Directory UMM :Data Elmu:jurnal:B:Biochemical Systematics and Ecology:Vol29.Issue1.Jan2001:
Biochemical Systematics and Ecology 29 (2001) 31}44
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 genus Strongylocentrotus (S. intermedius, S. nudus, S. pallidus) from the Sea
of Japan. S. pallidus and S. intermedius showed high mean values of expected heterozygosity,
H "0.223$0.072 (17 loci) and H "0.230$0.065 (19 loci), respectively. This estimate was
e
e
somewhat lower in S. nudus, H "0.126$0.043 (17 loci). Estimates of Nei's genetic distance
e
between S. nudus/S. intermedius (D"1.578, 17 loci) and S. nudus/S. pallidus (D"1.327, 15 loci)
were considerably higher than that between S. intermedius/S. pallidus (D"0.269, 17 loci).
Invoking the protein clock hypothesis and using Panamanian geminate sea urchins for protein
clock calibration, the time of divergence between S. intermedius and S. pallidus was estimated as
2.7 MY. The results obtained for S. intermedius and S. nudus by us di!er considerably from
results obtained for these species by Norimasa Matsuoka and coworkers. The revealed
discrepancies are discussed and the conclusion made that Matsuoka and coworkers' data on
echinoderm biochemical genetics and systematics should be used with caution. ( 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Strongylocentrotus intermedius; Strongylocentrotus nudus; Strongylocentrotus pallidus; Echinoidea;
Sea urchins; Allozymic variation; Genetic distance
1. Introduction
Taxonomy and phylogeny of echinoids is mainly based on data obtained using
methods of comparative morphology and paleontology. Because evolutionary change
* Corresponding author. Tel.: #7-4232-310905; Fax: #7-4232-310900.
E-mail address: [email protected] (G.P. Manchenko).
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 2 7 - 2
32
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
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 genus Strongylocentrotus demonstrate high
level of intraspeci"c morphological variation which, in some cases, overlaps interspeci"cally and generates problems in unequivocal identi"cation of and discrimination 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, and S. 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 relationships 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 spatial 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 genus Strongylocentrotus were studied using both DNA and enzyme
electrophoresis techniques.
The use of nuclear and mitochondrial DNA sequences to study evolutionary
relationships between seven sea urchin species of the family Strongylocentrotidae has
unexpectedly failed to resolve the branching order among four members of the genus
Strongylocentrotus including S. purpuratus, S. polyacanthus, S. droebachiensis and S.
pallidus (Biermann, 1998). At the same time, the results obtained in the cited study
provided evidence that two other sea urchin species of the family, Allocentrotus fragilis
and Hemicentrotus pulcherrimus, fall phylogenetically within the genus Strongylocentrotus. Mitochondrial DNA variation was used to assess intraspeci"c genetic diversity
and spatial genetic di!erentiation of populations in sea urchin species of the genus
Strongylocentrotus (Vawter and Brown, 1986; Palumbi and Wilson, 1990; Palumbi and
Kessing, 1991; Palumbi and Metz, 1991; Palumbi, 1995). The only (to our knowledge)
work that successfully combined the use of isozyme and mtDNA gene markers in the
study of genetic divergence in S. purpuratus along the coast of California and North
Mexico is that by Edmands et al. (1996). The results obtained in this work proved in
a sharp contrast with results of previous work on S. purpuratus that failed to detect
any genetic di!erence between geographic populations of the species separated by
a distance of 1500 km (Palumbi and Wilson, 1990; Palumbi, 1995).
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
33
The use of isozymes to study genetic divergence and variation in sea urchins of the
genus Strongylocentrotus is 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 between S. intermedius 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 of Strongylocentrotus and is rather a member of this same genus. Another pair
of species of Strongylocentrotus, S. pallidus and S. 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 between S. pallidus and S. intermedius as well as between S. pallidus
and S. nudus.
This work presents enzyme electrophoretic estimates of intraspeci"c genetic variation 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 were Strongylocentrotus intermedius, S. nudus and
S. pallidus from 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 of S. 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
electrophoresis, and isozyme loci scored are listed in Table 1. In our pilot electrophoretic runs we also detected activity bands of nine additional enzymes (alanine
transaminase, alcohol dehydrogenase, catalase, hexokinase, lactoylglutathione lyase,
inorganic pyrophosphatase, octanol dehydrogenase, peptidases, phe-ala and leu-val
34
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
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)
Arginine kinase (ARGK; EC 2.7.3.3)
Aspartate transaminase (ATA; EC 2.6.1.1)
Creatine kinase (CK; EC 2.7.3.2)
Cytosol non-speci"c dipeptidase
(PEP; EC 3.4.13.18), gly}leu as substrate
Formaldehyde dehydrogenase
(FDH; EC 1.2.1.1)
Glucose-6-phosphate isomerase
(GPI; EC 5.3.1.9)
Isocitrate dehydrogenase
(NADP) (IDH; EC 1.1.1.42)
Malate dehydrogenase
(MDH; EC 1.1.1.37)
Malate dehydrogenase (NADP)
(MDHP; EC 1.1.1.40)
Mannose-6-phosphate isomerase
(MPI; EC 5.3.1.8)
Methylumbelliferyl-acetate deacetylase
(EST; EC 3.1.1.56)
Phosphogluconate dehydrogenase
(PGD; EC 1.1.1.44)
Phosphoglucomutase (PGM; EC 5.4.2.2)
Ak
Argk
Ata-1,-2
Ck
Pep
M
G, M
G#M
M
G#M
TM
TEB
TC
TEB
TEB
Fdh
G#M
TC
Gpi
G#M
TC
Idh
G
TC
Mdh-1,-2
G#M
TC
Mdhp
G#M
TC
Mpi
G
TEB
Muad
M
TEB
Pgdh
G#M
TM
Pgm-1
Pgm-2
Sod-1
Sod-2
G
G#M
M
G
TEB
TEB
TEB
TEB
Superoxide dismutase (SOD; EC 1.15.1.1)
!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 ) and expected (H ) heterozygosity as
o
e
well as Nei's (1978) unbiased genetic identity (I) and genetic distance (D) coe$cients.
3. Results
In total, 15 isozyme loci coding for 11 enzyme systems were resolved and proved
scorable in all the three sea urchin species studied. Additional gene loci coding for
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
35
arginine kinase (Argk) and creatine kinase (Ck) proved scorable only in S. intermedius
and S. nudus, while loci coding for formaldehyde dehydrogenase (Fdh) and malic
enzyme (Me) were studied only in S. intermedius and S. 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 heterozygotes were revealed, at least in one of the studied species, at Mpi, 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 of S. intermedius. This isozyme demonstrated allozyme variation characteristic of monomeric
enzymes (i.e. heterozygotes were two-banded). It was detected only in S. 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-2 loci. Allozyme variants were also observed in Me, Muad, Pep, and
Pgdh loci 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 three Strongylocentrotus species
are given in Table 2. Based on these data estimates of mean observed (H ) and
o
expected (H ) heterozygosities per locus and the percentage (P
) of polymorphic
e
0.95
loci per species were calculated and are given in Table 3. H is known to be the best
e
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 "0.223$0.072), and S. nudus (17 loci,
e
H "0.126$0.043).
e
Genetic identity (I) and genetic distance (D) coe$cients calculated from allele
frequency data are given in Table 4. The values of genetic distance estimate obtained
for S. nudus/S. intermedius (17 loci, D"1.578) and S. nudus/S. pallidus (15 loci, D"
1.327) species pairs are considerably higher than that for S. 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. pallidus and S. 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 in S. 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 of H estimate and may therefore be considered only as
e
di!erences demonstrating a general trend in the relationship between the level of
intraspeci"c genetic variability and the breadth of geographic distribution of species.
36
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
Table 2
Allele frequencies for isozyme loci studied in the three sea urchin species of the genus Strongylocentrotus.
Number of individuals, N
Locus
Species
Allele
S. nudus
S. intermedius
S. pallidus
Loci studied in all the three species:
Ak
N
9
a
b
0.000
1.000
a
b
0.050
0.950
a
b
c
0.000
0.000
1.000
a
b
c
d
0.000
0.925
0.025
0.050
a
b
c
d
0.950
0.000
0.050
0.000
a
b
c
d
e
0.000
0.000
0.000
0.875
0.125
a
b
c
d
1.000
0.000
0.000
0.000
a
b
c
d
0.000
0.000
0.000
0.000
10
0.000
1.000
16
1.000
0.000
Ata-1
N
10
10
1.000
0.000
10
1.000
0.000
Ata-2
N
10
10
0.100
0.850
0.050
11
0.318
0.591
0.091
Gpi
N
20
19
0.079
0.921
0.000
0.000
21
0.024
0.976
0.000
0.000
Idh
N
10
10
0.000
0.150
0.800
0.050
21
0.000
0.119
0.881
0.000
Mdh-1
N
20
36
0.958
0.042
0.000
0.000
0.000
81
0.969
0.006
0.025
0.000
0.000
Mdh-2
N
20
36
0.014
0.986
0.000
0.000
81
0.000
0.290
0.580
0.130
Mpi
N
10
10
0.050
0.100
0.150
0.100
21
0.000
0.000
0.000
0.024
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
37
Table 2*continued
Locus
Species
Allele
S. nudus
S. intermedius
S. pallidus
e
f
g
h
I
j
k
l
m
0.000
0.000
0.000
0.050
0.000
0.000
0.950
0.000
0.000
0.250
0.100
0.150
0.000
0.050
0.050
0.000
0.000
0.000
0.048
0.262
0.071
0.000
0.381
0.143
0.000
0.048
0.024
a
b
c
d
0.000
0.000
0.000
1.000
a
b
c
0.000
0.400
0.600
a
b
c
0.000
1.000
0.000
a
b
0.950
0.050
a
b
c
d
e
f
g
0.000
0.050
0.425
0.525
0.000
0.000
0.000
a
b
0.000
1.000
a
b
c
1.000
0.000
0.000
Muad
N
20
19
0.000
0.974
0.026
0.000
43
0.012
0.953
0.035
0.000
Pep
N
10
9
1.000
0.000
0.000
22
1.000
0.000
0.000
Pgdh
N
10
10
0.050
0.150
0.800
16
0.000
1.000
0.000
Pgm-1
N
10
10
0.000
1.000
10
0.000
1.000
Pgm-2
N
20
19
0.000
0.079
0.289
0.316
0.132
0.053
0.132
42
0.083
0.429
0.286
0.119
0.036
0.048
0.000
Sod-1
N
20
19
1.000
0.000
43
1.000
0.000
Sod-2
N
20
36
0.250
0.736
0.014
81
0.981
0.019
0.000
38
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
Table 2*continued
Locus
Species
Allele
S. nudus
S. intermedius
S. pallidus
Loci studied only in two species:
Argk
N
40
a
b
0.813
0.188
a
b
1.000
0.000
a
b
c
d
e
not studied
*
*
*
*
a
b
c
not studied
*
*
39
0.000
1.000
42
not detected
*
Ck
N
40
39
0.000
1.000
42
not detected
*
Fdh
N
0
17
0.000
0.059
0.706
0.235
0.000
21
0.048
0.167
0.476
0.000
0.310
Mdhp
N
0
17
0.382
0.588
0.029
22
0.000
0.977
0.023
Table 3
Mean estimates of observed (H ) and expected (H ; Nei, 1978) heterozygosities and percentage of loci
o
e
polymorphic (P
) in the three Strongylocentrotus species studied. A locus was considered polymorphic if
0.95
the frequency of the most common allele did not exceed 0.95
Species
S. nudus
S. intermedius
S. pallidus
No. of loci per
species
17
19
17
Mean number of P
0.95
individuals
per locus
17.6
18.7
33.1
52.9
47.4
35.3
Mean heterozygosity ($SE)
H
o
H
e
0.128 ($0.043)
0.237 ($0.067)
0.238 ($0.078)
0.126 ($0.043)
0.230 ($0.065)
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 three Strongylocentrotus species 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
S. intermedius
S. pallidus
*
1.578 (17)
1.327 (15)
0.206 (17)
*
0.269 (17)
0.265 (15)
0.764 (17)
*
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
39
Table 5
Mean expected heterozygosity estimates (H ) obtained for two sea urchin and two sea star species from the
e
Sea of Japan studied electrophoretically by di!erent authors
Species
H $S.E.
e
Strongylocentrotus
intermedius
0.064$0.035
Strongylocentrotus nudus
Asterias amurensis
Asterina pectinifera
0.230$0.065
0.115$0.030
0.026$0.020
0.059$0.027
0.067$0.028
0.126$0.043
0.192$0.034
0.076$0.032
0.216$0.055
0.213$0.058
0.029$0.029
0.168$0.039
0.106$0.041
0.123$0.041
No. of loci
studies per
species
Sea urchins
21
19
41
20
33
33
17
Sea stars
36
29
22
22
15
23
23
23
References
No. of
individual
studied per
locus (locality)!
12 (MB)
Matsuoka (1987)
19
36
12
17
21
18
(VB)
(VB)
(MB)
(MB)
(FK)
(VB)
Present study
Manchenko (1985a)
Matsuoka (1987)
Matsuoka et al. (1995)
Matsuoka et al. (1995)
Present study
27 (VB)
20 (MB)
47 (MB)
46 (PI)
9-16 (FK)
241 (VB)
28-30 (FK)
28-30 (MB)
Manchenko (1986)
Matsuoka et al. (1994)
Ward (1994)
Ward (1994)
Matsuoka (1981)
Manchenko (1986)
Matsuoka et al. (1995)
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 that S. pallidus and S. intermedius are closely
related species, D"0.269. S. pallidus is known to be capable of hybridization with S.
droebachiensis (Strathmann, 1981; Roller and Stickle, 1985). Using enzyme electrophoresis, 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) electrophoretically studied three pairs of geminate species of regular sea urchins of the
genera Echinometra, Eucidaris and Diadema separated by the Isthmus of Panama
about 3.5 MY ago. Taking into account the criticism of this work by Vawter et al.
(1980), the protein clock calibration assuming the rate of D"1 accumulating in
10 MY (inferred from Nei's D"0.329 obtained for germinate species of Eucidaris)
seems to be the most adequate for regular sea urchins. Invoking the protein clock
hypothesis and the suggested protein clock calibration, the time of divergence between
S. intermedius and S. pallidus is estimated as 2.7 MY. This estimate is close to the lower
date of appearance of S. pallidus, S. droebachiensis and S. purpuratus (3}5 MY ago)
suggested from molecular and fossil evidences to be the best estimate of their
divergence times from one another (Palumbi and Kessing, 1991). The divergence
40
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
between S. intermedius and S. nudus (D"1.578) and between S. nudus and S. pallidus
(D"1.327) lines has occurred much earlier.
It should be stressed that values of H estimate obtained by Matsuoka and
e
coworkers for S. intermedius and S. nudus are 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 monomers 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 heterozygosities 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)
between S. intermedius and S. nudus obtained by Matsuoka (1987) is also considerably
lower than that obtained by us (D"1.578). Remarkably that the tendency to obtain
reduced H values is rather a common characteristic of works by Matsuoka and
e
coworkers. Some examples demonstrating this tendency are listed in Table 5 where
H values obtained for the same echinoderm species by Matsuoka and coworkers, by
e
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 in S. intermedius
at Hk (the per locus expected heterozygosity, h "0.496), Got-1 (h "0.121), Got-2
e
e
(h "0.219) and Mdh-1 (h "0.116) loci, while Matsuoka (1987) found these loci to be
e
e
monomorphic. However, the most demonstrative examples are those concerning sea
stars Asterina ("Patiria) pectinifera and Asterias amurensis studied independently by
the three groups of investigators. Manchenko (1976) described 6 alleles at Mdh-2 locus
(h "0.647) in A. pectinifera from the Vostok Bay of the Sea of Japan (Vladivostok
e
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 at Gpi (h "0.278), Hk-1 (h "0.262), Hk-2 (h "0.256) and
e
e
e
Lap (h "0.214) loci in this species, while Matsuoka (1981) revealed no intraspeci"c
e
variations of these enzymes. Ward (1994) has paid special attention that H values
e
obtained electrophoretically by Matsuoka et al. (1994) for A. amurensis are 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 monomorphic. Ward concluded that `2the reduced average heterozygosity found by Matsuoka 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 at Gpi
(h "0.414) and H (h "0.519) loci in A. amurensis (Ward, 1994), only by di!erent
e
k e
electrophoresis systems used by di!erent authors. Indeed, one of us (Manchenko,
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
41
1986) used electrophoresis system quite di!erent from those used by Matsuoka and
Ward, however, revealed allozymic polymorphisms at both these loci (Gpi, h "0.340;
e
Hk, h "0.431) in A. amurensis sample from the Vostok Bay of the Sea of Japan. The
e
same geographic population of A. amurensis was studied independently by Manchenko (unpublished data) and by Ward (1994) using starch gel and cellogel electrophoresis systems, respectively. Although electrophoretic bu!er systems used by
these authors were also di!erent, they obtained quite comparable values of expected
heterozygosity for Gpi (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-expressed 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 of S.
intermedius reared in the laboratory is 30 days (Naidenko, 1983). Similar, and even
higher, time estimates are characteristic of A. amurensis (S.S. Dautov, pers. commun.)
and of S. nudus and A. pectinifera (S.N. Yakovlev, unpublished data) larvae at natural
conditions. Such species commonly demonstrate low genetic divergence among geographic populations (for review see Palumbi, 1992, 1994; Avise, 1994). This is supported by results of population genetic study of A. 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 populations S. intermedius and A. amurensis (G.P. Manchenko, unpublished 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 allozyme variations (Buth, 1990) and can therefore result in the underestimation of
H values and in the overestimation of number of monomorphic isozyme loci. We are
e
sure, for example, that the four monomorphic MDH and the "ve monomorphic SOD
loci described in S. nudus by Matsuoka et al. (1995) resulted from inadequate genetic
interpretation of multiple MDH and SOD subbands displayed on PAG zymograms.
To our knowledge, there are no echinoderm species that express as many as four
MDH or "ve SOD loci. Using starch gel electrophoresis we never observed more than
two MDH or three SOD (commonly two) loci expressed in electrophoretically studied
42
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
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 supported by several strikingly unusual (anomalous) allozyme polymorphisms described
by these authors in echinoderms. For example, allozyme polymorphisms characteristic of monomeric enzymes (i.e. heterozygotes display two-banded phenotypes)
were described in the sea urchin S. intermedius for tetrazolium oxidase (recommended
name superoxide dismutase, SOD) (Matsuoka, 1987), and in the sea star A. 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 in A. 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 genus Strongylocentrotus from 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). Our D estimate for S. intermedius/S. nudus species pair di!ers considerably from that obtained by Matsuoka (1987). Considerable di!erences are also
characteristic of H estimates obtained for S. intermedius and S. nudus by Matsuoka
e
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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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 genus Strongylocentrotus (S. intermedius, S. nudus, S. pallidus) from the Sea
of Japan. S. pallidus and S. intermedius showed high mean values of expected heterozygosity,
H "0.223$0.072 (17 loci) and H "0.230$0.065 (19 loci), respectively. This estimate was
e
e
somewhat lower in S. nudus, H "0.126$0.043 (17 loci). Estimates of Nei's genetic distance
e
between S. nudus/S. intermedius (D"1.578, 17 loci) and S. nudus/S. pallidus (D"1.327, 15 loci)
were considerably higher than that between S. intermedius/S. pallidus (D"0.269, 17 loci).
Invoking the protein clock hypothesis and using Panamanian geminate sea urchins for protein
clock calibration, the time of divergence between S. intermedius and S. pallidus was estimated as
2.7 MY. The results obtained for S. intermedius and S. nudus by us di!er considerably from
results obtained for these species by Norimasa Matsuoka and coworkers. The revealed
discrepancies are discussed and the conclusion made that Matsuoka and coworkers' data on
echinoderm biochemical genetics and systematics should be used with caution. ( 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Strongylocentrotus intermedius; Strongylocentrotus nudus; Strongylocentrotus pallidus; Echinoidea;
Sea urchins; Allozymic variation; Genetic distance
1. Introduction
Taxonomy and phylogeny of echinoids is mainly based on data obtained using
methods of comparative morphology and paleontology. Because evolutionary change
* Corresponding author. Tel.: #7-4232-310905; Fax: #7-4232-310900.
E-mail address: [email protected] (G.P. Manchenko).
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 2 7 - 2
32
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
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 genus Strongylocentrotus demonstrate high
level of intraspeci"c morphological variation which, in some cases, overlaps interspeci"cally and generates problems in unequivocal identi"cation of and discrimination 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, and S. 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 relationships 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 spatial 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 genus Strongylocentrotus were studied using both DNA and enzyme
electrophoresis techniques.
The use of nuclear and mitochondrial DNA sequences to study evolutionary
relationships between seven sea urchin species of the family Strongylocentrotidae has
unexpectedly failed to resolve the branching order among four members of the genus
Strongylocentrotus including S. purpuratus, S. polyacanthus, S. droebachiensis and S.
pallidus (Biermann, 1998). At the same time, the results obtained in the cited study
provided evidence that two other sea urchin species of the family, Allocentrotus fragilis
and Hemicentrotus pulcherrimus, fall phylogenetically within the genus Strongylocentrotus. Mitochondrial DNA variation was used to assess intraspeci"c genetic diversity
and spatial genetic di!erentiation of populations in sea urchin species of the genus
Strongylocentrotus (Vawter and Brown, 1986; Palumbi and Wilson, 1990; Palumbi and
Kessing, 1991; Palumbi and Metz, 1991; Palumbi, 1995). The only (to our knowledge)
work that successfully combined the use of isozyme and mtDNA gene markers in the
study of genetic divergence in S. purpuratus along the coast of California and North
Mexico is that by Edmands et al. (1996). The results obtained in this work proved in
a sharp contrast with results of previous work on S. purpuratus that failed to detect
any genetic di!erence between geographic populations of the species separated by
a distance of 1500 km (Palumbi and Wilson, 1990; Palumbi, 1995).
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
33
The use of isozymes to study genetic divergence and variation in sea urchins of the
genus Strongylocentrotus is 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 between S. intermedius 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 of Strongylocentrotus and is rather a member of this same genus. Another pair
of species of Strongylocentrotus, S. pallidus and S. 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 between S. pallidus and S. intermedius as well as between S. pallidus
and S. nudus.
This work presents enzyme electrophoretic estimates of intraspeci"c genetic variation 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 were Strongylocentrotus intermedius, S. nudus and
S. pallidus from 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 of S. 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
electrophoresis, and isozyme loci scored are listed in Table 1. In our pilot electrophoretic runs we also detected activity bands of nine additional enzymes (alanine
transaminase, alcohol dehydrogenase, catalase, hexokinase, lactoylglutathione lyase,
inorganic pyrophosphatase, octanol dehydrogenase, peptidases, phe-ala and leu-val
34
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
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)
Arginine kinase (ARGK; EC 2.7.3.3)
Aspartate transaminase (ATA; EC 2.6.1.1)
Creatine kinase (CK; EC 2.7.3.2)
Cytosol non-speci"c dipeptidase
(PEP; EC 3.4.13.18), gly}leu as substrate
Formaldehyde dehydrogenase
(FDH; EC 1.2.1.1)
Glucose-6-phosphate isomerase
(GPI; EC 5.3.1.9)
Isocitrate dehydrogenase
(NADP) (IDH; EC 1.1.1.42)
Malate dehydrogenase
(MDH; EC 1.1.1.37)
Malate dehydrogenase (NADP)
(MDHP; EC 1.1.1.40)
Mannose-6-phosphate isomerase
(MPI; EC 5.3.1.8)
Methylumbelliferyl-acetate deacetylase
(EST; EC 3.1.1.56)
Phosphogluconate dehydrogenase
(PGD; EC 1.1.1.44)
Phosphoglucomutase (PGM; EC 5.4.2.2)
Ak
Argk
Ata-1,-2
Ck
Pep
M
G, M
G#M
M
G#M
TM
TEB
TC
TEB
TEB
Fdh
G#M
TC
Gpi
G#M
TC
Idh
G
TC
Mdh-1,-2
G#M
TC
Mdhp
G#M
TC
Mpi
G
TEB
Muad
M
TEB
Pgdh
G#M
TM
Pgm-1
Pgm-2
Sod-1
Sod-2
G
G#M
M
G
TEB
TEB
TEB
TEB
Superoxide dismutase (SOD; EC 1.15.1.1)
!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 ) and expected (H ) heterozygosity as
o
e
well as Nei's (1978) unbiased genetic identity (I) and genetic distance (D) coe$cients.
3. Results
In total, 15 isozyme loci coding for 11 enzyme systems were resolved and proved
scorable in all the three sea urchin species studied. Additional gene loci coding for
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
35
arginine kinase (Argk) and creatine kinase (Ck) proved scorable only in S. intermedius
and S. nudus, while loci coding for formaldehyde dehydrogenase (Fdh) and malic
enzyme (Me) were studied only in S. intermedius and S. 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 heterozygotes were revealed, at least in one of the studied species, at Mpi, 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 of S. intermedius. This isozyme demonstrated allozyme variation characteristic of monomeric
enzymes (i.e. heterozygotes were two-banded). It was detected only in S. 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-2 loci. Allozyme variants were also observed in Me, Muad, Pep, and
Pgdh loci 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 three Strongylocentrotus species
are given in Table 2. Based on these data estimates of mean observed (H ) and
o
expected (H ) heterozygosities per locus and the percentage (P
) of polymorphic
e
0.95
loci per species were calculated and are given in Table 3. H is known to be the best
e
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 "0.223$0.072), and S. nudus (17 loci,
e
H "0.126$0.043).
e
Genetic identity (I) and genetic distance (D) coe$cients calculated from allele
frequency data are given in Table 4. The values of genetic distance estimate obtained
for S. nudus/S. intermedius (17 loci, D"1.578) and S. nudus/S. pallidus (15 loci, D"
1.327) species pairs are considerably higher than that for S. 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. pallidus and S. 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 in S. 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 of H estimate and may therefore be considered only as
e
di!erences demonstrating a general trend in the relationship between the level of
intraspeci"c genetic variability and the breadth of geographic distribution of species.
36
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
Table 2
Allele frequencies for isozyme loci studied in the three sea urchin species of the genus Strongylocentrotus.
Number of individuals, N
Locus
Species
Allele
S. nudus
S. intermedius
S. pallidus
Loci studied in all the three species:
Ak
N
9
a
b
0.000
1.000
a
b
0.050
0.950
a
b
c
0.000
0.000
1.000
a
b
c
d
0.000
0.925
0.025
0.050
a
b
c
d
0.950
0.000
0.050
0.000
a
b
c
d
e
0.000
0.000
0.000
0.875
0.125
a
b
c
d
1.000
0.000
0.000
0.000
a
b
c
d
0.000
0.000
0.000
0.000
10
0.000
1.000
16
1.000
0.000
Ata-1
N
10
10
1.000
0.000
10
1.000
0.000
Ata-2
N
10
10
0.100
0.850
0.050
11
0.318
0.591
0.091
Gpi
N
20
19
0.079
0.921
0.000
0.000
21
0.024
0.976
0.000
0.000
Idh
N
10
10
0.000
0.150
0.800
0.050
21
0.000
0.119
0.881
0.000
Mdh-1
N
20
36
0.958
0.042
0.000
0.000
0.000
81
0.969
0.006
0.025
0.000
0.000
Mdh-2
N
20
36
0.014
0.986
0.000
0.000
81
0.000
0.290
0.580
0.130
Mpi
N
10
10
0.050
0.100
0.150
0.100
21
0.000
0.000
0.000
0.024
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
37
Table 2*continued
Locus
Species
Allele
S. nudus
S. intermedius
S. pallidus
e
f
g
h
I
j
k
l
m
0.000
0.000
0.000
0.050
0.000
0.000
0.950
0.000
0.000
0.250
0.100
0.150
0.000
0.050
0.050
0.000
0.000
0.000
0.048
0.262
0.071
0.000
0.381
0.143
0.000
0.048
0.024
a
b
c
d
0.000
0.000
0.000
1.000
a
b
c
0.000
0.400
0.600
a
b
c
0.000
1.000
0.000
a
b
0.950
0.050
a
b
c
d
e
f
g
0.000
0.050
0.425
0.525
0.000
0.000
0.000
a
b
0.000
1.000
a
b
c
1.000
0.000
0.000
Muad
N
20
19
0.000
0.974
0.026
0.000
43
0.012
0.953
0.035
0.000
Pep
N
10
9
1.000
0.000
0.000
22
1.000
0.000
0.000
Pgdh
N
10
10
0.050
0.150
0.800
16
0.000
1.000
0.000
Pgm-1
N
10
10
0.000
1.000
10
0.000
1.000
Pgm-2
N
20
19
0.000
0.079
0.289
0.316
0.132
0.053
0.132
42
0.083
0.429
0.286
0.119
0.036
0.048
0.000
Sod-1
N
20
19
1.000
0.000
43
1.000
0.000
Sod-2
N
20
36
0.250
0.736
0.014
81
0.981
0.019
0.000
38
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
Table 2*continued
Locus
Species
Allele
S. nudus
S. intermedius
S. pallidus
Loci studied only in two species:
Argk
N
40
a
b
0.813
0.188
a
b
1.000
0.000
a
b
c
d
e
not studied
*
*
*
*
a
b
c
not studied
*
*
39
0.000
1.000
42
not detected
*
Ck
N
40
39
0.000
1.000
42
not detected
*
Fdh
N
0
17
0.000
0.059
0.706
0.235
0.000
21
0.048
0.167
0.476
0.000
0.310
Mdhp
N
0
17
0.382
0.588
0.029
22
0.000
0.977
0.023
Table 3
Mean estimates of observed (H ) and expected (H ; Nei, 1978) heterozygosities and percentage of loci
o
e
polymorphic (P
) in the three Strongylocentrotus species studied. A locus was considered polymorphic if
0.95
the frequency of the most common allele did not exceed 0.95
Species
S. nudus
S. intermedius
S. pallidus
No. of loci per
species
17
19
17
Mean number of P
0.95
individuals
per locus
17.6
18.7
33.1
52.9
47.4
35.3
Mean heterozygosity ($SE)
H
o
H
e
0.128 ($0.043)
0.237 ($0.067)
0.238 ($0.078)
0.126 ($0.043)
0.230 ($0.065)
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 three Strongylocentrotus species 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
S. intermedius
S. pallidus
*
1.578 (17)
1.327 (15)
0.206 (17)
*
0.269 (17)
0.265 (15)
0.764 (17)
*
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
39
Table 5
Mean expected heterozygosity estimates (H ) obtained for two sea urchin and two sea star species from the
e
Sea of Japan studied electrophoretically by di!erent authors
Species
H $S.E.
e
Strongylocentrotus
intermedius
0.064$0.035
Strongylocentrotus nudus
Asterias amurensis
Asterina pectinifera
0.230$0.065
0.115$0.030
0.026$0.020
0.059$0.027
0.067$0.028
0.126$0.043
0.192$0.034
0.076$0.032
0.216$0.055
0.213$0.058
0.029$0.029
0.168$0.039
0.106$0.041
0.123$0.041
No. of loci
studies per
species
Sea urchins
21
19
41
20
33
33
17
Sea stars
36
29
22
22
15
23
23
23
References
No. of
individual
studied per
locus (locality)!
12 (MB)
Matsuoka (1987)
19
36
12
17
21
18
(VB)
(VB)
(MB)
(MB)
(FK)
(VB)
Present study
Manchenko (1985a)
Matsuoka (1987)
Matsuoka et al. (1995)
Matsuoka et al. (1995)
Present study
27 (VB)
20 (MB)
47 (MB)
46 (PI)
9-16 (FK)
241 (VB)
28-30 (FK)
28-30 (MB)
Manchenko (1986)
Matsuoka et al. (1994)
Ward (1994)
Ward (1994)
Matsuoka (1981)
Manchenko (1986)
Matsuoka et al. (1995)
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 that S. pallidus and S. intermedius are closely
related species, D"0.269. S. pallidus is known to be capable of hybridization with S.
droebachiensis (Strathmann, 1981; Roller and Stickle, 1985). Using enzyme electrophoresis, 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) electrophoretically studied three pairs of geminate species of regular sea urchins of the
genera Echinometra, Eucidaris and Diadema separated by the Isthmus of Panama
about 3.5 MY ago. Taking into account the criticism of this work by Vawter et al.
(1980), the protein clock calibration assuming the rate of D"1 accumulating in
10 MY (inferred from Nei's D"0.329 obtained for germinate species of Eucidaris)
seems to be the most adequate for regular sea urchins. Invoking the protein clock
hypothesis and the suggested protein clock calibration, the time of divergence between
S. intermedius and S. pallidus is estimated as 2.7 MY. This estimate is close to the lower
date of appearance of S. pallidus, S. droebachiensis and S. purpuratus (3}5 MY ago)
suggested from molecular and fossil evidences to be the best estimate of their
divergence times from one another (Palumbi and Kessing, 1991). The divergence
40
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
between S. intermedius and S. nudus (D"1.578) and between S. nudus and S. pallidus
(D"1.327) lines has occurred much earlier.
It should be stressed that values of H estimate obtained by Matsuoka and
e
coworkers for S. intermedius and S. nudus are 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 monomers 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 heterozygosities 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)
between S. intermedius and S. nudus obtained by Matsuoka (1987) is also considerably
lower than that obtained by us (D"1.578). Remarkably that the tendency to obtain
reduced H values is rather a common characteristic of works by Matsuoka and
e
coworkers. Some examples demonstrating this tendency are listed in Table 5 where
H values obtained for the same echinoderm species by Matsuoka and coworkers, by
e
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 in S. intermedius
at Hk (the per locus expected heterozygosity, h "0.496), Got-1 (h "0.121), Got-2
e
e
(h "0.219) and Mdh-1 (h "0.116) loci, while Matsuoka (1987) found these loci to be
e
e
monomorphic. However, the most demonstrative examples are those concerning sea
stars Asterina ("Patiria) pectinifera and Asterias amurensis studied independently by
the three groups of investigators. Manchenko (1976) described 6 alleles at Mdh-2 locus
(h "0.647) in A. pectinifera from the Vostok Bay of the Sea of Japan (Vladivostok
e
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 at Gpi (h "0.278), Hk-1 (h "0.262), Hk-2 (h "0.256) and
e
e
e
Lap (h "0.214) loci in this species, while Matsuoka (1981) revealed no intraspeci"c
e
variations of these enzymes. Ward (1994) has paid special attention that H values
e
obtained electrophoretically by Matsuoka et al. (1994) for A. amurensis are 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 monomorphic. Ward concluded that `2the reduced average heterozygosity found by Matsuoka 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 at Gpi
(h "0.414) and H (h "0.519) loci in A. amurensis (Ward, 1994), only by di!erent
e
k e
electrophoresis systems used by di!erent authors. Indeed, one of us (Manchenko,
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
41
1986) used electrophoresis system quite di!erent from those used by Matsuoka and
Ward, however, revealed allozymic polymorphisms at both these loci (Gpi, h "0.340;
e
Hk, h "0.431) in A. amurensis sample from the Vostok Bay of the Sea of Japan. The
e
same geographic population of A. amurensis was studied independently by Manchenko (unpublished data) and by Ward (1994) using starch gel and cellogel electrophoresis systems, respectively. Although electrophoretic bu!er systems used by
these authors were also di!erent, they obtained quite comparable values of expected
heterozygosity for Gpi (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-expressed 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 of S.
intermedius reared in the laboratory is 30 days (Naidenko, 1983). Similar, and even
higher, time estimates are characteristic of A. amurensis (S.S. Dautov, pers. commun.)
and of S. nudus and A. pectinifera (S.N. Yakovlev, unpublished data) larvae at natural
conditions. Such species commonly demonstrate low genetic divergence among geographic populations (for review see Palumbi, 1992, 1994; Avise, 1994). This is supported by results of population genetic study of A. 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 populations S. intermedius and A. amurensis (G.P. Manchenko, unpublished 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 allozyme variations (Buth, 1990) and can therefore result in the underestimation of
H values and in the overestimation of number of monomorphic isozyme loci. We are
e
sure, for example, that the four monomorphic MDH and the "ve monomorphic SOD
loci described in S. nudus by Matsuoka et al. (1995) resulted from inadequate genetic
interpretation of multiple MDH and SOD subbands displayed on PAG zymograms.
To our knowledge, there are no echinoderm species that express as many as four
MDH or "ve SOD loci. Using starch gel electrophoresis we never observed more than
two MDH or three SOD (commonly two) loci expressed in electrophoretically studied
42
G.P. Manchenko, S.N. Yakovlev / Biochemical Systematics and Ecology 29 (2001) 31}44
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 supported by several strikingly unusual (anomalous) allozyme polymorphisms described
by these authors in echinoderms. For example, allozyme polymorphisms characteristic of monomeric enzymes (i.e. heterozygotes display two-banded phenotypes)
were described in the sea urchin S. intermedius for tetrazolium oxidase (recommended
name superoxide dismutase, SOD) (Matsuoka, 1987), and in the sea star A. 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 in A. 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 genus Strongylocentrotus from 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). Our D estimate for S. intermedius/S. nudus species pair di!ers considerably from that obtained by Matsuoka (1987). Considerable di!erences are also
characteristic of H estimates obtained for S. intermedius and S. nudus by Matsuoka
e
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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Avise, J.C., 1994. Molecular Markers, Natural History, and Evolution. Chapman & Hall, New York.
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