Genes Genet. Syst. (1998) 73, p. 59–64 Genetic differentiation among local Japanese populations of the starfish Asterias amurensis inferred from allozyme variation Norimasa Matsuoka* and Toshihiko Hatanaka Department of Biofunctional Science, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan (Received 6 November 1997, accepted 16 February 1998) The starfish Asterias amurensis that is a common species in Japanese waters shows the remarkable morphological variation in several characters such as colour pattern of body between local populations. The genetic differentiation and relationships among seven local Japanese populations were investigated by allozyme analysis. From the allozyme variation observed in 25 genetic loci coding for 14 enzymes, Nei’s genetic distances between seven local populations were calculated and a biochemical dendrogram for seven populations was constructed. The dendrogram indicated that the Akkeshi (Hokkaido), Ushimado (Inland Sea), and Ise (Ise Bay) populations are much genetically differentiated from the other four populations, and that the degree of genetic differentiation between them was much higher than that between conspe- cific local populations. Judging from allozyme and morphological data, we conclude that the starfish A. amurensis from Japanese waters consists of at least three groups that are largely genetically divergent at subspecies or sibling species level. other populations. Populations from Mutsu Bay of north- INTRODUCTION ern Tohoku have the standard blue or purple body with In a previous study, we indicated using allozyme analy- slender arms. From the morphological study on geographi- sis that the tropical common sea-urchin Echinometra cal populations of the species, Hayashi (1974) considered mathaei from Okinawa Island of southern Japan consists that the populations distributing from the central region of four different species or sibling species (Matsuoka and to the southern region of Honshu in Japan may be a sub- Hatanaka, 1991). This study was strongly supported by species of A. amurensis, and classified those as A. amurensis non-molecular data from the morphological, ecological, versicolor, though this taxonomic system is not generally karyological, and embryological studies (Tsuchiya and accepted at present. Nishihira, 1984; Uehara and Shingaki, 1985). Naturally, The taxonomic problem as to whether these morphologi- it is well expected that there exist marine invertebrates cally variable populations of A. amurensis represent only consisting of some sibling species such as E. mathaei or intraspecific variation or subspecies and different species some subspecies, since the taxonomic and phylogenetic stud- merits biochemical and genetic scrutiny. At present, the ies of marine invertebrates are more backward than those molecular phylogenetic approach has been proved to be of vertebrates. reliable, effective and powerful for distinguishing species The starfish Asterias amurensis is one of the most com- and assessing their phylogenetic relationships (Ferguson, mon species in Japan, and has been widely used in various 1980; Ayala, 1982; Nei, 1987; Hillis and Moritz, 1990), and fields of biology. The starfish is extremely variable in thus it can provide useful information for such taxonomic morphology, and some local Japanese populations are dif- problems. One of the present authors (N.M.) has been ferentiated at the phenotypic level. Namely, the Akkeshi carrying out biochemical systematic studies of echinoderms population (Hokkaido) has very large-sized body with wider (echinoids and asteroids) using allozyme electrophoresis, arm and many spines. The Ushimado (Inland Sea) and immunological methods and enzyme kinetic methods. Ise (Ise Bay) populations have a deeper blue or purple body Through these studies, we have found that allozyme analy- with well developed dorsal spines. Populations from To- sis is a reliable method in the field of echinoderm taxonomy kyo Bay and the neighbouring regions have a yellow body, and phylogeny (see the review of Matsuoka, 1990, 1993). in contrast to the blue or purple bodied starfish of many In this study, we report the results of an allozyme study designed to clarify the genetic differentiation and relation- * Corresponding author. ships among seven local Japanese populations of the star- 60 N. MATSUOKA and T. HATANAKA fish A. amurensis of the family Asteriidae. dehydrogenase (XDH), glucose-6-phosphate isomerase (GPI), hexokinase (HK), superoxide dismutase (SOD), as- partate aminotransferase (AAT), alkaline phosphatase MATERIALS AND METHODS (ALK), peroxidase (PO), esterase (EST), and leucine amino Starfish materials. The starfish A. amurensis examined peptidase (LAP). Stain recipes for these enzymes have in this study were collected from seven different localities been described previously (Matsuoka and Hatanaka, 1991; in Japanese waters as shown in Figure 1. The number of Matsuoka et al., 1991). individuals assayed and the collecting sites were as follows; 12 from Akkeshi (Hokkaido), 20 from Asamushi in Mutsu RESULTS AND DISCUSSION Bay (Aomori Pref.), 11 from Oga Peninsula (Akita Pref.), 12 from Miyako (Iwate Pref.), nine from Misaki (Kanagawa Genetic variation within populations. From the Pref.), 24 from Ise Bay (Mi-e Pref.), and 28 from Ushimado allozyme variation observed in the 14 different enzymes, in the Inland Sea of Japan (Okayama Pref.). The total 25 genetic loci were inferred. The allele frequencies for number of individuals collected from the above seven lo- 25 genetic loci in seven populations of A. amuresis are shown calities and assayed in this study was 116. After collec- in Table 1. Thirteen loci (H6pd, Me, Odh, Sdh, Hk, Gpi, tion, pyloric ceacae were cut off and stored at –80°C until Aat, Alk-2, Po-1, Est-1, Lap-3, Lap-4, and Lap-5) were mono- used in the allozyme analysis. morphic in all population. The remaining 12 loci (Mdh, Xdh, Sod-1, Sod-2, Po-2, Alk-1, Alk-3, Est-2, Est-3, Est-4, Allozyme electrophoresis. Electrophoresis was per- Lap-1, and Lap-2) were polymorphic in at least one formed on 7.5% polyacrylamide gels as described previously population. In nine loci (Mdh, Xdh, Sod-1, Po-2, Alk-1, Alk- (Matsuoka, 1985). About one g of pyloric ceaca was indi- 3, Est-2, Lap-1, and Lap-2), single- and double-banded phe- vidually homogenized with two volumes of cold 20 mM phos- notypes were observed and these were interpreted as rep- phate buffer (pH 7.0) containing 0.1 M KCl and 1 mM EDTA resenting homozygous and heterozygous states controlled in an ice-water bath using a glass homogenizer of the Pot- by two or three different alleles at a single locus coding a ter-Elvehjem type. After centrifugation at 108,800 × g for monomeric protein. In two loci (Sod-2 and Est-4), single- 20 min, 0.01–0.1 ml of the clear supernatant was used for and triple-banded phenotypes were observed in most popu- electrophoretic analyses of enzymes. The electrode buffer lations, which were interpreted as representing homozy- was 0.38 M glycine-tris buffer, pH 8.3. After electrophore- gotes and heterozygotes at a single locus coding for a dimeric sis, the gels were stained for the following 14 different en- protein. In Est-2 of Ise and Ushimado populations, alle- zymes; hexose-6-phosphate dehydrogenase (H6PD), malate les were not scored, though the cause is unclear. dehydrogenase (MDH), malic enzyme (ME), octanol dehy- In the course of this study, the three enzymes (ADH, drogenase (ODH), sorbitol dehydrogenase (SDH), xanthine G6PD, and AMY) scored easily in sea-urchins could not be Fig. 1. Map showing the geographical locations of seven populations of the starfish Asterias amurensis from Japanese waters. Geographical divergence of starfish 61 Table 1. Allele frequencies at 25 genetic loci in seven local detected in the starfish studied here. On the other hand, Japanese populations of the starfish Asterias the starfish showed the stronger LAP activity than the sea- amurensis urchins. Non-detectability of AMY activity and the strong Locus Allele Akke Asa Oga Miya Misa Ise Ushi LAP activity in starfish may be closely related to the feed- H6pd a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 ing habits that the starfish is one of the predatory marine Me a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 animals in contrast with the sea-urchin. Odh a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Table 2 summarizes the extent of genetic variation in Sdh a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 seven local populations of A. amurensis. The number of Hk a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Gpi a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 alleles per locus was in the range of 1.16–1.50, with a mean Aat a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 of 1.36, the proportion of polymorphic loci (P), in the range Alk-2 a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 of 16.0–44.0%, with a mean of 31.8%, and the expected av- Po-1 a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 erage heterozygosity per locus (H), in the range of 4.3– Est-1 a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 16.4%, with a mean of 10.1%. In the previous paper, we Lap-3 a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Lap-4 a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 reported on the biochemical systematics of five asteroid spe- Lap-5 a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 cies of the family Asteriidae by allozyme electrophoresis Mdh a – 0.05 – – – – – (Matsuoka et al., 1994).