Molecular population structure of the kuruma shrimp <Emphasis Type="Italic">Penaeus</Emphasis><Emphasis Type="Italic"> japonicus</Emphasis> species complex in western Pacific

In a previous study on the kuruma shrimp Penaeus japonicus from the South China Sea, we detected high genetic divergence between two morphologically similar varieties (I and II) with distinct color banding patterns on the carapace, indicating the occurrence of cryptic species. In the present study, we clarify the geographical distribution of the two varieties in the western Pacific by investigating the genetic differentiation of the shrimp from ten localities. Two Mediterranean populations are also included for comparison. Based on the mitochondrial DNA sequence data, the shrimps are separated into two distinct clades representing the two varieties. Variety I comprises populations from Japan and China (including Taiwan), while variety II consists of populations from Southeast Asia (Vietnam, Singapore and the Philippines), Australia and the Mediterranean. Population differentiation is evident in variety II, as supported by restriction profiles of two mitochondrial markers and analysis of two microsatellite loci. The Australian population is genetically diverged from the others, whereas the Southeast Asian and Mediterranean populations show a close genetic relationship. Variety I does not occur in these three localities, while a small proportion of variety II is found along the northern coast of the South China Sea and Taiwan, which constitute the sympatric zone of the two varieties. The present study reveals high genetic diversity of P. japonicus. Further studies on the genetic structure of this species complex, particularly the populations in the Indian Ocean and Mediterranean, are needed not only to understand the evolutionary history of the shrimp, but also to improve the knowledge-based fishery management and aquaculture development programs of this important biological resource.     

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.     

Growth rate and reproductive differences in a widespread estuarine harpacticoid copepod (Scottolana canadensis)

We demonstrate the presence of significant genetically based differentiation in growth rate (g dry weight d^-1) and reproductive traits (percent reproductive females and mean clutch size g dry weight^-1) among females of an harpacticoid copepod (Crustacea),Scottolana canadensis (Willey), taken from a broad range of latitudes and reared in the laboratory under the same conditions. As temperature increases (15°–25° C), the growth rate of southern-derived copepods continues to increase, while that of northern-derived copepods levels off or decreases. Southern-derivedS. canadensis also have a higher percentage of reproducing females at high temperature (25°C) when rations (cells ml^-1) are reduced, while northern-derived females are at an advantage at low temperature (15°C). Both life-history traits indicate local adaptation to maximize scope for growth and reproduction at prevailing temperatures. The data support our hypothesis that evolution has occurred to maximize feeding minus metabolic energy expended, and that this maximization requires changes in feeding efficiency with differing temperatures.     

Geographic clines and stepping-stone patterns detected along the East Pacific Rise in the vetigastropod Lepetodrilus elevatus reflect species crypticism

Three different molecular markers (i.e. seven allozyme loci, two nuclear gene loci and, mtCOI DNA sequences) were used to assess the genetic structure of the vent gastropod Lepetodrilus elevatus collected from three vent fields along the East Pacific Rise (13°N, 9°50′N and 17°S). While allozymes and nuclear loci suggested a strong stepping-stone pattern, a multivariate analysis performed on allozymic frequencies showed the presence of two distinct evolutionary lineages: the first situated in the north from 13°N to 9°50′N and the second in the south from 9°50′N to 17°S. The analysis of mitochondrial DNA sequences confirmed the separation of L. elevatus into two distinct clades with a divergence of 6.5%, which is consistent with the interspecific level of sequence variation in other vent species. A divergence time of 6–14 Mya was estimated between the two clades from previous clock calibrations. Our results suggest that these taxa followed an allopatric speciation between the northern and southern parts of the EPR with a recent demographic expansion of the southern clade to the north and a subsequent secondary contact (clade hybridisation). This speciation was probably reinforced by a habitat specialisation of the two cryptic species because the southern clade was mainly found associated with mussel-dominated communities and the northern clade with tubeworm-dominated communities. However, the analysis of shell morphology failed to separate the two cryptic species based on this sole criterion although they differed from Lepetodrilus elevatus galriftensis (Galapagos population) by a higher shell elevation. Within each clade, genetic differentiation was not related to the distance across populations and could be within vent field as important as between fields. While both clades appear to be in expansion since their speciation, significant excesses in heterozygotes suggest a very recent and local bottleneck at 17°S, probably due to massive site extinction in this region.     

Population structure of the giant tiger prawn Penaeus monodon in Australian waters, determined using microsatellite markers

 We describe three highly polymorphic microsatellite loci which have been isolated from the giant tiger prawn Penaeus monodon. The number of alleles present among 312 samples at the loci Pmo9, Pmo25 and Pmo27 were 84, 34 and 35, respectively, with heterozygosities all >90%. Analyses of the distribution of length variation at three microsatellite loci among five Australian P. monodon populations revealed strong differentiation between populations from the west and those from the northern and eastern coasts. Tests for population differentiation (F st) values and an analogous measure for microsatellite loci (R st) all demonstrated that Western Australian P. monodon are a separate genetic stock which exhibits reduced genetic variation relative to the other populations. Reduced variability is consistent with a recent population bottleneck or colonization by a small founding population from the east when sea links between Indonesia, New Guinea and Australia were re-established following the last ice age. The results of this study are in agreement with previous surveys of P. monodon conducted with allozymes and mtDNA. Received: 18 December 1998 / Accepted: 27 August 1999     

Regional patterns of microsatellite variation in <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> from the Iberian Peninsula

The Almería-Oran Oceanographic Front (AOOF) has been proposed as an effective marine barrier to gene flow between the NE Atlantic Ocean and the Mediterranean Sea for several species. Previous studies using allozymes and mitochondrial DNA have reported a scenario of secondary intergradation between populations of Mytilus galloprovincialis from those basins, with the allelic frequencies of some loci showing abrupt clinal patterns across the AOOF. In this study, we aimed at testing the congruence between six neutral polymorphic microsatellites versus previous data on allozymes and mtDNA-RFLPs, at depicting the population structure of this species in the Iberian Peninsula. Microsatellite genotyping was scored on 17 samples of mussels collected in the Iberian coast, including some areas not sampled before. Microsatellites exhibited larger intrabasin diversity (F _SC = 1.72%, ), similar interbasin differentiation (F _CT = 2.81%) and fewer allelic clines than allozymes or mtDNA haplotypes. These results fully support the scenario of secondary intergradation with some ongoing gene flow between basins, as proposed in previous analyses. Moreover, this congruence between markers and analyses separated by a 12-year period (1988–2000) confirm the temporal stability of this marine barrier at shaping the Iberian phylogeographic break in M. galloprovincialis. In addition, the genetic continuity between the NE Atlantic (Portugal) and the Alboran Sea seems to be warranted across the Gulf of Cadiz and the Gibraltar strait after the present microsatellite data. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Population structure of red porgy, <Emphasis Type="Italic">Pagrus pagrus</Emphasis>, in the Atlantic Ocean

The red porgy, Pagrus pagrus (L.), is a protogynous sparid associated with reefs and hard bottom habitat throughout the warm-temperate Atlantic Ocean. In this study, the degree of geographic population differentiation in Atlantic populations was examined with microsatellite and mitochondrial DNA markers (mtDNA). Six microsatellite loci were amplified and scored in 690 individuals from the eastern North Atlantic (Crete, Madeira, and Azores), western North Atlantic (North Carolina to Florida, and the eastern Gulf of Mexico), and Brazil. At two loci, fixed allelic differences were found among the three major geographic areas, while frequency differences were observed at three other loci. The DNA of 371 individuals was amplified at the mtDNA control region, and 526 bp were sequenced. Tamura–Nei’s D was used as a measure of nucleotide diversity and divergence: diversity averaged 0.011 within samples, while the corrected divergence averaged 0 between samples within the same area and 0.061 between samples from different areas. Transversion haplotype minimum spanning networks, nucleotide divergence, and F _ST values all show that the western Atlantic samples were more closely related to each other than any was to samples from the eastern North Atlantic. Within the western North Atlantic, no significant population differentiation was observed, and within the eastern North Atlantic, only the Azores sample showed detectable differences from Crete and Madeira. These data indicate general homogeneity within large areas, and deep divisions between these areas. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

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.     

Genetic differentiation among populations of the Mediterranean hermaphroditic brooding coral<Emphasis Type="Italic"> Balanophyllia europaea</Emphasis> (Scleractinia: Dendrophylliidae)

Spatial models of genetic structure and potential gene flow were determined for five populations of Balanophyllia europaea, a simultaneous hermaphroditic and brooding coral, endemic to the Mediterranean. Six allozyme loci indicated a genetic structure that departed markedly from Hardy–Weinberg equilibrium, with a significant lack of heterozygotes. The genetic structure observed supports the hypothesis that self-fertilisation characterises the reproductive biology of B. europaea. Populations at small spatial scales (8–40 m) are genetically connected, while those at large scales (36–1,941 km) are genetically fragmented; the genetic differentiation of the populations is not correlated to geographic separation. This spatial model of genetic structure is compatible with an inbreeding mating system. Furthermore, it is also consistent with the expected dispersal potential of swimming larvae of brooding corals, i.e. larvae that are able to produce significant gene flows only within limited spatial scales.Communicated by R. Cattaneo-Vietti, Genova     

Effect of temperature on permeability and drinking rates of the metanauplii of the brine shrimp Artemia sp.

Diffusional permeabilities and drinking rates of 24 h-old Artemia sp. metanauplii incubated at 20 °C were studied at five temperatures: 15, 20, 25, 30, and 35 °C over a period of 6 h. Departures from the incubation temperature resulted in changes in both permeability and drinking rates which both reached their highest values at 35 °C. Metanaupliar permeability and drinking were more elevated than previously reported for adults.     

Phylogeography of surfclams,<Emphasis Type="Italic"> Spisula solidissima</Emphasis>, in the western North Atlantic based on mitochondrial and nuclear DNA sequences

The Atlantic surfclam, Spisula solidissima (Dillwyn), is broadly distributed in sandy sediments of the western North Atlantic between the Gulf of St. Lawrence and the Gulf of Mexico. In the United States, a substantial commercial fishery between Long Island and Cape Hatteras harvests offshore populations of one subspecies, S. s. solidissima. A smaller coastal form, S. s. similis Say (also known as S. s. raveneli Conrad), has a partially sympatric geographic distribution, but differs in several life-history characteristics. DNA sequence variation in mitochondrial cytochrome oxidase I (COI) and in introns at two nuclear calmodulin loci was examined to measure genetic divergence between the two subspecies and to test for population structure among populations of S. s. solidissima. Surfclams were collected from seven localities between 1994 and 2001. Based on both mitochondrial and nuclear DNA variation, the two subspecies of S. solidissima are reciprocally monophyletic, with a net COI divergence of 13.9%, indicating long-term reproductive isolation. The only significant differentiation among populations of S. s. solidissima (based on an AMOVA analysis of COI sequences) was between the Gulf of St. Lawrence and more southerly populations. A long internal branch in the S. s. solidissima genealogy coupled with low haplotype diversity in the northern-most population suggests that populations north and south of Nova Scotia have been isolated from each other in the past, with gene exchange more recently. Populations of S. s. similis from Atlantic and Gulf of Mexico coasts had a net COI divergence of 9.2%. Thus, diversification of Spisula spp. clams in the western North Atlantic involved an early adaptive divergence between coastal and offshore forms, with later barriers to dispersal emerging in the offshore form from north to south and in the coastal form between Atlantic and Gulf of Mexico populations.     

Genetic structure of dictyoceratid sponge populations on the western Coral Sea reefs

Allozyme variation at six polymorphic loci was examined in foliose dictyoceratid sponges from isolated reefs in the western Coral Sea. Four major genetic groups corresponding to the species Phyllospongia lamellosa, P. alcicornis, Carterospongia flabellifera and Collospongia auris were examined. A further two rare morphotypes from individual reefs formed genetic outliers to the P. lamellosa group, and may represent further taxa related to P. lamellosa. Gene frequencies in individual reef populations were largely in Hardy-Weinberg equilibrium, suggesting that random mating occurred in local populations of all four common species. Genetic variability was high and observed heterozygosities within populations ranged from 0.13 to 0.40. All four taxa showed significant genetic differentiation among populations (F _ST=0.05 to 0.36). Genetic distances (Nei's D) among populations within species ranged from 0 to 0.723 and increased with increasing geographical separation. There was evidence that genetic differentiation between populations to the north and to the south of the southern limit of the South Equatorial Current (SEC) divergence was greater than expected on the basis of their geographical separation. The SEC divergence may form a partial barrier to gene flow among populations of these ecologically important sponges on the submerged Queensland Plateau. Levels of migration among populations of three of the species was less than those required to prevent divergence of the populations through genetic drift (Nm<1). Restricted migration among populations may provide a mechanism to explain the occurrence of highly divergent populations of dictyoceratid sponges whose specific identity is not clear, and may allow them additionally to develop partial reproduction isolation from other populations.     

Genetic diversity and population structure of the Chinese mitten crab <Emphasis Type="Italic">Eriocheir sinensis</Emphasis> in its native range

The Chinese mitten crab Eriocheir sinensis is an indigenous and economically important species in China, but can also be found as invasive species in Europe and America. Mitten crabs have been exploited extensively as a food resource since the 1990s. Despite its ecological and economic importance, the genetic structure of native mitten crab populations is not well understood. In this paper, we investigated the genetic structure of mitten crab populations in China by screening samples from ten locations covering six river systems at six microsatellite loci. Our results provide further evidence that mitten crabs from the River Nanliujiang in Southern China are a genetically differentiated population within the native range of Eriocheir, and should be recognized as a separate taxonomic unit. In contrast, extremely low levels of genetic differentiation and no significant geographic population structure were found among the samples located north of the River Nanliujiang. Based on the reproductive biology of mitten crabs and the geography of their habitat we argue that both natural and human-mediated gene flow are unlikely to fully account for the similar allele frequency distributions at microsatellite loci. Large population sizes of mitten crabs suggest instead that a virtual absence of genetic drift and significant homoplasy of microsatellite alleles have contributed to the observed pattern. Furthermore, a coalescent-based maximum likelihood method indicated a more than two-fold lower effective population size of the Southern population compared to the Northern Group and low but significant levels of gene flow between both areas.     

Eurythoe complanata (Polychaeta: Amphinomidae), the ‘cosmopolitan’ fireworm, consists of at least three cryptic species

Eurythoe complanata (Pallas 1766) has been considered a cosmopolitan species with a great morphological similarity across its geographic range. To elucidate whether E. complanata is actually a single species, genetic (cytochrome oxidase subunit I and allozymes) and morphological differences were compared among specimens from the Pacific, Caribbean, and South Atlantic Oceans. Large levels of COI divergence (10–22%) and diagnostic allozyme loci identified three cryptic species: one in the eastern Pacific and two in the Atlantic, with one being morphologically differentiated and found only in islands. COI sequences between Pacific and Atlantic lineages were much more divergent than those of other transisthmian invertebrates, indicating their split before the Panama Isthmus closure or a faster evolutionary rate of COI for this species. The existence of two Atlantic species may be a consequence of parapatric speciation followed by a secondary invasion or even a sympatric speciation in the Atlantic oceanic islands.     

Genetic structure of natural populations of California red abalone (<Emphasis Type="Italic">Haliotis rufescens</Emphasis>) using multiple genetic markers

Mitochondrial cytochrome oxidase subunit one (COI) sequence, nuclear microsatellites, and amplified fragment length polymorphisms (AFLPs) were used to evaluate connectivity among nine red abalone (Haliotis rufescens) populations sampled between August 1998 and November 2003 along approximately 1,300 km of California coastline from Crescent City (41°46′N, 124°12′W) to San Miguel Island (34°02′N, 120°22′W). COI sequences and microsatellite genotypes did not show significant genetic divergence among nine sampled populations. A subset of five populations spanning the geographic range of the study was scored for 163 polymorphic AFLP markers. Of these, 41 loci showed significant divergence (P < 0.001) among populations. Still, no AFLP markers were diagnostic for any of the study populations, and assignment tests did not consistently assign individuals to the correct population. Although the AFLP data are the first to suggest there is significant genetic differentiation among California red abalone populations, the discordance between the different genetic markers needs further study before unambiguous conclusions can be drawn with respect to connectivity among the populations. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Morphological and genetic evidence for vicariance and refugium in Atlantic and Gulf of Mexico populations of the hermit crab Pagurus longicarpus

The number and wide variety of southeastern United States marine taxa with significant differentiation between Gulf of Mexico and Atlantic Ocean populations suggests that these taxa may have experienced major vicariance events, whereby populations were subdivided by geological or ecological barriers. The present study compared variation in morphology, allozymes, and mtDNA in Gulf of Mexico and western Atlantic populations of the longwrist hermit crab Pagurus longicarpus Say collected during 1997 and 1998. Combined Atlantic populations had significantly fewer denticles on the second segment of the third maxilliped than did Gulf of Mexico populations, and the mean ratio of dactyl length to propodus length was significantly greater in the Atlantic crabs than in the Gulf of Mexico crabs. Allozyme allele frequencies at three loci showed genetic differentiation between a Gulf of Mexico population and two Atlantic populations. Analysis of mtDNA sequence data revealed a clear reciprocal monophyly between Gulf and Atlantic populations, with an estimated divergence age of ~0.6 million years ago. This estimated age of divergence is significantly more recent than an age previously estimated for its congener Pagurus pollicaris (~4 million years ago), suggesting that species with a similar genetic break between Gulf and Atlantic populations may not necessarily share an identical history. Surprisingly, there is evidence of geographic subdivision within Atlantic populations of P. longicarpus along the east coast of North America. This differentiation is especially strong between Nova Scotia and southern populations, suggesting that the Nova Scotia population may represent survivors from a northern refugium during the last glacial maximum.     

Genetic differentiation between morphotypes in the Antarctic limpet <Emphasis Type="Italic">Nacella concinna</Emphasis> as revealed by inter-simple sequence repeat markers

The limpet Nacella concinna (Strebel 1908) was the focus of numerous studies dealing with Antarctic benthos. One of the main characteristics of the species is the presence of two distinguishable morphotypes, one inhabiting the intertidal (during summer) and the other inhabiting the subtidal. For a long time these forms were considered as an expression of phenotypic plasticity, since previous studies did not found genetic differences between them. In the present work, we performed both a morphometric and a genetic differentiation analysis (using ISSR-PCR markers) of these two forms in three stations sampled at Potter Cove, South Shetland Islands. The results confirmed the morphological differences between intertidal and subtidal forms reported in other Antarctic localities. The genetic differences detected indicate that the two forms can be considered as genetically distinct populations maintaining low levels of gene flow. The degree of reproductive isolation of the ecotypes is discussed, as well as the possible origin of the divergence. The genetic differentiation observed can also have behavioral and physiological correlates, pointing out the importance of taking into account the potential differences in the response of both populations to different conditions in future studies in this species. M. C. de Aranzamendi and R. Sahade contributed equally to this paper.     

Sibling species in interstitial flatworms: a case study using<Emphasis Type="Italic"> Monocelis lineata</Emphasis> (Proseriata: Monocelididae)

The genetic relationships between morphologically indistinguishable marine and brackish-water populations of Monocelis lineata (O.F. Müller, 1774) (Proseriata: Monocelididae) were analysed by means of allozyme electrophoresis. Fifteen samples of M. lineata (13 from the Mediterranean and two from the Atlantic) from coastal marine and brackish-water habitats were examined for variation at 18 loci. Eleven loci were polymorphic in at least one population of M. lineata. Low levels of within-population genetic variability were found, with average observed and expected heterozygosity values ranging from H_o=0.015±0.015 to 0.113±0.044, and from H_e=0.028±0.028 to 0.138±0.054, respectively. The occurrence of a number of private alleles indicated a marked genetic divergence among populations of M. lineata, with Rogers genetic distances ranging from D_R=0.003 to 0.676 and a highly significant F_ST value (0.918±0.012, P<0.001). UPGMA (unweighted pair-group method using arithmetic average) cluster analysis and multidimensional scaling showed a clear genetic divergence between marine and brackish-water populations. Moreover, Atlantic and Mediterranean populations were sharply separated. Our results suggest that M. lineata is a complex of sibling species.Communicated by R. Cattaneo-Vietti, Genova     

Geographical patterns in reproductive biology of the Pan-American sandy beach isopod<Emphasis Type="Italic"> Excirolana braziliensis</Emphasis>

Eleven populations of the Pan-American sandy beach isopod Excirolana braziliensis, distributed from tropical (9°N) to temperate (39°S) sandy beaches in Atlantic and Pacific Oceans, were analyzed to determine latitudinal variations in: breeding and recruitment patterns, sex ratios, size of ovigerous females and of juveniles and length–fecundity relationships. E. braziliensis exhibited strong latitudinal patterns in all reproductive traits throughout its distribution range. Breeding and recruitment shifted from continuous to seasonal from tropical to temperate beaches, having a predominance of females at higher latitudes. In agreement with the latitudinal gradient hypothesis, ovigerous females inhabiting tropical sandy beaches (low latitudes) were smaller, became sexually mature at smaller sizes and had lower individual fecundity than on temperate beaches. Juveniles were also smallest at low latitudes. Between-ocean comparisons showed very similar reproductive characteristics for roughly the same latitude. These linked reproductive parameters suggest that the intensity of breeding effort is associated with the duration of the breeding season and geographically size-related characteristics of the species. Geographic variations in the breeding and recruitment seasons, as well as in individual fecundity, size structure of mature females and sex ratios, are proposed to have major consequences in explaining local variations in population demography. Our paper also reinforces the notion that sandy beach animals are highly plastic.Communicated by O. Kinne, Oldendorf/Luhe