogen. Any difference in the two types of granulocytes could not be found in PAS reac- tion. Most of hyalinocytes were negative in the 328 PARKETAL. FIG. 1. Photomicrographs of the hemocytes of Meretrix lusoria. a, live granulocytes (Lg and Sg) and hyalinocyte (H) ovserved with a phase contrast microscope; b, large eosinophilic granulocytes (Lg), small granulocytes (Sg) and hyalinocyte (H) stained with May-Grünwald Giemsa. Bars indicate 10 um. TABLE 1. Morphology, cytohemistry and phagocytic ability of hemocytes of Meretrix lusoria Cytochemistry Shape Cell* ==: ¿Sudan Acid Phenol- Phago- Hemocytes size (um) Cell Nucleus blackB PAS phosphatase oxidase cytosis Lgs 16.2+3.3 Ovoid, round, Ovoid, = + Fe + à irregular round Granulocytes Sgs 13.1 + 2.6 Ovoid, round Round = 4 e = tb Halinocyte 11.3 + 2.8 Ovoid, round Round = 35 = = + *Mean + SD of 60 cells from ten individuals. Lgs, large eosinophilic granulocytes; Sgs, small eosinophilic granulocytes. PAS reaction, but a few cells showed positive reaction. In the PAS reaction small hyalino- cytes were weakly positive but large hyalino- cyes with a central round nucleus or cytoplas- mic vaculoes were all negative (Fig. 2a). Acid phosphatase was observed only in granules of granulocytes as black deposits (Fig. 2b). The number of positive cells was much higher in Lgs than in Sgs. Phenoloxidase activity was detected as brown deposits in the peripheral cytoplasm of Lgs (Fig. 2c). The positive reaction for phe- noloxidase was seen as a black deposit in the granules after staining with Giemsa solution. The activity was not detected in both Sgs and hyalinocytes. When Meretrix hemocytes were incubated with zymosan particles, both granulocytes and hyalinocytes phagocytized zymosan par- ticles (Fig. 2d). Granulocytes appeared to be more phagocytic than hyalinocytes, which ingested only a few zymosan particles. Just a few hyalinocytes exhibited phagocytic activity to zymosan particles. DISCUSSION Several authors have classified two types of hemocytes in bivalves based on morpho- logical, cytochemical and functional charac- teristics. These authors (Foley & Cheng, 1974; Löpez et al., 1997; Hunakoshi, 2000) reported two hemocytes type, granulocytes and agranulocytes on the basis of staining characteristics and morphological criteria. Granulocytes were reclassified into eosino- philic granulocytes and basophilic granulo- cytes on the basis of the staining affinity ofthe cytoplasmic granules. Eosinionophilic granu- locytes were divided into large and small eoninophilic guranulocytes depending on the granular size. Bivalve granulocytes vary in size or shape of the cytoplasmic granules in different species (Foley & Cheng, 1972; Carballal et al., 1997b). Agranulocytes were classified into hyalinocytes and fibrocytes based on the cell size and cytoplasmic char- acteristics. Fibrocytes were described as larger cells with slightly basophilic cytoplasm CHARACTERISTICS OF HEMOCYTES OF MERETRIX LUSORIA 329 FIG. 2. Photomicrographs of the hemocytes of Meretrix lusoria, showing cytochemical reactions (a-c) and phagocytosis (d). a, PAS reaction; b, acid phosphatase; c, phenoloxidase; d, phagocytic activity. Bars indi- cate 10 um. that had a few or no granules and in some cases containing many vacuoles in the cyto- plasm. Foley & Cheng (1972) also reported the presence of fibrocytes in Mercenaria mer- cenaria, but Cheng & Foley (1975) suggested that fibrocytes were considered to degranu- lated granulocytes with results of ultrastruc- tural observation of the cells. In our study, two types of hemocytes, thatis, granulocytes and hyalinocytes, occurring in M. lusoria are distinguishable by the presence or absence of cytoplasmic granules either under a phase contrast microscope or a light micro- scope. Under the phase contrast microscope, granulocytes fixed with Baker’s formol calcium revealed visible cytoplasmic granules and were easily differentiated from hyalinocytes, which had a few or no cytoplasmic granules. But when hemocytes were fixed with MAS which have been commonly used as an anti- coagulant for bivalve hemocytes, it was diffi- cult to observe the cytoplamic granules in granulocytes from preparations of live hemo- cytes. Baker’s formol calcium appeared to be an excellent fixative for bivalve hemocytes even though it could not be used for phago- cytic assay. To explain granulocytes matura- tion in bivalve, Cheng (1981) proposed an hypothesis that ganulocytes with small ba- sophilic granule would be younger granulo- cytes, and that the basophilic granules be- come eosinophilic and larger as they mature. But it is very interesting that no granulocytes with basophilic granules could be found in the present study as in observation of Wen et al. (1994). Very few large cells with large cyto- plasmic vaculoles and without any cytoplas- mic granules were also observed in this study. Lipid droplets and glycogen have been found in the hemocytes of Mytilus galloprovin- ciallis (Cajaraville & Pal, 1995). The presence of polysaharides in the cytoplasm of hemo- cytes were reported in Tridacna crocea (Nakayama et al., 1997) and Perna perna 330 PARKETAL. (Barracco et al., 1999). In P perna the PAS reaction was stronger in granulocytes than in hyalinocytes, and the PAS positive materials were different from glycogen since its staining affinity did not weaken with a diastase treat- ment. The occurrence of acid phosphatase has been demonstrated in granulocytes of some bivalve species, and the cytoplasmic granules showing acid phosphatase acivity were con- sidered as a form of lysosomes which partici- pate in phagocytosis (Carballal et al.,1997a; Nakayama et al., 1997). Löpez et al. (1997) reported that acid phosphatase is only found on the granules of granulocytes and can be used to distinguish granulocytes from hyalino- cytes. Phenoloxidase was detected in hemocytes of some bivalves (Coles & Pipe, 1994; Carballal et al., 1997a; Deaton et al., 1999). This enzyme is known to take part in defense reactions of arthropod (Söderhäll, 1992), but the roles of this enzyme in defense mecha- nisms of bivalve are still unclear. The phe- noloxidase activity was suggested to partici- pate in defense reactions against the etiological agent, Vibrio tapetis, of brown ring disease in Venerupis philippinarum (Paillard et al., 1994). Coles & Pipe (1994) reported that fixation of mussel hemocytes with 10% Baker’s formol calcium produces the best preservation ofthe phenoloxidase activity and the activity is seen in granules as a fine gray to black deposit. Both types of hemocytes of M. lusoria did not contain lipids, but showed positive reac- tions with Schiff's reagent. Acid phosphatase activity was detected only in granulocytes, especially Lgs in the present study. Phenoloxidase activity in hemocytes of M. lusoria was first found in the present study. The activity was detected only in Legs. The positive activity showing brown color was seen as black deposits in granules after coun- terstaining with Giemsa solution. The enzyme activity was detected in the peripheral cyto- plasm and a strong reaction was observed in large cells, whereas small cells showed neg- ative reaction. Granulocytes were always more phagocytic than hyalinocytes in many bivalves (Foley & Cheng, 1975; Tripp, 1992, Carballal et al., 1997c; Barracco et al., 1999). Both cell types in M. lusoria, especially in large eosinophilic granulocytes could actively pagocytose the zymosan particles. As most phagocytic hemo- cytes in М. lusoria were large eosinophilic granulocytes containing some enzymes such as acid phosphatase and phenoloxidase, it is suggested that those ezmymes might be in- volved in cellular defense mechanisms. And changes in the number and the enzyme activ- ities of hemocytes might be used for indicators to evaluate health condition in cultured or transplanted bivalves. ACKNOWLEDGEMENTS The authors are grateful to Dr. John B. Burch of the University of Michigan for helpful comments on the manuscript. 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VILLALBA, 1997b, Hemolymph cell types of the mussel Mytilus galloprovincialis. Diseases of Aquatic Organisms, 29: 127-135. CARBALLAL, M. J., C. LOPEZ, C. AZEVEDO £ A. VILLALBA, 1997c, In vitro study of phagocytic ability of Mytilus galloprovincialis. Diseases of Aquatic Organisms, 29: 127-135. CAJARAVILLE, M. P & 5. G. PAL, 1995, Morpholfunctional study of the hemocytes of the bivalve mollusc Mytilus galloprovincialis with emphasis on the endolysosomal compartment. Cell Strucuture and Function, 20: 355-367. CHENG, T. C., 1981, Invertebrate blood cells 1. Bivalves. Academic Press, pp. 233-300 CHENG, T. C. & D. A. FOLEY, 1975, Hemolymph cells of the bivalve mollusk Mercenaria merce- naria: an electron microscopical study. Journal of Invertebrate Pathology, 23: 341-351. COLES, y. A. 8 R. K. PIPE, 1994, Phenoloxidase CHARACTERISTICS OF HEMOCYTES OF MERETRIX LUSORIA 331 activity in the haemolymph and haemocytes of the marine mussel Mytilus edulis. Fish & Shellfish Immunology, 4: 337-352. DEATON, L.E., P. J. JORDAN & J. R. DANKERT, 1999, Phenoloxidase activity in the hemolymph of bivalve mollusks. Journal of Shellfish Research, 18: 223-226 FOLEY, D. A. & T. C. CHENG, 1972, Interaction of mollusks and foreign substances: the morphol- ogy and behavior of hemolymph cells of the American oyster, Crassostrea virginica, in vitro. Journal of Invertebrate Pathology, 19: 383-394. FOLEY, D. A. & T. C. CHENG, 1974, Morphology, hematology parameters, and behavior of hemolyph cells of quahaug clam, Mercenaria mercenaria. Biological Bulletin, 146, 343-356. FOLEY, D. A. & Т. С. CHENG, 1975, A quantitative study of phagocytosis by hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria. Journal of Invertebrate Pathology, 25: 189-197. HUNAKOSHI, S., 2000, Studies on the classifica- tion, strucuture and function of hemocytes in bivalves. Bulletin of National Research Institute of Aquaculture, 29: 1-103. LOPEZ, C., M. J. CARBALLAL, C. AZEVEDO & A. VILLALABA, 1997, Morphological characteriza- tion of the hemocytes of the clam Ruditapes philiphinarum (Mollusca: Bivalve). Journal of Invertebrate Pathology, 69: 51-57. NAKAYAMA, K., A. M. NOMOTO, M. NISHIJIMA & T. MARUYAMA, 1997, Morphological and func- tional characterization of hemocytes in the giant clam Tridacna crocea. Journal of Invertebrate Pathology, 69: 105-111. PAILLARD, C., Р. MAES & В. OUBELLA, 1994, Brown ring disease in clams. Annual Review of Fish Diseases, 4: 219-240. SAIT SUS ECHANGES НО, Main LIA, С. Y. PAN & J. Y. JAIR, 1996, Studies on the hemocytes of Meretrix lusoria. Biological Bulletin of National Taiwan Normal University, 31: 131- 139. SÖDERHÄLL, K.,1992, Biochemical and molecular aspects of cellular communication in arthropods. Biologie et Zoologie, 59: 141-151. TRIPP, M. R.,1992, Phagocytosis by haemocytes of the hard clam Mercenaria mercenaria. Journal of Invertebrate Pathology, 59: 222-227. WEN, C.M., G.H.KOU & S. N. CHEN, 1994. Light and electron microscopy of hemocytes of the hard clam, Meretrix lusoria (Röding). Comparative Biochemistry and Physiology, 180A: 279- 286. YOO, S. K., Т. Y. LEE, P. CHIN, $. К. CHUN 8 W. К. CHOE, 1975, Studies on the mortality of the hard clam Meretrix lusoria (Roding) in Buan framing area. Publication of Institute of Marine Science of National Fisheries University of Busan, 8: 39-52. Revised ms accepted 2 April 2002 MALACOLOGIA, 2002, 44(2): 333-347 POPULATION GENETICS, MICRO-PHYLOGEOGRAPHY, ECOLOGY, AND SUSCEPTIBILITY TO SCHISTOSOME INFECTION OF CHINESE ONCOMELANIA HUPENSIS HUPENSIS (GASTROPODA: RISSOOIDEA: POMATIOPSIDAE) IN THE MIAO RIVER SYSTEM Chao-Hui Shi', Thomas Wilke**, George M. Davis’, Ming-Yi Xia? & Chi-Ping Qiu® ABSTRACT Chinese Oncomelania h. hupensis from the flood plains of the Yangtze River have ribbed shells. However, populations living above the effects of annual floods usually are smooth- shelled. Previous allozyme studies of smooth-shelled populations not affected by flooding, and ribbed-shelled populations affected by flooding from the Miao River, Hubei Province, showed that they all belong to O. h. hupensis (Davis et al., 1999a). As the allozyme data were of limited use for assessing Oncomelania-population genetics, we re-examined the above populations to answer the following questions using mitochondrial COI sequence data. Will DNA sequences provide higher resolution for the analysis of population structure than allozyme data? Are there significant genetic differences among ribbed- and smooth-shelled populations? Do they differ in their susceptibility to infection with Schistosoma japonicum? Sequences from 59 individuals revealed four groups of haplotypes. Smooth- and ribbed- shelled individuals clustered together in two of the groups. The greatest sequence divergence between a smooth-shelled and ribbed-shelled population was 0.020, indicating that all populations fall within the range of variation expected for О. h. hupensis. Overall, the highest genetic diversity was found among downstream ribbed-shelled populations. The analysis of molecular variance (AMOVA) showed the following distribution of total variance: 69% (P < 0.0001) within populations, 9% (Р < 0.0025) among populations within the ribbed- and smooth-shelled groups, and 22% (P < 0.0569) between the ribbed- and smooth-shelled populations. Mismatch distributions indicated that downstream populations are aggregates of snails from different populations. Downstream populations also showed a higher infectivity rate and a higher susceptibility to infection with Schistosoma japonicum. This is probably due to the importation and mixture of snail and parasite strains in flooded areas increasing the probability that schistosomes encounter genetically suitable snails, and/ or the possibility of multiple infections by different parasite strains. The low infection rate in upstream populations is probably due to their relative isolation where there is equilibrium with low frequencies of infection. Key words: Oncomelania hupensis, Schistosoma japonicum, China, Red Queen, mtDNA, population genetics, AMOVA, infectivity. INTRODUCTION The dioecious snail genus Oncomelania is widespread throughout China, with a distribu- tion extending to Sulawesi, the Philippines, and Japan. There are three subspecies on the mainland of China (Davis et al., 1995, 1998). Oncomelania hupensis hupensis is found throughout the lower Yangtze River drainage below the Three Gorges, with an extension into Guangxi Province. Oncomelania hupensis robertsoni lives at high elevation on the plateaus ‘Chinese Academy of Medical Sciences, Institute of Laboratory Animal Science, 5 Pan Jia Yuan Nanli, Beijing 100021, China “The George Washington University Medical Center, Department of Microbiology and Tropical Medicine, 2300 Eye Street, N.W., Washington, DC 20037, U.S.A. “Institute of Parasitic Diseases, Chinese National Center of Systematic Medical Malacology, 2007 Rui Jin Er Lu, Shanghai 200025, China “To whom correspondence should be addressed; mtmtxw @ gwumc.edu 334 SHI ET AL. and mountains of Yunnan and Sichuan in far southwestern China. Oncomelania hupensis tangi occurs on the seaside of Fujian Province, removed from the Yangtze River drainage by a range of tall mountains. All three subspecies transmit the human blood fluke, Schistosoma japonicum, argu- ably the most serious parasitic disease problem in China now that malaria is highly controlled. Historically there has been the question of how to classify smooth-shelled Oncomelania living in the same geographic range of typical ribbed-shelled ©. h. hupensis. On the basis of allozymes, Davis et al. (1995) classified popu- lations with both shell types from the lower Yangtze River drainage as ©. h. hupensis. The latter study raised the question whether the character of shell sculpture with its two states (ribbed, smooth) was influenced by elevation of habitat above the reach of the annual flood of the Yangtze River and its tributaries. An experi- ment to test this hypothesis was provided by nature in the Miao River of Hubei Province. This is a short, small river only about 22 km long with flood waters reaching some 14 km up- stream. Allozyme data were obtained from four upstream populations (smooth-shelled) not af- fected by flooding, and three downstream populations (ribbed-shelled) affected by flood- ing each year. These populations were sepa- rated from each other by discrete stretches of river with no evidence of snails. The results showed that there was no genetic basis for dis- tinguishing between the two groups; they both belong to О. h. hupensis (Davis et al., 1999a). However, the results from both allozyme-based studies (Davis et al., 1995, 1999a) raised three important issues relative to population genetics of Oncomelania hupensis. (1) Polymorphism and heterozygosity are too low to enable use of marker alleles to track patterns of dispersal or gene flow. This is apparently the situation throughout rissooidean snails (reviewed in Davis et al., 1999a). (2) With increasing num- ber of populations, it becomes increasingly dif- ficult to determine homology of presumptive alleles of polymorphic loci. (3) The few poly- morphic loci are frequently not in Hardy- Weinberg equilibrium. In the present paper, we revisit the Miao River to re-examine the same populations studied by Davis et al. (1999a) to answer the following questions using sequence data of the mitochondrial cytochrome c oxidase subunit | (COI) gene: (1) Has the mitochondrial genome of Oncomelania hupensis accumulated enough substitutions to enable differentiation among populations that are separated by small geographic distances, such as those found between populations along the short Miao River? (2) Will COI sequence data provide higher resolution for the analysis of population structure than allozyme data? Can sequence data help to explain the departure from Hardy- Weinberg equilibrium? (3) Are there significant differences among ribbed- and smooth-shelled populations relative to genetic diversity and population structure? (4) Do ribbed-shelled and smooth-shelled populations differ in their susceptibility to infection with Schistosoma japonicum? METHODS Locality Data The distribution of the seven populations studied is shown in Figure 1 (adapted from Davis et al. 1999a). Detailed locality data are given in Davis et al. (1999a). Snails were collected by C. H. Shi and G. M. Davis in the fall of 1998 from the same localities (A-G) previously collected in 1994 for the allozyme study (Davis et al., 1999a). Sites A, B, and C are annually flooded by the Yangtze River. There is a dam between sites Band C. The sluices of the dam are closed part ofthe year to retain water at site C in order to drown snails. Sites D-G are never affected by flooding. All 28 specimens from sites A-C had ribbed shells, all 31 specimens from sites D-G had smooth shells. DNA Isolation and Sequencing The methods of Spolsky et al. (1996) and Davis et al. (1998) were used for isolating DNA from in- dividual snails. The primers to amplify a fragment of the COI gene are LCO1490 and HCO2198 (Folmer et al. 1994). The quality of PCR product was deter- mined by electrophoresis through a 1% agarose gel. Amplified DNA products were purified using Wizard PCR preps (Promega, Madison, Wis- consin). COI sequences were determined us- ing the LI-COR (Lincoln, Nebraska) Long ReadiR 4000 DNA sequencer and the Thermo Sequenase fluorescent labeled primer cycle se- GENETIC STRUCTURE AND INFECTIVITY OF ONCOMELANIAH. HUPENSIS 335 A IE a: = pe re = 5 er A à B 10) A Wuhan City = Е ai Sha Shi City | Г № eo‘ | ge 190 | с NE We e = DES E er | U A N A N > 2 р S ——_—— KT) PA nl 10 20 ee a m FIG. 1. Localities of Oncomelania h. hupensis populations studied. Sites A-C are affected by the annual flooding of the Yangtze River. The black bar between sites B and C indicates a dam. quencing kit (Amersham Pharmacia Biotech, Piscataway, New Jersey) according to the manufacturer's protocols. A total of 59 individuals with an average of 8.4 individuals per population were se- quenced. Individual codes, DNA sample numbers and GenBank accession numbers are provided in the Appendix. Data Analysis COI sequences, which do not have base in- sertions or deletions in Oncomelania, were un- ambiguously assembled by eye using the software program ESEE 3.0s (Cabot & Beckenbach, 1989). The first 2-10 base pairs behind the 3’ end of each primer are often diffi- cult to read. We therefore uniformly excluded the first and last ten bp of each sequence, leav- ing an alignment of 638 bp without any inser- tions or deletions. Mismatch distributions and parameters of DNA sequence polymorphism including haplo- type diversity (d,,; Nei, 1987), number of poly- morphic sites ($), nucleotide-sequence diversity (п; Nei, 1987), its stochastic variance (Vst ), and nucleotide-sequence divergence (Dxy; Nei, 1987), were calculated using the software program DnaSP 3.0 (Rozas & Rozas, 1999). The coefficient of correlation (r) of haplotype diversity and geographic dis- tance was calculated using the function CORREL of Microsoft Excel for Windows 95, version 7.0a. The genetic structure in our data set was in- vestigated by analyses of molecular variance (AMOVA) implemented in the computer pack- age ARLEQUIN ver. 2.001 (Schneider et al., 2000). The AMOVA approach is based on gene (haplotype) frequencies, but takes into account the number of mutations between haplotypes (Excoffier et al., 1992). Statistical significance of variance components was assessed with 20,000 random permutations. In order to test whether the COI data set as a whole exhibits phylogenetic signal, the relative apparent synapomorphy analysis (RASA 3.04Т; Lyons-Weiler, 2001) was used. The software compares the observed rate of increase in pairwise cladistic similarity per unit pairwise phe- netic similarity (В observed) to a null slope (В null), where cladistic support and phenetic simi- larities are randomly distributed among pairs of taxa (Lyons-Weiler et al., 1996). The data set showed a significant phylogenetic signal of t.,., = 44.71 (P < 0.05; B observed = 18.71; B null = 6.16) and was therefore considered to be suitable for further analyses. 336 SHI ET AL. Prior to the phylogenetic analyses we used the computer program Modeltest 3.0 (Posada & Crandall, 1998) in order to test which model of DNA substitution best fits our data. It per- forms hierarchical likelihood ratio tests among 56 possible models. The model se- lected was HKY85 (Hasegawa et al., 1985) with a Ti/Tv ratio of 3.942; base frequencies of А—=0-2270, С = 0.1823; © = 02208) and T= 0.3699; as well as а gamma distribution shape parameter of 0.0856. A maximum likelihood tree was then con- structed from all haplotypes using the computer program Tree-Puzzle 4.0.2 (Strimmer & Haeseler, 1999). The analysis was performed with the parameter of sequence substitution suggested by Modeltest. In addition, the num- ber of puzzling steps was set to 100,000 and the parameter estimates to “exact”. As evolu- tionary relationships above and below the spe- cies level are different in nature (Posada & Crandall, 2001), we also analysed the phyloge- netic relationships of our seven O. h. hupensis populations with a method that was specifically designed to infer intraspecific relationships — the statistical parsimony (SP) network ap- proach implemented in the computer program TCS v. 1.06 (Clement et al., 2000). Infectivity Study From each location between 59 and 464 snails (1253 total) were subjected to shed- ding conditions to determine the degree of infection (Table 5). Cercariae obtained from the shedding (populations A-C) were used to infect mice. Then, snails from upstream populations (D, F, G) and one downstream population (C) were infected with miracidia from the infected mice. In the infectivity experiment, uninfected snails were challenged with five miracidia per snail, and maintained in 10 cm petri dishes in culture using methods of Davis (1967). The prepatent period was deter- mined, using standard shedding tech- niques, along with the number of cercariae shed. Mortality was checked for on a daily basis. RESULTS Data on haplotype diversity, number of poly- morphic sites, nucleotide-sequence diversity and its variance for each population are pro- vided in Table 1. A total of 27 different haplotypes was found. The index of haplo- type diversity (h,) ranged from a high of 0.978 at site A to a low of 0.356 at site G. There is a clear trend of decreasing haplotype diversity from downstream to upstream populations (r = -0.71). Nucleotide-sequence diversity was > 0.015 at sites A and B; < 0.0085 at sites C and G. A matrix of pairwise comparisons of nucleotide- sequence divergence (Dxy) between populations (Table 2) shows that all values are rather similar with the lowest Dxy of 0.0099 being observed between the two smooth-shelled populations E and G and the highest Dxy of 0.0200 between the ribbed-shell population B and the smooth-shelled population D. Among the 28 ribbed-shell specimens (sites A-C), the total number of haplotypes found was 17 with a haplotype diversity of d, = 0.825; the total number of polymorphic sites was 41. In contrast, the total number of polymorphic sites TABLE 1. Sequence polymorphism in the Miao River populations of Oncomelania h. hupensis. ribbed smooth total A B C D E Е G (A-C) (D-G) (A-G) No. individuals 10 9 5 10 10 28 31 59 sequences No. haplotypes 9 7 3 5 3 4 2 17 10 27 Haplotype 0.978 0.917 0.417 0.933 0.700 0.644 0.356 0.825 0.692 0.878 diversity (ho) No. polymorphic 23 30 22 21 16 ТЯ 15 41 26 53 sites (S) Nucleotide- sequence diversity (п) Variance of x (Vst(») 0.0153 0.0159 0.0077 0.0110 0.0144 0.0130 0.0084 0.0131 0.0132 0.0161 0.0301 0.0321 0.0085 0.0156 0.0252 0.0221 0.0099 0.0232 0.0234 0.0344 GENETIC STRUCTURE AND INFECTIVITY OF ONCOMELANIA H. HUPENSIS 337 TABLE 2. Nucleotide-sequence divergence (Dxy) between the populations of Oncomelania h. hupensis studied. The population from GuiChi (Yangtze River, Anhui Province) was used for comparison. GuiChi A B GuiChi - A 0.0183 = B 0.0192 0.0152 - C 0.0182 0.0123 0.0117 D 0.0185 0.0180 0.0200 E 0.0173 0.0158 0.0174 Е 0.0170 0.0154 0.0170 G 0.0159 0.0138 0.0150 C D E 5 G 0.0180 = 0.0141 0.0148 5 0.0152 0.0150 0.0125 à 0.0127 0.0140 0.0099 0.0106 = TABLE 3. Summary of analysis of molecular variance (AMOVA) for smooth and ribbed-shelled populations of Oncomelania h. hupensis. Levels of significance are based on 20,000 permutations. Source of variation df Sum of squares Among groups 1 3.928 (ribbed vs. smooth) Among populations 5 3.563 within groups Within populations 52 17.967 among the 31 smooth-shelled individuals (sites D-G) was only 26 and the 10 ob- served haplotypes had a haplotype diversity of d,= 0.692. However, the nucleotide se- quence diversity within the ribbed-shell specimens (x = 0.0131) was almost identi- cal with the diversity within the smooth- shelled individuals (x = 0.0132). The analysis of molecular variance (AMOVA) showed that most of the variation is within populations (69%), and the respective fixation index, although relatively low at 0.114, is highly significant. The variation among populations within the ribbed- and smooth-shelled groups accounts for only 9% of the total variation. How- Variance components Absolute % Fixation index E 0.10826 2] 73 Rep = 0:27 < 0.0569 0.04453 8.94 Fsr = 0.307 < 0.0025 0.34551 69.34 Fsc = 0.114 < 0.0001 ever, the fixation index is relatively high and sig- nificant at 0.307. The differences between the ribbed- and smooth-shelled populations (22% of the total variation) resulted in a fixation index of 0.217. However, the significance limit of 0.050 was slightly exceeded with 0.057 (Table 3). A pairwise test of differences in population structure showed that eight out of twelve com- parisons between smooth- and ribbed shelled populations were significant, whereas only two out of twelve pairwise comparisons within the three ribbed-shelled and the four smooth shelled populations were significant (popu- lations D vs. F and D vs. G) (Table 4). A maximum likelihood tree shows the phy- TABLE 4. Pairwise test on significant differences between populations of Oncomelania h. hupensis from the Miao River based on molecular variance (AMOVA). Significance level = 0.05. A B A Ribbed B - © 2 E D À 3 Smooth E ь z 5 + + G + + C D E F G + + - + + - + + - - 338 SHI ET AL. D4C C7 B1 Ye B6 A6, 8 В5; 8, 9 | C1, 3, 4, 5, 6, 8, 9 FIG. 2. Phylogenetic tree for seven populations of Oncomelania В. hupensis from the Мао River based on maximum likelihood analysis. The circle sizes are proportional to the observed number of individuals with each haplotype. Missing haplotypes are indicated by small circled. Smooth-shelled individuals are represented by white circles, ribbed-shelled individuals by black circles. Nodes that have support values between 50 and 80% are marked with a *. All other branches have support values of > 80%. Main haplotype groups and lineages are marked with roman numerals. Alternative arrangements of haplotypes, as inferred with statistical parsimony (SP), are indicated by dashed arrows. Note that in the SP network clades I/II, Ш, and IV could not be connected in a parsimonious fashion as the three groups exceeded the 95% connection limit of 10 mutational differences. logenetic relationships of the COl- haplotypes (Fig. 2). The tree consists of four main haplotype groups and lineages (I-IV). Lineage Ill comprises a single individual (B3), which is separated by twelve nucle- otide differences (x = 0.0204, = 2.0%) from its nearest neighbor, A7. Groups 1, Il, and IV con- tain 19, 20, and 19 individuals, respectively. The greatest nucleotide-sequence diver- gence between individuals can be found be- tween a ribbed-shelled individual (C7) anda smooth-shelled individual (D4) with x = 0.0454. GENETIC STRUCTURE AND INFECTIVITY OF ONCOMELANIAH. HUPENSIS 339 ) Miao River Ä Er & 8 | Population G E D С В А | №. specimens 10 10 5 6 9 9 10 | | | №. haplotypes 2 4 3 5 3 7 9 | smooth-shelled no flooding ribbed-shelled flooding FIG. 3. Information on haplotype diversity for the Oncomelania h. hupensis populations studied. Each site is represented by a circle divided into areas proportional to the number of individuals that belong to haplotype groups I-IV (see Fig. 2). The black bar between sites Band C indicates a dam. The greatest nucleotide-sequence divergence between two ribbed-shelled individuals is 0.0392, and between smooth-shelled indi- viduals, 0.0376. The smallest difference be- tween a ribbed- and smooth-shelled individual is 0.0031. Two haplotype groups each contain a common haplotype that is shared by more than ten individuals. In group II, one haplo- type is shared by 17 smooth-shelled indi- viduals (54.8% of all smooth-shelled individuals); in group |, one haplotype is shared by 12 ribbed-shelled individuals (42.9% of all ribbed-shelled individuals). We consider these to be “core” haplotypes. Smooth-shelled upstream individuals com- prise only haplotypes of groups II and IV; ribbed-shelled downstream individuals comprise haplotypes of all four groups. Addi- tional information on haplotype diversity is given in Figure 3. Sites A and B near the mouth of the Miao River have the greatest haplotype diversity. Group IV individuals are to be found at every site; group II individuals are to be found at all sites except C and group | individuals are restricted to the sites that are affected by flooding. The relatively low haplotype diversity in upstream popula- tions is mostly due to the lack of haplotype group l-specimens in the smooth-shelled populations (Fig. 3). The phylogeny obtained with the statistical parsimony network approach was almost identical with the maximum likelihood phy- logeny shown in Figure 2, except for minor changes in the arrangement of three termi- nal haplotypes in clade IV (indicated by dashed arrows in Fig. 2) and that clades ИП, Ш, and IV could not be connected in a parsi- monious fashion as the three groups ex- ceeded the 95% connection limit of ten mutational differences. The different haplotype diversities in smooth vs. ribbed-shell populations are readily re- flected in the mismatch distributions (Fig. 4). With the exception of populations C and D (both are from sites close to the flooding boundary), there is a trend of decreasing the number of classes of mutational differences from downstream to upstream populations. At site A we find a wide spectrum of classes rang- ing from 0 to 19 mutational differences (only with the 5, 11, and 15 bp-classes missing). A similar spectrum can be found at site B, but now with a gap between 6 and 14 mutational differences. The number of classes is further decreased in site C-specimens, temporarily in- creases in site D-specimens and then degreases to two (0 and 15 bp) in the speci- mens from site G However, mismatch distribu- tions are sensitive to population size. Therefore, the results for sites D (only six indi- viduals studied) and for site E (only five indi- viduals studied) have to be treated with caution. Interestingly, the mismatch distribution for the ten ribbed-shell specimens from site A is very 340 Frequency FIG. 4. Mismatch distributions of pairwise number of mutational differences between individuals of popu- lations A to С, between all ribbed-shelled individuals (“ribbed”), between all smooth-shelled individuals SHI ET AL. 0.7 Inn. ah. tall, 10 12 14 16 18 20 22 24 26 0.6 0.51 04! 0.3 0.2 0.1 0.0 6 8 10 12 14 16 18 20 22 24 26 2 B thn. In. 8 10 12 14 16 18 20 22 24 26 0.7 0.6 G 0.5 0.4 0.3 0.2 0.1 0.0 072 4 678 10 12 14 16 18 20 22 24 26 с | 0.7 ribbed 0.6} 0.57 04! 0 2 4 6 8 10 12 14 16 18 20 22 24 26 0.3 0.2 0.1 0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 D 0.7 — smooth 0.6} 0.5 blu hu 10 12 14 16 18 20 22 24 26 0.4} 0.3 0.2 0.1 0.0 OZ 4 6 8 10 12 14 16 18 20 22 24 26 0.7 0.6} ribbed + smooth | 0.5 0.4 0.3! 0274 18 6 8 10 12 14 16 18 20 22 24 26 0.2 + 0.1 = 0 01284568 10 12 14 16 18 20 22 24 26 Pairwise number of mutational differences (“smooth”), and between all individuals studied (“ribbed + smooth’). GENETIC STRUCTURE AND INFECTIVITY OF ONCOMELANIA Н. HUPENSIS 341 TABLE 5. Natural infection rates of field-collected Oncomelania h. hupensis from the Miao River. Total A B C A-C No. snails 464 381 408 No. positive 25 49 0 74 % positive 5.38 12.86 0 5.91 1,253 Total D E F G D-G 212 59 295 278 844 0 0 0 0 0 0 0 0 0 0 TABLE 6. Prepatent period giving the average and standard deviation of the number of days from infection to shedding of field-collected individual Oncomelania h. hupensis snails. Average @ D F G D-G Prepatent time (days) 81.7 +5.4 92.8 + 1.5 86 82.8 + 3.9 87.6 +5.7 No. cercariae shed 44.2 + 18.7 17.8 + 12.8 21 19.0 + 11.5 18.7 + 10.6 similar to both, the mismatch distribution of the DISCUSSION combined 28 ribbed-shell specimens and the mismatch distribution of all 59 specimens (ribbed and smooth) in our study (Fig. 4). Infectivity data area given in Tables 5-7. Only downstream snails were infected, and these with a prevalence of 5.9% (Table 5). Infections in these snails were used to test the prepatent period for cercarial emer- gence. There was no significant difference between upstream and downstream groups with regard to average prepatent period (P > 0.05), that is, 82-83 days. However, there was a significant difference between groups regarding numbers of cercariae shed per snail; the ribbed-shelled downstream snails shed more (P < 0.05). Also, there was a sig- nificant difference between groups in per- centage of snails that could be experimentally infected (P < 0.01). Site C snails had 60.9% successful infections compared to an average 8.1% for the up- stream D, F, and G site snails. There was no significant difference in mortality (P > 0.05). Genetic Diversity There are a least three possible explana- tions for the observed cluster patterns of mi- tochondrial haplotypes in Figure 2: (1) methodological problems, (2) the presence of a cryptic species complex, (3) high in- traspecific variation. In order to test whether our results are based on sequencing and/or alignment er- rors, we reanalyzed the sequence for the distinct haplotype B3, and the same result was obtained. Also, the sequences were free of ambiguities and base insertions/de- letions, and a translation into protein se- quences did not result in premature stop codons. We therefore can likely exclude the possibility of having nuclear pseudocopies of the COI gene, though there is a slight chance of a recent jump to the nucleus. Other possibilities would be heteroplasmy or duplications within the mitochondrial ge- TABLE 7. Prevalence of laboratory infections and death rates of field-collected Oncomelania h. hupensis in the Miao River. C No. snails 48 42 No. dead 2 No. infected 28 Infection (%) 60.9 9.8 Mortality (%) 4.7 2.4 Total F G D-G 42 42 126 1 0 2 2 4 10 4.9 9.5 8.1 2.4 0 1.6 342 SHI ET AL. nome (Zhang & Hewitt, 1996). However, we can safely exclude the possi- bility of having a cryptic species complex. In the 59 specimens studied, we have found a total of 27 haplotypes with a nucleotide-se- quence diversity of m = 0.0161, a haplotype diversity of h, = 0.878, and total number of 53 polymorphic sites (Table 1). These num- bers are well within the known sequence variation for Oncomelania h. hupensis. Wilke et al. (2000) studied ten populations with 80 specimens of O. h. hupensis from the lower Yangtze River. They found a total of 45 COI- haplotypes with an average haplotype diver- sity of h, = 0.972, a nucleotide-sequence diversity of x = 0.0192, and a total of 85 poly- morphic sites. The highest nucleotide-se- quence divergence between those ten populations was Dxy = 0.0207. This is even slightly higher than the highest divergence between populations found in the present study (Dxy = 0.0200). Moreover, Wilke et al. (2000) showed that the nucleotide-se- quence divergence between the 80 specimens of O. h. hupensis and a representative of O. h. robertsoni (the subspecies that occurs in southwestern China) ranged from 0.129— 0.132. Using the same specimen of O. h. robertsoni (GenBank accession AF253074), the nucleotide-sequence divergence with the 59 specimens of O. h. hupensis studied here is very similar with Dxy = 0.132. Thus, the diver- gence between the two subspecies O. h. hupensis and O. h. robertsoni is more than eight times higher than the nucleotide se- quence diversity within specimens of the sub- species O. h. hupensis studied here. Phylogenetic trees of O. h. hupensis often show numerous cluster and lineages that are separated by two to about ten substitutions. The respective missing haplotypes are possibly due to one or more of the following factors: (1) Inadequate sampling: Considering the haplotype diversity in the present study (e.g., the fact that we have found nine haplotypes in ten individuals from site A), it is clear that we have not reached the end of variation. (2) Genetic history: Considering the close coevolved relationships between the snail host and the schistosome parasite (reviewed in Davis 1980, 1992), a series of extinction- events, bottle-necks and strong selection for certain haplotypes must have occurred. Also, it is possible that there are multiple substitution events at the same time, thus some of the missing haplotypes may never have existed. (3) Current demographic processes: Human activities have a profound effect on the population structure of Oncomelania hupensis. The massive use of molluscicides and drowning as measures to control snail populations and therefore schistosomiasis, often with only few surviving snails, create major bottle necks. These measures likely play a role in the extinction of haplotypes. The cluster pattern in the phylogenetic tree (Fig. 2) somewhat resembles the СО! tree for populations of the rissooidean snail species Peringia ulvae from the northern Atlantic (Wilke & Davis, 2000). The brackish- water P. ulvae also has a similar genetic diversity (in terms of nucleotide and haplotype diversities). These patterns are believed to be the result of a high dispersal capacity combined with past fragmentation/ secondary contact events caused by Pleistocene glaciations. By comparison, while Oncomelania В. hupensis probably was not directly affected by Pleistocene glaciations, it also has a very high dispersal capacity. Oncomelania h. hupensis has negative rheotropism, that is, snails will, along a river, gradually move upstream. Snails can float upside down at the surface of the water to feed; there is net transport downstream. Flooding lifts and transports them downstream, especially during the annual monsoon floods (reviewed in Davis et al., 1999b). Snails attached to insects, birds, and hoofs of water buffalos can be transported from one locality to another. Allozyme vs. COI Sequence Data Allozyme data from the Miao River popula- tions based on Wright's D were robust enough to show distinctive differences among popula- tions, to show that population A was unique due to the extent of divergence from all other popu- lations, and to reveal unique alleles at different sites (Davis et al., 1999a). However, the poly- morphic loci were not in Hardy-Weinberg equi- librium. Low heterozygosity and paucity of unique alleles reduced their usefulness for ana- lyzing genetic variation within populations, gene flow, or population structure. Our hypothesis to account for these facts is that the annual floods of the Yangtze River transport large numbers of snails from one locality to the next. The annual deposition of snails in any area cre- ates an aggregate that is not a true population, GENETIC STRUCTURE AND INFECTIVITY OF ONCOMELANIA H. HUPENSIS 343 that is, there is no chance to reach Hardy- Weinberg equilibrium. Such aggregates have been called “genetically unstable populations” (Davis et al., 1999a, b). The cluster patterns of COl-haplotypes in the phylogenetic tree (Fig. 2), combined with the observed sequence polymorphism (Table 1) and the inferred mis- match distribution (Fig. 4) support this hypoth- esis. The downstream sites А, В, С, and, to a lesser extent, D are clearly not true populations but assemblages of diverse genetically distinct units. These results are concordant with those in Davis et al. (1999a) and are most likely due to a combination of the Wahlund (1928) effect (especially in aggregates A, B, and C) and intense inbreeding in upstream popula- tions heavily impacted by man. In this regard it is interesting that, though 22% of the total AMOVA variation is attributed to differences between the ribbed- and smooth- shelled populations, the fixation index (F...) is only 0.217 and the differences between the two groups are not quite significant (Table 3). Though, part of the problem may be the low sample size, the analysis of population struc- ture indicates that the differences between ribbed- and smooth-shelled populations are not discrete but rather gradually changing from downstream to upstream populations. Thus, all pairwise comparisons between the three most- downstream populations A, B, and C (ribbed- shelled) and the two most-upstream populations F and G (smooth-shelled) are sig- nificant, whereas differences involving popula- tions C-E may or may not be significant no matter whether they are ribbed- or smooth- shelled (Table 4). The Question of Ribbing Ribbing in Oncomelania h. hupensis is only found in snails living along the flood plains of the Yangtze River and associated tributaries, where severe flooding is an annual event driven by the seasonal spring monsoons (Davis et al., 1995, 1999a, b). In slightly higher elevation and beyond the effects of flooding, the shells are smooth. Wilke et al. (2000) studied the phylogeography of smooth- and ribbed- shelled populations in eastern China. They found that smooth and ribbed conditions are not discrete, but the two extremes of a continu- ously varying character as specimens from one site in Anhui Province, which was removed from the Yangtze River flood plains and prob- ably only slightly affected by annual flooding, had only weak ribs. The data also sug- gested that the smooth-shelled condition results from relief from flooding, not eleva- tion per se. Davis (1979) demonstrated that the primitive conditions historically among rissooidean snails in general, and Asian pomatiopsid snails in particular, are shells both small in size and smooth (Attwood & Johnston, 2001). Sculpture and increased size are derived. Oncomelania snails from Yunnan and Sichuan provinces (Subspecies O. hupensis robertsoni) have these plesiomorphic character-states. It is hypoth- esized that the sculpture and large size of On- comelania h. hupensis in eastern China are derived, and apparently evolved under selective pressure of annual torrential floods to survive on the flood plains of the lower Yangtze River (Davis, 1979). According to Davis & Ruff (1973), ribbing is controlled by a single gene with ribbing domi- nant to smooth and with inheritance typically Mendelian. Ribbing, while a dominant gene, has multiple alleles controlling size and thick- ness of the ribs. If there is a founder event with a ribbed colony established beyond the affects of flooding, ribbing will persist for a time. We do not yet know how long it takes for the evolution- ary process of strong selection against ribbing to result in a smooth shell, or the converse. As these snails live and reproduce over a life span of up to five years, it may take decades for all traces of ribbing to be lost. In a controlled ex- periment, Urabe (2000) found for the freshwa- ter snail Semisulcospira reiniana (family Pleuroceridae) that phenotypic variation of shell sculpture (ribbed vs. smooth) is controlled by environmental factors although it has a genetic basis and that, after changing the ecological setting, shell sculpture frequencies already started to change in the F1 generation. Rapid morphological changes of shell sculp- ture based on developmental flexibility or true adaptation have been reported for various gas- tropod taxa. The significance of shell sculpture has been attributed to defense against preda- tors (e.g., Vermeij, 1976; West & Cohen, 1996; Quensen & Woodruff, 1997), to thermoregula- tion (Vermeij, 1973), to prevention from sinking into the substratum (Hutchinson, 1993), to bur- rowing habits (Palmer, 1980), or to an in- creased drag in environments affected by water currents (Linsley, 1978; Denny, 1988; Urabe, 2000). Considering the distinct ecology of On- comelania hupensis (reviewed in Davis et al., 344 SHIETAL. 1999b), the above functions of ribbing can likely be excludes: (1) Increased drag in aquatic environments. Not applicable: adult Oncomelania snails are not aquatic; they do not live in the Yangtze River. Only the young, newly hatched snails are aquatic; at about two months of age, the snails move to an ecotone, a land-water interface. When swept away by the floods, they can float upside down at the water surface for a certain time. They cannot withstand continual sub- mersion; they will drown. (2) Defense against predation. Adult On- comelania snails do not have any major preda- tors. (3) Burrowing habits; prevention from sinking into the substratum. Oncomelania prefers silt- rich soil supporting an abundance of soil dia- toms used for food. We have not found significant differences in ecotone soil quality (composition, coarseness) from sites with smooth- and ribbed-shelled individuals. More- over, we have not observed any differences in burrowing habits between the two forms; at temperatures below 10°C both, smooth- and ribbed-shelled specimens, dig down into soil and aestivate. (4) Thermoregulation. We did not study ther- moregulation in Oncomelania. However, given the short distance between sites with smooth- and ribbed-shelled populations, there are no major differences in micro-climate (soil and air temperature, humidity, radiation). In our study, all specimens from sites af- fected by floods (sites A-C) had ribbed shells, and all specimens from sites that were isolated from the ‘flooding (sites D-G) had smooth shells. We therefore attribute ribbing as a re- sponse to flooding, as stated by Lou et al. (1982), and Davis et al. (1999a, b). The two hypotheses that account for this are (1) ribbing increases surface area that is important for floatation; (2) ribbing increases shell strength, an important factor in surviving the annual flooding and being swept around in flood wa- ters. The possibly rapid nature of shell sculp- ture changes may be responsible for the lack of intermediate shell types in the Miao River, which were previously reported from few other locations in China (see above). The Question of Susceptibility to Schistosome Infection No natural infections were found from site C to the top of the river. Historically, these popu- lations were infected (records of the local pub- lic health offices). In addition to the man-made flooding of site C (see Methods), there is exten- sive use of molluscicides to kill snails along the edges of the river thus controlling schistosome transmission. Large infection prevalences were found near the mouth of the river at sites A and B. This is the hardest area to control against schistosomes because of annual importation of adult infected snails swept in by the floods of the Yangtze River. It is well Known to Chinese provincial anti- schistosomiasis control centers that snail populations with high infection rates, that is, > 1%, are to be found on the flood plains of the Yangtze River or its tributaries. These popula- tions have a high genetic diversity. In regionally isolated areas where populations have a lower genetic diversity, the infection rates are much lower, too (i.e., < 0.1%). Accordingly, the infec- tion rates in popuiations A and B at the mouth of the Miao River are very high. The site C population was used in the infection experi- ments because it is a ribbed-shelled population affected by flooding but snails were not natu- rally infected, an essential aspect of the infec- tivity study. We attribute the lack of natural infections partly to the snail control measure (routine drowning) that kills off the adult infected snails. In the experiment, group C snails were significantly more susceptible to infection than were the upstream smooth-shelled populations (Table 7). However, this difference has more to do with the genetic isolation of upstream popu- lations than with the smooth or ribbed nature of the shells. The argument for this assertion fol- lows. There is a coevolved interaction between the parasite Schistosoma japonicum and its snail hosts Oncomelania hupensis (reviewed in Davis, 1980, 1992) that is specific, at this point in time, at the regional level and frequently at the population level. The literature on this speci- ficity is quite large and involves regional differ- ences in snail infectivity by different geographic strains of S. japonicum (DeWitt, 1954; Yuan et al., 1984). The hypothesis to explain this coevolution is that infected snails evolve defense mecha- nisms that act to counter the parasite. The parasite then counters with an evolved re- sponse to overcome the host's defenses. This invokes the Red Queen hypothesis of Van Valen (1973). Genetic studies of freshwater snails (Dybdahl & Lively, 1998) and bumble bees GENETIC STRUCTURE AND INFECTIVITY OF ONCOMELANIA H. HUPENSIS 345 (Baer & Schmid-Hempel, 1999) showed that infections with parasites were significantly lower in populations with higher genetic diver- sity and that rare genotypes were also less sus- ceptible to infection. The theory is that parasites that can successfully infect the most common host genotype will be favored by natu- ral selection (Lively, 2001). The situation in Oncomelania appears to be different as genetically diverse snail popula- tions are more susceptible to infection. This could be due to the high immigration and emi- gration rates of snails and parasites in the floodplains of the Yangtze River. The low temporal stability of host-parasite interac- tions probably makes it impossible for the Red Queen to get engaged. Our hypothesis for the high infection rates in downstream Miao River population follows from this as- sumption. Flood-affected populations bring together snails and the schistosomes they carry coming from diverse locations (Wahlund effect). In these heterogeneous parasites and snails there is a higher prob- ability that parasite eggs hatching from a lo- cal definitive host (water buffalo, pig, man) will encounter genetically suitable snails that do not have a sufficient defense mechanism against this particular strain of parasite. More- over, it is conceivable that snail populations can effectively fight off only one or few parasite strains at atime. In cases of multiple infections by different parasite strains, which likely occur in the flood-affected areas, the defense system of the snail host might be inadequate. In isolated snail populations with low rates of immigration and emigration, the laws of the Red Queen probably apply. The outcome may be equilibrium with low frequencies of infection (observed for a number of isolated populations of Oncomelania hupensis with low heterogene- ity; see also Lively, 2001), fixation in the snails to resist all attacks of the parasite (seen in some populations in Taiwan), or snail popula- tion extinction (of course, not directly detect- able; but observed for a population of the rissooidean snail genus Hydrobia caused by bird trematodes; Davis et al., 1989). The comparatively low experimental infection rates obtained in the smooth-shelled Miao River localities is probably due to the relative isolation of those populations where there is equilibrium with low frequencies of infection. 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Individualcode DNA sample # GenBank accession # Al MG 301 AF306572 A2 MG 302 AF306573 A3 MG 303 AF306574 A4 MG 304 AF306575 A5 MG 305 AF306576 A6 MG 306 AF306577 A7 MG 307 AF306578 A8 MG 308 AF306579 A9 MG 309 AF306580 A10 MG 310 AF306581 B1 MG 341 AF306613 B2 MG 342 AF306614 B3 MG 343 AF306615 B4 MG 344 AF306616 B5 MG 345 AF306617 B6 MG 346 AF306618 B7 MG 347 AF306619 B8 MG 349 AF306620 B9 MG 350 AF306621 C1 MG 259 AF306622 C2 MG 260 AF306623 C3 MG 286 AF306624 C4 MG 287 AF306625 C5 MG 288 AF306626 C6 MG 289 AF306627 C7 MG 290 AF306628 C8 MG 291 AF306629 C9 MG 292 AF306630 D1 MG 335 AF306582 D2 MG 336 AF306583 Individual code DNA sample # GenBank accession # D3 MG 337 AF306584 D4 MG 338 AF306585 D5 MG 339 AF306586 D6 MG 340 AF306587 Ei MG 351 AF306598 E2 MG 352 AF306599 ЕЗ MG 353 AF306600 E4 MG 354 AF306601 E5 MG 355 AF306602 FA MG 321 AF306588 F2 MG 322 AF306589 ES MG 323 AF306590 F4 MG 324 AF306591 F5 MG 325 AF306592 F6 MG 326 AF306593 F7 MG 327 AF306594 F8 MG 328 AF306595 F9 MG 329 AF306596 F10 MG 330 AF306597 G1 MG 311 AF306603 G2 MG 312 AF306604 G3 MG 313 AF306605 G4 MG 314 AF306606 G5 MG 315 AF306607 G6 MG 316 AF306608 G7 MG 317 AF306609 G8 MG 318 AF306610 G9 MG 319 AF306611 G10 MG 320 AF306612 4 mA: ¡1 MALACOLOGIA, 2002, 44(2): 349-352 RESEARCH NOTES SYSTEMATIC IMPLICATIONS OF EXTREME LOSS OR REDUCTION OF MITOCHONDRIAL LSU rRNA HELICAL-LOOP STRUCTURES IN GASTROPODS Charles Lydeard', Wallace E. Holznagel', Rei Ueshima? & Atsushi Kurabayashi? Recent systematic studies on one of the largest recognized classes of animals —the Gastropoda —have resulted in the disman- tling of the traditional classification scheme, which had been firmly entrenched since the early 1900s (Haszprunar, 1985, 1988; Bieler, 1992; Ponder & Lindberg, 1997). This revision was precipitated by the recognition of a new clade currently referred to as the subclass Heterobranchia, which includes the pulmo- nates, opisthobranchs, and several groups of mostly small marine snails formerly placed in the now obsolete subclass, Prosobranchia. Heterobranchs presumably separated from its sister group the Caenogastropoda, which includes mostly marine, but some terrestrial and freshwater taxa from several major, po- tentially paraphyletic, taxonomic groups (e.g., Architaenioglossa, Neotaenioglossa, Pteno- glossa and Neogastropoda), about 350-400 million years ago (Bieler, 1992; Ponder & Lindberg, 1997). The monophyly of Hetero- branchia is strongly supported by many anatomical features, such as loss of a true ctenidium, simple esophagus, lack of odon- tophoral cartilages, and pigmented mantle organ (see Ponder & Lindberg, 1997, for de- tailed review). Given that these radical phylo- genetic hypotheses eliminated such tradi- tional groups as the Prosobranchia and Mesogastropoda, which were recognized for nearly a century (but still erroneously used by GenBank and many recent textbooks), it is imperative that they be further tested using in- dependent data sets. It is well documented that ribosomal RNA sequences fold into com- plex secondary structures based largely on in- tramolecular base pairing. The vast majority of the rRNA secondary structure models have been determined with comparative sequence analyses (Woese et al., 1980; Noller et al., 1981; Gutell et al., 1994). Based on a detailed comparative analysis of complete mitochon- drial (mt) large subunit (LSU) rRNA secondary structures among 10 molluscan taxa, Lydeard et al. (2000), discovered three putative struc- turally based synapomorphies (Lydeard et al., 2000; 98-99, fig. 7) uniting the stylom- matophoran gastropods, a group of terrestrial heterobranchs. Here, we show based on a more comprehensive analysis of 58 complete or near-complete mt LSU rDNA sequences and subsequently derived secondary struc- tures that the loss or reduction of the three substantial helical-loop structures described previously for stylommatophorans are molec- ular synapomorphies of the Heterobranchia. We sequenced 50 complete or near-com- plete mt LSU rDNA sequences and obtained four gastropod, two bivalves, one cephalo- pod, and one chiton sequence from GenBank. Sequencing primers and methods are de- scribed in detail in Lydeard et al. (2000) and Kurabayashi & Ueshima (2000). Most of the taxa (n = 32) that were examined were mem- bers of Cerithioidea for an examination of the phylogenetic relationships within the super- family and are dealt with in more detail else- where (Lydeard et al., 2002). We generated secondary structure models for a comparative analysis based on secondary structure mod- els described and determined elsewhere for ten representative mollusks (Lydeard et al., 2000; available online at http://www.rna. icmb.utexas.edu), and focused our efforts on the three relevant regions hypothesized to be synapomorphies of stylommatophorans based on the previous study. Exemplar struc- ‘Biodiversity & Systematics, Department of Biological Sciences, University of Alabama, Вох 870345, Tuscaloosa, Alabama 35487 USA; clydeard@ bama.ua.edu Department of Biological Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; rueshima O biol.s.u- tokyo.ac.up Sinstitute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan 350 tures representing the range of variation ob- served within mollusks for the three relevant regions of mt LSU rRNA are shown in Figure 1. Figure 2 shows the taxa that were exam- ined in a phylogenetic framework using a phy- logenetic hypothesis of the Gastropoda based on a Cladistic analysis of 117 morphological characters (Ponder & Lindberg, 1997). To il- lustrate the evolution of the three secondary structural characters, we have chosen to map the characters on the independently derived morphological-based tree (Fig. 2). The ab- sence or reduction of three substantial helical- loop structures was discovered for all hetero- branch taxa. Within Domain Il, a helical-loop structure is absent in all heterobranchs, with the exception of the basal heterobranch taxa Valvata hokkaidoensis and Psilaxis radiatus. All other gastropods and outgroup taxa, with the exception of Nerita atramentosa and Mytilus edulis, possess a helical-loop struc- ture that ranges from 42-62 bp in length. Val- vata, Psilaxis, Nerita, and Mytilus have a re- duced (15, 15, 19, 16 bp, respectively) helical-loop structure relative to other mol- lusks. The most parsimonious hypothesis ex- plaining the secondary structure variation de- a. b. Psilaxis radiatus Littorina saxatilis LYDEARD ET AL. picted in Domain II requires four steps. With the reduction of the helical-loop structure evolving independently on three separate oc- casions in Mytilus, Nerita, and the Hetero- branchia, followed by the complete loss of the structure in derived heterobranch lineages (Figs. 1, 2). The second structural-based character involves the entire Domain Ill. All outgroup taxa, basal gastropod lineages, and caenogastropods possess a helical-loop structure ranging from 30-79 bp; however, all heterobranchs lack the helical-loop structure (Figs. 1, 2). The third structural-based molec- ular character is the presence or absence of a bulge-stem-loop structure that is 41-43 bp, whereas all heterobranchs lack the bulge- stem-loop structure and only have a 20-30 bp structure. The indel in Domain V has been re- ported previously in a study of euthyneuran relationships (Thollesson, 1999). The most parsimonious hypothesis explaining the varia- tion found in Domains Ш and Vis that the loss of the helical-loop structures is the derived condition uniting all heterobranchs. Interest- ingly, the extreme reduction or loss of the three helical-loop structures reveals, in part, why heterobranchs have among the shortest С. Albinaria coerulea h Ayu - С UUUUAAG UUUU UUAU P ) СААСАА! А S DIU ied AS A TUE AAAAA AAUA - CGAAAUUC A СС и AGUUUUU U AG i E AG y À U A ‚С С U à Ale Al] U ñ Uy UL U U=A A mn ^ У U Ve is ar hs NA As yA А, < у А с UA . А AV “AceG С C : A Domain И USA ES Amu Domain II A = UN AT GC ven U—A >. NU U—A A=U N U A Ü uV =Ac D ain II U Sa UN oi Geu ЗА = UY U—A omaın < ze E Gel y NA ie A À A=Uc © “0 AS с Sie AUA NG AG Realty Uy s Ba Ar AU NA À = |) 5 U - А J Ù — À 5 | A U Am UV = J U=A | Domain Ш Domain ИГ ------- missing Domain II ------- missing CAUAA uuuuu® Sg Ne CA GAAG SAUNA - 4 . . U LET «I AE la los IT IS GUGUUGAA ¿A GGU GAGCCUUCA GUGUUGGAAAAAA y ‹ UC С UA Domain V Domain V Domain V FIG. 1. Representative gastropod secondary structure models for a, Psilaxis radiatus (Heterobranchia: Ar- chitectonicoidea); b, Littorina saxatilis (Caenogastropoda: Littorinioidea) and c, Albinaria coerulea (Hetero- branchia: Euthyneura). Additional secondary structure diagrams for other molluscan taxa can be found in Ly- deard et al. (2000). MITOCHONDRIAL LSU rRNA HELICAL-LOOP STRUCTURES IN GASTROPODS 351 a Domain b Domain II e . .ne = 5 ee Ш Ш Ш Katharina ex) Ш Ш Ш Pectn A ie о № m Mu ie Ш Ш Ш Loligo 42 - 62 bp eos Ш Ш Ш Cellana JPatellogastropoda > DY о DO MM \eria ]Neritopsina ee e So eo. 15-19 bp NE Ш Ш Ш Chpidina ]Vetigastropoda eos oP oe E Ш Ш m Pomacea/Tulotoma e Ye? 0% N° % Ш Ш E Coclophorus e o es о = A ee бо о Ш Ш Ш Cerithioidea ы W E E Campaniloidea = A o e aa Ш Ш E Apdrobia |Rissooidea 2 o e ee В = e e Ш Ш EM Serpulorbis | Vermetoidea = o e Nodilittorina Sa e o ини ERW [Littorinioides Le °e—e° Ш Ш Ш Litorina e—e Ш Ш Ш Busycotypus ]Neogastropoda Domain Ш 323 e—e 0b О О О Valvara |Valvatoidea 30-795 о 0000 р О О DO Psiaxis |Architectonicoidea P = O O © Cingulina|Pyramidelloidea = О О а Omalogyra |Omalogyroidea я e—e e Siph i 3 A ооо ue = Domain Y Albinaria с. | = O00 8 nan = 5 EYTYYYYYIYIYYT De a ®о О DD Albinariaı | Е = OTE IN e 41-43bp о са Cepaea 2 5 0000000000000, 00, e? О Оо Euhadra |2 00000 особо P ТЕТЕ III © 20-30bp ОО 9 pa 00000 00050 Ponder & Lindberg, 1997 FIG. 2. Evidence from mitochondrial LSU secondary structures uniting the Heterobranchia, a group that di- verged from the Caenogastropoda 350-400 mya. a, Phylogeny of gastropods inferred from a cladistic analy- sis of 117 morphological characters (Ponder & Lindberg, 1997) showing the taxa included in the present study. Mitochondrial LSU rRNA sequences for the outgroup taxa (Katharina tunicata, Pecten maximus, Mytilus edulis, Loligo bleekeri), the heterobranchs Albinaria coerulea, Albinaria turrita, Cepaea nemoralis, and Euhadra herklotsi, and the caenogastropod Littorina saxatilis (Wilding et al., 1999) were obtained from GenBank (U09810, X92688, M83756, AB009838, X83390, X71393, X71394, U23045, 271693, AJ132137). GenBank accession numbers for the remaining taxa are AB028237, AY10505-526, AF101007-08, AF100991, AYO10314-327, AY081770-772, AY081996-2000. The columns of boxes show the three sec- ondary structural characters: Domain II (character states = (1) 42-62 nucleotides in length — black box, (2) 15-19 nucleotides in length — gray box, (3) stem-loop structure absent —open box); Domain III (character states = (1) 30-79 nucleotides in length —black box, (2) stem-loop structure absent — open box); and Do- main V (character states = (1) 41-43 nucleotides in length — black box, (2) stem-loop structure absent — gray box). b, Schematic diagrams representing consensus secondary structure models for the three secondary structural characters showing the character states within Domains Il, Ill, and V (described above) of the ex- amined taxa (actual secondary structure models for the three Domain regions is available online (http://www.bama.ua.edu/~clydeard). The shade (black, gray or white) of the helical-loop structure corre- sponds to the character states shown at the tips of each terminal branch in 2a. The four open dots shown for Domain Il and III, respectively, indicate no helical-loop structure is found for the taxon. mitochondrial genomes documented for metazoans (Kurabayashi & Ueshima, 2000). The nature of secondary structural variation exhibited among gastropods is analogous to mative data for elucidating deep evolutionary nodes. conservative variation reported for mitochon- drial gene order among arthropods (Boore et al., 1995) and gastropods (Kurabayashi & Ueshima, 2000). It is possible comparative analyses of secondary structure of other mol- luscan taxa will yield phylogenetically infor- ACKNOWLEDGMENTS This work was funded, in part, with a grant from the National Science Foundation to CL and W. F. Ponder. We thank our many coop- erative colleagues for help in obtaining mate- 352 LYDEARD ET AL. rial for study. Don Colgan, Winston Ponder, and Brian Simison provided helpful comments on the manuscript. LITERATURE CITED BIELER, R. D., 1992, Gastropod phylogeny and systematics. Annual Review of Ecology and Sys- tematics, 23: 311-338. BOORE, J. L., Т. M. COLLINS, D. STANTON, L. L. DAEHLER & W. M. BROWN, 1995, Deducing the pattern of arthropod phylogeny from mitochon- drial DNA rearrangements. Nature, 376: 163- 165. GUTELL, В. В., М. LARSEN & С. В. WOESE, 1994, Lessons from an evolving rRNA: 16$ and 23$ rRNA structures from a comparative perspective. Microbiology Review, 58: 10-26. HASZPRUNAR, G., 1985, The fine morphology of the osphradial sense organs ofthe Mollusca. Part 1: Gastropoda-Prosobranchia. Philosophical Transactions of the Royal Society of London (B)307: 457-496. HASZPRUNAR, G., 1988, On the origin and evolu- tion of major gastropod groups, with special ref- erence to the Streptoneura (Mollusca). Journal of Molluscan Studies, 54: 367-441. KURABAYASHI, A. & R. UESHIMA, 2000, Com- plete sequence of the mitochondrial DNA of the primitive opisthobranch gastropod Pupa strigosa: systematic implication of the genome organiza- tion. Molecular Biology and Evolution, 17: 266-277. LYDEARD, C., W. E. HOLZNAGEL, M. GLAUB- RECHT & W. F. PONDER, 2002, Molecular phy- logeny of a circum-global, diverse gastropod superfamily Cerithioidea (Mollusca: Caeno- gastropoda): pushing the deepest phylogenetic limits of mitochondrial LSU rDNA sequences. Molecular Phylogenetics and Evolution,22: 399- 406. LYDEARD, C., W. E. HOLZNAGEL, M. N. SCHNARE & R. R. GUTELL, 2000, Phylogenetic analysis of molluscan mitochondrial LSU rDNA sequences and secondary structures. Molecular Phylogenetics and Evolution, 15: 83-102. NOLLER, H. F., J. КОР, V. WHEATON, J. BRO- SIUS; В. В. (GUTEEE A: М; KORYEOVAE DOHME & W. HERR, 1981, Secondary structure model for 23S ribosomal RNA. Nucleic Acids Re- search, 9: 6167-6189. PONDER, W. F. & D. R. LINDBERG, 1997, Towards a phylogeny of gastropod molluscs: an analysis using morphological characters. Zoological Jour- nal of the Linnean Society, 119: 83-265. THOLLESSON, M. 1999. Phylogenetic analysis of Euthyneura (Gastropoda) by means of the 16S rRNA gene: use of a ‘fast’ gene for ‘higher-level’ phylogenies. Proceedings of the Royal Society of London, (B)266: 75-83. WILDING, С. S., P. 4. MILL 4 J. GRAHAME, 1999, Partial sequence of the mitochondrial genome of Littorina saxatilis: relevance to gastropod phylo- genetics. Journal of Molecular Evolution, 48: 348-359. WOESE, С. R., |. J. MAGRUM, В. GUPTA, В. В. SIEGEL & D. A. STAHL, 1980, Secondary struc- ture model for bacterial 16S ribosomal RNA: phy- logenetic, enzymatic and chemical evidence. Nu- cleic Acids Research, 8: 2275-2293. Revised ms. accepted 5 March 2002 MALACOLOGIA, 2002, 44(2): 353-361 TWO NEW TROPHONINAE (GASTROPODA: MURICIDAE) FROM ANTARCTIC WATERS Guido Pastorino Museo Argentino de Ciencias Naturales, Av. Angel Gallardo 470 3° piso lab 57, C1405DJR Buenos Aires, Argentina; pastorin @ mail.retina.ar ABSTRACT Two new species of Trophon from Antarctic waters are described. Trophon emilyae, n. sp., from the northwestern Amundsen Sea, is characterized by a small, slender, almost smooth shell and a thin profile. Axial sculpture consists of regular, thin varices, and four weak cords on the first whorls are the only spiral ornamentation. It is similar to T. declinans Watson, 1882, but the radula and shell ornamentation are clearly different. Trophon arnaudi, п. sp., was collected off South Sandwich Island. It is a small species similar to T. scolopax Watson, 1882, from the Kerguelen Islands. The shell is chalky white, with 10-11 axial lamellae crossed by five spiral cords on the last whorl. Adult specimens of the new species are illustrated, described and compared with other living species of the same genus and of similar geographic distribution. Lectotypes of 7. septus Watson, 1882, and 7. declinans Watson, 1882, are here designated. Key words: Antarctica, Trophon arnaudi n. sp., Trophon emilyae n. sp., Muricidae, Trophoninae, systematics, biogeography. INTRODUCTION The subfamily Trophoninae is by far the larg- est group of marine living gastropods in Antarc- tica. A detailed survey shows more than 100 names published for this group in southern South America and Antarctica. From this total, approximately 30 belong to Patagonian species of the genus Trophon 5. I., 34 to Xymenopsis, and the rest to Antarctic species. Recently, Xymenopsis was show to have only four valid species by Pastorino & Harasewych (2000). In the last comprehensive revision of southern species of Trophon, Powell (1951) described T. cuspidarioides and included Watson’s species T. scolopax, T. septus, and T. acanthodes in the same group, because of their long anterior canals. However, he considered this feature not of sufficient taxonomic importance to warrant separation of this group of species. This is true, because T. acanthodes Watson, 1882, despite the similarities in the long anterior canal, has a very different radula and protoconch (Pastorino, in prep.). In this paper, two new living species of the subfamily Trophoninae are described from 353 Antarctic waters. Adult specimens, operculum, radula, protoconch, and shell ultrastructure are described for each. Due to the extraordinary morphological variation within this genus, comparison with all similar species from the same geographic area is mandatory. MATERIAL AND METHODS Specimens studied in this work are housed in the collections of: the National Museum of Natural History, Smithsonian In- stitution, Washington, D.C. (USNM); Museo Argentino de Ciencias Naturales “Bernar- dino Rivadavia”, Buenos Aires (MACN); The Natural History Museum, London, (NHM); and Zoological Institute of the Russian Acad- emy of Sciences, St. Petersburg (ZIN). Mate- rial housed at USNM, collected by the ship R/ V Eltanin belongs to the United States Ant- arctic Program (USAP) and material housed at ZIN was collected by the R/V Evrica. In ad- dition, type specimens from the Strebel col- lection housed in part at the Zoologisches Institut und Zoologisches Museum der Universität Hamburg was also studied. 354 PASTORINO Dissections were performed on ethanol- preserved specimens to study radulae and male reproductive system. Radulae were prepared according to the method described by Solem (1972) and observed using a LEO 440 scanning electron microscope (SEM) at the USNM. Radular terminology follows Kool (1993: fig. 6B). Shell ultrastructure data were procured from freshly fractured colabral sec- tions taken from the central portion of the lip on the last whorl of two individuals per taxon, whenever sufficient material was available. Photographs were taken using a digital scan- ning camera. Several images were scanned from black and white 35 mm negatives using a slide scanner. All images were digitally pro- cessed. SYSTEMATICS Class Gastropoda Cuvier, 1797 Order Neogastropoda Wenz, 1938 Family Muricidae Rafinesque, 1815 Subfamily Trophoninae Cossmann, 1903 Genus Trophon Montfort, 1810 Type species: Murex magellanicus Gmelin, 1791, = Trophon geversianus (Pallas, 1774), by original designation. Trophon emilyae Pastorino, new species (Figs. 1-14) Diagnosis Shell small, slender, almost smooth; siphonal canal very long, twisted, narrow. Axial sculpture of regular, thin, gentle varices; spiral sculpture of four very weak cords in the first whorls, then obsolete. Description Shell small in size (up to 16 mm), slender, fusiform, very thin, translucid; protoconch of one and a half whorls; teleoconch of five mod- erately convex whorls; spire 1/4 of total shell length; subsutural shelf faint. Spire angle about 40°, suture abutting; aperture small, its interior glossy white; anterior siphonal canal very long, twisted, narrow; umbilicus closed; outer lip rounded; columellar lip almost indistinct. Axial ornamentation of regular, thin varices, about 8— 11 on last whorl, slightly developed, running along whorl surface from adapical suture to siphonal fasciole, weakly projecting outwards along keel. Spiral ornamentation of low, rounded spiral cords, four in the first whorls then obsolete, the entire shell surface covered by regular threads. Regular growth lines cover- ing whorls and lamellae. Innermost shell layer very thin, comprising about 5% of shell, composed of aragonite (?), with the crystal planes oriented perpendicular to growing edge of shell; second layer arago- nitic crossed-lamellar, representing 95% of shell thickness. Operculum suboval, completely covering aperture, brownish in color, thin, with terminal nucleus; growth lines covering external surface curved at upper ends; attachment area with two or three horseshoe-shape scars. Rachiglossan radula with teeth very closely packed; rachidian two times wider than height; central cusp very thin; lateral cusps shorter but of similar thickness to central cusp, denticle between central and lateral cusp very large. Base of rachidian teeth curved, large; marginal area inclined, smooth. Lateral teeth large size, with a thick attached portion. Etymology This species is dedicated to Dr. Emily Vokes, of Tulane University, New Orleans, Louisiana (now retired), who helped me with my first steps on Trophon. Type Material & Locality Holotype: USNM 896438; R/V Eltanin Station 1343, 567-604 m, collected with rock dredge, 7 November 1964, 54°50’S/29°50’W. Paratypes: USNM 1003475, from type local- ity, 3 specimen (one with animal); USNM 896441, R/V Eltanin Station 1346, 549 m, 54°49’S-129°48’W, 4 specimens; USNM 898917, R/V Eltanin Station 1345, 915-1153 m, 7 November 1964, 54°50’S/129°48’W, 4 shells, collected with Menzies trawl. Distribution Known only from the vicinity of the type locality. Remarks The shell morphology of Т. етйуае is com- parable to some specimens of T. declinans Watson (Figs. 16, 17) despite the distance be- tween their distributions on the opposite sides of Antarctica (Fig. 44). The shell of T. emilyae NEW ANTARCTIC SPECIES OF TROPHON 355 FIGS. 1-3. Trophon emilyae, new species, three views of holotype, USNM 896438; scale bar = 0.5 cm. FIGS. 4, 5. Two views of a paratype, USNM 896441, uncoated SEM; same scale as in Figs. 1-8. FIG. 6. Apertural view of a paratype, USNM 896441; same scale as in Figs. 1-3. FIG. 7. External view of operculum of paratype in Figs. 4, 5; scale bar = 1 cm. FIGS. 8, 9. Two views of protoconch of paratype in Figs. 4-5; scales = 500 um. FIG. 10. Radula side view; scale bar = 10 um. FIG. 11. Radula front view; scale bar = 20 um. FIG. 12. Shell ultrastructure, uncoated SEM; scale bar = 30 um. FIG. 13. Trophon declinans Watson, 1882, MACN 34956, collected by P. Arnaud, February 3, 1975, from NW of Kerguelen Islands in 70 m, radula front view; scale bar = 30 um. FIGS. 14, 15. Two views of protoconch of same specimen; scale = 500 um. FIGS. 16, 17. Trophon declinans Watson, 1882, two views of lectotype, NHM 1887.2.9.573; scale Баг = 0.5 cm. 356 PASTORINO FIGS. 18-20. Trophon arnaudi, new species, three views of holotype, USNM 1003473; scale bar = 0.5 cm. FIG. 21. Apertural view of paratype ZIN 59775; same scale as in Figs. 18-20. FIGS. 22, 23. Holotype, two views of protoconch, uncoated SEM; scale bar = 400 um. FIG. 24. Radula frontal view; scale bar = 30 um. FIG. 25. Radula side view; scale bar = 30 um. FIGS. 26, 27. T. scolopax Watson, 1882. FIG. 26. Radula side view, MACN 34955; scale bar = 40 um. FIG. 27 radula frontal view, same specimen as in Fig. 26, scale bar = 80 um. NEW ANTARCTIC SPECIES OF TROPHON 357 is more slender, with a longer anterior canal. The axial lamellae in T. declinans are some- what protruding from the whorl side, whereas in T. emilyae they are never detached from the shell wall. The radulae are also different; the base of the rachidian in T. emilyae is some- what curved, with a long, thin intermediate den- ticle between the central and lateral cusps. The lateral cusps are thinner and longer in the new species (Figs. 10, 11). To fix the identity of the taxon (ICZN, 1999: Art. 74.7), the specimen of T. declinans illustrated here in Figures 16 and 17 (NHM 1887.2.9.573) is here designated lectotype. The other three lots with one speci- men each therefore become paralectotypes (NHM 1887.2.9.574, 1887.2.9.575, and 1985041). Cernohorsky (1977) mentioned the Magel- lanic T. ohlini Strebel, 1904, as being very simi- lar to T. declinans. The type material of T. ohlini, housed in the Zoologisches Institut und Zoologisches Museum der Universitat Ham- burg, was studied as part of the complete revi- sion of the genus in progress. Based on the morphology of its protoconch, it is apparently not related to any Antarctic species. Trophon arnaudi Pastorino, new species (Figs. 18-20) Diagnosis Shell small, thin, fusiform; axial ornamenta- tion of regular low lamellae, projecting out- wards along periphery; 2-3 spiral rounded cords on the first whorls become 4—5 on the last whorl. Description Shell small in size (~ 13 mm), thin, fusiform, chalky; protoconch of 1% whorls; teleoconch of four and a half, right-angled whorls; spire 1/5 of total shell length; subsutural shelf short but clearly defined. Spire angle about 45-50°, su- ture abutting; aperture small, rounded, its inte- rior glossy white; anterior siphonal canal long, curved backwards; umbilicus closed; outer lip rounded, with lirations from spiral ornament; columellar lip narrow. Axial ornamentation of regular, thin lamellae, about 8—11 on last whorl: lamellae moderately developed, running along whorl surface from adapical suture to base of last whorl, projecting outwards along periphery. Spiral ornamentation of rounded cords, slightly developed on the first whorls to 4—5 on the last. Regular growth lines covering whorls, cords and lamellae. Operculum and shell ultrastructure unknown. Radula rachiglossate, rachidian with central cusp very thin; lateral cusps slightly shorter but of similar thickness to central cusp, denticle between central and lateral cusp large, rising from the base. Base of the rachidian teeth curved; large, smooth marginal area. Lateral teeth large, with a thick attached portion. Etymology This species is dedicated to Dr. Patrick Arnaud from Endoume, Marseille, for his many contributions to Antarctic malacological studies. Type Material & Locality Holotype: USNM 1003473; R/V Islas Orcadas, Cruise 575, Station 53, 26 May 1975, off South Sandwich Island, 57°41.4’S/ 26°22.3’W, 355-468 m; one paratype: ZIN 59775 collected alive by R/V Evrica on 22 Janu- ary 1987, drag 2, in 370 m, 57°38’S/26°18’W, volcanic sand bottom. Distribution Known only from the vicinity of the type locality. Remarks Despite the 100° of longitude separating their distributions, 7. scolopax Watson, 1882, de- scribed from off the Kerguelen Islands and Crozet Island, is comparable, but it has differ- ent lamellae. In T. scolopax, after a large su- tural shelf, the lamellae end in vaulted spines in number of two or three plus the keel. In addi- tion, the 2-3 rounded spiral threads in T. scolopax are 4-5 real cords in 7. arnaudi. The anterior siphonal canal is shorter and curved in T. arnaudi and always measures less than half shell height. Trophon scolopax has a straight siphonal canal that is more than half the shell length. Trophon scolopax was described by Watson from material belonging from the Challenger Expedition. The holotype (Figs. 28-30) is housed in the NHM under the number 1887.2.9.580. There were very few literature citations after the original description. Cantera & Arnaud (1985) cited living specimens of T. scolopax between 60 to 620 m off the Kerguelen Islands. 358 PASTORINO FIGS. 28-30. Trophon scolopax Watson, 1882; three views of holotype, NHM 1887.2.9.580; FIGS. 31, 32. T. scolopax apertural and adapertural views, MACN 34955, collected by P. Arnaud on April 7, 1974, in 560-525 m, NE of Heard Island; FIGS. 33, 34. MACN 34955 apertural and adapertural view of a smooth specimen from same lot of specimens in Figs. 31, 32. Figs. 35-37. T. cuspidarioides Powell, 1951, three views of holotype, NHM 1961547. FIGS. 38, 39. T. septus Watson, 1882, paralectotype, NHM 1887.2.9.579. FIGS. 40-42, lectotype, NHM 1887.2.9.578; scale bar = 1 cm for all figs. NEW ANTARCTIC SPECIES OF TROPHON 359 Trophon cuspidarioides Powell, 1951, known only from the original material from off mouth of Cumberland Bay, South Georgia Island, resembles a somewhat smooth specimen of 7. arnaudi. However, Т. cuspidarioides (Holotype: NHM 1961547) (Figs. 35-37) never develops the spines, lamellae and the spiral cords that charac- terize T. arnaudi. Finally, T. septus Watson, 1882, the last com- parable Antarctic species, known from off the Kerguelen Islands, differs in having triangular upturned spines and smooth whorls. Three syntypes are in the NHM 1887.2.9.578, 1887.2.9.579 and 1887.2.9.579a, the first two 90°—1 1015 dry are illustrated here in Figures 38-42. The specimen illustrated in the Figures 40-42 is here selected as lectotype (ICZN. 1999: Art. 74.7). The remaining syntypes are there- fore paralectotypes. Cernohorsky (1977) mentioned some resem- blance of large specimens of T. septus with T. coronatus H. Adams & A. Adams, 1864, a bo- real species not related to the Antarctic groups and which belongs in Nodulotrophon. Previ- ously, Powell (1951) pointed out theories of bi- polarity of some species of gastropods. In fact, Egorov (1993) in a revision of Russian Trophoninae illustrated all living Russian trophonine species, and several of them show FIG. 43. Type localities of new species of Trophon and other species of the same genus with similar shell morphology: A = T. emilyae, new species; B = T. cuspidarioides Powell, 1951; C = T. arnaudi, new species; D = T. declinans Watson; 1882; E = T. scolopax Watson, 1882. The size is not relative. Arrows point to type localities. Thick line represents the Antarctic Convergence. 360 PASTORINO TABLE 1. Measurements (in mm), distribution, and depth range (in m) of Trophon emilyae, new species; T. arnaudi, new species; T. septus Watson, 1882; T. scolopax Watson, 1882; and T. cuspidarioides Powell, 1951. Length Width Whorls Depth range T. emilyae, new species USNM 896438 holotype T. arnaudi, new species USNM 1003473 holotype ZIN 59775 paratype T. septus Watson 12:1 3.2 5 13.5 5:9 4 NHM 1887.2.9.578 21:1 10.5 5-6 lectotype NHM 1887.2.9.579 20.0 9.9 5-6 paralectotype T. scolopax Watson NHM 1887.2.9.580 23.4 10.4 6-7 holotype T. declinans Watson NHM 1887.2.9.573 lectotype T. cuspidarioides Powell NHM 1961547 13 Bo 5 holotype 19.5 8.0 7 a striking similarity with Antarctic species — for example, T. barvicensis (Johnston, 1825) with T. arnaudi. However, when these spe- cies are carefully studied they present suffi- cient features to ensure a different generic allocation. Crame (1996) discussed from a paleontological point of view the evolution of bipolarity of several groups of mollusks, in- cluding the genus Trophon as the southern counterpart of the northern Boreotrophon and Trophonopsis. Despite the poor knowledge of fossil repre- sentatives of the genus Trophon, there is a good record of Paleogene and Neogene spe- cies in Patagonian deposits (Griffin 8 Pastorino, in prep.). The oldest record in Ter- tiary deposits from Patagonia is apparently early Oligocene in age. However, there is no formal citation of species of this genus in Ter- tiary deposits around Antarctica. Nevertheless, Jonkers (2000), in a non-taxonomic paper, il- lustrated a shell that apparently belongs to this genus. The living species belonging to Trophon could be easily split based on the morphology Distribution Source 549-1153 NW Amundsen This paper Sea This paper 355-468 Off South Sandwich Island 370 Off South Sandwich Island 30-620 Off Kerguelen & Cantera & Arnaud, Crozet Islands 1985 60-620 Off Kerguelen & Cantera & Arnaud, Heard Islands 1985 70-274 Marion, Kerguelen Watson, 1882 & Crozet Islands Arnaud (pers. com) 120-204 South Georgia Powell, 1951 Island of the soft parts into isolated Patagonian and Antarctic clades (Pastorino, in press). Further studies will show if the Antarctic clade justi- fies a new generic placement. ACKNOWLEDGEMENTS This work was possible thanks to the mate- rial of T. scolopax and T. declinans collected by Dr. Patrick Arnaud from the Kerguelen Is- lands that he kindly offered during a trip to the Marine Station of Endoume, Marseille. This specimens are housed at the MACN. | thank the following people for access to material in their institutions: K. Way and J. Pickering (NHM); P. Bouchet and V. Heros (MNHN); A. Tablado (MACN), B. Hausdorf (Zoologisches Institut — Hamburg) and Е. Egorova (ZIN). | am grateful to M. Griffin and F. Scarabino for thor- ough reviews and helpful suggestions that im- proved this paper. Finally R. Bieler, E. Coan and an anonymous reviewer proposed thought- ful suggestions that also improved it. Part of this study was conducted during a NEW ANTARCTIC SPECIES OF TROPHON 361 Post-Doctoral Fellowship granted by the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Argentina, to work at the National Museum of Natural History, Smithsonian Institution, Washington, DC. | gratefully appreciate the advise and friendship of J. Harasewych during my time at the USNM. It was supported in part by a Research Award from the NSF-USAP United States Antarctic Program [Contract No. OPP- 9509761], a grant in aid from the Concholo- gists of America and the Walter E. Sage Memorial Award and the project PICT No. 01- 04321 from the National Agency for Scientific and Technical Promotion, Argentina. LITERATURE CITED CANTERA, J. R. & P. M. ARNAUD, 1985, Les gastéropodes prosobranches des iles Kerguelen et Crozet (sud de l'Océan Indien) comparaison écologique et particularités biologiques. Comité National Français des Recherches Antarctiques, 56: 1-169. CERNOHORSKY, W. O., 1977, The taxonomy of some southern ocean Mollusca (Gastropoda) mainly Antarctic and subantarctic: Records of the Aukland Institute and Museum, 14: 105-119. CRAME, J. A., 1996, Evolution of high-latitude molluscan faunas. Pp. 119-131, in: J. TAYLOR, ed., Origin and evolutionary radiation of the Mollusca. Oxford University Press, Oxford. EGOROV, R., 1993, Trophoninae (Muricidae) of Russian and adjacent waters. Ruthenica, Supplement, 1: 1-49. ICZN, 1999, International Code of Zoological Nomenclature: London, The International Trust for Zoological Nomenclature, 306 pp. JONKERS, H. A., 2000, Gastropod predation patterns in Pliocene and Recent pectinid bivalves from Antarctica and New Zealand. New Zealand Journal of Geology & Geophysics, 43: 247-254. KOOL, S. P., 1993, Phylogenetic analysis of the Rapaninae (Neogastropoda: Muricidae). Malacologia 35: 155-260. PASTORINO, G., in press, Systematics and phylogeny of the genus Trophon Montfort, 1810 (Gastropoda: Muricidae) from Patagonia and Antarctica: morphological patterns. Bollettino Malacologico. PASTORINO, G. & М. С. HARASEWYCH, 2000, A revision of the Patagonian genus Xymenopsis Powell, 1951 (Gastropoda: Muricidae). The Nautilus, 114: 38-58. POWELL, A. W. B., 1951, Antarctic and subantarctic Mollusca: Pelecypoda and Gastropoda. Discovery Reports, 26: 47-196. SOLEM, A., 1972, Malacological application of scanning electron microscopy, Il. Radular structure and functioning. The Veliger, 14: 327-336. STREBEL, H., 1904, Beiträge zur Kenntnis der Molluskenfauna der Magalhaen-Provinz: Zoologischen Jahrbüchern. Abteilung für Systematik, Geographie und Biologie der Tiere, 21: 171-248. WATSON, В. B., 1882, Mollusca of H. М. S. ‘Challenger Expedition — Part 13. The Journal of The Linnean Society. Zoology, 16: 358-392. Revised Ms. accepted 1 May 2002 MALACOLOGIA, 2002, 44(2): 363 CORRECTION TO: COAN, EUGENE V., 2002, THE EASTERN PACIFIC RECENT SPECIES OF THE CORBULIDAE (BIVALVIA). MALACOLOGIA 44(1): 47-105 Eugene V. Coan Department of Invertebrate Zoology & Geology, California Academy of Sciences, Golden Gate Park, San Francisco, California 94118-4599, U.S.A.; gene.coan Osierraclub.org It has been pointed out to me that one of the figure captions in my paper on the Corbulidae (Coan, 2002) was incorrectly labeled. As per my text, the lectotype of Serracorbula tumaca Olsson is the right valve depicted in Fig. 6b; the left valve in Fig. 6a is a paralectotype, not the lectotype. 12 May 2002 363 MALACOLOGIA, 2002, 44(1-2): 365-378 INDEX Taxa in bold are new; pages in italic indicate amurensis, Corbula 49, 69, 71-72, 93 figures of taxa. abscissa, Melanthalia 2-3 acanthias, Squalus 135 acanthocarpa, Aglaophenia 3 acanthodes, Trophon 353 Acanthodoris 302 Acmaea 253 Acromobacter dermidis 325 acuta, Physa 252 acutirostra, Corbula 74 adamsi, Lampeia 108 aequalis, Corbula 66-67 Potamomya 66-67, 67, 70 aequivalvis, Corbula 64, 66 africana, Chromodoris 297 Agama 135 Aglaophenia acanthocarpa 3 Agriolimax 145, 150 reticulatus 150, 252 alabamiensis, Corbula 51, 57 alba, Corbula 53, 55, 57, 66 Albinaria 33-34, 35, 37, 38-39, 40, 41, 42-44, 351 coerulea 34, 36-37, 40, 42-44, 350-351 discolor 34, 36-37, 40, 42-43 turrita 34, 36-37, 40, 42-43, 351 voithii 34, 36-37, 40, 42-44 albopunctatus, Chromodoris 301 albopustulosa, Chromodoris 301 Aloides 50 guineensis 50 Aloidis (Aloidis) 89 (Aloidis), Aloidis 89 (Caryocorbula) 53, 58, 60-61, 64, 78, 82, 84,87 (Caryocorbula) nasuta 53 (Tenuicorbula) 53 Alopiinae 43 altirostris, Corbula 90 alveata, Gigartina 3 americana, Vallisneria 5 amethystina, Corbula 91 Corbula (Caryocorbula) 51 amoena, Ceratosoma 297 Amphibola cerenata 150 Amphisbetia bispinosa 3, 12 amplexa, Corbula 71 Ampullariidae 153, 273, 279 365 Corbula (Potamocorbula) 71-72 amythestina, Corbula 49, 52, 53 anagallis, Enhydra 276, 279, 281 Anaspidea 289 anatinum, Drepanotrema 273, 277-278, 282- 285 Ancylidae 273, 279 angillarum, Vibrio 325 angustifolium, Carpophyllum 2, 4, 12 Anisocorbula 50 annulata, Chromodoris 297, 301 Anodonta cellensis 140 anomala, Corbula 50 antiqua, Venus 17-21, 19 aquatica, Cabomba 5 Araceae 276 arbutus, Digidentis 293, 295, 297 Architectonicoidea 350-351 Arctica islandica 140 arcticus, Bathypolypus 175, 177-178, 179- 180, 181-187, 182, 190-191, 193-198, 200- 205, 207-210, 208-210, 212-214, 220 Octopus 175, 177, 187, 202, 204, 221 Polypus 187 Ardeadoris 299-300 Argopecten purpuratus 165 Ariolimax columbianus 237 Arion ater224, 236 lusitanicus 224 arnaudi, Trophon 353, 356, 357-360, 359 Asaphis 50 aspersa, Helix 146-147, 149 Chromodoris 297-298 Asterias rubens 135 Asthenothaerus 107, 112-114 diegensis 107 hemphillii 107 huanghaiensis 112 isaotakii 107-108, 131 sematana 107, 126 sematanus 126 sematensis 126 ater, Arion 224, 236 atramentosa, Nerita 350 atromarginata, Glossodoris 297 aurantionodulosa, Diversidoris 293, 295, 297, 301 aureopurpurea, Chromodoris 297 australis, Thorunna 297 366 INDEX Azara 66 (Azara), Corbula 66 bairdii, Bathypolypus 175, 181, 183-187, 188- 189, 190-198, 193, 201-214, 208-209, 211, 217 Octopus 175, 184-185, 187, 191, 194, 219 Balea perversa 43 banyulensis, Godiva 252 barrattiana, Corbula 57, 87 barvicensis, Trophon 360 Bathypolypodinae 175, 202, 204 Bathypolypus 175-176, 185, 191, 193-195, 201-202, 204-207, 208, 212-214 arcticus 175, 177-178, 179-180, 181-187, 182, 190-191, 193-198, 200-205, 207-210, 208-210, 212-214, 220 bairdii 175, 181, 183-187, 188-189, 190- 198, 193, 201-214, 208-209, 211,217 ergasticus 201, 205-207, 208, 212, 214 faeroensis 178, 181, 183-184, 186-187, 203-204, 220 grimpei 202, 206 lentus 191, 193 obesus 191-193 proschi 175, 187, 194-195, 204 pugniger 175, 187, 191, 195-196, 196-197, 198, 199-200, 200-204, 206-207, 208-209, 212-214, 222 salebrosus 204-205 sponsalis 194, 201-202, 204-207, 206, 208, 212-214 valdiviae 201-202, 204, 206-207, 212 Bedevina birileffi 254 Benthoctopus 175-176, 185, 194, 202, 204- 205, 207 berryi 202 ergasticus 185, 204, 206-207 januarii 204 normani 185 piscatorum 175, 178, 183-186, 202-203, 207,220 salebrosus 204-205, 207 sasakii 178, 183-186, 220 bentoniensis, Goniobasis 23, 29 berryi, Benthoctopus 202 biangulata, Corbula 51 bicarinata, Corbula 49, 51, 53, 57, 64, 65, 66, 87,92 Corbula (Juliacorbula) 64 binominata, Corbula 90 binza, Chromodoris 297, 300 Biomphalaria 273-274, 277, 282-283, 285 glabrata 252, 273 occidentalis 286 straminea 273, 277-278, 282-286 tenagophila 273, 277-278, 282-286 Biophalaria glabrata 17, 150 biradiata, Corbula 49, 51, 64, 74, 78, 79-80, 80-82, 94 birileffi, Bedevina 254 bispinosa, Amphisbetia 3, 12 blandiana, Corbula 87, 89-90 bleekeri, Loligo 351 Boettgerilla pallens 223-225, 228-229, 230- 233, 233-234, 235, 236-238 Boettgerillidae 223-224 boivinei, Corbula 90 Bombyx mori 19 Boreotrophon 360 Bosellia 290 Bothrocorbula 63 boyntoni, Corbula (Cuneocorbula) whitfieldi 58 bradleyi, Corbula 76 Bradybaena fruticum 43 brevicaudatum, Ceratosoma 297 bronni, Thais 254 buccinoidea, Melanopsis 307-308, 311, 313, 315-318, 316, 320-322 budapestensis, Tandonia 224 buenavistana, Corbula (Caryocorbula) 64 Bufo marinus 135 Bulinus 167 bullocki, Hypselodoris 293, 296-297 burnsii, Corbula engonata 89 Bushia 113 Busycotypus 351 Bythinella 319 Cabomba aquatica 5 Cadlina 299-301 luteomarginata 297 modesta 297 pellucida 297 Cadlinella 289-291, 297, 299-301 ornatissima 290, 297, 301 caimitica, Corbula (Cuneocorbula) 58 californica, Cumingia 90 Pleurobranchaea 135 californicus, Schoenoplectus 154 californiensis, Penaeus 19 caloosae, Corbula 75-76 Campaniloidea 351 canaliculata, Pomacea 153, 157, 159-161, 279 canaliculus, Рета 1, 2, 13 Cannaceae 276 Canna glauca 273, 276, 279, 281-285 Cardiomya 89 caribaea, Corbula 57 carinata, Corbula 57 carolina, Corbula chipolana 76 Carpophyllum angustifolium 2, 4, 12 carrizalana, Corbula 76 caruanae, Deroceras 150 Caryocorbula 51, 61 (Caryocorbula) 53, 58, 60-61, 82, 87 (Caryocorbula) amethystina 51 (Caryocorbula) prenasuta 58 (Hexacorbula) esmeralda 63 nasuta 53 (Caryocorbula), Aloidis 53, 58, 60-61, 64, 78, 82, 84, 87 Caryocorbula 53, 58, 60-61, 82, 87 Corbula 51, 53, 58, 60-61, 64, 77, 80, 82, 84, 87 casertanum, Pisidium 166-167 catenaria, Goniobasis 23-24, 25, 26-29 Goniobasis catenaria 23-24, 26, 26-28, 30- 31 Cellana 351 cellensis, Anodonta 140 Cepaea 351 nemoralis 320, 351 Ceratophyllum demersum 5 Ceratopteris thalictroides 5 Ceratosoma 299-300 amoena 297 brevicaudatum 297 magnifica 297 cercadica, Corbula (Cuneocorbula) 58 cerenata, Amphibola 150 Cerion 320 Cerithiacea 307 Cerithioidea 351 Champia laingii 3 Chara 154 Chicoreus torrefactus 255 chipolana, Corbula 76 chittyana, Corbula 57 chowanensis, Corbula 76 Chromodorididae 289-290, 294, 298-302 Chromodoris 289-291, 294, 296, 298-301 africana 297 albopunctatus 301 albopustulosa 301 annulata 297, 301 INDEX 367 aspersa 297-298 aureopurpurea 297 binza 297, 300 clenchi 297, 300 coi 297-298 collingwoodi 293-294, 297, 301 daphne 293-294, 297, 301 decora 301 elisabethina 293-294, 297 fidelis 301 galactos 301 geometrica 293-294, 296-301 inornata 297 kuiteri 293-294, 297 kuniei 293-294, 297-298, 301 leopardus 293-294, 297, 301 lineolata 297 lochi 293,295, 297 loringi 301 magnifica 297-298 marginata 301 obseleta 297 orientalis 290, 297-298 perola 297 preciosa 301 quadricolor 297-298 roboi 293, 295, 297-298, 301 rubrocornuta 301 strigata 293, 295, 297 thompsoni 301 tinctoria 293, 295, 297, 301 vibrata 301 willani297 woodwardae 297 cimex, Drepanotrema 273, 277-278, 282-285 cincta, Glossodoris 297 Cingulina 351 Cirroteuthis 183 mülleri 177 Clausiliidae 33 clavigera, Thais 254 clenchi, Chromodoris 297, 300 Clypidina 351 coerulea, Albinaria 34, 36-37, 40, 42-44, 350- 351 coi, Chromodoris 297-298 colensoi, Osmundaria 2, 12 colimensis, Corbula 47, 49, 66, 82, 83, 84, 94 collazica, Corbula 90 collingwoodi, Chromodoris 293-294, 297, 301 colorata, Corbicula 166 368 columbianus, Ariolimax 237 complanata, Corbula 50 Compositae 276 concinna, Eximiothracia 107 conradi, Corbula 58 Corbula (Caryocorbula) 82 constracta, Corbula 87 contectus, Viviparus 320 Corallina officinalis 3 Corbicula 165, 167 colorata 166 (Corbiculina) leana 165 fluminea 166 japonica 166 leana 166 papyracea 166 sandai 166 Corbiculidae 165 Corbiculoidea 165-166 Corbula 47, 49-50, 87, 90, 91-95 acutirostra 74 aequalis 66-67 aequivalvis 64, 66 aequivalvis stainforthi 66 alabamiensis 51, 57 alba 53, 55, 57, 66 altirostris 90 amethystina 91 amplexa 71 amurensis 49, 69, 71-72, 93 amurensis takatuayamaensis 71 amythestina 49, 52, 53 anomala 50 (Azara) 66 barrattiana 57,87 barrattiana leonensis 89 biangulata 51 bicarinata 49, 51, 53, 57, 64, 65, 66, 87, 92 binominata 90 biradiata 49, 51, 64, 74, 78, 79-80, 80-82, 94 blandiana 87, 89-90 boivinei 90 bradleyi 76 caloosae 75-76 caribaea 57 carinata 57 carrizalana 76 (Caryocorbula) 51, 53, 58, 60-61, 64, 77, 80, 82, 84, 87 (Caryocorbula) amethystina 51 (Caryocorbula) buenavistana 64 (Caryocorbula) conradi 82 (Caryocorbula) democraciana 82 (Caryocorbula) franci 88 (Caryocorbula) nasuta 50, 53 (Caryocorbula) otra 47, 58 (Caryocorbula) ovulata 60 (Caryocorbula) porcella 61 (Caryocorbula) retusa 82 (Caryocorbula) urumacoensis 82 (Caryocorbula) wakullensis 88 chipolana 76 chipolana carolina 76 chittyana 57 chowanensis 76 colimensis 47, 49, 66, 82, 83, 84, 94 collazica 90 complanata 50 conradi58 constracta 87 (Corbula) 89 (Corbulomya) 50, 53, 84 coxi57 crassa 47 cubaniana 64, 66 cuneata 89 (Cuneocorbula) 51, 53 (Cuneocorbula) caimitica 58 (Cuneocorbula) caribaea pergrata 58 (Cuneocorbula) cercadica 58 (Cuneocorbula) daphnis 58 (Cuneocorbula) helenae 58 (Cuneocorbula) ira 84 (Cuneocorbula) sarda 58 (Cuneocorbula) sericea 58 (Cuneocorbula) smithiana 58 (Cuneocorbula) swiftiana harrisii 58 (Cuneocorbula) whitfieldi boyntoni 58 (Cuneocorbula) whitfieldi stika 58 cymella 90 dautzenberg 57 dietziana 87, 89 disparilis 75 dominicensis 53 ecuabula 79, 80-82 engonata burnsii 89 (Erodona) 71 esmeralda 49, 62, 64, 92 fragilis 50, 53, 54, 55, 57, 61, 63 frequens 71 fulva 57 gatunensis 63 gibba 47, 76 INDEX 369 gibbiformis 76 rosea 85 gibbosa 90 rubra 79, 80-81 glypta 72, 73 sanctidominici 76 granti 78 scutata 64, 66 grovesi 75, 75-76, 78, 93 sematensis 71 heterogena 76 (Serracorbula) 53 (Hexacorbula) 63 speciosa 49, 87, 88, 89-90, 95 (Hexacorbula) cruziana 64 striata 47 (Hexacorbula) esmeralda 63 sulcata 50 (Hexacorbula) gatunensis 64 swiftiana 57, 87, 90 hexacyma 63-64 (Tenuicorbula) 72 inaequalis 58 (Tenuicorbula) tenuis 72 inaequalis mansfieldi 58 tenuis 49, 72, 73, 74, 90, 93 ira 49, 66, 70, 83, 84, 86, 90 tenuis lupina 74 islatrinitalis 76 uruguayensis 57 (Juliacorbula) 64, 80-81, 84 ustulata 70, 72 (Juliacorbula) bicarinata 64 (Varicorbula) 76, 89-90 kelseyi 90 (Varicorbula) granti76 kjoeriana 57 (Varicorbula) grovesi 47, 74 knoxiana 64, 66 (Varicorbula) obesa 76 knoxiana fossilis 66 ventricosa 49, 66, 67-69, 70-72, 82, 84, 92 krebsiana 75 vieta 76 laevis 72 viminea 63 lavalleana 57 vladivostokensis 71 (Lentidium) 50, 61, 84 waltonensis 76 limatula 90 waltonensis rubisiniana 76 luteola 49-50, 63, 84-87, 85, 94 zuliana 76 luteola rosea 84-85, 85 (Corbula), Corbula 89 lyrata 57 Corbulidae 47-48, 50, 363 macdonaldi68, 69, 70 Corbulinae 50 marmorata 49, 58, 84, 86-87, 88, 89-90, 95 Corbulomya 50 nasuta 49-51, 52, 53-55, 54, 56, 57-58, 63, (Corbulomya), Corbula 50, 53, 84 66, 74, 77-78, 84, 86-87, 91 coreanum, Pisidium 165-167, 166 nuciformis 53-54, 54, 57, 77-78 corneum, Sphaerium 166-167 obesa 49, 53, 63, 76-78, 77, 84, 93 corneus, Planorbarius 224, 252 operculata 75, 90 coronatus, Trophon 359 oropendula dolicha 58 costata, Melanopsis 307-308, 313, 315-317, otra 49, 59, 60-61, 91 320-322 ovulata 49, 58, 59, 60, 90, 91 Melanopsis costata 307, 310-311, 313, (Panamicorbula) 66-68, 70-71 315-316, 376, 3184321 (Panamicorbula) ventricosa 66, 72 costatum, Plocamium 3 patagonica 78 coxi, Corbula 57 philippi 75 crassa, Corbula 47 polychroma 80-81 crassipes, Eichhornia 273, 276, 279, 281-285 porcella 49-50, 55, 61, 62, 63, 70, 78, 87, Crateritheca insignis 3 92 Crenomytilus grayanus 135, 136-139, 139- porcellio 61 140-142, 141 (Potamocorbula) amurensis 71-72 Cristataria genezarethana 40, 43-44 prenucia 76 Cristilabrum 320 pustulosa 53, 55, 56, 57, 71 crocea, Tridacna 329 radiata 89 cruziana, Corbula (Hexacorbula) 64 revoluta 57 Victoria 276, 281 370 cubaniana, Corbula 64, 66 Cumingia californica 90 cuneata, Corbula 89 Parvithracia 109 Cuneocorbula 51 pelseneeri51 (Cuneocorbula), Corbula 51, 53 curta, Thracia 108 cuspidarioides, Trophon 353, 358-359, 359- 360 Cyathodonta 113 Cyclophorus 351 cylindrica, Panamicorbula 68, 69, 70 cymella, Corbula 90 Cyperus 154 Dalhousia 319 daniellae, Thorunna 297 daphne, Chromodoris 293-294, 297, 301 daphnis, Corbula (Cuneocorbula) 58 dautzenberg, Corbula 57 declinans, Trophon 353-354, 355, 357, 359, 360 decora, Chromodoris 301 decussata, Noumea 297 deflorata, Venus 50 demersum, Ceratophyllum 5 democraciana, Corbula (Caryocorbula) 82 densa, Elodea 5 Dermatomya 90 mactrioides 90 (Dermatomya), Poromya 90 dermidis, Acromobacter 325 Deroceras caruanae 150 laeve 224, 237 reticulatum 150, 224, 236-237 rodnae 224, 237 dichotama, Rhodymenia 2, 12 Dictyocladium moniliferum 3 diegensis, Asthenothaerus 107 dietziana, Corbula 87, 89 Digidentis 289, 295, 299-300 arbutus 293, 295, 297 discolor, Albinaria 34, 36-37, 40, 42-43 dislocata, Goniobasis catenaria 23-28, 26, 31 disparalis, Varicorbula 90 disparilis, Corbula75 Diversidoris 289, 295, 298-299 aurantionodulosa 293, 295, 297, 301 dolicha, Corbula oropendula 58 dominicensis, Corbula 53 Drepanotrema 273-274, 277, 282, 285 INDEX anatinum 273, 277-278, 282-285 cimex 273, 277-278, 282-285 kermatoides 277-278, 282-286 lucidum 273, 277-278, 282-285 Durvilledoris 299-300 Echinochloa polystachya 285 ecuabula, Corbula 79, 80-82 edulis, Mytilus 1, 17, 19-21, 140, 252, 350- 351 Eichhornia crassipes 273, 276, 279, 281-285 elata, Vestia 43 elatinoides, Myriophyllum 154 elenensis, Juliacorbula 80, 81-82 elephantipes, Panicum 273, 276, 279, 281- 285 Elimia 23 elisabethina, Chromodoris 293-294, 297 Elodea5 densa5 Elysia 290 Elysiidae 290 emarginata, Thais 255 emilyae, Trophon 353-354, 355, 357, 359, 360 emma, Hypselodoris 297 Enhydra anagallis 276, 279, 281 Enteromorpha 155 Eobania vermiculata 43 ergasticus, Bathypolypus 201, 205-207, 208, 212,214 Benthoctopus 185, 204, 206-207 Octopus 206 Polypus 206 Erodona 66 (Erodona), Corbula 71 Erodonidae 66 esmeralda, Caryocorbula (Hexacorbula) 63 Corbula 49, 62, 64, 92 Corbula (Hexacorbula) 63 Euhadra 351 herklotsi 351 Euperinae 167 Euthyneura 350-351 Eximiothracia 113 concinna 107 faeroensis, Bathypolypus 178, 181, 183-184, 186-187, 203-204, 220 Polypus 177,183, 185, 221 festiva, Hypselodoris 298 fidelis, Chromodoris 301 florens, Thorunna 297 floridensis, Goniobasis 25 fluminea, Corbicula 166 Fluvidona 319 fontandraui, Hypselodoris 300 fossilis, Corbula knoxiana 66 fragilis, Corbula 50, 53, 54, 55, 57, 61, 63 franci, Corbula (Caryocorbula) 88 frequens, Corbula 71 fruticum, Bradybaena 43 fulva, Corbula 57 Gadus morhua 135 gairdneri, Salmo 135 galactos, Chromodoris 301 galloprovincialis, Mytilus 140, 329 gatunensis, Corbula 63 Corbula (Hexacorbula) 64 Hexacorbula 64 Geloina proxima 150 genezarethana, Cristataria 40, 43-44 geometrica, Chromodoris 293-294, 296-301 geversianus, Trophon 354 ghardagana, Risbecia 298 gibba, Corbula 47, 76 Tellina 74 gibbiformis, Corbula 76 gibbosa, Corbula 90 Gigartina 3 alveata 3 marginifera 3 glabrata, Biomphalaria 252, 273 Biophalaria 17, 150 glacialis, Marthasterias 135 glauca, Canna 273, 276, 279, 281-285 Glossodoris 289, 295, 297-300 atromarginata 297 cincta 297 pallescens 290, 297 pallida 297 plumbea 297 sibogae 297 vespa 293, 295, 297 glypta, Corbula 72, 73 Godiva banyulensis 252 Goniobasis 23-24, 27-29 bentoniensis 23, 29 boykiniana viennaensis 23-24, 29 catenaria 23-24, 25, 26-29 catenaria catenaria 23-24, 26, 26-28, 30-31 catenaria dislocata 23-28, 26, 31 catenaria postelli 23-28, 26, 31 INDEX 371 floridensis 25 mutabilis 23, 29 mutabilis timida 23 postellii 23, 29 proxima 23-31, 25-26 suturalis 24, 29 timida 23, 29 Gramineae 276-277, 281 Graneledone pacifica 213 granti, Corbula 78 Corbula (Varicorbula) 76 granulatus, Octopus 176-177 grayanus, Crenomytilus 135, 136-139, 139- 142, 141 grimpei, Bathypolypus 202, 206 groenlandica, Sepia 176 groenlandicus, Octopus 177, 193 grovesi, Corbula 49, 75, 75-76, 78, 93 Corbula (Varicorbula) 47, 74 guineensis, Aloides 50 Gundlachia moricandi 279, 281 haliclona, Noumea 297 Haliotis 320 Haliptilon roseum 3 harrisii, Corbula (Cuneocorbula) swiftiana 58 helenae, Corbula (Cuneocorbula) 58 Heleobias 279, 281 Helicella pappi 43 Helicidae 146 Helix 145, 320 aspersa 146-147, 149 lucorum 145-147, 148, 149-150 hemphillii, Asthenothaerus 107 herklotsi, Euhadra 351 herzogii, Salvinia 276 heterogena, Corbula 76 Hexacorbula 63 gatunensis 64 (Hexacorbula), Corbula 63 hexacyma, Corbula 63-64 hokkaidoensis, Valvata 350 huanghaiensis, Asthenothaerus 112 hupensis, Oncomelania 259-261, 333-334, 342-345 Oncomelania hupensis 259-261, 268, 271, 333-334, 334, 336-337, 338-339, 341-343, 347 Hydrobia 319-320, 322, 345, 351 Hydrobiidae 273, 279 Hydrocotyle ranunculoides 273, 276, 279, 281-285 372 Hypselodoris 289, 293, 296, 298-300 bullocki 293, 296-297 emma 297 festiva 298 fontandraui 300 iacula 300 kanga 298 maculosa 298, 302 midatlantica 300 mouaci 298 obscura 293, 296, 298, 301 orsinii 300 purpureomaculosa 300 rudmani 300 tricolor 252 whitei 298 zebra 298-300 zephyra 293, 296, 298 iacula, Hypselodoris 300 ichyodermidis, Pseudomoans 325 llyanassa obsoleta 252 imperialis, Risbecia 300 inaequalis, Corbula 58 incisa, Lytocarpia 3 indica, Rotala 5 inflata, Potamomya 66, 68, 68, 70 inornata, Chromodoris 297 insignis, Crateritheca 3 intermedia, Spirodela 276, 281 isaotakii, Asthenothaerus 107-108, 131 Parvithracia (Pseudoasthenothaerus) 107, 119, 122, 126, 129, 131-132, 131 islandica, Arctica 140 islatrinitalis, Corbula 76 ira, Corbula 49, 66, 70, 83, 84, 86, 90 Corbula (Cuneocorbula) 84 Ixartia 113 januarii, Benthoctopus 204 japonica, Corbicula 166 Schistosoma 259 Thracidora 108 japonicum, Schistosoma 333-334, 344-345 jordanica, Melanopsis costata 307-309, 311, 313, 315-316, 316, 318, 321 Juliacorbula 64, 80-81, 84 elenensis 80, 81-82 (Juliacorbula), Corbula 64, 80-81, 84 kakumana, Thracia 108 kanga, Hypselodoris 298 INDEX Katharina 351 tunicata 351 kelseyi, Corbula 90 kermatoides, Drepanotrema 277-278, 282- 286 kjoeriana, Corbula 57 knoxiana, Corbula 64, 66 krebsiana, Corbula 75 kuiteri, Chromodoris 293-294, 297 kuniei, Chromodoris 293-294, 297-298, 301 laeve, Deroceras 224, 237 laevis, Corbula 72 laingii, Champia 3 Lampeia 107-108, 112-113 adamsi 108 posteroresecta 108 triangula 108 Lasaea 165 Laurencia thyrsifera 3 lavalleana, Corbula 57 leana, Corbicula 166 Corbicula (Corbiculina) 165 Lehmannia marginata 223-226, 228-229, 230- 233, 233-234, 235, 236-238 Lemnaceae 276 Lentidiinae 50 Lentidium 50 mediterraneum 47 (Lentidium), Corbula 61, 84 lentus, Bathypolypus 191, 193 Octopus 184-185, 187, 191, 219 Polypus 187 leonensis, Corbula barrattiana 89 leopardus, Chromodoris 293-294, 297, 301 Limacidae 223-224 Limacoidea 223-224 limatula, Corbula 90 lineolata, Chromodoris 297 Littorina 351 saxatilis 350-351 Littorinioidea 350-351 lochi, Chromodoris 293, 295, 297 Loligo 351 bleekeri 351 loringi, Chromodoris 301 lucida, Pterocladia 3 lucidum, Drepanotrema 273, 277-278, 282- 285 lucorum, Helix 145-147, 148, 149-150 Ludwigia 154-155 peploides 276, 281 INDEX 373 lukini, Parvithracia (Pseudo- meiostoma 307-311, 313, 315-318, 376, asthenothaerus) 107, 114, 115-118, 119- 320-321 120, 121, 122, 125-126, 128-129, 132 saulcyi 307-308, 310-311, 313, 315-318, Pseudoasthenothaerus 111 316, 320-322 lupina, Corbula tenuis 74 Melanthalia abscissa 2-3 lusitanicus, Arion 224 Mercenaria mercenaria 329 lusoria, Pachymenia 3 mercenaria, Mercenaria 329 Meretrix 243, 325-330, 328-329 Meretrix 326-328 luteola, Corbula 49-50, 63, 84-87, 85, 94 lusoria 243, 325-330, 328-329 luteomarginata, Cadlina 297 Mexichromis 299-300 Lymnaea stagnalis 142, 150, 224, 236-237, multituberculata 297 252 Miamira 297 lyrata, Corbula 57 midatlantica, Hypselodoris 300 Lytocarpia incisa 3 Minicorbula 50 modesta, Cadlina 297 macdonaldi, Corbula 68, 69, 70 moniliferum, Dictyocladium 3 Mactra 90 Montacuta triquetra 109 veneriformis 243, 252 montrouzieri, Thorunna 297 mactrioides, Dermatomya 90 morhua, Gadus 135 maculosa, Hypselodoris 298, 302 mori, Bombyx 19 Pisania 255 moricandi, Gundlachia 279, 281 magellanicus, Murex 354 mouaci, Hypselodoris 298 magnifica, Ceratosoma 297 mülleri, Cirroteuthis 177 Chromodoris 297-298 Mulinia pallida 90 Malacolimax tenellus 224, 237 multituberculata, Mexichromis 297 Manduca sexta 19 Murex magellanicus 354 mansfieldi, Corbula inaequalis 58 Muricidae 241, 251, 254-255, 353-354 mansoni, Schistosoma 273-274 Musculium securis 166-167 marginata, Chromodoris 301 mutabilis, Goniobasis 23, 29 Lehmannia 223-226, 228-229, 230-233, Mya 47,66 233-234, 235, 236-238 plana 66 marginifera, Gigartina 3 truncata 90 marinus, Bufo 135 Myoidea 47, 66 marmorata, Corbula 49, 58, 84, 86-87, 88, 89- myopsis, Thracia 108 90, 95 Myriophyllum elatinoides 154 Stenophysa 279, 281 verticillatum 5 Marthasterias glacialis 135 Mytilidae 167 maximus, Pecten 351 Mytilus 350-351 mediterranea, Tellina 50 edulis 1, 17, 19-21, 140, 252, 350-351 mediterraneum, Lentidium 47 galloprovincialis 140, 329 meiostoma, Melanopsis 307-311, 313, 315- 318, 316, 320-321 nasuta, Caryocorbula 53 melajoensis, Tenuicorbula 74 (Caryocorbula) Aloidis 53 Melanopsidae 307 Corbula 49-51, 52, 53-55, 54, 56, 57-58, 63, Melanopsis 307, 319-322 66, 74, 77-78, 84, 86-87, 91 buccinoidea 307-308, 311, 313, 315-318, Corbula (Caryocorbula) 50, 53 316, 320-322 nemoralis, Cepaea 320, 351 costata 307-308, 313, 315-317, 320-322 Nerita 350-351 costata costata 307, 310-311, 313, 315- atramentosa 350 316, 316, 318, 321 Neritopsina 351 costata jordanica 307-309, 311, 313, 315- Ninfaceae 276 316, 316, 318, 321 nobilis, Tyrinna 297 374 INDEX Nodilittorina 351 Nodulotrophon 359 norba, Noumea 293, 296-297, 301 normani, Benthoctopus 185 Polypus 185 Noumea 289, 296, 299-300 decussata 297 haliclona 297 norba 293, 296-297, 301 simplex 297 nuciformis, Corbula 53-54, 54, 57, 77-78 obesa, Corbula 49, 53, 63, 76-78, 77, 84, 93 Corbula (Varicorbula) 76 obesus, Bathypolypus 191-193 Octopus 184, 187, 191,219 obscura, Hypselodoris 293, 296, 298, 301 obseleta, Chromodoris 297 obsoleta, Ilyanassa 252 occidentale, Sphaerium 166-167 occidentalis, Biomphalaria 286 octopodia, Sepia 176 Octopodidae 175, 204 Octopodinae 202 Octopus 202, 204 arcticus 175, 177, 187, 202, 204, 221 bairdii 175, 184-185, 187, 191, 194, 219 ergasticus 206 granulatus 176-177 groenlandicus 177,193 lentus 184-185, 187, 191,219 obesus 184, 187, 191, 219 piscatorum 177, 184-185, 187, 194, 202, 204 profundicola 206-207 sponsalis 205 officinalis, Corallina 3 ohlini, Trophon 357 Omalogyra 351 Omalogyroidea 351 Onagraceae 276 Oncomelania 319-320, 333-335, 343-345 hupensis 259-261, 333-334, 342-345 hupensis hupensis 259-261, 268, 271, 333- 334, 334, 336-337, 338-339, 341-343, 347 hupensis robertsoni 259-261, 268, 271, 333, 342-343 hupensis tangi 334 operculata, Corbula 75, 90 orientalis, Chromodoris 290, 297-298 ornatissima, Cadlinella 290, 297, 301 orsinii, Hypselodoris 300 Osmundaria colensoi2, 12 otra, Corbula 49, 59, 60-61, 91 Corbula (Caryocorbula) 47, 58 ovulata, Corbula 49, 58, 59, 60, 90, 91 Corbula (Caryocorbula) 60 Pachymenia lusoria 3 pacifica, Graneledone 213 pallens, Boettgerilla 223-225, 228-229, 230- 233, 233-234, 235, 236-238 pallescens, Glossodoris 290, 297 pallida, Glossodoris 297 Mulinia 90 Panamicorbula 66-68, 70 cylindrica 68, 69, 70 trigonalis 70 (Panamicorbula) 66, 70 Corbula 66-68, 70-71 Pandalus 211 Panicum elephantipes 273, 276, 279, 281- 285 pappi, Helicella 43 papyracea, Corbicula 166 Parvithracia 107, 109, 112, 122, 129, 131, 133 cuneata 109 (Parvithracia) 113 (Parvithracia) suteri 109, 110, 111-112, 122 (Pseudoasthenothaerus) 112-114 (Pseudoasthenothaerus) isaotakii 107, 119, 122, 126, 129, 131-132, 131 (Pseudoasthenothaerus) lukini 107, 114, 115-118, 119-120, 121, 122, 125-126, 128- 129, 132 (Pseudoasthenothaerus) sematana 107, 119, 122, 126, 129, 130, 131, 132-133 (Pseudoasthenothaerus) sirenkoi 107, 119, 122-123, 123-124, 126-129, 128, 132 sematana 126 suteri 107-109 (Parvithracia), Parvithracia 113 Paspalum repens 285 patagonica, Corbula 78 Patellogastropoda 351 paulensis, Penaeus 19 Pecten 351 maximus 351 Pectenodoris 289, 296, 299-300 trilineata 293, 296-297 Pectinidae 167 pellucida, Cadlina 297 pelseneeri, Cuneocorbula 51 INDEX 375 Penaeus californiensis 19 paulensis 19 peploides, Ludwigia 276, 281 pergrata, Corbula (Cuneocorbula) caribaea 58 Peringia ulvae 342 Perna canaliculus 1, 2, 13 perna 17, 329-330 viridis 17, 19 perna, Perna 17, 329-330 perola, Chromodoris 297 perversa, Balea 43 philippii, Corbula 75 philippinarum, Ruditapes 243 Venerupis 326, 330 Pholadomyoida 109 Phragmorisma 113 Physa acuta 252 Physidae 279 Pisania maculosa 255 piscatorum, Benthoctopus 175, 178, 183-186, 202-203, 207, 220 Octopus 177, 184-185, 187, 194, 202, 204 Polypus 177, 185 Pisidiidae 165, 167 Pisidiinae 167 Pisidium 167 casertanum 166-167 coreanum 165-167, 166 Pistia stratiotes 273, 276, 279, 281-285 plana, Mya 66 Planorbarius corneus 224, 252 Planorbidae 273-274 Pleurobranchaea Californica 135 Pleuroceridae 23, 343 Plocamium costatum 3 plumbea, Glossodoris 297 Polybranchiidae 290 polychroma, Corbula 80-81 Polypus arcticus 187 ergasticus 206 faeroensis 177, 183, 185, 221 lentus 187 normani 185 piscatorum 177, 185 profundicola 206 salebrosus 204 polystachya, Echinochloa 285 Pomacea 160-161, 351 canaliculata 153, 157, 159-161, 279, 281 scalaris 281 Pomatiopsidae 259, 333 Pontederiaceae 276 porcella, Corbula 49-50, 55, 61, 62, 63, 70, 7919798 Corbula (Caryocorbula) 61 porcellio, Corbula 61 Poromya 71 (Dermatomya) 90 postellii, Goniobasis 23, 29 Goniobasis catenaria 23-28, 26, 31 posteroresecta, Lampeia 108 Potamocorbula 71 (Potamocorbula) 71 Potamogeton 154-155 Potamomya 66 aequalis 66-67, 67, 70 inflata 66, 68, 68, 70 triagonalis 67 trigonalis 67, 68, 70 preciosa, Chromodoris 301 prenasuta, Caryocorbula (Caryocorbula) 58 prenucia, Corbula 76 profundicola, Octopus 206-207 Polypus 206 proschi, Bathypolypus 175, 187, 194-195, 204 proxima, Geloina 150 Goniobasis 23-31, 25-26 Psammobiidae 50 Pseudoasthenothaerus 107, 111-112 lukini 111 (Pseudoasthenothaerus), Parvithracia 112-114 Pseudomoans ichyodermidis 325 Psilaxis 350-351 radiatus 350 Pterocladia capillacea 3 lucida 3 pugniger, Bathypolypus 175, 187, 191, 195- 196, 196-197, 198, 199-200, 200-204, 206- 207, 208-209, 212-214, 222 pulchella, Risbecia 298, 300 punicea, Thorunna 300 Pupa 351 purpuratus, Argopecten 165 purpureomaculosa, Hypselodoris 300 pusilla, Trigonothracia 108, 126 pustulosa, Corbula 53, 55, 56, 57, 71 Pyramidelloidea 351 quadricolor, Chromodoris 297-298 radiata, Corbula 89 radiatus, Psilaxis 350 ranunculoides, Hydrocotyle 273, 276, 279, 281-285 376 Rapana 254 venosa 241-244, 243, 245-246, 247, 248, 249-255, 250-251 reiniana, Semisulcospira 343 repens, Paspalum 285 reticulatum, Deroceras 150, 224, 236-237 reticulatus, Agriolimax 150, 252 retusa, Corbula (Caryocorbula) 82 revoluta, Corbula 57 Rhodymenia dichotama 2, 12 Risbecia 299-300 ghardagana 298 imperialis 300 pulchella 298, 300 tryoni 298, 300, 302 Rissooidea 333, 351 robertsoni, Oncomelania hupensis 259-261, 268, 271, 333, 342-343 roboi, Chromodoris 293, 295, 297-298, 301 rodnae, Deroceras 224, 237 rosea, Corbula 85 Corbula luteola 84-85, 85 roseum, Haliptilon 3 Rostanga 302 Rotala indica5 rotundifolia, Salvinia 276 rubens, Asterias 135 rubisiniana, Corbula waltonensis 76 rubra, Corbula 79, 80-81 rubrocornuta, Chromodoris 301 Ruditapes philippinarum 243 rudmani, Hypselodoris 300 salebrosus, Bathypolypus 204-205 Benthoctopus 204-205, 207 Polypus 204 Salmo gairdneri 135 Salvinaceae 276 Salvinia herzogii 276 rotundifolia 276 sanctidominici, Corbula 76 sandai, Corbicula 166 sarda, Corbula (Cuneocorbula) 58 sasakii, Benthoctopus 178, 183-186, 220 saulcyi, Melanopsis 307-308, 310-311, 313, 315-318, 316, 320-322 saxatilis, Littorina 350-351 Scaeurgus 213 scalaris, Pomacea 281 Schistosoma 259 Japonica 259 japonicum 333-334, 344-345 INDEX mansoni273-274 Schoenoplectus californicus 154 scolopax, Trophon 353, 356, 357, 358-359, 360 scutata, Corbula 64, 66 securis, Musculium 166-167 sematana, Asthenothaerus 107, 126 Parvithracia 126 Parvithracia (Pseudoasthenothaerus) 107, 119, 122, 126, 129 130131182485 Thracia 126 sematanus, Asthenothaerus 126 sematensis, Asthenothaerus 126 Corbula 71 seminuda, Thracia 108 Semisulcospira reiniana 343 senegalensis, Siratus 255 Sepia groenlandica 176 octopodia 176 septentrionalis, Thracia 108 septus, Trophon 353, 358, 359-360 sericea, Corbula (Cuneocorbula) 58 Serpulorbis 351 Serracorbula 51 tumaca 51, 53, 55, 56, 57, 363 (Serracorbula), Corbula 53 sexta, Manduca 19 sibogae, Glossodoris 297 simplex, Noumea 297 Siphonaria 351 Siratus senegalensis 255 sirenkoi, Parvithracia (Pseudo- asthenothaerus) 107, 119, 122-123, 123- 124, 126-129, 128, 132 smithiana, Corbula (Cuneocorbula) 58 Solidicorbula 50 speciosa, Corbula 49, 87, 88, 89-90, 95 Sphaeriidae 166 Sphaeriinae 167 Sphaerium corneum 166-167 occidentale 166-167 striatinum 165-167 spiralis, Vallisneria 5 Spirodela intermedia 276, 281 sponsalis, Bathypolypus 194, 201-202, 204- 207, 206, 208, 212-214 Octopus 205 Squalus acanthias 135 stagnalis, Lymnaea 142, 150, 224, 236-237, 252 Stagnicola 320 stainforthi, Corbula aequivalvis 66 INDEX 377 Stenophysa marmorata 279, 281 stika, Corbula (Cuneocorbula) whitfieldi 58 straminea, Biomphalaria 273, 277-278, 282-286 stratiotes, Pistia 273, 276, 279, 281-285 striata, Corbula 47 striatinum, Sphaerium 165-167 strigata, Chromodoris 293, 295, 297 sulcata, Corbula 50 suteri, Parvithracia 107-109 Parvithracia (Parvithracia) 109, 110, 111- 112122 suturalis, Goniobasis 24, 29 swiftiana, Corbula 57, 87, 90 takatuayamaensis, Corbula amurensis 71 Tandonia budapestensis 224 tangi, Oncomelania hupensis 334 Tapes waltingii 140 tapetis, Vibrio 330 Tectus 320 Tellina 50, 57 gibba 74 mediterranea 50 tenagophila, Biomphalaria 273, 277-278, 282- 286 tenellus, Malacolimax 224, 237 Tenuicorbula 72 melajoensis 74 (Tenuicorbula) 72 Aloidis 53 Corbula 72 tenuis, Corbula 49, 72, 73, 74, 90, 93 Corbula (Tenuicorbula) 72 Thais bronni 254 clavigera 254 emarginata 255 tissoti 254 thalictroides, Ceratopteris 5 thompsoni, Chromodoris 301 Thorunna 299-300 australis 297 daniellae 297 florens 297 montrouzieri 297 punicea 300 Thracia 107, 113, 118 curta 108 kakumana 108 myopsis 108 sematana 126 seminuda 108 septentrionalis 108 Thracidora 113 japonica 108 Thraciidae 107, 109 Thracioidea 109 Thraciopsis 113 thyrsifera, Laurencia 3 timida, Goniobasis 23, 29 Goniobasis mutabilis 23 tinctoria, Chromodoris 293, 295, 297, 301 tissoti, Thais 254 torrefactus, Chicoreus 255 triagonalis, Potamomya 67 triangula, Lampeia 108 tricolor, Hypselodoris 252 Tridacna crocea 329 trigonalis, Panamicorbula 70 Potamomya 67, 68, 70 Trigonothracia 108, 113 pusilla 108, 126 trilineata, Pectenodoris 293, 296-297 triquetra, Montacuta 109 Trochus 320 Trophon 353-354, 359-360 acanthodes 353 arnaudi 353, 356, 357-360, 359 barvicensis 360 coronatus 359 cuspidarioides 353, 358-359, 359-360 declinans 353-354, 355, 357, 359, 360 emilyae 353-354, 355, 357, 359, 360 geversianus 354 ohlini 357 scolopax 353, 356, 357, 358-359, 360 septus 353, 358, 359-360 Trophoninae 353-354 Trophonopsis 360 truncata, Mya 90 tryoni, Risbecia 298, 300, 302 Tulotoma 351 tumaca, Serracorbula 51, 53, 55, 56, 57, 363 tunicata, Katharina 351 turrita, Albinaria 34, 36-37, 40, 42-43, 351 Typha 154 Tyrinna 299-301 nobilis 297 ulvae, Peringia 342 Umbeliferae 276 uruguayensis, Corbula 57 urumacoensis, Corbula (Caryocorbula) 82 ustulata, Corbula 70, 72 378 valdiviae, Bathypolypus 201-202, 204, 206- 207,212 Polypus 206 Vallisneria americana 5 spiralis 5 Valvata 350-351 hokkaidoensis 350 Valvatoidea 351 Varicorbula 74, 76, 89-90 disparalis 90 (Varicorbula) 74 Corbula 76, 89-90 Veneridae 325 veneriformis, Mactra 243, 252 Veneroida 165 Veneroidea 167 Venerupis philippinarum 326, 330 venosa, Rapana 241-244, 243, 245-246, 247, 248, 249-255, 250-251 ventricosa, Corbula 49, 66, 67-69, 70-72, 82, 84, 92 Corbula (Panamicorbula) 66, 72 Venus antiqua 17-21, 19 deflorata 50 Verconia 298-300 verconis 297 verconis, Verconia 297 Vermetoidea 351 vermiculata, Eobania 43 verticillatum, Myriophyllum 5 INDEX vespa, Glossodoris 293, 295, 297 Vestia elata 43 Vetigastropoda 351 vibrata, Chromodoris 301 Vibrio 326 angillarum 325 tapetis 330 Victoria cruziana 276, 281 viennaensis, Goniobasis boykiniana 23-24, 29 vieta, Corbula 76 viminea, Corbula 63 viridis, Perna 17, 19 Viviparus 319-320 contectus 320 vladivostokensis, Corbula 71 voithii, Albinaria 34, 36-37, 40, 42-44 wakullensis, Corbula (Caryocorbula) 88 waltingii, Tapes 140 waltonensis, Corbula 76 whitei, Hypselodoris 298 willani, Chromodoris 297 woodwardae, Chromodoris 297 Xymenopsis 353 zebra, Hypselodoris 298-300 zephyra, Hypselodoris 293, 296, 298 zuliana, Corbula 76 Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Publication dates 34, No 35, No. 35, No. 36, No. 37, No. 37, No. 38, No. 39, No. 40, No. 41, No. 41, No. 42, No. 43, No. 44, No. . 1-2 . 1992 1993 . 1993 211995 - 1995 _ 1996 . 1996 1998 . 1998 1999 . 1999 . 2000 . 2001 2002 VOL. 44, NO. 2 MALACOLOGIA 2002 CONTENTS EE-YUNG CHUNG, SUNG-YEON KIM, KWAN HA PARK, & GAB-MAN PARK Sexual Maturation, Spawning, and Deposition of the Egg Capsules of the Female Purple Shell, Rapana venosa (Gastropoda: Muricidae) .......... 241 EUGENE V. COAN Correction to: Coan, Eugene V., 2002, The Eastern Pacific Recent Species of the Corbulidae (bivalvia). Malacologia 44(1): 47-105 ................ 363 ANDRZEJ FALNIOWSKI, JOSEPH HELLER, MAGDALENA SZAROWSKA, & KRYSTYNA MAZAN-MAMCZARZ Allozymic Taxonomy within the Genus Melanopsis (Gastropoda: Cerithi- acea) in Israel: A Case in which Slight Differences Are Congruent ........ 307 ANA MARIA LEAL-ZANCHET Ultrastructure of the Supporting Cells and Secretory Cells of the Alimentary Canal of the Slugs, Lehmannia marginata and Boettgerilla pallens (Pul- топа: Stylommatophora: Limacoidea): =... 0. ee ens ee ee 223 CHARLES LYDEARD, WALLACE E. HOLZNAGEL, REI UESHIMA, & ATSUSHI KURABAYASHI Systematic Implications of Extreme Loss or Reduction of Mitochondrial LSU FRNA Helical-Loop Structures in'Gastropods «05... three eas 332 BENT MUUS The Bathypolypus-Benthoctopus problem of the North Atlantic (Octopodidae Cephalopoda) ee nn ae 175 SUNG-WOO PARK, KANG-SOO LEE & EE-YUNG CHUNG Morphological and Cytochemical Characteristics of Hemocytes of Meretrix возопа (Е мама менее ав inn. RE ee ee a 325 GUIDO PASTORINO Two New Trophoninae (Gastropoda: Muricidae) from Antarctic Waters .... 353 A. RUMI, J. A. BECHARA, M. I. HAMANN, & M. OSTROWSKI DE NUNEZ Ecology of Potential Hosts of Schistosomiasis in Urban Environments of Ghaco Argentina Arte a ee re me antenne ae 273 EDMUND Y. W. SETO, WEIPING WU, DONGCHUAN QIU, HONGYUN LIU, XUEGUANG GU, HONGGEN CHEN, ROBERT C. SPEAR, & GEORGE M. 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There is a $4.50 handling charge per volume for all pur- chases of single volumes. Address inquiries to the Subscription Office. VOL. 44, NO. 2 MALACOLOGIA CONTENTS BENT MUUS The Bathypolypus-Benthoctopus problem of the North Atlantic (Setepedidas; Cophalapoda) us ta а wens er de an ANA MARIA LEAL-ZANCHET Ultrastructure of the Supporting Cells and Secretory Cells of the Alimentary Canal of the Slugs, Lehmannia marginata and Boettgerilla pallens (Pul- monata: Stylommatophora: Limacoidea) .......................... EE-YUNG CHUNG, SUNG-YEON KIM, KWAN HA PARK, & GAB-MAN PARK Sexual Maturation, Spawning, and Deposition of the Egg Capsules of the Female Purple Shell, Rapana venosa (Gastropoda: Muricidae) ......... EDMUND Y. W. SETO, WEIPING WU, DONGCHUAN QIU, HONGYUN LIU, XUEGUANG GU, HONGGEN CHEN, ROBERT C. SPEAR, & GEORGE M. DAVIS Impact of Soil Chemistry on the Distribution of Опсотеата hupensis (Gastropoda: Pomatiopsidae) in China ............................ A. RUMI, J. A. BECHARA, М. I. HAMANN, & М. OSTROWSKI DE NUNEZ Ecology of Potential Hosts of Schistosomiasis in Urban Environments of E A es Soe NERIDA G. WILSON Egg Masses of Chromodorid Nudibranchs (Mollusca: Gastropoda: Opistho- MA nak ee es oo age Hons grees on eta re apes ba ea ER ANDRZEJ FALNIOWSKI, JOSEPH HELLER, MAGDALENA SZAROWSKA, & KRYSTYNA MAZAN-MAMCZARZ Allozymic Taxonomy within the Genus Melanopsis (Gastropoda: Cerithi- acea) in Israel: A Case in which Slight Differences Are Congruent ....... SUNG-WOO PARK, KANG-SOO LEE & EE-YUNG CHUNG Morphological and Cytochemical Characteristics of Hemocytes of Meretrix Lusona (Bivalvia: VONOrdaS) 2.2... csc ca ses sun. dead goss a CHAO-HUI SHI, THOMAS WILKE, GEORGE M. DAVIS, MING-YI XIA, & CHI-PING QIU Population Genetics, Micro-Phylogeography, Ecology, and Susceptibility to Schistosome Infection of Chinese Oncomelania hupensis hupensis (Gastropoda: Rissooidea: Pomatiopsidae) in the Miao River System ..... RESEARCH NOTES CHARLES LYDEARD, WALLACE E. HOLZNAGEL, REI UESHIMA, & ATSUSHI KURABAYASHI Systematic Implications of Extreme Loss or Reduction of Mitochondrial LSU rRNA Helical-Loop Structures in Gastropods .......................... GUIDO PASTORINO Two New Trophoninae (Gastropoda: Muricidae) from Antarctic Waters .... EUGENE V. COAN Correction to: Coan, Eugene V., 2002, The Eastern Pacific Recent Species of the Corbulidae (bivalvia). Malacologia 44(1): 47-105 ............... 2002 175 223 241 259 273 289 307 325 333 | ur ag Ml u Bir! 2 1 alia ie nie EEE HATT у Y! ie я У} I | | | i rs 4 f stat me у а. a у | Е A | | | } | 1 : | | | 1] fn 1 y у т | у 1 u ah | u т i и y Zu | y 1 WI Ñ | № р rl | Ab úl tp AN À я ITNT AL A 3 2044 072 160 575 Mage Bee ieee oR ND na LA LAN A y - > Кая KA елена FR CRE EIERN TE An ET EC LESE TE fr gan Rep Rt STR = НИИ т EEE Kal