The origin, current diversity and future conservation of the modern lion (Panthera leo)

Understanding the phylogeographic processes affecting endangered species is crucial both to interpreting their evolutionary history and to the establishment of conservation strategies. Lions provide a key opportunity to explore such processes; however, a lack of genetic diversity and shortage of suitable samples has until now hindered such investigation. We used mitochondrial control region DNA (mtDNA) sequences to investigate the phylogeographic history of modern lions, using samples from across their entire range. We find the sub-Saharan African lions are basal among modern lions, supporting a single African origin model of modern lion evolution, equivalent to the 'recent African origin' model of modern human evolution. We also find the greatest variety of mtDNA haplotypes in the centre of Africa, which may be due to the distribution of physical barriers and continental-scale habitat changes caused by Pleistocene glacial oscillations. Our results suggest that the modern lion may currently consist of three geographic populations on the basis of their recent evolutionary history: North African-Asian, southern African and middle African. Future conservation strategies should take these evolutionary subdivisions into consideration.     

Species identification using a small nuclear gene fragment: application to sympatric wild carnivores from South-western Europe

Species identification is essential for non-invasive studies of elusive and rare animals, and for detecting illegal harvest or trade of wildlife species. However, most molecular tests identify only a limited number of species or require multiple laboratory steps to distinguish many taxa. Additionally, most protocols use mitochondrial DNA being, therefore, especially prone to problems such as nuclear insert copies, high intraspecific diversity or heteroplasmy. Here, we developed a molecular test based on the polymorphisms detected on a small nuclear gene fragment (221 bp of the IRBP -Interphotoreceptor Retinoid-Binding Protein- exon 1). This fragment revealed 51 variable sites (including 12 non-synonymous and 19 species-specific sites), which enabled the successful distinction of all 16 carnivore species native to South-western Europe. A SSCP (Single-Strand Conformational Polymorphism) gel electrophoresis technique was also optimized to allow the simple and inexpensive application of this molecular test. Sequences and SSCP profiles were consistent in identifying a total of 387 samples, including faeces (172) and hairs (17) collected non-invasively in the field. Due to its low cost, simplicity, and wide range of identifiable species, this test shows great promise to facilitate studies in molecular ecology, conservation genetics, and forensic analysis, as well as DNA bar-coding projects.     

扬子鳄分子遗传学与遗传多样性研究现状

扬子鳄 (Alligatorsinensis)是中国特有的珍稀物种 ,其遗传资源的保护已受到广泛关注和重视。本文简要介绍了近年来有关扬子鳄在分子系统学、遗传多样性与种群遗传结构、线粒体基因组、个体识别以及性别决定方面的分子遗传学研究状况。     

Genetic distinctiveness of the damselfly <Emphasis Type="Italic">Coenagrion puella</Emphasis> in North Africa: an overlooked and endangered taxon

North African odonates are facing conservation challenges, not only by increased degradation and loss of habitat, but also by having poorly understood taxonomy. Coenagrion puella is a widely distributed damselfly but there is debate about the taxonomic status of North African populations, where the species is very rare. We evaluate the genetic distinctiveness of North African C. puella using mitochondrial and nuclear genetic markers. We found a clear genetic differentiation between North African and European populations (3.4 % mtDNA) and a lack of shared haplotypes between individuals from the two continents. These results suggest that the damselfly C. puella comprises two genetically distinct phylogenetic lineages: one in Europe and one in North Africa, and re-invigorate the debate on the validity of the North African endemic C. puella kocheri. We propose that these two lineages of C. puella should be managed as distinct molecular operational taxonomic units. More generally, this study reinforces the important role of North Africa as centre of speciation and differentiation for odonates, and highlights the relevance of incorporating genetic data to understand the evolutionary history and taxonomy for effective biodiversity conservation.     

家马核基因组中线粒体核内插入序列分析

在各种真核生物核基因组中,存在一些由线粒体基因组转移进入核基因组中的DNA片段,这些被认为是分子化石的片段叫做线粒体核内插入序列(Numt)。由于Numt与真实的线粒体序列高度相似,因此它的存在必然会成为PCR扩增线粒体DNA的不利因素。利用已经公布的家马(Equus caballus)基因组序列(2007年9月公布,GenBank登录号为NC_009144-NC_009175)对家马Numt进行了深入分析,共发现200个可能的Numt,长度范围为29到3727bp,其中有10个的长度大于800bp。分析结果显示由于不存在线粒体控制区域的疑似Numt,因此对基于此区域的群体遗传学研究不会产生影响。本研究还发现在家马进化过程中,第1号和27号染色体更倾向于接受线粒体序列的转移。以上结果将为今后马科动物的研究提供重要的参考信息,有助于避免在线粒体DNA研究中由于Numt污染的存在而得出错误的实验结果。     

人类线粒体DNA异质性研究进展及其法医学应用

线粒体DNA(mitochondrial DNA mtDNA)的异质性自从被发现以来,一直被遗传学、进化学、发育遗传学以及法医遗传学、分子生物学领域所重视。由于线粒体异质性的存在,使得很多涉及疾病、进化、系统发育线粒体基因组与核基因组的相互作用关系、线粒体DNA复制机制以及法医学运用线粒体DNA进行实际案件评估的问题变得复杂化。此外线粒体DNA异质性的发生原因以及对线粒体异质性的检测方法标准化问题还没有一个统一的答案。针对线粒体DNA异质性带来的种种问题,近年来国内外取得了不少研究进展。     

Neglected taxonomy of rare desert fishes: congruent evidence for two species of leatherside chub

Conservation biologists rely heavily on taxonomy to set the scope for biological monitoring and recovery planning of rare or threatened species. Yet, taxonomic boundaries are seldom evaluated as falsifiable hypotheses that can be statistically tested. Here, we examine species boundaries in leatherside chub (Teleostei, Cyprinidae), an imperiled desert fish native to the Bonneville Basin and upper Snake River drainages of western North America. Recent molecular data hint that this fish could be composed of two distinct taxa that are geographically separated into northern and southern species. To formally test this hypothesis, we evaluated leatherside chub using several different categories of species concepts, including criteria dependent on phylogenetic, morphological, and ecological data. We found that leatherside chub is composed of two reciprocally monophyletic clades (candidate species) characterized by numerous fixed genetic differences for both mitochondrial and nuclear DNA markers; mtDNA sequence divergence between the two clades approached 8%. The candidate species also showed significant differences in cranial shape, revealed by morphometric analysis. Finally, controlled growth and foraging experiments using representative populations from each clade show that candidate species appear to be locally adapted to the thermal environments where they now occur. Combined, these three lines of evidence support the hypothesis that leatherside chub is composed of two species. Moreover, all lines of evidence place these two species within the genusLepidomeda, a group consisting of three additional species of endangered spinedace fishes, and one extinct species, all native to the Colorado River system. Hence, we elevate the two clades of leatherside chub to distinct species status (Lepidomeda copeiin the north andL. aliciaein the south), and argue that each warrants independent conservation and recovery action.     

Combining genetic and ecological data to assess the conservation status of the endangered Ethiopian walia ibex

Knowledge about the phylogenetic history, genetic variation and ecological requirements of a species is important for its conservation and management. Unfortunately, for many species this information is lacking. Here we use multiple approaches (phylogenetics, population genetics and ecological modelling) to evaluate the evolutionary history and conservation status of Capra walie , an endangered flagship species of wild goat endemic to Ethiopia. The analysis of mitochondrial cytochrome b and Y-chromosome DNA sequences suggests that C. walie forms a monophyletic clade with Capra nubiana , but potentially has been isolated for up to 0.8 million years from this closely related species. Microsatellite DNA analyses show that C. walie has very low genetic variation (mean heterozygosity=0.35) compared with other endangered mammals. This reduced variation likely derives from a prolonged demographic decline and small effective population size. Ecological niche modelling using the bioclimatic features of habitats occupied by C. walie , suggests ecological differences between C. walie and C. nubiana , and identifies the areas most suitable for future reintroductions of C. walie . The genetic and bioclimatic data suggest that C. walie is distinct and requires immediate conservation actions including genetic monitoring and reintroductions to establish independent populations. This study illustrates how combining noninvasive sampling along with genetic and ecological (bioclimatic) approaches can help assess conservation status of poorly known species.     

Distinct and Diverse: Range-Wide Phylogeography Reveals Ancient Lineages and High Genetic Variation in the Endangered Okapi (Okapia johnstoni)

The okapi is an endangered, evolutionarily distinctive even-toed ungulate classified within the giraffidae family that is endemic to the Democratic Republic of Congo. The okapi is currently under major anthropogenic threat, yet to date nothing is known about its genetic structure and evolutionary history, information important for conservation management given the species'' current plight. The distribution of the okapi, being confined to the Congo Basin and yet spanning the Congo River, also makes it an important species for testing general biogeographic hypotheses for Congo Basin fauna, a currently understudied area of research. Here we describe the evolutionary history and genetic structure of okapi, in the context of other African ungulates including the giraffe, and use this information to shed light on the biogeographic history of Congo Basin fauna in general. Using nuclear and mitochondrial DNA sequence analysis of mainly non-invasively collected samples, we show that the okapi is both highly genetically distinct and highly genetically diverse, an unusual combination of genetic traits for an endangered species, and feature a complex evolutionary history. Genetic data are consistent with repeated climatic cycles leading to multiple Plio-Pleistocene refugia in isolated forests in the Congo catchment but also imply historic gene flow across the Congo River.     

Phylogeography,Population Structure,and Conservation of the Javan Gibbon (<Emphasis Type="Italic">Hylobates moloch</Emphasis>)

An understanding of phylogeography and population genetics is needed for a comprehensive long-term conservation management strategy. The Javan gibbon (Hylobates moloch), an Endangered species endemic to the island of Java, has been protected since 1924 but is threatened by ongoing habitat loss, habitat degradation, and the wildlife trade. We studied the phylogeography and population genetic structure of the Javan gibbon, to define the number of Evolutionary Significant Units (ESUs) in the species, and the population genetic structure in each ESU. We sampled 47 individuals, analyzing 35 for variation in mitochondrial DNA control region, 41 for variation in 8 nuclear DNA microsatellites, and 13 for variation in 45 nuclear DNA single nucleotide polymorphisms (SNPs). We found support for two ESUs across the species range: a western ESU, extending from Ujung Kulon to Gunung Gede–Pangrango, and a central ESU, extending from Gunung Masigit–Simpang–Tilu to Gunung Slamet. Analysis of molecular variance and population structure analysis indicate significant structuring in the western ESU between Ujung Kulon and Gunung Halimun–Salak–Gede–Pangrango, and little to moderate structure in the central ESU, underscoring the importance of conserving as many populations as possible to preserve the full array of genetic diversity in this species. Our results will inform future more comprehensive population genetic surveys and the conservation genetic management of the Javan gibbon. This study demonstrates the importance of genetics when designing conservation management strategies for endangered primates.     

Phylogeography of the heavily poached African common pangolin (Pholidota,Manis tricuspis) reveals six cryptic lineages as traceable signatures of Pleistocene diversification

Knowledge on faunal diversification in African rainforests remains scarce. We used phylogeography to assess (i) the role of Pleistocene climatic oscillations in the diversification of the African common pangolin (Manis tricuspis) and (ii) the utility of our multilocus approach for taxonomic delineation and trade tracing of this heavily poached species. We sequenced 101 individuals for two mitochondrial DNA (mtDNA), two nuclear DNA and one Y‐borne gene fragments (totalizing 2602 bp). We used a time‐calibrated, Bayesian inference phylogenetic framework and conducted character‐based, genetic and phylogenetic delineation of species hypotheses within African common pangolins. We identified six geographic lineages partitioned into western Africa, Ghana, the Dahomey Gap, western central Africa, Gabon and central Africa, all diverging during the Middle to Late Pleistocene. MtDNA (cytochrome b + control region) was the sole locus to provide diagnostic characters for each of the six lineages. Tree‐based Bayesian delimitation methods using single‐ and multilocus approaches gave high support for ‘species’ level recognition of the six African common pangolin lineages. Although the diversification of African common pangolins occurred during Pleistocene cyclical glaciations, causative correlation with traditional rainforest refugia and riverine barriers in Africa was not straightforward. We conclude on the existence of six cryptic lineages within African common pangolins, which might be of major relevance for future conservation strategies. The high discriminative power of the mtDNA markers used in this study should allow an efficient molecular tracing of the regional origin of African common pangolin seizures.     

Phylogeographic patterns and conservation implications of the endangered Chinese giant salamander

Understanding genetic diversity patterns of endangered species is an important premise for biodiversity conservation. The critically endangered salamander Andrias davidianus, endemic to central and southern mainland in China, has suffered from sharp range and population size declines over the past three decades. However, the levels and patterns of genetic diversity of A. davidianus populations in wild remain poorly understood. Herein, we explore the levels and phylogeographic patterns of genetic diversity of wild‐caught A. davidianus using larvae and adult collection with the aid of sequence variation in (a) the mitochondrial DNA (mtDNA) fragments (n = 320 individuals; 33 localities), (b) 19 whole mtDNA genomes, and (c) nuclear recombinase activating gene 2 (RAG2; n = 88 individuals; 19 localities). Phylogenetic analyses based on mtDNA datasets uncovered seven divergent mitochondrial clades (A–G), which likely originated in association with the uplifting of mountains during the Late Miocene, specific habitat requirements, barriers including mountains and drainages and lower dispersal ability. The distributions of clades were geographic partitioned and confined in neighboring regions. Furthermore, we discovered some mountains, rivers, and provinces harbored more than one clades. RAG2 analyses revealed no obvious geographic patterns among the five alleles detected. Our study depicts a relatively intact distribution map of A. davidianus clades in natural species range and provides important knowledge that can be used to improve monitoring programs and develop a conservation strategy for this critically endangered organism.     

Targeted multiplex next‐generation sequencing: advances in techniques of mitochondrial and nuclear DNA sequencing for population genomics

Next‐generation sequencing (NGS) is emerging as an efficient and cost‐effective tool in population genomic analyses of nonmodel organisms, allowing simultaneous resequencing of many regions of multi‐genomic DNA from multiplexed samples. Here, we detail our synthesis of protocols for targeted resequencing of mitochondrial and nuclear loci by generating indexed genomic libraries for multiplexing up to 100 individuals in a single sequencing pool, and then enriching the pooled library using custom DNA capture arrays. Our use of DNA sequence from one species to capture and enrich the sequencing libraries of another species (i.e. cross‐species DNA capture) indicates that efficient enrichment occurs when sequences are up to about 12% divergent, allowing us to take advantage of genomic information in one species to sequence orthologous regions in related species. In addition to a complete mitochondrial genome on each array, we have included between 43 and 118 nuclear loci for low‐coverage sequencing of between 18 kb and 87 kb of DNA sequence per individual for single nucleotide polymorphisms discovery from 50 to 100 individuals in a single sequencing lane. Using this method, we have generated a total of over 500 whole mitochondrial genomes from seven cetacean species and green sea turtles. The greater variation detected in mitogenomes relative to short mtDNA sequences is helping to resolve genetic structure ranging from geographic to species‐level differences. These NGS and analysis techniques have allowed for simultaneous population genomic studies of mtDNA and nDNA with greater genomic coverage and phylogeographic resolution than has previously been possible in marine mammals and turtles.     

A European Melting Pot of Harbour Porpoise in the French Atlantic Coasts Inferred from Mitochondrial and Nuclear Data

Field surveys have reported a global shift in harbour porpoise distribution in European waters during the last 15 years, including a return to the Atlantic coasts of France. In this study, we analyzed genetic polymorphisms at a fragment of the mitochondrial control region (mtDNA CR) and 7 nuclear microsatellite loci, for 52 animals stranded and by-caught between 2000 and 2010 along the Atlantic coasts of France. The analysis of nuclear and mitochondrial loci provided contrasting results. The mtDNA revealed two genetically distinct groups, one closely related to the Iberian and African harbour porpoises, and the second related to individuals from the more northern waters of Europe. In contrast, nuclear polymorphisms did not display such a distinction. Nuclear markers suggested that harbour porpoises behaved as a randomly mating population along the Atlantic coasts of France. The difference between the two kinds of markers can be explained by differences in their mode of inheritance, the mtDNA being maternally inherited in contrast to nuclear loci that are bi-parentally inherited. Our results provide evidence that a major proportion of the animals we sampled are admixed individuals from the two genetically distinct populations previously identified along the Iberian coasts and in the North East Atlantic. The French Atlantic coasts are clearly the place where these two previously separated populations of harbour porpoises are now admixing. The present shifts in distribution of harbour porpoises along this coast is likely caused by habitat changes that will need to be further studied.     

A genetic survey of heavily exploited, endangered turtles: caveats on the conservation value of trade animals

Asian turtles face an extinction crisis, and so it is imperative that systematists accurately determine species diversity in order to guide conservation strategies effectively. We surveyed mitochondrial and nuclear DNA (mtDNA and nuDNA) variation of the heavily exploited Mauremys mutica complex, a clade of Asian turtles that contains the endangered M. mutica from Japan, Taiwan, China and Vietnam, and the critically endangered Mauremys annamensis from central Vietnam. We discovered extensive mtDNA and nuDNA variation among samples that did not correspond to the currently recognized taxonomy. Both nuDNA and mtDNA data suggest that M. mutica is paraphyletic with respect to M. annamensis . Surprisingly, M. annamensis exhibits a previously unknown mtDNA structure in the form of two clades that are paraphyletic to M. mutica . These data reveal that the currently recognized taxonomy of the mutica complex does not reflect the genetic diversity of our samples. Unfortunately, many conservation-oriented captive-breeding efforts for turtles are also based on trade samples such as the ones studied here. These efforts include plans to breed trade-rescued individuals and release their progeny into the wild. Because our genetic survey reveals that the taxonomic identity of these samples does not reflect genetic diversity, we raise serious questions about the efficacy of these programs. In order to address conservation issues and provide more accurate estimates of evolutionary lineages within Mauremys , we recommend continued surveys for wild populations of the mutica complex to provide new genetic material and additional distributional data, attempts to extract DNA from historic museum specimens and a shift in conservation focus to in situ preservation of wild populations and associated habitat.     

滇金丝猴线粒体D-loop 片段SNPs 位点的鉴别及初步分析

本文利用已测序的157 个滇金丝猴控制区(D-loop)片段,通过与参考序列比对,鉴别了线粒体D-loop 片段中的52 个SNP (Single Nucleotide Polymorphisms)位点,定义了30 种滇金丝猴单倍型,排除概率为0. 938。谱系及种群遗传结构分析结果与以前利用D-loop 片段的研究结果相似。同时表明基于粪便样品进行保护遗传学、谱系生物地理学、种群遗传学等研究时,与线粒体标记和微卫星标记相比,SNP 标记可能具有一定的优越性,并建议进一步分析滇金丝猴线粒体D-loop 全序列甚至线粒体全基因组上的SNPs 位点的信息,以促进滇金丝猴保护遗传学等研究的开展。     

Postglacial recolonization patterns and genetic relationships among whitefish (Coregonus sp.) populations in Denmark, inferred from mitochondrial DNA and microsatellite markers

The genetic relationships among morphologically and geographically divergent populations of whitefish (genus: Coregonus ) from Denmark and the Baltic Sea region were studied by analysis of microsatellites and polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis of mitochondrial DNA (mtDNA) segments. The endangered North Sea houting (classified as C. oxyrhynchus ) differs morphologically and physiologically from other Danish whitefish ( C. lavaretus ). However, limited divergence of North Sea houting was observed both at the level of mtDNA and microsatellites. The implications of these results for the conservation status of North Sea houting are discussed in the light of current definitions of evolutionary significant units. Both mtDNA and microsatellite data indicated that postglacial recolonization by C. lavaretus in Denmark was less likely to have taken place from the Baltic Sea. Instead, the data suggested a recent common origin of all Danish whitefish populations, including North Sea houting, probably by recolonization via the postglacial Elbe River system. Estimates of genetic differentiation among populations based on mtDNA and microsatellites were qualitatively different. In addition, for both classes of markers analyses of genetic differentiation yielded different results, depending on whether molecular distances between alleles or haplotypes were included.     

Population genetics of the endangered Wattled Curassow (Crax globulosa,Cracidae, Aves) of the Colombian–Peruvian Amazon using DNA microsatellites and ND2 mitochondrial sequences

The Wattled Curassow (Crax globulosa, Cracidae, Aves) is a large bird living in the Western Amazon basin and a critically endangered species in the Colombian and in the Peruvian Amazon. We carried out the first population genetics analysis of this species employing six nuclear microsatellite markers and sequences of the mtND2 gene. The main results are as follows. (1) The levels of gene diversity were high for the overall population as well as for each of the three islands for both microsatellites and mtDNA. (2) A small amount of genetic differentiation among populations was found with both types of markers (F_ST = 0.027 for microsatellites and N_ST = 0.17 for mitochondrial sequences). (3) Using microsatellites, the Geneclass 2.0 software detected a low correct assignment of individuals to their respective populations. The Structure software only detected one gene pool for the entire area studied. These results are relevant for conservation efforts of this critically endangered species.     

基因组文库构建及其在保护遗传学中的应用前景

基因组文库是进行分子克隆和基因组结构与功能研究的基础。完整的基因组文库的构建, 使任何DNA 片段的筛选和获得成为可能。随着不同克隆载体的相继出现, 基因组文库也经历了一系列的发展过程。其中, BAC文库因其转化效率高、嵌合体少、插入片段易回收, 以及容载量较大等优点而被广泛应用。近几十年来, 随着环境的日益恶化、分子遗传学技术的迅速发展, 以及保护生物学和分子遗传学的不断相互渗透, 孕育产生了保护遗传学这一分支学科。作为保护遗传学中物种保护策略的重要部分, 建立濒危野生动植物的基因组文库, 是保存其种质资源的最为有效的实际手段和方法, 并能为进一步开展与生殖、疾病和重要经济性状等方面有关的功能基因的研究, 提供可靠的材料保证。     

Recurrent introgression of mitochondrial DNA among hares (Lepus spp.) revealed by species-tree inference and coalescent simulations

Understanding recent speciation history requires merging phylogenetic and population genetics approaches, taking into account the persistence of ancestral polymorphism and possible introgression. The emergence of a clear phylogeny of hares (genus Lepus) has been hampered by poor genomic sampling and possible occurrence of mitochondrial DNA (mtDNA) introgression from the arctic/boreal Lepus timidus into several European temperate and possibly American boreal species. However, no formal test of introgression, taking also incomplete lineage sorting into account, has been done. Here, to clarify the yet poorly resolved species phylogeny of hares and test hypotheses of mtDNA introgression, we sequenced 14 nuclear DNA and 2 mtDNA fragments (8205 and 1113 bp, respectively) in 50 specimens from 11 hare species from Eurasia, North America, and Africa. By applying an isolation-with-migration model to the nuclear data on subsets of species, we find evidence for very limited gene flow from L. timidus into most temperate European species, and not into the American boreal ones. Using a multilocus coalescent-based method, we infer the species phylogeny, which we find highly incongruent with mtDNA phylogeny using parametric bootstrap. Simulations of mtDNA evolution under the speciation history inferred from nuclear genes did not support the hypothesis of mtDNA introgression from L. timidus into the American L. townsendii but did suggest introgression from L. timidus into 4 temperate European species. One such event likely resulted in the complete replacement of the aboriginal mtDNA of L. castroviejoi and of its sister species L. corsicanus. It is remarkable that mtDNA introgression in hares is frequent, extensive, and always from the same donor arctic species. We discuss possible explanations for the phenomenon in relation to the dynamics of range expansions and species replacements during the climatic oscillations of the Pleistocene.