*Corresponding author. Tel.:#7-4232-310905; Fax:#7-4232-310900.
E-mail address:[email protected] (G.P. Manchenko).
Genetic divergence between three sea urchin
species of the genus
Strongylocentrotus
from the Sea of Japan
Gennady P. Manchenko
*
, Sergei N. Yakovlev
Institute of Marine Biology, Vladivostok 690041, Russia
Received 13 August 1999; accepted 28 February 2000
Abstract
Intraspeci"c allozymic variation and interspeci"c genetic divergence were studied in three sea urchin species of the genusStrongylocentrotus (S. intermedius, S. nudus, S. pallidus)from the Sea of Japan.S. pallidusandS. intermediusshowed high mean values of expected heterozygosity, H
e"0.223$0.072 (17 loci) andHe"0.230$0.065 (19 loci), respectively. This estimate was somewhat lower inS. nudus,H
e"0.126$0.043 (17 loci). Estimates of Nei's genetic distance betweenS. nudus/S. intermedius(D"1.578, 17 loci) andS. nudus/S. pallidus(D"1.327, 15 loci) were considerably higher than that between S. intermedius/S. pallidus (D"0.269, 17 loci). Invoking the protein clock hypothesis and using Panamanian geminate sea urchins for protein clock calibration, the time of divergence betweenS. intermediusandS. palliduswas estimated as 2.7 MY. The results obtained forS. intermediusandS. nudusby us di!er considerably from results obtained for these species by Norimasa Matsuoka and coworkers. The revealed discrepancies are discussed and the conclusion made that Matsuoka and coworkers'data on echinoderm biochemical genetics and systematics should be used with caution. ( 2000 Else-vier Science Ltd. All rights reserved.
Keywords: Strongylocentrotus intermedius;Strongylocentrotus nudus;Strongylocentrotus pallidus; Echinoidea; Sea urchins; Allozymic variation; Genetic distance
1. Introduction
Taxonomy and phylogeny of echinoids is mainly based on data obtained using methods of comparative morphology and paleontology. Because evolutionary change
of morphological characters is known to be subject to convergent processes and because speciation is not always accompanied by clear-cut morphological di!erences, some problems persists in classi"cation and phylogeny of echinoids (Jensen, 1981). Some species of regular sea urchins of the genusStrongylocentrotusdemonstrate high level of intraspeci"c morphological variation which, in some cases, overlaps inter-speci"cally and generates problems in unequivocal identi"cation of and discrimina-tion between species of the genus (Jensen, 1974). As a consequence, the number of species and subspecies of Strongylocentrotus described at di!erent times from sea waters surrounding Russia varied from 12 to 6 and was recently suggested to be not more than 5 (Bazhin, 1998). Three species of the genus are known to be present in the Vladivostok area of the Sea of Japan, Russia. These are:S. nudus(Agassiz)*endemic to the Sea of Japan,S. intermedius(Agassiz) *occurs in the Sea of Japan and the Okhotsk Sea, andS. pallidus(Sars)*widely distributed in the Northern Hemisphere and thought to be of circumpolar distribution (Bazhin, 1998). Genetic divergence and phylogenetic relationships between these sea urchin species remain mainly uncertain. During the last three decades enzyme electrophoresis remained one of the most practical tools in taxonomic and phylogenetic studies (Murphy et al., 1996). It was successfully used to evaluate interspeci"c genetic divergence and phylogenetic rela-tionships in a variety of marine invertebrate groups including echinoids (see for example Thorpe and SoleH-Cava, 1994). There are a multitude of techniques presently available for examining genetic variation within and between species directly at the DNA level (Avise, 1994). Despite of virtually unlimited power and sensitivity of DNA techniques, they still remain more di$cult, costly, and time consuming than enzyme electrophoresis (Allendorf, 1994). Allozyme electrophoresis allows sampling on a spa-tial and temporal scale not practical yet for DNA (Edmands et al., 1996). Intraspeci"c genetic variation and divergence as well as interspeci"c genetic divergence of sea urchins of the genusStrongylocentrotus were studied using both DNA and enzyme electrophoresis techniques.
The use of isozymes to study genetic divergence and variation in sea urchins of the genusStrongylocentrotusis limited to two works from the Vladivostok area, Russia (Pudovkin et al., 1984; Manchenko, 1985a), two works from the North Japan (Matsuoka, 1987; Matsuoka et al., 1995) and one work from the eastern North Paci"c (Stickle et al., 1990). Matsuoka (1987) studied genetic divergence betweenS. inter-medius and S. nudus from the shore of Japan and found that Nei's genetic distance between these species is of the same magnitude as for many other animal congeneric species (Thorpe, 1983; Nei, 1987). From the obtained results Matsuoka also concluded that H. pulcherrimus is very closely related to the both species ofStrongylocentrotusand is rather a member of this same genus. Another pair of species ofStrongylocentrotus,S. pallidusandS. droebachiensis, which are capable of restricted hybridization (Strathmann, 1981; Roller and Stickle, 1985), demonstrated no pronounced genetic di!erence at all (Stickle et al., 1990). Allozyme variation was studied in S. intermedius and S. nudus using starch gel (Pudovkin et al., 1984; Manchenko, 1985a) and polyacrylamide gel (Matsuoka, 1987; Matsuoka et al., 1995) electrophoresis and the obtained results are controversial. Nothing is known about genetic divergence betweenS. pallidusandS. intermediusas well as betweenS. pallidus andS. nudus.
This work presents enzyme electrophoretic estimates of intraspeci"c genetic vari-ation and interspeci"c genetic divergence in three sea urchin species of the genus Strongylocentrotus (S. pallidus, S. intermedius and S. nudus) from the Sea of Japan (Vladivostok area, Russia). Some discrepancies between our and Matsuoka and coworkers'electrophoretic data are discussed.
2. Materials and methods
The sea urchins used in this study wereStrongylocentrotus intermedius, S. nudusand S. pallidusfrom the Sea of Japan. Samples of the"rst two species were collected from the depth of 3 m in the Vostok Bay near Nakhodka (132345AE; 42353AN) and immediately used for electrophoresis. The sample ofS. pallidus (from the depth of about 100 m) was collected from the locality situated about 300 km to the north from Nakhodka (135355AE; 44330AN) and stored frozen at !183C for a month before electrophoresis.
Lantern muscle and gut tissues were homogenized in two volumes of distilled water and crude enzyme-containing homogenates analyzed by horizontal 14% starch-gel electrophoresis as previously described (Manchenko, 1985a). Three continuous bu!er systems were used: TC (tris-citric acid, pH 7.0), TEB (tris-EDTA}boric acid, pH 8.5), and TM (tris-maleate, pH 7.4). The staining of electrophoretic gels followed standard procedures, using recipes from Manchenko (1994). Fifteen enzymes proved scorable
and were used in this survey. Enzymes assayed, bu!ers and tissues used for
Table 1
Enzymes (enzyme names and EC numbers given according to IUBMB NC, 1992), isozyme loci, tissue sources (G, gut; M, muscle), and bu!er systems used in enzyme electrophoretic survey of sea urchins Enzyme name (Abbreviation; EC number) Isozyme locus Tissue source Bu!er system!
Adenylate kinase (AK; EC 2.7.4.3) Ak M TM
Arginine kinase (ARGK; EC 2.7.3.3) Argk G, M TEB
Aspartate transaminase (ATA; EC 2.6.1.1) Ata-1,-2 G#M TC
Creatine kinase (CK; EC 2.7.3.2) Ck M TEB
Cytosol non-speci"c dipeptidase (PEP; EC 3.4.13.18), gly}leu as substrate
Pep G#M TEB
Phosphoglucomutase (PGM; EC 5.4.2.2) Pgm-1 G TEB
Pgm-2 G#M TEB
Superoxide dismutase (SOD; EC 1.15.1.1) Sod-1 M TEB
Sod-2 G TEB
!Continuous bu!er systems: TM, tris-maleate (pH 7.4); TEB, tris-EDTA}boric acid (pH 8.5); TC, tris-citric acid (pH 7.0).
dipeptides as substrates) but their patterns proved not suitable for comparison because of poor resolution or low staining intensity at least in two of the three sea urchin species studied. These enzymes were not used in our further electrophoretic analysis. Genetic interpretations of banding patterns developed on the stained gels (zymograms) were made according to Buth (1990) taking into account our previous results (Manchenko, 1985a).
The program BIOSYS-1 (Swo!ord and Selander, 1981) was used to compute allele frequencies and mean estimates of observed (H
o) and expected (He) heterozygosity as well as Nei's (1978) unbiased genetic identity (I) and genetic distance (D) coe$cients.
3. Results
arginine kinase (Argk) and creatine kinase (Ck) proved scorable only inS. intermedius and S. nudus, while loci coding for formaldehyde dehydrogenase (Fdh) and malic enzyme (Me) were studied only inS. intermediusandS. pallidus.
Two loci,Ak and Sod-2, were found to be monomorphic in all the three species studied. Allozyme variations with one-banded homozygotes and two-banded hetero-zygotes were revealed, at least in one of the studied species, atMpi, Pgm-1 and Pgm-2 loci providing evidence for the monomeric subunit structure of corresponding enzyme molecules. Besides monomorphic principal ARGK isozyme (presumably dimeric) we detected an additional ARGK isozyme in lantern muscle preparations ofS. inter-medius. This isozyme demonstrated allozyme variation characteristic of monomeric enzymes (i.e. heterozygotes were two-banded). It was detected only inS. intermedius and was therefore not involved in our further consideration. It should be stressed, however, that this is the "rst "nding of unusual monomeric ARGK in echinoids. Three-banded allozyme patterns characteristic of dimeric enzymes were observed in individuals heterozygous for Argk (principal), Ata-1, Ata-2, Fdh, Gpi, Idh, Mdh-1, Mdh-2, and Sod-2loci. Allozyme variants were also observed inMe, Muad, Pep, and Pgdhloci with heterozygotes showing broad di!use bands which were not resolved into separate allozymes under the used electrophoretic conditions.
The allele frequencies at isozyme loci studied in the threeStrongylocentrotusspecies are given in Table 2. Based on these data estimates of mean observed (H
o) and expected (H
e) heterozygosities per locus and the percentage (P0.95) of polymorphic loci per species were calculated and are given in Table 3.H
eis known to be the best single estimator for comparing genetic variation between species. The studied species
demonstrate high level of allozyme variation: S. intermedius (19 loci, H
e" 0.230$0.065), S. pallidus (17 loci, H
e"0.223$0.072), and S. nudus (17 loci, H
eGenetic identity ("0.126$0.043).I) and genetic distance (D) coe
$cients calculated from allele frequency data are given in Table 4. The values of genetic distance estimate obtained forS. nudus/S. intermedius(17 loci,D"1.578) andS. nudus/S. pallidus(15 loci,D"
1.327) species pairs are considerably higher than that forS. intermedius/S. pallidus(17 loci,D"0.269).
4. Discussion
The sea urchin species studied demonstrate high level of intraspeci"c genetic variation. Species with a wide geographic distribution,S. pallidusandS. intermedius, are the most variable. Very similar level of intraspeci"c variation was revealed in the results of allozyme electrophoretic survey of S. californianus, which is distributed along the shore of North America from British Columbia to Mexico (Edmands et al., 1996). Genetic variation inS. nudus, which is an endemic of the Sea of Japan, is about two times lower. These di!erences, however, are statistically not signi"cant because of high standard error values ofH
Table 2
Allele frequencies for isozyme loci studied in the three sea urchin species of the genusStrongylocentrotus. Number of individuals,N
Locus Species
Allele S. nudus S. intermedius S. pallidus
Loci studied in all the three species:
Ak
N 9 10 16
a 0.000 0.000 1.000
b 1.000 1.000 0.000
Ata-1
N 10 10 10
a 0.050 1.000 1.000
b 0.950 0.000 0.000
Ata-2
N 10 10 11
a 0.000 0.100 0.318
b 0.000 0.850 0.591
c 1.000 0.050 0.091
Gpi
N 20 19 21
a 0.000 0.079 0.024
b 0.925 0.921 0.976
c 0.025 0.000 0.000
d 0.050 0.000 0.000
Idh
N 10 10 21
a 0.950 0.000 0.000
b 0.000 0.150 0.119
c 0.050 0.800 0.881
d 0.000 0.050 0.000
Mdh-1
N 20 36 81
a 0.000 0.958 0.969
b 0.000 0.042 0.006
c 0.000 0.000 0.025
d 0.875 0.000 0.000
e 0.125 0.000 0.000
Mdh-2
N 20 36 81
a 1.000 0.014 0.000
b 0.000 0.986 0.290
c 0.000 0.000 0.580
d 0.000 0.000 0.130
Mpi
N 10 10 21
a 0.000 0.050 0.000
b 0.000 0.100 0.000
c 0.000 0.150 0.000
Table 2*continued
Locus Species
Allele S. nudus S. intermedius S. pallidus
e 0.000 0.250 0.048
f 0.000 0.100 0.262
g 0.000 0.150 0.071
h 0.050 0.000 0.000
I 0.000 0.050 0.381
j 0.000 0.050 0.143
k 0.950 0.000 0.000
l 0.000 0.000 0.048
m 0.000 0.000 0.024
Muad
N 20 19 43
a 0.000 0.000 0.012
b 0.000 0.974 0.953
c 0.000 0.026 0.035
d 1.000 0.000 0.000
Pep
N 10 9 22
a 0.000 1.000 1.000
b 0.400 0.000 0.000
c 0.600 0.000 0.000
Pgdh
N 10 10 16
a 0.000 0.050 0.000
b 1.000 0.150 1.000
c 0.000 0.800 0.000
Pgm-1
N 10 10 10
a 0.950 0.000 0.000
b 0.050 1.000 1.000
Pgm-2
N 20 19 42
a 0.000 0.000 0.083
b 0.050 0.079 0.429
c 0.425 0.289 0.286
d 0.525 0.316 0.119
e 0.000 0.132 0.036
f 0.000 0.053 0.048
g 0.000 0.132 0.000
Sod-1
N 20 19 43
a 0.000 1.000 1.000
b 1.000 0.000 0.000
Sod-2
N 20 36 81
a 1.000 0.250 0.981
b 0.000 0.736 0.019
Table 3
Mean estimates of observed (H
o) and expected (He; Nei, 1978) heterozygosities and percentage of loci
polymorphic (P
0.95) in the threeStrongylocentrotusspecies studied. A locus was considered polymorphic if the frequency of the most common allele did not exceed 0.95
Species No. of loci per species
Mean number of individuals per locus
P
0.95 Mean heterozygosity ($SE)
H
o He
S. nudus 17 17.6 52.9 0.128 ($0.043) 0.126 ($0.043)
S. intermedius 19 18.7 47.4 0.237 ($0.067) 0.230 ($0.065)
S. pallidus 17 33.1 35.3 0.238 ($0.078) 0.223 ($0.072)
Table 4
Matrix of Nei's (1978) genetic identity (above the diagonal) and genetic distance (below diagonal) unbiased coe$cients between pairs of the threeStrongylocentrotusspecies studied. Calculations are based on the allele frequency data presented in Table 2. Numbers of loci involved in the comparison are given in parentheses
Species S. nudus S. intermedius S. pallidus
S. nudus * 0.206 (17) 0.265 (15)
S. intermedius 1.578 (17) * 0.764 (17)
S. pallidus 1.327 (15) 0.269 (17) *
Table 2*continued
Locus Species
Allele S. nudus S. intermedius S. pallidus
Loci studied only in two species: Argk
N 40 39 42
a 0.813 0.000 not detected
b 0.188 1.000 *
Ck
N 40 39 42
a 1.000 0.000 not detected
b 0.000 1.000 *
Fdh
N 0 17 21
a not studied 0.000 0.048
b * 0.059 0.167
c * 0.706 0.476
d * 0.235 0.000
e * 0.000 0.310
Mdhp
N 0 17 22
a not studied 0.382 0.000
b * 0.588 0.977
Table 5
Mean expected heterozygosity estimates (H
e) obtained for two sea urchin and two sea star species from the
Sea of Japan studied electrophoretically by di!erent authors
Species H
e$S.E. No. of locistudies per
species
No. of individual studied per locus (locality)!
References
Sea urchins
Strongylocentrotus intermedius
0.064$0.035 21 12 (MB) Matsuoka (1987)
0.230$0.065 19 19 (VB) Present study
0.115$0.030 41 36 (VB) Manchenko (1985a)
Strongylocentrotus nudus 0.026$0.020 20 12 (MB) Matsuoka (1987)
0.059$0.027 33 17 (MB) Matsuoka et al. (1995)
0.067$0.028 33 21 (FK) Matsuoka et al. (1995)
0.126$0.043 17 18 (VB) Present study
Sea stars
Asterias amurensis 0.192$0.034 36 27 (VB) Manchenko (1986)
0.076$0.032 29 20 (MB) Matsuoka et al. (1994)
0.216$0.055 22 47 (MB) Ward (1994)
0.213$0.058 22 46 (PI) Ward (1994)
Asterina pectinifera 0.029$0.029 15 9-16 (FK) Matsuoka (1981)
0.168$0.039 23 241 (VB) Manchenko (1986)
0.106$0.041 23 28-30 (FK) Matsuoka et al. (1995) 0.123$0.041 23 28-30 (MB) Matsuoka et al. (1995)
!Abbreviated names of localities: MB*Mutsu Bay, Aomori Prefecture, Japan; FK*Fukaura, Aomori Prefecture, Japan; VB*Vostok Bay, Vladivostok area, Russia; PI*Popov Island, Vladivostok area, Russia.
The obtained results provide evidence thatS. pallidusandS. intermediusare closely related species,D"0.269.S. pallidusis known to be capable of hybridization withS. droebachiensis (Strathmann, 1981; Roller and Stickle, 1985). Using enzyme elec-trophoresis, it was shown that these species share the same alleles at isozyme loci compared and demonstrate very low interspeci"c allele frequency variance (Stickle et al., 1990). It may be concluded therefore that S. intermedius, S. pallidus and S. droebachiensis represent a group of closely related species. Lessios (1979) elec-trophoretically studied three pairs of geminate species of regular sea urchins of the
genera Echinometra, Eucidaris and Diadema separated by the Isthmus of Panama
betweenS. intermediusandS. nudus(D"1.578) and betweenS. nudusandS. pallidus (D"1.327) lines has occurred much earlier.
It should be stressed that values of H
e estimate obtained by Matsuoka and
coworkers forS. intermediusandS. nudusare several times lower than those obtained for these species by us (Table 5). The discrepancy between Matsuoka and coworkers' and our results cannot be readily explained by di!erent sets of enzymes used as gene markers by di!erent investigators. Indeed, monomeric enzymes are known to be more variable than oligomeric ones (Ward et al., 1992), however, the proportion of mono-mers in a set of enzymes used by us is not higher than that in a set of enzymes used by Matsuoka (1987). Because a high positive correlation exists between average hetero-zygosities and average distances of di!erent enzymes (Skibinski and Ward, 1981; Ward and Skibinski, 1985), it is not surprising that the genetic distance (D"0.464) betweenS. intermediusandS. nudusobtained by Matsuoka (1987) is also considerably lower than that obtained by us (D"1.578). Remarkably that the tendency to obtain
reduced H
e values is rather a common characteristic of works by Matsuoka and
coworkers. Some examples demonstrating this tendency are listed in Table 5 where H
evalues obtained for the same echinoderm species by Matsuoka and coworkers, by Manchenko, and by Ward are given. The resolving power of PAG electrophoresis used by Matsuoka and coworkers is at least not less as that of starch gel or cellogel electrophoresis methods used by Manchenko and by Ward, respectively (see for example Murphy et al., 1996). It is striking therefore that Matsuoka and coworkers commonly fail to reveal allozymic variations at obviously polymorphic isozyme loci. For example, Manchenko (1985a) revealed allozyme polymorphisms inS. intermedius at Hk (the per locus expected heterozygosity,h
e"0.496),Got-1 (he"0.121), Got-2 (h
e"0.219) andMdh-1(he"0.116) loci, while Matsuoka (1987) found these loci to be monomorphic. However, the most demonstrative examples are those concerning sea starsAsterina("Patiria) pectinifera andAsterias amurensisstudied independently by the three groups of investigators. Manchenko (1976) described 6 alleles atMdh-2locus (h
e"0.647) inA. pectiniferafrom the Vostok Bay of the Sea of Japan (Vladivostok area, Russia), while Matsuoka (1981) failed to detect any MDH variation in this sea star from the Fukaura, Aomori Prefecture (Japan). Manchenko (1986) reported allozymic polymorphisms atGpi(h
e"0.278),Hk-1(he"0.262),Hk-2(he"0.256) and Lap(h
e"0.214) loci in this species, while Matsuoka (1981) revealed no intraspeci"c variations of these enzymes. Ward (1994) has paid special attention thatH
e values obtained electrophoretically by Matsuoka et al. (1994) forA. amurensisare signi" -cantly lower than that obtained by him (Table 5), although the same population was studied and some enzyme systems used were the same. For example, Ward (1994) found that Gpi and Hk loci are clearly polymorphic in A. amurensis sample from Mutsu Bay where these loci were reported by Matsuoka et al. (1994) to be monomor-phic. Ward concluded that`2the reduced average heterozygosity found by
Mat-suoka et al. can be attributed not only to a di!erent suite of loci, but also to electrophoresis systems which failed to resolve some variationa. However, it is di$ -cult, if possible at all, to explain the failure to detect polymorphisms, like those atGpi (h
1986) used electrophoresis system quite di!erent from those used by Matsuoka and Ward, however, revealed allozymic polymorphisms at both these loci (Gpi,h
e"0.340; Hk,h
e"0.431) inA. amurensissample from the Vostok Bay of the Sea of Japan. The
same geographic population of A. amurensis was studied independently by
Man-chenko (unpublished data) and by Ward (1994) using starch gel and cellogel elec-trophoresis systems, respectively. Although electrophoretic bu!er systems used by these authors were also di!erent, they obtained quite comparable values of expected heterozygosity forGpi(0.332 and 0.200) and Hk (0.439 and 0.394) loci, respectively. Thus, the results obtained by Matsuoka and coworkers demonstrate the well-ex-pressed tendency of the per locus heterozygosity values to be diminished. The failure of these authors to reveal allozyme polymorphisms at some isozyme loci cannot be attributed to insu$cient resolving power of the used PAG electrophoretic method.
One can suggest that at least in some cases (e.g., when geographically distinct populations are studied) the observed di!erences in allozyme variations can be explained by high level of genetic divergence between conspeci"c populations studied by di!erent authors. We believe, however, that this is not a case for echinoderm species listed in Table 5 all of which have long-lived planktonic larvae capable of wide dispersal. The maximum time from fertilization to settling reported for larvae ofS. intermediusreared in the laboratory is 30 days (Naidenko, 1983). Similar, and even higher, time estimates are characteristic ofA. amurensis(S.S. Dautov, pers. commun.) and ofS. nudusandA. pectinifera(S.N. Yakovlev, unpublished data) larvae at natural conditions. Such species commonly demonstrate low genetic divergence among geo-graphic populations (for review see Palumbi, 1992, 1994; Avise, 1994). This is sup-ported by results of population genetic study ofA. pectinifera(Pudovkin et al., 1981) that demonstrated no signi"cant genetic divergence between geographic populations of this species separated by more than 750 km. Very similar results were obtained for geographic populationsS. intermedius andA. amurensis (G.P. Manchenko, unpub-lished data). It was shown that geographic patterns of population genetic structure in echinoderm species with planktonic larvae begin to be expressed only on geographic scales much larger than the Sea of Japan (Palumbi et al., 1997). We think therefore that the presence in the Sea of Japan of isolated populations of the considered echinoderm species with drastically low genetic variability at the considered isozyme loci is of very low probability.
We believe that inadequate genetic interpretation of electrophoretic variation detected on PAG zymograms is the most probable cause of the phenomenon in
question. PAG zymograms are known to have an important disadvantage* the
formation of secondary isozymes or subbands (Manchenko, 1994) which mask al-lozyme variations (Buth, 1990) and can therefore result in the underestimation of H
echinoderms including 12 sea star species, 9 sea urchin species, 5 holoturian species, and 2 sea lilian species (Manchenko, 1985a, b, 1986, 1989; Manchenko and Oleinik, 1985, present work). This is in a good agreement with data on the average per species number of MDH (1.75) and SOD (1.21) loci revealed in 342 and 258 invertebrate species, respectively, reviewed by Ward et al. (1992). The suggested inadequate genetic interpretation of electrophoretic variation by Matsuoka and coworkers is also sup-ported by several strikingly unusual (anomalous) allozyme polymorphisms described by these authors in echinoderms. For example, allozyme polymorphisms character-istic of monomeric enzymes (i.e. heterozygotes display two-banded phenotypes) were described in the sea urchinS. intermediusfor tetrazolium oxidase (recommended name superoxide dismutase, SOD) (Matsuoka, 1987), and in the sea starA. pectinifera for glutamic-oxaloacetic transaminase (recommended name aspartate transaminase, ATA) and for malate dehydrogenase (MDH) (Matsuoka et al., 1995). All these enzymes are known to be dimers in phylogenetically very distinct groups of living organisms (Manchenko, 1988). Three-banded heterozygote phenotypes of SOD were clearly resolved on starch gel zymograms in S. intermedius (Manchenko, 1985a). Similar heterozygote phenotypes of MDH and ATA were readily detected on starch gel zymograms inA. pectinifera(Manchenko, 1976; Manchenko and Priima, 1981).
In conclusion, we revealed high level of intraspeci"c allozyme variation in the three sea urchin species of the genusStrongylocentrotusfrom the Sea of Japan.S. intermedius and S. pallidus are genetically the most similar species (D"0.269), while S. nudus di!ers signi"cantly from the both S. intermedius (D"1.578) and S. pallidus (D"1.327). OurDestimate forS. intermedius/S. nudusspecies pair di!ers consider-ably from that obtained by Matsuoka (1987). Considerable di!erences are also characteristic ofH
e estimates obtained forS. intermediusandS. nudusby Matsuoka and coworkers and by us. The analysis of electrophoretic data obtained by Matsuoka and coworkers and the comparison of these data with those obtained by other authors, provide evidence that inadequate genetic interpretation of PAG zymograms in terms of number of gene loci and number of alleles at these loci is the most probable reason of the discrepancy between results obtained by Matsuoka and coworkers and by other authors. We recommend therefore to use critically the array of data on biochemical genetics and systematics of echinoderms published by these authors.
Acknowledgements
We wish to thank Alexander Pudovkin for critical reading draft manuscript and for valuable suggestions. This work was supported in part by a grant from the State Program in Science and Technology`Priority Frontiers of Geneticsa.
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