Detection of Low Genetic Variation in a Critically Endangered Chinese Pine, Pinus squamata, Using RAPD and ISSR Markers

With only 32 individuals in the northeastern corner of Yunnan Province, China, Pinus squamata is one of the most endangered conifers in the world. Using two classes of molecular markers, RAPD and ISSR, its very low genetic variation was revealed. Shannon's index of phenotypic diversity (I) was 0.030, the mean effective number of alleles per locus (A_e) was 1.032, the percentage of polymorphic loci (P) was 6.45, and the expected heterozygosity (H_e) was 0.019 at the species level based on RAPD markers. The results of ISSR were consistent with those detected by RAPD but somewhat higher (I = 0.048, A_e = 1.042, P = 12.3, H_e = 0.029). The genetic variation of the subpopulation on the southwest-facing slope was much higher than that of the subpopulation on the northeast-facing slope, which may be attributed to the more diverse environment on the southwest-facing slope. The genetic differentiation between the two subpopulations was very low. The between-subpopulation variabilities, Φ_ST, calculated from RAPD and ISSR data were 0.011 and 0.024. Because of the lack of fossil records and geological historical data, it was difficult to explain the extremely low genetic diversity of the species. We postulate that this ancient pine might have experienced strong bottlenecks during its long evolutionary history, which caused the loss of genetic variation. Genetic drift and inbreeding in post-bottlenecked small populations may be the major forces that contribute to low genetic diversity. Human activities such as logging may have accelerated the loss of genetic diversity in P. squamata.     

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.     

Genetic diversity analysis and conservation of the endangered Chinese endemic herb Dendrobium officinale Kimura et Migo (Orchidaceae) based on AFLP

Dendrobium officinale is a critically endangered perennial herb endemic to China. Determining the levels of genetic diversity and patterns of population genetic structure of this species would assist in its conservation and management. Data of 12 populations were used to assess its genetic diversity and population structure, employing the method of amplified fragment length polymorphism (AFLP). A high level of genetic diversity was detected (H (E) = 0.269) with POPGENE. As revealed by AMOVA analysis, there was moderate variation between pairs of populations with Phi(ST) values ranging from 0.047 to 0.578 and on average 26.97% of the genetic variation occurred among populations. Three main clusters were shown in UPGMA dendrogram using TFPGA, which is consistent with the result of principal coordinate ananlysis (PCO) using NTSYS. Keeping a stable environment is critical for the in situ conservation and management of this rare and endangered plant, and for ex situ conservation it is important to design an integrated germplasm bank.     

广东省五节芒遗传多样性的ISSR分析

采用ISSR分子标记技术,对广东省内分布的能源植物五节芒(Miscanthus floridulus)13个野生居群共215个个体的遗传多样性进行了研究.9个ISSR引物共扩增到了81个位点,其中76个是多态性位点.结果表明,广东五节芒的遗传多样性水平很高,物种水平上,多态位点百分率(PPB)为93.83％,Nei's...     

濒危物种四合木与其近缘种霸王遗传多样性的比较研究

 采用垂直板聚丙烯酰胺凝胶电泳技术,对西鄂尔多斯高原的濒危植物四合木(Tetraena mongolica)种群和生长于同一生境的普通种霸王(Zygophyllon xanthoxylon)种群的遗传多样性和遗传分化进行了比较研究。等位酶分析表明,与其他灌木相比,两种植物均维持较高的遗传多样性。但濒危植物四合木种群遗传多样性水平低于霸王种群,多态位点百分率P分别为60％和83.3％,等位基因平均数A为1.6和2.2,平均期望杂合度He为0.245和0.392。种群间遗传分化不明显,基因分化系数Gst为0.051和0.020。这一结果说明不同类型的濒危植物具有不同的遗传含义,采取的保护措施也不尽相同。     

濒危植物华东黄杉种群遗传多样性ISSR分析

采用ISSR分子标记技术分析了5个华东黄杉居群的遗传多样性水平和居群遗传结构。用10个引物对5个居群共76个样品进行扩增,共得到76条清晰的扩增带,其中69个位点为多态性位点。在物种水平上,多态性百分率（PPB）为90.79%,Nei’s基因多样性指数（H）为0.3361,Shannon’s信息多样性指数（I）为0.4974。在居群水平上,多态性位点百分率在56.58%～85.53%之间,Nei’s基因多样性指数为0.2084～0.2675,Shannon’s信息多样性指数为0.2695～0.4076。物种和居群遗传多样性水平都较高,居群间产生了一定的遗传分化（Gst=0.2366,Φst=26.88%）。由UPGMA聚类分析可知,在5个居群中,湖南阳明山两支与浙江临安先聚为一支,再与安徽休宁聚为一支,最后与江西三清山聚合。鉴于华东黄杉生境的特殊性和人为大量的砍伐,华东黄杉的数量逐渐减少,建议在华东黄杉分布区建立自然保护区,对其生存空间进行长期、有效的保护。     

云南金钱槭 等位酶 遗传多样性 生境片段化 保育策略

云南金钱槭（Dipteronia dyeriana）是我国特有的国家Ⅱ级重点保护珍稀濒危植物,仅分布于云南的文山、屏边和蒙自三县交界地区。采用水平切片淀粉凝胶电泳等位酶分析方法,对采自三个县的天然居群的63个样品进行了遗传多样性检测。8个酶系统的13个等位基因位点分析表明,云南金钱槭的遗传多样性水平低,其平均多态位点百分率P=15.4%,每位点平均等位基因数A=1.2,平均预期杂合度He=0.064,各居群都偏离Hardy-Weinberg平衡,杂合子过量,居群间的基因分化系数GST=0.099,遗传变异主要发生在居群内（90.1%）,居群间分化较小,居群间遗传一致度较高（I=0.985～1）,具有一定的基因流（Nm=2.669）。不加权对平均法（UPGMA）聚类可将4个居群分为两支,水头箐居群和马家寨居群亲缘关系最近,聚类后与黑洞居群形成一支再和中槽子居群聚类,这与其地理分布格局大致吻合。据此提出了关于云南金钱槭保育策略的建议。     

Development and characterization of microsatellite markers for Emmenopterys henryi (Rubiaceae), a rare tree from China

? Premise of the study: Compound microsatellite primers were developed for Emmenopterys henryi, an endangered deciduous tree endemic to China, to assess its genetic diversity and population structure as well as its evolutionary history. ? Methods and Results: Using the compound microsatellite marker technique, 10 pairs of polymorphic microsatellite primers were isolated and characterized in E. henryi. Levels of polymorphism were tested across a total of 63 individuals from three natural populations. Allele numbers varied from 10 to 20 per locus, with an average of 14.50 alleles per locus. The observed heterozygosity per locus ranged from 0.125 to 0.962, and the expected heterozygosity ranged from 0.377 to 0.903. ? Conclusions: The highly polymorphic markers developed and characterized in this study will facilitate evolutionary and population genetic studies in E. henryi.     

Genetic Structure Within and Among Populations of Saruma henryi, an Endangered Plant Endemic to China

The endangered perennial plant Saruma henryi Oliv. is endemic to China and has phylogenetic, ecological, and medicinal value. We used 10 microsatellite (SSR) loci to investigate genetic diversity and differentiation in 16 natural populations. Genetic diversity was high at the species level (H(E) = 0.9427, h = 0.9410, I = 3.0213) and low at the population level (H(E) = 0.4441, h = 0.4307, I = 0.6822). Pronounced genetic differentiation was detected among populations (G(ST) = 0.5428, F(ST) = 0.5524), in line with the limited among-population gene flow (Nm = 0.21). The significant isolation-by-distance pattern revealed by a Mantel test (r = 0.311, P = 0.001) suggested a clear geographic tendency in the distribution of genetic variability. Bayesian assignment and principal coordinates analyses supported the clustering of 16 populations into three groups. The present SSR results were also compared with previously published ISSR results. These results have significant implications for conservation of the species.     

珍稀濒危蕨类植物东方水韭的遗传多样性

采用ISSR分子标记对珍稀濒危蕨类植物东方水韭和中华水韭4个自然居群共72个样品进行了DNA多态性分析。从65个随机引物中筛选出11个有效引物,共产生98条DNA片段,其中68条为多态性条带,多态位点百分率(PPL)为66.33%。东方水韭两个居群的多态位点百分比率(PPL)较低(8.16%和7.14%)。中华水韭两个居群的PPL也较低(9.18%和23.47%)。中华水韭物种水平上的PPL(51.02%)高于东方水韭(9.18%)。AMO-VA分析结果表明,东方水韭遗传变异中主要存在于居群内(94.74%),而中华水韭绝大部分的遗传变异却存在于居群间(86.71%)。东方水韭和中华水韭个体间UPGMA聚类结果表明:同一物种的个体完全能聚在一起,中华水韭的两个居群也能明显分开,而东方水韭两居群的个体并不能完全聚在一起。探讨了可能造成上述居群遗传结构模式的主要因素,同时提出今后工作中需重点解决的问题。     

ISSR and SRAP Markers Reveal Genetic Diversity and Population Structure of an Endangered Slipper Orchid,Paphiopedilum micranthum (Orchidaceae)

Paphiopedilum micranthum is an endangered pink slipper orchid mainly distributed in the limestone areas of southwestern China. Wild populations of this species have been seriously threatened by excessive collections, rampant smuggling for export, and habitat destruction. We used 15 ISSR markers and 11 SRAP markers to investigate the genetic diversity and structure of 15 natural populations. A high degree of diversity was observed at the species level (ISSR: PPB=9166%, He=03839; SRAP: PPB=9929%, He=02806). Certain degree of genetic differentiation among populations (ISSR: Gst=02577; SRAP: Gst=02383) was detected maybe caused by low gene flow (ISSR: Nm=07201; SRAP: Nm=07991). Consistent with the results of Principal Coordinate Analysis, the UPGMA dendrogram analysis divided the 15 populations into two main clades. In addition to geographic distance, the difference in elevation was another natural factor contributing to this differentiation. Knowledge about genetic diversity and structure gained from our study will be beneficial for the development of reasonable and efficient strategies to conserve this endangered species.     

Allozyme variation in endangered Castanea pumila var. pumila

Allozyme genetic variation in 12 populations of the endangered Castanea pumila var. pumila (Allegheny chinkapin), sampled across the natural range of the species in the United States, was evaluated using 11 loci from seven enzyme systems. At the species level, the percentage of polymorphic loci (Ps) was 72.7 %, the mean number of alleles per locus (As) was 1.9, the mean number of alleles per polymorphic locus (APs) was 2.3, the effective number of alleles per locus (Aes) was 1.5 and the genetic diversity (Hes) was 0.296. At the population level, Pp = 49.2 %, Ap = 1.5, Aep = 1.4, APp = 2.1 and Hep = 0.21. Most of the allozyme variation (70 %) in C. pumila var. pumila occurred within populations. Wright's gene flow rate [Nm(W)] was as low as 0.57. Population differentiation along the species range was not detected. Populations of C. pumila var. pumila in Florida had the most variable levels of genetic diversity, but populations in Virginia and Mississippi also showed high levels. Based on the results of this study, conservation management strategies are discussed.     

Genetic variation within the endangered mangrove species Sonneratia paracaseolaris (Sonneratiaceae) in China detected by inter-simple sequence repeats analysis

Sonneratia paracaseolaris, is a critically endangered mangrove species in China. Using inter-simple sequence repeats (ISSR) markers, we compared the genetic variation of introduced populations with that of natural populations to check whether the genetic diversity has been conserved. At the species level, genetic diversity was relatively high (P = 81.37%, He = 0.2241, and SI = 0.3501). Genetic variation in introduced populations (P = 75.78%, He = 0.2291, and SI = 0.3500) was more than that in natural populations (P = 70.81%, He = 0.1903, and SI = 0.2980). Based on Nei's G_ST value, more genetic differentiation among natural populations was detected (G_ST = 0.3591). Our data show that the genetic diversity of S. paracaseolaris was conserved in introduced populations to some extent, however, owing to the small natural populations and the threats they encountered, more plants should be planted to enlarge and restore the populations.     

Genetic diversity of natural and cultivated populations ofOenanthe javanica in Korea

Enzyme electrophoresis was used to estimate genetic diversity and population structure in natural and cultivatedOenanthe javanica (Blume) DC. In the six natural populations, 8 of the 22 loci showed polymorphisms. Cultivated populations had fewer alleles per locus (1.84 vs. 1.91), fewer effective alleles per locus (1.47 vs. 1.52), a lower percentage of polymorphic loci (42.3 vs. 50.0), and lower diversity (0.210 vs. 0.228) than did natural populations. These parameters of genetic diversity indicate that the cultivated populations are genetically depauperate relative to their presumptive progenitor, and that the domestication process has partly eroded the level of genetic variation of this species. Nevertheless, the diversity of this species has higher-than-average values compared with other species having similar life-history traits. We propose that the mix-mating system; perennial, high gene flow; and large population sizes are possible factors contributing to this high diversity, which seemed to increase with distance from the coastlines.     

Genetic Variation in Remnant Populations of the Woolly Spider Monkey (Brachyteles arachnoides)

The muriqui or woolly spider monkey (Brachyteles arachnoids) is an endangered primate endemic to the Atlantic Forest of Brazil, <5% of which remains. The known muriqui population consists of <700 individuals separated into approximately 15 geographically isolated forest fragments. I present data on the distribution of genetic variation within and between two such remnant populations (FE and FBR) and summarize the implications of these results for long-range management of species genetic diversity. Eleven of 32 allozyme loci were polymorphic, representing an overall level of polymorphism of 34.4% and a mean heterozygosity per locus of 11%. Both values are among the highest reported for New World monkeys. Genetic differentiation between the two localities is highly significant (F_ST = 0.413, p < 0.001). Genetic distance between them is an order of magnitude greater than that between other populations of platyrrhine subspecies, but this could be an artifact of the small sample size from FBR. High levels of genetic diversity apparently characteristic of this species persist because (1) fragmentation and size reduction of muriqui populations has occurred very rapidly relative to the muriqui life span—although both polymorphism and heterozygosity were lost between generations in the largest population, the high genetic diversity present in the parent population was still in evidence; and (2) genetic diversity before population fragmentation by human activity was not distributed uniformly throughout the species' historic distribution. Thus, remnant muriqui populations are important genetic reservoirs of alleles that are unique or rare in the species gene pool as a whole. These results emphasize the need for the integration of conservation management efforts throughout the species range.     

Analysis of genetic diversity in the endangered Hucho taimen from China

Hucho taimen are listed as endangered in China. The population size has declined recently, prompting an increase in the level of listing from grade three in 2002 to grade five in 2006. We analyzed the genetic diversity of wild populations using 17 microsatellite markers to establish a scientific basis for conservation of this species. We collected tissue samples from four populations in the Heilongjiang River basin: Huma River (HM), Hutou (HT), Haiqing (HQ), and Zhuaji (ZJ). A total of 21 loci were amplified, 18 of which were polymorphic. The number of alleles per locus ranged from 2 to 9 (mean: 4.1905). There were 13 highly polymorphic loci and 5 moderately polymorphic loci. Analysis of five genetic diversity parameters (Na, Ne, Ho, He, and PIC) suggested moderate levels of diversity within the populations. The populations were ranked HT > HQ > ZJ > HM, but the differences in diversity were not statistically significant (P > 0.05). A comparison of variation among all four populations suggested Hardy–Weinberg disequilibrium at 20% of the loci. Genetic differentiation (Fst) was 0.0644 and the gene flow among populations was estimated at 3.36 individuals per generation. The majority of diversity (93.88%) occurred among individuals within a population. In contrast, relatively little (6.12%) of the genetic diversity was distributed between the populations. An analysis of genetic differentiation and genetic distance between pairs of populations revealed that both parameters were higher in comparisons of the HM population to the HT, HQ, and ZJ populations than among the three latter populations. This suggests that the HM population has a distinct genetic structure. We hypothesize that habitat degradation and excessive fishing, not low genetic diversity, has caused the decline in H. taimen populations. However, this species should be protected from further declines in genetic diversity.     

Genetic diversity in Nothofagus alessandrii (Fagaceae), an endangered endemic tree species of the coastal maulino forest of Central Chile

BACKGROUND AND AIMS: The endemic tree Nothofagus alessandrii (Fagaceae) has been historically restricted to the coastal range of Region VII of central Chile, and its forests have been increasingly destroyed and fragmented since the end of the 19th century. In this study, the patterns of within- and among-population genetic diversity in seven fragments of this endangered narrowly endemic tree were examined. METHODS: Allozyme electrophoresis of seven loci of N. alessandrii was used to estimate genetic diversity, genetic structure and gene flow. KEY RESULTS: High levels of genetic diversity were found as shown by mean expected heterozygosity (H(e) = 0.182 +/- 0.034), percentage of polymorphic loci (P(p) = 61.2 %), mean number of alleles per locus (A = 1.8) and mean number of alleles per polymorphic locus (A(p) = 2.3). Genetic differentiation was also high (G(ST) = 0.257 and Nm = 0.7). These values are high compared with more widespread congeneric species. CONCLUSIONS: Despite its endemic status and restricted geographical range N. alessandrii showed high levels of genetic diversity. The observed patterns of diversity are explained in part by historical processes and more recent human fragmentation.     

珍稀濒危植物硬叶兜兰的遗传多样性及遗传结构研究

由于人为采集、走私贩卖以及生境的破坏,分布于中国西南石灰岩地区的野生硬叶兜兰居群受到严重的干扰与威胁。为有效地保护这种珍稀野生植物,本研究采用ISSR和SRAP两种分子标记对15个硬叶兜兰野生居群进行遗传多样性及遗传结构的研究。结果表明,硬叶兜兰在物种水平上具有较高的遗传多样性（ISSR：PPB=91．66％,He=0．3839;SRAP：PPB=99．29％,Hc=0．2806）。硬叶兜兰居群间存在一定程度的遗传分化（ISSR：Gs1： 0．2577;SRAP：Gst=0．2383）,可能由于较低的基因流（ISSR：Nm=0．7201;SRAP：Nm=0．7991）所致。UPGMA聚类分析以及主成分分析均把15个居群分成2个主要分支。居群间的地理距离和海拔差距是引起居群遗传分化的自然因素。     

Genetic Variation in the Endangered Endemic Species Cycas fairylakea (Cycadaceae) in China and Implications for Conservation

Cycas fairylakea is an endangered endemic species in China. Genetic diversity within and among four natural populations of this species in China was investigated using amplified fragments length polymorphism (AFLP). A moderate to low level of intraspecific genetic diversity was detected in this species (at population level: P = 39.57 %, H₀ = 0.244; at species level: P = 60.22%, H₀ = 0.356). The among-population component accounted for, respectively, 25.7 and 31.5% of the genetic variation, according to AMOVA and Shannon’s index, indicating most of the genetic variation was found between individuals within populations. All four populations have opposite pyramid age structure, and few coning individuals, which is still decreasing. Possibly because of habitat degradation and environmental pollution, plant diseases and insect pests in the populations were extremely serious, suggesting that the main factors threatening the survival of C. fairylakea populations were not genetic variation, but human activities and the breeding system of this species.     

Isolation and characterization of polymorphic microsatellite markers in <Emphasis Type="Italic">Cupressus </Emphasis><Emphasis Type="Italic">chenggiana</Emphasis> S. Y. Hu (Cupressaceae)

Cupressus chenggiana S. Y. Hu (Cupressaceae) is an endemic and endangered conifer species in southwest China. In order to study the population genetics and design the effective conservation methods, we aimed to develop microsatellite primers for this species in the present study. We developed eight new microsatellite loci for this species through biotin capture method. Polymorphism of each locus was further assessed in 18 individuals from three geographically distant populations. The number of alleles per locus ranged from 6 to 11 with an average of 8.13. The observed and expected heterozygosities ranged from 0.219 to 0.296 and from 0.374 to 0.470, with averages of 0.254 and 0.417, respectively. We further found that three of nine microsatellite loci developed previously for another congeneric species showed polymorphic banding patters. We performed primer-crossing tests of these loci in the other two congeneric species which are closely related to C. chenggiana (C. gigantea and C. duclouxiana). These microsatellite markers would be effective for analyzing genetic diversity and population genetic structure of this species and its morphological differentiation with the close relatives.