_??_1993 The Japan Mendel Society Cytologia 58: 145 -149 , 1993 Polyploid Chromosome Numbers in the Torquis Group of the Freshwater Snail Genus Gyraulus (Mollusca: Pulmonata: Planorbidae)1 John B. Burch2 and Younghun Jung3 2 Museum of Zoology and Department of Biology , College of Literature, Science and the Arts, and School of Natural Resources , University of Michigan, Ann Arbor, Michigan 48109, U. S. A. 3 Department of Parasitology, College of Medicine , Inha University, 253 Yong Hyun-Dong, Nam-Gu, Inchon , 402-751, Korea Accepted December 28, 1992 The subclass Pulmonata is a very large group of land, freshwater and marine gastropods which use a lung rather than gills for respiration. All pulmonate snails are hermaphroditic, and many are known to be capable of propagation by self fertilization when the more normal biparental mating is prevented. In such a group, a large amount of polyploidy might be ex pected, but chromosome surveys have shown that such is not the case. Very few pulmonate snails have been found to be polyploid (Burch and Huber 1966). The first pulmonate snail in which polyploidy was detected was Gyraulus (Torquis) cir cumstriatus (Tryon 1866, Burch 1960a). The chromosome numbers have been determined for several other species belonging to other subgenera of Gyraulus (Table 1). In these, the only number found has been n=18, 2n=36, which is the basic chromosome number for the family Planorbidae to which they belong (Burch and Patterson 1978). The genus Gyraulus comprises a large group of very small species of freshwater snails belonging to the pulmonate family Planorbidae. Although the various species each have circumscribed and often limited distributions, the genus itself is worldwide in distribution, occurring abundantly and commonly in diverse aquatic habitats on all of the continents. Some of the species are implicated in the mediation of parasitic diseases to wild and domestic animals, and to man. Several groups of species (i.e., subgenera) comprise the genus Gyraulus. One of these groups, the subgenus Torquis, contains some of the smaller members of the genus (and of the family), and includes one of the most widespread and common of the North American fresh water snails, G. (Torquis) parvus (Say 1817). In addition to Gyraulus circumstriatus, two other species of the Gyraulus subgenus Torquis occur in North America, G. (T.) parvus (Say 1817) and G. (T.) huronensis Burch and Jung, 1990 (Fig. 1). Gyraulus huronensis has a limited distribution, but G. circumstriatus and G. parvus are widely distributed (Burch 1989). The purpose of this study was to determine if Gyraulus parvus and G. huronensis had the chromosome number found in most planorbid snails (n=18, 2n=36), or if they shared the polyploid condition of their Torquis relative, G. circumstriatus. Materials and methods The cytological preparations involved tissue fixation followed by acetic-orcein squash procedures. The organ used for chromosome number determinations was the ovotestis. 1 Contribution from the Museum of Zoology, Biological Station, and the School of Natural Resources, University of Michigan. 146 John B. Burch and Younghun Jung Cytologia 58 Fixation was with modified Carnoy's fluid (1 part glacial acetic acid, 3 parts of 95% ethanol), which, on some occasions, was taken to the field so that the snails could be fixed immediately. The shell of each snail was gently crushed with a dissecting pin and transferred with fine forceps to the fixative. Pieces of shell were removed, and the animals placed into fresh Carnoy's Table 1. Chromosome numbers in Gyraulus Fig. 1. Shells in three views of a, Gyraulus (Torquis) parvus, Hook Point lagoon, Douglas Lake, Cheboygan Co. (UMMZ cat. no. 250587); b, G. (T.) circumstriatus, woods pool near shore, Burt Lake, Cheboygan Co. (UMMZ cat. no. 250955); c. G. (T.) huronensis, shore of Lake Huron, north of Hammond Bay, Presque Isle Co. (UMMZ cat. no. 250571). Scale line with each set of shells= 1 mm. 1993 Polyploid Chromosome Numbers in the Torquis Group 147 fixative. Slides were prepared on returning to the laboratory , or the tissues were stored in a refrigerator for a short while until slides could be prepared . Observations were made with a Nikon compound microscope using 100•~ oil immersion objectives and 10•~ , 20•~ and 40•~ oculars. The localities from which we obtained snail specimens are as follows: Gyraulus circums triatus: Hook Point lagoon, North Fishtail Bay, Douglas Lake , Section 32, T. 37N., R. 3W., Munro Township, Cheboygan County, Michigan (oth Gyraulus circumstriatus and G . parvus occurred at this locality, but each species was restricted to its own special habitat, i.e., their local distributions did not overlap); a small tributary on the west side of the East Branch of the Maple River, southeastern corner of Section 25, T. 37N., R. 4W., McKinley Township, Emmet County, Michigan; woods pool next to the shore of Burt Lake at the Maple Bay Forest Camp Ground, Section 29, T. 36N., R. 3W., Burt Township, Cheboygan County, Michigan . Gyraulus huronensis: shore of Lake Huron, north of Hammond Bay, T. 37N., R. 2E., Section 14, Bearinger Township, Presque Isle County, Michigan. Gyraulus parvus: Pond on Liberty Road, near Ann Arbor, Section 28, T. 2S., R. 5E., Scio Township, Washtenaw County, Michi gan; Au Sable River, Middle Branch, 3 miles east of Grayling, Crawford County, Michigan; roadside drainage ditch and swamp along Levering Road, 0.35 mile east of crossroads (high ways US 31 and C 66) at Levering, northeast corner of Section 3, T. 37N., R. 4W., McKinley Township, Emmet County, Michigan; Hook Point lagoon, North Fishtail Bay, Douglas Lake, Section 32, T. 37N., R. 3W., Munro Township, Cheboygan County, Michigan. Voucher specimens of duplicate shells from the same populations from which chromosome number determinations were made are part of the collections of the Museum of Zoology, University of Michigan. Results All specimens of Gyraulus parvus, G. circumstriatus and G. huronensis in which chromo somes could be adequately observed were found to have 36 pairs of chromosomes during metaphase of the first meiotic division of spermatogenesis (Table 2, Fig. 2). All appeared as bivalents. No univalents or multivalents could be found. When spermatogonial metaphases could be observed, they could be seen to have approximately twice this number, i.e., ca. 72 chromosomes. The chromosome cycle in the polyploid Torquis species of Gyraulus is similar to that re ported for other pulmonate gastropods (Burch 1960c, Patterson and Burch 1978). Game togensis begins in gametogenic cells lining the lumen of the ovotestis acini. The chromosome cycle is the same for both female and male cell lines. However, the chromosome cycle is easiest to follow in the male line, and it is the male line on which we made most of our observa tions. Discussion Among the Gyraulus species previously studied cytologically, three species are from North America, two species are from East Asia, and one species is from South Africa (Table 1). The chromosome number reported for the East Asian and South African species of Gyraulus, and for the North American G. deflectus, was n=18, 2n=36, which is the chromosome number basic for the family Planorbidae (Burch and Patterson 1978). The chromosome number reported for G. circumstriatus by Burch (1960a) was n=36, 2n=72. The latter report was the first of polyploidy in pulmonate snails. Now, with the present study, we know that the other two species of the Torquis group in North America have this same tetraploid number. 148 John B. Burch and Younghun Jung Cytologia 58 From the numbers found in the three species of Torquis, two things are noteworthy. The first is that each of the three species is polyploid, which indicates that the tetraploid condi tion was first attained once, and then from this original tetraploid species the other tetraploid species evolved. It would seem very unlikely that the attainment of polyploidy among these three species was the result of three separate and independent events. The second interesting point is that once the tetraploid condition was reached, the stability of this number was main tained in each of the three species. Numbers other than n=36 were not observed. The origin of polyploidy in the Planorbidae is still speculative. Burch and Huber (1966) suggested that polyploidy came about in the African-Near Eastern Bulinus by hybridization, Table 2. Chromosome numbers of Gyraulus (Torquis) species in this study Fig. 2. Diakinesis chromosomes of a, Gyraulus (Torquis) circumstriatus, Burt Lake woods pool; b, G. (T.) huronensis, Hammond Bay. followed by doubling the chromosome number, i.e., allopolyploidy was involved. Cytological evidence presented by Goldman et al. (1983) tend to support this conclusion. Certainly the ecological conditions found where most of the ploidy states in Bulinus occur (the Ethiopian highlands, located near the equator) would provide the opportune physical characteristics in which polyploid events might be expected to occur (Patterson and Burch 1978). However , if, as our unpublished electrophoretic studies indicate, Torquis species are reproducing normally by automixis, or by apomictic parthenogenesis, then polyploidization may well have occurred by autopolyploidy. 1993 Polyploid Chromosome Numbers in the Torquis Group 149 Summary The first case of polyploidy in pulmonate snalis was found in the planorbid species Gy raulus (Torquis) circumstriatus (n=36, 2n=72). We now report the chromosome numbers of the other two members of the subgenus in North America, G. (T.) parvus and G. (T.) huronen sis. The latter two species have the same chromosome number, i.e., they are also tetraploid. The basic chromosome number for the family Planorbidae is x=18. The tetraploid condition found in the three polyploid species was probably originally the result of one single event. Once the tetraploid condition was reached, the stability of this number has been maintained in each of the three species.