Nuclear Microsatellite Loci Research Articles

Low genetic diversity and potential inbreeding in an isolated population of alder buckthorn (Frangula alnus) following a founder effect

Alder buckthorn (Frangula alnus) is one of Ireland’s rarest tree species, and in Northern Ireland the species is now restricted to a single population in Peatlands Park, Co. Armagh numbering ca. 140 mature trees. Genotyping of 95% of the trees at nine nuclear microsatellite loci revealed that levels of genetic diversity within this population were generally lower than those reported from larger populations in Spain. Analysis of six chloroplast microsatellite loci revealed no variation. The level of FIS was significantly higher than that in the Spanish populations, as well as in other populations across Europe, potentially indicating inbreeding. Spatial autocorrelation analysis indicated some evidence of fine-scale genetic structuring, most likely due to limited seed dispersal, but the overall level of differentiation between subpopulations was low, indicating high levels of gene flow, probably due to cross-pollination by bees. Our results are consistent with a gradual population expansion from a limited number of individuals. We suggest that more immediate conservation efforts might be best focused on ensuring suitable habitat for the continued recovery of this isolated population.

Parallel Patterns of Host-Specific Morphology and Genetic Admixture in Sister Lineages of a Commensal Barnacle.

Symbiotic relationships are often species specific, allowing symbionts to adapt to their host environments. Host generalists, on the other hand, have to cope with diverse environments. One coping strategy is phenotypic plasticity, defined by the presence of host-specific phenotypes in the absence of genetic differentiation. Recent work indicates that such host-specific phenotypic plasticity is present in the West Pacific lineage of the commensal barnacle Chelonibia testudinaria (Linnaeus, 1758). We investigated genetic and morphological host-specific structure in the genetically distinct Atlantic sister lineage of C. testudinaria. We collected adult C. testudinaria from loggerhead sea turtles, horseshoe crabs, and blue crabs along the eastern U.S. coast between Delaware and Florida and in the Gulf of Mexico off Mississippi. We find that shell morphology, especially shell thickness, is host specific and comparable in similar host species between the Atlantic and West Pacific lineages. We did not detect significant genetic differentiation related to host species when analyzing data from 11 nuclear microsatellite loci and mitochondrial sequence data, which is comparable to findings for the Pacific lineage. The most parsimonious explanation for these parallel patterns between distinct lineages of C. testudinaria is that C. testudinaria maintained phenotypic plasticity since the lineages diverged 4-5 mya.

Long-term genetic monitoring of a riverine dragonfly, Orthetrum coerulescens (Odonata: Libellulidae]: Direct anthropogenic impact versus climate change effects.

Modern conservationists call for long term genetic monitoring datasets to evaluate and understand the impact of human activities on natural ecosystems and species on a global but also local scale. However, long-term monitoring datasets are still rare but in high demand to correctly identify, evaluate and respond to environmental changes. In the presented study, a population of the riverine dragonfly, Orthetrum coerulescens (Odonata: Libellulidae), was monitored over a time period from 1989 to 2013. Study site was an artificial irrigation ditch in one of the last European stone steppes and “nature heritage”, the Crau in Southern France. This artificial riverine habitat has an unusual high diversity of odonate species, prominent indicators for evaluating freshwater habitats. A clearing of the canal and destruction of the bank vegetation in 1996 was assumed to have great negative impact on the odonate larval and adult populations. Two mitochondrial markers (CO1 & ND1) and a panel of nuclear microsatellite loci were used to assess the genetic diversity. Over time they revealed a dramatic decline in diversity parameters between the years 2004 and 2007, however not between 1996 and 1997. From 2007 onwards the population shows a stabilizing trend but has not reached the amount of genetic variation found at the beginning of this survey. This decline cannot be referred to the clearing of the canal or any other direct anthropogenic impact. Instead, it is most likely that the populations’ decay was due to by extreme weather conditions during the specific years. A severe drought was recorded for the summer months of these years, leading to reduced water levels in the canal causing also other water parameters to change, and therefore impacting temperature sensitive riverine habitat specialists like the O. coerulescens in a significant way. The data provide important insights into population genetic dynamics and metrics not always congruent with traditional monitoring data (e.g. abundance); a fact that should be regarded with caution when management plans for developed landscapes are designed.

Genetic variation and phylogeographic structure of the cotton aphid, Aphis gossypii, based on mitochondrial DNA and microsatellite markers

Aphis gossypii, one of the most important agricultural pests in the world, can cause serious economic losses in the main crop-producing areas. To clarify issues such as the genetic differentiation, genetic structure, and demographic history of A. gossypii populations, we used 10 nuclear microsatellite loci (SSR) and two mitochondrial gene sequences (COI and Cytb) to investigate genetic diversity and population structure of A. gossypii populations that were collected from 33 sampling sites in China from different climatic zones. SSR and mtDNA data suggested low to moderate levels of genetic diversity. A star-shaped network of mtDNA haplotypes indicated that the maternal ancestor of China cotton aphids likely originated in Xinjiang. The POPTREE, STRUCTURE and principal coordinate analysis (PCoA) revealed two genetic clusters: an eastern and a western region group. Isolation by distance (IBD) results showed a positive correlation between geographic distance and genetic distance in the vast eastern region but not in the western region. Neutrality testing and mismatch distribution analysis provided strong evidence for a recent rapid expansion in most populations. Genetic bottleneck was not detected in A. gossypii populations of China. The present work can help us to develop strategies for managing this pest.

Genetic identification of source and likely vector of a widespread marine invader.

The identification of native sources and vectors of introduced species informs their ecological and evolutionary history and may guide policies that seek to prevent future introductions. Population genetics provides a powerful set of tools to identify origins and vectors. However, these tools can mislead when the native range is poorly sampled or few molecular markers are used. Here, we traced the introduction of the Asian seaweed Gracilaria vermiculophylla (Rhodophyta) into estuaries in coastal western North America, the eastern United States, Europe, and northwestern Africa by genotyping more than 2,500 thalli from 37 native and 53 non‐native sites at mitochondrial cox1 and 10 nuclear microsatellite loci. Overall, greater than 90% of introduced thalli had a genetic signature similar to thalli sampled from the coastline of northeastern Japan, strongly indicating this region served as the principal source of the invasion. Notably, northeastern Japan exported the vast majority of the oyster Crassostrea gigas during the 20th century. The preponderance of evidence suggests G. vermiculophylla may have been inadvertently introduced with C. gigas shipments and that northeastern Japan is a common source region for estuarine invaders. Each invaded coastline reflected a complex mix of direct introductions from Japan and secondary introductions from other invaded coastlines. The spread of G. vermiculophylla along each coastline was likely facilitated by aquaculture, fishing, and boating activities. Our ability to document a source region was enabled by a robust sampling of locations and loci that previous studies lacked and strong phylogeographic structure along native coastlines.

High intra-ocean, but limited inter-ocean genetic connectivity in populations of the deep-water oblique-banded snapper Pristipomoides zonatus (Pisces: Lutjanidae)

While many studies have investigated connectivity and subdivision in marine fish occupying tropical, shallow water reef habitats, relatively few have been conducted on commercially important deep-water species in the Indo-Pacific region. Here, we examine spatial and temporal genetic variation in the deep-water oblique-banded snapper Pristipomoides zonatus, collected from eight locations across the Indian and Pacific Oceans. A total of 292 individuals were screened for genetic variation at six nuclear microsatellite loci and the cytochrome c oxidase subunit 1 (COI) mitochondrial DNA (mtDNA) gene. There was evidence of low, but significant genetic differentiation between ocean basins (FCT=0.009) and no significant divergences between sites within oceans. The lack of population structure within ocean basins suggests P. zonatus has a long pelagic larval duration with high levels of connectivity between populations over large geographical distances (>2000km). There was no evidence of temporal variation in allele frequencies within populations. However, ephemeral genetic divergences between sites were detected, along with a significant reduction in genetic diversity at one site, suggesting there may be low effective population sizes (Ne). Our results suggest that localized declines in genetic diversity could be offset by gene flow from other locations within ocean basins, though predicting the broader impacts of localized stock depletions requires further understanding of recruitment dynamics and life history characteristics of the species.

A new set of microsatellite primers for Coelogyne fimbriata (Orchidaceae) and cross-amplification in C. ovalis.

Premise of the study:Declining orchid populations have made it necessary to prioritize the study of population structure and genetic diversity for species including Coelogyne fimbriata (Orchidaceae).Methods and Results:A biotin-streptavidin capture method was used to construct a microsatellite library for C. fimbriata. A total of 15 polymorphic nuclear microsatellite loci were isolated and characterized using 47 C. fimbriata individuals from two natural populations in China. The number of alleles per locus for the two populations ranged from two to 17. The observed and expected heterozygosities ranged from 0.000 to 1.000 and from 0.000 to 0.867, respectively. Among these polymorphic primers, 11 loci were also successfully amplified in C. ovalis, and 10 loci showed moderate to high-level polymorphism. Cross-amplification of the 15 polymorphic loci was tested in five related species: C. cumingii, C. eberhardtii, C. mayeriana, C. peltastes, and C. velutina.Conclusions:Fifteen microsatellites in C. fimbriata and 10 in C. ovalis have moderate to high-level genetic variation, indicating their utility in population genetic studies, thus contributing to orchid conservation.

Inferences of genetic structure and demographic history of Rhododendron protistum var. giganteum—The world’s largest Rhododendron using microsatellite markers

Big tree rhododendron (Rhododendron protistum var. giganteum) is an extremely endangered plant with only two remnant populations distributed in the Gaoligong Mountains of northwestern Yunnan Province, China. However, the genetic structure, demographic history, and the population dynamics of this species have been rarely described. In the present study, data from 14 polymorphic nuclear microsatellite loci were used to assess the levels of genetic diversity, genetic structure, and demographic history of big tree rhododendron remnant populations. Results suggested that renmants maintained a moderate level of genetic diversity (NE=2.86, AR=5.726, Ho=0.510, He=0.602, I=1.174) despite their small population. Bayesian clustering and principal coordinate analysis indicated that sampled individuals were clustered into two distinct genetic groups. Demographic history analysis revealed high historical and low contemporary gene flow between two remnant populations. BOTTLENECK analysis under a two-phase mutation model showed that the populations could have experienced a recent historical bottleneck. The effective population sizes of the two populations were smaller than 30. Therefore, in situ conservation of this species should be prioritized. Furtheremore, ex situ cultivation of field collected seeds and introduction of saplings into the source site might be considered for the future conservation and restoration of big tree rhododendron.

Reproductive Strategy of the Polyploid Species Varronia curassavica Jacq. in Restinga Environment.

This study aimed to elucidate the breeding strategies of Varronia curassavica, an important medicinal species associated with Brazilian restinga. This was accomplished by combining phenological and genetic data. Every 2 weeks over a period of 2 years, we measured flowering and fruiting phenology to evaluate the activity and intensity of phenophases (n = 60). We evaluated the mating system, pollen ovule ratio and genotypes from progeny and mother plants using 8 nuclear microsatellite loci. We observed flowering and fruiting of V. curassavica at low intensity throughout the entire year, but with 2 distinct peaks, one of which was seasonal, corresponding to the period of gradual increase of temperature and photoperiod. Overlapping of flowering and fruiting strategies favors gene flow among different groups of individuals and between populations by attraction of fauna throughout the year. Analysis of the mating system indicates that V. curassavica is a typical outcrossed species (t^ = 0.98; pollen/ovule ratio = 7087.50). Combining phenology with genetic studies improved our understanding of the reproductive strategies of this species. The typical outcrossing system of V. curassavica reflects the existence of functional self-incompatibility mechanisms still unaffected by changes in genetic balance by polyploidy.

Tiger on the prowl: Invasion history and spatio-temporal genetic structure of the Asian tiger mosquito Aedes albopictus (Skuse 1894) in the Indo-Pacific.

BackgroundWithin the last century, increases in human movement and globalization of trade have facilitated the establishment of several highly invasive mosquito species in new geographic locations with concurrent major environmental, economic and health consequences. The Asian tiger mosquito, Aedes albopictus, is an extremely invasive and aggressive daytime-biting mosquito that is a major public health threat throughout its expanding range.Methodology/Principal findingsWe used 13 nuclear microsatellite loci (on 911 individuals) and mitochondrial COI sequences to gain a better understanding of the historical and contemporary movements of Ae. albopictus in the Indo-Pacific region and to characterize its population structure. Approximate Bayesian computation (ABC) was employed to test competing historical routes of invasion of Ae. albopictus within the Southeast (SE) Asian/Australasian region. Our ABC results show that Ae. albopictus was most likely introduced to New Guinea via mainland Southeast Asia, before colonizing the Solomon Islands via either Papua New Guinea or SE Asia. The analysis also supported that the recent incursion into northern Australia’s Torres Strait Islands was seeded chiefly from Indonesia. For the first time documented in this invasive species, we provide evidence of a recently colonized population (the Torres Strait Islands) that has undergone rapid temporal changes in its genetic makeup, which could be the result of genetic drift or represent a secondary invasion from an unknown source.Conclusions/SignificanceThere appears to be high spatial genetic structure and high gene flow between some geographically distant populations. The species' genetic structure in the region tends to favour a dispersal pattern driven mostly by human movements. Importantly, this study provides a more widespread sampling distribution of the species’ native range, revealing more spatial population structure than previously shown. Additionally, we present the most probable invasion history of this species in the Australasian region using ABC analysis.

Hybridization drives evolution of apomicts in Rubus subgenus Rubus: evidence from microsatellite markers.

Rubus subgenus Rubus is a group of mostly apomictic and polyploid species with a complicated taxonomy and history of ongoing hybridization. The only polyploid series with prevailing sexuality is the series Glandulosi , although the apomictic series Discolores and Radula also retain a high degree of sexuality, which is influenced by environmental conditions and/or pollen donors. The aim of this study is to detect sources of genetic variability, determine the origin of apomictic taxa and validate microsatellite markers by cloning and sequencing. A total of 206 individuals from two central European regions were genotyped for 11 nuclear microsatellite loci and the chloroplast trn L- trn F region. Microsatellite alleles were further sequenced in order to determine the exact repeat number and to detect size homoplasy due to insertions/deletions in flanking regions. The results confirm that apomictic microspecies of ser. Radula are derived from crosses between sexual series Glandulosi and apomictic series Discolores , whereby the apomict acts as pollen donor. Each apomictic microspecies is derived from a single distinct genotype differing from the parental taxa, suggesting stabilized clonal reproduction. Intraspecific variation within apomicts is considerably low compared with sexual series Glandulosi , and reflects somatic mutation accumulation. While facultative apomicts produce clonal offspring, sexual species are the conduits of origin for new genetically different apomictic lineages. One of the main driving forces of evolution and speciation in the highly apomictic subgenus Rubus in central Europe is sexuality in the series Glandulosi . Palaeovegetation data suggest that initial hybridizations took place over different time periods in the two studied regions, and that the successful origin and spread of apomictic microspecies of the series Radula took place over several millennia. Additionally, the cloning and sequencing show that standard evaluations of microsatellite repeat numbers underestimate genetic variability considering homoplasy in allele size.

Microsatellite analysis supports the existence of three cryptic species within the bumble bee Bombus lucorum sensu lato

Mitochondrial cytochrome oxidase I (COI) partial sequences are widely used in taxonomy for species identification. Increasingly, these sequence identities are combined with modelling approaches to delineate species. Yet the validity of species delineation based on such DNA ‘barcodes’ is rarely tested and may be called into question by phenomena such as ancestral polymorphisms in DNA sequences, phylogeographic divergence, mitochondrial introgression and hybridization, or distortion of mitochondrial inheritance through such factors as Wolbachia infection. The common and widespread European bumble bee Bombus lucorum s. lato contains three distinct mitochondrial DNA lineages that are assumed to represent three cryptic species, namely Bombus cryptarum, B. lucorum s. str. and B. magnus. To test whether nuclear gene pools of the three putative species were differentiated, we genotyped 304 sympatric members of the lucorum complex (54 B. cryptarum females, 168 B. lucorum s. str. females and 82 B. magnus females, as defined using mtDNA COI haplotypes) from 11 localities spread across the island of Ireland at seven nuclear microsatellite loci. Multilocus genotypes clustered into three discrete groups that largely corresponded to the three mtDNA lineages: B. cryptarum, B. lucorum s. str. and B. magnus. The good fit of mitochondrial haplotype to nuclear (microsatellite) genotypic data supports the view that these three bumble bee taxa are reproductively isolated species, as well as providing a vindication of species identity using so-called DNA barcodes.

Population structure and historical demography of Dipteronia dyeriana (Sapindaceae), an extremely narrow palaeoendemic plant from China: implications for conservation in a biodiversity hot spot.

Inferring past demography is a central question in evolutionary and conservation biology. It is, however, sometimes challenging to disentangle their roles of contemporary versus historical processes in shaping the current patterns of genetic variation in endangered species. In this study, we used both chloroplast microsatellite (cpSSR) loci and nuclear microsatellite (nSSR) loci to assess the levels of genetic differentiation, genetic effective population size, contemporary/historical levels of gene flow and demographic history for five populations sampled across the range of Dipteronia dyeriana, an endangered palaeoendemism from Southwestern China. We found that D. dyeriana had a mixed pattern of moderate genetic diversity and high inbreeding. Bayesian clustering divided D. dyeriana populations into two nSSR genetic clusters. Coalescent-based approximate Bayesian computation analyses suggest the western and eastern groups of D. dyeriana likely persisted in a long-term refuge in Southern China since the beginning of the last glacial period, whereas increasingly colder and arid climates at the onset of the last glacial maximum might have fostered the fragmentation of D. dyeriana within refugia. Following their divergence, the western group kept relatively stable effective population size, whereas the eastern group had experienced 500-fold population expansion during the Holocene. Although clear loss of genetic diversity by human activities was not suggested, recent habitat fragmentation has led to a reduction of population connectivity and increased genetic differentiation by ongoing genetic drift in isolated populations, possibly owing to decreased population size in recent dozen years. Finally, we discussed the implications of these results on conservation policies.

Genetic conservation of small populations of the endemic tree Swartzia glazioviana (Taub.) Glaz. (Leguminosae) in the Atlantic Forest

Swartzia glazioviana is a threatened legume tree species from the Brazilian Atlantic Forest characterized by aggregations of individuals and endemism to an area with extensive human occupation. It is critical to conduct studies on the species to conserve the remaining populations. Using ten nuclear microsatellite loci, we examined the genotypic and genetic diversity and structure, inbreeding, stand-level spatial genetic structure (SGS), effective population size, mating system, and pollen flow in three isolated remnant populations, aiming to inform conservation strategies. All adult individuals found in the populations were mapped and sampled and open-pollinated seeds were collected from two populations. The genotypic diversity (>0.85) indicates that sexual reproduction is predominant and the short distance between ramets indicates that asexual reproduction occurs by root development. In general, populations present SGS which is explained, in part, by root development. The genetic differentiation among populations was greater between more distant populations, suggesting a gene dispersal pattern of isolation by distance. Pollen flow (>27%) indicates that populations are not reproductively isolated, but fertilization followed an isolation by distance pattern. The outcrossing rate was high (\({t_m}\) > 0.8), but some mating occurred among related individuals (\({t_m}\; - \;{t_s}\) > 0.1) and were correlated (\({r_p}\) > 0.15), indicating inbreeding and varying levels of relatedness within families. Inbreeding was higher in seed cohorts than adults, suggesting selection against inbred individuals between seed and adult stages. The results are discussed considering in situ conservation and strategies for seed collection for environmental reforestation.

Population genetic structure and comparative diversity of smallmouth bass Micropterus dolomieu: congruent patterns from two genomes.

Genetic diversity and divergence patterns of smallmouth bass Micropterus dolomieu spawning groups are analysed across its northern native range with mtDNA cytochrome b gene sequences and eight unlinked nuclear DNA microsatellite loci. Results reveal high levels of genetic variability and significant differences in allelic representation among populations (mtDNA: mean ± s.e., HD = 0·50 ± 0·06, mean ± s.e., θST = 0·41 ± 0·02 and microsatellites: mean ± s.e. HO = 0·46 ± 0·03, mean ± s.e. θST = 0·25 ± 0·01). The distributions of 28 variant mtDNA haplotypes, which differ by an average of 3·94 nucleotides (range = 1-8), denote divergent representation among geographic areas. Microsatellite data support nine primary population groups, whose high self-assignment probabilities likewise display marked divergence. Genetic patterns demonstrate: (1) high genetic diversity in both genomes, (2) significant divergence among populations, probably resulting from natal site homing and low lifetime migration, (3) support for three post-glacial refugia that variously contributed to the current northern populations, which remain evident today despite waterway connectivity and (4) a weak yet significant genetic isolation by geographic distance pattern, indicating that other processes affect the differences among populations, such as territoriality and site fidelity.

Species Delimitation and Lineage Separation History of a Species Complex of Aspens in China.

Species delimitation in tree species is notoriously challenging due to shared polymorphisms among species. An integrative survey that considers multiple operational criteria is a possible solution, and we aimed to test it in a species complex of aspens in China. Genetic [four chloroplast DNA (cpDNA) fragments and 14 nuclear microsatellite loci (nSSR)] and morphological variations were collected for 76 populations and 53 populations, respectively, covering the major geographic distribution of the Populus davidiana-rotundifolia complex. Bayesian clustering, analysis of molecular variance (AMOVA), Principle Coordinate Analysis (PCoA), ecological niche modeling (ENM), and gene flow (migrants per generation), were employed to detect and test genetic clustering, morphological and habitat differentiation, and gene flow between/among putative species. The nSSR data and ENM suggested that there are two separately evolving meta-population lineages that correspond to P. davidiana (pd) and P. rotundifolia (pr). Furthermore, several lines of evidence supported a subdivision of P. davidiana into Northeastern (NEC) and Central-North (CNC) groups, yet they are still functioning as one species. CpDNA data revealed that five haplotype clades formed a pattern of [pdNEC, ((pdCNC, pr), (pdCNC, pr))], but most haplotypes are species-specific. Meanwhile, PCA based on morphology suggested a closer relationship between the CNC group (P. davidiana) and P. rontundifolia. Discrepancy of nSSR and ENM vs. cpDNA and morphology could have reflected a complex lineage divergence and convergence history. P. davidiana and P. rotundifolia can be regarded as a recently diverged species pair that experienced parapatric speciation due to ecological differentiation in the face of gene flow. Our findings highlight the importance of integrative surveys at population level, as we have undertaken, is an important approach to detect the boundary of a group of species that have experienced complex evolutionary history.

Tight species cohesion among sympatric insular wild gingers (Asarum spp. Aristolochiaceae) on continental islands: Highly differentiated floral characteristics versus undifferentiated genotypes.

The Amami Island group of the Ryukyu Archipelago, Japan, harbors extensive species diversity of Asarum in a small landmass. The fine-scale population genetic structure and diversity of nine insular endemic Asarum species were examined using nuclear DNA microsatellite loci and ITS sequences. High population genetic diversity (HS = 0.45–0.79) was estimated based on the microsatellites, implying outcrossing of Asarum species within populations accompanied by inbreeding. Bayesian clustering analyses revealed that species were divided into three robust genetic clusters and that the species within each cluster had a homogeneous genetic structure, indicating incomplete lineage sorting. This conclusion was supported by an ITS phylogeny. The degree of genetic differentiation among species was very low both within and between clusters (FST = 0.096–0.193, and 0.096–0.266, respectively). Although species can be crossed artificially to produce fertile hybrids, our results indicate that there is very little evidence of hybridization or introgression occurring among species in the wild, even within stands composed of multiple sympatric species. The highly differentiated floral morphology of the studied species is likely to impose reproductive isolation between them and maintain their integrity in the wild. A lack of genetic differentiation between sympatric species suggests that speciation within this group occurred rapidly and recently.

Genetic diversity and genetic structure of the endangered Manchurian trout, Brachymystax lenok tsinlingensis, 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.

Lack of introgressive hybridization by North African catfish (Clarias gariepinus) in native Vietnamese bighead catfish (Clarias macrocephalus) populations as revealed by novel nuclear and mitochondrial markers

The widespread farming of catfish (genus Clarias) hybrids between bighead catfish (C. macrocephalus, Cm) and the introduced North African Catfish (C. gariepinus, Cg) in the Mekong Delta, where flooding is common, has raised concerns of introgression of Cg into the native catfish due to backcrossing by escaped hybrids with wild Cm. This study employed novel PCR-RFLP analyses of a mitochondrial (Cytochrome c oxidase subunit I, COI) and two nuclear (Rhodopsin and Tropomyosin) markers and six microsatellite loci to differentiate hybrid individuals from their parental species, and evaluate whether introgression has occurred in wild and cultured Cm populations in the region. Results of marker screening showed that sizes and frequencies of microsatellite alleles differed between the two parental species. Four loci exhibited fixed species-specific differences in allele frequency (monomorphic in Cg and highly polymorphic in Cm). Levels of sequence variation varied at the three genes examined, ranging from 3 to 8% (sequence length 652bp for COI, 802bp for Rhodopsin and 925bp for Tropomyosin). Based on variable sites identified that were not shared between the two species, 3 restriction enzymes (SpeI, XcmI and PflMI) were selected to digest PCR products at species-specific sites within the COI, Rhodopsin and Tropomyosin genes, respectively. Results of PCR-RFLP analyses confirmed that Cm is the maternal lineage of cultured hybrids, while nuclear genes (Rhodopsin and Tropomyosin) and microsatellite loci revealed that hybrids possessed admixed multi-locus genotypes relative to the two parental species. Analyses of six microsatellite loci and PCR-RFLP of two nuclear genes in 473 individuals collected from 11 wild and three cultured populations revealed one F1 hybrid in the wild but no evidence of widespread introgression into native bighead catfish. These results differ from findings in Thailand where intensive catfish culture and deliberate species hybridization is practiced.

Variability of nuclear microsatellite loci in the populations of Siberian dwarf pine (Pinus pumila (Pallas) Regel) from the Russian part of the range

Variability of nuclear microsatellite loci was examined in Siberian dwarf pine. Six microsatellite loci (RPS2, RPS6, RPS12, RPS124, RPS127, Pc18) demonstrated different polymorphism levels in ten populations of Siberian dwarf pine. The average number of alleles per locus was 4.88, the average observed heterozygosity was 0.465, and the average expected heterozygosity was 0.510. About 13% of total genetic variability was explained by the genetic differences between the populations (F ST = 0.129). Genetic distances between the examined populations of Pinus pumila inferred from the data on the SSR marker frequencies statistically significantly correlated with the geographical distances between the population samples. The level of genetic variability of the populations from Kamchatka Peninsula was lower than that demonstrated by continental and island populations. The genetic differentiation of the Kamchatka–Magadan and other populations of Siberian dwarf pine observed in our study can be explained in terms of their formation from different Pleistocene refugial centers.