Genetic Differentiation Among Local Japanese Populations of the Starfish Asterias Amurensis Inferred from Allozyme Variation

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). According to it, the H values of b 1.00 0.85 0.83 0.75 0.94 0.98 0.98 the five asteroids (A. amurensis, Aphelasterias japonica, c – 0.10 0.17 0.25 0.06 0.02 0.02 Xdh a – 0.65 0.59 0.29 0.63 0.54 – Distolasterias nipon, Coscinasterias acutispina, and b 0.67 0.35 0.41 0.54 0.37 0.46 1.00 Plazaster borealis) of the family Asteriidae were in the range c 0.33 – – 0.17 – – – of 5.9–16.7%, with a mean of 9.0%. The present H values Sod-1 a – 0.40 – – 0.71 0.14 – were comparable to those of the five asteroids. On the b 1.00 0.60 1.00 1.00 0.29 0.86 1.00 other hand, the mean H value of 19 echinoid species re- Sod-2 a 0.83 0.37 0.05 0.08 0.31 0.12 0.12 b 0.17 0.63 0.95 0.92 0.69 0.88 0.88 ported previously was 3.4% (Matsuoka, 1987, 1989; Po-2 a 0.14 0.33 0.17 – – – – Matsuoka and Suzuki, 1989a; Matsuoka and Nakamura, b 0.86 0.67 0.83 1.00 1.00 1.00 1.00 1991). Namely, the asteroids showed the higher genetic Alk-1 a – 0.42 0.08 – 0.31 0.42 0.93 variability than the echinoids. With respect to the main- b 0.94 – 0.92 0.75 – – – c – 0.58 – 0.25 0.63 0.45 – tenance mechanism of genetic variability in populations, d 0.06 – – – 0.06 0.13 0.07 Nei (1983) and Nei and Graur (1984) examined the rela- Alk-3 a – 0.10 0.05 0.08 – 0.04 0.04 tionship between average heterozygosity and population b 1.00 0.90 0.95 0.92 1.00 0.81 0.96 size for 77 different species. As a result, they found a c – – – – – 0.15 – significant correlation between them. From their findings, Est-2 a 1.00 0.11 – 0.42 – – – b – 0.89 1.00 0.58 1.00 –– it may safely be considered that the difference in the ex- Est-3 a 1.00 0.70 0.64 1.00 0.12 0.83 1.00 tent of genetic variation in echinoderms verified by our se- b – 0.30 0.36 – 0.88 0.17 – rial allozyme studies is closely related to their population Est-4 a – – – – – – 0.02 sizes. Namely, most of the echinoids reported previously b – 0.17 0.19 0.08 0.42 0.31 0.27 c 1.00 0.83 0.81 0.92 0.58 0.69 0.71 are shallow water species, and thus the population size Lap-1 a – 0.30 – – 0.31 – 0.12 would be smaller than deep-sea echinoderms. On the other b 1.00 0.70 1.00 1.00 0.69 1.00 0.88 hand, many of the asteroids are widely distributed from Lap-2 a – 1.00 0.63 1.00 0.36 0.06 – shallow water to deep-sea water. In paticular, A. amurensis b 1.00 – 0.37 – 0.64 0.06 – is a common species which is widely distributed from the c – – – – – 0.88 1.00 North Pacific regions to Japanese waters. Thus, it may Alleles are correspondingly lettered from “a”, this being the be concluded that the asteroids including A. amurensis have allele of lowest electrophoretic mobility. A hyphen indicates the larger population sizes, and thus, can maintain higher zero frequency. Akke = Akkeshi, Asa = Asamushi, Oga = Oga, Miya = Miyako, Misa = Misaki, Ise = Ise, Ushi = Ushimado. genetic variability within population than the echinoids

Table 2. Genetic variation in seven local Japanese populations of the starfish Asterias amurensis

Parameter Akke Asa Oga Miya Misa Ise Ushi No. of alleles per locus 01.16 01.48 01.36 01.32 01.40 01.50 01.29 Proportion of polymorphic loci: P (%) 16.00 44.00 36.00 28.00 36.00 37.50 25.00 Expected average heterozygosity per locus: H (%) 4.3 16.40 10.50 9.1 14.00 11.80 4.5

Akke = Akkeshi, Asa = Asamushi, Oga = Oga, Miya = Miyako, Misa = Misaki, Ise = Ise, Ushi = Ushimado. 62 N. MATSUOKA and T. HATANAKA

from shallow water. Further extensive population surveys cline of allele frequency at some loci. However, the present in various marine invertebrates would be required for es- starfish populations did not show such a cline, and the fre- tablishing the validity of this prediction. quencies of the alleles described above remarkably differed With respect to the relationship between enzyme func- between populations. The difference of allelic composition tion and heterozygosity, Yamazaki (1977) showed, using verified by this study shows that the gene flow is remark- data from various Drosophila species, that the substrate ably inhibited between Akkeshi and others and between specific enzymes have lower heterozygosity than non-spe- Ushimado-Ise and others. This is a strong evidence for cific enzymes. Similar analysis was carried out by Gojobori that they are much genetically differentiated to one an- (1982) using data on 20 different proteins (mostly enzymes) other. from 14 Drosophila species, 14 Anolis species, and 31 other To quantify the degree of genetic differentiation among species. As a result, he found that enzymes with various seven populations of A. amurensis, the genetic identity (I) functional constraints tend to have low heterozygosity. and genetic distance (D) between each population were cal- This holds true for the asteroid enzymes studied here; the culated by the method of Nei (1972) from the allele fre- glucose metabolizing enzymes (the mean H = 2.7%) with quency data in Table 1. Table 3 shows the matrices of I high functional constraints were less variable than the non- and D values between all pairs of the seven populations. glucose metabolizing enzymes (the mean H = 12.5%) con- The D values ranged from 0.027 to 0.190. taining the non-specific enzymes. The similar results have To clarify their genetic differentiation and relationships, also been obtained in many other echinoderm species re- the biochemical dendrogram for seven populations was con- ported previously (Matsuoka, 1987; Matsuoka and Suzuki, structed from the Nei’s genetic distance matrix of Table 3 1989a; Matsuoka and Hatanaka, 1991; Matsuoka et al., by using the UPGMA clustering method of Sneath and 1994). These result can be explained by the neutral theory Sokal (1973). The dendrogram (Fig. 2) indicated the fol- of Kimura (1983). The more strictly functional constraint lowings: would decrease the neutral regions of the molecules and (a) The Akkeshi population is most genetically differ- the probability of a neutral mutation is smaller for the sub- entiated among the seven populations. The mean genetic strate specific enzymes than for nonspecific enzymes. distance between Akkeshi population and the cluster of other six populations was 0.147. Genetic differentiation between populations. The (b) The Ushimado and Ise populations are also geneti- allele frequencies data (Table 1) at 25 genetic loci show that cally differentiated from the cluster of other (Miyako-Oga- the allelic composition in Akkeshi population remarkably Asamushi-Misaki) populations. The mean genetic dis- differs from that in other populations. Namely, the allele tance between the two clusters was 0.132. c of Mdh, b of Est-2, b of Est-4, a of Xdh, a of Alk-1, a of Alk- (c) The Miyako, Oga, Asamushi, and Misaki populations 3, and a of Lap-2 were not observed in Akkeshi population, are closely related to one another and the genetic distances while most populations in Honshu shared these alleles. between them were lower, in the range of 0.027–0.080, with Furthermore, the allele frequencies at the three genetic loci a mean of 0.053. (Sod-2, Est-2, and Lap-2) were remarkably different be- In the previous papers, we reported on the D values be- tween Akkeshi population and other populations. In ad- tween conspecific local Japanese populations of some echi- dition, Ushimado and Ise populations also differ from other noids belonging to the same Echinodermata (Matsuoka and populations in the high frequency of the allele c at Lap-2 Suzuki, 1989b; Matsuoka, 1989). According to them, the that was not observed in other populations. Generally, D values between conspecific local populations were in the conspecific local populations tend to show a geographical range of 0.008–0.069 (the mean D = 0.038) for the six local

Table 3. Genetic identities (above diagonal) and genetic distances (below diago- nal) between seven local Japanese populations of the starfish Asterias amurensis Populations 1234567 1. Akkeshi – 0.848 0.894 0.910 0.827 0.854 0.849 2. Asamushi 0.165 – 0.946 0.947 0.950 0.900 0.863 3. Oga 0.112 0.056 – 0.973 0.920 0.895 0.866 4. Miyako 0.094 0.054 0.027 – 0.880 0.902 0.881 5. Misaki 0.190 0.051 0.083 0.128 – 0.879 0.828 6. Ise 0.158 0.105 0.111 0.103 0.129 – 0.973 7. Ushimado 0.164 0.147 0.144 0.127 0.189 0.027 –

Genetic identity (I) and genetic distance (D) were calculated by the method of Nei (1972). Geographical divergence of starfish 63

populations was 0.492. Further, the value between (Ushimado-Ise) and (Miyako-Oga-Asamushi-Misaki) popu- lations was 0.388. These values was much higher than those obtained between conspecific local populations in various animals reported previously (Nei, 1987). For ex-

ample, the GST value between four populations of horse- shoe crab of marine invertebrate was 0.072. All of these allozyme data indicate that Akkeshi, Ushimado-Ise, and other four populations are much geneti- cally differentiated to one another, and thus, it may be con- cluded that A. amurensis from Japanese waters consists of at least three groups that are largely genetically divergent at the subspecies or sibling species level. The present molecular results are strongly supported by Fig. 2. A biochemical dendrogram showing the genetic relation- the morphological studies. S. Saba (personal communica- ships among seven local Japanese populations of Asterias tion) found that Akkeshi population is much different from amurensis. It was constructed from Nei’s genetic distances by other populations in the following morphological charac- using the UPGMA clustering method. ters; the number of superomarginal spines, formula of adambulacral spines in the proximal of mouth, rows of intermarginal spines, and the number of dorsal spines out- populations of Anthocidaris crassispina, 0.001–0.007 (the lining papular areas. Further, Ushimado and Ise popula- mean D = 0.004) for three local populations of Diadema tions have deeper blue or purple body and more sharp dor- setosum, and 0.015 for two local populations of Echinothrix sal spines than other local populations. Based on the dif- calamaris. In the asteroids, Nishida and Lucas (1988) ference in such several morphological characters, Hayashi examined electrophoretically the degree of genetic differ- (1974) classified the population from Inland Sea (Ushimado entiation among 10 local populations of the tropical star- population in this study corresponds to the population) as fish Acanthaster planci from the Pacific region. As a re- the subspecies of A. amurensis versicolor. Furthermore, sult, the D values between them were in the range of 0– S. Saba also referred the population from Ise Bay to A. 0.098 (the mean D = 0.029). The present mean D value amurensis versicolor (personal communication). The (0.147) between Akkeshi population and the cluster of other subspecies system of Hayashi (1974) and S. Saba that six populations and that (D = 0.132) between the cluster of Ushimado and Ise populations of A. amurensis should be (Ise-Ushimado) and the cluster of (Miyako-Oga-Asamushi- classified as the subspecies, A. amurensis versicolor is well Misaki) were considerably higher than those between con- consistent with the present allozyme study. Although the specific local populations of other echinoderms. As de- subspecies system is not generally adopted by asteroid tax- scribed in introduction, we previously demonstrated by onomists at present, we propose that their subspecies sys- allozyme electrophoresis that there exist four sibling spe- tem should be accepted, based on both molecular and mor- cies within the tropical common sea-urchin Echinometra phological studies. Furthermore, in the future, Akkeshi mathaei (Matsuoka and Hatanaka, 1991). The D values population may also be classified as subspecies or different between four sibling species of E. mathaei were in the range species from molecular data, morphological evidence, hy- of 0.105–0.322 (the mean D = 0.184). The present D val- bridization test (this experiment may be very difficult be- ues described above were comparable to those obtained be- cause of different breeding seasons between local popula- tween four different or four sibling species of E. mathaei. tions of the starfish) and so on, and we expect that the new The similar D value was also observed between the two species or subspecies name would be given to Akkeshi popu- morphologically similar starfish, Acanthaster planci and A. lation. brevispinus (Nishida and Lucas, 1988). Generally, such Of the seven local populations studied here, Misaki popu- level of D values has been found between subspecies and lation has yellow body, in contrast with the blue or purple incipient species or very closely related species in many body of other populations. The starfish with yellow body other animal groups (Ayala, 1982; Nei, 1987). are also found in Tokyo Bay and the near regions. It has To confirm the genetic differentiation between popula- often been noticed by many marine biologists, and some tions of A. amurensis, we calculated the coefficient of gene workers suggested that the starfish with yellow body may differentiation (GST) which is another parameter of be different species from that with blue or purple body. interpopulational genetic differentiation by the method of However, the present molecular results clearly showed that

Nei (1987). As a result, the GST value between Akkeshi the degree of genetic differentiation between Misaki popu- and (Miyako-Oga-Asamushi-Misaki) populations was lation with the yellow body and Asamushi population with 0.377. The value between Akkeshi and (Ushimado-Ise) blue or purple body is considerably lower (D = 0.051), and 64 N. MATSUOKA and T. HATANAKA

furthermore, that between the cluster of (Misaki-Asamushi) species of the family Diadematidae from Japanese waters. and the cluster of (Oga-Miyako) populations with blue or Biochem. Syst. Ecol. 17, 423–429. Matsuoka, N. (1990) Evolutionary relationships of sea-urchins at purple body is also lower (the mean D = 0.080), and thus, the molecular level. Comp. Biochem. Physiol. 97B, 31–36. they are conspecific local populations and the yellow body Matsuoka, N. (1993) Molecular studies on the phylogeny and evo- of the starfish is only intraspecific variation, though it is lution of echinoderms. Trends Comparat. Biochem. Physiol. unclear why the starfish from Misaki or Tokyo Bay has yel- 1, 155–169. low body. Matsuoka, N., Fukuda, K., Yoshida, K., Sugawara, M., and Inamori, M. (1994) Biochemical systematics of five asteroids of the fam- A. amurensis is widely distributed from the North Pa- ily Asteriidae based on allozyme variation. Zool. Sci. (Tokyo) cific regions to Japanese waters. From the geographical 11, 343–349. distribution and the first divergence of the Akkeshi popu- Matsuoka, N., and Hatanaka, T. (1991) Molecular evidence for the lation from Hokkaido shown in the dendrogram (Fig. 2), it existence of four sibling species within the sea-urchin, may be speculated that A. amurensis groups originated in Echinometra mathaei in Japanese waters and their evolution- ary relationships. Zool. Sci. (Tokyo) 8, 121–133. the North Pacific regions. Matsuoka, N., and Nakamura, Y. (1991) Genetic distance and pro- Putting the present allozyme study and morphological tein polymorphism in two sea-urchin species of the order evidence together, it is concluded that A. amurensis from Arbacioida and implications for their evolution. Comp. Japanese waters consists of at least three groups that are Biochem. Physiol. 98B, 21–27. largely genetically divergent at subspecies or sibling spe- Matsuoka, N., and Suzuki, H. (1989a) Electrophoretic study on the phylogenetic relationships among six species of sea-urchins cies level. of the family Echinometridae found in the Japanese waters. Zool. Sci. (Tokyo) 6, 589–598. We thank S. Kubota, Hokkaido University, M. Isozaki, Ushimado Matsuoka, N., and Suzuki, H. (1989b) Genetic variation and dif- Marine Biological Station, Okayama University, S. Saba, Ise High ferentiation in six local Japanese populations of the sea- School, T. Oshida, Hirosaki University, the staff of Asamushi Ma- urchin, Anthocidaris crassispina: Electrophoretic analysis of rine Biological Station, Tohoku University, the staff of Misaki allozymes. Comp. Biochem. Physiol. 92B, 1–7. Marine Biological Station, University of Tokyo and Oga Aquarium Matsuoka, N., Yoshida, K., Fukuda, K., and Shigei, M. (1991) Ge- for their kind help in collecting starfish specimens. We also netic variation in the starfish Coscinasterias acutispina. thank S. Saba for his valuable advice. Comp. Biochem. 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