Applying molecular genetic tools to tiger conservation

The utility of molecular genetic approaches in conservation of endangered taxa is now commonly recognized. Over the past decade, conservation genetic analyses based on mitochondrial DNA sequencing and microsatellite genotyping have provided powerful tools to resolve taxonomy uncertainty of tiger subspecies, to define conservation units, to reconstruct phylogeography and demographic history, to examine the genetic ancestry of extinct subspecies, to assess population genetic status non-invasively, and to verify genetic background of captive tigers worldwide. The genetic status of tiger subspecies and populations and implications for developing strategies for the survival of this charismatic species both in situ and ex situ are discussed.     

Characterization of 18 polymorphic microsatellite loci in the red‐crowned crane (Grus japonensis), an endangered bird

Red‐crowned cranes (Grus japonensis) were classified as an endangered species by the International Union for Conservation of Nature, but recently, their population has decreased dramatically. For the purpose of conserving this endangered species, 18 microsatellite markers were developed, including 12 newly isolated ones from a genomic library and 6 modified from another crane species. The markers were characterized in 26 red‐crowned cranes. As a result, these markers displayed 3–13 alleles, and the observed and expected heterozygosities ranged from 0.462 to 1.000 and from 0.483 to 0.884, respectively. The marker suite averaged 6.390 alleles per locus with an average polymorphic information content of 0.631. The combined exclusion probability (PE‐1) was 0.9985, and the combined exclusion probability (PE‐2) was 0.9999. Three of the 18 microsatellite loci presented significant deviations from Hardy–Weinberg Equilibrium (P < 0.05), likely due to sampling bias and unknown founder relatedness in a semi‐free population. Our results show that microsatellite loci can provide a standard protocol for genetic information in red‐crowned crane populations upon which strategies for effective conservation and management may be based.     

Noninvasive genetics provides insights into the population size and genetic diversity of an Amur tiger population in China

Understanding population size and genetic diversity is critical for effective conservation of endangered species. The Amur tiger (Panthera tigris altaica) is the largest felid and a flagship species for wildlife conservation. Due to habitat loss and human activities, available habitat and population size are continuously shrinking. However, little is known about the true population size and genetic diversity of wild tiger populations in China. In this study, we collected 55 fecal samples and 1 hair sample to investigate the population size and genetic diversity of wild Amur tigers in Hunchun National Nature Reserve, Jilin Province, China. From the samples, we determined that 23 fecal samples and 1 hair sample were from 7 Amur tigers: 2 males, 4 females and 1 individual of unknown sex. Interestingly, 2 fecal samples that were presumed to be from tigers were from Amur leopards, highlighting the significant advantages of noninvasive genetics over traditional methods in studying rare and elusive animals. Analyses from this sample suggested that the genetic diversity of wild Amur tigers is much lower than that of Bengal tigers, consistent with previous findings. Furthermore, the genetic diversity of this Hunchun population in China was lower than that of the adjoining subpopulation in southwest Primorye Russia, likely due to sampling bias. Considering the small population size and relatively low genetic diversity, it is urgent to protect this endangered local subpopulation in China.     

Integrated tool for microsatellite isolation and validation from the reference genome and their application in the study of breeding turnover in an endangered avian population

Accurate individual identification is required to estimate survival rates in avian populations. For endangered species, non‐invasive methods of obtaining individual identification, such as using molted feathers as a source of DNA for microsatellite markers, are preferred because of less disturbance, easy sample preparation and high efficiency. With the availability of many avian genomes, a few pipelines isolating genome‐wide microsatellites have been published, but it is still a challenge to isolate microsatellites from the reference genome efficiently. Here, we have developed an integrated tool comprising a bioinformatic pipeline and experimental procedures for microsatellite isolation and validation based on the reference genome. We have identified over 95 000 microsatellite loci and established a system comprising 10 highly polymorphic markers (PIC value: 0.49–0.93, mean: 0.79) for an endangered species, saker falcon (Falco cherrug). These markers (except 1) were successfully amplified in 126 molted feathers, exhibiting high amplification success rates (83.9–99.7%), high quality index (0.90–0.97) and low allelic dropout rates (1–9.5%). To further assess the efficiency of this marker system in a population study, we identified individual sakers using these molted feathers (adult) and 146 plucked feathers (offspring). The use of parent and offspring samples enabled us to infer the genotype of missing samples (N = 28), and all adult genotypes were used to ascertain that breeding turnover is a useful proxy for survival estimation in sakers. Our study presents a cost‐effective tool for microsatellite isolation based on publicly available reference genomes and demonstrates the power of this tool in estimating key parameters of avian population dynamics.     

High genetic diversity in the endangered and narrowly distributed amphibian species Leptobrachium leishanense

Threatened species typically have a small or declining population size, which make them highly susceptible to loss of genetic diversity through genetic drift and inbreeding. Genetic diversity determines the evolutionary potential of a species; therefore, maintaining the genetic diversity of threatened species is essential for their conservation. In this study, we assessed the genetic diversity of the adaptive major histocompatibility complex (MHC) genes in an endangered and narrowly distributed amphibian species, Leptobrachium leishanense in Southwest China. We compared the genetic variation of MHC class I genes with that observed in neutral markers (5 microsatellite loci and cytochrome b gene) to elucidate the relative roles of genetic drift and natural selection in shaping the current MHC polymorphism in this species. We found a high level of genetic diversity in this population at both MHC and neutral markers compared with other threatened amphibian species. Historical positive selection was evident in the MHC class I genes. The higher allelic richness in MHC markers compared with that of microsatellite loci suggests that selection rather than genetic drift plays a prominent role in shaping the MHC variation pattern, as drift can affect all the genome in a similar way but selection directly targets MHC genes. Although demographic analysis revealed no recent bottleneck events in L. leishanense, additional population decline will accelerate the dangerous status for this species. We suggest that the conservation management of L. leishanense should concentrate on maximizing the retention of genetic diversity through preventing their continuous population decline. Protecting their living habitats and forbidding illegal hunting are the most important measures for conservation of L. leishanense.     

Identification of tamaraw (Bubalus mindorensis) from natural habitat‐derived fecal samples by PCR‐RFLP analysis of cytochrome b gene

Fecal DNA analysis is a useful tool for the investigation of endangered species. Tamaraw (Bubalus mindorensis) is endemic to the Philippine island of Mindoro but knowledge of its genetic and ecological information is limited. In this study, we developed a species identification method for tamaraw by fecal DNA analysis. Eighteen feces presumed to be from tamaraw were collected in Mount Iglit‐Baco National Park and species‐known feces from domestic buffaloes and cattle were obtained from a farm. Additionally, one species‐unknown fecal sample was obtained in Mount Aruyan Preserve, where the sighting of tamaraw has not been reported in recent years. Based on DNA sequence data previously reported, the genus Bubalus‐ and tamaraw‐specific primers for PCR of cytochrome b gene were newly designed. The Bubalus‐specific primer yielded a 976 bp fragment of cytochrome b for all fecal samples from tamaraw and domestic buffaloes, but not for cattle, whereas the tamaraw‐specific primer yielded a 582 bp fragment for all tamaraw fecal samples and for one of the four domestic buffalo samples. PCR‐RFLP (restriction fragment length polymorphism) analysis of the 976 bp PCR fragment with AvrII or BsaXI provided distinct differences between tamaraw and domestic buffalo. PCR‐RFLP analysis also showed that the species‐unknown sample obtained in Mount Aruyan Preserve, originates from tamaraw.     

A short note on seed dispersal by colobines: the case of the proboscis monkey

Although the role of primates in seed dispersal is generally well recognized, this is not the case for colobines, which are widely distributed in Asian and African tropical forests. Colobines consume leaves, seeds and fruits, usually unripe. A group of proboscis monkeys (Colobinae, Nasalis larvatus) consisting of 1 alpha‐male, 6 adult females and several immatures, was observed from May 2005 to May 2006. A total of 400 fecal samples from focal group members covering 13 months were examined, with over 3500 h of focal observation data on the group members in a forest along the Menanggul River, Sabah, Malaysia. Intact small seeds were only found in 23 of 71 samples in Nov 2005, 15 of 38 in Dec 2005 and 5 of 21 in Mar 2006. Seeds of Ficus (all <1.5 mm in length) were found in all 3 months and seeds from Antidesma thwaitesianum (all <3 mm) and Nauclea subdita (all <2 mm) only in Nov and Dec, which was consistent with members of the study group consuming fruits of these species mostly at these times. To our knowledge, these are the first records of seeds in the fecal samples of colobines. Even if colobines pass relatively few seeds intact, their high abundance and biomass could make them quantitatively significant in seed dispersal. The potential role of colobines as seed dispersers should be considered by colobine researchers.     

Who killed Porthos? Genetic tracking of a gorilla death

We describe a novel application of “forensic” genetics to study a key demographic event in a population of wild gorillas. We used microsatellite genotyping and non‐invasively collected DNA samples to identify one individual western lowland gorilla as being most likely to have caused the death of another in Lopé National Park, Gabon. Data from relatively few genotypes permitted the identification of female transfer events and the interpretation of individual behavior that was previously impossible using observational methods, thus providing rare behavioral data on an elusive forest‐dwelling species. Importantly, this study highlights the need for future studies of dispersal and local population structuring in forest populations, and more accurate population census methods. Genetic studies focusing on individual identification may play a valuable role in future gorilla conservation efforts.     

Lineages differentiation and population structure of the light‐vented bulbul Pycnonotus sinensis in China based on mitochondrial DNA and microsatellite analysis

The light‐vented bulbul Pycnonotus sinensis is a small resident passerine that is found widely in central and south China, North Vietnam, and some East Asian islands, including Hainan, Taiwan and the Ryukyu Islands. Its subspecies status has been under considerable debate. A total of 10 subspecies have been proposed, but only 4 are widely recognized, including 1 continental taxon, P. s. sinensis, and 3 insular subspecies: P. s. hainanus, P. s. formosae and P. s. orii. Two mitochondrial DNA genes and 9 microsatellite loci were used to investigate the genetic divergence of this species, to identify the evolutionary status of 2 insular subspecies (P. s. hainanus and P. s. formosae) and to uncover probable historical causes that shaped them. The results reveal that P. s. formosae has diverged significantly from the other 2 subspecies because of a substantial time of isolation for populations on Taiwan Island. There was no significant genetic differentiation between P. s. sinensis and P. s. hainanus. The notable morphological difference in P. s. hainanus might be attributed to a rapid, recent adaptation to the tropical environment of Hainan Island. The low genetic divergence between P. s. sinensis and P. s. hainanus might result from a recent divergence or gene flow between them. Two insular subspecies of light‐vented bulbul populations might have diverged recently from their continental relatives through a complex evolution history owing to island isolation, ecological isolation, and possibly even hybridization. Both P. s. hainanus and P. s. formosae should be considered important conservation units because of their morphological and genetic distinctiveness.     

Endozoochorous seed dispersal by golden snub-nosed monkeys in a temperate forest

Seed dispersal is essential for plant recruitment and the maintenance of biodiversity. Colobine monkeys are primarily folivorous, but they also consume fruits and are often assumed to be seed predators. Although they are known to be epizoochorous seed dispersers, their role as endozoochorous seed dispersers needs reassessment. We examined potential endozoochory in golden snub-nosed monkeys (Rhinopithecus roxellana) at Dalongtan in Shennongjia National Park, central China, by assessing potential germination of ingested seeds (n = 1806, 9 species) from fecal samples. Intact seeds were in almost all fecal samples (ranging from 5–130 seeds), and ingested seeds were from small seeded species (seed width <4.5 mm). The 2 most abundant species were Actinidia arguta (73%) and Rosa caudata (15%). The fruits of A. arguta were unripe when ingested (i.e. effective seed predation) and the ingested seeds did not germinate in the trials. Therefore, ingestion of unripe seeds does not lead to effective seed dispersal. However, germination rates of defecated R. caudata (9%) were greater than control seeds (6% and 0%), demonstrating potential endozoochorous seed dispersal. Thus, colobine monkeys do indeed disperse mainly small-seeded from multi-seeded fruits through potential endozoochory and this process enhances the recruitment of seedlings.     

东方白鹳微卫星遗传标记的筛选及其在种群遗传分析中的应用

东方白鹳(Ciconia boyciana)是分布于东亚的大型濒危涉禽,亟待深入开展其种群的保护遗传学研究.本研究采用微卫星标记技术筛选东方白鹤的微卫星位点,并进行种群的遗传多样性分析.所用的36对微卫星引物7对来自白鹳(Ciconia ciconia),12对来自朱鹮(Nipponia nippon),17对来自大蓝...     

An optimized high quality male DNA extraction from spermatophores in open thelycum shrimp species

The crucial step of most of the current genetic studies is the extraction of DNA of sufficient quantity and quality. Several genomic DNA isolation methods have been described to successfully obtain male DNA from shrimp species. However, all current protocols require invasive handling methods with males for DNA isolation. Using Aristeus antennatus as a model we tested a reliable non‐invasive differential DNA extraction method to male DNA isolation from spermatophores attached to female thelycum. The present protocol provides high quality and quantity DNA for polymerase chain reaction amplification and male genotyping. This new approach could be useful to experimental shrimp culture to select sires with relevant genetic patterns for selective breeding programs. More importantly, it can be applied to identify the mating pairs and male structure in wild populations of species as A. antennatus, where males are often difficult to capture. Our method could be also valuable for biological studies on other spermatophore‐using species, such as myriapods, arachnids and insects.     

Taxonomic status of Chinese blue sheep (Pseudois nayaur): new evidence of a distinct subspecies

The blue sheep is an endemic species to the Qinghai‐Tibet Plateau and surrounding regions. It has been regarded as having 2 subspecies: Pseudois nayaur nayaur and P. n. szechuanensis. However, such a classification remains controversial. Herein, we analyze 10 microsatellite loci and part of the mitochondrial control region for clarification in such taxonomic debates. We use samples from 168 individuals from 6 geographic populations covering almost all the distribution areas of the species in China to carry out comparisons. Phylogenetic trees derived from both the microsatellite and mitochondrial markers combined with the discriminant analysis of principal components (DAPC) and the STRUCTURE analysis reveal that the individuals in the Helan Mountains are well grouped with a distinct evolutionary lineage and are significantly different from the other populations of P. n. szechuanensis according to Fst values, implying that this isolated population should be categorized as a valid subspecies; namely, Pseudois nayaur alashanicus. The isolation‐by‐distance (IBD) analysis shows a significant positive relationship between genetic and geographical distances among the populations.     

长江三角洲白山羊群体遗传结构研究

应用结构基因座和微卫星DNA两种遗传标记探讨长江三角洲白山羊群体遗传结构。结果表明,检测的9个结构基因座位上7个存在多态性,座位的基因平均杂合度、多态信息含量及有效等位基因数分别为0.1767、0.1457和1.2837;7个微卫星位点上共检测到110个等位基因,位点的基因平均杂合度、多态信息含量及有效等位基因数分别为0.8867、0.8774和11.2907。群体内的遗传变异程度相对较高,反映出丰富的遗传多样性,而且微卫星DNA标记揭示的遗传变异高于结构基因座。     

Genetic structure of the Amur tiger (Panthera tigris altaica) population: Are tigers in Sikhote‐Alin and southwest Primorye truly isolated?

We used molecular genetic analyses to noninvasively identify individual Amur tigers and define subpopulations of tigers in the Russian Far East. We identified 63 individuals after genotyping 256 feces, 7 hair and 11 blood samples collected within southern, central and northern Sikhote‐Alin, as well as Southwest Primorye. Analysis of nuclear DNA at 9 microsatellite loci demonstrated greater genetic similarity between animals from southern and northern Sikhote‐Alin (some 500 km apart) than between animals from Ussuriskii State Nature Reserve and Southwest Primorye (less than 10 km apart at their nearest point), suggesting that a true barrier exists preventing movements of tigers between Southwest Primorye and the southern Sikhote‐Alin Mountains.     

Genetic variation and phylogenetics of Lanyu and exotic pig breeds in Taiwan analyzed by nineteen microsatellite markers

The Lanyu pig is an indigenous miniature pig breed on Lanyu Islet near Taiwan, with a mitochondrial DNA genetic lineage remote from Asian and European pig breeds. The unknown population genetic structure and increased inbreeding among the small population of conserved Lanyu pigs is now of great conservation concern. Additionally, the presence for more than a century of exotic pig breeds in Taiwan has made gene introgression from exotic pig breeds into Lanyu pigs very possible. The present study thus aimed to investigate nuclear genetic variation within the conserved Lanyu pigs and the phylogenetic relationship and possible genetic introgression between Lanyu and exotic pig breeds by determining the polymorphism of 19 microsatellite loci. In the neighbor-joining tree constructed from 7 pig breeds based on Cavalli-Sforza and Edward chord genetic distances, 3 major clades were recognized, in which the Asian and European breeds were separately clustered into 2 clades with a 93.0 and 99.9% bootstrap confidence value, respectively. All individuals of the Lanyu breed formed a unique subclade within the Asian clade based on the distance of the proportion of shared alleles, -ln(ps), suggesting that the Lanyu breed possesses a unique nuclear genetic structure and that no nuclear gene introgression from exotic breeds into the conserved Lanyu pigs has occurred in recent history. Fifteen of 19 microsatellite loci deviated from Hardy-Weinberg equilibrium (by Wright's statistic), suggesting a significant loss of heterozygosity in the conserved population. The valuable nuclear genetic structure and phylogenetic information should assist future conservation and population management of Lanyu pigs.     

Past climate change and recent anthropogenic activities affect genetic structure and population demography of the greater long‐tailed hamster in northern China

The genetic diversity and the spatial structure of a species are likely consequences of both past and recent evolutionary processes, but relevant studies are still rare in East Asia where the Pleistocene climate has unique influences. In this study, we examined the impact of past climate change and recent anthropogenic activities on the genetic structure and population size of the greater long‐tailed hamster (Tscherskia triton), an agricultural rodent pest species in northern China. DNA sequence data of 2 mitochondrial genes and genotypic data of 11 microsatellite DNA loci from 41 populations (545 individuals) were gathered. Phylogenetic and population genetic analyses, as well as species distribution modeling and coalescent simulations, were conducted to infer its historical and demographic patterns and processes. Two deeply diverged mitochondrial clades were recovered. A small one was restricted to the Shandong Peninsula while the main clade was further divided into 3 geographic clusters by their microsatellite DNA genotypes: Northwest, North‐center and Northeast. Divergence dating indicated a Middle‐to‐Late Pleistocene divergence between the 2 clades. Demographic analysis indicated that all 3 and pooled populations showed consistent long‐period expansions during last glacial period; but not during the Holocene, probably due to the impact of climate warming and human disturbances. Conflicting patterns between mtDNA and microsatellite markers imply an anthropogenic impact on North‐center populations due to intensified agricultural cultivation in this region. Our study demonstrated that the impact of past glaciation on organisms in East Asia significantly differs from that of Europe and North America, and human activity is an important factor in determining the genetic diversity of a species, as well as its spatial structure.     

Genetic variation within and relationships among populations of Asian goats (Capra hircus)

Genetic variation at 59 protein coding loci (16 polymorphic) and 25 microsatellite loci was analysed for 11 indigenous south-east Asian goat populations, and the Australian feral population, to determine the magnitude of genetic differentiation and the genetic relationships among the populations. Significant deviations from Hardy–Weinberg equilibrium were detected in one or more populations for eight of the nine protein loci with codominant alleles, and for microsatellites for all except the two Sri Lankan populations and for all but four loci. For both marker types, average inbreeding coefficients ( F _IS) were exceptionally high. Heterogeneity of deviations from Hardy–Weinberg equilibrium for the microsatellites showed no differences for among loci within populations as compared with among populations within loci. For protein loci, however, the former was higher, indicating selection affecting allele frequencies at some loci. The variance among protein loci was significantly higher than among microsatellite loci, further indicating selection at some protein loci. There was significant differentiation among populations for both protein and microsatellite loci, most likely reflecting the geography of south-east Asia, and the presumed spread of goats throughout the region. Phylogenies derived from pair-wise genetic distance estimates show some similar clustering for the microsatellite and protein based trees, but bootstrap support was generally low for both. A phylogeny based on the combined set of 38 protein and microsatellite loci showed better consistency with geography and higher bootstrap values. The genetic distance phylogeny and the Weitzman diversity tree derived from microsatellite data showed some identical clusters, and both identified the Ujung Pandang and Australia populations as contributing most to overall genetic diversity.     

Analysis of genetic diversity and phylogenetic relationship of red deer subspecies in XinJiang,China

Polymorphisms for seven microsatellite loci in three red deer subspecies (9 populations) found in XinJiang were detected by polymerase chain reaction (PCR), 12% nondenaturation polyacrylamide gel electrophoresis and the Sanguinetti silver staining method. Numbers of alleles, average effective numbers of alleles (E) and the average rate of homozygosity, allelic frequencies of seven microsatellite loci, polymorphism information content (PIC), mean heterozygosity (H) and genetic distances among the populations were calculated for each population. Dendrograms were constructed based on genetic distances by the neighbor‐joining method (NJ), utilizing molecular evolutionary genetics analysis software PHYLIP (3.6). The phylogenetic tree was constructed based on allelic frequencies using maximum likelihood (ML); the bootstrap value was estimated by bootstrap test in the tree. Lastly, phylogenesis was analyzed. The results showed that four of the seven microsatellite loci were highly polymorphic, but BMS2508 and Celjp0023 showed no polymorphism and BM5004 was a neutral polymorphism. It is our conclusion that the four microsatellite loci are effective DNA markers for the analysis of genetic diversity and phylogenetic relationships among the three red deer subspecies. The mean PIC, H and E‐values across the microsatellite loci were 0.5393, 0.5736 and 2.64, which showed that these microsatellite loci are effective DNA markers for the genetic analysis of red deer. C.e. songaricus populations from Regiment 104, 151 and Hami are clustered together. C.e. yarkandensis populations from Regiment 35, Xaya and Alaer are clustered together. These two clusters also cluster together. Lastly, C.e. sibiricus populations from Burqin, Regiment 188 and the first two clusters were clustered together. The phylogenetic relationship among different red deer populations is consistent with the known origin, history of breeding and geographic distributions of populations.     

Paternity determination of captive bottlenose dolphins (Tursiops truncatus) using microsatellite DNA analysis

We applied previously published PCR primer pairs to amplify alleles at three polymorphic microsatellite loci to determine the genetic relationship of 6 bottlenose dolphins (Tursiops truncatus) that were living together in a Japanese aquarium. The three microsatellite loci were sufficient to determine the haplotype relationships of the six dolphins, which represented three different generations. It was confirmed that this genotyping method is simple and economical for assessing, establishing and maintaining genetic diversity in captive populations and will become a very effective technique for ex situ conservation in aquariums and zoos.