Genetic variation of microsatellite loci in a bottlenecked species: the northern hairy-nosed wombat Lasiorhinus krefftii

We investigate the utility of hypervariable microsatellite loci to measure genetic variability remaining in the northern hairy-nosed wombat, one of Australia's rarest mammals. This species suffered a dramatic range and population reduction over the past 120 years and now exists as a single colony of about 70 individuals at Epping Forest National Park, central Queensland. Because our preliminary research on mitochondrial DNA and multilocus DNA fingerprints did not reveal informative variation in this population, we chose to examine variation in microsatellite repeats, a class of loci known to be highly polymorphic in mammals. To assess the suitability of various wombat populations as a reference for comparisons of genetic variability and subdivision we further analysed mitochondrial DNA cytochrome b sequence, using phylogenetic methods. Our results show that appreciable levels of variation still exist in the Epping Forest colony although it has only 41% of the heterozygosity shown in a population of a closely-related species. From museum specimens collected in 1884, we also assessed microsatellite variation in an extinct population of the northern hairy-nosed wombat, from Deniliquin, New South Wales, 2000 km to the south of the extant population. The apparent loss of variation in the Epping Forest colony is consistent with an extremely small effective population size throughout its 120-year decline.     

Female dispersal and male kinship-based association in southern hairy-nosed wombats (Lasiorhinus latifrons)

Generalizations about sociobiology require investigations of species with diverse ecological roles and phylogenetic affiliations. The southern hairy-nosed wombat ( Lasiorhinus latifrons ) is valuable here, in that it is a marsupial of semi-arid habitat, and one of the largest burrowing herbivores (commonly attaining 26 kg). Its sociobiology is poorly understood because the species is nocturnal, shy, and difficult to observe or capture nondisruptively. To investigate aspects of the species' sociobiology in continuous habitat, we applied high-intensity, temporally replicated, noninvasive sampling and genotyping of hairs to identify individuals and their sex. Spatial relatedness (kinship) structure was estimated, and related to patterns of burrow-use. To understand the association of kinship with burrow/warren-sharing and preferential colocation between wombats, > 100 genetically 'tagged' individuals at Brookfield Conservation Park (Murraylands, South Australia) were 'tracked' through multiple seasons between 1999 and 2001. Dispersal was female-biased, and may be performed by females after breeding. Conversely, males were philopatric. Male kin relationships were characterized by preferential burrow- and warren-sharing among closely related males, often in associations lasting for years. In contrast, females under-associated with their close female relatives and did not form matrilineal groupings with potential for favourable kin interactions. This fundamental departure from the predominant mammalian pattern raises questions about the origins and maintenance of the system, which is now known from all three species of wombat. The present study provides starting points to address those questions by adding to our knowledge of longitudinal spatiotemporal associations and habitat use of a marsupial with the unusual system of female-biased dispersal, and by outlining robust methodologies.     

Relatedness structure in Rhododendron metternichii var. hondoense revealed by microsatellite analysis

The relatedness structure of Rhododendron metternichii Sieb. et Zucc. var. hondoense Nakai was analysed in a 150 x 70-m quadrat in Hiroshima Prefecture, western Japan. The population of R. metternichii occurred as three subpopulations at the study site (A1-A3). Pairwise relatedness based on microsatellite genotypes at eight loci and Mantel tests revealed a hierarchical structure of relatedness within and among subpopulations: (i) relatedness between individuals within 10 m of one another was significantly positive; (ii) relatedness between individuals in the same subpopulation was significantly positive, but negative between individuals in distant subpopulations; and (iii) relatedness was not significantly different from zero among neighbouring subpopulations. In detail, however, relatedness within each subpopulation was significantly positive in subpopulation A1, relatively weak but significantly positive in subpopulation A2, and not significantly different from zero in subpopulation A3. Relatedness within each subpopulation was inversely related to correlations between interindividual distance and relatedness. The aggregation of related individuals at short distances from one another may lead to decreasing relatedness within subpopulations as a whole. Moreover, negative correlations between interindividual distance and relatedness corresponded to high flowering densities at less than 10-m distance, implying that high flowering densities reduce pollinator foraging distance and lead to stronger genetic structure within subpopulations. Small individuals (< 2.0 m in height) showed stronger genetic structure compared with that of large individuals (> or = 2.0 m in height). The different relatedness structure within and among subpopulations may be caused by various degrees of gene flow affected by distribution patterns of individuals and population density.     

Polymorphic microsatellites identified in an endangered dasyurid marsupial,the dibbler (Parantechinus apicalis)

Dibblers are carnivorous marsupials that were considered extinct for over 60 years. They now persist in small, highly fragmented populations. In this work we describe the characterization of seven polymorphic microsatellite loci that are being used to investigate population parameters. For 22 individuals, from the mainland population, these markers had allelic diversities from three to 11 (alleles per locus) and expected heterozygosities ranging from 0.53 to 0.89. Primers were also tested on a number of closely related dasyurids and in other marsupials, suggesting that their usefulness in population studies may go beyond investigations of the dibbler.     

Development of polymorphic microsatellite markers for Dalbergia nigra (Papilionoideae), an endangered tree from the Brazilian Atlantic Forest

Dalbergia nigra is an endangered tree restricted to the Brazilian Atlantic Forest. Ten polymorphic microsatellite loci were isolated and characterized in 47 trees from two populations. The total number of alleles per locus ranged from three to 12 alleles. The levels of observed and expected heterozygosities ranged from 0.304 to 0.740 and from 0.278 to 0.872, respectively. Significant deviations from Hardy-Weinberg were detected for only three loci in each population. No pair of loci exhibited significant linkage disequilibrium. These microsatellites provide an efficient tool to investigate genetic structure in forest remnants with the purpose of conservation of this species.     

Genetic diversity in the US ex situ populations of the endangered Puerto Rican Boa,Chilabothrus inornatus

The Puerto Rican Boa (Chilabothrus inornatus) was placed on the US Endangered Species List in 1970. Progress has been made since to clarify the recovery status of this species, though the design of a new recovery plan must include information regarding genetic variation within and among populations of this species. While measures of genetic diversity in wild populations of this species are finally becoming available, relative genetic diversity represented in ex situ populations is unknown, which hampers efforts to develop an ex situ species management plan. Here, we provide an analysis of genetic diversity in US public and private collections (zoos and breeders) using mitochondrial sequence data and five highly polymorphic nuclear microsatellite loci. We analyzed 50 boas from the US ex situ population and determined overall genetic diversity and relatedness among these individuals. We then compared these data to mitochondrial and microsatellite data obtained from 176 individuals from wild populations across the native range of the species. We found little inbreeding and a large amount of retained genetic diversity in the US ex situ population of C. inornatus relative to wild populations. Genetic diversity in the ex situ population is similar to that found in wild populations. Ours is only the second explicit attempt to characterize genetic diversity at the molecular level in ex situ populations of boid snakes. We anticipate that these results will inform current breeding strategies as well as offer additional information that will facilitate the continuation of ex situ conservation breeding or management in boas.     

Isolation and characterization of polymorphic microsatellite markers in the endangered Nothotsuga longibracteata (Pinaceae)

Twelve polymorphic microsatellite loci were isolated and characterized from an AC‐enriched genomic library of Nothotsuga longibracteata. The average allele number of these microsatellites was 8.3 per locus, ranging from two to 13. The ranges of observed and expected heterozygosities were 0.03–0.97 and 0.09–0.88, respectively. These polymorphic markers provide useful tools for the study of evolutionary history and conservation genetics of N. longibracteata.     

Isolation and characterization of microsatellite loci in an endangered tree species, Afzelia xylocarpa (Kurz) Craib (Caesalpinioideae)

Afzelia xylocarpa is listed as an endangered species on the International Union for the Conservation of Nature and Natural Resources World list of Threatened Trees, due to overexploitation for its valuable timber and habitat loss. We isolated eight polymorphic microsatellite loci from A. xylocarpa using dual suppression polymerase chain reaction technique. These loci provide microsatellite markers with high polymorphism, as the number of alleles ranged from two to seven, and the estimate of gene diversity was between 0.285 and 0.795. The markers are now available for more detailed investigation of population genetic structure and gene flow among A. xylocarpa populations.     

Gene flow and inbreeding depression inferred from fine-scale genetic structure in an endangered heterostylous perennial, Primula sieboldii

We estimated the gene dispersal distance and the magnitude of inbreeding depression from the fine-scale genetic structure in the endangered heterostylous perennial Primula sieboldii. We indirectly estimated the neighbourhood size (Nb) and the standard deviation of gene dispersal distance (sigma(g)) from the detected genetic structure by using 10 microsatellite markers. We also estimated the fitness reduction in mating among neighbouring individuals caused by biparental inbreeding according to the genetic structure. We found clear fine-scale genetic structure (a significantly positive kinship coefficient within 42.3 m), and the indirect estimates of sigma(g) and Nb were 15.7 m and 50.9, respectively. These indirect estimates were similar to the direct estimates (18.4 m and 44.0). The slightly larger indirect estimate of Nb may reflect that inbreeding depression and genetic structure or rare long-distance dispersal that were overlooked in the direct estimate have elongated the long-term average of gene dispersal distance. P. sieboldii is also likely to suffer about 19% fitness reduction in progenies from mating among individuals 5 m apart. Our results suggest that biparental inbreeding and genetic structure can affect the range of gene dispersal and seed reproductive success in P. sieboldii.     

Isolation and characterization of polymorphic microsatellite markers in the gray,short‐tailed opossum (Monodelphis domestica)

Twenty‐six polymorphic microsatellite markers were isolated from (AC)_n and (AG)_n microsatellite‐enhanced genomic libraries of the gray, short‐tailed opossum Monodelphis domestica. All 26 loci showed high allelic diversity, with allele numbers ranging from five to 11 in a subset of 35 animals. Normal Mendelian inheritance was confirmed for 24 loci by analysing allelic segregation in 10, two‐generation, families. Non‐amplifying (null) alleles were detected at two loci, which we recommend be used only if pedigree data are available. We conclude that all of these microsatellite markers would be useful for quantitative trait locus mapping and population genetic studies.     

Isolation and characterization of microsatellite loci in the southern brown bandicoot (Isoodon obesulus), and their applicability to other marsupial species

The southern brown bandicoot (Isoodon obesulus) was once widely distributed throughout coastal regions of Australia, however, populations have now become fragmented and the number of individuals are declining rapidly. As a result, this species is now classified as endangered in New South Wales and requires management. We have developed eight microsatellite markers to investigate levels of genetic variation and subdivision among these isolated populations. These microsatellite markers show levels of variability ranging from four to eight alleles and observed heterozygosities of 0.353–0.938. These microsatellite markers are also suitable for similar population studies on other related marsupials.     

Isolation and characterization of microsatellite loci in Psilopeganum sinense Hemsl (Rutaceae), an endangered herb endemic to Yangtze River valley

Twenty-nine primer pairs flanking microsatellite repeats were designed from an AC-enriched genomic library of Psilopeganum sinense and tested using 24 individuals derived from a natural population. A total of 11 microsatellite loci were found polymorphic. The number of alleles per locus varied from two to eight, with an average value of 3.7. The ranges of observed and expected heterozygosities were 0.000-0.750 and 0.365-0.800, respectively. These microsatellite loci have been directly applied to the ongoing conservation genetic studies of P. sinense.     

PERMANENT GENETIC RESOURCES: Development and characterization of 14 polymorphic microsatellite loci in the endangered tree Euptelea pleiospermum (Eupteleaceae)

Fourteen polymorphic microsatellite loci were developed and characterized for the endangered and tertiary relict tree, Euptelea pleiospermum. A genomic DNA enrichment protocol was used to isolate microsatellite loci and polymorphism was explored using 32 individuals from one natural population. The observed number of alleles ranged from two to nine. The ranges of observed and expected heterozygosities were 0.25-1.00 and 0.22-0.85, respectively. These microsatellite markers provide powerful tools for the ongoing conservation genetic studies of E. pleiospermum.     

Population structure of the yellow-footed rock-wallaby Petrogale xanthopus (Gray, 1854) inferred from mtDNA sequences and microsatellite loci

The yellow-footed rock-wallaby Petrogale xanthopus is considered to be potentially vulnerable to extinction. This wallaby inhabits naturally disjunct rocky outcrops which could restrict dispersal between populations, but the extent to which that occurs is unknown. Genetic differences between populations were assessed using mitochondrial DNA (control region) sequencing and analysis of variation at four microsatellite loci among three geographically close sites in south-west Queensland (P. x. celeris) and, for mtDNA only, samples from South Australia (P. x. xanthopus) as well. Populations from South Australia and Queensland had phylogenetically distinct mtDNA, supporting the present classification of these two groups as evolutionarily distinct entities. Within Queensland, populations separated by 70 km of unsuitable habitat differed significantly for mtDNA and at microsatellite loci. Populations separated by 10 km of apparently suitable habitat had statistically homogeneous mtDNA, but a significant difference in allele frequency at one microsatellite locus. Tests for Hardy-Weinberg equilibrium and micro-geographical variation at microsatellite loci did not detect any substructuring between two wallaby aggregations within a colony encircling a single rock outcrop. Although the present study was limited by small sample sizes at two of the three Queensland locations examined, the genetic results suggest that dispersal between colonies is limited, consistent with an ecological study of dispersal at one of the sites. Considering both the genetic and ecological data, we suggest that management of yellow-footed rock-wallabies should treat each colony as an independent unit and that conservation of the Queensland and South Australian populations as separate entities is warranted.