Population genetics of the family ostreidae. I. Intraspecific studies of Crassostrea gigas and Saccostrea commercialis

The levels of genetic variation and estimates of genetic similarity and distance were determined for populations of Crassostrea gigas (Thunberg) and Saccostrea commercialis (Iredale and Roughley). The proportion of polymorphic loci in Japanese populations of C. gigas ranged from 0.58 to 0.63, while the individual heterozygosities ranged from 0.20 to 0.22. The proportion of polymorphic loci in Australian populations of S. commercialis ranged from 0.43 to 0.46, the individual heterozygosities from 0.17 to 0.19. The genetic similarity between different geographical populations of both species was approximately 99%. The genetic distance and similarity data derived from this investigation has resulted in some provisional reclassifications. The Portuguese oyster C. angulata (Lamarck) appears to be more appropriately regarded as a recent colonized isolate of C. gigas. The New Zealand oyster S. glomerata (Gould) is considered a subspecies of the eastern Australian rock oyster S. commercialis. The Japanese Kumamoto population of C. gigas warrants reclassification as the non-sibling species C. sikamea.     

Molecular phylogenies help resolve taxonomic confusion with Asian <Emphasis Type="Italic">Crassostrea</Emphasis> oyster species

Identification of oyster species is still largely based on phenotypic characters that are highly plastic. Prompted by the proposed introduction of the Asian oyster species Crassostrea ariakensis into the Chesapeake Bay region of the U.S.A., this study uses molecular genetic information to understand the taxonomic framework surrounding C. ariakensis and to confidently distinguish among various sympatric oyster species. Putative samples of C. ariakensis and other species of cupped oysters from across Asia were collected and phylogenetic analyses were conducted on DNA sequences of both nuclear (ITS-1) and mitochondrial (COI) regions. Trees generated based on the two independent molecular datasets were highly congruent, and indicate that many oysters collected for this study as C. ariakensis were originally misidentified. Results also indicate that C. ariakensis, C. hongkongensis and C. nippona are distinct, but closely related species. There is strong support in both analyses for a close relationship between C. gigas and C. sikamea, as well as between C. belcheri and C. gryphoides, and between C. iredalei and C. madrasensis. The parsimony analyses based on these DNA markers, however, did not provide evidence to support C. angulata as a distinct species from C. gigas. Overall, the results emphasize the need for rigorous species identification, and additional extensive and intensive sampling to more accurately determine relationships among Crassostrea species, define their geographic distributions, and establish existing sympatry patterns.     

Population genetics of the American oyster Crassostrea virginica along the Atlantic coast and the Gulf of Mexico

An examination by protein-gel electrophoresis of 19 different geographical populations of the American oyster Crassostrea virginica (Gmelin) was conducted along the Atlantic coast and the Gulf of Mexico. Estimates were made of levels of genetic variation and similarity among the populations based on 32 structural loci. The percentage of loci polymorphic ranged from 46.9 to 65.6% along the Atlantic coast while the estimate ranged from 54.8% to 68.8% on the Gulf of Mexico. The percentage of loci heterozygous ranged from 18.6 to 23.6% along the Atlantic coast and from 20.0 to 25.4% in the Gulf of Mexico. The genetic similarities between all contiguous populations from Cape Cod, Massachusetts to Corpus Christi, Texas were estimated as 99%, while the same estimate between Corpus Christi and Brownsville, Texas was computed as 93%, indicating a major transition in genetic structure for the Brownsville population of the Laguna Madre. The study revealed that the migration of planktonic oyster larvae is predominantly in a westerly direction along the Gulf of Mexico and that gene flow appears to be disrupted in the region of the Laguna Madre. Evidence has been presented for single gene selection at the Lap-2 and Pgi loci in the form of macrogeographical clines in allele frequencies with changing environmental conditions.     

Gametic incompatibility and genetic divergence of Pacific and Kumamoto oysters,Crassostrea gigas and C. sikamea

The biological and taxonomic separation of the Pacific oyster Crassostrea gigas (Thunberg, 1793) from the Kumamoto oyster C. sikamea (Amemiya, 1928) is affirmed by three concordant lines of evidence: (1) fixed differences in 2% of a mtDNA sequence coding for large subunit rRNA; (2) a genetic distance of 0.440 based on 19 allozyme loci, including 5 diagnostic loci (Aat-1, Idh-1, Idh-2, Mpi-1, Mdh-2); (3) one-way gametic incompatibility resulting in partial reproductive isolation in interspecific crosses. C. gigas sperm x C. sikamea egg fertilizations form viable hybrid offspring, but C. sikamea sperm do not fertilize C. gigas eggs. Divergence between these two species is mediated by differing peaks in the periods for gamete release and by one-way sperm/egg incompatibility. Two attempts to recover C. sikamea from its place of origin in southern Japan have yielded only individuals with the mitochondrial haplotype that characterizes C. gigas. We thus identify a crucial need for careful screening, management, and conservation of the cultivated populations of C. sikamea on the US west coast.     

Allozyme variation in European populations of the oyster Ostrea edulis

Variations at 22 enzyme coding loci were surveyed in 11 populations of the oyster Ostrea edulis L., which were sampled between 1988 and 1990 along the Atlantic and Mediterranean coasts of Europe. Atlantic oyster beds suffered a steady decline during the last century, and restocking of beds with oysters of foreign origin has probably resulted in a high degree of interbreeding of natural oyster stocks from all Atlantic Europe. Our study confirms the low levels of genetic variability previously reported for the oyster populations from the Atlantic coasts, and extends it to the Mediterranean coasts. The locus arginine-kinase (ARK ^*) exhibited a high degree of interpopulation differentiation (F _ST=0.289), resulting from extensive variation in gene frequencies along a geographical cline. However, the overall genetic differentiation between populations was slight, and similar to that reported for other local populations of bivalves (mean genetic distance between populations is 0.010, mean F _ST=0.062). A general pattern of increasing differentiation along the coastline in an Atlantic-mediterranean direction emerged; but genetic differentiation among the Atlantic populations was not significantly lower than that observed among the Mediterranean populations. This and other results suggest that the effects of extensive transplantation of oysters among various areas in Europe are detectable only in some particular localities. The geographical distribution of low-frequency alleles suggests a restriction to gene flow outwards from the Mediterranean Sea, across the Straits of Gibraltar.     

Geographical patterns of variability at allozyme loci in the European oyster Ostrea edulis

The variability of 14 enzyme-coding genes has been analysed in samples from 19 populations of the oyster Ostrea edulis L., collected along the Atlantic and Mediterranean coasts of Europe. We found an abundance of clines, which appeared at 8 loci, including the most polymorphic (AP-2 ^*, ARK ^*, EST-4 ^*, MDH-2 ^*, ME-1 ^*, 6PGH ^*, PGI ^* and PGM ^*). Another 6 loci (ALDH ^*, EST-3 ^*, EST-5 ^*, IDH-2 ^*, MDH-1 ^*, ME-2 ^*) exhibited V-shaped patterns of gene-frequency variation, with clines at one or both sides of the Straits of Gibraltar. The observation of coincident clines at many loci can be explained by a model of secondary intergradation. The geographical location of the midpoints of the clines and V-shaped patterns suggests the existence of two ancient Atlantic and Mediterranean oyster stocks which became differentiated in allopatry and subsequently merged. Clines observed along Atlantic and/or Mediterranean coasts at the loci with V-shaped patterns must have arisen independently. The large heterogeneity observed in the levels of gene differentiation (G _ST ) across loci (G _ST ranged from 0.008 to 0.290) and important differences in estimates of gene flow obtained by different methods suggest that the populations of O. edulis are not in genetic equilibrium. Lack of population equilibrium can be due to natural selection and/or restrictions to gene flow. The average among-population variability was higher than in other oyster species that do not show incubatory habits, and represented 8.8% of the total heterozygosity. Levels of intrapopulation variability were lowest in populations from the North Atlantic, suggesting low population sizes in that area.     

Occurrence of the Kumamoto oyster Crassostrea sikamea in the Ariake Sea, Japan

The Kumamoto oyster Crassostrea sikamea is distinguished from the closely related Pacific oyster C. gigas by concordant differences in 16S rDNA, allozymes, and a one-way gametic incompatibility. After repeated failures to find this oyster in its native habitat, we speculated in 1994 that “the Kumamoto oyster may be extinct in Japan”. In September 1996, we sampled small, deep-cupped oysters from the Ariake Sea and typed these for 16S rDNA and ITS-1 DNA markers previously shown to be diagnostic for the three most common oysters in the Ariake Sea, C. gigas, C. sikamea and  C. ariakensis. Our earlier suggestion of the demise of  C. sikamea proved incorrect. Of the 256 oysters sampled, 181 (71%) were  C. gigas, 53 (21%) were C. sikamea, and 22 (9%) were  C. ariakensis; no interspecific hybrids were observed. The distributions of C. sikamea and  C. ariakensis are clumped in the Ariake Sea:  C. sikamea occurs on the eastern and northern shores,  C. ariakensis occurs only in the northern part. These results emphasize the value of molecular markers for discriminating these morphologically plastic species both in the field and in aquaculture. Received 6 July 1998 / Accepted 8 October 1998     

Genetic differentiation between morphotypes of the marine sponge Suberites ficus (Demospongiae: Hadromerida)

Sponges of three morphotypes of Suberites ficus (Johnston, 1842) were collected during February and March 1985 off the south-west of the Isle of Man, and were compared by using spicule size distributions and genetic allele frequencies of isozyme loci. The populations did not show any significant differences of spicule size or type, but could be easily differentiated into three separate species based on isozyme patterns. Samples of pale orange S. ficus growing on gastropod shells inhabited by hermit crabs (Pagurus spp.) were reproductively isolated from the redorange and the pale yellow colour morphs encrusting the bivalve Chlamys opercularis. These latter two colour morphs were genetically similar, but significant differences were observed at two of the 19 gene loci assayed. All the sponges studied were sympatric, and therefore the genetic differences, indicating reproductive isolation, are strong evidence for separate gene pools and, hence, that they are different species. The genetic identity between the two colour morphs of S. ficus on C. opercularis shells was 0.977, whilst between each of these and S. ficus on hermit crabs it was about 0.65. In all three species genetic variability was high, with mean expected and observed heterozygosity values per locus ranging from 0.17 to 0.36.     

Genetic evidence of the presence of two species of Crassostrea (Bivalvia: Ostreidae) on the coast of Brazil

 Although oysters are commercially very important in Brazil, there is still much dispute about the number of Crassostrea species occurring on the Brazilian coast. The dispute is centered around C. brasiliana, considered by some authors to be a junior synonym of C. rhizophorae. In this paper we compared, by allozyme electrophoresis, sympatric and allopatric populations of the two putative species. Of the 17 loci analysed, five were diagnostic for the two species in sympatry (gene identity = 0.46 to 0.47), clearly demonstrating that they are distinct biological species. Heterozygosity (h) levels were high for both species (h = 0.24 to 0.28), and no heterozygote deficiencies were observed in any population (local inbreeding, F _IS  = 0.141; P > 0.70). Levels of population structure in C. rhizophorae along 1300 km of coast were very low (population inbreeding, F _ST  = 0.026; P > 0.15), indicating that the planktonic, planktotrophic larvae of these species are capable of long-range dispersal. Received: 14 January 1999 / Accepted: 8 December 1999     

Separation of the two Atlantic species of Pomatoceros,P. lamarckii and P. triqueter (Annelida: Serpulidae) by means of biochemical genetics

In order to confirm the status of two proposed species of the serpulid Pomatoceros, the electrophoretic mobilities of 13 enzymes encoded at 19 loci in P. lamarckii (Quatrefages) and P. triqueter (L.) were compared using horizontal starch gel electrophoresis. The serpulids were sampled from Menai Bridge (North Wales), Plymouth (England) and the Isle of Man in the winter of 1981/1982. Six loci were monomorphic or nearly so and showed no useful or significant difference between species. At a further 7 loci, some of the allozymes common to both species were present, though usually at significantly different frquencies, while others were species-specific. The remaining 6 loci presented species-specific allozymes. In P. lamarckii, 61% of the loci were polymorphic compared with 50% in P. triqueter. The mean observed heterozygosity was 0.169 for P. lamarckii and 0.208 for P. triqueter. Calculation of genetic distance and identity gave values of 0.821 and 0.440, respectively. This study, therefore, confirms the existence of two separate species of Pomatoceros as has been previously proposed on the basis of opercular structure and supported by ecological separation where these species occur sympatrically.     

Population genetics of Crassostrea ariakensis in Asia inferred from microsatellite markers

Crassostrea ariakensis is an important aquacultured oyster species in Asia, its native region. During the past decade, consideration was given to introducing C. ariakensis into Chesapeake Bay, in the United States, to help revive the declining native oyster industry and bolster the local ecosystem. Little is known about the ecology and biology of this species in Asia due to confusion with nomenclature and difficulty in accurately identifying the species of wild populations in their natural environment. Even less research has been done on the population genetics of native populations of C. ariakensis in Asia. We examined the magnitude and pattern of genetic differentiation among 10 wild populations of C. ariakensis from its confirmed distribution range using eight polymorphic microsatellite markers. Results showed a small but significant global θ _ST (0.018), indicating genetic heterogeneity among populations. Eight genetically distinct populations were further distinguished based on population pairwise θ _ST comparisons, including one in Japan, four in China, and three populations along the coast of South Korea. A significant positive association was detected between genetic and geographic distances among populations, suggesting a genetic pattern of isolation by distance. This research represents a novel observation on wild genetic population structuring in a coastal bivalve species along the coast of the northwest Pacific.     

Is Syllis gracilis (Polychaeta: Syllidae) a species complex? An allozyme perspective

The genetic relationships between morphologically indistinguishable marine and brackish populations of Syllis gracilis Grube, 1840 (Polychaeta: Syllidae) were studied by means of allozyme electrophoresis. Samples of S. gracilis from marine coastal and brackish-water habitats were examined for variation at 13 presumptive loci. In addition, a sample of the closely related species S. prolifera (Krohn, 1852) was analysed. Five loci were multiallelic in at least one population of S. gracilis and eight loci in S. prolifera. Low to moderate levels of within-population genetic variability were found, with average expected heterozygosity values ranging from H = 0.068 (±0.043 SE) to 0.187 (±0.069 SE) in the populations of S. gracilis; higher values were found in S. prolifera (H = 0.325 ± 0.076). The presence of various private alleles indicated a marked genetic divergence among populations of S. gracilis, with Nei's genetic distances ranging from D = 0.000 to 0.833 and a highly significant F _ST value. Furthermore, evidence for strong genetic heterogeneity between two sympatric marine populations was found. UPGMA cluster analysis and multidimensional scaling pointed out a clear genetic divergence between brackish and marine populations. At least two genetically divergent entities occurred in marine and brackish habitats. This could be due to local adaptation of individuals coming from marine populations to brackish habitats, but more presumably to the occurrence of a species complex within S. gracilis. Received: 6 June 1999 / Accepted: 7 February 2000     

Biochemical systematics of fishes of the genus Stegastes (Pomacentridae) from the Southern Marianas

The biochemical systematics of the subset of species of Stegastes (Pisces: Pomacentridae) available from coral reefs surrounding Guam, USA, was studied using three species of Pomacentrus as the outgroup. Analyses of allozymic variation at 14 presumptive loci (10 of which were polymorphic) using various evolutionary tree-construction techniques identified S. fasciolatus as the sister species of an assemblage comprised of S. albifasciatus, S. nigricans, and S. lividus: within this assemblage, S. lividus is the sister species of S. albifasciatus and S. nigricans, the pair of species which shared the most synapomorphic alleles and consistently exhibited the lowest degree of genetic distance relative to estimates for the other pairs of species studied. The consistency of ingroup topologies produced by parsimony analyses using Manhattan rectilinear distances computed from allele frequencies with those produced using the distance Wagner procedure and UPGMA (unweighted pair-group method of arithmetic averages) clustering, substantiates strongly the proposed phylogeny. The systematic scheme inferred from molecular data is concordant with predictions based on morphologic differences, i.e., differences in numbers of dorsal spines, that have been used as evidence in support of the proposal to partition Stegastes into at least two subgenera.     

Comparing the effect of elevated pCO2 and temperature on the fertilization and early development of two species of oysters

This study compared the synergistic effects of elevated pCO₂ and temperature on the early life history stages of two ecologically and economically important oysters: the Sydney rock oyster, Saccostrea glomerata and the Pacific oyster, Crassostrea gigas. Gametes, embryos, larvae and spat were exposed to four pCO₂ (375, 600, 750, 1,000 μatm) and four temperature (18, 22, 26, 30°C) levels. At elevated pCO₂ and suboptimal temperatures, there was a reduction in the fertilization success of gametes, a reduction in the development of embryos and size of larvae and spat and an increase in abnormal morphology of larvae. These effects varied between species and fertilization treatments with S. glomerata having greater sensitivity than C. gigas. In the absence of adaptation, C. gigas may become the more dominant species along the south-eastern coast of Australia, recruiting into estuaries currently dominated by the native S. glomerata.     

Systematic status of two oyster populations of the genus Tiostrea from New Zealand and Chile

A systematic examination of Tiostrea chilensis endemic to the Pacific coast of South America and of T. lutaria endemic to new Zealand was conducted using protein-gel electrophoresis. Also, ecological and life-history traits were compared between these two documented species. Our study revealed that although three different ecomorphs could be distinguished from various environmental and ecological conditions, the life-history traits between the two species were remarkably similar. Estimates were made of levels of genetic variation and similary based on 29 structural loci among individuals from each documented species. The percentage of loci polymorphic was estimated at 17.2 and 17.6, while the percentage of loci heterozygous was 2.1 and 6.7 for T. chilensis and T. lutaria, respectively. The genetic similarity between the two species was computed as 0.991, where 1.0 indicates no genetic difference among structural loci. The overwhelming evidence of this study indicates that T. chilensis and T. lutaria are different geographical populations of one species and by priority this species should be T. chilensis.     

Inducible defenses in the eastern oyster Crassostrea virginica Gmelin in response to the presence of the predatory oyster drill Urosalpinx cinerea Say in Long Island Sound

The response of the eastern oyster C. virginica to the presence of the oyster drill Urosalpinx cinerea was examined from July to September 2011. Several aspects of oyster growth were measured, including wet weight, shell weight, and dorsal shell area for oysters collected near Groton, Connecticut (41.32036 N, −72.06330 W). Wet weight and shell weight growth were significantly higher in the presence of the predator U. cinerea, while tissue weight showed no difference from the control. The control group showed more shell area growth and a much lower ratio of shell weight growth to shell area growth. Differences in shell weight to area ratio indicated that C. virginica dramatically shifted from lateral shell growth to shell thickening in the presence of U. cinerea. This inducible defense has not been previously shown for C. virginica and could play an important role in the predator–prey interaction between these two species.     

The genetic mating system of a sex-role-reversed pipefish (Syngnathus typhle): a molecular inquiry

In the pipefish Syngnathus typhle as in other species of Syngnathidae, developing embryos are reared on the male's ventral surface. Although much laboratory research has been directed toward understanding sexual selection in this sex-role-reversed species, few studies have addressed the mating behavior of S. typhle in the wild, and none has capitalized upon the power of molecular genetic assays. Here we present the first direct assessment of the genetic mating system of S. typhle in nature. Novel microsatellite loci were cloned and characterized from this species, and employed to assay entire broods from 30 pregnant, field-captured males. Genetic analysis of 1340 embryos revealed that 1–6 females (mean = 3.1) contributed to each brooded clutch, the highest rate of multiple maternity yet documented in any pipefish. Evidence of multiple mating by females was also detected. Thus, this population of S. typhle displays a polygynandrous mating system, a finding consistent with previous field and laboratory observations. Our results, considered together with similar studies of other syngnathid species, provide preliminary support for the hypothesis that the genetic mating system is related to the evolution of sexual dimorphism in the fish family Syngnathidae. Received: 19 January 1999 / Received in revised form: 15 April 1999 / Accepted: 9 May 1999     

Present-day genetic composition suggests contrasting demographic histories of two dominant chaetognaths of the North-East Atlantic, <Emphasis Type="Italic">Sagitta elegans</Emphasis> and <Emphasis Type="Italic">S. setosa</Emphasis>

Sagitta elegans and S. setosa are the two dominant chaetognaths in the North-East (NE) Atlantic. They are closely related and have a similar ecology and life history, but differ in distributional ranges. Sagitta setosa is a typical neritic species occurring exclusively above shelf regions, whereas S. elegans is a more oceanic species with a widespread distribution. We hypothesised that neritic species, because of smaller and more fragmented populations, would have been more vulnerable to population bottlenecks resulting from range contractions during Pleistocene glaciations than oceanic species. To test this hypothesis we compared mitochondrial Cytochrome Oxidase II DNA sequences of S. elegans and S. setosa from sampling locations across the NE Atlantic. Both species displayed very high levels of genetic diversity with unique haplotypes for every sequenced individual and an approximately three times higher level of nucleotide diversity in S. elegans (0.061) compared to S. setosa (0.021). Sagitta setosa mitochondrial DNA (mtDNA) haplotypes produced a star-like phylogeny and a uni-modal mismatch distribution indicative of a bottleneck followed by population expansion. In contrast, S. elegans had a deeper mtDNA phylogeny and a multi-modal mismatch distribution as would be expected from a more stable population. Neutrality tests indicated that assumptions of the standard neutral model were violated for both species and results from the McDonald-Kreitman test suggested that selection played a role in the evolution of their mitochondrial DNA. Congruent with these results, both species had much smaller effective population sizes estimated from genetic data when compared to census population sizes estimated from abundance data, with a factor of ~10⁸–10⁹ difference. Assuming that selective effects are comparable for the two species, we conclude that the difference in genetic signature can only be explained by contrasting demographic histories. Our data are consistent with the hypothesis that in the NE Atlantic, the neritic S. setosa has been more severely affected by population bottlenecks resulting from Pleistocene range shifts than the more oceanic S. elegans.     

Genetic evidence for the existence of cryptic species in an endangered clam <Emphasis Type="Italic">Coelomactra antiquata</Emphasis>

Coelomactra antiquata is a commercially important bivalve species, but has been suffering from severe population decline due to over-exploitation and the deterioration of environmental conditions. Previous genetic survey of C. antiquata conducted with allozymes combined with morphology revealed high levels of genetic differentiation between northern and southern populations which suggests a cryptic species might exist in C. antiquata. To test this hypothesis, amplified fragment length polymorphisms (AFLPs) and 16S rRNA gene sequence were used to re-evaluate the spatial genetic structure of six populations of C. antiquata along the coast of China. Both genetic markers display a sharp genetic break between the four northern populations (northern lineage) and two southern population (southern lineage). Large numbers of private alleles (AFLP) were found within the northern or southern populations and a deep divergence of about 6.5% in 16S rRNA gene sequence between the northern and southern lineages suggests the occurrence of potential cryptic or sibling species of C. antiquata. Applying previously published rates of mutation, divergence between the two lineages is estimated to have occurred approximately 3 million years ago and may be due to allopatric isolation during the middle Pliocene times. While no genetic differentiation was found within the northern or southern populations in both AFLP and 16S mtDNA markers, the results indicate that the northern and southern lineage should be managed separately and any translocation between the two areas should be avoided.