Molecular Phylogeography and Population Genetic Structure of an Endangered Species Pachyhynobius shangchengensis (hynobiid Salamander) in a Fragmented Habitat of Southeastern China

The salamander Pachyhynobius shangchengensis (Hynobiidae) is a vulnerable species restricted to a patchy distribution associated with small mountain streams surrounded by forested slopes in the Mount Dabieshan region in southeastern China. However, molecular phylogeography and population genetic structure of P. shangchengensis remain poorly investigated. In this study, we explored the genetic structure and phylogeography of P. shangchengensis based on partial sequences of the mitochondrial DNA (mtDNA) cytochrome b and cytochrome c oxidase subunit I genes. Fifty-one haplotypes and four clades were found among 93 samples. Phylogenetic analyses revealed four deeply divergent and reciprocally monophyletic mtDNA lineages that approximately correspond to four geographic regions separated by complicated topography and long distances. The distinct geographic distributions of all lineages and the estimated divergence time suggest spatial and temporal separation coinciding with climatic changes during the Pleistocene. Analysis of molecular variance indicated that most of the observed genetic variation occurred among the four groups, implying long-term interruption of gene flow, and the possible separation of P. shangchengensis into four management units for conservation.     

Genetic Diversity and Genetic Structure of an Endangered Species, Trillium tschonoskii

The genetic diversity and genetic structure of Trillium tschonoskii (Maxim) were investigated using amplified fragment length polymorphism markers. Eight primer combinations were carried out on 105 different individuals sampled from seven populations. Of the 619 discernible DNA fragments generated, 169 (27.3%) were polymorphic. The percentage of polymorphic bands within populations ranged from 4.52 to 10.50. Genetic diversity (H_E) within populations ranged from 0.0130 to 0.0379, averaging 0.0536 at the species level. Genetic differentiation among populations was detected based on Nei's genetic diversity analysis (53.03%) and analysis of molecular variance (AMOVA) (52.43%). AMOVA indicated significant genetic differentiation among populations (52.43% of the variance) and within populations (47.57% of the variance) (p < 0.0002). Gene flow was low (0.4429) among populations. Species breeding system and limited gene flow among populations are plausible reasons for the high genetic differentiation observed for this species. We propose an appropriate strategy for conserving the genetic resources of T. tschonoskii in China.     

Reduced Genetic Diversity and Effective Population Size in an Endangered Atlantic Salmon (Salmo Salar) Population from Maine, USA

Atlantic salmon (Salmo salar) populations in Maine, USA, are listed as a Distinct Population Segment under the U.S. Endangered Species Act due to reduced spawning runs and juvenile densities. Whenever possible, optimal conservation strategies for endangered populations should incorporate both present and historical knowledge of genetic variation. We assayed genetic diversity at seven microsatellite loci and at the mitochondrial ND1 gene in an endangered wild population of Atlantic salmon captured from the Dennys River from 1963 to 2001 using DNA’s extracted from archival scale and tissue samples. We examined temporal trends of genetic diversity, population structure, and effective population size (Ne). Overall temporal trends of diversity and Ne show significant reductions from 1963 to 2001 raising the possibility that current restoration efforts may be impacted by historical loss of diversity potentially critical to adaptation. Although our results suggest genetic stability in this population from 1963 to 1981, significant differentiation was observed for both the 1995 and 2001 samples compared with all other temporal samples. The presence of an ND1 mtDNA haplotype in this population, historically observed only in European and Newfoundland stocks, may represent previously unrecognized local wild diversity or, alternatively, may represent introgression from non-native fish.     

Limited Population Structure,Genetic Drift and Bottlenecks Characterise an Endangered Bird Species in a Dynamic,Fire-Prone Ecosystem

Fire is a major disturbance process in many ecosystems world-wide, resulting in spatially and temporally dynamic landscapes. For populations occupying such environments, fire-induced landscape change is likely to influence population processes, and genetic patterns and structure among populations. The Mallee Emu-wren Stipiturus mallee is an endangered passerine whose global distribution is confined to fire-prone, semi-arid mallee shrublands in south-eastern Australia. This species, with poor capacity for dispersal, has undergone a precipitous reduction in distribution and numbers in recent decades. We used genetic analyses of 11 length-variable, nuclear loci to examine population structure and processes within this species, across its global range. Populations of the Mallee Emu-wren exhibited a low to moderate level of genetic diversity, and evidence of bottlenecks and genetic drift. Bayesian clustering methods revealed weak genetic population structure across the species'' range. The direct effects of large fires, together with associated changes in the spatial and temporal patterns of suitable habitat, have the potential to cause population bottlenecks, serial local extinctions and subsequent recolonisation, all of which may interact to erode and homogenise genetic diversity in this species. Movement among temporally and spatially shifting habitat, appears to maintain long-term genetic connectivity. A plausible explanation for the observed genetic patterns is that, following extensive fires, recolonisation exceeds in-situ survival as the primary driver of population recovery in this species. These findings suggest that dynamic, fire-dominated landscapes can drive genetic homogenisation of populations of species with low-mobility and specialised habitat that otherwise would be expected to show strongly structured populations. Such effects must be considered when formulating management actions to conserve species in fire-prone systems.     

High Genetic Diversity and Population Differentiation in the Critically Endangered Plant Species Trailliaedoxa gracilis (Rubiaceae)

Trailliaedoxa gracilis W. W. Smith et Forrest (Rubiaceae), a Chinese endemic monotypic genus belonging to the Alberteae (Rubiaceae), exhibits a narrow distribution in the dry valleys of the Jinsha River and Red River drainage area in southwestern China. The few sites at which T. gracilis occurs are fragmented and isolated, and several are highly vulnerable to human disturbance. As T. gracilis is a protected plant with a second-degree national priority, the genetic diversity and structure of the populations of this species should be investigated to determine the most suitable conservation strategy. In this study, two chloroplast regions and one nuclear region were used to investigate the genetic diversity, genetic structure, and demographic history of T. gracilis. We observed a high total genetic diversity (H _T?=?0.952 and 0.966) and low average within-population diversity (H _S?=?0.07 and 0.489) based on cpDNA and nDNA analyses. Thus, a strong genetic structure (F _ST?=?0.98049 and 0.59731) was detected. A phylogeographic structure was detected by nuclear DNA analysis (N _ST?>?G _ST, P?<?0.05); however, the chloroplast data did not show a significant phylogeographic structure (N _ST?<?G _ST, P?>?0.05). The Bayesian skyline plot and isolation with migration analysis were used to estimate the demographic history of T. gracilis. The results indicated that a marked bottleneck effect occurred during the glacial-interglacial of the Pleistocene. Among the extant populations of T. gracilis, the population found in ChunJiang, LuQuan, and YuXi showed the highest haplotype diversity based on cpDNA sequences and should be given priority for protection. According to the nDNA analysis, every population presented a high level of diversity and a high content of private haplotypes. Therefore, every population should be protected.

     

濒危植物南方红豆杉遗传多样性的RAPD分析

采用RAPD技术检测了山西南部南方红豆杉8个种群的遗传多样性。利用21个10聚寡核苷酸引物共检测出134个位点,其中多态性位点123个,占91.79%,8个种群的遗传多态位点百分率分别为67.16%(红豆峡)、67.91%(凤凰谷)、66.42%(小梯河)、66.42%(蟒河)、50.75%(历山西峡)、43.28%(云蒙山)、78.36%(长治宾馆)、50.75%(磨河)。南方红豆杉的遗传多样性分析结果显示：Shannon指数为2.180,其中31.7%的遗传多样性来自种群间,68.3%来自种群内;Nei指数为0.571,种群间的遗传分化系数(Gst)为0.181。8个种群间的遗传相似性分析结果显示：壶关红豆峡和陵川凤凰谷种群间的遗传距离最小(0.109 2),壶关红豆峡和陵川蟒河种群间遗传距离最大(0.55)。本研究结果揭示,南方红豆杉自然种群具有较高的遗传多样性,其遗传多样性不是导致该种群濒危的主要原因,导致南方红豆杉种群濒危的原因可能与南方红豆杉自然种群及群落所在生境的直接破坏及其本身生物学和生态学特性所导致的自然更新不良有着密切的联系。     

The Breeding Ecology of a Critically Endangered Salamander,Hynobius amjiensis(Caudata:Hynobiidae),Endemic to Eastern China

Hynobius amjiensis is a critically endangered salamander species(IUCN Red List) endemic to eastern China. It currently has three known populations: one in Longwangshan, Zhejiang Province(type locality), and two in Qingliangfeng between Anhui and Zhejiang Provinces. We examined the relatively unstudied breeding ecology of this species in the field and at laboratory from March 2007 to May 2014. Adult males and females were year-round terrestrial, except for the February–April breeding season. During this period, we captured only a total of 16 breeding adults(11 males and 5 females). As few as 100 breeding females were estimated based on the number of egg sacs observed since 2007. This number was significantly reduced from the estimated number between 1992 and 1998. Males(mean total length = 16.21 cm, mean body mass = 18.8 g) were slightly smaller than females(16.51 cm, 19.2 g). Size of breeding pools ranged from 0.2 m~2 to 1.2 m~2(0.1–1.2 m depths). Each female deposits a pair of egg sacs by attaching the adhesive tips of the sacs to aquatic plants or dead twigs. Fifteen pairs of egg sacs had an average length of 28.6 cm and a diameter of 3.3 cm. On average, each egg sac contained 75 eggs with a diameter of 0.3 cm. Our field survey revealed that H. amjiensis used oviposition sites in small, cool, and weakly acidic pools at high elevations(1 300–1 600 m) where peat moss was abundant. Reduction in wetland size and disappearance of suitable breeding pools suggest that this salamander species is under threat of extinction, particularly at Longwangshan, where 5 of the 9 breeding pools have either dried up or disappeared. Combined size of the remaining 4 pools is less than 2 m~2. We urge immediate implementation of more effective conservation measures and suggest that preservation priority should be given to habitat that contains suitable breeding pools.     

Molecular Evidence for an Extreme Genetic Bottleneck During Introduction of an Invading Grass to California

Although exotic species cause tremendous economic and ecological loss, we know relatively little about the post-introduction evolutionary dynamics of the invasive species themselves. Barbed goatgrass, Aegilops triuncialis L., is a cleistogamous annual grass with a native range throughout the Mediterranean Basin and Asia and introduced to California during the last century. It is considered a serious noxious range weed and is one of the few exotic plant species that is invading serpentine soil habitats. We examined whether patterns of molecular variation are consistent with a single or multiple introduction events into California and further, if individual populations show evidence for a genetic bottleneck during introduction. Fingerprinting patterns, using microsatellite loci derived from Triticum aestivum, were investigated for 57 Eurasian accessions, broadly spanning the native range and for 108 individuals from 11 localities in California. There is strong evidence for an extreme bottleneck in this species as it colonizes its new range because we detected only three multilocus genotypes occurring in California and 36 genotypes in Eurasia. In California one of the genotypes differs from one other by only one fragment and only occurs in one individual. This suggests two separate introductions. Each population is composed of highly uniform individuals and the two main genotypes are geographically separated. A. triuncialis is still expanding its range in California despite genomic uniformity after a strong bottleneck and its recently increased rate of spread is not correlated to a high within-population variability created by multiple introductions.     

珍稀濒危植物海南粗榧种群遗传多样性研究

利用RAPD技术对珍稀濒危植物海南粗榧（Cephalotaxus manniiHook.f.)遗传多样性水平,分布、濒危原因及物种保护等问题进行了探讨。结果表明：1、海南粗榧在海南岛的5个取样地点表现出低水平的遗传多样性,对环境变化物适应能力不强;2、海南粗榧种群内和种群间的遗传多样性所占比例有很大差异,绝大部分变异分布于种群内（DAN多样性为85.1%);种群间仅有较低程度的分化;3、人为砍伐,植被破坏,台风、被食用遗传漂变是海南粗榧遗传多样性低水平的主要原因,也是物种濒危的主要原因;4、对于呈零星分布的濒危植物海南粗榧的研究与保护,应充分考虑个体小环境之间的差异。考虑影响小种群的随机因素;5、应采取有力措施,就地保护现有种群,并寻求适当的方法迅速扩展种群,降低基因丧失率;选择遗传多样性较高且破坏相对较小的黎母岭种群作为保护重点;同时应加强对其他种群的保护与管理;6、海南粗榧种群内,种群音质遗传多样性在不同引物之间有较大差别。多态性位点百分率则是种群间的变化大于引物间的变化。     

Population Genetics and the Effects of a Severe Bottleneck in an Ex Situ Population of Critically Endangered Hawaiian Tree Snails

As wild populations decline, ex situ propagation provides a potential bank of genetic diversity and a hedge against extinction. These programs are unlikely to succeed if captive populations do not recover from the severe bottleneck imposed when they are founded with a limited number of individuals from remnant populations. In small captive populations allelic richness may be lost due to genetic drift, leading to a decline in fitness. Wild populations of the Hawaiian tree snail Achatinella lila, a hermaphroditic snail with a long life history, have declined precipitously due to introduced predators and other human impacts. A captive population initially thrived after its founding with seven snails, exceeding 600 captive individuals in 2009, but drastically declined in the last five years. Measures of fitness were examined from 2,018 captive snails that died between 1998 and 2012, and compared with genotypic data for six microsatellite loci from a subset of these deceased snails (N = 335), as well as live captive snails (N = 198) and wild snails (N = 92). Surprisingly, the inbreeding coefficient (F_is) declined over time in the captive population, and is now approaching values observed in the 2013 wild population, despite a significant decrease in allelic richness. However, adult annual survival and fecundity significantly declined in the second generation. These measures of fitness were positively correlated with heterozygosity. Snails with higher measures of heterozygosity had more offspring, and third generation offspring with higher measures of heterozygosity were more likely to reach maturity. These results highlight the importance of maintaining genetic diversity in captive populations, particularly those initiated with a small number of individuals from wild remnant populations. Genetic rescue may allow for an increase in genetic diversity in the captive population, as measures of heterozygosity and rarified allelic richness were higher in wild tree snails.     

Evaluating a Putative Bottleneck in a Population of Bowhead Whales from Patterns of Microsatellite Diversity and Genetic Disequilibria

A size-selected Balaena mysticetus genomic library was screened for clones containing simple sequence repeat, or microsatellite, loci. A total of 11 novel loci was identified. These loci were combined with a set of 9 published loci, for a total of 20 markers, and were scored across a sample of 108 bowhead whales from the Bering–Chukchi–Beaufort Seas population of bowhead whales. Genetic variability was measured in terms of polymorphism information content values and unbiased heterozygosity. From the latter, estimates of long-term effective population size were obtained. In addition, gametic phase disequilibrium among loci was investigated. Moderate to high levels of polymorphism were found overall, and the long-term effective size estimates were large relative to total population size. Tests of heterozygosity excess (Cornuet and Luikart 1996) and allele frequency distribution (Luikart et al. 1998) indicated that the possibility of a recent genetic bottleneck in the Bering–Chukchi–Beaufort Seas population of bowhead whales is highly unlikely. However, the fact that five loci displayed a statistically significant heterozygote deficiency remains to be explained. Received: 3 November 1998 / Accepted: 28 April 1999     

Estimating the Population Size and Genetic Diversity of Amur Tigers in Northeast China

Over the past century, the endangered Amur tiger (Panthera tigris altaica) has experienced a severe contraction in demography and geographic range because of habitat loss, poaching, and prey depletion. In its historical home in Northeast China, there appears to be a single tiger population that includes tigers in Southwest Primorye and Northeast China; however, the current demographic status of this population is uncertain. Information on the abundance, distribution and genetic diversity of this population for assessing the efficacy of conservation interventions are scarce. We used noninvasive genetic detection data from scats, capture-recapture models and an accumulation curve method to estimate the abundance of Amur tigers in Northeast China. We identified 11 individual tigers (6 females and 5 males) using 10 microsatellite loci in three nature reserves between April 2013 and May 2015. These tigers are confined primarily to a Hunchun Nature Reserve along the border with Russia, with an estimated population abundance of 9–11 tigers during the winter of 2014–2015. They showed a low level of genetic diversity. The mean number of alleles per locus was 2.60 and expected and observed heterozygosity were 0.42 and 0.49, respectively. We also documented long-distance dispersal (~270 km) of a male Amur tiger to Huangnihe Nature Reserve from the border, suggesting that the expansion of neighboring Russian populations may eventually help sustain Chinese populations. However, the small and isolated population recorded by this study demonstrate that there is an urgent need for more intensive regional management to create a tiger-permeable landscape and increased genetic connectivity with other populations.     

西双版纳地区流苏石斛遗传多样性的ISSR分析

采用ISSR分子标记技术,对西双版纳分布的兰科濒危植物流苏石斛（Dendrobium fimbriatum）5个居群共114个个体的遗传多样性进行了研究。从100条引物中筛选出了12条用于扩增,共检测到117个位点,其中105个为多态位点。分析结果表明,流苏石斛居群水平遗传多样性较低。在物种水平上,流苏石斛多态位点百分率PPB为89．74％,Nei’s基因多样性指数日为0．3227,Shannon’s多样性信息指数见。为0．4779;在居群水平上,各个居群的多态位点百分率PPB差异较大（6．84％～39．32％）,平均值为23．93％,Nei’s基因多样性指数H为0．0871,各个居群的Shannon’s多样性信息指数见平均为0．1290。AMOVA分析的结果显示,流苏石斛的遗传变异大多数存在于居群间,占总遗传变异的74．79％。基于Nei’s遗传多样性分析得出的居群间遗传分化系数Gst=0．7443。各居群间的Nei’s遗传一致度（I）范围为0．5882～0．8331。Mantel检测发现,居群间的遗传距离和地理距离之间无显著的正相关关系（r=0．2419,P=0．2416）。鉴于流苏石斛的遗传多样性现状和居群遗传结构,我们建议对流苏石斛居群所有个体实施及时的就地保护,同时建立迁地保护居群,促进基因交流。     

Genetic Diversity of Dalbergia monticola (Fabaceae) an Endangered Tree Species in the Fragmented Oriental Forest of Madagascar

There is an urgent need to maintain and restore a broad genetic base for the management of Dalbergia monticola, a very economically important but endangered tree species in Madagascar. Random amplified polymorphism DNAs (RAPDs) and chloroplast microsatellite markers were used to quantify the genetic variation and to analyse the geographic distribution of diversity. Ten locations covering most of the natural range were sampled. Sixty-three RAPD polymorphic and 15 monomorphic loci were obtained from 122 individuals. Genetic diversity was low and very close among populations and regions. The unrooted neighbour-joining tree exhibited 4 groups, representing 6% (p = 0.000) of the total variation. The greater part of the variance, 81%, was observed within populations. A Mantel test suggested that genetic distances between populations were weakly correlated with geographic distances (R = 0.46, p = 0.12). The three chloroplast microsatellite primers assayed on 100 individuals gave 13 chlorotypes. Most of the populations showed 2 or 3 haplotypes. Haplotype diversity for the total population was equal to He_Cp = 0.83 and ranged from 0.00 to 0.80 among the populations. The unrooted neighbour-joining tree exhibited 4 groups corresponding to the four regions representing 80% (p = 0.0000) of the total variation. Genetic diversity varies with regions, the north and south being less variable. Chlorotype distribution, the phylogenetic tree and historical information suggest that putative refugias in the centre-north region originating from the early Holocene could explain the pattern of variation observed today. By combining the results obtained at nuclear and organellar loci, a strategy of conservation based on evolutionarily significant units is proposed.     

濒危黑颈长尾雉圈养种群的RAPD遗传多样性分析

运用RAPD技术对黑颈长尾雉圈养种群的遗传多样性进行了分析。从50条随机引物中筛选出14条引物,对24个个体的基因组DNA进行了PCR扩增,从检测出的119个位点中有98个多态位点,占总位点的82.35%,标记的分子量大小范围是0.2～3kb。24个个体间的遗传距离幅度0.1597～0.4874,平均是0.2810;用软件NTsys2.10e构建了24个个体相互关系的分支图,24个个体可分为3个类群。实验表明:黑颈长尾雉圈养种群的遗传多样性水平较高,圈养种群内遗传差异性较大。     

珍稀濒危植物安徽羽叶报春遗传多样性的RAPD分析

利用RAPD分子标记对安徽特有濒危物种安徽羽叶报春（Primula merrilliana）6个自然居群的134个个体的遗传多样性进行了研究。从100个随机引物中筛选出12个RAPD引物,扩增共得到158条带,其中129个多态性位点（PPL）。POPGENE分析显示安徽羽叶报春具有较丰富的遗传变异（PPL=81.65%,He=0.2515,Ho=0.3849）。Nei′s基因多样性指数计算的居群间遗传分化系数（GST=0.5511）与Shannon信息指数（54.48%）基本一致。生境的片段化和基因流障碍可能是导致居群间遗传分化显著的主要原因。针对安徽羽叶报春的居群遗传变异提出了相应的保护措施：保护好自然生境和现有的居群及个体;加强居群间的基因流动;在迁地保护过程中,在尽可能多的居群中采样,以提高栽培居群的遗传多样性。     

珍稀濒危植物长叶红砂种群遗传多样性的ISSR分析

采用ISSR分子标记技术,对濒危小灌木长叶红砂(Reaumuria trigyna) 集中分布的5个种群的遗传多样性水平和遗传结构进行了研究。14条引物共检测到114个位点,其中99个为多态位点,多态位点比率为86.84％,长叶红砂种群具有较高的遗传多样性。物种水平上Shannon多样性指数(I)为0.468 8,Nei基因多样性指数(H)为0.308 4;种群水平上,多态位点比率P为77.89%,I为0.410 6,H为0.260 9,基因分化系数Gst为0.106 9,揭示了长叶红砂种群遗传变异多存在于种群内,种群间的遗传分化较小,占10.69%。 基因流(Nm)为4.178 7＞1,说明种群间的基因交流,防止了由于遗传漂变导致的遗传分化。聚类分析表明长叶红砂种群遗传距离与地理距离之间无显著的相关性。研究结果说明遗传多样性水平与物种本身特性和所处不同群落有关,濒危植物并不一定表现为遗传变异水平的降低。     

Molecular Evidence Shows Low Species Diversity of Coral-Associated Hydroids in Acropora Corals

A novel symbiosis between scleractinians and hydroids (Zanclea spp.) was recently discovered using taxonomic approaches for hydroid species identification. In this study, we address the question whether this is a species-specific symbiosis or a cosmopolitan association between Zanclea and its coral hosts. Three molecular markers, including mitochondrial 16S and nuclear 28S ribosomal genes, and internal transcribed spacer (ITS), were utilized to examine the existence of Zanclea species from 14 Acropora species and 4 other Acroporidae genera including 142 coral samples collected from reefs in Kenting and the Penghu Islands, Taiwan, Togian Island, Indonesia, and Osprey Reef and Orpheus Island on the Great Barrier Reef, Australia. Molecular phylogenetic analyses of the 16S and 28S genes showed that Acropora-associated Zanclea was monophyletic, but the genus Zanclea was not. Analysis of the ITS, and 16S and 28S genes showed either identical or extremely low genetic diversity (with mean pairwise distances of 0.009 and 0.006 base substitutions per site for the 16S and 28S genes, respectively) among Zanclea spp. collected from diverse Acropora hosts in different geographic locations, suggesting that a cosmopolitan and probably genus-specific association occurs between Zanclea hydroids and their coral hosts.     

Using Species Distribution Model to Estimate the Wintering Population Size of the Endangered Scaly-Sided Merganser in China

Scaly-sided Merganser is a globally endangered species restricted to eastern Asia. Estimating its population is difficult and considerable gap exists between populations at its breeding grounds and wintering sites. In this study, we built a species distribution model (SDM) using Maxent with presence-only data to predict the potential wintering habitat for Scaly-sided Merganser in China. Area under the receiver operating characteristic curve (AUC) method suggests high predictive power of the model (training and testing AUC were 0.97 and 0.96 respectively). The most significant environmental variables included annual mean temperature, mean temperature of coldest quarter, minimum temperature of coldest month and precipitation of driest quarter. Suitable conditions for Scaly-sided Merganser are predicted in the middle and lower reaches of the Yangtze River, especially in Jiangxi, Hunan and Hubei Provinces. The predicted suitable habitat embraces 6,984 km of river. Based on survey results from three consecutive winters (2010–2012) and previous studies, we estimated that the entire wintering population of Scaly-sided Merganser in China to be 3,561 ± 478 individuals, which is consistent with estimate in its breeding ground.