Conservation genetics of an endangered orchid in eastern Canada

Platanthera leucophaea, the Eastern Prairie Fringed-orchid, is a globally imperilled species native to the USA and Canada, with its Canadian distribution limited to the Province of Ontario. In Canada, where P. leucophaea is listed as endangered, approximately 40 % of populations have been extirpated in recent years, and many remaining populations have experienced substantial declines. In this study, we investigated whether reduced population sizes have led to low genetic diversity and inbreeding. We also investigated the extent to which hybridization with Platanthera psycodes may be threatening the genetic integrity of P. leucophaea populations. We found that overall, genetic diversity is low, and inbreeding is high. This is despite evidence of regular gene flow between proximate populations, although more distant populations show high levels of genetic differentiation. At sites where P. leucophaea is sympatric with P. psycodes, interspecific hybridization occurs in a bidirectional manner, i.e. with both parental species acting as either pollen donor or pollen recipient. Inbreeding and low genetic diversity in all populations, and hybridization in some populations, may pose future threats to P. leucophaea, and should be considered in the future by biodiversity managers.     

Conservation genetics of endangeredBrasenia schreberi based on RAPD and AFLP markers

Brasenia schreberi J.F. Gmelin is a declared endangered species found in the lakes and ponds of South Korea. For planning its conservation strategy, we examined the genetic diversity within and among six populations, using randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Polymorphisms were more frequently detected per loci with AFLP (69.3%) than RAPD (36.8%). High genetic diversity was recognized within populations: polymorphic loci (PPL) values ranged from 36.3% in the CJM population to 74.5% in the GGT population, with a mean value of 47.8% based on AFLP markers. Great genetic differentiation (θ^B) was detected among the six populations (0.670 on RAPD and 0.196 on AFLP), and we calculated a low rate of gene flow (N_em), i.e., 0.116 on RAPD and 0.977 on AFLP. Furthermore, a Mantel test revealed that no correlation existed between genetic distances and geographical distances among the six local populations, based on RAPD or AFLP markers. These results are attributed to a number of factors, including an insufficient length of time for genetic diversity to be reduced following a natural decline in population size and isolation, adaptation of the genetic system to small population conditions, and a restricted gene flow rate. Based on both its genetic diversity and population structure, we suggest that a strategy for conserving and restoringB. schreberi must focus on maintaining historical processes, such as high levels of outbreeding, while monitoring increased gene flow among populations. This is because a reduction in genetic diversity as a result of genetic drift is undesirable.     

Conservation genetics of Heritiera littoralis (Sterculiaceae), a threatened mangrove in China,based on AFLP and ISSR markers

AFLP and ISSR markers were used to determine the genetic variations in eight mangrove and non-mangrove populations of Heritiera littoralis (Sterculiaceae), a threatened species in China. Our results showed a moderate to high level of genetic variation in this species (P = 63.69%, H_T = 0.20 for AFLP; P = 76.07%, H_T = 0.22 for ISSR), and a relatively high level of genetic differentiation among populations (G_ST = 0.24 for AFLP and 0.27 for ISSR). Life history traits, long-distance dispersal of floating seeds, and local environments may play important roles in shaping the genetic diversity and genetic structure of this species. Investigation of the plant’s reproductive capacity showed that the natural seed production of H. littoralis was low, as was the germination rate and the transformation rate from juvenile to adult. H. littoralis is seriously threatened and is in urgent need of conservation in China.     

Identity and Specificity of Rhizoctonia-Like Fungi from Different Populations of Liparis japonica (Orchidaceae) in Northeast China

Mycorrhizal association is known to be important to orchid species, and a complete understanding of the fungi that form mycorrhizas is required for orchid ecology and conservation. Liparis japonica (Orchidaceae) is a widespread terrestrial photosynthetic orchid in Northeast China. Previously, we found the genetic diversity of this species has been reduced recent years due to habitat destruction and fragmentation, but little was known about the relationship between this orchid species and the mycorrhizal fungi. The Rhizoctonia-like fungi are the commonly accepted mycorrhizal fungi associated with orchids. In this study, the distribution, diversity and specificity of culturable Rhizoctonia-like fungi associated with L. japonica species were investigated from seven populations in Northeast China. Among the 201 endophytic fungal isolates obtained, 86 Rhizoctonia-like fungi were identified based on morphological characters and molecular methods, and the ITS sequences and phylogenetic analysis revealed that all these Rhizoctonia-like fungi fell in the same main clade and were closely related to those of Tulasnella calospora species group. These findings indicated the high mycorrhizal specificity existed in L. japonica species regardless of habitats at least in Northeast China. Our results also supported the wide distribution of this fungal partner, and implied that the decline of L. japonica in Northeast China did not result from high mycorrhizal specificity. Using culture-dependent technology, these mycorrhizal fungal isolates might be important sources for the further utilizing in orchids conservation.     

Conservation genetics assessment and phylogenetic relationships of critically endangered Hucho bleekeri in China

Hucho bleekeri is a critically endangered salmonid fish found in the Yangtze River drainage in China. In this study, the genetic diversity of a small population (n = 43) was first assessed with partial mitochondrial DNA sequences (D‐loop region and a cytochrome b gene [CYTB] gene fragment) and 15 microsatellite markers. Low levels of nucleotide diversity (Pi) were demonstrated in the H. bleekeri population based on the two mitochondrial DNA markers. The number of haplotypes (h) and the haplotype diversity (Hd) in the D‐loop region (12 haplotypes and Hd = 0.8208) were higher than in the CYTB gene fragment (three haplotypes and Hd = 0.0941). The number of microsatellite alleles (Na) ranged from 2 to 13 in these individuals. The mean observed heterozygosity (Ho) and the expected heterozygosity (He) were 0.59719 and 0.44735, respectively. Analysis of molecular variance showed that the degree of differentiation in the population was low (F_ST = 0.04041) and the coefficient of inbreeding (F_IS) was negative, indicating no obvious evidence of inbreeding in this population. A demographic assessment suggested that this species expanded a long time ago, but has suffered great losses in recent years. A molecular phylogenetic analysis clearly indicated that H. bleekeri is not introgressed by Brachymystax lenok tsinlingensis. The baseline population genetic information supplied by this study will be vital in monitoring this highly threatened species.     

Conservation genetics of endangered leaf-beetle Cheilotoma musciformis populations in Poland

Steppe-like habitats in Europe are seriously threatened as a result of fragmentation and anthropogenic degradation, at least in western and central parts. Considering the dramatic loss of steppe-like habitats, the evaluation of genetic variation in populations of steppe species is of immediate importance if appropriate conservation measures are to be undertaken. In this paper, we examine the genetic diversity of the highly endangered populations of the leaf-beetle Cheilotoma musciformis, which inhabits only a limited area in south-central Poland, which is geographically isolated from the continuous range of this species. Both mitochondrial and nuclear markers show that the Polish populations are distinct from Slovakian and Ukrainian ones. These regional populations should be considered independent conservation units. On the other hand, very little (mtDNA) or no (nuclear DNA) diversity has been found among the Polish subpopulations. This leads to the conclusion that this species has gone through a strong bottleneck leading to a drastic reduction in its genetic diversity prior to the establishment of present-day populations. Host plants have been identified for this species using barcodes, and the only hosts for the Polish and Ukrainian samples are sainfoins Onobrychis spp. while for the Slovakian sample it is either Dorycnium pentaphyllum or Lotus spp. (all Fabaceae). All of these data can be very valuable for the conservation of C. musciformis populations (e.g. for reintroductions).     

Phylogenetic and conservation status of the endangered terrestrial orchid Nervilia nipponica (Orchidaceae) in Korea

The putative Japanese endemic Nervilia nipponica was discovered recently on Jeju Island in Korea. However, Japanese populations of N. nipponica have since been shown to comprise two polyphyletic taxa. To confirm the taxonomic status of the Korean Nervilia populations, we compared ITS1, matK and trnL intron sequence data with that from the two cryptic Japanese taxa. Our results confirmed all Korean populations as belonging to the true N. nipponica s.s, which must shed its endemic status. Korean accessions showed no distinct genotypic patterning, with samples from all populations and representing two distinct colour forms forming a single monophyletic clade. The extremely low level of genetic diversity detected within N. nipponica on Jeju Island underscores the urgent need for measures to secure the species from local extirpation. We conclude that N. nipponica is a priority for conservation and recommend that systematic controls and sustained efforts are required to ensure its protection.     

濒危植物绵刺8个种群遗传多样性的AFLP分析

利用8对AFLP引物对我国绵刺的8个种群240份材料的基因组DNA进行扩增,得到大小在65～530bp之间的397条清晰显带,其中296(74.56%)条呈多态性,平均每对AFLP引物得到37条多态性带;用PopGen32软件将AFLP多态性数据进行分析,不同种群的Nei's基因多样性指数和Shannon信息指数变化范围分别在0.0845～0.1779和0.1280～0.2377之间,其中遗传多样性最高为上沙窝种群,最低为银根种群,可将上沙窝种群作为种质遗传中心之一进行保护.绵刺遗传变异有68.31%存在种群内,31.69%种群之间,说明变异主要存在于种群内部.8个种群的平均遗传距离为0.1341,按UPGMA进行聚类分析,结果表明绵刺种群具有明显的地域相关性和遗传类型趋同性,说明不同的种群可能有共同的起源,随机遗传漂变不是影响绵刺种群遗传多样性的主要过程.建议在迁地保护和取样时,不仅要在每个种群中取足够多的个体,而且要在尽可能多的种群中取样,最大限度地保护绵刺的遗传多样性,为进一步地系统演化研究奠定基础.     

Conservation genetics of two critically endangered island dwarf carnivores

Genetic diversity is crucial for conservation biology and for understanding evolutionary processes. Oceanic islands harbor a unique biota and high endemism, with populations frequently facing detrimental genetic processes (e.g. drift, bottlenecks). Human activities like habitat transformation further increase extinction risk of insular biota. Mammals comprise the most endangered group among insular fauna. Our aim was to evaluate the genetic and evolutionary patterns of two critically endangered dwarf carnivores from Cozumel Island, the pygmy raccoon (Procyon pygmaeus) and the dwarf coati (Nasua nelsoni), at both historical and contemporary evolutionary scales. We also reviewed their genetic relationships with their mainland counterparts (P. lotor, N. narica), not intended to describe their phylogeny but to ascertain their endemism. Our mitochondrial results support that both Cozumel carnivores are divergent from continental populations, strengthening their endemic status. Both species showed moderate levels of nuclear genetic diversity that were, as expected for island populations, lower in comparison with their mainland congeneric species; they also exhibited significantly low population sizes. We documented historical and contemporary bottleneck signals for P. pygmaeus, whereas N. nelsoni may be suffering the initial stages of a bottleneck not yet fully manifested. The pygmy raccoon is structured into two isolated genetic clusters likely due to interactions with humans on the north of the island, where most potential for disease transmission and health problems exist. We also add evidence about the introduction of the mainland species into the island, risking genetic introgression and hybridization. We discuss specific conservation measures that should include our genetic information, directed to the long-term viability of these endemic carnivores.

     

Conservation genetics of the endangered conifer Fitzroya cupressoides in Chile and Argentina

Intraspecific patterns of genetic variation can often be used toidentify biogeographic divisions which can be especially useful in thedesign of conservation strategies. Although abundant empirical evidenceexist on the genetic characteristics of plant species from the NorthernHemisphere as well as tropical endangered taxa, this information isparticularly limited on threatened species from endemism-rich areas inthe southern Andes of Argentina and Chile. The objective of the currentstudy was to analyze the levels and distribution of the isozymevariation in Fitzroya cupressoides (Mol.) Johnst.(Cupressaceae), a rare conifer restricted to temperate rainforests ofnorthern Patagonia, and to evaluate the role of current conservationareas protecting the gene pool of this valuable long-lived conifer.Sampling schedules consisted of fresh foliage collected from 30 randomlyselected trees at each of 24 different populations located along thegeographic range of the species. Extraction of enzymes followed standardprocedures and homogenates were loaded in 12% starch gels whichwere analyzed by horizontal electrophoresis. Eleven enzyme systems wereresolved using a combination of four different buffer solutions whichyielded information on 21 putative loci, 52% of them werepolymorphic in at least one population. Relatively low levels ofwithin-population genetic variability were scored in Fitzroyapopulations which were approximately half of the typical levelspublished for gymnosperms (percent of polymorphic loci, P = 23 vs.53% and expected heterozygosity, H_E = 0.077 vs. 0.155for Fitzroya and other conifers respectively). Substantialbetween-population variation was detected, and certain individualpopulations stand out as much more genetically variable than nearbypopulations, which in turn are located outside protected areas. Ourfindings suggest that if the objective is to protect key species likeFitzroya, spatially explicit genetic information can be auseful tool to attain this goal.     

Conservation genetics of the endangered Pampas deer (Ozotoceros bezoarticus)

The Pampas deer ( Ozotoceros bezoarticus L. 1758) is the most endangered neotropical cervid, and in the past occupied a wide range of open habitats including grassland, pampas, savanna, and cerrado (Brazil) from 5° to 41° S. To better understand the effect of habitat fragmentation on gene flow and genetic variation, and to uncover genetic units for conservation, we examined DNA sequences from the mitochondrial control region of 54 individuals from six localities distributed throughout the present geographical range of the Pampas deer. Our results suggest that the control region of the Pampas deer is one of the most polymorphic of any mammal. This remarkably high variability probably reflects large historic population sizes of millions of individuals in contrast to numbers of fewer than 80 000 today. Gene flow between populations is generally close to one migrant per generation and, with the exception of two populations from Argentina, all populations are significantly differentiated. The degree of gene flow was correlated with geographical distance between populations, a result consistent with limited dispersal being the primary determinant of genetic differentiation between populations. The molecular genetic results provide a mandate for habitat restoration and reintroduction of Pampas deer so that levels of genetic variation can be preserved and historic patterns of abundance can be reconstructed. However, the source of individuals for reintroduction generally should be from populations geographically closest to those now in danger of extinction.     

Planning the priority protected areas of endangered orchid species in northeastern China

We used endangered orchid species as a case study for establishing priority protected areas (PPAs). We conducted a detailed investigation of 2,884 plots in northeastern China and selected 6 nationally protected orchid species (the number of plots for each species ≥5) to plan PPAs in order to conserve these endangered plants. First, we estimated the potential habitat distributions of 6 orchid species using species distribution models (in the program Maxent). Second, we identified PPAs using the results of both Maxent and systematic conservation planning software (Zonation). We found that the PPAs we identified were all distributed in the eastern, northeastern, and northern parts of the study region on which we focused. We observed that existing nature reserves in those areas were much smaller than the PPAs we identified, suggesting the need for expansion of these zones. Finally, we identified some recommended protection areas based on our PPAs, which we believe are suitable areas of in situ conservation. These areas could assist in the formulation of protection policies for these endangered orchid species. Our results have broad implications for conservation biology and the redefinition of protective zones for at-risk species.     

Conservation genetics of the endangered endemic Hawaiian genus Brighamia (Campanulaceae)

The endemic Hawaiian genus Brighamia (Campanulaceae) comprises two federally endangered, morphologically similar species, B. insignis from Kaua`i and Ni`ihau and B. rockii from Moloka`i. To assist the design of conservation management programs for these taxa, isozyme analyses were performed to assess the levels of genetic diversity at the population and species levels, including comparisons within and among seven natural populations and one ex situ collection each of B. insignis and B. rockii. Our sampling (N = 80) represents ~41% of all known individuals in the wild. Isozyme analyses revealed levels of genetic variation comparable to those reported for other Hawaiian flowering plant taxa but low levels of genetic variation at the population and species levels when compared to flowering plants in general. Ex situ individuals (N = 61) were genetically representative of natural populations and hence may appropriately serve as stock for population augmentations. The two morphologically similar Brighamia species were highly distinct genetically. The combination of morphological and ecological similarity with allozymic dissimilarity observed in Brighamia is unique among the Hawaiian taxa studied to date.     

Conservation genetics of the rare and endangered Leucopogon obtectus (Ericaceae)

Leucopogon obtectus Benth. is a declared rare species found in the kwongan vegetation in Western Australia. Plants on a mineral sand mine and the rehabilitation area are subject to disturbance. Genetic diversity was examined within and among all known populations using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLPs) for conservation. Both molecular markers revealed a high percentage (> 89%) of polymorphic markers and a high mean genetic distance among individuals (D = 0.3). Analysis of molecular variance showed that 86.7% (RAPD) and 89.7% (AFLP) of variability was partitioned among individuals within populations. Exact tests showed no significant population differentiation. The analyses indicated that L. obtectus exhibits high levels of genetic diversity despite small population sizes. The high levels of variability among individuals and the lack of clear population differentiation suggest that this species comprises a single, genetically diverse group. Conservation and management of L. obtectus should concentrate on maintaining the high levels of genetic variability through mixing genotypes and promoting outcrossing.     

Chemical constituents from the whole plant of Liparis japonica

A new lignanoid (1), together with six known lignanoids (2–7) and four known flavonoids (8–11), were isolated from the EtOAc extract of Liparis japonica. Their structures were determined by analyzing the HRESIMS, NMR, CD spectroscopic data, and comparing with the reported literatures. Among them, 1 was a new compound, 2–11 were first isolated from L. japonica and the genus of Liparis. The chemotaxonomic significance of these isolates was comprehensively discussed.     

曲唇羊耳蒜在中国东北分布的真实性研究

曲唇羊耳蒜分布于日本、朝鲜和俄罗斯远东地区,中国东北的黑龙江、吉林和辽宁也有报道,但是《中国植物志》认为,该种在中国东北是否存在尚有很大的疑问。本研究证明曲唇羊耳蒜的确在中国东北有分布。     

微卫星分子标记在濒危动物保护遗传学研究中的应用

微卫星DNA广泛分布于真核生物基因组中,具有多态性高、共显性遗传、选择中性、易于操作等特点,是一种极具应用价值的分子遗传标记,近年来在濒危动物保护遗传学研究中得到越来越多的应用。微卫星DNA高度多态性提供的高分辨率遗传信启,使其不仅适合个体水平的亲子鉴定与交配系统研究,而且也已成为种群遗传结构与多样性分析的有效分子标记。微卫星分析所需的DNA量极少,用非损伤性方法获取的极少量样品或陈旧样品就能用于有效分析,方便了濒危动物野外调查工作的开展,并且可以利用年代久远的馆藏历史标本揭示种群的重要历史进程。另外,某些微卫星DNA大小在近缘物种间可相互区分,这使得部分物种的DNA分子鉴别将更为简便。但微卫星分子标记的座位筛选和特异引物开发耗时费力,一定程度上限制了其广泛应用。针对不同的研究目的选择合适的分子标记方法将有助于更好的揭示问题本质。