Polyploidy and Meiosis in the Freshwater Clam Sphaerium Striatinum (Lamarck) and Chromosome Numbers in the Sphaeriidae (Bivalvia, Veneroida)

C 1999 The Japan Mendel Society Cytologia 64: 247-252, 1999 Polyploidy and Meiosis in the Freshwater Clam Sphaerium striatinum (Lamarck) and Chromosome Numbers in the Sphaeriidae (Bivalvia, Veneroida) Taehwan Lee * Museum of Zoology and School of Natural Resources and Environment, University of Michigan, 1109 Geddes Ave., Ann Arbor, MI 48109, U.S.A. Accepted April 23, 1999 Summary Spermatogenetic meiosis as well as a very large number of chromosomes (n=76, 2n=ca. 152) were observed in a North American freshwater clam, Sphaerium striatinum. Sphaeriid species studied to date, except for one species, are all polyploids having high mitotic chromosome numbers, which range from ca. 150 to ca. 247. These results indicate that pronounced polyploidization may have played a major role in the evolution of the Sphaeriidae.This study also suggests that the basic chromosome number of the sphaeriid polyploids may be 19 and that a very significant variation in ploidy levels (2n to 13n) occur in the Sphaeriidae. Even though meiosis has been observed in a polyploid species, S. striatinum, and in the diploid S. corneum (n=18, 2n=36), it is not clear whether or not polyploidy in any of the other sphaeriid species is associated with asexuality. Key words Sphaerium striatinum, Sphaeriidae, Freshwater bivalve, Polyploidy, Meiosis. The Sphaeriidae are prominent and ubiquitous members of freshwater ecosystems (Herrington 1962, Burch 1975, Kuiper 1983), containing five widely recognized genera: Sphaerium, Musculium, Pisidium, Eupera and Byssanodonta. All sphaeriid clams studied to date are simultaneous hermaph- rodites and brood their direct-developing young within the inner demibranchs until they are re- leased as benthic juveniles (Gilmore 1917, Okada 1935a, b, Benetto and Ezcurra 1964, Heard 1965a, b, Mackie et al. 1974, Ituarte 1997). Experimental studies (Odhner 1929, 1951, Thomas 1959, Meier-Brook 1970) have demonstrated that some sphaeriid species can reproduce in isolation for multiple generations. It is generally believed that sphaeriids typically reproduce by cross-fertilization, but can facultatively self-fertilize (Heard 1965a, Kuiper 1983, Araujo and Ramos 1997). The few existing studies of chromosomes of sphaeriid clams have shown strikingly variable mitotic chromosome numbers, ranging from 36 to ca. 247 (Table 1). In his description of germ cell history using sectioned material, Woods (1931) reported spermatogenetic meiosis and roughly counted the chromosome numbers (n=ca. 32-46, 2n=ca. 68-98) in Sphaerium striatinum (Lamar- ck), although his paraffin section method was not satisfactory for chromosome study, as he men- tioned. The first sphaeriid clam studied cytogenetically was the European S. corneum (Linnaeus), for which Keyl (1956) demonstrated the chromosome numbers n=18 and 2n=36, and reported an abnormal meiosis, lacking chiasma formation, during spermatogenesis. Burch and Huber (1966) and Burch (1975) found much higher chromosome numbers on a cursory examination of several North American sphaeriid species, and they suggested that the origin of such high numbers was due to polyploidization. In 1996, Barsiene et al. reported that populations of Pisidium casertanum (Poli) inhabiting two different Spanish mountain springs have a large and variably numbered chromosome component, 2n=ca. 150 in one spring and 2n=ca. 180 in the other. Burch et al. (1998) found simi- larly high chromosome numbers in three species of North American sphaeriids: S. occidentale (Prime) [2n=ca. 209], Musculiumsecuris (Prime) [2n=ca. 247] and P casertanum (2n=ca. 190). * E-mail: [email protected] 248•@ Taehwan Lee Cytologia 64 Table 1. Chromosome numbers in the Sphaeriidae a b c d Fig. 1. Photomicrographs of the shells in three views of Sphaerium striatinum (Lamarck) collected from the Maple River, Michigan, U.S.A. (UMMZ 265688). Striae are faint on the umbonal region but become heavier toward the ventral part of shell. a) Side view of the left valve, b) front view of the shell, c) inside view of the left valve, d) right valve. The scale bar represents 5 mm. Hermaphroditism, the ability of self-fertilization and the possibility of pronounced polyploidy may indicate a complicated reproductive mode (or modes) in the Sphaeriidae. Asexual reproduction has been confirmed in other hermaphroditic, brooding, polyploid bivalves. Parthenogenetic deve- lopment is triggered by autosperm (autogynogenesis) in the polyploid marine genus, Lasaea (O Foighil and Thiriot-Quievreux 1991). Unusual androgenesis has been reported for the triploid freshwater clam, Corbicula leana Prime, in which only chromosomes derived from unreduced spermato- zoa are inherited while all maternal genome is extruded in polar bodies (Komaru et al. 1998). The possibility of asexual reproduction, however, has not been raised in the Sphaeriidae. In effect, no detailed study has been performed using genetic markers, or by direct cytological observations. In this study, the chromosomes of Sphaerium striatinum (Lamarck) were investigated to determine if this species is also polyploid, and if so, to determine if gametes are produced by meiosis as described by Woods (1931). The basic chromosomal complement of the family Sphaeriidae is also considered. 1999 Polyploidy in the Sphaeriidae 249 Materials and methods Sphaerium striatinum is one of the most commonly encountered sphaeriid species in our area, and is found in lakes and streams of all sizes throughout North America (Herrington 1962, Burch 1975). This species shows considerable ecophenotypic and allometric variation in shell shape (Bai- ley et al. 1983) and probably has distinct subspecies (Baker 1928, Herrington 1962). Specimens were collected from a sandy gravel bottom of a shallow river in northern Michigan (Dam Site, Maple River, Section 10, T. 36 N., R. 3 W, Maple River Township, Emmet County, Michigan, U.S.A.) periodically from May 1997 to July 1998. The shells of these samples show relatively weak striae which are distributed unevenly over the shell surface. Striae are weak on the umbonal region, but they become heavier toward the outer part of shell (see Herrington 1962 for detailed description of the species). Voucher specimens of the population from which chromosomes were studied have been deposited in the collections of the Museum of Zoology, University of Michigan (catalogue numbers UMMZ 265688, 265689, 265690). Chromosome squashes were made from the gonads in active stages of gametogenesis, and from juveniles brooded within the parents' gills. About 0.1 ml of 0.005% colchicine was injected directly into a portion of the gonad, and tissues were dissected 1.5 h after the injection. These tissues were placed in distilled water for 40 to 60 min and then fixed with modified Carnoy's fixative (1 part glacial acetic acid and 3 parts absolute ethanol). The fixed tissues were stained with acetic- orcein and squashed between coverslip and slide with thumb pressure. The chromosome squashes were observed and photographed with a Zeiss compound microscope. Results The squash preparations made from Sphaerium striatinum gonads show spermatogenetic meiosis as well as mitotic divisions. When mitotic metaphases could be observed from the preparations made from either gonads or juvenile tissues, ca. 152 chromosomes were observed (Fig. 2a). 76 chromosomal elements, presumably all bivalents, were counted during meiotic metaphase I of spermatogenesis from the cells in which chromosomes could be adequately observed (Fig. 2b). Discussion The chromosome numbers (n= 76, 2n= ca. 152) of Sphaerium striatinum counted in this study Fig. 2. Photomicrographs of a) mitotic metaphase chromosomes (2n=ca. 152), b) meiotic metaphase I chromosomes (n=76) of Sphaerium striatinum. Scale bars represent 5 ,um. 250•@ Taehwan Lee Cytologia 64 are much higher than that counted by Woods (1931) (n=ca. 32-46, 2n =ca. 68-98). However, as Woods (1931) himself, and later Burch (1960d), pointed out, paraffin section techniques are not satisfactory for studies of molluscan chromosomes. Therefore, this is the first accurate report of chromosome numbers for the species. The high chromosome number of S. striatinum is consistent with recent reports of North American and European sphaeriid clams by other workers (Table 1). Sur- prisingly, the chromosome numbers obtained to date in the Sphaeriidae are all very large (more than 150 mitotic chromosomes), except for the European S. corneum (2n=36). The large numbers of chromosomes in this family are obviously polyploid since not only are the numbers far above the normal range within the Bivalvia (Nakamura 1985), but aneuploidy or supernumerary chromosomes cannot adequately explain such large chromosome numbers. Frequently encountered polyploidy in this family suggests that pronounced polyploidization may be prevalent among sphaeriids. According to a recent review on molluscan chromosomes (Nakamura 1985), veneroid bivalves have from 24 to 48 diploid chromosomes, with the most frequent diploid number being 38. This is, however, not the case in the Sphaeriidae and it is indeed difficult to decide the basic chromosome number of the family. Keyl (1956) found the haploid number 18 in Sphaerium corneum, and Barsiene et al. (1996) suggested the basic number of the Sphaeriidae to be 15. Nevertheless, it is in- teresting to note that all the chromosome numbers of the North American sphaeriid species obtained to date are multiples of 19, suggesting that the basic chromosome number of these sphaeriid polyploids may be 19. Irrespective

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