Conservation genetics of the black rhinoceros, <Emphasis Type="Italic">Diceros bicornis bicornis</Emphasis>, in Namibia

Poaching and habitat destruction across sub-Saharan Africa brought the black rhinoceros (Diceros bicornis) close to extinction. Over the past few decades, however, one of four subspecies, D. b. bicornis, has experienced a significant population increase as a consequence of its protection within Etosha National Park (ENP), Namibia. We report here on the level and spatial distribution of black rhinoceros genetic diversity within ENP. Using nine microsatellite loci, genetic variation was assessed from 144 individuals. Our results are consistent with the observation of lower levels of genetic diversity in D. b. bicornis, when compared to D. b. michaeli, but greater diversity when compared to D. b. minor. We also showed that ENP’s black rhino genetic diversity is well represented in Waterberg National Park, originally founded with ENP individuals. We found no genetic signature of a recent bottleneck in ENP, however, suggesting that the genetic diversity within ENP has not been adversely affected by the recent severe population decline. Using Bayesian clustering methods, we observed no significant population structure within ENP, but positive spatial genetic correlation is observed at distances up to 25 km. This relationship exists in females but not males, suggesting reduced dispersal among females, the first evidence of limited female dispersal or philopatry in any species of rhinoceros.     

Phylogeography and conservation genetics of Eld's deer (Cervus eldi)

Eld's deer (Cervus eldi) is a highly endangered cervid, distributed historically throughout much of South Asia and Indochina. We analysed variation in the mitochondrial DNA (mtDNA) control region for representatives of all three Eld's deer subspecies to gain a better understanding of the genetic population structure and evolutionary history of this species. A phylogeny of mtDNA haplotypes indicates that the critically endangered and ecologically divergent C. eldi eldi is related more closely to C. e. thamin than to C. e. siamensis, a result that is consistent with biogeographic considerations. The results also suggest a strong degree of phylogeographic structure both between subspecies and among populations within subspecies, suggesting that dispersal of individuals between populations has been very limited historically. Haplotype diversity was relatively high for two of the three subspecies (thamin and siamensis), indicating that recent population declines have not yet substantially eroded genetic diversity. In contrast, we found no haplotype variation within C. eldi eldi or the Hainan Island population of C. eldi siamensis, two populations which are known to have suffered severe population bottlenecks. We also compared levels of haplotype and nucleotide diversity in an unmanaged captive population, a managed captive population and a relatively healthy wild population. Diversity indices were higher in the latter two, suggesting the efficacy of well-designed breeding programmes for maintaining genetic diversity in captivity. Based on significant genetic differentiation among Eld's deer subspecies, we recommend the continued management of this species in three distinct evolutionarily significant units (ESUs). Where possible, it may be advisable to translocate individuals between isolated populations within a subspecies to maintain levels of genetic variation in remaining Eld's deer populations.     

Subspecies of the Central American Squirrel Monkey (Saimiri oerstedii) as Units for Conservation

The accurate diagnosis of conservation units now typically includes recognition of genetic diversity and unique evolutionary lineages and is necessary to inform the conservation management of endangered species. We evaluated whether the two currently recognized subspecies of the endangered Central American squirrel monkey (Saimiri oerstedii) in Costa Rica are evolutionarily significant units (ESUs) that should be managed separately in conservation efforts. We used previously published sequences of 50 individuals of Saimiri oerstedii for 880 bp of the mtDNA d-loop and genotypes of 244 individuals for 16 microsatellites and conducted novel analyses to characterize genetic differentiation between subspecies of Saimiri oerstedii. We measured sequence differentiation and inferred an intraspecific molecular phylogeny and a haplotype network, and found consistent results supporting statistically significant divergence and reciprocal monophyly between subspecies. A population aggregation analysis also supported Saimiri oerstedii citrinellus and S. o. oerstedii as diagnosably distinct units. These results confirm previous genetic studies with smaller sample sizes and are consistent with other factors including differences in pelage and morphology and divergence at nuclear markers. Conservation managers should manage these subspecies separately to prevent the loss of genetic diversity via artificially induced outbreeding. High levels of genetic diversity may buffer populations against outside extinction pressures, to which Saimiri oerstedii are vulnerable because of their dwindling habitat and small population size.     

Genetic variation of complete mitochondrial genome sequences of the Sumatran rhinoceros (<Emphasis Type="Italic">Dicerorhinus sumatrensis</Emphasis>)

The Sumatran rhinoceros (Dicerorhinus sumatrensis) is the smallest and one of the most endangered rhinoceros species, with less than 100 individuals estimated to live in the wild. It was originally divided into three subspecies but only two have survived, D. sumatrensis sumatrensis (Sumatran subspecies), and D. s. harrissoni (Bornean). Questions regarding whether populations of the Sumatran rhinoceros should be treated as different management units to preserve genetic diversity have been raised, particularly in light of its severe decline in the wild and low breeding success in captivity. This work aims to characterize genetic differentiation between Sumatran rhinoceros subspecies using complete mitochondrial genomes, in order to unravel their maternal evolutionary history and evaluate their status as separate management units. We identified three major phylogenetic groups with moderate genetic differentiation: two distinct haplogroups comprising individuals from both the Malay Peninsula and Sumatra, and a third group from Borneo. Estimates of divergence time indicate that the most recent common ancestor of the Sumatran rhinoceros occurred approximately 360,000 years ago. The three mitochondrial haplogroups showed a common divergence time about 80,000 years ago corresponding with a major biogeographic event in the Sundaland region. Patterns of mitochondrial genetic differentiation may suggest considering Sumatran rhinoceros subspecies as different conservation units. However, the management of subspecies as part of a metapopulation may appear as the last resource to save this species from extinction, imposing a conservation dilemma.     

Species‐level view of population structure and gene flow for a critically endangered primate (Varecia variegata)

Lemurs are among the world's most threatened mammals. The critically endangered black‐and‐white ruffed lemur (Varecia variegata), in particular, has recently experienced rapid population declines due to habitat loss, ecological sensitivities to habitat degradation, and extensive human hunting pressure. Despite this, a recent study indicates that ruffed lemurs retain among the highest levels of genetic diversity for primates. Identifying how this diversity is apportioned and whether gene flow is maintained among remnant populations will help to diagnose and target conservation priorities. We sampled 209 individuals from 19 sites throughout the remaining V. variegata range. We used 10 polymorphic microsatellite loci and ~550 bp of mtDNA sequence data to evaluate genetic structure and population dynamics, including dispersal patterns and recent population declines. Bayesian cluster analyses identified two distinct genetic clusters, which optimally partitioned data into populations occurring on either side of the Mangoro River. Localities north of the Mangoro were characterized by greater genetic diversity, greater gene flow (lower genetic differentiation) and higher mtDNA haplotype and nucleotide diversity than those in the south. Despite this, genetic differentiation across all sites was high, as indicated by high average F_ST (0.247) and Φ_ST (0.544), and followed a pattern of isolation‐by‐distance. We use these results to suggest future conservation strategies that include an effort to maintain genetic diversity in the north and restore connectivity in the south. We also note the discordance between patterns of genetic differentiation and current subspecies taxonomy, and encourage a re‐evaluation of conservation management units moving forward.     

Small edge populations at risk: genetic diversity of the green lizard (<Emphasis Type="Italic">Lacerta viridis viridis</Emphasis>) in Germany and implications for conservation management

Edge and central populations can show great differences regarding their genetic variation and thereby also in their probability of extinction. This fact might be of great importance for the conservation strategies of endangered species. In this study we examine the level of microsatellite variability within three threatened edge populations of the green lizard subspecies Lacerta viridis viridis (Laur.) in Brandenburg (Germany) and compare the observed variation to other edge and central populations within the northern species range. We demonstrate that the northernmost edge populations contain less genetic variation in comparison to the central population. However, there were no observable significant differences to the other edge population included in this study. Surprisingly, we observed a high genetic differentiation in a small geographical range between the three endangered populations in Brandenburg, which can be explained by processes like fragmentation, isolation, genetic drift and small individual numbers within these populations. We also detected unique genetic variants (alleles), which only occurred in these populations, despite a low overall genetic variation. This study demonstrates the potential of fast evolving markers assessing the genetic status of endangered populations with a high resolution. It also illustrates the need for a comparative analysis of different regions within the species range, achieving a more exact interpretation of the genetic variation in endangered populations. This will aid future management decisions in the conservation of genetic diversity in threatened species.     

Conservation of deer: contributions from molecular biology, evolutionary ecology, and reproductive physiology

Molecular phylogenetics, interspecific comparisons, and assisted reproductive techniques are recent approaches to understanding and facilitating conservation of endangered species. This paper reviews the contribution of these approaches to a small but well-studied group of mammals, deer, many of which are endangered in the wild. Conservation efforts require a comprehensive understanding of the biology and history of these animals. The value of assisted reproductive technologies for conservation of deer has received increased awareness especially for captive populations. Such breeding programmes are designed to assist propagation of threatened species and to maximize genetic diversity within populations through the movement of genetic material across the globe, but will only be successful if we understand the genetic and reproductive potential of various lineages. Here we discuss the phylogenetic status of deer, the distinctiveness and evolution of their reproductive patterns, and current approaches for improving the success of controlled breeding programmes for the conservation of endangered lineages. Only by combining both theoretical and practical approaches to conservation efforts can we hope to salvage the remaining organismal diversity of our planet.     

Genetic diversity in fragmented populations of Berchemiella wilsonii var. pubipetiolata (Rhamnaceae)

BACKGROUD AND AIMS: Berchemiella wilsonii var. pubipetiolata (Rhamnaceae) is distributed in fragmented habitat patches in eastern China. It is highly endangered because of severe disturbance by anthropogenic activities. Information on genetic variation and structure is critical for developing successful conservation strategies for this species. METHODS: Allozyme variation of population genetic diversity and structure was investigated for a total of 98 individuals sampled from four extant populations using isoelectric focusing in thin-layer polyacrylamide slab gels. KEY RESULTS: Based on 20 loci scored from the nine enzymes examined, a high genetic diversity was detected at both the species and population level, while there was a loss of low frequency alleles (<0.1) in all populations. Most loci showed deviation from Hardy-Weinberg equilibrium due to excess of heterozygotes in all populations, suggesting that selection for heterozygotes has occurred in this species. The genetic diversity was mainly found within populations with a moderate genetic differentiation (F(ST) = 0.13), but the two geographically discontinuous population groups showed significant differences, with F-statistic values of 0.078 for the Zhejiang populations and 0.014 for the Anhui populations, respectively. CONCLUSIONS: It appears most likely that this species has experienced a recent decrease in population size, and genetic drift in small populations has resulted in a loss of alleles occurring at low frequency. The differentiation into two population groups reflects a population genetic consequence that has been influenced by the different land-use in the two regions. Some conservation concerns are discussed together with possible strategies for implementing in situ and ex situ conservation.     

Maintenance of genetic diversity in a small,isolated population of ancient tree Erythrophleum fordii

Ancient trees remaining in natural areas from once widespread forest due to overexploitation and habitat modification would naturally regenerate to form small isolated populations after the halting of disturbance. The genetic processes and consequence of this regeneration are generally unknown, which prevents the effective conservation and management of these populations. One such population, an Erythrophleum fordii population, is located in Dinghushan National Nature Reserve (China). Owing to the remarkably huge trunk of the sole ancient tree (KG) relative to the other individuals in the population, the local people and local tourism administration presume that this population has developed solely from KG. We found, using genetic diversity analyses, that contrary to this presumption, the population might have regenerated from several founders with diverse genetic backgrounds. Severe degradation of genetic diversity within the population did not take place, rather, it possessed the potential ability to maintain and recover gradually with population development. According to the results of simulations, we found that the longevity of tree species with iteroparity offers the potential to maintain genetic diversity in small isolated populations. The results from this study will benefit the restoration and conservation of the endangered species E. fordii, and of other congener species and tree species with similar life history traits.     

Allozyme variation and population genetic structure of common wild rice Oryza rufipogon Griff. in China

In order to determine the genetic diversity and genetic structure of populations in common wild rice Oryza rufipogon, an endangered species, allozyme diversity was analyzed using 22 loci in 607 individuals of 21 natural populations from the Guangxi, Guangdong, Hainan, Yunnan, Hunan, Jiangxi and Fujian provinces in China. The populations studied showed a moderate allozyme variability (A=1.33, P=22.7%, Ho=0.033 and He=0.068), which was relatively high for the genus Oryza. The levels of genetic diversity for Guangxi and Guangdong were significantly higher than those for the other regions, and thus South China appeared to be the center of genetic diversity of O. rufipogon in China. A moderate genetic differentiation (F_ST=0.310, I=0.964) was found among the populations studied. Interestingly, the pattern of population differentiation does not correspond to geographic distance. An estimate of the outcrossing rate (t=0.324) suggests that the species has a typical mixed-mating system. The deficit of heterozygotes (F=0.511) indicates that some inbreeding may have taken place in outcrossing asexual populations because of intra-clone outcrossing events and ”isolation by distance” as a result of human disturbance. In order to predict the long-term genetic survival of fragmented populations, further studies on gene flow among the remaining populations and the genetic effects of fragmentation are proposed. Finally, some implications for the conservation of endangered species are suggested. Received: 22 June 1999 / Accepted: 20 December 1999     

Limited structure and widespread diversity suggest potential buffers to genetic erosion in a threatened grassland shrub Pimelea spinescens (Thymelaeaceae)

Pimelea spinescens is a critically endangered species of the temperate grasslands of southeastern Australia. Two subspecies are recognised. Subspecies, P. spinescens subsp. spinescens, formerly common and widespread, is found in isolated remnants of previously extensive grasslands. The second subspecies, subsp. pubiflora, is thought to have been historically rare with only two geographically-isolated extant populations. The grassland communities exist now as fragmented remnants representing <1 % of their extent prior to European settlement in the early 1800s. Conservation management strategies for species in these critically endangered ecosystems rely on an understanding of genetic diversity and population structure to ensure long-term evolutionary potential. We used chloroplast DNA (cpDNA) and microsatellite markers to examine the population genetic structure of both subspecies. Analysis of cpDNA revealed 14 haplotypes with high divergence between the single haplotype found in subsp. pubiflora and most remaining haplotypes restricted to subsp. spinescens. Microsatellites also indicated high genetic differentiation between subspecies but little evidence of sub-structuring within either subspecies. Results suggest that seed dispersal has not been as limited as previously thought. In this fragmented habitat, a lack of genetic structure suggests buffers to genetic erosion, with current patterns reflecting genetic diversity prior to fragmentation. Plant longevity and the presence of seed banks may contribute to the maintenance of these patterns, resulting in a lag between fragmentation and genetic erosion. Whilst factors such as longevity and seed banks may be preserving historic genetic diversity, management is required to ensure the maintenance of this diversity into the future.     

Genetic variation and structure of the endangered Lady Fern Athyrium viridescentipes based on ubiquitous genotyping

To clarify the genetic status and provide effective information for the conservation of Athyrium viridescentipes, a critically endangered fern species with only 103 individuals remaining in the wild, we conducted ubiquitous genotyping to determine the genotypes of all remnant individuals of the target species. We analyzed the genetic variation of the 103 known individuals in four populations by using 13 microsatellite loci. The genotypes of single spores from a sporophytic individual were also determined in order to reveal the breeding system of this species. The level of allelic variation in A. viridescentipes was significantly lower than that of closely related Athyrium species. The genetic composition of the four populations was rather similar. Sixty-nine individuals (67%) possessed an identical pattern in the allele combinations at 13 microsatellite loci. The mean pairwise F (ST) among four populations was 0.018. The segregated pattern of alleles, determined by single-spore genotyping, revealed that allelic recombination occurs through meiosis. The results indicate that this species contains a low level of genetic variation, has low population differentiation, and maintains populations by sexual reproduction. These findings could lead to more effective conservation programs, the selection of the most appropriate individuals for ex situ conservation efforts, and separate management of extant populations.     

Conservation of rare species with island-like distributions: A case study of Lasthenia conjugens (Asteraceae) using population genetic structure and the distribution of rare markers

Californian vernal pools, a patchy, island-like habitat, are endangered as a result of habitat destruction. Conservation of the remaining vernal pool habitat is essential for the persistence of several endangered species. We present the first study examining DNA-level genetic diversity within and among populations of a vernal pool plant species. We investigated genetic variation across eight populations of the US federally endangered vernal pool endemic Lasthenia conjugens (Asteraceae) using intersimple sequence repeat (ISSR) markers. Genetic diversity within the species was high (Nei's gene diversity estimate was 0.37), with moderate differentiation among populations (Bayesian F _ST analog of 0.124). Using an amova analysis, we found that the majority of the genetic variation (84%) was distributed within populations. There is a significant relationship between geographical distance and pairwise genetic differentiation as measured by the Bayesian estimate θ^B. The alternative hypotheses of historic geological processes within the Central Valley and contemporary gene flow are discussed as explanations of the data. Because of the vulnerability of the populations, we calculated a probability of loss for rare alleles (fragments) in the populations. Calculations show that sampling only one of the eight populations for ex-situ conservation or restoration will capture approximately 54% of the sampled rare fragments. We believe that one of the sampled populations has become extinct since it was sampled. When removing this population from the above-mentioned calculations, sampling one population will capture only 41.3% of the sampled rare fragments. We recommend sampling strategies for future conservation and restoration efforts of L. conjugens.     

濒危物种山红树居群遗传结构的RAPD分析

利用RAPD分子标记检测了云南省3个山红树居群的遗传多样性和居群遗传结构。10个引物共检测到54个位点, 其中多态位点11个, 占20 37%。与其他的濒危物种相比, 山红树居群内的遗传多样性很低, 居群间的遗传分化很大(78 65%)。大量经济植物的种植加上人为的破坏, 使山红树的生境遭到严重破坏, 数量大为减少, 可能导致了山红树遗传多样性的丧失、居群间较高的遗传分化。基于以上结果, 探讨了山红树进一步的迁地保护措施。     

High RAPD but no cpDNA sequence variation in the endemic and endangered plant, Heptacodium miconioides Rehd. (Caprifoliaceae)

Heptacodium miconioides Rehd. is an endangered species endemic to China and has suffered rapid decrease of distribution range and population size. This species has been disappeared in central China where the modal specimen was collected. We analyzed the genetic variation of the remaining populations to reveal whether the genetic diversity also suffered decrease and to provide some suggestions for conservation. All the nine known remaining populations were sampled. Genetic variation was analyzed based on RAPD markers and two fragments of cpDNA sequence, intergenic spacers of petG-trnP and trnS-trnG. No variation was observed in the two fragments of cpDNA sequence. However, the species exhibited high level of RAPD variation compared to other threatened or rare plants. Measures of genetic diversity within populations were strongly related to the log of estimated population size, indicating that large populations usually have more genetic diversity than that of small ones. About 25% of the variation was partitioned among populations. Significant relationship was observed between differentiation and geographical distance, indicating a pattern of isolation-by-distance. Given for few populations remaining, all the populations should be protected and urgent efforts be paid on the small populations to avoid their local extinction.     

High genetic diversity vs. low genetic differentiation in Nouelia insignis (Asteraceae), a narrowly distributed and endemic species in China, revealed by ISSR fingerprinting

BACKGROUND AND AIMS: Nouelia insignis Franch., a monotypic genus of the Asteraceae, is an endangered species endemic in Yunnan and Sichuan Provinces of China. Most of the populations are seriously threatened. Some of them are even at the brink of extinction. In this study, the genetic diversity and differentiation between populations of this species were examined in two drainage areas. METHODS: DNA fingerprinting based on inter-simple sequence repeat polymorphisms was employed to detect the genetic variation and population structure in the species. KEY RESULTS: Genetic diversity at species level was high with P=65.05% (percentage of polymorphic loci) and Ht=0.2248 (total genetic diversity). The coefficient of genetic differentiation among populations, Gst, which was estimated by partitioning the total gene diversity, was 0.2529; whereas, the genetic differentiation between populations in the Jinsha and Nanpan drainage areas was unexpectedly low (Gst=0.0702). CONCLUSIONS: Based on the genetic analyses of the DNA fingerprinting, recent habitat fragmentation may not have led to genetic differentiation or the loss of genetic diversity in the rare species. Spatial apportionment of fingerprinting polymorphisms provides a footprint of historical migration across geographical barriers. The high diversity detected in this study holds promise for conservation and restoration efforts to save the endangered species from extinction.     

濒危植物毛柄小勾儿茶片断化居群的遗传多样性

采用扩增片段长度多态性(AFLP)标记对我国特有的濒危植物毛柄小勾儿茶(Berchemiella wilsonii var. pubipetiolata)现存于浙江和安徽的4个片断化居群中的89株个体进行了遗传多样性和遗传结构的研究。结果表明,与其它木本濒危植物相比,毛柄小勾儿茶具有与它们相当的遗传多样性,8对选择扩增引物共扩增出122条清晰的条带,居群的平均多态位点百分率为P_p=26.4%,其中马家河居群最高(29.5%)而湍口居群最低(23.8%),居群的平均基因多样度为H_ep=0.162 8(0.140 5~0.172 4);而在物种水平上的遗传多样性为P_s=36.9%, H_es=0.202 4。居群间的遗传分化系数F_ST=0.193 9,表明居群间有显著的遗传分化,进一步利用AMOVA软件对遗传变异进行等级剖分发现:24.88%的遗传变异存在于地理宗间(浙江地理宗和安徽地理宗),14.71%的遗传变异存在于居群间,60.42%存在于居群内。该研究结果表明,由于人为干扰引起的生境片断化和居群减小导致了毛柄小勾儿茶居群的遗传多样性丧失和遗传分化,并对毛柄小勾儿茶的生存造成潜在威胁。该文还就保育策略进行了讨论。     

Low genetic diversity and limited genetic structure across the range of the critically endangered Mexican howler monkey (Alouatta palliata mexicana)

Genetic diversity provides populations with the possibility to persist in ever-changing environments, where selective regimes change over time. Therefore, the long-term survival of a population may be affected by its level of genetic diversity. The Mexican howler monkey (Alouatta palliata mexicana) is a critically endangered primate restricted to southeast Mexico. Here, we evaluate the genetic diversity and population structure of this subspecies based on 83 individuals from 31 groups sampled across the distribution range of the subspecies, using 29 microsatellite loci. Our results revealed extremely low genetic diversity (H_O = 0.21, H_E = 0.29) compared to studies of other A. palliata populations and to other Alouatta species. Principal component analysis, a Bayesian clustering method, and analyses of molecular variance did not detect strong signatures of genetic differentiation among geographic populations of this subspecies. Although we detect small but significant F_ST values between populations, they can be explained by a pattern of isolation by distance. These results and the presence of unique alleles in different populations highlight the importance of implementing conservation efforts in multiple populations across the distribution range of A. p. mexicana to preserve its already low genetic diversity. This is especially important given current levels of population isolation due to the extreme habitat fragmentation across the distribution range of this primate.     

Low genetic diversity and high genetic divergence caused by inbreeding and geographical isolation in the populations of endangered species Loropetalum subcordatum (Hamamelidaceae) endemic to China

Loropetalum subcordatum (Hamamelidaceae) is one of the most endangered angiosperm species in China. It is narrowly distributed in a few localities in the evergreen broadleaved forest of southern China. Up to now only a few dozen remnant individuals have been found in the four extant populations. In this project, we studied its genetic diversity and population genetic structure using the high resolution molecular marker of amplified fragment length polymorphism. In total, 47 individuals from all the four populations (including all individuals in three populations) were analyzed. Comparably low genetic diversity within populations was revealed and significantly high genetic differentiation among the populations was detected. Four independent groups were identified which corresponded with their geographical ranges. Autogamy is considered to be the major factor contributing to the low genetic variation and high genetic divergence within this species. In addition, small population size, restricted distribution range, geographical isolation and limited seed dispersal may also contribute to the low genetic diversity and high population genetic differentiation. Clonal reproduction was inferred to occur in the two island populations. Suggestions for conservation strategies are provided to preserve the genetic resources of this species.     

A new oligonucleotide probe for the giant panda

Quantifying genetic diversity in populations is one of the fundamental measures for species conservation. This is far more important for critically endangered species like giant pandas, where there are few individuals remaining in the population. However, previous multilocus probes could not identify homozygous loci resulting from inbreeding of giant pandas, and produced few polymorphic loci. As a result, we have prepared a new oligonucleotide probe, which had the highest paternity probability and succeeded in identifying the homozygous loci and in discriminating giant panda individuals.