The malacologicalsocietymalacological society of Japan Malac.) "lteVENUS (Jap.jnur. J Vel, S4, Ne. 3 c199S}: 185 t93 Afv -vSd l/ Y R ta3ptofkth J: F sf Y } Imatlstera) 7i v A ,Z ng * " " " ･ ･ ･ op fi fi fa T･ *NiPfE re ssJ[[ ktt K" rp E \" ･ "ftecmrt<teG･rtwtn ･ "'JttamaJctpt<de･'}:s･s) (-*emaJk\mpt?ens Phylogeny of Three Corbicula Species and Isozyme Polymorphism Populations in the CoTbic2ela2'aponiea HOSOKAwA' Machiko HATsuMI', Mikio NAKAMuRA'", Muneyoshi and Shigeru NAKAO*** "Shimane ('Faculty of Science, Shimane University, Matsue, Shimane Pref. 690, Prefectural Fisheries Experimental Station, Mitoya, Shimane Pref, 690--24, *'"Faculty and ef Fisheries, Hokkaido University, Minato, Hakodate 041.) Abstract; Six populations of Corbicula J'aponica, thTee populations of C. ieana and one twelve isozyme coding loci population of C santlai were electrophoretically analyzed at in order to appToach the phylogenetic relationship among the three species and the level of intra-specific differentiation within C. japonica and C. Ieana, Among them, C, J'apenica was the most variable with most ef the examined loci being polymorphic. It was also most geographically differentiated. Genetic differentiation among populations of C. joponi- ea was not large enough te regard the geographic morphs as subspecies, C. sandai, endemic to Lake Biwa, was more variable than C. Ieana, a triploid species. Most of the examined loci of C. leana were monomorphic. A phylogenetic tree constructed from Nei's genetic distances between populations indicated that C. J'aponica diverged from the ancestral species first, with C sandai and C. leana subsequently differentiating from each other. Introduction Corbicula species are common bivalves, and an economically important aquatic resource in Japan. Two species of Corbicula, C. J'aponica and C. Ieana (Japanese names, Y17mato- shijimi, and Ma-shijimi, respectively), are widely distributed in Japan and on the Asian from continent. C. japonica is found in brackish lakes and at the mouths of rivers Hokkaido to Okinawa, C. leana lives usually in freshwater lakes and ponds from Honshu to Kyushu, and C. sandai (Japanese name, Seta-shijimD is endemic to Lake Biwa (Habe, 1977). There are two competing hypotheses on the phylogenetic relationship among these three species. One is that C. Ieana was branched off first from the ancestral species, followed by differentiation of C. sandai fTom C japonica (Takaya, 1963; Ishida, 1967). This hypothesis is supported by the fact that young shells have a radiating striation on the shell surface in C japonica and C. sandai, while C. ieana has black spots on the shell NII-Electronic Library Service The malacologicalsocietymalacological society of Japan 186 VENUS: Vol, S4, No.3(1995) surface. Their reproductive systems also differ. C. japoniea and C. sandei are dioecious and shed their eggs in water, while C. Ieana is an ovoviviparous hermaphrodite and employs self-fertilization (Ikematsu and Yamane, 1977). The other hypothesis is that C japonica diverged from the ancestral species first, with C. sundui and C. leana subsequently differen- tiated from each other (Okamoto and Arimoto, 1986). The latter hypothesis is supported by the following data: (1) C, sandui and C. leana live mainly in freshwater, while C. 1'aponica lives mainly in brackish water; (2) the haploid chromosome nurnbers of C. sanddi and the triploid species C. leana are identical (N=18), while the haploid chromosome number of C. japonica is N= 19 (Okamoto and Arimoto, 1986); (3) the color inside the shells is dark purple in C. sandui and C. teana, while it is whitish purple in C. juponiea, Recently the findings of Sakai et al. (1994) also supported the latter hypothesis. They examined isozyme polymorphism in these three species and found that C. Ieana had monomorphically the same alleles as C. sandoi at all twelve loci analyzed, and allelic dis- placement was observed at six loci between C. J'aponica and the other two species. In addition to the phylogenetic relationships between species, genetic differentiation be- tween populations should be elucidated in these species from the viewpoint of systematics. Population genetics has shown that the study of allezyme polymorphism is usefu1 in deter- mining not only phylogenetic relationships among species, but also the population structure of a species. since genetic differentiations are measurable using the genetic distance calculat- ed from allelic frequencies (Nei, 1972). In this study we analyzed isozyme polymorphisms of ten populations of three Corbicula species in order to estimate the degree of genetic differentiation within and between species and to construct a phylogenetic tree showing the eyolutionary relationship among the species. Materials and Methods Six populations of C J'aponica, the Lake Biwa population of C. sandui, and three populations of C. Ieana were analyzed. Table 1 shows the materials used, sites, dates and methods of collection and suppliers who provided samples. Fig. 1 is a map showing the sampling sites. Twenty-four individuals were analyzed from each population. Horizontal starch-gel electrophoresis and protein staining were carried out using methods similar to those employed by Aizawa et al. (1994). Live materials were frozen in liquid - nitrogen and maintained in a deepfreezer at 80eC before dissection. The mid-gut gland was removed from every individual, homogenized, centrifuged and stored at -80eC. Eight enzymes were examined using three buffer systems: isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH) and phosphogulucomutase (PGM) on citrate- aminopropyl morphorine buffer (pH6.2) (Clayton and Tretiak, 1972), aspartate amino transferase (AAT), leucine aminopeptidase (LAP), phosphogluconate dehydrogenase (6PGD) and superoxide dismutase (SOD) on Tris-citrate buffer (pH 7.0) (Fujio, 1984) and catalase (CAT) on Tris-EDTA-Borate buffer (pH8.0) (Selander et al., 1971). CAT was stained by the method described by Richardson et al. (1986), and the isozyme patterns of CAT were Tecorded immediately after the gels were stained. When one enzyme had two loci, each locus was numbered in order of low mobility to the anode. Each allele was alphabeti- cally labeled in order of increasing electrophoretic mobility. NII-Electronic Library Service The malacologicalsocietymalacological society ofJapanof Japan Hatsumi et al.: Phylogeny ef Corbicula species 187 Table1. Collection data of materials. HILift:MfiFe:mar;-6f'-a. SpeciesAbbreviations used in Locality Date MethodCollector this paper Teshio-cho) 1993 Dredge K. Sasaki C, J'aponicaTSRABL Teshio River (Hokkaido, .Oct Lake Abashiri (Hokkaido, Jul 1991 Dredge I. Sugano Abashiri-city) HNLNGRSHLJZLLake Hinuma (Ibaraki, Ibaraki-cho) Oct 1993 Dredge K. Fukazawa 1993 Nagara River (Mie, Kuwana-city) Oct Dredge K. Akita Lake Shinji (Shimane) Oct 1993 Dredge Y, Hara Lake Jinzai (Shimane, Izumo-city) Oct 1993 Dredge T. Kuwabara C, sandai BWLLake Biwa (Shiga) Oct 1993 Dredge T, Hiratsuka Dec C. ieana OKY Irrigation Pend {Okayama, 1993 Hand Hayasima-cho) MTYMYZ Waterway (Shimane, Mitoya-cho) Oct 1993 Hand Waterway (Miyazaki, Kobayashi-city) Nov 1993 HandM. Kuroki ABL / TSR f. .g HNL Fig. 1. A map of Japan showing sampling localities. Abbreviatien corresponds to the localities listed in Table 1. ptee±dea)i".W. ag"iTable 1tmaC, NII-Electronic LibraryMbrary Service The malacologicalsocietymalacological society of Japan 188 YENUS: Vol. 54, No. 3 (199S) Results Twelve loci were presumed from eight enzymes. The examples of zymograms are shown in Fig. 2. The Sod locus was monomorphic in al1 three species, and eleven loci had more than three variants. Two LAP patterns were found in C teana, the triploid species. Both patterns consisted of two bands of b and c, but the thickness of bands differed. One consisted of a thicker band of Lapb and a thinner band of LapC, and the other consisted of a thicker band of LapC and a thinner band of Lapb. The allelic frequency of Lap in C. teana was estimated by presuming that the thicker bands were produced by two genes and the thinner bands were produced by one gene. Table 2 shows the allelic frequency at each locus for each population. Chi-square tests of allelic frequencies in each locus in each population of C juponica and C. sandoi were applied to detect the deviation from the Hard"k Weinberg equilibrium, and there were no significant differences between expected and observed frequencies. a -- b ,- pm ---- i-- ---- M P-C <A:d .., e-mn- - l- - fM g - Jm- -- - - aSbF --m - aT-- - -- -- ----m-n- M--NM- Nc ± !ll b M--N-- c M-----M--MN -- - - - Cjaponica Csandai C. teana Fig. 2. Examples of zymograms of Corbicula species. VY}f,fi3pta)V'if Eti7AO-lijU. was the variable among C. ieana least the species studied. Only the Lap locus had variations as previeus]y described, and the other loci were monomorphic in all three populations. C. j'oponica was polymorphic at ten loci. Three loci, Cat. Lap and Rgm-l had rnore than three alleles in every population. Each population was characterized by the allelic frequencies of Cket and Pgm-l loci. CZiE in the Teshio River, the Lake Shinji and the Lake Jinzai populations, Clatd in the Lake Hinuma and the Nagara River populations, and CZitb in the Lake Abashiri population were the most frequent alleles, respectively. Ctrtd in the former three populations and CbE in the latter three populations were the second NII-Electronic Library Service The malaoologioalmalacological societysooiety of Japan Hatsumi et al．： Phylogeny of COrbicuia species 189 Table 2． Allelic