Directory UMM :Data Elmu:jurnal:B:Biochemical Systematics and Ecology:Vol28.Issue8.Oct2000:
Biochemical Systematics and Ecology 28 (2000) 737}750
High level of genetic divergence between
sympatric color morphs of the littoral sea
anemone Anthopleura orientalis
(Anthozoa: Actiniaria)
Gennady P. Manchenko*, Tatyana N. Dautova, Yury Y. Latypov
Institute of Marine Biology, Vladivostok 690041, Russia
Received 18 May 1998; received in revised form 6 October 1998; accepted 26 July 1999
Abstract
Using enzyme electrophoresis and nematocyst analysis, the sympatrically occurring `lighta
and `darka color morphs of the sea anemone Anthopleura orientalis from the Sea of Japan were
shown to be two valid species. The `lighta morph was identi"ed as A. orientalis (Averintsev,
1967 Issledovaniya fauny morei: Vyp. 5 (13). Nanka, Leningrad, pp. 62}77), while the `darka
morph was designated as Anthopleura sp. The analysis of 21 isozyme loci revealed high value of
Nei's genetic distance (D"1.284) between the two species, which are indistinguishable in their
external morphology. The mean values of observed and expected heterozygosities for A.
orientalis and Anthopleura sp. are high (H "0.252$0.061, H "0.250$0.061 and
0
%
H "0.327$0.052, H "0.351$0.054, respectively). The species di!er signi"cantly in the size
0
%
of spirocysts and nematocysts, among which the atrichs from acrorhagi and the basitrichs from
actinopharynx contribute most to the observed di!erence. Strong qualitative di!erence is
revealed between distributions of nematocysts in mesenteric "laments of the two sea anemone
species studied. The possible conspeci"city of Anthopleura sp. with Anthopleura artemisia (Dana,
1848) is discussed and the conclusion made that these are two separate species. ( 2000
Elsevier Science Ltd. All rights reserved.
Keywords: Actiniaria; Anthopleura orientalis; Anthozoa; Color morphs; Sibling species; Enzyme electrophoresis; Genetic divergence; Genetic variation
* Corresponding author. Tel.: #7-4232-310905; Fax: #7-4232-310900.
E-mail address: [email protected] (G.P. Manchenko)
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 9 9 ) 0 0 1 2 0 - 9
738
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
1. Introduction
Sea anemones are a very diverse group of lower marine invertebrates (Shick, 1991).
Because of relative structural simplicity, the taxonomically useful morphological
characters are few and taxonomic problems many in these organisms. The littoral sea
anemones of the genus Anthopleura are very common on sandy shores of the Russian
coast of the Sea of Japan, however, their taxonomic status was not carefully studied.
The only taxonomic study of Anthopleura from this region was carried out by
Averintsev (1967) who described new species A. orientalis. Two other members of the
genus, A. artemisia (Dana, 1848) and A. xanthogrammica (Brandt, 1835) described from
the Eastern North Paci"c, were also ideti"ed by Averintsev (1967, 1976)) and Kostina
(1989) in the Sea of Japan. Anthopleura from the Eastern North Paci"c are highly
polymorphic in body color and morphology and have no reliable species-speci"c
external characters (Hand, 1955). The same is true for Anthopleura from the Sea of
Japan, where a continuous range of sympatric color varieties can be found in samples
collected during "eld observations. The taxonomic status of such varieties is usually
uncertain.
Enzyme electrophoresis has proved a particularly useful method in distinguishing
morphologically cryptic species in various invertebrate groups (Knowlton, 1993;
Thorpe and SoleH -Cava, 1994). It is an especially powerful tool for discriminating
between sympatric sibling species. From conventional de"nitions according to the
biological species concept, such species should have di!erent allele frequencies or
di!erent "xed alleles at least at some gene loci, thus giving evidence that the species
under question are reproductively isolated. Enzyme electrophoretic studies have
clearly demonstrated that many sympatric `morphsa of sea anemone species are
reproductively isolated and thus are valid separate species (Carter and Thorpe, 1981;
Bucklin and Hedgekock, 1982; Haylor et al., 1984; SoleH -Cava et al., 1985; SoleH -Cava
and Thorpe, 1987, 1992; McFadden et al., 1997).
The objective of this work was to establish the taxonomic status of two sympatric
color morphs of A. orientalis from northern part of the Sea of Japan using enzyme
electrophoresis in conjunction with nematocyst analysis. In the text we refer to the
`lighta and the `darka color morphs of A. orientalis until the taxonomic status of each
morph has been determined.
2. Materials and methods
2.1. Collection of samples
Samples of two color morphs of A. orientalis, the `lighta morph and the `darka
morph, were collected from the shoals (less than 2 m deep) at Skrebtsov Island
(Amursky Bay, the Sea of Japan) near Vladivostok. Collections were made over the
period July}September 1995 from sites of gravely}rocky substratum covered by sand.
Some characters of external morphology of the two color morphs are listed in Table 1.
Oral and pedal disk diameters were measured using relaxed anemones treated with
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
739
Table 1
Some morphological and ecological characters of `Lighta and `Darka color morphs of Anthopleura
orientalis
Character
Oral disk diameter
Pedal disk diameter
Column color
Oral disk color
Pedal disc color
Veruccae
Substratum
Immersion in sand
Color morph
`Lighta
`Darka
18}34 mm
15}30 mm
Green}gray
Green}gray (light)
Gray}brown
Not extending to limbus
Fine}grained gravel, dead broken shells
Less than 1/2 of the column length
19}32 mm
15}30 mm
Green}gray
Black}gray (dark)
Gray}brown
Not extending to limbus
Coarse}grained gravel, small stones
More than 2/3 of the column length
magnesium chloride. All anemones were adult and dioecious as evidenced from the
inspection of their gonad preparations. After collection, the anemones were kept in the
laboratory in running sea water before electrophoresis and nematocyst analysis.
Voucher specimens of the `lighta and the `darka morphs are deposited in Zoological
Institute, St. Petersburg, Russia (registration number 9.208) and the Institute of
Marine Biology, Vladivostok, Russia (registration number 3175), respectively.
2.2. Enzyme electrophoresis
Before sample preparation the anemones were placed in a freezer (!123C) for 1 h.
For electrophoresis, longitudinal sections were cut from frozen anemones to include
tissues of the oral disc (with tentacles), internal organs, and the column. Tissue
samples were homogenized in two volumes of distilled water and crude homogenates
analyzed by horizontal 14% starch-gel electrophoresis as described by Manchenko
and Balakirev (1984). In total, 82 individuals of the `lighta morph and 19 individuals
of the `darka morph were available for electrophoretic analysis. Two continuous
bu!er systems were used: TEB (tris-EDTA-boric acid, pH 8.5) and TC (tris-citric acid,
pH 7.0). The staining of electrophoretic gels followed standard procedures, using
recipes from Manchenko (1994). Enzymes assayed, bu!ers used and isozyme loci
scorable in both color morphs are listed in Table 2. Gene loci coding for ASTA-2,
DHLDH-1, FDH-1, GLDH-2, IDH-1, and MD-2 isozymes proved not scorable
because of low activity and/or poor resolution of corresponding isozymes in one or
both the morphs. These loci were not taken into account in our further considerations
based on the use of allele frequency data.
Genetic interpretations of banding patterns developed on zymograms were made
according to Buth (1990) taking into account previously obtained electrophoretic data
for A. orientalis (Manchenko and Balakirev, 1984). Enzyme nomenclature followed
recommendations of the IUBMB NC (1992). The locus and allele designations were
formated according to principles recommended for protein-coding loci (Shaklee et al.,
740
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
Table 2
Enzymes assayed, electrophoretic bu!ers used and isozyme loci scored in `Lighta and `Darka color morphs
of Anthopleura orientalis
Enzyme
EC No.
Locus
Bu!er
Aconitate hydratase
Alanine transaminase
b-Alanopine dehydrogenase
Aspartate transaminase
Catalase
Dihydrolipoamide dehydrogenase
Formaldehyde dehydrogenase (glutathione)
Fumarate hydratase
Glutamate dehydrogenase (NADP)
Hexokinase
Isocitrate dehydrogenase (NADP)
Lactoylglutathione lyase
Leucyl aminopeptidase
Mannose-6-phosphate isomerase
Methylumbelliferyl-acetate deacetylase
Octopine dehydrogenase
Peptidase (detected with val}leu)
Phosphoglucomutase
Xanthine dehydrogenase
4.2.1.3
2.6.1.2
1.5.1.26
2.6.1.1
1.11.1.6
1.8.1.4
1.2.1.1
4.2.1.2
1.4.1.4
2.7.1.1
1.1.1.42
4.4.1.5
3.4.11.1
5.3.1.8
3.1.1.56
1.5.1.11
3.4.11... or 13...
5.4.2.2
1.1.1.204
Ah
Alta
b-Alpdh
Asta-1
Cat
Dhldh-2
Fdh-2
Fh
Gldh-1
Hk
Idh-2
Lgl
Lap
Mpi
Md-1, Md-3
Ondh
Pep-1, Pep-3
Pgm
Xdh
TC
TC
TC
TEB
TC
TC
TC
TC
TC
TEB
TC
TEB
TEB
TC
TC
TC
TEB
TEB
TEB
1990) with minor modi"cation (asterisks were omitted from designations of isozyme
loci).
2.3. Nematocyst and spirocyst analysis
The nematocyst types were identi"ed as atrichs, basitrichs, and microbasic pmastigophores following the terminology of Carlgren (1949) and Hand (1955). Thousands of nematocysts were visually inspected before "nal determination of the number
of di!erent nematocyst types was made. Twelve spirocysts of each size class and 12
nematocysts of each type and each size class were measured in macerated tissue
samples from 10 individuals of each of the two color morphs. Sea anemones with
approximately equal body size were chosen for nematocyst and spirocyst comparison
to avoid undesirable in#uence of the body size on the size of cnida (Chintiroglou and
Simsiridou, 1997).
2.4. Data analysis
Nematocyst and spirocyst data were compared between color morphs using Student's t-test provided by the SYSTAT 4.0 computer program (Wilkinson, 1987).
Genotype frequencies were inferred by direct count from banding patterns observed
on zymograms. Allele frequencies, genetic variability measures and unbiased (Nei,
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
741
1978) genetic identity (I) and genetic distance (D) estimates were calculated from
genotype frequencies using the BIOSYS-1 computer program of Swo!ord and Selander (1981).
Because of small sample sizes, the expected numbers of heterozygotes were in some
cases too small to allow computation of conventional Chi-square. Therefore, signi"cance of deviations of observed genotype frequencies from those expected under
Hardy}Weinberg equilibrium was estimated using the pseudo-probability test (the
CHIHW program by Zaykin and Pudovkin, 1993).
When testing for conformity of genotype distribution to the Hardy}Weinberg
equilibrium, a number of separate tests for individual loci are usually performed. To
avoid Type I errors, corrections for multiple tests were performed using Sidak's
multiplicative inequality for calculations of critical values of the Chi-square distribution (Rolf and Sokal, 1981, p. 101; Sokal and Rolf, 1981, p. 728). The program
MULTTEST (Zaykin and Pudovkin, 1991) was used to "nd the critical values of
Chi-square for each replicate test considering that it was part of a set of separate
independent tests.
3. Results
3.1. Genetic analysis
In total, 21 isozyme loci coding for 19 enzyme systems were resolved and proved
scorable in both color morphs (Table 3). Allozyme variations with one-banded
homozygotes and two-banded heterozygotes were revealed at Ah, b-Alpdh, Hk, ¸ap,
Mpi, Ondh, and Pgm loci providing evidence that catalytically active molecules of the
corresponding isozymes are monomers. Three-banded allozyme patterns characteristic of dimeric enzymes were observed in individuals heterozygous for Asta-1, Fdh-2,
Idh-2, Lgl, and Md-1 loci. Clearly resolved "ve-banded allozyme patterns in individuals heterozygous for the Cat locus give strong evidence that this enzyme is
a tetramer. Allozyme patterns in individuals heterozygous for Alta, Fh, Gldh-1, Pep-1,
Pep-2 and Xdh loci were displayed as broad di!use bands not resolved into separate
allozymes, thus suggesting that the corresponding enzymes are rather oligomers. No
allozyme variants were revealed in Dhldh-2 and Md-3 loci.
The allele frequencies for 21 isozyme loci studied in `lighta and `darka color
morphs of A. orientalis are given in Table 3. Eight loci (Ah, Alta, Fdh-2, Gldh-1, Idh-2,
Lgl, Md-1, and Pep-2) share no common alleles in the `lighta and `darka morphs
providing strong evidence that gene pools of these morphs are separated.
Genetic divergence between studied color morphs of A. orientalis is very high as is
evident from the genetic identity (I"0.277) and genetic distance (D"1.284) estimates. Mean values of observed and expected heterozygosities for `lighta morph
(H "0.252$0.061; H "0.250$0.061) and `darka morph (H "0.327$0.052;
0
%
0
H "0.351$0.054) are also very high.
%
We did not observe identical genotypes over all loci analyzed in any two or more
individuals either in `lighta or `darka color morphs. This provides evidence that no
742
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
Table 3
Allele frequencies at 21 isozyme loci in the `lighta and `darka color morphs of Anthopleura orientalis
Locus
Allele
Color morph
`Lighta
`Darka
Ah
(N)!
1
2
3
4
5
6
7
11
0.000
0.000
0.000
0.000
0.000
0.045
0.955
12
0.042
0.042
0.750
0.125
0.042
0.000
0.000
b-Alpdh
(N)
1
2
3
7
1.000
0.000
0.000
11
0.136
0.636
0.227
Alta
(N)
1
2
3
27
0.981
0.019
0.000
12
0.000
0.000
1.000
Asta-1
(N)
1
2
3
4
42
0.274
0.274
0.440
0.012
15
0.100
0.867
0.033
0.000
Cat
(N)
1
2
3
4
5
6
7
8
9
10
76
0.125
0.007
0.000
0.033
0.000
0.553
0.000
0.283
0.000
0.000
17
0.000
0.000
0.029
0.000
0.118
0.000
0.235
0.088
0.147
0.382
Dhldh-2
(N)
1
18
1.000
12
1.000
Fdh-2
(N)
1
2
3
4
5
76
0.000
0.000
0.007
0.987
0.007
19
0.816
0.184
0.000
0.000
0.000
Fh
(N)
1
2
3
4
18
0.861
0.000
0.056
0.083
12
0.000
0.792
0.208
0.000
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
743
Table 3 (continued)
Locus
Allele
Color morph
`Lighta
`Darka
Gldh-1
(N)
1
2
3
27
0.000
0.000
1.000
12
0.958
0.042
0.000
Hk
(N)
1
2
3
4
5
6
7
8
81
0.000
0.006
0.093
0.235
0.377
0.167
0.025
0.099
17
0.029
0.206
0.412
0.029
0.324
0.000
0.000
0.000
Idh-2
(N)
1
2
3
4
5
75
0.000
0.000
0.020
0.973
0.007
11
0.045
0.955
0.000
0.000
0.000
¸ap
(N)
1
2
3
4
5
6
82
0.000
0.000
0.104
0.854
0.024
0.018
17
0.029
0.794
0.147
0.029
0.000
0.000
¸gl
(N)
1
2
3
4
5
81
0.006
0.994
0.000
0.000
0.000
17
0.000
0.000
0.676
0.088
0.235
Mpi
(N)
1
2
3
4
5
6
7
8
9
28
0.018
0.107
0.161
0.054
0.054
0.339
0.089
0.179
0.000
12
0.000
0.042
0.125
0.000
0.000
0.542
0.125
0.083
0.083
Md-1
(N)
1
2
3
4
5
61
0.000
0.148
0.000
0.016
0.836
14
0.036
0.000
0.964
0.000
0.000
Table 3 (continued on next page)
744
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
Table 3 (continued)
Locus
Allele
Color morph
`Lighta
`Darka
Md-3
(N)
1
61
1.000
14
1.000
Ondh
(N)
1
2
3
4
7
0.000
0.071
0.714
0.214
10
0.150
0.350
0.500
0.000
Pep-1
(N)
1
2
3
81
0.981
0.000
0.019
17
0.000
0.500
0.500
Pep-2
(N)
1
2
3
4
5
81
0.000
0.000
0.006
0.981
0.012
17
0.794
0.206
0.000
0.000
0.000
Pgm
(N)
1
2
3
4
5
6
81
0.000
0.216
0.660
0.086
0.037
0.000
17
0.029
0.000
0.029
0.735
0.000
0.206
Xdh
(N)
1
2
3
42
0.012
0.798
0.190
15
0.033
0.833
0.133
!(N)"number of analyzed individuals.
asexually produced individuals (or clonemates) were involved in the analysis. The
genotype frequencies observed in both the color morphs corresponded well the
Hardy}Weinberg equilibrium when having applied Sidak's correction for the whole
set of tests.
3.2. Nematocyst and spirocyst analysis
Both color morphs have the cnidom composition (spirocysts, basitrichs, atrichs,
and microbasic p-mastigophores) characteristic of the genus Anthopleura (Hand,
1955). However, three size classes of basitrichs were found in mesenteric "laments of
the `lighta morph in contrast to only two such classes found in the `darka morph
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
745
Table 4
Nematocyst and spirocyst mean size (lm) and standard deviation (S.D.) in `lighta and `darka color morphs
of Anthopleura orientalis
Tissue
Tentacles:
Acrorhagi:
Mesenteric
"laments:
Actinopharynx:
Type of cnida
Spirocyst
Basitrich
Spirocyst
Atrich
Basitrich
Basitrich 1
Basitrich 2
Basitrich 3
Basitrich
`Lighta morph
`Darka morph
Mean
S.D.
Mean
S.D.
18.0
19.2
27.7
43.1
12.1
13.4
19.8
33.6
24.2
1.51
0.95
2.43
5.13
1.62
1.50
1.94
2.46
1.50
15.9
19.6
25.5
58.4
12.5
13.9
*
33.3
26.5
1.68
1.46
1.95
1.85
1.35
1.20
*
3.72
1.86
t!
p
3.30
1.31
4.63
13.39
3.69
3.48
0.005
0.212
0.006
0.000"
0.010
0.005
0.58
7.22
0.572
0.000"
!Student's t-test is calculated using individual means of the 10 individuals of each color morph (12
nematocysts or spirocysts of each type and/or size class are analyzed per individual).
"p"0.000 means p(0.0005.
*Data not available (basitrichs 2 are absent from mesenteric "laments of the `darka morph).
(Table 4). Nematocyst and spirocyst measurements summarized in Table 4 provide
additional evidence that we are dealing with two separate species: highly signi"cant
(p(0.05) di!erences in the size of nematocysts and spirocysts are revealed in six out
of eight comparisons made. Microbasic p-mastigophores were not included in our
statistical analysis because they proved very rare and we were unable to make
measurements of su$cient number of such cells per tissue and per individual.
4. Discussion
The results obtained through genetic and nematocyst analyses provide strong
evidence that the two studied color morphs of the sea anemone A. orientalis are indeed
two valid biological species. Because all the nematocyst and ecological characteristics
of the `lighta morph are identical with those described for A. orientalis by Averintsev
(1967), this morph will subsequently be referred to as A. orientalis and the `darka
morph as Anthopleura sp.
Two main features of the allele frequency data presented in Table 3 are: (1) high
levels of intraspeci"c allozymic variability within each species studied and (2) high
level of interspeci"c genetic divergence.
A high level of allozymic variation is characteristic of the majority of electrophoretically studied cnidarian species. The mean value of the H estimate calculated for 27
%
cnidarian species listed in review by SoleH -Cava and Thorpe (1991) is 0.197. A very
similar value of 0.200 was obtained from the same source of data for 6 sea anemone
species of the genus Anthopleura. Our estimates obtained for A. orientalis and Anthopleura sp. are even higher being 0.250 and 0.351, respectively. The mean expected
746
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
heterozygosity estimates obtained by Smith and Potts (1987) for A. artemisia (0.375)
and by us for Anthopleura sp. (0.351) are very similar and both are quite speci"c in that
they are the highest heterozygosity estimates so far reported for species of Anthopleura.
The only sea anemone species that demonstrates a higher H estimate (0.401) is
%
Urticina eques (SoleH -Cava and Thorpe, 1991). High levels of intraspeci"c genetic
variation in a majority of marine invertebrate phyla (Nevo et al., 1984; Manchenko,
1989; SoleH -Cava and Thorpe, 1991; Ward et al., 1992) seems to be a general rule with
few exceptions (e.g., Thorpe and Beardmore, 1981; Hedgecock et al., 1982). It should
be stressed that the value of expected heterozygosity (H "0.144$0.034) obtained
%
for A. orientalis in our previous survey (Manchenko and Balakirev, 1984) through the
analysis of 42 isozyme loci is considerably lower than that obtained in the present
work. This di!erence may be attributed to di!erent sets of isozyme loci used in these
surveys.
The level of genetic divergence between A. orientalis and Anthopleura sp. is very high
(I"0.277; D"1.284). It is several times higher than that revealed by SoleH -Cava and
Thorpe (1992) between species of the Actinia equina/prasina complex and comparable
with the level of genetic divergence between sea anemone species from di!erent
confamilial genera and even between species from di!erent families (SoleH -Cava et al.,
1994). Recently, even more drastic genetic divergence (I"0.11; D"2.25) was revealed between `reda Mediterranean and `orangea Isle of Man A. equina species
(Monterio et al., 1997). In general, genetic divergence between A. orientalis and
Anthopleura sp. is about two times higher than that characteristic of congeneric species
(I"0.540; D"0.616) and close to the level of genetic divergence between species
belonging to di!erent confamilial genera (I"0.273; D"1.298) (Thorpe, 1982). A high
level of genetic divergence between morphologically cryptic species is characteristic of
many lower marine invertebrates (e. g., Manchenko and Kulikova, 1988, 1996;
Monterio et al., 1997; SoleH -Cava et al., 1991; Manchenko and Radashevsky, 1993,
1998; present study). This has been explained by much lower rate of morphological
evolution in such species in comparison with the rate of their molecular evolution
(Manchenko and Kulikova, 1988). Such situations were suggested to be a common
phenomenon in species which have reached the peak of their morphological and
ecological adaptation (Palumbi and Benzie, 1991; Todaro et al., 1996).
Signi"cant di!erences were found in the mean size of some nematocyst types
between A. orientalis and Anthopleura sp. (Table 4). The atrichs from acrorhagi and
basitrichs from actinopharynx contribute most to the overall interspeci"c di!erence.
Spirocysts from tentacles and acrorhagi also demonstrate statistically signi"cant
di!erences (p(0.01) between the species studied. These results are in good accordance with those obtained by Averintsev (1967) who described di!erences between A.
orientalis and A. artemisia in the size of spirocysts from acrorhagi. However, spirocysts
are considered as being of little taxonomic signi"cance in Anthozoa in general
(Schmidt, 1974; Manuel, 1988).
The comparison of our data (Table 4) with those of Hand (1955) provides evidence
that the distribution of nematocysts in Anthopleura sp. is di!erent from that of A.
orientalis and A. xanthogrammica and most similar to that of A. artemisia. However,
Anthopleura sp. and A. artemisia demonstrate at least one signi"cant di!erence that
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
747
may be considered as interspeci"c one: we observed only two size classes of basitrichs
in mesenteric "laments of Anthopleura sp., while three such classes were revealed in A.
orientalis (Table 4) and reported in A. artemisia by Hand (1955) and in A. orientalis and
A. artemisia by Averintsev (1967). Our electrophoretic data provide additional evidences that Anthopleura sp. and A. artemisia are rather di!erent species. First, three
anodally migrating monomorphic bands of MDH activity were revealed by us in
Anthopleura sp. (data not included in this paper because MDH proved not scorable
in A. orientalis) in contrast to only one monomorphic band of MDH activity reported
in A. artemisia by Smith and Potts (1987). Second, the absence of individuals with
identical multilocus genotypes in samples of the sea anemone species studied in this
work indicates that individuals in these samples have originated through sexual
reproduction. By contrast, Smith and Potts (1987) reported that genotypic proportions in A. artemisia deviate markedly from equilibrium due to a mixed (asexual and
sexual) reproduction. Are electrophoretic di!erences su$cient to state that Anthopleura sp. and A. artemisia are separate species? We think that the "rst di!erence is
su$cient. Indeed, Smith and Potts (1987) and we in this study used electrophoretic
bu!er systems which were very similar in their pH values (7.4 and 7.0, respectively).
However, the results obtained in these two surveys were drastically di!erent. We
observed three-banded electrophoretic pattern of MDH with two of the most distant
bands separated by about 2 cm distance from each other. We believe that these bands
should also be resolvable in a bu!er system (pH 7.4) used by Smith and Potts (1987).
As they did not reveal these bands we think that they dealt with a species other than
Anthopleura sp. The fact that we did not "nd any evidence for asexual reproduction in
our sample of Anthopleura sp. does not mean that the species is not capable of
reproducing asexually under other conditions as it was found in some other species of
Anthopleura (Lin et al., 1992; Tsuchida and Potts, 1994a,b). Summarizing both
nematocyst and electrophoretic di!erences discussed above, we conclude that Anthopleura sp. and A. artemisia are separate species.
Preliminary discrimination between A. orientalis and Anthopleura sp. can be made
on the basis of the di!erences listed in Table 1. However, the most important of these,
the color of the oral disk, may prove to be of small diagnostic value in the sea
anemone samples taken from other geographic localities because this character is
highly variable in A. orientalis (E.E. Kostina, pers. commun.). It should also be
remembered that such characteristic as immersion in sand and the amount of
encrustation on the body column can be changed as a result of the photoadaptation
process (Dykens and Shick, 1984) and thus should be used with caution. Our present
study provides evidence that eight isozyme loci (see Table 3) and the basitrichs from
mesenteric "laments (Table 4) are diagnostic characters suitable for unequivocal
discrimination between A. orientalis and Anthopleura sp. It also provides evidence that
Anthopleura sp. and A. artemisia are di!erent species. It is unclear, however, whether or
not A. artemisia and A. xanthogrammica described from the Eastern North Paci"c are
indeed present in the Sea of Japan. A species complex including several morphologically cryptic species may be erroneously considered as a single widely distributed
species. Using methods of molecular systematics, such situations have been recently
distinguished in lower marine invertebrates (SoleH -Cava et al., 1991; Todaro et al.,
748
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
1996). The taxonomic integrity of cosmopolitan and widely distributed species of
lower marine invertebrates is therefore open to doubt and should be considered with
caution (Manchenko and Radashevsky, 1998). The combination of electrophoretic
and nematocyst comparisons of sympatric sea anemones of the genus Anthopleura,
collected from the same localities as those studied by Averintsev (1967), is expected to
be a powerful tool suitable for reliable identi"cation of and discrimination between
species of Anthopleura inhabiting the Sea of Japan.
Acknowledgements
This study was supported in part by Russian Foundation for Basic Research grant
no 94-04-12503. We wish to thank E.E. Kostina for useful consultations on the
morphology and systematics of Anthopleura and A.I. Pudovkin for reading the
manuscript and valuable comments. We also thank D.G. Buth and two anonymous
referees for their constructive suggestions and criticism.
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High level of genetic divergence between
sympatric color morphs of the littoral sea
anemone Anthopleura orientalis
(Anthozoa: Actiniaria)
Gennady P. Manchenko*, Tatyana N. Dautova, Yury Y. Latypov
Institute of Marine Biology, Vladivostok 690041, Russia
Received 18 May 1998; received in revised form 6 October 1998; accepted 26 July 1999
Abstract
Using enzyme electrophoresis and nematocyst analysis, the sympatrically occurring `lighta
and `darka color morphs of the sea anemone Anthopleura orientalis from the Sea of Japan were
shown to be two valid species. The `lighta morph was identi"ed as A. orientalis (Averintsev,
1967 Issledovaniya fauny morei: Vyp. 5 (13). Nanka, Leningrad, pp. 62}77), while the `darka
morph was designated as Anthopleura sp. The analysis of 21 isozyme loci revealed high value of
Nei's genetic distance (D"1.284) between the two species, which are indistinguishable in their
external morphology. The mean values of observed and expected heterozygosities for A.
orientalis and Anthopleura sp. are high (H "0.252$0.061, H "0.250$0.061 and
0
%
H "0.327$0.052, H "0.351$0.054, respectively). The species di!er signi"cantly in the size
0
%
of spirocysts and nematocysts, among which the atrichs from acrorhagi and the basitrichs from
actinopharynx contribute most to the observed di!erence. Strong qualitative di!erence is
revealed between distributions of nematocysts in mesenteric "laments of the two sea anemone
species studied. The possible conspeci"city of Anthopleura sp. with Anthopleura artemisia (Dana,
1848) is discussed and the conclusion made that these are two separate species. ( 2000
Elsevier Science Ltd. All rights reserved.
Keywords: Actiniaria; Anthopleura orientalis; Anthozoa; Color morphs; Sibling species; Enzyme electrophoresis; Genetic divergence; Genetic variation
* Corresponding author. Tel.: #7-4232-310905; Fax: #7-4232-310900.
E-mail address: [email protected] (G.P. Manchenko)
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 9 9 ) 0 0 1 2 0 - 9
738
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
1. Introduction
Sea anemones are a very diverse group of lower marine invertebrates (Shick, 1991).
Because of relative structural simplicity, the taxonomically useful morphological
characters are few and taxonomic problems many in these organisms. The littoral sea
anemones of the genus Anthopleura are very common on sandy shores of the Russian
coast of the Sea of Japan, however, their taxonomic status was not carefully studied.
The only taxonomic study of Anthopleura from this region was carried out by
Averintsev (1967) who described new species A. orientalis. Two other members of the
genus, A. artemisia (Dana, 1848) and A. xanthogrammica (Brandt, 1835) described from
the Eastern North Paci"c, were also ideti"ed by Averintsev (1967, 1976)) and Kostina
(1989) in the Sea of Japan. Anthopleura from the Eastern North Paci"c are highly
polymorphic in body color and morphology and have no reliable species-speci"c
external characters (Hand, 1955). The same is true for Anthopleura from the Sea of
Japan, where a continuous range of sympatric color varieties can be found in samples
collected during "eld observations. The taxonomic status of such varieties is usually
uncertain.
Enzyme electrophoresis has proved a particularly useful method in distinguishing
morphologically cryptic species in various invertebrate groups (Knowlton, 1993;
Thorpe and SoleH -Cava, 1994). It is an especially powerful tool for discriminating
between sympatric sibling species. From conventional de"nitions according to the
biological species concept, such species should have di!erent allele frequencies or
di!erent "xed alleles at least at some gene loci, thus giving evidence that the species
under question are reproductively isolated. Enzyme electrophoretic studies have
clearly demonstrated that many sympatric `morphsa of sea anemone species are
reproductively isolated and thus are valid separate species (Carter and Thorpe, 1981;
Bucklin and Hedgekock, 1982; Haylor et al., 1984; SoleH -Cava et al., 1985; SoleH -Cava
and Thorpe, 1987, 1992; McFadden et al., 1997).
The objective of this work was to establish the taxonomic status of two sympatric
color morphs of A. orientalis from northern part of the Sea of Japan using enzyme
electrophoresis in conjunction with nematocyst analysis. In the text we refer to the
`lighta and the `darka color morphs of A. orientalis until the taxonomic status of each
morph has been determined.
2. Materials and methods
2.1. Collection of samples
Samples of two color morphs of A. orientalis, the `lighta morph and the `darka
morph, were collected from the shoals (less than 2 m deep) at Skrebtsov Island
(Amursky Bay, the Sea of Japan) near Vladivostok. Collections were made over the
period July}September 1995 from sites of gravely}rocky substratum covered by sand.
Some characters of external morphology of the two color morphs are listed in Table 1.
Oral and pedal disk diameters were measured using relaxed anemones treated with
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
739
Table 1
Some morphological and ecological characters of `Lighta and `Darka color morphs of Anthopleura
orientalis
Character
Oral disk diameter
Pedal disk diameter
Column color
Oral disk color
Pedal disc color
Veruccae
Substratum
Immersion in sand
Color morph
`Lighta
`Darka
18}34 mm
15}30 mm
Green}gray
Green}gray (light)
Gray}brown
Not extending to limbus
Fine}grained gravel, dead broken shells
Less than 1/2 of the column length
19}32 mm
15}30 mm
Green}gray
Black}gray (dark)
Gray}brown
Not extending to limbus
Coarse}grained gravel, small stones
More than 2/3 of the column length
magnesium chloride. All anemones were adult and dioecious as evidenced from the
inspection of their gonad preparations. After collection, the anemones were kept in the
laboratory in running sea water before electrophoresis and nematocyst analysis.
Voucher specimens of the `lighta and the `darka morphs are deposited in Zoological
Institute, St. Petersburg, Russia (registration number 9.208) and the Institute of
Marine Biology, Vladivostok, Russia (registration number 3175), respectively.
2.2. Enzyme electrophoresis
Before sample preparation the anemones were placed in a freezer (!123C) for 1 h.
For electrophoresis, longitudinal sections were cut from frozen anemones to include
tissues of the oral disc (with tentacles), internal organs, and the column. Tissue
samples were homogenized in two volumes of distilled water and crude homogenates
analyzed by horizontal 14% starch-gel electrophoresis as described by Manchenko
and Balakirev (1984). In total, 82 individuals of the `lighta morph and 19 individuals
of the `darka morph were available for electrophoretic analysis. Two continuous
bu!er systems were used: TEB (tris-EDTA-boric acid, pH 8.5) and TC (tris-citric acid,
pH 7.0). The staining of electrophoretic gels followed standard procedures, using
recipes from Manchenko (1994). Enzymes assayed, bu!ers used and isozyme loci
scorable in both color morphs are listed in Table 2. Gene loci coding for ASTA-2,
DHLDH-1, FDH-1, GLDH-2, IDH-1, and MD-2 isozymes proved not scorable
because of low activity and/or poor resolution of corresponding isozymes in one or
both the morphs. These loci were not taken into account in our further considerations
based on the use of allele frequency data.
Genetic interpretations of banding patterns developed on zymograms were made
according to Buth (1990) taking into account previously obtained electrophoretic data
for A. orientalis (Manchenko and Balakirev, 1984). Enzyme nomenclature followed
recommendations of the IUBMB NC (1992). The locus and allele designations were
formated according to principles recommended for protein-coding loci (Shaklee et al.,
740
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
Table 2
Enzymes assayed, electrophoretic bu!ers used and isozyme loci scored in `Lighta and `Darka color morphs
of Anthopleura orientalis
Enzyme
EC No.
Locus
Bu!er
Aconitate hydratase
Alanine transaminase
b-Alanopine dehydrogenase
Aspartate transaminase
Catalase
Dihydrolipoamide dehydrogenase
Formaldehyde dehydrogenase (glutathione)
Fumarate hydratase
Glutamate dehydrogenase (NADP)
Hexokinase
Isocitrate dehydrogenase (NADP)
Lactoylglutathione lyase
Leucyl aminopeptidase
Mannose-6-phosphate isomerase
Methylumbelliferyl-acetate deacetylase
Octopine dehydrogenase
Peptidase (detected with val}leu)
Phosphoglucomutase
Xanthine dehydrogenase
4.2.1.3
2.6.1.2
1.5.1.26
2.6.1.1
1.11.1.6
1.8.1.4
1.2.1.1
4.2.1.2
1.4.1.4
2.7.1.1
1.1.1.42
4.4.1.5
3.4.11.1
5.3.1.8
3.1.1.56
1.5.1.11
3.4.11... or 13...
5.4.2.2
1.1.1.204
Ah
Alta
b-Alpdh
Asta-1
Cat
Dhldh-2
Fdh-2
Fh
Gldh-1
Hk
Idh-2
Lgl
Lap
Mpi
Md-1, Md-3
Ondh
Pep-1, Pep-3
Pgm
Xdh
TC
TC
TC
TEB
TC
TC
TC
TC
TC
TEB
TC
TEB
TEB
TC
TC
TC
TEB
TEB
TEB
1990) with minor modi"cation (asterisks were omitted from designations of isozyme
loci).
2.3. Nematocyst and spirocyst analysis
The nematocyst types were identi"ed as atrichs, basitrichs, and microbasic pmastigophores following the terminology of Carlgren (1949) and Hand (1955). Thousands of nematocysts were visually inspected before "nal determination of the number
of di!erent nematocyst types was made. Twelve spirocysts of each size class and 12
nematocysts of each type and each size class were measured in macerated tissue
samples from 10 individuals of each of the two color morphs. Sea anemones with
approximately equal body size were chosen for nematocyst and spirocyst comparison
to avoid undesirable in#uence of the body size on the size of cnida (Chintiroglou and
Simsiridou, 1997).
2.4. Data analysis
Nematocyst and spirocyst data were compared between color morphs using Student's t-test provided by the SYSTAT 4.0 computer program (Wilkinson, 1987).
Genotype frequencies were inferred by direct count from banding patterns observed
on zymograms. Allele frequencies, genetic variability measures and unbiased (Nei,
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
741
1978) genetic identity (I) and genetic distance (D) estimates were calculated from
genotype frequencies using the BIOSYS-1 computer program of Swo!ord and Selander (1981).
Because of small sample sizes, the expected numbers of heterozygotes were in some
cases too small to allow computation of conventional Chi-square. Therefore, signi"cance of deviations of observed genotype frequencies from those expected under
Hardy}Weinberg equilibrium was estimated using the pseudo-probability test (the
CHIHW program by Zaykin and Pudovkin, 1993).
When testing for conformity of genotype distribution to the Hardy}Weinberg
equilibrium, a number of separate tests for individual loci are usually performed. To
avoid Type I errors, corrections for multiple tests were performed using Sidak's
multiplicative inequality for calculations of critical values of the Chi-square distribution (Rolf and Sokal, 1981, p. 101; Sokal and Rolf, 1981, p. 728). The program
MULTTEST (Zaykin and Pudovkin, 1991) was used to "nd the critical values of
Chi-square for each replicate test considering that it was part of a set of separate
independent tests.
3. Results
3.1. Genetic analysis
In total, 21 isozyme loci coding for 19 enzyme systems were resolved and proved
scorable in both color morphs (Table 3). Allozyme variations with one-banded
homozygotes and two-banded heterozygotes were revealed at Ah, b-Alpdh, Hk, ¸ap,
Mpi, Ondh, and Pgm loci providing evidence that catalytically active molecules of the
corresponding isozymes are monomers. Three-banded allozyme patterns characteristic of dimeric enzymes were observed in individuals heterozygous for Asta-1, Fdh-2,
Idh-2, Lgl, and Md-1 loci. Clearly resolved "ve-banded allozyme patterns in individuals heterozygous for the Cat locus give strong evidence that this enzyme is
a tetramer. Allozyme patterns in individuals heterozygous for Alta, Fh, Gldh-1, Pep-1,
Pep-2 and Xdh loci were displayed as broad di!use bands not resolved into separate
allozymes, thus suggesting that the corresponding enzymes are rather oligomers. No
allozyme variants were revealed in Dhldh-2 and Md-3 loci.
The allele frequencies for 21 isozyme loci studied in `lighta and `darka color
morphs of A. orientalis are given in Table 3. Eight loci (Ah, Alta, Fdh-2, Gldh-1, Idh-2,
Lgl, Md-1, and Pep-2) share no common alleles in the `lighta and `darka morphs
providing strong evidence that gene pools of these morphs are separated.
Genetic divergence between studied color morphs of A. orientalis is very high as is
evident from the genetic identity (I"0.277) and genetic distance (D"1.284) estimates. Mean values of observed and expected heterozygosities for `lighta morph
(H "0.252$0.061; H "0.250$0.061) and `darka morph (H "0.327$0.052;
0
%
0
H "0.351$0.054) are also very high.
%
We did not observe identical genotypes over all loci analyzed in any two or more
individuals either in `lighta or `darka color morphs. This provides evidence that no
742
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
Table 3
Allele frequencies at 21 isozyme loci in the `lighta and `darka color morphs of Anthopleura orientalis
Locus
Allele
Color morph
`Lighta
`Darka
Ah
(N)!
1
2
3
4
5
6
7
11
0.000
0.000
0.000
0.000
0.000
0.045
0.955
12
0.042
0.042
0.750
0.125
0.042
0.000
0.000
b-Alpdh
(N)
1
2
3
7
1.000
0.000
0.000
11
0.136
0.636
0.227
Alta
(N)
1
2
3
27
0.981
0.019
0.000
12
0.000
0.000
1.000
Asta-1
(N)
1
2
3
4
42
0.274
0.274
0.440
0.012
15
0.100
0.867
0.033
0.000
Cat
(N)
1
2
3
4
5
6
7
8
9
10
76
0.125
0.007
0.000
0.033
0.000
0.553
0.000
0.283
0.000
0.000
17
0.000
0.000
0.029
0.000
0.118
0.000
0.235
0.088
0.147
0.382
Dhldh-2
(N)
1
18
1.000
12
1.000
Fdh-2
(N)
1
2
3
4
5
76
0.000
0.000
0.007
0.987
0.007
19
0.816
0.184
0.000
0.000
0.000
Fh
(N)
1
2
3
4
18
0.861
0.000
0.056
0.083
12
0.000
0.792
0.208
0.000
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
743
Table 3 (continued)
Locus
Allele
Color morph
`Lighta
`Darka
Gldh-1
(N)
1
2
3
27
0.000
0.000
1.000
12
0.958
0.042
0.000
Hk
(N)
1
2
3
4
5
6
7
8
81
0.000
0.006
0.093
0.235
0.377
0.167
0.025
0.099
17
0.029
0.206
0.412
0.029
0.324
0.000
0.000
0.000
Idh-2
(N)
1
2
3
4
5
75
0.000
0.000
0.020
0.973
0.007
11
0.045
0.955
0.000
0.000
0.000
¸ap
(N)
1
2
3
4
5
6
82
0.000
0.000
0.104
0.854
0.024
0.018
17
0.029
0.794
0.147
0.029
0.000
0.000
¸gl
(N)
1
2
3
4
5
81
0.006
0.994
0.000
0.000
0.000
17
0.000
0.000
0.676
0.088
0.235
Mpi
(N)
1
2
3
4
5
6
7
8
9
28
0.018
0.107
0.161
0.054
0.054
0.339
0.089
0.179
0.000
12
0.000
0.042
0.125
0.000
0.000
0.542
0.125
0.083
0.083
Md-1
(N)
1
2
3
4
5
61
0.000
0.148
0.000
0.016
0.836
14
0.036
0.000
0.964
0.000
0.000
Table 3 (continued on next page)
744
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
Table 3 (continued)
Locus
Allele
Color morph
`Lighta
`Darka
Md-3
(N)
1
61
1.000
14
1.000
Ondh
(N)
1
2
3
4
7
0.000
0.071
0.714
0.214
10
0.150
0.350
0.500
0.000
Pep-1
(N)
1
2
3
81
0.981
0.000
0.019
17
0.000
0.500
0.500
Pep-2
(N)
1
2
3
4
5
81
0.000
0.000
0.006
0.981
0.012
17
0.794
0.206
0.000
0.000
0.000
Pgm
(N)
1
2
3
4
5
6
81
0.000
0.216
0.660
0.086
0.037
0.000
17
0.029
0.000
0.029
0.735
0.000
0.206
Xdh
(N)
1
2
3
42
0.012
0.798
0.190
15
0.033
0.833
0.133
!(N)"number of analyzed individuals.
asexually produced individuals (or clonemates) were involved in the analysis. The
genotype frequencies observed in both the color morphs corresponded well the
Hardy}Weinberg equilibrium when having applied Sidak's correction for the whole
set of tests.
3.2. Nematocyst and spirocyst analysis
Both color morphs have the cnidom composition (spirocysts, basitrichs, atrichs,
and microbasic p-mastigophores) characteristic of the genus Anthopleura (Hand,
1955). However, three size classes of basitrichs were found in mesenteric "laments of
the `lighta morph in contrast to only two such classes found in the `darka morph
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
745
Table 4
Nematocyst and spirocyst mean size (lm) and standard deviation (S.D.) in `lighta and `darka color morphs
of Anthopleura orientalis
Tissue
Tentacles:
Acrorhagi:
Mesenteric
"laments:
Actinopharynx:
Type of cnida
Spirocyst
Basitrich
Spirocyst
Atrich
Basitrich
Basitrich 1
Basitrich 2
Basitrich 3
Basitrich
`Lighta morph
`Darka morph
Mean
S.D.
Mean
S.D.
18.0
19.2
27.7
43.1
12.1
13.4
19.8
33.6
24.2
1.51
0.95
2.43
5.13
1.62
1.50
1.94
2.46
1.50
15.9
19.6
25.5
58.4
12.5
13.9
*
33.3
26.5
1.68
1.46
1.95
1.85
1.35
1.20
*
3.72
1.86
t!
p
3.30
1.31
4.63
13.39
3.69
3.48
0.005
0.212
0.006
0.000"
0.010
0.005
0.58
7.22
0.572
0.000"
!Student's t-test is calculated using individual means of the 10 individuals of each color morph (12
nematocysts or spirocysts of each type and/or size class are analyzed per individual).
"p"0.000 means p(0.0005.
*Data not available (basitrichs 2 are absent from mesenteric "laments of the `darka morph).
(Table 4). Nematocyst and spirocyst measurements summarized in Table 4 provide
additional evidence that we are dealing with two separate species: highly signi"cant
(p(0.05) di!erences in the size of nematocysts and spirocysts are revealed in six out
of eight comparisons made. Microbasic p-mastigophores were not included in our
statistical analysis because they proved very rare and we were unable to make
measurements of su$cient number of such cells per tissue and per individual.
4. Discussion
The results obtained through genetic and nematocyst analyses provide strong
evidence that the two studied color morphs of the sea anemone A. orientalis are indeed
two valid biological species. Because all the nematocyst and ecological characteristics
of the `lighta morph are identical with those described for A. orientalis by Averintsev
(1967), this morph will subsequently be referred to as A. orientalis and the `darka
morph as Anthopleura sp.
Two main features of the allele frequency data presented in Table 3 are: (1) high
levels of intraspeci"c allozymic variability within each species studied and (2) high
level of interspeci"c genetic divergence.
A high level of allozymic variation is characteristic of the majority of electrophoretically studied cnidarian species. The mean value of the H estimate calculated for 27
%
cnidarian species listed in review by SoleH -Cava and Thorpe (1991) is 0.197. A very
similar value of 0.200 was obtained from the same source of data for 6 sea anemone
species of the genus Anthopleura. Our estimates obtained for A. orientalis and Anthopleura sp. are even higher being 0.250 and 0.351, respectively. The mean expected
746
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
heterozygosity estimates obtained by Smith and Potts (1987) for A. artemisia (0.375)
and by us for Anthopleura sp. (0.351) are very similar and both are quite speci"c in that
they are the highest heterozygosity estimates so far reported for species of Anthopleura.
The only sea anemone species that demonstrates a higher H estimate (0.401) is
%
Urticina eques (SoleH -Cava and Thorpe, 1991). High levels of intraspeci"c genetic
variation in a majority of marine invertebrate phyla (Nevo et al., 1984; Manchenko,
1989; SoleH -Cava and Thorpe, 1991; Ward et al., 1992) seems to be a general rule with
few exceptions (e.g., Thorpe and Beardmore, 1981; Hedgecock et al., 1982). It should
be stressed that the value of expected heterozygosity (H "0.144$0.034) obtained
%
for A. orientalis in our previous survey (Manchenko and Balakirev, 1984) through the
analysis of 42 isozyme loci is considerably lower than that obtained in the present
work. This di!erence may be attributed to di!erent sets of isozyme loci used in these
surveys.
The level of genetic divergence between A. orientalis and Anthopleura sp. is very high
(I"0.277; D"1.284). It is several times higher than that revealed by SoleH -Cava and
Thorpe (1992) between species of the Actinia equina/prasina complex and comparable
with the level of genetic divergence between sea anemone species from di!erent
confamilial genera and even between species from di!erent families (SoleH -Cava et al.,
1994). Recently, even more drastic genetic divergence (I"0.11; D"2.25) was revealed between `reda Mediterranean and `orangea Isle of Man A. equina species
(Monterio et al., 1997). In general, genetic divergence between A. orientalis and
Anthopleura sp. is about two times higher than that characteristic of congeneric species
(I"0.540; D"0.616) and close to the level of genetic divergence between species
belonging to di!erent confamilial genera (I"0.273; D"1.298) (Thorpe, 1982). A high
level of genetic divergence between morphologically cryptic species is characteristic of
many lower marine invertebrates (e. g., Manchenko and Kulikova, 1988, 1996;
Monterio et al., 1997; SoleH -Cava et al., 1991; Manchenko and Radashevsky, 1993,
1998; present study). This has been explained by much lower rate of morphological
evolution in such species in comparison with the rate of their molecular evolution
(Manchenko and Kulikova, 1988). Such situations were suggested to be a common
phenomenon in species which have reached the peak of their morphological and
ecological adaptation (Palumbi and Benzie, 1991; Todaro et al., 1996).
Signi"cant di!erences were found in the mean size of some nematocyst types
between A. orientalis and Anthopleura sp. (Table 4). The atrichs from acrorhagi and
basitrichs from actinopharynx contribute most to the overall interspeci"c di!erence.
Spirocysts from tentacles and acrorhagi also demonstrate statistically signi"cant
di!erences (p(0.01) between the species studied. These results are in good accordance with those obtained by Averintsev (1967) who described di!erences between A.
orientalis and A. artemisia in the size of spirocysts from acrorhagi. However, spirocysts
are considered as being of little taxonomic signi"cance in Anthozoa in general
(Schmidt, 1974; Manuel, 1988).
The comparison of our data (Table 4) with those of Hand (1955) provides evidence
that the distribution of nematocysts in Anthopleura sp. is di!erent from that of A.
orientalis and A. xanthogrammica and most similar to that of A. artemisia. However,
Anthopleura sp. and A. artemisia demonstrate at least one signi"cant di!erence that
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
747
may be considered as interspeci"c one: we observed only two size classes of basitrichs
in mesenteric "laments of Anthopleura sp., while three such classes were revealed in A.
orientalis (Table 4) and reported in A. artemisia by Hand (1955) and in A. orientalis and
A. artemisia by Averintsev (1967). Our electrophoretic data provide additional evidences that Anthopleura sp. and A. artemisia are rather di!erent species. First, three
anodally migrating monomorphic bands of MDH activity were revealed by us in
Anthopleura sp. (data not included in this paper because MDH proved not scorable
in A. orientalis) in contrast to only one monomorphic band of MDH activity reported
in A. artemisia by Smith and Potts (1987). Second, the absence of individuals with
identical multilocus genotypes in samples of the sea anemone species studied in this
work indicates that individuals in these samples have originated through sexual
reproduction. By contrast, Smith and Potts (1987) reported that genotypic proportions in A. artemisia deviate markedly from equilibrium due to a mixed (asexual and
sexual) reproduction. Are electrophoretic di!erences su$cient to state that Anthopleura sp. and A. artemisia are separate species? We think that the "rst di!erence is
su$cient. Indeed, Smith and Potts (1987) and we in this study used electrophoretic
bu!er systems which were very similar in their pH values (7.4 and 7.0, respectively).
However, the results obtained in these two surveys were drastically di!erent. We
observed three-banded electrophoretic pattern of MDH with two of the most distant
bands separated by about 2 cm distance from each other. We believe that these bands
should also be resolvable in a bu!er system (pH 7.4) used by Smith and Potts (1987).
As they did not reveal these bands we think that they dealt with a species other than
Anthopleura sp. The fact that we did not "nd any evidence for asexual reproduction in
our sample of Anthopleura sp. does not mean that the species is not capable of
reproducing asexually under other conditions as it was found in some other species of
Anthopleura (Lin et al., 1992; Tsuchida and Potts, 1994a,b). Summarizing both
nematocyst and electrophoretic di!erences discussed above, we conclude that Anthopleura sp. and A. artemisia are separate species.
Preliminary discrimination between A. orientalis and Anthopleura sp. can be made
on the basis of the di!erences listed in Table 1. However, the most important of these,
the color of the oral disk, may prove to be of small diagnostic value in the sea
anemone samples taken from other geographic localities because this character is
highly variable in A. orientalis (E.E. Kostina, pers. commun.). It should also be
remembered that such characteristic as immersion in sand and the amount of
encrustation on the body column can be changed as a result of the photoadaptation
process (Dykens and Shick, 1984) and thus should be used with caution. Our present
study provides evidence that eight isozyme loci (see Table 3) and the basitrichs from
mesenteric "laments (Table 4) are diagnostic characters suitable for unequivocal
discrimination between A. orientalis and Anthopleura sp. It also provides evidence that
Anthopleura sp. and A. artemisia are di!erent species. It is unclear, however, whether or
not A. artemisia and A. xanthogrammica described from the Eastern North Paci"c are
indeed present in the Sea of Japan. A species complex including several morphologically cryptic species may be erroneously considered as a single widely distributed
species. Using methods of molecular systematics, such situations have been recently
distinguished in lower marine invertebrates (SoleH -Cava et al., 1991; Todaro et al.,
748
G.P. Manchenko et al. / Biochemical Systematics and Ecology 28 (2000) 737}750
1996). The taxonomic integrity of cosmopolitan and widely distributed species of
lower marine invertebrates is therefore open to doubt and should be considered with
caution (Manchenko and Radashevsky, 1998). The combination of electrophoretic
and nematocyst comparisons of sympatric sea anemones of the genus Anthopleura,
collected from the same localities as those studied by Averintsev (1967), is expected to
be a powerful tool suitable for reliable identi"cation of and discrimination between
species of Anthopleura inhabiting the Sea of Japan.
Acknowledgements
This study was supported in part by Russian Foundation for Basic Research grant
no 94-04-12503. We wish to thank E.E. Kostina for useful consultations on the
morphology and systematics of Anthopleura and A.I. Pudovkin for reading the
manuscript and valuable comments. We also thank D.G. Buth and two anonymous
referees for their constructive suggestions and criticism.
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