Eighteen polymorphic microsatellite markers for the highly endangered Spanish imperial eagle (Aquila adalberti) and related species

Here we describe the development of 18 polymorphic microsatellite markers for the endangered Spanish imperial eagle (Aquila adalberti). Microsatellites were tested in five other raptor species. These markers were revealed as good molecular tools for genetic population studies, individual identification and parentage assessment in Spanish imperial eagle and closely related species.     

Minisatellite DNA profiling detects lineages and parentage in the endangered kakapo (Strigops habroptilus) despite low microsatellite DNA variation

An important goal of the conservationmanagement program of the critically endangeredground parrot, the New Zealand kakapo (Strigops habroptilus) is the determination ofparentage and levels of genetic diversitywithin the remaining population. Our previousmicrosatellite DNA studies have shown that allindividuals of this species except one arehomozygous at seven loci examined. Incontrast, we now show that a minisatellite DNAanalysis of kakapo provides sufficientvariation to conduct paternity analyses anddetect heterogeneity within the 86 livingkakapo. The sole remaining Fiordland kakapo,Richard Henry, is shown to be geneticallydivergent from individuals originating from theonly other remaining population on StewartIsland, suggesting that two lineages of kakapoare present in the extant population. This hasparticular significance for the conservationmanagement goal of maintenance of the maximumgenetic diversity in the species as a whole. The example of the kakapo illustrates thatminisatellite DNA markers can be useful incases where microsatellite DNA fails to showsufficient variation.     

Twenty-three microsatellite DNA loci for population genetic studies and parentage assignment in orangethroat darter, Etheostoma spectabile

The genus Etheostoma is a species-rich and ecologically important group of fishes in North America. The orangethroat darter (Etheostoma spectabile) is widely distributed and abundant in headwater streams throughout the central Midwest, and is an excellent model for ecological and mating system studies. We developed 23 novel, polymorphic, and independent microsatellite loci for E. spectabile. We found from two to 14 alleles per locus, and observed heterozygosities ranged from 0.39 to 1.0. These markers, in combination with others isolated from Etheostoma taxa, will be useful for ecological and evolutionary studies in the genus.     

Disentangling the impact of demographic factors on population differentiation of an endangered freshwater crayfish (Austropotamobius pallipes) using population density and microsatellite data

1. Habitat fragmentation of stream ecosystems often results in decreased connectivity between populations and lower population sizes. Hence, understanding how habitat fragmentation affects genetic erosion is important for the preservation of freshwater biodiversity, in particular, as small populations suffer from loss of genetic diversity through genetic drift and loss of fitness because of inbreeding, increasing the risk of extinction. 2. Here, we assess the impact of demographic factors on population differentiation in the endangered freshwater crayfish Austropotamobius pallipes by analysing population genetic structure, estimating effective population sizes and comparing levels of polymorphism at five microsatellite loci with density estimates of 10 populations within a small French catchment that has become progressively confined to headwaters over the last six decades. 3. Levels of expected heterozygosity and allelic richness per population were relatively low (0.214–0.396 and 1.6–2.6, respectively). We found strong genetic differentiation between these geographically close populations (F_ST = 0.283), with weak statistical evidence for a pattern of isolation by distance. Estimates of effective population size were low (<150) in most populations, but potentially reached several thousands in three populations. 4. Population density and allelic richness were strongly positively correlated. A robust relationship between population density and heterozygosity values was also noted, but only after discarding two populations for which significant genetic signatures of a recent bottleneck were found; these two populations displayed high expected heterozygosity compared with a very low density. Populations with the highest densities of individuals had the highest effective population size estimates and vice versa. 5. Our results clearly show the importance of demographic factors and genetic drift on A. pallipes populations. Furthermore, analysis of genetic and population density data is a pragmatic and efficient approach to corroborate inferences from genetic data and can be particularly useful in the identification of populations experiencing a bottleneck and therefore in conservation genetics studies aiming at identifying priority populations for conservation.     

A population genetic study of the endangered plant species Limonium dufourii (Plumbaginaceae) based on amplified fragment length polymorphism (AFLP)

Limonium dufourii ( Plumbaginaceae ) is a triploid species with obligate apomictic reproduction and is endemic to the East Mediterranean coast of Spain, where it is present in only six populations, most of which have a very low number of individuals. Genetic variation and population structure in this species was studied using amplified fragment length polymorphisms (AFLPs) as markers, using the same individuals as in a previous study with random amplified polymorphic DNA (RAPD). Three different primers provided 252 bands of which 51 were polymorphic among the 152 individuals analysed. Those polymorphic bands were able to define 65 different phenotypes, of which all but two were present in only one population. The comparative analyses of data from AFLPs with those from RAPDs show a high degree of concordance. Additionally, and given the nature of these markers, we propose the estimation of nucleotide divergences from AFLP patterns. Relationships among the different AFLP patterns and the estimates of population genetic parameters obtained with this evolutionary distance are in good agreement with previous results. These analyses show that substantial genetic variability and differentiation exist within and among populations of L. dufourii . Their higher reproducibility and the possibility of obtaining estimates of nucleotide divergence make AFLPs a much better DNA fingerprinting technique.     

Microsatellite analysis of genetic population structure in an endangered salmonid: the coastal cutthroat trout (Oncorhynchus clarki clarki)

The genetic population structure of coastal cutthroat trout ( Oncorhynchus clarki clarki ) in Washington state was investigated by analysis of variation in allele frequencies at six highly polymorphic microsatellite loci for 13 anadromous populations, along with one outgroup population from the Yellowstone subspecies ( O. clarki bouvieri) (mean heterozygosity = 67%; average number of alleles per locus = 24). Tests for genetic differentiation revealed highly significant differences in genotypic frequencies for pairwise comparisons between all populations within geographical regions and overall population subdivision was substantial ( F _ST = 0.121, R _ST = 0.093), with 44.6% and 55.4% of the among-population diversity being attributable to differences between streams ( F _SR = 0.054) and between regions ( F _RT = 0.067), respectively. Analysis of genetic distances and geographical distances did not support a simple model of isolation by distance for these populations. With one exception, neighbour-joining dendrograms from the Cavalli-Sforza and Edwards' chord distances and maximum likelihood algorithms clustered populations by physiogeographic region, although overall bootstrap support was relatively low (53%). Our results suggest that coastal cutthroat trout populations are ultimately structured genetically at the level of individual streams. It appears that the dynamic balance between gene flow and genetic drift in the subspecies favours a high degree of genetic differentiation and population subdivision with the simultaneous maintenance of high heterozygosity levels within local populations. Results are discussed in terms of coastal cutthroat trout ecology along with implications for the designation of evolutionarily significant units pursuant to the US Endangered Species Act of 1973 and analogous conservation units.     

Accounting for cryptic population substructure enhances detection of inbreeding depression with genomic inbreeding coefficients: an example from a critically endangered marsupial

Characterizing inbreeding depression in wildlife populations can be critical to their conservation. Coefficients of individual inbreeding can be estimated from genome‐wide marker data. The degree to which sensitivity of inbreeding coefficients to population genetic substructure alters estimates of inbreeding depression in wild populations is not well understood. Using generalized linear models, we tested the power of two frequently used inbreeding coefficients that are calculated from genome‐wide SNP markers, F_H and F^^III, to predict four fitness traits estimated over two decades in an isolated population of the critically endangered Leadbeater's possum. F_H estimates inbreeding as excess observed homozygotes relative to equilibrium expectations, whereas F^^III quantifies allelic similarity between the gametes that formed an individual, and upweights rare homozygotes. We estimated F_H and F^^III from 1,575 genome‐wide SNP loci in individuals with fitness trait data (N = 179–237 per trait), and computed revised coefficients, F_H^{by group} and F^^{IIIby group}, adjusted for population genetic substructure by calculating them separately within two different genetic groups of individuals identified in the population. Using F_H or F^^III in the models, inbreeding depression was detected for survival to sexual maturity, longevity and whether individuals bred during their lifetime. F^^{IIIby group} (but not F_H^{by group}) additionally revealed significant inbreeding depression for lifetime reproductive output (total offspring assigned to each individual). Estimates of numbers of lethal equivalents indicated substantial inbreeding load, but differing between inbreeding estimators. Inbreeding depression, declining population size, and low and declining genetic diversity suggest that genetic rescue may assist in preventing extinction of this unique Leadbeater's possum population.     

The power of genetic monitoring for studying demography,ecology and genetics of a reintroduced brown bear population

Genetic monitoring has rarely been used for wildlife translocations despite the potential benefits this approach offers, compared to traditional field‐based methods. We applied genetic monitoring to the reintroduced brown bear population in northern Italy. From 2002 to 2008, 2781 hair and faecal samples collected noninvasively plus 12 samples obtained from captured or dead bears were used to follow the demographic and geographical expansion and changes in genetic composition. Individual genotypes were used to reconstruct the wild pedigree and revealed that the population increased rapidly, from nine founders to >27 individuals in 2008 (λ = 1.17–1.19). Spatial mapping of bear samples indicated that most bears were distributed in the region surrounding the translocation site; however, individual bears were found up to 163 km away. Genetic diversity in the population was high, with expected heterozygosity of 0.74–0.79 and allelic richness of 4.55–5.41. However, multi‐year genetic monitoring data showed that mortality rates were elevated, immigration did not occur, one dominant male sired all cubs born from 2002 to 2005, genetic diversity declined, relatedness increased, inbreeding occurred, and the effective population size was extremely small (Ne = 3.03, ecological method). The comprehensive information collected through genetic monitoring is critical for implementing future conservation plans for the brown bear population in the Italian Alps. This study provides a model for other reintroduction programmes by demonstrating how genetic monitoring can be implemented to uncover aspects of the demography, ecology and genetics of small and reintroduced populations that will advance our understanding of the processes influencing their viability, evolution, and successful restoration.     

利用RAPD标记分析濒危植物白豆杉种群的遗传结构

以我国特有的濒危裸子植物白豆杉为材料,采用RAPD标记对其分布于浙江、江西、湖南和广西的11个天然种群予以检测,通过贝叶斯法估测了种群的遗传分化程度,并和由其他算法得出的结果进行了比较.求出的θ^B（Hickory）、GST（Nei）及φST（AMOVA）值分别为0.5018、0.5865和0.5436;而经由Shannon指数计算出的种群间遗传多样性所占比例为0.4839,同贝叶斯法估计出的结果最为接近.和其他松杉类植物相比,白豆杉种群间发生了极其显著的遗传分化,这可能是因为：（1）种群长期处于星散分布状态;（2）雌雄异株,而生于林下的雌株经常不能正常受粉;（3）种群取样跨越的地理范围宽广.Mantel检验表明,种群间的遗传分化程度和地理距离之间显著相关（r=0.719,P=0.003）.此外还发现白豆杉的遗传变异水平偏低,推测瓶颈效应和遗传漂变对小种群的作用是造成这一后果的重要因素.对白豆杉种群的保育和管理提出了建议.     

Viral gut metagenomics of sympatric wild and domestic canids,and monitoring of viruses: Insights from an endangered wolf population

Animal host–microbe interactions are a relevant concern for wildlife conservation, particularly regarding generalist pathogens, where domestic host species can play a role in the transmission of infectious agents, such as viruses, to wild animals. Knowledge on viral circulation in wild host species is still scarce and can be improved by the recent advent of modern molecular approaches. We aimed to characterize the fecal virome and identify viruses of potential conservation relevance of diarrheic free‐ranging wolves and sympatric domestic dogs from Central Portugal, where a small and threatened wolf population persists in a highly anthropogenically modified landscape. Using viral metagenomics, we screened diarrheic stools collected from wolves (n = 8), feral dogs (n = 4), and pet dogs (n = 6), all collected within wolf range. We detected novel highly divergent viruses as well as known viral pathogens with established effects on population dynamics, including canine distemper virus, a novel bocavirus, and canine minute virus. Furthermore, we performed a 4‐year survey for the six wolf packs comprising this endangered wolf population, screening 93 fecal samples from 36 genetically identified wolves for canine distemper virus and the novel bocavirus, previously identified using our metagenomics approach. Our novel approach using metagenomics for viral screening in noninvasive samples of wolves and dogs has profound implications on the knowledge of both virology and wildlife diseases, establishing a complementary tool to traditional screening methods for the conservation of threatened species.     

Heterozygote excess in ancient populations of the critically endangered Dioon caputoi (Zamiaceae,Cycadales) from central Mexico

Dioon caputoi is a long‐lived cycad known from only four populations that range in size from 50 to 120, mostly adult individuals. Dioon caputoi has the most narrow geographical range of all Dioon spp. (less than 10 km), existing completely within the boundaries of the Tehuacán–Cuicatlán Biosphere Reserve, Mexico. Negative inbreeding values were found in all four populations (F_IT = ?0.242) and within subpopulations (F_IS = ?0.379). Only c. 10% of the total genetic variation was partitioned among populations (F_ST = 0.099). We also found that most mean values of genetic variation (A = 1.91 ± 0.12; P = 78.9 ± 10.2; H_E = 0.35 ± 0.01) are within the range reported for other Dioon species with larger populations and with wider geographical ranges. These results support recent findings that rare plant species maintain high levels of genetic diversity. The heterozygote excess found at all loci is discussed in detail from a neutral evolutionary perspective, leaving arguments as working hypotheses for further research. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 436–447.     

Patterns of genetic diversity and migration in increasingly fragmented and declining orang-utan (Pongo pygmaeus) populations from Sabah, Malaysia

We investigated the genetic structure within and among Bornean orang-utans (Pongo pygmaeus) in forest fragments of the Lower Kinabatangan flood plain in Sabah, Malaysia. DNA was extracted from hair and faecal samples for 200 wild individuals collected during boat surveys on the Kinabatangan River. Fourteen microsatellite loci were used to characterize patterns of genetic diversity. We found that genetic diversity was high in the set of samples (mean H(E) = 0.74) and that genetic differentiation was significant between the samples (average F(ST) = 0.04, P < 0.001) with F(ST) values ranging from low (0.01) to moderately large (0.12) values. Pairwise F(ST) values were significantly higher across the Kinabatangan River than between samples from the same river side, thereby confirming the role of the river as a natural barrier to gene flow. The correlation between genetic and geographical distance was tested by means of a series of Mantel tests based on different measures of geographical distance. We used a Bayesian method to estimate immigration rates. The results indicate that migration is unlikely across the river but cannot be completely ruled out because of the limited F(ST) values. Assignment tests confirm the overall picture that gene flow is limited across the river. We found that migration between samples from the same side of the river had a high probability indicating that orang-utans used to move relatively freely between neighbouring areas. This strongly suggests that there is a need to maintain migration between isolated forest fragments. This could be done by restoring forest corridors alongside the river banks and between patches.     

Extremely low effective population sizes, genetic structuring and reduced genetic diversity in a threatened bumblebee species, Bombus sylvarum (Hymenoptera: Apidae)

Habitat fragmentation may severely affect survival of social insect populations as the number of nests per population, not the number of individuals, represents population size, hence they may be particularly prone to loss of genetic diversity. Erosion of genetic diversity may be particularly significant among social Hymenoptera such as bumblebees (Bombus spp.), as this group may be susceptible to diploid male production, a suggested direct cost of inbreeding. Here, for the first time, we assess genetic diversity and population structuring of a threatened bumblebee species (Bombus sylvarum) which exists in highly fragmented habitat (rather than oceanic) islands. Effective population sizes, estimated from identified sisterhoods, were very low (range 21-72) suggesting that isolated populations will be vulnerable to loss of genetic variation through drift. Evidence of significant genetic structuring between populations (theta = 0.084) was found, but evidence of a bottleneck was detected in only one population. Comparison across highly fragmented UK populations and a continental population (where this species is more widespread) revealed significant differences in allelic richness attributable to a high degree of genetic diversity in the continental population. While not directly related to population size, this is perhaps explained by the high degree of isolation between UK populations relative to continental populations. We suggest that populations now existing on isolated habitat islands were probably linked by stepping-stone populations prior to recent habitat loss.     

Rapid genetic differentiation between ex situ and their in situ source populations: an example of the endangered Silene otites (Caryophyllaceae)

Ex situ cultivation in botanic gardens could be one possibility to preserve plant species diversity and genetic variation. However, old ex situ populations are often sparsely documented. We were able to retrieve three different ex situ populations and their source in situ populations of the endangered plant species Silene otites after 20–36 years of isolation. Furthermore, three additional wild populations were included in the analysis. Population genetic diversity and differentiation were analysed using AFLP markers. Genetic variation in the ex situ populations was lower than the variation found in the in situ populations. Strong differentiation (F_ST = 0.21–0.36) between corresponding in situ and ex situ populations was observed. Bayesian clustering approach also showed a distinct genetic separation between in situ and ex situ populations. The high genetic differentiation and loss of genetic diversity during spatial and temporal isolation in the ex situ populations can be attributable to small population sizes and unconscious selection during cultivation. Therefore, adequate sampling prior to ex situ cultivation and large effective population sizes are important to preserve genetic diversity. Near‐natural cultivation allowing for generation overlap and interspecific competition without artificial selection is recommended as being best for the maintenance of the genetic constitution. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, ??, ??–??.     

(Not) far from home: No sex bias in dispersal,but limited genetic patch size,in an endangered species,the Spotted Turtle (Clemmys guttata)

Sex-biased dispersal is common in many animals, with male-biased dispersal often found in studies of mammals and reptiles, including interpretations of spatial genetic structure, ostensibly as a result of male–male competition and a lack of male parental care. Few studies of sex-biased dispersal have been conducted in turtles, but a handful of studies, in saltwater turtles and in terrestrial turtles, have detected male-biased dispersal as expected. We tested for sex-biased dispersal in the endangered freshwater turtle, the spotted turtle (Clemmys guttata) by investigating fine-scale genetic spatial structure of males and females. We found significant spatial genetic structure in both sexes, but the patterns mimicked each other. Both males and females typically had higher than expected relatedness at distances <25 km, and in many distance classes greater than 25 km, less than expected relatedness. Similar patterns were apparent whether we used only loci in Hardy–Weinberg equilibrium (n = 7) or also included loci with potential null alleles (n = 5). We conclude that, contrary to expectations, sex-biased dispersal is not occurring in this species, possibly related to the reverse sexual dimorphism in this species, with females having brighter colors. We did, however, detect significant spatial genetic structure in males and females, separate and combined, showing philopatry within a genetic patch size of <25 km in C. guttata, which is concerning for an endangered species whose populations are often separated by distances greater than the genetic patch size.     

Estimates of gene flow, genetic substructure and population heterogeneity in bracken (Pteridium aquilinum)

Bracken [ Pteridium aquilinum (L.) Kuhn] is a cosmopolitan species and is a noxious weed in many areas. Because of its abundance, particularly in Britain, bracken affords an ideal system for investigating various aspects of population genetics and evolution. High mobility of dispersal units (spores) suggests that rates of gene flow among distant populations should be high. Gene flow is a major evolutionary force that influences the genetic structure of populations. To examine the effects of gene flow on population heterogeneity and population substructuring in bracken, starch gel electrophoresis of enzymes was used to provide the necessary genetic database. Allele frequency data at 21 loci were obtained for seven British populations, one Majorcan and one from the eastern United States. A model was employed to estimate the amount of gene flow ( Nm ) at several levels. Gene flow among British populations was extremely high ( Nm = 36.51), one of the highest estimates reported for plants. Among eight European populations gene flow was lower (but still considered high) at Nm = 2.47. Trans-Atlantic gene flow was low ( Nm = 0.0926).
F -statistics were used to assess population heterogeneity and substructuring. The data indicate that, compared with other species, there is very little genetic differentiation among British populations of bracken. Indeed, it appears that the whole island is behaving as a single randommating population. This result is consistent with high levels of gene flow. Only one population (on the Isle of Arran) showed statistically significant genetic substructuring. Habitat heterogeneity on the island and age structure are hypothesized as possible causes of this result.
The data reported here support previous studies demonstrating that bracken is genetically polymorphic and is an outcrossing species.     

Using a reference population yardstick to calibrate and compare genetic diversity reported in different studies: an example from the brown bear

In species with large geographic ranges, genetic diversity of different populations may be well studied, but differences in loci and sample sizes can make the results of different studies difficult to compare. Yet, such comparisons are important for assessing the status of populations of conservation concern. We propose a simple approach of using a single well-studied reference population as a ‘yardstick'' to calibrate results of different studies to the same scale, enabling comparisons. We use a well-studied large carnivore, the brown bear (Ursus arctos), as a case study to demonstrate the approach. As a reference population, we genotyped 513 brown bears from Slovenia using 20 polymorphic microsatellite loci. We used this data set to calibrate and compare heterozygosity and allelic richness for 30 brown bear populations from 10 different studies across the global distribution of the species. The simplicity of the reference population approach makes it useful for other species, enabling comparisons of genetic diversity estimates between previously incompatible studies and improving our understanding of how genetic diversity is distributed throughout a species range.     

Genetic diversity in an endangered alpine plant, Eryngium alpinum L. (Apiaceae), inferred from amplified fragment length polymorphism markers

Eryngium alpinum L. is an endangered species found across the European Alps. In order to obtain base-line data for the conservation of this species, we investigated levels of genetic diversity within and among 14 populations from the French Alps. We used the amplified fragment length polymorphism (AFLP) technique with three primer pairs and scored a total of 62 unambiguous, polymorphic markers in 327 individuals. Because AFLP markers are dominant, within-population genetic structure (e.g. FIS) could not be assessed. Analyses based either on the assumption of random-mating or on complete selfing lead to very similar results. Diversity levels within populations were relatively high (mean Nei's expected heterozygosity = 0.198; mean Shannon index = 0.283), and a positive correlation was detected between both genetic diversity measurements and population size (Spearman rank correlation: P = 0. 005 and P = 0.002, respectively). Moreover, FST values and exact tests of differentiation revealed high differentiation among populations (mean pairwise FST = 0.40), which appeared to be independent of geographical distance (nonsignificant Mantel test). Founder events during postglacial colonizations and/or bottlenecks are proposed to explain this high but random genetic differentiation. By contrast, we detected a pattern of isolation by distance within populations and valleys. Predominant local gene flow by pollen or seed is probably responsible for this pattern. Concerning the management of E. alpinum, the high genetic differentiation leads us to recommend the conservation of a maximum number of populations. This study demonstrates that AFLP markers enable a quick and reliable assessment of intraspecific genetic variability in conservation genetics.