*Corresponding author. Tel.:#7-4232-310905; Fax:#7-4232-310900.
E-mail address:[email protected] (G.P. Manchenko)
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`darkacolor morphs of the sea anemoneAnthopleura orientalisfrom the Sea of Japan were shown to be two valid species. The`lightamorph was identi"ed asA. orientalis(Averintsev, 1967 Issledovaniya fauny morei: Vyp. 5 (13). Nanka, Leningrad, pp. 62}77), while the`darka
morph was designated asAnthopleurasp. 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"0.252$0.061, H%"0.250$0.061 and H
0"0.327$0.052,H%"0.351$0.054, respectively). The species di!er signi"cantly in the size 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 ofAnthopleurasp. withAnthopleura 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 elec-trophoresis; Genetic divergence; Genetic variation
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 genusAnthopleuraare 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 speciesA. orientalis. Two other members of the genus,A. artemisia(Dana, 1848) andA.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. Anthopleurafrom 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 forAnthopleurafrom 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 ofA. orientalis from northern part of the Sea of Japan using enzyme electrophoresis in conjunction with nematocyst analysis. In the text we refer to the `lightaand the`darkacolor morphs ofA. orientalisuntil the taxonomic status of each morph has been determined.
2. Materials and methods
2.1. Collection of samples
Table 1
Some morphological and ecological characters of`Lighta and `Darka color morphs of Anthopleura orientalis
Character Color morph
`Lighta `Darka
Oral disk diameter 18}34 mm 19}32 mm
Pedal disk diameter 15}30 mm 15}30 mm
Column color Green}gray Green}gray
Oral disk color Green}gray (light) Black}gray (dark)
Pedal disc color Gray}brown Gray}brown
Veruccae Not extending to limbus Not extending to limbus
Substratum Fine}grained gravel, dead broken shells Coarse}grained gravel, small stones Immersion in sand Less than 1/2 of the column length 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`lightaand the`darkamorphs 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`lightamorph 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.
Table 2
Enzymes assayed, electrophoretic bu!ers used and isozyme loci scored in`Lightaand`Darkacolor morphs ofAnthopleura orientalis
Enzyme EC No. Locus Bu!er
Aconitate hydratase 4.2.1.3 Ah TC
Alanine transaminase 2.6.1.2 Alta TC
b-Alanopine dehydrogenase 1.5.1.26 b-Alpdh TC
Aspartate transaminase 2.6.1.1 Asta-1 TEB
Catalase 1.11.1.6 Cat TC
Dihydrolipoamide dehydrogenase 1.8.1.4 Dhldh-2 TC
Formaldehyde dehydrogenase (glutathione) 1.2.1.1 Fdh-2 TC
Fumarate hydratase 4.2.1.2 Fh TC
Glutamate dehydrogenase (NADP) 1.4.1.4 Gldh-1 TC
Hexokinase 2.7.1.1 Hk TEB
Isocitrate dehydrogenase (NADP) 1.1.1.42 Idh-2 TC
Lactoylglutathione lyase 4.4.1.5 Lgl TEB
Leucyl aminopeptidase 3.4.11.1 Lap TEB
Mannose-6-phosphate isomerase 5.3.1.8 Mpi TC
Methylumbelliferyl-acetate deacetylase 3.1.1.56 Md-1, Md-3 TC
Octopine dehydrogenase 1.5.1.11 Ondh TC
Peptidase (detected with val}leu) 3.4.11... or 13... Pep-1, Pep-3 TEB
Phosphoglucomutase 5.4.2.2 Pgm TEB
Xanthine dehydrogenase 1.1.1.204 Xdh 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 p-mastigophores following the terminology of Carlgren (1949) and Hand (1955). Thou-sands 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 Stu-dent'st-test provided by the SYSTAT 4.0 computer program (Wilkinson, 1987).
1978) genetic identity (I) and genetic distance (D) estimates were calculated from genotype frequencies using the BIOSYS-1 computer program of Swo!ord and Selan-der (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 distribu-tion (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 atAh,b-Alpdh,Hk,¸ap, Mpi,Ondh, andPgmloci providing evidence that catalytically active molecules of the corresponding isozymes are monomers. Three-banded allozyme patterns character-istic of dimeric enzymes were observed in individuals heterozygous forAsta-1,Fdh-2, Idh-2, Lgl, and Md-1 loci. Clearly resolved "ve-banded allozyme patterns in indi-viduals heterozygous for the Cat locus give strong evidence that this enzyme is a tetramer. Allozyme patterns in individuals heterozygous forAlta,Fh,Gldh-1,Pep-1, Pep-2andXdhloci 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 inDhldh-2andMd-3loci.
The allele frequencies for 21 isozyme loci studied in `lighta and `darka color morphs ofA. orientalisare 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 `lightaand `darka morphs providing strong evidence that gene pools of these morphs are separated.
Genetic divergence between studied color morphs ofA. orientalisis very high as is evident from the genetic identity (I"0.277) and genetic distance (D"1.284) esti-mates. Mean values of observed and expected heterozygosities for `lighta morph (H
0"0.252$0.061; H%"0.250$0.061) and `darka morph (H0"0.327$0.052;
H
%"0.351$0.054) are also very high.
Table 3
Allele frequencies at 21 isozyme loci in the`lightaand`darkacolor morphs ofAnthopleura orientalis
Table 3 (continued)
Table 3 (continued)
Locus Allele Color morph
`Lighta `Darka
Md-3 (N) 61 14
1 1.000 1.000
Ondh (N) 7 10
1 0.000 0.150
2 0.071 0.350
3 0.714 0.500
4 0.214 0.000
Pep-1 (N) 81 17
1 0.981 0.000
2 0.000 0.500
3 0.019 0.500
Pep-2 (N) 81 17
1 0.000 0.794
2 0.000 0.206
3 0.006 0.000
4 0.981 0.000
5 0.012 0.000
Pgm (N) 81 17
1 0.000 0.029
2 0.216 0.000
3 0.660 0.029
4 0.086 0.735
5 0.037 0.000
6 0.000 0.206
Xdh (N) 42 15
1 0.012 0.033
2 0.798 0.833
3 0.190 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
Table 4
Nematocyst and spirocyst mean size (lm) and standard deviation (S.D.) in`lightaand`darkacolor morphs ofAnthopleura orientalis
Tissue Type of cnida `Lightamorph `Darkamorph t! p
Mean S.D. Mean S.D.
Tentacles: Spirocyst 18.0 1.51 15.9 1.68 3.30 0.005
Basitrich 19.2 0.95 19.6 1.46 1.31 0.212
Acrorhagi: Spirocyst 27.7 2.43 25.5 1.95 4.63 0.006
Atrich 43.1 5.13 58.4 1.85 13.39 0.000"
Basitrich 12.1 1.62 12.5 1.35 3.69 0.010
Mesenteric Basitrich 1 13.4 1.50 13.9 1.20 3.48 0.005
"laments: Basitrich 2 19.8 1.94 * *
Basitrich 3 33.6 2.46 33.3 3.72 0.58 0.572
Actinopharynx: Basitrich 24.2 1.50 26.5 1.86 7.22 0.000"
!Student'st-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 meansp(0.0005.
*Data not available (basitrichs 2 are absent from mesenteric"laments of the`darkamorph).
(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 anemoneA. orientalisare indeed two valid biological species. Because all the nematocyst and ecological characteristics of the`lightamorph are identical with those described forA. orientalisby Averintsev (1967), this morph will subsequently be referred to as A. orientalis and the`darka morph asAnthopleurasp.
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 electrophoreti-cally studied cnidarian species. The mean value of theH
% estimate calculated for 27
heterozygosity estimates obtained by Smith and Potts (1987) forA. artemisia(0.375) and by us forAnthopleurasp. (0.351) are very similar and both are quite speci"c in that they are the highest heterozygosity estimates so far reported for species ofAnthopleura. 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
forA. orientalisin 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 betweenA. orientalisandAnthopleurasp. 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 theActinia equina/prasinacomplex 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 re-vealed between `reda Mediterranean and `orangea Isle of Man A. equina species (Monterio et al., 1997). In general, genetic divergence between A. orientalis and Anthopleurasp. 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 betweenA. orientalisandAnthopleurasp. (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 accord-ance with those obtained by Averintsev (1967) who described di!erences betweenA. orientalisandA. artemisiain 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).
may be considered as interspeci"c one: we observed only two size classes of basitrichs in mesenteric"laments ofAnthopleurasp., while three such classes were revealed inA. orientalis(Table 4) and reported inA. artemisiaby Hand (1955) and inA. orientalisand A. artemisiaby Averintsev (1967). Our electrophoretic data provide additional evid-ences thatAnthopleura sp. and A. artemisia are rather di!erent species. First, three anodally migrating monomorphic bands of MDH activity were revealed by us in Anthopleurasp. (data not included in this paper because MDH proved not scorable inA. orientalis) in contrast to only one monomorphic band of MDH activity reported in A. artemisiaby 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 propor-tions inA. artemisiadeviate markedly from equilibrium due to a mixed (asexual and sexual) reproduction. Are electrophoretic di!erences su$cient to state that Anthop-leurasp. andA. 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 Anthopleurasp. 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 Anthop-leurasp. andA. artemisiaare separate species.
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 genusAnthopleura, 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 ofAnthopleurainhabiting 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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