First Insights into the Genetic Diversity of the Pinewood Nematode in Its Native Area Using New Polymorphic Microsatellite Loci

The pinewood nematode, Bursaphelenchus xylophilus, native to North America, is the causative agent of pine wilt disease and among the most important invasive forest pests in the East-Asian countries, such as Japan and China. Since 1999, it has been found in Europe in the Iberian Peninsula, where it also causes significant damage. In a previous study, 94 pairs of microsatellite primers have been identified in silico in the pinewood nematode genome. In the present study, specific PCR amplifications and polymorphism tests to validate these loci were performed and 17 microsatellite loci that were suitable for routine analysis of B. xylophilus genetic diversity were selected. The polymorphism of these markers was evaluated on nematodes from four field origins and one laboratory collection strain, all originate from the native area. The number of alleles and the expected heterozygosity varied between 2 and 11 and between 0.039 and 0.777, respectively. First insights into the population genetic structure of B. xylophilus were obtained using clustering and multivariate methods on the genotypes obtained from the field samples. The results showed that the pinewood nematode genetic diversity is spatially structured at the scale of the pine tree and probably at larger scales. The role of dispersal by the insect vector versus human activities in shaping this structure is discussed.     

Genetic diversity and population structure of the Black-faced Spoonbill (Platalea minor) among its breeding sites in South Korea: Implication for conservation

The endangered Black-faced Spoonbill Platalea minor has experienced drastic reductions in population size, geographic distribution, and habitat availability throughout East Asia. In the present study, we examined population genetic structure and genetic diversity of Black-faced Spoonbills inhabiting five sites off the west coast of South Korea encompassing a few of its major breeding sites. Ten microsatellite loci and the mitochondrial sequence were used to assess patterns of genetic variation based on 63 individuals. Three ND2 haplotypes were found among 61 individuals; the remaining two were identified as Eurasian Spoonbills, revealing an unexpected hybridization between these two species having different ecological niches in South Korea—the Eurasian Spoonbill overwinters in inland areas, whereas the Black-faced Spoonbill inhabits coastal areas during the summer. Analyses of microsatellite variation revealed no discrete population structure among the five breeding sites but very weak genetic differentiation among geographically distant regions. Assignment tests identified several possible migrants among sites. Our findings suggested that Black-faced Spoonbills from the five breeding sites could be managed as a single population and highlighted the importance of conserving the populations from Maedo, Suhaam, and Namdong reservoir, which are geographically close and have retained high levels of genetic diversity and large populations.     

Isolation and characterization of 12 microsatellite loci from Korean water deer (Hydropotes inermis argyropus) for population structure analysis in South Korea

The main goals of this study were to isolate microsatellites markers of Korean water deer (Hydropotes inermis argyropus) and understand the genetic status of the species in South Korea. Twelve new microsatellite loci were isolated and characterized to establish basic population genetic parameters for 45 H. i. argyropus specimens in South Korea. There were no significant regional or genetic structure differences between the mid-eastern and southwestern populations in South Korea according to the population genetic analyses. The number of alleles per locus ranged from two to 13 with an average of 6.08. Mean expected (H _E) and observed heterozygosity (H _O) were 0.622 and 0.533, respectively. Microsatellite variability was also not significant between the two regions (F _ST=0.012). These new markers should facilitate the future population genetics studies of Korean water deer and other closely related species.     

Differentiation at mitochondrial and nuclear loci between the blesbok (Damaliscus pygargus phillipsi) and bontebok (D. p. pygargus): implications for conservation strategy

Damaliscus pygargus is an endemic species in South Africa belonging to the contemporary antelope tribe Alcelaphine. The species has been subdivided into two subspecies based upon phenotypic differences and historical geographic isolation. We found D. p. pygargus (bontebok) and D. p. phillipsi (blesbok) to be significantly differentiated based on microsatellite and mtDNA control region markers. Bontebok genetic diversity was depauperate with just one mtDNA haplotype, not found in blesbok populations, and reduced heterozygosity and polymorphism at microsatellite DNA loci compared to the blesbok. Erosion of molecular genetic variation in bontebok may have been due to anthropogenic impact causing isolation and reduced population size. Our data based on 34 bontebok and 42 blesbok indicate that the classification of alpha taxonomy should be reconsidered in this genus (in support of earlier similar suggestions), and that careful management should seek to avoid hybridization and retain the remaining diversity in the bontebok.     

Conservation genetics of Amorpha georgiana (Fabaceae), an endangered legume of the Southeastern United States

Amorpha georgiana (Fabaceae) is an endangered legume species found in longleaf pine savannas in the Southeastern United States. Approximately 900 individuals and 14 populations remain, most of which are concentrated in North Carolina. Eleven microsatellite loci were used to explore genetic diversity, population structure and recent population bottlenecks using genotypic data from 132 individuals collected at ten different localities. Although A. georgiana is quite rare, it exhibited high levels of genetic diversity (17.7 alleles/locus; H _o = 0.65, H _E = 0.75). Most of the genetic variation was found within rather than between populations of this species. The single remaining Georgia population was well differentiated from populations of the Carolinas ( F _ST > 0.1), which had weaker structure among them ( F _ST < 0.1). Only a geographically disjunct population showed strong evidence of a recent population bottleneck, perhaps due to a recent founder event. Hybridization with A. herbacea was also detected. For conservation management plans, A. georgiana populations in each geographic region (North Carolina, South Carolina and Georgia) plus a disjunct population in North Carolina (Holly Shelter) should be treated as separate management units for which in situ conservation, including habitat restoration and use of prescribed burns, should ensure persistence of this species and preservation of its evolutionary potential.     

黑头酸臭蚁微卫星标记的分离与开发(英文)

【目的】本研究旨在分离黑头酸臭蚁Tapinoma melanocephalum基因组微卫星标记,确定这些微卫星位点的多态性。【方法】使用454 GS-FLX焦磷酸测序技术开发来自中国华南陆地和岛屿的11个黑头酸臭蚁地理种群基因组微卫星位点。从随机设计的100对微卫星引物中筛选出10对引物,用于确定黑头酸臭蚁4个地理种群［东澳岛(DAD)、荷包岛(HBD)、梅州(MZ)和山咀(SJ)］10个微卫星位点的多态性,分析种群遗传多样性和种群分化。【结果】从11个黑头酸臭蚁地理种群基因组中成功开发和分离10对微卫星引物。在DAD, HBD, MZ和SJ 4个地理种群中,10个微卫星位点中7个有高多态性,这10个位点均显著偏离Hardy-Weinberg平衡;每个位点的等位基因数量(A)是3.50~9.00个,每个地理种群每个位点等位基因丰富度(A_R)在1.992~12.938之间。岛屿地理种群(DAD和HBD)的AR和预期杂合度(H_E)与大陆地理种群(MZ和SJ)的相比差异不显著。4个地理种群均显示高水平遗传分化(F_ST=0.15969);HBD和MZ种群与其他配对地理种群相比,遗传分化较高(F_ST=0.185),基因流较低,说明这两个种群基因流被限制。此外,遗传变异来自种群内个体之间。【结论】筛选新的微卫星位点能够为研究黑头酸臭蚁种群结构和繁殖结构提供有效工具,以深入了解其传播机制。     

Genetic diversity and population structure of sickleweed (Falcaria vulgaris; Apiaceae) in the upper Midwest USA

Sickleweed (Falcaria vulgaris), an introduced species native to Europe and Asia, grows as an aggressive weed in some areas of the upper Midwest in the United States. We are reporting genetic diversity and population structure of sickleweed populations using microsatellite markers and nuclear and chloroplast DNA sequences. Populations showed high genetic differentiation but did not show significant geographic structure, suggesting random establishment of different genotypes at different sites was likely due to human mediated multiple introductions. Three genetic clusters revealed by microsatellite data and presence of six chlorotypes supported our hypothesis of multiple introductions. Chloroplast DNA sequence data revealed six chlorotypes nested into two main lineages suggesting at least two introductions of sickleweed in the upper Midwest. Some individuals exhibited more than two alleles at several microsatellite loci suggesting occurrence of polyploidy, which could be a post-introduction development to mitigate the inbreeding effects. High genetic variation in the introduced range attributable to multiple introductions and polyploidy may be inducing the evolution of invasiveness in sickleweed. Results of this study provide valuable insights into the evolution of sickleweed and baseline data for designing proper management practices for controlling sickleweed in the United States.     

Isolation and Characterization of Microsatellite Markers for <Emphasis Type="Italic">Viola websteri</Emphasis> (Violaceae) and Cross-Species Amplification within the Genus <Emphasis Type="Italic">Viola</Emphasis>

We isolated and characterized microsatellite loci in Viola websteri (Violaceae), an endangered species from Korea and endemic to Northeast Asia. A total of 27 microsatellite loci were developed and tested in Korean and Chinese populations. The number of alleles per locus varied from two to eight. The observed and expected heterozygosities within two populations were 0.000 to 1.000 and 0.080 to 0.816, respectively. Korean and Chinese populations were clearly distinguished by the private alleles from 16 loci. A total of 21 loci out of the 27 developed loci were successfully cross-amplified in 39 other Viola species. We believe that these microsatellite loci will be useful for future studies on genetic diversity and population structure of V. websteri, as well as other Viola species.     

Phylogeography and genetic ancestry of tigers (Panthera tigris)

Eight traditional subspecies of tiger (Panthera tigris),of which three recently became extinct, are commonly recognized on the basis of geographic isolation and morphological characteristics. To investigate the species' evolutionary history and to establish objective methods for subspecies recognition, voucher specimens of blood, skin, hair, and/or skin biopsies from 134 tigers with verified geographic origins or heritage across the whole distribution range were examined for three molecular markers: (1) 4.0 kb of mitochondrial DNA (mtDNA) sequence; (2) allele variation in the nuclear major histocompatibility complex class II DRB gene; and (3) composite nuclear microsatellite genotypes based on 30 loci. Relatively low genetic variation with mtDNA,DRB,and microsatellite loci was found, but significant population subdivision was nonetheless apparent among five living subspecies. In addition, a distinct partition of the Indochinese subspecies P. t. corbetti in to northern Indochinese and Malayan Peninsula populations was discovered. Population genetic structure would suggest recognition of six taxonomic units or subspecies: (1) Amur tiger P. t. altaica; (2) northern Indochinese tiger P. t. corbetti; (3) South China tiger P. t. amoyensis; (4) Malayan tiger P. t. jacksoni, named for the tiger conservationist Peter Jackson; (5) Sumatran tiger P. t. sumatrae; and (6) Bengal tiger P. t. tigris. The proposed South China tiger lineage is tentative due to limited sampling. The age of the most recent common ancestor for tiger mtDNA was estimated to be 72,000-108,000 y, relatively younger than some other Panthera species. A combination of population expansions, reduced gene flow, and genetic drift following the last genetic diminution, and the recent anthropogenic range contraction, have led to the distinct genetic partitions. These results provide an explicit basis for subspecies recognition and will lead to the improved management and conservation of these recently isolated but distinct geographic populations of tigers.     

SSR-based population structure, molecular diversity and linkage disequilibrium analysis of a collection of flax (Linum usitatissimum L.) varying for mucilage seed-coat content

Flax seed mucilage (SM) presents specific biological activities useful for the food and pharmaceutical industries. Understanding the population structure, genetic diversity and linkage disequilibrium (LD) of germplasm varying for mucilage content is pivotal for the identification of genes and quantitative trait loci underlying mucilage variation by association mapping (AM). In this study, 150 microsatellite loci were used to assess the population structure, genetic diversity and LD of a set of 60 flax cultivars/accessions capturing the breadth of SM variation in flax germplasm. STRUCTURE analysis and similarity-based methods revealed the presence of three populations reflecting mainly their geographic origins (South Asia, South America and North America), and the impact of germplasm exchange within and between North American flax breeding programs. Analysis of molecular variance showed that 78.32% of the genetic variation resided within populations and 21.68% among populations. The phi-statistic (??_st) value of 0.22 confirmed the presence of a strong population structure. A total of 408 alleles were detected, with the South American population capturing the highest overall diversity. However, the genetic diversity was narrow, as indicated by the small number of alleles per locus (2.72) and gene diversity (mean?=?0.34). LD was significant between 3.9% (r ²) and 36.2% (D??) of the loci pairs (FDR?<?0.05). The mean r ² and D?? were 0.26 and 0.53, respectively. The results suggest that the collection could be useful in AM studies aimed at the discovery of genes/alleles involved in SM; however a greater diversity may be required to improve the AM resolution.     

Genetic differentiation of the pine wilt disease vector Monochamus alternatus (Coleoptera: Cerambycidae) over a mountain range - revealed from microsatellite DNA markers

To study the dispersal process of the pine sawyer Monochamus alternatus (Hope) in frontier populations, a microsatellite marker-based genetic analysis was performed on expanding populations at the northern limit of its range in Japan. In Asian countries, M. alternatus is the main vector of pine wilt disease, the most serious forest disease in Japan. Sawyers were collected from nine sites near the frontier of the pine wilt disease damage area. A mountain range divides the population into western and eastern sides. Five microsatellite loci were examined and a total of 188 individuals was genotyped from each locus with the number of alleles ranged from two to nine. The mean observed heterozygosity for all loci varied from 0.282 to 0.480 in the nine sites, with an overall mean of 0.364. None of the populations have experienced a significant bottleneck. Significant differentiation was found across the mountain range, but the genetic composition was similar amongst populations of each side. It is believed that the mountain range acts as a geographical barrier to dispersal and that gene flow without a geographical barrier is high. On the west side of the mountain range, a pattern of isolation by distance was detected. This was likely to be caused by secondary contact of different colonizing routes on a small spatial scale. Based on these data, a process linking genetic structure at local (kilometres) and regional spatial scales (hundreds of kilometres) was proposed.     

Genetic diversity and genetic structure of the endangered Manchurian trout, <Emphasis Type="Italic">Brachymystax lenok tsinlingensis</Emphasis>, at its southern range margin: conservation implications for future restoration

The Manchurian trout, Brachymystax lenok tsinlingensis (family: Salmonidae), is a cold freshwater fish endemic to Northeast Asia. South Korean populations of this species, which comprise its southern range limit, have recently decreased markedly in size and are now becoming critically endangered. We assessed the current population status of this species in South Korea by estimating the levels of genetic diversity and genetic structure of five natural and four restored populations using mitochondrial DNA (mtDNA) control region sequences and eight nuclear microsatellite loci. Levels of within-population genetic diversity were low, suggesting that past effective population sizes (N _e) have been small. Each population had one or a maximum of two mtDNA haplotypes. Microsatellite allelic richness (AR) was significantly higher for natural populations (mean AR?=?3.51; 95% confidence interval, 3.00–4.03) than for restored populations (mean AR?=?2.61; 2.38–2.98). South Korean populations were significantly genetically isolated from one another, with private mtDNA haplotypes and microsatellite alleles, suggesting that limited gene flow has been occurring among populations. A mtDNA phylogeny revealed that South Korean lineages were more closely related to those of China than to those of North Korea and Russia. Overall, we suggest that future restoration efforts aimed at South Korean populations should consider the genetic characteristics reported here, which should help to fulfil effective conservation strategies for this highly cherished species. Our results will inform other conservation efforts, including assisted migration of freshwater fish populations at the equatorial end of the geographical range limit of the species.     

Environmental Variables Explain Genetic Structure in a Beetle-Associated Nematode

The distribution of a species is a complex expression of its ecological and evolutionary history and integrating population genetic, environmental, and ecological data can provide new insights into the effects of the environment on the population structure of species. Previous work demonstrated strong patterns of genetic differentiation in natural populations of the hermaphroditic nematode Pristionchus pacificus in its La Réunion Island habitat, but gave no clear understanding of the role of the environment in structuring this variation. Here, we present what is to our knowledge the first study to statistically evaluate the role of the environment in shaping the structure and distribution of nematode populations. We test the hypothesis that genetic structure in P. pacificus is influenced by environmental variables, by combining population genetic analyses of microsatellite data from 18 populations and 370 strains, with multivariate statistics on environmental data, and species distribution modelling. We assess and quantify the relative importance of environmental factors (geographic distance, altitude, temperature, precipitation, and beetle host) on genetic variation among populations. Despite the fact that geographic populations of P. pacificus comprise vast genetic diversity sourced from multiple ancestral lineages, we find strong evidence for local associations between environment and genetic variation. Further, we show that significantly more genetic variation in P. pacificus populations is explained by environmental variation than by geographic distances. This supports a strong role for environmental heterogeneity vs. genetic drift in the divergence of populations, which we suggest may be influenced by adaptive forces.     

Genome-wide effects of postglacial colonization in Arabidopsis lyrata

The perennial outcrossing Arabidopsis lyrata is becoming a plant model species for molecular ecology and evolution. However, its evolutionary history, and especially the impact of the climatic oscillations of the Pleistocene on its genetic diversity and population structure, is not well known. We analyzed the broad-scale population structure of the species based on microsatellite variation at 22 loci. A wide sample in Europe revealed that glaciations and postglacial colonization have caused high divergence and high variation in variability between populations. Colonization from Central Europe to Iceland and Scandinavia was associated with a strong decrease of genetic diversity from South to North. On the other hand, the Russian population included in our data set may originate from a different refugium probably located more to the East. These genome-wide patterns must be taken into account in studies aiming at elucidating the genetic basis of local adaptation. As shown by sequence data, most of the loci used in this study do not evolve like typical microsatellite loci and show variable levels of homoplasy: this mode of evolution makes these markers less suitable to investigate the between-continent divergence and more generally the worldwide evolution of the species. Finally, a strong negative correlation was detected between levels of within-population diversity and indices of differentiation such as F(ST). We discuss the causes of this correlation as well as the potential bias it induces on the quantification and interpretation of population structure.     

Genetic diversity and differentiation of the Korean starry flounder (Platichthys stellatus) between and within cultured stocks and wild populations inferred from microsatellite DNA analysis

The Korean starry flounder, Platichthys stellatus, is economically valuable coastal resident fish species. However, the annual catch of this fish has fluctuated and suffered major declines in Korea. We examined the genetic diversity and population structure for four wild populations and three hatchery stocks of Korean starry flounder to protect its genetic integrity using nine microsatellites. A group of 339 genotypes belonging to seven populations were screened. High degrees of polymorphism at the microsatellite loci were observed within both the wild and hatchery populations. Compared to the wild populations, genetic changes, including reduced genetic diversity and highly significant differentiation, have occurred in cultured stocks. Significant population differentiation was also observed in wild starry flounder populations. Similar degrees of inbreeding and significant Hardy–Weinberg equilibrium deviations were detected in both the wild and the hatchery populations. The genetic connectivity pattern identified four distinct metapopulations of starry flounder in Korea by clustering in the phylogenetic tree, Bayesian analyses, molecular variance analysis, PCA and multidimensional scaling analysis. A pattern of isolation-by-distance was not significant. This genetic differentiation may be the result of the co-effects of various factors, such as historic dispersal, local environment or anthropogenic activities. These results provide useful information for the genetic monitoring of P. stellatus hatchery stocks, for the genetic improvement of this species by selective breeding and for designing suitable management guidelines for the conservation of this species.     

Characterisation of 12 microsatellite loci in the Vietnamese commercial clam <Emphasis Type="Italic">Lutraria rhynchaena</Emphasis> Jonas 1844 (Heterodonta: Bivalvia: Mactridae) through next-generation sequencing

The marine clam Lutraria rhynchaena is gaining popularity as an aquaculture species in Asia. Lutraria populations are present in the wild throughout Vietnam and several stocks have been established and translocated for breeding and aquaculture grow-out purposes. In this study, we demonstrate the feasibility of utilising Illumina next-generation sequencing technology to streamline the identification and genotyping of microsatellite loci from this clam species. Based on an initial partial genome scan, 48 microsatellite markers with similar melting temperatures were identified and characterised. The 12 most suitable polymorphic loci were then genotyped using 51 individuals from a population in Quang Ninh Province, North Vietnam. Genetic variation was low (mean number of alleles per locus = 2.6; mean expected heterozygosity = 0.41). Two loci showed significant deviation from Hardy–Weinberg equilibrium (HWE) and the presence of null alleles, but there was no evidence of linkage disequilibrium among loci. Three additional populations were screened (n = 7–36) to test the geographic utility of the 12 loci, which revealed 100 % successful genotyping in two populations from central Vietnam (Nha Trang). However, a second population from north Vietnam (Co To) could not be successfully genotyped and morphological evidence and mitochondrial variation suggests that this population represents a cryptic species of Lutraria. Comparisons of the Qang Ninh and Nha Trang populations, excluding the 2 loci out of HWE, revealed statistically significant allelic variation at 4 loci. We reported the first microsatellite loci set for the marine clam Lutraria rhynchaena and demonstrated its potential in differentiating clam populations. Additionally, a cryptic species population of Lutraria rhynchaena was identified during initial loci development, underscoring the overlooked diversity of marine clam species in Vietnam and the need to genetically characterise population representatives prior to microsatellite development. The rapid identification and validation of microsatellite loci using next-generation sequencing technology warrant its integration into future microsatellite loci development for key aquaculture species in Vietnam and more generally, aquaculture countries in the South East Asia region.     

Cross-species amplification of Bovidae microsatellites and low diversity of the endangered Korean goral

The Korean goral (Nemorhaedus caudatus) is an endangered species of wild goat. The conservation and management of this species could benefit from a better understanding of its genetic diversity and structure. Cross-species amplification of 34 Bovidae microsatellite loci was tested on a panel of 6 Korean gorals and 10 domestic goats. After polymerase chain reaction (PCR) optimization, 29 (85.3%) microsatellite loci amplified successfully for the Korean gorals and 27 (79.4%) for the domestic goats. Of the amplified products, 16 (55.2%) were polymorphic in the Korean goral and 22 (81.5%) in domestic goats. Nei's unbiased mean heterozygosity and mean allele number per locus were, respectively, 0.356 and 2.6 in the Korean goral and 0.636 and 4.8 in domestic goats. Low genetic diversity in the Korean gorals observed in this preliminary microsatellite survey suggests an urgent need for further detailed study of genetic diversity in Korean goral populations and a population management strategy based on these studies. Current results of cross-species amplification of domestic Bovidae microsatellites could be employed for the necessary population genetic studies on the Korean goral and other endangered Caprinae species.     

Population genetic structure of Korean pen shell (Atrina pectinata) in Korea inferred from microsatellite marker analysis

The Korean pen shell Atrina pectinata is a commercially valuable species in Korea. The commercial catch of this fish has decreased continuously since 1990. However, its genetic characteristics have never been studied. In order to explore the population genetic structure of this species, 125 pen shells sampled from three major habitats along the western coast of Korea were genotyped at 21 microsatellite loci. Relatively high levels of genetic variability (mean number of allelic richness (A_R) = 14.74; mean hetrerozygosity (He) = 0.849) was found among localities. However, despite the potential of the Korean pen shell to exhibit genetic structure (adults are sedentary), none of the genetic tests applied in this study detected significant genetic differentiation among the samples. The lack of genetic differentiation among samples may be due to high levels of larval dispersal, through passive drift with ocean currents. Alternatively, populations may have diverged too recently for significant genetic differentiation to have become evident. Furthermore, small sample sizes and the limited number of samples may have hampered the detection of genetic structure. The results of this study therefore suggested a lack of genetic structure among the populations of pen shell of Korean waters and should be managed as a single unit. This information on the genetic characteristics of Korean pen shell populations has important implications for the sustainable exploitation of the fishing resources and the preservation of biodiversity.     

Influence of voltine ecotype and geographic distance on genetic and haplotype variation in the Asian corn borer

Diapause is an adaptive dormancy strategy by which arthropods endure extended periods of adverse climatic conditions. Seasonal variation in larval diapause initiation and duration in Ostrinia furnacalis may influence adult mating generation number (voltinism) across different local environments. The degree to which voltine ecotype, geographic distance, or other ecological factors influence O. furnacalis population genetic structure remains uncertain. Genetic differentiation was estimated between voltine ecotypes collected from 8 locations. Mitochondrial haplotypes were significantly different between historically allopatric univoltine and bivoltine locations, but confounded by a strong correlation with geographic distance. In contrast, single nucleotide polymorphism (SNP) genotypes show low but significant levels of variation and a lack of influence of geographic distance between allopatric voltine locations. Regardless, 11 of 257 SNP loci were predicted to be under selection, suggesting population genetic homogenization except at loci proximal to factors putatively under selection. These findings provide evidence of haplotype divergent voltine ecotypes that may be maintained in allopatric and sympatric areas despite relatively high rates of nuclear gene flow, yet influence of voltinism on maintenance of observed haplotype divergence remains unresolved.