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Malac.) "lteVENUS (Jap.jnur. J Vel, S4, Ne. 3 c199S}: 185 t93

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Phylogeny of Three Corbicula Species and Isozyme Polymorphism

in the CoTbic2ela 2'aponiea Populations

Machiko HATsuMI', Mikio NAKAMuRA'", Muneyoshi HOSOKAwA' 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 population of C santlai were electrophoretically analyzed at twelve isozyme coding loci 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 Hokkaido to Okinawa, C. leana lives usually in freshwater lakes and ponds from 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

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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.

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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 (Hokkaido, .Oct Lake Abashiri (Hokkaido, Jul 1991 Dredge I. Sugano Abashiri-city) HNLNGRSHLJZLLake Hinuma (Ibaraki, Ibaraki-cho) Oct 1993 Dredge K. Fukazawa

1993 (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,

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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

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.

C. ieana was the least variable among 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

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Hatsumi et al．： Phylogeny of COrbicuia species 189

Table 2． Allelic frequencies at eleven loci in ten populations of Corbicula species ． ． シ ジ ミ 類 10 集 団 に お け る 各 ア イ ソ ザ イ ム 遺 伝 了座 の 遺 伝 子頻 度 ． ’ C ．」aponica c ．sandai c ．leana LocusAllele TSRABL HNL NGR SHLJZLBWLOKY MTY MYZ

Aat − 1 010 0．1670 0 ，000 0．000 0 ．000 0．021 α α 001 00

δ ．8330 LOOO LOOO l ．000 0 ．979 α q1

C ．000 0rO國DO O，000 0 ．000 0 ．000 L ， 1サ − Aat 2 α 001 001 0 。000 0 ．000 0 ．000 0 ．OOO 0 ．5830 a1 0， 0 ロ

∂ O ．042 0 ．021 0 ．000 0 ．021 ，4170 ， L − ロ

C 0 ．958 0 ．979 1．000 0 ．979 ．OOO a α 0ロ

Cat 0 ，0000 O．OOOO O ．OOO O ．000 O ．OOO O ．OOO 1000 O100 0100 O100 ∂ ．08305210．4170 0．188 0 ．208 O ．229 0．208 c4 。3330 0 ．250 0 ．313 0 ．479 0，542 ．396 ．250 0563 0 ．479 0 ．292 0．250

Idh − 1 σ 0 ，0000 0．0000 0。000 0 ．000 0 ，000 0．000 O ．0210 O100 O100 O100 占 ，0000 ，0000 0，000 0 ．000 0 ．000 0，000 ．9790 C ．8960 ．6460 0．750 1，000 1，000 0．938 ，0000

」 04 ，354 0 ，250 0 ．000 0 ．000 0，063 ．OOO

Idh −2 α 0 」 040 0．021 0 ．042 0 ．000 0 ．042 α 0 ．0000 01 0 ．0000 ∂ L ．8960 0．979 0 ．958 1．000 0 ．958 α ．OOOLOOO“ ．OOO1 C α ．000 0 ．000 0 ．0（K） 0 ．000 0 ．000 L L ．000

Lap α 0 ．0000 0 ．0000 0 ．000 0，000 0 ．000 0 ．000 0．0830 0 ．0000 0 ，0000 0 ．0000 ウ ．0000 ．OOOO 0．000 0．000 0 ．000 0．000 ．33305830．3330 ．3330 ．6670 C ，0000 ．OOOO 0 ，000 0 ．000 0 ，000 0 ．000 ．6670 ．6670 ，3330 ゴ ．0000 ．0630 0 ．083 0．021 0 ．042 0 ．000 ．0000 ．0000 ，0000 ．0000

ε ．7500 ，6040 0 ，646 0．563 0 ．646 0 ，563 ．0000 ．0000 ．0000 ．OOOO ノ ．2500 ．3330 0 ．271 0．417 0 ．250 0 ．438 ．0000 ．0000 ．0000 ．0000 9 ．000 ．000 0 ．000 0．000 0 ，063 0 ．000 ．OOO ．OOO ．000 ．∞ 0

Mdh ＿1 α 0．1250 0，1460 O ．396 0 ．083 0 ．063 0 ．021 001 001 α a わ ．8750 ．8540 0 ．604 0 ．896 0 ．938 0 ，979 α α C ．000 ，000 0 ．000 0 ．021 0 ．000 0 ，0（》0 L L

Mdh −2 α 001 001 O．OOO O ．000 0 ●DOO O．000 0．5420 α O10 O10 ∂ 脚 0．OOO O ．OOO O ，000 0．OOO ．4580 LO C 1．OOO l．000 1．〔X）0 1●000 ．000 ，

6Pgd 0 ．0000 010コ O．OOO O ．028 0 ．028 0 ，056 0．0830 O10 0 。 010ワ

∂ ．9720 ワ LOOO O ．944 0 ．972 0 ，917 ．8060 LO ロ

C ．028 ロ 0．000 0 ．028 0 ．000 0 ．028 ．111 伽 ， コ

Pgm − 1 α 0 ．0000 0 ．0000 0．0000 O ．OOOO 0．0000 0 ．0000 0 ．6〔叫 10000 L 10000 わ ，0000 ．0000 ．0000 ．0000 ．0000 ．0000 0 ．3960 住 Cd ，8540 ．8130 ．2920 ．1670 ，3750 ．2920 ．0000 伽 α ，0210 ．0210 ．1460 ．1040 ．4380 ．5420 ．0000 α

ε ．125 ．167 ．563 ．729 ．188 ．167 ．000 α 伽 − Pgm 2 口 010 0 ， 0 ． 0． 0． 0 ． 且 ロ LOOO 1．000 L

∂ L 0 ．979 LOOO O ．938 0．938 00 ロ 0 ，000 0 ．000 α

C α 0 ．021 0．000 0 ，063 0，063 0 ．000 0 ．000 α

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190 VENUS: Vol, 54, No.3(1995)

most frequent, respectively. The frequency of Rgm-IC was highest in the Teshio River and the Lake Abashiri populations, while IRgm-IC showed the highest frequency after llgm-le in the Hinuma and the Nagara River populations, and after llgm-ld in the Lake Shinji and the Lake Jinzai populations. Average heterozygosity ranged from O.138 (the Teshio River population) to O.223 (the Hinuma Lake population) in C, japonica. These figures are within the standard range for bivalves (Powell, 1975). C. sandai, endemic to Lake Biwa, was more variable than C. teana, and six loci (Aat-2, Idh-1, Lap, Mdh-2, 6Pgd, and Ilgm-1) in C. sandoi were polymorphic. Allelic frequen-

cies of ldh-lb, 6Pgdb and ]Rgm-1" were highest in each locus in C. sanciai, and these three alleles were also fixed in C. Ieana. At four loci (Aat-L ldh-2. Mdh-1 and Iigm-2) showing monomorphic, alleles fixed at each locus in C. sandai and in C. Ieana were identi- cal. C. sandei and C. leana were fixed for alternative alleles at only the det locus. On -IC, the contrary, five alleles, Aat-2b, three 6LPgd alleles and Mdh were maintained in both `Aat-2b C. japonica and C sandai, but and Mdh-IC were rare variants in C. juponica.

Table 3. Nei's genetic identities (above diagonal) and Nei's genetic distances (below diagonal) between pairs of populations of three Corbicula species. if V l- faioeewrmaa)mefig'iiMfD(ff (SiFt-J'LSB) tEfrt .fretime (kYi'lrfirs).

C. japonica sandbi C. leana

TSRABLHNLNGRSHLJZLC. BWL OKY MTY MYZ

TSRABLHNLNGLSHLJZL - O.9g05 0.9581 O.9509 O.9757 O.9635 O.1790O.1904O.1925O.1830O.1826O.1815O.1879 O.1879 O.1879

O.O197 - O.9557 O.94oo O.9595 O.9520 O.2326 O.2326 O.2326

O.0428 O.0454 - O.9797 O.9629 O.9526 O.2149 O.2149 O.2149

O.0504 O.06!9 O.0205 - O.9716 0.9668 O.2040 O.2040 O,2040 O.0246 O,0413 O.0378 O,0288 - O.9953 O.20S4 O.2054 O.2054

O.0371 O.0491 O.0486 O,0338 0.oo47 - O.2024 O.2024 O.2024

BWL1.7202 1.6584 1.6475 1.6985 1.7003 1.7066 O.8310 O.8310 O.8231

OKYMTYMYZ 1.6716 1,4S8S 15377 1.5898 1,S826 1.5973 O.1851O.1851O.1947 - 1.0000 0,9903 - 1.6716 1.4S85 1.5377 1.5898 1.5826 1,5973 O.OOOO O.9903

1.6716 1.4S85 1.5377 1.5898 1.5826 1.S973 O.oo97 O.oo97 -

Nei's genetic identities and genetic distances were calculated to determine the genetic differentiation between populations and between species (Table 3). The genetic distances between C. J'uponica and C. sandui ranged from 1.6475 to 1.7202, between C. juponica and C. leana from 1.4585 to 1.6716, and between C. santiai and C. leana from O.1851

to O.1947, suggesting C. sandoi is more closely related to C, leana than to C. J'aponica. A phylogenic tree was constructed from the genetic distances by the unweighted pair group method of cluster analysis, UPGMA (Sneath and Sokcal, 1973) (Fig. 3).

Discussion

Genetic distances and the resultant phylogenetic tree in the present study confirm the results of Sakai et al. (1994), which showed C. sandai is more closely related to C. leana

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Hatsumi et al.: Phylogeny of Cerbicula species 191

TSRABLHNL

C.japonica NGRSHL

JZL BWL 1 C. santlai OKY C leana MTY MYZ

10 1 O.1 O.Ol O.OOI Geneticdistance

Fig. 3. UPGMA tree of Corbicula species based on Nei's genetic distances. ma tt,a)xxfl ftyvEent:g-:-] < upGMA?x[z .lt 6 i/ y a r,fimru a)kMret .

than te C, japonica, According to the estimation of divergence time proposed by Nei (1987), t = 5 x 106D, C J'aponica diverged from the ancestral species about 8 million years

ago, and the ancestral species diverged to C. sandai and C. teana about 900 thousand

years ago. Though the numbers of loci studied are small, our results appear to reject

the hypothesis that C. sandai diverged from C. japonica about one million years ago when Lake Biwa was situated near Osaka Bay (Takaya, 1963; Ishida, 1976). Our results are

consistent with the results of a chromosome study that concluded C. Ieana diverged from

C. sandai by polyploidy (Okamoto and Arimoto, 1986). It is hard to believe, however,

that C. leana originated from C. sanclai at the Lake Biwa and spread through the freshwater

waterways of Japan (Okamoto and Arimoto, 1986). It is more probable that the common

ancestor was widely distributed throughout the Asian continent, and produced C. teana by polyploidy,as suggested by Sakai et al. (1994). This is supported by the fact that a

Corbicula species which is morphologically similar to C. sandai has been reported from

the Asian continent (Morton, 1986). Further study on Corbicula species from the Asian continent will supply more useful inforrnation on the evolution of C. sandai and C. leana.

The six populations of C. J'aponica were more differentiated from each other than the three populations of C. leana. The genetic distances within species were larger in C.

juponica (O.oo47--O.0619) than in C leana (O.OOOO-O.oo97). Large genetic differentiation

among the C. juponica populations can suggest the existence of geographic morphs in this species. The relationship between genetic distance and geographical distance of C. japonica is shown in Fig. 4. Geographically distant populations tended to show large genetic distance C7b). (r =O.705; O.1{7b

other. However, genetic differentiation between populations of C japonica is not large enough to regard the geographic morphs as subspecies. The lowest genetic identity of C.

J'aponica was O.94oo between the Lake Abashiri and the Nagara River populations. This level of genetic similarity is usually found in geographic populations within a species

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192 VENUS ： Vo1 ．54 ， No ．3 （1995）

0 ．07

0 ．06

（ O．05 ）Ω Φ O ⊆ o ．04 一〇 皿ω OO 0 ．Q3

眉

仁 0 ．02 爪O ） O ．Ol

oo

500 1000 1500

Geographical Distance （km ） ’ Fig．4 ． Relationship between Nei s geQgraphical distance （horizontal axis ） ， and Nei s genetic distance （vertical axis ）in the Corbicula joponica populations ．ヤ マ ト シ ジ ミ 集 団 の 地 理 的 距離 （横 軸 ） と 遺 伝 的距 離 （縦 軸 ） の 関係 ．

（Thorpe ，1982）． On the other hand ， genetic variation was scarcely detected within and between the C ． leana populations， as Sakai et a1 ．（1994 ）has demonstrated． Only the Lap

locus was heterozygous in this specics 。 This was not a resu 互t of the reproductive system

of C ， leana， self −fertilization， because parthenogenetic morphospecies can possess abundant

Noer 1988 ・In snails genetic variation （Schultz，1977 ； Turner et a1 ．，1983 ； ， ） parthenogenetic ， a high level of genetic divergence between populations was shown despite the lack of

− 1983 ． More should bc analyzed to under − intrapopulation variation （Stoddart， ） populations ． Ecological study especially on vagility stand the genetic population structure of C ．teana ， in the larval stages ， may reveahhe reason why the leve互 of divergence among populations

is different in Co 厂bicula sPecies ・

− Fishcries for revising Acknowledgments ； We thank DL Harumi Sakai ， Shimonoseki University of ，

Mr ， Kouichi Sasaki Hokkaido Fisheries Experimental Station the manuscript ．Thanks are also due to ， ． Mr ．Katsuhisa Fukazawa Ibaraki− Mr ． Isao Sugano ， Hokkaido Abashiri Fisheries Experimental Station， ， Ken Fisheries Experimental Station， Mr ． Kiyone Akita ， Gifu Prefectural Fisheries Experimental Station， − Mrs ． Yuzo Hara and Tsuneo Kuwabara ， Shimane Prefecture， Mr ． Tadayuki Hiratsuka ， Shiga Prefecmr al Fisheries Experimental Station and ML Masaru Kuroki ， Miyazaki Prefectural Fisheries Station， for providing materia 且s ， We are gratefuho DL Katsumi Takayasu and DL Brian Daldorph ， Shimane University， for geological suggestion and grammatical check on this paper ， respectively ．

要 約

シ ジ ミ 類 3 種 ，ヤ マ トシ ジ ミ ，セ タ シ ジ ミ ，マ シ ジ ミの 系統 関 係 と ．種 内集 団間 の 遺 伝的 分 化 を 解 明 す る た め に ア ．イ ソ ザ イ ム 多 型 を 分 析 し た ．ヤ マ ト シ ジ ミ は 多 型 的 で 地 理 的 分 化 も 進 ん で い た が ，亜 種 ． の セ タ シ ジ ミ マ シ ミ に と し て 区 別 す る ほ ど の 分 化 で は な か っ た 、，琵 琶 湖 内 に 固 有 よ り 単 型的 な ジ は 地 ． マ ト シ ジ ミ の 理 的 分 化 が 見 ら れ な か っ た 、．t 根 井 の 遺伝 的距 離 を も と に 系統 樹 を作 成 し た 結 果 ，ヤ 祖 先

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Hatsumi et al.: Phylogeny of Corbicula species 193

-? waht 5i1[[k L k: the: le g v･ ); a t t- V -i Jii tz e'tLcr)M)twa DtbldFi L ft: [: 2: htfift ML k .

References

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[Received: March 24, 1994]

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