Geographic patterns of genetic variation in nuclear and chloroplast genomes of two related oaks (<Emphasis Type="Italic">Quercus aliena</Emphasis> and <Emphasis Type="Italic">Q. serrata</Emphasis>) in Japan: implications for seed and seedling transfer

In this study, we assessed geographic patterns of genetic variations in nuclear and chloroplast genomes of two related native oaks in Japan, Quercus aliena and Q. serrata, in order to facilitate development of genetic guidelines for transfer of planting stocks for each species. A total of 12 populations of Q. aliena and 44 populations of Q. serrata were analyzed in this study. Genotyping of nuclear microsatellites in Q. aliena was done with only nine populations (n = 212) due to limited numbers of individuals in two populations, while all 12 populations (n = 89) were used in sequencing chloroplast DNA (cpDNA). In Q. serrata, 43 populations (n = 1032) were genotyped by nuclear microsatellite markers, while cpDNA of 44 populations (n = 350) was sequenced. As anticipated, geographic patterns detected in the variations of Q. aliena’s nuclear genome and its chloroplast haplotype distribution clearly distinguished northern and southern groups of populations. However, those of Q. serrata were inconsistent. The geographic distribution of its chloroplast haplotypes tends to show the predicted differentiation between northern and southern lineages, but geographic signals in the genetic structure of its nuclear microsatellites are weak. Therefore, treating northern and southern regions of Japan as genetically distinct transferrable zones for planting stocks is highly warranted for Q. aliena. For Q. serrata, the strong NE-SW geographic structure of cpDNA should be considered.     

Variability of allozyme and cpSSR markers in the populations of Siberian spruce

The variability of 21 allozyme and three microsatellite loci of chloroplast DNA (cpDNA) was studied in the populations of Siberian spruce (Picea obovata Ledeb.) from Irkutsk oblast, Magadan oblast, Buryatia, and Mongolia. It was demonstrated that the highest level of genetic diversity among the examined populations at both allozyme and microsatellite loci was observed in the Tulyushka population from Irkutsk oblast. The lowest level of genetic diversity was observed in marginal isolated populations of Bogd Uul and Magadan. In the relict spruce population from Olkhon Island, differing from the other populations in the lowest allelic diversity of both types of markers, no expected decline of expected heterozygosity and haplotype diversity was observed. In this population, the variability parameters mentioned were close to the population mean. The obtained intrapopulation and intraspecific variability parameters of allozyme and microsatellite loci of chloroplast DNA and the data on the population differentiation at these loci indicate that the given markers can be used for the analysis of the population structure of Siberian spruce.     

Genetic diversity and differentiation of Siberian spruce populations at nuclear microsatellite loci

The results of the study of 21 populations of Siberian spruce (Picea obovata Ledeb.) from different parts of the species natural range by microsatellite (SSR) analysis of nuclear DNA are presented. Using nine loci developed for Picea abies (L.) Karst. and Picea glauca (Moench) Voss and detecting variation in Picea obovata, the parameters of intra- and interpopulation genetic diversity, as well as the degree of population differentiation, were determined. It was demonstrated that the population of Siberian spruce in the study was characterized by a relatively high average level of intrapopulation variability (H_o = 0.408; H_e = 0.423) and low interpopulation differentiation (F_st = 0.048, P = 0.001) at this class of DNA markers. The genetic distance between populations ranged from 0.009 to 0.167, averaging 0.039. The isolated Magadan population, located in the extreme Northeast of Russia at a considerable distance from the main species range and characterized by the lowest genetic diversity among the studied populations, was maximally differentiated from the rest of the spruce populations. In addition, the steppe Ubukun population from Buryatia and the population from the Bogd Khan Uul Biosphere Reserve, Mongolia, were considerably different in the genetic structure from most populations of Siberian spruce, although to a lesser extent than the Magadan population. These findings are consistent with the results of previous studies of this species carried out using allozyme and microsatellite loci of chloroplast DNA and point to the prospects of using nuclear microsatellites as DNA markers to analyze the population genetic structure of Siberian spruce.     

Molecular and genetic characterization of the allelic variants of Du215, Du281, Du323, and Du47G microsatellite loci in parthenogenetic lizard <Emphasis Type="Italic">Darevskia armeniaca</Emphasis> (Lacertidae)

A key issue in the study of unisexual (parthenogenetic) vertebrate species is the determination of their genetic and clonal diversity. In pursuing this aim, various markers of nuclear and mitochondrial genomes can be used. The most effective genetic markers include microsatellite DNA, characterized by high variability. The development and characterization of such markers is a necessary step in the genetic studies of parthenogenetic species. In the present study, using locus-specific PCR, for the first time, an analysis of allelic polymorphism of four microsatellite loci is performed in the populations of parthenogenetic species Darevskia armeniaca. In the studied populations, allelic variants of each locus are identified, and the nucleotide sequences of each allele are determined. It is demonstrated that allele differences are associated with the variation in the structure of microsatellite clusters and single nucleotide substitutions at fixed distances in flanking DNA regions. Structural allele variations form haplotype markers that are specific to each allele and are inherited from their parental bisexual species. It is established which of the parental alleles of each locus were inherited by the parthenogenetic species. The characteristics of the distribution and frequency of the alleles of microsatellite loci in the populations of D. armeniaca determining specific features of each population are obtained. The observed heterozygosity of the populations at the studied loci and the mutation rates in genome regions, as well as Nei’s genetic distances between the studied populations, are determined, and the phylogenetic relationships between them are established.     

Pollen-mediated gene flow promotes low nuclear genetic differentiation among populations of <Emphasis Type="Italic">Cycas debaoensis</Emphasis> (Cycadaceae)

Cycas debaoensis is a critically endangered cycad species endemic to China. This species is found on two kinds of habitats according to the edaphic differences, sand and karst. A previous chloroplast DNA (cpDNA) study indicated that C. debaoensis had low genetic variation within populations and high genetic differentiation among populations. Because maternally inherited cpDNA does not fully characterize genetic structure of the species, we screened seven low-copy nuclear genes and 17 nuclear microsatellite loci to detect the nuclear genetic diversity, differentiation, and the population structure of C. debaoensis. The nuclear genes revealed higher level of genetic diversity. There were both the same and region-specific haplotypes or alleles between the karst and sand regions. Nuclear gene flow among all the populations was much greater than that of cpDNA, which indicated that pollen-mediated gene flow was much greater than seed-mediated gene flow. This promoted low nuclear genetic differentiation among populations of C. debaoensis. The study suggests that both genetic and anthropogenic disturbances have resulted in the critically endangered status of C. debaoensis.     

Development of ten microsatellite markers from the keystone mistletoe <Emphasis Type="Italic">Tristerix corymbosus</Emphasis> (Loranthaceae) using 454 next generation sequencing and their applicability to population genetic structure studies

Tristerix corymbosus (Loranthaceae) is a keystone mistletoe from the South American temperate rainforests. As most mistletoes, T. corymbosus relies on biotic pollination and seed dispersal, which may cause population structure. For a better understanding of its ecology, we isolated and characterized ten polymorphic microsatellite loci for this species. We used 454 Next Generation Sequencing to build a microsatellite library from a high quality DNA sample. We tested 90 sequences from which we obtained ten polymorphic markers. In order to assess their variability, the novel markers were tested in 48 individuals from three locations of the Valdivian Coastal Reserve in Chile. We also estimated genetic differences between pairs of populations using the F_ST statistic. The mean number of alleles per locus in the 48 individuals studied was 7.1 (2–17 alleles per locus). The observed and expected heterozygosity ranged from 0.298 to 0.634 and from 0.310 to 0.881, respectively. There were genetic differences among the three populations assessed, according to the F_ST values (ranging from 0.048 to 0.100, all significant) and, the number of alleles per locus ranged from 3.9 to 5.1. These are the first microsatellite markers developed for T. corymbosus, and they arise as a powerful tool for studying population structure, genetic diversity and gene flow at the landscape scale, along its distribution.     

Nuclear microsatellite markers reveal the low genetic structure of <Emphasis Type="Italic">Pinus mugo</Emphasis> Turra (dwarf mountain pine) populations in Europe

Twenty-one populations (555 individuals) covering the entire native range of Pinus mugo Turra (dwarf mountain pine) were investigated for genetic variation scored at 13 nuclear microsatellite markers (nSSRs). The main objective of the present study was to determine the genetic structure across the present distribution of the species and locate populations of different genetic compositions. Most of the genetic variation was observed within the populations (95%). The assignment of populations based on Bayesian clustering methods revealed that the Sudeten populations of P. mugo form a separate genetic cluster. These stands have likely been established through the founder effects of Alpine migrants. The distribution and level of SSR polymorphisms, along with no evidence of isolation by distance or phylogeographic structure, indicate that the present populations of P. mugo have diverged relatively recently and originate from a larger glacial distribution of the species. One peripheral stand from Italy had the lowest values of most calculated genetic variation indices. This stand could therefore be more susceptible to genetic drift and a negative impact of predicted environmental changes. We discuss our findings with respect to previously published results on the genetic and morphological variation of P. mugo and with consideration for the conservation genetics of the species.     

Molecular phylogeography and population evolution analysis of <Emphasis Type="Italic">Citrus ichangensis</Emphasis> (Rutaceae)

Ichang papeda (Citrus ichangensis), a wild and endemic perennial plant in Rutaceae, is characterized by the existence of wild and natural populations in southwestern and middle-west China. We analyzed a total of 231 individuals across 16 natural populations using chloroplast SSR markers, nuclear SSR markers, and single-copy nuclear genes. Standard population genetic analyses as well as Bayesian and maximum likelihood models were used to clarify the genetic diversity, population differentiation, barriers to gene flow, bottleneck events, isolation by distance, history migration, demographic history among populations, and phylogeny evolution. The chloroplast and nuclear genome analyses revealed a low level of genetic diversity in C. ichangensis. Clear signals of recent bottlenecks and strong patterns of isolation by distance were detected among different subpopulations, indicating a low extent of historical gene flow for this species and that genetic drift would occur after population differentiation. Bayesian clustering analyses revealed a clear pattern of genetic structure, with one cluster spanning the potential refugia in Wuling Mountains and Ta-pa Mountains, and other two clusters covering a more limited distribution range. The demographic history also supported the scenario that two isolated clusters originated in parallel from the genetic diversity center. Taxonomically, Ichang papeda may be a member of subgenus Citrus. Owing to the complicated topography, the mountainous regions and the Yangtze River have provided long-term stable habitats for C. ichangensis and acted as main barriers for its expansion, which might facilitate the process of speciation. Statistical population models and genetic data indicated strong genetic structure in C. ichangensis, which might result from the restricted gene flow, genetic drift, and population bottlenecks.     

Genetic diversity and population structure in the narrow endemic Chinese walnut <Emphasis Type="Italic">Juglans hopeiensis</Emphasis> Hu: implications for conservation

The conservation of narrow endemic species relies on accurate information regarding their population structure. Juglans hopeiensis Hu (Ma walnut), found only in Hebei province, Beijing, and Tianjin, China, is a threatened tree species valued commercially for its nut and wood. Sequences of two maternally inherited mitochondrial markers and two maternally inherited chloroplast intergenic spacers, three nuclear DNA sequences, and allele sizes from 11 microsatellites were obtained from 108 individuals of J. hopeiensis, Juglans regia, and Juglans mandshurica. Haplotype networks were constructed using NETWORK. Genetic diversity, population differentiation, and analysis of molecular variance (AMOVA) were used to determine genetic structure. MEGA was used to construct phylogenetic trees. Genetic diversity of J. hopeiensis was moderate based on nuclear DNA, but low based on uniparentally inherited mitochondrial DNA and chloroplast DNA. Haplotype networks showed that J. hopeiensis haplotypes were different than haplotypes found in J. regia and J. mandshurica. Allelic variants in nuclear genes that were shared among J. hopeiensis populations were not found in J. regia or J. mandshurica. Sampled populations of J. hopeiensis showed clear genetic structure. The maximum parsimony (MP) tree showed J. hopeiensis to be distinct from J. mandshurica but threatened by hybridization with J. regia and J. mandshurica. J. hopeiensis populations are strongly differentiated from sympatric Juglans species, but they are threatened by small population sizes and hybridization.     

Unique and isolated: population structure has implications for management of the endangered New Zealand sea lion

Female otariids (eared seals) frequently display strong levels of philopatry, a behaviour that has the potential to influence population structure, particularly at the mitochondrial level. Conversely, male otariids often move between breeding colonies, likely facilitating nuclear gene flow between colonies. Such gender-specific movements have the potential to influence species population structure. Here we investigate the genetic population structure of the endangered New Zealand (NZ) sea lion, using nuclear (microsatellite) and mitochondrial molecular markers, with the intention to better inform conservation through identification of management units for the species. The strong levels of female philopatry in this species have potential to lead to population structure at the mitochondrial loci. In contrast, weak or no population structure is expected across nuclear loci. NZ sea lions were sampled from the main breeding areas across the species’ current distribution (three Auckland Islands sites, two Campbell Island sites, one Stewart Island site and one Otago Peninsula site). Individuals were screened for microsatellite (n?=?271; 16 loci) and mitochondrial (n?=?56; 1027 bp D-loop and 1189 bp cytb). Despite a small (c. 9880 individuals) population size, moderate levels of microsatellite variation are observed in the NZ sea lions, in contrast to low levels of mitochondrial genetic variation. Results from mitochondrial DNA analyses revealed no population structure, suggesting that the strong level of female philopatry in NZ sea lions alone is not sufficient to maintain genetic population structure. Due to the frequent male movements between breeding colonies, no population structure was detected across the nuclear loci either. The absence of genetic structure suggests that, from a genetic perspective, NZ sea lions can be considered to be a single population. Despite this, the differing impacts of threats (e.g. fisheries by-catch) to each individual breeding colony must also be taken into consideration when defining management units for this endangered species.     

On the origin and genetic variability of the two invasive biotypes of <Emphasis Type="Italic">Chromolaena odorata</Emphasis>

Chromolaena odorata (L.) R. M. King and H. Robinson (Asteraceae), originally from the Neotropics, has become a serious weed in the humid tropics and subtropics of Southeast Asia, Africa and Pacific Islands. In its introduced distributions, C. odorata has been recognised as two biotypes, the Asian/West African (AWA) biotype and South African (SA) biotype, with independent distribution, morphology and ecological characters. To characterise the genetic variability and identify the likely source regions in the native distributions of the two biotypes, we carried out an extensive phylogeographic study using chloroplast and nuclear DNA sequences and microsatellite DNA markers. The analysis of both DNA sequences and nuclear markers showed that native populations possessed high genetic diversity, while both the AWA and SA biotypes in invaded regions appeared to have low genetic diversity. The AWA and SA biotypes were genetically distinct. Strong competitive ability and environmental adaptability may have facilitated the invasion AWA and SA biotypes in its respective invasive regions. We conclude that the source of AWA biotype may be Trinidad and Tobago, while the SA biotype was from Cuba and Jamaica. For a better outcome of biocontrol, the potential biological control agents for the two biotypes should be collected from these native regions, respectively.     

Conserving the small milkwort, <Emphasis Type="Italic">Comesperma polygaloides</Emphasis>, a vulnerable subshrub in a fragmented landscape

The conservation of remnant grassland vegetation on the Victorian volcanic plain (VVP) is crucial for the persistence of local biodiversity. Recent habitat loss has restricted the grassland to only a small percentage of its former range. Along with grassland habitats, species that occur on the VVP are in decline and many are legally protected. Comesperma polygaloides is a grassland species of the VVP that also occurs outside of the region in woodland habitats. We use 12 neutral microsatellite loci and two chloroplast regions to understand genotypic patterns of C. polygaloides in southeastern Australia. We found separate genetic clusters but they do not follow geographic boundaries. There are fewer alleles (2.96) and effective alleles (2.01) than expected from 12 microsatellite markers compared to other species. Even with the low number of alleles per locus there was a moderate level of genetic diversity detected (I = 0.69; H_o = 0.43; H_e = 0.40). Populations of the VVP could not be differentiated from populations elsewhere using neutral markers or chloroplast analyses. The genetic structure discovered was not consistent with the level of fragmentation observed. There may be several reasons for the observed lack of genetic structure: the species is more common than perceived, plants are long-lived and can reproduce clonally, and the bioregion is relatively young, geologically. Results indicate that restoration projects and long-term viability of C. polygaloides will be improved by composite seed sourcing, alleviating the risk of insufficient genetic diversity posed by an over-emphasis on local provenancing.     

Genetic diversity and population structure of the northern snakehead (<Emphasis Type="Italic">Channa argus</Emphasis> Channidae: Teleostei) in central China: implications for conservation and management

Major threats to freshwater fish diversity now include loss of native genetic diversity as a consequence of translocations of fishes between sites and from hatcheries to sites, and small effective population sizes resulting from overfishing and/or habitat loss. Ten polymorphic microsatellite markers were employed to evaluate genetic diversity, population genetic structure and gene flow amongst nine populations of the ecologically and economically important fish, the northern snakehead (Channa argus), in three river systems in central China. Multiple analyses revealed evidence of high genetic diversity and pronounced subdivision based on both regional separation and on river systems. A lack of evidence of genetic bottleneck over recent generations was consistent with the long-term stability of population size and contemporary distribution. The effective population sizes for most C. argus populations were small, suggesting the need for future conservation efforts focusing on these populations. Different lines of evidence point to the local enhancement of stocks by both aquaculture-reared fish and the transfer of wild fish. This study illustrates how human activities may affect genetic diversity and population genetic structure of C. argus populations, and highlights the need for new management regimes to protect native freshwater fish genetic diversity.     

Conservation genetics of the short-beaked common dolphin (<Emphasis Type="Italic">Delphinus delphis</Emphasis>) in the Mediterranean Sea and in the eastern North Atlantic Ocean

Mediterranean Sea common dolphins have recently been listed as ‘endangered’ in the IUCN Red list, due to their reported decline since the middle of the 20th century. However, little is know about the number or distribution of populations in this region. We analysed 118 samples from the Black Sea, Mediterranean Sea and eastern North Atlantic at nine microsatellite nuclear loci and for 428 bps of the mtDNA control region. We found small but significant population differentiation across the basin between the eastern and the western Mediterranean populations at both nuclear and mtDNA markers (microsatellite F _ST = 0.052, mtDNA F _ST = 0.107, P values ≤ 0.001). This matched the differential distribution and habitat use patterns exhibited by this species in the eastern and the western parts of the Mediterranean Sea. The assignment test of a small number of samples from the central Mediterranean could not exclude further population structure in the central area of the basin. No significant genetic differentiation at either marker was observed among the eastern north Atlantic populations, though the Alboran population (inhabiting the Mediterranean waters immediately adjacent the Atlantic ocean) showed significant mtDNA genetic differentiation compared to the Atlantic populations. Directional estimates of gene flow suggested movement of females out of the Mediterranean, which may be relevant to the population decline. Phylogenetic analysis suggested that the observed population structure evolved recently.     

<Emphasis Type="Italic">Bruguiera hainesii</Emphasis>, a critically endangered mangrove species,is a hybrid between <Emphasis Type="Italic">B. cylindrica</Emphasis> and <Emphasis Type="Italic">B. gymnorhiza</Emphasis> (Rhizophoraceae)

Bruguiera hainesii (Rhizophoraceae) is one of the two Critically Endangered mangrove species listed in the IUCN Red List of Threatened Species. Although the species is vulnerable to extinction, its genetic diversity and the evolutionary relationships with other Bruguiera species are not well understood. Also, intermediate morphological characters imply that the species might be of hybrid origin. To clarify the genetic relationship between B. hainesii and other Bruguiera species, we conducted molecular analyses including all six Bruguiera species using DNA sequences of two nuclear genes (CesA and UNK) and three chloroplast regions (intergenic spacer regions of trnL-trnF, trnS-trnG and atpB-rbcL). For nuclear DNA markers, all nine B. hainesii samples from five populations were heterozygous at both loci, with one allele was shared with B. cylindrica, and the other with B. gymnorhiza. For chloroplast DNA markers, the two haplotypes found in B. hainesii were shared only by B. cylindrica. These results suggested that B. hainesii is a hybrid between B. cylindrica as the maternal parent and B. gymnorhiza as the paternal one. Furthermore, chloroplast DNA haplotypes found in B. hainesii suggest that hybridization has occurred independently in regions where the distribution ranges of the parental species meet. As the IUCN Red List of Threatened Species currently excludes hybrids (except for apomictic plant hybrids), the conservation status of B. hainesii should be reconsidered.     

Development of 13 microsatellite markers for <Emphasis Type="Italic">Castanopsis tribuloides</Emphasis> (Fagaceae) using next-generation sequencing

Catanopsis tribuloides is a climax tree species commonly distributed in evergreen forests and has been used to restore degraded areas in northern Thailand. To aid in study of genetic diversity of the species, microsatellite markers, which are specific to C. tribuloides, were developed using whole genome sequencing by next-generation sequencing technology. The primers for microsatellite were developed and screened for optimal annealing temperature by PCR assay. The loci primers specific with C. tribuloides, 13 polymorphic microsatellite primers were successfully developed. The results from genetic information analyzing showed the number of alleles presented were between 2 and 24. Accordingly, the expected and observed heterozygosity obtained were between 0.298 and 0.920 and 0.364 to 1.000, respectively. Null allele frequency was presented 0.000–0.199. Genetic information was generated 10 loci primers significantly deviated from Hardy–Weinberg Equilibrium. All 13 primer pairs of loci were not significant with linkage disequilibrium. A set of microsatellite markers in this study could be applied to gene flow, genetic structure and population genetic studies in the future.     

Genetic diversity of the Pacific walrus (<Emphasis Type="Italic">Odobenus rosmarus divergens</Emphasis>) in the western part of the Chukchi Sea

We study 117 Pacific walrus samples from three rookeries within the western part of Chukchi Sea (Cape Vankarem, Cape Serdtse-Kamen, and Kolyuchin Island). We analyze the variability of nuclear (20 microsatellite loci) and mitochondrial DNA (three fragments). Two microsatellite loci which are described as microsatellites for the first time are used in this study: repeated sequences within introns of Coro1c and Plod2 genes. A high degree of genetic diversity is demonstrated for both nuclear and mitochondrial markers compared to Atlantic walrus. A high degree of genetic diversity is preserved within populations of Pacific walrus, despite a strong decline in the recent past. We discover the absence of significant differentiation for microsatellite loci and the presence of weak differentiation for mtDNA (mainly for a D-loop fragment). Walrus specimens that use the rookeries of the western part of Chukchi Sea are thought to belong to a single reproductive group.     

Integrating genetics and suitability modelling to bolster climate change adaptation planning in Patagonian <Emphasis Type="Italic">Nothofagus</Emphasis> forests

We investigated the impact of past changes in habitat suitability on the current patterns of genetic diversity of two southern beeches (Nothofagus nervosa and Nothofagus obliqua) in their eastern fragmented range in Patagonian Argentina, and model likely future threats to their population genetic structure. Our goal was to develop a spatially-explicit strategy for guiding conservation and management interventions in light of climate change. We combined suitability modelling under current, past (Last Glacial Maximum ~ 21,000 bp), and future (2050s) climatic conditions with genetic characterization data based on chloroplast DNA, isozymes, and microsatellites. We show the complementary usefulness of the distribution of chloroplast haplotypes and locally common allelic richness calculated from microsatellite data for identifying the locations of putative glacial refugia. Our findings suggest that contemporary hotspots of genetic diversity correspond to convergence zones of different expansion routes, most likely as a consequence of admixture processes. Future suitability predictions suggest that climate change might differentially affect both species. All genetically most diverse populations of N. nervosa and several of N. obliqua are located in areas that may be most severely impacted by climate change, calling for forward-looking conservation interventions. We propose a practical spatially- explicit strategy to target conservation interventions distinguishing priority populations for (1) in situ conservation (hotspots of genetic diversity likely to remain suitable under climate change), (2) ex situ conservation in areas where high genetic diversity overlaps with high likelihood of drastic climate change, (3) vulnerable populations (areas expected to be negatively affected by climate change), and (4) potential expansion areas under climate change.     

Contribution of genetics for implementing population translocation of the threatened <Emphasis Type="Italic">Arnica montana</Emphasis>

Ecological restoration programmes aiming at population recovery of imperilled plant species increasingly involve plant translocations. Evaluating the genetic status of seed source and target populations is essential for designing plant translocation protocols and optimizing recovery success. We developed nine polymorphic microsatellite markers and used three plastid markers to investigate genetic variation and structure of the two last large and six small remaining populations of the self-incompatible, clonally-propagating Arnica montana in southern Belgium and bordering France. The aim of the study was to determine the genetic status of these remaining populations and whether the large populations can be used as seed source for translocations. Most small populations maintained high genetic diversity and showed no inbreeding or a heterozygote excess, which may be explained by high genet longevity thanks to clonal propagation, heterosis, inbreeding depression at early development stages and/or no recruitment. Genotypic diversity was low in small populations, with clonal propagation mainly contributing to rosette production. The number of genets, and therefore effective population size, was often very small, restricting compatible mate availability. The situation is therefore more critical than it seems on the field, and bringing new genetic variation is necessary. Although no polymorphism was found in plastid DNA markers, between-population differentiation based on microsatellite markers was moderate, except for very small populations, where it was greater (F_ST?>?0.200). These patterns of differentiation were likely due to genetic drift effects and demographic stochasticity. We recommend using mixed seed material from the two large populations for translocations, and before conducting reinforcements, to first implement crossing experiments and reintroductions of mixed and crossed material in ecologically restored sites to understand the long-term effects of combining genotypes from different locations.     

Development and Characterization of Microsatellite Markers for Genetic Analysis of the Swimming Crab, <Emphasis Type="Italic">Portunus trituberculatus</Emphasis>

This study isolated and characterized 11 novel microsatellite markers for the commercially important swimming crab species, Portunus trituberculatus. Genetic diversity and population structure of two populations of P. trituberculatus in the East China Sea were assessed using these loci. The microsatellite markers produced 242 alleles, varying from 17 to 26 alleles per locus. In all the samples, the range of heterozygosity was 0.6324–0.9403 (observed) and 0.8998–0.9547 (expected). An F-statistic analysis revealed low genetic differentiation between the populations (mean F _ST = 0.0197), with 98% of the variation resulting from the within-population component. In addition, cross-amplification was tested in two other portunid species, and we found that many loci yielded useful information. The high degree of polymorphism exhibited by the 11 microsatellites suggests that these markers will be useful for both aquaculture and studies of natural populations of the genus.