Genetic Diversity and Population Structure for the Conservation of Giant Spiny Frog(Quasipaa spinosa) Using Microsatellite Loci and Mitochondrial DNA

The giant spiny frog(Quasipaa spinosa) is an endangered species with a relatively small distribution limited to southern China and Northern Vietnam. This species is becoming increasingly threatened because of over-exploitation and habitat degradation. This study provides data on the genetic diversity and population genetic structure of the giant spiny frog to facilitate the further development of effective conservation recommendations for this economically important but threatened species. We examined 10 species-specific microsatellite loci and Cyt b genes(562 bp) collected from 13 wild populations across the entire range of this species. Results of 10 microsatellite loci analysis showed a generally high level of genetic diversity. Moreover, the genetic differentiation among all 12 populations was moderate to large(overall F_(ST) = 0.1057). A total of 51 haplotypes were identified for Cyt b, which suggests high haplotype nucleotide diversities. Phylogeographic and population structure analyses using both DNA markers suggested that the wild giant spiny frog can be divided into four distinct major clades, i.e., Northern Vietnam, Western China, Central China, and Eastern China. The clades with significant genetic divergence are reproductively isolated, as evidenced by a high number of private alleles and strong incidence of failed amplification in microsatellite loci. Our research, coupled with other studies, suggests that Q. spinosa might be a species complex within which no detectable morphological variation has been revealed. The four phylogenetic clades and some subclades with distinct geographical distribution should be regarded as independent management units for conservation purposes.     

Population structure and conservation genetics of wild rice Oryza rufipogon (Poaceae): a region-wide perspective from microsatellite variation

Oryza rufipogon Griff. is the most agriculturally important but seriously endangered wild rice species. To better estimate how genetic structure can be used to obtained a conservation perspective of the species, genetic variability at six polymorphic microsatellite DNA loci was examined. High levels of genetic variability were detected at six loci in 1245 individuals of 47 natural populations covering most of the species' range in China (overall RS = 3.0740, HO = 0.2290, HS = 0.6700). Partitioning of genetic variability (FST = 0.246) showed that most microsatellite variation was distributed within populations. Significant departures from Hardy-Weinberg expectations and very strong linkage disequilibrium indicate a high degree of inbreeding in the species and severe subdivision within populations. A mean Nm value of 0.7662 suggested a limited gene flow among the assayed populations. Our study suggests that conservation and restoration genetics should focus in particular on the maintenance of historically significant processes such as high levels of outbreeding and gene flow and large effective population size in the species.     

Genetic Diversity of Dalbergia monticola (Fabaceae) an Endangered Tree Species in the Fragmented Oriental Forest of Madagascar

There is an urgent need to maintain and restore a broad genetic base for the management of Dalbergia monticola, a very economically important but endangered tree species in Madagascar. Random amplified polymorphism DNAs (RAPDs) and chloroplast microsatellite markers were used to quantify the genetic variation and to analyse the geographic distribution of diversity. Ten locations covering most of the natural range were sampled. Sixty-three RAPD polymorphic and 15 monomorphic loci were obtained from 122 individuals. Genetic diversity was low and very close among populations and regions. The unrooted neighbour-joining tree exhibited 4 groups, representing 6% (p = 0.000) of the total variation. The greater part of the variance, 81%, was observed within populations. A Mantel test suggested that genetic distances between populations were weakly correlated with geographic distances (R = 0.46, p = 0.12). The three chloroplast microsatellite primers assayed on 100 individuals gave 13 chlorotypes. Most of the populations showed 2 or 3 haplotypes. Haplotype diversity for the total population was equal to He_Cp = 0.83 and ranged from 0.00 to 0.80 among the populations. The unrooted neighbour-joining tree exhibited 4 groups corresponding to the four regions representing 80% (p = 0.0000) of the total variation. Genetic diversity varies with regions, the north and south being less variable. Chlorotype distribution, the phylogenetic tree and historical information suggest that putative refugias in the centre-north region originating from the early Holocene could explain the pattern of variation observed today. By combining the results obtained at nuclear and organellar loci, a strategy of conservation based on evolutionarily significant units is proposed.     

Microsatellite marker polymorphism and mapping in pea (Pisum sativum L.)

This paper aims at providing reliable and cost effective genotyping conditions, level of polymorphism in a range of genotypes and map position of newly developed microsatellite markers in order to promote broad application of these markers as a common set for genetic studies in pea. Optimal PCR conditions were determined for 340 microsatellite markers based on amplification in eight genotypes. Levels of polymorphism were determined for 309 of these markers. Compared to data obtained for other species, levels of polymorphism detected in a panel of eight genotypes were high with a mean number of 3.8 alleles per polymorphic locus and an average PIC value of 0.62, indicating that pea represents a rather polymorphic autogamous species. One of our main objectives was to locate a maximum number of microsatellite markers on the pea genetic map. Data obtained from three different crosses were used to build a composite genetic map of 1,430 cM (Haldane) comprising 239 microsatellite markers. These include 216 anonymous SSRs developed from enriched genomic libraries and 13 SSRs located in genes. The markers are quite evenly distributed throughout the seven linkage groups of the map, with 85% of intervals between the adjacent SSR markers being smaller than 10 cM. There was a good conservation of marker order and linkage group assignment across the three populations. In conclusion, we hope this report will promote wide application of these markers and will allow information obtained by different laboratories worldwide in diverse fields of pea genetics, such as QTL mapping studies and genetic resource surveys, to be easily aligned.Electronic Supplementary Material Supplementary material is available for this article at     

Microsatellite markers confirm extensive population fragmentation of the endangered Balkan palaeoendemic Martino’s vole (<Emphasis Type="Italic">Dinaromys bogdanovi</Emphasis>)

The Martino’s vole is an endangered rodent endemic to the western Balkan Peninsula. Its range is fragmented, and populations are small due to high habitat specificity. The level of genetic variation within such populations is often low, and genetic differentiation between patchily dispersed populations is high. By scoring eight microsatellite loci in 110 individual Martino’s voles originating from 27 locations throughout the species range, we analysed genetic variation at both the intra- and interpopulation level. Factorial correspondence analysis, Bayesian analyses, and allele sharing distances divided individuals into three phylogroups (Northwestern, Central, and Southeastern), thus providing independent support for phylogeographic structuring, a pattern that has been described in previous studies based on mitochondrial DNA. Spatial genetic analyses showed that populations are highly fragmented, even in those areas with the highest population densities. The highest intrapopulation genetic variability and stable effective population sizes were found in Mount Zelengora (Bosnia and Herzegovina), which harbours a relatively large population of Martino’s voles. Populations in the Central and Southeastern lineages exhibited a significant isolation-by-distance pattern, indicating limited gene flow between them. Contrary to previous opinion, low effective population size and very limited gene flow between remaining populations suggest that the long-term existence of the Martino’s vole might not be secure, even in populations that live in optimal habitats. The only threat to the Martino’s vole identified thus far is competitive exclusion by the European snow vole. However, our results suggest that conservation problems associated with this paleoendemic rodent are more complex.     

Population genetic structure of the African elephant in Uganda based on variation at mitochondrial and nuclear loci: evidence for male-biased gene flow

A drastic decline has occurred in the size of the Uganda elephant population in the last 40 years, exacerbated by two main factors; an increase in the size of the human population and poaching for ivory. One of the attendant consequences of such a decline is a reduction in the amount of genetic diversity in the surviving populations due to increased effects of random genetic drift. Information about the amount of genetic variation within and between the remaining populations is vital for their future conservation and management. The genetic structure of the African elephant in Uganda was examined using nucleotide variation of mitochondrial control region sequences and four nuclear microsatellite loci in 72 individuals from three localities. Eleven mitochondrial DNA (mtDNA) haplotypes were observed, nine of which were geographically localized. We found significant genetic differentiation between the three populations at the mitochondrial locus while three out of the four microsatellite loci differentiated KV and QE, one locus differentiated KV and MF and no loci differentiated MF and QE. Expected heterozygosity at the four loci varied between 0.51 and 0.84 while nucleotide diversity at the mitochondrial locus was 1.4%. Incongruent patterns of genetic variation within and between populations were revealed by the two genetic systems, and we have explained these in terms of the differences in the effective population sizes of the two genomes and male-biased gene flow between populations.     

Remotely plucked hair genotyping: a reliable and non-invasive method for censusing pine marten (Martes martes,L. 1758) populations

We investigated the feasibility of using genetic techniques to census pine marten (Martes martes) populations by genotyping non-invasively collected samples (plucked hair and scats), with particular reference to the genetically depauperate Irish population. Novel real-time polymerase chain reaction methods were developed for species and sex identification, targeting short DNA sequences. Background genetic variation at 17 microsatellite loci was very low in the Irish population, with an average of 2.29 alleles per locus and expected heterozygosity of 0.35. Despite such low polymorphism, a panel of eight loci with a sibling probability of identity of 0.011 reliably identified individual pine marten and their gender, as determined by reference to genotypes of live trapped individuals. With high nuclear DNA amplification success rates (93.8%) and low genotyping error rates (1.8%), plucked hairs may represent a more reliable and cost-effective DNA source than scats for monitoring populations of this elusive carnivore, and similar taxa such as the sympatric stone marten Martes foina.

     

栓皮栎天然群体SSR遗传多样性研究

利用微卫星(SSR)标记对我国4个省内的5个栓皮栎（Quercus variabilis Bl.）天然群体的遗传多样性进行了研究。16对SSR标记揭示了栓皮栎丰富的遗传多样性：等位基因数（A）平均8.4375个,有效等位基因数(Ne)平均为5.9512个,平均期望杂合度(He)0.8059,Nei多样性指数(h)为0.8041。栓皮栎自然分布区中心地带的群体具有较高的遗传多样性,而人为对森林的破坏将降低林木群体的遗传多样性。栓皮栎群体的变异主要来源于群体内,群体间分化较小,遗传分化系数仅为0.0455。此外,栓皮栎群体间的遗传距离与地理距离之间存在显著的正相关。这些遗传信息为栓皮栎遗传多样性的保护和利用提供了一定依据。Abstract: Genetic diversity of five Quercus variabilis natural populations in four provinces of China was studied with microsatellite (SSR) markers. A relatively high level of genetic diversity was detected in Q. variabilis species with 16 polymorphic microsatellite loci. Average number of alleles (A) and effective number of alleles (Ne) were 8.4375 and 5.9512 respectively. The mean expected heterozygosity (He) was 0.8059 and Nei diversity index (h) was 0.8041. Higher diversity was found with the populations from the central range of the species in contrast to those from peripheral areas and human activities might decrease the genetic diversity of populations. The majority of genetic variation occurred within populations, which could be concluded from the low coefficient of genetic differentiation (Fst=0.0455). In addition, significant correlation was found between geographical distance and genetic distance. All these results present a basis to the conservation and utilization of genetic diversity of Quercus variabilis.     

Genetic Diversity and Population Structure of Acacia senegal (L) Willd. in Kenya

The level of genetic diversity and population structure of Acacia senegal variety kerensis in Kenya was examined using seven polymorphic nuclear microsatellite loci and two chloroplast microsatellite loci. In both chloroplast and nuclear datasets, high levels of genetic diversity were found within all populations and genetic differentiation among populations was low, indicating extensive gene flow. Analysis of population structure provided support for the presence of two groups of populations, although all individuals had mixed ancestry. Groups reflected the influence of geography on gene flow, with one representing Rift Valley populations whilst the other represented populations from Eastern Kenya. The similarities between estimates derived from nuclear and chloroplast data suggest highly effective gene dispersal by both pollen and seed in this species, although population structure appears to have been influenced by distributional changes in the past. The few contrasts between the spatial patterns for nuclear and chloroplast data provided additional support for the idea that, having fragmented in the past, groups are now thoroughly mixed as a result of extensive gene flow. For the purposes of conservation and in situ management of genetic resources, sampling could target a few, large populations ideally distributed among the spatial groups identified. This should ensure the majority of extant variation is preserved, and facilitate the investigation of variation in important phenotypic traits and development of breeding populations.     

三江源和祁连山国家公园雪豹种群的遗传结构分析

掌握遗传信息对濒危物种的保护和管理具有重要意义。本研究在我国雪豹重要分布区祁连山和三江源国家公园分别采集粪便样品,利用mtDNA的cyt b基因、微卫星多态性位点进行了雪豹的物种鉴定、个体识别和种群遗传结构评估。在采集286份疑似雪豹粪便样品中,成功的对86份雪豹样品进行了扩增鉴定,利用微卫星位点进行个体识别获得41只雪豹个体,其中祁连山国家公园26只,三江源国家公园15只。通过等位基因数、有效等位基因数、观测杂合度、期望杂合度、多态信息含量等指标进行种群遗传多样性评估,认为雪豹种群遗传多样性相对较低,但祁连山国家公园雪豹种群遗传多样性相对较高。STRUCTURE进行群体遗传结构分析表明,4个种群可以划分为3个遗传类群,祁连山国家公园的种群（YCW和QLS）与三江源国家种群(DC和SJ)的遗传差异,可能与种群间的地理隔离存在明显的相似性。     

The genetic impact of demographic decline and reintroduction in the wild boar (Sus scrofa): a microsatellite analysis

The reintroduction of wild boar from central Europe after World War II has contributed substantially to the range expansion of this species in Italy, where indiscriminate hunting in earlier times resulted in extreme demographic reduction. However, the genetic impact of such processes is not well-understood. In this study, 105 individuals from Italian and Hungarian wild boar populations were characterized for nine autosomal microsatellite loci. The Hungarian samples, and two central Italian samples from protected areas (parks) where reintroduction is not documented, were assumed to be representative of the genetic composition of the source and the target populations in the reintroduction process, respectively. Animals hunted in the wild in the Florence area of Tuscany (Italy) were then studied to identify the effects of reintroduction. The results we obtained can be summarized as follows: (i) none of the populations analysed shows genetic evidence of demographic decline; (ii) the three parental populations from Italy and Hungary are genetically distinct; however, the low level of divergence appears in conflict with the naming of the Italian and the European subspecies (Sus scrofa majori and Sus scrofa scrofa, respectively); in addition, the Italian groups appear to be as divergent from each other as they are from the Hungarian population; (iii) most of the individuals hunted near Florence are genetically intermediate between the parental groups, suggesting that hybridization has occurred in this area, the average introgression of Hungarian genotypes is 13%, but approximately 45% of the genetic pool of these individuals can not be directly attributed to any of the parental populations we analysed; (iv) analysis of microsatellite loci, though in a limited number, is an important tool for estimating the genetic effect of reintroduction in the wild boar, and therefore for the development of conservation and management strategies for this species.     

Definition of the USDA103 strain of channel catfish (Ictalurus punctatus)

Although there are differences in performance between genetic groups of channel catfish, identification and management of these groups is difficult because catfish strains look alike and individuals cannot be tagged efficiently. Thus, US catfish producers have not been able to objectively identify fish from different strains or populations, and it has been difficult for them to maintain the genetic purity of populations on the farm. We have developed a multiplexed microsatellite genotyping system to define catfish populations based on allelic frequency and exclusion. A commercial catfish genotype database was developed using catfish samples collected from 24 processing plants in the four main US catfish-producing states. The utility of the system was tested by the molecular characterization of the USDA103 research strain. Using eight microsatellite loci, the probability of falsely classifying an individual non-USDA103 catfish as a USDA103 was 0.0065. From a sample of 50 fish from a putative USDA103 pond, the probability of falsely including two non-USDA103 fish was 1 x 10(-105), and the conservative probability of falsely excluding two USDA103 fish was 1 x 10(-6). This genotyping system provides channel catfish producers with an objective mechanism for identification and management of genetically selected fish.     

Genetic diversity and population structure in the Barrens Topminnow (<Emphasis Type="Italic">Fundulus julisia</Emphasis>): implications for conservation and management of a critically endangered species

The Barrens Topminnow (Fundulus julisia) has undergone a rapid and dramatic decline. In the 1980s, at least twenty localities with Barrens Topminnows were known to exist in the Barrens Plateau region of middle Tennessee; currently only three areas with natural (not stocked) populations remain. The long-term survival of the Barrens Topminnow will depend entirely on effective management and conservation efforts. Captive propagation and stocking of captive-reared juveniles to suitable habitats have successfully established a handful of self-sustaining populations. However, very little is known about the genetic composition of source and introduced populations including levels of genetic diversity and structuring of genetic variation. Here we use both mitochondrial sequence data and genotypes from 14 microsatellite loci to examine patterns of genetic variation among ten sites, including all sites with natural populations and a subset of sites with introduced (stocked) populations of this species. Mitochondrial sequence analysis reveals extremely low levels of variation within populations and fixed differences between drainages. Microsatellite genotype data shows higher levels of genetic variability and a molecular signature consistent with a recent history of population bottlenecks. Measures of genetic diversity at microsatellite loci including allelic richness are similar within source and introduced populations. Bayesian assignment tests and analysis of molecular variation (AMOVA) support two distinct populations, consistent with drainage boundaries. Results from AMOVA analysis also suggest low levels of genetic connectivity between isolated populations within the same drainage. Here we propose two distinct evolutionary significant units (ESUs) and two management units that reflect this population substructure and warrant consideration in future management efforts.     

Variation in DNA microsatellites of the ferruginous pygmy-owl (Glaucidium brasilianum)

Eleven polymorphic microsatellite loci were used to assess genetic variation in the ferruginous pygmy owl (Glaucidium brasilianum) from North America. Analysis of genotypic variation suggests restricted gene flow between pygmy-owl populations in Arizona-Sonora and Sinaloa, and Texas and the remaining states in Mexico. The Arizona-Sonora population showed signs of a recent genetic bottleneck, an observation supported by low population estimates for Arizona (13–117 individuals). Heterozygosity in Arizona, however, was equal to levels recorded throughout Mexico and Texas. Congruent patterns revealed by nuclear (microsatellites) and mitochondrial DNA that indicate Arizona-Sonora and Texas populations are distinct from adjacent populations in Mexico, thus emphasizing need for the design and implementation of separate management plans for recovery and conservation efforts. Revealing evidence of distinct groups within the pygmy-owl populations in North America, results from this study may be used to make management decisions for the recovery and conservation of this species.     

Isolation and characterization of polymorphic microsatellite loci in the scarlet ibis (Eudocimus ruber— Threskiornithidae‐Aves)

Here we presented 10 polymorphic microsatellite loci obtained from scarlet ibis through an enriched genomic library. The analysis of 45 individuals from three Brazilian natural populations showed allelic diversity ranged from three to 17 alleles, observed heterozygosity ranged from 0.03 to 0.92, and expected heterozygosity ranged from 0.06 to 0.92. These highly variable microsatellite loci can provide means for assessing overall genetic variation in its remnant natural populations, which may help the development of effective conservation programs.     

Introgression in natural populations of bioindicators: a case study of Carabus splendens and Carabus punctatoauratus

The evolutionary importance of hybridization in wild plants and animals has become increasingly widely recognized in the last decade. In practical terms, hybridization provides an exceptionally tough set of problems for conservation biologists. We illustrate this in a case study of two Carabidae species widely used to evaluate the impact of human activities on biodiversity. These two species live in a complex mosaic of sympatry/allopatry and are known to hybridize in controlled conditions. Hybridization has not been quantified in natural populations to date due to the lack of a simple set of phenotypic traits for identifying hybrids. We thus screened for hybrids in natural populations, by multilocus genotyping at nine microsatellite loci. A high level of genetic differentiation between these two taxa was observed, as shown by allelic frequency distributions. Two Bayesian assignment procedures without obligatory pure taxon references were used to infer different classes of hybrids (F(1), F(2) and backcrosses) and mixture proportions between the two species. A low level of hybridization (F(1) genotypes) was observed in natural populations, contrasting with results obtained in controlled conditions. A high level of introgression was, however, detected at three of 12 sites, as revealed by the detection of backcrossed genotypes. This interspecific gene flow was detected in a limited zone of the common geographical range of the two species and was not related to the pattern of sympatry/allopatry. We then considered the origin and repercussions of this introgression, based on intraspecific genetic diversity and geographical structure.     

Microsatellite variation within and among populations of Oryza officinalis (Poaceae), an endangered wild rice from China

Oryza officinalis Wall. ex Watt. is an agriculturally important but seriously endangered species of wild rice. To obtain more accurate estimates of population structure for improved conservation planning of the species, genetic variability at 14 microsatellite DNA loci was examined in population samples covering most of the species' range in China. Considerable genetic variability (overall Na = 1.886, P = 62%, HO = 0.056, HE = 0.216, and HS = 0.277) was detected at the 14 loci in 442 individuals of the 18 natural populations. The evaluation of partitioning of genetic variability (FST = 0.442) suggested high genetic differentiation among the Chinese O. officinalis populations. An overall value of Nm = 0.316 suggested limited gene flow occurred among the sampled populations. Most of the populations showed heterozygote deficits in tests of Hardy-Weinberg equilibrium and significantly positive FIS values. This could be due to some inbreeding occurring in this predominantly outcrossing species. For effective in situ conservation and restoration genetics, maintenance of significant historical processes is particularly important, including high outbreeding, considerable gene flow, and large population effective sizes. The high FST values detected among populations in this study are instructive for adopting a conservation plan that includes representative populations with the greatest genetic variation for either in situ conservation management or germplasm collection expeditions.     

Differentiation of Alpine marmot populations traced by DNA fingerprinting.

As revealed by allozyme studies, the genetic variation of the Alpine marmot (Marmota m. marmota) has been reduced by a species-wide bottleneck at the end of the last glaciation. Therefore the more variable microsatellite loci were used as a genetic marker system to investigate variablility and differentiation of four autochthonous and four allochthonous populations founded by the release of small numbers of individuals during the last 150 years. The microsatellite loci detected by the DNA-probe (ATCC)₄ were found to be polymorphic in all populations, but the amount of variation was lower than in comparable mammalian species. In spite of founder effects the variation in the allochthonous populations was not significanlty reduced compared to the autochthonous populations. The autochthonous populations from Austria and from the eastern part of Switzerland were genetically similar, only the population from western Switzerland was clearly differentiated from the others. In the allochthonous populations similarities in the microsatellite patterns reveal genetic affinities to putative autochthonous source populations of the founder individuals.     

Genetic diversity and differentiation between the two remaining populations of the critically endangered Mediterranean monk seal

The Mediterranean monk seal Monachus monachus , is a critically-endangered species of which only two populations, separated by c . 4000 km, remain: the eastern Mediterranean (150–300 individuals) and the Atlantic/western Sahara populations (100–130 individuals). We measured current levels of nuclear genetic variation at 24 microsatellite loci in 12 seals from the eastern Mediterranean and 98 seals from the western Sahara population and assessed differences between them. In both populations, genetic variation was found to be low, with mean allelic richness for the loci polymorphic in the species of 2.09 and 1.96, respectively. For most loci, the observed allele frequency distributions in both populations were discontinuous and the size ranges similar. The eastern Mediterranean population had 14 private alleles and the western Sahara had 18, but with a much larger sample size. Highly significant differences in allele frequencies between the two populations were found for 14 out of 17 loci. F _ST between the two populations was 0.578 and the estimated number of migrants per generation was 0.046, both clearly indicating substantial genetic differentiation. From a conservation perspective, these results suggest that each population may act as a source for introducing additional genetic variation into the other population.     

Near panmixia at the distribution‐wide scale but evidence of genetic differentiation in a geographically isolated population of the Terek Sandpiper Xenus cinereus

Populations from different parts of a species range may vary in their genetic structure, variation and dynamics. Geographically isolated populations or those located at the periphery of the range may differ from those located in the core of the range. Such peripheral populations may harbour genetic variation important for the adaptive potential of the species. We studied the distribution‐wide population genetic structure of the Terek Sandpiper Xenus cinereus using 13 microsatellite loci and the mitochondrial DNA (mtDNA) control region. In addition, we estimated whether genetic variation changes from the core towards the edge of the breeding range. We used the results to evaluate the management needs of the sampled populations. Distribution‐wide genetic structure was negligible; the only population that showed significant genetic differentiation was the geographically isolated Dnieper River basin population in Eastern Europe. The genetic variation of microsatellites decreased towards the edge of the distribution, supporting the abundant‐centre hypotheses in which the core area of the distribution preserves the most genetic variation; however, no such trend could be seen with mtDNA. Overall genetic variation was low and there were signs of past population contractions followed by expansion; this pattern is found in most northern waders. The current effective population size (N_e) is large, and therefore global conservation measures are not necessary. However, the marginal Dnieper River population needs to be considered its own management unit. In addition, the Finnish population warrants conservation actions due to its extremely small size and degree of isolation from the main range, which makes it vulnerable to genetic depletion.